CRISPR/Cas9-induced disruption of wt1a and wt1b reveals their different roles in kidney and gonad development in Nile tilapia.

Wilms tumor 1 (Wt1) is an essential factor for urogenital system development. Teleosts have two wt1s, named as wt1a and wt1b. In this study, the expression pattern of wt1a and wt1b and their functions on the urogenital system were analyzed by in situ hybridization and CRISPR/Cas9. wt1a was found to be expressed in the glomerulus at 3 dah (days after hatching), earlier than wt1b. wt1a and wt1b were simultaneously expressed in the somatic cells of gonads at 3 dah, while their cell locations were similar, but not identical in adult fish gonads. The wt1a-/- fish displayed pericardial edema and yolk sac edema at 3 dah and subsequently expanded as general body edema at 6 dah, failed to develop glomerulus and died during 6-10 dah, whereas the wt1b-/- fish were phenotypically normal. Immunohistochemical analyses revealed that the germ cell marker Vasa was expressed, while somatic cell genes Cyp19a1a, Amh, Gsdf and Dmrt1 were not expressed in the wt1a-/- gonads at 6 dah. The sex phenotypes of XX and XY in the wt1b-/- fish were not affected. Real-time PCR revealed that the ovarian cyp19a1a expression was up-regulated in XX wt1b-/- fish, compared with XX control at 90 dah. Serum estradiol-17ß level was also up-regulated in XX wt1b-/- fish at 90 and 180 dah. The XY wt1b-/- fish had normal serum estradiol-17ß and 11-ketotestosterone levels and remained fertile. These results suggest that Wt1a and Wt1b have different functions in the kidneys and gonads of tilapia.

Transcriptome display during tilapia sex determination and differentiation as revealed by RNA-Seq analysis.

BACKGROUND: The factors determining sex in teleosts are diverse. Great efforts have been made to characterize the underlying genetic network in various species. However, only seven master sex-determining genes have been identified in teleosts. While the function of a few genes involved in sex determination and differentiation has been studied, we are far from fully understanding how genes interact to coordinate in this process. RESULTS: To enable systematic insights into fish sexual differentiation, we generated a dynamic co-expression network from tilapia gonadal transcriptomes at 5, 20, 30, 40, 90, and 180 dah (days after hatching), plus 45 and 90 dat (days after treatment) and linked gene expression profiles to both development and sexual differentiation. Transcriptomic profiles of female and male gonads at 5 and 20 dah exhibited high similarities except for a small number of genes that were involved in sex determination, while drastic changes were observed from 90 to 180 dah, with a group of differently expressed genes which were involved in gonadal differentiation and gametogenesis. Weighted gene correlation network analysis identified changes in the expression of Borealin, Gtsf1, tesk1, Zar1, Cdn15, and Rpl that were correlated with the expression of genes previously known to be involved in sex differentiation, such as Foxl2, Cyp19a1a, Gsdf, Dmrt1, and Amh. CONCLUSIONS: Global gonadal gene expression kinetics during sex determination and differentiation have been extensively profiled in tilapia. These findings provide insights into the genetic framework underlying sex determination and sexual differentiation, and expand our current understanding of developmental pathways during teleost sex determination.

Roles of anti-Müllerian hormone and its duplicates in sex determination and germ cell proliferation of Nile tilapia.

Duplicates of amh are crucial for fish sex determination and differentiation. In Nile tilapia, unlike in other teleosts, amh is located on X chromosome. The Y chromosome amh (amhΔ-y) is mutated with 5 bp insertion and 233 bp deletion in the coding sequence, and tandem duplicate of amh on Y chromosome (amhy) has been identified as the sex determiner. However, the expression of amh, amhΔ-y, and amhy, their roles in germ cell proliferation and the molecular mechanism of how amhy determines sex is still unclear. In this study, expression and functions of each duplicate were analyzed. Sex reversal occurred only when amhy was mutated as revealed by single, double, and triple mutation of the 3 duplicates in XY fish. Homozygous mutation of amhy in YY fish also resulted in sex reversal. Earlier and higher expression of amhy/Amhy was observed in XY gonads compared with amh/Amh during sex determination. Amhy could inhibit the transcription of cyp19a1a through Amhr2/Smads signaling. Loss of cyp19a1a rescued the sex reversal phenotype in XY fish with amhy mutation. Interestingly, mutation of both amh and amhy in XY fish or homozygous mutation of amhy in YY fish resulted in infertile females with significantly increased germ cell proliferation. Taken together, these results indicated that up-regulation of amhy during the critical period of sex determination makes it the sex-determining gene, and it functions through repressing cyp19a1a expression via Amhr2/Smads signaling pathway. Amh retained its function in controlling germ cell proliferation as reported in other teleosts, while amhΔ-y was nonfunctionalized.

Heterozygous mutation of eEF1A1b resulted in spermatogenesis arrest and infertility in male tilapia, Oreochromis niloticus.

Eukaryotic elongation factor 1 alpha (eEF1A) is an essential component of the translational apparatus. In the present study, eEF1A1b was isolated from the Nile tilapia. Real-time PCR and Western blot revealed that eEF1A1b was expressed highly in the testis from 90 dah (days after hatching) onwards. In situ hybridization and immunohistochemistry analyses showed that eEF1A1b was highly expressed in the spermatogonia of the testis. CRISPR/Cas9 mediated mutation of eEF1A1b resulted in spermatogenesis arrest and infertility in the F0 XY fish. Consistently, heterozygous mutation of eEF1A1b (eEF1A1b+/-) resulted in an absence of spermatocytes at 90 dah, very few spermatocytes, spermatids and spermatozoa at 180 dah, and decreased Cyp11b2 and serum 11-ketotestosterone level at both stages. Further examination of the fertilization capacity of the sperm indicated that the eEF1A1b+/- XY fish were infertile due to abnormal spermiogenesis. Transcriptomic analyses of the eEF1A1b+/- testis from 180 dah XY fish revealed that key elements involved in spermatogenesis, steroidogenesis and sperm motility were significantly down-regulated compared with the control XY. Transgenic overexpression of eEF1A1b rescued the spermatogenesis arrest phenotype of the eEF1A1b+/- testis. Taken together, our data suggested that eEF1A1b is crucial for spermatogenesis and male fertility in the Nile tilapia.