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 anam- niotes 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 infor- mation 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