<p> In my thesis I investigated whether loss of growth hormone receptor (GHR) signaling affects post-natal skeletal muscle development and regenerative ability in adult male mice, and whether the effect is via insulin-like growth factor-1 receptor (IGF-1R) signaling. Mouse models were used that had a post-natal muscle-specific loss of GHR signaling (<b>mGHRKO</b>), insulin-like growth factor receptor/ insulin receptor (IGF-1R/IR) signaling (<b>MKR </b>), and loss of both GHR and IGF-1R/IR signaling (<b>mGHRKO/MKR</b>). The loss of GHR signaling did not alter muscle <i>igf-1</i> expression, fiber cross sectional area, fiber type proportions, or regenerative fusion ability. Further more, loss of IGF-1R/IR signaling decreased regenerative ability. In conclusion, my data suggests that GHR signaling does not play a role in post-natal skeletal muscle development or regenerative fusion ability, and that IGF-1R/IR signaling has an effect that is independent of direct GH effects on muscle, though may be affected by systemic GH/IGF-1 function.</p>

<p> Anthropogenic contaminants have been well documented in Southern California coastal marine waters and are largely associated with point source pollution entities like waste water treatment plants (WWTPs). This study measured known and suspected contaminants capable of disrupting physiological and endocrine functions in marine organisms. Thyroid endocrine and hepatic contaminant concentrations were measured in English Sole (<i>Parophrys vetulus</i>) and Hornyhead Turbot (<i>Pleuronichthys verticalis</i>) residing at a WWTP outfall location and two other locations, up- and down-current from the outfall. Fish from the outfall and down current locations had higher levels of contaminant compounds and altered thyroid endocrine physiology compared to the up-current reference location. Selected contaminants were significantly correlated with thyroid endocrine parameters, consistent with observed differences in contaminant levels among sites.</p><p>

Endocrine disrupting chemicals (EDCs) such as pesticides and pharmaceuticals can upset amphibian development and contribute to worldwide amphibian population declines. The first objective of this doctoral research was to investigate if the widely used herbicide atrazine (ATZ; used on corn and soy crops) alters Lithobates (Rana) pipiens (Northern leopard frog) development using concentrations of ATZ reported in Canadian ecosystems. Chronic exposures to 0.1 and 1.8 mug/L ATZ on L. pipiens tadpoles were performed in semi-controlled mesocosms. Atrazine reduced metamorphosic success, induced female-biased sex ratio, altered the hepatic activity of steroid 5beta-reductase (srd5beta; an enzyme involves in the conversion of testosterone into 5beta-dihydrotestosterone) and affected the expression of estrogen receptor alpha in brain and deiodinase type 3 in tail. The second objective was to characterize the functions of the steroid 5alpha-reductase (srd5alpha; type 1, 2 and 3) and srd5beta in frogs. These enzymes represent a convergence in evolution: they share similar biological functions (e.g., testosterone reduction, bile acid biosynthesis and erythropoesis), but srd5alpha and srd5beta do not have a common ancestor. Using real-time RT-PCR analysis, these enzymes were detected throughout Silurana (Xenopus) tropicalis (Western clawed frog) early development. The prostate drug finasteride (a 5alpha-reductase type 2 and srd5beta inhibitor in humans) was used in short-term (25, 50 and 100 muM) and chronic (25 muM) exposures of S. tropicalis under laboratory conditions. Finasteride inhibited srd5beta and aromatase (cyp19) activities in whole embryos after short-term exposures. However, chronically exposed S. tropicalis until metamorphosis, revealed no effects of finasteride on cyp19 activity and an increase in male hepatic srd5beta activity. Furthermore, chronic treatment with finasteride induced testicular oocytes in developing males (also referred to as the intersex condition). In addition, real-time RT-PCR analysis showed that finasteride treatments altered sex steroid- and thyroid hormone-related gene expression. Alterations of thyroid hormone gene expression following the inhibition of srd5alpha and srd5beta suggest a complex relationship between the thyroid hormone-responsive genes and the androgen status in early frog development and at metamorphosis. In conclusion, real-time RT-PCR, enzymatic activity and histology analyses demonstrated that srd5alpha and srd5beta are important enzymes during frog development and are 'new' targets to EDCs.

Fibrate pharmaceuticals are peroxisome proliferator-activated receptor alpha (PPARalpha) ligands that are detected in sewage treatment plant effluents, surface water and groundwater. In a previous study, male goldfish ( Carassius auratus) exposed to the fibrate gemfibrozil (GEM) in the water exhibited decreased plasma testosterone concentrations. The following studies investigate the effects of GEM and other PPAR ligands on components of the hypothalamic-pituitary-gonadal axis of the male goldfish; the pituitary and testes. In vitro cultures of dispersed goldfish pituitary cells and testis fragments were employed to study these two tissues in isolation. The secretion of neither luteinizing hormone nor growth hormone from pituitary cells was affected in a manner that could explain decreases in circulating testosterone. Testosterone synthesis in testis fragments stimulated with human chorionic gonadotropin (hCG) was suppressed following treatment with GEM. This inhibition appeared to be the result of impaired mitochondrial cholesterol transport. However, changes in the expression of two proteins important for mitochondrial cholesterol transport, steroidogenic acute regulatory protein and peripheral benzodiazepine receptor, were not affected by GEM treatment. Investigations on the involvement of extracellular-regulated signal kinase 1/2 (ERK 1/2) in the steroidogenic pathway revealed that phosphorylated ERK 1/2 (P-ERK 1/2) is required for hCG-stimulated testosterone synthesis and that GEM treatment decreased levels of P-ERK 1/2 in the mitochondria of this tissue. The main conclusions from these studies include the following: (1) The effect of waterborne GEM on the HPG axis of the goldfish is a direct effect on the testis, (2) GEM impairs mitochondrial cholesterol transport in the goldfish testis, (3) Phospho-ERK 1/2 is required for hCG-stimulated steroidogenesis in the goldfish testis, and (4) the proposed mechanism by which GEM impairs mitochondrial cholesterol transport is through a reduction in mitochondrial phospho-ERK 1/2.

<p> Sexual receptivity in the female rat is dependent on dose and duration of estradiol exposure. A 2&micro;g dose of estradiol benzoate (EB) primes reproductive behavior circuits but without subsequent progesterone does not facilitate lordosis. However, 50&micro;g EB facilitates lordosis after 48 hours. Both EB doses activate membrane estrogen receptor-&alpha; complexed with metabotropic glutamate receptor-1a (mER&alpha;-mGluR1a), activating a multisynaptic circuit in the arcuate nucleus (ARH). I hypothesized that 50&micro;g EB downregulates ER&alpha; and mER&alpha;-mGluR1a complexes in the ARH. Total ARH ER&alpha; protein was reduced 48 hours after 50&micro;g EB, but the 2&micro;g dose was intermediate between oil and 50&micro;g EB. mER&alpha; that co-immunoprecipitated with mGluR1a were greater 48 hours after 2&micro;g EB treatment versus rats receiving 50&micro;g EB. Progesterone signals rapidly but not through progesterone receptor-dopamine D1 receptor complexes. These results indicate 2&micro;g EB maintains but 50&micro;g EB downregulates mER&alpha;-mGluR1a to regulate lordosis.</p>

<p> Industrialized and developed urban centers surround the aquatic ecosystem of San Francisco Bay, and are historic and current sources of anthropogenic contaminants to which resident wildlife is exposed and impacted. An indigenous fish species, the shiner perch (Cymatogaster aggregata), was demonstrated to exhibit significant reductions in plasma tetraiodothyronine (T4) concentrations when sampled from highly industrial Oakland Inner Harbor (OAK) as compared with less impacted locations like Redwood City (RED). This study combined histomorphological evaluation of thyroid follicular tissues and characterization of plasma thyroid hormones, T4 and triiodothyronine (T3). Fish from OAK, but not RED, exhibited reduced plasma T4, T3, and total thyroid hormones, changes that were significantly correlated with thyroid glandular changes including reduced follicular colloid sizes. The findings indicate environmental disruption of thyroid hormone biosynthesis and possible thyroid gland compensatory responses (to low thyroid hormone levels) given the reduced colloid volumes.</p><p>

Abstract not available.

The neurotransmitter gamma-aminobutyric acid (GABA) stimulates the release of luteinizing hormone (LH) by enhancing gonadotropin-releasing hormone (GnRH) release in the goldfish, Carassius auratus. Activin A is another protein that stimulates the release of LH. Activin A also stimulates the release of GnRH from the rat hypothalamus, but this effect has never been shown in fish. Using real-time RT-PCR, we have shown that an injection of baclofen, a GABAB receptor agonist, into sexually mature goldfish stimulates the expression of activin betaA subunits in the telencephalon and sGnRH in the hypothalamus. Baclofen also inhibits the expression of that activin receptor IIB and IB in the hypothalamus. Immunocytochemical studies show that activin betaA subunits and activin receptors are localised in the olfactory bulb, telencephalon, thalamus, hypothalamus and optic tectum. Activin receptors are colocalised with GnRH fibres in the hypothalamus. This study has provided further insight into the role of activin as a neuroendocrine factor controlling reproduction in the goldfish brain.

Neuroendocrine and reproductive systems are strictly regulated by a series of sex hormones, especially 17-beta estradiol (E2). Through specific membrane or nuclear receptors, E2 initiates a series of diverse signaling pathways that regulates the expression of target genes. This transcriptomic output shapes the specific spatial (cell or tissue level) and temporal (seasonal level) E2 actions. In this study, I attempted to define estrogen-related gene expression changes in the neuroendocrine and reproductive systems. Firstly, I targeted the physiological period of E2 action when gonad size is large just prior to spawning (March and April) in the goldfish ( Carassius auratus) model. The effect of the aromatase inhibitor fadrozole and the resultant decline in E2 on neuroendocrine gene expression and reproductive development was determined using microarray analysis. Several regulatory themes for physiological E2 action in fish brain have been revealed from these novel E2 regulated genes, including regulation of the calcium signaling pathway and auto-regulation of nuclear estrogen receptor action. Secondly, I aimed to define the seasonal gene expression characteristics that are associated with hormone profiles, typically E2 blood level change, during a breeding cycle in the goldfish. By using both theoretical and experimental strategies, I have identified a core set of genes in fish neuroendocrine brain that were differentially expressed between physiologically distinct stages including sexually mature prespawning, sexual regression, and early gonadal re-development. Moreover I demonstrated that gene expression changes between stages can be regulated by photoperiod. Thirdly, to further understand the mechanism underlying fadrozole effects on gonadal development, I used a frog model (Xenopus tropicalis) to show that a germline specific piRNA (piwi-interacting RNA) pathway may be involved in the E2/testosterone regulation of gonadal development in the tadpole. Here, I investigated the effect of fadrozole or finasteride (5 alpha-reductase inhibitor), which are known to influence gonadal development, on the gene expression of a piRNA-specific protein Maelstrom (MAEL) and showed both treatments increased MAEL mRNA expression. Moreover, since the specific function of MAEL is unknown, I conducted a bioinformatics analysis to infer its putative function and evolutionary history. This is one case study for our efforts to annotate some functionally unknown genes which are related to or regulated by E2 actions. In conclusion, the physiological stage or seasonal specific gene expression information defined in this study provides a series of new functional insights into regulatory mechanisms of E2 and related hormones in the vertebrate neuroendocrine and reproductive systems.

Secretoneurin (SN) is a major functional peptide derived from the secretogranin II (SgII) precursor, one of granin family members. Our previous studies revealed that SN had a stimulatory action on luteinizing hormone (LH) secretion from goldfish pituitary in vivo and in vitro. In my PhD thesis, I isolated and identified free SN peptide in goldfish brain and pituitary by using reversed phase HPLC and western blots. Immunocytochemical localization showed that SN immunoreactivity (IR) was found within magnocellular neurons of the hypothalamic nucleus preopticus, prolactin producing lactotrophs in rostral pars distalis (RPD) of the pituitary and nerve fibres in pituitary neurointennediate lobe (NIL). These studies indicated the production of SN in goldfish brain and pituitary. I furthermore explored SN production under the regulation of hypothalamic gonadotropin releasing hormone (GnRH). Treatment of dispersed goldfish pituitary cells in vitro with GnRH increased both gene and protein expression of SgII and SgII-derived peptides which were significantly associated with that of prolactin. In vitro treatment with SN stimulated LH release from dispersed goldfish pituitary cells. Immunoneutralization experiments indicated that pituitary-sourced endogenous SN not only plays a paracrine role but also acts as a potential transduction factor to mediate GnRH-induced LH release in goldfish pituitary. In mouse LbetaT2 gonadotrophs, SN is able to up-regulate both LH secretion and the mRNA levels of multiple gonadotropin subunits. In LbetaT2 cells, SN also activates a mitogen-activated protein kinase (MAPK) cascade leading to phosphorylation of extracellular signal-regulated kinase (ERK). Further studies should focus on the isolation and localization of SN receptors in pituitary to expand the hypothesis that SN is a newly-identified hormone in the neuroendocrine system.