Kisspeptin is a potent stimulator of GnRH secretion that has been implicated in the feedback actions of ovarian steroids. In ewes, the majority of hypothalamic kisspeptin neurons are found in the arcuate nucleus (ARC), with a smaller population located in the preoptic area. Most arcuate kisspeptin neurons express estrogen receptor-alpha, as do a set of arcuate neurons that contain both dynorphin and neurokinin B (NKB), suggesting that all three neuropeptides are colocalized in the same cells. In this study we tested this hypothesis using dual immunocytochemistry and also determined if kisspeptin neurons contain MSH or agouti-related peptide. To assess colocalization of kisspeptin and dynorphin, we used paraformaldehyde-fixed tissue from estrogen-treated ovariectomized ewes in the breeding season (n = 5). Almost all ARC, but no preoptic area, kisspeptin neurons contained dynorphin. Similarly, almost all ARC dynorphin neurons contained kisspeptin. In experiment 2 we examined colocalization of kisspeptin and NKB in picric-acid fixed tissue collected from ovary intact ewes (n = 9). Over three quarters of ARC kisspeptin neurons also expressed NKB, and a similar percentage of NKB neurons contained kisspeptin. In contrast, no kisspeptin neurons stained for MSH or agouti-related peptide. These data demonstrate that, in the ewe, a high percentage of ARC kisspeptin neurons also produce dynorphin and NKB, and we propose that a single subpopulation of ARC neurons contains all three neuropeptides. Because virtually all of these neurons express estrogen and progesterone re-ceptors, they are likely to relay the feedback effects of these steroids to GnRH neurons to regulate reproductive function.

Reproductive activity in sheep is seasonal, being activated by short-day photoperiods and inhibited by long days. During the nonbreeding season, GnRH secretion is reduced by both steroid-independent and steroid-dependent (increased response to estradiol negative feedback) effects of photoperiod. Kisspeptin (also known as metastin) and gonadotropin-inhibitory hormone (GnIH, or RFRP) are two RFamide neuropeptides that appear critical in the regulation of the reproductive neuroendocrine axis. We hypothesized that expression of kisspeptin and/or RFRP underlies the seasonal change in GnRH secretion. We examined kisspeptin and RFRP (protein and mRNA) expression in the brains of ovariectomized (OVX) ewes treated with estradiol (OVX+E) during the nonbreeding and breeding seasons. In OVX+E ewes, greater expression of kisspeptin and Kiss1 mRNA in the arcuate nucleus and lesser expression of RFRP (protein) in the dorsomedial nucleus of the hypothalamus were concurrent with the breeding season. There was also a greater number of kisspeptin terminal contacts onto GnRH neurons and less RFRP-GnRH contacts during the breeding season (compared with the nonbreeding season) in OVX+E ewes. Comparison of OVX and OVX+E ewes in the breeding and nonbreeding season revealed a greater effect of steroid replacement on inhibition of kisspeptin protein and Kiss1 mRNA expression during the nonbreeding season. Overall, we propose that the two RFamide peptides, kisspeptin and RFRP, act in concert, with opposing effects, to regulate the activity of GnRH neurons across the seasons, leading to the annual change in fertility and the cyclical seasonal transition from nonbreeding to breeding season.

We identified a gene in the ovine hypothalamus encoding for RFamide-related peptide-3 (RFRP-3), and tested the hypothesis that this system produces a hypophysiotropic hormone that inhibits the function of pituitary gonadotropes. The RFRP-3 gene encodes for a peptide that appears identical to human RFRP-3 homolog. Using an antiserum raised against RFRP-3, cells were localized to the dorsomedial hypothalamic nucleus/paraventricular nucleus of the ovine brain and shown to project to the neurosecretory zone of the ovine median eminence, predicating a role for this peptide in the regulation of anterior pituitary gland function. Ovine RFRP-3 peptide was tested for biological activity in vitro and in vivo, and was shown to reduce LH and FSH secretion in a specific manner. RFRP-3 potently inhibited GnRH-stimulated mobilization of intracellular calcium in gonadotropes. These data indicate that RFRP-3 is a specific and potent mammalian gonadotropin-inhibiting hormone, and that it acts upon pituitary gonadotropes to reduce GnRH-stimulated gonadotropin secretion.

We determined whether kisspeptin could be used to manipulate the gonadotropin axis and ovulation in sheep. First, a series of experiments was performed to determine the gonadotropic responses to different modes and doses of kisspeptin administration during the anestrous season using estradiol-treated ovariectomized ewes. We found that: 1) injections (iv) of doses as low as 6 nmol human C-terminal Kiss1 decapeptide elevate plasma LH and FSH levels, 2) murine C-terminal Kiss1 decapeptide was equipotent to human C-terminal Kiss1 decapeptide in terms of the release of LH or FSH, and 3) constant iv infusion of kisspeptin induced a sustained release of LH and FSH over a number of hours. During the breeding season and in progesterone-synchronized cyclical ewes, constant iv infusion of murine C-terminal Kiss1 decapeptide-10 (0.48 mumol/h over 8 h) was administered 30 h after withdrawal of a progesterone priming period, and surge responses in LH occurred within 2 h. Thus, the treatment synchronized preovulatory LH surges, whereas the surges in vehicle-infused controls were later and more widely dispersed. During the anestrous season, we conducted experiments to determine whether kisspeptin treatment could cause ovulation. Infusion (iv) of 12.4 nmol/h kisspeptin for either 30 or 48 h caused ovulation in more than 80% of kisspeptin-treated animals, whereas less than 20% of control animals ovulated. Our results indicate that systemic delivery of kisspeptin provides new strategies for the manipulation of the gonadotropin secretion and can cause ovulation in noncyclical females.

Many species express endogenous cycles in physiology and behavior that allow anticipation of the seasons. The anatomical and cellular bases of these circannual rhythms have not been defined. Here, we provide strong evidence using an in vivo Soay sheep model that the circannual regulation of prolactin secretion, and its associated biology, derive from a pituitary-based timing mechanism. Circannual rhythm generation is seen as the product of the interaction between melatonin-regulated timer cells and adjacent prolactin-secreting cells, which together function as an intrapituitary "pacemaker-slave" timer system. These new insights open the way for a molecular analysis of long-term timing mechanisms.