JOURNAL OF MORPHOLOGY 212:281-290 (1992)

Spermiophagy by the Spermathecal Epithelium of the Salamander Eurycea cirrigera

DAVID M . SEVER Department of Biology, Saint Mary$ College, Notre Dame, Indiana 46556

ABSTRACT The spermathecae of Eurycea cirrigera are exocrine glands in the cloaca that secrete a substance that bathes sperm stored in the lumen after mating and prior to oviposition. Many sperm remain in the spermathecae after oviposition, and the spermathecal epithelium becomes spermiophagic. Pseudopo- dia enclose sperm into endocytic vacuoles. The vacuoles become associated with primary lysosomes in the cytoplasm. Following formation of secondary lyso- somes and resulting condensation of the sperm fragments, residual bodies are exocytized into the surrounding connective tissue stroma. By the start of the next breeding cycle, most sperm remaining from the previous mating have been degraded, but some sperm remain in the lumen, and the viability of these sperm is unknown.

In females of most species of salamanders (Amphibia: Caudata), sperm storage organs, the spermathecae, occur in the dorsal roof of the cloaca (Sever, '87). Ova of oviparous spe- cies are fertilized by sperm released from the spermathecae during oviposition (Boisseau and Joly, '75). Sperm may remain in the spermathecae after oviposition, and some studies have suggested that these sperm may be capable of fertilizing ova in a subsequent breeding season (Houck and Schwenk, '84; Massey, '90). Detailed cytological studies on the storage or degradation of sperm remain- ing in the spermathecae in the months imme- diately following oviposition are lacking, how- ever.

Recently, Sever ('91) reported cyclic changes in the spermathecae of female two- lined salamanders, Eurycea cirrigera. He re- ported that after oviposition in late April or early May, many sperm remain in the sper- mathecae, but specimens examined from Sep- tember and October contain few or no sperm in spermathecae. In specimens collected im- mediately after oviposition, sperm occur in the lumina of the spermathecae, are encased in endocytic vacuoles, or have passed into the surrounding connective tissue stroma through gaps caused by selective desquama- tion of spermathecal epithelium (Sever, '91). Sever ('91) lacked specimens from late spring and the summer months (25 April-31 Au- gust). This paper reports the fate of sperm in the spermathecae of female E. cirrigera re- moved from their recently oviposited egg

masses in the field on 26-27 April, main- tained in the laboratory, and sacrificed at regular intervals between 29 April and 2 September.

MATERIALS AND METHODS

Female E. cirrigera attach their eggs to the undersides of rocks in streams and remain with them (Bishop, '47). The 10 females used in this study were found under rocks with their egg clutches on 26-27 April 1991 in a stream in Shades State Park, Montgomery County, Indiana. The females were main- tained in the laboratory at 16-20°C on a 12:12 light/dark cycle in 17 x 31 x 9 cm vinyl containers supplied with wet paper tow- els, and were fed ad libitum from vials of Drosophila. Animals were killed by immer- sion in 5% MS-222, and snout-vent length (SVL) was measured from tip of the snout to posterior end of the vent prior to fixation. Dates of sacrifice and SVLs were: 29 April, 47.1 mm; 15 May, 44.0 mm and 47.7 mm; 3 June, 41.4 mm; 6 June, 43.7 mm; 1 July, 44.3 mm and 44.4 mm; 1 August, 41.8 mm and 45.0 mm; and 2 September, 43.2 mm.

For comparative purposes, two animals from previous studies sacrificed after mat- ing, but prior to oviposition were examined. These were a specimen collected on 15 March 1977 in Sevier County, Tennessee, and em-

Addresti reprint requests t o Dr. David M Sever, Department of Biology, Saint Mary's C'ollegr, Notre Dame. Indiana 46556.

Q 1992 WILEY-LISS, INC.

282 D.M. SEVER

bedded in paraffin for light microscopy (Sever, ’87), and a specimen from Montgomery County, Indiana, sacrificed on 25 April 1990 (Sever, ’91).

Following measurement of SVL, the cloa- cal region was excised and the vent was cut to expose the spermathecal region. The block of tissue was placed in 2.5% glutaraldehyde in Millonig’s phosphate buffer a t pH 7.4 for 2 hr. The tissue was then rinsed and held over- night in Millonig’s buffer in a refrigerator (10°C). Subsequently, the spermathecae were dissected from surrounding tissue, postfixed in an aqueous solution of 2% osmium tetrox- ide and 10% sucrose, rinsed in water, stained en-block with 2% uranyl acetate in 50% etha- nol, further dehydrated in a graded series of ethanol solutions, cleared with propylene ox- ide, and embedded in epoxy resin (EM-BED- 812, Electron Microscopy Sciences, Fort Washington, PA).

Thick (250 nm) and ultrathin (65-70 nm) sections were cut on either a MT-6000XL or MT-7 RMC ultramicrotome using glass or

diamond knives. Following transfer to cop- per grids and staining with solutions of 2% uranyl acetate and 2.7% lead citrate, sections were viewed with a Hitachi H-300 transmis- sion electron microscope. Terminology for salamander sperm follows Fawcett (’70) and Picheral(’79).

RESULTS

The relationship of the spermathecae to the cloaca is shown in Figure 1A. A single duct, the common tube, extends from the roof of the cloaca and divides into narrow neck tubules that expand distally to form the spermathecal bulbs. Sperm are stored in the spermathecal bulbs after mating and prior to oviposition (Fig. 1B). Secretory material pro- duced by the spermathecal epithelium is re- leased into the lumen and bathes the stored sperm. Some sperm are embedded in the spermathecal epithelium; these may be dam- aged and undergoing degradation (Sever, ’91). Lysosomes, however, are not evident at this time (Fig. 1B).

Fig. 1. Spermathecae of E. cirrigera. These females were sacrificed after mating but prior to oviposition. The “box” around a portion of the distal bulb of the specimen in A corresponds to the area shown at higher magnifica- tion in B. A: Transverse section through the spermathe- cal region to show relationships with the cloaca. Paraffin sectinn stained with hematoxylin and eosin. B: Overview

of the fine structure of the spermathecal epithelium and luminal border during sperm storage. Scale bar = approx- imately 150 pm for A and 3.75 pm for B. AC, apical cytoplasm; CL, cloaca; CT, common tube; DB, distal bulb of a spermathecal tubule; EN, epithelial cell nucleus; NT, neck tubule; SE, secretory product after its release into the lumen; SLU, sperm in the lumen.

SPERMATHECAL

After oviposition, the spermathecal epithe- lium is involved in spermiophagy during late spring and summer. Interepithelial or lumi- nal macrophages are not evident, and the spermathecal epithelium seems responsible for all of the phagocytic activity observed. The overall appearance of a portion of a distal bulb in a specimen sacrificed 15 May is shown in Figure 2, and spermathecae from individu-

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als sacrificed throughout the summer are similar in cytology. Some sperm in the lumen appear normal, but others seem damaged or degraded. Notice the decreased electron den- sity of the nucleus, axoneme, axial fiber, and mitochondria associated with the middle piece of the tail. Pseudopodia extend from the epi- thelium into the narrowed lumen, and parts of spermatozoa and associated lysosomal

Fig. 2. Spermathecal epithelium and luminal border ofE. cirrzgeru. This female was collected on 26 April after oviposition of eggs and sacrificed on 15 May. Scale bar = approximately 4 pm. Labels same as for Figure 1, plus: PP, pseudopodia; PS, phagosomes; SL, secondary lysosomes.

284 D.M. SEVER

dense bodies are numerous in the epithelium (Fig. 2).

Sperm are enclosed in the apical epithe- lium of the spermathecae by pseudopodia extending from the cytoplasm (Fig. 2) or by slender cytoplasmic processes of the surface plasma membrane that enclose sperm that come in contact with the spermathecal epithe- lium (Fig. 3A). The resulting endocytic vacu- oles containing sperm fragments are referred to here as phagosomes. Microfilaments asso- ciated with the luminal epithelium (Fig. 3A) may be involved in movement of pseudopodia and vacuoles (Allison et al., '71). Mitochon- dria are numerous throughout the epithelial cells, but are especially prominent apically and at the base. Lipid droplets and smooth endoplasmic reticulum often are associated with the mitochondria, and some apical mito- chondria curl around lipid droplets (Fig. 3B).

Electron dense, membrane bound or- ganelles (Fig. 3) that are interpreted in this study to be primary lysosomes might be pro- duced by the Golgi complexes that are abun- dant in the perinuclear cytoplasm (Fig. 4). The vacuolated space around endocytized sperm disappears when the phagosomes be- come associated with primary lysosomes (Fig. 4A). Initially, while the axial fiber maintains a uniform electron density, a fine fibrous meshwork occurs around the sperm (Fig. 4A). As the central portions of the axial fiber collapse inward and the mitochondrial sheath around the middle piece disintegrates, the outer filamentous material disappears. The phagosome thus becomes a secondary lyso- some, a dense body that usually is less opaque centrally and in which parts of the sperm can no longer be discerned (Fig. 4B). Larger masses of dense material are occasionally seen in the lumen adjacent to the epithelium (Fig. 4B).

At the base of the epithelial cells, exocyto- sis of residual bodies is apparent (Fig. 5). As in the apical cytoplasm, mitochondria and microfilaments are numerous in the basal cytoplasm (Fig. 5). Small vesicles associated with the basement membrane also are abun- dant. The basement membrane is irregular and possesses cytoplasmic processes that ex- tend into the surrounding connective tissue stroma. The evidence for exocytosis is that residual bodes are present in the basal cyto- plasm of the spermathecal epithelium, and dense bodies with the same appearance occur in the connective tissue stroma surrounding the spermathecae (Fig. 5B).

Whether sperm are fragmented prior to (Moyer et al., '65) or after (Murakami et al., '85) endocytosis could not be determined. Longitudinal sections of endocytized sperm fragments observed in some cells indicate that degradation may occur more quickly in some areas than others (Fig. 4A). The nu- cleus is no more resistant to degradation than other parts of the sperm are. The acroso- mal sheath and the nuclear plasma mem- brane often appear disrupted in sperm heads observed in the lumen (Fig. 6A) and in phago- somes (Fig. 6B). The nuclear ridge, however, still is distinct in sperm heads observed in phagosomes (Figs. 3A, 6B).

Sections through some tubules of the in&- vidual sacrificed 2 September show a marked reduction in the number of spermatozoa in the lumen and changes in the spermathecal epithelium. These tubules are comparable to those described by Sever ('91) for reproduc- tively inactive specimens collected 13 August and sacrificed 31 August (compare Fig. 6C here with Fig. 1 of Sever, '91). The nuclei are large compared with the amount of cyto- plasm, and secretory organelles are not appar- ent. The most prominent feature in the cyto- plasm is the presence of small dense bodies, interpreted here as residual bodies (Fig. 6C).

DISCUSSION

Phagocytosis of sperm by the spermathe- cal epithelium previously has been reported for salamanders by Dent ('701, Pool and Hoage ('73), Boisseau and Joly ('75), Davitt and Larsen ('88), Brizzi et al. ('891, and Sever ('91). Only Sever ('91) reported the forma- tion of endocytic vacuoles, and none of the authors noted the involvement of lysosomes in sperm degradation or the exocytosis of residual bodies from the epithelium.

However, findings similar to those de- scribed here have been reported for degrada- tion of surplus sperm in the reproductive tracts of mammals. Sperm are known to be degraded in epithelia of the oviducts of white mice (Chakraborty and Nelson, '75) and cats (Murakami et al., '85b); vasa deferentia of rats (Cooper and Hamilton, '77), rabbits (Mu- rakami et al., '85a), and dogs (Murakami et al., '86); and seminiferous tubules of rats (Yokoyama et al., '87) and cats (Murakami et al., '88). Lysosomes are involved in sperm degradation by the epithelial cells according to some of these reports (Murakami et al., '85a,b, '86, '88; Yokoyama et al., '871, but exocytosis of residual bodies was not men- tioned. Invasion of phagocytic leukocytes is

SPERMATHECAL SPERMIOPHAGY 285

Fig. 3. The apical cytoplasm of the spermathecae of E. cirrigeru. These females were collected on 26 April after oviposition. A Specimen sacrificed 15 May. B: Specimen sacrificed 3 June. Scale bar = approximately 571 nm for A and 444 nm for B. Labels same as for Figures 1 and 2, plus: AF, axial fiber; AX, axoneme; CP, cytoplasmic process; FI, microfilaments; FL, flagellum;

LI, lipids; LU, lumen; MF, marginal filament; MI, mito- chondria; MTA, middle piece of the tail near attachment to the neck piece; MTB, middle piece of the tail distal to the neck piece; NR, nuclear ridge; PL, primary lysosome; PT, principle piece of the tail; PV, phagocytic vacuole; SER, smooth endoplasmic reticulum; SN, sperm nucleus; UM, undulating membrane.

286 D.M. SEVER

Fig. 4. The perinuclear cytoplasm of the spermathe- cae of E. cirrigeru, showing various stages of lysosome formation. This female was collected on 26 April after oviposition and sacrificed 15 May. Scale bar = approxi- mately 389 nm for A and 500 nm for B. Labels same as for Figures 1-3, plus: DA, degraded area of axial fiber

adjacent to areas that are still electron dense; DE, spot desmosome; FM, filamentous material; GO, Golgi bodies; IC, intercellular canaliculus; LDM, luminal dense mate- rial; PV, vacuolated space of a phagosome; RER, rough endoplasmic reticulum.

SPERMATHECAL SPERMIOPHAGY 287

Fig. 5. Basal epithelium of the spermathecae of E. cirrigeru, showing exocytosis of sperm. These females were collected on 26 April after oviposition, A: Specimen sacrificed 1 August. B: Specimen sacrifice 1 July. Scale bar = approximately 482 nm for A and 426 nm for B.

Labels same as for Figures 1-4, plus: BM, basement membrane; CF, collagen fiber; CS, connective tissue stroma; MG, melanin granule; RB, residual body; VE, vesicle.

288 D.M. SEVER

Fig. 6. Female E. cirrzgera collected on 26 April after oviposition and sacrificed on 2 September. A: Sperm nucleus in the lumen of a spermathecal tubule. B: Sperm nuclei in phagosomes in the apical cytoplasm of the same spermathecal tubule. C: Spermathecal epithelium of an- other tubule, showing reduction in cytoplasm and secre-

tory activity. Scale bar = approximately 248 nm for A, 300 nm for B, and 1.11 pm for C. Unlabeled arrows indicate disruptions in the plasma membrane of sperm nuclei. Other labels same as for Figures 1-5, plus: PM, plasma membrane.

SPERMATHECAL SPERMIOPHAGY 289

reported as a method of sperm removal in the epithelia and/or lumina of the uterus of ham- sters (Yanagimachi and Chang, '63) and bats (Mori and Uchida, '80); the uterine tubes or oviducts of rabbits (Moyer et al., '65) and cats (Murakami et al., '85b); vas deferens of the rat (Cooper and Hamilton, '77; Kennedy and Heidger, '79); and tubuli recti of cats (Mura- kami et al., '88).

Cooper and Hamilton ('77) reported masses of dense material in the lumen, as also noted here. They stated that sperm fragments in these masses are phagocytized by the epithe- lium, but that the dense material remains in the lumen (Copper and Hamilton, '77). Mura- kami et al. ('86) found lipid droplets in epithe- lial cells involved in sperm degradation, but did not mention an association of lipid drop- lets with mitochondria reported here.

In an intriguing abstract, Davitt and Larsen ('88) reported that the cytoplasmic droplets of sperm are phagocytized by the spermathe- cal epithelium of the salamander Rhyacotri- ton olympicus during sperm storage. A simi- lar phenomenon was not verified in this study. Cytoplasmic droplets in salamanders often contain osmiophilic bodies that may repre- sent lipid droplets (Murphy et al., '73). Thus, lipid found in the spermathecal epithelium might be exogenous in origin. Lipid droplets, however, were not observed in endocytic vac- uoles in this study. Smooth endoplasmic retic- ulum is abundant in the spermathecal epithe- lium and may be the source of the lipid droplets.

The spermathecal epithelium of the labora- tory-maintained specimen sacrificed 2 Sep- tember had the inactive appearance described by Sever ('91) for specimens collected and sacrificed at the end of August. The specimen sacrificed 2 September and the two animals sacrificed in August still possessed sperm in their spermathecae. In contrast, only one out of five wild-caught animals from August and September possessed sperm (Sever, '91).

These results indicate that the number of sperm decreases as a result of spermiophagy by the spermathecal epithelium, but sperm that look like freshly stored sperm may still occur in the lumina of the spermathecae four or five months after oviposition. The reten- tion of sperm is significant if these sperm can fertilize ova when the female next ovulates (Houck and Schwenk, '84). It is still un- known whether sperm can survive in the spermathecae of E. cirrigera from one breed- ing season to the next and remain capable of fertilizing ova. If so, the possibility of be-

tween-season sperm competition exists (Hal- Iiday and Verrell, '84).

ACKNOWLEDGMENTS

This is publication 2 from the Saint Mary's College Electron Microscopy Facility. Sup- port for this study came from National Sci- ence Foundation grant BSR 9024918. I thank Janet Libbing for help in preparation and sectioning of specimens, and Thomas Fogle for critically reading the manuscript.

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