Androgen receptor (AR) signalling is essential for the development and function of the male reproductive system. Conditional gene ablation using the Cre-loxP system has previously assisted in the elucidation of the role of AR in different cell types. The aim of this study was to examine the effects of the ablation of AR in previously untargeted cell types, with the hypothesis that this will have significant and novel effects on reproductive development and function that have not been previously documented by current models of androgen disruption. In these studies, three Cre recombinase lines were empirically validated for action in the male reproductive system, before being used to ablate AR and the phenotypes of the resulting lines were characterised. Endothelial-specific receptor tyrosine kinase (Tie2)-Cre was shown to target the vascular and endothelial cells of the testis, and used to ablate AR in these cells. The testes of the resulting Tie2-ARKO line were morphologically similar to controls, with normal spermatogenesis and mature spermatozoa present in the cauda epididymis. Aquaporin 2 (Aqp2)-Cre was shown to target the post-meiotic germ cells of the testis, and was used to ablate AR in these cells. The testes of the resulting Aqp2-ARKO line were morphologically similar to controls, with normal spermatogenesis and mature spermatozoa present in the cauda epididymis. It was concluded that the Ar gene was dispensable in the endothelial cells and post-meiotic germ cells of the testis for normal spermatogenesis. Forkhead box protein G1 (FoxG1)-Cre was shown to target the caput epididymal epithelium and pituitary, and used to ablate AR in these cells. d100 FoxG1-ARKO mice had a severe testicular phenotype, with sloughing of the seminiferous epithelium, atrophy of some seminiferous tubules and distension of the rete testis with spermatozoa. Despite the severe testis phenotype, ablation in the testis was incomplete and restricted to a small percentage of Leydig cells, with no ablation in Sertoli cells. Ablation of AR in the embryonic pituitary did not cause adult serum testosterone or LH concentrations to change, nor did it cause changes in other pituitary hormone transcripts. Mosaic ablation of AR in the caput epididymal epithelium was shown to impair epididymal development, with failure of initial segment (segment I) development and a significant decrease in epithelial cell height and lumen diameter in the remaining proximal caput epididymis (segment II). Dysfunction of the caput epididymis resulted in the failure of spermatozoa to transit the efferent ducts into the epididymis correctly: instead they were found to stall in the efferent ducts and produce a block. The testicular phenotype could be explained as the result of fluid backpressure effects resulting from the efferent duct block. Consequently, low concentration of spermatozoa in the cauda epididymis resulted in infertility in the FoxG1-ARKO, which represents a new model of obstructive azoospermia.

The shock prod defensive burying test has proven incredibly reliable and instrumental in determining the underpinnings of normal anxiety in rodents. Largely ignored in tests of defensive burying, however, is the capacity for individual animals to display marked variations in active and passive coping behaviors. To unmask the neurobiological correlates of this behavioral differentiation, rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. This design invited striking variations in defensive burying behavior levels, in which some rats either buried robustly or showed little to no levels of defensive burying. Furthermore, differences in burying behavior were associated with marked differences in the central expression of arginine vasopressin (AVP) and oxytocin (OT). Thus, relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying expressed higher levels of AVP mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses expressed relatively higher levels of AVP and OT mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. CRH mRNA levels did not vary as a function of burying activity in the central nucleus of the amygdala, the anterior division of the bed nuclei of the stria terminalis, nor in the paraventricular nucleus. These findings suggest a role for central AVP and OT in mediating differential defensive behaviors, and demonstrate the utility of using a pain free test of conditioned defensive burying as a framework for exploring individual differences in behavioral coping and neuroendocrine capacity.

The shock prod defensive burying test has proven incredibly reliable and instrumental in determining the underpinnings of normal anxiety in rodents. Largely ignored in tests of defensive burying, however, is the capacity for individual animals to display marked variations in active and passive coping behaviors. To unmask the neurobiological correlates of this behavioral differentiation, rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. This design invited striking variations in defensive burying behavior levels, in which some rats either buried robustly or showed little to no levels of defensive burying. Furthermore, differences in burying behavior were associated with marked differences in the central expression of arginine vasopressin (AVP) and oxytocin (OT). Thus, relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying expressed higher levels of AVP mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses expressed relatively higher levels of AVP and OT mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. CRH mRNA levels did not vary as a function of burying activity in the central nucleus of the amygdala, the anterior division of the bed nuclei of the stria terminalis, nor in the paraventricular nucleus. These findings suggest a role for central AVP and OT in mediating differential defensive behaviors, and demonstrate the utility of using a pain free test of conditioned defensive burying as a framework for exploring individual differences in behavioral coping and neuroendocrine capacity.

The shock prod defensive burying test has proven incredibly reliable and instrumental in determining the underpinnings of normal anxiety in rodents. Largely ignored in tests of defensive burying, however, is the capacity for individual animals to display marked variations in active and passive coping behaviors. To unmask the neurobiological correlates of this behavioral differentiation, rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. This design invited striking variations in defensive burying behavior levels, in which some rats either buried robustly or showed little to no levels of defensive burying. Furthermore, differences in burying behavior were associated with marked differences in the central expression of arginine vasopressin (AVP) and oxytocin (OT). Thus, relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying expressed higher levels of AVP mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses expressed relatively higher levels of AVP and OT mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. CRH mRNA levels did not vary as a function of burying activity in the central nucleus of the amygdala, the anterior division of the bed nuclei of the stria terminalis, nor in the paraventricular nucleus. These findings suggest a role for central AVP and OT in mediating differential defensive behaviors, and demonstrate the utility of using a pain free test of conditioned defensive burying as a framework for exploring individual differences in behavioral coping and neuroendocrine capacity. / Medicine, Faculty of / Graduate

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2008. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 87-114).

Afin de mettre en évidence les principaux éléments de la voie androgénique et œstrogénique dans l’épididyme du rat des sables adulte, capturé dans la région de Beni Abbès, en Algérie, l’aromatase, l’œstradiol, les récepteurs des androgènes (RA) et des œstrogènes (REα, REβ, GPR30) ont été recherchés chez des animaux en saison d’activité, en saison de repos sexuel, chez des animaux castrés, castrés puis traités par la testostérone et chez des animaux ayant subi la ligature des canaux efférents. En saison d’activité, les RA sont ubiquitaires, l’aromatase est cytoplasmique par contre l’œstradiol est nucléaire et cytoplasmique. Les REα et le GPR30 sont principalement dans le cytoplasme apical par contre les REβ sont nucléaires. En saison de repos sexuel, les RA, l’aromatase, l’œstradiol, les REα et le GPR30 persistent, cependant, les REβ subissent une translocation cytoplasmique. Chez les animaux castrés, les RA, l’aromatase et l’œstradiol sont réduits par contre les REα persistent avec une faible intensité. Le GPR30 est cytoplasmique et nucléaire. Chez les animaux castrés puis traités, les RA, l’aromatase, l’œstradiol, les REα, les REβ et le GPR30 sont restaurés. Chez les animaux ligaturés, le RA est faiblement conservé uniquement dans l’épididyme proximal. L’aromatase et l’œstradiol sont conservés. Le signal des REα, des REβ et du GPR30 est fortement exprimé dans le noyau et le cytoplasme dans l’épididyme proximal par contre il est fortement exprimé uniquement pour les REα dans l’épididyme distal. Par western blot, les RA, REα, REβ et GPR30 sont de 122, 64, 55 et 55 kDa respectivement. / In order to highlight the main elements of androgen and estrogen pathway in the epididymis of sand rat, captured in Beni Abbès area, in Algeria, androgen receptor (AR), aromatase, estradiol, estrogen receptors (ERα, ERβ and GPR30) were explored in breeding season, in resting season and in animals underwent castration, castration then testosterone treatment and ligation of efferent ducts. In breeding season, AR has a ubiquitous distribution, aromatase is exclusively cytoplasmic and estradiol is nuclear and cytoplasmic. The ERα and GPR30 were distributed with a high intensity in the apical cytoplasm contrarily to ERβ which were nuclear. In resting season, AR, aromatase, estradiol, ERα persist with lower staining. However, ERβ undergo cytoplasmic translocation and GPR30 persist in cytoplasm. In castrated animals, AR, aromatase and estradiol are reduced. ERα persist with low intensity in the apical cytoplasm. GPR30 is distributed in the cytoplasm and the nucleus. In castrated then treated animals, AR is restored; aromatase and estradiol reappear with a cytoplasmic localization for aromatase, nuclear and apical for ERα. ERβ and GPR30 are restored and have a cytoplasmic localization. In ligatured, RA is preserved in the caput, aromatase and estradiol persist caput and cauda. The signal of ERα, ERβ and GPR30 is highly expressed in the nucleus and cytoplasm of caput epididymis and highly expressed of ERα exclusively in cauda. By Western blot, RA, ERα, ERβ and GPR30 are found with molecular weights of 122, 64, 55 and 55 kDa respectively.

The sexual differentiation of the spinal nucleus of bulbocavernosus (SNB) and the bulbocavernosus (BC) and levator ani (LA) muscles that the SNB innervates, are masculinized by androgens acting on the BC/LA. The site of androgen receptors (AR) responsible for the masculinization of the neuromuscular system is not known. A potential site of action is satellite cells: proliferation of these cells is androgen-dependent and satellite cells seem to contribute to the size of the LA. Fluorescent immunohistochemistry and confocal microscopy were used to co-localize satellite cells and AR within the LA of postnatal day one and three male and female rats. Results indicate that satellite cells express AR and reveal a difference in proportion of satellite cells expressing AR between the LA and control muscle. Interpretations of these findings, including whether the relatively small proportion of AR accounted for by satellite cells is enough to masculinize the SNB system, are discussed.

The sexual differentiation of the spinal nucleus of bulbocavernosus (SNB) and the bulbocavernosus (BC) and levator ani (LA) muscles that the SNB innervates, are masculinized by androgens acting on the BC/LA. The site of androgen receptors (AR) responsible for the masculinization of the neuromuscular system is not known. A potential site of action is satellite cells: proliferation of these cells is androgen-dependent and satellite cells seem to contribute to the size of the LA. Fluorescent immunohistochemistry and confocal microscopy were used to co-localize satellite cells and AR within the LA of postnatal day one and three male and female rats. Results indicate that satellite cells express AR and reveal a difference in proportion of satellite cells expressing AR between the LA and control muscle. Interpretations of these findings, including whether the relatively small proportion of AR accounted for by satellite cells is enough to masculinize the SNB system, are discussed.