Emerging insights into hypothalamic-pituitary-gonadal axis regulation and interaction with stress signalling.

Reproduction and fertility are regulated via hormones of the hypothalamic-pituitary-gonadal (HPG) axis. Control of this reproductive axis occurs at all levels, including the brain and pituitary, and allows for the promotion or inhibition of gonadal sex steroid secretion and function. In addition to guiding proper gonadal development and function, gonadal sex steroids also act in negative- and positive-feedback loops to regulate reproductive circuitry in the brain, including kisspeptin neurones, thereby modulating overall HPG axis status. Additional regulation is also provided by sex steroids made within the brain, including neuroprogestins. Furthermore, because reproduction and survival need to be coordinated and balanced, the HPG axis is able to modulate (and be modulated by) stress hormone signalling, including cortiscosterone, from the hypothalamic-pituitary-adrenal (HPA) axis. This review covers recent data related to the neural, hormonal and stress regulation of the HPG axis and emerging interactions between the HPG and HPA axes, focusing on actions at the level of the brain and pituitary.

Kisspeptin neurones do not directly signal to RFRP-3 neurones but RFRP-3 may directly modulate a subset of hypothalamic kisspeptin cells in mice.

The neuropeptides kisspeptin (encoded by Kiss1) and RFamide-related peptide-3 (also known as GnIH; encoded by Rfrp) are potent stimulators and inhibitors, respectively, of reproduction. Whether kisspeptin or RFRP-3 might act directly on each other's neuronal populations to indirectly modulate reproductive status is unknown. To examine possible interconnectivity of the kisspeptin and RFRP-3 systems, we performed double-label in situ hybridisation (ISH) for the RFRP-3 receptors, Gpr147 and Gpr74, in hypothalamic Kiss1 neurones of adult male and female mice, as well as double-label ISH for the kisspeptin receptor, Kiss1r, in Rfrp-expressing neurones of the hypothalamic dorsal-medial nucleus (DMN). Only a very small proportion (5-10%) of Kiss1 neurones of the anteroventral periventricular region expressed Gpr147 or Gpr74 in either sex, whereas higher co-expression (approximately 25%) existed in Kiss1 neurones in the arcuate nucleus. Thus, RFRP-3 could signal to a small, primarily arcuate, subset of Kiss1 neurones, a conclusion supported by the finding of approximately 35% of arcuate kisspeptin cells receiving RFRP-3-immunoreactive fibre contacts. By contrast to the former situation, no Rfrp neurones co-expressed Kiss1r in either sex, and Tacr3, the receptor for neurokinin B (NKB; a neuropeptide co-expressed with arcuate kisspeptin neurones) was found in <10% of Rfrp neurones. Moreover, kisspeptin-immunoreactive fibres did not readily appose RFRP-3 cells in either sex, further excluding the likelihood that kisspeptin neurones directly communicate to RFRP-3 neurones. Lastly, despite abundant NKB in the DMN region where RFRP-3 soma reside, NKB was not co-expressed in the majority of Rfrp neurones. Our results suggest that RFRP-3 may modulate a small proportion of kisspeptin-producing neurones in mice, particularly in the arcuate nucleus, whereas kisspeptin neurones are unlikely to have any direct reciprocal actions on RFRP-3 neurones.

The role of kisspeptin and RFamide-related peptide-3 neurones in the circadian-timed preovulatory luteinising hormone surge.

Many aspects of female reproduction often require intricate timing, ranging from the temporal regulation of reproductive hormone secretion to the precise timing of sexual behaviour. In particular, in rodents and other species, ovulation is triggered by a surge in pituitary luteinising hormone (LH) secretion that is governed by a complex interaction between circadian signals arising in the hypothalamus and ovarian-derived oestradiol signals acting on multiple brain circuitries. These circadian and hormonal pathways converge to stimulate a precisely-timed surge in gonadotropin-releasing hormone (GnRH) release (i.e. positive-feedback), thereby triggering the preovulatory LH surge. Reflecting its control by afferent circadian signals, the preovulatory LH surge occurs at a specific time of day, typically late afternoon in nocturnal rodents. Although the specific mechanisms mediating the hormonal and circadian regulation of GnRH/LH release have remained poorly understood, recent findings now suggest that oestradiol and circadian signals govern specific reproductive neuropeptide circuits in the hypothalamus, including the newly-identified kisspeptin and RFamide-related peptide (RFRP)-3 neuronal populations. Neurones producing kisspeptin, the protein product of the Kiss1 gene, and RFRP-3 have been shown to provide excitatory and inhibitory input to GnRH neurones, respectively, and are also influenced by sex steroid and circadian signals. In the present review, we integrate classic and recent findings to form a new working model for the neuroendocrine regulation of the circadian-timed preovulatory LH surge in rodents. This model proposes kisspeptin and RFRP-3 neuronal populations as key nodal points for integrating and transducing circadian and hormonal signals to the reproductive axis, thereby governing the precisely-timed LH surge.

Gonadal and nongonadal regulation of sex differences in hypothalamic Kiss1 neurones.

The brains of males and females differ anatomically and physiologically, including sex differences in neurone size or number, synapse morphology and specific patterns of gene expression. Brain sex differences may underlie critical sex differences in physiology or behaviour, including several aspects of reproduction, such as the timing of sexual maturation (earlier in females than males) and the ability to generate a preovulatory gonadotrophin surge (in females only). The reproductive axis is controlled by afferent pathways that converge upon forebrain gonadotrophin-releasing hormone (GnRH) neurones, but GnRH neurones are not sexually dimorphic. Although most reproductive sex differences probably reflect sex differences in the upstream circuits and factors that regulate GnRH secretion, the key sexually-dimorphic factors that influence reproductive status have remained poorly defined. The recently-identified neuropeptide kisspeptin, encoded by the Kiss1 gene, is an important regulator of GnRH secretion, and Kiss1 neurones in rodents are sexually dimorphic in specific hypothalamic populations, including the anteroventral periventricular nucleus-periventricular nucleus continuum (AVPV/PeN) and the arcuate nucleus (ARC). In the adult AVPV/PeN, Kiss1 neurones are more abundant in females than males, representing a sex difference that is regulated by oestradiol signalling during critical periods of postnatal and pubertal development. By contrast, Kiss1 neurones in the ARC are not sexually differentiated in adult rodents but, in mice, the regulation of ARC Kiss1 cells by gonadal hormone-independent factors is sexually dimorphic during prepubertal development. These various sex differences in hypothalamic Kiss1 neurones may relate to known sex differences in reproductive physiology, such as puberty onset and positive feedback.

Evidence that the type-2 gonadotrophin-releasing hormone (GnRH) receptor mediates the behavioural effects of GnRH-II on feeding and reproduction in musk shrews.

Gonadotrophin-releasing hormone (GnRH) is a regulatory neuropeptide of which there are multiple structural variants. In mammals, a hypothalamic form (GnRH-I) controls gonadotrophin secretion whereas a midbrain form (GnRH-II) appears to have a neuromodulatory role affecting feeding and reproduction. In female musk shrews and mice, central administration of GnRH-II reinstates mating behaviour previously inhibited by food restriction. In addition, GnRH-II treatment also decreases short-term food intake in musk shrews. GnRH-II can bind two different mammalian GnRH receptors (type-1 and type-2), and thus it is unclear which receptor subtype mediates the behavioural effects of this peptide. Adult female musk shrews implanted with i.c.v. cannula were food restricted or fed ad lib and then tested for sexual behaviour or food intake. One hour before testing, animals were pretreated with vehicle or Antide, a potent type-1 GnRH receptor antagonist (at a dose that blocks GnRH-I or -II mediated ovulation). Twenty minutes before testing, females were infused a second time with either GnRH-II or vehicle. Additional females were tested after an infusion of 135-18, a type-1 receptor antagonist that displays agonist actions at the primate type-2 receptor. GnRH-II treatment increased sexual behaviour in underfed female shrews; pretreatment with Antide did not block this action, suggesting that the effects of GnRH-II are not mediated via the type-1 receptor. Similarly, the inhibitory effects of GnRH-II on short-term food intake were not prevented by pretreatment with Antide. The behavioural effects of the type-2 receptor agonist 135-18 were similar to those seen in GnRH-II-treated females, with 135-18 promoting sexual behaviour and decreasing food intake. Collectively, these results indicate that GnRH-II does not act via the type-1 GnRH receptor to regulate mammalian behaviour but likely activates the type-2 GnRH receptor.

Emerging functions of gonadotropin-releasing hormone II in mammalian physiology and behaviour.

Gonadotropin-releasing hormone (GnRH) is the central neuroendocrine regulator of the hypothalamic-pituitary-gonadal axis. Multiple structural variants of GnRH are present in vertebrates. The first isoform isolated in the mammalian brain (GnRH I) was shown to regulate the release of pituitary gonadotropins. Recently, a second form has been discovered in mammals (GnRH II), both in the brain and periphery. Although it is unlikely to be a primary regulator of gonadotropin release, the highly conserved GnRH II variant appears to have a wide array of physiological functions. In the periphery, GnRH I and II have similar roles in regulating cell proliferation and mediating hormonal secretion from the ovary and placenta in an autocrine/paracrine manner. In the brain, GnRH I and II apparently modulate mammalian reproductive behaviours in different but complementary ways: GnRH I stimulates luteinizing hormone/follicle-stimulating hormone secretion (and thus gonadal steroids) and promotes sexual behaviour in ad libitum fed animals. By contrast, GnRH II acts as a permissive regulator of female reproductive behaviour based on energy status, as well as a modifier of short-term food intake. GnRH II has also been implicated in the regulation of calcium and potassium channels in nervous systems of amphibians, functions which may also be present in mammals. Increasing evidence suggests that the effects of GnRH II in both the periphery and brain may be mediated by GnRH receptor subtypes distinct from the type-1 GnRH receptor. It is likely that this evolutionarily conserved peptide has been co-opted over evolutionary time to possess multiple regulatory functions in a broad range of biological aspects, including, but not limited to, reproduction. Here, the proposed actions of both neural and peripheral GnRH II in affecting physiology and behaviour are summarized, and an outline of critical directions for future research is proposed.

Termination of neuroendocrine refractoriness to melatonin in Siberian hamsters (Phodopus sungorus).

Siberian hamsters maintained from birth in a short day length (DL), unlike their long-day counterparts, fail to undergo reproductive development by 5 weeks of age. Instead, reproductive maturation of short-day males is delayed for approximately 20 weeks, at which point neuroendocrine refractoriness to the inhibitory effects of short DLs develops, resulting in growth of the gonads. To terminate refractoriness and re-establish responsiveness to short photoperiods, 10-15 weeks of long-day exposure is required. We assessed whether continuous exposure to long days is necessary to terminate refractoriness or whether the first few weeks of long days initiate a process that culminates several months later in the breaking of refractoriness. Male hamsters refractory to short DLs were transferred to a long-day photoperiod, pinealectomized (PINx) after 0, 3, 6 or 15 weeks, and subsequently infused for 6 weeks with a short-day melatonin signal. This melatonin treatment induces gonadal regression in photosensitive but not in photorefractory hamsters. Six percent of males PINx at week 0 and 88% of those PINx at week 15 underwent gonadal atrophy by the end of the melatonin infusion treatment initiated on week 15. Among hamsters PINx on week 6, 17% versus 76% underwent testicular involution in response to melatonin infusions initiated on week 6 and week 15, respectively. This finding indicates that a fraction of the long days that hamsters experience during spring and summer are sufficient to trigger the processes that restore responsiveness to short DLs. Additional groups of pineal-intact photorefractory animals were given 3, 6 or 15 weeks of long-day exposure and then returned to a short DL for several months; only those treated for 15 weeks terminated refractoriness. The breaking of refractoriness, once triggered by long-day melatonin signals, proceeds to completion only in the absence of short-day melatonin signals.

Torpor characteristics and energy requirements of furless Siberian hamsters.

After approximately 10 wk of exposure to decreasing day lengths, Siberian hamsters (Phodopus sungorus) begin to display spontaneous torpor bouts several times each week. Torpor is associated with reduced daily energy expenditure and lower food consumption and ameliorates the thermoregulatory challenges of winter. We tested the extent to which the energy savings conferred by daily torpor depend on the presence of an insulative pelage. Female hamsters were housed in a winter day length (8L:16D) at 5 degrees C; daily food intake and torpor characteristics were recorded for 5 wk in shaved (furless) or normal hamsters. Torpor-bout incidence decreased by 62% in furless hamsters, but the duration of individual bouts and the minimum body temperature attained during torpor were unaffected by loss of pelage. Body temperature declined more rapidly during entry into torpor and increased more slowly during arousal from torpor in furless than in control hamsters. Energy savings per torpor bout, assessed by the amount of food consumed on days that included a torpor bout, was substantially greater in normal than in furless hamsters (16.0% vs. 3.3%); this difference likely reflects the increased cost of thermoregulation during torpor, as well as the increased caloric expenditure incurred by furless hamsters during arousal from torpor. An insulative pelage may be a prerequisite for the energetic benefits derived from heterothermy in this species.

Energy intake and fur in summer- and winter-acclimated Siberian hamsters (Phodopus sungorus).

Few studies have directly addressed the impact of fur on seasonal changes in energy intake. The daily food intake of Siberian hamsters (Phodopus sungorus) was measured under simulated summer and winter conditions in intact animals and those with varying amounts of pelage removed. Energy intake increased up to 44% above baseline control values for approximately 2-3 wk after complete shaving. Increases in food intake varied with condition and were greater in hamsters housed in short than long day lengths and at low (5 degrees C) than moderate (23 degrees C) ambient temperatures. Removal of 8 cm(2) of dorsal fur, equivalent to 30% of the total dorsal fur surface, increased food intake, but removal of 4 cm(2) had no effect. An 8-cm(2) fur extirpation from the ventral surface did not increase food consumption. Food intake was not influenced differentially by fur removal from above brown adipose tissue hot spots. Fur plays a greater role in energy balance in winter- than summer-acclimated hamsters and conserves energy under a wide range of environmental conditions.