Ultrastructural Examination of Spermiogenesis Within the Testis of the Ground Skink, Scincella Laterale (Squamata, Sauria, Scincidae)

JOURNAL OF MORPHOLOGY 268:181–192 (2007)

Ultrastructural Examination of Spermiogenesis Within the Testis of the Ground Skink, Scincella laterale (Squamata, Sauria, Scincidae)

Kevin M. Gribbins,1* Erin M. Mills1, and David M. Sever2

1Department of Biology, Wittenberg University, Springﬁeld, Ohio 45501 2Department of Biological Sciences, Southeastern Louisiana University, Hammond, Louisiana 70402

ABSTRACT Although the events of spermiogenesis are velopment within this large diverse family. Most commonly studied in amniotes, the amount of research data collected on male reproduction in Scincidae available for lizards (Sauria) is lacking. Many studies have concentrated on the ultrastructure of their have described the morphological characteristics of spermatozoa (Furieri, 1970; Okia, 1990; Jamieson mature spermatozoa in lizards, but few detail the ultra- and Scheltinga, 1993, 1994; Jamieson et al., 1996) structural changes that occur during spermiogenesis. The purpose of this study was to gain a better understanding with a few accounts detailing specific events during of the subcellular events of spermiogenesis within the spermiogenesis (Carcupino et al., 1989; Dehlawi, temperate ground skink (Scincella laterale). The morpho- 1991; Dehlawi et al., 1992). Jamieson et al. (1996) logical data presented here represent the first complete describe a number of spermatozoal autapomorphies ultrastructural study of spermiogenesis within the Scinci- seen within the skink clade and provide a morpho- dae clade. Samples of testes from 20 specimens were pre- logical model of the mature spermatozoa. pared using standard techniques for transmission elec- The utility of spermatozoal ultrastructure as a tron microscopy. Many of the ultrastructural changes source of characters for phylogenetic analysis within occurring during spermiogenesis within the ground skink Chordata is well established (see review by Jamie- are similar to that of other saurians. However, there were son, 1991), and the ultrastructure of mature sperm a few unique characteristics that to date have not been described during spermiogenesis in other lizards. For diagrammed for Scincidae is applicable to many of example, during early round spermatid development these species (Jamieson et al., 1996). However, within the ground skink testis, proacrosomal granules within Reptilia, ultrastructural data for mature begin to form within the acrosomal vesicle before making spermatozoa are presently inadequate, and many contact with the apex of the nucleus. Also, a prominent view their potential as phylogenetic tools to be rela- microtubular manchette develops during spermiogenesis; tively limited. Kluge (1989) states, ‘‘databases must however, the circular component of the manchete is be expanded to include new and non-traditional absent in this species of skink. This developmental differ- sources to better resolve relationships among the ence in manchette formation may lead to the more robust major squamate groups.’’ Spermatozoon morphology and straight mature spermatozoa that are common is often considered as one of these nontraditional within the Scincidae family. These anatomical character differences may be valuable nontraditional sources that sources of data (Jamieson et al., 1996), and we also along with more traditional sources (i.e., mitochondrial believe that the developmental steps of spermiogen- DNA) may help elucidate phylogenetic relationships, esis could be considerable sources of nontraditional which are historically considered controversial at best, data that could help decipher the phylogenetic rela- among species within Scincidae and Sauria. J. Morphol. tionships within Sauria. 268:181–192, 2007. Ó 2006 Wiley-Liss, Inc. Theoretically, morphological similarities and differences of spermatozoa between species of lizards KEY WORDS: Scincella laterale; ground skink; sper- should parallel some similarity or difference ob- miogenesis; ultrastructure served during the developmental steps of spermiogenesis. Many similarities occur in spermatid mor- The Ground Skink, Scincella laterale, is a mem- phology among amniotes and the spermiogenic ber of the Scincidae family, which is considered the events of nuclear elongation, acrosome formation, largest family of lizards ( 1,200 species) and has and tail development are common to both reptiles representatives on every continent except Antarc- tica (European Molecular Biology Laboratory Rep- tile Database, 2006). Much is known about their *Correspondence to: Kevin M. Gribbins, Department of Biology, ecology and life history. Numerous studies world- Wittenberg University, P.O. Box 720, Springfield, OH 45501-0720. E-mail: [email protected] wide have concentrated on reproductive cycles in skinks, and a large percentage of skinks are known Published online 8 December 2006 in to be viviparous (45%) (Pough et al., 2001). How- Wiley InterScience (www.interscience.wiley.com) ever, little is known about the specifics of sperm de- DOI: 10.1002/jmor.10505

Ó 2006 WILEY-LISS, INC. 182 K.M. GRIBBINS ET AL. and non-reptilian amniotes. However, the ultra- phate buffer, pH 7.4. The samples were post-fixed in 2% osmium structural features of each event of spermiogenesis tetroxide in deionized water for 60 min. Following post-fixation, the tissues were dehydrated through a graded series of ethanol, can vary between different species. These develop- cleared in propylene oxide, infiltrated, and then polymerized in mental differences that occur during spermiogene- epoxy resin (Embed 812, EMS, Fort Washington, PA) within sis may lead to the morphological differences seen beem capsules overnight at 608C. between mature spermatozoa of different species Tissue blocks were faced and ultrathin sections (90 nm) were representing different reptilian taxa. Thus, under- cut using a diamond knife (DDK, Wilmington, DE) and a Reich- ert Ultracut E Ultramicrotome. Sections were placed on copper standing the ultrastructural events of spermiogene- grids (200 mesh) and stained with 2% uranyl acetate for 20 min sis between species could corroborate spermatozoal and Reynold’s lead citrate for 9 min. Ultrathin sections were data and may help build a stronger argument for viewed with a Zeis EM900 transmission electron microscope phylogenetic relationships. (Carl Zeis, Berlin, West Germany) and photographed using a Megaview II digital camera (Soft Imaging Systems, Lakewood, The purpose of this study was to examine the ul- CO). trastructural events of spermiogenesis within the testis of Scincella laterale. The ground skink is most commonly found in moist, wooded environ- RESULTS ments ranging from New Jersey to Nebraska and south to Texas and Florida. The reproductive cycle The beginning of spermiogenesis within Scincella of the male ground skink has been described by laterale is characterized by cohorts of round young Sever and Hopkins (2005). However, the light mi- spermatids connected by cytoplasmic junctions croscopic and subcellular events of spermatogenesis (Fig. 1A, *). These haploid spermatids have cen- and spermiogenesis have yet to be determined. To trally located nuclei, prominent smooth endoplas- our knowledge, this is not only the first ultrastruc- mic reticulum, multivesicular bodies, and numer- tural description of spermiogenesis in S. laterale, ous mitochondria. The most evident organelle is the but also is the first complete examination focusing Golgi apparatus (Fig. 1B,C, black arrowheads) and only on spermiogenesis within a species of skink. its juxtapositioned developing acrosomal vesicle The only other accounts of spermiogenic events in (AV) (Fig. 1B,C). The Golgi has multiple-layered species of skinks are brief ultrastructure descrip- concave cisternae and numerous transport vesicles tions of some of the spermatids within the testes of radiating off the most proximal (closest to the AV) Chalcides ocellatus (Dehlawi, 1991; Ismail and cistern (Fig. 1C, inset, white arrows; 1D, white Dehlawi, 1992), Chalcides ocellatus tiligugu (Fur- arrows). The Golgi transport vesicles travel to and ieri, 1970; Carcupino et al., 1989), and Eumeces fuse with the growing AV (Fig. 1D, white arrows), laticeps (Okia, 1990). The ultrastructural data pre- which increases in size during the early stages of sented here are compared with the typical mature spermiogenesis (acrosome phase). Dense granular spermatozoan morphology illustrated by Jamieson material, presumably from the Golgi transport et al. (1996) for Scincidae and to the relatively lim- vesicles, starts to accumulate in the AV and gives ited subcellular data described during spermiogene- rise to a prominent acrosomal granule (Fig. 1C, sis for other lizards. The present data combined inset, black arrowhead). Early in the growth of the with future ultrastructural studies of spermiogene- AV, contact is made with the nucleus, the contact sis within different species of skinks may also add being evident by a nuclear depression forming on to our current understanding of the phylogenetics the juxtapositioned nuclear envelope (Fig. 1D, black within the Scincidae family and within Sauria, arrow). Also, electron-dense material accumulates which historically are considered controversial at on the inside of the nuclear membrane where the best (Kluge, 1989; Vidal and Hedges, 2005). AV contacts the nucleus. The majority of AV growth occurs after contact with the nucleus (Fig. 2). An electron-lucent space MATERIALS AND METHODS develops between the rostral nuclear membrane and the inner acrosomal membrane (Fig. 2A bet- Twenty male ground skinks were collected during six sample ween the * and black arrowhead and B, between periods (April 2001, May 2001, October, 2001, March 2002, June 2002, and August 2002) at uncontaminated sites located within black arrowhead and white arrow), which remains the US Department of Energy’s Savannah River Site in Aiken present throughout much of spermiogenesis. The County, South Carolina. After euthanasia (with ether), speci- acrosomal granule also enlarges after contact with mens were slit midventrally, and all viscera were removed from the nucleus as more dense material is delivered to the peritoneal cavity excluding the reproductive systems. Whole the AV via transport vesicles. This granule, when carcasses were then preserved in 10% neutral buffered formalin and are housed in the research collections at Southeastern Loui- present, maintains contact with the inner acroso- siana State University, Hammond, LA. David M. Sever, South- mal membrane throughout acrosome development. eastern Louisiana State University, fixed the reproductive tracts Also apparent is the beginning of flagellar develop- in 2.5% glutaraldehyde and 2.5% formaldehyde in 0.1 M sodium ment (Fig. 2A,F), which begins in the anterior cyto- cacodylate buffer at a pH of 7.4 and then sent the tracts to the Kevin Gribbins for the current ultrastructural study. plasm of round spermatids directly opposite its Small portions of testes from each lizard were cut into approxi- eventual attachment site on the posterior nuclear mately 2–3 mm cubes and washed several times in 0.1 M phos- surface. The AV becomes deeply embedded in the

Journal of Morphology DOI 10.1002/jmor SPERMIOGENESIS IN THE GROUND SKINK 183

Fig. 1. Development of the acrosomal vesicle during the early stages of spermiogenesis in the seminiferous epithelium of Scin- cella laterale. TEM: (A) Round spermatids active in acrosome vesicle (AV) formation. The Golgi apparatus (black arrow) is very prominent and the acrosome vesicle has not made contact with the nucleus. Cytoplasmic bridges are visible (*). Also present are mitochondria (white arrowhead), endoplasmic reticulum (white arrows), and multivesicular bodies (black arrowhead). Bar ¼ 2 lm. (B) Higher power of the acrosomal vesicle (AV) and Golgi apparatus (black arrowhead) from (A). Transport vesicle (white arrow) can be seen coming off the lateral edge of a Golgi cisterna. Bar ¼ 0.3 lm. (C) A later stage round spermatid undergoing active vesicle formation as seen with the many transport vesicles (inset, white arrows) leaving the Golgi (black arrowhead; inset: black arrow) to fuse with the acrosomal vesicle (AV). Granular material presumably from the transport vesicles is starting to accumulate into a visible proacrosomal granule (inset, black arrowhead). Bar ¼ 2 lm; Inset: Bar ¼ 0.2 lm. (D) Later in development, the acrosomal vesicle (AV) makes contact with the nucleus as is evident by dense granular material (black arrow) accumulating at a small depression where the acrosomal vesicle meets the nuclear membrane. The Golgi (black arrowhead) is still actively producing transport vesicles (white arrows) that fuse with the acrosomal vesicle. Bar ¼ 0.6 lm. nucleus, and the nuclear membrane contacts the fossa (Fig. 2C, PC and DC). These centrioles will inner acrosomal membrane (Fig. 2D, black arrow- give rise to the flagellar axoneme during later de- head). Nuclear chromatin during the middle devel- velopment. Occasionally, microtubules are associ- opmental stage of round spermatids remains pro- ated with the centrioles and the nucleus and are fuse and evenly distributed within the nucleoplasm. most likely the early precursors of the manchette As the AV grows in size and the nuclear depression (Fig. 2C, black arrows). forms, the nucleus migrates from a central position Figure 3 represents the end stages of round sper- in the cell to a more peripheral location. The cyto- matid development. The shoulders of the acrosome plasm is forced to the caudal end of the round sper- begin to migrate around the apical surface of nu- matid as the outer acrosomal membrane and cleus (Fig. 3A, black arrows), and toward the end of plasma membrane become juxtapositioned (Fig. 2D, this migration, the nuclear depression has disap- white and black arrows). Figure 2C shows early de- peared and the nucleus returns to its round configu- velopment of the spermatid tail. The developing ration (Fig. 3B). Throughout acrosome migration, flagellum has migrated to a basal position opposite the acrosomal granule maintains its position on the to that of the acrosomal vesicle. The proximal and inner acrosomal membrane. The granule begins to distal centrioles are anchored within the nuclear decrease in size as its granular material is diffused

Journal of Morphology DOI 10.1002/jmor 184 K.M. GRIBBINS ET AL.

Fig. 2. Later developmental stages of round spermatids within the seminiferous epithelium of Scincella laterale. TEM: The acrosome vesicle (AV) now has made more direct contact with the nuclear head. A depression forms and deepens over the series of micrographs to accommodate the acrosome as it develops. The nucleus also migrates from a deep central position to a more peripheral location. This migration juxtapositions the outer acrosomal membrane to the plasma membrane. The acrosomal granule also becomes large and more prominent over the series of micrographs. Early in this stage, the flagellum begins to form within the anterior cytoplasm away from nucleus. Later in the stage, the proximal and distal centrioles have migrated to the basal nucleus and give rise to the flagellar anoxeme, which is anchored within the nuclear fossa. (A) White arrow, Golgi apparatus; black arrow, acrosomal granule; *, nuclear fossa; black arrowhead, inner acrosomal membrane; black arrowhead, outer acrosomal membrane; F, early flagellum formation. Bar ¼ 2 lm. (B) Black arrow, acrosomal granule; black arrowhead, inner acrosomal membrane; white arrow, nuclear fossa. Bar ¼ 0.3 lm. (C) White arrow, nuclear fossa; black arrows, microtubules in transverse section; PC, proximal centrioles; DC, distal centrioles. Bar ¼ 2 lm. (D) White arrow, plasma membrane; outer (black arrow) and inner acrosomal membranes (black arrowhead); AV, acrosomal vesicle. Bar ¼ 1 lm. throughout the acrosome (Fig. 3C, *). Also during Figure 4 represents the early developmental acrosome migration, the longitudinal microtubules stages of elongation. At the end of acrosomal of the manchette become visible for the first time shoulder migration, the acrosome vesicle com- and are positioned near the nucleus (Fig. 3B, black pletely envelops the entire apical surface of the nu- arrows). Chromatin condensation becomes more no- cleus (Fig. 4A white arrow; 4B, black arrows). Cyto- ticeable as the acrosome shoulders migrate to enve- plasm is completely forced away from this apical lope the apical nuclear surface. Elongation of the surface, and the acrosome-covered portion of the flagellar tail is prominent during acrosome migra- nucleus appears to extend outside of the cell but is tion as well. The deep nuclear fossa houses the ba- in fact still contained within the plasma membrane sal body, which anchors the flagellum to the nucleus (Fig. 4B, black arrowheads). This cytoplasm migra- (Fig. 2B, white arrow and 3D,*). Dense bodies begin tion results in most organelles moving to the poste- to accumulate at the neck of the flagellum (Fig. 3D, rior side of the elongating spermatids. As the nu- black arrows), and the central doublet of the 9 þ 2 cleus elongates, the DNA becomes condensed into flagellar arrangement is visible (Fig. 3D, black filamentous chords (Fig. 4B–D). The nucleus also arrowhead). A dense fibrous sheet surrounds the appears to be surrounded by several longitudinal elongating axoneme (Fig. 3D, white arrowhead). microtubules that run from the base of the acro-

Journal of Morphology DOI 10.1002/jmor SPERMIOGENESIS IN THE GROUND SKINK 185

Fig. 3. The termination of round spermatid development in Scincella laterale. TEM: The acrosome migrates across and flattens the apical surface of the nucleus. The deep nuclear depression that housed the acrosome early in development becomes very shal- low until its disappearance at the end of acrosomal vesicle (AV) migration. The large acrosomal granule begins to dissipate as it dif- fuses into the lumen of the acrosomal vesicle. Flagellar development is well underway. Distinct anoxeme extends from the basal body located in the nuclear fossa and dense material accumulates at the neck region of the flagella. A dense ring also forms around the elongating anoxeme. (A) White arrowhead, acrosomal granule; *, nuclear depression; black arrows, acrosomal vesicle shoulders; white arrow, basal body; black arrowhead, distal centriole of the anoxeme. Bar ¼ 2 lm. (B) Black arrowheads, acrosomal vesicle shoulders; black arrows, microtubules of the manchette; D, dense ring formation around the anoxeme; white arrow, basal body. Bar ¼ 2 lm. (C) White arrows, acrosomal vesicle shoulders; *, acrosomal granule dissipation; inner (black arrowhead) and outer (black arrowhead) acrosomal membranes; white arrowhead, plasma membrane. Bar ¼ 1 lm. (D) White *, nuclear fossa; black arrows, dense bodies; black arrowhead, central microtubule doublet; white arrowhead, outer dense ring sheath of flagellum. Bar ¼ 1 lm. some and extend past the most caudal end of the nucleus is covered by the acrosomal complex, which nucleus (Fig. 4A, black arrows; 4B, white arrow- contains the acrosome (Fig. 5B, AV), subacrosomal heads; 4D, black arrows). The acrosomal granule cone (Fig. 5B, white *), and many concentric Sertoli (Fig. 4C, *) continues to diffuse its granular mate- cell membrane layers (Fig. 5B, black*) superficially rial throughout the acrosome, with a great majority covering the acrosome. At this stage of elongation, of it accumulating near the outer acrosomal mem- nuclear lacunae are often present in cross sections brane (Fig. 4C, black arrowhead). At this point of of the nuclei (Fig. 5C, white arrow). The longitudi- spermiogenesis, the subacrosomal space is very visi- nal manchette runs the entire length of the nucleus ble between the nuclear membrane and inner acroso- (Fig. 5A, black arrowhead; 5D, black arrow). These mal membrane (Fig. 4B, *). The subacrosomal space longitudinal microtubules (Fig. 5D, inset: black continues to widen during early elongation and pro- arrow) are very prominent in Scincella laterale and duces a large cavity (Fig. 4D, 2) under the inner acro- are located in juxtaposition to the nuclear mem- somal membrane (Fig. 4D, white arrowhead). brane. At this stage of elongation, both the proxi- In the late stages of nuclear elongation, the nu- mal and distal centrioles are still visible and origi- clear DNA is at its most condensed state and the nate within the nuclear fossa (Fig. 5D, inset). nucleoplasm appears uniformly black (Fig. 5A). At At the end stage of elongation, the mature sper- the height of elongation, the apical surface of the matid contains three major sections: the acrosomal

Journal of Morphology DOI 10.1002/jmor 186 K.M. GRIBBINS ET AL.

Fig. 4. Early nuclear elongation in developing spermatids of Scincella laterale. TEM: The acrosomal vesicle (AV) envelops the entire apical nucleus. The DNA appears filamentous during its slow condensation into chromosomes within early elongating spermatids. A subacrosomal space forms between the acrosome and nuclear head and in many instances the acrosomal granule is still present. The granule’s material is diffusing and accumulating near the outer acrosomal membrane. Microtubules of the developing manchette are prominent within the cytoplasm. (A) Black arrows, microtubules; white arrow, lumen of acrosomal vesicle; Nu, nucleus. Bar ¼ 2 lm. (B) Black arrows, acrosomal vesicle shoulders; white arrowheads, microtubules; *, subacrosomal space; outer (1) and inner (2) acrosomal membranes; black arrowheads, plasma membrane. Bar ¼ 1 lm. (C) White arrow, plasma membrane; inner (white arrowhead) and outer (black arrowhead) acrosomal membrane with accumulating granular material from acrosomal granule (*). Bar ¼ 1 lm. (D) Black arrows, manchette microtubules; 2, subacrosomal space; 1, acrosomal vesicle; inner (white arrowhead) and outer (black arrowhead) acrosomal membranes. Bar ¼ 2 lm. complex, nucleus, and flagellar tail (Fig. 6). The api- run parallel along the nucleus’s entire length (Fig. cal nucleus maintains its cone shape and the tip of 6B–E). the nucleus tapers conically into a sharp point Nucleoplasm within the spermatid is electron within the subacrosomal space (Fig. 6A). A short dense and appears black throughout its length at but visible perforatorium rests within the nuclear the end of elongation (Fig. 6A). A nuclear lacuna is tip (Fig. 6A, black arrow). In cross section, the per- sometimes visible in cross section within this region foratorium, subacrosomal space, AV, and concentric of the spermatid (Fig. 6D, white arrowhead). The Sertoli cell membrane layers are all visible (Fig. lacuna does not form a rod within the nucleus be- 6B). The shoulders of the AV wrap around either cause it does not span the entire nuclear length. side of the nuclear cone and appear narrower than The manchette is extremely prominent across the the rostral AV in cross section (Fig. 6A–C). Moving entire length of the nucleus, and the number of dorsally along the apical nucleus reveals a clear microtubules appears to increase when moving from region in cross section within the concentric mem- the apical head to the dorsal end of the nucleus brane layers of the Sertoli cell that is associated (Fig. 6B–E). At the base of the nucleus, a well-devel- with the AV (Fig. 6C, *) and is most likely an arti- oped basal body is visible (Fig. 6A, white arrow), fact of shrinkage during embedding and dehydra- anchoring the flagellum to the spermatid head. tion. The longitudinal manchette microtubules sur- The flagellar tail is also fully developed and the round the entire perimeter of the apical head and proximal neck, midpiece, and principle piece are

Journal of Morphology DOI 10.1002/jmor SPERMIOGENESIS IN THE GROUND SKINK 187

Fig. 5. Late elongating phase of spermatid development in Scincella laterale. TEM: Nuclear DNA is condensed to form a uniform black nucleoplasm. The climax of elongation ends with a straight elongated spermatid that has a tapered nuclear apex. The acrosome envelops the apical nuclear head and the subacrosomal space is filled with a dense granular material. As development continues the nucleus will curve, forming concave and convex surfaces. Many superficial layers of Sertoli cell membrane surround the acrosomal complex and the longitudinal manchette is very prominent and is located along the entire length of the nucleus. In proper sagittal section, both centrioles are still visible at the base of the flagella. A single nuclear lacuna is present in the middle of the nucleus in cross section. (A) Black arrow, acrosome; white arrow, peripheral microtubules; black arrowhead, manchette microtubules. Bar ¼ 2 lm. (B) AV, acrosomal vesicle; outer (white arrowheads) and inner (black arrowheads) acrosomal membranes; white *, subacrosomal space; black *, Sertoli cell membrane layers. Bar ¼ 2 lm. (C) Black arrow, acrosome; white arrow, nuclear lacuna. Bar ¼ 2 lm. (D) Black arrow, microtubules of the manchette; white arrow, basal body; Inset: Black arrow, microtubules; PC, proximal centriole; DC, distal centriole. Bar ¼ 2 lm; Inset: Bar ¼ 0.3 lm. visible and recognizable by their components in erale are shared by other vertebrate taxa. However, cross section (Fig. 6F–H). The proximal neck (Fig. subtle developmental differences during spermio- 6A, white arrowhead) is nearest to the nucleus, and genesis to a structure shared by many vertebrates a dense granular material arranged into a single can lead to what seems a characteristic that is solid ring surrounds its axoneme. (Fig. 6F, DM). A exclusively seen, for example, only in squamates. cross section through the midpiece (Fig. 6G) reveals The best illustration of this is the multilayered the 9 þ 2 arrangement of microtubules within the acrosomal complex that sits on the conical apical axoneme. Also surrounding the flagellum is a thick head of the mature spermatid nucleus. The acro- fibrous ring (Fig. 6G,D) and a superficial ring of some is an enzyme-rich organelle that facilitates large mitochondria (Fig. 6D,M). The cross section spermatozoal penetration to the ovum, and thus its through the principle piece (Fig. 6H) is similar to presence in all vertebrate taxa is well understood. that of the midpiece except the mitochondria are In reptiles, particularly squamates, the acrosomal smaller and in a random order within the cytoplasm. complex is highly compartmentalized. This com- partmentalization has been suggested as an aid DISCUSSION to the sequential release of acrosomal enzymes (Talbot, 1991). The compartments (subacrosomal Many of the morphological characteristics dis- space, perforatorium, acrosomal vesicle, and super- played during spermiogenesis within Scincella lat- ficial Sertoli cell membrane layers) found in the

Journal of Morphology DOI 10.1002/jmor 188 K.M. GRIBBINS ET AL.

Fig. 6. (A) Sagittal section of a late elongating spermatid with all three major parts of the spermatid visible (acrosome, nucleus, and flagella) within Scincella laterale. TEM: Lines and represented letters show approximately where transverse sections occurred in order to obtain cross sections (B)–(H). Note the well developed basal body (white arrow) at the base of the flagellum, the neck region (white arrowhead), and the perforatorium (black arrow), and dense segments of the fibrous ring surrounding the flagellum (black arrowhead). (B)–(H) represent cross sections (CS) at or near the same stage of development as the spermatid represented in (A). Bar ¼ 2 lm. (B) CS through the acrosome and conical point of the apical nucleus. N, nucleus; SA, subacrosomal space; AV, acrosomal vesicle; P, perforatorium; CM, concentric membrane layers; Ma, manchette. (C) CS through the nucleus and acrosomal vesicle shoulders: N, Nucleus; AV, acrosomal vesicle; CM, concentric membrane layers; AM, acrosomal membrane; *, artifact from swelling; PM, plasma membrane. (D), (E) CS through nucleus proper: N, Nucleus; Ma, manchette; white arrowhead, nuclear lacuna. (F) CS through the distal neck of the flagella: Ax, Axoneme; DM, dense granular material; M, mitochondria. (G) CS through the midpiece: Ax, Axoneme; D, dense fibrous ring; M, mitochondria. (H) CS through the principle piece of the flagellum: D, Dense fibrous ring; M, mitochondria. CS B-H: Bar ¼ 0.2 lm. acrosomal complex of the ground skink are similar tus (Courtens and Depeiges, 1985; Dehlawi et al., to that of other squamates. The only missing layer 1992; Soley, 1996; Lin and Jones, 2000). In Iguana to S. laterale’s acrosomal complex is the epinuclear iguana, however, some of these vesicles originate space, which has already been reported missing in from the endoplasmic reticulum (Ferreira and the mature spermatozoa of Scincidae (Jamieson Dolder, 2002). No evidence was found in S. laterale et al., 1996). that vesicles from the endoplasmic reticulum aid in In Scincella laterale, early acrosome formation is AV formation. The AV remains relatively uniform in consistent with that of other amniotes. Like many its shape during early acrosome development. Its other species, the acrosome vesicle (AV) originates circular shape and the presence of a basally located from membrane-bound vesicles of the Golgi appara- acrosomal granule is consistent with that of other

Journal of Morphology DOI 10.1002/jmor SPERMIOGENESIS IN THE GROUND SKINK 189 lizard species (Clark, 1967; Da Cruz-Landim and spermatids. These data support Aire’s (2003) sug- Da Druz-Hofling, 1977; Butler and Gabri, 1984; gestion that the acrosomal granule may participate Dehlawi et al., 1992; Ferreira and Dolder, 2002, in perforatorium formation. The perforatorium is 2003). short and hard to see in S. laterale. The only time it Cytoplasmic bridges between cohorts of sperma- is readily visible is at the end of spermiogenesis tids at the same stage of development are observed and within the most mature elongated spermatids. most often during early acrosome development in The acrosome–nuclear complex has been shown Scincella laterale and are also common in other am- to migrate to a peripheral location in the developing niotic and anamniotic taxa (Da Cruz-Landim and spermatid and becomes associated with the Sertoli Da Druz-Hofling, 1977; Dehlawi et al., 1992; Lin cell plasma membrane (Da Cruz-Landim and Da and Jones, 2000; Smita et al., 2004). The function of Cruz-Hofling, 1977; Butler and Gabri, 1984; Deh- cytoplasmic bridges between spermatids of S. later- lawi et al., 1992). This movement causes the cyto- ale is assumed to serve the same function as those plasm to become displaced to the posterior pole of described for rats. Cytoplasmic junctions in rats the cell (Clark, 1967; Guraya, 1971; Vieira et al., serve as highways for intercellular communication 2001). In Scincella laterale as well as in Tropidurus between the cytoplasms of connected germs cells torquatus (Vieira et al., 2001), this relocation is im- and are termed mechanisms of haploid gene prod- portant because many of the cellular organelles, uct sharing (Ventela et al., 2003). Recent data on primarily the mitochondria and endoplasmic reticu- germ cell development strategies in temperate rep- lum, will become associated with the flagellar tail. tiles suggest that all germ cells within the seminif- Similar results also have been reported in non-liz- erous epithelium develop as a single population ard amniotes (Sprando and Russell, 1988; Soley, throughout the phases of spermatogenesis (Grib- 1997; Lin and Jones, 1993, 2000). bins et al., 2003; Gribbins and Gist, 2003, Gribbins Some authors also attribute this peripheral migra- et al., 2005; Gribbins et al., 2006). Thus, these cyto- tion to the flattening of the acrosomal vesicle over plasmic bridges may allow cell to cell communica- the anterior surface of the nucleus (Clark, 1967; But- tion that might synchronize the temporal develop- ler and Gabri, 1984; Courtens and Depeiges, 1985; ment seen during spermatogenesis in temperate Vieira et al., 2001). The peripheral relocation of the reptiles. round spermatids in Scincella laterale may be re- In electron-lucent acrosomes, dense material responsible for flattening the acrosomal vesicle and ferred to as proacrosomal granules (Russell et al., forming the acrosomal shoulders. Shoulder formation 1990) accumulates to form at least one large acroso- is most likely caused by the ‘‘sandwiching’’ of the AV mal granule. This occurs when the acrosome comes against the plasma membrane, which would cause in contact with the nuclear membrane in lizards the lateral movement of the shoulders over the nu- (Del Conte, 1976). However, in Scincella laterale clear head and result in the collapse of the vesicle the granule is seen within the AV before it makes onto the apical surface of the nucleus. contact with the nucleus, a feature that may be Nuclear condensation occurs with elongation and unique to the Ground Skink. It has been suggested is highly dependent on the species. In lizards, that the acrosomal granule is responsible for form- including Scincella laterale, Anolis carolinensis ing the perforatorium, a structure present in all liz- (Clark, 1967), Lemniscatus lemniscatus (Del Conte, ards and other squamates (Ferreira and Dolder, 1976), Tropidurus torquatus (Da Cruz-Landim and 2002), later in spermiogenesis (Aire, 2003). Most Da Cruz-Hofling, 1977; Vieira et al., 2001), Podarcis lizards, including S. laterale, show the formation of taurica (Butler and Gabri, 1984), Lacerta vivipara only one acrosomal granule. The Saudian lizard (Courtens and Depeiges, 1985), Agama adramitana Agama adramitana is the only lizard known to date (Dehlawi et al., 1992), and Iguana iguana (Ferreira to show the presence of two proacrosomal granules and Dolder, 2002), the chromatin condenses into during spermiogenesis (Dehlawi et al., 1992). long dense fibers. In I. iguana (Ferreira and Dolder, The granule in Scincella laterale remains at- 2002), these dense fibers are arranged helically tached to the inner acrosomal membrane during during early condensation and are later arranged acrosome formation. During the late round sperma- longitudinally. The same pattern is observed in tid stage and early in nuclear elongation, the gran- S. laterale. ule starts to diffuse at its anterior surface and a In Scincella laterale, we also observed a very light opaque area of diffused material surrounds prominent longitudinal manchette, especially in nu- the granule. This granular material accumulates on clear cross sections of elongating spermatids. The the inside of the outer acrosomal membrane during manchette is responsible for nuclear elongation early elongation. The significance of this accumu- (Russell et al., 1990), and is considered a common lating electron-dense material is not known and to structure of spermiogenesis in most reptiles (Fer- our knowledge has not been described in other spe- reira and Dolder, 2002). These microtubules can be cies of lizards. The granule also becomes less com- arranged helically and then align longitudinally pact during early elongation and is in the location (Courtens and Depeiges, 1985) or maintain a longi- where the perforatorium is seen in late elongating tudinal arrangement throughout nuclear elonga-

Journal of Morphology DOI 10.1002/jmor 190 K.M. GRIBBINS ET AL. tion (Da Cruz-Landim and Da Cruz-Hofling, 1977). which parallels that of other amniotes (McIntosh Scincella laterale microtubules are longitudinally and Porter, 1967; Lin and Jones, 1993, 2000; Ferre- arranged throughout elongation. Many bird species ira and Dolder, 2002). The developing flagellum ini- have circular and longitudinal microtubules associ- tially originates from both the proximal and distal ated with their manchette (Lin and Jones, 1993; centrioles within the cytoplasm and away from the Soley, 1996; Aire, 2003) as do many lizard species nucleus. During acrosome formation the flagellum (Da Cruz-Landim and Da Cruz-Hofling, 1977; But- then attaches to the nucleus within the nuclear ler and Gabri, 1984; Dehlawi et al., 1992). The fossa, which is similar to that of other lizards ground skink is missing this prominent circular (Courtens and Depeiges, 1985; Vieira et al., 2001). layer of microtubules. The significance of this ab- A dense ring of material that surrounds the distal sence is not known. Most lizard spermatozoa are centriole within the neck region of the ground skink considered slender, except in Eugongulus and Scin- flagellum has been called the pericentriolar mate- cidae (Jamieson and Scheltinga, 1993; Ferreira and rial (Vieira et al., 2001) and is common in most liz- Dolder, 2003). The spermatozoa of Scincidae are ards. A fibrous ring surrounds the axoneme of the wider and straighter than are those of other lizard mid and principle pieces of the flagella of Scincella groups. The reason for this morphological outcome laterale, which is also similar to that of all saurians may be the result of the lack of the circular microtu- studied to date. These fibrous rings in Ground bule component of the manchette during spermio- Skinks are laid down in thick blocks in longitudinal genesis. In other words, stretching of the nucleus section similar to the case in other lizards (Furieri, will result from the longitudinal microtubules, 1974; Depeiges et al., 1985; Vieira et al., 2001). which are assumed to be responsible for nuclear There are a few dense bodies that are associated elongation of the spermatids. However, constriction with the mitochondria of the mid and principle of the nucleus may not occur as efficiently in skinks pieces in S. laterale. This seems to be a major mor- because of the lack of the circular microtubules, phological difference between S. laterale and other which theoretically would constrict the diameter of lizard taxa, where dense bodies are commonly the nucleus resulting in a more slender mature found associated with mitochondria of the flagellum spermatozoa. (Clark, 1967; Furieri, 1974; Saita et al., 1988; Car- Once the nucleus reaches the end of elongation, cupino et al., 1989; Oliver et al., 1996; Vieira et al., its nucleoplasm becomes homogenously electron 2004). dense. This is common to all squamates including The mechanism of spermiogenesis in Scincella saurians (Jamieson et al., 1996; Teixeira et al., laterale appears to be very similar to that of other 1999a,b; Tavares-Bastos et al., 2002). Birds tend to amniotes and especially of other Sauria taxa. Many show a more sporadic arrangement of dark chroma- aspects of spermiogenesis are highly conserved in tin clumps (Thurston and Hess, 1987; Jamieson Sauria, with few differences existing among species and Tripepi, 2005). We also observed an electron- studied to date. In each species discussed here, lucent or clear zone within the main body of the spermiogenesis consists of acrosomal vesicle forma- elongating nucleus in Scincella laterale. This zone tion, nuclear indentation at the level of the acro- has been called lacunae (Jamieson et al., 1996; Teix- some, nuclear condensation and elongation, and eira et al., 1999a,b), the epinuclear electron-lucent flagellar maturation resulting in mature spermato- zone (Ferreira and Dolder, 2002), and electron- zoa. However, the ultrastructural events that occur lucent zone (Tavares-Bastos et al., 2002) in other during these processes can vary somewhat between species of squamates. A prominent clear zone has species. In S. laterale, there are a few of these var- also been seen in turtles and has been called an iations, which include no epinuclear space within electron-lucent intranuclear tubule in Testudo the acrosome, no circular microtubule component to graeca and Chrysemys picta (Ibargu¨ engoytıá et al., the manchette, the lack of dense bodies associated 1999). Jamieson et al. (1996) and Tavares-Bastos with mitochondria of the flagella, acrosomal gran- et al. (2002) suggest this space is located at the tip ule presence in the AV before nuclear attachment, of the nuclear apex, while Teixeira et al. (1999a,b) and accumulation of dense material underneath the suggest this space is in the main body of the nu- outer acrosome membrane. These differences may cleus. We use the term nuclear lacuna in reference be significant in the final outcome of the mature to the ground skink lucent zone because no evi- spermatozoon morphology. For example, the lack of dence was found that suggested it extended into a the circular manchette may be why Scincidae sper- rod-like structure within the nucleus. This endonu- matozoa are straighter and wider than other lizard clear canal or lacuna is also typical of non-passerine spermatozoa. Others may not be so relevant to birds, but is not present in Apus apus (Jamieson ultrastructure and may only be slight variations in and Tripepi, 2005). The function of this space in development during spermiogenesis. spermatids is still not known. This is the first extensive study of spermiogenesis The flagellar tail, including the axoneme and fi- within a scincidid lizard and adds data to the brous sheath, develops in the cytoplasm during scanty database that exists for spermiogenesis early spermatid formation in Scincella laterale, within Sauria. As additional families are studied,

Journal of Morphology DOI 10.1002/jmor SPERMIOGENESIS IN THE GROUND SKINK 191 more and more information will be gathered on the epithelium in the male slider turtle, Trachemys scripta. ultrastructural development of spermatids during J Morphol 255:337–346. Gribbins KM, Happ CS, Sever DM. 2005. Ultrastructure of the spermiogenesis within lizards. Accumulation of reproductive system of the black swamp snake (Seminatrix data, particularly recognizable variation in the de- pygaea). V. The temporal germ cell development strategy of velopmental steps of spermiogenesis as outlined the testis. Acta Zool 86:223–230. here for Scincella laterale may be phylogenetically Gribbins KM, Elsey RM, Gist DH. 2006. The cytological evalua- significant. Mature sperm have been used recently tion of the germ cell development strategy in the American alligator, Alligator mississippiensis. Acta Zool 87:59–69. for phylogenetic analysis (Jamieson et al., 1996; Guraya SS. 1971. Histochemical observations on lizard spermio- Teixeira et al., 1999b; Vieira et al., 2004; Vieira genesis. Acta Anat 79:270–279. et al., 2005) and differences that occur between liz- Ibargu¨ engoytıá NR, Pastor LM, Pallares J. 1999. A light and ul- ard taxa during spermiogenesis can improve these trastructural study of the testes of tortoise Testudo graeca (Testudinidae). J Submicrosc Cytol Pathol 31:221–230. databases by adding developmental and ontogenetic Ismail MF, Dehlawi GY. 1992. Studies on the ultrastructure of perspectives that supplement the present nontradi- the spermiogenesis of Saudian reptiles 5: The sperm tail dif- tional data used for phylogenetic analysis. These ferentiation in Chalcides ocellatus. Histol Histochem 7(C): data may ultimately help in our overall under- 211–222. standing of the phylogenetic relationships in Sauria Jamieson BGM. 1991. Fish Evolution and Systematics: Evi- dence From Spermatozoa. Camridge, UK: Cambridge Univer- and give us better perspective on the placement of sity Press. lizard taxa within the order Squamata and the Jamieson BGM, Scheltinga DM. 1993. The ultrastructure of the class Reptilia. spermatozoa of Nangura spinosa (Scincidae, Reptilia). Mem- oirs of the Queensland Museum 12:169–179. Jamieson BGM, Scheltinga DM. 1994. The ultrastructure of LITERATURE CITED the spermatozoa of the Australian skinks, Cenotus taeniola- tus, Carlia pectoralis and Tilqua scincoides scincoides (Scin- Aire TA. 2003. Ultrastructural study of spermiogenesis in the cidae, Reptilia). Memoirs of the Queensland Museum 37: turkey, Meleagris gallopavo. Br Poult Sci 44:674–682. 181–193. Butler RD, Gabri MS. 1984. Structure and development of the Jamieson BGM, Tripepi S. 2005. Ultrastructure of the sperma- sperm head in the lizard Podarcis (Lacerta) taurica. J Ultra- tozoon of Apus apus (Linneaus 1758), the common swift struct Res 88:261–274. (Aves; Apodiformes; Apididae), with phylogenetic implications. Carcupino M, Corso G, Pala M. 1989. Spermiogenesis in Chal- Acta Zool 86:239–244. cides ocellatus (Gmelin) (Squamata, Scincidae): An electron Jamieson BGM, Oliver SC, Scheltinga DM. 1996. The ultra- microscope study. Boll Zool 56:119–124. structure of the spermatozoa of Squamata I. Scincidae, Gek- Clark AW. 1967. Some aspects of spermiogenesis in a lizard. Am konidae, and Pygopididae (Reptilia). Acta Zool 77:85–100. J Anat 121:369–400. Kluge AG. 1989. Progress in squamate classification. Herpetol Courtens JL, Depeiges A. 1985. Spermiogenesis of Lacerta 45:368–379. vivipara. J Ultrastruct Res 90:203–4. Lin M, Jones RC. 1993. Spermiogenesis and spermiation in the Da Cruz-Landim C, Da Cruz-Hofling MA. 1977. Electron micro- Japanese quail (Coturnix coturnix japonica). J Anat 183:525– scope study of lizard spermiogenesis in Tropidurus torquatus 535. (Lacertilia). Caryologia 30:151–162. Lin M, Jones RC. 2000. Spermiogenesis and spermiation in a Dehlawi GY. 1991. Studies on the ultrastructure of the spermio- monotreme mammal, the platypus, Ornithorhynchus anatinus. genesis of Saudian reptiles 4. The sperm head differentiation J Anat 196:217–232. in Chalcides ocellatus. Histol Histochem 7(C):331–347. McIntosh JR, Porter KR. 1967. Microtubules in the spermatids Dehlawi GY, Ismail MF, Hamdi SA, Jamjoom MB. 1992. Ultra- of the domestic fowl. J Cell Bio 35:153–173. structure of spermiogenesis of Saudian reptiles 6. The sperm Okia NO. 1990. The ultrastructure of the spermatozoon of the head differentiation in Agama adramitana. Arch Androl 28: lizard Cnemidophorus sexlineatus (Sauria: Teiidae). Herpetol 223–234. 48:330–343. Del Conte E. 1976. The subacrosomal granule and its evolution Oliver SC, Jamieson BGM, Scheltinga DM. 1996. The ultra- during spermiogenesis in a lizard. Cell Tissue Res 171:483–498. structure of spermatozoa of Squamata II. Agamidae, Varnai- Depeiges A, Betail G, Coulet M, Dufaure JP. 1985. Histochemis- dae, Colubridae, Elapidae, and Boidae (Reptilia). Herpetol 52: try study of epididymal secretions in the lizard, Lacerta vivipara. 216–241. Localization of lectin binding sites. Cell Tissue Res 239:463– Pough FH, Andrews RM, Cadle JE, Crump ML, Savitzky AH, 466. Wells KD. 2001. Herpetology, 2nd ed. New Jersey: Prentice European Molecular Biology Laboratory Reptile Database. Hall. pp124–125. 2006. Species numbers.http://www.reptile-database.org. Russell LD, Ettlin RA, Hikim AMP, Clegg ED. 1990. Histologi- Ferreira A, Dolder H. 2002. Ultrastructural analysis of spermio- cal and Histopathological Evaluation of the Testis. Clear- genesis in Iguana iguana (Reptilia: Sauria: Iguanidae). Eur J water, Florida: Cache River Press. Morphol 40:89–99. Saita A, Tripepi S, Longo OM. 1988. Comparative observations Ferreira A, Dolder H. 2003. Sperm ultrastructure and spermato- on spermiogenesis 2. Nuclear shaping in the absence of a genesis in the lizard Tropidurus itambere. Biocell 27:33–362. microtubular manchette in the spermatids of the bird Croto- Furieri P. 1970. Comparative spermatology: Proceedings of the phaga ani (Cuculiformes). Boll di Zool 49:115–124. International Symposium held in Rome and Siene, 1–5 July Sever DM, Hopkins WA. 2005. Renal sexual segment of the 1969. Academic Press: New York. pp. 1–20. ground skink, Scincella laterale (Reptilia, Squamata, Scinci- Furieri P. 1974. Spermi e spermatogenesis in alcuni iguanidi dae). J Morphol 266:46–59. Argentina. Riv di Biol 67:233–279. Smita M, George JM, Girija R, Akbarsha MA, Oommen OV. Gribbins KM, Gist DH. 2003. The cytological evaluation of sper- 2004. Spermiogenesis in Caecilians Ichthyophis tricolor and matogenesis within the germinal epithelium of the male Uraeotyphlus cf. narayani (Amphibia: Gymnophiona): Analy- European wall lizard, Podarcis muralis. J Morphol 258:296– sis by light and transmission electron microscopy. J Morphol 306. 262:484–499. Gribbins KM, Gist DH, Congdon JD. 2003. The cytological eval- Soley JT. 1996. Differentiation of the acrosomal complex in uation of spermatogenesis and organization of the germinal ostrich (Struthio camelus) spermatids. J Morphol 227:101–111.

Journal of Morphology DOI 10.1002/jmor 192 K.M. GRIBBINS ET AL.

Soley JT. 1997. Nuclear morphogenesis and the role of the man- Thurston RJ, Hess RA. 1987. Ultrastructure of spermatozoa from chette during spermiogenesis in the ostrich (Struthio cam- domesticated birds: Comparative study of turkey, chicken, and elus). J Anat 190:563–576. guinea fowl. Scanning Microsc 1:1829–1838. Sprando RL, Russell LD. 1988. Spermiogenesis in the red-ear Ventela S, Toppari J, Parvinen M. 2003. Intercellular organelle turtle (Pseudemys scripta) and the domestic fowl (Gallus domes- traffic through cytoplasmic bridges in early spermatids of the ticus): A study on cytoplasmic events including cell volume rat: Mechanisms of haploid gene product sharing. Mol Biol changes and cytoplasmic elimination. J Morphol 198:95–118. Cell 14:2768–2780. Talbot P. 1991. Compartimentalization in the acrosome. In: Bac- Vidal N, Hedges SB. 2005. The phylogeny of squamate reptiles cetti B, editor. Comparative Spermatology–20 Years After. (lizards, snakes, and amphisbaenids) inferred from nine nu- New York: Raven Press. pp 255–259. clear protein-coding genes. C R Biol 328:1000–1008. Tavares-Bastos L, Teixeira RD, Colli GR, Baó SN. 2002. Poly- Vieira GHC, Wiederhecker HC, Colli GR, Baó SN. 2001. Sper- morphism in the sperm ultrastructure among four species of miogenesis and testicular cycle of the lizard Tropidurus lizards in the genus Tupinambis (Squamata: Teiidae). Acta torquatus (Squamata, Tropiduridae) in the Cerrado of central Zool 83:297–307. Brazil. Amphibia-Reptilia 22:217–233. Teixeira RD, Colli GR, Baó SN. 1999a. The ultrastructure of the spermatozoa of the lizard Micrablepharus maximiliani (Squa- Vieira GHC, Colli GR, Baó SN. 2004. The ultrastructure of the mata, Gymnophthalmidae), with considerations on the use of spermatozoon of the lizard Iguana iguana (Reptilia, Squa- sperm ultrastructure characters in phylogenetic reconstruc- mata, Iguanidae) and the variability of sperm morphology tion. Acta Zool 80:47–59. among iguanian lizards. J Anat 204:451–464. Teixeira RD, Vieira GHC, Colli GR, Baó SN. 1999b. Ultrastruc- Vieira GHC, Colli GR, Baó SN. 2005. Phylogenetic relationships tural study of spermatozoa of the neotropical lizards, Tropidu- of corytophanid lizards (Iguania, Squamata, Reptilia) based rus semitaeniatus and Tropidurus torquatus (Squamata, on partitioned and total evidence analyses of sperm morphol- Tropiduridae). Tissue Cell 31:308–317. ogy, gross morphology, and DNA data. Zool Scri 34:605–625.

Journal of Morphology DOI 10.1002/jmor