Role of the Melanocortin System in Gonadal Steroidogenesis of Zebrafish

Abstract

In teleost, as in other vertebrates, stress affects reproduction. A key component of the stress response is the pituitary secretion of the adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. In zebrafish, Mc2r was identified in male and female gonads, while ACTH has been shown to have a physiological role in modulating reproductive activity. In this study, the hypothesis that other melanocortins may also affect how the zebrafish gonadal function is explored, specifically steroid biosynthesis, given the presence of members of the melanocortin signaling system in zebrafish gonads. Using cell culture, expression analysis, and cellular localization of gene expression, our new observations demonstrated that melanocortin receptors, accessory proteins, antagonists, and agonists are expressed in both the ovary and testis of zebrafish (n = 4 each sex). Moreover, melanocortin peptides modulate both basal and gonadotropin-stimulated steroid release from zebrafish gonads (n = 15 for males and n = 50 for females). In situ hybridization in ovaries (n = 3) of zebrafish showed mc1r and mc4r in follicular cells and adjacent to cortical alveoli in the ooplasm of previtellogenic and vitellogenic oocytes. In zebrafish testes (n = 3), mc4r and mc1r were detected exclusively in germ cells, specifically in spermatogonia and spermatocytes. Our results suggest that melanocortins are, directly or indirectly, involved in the endocrine control of vitellogenesis in females, through modulation of estradiol synthesis via autocrine or paracrine actions in zebrafish ovaries. Adult zebrafish testes were sensitive to low doses of ACTH, eliciting testosterone production, which indicates a potential role of this peptide as a paracrine regulator of testicular function.

Keywords: ASIP; MSH; POMC; agouti-signaling protein; fish; ovarian follicles; reproduction; spermatogenesis.

Figure 1

Relative expression levels of melanocortin-related genes in zebrafish ovaries and testes. Expression of the 18s rRNA gene was used as the reference gene for normalization of cDNA levels. Asterisk indicates significant variation after a two-way ANOVA analysis followed by Sidak’s multiple comparison test (*** p < 0.001, **** p < 0.0001). Data are expressed as mean ± SEM.

Figure 2

Localization of mcr1 and mcr4 transcripts expression in the zebrafish ovary by in situ hybridization. Panels (A,B) show mc1r and mc4r expression in the follicular cells, respectively. Panels (C,D) display mc1r and mc4r expression in the previtellogenic and vitellogenic follicles of the zebrafish ovary. (E,F) are representative tissue sections of (C,D) stained with haematoxylin-eosin, respectively. Signal was absent in the interstitial region.

Figure 3

Localization of mcr1 and mcr4 transcripts expression in zebrafish testes by in situ hybridization. Panels (A,B) show mc1r and mc4r expression in germ cells, respectively. (C,D) are magnified views of (A,B) for detail. (E,F) are representative tissue sections of (A,B) stained with toluidine blue, respectively.

Figure 4

Effects of melanocortin peptides on basal and human chorionic gonadotropin (hCG)-induced estradiol (E2) secretion in previtellogenic and vitellogenic ovarian follicles of zebrafish. (A) Effects of ACTH, (B) MSH chemical analogues (MTII), (C) human ASIP, and (D) chemical antagonist (SHU9119). Experiments were performed in triplicate and repeated at least three times. Data is expressed as a percentage of the control (mean ± SEM). Different letters indicate significant variations after a one-way ANOVA analysis, followed by Tukey’s post-hoc test, p < 0.05.

Figure 5

Effect of melanocortin peptides on basal and human chorionic gonadotropin (hCG)-induced testosterone (T) in primary cell cultures of dispersed testicular cells of zebrafish. (A) Effects of ACTH, (B) MSH chemical analogues (MTII), (C) human ASIP and (D) chemical antagonist (SHU9119). Experiments were performed in triplicate and repeated at least three times. Data is expressed as a percentage of the control (mean ± SEM). Different letters indicate significant variations after a one-way ANOVA analysis, followed by Tukey’s post-hoc test, p < 0.05.