V. the Temporal Germ Cell Development Strategy of the Testis Kevin M

Acta Zoologica (Stockholm) 86: 223–230 (October 2005)

UltrastructureBlackwell Publishing, Ltd. of the reproductive system of the black swamp snake (Seminatrix pygaea). V. The temporal germ cell development strategy of the testis Kevin M. Gribbins,1 Carrie S. Happ1 and David M. Sever2

Abstract 1Department of Biology, Wittenberg Gribbins, K.M., Happ, C.S. and Sever, D.M. 2005. Ultrastructure of the University, Springfield, OH 45501–0720, reproductive system of the black swamp snake (Seminatrix pygaea). V. The 2 USA; Department of Biological Sciences, temporal germ cell development strategy of the testis. — Acta Zoologica Southeastern Louisiana University, (Stockholm) 86: 223–230 Hammond, LA 70402, USA The germ cell development strategy during spermatogenesis was investigated Keywords: in the black swamp snake (Seminatrix pygaea). Testicular tissues were collected, Spermatogenesis, germ cell cycle, testis, embedded in plastic, sectioned by ultramicrotome, and stained with methylene black swamp snake, seminiferous blue and basic fuchsin. Black swamp snakes have a postnuptial pattern of epithelium development, where spermatogenesis occurs from May to July and spermiation is completed by October. Though spatial relationships are seen between germ Accepted for publication: cells within the seminiferous epithelium during specific months, accumulation 8 September 2005 of spermatogonia and spermatocytes early in spermatogenesis and the depletion of spermatocytes and accumulation of spermatids late in spermatogenesis prevent consistent cellular associations. This temporal germ cell development within an amniotic testis is consistent with that seen in other recently studied temperate reptiles (slider turtle and wall lizard). These reptiles’ temporal development is more similar to the developmental strategy found in anamniotes than the spatial germ cell development that characterizes birds and mammals. Our findings also imply that a third germ cell development strategy may exist in temperate breeding reptiles. Because of the phylogenetic position of reptiles between anamniotes and other terrestrial amniotes, this common germ cell development strategy shared by temperate reptiles representing different orders may have significant implications as far as the evolution of sperm development within vertebrates. Kevin M. Gribbins, Department of Biology, Wittenberg University, PO Box 720, Springfield, OH 45501–0720, USA. E-mail: [email protected]

advance through development in association with Sertoli Introduction cells, which make up the amniotic seminiferous epithelium. Two distinctive germ cell development strategies have been Sections of seminiferous epithelia within mammals and described in vertebrates. Anamniotic vertebrates possess a birds will contain three to ﬁve generations of germ cells that temporal germ cell development strategy in which germ cells are consistently found together. These consistent spatial progress through spermatogenesis as a single cohort within relationships among germ cells represent stages or cellular cysts that line the testis (Lofts 1964; Van Oordt and Brands associations (Russell et al. 1990). This spatial germ cell 1970). Upon the completion of spermatogenesis, cysts rupture development leads to multiple spermiation events throughout and release spermatozoa in a single spermiation event to the spermatogenesis within continually and seasonally breeding lumina of the tubules or lobules of the testis. Within the mammals and birds (Roosen-Runge 1977). amniotic testis, germ cells proceed through the phases of Recent studies on the germ cell development strategy spermatogenesis within seminiferous tubules (Lebonde and within seasonally breeding reptiles have suggested that a third Clermont 1952; Lofts 1977; Kumar 1995). Germ cells germ cell development strategy exists within an amniotic

© 2005 The Royal Swedish Academy of Sciences  Germ cell development strategy in S. pygaea • Gribbins et al. Acta Zoologica (Stockholm) 86: 223–230 (October 2005) testis (Gribbins and Gist 2003; Gribbins et al. 2003). Germ within a snake testis. We relate these observations to the cells within the seminiferous epithelia of the temperate lizard accumulating data that suggest temperate reptiles retain a (Podarcis muralis) and turtle (Trachemys scripta) progress temporal germ cell development pattern similar to anamniotes through the phases of spermatogenesis as a single population. and to the context of sperm storage, the oviductal cycle, and Their temporal germ cell development strategy leads to a single the renal segment cycle previously described for the black major spermiation event towards the end of spermatogenesis swamp snake. that is reminiscent of that seen in anamniotic testes. Reptiles phylogenetically represent the most ancestral amniotes and it Materials and methods has been suggested that they are the bridge between anamniotes and the more derived amniotes, birds and mammals Animal collection (Robert 1975). Therefore, this temporal germ cell development strategy within a structurally amniotic testis may be transi- Adult male black swamp snakes (Seminatrix pygaea), over tional and evolutionarily significant. However, this episodic 23 cm in snout–vent length, were captured using unbaited germ cell development has been documented in only these minnow traps and from under cover boards along the shore- two temperate reptiles, which represent only two of the major line of Ellenton Bay, which is located on the Department of reptilian clades (Chelonia and Sauria). Furthermore, specific Energy’s Savannah River Site in Aiken County, SC. The information on the germ cell development within tropical, population of black swamp snakes occupying this shallow continuously breeding reptiles has not been documented. bay is thought to be the largest known for this species Thus, the reality of these new histological data is that more (Gibbons and Semlitsch 1991). Fifteen black swamp snakes species have to be studied from both tropical and temperate were collected during five different months, 4 months in environments to determine if this germ cell development is 1998 (May 10, June 7, July 22–24, and October 8) and from only employed by seasonally breeding species or is a common one month in 1999 (March 17–22). strategy utilized by most reptilian taxa. A complete series of specimens representing each month The present study is a continuation of this ongoing series of of the year is not presented in this study. However, the studies examining the cytological events of spermatogenesis in months represented do cover at least one complete cycle of temperate reptilian testes. The black swamp snake, Seminatrix spermatogenesis and allow for a detailed analysis of the germ pygaea, was chosen for this study to complete an initial survey cell development strategy. The focus here is on the germ cell of two temperate reptiles representing the two major sub- development strategy and not the histological details of the orders within the order Squamata. The back swamp snake entire reproductive cycle, such as the beginning and ending represents Serpentes and the recently studied European wall of spermatogenesis. Sever et al. (2002) provides more lizard (Podarcis muralis) (Gribbins and Gist 2003) represents complete information on the male reproductive cycle of Sauria. black swamp snakes and includes a description of annual The testicular samples used here are from tissues collected changes to the testis and sexual segment of the kidney. and used in a sequence of articles detailing the ultrastructure of the black swamp snake’s reproductive system (Sever and Tissue preparation for light microscopy Ryan 1999; Sever et al. 2000, 2002; Sever 2004). The information provided in the present study will also be included Snakes were killed by a lethal injection (3–5 mL) of Nembutal in this sequence and will add to the overall understanding of (Abbott Laboratories, North Chicago, IL). The reproductive the histology of the reproductive system in this snake. To our tracts were removed and testicular tissues were fixed in either knowledge this is the only comprehensive sequence of studies 10% neutral buffered formalin or 2.5% glutaraldehyde and that details the histology of the major reproductive organs of 3.7% formaldehyde in Millonig’s phosphate buffer (pH 7.2). both sexes within a species of Squamate. The 1-mm pieces of fixed testis were dehydrated through a There are two classic studies that explore the histology of graded series of alcohols. Then they were either infiltrated the snake testis and specifically detail germ cell development. with a catalysed acrylic monomer prior to embedding in glycol Volsøe (1944) describes seasonal variation in the reproductive methacrylate plastic (Electron Microscopy Sciences, Port tract of Vipera berus and includes a thorough description Washington, PA) or cleared in propylene oxide and polymerized of germ cell morphology. Fox (1952) also describes in some in epoxy resin (Embed 812, Electron Microscopy Sciences, detail the cytology of germ cells during seasonal changes Port Washington, PA). Sections (1–2 µm) were cut and then occurring in the testis in the genus Thamnophis. However, stained with methylene blue and basic fuchsin. both studies are fairly dated and fail to reference germ cell organization within the seminiferous epithelium and whether Histological analysis the germ cell development strategy is temporal or spatial. In this article, we provide the first study that describes not only Stained sections were examined using a Nikon compound the morphology of the complete germ cell cycle during microscope, and digital pictures were taken with a high- spermatogenesis but also the germ cell development strategy resolution Spot digital camera. Digital pictures were evaluated

 © 2005 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 86: 223–230 (October 2005) Gribbins et al. • Germ cell development strategy in S. pygaea for cytological changes occurring to germ cells during spermatogenesis. Cytological changes accompanying spermatogenesis are common in the testes of all vertebrates (Russell et al. 1990). Thus, the black swamp snakes’ germ cells were scored for changes to the acrosomal granule and vesicle, changes to the chromatin of the nucleus, and nuclear elongation and condensation (reviewed by Hess 1990). Once the morphologies of the germ cells were determined, trans- verse sections of the seminiferous epithelium were evaluated for consistent spatial cellular associations or stages for each month sampled.

Results

Germ cell morphology and development The testes of black swamp snake contain seminiferous tubules lined with a continuous seminiferous epithelium. This typical amniotic epithelium consists of Sertoli cells, which envelope the developing germ cells in highly convoluted cellular pro- jections or processes. Germ cells develop through the phases of spermatogenesis within the seminiferous epithelium and spermatozoa are shed at the completion of spermiation to Fig. 1—Cell types found within the seminiferous epithelia of Seminatrix pygaea. Scale bar = 20 µm. SpA, type A spermatogonia; the lumina of the seminiferous tubules. The morphological SpB, type B spermatogonia; PL, pre-leptotene spermatocyte; changes occurring to germ cells in black swamp snakes LP, leptotene spermatocyte; ZY, zygotene spermatocyte; during development are grouped together into three major PA, pachytene spermatocyte; DI, diplotene spermatocyte; phases of spermatogenesis within this study. These groupings M1, meiosis I; SS, secondary spermatocyte; M2, meiosis II; of germ cells, proliferative (pre-meiotic), meiotic and S1, step 1 spermatid; S2, step 2 spermatid; S3, step 3 spermatid spermiogenic (maturational) phases, simplify the analysis of (black arrow: acrosome granule); S4, step 4 spermatid (black arrow: the germ cell cycle and developmental strategy. acrosome granule); S5, step 5 spermatid; S6, step 6 spermatid (black arrowhead: apical extension with acrosome); S7, step 7 spermatid (white arrow: attachment site of the flagellum); MS, mature sperm. Pre-meiotic (proliferative) cells Two morphologies of spermatogonia are present within the Meiotic cells seminiferous epithelium of the black swamp snake; they are delineated type A and type B spermatogonia, respectively. Meiotic cells are characterized by increasing nuclear and Type A spermatogonia (Fig. 1, SpA) are observed within the cytoplasmic size and the gradual condensation of nuclear epithelium in all collected samples. They represent the chromatin into distinct chromosomes. Spermatogonia B majority of the germ cell population during March and are undergo mitotic divisions beginning in March and continue most frequently observed dividing in the seminiferous to produce pre-leptotene cells (Fig. 1, PL) in large numbers epithelia taken in March and May. Spermatogonia A are in the collected samples up to May Although pre-leptotene ovoid in shape and have one flattened cellular surface resting cells are still present within the seminiferous epithelium of directly on the basement membrane. Their nuclei contain the June samples, their numbers have dropped substantially prominent nucleoli and very little heterochromatin. from that seen in May Their small size easily distinguishes The nuclei of type B spermatogonia (Fig. 1, SpB) are them from the larger type A and B spermatogonia within the round and contain several large globules of heterochromatin basal region of the seminiferous epithelium. within the nucleoplasm. Spermatogonia B appear more Leptotene (Fig. 1, LP) and zygotene (Fig. 1, ZY) sper- round in shape and contain more membrane-associated matocytes are slightly larger and stain more intensely than heterochromatin than type A spermatogonia. Type B pre-leptotene cells and chromatin fibres pack their nuclei. spermatogonia also are found in the basal portion of the The major morphological difference between the two is that seminiferous epithelium throughout our sample range. The zygotene cells have more condensed globular chromatin proliferative phase of spermatogenesis produces a large compared to the fine filamentous chromatin found in population of these spermatogonia, which accumulate above leptotene cells. They appear together and constitute a large the basement membrane in the seminiferous epithelia proportion of the primary spermatocyte population within sampled in March and May. the seminiferous epithelium of the May samples.

Pachytene cells (Fig. 1, PA) first appear in the samples of spermiogenesis, which involves the development of the taken in May and remain in the seminiferous epithelium acrosome. within all the samples collected in later months in this study. Extreme elongation and condensation of chromatin within They make up a large percentage of the germ cell population the nucleus characterizes step 5, step 6 and step 7 spermatids in May and June These germ cells undergo a substantial size (elongating spermatids). Step 5 spermatids (Fig. 1, S5) increase and their nuclei contain very thick chromatin fibres are longer and more tubular in shape compared to the that are interspersed with areas of open nucleoplasm round spermatids (steps 1–4) and mark the beginning of the Diakinesis (Fig. 1, DI), metaphase I and II (Fig. 1, M1 elongation phase of spermiogenesis. The acrosome of step 5 and M2), and secondary spermatocytes (Fig. 1, SS) are most spermatids is deeply embedded into the apex of the nucleus. often observed in the May and June samples. These four Step 6 spermatids (Fig. 1, S6) undergo further elongation and transitional germ cells are usually found together within the condensation and are much thinner than step 5 spermatids. seminiferous epithelium. Diakinesis cells are characterized Late developing step 6 spermatids represent the termination by thick fully condensed chromosomal fibres that are inter- of nuclear elongation and the nuclear head has reached its spersed with large open areas of nucleoplasm. The nuclear maximum length of up to 30 µm. The acrosome, in many membrane is indistinguishable and their spoke-like chromo- instances, is still present and is found associated with a thin somal fibres are arranged in a circular pattern. Metaphase I extension of the apical nucleus (Fig. 1, S6 black arrowhead). and II cells contain a condensed clump of chromosomes that Step 5, 6 and 7 spermatids are commonly observed in is located on the equatorial plate with no apparent nuclear bundles within the seminiferous epithelium and typically boundaries. Metaphase II cells are smaller in size than have flagella projecting into the lumen in July and October metaphase I cells and visually appear to have approximately seminiferous tubules. half the amount of chromatin. Secondary spermatocytes are Step 7 (Fig. 1, S7) spermatids are completing nuclear usually dispersed randomly between metaphase I and II condensation. Their nuclei stain more intensely than those of cells. Secondary spermatocytes have lightly stained centrally any of the previous steps. Their nuclei are slightly concave and located nuclei that are roughly 15% larger than subsequent at their base show an indentation (Fig. 1, S7 white arrow) step 1 spermatids. where the prominent flagellum attaches to the nucleus. Once step 7 spermatids complete spermiogenesis, they are released as mature spermatozoa (Fig. 1, MS). The mature spermatozoa Spermiogenic cells are vermiform in shape with very long nuclear heads (up to Spermiogenesis is divided into seven recognizable steps 25 µm). Spermiation starts in July and terminates in the based on the terminology of Russell et al. (1990) for October sample. mammalian species and includes the development of the acrosomic system, elongation of the nucleus, and condensa- Germ cell development strategy within the seminiferous epithelium tion of chromatin material. The appearance of step 1 (Fig. 1, S1) and 2 (Fig. 1, S2) spermatids in the black swamp snake Spermatogonial proliferation is just beginning in the March testis marks the beginning of spermiogenesis. Their small size testis. The majority of the germ cell population in March and lightly stained Golgi zone (early development of the (Fig. 2) is spermatogonia (types A and B) with a few germ acrosomic system) that lies in juxtaposition to the nuclear cells advancing as far as the pre-leptotene stage of meiosis 1. surface characterize these spermatids. Their spherical nuclei No spermatids (representing spermiogenesis) and late devel- are centrally located and contain one or more chromatin oping spermatocytes (representing meiosis) are found within bodies and lightly staining perfuse chromatin. Steps 1 and 2 the March seminiferous epithelium. Sertoli cell nuclei are first appear together within the May testes of black swamp located in close association with the basement membrane. snakes. The majority of the germ cell population is completing A distinct acrosomic vesicle in direct contact with the proliferation (mitosis) by May (Fig. 3). Most of the germ cell nuclear envelope characterizes step 2, step 3 and step 4 population has advanced as a single cohort into the phases of spermatids (Fig. 1, S2, S3, S4). A proper section through the meiosis 1 and early spermiogenesis. Though type A and B acrosomic vesicle reveals a prominent staining acrosomic spermatogonia are found throughout the months of sperma- granule (Fig. 1, S3 and S4, black arrows) that is basally togenesis, they are typically only observed near the basement located within the vesicle. A large, towering acrosome, which membrane and mitotic divisions of these germ cells are extends far away from the apex of each nucleus, distinguishes limited to the March and May testes. The dominant cell types step 3 from steps 2 and 4. The acrosomes of step 4 spermatids found within the May seminiferous epithelia are primary begin to flatten, giving their acrosomes an oval shape. These spermatocytes that are sequentially developing through the three spermatids along with step 1 are observed together phases of meiosis I. Also, a small part of the germ cell within the seminiferous epithelium in July and represent population has progressed as far as the early events of sper- most of the spermatid population at this time. These four miogenesis as is evident by round spermatids found near the steps (round spermatids) represent the acrosomic phase lumen of individual seminiferous tubules. The accumulation

Fig. 4—Sections of June seminiferous epithelia with higher magnifications of represented cell types. Scale bar = 50 µm for Fig. 2—Sections of March seminiferous epithelia with higher seminiferous epithelium section and 10 µm for magnified germ cell magnifications of represented cell types. Scale bar = 50 µm for types. S3, step 3 spermatid; S2, step 2 spermatid; S1, step 1 seminiferous epithelium section and 10 µm for magnified germ cell spermatid; SS, secondary spermatocyte; DI, diplotene; types. PL, pre-leptotene; SpB, type B spermatogonia; SpA, type A PA, pachytene; LP, leptotene; PL, pre-leptotene spermatocytes; spermatogonia. SpB, type B spermatogonia; SpA, type spermatogonia.

Fig. 3—Sections of May seminiferous epithelia with higher magnifications of represented cell types. Scale bar = 50 µm for seminiferous epithelium section and 10 µm for magnified germ cell types. S1, step 1 spermatid; SS, secondary spermatocyte; DI, diplotene spermatocyte; PA, pachytene spermatocyte; Fig. 5—Sections of July seminiferous epithelia with higher ZY, zygotene spermatocyte; LP, leptotene spermatocyte; magnifications of represented cell types. Scale bar = 50 µm for PL, pre-leptotene spermatocyte; SpB, type B spermatogonia; seminiferous epithelium section and 10 µm for magnified germ cell SpA, type A spermatogonia. types. White arrowhead represents spermiated mature spermatozoa within the lumen of the seminiferous tubule. S6, step 6 spermatid; S5, step 5 spermatid; S4, step 4 spermatid; S3, step 3 spermatid; of leptotene, zygotene, pachytene and diplotene spermato- S2, step 2 spermatid; S1, step 1 spermatid; SS, secondary cytes, the depletion of the spermatogonial population, and spermatocyte; M2 and M1, meiosis 1 and 2; DI, diplotene the absence of elongating spermatids prevent consistent spermatocyte; PA, pachytene spermatocytes; cellular associations between early and late developing germ SpB, type B spermatogonia; SpA, type A spermatogonia. cells within the May seminiferous epithelium. By June (Fig. 4), the major portion of the germ cell population is undergoing spermiogenesis and finishing meiosis. represented within the seminiferous epithelium and constitute Most of the germ cell population will spend the rest of its the largest portion of the germ cell population during this development in spermiogenesis. The mitotic and meiotic month. Meiosis is seen in only a small portion of the germ cell events of spermatogenesis are nearly complete by June and population and five to six spermatids can appear within the will continue in only small portions of the germ cell population same cross-section of the seminiferous epithelium. Pockets through October The majority of the germ cell population in of spermatozoal release can be seen in many seminiferous June is represented by the round (early) spermatids that are tubules as is evident by the presence of luminal spermatozoa actively developing the acrosomic system. (Fig. 5, white arrowhead). The accumulation and layering The later stages of spermiogenesis are observed within the of sequential spermatid steps prevent consistent cellular July seminiferous epithelia (Fig. 5). Round spermatids (steps associations between germ cell generations within the testis 1, 2, 3) and elongating spermatids (steps 4, 5, 6) are equally at this time.

morphology and in some instances when and where germ cell types are seen within the snake testis during different months of the year. Neither study looked specifically at the mode of germ cell development compared to that of other temperate reptiles and also to that of more derived amniotic taxa. More recent information on temperate snake spermatogenesis is tied to reproductive cycles of both sexes and focuses on the beginning and end of the germ cell cycles, how spermatozoa are stored within individual species, and the steroidogenic activity of the testis (Krohmer et al. 1987; Aldridge et al. 1990; Hondo et al. 1994; Schuett et al. 1997; Santos and Llorente 2001; Clesson et al. 2002). Fig. 6—Sections of October seminiferous epithelia with higher The majority of the black swamp snake germ cell popula- magnifications of represented cell types. Scale bar = 50 µm for tion progresses through the major phases of spermatogenesis seminiferous epithelium section and 10 µm for magnified germ cell (proliferation, meiosis, spermiogenesis) as a single cohort types. MS, mature spermatozoa; S7, step 7 spermatid; S6, step 6 within the seminiferous epithelium. This temporal germ cell spermatid; S5, step 5 spermatid; S4, step 4 spermatid; S2, step 3 development strategy is considered ancestral and occurs in spermatid; S1, step 2 spermatid; PA, pachytene spermatocyte; almost all fish and amphibian species that have been studied SpB, type B spermatogonia; SpA, type A spermatogonia. to date (Dodd and Sumpter 1984; Lofts 1984). Historically, studies examining germ cell development strategies within amniotes have been limited almost exclusively to mammals The completion of spermiation and lingering spermatids with a few studies involving different bird species. All mammals in the process of finishing spermiogenesis are seen within the studied to date possess a spatial germ cell development October seminiferous epithelium (Fig. 6). Most of the germ strategy with consistent cellular associations, independent of cell population is represented by late elongating spermatids seasonality, which leads to multiple spermiation events during and mature spermatozoa found within the lumina of most their reproductive cycles (Roosen-Runge 1977; Russell et al. seminiferous tubules. There are a number of tubules found in 1990). the October testis that have fully completed both spermio- Three recent studies on temperate reptiles representing genesis and spermiation. Their seminiferous epithelia are different orders have shown a similar temporal germ cell devoid of most germ cell types except type A and B spermato- development to that of the black swamp snake. The European gonia and a few remnant spermatids that have not completed wall lizard (Gribbins and Gist 2003), the red-eared slider turtle spermiogenesis. One cycle of spermatogenesis is completed (Gribbins et al. 2003), and the American alligator (Gribbins by October within the samples collected in this study. and Gist, submitted for publication) represent distinctly different taxa (Squamata, Testudines and Crocodilia) within the class Reptilia, and they all share this common temporal Discussion germ cell development strategy. Phylogenetic relationships Spermatogenesis within male black swamp snakes (Seminatrix among the Reptilia are controversial, and the class is widely pygaea) closely parallels that seen in the previously studied considered to be polyphyletic (Jefferies 1986). However, slider turtle, Trachemys scripta (Gribbins et al. 2003). Germ ancestors of modern reptiles were the first amniotes to evolve, cell development starts in early spring, continues through the and within the Reptilia they shared many derived reproductive summer, and is completed by autumn. Most temperate turtles characters that allowed for their successful invasion of the are postnuptial in their sperm development (Moll 1979), as are terrestrial environment. Most of these adaptations protect most temperate snakes (Licht 1984). Thus in the temperate and store gametes away from the extreme terrestrial environ- black swamp snake, sperm development seems to follow this ment and allow for safe transfer of spermatozoa directly postnuptial pattern, which is consistent with previous to the female reproductive tract. These male reproductive research. This male gamete production pattern is the opposite adaptations include tubular testes, internal fertilization with its of what has been observed in the female reproductive system, associated copulatory organs, organs for male sperm storage which shows a prenuptial pattern to egg production and (epididymis and/or ductus deferens), and sperm behaviour ovulation (Seigel et al. 1995). The significance of these (Pudney 1990; Gist and Congdon 1998). differences between the timing of male and female gamete Since this temporal germ cell development strategy is development will be discussed later in this section. found in a large number of seasonally breeding vertebrate To our knowledge this is the first complete study of a snake taxa (fish, amphibians and now reptiles); many could argue species that focuses specifically on the germ cell development that this newly described temporal spermatogenesis is a strategy employed during spermatogenesis. Volsøe (1944) strategy utilized by all temperate breeding vertebrates including and Fox (1952) provide detailed analysis of male germ cell amniotes and not a primitive reproductive character shared by

 © 2005 The Royal Swedish Academy of Sciences Acta Zoologica (Stockholm) 86: 223–230 (October 2005) Gribbins et al. • Germ cell development strategy in S. pygaea only temperate reptiles within the amniotic clade. However, breeding events by alternating the insertion of hemipeni, germ cell development within temperate breeding birds and which is a described behaviour in snakes that engage in mammals is consistent with the spatial development of multiple matings within a three-day period (Olsson and continually breeding mammals and birds (Yamamoto et al. Madsen 1998). This may ensure adequate sperm deposition 1967; Roosen-Runge 1977; Tait and Johnson 1982; Tsubota in the female reproductive tract during promiscuous spring and Kanagawa 1989; Tiba and Kita 1990, 1991; Kumar matings even though sperm release only occurs in the autumn. 1995; Foreman 1997). Therefore, these three reptiles and the This study on black swamp snakes completes an initial black swamp snake contain amniotic testes with an ancestral survey examining germ cell development in temperate episodic mode of germ cell development, which is different Squamates. The phylogenetic implications of this conserved from the spatial development seen within the rest of the germ cell development strategy within the most ancestral amniotes. amniotes, reptiles, could be significant. However, few This temporal sperm production not only has phylogenetic comparative germ cell development data exist for specific implications on the reproductive systems of amniotes but species of seasonally breeding reptiles and information on also further elucidates our understanding of the reproductive germ cell development strategies in tropical species is biology of black swamp snakes. The female reproductive lacking. How does seasonality affect germ cell development in system follows a prenuptial pattern of egg development, continuous versus seasonally breeding reptiles? This question where eggs are produced after emergence from hibernation and other comparative and phylogenetic questions cannot be in the early spring (Seigel et al. 1995; Sever and Ryan 1999). answered unless more data are collected on germ cell devel- Therefore, asynchrony exists between the female and male opment strategies and sperm development in tropical and production of viable gametes because male black swamp temperate species. We feel, however, that our results, along snakes have a postnuptial development where spermatozoa with the accumulating data suggesting that this mode of are produced over the summer months and spermiated to the spermatogenesis is a common strategy shared among epididymis and ductus deferens between July and October temperate breeding reptiles, can be used as a model system This difference in gamete production is not unheard of in for future comparative work on reptilian spermatogenesis reptiles (Licht 1984) and facilitates the need for sperm storage that will facilitate our understanding of the anatomy, (Gist and Congdon 1998; Sever et al. 2002). Indeed, sperm physiology and evolution of the amniotic testis. storage occurs in the male ductus deferens/ampulla (Sever 2004) and within sperm storage tubules (SSTs) in the Acknowledgements oviducts of female black swamp snakes (Sever and Ryan 1999). Black swamp snakes apparently breed in the late spring We thank Travis J. Ryan for the collection of the black swamp and early summer (Sever and Ryan 1999). Spermatozoa are snakes used in this study found in the ductus deferens throughout the year (Sever 2004) and the renal sexual segment shows little seasonal variation References (Sever et al. 2002), indicating that male snakes can breed at any time of the year. However, Sever and Ryan (1999) found Aldridge, R. D., Greenhaw, J. J. and Plummer, M. V. 1990. The evidence that autumn matings (when spermiation is male reproductive cycle of the rough green snake (Opheodrys commencing) do not occur, as spermatozoa are absent from aestivus). – Amphibia and Reptilia 11: 165–172. the female oviducts at this time. Sever and Ryan (1999) also Clesson, D., Bautista, A., Baleckaitis, D. D. and Krohmer, R. W. 2002. Reproductive biology of male eastern garter snakes reported spermatozoa in the SSTs of a female collected in (Thamnophis sirtalis sirtalis) from a denning population in central March and in the anterior vagina of a female taken in June, Wisconsin. – American Midland Natura1ist 47: 376–386. which provides evidence for recent breeding activity. Also, Dodd, J. M. and Sumpter, J. P. 1984. Fishes. In Lamming, G.E. Sever (2004) found that fewer spermatozoa are located in the (Ed.): Marshall’s Physiology of Reproduction Volume 1 Reproductive ampulla ductus deferens in March than any other month of Cycle of Vertebrates, pp. 1–127. Churchill Livingstone, New York. the year. This could be interpreted as recent sperm evacuation Foreman, D. 1997. Seminiferous tubule stages in the prairie dog (Cynomys ludovicianus) during the annual breeding cycle. – and evidence for recent mating. Furthermore, female black Anatomical Record 247: 355–367. swamp snakes collected in June had undergone ovulation Fox, W. 1952. Seasonal variation in the male reproductive system and females killed in July contained developing fetuses of pacific coast garter snakes. – Journal of Morphology 90: 481– (Sever and Ryan 1999; Sever et al. 2000). 553. Thus, without the ability to store spermatozoa in the ductus Gibbons, J. W. and Semlitsch, R. D. 1991. Guide to the Reptiles and deferens over the winter (assuming one cycle of temporal Amphibians of the Savannah River Site, Athens, GA. University of sperm development and a postnuptial production of sperma- Georgia Press, Athens, GA. Gist, D. H. and Congdon, J. D. 1998. Oviductal sperm storage as a tozoa in the autumn), males would not be able to furnish reproductive tactic of turtles. – Journal of Experimental Zoology viable spermatozoa to females during subsequent early spring 282: 526–534. matings. 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