A synthetic kisspeptin analog that triggers ovulation and advances puberty.

The neuropeptide kisspeptin and its receptor, KiSS1R, govern the reproductive timeline of mammals by triggering puberty onset and promoting ovulation by stimulating gonadotrophin-releasing hormone (GnRH) secretion. To overcome the drawback of kisspeptin short half-life we designed kisspeptin analogs combining original modifications, triazole peptidomimetic and albumin binding motif, to reduce proteolytic degradation and to slow down renal clearance, respectively. These analogs showed improved in vitro potency and dramatically enhanced pharmacodynamics. When injected intramuscularly into ewes (15 nmol/ewe) primed with a progestogen, the best analog (compound 6, C6) induced synchronized ovulations in both breeding and non-breeding seasons. Ovulations were fertile as demonstrated by the delivery of lambs at term. C6 was also fully active in both female and male mice but was completely inactive in KiSS1R KO mice. Electrophysiological recordings of GnRH neurons from brain slices of GnRH-GFP mice indicated that C6 exerted a direct excitatory action on GnRH neurons. Finally, in prepubertal female mice daily injections (0.3 nmol/mouse) for five days significantly advanced puberty. C6 ability to trigger ovulation and advance puberty demonstrates that kisspeptin analogs may find application in the management of livestock reproduction and opens new possibilities for the treatment of reproductive disorders in humans.

Evidence for RGS4 modulation of melatonin and thyrotrophin signalling pathways in the pars tuberalis.

In mammals, the pineal hormone melatonin is secreted nocturnally and acts in the pars tuberalis (PT) of the anterior pituitary to control seasonal neuroendocrine function. Melatonin signals through the type 1 Gi-protein coupled melatonin receptor (MT1), inhibiting adenylate cyclase (AC) activity and thereby reducing intracellular concentrations of the second messenger, cAMP. Because melatonin action ceases by the end of the night, this allows a daily rise in cAMP levels, which plays a key part in the photoperiodic response mechanism in the PT. In addition, melatonin receptor desensitisation and sensitisation of AC by melatonin itself appear to fine-tune this process. Opposing the actions of melatonin, thyroid-stimulating hormone (TSH), produced by PT cells, signals through its cognate Gs-protein coupled receptor (TSH-R), leading to increased cAMP production. This effect may contribute to increased TSH production by the PT during spring and summer, and is of considerable interest because TSH plays a pivotal role in seasonal neuroendocrine function. Because cAMP stands at the crossroads between melatonin and TSH signalling pathways, any protein modulating cAMP production has the potential to impact on photoperiodic readout. In the present study, we show that the regulator of G-protein signalling RGS4 is a melatonin-responsive gene, whose expression in the PT increases some 2.5-fold after melatonin treatment. Correspondingly, RGS4 expression is acutely sensitive to changing day length. In sheep acclimated to short days (SP, 8 h light/day), RGS4 expression increases sharply following dark onset, peaking in the middle of the night before declining to basal levels by dawn. Extending the day length to 16 h (LP) by an acute 8-h delay in lights off causes a corresponding delay in the evening rise of RGS4 expression, and the return to basal levels is delayed some 4 h into the next morning. To test the hypothesis that RGS4 expression modulates interactions between melatonin- and TSH-dependent cAMP signalling pathways, we used transient transfections of MT1, TSH-R and RGS4 in COS7 cells along with a cAMP-response element luciferase reporter (CRE-luc). RGS4 attenuated MT1-mediated inhibition of TSH-stimulated CRE-luc activation. We propose that RGS4 contributes to photoperiodic sensitivity in the morning induction of cAMP-dependent gene expression in the PT.

Effect of photoperiod on the thyroid-stimulating hormone neuroendocrine system in the European hamster (Cricetus cricetus).

Recent studies have characterised a retrograde mechanism whereby the pineal hormone melatonin acts in the pars tuberalis (PT) of the pituitary gland to control thyroid hormone action in the hypothalamus, leading to changes in seasonal reproductive function. This involves the release of thyroid-stimulating hormone (TSH) from PT that activates type II deiodinase (DIO2) gene expression in hypothalamic ependymal cells, locally generating biologically active T3, and thus triggering a neuroendocrine cascade. In the present study, we investigated whether a similar regulatory mechanism operates in the European hamster. This species utilises both melatonin signalling and a circannual timer to time the seasonal reproductive cycle. We found that expression of betaTSH RNA in the PT was markedly increased under long compared to short photoperiod, whereas TSH receptor expression was localised in the ependymal cells lining the third ventricle, and in the PT, where its expression varied with time and photoperiod. In the ependymal cells at the base of the third ventricle, DIO2 and type III deiodinase (DIO3) expression was reciprocally regulated, with DIO2 activated under long and repressed under short photoperiod, and the reverse case for DIO3. These data are consistent with recent observations in sheep, and suggest that the PT TSH third ventricle-ependymal cell relay plays a conserved role in initiating the photoperiodic response in both long- and short-day breeding mammals.

Does a melatonin-dependent circadian oscillator in the pars tuberalis drive prolactin seasonal rhythmicity?

The pars tuberalis (PT) of the adenohypophysis expresses a high density of melatonin receptors and is thought to be a crucial relay for the actions of melatonin on seasonal rhythmicity of prolactin secretion by the pars distalis (PD). In common with the suprachiasmatic nucleus of the hypothalamus and most other peripheral tissues, the PT rhythmically expresses a range of 'clock genes'. Interestingly, this expression is highly dependent upon melatonin/photoperiod, with several aspects unique to the PT. These observations led to the establishment of a conceptual framework for the encoding of seasonal timing in this tissue. This review summarises current knowledge of the morphological, functional and molecular aspects of the PT and considers its role in seasonal timing. The strengths and weaknesses of current hypotheses that link melatonin action in the PT to its seasonal effect on lactotrophs of the PD are discussed and alternative working hypotheses are suggested.

MT1 melatonin receptor mRNA expressing cells in the pars tuberalis of the European hamster: effect of photoperiod.

Melatonin, secreted only during the night by the pineal gland, transduces the photoperiodic message to the organism. One important target for the hormone is the pars tuberalis (PT) of the adenohypophysis which displays a very high number of melatonin binding sites in mammals and is implicated in the seasonal regulation of prolactin secretion. To gain insight into the mechanism by which the melatonin signal is decoded in the PT, we studied the effect of photoperiod on the PT cells expressing the MT1 melatonin receptor in a highly photoperiodic species, the European hamster. Recently, we showed that, in the rat, the MT1 receptor mRNA is expressed in PT-specific cells characterized by their expression of beta-thyroid stimulating hormone (beta-TSH) along with the alpha-glycoprotein subunit (alpha-GSU). As the cellular composition of the PT shows variability among species, we first identified the cell type expressing the MT1 receptor in the European hamster by combining immunocytochemistry and nonradioactive in situ hybridization for the MT1 receptor mRNA. Our results show that, in the European hamster, as in the rat, the MT1 receptor is only expressed by the PT-specific-cells, beta-TSH and alpha-GSU positive. In a second step, we analysed the effects of photoperiod on the MT1 mRNA, and on beta-TSH and alpha-GSU both at the mRNA and protein levels. Our data show that, compared to long photoperiod, short photoperiod induces a dramatic decrease of MT1, beta-TSH and alpha-GSU expression. Protein levels of beta-TSH and alpha-GSU were also dramatically reduced in short photoperiod. Together, our data suggest that melatonin exerts its seasonal effects in the PT by signalling to PT specific-cells through the MT1 receptor subtype.