annual cycle of sperm storage in spermathecae of the red

JOURNAL OF MORPHOLOGY 227:155-170 (1996)

Annual Cycle of Sperm Storage in Spermathecae of the Red-Spotted Newt, Notophthalmus viridescens (Amphibia: S al a m a n d r i d ae)

DAVID M. SEVER, LISA C. M I A , AND JOHN D. KRENZ Department of Biology, Saint Mary's College, Notre Dame, Indiana 46556 (D.M.S., L. C.R.); Savannah River Ecology Laboratory, Drawer E, Aiken, South Carolina 29802 (L.C.R., J.D.K.); Institute of Ecology, University of Georgia, Athens, Georgia 30602 (J.D.K.)

ABSTRACT Female sperm storage was studied in a population of Notophthal- mus viridescens from South Carolina. Spermathecae initiate production of a glycoprotein secretory product in October. At this time ovarian follicles are immature (0.5-0.9 mm dia), and mating does not occur despite spermiation in males. Six of the 10 females collected in December had sperm in their spermathecae, indicating onset of mating. Unmated females collected in October and sacrificed in February and March possessed mature ovarian follicles (1.3-1.4 mm dia), and the spermathecae contained large secretory vacuoles 2-3 km dia. Release of secretory product is concomitant with the appearance of sperm in the spermathecae. Thus mated females lack secretory vacuoles in the spermathecal epithelium, and additional synthesis of secretory product does not occur. All females collected in February and March have mated. Sperm are embedded in the spermathecal epithelium and are undergoing degradation in February. Degradation of sperm in the lumen and epithelium is evident in specimens examined from May and June. Atresia of ovarian follicles begins in April in captive specimens, and specimens captured from the bay in May are spent. A general postbreeding emigration from the pond occurs in summer. Fourteen females collected 7 March were injected with human chorionic gonadotropin (hCG) on 9 March and laid fertile eggs 10-18 March. Two of these females were sacrificed each month from April-September; all retained some sperm in their spermathecae, but further oviposition did not occur. Four females were kept 1 year after oviposition of fertile eggs, and oviposition again was induced with hCG; these eggs were infertile, and spermathecae lacked sperm. Spermathecae are inactive from June-September in captive and wild-caught specimens. Sperm may be stored effectively up to 6 months (December-May), and no evidence was found for retention of viable sperm from one breeding season to the next. Q 1996 Wiley-Liss, Inc.

The red-spotted newt, Notophthalmus viridescens Rafinesque, is widespread in eastern North America (Mecham, '67). Like other salamanders in the seven families compris- ing the suborder Salamandroidea, males pro- duce a spermatophore that becomes lodged in the female's cloaca during courtship (Verrell, '82). Sperm from the spermatophore cap migrate into exocrine glands, spermathecae, in the cloaca of the female (Hardy and Dent, '86). These glands are simple tubuloalveolar glands in all families except Plethodontidae, in which spermathecae are compound alveo-

lar glands (Sever, '94). During oviposition, eggs are fertilized internally during their pas- sage through the cloaca by sperm released from the spermathecae (Jordan, 1893; Bois- seau and Joly, '75).

Dent ('70) used transmission electron microscopy to examine the spermathecae of immature N. uiridescens and those of mated and unmated mature females from various populations in central Virginia, middle Ten-

Address reprint requests to David M. Sever, Department of Biology, Saint Mary's College, Notre Dame, IN 46556.

o 1996 WILEY-LISS, INC.

156 D.M. SEVER ET AL

nessee, and western Massachusetts. How- ever, he did not follow the annual cycles of spermathecal cytology and sperm storage in any single population. These annual cycles are of interest due to the considerable amount of intraspecific variation that has been reported in the reproductive biology of N. viridescens.

For example, in a population from Massa- chusetts, Adams ('40) found sperm in the spermathecae of females examined in every month of the year, although sperm were especially abundant in the fall and spring. Mat- ings in Adam's population occurred in fall during a "false breeding season" (Gage, 1891; Jordan, 1893; Pope, '24) as well as spring; males had sperm in their vasa deferentia from September through May, and some males possessed sperm during the midsummer months (Adams, '40). Thus, Adams found a prolonged mating season, which could account for the presence of sperm in spermathecae throughout much of the year.

In contrast, Massey ('88, '90) reported matings from mid-March to late June in newts from the mountains of Virginia, and no fall breeding season existed. An August-Septem- ber emigration to terrestrial hibernacula occurs, and adults do not migrate back to the ponds until the following March-April. A year after breeding, eight marked females were recaptured in pit traps around a partially fenced pond (Massey, '90). Seven of the females had sperm in their spermathecae, although they were not abundant, indicating no recent insemination. Massey ('90) con-

cluded that the sperm were retained from a previous breeding season, and these females had stored sperm for at least 10 months. Thus, Massey ('90) found a relatively short mating period, but a prolonged period of sperm storage.

In this report, we present the first cytologi- cal observations on the annual cycle of sperm storage and spermathecal secretions in a single population of Notophthalmus viridescens. We extend the observations of Dent ('70) and compare our findings to those on the cytology of spermathecae in other salamanders (Pool and Hoage, '73; Boisseau and Joly, '75; Brizzi et al., '89, '95; Sever, '91a, '92a, '94, '95; Sever and Brunette, '93; Sever and Kloepfer, '93; Sever et al., '95).

MATERIALS AND METHODS

All newts were collected from Castor Bay (Wood Duck Bay), a 3.5 ha Carolina bay on the Department of Energy's Savannah River Site (Aiken County, SC). The bay held water throughout the period of the study. Collec- tions were made on 24 October 1993, and 18 February, 10 March, 17 May, 20 June, 27 June, 2 August, and 3 December 1994 (Tables 1-3). Some specimens were sacrificed within 2 days after capture, whereas others were maintained in the lab for various periods of time prior to sacrifice, as described below.

Females from the October and March collections were separated from males based upon their swollen bodies, indicating presence of enlarged ovarian follicles, and/or the

TABLE 1. Branchiated (Br) and metamorphosed (Me) specimens collected in October a n d December 1993 a n d Februaw 1994

Specimens collected 24 October 1993 Ovarian follicles > 0.5 mm

~

Sacrificed BrlMe SVL (mm) Saerm' N Mean dia (mm)

28 Oct 93 5 Br Br 4 Me

18 Feb 94 2 Me 10 Mar 94 Me 10 Mar 94 Me Specimens collected 3 December 5 Dec 94 Br 5 Dec 94 Br 5 Dec 94 5 Me 5 Dec 94 3 Me Specimens collected 16 February 18 Feb 94 Me 18 Feb 94 Me

30.744.0 37.1

34.6-38.0 36 .94 1.0

40.5 40.6

30.5 35.0

33.9-37.7 32.5-33.0

38.3 39.7

-

+

133-192 202

94 85-232

0 134

++ 174 73

++ 31-127 - 2 2 4 7

-

+ 133 ++ 127

0.5-0.9 0.6 0.7

1.3-1.4 0 1.3

0.6 0.9

0.8-1.2 0.9-1.0

1.4 1.4

l + +, abundant; +, scant; -,absent.

SPERM STORAGE IN THE NEWT 157

TABLE 2. Branchiated (Br) and metamorphosed (Me) specimens collected 7March, injected with hCG 9 March, and ouiposited 10-18 March 199-2’

Sacrificed Ovarian follicles Eggs ( 1994) Br/Me SVL Sperm2 N Mean dia Atretic NOviduct NOviposit N Hatch

10 Mar3 Me 40.6 + 77 1.4 0 15 10 Mar3 Br 38.0 ++ 101 1.5 0 2 12 Apr Br 42.1 ++ 65 1.3 3 0 20 19 12 Apr Br 44.2 ++ 131 1.4 0 0 6 0 17 May Me 38.6 + 28 1.4 27 0 11 0 17 May Me 45.9 + 208 1.5 0 0 28 23 23 June Me 37.9 ++ 0 0 11 0 12 12 23 June Me 42.2 + 28 1.4 0 0 6 1 3 Aug Br 43.0 + 0 0 1 0 11 8 3 Aug Br 43.6 ++ 95 1.5 9 0 2 1 22 Sep Me 41.9 + 72 0.9 0 0 4 2 22 Sep Me 44.3 + 74 1.0 0 0 12 4

’All measurements are in mm. 2 + + , abundant; +,scant; -,absent. 3Not injected with hCG.

absence of male external sexual differences including the presence of a heightened tail fin, tarsal pads, and the yellow crescent posterior to the cloaca that represents the presence of a dorsal gland from the cloacal gland complex (Sever, ’91b, ’92b). Males were sacrificed and females were maintained in 25 x 75 x 30 cm glass aquaria, supplied with local well water and a liberal diet of bloodworms (Chironomidae).

Specimens were sacrificed 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. For males, “tail height” was the height of the tail and tail fin in transverse section measured at the widest point. 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 speci-

mens, tissues were initially fixed in 10% neutral-buffered formalin (NBF, for LM) or in a 1:l solution of 2.5% glutaraldehyde in Mil- lonig’s phosphate buffer at pH 7.4 and 3.7% formaldehyde buffered to pH 7.2 with mono- basic and dibasic phosphate (TEM). Car- casses of all specimens are stored in NBF in the research collections at Saint Mary’s Col- lege.

For paraffin infiltration prior to sectioning for LM, the tissue was rinsed in water after fixation, dehydrated in ethanol, cleared in Histosol (National Diagnostics, Manville, NJ), and embedded in paraffin. Sections (10 km) were cut with a rotary microtome, affixed to albuminized slides, and alternate slides were stained with hematoxylin-eosin (HE, for general cytology) or alcian blue at pH 2.5 (AB, for glycosaminoglycans) followed by the peri- odic acid-Schiff method (PAS, neutral carbo- hydrates). 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,

TABLE 3. Branchiated (Br) and metamorphosed (Me) specimens collected May, June, and August 1994l

Ovarian follicles

Collected Sacrificed Br/Me SVL Spermz N Mean dia Atretic

16 May 16 May 20 June 20 June 27 June 2 Aug 2 Aug 2 Aug

17 May 17 May 23 June 23 June 28 June 3 Aug 3 Aug 3 Aug

Br Me Me Me Br Me Br Me

41.5 39.8 38.3 38.9 38.2 40.9 40.1 34.4

++ 9 ++ 28 + 17

3 + 22 i 7

1 0

-

- -

1.4 1.4 1.3 1.3 1.3 1.3 1.1 0

6 6 6 3

12 15 14 4

‘All measurements are in mm. 2 + + = abundant, + = scant, - = absent.


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 Mi- croscopy Sciences, Fort Washington, PA). Thin sections (0.5-1 km) for light microscopy 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.

Fourteen females collected 7 March 1994 were injected on 9 March and 15 March 1994 with 0.2 ml of human chorionic gonadotropin (hCG, Sigma Chemical Co., Saint Louis, MO). These animals were kept in separate 17 x 31 x 9 cm plastic containers containing local well water and Elodea. All animals oviposited some eggs between 10-18 March 1994, at which time the females were removed, toe- clipped for individual identification, and placed in aquaria. For the next 5 months, two females per month were removed for sacrifice and tissue preparation for LM and TEM (Table 2). Four of these individuals were maintained together without males in a 25 x 75 x 30 cm aquarium until March 1995. These specimens were injected with 0.2 ml of hCG on 9 March and 15 March 1995, and again, all females deposited some eggs between 13-18 March, when the specimens were sacrificed and preserved in NBF (Table 4). Cloacae from these specimens were prepared by the paraffin method for LM and stained with HE.

RESULTS Breeding season

Field observations Mature, adult newts were abundant in the

bay from October-May. Despite considerable

effort expended during a minimum of two collecting trips each month, no adult specimens were collected at the breeding site in July and September, and few specimens were found in June (3 males, 3 females) and Au- gust (3 females). Thus a summer, postbreeding emigration from the pond is indicated. Amplexus was observed in the field or in the lab among males and females collected De- cember-March but not from those collected in October or from May, June, and August.

A terrestrial eft stage, estimated to last 1-2 years, is known to occur a t SRS (Gibbons and Semlitsch, '91). However, 43% of the females used in this study are branchiated, and these individuals must have matured in the bay without a terrestrial eft stage. If a general exodus of mature newts occurs during the summer, branchiated individuals must metamorphose after breeding. Thus the breeding population is composed of branchiated individuals that matured without leaving the bay and metamorphosed individuals with a more complicated life history. The metamorphosed individuals may have (1) metamorphosed without ever leaving the bay, (2) migrated into the bay in October for their first breeding after spending one or more years in an immature, terrestrial eft stage, or (3) have gone through more than one breeding season and migration to and from the bay.

Male reproductive cycle Fourteen males, 33.4-39.0 mm SVL, were

examined from October, February, March, May, and June. Only one male, a 36.4 mm SVL specimen from the October collection, is branchiated, and just one male, a 37.4 mm SVL mm metamorphosed specimen, also from the October collection, has two testicular lobes; the remaining specimens have simple testes. The yellow crescent posterior to the cloacal orifice, representing the position of the dorsal gland, is conspicuous in all speci-

TABLE 4. Metamorphosed specimens collected 7 March 1994, induced by HCG injections to oviposit fertile eggs i n March 1994, and maintained isolated from males for one

Eggs oviposited 1994 Eggs oviposited 1995 Ovarian follicles SVL N Oviposited N Hatch N Oviposited N Hatch N Mean dia Sperm2 38.8 15 15 23 0 61 1.3 - 39.2 6 4 69 0 66 1.4 40.9 5 4 16 0 21 1.5 44.9 19 13 114 0 63 1.4

'After induced oviposition of infertile eggs in March 1995, specimens were sacrificed 18 March 1995. All measurements are in mm. 2- , absent.

- - -


mens, but is more apparent in life than in specimens that have been preserved and stored in NBF.

Testes and vasa deferentia were examined from six males collected 25 October and sacrificed 28 October. In these individuals, spermiogenesis occurs throughout the testes, and testicular lobules are filled with mature and maturing sperm (Fig. 1A). The vasa deferentia also contain whorls of mature spermatozoa. Certain secondary sexual characters, however, are not fully developed. Tarsal pads are conspicuous externally in only two of the specimens, and a heightened dorsal fin is not remarkable in any of the specimens (tail height, 5.1-6.9 mm).

In two males collected 16 February and sacrificed 18 February and two males collected 7 March and sacrificed 10 March, both the tarsal pads and the heightened dorsal fin are well-developed (tail height, 7.5-8.3 mm). Spermiogenesis is occurring in the testes, although some evacuated lobules are apparent (Fig. lB), and the vasa deferentia are filled with sperm (Fig. 1C).

In specimens collected 16 May and sacrificed 17 May and specimens collected 20 June and sacrificed 23 June, the tarsal pads are not developed and the tail fins are not heightened as in the specimens from March (tail height, 5.3-6.5 mm). These specimens, however, still contain some mature sperm in the testis and vasa deferentia, although most testicular lobules are evacuated (Fig. 1D). Female reproductive cycle

Females from February and March contain 65-232 ovarian follicles 1.3-1.5 mm mean dia, corresponding to the size of recently oviposited eggs (Bishop, '47). Some follicles this size are present May-August, but females generally are spent by May, and many of the follicles remaining in summer are atretic (Tables 2, 3). Abundant sperm indicative of a recent mating first appear in the spermathecae of 6 of 10 females collected in December (Table 1). Sperm are abundant in all females examined subsequently from February-May, including those that are largely spent (Tables 1-3). Sperm are absent or generally scant in specimens from June- October (Tables 2, 31, including some individuals that had been isolated from males since oviposition of fertile eggs in March (Table 2 ) . One specimen collected in October 1993 and isolated from males until sacrificed in March 1994 had two spermatozoa present in a spermathecal tubule, and these must

represent remnants from the previous breeding season, since no female from October possessed the abundant sperm characteristic of a recent mating (Table 1).

CytoZogy and secretory products of the spermathecae

In females collected in October, the spermathecal epithelium is filled with small (0.5- 0.8 pm) condensing vacuoles (Fig. 2A) that are responsible for a PAS+ reaction in paraffin sections. The epithelium is cuboidal to columnar, and intercellular canaliculi and lumina are narrow (Fig. 2A,B). Golgi complexes (Fig. 2B) are abundant around the luminal borders of the elongate, euchromatic nuclei that are oriented along the long axes of the cells. Of 10 females collected and sacrificed in October, only one contained sperm, and only two sperm cells occurred in a single tubule in a paraffin section.

Four specimens collected in October were kept isolated from males and were sacrificed in February and March (Table 1). In these specimens the apical cytoplasm of the spermathecal epithelium is filled with large secretory vacuoles 2-3 pm in diameter (Figs. 2C,D, 3A,B), and Golgi complexes as well as cister- nae of rough endoplasmic reticulum (Rer) are abundant in the cytoplasm (Fig. 2D). The lumen is a narrow slit (Figs. 2C, 3A), and the myoepithelial sheath is thick (Fig. 3A, cf., Dent, '70). Large secretory vacuoles are present in a 40.5 mm SVL female collected in October and sacrificed in March that lacks ovarian follicles > 0.5 mm dia (Fig. 3A). Another specimen (Fig. 3B), 40.6 mm SVL, collected in October and sacrificed in March contained 134 follicles 1.3 mm mean dia; two sperm cells occur in a paraffin section of one spermathecal tubule.

Of 10 females collected and sacrificed in December, six had abundant sperm in their spermathecae, indicating a recent mating (Table 1). All specimens collected and sacrificed in February and March possessed sperm in their spermathecae (Tables 1,2) , and 12 of 14 females collected in March and induced to oviposit with hCG injections had one or more eggs hatch (Tables 2,4). These females generally laid small clutches (2-28 eggs), and those sacrificed April-September 1994 retained 28- 208 large follicles (0.9-1.5 mm mean dia) in their ovaries except for two females that were completely spent after ovipositing 11 (8 viable) and 12 (all viable) eggs (Table 2) .

All females collected and sacrificed in Feb- ruary and March had mated, and secretory


Fig. 1. Male Notophthalmus uiridescens. Sagittal paraffin sections stained with hematoxylin-eosin. A: Testis of a 36.4 mm SVL specimen collected 24 October and sacrificed 28 October. Lobules are uniformly filled with sperm (Sp). B: Testis of a 33.4 mm SVL specimen collected 7 March and sacrificed 10 March. Sperm occur in

anterior lobules and evacuated lobules (El) are found posteriorly. C : Same specimen as B, showing vas defer- ens filled with sperm. D: 36.4 mm SVL specimen collected 20 June and sacrificed 23 June. Most lobules have regressed and lack sperm, but some sperm remain in lobules indicated.


Fig. 2. Female NotophthaZrnus uzridescerzs. Ultrathin ficed 18 February, unmated, with 232 follicles 1.4 mm sections of spermathecae. A: 44.0 mm SVL specimen mean dia. Note large secretory vacuoles (Sv). D: Same collected 24 October and sacrificed 28 October, unmated, specimen as C, detailing organelles associated with secre- with 133 ovarian follicles 0.9 mm mean dia. Overview of tory vacuoles. Go, Golgi complex; Ic, intercellular cana- apical cytoplasm of epithelium. Note numerous small liculi; Lu, lumen; Mi, mitochondria; Nu, nucleus of sper- condensing vacuoles (Cv). B: Same specimen as A, detail- mathecal epithelial cell; Rer, rough endoplasmic ing organelles associated with condensing vacuoles. C: reticulum. 41.0 mm SVL. specimen collected 24 October and sacri-


Fig. 3. Female Notophthalmus uiridescens. Ultrathin sections of spermathecae. A 40.5 mm SVL specimen collected 24 October and sacrificed 10 March, unmated, and lacking ovarian follicles > 0.5 mm. Note abundant secretory vacuoles (Sv) and thick myoepithelial sheath (My). B: 40.6 mm SVL specimen collected 24 October and sacrificed 10 March, unmated, with 134 ovarian follicles 1.3 mm mean dia. Detail of secretory vacuoles in apical cytoplasm. C: 39.7 mm SVL specimen collected 16 Febru-

ary and sacrificed 18 February, mated, with 127 ovarian follicles 1.3 mm mean dia. Note absence of secretory vacuoles in epithelium and abundant sperm (Sp) in the lumen; also, some sperm are embedded in the cytoplasm (Es). D: Same specimen as C. Luminal diameter in- creases from coalescence of vacuolated spaces remaining in the cytoplasm after release of the secretory product. Cf, collagen fibers; Lu, lumen; Nu, nucleus of spermathecal epithelial cell.


vacuoles are absent from the spermathecal epithelium (Fig. 3C,D). The release of the secretory product creates vacuolated spaces along the luminal border (Fig. 3C,D). Also, clear, multivesicular structures are common in the lumen, especially along the luminal border (Fig. 4B). The sperm in the lumen (Fig. 4A) and along the luminal border (Fig. 4B) appear normal in cytology and are ran- domly oriented, since different portions of the sperm cells appear in the same section. In a specimen collected in February, portions of sperm cells are embedded in the cytoplasm (Figs. 3C, 4C). These sperm are undergoing degradation as indicated by abnormal confor- mation of the axial filament (Fig. 4C). Small (0.3-0.5 km), membrane-bound, and electron-dense bodies that likely are primary lysosomes (cf., Sever, '92a) occur in the spermathecal epithelium (Fig. 4C,D).

The females induced to oviposit eggs in March 1994 by injections of hCG were maintained isolated from males, and two females per month were sacrificed between April- September (Table 2). Four were kept until March 1995, when again they were injected with hCG to induce oviposition of eggs. These four females all contained large ovarian follicles (1.3-1.5 mm mean dia), and all laid eggs (16-114) and retained large follicles (21- 66) in the ovaries (Table 4). None of the eggs laid by these four females were viable.

In addition to captives retained isolated in the lab since oviposition, specimens were collected from the bay in May, June and August. In specimens sacrificed between May-Sep- tember, some sperm occurred in the spermathecae except for one field-collected specimen from June and two from August (Tables 2, 3). When sperm are present after April (lab-retained specimens) or May (field-collected specimens), sperm usually are scant as viewed by LM. After the May samples, no sperm were observed ultrastructurally in any spermathecal tubules.

In specimens from May, secretory vacuoles are absent in the spermathecal epithelium (Fig. 5A), and further evidence of sperm degradation is present (Fig. 5B-D). Mitochon- dria are the most conspicuous organelles in the epithelium (Fig. 5A). Fusions occur between the plasma membranes of sperm and the spermathecal epithelium (Fig. 5B,C). Also, some sperm in the lumen do not appear normal; few mitochondria occur in axial sheaths, and we observed fragments of marginal filaments (Fig. 5D). Microvilli are more

numerous around the luminal border than in February and March.

Specimens examined from June-Septem- ber have spermathecal tubules that are inactive, virtually atrophied (Fig. 6). No secretory activity is apparent, the cytoplasm is scant, and lumina are narrow. Large hetero- chromatic nuclei largely fill the cells in which the most conspicuous organelles are elongate mitochondria (Fig. 6A). Occasionally, however, small electron-dense clusters of secondary lysosomes occur, perhaps containing re- siduals from sperm degeneration (Fig. 6B).

DISCUSSION Life history

A great deal of literature exists on the life history of the red-spotted newt. Much of this interest was sparked by the discovery that many populations have pond-dwelling, aquatic adults and larvae, but the larvae metamorphose into an immature eft stage that leaves the water and assumes a terrestrial existence for anywhere from 1 (Noble, '26, '29) to 7 (Healy, '74) years before return- ing to ponds as mature individuals and as- suming the adult morphology. However, some populations contain individuals that become mature and breed without completing metamorphosis (i.e., larval gills are retained) and may spend their entire life in a permanent pond (Noble, '26, '29; Brandon and Beemer, '66; Healy, '74; Reilly, '86, '87).

The virtual absence of adult specimens during the summer in the bay used in our study indicates that a post-breeding emigration from the pond occurs among adults as reported in several other, more northern populations (Hurlbert, '69; Gill, '78; Massey, '90). Apparently, branchiated individuals that are in mating condition during the fall and spring have never emigrated from the pond and have reached maturity without passing through an eft stage (Healy, '74). A postbreeding terrestrial emigration, therefore, im- plies that branchiated individuals resorb their gills and fully metamorphose after breeding. The eft stage does occur at SRS (Gibbons and Semlitsch, '91). Branchiated specimens in the breeding population may be significantly younger (perhaps 1 year old at first breeding) than specimens that have spent time in the immature eft terrestrial stage (Healy, '73, '74), but the proportion of the breeding population at SRS that has passed through the eft stage is unknown.

Data on other populations indicate that mortality among adults is high; in the moun-

164 D.M. SEVER ET Az,

Fig. 4. Female Notophthalmus uiridescens. Ultrathin sections of spermathecae from a 39.7 mm SVL specimen collected 16 February and sacrificed 18 February, mated, with 127 ovarian follicles 1.3 mm mean dia. A: Overview of sperm in the lumen. Sperm appear normal, and their orientation is random, since sperm nuclei (Sn), middle pieces of the tail (Mt), and principal pieces of the tail (Pt) appear in the same section. B: Sperm near apical cytoplasm (Ac). Multivesicular structures (Ms) are numer-

ous. C: Sperm embedded in the cytoplasm. Note abnormal appearance of axial filaments (Af, compare to normal cytology in B) and association with microfilaments (MI) and primary lysosome (PI). D: Detail of primary lysosomes in perinuclear cytoplasm. Ax, axoneme; Mi, mitochondria; Mf, marginal filament; Nu, nucleus of spermathecal epithelial cell; Rer, rough endoplasmic reticulum; Um, undulating membrane.


Fig. 5 . FemaleNotophthalrnus uzrzdescens. Ultrathin sections of spermathecae. A: 45.9 mm SVL specimen collected 7 March, induced to lay 23 fertile eggs in March, and sacrificed 17 May with 208 ovarian follicles 1.5 mm mean dia. Note again absence of secretory vacuoles in the cytoplasm. B, C: Same specimen as A, showing cytoplas- mic fusions (Fu) between sperniathecal epithelium and luminal sperm. D: 41.5 mm SVL specimen collected 16

May and sacrificed 17 May, mated, with 15 ovarian follicles (9 atretic) 1.4 mm mean dia. Note loss of mitochondria (Mi) from axial sheath and fragments of marginal filaments (Mf, detail in inset). Ac, apical cytoplasm of spermathecal epithelium; Ic, intercellular canaliculi; Lu, lumen; Ms, multivesicular structures; Mt, middle piece of tail; Mv, microvilli; Nu, nucleus of spermathecal epithelial cell.

166 D.M. SEVER ET AL.

Fig. 6. Female Notophthalrnus uzridescens. Ultrathin sections of spermathecae. A 38.3 mm SVL female collected 20 June and sacrificed 23 June, mated, with 34 ovarian follicles (12 atretic) 1.3 mm mean dia. Spermathe- cal cytoplasm is scant and most conspicuous organelles are mitochondria (Mi). B. Same specimen as A, showing secondary lysosomes (S1) in perinuclear cytoplasm. C: 40.9 mm SVL specimen collected 2 August and sacrificed 3 August, mated, with 22 ovarian follicles (15 atretic) 1.3

mm mean dia. Spermathecal epithelium shows no indication of synthetic activity. D: 41.9 mm SVL specimen collected 7 March, induced to oviposit 2 fertile eggs in March, and sacrificed 22 September with 72 ovarian follicles 0.9 mm mean dia. Spermathecal epithelium is inactive. B1, basal lamina; Lu, lumen; M1, microfilaments; My, myoepithelial cells; Nu, nucleus of spermathecal epithelial cell; Rer, rough endoplasmic reticulum.


tains of Virginia, the “average female” breeds only 1.3 times (Gill, ’781, and Massey (’90) found that 75% of the females in 1 year’s breeding population did not appear the following year. Only one male examined in this study had more than one testicular lobe; N. viridescens, like other salamandrids, has multiple testes, and the presence of more than one testicular lobe is an indication of age (Sever, ’74). Thus the males observed in this study may, in general, represent a young cohort, perhaps breeding for the first time.

Mating season One aspect of the life history of the red-

spotted newt that has come under much scru- tiny is the timing of the breeding season, especially if an autumnal “false breeding season” exists. A false breeding season is one in which males and females engage in courtship and mating, but the females do not oviposit eggs. This period has been reported in the fall in northern populations (mostly from Massa- chusetts, New York, and Pennsylvania) in which adults overwinter in the ponds (Gage, 1891; Jordan, 1891,1893; Pope, ’24; Adams, ’40; Bishop, ’41). Mating activity in the fall does not approach the “outbreak of sexual susceptibility” that occurs in spring (Jordan, 1893), and Pope considered the fall mating “quite superfluous” since no eggs are laid.

Jordan (1893) reported that the spermathecae of females captured in autumn usually contain sperm “although not in the same abundance as in spring,” and he suggested that the presence of sperm in the fall was due to a recent mating (during the false breeding season) or storage from a previous spring. Jordan (1891, 1893) noted that one captive female laid 96 eggs between 24 April and 13 May after being isolated from males. Jordan (1893) thus reasoned that Gage (1891) erred by stating that only 6-7 eggs could be fertilized by one mating: “It does not seem to me necessary that more than one mating should occur in a single season, but I agree with Gage that in a state of nature several matings of the same female may and frequently do take place.”

Adams (’40) reported that male newts contain abundant sperm in their vasa deferentia September-May, and some specimens retain a few sperm during the midsummer months. Sperm occur in spermathecae of females “. . . examined in every month of the year but they are especially abundant in the fall and spring” (Adams, ’40). Oviposition, however, is strictly limited to the spring; out of hundreds dis-

sected, only one female collected during the fall and winter had an oviducal egg, whereas in AprilJune, eggs are found in the oviducts of some newts from every collection (Adams, ’40). In both males and females, the “. . . gonads are in a preparatory growth state during the summer and by October have attained the mature condition which results in a false breeding season. . . . The true breeding season of the spring and the false one of the autumn were both observed as Gage (18911, Jordan (1891), and Pope (’24) have described them” (Adams, ’40).

The breeding season of N . viridescens was characterized as winter and early spring by Smith (’61) in Illinois and Mount (’75) in Alabama, January-May by Minton (’72) in Indiana, and February-May by Gibbons and Semlitsch (’91) on the Savannah River Site in South Carolina. Although some workers state that in the northern states newts are active under the ice all winter (Bishop, ’41; Minton, ’721, others report that they hiber- nate in terrestrial hibernacula or in the muddy bottoms of dried-up ponds (Gill, ’78; Smith and Pfingsten, ’89). One would expect differences in breeding activity between northern and southern populations related to temperature, and it is interesting to note that a fall “false breeding season” has not been reported for any southern populations.

In many populations in the north, some or all of the adult newts leave the ponds in the summer (sometimes because the ponds have dried up) and spend the fall and winter on land. In New York, Hurlbert (’69) found a fall and a spring migration occurs to the breeding sites, but the fall migrations almost exclu- sively involved efts, maturing individuals re- turning to the water for the first time. Whether mating occurs in fall or not was not mentioned by Hurlbert (’69). Gill (’78) and Massey (’90) in the mountains of Virginia, reported a breeding immigration March- April to the ponds, and an August-Septem- ber emigration to terrestrial hibernacula. Breeding occurs from mid-March to late June (Massey, ’881, and no fall breeding season exists (Massey, ’90).

We found no evidence of a false breeding season in our population. A summer exodus and subsequent fall immigration occur to the breeding pond. Mature males and females enter the pond in October, and males possess sperm in their vasa deferentia. Male secondary sexual characters, however, are not fully developed, and females contain follicles


smaller than ovulatory size. Amplexus was not observed until December, when 60% of the females collected had recently mated. All females collected in February and March have mated. In March, males exhibit maximal development of secondary sexual characters, and females can be induced to lay fertile eggs. Thus it does not appear that mating occurs until females are near (December) or in (Feb- ruary-March) ovulatory condition. Females coIlected in May are spent, and sperm are degrading. Thus the effective period of sperm storage in our population is from two (Decem- ber-February) to possibly six (December- May) months, and sperm that were stored for shorter periods may be used in fertilization.

Sperm storage between breeding seasons Retention of sperm in the spermathecae of

N. viridescens between breeding seasons was first suggested by Jordan (1893), and more recently proposed by Massey (’90). As noted in the introduction, Massey (’90) recaptured eight females immigrating into a partially fenced pond in which they had bred the previous year. Seven of the females had some sperm in their spermathecae, and Massey (’90) concluded that the sperm had been retained at least 10 months. The viability of the sperm and whether they were in sufficient quantity to fertilize eggs was unknown, but Massey suggested the possibility of between- breeding season sperm competition.

However, the pond used in the study by Massey was only partially fenced, and as noted by Hulbert (’69), newts following their ar- rival a t a pond move back and forth between water and land, a phenomenon also recorded in other species, and termed “wandering behavior” (Pimentel, ’60; Hasumi and Iwasawa, ’92). Thus the female newts could have mated in the current season in their “home” pond or another pond. Also, if the males in this population are typical of others reported from northern states during the fall and winter (Adams, ’401, the males possess sperm in their vasa deferentia from September on- ward, and one cannot definitely rule out a fall mating.

In some females collected in summer and in fall for this study, we found a few sperm in the spermathecae, and these sperm likely are remnants from the previous breeding season. However, we do not believe that these sperm would be capable of fertilizing eggs. First, fertilization in N. viridescens requires polyspermy (Fankhauser and Moore, ’41; Humphries, ’66; McLaughlin and Humphries,

’78), and too few sperm (only two sperm cells in some specimens) were noted to make fertilization possible. Second, sperm were embedded in the spermathecal epithelium and degrading early in the mating period (February), and degradation of sperm in the lumen and epithelium was evident in specimens examined cytologically from May and June. Thus the fate of remnant sperm is degradation. Also, the spermathecae are inactive from June-September, providing no maintenance for sperm. Finally, four females that had mated and laid fertile eggs were kept isolated from males and induced to oviposit one year later. All had mature follicles, but none of the eggs oviposited were fertile, and subsequent examination of the spermathecae found sperm lacking.

Comparative cytology of the spermathecae Dent (’70) did not report the presence of

large secretory vacuoles in the spermathecal epithelium of N. viridescens, but glycoprotein globules, such as those observed herein, have been reported from other salamanders in which the spermathecae have been studied ultrastructurally (Pool and Hoage, ’73; Brizzi et al., ’89, ’95; Sever and Brunette, ’93; Sever and Kloepfer, ’93; Sever, ’94, ’95). Dent ( ’70) was the first to note degenerating sperm in the spermathecal epithelium, although he also proposed that the epithelial cells provide nourishment for sperm. Our results confirm those of Dent (’70) and other recent studies (Sever, ’92a; Sever and Kloepfer, ’93; Brizzi et al., ’95) that sperm that come in contact with the epithelium are phagocytized.

In addition to Dent (’70), a “nutritional” role for spermathecal secretions was proposed by Benson (’68) and Boisseau and Joly (’75). We found that the secretions in N. viridescens are released at onset of mating and not replenished during sperm storage, so “nutrition” does not seem a likely function. In Ambystoma tigrinum, in which sperm are stored for only a few days, Sever (’95) found that the secretions are released during oviposition, and the secretions serve to flush sperm from the glands. The secretions of the spermathecae of N. viridescens clearly could not have a “flushing” role since the secretions occur with mating and not with oviposition, which is spread out over more days in N. viridescens than A. tigrinum. The duration of sperm storage and role of the secretion as found in A. tigrinum are considered ances- tral for salamanders (Sever, ’951, and N. uiri-


contract DE-AC09-76SR00-819 between the University of Georgia and the D.O.E. We thank Brandi Rose for her help in the laboratory on earlier stages of this study, and Michelle Boone for her help in the field. This work is publication number eight from the Saint Mary's College Electron Microscopy Facility.

descens appears to be more specialized in these regards.

We found that in the breeding season unmated females have large secretory vacuoles in their spermathecal epithelium, and these vacuoles are absent in all mated females, indicating release of the secretion with some aspect of mating. Hardy and Dent ('86) dis- counted a sperm attraction function for the secretion. However, the secretory product is not continual1y during the breeding

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ACKNOWLEDGMENTS

This work received support from National Science Foundation grant DEB-9024918 to D.M.S., although latter stages of the research lacked support from NSF. D.M.S. also received support from U.S. Department of Energy (D.O.E.) contract DE-AC05-760R00- 033. LCR was supported by the Undergradu- ate Research Program of the Savannah River Ecology Laboratory. J.D.K. was supported by

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