Targeted Mutagenesis of the Hypophysiotropic Gnrh3 in Zebrafish (Danio rerio) Reveals No Effects on Reproductive Performance

Abstract

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.

Stathmin-like 4 is critical for the maintenance of neural progenitor cells in dorsal midbrain of zebrafish larvae

“…Other stathmin family isoforms might be functional complementary to Stmn4. The loss of Stmn4 might also be compensated by other molecules sharing similar properties or acting downstream of mutated factors21 that is a common features in lower vertebrates31. We addressed these issues by demonstrating the upregulation of stmn1b in stmn4Δ5 −/− embryos.…”

Cell-type heterogeneity in the early zebrafish olfactory epithelium is generated from progenitors within preplacodal ectoderm

“…Like GnRH1 neurons in amniotes, zebrafish Gnrh3 neurons leave the proximity of the olfactory epithelium and can be found along a migratory tract from the terminal nerve to the ventral telencephalon at late larval stages. While laser-ablating Gnrh3+ cells leads to sterility, animals homozygous for TALEN-induced mutations of gnrh3 are fertile, pointing to the need for identifying other genes expressed in these cells that might underlie the differences between these phenotypes ( Abraham et al, 2010 ; Spicer et al, 2016 ).…”

Expression patterns of kiss2 and gpr54‐2 in Monopterus albus suggest these genes may play a role in sex reversal in fish

“…Although these findings indicate that Kisspeptin/GPR54 system might indeed play a role in the sex reversal of M. albus, interpretation of these data and comparisons between species are rendered exceptionally complex by the existence of multiple paralogues in other fish species, by their species-specific functions, diverse reproductive strategies, and differences in experimental approaches between studies [10,16]. Additionally, as targeted mutations of kiss and gpr54 do not disrupt gonadal development and reproductive performance in zebrafish, this is a strong indication of the existence of a compensatory mechanism that ensures reproductive success [19,40,41]. This suggests that, as opposed to mammals, kisspeptin may not be a central and absolute upstream regulator of the HPG axis in fish [18].…”