Seasonal changes in serum gonadal steroids associated with migration, mating, and nesting in the loggerhead sea turtle (Caretta caretta)

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

79, 1%-16‘t (I!?%$

Seasonal Changes in Serum Gonadal Steroids Associated with Migration, Mating, and Nesting in the Loggerhead Sea Turtle (Caretfa caretfa) THANE

WIBBELS,*”

DAVID

WM.

OWENS,* COLIN J. LIMPUS,? MAX S. AMOSS, JR.*

PHILIP

C. REED,? AND

*Department of Biology and SDepartment of Veterinary Physiology and Pharmacology, Texas A & M University, College Station, Texas 77843, and fQueensland National Parks and Wildlife Service, Pallarenda, Townesville 4810, Austraiia Accepted March 17, 1988 Adult male loggerhead sea turtles, Caretta caretta, exhibited a “prenuptial” spermatogenie cycle that was coincident with increased concentrations of serum testosterone (T). Serum T was high during the months when migration and mating have been recorded for males. In contrast to females, males appear to be annual breeders. Nine reproductively active female C. caretta (as verified through laparoscopy) were tagged with sonic transmitters and were repeatedly bled prior to migration. Four months prior to the nesting season, the ovaries of reproductively active females had hundreds of vitellogenic follicles of approximately 1.5 cm in diameter (i.e., half the size of ovulatory follicles). Approximately 4-6 weeks prior to migration from feeding grounds to mating and nesting areas, serum estradiol-17f3 (E,) concentrations increased significantly and remained high for approximately 4 weeks, suggesting a period of increased vitellogenesis. During a 1- to 2-week period prior to migration, serum E, decreased significantly, while serum T concentrations increased (at least) until the time of migration. Serum T, E,, and progesterone (PRO) were elevated during nesting if a turtle was going to nest again during that nesting season. During the last nesting of a season, turtles had low serum concentrations of T, E,, and Pro. The prenuptial pattern of gonadal recrudescence and gonadal steroid production in both male and female C. caretta contrasts with those of many temperate freshwater turtles, and this type of reproductive pattern may have been facilitated by adaptation to a tropical marine environment. 0 1990 Academic Press. 1~

Sea turtles represent one of the few reptilian groups adapted to a marine environment. An understanding of the hormonal control of sea turtle reproduction could suggest possible effects of marine adaptation on a reptilian endocrine system. Three previous studies have reported seasonal changes in gonadal steroid concentrations (Licht et al., 1979, 1985b; Wibbels et al., 1987). Licht et al. (1979, 1985b) recorded gonadotropin and gonadal steroid concentrations of a captive group of green sea turtles (Chelonia my&s) at the Cayman ’ Current address: Department of Zoology, University of Texas at Austin, Austin, TX 78712.

Turtle Farm, LTD. The data on C. mydus have provided a model of seasonal gonadal steroid dynamics associated with sea turtle reproduction, but were limited to a single species, maintained in captivity. Factors associated with captivity, such as increased food availability, altered nutritional composition, decreased seasonal thermal fluctuation (Licht et al., 1985b), and lack of migration, could affect reproduction. For example, the interval between nesting seasons is shorter, and the number of nests per season as well as the total egg production, per female C. mydus at the Cayman Turtle Farm, LTD, is two to five times greater than that of C. mydus natural populations (Wood and 154

0016~6480190 $1.50 Copyright 8 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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HORMONES

Wood, 1980). Thus, studies of sea turtles from natural populations as well as studies of other sea turtle species would enhance our present understanding of their reproductive endocrinology and provide a means of evaluating the comparative value of previous data on captive sea turtles. While two studies have recorded gonadal steroid titers associated with specific reproductive events (i.e., mating and nesting) in naturally occurring sea turtles (Licht et al., 1980, 1982), there has been only one seasonal study of this sort (Wibbels et al., 1987). However, that study reported only serum testosterone (T) concentrations and was limited by the fact that the specific reproductive status of most turtles was unknown. In addition, no previous study has been able to address hormonal changes during the time period leading up to migration. The following study reports seasonal changes in gonadal steroid concentrations of loggerhead sea turtles, Caretta curettu, from a natural population whose reproductive status was determined by laparoscopic examination or by behavioral observations (i.e., nesting). MATERIALS

AND METHODS

Study sites. Three study sites were utilized. The first two sites, Heron Island Reef and Mon Repos, were located in southern Queensland, Australia. Southern Queensland supports one of the world’s major breeding areas for C. caretta with over 3000 turtles nesting each year (Limpus, 1981). The third study site was Melbourne Beach on the Atlantic coast of Florida which supports the densest nesting of C. caretta in the North Atlantic (Bjomdal and Meylan, 1983). Heron Island Reef is in the Capricorn Group of coral reefs at the southern end of the Great Barrier Reef. It is an elongate lagoonal platform reef approximately 11 km long and 5 km wide at its eastern end (Mather and Bennet, 1984). A small sand island, Heron Island, is located on the western end of this reef. Relatively large numbers of three sea turtle species, C. caretta, C. mydas, and Eretmochelys imbricata, inhabit the coral reefs in this area, but C. cnretta was chosen as the species of study because of a preexisting data base documenting its reproductive ecology in the area (Limpus, 1985). Heron Island Reef is a year-round

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155

feeding ground for resident immature and adult C. cnretta. During the nesting season (November through January) nonresident C. caretta migrate onto the reef and nest on Heron Island (Limpus, 1985). The second study site was a major C. caretta nesting beach located at Mon Repos, which is on the mainland of southern Queensland, Australia (Limpus, 1980; 1981). The turtles nesting at Mon Repos include some individuals that use Heron Island Reef as a feeding ground (Limpus, 1985). The peak nesting season at Mon Repos occurs from November through January. Close surveillance of the nesting beachs at Mon Repos and at Heron Island (Limpus, 1985) facilitated the documentation of complete nesting records for specific turtles. The third study site was Melbourne Beach, Florida. The turtles nesting on that beach are members of the population of C. caretta that inhabit the Atlantic and Gulf waters of the southeastern United States (Bjomdal and Meylan, 1983). This group of turtles is believed to be the second largest population of C. caretnr in the world (Bjomdal and Meylan, 1983). Turtles nesting at Melbourne beach have been recaptured as far north as Cape Island, North Carolina, and as far south and west as Sanibel Island on the Gulf coast of Florida (Bjomdal and Meylan, 1983). The nesting season for C. caretta in this area normally begins during May and ends during late August (Ehrhart, 1980). Capture and bleeding. On Heron Island Reef a turtle rodeo technique (described by Limpus, 1981) was used to capture turtles. This technique was facilitated by the clear and shallow water covering the reef. Briefly, the capture method involved chasing the turtles with a 4.2-m speedboat, diving onto the turtle from the moving boat, grasping the turtle’s carapace firmly, and then steering the turtle to the surface. The turtle was pulled into the boat with the aid of ropes which where looped around its front flippers. Blood samples were taken within 5 min of capture. Nesting turtles at all three study sites were temporarily restrained and bled after they had oviposited. All turtles were tagged with two titanium flipper tags as described by Limpus and Reed (1985). A 3.8-cm 21-gauge needle, needle holder, and a sterile vacuum tube were used to obtain blood from the bilateral cervical sinus (Owens and Ruiz, 1980). Blood samples were kept on ice for up to 5 hr until centrifugation at 3400 rpm for 5 min. Serum was pipetted into cryotubes and frozen in liquid nitrogen or in a - 20” or below freezer. The blood sampling of adult turtles on Heron Island Reef was conducted from July 5, 1985 through March 3 1, 1986. Blood samples were also obtained from two turtles during their first nestings of the season on Heron Island on the 16th and the 20th of November 1985, respectively. Blood samples were taken from turtles nesting at Mon Repos from November 22, 1985 to January 29, 1986 and from turtles nest-

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WIBBELS

ing on Melbourne Beach during July and August of 1986 and during June and August of 1987. Laparoscopic examinations. Turtles captured on Heron Island Reef were taken to Heron Island for laparoscopic examination (Wood et al., 1983; Limpus and Reed, 1985). The majority of adult males and all adult females were laparoscopically examined at least once during the study to determine if they were reproductively active. In males, the general appearance of the testes (e.g., seminiferous tubules engorged, seminiferous tubules not engorged) and epididymides (e.g, tubule obvious, tubule not obvious) was recorded. Testicular biopsies were taken with a laparoscopic biopsy forceps during double puncture laparoscopy. The tissue biopsies were fixed in buffered formalin, infdtrated with paraffin, sectioned, and stained. The tissues were then classified into stages of spermatogenesis as described by Licht (1967) and McPherson et al. (1982). The majority of reproductively active females were laparoscopically examined early in the study, approximately 3-5 months prior to the nesting season. Additionally, some females were examined after their final nesting of the season to verify that their ovaries did not have enough mature follicles to support an additional nesting. Sonic tracking. To facilitate repeated capture and blood sampling of reproductively active females on Heron Island Reef, sonic transmitters were attached to nine reproductively active females. Sonic transmitters were obtained from Custom Telemetry and Consulting, Inc. (Athens, GA). Each transmitter could be identified by its unique operating frequency (ranging from 20 to 80 kHz). Two sizes of cylindrical transmitters were used (5 cm diameter x 20 cm length and 2 cm diameter x 10 cm length), the larger giving off a more powerful signal. A single transmitter was attached under the posterior portion of each turtle’s carapace. The transmitters were attached to the turtles by monofilament fishing line, threaded through 3-mm holes drilled in the rear marginal scutes and through holes in the ends of the transmitter. A USR-5 sonic receiver and hydrophone (Sonotronics, Tucson, AZ) were used from the boat to locate the turtles. An attempt was made to recapture each turtle at approximately 1Cday intervals to obtain blood samples. Steroid radioimmunoassays (RIA). Radioimmunoassay procedures similar to those described by Coyotupa et al. (1972) were used to determine serum T, progesterone (PRO), and estradiol-178 (E2) concentrations. Tritiated steroids were obtained from New England Nuclear (Boston, MA) and each was diluted with TrYgelatin assay buffer to yield approximately 7000 cpm per 100 pl. The T antiserum was obtained from Cambridge Medical Diagnostics and was diluted 11 4500 with Tris/gelatin assay buffer; it cross-reacts 100% with T, 21.0% with dihydrotestosterone (DHT), 3.6% with 3B,17B-dihydroxy-5a-androstane, 0.5%

ET AL.

with androstenedione, and less than 0.5% with androstenediol, androsterone, and dehydroepiandrosterone. Cross-reaction with DHT in the serum samples was presumed to be minimal since previous studies of green sea turtles, C. mydas (Licht et al., 1979), as well as freshwater turtles (Callard et al., 1976; McPherson et al., 1982) indicated that DHT accounts for less than 1.5% of total androgens in chelonians. The E, antiserum was obtained from D. C. Collins (Emory University School of Medicine, Atlanta, GA) and was diluted l/10,000 with TrYgelatin assay buffer. The procedure for generating this antiserum and the validation of the E2 RIA were reported by Wright et al. (1973). The E, antiserum cross-reactivity was consistent with that reported by Wright et al. (1973) (e.g., 1.8% with estrone, 0.8% with estriol, 0.4% with PRO, 0.4% with T, and less than 0.1% with cortisol and dehydroepiandrosterone). The PRO antiserum was produced by a standard in vivo rabbit immunization technique (as described by Franchimont et al., 1983) and was diluted l/4000 with Tris/gelatin assay buffer. This antiserum cross-reacted 0.9% with deoxycorticosterone, 0.6% with corticosterone, and less than 0.5% with aldosterone, androstenedione, E,, estrone, 17u-hydroxypregnenolone, 17a-hydroxyprogesterone, pregnenolone, and testosterone. The sensitivities of the T, PRO, and E, assays (at 80% BIB,) were 2.3, 9.9, and 5.5 pg/tube, respectively. Two to six control samples were included in each assay. The intraassay coefftcients of variation for T, PRO, and E, assays were 7.5, 7.4, and 7.1%, respectively, and the interassay coefftcients of variations were 9.0, 15.7, and 12.0%, respectively. Sea turtle sera (from adult male C. caretta, immature male and immature female c. caretta and Lepidochelys kempI> at five different dilutions were tested in duplicate in each assay and the response curve was parallel to the standard curve. Approximately 30 samples were extracted for each assay. Depending on the sensitivity of the assay and the serum titer of a specific steroid, an aliquot ranging from 250 to 2000 ~1 of each serum sample was extracted for each steroid RIA with 4 ml of anhydrous ether. Extraction efficiencies for T, Pro, and E, were 79,85, and 74%, respectively. The aqueous phase was frozen using liquid nitrogen and the ether phase was decanted, evaporated under nitrogen gas, and the residue reconstituted with 1 ml of acetone. Two 400~p1 aliquots of the acetone were pipetted into assay tubes and evaporated overnight. Following evaporation the residue was reconstituted with 100 pl of Tris/gel buffer. Specific steroids for preparation of standards were obtained from Steraloids Inc. (Wilton, NH). Standard tubes with 100 u1 of a known concentration of the specific steroid being assayed (ranging from 15.6 to 400 pg/ml) were prepared. Tritiated hormone and antisera (100 ul of each) were added to all standard and sample tubes. The tubes were incubated for 24 hr at 4”.

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HORMONES

After incubation, 1.0 ml of dextran-coated charcoal (0.313 g T 70 Dextran (Sigma) and 3.125 g Norit A charcoal (Sigma) per 500 ml Tris/gel buffer) was added to each tube to absorb unbound hormone. All tubes were vortexed, incubated for 15 min at 4”, and then centrifuged for 15 min at 12OOg. The supematant was poured into sealable plastic bags with 5 ml of scintillation cocktail. Samples were counted for 5 min in a scintillation spectrometer. Statistical analyses. Turtles were grouped for statistcial analysis relative to their date of capture. Males were grouped into 30&y intervals starting with July 1, 1985. The sonic transmitter-tagged females were recaptured approximately every 2 weeks and were therefore grouped into 15-day intervals. Statistical analyses were performed on raw and logtransformed data using the SASISTAT personal computer program (SAS Institute Inc.). Two-way ANOVA was performed to determine if significant changes were detectable in hormone concentrations of the repeatedly sampled females over time (Sokal and Rohlf, 1981). One-way ANOVA was used for analysis of the male and nesting female concentrations over time. A Duncan’s multiple range test was performed if significant changes were detected in the above analyses.

RESULTS Figure 1 shows mean serum T concentrations of the male c. caretta captured on

JUL

SEP

NOV

JAN

MAR

1. Mean serum testosterone concentrations of adult male loggerheads, C. caretta, captured on Heron Island Reef. Vertical bars are standard errors of the mean. Serum concentrations during January, February, and March were significantly lower than those during the months of July through November (P < 0.001). FIG.

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157

Heron Island Reef. Circulating T exhibited significant changes over time with concentrations from June, July, August, September, October, and November being significantly greater than those of January, February, and March (P < 0.001). Table 1 lists the months during which specific spermatogenie stages were observed during the study. Visual assessment of the epididymides via laparoscopy indicated that from January through March each was a regressed pendulous mass with no distinct tubule, whereas from July through early November, a white convoluted tubule was obvious . Four to thirteen reproductively inactive adult females were captured and laparoscopically examined each month from July through November on Heron Island Reef. All of these turtles had relatively low or nondetectable concentrations of PRO (~71.3 pglml), Ez (~81.4 pgfml), and T (~56.0 pg/ml). No significant seasonal changes in gonadal steroids were detected in these females. By July, the ovaries of reproductively active females contained hundreds of approximately 1 S-cm (diameter) vitellogenic follicles. Of the nine sonic transmitter-tagged females, five were bled six to nine times over a 3- to 4-month period and one was sampled four times over a 2month period. The other three turtles either had transmitter malfunctions or they left the reef shortly after their initial capture and tagging. The six females which were repeatedly bled over at least a 2-month period migrated away from Heron Atoll during a period ranging from mid-October through early November. Figure 2 shows the mean E, and T concentrations of the transmitter-tagged female C. caretta prior to migration. Both E, and T concentrations exhibited significant changes relative to time (P < 0.01). Serum E, was significantly greater during September and the first half of October than concentrations during the last half of August (P < 0.05). Serum T concentrations during October were signifi-

158

WIBBELS TABLE

SPERMATOGENIC

STAGES OBSERVED

IN LOGGERHEAD

ET AL. 1 SEA TURTLES

CAPTURED

ON HERON

ISLAND

REEF“

Month and number of turtles observed

Stage

Seminiferous tubules

1

Involuted with only spermatogonia; spermatozoa may be present in lumen Primary spermatocytes present; spermatogonia becoming abundant Secondary spennatocytes and early spermatids abundant Transforming spermatids and some spermatozoa Spermatids and spermatozoa abundant Maximal spermiogenesis Spermatozoa abundant; spermatids and spennatocytes reduced Few spermatozoa; spermatids and spermatocytes may be absent; spermatozoa may be abundant in lumen

8

JFMAMJJASOND 5 1

2

5 3 2 6 4

4

1 2 7 4

3 1

3

a Adapted from Licht (1967) and McPherson et al. (1982).

cantly greater than concentrations from mid-July through September (P < 0.05). The five turtles that were most comprehensively sampled exhibited similar patterns of changes in T and E, concentrations as exemplified by turtle X54 (Fig. 3); circulating E, increased approximately 4 to 6 weeks prior to migration and then decreased as circulating T increased until (at least) the time of migration. In contrast to E, and T, PRO remained low or nondetectable (~65.0 pg/ml) throughout the premigratory period and significant fluctuations were not detected. Serum gonadal steroid concentrations during each nesting of turtle X54 are shown in Fig. 4. Serum E,, T, and PRO were relatively high in this turtle during the first three nestings of the season, but were low or nondetectable during the last nesting. Blood samples were obtained from five turtles at Mon Repos or Heron Island during their first nesting of the season and from seven turtles sampled during their last nesting of the season. Concentrations of the three steroids during the first nesting of the season (E,, Sz= 168.8 f 48.7 pg/ml; T, X = 528.2 2 81.2 pglml; PRO, X = 799.2 f 261.2 pg/ml) were significantly higher than con-

centrations during the last nesting of the season (P < O.OOl), when E2 and T were low or nondetectable (~50 pglml) and PRO ranged from nondetectable to 176.3 pg/ml. Concentrations of T, E,, and PRO of nesting C. caret& from Melbourne Beach, Florida, are shown in Fig. 5. The nesting concentrations of all three of these steroids exhibited significant changes over the nesting season (P < 0.05). Serum E, and T concentrations during August were significantly lower than in June and July (P < 0.05). Progesterone concentrations during the latter part of August (i.e., the end of the nesting season) were significantly lower than those during June and July (P < 0.05). DISCUSSION

The results indicate that male C. caret& possess a prenuptial spermatogenic pattern that is coincident with increased circulating testosterone. Sperm are produced during the winter and early spring and are stored in the epididymides until spring mating. Similar data have been recorded for a captive sea turtle, C. mydus (Licht et al., 1979; 1985b). Additionally, the serum T and lap-

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HORMONES

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TURTLES

, TESTOSTERONE A ESTRADIOL 0

JUL

SEP

NOV

3. Serum estradiol and testosterone concentrations of turtle X54 prior to migration. The asterisks indicate points represented by lowest sensitivity of the assay; actual values were below assay sensitivity. FIG.

\z v

~1000 a

ter turtles (Callard et al., 1976; McPherson et al., 1982; Mendonca and Licht, 1985). Serum T in males remained high through October and into November, corresponding to the courtship and mating period for these turtles (Limpus, 1985). Several past studies have also recorded circulating T in male chelonians during the mating season. Serum T in male C. carettu along the At-

1

E: z g

50.0

-

2

0.0 1

JUN

JUL

AUG

SEP OCT

NOV

FIG. 2. Mean serum estradiol and testosterone concentrations of adult female loggerheads, C. caretru, captured on Heron Island Reef. These females were vitehogenic in preparation for the approaching breeding season. Vertical bars are standard errors of the mean. Progesterone was low (<65.0 pg/ml) during this period and did not exhibit significant changes. Both estradiol and testosterone exhibited significant changes over time (P < 0.01).

aroscopy data suggest that all of the male C. caretta sampled were reproductively active, indicating that in contrast to females, male C. carettu from Heron Island Reef can be annual breeders. Serum T in males remained high during spermatogenic stages four through seven and then dropped to significantly lower concentrations during stages eight, one, two, and three. These results are consistent with those for freshwa-

~600., ho a

m

TESTOSTERONE

0

ESTRADIOL

legal PROGESTERONE

-5000 5 F: 2 4000 E e

300.0

5 u w 2000 5 2o

100.0

3: 00

FIG. 4. Serum estradiol, testosterone, and progesterone concentrations during each nesting of turtle x54.

160

WIBBELS

n=7

n=4

o.oi

fL

6000

400 0 /2 z E 2

2000

5 E

-L

0.0 2000 n=7 T

<-

150.0

1

2 1 500

n=lO

T

ril

n=16

n=4

0.0

.$+

3

\&

+

2’

i -2 ,5”

+

9

ET AL.

lantic coast of Fiorida appears high during the start of the suspected courtship and mating season (Wibbels et al., 1987). In contrast to our results, serum T decreases during the start of mating season in captive male C. my&s, and maximal mating occurs when T concentrations are at their nadir (Licht et al., 1985b). Nevertheless, Licht et al. (1985b) detected a positive correlation between the duration of mating and the maximal T concentrations approximately 1 month prior to the mating season. Serum T concentrations of male C. my&s captured while mating off the Pacific coast of Mexico were not consistent with those of the captive males since they were relatively high (21.3 + 3.4 rig/ml; Licht et al., 1980) as were the serum T concentrations of two male olive ridley sea turtles, Lepidochelys olivuceu, captured while mating off the Pacific coast of Mexico (Licht et al., 1982). Additionally, Owens (1976) has shown that T injections stimulate mounting behavior in immature C. mydus. Thus, T appears to be a likely candidate for stimulating mating behaviors in male sea turtles. Several male freshwater and terrestrial chelonians also mate when circulating T is high (Callard et al., 1976; McPherson et al., 1982; Ganzhorn and Licht, 1983; Licht et al., 1985a; Mendonca and Licht, 1985); however, in a few chelonians, mating may occur when T is low (Lofts and Tsui, 1977; Silva et al., 1984). The high T concentrations during October and early November also coincide with the migration of some males to specific breeding locations; however, some males appear to be year-round residents at Heron Island Reef (Limpus, 1985). Past studies have recorded elevated T concentrations in other chelonians during migratory periods.

d+

FIG. 5. Serum estradiol, testosterone, and progesterone concentrations of loggerhead sea turtles, C. caretta, nesting at Melbourne Beach, Florida. Vertical bars are standard errors of the mean. Serum estradiol and testosterone concentrations during June and July

were significantly greater than those in August (P < 0.05). Serum progesterone concentrations during June and July were significantly greater than those during the latter half of August (P < 0.05).

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HORMONES

Male C. caretta from the Atlantic coast of Florida have high serum T when they migrate into an area adjacent to a major nesting beach during early spring (Wibbels er al., 1987). In freshwater red-ear turtles, Chrysemys picta, the spring peak recorded for males (Callard et al., 1976; Ganzhorn and Licht, 1983; Licht et al., 1985a) may coincide with interpond breeding migrations (Gibbons, 1968). Thus, the results indicate that T could affect a variety of reproductive events, including spermatogenesis, migration, courtship, and mating. Female C. caretta have a multiannual reproductive cycle (Limpus, 1985). Generally speaking, about every 3 years, a female will nest approximately four times at 2-week intervals. The results indicate that adult females that were not going to nest in the upcoming nesting season had relatively low concentrations (compared to turtles that were going to nest) of all three steroids. However, a few of these females had E, and T concentrations that were notably higher than the others, suggesting that there may be some ovarian steroid activity during nonnesting years. Reproductively active females had hundreds of relatively large vitellogenic follicles in July. Precisely when vitellogenesis began was not addressed in this study; however, observations by Limpus (1985) suggest that vitellogenesis was occurring over the previous 4 months. Approximately 1-2 months prior to migration the females exhibited increased serum E, concentrations. Estradiol-178 appears to be the primary stimulus for vitellogenesis in reptiles (reviewed by Ho, 1977), and Owens (1976) has shown that E, injections stimulate vitellogenesis in the sea turtle C. mydas. Collectively, these data suggest that, in C. caretta, vitellogenesis occurs during a period of approximately 7-8 months prior to migration, with at least one period of high activity about l-2 months prior to migration. The laparoscopy data support this hypothesis since vitellogenic follicles during July and August (approximately 4 to 5

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months prior to nesting) appear to be approximately 1 cm in diameter less than those at the time of nesting (Limpus, 1985). In the reproductively active females, serum T increased as E, decreased prior to migration. Thus, as with males, migratory physiology and/or behavior may be affected by changes in serum T. Since evidence suggests that mating occurs during and/or immediately after migration in C. caretta (Limpus, 1985; Wibbels et al., 1987), T could also influence courtship and mating behavior. Licht et al. (1979) found that T concentrations were high during heightened receptivity in female C. mydas. High circulating T has also been recorded in female freshwater turtles during the mating season (Callard et al., 1976; McPherson ef al., 1982).

The nesting concentrations of E,, T and PRO of turtle X54 (Fig. 4) indicate that during all but the last nesting of the season, serum concentrations of these steroids are relatively high. Similar data were obtained for turtles during their first or Iast nestings at Mon Repos and Heron Island, and for turtles nesting at Melbourne Beach. The relatively high E, concentrations during early season nestings suggest that vitellogenesis may continue into the nesting season. Serum PRO as well as serum T concentrations at the time of nesting may be related to ovulation, which has been shown to occur approximately 2-3 days after nesting (Licht et al., 1979, 1980, 1982). Licht et al. (1979) suggest that PRO may initiate the periovulatory surge in serum-luteinizing hormone (LH) in sea turtles. By the final nesting of a season, ovarian production of E, and T is minimal, and although circulating PRO is lower than during previous nestings, it is generally higher than before migration, suggesting that the ovary is still actively producing PRO. If PRO mediates the periovulatory LH surge then the concentrations near the time of the last nesting must be lower than the threshold concentration necessary to trigger LH

162

WIBBELS

release. On the other hand, T may be a more likely candidate for this function. The results indicate that T is high during nestings that will be followed by ovulation, but it is minimal during the final nesting of the season. Furthermore, in C. mydas, it has been shown that serum T is relatively low during much of the nesting cycle, but increases during a period that includes nesting and immediately precedes the periovulatory LH surge (Licht et al., 1979). The reproductive patterns recorded for both male and female C. caretta are generally similar to those recorded for captive C. mydas (Licht et al., 1979; 1985b); however, two differences are notable. In contrast to captive female C. mydas, the females in this study exhibit a significant decrease in serum Ez approximately 2 weeks prior to migration. The lack of a premigratory decrease in E, could represent an interspecific difference in steroid hormone dynamics or it could relate to the absence of migration in captivity. In males, serum T in C. caretta was at its peak during mating season in contrast to captive C. mydas. However, it is interesting to note that the serum concentrations of T were generally much higher in captive male C. mydas, peaking at approximately 40 rig/ml in contrast to 8 rig/ml for the C. caretta in this study. Furthermore, although serum T had decreased from its peak concentrations, during the middle of the mating period for captive C. mydas, mean concentrations were still approximately IO-20 rig/ml. Thus, serum T concentrations may still have been high enough to stimulate mating behavior. In the study by Licht et al. (1985b), mating intensity was the measure of the number of successful mounts and time spent mounted. However, their data may not take into account the receptivity of the females. It is possible that males may have attempted to mount other turtles when their serum T was maximal, but, due to the lack of receptive females, they may not have been successful.

ET AL.

The observed differences between the serum T concentrations of male C. caretta and the captive male C. mydas could relate to captivity or to interspecific variation. The mean serum T concentration of 27 mating C. mydas from a natural population was 21.3 rig/ml late in the nesting season (Licht et al., 1980), suggesting that indeed, C. mydas may naturally have higher concentrations of T than C. caretta. However, the fact that those 27 males were at a mean of 21.3 rig/ml suggests that their serum concentrations may still have been relatively high late in the mating season. Thus, the androgen cycle of the captive C. mydas may be shifted forward in time relative to naturally occurring males and possibly relative to naturally occurring (or even captive) females. It is plausible that a factor such as decreased thermal fluctuation in captivity (Licht et al., 1985b) could affect the seasonal timing of the gonadal recrudescence and T production. Temperature has been shown to be a major cue for gonadal recrudescence in freshwater turtles (Ganzhorn and Licht, 1983; Mendonca 1987a, b). In contrast to captive male C. mydas, seasonal changes in serum steroids of captive females appear temporally similar to those of naturally occurring females sea turtles. Although captive females are exposed to the same environmental stimuli as the captive males, it may not be possible for females to accelerate their gonadal recrudescence to the extent males can due to the energy requirements necessary to develop hundreds of large vitellogenic follicles. In summary, the results of this study indicate that both male and female C. caretta have prenuptial patterns of gonadal recrudescence with temporally associated gonadal steroid production and reproductive behaviors. Crews (1987) indicates that in mild environments it is common to find vertebrates with this type of reproductive cycle. In contrast, many temperate-zone freshwater and terrestrial chelonians exhibit postnuptial gonadal recrudescence

STEROID

HORMONES

(Callard et al., 1976; Lofts and Tsui, 1977; Kuchling et al., 1981; Licht, 1982; McPherson er al., 1982; Silva et al., 1984; Licht et al., 1985; Mendonca and Licht, 1985). It is plausible that cold winters in temperate zones may have selected for the postnuptial reproductive pattern in many chelonians, whereas the mild temperatures in tropical and subtropical oceans may have facilitated the retention of a prenuptial reproductive pattern in sea turtles. Consistent with this hypothesis is that fact that at least one tropical freshwater turtle, Lissemys punctata, also exhibits a prenuptial reproductive pattern (Singh, 1977). Finally, the results of this study indicate that serum steroid concentrations could be used as a conservational tool for studying sea turtle populations. At present there are conservation programs throughout the world intensely studying the reproductive ecology of sea turtles in endangered and threatened populations (Bjorndal, 1981). Analysis of gonadal steroid concentrations of turtles captured in those programs could indicate the reproductive status of individuals and thus provide a method of predicting the percentage of a given population that is reproductively active during a specific year. ACKNOWLEDGMENTS The study of sea turtles in southern Queensland is conducted as part of the Queensland Sea Turtle Research Project of the Queensland National Parks and Wildlife Service. The Queensland National Parks and Wildlife Service rangers on Heron Island and the faculty and staff at the University of Queensland Heron Island Research Station graciously provided assistance upon request. We would also like to acknowledge Diana Crowell-Comuzzie, Tony Comuzzie, Robert Figler, Nancy Figler, Valona Baker, John Parmenter, and Leisa Fein for their dedicated assistance during turtle capture and laparoscopy. The research at Melbourne Beach, Florida, would not have been possible without the collaboration of Lewellyn M. Ehrhart and his colleages Paul Raymond, Cori Etchberger, Lawrence Luepschen, and Cleve Doe. This study was made possible through grants from the National Science Foundation (Grant BNS-8418538), the Texas

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A&M Sea Grant College (U.S. Department of Commerce) (Grant NA85AA-D-SGl28), and through Queensland National Parks and Wildlife Service and Marine Science and Technology Grant funding for the Queensland Sea Turtle Research Project. We would like to acknowledge the Texas A&M Sea Grant College for providing a University Marine Fellowship for T. Wibbels.

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hi&chemical changes in gonads and epididymides of the male softshell turtle, Trionyx sinensis. .I. Zool. 181, 57-58. Mather, P., and Bennett, I. (1984). “A Coral Reef Handbook.” The Australian Coral Reef Society, Brisbane, Australia. McPherson, R. J., Boots, L. R., MacGregor, R., III, and Marion, K. R. (1982). Plasma steroids associated with seasonal reproductive changes in a multiclutched freshwater turtle, Sternotherus odoratus. Gen. Camp. Endocrinol. 48, 440-451. Mendonca, M. T. (1987a). Photothermal effects on the ovarian cycle of the musk turtle, Sternotherus odoratus. Herpetologica 43, 82-90. Mendonca, M. T. (1987b). Timing of reproductive behaviour in male musk turtles, Sterotherus odoratus: Effects of photoperiod, temperature and testosterone. Anim. Behav. 35, 1002-1014. Mendonca, M T., and Licht, P. (1985). Seasonal cycles in gonadal activity and plasma gonadotropin in the musk turtle, Sternotherus odoratus. Cert. Comp. Endocrinol. 62,459-469. Owens, D. W. (1976). “Endocrine Control of Reproduction and Growth in the Green Sea Turtle, Chelonia mydas.” Ph.D dissertation, Univ. of Arizona, Tucson. Silva, A. M. R., Moraes, G. S., and Wasserman, G. F. (1984). Seasonal variations of testicular morphology and plasma levels of testosterone in the turtle Chrysemys dorbingni. Comp. Biochem. Physiol. 78, 153-157. Singh, D. P. (1977). Annual sexual rhythm in relation to environmental factors in a tropical pond turtle, Lissemys punctata granosa. Herpetologica 33, 190-194. Sokal, R. S., andRohlf, F. J. (1981). “Biometry,” 2nd ed. Freeman, San Francisco. Wibbels, T., Owens, D. W., and Amoss, M. (1987). Seasonal changes in serum testosterone of loggerhead sea turtles captured along the Atlantic coast of the United States. In “Ecology of East Florida Sea Turtles” (W. WitzelI, Ed.), pp. 59-64. U.S. Dept. Commer., NOAA Tech., Rep. NMFS 53. Wood, J. R., and Wood, F. E. (1980). Reproductive biology of captive green sea turtles, Chelonia mydas. Amer. Zool. 20, 499-505. Wood, J. R., Wood, F. E., Critchley, K. H., Wildt, D. E., and Bush, M. (1983). Laparoscopy of the green sea turtle, Chelonia mydas. Brit. J. Herpatol. 6, 323-327.