GHRH/PACAP-GH-IGF Axis in the Ovary of Zebrafish, Danio rerio

Growth and reproduction are two major physiological processes, which have been proven to be closely related in vertebrates. The process of growth is governed by the brain-pituitary-liver axis involving growth hormone releasing hormone/pituitary adenylate cyclase-activating polypeptide (GHRH/PACAP), growth hormone (GH) and insulin-like growth factors (IGFs). Interestingly, the expression of all the genes involved in this axis has been reported in the ovary, which indicates that a functional mini-axis might exist in the ovary. In this study, we focus on the characterization of PACAP, the upstream element of the axis, to reveal the existence and functional roles of this intraovarian mini-axis.;PACAP is a neuropeptide originally purified from ovine hypothalamus for its potent activity to stimulate cAMP production. However, its presence and actions have also been demonstrated in various peripheral tissues including the ovary. In the zebrafish, two forms of PACAP (PACAP38-1, adcyap1a; and PACAP38-2, adcyap1b) and three PACAP receptors (PAC1-R, adcyap1r1; VPAC1-R, vipr1 and VPAC2-R, vipr2) were all expressed in the ovary. To provide clues to the importance of the PACAP system in the function of zebrafish ovary, we first investigated the spatial distribution of both PACAP ligands and the three potential receptors in the somatic follicle layer and denuded oocytes. We also analyzed the temporal expression profiles of PACAP ligands and receptors during follicle growth and maturation. Spatiotemporal expression data of PACAP system suggested that PACAP might play dual roles in regulating follicle growth and maturation through different receptors located in different compartments. This hypothesis was further supported by the observation that PACAP promoted maturation of follicle-enclosed oocytes but suppressed spontaneous maturation of denuded oocytes in vitro..;As the expression of PACAP (PACAP38-2) was significantly stimulated by pituitary gonadotropins (hCG) in cultured zebrafish follicle cells, PACAP is therefore likely a downstream mediator or modulator of pituitary gonadotropins in controlling ovarian functions. We illustrated from several aspects to verify our hypothesis. Firstly, we tested the regulation of PACAP on the expression of activin/follistatin system for its well characterized roles in mediating pituitary gonadotropins (hCG). According to our results, PACAP could not only mimic gonadotropin-regulated expression of the activin/follistatin system, but also stimulated activin-mediated oocyte maturation. The same cAMP-dependent signal pathways PACAP and hCG chose towards the differential regulation of activin/follistatin system further confirm PACAP’s role as a mediator or even an amplifier. Secondly, EGF, the ovary-derived growth factor, was administrated to study its effects on PACAP regulated expression of activin/follistatin system. Similar with its influences on hCG regulated genes expression of activin system, EGF could work on its membrane receptors using a MEK pathway to regulate the effects of PACAP. Thirdly, the effect of PACAP on steroidogenesis was also studied. PACAP could stimulate the expression of aromatase, one of the steroidogenic enzymes that could convert testosterone to E2, in cultured zebrafish follicle cells. Its effect on aromatase expression could be mimicked by drugs that increase intracellular cAMP levels such as forskolin and db-cAMP. PACAP induced aromatase expression was totally abolished by a PKA inhibitor H89, which indicated that PACAP worked through a cAMP-PKA dependent pathway to regulate aromatase expression. As gonadotropins also use the same cAMP-PKA pathway to regulate the expression of aromatase, it was further confirmed that PACAP could mediate or amplify the effects of gonadotropins, even in steroidogenesis.;We also studied the regulatory effects of PACAP on other components of this mini-axis to find out whether this hypothetical GHRH/PACAP-GH-IGF axis in the ovary work the same way as the systemic somatotrophic one. We carried out a series of regulatory studies using the primary zebrafish follicle cell culture system. Interestingly, PACAP up-regulated the expression of gh and its receptors ghra and ghrb. In addition, it also increased the expression of igf1 but not igf2a and igf2b. Accompanied with the fact that recombinant zfGH could stimulate the expression of igf1 , we have reason to believe that a functional intraovarian axis exited in zebrafish ovary. It seems that the regulatory effects of PACAP on this axis also mediated through a cAMP-PKA pathway.;The present study not only increases our understanding of the GHRH/PACAP-GH-IGFs axis and its actions in the ovary, but also provides valuable information on the coordination of growth and reproduction in fish, which will surely benefit the manipulation of fish growth and breeding in aquaculture. Since fish represent the most primitive and diverse group of vertebrates, the information obtained from the present study will serve as important reference for the studies in other organisms.