| Hoary bamboo rat(Rhizomys pruinosus)lived in underground tunnels for a lifetime.It had evolved many characteristics different from ground rats in morphology and physiology.One of its typical characteristics was the decline in fecundity,and the average litter size was only 2-4.In terms of life history strategies,subterranean rodents choosed r strategies to cope with resource-limited cave life.Therefore,even if the litter size of the underground rat was significantly lower than that of the ground rat,it can still ensure that the population level was basically stable due to its longer growth cycle and lifespan and fewer natural enemies.However,the research on the mechanism of the low reproduction rate of subterranean rodents was still very insufficient.In this study,the ground rat SD rats with high reproduction rate were used as the control to compare and study the molecular mechanism of the low fertility rate of Hoary bamboo rat.First,observed the tissue structure of the ovary of Hoary bamboo rats and SD rats by histological methods to determine the ovarian development status at different stages.Based on the results of histomorphology,both species were grouped according to the same level of ovarian development(Hoary bamboo rats 1,3,4,5,6,7 months old;SD rats 7,15,25,30,40,60 days old)for physiological and molecular biology experiments.Used enzyme-linked immunoassay to detect both species serum hormones estradiol(E2),testosterone(T),follicle stimulating hormone(FSH)and luteinizing hormone(LH).Real-time fluorescent quantitative PCR technology was used to detect the relative expression of mRNAs related to the development and maturation of ovarian follicles.Western blot method to detect the expression of related proteins(FSHR,ESR2,AR,FOXO1 and IGFBP4).The main results of this research were as follows:1.The histological results of the ovary in different developmental stages of Hoary bamboo rat and SD rat showed:there were no antral follicles in the ovaries at 2-month-old,indicating that the ovaries of Hoary bamboo rat developed to 2 months old was still in the early stages.There were various stages of follicles in the ovaries of the 5 months old Hoary bamboo rat,and the corpus luteum was not seen.The corpus luteum appeared in the ovaries of the 6-month and 7-month-old of Hoary bamboo rat,indicating that Hoary bamboo rat has reached fully maturity at the age of 7 months,that is,the time to enter the childbearing age was later.Counted the number of follicles during ovarian development,the number of secondary follicles of Hoary bamboo rat reached the highest peak and was close to the level of SD rats.The number of antral follicles and the number of mature follicles were significantly lower than SD rats when they reached the maximum(P<0.01),and the number of mature follicles was significantly lower than that of SD rats at different stages of the ovarian maturation(P<0.01).The level of atretic follicles increased significantly after 2 months of age and was extremely significantly higher than that of SD rats(P<0.01).2.Serum hormones(FSH,E2,LH,T)levels,the FSH level of Hoary bamboo rat was significantly lower than that after the ovarian development and maturity(P<0.05),and it was extremely significantly lower than that in SD rat(P<0.01),There was no obvious peak of E2 level during ovarian development,and then maintained a stable level,and significantly decreased at 7 months of age(P<0.05).E2 level was significantly lower than SD rats during the ovarian development period of 4-7 months(P<0.01).The LH level was lower in 4-7 months of age,and it was significantly lower than that of SD rats(P<0.05).T maintained a stable level before ovarian development reached maturity,and was lower than SD rats during the entire development period.3.The relative expression of genes involved in ovarian follicle development and maturation:Fshr,Esr2,Lhcgr,Ar mRNA expression:the expression of Fshr mRNA was significantly up-regulated in the ovary of Hoary bamboo rat(P<0.05).The expression of Esr2 mRNA was significantly up-regulated at 5 months of age(P<0.05).After the ovarian matured,and the expression of Esr2 mRNA was significantly down-regulated(P<0.05),and the expression of Lhcgr mRNA was significantly down-regulated at 7 months of age(P<0.05).At different stages of ovarian development,the expression of Fshr,Esr2 and Ar mRNA in Hoary bamboo rats were significantly lower than those in SD rats(P<0.01).After the ovarian development enters the mature stage,the expression of Lhcgr mRNA in Hoary bamboo rat was significantly lower than that in SD rat(P<0.01).Igf1,Igfr1 and Igfbp4 mRNA expression:the relative expression of Igf1 mRNA in the ovary of Hoary bamboo rat was the highest at 3 months old,and was significantly higher than that of SD rats at 15 days.But the expression of Igf1 mRNA at the age of 6 months was significantly lower than that of SD rats at 40 days.The expression of Igfr1 mRNA in ovarian development of 4-7 months of age wad significantly lower than that of SD rats of 25-60 days of age(P<0.01).The expression of Igfbp4 mRNA continued to be down-regulated during ovarian development,but it was up-regulated at 6 months of age.During ovarian development was significantly higher than that of SD rats(P<0.01).The expression of Cyp19a1,Bmp-15 and Foxol mRNA:the expression of Cyp19a1 mRNA in the ovary of the bamboo rat was significantly up-regulated at the age of 6 months(P<0.05).The expression of 1,5,and 7 months of age was significantly lower than that of SD rats of 7,30,and 60 days of age(P<0.01).The expression of Bmp-15 mRNA was highest at 4 months of age,and during ovarian development was significantly higher than that of SD rats(P<0.01).The expression of Foxol mRNA is highest at 6 months of age,and was significantly higher than that of SD rats(P<0.01).FSHR,ESR2,AR,FOXO1 and IGFBP4 protein expression:the expression of FSHR protein in the ovary of Hoary bamboo rat was significantly lower at 4 months of age(P<0.05).There was no significant change in the expression of ESR2 protein at the age of 1-6 months,and the expression at the age of 7 months decreased significantly(P<0.05).During ovarian development,the protein expressions of FSHR and ESR2 of Hoary bamboo rat were lower than those of SD rats,and the protein expressions of FSHR and ESR2 of 4-7 months old were significantly lower than those of 25-60 days old SD rats(P<0.01).AR protein expression level was highest at 5 and 7 months of age.The expression level of AR protein in Hoary bamboo rats aged 1-6 months was extremely significantly higher than that of SD rats aged 7-40 days(P<0.01).The expression of FOXO1 protein reached the highest at the age of 6 months,and was significantly higher than that of SD rats at 40 days of age(P<0.01).The expression of IGFBP4 protein was significantly higher than that of SD rats during the development of the ovary(P<0.01).The above results indicate that,compared with SD rats,in Hoary bamboo rat ovarian maturation before low FSH levels,and the E2,LH,and T levels were significantly low after the ovarian matures,resulting in a decrease in the number of antral follicles,and then the number of mature follicles is few.The main reason for the above result was that the expression of genes regulating the development and maturation of ovary follicles in different developmental stages of Hoary bamboo rats was significantly different from that of SD rats.Highly expressed Bmp-15 inhibited the level of FSH,resulting in a weakened effect of lower levels of FSH on FSHR activation.At same time,AR weakened the promotion of FSHR.During ovarian development,thereby inhibiting the expression of IGF-1 and Cyp19a1,which were downstream genes that promote follicular development and maturation,and LH reactivity.The decrease of IGF-1 results in the suppression of the expression of LHCGR and ESR2,and low levels of LH and LHCGR induced membrane cells to release a small amount of T.Low Cyp19al catalyzed the synthesis of a small amount of T into E2,E2 level and the expression of receptor ESR2 were low,which inhibited follicle development and maturity.The expression of FoxO1,which induces follicular atresia,was significantly higher than that of SD rats during the ovarian development and maturity of Hoary bamboo rats,and the expression level of IGFBP4 during the entire ovarian development process was extremely significantly higher than that of SD rats.This resulted in a large number of follicles atresia in Hoary bamboo rats.In summary,compared with SD rats,the negative regulatory factor Bmp-15 was highly expressed during the development and maturation of the ovarian follicles,and the positive regulatory factors FSHR,AR,IGF-1R,LHCGR,Cyp19a1 and ESR2 were lower.Expression inhibits the level of hormones that promote follicle maturation,resulting in a decrease in the number of mature follicles.In addition,the genes IGFBP4 and FoXO1 that induce follicular atresia were highly expressed,leading to serious follicular atresia,which in turn leads to fewer ovulations,fewer litters,and reduced fertility in Hoary bamboo rat. |
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