A Hormone That Lost Its Receptor: Anti-Müllerian Hormone (AMH) in Zebrafish Gonad Development and Sex Determination

Abstract

Fetal mammalian testes secrete Anti-Müllerian hormone (Amh), which inhibits female reproductive tract (Müllerian duct) development. Amh also derives from mature mammalian ovarian follicles, which marks oocyte reserve and characterizes polycystic ovarian syndrome. Zebrafish (Danio rerio) lacks Müllerian ducts and the Amh receptor gene amhr2 but, curiously, retains amh. To discover the roles of Amh in the absence of Müllerian 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 nonvitellogenic follicles in exceedingly large but sterile ovaries, showing that Amh helps control ovarian follicle maturation and proliferation. RNA-sequencing data partitioned juveniles at 21 days postfertilization (dpf) into two groups that each contained mutant and wild-type fish. Group21-1 upregulated ovary genes compared to Group21-2, which were likely developing as males. By 35 dpf, transcriptomes distinguished males from females and, within each sex, mutants from wild types. In adult mutants, ovaries greatly underexpressed granulosa and theca genes, and testes underexpressed 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. KEYWORDS germ cells; PGC; male fertility; female fertility; gonad development; Genetics of Sex D EVELOPING mammalian embryos form the rudiments of both male and female sex ducts, the Wolffian and Müllerian ducts, respectively. Over 70 years ago, Alfred Jost conducted remarkable experiments to learn if gonads control sex duct development (Jost 1947). He removed undifferentiated gonads from rabbit fetuses and reimplanted them into the uterus of surrogate rabbit hosts. Gonadectomized kits lost male sex ducts but retained female sex ducts. He concluded that developing testes maintain male ducts (epididymis, seminal vesicles, and vas deferens) but destroy female sex duct anlagen (fallopian tubes and uterus). In contrast, developing ovaries neither maintain male ducts nor destroy female ducts. Subsequent experiments showed that one testisderived substance (testosterone) maintains male sex duct rudiments and another [anti-Müllerian hormone (AMH), also called Müllerian Inhibiting Substance (MIS)], inhibits female reproductive duct anlagen (Elger 1966; Josso 1972). Although AMH from testes represses female duct d...

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