A Transgenic Boar Model to Elucidate the Role of Gonadotropin-Releasing Hormone 2 (GnRH2) and Its Receptor in Regulating Testes and Sperm Function

Boar subfertility represents a major limitation to swine production, reducing conception rate and litter size. Critical to reproductive function, the classical form of GnRH (GnRH1) promotes secretion of the gonadotropins; however, the second mammalian isoform (GnRH2) is a poor stimulator of gonadotropin release. A receptor specific to GnRH2 (GnRHR2) has been identified in mammals, although gene coding errors prevent the production of a full-length protein in most species. In contrast, the GnRHR2 is functional in swine, allowing us to establish in the boar that: 1) GnRH2 and its receptor are more abundant within the testis compared to the hypothalamus and anterior pituitary gland; 2) GnRHR2s are present on Leydig cells; 3) exogenous GnRH2 stimulates testosterone secretion from testicular explants; and 4) GnRH2 stimulates testosterone release in vivo as effectively as GnRH1, despite minimal secretion of the conventional androgen stimulator, luteinizing hormone (LH). In addition, we have found GnRH2 on developing germ cells, immunolocalized GnRHR2 to the connecting piece of mature sperm and detected GnRH2 in seminal plasma. To further examine the function of GnRH2 and its receptor within the porcine testis, we produced a swine line with reduced endogenous GnRHR2 levels [GnRHR2 knockdown (KD)]. During pubertal development, hemizygous GnRHR2 KD boars tended (P < 0.06) to have lower serum testosterone concentrations than littermate controls (1.6 vs. 4.2 ng/mL), although LH levels were similar (P > 0.90). Predicted testis volumes of GnRHR2 KD males were smaller than controls (331.8 vs. 374.8 cm³; P < 0.05), despite similar body weights (P > 0.05). In mature boars, diurnal testosterone secretion was reduced by 80% in GnRHR2 KD compared to control animals (0.8 vs. 4.1 ng/mL; P < 0.05). Further, GnRHR2 mRNA levels were diminished by 70% in transgenic vs. control testes (P < 0.001). These data suggest that GnRH2 acts directly on Leydig cells to stimulate steroidogenesis, independent of LH secretion. In addition, computer-assisted sperm analysis indicated that ejaculates from GnRHR2 KD boars tended to have less motile sperm (P = 0.10) and reduced sperm concentration (P < 0.10) than littermate control males, potentially due to lower testosterone levels and/or fewer GnRHR2s on germ cells. A better understanding of how GnRH2 and its receptor regulate testicular function will lead to new technologies to improve fertility in boars, enhancing the sustainability/profitability of pork producers. Partially supported by NIFA Hatch (NEB-26-199; BRW) and AFRI (2011-67015; CAL) funds. USDA is an equal opportunity provider and employer.