The roles of Amh in zebrafish gonad development and sex determination

Abstract

Fetal mammalian testes secrete Amh (Anti-Mullerian hormone), which inhibits female reproductive tract (Mullerian duct) development. Amh also derives from mature mammalian ovarian follicles, which marks oocyte reserve and characterizes PCOS (polycystic ovarian syndrome). Zebrafish (Danio rerio) lacks Mullerian ducts and the Amh receptor gene amhr2 but, curiously, retains amh. To discover the roles of Amh in the absence of Mullerian ducts and the ancestral receptor gene, we made amh null alleles in zebrafish. Results showed that normal amh prevents female-biased sex ratios. Adult male amh mutants had enormous testes, half of which contained immature oocytes, demonstrating that Amh regulates male germ cell accumulation and inhibits oocyte development or survival. Mutant males formed sperm ducts and some produced a few offspring. Young female mutants laid a few fertile eggs, so they also had functional sex ducts. Older amh mutants accumulated non-vitellogenic follicles in exceedingly large but sterile ovaries, showing that Amh helps control ovarian follicle maturation and proliferation. RNA-seq data partitioned 21-day post-fertilization (dpf) juveniles into two groups that each contained mutant and wild type fish. Group21-1 up-regulated ovary genes compared to Group21-2, which were likely developing as males. By 35dpf, transcriptomes distinguished males from females and, within each sex, mutants from wild types. In adult mutants, ovaries greatly under-expressed granulosa and theca genes and testes under-expressed Leydig cell genes. These results show that ancestral Amh functions included development of the gonadal soma in ovaries and testes and regulation of gamete proliferation and maturation. A major gap in our understanding is the identity of the gene encoding a zebrafish Amh receptor; we show here that the loss of amhr2 is associated with the breakpoint of a chromosome rearrangement shared among cyprinid fishes.\n\nArticle SummaryAnti-Mullerian hormone (Amh) inhibits female reproductive duct development, signals oocyte reserve, and marks polycystic ovarian syndrome. Zebrafish lacks Mullerian ducts and the typical Amh receptor, questioning evolving roles of Amh. We made knockout mutations in zebrafish amh. Most mutants were female and the few males often had oocytes in their testes, showing that Amh promotes male development. Mutant reproductive ducts functioned, but testes were enormous and ovaries accumulated immature oocytes, showing that Amh regulates germ cell proliferation and maturation. Transcriptomics revealed that Amh controls development of steroid-producing gonad cells. Amh in zebrafish preserved ancestral roles despite losing Mullerian ducts and the Amh receptor.