Ultrastructure of the Annual Cycle of Female Sperm Storage in Spermathecae of the Torrent Salamander, Rhyacotriton Variegatus (Amphibia: Rhyacotritonidae)
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JOURNAL OF MORPHOLOGY 261:1–17 (2004) Ultrastructure of the Annual Cycle of Female Sperm Storage in Spermathecae of the Torrent Salamander, Rhyacotriton variegatus (Amphibia: Rhyacotritonidae) David M. Sever,1* Cynthia K. Tait,2 Lowell V. Diller,3 and Laura Burkholder3 1Department of Biology, Saint Mary’s College, Notre Dame, Indiana 46556 2Bureau of Land Management, Vale, Oregon 97918 3Simpson Timber Company, Arcata, California 95521 ABSTRACT This study is the first report on the ultra- spermathecae. The ancestral condition for structure of the sperm storage glands (spermathecae) in salamanders is lack of sperm storage glands, a con- the salamander Rhyacotriton variegatus. The population dition found in three families with external fertili- studied is associated with cold-water, rocky streams of the zation, Sirenidae, Hynobiidae, and Cryptobranchi- redwood (Sequoia) zone in northern California. Males pos- sess sperm in their vasa deferentia and undergo spermi- dae (Sever, 1991b). ation throughout the year, but mating is seasonal. Most The anatomy of sperm storage glands has been females with large, vitellogenic follicles (2.0–3.9 mm extensively studied in salamanders and was most mean dia.) collected from February–June contain sperm in recently reviewed by Sever (2003). Much work has their spermathecae, although some females with large been done on spermathecae at the light microscopy follicles lack sperm. Other mature-size females collected level, but ultrastructural studies using transmission during this period have small ovarian follicles (0.9–1.2 electron microscopy are the best means for studying mm mean dia.) and lack stored sperm. All females col- cytology of sperm/epithelial interactions and the se- lected from September–November have small follicles cretory cycle of spermathecal epithelium. (0.6–1.6 mm mean dia.) and lack sperm, except in one instance in which a female collected in November had a In this article, we present the first ultrastructural small amount of degraded sperm, apparently retained observations on sperm storage in females of a spe- from the previous breeding season. The spermathecae con- cies of the Rhyacotritonidae, using Rhyacotriton var- sist of simple tubulo-alveolar glands in which the neck iegatus (Fig. 1A). Rhyacotriton variegatus is one of tubules produce a mucoid secretory product, and the distal four species in the family Rhyacotritonidae, which is bulbs, where sperm are stored, contain secretory vacuoles restricted to the Pacific Northwest region of the of uniform density that stain positively for glycosamino- United States (Good and Wake, 1992). Sister-group glycans. In specimens containing sperm, some bulbs have relationships of Rhyacotritonidae to other abundant sperm and others lack sperm, but the ultra- salamander families are obscure (Larson et al., structure is similar in both conditions. The acini contain columnar epithelial cells with wide intercellular canalic- 2003). uli, and a merocrine process releases the secretion. Sper- Members of this family occupy springs, seeps, and miophagy occurs. In specimens from spring and summer the edges of swift-flowing, permanent streams with with small ovarian follicles, the neck tubules are similar coarse substrates (Petranka, 1998). In Oregon, ovi- to those of breeding females, but the distal bulbs are position can occur at almost any time, although reduced to cords of cells lacking a discernible lumen. Se- there may be a peak of courtship and egg-laying cretory activity in the distal bulbs is initiated in the fall. activity in spring or early summer (Nussbaum and Spermathecae of R. variegatus are most similar to those of Tait, 1977). The few nests reported for any of the a stream-dwelling plethodontid, Eurycea cirrigera. J. Mor- species were found submerged in rock crevices or phol. 261:1–17, 2004. © 2004 Wiley-Liss, Inc. under gravel in first order streams and springs. In KEY WORDS: Amphibia; Urodela; Rhyacotritonidae; these nests, eggs were apparently laid in summer or Rhyacotriton; reproduction; sperm storage; spermathecae; fall (Nussbaum, 1969; Karraker, 1999; Russell et al., ultrastructure 2002). A description of the cloaca and spermathecae Sperm storage occurs in all females of the seven *Correspondence to: David M. Sever, Department of Biology, Saint families of salamanders comprising the suborder Mary’s College, Notre Dame, IN 46556. Salamandroidea (Sever, 1991a, 1994). Instead of E-mail: [email protected] oviductal sperm storage, as known in other female Published online in vertebrates that store sperm, sperm storage in fe- Wiley InterScience (www.interscience.wiley.com) male salamanders occurs in cloacal glands called DOI: 10.1002/jmor.10149 © 2004 WILEY-LISS, INC. 2 D.M. SEVER ET AL. Fig. 1. Rhyacotriton variega- tus. A: Dorsal view of a 60.8 mm SVL female collected 10 April with ovarian follicles 3.5 mm mean dia. B: Ventral view, dis- sected to show reproductive tract. Od, oviduct; Ov, ovary; St, spermathecae. of Rhyacotriton variegatus at the light microscopy sample from March 2001 was used in a separate study involving level was provided by Sever (1992b). scanning electron microscopy (SEM) of sperm packing in the spermathecae (Sever et al., in prep.). The purpose of this study is three-fold. First, we Specimens were killed by immersion in 10% aqueous solution of provide descriptions of the seasonal variation in ul- MS-222 (3-aminobenzoic acid ethyl ester). The Animal Care and trastructure of the spermathecae of Rhyacotriton Use Committee of Saint Mary’s College approved this procedure. variegatus. Second, we compare our findings on R. After death, snout–vent length (SVL) was measured from the tip variegatus to those on ultrastructural characteris- of the snout to the posterior end of the cloacal orifice. Vitellogenic follicles were counted and their diameters measured to the near- tics of sperm storage in other salamanders. Finally, est 0.1 mm. Based upon the size of the follicles and appearance of we provide preliminary observations on other as- the oviducts, females were separated into three categories: 1) pects of the male and female reproductive cycles of Gravid—females with large yolked follicles (Ͼ2.0 mm mean dia.) R. variegatus in northern California. This study is and thick, convoluted oviducts (Fig. 1); 2) Spent—females with part of a more extensive project coordinated by LVD smaller follicles but flaccid, thick, convoluted oviducts indicative of recent stretching; and 3) First yolkers—females with small on the reproductive biology of R. variegatus. follicles and thin, straight or wavy oviducts. First yolkers are females reaching sexual maturity, beginning to yolk eggs for the first time, but are not yet ready to breed. MATERIALS AND METHODS The cloacal area and an associated oviduct were removed from The specimens of Rhyacotriton variegatus used in this study each specimen. The spermathecal area is pigmented (Fig. 1B), came from the North Fork Mad River watershed on lands owned and this region was bisected. One-half was prepared for TEM and by the Simpson Timber Company in western Humboldt County, the other half for LM. For TEM, a 1-mm block of pigmented tissue California, in the north coast redwood (Sequoia sempervirens) was removed and fixed in a 1:1 solution of 2.5% glutaraldehyde zone. Rhyacotriton variegatus is considered a “species of concern” and 3.7% formaldehyde in cacodylate buffer at pH 7.2. After by the state of California, and permits for the collection of a initial fixation, tissues were rinsed in distilled-deionized water, limited sample were granted to Lowell V. Diller from the Califor- postfixed in 2% osmium tetroxide, dehydrated through a graded nia Department of Fish and Game. Collection dates and other series of ethanol, cleared in propylene oxide, and polymerized in data on the 25 female specimens used in this study are shown in an epoxy resin (Embed 812, Electron Microscopy Sciences, Port Table 1. Animals generally were sacrificed and preserved within Washington, PA). Plastic sections were cut with an RMC MT7 24 h of collection. Collections were made in February 2002, April ultramicrotome (Research and Manufacturing Co., Tucson, AZ) 2000, 2001, June 1999, 2001, September 2001, and November and DiATOME (Biel, Switzerland) diamond knives. Semithin 1999, 2001. The spermathecae of these specimens were examined sections (0.5–1 m) for LM were placed on microscope slides and by light (LM) and transmission electron microscopy (TEM). A stained with toluidine blue. Ultrathin sections (70 nm) for TEM TORRENT SALAMANDER SPERM STORAGE 3 TABLE 1. Female Rhyacotriton variegatus used in this study1 Follicles Date SVL N Range Mean SE Cond2 Sperm 10 Feb 52.2 8 1.5–2.5 2.0 0.11 GD 0 10 Feb 54.2 10 1.6–3.0 2.4 0.11 GD ϩ 10 Feb 54.2 6 2.1–3.1 2.8 0.16 GD ϩ 10 Feb 54.4 7 1.8–2.6 2.1 0.11 GD ϩ 10 Feb 56.8 11 1.9–2.7 2.3 0.08 GD ϩ 1 April 45.3 10 0.8–1.1 0.9 0.03 FY 0 1 April 55.0 9 3.0–3.5 3.2 0.06 GD ϩ 10 April 48.6 8 0.8–1.6 1.1 0.05 FY 0 10 April 60.8 12 3.2–3.9 3.5 0.07 GD ϩ 18 June 45.1 7 0.6–1.2 1.0 0.06 FY 0 18 June 48.1 7 1.0–1.5 1.2 0.07 FY 0 18 June 50.3 6 1.4–3.3 2.8 0.16 GD ϩ 18 June 53.0 9 3.0–4.4 3.5 0.16 GD ϩ 23 Jun 45.8 eggs not yolked; count unreliable FY 0 23 Jun 52.1 7 3.8–4.0 3.9 0.03 GD 0 23 Jun 57.4 8 2.7–3.7 3.1 0.15 GD ϩ3 30 Jun 50.6 6 2.1–3.2 2.7 0.17 GD 0 7 Sep 52.6 11 1.0–1.4 1.2 0.04 ? 0 1 Nov 43.4 5 0.5–0.7 0.6 0.03 FY 0 1 Nov 44.3 5 0.7–1.0 0.9 0.04 FY 0 1 Nov 47.9 16 0.6–1.0 0.8 0.04 FY 0 1 Nov 52.2 9 1.2–1.9 1.6 0.08 ST 0 5 Nov 52.6 14 1.1–1.8 1.4 0.04 ST ϩ 5 Nov 53.7 10 1.0–1.5 1.3 0.06 ST 0 5 Nov 55.0 12 1.1–1.7 1.4 0.07 ST 0 1Measurements for SVL and the range and mean of follicle dia.