Role Of Dnd And Aldh1a2in Germ Cell Development Of Nile Tilapia

After the primordial germ cells (PGCs) enter the genital ridge, they continue to proliferate. At this point, they commit to a developmental pathway that will lead them to become either eggs or sperm, depending on whether the genital ridge has begun to develop into an ovary or a testis, respectively. PGCs play a critical role in assignment of gonad determination in fish. Study on germ cells could elucidate the mechanism of sex determination and differentiation. As a germ cells marker, dnd (dead end) is essential for PGCs development and gametogenesis in vertebrates. PGCs undergo meiosis to produce sperm and oocyte. In addition, meiotic initiation time displays sexual dimorphism. Recent studies have implicated that retinoic acid (RA) signal pathway played a critical role in meiosis initiation in mammals, birds and amphibians. However, the role of RA in meiosis initiation of teleosts remains to be elucidated. In tilapia, meiotic germ cells first appear in ovaries of fry at around30dah, while meiosis initiation does not occur until75dah in testes. Moreover, our previous study showed that RA signal pathway is indispensable for meiosis initiation in the Nile tilapia. In the present study, CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated9) system was used to study the role of dnd and aldhla2in the germ cell development of Nile tilapia. The results are as follows:Multiple alignment of amino acid sequences showed that dnd is highly conserved during evolution. Tissue distribution analysis by RT-PCR demonstrated that dnd was only expressed in the gonads, and its expression in the ovary is higher than that in the testis.Disruption of dnd in tilapia through CRISPR/Cas9system was achieved with low efficiency. Histological observations of dnd deficiency XX gonad exhibited a small number of oogonium and a large cavity. The morphology of dnd deficiency XY testis was similar to the control testis. However, germ cell was not detected in the dnd deficiency XY testis. Immunohistochemistry analysis showed that the signal of Vasa was not detected in dnd mutant testis and weak dispersed were detected in dnd mutant ovary. The results indicated that germ cells were not lose complete. Cyp19a1a, which was confined to interstitial cells and theca cells surrounding oocytes in control ovaries, was found to be expressed in a small region adjacent to the blood vessel. Meanwhile, weak signal of Cypllb2(11-hydroxylase, the key enzyme responsible for the production of the androgen11-KT) and Dmrtl (the key factor involved in testicular differentiation and maintenance) were detected. These results indicated that somatic cells in germ cell-deficient XX were masculinization. Expressions of Cypllb2and Dmrtl in dnd deficiency XX gonad were similar to those of the control testis. In addition, neither Cypl9ala nor Vasa was detected in dnd deficiency XY testis. Therefore, germ cell-deficient in dnd deficiency XY testis did not influence the testicular differentiation.The polyclonal antibody of Aldh1a2of Nile tilapia are prepared in our laboratory, it was used to investigate the expression profiles of Aldhla2during development of gonads. Immunohistochemistry analysis showed that, A1dh1a2was expressed in somatic cells of both XX and XY gonads.In order to inhibit RA synthase, the inhibitor of RA synthase enzyme aldhla family-DEAB (4-diethylaminobenzaldehyde) and disruption of aldhla2in XX tilapia through CRISPR/Cas9system were used. Histological observation showed that the treated and knock-out groups both have undifferentiated gonads, while the control group began to enter meiosis at30dah; Meiosis was not initiated till60dah in DEAB treated and knock out groups, while phase â…¡ oocytes were detected in control group. At75dah, few oocytes were observed in knock out group, same as the treated group. At90dah, only few oocytes were detected in treated gonads. The results implied meiosis initiation requires RA in Nile tilapia ovary. RA deficiency influence ovary development and could reduce the amount of spawned eggs in fish.Immunohistochemistry analysis indicated that the expression of Aldhla2in dnd deficiency gonads is similar to that of control gonads. The results indicated the expression of Aldhla2in somatic cells is independent of germ cells. In summary, dnd is highly conserved during evolution and essential for germ cells development. Masculinization of somatic cells in germ cell-deficient XX indicated that germ cells are essential for the differentiation of ovary. In addition, Aldhla2through synthesis of RA also plays an important role in the meiotic initiation of germ cells in ovary. The expression of Aldhla2in somatic cells is independent of germ cells.