4.1. Effect of rhGH on spermatogenesis and steroidogenesis in immature rats
Correlations between GH and sexual maturation have been reported in both sexes (Zaczek et al., 2002; Sanchez-Cardenas et al., 2010). In immature male rats, puberty was significantly advanced, together with an increase in total body weight and reproductive organ weight, following rhGH administration (Ronis et al., 1998). In this study, the testis weights of the 2 IU/kg rhGH rats were significantly lower than those of the control rats on PND 24, although that difference was mitigated on PND 31. Similarly, in immature male dogs, a high dose of rhGH (25 IU/kg/day) induced atrophy of the testes and accessory organs (Sjögren et al., 1998). GH can induce apoptosis and atretic changes in the ovaries and testes of mammals, including humans, by activating the PI3K-Akt pathway (Santos et al., 1999; Sirotkin, 2005). Though no explanation for the reduced testis weights in rhGH rats during the early prepubertal period has been confirmed, high doses of rhGH in the early prepubertal period might transiently reduce testis development in a way that is impermanent and recoverable. In histology, the diameter and luminal areas of the seminiferous tubules were visibly increased in the 2 IU/kg rhGH rats on PND 31. In parallel, testicular mRNA levels of spermatogenic marker genes in the rhGH rats were significantly higher than those in the control rats on PND 31, indicating that rhGH activated spermatogenesis in the immature rats. Similarly, in immature rats, GH treatment for four weeks induced testicular growth and germ cell differentiation (Ipsa et al., 2019). In the 2 IU/kg rhGH rats, blood testosterone levels were significantly higher than in the control rats on PNDs 24 and 31. Similarly, rhGH injection stimulated testosterone synthesis in both immature and adult rodents (Colón et al., 2005; Sriraman et al., 2005). Given that circulating LH levels did not differ significantly between the control and rhGH rats, the activation of androgen production might indicate that rhGH has a direct effect on Leydig cells. In Leydig cell cultures, rhGH directly activated steroidogenesis (Wang & Hardy, 2004; Ipsa et al., 2019). In rat testes, HSD3B(+) progenitor Leydig cells are typically observed on PND 21 before they increase in number and become HSD17B(+) immature Leydig cells that go on to differentiate into functional adult Leydig cells (Hu et al., 2010). In light of the observed mRNA levels of steroidogenic pathway genes, increases in the number and mean size of HSD17B(+) Leydig cells in rhGH rat testes on PNDs 24 and 31, and the increased number and mean size of HSD3B(+) Leydig cells in rhGH rat testes on PND 24, prepubertal rhGH administration might have accelerated the functional differentiation of progenitor Leydig cells (PLCs) into immature, testosterone-producing Leydig cells. In the testes, GH and IGF1 modulate the proliferation and steroidogenesis of Leydig cells via the MAPK-ERK, JAK-STAT, and PI3K-AKT pathways (Rotwein, 2012; Ipsa et al., 2019). In the HSD3B(+) progenitor Leydig cells isolated on PND 21, 10 µg/L of rhGH treatment increased the expression of steroidogenic pathway gene mRNA (Igf1, Sf1, Cyp11a1, Cyp17a1, Cyp19a1, Hsd3b1, and Hsd17b3) and testosterone secretion. This suggests that rhGH-treatment directly activates steroidogenesis in progenitor Leydig cells, which is consistent with the results of previous studies (Dufau, 1988; Maran et al., 2000). The increase in circulating testosterone found in rhGH rats on PNDs 24 and 31 might be attributable to greater steroidogenic differentiation of PLCs to testosterone-producing Leydig cells, activating the spermatogenesis.
4.2. rhGH triggered changes in kisspeptin, GnRH, and LH in immature male rats
During the prepubertal period, kisspeptin activates the HPG axis and increases sex steroids (Soliman et al., 2014; Devesa & Caicedo, 2019). In male rats, hypothalamic kisspeptin and GnRH concentrations were elevated after PND 7 (Semaan et al., 2013; Luo et al., 2016). In this study, hypothalamic Kiss1 and Gnrh1 mRNA levels in the rhGH rats were significantly higher than those in the control rats on PND 24, when kisspeptin is elevated in male rats. Thus, prepubertal rhGH administration might potentiate hypothalamic kisspeptin and GnRH production. In adult rodents, hypothalamic Kiss1 and Gnrh1 mRNA are downregulated by sex steroids (Smith, 2008; Ng et al., 2009). In this study, circulating testosterone levels in the rhGH rats were significantly higher than those in the control rats on PNDs 24 and 31. Although the circulating LH levels of the rhGH rats did not differ from those of the control rats on PND 24 or 31, the pituitary Lhb mRNA levels in the rhGH rats were significantly lower than those in the control rats on PNDs 24 and 31, suggesting that elevated testosterone levels provide negative feedback for the expression of pituitary Lhb.
4.3. rhGH triggered IGF1 changes in the hypothalamus, liver, and testes of immature rats
IGF1 mediates GH-dependent and GH-independent anabolism and growth (Cheng & Chen, 1995; Kolodziejczyk et al., 2003). In rodents and primates, hypothalamic IGF1 expression increases at puberty, which activates kisspeptin-GnRH neurons (Hiney et al., 1996; Wolfe et al., 2014; Huh et al., 2021). In human and mouse brains, hypothalamic GnRH neurons express the IGF1 receptor (D’Ercole et al., 1996; Divall et al., 2010). In prepubertal female rats, an intracerebroventricular infusion of IGF1 stimulated the secretion of GnRH and might advance the onset of puberty (Hiney et al., 1991 and 1996). In prepubertal male rats, a central infusion of IGF1 antiserum delayed pubertal development (Pazos et al., 1999). Altogether, brain IGF1 is an important factor in the initiation of puberty. In this study, hypothalamic Igf1 mRNA was increased in the rhGH rats on PND 24, suggesting that the activation of kisspeptin-GnRH expression can be attributed to the activation of hypothalamic IGF1. In addition, in the hypothalamus, sex steroids activate the GH-IGF1 axis, accelerating the growth and maturation of reproductive organs for puberty (Veldhuis, 1997). Together, increased hypothalamic Igf1 mRNA in the rhGH rats on PND 24 might be attributable to the elevated testosterone levels, as well as the direct action of rhGH on hypothalamic Igf1 expression. In contrast, on PND 31, hypothalamic Igf1 mRNA levels in the rhGH rats were significantly lower than those in the control rats, which could be a result of negative feedback from elevated androgens at puberty in the rhGH rats. Stimulation of IGF1 production in the liver has been considered to be a major effect of GH (LeRoith & Roberts Jr, 2003; Bielohuby et al., 2009). Systemic IGF1 plays a major modulatory role in testicular endocrine function. In the rhGH rats in this study, however, liver Igf1 mRNA and blood IGF1 levels tended to increase, though not to a statistically significant degree, by PNDs 24 and 31. Similarly, in immature mice given rhGH, circulating IGF1 and hepatic cell Igf1 mRNA levels did not increase (Mireuta et al., 2014). Of note, in the rhGH rats in this study, testicular Igf1 mRNA levels were significantly higher than those in the control rats on both PND 24 and 31. Similarly, in immature hypophysectomized rats, rhGH increased testicular IGF1 levels (Grizard, 1994; Laron & Klinger, 1998; Diez-Caballero et al., 2006). Given that IGF1 and cognate receptor are expressed in germ cells, Leydig cells and Sertoli cells (Grizard, 1994; Wang & Hardy, 2004; Diez-Caballero et al., 2006; Griffeth et al., 2014), intratesticular IGF1 could regulate various aspects of testicular function in both autocrine and paracrine manners. In rodents, IGF1 can promote the proliferation, maturation, and steroidogenesis of Leydig cells by means of para- and autocrine action (Khan et al., 1992; LeRoith & Roberts Jr, 2003; Diez-Caballero et al., 2006). In mouse Leydig cells, the expression of the LH receptor and response to LH are potentiated by IGF1 (Lin et al., 1986; Kasson & Hsueh, 1987). In the rhGH rat testes in this study, the number and size of HSD3B(+) and HSD17B(+) Leydig cells increased significantly. In addition, in the HSD3B(+) progenitor Leydig cells isolated on PND 21, rhGH treatment increased the expression of Igf1 mRNA. Therefore, testicular increases in IGF1 could mediate functional differentiation of testosterone-producing Leydig cells in rhGH rats, and the increased circulating testosterone in rhGH rats may be due to the increased LH response in Leydig cells by the testicular IGF1. In the rhGH rats in this study, the elevated testicular IGF1 levels might be responsible for the activation of testosterone production and spermatogenesis (Fig. 9). In gonadotropin-independent precocious puberty (GIPP), sexual maturation is induced by sex steroids that increase through a gonadotropin-independent mechanism such as testotoxicosis, tumors, or environmental hormones (Kremer et al., 1993; Traggiai and Stanhope, 2003). The elevation of testicular IGF1 through prepubertal administration of rhGH could thus evoke the early onset of sexual maturation without increasing circulating LH levels, resembling GIPP.