Title: Molecular mechanisms regulating reproduction in the presence and absence of the hypophysiotropic GnRH in zebrafish females

Speaker: Sakura Tanaka (PhD candidate, UMBC-IMET)

Host: Dr. Yonathan Zohar

Abstract: Gonadotropin-releasing hormone (GnRH) is a hypothalamic neuropeptide essential for inducing luteinizing hormone (LH) secretion required during the later stages of gonadal development and ovulation in all vertebrates. Most vertebrates possess two or three GnRH isoforms (GnRH1–3). GnRH1 is the pivotal regulator of the pituitary gonadotropes (LH producing cells) and the pre-ovulatory LH surge. The zebrafish is an established model organism that lost GnRH1 during evolution and contains only two GnRHs (GnRH2 & 3). GnRH3 in zebrafish is distributed throughout the same brain nuclei as GnRH1, and its neurons reach the pituitary. Therefore, it is considered to function as GnRH1 in regulating LH secretion. However, unlike loss of GnRH1 models in mammals and medaka, recent studies have shown that GnRH3 knockout zebrafish (gnrh3 ^–/– ) display normal reproduction and fertility. This finding may be explained by functional compensation, in which other factors kick in when GnRH3 is absent, or redundancy, in which GnRH3 is not the only factor regulating LH secretion. This dissertation presents evidence supporting the functional redundancy theory, indicating that GnRH3 is dispensable in modulating the LH surge in female zebrafish. To determine the role of GnRH3 and its neurons in controlling ovulation in zebrafish, conditional chemogenetic GnRH3 neuronal ablation was performed in adult fish, which resulted in an 85.3% average reduction in GnRH3 neurons innervating the pituitary. However, again, no change was observed in the reproductive capacity of these fish. In the frame of testing the redundancy theory, the role of another brain neuropeptide, vasoactive intestinal peptide (Vip), in inducing LH secretion in zebrafish females was demonstrated. A combination of in vitro pituitary incubation trials and in vivo studies on wild-type (WT) and gnrh3 ^–/– females demonstrated that Vip induces LH secretion independently and indirectly of GnRH3, supporting the redundancy theory. Finally, to understand the role of GnRH3 and identify additional potential factors pertinent to LH secretion, expression profiles of individual pituitary cells of WT and gnrh3 ^–/– adult female zebrafish were analyzed by single-cell RNA sequencing. In LH gonadotropes, there were no significant differences in LH secretion pathway-related gene expressions between the genotypes. However, some non-reproductive hormone gene expression was significantly increased in gnrh3 ^–/– . Furthermore, a potential novel cell type was detected in WT, which was barely detectable in the gnrh3 ^–/– , and a subpopulation of somatotropes in the gnrh3 ^–/– pituitary. These changes indicate that GnRH3 suppresses the expression of the other hormones, which contributes to the specialization of LH gonadotropes. Taken altogether, the primary roles of GnRH3 and its neurons may not be inducing the pre-ovulatory LH secretion in zebrafish, likely stimulated by redundant factors. Unlike GnRH1 in other vertebrates, GnRH3 plays a key role in neuromodulation rather than as a regulator of the LH surge. For the first time, the current comprehensive study provides novel evidence that supports the concept of a GnRH-independent system in vertebrate reproduction.

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