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Undifferentiated spermatogonia (Aund) or spermatogonial stem cells (SSCs) are committed to the establishment and maintenance of spermatogenesis and fertility throughout a male’s life and are located in a highly specialized microenvironment called niche that regulates their fate. Although several studies have been developed on SSCs in mammalian testis, little is known about other vertebrate classes. The present study is the first to perform a more detailed investigation on the spermatogonial cells and their niche in a reptilian species. Thus, we characterized Aund/SSCs and evaluated the existence of SSCs niche in the Kinosternon scorpioides, a freshwater turtle found from Mexico to northern and central South America. Our results showed that, in this species, Aund/SSCs exhibited a nuclear morphological pattern similar to those described for other mammalian species already investigated. However, in comparison to other spermatogonial cell types, Aund/SSCs presented the largest nuclear volume in this turtle. Similar to some mammalian and fish species investigated, both GFRA1 and CSF1 receptors were expressed in Aund/SSCs in K. scorpioides. Also, as K. scorpioides Aund/SSCs were preferentially located near blood vessels, it can be suggested that this niche characteristic is a well conserved feature during evolution. Besides being valuable for comparative reproductive biology, our findings represent an important step towards the understanding of SSCs biology and the development of valuable systems/tools for SSCs culture and cryopreservation in turtles. Moreover, we expect that the above-mentioned results will be useful for reproductive biotechnologies as well as for governmental programs aiming at reptilian species conservation.

Prolactin (PRL) plays crucial roles in many physiological and pathological processes through activating its specific membrane-anchored receptor (PRLR). Although this ligand-receptor pair has been extensively studied in mammals, birds and fishes, researches examining their significance is rather scarce in reptiles. Additionally, the interaction mechanism of PRL-PRLR has abortively understood across vertebrates, since two tandem repeated ligand-binding domains of PRLR have been identified in birds and few reptiles. To lay the foundation to clarify their roles and ligand-receptor interaction in reptiles, using Chinese soft-shelled turtle as model, the cDNAs containing open reading frame of PRL and PRLR were cloned. The cloned PRL consisted of 710 bp and encoded a precursor of 228 amino acid (-aa), while PRLR was 2658 bp in length and predicted to generate a 828-aa precursor. Furthermore, the recombinant PRL protein with high-purity was prepared from Escherichia coli (E. coli) strain Rosetta gamiB (DE3) by using cobalt resin. Using the 5 × STAT5-Luciferase reporter system, we found PRLR and PRLR-M2 (the PRLR-mutant lacking membrane-distal ligand-binding domain) could be dose-dependently activated by recombinant PRL, thereby triggering the intracellular JAK2-STAT5 signaling cascade, suggesting PRL-PRLR is functional in Chinese soft-shelled turtle, and the membrane-proximal ligand-binding domain of PRLR is the critical domain involving in PRL-binding. Quantitative real-time PCR revealed that PRL was predominantly and abundantly expressed in pituitary, while PRLR exhibited ubiquitous expression in all of the tissues examined. Collectively, our data indicate the PRL-PRLR pair may function in reptiles including Chinese soft-shelled turtle, in a way similar to that in birds.

Seasonal changes in glucocorticoids and their influence on the immune system are widespread in vertebrates, but whether this occurs in lesser studied taxa like turtles is unclear. The purpose of this study was to test for seasonal changes in baseline corticosterone (CORT), innate immunity, body condition, and metabolic factors (triglycerides, uric acid) in free-ranging Eastern Box Turtles (Terrapene carolina carolina), a species which is in decline across its range. In addition, the effect of handling and confinement on CORT levels was measured. In both years of the seasonal study, baseline CORT concentrations were significantly higher in fall than in spring, but summer CORT levels differed between the years. The annual variation in CORT levels may be related to weather conditions or associated with opportunistic mating. CORT levels of turtles confined for one hour were higher than turtles bled as quickly as possible, and unlike the seasonal study, females had higher baseline and post-stressor CORT levels than males. Baseline CORT levels were positively correlated with hemolysis titer, indicating a possible immunoenhancing effect of CORT. Triglycerides were positively correlated with body condition and were higher in females than males. The higher triglyceride level in females was likely associated with the energetic demands of reproduction and nesting. Males had a lower body condition in the fall than in both spring and summer, while females exhibited no seasonal differences in body condition. Uric acid and innate immunity measures failed to exhibit significant seasonal or sex related differences. Overall our findings of substantial annual, seasonal, and sexual variation in turtle physiology are consistent with the findings reported for other vertebrates and indicate the importance of incorporating these sources of variation into the design of future studies.

Chinese soft-shelled turtle Pelodiscus sinensis is an important aquaculture species in China, the male individual being more valuable in aquaculture because of its larger body size, higher growth rate and less fat compared with females. Understanding the mechanism of ovarian differentiation and development is crucial for the production of mono-sex male offspring. However, little is known about the molecular mechanism underlying turtle ovarian differentiation. Here, we characterized the Rspo1 gene, an upstream regulator of vertebrate female sexual differentiation, in P. sinensis. The messenger RNA of Rspo1 was initially expressed at stage 14, preceding gonadal sex differentiation, and exhibited a sexually dimorphic expression pattern throughout the sex determination and gonadal differentiation periods. Rspo1 was rapidly downregulated during aromatase inhibitor-induced female-to-male sex reversal, which occurred prior to gonadal differentiation. Rspo1 loss of function by RNA interference led to partial female-to-male sex reversal, with masculinized changes in the phenotype of gonads, the distribution of germ cells and the expression of testicular regulators. Collectively, these findings suggest that Rspo1 is necessary for primary female sexual differentiation in P. sinensis. This study demonstrates for the first time the functional role of Rspo1 in reptilian sex determination, and is of fundamental significance for the production of fertile pseudo-female parents and mono-sex male offspring of P.sinensis.

Populations of wide ranging ectotherms often exhibit variation in traits that are influenced by local environmental conditions. Although the gopher tortoise, Gopherus polyphemus , is well studied in pine flatwoods habitats across their range, little attention has been given to coastal populations existing in the southern extreme portion of the range. We examined the reproductive physiology of a coastal dune population in southwest Florida to determine if reproductive cycles vary across populations. Here we present the first year-round sex hormone profiles for a wild population of gopher tortoises. Male testosterone concentrations varied across the year ( F 11,54 = 2.52, P = 0.015) with elevated values from September to December and minimal levels from April to July, with the exception of a secondary peak during the month of June. Female testosterone and estradiol concentrations varied across the sampling period (T: F 11,66 = 8.54, P F 11,66 = 4.57, P F 11,64 = 3.29, P = 0.002) and increased in late fall with a peak in March. These data suggest this population has an extended breeding season from fall through spring with mating likely occurring from September through March, and nesting in winter through spring. This pattern is similar to reproductive patterns described for tropical and sub-tropical chelonians but differs from that of gopher tortoise populations in northern portions of the range where hibernation may last for five months and a single clutch of eggs are deposited in late spring.

Sex determination in vertebrates depends on the expression of a conserved network of genes. Sea turtles such as Lepidochelys olivacea have temperature-dependent sex determination. The present work analyses some of the epigenetic processes involved in this. We describe sexual dimorphism in global DNA methylation patterns between ovaries and testes of L. olivacea and show that the differences may arise from a combination of DNA methylation and demethylation events that occur during sex determination. Irrespective of incubation temperature, 5-hydroxymethylcytosine was abundant in the bipotential gonad; however, following sex determination, this modification was no longer found in pre-Sertoli cells in the testes. These changes correlate with the establishment of the sexually dimorphic DNA methylation patterns, down regulation of Sox9 gene expression in ovaries and irreversible gonadal commitment towards a male or female differentiation pathway. Thus, DNA methylation changes may be necessary for the stabilization of the gene expression networks that drive the differentiation of the bipotential gonad to form either an ovary or a testis in L. olivacea and probably among other species that manifest temperature-dependent sex determination.

Steroids hormones such as estradiol-17β (E2) and testosterone (T) are involved in gonadal differentiation of oviparous animals with temperature-dependent sex determination (TSD), and are greatly distributed. This hypothesizes that these embryonic steroid hormones probably accumulate in the eggshell throughout blood or/and chorioallantoic fluid in sea turtle species with TSD, producing females at higher temperature. To demonstrate this hypothesis, concentrations of E2 and T in the blood plasma from the hatchling loggerhead sea turtle (Caretta caretta) and in their eggshells were measured by radioimmunoassay. In the present study we propose that both concentrations of E2 and T in the blood plasma are correlated with amounts of these sex steroids in the eggshell. Moreover, contents of E2 in the eggshell showed a significant positive correlation with mean incubation temperatures during a thermosensitive period in the experimental nests, whereas T contents in the eggshell did not. Taken together, these findings indicated that embryonic E2 and T that accumulated in the eggshell can be extracted and measured. Furthermore, the present study suggested that contents of E2 in the eggshell may differ between male and female, and monitoring of these steroids is a useful method to identify the sex of loggerhead sea turtle hatchling.

Environmental chemicals can disrupt endocrine signaling and adversely impact sexual differentiation in wildlife. Bisphenol A (BPA) is an estrogenic chemical commonly found in a variety of habitats. In this study, we used painted turtles (Chrysemys picta), which have temperature-dependent sex determination (TSD), as an animal model for ontogenetic endocrine disruption by BPA. We hypothesized that BPA would override TSD and disrupt sexual development. We incubated farm-raised turtle eggs at the male-producing temperature (26°C), randomly assigned individuals to treatment groups: control, vehicle control, 17β-estradiol (E2, 20ng/g-egg) or 0.01, 1.0, 100μgBPA/g-egg and harvested tissues at hatch. Typical female gonads were present in 89% of the E2-treated "males", but in none of the control males (n=35). Gonads of BPA-exposed turtles had varying amounts of ovarian-like cortical (OLC) tissue and disorganized testicular tubules in the medulla. Although the percentage of males with OLCs increased with BPA dose (BPA-low=30%, BPA-medium=33%, BPA-high=39%), this difference was not significant (p=0.85). In all three BPA treatments, SOX9 patterns revealed disorganized medullary testicular tubules and β-catenin expression in a thickened cortex. Liver vitellogenin, a female-specific liver protein commonly used as an exposure biomarker, was not induced by any of the treatments. Notably, these results suggest that developmental exposure to BPA disrupts sexual differentiation in painted turtles. Further examination is necessary to determine the underlying mechanisms of sex reversal in reptiles and how these translate to EDC exposure in wild populations.

Steroid hormones affect sex determination in a variety of vertebrates. The feminizing effects of exposure to estradiol and the masculinizing effects of aromatase inhibition during development are well established in a broad range of vertebrate taxa, but paradoxical findings are occasionally reported. Four independent experiments were conducted on two turtle species with temperature-dependent sex determination (Chrysemys picta and Chelydra serpentina) to quantify the effects of egg incubation temperature, estradiol, and an aromatase inhibitor on offspring sex ratios. As expected, the warmer incubation temperatures induced female development and the cooler temperatures produced primarily males. However, application of an aromatase inhibitor had no effect on offspring sex ratios, and exogenous applications of estradiol to eggs produced male offspring across all incubation temperatures. These unexpected results were remarkably consistent across all four experiments and both study species. Elevated concentrations of estradiol could interact with androgen receptors or inhibit aromatase expression, which might result in relatively high testosterone concentrations that lead to testis development. These findings add to a short list of studies that report paradoxical effects of steroid hormones, which addresses the need for a more comprehensive understanding of the role of sex steroids in sexual development.

The Eastern Box Turtle ( Terrapene carolina carolina ) is a widespread species that has recently experienced precipitous declines throughout its range. Although many studies have documented aspects of reproduction in box turtles, reproductive physiology of free-ranging animals is unknown and can be crucial in this species’ recovery. Over a two-year period, we measured reproductive parameters, (vitellogenin [Vtg], estradiol-17β [E2], and total testosterone [TT]), in plasma of 116 free-ranging Eastern Box Turtles across their active season. We found similar seasonal variations of Vtg and E2 within females. Mid-season, females showed a sharp peak in E2 that correlates with the putative beginning of the ovarian cycle. Individual females lacking these expected peaks of both Vtg and E2 suggest that some female T. c. carolina may not reproduce annually. Females typically expressed undetectable levels of TT, yet there was a small peak in TT early in the active season. Male Eastern Box Turtles exhibited a dual peak in TT. Elevated TT in males was significantly associated with observed sexual behaviors and smaller home ranges. Body condition had no effect on the concentration of TT or E2 in either sex. This is the first study to (1) document Vtg and sex steroid hormones in free-ranging animals of this genus, and (2) relate those metrics to individuals, the population, the purported annual cycle, and to other chelonian species.

The critically endangered Radiated Tortoise (Astrochelys radiata) is endemic to the southern coastlines of Madagascar. Once common, wild populations of this tortoise have undergone dramatic declines in recent years. Although there have been studies documenting reproductive activities, reproductive physiological parameters are unknown yet may be crucial in the recovery of the species. Over four research seasons in remote field locations native to A. radiata, we surveyed for, radio-tracked, and sampled wild, free ranging tortoises. We sampled and measured stress and reproductive parameters (corticosterone [CORT], testosterone [T], estradiol-17β [E2], and progesterone [P]) in 311 plasma samples from 203 wild A. radiata, capturing their active period. Generally, hormone concentrations were associated with body condition, temperature, and humidity. There was wide variation in CORT that varied monthly and by group. Juvenile tortoises maintained more than twice the mean basal CORT concentrations than either adult sex, with the most dramatic distinctions in the middle of the wet season. For adult sex hormones, the last months of the dry season and into the wet season when ground humidities are low and just begin to rise prior to temperature declines, male T concentrations gradually increased to a peak before returning to near undetectable values into the dry season. We had limited data for T concentrations in females, but found average T concentrations were much lower than in males and positively correlated with larger female home range sizes. For female hormone cycles, E2 also peaked in the early 1/3 of the wet season along with male T, and was followed by an uptick in P which correlates to the putative ovulatory cycle. Females tracked over four years showed variation in patterns of P, indicating that number and frequency of clutches vary. Our results suggest that 1) there is high species plasticity in response to stress; 2) A. radiata reproductive cycling is somewhat dissociated with courtship timing and is instead triggered by environmental cues; and 3) individual female reproductive output is irregular. This study is oone of the first to document and describe multi-year seasonal stress and reproductive hormones in a free-ranging Malagasy chelonian. These data may be used to identify key high-production habitats for conservation, and aide in captive management and reproduction in assurance colonies for species health and survival.

The stimulatory effect of testosterone on male sexual activity is one of the clearest examples linking hormones and behaviors. However, this relationship is complex in Chelonians. We experimentally studied the influence of testosterone levels on the activity budget and space use in male Greek tortoises (Testudo graeca graeca) during the spring mating season. We first described the annual pattern of changes in plasma testosterone levels in free-ranging animals in Morocco. Two peaks, one in winter and one in summer, corresponded to periods of inactivity; whereas mating periods in spring and to a lesser extent in autumn were associated with low plasma testosterone levels. Second, we experimentally manipulated plasma testosterone levels in free-ranging males, and analyzed the behavioral consequences. The strong contrasts in plasma hormone levels induced by the experimental treatments did not result in changes in activity budget or space use, both in the short-term or more than one month after the beginning of the hormonal treatment. Our results suggest that testosterone levels did not influence directly behavioral activity in this species, either immediately or after a time delay of one month.

Maternally derived yolk steroids have been found to elicit both short-term and long-term effects on offspring phenotype. Paradoxically, their effects can be strikingly specific given the often substantial concentrations present at oviposition, and they do not appear to uniformly affect all steroid-sensitive processes. To better understand the dynamics of yolk steroids across embryonic development, we quantified levels of progesterone, testosterone, and estradiol at 5-day intervals throughout development in eggs of the red-eared slider turtle (Trachemys scripta) incubated at both male- and female-producing temperatures. We also assessed the effect of season on yolk steroid levels. For all steroids assayed, the concentrations in yolk declined significantly by day 15 of embryonic development despite large differences in initial concentrations among steroids. We found that estradiol was the only steroid whose initial concentration varied significantly with season, while only the decline in testosterone was affected by incubation temperature. These findings illustrate the complex nature of yolk steroid dynamics and suggest that maternal steroids may be rapidly degraded or subject to embryonic processing, emphasizing the need for studies aimed at understanding the mechanisms through which yolk steroids may elicit their effects.

Plasma concentrations of stress hormones [adrenaline (ADR), noradrenaline (NR)], lactate, glucose and CO2 were monitored during peak nesting period (May-October) at different phases of nesting in the green turtle, Chelonia mydas. These include, emergence from sea, excavating body and nest chambers, oviposition, covering and camouflaging the nest and then returning to sea. Turtles that completed all phases of nesting including oviposition before returning to sea were considered "successful" turtles, while those that completed all phases but failed to lay their eggs were "unsuccessful". Blood samples were taken from the cervical sinus within 5min of capture to avoid stress due to handling. The turtles were usually sampled for blood between 20:00 and 1:00h of nesting time to ensure uniformity in the sampling. Plasma ADR and NR values were highly significant (P

Portions of the reproductive tract of the male (Trachemys scripta) turtle were examined by immunocytochemistry for evidence of the capacity to produce and respond to estrogen hormones (via the expression of P450 aromatase and estrogen receptors). Aromatase was detected in both the Sertoli and Leydig cells of the testis and was expressed at different levels during the spermatogenic cycle, being highest in the quiescent testis and lowest during germ cell meiosis. ERα was found in the Leydig cells surrounding the seminiferous tubules as well as in the epithelial cells of the excurrent canals (rete testis, efferent ductule, and epididymis). ERβ immunoreactivity was found in both the spermatogonia and Sertoli cells in the testis, and in the epithelial cells of excurrent canals.

Anthropogenic stressors such as habitat loss are a global problem for wildlife. Coastal development in the United States has replaced estuary shorelines with hard erosion barriers. In Barnegat Bay, New Jersey, the diamondback terrapin (Malaclemys terrapin) encounters these barriers when approaching upland beaches for nesting. To determine the effects of shoreline barriers on this threatened species' nesting abilities, we measured adrenocortical response (i.e., stress response) by comparing natural corticosterone and testosterone levels of 91 terrapins following in situ exposure to either an experimentally blocked, or open nesting beach. In addition, we exposed 15 individuals, from various nesting beaches, to handling stress to identify acute corticosterone secretion, finding a significant increase over 60min to 8ng/ml. Corticosterone did not reach this level in terrapins exposed to barriers. Corticosterone and testosterone levels were not significantly higher among terrapins exposed to barriers compared to those at open reference beaches. This lack of a stress response suggests that terrapins do not physiologically respond to barriers when they approach nesting beaches and thus are not stressed. This may be due to an adaptive trait to help female turtles complete the nesting process despite the natural stresses inherent to coming on land. Our study suggests that this lack of stress response is also applied to non-natural, human made nesting barriers. If terrapins are not physiologically capable of adapting to shoreline barriers, future erosion control structures could support terrapin nesting with periodic upland access points. This endocrinological study provides a more quantitative approach to guiding management of anthropogenic stressors upon wildlife.

A cDNA encoding thyroid stimulating hormone beta-subunit (TSHbeta) was cloned from pituitary of the Chinese soft-shell turtle, Pelodiscus sinensis, and its regulation of mRNA expression was investigated for the first time in reptile. The Chinese soft-shell turtle TSHbeta cDNA was cloned from pituitary RNA by reverse transcription and polymerase chain reaction (RT-PCR), and rapid amplification cDNA end (RACE) methods. The Chinese soft-shell turtle TSHbeta cDNA consists of 580-bp nucleotides, including 67-bp nucleotides of 5'-untranslated region (UTR), 402-bp of the open reading frame, and 97-bp of 3'-UTR followed by a 14 poly (A) trait. It encodes a precursor protein molecule of 133 amino acids with a putative signal peptide of 19 amino acids and a putative mature protein of 114 amino acids. The number and position of 12 cysteine residues, presumably forming six disulfide bonds, one putative asparagine-linked glycosylation site, and six proline residues that are found at positions for changing the backbone direction of the protein have been conserved in the turtle as in other vertebrate groups. The deduced amino acid sequence of the Chinese soft-shell turtle TSHbeta mature protein shares identities of 82-83% with birds, 71-72% with mammals, 49-57% with amphibians, and 44-61% with fish. The Chinese soft-shell turtle pituitaries were incubated in vitro with synthetic TRH (TSH-releasing hormone), thyroxine and triiodothyronine at doses of 10(-10) and 10(-8)M. TRH stimulated, while thyroid hormones suppressed, TSHbeta mRNA levels in dose-related manner. The sequences of cDNA and its deduced peptide of TSHbeta as well as the regulation of its mRNA level were reported for the first time in reptile.

Follicle-stimulating hormone (FSH) is a member of the pituitary glycoprotein hormone family. These hormones are composed of two dissimilar subunits, alpha and beta. Very little information is available regarding the nucleotide and amino acid sequence of FSHbeta in reptilian species. For better understanding of the phylogenetic diversity and evolution of FSH molecule, we have isolated and sequenced the complementary DNA (cDNA) encoding the Chinese soft-shell turtle (Pelodiscus sinensis, Family of Trionychidae) FSHbeta precursor molecule by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The cloned Chinese soft-shell turtle FSHbeta cDNA consists of 602-bp nucleotides, including 34-bp nucleotides of the 5'-untranslated region (UTR), 396-bp of the open reading frame, and 3'-UTR of 206-bp nucleotides. It encodes a 131-amino acid precursor molecule of FSHbeta subunit with a signal peptide of 20 amino acids followed by a mature protein of 111 amino acids. Twelve cysteine residues, forming six disulfide bonds within beta-subunit and two putative asparagine-linked glycosylation sites, are also conserved in the Chinese soft-shell turtle FSHbeta subunit. The deduced amino acid sequence of the Chinese soft-shell turtle FSHbeta shares identities of 97% with Reeves's turtle (Family of Bataguridae), 83-89% with birds, 61-70% with mammals, 63-66% with amphibians and 40-58% with fish. By contrast, when comparing the FSHbeta with the beta-subunits of the Chinese soft-shell turtle luteinizing hormone and thyroid stimulating hormone, the homologies are as low as 38 and 39%, respectively. A phylogenetic tree including reptilian species of FSHbeta subunits, is presented for the first time. Out of various tissues examined, FSHbeta mRNA was only expressed in the pituitary gland and can be up-regulated by gonadotropin-releasing hormone in pituitary tissue culture as estimated by fluorescence real-time PCR analysis.

We purified ghrelin peptide and determined the cDNA sequence encoding the precursor protein from the stomach of the red-eared slider turtle, Trachemys scripta elegans. The Trachemys ghrelin is comprised of 25-amino acids and has the sequence GSSFLSPEYQNTQQRKDPKKHTKLN. The third serine residue was modified by n-octanoic (C8:0), decanoic (C10:0) or unsaturated decanoic acid (C10:1). The carboxyl-terminal end of the peptide was not amidated, as seen in the ghrelins of other land vertebrates. Quantitative real-time PCR analysis revealed high levels of gene expression in the stomach and moderate levels in the large intestine and pancreas. Histochemical studies of turtle stomach revealed that ghrelin-immunopositive (ghrelin-ip) cells, which were small and round, were observed in the mucosal layer of the stomach but not in the myenteric plexus, and ghrelin-mRNA-expressing (ghrelin-ex) cells detected by in situ hybridization were scattered in a similar distribution as ghrelin-ip cells. These results indicate that ghrelin is present in reptiles.

Plasma concentrations of sex steroids (testosterone and progesterone), proteins (total protein and albumin), lipids (phospholipids, cholesterol, and triglycerides), and minerals (calcium and phosphorus) were measured in wild Testudo horsfieldi in Uzbekistan, during the short, 3-month activity period (March-May, 1998). Testosterone concentration in males was highest (52 ng/ml) when they had just emerged from brumation (hibernation) in mid-March, which was also the beginning of the mating period, and fell in April. In females, progesterone peaked in mid-April (at 10 ng/ml), just before ovulation of the first clutches at the end of April and beginning of May. Testosterone levels in females and progesterone levels in males were low (

In reptiles with temperature-dependent sex determination (TSD), the temperature at which the eggs are incubated determines the sex of the offspring. The molecular switch responsible for determining sex in these species has not yet been elucidated. We have examined the dynamics of yolk steroid hormones during embryonic development in the snapping turtle, Chelydra serpentina, and the alligator, Alligator mississippiensis, and have found that yolk estradiol (E(2)) responds differentially to incubation temperature in both of these reptiles. Based upon recently reported roles for E(2) in modulation of steroidogenic factor 1, a transcription factor known to be significant in the sex differentiation process, we hypothesize that yolk E(2) is a link between temperature and the gene expression pathway responsible for sex determination and differentiation in at least some of these species. Here we review the evidence that supports our hypothesis.

Reptilia is the only vertebrate class in which cDNA for the gonadotropin beta subunit precursor molecule has not been cloned. We have isolated the full-length cDNA clone encoding the LH beta subunit precursor molecule and a partial cDNA clone encoding the FSH beta subunit precursor molecule from a pituitary cDNA library of Reeves's turtle. We further clarified the nucleotide sequence of the remaining part of the turtle FSH beta cDNA and that of full-length cDNA encoding the LH beta subunit precursor molecule of the Japanese grass lizard, by means of the 5' rapid amplification of cDNA end (RACE) and 3' RACE. The nucleotide sequence of the turtle FSH beta cDNA we determined was 584 bp long and contained the coding sequence, 5' untranslated region (UTR) and 3' UTR of 396, 34, and 154 bp, respectively. The nucleotide sequence of the turtle LH beta we isolated was 498 bp long and contained the coding sequence, 5' UTR and 3' UTR of 420, 7, and 71 bp, respectively. The nucleotide sequence of the lizard LH beta we determined was 537 bp long and contained the coding sequence, 5' UTR and 3' UTR of 441, 35, and 61 bp, respectively. Amino acid sequences deduced from coding regions of the turtle FSH beta, LH beta and the lizard LH beta were 131, 139, and 146 residues, respectively. Referring to the amino acid sequences of the bullfrog FSH and LH beta subunit molecules determined chemically, we deduced the amino acid sequences of mature peptide. Amino acid sequences of mature peptides of the turtle FSH, turtle LH, and the lizard LH were 111, 112, and 112 residues, respectively. Amino acid sequences of the mature peptides were compared with those of other vertebrates. The amino acid sequence of the turtle FSH beta subunit molecule was 84.7-85.6, 67.8-71.4, and 61.3-62.2% identical to the FSH sequence of birds, mammals, and amphibians, respectively. The amino acid sequence of the turtle LH beta subunit molecule was 51.6-54.6, 36.2-48.7, and 56.3-57.5% identical to the LH sequence of birds, mammals, and amphibians, respectively. The amino acid sequence of the lizard LH beta subunit molecule was 39.1-47.1, 32.9-43.0, and 46.0-47.3% identical to the LH sequence of birds, mammals, and amphibians, respectively. These identity values suggest that the turtle or reptilian FSH beta subunit molecule is more closely related to avian and mammalian FSH beta subunit molecules than to amphibian FSH beta subunit molecules but reptilian LH beta subunit molecules are more closely related to amphibian LH beta subunit molecules than to avian and mammalian LH beta subunit molecules. This discrepancy in the molecular similarity relationship found in the reptilian FSH and LH beta subunit molecules can be interpreted by assuming that evolution speed was not the same among hormone species and also among vertebrate groups.

In the freshwater turtle, the homeostatic control of plasma lipids and lipid-transporting proteins may be coordinately regulated by ovarian steroids and pituitary hormones such as growth hormone (GH). In order to elucidate the role of these hormones in the regulation of vitellogenesis and ovarian growth, we have investigated lipid metabolic changes in normal male and female turtles, and in hypophysectomized females with and without GH injections, in response to combinations of exogenously administered gonadal steroids (estradiol (E2), progesterone (P), and testosterone (T)). Determinations of total plasma triglycerides, cholesterol, vitellogenin, and apoA-I were performed. We have demonstrated that E2 alone and in combination with P significantly increased plasma apoA-I and triglyceride levels in both intact female and male turtles. Testosterone administered alone to males had no effect on any of the parameters measured. In hypophysectomized females, plasma apoA-I, vitellogenin, and triglyceride levels were all significantly elevated in animals which received GH and E2, compared to controls (sham and hypox) and those which received GH alone.

Plasma levels of sex steroids in both males and females of the endangered Hermann’s tortoise ( Testudo hermanni hermanni ) were measured throughout their active period in a wild population in the Massif des Maures, France, and in a nearby captive population at Le Village des Tortues in Gonfaron. Both plasma progesterone and testosterone were elevated in males at emergence from winter dormancy, and plasma progesterone levels were significantly higher in wild than in captive males. Plasma testosterone in males then fell to the lowest levels (10 ng ml −1 ) during the nesting season from April to June followed by an elevated plateau during summer, with levels reaching 80 ng ml −1 , presumably concomitant with spermiogenesis. Plasma testosterone increased in all females during autumn, an indication of follicular growth, and remained high on emergence from hibernation, to peak during April, although levels were lower in the captive population. Plasma progesterone also peaked during April and May, presumably related to ovulation, but, again, these changes were less marked in the captive than in the wild females. Measurements of testosterone, progesterone and 17β-oestradiol in the captive females during their period of oviposition in spring suggested that some females did ovulate and lay eggs, whereas others did not. Differences in sex steroid levels between captive and wild populations of Hermann’s tortoise may indicate a problem with ovulation and/or with stress in a proportion of captive females.

This study investigates the potential effects of maternally derived hormones present in the yolk of reptile eggs. Specifically, we ask when are these hormones utilized by developing red-eared slider turtles (Trachemys scripta elegans), a species with temperature-dependent sex determination (TSD). Eggs were incubated at 27 degrees C, a male-producing temperature, and at 31 degrees C, a female-producing temperature. Concentrations of progesterone, testosterone, and 17beta-estradiol were measured at four points during development: at oviposition, at the start of the temperature sensitive period (TSP), at the end of the TSP, and at hatching. No effects of incubation temperature on yolk hormone concentrations were detected. The highest concentrations of all three hormones were measured at oviposition. Hormone-specific patterns of decline occurred throughout development. Each hormone declined between oviposition and the early TSP. Although estradiol was present in detectable quantities at oviposition, it was virtually undetectable by the early TSP. Testosterone showed no further decline after the early TSP. Progesterone continued to decline between the early and post-TSP. These results demonstrate that maternally derived yolk hormones decline at different rates. Alternative explanations for the disappearance of these yolk hormones are presented.

Previous studies using the fresh water turtle Chrysemys picta have demonstrated that progesterone (P) inhibits estradiol (E)-induced vitellogenin (vtg) secretion in this species. Further, there is evidence for the differential expression of the two P receptor isoforms (PRA and PRB) in the liver during the turtle seasonal cycle, correlating with hepatic vitellogenesis. In this study we report changes in the hepatic PR mPNA, ER mRNA, and vitellogenin (vtg) mRNA transcripts during the reproductive cycle of the turtle. Fragments of the turtle hepatic PR and ER cDNAs were cloned and sequenced and a previously cloned turtle vtg cDNA were used as probes in Northern blotting. No 3.7-kb PR mRNA, corresponding to the smaller PR transcript, PRA of other species was found, although, a smaller 1.8-kb transcript (putative PRC mRNA) was present. These observations suggest that the turtle as in the chicken and human, the 4.5-kb PR mRNA transcript encodes both PRA and PRB proteins. Only the larger PR mRNA transcript (4.5-kb), was found to vary significantly during the annual cycle, being highest when vitellogenesis was inhibited in winter and summer. Vtg mRNA could not be detected during the summer or winter, was highest during vitellogenesis in the spring, and reappeared during the fall period of vitellogenesis and ovarian recrudescence. ER mRNA followed a similar pattern, being highest during spring and early fall, when vtg synthesis is high. The data suggest that P/PR, as well as E/ER, may be involved in the seasonal regulation of hepatic vitellogenesis in this species.

Many oviparous reptiles exhibit temperature-dependent sex determination (TSD); i.e., the temperature at which the egg is incubated determines the sex of the offspring. In TSD reptiles, yolk steroids not only may influence sex determination, but also may mediate hormonal effects on subsequent growth and behavior, as in some avian species. We investigated changes in the levels of estradiol (E(2)) and testosterone (T) during development in yolks of snapping turtle eggs, examined how incubation temperature affects hormone levels, and determined how hormones in turtle eggs are influenced by individual females (=clutch effects). Results indicate significant decreases in both hormones (50% decline) by the end of the sex-determining period, when two-thirds of the development is complete. The declines in both E(2) and T were significantly affected by incubation temperature, but in different ways. Eggs incubated at female-producing temperatures maintained high levels, those incubated at male-producing temperatures had low E(2) values, and eggs incubated at pivotal temperatures had intermediate levels of E(2). At all three temperatures, T values underwent significant but approximately equal declines, except during the developmental stages just after the sex-determining period, when T levels decreased more at the male-producing temperature than at either of the other two temperatures. Initially, there were significant clutch effects in both hormones, but such differences, attributable to individual females, were maintained only for E(2) later in development. Here we report for the first time that incubation temperature significantly affects the hormonal environment of the developing embryo of a turtle with temperature-dependent sex determination. Based on this and related findings, we propose that yolk sex steroids influence sexual differentiation in these TSD species and play a role in sex determination at pivotal temperatures.

Although yolk steroids appear to play important roles in the development, growth, and behavior of some birds, their effects in oviparous reptiles are largely unknown. These investigations were initiated to determine initial levels of steroid hormones in the yolks of eggs from two turtle species. Clutches of snapping turtle (Chelydra serpentina) and of painted turtle (Chrysemys picta) eggs were collected and individual egg yolks were analyzed for estradiol-17beta (E(2)) and testosterone (T) using specific RIAs. E(2) and T levels differed significantly between species, the mean E(2) value in snapping turtles was 2.78+/-0.095 (ng/g) compared to 0.89+/-0.064 (ng/g) for painted turtles, and the mean value for T in snapping turtle yolks was 2.56+/-0.098 (ng/g) compared to 0.68+/-0.045 (ng/g) for painted turtles. In addition, E(2) levels were greater than T levels in both species. Within each species, there were significant differences among clutches from different females. E(2) levels in the snapping turtle yolks varied from a clutch mean of 1.38 to 4.55 ng/g and in painted turtles, the clutch means for E(2) varied from 0.34 to 1.34 ng/g. T levels demonstrated similar phenomena within species, with levels in snapping turtles varying from a clutch mean of 0.68 to 4.71 ng/g. Painted turtle levels of T varied from a clutch mean of 0.22 to 0.72 ng/g. There were also significant differences in the E(2)/T ratio, however, E(2)/T ratios did not differ between species. Painted turtle follicles of different sizes showed significant differences in levels of both E(2) and T, and these differences may reflect differing deposition patterns of these steroids in the egg yolk of this turtle during vitellogenesis. The differences in E(2) and T concentration reported here could have important implications for development, growth, and behavior in oviparous reptilian species.

Plasma corticosterone, glucose, and testosterone concentrations were measured in wild, immature specimens of the highly endangered Kemp's ridley sea turtle ( Lepidochelys kempii ) to determine effects of acute handling stress. Thirty-nine free-ranging turtles were captured by entanglement net near the Cedar Keys, Florida. Blood samples were collected immediately after retrieval from the net, and at 30 min ( n = 15) and at 60 min ( n = 29) thereafter. Mean plasma corticosterone and glucose concentrations increased significantly with time. No significant difference was observed over time for mean testosterone concentrations. Approximately half of the turtles demonstrated an increase in plasma testosterone after 60 min of captivity while the others demonstrated a decrease. Initial testosterone concentrations were used to determine the sex of individual turtles. Fifty-nine percent of turtles were classified as female, 33% as male, and 8% as indeterminant. The results of this study demonstrate a responsive hypothalamic-pituitary-adrenal axis and hyperglycemia in immature Kemp's ridley turtles during acute handling stress

The reproductive endocrinology of nesting leatherback turtles (Dermochelys coriacea) was studied during the 1996-1997 and 1997-1998 nesting seasons at Los Baulas National Park, Playa Grande, Costa Rica. Blood samples were collected from nesting females throughout the season. Females were observed to nest up to 10 times during the nesting season. Plasma steroids were measured by radioimmunoassay and total plasma calcium was measured by flame atomic absorption spectrophotometry. Plasma testosterone and plasma estradiol levels declined throughout the nesting cycle of the female. Testosterone declined from a mean of 10.18 +/- 0.77 ng/ml at the beginning of the nesting cycle to 1.73 +/- 0.34 ng/ml at the end of the nesting cycle. Estradiol declined in a similar manner, ranging from a mean of 190.95 +/- 16.80 pg/ml at the beginning of the nesting cycle to 76.52 +/- 12.66 pg/ml at the end of the nesting cycle. Plasma progesterone and total calcium levels were relatively constant throughout the nesting cycle. Lack of fluctuation of total calcium levels, ranging from a mean high of 97.46 +/- 11.37 microg/ml to a mean low of 64.85 +/- 11.20 microg/ml, further suggests that vitellogenesis is complete prior to the arrival of the female at the nesting beach. Clutch size (both yolked and yolkless eggs) did not vary over the course of the nesting cycle.

The aim of the investigation was to study the influence of high ambient temperature on adrenomedullary activity and blood glucose levels in adult female soft-shelled turtles (Lissemys punctata punctata). Experiments were carried out at 25 degrees, 35 degrees, and 38 degrees, and one group was exposed to 38 degrees for 15 days and then maintained at 25 degrees for another 15 days. Exposure to a low ambient temperature of 25 degrees had no clear effect on adrenomedullary function with respect to histology (nuclear diameter), epinephrine and norepinephrine concentrations, and blood glucose level of turtles, but higher temperatures of 35 degrees and 38 degrees stimulated adrenomedullary activity as well as blood glucose level in turtles compared with controls (30 degrees ). The extent of these changes was greater at 38 degrees than that at 35 degrees, and withdrawal from high ambient temperature reversed the effect in turtles.

To characterize seasonal changes in thyroid function in a terrestrial reptile, thyroid hormones were measured over a period of 2 years in desert tortoises, Gopherus agassizii, maintained at the Desert Tortoise Conservation Center in Las Vegas, Nevada. In all samples, triiodothyronine was nondetectable (less than 0.1 ng/ml). Thyroxine (T(4)) exhibited distinct cycles in both sexes, being lowest during hibernation and rising toward the time of emergence. Females exhibited only one peak in T(4), during the early spring. In males, T(4) levels peaked in early spring and again in late summer. The desert tortoise has distinct activity patterns that include increased feeding, mating, and locomotor activity in the early spring and increased mating and combat in the late summer. In an experiment to determine whether food intake influences T(4), food was withheld for 2 weeks. Compared to continuously fed controls, T(4) declined significantly in unfed tortoises, but increased significantly within 36 h of refeeding, indicating that thyroid activity is responsive to nutrient intake. The second seasonal peak of T(4) only in males suggests that male reproductive activity in late summer is associated with thyroid activation. To evaluate this possibility, adult, subadult, and juvenile males were sampled during the months of the second seasonal peak in T(4). Although all three age groups showed similar foraging and thermoregulatory behaviors, T(4) peaked in July only in the reproductively active adults, which also exhibited significantly higher testosterone levels. Elevated T(4) in desert tortoises is thus associated with periods of increased feeding and reproductive activity, supporting a role for thyroid hormones in these energy-demanding activities.

The actions of circulating hormones, although relatively well understood for adults, are largely unknown for their developing embryos. Transfer of maternal hormones to the egg is known to occur in oviparous species, and recently the presence of hormonally heterogeneous yolk layers has been described in two avian species. To investigate the possibility of a similar phenomenon occurring in chelonian species, egg yolk layers were analyzed in the painted turtle (Chrysemys picta marginata) and the red-eared slider turtle (Trachemys scripta elegans), two species that exhibit temperature-dependent sex determination. There was a similar pattern of hormonally heterogeneous yolk layers in both species: concentrations of progesterone and testosterone were significantly higher in the external yolk layer while concentrations of 17beta-estradiol were significantly higher in the intermediate and internal layers. This pattern of hormone deposition concurs with previously published studies of plasma hormone profiles from females of temperate-zone turtle species. Yolks of freshly laid eggs were also sampled using a biopsy technique to examine the concordance of early yolk hormone concentrations and offspring sex. No relationship was found between yolk hormone concentrations and individual offspring sex. Previous work showing that maternally derived yolk estradiol concentrations are correlated with female-biased sex ratios was, however, replicated. These findings suggest that offspring sex is influenced, in part, by the maternal hormone environment.

From April to October of 1996 and 1997, we measured seasonal changes in plasma testosterone (T) in male and plasma T and estradiol-17beta (E(2)) in female yellow-blotched map turtles, Graptemys flavimaculata, from the Pascagoula River, Mississippi. In 1996, plasma from females was analyzed for progesterone. In 1997, ovaries of adult females were ultrasound imaged to determine stages of follicular development. Males exhibited peak T levels in September and October of both years, indicating fall gonadal activity. Males also exhibited a low level spring peak in T during April. Female E(2) levels increased significantly in May and June, the primary period of ovarian development. Females with preovulatory follicles did not have significantly higher E(2) levels than females with medium- or small-sized follicles. When compared to other freshwater turtle species, peak levels of E(2) were low. Testosterone levels did not follow a distinct yearly pattern in females. Progesterone levels were elevated in June when peak nesting was observed. Ultrasound and hormone data indicate that females lack ovarian development in the fall and produce on average only one clutch per year.

Mortality of breeding sea turtles due to excessive heat exposure after nesting activities is an unusual feature of the Raine Island green turtle rookery. Breeding turtles that fail to return to the ocean after oviposition can experience increasing body temperatures that exceed lethal limits (39 degrees C) as ambient temperatures rise after sunrise. We investigated how acute increases in body temperature influenced plasma corticosterone (B) concentrations of individual turtles. Furthermore, interactions between progesterone (P) and testosterone (T) and increasing body temperature and the glucocorticoid corticosterone were examined for negative correlations. Breeding green turtles exhibited a 16-fold mean increase in plasma corticosterone concentration as body temperature (cloacal) rose from 28.2 to 40.7 degrees C in less than 6 h. However, the absolute increase in plasma B was small and much less than expected, despite the lethal stressor. Comparatively, the maximal B response to lethal heat stress was similar to plasma B concentrations obtained from breeding female turtles exposed to 8 h of capture stress. However, the maximal B response of breeding turtles exposed to heat and capture stressors was significantly less than the B response of nonbreeding adult female turtles subjected to an 8-h capture stressor. No negative correlations were observed between plasma T and plasma B, between plasma T and body temperature, between plasma P and plasma B, or between plasma P and body temperature. Our findings provide further evidence that reduced adrenocortical function operates in breeding green turtles in the presence of even the most pervasive of environmental stressors.

The endocrine pancreatic cells of Pseudemys scripta elegans were investigated immunocytochemically by light and electron microscopy. Insulin-, somatostatin (SST)-1, SST-28 (1-12)-, salmon (s)SST-25-, glucagon-, pancreatic polypeptide (PP)-, peptide tyrosine tyrosine (PYY)-, and neuropeptide tyrosine (NPY)-like immunoreactivities were observed. Insulin cells were immunogold labeled with bonito insulin antiserum and secretory granules were characterized by a wide halo and a dense core of varying shape. Consecutive PAP-immunostained sections showed that SST-28 (1–12), SST-14, and sSST-25 immunoreactivities occurred in the same cells. However, preabsorption tests demonstrated that anti-sSST-25 serum detected the invariant SST-14 molecule. The SST-28 (1–12)/SST-14-immunogold-labeled cells mainly had round or ovoid medium electron-dense granules. Glucagon-IR cells were characterized by round secretory granules with an electron-dense core, with or without a narrow clear halo. There were PP, PYY, and NPY (NPY-like) immunoreactivities in a population of glucagon-IR cells in the pancreatic duodenal region (glucagon/NPY cells). Most of the secretory granules of these glucagon/NPY-like cells had an electron-dense content and were round, although there were also pyriform or ovoid secretory granules which were smaller than those of glucagon-IR cells. Preabsorption tests proved that the NPY-like peptides detected in the endocrine pancreas of P. scripta elegans were more similar to NPY or PYY than to PP.

We sampled a population of gopher tortoises (Gopherus polyphemus) from May to October 1997 to determine seasonal cycles of steroid hormones (testosterone, T; 17β-estradiol, E; and progesterone, P) and related them to observations of mating behavior. In males, plasma T levels peaked in July and August and remained elevated through October. This coincides with the reported time of peak mating and spermatogenesis, indicating that males display an associated pattern of reproduction. In females, E levels were high in September and October. Plasma T levels in females were elevated in May, decreased to basal levels in June and July, and rose again in August and September. Elevated E and T levels correspond to the reported time of peak vitellogenic activity, indicating that females also display an associated cycle. Plasma P in females remained basal throughout the active season, suggesting that ovulation occurs in late winter. We also determined levels of corticosterone (B) to assess the influence of capture stress on tortoises and correlated B levels with tortoise activity patterns and sex steroid levels. We found no seasonal variation in levels of B in males or females. Plasma B levels were not correlated with levels of T or E, but were positively correlated with female P levels. Further, we found no relationship between plasma B levels in males and mean distance moved, mean number of burrows used, or mean home range size. However, there was a significant negative correlation between plasma B levels and male body size. In females, there was no relationship between B levels and mean distance moved, but B levels were significantly negatively correlated with the number of burrows females occupied. Lastly, there was no relationship between levels of B and the number of minutes required to obtain blood from an animal. However, B levels increased with the length of time that a tortoise spent in a trap, suggesting that trapped tortoises do exhibit capture stress.

Xenobiotics suspected of being estrogenic-the PCB aroclor 1242 and the pesticides toxaphene, dieldrin, p,p'-DDD, cis-Nonachlor, trans-Nonachlor, p,p'-DDE, and chlordane-were examined for their ability to override a male-producing incubation temperature and result in female hatchlings in the red-eared slider, a turtle with temperature-dependent sex determination. Compounds were assayed in the environmentally relevant concentrations detected in alligator eggs from Lake Apopka, Florida, singly, in concert with one another, and with estradiol. Compounds assayed alone and resulting in significant sex reversal were trans-Nonachlor, cis-Nonachlor, aroclor 1242, p,p'-DDE, and chlordane. When administered with estradiol, only one of the compounds, chlordane, caused sex reversal at significant levels. When applied together, however, the eight compounds assayed resulted in significant sex reversal.

The seasonal reproductive cycle of the Kemp's ridley sea turtle (Lepidochelys kempi) was studied under seminatural conditions at the Cayman Turtle Farm, Grand Cayman, British West Indies, from June 1987 to July 1988. Male L. kempi displayed a prenuptial rise in serum testosterone 4 to 5 months prior to the mating period (March). Male testosterone then declined sharply during the mating period. Female L. kempi also displayed a prenuptial rise in serum testosterone, estradiol, and total calcium 4 to 6 months prior to the mating period (March). Female testosterone and estradiol declined during the nesting period (April to July) immediately following the mating period (March). Elevated levels in female estradiol and total calcium corresponded with the period of vitellogenesis as determined from gel electrophoresis and ultrasonography. Serum thyroxine also fluctuated seasonally with elevated levels observed in females associated with the period of vitellogenesis. L. kempi displayed a distinct seasonal reproductive cycle in captivity. Nesting in the captive study group corresponded with nesting in the wild population at Rancho Nuevo, Mexico (April to July). Female endocrine cycles during the nesting period were similar to those observed in the wild population.

The corticosterone response to capture and handling was measured in free-living red-eared slider turtles, Trachemys scripta elegans. To determine the ability of this species to exhibit this endocrine response, slider turtles were bled at the time of removal from hoop nets and again at 30 and 60 min following capture to create plasma profiles of acute corticosterone secretion from individuals. Plasma corticosterone concentration increased significantly with handling time. The greatest rise in corticosterone was within the first 30 min following capture and handling, with this rate of increase declining over the next 30 min of restraint. There was no correlation between corticosterone levels at the time of capture and the length of time it took to get the sample if the sample was taken within the first 10 min after capture. However, when these samples were included with those taken from other turtles sampled 11 to 25 min after capture, hormone levels were significantly correlated with handling time. This suggests that the critical time to obtain an initial sample that best represents the predisturbance level in slider turtles is within 10 min. There was no correlation between the turtles' energetic condition and initial corticosterone concentrations. Plasma corticosterone values at all sampling times were comparable to those observed in other reptile species. The results from this study may be used to investigate the effects of unpredictable resources on reproductive success and survival in freshwater turtles.

The distribution of receptors for atrial natriuretic peptide (ANP) and angiotensin II (ANG II) in the kidney and adrenal gland of the freshwater turtle, Amyda japonica, was examined by quantitative in vitro autoradiography using 125 I-labeled rat (r)ANP 1–28 and 125 I-labeled human ANG II as labeled ligands. Receptor subtypes were characterized by competition with des[Gln 18 ,Ser 19 ,Gly 20 , Leu 21 , Gly 22 ] ANP (4–23) (C-ANP) as a selective ligand of the clearance receptors to specific 125 I-rANP (1–28) binding, and with DuP 753 and PD 123319 as nonpeptide antagonists for ANG II receptors type 1 (AT 1 ) and type 2 (AT 2 ) subtypes, respectively, to 125 I-ANG II binding. Specific 125 I-rANP (1–28) binding with a single binding site was found overlying glomeruli of the kidney and the outer zone of the adrenal gland with dissociation constants ( K d ) of 4.39 ± 0.33 and 6.07 ± 1.36 n M, respectively. C-ANP (10 μ M ) inhibited about 90% of the glomerular and adrenal 125 I-rANP (1–28) binding. Specific 125 I-ANG II binding was also localized in the glomeruli of the kidney and the outer zone of the adrenal gland with K d of 1.02 ± 0.22 and 0.37 ± 0.04 n M, respectively. DuP 753 (10 μ M ) potently inhibited about 80% of glomerular and 90% of adrenal 125 I-ANG II binding, whereas PD 123319 (10 μ M ) was very weak in competing for specific 125 I-ANG II binding in both tissues. Therefore, these results indicate that specific ANP and ANG II receptors with high affinities have an overlapping distribution in glomeruli of the kidney and the outer zone of the adrenal gland of the freshwater turtle. They also suggest that biological and clearance ANP receptor-like subtypes coexist in both tissues, and the predominant ANG II receptor subtype in these tissues is the AT 1 -like receptor. The overlapping distribution of specific receptors for both peptides in these tissues provides the basis for possible receptor interactions to exert a functional antagonism between ANP and ANG II in the freshwater turtle.

Plasma corticosterone concentrations were measured in wild loggerhead sea turtles (Caretta caretta) in response to acute captivity (capture, serial bleeding, and restraint up to 6 hr). In general, concentrations of corticosterone dramatically increased 1 hr after capture, peaked at 3 hr, and decreased by 6 hr. Initial corticosterone concentrations were significantly lower in animals captured by tangle net than in those captured by trawl and were thought to more closely represent baseline levels. Significant effects of season and size class on corticosterone concentrations were found for turtles captured by trawl. Corticosterone concentrations of small turtles captured in summer were higher than those of large turtles captured in the same season and of all turtles captured during winter. In winter, corticosterone concentrations for small turtles were higher than those for large turtles at 3 hr after capture. Large turtles captured during winter experienced the slowest rate of increase in plasma corticosterone and a decline at 3 hr after capture. Although cloacal temperatures were significantly higher in summer samples, corticosterone concentrations of large turtles did not differ between seasons until 1 hr after capture. In addition, several large turtles during summer did not experience an increase in corticosterone concentrations 1 hr after capture. It is possible that the lower corticosterone response of large turtles captured during summer may be associated with reproductive condition.

Vitamin D binding protein (DBP) was isolated from the plasma of the snapping turtle, Chelydra serpentina (mixed sex adult), and compared with the previously characterized dual function binding plasma protein that binds both thyroxine (T4) and vitamin D (tsTDBP) in an emydid turtle, Trachemys scripta. Purification of Chelydra serpentina DBP (csDBP) was accomplished by ion exchange chromatography, preparative polyacrylamide gel electrophoresis, and reverse-phase high-performance liquid chromatography. Estimates of increased purification (ca. 71-fold), recovery (ca. 1.5%), and corresponding plasma concentration (ca. 0.6 mg/ml) are confounded by interference with other proteins. A protein was identified that showed the same high affinity for 25-OH-cholecalciferol (vitamin D3) as the tsTDBP, and the two exhibited similar heat resistances in binding. The csDBP and tsTDBP had similar molecular weights by SDS-PAGE (ca. 58 kDa), showed immunological cross reactivity, and differed by only three residues, representing conservative substitutions, in the 30 NH2-terminal amino acid sequence. A slightly lesser homology (up to 89% similarity based on conservative substitutions) was seen with three mammalian DBPs. However, unlike tsTDBP, the DBP from the snapper did not bind T4. These data support the view that the DBP of emydid turtles secondarily evolved a second T4 binding site, and this is probably independent of the D3 binding domain.

In embryos of Emys orbicularis, the sexual differentiation of gonads is influenced by the incubation temperature of eggs. Estrogens administered during the thermosensitive period result in the feminization of gonads at 25 degrees (male-producing temperature), whereas an antiestrogen or aromatase inhibitors masculinize the gonads at 30 degrees (female-producing temperature). The nonsteroidal aromatase inhibitor Letrozole induces gonads with different degrees of masculinization, from ovary-like to testis-like. The present study examines the endocrine function of such masculinized gonads, at the end of embryonic life. Aromatase activity (which is involved in estrogen synthesis in ovary) and the status of Müllerian ducts (the regression of which reflects the secretion of a putative anti-Müllerian hormone by the Sertoli cells) were examined. One month after treatment with Letrozole, the gonads of embryos presented various levels of aromatase activity. There was a strong correlation among aromatase activity, gonadal structure, and Müllerian duct status; high levels of aromatase (similar or close to those in control females) were found in ovary-like gonads; intermediate levels were found in gonads (masculinized ovaries or ovotestes?) exhibiting a cortex and a composite medulla containing a mixture of ovarian lacunae and testicular cord-like structures; low levels (similar or close to those in control males) were found in strongly masculinized gonads (testis-like or ovotestes). Müllerian ducts were regressing in the majority of embryos with gonads containing low levels of aromatase activity. In these individuals, gonads functioned as embryonic testes. These results confirm the implication of estrogens in gonadal differentiation. The origin of these hormones is controversial, so that the aromatase activity was compared in gonads, in the undissociated adrenal-mesonephric complex (AM), and in different parts of this complex during the thermosensitive period. At the female-producing temperature, the aromatase activity per unit of tissue increased in differentiating ovaries but it was low in AM and similar to that found in AM at male-producing temperature. In embryos whose gonads had been masculinized by early treatment with Letrozole, aromatase activity was unchanged in AM. These results suggest that the main source of estrogens involved in ovarian differentiation is the gonad itself.

In many turtles the temperature during the middle of incubation determines the gonadal sex of the hatchling. Sex steroid hormones have been implicated in temperature-dependent sex determination in the red-eared slider turtle, Trachemys scripta ; androgen is involved in male sex determination and estradiol in female sex determination. Administration of exogenous estradiol and its agonists to eggs incubating at a male-producing temperature can overcome the effect of temperature and result in all-female offspring. Exogenous testosterone will also result in some female hatchlings if administered to eggs incubated at a male-producing temperature, an effect due to the aromatization of testosterone to estradiol. This study demonstrates that in the red-eared slider, androstenedione, the precursor to both testosterone and estradiol, has a similar effect. In addition, both testosterone and androstenedione synergize with incubation temperature to exert a greater effect at intermediate incubation temperatures that normally produce mixed sex ratios, indicating that as with estradiol, androstenedione and testosterone are involved in the final common pathway of sex determination in this species. At the single dosage administered, estrone and estradiol produced all females at a male-producing incubation temperature.

A major difficulty in sea turtle conservation is the inability to nonlethally and noninvasively identify the sex of hatching sea turtles. Traditional sexing techniques such as plasma sex steroid quantification cannot be applied to hatchlings without sacrificing the hatchlings or utilizing invasive procedure. This paper presents a technique for sexing hatchling sea turtles by analysis of sex steroid concentrations in egg chorioallantoic/amniotic fluid (CAF). Metabolites of estradiol-17 beta (E) and testosterone (T) in CAF are best expressed as an index or E:T ratio. Chorioallantoic/amniotic fluid E:T ratios for males (0.5 +/- 0.1) were significantly lower than those for females (2.2 +/- 0.3). When separated by utilizing an E:T ratio of 1.25 as the determinant index value, 27 of 28 hatchlings were designated correctly as males (E:T1.25) or females (E:Tor = 1.25). Sex was verified for all hatchlings by gonadal histology. This study shows significant concentrations of T and E metabolites in CAF and plasma of hatchling loggerhead turtles and illustrates the use of a nonlethal, noninvasive method for determining sex, which could be potentially utilized for other endangered reptile and avian species.

Radioimmunoassays (RIAs) of insulin-like growth factor-I (IGF-I) in nonmammals are complicated by two problems: decreased specificity due to heterologous assays and assay interference from IGF binding proteins. We have developed and validated an RIA for the detection of IGF-I in reptilian plasma that circumvents these problems. Biochemical validations show parallelism between standard curves, plasma dilutions, and internal standards. Further, biological validations indicate that (1) plasma IGF-I levels are significantly reduced in nonreproductive, female American alligators (Alligator mississippiensis) with high plasma estradiol-17 beta concentrations compared to individuals with low plasma estradiol-17 beta concentrations and (2) plasma IGF-I concentrations are elevated in juvenile Trachemys scripta elegans turtles fed higher quality diets (defined by protein and energy content). These results provide impetus for future studies of IGF-I in reptiles.

We examined changes in serum protein composition after estradiol-17 beta treatment of ovariectomized female Trachemys scripta, with the objective of identifying proteins that are repressed by estrogen. The experimental protocol was validated by measuring serum estradiol-17 beta levels with a specific radioimmunoassay. Control turtle sera contained little or no estradiol-17 beta (mean = 25.8 pg/ml) while estrogen-treated turtle sera had elevated estradiol-17 beta levels (mean = 333.3 pg/ml). Estrogen treatment resulted in a significant increase in serum protein concentration. The increase was due largely to a 213-kDa protein that was abundant in estrogen-treated animal sera but was low or absent in control animal sera. This protein was identified as vitellogenin based on its biochemical characteristics (molecular weight, elution profile from DEAE, precipitation in the presence of Mg2+/EDTA). Several proteins were decreased in the sera of estrogen-treated animals. One of these had a molecular weight of 66 kDa and was determined to be serum albumin. This protein crossreacted in Western blot analyses with polyclonal antisera against chicken and human serum albumins. Also, amino acid sequence analysis revealed substantial homology between the 66-kDa turtle protein and serum albumins from other vertebrate species. Another protein decreased by estrogen treatment had a molecular weight of 170 kDa. Both albumin and the 170-kDa protein were reduced in estrogen-treated turtles to levels about 50% of those present in control turtles. Using [35S]methionine-labeling and denaturing (SDS) polyacrylamide gel electrophoresis, we detected the presence of newly labeled albumin and the 170-kDa protein in T. scripta liver cube cultures, indicating that these proteins are derived from the liver. Data from this study reveal that estrogen downregulates several serum proteins in T. scripta, while upregulating serum vitellogenin. The response to estrogen in this reptile is similar to that in the amphibian Xenopus laevis, suggesting that estrogen downregulation of serum proteins during vitellogenesis may be widespread among oviparous vertebrates.