Abstract
In the mammalian ovary a small number of follicles are steadily recruited from the quiescent pool to undergo development. Follicle loss, maintenance and growth are strictly controlled by complex molecular interactions including the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signalling pathway. Stimulation of PI3K promotes phosphorylation of Akt resulting in follicle survival and activation of growth whereas this pathway is suppressed by the actions of the phosphatase and tensin homologue (PTEN). The aim of this study was to determine the effect of dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate (bpV), a reversible inhibitor of PTEN, on the activation, survival and development of human ovarian follicles in vitro. Biopsied ovarian tissue fragments were obtained from 17 women aged 23–46 years and exposed to 1 µM bpV(HOpic) (n = 146) or control medium (n = 128) for 24 h. Media were then replaced with control medium and all tissue incubated for a further 5 days. Ovarian tissue from each treatment group was fixed after the initial 24 h culture period and phosphorylated Akt was quantified by western blotting. After 6 days incubation all tissue fragments were inspected under light microscopy and any secondary follicles ≥100 µm isolated. Isolated follicles were cultured individually in control medium supplemented with 100 ng/ml recombinant human activin A. Tissue fragments without follicles suitable for isolation were fixed and processed for histological and immunohistochemical analysis. During 6 days culture, follicle activation occurred in tissue samples from both treatment groups but with significantly more follicles progressing to the secondary stage of development in the presence of 1 µM bpV(HOpic) compared with control (31 versus 16%; P < 0.05). Increased activation was associated with increased Akt phosphorylation and increased nuclear export of FOXO3. However isolated and cultured follicles that had been exposed to bpV(HOpic) showed limited growth and reduced survival compared with follicles from control fragments (P < 0.05). This study demonstrates that inhibition of PTEN with bpV(HOpic) affects human ovarian follicle development by promoting the initiation of follicle growth and development to the secondary stage, as in rodent species, but severely compromises the survival of isolated secondary follicles.
Highlights
Human ovarian follicles largely exist as a quiescent population, of which a small number daily initiate growth throughout reproductive life
This study demonstrates that inhibition of phosphatase and tensin homologue (PTEN) with bpV(HOpic) affects human ovarian follicle development by promoting the initiation of follicle growth and development to the secondary stage, as in rodent species, but severely compromises the survival of isolated secondary follicles
In this study we investigated the ability of bpV(HOpic) a pharmacological inhibitor of PTEN, a major negative regulator of the phosphoinositide 3-kinase (PI3K) pathway, to affect human ovarian follicle activation and development in both tissue fragments and isolated follicles in vitro
Summary
Human ovarian follicles largely exist as a quiescent population, of which a small number daily initiate growth throughout reproductive life. Remaining follicles degenerate either from the dormant state (Tingen et al, 2009) or after growth has been initiated (Kaipia and Hsueh, 1997). Development of growing follicles is controlled by the coordinated actions of multiple complex, integrated signalling pathways regulated by local and systemic hormonal signals (Pangas, 2007; Sobinoff et al, 2013) and reproductive senescence occurs when the quiescent follicle population is exhausted through activation and degeneration. Prior to exhaustion of the follicle pool, the overwhelming majority of human follicles are dormant and can persist in this state for decades. The ability to recruit these dormant follicles into the growing pool and support their complete development in vitro would
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