Exogenous secreted frizzled-related protein-4 modulates steroidogenesis of rat granulosa cells through Wnt/beta-catenin and PI3K/AKT signaling pathways

Background: It has been reported that secreted frizzled-related protein-4 known as an antagonist of Wnt signaling pathway plays a role in luteinization process of rodent granulosa cells. The purpose of this study was twofold: 1] to determine whether recombinant human secreted frizzled-related protein-4 [rhSFRP-4] could directly induce terminal differentiation of rat Granulosa Cells [GCs] and 2] to understand how the modulation of beta-catenin and Protein Kinase B [PKB]/AKT activity by exogenous SFRP-4 could be involved in steroidogenesis

Methods: GCs were firstly stimulated with Follicle-Stimulating Hormone [FSH] named as FSH-primed cells then were treated with luteinizing hormone [LH]. Then estradiol [E[2]] and progesterone [P[4]] production levels were assessed in the absence or presence of rhSFRP-4 treatment. The expression levels of activated beta-catenin, pAKTser[473], pGSK3 beta ser[9] were assessed by western blot or immuno-fluoresence

Results: In the presence of rhSFRP-4, there was 38% decreased E[2] levels compared to untreated FSH-primed cells [p<0.05], and P[4] production subsequently decreased. However, in GCs pre-treated with rhSFRP-4 prior to addition of FSH, P[4] levels increased 2-fold compared with untreated cells [p<0.05]. Unexpectedly, treatment with rhSFRP-4 prior to LH stimulation inhibited LH-induced P[4] secretion. Treatment with low [0.5 ng/ml] but not high [50 ng/ml] concentrations of rhSFRP-4 led to significantly increased levels of pGSK3 beta ser[9] [1.6-fold] and nuclear active beta-catenin [2.8-fold] in GCs compared with untreated cells. Interestingly, pre-treating GCs with rhsFPR4 prior to LH stimulation resulted in a 38% decrease in pAKTser[473] levels compared with those in LH-treated cells [p<0.05]

Conclusion: Taken together, our results showed that rhSFRP-4 could directly induce terminal differentiation in GCs via the modulation of beta-catenin and PKB/AKT pathways and that it does so in a dose-dependent manner

Combined treatment of androgen-independent prostate cancer cell line DU145 with chemotherapeutic agents and lithium chloride: effect on growth arrest and/or Apoptosis

Hormone-independent prostate cancer cell lines are resistant to antineoplastic drugs, this study sought to determine the usefulness of lithium chloride as an inhibitor of glycogen synthase kinase-3 beta to increase the cytotoxic effect of doxorubicin, etoposide or vinblastine antineoplastic drugs on DU145 cells. Combination effect was assessed by using low and IC50 doses of drugs+lithium chloride. Subsequently, cell cycle analysis and p53 levels and its subcellular localization as a key regulator of cell cycle were assessed. Lithium chloride showed cytotoxic effect in a dose and time dependent manner [p<0.001]. Both drugs doxorubicin and etoposide in combination with lithium chloride [LiCl] showed higher percent of cells in SubG1 compared to control [p<0.001]. Combination of IC50 dose of doxorubicin and lithium chloride led to S phase arrest [p<0.001, compared to control, lithium chloride or doxorubicin alone]. Moreover, G2/M arrest was significantly increased when low dose of doxorubicin and vinblastine were combined with lithium chloride [p<0.001, com-pared to control and lithium chloride alone]. DU145 cells were highly sensitive to vinblastine and no significant changes were observed when combined with lithium chloride. The IC50 doses of all three drugs combined with lithium chloride demonstrated decreased cell percent in G1 phase compared to control or lithium chloride alone [p<0.001]. Moreover, in the presence of lithium chloride there were increased levels of p53 in cytoplasm and nucleus [p<0.05]. Our results suggest that combination of lithium chloride with chemotherapeutic agents may increases their cytotoxic effect on hormone non-responsive human prostate cancer cells

[Mechanism of lithium's effect on follicular development of the rat ovary]

It has been shown that lithium chloride [LiCl], an effective drug for the treatment of bipolar disorder, has side effects on the female reproductive system. In this study, cellular and histological effects of lithium chloride on the development of ovarian follicles in immature female rats were investigated. To induce ovarian follicular development, twenty-three day old immature female rats were injected with 10 IU of pregnant mare serum gonadotropin [PMSG], followed by four doses of LiCl [250 mg/kg/dose] each injected every 12 hours, starting from the time of the PMSG injection. The rat ovaries were removed 48 hours after the PMSG injection and prepared for histological, immunohistochemical, and DNA laddering studies. Control immature female rats received only PMSG, while sham treated rats received PMSG and physiological serum [lithium vehicle]. Our results showed that in the ovaries of LiCl-treated rats there were neither large antral follicles [800-1000 micro m] nor fewer medium sized follicles [400-800 micro m] but a increased number of atretic follicles compared to those in the control rats. The induction of atresia in the ovaries of LiCl-treated rats was further confirmed by the presence of DNA fragmentation. Looking at the cellular levels, lithium extremely significant [p<0.0001] increased the number of TUNEL-positive cells in the granulosa layer of the antral follicles. Taken together, our results suggest that lithium may decrease folliculogenesis by inducing apoptosis in the antral follicles

Comparing serum basal and follicular fluid levels of anti-Mullerian hormone as a predictor of in vitro fertilization outcomes in patients with and without polycystic ovary syndrome

The prediction of in vitro fertilization [IVF] outcomes by anti-Mllerian hormone [AMH] measurement is getting increasing attention from clinicians. This study compares the relationship between serum or intrafollicular AMH levels and IVF outcomes in women with and without polycystic ovary syndrome [PCOS]. This prospective study was carried out in two university-based fertility clinics. Serum samples were collected on cycle day 3 and follicular fluid [FF] was collected on the day of oocyte retrieval from 26 women with PCOS and 42 normo-ovulatory controls. AMH levels were measured in the samples using immunoenzymatic assay. The relationship between serum or FF AMH levels and IVF outcomes, including number of oocytes retrieved, oocyte maturation rate, fertilization rate, implantation rate, high quality grade embryo rate, and bio-chemical and clinical pregnancy rates were further assessed. Median serum basal AMH and FF AMH levels were significantly higher in the PCOS group as compared to controls, the values being 14.2 ng/mL vs. 3.2 ng/mL [P<0.001] and 8.2 ng/g protein vs. 4.7 ng/g protein [P,.01], respectively. In both groups, serum basal AMH levels showed a positive correlation with number of positive relationship between serum basal AMH levels and percentage of matured oocytes [r=0.331; P=0.32] and implantation rate [r=0.305; P=.05]. Serum basal, and not intrafollicular, AMH levels may be a good predictive factor for quantitative and qualitative IVF outcomes in normo-ovulatory, but not in PCOS patients

[ relationship between GSK3 beta and beta-catenin proteins with apoptotic events in normal and induced polycystic ovaries in rats]

Wnt signaling pathway plays an important role in folliculogenesis of rodent ovaries; however, its involvement in ovarian apoptotic events remains undetermined. With respect to the importance of apoptosis in homeostasis and ovarian biological function, this experimental study was conducted to determine the effects of Wnt/ beta -catenin signaling on follicular growth arrest and apoptosis in polycystic ovary [PCO] models of rats. The experiments were performed in three independent series and with each set of experiments, 8 rats were allocated to the group, half of them as the controls and the other four as the testosterone propionate [TP]-treated rats for the indicated period of time [1 and 4 weeks]. Induction of PCO in immature rats was performed by daily injection of testosterone propionate [TP] dissolved in sesame oil over 1 and 4 weeks in the experimental group but to the control group solvent was injected. At the end of the experiments, the ovaries were fixed and sequential paraffin slices were prepared for immunohistochemical analyses of GSK3 beta, beta -catenin and pGSK3 beta ser9 proteins. Assessment of Sfrp4 expression as an antagonist of Wnt signaling pathway was performed by Western blot test. Analysis of apoptosis was done by TUNEL staining, followed by quantification of apoptotic follicles in the different groups. The data were analyzed by using Mann-Whitney U-test and a p-value <0.05 was considered significant. Histological analysis of TP-treated rats showed cystic follicles, absence of corpus luteum and anovulation. GSK3 beta expression in apoptotic follicles of PCO-induced and control groups was observed. In addition, co-localization of nuclear beta -catenin and pGSK3 beta ser9 in 1-week-treated rats was detected. In long-term TP-treated rats, there was an increase in apoptosis and GSK3 beta expression and a 5.1 fold increase in Sfrp4 expression in granulosa cells, compared with the control group, which may explain the absence of nuclear beta -catenin in these cells. The results show testosterone propionate injections induces PCO in immature rats. in long-term treatment Furthermore, the increased expression of Sfrp4 and GSK3 beta with TP was associated with apoptosis. These results may reveal Wnt signaling inhibition in apoptotic events of rodent ovaries