Anti-Müllerian hormone (AMH) actions during ovarian follicular development appear to be stage-dependent. AMH expression increases during preantral follicle growth, which regulates granulosa cell proliferation. Preantral follicles are located in the ovarian cortex, an avascular region with limited oxygen supply. Therefore, experiments were performed to determine the mechanism of AMH in promoting cell cycle progression and cellular energy production of granulosa cells under hypoxia in preantral follicles. Ovaries were collected from macaques. Paraffin sections were obtained for immunohistochemistry to detect carbonic anhydrase IX. Preantral follicles were isolated for culture at 5% oxygen in two groups: (a) control media and (b) recombinant human AMH supplementation. Follicle diameters were measured on days 2 and 8. Control media samples were collected on day 2 for no-grow and growing follicles and analyzed using enzyme-linked immunosorbent assay and liquid chromatography tandem mass spectrometry. Selected control follicles were stained with Image-iT Hypoxia Reagent. Follicles from the control and AMH-treatment groups were pooled for each animal on day 8. RNA was extracted for RNA sequencing and RT-PCR. Culture media samples from these follicles were pooled accordingly for liquid chromatography tandem mass spectrometry. Data were analyzed using mixed models. Carbonic anhydrase IX immunostaining was identified in granulosa cells of preantral follicles developed in vivo. Granulosa cells of cultured preantral follicles were positive for Hypoxia Reagent staining. Although diameters of no-grow and growing follicles were comparable on day 2, growing follicles secreted higher levels of AMH, guanidinoacetate, and creatine. When cultured with AMH supplementation, follicle diameters were larger than those of the control group on day 8. The mRNA levels of minichromosome maintenance proteins 2, 3, 5, and 7, but not hypoxia-inducible factor-1 A, increased in AMH-treated follicles. AMH supplementation increased follicular secretion of guanidinoacetate, creatine, and creatinine. Granulosa cells of preantral follicles experience physiological hypoxia which can be mimicked using 5% oxygen culture. AMH may inhibit hypoxia-inducible factor-1 protein action via elevating minichromosome maintenance protein expression which stimulates cell cycle progression of granulosa cells under hypoxia. Granulosa cell proliferation is also supported by creatine-mediated adenosine triphosphate production which is promoted by AMH. Not applicable.
Xu et al. (Fri,) studied this question.