Browsing by Subject "Atlantic croaker"
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Item Alterations in thyroid hormone status in Atlantic croaker (Micropogonias undulatus) exposed to Aroclor 1254 and selected PCB congeners(2006-05) LeRoy, Kimberly Dale; Khan, Izhar A.Many studies in animals and humans have demonstrated that exposure topolychlorinated biphenyls (PCBs) can interfere with the normal function of the thyroid system. In this study, Atlantic croaker (Micropogonias undulatus) were exposed to a PCB mixture (Aroclor 1254) or one of three individual congeners (ortho-PCB 153, ortho-PCB 47 or planar PCB 77) in the diet for 30 days to investigate the effects of PCBs on thyroidal status. Thyroid hormones, thyroxine (T4) and triiodothyronine (T3) were extracted from plasma samples and measured with an enzyme-linked immunosorbent assay. Both male and female croaker exposed to the PCB mixture Aroclor 1254 (0.2 and 1.0 mg/kg body wt/day) had decreased plasma levels of total T3 when compared to the parallel control groups, but the effects on total T4 levels were inconsistent. Exposure to PCB 153 (0.1 and 1.0 mg/kg body wt /day) significantly lowered both T4 and T3, while PCB 47 at the same doses had no effect on thyroid hormone levels. Fish exposed to PCB 77 had no effect on T4 or T3 levels except an increase in T4 levels at the highest dose employed (0.1 mg/kg body weight/day). However, this dose of PCB 77 caused partial loss of appetite and may be too high to be relevant for the effects observed after exposure to Aroclor 1254 considering the fact that it constitutes only 0.31% of the PCB mixture. The results of the present study demonstrate that exposure to synthetic endocrine disrupting chemicals, such as PCBs at the higher end of environmentally realistic concentrations can have profound effects on the thyroidal status of Atlantic croaker. The ability of Aroclor 1254, as well as ortho-PCB 153, to consistently disrupt the natural homeostasis of the thyroid system in croaker is an important finding that contributes to a better understanding of PCB endocrine toxicity in teleosts.Item The effects of androgens on steroidogenesis in the ovary of Atlantic croaker (Micropogonias undulatus) : mechanism of action, the biochemical characterization of a membrane androgen receptor, and the chronic effects of exposure to an environmental antiandrogen(2002-05) Braun, Alyssa Maria, 1974-; Thomas, P. (Peter)Item Elucidating the signal cascades induced by progestins that mediate sperm hypermotility in Atlantic croaker (Micropogonias undulatus) and southern flounder (Paralichthys lethostigma)(2013-12) Tan, Wenxian, active 21st century; Thomas, P. (Peter)The overall goal of this research was to verify the involvement of membrane progestin receptor alpha (mPRα) in mediating progestin-stimulated sperm hypermotility in the Atlantic croaker and southern flounder. Sperm motility in Atlantic croaker and southern flounder were tested with both the endogenous progestin, 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) or the selective mPRα agonist, 10-ethenyl-19-norprogesterone (Org OD 02-0). In croaker, the Pi3k/Akt/Pde and ErbB2/Mapk intracellular signaling pathways were examined. The role of mPRα in mediating sperm hypermotility and fertility in southern flounder was also studied. The effects of seasonal hypoxia on sperm motility in croaker were investigated in a field study in the northern Gulf of Mexico in the fall of 2010. Finally, the effects of acidified activator solution (simulating ocean acidification) were studied in the laboratory. In vitro, Org OD 02-0 mimicked the stimulatory actions of 20β-S in inducing sperm hypermotility and intracellular signaling cascades in croaker and flounder sperm, indicating that mPRα is the mediator of progestin signaling in the sperm of these species. In croaker sperm, both the Pi3k/Akt/Pde and ErbB2/Mapk intracellular signaling pathways were shown to be important mediators of progestin-induced sperm hypermotility, suggesting novel functions of G [subscript olf] βγ-subunits in teleost sperm. In flounder sperm, mPRα was shown to be important in mediating sperm hypermotility as only high motility sperm with high expression of mPRα were responsive to progestin stimulation, resulting in higher fertilization success compared to low motility sperm. A single LHRHa injection resulted in increased sperm motility and fertility, associated with an increase in mPRα expression in the sperm plasma membrane. The results also suggest that the mPRα/Acy/cAMP pathway first described in croaker sperm is present in flounder sperm. Field studies of male Atlantic croaker exposed to chronic seasonal hypoxia showed that hypoxia exposure resulted in smaller gonads, lower spermatogenesis, reduced testicular mPRα expression, and in some sites, reduced sperm motility. Studies with croaker sperm using acidified activator solution to simulate ocean acidification indicated that croaker sperm were sensitive to environmental insult. Furthermore, the results suggested that the progestin signaling mechanism is more sensitive to changes in ocean pH levels than the mechanism that controls sperm motility.Item The identification and characterization of three distinct estrogen receptor subtypes in a teleost fish, the Atlantic croaker (Micropogonias undulatus)(2002) Hawkins, Mary Beth; Thomas, P. (Peter)Item Mechanisms of progestin-stimulated sperm hypermotility in two teleosts: the Atlantic croaker (Micropogonias undulatus) and the southern flounder (Platylicthys lethigstomata)(2007) Tubbs, Christopher William, 1979-; Thomas, P. (Peter)The goal of this research was to examine the role of the novel membrane progestin receptor alpha (mPR[alpha]) in the stimulation of sperm hypermotility by the progestin 17,20[beta],21-trihydroxy-4-pregnen-3-one (20[beta]-S) in two teleosts; the Atlantic croaker (Micropogonias undulatus) and the southern flounder (Platylicthys lethigstomata). In croaker, the expression, localization and hormonal regulation of mPR[alpha] in testis and sperm were investigated, as were the intracellular signaling pathways activated by 20[beta]-S and mPR[alpha] to induce croaker sperm hypermotility. In flounder, stimulation of sperm hypermotility by 20[beta]-S and binding of this steroid to flounder sperm membranes were examined. Finally, expression of mPR[alpha] was investigated in flounder testes and the expression and localization of this receptor in flounder testis and sperm was examined. In croaker sperm, mPR[alpha] was expressed on the plasma membrane and localized to the midpiece. Expression of mPR[alpha] was also shown to be associated with high sperm motility and regulated by gonadotropin. The signaling pathways activated by 20[beta]-S in croaker sperm were shown to involve activation of olfactory G-proteins (Golf). Subsequent activation of membrane adenylyl cyclases was also demonstrated and shown to be necessary for 20[beta]-S-stimulated cAMP production and 20[beta]-S-induction of sperm hypermotility. Furthermore, co-immunoprecipitation studies show mPR[alpha] and Golf physically associate with one another, establishing mPR[alpha] as the mediator of 20[beta]-S actions in croaker sperm. Finally, evidence was obtained for progestin-stimulation of sperm hypermotility and the presence of mPR[alpha] on sperm membranes in another marine teleost species belonging to a different family, the southern flounder. In addition, mPR[alpha] was shown to be expressed on flounder sperm membranes and also localized to the sperm midpiece. Results from the following studies support the hypothesis that mPR[alpha] is the mediator of 20[beta]S-stimulated sperm hypermotility in croaker and is a likely intermediary in southern flounder. Furthermore, these data provide a plausible mechanism by which 20[beta]-S and mPR[alpha] stimulate croaker sperm hypermotility. In addition, these results provide the first evidence of hormonal activation of Golf proteins for any species. Finally, mPR[alpha]-mediated mechanisms to increase sperm motility are suggested to be evolutionarily conserved in teleosts since they also likely exist in a non-sciaenid species, the southern flounder.Item Membrane progestin receptor expression, signaling and function in reproductive somatic cells of female vertebrates(2008-05) Dressing, Gwen Ellen, 1980-; Thomas, P. (Peter)The goal of the current research was to examine the expression, signaling and function of the membrane progestin receptors (mPRs) in the ovarian follicular cells of the Atlantic croaker (Micropogonias undulatus) and in human breast cancer cells. Multiple studies have examined the role of mPRs in the germ cells of several vertebrate classes, yet few studies have examined the role of the mPRs in the somatic cells of reproductive tissues. Therefore this research examines the mechanism of mPR action and its function in somatic cells of female reproductive tissues. Results from studies on the expression, localization and signaling of the mPR[alpha] in co-cultures of granulosa and theca cells from the croaker suggest that the mPR[alpha] is localized to the plasma membrane of both cell types and that the mPR[alpha] is associated with and signals via pertussis toxin-sensitive inhibitory G proteins to decrease intracellular cAMP and activate ERK. In addition, exposure of follicular co-cultures to progestins that activate the mPR[alpha] results in a decrease in serum starvation-induced cell death which is not replicated by progestins which activate the nuclear progestin receptor (nPR), indicating mPR mediation. Similar studies in two immortalized human breast cancer cell lines, MDA-MB-468 and SKBR3, suggest that the mPR[alpha] is also present in the membranes of these cells and signals in human breast cancer cell lines via activation of a pertussis toxin-sensitive G protein to significantly decrease in intracellular cAMP and activate ERK. Progesterone exposure also decreased serum starvation-induced cell death in SKBR3 cells which are nPR positive and in MDA-MB-468 cells which are nPR negative. Synthetic progestins which activate the nPR but not the mPR were ineffective in inhibiting death in either cell type suggesting that the mPR is the mediator of this progestin action. mPR[alpha], mPR[beta] and mPR[gamma] expression analysis of paired normal and malignant breast tissue biopsies from thirteen women revealed that at least one mPR isoform was upregulated in the malignant tissue of 70% of the women. In addition the expression of mPR[gamma] was positively correlated with the expression of the nPR and CK19, a breast epithelial cell marker.Item Modulation by hypoxia of membrane steroid receptor expression and functions in ovaries of Atlantic croaker (Micropogonias undulatus)(2015-12) Ondricek, Kathryn Elise; Thomas, P.(Peter),; Black, Bryan; Walther, Ben; Rahman, Md SaydurHypoxia is an endocrine disruptor, altering estrogen, testosterone, and progestin hormone levels and stunting gonadal growth in Atlantic croaker. Steroids act through specific hormone receptors to alter reproductive functions, and the hormonal response is dependent on the concentrations of these receptors. However, information is currently lacking on the effects of hypoxia on expression and functions of membrane receptors mediating rapid, non-genomic steroid actions such as final oocyte maturation and apoptosis. Atlantic croakers were exposed to normoxia (7.0 mg DO/L) or hypoxia (1.7 mg DO/L) for 6 weeks during their period of gonadal recrudescence (October-December). Relative gene expression was quantified using quantitative real-time PCR (qRT-PCR). mRNA expression of the membrane androgen receptor, ZIP9, was increased in hypoxia-exposed fish compared to normoxia-exposed controls, whereas mRNA expression of the membrane estrogen receptor, GPER, and membrane progestin receptor, mPRα, was decreased in hypoxia-exposed fish compared to controls. mRNA expression of pro-apoptotic factors Bax and p53 was also measured and expression of both genes was increased in hypoxia-exposed fish compared to controls. Relative protein expression of these receptors was quantified using Western blotting and the results were consistent with the qRT-PCR findings. Oocytes from both hypoxia-exposed and control fish were tested in an in vitro final oocyte maturation (FOM) assay to examine possible alterations in receptor functions. When oocyte maturation was stimulated with progestin, which acts through mPRα, significantly fewer oocytes of hypoxia-exposed fish underwent FOM compared to oocytes of normoxia-exposed controls. These results are consistent with the decrease in mPRα expression following hypoxia exposure. Ovaries were sectioned and stained with hematoxylin and eosin, and the proportions of perinucleolar stage, tertiary yolk stage, and atretic oocytes were determined. Ovaries from fish exposed to hypoxia showed an increase in the proportion of perinucleolar stage and atretic oocytes and a decrease in the proportion of tertiary yolk stage oocytes compared to controls. Finally, apoptotic cells in ovarian tissue sections were labeled using in situ TUNEL staining. Ovaries from fish exposed to hypoxia showed an increased proportion of TUNEL-positive ovarian follicle cells compared to controls. Collectively, these results show the concurrence of increased ZIP9 expression and apoptotic follicle cells in ovaries of Atlantic croaker exposed to hypoxia in vivo.Item Signal transduction pathways regulating steroidogenesis in the ovary of Atlantic croaker (Micropogonias undulatus)(2004-08) Benninghoff, Abby Diane, 1975-; Thomas, P. (Peter)The overall aim of this research was to study signal transduction pathways regulating ovarian steroid production in a teleost model, Atlantic croaker (Micropogonias undulatus). The potential roles of calcium-, adenylyl cyclase- and/or mitogen-activated protein kinase (MAP kinase)-dependent signaling pathways in mediating steroidogenesis induced by gonadotropin, the steroid hormone 20β-S or the polychlorinated biphenyl mixture Aroclor 1254 were investigated. Two experimental incubation systems were utilized for the measurement of steroidogenesis in vitro: static ovarian tissue incubation and a primary co-culture system of theca and granulosa cells developed specifically to investigate signaling pathways in steroidogenic cells. Multiple calcium-dependent sites of regulation of steroidogenesis were identified, including voltage-sensitive calcium channels (VSCCs), inositol-1,4,5- triphosphate receptors, calmodulin, calcium/calmodulin-dependent protein kinase II (CaMK II) and aromatase. Gonadotropin-induced testosterone synthesis involves the rapid synthesis of cAMP and the activity of the cAMP-dependent protein kinase (PKA). Furthermore, evidence was obtained for an involvement of mitogen-activated protein kinase (MAP kinase) in gonadal steroidogenesis in a lower vertebrate model. Treatment with hCG induced MEK-dependent phosphorylation of ERK1/2 in a concentration- and time-dependent manner in co-cultured croaker theca and granulosa cells. Few interactions among the signaling pathways were observed; however, there is evident cross-talk between the adenylyl cyclase and MAP kinase pathways mediated by cAMP. Furthermore, inhibitors of VSCCs, calmodulin, CaMK II and MEK reduced forskolin- and dbcAMP-induced testosterone synthesis. Acute in vitro exposure to lead cause moderate decreases in basal testosterone and estradiol synthesis by whole ovarian follicles, while o,p’-DDT increased both basal and hCG-stimulated steroid production. Aroclor 1254 induced a dramatic increase in gonadotropin-stimulated estradiol synthesis; this stimulatory effect was attenuated by inhibitors of VSCCs and calmodulin, thus providing preliminary evidence for a novel mechanism of endocrine disruption in vertebrates. Finally, a stimulatory effect of the maturation-inducing steroid 17α,20β,21- trihydroxy-4-pregnen-3-one (20β-S) on testosterone synthesis by co-cultured theca and granulosa cells was characterized. Physiological concentrations of 20β-S augmented both basal and gonadotropin-stimulated testosterone synthesis by cells from mature ovarian follicles. The stimulatory action of 20β-S is independent of calcium-dependent signaling, and 20β-S does not alter adenylyl cyclase activity. However, 20β-S stimulation of testosterone synthesis may be mediated by the MAP kinase pathway.Item Tracing organic matter pathways in marine food webs using fatty acids and compound specific stable isotope analysis(2015-08) Smith, Stephanie Denise; McClelland, James W.; Dunton, Kenneth H; Walther, Benjamin DOrganic matter inputs to the marine environment vary over seasonal and spatial scales, altering the type and availability of food sources for marine consumers. It is important to identify diet in order to understand basic ecology, characterize trophic interactions, and predict consequences of biotic and abiotic change within a community. Methods of direct observation of diet and feeding can be difficult, so indirect methods have been developed such as analysis of gut contents and fecal pellets. However, these methods only represent a snapshot of the last meal, and provide information about what was ingested, but not what was actually incorporated into consumer tissues. Therefore, biogeochemical approaches such as fatty acid (FA) and stable isotope analyses have been developed, which provide a time-integrated measure of diet. Further, stable isotope measurements of specific FA markers can be used to identify carbon sources, and can be applied to a variety of food web studies (Iverson et al., 2004). The purpose of this research is to examine the linkages between organic carbon sources and trophic transfer by consumers. To achieve this, we use FA biomarkers and compound specific stable isotope analysis (CSIA) to trace carbon cycling. This study has two main components: environmental sampling and experimental research. Chapter 1 demonstrates the use of these tools for elucidating seasonal trophic linkages in invertebrates collected from the Alaskan Arctic coast. Overall, invertebrate diets were characterized by terrestrial, detrital, and carnivorous sources in winter and spring, with a shift toward autochthonous diatom-based diets in summer. Our results demonstrate the importance of terrestrial organic carbon as a subsistence food source in winter, whereas in situ production in summer was critical for accumulating FA stores rich in essential FAs. Chapter 2 is an experimental feeding study designed to quantify the incorporation rates of 18:2n-6 from diet to tissue in Atlantic croaker. Liver tissues accumulated FAs more quickly than muscle tissues, but both tissues reached equilibrium at 5 to 7 weeks. From these experiments, quantitative assessments of diet sources can be made with confidence when using FAs to understand trophic interactions of Atlantic croaker and other similar species.