Adamts9 is critical for the development of primary ovarian follicles

Adamts9 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, \r\nmember 9) is an extracellular matrix metalloprotease that is highly evolutionarily conserved and \r\ncritical for development in vertebrates. Knockouts of ADAMTS9 in mice and AdamTS-A in \r\nDrosophila melanogaster are embryonic lethal\; and abnormal ADAMTS9 activity has also been \r\nlinked to various human disorders including ovarian and uterine disease. Our previous work has \r\ndemonstrated that Adamts9 is necessary for ovarian development in zebrafish. Adamts9 KO \r\nzebrafish had a heavily male-biased sex ratio as 6–7-month-old adults, and female Adamts9 KOs\r\nwere infertile. Furthermore, a novel intersex phenotype was also discovered in Adamts9 KOs\r\nzebrafish. These fish lacked clearly defined ovary or testis structure. To further understand the \r\nroles of Adamts9 in ovarian development and maintenance in zebrafish, we investigated \r\nAdamts9’s expression, role in primordial germ cell (PGC) migration, gonad development, sexual \r\ndifferentiation, and development of primary ovarian follicles in zebrafish. We found adamts9\r\nwas widely expressed in various tissues during embryonic and larval development and transcripts \r\nare also maternally deposited. We found strong expression in the developing retina at 48 hours \r\npost fertilization (hpf), that shifted to the ciliary marginal zone at 72hpf. We also found \r\nexpression in somites surrounding the PGCs during migration, in primary follicles in juveniles, \r\nand preovulatory follicular cells in adult ovaries. In contrast to its essential role in PGC \r\nmigration in invertebrate models, we only observed migration delay of PGCs in Adamts9 KOs. \r\nBut interestingly, we observed slower and under development of juvenile gonads in Adamts9 \r\nKO, and significantly reduced size and number of primary oocytes in Adamts9 KO zebrafish. \r\nSurprisingly, Adamts9 KO had a negligible effect on primary sex determination, but in female \r\nAdamts9 KOs the ovary remained dramatically underdeveloped compared to wildtype control \r\nsiblings. Rescuing global growth defects by overfeeding and lower rearing density did increase \r\nfemale percentage but did not rescue the underdeveloped ovary phenotype. Further, follicles in\r\nrescued Adamts9 KO females remained at Stage IB and only few follicles could continue \r\ndevelopment into late stages including mature follicles. As the fish continued to age, the male \r\nbiased sex ratio continued to increase in mid- or late-juvenile stages and even in adults, \r\nindicating that female Adamts9 KOs are sex reversing into males throughout their life. We also \r\nfound morphological evidence for sex reversal in Adamts9 KO at 90 days old adults, including \r\ncoexistence of Stage IB oocytes and sperm in the same tissue section. Taken together, we show\r\nthat Adamts9 is essential for proper ovarian development and maintenance and that loss of \r\nAdamts9 leads to folliculogenesis deficiency, follicle arrest, loss of ovarian follicles and eventual \r\nfemale to male sex reversal after primary sex determination in juvenile and adult zebrafish