Effects Of Telomerase Activity, Apoptosis-related MRNA And Protein Expression Of Human Luteined Granulosa Cells With Ovarian Response And Embryo Quality

Endocrine function and ovulation are the physiological function of ovarian. Follicle is the basic functional unit in ovarian including not only occyte development, maturity and ovulation but also production of hormones and certain cytokines. Follicle may be the key in reproduction. With the gap junction granulosa cells can contact with oocyte closely and play an important role in follicle development and oocyte maturation. Granulosa cells are the main functional cells in synthesis and secretion of inhibin, estradiol and progesterone with high proliferation potential. From the growth, atresia of primaril follicle or ovulation to corpus luteum, granulosa cells have changed in form and function. And the mechanisms of granulosa cells proliferation, differentiation and apoptosis are extremely complex, including complex molecular mechanisms and signal transduction pathways. So the study in granulosa cells may be an excellent starting point in studying ovarian function and luteinized granulosa cells in vitro cultured may be a good method to value ovarian function and the quality of embryos.Ovarian dysfunction may lead to a variety of gynecological endocrine diseases and infertility. Adjustment of ovarian function, controlled ovarian hyperstimulation and treatment of infertility may be important in gynecology. Decreased oocytes and embryos quality because of aging or ovarian dysfunction may increase follicular atresia and lead to the lower response and pregnancy rate in controlled ovarian hyperstimulation. Hormone drugs has made significant effect, but still have limitations and side effects, especially in controlled ovarian hyperstimulation with young but lower ovarian response. In previous studies of teacher it was found that in the signal transduction pathway critical regulator p53, p19ARF, Rb and p16 changes in the collaborative in the aging of entire gonadal axis(HPOA). Preventive application of Hswy may play the role of anti-aging by inhibiting the key factor of aging in the gonadal axis. Hswy may improve and adjust the function of HPOA. However, there was no evidence of a direct impact on ovarian function.Follicular atresia may be correlated with granulosa cells apoptosis. Telomerase activity, apoptosis-related mRNA and protein expression may play important roles in granulosa cells apoptosis. But the effects of them to ovarian function and quality of embryos were not very clear. The electrical activity of granulosa cells may be a new method to study the regulation of cell function. It may represent an important signal transduction pathway through the electrical connected link between granulosa cells and oocyte. Potassium ion channel is the largest number of subtypes found in a class of membrane ion channels. It plays an important role in regulating the formation of cells action potential, membrane depolarization and maintaining the level of resting potential as well as a series of physiological activities such as secretion and nervous of muscle cells. So far, the research of potassium ion channels in proliferation, differentiation in nerve-muscle cells and tumor cell is more fully, but the research in human ovarian granulosa cell and the linked function are still not been very clear.In this study, we first isolated luteinized granulosa cells from follicular fluid of in vitro fertilization(IVF) patients. By using TRAP-ELISA, RT-PCR, immunocytochemical methods, we have detected telomerase activity and apoptosis-related genes bcl-2 and protein expression, and compared these factors with ovarian response and embryo quality. To understand Hswy on ovarian function deeply, in the second part of this study, we have used the method of serum pharmacology to study SD rats granulosa cells from preovulatory follicles in vitro. TRAP-ELISA, RT-PCR, Western blotting were used to detect telomerase activity, apoptosis rate and apoptosis-related genes bcl-2 and protein expression to explore Hswy's interventional role. Finally, by RT-PCR we found the expression of voltage-gated potassium channels (Kv) mRNA in human luteinized granulosa cell based on a small number of researches in potassium ion channel in human ovarian granulosa cells. Chemoluminescence method, MTT method, flow cytometry, spectrophotometric analysis methods were used to study the influence of proliferation, differentiation, and apoptosis by 4-aminopyridine(4-AP) through inhibiting Kv in human ovarian luteinized GC with a view to provide some theoretical basis of granulosa cell function in regulating. Our findings may provide an essential background for experimental definition of granulosa cell and may represent novel targets for assisted reproduction.PartⅠStudy on the relationship between telomerase activity, apoptosis-related mRNA and protein expression of human luteined granulosa cells with ovarian response and embryo qualityObjectiveTo explore the relationship between telomerase activity, bcl-2, p53 mRNA and protein expression of human luteined granulosa cells with ovarian response and embryo quality.MethodsOvarian luteinized granulosa cells were recovered from 28 women underwent in vitro fertilization. The following procedures were carried out: 1) luteined granulosa cells were isolated from follicle fluid and were cultured 24 hours soon. 2) tests of the telomerase activity with TRAP-ELISA. 3) tests of the expression of apoptosis-related bcl-2, p53 proteins by using immuocytochemistry. 4) tests of the expression of apoptosis-related bcl-2, p53 mRNA by using RT-PCR. Comparisons of age, number of ampules of Gn administered, number of retrieved oocytes, ratio of high quality embryo and telomerase activity, protein and mRNA expression of bcl-2, p53 were made in different ovarian response and embryo quality groups.Results1.Isolated and purified GCs were cultured and adherenced well in 24 hours.2.TRAP-ELISA showed: there were negative correlation between telomerase activity with age, number of ampules of Gn administered and positive correlation with number of retrieved oocytes (r=-0.4931, P<0.05; r=-0.4238, P<0.05; r=0.4906, P<0.05 respectively). There were significant difference of telomerase activity in different ovarian response groups (F=6.8693, P<0.01). Low response group had lower telomerase activity compared to medium and high response groups (q=4.7172, P<0.01; q=4.8770, P<0.01 respectively). There were positive correlation between telomerase activity with ratio of high embryo quality (r=0.6968, P<0.01). There were significant difference of telomerase activity in different embryo quality groups (t=3.2983, P<0.01).3.Immuocytochemistry showed: there were positive correlation between the score of bcl-2 protein expression(HSCbcl-2) and the number of retrieved oocytes (r=0.6183, P<0.01); there were positive correlation between the score of p53 protein expression(HSCp53) with the number of ampules of Gn administered and negative correlation with the number of retrieved oocytes (r=0.4237, P<0.05; r=-0.5954, P<0.05 respectively). There were difference of HSCbcl-2 and HSCp53 in different ovarian response groups(F=5.2058, P<0.05; F=13.757, P<0.01). There were positive correlation between the HSCbcl-2 and negative correlation between the HSCp53 with ratio of high embryo quality(r=0.5888, P<0.01; r=-0.6343, P<0.01 respectively). There were difference of the HSCbcl-2 and HSCp53 in different embryo quality groups (t=2.2325, P<0.05; t=3.070, P<0.01 respectively).4.RT-PCR showed: there were negative correlation between bcl-2 mRNA expression with age and positive correlation with the number of retrieved oocytes(r=-0.5667, P<0.01; r=0.4584, P<0.05 respectively); there were positive correlation between p53 mRNA expression with age, the number of ampules of Gn administered and negative correlation with the number of retrieved oocytes(r=0.4370, P<0.05; r=0.5305, P<0.05; r=-0.4439, P<0.05 respectively). There were difference of bcl-2 and p53 mRNA expression in different ovarian response groups(F=3.5653, P<0.05; F=3.4913, P<0.05 respectively). There were positive correlation between the HSCbcl-2 and negative correlation between the HSCp53 with ratio of high embryo quality significantly(r=0.6861, P<0.01; r=-0.6238, P<0.01 respectively). There were difference of bcl-2 and p53 mRNA expression in different embryo quality groups (t=3.2114, P<0.01; t=2.4763, P<0.05 respectively).Conclusions:1.Telomerase activity could be tested in human luteinized granulosa cells after in vitro cultured.2.Telomerase activity in granulosa cells were negatively correlated with age, the number of ampules of Gn administered and positively correlated with the number of retrieved oocytes. Those may suggest that telomerase activity decreased with the decline of ovarian function.3.There were obviously relevant between telomerase activity and ratio of high quality embryo, the apoptosis of granulosa cells directly affect embryo quality. There were clear correlation between apoptosis-related bcl-2, p53 mRNA and protein expression with ovarian response and embryo quality, and significant correlation with telomerase activity. Bcl-2, p53 gene which may regulate the expression of telomerase activity could be used as a bridge to study the relationship between ovarian function in our research.PartⅡEffects of serum containing Hswy on telomerase activity, apoptosis ratio and apoptosis-related protein expression of rat ovarian granulosa cellsObjectiveTo investigate the effect of Hswy decoction on function in cultured rat ovarian granulosa cells, which in the purpose to explain the mechanism that the decoction is to promote follicular development.MethodsPMSG treated female S.D. rats were used as a model and ovarian granulosa cells were prepared. Granulosa cells were cultured in groups. Two groups were set up: study group with rat serum contained Hswy decoction in different dosage and blank group with normal serum. Granulosa cells were cultured in the medium containing 10% different rat serum at 37℃. After 24 hours, telomerase activity were measured by telomeric repeat amplification protocol-enzyme linked immunoadsordent assay(TRAP-ELISA), the percentage of apoptosis and death of granulosa cells were quantitatively assessed by flow cytometry(FCM) analysis, bcl-2 and bax protein expression were assessed by Western blotting and the activity of cellular caspase-3 were measured by colorimetric method.Results1.TRAP-ELISA illustrated: the serum with medicine could increase telomerase activity, there were different in groups(F=8.4996, P<0.01 respectively); compared with blank serum group, medium and high dosage groups could increase telomerase activity obviously (q=3.9745, P<0.05; q=6.5839, P<0.01 respectively).2.FCM showed: the percentage of apoptosis in study group was less than that in blank serum group(F=13.2252, P<0.01); and the percentage of death in study group was same as that in blank serum group(P>0.05); compared with blank serum group, medium and high dosage serum groups could decrease the apoptosis of granulosa cells significantly(q=5.5814, P<0.01; q=8.3490, P<0.01 respectively).3.Western blotting showed: the serum with medicine could increase the expression of bcl-2 protein and decrease the expression of bax protein (F=20.371, P<0.01; F=13.8507, P<0.01 respectively); compared with blank serum group, medium and high dosage groups could obviously increase bcl-2 protein and decrease bax protein expression (q=5.4043, P<0.01; q=10.0812, P<0.01 and q=6.1898, P<0.01; q=7.4314, P<0.01 respectively).4.Colorimetric method illustrated: the activity of caspase-3 of granulosa cells in study groups may be lower than the blank serum group(F= 9.2098, P<0.01); compared with blank serum group, medium and high dosage serum groups could decrease the activity of caspase-3 obviously(q=5.4043, P<0.01; q=10.0812, P<0.01).Conclusions1. Hswy decoction may increase telomerase activity and decrease ratio of apoptosis of rat granulosa cells in vitro cultured.2.Hswy decoction may increase the protein expression of bcl-2 and decrease the protein expression of bax. This may be one possible mechanism inhibiting granulosa cells apoptosis and follicle atresia.3.Hswy decoction may decrease caspase-3 protein expression which may be in the earlier stages of apoptosis, by blocking the cascade of caspase-3. 4.Adjustment of follicular local micro-environment may be one target of Hswy decoction function in hypothalamus - pituitary - ovarian axis.PartⅢInfluence of 4-aminopyridine on human ovarian luteinized granulosa cells proliferation, differentiation, and apoptosis through inhibiting voltage-gated K+ channelObjectiveTo study the influence of proliferation, differentiation, and apoptosis by 4-aminopyridine(4-AP) through inhibiting voltage-gated K+ channel(Kv) in human ovarian luteinized granulosa cells.MethodsOvarian luteinized granulosa cells were recovered from 25 women with regular menses who underwent in vitro fertilization programmed. The cultured granulosa cells were divided in 4 groups:blank group, 4-AP treated group, human chorionic gonadotropin(hCG)-induced group and hCG+4-AP co-treated group, and the final concentration of hCG and 4-AP were 1250 IU/L, 5 nmol/L respectively. The progesterone production was detected by chemoluminescence method. The expression of Kv mRNA on human ovarian luteinized granulosa cell was detected by reverse transcription polymerase chain reaction(RT-PCR). The influence on the early apoptosis of granulosa cells by 4-AP was observed by flow cytometry, cellular caspase-3 activities were observed with colorimetric method and the inhibition of the cell proliferation was studied using methyl thiazolyl tetrazolium(MTT) method.Results1.Kv mRNA were expressed in human luteinized granulosa cell of the blank group, 4-AP treated group, hCG-induced group and hCG+4-AP co-treated group.2.The progesterone production of the blank group, 4-AP treated group, hCG-induced group and hCG+4-AP co-treated group were (173.9933±15.9642), (64.3867±8.7411), (628.2333±54.1370) and (240.5867±24.8367)ng/ml respectively after 24 h cultured. Exposure of the granulosa cells to 4-AP reduced the differentiation of progesterone in blank and hCG-induced granulosa cells.3.24h after treated with 4-AP and hCG, the A value of MTT method of blank group, 4-AP treated group, hCG-induced group and hCG+4-AP co-treated group were 0.633±0.138, 0.508±0.116, 0.661±0.159 and 0.432±0.145 respectively. The inhibitory rate by MTT method of cultured granulosa cells of 4-AP treated group was higher than the blank group (19.7% vs. 0) (t=2.6871, P<0.05), hCG+4-AP co-treated group was higher obviously than hCG-induced group (34.6% vs. 0) (t=4.1422, P<0.01).4.The flow cytometry analysis and the cellular caspase-3 A405 showed that 4-AP increased the percentage of early phase apoptosis. 4-AP treated group vs. blank group [(40.13±4.85)% and 0.0494±0.00939] vs. [(17.24±3.69)% and 0.0286±0.00759] (t=14.5630, P<0.01; t=6.6594, P<0.01), 4-AP+hCG co-treated group vs. hCG-induced group[(24.86±4.13)% and 0.0387±0.00794] vs. [(14.55±3.19)% and 0.0219±0.00663] (t=7.6481, P<0.01; t=6.2742, P<0.01).Conclusions1.The voltage-gated K+ channels mRNA may be expressed in human ovarian luteinized granulosa cell.2.4-AP may inhibit production of progesterone in base and hCG-induced granulosa cells.3.4-AP may inhibit the proliferation of granulose cells and induce apoptosis of granulose cells. The effect of 4-AP to production of progesterone may be due to the inhabitation of proliferation and induction of apoptosis.4.4-AP may play an important role in cell proliferation, differentiation and apoptosis.To sum up, our study found that: (1) Telomerase activity could be tested in human luteinized granulosa cells after in vitro cultured. These may suggest that telomerase activity, apoptosis-related bcl-2 mRNA and protein expression decreased with the decline in ovarian function and embryo quality. (2) Hswy decoction could improve ovarian function by inhibiting apoptosis, increasing telomerase activity, the protein expression of bcl-2 and decreasing the expression of bax. (3) The voltage-gated K+ channels mRNA may be expressed in human ovarian luteinized granulosa cell. Due to the inhabitation of proliferation and induction of apoptosis of granulosa cells, 4-AP may inhibit production of progesterone in hCG-induced granulosa cells. Voltage-gated K+ channels may play an important role in granulosa cells proliferation, differentiation and apoptosis.