Adamts9 is critical for the development of primary ovarian follicles

Abstract

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