Ultrastructure of the Reproductive System of the Black Swamp Snake (Seminatrix Pygaea). II. Annual Oviducal Cycle

JOURNAL OF MORPHOLOGY 245:146–160 (2000)

Ultrastructure of the Reproductive System of the Black Swamp Snake (Seminatrix pygaea). II. Annual Oviducal Cycle

David M. Sever,1* Travis J. Ryan,2,3 Terasa Morris,1 Deborah Patton,1 and Shannon Swafford1

1Department of Biology, Saint Mary’s College, Notre Dame, Indiana 2Division of Biological Sciences, University of Missouri, Columbia, Missouri 3Savannah River Ecology Laboratory, Aiken, South Carolina

ABSTRACT This article is the ﬁrst ultrastructural nar, and cells with elongate cilia alternate with secretory study on the annual oviducal cycle in a snake. The ultra- cells. The secretory product of the infundibulum consists structure of the oviduct was studied in 21 females of the largely of lipids, whereas a glycoprotein predominates in viviparous natricine snake Seminatrix pygaea. Specimens the vagina; however, both products are found in these were collected and sacriﬁced in March, May, June, July, regions and elsewhere in the oviduct. In the SST area and and October from one locale in South Carolina during the anterior vagina, tubular glands are compound as well 1998–1999. The sample included individuals: 1) in an as simple. The epithelium of the SST is most active after inactive reproductive condition, 2) mated but prior to ovu- mating, and glycoprotein vacuoles and lipid droplets are lation, and 3) from early and late periods of gravidity. The equally abundant. When present, sperm form tangled oviduct possesses four distinct regions from cranial to masses in the oviducal lumen and glands of the SST area. caudal: the anterior infundibulum, the posterior infundib- The glands of the uterus are always simple. During sperm ulum containing sperm storage tubules (SSTs), the migration, a carrier matrix composed of sloughed epithe- uterus, and the vagina. The epithelium is simple through- lial cells, a glycoprotein colloid, lipids, and membranous out the oviduct and invaginations of the lining form tubu- structures surround sperm in the posterior uterus. During lar glands in all regions except the anterior infundibulum gravidity, tubular glands, cilia, and secretory products and the posterior vagina. The tubular glands are not al- diminish with increasing development of the fetus, and veolar, as reported in some other snakes, and simply rep- numerous capillaries abut the basal lamina of the atten- resent a continuation of the oviducal lining with no addi- uated epithelial lining of the uterus. J. Morphol. 245: tional specializations. The anterior infundibulum and 146–160, 2000. © 2000 Wiley-Liss, Inc. vagina show the least amount of variation in relation to season or reproductive condition. In these regions, the KEY WORDS: Reptilia; Serpentes; Seminatrix pygaea; epithelium is irregular, varying from squamous to colum- ultrastructure; oviduct

Numerous articles exist on the oviducal anatomy gions throughout the reproductive cycle. Other con- of squamate reptiles (see reviews by Fox, 1977; tributions in this series will deal with ultrastructure Blackburn, 1998), but relatively few of these studies of the male reproductive system. have speciﬁcally addressed the ultrastructure of the Seminatrix pygaea is a small (20–40 cm snout– oviduct in squamates (Hoffman, 1970; Hoffman and vent length as adults), highly aquatic snake limited Wimsatt, 1972; Bou-Resli et al., 1981; Perkins and to the southern Atlantic coastal plain (Dorcas et al., Palmer, 1996; Girling et al., 1997, 1998). As a part of 1998). The species ovulates large, yolky eggs and is a comprehensive study of reproduction in the Black viviparous by lecithotrophy, i.e., most if not all nu- Swamp Snake (Seminatrix pygaea Cope), we have trients for fetal development are supplied via the undertaken an analysis of various ultrastructural yolk of the ovulated ovum (Stewart, 1992; Stewart et features of the oviduct of this viviparous snake. Part al., 1990). The population of the species that we I dealt with transport of sperm and sperm storage in sampled is the largest known for the species (Gib- the oviduct (Sever and Ryan, 1999). Ultrastructural details of a carrier matrix in the uterus and sperm storage tubules (SSTs) in the posterior infundibulum (uterine tube) were described (Sever and Ryan, Contract grant sponsor: U.S. Department of Energy to the Univer- 1999). Another part will deal with the cytological sity of Georgia Research Foundation; Contract grant number: DE- relationships between the uterine lining and the FCO9-96SR18546. embryonic membranes. In this article, we report *Correspondence to: David M. Sever, Department of Biology, Saint more general aspects of oviducal cytology, with an Mary’s College, Notre Dame, IN 46556. emphasis on the ultrastructure of the various re- E-mail: [email protected]

TABLE 1. Specimens used in this study

Date Follicles/Eggs sacrificed SVL (cm) Location N Range Mean (mm) SE 31 March 22.2 Ovaries 5R 5L 2.0–2.9 2.5 0.09 25.3 Ovaries 5R 5L 1.9–2.8 2.5 0.11 29.3 Ovaries 8R 11L 1.8–3.5 2.6 0.12 31.0 Ovaries 9R 8L 2.2–3.6 3.0 0.10 32.9 Ovaries 11R 11L 2.4–3.9 3.2 0.10 14 May 98 23.7 Ovaries 6R 6L 1.8–3.1 2.4 0.11 28.0 Ovaries 5R 3L 5.0–7.2 5.8 0.35 32.0 Ovaries 10R 7L 1.8–3.5 2.7 0.12 33.0 Ovaries 5R 4L 7.0–11.2 9.6 0.47 9 June 26.0 Uteri 3R 2L 7.4–11.2 9.2 0.78 29.0 Uteri 3R 1L 17.5–18.2 17.9 0.14 30.2 Uteri 5R 3L 13.0–15.3 13.9 0.31 35.5 Ovaries 5R 5L 2.0–3.6 2.7 0.14 30 July 29.0 Ovaries 7R 3L 1.7–2.2 2.0 0.05 Uteri 2R 2L* 27.5 Ovaries 6R 5L 1.8–2.6 2.2 0.08 27.5 Ovaries 6R 4L 1.8–2.3 2.0 0.06 24.0 Ovaries 5R 3L 1.4–2.0 1.7 0.06 8 October 26.0 Ovaries 7R 7L 2.0–3.8 2.9 0.14 29.1 Ovaries 7R 9L 1.5–3.0 2.2 0.11 30.1 Ovaries 13R 10L 1.2–3.3 2.4 0.12 33.0 Ovaries 11R 7L 1.5–2.6 2.1 0.08 37.4 Ovaries 14R 11L 1.8–3.6 2.7 0.10 *Four fully formed fetuses, two of which were preserved in situ; other two measured 95 mm and 97 mm SVL. bons and Semlitsch, 1991), and other aspects of the preserved in 10% neutral buffered formalin (NBF) female reproductive cycle of this population were and housed in the research collections at Saint described by Seigel et al. (1995). They reported rapid Mary’s College. follicle growth in spring after hibernation, ovulation After death, the cloaca and both oviducts were in early June, and parturition in early August (Sei- removed in their entirety and fixed in a 1:1 solution gel et al., 1995). Because the reproductive cycle has of 2.5% glutaraldehyde in Millonig’s phosphate been well-studied (and continues to be monitored) buffer at pH 7.4 and 3.7% formaldehyde buffered to and seasonal samples are readily obtainable, fe- pH 7.2 with monobasic and dibasic phosphate. Fol- males from this population of S. pygaea are ideal for lowing the criteria of Blackburn (1998), three basic the first comprehensive studies on the oviducal ul- divisions of the oviduct could be recognized grossly: trastructure of a viviparous snake. infundibulum (with a posterior region of sperm storage tubules, SSTs), uterus, and vagina. The posterior infundibulum where SSTs are found has been MATERIALS AND METHODS referred to as the uterine tube by some authors (e.g., Specimens of Seminatrix pygaea were collected at Perkins and Palmer, 1996). Ellenton Bay, located on the Department of Energy’s For transmission electron microscopy (TEM), sec- Savannah River Site in Aiken County, South Caro- tions of the oviduct 2 mm in length were removed lina. Ellenton Bay is a shallow (2 m maximum from each division of the right oviduct. Tissue was depth), 10 ha freshwater “Carolina bay” that is rel- taken from the anterior portion of the infundibulum atively permanent (Gibbons and Semlitsch, 1991). as well as the SST area. From the uterine area, Collections were made during four periods in 1998 tissue was taken from both the anterior and poste- (10 May, 7 June, 22–24 July, and 29 September–2 rior ends of the uterus in nongravid snakes, whereas October), and one period in 1999 (17—22 March). in gravid snakes tissue was taken from the uterine Specimens were sacrificed within a week of capture lining surrounding an embryo (incubation chamber) (Table 1). Specimens were collected in unbaited min- and from the interembryonic area between eggs. now traps and from under coverboards alongside the After initial fixation, tissues were rinsed in water, bay. The reptiles of Ellenton Bay are the subjects of postfixed in 2% osmium tetroxide, and dehydrated long-term monitoring studies. In order to minimize through a graded series of alcohol. the ecological effects caused by removal of snakes, Tissues were subsequently cleared in propylene we limited each sample to a maximum of five adult oxide and immersed in increasing concentrations of female snakes. Specimens were killed by a lethal an epoxy resin (EmBed 812, Electron Microscopy injection (3–5 ml) of Nembutal (Abbott Laboratories, Sciences, Fort Washington, PA) in absolute ethanol North Chicago, IL). Carcasses of all specimens were before polymerization in pure resin for 36 h at 60°C. 148 D.M. SEVER ET AL. Plastic sections were cut with an RMC MT7 ultra- study. Some evidence exists, therefore, for at least a microtome (Research and Manufacturing Co., Tuc- biennial reproductive cycle among females in this son, AZ) and DiATOME diamond knives (Di- population, rather than an annual cycle as sug- ATOME, Biel, Switzerland). Semithin sections (0.5 gested by Seigel et al. (1995). Sperm only were found ␮m) for light microscopy were placed on microscope in the oviducts of the two specimens mentioned slides and stained with toluidine blue. Ultrathin above that were collected in May and June, so no sections (70 nm) were collected on uncoated copper evidence was found for a late summer or fall mating, grids and stained with solutions of uranyl acetate as is known in some snakes (Halpert et al., 1982; and lead citrate. Ultrathin sections were viewed Schuett, 1992). with a Hitachi H-300 transmission electron microscope (Nissei Sangyo America, Mountain View, CA). The left oviduct was prepared for light microscopy Light Microscopy by the standard paraffin method. Following fixation Basic oviducal histology is illustrated by sagittal in the 1:1 glutaraldehyde:formalin solution used for paraffin sections through the oviduct of a nonvitel- electron microscopy, tissue was dehydrated in etha- logenic female collected and killed in October (Fig. nol, cleared in Histosol (National Diagnostics, Man- 1). Four regions are histologically distinct and can ville, NJ), and embedded in paraffin. Sections were be characterized by their glandular epithelium. The cut at 10 ␮m and placed on albumin-coated slides. anterior infundibulum lacks tubular glands (Fig. Alternate slides were stained with hematoxylin- 1A). The SST area in the posterior infundibulum eosin (general cytology), Alcian blue 8GX at pH 2.5 possesses both simple and compound tubular glands (primarily carboxylated glycosaminoglycans) counter- (Fig. 1B). The uterus has only simple tubular glands stained using the periodic acid-Schiff’s (PAS) proce- (Fig. 1C). The anterior end of the vagina is defined dure (neutral carbohydrates), brilliant indocyanine 6B by the reappearance of compound tubular glands (proteins), and Cason’s trichrome (connective tissue). (Fig. 1D), although gland tubules become absent in Staining procedures followed Kiernan (1990). the posterior vagina. The oviduct has three basic layers which are, from RESULTS superficial to deep: the visceral pleuroperitoneum, Reproductive Condition of the Sample the muscularis, and the mucosa (Fig. 1). The visceral pleuroperitoneum is the same throughout the length If a female is reproductively active, vitellogenesis of the oviduct, and consists of squamous mesothe- becomes evident by May, ovulation occurs in June, lium with a thin subsurface layer of loose connective and parturition takes place in late July and August. tissue bound by collagen fibers. The muscularis con- Follicles in reproductively inactive females are typ- sists mostly of longitudinal fibers (Fig. 1C) and is ically 2.0–2.7 mm in diameter (Table 1). Only two of thickest in the walls of the posterior vagina. Re- the five specimens collected in March showed indi- gional and seasonal differences in the mucosa will be cations of incipient vitellogenesis (follicles 3.0–3.2 described at the ultrastructural level in subsequent mm dia), and just two of the four females from the sections of this article. May collection were vitellogenic (follicles 5.0–11.2 Histochemically, the mucosal lining of the entire mm dia). One of the preovulatory, vitellogenic fe- oviduct and the exocrine glands is PAS positive for males (33 cm SVL) collected in May had mated neutral carbohydrates and slightly positive with (sperm in the SSTs) but the other lacked sperm in brilliant indocyanine 6B for proteins. Scattered ar- the oviduct. eas are positive with Alcian blue 8GX at pH 2.5, an Three out of four females collected in early June indicator of carboxylated glycosaminoglycans. had ovulated and possessed eggs in the uterus in an early stage of development (Table 1). The other female from June was in an inactive reproductive Ultrastructure state (follicles 2.7 mm mean dia) but had sperm in Anterior infundibulum. The epithelium of the the vagina and posterior uterus, indicating a recent anterior infundibulum is characterized throughout mating. One of the four females collected in late July its cycle by squamous to columnar cells with euchro- possessed nearly full-term fetuses, but the other matic nuclei, scant cytoplasm, elongate cilia, narrow three females apparently had not bred in the current and labyrinthine intercellular canaliculi, and nu- season, since the oviducts were not distended as one merous lipid droplets (Figs. 2, 3). A filamentous ma- may expect after housing fetuses (Blackburn, 1998). terial is found in the oviducal lumina of all speci- The five females collected in October showed no ev- mens (Figs. 2A, 3A) but secretory vacuoles such as idence of recent parturition and may have been re- those found elsewhere in the oviduct are few. Syn- productively inactive during the past summer, al- thetic activity associated with production of glycop- though one cannot be certain that these females roteins as well as lipids is indicated even in nonvitel- skipped the last breeding season. logenic females by the occurrence of rough and Thus, only nine of 23 specimens examined were smooth endoplasmic reticulum (Fig. 2C). Lipid drop- definitely producing eggs during the period of this lets vary greatly in density (Figs. 2B,C, 3D), and are Fig. 1. Seminatrix pygaea. Light micrographs of sagittal paraffin sections through the oviduct of a 37.4 cm SVL nonvitellogenic female sacrificed 8 October 1998. Stained with hematoxylin-eosin. A: Anterior infundibulum. B: Sperm storage tubule area in the posterior infundibulum. C: Uterus. D: Vagina. Bv, blood vessels; Ep, epithelium; Lp, lamina propria; Mu, muscularis; Ol, oviducal lumen; Os, ostium; Sst, sperm storage tubules; Ug, uterine glands; Vg, vaginal glands. Fig. 2. Transmission electron micrographs of the anterior infundibulum of the oviduct in nonvitellogenic Seminatrix pygaea. A: Mucosa of a 35.5 cm SVL female sacrificed 9 June 1998. B: Same specimen as A, illustrating epithelial lipid droplets of different densities. C: Organelles in the infundibular epithelium of a 28.0 cm SVL female sacrificed 14 May 1998. Bb, basal bodies; Bl, basal lamina; Cf, collagen fibers; Ci, cilia; Fm, filamentous material; Ic, intercellular canaliculi; Ld, lipid droplets; Mi, mitochondria; Nu, nucleus of an epithelial cell; Np, nuclear pores; Ol, oviducal lumen; Rer, rough endoplasmic reticulum; Ser, smooth endoplasmic reticulum. Fig. 3. Transmission electron micrographs of the anterior infundibulum of the oviduct in mated and gravid Seminatrix pygaea. A: Oviducal mucosa of a mated but preovulatory female 33.0 cm SVL sacrificed 14 May 1998. B: Same specimen as A, illustrating apical cytoplasm of a ciliated cell. C: Apical cytoplasm of a 30.2 cm SVL female sacrificed 9 June in early gravidity. D: Apical cytoplasm of a 29.0 cm SVL female sacrificed 30 July 1998 in late gravidity. Cd, cellular debris; Cf, collagen fibers; Ci, cilia; Fm, filamentous material; Fmol, filamentous material in the oviducal lumen; Ic, intercellular canaliculi; Ld, lipid droplets; Mi, mitochondria; Nu, nucleus of an epithelial cell; Ol, oviducal lumen. 152 D.M. SEVER ET AL. most numerous in a nonvitellogenic female collected yolk and a small embryonic disc adjacent to the in June that had recently mated (Fig. 2B). During portion of the uterus associated with the mesome- gravidity, cellular debris is occasionally observed in trium. During this stage, the interembryonic region the lumen (Fig. 3C), perhaps resulting from slough- consists mostly of ciliated cells containing lipid drop- ing or cycling of the epithelium. lets, and sloughing of the epithelium is apparent Posterior infundibulum (uterine tube, SST (Fig. 6A). Nuclei are generally euchromatic but vary area). The epithelium of the SST area in reproduc- in electron density (Fig. 6B). Over the developing tively inactive females again possesses irregularly eggs, the epithelium consists of cuboidal cells that shaped cells, but nuclei are heterochromatic and lack cilia and possess many mitochondria but few cilia reduced (Fig. 4A) or absent (Fig. 4B). Scattered secretory vacuoles (Fig. 6C,D). Some lipid droplets lipid droplets and apical secretory vacuoles contain- occur, and Golgi complexes and microfilaments are ing a flocculent material occur in a non-vitellogenic found in the epithelium surrounding the embryo female collected in May (Fig. 4A), but minimal se- (Fig. 6D). cretory activity characterizes a nonvitellogenic fe- Later in gravidity, at the end of July when fetuses male from July (Fig. 4B). are nearly fully developed (95–97 mm SVL), the In a vitellogenic female sacrificed after mating but oviducts are thin, clear, hypertrophied tubes prior to ovulation, sperm are numerous in the lumen through which we could clearly observe that adja- of the oviduct and the SSTs (Fig. 4C,D). The cytology cent fetuses were nearly in contact with one another. of this area was described in detail by Sever and Although tissue can be obtained from the region Ryan (1999). The epithelium is again irregular, and between developing snakes, the interembryonic area alternation of ciliated and secretory cells is distinct is no longer distinct ultrastructurally from the uter- (Fig. 4C). Lipid droplets also are numerous, espe- ine epithelium surrounding the embryo (Fig. 7). Cil- cially basally. Sperm are irregularly oriented in both iated cells are sparse, cytoplasm is scant, and the the lumen of the oviduct and that of SSTs (Fig. nuclei are oval, euchromatic, and nearly fill the en- 4C,D). The SSTs simply represent a continuation of tire cells (Fig. 7A). Small lipid droplets are numer- the oviducal lining and do not possess a specialized ous (Fig. 7B,C); the only conspicuous organelles are cytology (Fig. 4D). Occasional “light cells” exhibiting mitochondria (Fig. 7D). Intercellular canaliculi are deteriorated cytoplasm are found between “dark narrow and straight apically and labyrinthine ba- cells” with normal cytology, indicating a pro- sally (Fig. 7D). Capillaries abut closely on the basal grammed cycling of the epithelium (Fig. 4D). lamina of the uterine epithelium, resulting in an Uterus. In a reproductively inactive female sac- epithelial interface of only 2–5 ␮m between the con- rificed in July, the uterine epithelium lining the tents of the capillaries and the uterine lumen (Fig. oviduct and the simple tubular uterine glands pos- 7A–C). sess ciliated and secretory cells, but secretory vacu- Vagina. The epithelium of the vagina is charac- oles are not numerous (Fig. 5A,B). Lipid droplets terized in all stages of the cycle by secretory cells also are scant and very electron-dense (Fig. 5A). The alternating with cells possessing elongate cilia (Figs. nuclei of epithelial cells of the anterior uterus are 8, 9). Vaginal glands in the anterior vagina are, like more heterochromatic (Fig. 5A) than those of the other oviducal glands, simple continuations of the posterior uterus (Fig. 5B). oviducal lining (Fig. 8A–C). In a female sacrificed in June after a recent mat- Throughout the vagina, ciliated cells seem to pre- ing, sperm are present in the posterior uterus and dominate in all reproductive stages, and secretory are associated with a carrier matrix (Fig. 5C,D), as vacuoles are limited to small areas of apical cyto- described previously (Sever and Ryan, 1999). The plasm. The secretory vacuoles contain a flocculent matrix consists of a colloid emanating from secre- material as noted elsewhere in the oviduct. In addi- tory vacuoles of the uterine epithelium (Fig. 5C) tion, an eccentric electron-dense particle occurs in associated with desquamated epithelium, lipid drop- some secretory vacuoles (Figs. 8D, 9D), and such lets, and membranous structures (Fig. 5D). particles are not prominent in secretory vacuoles in During gravidity, portions of the uterine lining other regions of the oviduct. Golgi complexes and surround the developing embryos while other, more RER indicate synthetic activity in the secretory cells constricted portions occur between developing em- (Fig. 8D), and microfilaments and mitochondria are bryos (Sever and Ryan, 1999; Figs. 6, 7). The portion numerous in ciliated cells (Fig. 9D). Nuclei are typ- of the uterus surrounding the developing embryo is ically euchromatic and intercellular canaliculi are referred to as the incubation chamber (Stewart, narrow and basally convoluted (Figs. 9C,D). 1990). Early in gravidity, the ultrastructure of the incubation chamber differs from the interembryonic DISCUSSION regions (Fig. 6), but the interembryonic regions be- Comparisons With Other Squamates come similar to the incubation chamber as gravidity advances (Fig. 7). The Squamata consists of the snakes (Serpentes), Three specimens collected in June are in early amphisbaenids (Amphisbaenia), and lizards (Sau- stages of gravidity. Eggs contain a large amount of ria). Aside from our initial study (Sever and Ryan, Fig. 4. Transmission electron micrographs of the sperm storage tubule (SST) area in the posterior infundibulum of the oviduct in Seminatrix pygaea. A: Mucosa of a 28.0 cm SVL nonvitellogenic female sacrificed 14 May 1998. B: SST of a 27.5 cm SVL nonvitellogenic female sacrificed 30 July 1998. C: Mucosa and sperm in the oviducal lumen of a 33.0 cm SVL preovulatory female sacrificed 14 May 1998. D: Same specimen as C, illustrating distal portion of a SST. Bl, basal lamina; Cf, collagen fibers; Ci, cilia; Dc, dark cell; Gl, gland lumen; Ic, intercellular canaliculi; Ld, lipid droplets; Ly, lysosomes; Nu, nucleus of an epithelial cell; Lc, light cell; Ol, oviducal lumen; Sp, sperm; Spol, sperm in the oviducal lumen; Sv, secretory vacuoles. Fig. 5. Transmission electron micrographs of the uterus of nongravid females of Seminatrix pygaea. A: Anterior uterine mucosa of a 27.5 cm SVL female sacrificed 30 July 1998. B: Same specimen as A, illustrating the uterine epithelium in the posterior uterus. C: Posterior uterus of a recently mated 35.5 cm SVL female sacrificed 9 June 1998. D: Same specimen as C, illustrating the carrier matrix in the lumen of the uterus. Cf, collagen fibers; Ci, cilia; Co, colloid; Ds, desquamated epithelial cell; Ld, lipid droplets; Mi, mitochondria; Mpt, middle piece of a sperm tail; Ms, membranous structures; Nu, nucleus of an epithelial cell; Ol, oviducal lumen; Sn, sperm nucleus; Sv, secretory vacuoles; Va, vacuole. Fig. 6. Transmission electron micrographs of the uterus of a 30.2 cm SVL Seminatrix pygaea sacrificed 9 June 1998 during early gravidity. A: Interembryonic region, illustrating uterine mucosa with dissociated epithelial cells. B: Interembryonic region, showing detail of the extremely electron-dense epithelium. C: Incubation chamber, illustrating the interface between the epithelium, oviducal lumen, and developing egg. D: Incubation chamber, showing organelles in the uterine cytoplasm. Ci, cilia; Ds, desquamated epithelial cells; Ep, uterine epithelium; Go, Golgi complex; Ld, lipid droplets; Mi, mitochondria; Mf, microfilaments; Mv, microvilli; Nu, nucleus of an epithelial cell; Ol, oviducal lumen; Sm, shell membrane; Sv, secretory vacuoles; Yo, yolk. Fig. 7. Transmission electron micrographs of the uterus of a 29.0 cm SVL Seminatrix pygaea sacrificed 30 July 1998 during late gravidity. A: Interembryonic region, showing capillaries abutting the uterine epithelium. B: Interembryonic region, showing detail of the capillary/uterine epithelium interface. C: Incubation chamber, again illustrating capillaries abutting the uterine epithelium (similar to A). D: Incubation chamber, showing detail of the uterine epithelium. Bd, blood plasma; Bl, basal lamina; Cf, collagen fibers; Ci, cilia; Cp, capillaries; Enc, endothelial cell cytoplasm; Enu, endothelial cell nucleus; Ic, intercellular canaliculi; Ld, lipid droplets; Mv, microvilli; Nu, nucleus of an epithelial cell; Ol, oviducal lumen; Rb, red blood cells. Fig. 8. Transmission electron micrographs of the vaginal glands of nonvitellogenic females of Seminatrix pygaea. A: Unmated 28.0 cm SVL female sacrificed 14 May 1998. B: Unmated 35.5 cm SVL female sacrificed 30 July 1998. C: Mated 35.5 cm SVL female sacrificed 9 June 1998. D: Same specimen as C, showing detail of the apical epithelium. Bb, basal bodies; Cf, collagen; Ci, cilia; Gl, gland lumen; Go, Golgi complex; Mf, microfilaments; Mi, mitochondria; Mpt, middle piece of a sperm tail; Nu, nucleus of an epithelial cell; Ol, oviducal lumen; Or, orifice of a vaginal gland; Rer, rough endoplasmic reticulum; Sn, sperm nucleus; Sv, secretory vacuoles. Fig. 9. Transmission electron micrographs of the vagina during gravidity of Seminatrix pygaea. A: Vaginal mucosa of a 30.2 cm SVL female sacrificed 9 June 1998 during early gravidity. B: Same specimen as A, showing detail of apical epithelium. C: Vaginal epithelium of a 29.0 cm SVL female sacrificed 30 July 1998 during late gravidity. D: Same specimen as C, illustrating luminal border of the vaginal epithelium. Bb, basal bodies; Bl, basal lamina; Cf, collagen fibers; Ci, cilia; Cp, capillary; Ds, desmosome; Ic, intercellular canaliculi; Inu, irregular nucleus; Ld, lipid droplets; Mf, microfilaments; Mi, mitochondria; Mv, microvilli; Nu, nucleus of an epithelial cell; Ol, oviducal lumen; Rb, red blood cell; Sv, secretory vacuoles; Tj, tight junction. REPRODUCTIVE SYSTEM OF SEMINATRIX PYGAEA 159 1999) on the ultrastructure of sperm storage in fe- The taxa examined so far, however, represent just a males of Seminatrix pygaea, the only TEM studies fragment of total squamate diversity, since some 40 on snakes of which we are aware concern placenta- families, 880 genera, and 5,100 species of squamates tion (Hoffman, 1970) and SSTs (Hoffman and Wim- currently are recognized (Pough et al., 1998). satt, 1972) of Thamnophis sirtalis. In addition, Per- As noted above, few ultrastructural studies exist kins and Palmer (1996) used scanning electron on the oviducts of any squamate. We find it difficult microscopy to study some aspects of oviducal cytol- to compare our results with those limited to light ogy in Diadophis punctatus. Among other squa- microscopy of the oviduct of distantly related taxa. mates, nothing has been published on oviducal ul- Any attempt to conduct a comparative phylogenetic trastructure in amphisbaenids and such studies on analysis of squamate oviducal anatomy that in- lizards are limited to the Gekkonidae (Bou-Resli et cludes ultrastructural characters certainly is pre- al., 1981; Girling et al., 1997, 1998). mature. We feel, however, our results can form a The most comparable studies are by Girling et al. basis (i.e., a “model system,” called for by Blackburn, (1997, 1998), who used light and electron microscopy 1998) for future ultrastructural studies on the squa- to study the oviducal cycle in the viviparous gecko mate oviduct that will facilitate comparative work Hoplodactylus maculatus (Girling et al., 1997) and on the anatomical, physiological, and phylogenetic subsequently reported observations on three addi- significance of oviducal variation. tional species of gecko, two of which are oviparous (Girling et al., 1998). They recognized five oviducal Implications for Mode of Parity regions: the infundibulum, uterine tube, isthmus, uterus, and vagina. The uterine tube corresponds to Much of the variation in oviducal anatomy docu- the SST area of the posterior infundibulum as rec- mented among squamates is related to variation in ognized by Blackburn (1998) and used herein. The eggshell structure and parity mode and is largely isthmus is described as a short region separating the limited to the uterine region (Girling et al., 1998). As uterine tube and uterus; the isthmus differs from noted by Blackburn (1998), nearly one-fifth of squa- the uterus by lacking a carbohydrate secretion mate taxa are viviparous. All viviparous snakes and (Girling et al., 1997). An isthmus was not found in most lizards are predominately lecithotropic (nutri- Seminatrix pygaea. ents supplied by yolk), while a few lizards are pri- Some other differences between the oviducts of marily placentotrophic (matrotrophic). No vivipa- geckos and Seminatrix pygaea are noteworthy. rous squamates that are exclusively lecithotropic or Girling et al. (1998) reported areas of oviducal epi- matrotrophic are known (Blackburn, 1998; J.R. thelium that are PAS positive and/or positive with Stewart, pers. comm.). Although viviparity is Alcian blue at pH 2.5, as we observed in S. pygaea, thought to have evolved independently nearly 100 but they did not observe lipid droplets, which were times in squamates (Shine, 1985; Blackburn, 1992), numerous throughout the oviduct of S. pygaea. viviparous forms that have been examined possess Girling et al. (1997, 1998) also reported nonciliated the same microscopic modifications of the uterus bleb-like cells in the infundibulum of geckos, and that include attenuation of the uterine epithelium, these were not observed in S. pygaea. Finally, al- reduction of uterine (shell) glands, and increased though Girling et al. (1997, 1998) observed sperm in vascularity (Hoffman, 1970; Mead et al. 1981; Guil- the posterior oviducts of several species, they did not lette, 1987; Masson and Guillette, 1987; Blackburn, find a carrier matrix associated with sperm in the 1998; Girling et al., 1998). As reported herein, the posterior regions of the oviduct. uterus of Seminatrix pygaea exhibits these changes As we observed in Seminatrix pygaea, the vivipa- during gravidity. rous geckos show reduction of uterine glands during Our observations on the proximity of capillary gravidity (Girling et al., 1997, 1998). The uterine beds to the uterine lumen during late gravidity con- glands secrete eggshell fibers in oviparous reptiles stitute the first description of the ultrastructure of (Palmer et al., 1993). In viviparous squamates, the this interface in a viviparous snake. We did not eggshell is reduced to a thin membrane that may determine in this study whether the uterine lining persist throughout gestation (Hoffman, 1970; Stew- around the embryo is similar throughout or whether art, 1990, 1992; Girling et al., 1997). differences occur at the embryonic and abembryonic poles or in the area of the omphalallantoic placenta, such as reported at the light microscopy level in Phylogenetic Considerations Virginia striatula (Stewart, 1990). We plan to de- Blackburn (1998) listed in his Table 1 all the scribe ultrastructure of placentation in another ar- known descriptions of squamate histology, amount- ticle in this series after a more extensive series of ing to studies on 20 genera and 12 families of liz- gestational stages are examined. ards, and 12 genera and 4 families of snakes. To this Our observations clearly illustrates that tissue he added new data based on the examination of an layers Ͻ5 ␮m wide separate the contents of the additional 19 genera and two families of lizards and capillaries from the developing fetus in many areas. one genus and family of snakes (Blackburn, 1998). The plasma membranes of the uterine epithelium 160 D.M. SEVER ET AL. and capillary endothelium remain unbroken barri- Jones RE, editors. Hormones and reproduction in fishes, am- ers, and fetal and material tissues are not highly phibians, and reptiles. New York: Plenum Press. p 523–562. interdigitated. The narrow interface nevertheless Halpert AP, Garstka WR, Crews D. 1982. Sperm transport and storage and its relation to the annual cycle of the female red- provides a morphology that would allow physiologi- sided garter snake, Thamnophis sirtalis parietalis. J Morphol cal exchange of substances between the maternal 174:149–159. bloodstream and the uterine lumen, as known from Hoffman LH. 1970. Placentation in the garter snake, Thamno- experimental data on other viviparous squamates phis sirtalis. J Morphol 131:57–88. (Stewart et al., 1990). As noted by various previous Hoffman LH, Wimsatt WA. 1972. Histochemical and electron workers, few morphological modifications of the uterus microscopic observations on the sperm receptacles in the garter snake oviduct. Am J Anat 134:71–96. are required under lecithotrophy to achieve increased Kiernan JA. 1990. Histological and histochemical methods: the- placentotrophy (Weekes, 1935; Boyd, 1942; Guillette, ory and practice. New York: Pergamon Press. p 513. 1987; Stewart, 1992; Blackburn, 1998). Masson GR, Guillette LJ Jr. 1987. Changes in oviducal vascularity during the reproductive cycle of three oviparous lizards (Eumeces obsoletus, Sceloporus undulatus and Crotaphytus col- ACKNOWLEDGMENTS laris). J Reprod Fert 80:361–371. Mead RA, Eroschenko VP, Highfill DR. 1981. Effects of proges- We thank Sean M. Poppy and Perri Mason for aid terone and estrogen on the histology of the oviduct of the garter in collecting specimens. We thank Melissa Maykuth snake, Thamnophis elegans. Gen Comp Endocrinol 45:345–354. and Patricia Michalski of Saint Mary’s College for Palmer BD, DeMarco VD, Guillette LJ Jr. 1993. Oviductal mor- help in tissue preparation. We thank James R. Stew- phology and eggshell formation in the lizard, Sceloporus woodi. art for valuable critique of an earlier version of the J Morphol 217:205–217. Perkins MJ, Palmer BD. 1996. Histology and functional morphol- manuscript. ogy of the oviduct of an oviparous snake, Diadophis punctatus. J Morphol 227:67–79. LITERATURE CITED Pough FH, Andrews RM, Cadle JE, Crump ML, Savitzky AH, Wells KD. 1998. Herpetology. Upper Saddle River, NJ: Blackburn DG. 1992. Convergent evolution of viviparity, matro- Prentice-Hall. trophy, and specializations for female nutrition in reptiles and Schuett GW. 1992. Is long-term sperm storage an important other vertebrates. Am Zool 32:313–321. component of the reproductive biology of temperate pitvipers? Blackburn DG. 1998. Structure, function, and evolution of the In: Campbell JA, Brodie ED Jr, editors. Biology of the pitvipers. oviducts of squamate reptiles, with special reference to vivipar- Austin, TX: Selva. p 169–184. ity and placentation. J Exp Zool 282:560–617. Seigel RA, Loraine RK, Gibbons JW. 1995. Reproductive cycles Bou-Resli MN, Bishay LF, Al-Zaid NS. 1981. Observations on the and temporal variation in fecundity in the black swamp snake, fine structure of the sperm storage crypts in the lizard Acantho- Seminatrix pygaea. Am Midl Nat 134:371–377. dactylus scutellatus hardyi. Arch Biol (Bruxelles) 92:287–298. Sever DM, Ryan TJ. 1999. Ultrastructure of the reproductive Boyd MMM. 1942. The oviduct, foetal membranes, and placenta- system of the black swamp snake (Seminatrix pygaea). I. Evi- tion in Hoplodactylus maculatus Gray. Proc Zool Soc London dence for oviducal sperm storage. J Morphol 241:1–18. Ser A 112:65–104. Shine R. 1985. The evolution of viviparity in reptiles: an ecolog- Dorcas ME, Gibbons JW, Dowling HG. 1998. Seminatrix, S. pyg- ical analysis. In: Gans C, Billet F, editors. Biology of the Rep- aea. Cat Am Amphib Rept 679:1–5. tilia, vol. 15. New York: John Wiley & Sons. p 605–694. Fox H. 1977. The urogenital system of reptiles. In: Gans C, Stewart JR. 1990. Development of the extraembryonic mem- Parsons TS, editors. Biology of the reptilia, vol. 6. London: branes and histology of the placentae in Virginia striatula Academic Press. p 1–157. Gibbons JW, Semlitsch RD. 1991. Guide to the reptiles and am- (Squamata: Serpentes). J Morphol 205:33–43. phibians of the Savannah River site. Athens: University of Stewart JR. 1992. Placental structure and nutritional provision Georgia Press. p 313. to embryos in predominately lecithotrophic viviparous reptiles. Girling JE, Cree A, Guillette LJ Jr. 1997. Oviductal structure in Am Zool 32:303–312. a viviparous New Zealand gecko, Hoplodactylus maculatus.J Stewart JR, Blackburn DG, Baxter DC, Hoffman LH. 1990. Nu- Morphol 234:51–68. tritional provision to embryos in a predominately lecithotrophic Girling JE, Cree A, Guillette LJ Jr. 1998. Oviducal structure in placental reptile, Thamnophis ordinoides (Squamata: Ser- four species of gekkonid lizard differing in parity mode and pentes). Physiol Zool 63:722–734. eggshell structure. Reprod Fertil Dev 10:139–154. Weekes CH. 1935. A review of placentation among reptiles, with Guillette LJ Jr. 1987. The evolution of viviparity in fish, amphib- particular regard to the function and evolution of the placenta. ians, and reptiles: an endocrinological approach. In: Norris DO, Proc Zool Soc (Lond) 2:625–645.