Studies on the role of gonadotropininhibitory hormone (GnIH) in the neuroendocrine regulation of reproduction in the sheep

The brain peptide GnRH provides the primary stimulus for the reproductive axis, through hypophysiotropic action on the pituitary gonadotropes. There is now strong evidence for the existence of an inhibitory factor named gonadotropin inhibitory hormone (GnIH).This thesis presents the results of a series of experiments which demonstrate that GnIH is produced in the hypothalamus of the ovine brain and acts on the pituitary gland, inhibiting gonadotropin synthesis and secretion. In addition, it is suggested that GnIH counteracts the positive feedback effect of estrogen in the pituitary gonadotropes. A demonstrated reduction in expression of GnIH in the late follicular phase of the estrous cycle may be permissive of the positive feedback effect of estrogen to cause the preovulatory LH surge. Initial studies in birds and rats suggested that GnIH‐3 acts in both the brain and pituitary inhibit gonadotropin secretion. It was hypothesized that GnIH is an hypophysiotropic hormone in the sheep which acts negatively for gonadotropin synthesis and secretion. In Chapter 2, studies are reported to show that GnIH‐3 producing cells are localized in the dorsomedial nucleus (DMN) and paraventricular nucleus (PVN) of the hypothalamus of the ovine brain, with GnIH terminals projecting to the neurosecretory zone of the median eminence (ME). GnIH‐3 inhibited GnRHstimulated LH and FSH secretion, but did not inhibit the basal gonadotropin secretion in the pituitary gonadotropes. The intravenous infusion of GnIH‐3 also reduced LH pulse amplitude in the OVX ewes. This negative effect of GnIH‐3 was specific to the gonadotropes, with no effect on growth hormone (GH), cortisol and prolactin (PRL) levels. In the pituitary gonadotropes, GnIH‐3 inhibited GnRH‐generated calcium signals, indicating at least one mechanism for reduced secretory response. The second study (Chapter 3) aimed to test the hypothesis that GnH‐3 is able to inhibit gonadotropin subunit synthesis in the gonadotropes. An in vitro model of repeated stimulation of ovine pituitary cells in primary culture was established. GnIH‐3 reduced both LHβ and FSHβ mRNA levels in the both sexes of the sheep. Consistent with the finding in the first study, there was no effect on the expression of genes for other pituitary hormones (adrenocorticotropin, growth hormone and prolactin). GnIH‐3 inhibited the GnRH‐induced phosphorylation of ERK‐1/2, suggesting this as a possible intermediary in the action of GnIH to inhibit the synthesis of gonadotropin subunit genes. This finding provided evidence that GnIH‐3 acts in the level of pituitary negatively regulates gonadotropin synthesis in the sheep. The studies conducted in the Chapter 4 aimed to examine the effect of GnIH‐3 on the positive feedback of estrogen in the pituitary gonadotropes. Studies carried out with ovine pituitary cells in culture showed that estradiol‐17β (E2) has a ‘priming’ effect on the response of gonadotropes to GnRH, as seen in vivo. The priming effect of E2 was blocked by GnIH treatment. In Chapter 5, expression of GnIH during luteal and follicular phases of the estrous cycle were measured by in situ hybridisation. GnIH mRNA expression was lower during the late follicular phase than during the luteal phase. Artificial elevation of GnIH‐3 levels in the mid‐follicular phase (by iv infusion) reduced mean LH levels as well as LH pulses. In a model of the E2 benzoate (EB)‐induced LH surge, iv infusion of GnIH‐3 had a powerful negative effect. Thus, GnIH blocked the surge in 4/6 treated animals with minor surges occurring in the other two animals. Taken together, the body of the work presented in this thesis provides strong evidence that GnIH‐3 plays a role in negatively regulating reproductive function in the sheep. The data show that GnIH‐3 is an hypophysiotropic hormone which acts negatively in the synthesis and secretion of gonadotropins. Reduction in expression of GnIH gene expression in the late follicular phase of the estrous cycle may be permissive of the positive feedback effect of EB to cause the preovulatory LH surge. Understanding the role of GnIH in the hypothalamo‐pituitary axis will lead to therapeutic uses for GnIH and analogues in the management of reproduction.