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JOURNAL OF MORPHOLOGY 22391-33 (1995)

S perm at h ecae of Salarnandrina terdigita ta (Am p h i 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-531 00 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 degradation 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 desquamation from the spermathecal epithelium and enter the lumen together with residual sperm. Desquamated cells, together with residual sperm, are a common 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 biosynthetic activity. o 1995 Wiley-Liss, Inc.

Females of many taxa, including the Am- phibia, are known to posses sperm storage organs associated with their reproductive tracts, which allow the spermatozoa to re- main functional for some time before fertilization occurs (Joly, '60; Boisseau and Joly, '75; Duellman and Trueb, '86; Selmi, '93; Sever, '91c, '92b). Among Urodela, the occurrence of sperm storage structures (usually defined as receptacula seminis or spermathecae; cf. Dent, '70 for a historical review of these terms) characterizes the female genital apparatus of the Salamandroidea (the salamanders which practice internal fertilization: Amphiu- midae, Dicamptodontidae, Proteidae, Am- bystomatidae, Plethodontidae, and Salaman- dridae). Females of the Cryptobranchoidea (Cryptobranchidae and Hynobiidae) and Sire- noidea (Sirenidae) undergo external fertilization and lack spermathecae (Sever, '78; Duell- man and Trueb, '86; Sever, '87, '88, '91a,b). The presence of spermathecae and male cloa-

cal glands involved in internal fertilization were the key characters in establishing the monophyly of the Salamandroidea in a recent analysis using 209 phylogenetically informa- tive characters (Larson and Dimmick, '93).

Sperm storage structures have been inves- tigated by light andlor electron microscopy in many salamanders (Dent, '70; Boisseau and Joly, '75; Pool and Hoage, '73; Brizzi et al., '89; Sever, '91a,c, '92a,b; Sever and Brunette, '93; Sever and Kloepfer, '93) and the results show remarkable similarities both in the tubule morphology and storage patterns. During storage, a close contact between sperm and spermathecal epithelium occurs and, as a rule, sperm storage does not last long after oviposition. Indeed, following this reproductive stage, most residual sperm seem to undergo degenerative processes and, shortly afterwards, the spermathecae exhibit reduced lumina devoid of secretory product 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 the following reproductive season, but the viability of these sperm is not known (Sever, '91~' '92b).

Apart from the patterns of sperm storage, however, cytological details on spermiophagy have only recently been reported for the spermathecal tubules of Eurycea cirrigena (Sever, '91c, '92b; Sever and Brunette, '93) and Am- bystoma opacum (Sever and Kloepfer, '93). In our opinion, this latter process represents a crucial step in storage activity by the spermathecae and is worthy of thorough investi- gation in other salamanders.

With this aim, we report histological and cytological findings on the spermathecal tubules in an European salamandrid, Salaman- drina terdigitata, collected at different stages of its reproductive cycle, with especial empha- sis on the degradative processes observed after the egg-laying season. A major goal of this study is to provide further data on sperm storage and degradation, which may help to elucidate the main characteristics of these processes in the Salamandroidea as well as to identify the specific morphofunctional traits of different taxa.

MATERIALS AND METHODS

Nineteen adult females of Salamandrina terdigitata were collected in the outskirts of Florence (Tuscany, Italy) in different seasons of the years 1992 and 1993 (four in autumn, three in winter, eight in spring, and four in summer). The animals were sacrificed within a few hours of collection to avoid any degenerative processes inside the spermathecae due to captivity. Specimen numbers, snout-vent lengths (from the tip of the snout to the posterior edge of the vent: SVL), ovarian follicle size, and collection date are listed in Table 1. Owing to the dissection of the entire cloacal region, the remaining trunk and head portions of the above specimens were consid- ered unfit for museum collection; voucher specimens from the same locality were depos- ited in the Museum of Natural History of Florence (collection numbers: MZUF 20751- 20757). Following sacrifice in 0.2% chlorbu- tol, specimens of each collection were pro- cessed both for light and transmission electron microscope observations. For light microscope procedures, the cloacal segments containing the spermathecal tubules were fixed in Carnoy's fluid after removal of the hindlimbs, vertebrae, and gut. These specimens were embedded in polystyrene, and transverse sections were cut at 10 km and

TABLE 1. Data on specimens utilized'

Breeding Collection Follicle Sperm in condition SVL date size swrmathecae

Unmated no. 1 41.1 20.1X.92 After mating/prior to oviposition no. 2 43.0 15.X1.92 no. 3 42.1 15.XI.92 no. 4 42.3 15.XI.92 no. 5 41.7 25.XI.93 no. 6 43.2 10.1.93 no. 7 41.8 10.1.93 no. 8 42.8 7.111.92 no. 9 40.8 26.111.93 no. 10 43.3 6.IV.92 no. 11 41.0 6.IV.92 no. 12 44.1 20.1V.93 After oviposition no. 13 40.8 30.1V.93 no. 14 42.7 15.V.92 no. 15 42.2 27.V.93 no. 16 43.0 27.V.93 no. 17 42.8 25.VI.93 no. 18 40.5 8.VII.92 no. 19 41.3 8.VII.92

0.50

0.62 0.65 0.50 0.70 0.82 0.70 0.80 0.97 1.10 0.95 1.35

S S S

0.30 0.42 0.45 0.40

N

Y Y Y Y Y Y Y Y Y Y Y

Y Y Y Y N N N

'Measurements are in mm; follicle size is the mean obtained from ten follicles; N, no; Y, yes; S, spent.

stained using the Mallory-Galgano trichrome method (Mazzi, '77) for general cytological studies.

For electron microscope observations, the cloacal segments were cut into smaller blocks, prefixed in a formaldehyde-glutaraldehyde mixture (Karnovsky, '65) and postfixed in 1% OsO4 (both in a 0.1 M cacodylate buffer). After dehydration, the fragments were embedded in Epon 812. Semithin (2 pm) sections, obtained with a Nova Ultratome LKB, were counterstained through OsO4 reduction over a Bunsen burner and observed under light microscopy to localize the spermathecal tubules. Ultrathin sections, corresponding to the spermathecal secretory units, were collected on uncoated copper grids, stained with uranyl acetate followed by lead citrate, and observed under a Philips 400 electron microscope.

RESULTS

Light microscopy observations Brizzi et al. ('89) reported that the sper-

mathecae of Salamandrina terdigitata are simple, tubular glands contained in the tu- nicapropria of the mucosa lining the cloacal cavity. Each tubule consists of a monolayered epithelium (Fig. 1A) surrounded by a sheath of myoepithelial cells. The spermathecal tubules open individually into the cloacal lu-

SPERMATHECAE OF SALAMANDRINA TERDIGITATA 23

Fig. 1. Salamandrina terdigitata. Light microscopy of the spermathecae. A: Specimen 3, collected in Novem- ber, at the onset of sperm storage. B: Female 13, collected in April, shortly after oviposition. C: Pattern of mitotic activity (arrows) in the spermathecal epithelium of specimen 13. D Specimen 1, from late September. Although

sperm are not yet stored inside the tubules, secretory product already occurs in the cytoplasm. Scale bars: A and C = 20 bm; B and D = 30 Fm. ec, epithelial cells; slu, sperm in the lumen; sp, secretory product; tp, tunica propria.

men and, if observed over the year, exhibit different cytological features related to the different phases in the reproductive cycle (Brizzi et al., '89).

Sperm storage occurs from autumn to spring, followed by a short phase after oviposition characterized by degradation of residual sperm. In the latter period, scattered spermatozoa are recognizable in the lumina mixed with deteriorated cells (Fig. 1B). Along with desquamation of the epithelial cells, pat-

terns of mitotic activity are often evident in the tubule wall (Fig. 10, which may account for the rapid cytological restoration of the spermathecal epithelium. By the beginning of the summer, each tubule consists of inactive, cuboidal cells, arranged around very reduced lumina devoid of sperm and secretory product (Brizzi et al., '89). In summer, secretory inactivity in the spermathecae coin- cides with female reproductive stasis, as con- firmed by the small follicle size (see Table 1).

24 R. BRIZZI ET AL.

In the specimen from September, the spermathecal cells exhibited resumption of biosynthetic activity, recognized by the presence of secretory product in the high supranuclear cytoplasm (Fig. 1D). From early autumn on- ward, bundles of sperm occur inside the spermathecae, mixed with secretory product (Fig. 1A). During autumn, the ovarian follicles reach 0.70 mm in diameter, which, however, does not correspond to the largest follicles observed during the spring egg-laying season. In this latter phase, oocytes of 1.35 mm in diameter occur in the oviducts just before oviposition.

Apart from the arrangement of sperm in the lumina and the shapes of the epithelial cells, few cytological details can be observed by light microscopy during the cyclic changes of the spermathecal epithelium. The epithelial cells contain secretory granules (during sperm storage), or are involved in desquamation processes followed by biosynthetic stasis (during late spring and early summer).

Electron microscopy observations Patterns of biosynthetic activity

During the initial steps of sperm storage (early autumn), the spermathecal cells are high columnar, with round, basal nuclei that are relatively small in comparison to the amount of cytoplasm. Inside the nuclei, het- erochromatin mostly occurs around the borders (Fig. 2A) and nucleoli are not prominent. Secretory vesicles fill the supranuclear cytoplasm, where conspicuous Golgi stacks (dictyosomes), rough endoplasmic reticulum profiles (rer), and elongate mitochondria also are visible (Fig. 2B,C). In many instances, condensing vesicles are common in the supranuclear as well as in the basal cytoplasm, giving rise to numerous secretory granules. The granules accumulate along the luminal borders before being exported through mero- crine secretion into the lumina, where the product occurs as a fine vesicular product (Fig. 2D). Inside the cytoplasm, most secretory vesicles contain a sparse, flocculent material as well as an eccentric electron-dense core (Fig. 2B-2D). Brizzi et al. ('89) stated that acid mucopolysaccharides (glycosamino- glycans), possibly associated with proteins (forming proteoglycans), compose the secretory product, which is consistent with rer profiles associated with the condensing vesicles (Fig. 2C).

Further ultrastructural characteristics of the active spermathecal cells consist of tightly arranged, sometimes concentric membranes,

in the form of lamellar bodies, and intercellular canaliculi between contiguous cells often associated with desmosomes. Similar relationships between plasmmembranes occur between epithelial (Fig. 3A) and myoepithelial cells (Fig. 3B; for further details on the myoepithelial cells in the spermathecae of Salu- mandrina terdigitutu see Brizzi et al., '89).

As noted previously, patterns of biosynthetic activity are evident in the spermathecae of females collected from autumn to spring, after mating but before oviposition. In the next phase, a short period after egg- laying (between late April and mid-May), the production of secretory material by the spermathecal cells rapidly decreases and the cytoplasm appears much reduced in comparison with the size of the nuclei (Fig. 3C). The nuclei are now characterized by dense hetero- chromatin and prominent nucleoli. Speci- mens in this phase have wide and irregular interstices between contiguous cells that occasionally contain sperm (Fig. 3D). A pale, non specific secretory product, mainly consist- ing of lipid droplets, is detectable both in the basal and supranuclear cytoplasm (Fig. 3C,D). Microvilli are scarce and short along the apical border, and sparse residual sperm occur inside the lumina (Fig. 3C). In females sacrificed in June and July, at the culmination of the reproductive stasis, the spermathecal tubules appear empty (see below), biosyntheti- cally inactive, and characterized by a very high N/P ratio (Fig. 4A). In some specimens, nevertheless, the approaching secretory condition already is recognizable. Although the epithelial cells are still devoid of condensing vesicles and contain large nuclei, imminent resumption of biosynthetic activity is as- sumed from the increased area of supranuclear cytoplasm (Fig. 4B), where a rich machinery of biosynthetic organelles occurs contiguous to elongate mitochondria. Be- tween neighbouring cells, narrow and labyrin- thine canaliculi are now detectable, which correspond to the patterns of close contiguity observed between epithelial cells during secretory activity and sperm storage. Patterns of sperm storage and degradation

Ultrastructural investigations of spermathecae storing sperm revealed the occurrence of a wide range of relationships between the gametes and the spermathecal epithelium. In all the females we observed in autumn (slightly after the onset of the mating season) spermatozoa were abundant inside the spermathecal lumina. In this phase,

SPERMATHECAE OF SAL.AMAh'DRINA TERDIGITATA 25

Fig. 2. Salamandrina terdigitatu. Ultrastructural features of the spermathecae observed after mating. Female 2, collected in November. The epithelial cells appear biosinthetically active (A-C) and stored sperm are evident in the lumina (D). Scale bars: A = 2 IJ-m; €3 = 250

nm; C and D = 500 nm. am, apical microvilli; en, epithelial cell nucleus; Go, Golgi apparatus; mi, mitochondria; mpt, middle piece of a sperm tail; rer, rough endoplasmic reticulum; se, secretory product in the lumen; sg, secretory granules; sh, sperm head.

26 R. BRIZZI ET AL.

Fig. 3. Salarnandrzna terdzgztata. Patterns of close (A,B) and loose (C,D) relationships between contiguous cells in the spermathecae collected in different seasons. A,B: Specimen 5, from November. Occurrence of tightly adhering plasmmembranes in the form of narrow and tortuosus canaliculi between contiguous epithelial (A) and myoepithelial cells (B). C,D: Female 14, collected in

May, after oviposition. Notice the wide interstices between contiguous cells and the occurrence of sperm in the intercellular space (D). Scale bars: A, B, and D = 500 nm; C = 2 p,m. Labels as in Figure 2, plus: de, desmo- some; i, interstice; ic, intercellular canaliculi; Ib, lamellar body; Id, lipid droplet; mcn, myoepithelial cell nucleus; se, stromal environment.


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, mostly against the luminal border, and bathed by a fine, vesicular secretory product (Figs. 2D, 5A,B). At higher magnifications, some sperm are visible in close contiguity with the spermathecal epithelial cells, which exhibit elongate apical microvilli (Figs. 2D, 5B) containing thin, tightly bundled microfilaments (Fig. 5B).

In females sacrificed between January and the beginning of April, some months after mating but prior to oviposition, sperm are still abundant in the lumina and in some instances they appear embedded in the spermathecal epithelium. This condition possibly results from progressive sperm capture by the apical cytoplasmic processes (Fig. 5C- 5E). As a result, some sperm appear enclosed within endocytic vacuoles occurring near the luminal border (Fig. 6A) as well as in the basal cytoplasm (Fig. 6B). The endocytic vacuoles (or phagosomes) are bounded by evident membranes and also can contain traces of secretory material. In many instances the embedded sperm appear damaged, as indi- cated by alteration or loss of some compo- nents (Figs. 5C,E, 6B).

In females observed in early April, slightly before oviposition, the phagosomes are more numerous and mostly contain sperm fragments resulting from a degradation process initiated by primary lysosomes (Sever, '92b). These lytic organelles originate from the activity of associated Golgi stacks and RER profiles (Golgi-RER-lysosomes or GERL; Sever and Brunette, '93), which pervade the supranuclear cytoplasms. In addition, primary lysosomes can be seen merging together (Fig. 6C,D) or with the phagosomes containing sperm fragments. In a later stage, resulting from the association between primary lysosomes and endocytic vacuoles, the space around the embedded sperm progressively contracts, which gives rise to more compact, electron-dense structures (secondary lysosomes), often characterized by the occurrence of pseudomyelinic figures (Fig. 6D). Steps of this process are evident in the spermathecae of females collected in early April, before oviposition, but become more prominent in specimens sacrificed slightly after egg-laying.

During sperm degradation, the axial fibers progressively collapse and lose their usual

28 R. BRIZZI ET AL.

Fig. 5. Sdarnandririu terdigztatu. Pattern of sperm storage (spermathecae from female 2, collected in Novem- ber: A,B) and sperm “capture” by the epithelial cells (specimen 9, from March: C- D- E). Scale bars: A = 2 pm;

B E = 250 nm. Labels as in previous figures, plus: af, axial fiber; ax, axoneme; cp, apical cytoplasmic process; ev, endocytic vacuole; mf, marginal filament; mif, microfilaments; urn, undulating membrane.


Fig. 6. Salamandrina terdzgitata. Patterns of sperm inclusion (specimen 10, from April: A,B) and sperm degradation (specimen 15, collected in late May: C,D) in the spermathecae observed at the end of the sperm storage phase. Double head-arrows in C and D point to

confluences between primary lysosomes. Scale bars: A-C = 500 nm; D = 250 nm. Labels as in previous figures, plus: pl, primary lysosome; rb, residual bodies; sl, secondary lysosome.

30 R. BRIZZI ET AL.

electron density, while the mitochondria1 sheath around the middle piece, the axoneme, and plasma membrane disintegrate and disappear (Fig. 7A). Sperm are no longer recognizable inside advanced phagosomes, which only hold residual bodies. The fate of the degraded spermatozoa seems to be exocytosis through the apical cytoplasm of the spermathecal epithelium, where secondary lysosomes and residual bodies accumulate (Fig. 7A). Residual sperm, not evacuated during oviposition, also are visible inside the lumen in females collected during late April and May (Figs. 7A,B). In Salamandrina we never observed sperm exocytosis from the basal cytoplasms of the spermathecae (as reported by Sever, '92b in Eurycea cirrigera), although occasionally the epithelial cells reach the stromal environment directly by means of foot processes through gaps in the myoepithelial sheath.

In regards to apical exocytosis, cells filled with vacuolized spermatozoa andlor residual bodies in many instances undergo desquamation from the spermathecal epithelium and enter the lumen together with residual sperm (Fig. 7B). In addition, cells (presumed macrophages) move into the epithelium and lumen from the stroma and contribute to the degradation processes, both of the sperm and desquamated cells (Fig. 7 0 . The phagocytic activity of the interepithelial or luminal macrophages is revealed by the presence of primary and secondary lysosomes in their cytoplasm, often recognizable in close contiguity to conspicuous Golgi bodies (Fig. 7 0 . In addition, the cytoplasm of the presumptive macrophages lacks secretory vesicles, and the cell profiles do not exhibit any junctional specializations.

The intense phagocytic activity occurring in spermathecae slightly after the egg-laying season produces a sharp and massive destruc- tion of residual sperm and desquamated celIs. Therefore, in females collected in summer during reproductive stasis, the tubule lumina appear devoid of both sperm and secretory product (Fig. 4A,B).

DISCUSSION

Brizzi et al. ('89) described seasonal changes inside the spermathecal epithelium of Salamandrina terdigitata and patterns of sperm degradation observed under light microscopy. Following the study by Brizzi et al. ('891, Sever ('91c, '92b), and Sever and Bru- nette ('93) reported their ultrastructural findings on the spermathecae of the plethodontid

Eurycea cirrigera, including the massive spermiophagic activity by the epithelium after the egg-laying season. In a further paper, Sever and Kloepfer ('93) described the spermathecal cytology of the ambystomatid Am- bystoma opacum, in which they observed only a few sperm embedded in the epithelium and undergoing degradation. These differences in sperm storage and phagocytosis, observed in urodeles of different taxa, stimulated this study in Salamandrina terdigitata, to clarify the mode of sperm degradation previously observed under light microscopy in this salamandrid, and to complete the ultrastructural observations on the changes in its spermathecal epithelium through the year.

As in the other Salamandroidea except the Plethodontidae, the spermathecae of the Sala- mandridae consist of numerous simple tubules, which open individually onto the roof of the cloacal cavity (Sever and Brunette, '93; Sever and Kloepfer, '93). In Salamandrina, this arrangement was observed by Brizzi et al. ('89), who also noticed areas of ciliated epithelium associated with the spermathecal pores in the cloaca. The occurrence of such cellular specializations, together with other mechanisms for attracting the sperm to- wards the spermathecae, i.e., contraction of the smooth muscles of the cloacal walls and sperm thigmotaxis (Hardy and Dent, '86), seem to account for the ability of sperm to reach the spermathecal tubules, where they are stored until oviposition.

Unlike the other families of the suborder Salamandroidea, the spermathecae of the Plethodontidae consist of a group of com- pound alveolar glands (bulbs and neck tubules) which pass into a medial common tube opening into the roof of the cloaca (Sever, '87; Sever and Brunette, '93). In this connec- tion, Sever and Brunette ('93) and Sever and Kloepfer ('93) hypothesized that the different anatomy of the spermathecae of Eurycea cirrigera and Ambystoma opacum may account for the different amount of sperm and degree of spermiophagy observed in these urodeles during and after oviposition. Al- though Salamandrina terdigitata is similar to A. opacum in spermathecal morphology (i.e., it has simple spermathecae), in this species we observed a great concentration of sperm in the spermathecae of all females collected from fall to spring. In addition, the ultrastructural observations revealed consistent patterns of spermiophagy in S. terdigi-

Fig. 7. Salamandrim terdigitata. Female 16, collected in late May. A: Advanced phagosomes accumulate along the apical cytoplasm before exocytosis. B: Desqua- mation of the spermathecal epithelium observed in the same specimen. C: Occurrence of a macrophage in an

area of massive sperm degradation and cell desquamation. Scale bars: A = 500 nm; B = 2 pm; C = 1 pm. Labels as in previous figures, plus: dc, desquamated cell; pmn, nucleus of a presumptive macrophage.

32 R. BRIZZI ET AL.

tata, as observed by Sever ('91c, '92b) and Sever and Brunette ('93) in E. cirrigera.

Spermiophagy in Salamandrina terdigitata strongly resembles that reported in Eu- rycea cirrigera. In both these salamanders, the spermathecal epithelium is actively spermiophagic as long as sperm are present, and lysosomes play an important role in the process. In addition, owing to the intense spermiophagy, some epithelial cells undergo desquamation and occur in the spermathecal lumina together with residual sperm after the egg-laying season. In Salamandrina, nevertheless, we also observed interepithelial leukocytes, possibly macrophages moving from the stroma, which, as reported by Sever and Kloepfer ('93) in Ambystoma opacum, seem to contribute to spermiophagic activity. Thus, present findings in Salamandrina do not agree with the clear ultrastructural differences previously noticed between members of different families (Sever and Brunette, '93; Sever and Kloepfer, '93) and suggest a pos- sible convergence toward similar mechanisms in sperm storage and degradation. Among the methods of sperm removal, the invasion of phagocytic leukocytes has also been reported in the female reproductive organs of many mammals (see Sever, '92b and Sever and Kloepfer, '93).

One common feature in the spermathecae of the urodeles is the production by the epithelial cells of an apical secretion for export into the lumen, where it bathes the sperm. Al- though some differences exist in the chemical composition (Lemaitre-Lutz, '68; Dent, '70; Pool and Hoage, '73; Boisseau and Joly, '75; Sever, '87; Brizzi et al., '89; Davitt and Larsen, '90; Sever et al., '90; Sever, '91c; Sever and Brunette, '93; Sever and Kloepfer, '93) and regionalization of this secretion (Sever and Brunette, '93), its constant occurrence inside the spermathecae observed during sperm storage indicates a role in this process. Many hypotheses have been proposed regarding the function of the apical secretion in the spermathecae of urodeles. According to Jordan (18931, Noble and We- ber ('29), and Lemaitre-Lutz ('681, the secretion inside the lumina may function as sperm attractant, a role, however, which does not agree with the biosynthesis of the product over the entire sperm storage period. In addition, Hardy and Dent ('86) related the entry of sperm in the spermathecae chiefly to thigmotaxis. Dent ('70) and Boisseau and Joly ('75) proposed that spermathecal secretion

provides nourishment for the stored sperm, but no cytological evidence exists for such a process. Finally, Sever and Kloepfer ('93) hypothesized that the spermathecal secretory product may represent an appropriate chemicallosmotic environment for sperm quiescence. Neither the bibliographical data nor present findings resolve the question, but they do suggest that the luminal secretion plays a role through the entire sperm storage phase, possibly in sperm nourishment and/or sperm quiescence.

This study confirms the observations by Sever ('91c, '92b) and Sever and Brunette ('93) on Eurycea cirrigera and that of Sever and Kloepfer ('93) on Ambystoma opacum, i.e., that sperm embedded in the spermathecal epithelium undergo degradation rather that receive nourishment. According to these reports, most of the embedded sperm appear damaged, possibly due to degradation activity by the lysosomes inside the epithelial cells. Dent ('70) also noticed in Notophthalmus uiridescens that most sperm embedded in the spermathecal cells were degenerating, whereas Boisseau and Joly ('75) related the close contact between sperm and epithelium in Salamandra salamandra to nutritional processes. In addition, Boisseau and Joly ('75) observed "sperm capture" by the epithelial cells similar to that which we observed in Salamandrina terdigitata. In our opinion, however, these patterns represent, at least in Salamandrina, the initial steps of a process which culminates in sperm degradation. Why the spermathecal epithelium is spermiophagic during sperm storage is an open question. We believe this process may represent a control mechanism regulating sperm surplus inside the spermathecal tubules andlor damaged sperm. In addition, this process may ensure that "old" sperm are removed from the spermathecae prior to the next breeding season. Some reports, however, have suggested that sperm may be retained in the spermathecae of salamanders between successive breeding seasons (Baylis, '39; Hafeli, '71; Massey, '901, leading to the potential for long-term sperm competition (Houck and Schwenk, '84). Our results agree with other reports in which no evidence was found for storage of residual sperm between breeding seasons (Peccio, '92; Sever and Brunette, '93; Sever and Kloepfer, '93; Verrell and Sever, '88).

In summary, further ultrastructural studies are needed to clarify the characteristics of sperm storage and degradation and the most

SPERMATHECAE OF SALAb4ANDRINA TERDIGITATA 33

significant differences among salamanders of different lineages. These, in turn, may help determine whether polyphyly is a likely hy- pothesis for origin of sperm storage organs in the Caudata (Sever, '94; Sever and Kloepfer, '93).

ACKNOWLEDGMENTS

The research of R. Brizzi, G . Delfino, and G. Selmi was supported by grants from the Minister0 Dell'Universita e della Ricerca Scientifica e Tecnologia. Support for the research of D.M. Sever came from U.S. National Science Foundation grant DEB- 9024918.

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Davitt, C.M., and J.H. Larsen Jr. (1990) Morphology of the principle cell types of the plethodontid salamander spermatheca following treatment with gonadotropin. Am. Zool.30:38A.

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