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

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.

ORIENTATIONAL DEVELOPMENT AND DIFFERENTIATION OF MOUSE PRIMORDIAL GERM CELLS INTO HEPATOCYTES IN VITRO / 解剖学报

Objective To investigate the better factor for inducing the primordial germ cells to differentiate into hepatocytes in vitro. Methods The primordial germ cells(PGCs) from the gonadal ridges of the mouse embryos at 11 days postcoitum(dpc) from Kunming pregnancy mice were cultured and proliferated in vitro.An aliquot PGCs suspension was cultured in 6-well dishes for 24?h.Then bFGF, EGF,?-NGF,RA and hepatocyte abstract were added into the dishes respectively,Anather aliquot PGCs were co-cultured with mouse embryonic hepatocytes for directional differentiation.The morphological changes and indiocyanine green(ICG) intake of the PGCs were observed;albumin(ALB) and ?-1-antitrypsin(AAT) were assayed by immunocytochemistry. Results After the addition of ?-NGF,hepatocyte abstract and CM-EH the ICG was intaken by the differentiated cells which like star and ovum,the ALB and AAT immune positive expression were detected in those differentiated cells.The ratio of positive cells was above 60% in 2 weeks.But bFGF,EGF and RA had no same as the effects.However RA could improve the ratio of differentiated cells induced by ?-NGF and hepatocyte abstract.Conclusion ?-NGF and some factors from hepatocytes and embryonic hepatocytes could effectively induce PGCs to differentiate into hepatocytes.

THE EFFECT OF TRANSPLANTATION OF MOUSE PRIMORDIAL GERM CELLS ON THE ACUTE DAMAGED LIVER / 解剖学报

Objective To investigate the milieu-dependent differentiation of primordial germ cells(PEGs) in the acute damaged liver microenviroment. Methods After PGCs were cultured and proliferated,these cells were labelled with 5-bromo-2-deoxyuridine(BrdU),then transplanted into the acute damaged liver by CCl_4 through tail vein.Two and four weeks later,the liver was extracted and 10?m-cryostat continuous sections were obtained.The existing and differentiation of the transplanted cells were identified by immunohistochemistry,immunofluorescence double staining and histochemistry for BrdU and hepatic-specific ALB,and the glycogen. Results Transplanted PGCs were found to be incorporated into the acute damaged liver and differentiated into hepatocytes,compensating for acute liver failure.Conclusion PGCs can be induced to differentiate into hepatocytes in the acute damaged liver microenvironment,and can be used for cellular hepatoplasty to treat severe liver disease.