It is generally thought that in amphibians, thyroid hormones (THs) regulate metamorphosis, while sex steroids (estrogens and androgens) regulate gonadal differentiation. However, inhibition of TH synthesis in frogs alters gonadal differentiation, suggesting instead that these two endocrine axes interact during development. Specifically, THs may be involved in male development, while estrogens may inhibit tadpole metamorphosis. However, we do not currently know the mechanisms that account for these interactions, let alone how such mechanisms may differ between species. To develop and test new hypotheses on the roles of sex steroids and THs, I first examined transcriptional profiles (mRNA) of enzymes and receptors related to sex steroids and THs during embryogenesis and metamorphosis in Silurana tropicalis. Tadpoles were exposed to either an estrogen synthesis inhibitor (fadrozole) or TH (triiodothyronine, T3) during early larval or tadpole development. Acute exposures of S. tropicalis to fadrozole or T3 during early development resulted in increased expression of androgen- and TH-related genes in whole body larvae, while chronic exposure to fadrozole during metamorphosis affected gonadal differentiation but did not affect tadpole development. On the other hand, acute exposure to T3 during metamorphosis increased the expression of androgen-related transcripts both in the brain and gonad. In S. tropicalis, the results suggested that cross-talk is primarily in one direction (i.e., effect of THs on the reproductive axis) with a strong relationship between TH and androgen status. Lastly, I established developmental transcript profiles and investigated T3 regulation of brain and gonad transcripts in Engystomops pustulosus. I then compared these results with S. tropicalis and an earlier study in Lithobates pipiens. While each species developed with similar profiles, they differed in their response to T3. Exposure to T3 resulted in either an increase in androgen-related genes (S. tropicalis) or a decrease in estrogen-related genes (E. pustulosus and L. pipiens). In conclusion, these data demonstrated that cross-talk mechanisms differ among these three evolutionary separate species, but in all cases, T3 appears to affect the balance of sex steroids, stimulating the androgen system and providing potential mechanisms of the masculinising effects of THs. These results will contribute to understanding the mechanisms of hormone interactions and their evolutionary basis in frogs.

The sex-role reversal and male pregnancy found in syngnathids are highly unusual traits in vertebrates. Reproductive hormones likely influence development and regulation of these traits. However, very few studies have examined the underlying hormonal mechanisms that mediate female competitive behavior and male pregnancy. New methodologies and better husbandry practices have made such studies more feasible in recent years. Research on a relatively small number of species has suggested that androgens are likely regulators of spermatogenesis and the development of the male brood pouch prior to pregnancy. Androgens are also potential candidates for mediating sex-role reversed behavior in female syngnathids. The goal of this dissertation was to examine the role of androgens in the male reproductive cycle and female intrasexual competitive behavior in the sex-role reversed Gulf pipefish, Syngnathus scovelli. From review of the literature, I developed a model for the hormonal regulation of the male reproductive cycle in seahorses. I predicted that androgens would be low during the early stages of pregnancy and increase during the end of pregnancy as males go through another cycle of spermatogenesis in preparation for the next mating event. My study of 11-ketotestosterone and testis mass across the reproductive cycle in male S. scovelli supported this model. I also conducted several studies on the role of androgens in female competitive behavior. I determined that treatment with 11-ketotestosterone the evening prior to an intrasexual interaction resulted in an increase in competitive behavior in large over small test females. Conversely, treatment with 11-ketotestosterone one hour prior to an intrasexual interaction resulted in a decrease in competitive behavior in large over small females when stimulus female behavior was controlled. A comparative study of competitive and courtship behavior in S. scovelli and the closely related S. floridae suggested that sexual selection has affected competitive and courtship behavior in both males and females of these species. The diversity of reproductive patterns exhibited by syngnathids suggests that they will provide a unique opportunity to assess how hormonal regulation of reproductive behavior and function has evolved within this lineage.

As apex predators in coastal systems, bottlenose dolphins (Tursiops truncatus) are susceptible to persistent organic pollutant (POP) accumulation and retention over time, which has prompted continued interest in understanding the extent to which contaminant body burdens or other stressors are sufficient to cause adverse sublethal effects on energetic fitness, immune function, or reproduction. Increasing our knowledge of reproductive endocrinology in bottlenose dolphins may provide insight into changes in reproductive rates, thereby expanding the capacity to assess conservation status. This study used the Enzyme-Linked ImmunoSorbent Assay (ELISA) technique to examine peptide fertility hormones [inhibin A, inhibin B and anti-Müllerian hormone (AMH)] measured in serum of free-ranging dolphins (n = 129) of varying age, gender, and maturity status from three locations (Sarasota Bay, FL, Indian River Lagoon, FL, and southern Georgia). The primary research objectives were to establish hormone baselines, investigate AMH and inhibin use as reproductive biomarkers, and examine the potential use of these hormones as biomarkers of toxicant or other stressor effects on reproduction. AMH secretion differed significantly with gender (p < 0.001), where levels were approximately 1,000-fold higher in males than females (1,122 ± 427 ng mL-1 and 1.15 ± 1.25 ng mL-1, mean ± SD). Male AMH levels were related to maturity status, and linear regression analysis revealed a significant, negative relationship between male AMH and age, body length, body weight, and maximum girth in all populations. Of the parameters assessed, age was the best indicator of AMH levels in males. AMH concentrations in females did not vary significantly over time or with maturity status, but exhibited a decrease in some older individuals, potentially indicating an AMH decline in long-lived female dolphins. Inhibins did not differ significantly between age classes in males, but appeared to be an estrous cycle indicator in females, where inhibin peaks were likely related to follicular and luteal phases. These data provide new information on circulating serum AMH and inhibin levels in bottlenose dolphins, which appear to reflect a degree of gonadal function and show promise as reproductive biomarkers. Our findings suggest the possibility of toxicant effects on AMH and inhibin production, but not conclusively. Further investigation of mechanism(s) of action for contaminant-related reproductive toxicity will elucidate the diagnostic value of these hormones to assess the effects of POPs on fertility potential in bottlenose dolphins.

This dissertation investigates the relationship between Madagascar’s environment and the stress physiology of the red-bellied lemur, Eulemur rubriventer. Unique lemurid traits are proposed to have evolved in response to selection pressures unlike those in other primate habitats, and appear to be particularly suited to helping animals cope with harsh and unpredictable environments. Several hypotheses as to why lemurs evolved characteristics such as small group sizes, low basal metabolic rates, and cathemerality rest upon the untested assumption that these species are ecologically and/or reproductively stressed. This study simultaneously analyzes seasonal changes in climate, ecology, and the behavior and stress hormones (cortisol) of Eulemur rubriventer, as well as differences in these parameters across habitats with different ecological matrices. The goals of this dissertation are to: (1) evaluate the influences of seasonally varying food availability and climate upon lemur behavior and physiology, to determine whether such changes are sufficient to exert strong selective pressure; and (2) assess the additional influences of habitat composition and quality to evaluate the effects of unpredictability and habitat disturbance. Eulemur rubriventer are sensitive to seasonal environmental changes, as indicated by their time budgets, diets, and fecal cortisol levels. Overall they adopt a time minimizing strategy whereby energy is conserved by resting a majority of the time. Ripe fruit scarcity periods elicit the launch of an energy maximizing strategy whereby fecal cortisol levels and time feeding increase, and time resting decreases. Sensitivity to these influences varies across habitats. The behavioral and stress responses of groups in the undisturbed habitat (UND) were more seasonal and pronounced than those from their disturbed habitat (DIST) counterparts. Lower cortisol levels in DIST may result from a less seasonally predictable environment requiring frequent short-term responses (with possible energy deficits during critical reproductive stages yielding 3 deaths out of 5 births and out-of-season reproduction). Alternatively, the attenuated behavioral and hormonal response to environmental change in DIST may indicate a severely stressed population with insufficient energy to launch an appropriate coping response. The hypothesis that animals in DIST have adapted to frequent unpredictability due to disturbance is rejected because all animals behaviorally and hormonally respond to fruit declines, indicating that this species undergoes ecological stress. / text

It is generally thought that in amphibians, thyroid hormones (THs) regulate metamorphosis, while sex steroids (estrogens and androgens) regulate gonadal differentiation. However, inhibition of TH synthesis in frogs alters gonadal differentiation, suggesting instead that these two endocrine axes interact during development. Specifically, THs may be involved in male development, while estrogens may inhibit tadpole metamorphosis. However, we do not currently know the mechanisms that account for these interactions, let alone how such mechanisms may differ between species. To develop and test new hypotheses on the roles of sex steroids and THs, I first examined transcriptional profiles (mRNA) of enzymes and receptors related to sex steroids and THs during embryogenesis and metamorphosis in Silurana tropicalis. Tadpoles were exposed to either an estrogen synthesis inhibitor (fadrozole) or TH (triiodothyronine, T3) during early larval or tadpole development. Acute exposures of S. tropicalis to fadrozole or T3 during early development resulted in increased expression of androgen- and TH-related genes in whole body larvae, while chronic exposure to fadrozole during metamorphosis affected gonadal differentiation but did not affect tadpole development. On the other hand, acute exposure to T3 during metamorphosis increased the expression of androgen-related transcripts both in the brain and gonad. In S. tropicalis, the results suggested that cross-talk is primarily in one direction (i.e., effect of THs on the reproductive axis) with a strong relationship between TH and androgen status. Lastly, I established developmental transcript profiles and investigated T3 regulation of brain and gonad transcripts in Engystomops pustulosus. I then compared these results with S. tropicalis and an earlier study in Lithobates pipiens. While each species developed with similar profiles, they differed in their response to T3. Exposure to T3 resulted in either an increase in androgen-related genes (S. tropicalis) or a decrease in estrogen-related genes (E. pustulosus and L. pipiens). In conclusion, these data demonstrated that cross-talk mechanisms differ among these three evolutionary separate species, but in all cases, T3 appears to affect the balance of sex steroids, stimulating the androgen system and providing potential mechanisms of the masculinising effects of THs. These results will contribute to understanding the mechanisms of hormone interactions and their evolutionary basis in frogs.

Hepatic and intestinal endocrine cells are potentially helpful targets for recombinant insulin expression. As the two cell types exhibit different secretion kinetics,it has been hypothesized that a combination of the two would better approximate insulin secretion kinetics from normal, functioning beta-cells than either cell type alone. This hypothesis was tested using two hepatic cell lines transiently transduced with one of three adenoviruses for insulin expression along with a stably transfected recombinant intestinal L cell line. The insulin secretion kinetics were analyzed for both the hepatic and intestinal cells to determine the potential of combining them to reproduce the insulin secretion kinetics of a normal, functioning beta-cell. It was observed that the two recombinant hepatic cell lines secreted insulin in a more sustained manner exhibiting slower release kinetics. They also exhibited an increase in insulin secretion when stimulated by the cocktail of nutrient secretagogues (glucose and meat hydrolysate) versus stimulating with only glucose. The cells transduced with the adenovirus containing an additional cytomegalovirus (CMV) promoter and green fluorescent protein (GFP) exhibited the highest insulin secretion after stimulation, whereas the cells transduced with an adenovirus encoding for destabilized preproinsulin mRNA exhibited the lowest secretion rates. The recombinant intestinal cell line (GLUTag-INS) secreted insulin with rapid kinetics upon stimulation, apparently due to the presence of secretory granules containing pre-synthesized insulin. The experiments demonstrated that the cells stimulated with medium containing only meat hydrolysate exhibited a significantly higher insulin secretion relative to secretagogue-free controls. The insulin secretion was not further enhanced when meat hydrolysate was combined with glucose.

En esta tesis se desarrolla una metodología estadística que permite obtener clasificaciones de modelos linalizables a través de una distancia basada en el concepto de información de Shamon. Se han obtenido explícitamente expresiones algebraicas de los estimadores que nos sirvieron para comparar dos o mas de dichas curvas estableciendo su relación con los contrastes de hipótesis propias del análisis de la varianza en el caso de que las curvas fueran asociadas a un modelo lineal normal de varianzas iguales. Finalmente, y como aplicación, se hace un análisis y una clasificación de distintos tipos de respuesta al TTOG en una muestra de niños, dando posteriormente un método de diagnostico automatizado que cuantifica sus errores.

Typescript. / "April 1987." / Includes bibliograpical references (leaves 160-194). / xii, 194 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Presents direct comparative information on growth and endocrinological aspects of reproductive development in intact and gonadectomized male and female Merino lambs born in autumn and treated prepubertally with either a non-aromatizable androgen (trenbolone acetate) with higher androgenic potency than testosterone, oestradiol -17beta(oestradiol), or a combination of these two steroid hormones. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, 1988

Coronary heart disease is the single greatest contributor to mortality within developed countries in our time. Type 2 diabetes mellitus and obesity, both of which are potent risk factors for coronary heart disease, are common conditions with rapidly rising rates of prevalence both globally and in Australia. Within Australia, Indigenous Australians suffer from disproportionately high rates of diabetes, obesity and coronary heart disease, the reason for which lies, in part, with changes in dietary intake and decreased levels of physical activity over the past 200 years. These disease trends have led to a substantially reduced life expectancy from an increase in excess cardiovascular deaths in Indigenous Australians. Assessment of cardiovascular risk in Indigenous Australians is complicated by ethnic differences in cardiovascular risk factors and their prevalence. There is also evidence that Indigenous Australians are genetically predisposed to diabetes. Current cardiovascular risk stratification algorithms are suboptimal, significantly underestimating risk in Indigenous Australians. This thesis aimed to address these issues. Firstly, to evaluate the efficacy of a lifestyle intervention programme to sustainably improve markers of health outcome in a cohort of Indigenous Australians who were overweight or had type 2 diabetes mellitus by improving diet and physical activity levels. Secondly, to evaluate the utility of anthropometric markers of obesity, ultrasound markers of atherosclerotic burden and serum C-reactive protein as additional cardiovascular risk stratification tools. The Healthy Lifestyle Programme (HELP) was a two year prospective intervention study designed and implemented with substantial input from the Indigenous community. The primary intervention consisted of a series of educational workshops delivered predominantly by Indigenous health workers advocating improving diet and increasing physical activity. To complement these workshops, facilities were established within the community to support dietary improvement and increased physical activity. Sustainable changes to markers of cardiovascular outcome, including significant reductions in central obesity and blood pressure were demonstrated over time. A slowing in the deterioration of renal function as marked by albuminuria was also detected. These beneficial changes were accompanied by increased physical activity and improved dietary intake. In a substudy, individuals with the apolipoprotein E4 genotype, who were dyslipidaemic at baseline, benefited from lipid profiles that improved significantly during the course of the study. Changes in blood pressure and central obesity detected during the course of the project are likely to have significant clinical impact on health outcomes considering their sustained nature. Evaluating ultrasound imaging based markers of cardiovascular risk, carotid artery intimal medial thickness was a more reliable measure of risk based on its firm correlation with established cardiovascular risk factors compared to brachial artery flow mediated vasodilatation. Carotid artery intimal medial thickness may be the better surrogate marker to improve cardiovascular risk stratification in the Indigenous population. Examining anthropometric markers of obesity, body mass index and waist circumference but not waist hip ratio correlated well with traditional cardiovascular risk factors. Furthermore, both body mass index and waist circumference predicted insulin resistance fairly well using threshold values derived from receiver operating characteristics curve analyses. Both body mass index and waist circumference were simple to perform with a high degree of reproducibility between observers and will likely be valuable additional cardiovascular risk stratification tools. Raised inflammatory stress, as marked by elevated serum C-reactive protein levels, was found in individuals with diabetes or obesity. C-reactive protein levels did not correlate with carotid artery intimal medial thickness, suggesting that C-reactive protein raises cardiovascular risk by means other than increasing atherosclerotic burden. Genotyping a small random sample of our the C-reactive protein gene promoter region. This SNP was found to independently increase serum C-reactive protein in the Framingham cohort. A genetic basis for increased inflammatory stress may contribute to coronary heart disease in Indigenous Australians. In summary, a community centred lifestyle intervention programme was efficacious in producing sustainable improvement in markers of health outcome. Examination of additional cardiovascular risk markers in the Indigenous community have provided evidence for using carotid artery intimal medial thickness, waist circumference, body mass index and serum C-reactive protein levels to improve cardiovascular risk stratification. These additional tools would allow for more specific resource allocation to target individuals at highest cardiovascular risk for preventative management. C-reactive protein gene polymorphisms, their contribution to circulating C-reactive protein levels and cardiovascular outcomes warrants further investigation in the Indigenous population. A more proactive risk management plan, which includes a lifestyle intervention component, may yield the greatest improvement in long term health outcomes if targeted at younger members of the Indigenous community.

The prepartum surge in fetal plasma cortisol, essential for the maturation of organs in mammals and the normal timing of parturition in some species, including sheep, may result from an increase in the molar ratio of adrenocorticotropin (ACTH) to pro-opiomelanocortin (POMC) in the fetal circulation. Related to this, the cleavage of POMC to ACTH by the enzyme, prohormone convertase 1 (PC1), may be influenced by corticotrophin releasing hormone (CRH) stimulation. Accumulating evidence suggests that the capacity of individual corticotrophs to process POMC to ACTH may vary and individual corticotrophs are differentially responsive to CRH. It is not known, however, if there are separate corticotroph subpopulations in the fetal sheep pituitary which can be identified by differential colocalisation of POMC, ACTH and the CRH receptor 1, CRHR₁, nor if changes in the relative proportions of such subpopulations play a role in the molecular mechanisms underlying the overall changes in pituitary function described previously during gestation and in response to suboptimal uterine environments. To investigate these hypotheses, it was first necessary to develop novel methods for the simultaneous immunohistochemical labelling of POMC, ACTH and CRHR₁ in individual cells on sections of fetal sheep pituitary. In addition, I developed and validated an automated method to categorise and count individual cells to increase the quantitative power of this study. Pituitary tissue was collected from control fetuses at 53-55 (n=6), 63-85 (n=6), 110 (n=4), 139-141 (n=4) and 144-145 (n=6) days gestation. Two animal models, known to alter pituitary function in the fetal sheep, were used to investigate corticotrophic adaptations to suboptimal uterine environments. For the maternal periconceptional undernutrition (PCUN) model, maternal feed was reduced to 70% of maintenance requirements from at least 45 days before to 7 days after mating and fetal tissues were collected at 53-55 days gestation (n=7). For the placental restriction (PR) model, the majority of the placental attachment sites were removed in five ewes before mating and fetal tissues were collected at 140 (n=4) and 144 (n=4) days gestation. Pituitary sections were simultaneously labelled with antisera raised against full length POMC, ACTH and CRHR₁ and the proportions of pituitary cells with combinations of antisera were quantified. Four subpopulations of corticotrophs were identified, which expressed either: POMC+ACTH+CRHR₁, ACTH+CRHR₁, POMC+ CRHR₁ or POMConly. There was a significant decrease in the proportion of pituitary cells expressing POMC+ACTH+CRHR₁ between 53-55 and 65-85 days gestation, before an increase at 110 days gestation and a further marked decrease between 139-141 and 144-145 days gestation. In fetuses from the PCUN group, the proportion of pituitary cells expressing POMC+ACTH+CRHR₁ in early gestation was reduced. PR resulted in a significantly higher proportion of corticotrophs expressing POMC+ACTH+CRHR₁ during the prepartum period. This work represents the discovery of the differential expression of POMC, ACTH and CRHR₁ in individual corticotrophs of the fetal sheep pituitary and the first insights into the pituitary adaptations to periconceptional nutrient restriction and placental restriction at the level of individual corticotrophs. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337370 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008