Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction

Abstract

Background AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. Results We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/− mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. Conclusions By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

In Search of a Target Gene for a Desirable Phenotype in Aquaculture: Genome Editing of Cyprinidae and Salmonidae Species

“…Females and males with the mutant of the wnt4a gene were sterile due to genital tract malformations [ 319 ]. The genes responsible for ovarian differentiation, oocyte maturation, and folliculogenesis are myoc [ 320 ], egf , egfra , egfrb [ 321 ], nobox [ 322 ], parn [ 323 ], ambra1b [ 324 ], scg2a , scg2b [ 325 ], avp [ 326 ], and bmp15 [ 300 , 310 ]. Disruptions in the latter two genes affected, among other things, the mating behavior of fish.…”

Editorial: Molecular and cytogenetic research advances in human reproduction - volume II

“…Recently, pathogenic genetic mutations have been identified in individuals suffering from reproductive disorders; these mutations are generally detected in the genes mainly involved in germ cell development and other reproductive processes ( 18 – 21 ). Nevertheless, thousands of functionally relevant genes are expressed in human testes and ovaries, and malfunctioning of even a single gene can potentially cause infertility ( 22 – 24 ). Although the overall mechanistic contribution of specific genes has been studied in animal models, more attention is now being paid to clinically detected alterations in DNA, which include mutations potentially affecting gene regulatory sequences such as the 3’UTR ( 25 , 26 ).…”