The Effects Of Testosterone Concentrations On Oocytes And Embryo Development Of Rats

Objectives This thesis targets to analyze the effects of changes in exogenous testosterone concentrations on the development of rat oocytes and subsequent embryos,explore the appropriate exogenous testosterone concentrations for high-quality oocytes and embryos,and evaluate the predictive effect of testosterone on in-vitro fertilization-embryo transfer（IVF-ET）pregnancy outcomes and Clinical Value.Methods 1 6-week-old healthy SD rats were selected as model animals,including 71female rats and 13 male rats.After adaptive feeding,the estrous cycle was investigated.2The pre-experiment combined with the literature to investigate the time and level range of the effect of testosterone propionate on rats.3 The formal experiment was conducted by randomly dividing the rats into 5 groups including T₀,T_0.25,T_0.5,T₁,T_1.5,and the testosterone propionate peanut oil solution was subcutaneously injected through the back of the neck for 10 consecutive days to construct rat models with different testosterone concentrations,and the control group was injected with the same amount of peanut oil.Blood was collected from the inner canthus the next morning,and serum testosterone levels were continuously monitored by ELISA.4 After the model was successful,the ovarian tissue was taken out to observe the general morphology,and the follicle morphology was analyzed and counted by HE staining.5 After controlled ovulation induction,the second meiotic metaphase II（MII）oocytes were collected from 5 female mice in each group.Mito-Tracker Red,Acetate（DCFH-DA）fluorescent probe,JC-1fluorescent probe,and Calcein AM fluorescent probe were used for fluorescent staining of oocytes,and the fluorescence intensity of oocytes under T₀,T_0.25,T_0.5,T₁ and T_1.5conditions was analyzed,and the number and distribution of mitochondria,mitochondrial reactive oxygen species（ROS）,mitochondrial membrane potential and mitochondrial membrane transfer permeability in oocytes under specific exogenous testosterone concentrations were analyzed.6 The other groups of female mice were caged with males,and after in-vivo fertilization,the fertilized eggs at the cell stage were collected for in-vitro culture to observe the embryonic development.Results 1 Different concentrations of testosterone have different effects on the body weight and ovarian weight of female SD rats.The weight of the rats in the T_0.25 group was higher,while the weight of the rats in the T_1.5 group was lower,the difference was statistically significant,P<0.05,and the difference of the weight of the rats in the T_0.5 and T₁ groups to the control group T₀ was not statistically significant.2 The internal environment of female SD rats finally kept the testosterone level within a range of fluctuations.The effect of exogenous testosterone concentration on the serum testosterone level of female rats in the first 7 days of the observation period was statistically significant,P<0.05.At the same time,the serum testosterone concentration of rats showed a downward trend with time,and the change trends of T_0.25,T_0.5,T₁,and T_1.5 groups were consistent.The difference in the average testosterone level of SD rats in each group on the last 3 days of the observation period was positively correlated with the exogenous testosterone concentration.higher than the blank control group T₀,the difference was statistically significant,P<0.05.3 Compared with the T₀ group,the number of growing follicles in the T_0.25 group increased,and the number of cystic follicles and atretic follicles in the T_1.5group increased,the differences were statistically significant,P<0.05.4 Mito-Tracker Red fluorescent probe was used to characterize the number and distribution of mitochondria.The staining results showed that,compared with the T₀ group,the fluorescence intensity of the T_0.25 group became stronger,and the difference was statistically significant,P<0.05,the fluorescence intensity in T_0.5,T_1,T_1.5 groups was decreased compared with the T₀ group,and all the differences were statistically significant,P<0.05.5 The results of DCFH-DA fluorescent probe,which was used to characterize the concentration of reactive oxygen species in oocytes,showed that,compared with the T₀ group,the oocytes in the T_0.25 group had higher fluorescence intensity and higher ROS concentration,while the fluorescence intensity of oocytes in T_0.5,T_1,T_1.5 group decreased,revealing that the ROS concentration gradually decreased,and the difference was statistically significant,P<0.05.6 The results of using JC-1 fluorescent dye to characterize the mitochondrial membrane potential of oocytes showed that,compared with the blank control group T₀,the mitochondrial membrane potential of the oocytes in the T_0.25 group increased with the incremental characteristic value of the membrane potential fluorescence intensity,and in the T_0.5 and T₁groups,the mitochondrial membrane potential of oocytes decreased with lower characteristic value of membrane potential fluorescence intensity.The difference was statistically significant,P<0.05.7 The results of using Calcein AM fluorescent dye to characterize mitochondrial membrane permeability showed that the fluorescence intensity of oocytes in the T_0.25 group was increased compared to the blank control group T₀,and the fluorescence intensity of the oocytes in the T₁ group was decreased compared to T₀,and the difference was statistically significant.P<0.05.8 The concentration of exogenous testosterone has a significant effect on the embryonic development of SD rats.Among them,the T_0.25 group has normal embryonic development and a higher rate of excellent embryos,while the T₁ group has relatively unhealthy embryonic development and a lower rate of excellent embryos.The difference is statistically significant,P<0.05.Conclusions 1 Injection of moderate concentrations of exogenous testosterone（about0.25mg/m L）into rats can promote the activity of ovaries,oocytes and their mitochondria,and maintain the healthy development of embryos;2 Injection of excessively high concentrations of exogenous testosterone（>1mg/m L）negatively affects ovaries,oocytes,mitochondria activities and embryonic development in female rats;3 Injection of exogenous testosterone at middle concentrations（0.5mg/m L）does not obviously affects relevant traits of the rats,and the difference to the control group does not show statistical significance;4 Based on the experimental results in the SD rats,it is suggested that maintaining a moderate testosterone concentration to improve the development of oocytes and embryos in infertile patients may be a more effective treatment method in clinical IVF-embryo transfer technology.Figure 16;Table 5;Reference 147...