Puberty is a tightly regulated process that leads to reproductive capacity. Kiss1 neurons are crucial in this process by stimulating GnRH, yet how Kiss1 neurons are regulated remains unknown. Substance P (SP), an important neuropeptide in pain perception, induces gonadotropin release in adult mice in a kisspeptin-dependent manner. Here, we assessed whether SP, through binding to its receptor NK1R (neurokinin 1 receptor), participates in the timing of puberty onset and fertility in the mouse. We observed that 1) selective NK1R agonists induce gonadotropin release in prepubertal females; 2) the expression of Tac1 (encoding SP) and Tacr1 (NK1R) in the arcuate nucleus is maximal before puberty, suggesting increased SP tone; 3) repeated exposure to NK1R agonists prepubertally advances puberty onset; and 4) female Tac1(-/-) mice display delayed puberty; moreover, 5) SP deficiency leads to subfertility in females, showing fewer corpora lutea and antral follicles and leading to decreased litter size. Thus, our findings support a role for SP in the stimulation of gonadotropins before puberty, acting via Kiss1 neurons to stimulate GnRH release, and its involvement in the attainment of full reproductive capabilities in female mice.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) is a neuromodulator implicated in anxiety, metabolism and reproductive behavior. PACAP global knockout mice have decreased fertility and PACAP modulates LH release. However, its source and role at the hypothalamic level remain unknown. We demonstrate that PACAP-expressing neurons of the ventral premamillary nucleus of the hypothalamus (PMVPACAP) project to, and make direct contact with, kisspeptin neurons in the arcuate and AVPV/PeN nuclei and a subset of these neurons respond to PACAP exposure. Targeted deletion of PACAP from the PMV through stereotaxic virally mediated cre- injection or genetic cross to LepR-i-cre mice with Adcyap1fl/fl mice led to delayed puberty onset and impaired reproductive function in female, but not male, mice. We propose a new role for PACAP-expressing neurons in the PMV in the relay of nutritional state information to regulate GnRH release by modulating the activity of kisspeptin neurons, thereby regulating reproduction in female mice.

The tachykinins substance P (SP) and neurokinin A (Tac1) have emerged as novel regulators of kisspeptin/GnRH release. Recently, we documented that SP modulates reproductive function in the female mouse. Here, we extended this characterization to the male mouse. Tac1-/- male mice showed delayed puberty onset. They also presented significantly decreased expression levels of Pdyn (dynorphin) and Nos1 (nitric oxide synthase) in the mediobasal hypothalamus and elevated Gnrh1 levels. Unexpectedly, the response of Tac1-/- mice to central kisspeptin or senktide (neurokinin B receptor-agonist) administration was significantly decreased compared with controls, despite the preserved ability of GnRH neurons to stimulate luteinizing hormone release as demonstrated by central N-methyl-D-aspartate receptor administration, suggesting a deficit at the GnRH neuron level. Importantly, we demonstrated that kisspeptin receptor and SP receptor (NK1R) heterodimerize, indicating that changes in the SP tone could alter the responsiveness of GnRH neurons to kisspeptin. Finally, electrophysiological recordings from arcuate Kiss1 neurons showed that, although virtually all Kiss1 neurons responded to NKB and senktide, only half responded to an NK1R agonist and none to the neurokinin A receptor agonist at a 1-μM dose. In summary, we provide compelling evidence for a role of Tac1 in the control of reproductive function in the male mouse, suggesting a predominant central action that may involve a change in the balance of neural factors that control GnRH expression.