Inhibition of Phosphatase and Tensin Homologue (PTEN) in Human Ovary In Vitro Results in Increased Activation of Primordial Follicles But Compromises Development of Growing Follicles

Abstract

The overwhelming majority of human ovarian follicles exist as a quiescent population from fetal life until puberty. Only a small number of follicles from this dormant population are recruited, undergo development, go on to complete growth, and release a mature fertilizable oocyte. The phosphoinositide 3-kinase (PI3K)–protein kinase B (Akt) signaling pathway has been identified as a key mechanism involved in the maintenance of follicle growth and loss. A number of growth factors stimulate PI3K to promote phosphorylation of Akt and downstream transcription factors including the forkhead winged helix box O1 (FOXO1); this results in follicle survival and activation of growth. These events are reversed by the action of phosphatase and tensin homolog (PTEN). In murine models, the effect of PTEN can be inhibited by the vanadate derivative, dipotassium bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate (V) (bpV(HOpic), and thereby promote the downstream phosphorylation of Akt. Previous studies suggest that PTEN inhibitors could be used to generate mature oocytes in women whose oocyte reserve is impaired because of illness or treatment. There are few or no data on the response of human follicles to bpV(HOpic) in vitro. It is not known whether selective promotion of the PI3K pathway by pharmacological inhibition of PTEN in vitro would promote initiation of growth in quiescent human ovarian follicles within tissue fragments and could provide a population of biopsy-derived growing secondary follicles of sufficient quality for in vitro maturation. The aim of this study was to investigate the effect of bpV(HOpic), a reversible inhibitor of PTEN, on the activation, survival, and development of human ovarian follicles in vitro. Ovarian cortical tissue was obtained by biopsy from a total of 17 adult women aged 23 to 46 years. Fragments of the biopsied tissue were cultured for 24 hours in control medium (n = 128) or medium supplemented with 1 μM bpV(HOpic (n = 146). Media in both cultures were then replaced with control medium, and tissues were incubated for a further 5 days. After the initial 24-hour culture, half of the tissue fragments from both groups were fixed, and phosphorylated Akt was quantified by Western blot analysis. Following 6 days of incubation, tissue fragments were examined under light microscopy, and any secondary follicles 100 μm in diameter or greater were isolated. Then, follicles isolated from tissue fragments were cultured individually in control medium supplemented with 100 ng/mL recombinant human activin A. Tissue fragments without detectable follicles or with only unilaminar follicles were fixed and processed for histological and immunohistochemical analysis. Microscopic examination showed that after 6 days incubation, initiation of follicle growth had occurred in both treatment groups, but significantly more follicles progressed to the secondary stage of development in the presence of 1 bpV(HOpic) than in the control (31% vs 16%; P < 0.05). Increased activation of the PI3K pathway in tissue exposed to bpV(HOpic) was confirmed by an increase in Akt phosphorylation and nuclear export of FOXO3. However, isolated and cultured secondary follicles treated with bpV(HOpic) exhibited limited growth and reduced survival compared with follicles from control fragments (P < 0.05). These data demonstrate that inhibition of PTEN with bpV(HOpic) promotes activation and development of isolated follicles to the secondary stage, as in rodents, but severely compromises their continued growth and survival.