| Ovarian aging is a biological phenomenon that interacts and gradually accumulates by a variety of physiological and external factors.The constant decline in the number of follicles caused by complex physiology and external factors is a key cause of physiological and pathological ovarian senility,which can lead ovarian infertility and menopause.According to the prediction of the World Health Organization,the reproduction will become the third most important disease in the world.Ovarian aging can also directly affect reproductive health,triggering menopause and chronic diseases such as cardiovascular,cerebrovascular and neurodegenerative diseases.However,ovarian function is not only regulated by the hypothalamus-pituitary-ovarian axis,but also by the complex regulatory network composed of numerous growth factors,cytokines and hormones inside and outside the body,as affecting the growth and development of ovarian follicles,and the degradation of steroid hormones.Synthesis and so on.Recently,a large number of studies have shown that members of the transforming growth factor-?(TGF-?)superfamily participate in the regulation of many physiological and pathological processes in the body,and the TGF-?/Smad signaling pathway is closely related to ovarian follicular development.However,there has been no report on the relationship between TGF-?/Smad signaling pathway and physiological and pathological ovarian function decline.This topic has become a research hotspot in reproductive medicine by observing the expression and dynamic changes of the main factors of TGF-?/Smad signaling pathway in physiological and pathological ovarian function decline,in order to improve and delay ovarian function decline.Lay the foundation for delaying fertility.In this study,vaginal exfoliated cell smears were used to select 3 months old(10 rats)with normal estrous cycle and >12 month old(5 rats)Sprague-Dawley(SD)rats with disordered estrus cycle.Randomly divided into control group(C group,5),chemotherapy group(H group,5)and menopause group(G group,5).Rats in H group were intraperitoneally injected with cisplatin 2 mg/kg for 7 days.Rats in groups C and G were injected intraperitoneally with an equal volume of normal saline for 7 days.The animals were weighed and sacrificed at the later stage of estrus.The wet weights of both ovarian tissues were weighed.The ovarian index was calculated for each group.Left ovarian tissue was fixed for paraffin sectioning.HE staining for observing the changes in ovarian morphology and sinusoidal follicles to evaluate ovarian function in each group.The expression of TGF-?/Smad signaling pathway in each group of ovarian tissue proteins was detected by immunohistochemistry.The right overian tissue was rapidly frozen in liquid nitrogen to extract total RNA from tissues.The factors in the TGF-?/Smad pathway were detected by real-time fluorescence quantitative PCR.According to the results of ovarian organ index in each group,compared with group C,the index of ovarian organ in group H(P<0.05)and group G(P>0.05)decreased;compared with group G,group H(P>0.05).Ovarian organ index also decreased.ELISA examination of serum E2 and FSH levels in rats showed that serum E2 levels in H group and G group were significantly lower than those in group C(P<0.01),and FSH levels were significantly higher(P<0.01);In comparison,serum E2 levels in group G decreased(P<0.05),and FSH levels increased(P<0.01).HE staining of ovarian tissue paraffin sections showed differences in ovarian structure among the groups.In group C,follicles at all levels were actively growing,a small number of closed follicles were observed,and a corpus luteum was seen in the ovarian cortex.Compared with group C,the number of growth follicles in group H and group G was significantly reduced,the connective tissue of ovarian interstitial fibrosis was proliferated,and atresia follicles in the ovary were significantly increased,but there were fewer atresia follicles in group G than in group H.Luteal reduction.At the same time,the results of follicle count analysis showed that the number of follicles and corpus luteum in group C,group H,and group G decreased gradually,and that in group H and group G was significantly less than that in group C(P<0.01),the number of atresia follicles in group H was higher than that in group C(P <0.01).The TGF-?1,TGF-?RI(T?RI),Smad2(P-Smad2),Smad3(P-Smad3),Smad4,and Smad7 TGF-?1/Smad signaling pathways were all expressed in rat ovarian tissues.Proteins were positively expressed in follicular granulosa cells and cytoplasm of oocytes,and showed brown-yellow positive products in the cytoplasm of various follicle cells.Smad3 and P-Smad3 were positively expressed in the nucleus of the corpus luteum cells.Compared with group C,the expression of TGF-?1 protein and m RNA in the ovary of group H and group G was significantly lower(P<0.01).The difference between group H and group G was not statistically significant.Compared with group C,the expression of TGF-?RI protein and m RNA in the ovary of group H was decreased(P<0.05),and it was higher in group G than in group C(P<0.05)and group H(P<0.01).Compared with group C,the expression of Smad2 protein and m RNA in the ovary of group H was significantly increased(P<0.01),while the difference in group G was not statistically significant,and group G was higher than group H(P<0.01).),m RNA decreased(P>0.05).Compared with group C,the expression of Smad3 protein and m RNA in the ovary of group H was significantly lower(P<0.05).The difference was not statistically significant in group G.There was no statistically significant difference between group G and group H protein..Compared with group C,the expression of Smad4 protein and m RNA was significantly decreased in group H(P<0.01)and in group G(P>0.05).The protein and m RNA of group G was higher than that of group H(P<0.01).).Compared with group C,the expression of Smad7 protein and m RNA in the ovary of group H was significantly lower(P<0.05),and protein and m RNA in group G was higher than that in group H(P<0.01).Compared with group C,the levels of P-Smad2 and P-Smad3 protein in group H were increased(P<0.05),the level of P-Smad2 protein in group G was decreased(P<0.05),and the level of P-Smad3 protein was increased(P<0.05).The P-Smad2 protein level in the G group was lower than that in the H group(P<0.01),and the P-Smad3 protein level was increased(P<0.01).The results of this experiment showed that cisplatin,a chemotherapeutic agent,caused damage to the ovary,leading to impaired ovarian tissue structure and follicular function.Physiological and pathological ovarian endocrine function are reduced,and then affect the regulation of ovarian function.TGF-?1/Smad major effector molecules are widely expressed in ovarian tissues and participate in the intrinsic regulation of ovarian function decline,and are related to the growth,development and apoptosis of follicles.During the decline of ovarian function,TGF-?1/Smad signaling pathway participates in the pathological ovarian function decline,mainly through down-regulating the expression of TGF-?1/Smad3 signaling pathway factors and activating the expression of Smad2 in the follicle cytoplasm.The TGF-?1/Smad signaling pathway may be involved in the regulation of Smad7 compensatory inhibition pathway in physiological ovarian function decline. |
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