Vasoactive intestinal peptide excites GnRH neurons via KCa3.1, a potential player in the slow afterhyperpolarization current.

Vasoactive intestinal peptide (VIP) is an important component of the suprachiasmatic nucleus (SCN) which relays circadian information to neuronal populations, including GnRH neurons. Human and animal studies have shown an impact of disrupted daily rhythms (chronic shift work, temporal food restriction, clock gene disruption) on both male and female reproduction and fertility. To date, how VIP modulates GnRH neurons remains unknown. Calcium imaging and electrophysiology on primary GnRH neurons in explants and adult mouse brain slice, respectively, were used to address this question. We found VIP excites GnRH neurons via the VIP receptor, VPAC2. The downstream signaling pathway uses both Gs protein/adenylyl cyclase/protein kinase A (PKA) and phospholipase C/phosphatidylinositol 4,5-bisphosphate (PIP2) depletion. Furthermore, we identified a UCL2077-sensitive target, likely contributing to the slow afterhyperpolarization current (IAHP), as the PKA and PIP2 depletion target, and the KCa3.1 channel as a specific target. Thus, VIP/VPAC2 provides an example of Gs protein-coupled receptor-triggered excitation in GnRH neurons, modulating GnRH neurons likely via the slow IAHP. The possible identification of KCa3.1 in the GnRH neuron slow IAHP may provide a new therapeutical target for fertility treatments.

Collection and Analysis of Vaginal Smears to Assess Reproductive Stage in Mice.

An increasing number of scientific studies include female mice to assess possible sex differences. As such, for reproducibility by others, it is important to consider hormonal levels, i.e., report the reproductive status of the female mice used. The mouse estrous cycle can be divided in 4 stages, all characterized by a different proportion of 3 cell types found in vaginal secretions. Observation of the mouse vaginal opening and collection of vaginal smears for analysis of cytology can be done in order to determine puberty onset and estrus stage. This protocol describes the characteristics of each estrus stage and details a quick and low-invasive method for collection of vaginal secretions. Examples of estrous cycle stages are included to help the investigator visualize patterns of cyclicity, which can provide important information about the reproductive health of the mice. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Visual assessment of vaginal opening Basic Protocol 2: Collection of vaginal secretion (smears).

Environmental disruption of reproductive rhythms.

Reproduction is a key biological function requiring a precise synchronization with annual and daily cues to cope with environmental fluctuations. Therefore, humans and animals have developed well-conserved photoneuroendocrine pathways to integrate and process daily and seasonal light signals within the hypothalamic-pituitary-gonadal axis. However, in the past century, industrialization and the modern 24/7 human lifestyle have imposed detrimental changes in natural habitats and rhythms of life. Indeed, exposure to an excessive amount of artificial light at inappropriate timing because of shift work and nocturnal urban lighting, as well as the ubiquitous environmental contamination by endocrine-disrupting chemicals, threaten the integrity of the daily and seasonal timing of biological functions. Here, we review recent epidemiological, field and experimental studies to discuss how light and chemical pollution of the environment can disrupt reproductive rhythms by interfering with the photoneuroendocrine timing system.

Thyroid hormone receptors are required for the melatonin-dependent control of Rfrp gene expression in mice.

Mammals adapt to seasons using a neuroendocrine calendar defined by the photoperiodic change in the nighttime melatonin production. Under short photoperiod, melatonin inhibits the pars tuberalis production of TSHß, which, in turn, acts on tanycytes to regulate the deiodinase 2/3 balance resulting in a finely tuned seasonal control of the intra-hypothalamic thyroid hormone T3. Despite the pivotal role of this T3 signaling for synchronizing reproduction with the seasons, T3 cellular targets remain unknown. One candidate is a population of hypothalamic neurons expressing Rfrp, the gene encoding the RFRP-3 peptide, thought to be integral for modulating rodent's seasonal reproduction. Here we show that nighttime melatonin supplementation in the drinking water of melatonin-deficient C57BL/6J mice mimics photoperiodic variations in the expression of the genes Tshb, Dio2, Dio3, and Rfrp, as observed in melatonin-proficient mammals. Notably, we report that this melatonin regulation of Rfrp expression is no longer observed in mice carrying a global mutation of the T3 receptor, TRα, but is conserved in mice with a selective neuronal mutation of TRα. In line with this observation, we find that TRα is widely expressed in the tanycytes. Altogether, our data demonstrate that the melatonin-driven T3 signal regulates RFRP-3 neurons through non-neuronal, possibly tanycytic, TRα.

Functional Implications of RFRP-3 in the Central Control of Daily and Seasonal Rhythms in Reproduction.

Adaptation of reproductive activity to environmental changes is essential for breeding success and offspring survival. In mammals, the reproductive system displays regular cycles of activation and inactivation which are synchronized with seasonal and/or daily rhythms in environmental factors, notably light intensity and duration. Thus, most species adapt their breeding activity along the year to ensure that birth and weaning of the offspring occur at a time when resources are optimal. Additionally, female reproductive activity is highest at the beginning of the active phase during the period of full oocyte maturation, in order to improve breeding success. In reproductive physiology, it is therefore fundamental to delineate how geophysical signals are integrated in the hypothalamo-pituitary-gonadal axis, notably by the neurons expressing gonadotropin releasing hormone (GnRH). Several neurochemicals have been reported to regulate GnRH neuronal activity, but recently two hypothalamic neuropeptides belonging to the superfamily of (Arg)(Phe)-amide peptides, RFRP-3 and kisspeptin, have emerged as critical for the integration of environmental cues within the reproductive axis. The goal of this review is to survey the current understanding of the role played by RFRP-3 in the temporal regulation of reproduction, and consider how its effect might combine with that of kisspeptin to improve the synchronization of reproduction to environmental challenges.