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Publisher Summary The endocrine system controls nearly every aspect of vertebrate life and is instrumental in regulating physiological processes such as metabolism, development, reproduction, tissue function, and behavior. The vertebrate endocrine system regulates many aspects of development, growth, and reproduction. Endocrine-disrupting chemicals (EDCs) are exogenous chemicals that mimic hormones or alter hormonal-signaling processes. These chemicals modify regulation by affecting hormone synthesis, transport, or clearance, or by altering receptor number or binding. Depending on concentration, timing, and duration of exposure, effects on physiological processes can be permanent or transient. Initially, EDC research on reptiles focused on internal concentrations of EDCs, but reptiles are now considered ideal species for the study of endocrine disruption because of their wide range of habitats, diet, parity mode, and modes of sex determination. The majority of research on reptiles focuses on species from aquatic habitats. There is a large and growing body of research on EDC actions in alligators and turtles. Little is known about the effects of EDCs on squamates. The value of reptiles as model species for endocrine disruption is broadening the knowledge of EDCs and how these chemicals affect wildlife and human public health.

he birth of a relatively welldeveloped neonate-viviparity-is commonly considered a characteristic unique to mammals. However, this reproductive mode is exhibited by some species of all vertebrate classes except the jawless fishes (agnathans) and the birds. Among the reptiles, viviparity has evolved almost 100 times among the 5500 or so species of squamates (lizards and snakes). In contrast, all of the approximately 250 species of turtles, 21 species of crocodilians, and 2 species of tuatara are oviparous, producing only shelled eggs (Blackburn 1982, 1985, Shine 1985). In this article, I briefly discuss current work examining the ecological factors correlated with the evolu

In many vertebrates, steroid hormones are essential for ovarian differentiation during a critical developmental stage as well as promoting the growth and differentiation of the adult female reproductive system. Although studies have been extensively conducted in mammals and a few fish, amphibians, and bird species, the molecular mechanisms of sex steroid hormone (estrogens) action have been poorly examined in reptiles. Here, we evaluate hormone receptor and ligand interactions in two species of snake, the Okinawa habu (Protobothrops flavoviridis, Viperidae) and the Japanese four-striped rat snake (Elaphe quadrivirgata, Colubridae) after the isolation of cDNAs encoding estrogen receptor α (ESR1) and estrogen receptor β (ESR2). Using a transient transfection assay with mammalian cells, the transcriptional activity of reptilian (Okinawa habu, Japanese four-striped rat snake, American alligator, and Florida red-belly freshwater turtle) ESR1 and ESR2 was examined. All ESR proteins displayed estrogen-dependent activation of transcription via an estrogen-response element-containing promoter; however, the responsiveness to various estrogens was different. Further, we determined the chromosomal locations of the snake steroid hormone receptor genes. ESR1 and ESR2 genes were localized to the short and long arms of chromosome 1, respectively, whereas androgen receptor was localized to a pair of microchromosomes in the two snake species examined. These data provide basic tools that allow future studies examining receptor-ligand interactions and steroid endocrinology in snakes and also expands our knowledge of sex steroid hormone receptor evolution.

Anti-Mullerian hormone (AMH) plays an important role in male sex differentiation in vertebrates. AMH produced by Sertoli cells of the fetal testis induces regression of the Mullerian duct in mammalian species. In alligators, sexual differentiation is controlled by the temperature during egg incubation, termed temperature-dependent sex determination (TSD). The TSD mechanism inducing sex differentiation is thought to be unique and different from that of genetic sex determination as no gene such as the SRY of mammals has been identified. However, many of the genes associated with gonadal differentiation in mammals also are expressed in the developing gonads of species exhibiting TSD. To clarify the molecular mechanisms associated with gonad formation during the temperature-sensitive period (TSP), we have cloned the full length AMH gene in the alligator, and quantitatively compared mRNA expression patterns in the gonad–adrenal–mesonephros (GAM) complex isolated from alligator embryos incubated at male and female producing temperatures. The deduced amino acid sequence of the alligator AMH cDNA showed high identity (59–53%) to avian AMH genes. AMH mRNA expression was high in the GAM of male alligator embryos at stage 24 (immediately after sex determination) and hatchlings, but suppressed in the GAM of estrogen-exposed hatchlings incubated at the male-producing temperature. In the alligator AMH proximal promoter, a number of transcriptional factors (for SF-1. GATA, WT-1 and SOX9) binding elements were also identified and they exhibit a conserved pattern seen in other species. SOX9 up-regulates transcriptional activity through the amAMH promoter region. These results suggested that AMH and SOX9 play important roles in TSD of the American alligator.

The egg yolk serves as a significant source of maternally derived steroids that are available to the embryo during early development. Altered deposition of yolk steroids can change the developmental trajectory of the embryo and have long lasting or permanent consequences. Alligators from contaminated environments have shown significant reproductive and developmental dysfunction, and it is unclear if altered deposition of yolk steroids could be a contributing factor. Alligator eggs were collected from Lake Woodruff (a reference lake), Lake Apopka (a site of known agricultural contamination), and the Merritt Island National Wildlife Refuge (MINWR) (home of the Kennedy Space Center and a site of heavy metal contamination). The yolks of eggs at embryonic stages 12 (prior to sex determination) and 24 (post-sex determination) were evaluated for concentrations of progesterone, 17-beta estradiol, and testosterone. Yolk concentrations of progesterone were significantly lower at embryonic stage 12 in eggs from Lake Apopka and MINWR when compared to eggs from Lake Woodruff. Yolk concentrations of 17-beta estradiol were significantly lower at embryonic stage 12 in eggs from MINWR when compared to the other two sites. Reductions in yolk 17-beta estradiol concentrations from embryonic stage 12 to 24 were significantly attenuated in eggs from MINWR versus that of Lakes Woodruff and Apopka. This study suggests that altered deposition of yolk steroids, and possibly differential utilization by the embryo, could be a contributory mechanism in the reproductive and developmental abnormalities seen in alligators from contaminated locales.

Sex-steroid hormones are essential for normal reproductive activity in both sexes in all vertebrates. Estrogens are required for ovarian differentiation during a critical developmental stage and promote the growth and differentiation of the female reproductive system following puberty. Recent studies have shown that environmental estrogens influence the developing reproductive system as well as gametogenesis, especially in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor-ligand interactions in Elasmobranchii, we cloned a single estrogen receptor (ESR) from two shark species, the cloudy catshark (Scyliorhinus torazame) and whale shark (Rhincodon typus) and used an ERE-luciferase reporter assay system to characterize the interaction of these receptors with steroidal and other environmental estrogens. In the transient transfection ERE-luciferase reporter assay system, both shark ESR proteins displayed estrogen-dependent activation of transcription, and shark ESRs were more sensitive to 17beta-estradiol compared with other natural and synthetic estrogens. Further, the environmental chemicals, bisphenol A, nonylphenol, octylphenol and DDT could activate both shark ESRs. The assay system provides a tool for future studies examining the receptor-ligand interactions and estrogen disrupting mechanisms in Elasmobranchii.

Transforming growth factor-beta (TGFbeta) homologues form a diverse superfamily that arose early in animal evolution and control cellular function through membrane-spanning, conserved serine-threonine kinases (RII and RI receptors). Activin and inhibin are related dimers within the TGFbeta superfamily that share a common beta-subunit. The evolution of the inhibin alpha-subunit created the only antagonist within the TGFbeta superfamily and the only member known to act as an endocrine hormone. This hormone introduced a new level of complexity and control to vertebrate reproductive function. The novel functions of the inhibin alpha-subunit appear to reflect specific insertion-deletion changes within the inhibin beta-subunit that occurred during evolution. Using phylogenomic analysis, we correlated specific insertions with the acquisition of distinct functions that underlie the phenotypic complexity of vertebrate reproductive processes. This phylogenomic approach presents a new way of understanding the structure-function relationships between inhibin, activin, and the larger TGFbeta superfamily.

Activins and estrogens participate in regulating the breakdown of ovarian germ cell nests and follicle assembly in mammals. In 1994, our group reported elevated frequencies of abnormal, multioocytic ovarian follicles in 6 month old, environmental contaminant-exposed female alligators after gonadotropin challenge. Here, we investigated if maternal contribution of endocrine disrupting contaminants to the egg subsequently alters estrogen/inhibin/activin signaling in hatchling female offspring, putatively predisposing an increased frequency of multioocytic follicle formation. We quantified basal and exogenous gonadotropin-stimulated concentrations of circulating plasma steroid hormones and ovarian activin signaling factor mRNA abundance in hatchling alligators from the same contaminated (Lake Apopka) and reference (Lake Woodruff) Florida lakes, as examined in 1994. Basal circulating plasma estradiol and testosterone concentrations were greater in alligators from the contaminated environment, whereas activin/inhibin betaA subunit and follistatin mRNA abundances were lower than values measured in ovaries from reference lake animals. Challenged, contaminant-exposed animals showed a more robust increase in plasma estradiol concentration following an acute follicle stimulating hormone (FSH) challenge compared with reference site alligators. Aromatase and follistatin mRNA levels increased in response to an extended FSH challenge in the reference site animals, but not in the contaminant-exposed animals. In hatchling alligators, ovarian follicles have not yet formed; therefore, these endocrine differences are likely to affect subsequent ovarian development, including ovarian follicle assembly.

The Vo2 (standard metabolic rate; cc · h−1) of pregnant Sceloporus jarrovi increases to a maximum near term. Immediately following parturition, Vo2 decreases sharply. Approximately 93.6% of the Vo2 of late pregnant S. jarrovi is due to litter and non-reproductive female Vo2. The difference between measures of the Vo2 of late pregnant lizards and late pregnant Vo2 estimated by summing litter and nonreproductive Vo2 (6.4%) represents the physiological cost of maintaining a litter. This estimate of the physiological cost of pregnancy in S. jarrovi is similar to that reported for mammals; however, it is lower than other estimates of the cost of pregnancy in reptiles. Estimates of litter metabolism were obtained by measuring maximum embryo metabolism, using an in vitro technique and multiplying embryo Vo2 by litter size. These results suggest that others have overestimated the physiological cost of pregnancy in reptiles because maximum litter Vo2 has been underestimated. Others have suggested that viviparity is an expensive mode of reproduction because the physiological cost of supporting a litter is an important component of an individual's reproductive effort. Data presented here support the alternative hypotheses that the cost of supporting a litter is not high and constitutes a small fraction of the energy apportioned into reproduction. © 1992 Wiley-Liss, Inc.

The morphology of the female reproductive tract and corpus luteum is examined in Sphenomorphus fragilis, a lizard from the lowland regions of New Guinea exhibiting incipient viviparity. Females oviposit eggs that hatch either immediately or within a few hours. Corpora lutea form from ovulated follicles and decrease in diameter as embryonic development progresses. The oviduct from vitellogenic females is sparsely populated with well developed uterine glands containing secretory granules. The eggs are covered with a relatively thin shell (10 μm thick) composed of an inner boundary layer and proteinacous fibers. The secreted shell is complete by early neurulation. Shell morphology does not change throughout the remainder of the in utero incubation period. A well vascularized uterus and chorioallantoic membrane provide simple placentation. These findings suggest that the reduction in shell thickness associated with the evolution of a placenta is due to a decrease in the number of shell glands in the uterus and is not a delay or inhibition of the shelling process per se. This hypothesis further suggests that the selective forces favoring shell gland loss act on the vitellogenic female during gland recruitment which occurs prior to ovulation and not on the pregnant female. © 1992 Wiley-Liss, Inc.

Cycles in gonadal activity and plasma sex steroid concentrations were investigated in wild female and male tuatara on Stephens Island, New Zealand. Females nest once every four years on average. Vitellogenesis is spread over the first three years, and mating, ovulation and nesting occur in the fourth. Oviducal eggs are carried for6–8 months before nesting. Although the length of this ovarian cycle is unparalleled among oviparous reptiles, the associated cycles in plasma concentrations of sex steroids are similar to those in other reptiles. Mean plasma concentrations of oestradiol increase during vitellogenesis, peak at mating, fall rapidly before or around ovulation, are low during most of gravidity, and rise slightly during late gravidity-nesting. Plasma concentrations of testosterone show a similarcycle. Mean plasma concentrations of progesterone are low during vitellogenesis and peak around ovulation. This periovulatory surge falls within1–2 months, and mean concentrations are low during the final 5–6 months of gravidity. Male tuatara show an annual, pre-nuptial reproductive cycle. Mean plasma concentrations of testosterone are low during winter, rise during spring, peak during midsummer-early autumn, when pre-nuptial displays and mating occur, and fall in mid autumn. Limited histological data indicate that spermiogenesis occurs during midsummer-early autumn, and also support a previous study suggesting that there may be no period of complete testicular and epididymal regression. In comparison with the pre-nuptial cycles of other temperate-zone reptiles, the cycle in male tuatara shows a more prolonged duration of testicular activity and of rising or elevated plasma testosterone concentrations. The prolonged reproductive cycles of male and female tuatara may be adaptations to a temperate environment with cool summers and cool, but not freezing, winters.

The female reproductive tract of birds is different from that of other oviparous amniotes in that the eggshell membranes and calcareous layer are formed in separate regions of the uterus; the isthmus and shell gland, respectively. Phylogenetically, birds are included among the archosaurs, along with crocodilians and dinosaurs. Many dinosaurs were oviparous, producing hard-shelled eggs, yet the reproductive system of dinosaurs has proven difficult to investigate, due to poor preservation of soft anatomy. In this study, we examined functional morphology and eggshell formation in a reptilian archosaur, the American alligator, and demonstrated that the crocodilian reproductive tract has separate uterine regions for formation of the eggshell membranes and calcareous layer. These uterine regions are ultrastructurally comparable to the isthmus and shell gland of birds, and may be homologous. This similarity of reproductive functional morphology between crocodilians and birds may implicate the evolution of an archosaurian mode of oviparity that may shed light on dinosaur reproduction.

We investigated ovary and testis development of Alligator mississippiensis during the first five months post-hatch. To better describe follicle assembly and seminiferous cord development, we employed histochemical techniques to detect carbohydrate-rich extracellular matrix components in one-week, one-month, three-month, and five-month-old gonads. We found profound morphological changes in both ovary and testis. During this time, oogenesis progressed up to diplotene arrest and meiotic germ cells increasingly interacted with follicular cells. Concomitant with follicles becoming invested with full complements of granulosa cells, a periodic acid Schiff’s (PAS)-positive basement membrane formed. As follicles enlarged and thecal layers were observed, basement membranes and thecal compartments gained periodic acid-methionine silver (PAMS)-reactive fibers. The ovarian medulla increased first PAS- and then PAMS-reactivity as it fragmented into wide lacunae lined with low cuboidal to squamous epithelia. During this same period, testicular germ cells found along the tubule margins were observed progressing from spermatogonia to round spermatids located within the center of tubules. Accompanying this meiotic development, interstitial Leydig cell clusters become more visible and testicular capsules thickened. During the observed testis development, the thickening tunica albuginea and widening interstitial tissues showed increasing PAS- and PAMS-reactivity. We observed putative inter-sex structures in both ovary and testis. On the coelomic aspect of testes were cell clusters with germ cell morphology and at the posterior end of ovaries, we observed “medullary rests” resembling immature testis cords. We hypothesize laboratory conditions accelerated gonad maturation due to optimum conditions, including nutrients and temperature. Laboratory alligators grew more rapidly and with increased body conditions compared to previous measured, field-caught animals. Additionally, we predict the morphological maturation observed in these gonads is concomitant with increased endocrine activities.

Lake Apopka in central Florida (USA) is significantly contaminated with a variety of chemicals including anthropogenic nutrients, organochlorine pesticides like dichlorodiphenyltrichloroethane (DDT) and dichlorodiphenyldichloroethylene (DDE), and multiple congeners of polychlorinated biphenyls (PCBs). Our laboratory has previously documented a number of reproductive abnormalities in alligators from Lake Apopka, compared with alligators captured from Lake Woodruff, a nearby reference lake. We conducted the present study to investigate if another native vertebrate, Gambusia holbrooki (eastern mosquitofish), is similarly affected. Adult female mosquitofish were collected from Lake Apopka and Lake Woodruff monthly for 16 months to document seasonal and lake-associated variation in reproductive patterns. In contrast to fish from Lake Woodruff (reference), females from Lake Apopka exhibited earlier and more synchronized spring ovarian recrudescence, increased body size, increased fecundity, increased adjusted hepatic weight, and more extreme fluctuations in muscle estradiol concentrations in most months. Endocrine disruption, consistent with other studies and Lake Apopka's pollution profile, is one explanation for these findings. Other environmental and physiological factors are also addressed. However, the higher fecundity among Apopka females suggests that, unlike Apopka alligators, Apopka mosquitofish are not impacted at the population level.

Paracrine factor signaling regulates many aspects of vertebrate gonadal development. We investigated key ovarian and testicular morphological markers of the American alligator (Alligator mississippiensis) during the first 5 months post-hatching and correlated gonadal development with mRNA expression levels of a suite of regulatory factors. In both sexes, we observed significant morphology changes, including ovarian follicle assembly and meiotic progression of testicular germ cells. Concomitant with these changes were sexually dimorphic and ontogenetically variable mRNA expressions. In ovaries, FOXL2, aromatase, and follistatin mRNA expression was greater than in testes at all ages. At one week after hatching, we observed ovarian medullary remodeling in association with elevated activin/inhibin βA subunit, follistatin, and aromatase mRNA expressions. Three and 5 months following hatching and concomitant with follicle assembly, ovaries showed increased mRNA expression levels of GDF9 and the mitotic factor PCNA. In testes, the activin/inhibin α and βB subunit transcript levels were greater than in ovaries at all ages. Elevated testicular expression of GDF9 mRNA levels at 5 months after hatching aligned with increased spermatogenic activity. We propose that the mRNA expression levels and concomitant morphological changes observed here affect the establishment of alligator reproductive health and later fertility.

BackgroundDuring development, all amniotes (mammals, reptiles, and birds) form extraembryonic membranes, which regulate gas and water exchange, remove metabolic wastes, provide shock absorption, and transfer maternally derived nutrients. In viviparous (live-bearing) amniotes, both extraembryonic membranes and maternal uterine tissues contribute to the placenta, an endocrine organ that synthesizes, transports, and metabolizes hormones essential for development. Historically, endocrine properties of the placenta have been viewed as an innovation of placental amniotes. However, an endocrine role of extraembryonic membranes has not been investigated in oviparous (egg-laying) amniotes despite similarities in their basic structure, function, and shared evolutionary ancestry. In this study, we ask whether the oviparous chorioallantoic membrane (CAM) of chicken (Gallus gallus) has the capability to synthesize and receive signaling of progesterone, a major placental steroid hormone.Methodology/principal findingsWe quantified mRNA expression of key steroidogenic enzymes involved in progesterone synthesis and found that 3beta-hydroxysteroid dehydrogenase, which converts pregnenolone to progesterone exhibited a 464 fold increase in the CAM from day 8 to day 18 of embryonic development (F(5, 68) = 89.282, pConclusions/significanceCollectively, our data indicate that the chick CAM is steroidogenic and has the capability to both synthesize progesterone and receive progesterone signaling. These findings represent a paradigm shift in evolutionary reproductive biology by suggesting that endocrine activity of extraembryonic membranes is not a novel characteristic of placental amniotes. Rather, we hypothesize that these membranes may share an additional unifying characteristic, steroidogenesis, across amniotes at large.

Xenobiotic compounds introduced into the environment by human activity have been shown to adversely affect the endocrine system of wildlife. Various species exhibit abnormalities of (1) plasma sex steroid hormones, (2) altered steroid synthesis form the gonad in vitro and (3) altered steroidogenic enzyme function. These endpoints are sensitive and relatively easy to measure quantitatively with reliability and precision. These observations have led to the conclusion that sex steroid hormones could be markers of exposure to, and altered function from, endocrine disrupting contaminants (EDCs). However, there are serious limitations in the use of steroid hormones as generalized markers of EDC exposure. Steroid hormones exhibit seasonal, ontogenetic, gender and species-specific variation. Moreover, the regulation of sex steroid plasma concentrations is a relatively complex phenomenon capable of short-term (minutes-hours) alteration due to environmental inputs, such as acute stress--an activational response. Alterations in steroids synthesis and degradation also can be a response to altered embryonic development due to EDC exposure--an organizational response. If steroid hormones are to be used as biomarkers, then closely controlled, well designed sampling has to be performed. Additionally, an appreciation of the variation possible in endocrine responses among the species to be studied must be obtained.

This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI., BACKGROUND In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. OBJECTIVES We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. DISCUSSION Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., “good science”). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. CONCLUSIONS Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

Gravid females of four different species of oviparous lizard were treated in vivo with varying doses of prostaglandin F 2 α (PGF 2 α ), prostaglandin E 2 or arachidonic acid (AA). In contrast to previous studies examining birds and viviparous lizards, no dosage induced oviposition in any of the treated females. All females, however, did exhibit behaviors associated with oviposition. Intact oviducts removed from gravid females and placed in organ culture did oviposit when treated with 30 or 100 ng PGF 2 α /ml of culture media. Arachidonic acid at similar concentrations also was effective in stimulating birth. These data suggest that prostaglandins can stimulate oviposition in oviparous lizards but further suggest that their action may be inhibited by oviducal innervation until just prior to natural birth.

Males of the Puerto Rican frog Eleutherodactylus coqui exhibit prolonged parental care (nest attendance and defense) of direct-developing eggs in nonaquatic nest sites. Earlier work showed that plasma androgen levels are significantly lower in males caring for eggs than in noncaring, calling males. In this study, we investigated the effects of elevated plasma androgens on parental care behavior by implanting either testosterone (T) or placebo (P) pellets in parental males brooding eggs in the field. Plasma androgen levels, assessed 4 days after pellet implantation, were six times higher in T males than in P males. Despite this increase, T males exhibited no changes in nest fidelity or brooding behavior. The results indicate that lower androgen levels, at least those corresponding to a decline in T during parental care, are not necessary to maintain parental behavior in this tropical frog.

The vitellogenic response to exogenous estradiol-17β in wild male and female tuatara (Sphenodon punctatus) is extremely slow to induce. Tuatara given slowrelease implants containing 4-11 μg estradiol/g body mass showed a marked elevation in plasma estradiol concentrations within 2 d (to levels of up to 2,967 pg/mL) but no marked increases in plasma concentrations of calcium, total protein, or inorganic phosphate within 11 d. A small increase in the amount of electrophoretically detectable vitellogenin in the plasma (expressed as a percentage of total plasma protein) was evident by 11 d. Plasma concentrations of progesterone in individual females and testosterone in individual males were low (

Phosphoinositide hydrolysis, resulting in inositol trisphosphate (IP3) and diacylglycerol (DG) formation, has been implicated in oxytocin-stimulated pulsatile secretion of prostaglandin F2 alpha (PGF2 alpha) from uterine endometrium of sheep and other mammals. In reptiles, arginine vasotocin (AVT) stimulates uterine secretion of PGF2 alpha. These studies investigated the ability of AVT to stimulate incorporation of [3H]inositol into inositol mono-, bis-, and trisphosphates in two reptilian species. In Experiment 1, AVT stimulated (P less than 0.01) IP formation in uterine from late-gravid (150 to 291%) and postpartum (104 to 363%) Yarrow's spiny lizards (Sceloporus jarrovi). Inositol phosphate formation, in response to AVT, was greater (P less than 0.01 for IP3 and P less than 0.05 for total IP) for gravid (stage 40 embryos) and postpartum (1-2 days) lizards near the time of parturition than for lizards at other stages (stage 38-39 embryos or 7 days postpartum). Inositol phosphate formation was greater (P less than 0.05) in uterine tissue from gravid than from postpartum lizards. In Experiment 2, basal IP formation and the response to AVT were the greatest (P less than 0.01) in endometrium from late-gravid American alligators (Alligator mississippiensis). AVT stimulated (P less than 0.02) IP synthesis in uterine endometrium from vitellogenic (136 to 394%), early-gravid (7 to 270%), late-gravid (315 to 1002%), postpartum (86 to 313%), and nonreproductive (292 to 322%) alligators. Results indicate that (1) AVT stimulates IP formation in the uterus of reptiles; (2) uterine IP formation increases in late-gravid reptiles; and (3) increased responsiveness to AVT may occur at parturition or oviposition.

Changes in plasma concentrations of steroids and prostaglandins (PGs) during natural nesting and oviposition in the loggerhead turtle were studied. Blood samples were obtained during nine distinct behavioral stages of oviposition. Emerging females had no detectable prostaglandin F (PGF) or prostaglandin E 2 (PGE 2 ) whereas plasma estradiol-17β averaged 255 pg/ml and mean plasma progesterone was 395 pg/ml. Plasma steroid concentrations did not vary significantly during nesting. In contrast, plasma PGF and PGE 2 exhibited significant elevations during nest digging about 15 min after emergence. A further significant increase in plasma PGs was observed 10 min later during early oviposition. Plasma PGE 2 peaked during mid oviposition whereas maximal plasma PGF levels occurred during nest covering although mean values were not significantly different than those observed during oviposition. Both PGs showed an abrupt decline (within 10 min) during body pit covering to concentrations similar to those observed during nest construction. Our data suggest that PGs have an active role during oviposition and nesting in the loggerhead turtle and are consistent with hypotheses that PGF 2α stimulates uterine contractions promoting egg expulsion while PGE 2 may be more important in promoting cervical relaxation.

The effects of beta-adrenergic stimulation on uterine contractions occurring in response to arginine vasotocin (AVT) and prostaglandin F2 alpha (PGF2 alpha) were compared during late pregnancy in the viviparous gecko Hoplodactylus maculatus. High doses of AVT (150 or 1,500 ng/g body weight) induced birth in vivo, but PGF2 alpha at doses of up to 2,000 ng/g did not induce birth. The effect of AVT (150 ng/g) on birth rate in vivo was not enhanced by pretreatment 20 min beforehand with the beta-adrenoreceptor antagonist dichloroisoproterenol (2 micrograms/g), whereas the effect of PGF2 alpha (200 ng/g) was markedly enhanced: geckos treated with dichloroisoproterenol and then with PGF2 alpha showed rapid birth-related behavior and gave birth. Isolated uteri showed a tonic contraction in response to AVT (100 ng/ml) and to PGF2 alpha (1,000 ng/ml). Pre-exposure of isolated uteri to the beta-adrenoreceptor agonist isoproterenol (1 microgram/ml) caused relaxation; this pre-exposure did not block the tonic contraction occurring in response to AVT, whereas it completely blocked the tonic contraction induced by PGF2 alpha. We conclude that in H. maculatus, beta-adrenergic stimulation inhibits uterine contractions induced by PGF2 alpha but not those induced by AVT. These data are the first to show that beta-adrenergic stimulation inhibits uterotonic responses to PGF2 alpha in a reptile, and they suggest that the cellular mechanisms by which AVT and PGF2 alpha induce contraction may differ in this species. They also provide further evidence for similarities between mammals and reptiles in the effects of beta-adrenergic stimulation on uterine relaxation.

Female Sceloporous jarrovi in late pregnancy given an ip injection of progesterone (50 ng/g body wt) or indomethacin (4 micrograms/g body wt) exhibited no change in the length of pregnancy when compared to saline-treated controls. In contrast, pregnant females given subcutaneous implants (constant release pellets) containing progesterone or indomethacin exhibited significantly delayed parturition when compared with females given control implants. Indomethacin treatment also disrupted the normal birth process when it occurred, as all females receiving this compound exhibited parturition of only a portion (24-32%) of the total clutch. A similar phenomenon occurred in one experiment following implantation of progesterone pellets. Histological examination of the corpora lutea and oviduct indicated no obvious difference in structure among any of the treatment groups.

1. 1. Plasma concentrations of the neurohypophysial arginine vasotocin (AVT) were measured during three stages of natural nesting [nest digging (N = 16), oviposition (N = 6), nest guarding (N = 6)] in the rare New Zealand reptile, the tuatara (Sphenodon punctatus). 2. 2. Nest digging females (X ± 1 SE: 134.9 ± 15.0 pg/ml) exhibited elevated but significantly lower concentrations of plasma AVT compared to those observed during oviposition (216.0 ± 28.1 pg/ml). Nest guarding females (54.6 ± 29.8 pg/μl) had plasma AVT concentrations significantly lower than other stages. 3. 3. These data are similar to those reporting elevated plasma AVT concentrations during oviposition in sea turtles but tuatara exhibit significantly lower plasma AVT values (10-fold difference). Plasma AVT concentrations reported for tuatara and birds are comparable.

We observed the distance moved, number of movements, and frequency of displays in ten minute intervals for two male color morphs, coral and yellow, and for brightly colored females, of Holbrookia propinqua. We also measured body sizes and, for males, plasma androgen concentrations. Distance moved, number of movements, and number of social displays were positively correlated in males. Females exhibited similar correlations, but the relationship between distance moved and number of displays was not significant. Males moved more often and greater distances than females. Females performed displays as frequently as did males, perhaps because we selected large, dominant females for observation. Distance moved, number of movements, and number of displays increased with body size in males. That these relationships did not hold in females may indicate a sex difference or reflect the small sample size and body size range of females. Plasma androgen concentrations increased with male body size, suggesting a possible androgenic mechanism of sexual selection for large size. Androgen level did not affect display frequency or distance moved. Distance moved increased with plasma androgen concentration, but when effects of body size were held constant, this relationship vanished. Coral and yellow males did not differ in androgen concentration, size, or in the observed behaviors.

Objective To evaluate the possible role of endocrine-disrupting compounds (EDCs) on female reproductive disorders emphasizing developmental plasticity and the complexity of endocrine-dependent ontogeny of reproductive organs. Declining conception rates and the high incidence of female reproductive disruptions warrant evaluation of the impact of EDCs on female reproductive health. Design Publications related to the contribution of EDCs to disorders of the ovary (aneuploidy, polycystic ovary syndrome, and altered cyclicity), uterus (endometriosis, uterine fibroids, fetal growth restriction, and pregnancy loss), breast (breast cancer, reduced duration of lactation), and pubertal timing were identified, reviewed, and summarized at a workshop. Conclusion(s) The data reviewed illustrate that EDCs contribute to numerous human female reproductive disorders and emphasize the sensitivity of early life-stage exposures. Many research gaps are identified that limit full understanding of the contribution of EDCs to female reproductive problems. Moreover, there is an urgent need to reduce the incidence of these reproductive disorders, which can be addressed by correlative studies on early life exposure and adult reproductive dysfunction together with tools to assess the specific exposures and methods to block their effects. This review of the EDC literature as it relates to female health provides an important platform on which women's health can be improved.

A variety of human land uses involve the release of toxins into the environment. Wildlife live alongside humans across this array of land uses and thus, are exposed to varying chemical milieus. Kidneys are the principle excretory organs for vertebrates and excessive or chronic exposure to exogenous toxins can lead to renal pathology and renal failure. Although studies have linked chemical exposure to specific renal diseases across diverse taxa, none compare renal lesions occurring in wildlife living in different types of human-modified landscapes. We identify lesions characteristic of renal stress, including toxin exposure, in 82 giant toad (Bufo marinus) males living in habitats ranging from suburban to agricultural. In a previous study [McCoy K.A., Bortnick L.J., Campbell C.M., Hamlin H.J., Guillette L.J., Jr., St. Mary C.M. Agriculture Alters Gonadal Form and Function in Bufo marinus. Environ Health Persp; in press.], these individuals were examined for gonadal abnormalities, which were significantly and positively associated with percentage of agriculture at the collection site. Thus, we hypothesized the same association for renal abnormalities. We scored gross anatomical abnormalities and used light microscopy to identify tubular and interstitial lesions that have been associated with toxicant exposure in other organisms, including humans. Renal lesions indicative of tubular disease were observed at one suburban and two agricultural sites, whereas interstitial lesions were most severe at one agricultural site. Although there was no relationship between frequency of renal disease and proportion of agriculture in the collection vicinity, the renal lesions we identify are consistent with toxin exposure and are similar to those found in human drug abusers and patients suffering medication-induced nephropathy. This is the first study to describe renal lesions in a wild amphibian species and investigate the distribution of renal lesions across human altered landscapes. Indentifying the chemicals inducing renal lesions across these landscapes, their toxicological mechanisms, and their implications on wildlife health will help us devise strategies to mitigate the impacts of toxins on humans and animals living in human-modified environments.

Evidence of significant effects of endocrine disrupting chemicals on human health has grown considerably in recent years. However, this new science of endocrine disruption presents considerable challenges to epidemiology, challenges that make identification of such effects in humans slow and difficult. We suggest several steps to develop a more ‘environmentally sensitive epidemiology’ and illustrate these concepts using data from two recent studies. These studies used biomarkers of exposure to low environmental levels of two classes of endocrine disrupting chemicals (phthalates and several current use pesticides) and precise outcome measures in order to examine the impacts of these chemicals on male reproductive human health.

Numerous chemicals used in pesticides, plastic, and other products have been shown to have the capacity to disrupt the endocrine system in wildlife and laboratory animals. These chemicals can permanently disrupt development, resulting in abnormal organ function and disease. Effects that have been observed in wildlife and in controlled experiments conducted in laboratory animals exposed to pesticides, plastics, and other chemicals include abnormal gonadal function and reduced fertility.

In a series of three articles we provide an overview of the impacts of endocrine disrupting chemicals (EDCs) on human health, laboratory animals, and wildlife. We begin with a brief definition of endocrine disruption and a consideration of several interrelated developments in the field, which necessitate important conceptual shifts in the theory and practice of toxicology. We then examine some of the data behind these conceptual shifts, first from studies of wildlife, then from studies of laboratory animals. Finally, we shift to a consideration of the considerable challenges this new science of endocrine disruption presents to epidemiology. We then briefly describe the progress being made toward a more ‘environmentally sensitive epidemiology’ and illustrate these concepts with data from a recent study that uses biomarkers of low environmental levels of EDCs to identify some of the impacts of these chemicals on human health.

Plasma concentrations of estradiol, progesterone, prostaglandin (PG) E2 and PGF were measured during three stages of the natural nesting cycle of the tuatara (Sphenodon punctatus), a rare New Zealand reptile. Specifically, we tested whether nesting activity in this species is correlated with significant modifications in plasma concentrations of these hormones. Tuatara dig a nest over a period of several nights, oviposit a complete clutch on a single night, and then guard that nest for several nights. We observed no significant changes in mean plasma concentrations of estradiol or progesterone among the different stages of nesting. In contrast, PGE2 was elevated during nest digging and oviposition but declined significantly during nest guarding. Plasma PGF was elevated during nest digging, rose significantly during oviposition, and declined to basal levels during nest guarding. These data indicate that oviposition in reptiles is associated with elevated plasma concentrations of PGs, suggesting that these hormones function in causing oviducal contractions and/or luteolysis. Moreover, these data suggest that the role of PGs in oviducal function may have been conserved throughout evolution of the amniote vertebrates.

The possible existence of daily cycles in plasma concentrations of sex steroids was examined in wild male and female tuatara (Sphenodon punctatus). Samples were collected from freshly captured animals at dusk, middle of the night, dawn, and middle of the day in January (summer) and July (winter). Males showed daily cycles in mean body temperature (Tb) in both seasons but no daily cycle in mean plasma testosterone concentration in either season. Vitellogenic female tuatara in January and females in mixed reproductive condition in July also showed significant daily variation in Tb. However, there were no daily cycles in mean plasma concentrations of estradiol, testosterone, or progesterone in either group of females. Vitellogenic female tuatara subjected to an acute capture stress (3-hr confinement) in January had mean plasma concentrations of estradiol and testosterone that did not differ from those of free-roaming females. However, progesterone and Tb were significantly higher in captives than in free-roaming females. The elevation in progesterone may result from physical confinement, the difference in Tb, or both. These data suggest that seasonal fluctuations in circulating concentrations of plasma sex steroids in tuatara can be determined using samples collected at different times of the 24-hr cycle. However, the effects of acute capture stress and/or changes in Tb on plasma progesterone concentrations need to be considered in future studies on this and possibly other female reptiles.