As the close paralog of adiponectin we

As the close paralog of adiponectin, we hypothesized that CTRP9 could participate in regulation of reproduction. In order to evaluate whether Ctrp9 plays a role in the control of reproduction in tilapia, i.p. injection experiment was performed. HPG (Hypothalamic/pituitary/gonadal) axis plays a key role in the regulation of reproduction across vertebrates. The secretion of GNRH from hypothalamus stimulates the release of LH and FSH from pituitary, and LH and FSH perform biological functions through the LHR and FSHR in gonad (Cheng et al., 2011, Levavi-Sivan et al., 2010). In present study, our results showed that gnrh2 and gnrh3 were markedly suppressed after injection of gCtrp9 in male and female tilapia. Previous studies demonstrate that adiponectin and globular adiponectin significantly reduced GNRH secretion from GT1-7 hypothalamic GnRH neuron α-Bungarotoxin sale through activation of AMPK and ERK 1/2 (extracellular signal-regulated kinase) pathway (Cheng et al., 2011, Martin, 2014, Wen et al., 2012, Wen et al., 2008). An inhibitory effect of adiponectin on Gnrh was also observed in rat in vivo studies (Cheng et al., 2011). After injection of Ctrp9, the expression of adipor1 was dramatically inhibited, while it was lack of effect of gCtrp9 on adipor2 expression in both sexes (Fig. S3B), suggesting that CTRP9 performed biological functions in an AdipoR1 dependent manner (Cheng et al., 2016, Kambara et al., 2015, Zheng et al., 2011). Knockdown or ablation of Adipor1 blocked the effects of CTRP9, but knockout of Adipor2 had no effect on CTRP9 function (Cheng et al., 2016, Kambara et al., 2015, Zheng et al., 2011). It may be hypothesized that the effects of Ctrp9 on reproduction of tilapia was related to adipor1. In male and female tilapia pituitary, the expression of gthα was inhibited at 12h post-injection, and the expression of fshβ and lhβ were also significantly suppressed after injection of gCtrp9. Similarly, recombinant adiponectin inhibited basal and GNRH-induced LH secretion in rat pituitary cells and mouse LβT2 gonadotrope cells (Lu et al., 2008, Rodriguez-Pacheco et al., 2007). In vivo, the plasma LH concentrations were decreased after i.c.v. (intraventricular) injection of adiponectin in rat (Cheng et al., 2011). Adiponectin caused a decrease in insulin- as well as GNRH- and insulin-stimulated LH secretion during the late luteal phase in porcine (Kiezun et al., 2014). And during estrous cycle, adiponectin reduced insulin- as well as GNRH- and insulin-stimulated FSH release (Kiezun et al., 2014). LH and FSH are complex heterodimer glycoproteins, and two of hormones are composed of a common α-subunit (GTHα) and a specific β-subunit (LHβ or FSHβ) (Li and Ford, 1998). In the gonads of both sexes, the expression levels of lhr and fshr were evidently decreased after injection of gCtrp9. Generally, LHR and FSHR are the important receptors for gonadotropins. The studies in mammals showed that adiponectin decreased LHR expression in granulosa and theca cells (Lagaly et al., 2008). ELISA (enzyme-linked immuno sorbent assay) results declared that the serum E2 levels were markedly decreased in female tilapia and serum T levels had an obvious decrease trend after 6 and 12h injection of gCtrp9. In cultured porcine granulosa cells, adiponectin decrease the CYP19 gene expression and increase the gene expression of STAR (Ledoux et al., 2006). Adiponectin suppressed the gene experssion of Cyp11a1 but not Star in mice cumulus cell-oocyte complexes (COCs) (Richards et al., 2012). It suggests that adiponectin might affect steroidogenesis through regulation of steroidogenic genes expression. It has been shown that recombinant adiponectin has a significant inhibitory effect on basal and hCG (Human Chorionic Gonadotropin)-stimulated testosterone secretion in rat testis (Chabrolle et al., 2009, Martin, 2014). Furthermore, adiponectin dramatically inhibited LH- and insulin-induced progesterone and androstenedione production in large-follicle theca cells (Lagaly et al., 2008). The serum adiponectin level was negatively correlated with serum androgen in human (Bottner et al., 2004). As the close paralog of adiponectin, our study showed that Ctrp9 could decrease the serum E2 and T levels in tilapia.