Gonadotropin-releasing hormone (Gnrh), as a critical factor in the hypothalamic-pituitary-gonad axis of vertebrates, is well-known in gonadotropin release, gonadal development, maturation, and reproduction. As a fish specific form of Gnrh, Gnrh3 is considered to be an important regulator in the reproduction of teleost. However, recent studies demonstrated that knockout of Gnrh3 had no obvious effects on the reproduction in adult zebrafish.

In a study published in Endocrinology, using gene editing technology, a research group led by Prof. HU Wei from Institute of Hydrobiology (IHB) of the Chinese Academy of Sciences generated a new line of gnrh3-null zebrafish by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated system (CRISPR/Cas9).

Adult gnrh3-null zebrafish showed a significant male-biased sex ratio. Primordial germ cell (PGC) numbers in gnrh3-null zebrafish embryos were less than those in wild-type embryos after the dome stage during early embryonic development.

Through immunofluorescence and confocal analysis, the researchers found that there was no proliferation signals of PGCs in adult gnrh3-null zebrafish embryos. The migration of PGCs was normal but no significant proliferation in mutant embryos observed with time-lapse imaging. The decreased PGC number in gnrh3-null zebrafish could be partially rescued by gnrh3 overexpression with gnrh3 mRNA microinjection and crossed with homozygous gnrh3 overexpression transgenic line. 

Besides, the researchers provided evidence in vivo that Gnrh3 regulates PGC proliferation through a mitogen-activated protein kinase (MARK) signaling pathway. At the key period of sex differentiation, the expression of male sex markers was up-regulated significantly, and a female sex marker was down-regulated significantly in gnrh3^-/^- zebrafish.  

These results indicated that Gnrh3 plays an important role in early sex differentiation by regulating the proliferation of PGCs through a MAPK-dependent path in the zebrafish model system.  

This study uncovered a new function of Gnrh3 in PGC proliferation and sex differentiation in any species for the first time. 

PGC distribution in different lines (Image by IHB)