From a Sperm's Eye View—Revisiting Our Perception of This Intriguing Cell

MILNE LECTURE

From a Sperm’s Eye View—Revisiting Our Perception of This Intriguing Cell

Dickson D. Varner, DVM, MS, Diplomate ACT; and Larry Johnson, MS, PhD

Authors’ address: Departments of Large Animal Clinical Sciences and Veterinary Integrative Bio- sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4475; e-mail: [email protected]. © 2007 AAEP.

1. Introduction and the events that accompany a spermatozoon’s The stallion represents one half of the breeding sojourn through the female reproductive tract, fol- equation; however, apportioned time and funding lowed by a discussion of some potential clinical ram- has been considerably less for efforts to advance the ifications. The lecture that accompanies this text discipline of stallion reproduction compared with will be directed, almost exclusively, to practical ap- that of mare reproduction. Modern-day fathers of plications and advancements in the discipline of stallion reproduction, such as Robert M. Kenney, stallion reproduction. Not all published work could Bill W. Pickett, and Marion Tischner, made signifi- be represented in this short review, and we preface cant strides in our understanding of stallion repro- this reading with an apology for unintentional ex- ductive physiology and clinical disease, but this clusion of pertinent data. Although we direct the fascinating area of study begs for individuals with reader to some relevant studies conducted in equids, the aptitude and tenacity required to take the disci- most of the information provided in this manuscript pline to new heights. In recent years, the level of stems from studies conducted in other species, interest in research directed toward the breeding where the process of discovery has been most pro- stallion has increased noticeably, as evidenced by nounced. Although this comparative approach can the 2006 Congress of the International Society of be quite enlightening, a drawback is that the rele- Equine Reproduction (ISER). The stallion was rep- vance to stallions of findings unveiled in other spe- resented in the largest section of papers presented cies will require documentation. and published at this meeting, a first since the in- A manuscript directed exclusively at the sperma- ception of the ISER in 1975. tozoon would seem to represent a miniscule part of This paper will be directed primarily at the male the total reproduction picture. On close scrutiny, gamete, the spermatozoon, and all the fascination however, one becomes enlightened about the level of that it bestows on those of us that have dedicated a scientific interest in this cell. As an example, large portion of our professional lives eavesdropping ϳ50,000 entries are logged in response to a query for on its microcosm. We will begin with a detailed the keywords, spermatozoa or spermatozoon, in overview of spermatozoal structure and function PubMed, a database of indexed citations offered by

NOTES

104 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE the National Institutes of Health. An uninformed tion of the outer acrosomal and overlying individual might describe a spermatozoon as a plasma membranes necessary for fusion and highly specialized, but simple, cell with only one role vesiculation) to fulfill—that of fertilization. Although fertiliza- 11. Penetration of the zona pellucida (release of tion is the endpoint of spermatozoal function, this acrosomal proteins with enzymatic activity cell must be extremely sophisticated and adaptable is required for this event to occur) to achieve this task, and the process involves a se- 12. Binding and fusion with the oolemma (re- ries of highly coordinated cellular and molecular quires specific region-dependent molecular events. The following is a list of some require- interactions) ments ascribed to a mammalian spermatozoon. 13. Dispersion of nuclear contents (requires specific fusogenic alterations of the lipid mem- 1. Loss of most organelles and cytoplasm dur- branes of the spermatozoon and oocyte) ing formation in the testis and maturation in 14. Oocyte activation (a spermatozoon-derived the epididymis (requires a host of intracellu- factor is required for activation of the oocyte lar and intercellular signaling events) and embryonic development) 2. Remodeling of spermatozoal chromatin 15. Pronucleus formation (requires decondensa- within the epididymis as a protective mech- tion of the highly compact spermatozoal anism against environmental injury (re- nucleus) quires repackaging of nuclear DNA into a 16. Organization of the mitotic spindle after pro- highly condensed form through the aid of nuclear formation (requires contribution of specialized proteins termed protamines) proximal centriole from the spermatozoon) 3. Plasma membrane alterations within the epididymis to yield proteins important to fertil- After viewing this lengthy list of functions, one ization (requires various enzymatic-linked becomes quite appreciative of the highly complex alterations of existing proteins and uptake of and specialized features of a spermatozoon. In fact, proteins from epididymal fluid or from the the biochemical and biophysical features are so so- epididymal epithelium) phisticated that many of the cellular and molecular 4. Passage through the uterus and uterotubal mechanisms remain unresolved to this day. Fur- junction of the female at the time of insemi- thermore, spermatozoa (and oocytes) represent nation (requires activated flagellar move- some of the most highly differentiated cells in the ments and protection against immunologic mammalian body; yet, when sperm and oocyte are attack) combined, they retain their potential for totipotency 5. Binding to oviductal epithelial cells to form a (ability to divide and produce all cell types of the spermatozoal reservoir (requires specific cell- body) through creation of a zygote. cell attachment, possibly mediated through spermatozoal surface carbohydrate-binding 2. Origin of the Spermatozoon proteins, termed lectins) The life of a spermatozoon begins within the testes, 6. Acquisition of additional maturational unless, of course, one wishes to consider the embry- changes, collectively termed capacitation, onic origin of the primordial germ cells. The testis, that permit a spermatozoon to fertilize an an elaborately designed organ, is classically consid- oocyte (requires an assortment of signal ered to possess two functions: (1) exocrine—sper- transduction cascades) matogenesis, and (2) endocrine—production of 7. Release from oviductal epithelial cells and hormones important to spermatogenesis, sexual dif- passage to the vicinity of the oocyte at the ferentiation, development of secondary sex charac- isthmic-ampullar junction of the oviduct (re- teristics, and libido. Although this simplistic quires a coordinated spermatozoal-release description provides one with the general concept of mechanism, hyperactivated motility, and testicular function, it does not portray the extremely probably chemotaxis) complex nature and elegant interplay of these two 8. Penetration through the extracellular matrix processes. Conventional descriptions convey the of the oocyte cumulus (possibly mediated by role of hypothalamic- and pituitary-derived hor- hyperactivated motility and redistribution/ mones on regulation of testicular function, as well as unmasking of surface-associated hyaluroni- feedback mechanisms required for homeostasis. dase, because the cumulus matrix is rich in Although such pathways are undoubtedly the key hyaluronic acid) orchestrators of testicular function, emerging infor- 9. Binding to the zona pellucida, a highly gly- mation is revealing a multitude of subcellular, mo- cosylated protein matrix surrounding the oo- lecular-mediated events that “cloud” our cyte (seems to involve specific affinity understanding of the events that actually occur between spermatozoal surface molecules and within the testes. As with any area of study, the the zona pellucida components) more learned we become about a topic, the more 10. Acquisition of the acrosome reaction, a regu- queries surface that require additional clarification. lated form of exocytosis (requires reorganiza- Such is the case with testicular function. Without

AAEP PROCEEDINGS ր Vol. 53 ր 2007 105 MILNE LECTURE question, a thorough understanding of testicular function will require a keen appreciation of the mechanisms by which genes and gene products are expressed and repressed.2–8 As an example of the genetic complexity surrounding control of testicular function, new information has revealed that single nucleotide polymorphisms (SNPs) have been identified in the follicle-stimulating hormone (FSH) receptor gene of humans, resulting in a mutation of the FSH receptor (as is found on the surface of the Sertoli cell) that can influence its activity.9 As another example, use of transgenic mice deficient in estrogen receptor genes has shown that estrogens are likely to play a more important role in testicular function than was once thought to be the case.10 Similarly, use of mice with a selective androgen receptor knockout in Sertoli cells revealed that the androgen receptor in the Sertoli cell is an absolute requirement for normal spermatogenesis.11,12 Furthermore, experimentation with germ cell–specific androgen receptor knockout mice revealed normal spermatogenesis, suggesting that germ cell androgen receptors may play different roles as the germ cells progress through spermatogenesis.13 As seen here, to more fully understand what makes the testes tick, we must capitalize on the powerful molecular tools that have been developed in this capacity. Most of these types of studies are conducted in human and laboratory specimens, so it will be important to test the relevance to the horse.

Organization of the Testis The testes are composed of testicular parenchyma encapsulated by the thick fibrous tunica albuginea. Extensions of the tunica albuginea penetrate the underlying testicular parenchyma, dividing it into numerous lobules (Figs. 1 and 2).14 The tunica albuginea is composed of collagen and elastic fibers, Fig. 1. Drawings of the left equine testis with attached epididymis and spermatic cord in lateral view (A) and medial view myoid cells, and a network of blood vessels (Fig. (B). The vascular pattern over the surface of the testis is 3).15–17 Testicular parenchyma occupies nearly 16 shown. The testis is normally oriented in the scrotum with the 90% of the total testicular mass in adult horses, long axis directed horizontally. The epididymis courses over the and Ͼ70% of the testicular parenchyma is occupied dorsolateral aspect of the testis. (a) Cranial pole of testis (ex- 18 by seminiferous tubules. The interstitial compo- tremitas capitata). (b) Caudal pole of testis (extremitas caudata). nent of the testis is located between seminiferous (c) Appendix testis (vestigial remnant of the embryologic Mu¨ lle- tubules and consists primarily of the Leydig cells rian [paramesonephric] duct). (d) Epididymal border of the testis, intermingled with fibroblasts, lymphocytes, mast showing the entrance to the testicular bursa. (e) Proper ligament cells, blood vessels, lymphatic vessels, and extracel- of the testis. (f) Ligament of the tail of the epididymis. (g) Re- lular matrix (Figs. 4–10).17–19 Nerves are rarely flected parietal tunic (tunica vaginalis parietalis). (h) Ductus deferens.14 seen in the testicular interstitium,17 but they are well established in the area of the spermatic cord leading to the testis. ous tubules, with a combined average length of Spermatogenesis 2419 m per testis in the stallion,20 are comprised of Spermatozoal production, i.e., spermatogenesis, oc- an epithelial wall, termed the seminiferous or ger- curs within the seminiferous tubules. Both ends of minal epithelium, and a lumen. The seminiferous these highly coiled tubules open directly into the epithelium consists of germ cells in various steps of rete testis, such that the products (both cellular and development intermingled with Sertoli cells that non-cellular) of the seminiferous tubules are ex- serve to provide structural support and a nurturing creted into the rete testis and delivered to the ex- source to the germ cells.17 The Sertoli cells are current duct system (e.g., the epididymis and ductus anchored to the basement membrane, and extend to deferens). The numerous and tortuous seminifer- the lumen, of the seminiferous tubules. The semi-

106 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

Fig. 2. Schematic representation of the testis and associated epididymis. The thick tunica albuginea (a) covers the surface of the testis and also extends into the testicular parenchyma, thereby separating the parenchyma into lobules that contain both seminiferous tubules (b) and interstitial tissue. The luminae of the seminiferous tubules empty into the interconnecting tubules of the rete testis (f). The tubules of the extra-testicular portion of the rete testis connect to the epididymis through several efferent ducts (c) on the dorsocranial aspect of the testis. The epididymis forms a single duct that spans up to 45 m in length and consists of an initial segment (d), a caput (or head; e), a corpus (or body; g), and a cauda (or tail; h), which, in turn, connects with the ductus deferens (i). niferous tubules are bordered by peritubular myoid the female. In the female, development of primary cells (myofibroblasts) that, through peristaltic con- oocytes occurs before the immune system recognizes tractions, may aid in evacuation of luminal contents self, and the female does develop the counterpart to into the rete testis. These myoid cells are also con- spermatids (i.e., ootids). In the male, germ cell de- sidered to be involved in paracrine signaling velopment to spermatocytes, and spermatids does events.21–23 not occur until puberty. This is well after the A critical feature of the seminiferous epithelium is immune system recognizes self and considers the formation of tight junctional complexes that de- these cells as foreign. Especially intriguing is velop between Sertoli cells, thereby physically divid- the molecular control over the transient disassem- ing the seminiferous epithelium into basal and bly of the blood–testis barrier to facilitate migration adluminal compartments (Fig. 11).17 This struc- of germ cells from the basal into the adluminal tural complex is termed the blood–testis barrier be- compartment.24,25 cause it restricts direct access of blood-borne Spermatogenesis is an extremely complex process substances into the adluminal compartment. The that involves germ cell proliferation, germ cell dif- described actions of the blood–testis barrier are (1) ferentiation, and, paradoxically, programmed germ to segregate meiotic (except the earliest prelepto- cell death (termed apoptosis). This lengthy pro- tene spermatocytes) and post-meiotic germ cells cess, 57 days in the stallion,20,26 is controlled by a from immunologic attack, because these germ cells vast array of messengers acting through endocrine, are considered to be in an immunologically privi- paracrine, and autocrine pathways.27–31 leged site, and (2) to provide a unique microenviron- Spermatogenesis not only involves transformation ment for the final stages of germ cell development of undifferentiated diploid germ cells into highly within the adluminal compartment.24 Although differentiated and specialized haploid spermatozoa several blood barriers exist in the general body, (Figs. 12 and 13),17,26 but it also involves profound there is no counterpart to the blood–testis barrier in transcriptional modifications within the cells.3,32

AAEP PROCEEDINGS ր Vol. 53 ր 2007 107 MILNE LECTURE

Fig. 4. Micrograph of cross-sections of equine seminiferous tubules and interstitial tissue, after toluidine blue staining of 0.5-␮m Epon section. Stages I, III, VI, and VII of the seminiferous epithelial cycle are depicted, and show respective groupings of spermatogonia (A and B), primary spermatocytes (L, Z, and P),

and spermatids (Sa, Sb1, Sc, Sd1, and Sd2) that are intimately associated with Sertoli cells (SCs) and contained by myoid cells (MCs) marking the outer limits of the seminiferous tubules. The interstitium between tubules is characterized by robust Leydig cells (LCs), lymphatic ducts (LDs), and blood capillaries (BCs). Scale bar ϭ 10 ␮m.

tinually replenished for most of a stallion’s adult life. Under normal circumstances, this is a highly productive process, yielding on the order of 5–6 billion spermatozoa per day for an adult stallion. This translates into 30–40 trillion spermatozoa produced over the course of a stallion’s life. From an

Fig. 3. The equine testis viewed in a gross cross-section (A), in a low-magniﬁcation histologic section (B), and a higher-magniﬁca- tion histologic section (C). (A) The testis is compromised of the tunica albuginea (capsule; TA) and testicular parenchyma (TP). The central vein (CV) has associated connective tissue. (B) The testicular parenchyma contains numerous tightly coiled seminiferous tubules (STs). (C) The interstitium between seminiferous tubules (STs) contains numerous Leydig cells (LCs), blood vessels (BVs), and lymphatic vessels (LVs). Scale bars ϭ 10 mm inA,1mminB,and30␮minC.15–17

As a partial list, the ﬁnished product of spermatogenesis has (1) a 1ϫ chromosomal complement that is profoundly repackaged, (2) a newly formed and intricately designed ﬂagellum, (3) the biogenesis of a highly complex secretory vesicle, the acrosome,33 and (4) retention of some mRNA (in a largely depleted cytoplasmic package) that likely has implications in fertilization and post-fertilization Fig. 5. Scanning electron micrograph of the equine testis reveal- 32,34,35 events. ing seminiferous tubules (STs) and Leydig cells (LCs). Tails of Spermatogenesis is initiated by the differentiation developing spermatids (T) can be seen projecting into the lumen of spermatogonia from a stem cell pool that is con- of seminiferous tubules. Scale bar ϭ 50 ␮m.19

108 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE tion that includes nuclear reshaping through chromatin compaction, creation of a ﬂagellum, development of the acrosome, and considerable loss of cytoplasm (Figs. 13 and 14). The fully developed spermatids are released as spermatozoa into the lumen of the seminiferous tubules by a process termed spermiation (Fig. 15).19,37 It is during spermiogenesis that the germ cells may be most vulnerable to both structural and genetic defects.34

Cellular Associations During Spermatogenesis The spermatogenic cycle, or cycle of the seminiferous epithelium, represents the series of changes in a given region of a seminiferous tubule between two appearances of the same developmental stages.38–41 Fig. 6. Transmission electron micrograph of equine Leydig cells For instance, if spermiation were used as a reference found in testicular interstitium. Equine Leydig cells have an point, the cycle would consist of all the cellular as- abundance of cytoplasm containing smooth endoplasmic reticulum (SER) and a large number of mitochondria (M). Gap junc- sociations occurring within a given cross-section of a tions (GJs) for intercellular communication are located in the seminiferous tubule between two consecutive sper- plasma membranes of two adjacent cells. The nucleus (N) is miations (Fig. 16). These cross-sectional cellular spherical and largely euchromatic, and has a distinct nucleolus associations can be divided into eight distinct stages (No). Scale bar ϭ 3 ␮m.18 (Figs. 16–19).19,42,43 The spermatogenic cycle length is constant at 12.2 days in the stallion.20,26 The spermatogenic wave, on the other hand, refers equally impressive view, an average healthy stallion to the spatial, sequential order of stages along the length of a seminiferous tubule at a given point in produces 60,000–70,000 spermatozoa per second. 42 These newly formed spermatogonia enter a prolifer- time (Fig. 20). These two characteristics of the ative phase, whereby continuous mitotic amplifica- seminiferous epithelium can be defined because of tions yield a dramatic increase in spermatogonial the synchronous nature of development for cohorts numbers. Interestingly, the cytoplasmic compo- of differentiating germ cells along the length of the nent of mitotic divisions is often incomplete, result- seminiferous tubules. Histologic evaluation of the stages of the seminiferous epithelial cycle permits ing in daughter cells that remain connected by 39,44 intracytoplasmic bridges (Fig. 14).36 Such an ar- one to assess the efficiency of spermatogenesis. rangement permits direct communication within Germ Cell Degeneration this syncytium of developing germ cells, thereby assisting in their synchronous development. After Germ cell degeneration can be amplified in stallions with abnormal testicular function, but it is also a completion of spermatocytogenesis, the spermatozoa 45 enter a meiotic phase, characterized by duplication normal phenomenon in spermatogenesis. In fact, and exchange of genetic information (i.e., genetic “normal” spermatogenesis is a relatively inefficient recombination) and two meiotic divisions that re- process and has been reported to result in an esti- duce the chromosome complement to form haploid mated loss of 25–75% of the potential number of spermatozoa produced by spermiation in the round spermatids. It is during the meiotic stage 46,47 that germ cells pass through the blood–testis barrier rat. In the stallion, germ cell degeneration is more profound during the physiologic breeding sea- to enter the adluminal compartment. During the 41 final phase of development, spermiogenesis, the son than during the non-breeding season. Devel- round spermatids undergo a dramatic transforma- oping spermatogonia are the most vulnerable to apoptotic degeneration (Fig. 21).43 It is possible that the “physiologic” form of germ cell degeneration is a homeostatic mechanism that prevents overload- ing the Sertoli cells, i.e., to maintain a fine balance in the germ cell:Sertoli cell ratio. Apoptosis is a well-defined physiological process of cell elimination,48 and the apoptotic process is required for normal spermatogenesis in mammals.49,50 A protective role of luteinizing hormone (LH) against germ cell apoptosis has been reported in rats, based on expression of various apoptotic genes following Fig. 7. The effect of stallion age on pigmentation of unfixed equine 51 testicular parenchyma, as observed grossly. (A) Light parenchyma is immunoneutralization of LH in germ cells. Ap- from a post-pubertal stallion (2–3 yr). (B) Moderately dark paren- optosis of spermatogonia and spermatocytes has chyma is characteristic of adult (4–5 yr) stallions. (C) Dark paren- been reported in normal stallion testes, a finding chyma characterizes aged stallions (13–20 yr).18 that is consistent with the expected time that germ

AAEP PROCEEDINGS ր Vol. 53 ր 2007 109 MILNE LECTURE

Fig. 8. Composite of the interstitium between seminiferous tubules (STs) and Leydig cells (LCs) in stallions at different ages in the middle (A, C, and E) or onset (B, D, and F) of the breeding season. (A and B) The post-pubertal (2–3 yr) stallion has large Leydig cell clusters in the interstitium. (C and D) The young adult stallion (4–5 yr) has larger clusters of Leydig cells that increase the density. (E and F) The aged stallion testis (13–20 yr) has mostly Leydig cells in its interstitium; however, some Leydig cells may contain a large accumulation of lipofuscin (Lf). No difference in Leydig cell structure is noted between the onset and middle of the breeding season. Scale bars ϭ 75 ␮minA,C,andEand25␮minB,D,andF.18

cells may be susceptible to removal from the sys- (Fig. 2). The anatomical features of the rete testis tem.43 Of interest, formation of the developing and efferent ductules have been well characterized seminiferous tubules and the Sertoli cell (i.e., blood– in the stallion (Figs. 22–24).15,57 For descriptive testis) barrier coincides with increased germ cell purposes, each epididymis is typically divided ana- apoptotic rates in stallions, providing evidence that tomically into a caput (or head), a corpus (or body), apoptosis may play an intricate role in initiation of and a cauda (or tail; Fig. 2). The caput is closely spermatogenesis.52 As expected, these changes are applied to the cranial pole of the testis, the corpus coincident with gene expression patterns.53 Stud- courses over the dorsolateral surface of the testis, ies involving rats indicate that FSH is an important and the bulbous cauda is attached loosely to the regulator of this event.54,55 caudal pole of the testis through the proper ligament of the testis (Fig. 1). An initial segment and an 3. Epididymal Transit and Maturation intermediate zone have also been ascribed to the Mammalian spermatozoa are incapable of in vivo beginning portion of the epididymal duct within the fertilization on exiting the testis. The cells must caput.58 undergo considerable post-testicular remodeling On average, 90 billion spermatozoa reside in the within the epididymis to acquire this ability. Each excurrent ducts of reproductively mature stallions epididymis is an unbranched tortuous duct that when sexually rested, with a lower number (60 bil- spans 70–80 m in the stallion.56 The epididymis is lion) reported for 2- to 4-yr-old stallions.59 On av- connected to the rete testis by several efferent ducts erage, 62% of the spermatozoa within the excurrent

110 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

Fig. 10. Scanning electron micrograph of the equine testicular interstitium and seminiferous tubule. Clusters of Leydig cells (LCs) are seen in the interstitium between seminiferous tubules (STs). One seminiferous tubule contains spermatids (Sperma- tids) with spherical nuclei and spermatids whose tails already project into the lumen. Spermatozoa (Sperm) in transit are seen in the lumen. The open arrows mark the height of the seminiferous epithelium. Scale bar ϭ 20 ␮m.17 Fig. 9. Transmission electron micrograph of Leydig cells in young adult (4–5 yr; A) and aged stallions (13–20 yr; B). In both cases, Leydig cells contain an abundance of smooth endoplasmic reticulum (SER) and mitochondria (M). In aged stallions (B), sexually rested stallions, rhythmic contractions of Leydig cells may have an accumulation of lipofuscin (lf). Scale the smooth musculature in the caudae epididymides 18 bar ϭ 1 ␮m. and deferent ducts are thought to result in spontaneous emission of spermatozoa into the urethra. Some stallions are known to accumulate excessive numbers of spermatozoa in the caudae epididymi- duct system are in the caudae epididymides, and 7% des, deferent ducts, and ampullar luminae (the am- are in the ductus deferens.59 Based on radiolabeled pullae surround the terminal segment of the ductus studies, the average time required for spermatozoal deferens). This condition, termed “spermiosta- transit through the epididymis of the stallion is re- sis”64 or “plugged ampullae”65 is thought to be asso- ported to be 8–11 days.60 Spermatozoal transit ciated with an intrinsic dysfunction in contractility time through the caput and corpus segments is con- of the smooth musculature surrounding the cauda stant at 4 days and is unaffected by frequency of epididymis and ductus deferens. It leads to a ejaculation, age, or season,60–62 whereas transit marked reduction in spermatozoal quality or time through the cauda epididymis can be acceler- azoospermia if the ductal luminae become ob- ated by increased ejaculation frequency.61,63 Sper- structed completely.14 A separate subset of stal- matozoal number in the caudae epididymides and lions is known to experience reduced semen quality ductus deferens (i.e., spermatozoa available for ejac- and fertility if sexually rested for Ͼ12–24 h (unpub- ulation) may be reduced by ϳ30% when semen is lished observations). In this instance, the microen- collected once every 2 days compared to sexually vironment within the cauda epididymis may impart rested stallions.59 The normal spermatozoal tran- factors that impede spermatozoal survival rather sit time through the cauda epididymis of the stallion than favoring maximal survival time. Because ep- ranges from 4 to 7 days. Spermatozoa enter the ididymal spermatozoa are known to be susceptible epididymis at a constant rate in reproductively nor- to oxidative injury,66,67 it is possible that reduced mal stallions (average of Ͼ5 billion spermatozoa per elimination of potentially harmful agents or reduced day). Spermatozoal phagocytosis within the epi- synthesis and secretion of antioxidants contributes didymis is considered to be rare,58,63 so spermatozoa to this disorder.58 Alternatively, spermatozoal must also exit the epididymis at a similar rate to membranes that are overly abundant in polyunsat- prevent epididymal overload. The length of time urated fatty acids may be at increased risk of oxida- that spermatozoa can remain in the cauda epididy- tive injury.67 mis without suffering storage-related injury has not As is the case with many other species studied, been critically studied. Under normal conditions in stallion spermatozoa are immotile upon entering the

AAEP PROCEEDINGS ր Vol. 53 ր 2007 111 MILNE LECTURE

Fig. 11. Transmission electron micrograph of Sertoli cells and germ cells and Sertoli cell–Sertoli cell junctional complexes (inset). Junctional complexes (JCs) between adjacent Sertoli cells (SCs) separate the basal compartment that contains spermatogonia (Sg) from the adluminal compartment that contains primary spermatocytes (PSs) and spermatids (St). Junctional complexes between adjacent Sertoli cells in the stallion are similar to those of other mammals. These include fusion (F) of outer leaflets of the plasma membranes of two Sertoli cells and the presence of juxtaposed endoplasmic reticulum (ER) and bundles of fine filaments (BFs). Scale bars ϭ 1 and 2 ␮m (inset).17

epididymis and do not gain motility until reaching oxytocin exposure.74 Although the epididymis is the distal corpus. A motility pattern consistent considered to be an androgen-dependent tissue,75,76 with ejaculated spermatozoa is not acquired until estrogens seem to play a more important role than spermatozoa reach the cauda epididymidis.68 As androgens in promotion of epididymal motility. such, movement of spermatozoa through the epidid- This is achieved through estrogen-mediated up-reg- ymis is primarily attributed to rhythmic contrac- ulation of both the oxytocin receptor gene and the tions of the smooth musculature that surrounds the receptor protein.77 epididymal duct (Figs. 25–27).57,58 In men, both In addition to serving as a conduit, the epididymis the thickness of the smooth musculature, and its bestows on spermatozoa specific structural and adrenergic innervation, increase from the proximal physiologic alterations, the result of which yields to the distal end of the epididymis, and into the acquisition of fertilizing potential. Maturational ductus deferens.69 Autonomic drugs, both cholin- changes in spermatozoa occur predominantly within ergic and adrenergic, increase contractility of all the caput and corpus, because spermatozoa recov- segments of the epididymis.70 Prostaglandins ered from the cauda have attained a fertilizing (PGF2␣ and PGE) have been isolated from the testes capacity similar to that of ejaculated spermatozoa and excurrent ducts of male rats, with a concentra- for most species studied.58,78,79 The cauda epidid- tion noted to be lowest in the testes, intermediate in ymis is considered to be a storage reservoir for sper- the epididymides, and highest in the ductus defer- matozoa, but this capacity extends into the ductus ens.71 These same authors showed that aspirin, deferens and ampullae. In one study, sufficient which suppresses production of PGF2␣, strongly in- spermatozoa were present in the ductus deferens hibited contractility of the caput epididymidis in and ampullae of stallions to account for average vitro. Oxytocin receptors are also present in the spermatozoal numbers in an ejaculate.62 epididymal epithelium,72,73 and the frequency of ca- A paucity of information exists with regard to the put epididymal contractions increases and tubule specific mechanisms that control spermatozoal mat- diameter decreases in a dose-responsive manner to uration. Studies to date indicate that a salient fea-

112 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

Fig. 12. Equine spermatogonia and spermatocytes viewed by bright-ﬁeld microscopy of 5-␮m methacrylate sections stained with toluidine blue. Small arrows designate cells of interest. The most primitive spermatogonia have small, oval, or ﬂattened nuclei (A1). Other subtypes include those with a light center (A2), a large single nucleolus (A3), two or three nucleoli (A3), or large nucleoli plus fragmented nucleoli (B1), or with a small spherical nucleus (B2). Preleptotene (Pl), leptotene (L), zygotene (Z), early pachytene (EP), late pachytene (LP), diplotene primary spermatocytes (D; open arrow), and secondary spermatocytes (SSs; closed arrow) are indicated. Scale bar ϭ 10 ␮m.17,26

ture of the epididymis is a continual change in the males. The corpus may be the most vulnerable seg- microenvironment to which spermatozoa are ex- ment of the epididymis to age-related barrier posed during epididymal transit. This is portrayed dysfunction.92 by the variation in histomorphologic characteristics Barrier-mediated isolation of the epididymal lu- of the numerous epididymal cell types that line the men from the blood plasma probably necessitates various regions of the duct (Figs. 24–27),57,58,80 as more reliance on alternative signaling pathways. well as the distinct regional and cell-speciﬁc pat- Paracrine pathways (where target cells in the epi- terns of gene expression and product secretion.81–84 didymis are issued signals from secretions of neigh- Although a variety of such localization studies have boring cells) and juxtacrine pathways (where target been conducted, these must be followed by mecha- cell membrane receptors are activated by molecules nistic and functional approaches before we can fully on the plasma membrane of neighboring cells) are appreciate the molecular interactions that impart likely to exist.93 Lumicrine pathways (where signal- fertilizing power to a spermatozoon.85–87 ing molecules originate in the testes and are trans- The unique composition of epididymal plasma can ported through the rete testis into the epididymal be attributed, in large part, to the formation of a duct or originate in one region of the epididymis and blood–epididymis barrier.88,89 This barrier is have an effect in another region of the excurrent formed by tight junctional complexes near the lumi- duct system) are also key to epididymal func- nal border between adjacent principal cells of the tion.58,93,94 Testosterone (synthesized by the Ley- epididymal epithelium.90 This mechanism re- dig cells) and androgen binding protein (synthesized stricts entry of various blood-borne molecules into by the Sertoli cells) represent prime examples of a the epididymal lumen, thereby allowing spermato- lumicrine-derived mechanism of action in the epi- zoa to be bathed in a milieu that is controlled locally, didymis. Of interest, androgen binding protein can and affording spermatozoal protection against im- also be synthesized and secreted by principal cells munologic attack.58 Disruption of the blood–testis along the entire length of the epididymis, possibly barrier91 and the blood–epididymis barrier92 may be exerting a paracrine- or lumicrine-related ac- a contributor to reduced semen quality in older tion.58,95 An assortment of growth factors derived

AAEP PROCEEDINGS ր Vol. 53 ր 2007 113 MILNE LECTURE

Fig. 13. Transmission electron micrographs of equine spermatids representing different steps of development during spermiogenesis. (A–E) The Sa spermatids (Sa) are characterized by a large Golgi apparatus (GA) that produced vesicles (Vs) that fuse to form larger vesicles and ultimately to form the acrosomic vesicle (AV). The acrosomic vesicle indents the nuclear envelope and flattens over the nucleus. (B) Developing flagella (F) from neighboring Sa spermatids have extended away from the spermatid cell body into the extracellular space between the Sertoli cell (SC) and spermatid. (C) Cytoplasmic bridges connect adjacent spermatids. (F) In the Sb1 spermatids (Sb1), the acrosome had formed a head cap over the nucleus. (G and H) The Sc spermatid (Sc) has a distinct manchette (Mn), elongating and condensing nucleus, attached flagellum with distinct annulus (An), and well-defined acrosome (A) over the anterior portion of the nucleus. (I and J) The Sd spermatids (Sd1 and Sd2) have lost their manchette and have further condensation of the nucleus. (B) The formation of the flagellum begins in the early Sa spermatid (G and H) appears as a growing axoneme in Sc spermatids, but (I) develops outer dense fibers (DFs), the fibrous sheath (FS), and has mitochondrial migration around the flagellum in early Sd1 spermatids. (J) Late Sd2 spermatids are mostly extended into the lumen (TL) and have a completely formed flagellum, with mitochondria (M) surrounding the middle piece and a distinct fibrous sheath (FS). Excess cytoplasm of the spermatids remains in the proximal region of the middle piece as a cytoplasmic droplet (CD). Scale bar ϭ 2 ␮m.17,26 from the testis have been proposed to have an action ferent ductules and proximal caput and, to a much on the epididymis in a lumicrine fashion,93 and lesser extent, in the remainder of the epididymal many growth factors are also produced locally duct leads to a 105 increase in spermatozoal concen- within the epididymis.93 Close interactions be- tration in the cauda epididymidis compared with the tween spermatozoa and the epididymal epithelium rete testis.58 Observable changes in spermatozoa are crucial for the maturation process to occur, so it during epididymal maturation, based on light micro- is not surprising that spermatozoa may also serve a scopic analysis, include (1) acquisition of flagellar lumicrine role within the epididymis.96 movement within the corpus epididymis, followed by In summary, the epididymis serves to concen- a progressive pattern of spermatozoal motility trate, mature, transport, and store spermatozoa. within the cauda epididymis, and (2) translocation Massive resorption of luminal water within the ef- and shedding of the cytoplasmic droplet. The cyto-

114 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE plasmic droplet of caput spermatozoa is consistently located in a proximal position near the neck of the middle piece. A few spermatozoa have bent middle pieces associated with translocation of the cytoplasmic droplet and spermatozoal maturation. When spermatozoa reach the proximal cauda epididymidis, most have completed translocation of the cytoplasmic droplet, as noted by droplets located near the junction of the middle piece and the principal piece. Under normal circumstances, few ejaculated spermatozoa have cytoplasmic droplets.68 Other more subtle, but necessary, changes acquired by spermatozoa during epididymal maturation include remodeling of the surface lipids, proteins, and gly- cocalyx97–104; reorganization of acrosomal mole- cules105; further changes in the structural stability of the nuclear chromatin (Fig. 28)68,106; development of signaling pathways107; and alterations in spermatozoal metabolism.108 The collective biochemical changes, both intracellular and extracellular, incurred by spermatozoa during epididymal maturation remain a mystery, but experimentation is gradually revealing new ﬁndings.

4. Spermatozoal Structure: Form to Function The general form of the spermatozoon was first described by Leeuwenhoek 330 yr ago (1677). Gen- Fig. 14. High-voltage electron micrographs of equine Sertoli and eral acceptance of his proposal that this tadpole- germ cells embedded in Epon 812 and sectioned at 1.0 ␮m. The shaped form contributed to fertilization required basal (A) and apical (B) regions of the Sertoli cells are character- ϳ200 years. Thereafter, the structure of the sper- ized. (A) The close association between Sertoli cells and germ cells matozoon received mounting attention. Resolving representing different developmental steps is evident. Laminar power of early microscopes hampered the ability to processes (LPs) extend from the main body of a Sertoli cell and provide an exquisite description of spermatozoal can be seen between germ cells. One forms a rim of Sertoli cell anatomy, but introduction of electron microscopes in cytoplasm from the main body of the Sertoli cell to the site of an the middle of the 20th century paved the way for intercellular bridge (IB) between two primary spermatocytes intricate viewing of the spermatozoon and its sub- (PSs). Long slender mitochondria (arrows) oriented with the structure. Excellent descriptions of spermatozoal long axis of the Sertoli cell characterize the main body cytoplasm, ultrastructure were provided by Saacke and while numerous dense lipid and lipofuscin granules are found in 109,110 111 the basal cytoplasm. Mitochondria (M) of germ cells are con- Almquist in 1964, and by Fawcett in 1975. trasted with those of Sertoli cells. Rough endoplasmic reticulum More recently, Eddy provided a exceptional review (RER) can be seen in the cytoplasm of germ cells. Portions of of the topic, and included molecular-related insights Sertoli cell nuclei (N) depict the highly indented nature of the that have been acquired in recent years.112 nuclear surface. An A spermatogonium shares the intercellular The spermatozoon is typically divided anatomi- space (IS) with a Sertoli cell. Although Golgi-phase spermatids cally into a head and a flagellum (or tail). The head (Sa) have not yet developed an acrosomic vesicle, the developing contains the nucleus, overlying acrosome, and a re- flagellum (F) from the centriole (Ct) can be seen within the duced complement of cytosolic elements. The head cytoplasm of one such spermatid. (B) Apical region of a Sertoli cell can be subdivided into an acrosomal region, equato- containing long slender mitochondria (arrows) and four embedded Sd1 spermatids with elongated nuclei are shown in this rial segment, post-acrosomal region, and posterior profile. A portion of the nucleus and three mitochondria of a Sa ring, which demarcates the junction between the spermatid (Sa) are present. By following Sertoli cell cytoplasm head and flagellum. The posterior ring is the site of marked by intercellular space (IS) between Sd1 spermatids and plasma membrane anchoring to the nuclear enve- the Sertoli cell, it is evident that Sertoli cell cytoplasm is located lope and is thought to produce a tight seal that sepa- between each spermatid. These Sd1 spermatids are deeply em- rates cytosolic components of the head and flagellum. bedded in the apex of the Sertoli cell through tubular indenta- The flagellum can be subdivided into a connecting tions in the Sertoli cell plasma membrane. The annulus (An), piece, middle piece (or midpiece), principal piece, and manchette (Mn), and mitochondria (M) of the Sd1 spermatids are end piece (Figs. 29 and 30).19,26 These various parts indicated. The flagellar canal (FC) is located between the develop- of the spermatozoon are surrounded by a common ing flagellum and surrounding manchette. The manchette is situated to provide structural support to the flagellar canal. This canal plasma membrane, although the composition of the is lined by the plasma membrane enfolded from the surface of the plasma membrane can be subdivided into regional do- spermatid at the lumen into which the flagellum extends. Scale mains that impact its multiple functions, such as bars ϭ 2 (A) and 1 ␮m (B).36 sperm-oviductal adhesion, penetration of the cumulus-

AAEP PROCEEDINGS ր Vol. 53 ր 2007 115 MILNE LECTURE

Fig. 15. Scanning electron micrograph of stage-VII (A), early stage-VIII (B), and late stage-VIII tubules (C and D). Spermatid tails project into the lumen in stages VII and VIII, but the entire spermatid projects into the lumen in late stage VIII, attached only by cytoplasmic stalks (CS). (C and D) An enlarged luminal view of C is seen in D. Maturation-phase spermatids are located in the lumen; however, tails (Ts) of Sa spermatids with round nuclei already project into the lumen. One luminal spermatozoon, recently released, has its characteristic cytoplasmic droplet (CD) in the proximal position where the middle piece attaches to the head. Scale bars ϭ 10 (A and C), 5 (B), and 2.6 ␮m (D).19

oophorus matrix; sperm-zona adhesion; the acrosome average lengths of the head, midpiece, principal reaction, acquisition of activated motility and hyper- piece, and end piece reported as 5.8–7.0, 9.8–10.5, activated motility; and sperm-oocyte adhesion and 43.8–67, and 2.79 ␮m, respectively.113–115 The fusion.112 maximum width of the head is reported to be 2.9– The equine spermatozoon has similar general 3.9 ␮m.113–115 The size of an equine spermatozoon, structures to that described for the bull, ram, boar, relative to its body size, is remarkably smaller than dog, and human. The average length of an equine that of some other species, such as the mouse, rat, spermatozoon is reported to be 61–86 ␮m, with the hamster, and honey possum, where spermatozoal

116 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

Fig. 16. Drawing that portrays the kinetics of equine spermatogenesis and the stages of the cycle of the seminiferous epithelium. Three major divisions of spermatogenesis (spermatocytogenesis, meiosis, and spermiogenesis) combine to constitute the eight stages of the cycle of the equine seminiferous epithelium (I–VIII). During the 19 days of spermatocytogenesis, A1 spermatogonia enter cyclic activity during stage III and undergo mitotic division to produce B1 and B2 spermatogonia, followed by production of preleptotene primary spermatocytes (Pl). During the 20 days of meiosis, preleptotene primary spermatocytes differentiate through zygotene meiotic division to produce Sa spermatids (Sa). During the 18 days of spermiogenesis, Sa spermatids differentiate through Sb1,Sb2, Sc, Sd1, and Sd2 steps of development before spermiation (i.e., release from the seminiferous epithelium) as spermatozoa. The letters indicate the development step; the numbers associated with each germ cell step indicate the developmental age (in days) of each cell type in the middle of each spermatogenic stage. The cycle length is 12.2 days, and the duration of spermatogenesis is 57 days in stallions. lengths are 123, 190, 189, and 356 ␮m, respective- termed protamines, which predominate as nucleopro- ly.113 For further comparison, the average length teins during spermatozoal maturation in the epididy- of a human spermatozoon is 57 ␮m, with an average mis. The cysteine residues of this protein establish head length of ϳ4.5 ␮m.113 intramolecular and intermolecular disulfide linkages The equine spermatozoon head is defined as splatu- that result in compaction and stabilization of the as- late-shaped, in contrast to the falciform-shaped sperm sociated DNA. This design is thought to provide pro- heads characteristic of some species (e.g., the mouse, tection to the chromosomes during their perilous rat, and hamster). Of the laboratory animal species, journey within the female reproductive tract and to the equine spermatozoon most closely resembles that streamline the spermatozoon to enhance its mobility of the rabbit. The spermatozoal head is somewhat on activation at the time of ejaculation. elliptical in shape, is flattened in one plane (dorsoven- The nuclear envelope, consisting of a double mem- trally) and is thicker in the posterior portion of the brane (each with a lipid bilayer), separates the con- head than in the apical portion of the head (Figs. 30 tents of the nucleus from the surrounding and 31).14,19,26 The nucleus, which occupies the ma- cytoplasm. Although the nuclear envelope is regu- jority of space within the spermatozoon head, contains larly perforated by nuclear pore complexes in so- the paternal genetic material. Specifically, the male genome is compromised of the X or Y chromosome and matic cells, such pores are absent over most of the a haploid number of somatic chromosomes. This ge- spermatozoal nuclear envelope, i.e., in the area un- netic material is packaged for delivery to the oocyte for der the acrosome and in the post-acrosomal region. fertilization, where two haploid (male and female) ge- The exception is the region of the redundant nuclear nomes are combined to produce a diploid offspring. envelope, a portion of the nuclear envelope posterior The chromatin (i.e., the DNA and associated proteins) to the chromatin that folds and extends back into within the nucleus of the mature spermatozoon is the neck region. This portion of the nuclear enve- highly condensed, resulting in a volume that may ap- lope contains abundant hexagonally arranged pores. proach only 5–10% of that of a somatic cell. This The caudal portion of the nuclear envelope forms a packing is a result of a marked alteration in the com- concavity, termed the implantation fossa, which is position of nucleoproteins that occurs during epididy- the site of attachment with the flagellum. This re- mal transit. The haploid genome of the round gion of the nuclear envelope is overlain with a thick spermatid encodes for unique spermatozoal proteins, sheet of material, termed the basal plate.

AAEP PROCEEDINGS ր Vol. 53 ր 2007 117 MILNE LECTURE

Fig. 17. Bright-field micrographs depicting the eight stages of the cycle of the seminiferous epithelium in paraffin sections stained with PAS and toluidine blue. Stage I, characterized by two generations of leptotene (L) and pachytene (P) primary spermatocytes. Sb1 round spermatids can be observed with a well-developed and easily recognizable acrosomal cap. Developing tails are sometimes visible, although these structures can be difficult to distinguish in paraffin sections. Stage II, two generations of L and P primary spermatocytes. Sb2 spermatids become irregularly shaped as elongation and orientation begins. Acrosomal caps are plainly evident. Stage III, two generations of zygotene (Z) and pachytene spermatocytes are evident, as are characteristic Sc spermatids which are oriented with acrosomal caps toward the basement membrane. No primary spermatocytes with meiotic figures are evident. Stage IV contains the most germ cell types of any state. B1 spermatogonia are sometimes evident (data not shown) along the basement membrane next to zygotene primary spermatocytes. In the adluminal compartment, large pachytene spermatocytes begin their divisions and are seen with numerous meiotic figures (MFs; metaphases I and II). Secondary spermatocytes (SSs), newly formed Sa round spermatids, and Sc elongated spermatids are also evident. Stage V, B1 spermatogonia are still evident. Only one generation of pachytene primary spermatocytes may be found. The newly formed Sa round spermatids have not yet begun formation of the acrosomal cap over the nucleus. Sd1 elongated spermatids with densely packed chromatin may be found stacked in clusters, deeply embedded within the seminiferous epithelium. Stage VI: small, round B2 spermatogonia with small clumps of heterochro- matin have formed and one generation of pachytene spermatocytes may be found near the basement membrane. Sa round spermatids may be observed with an acrosomic vesicle not yet touching the nucleus. Sd1 elongated spermatids begin their migration toward the lumen. Stage VII, B2 spermatogonia and one generation of pachytene primary spermatocytes may be found. Acrosomal caps of Sa round spermatids begin to flatten and cover the surface of the nucleus. Densely packed Sd2 elongated spermatids are migrating closer to the lumen. Stage VIII, B2 spermatogonia have divided to form small round preleptotene (Pl) primary spermatocytes along the basement membrane. One generation of pachytene primary spermatocytes and late Sa round spermatids with developing acrosomal caps can be found. Residual bodies (Rb) accumulate as mature spermatozoa are being released into the lumen as spermiation occurs. Scale bar ϭ 30 ␮m.43

The acrosome is a membrane-bound exocytotic or- Anatomically, the membrane is subdivided into an ganelle that overlies the rostral two thirds of the inner acrosomal membrane that is continuous with nucleus, with a ﬁt resembling that of a bathing cap. an outer acrosomal membrane. These connecting

118 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

Fig. 18. Bright-ﬁeld light (A, C, and E) and scanning electron (B, D, and F) micrographs of different stages of the equine spermatogenic cycle. (A and B) Stages I and II are shown. (C and D) Stages III, IV, and V are represented. (E and F) Stages VI and VII reveal changes in preparation of the release of spermatozoa in stage VIII. Scale bar ϭ 20 ␮m.19

membranes enclose a narrow heterogenous com- and hydrolytic enzymes, are thought to be important partment, termed the acrosomal matrix. The inner for adhesion to, and penetration of, the zona pellu- acrosomal membrane is closely apposed to the nu- cida and sperm–oolemma interactions.116–119 Dur- clear envelope whereas the outer acrosomal mem- ing the course of the acrosome reaction, the outer brane underlies the plasma membrane. These two acrosomal membrane fuses with the overlying membranes converge at the level of the equatorial plasma membrane, thereby creating hybrid vesicles segment. The acrosome originates from the Golgi and pores that lead to release and exposure of acro- apparatus in round spermatids during spermiogen- somal contents (Fig. 32).116 Although the sperma- esis. Light microscopic changes of the developing tozoal acrosome contains several enzymes acrosome can be visualized in cross sections of the characteristic of a typical cellular lysosome (in addi- seminiferous tubules (Figs. 4 and 13). Reﬁnement tion to those enzymes speciﬁc to the spermatozoon), in acrosomal morphology and biochemical composi- the enzymes are not used within the cell as in auto- tion continue as spermatozoa traverse the epididy- phagy for cellular organelle remodeling and renew- mis.116 In the mature spermatozoon, the acrosome ing, or in heterophagy as occurs in phagocytic cells. contains a bountiful supply of active molecules, both The cytosolic compartment of the mature sperma- within the acrosomal matrix and as components of tozoal head is virtually free of organelles other than the inner acrosomal membrane. These molecules, the acrosome, and the mature spermatozoon is con- which include an assortment of protein receptors sidered to be transcriptionally quiescent. Nonethe-

AAEP PROCEEDINGS ր Vol. 53 ր 2007 119 MILNE LECTURE

Fig. 19. A clockwise arrangement of eight stages of the cycle of the equine seminiferous epithelium observed by Nomarski optics in unstained, 20-␮m Epon histologic sections. Both nuclear and cytoplasmic details are revealed in Leydig cells (LCs), Sertoli cells (SCs), various types of germ cells, and myoid cells (MCs). A spermatogonia (A) of different types and Sertoli cells are found in all stages of the cycle. Classification of spermatid development was based on that described for humans and used in the horse. Briefly, the Sa spermatid is the earliest form of spermatid, as it contains a spherical nucleus and a large Golgi with either no acrosomic vesicle, a developing acrosomic vesicle, or an acrosomal cap. The Sb1 spermatid has a spherical nucleus but also has an attached flagellum and a distinct acrosome covering half of the nuclear surface. The Sb2 spermatid has begun nuclear elongation with the appearance of the manchette. The Sc spermatid has a distinct manchette and a more elongated nucleus than the Sb1 spermatid. The Sd1 spermatid is undergoing final maturation with the disappearance of the manchette and migration of mitochondria around the tail.

The Sd2 spermatid is the final form with a large portion of the cell, including the head, projecting into the tubular lumen in Stage VIII. Stage I (I) is characterized by preleptotene or leptotene (L) and pachytene (P) primary spermatocytes as well as Sb1 spermatids (Sb1). These spermatids are characterized by spherical nuclei, attached acrosomal caps (AC), and developing flagellum (F). Stage II (II) is characterized by leptotene primary spermatocytes (L), pachytene primary spermatocytes (P), and Sb2 spermatids (Sb2). The labeled pachytene primary spermatocyte is displaying its large spherical Golgi apparatus. The Sb2 spermatid has an elongating nucleus, manchette (Mn), and annulus (An). Stage III (III) has zygotene (Z) and pachytene (P) primary spermatocytes, and bundles of Sc spermatids (Sc). The Sc spermatid has an elongating and condensing nucleus, distinct manchette (Mn), and annulus (An). Stage IV (IV) is characterized by the presence of zygotene primary spermatocytes (Z), diplotene primary spermatocytes or secondary spermatocytes (SS), meiotic figures (MF), and Sc spermatids (Sc). Stage V (V) is composed of pachytene primary spermatocytes (P); newly formed

Sa spermatids (Sa) and Sd1 spermatids (Sd1). The Sa spermatid has a distinct Golgi apparatus (GA), developing acrosomic vesicle (AV), and chromatoid body (CB); and Sd1 spermatids form bundles which are deeply embedded in the seminiferous epithelium. Stage VI (VI) has type B spermatogonia (B), pachytene primary spermatocytes (P), Sa spermatids (Sa) with their grouped mitochondria (GM) and Sd1 spermatids (Sd1) with their annulus (An) and less distinct manchette. Stage VII (VII) is characterized by type B spermatogonia (B), pachytene primary spermatocytes (P), Sa spermatids (Sa), and Sd2 spermatids (Sd2). Sd2 spermatids, the most advanced form, are migrating toward the tubular lumen in this stage. Stage VIII (VIII) has newly formed preleptotene (Pl) and pachytene (P) primary spermatocytes, late Sa spermatids (Sa) with their acrosomic granule (AG) and newly attached developing ﬂagellum, and Sd2 spermatids lining the lumen with their migrated annulus, enlarged middle pieces (MP) and attached cytoplasmic droplet (CD). Residual bodies (RB) left behind by spermiation of spermatids can be seen near the luminal surface and in transit toward the base within Sertoli cell (SC) cytoplasm. Scale bar ϭ 10 ␮m.42

120 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

Fig. 21. Light microscopic view of a cross-section of an equine seminiferous tubule, showing the nuclei of some spermatogonia that label positive (brown-black) with an immunohistochemical apoptotic detection assay (termed the TUNEL assay). The background tissue stain is PAS-toluidine blue. The germ-cell associations in the cross sectional view are consistent with stage V of the seminiferous epithelial cycle.43

less, the cytoplasm of the head region contains cytoskeletal proteins that are important to spermatozoal function. Structural changes occur in the cytoskeleton (both microfilaments and microtubules) during spermiogenesis that lead to the reshaping of the spermatid head to an elongated form. This appears to be primarily mediated by the acrosome-acroplaxome-manchette complex and the perinuclear theca.120–122 The acroplaxome is formed at the leading edge of the developing acrosome. This structure contains both filamentous actin and kera- tin, and anchors the developing acrosome to the underlying nuclear envelope. The manchette consists of a circumferential bundle of microtubules that assemble posterior to the developing acrosome at the perinuclear ring, and extend along the devel- Fig. 20. The spermatogenic wave revealed by an enzymatical- oping flagellum to a point where the fibrous sheath ly-isolated equine seminiferous tubule that was fixed in glu- begins (Figs. 13 and 14).123 The manchette may taraldehyde, followed by osmium tetroxide, infiltrated with aid nuclear elongation, as well as directional migra- Epon, mounted in toto in Epon, and observed by bright field tion of molecules necessary for both nuclear conden- microscopy. The corresponding drawing reveals the consecu- sation and tail formation.120,121 The perinuclear tive stages along the length of the tubule as determined by theca is located between the inner acrosomal mem- observation with Nomarski optics. Although modulations (re- brane and the nuclear envelope (i.e., the subacroso- versal of order) occur, adjacent stages are in consecutive order. Latter stages (more developed; higher numbers) are observed in the same mal region), as well as between the nuclear envelope direction of two sets of all stages I–VIII. Scale bar ϭ 200 ␮m.42 and the plasma membrane posterior to the developing acrosome (i.e., the postacrosomal region). This theca is composed of a complex of highly condensed

AAEP PROCEEDINGS ր Vol. 53 ր 2007 121 MILNE LECTURE that connect the capitulum to the basal plate. The segmented columns anchor the dense fibers of the flagellum (Fig. 31). The centrioles, oriented at right angles to each other, are involved in development of the connecting piece and the axoneme. The proximal centriole remains attached at the implantation fossa, but the distal centriole gives rise to the axoneme during tail development. The primary structural elements of the flagellum include the axoneme, the outer dense fibers, the fibrous sheath, and the mitochondria (Figs, 29–31 and 33). The midpiece is characterized by the presence of an axoneme, an array of outer dense fibers, and an overlying sheath of helically arranged mitochondria. The midpiece joins the principal piece at the annulus, a point where the mitochondrial sheath is replaced with a fibrous sheath. The principal piece thereby consists of a centralized axoneme, outer dense fibers of variable length, and a fibrous sheath. The fibrous sheath terminates at the junction of the principal piece and end piece, with the end piece consisting of a small extension of the axoneme (or individually arranged acrosomal microtubules) past the termination site of the fibrous sheath. The entire flagellum is enveloped by a plasma membrane. The axonome is a cylindrical array of nine doublet microtubules that surround two singlet microtubules (termed the central pair) connected by reg- ularly spaced bridges, thus forming the “9 ϩ 2” configuration that is characteristic of both cilia and flagella throughout the plant and animal kingdoms. The microtubules are composed primarily of the tubulin family of proteins. Each of the nine outer microtubule doublets consists of an “A” subunit, which is completely cylindrical and composed of 13 protofilaments, and a “B” subunit, Fig. 22. Scanning electron microscopic view of the equine tes- which is C-shaped and composed of 10–11 proto- ticular parenchyma, showing (A) the rete testis complex (RTC) filaments (Fig. 33). The A subunits serve as an housed in the connective tissue of the mediastinum around the anchor for the outer dynein arms and inner dynein central vein (CV) of the equine testis, and (B) a cross support arms that possess ATPase activity and generate within the complex. (A) Seminiferous tubules (STs) are adjacent the force required for axonemal and thereby, to the connective tissue around the rete testis complex. Cross flagellar motion, through an attachment-detach- supports (open arrow) are found in the rete testis complex and in ment cycle between A- and B-subunits of adjacent rete testis tubules (RTT). (B) The cross supports are comprised doublets. Filamentous nexin links or nexin arms of a simple cuboidal epithelium overlying a connective tissue connect adjacent doublets, and radial spokes con- core. Testicular spermatozoa may be attached to this epitheli- nect the axonemal doublets to a helical sheath um. Scale bars ϭ 500 (A) and 4.6 ␮m (B).15 surrounding the central pair of microtubules. The mechanism of axonemal action is discussed below under spermatozoal motility. cytotoskeletal proteins that have actin-binding The outer dense fibers course from the connecting properties and seems to be involved in reshaping of piece through the midpiece into the principal piece. the spermatozoal head.121 These fibrous structures are thought to provide The connecting piece of the flagellum consists pri- structural support and passive elasticity during marily of a capitulum, segmented columns of fibers, flagellar bending. Nine irregularly shaped outer and the proximal and distal centrioles. The con- dense fibers occupy sites overlying the nine axon- necting piece serves to attach the flagellum to the emal doublets along the entire length of the mid- head, and also functions to produce and stabilize piece. The outer dense fibers taper at varying some structural components of the flagellum. The locations within the principal piece. Two of the capitulum articulates with the head at the level of outer dense fibers terminate in the proximal portion the implantation fossa, attaching by fine filaments of the principal piece, and the structural support of

122 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

Fig. 23. Transmission (A and B) and scanning (C and D) electron microscope views of the efferent ducts. (A and B) Cells lining the efferent ducts are composed of pseudostratiﬁed columnar epithelial cells and basal cells (BCs). Ciliated cells (CCs) provide a mechanism to move the spermatozoa through the duct, whereas nonciliated cells (NcC) contain microvilli that enhance absorption of ﬂuid by endocytosis and pinocytosis. Vesicles (Vs) located at the apex of the cells near the lumen are evidence of their absorptive functions. (C and D) Spermatozoa in the efferent ducts still have proximal droplets characteristic of spermatozoa in the testis and efferent ducts. Both ciliated and nonciliated epithelial cells can be seen. Scale bars ϭ 4.7 (A), 1.33 (B), 5 (C), and 2.2 ␮m (D).57

these two fibers is replaced by inward extensions of dense fibers are fully formed. The axoneme of the the fibrous sheath. flagellum in the developing spermatid originates from The mitochondrial sheath forms near the end of the centriolar apparatus and extends outwardly in the spermiogenesis, i.e., after the axoneme and outer flagellar canal (Fig. 14). This canal is created by

Fig. 24. Low-magnification scanning electron micrographs of an efferent duct (A) and the various profiles of the same single duct in the caput epididymidis (B). (B) A cluster of spermatozoa (arrow) is seen filling the lumen of one cross-section of this duct. Sparse smooth muscle surrounds the efferent ducts (A) and caput epididymidis (B). Scale bars ϭ 10 (A) and 112 ␮m (B).57

AAEP PROCEEDINGS ր Vol. 53 ր 2007 123 MILNE LECTURE

Fig. 25. Scanning (A) and transmission (B) electron micrographs of the epithelium of the caput epididymidis. Tall columnar cells are lined with stereo cilia (extremely long microvilli; Sc). (A) The cytoplasm (C) and luminal surface of these cells are revealed. (B) Section through the apex of these cells reveals forming vesicles (FVs) in the process of endocytosis. Scale bars ϭ 10 (A) and 2 ␮m (B).57 movement of the annulus and attached plasma mem- prevents attachment of the mitochondria to the mid- brane to the posterior aspect of the nucleus, resulting piece of the spermatid until the annulus migrates dis- in an infolding of the plasma membrane toward the tally to the junction of the midpiece and principal posterior aspect of the nucleus. The ﬂagellar canal piece.26,124 Coincidently, the mitochondria assume

Fig. 26. Scanning electron micrographs of spermatozoa in the caput epididymidis with their proximally located cytoplasmic droplets (CD; A), epithelium of the corpus epididymidis with its tall columnar cells with shorter stereo cilia than found in the caput (B), corpus epididymidis spermatozoa undergoing translocation of the cytoplasmic droplet (C), and epithelium and thick smooth muscle (SM) wall of the proximal cauda epididymidis (D). (C) The corpus epididymidis is the site of droplet translocation from the proximal to the distal position. The translocation process appears to occur by bending (arrow) of the tail around the droplet followed by re-straightening of the tail. Scale bars ϭ 10 (A), 45 (B), 10 (C), and 240 ␮m (D).57

124 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

Fig. 27. Scanning electron micrographs of epithelium and part of the smooth muscle (SM) wall of the proximal cauda epididymidis (A), spermatozoa of the cauda epididymidis (B), cross-section of the distal cauda epididymidis revealing the epithelium, supportive connective tissue, and thick smooth muscle (SM) wall (C), and ejaculated spermatozoa, most of which have lost their cytoplasmic droplets (D). Scale bars ϭ 50 (A), 10.8 (B), 556 (C), and 4.5 ␮m (D).57 their final position in an end-to-end spiral arrange- didymal reservoir (cauda epididymis and ductus ment around the outer dense fibers of the midpiece. deferens)—suppression of motility; ejaculation—ac- The length of the midpiece, and the number of mito- tivation of motility; and oviductal reservoir—hyper- chondrial gyres, varies greatly among mammalian activation of motility. Spermatozoa in the cauda species, but is relatively constant within a given spe- epididymis are intrinsically capable of motility,68,126 cies. The equine spermatozoon contains 40–50 mito- but do not exhibit motility until released from the chondrial gyres. The order and symmetry of the epididymis. A threshold level of cyclic adenosine mitochondria along the midpiece may convey a func- monophosphate (cAMP) is present in spermatozoa tional effect, as disruptions in this organization have within the cauda epididymis.127 Specific motility- 125 been associated with reduced fertility of stallions. inhibiting proteins have been identified in rat cauda The fibrous sheath extends along the entire length epididymal fluid that, when removed, allow initia- of the principal piece, i.e., from the annulus to the end tion of motility.128,129 A pH-dependent inhibitory piece. It is composed of two longitudinally arranged factor has been reported in bulls.130,131 Although fibrous columns that are bridged by a series of inter- such inhibitory factors may exist, it is possible that connecting, circumferentially arranged fibrous ribs. sperm motility may simply be suppressed by the The fibrous sheath is thought to provide rigid struc- acidic pH of the epididymal environment. The pH tural support and elasticity to the flagellum, but it also of bull cauda epididymal fluid is reported to be 5.5, serves an important role as a scaffold for a variety of and the cytosolic pH of bull epididymal spermatozoa cell-signaling and metabolic events. is reported to be 6.5–6.6.130,132 Caudal epididymal 5. Physiological Considerations fluid of bulls, rams, boars, and stallions does not contain measurable quantities of bicarbonate Ϫ 133 Ϫ Spermatozoal Motility (HCO3 ), and HCO3 is known to be a key effec- Under natural conditions, regulation of spermato- tor of spermatozoal motility.134,135 Bicarbonate is zoal motility occurs at three critical points: epi- present at fairly high concentrations in seminal

AAEP PROCEEDINGS ր Vol. 53 ր 2007 125 MILNE LECTURE

Fig. 28. Transmission electron micrographs of spermatozoa treated with the ionic detergent, sodium codicil sulfate, from the proximal corpus epididymidis (A) and distal corpus epididymidis (B). (A) Spermatozoa from the testis to the proximal corpus epididymidis have little resistance to detergent treatment, as noted by loss of all tail components and membranes and the deconden- sation of the nucleus (DN). (B) Spermatozoa from the distal corpus epididymidis, through the cauda epididymidis and in the ejaculate have acquired some resistance to this detergent treatment during maturation in the epididymidis as noted by the presence of the outer membrane of mitochondria (OM), the outer dense fibers (DF) of the tail, and nucleus (N) that have not decondensed. Scale bars ϭ 0.6 (A) and 1 ␮m (B).68 Fig. 29. Drawings showing magnified views of an equine spermatozoon, represented by an uncut view (center), a mid-sagittal view (left), and a partially resected view (right). The various lengthwise divisions of the spermatozoon are represented as plasma and may be higher in seminal plasma of head, flagellum (tail), midpiece, principal piece, and end piece stallions than some other mammals studied.136 (end). (A) Acrosome. (B) Plasma membrane. (C) Nucleus. (D) Simple exposure of normal spermatozoa to seminal Mitochondria. (E) Axoneme. (F) Outer dense fiber. (G) Fibrous plasma or physiologic fluids will activate spermato- sheath. (H) Axonemal microtubules. zoal motility that is characterized by a moderate- amplitude and symmetrical flagellar beat leading to a forward propulsive trajectory.137,138 This form of gradient permits the Naϩ/Kϩ exchangers to catalyze spermatozoal movement is defined as activated mo- the coupled exchange of extracellular Naϩ for intracel- tility and is to be differentiated from hyperactivated lular Hϩ.143 A sperm-specific Naϩ/Kϩ exchanger has motility discussed below. Activated motility is con- been localized to the principal piece and likely plays 142,145,145 sidered necessary for propelling sperm into the ovi- an important role in regulating [pH]i. Acti- ductal reservoir. vation of motility requires that this exchanger is func- 145 ϩ Ϫ Environmental cues activate spermatozoal motility tional. It is also likely that a Na /HCO3 co- though a signal transduction mechanism. Although transporter plays a role in controlling cytosolic pH the signaling pathway(s) of activated spermatozoal (Fig. 34).140 motility are not resolved completely, an ever-increas- Although increased alkalinization of the sper- Ϫ ing body of information indicates that HCO3 , calcium matozoal cytosol is known to activate membrane ions (Ca2ϩ), and cAMP are key signaling compo- Ca2ϩ channels, this may be of primary importance 112,139 Ϫ nents. Transmembrane movement of HCO3 in hyperactivation of spermatozoal motility where into the spermatozoal cytosol is associated with an increased cytosolic Ca2ϩ is required.141,147–152 increase intracellular pH ([pH]i), leading to regulation Spermatozoal motility can be activated and main- of cAMP.140,141 Activity of Na,K-ATPase and tained for a short time in Ca2ϩ-free media for Naϩ/Kϩ exchangers are also of central importance in many species,126,138,153 but presence of extracellu- 142–146 2ϩ 154–156 regulating [pH]i and sperm motility. Sperma- lar Ca maximizes spermatozoal motility. tozoal Na,K-ATPase is important for generation of the The flagellum is known to contain a variety of Naϩ gradient required for ion transport. This Naϩ Ca2ϩ membrane transport channels, including

126 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

Fig. 30. Phase contrast (J), scanning electron (B), and transmission electron (A and C–I) micrographs of equine spermatozoa. (A) The plasma membrane (PM) encloses the entire head and tail. The head is composed of the nucleus (N), the overlying acrosome (Ac), and postacrosomal region (PR). The acrosome can be divided into the apical segment (ASA), principal segment (PSA), and equatorial segment (ESA). The inner acrosomal membrane (IAM) is in juxtaposition with the nuclear membrane (NM). The outer acrosomal membrane (OAM) fuses with the plasma membrane during the acrosome reaction to discharge the acrosomal contents and expose molecules attached to the inner acrosomal membrane. (J) The tail is composed of the middle piece (MP) that houses the mitochondria (M), the principal piece (PP) that contains the ﬁbrous sheath (FS), and the end piece (EP). (C) The tail attaches at the implantation fossa (IF) (D–I) The distal centriole gives rise to the “9ϩ2”-arranged microtubular complex of the axoneme, with the nine outer doublets (DIs) surrounding the central pair (CP) of microtubules. The nine dense ﬁbers (DFs) parallel the axoneme and extend to different lengths within the principal piece. In the end piece, the axonemal doublets become disorganized and ultimately separate into 20 single microtubules (SM), but still are enclosed in the plasma membrane. The cytoplasmic droplet (CD), located at the proximal end of the middle piece in spermatozoa recovered from the equine efferent ducts (B and C) is characteristic of spermatozoa that have not yet matured in the epididymis. These cytoplasmic droplets contain remnants of spermitid cytoplasm that are not lost in the residual body on spermiation. Contents may include endoplasmic reticulum, mitochondria, and microtubules, including remnants of manchettes. Scale bars ϭ 0.5 (A and B), 0.75 (C), 0.24 (D–I), and 1.28 ␮m (J).19,26

voltage-gated, cyclic nucleotide–gated, transient suggests that they probably contribute in some 2ϩ 2ϩ receptor potential, Ca release, and CatSper manner. Sperm [Ca ]i is also known to be reg- channels. Although the roles of some of these ulated by Ca2ϩ-ATPases, Naϩ/Hϩ exchangers, and channels remain unknown, their mere presence Ca2ϩ/Hϩ exchangers.157

AAEP PROCEEDINGS ր Vol. 53 ր 2007 127 MILNE LECTURE

Fig. 33. Magnified illustrations of cross-sections through the middle piece (midpiece) and principal piece regions of equine spermatozoa. (A) Plasma membrane. (B) Mitochondria. (C) Outer dense fiber. (D) Axonemal doublet. (E) Central pair of Fig. 31. Further magnified illustrations of Fig. 29, revealing axonemal microtubules. (F) Sheath surrounding central pair of mid-sagittal and partially resected views of equine spermatozoa. axonemal microtubules. (G) Radial arm. (H) Fibrous sheath. (I) (A) Plasma membrane. (B) Acrosome. (C) Outer acrosomal mem- Outer dynein arm. (J) Inner dynein arm. (K) Longitudinal brane. (D) Inner acrosomal membrane. (E) Nuclear envelope. (F) column of fibrous sheath. (L) Connecting bridge between cen- Post-acrosomal lamina. (G) Nucleus. (H) Basal lamina and un- tral pair of axonemal microtubules. (M) Nexin links. (N) An- derlying capitulum. (I) Proximal centriole. (J) Segmented column. nulus. (K) Outer dense fibers. (L) Outer doublets of axoneme. (M) Center pair of microtubules within the axoneme. (N) Mitochondria.14

production of cAMP, as it relates to motility, is cat- The signaling pathway for activated spermatozoal alyzed by soluble adenylyl cyclase (sAC). sAC has motility involves the cAMP-dependent protein ki- been identiﬁed in the ﬂagellum of spermatozoa and nase A (PKA) pathway.156,158–165 Intracellular is required for activated motility166 and capacitation

Fig. 32. Transmission electron micrograph revealing sagittal views of adjacent equine spermatozoal heads. One spermatozoon has an intact acrosome (solid arrow) and one has undergone the acrosome reaction induced by the calcium ionophore, A23187 (open arrow).

128 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

Fig. 34. A schematic model of some documented and hypothesized molecular events associated with activated and hyperactivated forms of spermatozoal motility. Please refer to the text for an explanation of these signaling pathways and for ﬁgure abbreviations.

(see below). Interestly, the cAMP-sAC-PKA path- central to dynein function. One study indicated way does not seem to be required for hyperactivated that CaM can also interact with T-type voltage- motility150 or the acrosome reaction.166 sAC is gated Ca2ϩ channels by a mechanism independent Ϫ167,168 2ϩ 169,170 stimulated directly by HCO3 or Ca , of PKA, CaMK, or CaM-dependent protein phospha- thereby catalyzing phosphorylation of PKA, which tase.189 In addition, CaM may be involved in reg- leads to phosphorylation of flagellar proteins that ulation of adenylyl cyclase.182 are central to activated motility.138,171–174 One A substantial and continuous supply of energy, such protein is A kinase anchoring protein (AKAP). in the form of ATP, is required for activated AKAP has been detected at the level of the axonemal spermatozoal motility, and this requirement is central pair, the fibrous sheath (in the principal heightened for hyperactivated motility.150 The piece), and the outer dense fibers (in the middle mechanisms by which ATP is generated and piece).112,138,171,175,176 PKA is known to bind to transferred in the flagellum remain unsolved. AKAP, thus suggesting that the action of PKA can Certainly, oxidative respiration within the mito- be regionalized, and its substrate specificity aided, chondria yields a plentiful supply of ATP. Some through this anchoring mechanism.112,138,171,177,178 investigators propose that the ATP produced in Although PKA is known to be located in the vicinity this manner is capable of diffusing along the en- of the outer dynein arms,175 the need for a localized tire length of the flagellum in a manner suitable pattern of PKA distribution remains unresolved.179 for initiation and maintenance of spermatozoal In addition to regulating the cAMP-sAC-PKA motility.138,190–192 Possibly, an adenylate kinase pathway, Ca2ϩ affects dynein activity through direct shuttle may assist in the rapid distribution of control of the central pair of microtubules within the mitochondrial-produced ATP.192 Recently, ade- axoneme.180 As Ca2ϩ can inhibit the activity of the nylate kinases were regionalized to the principal axonemal central pair to regulate dynein arm func- piece of mice spermatozoa.193 Others contend tion,180 excessive Ca2ϩ can lead to cessation of sper- that local glycolysis within the principal piece is matozoal motility.181 critical to the timely generation and distribution Calmodulin (CaM), a Ca2ϩ-binding and sensor of ATP required for spermatozoal motili- protein, is a key component of spermatozoal signal- ty.171,194,195 Certainly, a variety of glycolytic en- ing mechanisms.181–186 CaM associates with the zymes have been identified in association with the outer dynein arms and the radial spokes, and may fibrous sheath and/or outer dense fibers,196–202 play a role in Ca2ϩ-dependant propagation of flagel- and it is well known that the glycolytic pathway to lar bending.172,175 CaM kinase II (CaMK)151 and energy production is essential under anaerobic CaM-dependent protein phosphatase172,187,188 are conditions. Arguments in favor of oxidative res-

AAEP PROCEEDINGS ր Vol. 53 ր 2007 129 MILNE LECTURE piration and of glycolysis exist for generation and flagellum to bend, and also provides elastic supply of ATP for spermatozoal motility. A recoil.210,211 sperm-specific glycolytic enzyme, glyceraldehyde- Bending of the flagellum requires a patterned ac- 3-phosphate dehydrogenase (GAPDH), has been tivation of microtubule sliding around the circum- localized to the principal piece, and targeted dele- ference, and along the length, of the cylindrical tion of this enzyme is reported to lead to a severe array of microtubules that compose the axoneme. reduction in motility of mouse spermatozoa.194 This dynein-generated activity is regulated by the In contrast, another study revealed that chemical central pair of microtubules and their associated inhibition of GAPDHs did not impact spermato- structures, collectively called the central apparatus. zoal motility or ATP concentration.192,203 At The asymmetry of components in the central appa- present, it seems possible that either oxidative ratus forms the basis of a “timing device” that cre- respiration or glycolysis, or both, can support the ates the spatially timed sliding of various 180,212 ATP generation and availability needed to drive microtubules. As such, the central apparatus spermatozoal motility. The length of the princi- both constrains and activates the dynein arms pal piece in stallions is similar to that in bulls, through communication through the radial 137,213 boars, rams, and dogs. It is also considerably spokes. shorter than the principal piece of mice, rats, ham- Spermatozoal Metabolism sters, and guinea pigs, where many experimental studies are conducted.113,192 This may explain Spermatozoa require a constant supply of energy for some of the inconsistencies among laboratories. maintenance of cellular order and functions needed The axoneme, dynein arms, outer dense fibers, for survival. This energy requirement increases mitochondrial sheath, and fibrous sheath are often significantly with the onset of activated motility,214,215 and becomes even more pronounced when portrayed as the fundamental elements of the fla- 150 gellum. Although each is vital to flagellar function, hyperactivated motility is initiated. Approxi- one must also be aware that these elements are mately 500 common metabolic reactions are known to occur in most somatic cells, many of which require embedded in a network of other molecules that are 216 equally important. Bending of the flagellum is energy. A spermatozoon is a “stripped-down” cell that is designed expressly for propagation of caused by reciprocal sliding between doublet micro- genes, and it is one of the smallest cells in the body, tubules within the axoneme. The dynein arms are but it accomplishes its mission at a distance far from permanently attached to one doublet microtubule its origin in the epididymis. While spermatozoa with arms extending to intermittently engage an require a relatively small amount of energy for adjacent doublet microtubule. The dynein arms “housekeeping” purposes,197,214 a plentiful supply of are situated at 24- (outer dynein arms) to 96-nm substrate is required to provide a spermatozoon (inner dynein arms) intervals along the entire 204,205 with the stamina required for this arduous journey. length of the axoneme. When the cross- Spermatozoa are also capable of prolonged survival bridges created by the dynein arms are complete both within and outside the body. Exogenously de- between neighboring doublets, the corresponding rived nutrients are needed to fulfill the energy de- microtubules are prevented from sliding. Dynein is mands of these very metabolically active cells. high molecular weight ATPase, so the dynein arms These nutrients (or substrates) are metabolized in- have the capacity to transform chemical energy into tracellularly, resulting in the release of chemical unidirectional mechanical force. Each dynein arm bond energy. Useable energy is made available for has three ATP-sensitive binding sites, and all sites cellular processes primarily in the form of the acti- must bind with ATP for the dynein arm to detach vated carrier molecule, ATP, with other macromol- from the microtubule,206 i.e., the crossbridge attach- ecules such as NADH, NADPH, FADH2, and acetyl ment-detachment cycle is dependent on binding and CoA also providing vital high-energy intracellular hydrolysis of ATP. The conformational change in linkages.216,217 the dynein arms results in the force generation re- Spermatozoa possess the metabolic machinery required for microtubule sliding. The microtubules quired for glycolysis, the citric acid cycle, and oxida- of the axoneme enhance the rate of ADP release tive phosphorylation.218 Glycolysis occurs in the after the dephosphorylation to aid the reactivation cytosol and has both an anaerobic and aerobic step.207 The nexin arms also undergo periods of mode,219 whereas the citric acid cycle and oxidative displacement to allow sliding of microtubules.137 phosphorylation are strictly aerobic and proceed The basic mechanism is similar to that of muscle within the mitochondria. A variety of substrates can myosin; however, the loss and rebinding of ATP be garnered for energy extraction, including products occurs at two to three orders of magnitude monosaccharides, pyruvic acid, lactic acid, and even faster for dynein than for myosin.208 Bending of fatty acids and amino acids, the last four of which the tail occurs during axonemal sliding because the require an aerobic environment. Sorbitol, a sugar microtubules are fixed at the base of the flagel- alcohol obtained by reduction of glucose through the lum.209 The passive elastic nature of the overlying polyol pathway, is thought to serve as a substrate for outer dense fibers and fibrous sheath permits the metabolism.218 Although the seminal plasma of

130 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE stallions does contain this product,136 stallion sper- creased responsiveness to both fructose and matozoa seem to be incapable of metabolizing it to glucose.226 produce ATP.218,220 Either glucose or fructose can increase motility Generally speaking, glycolysis is considered the and ATP concentration of human spermatozoa.227 “backbone” of the energy-procurement pathways be- One study with human semen revealed that sper- cause it can proceed in situations of low oxygen matozoal motility was maximized in medium con- tension and its product, pyruvate, can be further taining 1 mM glucose or 15 mM fructose; however, catabolized to CO2 and H2O by oxidative respiration. motility was further enhanced when both monosac- Efficiency of energy production is lowest in the gly- charides were provided.228 Conversely, in vitro fertil- colysis pathway, where one molecule of a monosac- ization in mice and rats is not attainable when fructose charide can generate a net increase of two molecules is the only available substrate but high when glucose 229–232 of ATP. Conversely, complete oxidation of glucose is included in the media. Other studies cer- through the citric acid cycle and oxidative phosphor- tainly support the importance of glucose in gamete 233–235 ylation generates an additional 34 molecules of ATP interaction. per monosaccharide molecule. Despite the in- Transport of monosaccharides across the plasma creased efficiency of oxidative metabolism, a membrane requires the presence of carrier proteins monosaccharide cannot directly enter the citric acid and is an ATP-dependent process. One report in- cycle, but must first be metabolized to pyruvate; volving bull spermatozoa indicated that the glucose hence, the need for glycolysis as the first stage of transport protein has only a limited ability to trans- energy production where monosaccharides are port fructose and that another carrier protein may 216 be the primary means by which fructose gains access concerned. 214,236 Glycolysis involves a sequence of 10 separate re- into the cytosol. This carrier protein may be actions, the first of which is catalyzed by hexokinase absent in stallion spermatozoa, thus explaining why fructose may not be a primary, or even a suitable, in an ATP-consuming manner. This enzyme can 214 phosphorylate several types of monosaccharides, in- energy source in this species. cluding glucose, fructose, mannose, and galactose. The extent to which spermatozoa use glycolysis or The affinity of hexokinase for glucose is reported to oxidative respiration for generation of energy varies considerably among mammalian species and proba- be 20 times higher than its affinity for fructose.221 bly with the profile of spermatozoal motility, and Another account indicated that, in mammalian sper- environmental conditions.214,218 Few studies have matozoa, glucose is used in preference to fructose, been directed toward stallion spermatozoa so much but that mannose has the highest affinity of the of the information must be extrapolated from infor- three monosaccharides to hexokinase.218 Of inter- mation garnered from other species. This approach est, a study involving astroglial cells revealed that may be precarious, however, because of the known mannose-phosphorylating ability is only 40% of glu- 222 species variation in spermatozoal metabolic prefer- cose-phosphorylating ability. ences. One report suggests that bull and ram sper- Fructose was identified as the “seminal sugar” in 223 matozoa use the glyocolytic pathway at a much 1946. Subsequent studies have revealed that higher rate than stallion spermatozoa and that stal- the concentration of this sugar is high in seminal lion spermatozoa rely more heavily on aerobic me- plasma of bulls, rams, and man (range of 40–1000 tabolism for energy production.214,220 Others mg/100 ml) but extremely low (Ͻ1 mg/100 ml) in 218 report that ram and bull spermatozoa oxidize acetic seminal plasma of stallions. Interestingly, an- acid in preference to fructose and glucose.218 Hu- other report indicated that fructose concentration in 224 man, boar, and mouse spermatozoa seem to obtain a stallion semen ranged from 7 to 11 mg/100 ml. high proportion of their ATP by glycolysis.237–239 Seminal plasma concentrations of some other me- Although more intensive study with stallion sper- tabolizable substrates, such as citric acid, lactic acid, matozoa is required to more fully elucidate meta- and pyruvic acid (used exclusively by oxidative res- bolic pathway preferences, glycolytic breakdown of piration), are also much lower in stallion seminal glucose is currently considered to be a major source 218 plasma than that of bulls, rams, or man. Bull of ATP production.214 and ram spermatozoa use fructose at a rate of 2 The minimal oxygen tension required to maintain mg/109 spermatozoa/h under anaerobic conditions at a linear rate of O2 uptake by rabbit spermatozoa has 37°C, whereas boar spermatozoa use fructose at a been reported to be ϳ10 mmHg.218 As such, the rate that is 10 times lower.218 One report suggests luminal environments of the uterus and oviduct are that stallion spermatozoa have a limited capacity to thought to be sufficiently high in oxygen content to use fructose.220 Interestingly, fructose is not found allow aerobic respiration to proceed.218,240,241 in seminal plasma of dogs and glucose is used pref- Storage of spermatozoa outside the body cavity, erentially to fructose by dog spermatozoa; yet, fruc- however, can impact availability of oxygen and met- tose maintains higher spermatozoal motility than abolic processes. If raw or extended semen is left does glucose under in vitro storage conditions.225 undisturbed in a laboratory setting, use of dissolved Heterogeneity may also exist among motile dog O2 by aerobic respiration leads to depletion of O2 and spermatozoa, with subpopulations experiencing in- the need to resort to glycolysis for meeting energy

AAEP PROCEEDINGS ր Vol. 53 ր 2007 131 MILNE LECTURE demands.214 For oxidative metabolism, bull and ram Spermatozoa are exposed to ROS that are derived ϳ ␮ 8 spermatozoa use O2 at a rate of 10–20 l/h/10 both intracellularly and extracellularly. Produc- spermatozoa at 37°C.218 A potential drawback of ox- tion of superoxide occurs continuously within the idative respiration is the production of reactive oxygen mitochondrial electron transport chain, where O2 species that can lead to a disruption of ATP produc- acts as an electron carrier during oxidative respiration, lipid peroxidation, or other cellular mechanisms tion. A molecule of O2 must pick up a total of four 242–246 ϩ ϩ ϩ Ϫ 3 that result in spermatozoal dysfunction. electrons to form water (i.e.,O2 4H 4e 2 Metabolism of endogenous substrates by sperma- H2O). During this process, the reactive oxygen tozoa for the production of energy is thought to be forms are generally caged within the respiratory minimal, possibly representing 10% or less of total enzyme complexes. A small percentage of the su- energy production, based on calorimetric and carbon peroxide produced does not stay within the mito- balance techniques.247 For bull spermatozoa, en- chondrial respiratory chain but escapes to exert dogenous metabolism occurs at a constant rate at actions on other cellular components, including lip- storage temperatures between 20°C and 35°C.248 ids, sugars, proteins, and nucleic acids.255,258,261–264 Recently, presumed non-mitochondrial production Spermatozoal capability for glycogen synthesis Ϫ• and storage was once thought to be nonexistent. of O2 has been shown to occur in spermatozoa of 265 266 Recent reports, however, indicate that spermatozoa both men and stallions. Evidence is mount- of dogs, boars, rams, and horses do contain glycogen, ing that spermatozoa contain NADPH oxidase that as well as the enzymatic machinery required for its catalyzes the formation of superoxide, presumably 267–270 production.192,249,250 for physiologic reasons, although some work- The pentose phosphate pathway does not seem to ers conclude that spermatozoal-derived NADPH ox- 254,271,272 be directly involved with spermatozoal motility,218 idase activity is insigniﬁcant. An but may be central to sperm–oocyte interactions,251 aromatic amino acid oxidase, released from dead possibly through production of NADPH and the re- spermatozoa or present in seminal plasma, has also sultant generation of radical oxygen species re- been reported to generate production of H2O2 pro- 273–276 quired for sperm–oocyte fusion.252 Furthermore, duction in semen. the pentose phosphate pathway may aid in protec- Generation of ROS is known to be more pro- tion against oxidative injury through generation of nounced in morphologically abnormal spermatozoa, a feature that can be associated with retention of NADPH, which is required to return expended glu- 258,277–281 tathione peroxidase to its reduced (protective) form excessive amounts of residual cytoplasm. (see Reactive Oxygen Species).253,254 Damaged or morphologically abnormal equine spermatozoa are known to generate more ROS than do morphologically normal sperm.282 Reactive Oxygen Species Neutrophils are known to be a primary exogenous A discussion directed at reactive oxygen species source of ROS in semen of men, where a relatively (ROS) at this point in the text seems appropriate high concentration of leukocytes is common- because of the pathologic consequences that ROS place.283,284 Although spiking equine semen with can have on spermatozoal motility and spermatozoal activated neutrophils can lead to reduced motili- metabolism. Reactive oxygen species are products ty,285 contamination of equine semen with sufﬁcient derived from the reduction of (i.e., addition of elec- neutrophils to affect spermatozoal motility is quite trons to) diatomic oxygen (O2) and include radicals uncommon. and other reactive products. Radicals are atomic or Another source of ROS is the uterine environ- molecular species that have unpaired electrons in ment, such as the ROS generated from estrogen- their orbits, thus making them quite unstable, i.e., induced uterine NADPH oxidase of the endometrial highly reactive and likely to participate in a variety epithelium.276,286 Cycle-regulated synthesis of of chemical reactions to displace, receive, or share uterine NO has also been described.287,288 Leuko- electrons so that they may once again become sta- cytes of mares produce H O in response to sperma- 255 2 2 ble. Other O2 derivatives are not radicals but tozoa or bacteria, and this activity is heightened by are highly reactive; hence, these products are collec- leukocyte exposure to seminal plasma,289 suggesting • tively termed ROS. Examples of ROS (where im- a means for uterine clearance of bacteria and sper- plies the presence of an unpaired electron) include matozoa. Others, however, report a protective ef- Ϫ• • 280,290,291 superoxide radical (O2 ), hydroxyl radical (OH ), fect of seminal plasma against ROS. • • hydroperoxyl radical (HO2 ), peroxyl radical (RO2 ), Seminal plasma of some subfertile men is known to • 292,293 alkoxyl radical (RO ), hydrogen peroxide (H2O2), possess reduced antioxidant capacity. and subclasses of ROS that contain reactive nitro- Unsaturated lipids are quite susceptible to peroxi- • gen or chlorine species, such as nitric oxide (NO ), dative injury when exposed to various ROS, and Ϫ • ϩ Ϫ• 3 peroxynitrite (ONOO ; where NO O2 mammalian spermatozoa are especially susceptible ONOOϪ), or hypochlorous acid (HOCl).255 ROS because their plasma membranes are rich in poly- may assume physiologic roles, as discussed under unsaturated fatty acids.242,245,257,294–299 A recent Capacitation; however, their presence can also lead report indicates that the spermatozoa of individual to spermatozoal demise.66,256–260 men vary in their membranous unsaturated fatty

132 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE acid content and that a superabundance of polyun- Glutathione reductase (GRD) reduces GSSG to GSH saturated fatty acids predisposes spermatozoa to ox- to complete the cycle as follows: idative injury.300 Peroxidative injury has been 258 thought to adversely affect membrane fluidity, ϩ ϩ although some work suggests that the oxidative ac- GSSG ϩ NADPH ϩ H O¡ 2 GSH ϩ NADP tion may not occur through lipid peroxidation.301 GRD Oxidation may directly affect proteins and membrane permeabilization (possibly through oxidation Superoxide dismutase is considered to be preva- lent in spermatozoa of many species261,311–313 and of sulphydryl groups on enzymes and membrane Ϫ• 302 rapidly reduces O2 to H2O2. The direct cellular proteins), as opposed to disturbing lipid fluidity. Ϫ• Others have described an effect of ROS on the sperm action of O2 is thought to be minimal, with its major effects produced indirectly through conversion to axoneme and ATP generation that can lead to an 243,256,260,274,294,301,307,310,314 255 irreversible loss of motility.303,304 H2O2 or other radicals. In stallion spermatozoa, a ROS-associated reduc- Spermatozoa of stallions are thought to have limited tion in motility was not associated with a detectable intrinsic SOD, with SOD activity derived primarily from the seminal plasma through adsorption to the increase in lipid peroxidation or a decrease in via- 315 bility or mitochondrial membrane potential.260 plasma membrane. Glutathione peroxidase has also been identified in This finding suggests that ROS-related effects on 311,313,316 equine spermatozoa may occur through a mecha- spermatozoa or seminal plasma and is considered to impart protection against oxidative injury nism unrelated to lipid peroxidation and also indi- incurred by H O 316,317; however, a primary action cates that motility may be a more sensitive indicator 2 2 of glutathione may be protection against peroxyl of ROS-related injury than the other experimental • radicals of polyunsaturated fatty acids (RO ) endpoints, i.e., viability, mitochondrial membrane 2 through conversion to relatively inert hydroxyl fatty potential, and lipid peroxidation. Others report acids.313,318 Glutathione also plays a structural that lipid peroxidation is a good predictor of sperm role, as shown by sulfhydryl oxidation and cross- motility and consider a lipid peroxidation assay to be 305 linking associated with nuclear condensation and a potential clinical test. assembly of the midpiece.317,319 Glutathione is The mechanisms of ROS-induced damage to DNA 315,320 306 present in stallion semen, but the source in have been evaluated intently. Sperm DNA is semen seems to be primarily of seminal plasma known to be susceptible to oxidative injury, result- origin.315 ing in reduced fertility and perhaps even pregnancy 307 Spermatozoa from most species contain little to loss or a variety of pathologic entities in offspring. no catalase243,245,321–324; however, seminal plasma Aberrant DNA repair by spermatozoa is thought to is generally high in catalase activity.67 The amount increase the mutagenic load of any resulting concep- of catalase in rabbit semen is genetically influenced tus, thereby leading to post-fertilization cri- (estimated heritability value of 0.48).325 Stallion 281,308 ses. Mitochondrial DNA is more susceptible semen contains catalase activity311,326 that is pri- 326 to H2O2-induced damage than is nuclear DNA, pos- marily derived from prostatic secretions. Several 309 sibly making it a good marker of oxidative injury. studies have shown that addition of catalase to se- Spermatozoa of stallions are susceptible to ROS- men of various species will help neutralize the det- induced DNA fragmentation.310 rimental effects of H2O2 on spermatozoa after Protection against the effects of ROS in sperma- in vitro storage.260,285,290,301,310,327–330 Similarly, tozoa is afforded by an assortment of scavenging oviductal fluids may contain catalase in sufficient molecules, including three enzyme systems: (1) su- quantity to protect sperm from oxidative injury.330,331 peroxide dismutase (SOD), (2) catalase (CAT), and Surprisingly, stallion semen with higher initial activ- (3) the glutathione peroxidase system (GPX), as in- ity of CAT, SOD, and GSH did not lead to improved dicated by the following reactions: retention of spermatozoal motility or membrane integrity after cooled storage.311 Addition of CAT to ex- Ϫ• ϩ Ϫ• ϩ ϩ O¡ ϩ O2 O2 2H SOD H2O2 O2 (1) tended equine semen did not improve maintenance of motility after cooled storage.332 Similarly, addition of CAT, SOD, or GSH, ascorbic acid, or ␣-tocopherol to O¡ ϩ 2H2O2 2H2O O2 (2) semen extender did not improve post-thaw quality of CAT stallion spermatozoa.333 Studies with ram spermatozoa suggest that CAT, SOD, and GSH are ineffective ϩ 3 ϩ 302,334 2 GSH H2O2 GSSG 2H2O (3) at preventing acute peroxidative injury to lipids, and incorporation of CAT in extenders for sex-sorted where GSH represents monomeric glutathione and or non-sorted semen does not improve post-thaw qual- GSSG represents oxidized glutathione disulfide. ity of ram spermatozoa.335

AAEP PROCEEDINGS ր Vol. 53 ր 2007 133 MILNE LECTURE Overall, spermatozoa have limited endogenous ported into the oviduct as early as 30 min after CAT, SOD, and GPX activity; therefore, spermato- insemination, based on extensive lavage of the zoa depend on extracellular availability of these en- uterus post-insemination with an iodine-based solu- zymes to counter oxidative stress, namely from tion to immobilize intrauterine spermatozoa. Preg- seminal plasma. Seminal plasma contains these nancy rates are maximized by delaying the uterine enzymes, in addition to a host of other free radical lavage until 4 h after insemination.365 Location of scavengers.276 Molecules with antioxidant activity intrauterine insemination seems to have a signifi- include, but are not limited to, ascorbic acid,336,337 cant impact on the spermatozoal number that gains ␣-tocopherol,246,334,336,338–340 taurine,341,342 hypo- access into the oviduct. In one study, a higher taurine,342 butylated hydroxyanisole (BHA; an anti- number of spermatozoa were recovered from the oxidant used for long-term preservation of food, ipsilateral oviduct after deep uterine-horn insemi- cosmetics, and pharmaceuticals),246,334,339 albu- nation than resulted from uterine body insemina- min,261,342,343 cysteine,344,345 lipoic acid,346–348 tion. In the same study, the number of oviductal xanthurenic acid,349–351 carnitine,352,353 ergothion- spermatozoa recovered from reproductively normal eine,354 and pyruvate.349,355 Many of these have mares and mares susceptible to post-mating endo- been used as dietary supplements, or as direct semen metritis were similar after artificial insemination of treatments, with mixed results.267,299,332,340,356–358 500 million total spermatozoa.366 Others have As the end product of glycolysis, pyruvate serves as an found more spermatozoa, and a higher percentage of important substrate for oxidative phosphorylation, motile spermatozoa, in the isthmic oviductal region but when present at relatively high concentrations, of reproductively normal mares than in mares sus- pyruvate can also exhibit antioxidant properties.355,359 ceptible to chronic uterine infection.367 The same Addition of pyruvate to milk-based extender at a con- investigators reported more spermatozoa in the ovi- centration of 2 mM is reported to improve equine sper- ducts when mares received semen from a fertile matozoal motility after cooled storage.349 stallion as opposed to a subfertile stallion.367 An- Presently, there is much controversy over types, other group reported an effect of stallion on sperma- doses, and delivery methods for antioxidants as they tozoal numbers in recovered oviducts of mares after relate to semen processing. Further study is re- artificial insemination, although the fertility or se- quired to determine whether antioxidant therapy men quality of these stallions was not provided.368 will maximize spermatozoal function, especially af- Administration of oxytocin to mares immediately ter cooled or frozen storage. after insemination did not improve pregnancy rates in mares bred by either fertile or subfertile stal- Spermatozoal Transport lions.369 This may be because of a directional pat- Spermatozoa are virtually immotile in the epididy- tern of luminal flow toward the cervix after oxytocin mis but develop motility (or, more precisely, acti- administration, as occurs naturally during the ex- vated motility) on ejaculation. Deposition of pulsive stage of labor (i.e., uterine evacuation).370,371 spermatozoa is intrauterine when artificial insemi- It is interesting to note, however, that dynamic scin- nation is used, and a large portion of an ejaculate is tigraphic analysis of radiolabeled spermatozoa in deposited directly into the uterine body at the time the mare uterus revealed radioactivity in the tips of of natural coitus if the mare’s cervix is dilated at the the uterine horns as early as 8 min after uterine time of breeding and if the stallion does not dis- body insemination (5-ml volume).360 In this study, mount prematurely during the ejaculatory process. uterine contractions did not propagate spermatozoa After intrauterine deposition of semen, the sperma- in one direction only; rather, movement continued in tozoa are rapidly transported to the oviduct, where a both tubal and cervical directions. Similarly, ultra- spermatozoal reservoir forms and the spermatozoa sonographic evaluation of the uterus after natural gain fertilizing potential before interaction with the mating of mares revealed similar patterns of uterine vestments of the oocyte near the ampullar–isthmic contractions in cervio-tubular and tubo-cervical di- junction. rections.362 The peristaltic contractions were in- Spermatozoal migration to the oviducts is depen- creased in frequency, amplitude, and duration in dent, to a large part, on uterine contractions.360 comparison with the pre-coital findings. It seems The effect of insemination volume on the frequency that bidirectional myogenic activity in the early pe- of these uterine contractions seems variable,361,362 riod after insemination assists spermatozoal trans- but, within the range tested (5–50 ϫ 106 spermato- port to the utero-tubal junction, and it is possible zoa/ml), spermatozoal concentration had no appar- that this mechanism is impeded by administration ent effect on spermatozoal numbers recovered from of supraphysiologic doses of oxytocin or other drugs mare oviducts at 4 hours after insemination.363 with oxytocic properties. Studies in pigs support The time required for spermatozoa to gain access this line of thought, because intrauterine infusion of into the oviduct has not been studied extensively. cloprostenol before insemination reduced oviductal Spermatozoa have been detected in the oviducts as recovery rates of spermatozoa and increased cervical early as 2 h after insemination, based on recovery of reflux of inseminates.372 In another report involv- spermatozoa in abattoir specimens.364 Sufficient ing pigs, spiking a semen dose with 10 IU oxytocin spermatozoa to establish pregnancy may be trans- reduced fertilization rate, whereas IV administra-

134 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE tion of the same dose at 5 min after insemination ond insemination were placed in an inflamed uterus improved fertilization rate.373 The addition of an- induced by the first insemination. Furthermore, it ␣ other uterotonic agent, prostaglandin F2 ,toex- could potentially impact semen transport in mares tended semen (final concentration of 125 ␮g/ml) did bred for the first time in an estrous cycle if the mare not improve the pregnancy rate in a small group had a pre-existing acute form of endometritis. To of mares.374 that effect, pregnancy rates in mares were dramat- The involvement of other factors that may impact ically increased on the second insemination of an spermatozoal transport in horses requires further estrous cycle (i.e., 12 h after insemination of 1 ϫ 109 examination. As an example, studies involving killed spermatozoa in semen extender or infusion of pigs revealed that the mere presence of a boar (i.e., semen extender only to induce an endometritis) non-tactile presence) could increase peripheral when the inseminate contained washed spermato- plasma oxytocin concentration in the sow,375 and zoa in seminal plasma (17/22; 77%) compared to washed spermatozoa in semen extender only (1/22; uterine activity after boar exposure was most en- 389 390 hanced in sows that initially had a reduced fre- 5%). Clement et al. reported embryo-recovery quency of uterine contractions.376,377 Similar statistics for mares inseminated two to three times studies involving horses are contradictory, with in an estrous cycle (at 2-day intervals) until ovula- some reports of increased myometrial activity and tion occurred, using different stallions for each in- plasma oxytocin concentrations after simple stallion semination. For mares inseminated twice in an exposure,378–382 and another report indicating no estrous cycle, 14 of 17 embryos recovered resulted from the first insemination (82%), whereas when increase in uterine contractions until mechanical mares were inseminated three times in an estrous stimulation of the vagina and cervix occurs.383 cycle, only 1 of 6 embryos recovered resulted from It seems logical that seminal plasma contributes the first insemination (17%). Therefore, the first to uterine transport of spermatozoa after natural insemination was not uniformly the most fertile. cover, because it provides a medium to aid peristal- This study was not designed to evaluate the effect of tic movement of spermatozoa toward the oviducts. seminal plasma on pregnancy rates, and separation Mann et al.384 showed the presence of two chemical of insemination times by 48 h may have resulted in markers of seminal plasma in the uterine horns at a uterine environment that was not as hostile as 50 min after mating, with concentrations similar to that reported by Troedsson et al.389 Seminal those detected in fresh ejaculates. These data are plasma concentration of inseminates was not re- consistent with the concept that most of the post- ported in this study but is presumed to be Ͼ5%. coital fluid in the uterus is derived from the seminal In a study reported by Metcalf,391 mares were in- plasma. Using the same chemical markers, these sci- seminated with frozen-thawed semen from two dif- entists also showed that seminal plasma may also gain ferent stallions during an estrous cycle at a 4- to access into the oviducts. The physiologic significance 10-h interval, followed by parentage determination of seminal plasma in spermatozoal transport extends of the resulting foals. Of nine foals born, four were beyond that of a simple vehicle. Seminal plasma con- the result of the first insemination (44%) and five tains a rich assortment of both organic and inorganic 136 were the result of the second insemination (56%). constituents. Prostaglandins have been identified Although the second inseminate likely contained in relatively high concentration in the seminal plasma some seminal plasma, the amount would have been of humans, monkeys, and the great apes, whereas the relatively low if the semen was processed for freez- concentration in seminal plasma of stallions is minis- ing in a standardized manner. The seminal plasma 136 cule. Of interest, the seminal plasma of boars con- concentration of the frozen semen was not provided tains an appreciable concentration of estrogens (up to in the report. Based on the combined data from 12 ␮g per ejaculate), and this steroid is thought to these three reports, it would appear that seminal stimulate myometrial contractions by inducing the re- plasma does provide some protective effect for sper- ␣ 376,385 lease of PGF2 from the endometrium. Spiking matozoa entering an inflamed uterine environment, boar semen with estradiol-17␤, however, did not im- but the seminal plasma concentration in the insem- prove pregnancy rate or fetal number after artificial inate can be quite low and still achieve this effect. insemination of gilts.386 We are unaware of reports Regardless of the method(s) used to promote uter- relating to estrogens in seminal plasma of stallions. ine transport of equine spermatozoa, in the end, Seminal plasma is a well-known modulator of only a small fraction of inseminated sperm reach spermatozoon-induced uterine inflammation, a fea- the oviductal luminae. In one report, only 0.0006– ture that likely plays an integral role in removal of 0.0007% of inseminated sperm was recovered from spermatozoa from the uterus.387,388 Troedsson et oviductal flushings of mares at 18 h after intrauter- al.388 reported that some proteins in equine seminal ine insemination.366 plasma can protect viable, but not damaged, sper- Barring oviductal occlusion, deposition of spermatozoa from binding to neutrophils. Although matozoa in the uterine body of mares leads to this finding may not impact spermatozoal transport similar spermatozoal numbers entering either ovi- at the first breeding of an estrous cycle, it could duct.366,368 This finding suggests that chemotactic affect spermatozoal transport mechanisms if a sec- effects are not present to attract uterine spermato-

AAEP PROCEEDINGS ր Vol. 53 ր 2007 135 MILNE LECTURE zoa to the oviduct ipsilateral to a periovulatory fol- can persist in the mare for up to 6 days before licle, nor is a signal elicited to induce differential ovulation, yet result in establishment of pregnan- control of the oviductal papillae. The process of cy.404,405 The mechanism of spermatozoal attach- spermatozoal passage into the oviduct, however, ment to the oviductal epithelial cells has received does not seem to be a passive one, i.e., controlled considerable study and seems to consist of a specific only through the reproductive tract of the female. sperm–ligand interaction involving glycoconjugates Studies to date suggest that a dynamic interaction (i.e., lectin-like molecules on the surface of spermato- occurs among spermatozoa, reproductive fluids, and zoa interacting with carbohydrate-containing moieties the epithelial surface of the utero-tubal junction on the surface of oviductal epithelial cells).406–412 (UTJ) and caudal isthmus of the oviduct.392 Based Recently, galactose-binding proteins were character- on studies with rats, it seems that only motile sper- ized on equine spermatozoa that may prove to be in- matozoa can negotiate passage through the UTJ.393 volved in the sperm–oviductal binding mechanism.413 Spermatozoa tend to associate closely with the epi- This intimate oviductal cell contact with sperma- thelium while traversing through the oviductal pa- tozoa in the oviductal reservoir seems to play two pilla of the mare,392,394 a phenomenon also observed divergent roles: assisting with the final matura- with other species.395 Cross-talk between sperma- tional events of spermatozoa that must occur before tozoa and the epithelium is further manifested by fertilization of an oocyte,414–416 and also maintain- studies with transgenic mice lacking a gene for two ing spermatozoa in a viable quiescent state to allow different spermatozoal surface proteins. In these for an extended storage period.417–420 Binding of males, all functional characteristics of the sperma- equine spermatozoa to oviductal epithelial cells un- tozoa appear normal, except that spermatozoal mi- der in-vitro culture conditions results in both a gration into the oviducts is hampered.396,397 quantitative and a qualitative change in protein A preferential selection process occurs for the few synthesis and secretion by the epithelial cells.421 spermatozoa that successfully pass through the UTJ Purified oviductal glycoprotein and polypeptides into the protective confines of the caudal isthmus. secreted by oviductal epithelial cells have been Scott et al.394 reported that Ͼ90% of spermatozoa shown to have positive effect on spermatozoal capac- visualized by scanning electron microscopy at the itation, including sperm–oocyte interactions.422,423 UTJ were morphologically normal, even when in- Peripheral proteins of the oviductal membrane seem seminates contained a high percentage of spermato- to contribute to maintenance of boar spermatozoal zoa with morphologic abnormalities. The most viability.424 Stallion spermatozoa that are bound common morphologic abnormality noted in the to oviductal epithelial cells in culture exhibit flagel- bound spermatozoa was a proximal cytoplasmic lar motion for up to 4 days, with gradual release of droplet. All of the spermatozoa were noted to have spermatozoa during that time frame.425 The ovi- normal head morphology. A high incidence of nor- ductal support system and gradual spermatozoal- mal morphologic features in oviductal spermatozoa release pattern are consistent with the concept of has also been reported in cattle,398,399 even when the ensuring that appropriately prepared spermatozoa inseminate contained a high proportion of sperma- are available to “greet” an ovulated oocyte soon after tozoa with abnormal heads.399 A recent report con- its arrival in the oviduct. veyed that cytoplasmic droplets in boar spermatozoa Spermatozoa attached to the isthmic epithelium may impede binding to the oviductal epithelium un- will subsequently detach and traverse the oviduct to der in vitro conditions.400 Thomas et al.401 showed the vicinity of an oocyte. The mechanism for de- that co-culture of equine spermatozoa with oviductal tachment is speculative but likely involves acquisi- epithelial cell monolayers yielded higher percent- tion of hyperactivated spermatozoal motility to ages of bound morphologically normal and motile break the connection with the oviductal epithelial spermatozoa than were present in the neat semen. cells.426 The lectin-like molecules on the spermato- When using oviductal explants to assess sperm– zoal surface responsible for specific binding with the oviductal interactions, this team of investigators oviductal cells may also be released during the ca- also found that more equine spermatozoa were pacitation process, thereby assisting with spermato- bound to isthmic explants than to ampullar im- zoal detachment.427 Detachment of spermatozoa plants and that more spermatozoa bound to explants probably yields cells that are both hypermotile and procured during the periovulatory period compared primed for spermatozoon-oocyte interaction. with luteal phase of the estrous cycle.402 The caudal isthmus is generally considered to be Spermatozoal Capacitation the reservoir site for oviductal spermatozoa,367 al- In mammals, freshly ejaculated spermatozoa are not though the UTJ also appears to harbor a consider- immediately capable of fertilizing an oocyte. Early able number of spermatozoa.403 The effect of the studies showed that spermatozoa require residence harsh post-inseminate conditions within the uterus time in the female reproductive tract to gain this on these spermatozoa requires further study. The capability,428,429 later termed capacitation.430 existence of a spermatozoal reservoir in the mare is Subsequent studies have revealed that similar further supported by the documentation in the lit- changes are required by spermatozoa subjected to erature that spermatozoa of some fertile stallions fertilization by in vitro methods (i.e., conventional in

136 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

Fig. 35. Examples of traditional and alternative biomarkers for assessing spermatozoal capacitation.

vitro fertilization [IVF]), as reviewed by Yanagima- etrating and fertilizing eggs443; thus, hyperactivated chi.431 As such, capacitation is considered to be an motility and the acrosome reaction would both be absolute requirement of mammalian spermatozoa considered components of capacitation, based on its destined to fertilize an oocyte without mechanical original meaning. Even though the acrosome reac- assistance (i.e., by intracytoplasmic sperm injection tion116,444 and hyperactivated motility441,445–448 are [ICSI]). The process involves a series of prepara- important for fertilization, the kinetic and temporal tive biochemical and biophysical changes in the relationships of these two features are not well un- spermatozoon that prime it to respond to signals derstood.449,450 The signaling pathways for hyper- originating from the oocyte and its surrounding cu- activated motility and the preparative changes mulus. If capacitation is incomplete, spermatozoa required for the acrosome reaction seem to be diver- are unable to penetrate the cumulus matrix,432,433 gent.150,171,441,451,452 Interestingly, human sper- and have greatly reduced ability to penetrate the matozoa that undergo hyperactivated motility are zona pellucida, undergo a zona-induced acrosome predominantly cells with normal morphology.453 reaction, and fuse with the oolema.434,435 In recent years, a variety of molecular markers have Two early laboratory hallmarks for verifying com- been identified that allow more critical evaluation of pletion of capacitation were acquisition of a hyper- the molecular events that emerge during capacita- activated pattern of motility and induciblity of the tion (Fig. 35).454–477 Unfortunately, many of these acrosome reaction by various stimulants (Fig. 35). assays can be technically challenging or have not Although capacitation has been defined by some as become feasible for clinical use.478 the changes that render a spermatozoon capable of Neither the precise signaling mechanisms that gov- undergoing the acrosome reaction,436–439 spermato- ern the attainment of capacitation, nor the exact cel- zoa are also known to exhibit a hyperactivated state lular characteristics of a capacitated spermatozoon, of motility when exposed to capacitating condi- are fully elucidated despite considerable study in this tions.440–442 The term was originally defined as area over the past few decades. Nonetheless, some the physiological changes of the spermatozoa in the key signaling pathways and cellular events have been female genital tract before they are capable of pen- identified and include surface (membrane) alterations,

AAEP PROCEEDINGS ր Vol. 53 ր 2007 137 MILNE LECTURE

Fig. 36. A schematic model of some documented and hypothesized molecular events associated with spermatozoal capacitation. Please refer to the text for an explanation of these signaling pathways and for ﬁgure abbreviations.

cytoskeletal modifications, influx and efflux of cytosolic matozoal lifespan is shortened appreciably by the constituents, and a myriad of enzymatic processes.479 capacitation process. The reader is directed to several reviews on this Spermatozoa are bathed in epididymal and ejacu- topic.431,451,452,479–490 latory fluids that contain “decapacitation factors”491 Interestingly, mature spermatozoa (i.e., those in that become adsorbed to the surface of the plasma the caudae epididymis or in ejaculated semen) seem membrane.450,492–496 Although these factors and to be pre-programmed to undergo capacitation, and their regulatory roles have not been fully elucidated, the process can be induced by a variety of signals, as they aid in suppressing premature initiation of the opposed to a specific molecular event. This is ex- capacitation process during spermatozoal passage to emplified by the fact that in vitro culture of sperma- the isthmic portion of the oviduct.479 Desorption of tozoa in many different media types can evoke these extracellular factors may unmask some key capacitation. It seems that nature has provided signaling mechanisms for capacitation.497,498 A spermatozoa with a broad array of capacitation in- cysteine-rich protein family, termed CRISP, associ- duction alternatives to ensure that they may retain ate with spermatozoa in the epididymis as well as fertilizing potential even if some evokers are ren- through the seminal plasma.104,499–504 These pro- dered nonfunctional. Figure 36 provides the reader teins have been shown to inhibit tyrosine phosphor- with some of the proposed signal transduction path- ylation and capacitation of spermatozoa,505 possibly ways for spermatozoal capacitation. Although the through their ability to block ion channels within literature is replete with potential activation mech- the plasma membrane.104 anisms, in our view, those presented in this figure Alterations in membrane-associated cholesterol, represent an accurate reflection of the most widely and cytosolic concentrations of bicarbonate and cal- Ϫ 2ϩ accepted signaling pathways. Species differences cium ions ([HCO3 ]i and [Ca ]i, respectively) seem to are known to exist in these regulatory pathways, be central evokers of capacitation in spermatozoa stud- even among eutherian mammals. Few in-depth ied under in vitro conditions,141,183,454,458,470,488,506–523 studies have been conducted with stallion sperma- and the same likely applies in vivo. Efflux of choles- tozoa, so much of our predictions must be extrapo- terol from the plasma membrane requires the pres- lated from studies performed with other species. ence of sterol acceptors in the milieu, such as albumin Conclusions drawn from work in non-equids may be or lipoproteins.483,497,508,514,523 Under laboratory misleading and thereby require verification through conditions, bovine serum albumin483,514,523 or experiments involving stallion spermatozoa. It is ␤-methyl cyclodextrin524–526 are generally used to also important to point out that ejaculates contain a achieve this effect. The net result of cholesterol efflux heterogenous population of spermatozoa, and the seems to be destabilization and increased fluidity of cells do not undergo capacitation in a highly syn- the plasma membrane, combined with externalization chronous fashion. An advantage gained by such an of key surface receptors.478,478,508,515,527,528 The cho- arrangement is the continuous replenishment of ca- lesterol efflux is hypothesized to result in an increased Ϫ 2ϩ 520 pacitating spermatozoa available in the oviduct to permeability of the membrane to HCO3 and Ca . await exposure to an ovulated oocyte, because sper- Both of these ions are thought to enter the cytosol from

138 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE the extracellular space through ion-specific chan- Indeed, tyrosine phosphorylation of spermatozoal nels.529,530 A variety of channels, transporters, and proteins seems to be crucial to acquisition of a ca- stores exist in spermatozoa that aid in regulation of pacitated state.489 Tyrosine phosphorylation of 2ϩ 480,529 2ϩ [Ca ]i. Voltage-dependent Ca channels head proteins occurs during capacitation, but this (VDCCs) exist in the plasma membrane of spermato- activity is even more pronounced in the flagel- zoa, and membrane depolarization mediates Ca2ϩ en- lum,477,553 thereby suggesting an importance of try through these channels.529,531 Additionally, an these proteins in hyperactivation.554 Two mem- increase in cytosolic pH ([pH]i) markedly increases the bers of a tyrosine-containing family of proteins, 532 Ϫ activity of VDCC. Internalization of HCO3 has termed AKAPs, are localized in the fibrous sheath been shown to further enhance Ca2ϩ entry through a and seem to tether PKA and various other signaling PKA-dependent mechanism.533,534 Interestingly, enzymes of the flagellum. These AKAP members 2ϩ 535,536 ϩ Ϫ Ca -Pi symporters and Na -HCO3 co-trans- are phosphorylated during capacitation and are porters140 are also known to exist in mammalian sper- thought to provide regional control of signal trans- matozoal membranes and are thought to be involved duction.187,523,554,555 Similarly, a calcium-binding in the capacitation process. Plasma membrane Ca2ϩ- protein, CABYR, has been localized to the fibrous ATPase (PMCA), an enzyme pump that extrudes sheath and is known to be tyrosine phosphorylated Ca2ϩ, is activated by the presence of decapacitation during capacitation.556 Phospholipid hydroperox- factors. In their absence, PMCA activity is de- ide glutathione peroxidase (PHGPx) has been iden- 2ϩ 537–539 creased, resulting in a net increase in [Ca ]i. tified in the mitochondrial capsule of the A family of transmembrane Ca2ϩ-selective ion chan- spermatozoal midpiece, and tyrosine phosphoryla- nels, called CatSpers, has been identified in recent tion of this enzyme during capacitation may play a years. These channels are thought to play an impor- role in hyperactivated motility.452 The phosphoty- tant role in both activated (non-capacitated) and hy- rosine protein, valosin-containing protein, is known peractivated (capacitated) motility.540,541 CatSper2 to redistribute from the neck region to the anterior channels have been localized to the flagellum,542 and head region and may be involved with the acrosome CatSper1 channels have been detected in the principal reaction.552 Other phosphotyrosine proteins may piece of the flagellum.543 be involved with recognition of the zona pellucida Receptors for adenosine, calcitonin, and fertiliza- and sperm–zona binding.557 Another spermatozo- tion promoting peptide (seminal plasma constitu- on-specific enzyme, scramblase, may be activated ents) have been identified in the plasma membrane through the PKA-tyrosine phosphorylation path- of spermatozoa and seem to play a modulating role way, leading to a collapse of the asymmetric bilayer in capacitation through regulation of membrane-as- distribution of phospholipids, lipid packing disor- sociated adenylyl cyclase (mAC). These molecules ders, and phospholipase activation.481,513,557–559 Ϫ activate mAC in uncapacitated spermatozoa but Some workers consider the HCO3 -cAMP-PKA- down-regulate mAC in capacitated cells, possibly to tyrosine phosphorylation (scramblase) pathway to prevent “overcapacitation” of cells.544 Another precede an efflux of cholesterol from the plasma method of preventing overcapacitation of spermato- membrane.472,481,513,557–560 Flippases, floppases, zoa is the polymerization of actin.545 These mole- and an amnophospholipid transporter localized to the cules have been identified in many areas of the acrosomal region of mouse spermatozoa are thought to spermatozoon, including the peri-acrosomal space, be important in maintaining the lipid asymmetry of the equatorial and post-acrosomal regions, and the the lipid bilayers before capacitation by establishing flagellum.546–548 Actin polymerization is a phos- higher concentrations of phosphatidylserine and phospholipase D (PLD)-dependent event, and this en- phatidylethanolamine in the inner leaflet and higher zyme is regulated by both PKA and protein kinase C concentrations of sphingomyelin and phosphatidylcho- (PKC).549,550 The formation of a filamentous form line in the outer leaflet.561 of actin in the peri-acrosomal space may prevent Another probable pathway to tyrosine phosphory- premature fusion of outer acrosomal and overlying lation involves formation of ROS that trigger down- plasma membranes when the properties of these two stream events that lead to capacitation. The effects ؊ membranes become more fusogenic during the ca- of superoxide anion (O2 ) and H2O2 on spermatozoal pacitation process. The actin must de-polymerize capacitation were first described 15 yr ago.562,563 for the acrosome reaction to occur.545 More recently, NOϪ has been shown to regulate Ϫ 2ϩ 564,565 An increase in [HCO3 ]i and [Ca ]i activate a capacitation. These findings have stimulated soluble cytosolic form of adenylyl cyclase (sAC), intensive studies regarding the physiologic roles of leading to elevated intracellular concentration of ROS in this process.266,268,272,485,566–576 Current cAMP.167,463,551 This nucleotide subsequently acti- evidence supports the concept that ROS induce the vates cAMP-dependent PKA activity, eventually cAMP-mediated tyrosine-phosphorylation cas- leading to an up-regulation in protein tyrosine phos- cade,577 but additional routes of action are also quite phorylation.520 A large number of proteins are likely.485,578 known to be typrosine phosphorylated during capac- As indicated above, the plasma membrane under- itation, but the roles of many remain unclear.552 goes considerable remodeling during capacitation,

AAEP PROCEEDINGS ր Vol. 53 ր 2007 139 MILNE LECTURE

Fig. 37. A schematic model of some documented and hypothesized molecular events associated with the spermatozoal acrosome reaction. Please refer to the text for an explanation of these signaling pathways and for ﬁgure abbreviations.

with an appreciable efflux of sterols and transverse tion, followed by spermatozoon–oocyte recognition, asymmetry of phospholipids. These events in- spermatozoon–zona binding, and elicitation of sig- crease the fluidity of the membrane, thereby allow- naling pathways.431,599–601 An excellent review of ing horizontal redistribution of membrane the acrosome reaction was recently provided by molecules. Recent studies have revealed that lipid Bailey,554 and the reader is referred to this source rafts participate in this redistribution process.579 for an in-depth account of the biochemical mecha- These cholesterol-rich microdomains are laden with nisms involved in the process. Other resources are signaling molecules that are known to be involved also available for those that desire a thorough grasp with capacitation and zona binding.580,581 The ca- of the subject.116,444,451,481,484,602 Capacitation pacitation process can increase the number of mem- primes a spermatozoon for the acrosome reaction brane rafts, as well as their affinity for zona through alterations in the membrane lipids; mem- binding.582 Actin polymerization may play a role in brane hyperpolarization; relocation, aggregation, association/dissociation of the membrane rafts.583 and externalization of membrane-signaling mole- This has become an intense area of study, as these cules; and changes in cytosolic and cytoskeletal com- signal-carrying lipid rafts appear to play an increas- ponents. A trigger, however, is required for ingly important role in capacitation and spermato- induction of the acrosome reaction, and most exper- zoon–oocyte interaction.584,585 imental evidence suggests that the zona pellucida The list of other potential endogenous signaling provides the inciting factor.602–604 Spermatozoal mechanisms is extensive, including involvement of interaction with the zona seems to involve a precise mitogen-activated protein kinase (MAPK),566 extra- species-specific receptor–ligand interaction, al- cellular signal-regulated kinases (ERKs),586 calmod- though the exact identity of the spermatozoal recep- ulin-dependent kinases,182,587 protein tyrosine K tor remains elusive. Evidence abounds, however, (PTK),485,588 PKC,549 Naϩ-Ca2ϩ exchangers,589–591 that designated glycoproteins of the zona pellucida, ϩ ϩ 482 592 Na /K ase, KATP channels, and the renin-an- termed ZP3, are responsible for initiating the giotensin system.593–596 This list is not meant to be signaling cascade(s) that lead(s) to the acrosome all inclusive, but to show that capacitation involves reaction.1,444,601,603,605–611 The exact signal trans- an extremely complex series of molecular events. duction pathway(s) for acquisition of the acrosome Future studies will more clearly define the precise reaction is (are) not fully understood, but decades of molecules and mechanisms that control capacita- intensive study have yielded much enlightening in- tion, including spatial, temporal, and kinetic formation on the subject. Figure 37 conceptualizes patterns. possible pathways involved. As with capacitation, it is quite possible that more than one reaction cas- Acrosome Reaction cade can elicit the acrosome reaction. Regulated acrosomal exocytosis is a fundamental The zona pellucida (ZP) is an extracellular glyco- element of fertilization and might be a more appro- protein matrix surrounding the oocyte. In most priate defining term than “acrosome reac- mammalian species studied, excluding hu- tion.”116,597,598 It must be preceded by a complex mans,612,613 the matrix is composed of only three series of events, including spermatozoal capacita- glycoproteins, typically termed ZP1, ZP2, and

140 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE ZP3.609,611,614 The peptide component of these gly- a role in both the acrosome reaction and in hyper- coproteins seems to be well conserved across species, activated spermatozoal motility.640 but post-translational modifications (i.e., glyco- Receptor-activated Gi proteins seem to play a cen- sylation) lead to species heterogeneity and hence tral role in the acrosome reaction, as shown by their species-specific binding affinity.615–619 The inter- activation by ZP3–GalT-I binding641 and their inac- actions seems to involve recognition of specific car- tivation by pertussis toxin (which inactivates Gi pro- bohydrate moieties present on the polypeptide teins).642,643 Activation of Gi proteins leads to a ϩ backbone of ZP3 by complementary zona-binding rise in [Ca2 ] .642 Spermatozoon–ZP3 adhesion di- i ϩ proteins of the plasma membrane of the spermato- rectly evokes Ca2 influx through low-voltage-acti- zoon.601,615,620,621 In the mouse, ␤1,4-galactosyl- vated calcium channel (T-type channel) activation in transferase I (GalT I) is the putative receptor on the membranes. Hyperpolarization of the membrane spermatozoal surface for a glycoside ligand of ZP3, potential, as occurs during capacitation, is thought the binding of which leads to induction of the acro- to release the channels from inactivation in order some reaction.621–627 A complex of spermatozoal that they may respond effectively to a stimulus de- 462,480,592,644 surface molecules is likely involved in spermatozo- rived from the zona pellucida, i.e., mem- 628 brane potential changes are translated into on–zona interaction. Whereas ZP3 can be con- 2ϩ 531 sidered the most likely signaling molecule of the ZP intracellular Ca signals. A ZP-mediated infor induction of the acrosome reaction, various ex- crease in membrane adenylyl cyclase (mAC) has also been reported to occur through Gi protein activa- periments also suggest that ZP2 may be involved in 645 the anchoring of acrosome-reacted spermatozoa to tion, presumably resulting in the increase in 605,620 cAMP that is known to occur after spermatozoal the zona. The ZP2 and ZP3 exist as het- 645,646 erodimers in filamentous arrangement cross-linked exposure to the ZP. Evidence for a cAMP/ PKA-mediated acrosome reaction exists after Ca2ϩ by ZP1, so it seems a fitting motif for integrated 647,648 roles of ZP2 and ZP3 in spermatozoon–oocyte inter- entry into the cell, but little is known about 444,606,629–631 the role of this pathway in the acrosome reaction. action. As evidence that the intrica- 634,649,650 cies of spermatozoon–zona interactions are not yet Breitbart and coworkers suggested that PKA may activate a voltage-dependent channel in resolved, a recent report revealed that, in bovine the outer acrosomal membrane that leads to release spermatozoa, glycosylation of ZP3 may not be an ϩ of Ca2 stores from the acrosome. Zona-induced absolute requirement for induction of the acrosome acrosomal exocytosis can be blocked by an inhibitor reaction.632 of PKA.651 Extensive literature indicates that elevated intra- ϩ The phospholipase (PLC) family seems to be cen- cellular Ca2 is a steadfast requirement of the acro- tral to the events leading to the acrosome reaction, some reaction. Spermatozoa use a range of and these enzymes have been localized to the sper- intracellular messengers for various functions. 652 2ϩ matozoal head region. Activation of PLC re- Cytosolic Ca is certainly a key regulator in these quires Ca2ϩ,604,653,654 but at relatively low processes and is subject to complex and sophisti- 451 2ϩ concentrations, as might be produced by the path- cated spatio-temporal control so that Ca -sensitive ways in the preceding paragraph. Zona-mediated responses can be activated discretely within various co-activation of membrane-bound PLC␤1 (through a compartments of the cell.529,633 A capacitated sper- 2ϩ 484 Gi-protein–coupled receptor) and membrane-bound matozoon has an increased [Ca ]i, created pri- PLC␥ (through a tyrosine kinase [TK] receptor) has marily by influx of transients through voltage- 634,652,655 2ϩ been proposed. Activation of PLC is operated Ca channels in the plasma membrane. thought to promote the acrosome reaction through a Exposure of spermatozoa to solubilized ZP has been 2ϩ couple of routes. First, PLC activates hydrolysis of shown to result in a further increase in [Ca ]i tran- phosphatidyl inositol 4,5-biphosphate (PIP ) within 2ϩ 2 sients. Although intracellular Ca stores are the membrane, resulting in production of two active thought to be minimal, the acrosome may serve as intracellular messengers: diacylglycerol (DAG) and an intracellular Ca2ϩ depot that is subsequently inositol 1,4,5-triphosphate (IP3). DAG mediates mobilized during the acrosomal reaction cas- the activation of PKC, and IP releases Ca2ϩ from 484,634 3 cade. The efflux of such intracellular stores intracellular stores, i.e., the acrosome.451,655 Acti- may then activate store-operated channels (SOCs) vation of PLC may also lead to disassembly of F- in the plasma membrane,529 probably transient re- actin filaments that are formed between the outer ceptor potential (TRP) channels,635 thus creating acrosomal membrane and overlying plasma mem- ϩ the sustained rise in intracellular Ca2 that is re- brane during capacitation, presumably to separate quired to drive the acrosome reaction.636–638 The these increasingly fusogenic membranes.484,634,656 mechanism of TRP channel activation is unresolved, PIP is purported to inhibit actin-severing proteins. ϩ 2 but both depletion of Ca2 from internal stores and Another member of the PLC family, PLC␦4, plays a direct receptor activation have been proposed meth- central role in the acrosome reaction. This enzyme ods of activation.636,639 The TRP channels have seems to be important for mobilizing Ca2ϩ stores in been expressed in both the head and flagellar re- the acrosome and also activating SOC in the plasma gions of spermatozoa, suggesting that they may play membrane, thereby creating a sustained increase in

AAEP PROCEEDINGS ր Vol. 53 ր 2007 141 MILNE LECTURE

2ϩ 484,634,655–659 cytosolic Ca concentration. The IP3 has revealed that a similar mechanism of action receptor and PLC␦4 have been localized to the acro- occurs between the outer acrosomal and plasma some.655,657 and both probably play a role in TRP- membranes,672,690–692 culminating in release of ac- channel regulation of Ca2ϩ entry into the rosomal contents and exposure of previously hidden cell.635,660–664 domains on the inner acrosomal membrane. Stud- The role of PKC, as a signaling pathway in the ies to date indicate that the machinery required for acrosome reaction, remains somewhat unclear. The this exocytotic event (which requires the finely reg- PKC activity of spermatozoa is dramatically lower ulated merger and fusion of two membranes) in- than that detected in some other tissues,665–667 but cludes several core components: (1) soluble nonetheless, has been identified in various regions of N-ethylmalameide-sensitive factor attachment pro- the spermatozoa.666,668 Certainly, DAG, produced tein receptor (SNARE) proteins; (2) N-ethylmalam- through the activity of PLC, is known to be a potent eide-sensitive factor (NSF); (3) soluble NSF activator of PKC,665,669 possibly leading to opening of a attachment protein (␣-SNAP); (4) Rab-GTPase; (5) Ca2ϩ channel in the plasma membrane.634,649,650 synaptotagmin; and (6) calcium ions. This basic DAG is also a membrane destabilizing molecule that machinery is considered obligate for acrosomal exo- 690,693 increases membrane fusibility.451,670,671 Experimenta- cytosis. Of interest, the SNARE proteins, tion has revealed that phosphorylation of spermato- NSF, and synaptotagmin have recently been identi- zoal proteins occurs on spermatozoal stimulation by fied immunocytochemically in stallion spermatozoa, progesterone, a known inducer of the acrosome reac- with predominant staining in the regions of the ac- 694 tion, and the activity can be mimicked with various rosomal cap and equatorial segment. activators of PKC and inhibited by various inhibitors The SNARE proteins are functionally classified as of PKC.672 Synaptogagmins are a family of calcium- v-SNAREs and t-SNAREs, and membrane fusion is binding transmembrane proteins purported to be cal- executed when v-SNAREs on the acrosomal (i.e., cium sensors responsible for triggering exocytosis in vesicle) membrane pair the complementary various cells.673,674 Synaptotagmin VI has been lo- t-SNARES on the plasma (i.e., target) membrane to calized to the outer acrosomal membrane of human form trans-SNARE complexes or SNAREpins. Both spermatozoa.675 Recently, PKC-mediated phosphor- v- and t-SNARE proteins are thought to be secured to their respective membranes by hydrophobic anchors ylation of spermatozoal synaptogagmin VI has been 689,695,696 672 that span both leaflets of the lipid bilayers. shown. These workers proposed a model by which ␣ PKC has a regulatory role in synaptotagmin function NSF and its co-factor, -SNAP, are cytosolic proteins that act cooperatively to regulate SNARE through phosphorylation of synaptotagmin C2 do- ␣ mains672,673; hence, PKC may play a vital role in con- protein function. The -SNAPs bind to the trolling the molecular machinery that mediates SNARE protein so as to correctly position NSF, and membrane fusion (discussed below). are responsible for stimulation of the ATPase activity of NSF that dissociates trans-SNARE com- Phospholipase A (PLA ) is recognized as an im- 2 2 plexes.697–701 This is a mechanism for recycling of portant enzyme for the acrosome reaction. Cer- components of the fusion machinery in other cell types, tainly, indirect evidence suggests that treatment of but would not seem necessary in the acrosome, where spermatozoa with PLA inhibitors will impede this 2 the reaction is a one-time event. Recent work with event.676–679 Both Ca2ϩ and DAG (in the presence ϩ spermatozoa indicate that they also serve essential 2 654,680–683 691,692 of Ca ) are capable of activating PLA2. pre-fusion roles, probably by inducing conforma- The resulting enzymatic action is deacylation of tional changes in the SNARE proteins complexes, i.e., fatty acids from phospholipids, producing fatty acids catalyzing disassembly of an inactive cis SNARE com- (such as arachidonic acid and lyophospholipids). plexes, to allow re-association as active trans complex- A key target is phosphatidylcholine, yielding lyso- es.691 The NSF has also been reported to act in a phosphatidylcholine. This “metabolite” is a likely pre-fusion step in non-spermatozoal cells by catalyzing contributor to the acrosome reaction, because it is SNARE protein-induced membrane rearrangement known to trigger the reaction in capacitated sperma- that is more suitable for Ca2ϩ-mediated fusion.702 tozoa.684–687 Exposure of capacitated spermatozoa Rabs are a family of proteins that are included in to solubilized zona results in increased metabolism the Ras superfamily of G proteins. They are an- of phosphatidylcholine to arachidonic acid and lyso- chored to the acrosomal membrane, a process aug- phosphatidylcholine, resulting in a high proportion mented by cholesterol efflux,703 and, on activation of acrosome-reacted cells. The PLA activation is (through guanosine triphosphate [GTP binding]), regulated by a signal transduction pathway involv- can interact with Rab effector molecules on the ing Gi protein and DAG.682 Lysophosphatidylcho- plasma membrane to form an initial physical link, line, or similar metabolites of PLA2 activity, seem to allowing the v-SNARE and t-SNARE proteins to be important to the final stages of cAMP-mediated interact to form a trans-SNARE complex.689,704,705 acrosomal exostosis.688 The assembly of the trans-SNARE complex is Regulated exocytosis, as it relates to membrane thought to generate sufficient energy to overcome fusion at neural synapses, has been studied exten- the repulsive forces generated by the polar head sively.689 Expanding these studies to spermatozoa groups of the two membranes.689 In essence, the

142 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE acrosome becomes tethered to the plasma mem- epithelium and subsequent passage through the brane. It is possible that that the filamentous F- mucinous environment of the oviduct) and to pene- actin interspersed between the outer acrosomal and trate the cumulus matrix and zona pellucida sur- plasma membranes could dampen the repulsive rounding the oocyte.149,152,441,445 It is of interest forces.451 Epac, a known guanine nucleotide ex- that hyperactivated spermatozoa tend to have nor- change factor (i.e., catalyzes replacement of GDP mal morphologic characteristics.453 When ob- with GTP on Rab proteins) is activated by cAMP, served in aqueous media, the motility pattern of a resulting in induction of the acrosome reaction706; hyperactivated spermatozoon is characterized by a hence, another signaling cascade enters the mix of high-amplitude, often asymmetric, whiplash flagel- possible regulatory mechanisms for the acrosome lar beat pattern, leading to a circular or non-pro- reaction. gressive trajectory.149,152,441 Studies have revealed Ca2ϩ-binding synaptotagmin, also a major Ca2ϩ that this flagellar wave form actually improves the sensor, is central to both the spatial and the kinetic progressivity of spermatozoa over that of activated elements of the fusion process. Synaptotagmin is motility when spermatozoa are exposed to viscoelas- known to have direct interaction with t-SNARE and tic conditions such as those that exist in the SNAP proteins, a feature augmented by Ca2ϩ, and oviduct.441,713 may actually assist in SNARE complex forma- Activated motility and hyperactivated motility tion.673,707 This protein is a large transmembrane may require different environmental signals. Pre- protein whose cytosolic component is primarily C2 sumably, a signal for hyperactivated motility is elic- domains, similar to that of pKC isoforms.673 In ited within the oviduct under natural conditions to other cell types, the protein is thought to facilitate initiate the event at a time that is conducive to docking of secretory vesicles close to Ca2ϩ channels fertilization.441 Although the precise signal(s) for where they might be exposed to high local concen- initiation of hyperactivation remain(s) unsolved, it trations of this ion.673,708,709 As mentioned above, is possible that chemotactic and/or thermotactic fac- studies with spermatozoa indicate that synaptotag- tors serve in this capacity.714 It is also possible min may also inhibit spontaneous fusion, through that spermatozoal exposure to alkalinizing condi- regulation by PKC. This control is likely to be tions, as exists in the oviduct and above that which 2ϩ driven by [Ca ]i. initiates activated motility, is the primary initiator Without doubt, Ca2ϩ is the driving force for the of hyperactivated motility.150,152,715,716 final stages of acrosomal preparedness for reaction, The signaling pathway is also dissimilar between from Rab3 activation through final pore forma- activated and hyperactivated forms of motility. tion.691 It seems that Ca2ϩ is maintained in locally Activated motility primarily uses the cAMP-sAC- low concentrations during SNARE complex formation, PKA pathway and requires a relatively low concen- possibly through synaptotagmin control. When trans tration of Ca2ϩ for phosphorylation of flagellar SNARE complexes are fully assembled for the final proteins (Fig. 34). Conversely, hyperactivated mo- fusion event, synaptotagmin likely triggers release of tility seems to require an elevated concentration of Ca2ϩ.673,691 Studies involving non-spermatozoal Ca2ϩ,148,164,717–719 and no cAMP149,150 or sAC.166 cells reveal that the docking mechanism pulls the pre- The precise mechanisms controlling hyperacti- fusion membranes to within a distance of 2–3 Å.691,710 vated motility are subject to continued study, but an Subsequent bridging of adjacent phosphate head increasing body of literature suggests that an in- groups by Ca2ϩ allows regional intermembranous dis- crease in extracellular pH leads to an increase in 2ϩ placement of water (i.e., by calcium hydration) so that [pH]i, thereby potentiating the action of Ca chan- the resulting anhydrous complex is amenable to lipid nels.147,467,716,718,720 The most likely mechanism mixing.691,710–712 The regions of fusion are restricted for an increase in cytosolic Ca2ϩ is through CatSper by the circular array of SNARE complexes.691,711 channels and through release from internal stores. Similar mechanisms probably exist for spermatozoa Four CatSperm channels (CatSper1–4) have been because the basic machinery is similar to that of so- localized to the principal piece, and they seem to matic secretory cells. have a physical interaction.716 One report suggests As with capacitation, the precise pathways in- that all four CatSper channels must be operational volved in the acrosome reaction are not fully eluci- for hyperactivation to occur716; however, others pro- dated despite considerable study in this area, and vide evidence that this may not be the case.721 species differences are likely. The potential for in- CatSper1 may serve as an internal pH sensor, as volvement of signaling pathways and endogenous evidenced by the composition of its N termi- molecules, other than those listed above, is exempli- nus.147,543 The redundant nuclear envelope (RNE) fied by the potential role of angiotensin II in the at the base of the flagellum has been identified as a acrosome reaction of stallion spermatozoa.593,594 possible internal store for Ca2ϩ. This structure represents the remnants of the nuclear membrane Hyperactivated Motility following spermiogenesis. The RNE is reported to 2ϩ A hyperactivated form of motility is required to free contain IP3 receptor-gated Ca channels that are the spermatozoa from the oviductal reservoir thought to release Ca2ϩ stores and regulate hyper- (through spermatozoal release from the oviductal activated motility independent of similar channels

AAEP PROCEEDINGS ր Vol. 53 ր 2007 143 MILNE LECTURE in the acrosome.719,722 Because the RNE is located migration of oviductal spermatozoa.729–741 Some at the base of the flagellum, where flagellar bending investigators assert that chemotactic responsive- is propagated, it seems a logical location for an in- ness is also a part of the capacitation process.729,730 ϩ ternal Ca2 store.138,721 A specific activator of PKC Spermatozoa possess specific chemotactic recep- has been shown to induce hyperactivated motility in tors.478 Olfactory receptors are considered to rep- human spermatozoa723; thus, a PKC signal trans- resent the largest family of genes in the human duction pathway may also be important to stimula- genome, with up to 1000 members,742,743 and odor- ϩ tion of hyperactivated motility. Although Ca2 is ant receptors have been identified, and genes encod- known to affect the bending pattern of the flagellum ing for olfactory receptors have been expressed, in and added Ca2ϩ is required for hyperactivated mo- 744–750 ϩ the testes of mammals. Olfactory receptor tility, the mechanism by which Ca2 controls this genes have even been identified in the primordial event remains unknown. germ cells of fetuses.751 Odorant receptors have The signaling pathways and cellular events lead- been localized to the flagellar base and proximal ing to hyperactivated motility and to the acrosome midpiece of spermatozoa,752,753 suggesting that reaction are different, and the events of each can odorant-like molecules emanating from the female 138,148 occur independently. Nonetheless, internal reproductive tract might induce hyperactivated alkalinization seems to be a common inciter for the spermatozoal motility, possibly by release of Ca2ϩ two events. The pathways, although not the same, from internal stores152,747 or another signal-trans- achieve the critical spermatozoal priming required duction pathway.754 Spermatozoa do not seem to for interaction with the oocyte. Because capacita- acquire a capacitated state, complete with chemo- tion was originally defined with this endpoint in tactic competence, in a synchronous manner; rather, mind, it seems appropriate to consider hyperacti- a small percentage of a spermatozoal population is vated motility as a component of the capacitation thought to achieve this potential at any given time. process. It has been quite difficult, however, to un- This is thought to serve as a safeguard to ensure derstand the interplay of the various components of that capacitated spermatozoa, which are short-lived, capacitation. Indeed, the various events of the ca- are available over a protracted period for prompt pacitation process are not tightly coupled. Pen- interaction with an ovulated oocyte.729,741 etration of zona-free hamster eggs by human Oriented migration of spermatozoa to follicular spermatozoa is maximal at 18 h under in vitro con- factors has been shown under in vitro condi- ditions, whereas changes in tyrosine phosphoryla- tions,714,741,755 and progesterone is thought to be an tion occur after 1–2 h, and hyperactivation is 714,739 maximal after3hofincubation.187,449,598,724,725 important mediator of this event. Follicular Furthermore, different groups have shown variabil- fluid is not thought to pass down the oviduct to the ity among men in spermatozoal capacitation caudal isthmic region, however, and migrating capac- 598,726,727 itated sperm can be detected in the more proximal time. An uncoupling of the acrosome re- 738 action and hyperactivated spermatozoal motility regions of the oviduct before ovulation. Studies has also been shown in hamsters and bulls.506,719,728 involving oocyte transfer in horses have also shown Indeed, we now know a great deal about the molec- that fertilization can occur when oocytes are trans- ular control of various facets of the capacitation ferred to the oviduct contralateral to an ovary contain- process, but many more discoveries are needed to ing a preovulatory follicle or when transferred to uncover the secrets of the spermatozoon. oviducts of non-cyclic mares supplemented with exogenous steroids.756 One working hypothesis is that Sperm–Oocyte Interaction spermatozoa are transported to the general site of Although spermatozoal capacitation is not a site- fertilization by thermotaxis and contractions of the specific phenomenon and can be induced in a variety oviduct and, when in the direct vicinity of the oocyte, of artificial media, the caudal segment of the oviduct are directed by chemoattractants secreted by the cu- 714,757,758 seems to be the principle location for spermatozoal mulus cells. Certainly, studies regarding capacitation and storage under in vivo conditions.436 spermatozoal chemotaxis remain scanty at this junc- Interactions with an ovulated oocyte, however, re- ture, but continued study may divulge a specific role of quire spermatozoal migration to the ampullar re- chemoattractants in spermatozoal migration patterns. gion of the oviduct, and only a small percentage of Before a spermatozoon can interact directly with spermatozoa that gain access into the oviduct will the oocyte, it must first negotiate passage through eventually arrive at this fertilization site.436 Such the two sizable extracellular matrices which sur- spermatozoa are thought to have achieved full fer- round the oocyte, i.e., the cumulus complex and the tilizing potential. The precise mechanisms by zona pellucida. An excellent description of the which spermatozoa migrate to the ampullar region three-dimensional structure of the zona pellucida of the oviduct remain speculative, but contractile was recently provided.759 Acquisition of hyper- movements of the oviduct and hyperactivated sper- activated motility and translocation/exposure of gly- matozoal motility are thought to play key roles in cosylphosphatidylinositol-anchored surface hyal- this migratory phase. Evidence is mounting that uronidase (termed PH-20) likely permit penetration chemotactic factors are also important to directional of the cumulus matrix.760–762 Binding of acrosome-

144 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

Fig. 38. Conceptualization of spermatozoon–oocyte interactions. (A) Spermatozoon negotiates passage through the intercellular matrix of the cumulus oophorus. (B) Initial adhesion of the equatorial region of the spermatozoon with the zona pellucida. (C) Induction of the acrosome reaction. (D) Spermatozoal penetration of the zona pellucida at an oblique angle, with entry into the perivitelline space. (E) fusion of the post-acrosomal spermatozoal membrane with the microvillar region of the oolemma. (F) Establishment of a gateway for spermatozoal nucleus entry into the cytoplasm of the oocyte, as well as entry of spermatozoon cytosolic molecules (presumably spermatozoon-speciﬁc phospholipase C) that serve as a signaling mechanism for activation of the oocyte and exocytosis of oocyte cortical granules. Modiﬁed from Primakoff et al. 2002 and Rubinstein et al. 2006.760,789

intact spermatozoa to the zona pellucida through lin-F, to promote spermatozoal-zona binding.780 species-specific carbohydrate moieties of the zona Not surprisingly, the zona pellucida of ovulated oo- pellucida and corresponding receptors on the sper- cytes is also known to contain products secreted by matozoal surface lead to induction of the acrosome the oviducts that may play a role in initial sperm reaction.605,609,610,763–766 An assortment of sper- adhesion.781,782 Further research may lead to iden- matozoal surface lectins and glycoenzymes have tification of a multitude of receptors that are in- been shown to have zona-binding ability.767–774 volved with spermatozoal–zona binding. Previous studies involving mouse spermatozoa sug- The cutting thrust issued by hyperactivated sper- gest that the binding of the spermatozoal surface matozoa,783 possibly combined with externalization/ receptor, GalT-I, to specific oligosaccharide moieties release of proteases and glycosidases within the of ZP3 on the zona pellucida is important to induc- acrosome,598,760,784,785 allow spermatozoal penetration of the acrosome reaction.608,775 More recent tion of the zona pellucida. Studies involving ham- work, however, indicates that other mechanisms ster spermatozoa indicate that the process of may be responsible for initial adhesion of spermato- spermatozoal adhesion to, and penetration of, the zoa to the zona pellucida.775–777 A newly voiced zona pellucida requires only 10–15 min.598,786,787 theory is that the zona pellucida acts as a sperma- Once situated within the perivitelline space, the tozoal scaffold, with ZP2 acting to orchestrate zona spermatozoon binds to, and fuses with, the egg permissibility to zona penetration by a spermato- plasma membrane (or oolemma), leading to inter- zoon, and initial zona penetration by the spermato- nalization of the sperm haploid chromosomal com- zoon activating the acrosome reaction through plement by the oocyte. Only spermatozoa that “mechanosensory” signals.778 One report involving have undergone an acrosome reaction are thought to horses suggests that spermatozoal surface galacto- be capable of fusion with the plasma membrane of syltransferase (GalT) and zona glycoprotein ZPC the oocyte,788 and this binding/fusion process in- (probably similar to ZP3 in the mouse) are not re- volves specific domains of the post-equitorial region quired for spermatozoal binding to the zona pellu- of the sperm head and the microvillar region of the cida.779 Acquisition of the acrosome reaction was oolemma (Fig. 38).760,789 The specific sperm pro- not an experimental endpoint in this study, so it teins that are involved with spermatozoal–oocyte remains possible that a GalT-ZPC–independent binding remains an enigma, although several candi- mechanism is responsible for initial adhesion, but date proteins have been identified.504,760,789,790 that a GalT-ZPC–dependent interaction may be re- A family of sperm-associated cysteine-rich secretory quired for the acrosome reaction. Passage through proteins (CRISPs) have been characterized in many the cumulus may also displace the surface-associ- mammalian species. These proteins are synthe- ated sperm glycoproteins, glycodelin-A and glycode- sized and secreted by the epididymis and associate

AAEP PROCEEDINGS ր Vol. 53 ր 2007 145 MILNE LECTURE with the spermatozoal membrane, most promi- tical granules (to prevent polyspermy); release of the nently in the distal corpus and cauda epididy- oocyte from meiotic arrest; pronuclear formation; mis.504,791 CRISPs have been localized to the mediation of genomic union; progression into mito- equatorial region of capacitated and acrosome-re- sis with reorganization of both nuclear and cytoskel- acted spermatozoa.792 Interestingly, stallion sper- etal components; and stimulation of oocyte matozoa possess an abundance of CRISP, with mitochondrial respiration.804–810 The factor re- largest amounts detected in ejaculated spermato- sponsible for this activation is derived from the sper- zoa.503 Three members of the CRISP family have matozoal cytosol, and a spermatozoon-specific PLC been identified in the testis, epididymis, ampullae, is considered to be the most likely candidate mole- or seminal vesicles of stallions.499,503 A portion of cule.811–813 In a manner similar to that of the ac- CRISP is tightly associated with the spermatozoa rosome reaction, PLC might activate hydrolysis of and is localized to the post-acrosomal and equitorial phosphatidyl inositol 4,5-biphosphate (PIP2) within regions of the sperm head, as well as the mid- the membrane, resulting in production of inositol 503 piece. The regionalized distribution of the pro- 1,4,5-triphosphate (IP3), which, in turn, could medi- tein hints to a potential role in spermatozoon–oocyte ate release of Ca2ϩ from intracellular (i.e., endoplas- interaction; however, complementary molecules mic reticulum) stores.814 have not been characterized on the oocyte mem- Internal energy generation is required for sperma- brane of any species studied.504 In addition, the tozoon–oocyte interactions. In the mouse, sperma- lack of hydrophobic domains in this molecule sug- tozoon–oocyte fusion is inhibited in the absence of gests that it may not be directly involved in mem- glucose.233 In this species, glucose seems to medi- brane fusion events.598,790 ate tyrosine phosphorylation of proteins in mid- The spermatozoal ADAM (ADisintegrin And Met- piece-specific sites.234 Sperm entry into the mouse alloprotease) family of proteins (including fertilin ␣ oocyte is characterized by pentose phosphate path- [ADAM1], fertilin ␤ [ADAM2], and cyritestin way activity and redox regulation, in addition to [ADAM3]) have been implicated in spermatozoon– glycolysis.251,252 Interestingly, oocytes are incapa- oocyte binding504,760 and are known to possess fuso- ble of metabolizing glucose; thus, pyruvate or cumu- genic potential.793 Nonetheless, gene disruption lus cells (which convert glucose or lactate to data do not corroborate an essential role of these pyruvate) must be present in IVF medium.598,815 proteins in fertilization.760,794 The protein, Izumo, has withstood the spatio-temporal, immunological, 6. Clinical Considerations: Semen Evaluation and gene-knockout tests required to consider it as a Given the lengthy and detailed nature of the above probable spermatozoal candidate in gamete fu- text, one may wonder about it’s relevance to the sion.789,795 Treatment of spermatozoa with anti- clinical arena. Assisted stallion reproduction bodies to integrins or osteopontin also reduce spans hundreds of years, with references to the topic spermatozoon–oocyte binding and fertilization in dating back to Arabic texts (circa 1300) and scien- vitro.796 Recently, proteomic analysis of the sper- tific reports issued from the late 1700s to the early matozoal membrane regions involved in spermato- 1900s by noted authorities such as Spallanzani, zoon–oocyte interactions has uncovered an Repiquet, Hoffman, and Ivanoff.816 Advancements abundance of proteins that could be participants, were furthered by the works of investigators such as including some previously uncharacterized Bielan´ ski, Day, Mann, Merkt, and Nishikawa, and proteins.797 the next generation of dedicated scientists, includ- A tetraspanin protein, CD9, has been implicated ing Amann, Foote, Hughes, Kenney, Palmer, Pick- as a very viable candidate oocyte pro- ett, and Tischner. These individuals took the tein,608,760,788,789,798 for spermatozoal adhesion and discipline of stallion reproduction to new heights. fusion, although additional proteins might also be As seen by the information provided in the previous involved.798 The microvilli of the oolemma seem to sections, we continue to garner a deeper under- be key to spermatozoon–oocyte fusion, and CD9 is standing of spermatozoal structure and function. enriched in the microvillar portion of the oolem- Although progress in equine reproduction is curbed ma.799 In addition, CD9 may be involved in coor- by funding limitations, our discipline has gained dination of microvillar shape and distribution along considerable insights from work conducted in hu- the oolemma.799 Membrane lipid molecules are mans, laboratory animals, and food-producing ani- also of fundamental importance to spermatozoon– mals. An appreciation of the molecular basis of oocyte fusion.800 In addition, polymerization of ac- spermatozoal function, spermatozoal–oviductal in- tin filaments may be critical to spermatozoal teractions, and spermatozoon–oocyte engagement incorporation into the oocyte cytoplasm, deconden- will undoubtedly lead to many practical applications sation of the nucleus, and activation of the oocyte in the clinical front, such as assemblage of a battery block to polyspermy.545,801–803 of in-depth laboratory tests to assess spermatozoal Entry of the sperm into the ooplasm elicits an function, expanded treatment options for subfertile initial increase in intracellular Ca2ϩ concentration, stallions, improved methods for preservation of se- followed by repetitive Ca2ϩ oscillations. This sig- men, and heightened applications for assisted repro- naling mechanism induces exocytosis of oocyte cor- ductive technologies such as conventional in vitro

146 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE fertilization and intracytoplasmic sperm injection tory parameters such as spermatozoal motility and techniques. The bottom line is that, the more we membrane integrity, but may induce post-fertiliza- learn, the more informed we can be in decision mak- tion embryonic failure.852 Because of the highly ing regarding diagnostic and therapeutic strategies condensed nature of the spermatozoal chromatin, as they relate to spermatozoal function and repro- mature spermatozoa are known to be transcription- ductive health in stallions. It becomes incumbent ally inactive,35 so it is logical that DNA damage on us, and future clinicians and academicians, to might not be expressed until mitosis occurs at the convert these opportunities into practical time of spermatozoon–oocyte fusion. This becomes applications. quite important clinically as it represents a poten- Conventional laboratory tests for assessment of tial noncompensible defect, i.e., affected spermato- spermatozoal quality have included light micro- zoa in an ejaculate may not be impaired for scopic evaluation of spermatozoal morphologic char- fertilization, so increasing the insemination number acteristics and estimation of spermatozoal motility will not increase pregnancy rate. Ejaculated sper- (including percentages of motile and progressively matozoa are known to retain a cohort of cytoplasmic motile sperm; velocity of spermatozoal movement; mRNA and translational ability,32,35 so it is also and longevity of spermatozoal motility following in possible that fragmentation of mRNA could have a vitro storage).17,817–820 Although other features of negative impact on some other spermatozoal func- semen quality are also considered, including sper- tions leading to reduced fertilization potential. matozoal concentration, semen volume, and pres- Assays other than the SCSA are available to mea- ence of blood, urine, or potentially pathogenic sure spermatozoal DNA fragmentation/chromatin bacteria, the basic features of this examination pro- disruption, including a TdT-mediated-dUTP nick cess date back several decades. The value of the end labeling (TUNEL) assay, an in situ nick trans- examination is predicated, to a large extent, on relation (NT) assay, a sperm chromatin dispersion liable equipment and personnel with good observa- (SCD) assay, and an electrophoresis-based Comet tional power. Even when these stipulations are in Assay.310,844,845,853–857 Although these assays effect, the predictive value of the examination can be have not been used to the same extent as the SCSA limited.114,115,125,821–833 The same holds true for in the equine arena, they are commonly applied in spermatozoa of other mammalian species.834–837 the human field. An immunofluorescence assay After viewing the previous sections of this paper, has also been developed for evaluation of human one can appreciate that expanded analysis of equine spermatozoal protamine levels,846 and a similar as- spermatozoal populations might improve the predic- say for equine spermatozoa could have diagnostic tive value of the testing process. In fact, additional value. tests shown to be of diagnostic value are presently Another assay that we now commonly use in our incorporated into the spermatozoal examination battery of tests is the acrosomal responsiveness as- process within some reference laboratories today. say (ARA). This assay is directed at testing the One of these tests is the sperm chromatin struc- functionality of the spermatozoal acrosome, i.e., its ture assay (SCSA). This assay, introduced by ability to acrosome react when challenged with a Evenson et al. in 1980,838 has been applied to sper- potent inducer of the event, the Ca2ϩ ionophore, matozoa from a number of species, including hors- A23187. Conventional tests are unable to predict es.839–841 The SCSA tests a compartment of the fertility of a subset of stallions with acrosomal spermatozoa that is not monitored by conventional dysfunction, because these stallions present with methods, i.e., nuclear chromatin. The SCSA is a normal spermatozoal morphology and motility, as flow cytometric procedure that uses the metachro- well as normal chromatin quality (based on SCSA matic fluorochrome, acridine orange, and tests the testing) and normal acrosomal structure (based on denaturability of spermatozoal chromatin chal- fluorescent light microscopy and transmission elec- lenged with acid treatment. The literature pro- tron microscopy). Meyers et al.858 first reported vides variable results regarding the relationship of that the acrosomes of subfertile stallions with poor stallion spermatozoal chromatin denaturation to the spermatozoal motility did not react readily in re- extent of disulfide bonding within and between pro- sponse to progesterone exposure. Fertile stallions tamine molecules;842,843 however, chromatin suscep- averaged a 17% acrosomal reaction rate after5hof tibility to denaturation is correlated with the level of incubation in capacitating conditions followed by ex- actual DNA strand breaks.841 The DNA strand posure to progesterone, whereas subfertile stallions breaks can be associated with a myriad of factors, averaged only a 6% response rate. This study in- including idiopathic apoptosis, oxidative stress, heat dicated that progesterone was capable of stimulat- stress, radiation injury, or protamine deficien- ing the acrosome reaction in equine spermatozoa cy,843–850 and may involve double-stranded or sin- exposed to capacitating conditions, and the response gle-stranded DNA fragmentation or oxidized in subfertile stallions was reduced. Others have nucleosides.844,851 Such lesions could create genet- reported that the plasma membrane of stallion sper- ically defective spermatozoa, leading to germ-line matozoa contains progesterone receptors and indi- mutations. Interestingly, spermatozoa affected by cate that this may be a pathway for induction of the such damage may appear normal, based on labora- acrosome reaction.859 In this regard, the percent-

AAEP PROCEEDINGS ր Vol. 53 ր 2007 147 MILNE LECTURE age of spermatozoa with exposed progesterone re- lying acrosome, (3) the fibrous and microtubular ceptors has been shown to be highly correlated with components of the flagellum, (4) a mitochondrial fertility of stallions.860 sheath, and (5) an enveloping plasma membrane. Other workers identified a subset of subfertile With this in mind, one can appreciate that a battery stallions whose only distinguishing spermatozoal of tests devised to evaluate these various compart- characteristic was the drastically reduced ability to ments might aid in improved localization of cellular acrosome react when exposed to A23187 for up to 3 h dysfunction and might also improve the predictive (i.e., mean reaction rate of 84% in fertile stallions value of a laboratory-based semen evaluation. As- versus mean reaction rate of 6% in subfertile stal- says developed for the nucleus and the acrosome lions),861 thereby suggesting the spermatozoal de- were discussed above. In addition, techniques spe- fect may be isolated to the acrosome. Measurement cific for mitochondrial function, flagellar substruc- of the cholesterol–phospholipid ratio in semen of ture, and plasma membrane integrity are available. these stallions revealed that the ratio was signifi- Although the importance of mitochondrial-based ox- cantly greater in both seminal plasma and whole idative metabolism for ATP production in spermato- sperm from the subfertile stallions compared with zoa has been challenged,237,877 a key role of the fertile stallions.862 Further studies are required to mitochondria in sustenance of spermatozoal func- dissect the underlying etiology of this condition and tions remains quite likely.192 Mitochondria-selec- to determine appropriate treatment strategies. tive fluorescent markers abound and include A keen understanding of the molecular pathways of rhodamine 123; an assortment of MitoTracker capacitation and the acrosome reaction, as discussed probes (which differ in spectral characteristics, above, provides us with logical investigative adaptability to permeabilization and fixation, and approaches. reliance on respiration state of the mitochondria); Although transmission electron microscopy re- and 5,5Ј,6,6Ј-tetrachloro-1,1Ј,3,3Ј-tetraethylben- mains a decisive means for detecting the acrosome zimidazolylcarbocyanine (JC-1). These reporter reaction in stallion spermatozoa,863 acrosomal sta- molecules passively diffuse across the plasma mem- tus can be assessed by bright-field microscopy using brane and accumulate in the mitochondria. Rho- Commassie blue stain.864,865 Fluorescence-tagged damine 123 was one of the original mitochondrial lectins or acidotrophic probes can be used with light probes, but has largely been replaced by other mark- microscopy or flow cytometric methods to assess the ers because of its reduced photostability and its ten- acrosome reaction or acrosomal damage.858,866–873 dency to lose retention in the mitochondria following Although the more popular fluoresceinated lectin a loss in membrane potential. Some of the Mito- markers have generally replaced chlortetracycline- Tracker probes do not fluoresce until oxidized, so based assays for detecting the acrosome reaction, these markers permit easy assessment of the respi- the latter assay remains useful because of its ability ration state of the mitochondria. The JC-1 may be to gage capacitation, as well as the acrosome reac- optimally suited for evaluation of the energetic state tion.863,874 Another marker shown to be useful for of mitochondria because it produces a membrane detection of capacitation in stallion spermatozoa is potential-sensitive shift in color pattern, i.e., the merocyanine 540, an impermeant lipophilic probe color changes from green to red-orange as mem- that permits evaluation of the architecture and dis- brane polarization increases. This is because of the order of lipids in the outer leaflet of the plasma reversible formation of red-orange J-aggregates in a membrane bilayer.455,481,513,560 Other probes, such state of high membrane potential compared with as fluorochrome-conjugated Annexin V or Ro-09- green monomeric JC-1 in a state of low to medium 0198, are being used increasingly to monitor mem- membrane potential.873,878–881 brane asymmetry. These two probes bind with The semi-permeable plasma membrane, which is phosphatidylserine and phosphatidylethanolamine, composed of a wide assortment of lipids, proteins, respectively. Surface exposure of these phospholip- and carbohydrates, envelops the entire spermato- ids is considered to represent spermatozoal capaci- zoon. This structure is vital to regulation of sper- tation because they generally reside in the matozoal functions by establishing ion gradients, cytoplasmic leaflet of the lipid bilayer in uncapaci- facilitating cytosolic entry of larger molecules and tated spermatozoa.472,875,876 orchestrating various cell-signaling events, to name We know from the information provided in the a few. Thus, assessment of its integrity would previous sections that a spermatozoon is laden with seem an important component of a semen evalua- special molecular domains that are designed, and tion. To this end, a variety of laboratory proce- modulated, for a multitude of functions within the dures have been used to evaluate the integrity of the female reproductive tract. In some respects, how- plasma membrane. One method is to evaluate the ever, the spermatozoon might be considered a rela- ability of spermatozoa to exclude extracellular dyes, tively simple structure, because it is only composed such as eosin Y, which are nonpermeable when the of a head and tail, with a dramatic reduction in membrane is intact. Another approach is to expose cellular organelles in comparison with somatic cells spermatozoa to hypotonic media (50- to 100-mOsm or its homolog, the ovum. As such, a spermatozoon range) to test their osmoregulatory function (termed might be subdivided into (1) a nucleus, (2) an over- the hypo-osmotic swelling test [HOST]).882,883

148 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE With this assay, membrane intact spermatozoa the- high throughput and objectivity associated with oretically permit excessive water entry into the cy- this approach.870,871,881,894,896 Use of a fluores- tosol, resulting in a variety of morphological changes cence microplate reader assay is also reported for in the flagellum associated with the cytosolic use with JC-1.897 swelling. Conversely, osmoregulation-incompetent Considerable effort has been directed toward iden- spermatozoa will not experience noticeable changes tification of biochemical markers of spermatozoal in flagellar shape. While these tests can serve an function that might aid in laboratory-based detec- adjunctive role in semen evaluation, they are not tion of fertility by targeting specific subcellular com- widely applied in the clinical setting because of the partments or domains. Many of these methods potential for misinterpretation. For instance, hu- have been devised for use with non-equine subjects, midity impacts the cellular permeability of eosin but several have been proposed for potential use Y.884 The incidence of spermatozoa with a bent with stallion spermatozoa. Although the value of flagellum prior to HOST will also confound the in- such tests requires further scrutiny and standard- terpretation of results after exposure of a spermato- ization, Table 1 provides examples of assays that zoal population to hypo-osmotic media. may prove valuable as diagnostic tools: A broad array of membrane-impermeable fluores- Incorporation of in-depth tests, such as those cent dyes is available commercially and can be used above, for semen evaluation does not replace or di- to test membrane intactness. Examples include minish the value of the classical measurements of the DNA dyes, propidium iodide, bis-benzimide spermatozoal motility or morphology, and these two (Hoechst 33258), YO-PRO-1, TOTO-1, and ethidium methods are likely to remain the hallmarks for se- homodimer-1. As an alternative, spermatozoa can be men evaluation. Evaluation of spermatozoal motil- bathed in cell-permeable probes that become hydro- ity in both raw and extended forms is considered to lyzed to form membrane-impermeant fluorescent be a fundamental laboratory test for assessing the products in the cytosol. Examples include carboxy- fertilizing capacity of spermatozoa in an ejaculate. fluorescein diacetate (hydrolyzed by nonspecific cy- Evaluation of raw (undiluted or neat) semen gives tosolic esterases to form carboxyfluorescein); calcein one an idea of how well spermatozoa perform in AM or dihydrocalcein AM (hydrolyzed by esterases their natural fluid milieu. Determining motility in to form calcein, which, in turn, forms fluorescent the raw form can be hampered by higher sperm complexes with Ca2ϩ, and other metals), and concentrations and spermatozoal agglutination to SYBR-14 (deacylated in the cytosol, with the result- the cover glass, making it difficult for the evaluator ing product expressing strong fluorescence when to discern individual motility patterns. To over- complexed with nucleic acids). Of interest, stain- come this limitation, an aliquot of semen can also be ing of porcine spermatozoa with SYBR-14, which appropriately diluted (e.g., to 25 ϫ 106 sperm/ml) in complexes with DNA, does not affect their ability to a good-quality semen extender that is free of debris fertilize oocytes.885 Certain membrane-imperme- when visualized microscopically. The extender able and membrane-permeable dyes can be com- may slightly alter motility pattern, usually by in- bined in solution before spermatozoal exposure in an creasing the velocity measures. After initial exten- effort to provide a more accurate reflection of mem- sion, a high percentage of spermatozoa may exhibit brane integrity. For instance, a combination of a circular motility pattern; however, this behavior SYBR-14 and propidium iodide yields three popula- usually resolves after 5–10 min of exposure time in tions of stained spermatozoa: (1) membrane-in- the extender. Spermatozoal motility is exception- tact, SYBR-14–stained cells (green); (2) membrane- ally susceptible to environmental conditions (such damaged, propidium-iodide-stained cells (red); and as excessive heat or cold, lubricants, light, disinfec- (3) moribund cells (double-stained).886,887 In one tants, and osmolarity/pH of semen extender), so it is study, the percentage of motile stallion spermatozoa necessary to protect the semen from injurious (based on computerized motility analysis) was agents or conditions before analysis. To enhance highly correlated (r ϭ 0.98) with the percentage of the reliability of motility estimation, the procedure spermatozoa with intact plasma membranes, based should be performed by an experienced person using on staining with SYBR-14 and propidium iodide.888 a properly equipped microscope, i.e., one with a Some fluorescent plasma-membrane dyes can also built-in stage warmer and properly adjusted phase- be combined with certain mitochondrial dyes888,889 contrast optics. Estimations of the percentages of or acrosomal dyes890–894 to provide more thorough motile and progressively motile spermatozoa are compartmental coverage in the assay. The litera- generally determined, in addition to an estimation of ture reports triple-stain fluorescent techniques for spermatozoal velocity (based on an arbitrary scale of use with stallion spermatozoa: propidium iodide/ 0 [stationary] to 4 [fast]). Subjective assessment SYBR-14/ JC1888 and propidium iodide/FITC-PNA of motility is generally quite acceptable, provided (an acrosomal lectin probe)/carboxy-SNARF-1 (an personnel are experienced in analysis of spermato- intracellular pH indicator).890,895 Although images zoal motility. can be ascertained with the various fluorophores Several different techniques and instruments described above by using fluorescence microscopy, have been developed in an effort to secure an objec- flow cytometry is typically applied because of the tive (i.e., unbiased) evaluation of spermatozoal mo-

AAEP PROCEEDINGS ր Vol. 53 ր 2007 149 MILNE LECTURE

Table 1. Biochemical Markers for Assessing Spermatozoal Function

Biochemical Marker Potential Value of Test

Acrosin/amidase activity898–900 Acrosome integrity SNARE proteins694,901 Acrosome reaction Caspases902–905 Apoptosis Heparin-binding proteins499,906 Capacitation Protein phosphotyrosine activity907–909 Capacitation Superoxide anion266 Capacitation 910–913 Chromomycin A3 Chromatin packing Acetylcarnitine/carnitine Motility/morphology Phospholipase C␨914,915 Oocyte activation 340,916–920 C11-BODIPY(581/591) Oxidative injury Malondialdehyde921–928 Oxidative injury Glutathione peroxidase320,929–932 Oxidative injury Reactive oxygen species257,282,835,928,931,933–938 Oxidative injury Lactate dehydrogenase939–942 Spermatozoal motility Creatine kinase/heat shock protein A2943–946 Spermatozoal maturity CRISP proteins104,499,503,790,947–949 Sperm–oocyte binding SP20/hyaluronidase456,761,950 Sperm–oocyte interaction AWN spermadhesion protein499,951–953 Sperm–zona binding P34H protein954–957 Sperm–zona binding Zonadhesin958–964 Sperm–zona binding SP22 protein965–968 Spermatozoal fertility

tility; however, these methods (e.g., time-lapse environment (at a veterinary hospital or an equine photomicrography, frame-by-frame playback video- breeding operation) is the ability to garner objective micrography, spectophotometry, or computerized results for a variety of motility variables. Confu- analysis) are generally considered to be too tedious sion arises, however, regarding the relationship of or expensive for routine use. Computerized sys- the myriad of obtainable CASA variables to fertility tems are currently in place in many reference labo- of the sample. As an example, we recently con- ratories with the intent to objectively assess motion ducted a fertility trial with a subfertile stallion characteristics of spermatozoa. Despite the com- whose semen was subjected to density-gradient cen- mercial availability of various generations of com- trifugation in an effort to improve semen quality puter-assisted spermatozoal analysis (CASA) before insemination. Values for percent motility systems for Ͼ20 yr, their presence has not provided (MOT), percent progressive motility (PMOT), and the deﬁnitive assay for measuring spermatozoal fer- mean curvilinear velocity (VCL; ␮m/s) before, and tilizing potential.969 Such an expectation, however, after, semen processing for the subfertile stallion is unrealistic given the numerous independent sper- and a fertile control stallion are listed in Table 2. matozoal attributes that are required for a spermato- Based on these results, it would seem that semen zoon to possess fertilization competence. What these treatment for the subfertile stallion yielded a sper- systems do provide is the prospect of objective mea- matozoal population with quality similar to, or ex- surement and protocol standardization. These in- ceeding (based on velocity values), that of the fertile struments permit customized selection of various control stallion. Nonetheless, when fertile mares features, including frequency and length of frame cap- were inseminated hysteroscopically with 20 ϫ 106 ture; threshold demarcations for presence of sperma- progressively motile spermatozoa (100-␮l volume), tozoal motion, progressivity of motion, and velocity the resulting pregnancy rates were 15/20 (75%) for measures; and gating freedom for both size and lumi- nocity representative of spermatozoal heads, as a means to maximize capture of spermatozoa while minimizing capture of non-spermatozoal material in Table 2. Effect of Equine Spermatozoal Centrifugation Through a Si- the sample of interest. Such manipulations are im- lanized Silica-Particle Solution on Spermatozoal Motion Characteristics portant for improving measurement accuracy and Semen repeatability within a given laboratory, but make it Stallion Treatment MOT (%) PMOT (%) VCL (␮m/s) virtually impossible for reliable comparisons among laboratories or among different CASA brands.970,971 Fertile Before 80 63 205 Computerized analysis of spermatozoa is primarily Fertile After 91 78 209 reserved for the research setting, where standard- Subfertile Before 63 48 251 ization, accuracy, and precision are a prerequisite to Subfertile After 90 79 259 measurement of experimental endpoints. A dis- MOT ϭ total motility; PMOT ϭ progressive motility; VCL ϭ tinct value of CASA instruments in the commercial curvilinear velocity.

150 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE the fertile stallion compared with 7/20 (35%) for the some morphologic abnormalities like detached subfertile stallion (p ϭ 0.01). This showed that heads can be primary, secondary or tertiary in na- spermatozoal motility does not provide absolute dis- ture, thereby introducing the possibility of error crimination power, again emphasizing that sperma- when using the traditional classification system ex- tozoal attributes other than motility play critical clusively. The reader is directed to Figure 39 for roles in spermatozoal fertilizing ability. Some drawings of normal and abnormal sperm morpho- CASA systems can be outfitted with fluorescence logic features, as might be viewed when using DIC optics, and the predictive value of CASA might be microscopy for analysis. improved by incorporation of fluorescent dyes in the The percentage of morphologically normal sper- media such that motility variables can be segregated matozoa is positively correlated with spermatozoal by presence or absence of membrane intactness.972 motility. Moreover, close morphological inspection Further studies are required to determine if the provides additional information about characteris- predictive value of spermatozoal motility can be im- tics of individual spermatozoa. This information is proved by this approach. A study involving boars important because semen can possess good sperma- suggests that CASA analysis might have an im- tozoal motility, yet have a relatively high incidence proved relationship with fertility if the spermatozoa of spermatozoal morphologic abnormalities. Fur- are also incubated under capacitating conditions to thermore, stallions can have many spermatozoal ab- observe changes in spermatozoal velocity over normalities, yet display normal fertility. Some time.973 morphologic defects (e.g., cytoplasmic droplets, bent The morphology or structure of spermatozoa is tails) seem to have a minor effect on fertility of typically examined with a light microscope at ϫ1000 stallions bred by natural cover, whereas other de- magnification. Standard bright-field microscope fects (e.g., detached heads, abnormally shaped optics can be used to examine air-dried semen heads, abnormally shaped midpieces, coiled tails, smears, provided appropriate stains are used in and premature germ cells) have a deleterious effect slide preparation. Specific sperm stains include on fertility.125 In general, morphologically abnor- those developed by Williams974 and Casarett.975 mal spermatozoa do not exert a direct negative in- General purpose cellular stains (e.g., Wright’s, Gi- fluence on normal spermatozoa. Therefore, the emsa, hematoxylin-eosin) also have been used to total number of morphologically normal sperm in accent both germinal and somatic cells in semen ejaculates may provide more information regarding smears. Background stains (e.g., eosin-nigrosin, the fertility of a stallion than the percentage or India ink) probably are the most widely used stains absolute number of morphologically abnormal sper- because of their ease of application. Visualization matozoa. Generally speaking, the percentage of of the structural detail of spermatozoa can be morphologically normal sperm in a semen sample is greatly enhanced by fixing the cells in buffered for- similar to the percentage of progressively motile mol saline or a similar fixative and viewing the spermatozoa. If spermatozoal motility is low and unstained cells as a wet mount with either phase- the percentage of morphologically normal sperm is contrast or, preferably, DIC microscopy.884 In ad- high, it suggests that laboratory errors occurred that dition, the incidence of artifactual changes is led to a lowering of spermatozoal motility. One can reduced in comparison with stained smears. not discount, however, a potentially negative effect At least 100 spermatozoa should be evaluated for of seminal plasma on spermatozoal motion evidence of morphological defects. The type and characteristics. incidence of each defect should be recorded. Abnor- As seen in the previous paragraphs, a variety of malities in spermatozoal morphology traditionally techniques and protocols are available for evalua- have been classified as primary, secondary, or ter- tion of the spermatozoon. Application of newly de- tiary. Primary abnormalities are considered to be veloped assays can be cumbersome and expensive. associated with a defect in spermatogenesis and, Nonetheless, incorporation of some of these diagnos- therefore, are of testicular origin. Secondary ab- tic tools into a standard semen analysis may yield normalities are created in the excurrent duct sys- improved discrimination ability regarding the compe- tem. Tertiary abnormalities, as opposed to the tence of these complex, yet intriguing, cells. Similar previous two types, develop in vitro as a result of applications would be valuable for critical evaluation improper semen collection or handling procedures. of laboratory techniques applied to liquid preservation The current trend is to record the numbers of or cryopreservation of spermatozoa. specific morphologic defects, such as knobbed acrosomes, proximal protoplasmic droplets, swollen mid- 7. Concluding Remarks pieces, and coiled tails. This method of Although a plethora of scientific information sur- classification is considered superior to the tradi- rounds spermatozoal structure and function, many tional system because it reveals more specific infor- unresolved issues remain, even with human and mation regarding a population of sperm while rodent spermatozoa where the largest body of infor- avoiding erroneous assumptions about the origin of mation has been assimilated. Only when we know these defects. The origin of some spermatozoal precisely the specific molecular interactions re- morphologic defects is unknown. Additionally, quired for attaining full spermatozoal fertilizing po-

AAEP PROCEEDINGS ր Vol. 53 ր 2007 151 MILNE LECTURE

Fig. 39. Drawings of spermatozoa to resemble images of normal and abnormal equine spermatozoal morphology, as viewed by differential-interference contrast (DIC) microscopy of a ﬁxed and unstained wet-mount semen specimen. The DIC optics provide a three-dimensional image of spermatozoa. (A) Normal spermatozoal morphology, in dorsoventral (A1 and A2) and side (A3) views. Abaxial midpieces (A1) are considered to be morphologically normal. (B) Variations in abnormal head morphology, including macrocephalic (large head) morphology (B1), microcephalic (small head) morphology (B2), nuclear vacuoles (or crater defects; considered by our laboratory to be normal if low in number and size, and located randomly over acrosomal region; B3), tapered head (B4), pyriform head (B5), constricted or hour-glass head (B6), and degenerate head (B7). (C) Acrosomal defect in dorsoventral (C1 and C2) and side (C3) views. This morphologic defect is termed “knobbed acrosome.” (D) Proximal (D1) and distal (D2 and D3) cytoplasmic droplets. (E) Abnormalities of the midpiece, including segmental aplasia of the mitochondrial sheath (E1), roughed midpiece from uneven distribution of mitochondria (E2), enlarged mitochondrial sheath (E3), bent midpiece (E4–E6), and double midpiece/double head (E7). (F) Bent tail (or hairpin tail), involving the mid-region of the principal piece (F1–F4) or with a singular bend (F5) or proximal bend (F6) involving the midpiece-principal piece junction. (G) Coiled tail, with the tail tightly encircling the head (G1) or the tail coil not encircling the head (G2 and G3). (H) Fragmented sperm, including head detachment (termed detached heads or tailless heads; H1) or fragmentation at the level of the annulus (H2). Fragmentation can also occur at other sites, as shown in F4. (I) Premature germ cells with a single nucleus (I1) or multiple nuclei (I2).

152 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE tential, including the spatial and temporal changes, profiling and its biologic significance, however, can be energetics, and gaseous environment involved, will a quagmire, and continued effort is required to hurdle we be able to reliably manipulate spermatozoa to this obstacle.988 Given the intensity of study in this meet the growing needs within the equine breeding area, the basic mechanisms that control gene expres- industry. Goals might include devising methods sion will emerge in the years ahead, and genetic mark- for long-term cooled semen preservation, improving ers for stallion fertility may one day abound.989 the fertility of cryopreserved semen, and incorpora- Robust mass spectrometry proteomics methodologies tion of in vitro fertilization (both conventional in allow inventory of proteins in cells, organelles, and vitro fertilization and intracytoplasmic sperm injec- body fluids990–992 and offer the potential for identification) into commercial programs. Although these tion of biomarkers for diagnostic tests.993–995 might seem to be lofty goals, a more absolute under- As with humans and other animal species, the standing of spermatozoal structure and function impacts that environmental chemicals have on stal- would certainly take a lot of the “guess work” out of lion fertility deserve attention. An apparent de- current approaches to analysis and manipulation of cline in spermatozoal quality and quantity of men equine spermatozoa. has been described in recent years.996–998 In- Attempts to gain a better understanding of equine creased rates of other reproductive tract problems, spermatozoa solely from extrapolation of data ac- such as cryptorchidism and testicular cancer, have quired from other mammalian species are likely des- also been reported.999,1000 Environmental repro- tined to failure because of the well-known species ductive toxicants are considered to be a major con- differences in spermatozoal attributes and physiol- tributor to these patterns.1001–1003 Both cDNA ogy. Nonetheless, much information derived from microarray and real-time reverse transcriptase- other species may have relevance to equids and polymerase chain reaction approaches have re- should be investigated for applicability. Examples vealed differential expression profiles in some include identification of candidate genes for specific spermatogenesis-related genes (i.e., heat shock pro- spermatozoal traits, targeted mutation of genes for tein 70–2, insulin growth factor binding protein 3, specific proteins to study the resulting effect on re- and glutathione S transferase pi) after toxicant ex- productive function, and use of gene silencing agents posure, so genes such as these may serve as useful for regulatable ablation of gene function. Incorpo- biomarkers when screening for testicular toxicity.1004 ration of molecular techniques would seem to be the A better understanding of the molecular mecha- key to elucidation of mechanisms that control sper- nisms regulating spermatozoal development and matozoal development and function in stallions. function will likely lead to new diagnostic tech- Indeed, the “omics” era (e.g., genomics, toxicog- niques and therapeutic strategies for reduced fertil- enomics, transcriptomics, metabonomics, and pro- ity in stallions and may possibly translate to teomics) is on us. Technological progress in the methodologies designed to curb gonadal aging. field of molecular genetics has been remarkable and Such progress can only be curtailed by funding def- has opened the door to new paradigms for the study icits or diverted interests of investigators in the of spermatozoal function and dysfunction. Signifi- years ahead. Undoubtedly, future generations will cant inroads have been made in horse genome map- look beyond descriptive morphology and motility in ping in recent years, including availability of characterizing structural and functional features synteny, linkage, radiation-hybrid, cytogenetic, and and deficiencies of spermatozoa. In addition, they human-horse comparative maps.976–981 The Na- will likely use the spermatozoon as a vehicle for tional Institutes of Health recently reported (www. production of transgenic horses,1005,1006 as well as nih.gov/news/pr/feb2007/nhgri-07.htm) that the 2.7- spermatogonial stem cell transplantation or testicu- billion DNA base-pair horse genome has been se- lar grafting techniques to study testicular function quenced and partially assembled, and the first draft or to propagate a genetic line.1007–1014 of the sequence is now accessible through public It seems befitting to conclude this manuscript databases; hence, the importance of bioinformatics to with a quote from Professor Storey, as presented in biological research. Continued progress in genomic the first reference entitled “Interactions between ga- research will lead to isolation and identification of metes leading to fertilization: the sperm’s eye genes responsible for spermatozoal development and view”....“the reactions that lead to fertilization of function, as well as genetic factors that underlie vari- the mammalian egg by its conspecific sperm are ous types of reproductive failure. High-throughput many, complex, and exquisitely coordinated. One microarray technology (cDNA and oligonucleotide) is might add that they are also vital.”1 being used with increased intensity to study differential gene expression.982 Reverse transcription and Financial assistance was provided by the Aber- amplification steps are being applied to quantify gene crombie Foundation, the Patsy Link Endowment activity in targeted cells or processes and to unravel Fund, Texas A&M University; the Shining Spark- the complexity of genes.983,984 The large-scale infor- Carol Rose Stallion Reproduction Fund (through the mation obtained from these emergent technologies is American Quarter Horse Association); the Azoom, being applied to male fertility,985 including that of Corona Cartel, and Teller Cartel syndicates horses.53,986,987 Interpretation of such expression (through Lazy E Ranch); Black Rock Ranch; Cool-

AAEP PROCEEDINGS ր Vol. 53 ր 2007 153 MILNE LECTURE more, and Darley. Special thanks go to Ricardo 10. Akingbemi BT. Estrogen regulation of testicular function. Levario, Nicole Hilborn, Victoria Star Varner, Vince Reprod Biol Endocrinol 2005;3:51. 11. De Gendt K, Swinnen JV, Saunders PTK, et al. A sertoli Hardy, and Dr. Noah Heninger for assistance with cell-selective knockout of the androgen receptor causes manuscript figures and Sarah Chellappan for refer- spermatogenic arrest in meiosis. Proc Natl Acad Sci USA ence management. 2004;101:1327–1332. After lengthy pondering about both the subject 12. Verhoeven G. A sertoli cell-specific knock-out of the an- area and the title of this manuscript, I arrived at drogen receptor. Andrologia 2005;37:207–208. 13. Tsai M, Yeh SD, Wang RS, et al. Differential effects of that listed above. Much to my chagrin, while con- spermatogenesis and fertility in mice lacking androgen ducting a literature search during the preparation of receptor in individual testis cell. Proc Natl Acad Sci USA this paper, I found that one of my mentors from the 2006;103:18975–18980. University of Pennsylvania, Dr. Bayard T. Storey, 14. Varner DD, Blanchard TL, Brinsko SP, et al. Techniques authored a paper over a decade ago with a similar for evaluating selected reproductive disorders of stallions. Anim Reprod Sci 2000;60–61:493–509. title. Rather than change the title, I elected to retain 15. Amann RP, Johnson L, Pickett BW. Connection between it as a tribute to Dr. Storey. Now a Professor Emer- the seminiferous tubules and the efferent ducts in the itus of Reproductive Biology and Physiology at the stallion. Am J Vet Res 1977;38:1571–1579. University of Pennsylvania, Dr. Storey established 16. Johnson L, Thompson DL Jr. Age-related and seasonal himself as an international leader in the area of variation in the Sertoli cell population daily sperm produc- spermatozoal physiology. With hundreds of master- tion and serum concentrations of follicle-stimulating hormone, luteinizing hormone and testosterone in stallions. ful manuscripts to his credit and an exhaustive list Biol Reprod 1983;29:777–789. of trainees that have emerged to become authorities 17. Varner DD, Schumacher J, Blanchard TL, et al. Diseases in their own right, Dr. Storey is a scientist and and management of breeding stallions. Goleta, CA: educator of the highest order. As one trainee that American Veterinary Publications, 1991. spent only a short time in his laboratory, I would 18. Johnson L, Neaves WB. Age-related changes in the Ley- dig cell population, seminiferous tubules, and sperm pro- like to honor Dr. Storey’s professional accomplish- duction in the stallion. Biol Reprod 1981;24:703–712. ments through this writing. Likewise, many of my 19. Johnson L, Amann RP, Pickett BW. Scanning electron colleagues and I received our residency training and light microscopy of the equine seminiferous tu- through the watchful eye and nurturing environ- bule. Fertil Steril 1978;29:208–215. ment offered by Dr. Robert M. Kenney, University of 20. Swierstra EE, Gebauer MR, Pickett BW. Reproductive physiology of the stallion I. Spermatogenesis and testis Pennsylvania. Dr. Kenney has not only touched our composition. J Reprod Fertil 1974;40:113–123. lives in such meaningful fashion, but opened his 21. Maekawa M, Kamimura K, Nagano T. Peritubular myoid laboratory and offered his wisdom to virtually any- cells in the testis: their structure and function. Arch one, in any part of the world, who sought to gain a Histol Cytol 1996;59:1–13. more thorough understanding of equine reproduc- 22. Skinner MK, Fritz IB. Identification of a non-mitogennic paracrine factor involved in mesenchymal-epithelial cell tion. His personable nature, combined with an en- interactions between testicular peritubular cells and Ser- cyclopedic knowledge base, is praised by all the toli cells. Mol Cell Endocrinol 1986;44:85–97. individuals that he has touched. We thank you both 23. Hettle JA, Balekjian E, Tung PS, et al. Rat testicular for your diligence in the field, your tenacity for in- peritubular cells in culture secrete an inhibitor of plasmin- tellectual growth, and your amiable approach to the ogen activator activity. Biol Reprod 1988;38:359–371. 24. Wong C, Cheng CY. The blood-testis barrier: its biology, education of others! regulation, and physiological role in spermatogenesis. Curr Top Dev Biol 2005;71:263–296. References 25. Li MWM, Xia W, Mruk DD, et al. Tumor necrosis factor (alpha) reversibly disrupts the blood-testis barrier and im- 1. Storey BT. Interactions between gametes leading to fer- pairs Sertoli-germ cell adhsion in the seminiferous epithe- tilization: the sperm’s eye view. Reprod Fertil Dev 1995; 7:927–942. lium of adult rat testes. J Endocrinol 2006;190:313–329. 2. MacLean JA II, Wilkinson MF. Gene regulation in sper- 26. Johnson L. Spermatogenesis. In: PT Cupps, ed. matogenesis. Curr Top Dev Biol 2005;71:131–197. Reproduction in domestic animals. San Diego, CA: Aca- 3. Tanaka H, Baba T. Gene expression in spermiogenesis. demic Press, 1991;173–219. Cell Mol Life Sci 2005;52:344–354. 27. Huleihel M, Lunenfeld E. Regulation of spermatogenesis 4. Rossi P, Dolci S, Sette C, et al. Analysis of the gene by paracrine/autocrine testicular factors. Asian J Androl expression profile of mouse male meiotic germ cells. Gene 2004;6:259–268. Expr Patterns 2004;4:267–281. 28. O’Donnell L, Meachem SJ, Stanton PG, et al. Endocrine 5. Elliott D. Pathways of post-transcriptional gene regula- regulation of spermatogenesis. In: Neill JD, ed. Knobil tion in mammalian germ cell development. Cytogenet Ge- and Neill’s physiology of reproduction. Boston, MA: nome Res 2003;103:210–216. Elsevier, 2006;1017–1069. 6. Ronfani L, Bianchi ME. Molecular mechanisms in male 29. Holdcraft RW, Braun RE. Hormonal regulation of sper- determination and germ cell differentiation. Cell Mol Life matogenesis. Int J Androl 2004;27:335–342. Sci 2004;61:1907–1925. 30. Perrard M-H, Vigier M, Damestoy A, et al. Beta-nerve 7. Grimes SR. Testis-specific transcriptional control. Gene growth factor participates in an autocrine/paracrine path- 2004;343:11–22. way of regulation of the meiotic differentiation of rat sper- 8. van der Weyden L, Arends MJ, Chausiaux OE, et al. matocytes. J Cell Physiol 2007;210:51–62. Loss of TSLC1 causes male infertility due to a defect at the 31. Abo-Elmaksoud A, Sinowatz F. Expression and localiza- spermatid stage of spermatogenesis. Mol Cell Biol 2006; tion of growth factors and their receptors in the mamma- 26:3595–3609. lian testis. Part I: fibroblast growth factors and insulin- 9. Gromoll J, Simoni M. Genetic complexity of FSH receptor like growth factors. Anat Histol Embryol 2005;34:319– function. Trends Endocrinol Metab 2005;16:368–373. 334.

154 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

32. Miller D, Ostermeier GC. Towards a better understand- isations between rat Sertoli cells in vitro and in vivo. J ing of RNA carriage by ejaculate spermatozoa. Hum Re- Endocrinol 2006;189:381–395. prod Update 2006;12:757–767. 56. Nickel R, Schummer A, Seiferle E, et al. The viscera of 33. Moreno RD, Alvarado CP. The mammalian acrosome as a domestic animals. New York: Springer-Verlag, 1973. secretory lysosome: new and old evidence. Mol Reprod 57. Johnson L, Amann RP, Pickett BW. Scanning electron Dev 2006;73:1430–1434. microscopy of the epithelium and spermatozoa in the 34. Dadoune JP, Siffrol JP, Alfonsi MF. Transcription in hap- equine excurrent duct system. Am J Vet Res 1978;39: loid male germ cells. Int Rev Cytol 2004;237:1–56. 1428–1434. 35. Miller D, Ostermeier GC. Spermatozoal RNA: why is it 58. Robaire B, Hinton BT, Orgebin-Crist M-C. The epididy- there and what does it do? Gynecol Obstet Fertil 2006;34: mis. In: Neill JD, ed. Knobil and Neill’s physiology of 840–846. reproduction. New York: Elsevier, 2006;1071–1148. 36. Johnson L. A new approach to quantification of Sertoli 59. Amann RP, Thompson DL Jr, Squires EL, et al. Effects of cells that avoids problems associated with the irregular age and frequency of ejaculation on sperm production and nuclear surface. Anat Rec 1986;214:231–237. extragonadal sperm reserves in stallions. J Reprod Fertil 37. Holstein AF, Schulze W, Davidoff M. Understanding Suppl 1979;27:1–6. spermatogenesis is a prerequisite for treatment. Reprod 60. Swierstra EE, Pickett BW, Gebauer MR. Spermatogen- Biol Endocrinol 2003;1:107. esis and duration of transit of spermatozoa through the 38. Lebland CP, Clermont Y. Definition of the stages of the excurrent ducts of stallions. J Reprod Fertil Suppl 1975; cycle of the seminiferous epithelium in the rat. Ann NY 23:53–57. Acad Sci 1952;55:548–573. 61. Amann RP. A review of anatomy and physiology of the 39. Johnson L. Efficiency of spermatogenesis. Microsc Res stallion. J Equine Vet Sci 1981;1:83–105. Tech 1995;32:385–422. 62. Gebauer MR, Pickett BW, Swierstra EE. Reproductive 40. Clermont Y. The cycle of the seminiferous epithelium in physiology of the stallion. III. Extra-gonadal transit man. Am J Anat 1963;112:35–51. time and sperm reserves. J Anim Sci 1974;39:737–742. 41. Johnson L. Increased daily sperm production in the 63. Amann RP. A critical review of methods for evaluation of breeding season of stallions is explained by an elevated spermatogenesis from seminal characteristics. J Androl population of spermatogonia. Biol Reprod 1985;32:1190. 1981;2:37–58. 42. Johnson L, Hardy VB, Martin MT. Staging equine semi- 64. Varner DD, Schumacher J, Blanchard TL, et al. Diseases niferous tubules by Normarski optics in unstained histo- of the epididymis. In: Varner DD, Schumacher J, Blan- logic sections and in tubules mounted in toto to reveal the chard TL, Johnson L, eds. Diseases and management of spermatogenic wave. Anat Rec 1990;227:167–174. breeding stallions. Goleta, CA: American Veterinary 43. Heninger NL, Staub C, Blanchard TL, et al. Germ cell Publications, 1991;233–240. apoptosis in the testes of normal stallions. Theriogenology 65. Love CC. Ultrasonographic evaluation of the testis, epi- 2004;62:297. didymis, and spermatic cord of the stallion. In: Blan- 44. Johnson L, Blanchard TL, Varner DD, et al. Factors af- chard TL, Varner DD, eds. Veterinary clinics of North fecting spermatogenesis in the stallion. Theriogenology America—equine practice (stallion management). Phila- 1997;48:1199–1216. delphia, PA: W.B. Saunders, 1992;167–182. 45. Blanchard TL, Johnson L. Increased germ cell degenera- 66. Alvarez JG, Storey BT. Spontaneous lipid peroxidation in tion and reduced germ cell:Sertoli cell ratio in stallion with rabbit epididymal spermatozoa: its effects on sperm mo- low sperm production. Theriogenology 1997;47:665–677. tility. Biol Reprod 1982;27:1102–1108. 46. Clermont Y. Quantitative analysis of spermatogenesis of 67. Vernet P, Aitken RJ, Drevet JR. Antioxidant strategies in the rat: a revised model for the renewal of spermatogonia. Am J Anat 1962;111:111–129. the epididymis. Mol Cell Endocrinol 2004;216:31–39. 47. Huckins C. The morphology and kinetics of spermatogo- 68. Johnson L, Amann RP, Pickett BW. Maturation of equine nial degeneration in normal adult rats: an analysis using a epididymal spermatozoa. Am J Vet Res 1980;41:1190– simplified classification of the germinal epithelium. Anat 1196. Rec 1978;190:905–926. 69. Baumgarten HG, Holstein AF, Rosengren E. Arrange- 48. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic bio- ment, ultrastructure and adrenegeric innervation of logical phenomenon with wide-ranging implications in tis- smooth musculature of the ductuli efferentes, ductus epi- sue kinetics. Br J Cancer 1972;26:239–257. didymis and ductus deferens of man. Z Zellforsch Mik- 49. Cisternas P, Moreno RD. Comparative analysis of apopto- rosk Anat 1971;120:37–79. tic pathways in rat, mouse, and hamster spermatoza. Mol 70. Pholpramool D, Triphrom N. Effects of cholinergic and Reprod Dev 2006;73:1318–1325. adrenergic drugs on intraluminal pressures and contractil- 50. Allan DJ, Harmon BV, Roberts SA. Spermatogonial apo- ity of the rat testis and epididymis in vivo. J Reprod Fertil ptosis has three morphologically recognizable phases and 1984;71:181–188. shows no circadian rhythm during normal spermatogenesis 71. Cosentino MJ, Takihara H, Burhop JW, Crockett AT. in the rat. Cell Prolif 1992;25:241–250. Regulation of rat caput epididymides contractility by pros- 51. Joshi AR, Dighe RR. Expression of apoptotic genes in taglandins. J Androl 1984;5:216–222. testicular germ cells and their modulation by luteinizing 72. Filippi S, Vannelli GB, Granchi S, et al. Identification, hormone. J Steroid Biochem Mol Biol 2006;101:22–30. localization and functional activity of oxytocin receptors in 52. Heninger NL, Staub C, Johnson L, et al. Testicular germ epididymis. Mol Cell Endocrinol 2002;193:89–100. cell apoptosis and formation of the Sertoli cell barrier during 73. Whittington K, Assinder SJ, Parkinson T, et al. Function the initiation of spermatogenesis in pubertal stallions. and localization of oxytocin receptors in the reproductive Anim Reprod Sci 2006;94:127–131. tissue of rams. Reproduction 2001;122:317–325. 53. Ing NH, Laughlin AM, Varner DD, et al. Gene experssion 74. Studdard PW, Stein JL, Cosentino MJ. The effects of in the spermatogenically inactive “dark” and maturing oxytocin and arginine vasopressin in vitro on epididymal “light” testicular tissues of the prepubertal colt. J Androl contractility in the rat. Int J Androl 2002;25:65–71. 2004;25:535–544. 75. Ganjam VK, Amann RP. Testosterone and dihydrotestos- 54. Meachem SJ, Ruwanpura SM, Siolkowski J, et al. Devel- terone concentrations in the fluid milieu of spermatozoa in opmentally distinct in vivo effects of FSH on proliferation the reproductive tract of the bull. Acta Endocrinol and apoptosis during maturation. J Endrocrinol 2005; (Copenh) 1973;74:186–200. 186:429–446. 76. Turner TT, Jones CE, Howards SS, et al. On the andro- 55. Sluka P, O’Donnell L, Bartles JR, et al. FSH regulates the gen microenvironment of maturing spermatozoa. Endo- formation of adherens junctions and ectoplasmic special- crinology 1984;115:1925–1932.

AAEP PROCEEDINGS ր Vol. 53 ր 2007 155 MILNE LECTURE

77. Filippi S, Luconi M, Granchi S, et al. Estrogens, but not sperm function during epididymal maturation or fertiliza- androgens, regulate expression and functional activity of tion. J Cell Sci 2001;114:1787–1794. oxytocin receptor in rabbit epididymis. Endocrinology 101. Schroter S, Osterhoff C, McArdle W, et al. The glycocalyx 2002;143:4271–4280. of the sperm surface. Hum Reprod Update 1999;5:302– 78. Amann RP, Hammerstedt RH, Veeramachaneni DNR. 313. The epididymis and sperm maturation: a perspec- 102. Tulsiani DRP. Glycan modifying enzymes in luminal tive. Reprod Fertil Dev 1993;5:361–381. fluid of rat epididymis: are they involved in altering 79. Bedford JM. Report of a workshop: maturation of the sperm surface glycoproteins during maturation? Microsc fertilizing ability of mammalian spermatozoa in the male Res Tech 2003;61:18–27. and female reproductive tract. Biol Reprod 1974;11:346– 103. Tulsiani DRP. Glycan-modifying enzymes in luminal 362. fluid of the mammalian epididymis: an overview of their 80. Nicander L. Studies on the regional histology and cyto- potential role in sperm maturation. Mol Cell Endocrinol chemistry of the ductus epididymidis in stallions, rams and 2006;250:58–65. bulls. Acta Morphol Neerl Scand 1958;1:337–362. 104. Roberts KP, Ensrud KM, Wooters JL, et al. Epididymal 81. Robaire B, Viger RS. Regulation of epididymal epithelial secreted protein CRISP-1 and sperm function. Mol Cell cell functions. Biol Reprod 1995;52:226–236. Endocrinol 2006;250:122–127. 82. Gatti J-L, Castella S, Dacheux F, et al. Post-testicular 105. Yoshinaga K, Toshimori K. Organization and modifica- sperm environment and fertility. Anim Reprod Sci 2004; tions of sperm acrosomal molecules during spermatogene- 82–83:321–339. sis and epididymal maturation. Micros Res Tech 2003;61: 83. Dacheux J-L, Gatti JL, Dacheux F. Contribution of epidid- 39–45. ymal secretory proteins for spermatozoa maturation. Mi- 106. Rodriguez CM, Kirby JL, Hinton BT. Regulation of gene crosc Res Tech 2003;61:7–17. 84. Dacheux JL, Belghazi M, Lanson Y, et al. Human epidid- transcription in the epididymis. Reproduction 2001;122: ymal secretome and proteome. Mol Cell Endocrinol 2006; 41–48. 250:36–42. 107. Lin M, Lee YH, Xu W, et al. Ontogeny of tyrosine phopho- 85. Lye RJ, Hinton BT. Technologies for the study of epidid- rylation-signaling pathways during spermatogenesis and ymal-specific genes. Mol Cell Endocrinol 2004;216:23–30. epididymal maturation in the mouse. Biol Reprod 2006; 86. Suzuki K, Drevet J, Hinton BT, et al. Epididymis-specific 75:588–597. promoter-driven targeting: a new approach to control ep- 108. Yeung C-H, Cooper TG. Developmental changes in sig- ididymal function. Mol Cell Endocrinol 2004;216:15–22. nalling transuction factors in maturing sperm during epi- 87. Turner TT, Johnston DS, Jelinsky SA. Epididymal didymal transit. Cell Mol Biol 2003;49:341–349. genomics and the search for a male contraceptive. Mol 109. Saacke RG, Almquist JO. Ultrastructure of bovine sper- Cell Endocrinol 2006;250:178–183. matozoa. I. The head of normal, ejaculated sperm. Am J 88. Cyr DG, Finnson K, Dufresne J, et al. Cellular interac- Anat 1964;115:143–161. tions and the blood-epidymis barrier. In: Robaire B, 110. Saacke RG, Almquist JO. Ultrastructure of bovine sper- Hinton BT, eds. The epididymis: from molecules to clin- matoza: II. The neck and tail of normal, ejaculates ical practice. New York: Kluwer Academic/Plenum, 2002; sperm. Am J Anat 1964;115:163–183. 103–118. 111. Fawcett DW. The mammalian spermatozoon. Dev Biol 89. Agarwal A, Hoffer AP. Ultrastructural studies on the de- 1975;44:394–436. velopment of the blood-epididymis barrier in immature 112. Eddy EM. The spermatozoon. In: Neill JD, ed. Kno- rats. J Androl 1989;10:425–431. bil and Neill’s physiology of reproduction. San Diego, CA: 90. Cyr DG, Robaire B, Hermo L. Structure and turnover of Elsevier Academic Press, 2006;3–54. junctional complexes between principal cells of the rat ep- 113. Cummins JM, Woodall PF. On mammalian sperm dimen- ididymis. Microsc Res Tech 1995;30:54–66. sions. J Reprod Fertil 1985;75:153–175. 91. Levy S, Serre V, Hermo L, Robaire B. The effects of aging 114. Bielanski W, Kaczmarski F. Morphology of spermatozoa on the seminiferous epithelium and the blood-testis barrier in semen from stallions of normal fertility. J Reprod Fer- of the Brown Norway rat. J Androl 1999;20:356–365. til Suppl 1979;27:39–45. 92. Levy S, Robaire B. Segment-specific changes with age in 115. Dott HM. Morphology of stallion spermatozoa. J Reprod the expression of junctional proteins and the permeability Fertil Suppl 1975;23:41–46. of the blood-epididymis barrier in rat. Biol Reprod 1999; 116. Gerton GL. Function of the sperm acrosome. In: 60:1392–1401. Hardy DM, ed. Fertilization. San Diego, CA: Aca- 93. Tomsig JL, Turner TT. Growth factors and the epididy- demic Press, 2002;265–302. mis. J Androl 2006;27:348–357. 117. Allison AC, Hartree EF. Lysosomal enzymes in the acro- 94. Hinton BT, Lan ZJ, Rudolph DB, et al. Testicular regu- some and their possible role in fertilization. J Reprod lation of epididymal gene expression. J Reprod Fertil Fertil 1970;21:501–515. Suppl 1998;53:47–57. 118. Abou-Haila A, Tulsiani DRP. Mammalian sperm acro- 95. Hermo L, Barin K, Oko R. Androgen binding protein se- some: formation, contents, and function. Arch Biochem cretion and endocytosis by principal cells in the adult rat epididymis and during postnatal development. J Androl Biophys 2000;379:173–182. 1998;19:527–541. 119. Herr JC. The acrosome. Proc Havemeyer Foundation 96. Garrett JE, Garrett SH, Douglass JA. A spermatozoa- Workshop Stallion Reprod 2007 (in press). associated factor regulates proenkephalin gene expression 120. Kierszenbaum AL, Tres LL. The acrosome-acroplaxome- in the rat epididymis. Mol Endocrinol 1990;4:108–118. manchette complex and the shaping of the spermatid head. 97. Jones R. Plasma membrane structure and remodeling Arch Histol Cytol 2004;67:271–284. during sperm maturation in the epididymis. J Reprod 121. Toshimori K, Ito C. Formation and organization of the Fertil Suppl 1998;53:73–84. mammalian sperm head. Arch Histol Cytol 2003;66:383– 98. Belmonte SA, Romano PS, Sosa MA. Mannose-6-phos- 396. phate receptors as a molecular indicator of maturation of 122. Tachibana M, Terada Y, Murakawa H, et al. Dynamic epididymal sperm. Arch Androl 2002;48:53–63. changes in the cytoskeleton during human spermiogenesis. 99. Martin-DeLeon PA. Epididymal SPAM1 and its impact Fertil Steril 2005;84:1241–1247. on sperm function. Mol Cell Endocrinol 2006;250:114– 123. Holstein AF, Roosen-Runge EC. Atlas of human spermat- 121. ogenesis. Berlin, Germany: Grosse Verlag, 1981. 100. Zhu G-Z, Myles DG, Primakoff P. Testase 1 (ADAM 24) a 124. Phillips DM. Spermiogenesis. New York: Adademic plasma membrane-anchored sperm protease implicated in Press, 1974.

156 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

125. Love CC, Varner DD, Thompson JA. Intra- and inter- 147. Kirichok Y, Navarro B, Clapham DE. Whole-cell patch- stallion variation in sperm morphology and their relation- clamp measurements of spermatozoa reveal an alkaline- ship with fertility. J Reprod Fertil Suppl 2000;56:93–100. activated Ca2ϩ channel. Nature 2006;439:737–740. 126. Storey BT. Energy metabolism of spermatozoa. IV. 148. Marquez B, Suarez SS. Different signaling pathways in bo- Effect of calcium on respiration of mature epididymal vine sperm regulate capacitation and hyperactivation. Biol sperm of the rabbit. Biol Reprod 1975;13:1–9. Reprod 2004;70:1626–1633. 127. Hoskins DD, Casillas ER. Function of cyclic nucleotides 149. Suarez SS, Ho HC. Hyperactivated motility in sperm. in mammalian spermatozoa. In: Hamilton DW, Greep Reprod Domest Anim 2003;38:119–124. RO, eds. Handbook of physiology. Bethesda, MD: 150. Ho HC, Granish KA, Suarez SS. Hyperactivated motility American Physiological Society, 1975;453–460. of bull sperm is triggered at the axoneme by Ca2ϩ and not 128. Turner TT, Giles RD. A sperm motililty-inhibiting factor cAMP. Dev Biol 2002;250:208–217. in the rat epididymis. Am J Physiol 1982;242:R199– 151. Ignotz GG, Suarez SS. Calcium/calmodulin and calmod- R203. ulin kinase II stimulate hyperactivation in demem- 129. Usselman MC, Cone RA. Rat sperm are mechanically branated bovine sperm. Biol Reprod 2005;73:519–526. immobilized in the cauda epididymis by “immobilin,” a 152. Ho HC, Suarez SS. Hyperactivation of mammalian sper- high molecular weight glycoprotein. Biol Reprod 1983;29: matozoa: function and regulation. Reproduction 2001; 1241–1253. 122:519–526. 130. Carr DW, Acott TS. Inhibition of bovine spermatozoa by 153. Morita Z, Chang MC. The motility and aerobic metabo- caudal epididymal fluid: I. studies of a sperm motility lism of spermatozoa in laboratory animals with special quiescence factor. Biol Reprod 1984;30:913–925. reference to the effects of cold shock and the importance of 131. Acott TS, Carr DW. Inhibition of bovine spermatozoa by calcium for the motility of hamster spermatozoa. Biol caudal epididymal fluid: II. interaction of pH and a qui- Reprod 1970;3:169–179. escence factor. Biol Reprod 1984;30:926–935. 154. Morton BE, Sagadraca R, Fraser C. Sperm motility 132. Babcock DF. Examination of the intracellular ionic envi- within the mammalian epididymis: species variation and ronment and of ionophore action by null point measure- correlation with free calcium levels in epididymal plasma. ments employing the fluorescein chromophore. J Biol Fertil Steril 1978;29:695–698. Chem 1983;258:6380–6389. 155. Chinoy JJ, Verma RJ, Patel KG. Effect of calcium on 133. Zaneveld LJD. The epididymis. In: Zaneveld LJD, sperm motility of cauda epididymis in vitro. Acta Eur Chatterton RT, eds. Biochemistry of mammalian repro- Fertil 1983;14:421–423. duction. New York: John Wiley and Sons, 1982;37–63. 156. Aoki F, Sakai S, Kohmoto K. Regualtion of flagellar bend- 134. Holt WV, Harrison RA. Bicarbonate stimulation of boar ing by cAMP and Ca2ϩ in hamster sperm. Mol Reprod sperm motility via a protein kinase a-dependent pathway: Dev 1999;53:77–83. between-cell and between ejaculate differences are not due 157. Fraser LR. Cellular biology of capacitation and the acro- to deficiencies in protein kinase A activation. J Androl some reaction. Hum Reprod 1995;10:22–30. 2002;23:557–565. 158. Tash JS. Protein phosphorylation: the second messen- 135. Okamura N, Tajima Y, Soejima A, et al. Sodium bicar- ger signal transducer of flagellar motility. Cell Motil Cy- bonate in seminal plasma stimulates the motility of mam- toskeleton 1989;14:332–339. malian spermatozoa through direct activation of adenylate 159. Vijayaraghavan S, Hoskins DD. Regulation of bovine cyclase. J Biol Chem 1985;260:9699–9705. sperm motility and cyclic adenosine 3Ј,5Ј-monophophate by 136. Mann T, Lutwak-Mann C. Biochemistry of seminal adenosine and its analogues. Biol Reprod 1986;34:468– plasma and male accessory fluids: application to andrologi- 477. cal problems. In: Mann T, Lutwak-Mann C, eds. Male 160. Skalhegg BS, Huang Y, Idzerda RL, et al. Mutation of the reproductive function and semen. New York: Springer- Calpha subunit of PKA leads to growth retardation and Verlag, 1981;269–336. sperm dysfunction. Mol Endocrinol 2002;16:630–639. 137. Gagnon C. Regulation of sperm motility at the axonemal 161. Brandt H, Hoskins DD. A cAMP-dependent phosphory- level. Reprod Fertil Dev 1995;7:847–855. lated motility protein in bovine epididymal sperm. J Biol 138. Luconi M, Baldi E. How do sperm swim? Molecular Chem 1980;255:982–987. mechanisms underlying sperm motility. Cell Mol Biol 162. Garbers DL, Kopf GS. The regulation of spermatozoa by 2003;49:357–369. calcium and cyclic nucleotides. In: Greengard P, Robison 139. Tash JS, Means AR. Regulation of protein phosphoryla- GA, eds. Advances in cyclic nucleotide research. New York: tion and motility of sperm by cyclic adenosine monophos- Raven Press, 1980;251–306. phate and calcium. Biol Reprod 1982;26:745–763. 163. Tash JS, Means AR. Cyclic adenosine 3Ј,5Ј monophos- 140. Demarco IA, Espinosa F, Edwards J, et al. Involvement of phate, calcium and protein phosphorylation in flagellar aNaϩ/HCO-3 cotransporter in mouse sperm capacitation. motility. Biol Reprod 1983;28:75–104. J Biol Chem 2003;278:7001–7009. 164. Lindermann CB, Kanous KS. Regulation of mammalian 141. Garbers DL, Tubb TJ, Hyne RV. A requirement of bicar- sperm motility. Arch Androl 1989;23:1–22. bonate for Ca2ϩ-induced elevations of cyclid AMP in guinea 165. Luconi M, Proazz I, Ferruzzi P, et al. Tyrosine phosphor- pig spermatozoa. J Biol Chem 1982;257:8980–8984. ylation of the a kinase anchoring protein 3 (AKAP3) and 142. Quill TA, Wang D, Garbers DL. Insights into sperm cell soluble adenylate cyclase are involved in the increase of motility signaling through sNHE and the CatSpers. Mol human sperm motility by bicarbonate. Biol Reprod 2005; Cell Endocrinol 2006;250:84–92. 72:22–32. 143. Woo AL, James PF, Lingrel JB. Roses of the Na,K-AT- 166. Hess KC, Jones BH, Marquez B, et al. The “soluble” ad- Pase alpha 4 isoform and the Naϩ/Hϩ exchanger in sperm enylyl cyclase in sperm mediates multiple signaling events motility. Mol Reprod Dev 2002;62:348–356. required for fertilization. Dev Cell 2005;9:249–259. 144. Woo AL, James PF, Lingrel JB. Sperm motility is depen- 167. Chen Y, Cann MJ, Litvin TN, et al. Soluble adenylyl dent on a unique isoform of the Na,K-ATPase. J Biol cyclase as an evolutionarily conserved bicarbonate sensor. Chem 2000;275:20693–20699. Science 2000;289:625–628. 145. Wang D, King SM, Quill TA, et al. A new sperm-specific 168. Wuttke MS, Buck J, Levin LR. Bicarbonate-regulated Naϩ/Hϩ exchanger required for sperm motility and fertil- soluble adenylyl cyclase. JOP 2001;2:154–158. ity. Nat Cell Biol 2003;5:1117–1122. 169. Litvin TN, Kamenetsky M, Zarifyan A, et al. Kinetic 146. Malo ME, Fliegel L. Physiological role and regulation of properties of “soluble” adenylyl cyclase; synergism between the Naϩ/Hϩ exchanger. Can J Physiol Pharmacol 2006; calcium and bicarbonate. J Biol Chem 2003;278:15922– 84:1081–1095. 15926.

AAEP PROCEEDINGS ր Vol. 53 ր 2007 157 MILNE LECTURE

170. Jaiswal BS, Conti M. Calcium regulation of the soluble ad- 190. Nevo AC, Rikmenspoel R. Diffusion of ATP in sperm fla- enylyl cyclase expressed in mammalian spermatozoa. Proc gella. J Theor Biol 1970;26:11–18. Natl Acad Sci USA 2003;100:10676–10681. 191. Adam DE, Wei J. Mass-transport of ATP within motile 171. Turner RM. Moving to the beat: a review of mammalian sperm. J Theor Biol 1975;49:125–145. sperm motility regulation. Reprod Fertil Dev 2006;18:25– 192. Ford WCL. Glycolysis and sperm motility: does a spoon- 38. ful of sugar help the flagellum go round? Hum Reprod 172. Tash JS. Protein phosphorylation: the second messen- Update 2006;12:269–274. ger signal transducer of flagellar motility. Cell Motil Cy- 193. Cao W, Haig-Ladewig L, Gerton GL, et al. Adenylate toskeleton 1989;14:332–339. kinase 1 and 2 are part of the accessory structures in the 173. Leclerc P, deLamirande E, Gagnon C. Cyclic adenosine mouse sperm flagellum. Biol Reprod 2006;75:492–500. 3Ј,5Ј monophosphate-dependent regulation of protein ty- 194. Miki K, Qu W, Goulding EH, et al. Glyceraldehyde rosine phosphorylation in relation to human sperm capac- 3-phosphate dehydrogenase-S, a sperm-specific glycolytic itation and motility. Biol Reprod 1996;55:684–692. enzyme, is required for sperm motility and male fertili- 174. Tash JS, Bracho GE. Regulation of sperm motility: ty. Proc Nat Acad Sci USA 2004;101:16501–16506. emerging evidence for a major role for protein phophatases. 195. Muka C, Okuno M. Glycolysis plays a major role for aden- J Androl 1994;15:505–509. osine triphosphate supplementation in mouse sperm 175. Inaba K. Molecular architecture of the sperm flagella flagellar movement. Biol Reprod 2004;71:540–547. molecules for motility and signaling. Zoolog Sci 2003;20: 196. Eddy EM, Toshimori K, O’Brien DA. Fibrous sheath of 1043–1056. mammalian spermatozoa. Microsc Res Tech 176. Visconti PE, Johnson LR, Oyaski M, et al. Regulation, 2003;61:103–115. localization, and anchoring of protein kinase A subunits 197. Storey BT, Kayne FJ. Properties of pyruvate kinase and during mouse sperm capacitation. Dev Biol flagellar ATPase in rabbit spermatozoa: relation to met- 1997;192:351–363. abolic strategy of the sperm cell. J Exp Zool 1980;211: 177. Hall DD, Davare MA, Shi M, et al. Critical role of cAMP- 361–367. dependent protein kinase anchoring to the L-type calcium 198. Travis AJ, Foster JA, Rosenbaum NA, et al. Targeting of channel Cav 1.2 via A-kinase anchor protein 150 in neu- a germ cell-specific type 1 hexokinase lacking a porin- rons. Biochemistry 2007;46:1635–46. binding domain to the mitochondria as well as to the head 178. Turner RMO, Casas-Dolz R, Schlingmann KL. Charac- and fibrous sheath of murine spermatozoa. Mol Biol Cell terization of an A-kinase anchor protein in equine sperma- 1998;9:263–276. tozoa and examination of the effect of semen cooling and 199. Westhoff D, Kamp G. Glyceraldehyde 3-phosphate dehy- cryopreservation on the binding of that protein to the regu- drogenase is bound to the fibrous sheath of mammalian larory subunit of protein kinase-A. Am J Vet Res 2005; spermatozoa. J Cell Sci 1997;110:1821–1829. 66:1056–1064. 200. Mori C, Nakamura N, Welch JE, et al. Mouse spermato- 179. Burton KA, Treash-Osio B, Muller CH, et al. Deletion of genic cell-specific type 1 hexokinase (mHk1-s) transcripts type II alpha regulatory subunit delocalizes protein kinase are expressed by alternative splicing from the mHk1 gene a in mouse sperm without affecting motility or fertilization. and the HK1-S protein is localized mainly in the sperm tail. J Biol Chem 1999;274:24131–24136. Mol Reprod Dev 1998;49:374–385. 180. Nakano I, Kobayashi T, Yoshimura M, et al. Central- 201. Bunch DO, Welch JE, Magyar PL, et al. Glyceraldehyde pair-linked regulation of microtubule sliding by calcium in 3-phosphate dehydrogenase-S protein distribution during flagellar axonemes. J Cell Sci 2003;116:1627–1636. mouse spermatogenesis. Biol Reprod 1998;58:834–841. 181. Bannai H, Yoshimura M, Takahashi K, et al. Calcium 202. Bradley MP, Geelan A, Leitch V, et al. Cloning, sequenc- regulation of microtubule sliding in reactivated sea urchin ing, and characterization of LDH-C4 from a fox testis sperm flagella. J Cell Sci 2000;113:831–839. cDNA library. Mol Reprod Dev 1996;44:452–459. 182. Gross MK, Toscano DG, Toscano WA Jr. Calmodulin- 203. Ford WCL, Harrison A. The effect of 6-chloro-6-deoxysug- mediated adenylate cyclase from mammalian sperm. J Biol ars on adenine nucleotide concentrations in and motility of Chem 1987;262:8672–8676. rat spermatozoa. J Reprod Fertil 1981;63:75–79. 183. White DR, Aitken RJ. Relationship between calcium, cy- 204. Nicastro D, Schwartz C, Pierson J, et al. The molecular clic AMP, ATP, and intracellular pH and the capacity of architecture of axonemes revealed by cryoelecron tomogra- hamster spermatozoa to express hyperactivated motili- phy. Science 2006;313:944–948. ty. Gamete Res 1989;22:163–177. 205. Lupetti P, Lanzavecchia S, Mercati D, et al. Three-di- 184. Si Y, Olds-Clark P. Evidence for the involvement of cal- mensional reconstruction of axonemal outer dynein arms modulin in mouse sperm capacitation. Biol Reprod 2000; in situ by electron tomography. Cell Motil Cytoskeleton 62:1231–1239. 2005;62:69–83. 185. Ahmad K, Bracho GE, Wolf DP, et al. Regulation of hu- 206. Shimizu T, Johnson KA. Kinetic evidence for multiple dy- man sperm motility and hyperactivation components by nein ATPase sites. J Biol Chem 1983;258:13841–13846. calcium, calmodulin, and protein phophatases. Arch An- 207. Holzbaur EL, Johnson KA. Microtubules accelerate ADP drol 1995;35:187–208. release by dynein. Biochemistry 1989;28:7010–7016. 186. Carafoli E, Santella L, Branca D, et al. Generation, con- 208. Johnson KA. Pathway of the microtubule-dynein ATPase trol, and processing of cellular calcium signals. Crit Rev and the structure of dynein: a comparison with actomyo- Biochem Mol Biol 2001;36:107–260. sin. Annu Rev Biophys Biophys Chem 1985;14:161–188. 187. Carrera A, Moos J, Ning XP, et al. Regulation of protein 209. Fujimura M, Okuno M. Requirement of the fixed end for tyrosine phosphorylation in human sperm by a calcium/ spontaneous beating in flagella. J Exp Biol calmodulin-dependent mechanism: Identification of A ki- 2006;209:1336–1343. nase anchor proteins as major substrates for tyrosine 210. Baltz JM, Willams O, Cone RA. Dense fibers protect phosphorylation. Dev Biol 1996;180:284–296. mammalian sperm against damage. Biol Reprod 1990;43: 188. Tash JS, Bracho GE. Identification of phosphoproteins 485–491. coupled to initiation of motility in live epididymal mouse 211. Hinsch KD, DePinto V, Aires VA, et al. Voltage-depen- sperm. Biochem Biophys Res Commun 1998;251: dent anion-selective channels VDAC2 and VDAC3 are 557–563. abundant proteins in bovine outer dense fibers, a cytoskel- 189. Lopez-Gonzalez I, DeLaVega-Beltran JL, Santi CM, et al. etal component of the sperm flagellum. J Biol Chem 2004; Calmodulin antagonists inhibit T-type Ca2ϩ currents in 279:15281–15288. mouse spermatogenic cells and the zona pellucida-induced 212. Wargo MJ, Smith EF. Asymmetry of the central appara- sperm acrosome reaction. Dev Biol 2001;236:210–219. tus defines the location of active microtubule sliding in

158 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

Chlamydomonas flagella. Proc Nat Acad Sci USA 2003; 235. Urner F, Sakkas D. Protein phosphorylation in mamma- 100:137–142. lian spermatozoa. Reproduction 2003;125:17–26. 213. Porter ME, Johnson KA. Dynein structure and function. 236. Hiipakka RA, Hammerstedt RH. 2-deoxyglucose trans- Annu Rev Cell Biol 1989;5:119–151. port and phosphorylation by bovine sperm. Biol Reprod 214. Amann RP, Graham JK. Spermatozoal function. In: 1978;19:368–379. DiRienzi D, ed. Equine reproduction. Philadelphia: 237. Mukai C, Okuno M. Glycolysis plays a major role for Lea & Febiger, 1993;715–745. adenosine triphosphate supplementation in mouse sperm 215. Hammerstedt RH, Volonte C, Racker E. Motility, heat, flagellar movement. Biol Reprod 2004;71:540–547. and lactate production in ejaculated bovine sperm. Arch 238. Ford WCL, Rees JM. The bioenergetics of mammalian Biochem Biophys 1988;266:111–123. sperm motility. In: Gagnon C, ed. Controls of sperm 216. Alberts B, Johnson A, Lewis J, et al. Cell chemistry and motility: biological and clinical aspects. Boca Raton, FL: biosynthesis. In: Alberts B, Johnson A, Lewis J, Raff CRC Press, 1990;175–202. M, Roberts K, Walter P, eds. Molecular biology of the 239. Marin S, Chiang K, Bassilian S, et al. Metabolic strategy cell. New York: Garland Science, 2002;47–127. of boar spermatozoa revealed by a metabolomic character- 217. Brooks DE, Mann T. Relation between the oxidation state ization. FEBS Lett 2003;20:342–346. of nicotinamide-adenine dinucleotide and the metabolism 240. Bishop DW. Oxygen concentration in the rabbit genital of spermatozoa. Biochem J 1972;129:1023–1034. tract, in Proceedings. 3rd Intern Congr Anim Reprod 218. Mann T, Lutwak-Mann C. Biochemistry of spermatozoa: 1956;53. chemical and functional correlations in ejaculated semen, 241. Mastroianni L, Jones R. Oxygen tension within the fallo- andrological aspects. In: Mann T, Lutwak-Mann C, eds. pian tube. J Reprod Fertil 1956;9:99. Male reproductive function and semen. New York: 242. Jones R, Mann T, Sherins R. Peroxidative breakdown of Springer-Verlag, 1981;195–268. phospholipids in human spermatozoa, spermicidal proper- 219. Storey BT, Kayne FJ. Energy metabolism of spermato- ties of fatty acid peroxides, and protective action of seminal zoa. V. The Embden-Myerhof pathway of glycolysis: plasma. Fertil Steril 1979;31:531–537. activities of pathway enzymes in hypotonically treated 243. Holland MK, Storey BT. Oxygen metabolism of mamma- rabbit epididymal spermatozoa. Fertil Steril lian spermatozoa. Generation of hydrogen peroxide by 1975;26:1257–1265. rabbit epididymal spermatozoa. Biochem J 1981;198: 220. Mann T. Metabolism of semen: fructolysis, respiration 273–280. and sperm energetics. In: Mann T, ed. The biochemis- 244. Alvarez JG, Storey BT. Assessment of cell damage try of semen and of the male reproductive tract. New York: caused by spontaneous lipid peroxidation in rabbit sper- Barnes and Noble, 1964;265–307. matozoa. Biol Reprod 1984;30:323–331. 221. Stryer L. Glycolysis. In: Stryer L, ed. Biochemistry. 245. Alvarez JG, Storey BT. Lipid peroxidation and the reac- New York: W.H. Freeman and Company, 1988;349–372. tions of superoxide and hydrogen peroxide in mouse sper- 222. Dringen R, Bergbauer K, Wiesinger H, et al. Utilization matozoa. Biol Reprod 1984;30:833–841. of mannose by astroglial cells. Neurochem Res 1994;19: 246. Aitken RJ, Clarkson JS. Significance of reactive oxygen 23–30. species and antioxidants in defining the efficacy of sperm 223. Mann T. Studies on the metabolism of preparation techniques. J Androl 1987;9:367–376. semen. 3. Fructose as a normal constituent of seminal 247. Hammerstedt RH, Lovrien RE. Calorimetric techniques plasma. Site of formation and function of fructose in semen. for metabolic studies of cells and organisms under normal Biochem J 1946;40:481. conditions and stress. J Exp Zool 1983;228:459–469. 224. Kosiniak K. Characteristics of the successive jets of ejac- 248. Inskeep PB, Hammerstedt RH. Endogenous metabolism ulated semen of stallions. J Reprod Fertil Suppl 1975;23: by sperm in response to altered cellular ATP requirements. 59–61. J Cell Physiol 1985;123:180–190. 225. Ponglowhapan S, Essen-Gustavsson B, Forsberg CL. 249. Ballester J, Fernandez-Novell JM, Rutlant J, et al. Evi- Influence of glucose and fructose in the extender during dence for a functional glycogen metabolism in mature long-term storage of chilled canine semen. Theriogenol- mammalian spermatozoa. Mol Reprod Dev 2000;56:207– ogy 2004;62:1498–1517. 219. 226. Rigau T, Farre M, Ballester J, et al. Effects of glucose and 250. Palomo MJ, Fernandez-Novell JM, Pena A, et al. Glu- fructose on motility patterns of dog spermatozoa from fresh cose- and fructose-induced dog-sperm glycogen synthesis ejaculates. Theriogenology 2001;56:801–815. shows specific changes in the location of the sperm glyco- 227. Williams AC, Ford WC. The role of glucose in supporting gen deposition. Mol Reprod Dev 2003;64:349–359. motility and capacitation in human spermatozoa. J An- 251. Urner F, Sakkas D. Characterization of glycolysis and drol 2001;22:680–695. pentose phosphate pathway activity during sperm entry 228. Nagai T, Yamaguchi K, Moriwaki C. Studies on the ef- into the mouse oocyte. Biol Reprod 1999;60:973–978. fects of sugars on washed human sperm motility. J Phar- 252. Urner F, Sakkas D. Involvement of the pentose phos- macobiodyn 1982;5:564–567. phate pathway and redox regulation in fertilization in the 229. Hoppe PC. Glucose requirement for mouse sperm capac- mouse. Mol Reprod Dev 2005;70:494–503. itation in vitro. Biol Reprod 1976;15:39–45. 253. Ford WCL, Whittington K, Williams AC. Reactive oxygen 230. Niwa K, Iritani A. Effect of various hexoses on sperm species in human sperm suspensions: production by leu- capacitation and penetration of rat eggs in vitro. J Re- kocytes and the generation of NADPH to protect sperm prod Fertil 1978;53:267–271. against their effects. Int J Androl 1997;20(Suppl 3):44– 231. Fraser LR, Quinn PJ. A glycolytic product is obligatory 49. for initiation of the sperm acrosome reaction and whiplash 254. Williams AC, Ford WCL. Functional significance of the motility required for fertiliziton in the mouse. J Reprod pentose phosphate pathway and glutathione reductase in Fertil 1981;61:25–35. the antioxidant defenses of human sperm. Biol Reprod 232. Sakkas D, Urner F, Menezo Y, et al. Effects of glucose 2004;71:1309–1316. and fructose on fertilization, cleavage, and viability of 255. Halliwell B, Gutteridge JMC. The chemistry of free rad- mouse embryos in vitro. Biol Reprod 1993;49:1288–1292. icals and related “reactive species”. In: Halliwell B, 233. Urner F, Sakkas D. Glucose participates in sperm-oocyte Gutteridge JMC, eds. Free radicals in biology and medi- fusion in the mouse. Biol Reprod 1996;55:917–922. cine. Oxford: Oxford University Press, 1999;36–104. 234. Urner F, Leppens-Luisier G, Sakkas D. Protein tyrosine 256. Alvarez JG, Storey BT. Role of glutathione peroxidase in phosphorylation in sperm during gamete interaction in the protecting mammalian spermatozoa from loss of motility mouse: the influence of glucose. Biol Reprod 2001;64: caused by spontaneous lipid peroxidation. Gamete Res 1350–1357. 1989;23:77–90.

AAEP PROCEEDINGS ր Vol. 53 ր 2007 159 MILNE LECTURE

257. Aitken RJ, Clarkson JS, Fishel S. Generation of reactive generation of reactive oxygen species. Mol Reprod Dev oxygen species lipid peroxidation and human sperm func- 1994;39:268–279. tion. Biol Reprod 1989;40:183–197. 279. Rao B, Soufir JC, Martin M, et al. Lipid peroxidation in 258. Aitken RJ. Free radicals, lipid peroxidation and sperm human spermatozoa as related to midpiece abnormalities function. Reprod Fertil Dev 1995;7:659–668. and motility. Gamete Res 1989;24:127–134. 259. Aitken RJ. Oxidative considerations relating to capacita- 280. Iwasaki A, Gagnon C. Formation of reactive oxygen spe- tion and the acrosome reaction: from studs to sterility. cies in spermatozoa of infertile patients. Fertil Steril Proc Havemeyer Foundation Workshop Stallion Reprod 1992;57:409–416. 2007;(in press). 281. Aitken RJ. Founders’ lecture. Human spermatozoa: 260. Baumber J, Ball BA, Gravance CG, et al. The effect of fruits of creation, seeds of doubt. Reprod Fertil Dev 2004; reactive oxygen species on equine sperm motility, viability, 16:655–664. acrosomal integrity, mitochondrial membrane potential, 282. Ball BA, Vo AT, Baumber J. Generation of reactive oxy- and membrane lipid peroxidation. J Androl 2000;21:895– gen species by equine spermatozoa. Am J Vet Res 2001; 902. 62:508–515. 261. Ford WCL. Reactive oxygen species and sperm. Hum 283. Krausz C, West K, Buckingham D, et al. Development of Fertil 2001;4:77–78. a technique for monitoring the contamination of human 262. Agarwal A, Prabakaran S, Allamaneni S. What an an- semen samples with leukocytes. Fertil Steril drologist/urologist should know about free radicals and 1992;57:1317–1325. why. Urology 2006;67:2–8. 284. Aitken RJ, West K, Buckingham D. Leukocytic infiltra- 263. Chandel NS, Budinger GRS. The cellular basis for dition into the human ejaculate and its association with verse responses to oxygen. Free Radic Biol Med 2007;42: semen quality, oxidative stress, and sperm function. J 165–174. Androl 1994;15:343–352. 264. Sikka SC, Rajasekaran M, Hellstrom WJG. Role of oxi- 285. Baumber J, Vo A, Sabeur K, et al. Generation of reactive dative stress and antioxidants in male infertility. J An- oxygen species by equine neutrophils and their effect on drol 1995;16:464–468. motility of equine spermatozoa. Theriogenology 2002;57: 265. De Iuliis GN, Wingate JK, Kopers AJ, et al. Definitive 1025–1033. evidence for the nonmitochondrial production of superox- 286. Jain S, Saxen D, Kumar PG, et al. NADPH dependent ide anion by human spermatozoa. J Clin Endrocrinol superoxide generation in the ovary and uterus of mice Metab 2006;91:1968–1975. during estrous cycle and early pregnancy. Life Sci 2000; 266. Burnaugh L, Sabeur K, Ball BA. Generation of superox- 66:1139–1146. ide anion by equine spermatozoa as detected by dihydro- 287. Norman JE, Cameron IT. Nitric oxide in the human ethidium. Theriogenology 2007;67:580–589. uterus. Rev Reprod 1996;1:61–68. 267. Ball BA, Baumber J, Sabeur K. Role of reactive oxygen 288. Huang Y, Roby KF, Pace JL, et al. Cellular localization species on normal and abnormal function of equine sper- and hormonal regulation of inducible nitric oxide synthase matozoa. Theriogenology 2002;58:299–300. in cycling mouse uterus. J Leukoc Biol 1995;57:27–35. 268. Banfi B, Molnar G, Maturana A, et al. A Ca2ϩ-activated 289. Hansen PJ, Hoggard MP, Rathwell AC. Effects of stallion NADPH oxidase in testis, spleen, and lymph nodes. J Biol seminal plasma on hydrogen peroxide release by leuko- Chem 2001;276:37594–37601. cytes exposed to spermatozoa and bacteria. J Reprod Im- 269. Armstrong JS, Bivalacqua TJ, Chamulitrat W, et al. munol 1987;10:157–166. A comparison of the NADPH oxidase in human sperm and 290. Kovalski NN, de Lamirande E, Gagnon C. Reactive oxy- white blood cells. Int J Androl 2002;25:223–229. gen species generated by human neutrophils inhibit sperm 270. Sabeur K, Ball BA. Detection of superoxide anion gener- motility: protective effect of seminal plasma and scaven- ation by equine spermatozoa. Am J Vet Res 2006;67:701– gers. Fertil Steril 1992;58:809–816. 706. 291. Smith R, Vantman D, Ponce J, et al. Total antioxidant 271. Richer SC, Ford WC. A critical investigation of NADPH capacity of human seminal plasma. Hum Reprod 1996;11: oxidase activity in human spermatozoa. Mol Hum Reprod 1655–1660. 2001;7:237–244. 292. Lewis SEM, Boyle PM, McKinney KA, et al. Total anti- 272. Ford WCL. Regulation of sperm function by reactive oxy- oxidant capacity of seminal plasma is different in fertile gen species. Hum Reprod Update 2004;10:387–399. and infertile men. Fertil Steril 1995;64:868–870. 273. Upreti GC, Jensen K, Munday R, et al. Studies on ram 293. Lewis SE, Steriling ESL, Young IS, et al. Comparison of spermatozoal aromatic amino acid oxidase. Proc Aust Soc individual antioxidants of sperm and seminal plasma in Reprod Biol 1994;26:115. fertile and infertile men. Fertil Steril 1997;67:142–147. 274. Tosic J, Walton A. Metabolism of spermatozoa. The for- 294. Aitken RJ, Clarkson JS. Cellular basis of defective sperm mation and elimination of hydrogen peroxide by spermato- function and its association with the genesis of reactive zoa and effects on motililty and survival. Biochem J 1950; oxygen species by human spermatozoa. J Reprod Fertil 47:199–212. 1987;81:459–469. 275. Shannon P, Curson B. Kinetics of the aromatic L-amino 295. Jones R, Mann T. Lipid peroxides in spermatozoa: acid oxidase from dead bovine spermatozoa and the effect formation, role of plasmalogen and physiological signifi- of catalase on fertility of diluted bovine semen stored at 5C cance. Proc R Soc B 1976;193:235–243. and ambient temperatures. J Reprod Fertil 1982;64:463– 296. Jones R, Mann T. Toxicity of exogenous fatty acid perox- 467. ides towards spermatozoa. J Reprod Fertil 1977;50:255– 276. Wai-sum O, Chen H, Chow PH. Male genital tract anti- 260. oxidant enzymes - Their abiilty to preserve sperm DNA 297. Jones R, Mann T, Sherins R. Peroxidative breakdown of integrity. Mol Cell Endocrinol 2006;250:80–83. phospholipids in human spermatozoa, spermicidal proper- 277. Gomez E, Buckingham DW, Brindle J, et al. Development ties of fatty acid peroxides and protective action of seminal of an image analysis system to monitor the retention of plasma. Fertil Steril 1979;31:531–537. residual cytoplasm by human spermatozoa: correlation 298. Lenzi A, Picardo M, Gandini L, et al. Lipids of the sperm with biochemical markers of the cytoplasmic space, oxida- plasma membrane: from polyunsaturated fatty acids con- tive stress, and sperm function. J Androl 1996;17:276– sidered as markers of sperm function to possible scavenger 287. therapy. Hum Reprod Update 1996;2:246–256. 278. Aitken RJ, Krausz C, Buckingham DW. Relationship be- 299. Ball BA, Vo A. Detection of lipid peroxidation in equine tween the biochemical marker for human sperm quality, spermatozoa based upon the lipophilic fluorescent dye C11- creatine kinase, and the mechanisms responsible for the BODIPY581/591. J Androl 2002;23:259–269.

160 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

300. Aitken RJ, Wingate JK, De Iuliis GN, et al. Cis-Unsatur- 323. Alvarez JG, Touchstone JC, Blasco L, et al. Spontaneous ated fatty acids stimulate reactive oxygen species generation lipid peroxidation and production of hydrogen peroxide and and lipid perooxidation in human spermatozoa. J Clin En- superoxide in human spermatozoa. J Androl 1987;8:338– docrinol Metab 2006;91:4154–4163. 348. 301. Armstrong JS, Rajasekaran M, Chamulitrat W, et al. 324. Bilodeau JF, Chatterjee S, Sirard MA, et al. Levels of Characterization of reactive oxygen species induced effects antioxidant defenses are decreased in bovine spermatozoa on human spermatozoa movement and energy metabolism. after a cycle of freezing and thawing. Mol Reprod Dev Free Radic Biol Med 1999;26:869–880. 2000;55:282–288. 302. Christova Y, James PS, Jones R. Lipid diffusion in sperm 325. Foote RH, Hare E. High catalase content of rabbit semen plasma membranes exposed to peroxidative injury from appears to be inherited. J Androl 2000;21:664–668. oxygen free radicals. Mol Reprod Dev 2004;68:365–372. 326. Ball BA, Gravance CG, Medina V, et al. Catalase activity 303. de Lamirande E, Gagnon C. Reactive oxygen species and in equine semen. Am J Vet Res 2000;61:1026–1030. human spermatozoa. I. Effects on the motility of intact 327. Aitken RJ, Buckingham D, Harkiss D. Use of a xanthine spermatozoa and on sperm axonemes. J Androl 1992;13: oxidase oxidant generating system to investigate the cyto- 368–378. toxic effects of reactive oxygen species on human sperma- 304. de Lamirande E, Gagnon C. Reactive oxygen species and tozoa. J Reprod Fertil 1993;97:441–450. human spermatozoa. II. Depletion of adenosine 328. Griveau JF, Dumont E, Renard P, et al. Reactive oxygen triphophate plays an important role in the inhibition of species, lipid peroxidation and enzymatic defence systems sperm motility. J Androl 1992;13:379–386. in human spermatozoa. J Reprod Fertil 1995;103:17–26. 305. Williams AC, Ford WCL. Relationship between reactive 329. Bilodeau JF, Blanchette S, Cormier N, et al. Reactive oxygen species production and lipid peroxidation in human oxygen species-mediated loss of bovine sperm motility in sperm suspensions and their association with sperm func- egg yolk Tris extender: protection by pyruvate, metal ch- tion. Fertil Steril 2005;83:929–936. elators and bovine liver or oviductal fluid catalase. Ther- 306. Dizdaroglu M, Jaruga P, Birincioglu M, et al. Free radi- iogenology 2002;57:1105–1122. cal-induced damage to DNA: mechanisms and measure- 330. Lapointe S, Sirard M-A. Catalase and oviductal fluid re- ment. Free Radic Biol Med 2002;32:1102–1115. verse the decreased motility of bovine sperm in culture 307. Aitken RJ, Baker MA. Oxidative stress, sperm survival medium containing specific amino acids. J Androl 1998;19: and fertility control. Mol Cell Endocrinol 2006;250:66– 31–36. 69. 331. Lapointe S, Sullivan R, Sirard M-A. Binding of a bovine 308. Aitken RJ, Koopman P, Lewis SE. Seeds of concern. oviductal fluid catalase to mammalian spermatozoa. Biol Nature 2004;432:48–52. Reprod 1998;58:747–753. 309. Sawyer DE, Mercer BG, Wiklendt AM, et al. Quantitative 332. Ball BA, Medina V, Gravance CG, et al. Effect of antioxidants on preservation of motility, viability and acrosomal analysis of gene-specific DNA damage in human sperma- integrity of equine spermatozoa during storage at tozoa. Mutat Res 2003;529:21–34. 5C. Theriogenology 2001;56:577–589. 310. Baumber J, Ball BA, Linfor JJ, et al. Reactive oxygen 333. Baumber J, Ball BA, Linfor JJ. Assessment of the cryo- species and cryopreservation promote deoxyribonucleic preservation of equine spermatozoa in the presence of en- acid (DNA) damage in equine sperm. Theriogenology zyme scavengers and antioxidants. Am J Vet Res 2005; 2003;58:301–302. 66:772–779. 311. Pagl R, Aurich C, Kankifer M. Anti-oxidative status and 334. Christova Y, James PS, Jones R. Lipid diffusion in sperm semen quality during cooled storage in stallion. J Vet plasma membranes exposed to peroxidative injury from Med A Physiol Pathol Clin Med 2006;53:486–489. oxygen free radicals. Mol Reprod Dev 2004;68:365–372. 312. Holland JK, Alverez JG, Storey BT. Production of super- 335. de Graaf SP, Evans G, Gillan L, et al. The influence of oxide and activity of superoxide dismutase in rabbit epi- antioxidant, cholesterol and seminal plasma on the in vitro didymal spermatozoa. Biol Reprod 1982;27:1109–1118. quality of sorted and non-sorted ram spermatozoa. Ther- 313. Storey BT. Biochemistry of the induction and prevention iogenology 2007;67:217–227. of lipoperoxidative damage in human spermatozoa. Mol 336. Krishnamoorthy G, Venkataraman P, Arunkumar A, et al. Hum Reprod 1997;3:203–213. Ameliorative effect of vitamins (alpha-tocopherol and 314. McLeod J. The role of oxygen in the metabolism and ascorbic acid) on PCB (Aroclor 1254) induced oxidative motility of human spermatozoa. Am J Physiol 1943;138: stress in rat epididymal sperm. Reprod Toxicol 2006;23: 512–518. 239–45. 315. Baumber J, Ball BA. Determination of glutathione perox- 337. Yazama F, Furuta K, Fujimoto M, et al. Abnormal spermat- idase and superoxide dismutase-like activities in equine sper- ogenesis in mice unable to synthesize ascorbic acid. Anat matozoa, seminal plasma, and reproductive tissues. Am J Sci Int 2006;81:115–125. Vet Res 2005;66:1415–1419. 338. Hatamoto LK, Baptista Sobrinho CA, Nichi M, et al. 316. Li T. The glutathione and thiol content of mammalian Effects of dexamethasone treatment (to mimic stress) and spermatozoa and seminal plasma. Biol Reprod 1975;12: Vitamin E oral supplementation on the spermiogram and 641–646. on seminal plasma spontaneous lipid peroxidation and an- 317. Drevet JR. The antioxidant glutathione peroxidase fam- tioxidant enzyme activities in dogs. Theriogenology 2006; ily and spermatozoa: a complex story. Mol Cell Endocri- 66:1610–1614. nol 2006;250:70–79. 339. Upreti GC, Jensen K, Oliver JE, et al. Motility of ram 318. Chance B, Sies H, Boveris A. Hydrogen peroxide metab- spermatozoa duirng storage in a chemically-defined di- olism in mammalian organs. Physiol Rev 1979;59:527– luent containing antioxidants. Anim Reprod Sci 1997;48: 605. 269–278. 319. Maiorino M, Ursini F. Oxidative stress, spermatogenesis 340. Almeida J, Ball BA. Effect of alpha-tocopherol and to- and fertility. Biol Chem 2002;383:591–597. copherol succinate on lipid peroxidation in equine sperma- 320. Stradaioli G, Rubei M, Zamparini M, et al. Enzymatic tozoa. Anim Reprod Sci 2005;87:321–337. evaluation of phospholipid hydroperoxide glutathione per- 341. Hansen SH, Andersen MD, Birkedal H, et al. The impor- oxidase (PHGPx) in equine spermatozoa. Anim Reprod tant role of taurine in oxidative metabolism. Adv Exp Sci 2006;94:29–31. Med Biol 2006;583:129–135. 321. Foote RH. Catalase content of rabbit, ram, bull and boar 342. Alverez JG, Storey BT. Taurine, hypotaruine, epineph- semen. J Anim Sci 1962;21:966–968. rine and albumin inhibit lipid peroxidation in rabbit sper- 322. White IG. Studies on semen. Aust J Biol Sci 1959;21: mtozoa and protect against loss of motility. Biol Reprod 208–214. 1983;29:548–555.

AAEP PROCEEDINGS ր Vol. 53 ր 2007 161 MILNE LECTURE

343. Twigg J, Fulton N, Gomez E, et al. Analysis of the impact uterine inflammatory response in the mare. Theriogenol- of intracellular reactive oxygen species generation on the ogy 2007;67:556–562. structural and functional integrity of human spermatozoa: 364. Bader H. An investigation of sperm migration into the lipid peroxidation, DNA fragmentation and effectiveness of oviducts of the mare. J Reprod Fertil Suppl 1982;32:59– antioxidants. Hum Reprod 1998;13:1429–1436. 64. 344. Funahashi H, Sano T. Select antioxidants improve the 365. Brinsko SP, Varner DD, Blanchard TL. The effect of uter- function of extended boar semen stored at 10 degrees ine lavage performed four hours post insemination on preg- C. Theriogenology 2005;63:1605–1616. nancy rate in mares. Theriogenology 1991;35:1111–1117. 345. Bilodeau JF, Blanchette S, Gagnon C, et al. Thiols pre- 366. Rigby S, Derczo S, Brinsko SP, et al. Oviductal sperm vent hydrogen peroxide-mediated loss of sperm motility in numbers following proximal uterine horn or uterine body cryopreserved bull semen. Theriogenology 2001;56:275– insemination. In Proceedings. Am Assoc Equine Practi- 286. tioners 2000;46:332–334. 346. Yang R, Le G, Li A, et al. Effect of antioxidant capacity on 367. Scott MA, Liu IKM, Overstreet JW. Sperm transport to blood lipid metabolism and lipoprotein lipase activity of oviducts: abnormalities and their clinical implications. rats fed a high-fat diet. Nutrition 2006;22:1185–1191. In Proceedings. Am Assoc Equine Practitioners 1995;41: 347. Prahalathan C, Selvakumar E, Varalakshmi P. Modula- 1–2. tory role of lipoic acid on adriamycin-induced testicular 368. Parker WG, Sullivan JJ, First NL. Sperm transport and injury. Chem Biol Interact 2006;160:108–114. distribution in the mare. J Reprod Fertil Suppl 1975;23: 348. Selvakumar E, Prahalathan C, Sudharsan PT, et al. 63–66. Chemoprotective effect of lipoic acid against cyclophosph- 369. Rigby S, Hill J, Miller C, et al. Administration of oxytocin amide-induced changes in the rat sperm. Toxicology immediately after insemination does not improve preg- 2006;217:71–78. nancy rates in mares bred by fertile or subfertile stallions. 349. Bruemmer JE, Coy RC, Squires EL, et al. Effect of pyru- Theriogenology 1998;51:1143–1150. vate on the function of stallion spermatozoa stored for up to 370. Haluska GJ, Currie WB. Variation in plasma concentra- 48 hours. J Anim Sci 2002;80:12–18. tions of oestradiol-17 beta and their relationship to those of 350. Denniston DJ, Squires EL, Bruemmer JE, et al. The ef- progesterone, 13,14-dihydro-15-keto-prostaglandin F2 al- fect of antioxidants on the motility and viability of cooled pha and oxytocin across pregnancy and at parturition in stallion spermatozoa. J Reprod Fertil 2000;56(Suppl): pony mares. J Reprod Fertil 1988;84:635–646. 121–126. 371. Allen WE, Chard T, Forsling ML. Peripheral plasma lev- 351. Lopez-Burillo S, Tan DX, Mayo JC, et al. Melatonin, xan- els of oxytocin and vasopressin in the mare during partu- thurenic acid, resveratrol, EGCG, vitamin C and alpha- rition. J Endocrinol 1973;57:175–176. lipoic acid differentially reduce oxidative DNA damage 372. Langendijk P, Bouwman EG, Kidson A, et al. Role of myometrial activity in sperm transport through the genital induced by Fenton reagents: a study of their individual tract and in fertilization in sows. Reproduction 2002;123: and synergistic actions. J Pineal Res 2003;34:269–277. 683–690. 352. Dokmeci D. Oxidative stress, male infertility and the role 373. Stratman FW, Self HL, Smith VR. The effect of oxytocin of carnitines. Folia Med (Plovdiv) 2005;47:26–30. on the fertility in gilts artificially inseminated with a low 353. Stradaioli G, Sylla L, Zelli R, et al. Effect of L-carnitine sperm concentration and semen volume. J Anim Sci administration on the seminal characteristics of oligoast- 1959;18:634–640. henospermic stallions. Theriogenology 2004;62:761–777. 374. Penrod L, Adams K, Arns MJ. The influence of exogenous 354. Mann T. Biochemistry of stallion semen. J Reprod Fer- prostaglandin F2-alpha on spermatozoa motility and fertil Suppl 1975;23:47–52. tility. Anim Reprod Sci 2006;94:36–38. 355. Upreti GC, Jensen K, Munday R, et al. Studies on aro- 375. Claus R, Schams D. Influence of mating and intra-uterine matic amino acid oxidase activity in ram spermatozoa: oestradiol infusion on peripheral oxytocin concentration in role of pyruvate as an antioxidant. Anim Reprod Sci the sow. J Endocrinol 1990;126:361–365. 1998;29:275–287. 376. Langendijk P, Bouwman EG, Soede NM, et al. Myome- 356. Agarwal A, Sushil A, Prabakaran A, et al. Prevention of trial activity around estrus in sows: spontaneous activity oxidative stress injury to sperm. J Androl 2005;26:654– and effects of estrogens, cloprostenol, seminal plasma and 660. clenbuterol. Theriogenology 2002;57:1563–1577. 357. Ford WCL, Whittington K. Antioxidant treatment for male 377. Langendijk P, Soede NM, Kemp B. Uterine activity, subfertility: a promise that remains unfulfilled. Hum Re- sperm transport, and the role of boar stimuli around in- prod 1998;13:1416–1419. semination in sows. Theriogenology 2005;63:500–513. 358. Donnelly ET, McClure N, Lewis SE. The effect of ascorbate 378. Stecco R, Paccamonti D, Gutjahr S, et al. Day of cycle and alpha-tocopherol supplementation in vitro on DNA integ- affects changes in equine intrauterine pressure in response rity and hydrogen peroxide-induced DNA damage in human to teasing. Theriogenology 2003;60:727–733. spermatozoa. Mutagenesis 1999;14:505–512. 379. Nikolakopoulos E, Kindahl H, Gilbert CL, et al. Release 359. Milzer E, Schmidt HL. Carbon isotope effects on the de- of oxytocin and prostaglandin F2 alpha around teasing, carboxylation of carboxylic acids: comparison of the lac- natural service and associated events in the mare. Anim tate oxidase reaction and the degradation of pyruvate by Reprod Sci 2000;63:89–99. hydrogen peroxide. Biochem J 1988;252:913–915. 380. Madill S, Troedsson MHT, Alexander SL, et al. Simulta- 360. Katila T, Sankari S, Ma¨kela O. Transport of spermatozoa neous recording of pituitary oxytocin secretion and myome- in the reproductive tracts of mares. J Reprod Fertil Suppl trial activity in oestrous mares exposed to carious breeding 2000;56:571–578. stimuli. J Reprod Fertil Suppl 2000;56:351–361. 361. Sinnemma L, Jarvimaa T, Lehmonen N, et al. Effect of 381. Alexander SL, Irvine CHG, Shand N, et al. Is luteinizing insemination volume on uterine contractions and inflam- hormone secretion modulated by endogenous oxytocin in matory response and on elimination of semen in the mare the mare? Studies on the role of oxytocin and factors af- uterus- scintigraphic and ultrasonographic studies. J Vet fecting its secretion in estrous mares. Biol Reprod Med 2005;52:466–471. Monogr 1995;1:361–371. 362. Campbell MLH, England GCW. Effects of coitus and the 382. Taverne MA, Van der Weyden GC, Rontijne P, et al. artificial insemination of diffrent volumes of fresh semen In vivo myometrial electrical activity in the cyclic mare. on uterine contractions in mares. Vet Rec 2006;159:843– J Reprod Fertil 1979;56:521–532. 849. 383. Campbell MLH, England GCW. M-mode ultrasound im- 363. Fiala SM, Pimentel CA, Mattos ALG, et al. Effect of aging of the contractions of the equine uterus. Vet Rec sperm numbers and concentration on sperm transport and 2002;150:575–577.

162 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

384. Mann T, Polge C, Rowson LEA. Participation of seminal 404. Wood J, Bergfelt DR, Ginther OJ. Effects of time of in- plasma during the passage of spermatozoa in the female semination relative to ovulation on pregnancy rate and reproductive tract of the pig and horse. J Endocrinol embryonic-loss rate in mares. Equine Vet J 1990;22:410– 1956;13:133–140. 415. 385. Claus R, Hoang-Vu C, Ellendorf F, et al. Seminal oestro- 405. Bain AM. Thoroughbred mare in australasia. JAmVet gens in the boar: original and functions in the sow. J Med Assoc 1957;131:179–185. Steroid Biochem 1987;27:331–335. 406. Dobrinski I, Ignotz GG, Thomas PGA, et al. Role of car- 386. Willenburg KL, Miller GM, Rodriguez-Zas SL, et al. bohydrates in the attachment of equine spermatozoa to Influence of hormone supplementation to extended semen uterine tubal (oviductal) epithelial cells in vitro. Am J Vet on artificial insemination, uterine contractions, establish- Res 1996;57:1635–1639. ment of a sperm reservoir, and fertility in swine. J Anim 407. Ekhasi-Hundrieser M, Gohr K, Wagner A, et al. Sper- Sci 2003;81:821–829. madhesin AQN1 is a candidate receptor molecule involved in 387. Troedsson MHT, Liu IKM, Crabo BG. Sperm transport the formation of the oviductal sperm reservoir in pig. Biol and survival in the mare: a review. Theriogenology Reprod 2005;73:536–545. 1998;50:807–818. 408. Gwathmey TYM, Ignotz GG, Suarez SS. PDC-109 (BSP- 388. Troedsson MHT, Desvousges A, Alghamdi AS, et al. A1/A2) promotes bull sperm binding to oviductal sperm Components in seminal plasma regulating sperm transport release. Biol Reprod 2003;69:809–815. and elimination. Anim Reprod Sci 2005;89:171–186. 409. Marini PE, Cabada MO. One step purification and bio- 389. Troedsson MHT, Alghamdi AS, Mattisen J. Equine sem- chemical characterization of a spermatozoa-binding pro- inal plasma protects the fertility of spermatozoa in an tein from porcine oviductal epithelial cells. Mol Reprod inflamed uterine environment. Theriogenology 2002;58: Dev 2003;66:383–390. 453–456. 410. Perez FA, Roma SM, Cabada MO, et al. Sperm binding 390. Clement F, Vincent P, Mahla R, et al. Which insemina- glycoprotein is differentially present surrounding the lution results in fertilization when several are performed men of isthmus and ampulla of the pig’s oviduct. Anat before ovulation? J Reprod Fertil Suppl 2000;56:579–585. Embryol (Berl) 2006;211:619–624. 391. Metcalf ES. The effect of postinsemination endometritis 411. Ignotz GG, Lo MC, Perez CL, et al. Characterization of a on fertility of frozen stallion semen. In Proceedings.Am fucose-binding protein from bull sperm and seminal Assoc Equine Practitioners 2000;46:330–331. plasma that may be responsible for formation of the ovi- 392. Scott MA. A glimpse at sperm function in vivo: sperm ductal sperm reservoir. Biol Reprod 2006;64:1806–1811. transport and epithelial interaction in the female reproduc- 412. Gwathmey TYM, Ignotz GG, Mueller JL, et al. Bovine tive tract. Anim Reprod Sci 2000;60–61:337–348. seminal plasma proteins PDC-109, BSP-A3, and BSP-30- 393. Gaddum-Rosse P. Some observations on sperm transport kDa share functional roles in storing sperm in the oviduct. through the uterotubal junction of the rat. Am J Anat Biol Reprod 2006;75:501–507. 1981;160:333–341. 413. Sabeur K, Ball BA. Characterization of galactose-binding 394. Scott MA, Liu IKM, Overstreet JW, et al. The structural proteins in equine testis and spermatozoa. Anim Reprod morphology and epithelial association of spermatozoa at Sci 2007;101:74–84. the uterotubal junction: a descriptive study of equine 414. Ellington JE, Ignotz GG, Miller PG, et al. Oviduct epithe- spermatozoa in situ using scanning electron microsco- lial cell co-culture modifies stallion and bull sperm cell py. J Reprod Fertil Suppl 2000;56:415–421. surface proteins. Biol Reprod 1993;48(Suppl 1):107. 395. Nilsson O, Reinius S. Light and electron microscope 415. Ellington JE, Ball BA, Blue BJ, et al. Capacitation-like structure of the oviduct. In: Hafez ESE, Blandau RJ, membrane changes and prolonged viability in vitro of eds. The mammalian oviduct: comparative biology and equine spermatozoa cultured with uterine tube epithelial methodology. Chicago: University of Chicago Press, cells. Am J Vet Res 1993;54:1505–1510. 1969;57–83. 416. Ellington JE, Ball BA, Yang X. Binding of stallion sper- 396. Cho C, Bunch DO, Faure JE, et al. Fertilization defects in matozoa to the equine zona pellucida after coculture with sperm from mice lacking fertilin b. Science oviductal epithelial cells. J Reprod Fertil 1993;98:203– 1998;281:1857–1859. 208. 397. Hagaman JR, Moyer JS, Bachman ES, et al. Angiotensin- 417. Smith TT, Yanagimachi R. Attachment and release of converting enzyme and male fertility. Proc Natl Acad Sci spermatozoa from the caudal isthmus of the hamster ovi- USA 1998;95:2552–2557. duct. J Reprod Fertil 1991;91:567–573. 398. Hunter RHF, Flechon B, Flechon JE. Distribution, mor- 418. Pollard JW, Plante C, King WA, et al. Fertilizing capacity phology and epithelial interactions of bovine spermatozoa of bovine sperm may be maintained by binding of oviductal in the oviduct before and after ovulation: a scanning elec- epithelial cells. Biol Reprod 1991;44:102–107. tron microscope study. Tissue Cell 1991;23:641–656. 419. Dobrinski I, Smith TT, Suarez SS, et al. Membrane contact 399. Larsson B. Distribution of spermatozoa in the genital with oviductal epithelium modulates the intracellular cal- tract of heifers inseminated with large numbers of abnor- cium concentration of equine spermatozoa in vitro. Biol Re- mal spermatozoa. J Vet Med 1988;35:721–728. prod 1997;56:861–869. 400. Waberski D, Magnus F, Ardon F, et al. Binding of boar 420. Smith TT, Nothnick WB. Role of direct contact between spermatozoa to oviductal epithelium in vitro in relation to spermatozoa and oviductal epithelial cells in maintaining sperm morphology and storage time. Reproduction 2006; rabbit sperm viability. Biol Reprod 1997;56:83–89. 131:1741–7899. 421. Thomas PG, Ignotz GG, Ball BA, et al. Effect of coculture 401. Thomas PGA, Ball BA, Miller PG, et al. A subpopulation with stallion spermatozoa on de novo protein synthesis and of morphologically normal, motile spermatozoa attach to secretion by equine oviduct epithelial cells. Am J Vet Res equine oviductal epithelial cell monolayers. Biol Reprod 1995;56:1657–1662. 1994;51:303–309. 422. Killian GJ. Evidence for the role of oviduct secretions 402. Thomas PGA, Ball BA, Brinsko SP. Interaction of equine in sperm function, fertilization and embryo development. spermatozoa with oviduct epithelial cell explants is afffected Anim Reprod Sci 2004;82–83:82–83. by estrous cycle and anatomic orgin of explant. Biol Reprod 423. Ellington JE, Ignotz GG, Ball BA, et al. De novo protein 1994;51:222–228. synthesis by bovine uterine tube (oviduct) epithelial cells 403. Scott MA, Varner DD, Liu IKM, et al. Presumptive evi- changes during co-culture with bull spermatozoa. Biol dence of a preovulatory sperm reservoir in the mare: mor- Reprod 1993;48:851–856. phological investigations using scanning electron 424. Fazeli A, Elliott RMA, Duncan AE, et al. In vitro main- microscopy. Theriogenology 2002;58:639–642. tenance of boar sperm viability by a soluble fraction ob-

AAEP PROCEEDINGS ր Vol. 53 ր 2007 163 MILNE LECTURE

tained from oviductal apical plasma membrane 452. Nagdas SK, Winfrey VP, Olson GE. Tyrosine phosphory- preparations. Reproduction 2003;125:509–517. lation generates multiple isoforms of the mitochondrial 425. Ellington JE, Ignotz GG, Varner DD, et al. In vitro inter- capsule protein, phospholipid hydroperoxide glutathione per- action between oviduct epithelia and equine sperm. Arch oxidase (PHGPx), during hamster sperm capacitation. Biol Androl 1993;31:79–86. Reprod 2004;72:164–171. 426. Pacey AA, Davies N, Warren MA, et al. Hyperactivation 453. Green S, Fishel S. Morphology comparison of individually may assist human spermatozoa to detach from intimate selected hyperactivated and non-hyperactivated human association with the endosalpinx. Hum Reprod 1995;10: spermatozoa. Hum Reprod 1999;14:123–130. 2603–2609. 454. Harrison RA, Ashworth PJ, Miller NG. Bicarbonate/CO2, 427. Topfer-Petersen E. Molecules on the sperm route to fer- an effector of capacitation, induces a rapid and reversible tilization. J Exp Zool 1999;285:259–266. change in the lipid architecture of boar sperm plasma 428. Austin CR. Observations on the penetration of the sperm membranes. Mol Reprod Dev 1996;45:378–391. into the mammalian egg. Aust J Biol Sci 1951;4:581–596. 455. Rath R, Colenbrander B, Bevers MM, et al. Evaluation on 429. Chang MC. Fertilizing capacity of spermatozoa deposited in vitro capacitation of stallion spermatozoa. Biol Reprod into fallopian tubes. Nature 1951;168:697–698. 2001;65:462–470. 430. Austin CR. Capacitation and the release of hyaluronidase 456. Meyers SA, Rosenberger AE. A plasma membrane-asso- from spermatozoa. J Reprod Fertil 1960;3:310–311. ciated hyaluronidase is localized to the posterior acrosomal 431. Yanagimachi R. Mammalian fertilization. In: Knobil region of stallion sperm and is associated with spermatozal E, Neill JD, eds. The physiology of reproduction. New function. Biol Reprod 1999;61:444–451. York: Raven Press, 1994;189–317. 457. Jimenez I, Gonzalez-Marquez H, Ortiz R, et al. Changes 432. Talbot P. Sperm penetration through oocyte investments in the distribution of lectin receptors during capacitation in mammals. Am J Anat 1985;174:331–346. and acrosome reaction in boar spermatozoa. Theriogenol- 433. Corselli J, Talbot P. In vitro penetration of hamster oo- ogy 2003;59:1171–1180. cyte-cumulus complexes using physiological numbers of 458. Cross NL. Decrease in order of human sperm lipids dur- sperm. Dev Biol 1987;122:227–242. ing capacitation. Biol Reprod 2003;69:529–534. 434. Cardullo RA, Thaler CD. Function of the egg’s extracel- 459. Smith TT, McKinnon-Thompson CA, Wolf DE. Changes lular matrix. In: Hardy DH, ed. Fertilization. San in lipid diffusibility in the hamster sperm head plasma Diego: Academic Press, 2002;119–152. membrane during capacitation in vivo and in vitro. Mol 435. Visconti PE, Bailey JL, Moore GD, et al. Capacitation of Reprod Dev 1998;50:86–92. mouse spermatozoa. I. Correlation between the capaci- 460. Benoff S. Carbohydrates and fertilization: an overview. tation state and protein tyrosine phosphorylation. Devel- Mol Hum Reprod 1997;3:599–637. opment 1995;121:1129–1137. 461. Cross NL, Overstreet JW. Glycoconjugates of the human sperm surface: distribution and alterations that accom- 436. Yanagimachi R. Sperm capacitation and gamete interac- pany capacitation in vitro. Gamete Res 1987;16:23–35. tion. J Reprod Fertil 1989;38:27–33. 462. Zeng Y, Clark EN, Florman HM. Sperm membrane pro- 437. Austin CR. Capacitation of spermatozoa. Int J Fertil tential: hyperpolarization during capacitation regulates 1967;12:25–31. zona pellucida-dependent acrosomal secretion. Dev Biol 438. Bedford JM. Sperm capacitation and fertilization in 1995;171:554–563. mammals. Biol Reprod Suppl 1970;2:128–158. 463. Visconti PE, Moore GD, Bailey JL, et al. Capacitation of 439. Yanagimachi R. Mechanisms of fertilization in mammals. mouse spermatozoa. II. Protein tyrosine phosphoryla- In: Mastroianni L, Biggers JD, eds. Fertilization and tion and capacitation are regulated by a cAMP-dependent embryonic development in vitro. New York: Plenum pathway. Development 1995;121:1139–1150. Press, 1981;81–182. 464. Piehler E, Petrunkina AM, Ekhlasi-Hundrieser M, et al. 440. Yanagimachi R. In vitro capacitation of hamster sperma- Dynamic quantification of the tyrosine phosphorylation of tozoa by follicular fluid. J Reprod Fertil 1969;18:275–286. the sperm surface proteins during capacitation. Cytom- 441. Suarez SS, Ho HC. Hyperactivation of mammalian etry A 2006;69:1062–1070. sperm. Cell Mol Biol 2003;49:351–356. 465. Linares-Hernandez L, Guzman-Grenfell AM, Hicks-Gomez 442. Mortimer ST, Maxwell WM. Kinematic definition of ram JJ, et al. Voltage-dependent calcium influx in human sperm hyperactivation. Reprod Fertil Dev 1999;11:25–30. sperm assessed by simultaneous optical detection of intra- 443. Chang MC. The meaning of sperm capacitation: a his- cellular calcium and membrane potential. Biochim Bio- torical perspective. J Androl 1984;5:45–50. phys Acta 1998;1372:1–12. 444. Kopf GS. Signal transduction mechanisms regulating 466. Bray C, Son JH, Meizel S. Acetylcholine causes an in- sperm acrosomal exocytosis. In: Hardy DM, ed. Fertil- crease of intracellular calcium in human sperm. Mol ization. San Diego: Academic Press, 2002;181–223. Hum Reprod 2005;11:881–889. 445. DeMott RP, Suarez SS. Hyperactivated sperm progress 467. Fraire-Zamora JJ, Gonzalez-Martinez MT. Effect of in- in the mouse oviduct. Biol Reprod 1992;46:779–785. tracellular pH on depolarization-evoked calcium influx in 446. Stauss CR, Suarez SS. Sperm motility hyperactivation human sperm. Am J Physiol Cell Physiol 2004;287:1688– facilitates penetration of the hamster zona pellucida. Biol 1696. Reprod 1995;53:1280–1285. 468. Neri-Vidaurri PD, Torres-Flores V, Gonzalez-Martinez 447. Suarez SS, Dai X. Intracellular calcium reaches different MT. A remarkable increase in the pHi sensitivity of volt- levels of elevation in hyperactivated and acrosome reacted age-dependent calcium channels occurs in human sperm hamster sperm. Mol Reprod Dev 1995;42:325–333. incubated in capacitating conditions. Biochem Biophys 448. Suarez SS, Katz DF, Owen DH, et al. Evidence for the Res Commun 2006;343:105–109. function of hyperactivated motility in sperm. Biol Reprod 469. Garcia MA, Meizel S. Regulation of intracellular pH in ca- 1991;44:375–381. pacitated human spermatozoa by a Naϩ/Hϩ exchanger. Mol 449. Robertson L, Wolf DP, Tash JS. Temporal changes in Reprod Dev 1999;52:189–195. motility parameters related to acrosomal status: identifi- 470. Harrison RA, Mairet B, Miller NG. Flow cytometric stud- cation and characterization of populations of hyperacti- ies of bicarbonate-mediated Ca2ϩ influx in boar sperm pop- vated human sperm. Biol Reprod 1988;39:797–805. ulations. Mol Reprod Dev 1993;35:197–208. 450. Mortimer ST, Swan MA, Mortimer D. Effect of seminal 471. Magistrini M, Guitton E, Levern Y, et al. New staining plasma on capacitation and hyperactivation in human methods for sperm evaluation estimated by microscopy and spermatozoa. Hum Reprod 1998;13:2139–2146. flow cytometry. Theriogenology 1997;48:1229–1235. 451. Breitbart H. Signaling pathways in sperm capacitation 472. Gadella BM, Harrison RAP. Capacitation induces cyclic and acrosome reaction. Cell Mol Biol 2003;49:321–327. adenosine 3Ј,5Ј-monophosphate-dependent, but apoptosis-

164 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

unrelated, exposure of aminophospholipids at the apical 494. Villemure M, Lazure C, Manjunath P. Isolation and char- head plasma membrane of boar sperm cells. Biol Reprod acterization of gelatin-binding proteins from goat seminal 2002;67:340–350. plasma. Reprod Biol Endocrinol 2003;1:39. 473. Colenbrander B, Brouwers JFHM, Neild DM, et al. Ca- 495. Parks JE, Hough SR. Platelet-activating factor acetylhy- pacitation dependent lipid rearrangements in the plasma drolase activity in bovine seminal plasma. J Androl 1993; membrane of equine sperm. Theriogenology 2002;58:341– 14:335–339. 345. 496. Huang YH, Chen YH, Lin CM, et al. Suppression effect of 474. Flesch FM, Brouwers JFHM, Nievelstein PFEM, et al. seminal vesicle autoantigen on platelet-activating factor- Bicarbonate stimulated phospholipid scrambling induces induced mouse sperm capacitation. J Cell Biochem 2006. cholesterol redistribution and enables cholesterol depletion 497. Therien I, Moreau R, Manjunath P. Major proteins of in the sperm plasma membrane. J Cell Sci 2001;114: bovine seminal plasma and high-density lopoprotein in- 3543–3555. duce cholesterol efflux from epididymal sperm. Biol Re- 475. Flesch FM, Colenbrander B, vanGolde LMG, et al. Ca- prod 1998;59:768–776. pacitation induces tyrosine phosphorylation of proteins in 498. Lusignan M-F, Bergeron A, Crete M-H, et al. Induction of the boar sperm plasma membrane. Biochem Biophys Res epididymal boar sperm capacitation by pB1 and BSP- Commun 1999;262:787–792. A1/-A2 proteins, members of the BSP protein family. Biol 476. Gadella BM, Lopes-Cardozo M, vanGolde LMG, et al. Reprod 2006;76:424–32. Glycolipid migration from the apical to the equatorial sub- 499. Topfer-Petersen E, Ekhasi-Hundrieser M, Kirchhoff C, domains of the sperm head plasma membrane precedes the et al. The role of stallion seminal proteins in fertilisa- acrosome reaction. Evidence for a primary capacitation tion. Anim Reprod Sci 2005;89:159–170. event in boar spermatozoa. J Cell Sci 1995;108:935–946. 500. Nolan MA, Wu L, Bang HJ, et al. Identification of rat 477. Pommer AC, Rutlant J, Meyers SA. Phosphorylation of cysteine-rich secretory protein 4 (Crisp4) as the Ortholog to protein tyrosine residues in fresh and cryopreserved stal- human CRISP1 and mouse CRISP4. Biol Reprod 2006; lion spermatozoa under capacitating conditions. Biol Re- 74:984–991. prod 2003;68:1208–1214. 501. Jalkane J, Huhtaniemi I, Poutanen M. Mouse systeine- 478. Jaiswal BS, Eisenbach M. Capacitation. In: Hardy rich secretory protein 4 (CRISP4): a member of the DM, ed. Fertilization. San Diego: Academic Press, CRISP family exclusively expressed in the epididymis in an 2002;57–117. androgen-dependent manner. Biol Reprod 2005;72:1268– 479. Florman HM, Ducibella T. Fertilization in mammals. 1274. In: Neill JD, ed. Knobel and Neill’s physiology of repro- 502. Udby L, Bjartell A, et al. Characterization and localiza- duction. Boston: Elsevier, 2006;55–112. tion of cysteine-rich secretory protein 3 (CRISP-3) in the 480. Felix R. Molecular physiology and pathology of Ca2ϩ-con- human male reproductive tract. J Androl 2005;26:333. ducting channels in the plasma membrane of mammalian 503. Schambony A, Hess O, Gentzel M, et al. Expression of sperm. Reproduction 2005;129:251–262. CRISP proteins in the male equine genital tract. J Re- 481. Gadella BM, Rathi R, Brouwers JFHM, et al. Capacita- prod Fertil Suppl 1998;53:67–72. tion and the acrosome reaction in equine sperm. Anim 504. Evans JP. The molecular basis of sperm-oocyte mem- Reprod Sci 2001;68:249–265. brane interactions during mammalian fertilization. Hum 482. Thundathil JC, Anzar M, Buhr MM. Naϩ/Kϩ ATPase as a Reprod Update 2002;8:297–311. signaling molecule during bovine sperm capacitation. Biol 505. Roberts KP, Wamstad JA, Ensrud KM, et al. Inhibition of Reprod 2006;75:308–317. capacitation-associated tyrosine phosphorylation signaling 483. Langlais J, Roberts KD. A molecular membrane model of in rat sperm by epididymal protein CRISP-1. Biol Reprod sperm capacitation and the acrosome reaction of mamma- 2003;69:572–581. lian spermatozoa. Gamete Res 1985;12:183–224. 506. Boatman EE, Robbins RS. Bicarbonate: Carbon-dioxide 484. Aitken RJ. Molecular mechanisms regulating human regulation of sperm capacitation, hyperactivated motility, sperm function. Mol Hum Reprod 1997;3:169–173. and acrosome reactions. Biol Reprod 1991;44:806–813. 485. O’Flaherty C, deLamirande E, Gagnon C. Positive role 507. Cross NL. Effect of cholesterol and other sterols on hu- of reactive oxygen species in mammalian sperm capaci- man sperm acrosomal responsiveness. Mol Reprod Dev tation: triggering and modulation of phosphorylation 1996;45:212–217. events. Free Radic Biol Med 2006;41:528–540. 508. Cross NL. Role of cholesterol in sperm capacitation. 486. deLamirande E, Leclerc P, Gagnon C. Capacitation as a Biol Reprod 1998;59:7–11. regulatory event that primes spermatozoa for the acrosome 509. DasGupta S, Mills EL, Fraser LR. Ca2ϩ-related changes reaction and fertilization. Mol Hum Reprod 1997;3:175– in the capacitation state of human spermatozoa assessed 194. by a chlortetracycline fluorescence assay. J Reprod Fertil 487. Darszon A, Acevedo JJ, Galindo BE, et al. Sperm channel 1993;99:135–143. diversity and functional multiplicity. Reproduction 2006; 510. Davis BK, Byrne R, Hungund B. Studies on the mecha- 131:977–988. nism of capacitation. II. Evidence for lipid transfer be- 488. Jha KN, Kameshwari DB, Shivaji S. Role of signaling tween plasma membrane of rate sperm and serum albumin pathways in regulating the capacitation of mammalian during capacitation in vitro. Biochem Biophys Acta 1979;- spermatozoa. Cell Mol Biol 2003;49:329–340. 257. 489. Visconti PE, Kopf GS. Regulation of protein phosphory- 511. Davis BK, Byrne R, Bedigian K. Studies on the mecha- lation during sperm capacitation. Biol Reprod 1998;59: nism of capacitation: albumin-mediated changes in 1–6. plasma membrane lipids during in vitro incubation of rat 490. Mitra K, Shivaji S. Proteins implicated in sperm capaci- sperm cells. Proc Natl Acad Sci USA 1980;77:1546–1550. tation. Indian J Exp Biol 2005;43:1001–1015. 512. Davis BK. Timing of fertilization in mammals: Sperm 491. Chang MC. A detrimental effect of seminal plasma on the cholesterol/phospholipid ratio as a determinant of the ca- fertilizing capacity of sperm. Nature 1957;175:258–259. pacitation interval. Proc Natl Acad Sci USA 492. Fraser LR. Interactions between a decapacitation factor 1981;78:7560–7564. and mouse spermatozoa appear to involve fucose residues 513. Gadella BM, Harrison RAP. The capacitating agent bi- and GPI-anchored receptor. Mol Reprod Dev carbonate induces protein kinase A-dependent changes in 1998;51:193–202. phospholipid transbilayer behavior in the sperm plasma 493. Lopes CH, Mazzini MN, Tortorella H, et al. Isolation, membrane. Development 2000;127:2407–2420. partial characterization and biological activity of mannosyl 514. Go KJ, Wolf DP. Albumin-mediated changes in sperm glycopeptides from seminal plasma. Glycoconj J 1998;15: sterol content during capacitation. Biol Reprod 1985;32: 477–481. 145–153.

AAEP PROCEEDINGS ր Vol. 53 ր 2007 165 MILNE LECTURE

515. Harrison RAP. Capacitation mechanisms, and the role of 534. Wennermuth G, Babcock DF, Hille B. Calcium clearance capacitation as seen in eutherian mammals. Reprod Fer- mechanisms of mouse sperm. J Gen Physiol 2003;122: til Dev 1996;8:581–594. 115–128. 516. Lee MA, Storey BT. Bicarbonate is essential for fertiliza- 535. Breitbart H, Webbie R, Lardy H. Regulation of calcium tion of mouse eggs: mouse sperm require it to undero the transport in bovine spermatozoa. Biochim Biophys Acta acrosome reaction. Biol Reprod 1986;34:349–356. 1990;1027:72–78. 517. Okamura N, Tajima Y, Soejima A, et al. Sodium bicar- 536. Zarca A, Rubinstein D, Breitbart H. Transport mecha- bonate in seminal plasma stimulates the motility of mam- nism for calcium and phosphate in ram spermatozoa. Bio- malian spermatozoa through the direct activation of chim Biophys Acta 1988;994:351–358. adenylate cyclase. J Biol Chem 1985;260:9699–9705. 537. DasGupta S, Mills CL, Fraser LR. A possible role for 2ϩ 518. Shi Q-X, Roldan ERS. Bicarbonate/CO2 is not required Ca -ATPase in human sperm capacitation. J Reprod for zona pellucida- or progesterone-induced acrosomal exo- Fertil 1994;102:107–116. ϩ cytosis of mouse spermatozoa but is essential for capacita- 538. Roldan ERS, Flemming AD. Is a Ca2 -ATPase involved ϩ tion. Biol Reprod 1995;52:540–546. in Ca2 regulation during capacitation and the acrosome 519. Suzuke F, Yanagimachi R. Changes in the distribution of reaction of guinea-pig spermatozoa. J Reprod Fertil 1989; intramembranous particles and filipin-reactive membrane 85:297–308. 2ϩ sterols during in vitro capacitation of golden hamster sper- 539. Fraser LR, Abeydeera LR, Niwa K. Ca -regulating matozoa. Gamete Res 1989;23:335–347. mechanisms that modulate bull sperm capacitation and 520. Visconti PE, Galantino-Homer H, Ning X, et al. Choles- acrosomal exocytosis as determined by chlortetracycline terol efflux-mediated signal transduction in mammalian analysis. Mol Reprod Dev 1995;40:233–241. sperm. Beta-cyclodextrins initiate transmembrane sig- 540. Quill TA, Sugden SA, Rossi KL, et al. Hyperactivated naling leading to an increase in protein tyrosine phosphor- sperm motility driven by CatSper2 is required for fertili- ylation and capacitation. J Biol Chem 1999;274:3235– zation. Proc Natl Acad Sci USA 2003;100:14869–14874. 3242. 541. Nikpoor P, Mowla SJ, Movahedin M, et al. CatSper gene 521. Visconti PE, Stewart-Savage J, Blasco A, et al. Roles of expression in postnatal development of mouse testis and in bicarbonate, cAMP, and protein tyrosine phosphorylation subfertile men with deficient sperm motility. Hum Re- on capacitation and the spontaneous acrosome reaction of prod 2004;19:124–128. hamster sperm. Biol Reprod 1999;61:76–84. 542. Quill TA, Ren D, Clapham DE, et al. A voltage-gated ion 522. Visconti PE, Ning X, Fornes MW, et al. Cholesterol ef- channel expressed specifically in spermatozoa. Proc Natl flux-mediated signal transduction in mammalian sperm: Acad Sci USA 2001;98:12527–12531. cholesterol release signals an increase in protein tyrosine 543. Ren D, Navarro B, Perez G, et al. A sperm ion channel phosphorylation during mouse sperm capacitation. Dev required for sperm motility and male fertility. Nature 2001;413:603–609. Biol 1999;214:429–443. 544. Fraser LR, Adeoya-Osiguwa S, Baxendale RW, et al. 523. Visconti PE, Westbrook VA, Chertihin O, et al. Novel First messenger regulation of mammalian sperm function signaling pathways involved in sperm acquistion of fertil- via adenylyl cyclase/cAMP. J Reprod Dev 2005;51:37–46. izing capacity. J Reprod Immunol 2002;53:133–150. 545. Breitbart H, Cohen G, Rubinstein S. Role of actin cy- 524. Choi YH, Toyoda Y. Cyclodextrin removes cholesterol toskeleton in mammalian sperm capacitation and the ac- from mouse sperm and induces capacitation in a protein rosome reaction. Reproduction 2005;129:263–268. free medium. Biol Reprod 1998;59:1328–1333. 546. Clarke GN, Clarke FM, Wilson S. Actin in human sper- 525. Cross NL. Effect of methyl-beta-cyclodextrin on the acro- matozoa. Biol Reprod 1982;26:319–327. somal responsiveness of human sperm. Mol Reprod Dev 547. Ochs D, Wolf DP. Actin in ejaculated human sperm cells. 1999;53:92–98. Biol Reprod 1985;33:1223–1226. 526. Osherroff JE, Visconti PE, Valenzuela JP, et al. Regula- 548. Virtanen I, Badley RA, Paasivuo R, et al. Distinct cy- tion of human sperm capacitation by a cholesterol efflux- toskeletal domains revealed in sperm cells. J Cell Biol stimulated signal transduction pathway leading to protein 1984;99:1083–1091. kinase A-mediated up-regulation of protein tyrosine phos- 549. Breitbart H, Rubinstein S, Etkovitz N. Sperm capacita- phorylation. Mol Hum Reprod 1999;5:1017–1026. tion is regulated by the crosstalk between protein kinase a 527. Nolan JP, Hammerstedt RH. Regulation of membrane and c. Mol Cell Endocrinol 2006;252:247–249. stability and the acrosome reaction in mammalian sperm. 550. Cohen G, Rubinstein S, Gur Y, et al. Crosstalk between FASEB J 1997;11:670–682. protein kinase a and c regulates phospholipase d and f- 528. Shinitzky M. Membrane fluidity and cellular functions. actin formation during sperm capacitation. Dev Biol In: Shinitzky M, ed. Physiology of membrane fluidity. 2004;267:230–241. Boca Raton, FL: CRC Press, 1984;1–51. 551. Cross NL, Overstreet JW. Glycoconjugates of the human 529. Jimenez-Gonzalez C, Michelangeli F, Harper CV, et al. sperm surface; distribution and alterations that accom- Calcium signalling in human spermatozoa: a specialized pany capacitation in vitro. Gamete Res 1987;16:23–35. ‘toolkit’ of channels, transporters and stores. Hum Re- 552. Ficarro S, Chertihin O, Westbrook VA, et al. Phosphopro- prod Update 2006;12:253–267. teome analysis of capacitated human sperm. Evidence of 530. Spira B, Breitbart H. The role of anion channels in the tyorsine phosphorylation of a kinase-anchoring protein 3 mechanism of acrosome reaction in bull spermatozoa. Bio- and valosin-containing protein/p97 during capacita- chim Biophys Acta 1992;1109:65–73. tion. J Biol Chem 2003;278:11579–11589. 531. Darszon A, Lopez-Martinez P, Acevedo JJ, et al. T-type 553. Urner F, Sakkas D. Protein phosphorylation in mamma- ϩ Ca2 channels in sperm function. Cell Calcium 2006;40: lian spermatozoa. Reproduction 2003;125:17–26. 241–252. 554. Bailey JL. Cell signalling during capacitation and the 532. Neri-Vidaurri PD, Torres-Flores V, Gonzalez-Martinez acrosome reaction. Proc Havemeyer Foundation Workshop MT. A remarkable increase in the pHi sensitivity of volt- Stallion Reprod 2007;(in press). age-dependent calcium channels occurs in human sperm 555. Vijayaraghavan S, Liberty GA, Mohan J, et al. Isolation incubated in capacitating conditions. Biochem Biophys and molecular characterization of AKAP110, a novel, Res Commun 2006;343:105–109. sperm-specific protein kinase A-anchoring protein. Mol 533. Wennermuth G, Westenbroek RE, Xu T, et al. Cav2.2 and Endocrinol 1999;13:705–717. Cav2.3 (N- and R-type) Ca2ϩ channels in depolarization- 556. Naaby-Hansen S, Mandal A, Wolkowicz MJ, et al. CABYR, evoked entry of Ca2ϩ into mouse sperm. J Biol Chem a novel calcium-binding tyrosine phosphorylation- regulation 2000;275:21210–21217. of tyrosine phosphorylation in human spermatozoa and its

166 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

role in the control of human sperm function. Dev Biol 2002; 577. Lewis B, Aitken RJ. A redox-regulated tyrosine phos- 242:236–254. phorylation cascade in rat spermatozoa. J Androl 2001; 557. Aisquith KL, Baleato RM, McLaughlin EA, et al. Ty- 22:611–622. rosine phosphorylation activates surface chaperones facil- 578. Aitken RJ, Harkiss D, Knox W, et al. A novel signal itating sperm-zona recognition. J Cell Sci 2004;117:3657. transduction cascade in capacitating human spermatozoa 558. Brewis IA, Moore HD, Fraser LR, et al. Molecular mech- characterized by a redox-regulated, cAMP-mediated induc- anisms during sperm capacitation. Hum Fertil 2005;8: tion of tyrosine phosphorylation. J Cell Sci 1998;111:645– 253–261. 656. 559. Harrison RAP, Miller NG. cAMP-dependent protein ki- 579. Cross NL. Reorganization of lipid rafts during capacita- nase control of plasma membrane lipid architecture in boar tion of human sperm. Biol Reprod 2004;71:1367–1373. sperm. Mol Reprod Dev 2000;55:220–228. 580. van Gestel RA, Brewis IA, Ashton PR, et al. Capacitation- 560. Harrison RA, Gadella BM. Bicarbonate-induced mem- dependent concentration of lipid rafts in the apical ridge brane processing in sperm capacitation. Theriogenology head area of porcine sperm cells. Mol Hum Reprod 2005; 2005;63:342–351. 11:583–590. 561. Wang L, Beserra C, Garbers DL. A novel aminophospho- 581. Weerachatyanukul W, Probodh I, Kongmanas K, et al. lipid transporter exclusively expressed in spermatozoa is Visualizing the localization of sulfoglycolipids in lipid raft required for membrane lipid asymmetry and normal fertil- domains in model membranes and sperm membrane ex- ization. Dev Biol 2004;267:203–215. tracts. Biochim Biophys Acta 2006;1768:299–310. 562. deLamirande E, Gagnon C. A positive role for the super- 582. Khalil MB, Chakrabandhu K, Xu H, et al. Sperm capac- oxide anion in triggering hyperactivation and capacitation itation induces an increase in lipid rafts having zona of human spermatozoa. Int J Androl 1993;16:21–25. pellucida binding ability and containing sulfogalacto- 563. Bize I, Santander G, Cabello P, et al. Hydrogen peroxide sylgycerolipid. Dev Biol 2006;290:220–235. is involved in hamster sperm capacitation in vitro. Biol 583. Thaler CD, Thomas M, Ramalie JR. Reorganization of Reprod 1991;44:398–403. mouse sperm lipid rafts by capacitation. Mol Reprod Dev 564. Kameshwari DB, Siva AB, Shivaji S. Inhibition of in vitro 2006;73:1541–1549. capacitation of hamster spermatozoa by nitric oxide syn- 584. Sleight SB, Miranda PV, Plaskett NW, et al. Isolation thase inhibitors. Cell Mol Biol 2003;49:421–428. and proteomic analysis of mouse sperm detergent-resistant 565. Herrero MB, de Lamirande E, Gagnon C. Nitric oxide membrane fractions: evidence for dissociation of lipid regulates human sperm capacitation and protein tyrosine rafts during capacitation. Biol Reprod 2005;73:721–729. phosphorylation in vitro. Biol Reprod 1999;61:575–581. 585. Tanphaichitr N, Carmona E, Bou Khalil M, et al. New 566. O’Flaherty C, deLamirande E, Gagnon C. Reactive oxy- insights into sperm-zona pellucida interaction: involve- gen species modulate independent protein phosphorylation ment of sperm lipid rafts. Front Biosci 2007;12:1748– pathways during human sperm capacitation. Free Radic 1766. Biol Med 2006;40:1045–1055. 586. deLamirande E, Gagnon C. The extracellular signal-reg- 567. Aitken RJ, Paterso M, Fisher H, et al. Redox regulation of ulated kinase (ERK) pathway is involved in human sperm tyrosine phosphorylation in human spermatozoa and its function and modulated by the superoxide anion. Mol role in the control of human sperm function. J Cell Sci Hum Reprod 2002;8:124–135. 1995;108:2017–2025. 587. Wasco WM, Orr GA. Function of calmodulin in mamma- 568. Aitken RJ, Fisher HM, Fulton N, et al. Reactive oxygen lian sperm: presence of a calmodulin-dependent cyclic species generation by human spermatozoa is induced by nucleotide phosphodiesterase associated with demem- exogenous NADPH and inhibited by the ﬂavoprotein inhib- branated rat caudal epididymal sperm. Biochem Biophys itors diphenylene iodonium and quinacrine. Mol Reprod Res Commun 1984;118:636–642. Dev 1997;47:482. 588. Luconi M, Krausz C, Forti G, et al. Extracellular calcium 569. Aitken RJ, Harkiss D, Knox W, et al. A novel signal negatively modulates tyrosine phosphorylation and ty- transduction cascade in capacitating human spermatozoa rosine kinase activity during capacitation of human sper- characterised by a redoxregulated cAMP-mediated induc- matozoa. Biol Reprod 1996;55:207–216. tion of tyrosine phosphorylaton. J Cell Sci 1998;111:645– 589. Ashraf M, Peterson RN, Russell LD. Evidence for Ca2ϩ/Naϩ 656. antiport in boar sperm plasma membrane vesicles. Biol Re- 570. Natarajan V, Forman HJ. Introduction to serial reviews prod 1982;26:37A. on redox regulation of phospholipases and sphingomyeli- 590. Bradley MP, Forrester IT. A sodium-calcium exchange nase in cell signaling. Free Radic Biol Med 2006;40:363. mechanism in plasma membrane vesicles isolated from 571. Thundathil J, de Lamirande E, Gagnon C. Nitric oxide ram sperm ﬂagella. FEBS Lett 1980;121:15–18. regulates the phosphorylation of the threonine-glutamine- 591. Rufo BA, Schoff PK, Lardy HA. Regulation of calcium tyrosine motif in proteins of human spermatozoa during content in bovine spermatozoa. J Biol Chem 1984;259: capacitation. Biol Reprod 2003;68:1291–1298. 2547–2552. 572. Rodriguez PC, O’Flaherty CM, Beconi MT, et al. Nitric 592. Acevedo JJ, Mendoza-Lujambio I, Vega-Beltran JL, et al.

oxide-induced capacitation of cryopreserved bull sperma- KATP channels in mouse spermatogenic cells and sperm, tozoa and assessment of participating regulatory path- and their role in capacitation. Dev Biol 2006;289:395– ways. Anim Reprod Sci 2005;85:231–242. 405. 573. O’Flaherty C, de Lamirande E, Gagnon C. Reactive oxy- 593. Ball BA, Gravance CG, Wessel MT, et al. Activity of an- gen species and protein kinases modulate the level of phos- giotensin-converting enzyme (ACE) in reproductive tissues pho-MEK-like proteins during human sperm capacitation. of the stallion and effects of angiotensin II on sperm mo- Biol Reprod 2005;73:94–105. tility. Theriogenology 2003;59:901–914. 574. Baumber J, Sabeur K, Vo A, et al. Reactive oxygen spe- 594. Sabeur K, Vo A, Ball BA. Affects of angiotensin II on the cies promote capacitation and tyrosine phosphorylation in acrosome reaction in equine spermatozoa. J Reprod Fertil equine sperm. Anim Reprod Sci 2001;68:337–338. 2000;120:135–142. 575. Baker MA, Atiken RJ. The importance of redox regulated 595. Fraser LR, Adeoya-Osiquwa SA, Baxendale RW, et al. pathways in sperm cell biology. Mol Cell Endocrinol Regulation of mammalian sperm capacitation by endoge- 2004;216:47–54. nous molecules. Front Biosci 2006;11:1636–1645. 576. Baumber J, Sabeur K, Vo A, et al. Reactive oxygen spe- 596. Mededovic S, Fraser LR. Mechanisms of action of angio- cies promote tyrosine phosphorylation and capacitation in tensin II on mammalian sperm function. Reproduction equine spermatozoa. Theriogenology 2003;60:1239–1247. 2005;129:211–218.

AAEP PROCEEDINGS ր Vol. 53 ր 2007 167 MILNE LECTURE

597. Hutt DM, Cardullo RA, Baltz JM, et al. Synaptotagmin 621. Miller DJ, Macek MB, Shur BD. Complementarity be- VIII is localized to the mouse sperm head and may function tween sperm surface beta-1,4-galactosyltransferase and in acrosomal exocytosis. Biol Reprod 2002;66:50–56. egg-coat ZP3 mediates sperm-egg binding. Nature 1992; 598. Olds-Clark P. Unresolved issues in mammalian fertiliza- 357:589–593. tion. Int Rev Cytol 2003;232:129–184. 622. Shi Q-X, Amindari S, Paruchuru K, et al. Cell surface beta- 599. Miller DJ, Shur BD. Molecular basis of fertilization in the 1,4-galactosyltransferase-I activates G protein-dependent mouse. Dev Biol 1994;5:255–264. exocytotic signaling. Development 2001;128:645–654. 600. Snell WJ, White JM. The molecules of mammalian fertil- 623. Rodeheffer C, Shur BD. Sperm from ␤1,4-galactosyltrans- ization. Cell 1996;85:629–637. ferase I-null mice exhibit precocious capacitation. Develop- 601. Wassarman PM. Mammalian fertilization: molecular ment 2004;131:491–501. aspects of gamete adhesion, exocytosis and fusion. Cell 624. Lopez LC, Bayna EM, Litoff D, et al. Receptor function of 1999;96:175–183. mouse sperm surface galactosyltransferase during fertili- 602. Kopf GS, Ning XP, Visconti PE, et al. Signaling mecha- zation. J Cell Biol 1985;101:1501–1510. nisms controlling mammalian sperm fertilization compe- 625. Scully NF, Shaper JH, Shur BD. Spatial and temporal tence and activation. In: Gagnon C, ed. The male expression of cell surface galactosyltransferase during gamete: from basic science to clinical applications. Vi- mouse spermatogenesis and epididymal maturation. Dev enna, IL: Cache River Press, 1999;105–118. Biol 1987;124:111–124. 603. Florman HM, Storey BT. Mouse gamete interactions: 626. Shur BD, Hall NG. A role for mouse sperm surface galac- the zona pellucida is the site of the acrosome reaction tosyltransferase in sperm binding to the egg zona pellu- leading to fertilization in vitro. Dev Biol 1982;91:121– cida. J Cell Biol 1982;95:574–579. 130. 627. Shur BD, Neely CA. Plasma membrane association, pu- 604. Roldan ERS, Murase T, Qi-Xian S. Exocytosis in sper- rification, and partial characterization of mouse sperm matozoa in response to progesterone and zona pellucida. Beta 1,4-galactosyltransferase. J Biol Chem 1988;263: Science 1994;266:1578–1581. 17706–17714. 605. Wassarman PM. Contribution of mouse egg zona pellu- 628. Rodeheffer C, Shur BD. Characterization of a novel ZP3- cida glycoproteins to gamete recognition during fertiliza- independent sperm-bining ligand that facilitates sperm ad- tion. J Cell Physiol 2005;204:388–391. hesion to the egg coat. Development 2004;131:503–512. 606. Wassarman P, Chen J, Cohen N, et al. Structure and 629. Jovine L, Darie CC, Litscher E, et al. Zona pellucida function of the mammalian egg zona pellucida. J Exp domain proteins. Annu Rev Biochem 2005;74:83–114. Zool 1999;285:251–258. 630. Wassarman PM. Fertilization. Mol Biol 2003;16:117–204. 607. Wassarman PM. Sperm receptors and fertilization in 631. Greve JM, Wassarman PM. Mouse egg extracellular coat mammals. Mt Sinai J Med 2002;69:148–155. is a matrix of interconnected filaments posessessing a 608. Wasserman PM, Jovine L, Litscher E. A profile of fertil- structural repeat. J Mol Biol 1985;181:253–264. ization in mammals. Nat Cell Biol 2001;3:E59–E64. 632. Hinsch E, Aires VA, Hedrich F, et al. A synthetic de- 609. Bleil JD, Wassarman PM. Structure and function of the capeptide from a conserved ZP3 protein domain induces zona pellucida: identification and characterization of the the G protein-regulated acrosome reaction in bovine sper- proteins of the mouse oocyte’s pona pellucida. Dev Biol matozoa. Theriogenology 2005;63:1682–1694. 1980;76:185–202. 633. Berridge MJ, Lipp P, Bootman MD. The versatility and 610. Bleil JD, Wassarman PM. Mammalian sperm-egg inter- unversality of calcium signaling. Nat Rev Mol Cell Biol action: identification of a glycoprotein in mouse egg zonae 2000;1:11–21. pellucidae possessing receptor activity for sperm. Cell 634. Breitbart H, Spungin B. The biochemistry of the acro- 1980;20:873–882. some reaction. Mol Hum Reprod 1997;3:195–202. 611. Bleil JD, Wassarman PM. Sperm-egg interactions in the 635. Jungnickel MK, Marrero H, Birnbaumer L, et al. Trp2 mouse: sequence of events and induction of the acrosome regulates entry of Ca2ϩ into mouse sperm triggered by egg reaction by a zona pellucida glycoprotein. Dev Biol 1983; ZP3. Nat Cell Biol 2001;3:499–502. 95:317–324. 636. O’Toole CM, Arnoult C, Darszon A, et al. Ca2ϩ entry 612. Caballero-Campo P, Chirinos M, Fan XJ, et al. Biological through store-operated channels in mouse sperm is initi- effects of recombinant human zona pellucida proteins on ated by egg ZP3 and drives the acrosome reaction. Mol sperm function. Biol Reprod 2006;74:760–768. Biol Cell 2000;11:1571–1584. 613. Conner SJ, Lefievre L, Hughes DC, et al. Cracking the 637. Gonzalez-Martinez MT, Galindo BE, De La Torre L, et al. egg: increased complexity in the zona pellucida. Hum A sustained increase in intracellular Ca2ϩ is required for Reprod 2005;20:1148–1152. the acrosome reaction in sea urchin sperm. Dev Biol 614. Wassarman PM. Fertilization in mammals. Sci Am 2001;236:220–229. 1988;259:78–84. 638. Hirohashi N, Vacquier D. Store-operated calcium channels 615. Florman HM, Wassarman P. O-linked oligosaccharides of trigger exocytosis of the sea urchin sperm acrosomal vesicle. mouse egg ZP3 account for its sperm receptor activity. Cell Biochem Biophys Res Commun 2003;304:285–292. 1985;41:313–324. 639. Hartneck C, Plant TD, Schultz G. From worm to man: 616. Epifano O, Liang LF, Familiari M, et al. Coordinate ex- three subfamilies of TRP. Trends Neurosci 2000; pression of the three zona pellucida genes during mouse 159–166. oogenesis. Development 1995;121:1947–1956. 640. Castellano LE, Trevino CL, Rodriguez D, et al. Transient 617. McLeskey SB, Dowds C, Carballada R, et al. Molecules receptor potential (TRPC) channels in human sperm: ex- involved in mammalian sperm-egg interaction. Int Rev pression, cellular localization and involvement in the reg- Cytol 1998;177:57–113. ulation of flagellar motility. FEBS Lett 2003;541:69–74. 618. Florman HM, Bechtol KB, Wassarman PM. Enzymatic 641. Gong X, Dubois DH, Miller DJ, et al. Activation of a G digestion of the functions of the mouse egg’s receptor for protein complex by aggregation of beta-1,4-galactosyltrans- sperm. Dev Biol 1984;106:243–255. ferase on the surface of sperm. Science 1995;269:1718– 619. Sinowatz F, Topfer-Petersen E, Kolle S, et al. Functional 1721. morphology of the zona pellucida. Anat Histol Embryol 642. Bailey JL, Storey BT. Calcium influx into mouse sperma- 2001;30:257–263. tozoa activated by solubilized mouse zona pellucida, mon- 620. Bleil JD, Wassarman PM. Galactose at the nonreducing itored with the calcium fluorescent indicator, fluo-3. terminus of O-linked oligosaccharides of mouse egg zona Inhibition of the influx by three inhibitors of the zona pellucida glycoprotein ZP3 is essential for the glycopro- pellucida induced acrosome reaction: tyrphostin A48, per- tein’s sperm receptor activity. Proc Natl Acad Sci USA tussis toxin, and 3-quinuclidinyl benzilate. Mol Reprod 1988;85:6778–6782. Dev 1994;39:297–308.

168 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

643. Lee MA, Check JH, Kopf GS. A guanine nucleotide-bind- 665. O’Toole CM, Roldan ER, et al. Protein kinase C activation ing regulatory protein in human sperm mediates acroso- during progesterone-stimulated acrosomal exocytosis in mal exocytosis induced by the human zona pellucida. Mol human spermatozoa. Mol Hum Reprod 1996;2:921–927. Reprod Dev 1992;31:78–86. 666. Breitbart H, Lax J, Rotem R, et al. Role of protein kinase C 644. Arnoult C, Kazam IG, Visconti PE, et al. Control of the in the acrosome reaction of mammalian spermatozoa. Bio- low voltage-activated calcium channel of mouse sperm by chem J 1992;281:473–476. egg ZP3 and by membrane hyperpolarization during capac- 667. Rotem R, Paz GF, Homonnai ZT. Further studies on the itation. Proc Natl Acad Sci USA 1999;96:6757–6762. involvement of protein kinase C in human flagellar motil- 645. Leclerc P, Kopf GS. Mouse sperm adenylyl cyclase: gen- ity. Endocrinology 1990;127:2571–2577. eral properties and regulation by the zona pellucida. Biol 668. Rotem R, Paz GF, Homonnai ZT, et al. Protein kinase C is Reprod 1995;52:1227–1233. present in human sperm: possible role in flageller motil- 646. Noland TD, Garbers DL, Kopf GS. An elevation in cyclic ity. Proc Nat Acad Sci USA 1990;87:7305–7308. AMP concentration precedes the zona pellucida-induced 669. Naor Z, Breitbart H. Protein kinase C and mammalian acrosome reaction of mouse spermatozoa. Biol Reprod spermatozoa acrosome reaction. Trends Endocrin Metab 1998;38(Suppl 1):138. 1997;8:337–342. 647. Garde J, Roldan ER. Rab 3-peptide stimulates exocytosis 670. Luk AS, Kaler EW, Lee SP. Phospholipase C-induced ag- of the ram sperm acrosome via interaction with cyclic AMP gregation and fusion of cholesterol-lecithin small unilamel- and phospholipase A2 metabolites. FEBS Lett 1996;39: lar vesicles. Biochemistry 1993;32:6965–6973. 263–268. 671. Siegel DP, Banschbach J, Alford D, et al. Physiological 648. De Jonge CJ, Han J-L, Lawrie H, et al. Modulation of levels of diacylglycerols in phospholipid membranes induce human sperm acrosome reaction by effectors of the adenyl- membrane fusion and stabilize inverted phases. Bio- ate cyclase/cAMP second messenger pathway. J Exp Zool chemistry 1989;28:3703–3709. 1991;258:113–125. 672. Roggero CM, Tomes C, deBlas GA, et al. Protein kinase 649. Spungin B, Breitbart H. Calcium mobilization and influx C-mediated phosphorylation of the two polybasic regions of during sperm exocytosis. J Cell Sci 1996;109:1947–1955. synaptotagmin VI regulates their function in acrosomal 650. Breitbart H, Naor Z. Protein kinases in mammalian exocytosis. Dev Biol 2005;285:422–435. sperm capacitation and the acrosome reaction. Rev Re- 673. Tucker WC, Chapman ER. Role of synaptotagmin in prod 1999;4:151–159. Ca2ϩ-triggered exocytosis. Biochem J 2002;366:1–13. 651. Bielfeld P, Faridi A, Zaneveld LJD, et al. The zona pellu- 674. Sudhof TC. Synaptotagmins: why so many? J Biol cida-induced acrosome reaction of human spermatozoa is Chem 2002;277:7629–7632. mediated by protein kinases. Fertil Steril 1994;61:536– 675. Michaut M, deBlas GA, Tomes CN, et al. Synaptotagmin 554. VI participates in the acrosome reaction of human sperma- 652. Tomes CN, McMaster CR, Saling PM. Activation of tozoa. Dev Biol 2001;235:521–529. mouse sperm phosphatidylinositol-4,5 bisphosphate-phos- 676. Ono K, Yanagimachi R, Huang TTF. Phospholipase A of pholipase C by zona pellucida is modulated by tyrosine guinea pig spermatozoa: Its preliminary characterization phosphorylation. Mol Reprod Dev 1996;43:196–204. and possible involvement in the acrosome reaction. Dev

653. Roldan ER, Fragio C. Phospholipase A2 activity and exo- Growth Diff 1982;24:305–310. cytosis of the ram sperm acrosome: regulation by bivalent 677. Lui CW, Meizel S. Further evidence in support of a role cations. Biochem Biophys Acta 1993;1168:108–114. for hamster sperm hydrolytic enzymes in the acrosome 654. Roldan ER. Role of phospholipases during sperm acroso- reaction. J Exp Zool 1979;207:173–186. mal exocytosis. Front Biosci 1998;3:1109–1119. 678. Singleton CL, Killian GJ. A study of phospholipase in 655. Walensky LD, Snyder SH. Inositol 1,4,5-trisphosphate albumin and its role in inducing the acrosome reaction of receptors selectively localized to the acrosomes of mamma- guinea pig spermatozoa in vitro. J Androl 1983;4:150– lian sperm. J Cell Biol 1995;130:857–869. 156. 656. Spungin B, Margalit I, Breitbart H. Sperm exocytosis 679. Kyono K, Hoshi K, Saito A, et al. Effects of phospholipase reconstructed in a cell-free system: evidence for the in- A, lysophosphatidylcholine, and fatty acid on the acrosome volvement of phospholipase C and actin ﬁlaments in mem- reaction of human spermatozoa. Tohoku J Exp Med 1984; brane fusion. J Cell Sci 1995;108:2525–2535. 144:257–263.

657. Fukami K, Nakao K, Inoue T, et al. Requirement of phos- 680. Roldan ER, Vazquez JM. Bicarbonate/CO2 induces rapid pholipase Cdelta4 for the zona pellucida-induced acrosome activation of phospholipase A2 and renders boar spermato- reaction. Science 2001;292:920–923. zoa capable of undergoing acrosomal exocytosis in response 658. Fukami K, Yoshida M, Inoue T, et al. Phospholipase C-⌬4 to progesterone. FEBS Lett 1996;396:227–232. is required for Ca2ϩ mobilization essential for acrosome 681. Roldan ER, Fragio C. Phospholipase A2 activation and reaction in sperm. J Cell Biol 2003;161:79–88. subsequent exocytosis in the Ca2ϩ/ionophore-induced acro- 659. Putney JWJ. Capacitative calcium entry revisited. some reaction of ram spermatozoa. J Biol Chem 1993; Cell Calcium 1990;11:611–624. 268:13962–13970. 660. Hikida M, Kurosaki T. Regulation of phospholipase C-␥2 682. Yuan YY, Che WY, Shi QX, et al. Zona pellucida induces

networks in B lymphocytes. Adv Immunol 2005;88:73– activation of phospholipase A2 during acrosomal exocytosis 96. in guinea pig spermatozoa. Biol Reprod 2003; 661. Montell CL, Birnbaumer L, Flockerzi V. The TRP chan- 68:904–913. nels, a remarkably functional family. Cell 2002;108:595– 683. Roldan ER, Fragio C. Diradylglycerols stimulate phos-

598. pholipase A2 and subsequent exocytosis in ram spermato- 662. Kiselyov K, Mignery GA, Zhu MX, et al. The N-terminal zoa. Biochem J 1994;297:225–232.

domain of the IP3 receptor gates store-operated hTrp3 684. Flemming AD, Yanagimachi R. Effects of various lipids channels. Mol Cell 1999;4:423–429. on the acrosome reaction and fertilizing capacity of guinea 663. Boulay G, Brown DM, Quin N, et al. Modulation of Ca2ϩ pig spermatozoa with special reference to the possible in- entry by polypeptides of the inositol 1,4,5-triphosphate re- volvement of lysophospholipids in the acrosome reac-

ceptor (IP3R) that bind transient receptor potential (TRP): tion. Gamete Res 1981;4:253–273. evidence for roles of TRP and IP3R in store depletion- 685. Ohzu E, Yanagimachi R. Acceleration of acrosome reac- activated Ca2ϩ entry. Proc Natl Acad Sci USA 1999;96: tion in hamster spermatozoa by lysolecithin. J Exp Zool 14955–14960. 1982;224:259–263. 664. Xu XZ, Sternberg PW. A C. elegans sperm TRP protein re- 686. Llanos MN, Morales P, Riffo MS. Studies of lysophospho- quired for sperm-egg interactions during fertilization. Cell lipids related to the hamster sperm acrosome reaction. 2003;114:285–297. J Exp Zool 1993;267:209–216.

AAEP PROCEEDINGS ր Vol. 53 ր 2007 169 MILNE LECTURE

687. Parrish JJ, Susko-Parish JL, Winer MA, et al. Capacita- 709. Catterall WA. Interactions of presynaptic Ca2ϩ channels tion of bovine sperm by heparin. Biol Reprod 1988;38: and snare proteins in neurotransmitter release. Ann NY 1171–1180. Acad Sci 1999;868:144–159. 688. Garde J, Roldan ER. Stimulation of Ca2ϩ-dependent exo- 710. Leabu M. Membrane fusion in cells: molecular machin- cytosis of the sperm acrosome by cAMP acting downstream ery and mechanisms. J Cell Mol Med 2006;10:423–427. of phopholipase A2. J Reprod Fertil 2000;118:57–68. 711. Jeremic A, Kelly M, Cho JA, et al. Calcium drives fusion 689. Sollner TH. Regulated exocytosis and SNARE fuction of SNARE-apposed bilayers. Cell Biol Int 2004;28:19–31. (Review). Mol Membr Biol 2003;20:209–220. 712. Portis A, Newton C, Pangborn W, et al. Studies on the 690. Ramalho-Santos J, Moreno RD, Sutovsky P, et al. SNAREs mechanism of membrane fusion: evidence for an inter- 2ϩ in mammalian sperm: Possible implications for fertiliza- membrane Ca -phospholipid complex, synergism with 2ϩ tion. Dev Biol 2000;223:54–69. Mg , and inhibition by spectrin. Biochemistry 1979;18: 691. deBlas GA, Roggero CM, Tomes C, et al. Dynamics of 780–790. SNARE assembly and disassembly during sperm acroso- 713. Suarez SS, Dai X. Hyperactivation enhances mouse mal exocytosis. PLoS Biol 2005;3:1801–1812. sperm capacity for penetrating viscoelastic media. Biol 692. Tomes CN, deBlas GA, Michaut MA, et al. Alpha-SNAP Reprod 1992;46:686–691. and NSF are required in a priming step during the human 714. Eisenbach M, Giojalas LC. Sperm guidance in mammals—an unpaved road to the egg. Nat Rev Mol Cell Biol sperm acrosome reaction. Mol Hum Reprod 2004;11:43– 2006;7:276–285. 51. 715. Suarez SS, Vincenti L, Ceglia MW. Hyperactivated mo- 693. Katafuchi K, Mori T, Toshimori K, et al. Localization of a tility induced in mouse sperm by calcium ionophore syntaxin isoform, syntaxin 2, to the acrosomal region of A23187 is reversible. J Exp Zool 1987;244:331–336. rodent spermatozoa. Mol Reprod Dev 2000;57:375–383. 716. Qi H, Moran MM, Navarro B, et al. All four CatSper ion 694. Gamboa S, Ramalho-Santos J. SNARE proteins and channel proteins are required for male fertility and sperm caveolin-1 in stallion spermatozoa: possible implications cell hyperactivated motility. Proc Natl Acad Sci USA for fertility. Theriogenology 2005;64:275–291. 2007;104:1219–1223. 695. McNew JA, Weber T, Parlati F, et al. Close is not enough: 717. Lindemann CB, Goltz JS. Calcium regulation of flagellar SNARE-dependent membrane fusion requires an active curvature and swimming pattern in triton X-100–ex- mechanism that transduces force to membrane an- tracted rat sperm. Cell Motil Cytoskeleton 1988;10:420– chors. J Cell Biol 2000;150:105–117. 431. 696. Grote E, Baba M, Ohsumi Y, et al. Geranyl-geranylated 718. Marquez B, Suarez SS. Bovine sperm hyperactivation is SNAREs are dominant inhibitors of membrane fu- promoted by alkaline-stimulated Ca2ϩ influx. Biol Reprod sion. J Cell Biol 2000;151:453–466. 2006. 697. He P, Southard C, Chen D, et al. Role of alpha-SNAP in 719. Ho HC, Suarez SS. An inositol 1,4,5-trisphosphate recep- promoting efficient neurotransmission at the crayfish neu- tor-gated intracellular Ca2ϩ store is involved in regulating romuscular junction. J Neurophysiol 1999;82:3406–3416. sperm hyperactivated motility. Biol Reprod 2001;65: 698. Barnard RJ, Morgan A, Burgoyne RD. Stimulation of 1606–1615. NSF ATPase activity by alpha-SNAP is required for 720. Santi CM, Santos T, Hernandez-Cruz A, et al. Properties SNARE complex dissembly and exocytosis. J Cell Biol of a novel pH-dependent Ca2ϩ permeation pathway present 1997;139:875–883. in male germ cells with possible roles in spermatogenesis 699. Clary DO, Griff IC, Rothman JE. SNAPs, a family of NSF and mature sperm function. J Gen Physiol 1998;112:33– attachment proteins involved in intracellular membrane 53. fusion in animal and yeast. Cell 1990;61:709–721. 721. Marquez B, Ignotz GG, Suarez SS. Contributions of ex- ϩ 700. Nagiec EE, Bernstein A, Whiteheart SW. Each domain of tracellular and intracellular Ca2 to regulation of sperm the N-ethylmaleimide-sensitive fusion protein contributes motility: Release of intracellular stores can hyperactivate to its transport activity. J Biol Chem 1995;270:29182– CatSper1 and CatSper2 null sperm. Dev Biol 2006. 29188. 722. Ho HC, Suarez SS. Characterization of the intracellular 701. Weidman PJ, Melanncon P, Block MR, et al. Binding of calcium store at the base of the sperm flagellum that reg- an N-ethylmaleimide-sensitive fusion protein to Golgi ulates hyperactivated motility. Biol Reprod 2003;68: membranes requires both a soluble protein(s) and an inte- 1590–1696. gral membrane receptor. J Cell Biol 1989;108:1589–1596. 723. Zhang Z. The effect of 12-myristate 13-acetate phorbol 702. Banerjee A, Barry VA, DasGupta BR, et al. N-Ethylma- ester on human sperm hyperactivation. Fertil Steril leimide-sensitive factor acts at a prefusion ATP-dependent 1997;67:1140–1145. step in Ca2ϩ-activated exocytosis. J Biol Chem 1996;271: 724. Burkman LJ. Discrimination between nonhyperactivated and classical hyperactivated motility patterns in human 20223–20226. spermatozoa using computerized analysis. Fertil Steril 703. Belmonte SA, Lopez CI, Roggero CM, et al. Cholesterol 1991;55:363–371. content regulates acrosomal exocytosis by enhancing 725. Rogers BJ. The sperm penetration assay: Its usefulness Rab3A plasma membrane association. Dev Biol 2005;285: reevaluated. Fertil Steril 1985;43:821–840. 393–408. 726. Wramsby H, Liedholm P. Importance of the use of short 704. Pfeffer SR. Transport-vesicle targeting: thethers before and long sperm pre-incubation periods in assessing the SNAREs. Nat Cell Biol 2001;1:E17–E22. fertilizing capacity of human spermatozoa by hamster test. 705. Waters MG, Hughson FM. Membrane tethering and fu- Human Reprod 1986;1:171–173. sion in the secretory and indocytic pathways. Traffic 727. Muller CH. Rationale, interpretation, validation and use 2000;1:588–597. of sperm fucntion tests. J Androl 2000;21:10–30. 706. Branham MT, Mayorga LS, Tomes CN. Calcium-induced 728. DeMott RP, Lefebvre R, Suarez SS. Carbohydrates medi- acrosomal exocytosis requires cAMP acting through a ate the adherence of hamster sperm to oviductal epithe- protein kinase A-independent, Epac-mediated pathway. lium. Biol Reprod 1995;52:1395–1403. J Biol Chem 2006;281:8656–8666. 729. Eisenbach M, Ralt D. Precontact mammalian sperm-egg 707. Littleton JT, Bai J, Vyas B, et al. Synaptotagmin mutants communication and role in fertilization. Am J Physiol ϩ reveal essential functions for the C2B domain in Ca2 - 1992;262:C1095–C1101. triggered fusion and recycling of SVs in vivo. J Neurosci 730. Cohen-Dayag A, Tur-Kaspa I, Dor J, et al. Sperm capac- 2001;21:1421–1433. itation in humans is transient and correlates with chemo- 708. Augustine GJ, Neher E. Neuronal Ca2ϩ signaling takes tactic responsiveness to follicular factors. Proc Nat Acad the local route. Curr Opin Neurobiol 1992;2:302–307. Sci USA 1995;92:11039–11043.

170 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

731. Thomas MB, Haines SL, Akeson RA. Chemoreceptors 755. Giojalas LC, Rovasio RA. Mouse spermatozoa modify expressed in taste, olfactory and male reproductive tis- their motility parameters and chemotactic response to fac- sues. Gene 1996;178:1–5. tors from the oocyte microenvironment. Int J Androl 732. Fabro G, Rovasio RA, Civalero S, et al. Chemotaxis of 1998;21:201–206. capacitated rabbit spermatozoa to follicular fluid revealed 756. Carnevale EM, Maclellan LJ, Coutinho da Silva MA, et al. by a novel directionality-based assay. Biol Reprod 2002; Equine sperm-oocyte interaction: results after intraovi- 67:1565–1571. ductal and intrauterine inseminations of recipients for oo- 733. Kirkman-Brown JC, Sutton KA, Florman HM. How to cyte transfer. Anim Reprod Sci 2001;68:305–314. attract a sperm. Nat Cell Biol 2003;5:93–96. 757. Sun F, Bahat A, Gakamsky A, et al. Human sperm che- 734. Riffell JA, Krug PJ, Zimmer RK. The ecological and evo- motaxis: both the oocyte and its surrounding cumulus lutionary consequences of sperm chemoattraction. Proc cells secrete sperm chemoattractants. Hum Reprod 2005; Natl Acad Sci USA 2004;101:4501–4506. 20:761–767. 735. Eisenbach M. Towards understanding the molecular 758. Suarez SS, Pacey AA. Sperm transport in the female mechanism of sperm chemotaxis. J Gen Physiol 2004;124: reproductive tract. Hum Reprod Update 2006;12:23–37. 105–108. 759. Familiari G, Relucenti M, Heyn R, et al. Three-dimen- 736. Ralt D, Manor M, Cohen-Dayag A, et al. Chemotaxis sional structure of the zona pellucida at ovulation. Micros and chemokinesis of human spermatozoa to follicular Res Tech 2006;69:415–426. factors. Biol Reprod 1994;50:774–785. 760. Primakoff P, Myles DG. Penetration, adhesion, and fu- 737. Cohen-Dayag A, Ralt D, Tur-Kaspa I, et al. Sequential sion in mammalian sperm-egg interaction. Science 2002; acquisition of chemotactic responsiveness by human sper- 296:2183–2185. matozoa. Biol Reprod 1994;50:786–790. 761. Meyers SA. Equine sperm-oocyte interaction: the role of 738. Hunter RHF, Petersen HH, Greve T. Ovarian follicular sperm surface hyaluronidase. Anim Reprod Sci 2001;68: fluid, progesterone and Ca2ϩ ion influences on sperm re- 291–303. lease from the fallopian tube reservoir. Mol Reprod Dev 762. Olds-Clarke P. How does poor motility alter sperm fertil- 1999;54:283–291. izing ability. J Androl 1996;17:183–186. 739. Villanueva-Diaz C, Arias-Martinez J, Bermejo-Martinez 763. Wassarman PM. Role of carbohydrates in receptor-medi- L, et al. Progesterone induces human sperm chemo- ated fertilization in mammals. Ciba Found Symp 1989; taxis. Fertil Steril 1995;64:1183–1188. 145:135–149. 740. Sliwa L. Chemotaction of mouse spermatozoa induced by 764. Vierira A, Miller DJ. Gamete interaction: is it species- certain hormones. Arch Androl 1995;35:105–110. specific? Mol Reprod Dev 2006;73:1422–1429. 741. Eisenbach M, Tur-Kaspa I. Do human eggs attract sper- 765. Velasquez JG, Canovas S, Barajas P, et al. Role of sialic matozoa? Bioessays 1999;21:203–210. acid in bovine sperm-zona pellucida binding. Mol Reprod 742. Feldmesser E, Olender T, Khen M, et al. Widespread Dev 2007;74:614–628. ectopic expression of olfactory receptor genes. BMC 766. Skutelsky E, Ranen E, Shalgi R. Variations in the distri- Genomics 2006;7:121. bution of sugar residues in the zona pellucida as possible 743. Vanderhaeghen P, Schurmans S, Vassart G, et al. Spe- species-specific determinants of mammalian oocytes. J cific repertoire of olfactory receptor genes in the male germ Reprod Fertil 1994;100:35–41. cells of several mammalian species. Genomics 1997;39: 767. Tantibhedhyangkul J, Weerachatyanukul W, Carmona E, 239–246. et al. Role of sperm surface arylsulfatase A in mouse 744. Spehr M, Schwane K, Riffell JA, et al. Odorant receptors sperm-zona pellucida binding. Biol Reprod 2002;67:212– and olfactory-like signaling mechanisms in mammalian 219. sperm. Mol Cell Endocrinol 2006;250:128–136. 768. Cheng A, Le T, Placios M, et al. Sperm-egg recognition in 745. Fukuda N, Touhara K. Developmental expression pat- the mouse: characterization of sp56, a sperm protein hav- terns of testicular olfactory receptor genes during mouse ing specific affinity for ZP3. J Cell Biol 1994;125:867– spermatogenesis. Genes Cells 2006;11:71–81. 878. 746. Walensky LD, Ruat M, Bakin RE, et al. Two novel odor- 769. Mori E, Mori T, Takasaki S. Binding of mouse sperm to ant receptor families expressed in spermatids undergo 5Ј- beta-galactose residues on egg zona pellucida and asialofe- splicing. J Biol Chem 1998;273:9378–9387. tuin-coupled beads. Biochem Biophys Res Commun 1997; 747. Spehr M. Identification of a testicular odorant receptor 238:95–99. mediating human sperm chemotaxis. Science 2003;299: 770. Nixon B, Lu Q, Wassler MJ, et al. Galactosyltransferase 2054–2058. function during mammalian fertilization. Cells Tissues 748. Fukuda M, Yomogida K, Okabe M, et al. Functional char- Organs 2001;168:46–57. acterization of a mouse testicular olfactory receptor and its 771. Thaler CD, Cardullo RA. Defining oligosaccharide speci- role in chemosensing and in regulation of sperm motili- ficity for initial sperm-zona pellucida adhesion in the ty. J Cell Sci 2004;117:5835–5845. mouse. Mol Reprod Dev 1996;45:535–546. 749. Vosshall LB. Olfaction: attracting both sperm and the 772. Cornwall GA, Tulsiani DRP, Orgebin-Crist MC. Inhibi- nose. Curr Biol 2004;14:R918–R920. tion of the mouse sperm surface alpha-d-mannosidase in- 750. Tatsura H, Nagao H, Tamada A, et al. Developing germ hibits sperm-egg binding in vitro. Biol Reprod 1991;44: cells in mouse testis express pheromone receptors. FEBS 913–921. Lett 2001;488:139–144. 773. Tulsiani DRP, Abou-Haila A. Mammalian sperm mole- 751. Goto T, Salpekar A, Monk M. Expression of a testis-spe- cules that are potentially important in interaction with cific member of the olfactory receptor gene family in human female genital tract and egg vestments. Zygote 2001;9: primordial germ cells. Mol Hum Reprod 2001;7:553–558. 51–69. 752. Walensky LD, Roskams AJ, et al. Odorant receptors 774. Rattanachaiyanont M, Weerachatyanukul W, Leveille and desensitization proteins colocalize in mammalian M-C, et al. Anti-SLIP1-reactive proteins exist on human sperm. Mol Med 1995;1:130–141. sperm and are involved in zona pellucida binding. Mol 753. Vanderhaeghen P, Schurmans S, Vassart G, et al. Olfac- Human Reprod 2001;7:633–640. tory receptors are displayed on dog mature sperm 775. Rodeheffer C, Shur BD. Characterization of a novel ZP3- cells. J Cell Biol 1993;123:1441–1452. independent sperm-binding ligand that facilitates sperm 754. Spehr M, Schwane K, Riffell JA, et al. Particulate ade- adhesion to the egg coat. Development 2004;131:503–512. nylate cyclase plays a key role in human sperm olfactory 776. Shur BD, Rodeheffer C, Ensslin MA, et al. Identification receptor-mediated chemotaxis. J Biol Chem 2004;279: of novel gamete receptors that mediate sperm adhesion to 40194–40203. the egg coat. Mol Cell Endocrinol 2006;250:137–148.

AAEP PROCEEDINGS ր Vol. 53 ր 2007 171 MILNE LECTURE

777. Dean J. Reassessing the molecular biology of sperm-egg 799. Runge KE, Evans JE, He Z-Y, et al. Oocyte CD9 is en- recognition with mouse genetics. Bioessay 2004;26:29– riched on the microvillar membrane and required for nor- 38. mal microvillar shape and distribution. Dev Biol 2007; 778. Baibakov B, Gauthier L, Talbot P, et al. Sperm binding to 304:317–325. the zona pellucida is not sufficient to induce acrosome 800. Atif SM, Hasan I, Ahmad N, et al. Fusogenic potential of exocytosis. Development 2007;134:933–943. sperm membrane lipids: nature’s wisdom to accomplish 779. Mugnier S, Lahuec C, Yvon JM, et al. Equine sperm targeted gene delivery. FEBS Lett 2006;580:2183–2190. surface galactosyltransferase and zona glycoprotein ZPC 801. Tsaadon A, Eliyahu E, Shtraizent N, et al. When a sperm are not necessary for sperm binding to the zona pellucida. meets an egg: block to polyspermy. Mol Cell Endocrinol Anim Reprod Sci 2006;94:331–332. 2006;252:107–114. 780. Chiu PCN, Chung M-K, Koistinen R, et al. Cumulus oo- 802. Kumakiri J, Oda S, Kinoshita K, et al. Involvement of phorus-associated glycodelin-C displaces sperm bound gly- Rho family G protein in the cell signaling for sperm incor- codelin-A and -F and stimulates spermatozoa-zona poration during fertilization of mouse eggs: inhibition by pellucida binding. J Biol Chem 2006;282:5378–5388. Clostridium difficile toxin B. Dev Biol 2003;260:522–535. 781. O’Day-Bowman MB, Mavrogianis PA, Minshall RD, et al. 803. Sanchez-Gutierrez M, Contreras RG, Mujica A. Cytocha- In vivo versus in vitro oviductal glycoprotein (OGP) asso- lasin-D retards sperm incorporation deep into the egg cy- ciation with the zona pellucida (ZP) in the hamster and toplasm but not membrane fusion with the egg plasma baboon. Mol Reprod Dev 2002;62:248–256. membrane. Mol Reprod Dev 2002;63:518–528. 782. Verhage HG, Mavrogianis PA, O’Day-Bowman MB, et al. 804. Tremoleda JL, Colenbrander B, Stout TAE, et al. Reorgani- Characteristics of an oviductal glycoprotein and its poten- zation of the cytoskeleton and chromatin in horse oocytes tial role in the fertilization process. Biol Reprod 1998;58: following intracytoplasmic sperm injection. Theriogenology 1098–1101. 2002;58:697–700. 783. Bedford JM. Mammalian fertilization misread? Sperm 805. Bedford SJ, Kurokawa M, Hinrichs K, et al. Patterns of 2ϩ penetration of the eutherian zona pellucida is unlikely to [Ca ]i oscillations after intracytoplasmic sperm injection be a lytic event. Biol Reprod 1998;59:1275–1287. of in vitro and in vivo matured horse oocytes. Theriog- 784. Ohmura K, Kohno N, Kobayashi Y, et al. A homologue of enology 2002;58:701–704. pancreatic trypsin is localized in the acrosome of mamma- 806. Comizzoli P, Urner F, Sakkas D, et al. Up-regulation of lian sperm and is released during acrosome reac- glucose metabolism during male pronucleus formation tion. J Biol Chem 1999;274:29426–29432. determines the early onset of the s phase in bovine 785. Tulsiani DRP, Abou-Haila A, Loeser CR, et al. The bio- zygotes. Biol Reprod 2003;68:1934–1940. logical and functional significance of the sperm acrosome 807. Gardner AJ, Evans JP. Mammalian membrane block to and acrosomal enzymes in mammalian fertilization. Exp polyspermy: new insights into how mammalian eggs pre- Cell Res 1998;240:151–164. vent fertilisation by multiple sperm. Reprod Fertil Dev 786. Drobnis EZ, Yudin AI, Cherr GN, et al. Hamster sperm 2006;18:53–61. penetration of the zona pellucida: Kinematic analysis and 808. Yanagimachi R. Male gamete contibutions to the embryo. mechanical implications. Dev Biol 1988;130:311–323. Ann NY Acad Sci 2005;1061:203–207. 787. Katz DF, Cherr GN, Lambert H. The evolution of ham- 809. Dumollard R, Duchen M, Sardet C. Calcium signals and ster sperm motility during capacitation and interaction mitochondria at fertilisation. Semin Cell Dev Biol 2006; with the ovum vestments in vitro. Gamete Res 1986;14: 17:314–323. 333–346. 810. Kline D, Kline JT. Repetitive calcium transients and the 788. Wassarman PM, Jovine L, Qi H, et al. Recent aspects of role of calcium in exocytosis and cell cycle activation in the mammalian fertilization research. Mol Cell Endocrinol mouse egg. Dev Biol 1992;149:80–89. 2005;234:95–103. 811. Kurokawa M, Sato K, Wu H, et al. Functional, biochem- 789. Rubinstein E, Ziyyat A, Wolf JP, et al. The molecular ical, and chromatographic characterization of the complete 2ϩ players of sperm-egg fusion in mammals. Semin Cell Dev [Ca ]i oscillation-inducing activity of porcine sperm. Dev Biol 2006;17:254–263. Biol 2005;285:376–392. 790. Cuasnicu PS, Ellerman DA, Cohen DJ, et al. Moecular 812. Swann K, Saunders CM, Lai FA. The cytosolic sperm mechanisms involved in mammalian gamete fusion. Arch factor that triggers Ca2ϩ oscillations and egg activation in Med Res 2001;32:614–618. mammals is a novel phospholipase C: PLCzeta. Repro- 791. Cameo MS, Blaquier JA. Androgen-controlled specific duction 2004;127:431–439. proteins in rat epididymis. J Endrocrinol 1976;69:47–55. 813. Saunders CM, Swann K, Lai FA. PLCzeta, a sperm-spe- 792. Rochwerger L, Cuasnicu PS. Redistribution of a rat cific PLC and its potential role in fertilization. Biochem sperm epididymal glycoprotein after in vitro and in vivo Soc Symp 2007;74:23–36. capacitation. Mol Reprod Dev 1992;31:34–41. 814. Evans JP, Florman HM. The state of the union: the cell 793. Gan SW, Xin L, Torres J. The transmembrane homotri- biology of fertilization. Nat Cell Biol 2002;4(Suppl):57–63. mer of ADAM 1 in model lipid bilayers. Protein Sci 2007; 815. Leese HJ, Barton AM. Production of pyruvate by isolated 16:285–292. mouse cumulus cells. J Exp Zool 1985;234:231–236. 794. Kim E, Yamashita M, Nakanishi T, et al. Mouse sperm 816. Brinsko SP, Varner DD. Artificial insemination and preser- lacking ADAM1b/ADAM2 fertilin can fuse with the egg vation of semen. In: Blanchard TL, Varner DD, eds. The plasma membrane and effect fertilization. J Biol Chem veterinary clinics of North America—equine practice (stal- 2006;281:5634–5639. lion management). Philadelphia: W.B. Saunders, 1992; 795. Inoue N, Ikawa M, Isotani A, et al. The immunoglobulin 205–218. superfamily protein Izumo is required for sperm to fuse 817. Kenney RM, Hurtgen J, Pierson R, et al. Theriogenology with eggs. Nature 2005;434:234–238. and the equine. Part II. The stallion. Hastings, NE: 796. Goncalves RF, Wolinetz CD, Killian GJ. Influence of argi- Society for Theriogenology, 1983. nine-glycine-aspartic acid (RGD), integrins (alpha-v and 818. Blanchard TL, Varner DD, Schumacher J, et al. Exami- alpha-5) and osteopontin on bovine sperm-egg binding, and nation of the stallion for breeding soundness. In: Blan- fertilization in vitro. Theriogenology 2007;67:468–474. chard TL, Varner DD, Schumacher J, Love CC, Brinsko SP, 797. Stein KK, Go JC, Lane WS, et al. Proteomic analysis of Rigby SL, eds. Manual of equine reproduction. St. Lou- sperm regions that mediate sperm-egg interactions. Pro- is: Mosby, 2003;143–164. teomics 2006;6:3533–3543. 819. Jasko DJ: Evaluation of stallion semen. In: Blanchard 798. Stein KK, Primakoff P, Myles D. Sperm-egg fusion: TL, Varner DD, eds. The veterinary clinics of North Amer- events at the plasma membrane. J Cell Sci 2004;177: ica—equine practice (stallion management). Philadel- 6269–6274. phia: W.B. Saunders, 1992;129–148.

172 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

820. Pickett BW. Reproductive evaluation of the stallion. In: 844. Makhlouf AA, Niederberger C. DNA integrity tests in McKinnon AO, Voss JL, eds. Equine reproduction. Phila- clinical practice: it is not a simple matter of black and delphia: Lea and Febiger, 1993;755–768. white (or red and green). J Androl 2006;27:316–323. 821. Jasko DJ, Little TV, Lein DH, et al. Determination of 845. O’Brien J, Zini A. Sperm DNA integrity and male infer- stallion semen quality and its relationship with fertili- tility. Urology 2005;65:16–22. ty. J Reprod Fertil Suppl 1991;44:649–650. 846. Aoki VW, Emery BR, Liu L, et al. Protamine levels vary 822. Jasko DJ, Little TV, Lein DH, et al. Comparison of sper- between individual sperm cells of infertile human males matozoal movement and semen characteristics with fertil- and correlate with viability and DNA integrity. J Androl ity in stallions: 64 cases (1987–1988). J Am Vet Med 2006;27:890–898. Assoc 1992;200:979–985. 847. Lewis SEM, Aitken RJ. DNA damage to spermatozoa has 823. Kenney RM, Kingston RS, Rajamannon AH, et al. Stal- impacts on fertilization and pregnancy. Cell Tissue Res lion semen characteristics for predicting fertility, in Pro- 2005;322:33–41. ceedings. Am Assoc Equine Pract 1971;17:53–67. 848. Aoki VW, Moskovtsev SI, Willis J, et al. DNA integrity is 824. Colenbrander B, Puyk H, Zandee AR, et al. Evaluation of compromised in protamine-deficient human sperm. J An- the stallion for breeding. Acta Vet Scand Suppl 1992;88: drol 2005;26:741–748. 29–37. 849. Sailer BL, Sarkar LJ, Bjordahl JA, et al. Effects of heat 825. Amann RP. Can the fertility potential of a seminal sam- stress on mouse testicular cells and sperm chromatin ple be predicted accurately? J Androl 1989;10:89–98. structure. J Androl 1997;18:294–301. 826. Bielanski W, Dudek E, Bittmar A, et al. Some character- 850. Oliva R. Protamines and male infertility. Hum Reprod istics of common abnormal forms of spermatozoa in highly Update 2006;12:417–435. fertile stallions. J Reprod Fertil Suppl 1982;32:21–26. 851. Fraga CG, Motchnik PA, Shigenaga MK, et al. Ascorbic 827. David JSE. Semen examination for fertility potential in acid protects against endogenous oxidative DNA damage in stallions. J Reprod Fertil Suppl 1982;32:635. human sperm. Proc Natl Acad Sci USA 1991;88:11003– 828. Voss JL, Pickett BW, Loomis PR. The relationship be- 11006. tween seminal characteristics and fertility in thoroughbred 852. Fatehi AN, Bevers MM, Schoevers E, et al. DNA damage stallions. J Reprod Fertil Suppl 1982;32:635–636. in bovine sperm does not block fertilization and early em- 829. Rousset H, Chanteloube P, Magistrini M, et al. Assessment bryonic development but induces apoptosis after the first of fertility and semen evaluations of stallions. J Reprod cleavages. J Androl 2006;27:176–188. Fertil Suppl 1987;35:25–31. 853. Agarwal A, Said TM. Role of sperm chromatin abnormal- 830. Dowsett KF, Pattie WA. Characteristics and fertility of ities and DNA damage in male infertility. Hum Reprod stallion semen. J Reprod Fertil Suppl 1982;32:1–8. Update 2003;9:331–345. 831. Voss JL, Pickett BW, Squires EL. Stallion spermatozoal 854. Chohan KR, Griffin JT, Lafromboise M, et al. Compari- morphology and motility and their relationships to fertil- son of chromatin assays for DNA fragmentation evaluation ity. J Am Vet Med Assoc 1981;178:287–289. in human sperm. J Androl 2006;27:53–59. 832. Clement F, Ladonnet Y, Magistrini M. Sperm morphol- 855. Erenpreiss J, Spano M, Erenpreisa J, et al. Sperm chro- ogy and fertility. Anim Reprod Sci 2001;68:362–363. matin structure and male fertility: biological and clinical 833. Kenney RM, Evenson DP, Garcia MC, et al. Relation- aspects. Asian J Androl 2006;8:11–29. ships between sperm chromatin structure, motility, and 856. Evenson DP, Wixon R. Comparison of the Halosperm test morphology of ejaculated sperm, and seasonal pregnancy kit with the sperm chromatin structure assay (SCSA) in- rate. Biol Reprod Monogr 1 1995;6:647–653. fertility test in relation to patient diagnosis and prognosis. 834. Wang C, Chan SYW, Ng M, et al. Diagnostic value of Fertil Steril 2005;84:846–849. sperm function tests and routine semen analyses in fertile 857. Linfor JJ, Meyers SA. Detection of DNA damage in re- and infertile men. J Androl 1988;9:384–389. sponse to cooling injury in equine spermatozoa using sin- 835. Aitken RJ. Sperm function tests and fertility. Int J An- gle-cell gel electrophoresis. J Androl 2002;23:107–113. drol 2006;29:69–75. 858. Meyers SA, Overstreet JW, Liu IKM, et al. Capacitation 836. Rodriguez-Martinez H. Can we increase the estimative in vitro of stallion spermatozoa: Comparison of progest- value of semen assessment? Reprod Domest Anim 2006;41: erone-induced acrosome reactions in fertile and subfertile 2–10. males. J Androl 1995;16:47–54. 837. Graham JK, Moce E. Fertility evaluation of frozen/ 859. Cheng FP, Gadella BM, Voorhout WF, et al. Progesterone- thawed semen. Theriogenology 2005;64:492–504. induced acrosome reaction in stallion spermatozoa is medi- 838. Evenson DP, Darzynkiewica Z, Melamed MR. Relation ated by a plasma membrane progesterone receptor. Biol of mammalian sperm chromatin heterogenecity to fertil- Reprod 1998;59:733–742. ity. Science 1980;210:1131–1133. 860. Rathi R, Nielen M, Cheng FP, et al. Exposure of proges- 839. Love CC. The sperm chromatin structure assay: a re- terone receptors on the plasma membranes of stallion sper- view of clinical applications. Anim Reprod Sci 2005;89: matozoa as a parameter for prediction of fertility. 39–45. J Reprod Fertil Suppl 2000;56:87–91. 840. Love CC, Kenney RM. The relationship of increased sus- 861. Varner DD, Brinsko SP, Blanchard TL, et al. Subfertility ceptibility of sperm DNA to denaturation and fertility in in stallions associated with acrosome dysfunction. In Pro- the stallion. Theriogenology 1998;57:955–972. ceedings. Am Assoc Equine Pract 2001;47:227–228. 841. Evenson DP, Sailer BL, Jost LK. Relationship between 862. Brinsko SP, Love CC, Bauer JE, et al. Cholesterol-to- stallion sperm deoxyribonucleic acid (DNA) susceptibility phospholipid ratio in whole sperm and seminal plasma to denaturation in situ and presence of DNA strand breaks: from fertile stallions and stallions with unexplained sub- implications for fertility and embryo viability. Biol Re- fertility. Anim Reprod Sci 2007;99:65–71. prod Mono 1 1995;6:655–659. 863. Varner DD, Ward CR, Storey BA, et al. Induction and 842. Evenson DP, Jost LK, Varner DD. Relationship between characterization of the acrosome reaction in equine sper- sperm nuclear protamine free -SH status and susceptibility matozoa. Am J Vet Res 1987;48:1383–1389. to DNA denaturation. J Reprod Fertil Suppl 2000;56: 864. Brum AM, Thomas AD, Sabeur K, et al. Evaluation of 401–406. coomassie blue staining of the acrosome of equine and 843. Love CC, Kenney RM. Scrotal heat stress induces altered canine spermatozoa. Am J Vet Res 2006;67:358–362. sperm chromatin structure associated with decrease in 865. Cardullo RA, Florman HM. Strategies and methods for protamine disulfide bonding in the stallion. Biol Reprod evaluating acrosome reaction. Methods Enzymol 1993;225: 1999;60:615–620. 136–153.

AAEP PROCEEDINGS ր Vol. 53 ր 2007 173 MILNE LECTURE

866. Bosard T, Love C, Brinsko SP, et al. Evaluation and diag- 888. Love CC, Thompson JA, Brinsko SP, et al. Relationship nosis of acrosome function/dysfunction in the stallion. Anim between stallion sperm motility and viability as detected Reprod Sci 2005;89:215–217. by two fluorescence staining techniques using flow cytom- 867. Varner DD, Thompson JA, Blanchard TL, et al. Induction etry. Theriogenology 2003;60:1127–1138. of the acrosome reaction in stallion spermatozoa: effects 889. Garner DL, Thomas CA. Organelle-specific probe JC-1 of incubation temperature, incubation time, and ionophore identifies membrane potential differences in the mitochon- concentration. Theriogenology 2002;58:303–306. drial function of bovine sperm. Mol Reprod Dev 1999;53: 868. Bosard TS. Flow cytometric evaluation of acrosome func- 222–229. tion/dysfunction in the stallion. College Station, TX: 890. Kavak A, Johannisson A, Lundeheim N, et al. Evaluation Texas A&M University; 2006. MS thesis. of cryopreserved stallion semen from Tori and Estonian 869. Carrell BP. Validation of pisum sativum agglutinin fluo- breeds using CASA and flow cytometry. Anim Reprod Sci rescent marker for stallion spermatozoal acrosomes with 2003;76:205–216. transmission electron microscopy. College Station, TX: 891. Kirk ES, Graham JK, Bruemmer JE, et al. Evaluating Texas A&M University; 2001. MS thesis. frozen equine semen by flow cytometry. Anim Reprod Sci 870. Graham JK. Assessment of sperm quality: a flow cyto- 2001;68:348–349. metric approach. Anim Reprod Sci 2001;68:239–247. 892. Cross NL, Morales P, Overstreet JW, et al. Two simple 871. Silva PFN. Detection of damage in mammalian sperm methods for detecting acrosome-reacted human sperm. Ga- cells. Theriogenology 2006;65:958–979. mete Res 1986;15:213–226. 872. Farlin ME, Jasko DJ, Graham JK, et al. Assessment of 893. Cross NL, Meizel S. Methods for evaluating the acroso- Pisum sativum agglutinin in identifying acrosomal damage mal status of mammalian sperm. Biol Reprod 1989;41: in stallion spermatozoa. Mol Reprod Dev 1992;32:23–27. 635–641. 873. Thomas CA, Garner DL, DeJarnette JM, et al. Effect of 894. Graham JK, Kunze E, Hammerstedt RH. Analysis of cryopreservation on bovine sperm organelle function and sperm cell viability, acrosomal integrity, and mitochondrial viability as determined by flow cytometry. Biol Reprod function using flow cytometry. Biol Reprod 1990;43:55– 1998;58:786–793. 64. 874. Ward CR, Storey BT. Determination of the time course of 895. Kavak A, Johannisson A, Malmgren L, et al. Post-thaw capacitation in mouse spermatozoa using a chlortetracy- evaluation of stallion spermatozoa using triple fluorescent cline fluorescence assay. Dev Biol 1984;104:287–296. staining and flow cytometry. Anim Reprod Sci 2001;68: 875. de Vries KJ, Wiedmer T, Sims PJ, et al. Caspase-inde- 349–350. pendent exposure of aminophospholipids and tyrosine 896. Cai K, Yang J, Guan M, et al. Single UV excitation of phosphorylation in bicarbonate responsive human sperm Hoechst 33342 and propidium iodide for viability assess- cells. Biol Reprod 2003;68:2122–2134. ment of rhesus monkey spermatozoa using flow cytometry. 876. Gadella BM, Miller NGA, Colenbrander B, et al. Flow Arch Androl 2005;51:371–383. cytometric detection of transbilayer movement of fluores- 897. Gravance CG, Garner DL, Baumber J, et al. Assessment cent phospholipid analogues across the boar sperm plasma of equine sperm mitochondrial function using JC-1. The- membrane: elimination of labelling artefacts. Mol Re- riogenology 2000;53:1691–1703. prod Dev 1999;53:108–125. 898. Ball BA, Fagnan MS, Dobrinski I. Determination of acro- 877. Miki K, Qu W, Goulding EH, et al. Glyceraldehyde sin amidase activity in equine spermatozoa. Theriogenol- 3-phosphate dehydrogenase-S, a sperm-specific glycolytic ogy 1997;48:1191–1198. enzyme, is required for sperm motility and male fertili- 899. Kennedy WP, Kaminski JM, Van der Ven HH, et al. ty. Proc Natl Acad Sci USA 2004;101:16506. A simple, clinical assay to evaluate the acrosin activity of 878. Smiley ST, Reers M, CMottola-Hartshorn C, et al. Intra- cellular heterogeneity in mitochondrial membrane poten- human spermatozoa. J Androl 1989;10:221–231. tials revealed by a J-aggregate-forming lipophilic cation 900. Cross NL. A modified and improved assay for sperm ami- JC-1. Proc Natl Acad Sci USA 1991;88:3671–3675. dase activity. J Androl 11:409413, 1990. 879. Cossarizza A, Ceccarelli D, Masini A. Functional heteroge- 901. Sousa APM, Gomes-Santos CSS, Ramalho-Santos J. neity of an isolated mitochondrial population revealed by Localization of snares, NSF and Caveolin 1 in human sper- cytofluorometric analysis at the single organelle level. Exp matozoa: relationship with seminal parameters. Arch Cell Res 1996;222:84–94. Androl 2006;52:347–353. 880. Garner DL, Thomas CA, Joerg HW, et al. Fluorometric 902. Desvousges AL, Dow CA, Hayna JT, et al. Heat shock assessments of mitochondrial function and viability in induces apoptosis in equine spermatozoa. Anim Reprod cryopreserved bovine spermatozoa. Biol Reprod 1997;57: Sci 2006;94:125–126. 1401–1406. 903. Brum A, Sabeur K, Ball BA. Apoptotic-like changes in 881. Gravance CG, Garner DL, Miller MG, et al. Fluorescent equine spermatozoa after separation by density-gradient probes and flow cytometry to assess rat sperm integrity centrifugation or after cryopreservation. Anim Reprod and mitochondrial function. Reprod Toxicol 2001;15:5– Sci 2006;94:138–139. 10. 904. Wang X, Sharma RK, Sikka SC, et al. Oxidative stress is 882. Neild D, Chaves G, Flores M, et al. Hypoosmotic test in associated with increased apoptosis leading to spermatozoa equine spermatozoa. Theriogenology 1999;51:721–727. DNA damage in patients with male factor infertility. Fer- 883. Jeyendran RS, Van der Ven HH, et al. The hypoosmotic til Steril 2003;80:531–535. swelling test: an update. Arch Androl 1992;29:105–116. 905. Said TM, Paasch U, Glander H-J, et al. Role of caspases 884. Kenney RM. Manual for clinical fertility evaluation of the in male infertility. Hum Reprod Update 2004;10:39–51. stallion. Hastings, NE: Society for Theriogenology, 906. Bellin ME, Oyarzo JN, Hawkins HE, et al. Fertility-as- 1983. sociated antigen on bull sperm indicates fertility potential. 885. Garner DL, Dobrinsky JR, Welch GR, et al. Porcine J Anim Sci 1998;76:2032–2039. sperm viability, oocyte fertilization and embryo develop- 907. Buffone MG, Calamera JC, Verstraeten SV, et al. Capaci- ment after staining spermatozoa with SYBR-14. Theriog- tation-associated protein tyrosine phosphorylation and enology 1996;45:1103–1113. membrane fluidity changes are impaired in the spermato- 886. Garner DL, Johnson LA. Viability assessment of mam- zoa of asthenozoospermic patients. Reproduction 2005; malian sperm using SYBR-14 and propidium iodide. Biol 129:697–705. Reprod 1995;53:276–284. 908. Yunes R, Doncel GF, Acosta AA. Incidence of sperm-tail 887. Garner DL, Thomas CA, Allen CH. Effect of semen dilu- tyrosine phophorylation and hyperactivated motility in tion on bovine sperm viability as determined by dual-DNA normozoospermic and asthenozoospermic human sperm staining and flow cytometry. J Androl 1997;18:324–331. samples. Biocell 2003;27:29–36.

174 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

909. Tardif S, Dube C, Chavalier S, et al. Capacitation is as- 929. Foresta C, Flohe L, Garolla A, et al. Male fertility is sociated with tyrosine phosphorylation and tyrosine ki- linked to the selenoprotein phospholipid hydroperoxide nase-like activity in pig sperm proteins. Biol Reprod glutathione peroxidase. Biol Reprod 2002;67:967–971. 2001;65:784–792. 930. Stradaioli G, Parillo F, Sylla L, et al. Enzymatic and 910. Bianchi PG, Manicardi GC, Urner F, et al. Chromatin immunohistochemical evaluation of phospholipid hy- packaging and morphology in ejaculated human sprmato- droperoxide glutathione peroxidase (PHGPx) in bovine zoa: evidence of hidden anomalies in normal spermato- spermatozoa. Proc Int Cong Anim Reprod 2004;I:192. zoa. Mol Hum Reprod 1996;2:139–144. 931. Weir CP. Spermatozoa have decreased antioxidant enzy- 911. Nasr-Esfahani MH, Razavi S, Mardani M. Relation be- matic capacity and increased reactive oxygen species pro- tween different human sperm nuclear maturity tests and duction during aging in the brown Norway rat. J Androl in vitro fertilization. J Assist Reprod Genet 2001;18:219– 2007;28:229–240. 225. 932. Mesequer M, de los Santos MJ, Simon C, et al. Effect of 912. Iranpour FG, Nasr-Esfahani MH, Yalojerdi MR, et al. sperm glutathione peroxidases 1 and 4 on embryo asymmetry Chromomycin A3 staining as a useful tool for evaluation of and blastocyst quality in ooctye donation cycles. Fertil Steril male fertility. J Assist Reprod Genet 2000;17:60–66. 2006;86:1376–1385. 913. Franken DR, Franken CJ, de la Guerre H, et al. Normal 933. Aitken RJ, Clarkson JS, Hargreave TB, et al. Analysis of sperm morphology and chromatin packaging: comparison the relationship between defective sperm function and the between aniline blue and chromomycin A3 statining. An- generation of reactive oxygen species in cases of oligozo- drologia 1999;31:361–366. ospermia. J Androl 1989;10:214–220. 914. Gradil C, Yoon S-Y, Brown J, et al. PLC-Zeta: a marker 934. Ball BA, Baumber J, Vo A, et al. Effect of oxidative stress of fertility for stallions. Anim Reprod Sci 2006;94:23–25. on equine spermatozoa. Anim Reprod Sci 2001;68:364. 915. Swann K, Saunders CM, Rogers NT, et al. PLCzeta: 935. Guthrie HD, Welch GR. Determination of intracellular a sperm protein that triggers calcium ion oscillations and reactive oxygen species and high mitochondrial membrane agg activation in mammals. Semin Cell Dev Biol 2006;17: potential in Percoll-treated viable boar sperm using fluo- 264–273. rescence-activated flow cytometry. J Anim Sci 2006;84: 916. Neild DM, Gadella BM, Colenbrander B, et al. Lipid per- 2089–2100. oxidation in stallion spermatozoa. Theriogenology 2002; 936. Said TM, Agarwal A, Sharma RK, et al. Human sperm 58:295–298. superoxide anion generation and correlation with semen 917. Neild DM, Brouwers JFHM, Colenbrander B, et al. Lipid quality in patients with male infertility. Fertil Steril peroxide formation in relation to membrane stability of 2004;82:871–877. fresh and frozen thawed stallion spermatozoa. Mol Re- 937. Aitken RJ, Irvine DS, Wu FC. Prospective analysis of prod Dev 2005;72:230–238. sperm-oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am J Obstet 918. Guthrie HD, Welch GR. Use of fluorescence-activated Gynecol 1991;164:542–551. flow cytometry to determine membrane lipid peroxidation 938. Whittington K, Harrison SC, Williams KM, et al. Reac- during hypothermic liquid storage and freeze-thawing of tive oxygen species (ROS) production and the outcome of viable boar sperm loaded with 4, 4-difluoro-5-(4-phenyl- diagnostic tests of sperm function. Int J Androl 1999;22: 1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-unde- 236–242. canoic acid. J Anim Sci 2007;85:1402–1411. 939. Pesch S, Bergmann M, Bostedt H. Determination of some 919. Brouwers JFHM, Gadella BM. In situ detection and lo- enzymes and macro- and microelements in stallion seminal calization of lipid peroxidation in individual bovine sperm plasma and their correlations to semen quality. Theriog- cells. Free Radic Biol Med 2003;35:1382–1391. enology 2006;66:307–313. 920. Brouwers JF, Silva PFN, Gadella BM. New assays for 940. Mollova M, Atanassov B, Nedkova R, et al. Biochemical detection and localization of endogenous lipid peroxidation and immunochemical characterization of boar sperm products in living boar sperm after BTS dilution or after flagellar protein with role in hyperactivation/capacitation freeze-thawing. Theriogenology 2005;63:458–469. process. Reprod Biol 2006;6:79–94. 921. Roca J, Gil MA, Hernandez M, et al. Survival and fertility 941. Yousef MI, Esmail AM, Baghdadi HH. Effect of isofla- of boar spermatozoa after freeze-thawing in extender sup- vones on reproductive performance, testosterone levels, plemented with butylated hydroxytoluene. J Androl lipid peroxidation, and seminal plasma biochemistry of 2004;25:397–405. male rabbits. J Environ Sci Health B 2004;39:819–833. 922. Stradaioli G, Gennevieve A, Magistrini M. Evaluation of 942. Noquera Velasco JA, Tovar Zapata I, Martinez Hernandez lipid peroxidation by thiobarbituric acid test in equine P, et al. Lactic dehydrogenase-C4 activity in seminal spermatozoa. Anim Reprod Sci 2001;68:320–322. plasma and male infertility. Fertil Steril 1993;60:331– 923. Stradaioli G, Magistrini M. A comparative evaluation 335. of thiobarbituric acid methods for determination of malon- 943. Huszar G, Corrales M, Vigue L. Correlation between dialdehyde in equine spermatozoa. Theriogenology 2002; sperm creatine phosphokinase activity and sperm concen- 58:347–350. tration in normospermic and oligospermic men. Gamete 924. Aitken RJ, Harkiss D, Buckingham DW. Analysis of lipid Res 1988;19:67–75. peroxidation mechanisms in human spermatozoa. Mol 944. Huszar G, Vigue L. Incomplete development of human sper- Reprod Dev 1993;35:302–315. matozoa is associated with increased creatine phosphokinase 925. Martinez P, Proverbio F, Camejo MI. Sperm lipid peroxi- concentration and abnormal head morphology. Mol Reprod dation and pro-inflammatory cytokines. Asian J Androl Dev 1993;34:292–298. 2007;9:102–107. 945. Huszar G, Stone K, Dix D, et al. Putative creatine kinase 926. Agarwal A, Prabakaran SA. Mechanism, measurement, M-isoform in human sperm is identified as the 70-kilodal- and prevention of oxidative stress in male reproductive ton heat shock protein HspA2. Biol Reprod 2000;63:925– physiology. Indian J Exp Biol 2005;43:963–974. 932. 927. Peris SI, Bilodeau JF, Dufour M, et al. Impact of cryo- 946. Huszar G, Ozkavukcu S, Jakab A, et al. Hyaluronic acid preservation and reactive oxygen species on DNA integrity, binding ability of human sperm reflects cellular maturity lipid peroxidation, and functional parameters in ram and fertilizing potential: selection of sperm for intracyto- sperm. Mol Reprod Dev 2007;74:878–892. plasmic sperm injection. Curr Opin Obstet Gynecol 2006; 928. Kumar TR, Muralidhara. Induction of oxidative stress by 18:260–267. organic hydroperoxides in testis and epididymal sperm of 947. Ellerman DA, Cohen DJ, Da Ros VG, et al. Sperm protein rats in vivo. J Androl 2007;28:77–85. “DE” mediates gamete fusion through an evolutionarily

AAEP PROCEEDINGS ր Vol. 53 ր 2007 175 MILNE LECTURE

conserved site of the CRISP family. Dev Biol 2006;297: 969. Amann RP, Katz DF. Reflections on CASA after 25 years. 228–237. J Androl 2004;25:317–325. 948. Reineke A, Hess O, Schambony A, et al. Sperm-assoicated 970. Jasko DJ, Lein DH, Foote RH. A comparison of two com- seminal plasma proteins—a novel approach for the evalua- puter-automated semen analysis instruments for the eval- tion of sperm fertilizing ability of stallions. Pferdcheikunde uation of sperm motion characteristics in the stallion. J 1999;6:531–537. Androl 1990;11:453–459. 949. Jude R, Giese A, Piumi F, et al. Molecular characteriza- 971. Varner DD, Vaughan SD, Johnson L. Use of a computer- tion of the CRISP 1 gene-a candidate gene for stallion ized system for evaluation of equine spermatozoal motility. fertility. Theriogenology 2002;58:417–420. Am J Vet Res 1991;52:224–230. 950. Meyers SA, Rosenberger A, Orpneck K. Localization and 972. Wessel MT, Althouse GC. Validation of an objective ap- cellular distribution of a unique hyaluronidase in stallion proach for simultaneous assessment of viability and motil- spermatozoa during epididymidal transit. J Reprod Fertil ity of fresh and cooled equine spermatozoa. Anim Reprod Suppl 2000;56:79–86. Sci 2006;94:21–22. 951. Jonakova V, Manaskova P, Kraus M, et al. Sperm surface 973. Holt C, Holt WV, Moore HDM, et al. Objectively mea- proteins in mammalian fertilization. Mol Reprod Dev sured boar sperm motility parameters correlate with the 2000;56(Suppl 2):277. outcomes of on-farm inseminations: results of two fertil- 952. Rodriguez-Martinez H, Iborra A, Martinez P, et al. Im- ity trials. J Androl 1997;18:312–323. munoelectronmicroscopic imaging of spermadhesin AWN 974. Williams WW. Cytology of the human spermatozoon. epitopes on boar spermatozoa bound in vivo to the zona Fertil Steril 1950;1:199–215. pellucida. Reprod Fertil Dev 1998;10:491–497. 975. Casarett GW. A one-solution stain for spermatozoa. 953. Jonakova V, Kraus M, Veselsky L, et al. Spermadhesins Stain Technol 1953;28:125–127. of the AQN and AWN families, DQH sperm surface protein 976. Penedo MC, Millon LV, Bernoco D, et al. International and HNK protein in the heparin-binding fraction of boar equine gene mapping workshop report: a comprehensive seminal plasma. J Reprod Fertil 1998;114:25–34. linkage map constructed with data from new markers and 954. Boue F, Sullivan R. Cases of human infertility are asso- by merging four mapping resources. Cytogenet Genome ciated with the absence of P34H an epididymal sperm Res 2005;111:5–15. antigen. Biol Reprod 1996;54:1018–1024. 977. Chowdhary BP, Taudsepp T, Kata SR, et al. The first- 955. Sullivan R, Legare C, Villeneuve M, et al. Levels of P34H, generation whole-genome radiation hybrid map in the a sperm protein of epididymal origin, as a predictor of horse identifies conserved seigments in human and mouse conventional in vitro fertilization outcome. Fertil Steril genomes. Genome Res 2003;13:742–751. 2006;85:1557–1559. 978. Chowdhary BP, Bailey E. Equine genomics: galloping to 956. Xia WY, Huang YF, Xu XF. Epididymal sperm protein new frontiers. Cytogenet Genome Res 2003;102:184–188. 979. Tozaki T, Hirota KI, Hasegawa T, et al. Whole-genome P34H and male reproduction. Zhonghua Nan Ke Xue linkage disequilibrium screening for complex traits in 2002;8:356–358. horses. Mol Genet Genomics 2007;277:663–672. 957. Sullivan R. Male fertility markers, myth or reality. 980. Tozaki T, Swinburne J, Hirota KI, et al. Improved reso- Anim Reprod Sci 2004;82–83:341–347. lution of the comparative horse-human map: investigat- 958. Bailey LB, Brady HB, Tardif S, et al. Zonadhesin expres- ing markers with in silico and linkage mapping sion and localization in stallion testes. Anim Reprod Sci approaches. Gene 2007;392:181–186. 2006;94:56–59. 981. Shiue Y-L, Bickel LA, Caetano AR, et al. A synteny map 959. Breazeale KR, Brady HA, Bi M, et al. Biochemical prop- of the horse genome comprised of 240 microsatellite and erties and localization of zonadhesin in equine spermato- RAPD markers. Anim Genet 1999;30:1–9. zoa. Theriogenology 2002;58:359–362. 982. Wrobel G, Priming M. Mammalian male germ cells are 960. Hardy DM, Garbers DL. A sperm membrane protein that fertile ground for expression profiling of sexual reproduc- binds in a species-specific manner to the egg extracellular tion. Reproduction 2005;129:1–7. matrix is homologous to von Willebrand factor. J Biol 983. Chan W-Y, Wu SM, Ruszczyk L, et al. The complexity of Chem 1995;270:26025–26028. antisense transcription revealed by the study of developing 961. Bi M, Hickox JR, Winfrey VP, et al. Processing, localiza- male germ cells. Genomics 2006;87:681–692. tion and binding activity of zonadhesin suggest a dfunction 984. Martins RP, Krawetz SA. RNA in human sperm. Asian in sperm adhesion to the zona pelucida during exocytosis. J Androl 2005;7:115–120. Biochem J 2003;375:477–488. 985. He Z, Chan WYC, Dym M. Microarray technology offers a 962. Lea IA, Sivashanmuqam P, O’Rand MG. Zonadhesin: novel tool for the diagnosis and identification of therapeu- characterization, localization, and zona pellucida bind- tic targets for male infertility. Reproduction 2006;132: ing. Biol Reprod 2001;65:1691–1700. 11–19. 963. Gao A, Garbers DL. Species diversity in the structure of 986. Laughlin AM, Forrest DW, Varner DD, et al. DNA mi- zonadhesin, a sperm-specific membrane protein containing croarray analysis of gene expression in testicular tissue of multiple cell adhesion molecule-like domains. J Biol stallions. Theriogenology 2002;58:413–415. Chem 1998;273:3415–3421. 987. Turner RM, Casas-Dolz R. Differential gene expression 964. Eager FE, Brady HA, Thompson LD, et al. Biochemical in stallions with idiopathic testicular degeneration. The- properties of zonadhesin in spermatozoa of fertile and sub- riogenology 2002;58:421–424. fertile stallions. Proc Equine Sci Soc 2005;19:95–100. 988. Smith GW, Rosa GJM. Interpretation of microarray data: 965. Klinefelter GR, Welch JE, Perreault SD, et al. Localiza- trudging out of the abyss towards elucidation of biological tion of the sperm protein SP22 and inhibition of fertililty in significance. J Anim Sci 2007;85(E Suppl):E20–E23. vivo and in vitro. J Androl 2002;23:48–63. 989. Leeb T, Sieme H, Topfer-Petersen E. Genetic markers for 966. Miller LMJ, Greene ES, Roberts KP, et al. Expression of stallion fertility-lessons from humans and mice. Anim equine sperm protein 22kDa (SP22) in testicular and epi- Reprod Sci 2005;89:21–29. didymal tissue. Anim Reprod Sci 2006;94:54–55. 990. Andersen JS, Mann M. Organellar proteomics: turning 967. Miller LT, Troedsson MH, Duoos LA, et al. Immunocyto- inventories into insights. EMBO Rep 2006;7:874–879. chemical detection and localization of sperm protein 22 991. Pilch B, Mann M. Large-scale and high-confidence pro- (SP22) in fresh and cryopreserved equine semen. Biol teomic analysis of human seminal plasma. Genome Biol Reprod 2003;68(Suppl 1):166–167. 2006;7:R40. 968. Wrench N, Pinto CRF, Klinefelter GR, et al. Seasonal 992. Martinez-Heredia J, Estanyol JM, Ballesca JL, et al. expression and pattern of SP22 immunolocalization in stal- Proteomic identification of human sperm proteins. Pro- lion semen. Anim Reprod Sci 2006;94:32–35. teomics 2006;6:4356–4369.

176 2007 ր Vol. 53 ր AAEP PROCEEDINGS MILNE LECTURE

993. Chu DS, Liu H, Nix P, et al. Sperm chromatin proteomics netic response at fetal and prepubertal ages. Toxicol Sci identifies evolutionarily conserved fertility factors. Na- 2006;93:369–381. ture 2006;443:101–105. 1004. Fukushima T, Yamamoto T, Kikkawa R, et al. Effects of 994. Bailey JL, Tardif S, Dube C, et al. Use of phosphopro- male reproductive toxicants on gene expression in rat tes- teomics to study tyrosine kinase activity in capacitating tes. J Toxicol Sci 2005;30:195–206. boar sperm. Kinase activity and capacitation. Theriog- 1005. Ball BA, Sabeur K, Allen WR. Uptake of exogenous DNA enology 2005;63:599–614. by equine spermatozoa and applications in sperm-medi- 995. Lalancette C, Faure RL, Leclerc P. Identification of the ated gene transfer. Anim Reprod Sci 2006;94:115–116. proteins present in the bull sperm cytosolic fraction en- 1006. Robl JM, Wang Z, Kasinathan P, et al. Transgenic ani- riched in tyrosine kinase activity: a proteomic approach. mal production and animal biotechnology. Theriogenol- Proteomics 2006;6:4523–4540. ogy 2007;67:127–133. 996. Swan SH, Elkin EP, Fenster L. The question of declining 1007. Dobrinski I. Germ cell transplantation and testis tissue sperm density revisited: an analysis of 101 studies pub- xenografting in domestic animals. Anim Reprod Sci 2005; lished 1934–1996. Environ Health Perspect 2000; 89:137–145. 108:961–966. 1008. Hamra FK, Chapman KM, Nguyen DM, et al. Self re- 997. Carlsen E, Giwercman A, Keiding N, et al. Evidence for newal, expansion, and transfection of rat spermatogonial decreasing quality of semen during past 50 years. Br stem cells in culture. Proc Natl Acad Sci USA 2005;102: Med J 1992;305:609–613. 17430–17435. 998. Swan SH, Elkin EP, Fenster L. Have sperm densities 1009. Honaramooz A, Li MW, Penedo MC, et al. Accelerated mat- declined? A reanalysis of global trend data. Environ uration of primate testis by xenografting into mice. Biol Health Perspect 1997;105:1228–1232. Reprod 2004;70:1500–1503. 999. Huyghe E, Matsuda T, Thonneau P. Increasing incidence 1010. McLean DJ. Spermatogonial stem cell transplantation of testicular cancer worldwide: a review. J Urol 2003; and testicular function. Cell Tissue Res 2005;322:21–31. 170:5–11. 1011. Rathi R, Honaramooz A, Zeng W, et al. Germ cell devel- 1000. Paulozzi L. International trends in rates of hypospadias opment in equine testis tissue xenografted into mice. Re- and cryptorchidism. Environ Health Perspect 1999;107: production 2006;131:1091–1098. 297–302. 1012. Turner RM, Rathi R, Zeng W, et al. Xenografting of de- 1001. Swan SH. Does our environment affect our fertility? generate stallion testis onto a mouse host does not rescue Some examples to help reframe the question. Semin Re- the testicular degeneration phenotype. Anim Reprod Sci prod Med 2006;24:142–146. 2005;89:253–255. 1002. Hauser R. The environment and male fertility: recent 1013. Turner RM, Rathia R, Zeng W, et al. Xenografting to research on emerging chemicals and semen quality. Se- study testis function in stallions. Anim Reprod Sci 2006; min Reprod Med 2006;24:156–167. 94:161–164. 1003. Lahousse SA, Wallace DG, Liu D, et al. Testicular gene 1014. Zeng W, Avelar GF, Rathi R, et al. The length of the expression profiling following prepubertal rat mono-(2-eth- spermatogenic cycle is conserved in porcine and ovine tes- ylhexyl) phthalate exposure suggests a common initial ge- tis xenografts. J Androl 2006;27:527–533.

AAEP PROCEEDINGS ր Vol. 53 ր 2007 177