A Study Of The Effect Of Leptin/GnRH On Pubertal Onset In High-fat Diet-induced Obesity Female Mice

Objective:In this study the newborn C57BL/6J female mice were selected,and the mice were exposed to high-fat-deit after birth.We want to explore the effect of obesity on the puberty development in female mice and the role of Leptin/GnRH by examining the vaginal opening time,the pathological changes of the ovaries,serum neutral hormone levels,hypothalamus and ovarian gonadotropin releasing hormone,the gene and protein expression of leptin receptors and GnRH.Methods:12 C57BL/6J male and 24 female mice were selected.After one week of adaptive feeding,they were matched by 1:2.On the day of birth,24 female pups were selected and randomly divided them into high-fat-deit group（HFD）and control group（CON）,n=12/group.The female mice in HFD group were fed with high-fat diet during the lactation period,and the offspring were continued to be fed after weaning;the female mice in the control group were fed with normal diet during the lactation period,and the offspring continued to be fed with common diet after weaning for a total of 45 days.On the 28^thday,vaginal opening were detected daily;On the 45^thday,liver,kidney,peripheral fat,ovaries,uterus,and peripheral fat tissues of mice were separated and weighed;light microscope and electron microscope were used to observe the pathological changes of ovaries;ELISA method was used to detect serum Leptin,LH,FSH,E2,T levels;immunohistochemical pathology was used to detect GnRH protein expression,and western blot method was used to detect Ob-R protein expression,and real-time quantitative PCR method was used to detect Gene expression of GnRH and Ob-R in hypothalamus and ovary.Result:On the 21^stday,the body weight of the two groups of mice was different（P<0.05）.On the 45^thday,the body weight of the mice in the HFD group was significantly higher than that in the CON group,and the difference was statistically significant（P<0.05）.At the end of the experiment,the body fat weight,body fat percentage and lean mass of the mice in the HFD group were significantly higher than those in the CON group,and the difference was statistically significant（P<0.05）.The lean mass percentage of the mice in the HFD group were significantly lower than those in the CON group,and the difference was statistically significant（P<0.05）.On the 45^th day,the peri-renal fat,peri-uterine fat,peri-renal fat coefficient,and peri-uterine fat coefficient of the HFD group mice were significantly higher than those of the CON group,the difference was statistically significant（P<0.05）.The liver weight,liver coefficient,kidney weight,kidney coefficient,uterine weight,uterine coefficient of mice in the HFD group were significantly higher than those in the CON group,and the difference was statistically significant（P<0.05）.The age of vaginal opening time in the HFD group was significantly earlier than that in the CON group,and the difference was statistically significant（P<0.05）.The number of follicles in the ovary of the HFD group were higher than that in the CON group;the ovarian surface epithelial cells of the HFD group were tightly arranged with regular cell shapes and sizes,and the ovarian epithelial cells developed well;the ovarian epithelial cells of the CON group were loosely arranged with irregular shape.More ovarian mesenchymal cells were found in the HFD group than in the CON group.More fat droplets around the cells were found in the HFD group than in the CON group,and the fat droplets were larger in the HFD group than in the CON group.Comparison of pathological changes of ovary under electron microscopy shows that in the CON group,the nucleus chromatin of ovarian granulosa cells was uneven,the nuclear membrane was unclear,the number of mitochondria was less,and the organelles such as mitochondria,lysosomes,and endoplasmic reticulum were blurred.In the HFD group,the nucleus chromatin of ovarian granulosa cells was uniform,clear nuclear membrane,rich mitochondria,uniform mitochondrial staining,obvious mitochondrial cristae,and clear mitochondria,endoplasmic reticulum,lysosomes and other organelles.Fat droplets in the corpus luteum in the CON group were less than in the HFD group.More fat droplets in the corpus luteum were found in the HFD group than in the CON group.The levels of serum leptin,estradiol,and follicle stimulating hormone in the HFD group were higher than those in the CON group（P<0.05）,but there was no significant difference in serum luteinizing hormone and testosterone levels between the HFD group and the CON group.The expression level of GnRH protein in the hypothalamus of the HFD group was significantly lower than that of the CON group（P<0.05）.The mRNA expression level of GnRH in the hypothalamus of the HFD group was significantly lower than that of the CON group（P<0.05）.The expression level of GnRH protein in the ovary of the HFD group was significantly lower than that of the CON group（P<0.05）.The expression level of GnRH mRNA in the ovary of the HFD group was significantly lower than that of the CON group（P<0.05）.The expression levels of Ob-R protein and mRNA in the hypothalamus and ovary of the HFD group were significantly lower than those of the CON group,and the difference was statistically significant（P<0.05）.Conclusion:Earlier puberty were found in obese female mice than in normal weight mice;Serum FSH level in obese mice increased which lead to the increase of estrogen levels,which may result in early puberty in female obese mice;There was a decreased expression of GnRH which may be related to the negative feedback of high level of FSH and estrogen.