Spermathecae of Salamandrina Terdigitata 23
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JOURNAL OF MORPHOLOGY 22391-33 (1995) S permat hecae of Salarnandrina terdigitata (Am p hi b i a: Salamandridae): Patterns of Sperm Storage and Degradation ROSSANA BRIZZI, GIOVANNI DELFINO, MARIA GLORIA SELMI, AND DAVID M. SEVER Department of Animal Biology and Genetics, University of Florence, 1-50125 Firenze, Italy (R.B.,G.D.); Department of Evolutionary Biology, University of Siena, I-53100 Siena, Italy W.G.S.); and Department of Biology, Saint Mary's College, Notre Dame, Indiana 46556 (D.M.S.) ABSTRACT The spermathecae of female Salamandrina terdigitata were observed using light and transmission electron microscopy during the fall- spring period of sperm storage and secretory activity and during the summer stasis. When sperm are stored inside the spermathecae, the product synthe- sized by spermathecal epithelial cells is exported into the lumen, where it bathes the sperm. During sperm storage some spermatozoa undergo degrada- tion by the spermathecal epithelium. This process, which includes sperm capture by the apical microvilli, formation of endocytic vacuoles and production of lysosomes, becomes prominent shortly after oviposition. In many instances, cells filled with vacuolized spermatozoa andlor residual bodies undergo desqua- mation from the spermathecal epithelium and enter the lumen together with residual sperm. Desquamated cells, together with residual sperm, are a com- mon feature in the spermathecal lumina at the end of the egg-laying season. Concomitant to the activity of the spermathecal epithelium, macrophages move into the spermathecae from the stroma and contribute to the degradation of both the residual sperm and desquamated epithelial cells. As a result of this degradation activity, spermathecae observed during the short summer stasis appear devoid of secretory product and sperm. By late summer, however, the spermathecae already show early signs of an imminent resumption of biosyn- thetic activity. o 1995 Wiley-Liss, Inc. Females of many taxa, including the Am- cal glands involved in internal fertilization phibia, are known to posses sperm storage were the key characters in establishing the organs associated with their reproductive monophyly of the Salamandroidea in a recent tracts, which allow the spermatozoa to re- analysis using 209 phylogenetically informa- main functional for some time before fertiliza- tive characters (Larson and Dimmick, '93). tion occurs (Joly, '60; Boisseau and Joly, '75; Sperm storage structures have been inves- Duellman and Trueb, '86; Selmi, '93; Sever, tigated by light andlor electron microscopy '91c, '92b). Among Urodela, the occurrence in many salamanders (Dent, '70; Boisseau of sperm storage structures (usually defined and Joly, '75; Pool and Hoage, '73; Brizzi et as receptacula seminis or spermathecae; cf. al., '89; Sever, '91a,c, '92a,b; Sever and Dent, '70 for a historical review of these Brunette, '93; Sever and Kloepfer, '93) and terms) characterizes the female genital appa- the results show remarkable similarities both ratus of the Salamandroidea (the salamanders in the tubule morphology and storage pat- which practice internal fertilization: Amphiu- terns. During storage, a close contact be- midae, Dicamptodontidae, Proteidae, Am- tween sperm and spermathecal epithelium bystomatidae, Plethodontidae, and Salaman- occurs and, as a rule, sperm storage does not dridae). Females of the Cryptobranchoidea last long after oviposition. Indeed, following (Cryptobranchidae and Hynobiidae) and Sire- this reproductive stage, most residual sperm noidea (Sirenidae) undergo external fertiliza- seem to undergo degenerative processes and, tion and lack spermathecae (Sever, '78; Duell- shortly afterwards, the spermathecae exhibit man and Trueb, '86; Sever, '87, '88, '91a,b). reduced lumina devoid of secretory product The presence of spermathecae and male cloa- and stored sperm. Only in some cases do a D 1995 WILEY-LISS, INC. 22 R. BRIZZI ET AL. few sperm survive to at least the beginning of TABLE 1. Data on specimens utilized' the following reproductive season, but the Breeding Collection Follicle Sperm in viability of these sperm is not known (Sever, condition SVL date size swrmathecae '91~''92b). Unmated Apart from the patterns of sperm storage, no. 1 41.1 20.1X.92 0.50 N however, cytological details on spermiophagy After mating/prior to oviposition have only recently been reported for the sper- no. 2 43.0 15.X1.92 0.62 Y no. 3 42.1 15.XI.92 0.65 Y mathecal tubules of Eurycea cirrigena (Sever, no. 4 42.3 15.XI.92 0.50 Y '91c, '92b; Sever and Brunette, '93) and Am- no. 5 41.7 25.XI.93 0.70 Y bystoma opacum (Sever and Kloepfer, '93). no. 6 43.2 10.1.93 0.82 Y In our opinion, this latter process represents no. 7 41.8 10.1.93 0.70 Y a crucial step in storage activity by the sper- no. 8 42.8 7.111.92 0.80 Y no. 9 40.8 26.111.93 0.97 Y mathecae and is worthy of thorough investi- no. 10 43.3 6.IV.92 1.10 Y gation in other salamanders. no. 11 41.0 6.IV.92 0.95 Y With this aim, we report histological and no. 12 44.1 20.1V.93 1.35 Y After oviposition cytological findings on the spermathecal tu- no. 13 40.8 30.1V.93 S Y bules in an European salamandrid, Salaman- no. 14 42.7 15.V.92 S Y drina terdigitata, collected at different stages no. 15 42.2 27.V.93 S Y of its reproductive cycle, with especial empha- no. 16 43.0 27.V.93 0.30 Y no. 17 42.8 25.VI.93 0.42 N sis on the degradative processes observed no. 18 40.5 8.VII.92 0.45 N after the egg-laying season. A major goal of no. 19 41.3 8.VII.92 0.40 N this study is to provide further data on sperm 'Measurements are in mm; follicle size is the mean obtained from storage and degradation, which may help to ten follicles; N, no; Y,yes; S, spent. elucidate the main characteristics of these processes in the Salamandroidea as well as to identify the specific morphofunctional traits of different taxa. stained using the Mallory-Galgano trichrome method (Mazzi, '77) for general cytological MATERIALS AND METHODS studies. Nineteen adult females of Salamandrina For electron microscope observations, the terdigitata were collected in the outskirts of cloacal segments were cut into smaller blocks, Florence (Tuscany, Italy) in different seasons prefixed in a formaldehyde-glutaraldehyde of the years 1992 and 1993 (four in autumn, mixture (Karnovsky, '65)and postfixed in 1% three in winter, eight in spring, and four in OsO4 (both in a 0.1 M cacodylate buffer). summer). The animals were sacrificed within After dehydration, the fragments were em- a few hours of collection to avoid any degen- bedded in Epon 812. Semithin (2 pm) sec- erative processes inside the spermathecae due tions, obtained with a Nova Ultratome LKB, to captivity. Specimen numbers, snout-vent were counterstained through OsO4 reduction lengths (from the tip of the snout to the over a Bunsen burner and observed under posterior edge of the vent: SVL), ovarian light microscopy to localize the spermathecal follicle size, and collection date are listed in tubules. Ultrathin sections, corresponding to Table 1. Owing to the dissection of the entire the spermathecal secretory units, were col- cloacal region, the remaining trunk and head lected on uncoated copper grids, stained with portions of the above specimens were consid- uranyl acetate followed by lead citrate, and ered unfit for museum collection; voucher observed under a Philips 400 electron micro- specimens from the same locality were depos- scope. ited in the Museum of Natural History of Florence (collection numbers: MZUF 20751- RESULTS 20757). Following sacrifice in 0.2% chlorbu- tol, specimens of each collection were pro- Light microscopy observations cessed both for light and transmission Brizzi et al. ('89) reported that the sper- electron microscope observations. For light mathecae of Salamandrina terdigitata are microscope procedures, the cloacal segments simple, tubular glands contained in the tu- containing the spermathecal tubules were nicapropria of the mucosa lining the cloacal fixed in Carnoy's fluid after removal of the cavity. Each tubule consists of a monolayered hindlimbs, vertebrae, and gut. These speci- epithelium (Fig. 1A) surrounded by a sheath mens were embedded in polystyrene, and of myoepithelial cells. The spermathecal tu- transverse sections were cut at 10 km and bules open individually into the cloacal lu- SPERMATHECAE OF SALAMANDRINA TERDIGITATA 23 Fig. 1. Salamandrina terdigitata. Light microscopy sperm are not yet stored inside the tubules, secretory of the spermathecae. A: Specimen 3, collected in Novem- product already occurs in the cytoplasm. Scale bars: A ber, at the onset of sperm storage. B: Female 13,collected and C = 20 bm; B and D = 30 Fm. ec, epithelial cells; in April, shortly after oviposition. C: Pattern of mitotic slu, sperm in the lumen; sp, secretory product; tp, tunica activity (arrows) in the spermathecal epithelium of speci- propria. men 13. D Specimen 1, from late September. Although men and, if observed over the year, exhibit terns of mitotic activity are often evident in different cytological features related to the the tubule wall (Fig. 10,which may account different phases in the reproductive cycle for the rapid cytological restoration of the (Brizzi et al., '89). spermathecal epithelium. By the beginning Sperm storage occurs from autumn to of the summer, each tubule consists of inac- spring, followed by a short phase after ovipo- tive, cuboidal cells, arranged around very sition characterized by degradation of re- reduced lumina devoid of sperm and secre- sidual sperm. In the latter period, scattered tory product (Brizzi et al., '89). In summer, spermatozoa are recognizable in the lumina secretory inactivity in the spermathecae coin- mixed with deteriorated cells (Fig. 1B). Along cides with female reproductive stasis, as con- with desquamation of the epithelial cells, pat- firmed by the small follicle size (see Table 1).