Kisspeptin (a product of the Kiss1 gene) and its receptor (GPR54 or Kiss1r) have emerged as key players in the regulation of reproduction. Mutations in humans or genetically targeted deletions in mice of either Kiss1 or Kiss1r cause profound hypogonadotropic hypogonadism. Neurons that express Kiss1/kisspeptin are found in discrete nuclei in the hypothalamus, as well as other brain regions in many vertebrates, and their distribution, regulation, and function varies widely across species. Kisspeptin neurons directly innervate and stimulate GnRH neurons, which are the final common pathway through which the brain regulates reproduction. Kisspeptin neurons are sexually differentiated with respect to cell number and transcriptional activity in certain brain nuclei, and some kisspeptin neurons express other cotransmitters, including dynorphin and neurokinin B (whose physiological significance is unknown). Kisspeptin neurons express the estrogen receptor and the androgen receptor, and these cells are direct targets for the action of gonadal steroids in both male and female animals. Kisspeptin signaling in the brain has been implicated in mediating the negative feedback action of sex steroids on gonadotropin secretion, generating the preovulatory GnRH/LH surge, triggering and guiding the tempo of sexual maturation at puberty, controlling seasonal reproduction, and restraining reproductive activity during lactation. Kisspeptin signaling may also serve diverse functions outside of the classical realm of reproductive neuroendocrinology, including the regulation of metastasis in certain cancers, vascular dynamics, placental physiology, and perhaps even higher-order brain function.

Stress-like elevations in plasma glucocorticoids suppress gonadotropin secretion and can disrupt ovarian cyclicity. In sheep, cortisol acts at the pituitary to reduce responsiveness to GnRH but does not affect GnRH pulse frequency in the absence of ovarian hormones. However, in ewes during the follicular phase of the estrous cycle, cortisol reduces LH pulse frequency. To test the hypothesis that cortisol reduces GnRH pulse frequency in the presence of ovarian steroids, the effect of cortisol on GnRH secretion was monitored directly in pituitary portal blood of follicular phase sheep in the presence and absence of a cortisol treatment that elevated plasma cortisol to a level observed during stress. An acute (6 h) cortisol increase in the midfollicular phase did not lower GnRH pulse frequency. However, a more prolonged (27 h) increase in cortisol beginning just before the decrease in progesterone reduced GnRH pulse frequency by 45% and delayed the preovulatory LH surge by 10 h. To determine whether the gonadal steroid milieu of the follicular phase enables cortisol to reduce GnRH pulse frequency, GnRH was monitored in ovariectomized ewes treated with estradiol and progesterone to create an artificial follicular phase. A sustained increment in plasma cortisol reduced GnRH pulse frequency by 70% in this artificial follicular phase, in contrast to the lack of an effect in untreated ovariectomized ewes as seen previously. Thus, a sustained stress-like level of cortisol suppresses GnRH pulse frequency in follicular phase ewes, and this appears to be dependent upon the presence of ovarian steroids.

Kisspeptin (Kiss1) signaling to GnRH neurons is widely acknowledged to be a prerequisite for puberty and reproduction. Animals lacking functional genes for either kisspeptin or its receptor exhibit low gonadotropin secretion and infertility. Paradoxically, a recent study reported that genetic ablation of nearly all Kiss1-expressing neurons (Kiss1 neurons) does not impair reproduction, arguing that neither Kiss1 neurons nor their products are essential for sexual maturation. We posited that only minute quantities of kisspeptin are sufficient to support reproduction. If this were the case, animals having dramatically reduced Kiss1 expression might retain fertility, testifying to the redundancy of Kiss1 neurons and their products. To test this hypothesis and to determine whether males and females differ in the required amount of kisspeptin needed for reproduction, we used a mouse (Kiss1-CreGFP) that has a severe reduction in Kiss1 expression. Mice that are heterozygous and homozygous for this allele (Kiss1(Cre/+) and Kiss1(Cre/Cre)) have ∼50% and 95% reductions in Kiss1 transcript, respectively. We found that although male Kiss1(Cre/Cre) mice sire normal-sized litters, female Kiss1(Cre/Cre) mice exhibit significantly impaired fertility and ovulation. These observations suggest that males require only 5% of normal Kiss1 expression to be reproductively competent, whereas females require higher levels for reproductive success.

Our laboratory has developed a paradigm of psychosocial stress (sequential layering of isolation, blindfold, and predator cues) that robustly elevates cortisol secretion and decreases LH pulse amplitude in ovariectomized ewes. This decrease in LH pulse amplitude is due, at least in part, to a reduction in pituitary responsiveness to GnRH, caused by cortisol acting via the type II glucocorticoid receptor (GR). The first experiment of the current study aimed to determine whether this layered psychosocial stress also inhibits pulsatile GnRH release into pituitary portal blood. The stress paradigm significantly reduced GnRH pulse amplitude compared with nonstressed ovariectomized ewes. The second experiment tested if this stress-induced decrease in GnRH pulse amplitude is mediated by cortisol action on the type II GR. Ovariectomized ewes were allocated to three groups: nonstress control, stress, and stress plus the type II GR antagonist RU486. The layered psychosocial stress paradigm decreased GnRH and LH pulse amplitude compared with nonstress controls. Importantly, the stress also lowered GnRH pulse amplitude to a comparable extent in ewes in which cortisol action via the type II GR was antagonized. Therefore, we conclude that psychosocial stress reduces the amplitude of GnRH pulses independent of cortisol action on the type II GR. The present findings, combined with our recent observations, suggest that the mechanisms by which psychosocial stress inhibits reproductive neuroendocrine activity at the hypothalamic and pituitary levels are fundamentally different. (Endocrinology 150: 762-769, 2009) V arious types of stress potently stimulate the hypothalamic-pituitary-adrenal axis and simultaneously suppress reproductive neuroendocrine activity. For example, psychosocial stress increases circulating levels of glucocorticoids and inhibits pulsatile LH secretion (1-4). Recent studies in ovariectomized sheep have demonstrated that a stress-like elevation of plasma cortisol decreases pulsatile LH secretion in the absence of stress and that this occurs via suppression of pituitary responsiveness to GnRH (5-7). This effect of cortisol, which reflects a direct action on the pituitary and mediation by the type II glucocorticoid receptor (GR), occurs without concurrent inhibition of pulsatile GnRH secretion (6,8,9). Our laboratory has recently described a paradigm of psychosocial stress that consists of sequential layering of isolation, restraint, blindfold, and predator cues (barking dog sound and odor) (10). This model of psychosocial stress ("layered stress paradigm") causes a robust elevation in circulating cortisol along with a profound decrease in LH pulse amplitude and steroidinduced sexual behavior in ovariectomized ewes (10, 11). The decrease in LH pulse amplitude was observed not only in ovariectomized ewes in which LH pulses were driven by endogenous GnRH pulses, it was also evident in a pituitary-clamp model in which endogenous GnRH pulses were blocked and LH pulses were driven by fixed hourly boluses of exogenous...

This study assessed the importance of cortisol in mediating inhibition of pulsatile LH secretion in sheep exposed to a psychosocial stress. First, we developed an acute psychosocial stress model that involves sequential layering of novel stressors over 3-4 h. This layered-stress paradigm robustly activated the hypothalamic-pituitary-adrenal axis and unambiguously inhibited pulsatile LH secretion. We next used this paradigm to test the hypothesis that cortisol, acting via the type II glucocorticoid receptor (GR), mediates stress-induced suppression of pulsatile LH secretion. Our approach was to determine whether an antagonist of the type II GR (RU486) reverses inhibition of LH pulsatility in response to the layered stress. We used two animal models to assess different aspects of LH pulse regulation. With the first model (ovariectomized ewe), LH pulse characteristics could vary as a function of both altered GnRH pulses and pituitary responsiveness to GnRH. In this case, antagonism of the type II GR did not prevent stress-induced inhibition of pulsatile LH secretion. With the second model (pituitary-clamped ovariectomized ewe), pulsatile GnRH input to the pituitary was fixed to enable assessment of stress effects specifically at the pituitary level. In this case, the layered stress inhibited pituitary responsiveness to GnRH and antagonism of the type II GR reversed the effect. Collectively, these findings indicate acute psychosocial stress inhibits pulsatile LH secretion, at least in part, by reducing pituitary responsiveness to GnRH. Cortisol, acting via the type II GR, is an obligatory mediator of this effect. However, under conditions in which GnRH input to the pituitary is not clamped, antagonism of the type II GR does not prevent stress-induced inhibition of LH pulsatility, implicating an additional pathway of suppression that is independent of cortisol acting via this receptor.