(313) Anti-Müllerian Hormone Promotes Cell Cycle Progression and Cellular Energy Production of Primate Preantral Follicles under Hypoxia

Jing Xu¹, Shally Wolf², Fuhua Xu³

1. Department of Biology & Chemistry, School of Health Sciences, Liberty University, Lynchburg, VA, USA
2. Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR, USA
3. Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Baltimore, MD, USA

Abstract Text:

After primordial follicle activation, folliculogenesis progresses into preantral follicle growth. Preantral follicles are located in the ovarian cortex, an avascular region without ready access to the circulation. Evidence suggests that minichromosome maintenance proteins expressed by preantral follicles are important for preventing cell cycle inhibition by hypoxia. Minichromosome maintenance protein 2, 3, 5, and 7 inhibit the function or enhances the degradation of hypoxia-inducible factor-1, a protein expressed in granulosa cells of preantral follicles that causes cell cycle arrest under oxygen deprivation. In addition, creatine metabolism is essential for ATP production under hypoxia to support cell cycle progression of granulosa cells in preantral follicles. Anti-Müllerian hormone (AMH) is a member of the transforming growth factor beta family produced by granulosa cells of growing follicles in the ovary. AMH expression increases during preantral follicles growth and promotes granulosa cell proliferation. Therefore, experiments were performed to test the hypothesis that AMH promotes granulosa cell cell cycle progression and cellular energy production under hypoxia, which leads to its stimulatory actions in preantral follicles. Ovaries were collected from rhesus macaques (Macaca mulatta, 6-10 years old, n = 6) by laparoscopy at early follicular phase. Preantral follicles (diameter = 125-225 μm) isolated from each animal were randomly assigned into two groups (48 follicles/group) for individual culture in ultra-low attachment microplates: (a) control medium and (b) 100 ng/ml recombinant human AMH protein supplementation. At culture day 7, follicle survival was assessed by microscopy. Diameters of surviving follicles were measured using an Image J software. Follicles from the control and AMH-treatment groups (10 follicles/group) were pooled for each animal. RNA was extracted for RNA sequencing and RT-PCR to evaluate mRNA levels of hypoxia-inducible factor-1A, as well as minichromosome maintenance protein 2, 3, 5, and 7. Culture medium samples from these follicles were also pooled accordingly for metabolomics analysis. Data were analyzed using mixed models. At culture day 7, follicle survival rates were comparable between the control and AMH-treatment groups. Diameters of survived preantral follicles in the AMH-treatment group were larger (P < 0.05) than those of the control group. AMH treatment during preantral follicle culture did not alter the mRNA levels of hypoxia-inducible factor-1A. However, the mRNA levels of minichromosome maintenance protein 2, 3, 5, and 7 increased (P < 0.05) in AMH-treated follicles. AMH treatment also increased (P < 0.05) follicular secretion of creatine and its precursor guanidinoacetate. Data suggest that, instead of altering hypoxia-inducible factor-1A gene expression, AMH inhibits hypoxia-inducible factor-1 protein action via elevating minichromosome maintenance protein levels which stimulates granulosa cell cell cycle progression in primate preantral follicles under hypoxia. Granulosa cell proliferation is also supported by creatine-mediated ATP production which is promoted by AMH. The increased AMH expression is crucial for primate preantral follicle growth to reach the antral stage. Supported by NIH/NICHD R01HD082208, NIH/OD P51OD011092.