The ventromedial hypothalamic nucleus (VMH) is an important central effector site involved in activating brown adipose tissue (BAT) or non-shivering thermogenesis. VMH neurons have previously been shown to be thermally responsive to changes in local temperature of the preoptic area/anterior hypothalamus (PO/AH). However, the thermoresponsiveness of VMH neurons of room temperature acclimatized and cold acclimatized rats to peripheral thermal stimulation has not been tested. In this thesis, a series of studies was designed to determine the thermoresponsiveness of VMH neurons to peripheral (scrotal) thermal stimulation of rats. Extracelluiar VMH neuronal activity was recorded from urethane anaesthetized male Sprague-Dawley rats, which were acclimatized either to room temperature (21°C for 4 weeks) or to cold (4°C for 4 weeks) prior to testing, during scrotal cooling and heating with glass-micropipettes filled with 0.5 M sodium acetate containing 2% pontamine sky blue. The rats' colonic temperatures were kept at normothermia (37°C), or hypothermia (33-35°C) during scrotal thermal stimulation. In the room temperature acclimatized rats, VMH neurons were temperature responsive to scrotal heating and cooling and were classified as warm responsive (WRN), cold responsive (CRN) and temperature non-responsive neurons (TNRN), based on their thermal coefficients. The ratio of VMH WRNs and CRNs was similar to that of thermoresponsive neurons observed in other brain regions (eg, the PO/AH and thalamus). VMH WRNs and CRNs were further classified as biphasic or monophasic in nature according to their thermal responses to scrotal heating and cooling. VMH neurons sustained their thermoresponsiveness to repeated trials of scrotal thermal stimulation with colonic temperatures maintained at 37°C or when colonic temperatures were acutely lowered from 37°C to 35°C and 33°C. In addition, scrotal thermal signals specifically altered neuronal activity of VMH thermoresponsive neurons, as changes in EEG activity did not occur with changes in VMH neuronal activity. Scrotal thermal inputs were functionally shown to be transmitted via the medial preoptic nucleus (MPO) prior to reaching the VMH nucleus because thermoresponsive VMH neuronal activity was blocked with the pretreatment of lidocaine into the MPO. Scrotal heating or cooling to 21°C-acclimatized rats did not increase IBAT temperatures, inferring that scrotal cooling had not evoked IBAT thermogenesis in this group. In cold acclimatized (CA) rats, prolonged (over 2 hours) and transient localized scrotal cooling caused IBAT temperatures to increase, inferring that scrotal cooling activated BAT thermogenesis. Mean basal firing rates of all recorded VMH neurons of CA-groups significantly increased, compared to those of VMH neurons observed in room temperature acclimatized (RA) groups. More VMH CRNs than WRNs were recorded in the CA-group and the thermoresponsiveness (ie, thermal coefficient) of VMH CRNs significantly increased during localized scrotal cooling in the cold acclimatized group, compared to thermal coefficients of VMH CRNs of the RA-groups. (Abstract shortened by UMI.)

Endocrine-disrupting chemicals (EDCs) are environmental pollutants known to perturb hormone systems and interfere with normal endocrine function. Exposure to EDCs during hormone-sensitive developmental periods can result in profound dysfunction in reproductive physiology and behavior. In this dissertation, effects of gestational exposure to a class of EDCs called polychlorinated biphenyls (PCBs) were examined in the developing hypothalamus, which is known to control reproductive physiology and behavior in vertebrates. The specific hypothesis was that PCBs caused changes in sexually dimorphic hypothalamic nuclei, resulting in perturbation of adult sociosexual behaviors and alteration of neural networks with changes in expression of microRNAs and genes during development and in adulthood. This research focused on two brain areas relevant to understanding the PCB effects on the developing hypothalamus: 1) microRNA and related target gene expression during postnatal development, 2) adult sociosexual behaviors and gene expression. In both sections, molecular changes were examined in two sexually dimorphic hypothalamic nuclei, medial preoptic nucleus (MPN) and ventromedial nucleus (VMN), known for their role in regulation of sociosexual behavior. In the first section of the dissertation, the effects of PCBs were examined on the expression of microRNAs and target genes at four ages during postnatal [P] development (P15, P30, P45, and P90). Age and sex specific effects were observed in both MPN and VMN, with greater effects in the MPN. The second research section of the dissertation explored whether sociosexual behaviors, namely ultrasonic vocalizations and sociosexual preference behaviors, were altered by gestational PCBs. Expression of forty-eight neuroendocrine candidate genes was also examined in the MPN and VMN of the same animals. Several sociosexual behaviors were affected, including number and acoustic properties of ultrasonic vocalizations, and nose-touching with opposite-sex animals. Gene expression was altered in sex and region-specific manner in the brains of behaviorally affected rats. Taken together, these findings suggest that gestational PCBs have lasting effects on molecular mechanisms during postnatal development and in adulthood, and could result in altered sociosexual behavior. These results have implications for human health and disease, as early life exposures to EDCs have been linked to reproductive decline in humans. / text

Endocrine disrupting chemicals (EDCs) are a class of environmental toxicants, of both natural and synthetic origin, that interfere with normal endocrine function. Exposure to EDCs during susceptible periods of development, particularly embryogenesis, can result in profound neurological and reproductive deficits. While the impact of developmental exposure to EDCs on reproductive function and behavior has been much studied, the underlying mechanisms responsible for these observed effects are not well understood. The goal of the research detailed in this dissertation is to elucidate the cellular and molecular targets by which a representative class of EDCs, polychlorinated biphenyls (PCBs), disrupts normal reproductive neuroendocrine function. My specific hypothesis is that PCBs cause changes in sexually dimorphic brain regions underlying sex-specific reproductive physiology and behavior through the perturbation of normal developmental apoptosis, with long-term consequences for reproductive success. The studies detailed herein focus on three areas which contribute to an understanding of the effects of PCBs on neuroendocrine reproductive function: (1) the in vitro effects of PCBs on a neuroendocrine cell line, (2) developmental effects of PCBs on the gestationally exposed F1 generation, and (3) the physiological consequences of these developmental alterations for adult reproductive function. In the first section of this dissertation, the neurotoxic and endocrine disrupting effects of PCBs on a representative developing neuroendocrine cell model, the GT1-7 GnRH cell line, are investigated in time- and dose-response experiments. Treatment and dose-dependent effects are observed for GnRH peptide concentrations, cell viability, apoptotic and necrotic cell death, and caspase activation. In general, GnRH peptide levels are suppressed by high doses and longer durations of PCBs, and elevated at low doses and shorter time points. The suppression of GnRH peptide levels was partially reversed in cultures co-treated with the estrogen receptor antagonist ICI 182,780. All PCBs tested reduced viability and increased both apoptotic and necrotic cell death. The second section of this dissertation examines whether prenatal PCB exposure alters normal neuroendocrine development in the F1 generation, including sexual differentiation of the brain. Disruption of hypothalamic development is detectable as early as the day after birth (postnatal day (P) 1), as indicated by abnormal programmed cell death, and alterations in neuroendocrine gene and protein expression. The third section discusses the physiological impact of developmental PCB exposure on reproductive maturation and adult neuroendocrine function. Pubertal onset is advanced and estrous cyclicity irregular in PCB endocrine-disrupted females. Furthermore, sexual differentiation of female neuroendocrine systems is masculinized/defeminized. Collectively, these results suggest that the disrupted sexual differentiation of the POA can be detected as early as the day after birth, effects that may underlie the adult reproductive phenotype. / text

published_or_final_version / Physiology / Master / Master of Philosophy

published_or_final_version / Physiology / Master / Master of Philosophy

No description available.

国立情報学研究所で電子化したコンテンツを使用している。

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.