189 ESTRADIOL PRODUCTION AND GENE UPREGULATION FOLLOWING PRIMORDIAL FOLLICLE ACTIVATION IN VITRO

Abstract

Activation and growth of dormant follicles completely in vitro, with the production of competent oocytes, would provide not only the opportunity to investigate control mechanisms of follicle growth, but also an instrument by which fertility might be preserved in women with cancer, premature ovarian failure, or advanced maternal age. The objective of this study was to assess the physical culture parameters, as well as the addition of an antibody to the anti-angiogenic isoform of VEGFA (VEGFAxxxb), for the ability to support activation of primordial follicles and subsequent functional follicular growth in vitro. The phosphatase and tensin homologue (PTEN) inhibitor, bpV(pic), was used to activate growth of primordial follicles in neonatal Day 3 mouse ovaries. BpV(pic) (100 µM) was used either alone, or in combination with FSH (0.3 IU mL^–1), IGF-1 (10 ng mL^–1), EGF (10 ng mL^–1), and ITS (insulin-transferrin-selenium; 1x). Ovaries were treated for 24 h and then cultured for an additional 12 to 20 days, either suspended on a membrane or encapsulated in an alginate bead, with or without VEGFAxxxb during culture. Oestradiol (E2) production and expression of Ahr, Amh, Bmp15, Cyp19, and Esr1, genes known to be involved in follicular growth, were evaluated to assess growth and functionality of follicles. The E2 data were analysed with ANOVA; the qPCR data were analysed using the non-parametric Mann–Whitney U t-test (significance at P < 0.05). Forty-eight hours after treatment with bpV(pic), alone or with FSH, IGF-1, EGF, and ITS, resulted in an upregulation of Amh and Esr1, and a tendency for upregulation of Ahr compared with untreated control ovaries. There were no differences in gene expression between the 2 bpV(pic) treatments. Following treatment and in vitro culture, expression of Ahr, Amh, and Bmp15 was upregulated in ovaries treated and cultured in a bead with VEGFAxxxb compared with ovaries treated and cultured in a bead without VEGFAxxxb, and ovaries treated on a membrane and cultured in a bead with VEGFAxxxb. Although ovaries treated on a membrane with bpV(pic) produced more E2 than untreated ovaries (5.1 ± 1.9 pg mL^–1 and 2.5 ± 0.8 pg mL^–1, respectively), this was not significantly different. Ovaries treated and cultured on a membrane without VEGFAxxxb produced more E2 (22.5 ± 12.2 pg mL^–1) than ovaries treated on a membrane and cultured in an alginate bead, with (3.9 ± 1.9 pg mL^–1) or without (1.8 ± 1.2 pg mL^–1) VEGFAxxxb; this was not significantly different from ovaries treated and cultured on a membrane with VEGFAxxxb (4.6 ± 4.6 pg mL^–1). In summary, primordial follicles can be induced to activate and grow in vitro using a PTEN inhibitor, as evidenced by the upregulated expression of follicle growth genes; VEGFAxxxb also increases expression of these genes. Treatment in a bead compared with a membrane appears to be advantageous for gene expression. However, culture on a membrane best supported oestradiol production, regardless of the presence of VEGFxxxb. These are critical pieces in the development of a successful culture system in which primordial follicles can be activated and functional growth supported in vitro.