Enhancing anti-Müllerian hormone processing reduces preantral follicle survival but spares female reproduction in mice

Anti-Müllerian hormone (AMH) is produced by granulosa cells within growing ovarian follicles and limits the number of follicles reaching ovulation. AMH is synthesized as a precursor protein comprising N-terminal prodomains and C-terminal mature domains, separated by a furin-like cleavage motif (RXXR). Proteolytic maturation of AMH (140 kDa) is required to release the bioactive mature dimer (25 kDa), which potentiates signaling via AMH receptors (AMHR2 and ALK2/3). However, the abundance of unprocessed AMH in human follicular fluid suggests that cleavage within the ovary is inefficient. This study hypothesized that enhancing AMH maturation would increase AMH activity in vitro and in vivo. Using targeted mutagenesis, we optimized the murine AMH cleavage site (from wild-type (WT) 443RTGR445 to 443RKKR445) and showed in vitro that this favored production of bioactive AMH. We then introduced this mutation into the Amh gene in C57Bl6/J mice using CRISPR/Cas9 and assessed the consequences for female reproduction. Analyses of 12-week-old AmhRKKR/RKKR mice revealed that the ovaries were significantly lower in mass (-25%, P < .05) relative to AmhWT/WT controls. Despite differences in ovarian masses, estrous cyclicity, and fertility were unaltered. Although maturing follicle numbers did not differ, ovaries from 12- and 24-week-old AmhRKKR/RKKR females contained a greater proportion of atretic secondary follicles (1.6- to 4-fold more, P < .05), underscoring AMH's role in preantral follicle survival. Analyses of adult male AmhRKKR/RKKR mice indicated that testis mass and morphology were unaltered. These findings support a physiological role for ovarian AMH in limiting preantral follicle survival and indicate that enhancing AMH maturation is otherwise nondisruptive to female reproduction.