| The ovary development of female animals is closely related to their reproductive performance.Previous studies of our research group have found that energy level affects the activation of primordial follicles of sows.Glucose is the primary metabolic substrate of diet energy,and its effect and regulatory mechanism on the activation and development of primordial follicle are still unclear.Studies on different type of animal cells have shown that energy stress induced by glucose starvation activates AMPK and participates in a variety of cellular processes by regulating Hippo and mTOR signaling pathways.However,whether glucose can affect primordial follicle activation through the above pathways has not been reported.Therefore,the mouse ovary was used as the model in this study.Firstly,a culture system of mouse ovary in vitro was established to investigate the effects of different glucose levels on the activation of primordial follicle in vitro.On this basis,the signal pathway of glucose affecting the activation of primordial follicle in vitro and in vivo was further explored.This study consists of the following three parts: Exp.1 Effects of glucose levels on the activation of primordial follicles in ovary cultured in vitroThe 3-day old SPFKM mice were selected to establish an ovary culture system in vitro to investigate the effects of glucose levels of 0,5.6,25,50 and 75 mM on the activation of ovarian primordial follicle,as well as the effects on the content of metabolites of glycolysis pathway in the ovary cultured in vitro.The results are as follows:1.With the ovaries of 3d and 5d mice as controls,the ovaries of 3d mice were cultured in vitro for 2 days,the ovarian tissue structure was still intact,and part of the primordial follicles began to activate,suggesting that the ovary culture system in vitro was successfully established.2.Compared with the control group,the proportion of primordial follicles with different levels of glucose was decreased(P<0.05),and the proportion of primary follicles was increased(P<0.05).There was no significant difference in the proportion of primordial,primary and secondary follicles among 25 mM,50mM and 75 mM glucose treatment groups(P>0.05).In vitro culture for 24 h,the proportion of secondary follicles in the 25 mM glucose treatment group was higher than that in the 5.6mm treatment group(P<0.05).In vitro culture for 48 h,the proportion of primordial follicles in the 25 mM glucose treatment group was lower than that in the 5.6mm treatment group(P<0.05),but its ratio of primary/primordial follicles was higher than that in the 5.6mm treatment group(P<0.05).3.Compared with the control group,the total protein content of ovary in each glucose treatment group was increased(P<0.05),but there was no significant difference among different glucose levels(P>0.05).Compared with the control group,the addition of 5.6mM,25 mM and 50 mM glucose increased PCNA protein expression(P<0.05).4.In vitro culture for 24 h,the pyruvate content in the culture medium of the 75 mM glucose treatment group was higher than that of the control group,the 25 mM and 50 mM glucose treatment groups(P<0.05).The lactic acid content of the culture medium in each glucose treatment group was higher than that in the control group(P<0.05),and the lactic acid content of the culture medium in the 25 mM and 75 mM glucose treatment groups was higher than that in the 5.6mM glucose treatment group(P<0.05).In vitro culture for 48 h,the pyruvate content of the culture medium in each glucose treatment group was higher than that in the control group(P<0.05);the difference in pyruvate content among groups with a glucose level lower than 75 mM was extremely significant(P<0.05);the lactic acid content of the culture medium in each glucose treatment group was higher than that in the control group(P<0.05),and the lactic acid content of the culture medium in the 25 mM,50mm and 75 mM glucose treatment groups was higher than that in the 5.6mm glucose treatment group(P<0.05).5.The content of ovarian ATP in the 75 mM glucose treatment group was higher than that in the control group and in the other glucose treatment groups(P<0.05).The ovarian HK activity of the 50 mM glucose treatment group was higher than that of the control group and other glucose treatment groups(P<0.05),and the ovarian HK activity of the 75 mM glucose treatment group was higher than that of the 25 mM glucose treatment group(P<0.05).The ovarian PFK activity was increased in the control group(P<0.05).The ovarian PK activity in the 5.6mM glucose treatment group was higher than that in the control group and other glucose treatment groups(P<0.05),the ovarian PK activity in the control group was higher than that in the 75 mM and 25 mM glucose treatment group(P<0.05),and the the ovarian PK activity in the 75 mM and 50 mM glucose treatment group was higher than that in the 25 mM glucose treatment group(P<0.05).The above results showed that glucose was an essential nutrient for the activation of primordial follicles,25 mM glucose was the optimal level for the activation of primordial follicles in this study,and glycolysis pathway was involved in the activation of primordial follicles in vitro.Exp.2 Mechanism of effects of glucose on the activation of primordial follicles in ovary cultured in vitroThe optimum glucose level was 25 mM.Further,protein expression of AMPK-Hippo-YAP and AMPK-mTOR pathway was investigated under glucose starvation and optimal glucose level treatment.Finally,ovaries were treated with AICAR or Compound C in vitro to investigate the effect of activation or inhibition of AMPK on primordial follicle activation,as well as the changes of AMPK-Hippo-YAP and AMPK-mTOR signaling pathways.The results are as follows:1.No significant differences in p-AMPK/AMPK,p-YAP(Ser127)/YAP were observed in ovaries starved of glucose or treated with 25 mM glucose for 12 and 24h(P>0.05).Glucose starvation for 48 h,p-AMPK/AMPK,p-MST/MST were increased(P<0.05),while p-LATS1/LATS1,p-YAP(Ser127)/YAP,p-YAP(Ser94)/YAP showed no significant difference compared with 25 mM glucose treatment(P>0.05).There was no significant difference of p-mTOR/mTOR and p-S6/S6 after ovaries treated with glucose starvation or 25 mM glucose for 24 or 48h(P>0.05).2.Ovaries were treated with 1mM or 2mM AICAR.The proportion of primordial follicles in the 1mM AICAR treatment group was higher than that in the control group and the 2mM AICAR treatment group(P<0.05).The proportion of primary follicles and ratio of primary/primordial follicles in the control group were higher than that in the 1mM AICAR and 2mM AICAR treatment groups(P<0.05).The proportion of secondary follicles in the 2mM AICAR treatment group was higher than that in the control group and the 1mM AICAR treatment group(P<0.05).3.Ovaries treated with 2mM AICAR for 4h increased p-AMPK/AMPK(P<0.05)and p-LATS1/LATS1(P<0.01),while decreased p-YAP(Ser127)/YAP(P<0.05),decreased p-mTOR/mTOR(P<0.05)and p-S6/S6(P<0.01).4.Ovaries were treated with 10μM or 100μM Compound C.10μM Compound C treatment group had lower primordial follicle percentage than the control group and the 100μM Compound C treatment group(P<0.05),its primary follicle percentage and ratio of primary/primordial follicles were higher than that in the control group and the 100μM Compound C treatment group(P<0.05).The control group had higher percentage of primary follicles than the 100μM Compound C treatment group(P<0.05).5.Ovaries treated with 20μM Compound C for 6h,resulted in reduced p-AMPK/AMPK(P<0.01),insignificant differences in p-LATS1/LATS1 and p-YAP(Ser127)/YAP(P>0.05),but increased in p-mTOR/mTOR and p-S6/S6 compared to the control group(P<0.05).The above results indicated that glucose affects the activation of primordial follicles in vitro through AMPK/mTOR signaling pathway.Exp.3 Effects of glucose on the activation of primordial follicles in ovary in vivoOn the basis of the studies in vitro of experiment 1 and experiment 2,further experiments in vivo were carried out to investigate the effect of blood glucose changes on the activation of primordial follicles in ovary in vivo and the mechanism.The results are as follows:1.After starvation for 24 h,the blood glucose level of newborn 4d female mice was lower than that of normal breastfeeding newborn 4d female mice(P<0.01).2.After starvation for 24 h,the proportion of primordial follicles of newborn 4d female mice was higher than that of normal breastfeeding newborn 4d female mice,but its proportion of primary follicles and ratio of primary/primordial follicles were lower than that of normal breastfeeding newborn 4d female mice(P<0.01).3.After starvation for 24 h,ovary p-AMPK/AMPK and p-YAP(Ser127)/YAP of newborn 4d female mice were not significantly different from that of normal breastfeeding newborn 4d female mice(P>0.05),but its p-mTOR/mTOR and p-S6/S6 were reduced(P<0.01).The above results showed that glucose affects the primordial follicle activation of ovary in vivo via mTOR signaling pathway.To sum up,the study draws the following conclusions:1.Glucose is an essential nutrient for the activation of primordial follicles,and 25 mM glucose is the optimal level for the in vitro activation of primordial follicles in this study.Glycolysis pathway is involved in the primordial follicle activation of ovary cultured in vitro.2.Glucose affects the primordial follicle activation of ovary through AMPK/mTOR signaling pathway. |
PDF Full Text Request