| Chapter1 Establishing a fluorouracil damage model of rat ovariesObjective:To establish a fluorouracil damage model of rat ovarian for fllow-up research.Methods:Twenty Sprague Dawley rats with normal estrous cycle, female, 8-10 week-old, were randomly divided into four groups: normal saline control, low-dose 5-FU, middle-dose 5-FU and high-dose 5-FU. Individuals in four groups separately received a 14-day intraperitoneal injection according to 1mg·kg-1·d-1(normal saline), 5mg·kg-1·d-1(5-FU), 10mg·kg-1·d-1(5-FU), 20mg·kg-1·d-1(5-FU). To detect the serum hormone levels of AMH, FSH and E2, samples of 1ml blood were collected from each individual via its tail at the time of pre-dose, drug withdrawal and two weeks after dose. Ovaries for each group were obtained, wet weighed, embedded in paraffin, sliced, and stained with hematoxylin-eosin separately at withdraw and two weeks after dose. Then the number of follicles were counted under the optical microscope.We observed changes of rats’ weight, activity, diet, water,and urine. Before administration, the withdrawal and withdrawal two weeks,we collected 1ml blood by tails to detect the serum hormone levels of AMH, FSH and E2.At the same time, Items including weight, motor activity, intake of food and water, urination, and defecation were observed and recorded. And after stopping 5-FU two weeks and the withdrawal, rats’ ovaries were obtained and weighed.one was embedded in paraffin, sliced and then stained by HE. And the number of follicles were counted under an optical microscope.Results : Weight and food intake decreased significantly in 5-FU groups. Averages of food intake of low-dose 5-FU, medium-dose 5-FU, and high-dose 5-FU groups reduced from normal 18g~20g to 15 g, 12 g and 9 g, while water intake and urine volume increased. The individuals in saline group had a normal estrous cycle of 4~5days. However, individuals in low-dose 5-FU group had an extended estrous cycle of 7~8 days. Estrous cycle disorders occurred in middle-dose 5-FU group on the ninth day and high-dose group dose 5-FU group on the fifth day: the estrous diestrus was prolonged and estrus was shortened or even disappeared. The serum sex hormone levels for four goups showed no significant difference before administration(P>0.05), while the serum sex hormones in low-dose, medium-dose and high-dose groups became significantly different levels from the one in saline group at drug withdraw. FSH increased significantly(P<0.05), while E2, AMH and the number of primordial follicles and growing follicles were significantly decreased(P<0.05). In addition, the ovarian wet weight decreased more obviously in the low-dose group(P<0.05), while no significant change in the high-dose group(P>0.05). Compared to normal saline group, the serum sex hormones in all 5-FU groups showed significant difference two weeks after withdrawal except that FSH in low dose group returned to normal level(P<0.05), and the number of primordial and growing follicles reduced significantly(P<0.05). In high dose group, ovarian wet weight reduced significantly(P<0.05). Changes of the serum sex hormones and the primordial and growing follicles in each group were not statistically significant(P>0.05), except that ovarian wet weight in high-dose group reduced significantly(P<0.05).Conclusions:5-FU can change serum E2, FSH and AMH levels, resulting in estrous cycle disorders or even its disappearance. We successfully established an animal model of ovarian damage and 5-FU 20mg/kg can be applied in follow-up research.5 Chapter2 The protective effect of Gn RHa on chemotherapy-induced ovarian damageObjetive: To explore the protective mechanisms of Gn RHa on chemotherapyinduced ovarian damage.Methods: Female Sprague Dawley rats with normal estrous cycle were randomly divided into four groups: group A, normal saline control, 10 individuals; group B, 5-FU, 15 individuals; group C, triptorelin +5-FU,10individuals; group C, triptorelin +saline, 10 individuals. Group A: individuals were given total 14 times of lml/d saline intraperitoneal injection at an interval of 24 hours. Group B: individuals were administered with 14 times of 5-FU 10mg/kg intraperitoneal injection at an interval of 24 hours. Group C: 14 days before the experiment and after 5-FU for 14 days, individuals were given triptorelin 0.1mg/kg using deep intramuscular injection with the same usage and dosage of 5-FU as that in group B. Group D: triptorelin was the same as group C, and saline was the same as the group A. Vaginal smears were observed to evaluate the estrous cycle. Blood samples of each group were collected for determination of the serum AMH, FSH and E2 on the 14 th day before the experiment, on the 28 th day after administration, 1 week and 2 weeks after withdrawal. When individuals were killed in the first and second week, the drug was stopped and the estrous cycle was during the estrus interval. Both ovaries were obtained for wet weight. One batch were fixed in 10% neutral solution of formalin, then the other were embedded by paraffin wax and sliced continuously in 5μm sections. The number of follicles at different stages was counted under light microscopy after the sections were stained with haematoxylin and eosin in four groups. The expression of Bcl-2 protein and Bax protein were detected with immunohistochemistry. The other side of the ovarian tissue was used to extract protein to perform Western-Blot.Results: ① The changes of estrous cycle: estrous cycle in group A was normal. After 5-FU was given, estrous cycle disorders occured after a complete estrogen estrous cycle. When 5-FU was continued, estrous cycle disappeared and only estrogen interphase remained. Estrus cycle came to normal when drugs was absent for 7days. After drugs was absent for 14 days, a normal estrus cycle returned by only 21.6%(3/14) in Group B. Estrous cycle in group C and group D disappeared after a normal estrous cycle when triptorelin was used. The estrous cycle in group C was extended to ten days on the fourth day occuring first estrous cycle after the drug was stopped, and estrous cycle gradually returned to normal. In group D estrous cycle resumed normal four to five days after the drug was stopped. ② The changes of serum hormone levels: there were no significant difference(P>0.05) in four groups of serum AMH, E2 and FSH before the experiment. The serum concentration of AMH and E2 in group B were significantly lower than the other three groups(P<0.05) on the 14 th day, and there was no significant difference of the serum concentration of FSH(P>0.05) despite it increased. The level of AMH in group C decreased significantly compared to group A(P<0.05). And the serum concentrations of AMH, E2 and FSH in group B were significantly different compared with other three groups on the 28 th day(P<0.05). While the level of serum AMH in group C decreased significantly compared with group A(P<0.05), and the levels of E2 and FSH did not decrease significantly(P>0.05). In the first week and second week after stopping drugs, the serum concentrations of FSH declined in group B, while the change was still significant compared with other three groups(P<0.05). However, there were relatively insignificant difference in group C and D compared with group A(P>0.05). In the first week after the drug was stopped, despite that the serum levels of AMH and E2 incresed in group B, but the significant difference was still found compared with other three groups(P<0.05). ③ The comparison of ovarian wet weight: the ovarian wet weight of group B was significantly reduced on the first week(P<0.05) compared with group A after stopping drugs. In the second week after stopping drugs, although the ovarian wet weight in group B increased, it was still lighter than other three groups(P<0.05). While comparing weight before and after each group no change was significant(P>0.05). ④ The chages of the number of primordial follicles and growing follicles: In the first week after stopping drugs, primordial follicles in group B was significantly less than those in group A(P<0.05). And compared with group A, growing follicles in group B, group C and group D significantly reduced(P<0.05). Primordial follicles in group C and group D were significantly more than that in group A(P<0.05). In the second week after stopping drugs, although the number of primordial follicles in group B had increased compared with the first week, there was still significant differences compared with other three groups(P<0.05). While the number of primordial follicles in group C and D was decreased and close to group A( P>0.05). The number of growing follicles in each group increased compared with the first week after stopping drugs, but the number of growing follicles in group B was significantly lower than that in group A(P<0.05), and group C and D were close to normal(P>0.05). The primordial and growing follicles before and after drug between the groups did not change significantly(P>0.05). ⑤ The expression of Bcl-2 and Bax protein in ovarian tissue: In group B, the expression of Bax protein was up-regulated, while Bcl-2 protein was down-regulated, so that the ratio of Bcl-2/Bax was lower. Group C was just the opposite: the expression of Bcl-2 protein increased, the Bax protein expression was down resulting in the increase of Bcl-2/Bax ratio.Conclusion: ① In early ovarian dysfunction, changes in serum AMH are more sensitive than E2 and FSH, and AMH can be used as an indicator to determine early ovarian function injury. ② Gn RHa can reserve primordial follicles, thereby reducing the sensitivity of the developing follicles to chemotherapeutic drugs to protect ovarian function. ③ Gn RHa maybe upregulate the Bcl-2 protein expression and downregulate Bax protein expression to regulate the ratio of Bcl-2/Bax, which plays a key role in anti-apoptotic or pro-apoptotic process in ovarian tissue. |
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