In male mice that lack the brain chemical PACAP, the cells that make the hormone GnRH (which tells the testes to produce testosterone) are fewer, while other hormone‑related cells like kisspeptin increase. This shift also changes the balance of estrogen and androgen receptors in key brain areas, which could explain why these mice have fertility problems.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal peptide (VIP) neuropeptide family and plays a role in the regulation of several releasing hormones and tropic hormones. The hypothalamic-pituitary-gonadal (HPG) axis governs the synthesis and the release of sex hormones and the gametogenesis in all mammals. While the effects of PACAP on fertility is well-documented in females, much less data are available in males. The aim of our study was to examine potential structural and expressional changes in the hypothalamus that might underlie the fertility deficits observed in male PACAP knockout (KO) mice. To this end, we performed immunofluorescent, immunohistochemical and RNAscope <i>in situ</i> hybridization stainings to detect the protein and/or mRNA expression of gonadotropin-releasing hormone (GnRH), kisspeptin, estrogen receptor alpha (ER&#x3b1;) and androgen receptor (AR) in the hypothalamus. Our results revealed that the number and immunoreactivity of GnRH neurons were lower in the medial preoptic area (MPOA) in PACAP KO mice. In contrast, the number of kisspeptin neurons was higher in the rostral periventricular region of the third ventricle (RP3V) and the mid arcuate nucleus (ARC). Furthermore, higher number of <i>Esr1-</i>positive cells was found in the kisspeptin-rich RP3V and the ARC. Notably, less AR-positive cells, and more ER&#x3b1;-positive cells were detected in the MPOA demonstrating a possible misbalance between estrogenic and androgenic signaling. Our results suggest that neuroendocrine changes induced by PACAP deficiency in the hypothalamus might contribute to the development of reproductive dysfunction in PACAP-deficient males by disrupting normal HPG axis function.