JOURNAL OF MORPHOLOGY 22391-33 (1995)

S permat hecae of Salarnandrina terdigitata (Am p hi b i a: ): Patterns of Sperm Storage and Degradation

ROSSANA BRIZZI, GIOVANNI DELFINO, MARIA GLORIA SELMI, AND DAVID M. SEVER Department of 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 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 (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, , Am- tween sperm and spermathecal epithelium bystomatidae, , 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 () 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 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). 24 R. BRIZZI ET AL. In the specimen from September, the sper- in the form of lamellar bodies, and intercellu- mathecal cells exhibited resumption of bio- lar canaliculi between contiguous cells often synthetic activity, recognized by the presence associated with desmosomes. Similar relation- of secretory product in the high supranuclear ships between plasmmembranes occur be- cytoplasm (Fig. 1D). From early autumn on- tween epithelial (Fig. 3A) and myoepithelial ward, bundles of sperm occur inside the sper- cells (Fig. 3B; for further details on the myo- mathecae, mixed with secretory product (Fig. epithelial cells in the spermathecae of Salu- 1A). During autumn, the ovarian follicles mandrina terdigitutu see Brizzi et al., '89). reach 0.70 mm in diameter, which, however, As noted previously, patterns of biosyn- does not correspond to the largest follicles thetic activity are evident in the spermathe- observed during the spring egg-laying sea- cae of females collected from autumn to son. In this latter phase, oocytes of 1.35 mm spring, after mating but before oviposition. in diameter occur in the oviducts just before In the next phase, a short period after egg- oviposition. laying (between late April and mid-May), the Apart from the arrangement of sperm in production of secretory material by the sper- the lumina and the shapes of the epithelial mathecal cells rapidly decreases and the cyto- cells, few cytological details can be observed plasm appears much reduced in comparison by light microscopy during the cyclic changes with the size of the nuclei (Fig. 3C). The of the spermathecal epithelium. The epithe- nuclei are now characterized by dense hetero- lial cells contain secretory granules (during chromatin and prominent nucleoli. Speci- sperm storage), or are involved in desquama- mens in this phase have wide and irregular tion processes followed by biosynthetic stasis interstices between contiguous cells that oc- (during late spring and early summer). casionally contain sperm (Fig. 3D). A pale, Electron microscopy observations non specific secretory product, mainly consist- Patterns of biosynthetic activity ing of lipid droplets, is detectable both in the basal and supranuclear cytoplasm (Fig. 3C,D). During the initial steps of sperm storage Microvilli are scarce and short along the api- (early autumn), the spermathecal cells are cal border, and sparse residual sperm occur high columnar, with round, basal nuclei that inside the lumina (Fig. 3C). In females sacri- are relatively small in comparison to the ficed in June and July, at the culmination of amount of cytoplasm. Inside the nuclei, het- the reproductive stasis, the spermathecal tu- erochromatin mostly occurs around the bor- bules appear empty (see below), biosyntheti- ders (Fig. 2A) and nucleoli are not promi- cally inactive, and characterized by a very nent. Secretory vesicles fill the supranuclear high N/P ratio (Fig. 4A). In some specimens, cytoplasm, where conspicuous Golgi stacks nevertheless, the approaching secretory con- (dictyosomes), rough endoplasmic reticulum dition already is recognizable. Although the profiles (rer), and elongate mitochondria also epithelial cells are still devoid of condensing are visible (Fig. 2B,C). In many instances, vesicles and contain large nuclei, imminent condensing vesicles are common in the supra- resumption of biosynthetic activity is as- nuclear as well as in the basal cytoplasm, sumed from the increased area of supra- giving rise to numerous secretory granules. nuclear cytoplasm (Fig. 4B), where a rich The granules accumulate along the luminal machinery of biosynthetic organelles occurs borders before being exported through mero- contiguous to elongate mitochondria. Be- crine secretion into the lumina, where the tween neighbouring cells, narrow and labyrin- product occurs as a fine vesicular product thine canaliculi are now detectable, which (Fig. 2D). Inside the cytoplasm, most secre- correspond to the patterns of close contiguity tory vesicles contain a sparse, flocculent ma- observed between epithelial cells during secre- terial as well as an eccentric electron-dense tory activity and sperm storage. core (Fig. 2B-2D). Brizzi et al. ('89) stated that acid mucopolysaccharides (glycosamino- Patterns of sperm storage and degradation glycans), possibly associated with proteins Ultrastructural investigations of sper- (forming proteoglycans), compose the secre- mathecae storing sperm revealed the occur- tory product, which is consistent with rer rence of a wide range of relationships be- profiles associated with the condensing tween the gametes and the spermathecal vesicles (Fig. 2C). epithelium. In all the females we observed in Further ultrastructural characteristics of autumn (slightly after the onset of the mat- the active spermathecal cells consist of tightly ing season) spermatozoa were abundant in- arranged, sometimes concentric membranes, side the spermathecal lumina. In this phase, SPERMATHECAE OF SAL.AMAh'DRINA TERDIGITATA 25

Fig. 2. Salamandrina terdigitatu. Ultrastructural fea- nm; C and D = 500 nm. am, apical microvilli; en, epithe- tures of the spermathecae observed after mating. Female lial cell nucleus; Go, Golgi apparatus; mi, mitochondria; 2, collected in November. The epithelial cells appear mpt, middle piece of a sperm tail; rer, rough endoplasmic biosinthetically active (A-C) and stored sperm are evi- reticulum; se, secretory product in the lumen; sg, secre- dent in the lumina (D). Scale bars: A = 2 IJ-m;€3 = 250 tory granules; sh, sperm head. 26 R. BRIZZI ET AL.

Fig. 3. Salarnandrzna terdzgztata. Patterns of close May, after oviposition. Notice the wide interstices be- (A,B) and loose (C,D) relationships between contiguous tween contiguous cells and the occurrence of sperm in cells in the spermathecae collected in different seasons. the intercellular space (D). Scale bars: A, B, and D = 500 A,B: Specimen 5, from November. Occurrence of tightly nm; C = 2 p,m. Labels as in Figure 2, plus: de, desmo- adhering plasmmembranes in the form of narrow and some; i, interstice; ic, intercellular canaliculi; Ib, lamellar tortuosus canaliculi between contiguous epithelial (A) body; Id, lipid droplet; mcn, myoepithelial cell nucleus; se, and myoepithelial cells (B). C,D:Female 14, collected in stromal environment. SPERMATHECAE OF SALAMANDRINA TERDIGITATA 27

Fig. 4. Salarnandrina lerdlgitata. Spermathecae of females collected in summer. A Sptbci- men 17, collected in late June, at the culmination of reproductive stasis. B Female 18, from July. Observe the increase in the relative quantity of supranuclcar cytoplasms in comparison with -4. Scale bars in A and B = 3 pm. Labels as in previous figures, plus: lu, lumen. the sperm appear tightly packed together, In females observed in early April, slightly mostly against the luminal border, and bathed before oviposition, the phagosomes are more by a fine, vesicular secretory product (Figs. numerous and mostly contain sperm frag- 2D, 5A,B). At higher magnifications, some ments resulting from a degradation process sperm are visible in close contiguity with the initiated by primary lysosomes (Sever, '92b). spermathecal epithelial cells, which exhibit These lytic organelles originate from the ac- elongate apical microvilli (Figs. 2D, 5B) con- tivity of associated Golgi stacks and RER taining thin, tightly bundled microfilaments profiles (Golgi-RER-lysosomes or GERL; (Fig. 5B). Sever and Brunette, '93), which pervade the In females sacrificed between January and supranuclear cytoplasms. In addition, pri- the beginning of April, some months after mary lysosomes can be seen merging to- mating but prior to oviposition, sperm are gether (Fig. 6C,D) or with the phagosomes still abundant in the lumina and in some containing sperm fragments. In a later stage, instances they appear embedded in the sper- resulting from the association between pri- mathecal epithelium. This condition possibly mary lysosomes and endocytic vacuoles, the results from progressive sperm capture by space around the embedded sperm progres- the apical cytoplasmic processes (Fig. 5C- sively contracts, which gives rise to more 5E). As a result, some sperm appear enclosed compact, electron-dense structures (second- within endocytic vacuoles occurring near the ary lysosomes), often characterized by the luminal border (Fig. 6A) as well as in the occurrence of pseudomyelinic figures (Fig. basal cytoplasm (Fig. 6B). The endocytic vacu- 6D). Steps of this process are evident in the oles (or phagosomes) are bounded by evident spermathecae of females collected in early membranes and also can contain traces of April, before oviposition, but become more secretory material. In many instances the prominent in specimens sacrificed slightly embedded sperm appear damaged, as indi- after egg-laying. cated by alteration or loss of some compo- During sperm degradation, the axial fibers nents (Figs. 5C,E, 6B). progressively collapse and lose their usual 28 R. BRIZZI ET AL.

Fig. 5. Sdarnandririu terdigztatu. Pattern of sperm BE = 250 nm. Labels as in previous figures, plus: af, storage (spermathecae from female 2, collected in Novem- axial fiber; ax, axoneme; cp, apical cytoplasmic process; ber: A,B) and sperm “capture” by the epithelial cells ev, endocytic vacuole; mf, marginal filament; mif, micro- (specimen 9, from March: C- D- E). Scale bars: A = 2 pm; filaments; urn, undulating membrane. SPERMATHECAE OF SALAMANDRINA TERDIGITATA 29

Fig. 6. Salamandrina terdzgitata. Patterns of sperm confluences between primary lysosomes. Scale bars: inclusion (specimen 10, from April: A,B) and sperm A-C = 500 nm; D = 250 nm. Labels as in previous degradation (specimen 15, collected in late May: C,D) in figures, plus: pl, primary lysosome; rb, residual bodies; sl, the spermathecae observed at the end of the sperm stor- secondary lysosome. age phase. Double head-arrows in C and D point to 30 R. BRIZZI ET AL. electron density, while the mitochondria1 Eurycea cirrigera, including the massive sper- sheath around the middle piece, the axo- miophagic activity by the epithelium after neme, and plasma membrane disintegrate the egg-laying season. In a further paper, and disappear (Fig. 7A). Sperm are no longer Sever and Kloepfer ('93) described the sper- recognizable inside advanced phagosomes, mathecal cytology of the ambystomatid Am- which only hold residual bodies. The fate of bystoma opacum, in which they observed only the degraded spermatozoa seems to be exocy- a few sperm embedded in the epithelium and tosis through the apical cytoplasm of the undergoing degradation. These differences in spermathecal epithelium, where secondary sperm storage and phagocytosis, observed in lysosomes and residual bodies accumulate urodeles of different taxa, stimulated this (Fig. 7A). Residual sperm, not evacuated dur- study in Salamandrina terdigitata, to clarify ing oviposition, also are visible inside the the mode of sperm degradation previously lumen in females collected during late April observed under light microscopy in this sala- and May (Figs. 7A,B). In Salamandrina we mandrid, and to complete the ultrastructural never observed sperm exocytosis from the observations on the changes in its spermathe- basal cytoplasms of the spermathecae (as re- ported by Sever, '92b in Eurycea cirrigera), cal epithelium through the year. although occasionally the epithelial cells reach As in the other Salamandroidea except the the stromal environment directly by means Plethodontidae, the spermathecae of the Sala- of foot processes through gaps in the myoepi- mandridae consist of numerous simple tu- thelial sheath. bules, which open individually onto the roof In regards to apical exocytosis, cells filled of the cloacal cavity (Sever and Brunette, '93; with vacuolized spermatozoa andlor residual Sever and Kloepfer, '93). In Salamandrina, bodies in many instances undergo desquama- this arrangement was observed by Brizzi et tion from the spermathecal epithelium and al. ('89), who also noticed areas of ciliated enter the lumen together with residual sperm epithelium associated with the spermathecal (Fig. 7B). In addition, cells (presumed macro- pores in the cloaca. The occurrence of such phages) move into the epithelium and lumen cellular specializations, together with other from the stroma and contribute to the degra- mechanisms for attracting the sperm to- dation processes, both of the sperm and de- wards the spermathecae, i.e., contraction of squamated cells (Fig. 70. The phagocytic the smooth muscles of the cloacal walls and activity of the interepithelial or luminal mac- sperm thigmotaxis (Hardy and Dent, '86), rophages is revealed by the presence of pri- seem to account for the ability of sperm to mary and secondary lysosomes in their cyto- reach the spermathecal tubules, where they plasm, often recognizable in close contiguity are stored until oviposition. to conspicuous Golgi bodies (Fig. 70.In addi- Unlike the other families of the suborder tion, the cytoplasm of the presumptive mac- Salamandroidea, the spermathecae of the rophages lacks secretory vesicles, and the cell Plethodontidae consist of a group of com- profiles do not exhibit any junctional special- pound alveolar glands (bulbs and neck tu- izations. bules) which pass into a medial common tube The intense phagocytic activity occurring opening into the roof of the cloaca (Sever, in spermathecae slightly after the egg-laying '87; Sever and Brunette, '93). In this connec- season produces a sharp and massive destruc- tion, Sever and Brunette ('93) and Sever and tion of residual sperm and desquamated celIs. Therefore, in females collected in summer Kloepfer ('93) hypothesized that the differ- during reproductive stasis, the tubule lu- ent anatomy of the spermathecae of Eurycea mina appear devoid of both sperm and secre- cirrigera and Ambystoma opacum may ac- tory product (Fig. 4A,B). count for the different amount of sperm and degree of spermiophagy observed in these DISCUSSION urodeles during and after oviposition. Al- Brizzi et al. ('89) described seasonal though Salamandrina terdigitata is similar changes inside the spermathecal epithelium to A. opacum in spermathecal morphology of Salamandrina terdigitata and patterns of (i.e., it has simple spermathecae), in this sperm degradation observed under light mi- species we observed a great concentration of croscopy. Following the study by Brizzi et al. sperm in the spermathecae of all females ('891, Sever ('91c, '92b), and Sever and Bru- collected from fall to spring. In addition, the nette ('93) reported their ultrastructural find- ultrastructural observations revealed consis- ings on the spermathecae of the plethodontid tent patterns of spermiophagy in S. terdigi- Fig. 7. Salamandrim terdigitata. Female 16, col- area of massive sperm degradation and cell desquama- lected in late May. A: Advanced phagosomes accumulate tion. Scale bars: A = 500 nm; B = 2 pm; C = 1 pm. Labels along the apical cytoplasm before exocytosis. B: Desqua- as in previous figures, plus: dc, desquamated cell; pmn, mation of the spermathecal epithelium observed in the nucleus of a presumptive macrophage. same specimen. C: Occurrence of a macrophage in an 32 R. BRIZZI ET AL. tata, as observed by Sever ('91c, '92b) and provides nourishment for the stored sperm, Sever and Brunette ('93) in E. cirrigera. but no cytological evidence exists for such a Spermiophagy in Salamandrina terdigi- process. Finally, Sever and Kloepfer ('93) tata strongly resembles that reported in Eu- hypothesized that the spermathecal secre- rycea cirrigera. In both these salamanders, tory product may represent an appropriate the spermathecal epithelium is actively sper- chemicallosmoticenvironment for sperm qui- miophagic as long as sperm are present, and escence. Neither the bibliographical data nor lysosomes play an important role in the pro- present findings resolve the question, but cess. In addition, owing to the intense sper- they do suggest that the luminal secretion miophagy, some epithelial cells undergo de- plays a role through the entire sperm storage squamation and occur in the spermathecal phase, possibly in sperm nourishment and/or lumina together with residual sperm after sperm quiescence. the egg-layingseason. In Salamandrina, nev- This study confirms the observations by ertheless, we also observed interepithelial leu- Sever ('91c, '92b) and Sever and Brunette kocytes, possibly macrophages moving from ('93) on Eurycea cirrigera and that of Sever the stroma, which, as reported by Sever and and Kloepfer ('93) on Ambystoma opacum, Kloepfer ('93) in Ambystoma opacum, seem i.e., that sperm embedded in the spermathe- to contribute to spermiophagicactivity. Thus, cal epithelium undergo degradation rather present findings in Salamandrina do not that receive nourishment. According to these agree with the clear ultrastructural differ- reports, most of the embedded sperm appear ences previously noticed between members damaged, possibly due to degradation activ- of different families (Sever and Brunette, '93; ity by the lysosomes inside the epithelial cells. Sever and Kloepfer, '93) and suggest a pos- Dent ('70) also noticed in Notophthalmus sible convergence toward similar mecha- uiridescens that most sperm embedded in the nisms in sperm storage and degradation. spermathecal cells were degenerating, Among the methods of sperm removal, the whereas Boisseau and Joly ('75) related the invasion of phagocytic leukocytes has also close contact between sperm and epithelium been reported in the female reproductive or- in Salamandra salamandra to nutritional gans of many mammals (see Sever, '92b and processes. In addition, Boisseau and Joly ('75) Sever and Kloepfer, '93). observed "sperm capture" by the epithelial One common feature in the spermathecae cells similar to that which we observed in of the urodeles is the production by the epithe- Salamandrina terdigitata. In our opinion, lial cells of an apical secretion for export into however, these patterns represent, at least in the lumen, where it bathes the sperm. Al- Salamandrina, the initial steps of a process though some differences exist in the chemical which culminates in sperm degradation. Why composition (Lemaitre-Lutz, '68; Dent, '70; the spermathecal epithelium is spermiophagic Pool and Hoage, '73; Boisseau and Joly, '75; during sperm storage is an open question. We Sever, '87; Brizzi et al., '89; Davitt and believe this process may represent a control Larsen, '90; Sever et al., '90; Sever, '91c; mechanism regulating sperm surplus inside Sever and Brunette, '93; Sever and Kloepfer, the spermathecal tubules andlor damaged '93) and regionalization of this secretion sperm. In addition, this process may ensure (Sever and Brunette, '93), its constant occur- that "old" sperm are removed from the sper- rence inside the spermathecae observed dur- mathecae prior to the next breeding season. ing sperm storage indicates a role in this Some reports, however, have suggested that process. Many hypotheses have been pro- sperm may be retained in the spermathecae posed regarding the function of the apical of salamanders between successive breeding secretion in the spermathecae of urodeles. seasons (Baylis, '39; Hafeli, '71; Massey, '901, According to Jordan (18931, Noble and We- leading to the potential for long-term sperm ber ('29), and Lemaitre-Lutz ('681, the secre- competition (Houck and Schwenk, '84).Our tion inside the lumina may function as sperm results agree with other reports in which no attractant, a role, however, which does not evidence was found for storage of residual agree with the biosynthesis of the product sperm between breeding seasons (Peccio, '92; over the entire sperm storage period. In addi- Sever and Brunette, '93; Sever and Kloepfer, tion, Hardy and Dent ('86) related the entry '93; Verrell and Sever, '88). of sperm in the spermathecae chiefly to thig- In summary, further ultrastructural stud- motaxis. Dent ('70) and Boisseau and Joly ies are needed to clarify the characteristics of ('75) proposed that spermathecal secretion sperm storage and degradation and the most SPERMATHECAE OF SALAb4ANDRINA TERDIGITATA 33 significant differences among salamanders of Massey, A. (1990) Notes on the reproductive ecology of different lineages. These, in turn, may help red-spotted (Notophthalmus uiridescens). Co- peia 1990:106-107. determine whether polyphyly is a likely hy- Mazzi, V. (1977) Manuale di Tecniche Istologiche e Isto- pothesis for origin of sperm storage organs in chimiche. Piccin, Padova. the (Sever, '94; Sever and Kloepfer, Noble, G.K., and J.A. Weber (1929) The spermatophores '93). of Desmognathus and other plethodontid salamanders. Amer. Mus. Novitates 351:l-15. ACKNOWLEDGMENTS Peccio, A. (1992) Insemination and egg laying dynamics in the smooth , Triturus uulguris in the labora- The research of R. Brizzi, G. Delfino, and tory. Herpetologica J. 2:5-7. G. Selmi was supported by grants from the Pool, T.B., and T.R. Hoage (1973) The ultrastructure of Minister0 Dell'Universita e della Ricerca secretion in the spermatheca of the Mancu- Scientifica e Tecnologia. Support for the lus quudridigitatus (Holbrook).Tissue Cell 5.303-313. Selmi, G. (1993) Sperm storage and capacitation. In R. research of D.M. Sever came from U.S. Dallai (ed):Sex Origin and Evolution: Selected Sympo- National Science Foundation grant DEB- sia and Monographs. UZI 6 Mucchi Modena, pp. 251- 9024918. 265. Sever, D.M. (1978) Male cloacal glands of Plethodon LITERATURE CITED cinereus and Plethodon dorsalis (Amphibia: Plethodon- Baylis, H.A. (1939) Delayed reproduction in the spotted tidae). Herpetologica 34:l-20. salamander. Proc. Zool. Soc., London 109A:243-246. Sever, D.M. (1987) Hemiductylium scutatum and the Boisseau, C., and J. Joly (1975) Transport and survival of phylogeny of cload anatomy in female salamanders. spermatozoa in female Amphibia. In E.S.E. Hafez and Herpetologica 43~105-116. C.G. Thibault (eds): The Biology of Spermatozoa: Sever, D.M. (1988) The ventral gland in female sala- Transport, Survival, and Fertilizing Ability. Basel: mander Eurycea bislineata (Amphibia: Plethodonti- Karger, pp. 94104. dae). Copeia 1988r572-579. Brizzi, R., G. Delfino, and C. Calloni (1989) Female Sever, D.M. (1991a) Comparative anatomy and phylog- cloacal anatomy in the , Sala- eny of the cloacae of salamanders (Amphibia: Cau- mundrina terdigitutu (Amphibia: Salamandridae). Her- data). l. Evolution at the family level. Herpetologica petologica 45:310-322. 47:165-193. Davitt, C.M., and J.H. Larsen Jr. (1990) Morphology of Sever, D.M. (1991b) Comparative anatomy and phylog- the principle cell types of the plethodontid salamander eny of the cloacae of salamanders (Amphibia: Cau- spermatheca following treatment with gonadotropin. data). 11. Cryptobranchidae, Hynobidae, and Sirenidae. Am. Zool.30:38A. Morphol. 207:283-301. Dent, J.N. (1970) The ultrastructure of the spermatheca J. in the red-spotted newt. J. Morphol. 132397424, Sever, D.M. (1991~)Sperm storage and degradation in Duellman W.E., and L. Trueb (1986) Biology of Amphib- the spermathecae of the salamander Eurycea cirrigera. ians. McGraw-Hill, New York. J. Morphol. 210:71-84. Hafeli, H.P. (1971) Zur Fortplantzungsbiologie des Alpen- Sever, D.M. (1992a) Comparative anatomy and phylog- salamanders (Salamundra atra Laur.). Rev. Suisse eny of the cloacae of salamanders (Amphibia: Cau- Zool. 78:235-293. data). IV. Salamandridae. Anat. Rec. 232:229-244. Hardy, M.P., and J.N. Dent (1986) Transport of sperm Sever, D.M. (1992b) Spermiophagy by the spermatha1 within the cloaca of the female red-spotted newt. J. epithelium of the salamander Eurycea cirrigeru. J. Morphol. 190r259-270. Morphol. 212t281-290. Houck, L.D.,and K. Schwenk (1984) The potential for Sever, D.M. (1994) Observations on regionalization of long-term sperm competition in a plethodontid sala- secretory activity in the spermathecae of salamanders mander. Herpetologica 40:410-415. and comments on phylogeny of sperm storage in female Joly, J. (1960) La conservation des spermatozoides et les . Herpetologica 50:in press. particularit& histophysiologiques du receptacle semi- Sever, D.M., and N.S. Brunette (1993) Regionalization of nal chez la Salamandre Salamandra salamandru tae- eccrine and spermiophagic activity in spermathecae of niata. C.R. Acad. Sci. 250:2269-2271. the salamander Eurycea cirrigera (Amphibia: Pleth- Jordan, E.O. (1893) The spermatophores ofDiemyctylus. odontidae). J. Morphol. 21 7:161-170. J. Morphol. 5:263-270. Sever, D.M., and N.M. Kloepfer (1993) Spermathecal Karnovsky, M.J. (1965) A formaldehyde-glutaraldehyde cytology of Ambystoma opacum (Amphibia: Ambysto- fixative of high osmolality for use in electron micros- matidae) and the phylogeny of sperm storage organs in copy. J. Cell Biol. 273137A. female salamanders. J. Morphol. 21 7:115-127. Larson, A., and W.W. Dimmick (1993) Phylogenetic rela- tionships of the salamander families: an analysis of Sever, D.M., E.A. Heinz, P.A. Lempart, and M.S. Taghon congruence among morphological and molecular char- (1990) Phylogenetic significance of the cloacal anatomy acters. Herpetol. Monogr. 7:77-93. of female bolitoglossine salamanders (Plethodontidae: Lemaitre-Lutz, F. (1968) Anatomie des glandes pelvi- tribe Bolitoglossini). Herpetologica 46~431-446. ennes de la femelle de Pleurodeles waltlii Michah: Leur Verrell, P.A., and D.M. Sever (1988) The cloaca and rBle de receptacle seminal. Ann. Embryol. Morphol. spermatheca of the female smooth newt, Triturus uul- 1:409-416. garis (Amphibia: Salamandridae). Act. 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