Louis J. Guillette

Biology of stress: interactions with reproduction, immunology and intermediary metabolism

Animals must maintain a steady physiological state, homeostasis, in response to a changing environment. An understanding of the stress response is essential if one is to appreciate the complex physiological mechanisms maintaining homeostasis in vertebrates. Moreover, a comprehensive understanding of the biology of stress is essential if we are to reduce stress in the care of any vertebrate species, including those that are not domesticated and traditional laboratory animals.

Ecoestrogens and embryos—Is there a scientific basis for concern?

Abstract A variety of estrogenic compounds exist in the environment, both natural (phyto- and fungal estrogens) and synthetic (e.g. pesticides, industrial contaminants). Recent studies have suggested that a number of reproductive abnormalities in wildlife and human populations could be due to embryonic exposure to ecoestrogens. These abnormalities include modifications in gonadal structure and functioning, abnormalities in genital morphology and size, depressed plasma sex steroid concentrations and various reproductive organ cancers. We review briefly some of these studies as well as those providing data that identify various ecoestrogens. The data to date suggest that a wide variety of compounds can interact with the estrogen receptor and can stimulate estrogen-associated responses. Compared with 17β-estradiol, ecoestrogens have a weak binding affinity for the estrogen receptor and thus have been described as weak estrogens. However, we hypothesize that the estrogenic action of these compounds is augmented by their bioavailability and persistence. Initial testing demonstrates that some ecoestrogens show relatively little affinity for plasma binding proteins; thus, the majority of the estrogenic compound in the plasma is available for translocation into the cell. Likewise, many synthetic ecoestrogens appear to persist for months or years, stored in body fat. Future research needs to examine in more detail the relative roles of receptor affinity, cellular availability, and nuclear persistence in determining the estrogenicity of ecoestrogens. Only then, will we begin to understand the true role of these compounds in ecosystems.

The evolution of viviparity in reptiles: a physiological model and its ecological consequences

Viviparity has evolved almost 100 times among lizards and snakes. Most scientific interest in this phenomenon has focussed on environmental variables that may have stimulated the evolution of viviparity via intermediate stages of prolonged oviductal retention of eggs (egg retention, or ER). Little attention has been paid to more proximate questions (e.g. what endocrine mechanisms control the duration of ER?) and their evolutionary consequences (e.g. is the degree of ER in a given female fixed or labile? Are there specific features of endocrine control systems for ER which may preadapt a taxon to the evolution of viviparity?). We develop a recently-proposed physiological model for prolonged uterine retention of eggs (Guillette, 1985), and investigate its implications for the evolution of reptilian viviparity. Our model is unusual in combining proximate mechanisms with ultimate (evolutionary) processes. We suggest that the duration of ER is controlled by circulating levels of progesterone, and that progesterone secretion by the adrenals in response to environmental cues may prolong ER. Hence, there may be a large phenotypic component to variance in ER. This mechanism may facilitate the evolution of viviparity in some taxa and in some habitats, by increasing variance in ER and hence (1) expediting the rate of change in ER inducible by selection, and (2) overcoming the necessity for very brief ER to confer a selective advantage. In situations in which prolonged ER confers a higher fitness to the reproducing organism, this character may evolve through genetic assimilation (“fixing” of a phenotypic response, from facultative to obligate). Our model generates several testable predictions, in terms of both endocrine mechanisms and evolutionary pathways.

Water temperature and concomitant waterborne ethinylestradiol exposure affects the vitellogenin expression in juvenile brown trout (Salmo trutta)

Environmental estrogens have the potential to considerably affect the reproduction and development of aquatic vertebrates by interfering with the endocrine system. In addition to the potential risk of environmental estrogens, increasing water temperatures as a result of global warming have become a serious problem in many rivers and streams. To assess the degree of estrogenic exposure, the analysis of the estrogen-dependent protein vitellogenin (Vtg) is a frequently used biomarker in field studies. Little, however, is known regarding the potential interaction between ambient water temperature and the Vtg production induced by waterborne environmental estrogens. In order to test the influence of temperature on Vtg synthesis, we exposed juvenile brown trout to an environmentally relevant concentration of ethinylestradiol (EE(2)) and held them either at low or high temperatures (12 and 19 degrees C, respectively), but also at temperature cycles of 12-19 degrees C in order to simulate the field situation. The EE(2) exposure caused a 7-74-fold increase of hepatic Vtg mRNA. The synthesis of Vtg mRNA was clearly stimulated in fish held at higher water temperatures (12-19 degrees C and 19 degrees C, respectively). On the protein level, Vtg showed a similar pattern; the higher the temperature, the higher the concentration of Vtg in the plasma. The experiment further revealed a temperature-dependent increasing amount of hepatic estrogen receptor alpha mRNA (ERalpha) after exposure to waterborne EE(2). The gene expression of estrogen receptor beta-1 (ERbeta-1) and the glucocorticoid receptor (GR) in the liver of EE(2) exposed fish, however, showed no treatment-related alterations. In line with observed constant bile cortisol concentrations, our data do not indicate corresponding stress related effects on hepatic Vtg production. The present survey, however, clearly demonstrates that increased temperature significantly elevates the estrogen-induced expression of Vtg and therefore has to be considered when interpreting environmental monitoring studies.

Absence of daily cycles in plasma sex steroids in male and female tuatara (Sphenodon punctatus), and the effects of acute capture stress on females

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.

Exogenous progesterone or indomethacin delays parturition in the viviparous lizard Sceloporus jarrovi.

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.

The Evolution of Viviparity in Fishes, Amphibians and Reptiles: An Endocrine Approach

Vertebrate viviparity has been of interest to biologists for many years. In fishes, amphibians and reptiles, studies have focused primarily on the anatomical and ecological aspects of this parity mode (for recent reviews, see Wourms,170 Wake,174,175 Shine150). Historically, few studies have examined the physiological adaptations required for the change from oviparity to viviparity in these vertebrate groups. In recent years, a resurgence of interest has occurred, and many studies have begun to focus on physiological and endocrine adaptations related to the evolution of viviparity.

Gene expression patterns in juvenile American alligators (Alligator mississippiensis) exposed to environmental contaminants.

Reproductive and developmental abnormalities have been reported in the American alligator (Alligator mississippiensis) population from Lake Apopka, FL, that is chronically exposed to a complex mixture of environmental contaminants. To begin to understand the molecular mechanisms that could lead to the observed abnormalities of the reproductive and endocrine system, we quantified concentrations of the steroid hormones testosterone (T) and estradiol-17beta (E(2)) and expression of steroid hormone receptors and genes relating to steroidogenesis in gonadal tissue from juvenile alligators from three lakes in Florida using enzyme immunoassay and quantitative real-time polymerase chain reaction. Alterations of ESR2 (estrogen receptor beta) and SF1 (steroidogenic factor 1) mRNA expression in male gonadal tissue, without an observed difference in plasma concentrations of T, from the different lakes, begin to provide insight into potential mechanisms underlying the alterations of the reproductive system previously observed. Likewise, alterations in P450 aromatase and DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1) mRNA expression, with elevated plasma E(2) concentrations in females, provide leads to the potential mechanisms modifying folliculogenesis and ovarian development. The investigation of these genes also helps clarify normal endocrine and reproductive system function in the American alligator.

Plasma estradiol-17β, progesterone, prostaglandin F, and prostaglandin E2 concentrations during natural oviposition in the loggerhead turtle (Caretta caretta)

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 E2 (PGE2) whereas plasma estradiol-17 beta 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 PGE2 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 PGE2 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 PGF2 alpha stimulates uterine contractions promoting egg expulsion while PGE2 may be more important in promoting cervical relaxation.

In vitro assessment of transcriptional activation of the estrogen and androgen receptors of mosquitofish, Gambusia affinis affinis

Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in many vertebrates and promote the growth and differentiation of the female reproductive system. Androgens play essential roles in the development and functioning of the vertebrate male reproductive system as well as actively supporting spermatogenesis. Importantly, recent studies suggest that androgens and estrogens have important reproductive roles in both males and females. To understand the molecular mechanisms of estrogen and androgen actions and to evaluate estrogen and androgen receptor-ligand interactions in the mosquitofish, Gambusia affinis affinis, we used degenerate primer sets and PCR techniques to isolated DNA fragments encoding estrogen receptor alpha (ERalpha; ESR1), ERbeta1 (ERbeta1) and ERbeta2 from the ovary. Full-length mosquitofish ER (mfER) cDNAs were obtained using cDNA library screening and RACE techniques. Amino acid sequences of mfERs showed over-all homology of 46% (alpha versus beta1), 43% (alpha versus beta2), and 52% (beta1 versus beta2). We applied the ERE-luciferase reporter assay system to characterize these receptors. In this transient transfection assay system using mammalian cells, the mfER proteins displayed estrogen-dependent activation of transcription. In addition to ERs, the transactivation of mosquitofish ARs (mfARs) previously isolated by our group, were examined using an androgen-responsive MMTV-luciferase assay system. Mosquitofish ARs showed androgen-dependent activation of transcription from the MMTV promoter. These data provide a basic tool allowing future studies examining the receptor-ligand interactions and endocrine disrupting mechanisms in mosquitofish and also expands our knowledge of estrogen and androgen receptor evolution.