Gnrh is the major neuropeptide regulator of vertebrate reproduction, triggering a cascade of events in the pituitary-gonadal axis that result in reproductive competence. Previous research in mice and humans has demonstrated that Gnrh/GNRH null mutations result in hypogonadotropic hypogonadism and infertility. The goal of this study was to eliminate gnrh3 (the hypophysiotropic Gnrh form) function in zebrafish (Danio rerio) to determine how ontogeny and reproductive performance are affected, as well as factors downstream of Gnrh3 along the reproductive axis. Using the TALEN technology, we developed a gnrh3^-/- zebrafish line that harbors a 62 bp deletion in the gnrh3 gene. Our gnrh3^-/- zebrafish line represents the first targeted and heritable mutation of a Gnrh isoform in any organism. Using immunohistochemistry, we verified that gnrh3^-/- fish do not possess Gnrh3 peptide in any regions of the brain. However, other than changes in mRNA levels of pituitary gonadotropin genes (fshb, lhb, and cga) during early development, which are corrected by adulthood, there were no changes in ontogeny and reproduction in gnrh3^-/- fish. The gnrh3^-/- zebrafish are fertile, displaying normal gametogenesis and reproductive performance in males and females. Together with our previous results that Gnrh3 cell ablation causes infertility, these results indicate that a compensatory mechanism is being activated, which is probably primed early on upon Gnrh3 neuron differentiation and possibly confined to Gnrh3 neurons. Potential compensation factors and sensitive windows of time for compensation during development and puberty should be explored.

High frequency production of zebrafish germline chimeras was achieved by transplanting ovarian germ cells into sterile Danio hybrid recipients. Ovaria

Gonadotropin-releasing hormone (GNRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3^−/−). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus–pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a noncell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.

It is widely believed that FSH and LH, which are known to play key roles in controlling the production of functional oocytes in vertebrates, are synthesized and secreted exclusively by the anterior pituitary. Here we present evidence for the novel expression of FSHβ, LHβ, and the common glycoprotein-α (Cgα) in the gilthead seabream ovary. Using in situ hybridization and immunocytochemistry, FSHβ was detected in primary-growth and secondary-growth-I oocytes, LHβ was found in secondary-growth oocytes, and Cgα was observed in both primary and secondary-growth oocytes. Northern blot analyses demonstrated that Fshβ transcript is 0.6 kb in both pituitary and ovary, whereas the ovarian Lhβ transcript (1.1 kb), unexpectedly, is longer than the known pituitary Lhβ transcript (0.6 kb). Sequence analyses revealed that ovarian Lhβ is driven by a different promoter than pituitary Lhβ, which generates an additional 459 bases at the distal portion of the 5′-untranslated region of the ovarian Lhβ. Furthermore, using in vitro ovarian fragment incubation, we demonstrated that mammalian GnRH analog agonist enhanced the expression of ovarian Fshβ (up to 2.7-fold), Lhβ (up to 1.4-fold), Cgα (up to 1.8-fold), and the secretion of ovarian LH (up to 2.2-fold). In contrast, GnRH antagonist, analog E, suppressed the secretion of ovarian LH. Our findings suggest that a GnRH-gonadotropin axis is present in the gilthead seabream ovary and that FSH and LH, the well-characterized pituitary hormones, may have prominent novel roles in teleost intraovarian communication between oocytes and ovarian follicle cells.

We developed a novel bath-immersion technology to produce large numbers of infertile fish. As seafood consumption shifts from fishery harvests towards artificially propagated species, optimization of aquaculture practices will be necessary to maximize food production and minimize ecological impact. Farming infertile fish is the most effective genetic-containment strategy to support the development of environmentally-responsible aquaculture. We discovered that a molecular transporter, Vivo, can effectively carry the Morpholino oligomer (MO) across the chorion, enter the embryo and reach target cells. Vivo-conjugated MO against zebrafish deadend (dnd-MO-Vivo) effectively caused primordial germ cell mis-migration and differentiation into somatic cells, which resulted in generation of infertile fish. Optimal conditions were achieved when embryos, immediately after fertilization, were immersed with dnd-MO-Vivo at the initial concentration of either 60 or 40 μM followed by a lower serially diluted concentration. Under these conditions, 100% induced sterility was achieved even when the total immersion time was reduced from 24 to 5 hours. In 8 independent experiments, 736 adults developed from these conditions were all found to be infertile fish that possessed minimally-developed gonads that lacked any gametes. The results demonstrate that dnd-MO-Vivo bath immersion is an effective strategy to produce infertile fish without introducing transgenic modifications.