Densitometric analysis of GnRH and IBA1 immunocytochemistry in the basal ventromedial hypothalamus of the ewe

Gonadotropin releasing hormone (GnRH) synthesis and secretion regulates seasonal fertility. In the brain, the distribution of GnRH-positive neurons is diffuse, hindering efforts to monitor variations in its cellular and tissue levels. Here, we aim at assessing GnRH immunoreactivity in nuclei responsible for seasonal fertility regulation (SFR) within the posterior, anterior, and preoptic areas of the basal hypothalamus during estrous in ewes. We detected reaction products in the ventromedial basal hypothalamus in neurons, nerve fibers, non-neuronal immunoreactive bodies, and diffuse interstitial areas. Immunoreactivity correlated with the distribution of the main SFR nuclei in the arcuate, retrochiasmatic, periventricular, medial preoptic, supraoptic, and preoptic areas. By independent component analysis density segmentation and by interferential contrast, we identified GnRH non-neuronal positive bodies as microglial cells encapsulated within a dense halo of reaction products. These GnRH-positive microglial cells were distributed in patches and rows throughout the basal ventromedial hypothalamus, suggesting their role in paracrine or juxtacrine signaling. Moreover, as shown by ionized calcium-binding adaptor molecule 1 (IBA1) immunocytochemistry, the distribution of GnRH reaction products overlapped with the microglial dense reactive zones. Therefore, our findings support the assertion that a combined densitometric analysis of GnRH and IBA1 immunocytochemistry enables activity mapping for monitoring seasonal changes following experimental interventions.