The Protective Effect Of Anti-Growth Differentiation Faotor-9 On Chemotherapy-induced Ovarian Damage In Rats

BackgroundPremature ovarian failure (POF) is defined as amenorrhea (>12 months), elevated gonadotropin level(FSH>40IU/L) and decreased estrogen level(<25pg/ml) before 40 years old. The patients may have the symptoms of estrogen deficiency: such as loss of libido, hot flashes, sweating, osteoporosis, amenorrhea, and even infertility. The morbility of POF is 1% before 40 years old, and 1‰before 30 years old. POF accounts for 20% to 25% in primary amenorrhea and accounts for 10% to 20% in secondary amenorrhea. The mechanism is not clear yet. But there are some potential reasons:①Abnormal genes:such as the deletion, translocation, and point mutations of x chromosome;②Immunization:about 10% to 15% of POF are related to immune factors;③Infection:such as pelvic inflammatory disease, mumps, rubella and measles virus infection;④Enzymes deficiency:studies confirmed that lacking of 7-αhydroxylase or 17,20 carbon-chain cleavage enzyme was associated with POF;⑤atrogenic factors:radiotherapy, chemotherapy, etc.;⑥Poisons;⑦Pychological factors;⑧Other reasons.The patients who have malignant tumors or autoimmune diseases can survive because of the widely used of chemotherapy drugs.But the ovarian damage caused by chemotherapy will lead to a series of physical, psychological and social problems to the patients especially to the young women. The chemotherapy-induced POF is focused by researchers, and how to protect the ovarian function is a hot spot in clinic. The mechanism of ovarian damage caused by chemotherapeutics is not clear yet. Some recent researches have proved that it was closely related to the apoptosis of ovarian granulosa cells and oocytes. The severity of ovarian damage caused by chemotherapy is closely related to category, total dose of chemotherapeutics and the patient s age. The studies found that chemotherapeutics mainly damaged the active cells in mitosis. The alkylating agents are the mainly drugs that damage the ovaries. Cyclophosphamide (CTX) is the most commonly used drug of the alkylating agents. The damage to the ovary is more serious by the greater dose and cumulative dose of the chemotherapy drugs. Leading to POF required larger dose of chemotherapy drugs to the younger patients.There are some methods to prevent the chemotherapy-induced POF:①Drugs:for example, the GnRH-a, oral contraceptives, and so on;②Frozen ovaries or embryos or oocytes. The gonadotropin releasing hormone analogues (GnRH-a) is mainly used to prevent the chemotherapy-induced ovarian damage, not cure. It can not be long-term used because of the complications. And it is expensive, so it can not be widely used. Currently the hormone replacement is the main method of treatment, but estrogen can not be used in the patients with hormone sensitivity tumors such as breast cancer and endometrial carcinoma. The cryopresevation technique remains immature.Growth differentiation factor-9 (GDF-9) is a cytokine that secreted by oocytes. It is confirmed that GDF-9 plays an indispensable role in follicle development. Studies confirmed that GDF-9 mainly make effect in the primary to secondary follicle transition. GDF-9 plays an important role in follicle growth and differentiation through paracrine. It decreased the growth and the mitogenic activity of oocytes. One study has found that cultured human ovarian follicles within slices of ovarian cortical tissue in the presence or absence of recombinant rat GDF-9, in serum-free cultures after 7 d,53% of the follicles had reached the secondary stage with GDF-9 vs.31% in the control group, follicle viability was also improved in the presence of GDF-9 after 7 d in culture. Recent researches have proved that GDF-9 can make a great quantity of primordial follicles to start growth, decrease follicular atresia and make follicles to survive. Shimizu T found that injected porcine GDF-9 gene fragments into the ovary in gilts resulting in an increase in the number of primary, secondary and tertiary follicles, concomitant with a decrease in the number of primordial follicles. Reduced the expression of GDF-9 in preantral follicle to antral follicle transition period by RNA interference, the development of preantral follicles and the follical growth induced by FSH were blocked. Follicles blocked at the primary stage in mice when the GDF-9 gene knockout. It could not form a dominant follicle and resulte in infertility. Gilchrist found that a GDF-9 neutralizing antibody, mAb-GDF9-53, could decrease the mitogenic activity of oocytes and make ovarian follicles stagnate in the primary follicle stage. The relationship between GDF-9 and POF is a hot spot in clinic. A missense mutation has been found in POF patients, so the missense mutation of GDF9 may result in POF.The cytotoxic of the chemotherapeutics is larger in the follicles that in the division and proliferation stage than in the quiescent stage. The alkylating agents are the most commonly used drugs that damage the ovaries especially cyclophosphamide (CTX). Glyciphosphoramide(PM) is the final metabolite of CTX. PM is active in vitro. So we study that when chemotherapy is priticed, give the GDF-9 neutralizing antibody to block the follicles at the primary follicle stage can against chemotherapy-induced ovarian damage of rats in vitro or not.Chapter 1 Study an appropriate dose of glyciphosphoramide in rat ovaries in vitroObjective:To find an appropriate dose of Glyciphosphoramide(PM) that will cause ovarian damage of female rats in vitro. Method:There were four groups, normal control group and three experimental groups,12 ovaries each group. Ovaries were obtained from 8 week wistar rats(weighting 180 to 200 g). To culture the ovaries in 24 hole culture plates, one ovary each hole. All ovaries of the four groups were added culture medium (a-MEM+10%FBS+0.075IU/ml hMG+1%ITS+1% penicillin and streptomycin) only first, then replaced culture medium after 24 h Now incubated with PM at concentrations of 10,20, and 50 micrograms/ml to experimental groups, normal control group ovaries were also added with culture medium only, then replaced culture medium(no PM) every two days. All ovaries were cultured for 7 days(6 days after given PM). Estradiol(E2) were measured by radio-immunity method before and at day 2,6 after given PM. Weighting, HE staining and counting the number of follicles of each ovary at day 7after culture(6 days after given PM). Result:①There were no difference in E2 lever between every two groups of the four groups before given PM (P>0.05). There showed significant difference in E2 lever between every two groups at day 2 after given PM (P<0.05) The E2 lever of the three experimental groups(11.99±0.82 pg/ml; 9.32±0.65pg/ml; 4.53±1.04 pg/ml) were lower than the normal control group (77.27±2.13 pg/ml). At day 6 after given PM, there also showed significant difference in E2 lever between every two groups (P<0.05). The E2 lever of the three experimental groups (20.90±5.48 pg/ml; 16.24±6.07 pg/ml; 7.47±2.00 pg/ml)were lower than the normal control group (89.23±5.96 pg/ml) too.②There were obvious difference in follicular numbers between every two groups at day 6 after given PM (P<0.05). The follicular numbers of the three experimental groups (64.50±16.17; 28.63±13.15; 19.42±8.39) were fewer than the normal control group (100.15±9.18). Conclusion:The lowest concentration of the PM (10μg/ml) in our study can cause ovarian damage of female rats in vitro.Chapter 2 The treatment of Anti-Growth differentiation faotor-9 on chemotherapy-induced ovarian damage in vitro.Objective:To study the treatment of Anti-Growth differentiation faotor-9 (Anti-GDF-9) on Glyciphosphoramide(PM) induced ovarian damage in female rats in vitro. Methods:28 ovaries (14 Wistar rats) were divided into four groups, such as normal control group(n= 6), PM group(n= 6),saline group(n= 6), PM+Anti-GDF-9 group(n=10). To culture the ovaries in 24 hole culture plates? one ovary each hole. The ovaries of the normal control group were added culture medium only, the ovaries of the saline group were added saline(containd 0.075IU/mlhMG). The ovaries of the PM+Anti-GDF-9 group were added culture medium(containd 10μg/ml PM) first then given Anti-GDF-9 (5μg/ml) after 24h. Replaced culture medium(no PM) or saline every other day. The ovaries of the normal control group, the PM group and the saline group were cultured for 8 days. The ovaries of the PM+Anti-GDF-9 group were cultured for 8days(6 ovaries) or 14 days(4 ovaries). Ovaries were removed at day 8 or day 14, HE staining and counting the number of follicles of each ovary Results:①There were obvious difference in follicular numbers between every two groups at day 8 after cultured (P<0.05). The follicular numbers of the PM+ Anti-GDF-9 group (39.90±7.52) were fewer than the normal control group (55.92±11.78) but more than the PM group(32.38±13.37) and the saline group(12.21±4.82).②There were no difference in follicular numbers between day 8(39.67±7.32)and at day 14(40.37±8.59) of the Anti-GDF-9 group. Conclusion: Anti-GDF-9 administerd after PM for 24h can not diminished PM induced ovarian damage in ovaries in vitro. And the ovarian function can not restore by time.Chapter 3 The protective effect of Anti-Growth differentiation faotor-9 on PM-induced ovarian damage in rats.Objective:To study the protective effect of Anti-Growth differentiation faotor-9 (Anti-GDF-9) against Glyciphosphoramide(PM) induced ovarian damage on rats in vitro. Through above experiment we know that Anti-GDF-9 administerd after PM can not diminished PM induced ovarian damage, so next experiment we administer Anti-GDF-9 before PM for 24h. Then study the Anti-GDF-9 can against PM induced ovarian damage in rats or not. Methods:96 ovaries (48Wistar rats) were randdomly divided into three groups,32 ovaries each group, such as normal control group, PM group, experimental group. To culture the ovaries in 24 hole culture plates, one ovary each hole. First, normal control group and PM group ovaries were added with culture medium only, experimental group ovaries were added culture medium containing Anti-GDF-9(5μg/ml). Replaced culture medium of the three groups after 24h. Then normal control group ovaries were also added culture medium only, PM group and experimental group ovaries were added culture medium containing 10 micrograms/ml PM. After that replaced culture medium(no PM) every two days for all ovaries. All ovaries were cultured for 7 days(6 days after given PM). Estradiol(E2) were measured by radio-immunity method before and at day 2,4,6 after given PM. At the same time,8 ovaries were removed separately, HE staining and counting the number of follicles of each ovary.Results:①There was no difference in E2 lever among three groups before given PM (P>0.05). There showed no difference in E2 lever between the experimental group (94.82±85.24 pg/ml) and the normal control group(85.66±20.57 pg/ml) (P=0.987), but there was obvious difference between the experimental group and the PM group(7.63±7.01 pg/ml) (P=0.000) at day 2 after given PM. At day 4 there showed no difference in E2 lever between the experimental group (43.39±10.45 pg/ml) and the normal control group(66.88±11.33 pg/ml) (P=0.050), but there was sinificant difference between the experimental group and the PM group(6.29±4.84 pg/ml) (P=0.000). There showed no difference in E2 lever between the experimental group (41.71±17.03 pg/ml) and the normal control group(52.36±15.00 pg/ml) (P=0.203), but there was difference between the experimental group and the PM group(11.24±8.39 pg/ml) (P=0.000) at day 6 too.②There showed no difference in the number of follices among three groups before given PM (P>0.05). There was obvious difference between the experimental group(67.50±9.90)and the PM group(20.50±7.83) at day 2 after given PM, there also showed difference between the experimental group and the normal control group(67.50±9.90) at the same time. At day 4 there showed no difference between the experimental group (56.50±14.57) and the normal control group(66.88±11.33) (P=0.088), but there was sinificant difference between the experimental group and the PM group(20.50±7.83) (P=0.000). There showed no difference in the number of follices between the experimental group (49.25±9.08) and the normal control group(48.75±11.51) (P=1.000), but there was sinificant difference between the experimental group and the PM group(12.88±5.87) (P=0.000) at day 6.Conclusion: Anti-GDF-9 administerd before PM for 24h can diminished PM induced ovarian damage in ovaries in vitro. But we don't know that Anti-GDF-9 has the same effect or not when administerd a larger doses of PM. And Anti-GDF-9 whether has the same effect in vivo is not clear now. We also don't know that Anti-GDF-9 can be used in people or not. So further studies are needed.