Sperm Aggregations in the Spermatheca of FemaleDesmognathine Salamanders (Amphibia: Urodela:Plethodontidae)

DAVID M. SEVER1* AND WILLIAM C. HAMLETT2

1Department of Biology, Saint Mary’s College, Notre Dame, Indiana2Indiana University School of Medicine, South Bend Center for MedicalEducation, Notre Dame, Indiana

ABSTRACT The alignment of sperm in a cloacal sperm storage gland, thespermatheca, was studied in female desmognathine salamanders by scanningand transmission electron microscopy. Females representing nine species andcollected in spring, late summer, and fall in the southern Appalachian Moun-tains contained abundant sperm in their spermathecae. The spermatheca is acompound tubuloalveolar gland connected by a single common tube to themiddorsal wall of the cloaca. Sperm enter the common tube in small groupsaligned in parallel along their axes, and continue in a straight course untilencountering divisions of the common tube (neck tubules) or luminal bordersof distal bulbs, which can act as a barriers. Sperm may form tangles, in whichsmall clusters retain their mutual alignment, at the branches of the necktubules from the common tube, or in the lumen of the distal bulbs, wheresubsequent waves of sperm collide with sperm already present. The nuclei ofsome sperm from the initial group to encounter the walls of the distal bulbsappear to become embedded in secretory material on the luminal border or inthe apical cytoplasm of the spermathecal epithelial cells. We propose thatthese sperm become trapped in the spermatheca and are ultimately degraded.J. Morphol. 238:143–155, 1998. r 1998 Wiley-Liss, Inc.

KEY WORDS: Amphibia; Desmognathus; ultrastructure; spermatheca; sperm

The seven families of salamanders com-prising the suborder Salamandroidea arecharacterized by the presence in the cloacaeof females of one or more exocrine glands,respectively, the spermatheca or spermathe-cae, that store sperm (Sever, ’91). Duringmating, males produce a spermatophore, thecap of which contains sperm and becomeslodged in the female cloacal orifice (Gasco,1881; Sever and Houck, ’85) (Fig. 1A). Spermmigrate from the spermatophore cap intothe spermathecae where they are stored inthe spermathecal lumina before oviposition(Hardy and Dent, ’86a; Sever and Brizzi,’98). As eggs pass through the cloaca, spermare released from the spermathecae onto theeggs, providing the conditions for internalfertilization (Jordan, 1893; Boisseau andJoly, ’75).

A number of studies exist on the ultra-structure of the annual cycle of sperm stor-age in species representing five of the sevenfamilies containing spermathecae, and data

from these observations were recently syn-thesized by Sever and Brizzi (’98). One as-pect of sperm storage, however, that has notreceived much attention is the migration ofsperm from the spermatophore into the sper-mathecae and the subsequent arrangementof sperm during storage.

In the newts Notophthalmus viridescensand Triturus vulgaris in the family Salaman-dridae, sperm migrate from the spermato-phore cap into the spermathecae within 1 hrafter mating (Hardy and Dent, ’86a; Sever etal., ’98), and this movement has been attrib-uted to an undirected, thigmotactic response(Hardy and Dent, ’86a). Females in the Sala-mandridae, like those in five of the remain-ing six families in the Salamandroidea, pos-sess simple spermathecae, consisting of

*Correspondence to: David M. Sever, Department of Biology,Saint Mary’s College, Notre Dame, IN 46556.E-mail: dsever@saintmarys.edu

JOURNAL OF MORPHOLOGY 238:143–155 (1998)

r 1998 WILEY-LISS, INC.

Fig. 1. Spermatheca of female desmognathine sala-manders. A: Transverse paraffin section showing a sper-matophore cap in the cloacal orifice of a 45-mm SVLDesmognathus ochrophaeus stained with the periodicacid–Schiff procedure and counterstained with fast greenFCF. (After Sever and Houck, ’85.) B: Three dimensionalreconstruction of the cloacal walls and spermatheca inD. brimleyorum. (After Sever and Trauth, ’90.) A rightlateral view rotated 25° clockwise. ‘‘A’’ indicates theanalogous location of the transverse section shown in

Fig. 1A. C: Transverse paraffin section through thecloaca of a 49-mm SVL D. fuscus, stained with hematoxy-lin-eosin. (After Sever and Trauth, ’90.) D: Transverseparaffin section through the common tube and necktubules of a 71- mm SVL D. marmoratus stained withhematoxylin & eosin. (After Sever and Trauth, ’90.) Cc,cloacal chamber; Cmt, common tube; Co, cloacal orifice;Ct, cloacal tube; Db, distal bulbs; Nt, neck tubules; Sc,spermatophore cap; Sk, skeletal muscle; Tp, tunica pro-pria.

numerous simple, tubuloalveolar glandsregularly spaced in the dorsal and lateralwalls of the anterior end of the cloaca (Severand Klopefer, ’93; Sever, ’94a; Sever andBrizzi, ’98).

Sperm typically occur as tangled massesin the spermathecae of Notophthalmus viri-descens (Dent ,’70; Sever et al.,’96a) andTriturus vulgaris (Sever et al., ’98). In somesalamanders in which simple spermathecaeare found, however, luminal areas wheresperm are arranged in parallel arrays show-ing the same orientation have been noted(Brizzi et al, ’95; Sever and Klopefer, ’93;Sever et al., ’96b).

In one family of the Salamandroidea,Plethodontidae, the spermathecae consist ofa single compound tubuloalveolar gland(Sever, ’94a,b; Sever and Brizzi, ’98). Distalbulbs connect to narrower neck tubules thatconverge into a common duct that opens intothe middorsal roof of the cloaca (Fig. 1B–D).In female plethodontids in which sperm stor-age has been studied, areas of the spermathe-cal lumina invariably have been noted inwhich clusters of sperm are aligned alongtheir entire lengths in parallel arrays (Mary-nick, ’71; Pool and Hoage, ’73; Davitt andLarsen, ’88; Sever and Brunette, ’93; Sever,’97), although in the distal bulbs of Euryceacirrigera, where spermiophagy is extensive,sperm possess no patterned arrangement(Sever and Brunette, ’93).

The Plethodontidae is the largest familyof salamanders, containing some 350 speciesand two subfamilies (Duellman and Trueb,

’86). The subfamily Desmognathinae con-sists of some 18 species of Desmognathusand monotypic Phaeognathus hubrichti(Tilley and Mahoney, ’96; Titus and Larson,’96), whereas the subfamily Plethodontinaeand its three tribes (Plethodontinae, Bolito-glossini, and Hemidactyliini) contain the re-maining taxa (Wake, ’66). The Desmog-nathinae usually are considered closer tothe ancestral plethodontid morphology thanthe Plethodontinae (Lombard and Wake, ’86).

While conducting a survey on spermathe-cal cytology in the Desmognathinae, observa-tions were made on the ultrastructure ofstored sperm. In this paper, sperm aggrega-tions in several species of desmognathinesare illustrated by scanning and transmis-sion electron microscopy, and the possiblesignificance of the sperm clusters in regardto sperm movements and sperm competitionare considered.

MATERIALS AND METHODS

The species used, collection data, and re-productive conditions are listed in Table 1.The sample contains species representingeach of the four major adaptive zones occu-pied by desmognathines (Titus and Larson,’96), and the species examined vary from theminiature, fully terrestrial species, Desmog-nathus wrighti, to robust, highly aquaticD. marmoratus (Conant and Collins, ’91).Desmognathus conanti and D. monticola col-lected 11 April 1997 came from Spivey Cove,Monroe County, Tennessee (elevation 750 m).Desmognathus marmoratus and D. monti-

TABLE 1. Specimens of Desmognathus used in this study1

Adaptive zone/species Locality Date SVL

Ovarian follicles

Sperm2N Dia

AquaticD. marmoratus Graham Co, NC 20/X/97 65.6 23 2.6 0

Stream sideD. monticola Monroe Co, TN 11/IV/97 63.0 34 3.1 1

Graham Co, NC 20/X/97 62.0 33 3.0 1Graham Co, NC 20/X/97 62.9 47 2.9 0

SeepD. conanti Monroe Co, TN 11/IV/97 46.0 15 2.6 1

Monroe Co, TN 11/IV/97 51.3 30 2.3 1D. ocoee Graham Co, NC 11/IV/97 40.8 17 2.3 1

Graham Co, NC 11/IV/97 43.2 27 2.2 1Macon Co, NC 21/X/97 42.0 16 2.2 0Macon Co, NC 21/X/97 42.8 20 2.2 1

D. santeetlah Graham Co, NC 20/X/97 43.8 23 1.8 1Forest floor

D. wrighti Sevier Co, TN 3/VIII/96 28.3 11 1.8 1

1All measurements, in mm.20, absent; 1, present.

SALAMANDER SPERM STORAGE 145

cola collected 20 October 1997 came fromSanteetlah Creek, Graham County, NorthCarolina (elevation 580 m). Desmognathusocoee collected 11April 1997 came from Hoop-er’s Bald, Graham County, North Carolina(elevation 1,600 m), whereas those collected21 October 1997 came from Wayah Bald,Macon County, North Carolina (elevation1,560 m). Desmognathus santeetlah camefrom Straton Meadows, Graham County,North Carolina (elevation 1,500 m), andD. wrighti came from Mt. Le Conte, SevierCounty, North Carolina (elevation 1,900 m).Collecting permits were issued by the Na-tional Park Service for the Great SmokyMountains and by the North Carolina Wild-life Resources Commission (license 110).

Specimens were killed by immersion in5% benzocaine, and snout-vent length (SVL)was measured from the tip of the snout tothe posterior end of the vent. The cloacalorifice was bisected, and the spermathecawas removed and fixed for either transmis-sion electron microscopy (TEM)(10 speci-mens representing all species) or scanningelectron microscopy (SEM)(2 specimens ofDesmognathus ocoee). Carcasses of all speci-mens were preserved in 10% neutral buff-ered formalin (NBF) and are housed in theresearch collections at Saint Mary’s College.

The spermathecae were fixed in a 1:1 solu-tion of 2.5% glutaraldehyde in Millonig’sphosphate buffer at pH 7.4 and 3.7% formal-dehyde buffered to pH 7.2 with monobasicand dibasic phosphate. For TEM, the tissueswere trimmed into 1-mm blocks. After initialfixation, tissues were rinsed in Millonig’sbuffer, postfixed in 2% osmium tetroxide,and dehydrated through a graded series ofethanol.

For SEM, the tissue was then subjected tocritical point drying, mounted on a metalstub with adhesive tape, and sputter-coatedwith gold in a Denton Desk II. The speci-mens were examined with a JEOL JSM-T300scanning electron microscope. For TEM, af-ter dehydration, tissues were immersed inincreasing concentrations of Epon epoxyresin in absolute ethanol before polymeriza-tion in pure Epon for 12 hr at 60°C. Plasticsections were cut with an RMC MT7 ultrami-crotome and Diatome diamond knives. Semi-thin sections (0.5–1 mm) for light micros-copy were placed on microscope slides andstained with toluidine blue. Ultra-thin sec-tions (70 nm) for TEM were collected onuncoated copper grids and stained with solu-

tions of uranyl acetate and lead citrate. Ul-tra-thin sections were viewed with a HitachiH-300 transmission electron microscope. Ter-minology for sperm ultrastructure followsPicheral (’79).

Ovaries and oviducts were removed fromthe carcasses, and the number of vitello-genic follicles of similar size was counted. Atleast 11 follicles (the minimum observed)from the ovaries of each specimen were mea-sured to the nearest 0.01 mm with an ocularmicrometer in a dissecting microscope (Ta-ble 1).

RESULTSReproductive condition

Specimens were examined from threemonths, April, August, and October, and allspecimens contained large vitellogenic ovar-ian follicles (Table 1). Only three specimenslacked sperm in their spermathecae, andthese specimens represented three differentspecies collected in October (Table 1).

Ultrastructure of spermand the spermatheca

The ultrastructure of the spermatheca andthe relationship of stored sperm to the lin-ings of the gland were the same for all spe-cies. Where sperm were crowded, the spermwere in arrays featuring differing orienta-tions, and where sperm were not crowded,they were arranged in parallel arrays.

Figure 2A shows seemingly disorderedclusters of sperm in the common tube at thejunction with the narrow neck tubules (cov-ered by the sperm mass). Closer examina-tion, however, reveals that smaller groups ofsperm within the larger cluster exhibit simi-lar orientations (Fig. 2B). In areas of thedistal bulb were sperm are not crowded, thesperm cells align themselves in parallel ar-rays (Fig. 3A) with the sperm nuclei oftenadjacent to, or embedded in, secretory mate-rial coating the luminal border of the sper-mathecal epithelium (Figs. 3B, 4) or actuallyembedded in the epithelial cell cytoplasmitself (Esn, Fig. 4C). The secretory material

Fig. 2. Scanning electron micrographs of the sper-matheca of a 43.2 mm SVL Desmognathus ocoee col-lected 11 April 1997. A: Overview of distal bulbs withsperm masses in the common tube. B: Detail of spermmass. Although tangled, groups of sperm possess thesame axial alignment. Db, distal bulbs; Sp Cmt, spermin the common tube; Sp Db, sperm in the distal bulb; Tp,tunica propria.

146 D.M. SEVER AND W.C. HAMLETT

Figure 2

SALAMANDER SPERM STORAGE 147

Figure 3

148 D.M. SEVER AND W.C. HAMLETT

apparently arises from coalescence of secre-tory vacuoles in the apical cytoplasm, whichthen cleaves off the luminal border in anapocrine fashion (Fig. 4C).

After escaping from clusters that form atthe junction of the common tube with necktubules, sperm pass in parallel groupsthrough the neck tubules (Fig. 5B) and keepmoving until the forward-most cells hit theluminal border of the spermathecal epithe-lium with its layer of secretory material(Fig. 5B). This results in some sperm becom-ing embedded in the secretory material orthe epithelium, and causes the tails of thesperm to be curved (Fig. 6C). Sperm thatfollow the initial groups into the distal bulbshave their forward progress halted by thesperm already present, so that an overallpattern of disorder is present, but smallgroups of luminal sperm exhibit the sameorientation (Figs. 6A,B, 7).

In one specimen of Desmognathus co-nanti, two size classes of sperm were appar-ent in the distal bulbs (Fig. 6B), perhapsrepresenting ejaculates from different malesof the same or a closely related species. Mini-mal variation in sperm size occurs in thesperm from a single male, but intraspecificvariation in sperm length among desmog-nathines can be as much as 40 mm, andinterspecific variation can exceed 100 mm(Wortham et al., ’77). The groups of sperm ofdifferent sizes are separate.

DISCUSSION

Much past work on movements of meta-zoan sperm was reviewed by Miller (’85).Basically, he concluded that vertebratesperm do not possess the sensory and locomo-tory capabilities required for as complex abehavior as following a chemical gradient,and that sperm, although undirected, tendto move in a straight line until hitting abarrier. Recent work on mammals also failedto find support for sperm chemotaxis, al-though evidence for chemokinesis, an alter-ation in speed of travel due to extrinsic fac-tors, is well established (Mortimer, ’95).

Hardy and Dent (’86a) studied transport ofsperm in the newt, Notophthalmus viride-scens, which has numerous simple tubularspermathecae in the roof of the cloaca, ratherthan a compound tubuloalveolar gland asfound in Desmognathus and other plethodon-tids (Sever, ’94b). Hardy and Dent (’86a)proposed that sperm from a spermatophorecap move thigmotactically along the cloacalepithelium, and, upon encountering theopenings of the tubules, are carried throughthose openings by a continuation of the thig-motactic response. The openings to the sper-mathecae of N. viridescens are so minute(4 mm) and the sperm so elongate (590 mm)that Hardy and Dent (’86a) proposed thatsperm enter these tubules singly.

Our observations support thigmotaxis asa sufficient explanation for passage of sperminto the spermatheca and divisions of thegland in desmognathines. The common tubein desmognathines, however, is certainlylarger than the diameter of an individualsperm cell (Figs. 1, 2), so we further proposethat sperm enter the common tube in smallclusters in parallel arrays.

Sperm then may form what appear to betangled masses because their movement be-comes restricted, as at the branches of theneck tubules or by the walls of the distalbulbs. But close examination of these‘‘tangles’’ always reveals smaller groups ofsperm aligned along their long axes.

The results of collision of groups of spermfrom different males are unpredictable butcould include displacement of sperm, inter-mixing of the ejaculates, or pushing the ini-tial ejaculate deeper into the epithelium orsecretory matrix. These phenomena haveconsequences for sperm competition, thecompetition between the ejaculates of differ-ent males to fertilize the ova of a female(Houck and Schwenk, ’84; Halliday, ’98). In-teractions among sperm from multiple mat-ings are probably most intense in the distalbulbs, whose walls ultimately hinder spermmovements and influence the interactionslisted above. If movement into the distalbulbs becomes restricted, the more proximalportions of the spermatheca should be packedwith sperm from the most recent mating. Asa working hypothesis, therefore, we proposethat last male paternity would be favored inplethodontids. However, in the only studythat related genotypes of offspring to thesequence of matings by different males of aplethodontid (Desmognathus ochrophaeus),

Fig. 3. Scanning electron micrographs of the sper-matheca of a 43.2-mm SVL Desmognathus ocoee col-lected April 11,1997 showing sperm clusters in a distalbulb (A) and proximal ends of two bundles of sperm (B)associated with secretory material. Db, distal bulb; Lu,lumen; Rb, red-blood cell; Sm, secretory material; Sn,sperm nucleus; Sp, sperm; Tp, tunica propria.

SALAMANDER SPERM STORAGE 149

Fig. 4. Sperm embedded in secretory material andthe spermathecal epithelium (Esn). A: Same specimenof Desmognathus ocoee used for Figs. 2 and 3, showing ascanning electron micrograph of sperm nuclei embeddedin secretory material. B, C: Transmission electron-micrographs of the luminal border of the spermatheca in

a 46.0-mm SVL Desmognathus conanti collected April11, 1997. Unlabeled arrows, apocrine sloughing of secre-tory material. Ac, apical cytoplasm; Esn, embeddedsperm nucleus; Ic, intercellular canaliculi; Lu, lumen;Mpt, middle piece of the tail; Sm, secretory material; Sn,sperm nuclei; Sv, secretory vacuoles.

150 D.M. SEVER AND W.C. HAMLETT

Fig. 5. Transmission electron-micrographs showingpatterns of sperm alignment in a neck tubule (A) anddistal bulb (B) of a 63-mm SVL Desmognathus monti-cola collected April 11, 1997. Ac, apical cytoplasm; Esn,

embedded sperm nuclei; Lu, lumen; Mpt, middle piece ofthe tail; Nu, nucleus of spermathecal epithelial cell; Sm,secretory material; Sn, sperm nuclei; Tp, tunica propria.

SALAMANDER SPERM STORAGE 151

Fig. 6. Electron micrographs showing alignment ofluminal sperm in distal bulbs of female desmognathinesalamanders. A: Desmognathus wrighti, 28-mm SVL,collected August 3, 1996. B: Desmognathus conanti, 51.3mm SVL, collected April 11, 1997. Note what appears to

be two size classes of sperm (Ls, Ss) on the right and leftsides of the micrograph. Ac, apical cytoplasm; Lu, lu-men; Mpt, middle piece of the tail; Nu, nucleus of sper-mathecal epithelial cell; Sm, secretory material; Sn,sperm nuclei.

Fig. 7. Sperm alignment in a portion of the sper-mathecal lumen of a Desmognathus ocoee (40.8-mm SVL)collected April 11, 1997. Except for a few sections in the

upper right corner through the principal piece of the tail(unlabeled arrows), all sections are through the sameportion of the middle piece of the tail.

SALAMANDER SPERM STORAGE 153

no last male advantage was found (Houck etal., ’85).

We observed, as reported previously nu-merous times in the literature (see Severand Brizzi, ’98, for review), that the nuclei ofsome sperm are embedded in secretory mate-rial or in the apical cytoplasm of the epithe-lium. We suggest that these embedded spermwere the initial ones to enter a blind-endedtubule and thus be halted after striking thebarrier presented by the epithelium. Sever(’94b) reported that the secretory materialin most desmognathines stains positively forboth neutral carbohydrates (positive follow-ing the periodic-acid Schiff procedure) andsulfated or carboxylated glycosaminogly-cans (positive in Alcian blue 8GX at pH 2.5).Although some investigators have suggesteda ‘‘nutritive’’ function for these secretions(Benson, ’68; Boisseau and Joly, ’75), a mech-anism by which such nutrition could occurhas not been described. Hardy and Dent(’86b) found that sperm were immobile dur-ing storage; inactive sperm do not requiremetabolic fuel. Instead, Hardy and Dent(’86b) and Sever and Klopefer (’93) proposedthat the secretions provide the sperm withthe chemical/osmotic environment for quies-cence.

All detailed studies on the annual cycle ofsperm storage in the spermathecae of sala-manders report some sperm left in the sper-mathecae after oviposition (Sever and Brizzi,’98). All these studies also report degenera-tion of sperm remaining in the lumen, and ina number of species, the spermathecal epi-thelium is spermiophagic (Sever, ’92, ’97;Sever and Kloepfer, ’93; Brizzi et al., ’95;Sever et al., ’96a). We hypothesize that spermthat become embedded in the secretory ma-trix or in the spermathecal epithelium itselfdo not escape, but eventually undergo degra-dation in the spermatheca.

In mammals, however, Suarez (’98) pro-posed that sperm in isthmic reservoirs bindto carbohydrate moieties on glycoproteinson the surface of the oviducal epithelium vialectin-like molecules. During the period ofadherence, sperm viability is maintained(Smith, ’98). The lectins are believed to belost or modified during capacitation, allow-ing sperm to release (Suarez, ’98). This pro-cess has not been experimentally verified inmammals, and, as indicated above, we be-lieve it is unlikely that embedded sperm inthe spermatheca of salamanders await ca-pacitation. Certainly, however, a search for

lectins or other adhesive molecules on thesurface of salamander sperm could enhanceour understanding of sperm–epithelial inter-actions.

Obviously, much more study is needed totest hypotheses posed in this paper and toaddress other issues, such as the movementof sperm out of the spermathecal tubulesduring oviposition (Hardy and Dent, ’87).Desmognathine salamanders are numerous,easily to collect and maintain, and, as shownin Table 1, possess an extended reproductiveseason. Thus, desmognathines are excellentmodel organisms on which to extend observa-tions of sperm storage in complex spermathe-cae.

ACKNOWLEDGMENTS

We thank W. Archer for his assistancewith scanning electron microscopy. We thankR. Hartzell for her aid with transmissionelectron microscopy, especially specimenpreparation for samples collected in April1997. This is publication 14 from the SaintMary’s College Electron Microscopy Facility.

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