Implication of Oxygen level on the regulation of follicle growth in cultured mice ovaries in vitro

Abstract

Objectives: Mammalian ovaries contain a large pool of non-growing, primordial follicles. The mechanisms that regulate the gradual exit of ovarian follicles from the non-growing, primordial pool are very poorly understood. One of the critical molecules used to manage follicle development is oxygen. Follicle development occurs commensurate with neo-vascularization of the theca-interstitial cell compartment and the accompanying relative relief from the hypoxic conditions that exist in primordial follicles. This study was conducted to evaluate implication of Oxygen level on the regulation of follicle growth using in vitro cultured immature mice ovaries.

Methods: Ovaries were obtained from seven-day old immature mice and dissected for organ culture. Three different oxygen tensions, such as 1.5%, 5%, 20%, were used for this study. Each three ovaries per group were placed on the organ culture dish in basal medium (without serum) for 3 days. At the end of culture, ovaries were fixed in buffered formalin for histological analysis, or were frozen for DNA extraction. The slide sections from cultured ovaries were stained with H&E for histological analyses and follicle growth was further confirmed by immuno-localization of proliferating cell nuclear antigen (PCNA), a marker for cell growth and proliferation. In addition, we analyzed DNA fragmentation from cultured mice ovaries to evaluate the effect of oxygen tension and nerve growth factor (NGF) on apoptosis.

Result: Histo-morphological analysis showed that 1.5% was not proper for follicle growing. Under the higher oxygen tension, more follicles were growing (more cellular proliferation). However, more DNA fragmentation was also appeared in cultured ovaries under the 1.5% and 20% of oxygen than those of 5% of oxygen. And this apoptosis was remarkably decreased by addition of NGF in cultured ovaries only under the 20% oxygen, not in those of 1.5 or 5%.

Conclusion: This study represents that initial follicle growing and survival definitely need oxygen. Higher oxygen caused more growing follicles and more apoptosis as well. Based on this result, 5% oxygen likely seems to proper tension for growing and less apoptosis. However, in the presence of NGF, 20% oxygen may be the most optimal tension for more growing, but little apoptosis. From this research, a better understanding about the regulation of this pool of small, growing follicles may have a great impact on the ability to manipulate fertility as well as the timing of menopause.