| With the approaching of aging society, the healthy issues of the aged women have drawn more and more attentions these years. Especially for women, ovarian aging is obviously faster than other organs.Ovarian aging can have profound effect on reproductive health, inducing early menopause, cancer, cardiovascular diseases, neurodegenerative diseases and some other chronic problems. Furthermore, with rapid economic development and society transfiguration dynamics, such as the implementation of two-child policy, elder women have higher expectations for beter life quality, longer life span as well as better fecundity. Therefore, it has become a research impendency to clarify the ovarian physiological as well as the pathological hypofunction/dysfunction and then construct an efficient intervention means. Expecially, the discovery of ovarian germline stem cells brings hopes for the effective increasing of primordial pool, improvement of the ovarian function and slowing down the ageing pace of ovaries as well.Hippo signaling pathway is an important way to regulate the proliferation and differentiation of the stem cells. In our previous research, we found that the main effector molecules of the Hippo signaling pathway can trigger the primordial follicle proliferation. But it still remains unknown whether the Hippo signaling pathway can play any role in the regulation of ovarian germline stem cells(OGSCs) and if it has a close relationship with the ovarian physiological and pathological ageing. So in this study, KM mice and human ovarian cortex tissue were choosed as our experimental subjects:(1) analyze the expression and dynamic change of the main effector molecules of the Hippo signaling pathway in the ovarian surface epithelium(OSE) as well as the relationship between the dynamic changes and the physiological and pathological aging of the ovarian functions;(2) Observe the function of the main effector molecules YAP1 of Hippo signaling pathway during the OGSCs’ proliferation and differentiation in vitro;(3) Then analyze the how YAP1 can help the ovarian physiological and pathological after it is microinjected into the ovary in vivo. According to the experimental statistics, we can get more evidence on the mature mammal’s ovarian germline stem cells’ self-renewal, differentiation, and senescence. Also, it helps to provide new treatment and drug targets to the infertility, remodeling and protection of ovaries.Chapter 1 The ovarian germline stem cells(OGSCs) and Hippo signaling pathway association with physiological and pathological ovarian aging in mice and womenObjectiveTo clarify the expression and dynamic changes of the main effector molecules of Hippo in the development of OGSCs during physiological and pathological ovarian aging in mice and women.MethodsSelect mice ovaries from the 7 day’s(7D), 2 months’(2M), 10 months’(10M) and 20 months’(20M) and woman ovarian cortex samples from the child-bearing period’s(adolescence, puberty-38 years old), the middle ages’(middlescence, 36-50 years old) and the senescence period’s(51-60 years old) as the physiological ovarian aging samples. Mice were given a gavage of Tripterygium Glycosides(TPT) and intraperitoneal injected with Cyclophosphamidum/Busulfan(CY-BUS) to construct pathological retrogressed ovaries models. Compare the expression and dynamic changes of different effector molecules of the Hippo signaling pathway(LATS2, MST1 and YAP1) in the OSE cells as well as the relationship between them.Results(1) Relative Hippo signaling pathway molecules and OGSCs markers(MVH and OCT4) can both express in the mice’s OSE cells from different ages. The expression of tumor suppressor gene LATS2, MST1 and MVH/OCT4 will be lower with the increasing age; proto-oncogene YAP1 express most in the OSE cells of 2M mice, then 7D mice and least in the 20 M mice; The expression of p YAP1 are at the highest/summit in the 7D mice. What’s more, the value of the p YAP1/ YAP1 will be gradually decreased with the increasing age;(2) In the retrogressed ovaries of mice models, Hippo signaling pathway relative molecules and OGSCs markers(MVH and OCT4) can also express together. The expressions of YAP1, MST1 and MVH/OCT4 are all lower gradually after TPT and CY/BUS treatment. However, the expression of LATS2 is in the crosscurrent. The expression of p YAP1 is very low in the TPT- and CY/BUS-treated groups, while the value p YAP1/ YAP1 increase somehow.(3) In the human ovarian cortex OSE cells from different age periods, Hippo signal pathway relevant molecules and OGSCs markers(MVH and OCT4) can also express themselves together. The value of MVH/ OCT4 will decrease gradually with the increasing age. LATS2 expresses most during the adolescence period. However, YAP1 and p YAP1 express at the highest during the middlescence groups while MST1 express at the lowest in the senescence group. One on mention, experimental statistics shows that p YAP1/YAP 1decreases gradually from adolescence to senescence.ConclusionHippo signaling pathway relevant molecules LATS2, MST1, YAP1 and OGSCs markers(MVH and OCT4) were found all expressed in the mice’s OSE cells as well as the women’s. Both expression changes in two species are relevant with time and sequences as well as ovarian physiological recession and pathological recession. Based on statistics, Hippo signaling pathway may be involved in the development schedule of OGSCs during physiological and pathological ovarian aging in women and mice.Chapter 2 Hippo signaling pathway in the ovarian germline stem cells’ proliferation and differentiationObjectiveTo get a better idea about separating, identifying and cultivating ovarian germline stem cells(OGSCs); exploring the function of Hippo signaling pathway in the OGSCs’ proliferation and differentiation in vitro.MethodsThe two-step enzymatic separation processes and mangnetic separation were used to isolate and identify OGSCs by determining the expression of MVH, OCT4, Nango, Fraglis and Stella markers. Then, isolated OGSCs were transfected onto the high-expression/ interference lentiviral vector YAP1 to observe the proliferation and differentiation of OGSCs.Results(1) The specific expression of MVH, OCT4, Nango, Fraglis and Stella markers were assessed in isolated stem cells. Thus, the isolated cells were preliminary identified for OGSCs.(2) The co-expression of LATS2, MST1, YAP1 and MVH were observed on isolated OGSCs. After the OGSCs are transfected with over-expressed YAP1, the expressions of the specific markers MVH and OCT4 are obviously increased. While OGSCs are transfected with YAP1 sh RNA interfering lentivirus, the expressions of MVH and OCT4 are decreased.ConclusionThe isolation, identification and culture methods of OGSCs were successfully established. These results indicated that the isolated OGSCs can specifically recognize Hippo signaling molecules, which can take part in the proliferation and differentiation of OGSCs.Chapter 3 The first exploration of the Hippo signaling pathway in the mice ovarian function remodeling by regulating the function of ovarian germline stem cellsObjectiveTo elucidate of Hippo signaling pathway in regulating the OGSCs’ proliferation and differentiation and the remodeling function.MethodsTake the main Hippo signaling pathway effector molecule YAP1 as the target gene, then inject the vectors with over-expressed YAP1 and relevant sh RNA interfering lentivirus both into the infertility models as well as the mice ovaries in the child-bearing period. Finally observe the reproductive functions and endocrine functions of the ovaries.Results(1) The follicles’ regeneration and the primordial follicles’ activation are detected in the ovaries after the over-expressed YAP1 is transfected in the infertility models in 30-75 days, which can induce offspring and the high level of E2 and FSH. Meanwhile, the level of MVH, OCT4, YAP1 and p YAP1 were higher in mice OSE with YAP1 overexpression.(2) The ovarian follicles decrease after the YAP1 sh RNA interfering lentivirus vectors are transfected in the mice of 30-75 days, which show high fecundity in default. The littering ability also decreases as well as the level of E2 and FSH in the serum. Furthermore, the expression of YAP1, p YAP1, MVH and OCT4 all are decreasing obviously.ConclusionThe Hippo signaling pathway key player, YAP1, can regulate the proliferation and differentiation of the OGSCs and thus it can play an important part in the ovarian function remodeling. |
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