Puberty is a critical period of development that is characterized by significant remodeling and reorganization of neuronal connections. Additionally, this period is marked by the transition to a fertile state and the development of secondary sex characteristics driven by a surge in gonadal steroid hormones. Puberty is also vulnerable to stress exposure, as pubertal stress during this period results in negative enduring changes to the brain and behavior. Treatment with the bacterial endotoxin lipopolysaccharide (LPS) results in enduring dysfunction of the hypothalamic-pituitary-gonadal (HPG) axis in male and female mice. However, the mechanism underlying this dysfunction was unclear. Thus, this thesis was designed to investigate possible mechanisms through which pubertal stress could alter HPG axis functioning. This work first examined the acute effects of LPS treatment on various components of the HPG axis, such as kisspeptin (Kiss1) and kisspeptin receptor (Kiss1R) expressions in the brain, and luteinizing (LH) and follicle stimulating hormone (FSH) concentrations in the blood (Study 1). The next study examined the enduring effects of LPS treatment on inflammation as well as on Kiss1 and Kiss1R expressions in the brain, and LH and FSH concentrations in the blood (Study 2). The findings showed that Kiss1 and Kiss1R were downregulated in an acute and enduring manner following LPS treatment and are likely responsible for HPG axis downregulation. The final study amalgamates the novel discoveries of the previous findings and hypothesizes that pharmaceutical Kiss1 treatment will prevent adult sexual behavior dysfunction after pubertal LPS treatment. Findings from this study will inform us whether the enduring HPG axis downregulation following pubertal LPS treatment is reversible in adulthood and could provide a viable prospective intervention for fertility complications across species.

The neuroendocrine basis of paternal behaviour is hypothesised to be homologous to that of maternal behaviour, with the expression of paternal behaviour resulting from the activation of estrogen-sensitive pathways in the brain. However, despite the fact that Phodopus campbelli fathers are highly paternal, previous studies have failed to identify an activational role for estradiol in the expression of this behaviour. The goal of this thesis was to exclude or define a role for estradiol in P. campbelli paternal behaviour. In the first study, estrogen receptor alpha (ERα) expression was examined in three brain regions relevant to maternal behaviour (medial preoptic area, bed nucleus of the stria terminalis, and medial amygdala). Male and female P. campbelli and its close relative P. sungorus were sampled as sexually naïve adults, following mating to satiety, or as new parents. As predicted, new fathers of either species did not upregulate ERα expression in these brain regions. Mating also had no effect on ERα expression in males or females from either species. However, ERα expression was also not upregulated in any of the three brain regions in new mothers. Thus, it is possible that estradiol does not play a primary role in activating maternal behaviour in Phodopus females. In the second study, P. campbelli males were administered the aromatase inhibitor letrozole at three different developmental stages (independence from the mother, testicular maturation, and sexually naïve adult) and tested for adult sexual and paternal behaviour. Aromatase inhibition during juvenile development resulted in diminished expression of adult paternal and sexual behaviour, suggesting that estradiol acts at critical periods in development to organize the paternal brain for subsequent behavioural responses. Treatment of sexually naïve adults did not alter male behaviour, which further supports previous evidence that estradiol does not activate paternal behaviour in P. campbelli males, although sexual behaviour in females is completely estradiol-dependent in this species. These findings suggest that the activational roles of estradiol in maternal and male sexual behaviour identified in the rat do not generalize to Phodopus, and imply greater species to species variation in the neuroendocrine control of these behaviours than previously suspected. / Thesis (Ph.D, Biology) -- Queen's University, 2008-09-15 10:52:00.161

Thesis (M.Sc.)(Entomology)--University of Pretoria, 2006. / Includes summary. Includes bibliographical references. Available on the Internet via the World Wide Web.

Water balance in insects is under neuroendocrine control, and both diuretic and antidiuretic factors are thought to be involved. Despite being the largest and most diverse order of insects, the Coleoptera have been largely neglected in studies of hormonal control of excretion. However, the only insect from which both diuretic and antidiuretic peptides, acting on Malpighian tubules, have been isolated, is a beetle: the mealworm Tenebrio molitor. This study reports the effects of different diuretic factors on fluid secretion rates by mealworm tubules and briefly explores cross-reactivity of factors between beetle species, using the scarabaeid Onthophagus gazella. Attempts to localise an antidiuretic factor and a diuretic factor in the mealworm nervous system are also described. The study showed that calcitonin(CT)-like peptides from silkmoth and mosquito increased fluid secretion in mealworm tubules in a dose-dependent manner, but the cockroach CT-like peptide, Dippu-DH31, had no effect. The pharmacological agent thapsigargin, which mimics the action of kinins, caused a small but significant increase in tubule secretion rates. Since mealworm tubules are affected by more than one diuretic factor, the possibility of synergism between different factors was explored by testing CT-like peptides, thapsigargin and the mealworm corticotropin-releasing factor (CRF)-related diuretic peptide Tenmo-DH37, in various combinations and at different concentrations. No synergistic or additive effects were observed. C-terminal fragments of the locust and cockroach CRF-related diuretic peptides, Locmi-DH46 and Dippu-DH46 respectively, had no significant effect on fluid secretion. This is consistent with previous studies which have shown that the receptor binding and activation sites have to be linked in the same molecule for biological activity. However, the corresponding whole peptides increased fluid secretion, revealing the high degree of cross-reactivity of CRF-related peptides across insect orders. The mealworm diuretic factor Tenmo-DH37 increased fluid secretion in isolated tubules of O. gazelle in a dose-dependent manner, demonstrating cross-reactivity with this distantly related beetle species. However, brain homogenates from O. gazelle significantly inhibited fluid secretion in mealworm tubules. This is unusual, because extracts of insect nervous tissue have generally been shown to be a source of diuretic activity. Immunohistochemical techniques were used in an attempt to localise the mealworm antidiuretic factor Tenmo-ADFa in the nervous system of T. molitor. The antiserum developed against Tenmo-ADFa was found to be inadequately sensitive, and so these efforts were unsuccessful. The same techniques were employed in an attempt to locate the tissue containing a putative beetle CT-like peptide in the mealworm, using antiserum raised against the cockroach CT-like peptide Dippu-DH31. This too did not lead to a result. Experiments were repeated with various modifications, without success. It remains unclear whether the lack of results is due to degradation of the antiserum or if CT-like peptides are not present in detectable amounts in the mealworm nervous system. / Dissertation (MSc (Entomology))--University of Pretoria, 2007. / Zoology and Entomology / MSc / unrestricted

The physiological mechanisms through which nutrition mediates its effects in controlling reproduction are not well characterized. Both neural and endocrine components have been implicated in the communication of nutritional status to the central nervous system. Leptin, a hormone synthesized and secreted mainly by adipocytes, is heavily involved in this communication network. The objectives of studies reported herein were 1) to determine the effects of short-term restriction of nutrients on circulating leptin, leptin gene expression in adipose tissue, and leptin receptor (LR) gene expression in the adenohypophysis of ovariectomized cows; and 2) to investigate the responsiveness of the hypothalamic-adenohypophyseal (AP) axis of fasted and non-fasted cattle to leptin. Studies demonstrated that circulating concentrations of leptin and leptin gene expression in subcutaneous adipose tissue are decreased by fasting. Although 2 to 3 days of fasting did not affect patterns of release of luteinizing hormone (LH), cerebroventricular infusions of leptin increased mean circulating concentrations of LH in fasted, but not normal-fed cows, without affecting frequency or amplitude of pulses of LH. In vitro studies were conducted to determine whether the in vivo effects of leptin could be accounted for at the hypothalamic and/or AP levels. Leptin did not affect the release of gonadotropin-releasing hormone (GnRH) from hypothalamic-infundibular explants from either normal-fed or fasted cattle. Moreover, leptin did not affect the basal release of LH from bovine AP cells or AP explants from normal-fed cows. However, leptin induced a higher basal release of LH from AP explants of fasted cows and increased GnRH-stimulated release of LH from AP explants of normal-fed cows. Results demonstrate that leptin acts directly at the AP level to modulate the secretion of LH, and its effects are dependent upon nutritional status. Cellular mechanisms associated with the increased responsiveness of gonadotropes to leptin in fasted cows were investigated. Expression of LR and suppressor of cytokine signaling-3 (SOCS-3) in the adenohypophysis did not account for the increased responsiveness of fasted cows to leptin. Therefore, although leptin clearly stimulates the hypothalamic-gonadotropic axis in nutrient-restricted cattle, it is unclear why cattle maintained under neutral or positive energy balance are resistant to leptin.

Canadians live in a society where the sun does not dictate the workday. Our lifestyles must shift to cater to the 24-hour demands of a fast paced global community. As a result our circadian rhythms are altered, leading to dysregulation of key physiological processes responsible for the maintenance of essential functions like energy homeostasis. Energy homeostasis is controlled by neuropeptide-expressing neurons within the hypothalamus. These neurons are affected by circulating hormone and nutrient levels in addition to their endogenous molecular clock machinery that controls cellular processes. Therefore, hypotheses were generated that non-SCN hypothalamic neurons express orexigenic neuropeptides in a rhythmic fashion without external influence from the SCN as a result of internal rhythmicity; and that elevated concentrations of palmitate, a ubiquitous saturated FFA common in a high fat diet, have direct influence on the mRNA levels of circadian clock components Bmal1, Clock, Per2, Rev-erbα and the potent orexigenic neuropeptides NPY, AgRP and ppGhrelin through mechanisms related to HAT, SIRT1 and AMPK. Using the mHypoE-44 neurons, a well characterized cell line that expresses the molecular clock and specific neuropeptides these hypotheses were explored in four studies. Neuropeptide expression within the mHypoE-44 neurons was determined to be rhythmic. NPY and NT demonstrate significant 24-hour rhythms. CRH and ppGhrelin mRNA cycled significantly in an ultradian fashion, oscillating approximately every 18 h. AgRP mRNA did not show a significant rhythm. We identified rhythmic binding of BMAL1 to the NPY promoter, suggesting clock-mediated control of neuropeptide expression. Bmal1 and Clock mRNA levels were elevated with palmitate, whereas Per2 and Rev-erbα mRNA showed significant decreases following palmitate treatment. Palmitate increased the acetylation of both BMAL1 and PER2 proteins. Alteration of AMPK activity altered the mRNA levels of all clock genes assayed and AMPK activation diminished the palmitate-induced changes in Bmal1 mRNA. Palmitate significantly elevated both NPY and ppGhrelin mRNA levels. Chemical modifiers that decrease acetylation altered these systems. AMPK activation reduced the palmitate-induced changes in NPY mRNA levels. These findings demonstrate that non-SCN neurons have rhythmic neuropeptide transcript levels. This thesis elucidates a direct effect of palmitate on the molecular clock and neuropeptide expression at the level of the hypothalamic neuron; and these findings highlight a role for HAT/SIRT1 activation and AMPK in these important processes, which ultimately contribute to the understanding of circadian dysregulation and energy balance.

Canadians live in a society where the sun does not dictate the workday. Our lifestyles must shift to cater to the 24-hour demands of a fast paced global community. As a result our circadian rhythms are altered, leading to dysregulation of key physiological processes responsible for the maintenance of essential functions like energy homeostasis. Energy homeostasis is controlled by neuropeptide-expressing neurons within the hypothalamus. These neurons are affected by circulating hormone and nutrient levels in addition to their endogenous molecular clock machinery that controls cellular processes. Therefore, hypotheses were generated that non-SCN hypothalamic neurons express orexigenic neuropeptides in a rhythmic fashion without external influence from the SCN as a result of internal rhythmicity; and that elevated concentrations of palmitate, a ubiquitous saturated FFA common in a high fat diet, have direct influence on the mRNA levels of circadian clock components Bmal1, Clock, Per2, Rev-erbα and the potent orexigenic neuropeptides NPY, AgRP and ppGhrelin through mechanisms related to HAT, SIRT1 and AMPK. Using the mHypoE-44 neurons, a well characterized cell line that expresses the molecular clock and specific neuropeptides these hypotheses were explored in four studies. Neuropeptide expression within the mHypoE-44 neurons was determined to be rhythmic. NPY and NT demonstrate significant 24-hour rhythms. CRH and ppGhrelin mRNA cycled significantly in an ultradian fashion, oscillating approximately every 18 h. AgRP mRNA did not show a significant rhythm. We identified rhythmic binding of BMAL1 to the NPY promoter, suggesting clock-mediated control of neuropeptide expression. Bmal1 and Clock mRNA levels were elevated with palmitate, whereas Per2 and Rev-erbα mRNA showed significant decreases following palmitate treatment. Palmitate increased the acetylation of both BMAL1 and PER2 proteins. Alteration of AMPK activity altered the mRNA levels of all clock genes assayed and AMPK activation diminished the palmitate-induced changes in Bmal1 mRNA. Palmitate significantly elevated both NPY and ppGhrelin mRNA levels. Chemical modifiers that decrease acetylation altered these systems. AMPK activation reduced the palmitate-induced changes in NPY mRNA levels. These findings demonstrate that non-SCN neurons have rhythmic neuropeptide transcript levels. This thesis elucidates a direct effect of palmitate on the molecular clock and neuropeptide expression at the level of the hypothalamic neuron; and these findings highlight a role for HAT/SIRT1 activation and AMPK in these important processes, which ultimately contribute to the understanding of circadian dysregulation and energy balance.

The physiological mechanisms through which nutrition mediates its effects in controlling reproduction are not well characterized. Both neural and endocrine components have been implicated in the communication of nutritional status to the central nervous system. Leptin, a hormone synthesized and secreted mainly by adipocytes, is heavily involved in this communication network. The objectives of studies reported herein were 1) to determine the effects of short-term restriction of nutrients on circulating leptin, leptin gene expression in adipose tissue, and leptin receptor (LR) gene expression in the adenohypophysis of ovariectomized cows; and 2) to investigate the responsiveness of the hypothalamic-adenohypophyseal (AP) axis of fasted and non-fasted cattle to leptin. Studies demonstrated that circulating concentrations of leptin and leptin gene expression in subcutaneous adipose tissue are decreased by fasting. Although 2 to 3 days of fasting did not affect patterns of release of luteinizing hormone (LH), cerebroventricular infusions of leptin increased mean circulating concentrations of LH in fasted, but not normal-fed cows, without affecting frequency or amplitude of pulses of LH. In vitro studies were conducted to determine whether the in vivo effects of leptin could be accounted for at the hypothalamic and/or AP levels. Leptin did not affect the release of gonadotropin-releasing hormone (GnRH) from hypothalamic-infundibular explants from either normal-fed or fasted cattle. Moreover, leptin did not affect the basal release of LH from bovine AP cells or AP explants from normal-fed cows. However, leptin induced a higher basal release of LH from AP explants of fasted cows and increased GnRH-stimulated release of LH from AP explants of normal-fed cows. Results demonstrate that leptin acts directly at the AP level to modulate the secretion of LH, and its effects are dependent upon nutritional status. Cellular mechanisms associated with the increased responsiveness of gonadotropes to leptin in fasted cows were investigated. Expression of LR and suppressor of cytokine signaling-3 (SOCS-3) in the adenohypophysis did not account for the increased responsiveness of fasted cows to leptin. Therefore, although leptin clearly stimulates the hypothalamic-gonadotropic axis in nutrient-restricted cattle, it is unclear why cattle maintained under neutral or positive energy balance are resistant to leptin.

Endocrine disrupting chemicals (EDCs) are compounds in the environment that interfere with hormone systems in the body. I investigated if gestational exposure to a known class of EDCs, polychlorinated biphenyls (PCBs), resulted in life long alterations in neuroendocrine function. My overall hypothesis was that prenatal PCB exposure would cause molecular and cellular changes to the developing hypothalamus that would manifest across development through differences in hypothalamic gene expression, molecular epigenetic modifications, and corresponding effects on sexual development. To perform this work, I characterized changes in gene expression in two regions of the hypothalamus required for reproductive function throughout the life cycle and measured changes in somatic markers associated with reproductive physiology and development. This approach allowed me to relate specific neuroendocrine changes back to altered reproductive function. First, I present normative data showing gene and hormone changes throughout development in male and female rats to use as a basis of comparison for my further studies on EDCs. Second, I investigated how gestational exposure to PCBs on embryonic day 16 and 18 affected development of the hypothalamus through adulthood and caused corresponding changes in physiological functions. PCBs altered estrous cyclicity in females and delayed the timing of puberty in males. Developmental changes in gene expression were associated with sex, age and region of the hypothalamus. As a whole, the data suggested that gestational exposure to PCBs altered a network of hypothalamic genes and was associated with altered reproductive physiology. Finally, I extended my study farther along the life cycle to investigate if gestational exposure to PCBs altered the timing of reproductive aging in male and female rats. Few effects in males were observed. However, females exposed to PCBs had lower serum concentrations of LH on proestrus, and altered expression of numerous genes in the hypothalamus. These changes in gene expression were specific to the females’ cycle status and the results provided novel insight into the molecular mechanisms underlying reproductive aging. Taken together, my dissertation resulted in a comprehensive profile of both normal hypothalamic developmental changes, as well as providing insight into endocrine disruption of hypothalamic gene networks from birth through aging. / text

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