Abstract EN:

To ensure the birth of the offspring at the most favourable time of year, photoperiod (or day-Iength) is "read" by most seasonal breeders to reslrict their fertility 10 a particular season. Annual variations of the photoperiod are translated into the release of the pineal hormone melatonin, the noctumal production of which relies on night-Iength. Previous experiments have demonstrated that the annual variations of melatonin secretion synchronise reproductive activity with seasons. For instance, exposure to a long nocturnal peak of melatonin for 8 to 10 weeks (like in short days conditions, SD) induces a marked gonadal atrophy in male Syrian hamsters (Mesocricetus auratus). Conversely, when transferred back ta long days (LD), a condition in which the nocturnal peak of melatonin is short, hamsters become sexually active within 6 weeks. Despite ils strong action on the reproductive axis, the precise sites and mechanisms of action of melatonin are still unclear. We recentiy reported that in the Syrian hamster, melatonin inhibits the expression of several hypothalamic genes, including Kissl, which might mediate the photoperiodic message to the reproductive axis. Kiss 1 encodes a family of peptides, the kisspeptins (Kp), that ail bind to the same receptor, Kiss1 r. Kp play a crucial role in the activation of the gonadotropic since a loss-of-function mutation in Kiss 1 r prevents puberty humans and rodents. My thesis work consisted in studying the implication of Kiss l-encoded peptides (kisspeptins, Kp) in the seasonal cycles of the reproductive activity. In a first part, the anatomy of the kisspeptinergic system was characterised in the Syrian hamster's brain in bath LD sexually active and SD sexually inactive individu ais. In a second part, investigated how photoperiod affects Kiss 1 expression and finally, in a third part, analysed the effects of exogenous administration of Kp and its site of action on the reproductive axis First of ail, the distribution of Kp fibres and cell bodies was characterised in the Syrian hamster's by immunohistochemistry and non-radioactive in situ hybridlsation. Two populations of Kiss 1 neurones were identified in the Arcuate nucleus (ARC) and in the Anteroventral periventricular nucleus (AVPV). Kp immunoreactive fibres were observed in both nucleus and in other hypothalamic (preoptic median nucleus, preoptic median area, anterior hypothalamic area, paraventricular nucleus, dorsomedial hypothalamus) and extra hypothalamic structures (Bed nucleus of stria terminalis, paraventricular nucleus of the thalamus, central amygdala). Since Kiss 1 expression is highly dependent on photoperiod, the distribution of Kp immunoreactive fibres was also analysed in SDadapted hamsters. The number of Kissl and Kp neurones is strongly decreased in SD sexually inactive hamsters. Fibres distribution is identical as in LD but fibres density is higher in SD, probably because of an accumulation of Kp into the fibres. To determine wether these fibres innervate fibres-containing structures or just go through them, we analysed the expression of cFos (a marker of cellular activity) alter an intracerebroventricular (ICV) injection of Kp and we found that cFos expression is increased in the preoptic median nucleus, the preoptic medial area, the paraventricular nucleus, the dorsomedial hypothalamus, the bed nucleus of stria terminalis and the paraventricular nucleus of the thalamus. This indicates that these brain structures areIrlnervalea Dy Kp libres and aclivated upon Kp release. The phenotype of the target neurones however remains to be determined. Ln a second part, 1 analysed how photoperiod affects Kiss 1 expression in the Syrian hamster. First of ail, we demonstrated that Kissl mRNA is highly expressed in the ARC and AVPV in LD-adapted male or female hamsters, while exposition to SD condition drastically reduces the number of Kiss 1 neurones in both structure and sex. Dally melatonin injections mimicking SD-like melatonln levels reduces Kissl expression in ARC and AVPV. By contrast pinealectomy in SD-adapted hamsters Increases the number of Kissl neurones in both nuclei, showing that melatonin mediates the effect of photoperiod on Kiss 1 neurones. The kinetic of melatonin effect on Kiss 1 neurones was also analysed and one or three weeks of daily melatonin injections do not affect Kissl expression whereas alter eight weeks of treatment, Kiss 1 expression reaches its minimal level. Noteworthy, SD exposure induces gonadal regression as weil as a massive reduction in testosterone levels. In rats and mice, testosterone strongly regulates Kissl expression in both ARC and AVPV and the SD- or melatonin-induced reduction in the number of Kissl neurones could be secondary to variations in testosterone plasmatic concentration. As a consequence, 1 also analysed the effect of gonadal hormones on Kissl expression in the Syrian hamster and 1 observed that testosterone up-regulates Kissl expression in the AVPV and down-regulates the number of ARC Kissl neurones. Thus, the SD- or melatonin-induced reduction in the number of ARC Kissl neurones is not secondary to a reduction in testosterone level. By contrast, in the AVPV where testosterone is stimulatory, the SD- or melatonin-induced down-regulation of Kiss 1 expression was found to be mediated by the SD- or melatonin-induced decrease in gonadal hormones levels. Ali together these data indicate that the SD-induced reduction in the number of ARC Kissl neurones is due to a direct effect of melatonin on the hypothalamus whereas the decrease in the number of AVPV Kiss 1 neurones is secondary to a reduction of testosterone levels. The AVPV is a sexually dimorphic brain nucleus with Kissl expression being higher in females. Thus, the effects of melatonin and oestradiol were analysed in females hamsters as weil and similar results were observed. Finally, in a third part, 1 analysed peripheral Kp sites of action. In the lab, we demonstrated that Kp chronic intracerebroventricular treatment for 4 weeks re-activates gonadal activity in SD-adapted male hamsters. This indicates that Kp overpass melatonin inhibitory effect on the reproductive axis. Previous studies showed that Kp trigger LH, FSH and sex steroids release when given centrally or peripherally. 1 thus studied the effect of several protocols of peripheral Kp long-term administration to SDacclimated sexually inactive Syrian hamsters. We demonstrated that 2 daily IP injections of Kp during 5 weeks restore gonadal activity. To identify the structure(s) involved in the peripheral effect of Kp, we studied the expression of cFos alter IP Injection of Kp. The anterior pituitary was found to strongly express cFos and this Kp-induced activation is GnRH-dependent since a GnRH antagonist prevented il. To conclude, this PhD work not only proved that the Kp system is strongly regulated by photoperiod and melatonin (via distinct mechanisms involving both direct effects and seasonal variations in gonadal hormones levels), but also that Kiss1 neurones impacts the photoperiodic message on the reproductive axis, probably at the level of GnRH nerve terminais of the median eminence.