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

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.

Keywords: PTEN; follicle; human; in vitro; ovary.

Figure 1

(A) Photomicrograph of 0 h human ovarian cortex; all follicles are at the earliest stages of development. (B and C) Photomicrographs of cultured ovarian cortex at Day 6 showing activation of follicle growth coincident with non-growing follicles in control (B), and in bpV(HOpic) treated cortical tissue (C) showing multiple secondary follicles. (D) Distribution of follicles in adult human ovarian cortical tissue by stage of development in uncultured tissue (0 h) and at Day 6. Follicle distribution is shown as a percentage of the total. Arrows indicate in vitro grown secondary follicles. Scale bar = 50 microns.

Figure 2

(A) Western blot showing enhanced phosphorylation of Akt in tissue exposed to bpV(HOpic) compared with control cultured tissue, indicating PTEN inhibition is associated with increased activation of the PI3K pathway; β-actin used as loading control. (B) The fold change in expression of pAkt in human ovarian tissue in control and bpV(HOpic) treated tissue, indicating significantly higher AKT phosphorylation in bpV(HOpic) treated tissue (*P < 0.01, n = 5). (C) Photomicrographs showing immunohistochemical detection of FOXO3 in human ovarian cortex. (i) Brown staining indicating nuclear enclosed, inactivated FOXO3 in a non-growing follicle exposed to control medium. Arrow indicates discrete brown staining in germinal vesicle; (ii) export of FOXO3 from the nucleus indicated by brown staining in the ooplasm of in vitro grown secondary follicle exposed to bpV(HOpic), indicating activation of PI3K pathway with Akt phosphorylation. Arrow indicates absence of staining in the germinal vesicle. (iii) Negative control. Scale bar = 30 microns. (D) Oocyte nuclear export of FOXO3 in control and bpV(HOpic) treated tissue: a significantly greater percentage of Oocytes showed FOXO3 nuclear export in tissue exposed to bpV(HOpic) compared with control (*P = 0.0019). Eighty-nine follicles analysed in total, 48 from control and 41 from bpV(HOpic) exposed tissue.

Figure 3

(A) The percentage of morphologically normal follicles observed in tissue fragments in uncultured tissue (0 h) and after 6 days culture in control or bpV(HOpic) medium. Percentages of healthy follicles are shown by developmental stage. (B) Mean diameter (µm) of follicles isolated from control (green) and bpV(HOpic) (red) exposed tissue over a further 6 days in vitro. At the end of the culture period follicles isolated from control tissue were significantly larger than bpV(HOpic) treated follicles (*P < 0.001). (C) On completion of the isolated follicle culture period a significantly greater percentage of control exposed follicles were morphologically normal compared with those exposed to bpV(HOpic) (*P < 0.01). (D) Photomicrographs of isolated follicles after a total of 12 days in culture. (i) Morphologically normal IVG follicle from control cultured tissue, and (ii) morphologically abnormal IVG follicle isolated from bpV(HOpic) exposed tissue. Scale bar = 50 microns.