Sperm Storage in Spermathecae of the Great Lamper Eel, Amphiuma tridacfyhm (Caudata: Amp h i u m idae)

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1 JOURNAL OF MORPHOLOGY 230:79-97 (1996) Sperm Storage in Spermathecae of the Great Lamper Eel, Amphiuma tridacfyhm (Caudata: Amp h i u m idae) DAVID M. SEVER, J. SEAN DOODY, COURTNEY A. REDDISH, MICHELLE M. WENNER, AND DON R. CHURCH Department of Biology, Saint Mary's College, Notre Dame, Indiana (D.M.S., C.A.R., M.M. W.); Department of Biological Sciences, Southeastern Louisiana University, Hammond, Louisiana (J.S.D., D.R.C.) ABSTRACT The spermathecae of ten female Amphiuma tridactylum were examined by light and electron microscopy during the presumed mating and ovipository seasons (March-August) in Louisiana. Spermathecae were simple tubuloalveolar glands in the dorsal wall of the cloaca. Six of the ten specimens were vitellogenic, and all of these specimens contained sperm in their spermathecae and had secretory activity in the spermathecal epithelium. Two nonvitellogenic females also had sperm in their spermathecae and active epithelial cells, whereas the other nonvitellogenic females lacked stored sperm and secretory activity in the spermathecae. In specimens storing sperm from March-May, the sperm were normal in cytology, and secretory vacuoles were contained within the epithelium. In the August sample, however, evidence of sperm degradation was present, and secretory material had been released into the lumen by an apocrine process. We therefore hypothesize that the spermathecal secretions function in sperm degeneration. Q 1996 Wiley-Liss, Inc. The Amphiumidae consists of three species of Amphiuma, elongate, fossorial salamanders with reduced limbs and one external gill slit, a paedomorphic character (Duellman and Trueb, '86). Two of the species are aquatic and often exceed 70 cm snoutvent length (SVL); A. means occurs in the coastal plain from Virginia to Louisiana, and A. tridactylum occurs from western Alabama to Texas and up the Mississippi River valley to western Kentucky. A. phloleter is a dwarf (20-25 cm SVL), terrestrial species limited to the Florida panhandle and southern Alabama (Conant and Collins, '91). In the Mississippi River valley, A. tridactylum is commonly called the "great lamper eel" (Parker, '37; Baker, '45; Dundee and Rossman, '89). A number of reports exist on the reproductive biology of Amphiuma, but most of these are anecdotal and some are contradictory (Table 1). The majority of the literature concerns A. tridactylum. Sperm in the female cloaca was first observed by Davison (1895). The only report of mating behavior is an observation of copulation by cloacal apposition (Baker et al., '47). Sperm storage glands, spermathecae, in the female cloaca were first described by Kreeger ('42), and Sever ('92a) extended her observations on spermathecae of A. tridactylum and described these glands in A. means and A. phloleter. In addition, Sever ('gla, '94) reported on the phylogeny of the spermathecae and other cloacal glands in the Amphiumidae. Kreeger ('42) stated that female A. tridactylum could store sperm in their spermathecae for 7 or 8 months, and Baker ('62) found that isolated females could retain living sperm for 15 months. In females of other salamanders, the longest period that Sever ('95) felt was reliable for duration of sperm storage was 6 months. The best way to ascertain the cytological condition of sperm during storage is by use of electron microscopy, but no ultrastructural studies of the spermathecae of Amphiuma have heretofore existed. We recently had the opportunity to cytologically examine the spermathecae of ten females sacrificed during the mating and ovipository period, and herein we present our findings on the ultrastructure of sperm storage in this species. These results are then compared with those on the ultrastructure of the spermathecae of other salamanders (for recent reviews see Sever, '95; Sever et al., '96). Address reprint requests to David M. Sever, Department of Biology, Saint Mary's College, Notre Dame, IN o 1996 WILEY-LISS, INC.

2 80 D.M. SEVER ET AL MATERIALS AND METHODS Great lamper eels were collected from roadside ditches at three sites, each within 10 km of Hammond, Tangipahoa Parish, Louisiana. We obtained specimens monthly from February-May We captured most individuals by hand but also used a dipnet, and a few individuals were caught in funnel traps baited with chicken necks. We concentrated collecting efforts at night after rain, when lamper eels were more active; many were obtained from ditches that were dry prior to rainfall. Because females become scarce in summer, presumably as a result of nesting (Cagle, '48; Fontenot, '901, we placed 22 presumed adult females captured between 9 and 13 May into two outdoor cattle tanks (2.0 m diameter, 0.7 m sidewall height) in Hammond, Louisiana. Mature females were considered those >33 cm total length (Fontenot, 'go), and females were identified by black pigmentation in the cloaca1 wall (Baker, '37). Cattle tanks were filled with water to a level of approximately 15 cm and contained mud from a ditch at one of the collecting sites. Eight plastic nesting boxes (25 x 50 x 20 cm) containing mud were placed at staggered heights in each tank, and some individuals entered the boxes. Plywood sheets were placed over the cattle tanks to prevent further filling due to rainfall. Tanks were examined for eggs every few days until 25 July when other circumstances dictated that the observations must end. Table 2 presents the data on the specimens from which spermathecae were examined by light and electron microscopy. Those lulled on 22 August were specimens that had been maintained in the cattle tanks and subsequently kept in a living stream (180 cm x 50 cm x 60 cm; Frigid Units, Inc., Toledo, Ohio) at Saint Mary's College until time of sacrifice. The number of yolky ovarian oocytes was counted in each female, and 11 such oocytes from each female were measured to the nearest 0.1 mm with a dial caliper under 5 x magnification. In addition to the females, single males were respectively collected (sacrificed) on 19 February (3 March), 6 March (14 March), 21 April (5 May), and 9-13 May (18 October). Specimens were killed by immersion in 10% MS-222, and snout-vent length (SVL) was measured from the tip of the snout to the posterior end of the vent. Tissues were excised from freshly killed specimens and fixed for preparation by paraffin infiltration for light microscopy (LM) or for embedding in epoxy resin for thin (LM) or ultrathin sections for transmission electron microscopy (TEM). Testes and vasa deferentia were removed from males, and cloacae were dissected from females. For all specimens, tissues were initially fixed in 10% neutral buffered formalin (NBF) (for LM) or in a 1:l solution of 2.5% glutaraldehyde in Millonig's phosphate buffer at ph 7.4 and 3.7% formaldehyde buffered to ph 7.2 with monobasic and dibasic phosphate (TEM). Carcasses of all specimens are stored in NBF in the research collections at Saint Mary's College. For paraffin infiltration prior to sectioning for LM, the tissue was rinsed in water after fixation, dehydrated in ethanol, cleared in Histosol (National Diagnostics, Inc., Manville, NJ), and embedded in paraffin. Sections (10 km) were cut with a rotary microtome, and a&ed to albuminized slides, and alternate slides were stained with hematoxylineosin (HE) (for general cytology) or alcian blue 8GX (AB) (for primarily carboxylated glycosaminoglycans) at ph 2.5 followed by the periodic acid-schiff method (PAS) (neutral carbohydrates and sialic acids). Procedures followed Kiernan ('90). After initial fixation, tissues prepared for plastic infiltration prior to sectioning for LM and TEM were trimmed into 1.5 mm blocks, rinsed in Millonig's buffer, postfixed in 2% osmium tetroxide, dehydrated in ethanol, cleared in propylene oxide, and embedded in an epoxy resin (EMBED-812; Electron Microscopy Sciences, Fort Washington, PA). Semithin sections (0.5-1 km) for LM were cut with glass knives, placed on microscope slides, and stained with toluidine blue. Ultrathin sections (70 nm) for TEM were collected on uncoated copper grids and stained with solutions of uranyl acetate and lead citrate. These sections were cut with RMC XLlOOO and RMC MT7 ultramicrotomes, and thin sections were viewed with a Hitachi H-300 transmission electron microscope. Terminology for sperm ultrastructure follows Picheral ('79). RESULTS Specimens maintained in cattle tanks During the time lamper eels were kept in cattle tanks, seven individuals died. One of the specimens retrieved from the cattle tanks later proved to be a male (and was sacrificed on 18 October so the testes and vasa deferentia could be prepared for histological examination). None of the females kept in the cattle

3 TABLE 1. Literature on the reproductive biology ofamphiuma Species State Spermiogenesis Vitellogenesis Mating Sperm storage Oviposition Nesting Hatching Reference Not specified October Midsummer McGregor (1889) Winter Sturdivant ('49) A. means AL Nov Ultsch and Arce- A. tridactylum AK LA LA LA LA LA LA LA neaiix. ~, ('88). NC FL FL FL sc October-May 15 months July July January-February July Brimley ('39) Brimley ('10) Weber ('44) Baker ('62) Hildebrand ('10) August-September Hay (1888) August-March NovemberJuly February-April July-September Wilson ('40) January-March Wilson ('41) March-December Kreeger ('42) January-May MayJune Nov Cagle ('48) October-May 15 months April-May Fall Baker ('62) December-February November-April February-winter Rose ('67) September-May June June-November November- Fontenot ('90) December TN May August- Davison (1895) September TN August-winter Parker ('37) TN July August August-September Baker ('37) TN July Baker et al. ('47)

4 82 TABLE 2. SDecimens used in this study' Collected Sacrificed SVL Sperm2 N Ovarian oocytes D.M. SEVER ET AL. Mean diameter SD 19Feb 3Mar Mar 14 Mar Mar 14Mar l0ap 21Ap Ap 24Ap Ap 5May Ap 5 May May 22Aug May 22Aug May 22Aug 'Measurements are in millimeters. 'Sperm present ( +) or sperm absent ( - 1 tanks oviposited eggs while in the tanks or when subsequently maintained in a living stream. Male reproductive cycle Since the male spermatogenic cycle has been described elsewhere in detail (Wilson, '40; Baker, '62; Rose, '67), we examined only males sacrificed in March, May, and October to verify the pattern. We found sperm in the vasa deferentia of all males, and sperm were relatively most numerous in vasa deferentia of specimens sacrificed in March. The testes of the specimen examined from October contained lobules filled with spermatids and spermatozoa, while testes of the specimens from March contained few sperm and those of the specimen sacrificed in May were totally evacuated of sperm. Female reproductive cycle Data on the presence of sperm and vitellogenesis (presence of enlarged yolky ovarian oocytes) are given in Table 2 for females sacrificed between 3 March and 22 August. Females containing vitellogenic follicles possessed sperm in their spermathecae, and oocytes in specimens sacrificed March-May were smaller than in those sacrificed in August (Table 2; Fig. 1A,B). Four females lacked vitellogenic follicles. Two of these females, one sacrificed 14 March (Fig. 1C) and the other sacrificed 22 August (Fig. ld), contained sperm in their spermathecae (Table 2). Gravid females sacrificed March-May Four females ( cm SVL) collected 19 February-21 April and sacrificed 3 March-5 May possessed yolky ovarian oocytes mm mean diameter and contained abundant sperm in their sper- mathecae, indicative of recent mating. AS noted by Sever ('92a), the spermathecae were numerous, simple, tubuloalveolar exocrine glands opening into the roof of the cloaca. Sperm were not present in all spermathecae observed in a given section and seemed most abundant in the widened lumina of the alveolar end of the glands (Fig. 2A). The epithelial cells were cuboidal to columnar, and the largely euchromatic nuclei were basal and oriented to the long axes of the cells. Round to oval secretory vacuoles ( km) were abundant in the apical cytoplasm, and intercellular canaliculi were narrow (Fig. 2B). Abundant vesicles, ribosomal complexes, microfilaments, and elongate mitochondria with tubular cristae were associated with secretory vacuoles in the perinuclear cytoplasm (Fig. 2C). Mature secretory vacuoles usually contained a circular, electron-dense area ( km) along one portion of the circumference and a less electron-dense (more flocculent) area that was often larger and more ovate (Fig. 20. Immature condensing vacuoles were composed entirely of the flocculent material, so the electron-dense portion may result from compaction of the flocculent material. Since alpha and beta particles of glycogen were absent, the secretory vacuoles were responsible for the PAS+ reaction in paraffin sections. The combination of ribosomal organelles responsible for peptide synthesis and a PAS+ reaction indicates that the product contained glycoproteins (Kiernan, '90). No evidence of release of the secretory product was found in specimens sacrificed March-May. Portions of sperm cells were sometimes found embedded in the apical cytoplasm of the spermathecal epithelium (Fig. 3A), but most sperm were observed in the lumen in small clusters in which sperm had a similar orientation (Fig. 3B,C). These small clusters were often adjacent to other groups of sperm with different orientations. The sperm appeared normal in cytology (Fig. 3B-D). Mitochondria were numerous around the axial rod of the principal piece of the tail and were characterized by lamellar cristae (Fig. 3D). Gravid females sacrificed in August Two females (59.0 and 68.8 cm SVL) collected between 9 and 13 May, maintained in a cattle-tank until 25 July, and subsequently kept in a living stream until sacrificed on 22 August contained yolky ovarian follicles mm mean diameter and had

5 SPERM STORAGE IN AMPHIUMA 83 Fig. 1. Female Amphiumu triductylum showing relative development of oocytes in specimens in which spermathecae contained sperm. A: Specimen 40.4 mm SVL collected 10 April and sacrificed 24 April with 93 yolky oocytes 4.0 mm mean diameter. B: Specimen 59.0 mm SVL collected between 9 and 13 May and sacrificed 22 August with 137 yolky oocytes 5.8 mm mean diameter. C: Specimen 59.6 mm SVL collected 6 March and sacrificed 14 March lacking enlarged yolky oocytes. D: Specimen 51.0 mm SVL collected between 9 and 13 May and sacrificed 22 August lacking enlarged yolky oocytes. Fb, fat bodies; In, intestines; Lg, lung; 00, enlarged yolky oocytes; Ov, ovary; So, stomach; Ub, urinary bladder.

6 84 D.M. SEVER ET AL Fig. 2. FemaleAmphiuma tridactylum, 40.4 mm SVL. Specimen collected 10 April and sacrificed 24 April with 93 yolky ovarian oocytes 4.0 mm mean diameter. A Light micrograph of plastic semithin section stained with toluidine blue showing overview of spermathecal tubules containing sperm and surrounding connective tissue. B: Electron micrograph showing supranuclear cytoplasm with numerous secretory vacuoles. C: Electron micro- graph showing detail of cytoplasm containing synthetic organelles and the relative density of secretory vacuoles. Cr, cristae; Cv, condensing vacuole; Dm, dense material; Fm, flocculent material; Ic, intercellular canaliculi; Lu, lumen; Mf, microfilaments; Mi, mitochondria; Nu, epithelial cell nucleus; Po, polyribosomes; Se, spermathecal epithelium; Spl, sperm in the lumen; Sv, secretory vacuoles; Tp, tunica propria; Ve, vesicles.

7 Fig. 3. Female Amphiuma tridactylum, electron micrographs. A Specimen 48.8 mm SVL collected 21 April and sacrificed 5 May with 152 yolky ovarian oocytes 4.7 mm mean diameter, showing sperm in contact with the apical spermathecal epithelium. B-D: Specimen 40.4 mm SVL collected 10 April and sacrificed 24April with 93 yolky ovarian oocytes 4.0 mm mean diameter, showing portions of sperm cells in lumen of a spermathecal tubule. Clusters of sperm have similar orientations. B: Shows mostly middle pieces of the tail. C: Illustrates primarily principal pieces of the tail in adjacent groups of sperm. D: Shows detail of the middle piece of the tail, and the inset depicts the lamellar cristae characteristic of mitochondria associated with the middle piece of the tail. Ac, apical cytoplasm of the spermathecal epithelium; Af, axial fiber; Ax, axonemal complex; Cr, cristae; Lu, lumen; Mi, mitochondria; Mpt, middle piece of the tail; Mt, marginal filament; Ppt, principal piece of the tail; Sn, sperm nucleus; Um, undulating membrane.

8 86 D.M. SEVER ET AL abundant sperm in their spermathecae. The apical epithelial cytoplasm contained numerous secretory vacuoles (Fig. 4A). Golgi bodies and ribosomal complexes, however, were no longer observed in the perinuclear cytoplasm (Fig. 4B). Secretory material was found in association with sperm in the lumen (Fig. 4C,D). This secretory material resembled the flocculent portion of the secretory vacuoles (Fig. 4C,D). Some sperm in contact with the epithelium seemed to be undergoing degradation as evidenced by loss of mitochondria around the axial rod (Fig. 1C). In the lumen, mitochondria around the axial fiber were invested with secretory material, and the axonema1 complexes of sperm surrounded by secretory material appeared abnormal (Fig. 4D). Nongravid females containing sperm One female (59.6 cm SVL) collected 6 March and sacrificed 14 March and another (51.0 cm SVL) collected between 9 and 13 May and sacrificed 22 August contained numerous sperm in their spermathecae but did not possess large yolky oocytes. The stored sperm and the spermathecal epithelium were similar in cytology to those of gravid specimens sacrificed during the same periods (Figs. 2-6). In the nongravid specimen collected 6 March and sacrificed 14 March, secretory vacuoles were numerous, and Golgi profiles and mitochondria were abundant in the perinuclear cytoplasm (Fig. 5A,B). Some sperm were embedded in the cytoplasm (Fig. 5A,D), but sperm in the lumen and adjacent to the luminaliepithelial border were normal in cytology (Fig. 5A,C). In the nongravid specimen containing sperm that was collected 5 May and sacrificed 22 August, sperm were still numerous in some luminal areas (Fig. 6A), but degradation of luminal sperm was evidenced by disruption of the mitochondria1 sheath of the axial rod (Fig. 6B). Secretory vacuoles were abundant in the apical cytoplasm of the spermathecal epithelium (Fig. 6A), but organelles (Golgi bodies, ribosomal complexes) involved in further synthetic activity were not observed. In several instances, sperm were observed embedded in epithelial cells, and these cells themselves sometimes appeared desquamated into the lumen. Both the epithelial cells, as indicated by vacuolated spaces, and the sperm contained within the epithelium appeared to be degenerating (Fig. 6C). The axial rods of the sperm were distorted and had lost electron density, and the mito- chondrial sheaths of the sperm were disrupted (Fig. 60. Release of secretory product into the lumen was apparent in the nongravid female sacrificed 22 August, as in gravid specimens sacrificed at the same time. Some cytological details associated with release of the secretory product, however, were especially apparent in sections from the nongravid female (Fig. 7). The release of secretory material apparently involved the loss of apical cytoplasm, making the mode apocrine (Fig. 7). Clusters of secretory granules, either free in the lumen or still surrounded by cell membranes, were observed in lumina (Fig. 7A). The flocculent portion of the secretory vacuoles dissociated from the more electrondense portion during apocrine release of the material (Fig. 7A,B). Apical areas of the spermathecal cytoplasm containing dissociated secretory vacuoles were separated from the nuclear regions of the cells by intracellular canaliculi that served as cleavage lines for the apocrine release of these portions of the cytoplasm (Fig. 7B). Nongravid females lacking sperm in the spermathecae Two females (53.0 and 56.2 cm SVL) sacrificed 21 April and 5 May lacked both large yolky ovarian oocytes and sperm in the spermathecae (Fig. 8A). Spermathecae of these specimens possessed heterochromatic nuclei and scant cytoplasm (Fig. 8B), and electrondense secretory vacuoles were absent from the apical cytoplasm. The most abundant organelles in the cytoplasm were mitochondria, although scattered vesicles and areas rich in microfilaments also were numerous (Fig. 8C). DISCUSSION Reproductive cycles A summary of the chronology of reproductive events in Amphiuma based upon the literature is presented in Table 1. In Louisiana, mating in A. tridactylum generally has been restricted to January-May, with most activity January-March when the male cloacal glands reach their maximal development (Wilson, '41; Cagle, '48). In Tennessee, however, mating has been ascribed to the summer months, and, indeed, the only actual observation of presumed copulation in Amphiuma was during the last 10 days of July (Baker et al., '47). Oviposition occurs in summer (Table I), but nests on land have been found from

9 SPERM STORAGE IN AMPHIUMA 87 Fig. 4. FemaleAmphzuma tridactyhm, 68.8 mm SVL, electron micrographs. Specimen collected between 9 and 13 May and sacrificed 22 August with 254 yolky ovarian follicles 6.0 mm mean diameter. A Overview of spermathecal epithelium. B: Detail of spermathecal cytoplasm. C: Sperm and secretory material in the lumen. D Detail of principal piece of the tail of sperm in the lumen in contact with secretory material. Aax, abnormal axo- neme; Ac, apical cytoplasm of the spermathecal epithelium; Af, axial fiber; Ax, axonemal complex; Lu, lumen; Me, myoepithelial cell nucleus; Mf, microfilaments; Mi, mitochondria; Mpt, middle piece of the tail; Mt, marginal filament; Nu, epithelial cell nucleus; Sm, secretory material; Spl, sperm in the lumen; Sv, secretory vacuoles; Um, undulating membrane; Vs, vacuolated spaces.

10 88 D.M. SEVER ET AL. Fig. 5. FemaleAmphiuma tridactylum, 59.6 mm SVL, electron micrographs. Specimen collected 6 March and sacrificed 14 March lacking enlarged yolky oocytes. A Overview of spermathecal epithelium and adjacent luminal areas. Areas similar to those shown in detail in B-D indicated by arrowheads. B: Supranuclear cytoplasm of the spermathecal epithelium. C: Sperm in lumen adja- cent to the spermathecal epithelium. D: Sperm nucleus embedded in the spermathecal epithelium. Ev, endocytic vacuole; Go, Golgi bodies; Ic, intercellular canaliculi; Lu, lumen; Mi, mitochondria; Mpt, middle piece of the tail; Mv, microvilli; Nu, epithelial cell nucleus; Ppt, principal piece of the tail; Sn, sperm nucleus; Sv, secretory vacuoles.

11 Fig. 6. FemaleAmphiuma triductylum, 51.0 mm SVL, electron micrographs. Specimen collected between 9 and 13 May and sacrificed 22 August lacking enlarged yolky oocytes. A: Apical spermathecal epithelium and adjacent lumen containing sperm. B: Detail of the middle piece of a sperm cell in the lumen. C: Degradation of sperm in spermathecal epithelium. Aaf, abnormal axial fiber; Af, axial fiber; Ax, axoneme; Dmi, disrupted mitochondria from axial fiber; Lu, lumen; Mi, mitochondria; Mt, marginal filament; Nu, epithelial cell nucleus; Se, spermathecal epithelium; Spl, sperm in the lumen; Sv, secretory vacuoles; Um, undulating membrane; Vs, vacuolated spaces.

12 90 D.M. SEVER ET AL. Figure I

13 June-February (Table 1). Baker ('45), however, notes that "every record of nests available indicates that water had previously covered the area where nests were found. It is therefore believed that Amphiumae burrow into the mud or crawl under a log or other protective object in the water shortly before depositing eggs.... The water may recede due to the lack of rain in the late summer or from drainage control and the nests and Amphiuma are... left in a dry area of a swamp.... The abundance of Amphiumae would indicate that many nests are formed each year but are normally hidden under logs or in the mud and remain below the water level." Baker ('45) believed that water may be necessary to initiate hatching (as in the ambystomatid Ambystoma opacum). Thus, in the absence of fall rains, eggs in nests stranded on land may carry over from the normal hatching time in November into later in the winter wet season in the southeastern United States, perhaps accounting for the reports of nests in midwinter (Parker, '37; Weber, '44). Due to the long period of vitellogenesis (September-May) and nesting period (June- November), A. tridactylum probably is unable to develop the follicles necessary for annual reproduction in Louisiana (Fontenot, '90). Only 35-48% of adult females examined by various authors were gravid in any given year, leading to predictions of biennial (Wilson, '42; Cagle, '48) or even triennial (Fontenot, '90) reproduction in females. Males, however, breed every year (Wilson, '42). Despite the restriction of mating to the spring (Louisiana) or summer (Tennessee), the capability for mating and fertilization exists over a longer period. For A. means from Florida and A. tridactylum from Louisiana, Baker ('62) noted that mature sperm Fig. 7. Female Amphiuma triductylum, 51.0 mm SVL, electron micrographs. Specimen collected between 9 and 13 May and sacrificed 22 August lacking enlarged yolky oocytes, showing apocrine mode of secretion. A Apical cytoplasm and adjacent lumen showing release of secretory vacuoles and secretory material. B: Evagination of apical cytoplasm of the spermathecal epithelium into the lumen. Acl, apical cytoplasm containing secretory vacuoles; C1, cleavage line; De, desmosome along an intercellular canaliculus; Dm, dense material; Dml, dense material free in the lumen; Eac, evagination of apical cytoplasm; Fm, flocculent material; Fml, flocculent material free in the lumen; Lu, lumen; Mf, microfilaments; Mv, microvilli; Nu, epithelial cell nucleus; Sv, secretory vacuoles; Tj, tight junction. SPERM STORAGE IN AMPHIUMA 91 can be obtained from the testes October- April and from the sperm ducts November- May; in addition, mature sperm can usually be found in the cloaca of a female at any time. Kreeger ('42) found that female A. tridactylum collected and isolated in March and April still possessed active sperm in their cloacae in April and December, respectively. Baker ('62) observed sperm in a female's cloaca 15 months after complete isolation from males. Such females never lay eggs in captivity, and, if eggs are present, they are probably resorbed (Baker, '62). Pituitary injections generally have been ineffective in inducing oviposition in Amphiuma (Baker, '37; Kammeraad, '42)' although one female A. tridactylum was induced to lay 49 eggs following intramuscular injection of bovine pituitary (Kammeraad, '42). Baker ('45) stated that 31% of those eggs were fertile, but this information is not in the original paper (Kammeraad, '42). Finally, we should note Dundee and Rossman's ('89) report that Percy Viosca, an accurate chronicler of natural history, wrote in his field notes that a female A. tridactylum collected April-May 1937 contained "two quarts of eggs with active embryos," which implies ovoviviparity. In the present study, males were sacrificed from October-May, and we found sperm in the vasa deferentia from males in each sample. The testes are filled with mature and maturing sperm in October and are largely evacuated by March, as found by Baker ('62). Four of the ten females used were not gravid; these females were considered mature since they were larger in SVL than some gravid females (Table 2). Even though our sample is small, the observation that 40% of our females were not gravid lends support to the notion of a biennial or triennial reproductive cycle (Fontenot, '90). The number of large, yolky oocytes in gravid females was , with the smallest oocytes in the female sacrificed 3 March (3.7 mm mean diameter) and the largest in females kept in the cattle tanks and sacrificed on 22 August ( mm mean diameter). All of the gravid females possessed sperm in their spermathecae. These results are consistent with reports of Wilson ('40) and Rose ('67) that vitellogenesis begins in late fall and winter and with findings by Wilson ('40, '4l), Cagle ('48), and Rose ('67) that mating occurs winter and spring. Two of the nongravid females contained sperm in their spermathecae. Either these

14 92 D.M. SEVER ET AL. Fig. 8. Female Amphiurna tridactylum, 53.0 mm SVL. Specimen collected 10 April and sacrificed 21 April lacking enlarged yolky ovarian oocytes and possessing inactive spermathecae lacking sperm. A: Light micrograph of plastic semithin section stained in toluidine blue showing overview uf spermathecal tubules and surrounding connective tissue. B: Electron micrograph showing overview of perinuclear cytoplasm of spermathecal epithelial cells. C: Electron micrograph showing detail of the cytoplasm of a spermathecal epithelial cell. Cr, cristae; Cv, condensing vacuole; Ic, intercellular canaliculi; Lu, lumen; Mf, microfilaments; Mi, mitochondria; Nu, epithelial cell ilildclib, Zer. rough endoplasmic reticulum; Se, spermathecal epithelium; Tp, tunica propria; Va, vacuoles.

15 SPERM STORAGE IN AMPHZUMA 93 individuals stored sperm from a previous breeding season when they were gravid, or mating had occurred in the current breeding season even though the females were not vitellogenic. These alternatives cannot be resolved based upon available data. However, no differences were observed in the cytology of the spermathecae and stored sperm between gravid and nongravid individuals. At least some of the sperm in the gravid and nongravid females sacrificed 22 August were degenerating, and further production of secretory vacuoles was not occurring. Sperm storage between breeding seasons has not been found in any salamander in which the annual cycle of sperm storage has been studied (Sever et al., '96). Sever et al. ('96) observed hypertrophy of the spermathecal epithelium in a nonvitellogenic female Notophthalmus viridescens during the breeding season similar to the extent found in gravid females. The coupling among vitellogenesis, hypertrophy of secondary sexual characters (including the spermathecae), and mating behavior in salamanders needs further study (Houck and Woodley, '95). Comparative cytology of the spermathecae The seven families of salamanders comprising the Salamandroidea are unique among vertebrates in the possession of a distinct set of male cloacal glands that make spermatophores and female cloacal glands that store sperm (Sever, '94). Sever and Brunette ('93) noted that two main structural types of spermathecae exist in salamanders. One type, called simple spermathecae, consists of numerous simple tubuloalveolar glands opening individually into the roof of the cloaca; simple spermathecae are found in all females in the suborder Salamandroidea except Plethodontidae. Plethodontids are characterized by complex spermathecae composed of compound alveolar glands. No structural or developmental reasons currently exist to consider simple spermathecae homologous among the families that possess them (Sever and Kloepfer, '93). The identification and analysis of characters associated with sperm storage in female salamanders may help resolve questions concerning the evolution of spermathecae and the phylogeny of salamanders. Duration of sperm storage in the spermathecae Cytologically and experimentally verified data are needed on the length of time sala- manders can store functional sperm in their spermathecae. Claims that salamanders store viable sperm for several years or between successive breeding seasons are largely anecdotal (Jordan, 1893; Baylis, '39; Adams, '40; Pool and Hoage, '73; Houck and Schwenk, '84; Massey, '90). On the other hand, much evidence exists for relatively short periods of sperm storage in certain salamanders. European newts (Triturus) start to lay eggs within a few days of mating (Halliday and Verrell, '84; Pecio, '92), and Wilbur ('77) reported that four species of Ambystoma (A. laterale, A. maculatum, A. tigrinum, and A. tremblayi) lay their eggs within 2 days after mating. Under laboratory conditions, the axolotl (A. mexicanum) starts laying eggs within a few hours after mating (Armstrong and Duhon, '89), and sperm can survive no more than 2 weeks in the spermathecae of the axolotl (Humphrey, '77). In studies containing critical analyses of the annual cycle of sperm storage, the longest periods are 6 months for the salamandrid Salamandrina terdigitata (Brizzi et al., '95) and the proteid Necturus beyeri (Sever and Bart, in press) and 3 or 4 months in plethodontids such as Desmognathus "fuscus" from Louisiana (Marynick, '71) and Eurycea quadridigitata from Alabama (Trauth, '83). Sever ('95) proposed that short-term sperm storage (2 days or less) as reported for some Ambystoma (Wilbur, '77; Armstrong and Duhon, '89) and Triturus (Halliday and Verrell, '84; Pecio, '92) should be regarded as more plesiomorphic than long-term sperm storage (several weeks or more) documented (as reported above) for some species. The total length of time over which Amphiuma tridactylum can store viable sperm was not determined in this study or in any previous study. The data indicate that the potential for long-term sperm storage is present, perhaps as long or longer than the 15 months suggested by Baker ('62). Some characters mentioned below are no doubt related to the duration of sperm storage. Staining reactions of the spermathecal epithelium Sever ('94) reviewed the literature on the reactions of spermathecal secretions to various stains at the light microscopy level and presented results for 71 species in which spermathecae were stained with carbohydrate stains. As in the current study, the procedures used were the periodic acid- Schiff (PAS), which stains neutral carbohy-

16 94 D.M. SEVER ET AL. drates (such as glucose, mannose, galactose) and sialic acids, and alcian blue (AB) at ph 2.5, which stains primarily carboxylated glycosaminoglycans (Kiernan, '90). In the Amphiumidae, Sever ('94) reported that the spermathecae ofa. tridactylum, as verified herein, was PAS+, but that of A. pholeter was both PAS + and AB +. In other species with simple spermathecae, the staining reactions were AB+ and PAS- in 25 of 28 species, including all 9 species of Ambystoma and 13 species of Salamandridae examined (Sever, '94). Thus, A. tridactylum may be relatively unusual among salamanders with simple spermathecae by having a PAS+ secretion. Closer examination of histochemical reactions, however, may reveal the presence of both neutral and acidic carbohydrates in the spermathecae of most if not all salamanders, although one type clearly may dominate. For example, Sever ('95) reported a PAS+ secretion in Ambystoma tigrinum, and Sever et al. ('96) found PAS+ secretions in the salamandrid Notophthalmus viridescens. The intensity of staining reactions to the dominant type of secretion may obscure reactions to other types of compounds, especially when using multiple stains on the same tissue sample. Secretory vacuoles of varying or uniform density In the few species whose spermathecal secretions have been studied by TEM, a dichotomy in the appearance of the secretory vacuoles is apparent. In the plethodontid Eurycea cirrigera (Sever, '91a) and in the salamandrid Notophthalmus viridescens (Dent, '70; Sever et al., '96), the secretory vacuoles are uniformly electron dense. In the ambystomatids Ambystoma opacum (Sever and Kloepfer, '93; Sever et al., '95) and A. tigrinum (Sever, '951, the proteid Necturus beyeri (Sever and Bart, in press), and the salamandrid Salamandrina terdigitata (Brizzi et al., '89, '951, the secretory vacuoles consist of an electron-dense particle surrounded (although not always centered) by a more electronlucent area called the "flocculent material" by Sever and Kloepfer ('93). This is the type of secretory vacuole we found in Amphiuma tridactylum. Whether the solid or the varying secretory vacuole is more plesiomorphic cannot be resolved with current data. The staining reaction of the vacuole seems independent of the density of the vacuole, since, for example, vacuoles of varying density in A. opacum are AB+ while those of A. tigrinum and N. beyeri are generally PAS+. In A. opacum, Sever and Kloepfer ('93) observed both the electron-dense particle and the flocculent material released into the lumen during sperm maintenance, but in A. tigrinum Sever ('95) reported that the flocculent material resulted from dissociation of the electron-dense particle, and only the vacuole containing the dissociated material was released (by exocytosis) into the lumen (during oviposition). In Amphiuma tridactylum, the fate of the electron-dense particle of the secretory vacuole is unknown, but perhaps it dissociates into the finer product, as suggested by Sever ('95) for the secretory product of Ambystoma tigrinum. Release of the secretory product in Amphiuma tridactylum is apocrine, which so far is unique among salamanders; the process has been described as merocrine in other species. Again, the phyletic significance of the secretory process cannot be determined. Functions of the spermathecal secretions In Ambystoma tigrinum, Sever ('95) found that the spermathecal secretions were not released concomitant with the appearance of sperm in the spermathecae but with the act of oviposition. He proposed, therefore, that the secretions serve to help flush sperm from the spermathecae as eggs pass through the cloaca (contraction of myoepithelial cells has also been implicated in this process [Hardy and Dent, '871). Sever ('95) believed that the flushing of sperm is an ancestral state for spermathecal secretions in which duration of sperm storage was short. Providing the conditions for maintenance of sperm could be a derived state to be searched for in species in which sperm are retained for extended periods. Maintenance could involve either nourishment (Benson, '68; Boisseau and Joly, '75) or providing the environment for sperm quiescence (Hardy and Dent, '86). In Amphiuma tridactylum, the secretory product was being produced during the mating season (March-May), but release of the product into the lumen was not observed at that time. Secretory material was found in the lumen only in those specimens sacrificed in August, and the product was often associated with degeneration of sperm. We did not observe the spermathecae of any females in a postovipository state. The gravid females sacrificed in August had presumably retained eggs through the normal ovipository period, and, thus, sperm storage in these individuals may not have been following the pattern

17 found in the natural environment. The nongravid specimens that contained sperm, however, also showed signs of release of the secretory material and degeneration of sperm in the lumen. The role of the secretory product in A. tridactylum still needs elucidation, but a hypothesis of a function in sperm degeneration is hereby tendered, which would be the first proposal of this function for spermathecal secretions. Occurrence of spermiophagy within the spermathecae Spermiophagy by the spermathecal epithelium was suggested in the newt Notophthalmus viridescens by Dent ('70) but conclusively demonstrated first in salamanders in the plethodontid Eurycea cirrigera by Sever ('91b, '92b) and Sever and Brunette ('93). Sever and Kloepfer ('93) then reported on spermiophagy by the spermathecal epithelium of Ambystoma opacum, and Brizzi et al. ('95) followed with a report of spermiophagy in the spermathecae of the salamandrid Salamandrina terdigitata. Finally, Sever et al. ('96) extended the observations of Dent ('70) and confirmed that spermiophagy does indeed occur in the spermathecae of N. uiridescens. Spermiophagy may be initiated early in the breeding season before oviposition begins but ultimately results in the elimination of excess sperm within 2-3 months following oviposition (Sever, '92b; Sever and Kloepfer, '93; Sever et al., '96). However, TEM studies of two salamanders, Ambystoma tigrinum (Sever, '95) and Necturus beyeri (Sever and Bart, in press), failed to find evidence of spermiophagy before or after oviposition, so the phenomenon may not be universal or may simply need more careful study. The evidence for spermiophagy in Amphiuma tridactylum is limited. Portions of sperm were occasionally seen embedded in the spermathecal epithelium in all seasons, but the only instance in which the sperm certainly were deteriorating was in a nongravid specimen sacrificed in August, in which abnormal appearing sperm was found in epithelial cells along the luminal border and in cells that seemingly had broken free into the lumen (Fig. 6C). Degradation of sperm in association with secretions into the lumen is more prevalent. Sperm ultrastructure The ultrastructure of the sperm stored in the spermathecae of A. tridactylum differed SPERM STORAGE IN AMPHIUMA 95 in some aspects from sperm observed in the testes, vasa deferentia, or spermathecae of other salamanders (cf. Picheral, '79; Sever, %la, '95). As opposed to other salamanders, the axial fiber in the middle piece of the tail in transverse section has two horseshoe-shaped ends connected by a slender filament, and all portions are ensheathed by mitochondria. In other species of salamanders, only one horseshoe-shaped end is present in this region. More work on sperm ultrastructure in salamanders is necessary to determine whether such variation has functional or phyletic significance. In summary, we have studied some aspects of sperm storage in female A. tridactylum, especially during the mating period, but much more work is needed before the process is fully understood in this enigmatic species. Efforts to induce lamper eels to mate under laboratory conditions have been totally unsuccessful, and oviposition has been observed only once (Baker, '37; Kammeraad, '42). Thus, we still lack any data on the maximal duration of time between last mating and the oviposition of a fertile clutch of eggs. Although the species has proven to be a frustrating one on which to conduct research on reproductive biology, additional data on sperm storage, especially after fertilization of a clutch, would be most worthwhile, as it would help address some of the issues raised in this paper concerning the functional and phylogenetic significance of sperm storage in salamanders. ACKNOWLEDGMENTS This work received support from National Science Foundation grant DEB to D.M.S., although latter stages of the research lacked support from NSF. This paper is publication 10 from the Saint Mary's College Electron Microscopy Facility. LITERATURE CITED Adams, D.E. (1940) Sexual conditions in Triturus uirzdewens 111. The reproductive cycle of' the adult aquatic form of both sexes. Am. J. hat. 66: Armstrong, J.B., and S.T. Duhon (1989) Induced spawnings, artificial insemination, and other genetic manipulations. In J.M. Armstrong and G.M. Malacinski (eds): Developmental Biology of the Axolotl. London: Oxford University Press, pp Baker, C.L. (1945) The natural history and morphology ofamphiuma. Rep. Reelfoot Lake Biol. Sta Baker, C.L. (1962) Spermatozoa of Amphiumae: Spermateleosis, helical motility and reversibility. J. Tenn. Acad. Sci. 37: Baker, C.L., L.C. Baker, and M.F. Caldwell(1947) Observation of copulation in Amphiuma tridactylum. J. Tenn. Acad. Sci. 22:87-88.

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