Histological Analysis of Spermatogenesis and the Germ Cell

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ARTICLE IN PRESS

Ann Anat 190 (2008) 461—476

www.elsevier.de/aanat

RESEARCH ARTICLE Histological analysis of spermatogenesis and the germ cell development strategy within the testis of the male Western Cottonmouth Snake, Agkistrodon piscivorus leucostoma

Kevin M. Gribbinsa,Ã, Justin L. Rheuberta, Matthew H. Colliera, Dustin S. Siegelb, David M. Severc aDepartment of Biology, Wittenberg University, PO Box 720, Springﬁeld, OH 45501-0720, USA bDepartment of Biology, Saint Louis University, St. Louis, MO 63103, USA cDepartment of Biological Sciences, Southeastern Louisiana University, Hammond, LA 70402, USA

Received 7 December 2007; received in revised form 25 June 2008; accepted 1 July 2008

KEYWORDS Summary Cottonmouth; Cottonmouth (Agkistrodon piscivorus leucostoma) testes were examined histologi- Agkistrodon; cally to determine the germ cell development strategy employed during Spermatogenesis; spermatogenesis. Testicular tissues from Cottonmouths were collected monthly Testes; from swamps around Hammond, Louisiana. Pieces of testis were fixed in Trump’s Reproduction; fixative, dehydrated in ethanol, embedded in Spurr’s plastic, sectioned with an Bimodal ultramicrotome, and stained with toluidine blue and basic fuchsin. Spermatogenesis within Cottonmouths occurs in two independent events within a single calendar year. The testes are active during the months of March–June and August–October with spermiation most heavily observed during April–May and October. To our knowledge, this is the first study that describes bimodal spermatogenesis occurring in the same year within the subfamily Crotalinae. During spermatogenesis, no consistent spatial relationships are observed between germ cell generations. Typically, either certain cell types were missing (spermatocytes) or the layering of 3–5 spermatids and/or spermatocytes within the same cross-section of seminiferous tubule prevented consistent spatial stages from occurring. This temporal pattern of sperm development is different from the spatial development found within birds and mammals, being more reminiscent of that seen in amphibians, and has now been documented within every major clade of reptile (Chelonia, Serpentes, Sauria, Crocodylia). This primitive-like sperm development, within a testis structurally similar to mammals

ÃCorresponding author. E-mail address: [email protected] (K.M. Gribbins).

462 K.M. Gribbins et al.

Introduction to date have testes with typical amniotic structures (seminiferous tubules, seminiferous epithelium, Two different germ cell development strategies Sertoli cells). However, their germ cell develop- exist within vertebrates. Anamniotic vertebrate ment strategy is more reminiscent of that found in testes are composed of tubules or lobules that are anamniotes. This germ cell development may be lined with cysts. Germ cells develop as a single conserved and considered primitive compared with cohort through mitosis, meiosis, spermiogenesis, the derived spatial spermatogenesis seen in birds and upon completion of spermatogenesis mature and mammals. The basal position of reptiles within spermatozoa are released from these cysts into the the amniotic clade and the reoccurring theme of a lumina of the tubules or lobules in a single temporal germ cell development strategy in every spermiation event (Lofts, 1964; Van Oordt and temperate reptile studied to date suggests that this Brands, 1970). Amniotic testes contain seminifer- pleisomorphic-like spermatogenic cycle within a ous tubules that are lined with seminiferous structurally amniotic testis may be evolutionary epithelia (Leblond and Clermont, 1952). Sperma- signiﬁcant. togenesis occurs in association with Sertoli cells, The present study on Agkistrodon piscivorus which make up the seminiferous epithelium of leucostoma is a continuation of this long-term amniotic testes. investigation on germ cell development in reptiles. Throughout the spermatogenic cycle in most The Cottonmouth is a viviparous snake that can be amniotes studied to date (limited mostly to found within lowlands of the southeastern United temperate and continually breeding birds and States (Conant and Collins, 1991). Cottonmouths mammals), germ cells within the seminiferous are semi-aquatic snakes that in many cases occupy epithelium are organized in consistent spatial permanent water sources in abundant numbers arrangements as generations of germ cells move within their geographic range. Pairings of males and from the periphery of the epithelium to the lumen females have been seen in Cottonmouths by of the seminiferous tubules. This type of synchro- Wharton (1966) throughout the entire year except nized germ cell development strategy results in January with female ovulation occurring in the consistent cellular associations that can be seen spring (Zaidan et al., 2003). Spermatogenesis and predicted. These stages are typically numeri- begins in April and spermiogenesis is completed cally arranged within large segments of seminifer- by late October (Johnson et al., 1982) for popula- ous tubules, which lead to waves of sperm release tions of Cottonmouths found in Alabama. The throughout the reproductively active months in climax of spermatogenesis, along with increased mammals (Russell et al., 1990). testosterone levels, occurs in mid July in all Current research provides evidence that most Cottonmouth populations studied to date (Johnson temperate pitvipers mate, have a period of et al., 1982; Zaidan et al., 2003; Graham, 2006). testicular quiescence, and then begin spermato- The purpose of this study is to provide a detailed genesis during the late spring/early summer histological evaluation of the major events asso- (Srivastava and Thapliyal, 1965; Saint Girons, ciated with spermatogenesis so that the germ cell 1982; Aldridge, 2002). Although spermatogenesis development strategy of a southeastern Louisiana generally starts during the late spring/early sum- Cottonmouth population (Johnson et al. (1982) did mer, many variations and testicular cycles exist. not provide information about the germ cell These cycles differ from species to species and are development strategy within Alabama male Cotton- associated with internal and external androgenic mouths) can be compared with that of the Swamp cues (Shea, 2001; Bertona and Chiaraviglio, 2003). Snake and other temperate reptiles. Cottonmouths Recent research has also shown that temperate are unique to the other temperate species studied snakes, like the Black Swamp Snake (Seminatrix to date in that there is evidence that spermatogen- pygaea), may rely on a completely different germ esis is seasonal, but breeding may occur any time cell development strategy than that observed in during the year (assuming intersexual pairings are birds and mammals. Black Swamp Snakes (Gribbins indicative of mating activity). Furthermore, within et al., 2005) and other temperate reptiles (Gribbins the population of Cottonmouths from this study and Gist, 2003; Gribbins et al., 2003, 2006) studied mature sperm can be found in the distal regions of ARTICLE IN PRESS

Spermatogenesis in Agkistrodon piscivorus leucostoma 463 the female reproductive tract in late spring and fall 0.2–0.5 ml intraperitoneal injection of sodium suggesting that there may be two major breeding pentobarbital (1 g sodium pentobarbital in 10% seasons in Louisiana (Siegel and Sever, 2008). This ethanol/40% propylene glycol solution) and the information leads to the hypothesis that male testes were removed and fixed in Trump’s fixative Cottonmouths in Louisiana either store sperm that (EMS, Hatfield, PA). The testes were then cut is produced from one seasonal peak of spermato- into transverse sections and stored in 70% ethanol genesis as observed by Johnson et al. (1982) or at 4 1C. there may be more than one spermatogenic event (i.e. early summer and late fall) that would provide Tissue preparation for light microscopy sperm for the majority of matings, which are assumed to occur most often during the warmer Sections of each testis were cut into approxi- months of the year. This type of bimodal sperma- mately 3 mm cubes, dehydrated in a graded series togenesis has not been documented within a of ethanol (70%, 85%, 2 95%, 2 100%), incubated species of snake belonging to the subfamily in 1:2 Spurr’s plastic (EMS, Hatfield, PA): 100% Crotalinae. However, a similar biannual germ cell ethanol for 30 min, and then once in 1:1 Spurr’s development in males has been previously de- plastic: 100% ethanol for 30 min. The tissues were scribed during the summer months for species then infiltrated in pure Spurr’s plastic overnight. within Colubridae (Goldberg, 1995, 1998). If a New plastic was prepared and the testes were bimodal type of spermatogenesis occurs in this embedded and cured at 60 1C for 48 h in a Fisher Louisiana population of Cottonmouths, then this isotemperature vacuum oven (Fisher Scientific, new annual pattern of reproduction would be quite Pittsburg, PA). Sections (2–3 mm) were cut from different from the postnuptial pattern of sperma- the plastic blocks using a dry glass knife and an LKB- togenesis typical of temperate-zoned snakes that Ultramicrotome III (LKB Produkter AB, Bromma, was previously described by Johnson et al. (1982) Sweden). The tissues were visualized using a basic for Alabama Cottonmouths. A biannual pattern of fuchsin and toluidine blue composite stain as spermatogenesis within Louisiana Cottonmouths described by Hayat (1993). would provide the opportunity to retest the hypothesis that reproductive strategy, which has already been examined in postnuptial (Seminatrix Histological analysis pygaea, Gribbins et al., 2005), prenuptial (Alligator mississippiensis, Gribbins et al., 2006), and mixed The testicular tissues were examined using an (Podarcis muralis, Gribbins and Gist, 2003) repro- Olympus compound microscope (Olympus America, ductive cycles, may predetermine the type of germ Center Valley, PA) to determine the cytological cell development strategy utilized by seasonally changes that occurred during spermatogenesis. breeding reptiles. Sagittal and cross-sectional areas of the seminiferous tubules were selected at random and germ cell morphologies and the presence or absence of spatial stages were determined. Photographs were Materials and methods taken with a SPOT digital camera (Diagnostic Systems Laboratories, Webster, TX) and were Animal collection viewed and edited using Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA). Twenty-two adult male Cottonmouth snakes, A. piscivorus leucostoma, were collected during Data analysis, measurements, the months of January 2005 through November and morphometrics 2006 (2 snakes were captured per month except: 1 specimen per month for September, November, and Upon capture, each snake’s snout-to-vent length January and 5 specimens for June) from three (SVL) was determined and ranged from 36.0 to localities; the Amite River Diversion Canal (North 73.1 cm. The width and length (in mm) were 30122.616/West 090168.506, Livingston Parish, LA), measured for the right and left testes shortly after Turtle Cove Environmental Research Station on Pass dissection and testis volumes were estimated using Manchac (North 30129.426/West 090135.592, Tan- the formula for a prolated spheroid: V ¼ 4/3p gipahoa Parish, LA), and the private residence of (1/2 L)(1/2 W)2 (Selby, 1965; Ramirez-Bautista Dr. Clifford Fontenot, 10 km Northwest of New and Gutierrez-Mayen, 2003). Right and left testi- Albany (North 30130.871/West 090136.202, Living- cular volumes were pooled in order to increase ston Parish, LA). The snakes were sacrificed with a data collection for each month of the year. Thirty ARTICLE IN PRESS

464 K.M. Gribbins et al. cross-sections of seminiferous tubules were chosen shown that SVL can bias testicular volume data; for each represented month and the tubular therefore, linear regression was first used to diameter and germinal epithelial heights were determine whether mean testicular volumes were measured using an ocular micrometer. significantly affected by variations in male mean Data analysis was performed using Minitab 15 SVL. Linear regression analysis indicated that (Minitab Inc., State College, PA) for Windows. testicular volumes were not significantly correlated Results were deemed significant if Pp0.005. with variations in mean male SVL (right testicular Testicular volume, tubular diameter, and germinal volumes: R2 ¼ 0.217, Pp0.05; left testicular epithelial height data were tested for normality volumes: R2 ¼ 0.261, Pp0.05). Conversely, mean and homogeneity of variances using the Kolmogor- testicular volumes in these Cottonmouths do ov–Smirnov and Bartlett’s tests, respectively, indeed show a significant annual trend (Kruskal– before statistical analyses were performed (Sokal Wallis; H ¼ 27.7, df ¼ 10, P ¼ 0.002, Figure 1). and Rohlf, 1995; Flemming, 1993). These data did Testis volumes were at a minimum during the not meet assumptions of normality; thus the months of quiescence (November, 59.00 cm3; Jan- Kruskal–Wallis analysis of variance was used to test uary, 16.6 cm3; February, 70.6 cm3; July, 50.15 cm3) for significant seasonal variation in testicular and during the month of September (61.77 cm3). volume, seminiferous tubular diameter, and germi- The specimen caught during September should nal epithelial height. have a high testis volume and also show spermiogenic activity similar to the August and September testis. However, there are only spermatogonia present in the seminiferous epithelium and the Results testis volume is low. It also should be noted that this Cottonmouth was caught 2 weeks after Germ cell morphology and the cell cycle hurricane Katrina and outside of its normal habitat. Testicular volumes (Figure 1) increase steadily as The A. piscivorus leucostoma testis contains spermatogenic activity increases (April, 68.42 cm3; seminiferous tubules lined with seminiferous May, 215.30 cm3) and volumes peak during the epithelia consisting of Sertoli cells and developing climax of spermiogenesis and spermiation in June germ cells. Germ cells develop spermatogenically (292.60 cm3). Testicular volume then drops signifi- in close association with the Sertoli cells of this cantly during July (50.15 cm3) when the testis is epithelium. Histological examination shows that quiescent and then increases again as spermato- testes of this southeastern Louisiana population of genesis and spermiogenesis increases during August Cottonmouths are spermatogenically active during (142.60 cm3) and October (153.60 cm3). Testis two periods within the same year, the months of volume is much larger during the spring spermato- March–June and again in August–October. Sperma- genic climax (292.60 cm3) when compared with the togenesis commences in March and after a brief fall peak of sperm development (153.60 cm3). quiescence in July starts again in August with the proliferation of spermatogonia A and B near the basal lamina (basement membrane) of the semi- Pre-meiotic cells niferous epithelia. The majority of spermatogonia undergo meiosis during the months of April and The seminiferous epithelium contains two August/October and continue through spermiogen- morphologies of pre-meiotic cells (Spermatogonia esis in May–June and then again in October. A and B) (Figure 2; SpA and SpB) during all months Spermatogonia are present and undergo mitosis in of the year. These cells are characterized by nuclei all months of the year; however, divisions are with random clumps of heterochromatin. The slowed during the months of June/July, November, major morphological differences between the two and January/February. Although there is a decrease types of spermatogonia are that the A type is ovoid in mitotic activity, there are no periods during the in shape with one large nucleolus and B type is year in which the testes of these Cottonmouths are more round in shape and usually lacks a prominent inactive in terms of proliferation. nucleolus. Both spermatogonial types are generally Testicular volume has been used in reproductive found near the basement membrane of the epithe- studies performed on reptiles as an indicator of lium away from the lumen and associated with the spermatogenic activity (Srivastava and Thapliyal, basal compartments formed by Sertoli cells. During 1965; Flemming, 1993; Ramirez-Bautista and the spermatogenic cycle both types of spermato- Gutierrez-Mayen, 2003). However, previous studies gonia undergo mitosis to maintain the spermatogo- (see Brown and Bomberger Brown, 2003) have nial population and many of the B spermatogonia ARTICLE IN PRESS

Spermatogenesis in Agkistrodon piscivorus leucostoma 465

Figure 1. Variation in testicular volume (mean71 SE) during the annual reproductive cycle of the male Cottonmouth, Agkistrodon piscivorus leucostoma. divide to form pre-leptotene spermatocytes that increasingly thick chromatin fibers. Zygotene cells then enter meiosis. Although spermatogonia A and are found within the germinal epithelium of the B can be seen throughout all months of the year, March and August testis and are the most infre- they are most abundant during the months of quently seen spermatocytes in the germinal epithe- March, April, and August. lium of the Cottonmouth. Pachytene spermatocytes (Figure 2, PA) are the largest and most commonly observed meiocyte within the Cottonmouth testis. Meiotic cells They are similar in morphology to zygotene cells; however, their nuclei have almost double the Meiotic cells are characterized by an increase in volume compared with zygotene cells and there nuclear size and a condensation of chromatin into are much more open nucleoplasm and thicker chromosomes. Spermatogonia B undergo mitotic chromatin fibers. Pachytene cells are present in divisions and enter prophase of meiosis I in March March–June and August–October. and then again in August. These pre-leptotene Diplotene spermatocytes (Figure 2, DI), meta- spermatocytes (Figure 2, PL) contain a nucleus phase I (Figure 2, M1), secondary spermatocytes with a well-defined dark staining nucleolus. (Figure 2, SS), and metaphase II (Figure 2, M2) cells Pre-leptotene cells along with step 1 spermatids can be found within the seminiferous epithelium (Figure 2, S1) are the smallest of the developing throughout all active months of spermatogenesis germ cells. The pre-leptotene spermatocyte small (March–June and August/October). These germ size (1/2 the size of spermatogonia B) allows them cells are typically found together in tight clusters to be easily distinguished from spermatogonia B within the germinal epithelium. In diplotene within the basal compartment of the seminiferous spermatocytes, the nuclear membrane begins to epithelium. degenerate and the almost fully condensed chro- Leptotene spermatocytes (Figure 2, LP) are close mosomal fibers form a tight circle just under this in size to pre-leptotene cells and are more easily degenerating membrane. Metaphase 1 cells have distinguished based on their darker staining fila- fully condensed chromosomes that aggregate on mentous chromatin. Leptotene cells are present in the metaphase plate. The results of meiosis I March–June and then again in August/October. are the secondary spermatocytes. The chromatin Their largest numbers are seen in April and August. fibers of secondary spermatocytes are dispersed Zygotene spermatocytes (Figure 2, ZY) are larger randomly throughout the nucleoplasm. These cells and stain less intensely than leptotene cells (more are about twice the size of Step 1 spermatids, open nucleoplasm). Their nuclei are filled with which are typically clumped with secondary ARTICLE IN PRESS

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Figure 2. Germ cell types found within the seminiferous epithelium Agkistrodon piscivorus leucostoma. Bar ¼ 15 mm. SpA, type A spermatogonia; SpB, type B spermatogonia; PL, pre-leptotene spermatocyte; LP, leptotene spermatocyte; ZY, zygotene spermatocyte; PA, pachytene spermatocyte; DI, diplotene spermatocyte; M1, meiosis I; SS, secondary spermatocyte; M2, meiosis II; S1, step 1 spermatid; S2, step 2 spermatid; S3, step 3 spermatid; S4, step 4 spermatid; S5, step 5 spermatid; S6, step 6 spermatid; S7, step 7 spermatid; MS, mature spermatozoa. spermatocytes within the seminiferous epithelium. metaphase 2 is the germ cell size and the amount During metaphase 2, chromosomes aggregate of chromatin present. The metaphase 2 cells are around the metaphase plate again. The only slightly smaller and contain about half the amount differential factor between metaphase 1 and of chromatin found in metaphase 1 cells. ARTICLE IN PRESS

Spermatogenesis in Agkistrodon piscivorus leucostoma 467

Figure 3. Top: variation in seminiferous tubule diameter (mean71 SE) and bottom: variation in germinal epithelial height (mean71 SE) during the annual reproductive cycle of the male Cottonmouth, Agkistrodon piscivorus leucostoma.

Spermiogenic cells matids (Figure 2, S2). These germ cells have chromatin that is interspersed throughout the Spermiogenesis can be divided into seven steps in nucleoplasm. Step 2 spermatids are present in the Cottonmouth germinal epithelium based on the large numbers in May and October. terminology of Russell et al. (1990) for mammalian Step 3 spermatids (Figure 2, S3) and Step 4 species. Steps of spermiogenesis are defined based spermatids (Figure 2, S4) are often present at the on acrosomal formation, nuclear elongation, and same time as step 2 spermatids. As step 3 and 4 chromosomal condensation. The presence of step 1 spermatids continue to develop, the acrosome spermatids (Figure 2, S1) in April and August marks begins to widen and envelope the nuclear the beginning of spermiogenesis. Step 1 spermatids head, which flattens the apex of the nucleus. A are small in size, have a well-defined nuclear centrally located acrosome granule is often membrane, and no definable acrosomal vesicle. present during this stage of spermatid develop- A well-defined acrosomal vesicle in contact with ment. Step 5 spermatids (Figure 2, S5) mark the nuclear membrane characterizes step 2 sper- the transition between round and elongating ARTICLE IN PRESS

468 K.M. Gribbins et al. spermatids. Elongation begins at the opposite end other elongating steps. Once spermiogenesis is of the nucleus away from the acrosome creating a complete, the mature spermatozoa (Figure 2, MS) nucleus that is stretched in its dorsoventral plane. are released into the lumen of the seminiferous As elongating spermatids undergo development, tubules where they will be transported to the they also begin to accumulate near the apical excurrent ducts of the male reproductive system. surfaces of the Sertoli cells with their tails The late steps of spermiogenesis are seen in stretching out into the lumen and their nuclear May–June and also in October. heads facing the basement membrane. The nuclei of step 6 spermatids (Figure 2, S6) are longer than they are wide. The acrosomal vesicle Seasonal development and germ cell continues to extend over the head of the nucleus development strategy of the seminiferous when visible. Step 7 spermatids (Figure 2, S7) epithelium represent the climax of elongation. They have undergone nuclear condensation and cytoplasmic From the 22 samples of A. piscivorus leuco- elimination, resulting in nuclei more intensely stoma testes taken over an entire year (excluding stained and thinner in diameter than any of the December), two distinct waves of spermatogenesis

Figure 4. (A) Sagittal section (40 ) of a January seminiferous tubule. Bar ¼ 100 mm. (B) The cell types represented within the January germinal epithelium (100 ). Bar ¼ 20 mm. Labeled cell types: SpA, type A spermatogonia; SpB, type B spermatogonia. Note: Germ cells from the previous cycle are being shed into the lumen of the seminiferous tubule, * and many of the spermatocytes appear hypertrophic (white arrow).

Figure 5. (A) Sagittal section (40 ) of a February seminiferous tubules. Bar ¼ 100 mm. Germ cells from the previous cycle are being shed into the lumen of the seminiferous tubule, *. (B) The cell types represented within the February germinal epithelium (100 ). Bar ¼ 20 mm. Labeled cell types: SpA, type A spermatogonia; SpB, type B spermatogonia. Note: High vacuolation of the germinal epithelium. ARTICLE IN PRESS

Spermatogenesis in Agkistrodon piscivorus leucostoma 469 are observed histologically within the seminiferous seminiferous epithelium sloughed off into the epithelium. This biannual type of spermatogenesis lumen (Figures 4B and 5A, *) at this time of the is supported by seminiferous tubule diameter and year. germinal epithelial height data. Seasonal variation The ﬁrst wave of spermatogenesis begins in in seminiferous tubule diameter (Kruskal–Wallis; March and April (Figures 6 and 7) with an increase H ¼ 287.22, df ¼ 10, P ¼ 0.000, Figure 3) and in spermatogonial proliferation and the early germinal epithelial height (Kruskal–Wallis; events of meiosis I dominating the seminiferous H ¼ 270.93, df ¼ 10, P ¼ 0.000, Figure 3) parallel epithelia. The increase in mitosis and meiosis leads each other and show a signiﬁcant monthly trend. to larger seminiferous tubule diameters (March, Prior to March the seminiferous tubules are in a 126.05 mm; April, 130.75 mm) and germinal epithe- quiescent phase of development (January and lial heights (March, 28.20 mm; April, 29.15 mm). No February, Figures 4 and 5) and spermatogonia A consistent cellular associations are observed bet- and B are the only major germ cell types present ween early and late developing generations of germ within the seminiferous epithelium. Seminiferous cells because the later events of spermiogenesis tubule diameters (January, 101.25 mm; February, are missing from the seminiferous tubules of 99.80 mm) and germinal epithelial heights (January, January–April Cottonmouth testes. 21.55 mm; February, 21.65 mm) are also close to Spermatogenesis continues to advance in the May their smallest values during these two months. It is testis (Figure 8). The majority of the population of not uncommon to observe large portions of the germ cells has completed meiosis II and has entered

Figure 6. (A) Sagittal section (40 ) of a March seminiferous tubule. Bar ¼ 100 mm. (B) The cell types represented within the March germinal epithelium (100 ). Bar ¼ 20 mm. Labeled cell types: SpA, type A spermatogonia; SpB, type B spermatogonia; LP,leptotene spermatocytes; ZY, zygotene spermatocytes; PA, Pachytene spermatocytes; SS, secondary spermatocytes; S1, step 1 spermatid.

Figure 7. (A) Sagittal section (40 ) of an April seminiferous tubule. Bar ¼ 100 mm. (B) The cell types represented within the April germinal epithelium (100 ). Bar ¼ 20 mm. Labeled cell types: SpA, type A spermatogonia; SpB, type B spermatogonia; PL, pre-leptotene spermatocytes; LP, leptotene spermatocytes; PA, Pachytene spermatocytes; M1, meiosis 1; M2, meiosis 2; SS, secondary spermatocytes; S1, step 1 spermatid; S3, step 3 spermatid; S4, step 4 spermatid. ARTICLE IN PRESS

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Figure 8. (A) Cross-section (40 ) of a May seminiferous tubule. Bar ¼ 100 mm. Note, mature spermatozoa (white arrows) in the lumen suggest spermiation has started. (B) The cell types represented within the May germinal epithelium (100 ). Bar ¼ 20 mm. Labeled cell types: SpA, type A spermatogonia; SpB, type B spermatogonia; PL, pre- leptotene spermatocytes; S1, step 1 spermatid; S3, step 3 spermatid; S5, step 5 spermatid; S6, step 6 spermatid; S7, step 7 spermatid.

Figure 9. (A) Cross-section (40 ) of a June seminiferous tubule. Bar ¼ 100 mm. (B) The cell types represented within the June germinal epithelium (100 ). Bar ¼ 20 mm. Labeled cell types: SpA, type A spermatogonia; SpB, type B spermatogonia; MT, mitosis; LP, leptotene spermatocytes; PA, pachytene spermatocytes; SS, secondary spermatocytes; S1, step 1 spermatid; S4, step 4 spermatid; S5, step 5 spermatid; S6, step 6 spermatid; S7, step 7 spermatid; MS, mature spermatozoon. the early phases of spermiogenesis. It is common to increase in size as mature sperm are dumped into see 4–5 different spermatids layered together the seminiferous tubules leads to a climax in within the adluminal compartment of the semini- spring/early summer seminiferous tubule diameter ferous epithelium. The multiplying spermatid po- (180.85 mm) and the accumulation of generations of pulation causes the formation of large columns of elongating spermatids causes an enlargement in seminiferous epithelium, which leads to a substan- the germinal epithelial height (43.80 mm). The lack tial size increase in seminiferous tubule diameter of early meiotic cells and accruing number of (150.05 mm) and germinal epithelium height elongating spermatids prevents consistent cellular (38.10 mm). The abundance of spermatids and the association between germ cell types. By July absence of early meiotic I cells again preclude (Figure 10), spermiation is complete and the consistent cellular associations from forming be- seminiferous tubules have entered their second tween late and early generations of germ cells. phase of quiescence, which leads to a dramatic June samples of testis (Figure 9) represent the decrease in seminiferous tubule diameter climax of spermiogenesis and an increase in (92.75 mm) and germinal epithelial height spermiation. Most of the population of germ (20.05 mm). The only cell types found within the cells is completing spermiogenesis and entering highly vacuolated germinal epithelium are a single the lumen as mature spermatozoa. The luminal row of spermatogonia A and B located against the ARTICLE IN PRESS

Spermatogenesis in Agkistrodon piscivorus leucostoma 471

Figure 10. (A) Sagittal section (40 ) of a July seminiferous tubule. Bar ¼ 100 mm. (B) The cell types represented within the July germinal epithelium (100 ). Bar ¼ 20 mm. Labeled cell types: SpA, type A spermatogonia; SpB, type B spermatogonia. Note: Germ cells from the previous cycle are being shed into the lumen of the seminiferous tubule, white arrow.

Figure 11. (A) Cross-section (40 ) of an August seminiferous tubule. Bar ¼ 100 mm. (B) The cell types represented within the August germinal epithelium (100 ). Bar ¼ 20 mm. Labeled cell types: SpA, type A spermatogonia; SpB, type B spermatogonia; PL, pre-leptotene; PA, pachytene spermatocytes; SS, secondary spermatocytes; S1, step 1 spermatid; S2, step 2 spermatid; S3, step 3 spermatid; S6, step 6 spermatid; S7, step 7 spermatid; MS, mature spermatozoon. basement membrane. The lumina of these tubules spermatogenesis are observed in August, no con- are void of most spermatozoa and have pieces of sistent cellular association are formed because the seminiferous epithelium with left over germ of the 4–5 different spermatids occupying the cells (Figure 10B, white arrow) from the spring apical portion of the seminiferous epithelium. The cycle of spermatogenesis. September testis (Figure 12) represents an aberra- The second wave of spermatogenesis has begun tion in the sequence of events that are occurring in the August Cottonmouth testis (Figure 11). The during spermatogenesis. The seminiferous tubules early stages of proliferation and meiosis are similar within this testis are shriveled and only A and B to the March and April samples; however, spermio- type spermatogonia are present in the seminiferous genic cells are in more advanced stages than May epithelium that is very thin in comparison to the testes. August is the only month in which prolif- other represented months (seminiferous tubule erative, meiotic, and spermiogenic cells are found diameter, 89.35 mm; germinal epithelial height, together regularly in the seminiferous epithelium, 19.30 mm). Large numbers of immature spermatids leading to the largest increase in seminiferous have been sloughed into the seminiferous tubules in tubule diameter (227.70 mm) and germinal epithe- the September samples (Figure 12B, Sp). Sperma- lial height (46.10 mm). Although all three stages of togenesis in the October seminiferous epithelium ARTICLE IN PRESS

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Figure 12. (A) Cross-section (40 ) of a September seminiferous tubule. Bar ¼ 100 mm. Note: Many shed generations of germ cells are found in the lumen of the seminiferous tubules (black arrows). (B) The cell types represented within the September germinal epithelium (100 ). Bar ¼ 20 mm. Labeled cell types: SpA, type A spermatogonia; SpB, type B spermatogonia. Note: Many shed generations of spermatids are found in the lumen of the seminiferous tubules (Sp).

Figure 13. (A) Cross-section (40 ) of an October seminiferous tubule. Bar ¼ 100 mm. (B) The cell types represented within the October germinal epithelium (100 ). Bar ¼ 20 mm. Labeled cell types: SC, Sertoli cell nucleus; SpA, type A spermatogonia; SpB, type B spermatogonia; PL, pre-leptotene; S1, step 1 spermatid; S3, step 3 spermatid; S4, step 4 spermatid; S5, step 5 spermatid; S6, step 6 spermatid; S7, step 7 spermatid; MS, mature spermatozoon.

Figure 14. (A) Sagittal section (40 ) of a November seminiferous tubule. Bar ¼ 100 mm. (B) The cell types represented within the November germinal epithelium (100 ). Bar ¼ 20 mm. Labeled cell types: SpA, type A spermatogonia; SpB, type B spermatogonia; PL, pre-leptotene spermatocyte. Note: Remnant deteriorating germ cells are being shed into the lumen of the seminiferous tubule (white arrows: spermatocytes; white arrowhead: elongating spermatid).

(Figure 13) has advanced into spermiogenesis togonia A and B are found near the base- with round and elongating spermatids represented. ment membrane of the seminiferous tubules. The Spermatocytes have been exhausted and sperma- loss of meiotic cells has slightly decreased the ARTICLE IN PRESS

Spermatogenesis in Agkistrodon piscivorus leucostoma 473 seminiferous tubule diameter (215.40 mm) and demonstrated in stress-induced Cottonmouths germinal epithelial (41.55 mm) height when com- (Graham, 2006) and supported by the large number pared with August seminiferous tubules. Many of of detached spermatids found in the September the developing spermatids have completed sper- seminiferous tubular lumen (Figure 12B, Sp). The miogenesis and are being shed to the lumina of the August, October, and November samples all show a seminiferous tubules as mature spermatozoa. Like similar histological sequence to the fall spermato- the July sample, the November seminiferous genic cycle described by Johnson et al. (1982) for tubules (Figure 14) are in a state of quiescence A. piscivorus in Alabama. with spermatogonia A and B making up the majority The two spermatogenic events within this Cot- of germ cells and lipid-rich vacuoles dominating the tonmouth population are separated by a quiescent seminiferous epithelium. The seminiferous tubule period (July) in which the testes are inactive and diameter (94.25 mm) and germinal epithelial height only spermatogonia type A and B are present and (20.15 mm) in November also mirror that of July the seminiferous tubule diameter, germinal epithe- testes. lial height and testicular volume are smaller than all other measurements except for the months of September and January (testicular volume only). To Discussion our knowledge, this is the first evidence of bimodal spermatogenesis described for a temperate species The testes of the A. piscivorus leucostoma within Crotalinae. Like other crotalids, most consist of seminiferous tubules lined by seminifer- temperate snakes have a single annual spermato- ous epithelia. Sertoli cells are present at all times genic cycle that typically follows a postnuptial of the year and are associated with a continuous pattern where spermatogenesis commences after supply of spermatogonia. This overall testicular spring mating (Licht, 1984; Saint Girons, 1982). structure is consistent with nonmammalian amnio- Other studies (Johnson et al., 1982)onA. pisci- tic testes (Pudney, 1995) and the testes of Black vorus have shown that spermatogenesis starts in Swamp Snakes (Gribbins et al., 2005). the spring, peaks in late summer, and terminates in Spermatogenesis takes place during two inde- the fall. The populations of Cottonmouths studied pendent events between the months of March–June by Johnson et al. (1982) were found in Alabama and August–October in this Louisiana population of (at a more northern latitude versus southern A. piscivorus leucostoma. Monthly morphometric Louisiana). The further north a population of snakes data, testicular volume measurements, and histo- resides, the shorter the number of warm months, logical analysis of the Cottonmouth testis all which may limit energy sources and the metabolism provide strong support for a biannual/bimodal type needed to maintain sperm development. This may of spermatogenesis. However, it should be noted be the reason why the early (spring) spermatogenic that the September testis does not fall into cycle is absent in the Alabama Cottonmouth sequence as far as spermatogenesis with that of population. the other fall samples. In September, only sperma- Interestingly, Johnson et al. (1982) and Sever togonia are seen within the seminiferous epithe- et al. (2008) both provide data for hypertrophic lium and this snake has the smallest seminiferous renal sexual segments (RSSs) during spring and late tubule diameter (89.35 mm) and germinal epithelial summer/early fall even though there is only one height (19.30 mm) measured during this entire late summer testosterone peak (Johnson et al., study. August testes show the early events of 1982; Graham, 2006). Though testosterone levels spermatogenesis and October seminiferous tubules were not taken during the present study, it is worth show the later events of spermiogenesis and noting that the literature to date suggests that spermiation. September seminiferous epithelia hypertrophic RSSs in the Cottomouth do not should be dominated by spermatocytes and early parallel the timing of peak testosterone as pre- round spermatids, similar to the September testis cisely as those found in other squamates (Bishop, described for populations of Cottonmouths in 1959; Misra and Prasad, 1965; Prasad and Sanyal, Alabama (Johnson et al., 1982). This particular 1969; Krohmer, 1986). Nevertheless, RSSs do snake was caught outside of its normal habitat and correlate strongly with the timing of spermatogen- 2 weeks after hurricane Katrina. Thus, this snake esis and when breeding may occur. Hypertrophic was exposed to extreme stress and the spermato- RSSs are often good indicators of breeding because genic cycle may have been shut down in response to sexual segments in reptiles are presumed to this environmental catastrophe due to conflict produce the seminal fluid added to spermatozoa between the hypothalamic–pituitary–gonadal axis during ejaculation (Prasad and Reddy, 1972). and the hypothalamic–pituitary–adrenal axis as Furthermore, sperm aggregates found in the ARTICLE IN PRESS

474 K.M. Gribbins et al. posterior uterus, alleged artifacts of recent breed- testes that are structurally similar to that of ing (Saint Girons, 1957, 1962a, b), of female derived amniotic lineages (birds and mammals). Cottonmouths, also occur during the spring and Thus, reptiles might represent a better transitional fall in Louisiana (Siegel and Sever, 2008). intermediary in terms of testicular organization In light of the breeding/endocrine/RSSs data, between anurans and the derived avian and we suggest that the two spermatogenic cycles mammalian taxa. observed in A. piscivorus leucostoma from south- Temperate reproductive strategies seem to have eastern Louisiana provide mature spermatozoa for no effect on the type of germ cell development the two major breeding periods suggested by Siegel strategy employed by the reptiles studied to date. and Sever (2008) and supported by previous studies Previously studied prenuptial (Gribbins et al., (Beyer, 1898; Martin, 1984). The majority of female 2006), postnuptial (Gribbins et al., 2003, 2005), A. piscivorus practice biennial breeding, where mixed (Gribbins and Gist, 2003), and the present copulation initially begins in late summer and results on bimodal spermatogenesis all express the continues through early fall (Burkett, 1966; Whar- same temporal type of germ cell development. It ton, 1966; Ford, 2002; Ford et al., 2004). Also, would be interesting to test whether continually during this late summer period, vitellogenesis (yolk reproducing populations of reptiles, such as those accumulation in the developing oocyte) presumably in the tropics, would show this same temporal germ begins and is subsequently halted during hiberna- cell development or if they have a more spatial tion (Aldridge and Duvall, 2002). The ﬁrst sperma- germ cell development strategy as observed in togenic cycle ending in June would provide continually breeding mammals and birds. Unfortu- spermatozoa for these late summer/fall breeding nately, data on the details of spermatogenesis for events. Vitellogenesis in females resumes again in tropical and temperate reptilian species are lacking the following early spring, when the second mating and need to be addressed in order for such season starts, and is completed by late spring/early comparative models to be tested. These types of summer when mature ovarian follicles are ready for comparative, phylogenetic, and anatomical ques- ovulation (Burkett, 1966). The second (fall) sper- tions on spermatogenesis and the morphology of matogenic cycle is completed before hibernation the testis cannot be answered conclusively until and sperm is stored in the excurrent duct system further information is collected from a variety of until emergence from hibernation in the following tropical and temperate species representing the spring, which is when the second mating season major taxa within Reptilia. begins (Johnson et al., 1982). Recent studies on temperate squamates (Gribbins and Gist, 2003; Gribbins et al., 2005) Acknowledgments have shown a similar temporal germ cell development strategy described here for the Cottonmouth, We thank Robert Aldridge for his helpful com- which is different from the spatial germ cell ments on this manuscript. This study is funded in development seen in seasonally and continually part by competitive research grants from Witten- breeding birds and mammals (Yamamoto et al., berg University. 1967; Rossen-Runge, 1977; Tait and Johnson, 1982; Tsubota and Kanagawa, 1989; Tiba and Kita, 1990; Foreman, 1997). This episodic germ cell develop- References ment has also been observed within the other reptilian orders (Gribbins et al., 2003: Trachemys Aldridge, R.D., 2002. The link between mating season scripta, Chelonia; Gribbins et al., 2006: Alligator and male reproductive anatomy in the rattlesnakes mississippiensis, Crocodylia) and is very similar to Crotalus viridis oreganus and Crotalus viridis helleri. the temporal germ cell development strategy of J. Herpetol. 36, 295–300. derived amphibians such as anurans (Lofts, 1964; Aldridge, R.D., Duvall, D., 2002. Evolution of the mating Van Oordt and Brands, 1970). Furthermore, anuran season in the pitvipers of North America. Herpetol. amphibians have been described as a transitional Monogr. 16, 1–25. taxon between the anamniotes and amniotes in Bertona, M., Chiaraviglio, M., 2003. Reproductive biology, mating aggregations, and sexual dimorphism terms of testicular organization (Van Oordt, 1955). of the Argentine Boa Constrictor (Boa constrictor Yet, the seminiferous tubules in anurans are lined occidentalis). J. Herpetol. 37, 510–516. with seasonal cysts and not a continuous epithelium Beyer, G.E., 1898. Contribution on the life histories of like that of other amniotes. Extant reptiles and certain snakes. Am. Nat. 32, 17–24. presumably their ancestors are considered the Bishop, J.E., 1959. A histological and histochemical study most primitive amniotes phylogenetically and have of the kidney tubule of the common Garter Snake, ARTICLE IN PRESS

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