In vitro follicle culture systems allow for the development of cryopreserved ovarian tissue, and therefore may provide reproductive options to women facing premature infertility due to cancer therapies. The ovarian follicle is the reproductive unit of the ovary, and consists of an oocyte and its associated somatic cells. Previous studies have demonstrated that follicle growth in a 3D alginate culture system is regulated by the matrix stiffness. Follicle growth, oocyte quality, embryonic development, steroid biosynthesis, and development of the fluid-filled antral cavity were all regulated by alginate stiffness. The antral cavity is a defining characteristic of mature follicles, yet the mechanisms regulating its formation are not understood. We hypothesized that aquaporins, transmembrane proteins that facilitate water transport across cell membranes, play a role in development of the antral cavity, and may be regulated by matrix stiffness. Multilayered secondary follicles (150-180 μm diameter) were isolated from mice at 16 days of age and encapsulated in 0.5% (permissive) and 1.5% (non-permissive) alginate matrices. Follicles were cultured for 6-8 days, at which time surviving follicles were removed from the alginate for RNA isolation. In addition, RNA was isolated from freshly dissected follicles of sizes 150-180 μm (preantral) as well as 300-400 μm (antral) from both untreated and pregnant mare serum gonadotropin- (PMSG-) stimulated mice.

Growth and antrum formation were regulated by the matrix stiffness in these culture conditions, and aquaporin 7 and 8 expression varied with follicle stage and matrix stiffness. Follicles cultured in 0.5% alginate increased in diameter 83.0±5.5% by day 8, while those cultured in 1.5% alginate increased in diameter 39.4±2.6%. By day 8 of culture, 46.4% of follicles cultured in 0.5% alginate and only 4.2% of follicles cultured in 1.5% alginate had formed antral cavities. Aquaporins 7, 8, and 9 were examined by RT-PCR, and aquaporins 7 and 8 were found to be present at detectable levels in both freshly isolated and cultured follicles, indicating that aquaporin 9 is not involved in follicle development. In cultured follicles, the highest level of aquaporin 7 and 8 expression was observed in follicles collected at day 6 of culture from 1.5% alginate. These results may indicate that aquaporins are upregulated in non-permissive matrices to compensate for the increased resistance the follicle encounters in trying to grow in the relatively stiff matrix. For freshly isolated follicles, lowest expression of both aquaporins 7 and 8 was observed in the PMSG-stimulated antral follicles. This result demonstrates that these aquaporins are not induced by gonadotropins, and indicates that other gonadotropin-responsive factors may play a role in antrum formation in these externally-stimulated follicles. Together, these results demonstrate that antrum formation is regulated by the stiffness of the matrix surrounding the follicle in vitro, and that aquaporins 7 and 8, as well as additional factors are responsible for antrum formation. Further studies using this system may provide insight into ovarian disorders such as polycystic ovary syndrome, in which the ovarian stroma thickens causing suspension of follicle growth in the early antral stage, often causing infertility.

Supported by NIH U54HD41857