Developmental effects of imatinib mesylate on follicle assembly and early activation of primordial follicle pool in postnatal rat ovary.

Imatinib mesylate is an anti-cancer agent that competitively inhibits several receptor tyrosine kinases (RTKs). RTKs play important roles in the regulation of primordial follicle formation, the recruitment of primordial follicles into the pool of growing follicles and maturation of the follicles. In the present study, we investigated the effects of the tyrosine kinase inhibitor imatinib on primordial follicle assembly and early folliculogenesis in postnatal rats. Female Sprague-Dawley rats were treated with either imatinib (150mg/kg) or placebo (water) on postnatal days 2-4. Bilateral ovariectomy was performed on postnatal day 2 and 5. Histology, immunohistochemistry, and mRNA analysis were performed. Imatinib treatment was associated with increased density of the multi-oocyte follicles (P<0.01), oogonia (p<0.01) and germline clusters (P<0.05), decreased activation of primordial follicles, increased expression of c-Kit and AMH, and decreased protein expression of Kit-ligand and GDF9 when compared to age-matched controls. In conclusion, imatinib affects folliculogenesis in postnatal rat ovaries by delaying the cluster breakdown, follicular assembly and early activation of the primordial follicle pool.

Growth and differentiation factor-9 promotes adhesive and motile capacity of prostate cancer cells by up-regulating FAK and Paxillin via Smad dependent pathway.

The majority of advanced prostate cancers metastasis to the bone. Mediators of bone remodelling, the bone morphogenetic proteins have extensively been implicated in the progression and metastasis of prostate cancer. The present study investigated the function of BMP member GDF-9, in prostate cancer. We overexpressed GDF9 in PC-3 cells using a mammalian expression construct, and knocked-down with the use of ribozyme transgenes. These cells were further used in in vitro adhesion and motility assays, in order to determine the effect of GDF-9 on these properties. Recombinant GDF-9 was generated to treat PC-3 WT cells before further analysing the effect on adhesion. The GDF-9 overexpressing PC-3 cells demonstrated a significantly enhanced adhesive and motile capacity compared to their controls. The opposite effect was seen in the GDF-9 knock-down cells. In addition, treating PC-3 cells with rh-GDF-9 resulted in them becoming more adhesive. Both endogenous and exogenous GDF-9 was demonstrated to up-regulate focal adhesion associated proteins FAK and paxillin which contribute to promoted cell adhesion and motility. With the use of a Smad3 inhibitor, this effect was inhibited suggesting that GDF-9 signals via Smad3 to up-regulate expression of these proteins. This study shows that GDF-9 can promote the motile and adhesive capacity of PC-3 prostate cancer cells by up-regulating expression of FAK and paxillin in a Smad dependent manner, suggesting a pro-tumourigenic role for GDF-9 in prostate cancer.

Growth differentiation factor 9 reverses activin A suppression of steroidogenic acute regulatory protein expression and progesterone production in human granulosa-lutein cells.

BACKGROUND: We have reported that growth differentiation factor 9 (GDF9) can enhance activin A (ß(A)ß(A))-induced inhibin B (αß(B)) secretion in human granulosa-lutein (hGL) cells, but its effects on steroidogenic acute regulatory protein (StAR), ovarian steroidogenic enzymes, and progesterone production are unknown. We undertook this study to further evaluate GDF9 in this regard. METHODS: hGL cells from women undergoing in vitro fertilization treatment were cultured with and without small interfering RNA (siRNA) transfection targeted at inhibin α-subunit or GDF9 before treatment with GDF9, activin A, FSH, or combinations. We compared StAR, P450 side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase expression in hGL cells and progesterone levels in culture media after these treatments. mRNA, protein, and hormone levels were assessed with real-time RT-PCR, immunoblotting, and ELISA, respectively. Data were analyzed by ANOVA followed by Tukey's test. RESULTS: Activin A alone reduced basal and FSH-induced progesterone production by decreasing the expression of StAR protein, which regulates the rate-limiting step in steroidogenesis but not P450 side-chain cleavage enzyme and 3ß-hydroxysteroid dehydrogenase. GDF9 attenuated these activin A effects on StAR and progesterone. After transfection of α-subunit siRNA, activin A level increased (P < 0.001), whereas basal and activin A-induced inhibin B levels (with and without GDF9) decreased. Furthermore, the effects of GDF9 in reversing activin A suppression of progesterone production were attenuated (P < 0.001). Transfection of GDF9 siRNA decreased GDF9 as expected and led to lower StAR expression and progesterone secretion than those observed with activin A treatment alone. CONCLUSION: GDF9 attenuates the suppressive effects of activin A on StAR expression and progesterone production by increasing the expression of inhibin B, which acts as an activin A competitor.

Inhibition of oocyte growth factors in vivo modulates ovarian folliculogenesis in neonatal and immature mice.

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are among the key regulators transmitting the signaling between the oocyte and the surrounding granulosa cells. Previously, it has been shown that a recombinant BMP type II receptor ectodomain-Fc fusion protein (BMPR2ecd-Fc) is able to inhibit the actions of GDF9 and BMP15 in vitro. Here, we have produced bioactive BMPR2ecd-Fc, which was injected i.p. into neonatal mice. Early folliculogenesis was first studied by injecting mice five times with various doses of BMPR2ecd-Fc during the postnatal days 4-12. Folliculogenesis was affected dose dependently, as evidenced by a decreased mitogenesis of granulosa cells of the growing follicles. Furthermore, we also noticed a decrease in the number of secondary and tertiary follicles as well as an increase in the oocyte size. Electron microscopic analysis revealed that the ultrastructure of the granulosa cells of the primary follicles was not affected by the BMPR2ecd-Fc treatment. A second study was conducted to investigate whether a longer treatment with 12 injections during postnatal days 4-28 would inhibit folliculogenesis. Similar effects were observed in the two studies on the early follicular developmental stages. However, in the long-term study, later stages of folliculogenesis were not blocked but rather increased numbers of antral follicles, preovulatory follicles, and corpora lutea were found. We conclude that BMPR2ecd-Fc is a potent modulator of ovarian folliculogenesis in vivo, and thus, is a valuable tool for studying the physiology and downstream effects of oocyte-derived growth factors in vivo.

Expression of growth differentiation factor-9 and bone morphogenetic protein-15 in oocytes and cumulus granulosa cells of patients with polycystic ovary syndrome.

OBJECTIVE: To evaluate the effect of growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) on the development of follicles among patients with polycystic ovary syndrome (PCOS). DESIGN: Case-control study. SETTING: University Hospital. PATIENT(S): Twenty-two oocytes were obtained from 15 patients with PCOS and 67 oocytes from 58 controls. Cumulus granulosa cells (GC) were obtained from 16 patients with PCOS and controls treated with intracytoplasmic sperm injection. INTERVENTION(S): Immunofluorescence combined with laser scanning confocal microscopy and immunocytochemistry were used to analyze the expression of GDF-9 and BMP-15 in oocytes and cumulus GCs. MAIN OUTCOME MEASURE(S): To detect the protein expression levels. RESULT(S): No significant difference was found in the expression of GDF-9 and BMP-15 in the oocytes and BMP-15 in the cumulus GCs of patients with PCOS and controls. However, the expression of GDF-9 in cumulus GCs of patients with PCOS was decreased significantly compared with controls (8.88 +/- 1.52 vs. 5.01 +/- 0.83). CONCLUSION(S): The expression of GDF-9 and BMP-15 in the oocytes of patients with PCOS who received ovulation induction treatment was in the normal range, but the GDF-9 expression in cumulus GCs from patients with PCOS was significantly lower than the normal. Reduced GDF-9 expression in cumulus GCs of patients with PCOS appears to be associated with decreased long-term developmental potential of the oocytes of patients with PCOS.