The neuroendocrine sites and mechanisms of action whereby testosterone (T) regulates LH, FSH and prolactin signals in male rats were investigated by determining the effects of orchidectomy and graded T replacement on the secretion patterns of these hormones in blood. A chronic jugular catheter system was established to obtain blood samples at 5-10 min intervals from unrestrained, unanesthetized rats; this enabled moment to moment changes in plasma hormone concentrations, and thus hormone patterns, to be determined. In intact rats the patterns of LH and prolactin in plasma are pulsatile, but exhibit no synchrony; the FSH pattern shows no pulses. Post-orchidectomy LH concentrations rise due to increases in pulse amplitude and frequency; FSH also rises, but still no pulses are detected. Decreased prolactin pulse amplitudes result in lower plasma concentrations, despite an increase in pulse frequency. Treatment of orchidectomized rats with low doses of T increases mean LH and FSH concentrations, thus demonstrating that T is capable of exerting a positive feedback effect in males. The effect on LH is due to a stimulation of LH pulse amplitude which is not mediated at the pituitary. The FSH pattern becomes pulsatile and is synchronized with that of LH. Higher doses of T exert negative feedback on LH and FSH; LH pulse amplitude and frequency are decreased and the FSH pattern once again shows no pulses. These effects of T are partially due to inhibition of the pituitary response to LHRH. Low doses of T completely block the effects of orchidectomy on prolactin pulse amplitude, but T levels similar to those in intact animals are required to reverse the accelerated pulse frequency. The results suggest that gonadotropins and prolactin are regulated by separate pulse generators, each responsive to changes in plasma T. Furthermore, T, depending on its concentration, regulates gonadotropin and prolactin secretion by exerting stimulatory and/or inhibitory effects on various neur

The Ca$ sp{2+}$ binding protein, calmodulin, has been implicated in several Ca$ sp{2+}$ dependent processes during secretion in many different secretory systems. One area where calmodulin is suggested to play a role is the fusion of the secretory granule with the plasma membrane during exocytosis. Calmodulin may mediate the interaction or fusion through specific calmodulin-binding proteins (CMBPs) present in these two membranes. / CMBPs present in bovine chromaffin cell granule membranes were characterized using the techniques of calmodulin affinity chromatography and $ sp{125}$I calmodulin overlay. Several CMBPs were detected in these membranes. One of these proteins, of molecular mass 65 kilodaltons (65-CMBP), was found to be immunologically identical to a protein previously identified in rat brain synaptic vesicles termed "p65". / Recent studies have debated the subcellular localization of 65-CMBP (p65) as well as another synaptic vesicle protein, synaptophysin (p38). A controversial question surrounding these proteins is whether or not they are present in large dense core secretory granules of neurons and endocrine cells, or exclusively localized on small synaptic or synaptic-like vesicles present in these tissues. Subcellular fractionation studies of adrenal medulla showed that both 65-CMBP and p38 were present in fractions corresponding to granule membranes and intact granules. However, an additional membrane fraction equilibrating near the upper portion of the sucrose gradient, also showed strong immunoreactivity with an antibody to p38. / CMBPs were also isolated from bovine posterior pituitary neurosecretory granules and rat brain synaptic vesicles. These membranes were also found to contain the 65-CMBP (63 kDa in rat brain synaptic vesicles). / Chromaffin cell membranes were isolated using positively charged microcarriers. The 65-CMBP (p65) was also identified in this structure. In addition, immunoblots of plasma and granule membranes showed that the 65-CMBP was a component of both membranes, whereas p38 was only present in granule membranes. Thus, there appears to be a different subcellular localization between the 65-CMBP and p38 in chromaffin cells. / These findings on the 65-CMBP are discussed in relation to its possible role as a mediator of the fusion step of the exocytotic process.

The goal of this thesis was to move toward defining the molecular mechanism(s) of Vitamin A-induced limb malformations. Vitamin A and its biologically active metabolites, the retinoids, are potent limb teratogens, inducing reductive defects, preceded by decreased chondrogenesis and upregulated apoptosis. To target the origins of these effects, we focused initially on characterizing the pathway of Vitamin A-induced apoptosis. Our first objective was to identify mediators of the degradation and commitment phases of this process. We show dose-dependent activation of the effector caspase, caspase-3, and increased mitochondrial cytochrome-c release after treatment. The next objective was to determine whether these changes were receptor-mediated. Both pan-RAR (BMS 189453) and, surprisingly, pan-RXR (HX603) antagonists were able to ameliorate Vitamin A-induced limb malformations and apoptosis, indicating both receptor subtypes are important mediators of these processes. Interestingly, exposure to either antagonist alone also upregulated apoptosis. Each compound induced a specific, reproducible pattern of limb bud apoptosis, reiterating the fact that proper regulation of this process is crucial for normal limb morphology. Our final objective was to identify genes that may be responsible for the apoptosis and other pathologies observed after retinoid exposure. Vitamin A treatment significantly upregulated 81 genes, including key limb development signaling molecules, oncogenes, extracellular matrix/cell adhesion molecules, and transcriptional regulators such as Id3, Snai1, Hes1 and Eya2. To link these expression changes to teratogenic outcome, the response of these 4 genes was assessed after Vitamin A exposure, and after limb bud rescue with BMS 189453 and HX603; expression levels were correlated with limb malformations. Pathway analysis of the significantly upregulated genes reveals that many that are associated with cell-cycle, apoptosis and chondrogenesis are functionally linked. Moreover, members of these cascades cross-talk with one another, indicating that retinoids affect multiple cellular processes in a coordinated fashion to induce their teratogenic effects. Collectively, these studies have advanced our understanding of Vitamin A-mediated apoptotic pathways. In addition, we have proposed several mechanisms for retinoid-induced limb malformations, identified novel retinoid targets, and revealed multiple avenues for future research.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN. / CHAIRMAN: VINCENT G. ZANNONI.

Thesis (Ph. D.)--University OF MICHIGAN.