AMH regulates ovary size by counteracting the positive influence of clustered ovarian follicle growth.

study questionDoes anti-Müllerian hormone (AMH) influence preantral follicle development within a short range of AMH-secreting antral follicles? SUMMARY ANSWER: Immunization of sheep against AMH leads to increased follicle survival, specifically in regions close to small-medium antral follicles. WHAT IS KNOWN ALREADY: Serum AMH is known to inhibit the survival of immature ovarian follicles, but its biological role remains poorly understood. Mammalian ovaries contain many more developing ovarian follicles than are needed for ovulation, but how these systems operate remains unclear. STUDY DESIGN, SIZE, DURATION: Cross-sectional-control versus treatment studies examining the effects in the presence or absence of AMH. PARTICIPANTS/MATERIALS, SETTING,

methodsAnti-AMH immunization experiments were conducted in sheep followed by histological 2D and 3D tissue analysis. Microdialysis was conducted on ex vivo human and sheep ovaries. Live-imaging of a fluorogenic enzyme-responsive reagent was conducted on ex vivo mouse ovaries. Organ/follicle culture was conducted on Amh+/+ and Amh-/- ovary tissues to measure follicle activation and growth rates. MAIN RESULTS AND THE ROLE OF CHANCE: The likely site of action of AMH was shown to be ovarian stroma adjacent to large follicles as microdialysis determined that receptor-activating concentrations of AMH were only observed within a short range of the follicle. The AMH-activating enzymes were also shown to be primarily located in stroma using live-imaging of a fluorogenic enzyme-responsive reagent. Sheep were immunized against AMH protein to inhibit extracellular signalling and the ovaries were examined using 3-dimensional reconstructions of ovarian follicle positions. This showed that inhibition of AMH signalling caused an increase in preantral follicle survival, but almost entirely in proximity to large developing follicles. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Limited studies were conducted on human tissue but the results concur with the sheep experiments. Sheep ovaries provide a useful large-animal model for comparative anatomy with humans but there will be interspecies differences. WIDER IMPLICATIONS OF THE

findingsThese results add to the evidence that small growing follicle survival is influenced by the proximity of large follicles. This has relevance for conditions where large follicles are lacking (e.g. primary ovary insufficiency) or where follicle growth is excessive (e.g. PCOS). STUDY FUNDING/COMPETING INTEREST(S): This research was funded by a Sir Charles Hercus Research Fellowship from the Health Research Council of New Zealand (grant number 18-027). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.