Lepr Gene Defect And Exogenous Estrogen Stimulation Cause Reproductive Disorders In Adult Mice

With the economic development and social progress, obesity has become a universal factors affecting human health, which could lead to many diseases, such as cardiovascular disease, diabetes, etc., at the same time, cause birth defects and even infertility. Leptin is recognized in1994, which is the expression product of the obesity gene, has a role in regulating body weight and fat distribution, and is closely related to obesity and reproduction. Patients with polycystic ovary syndrome (PCOS) are not only associated with obesity, abnormal ovulation, and even sterility. Part of the PCOS patients are detected with Leptin gene or leptin receptor gene defects, and with the insulin resistance. It has been confirmed in animal models that leptin-deficient mice (ob/ob) and leptin receptor defect (db/db) mice showed obesity and infertility. In order to further study the relationship between leptin and infertility, Y123F LepR mutation mouse model, similar to the db/db mice, but in which the three key tyrosine sites of the leptin receptor Ob-Rb are mutated to phenylalanine, resulting in downstream signaling pathways mediated by the three points been blocked. The adult female of Y123F model shows infertility and obesity, which is closer to pathological characteristics of human PCOS, and is therefore an ideal object for the study of obesity and infertility.Compared with the wild-type mice, body weight and abdominal fat of Y123F homozygous mice was increased sharply, but the quality of the ovaries and uteri are reduced significantly. The histological phenotype analysis showed that there were only three stages of ovarian follicles, not mature follicles and corpus luteum in the ovary of the mature Y123F homozygous mouse, suggesting no normal ovulation in Y123F homozygous mice; the endometrial stroma, epithelia and gland shows degradated appearance, and with some obvious cavity in the stroma. Through observating vaginal smears on consecutive12days, we found that Y123F homozygous mice don’t have estrous cycle, and the estrogen concentration in serum keep at low level. Gene PPARgamma, related to the fat metabolism, is confirmed by RT-PCR and Western-blot analysis, that its levels of ovarian and uterine mRNA and protein are lower in Y123F homozygous mice than in wild-type mice. By immunohistochemistry, we found that PPARgamma protein was expressed in ovarian granulosa cells and the oocyte plasma membrane in both Y123F homozygous mice and wild-type mice, but its amount in granulosa cells was less in Y123F homozygous mice, suggesting that PPARgamma contributued to the ovarian defects caused by leptin receptor, mutation.The gene micrarray chip technology helps select the most significant changes in the ovary and uterus of the three genotypes. Up to now, it have not been reported the ovarian or uterine gene expression profile changes resulted from the gene defects of Leptin and its receptor. By comparing cDNA microarray results in ovaries and uteri among the Y123F homozygous, heterozygous and wild-type mice,179genes related to ovary and4701genes related to uterus were found different expression levels between Y123F homozygous mice and wild-type mice ovarian, at the same time, there are75genes related to ovary and1921genes related to uterus between Y123F heterozygous mice and wild-type mice. Many metabolic pathways, including41signaling pathways in the ovary and173in uterus between Y123F homozygous wild type mice, and including15signal transduction pathways in the ovary and139in uterus between Y123F heterozygous and wild type mice. The primers are designed for genes related to fat metabolism and steroid hormone synthesis, including Leptin, Prlr, Gadd45a, GADD45b, Gadd45g, Jun, FABP3, ACSL4, Foxa3and Cish, to check the microArray chip results by RT-PCR and real-time quantitativeRT-PCR methods. Results showed that the ovarian and uterine dysfunction in receptor-deficient mice is due to the complex interaction of many genes.Estrogen plays a key role to the ovarian and uterine normal function. For Y123F homozygous mice, the levels of estrogen in serum reduced, and the microArray chip results also showed that the expression of genes related to estrogen receptor signaling pathways changed in both ovaries and uterus, and showed that the PCOS symptoms can be caused by excess estrogen stimulation. It is reported that soybean peptides suppress appetite and intestine nutrient absorption. In the third part of this study, immature mice were irritated by an excess estrogen, and then fed soy peptide for two moths continuously with water as control, and in parallel with Y123F homozygous mice as a comparison study. At the age of12weeks, compared with the control group, although the weight of the excess estrogen group does not increase, there were great morphological changes in the ovaries and uterus. Ovaries enlarged, tertiary follicles increased, but corpus luteum was not observed, suggesting that ovaries have not normal ovulation; and similarly, it could be seen hyperplasia of endometrial stroma and a large number of blood vessels in the enlarged uterus. Soybean peptide, which could reduce the body weight of the Y123F homozygous mice, but does not play a significant role improving the estrogen concentration in serum, and repairing ovarian and uterine tissue morphology, in both excessive estrogen treated and Y123F homozygous mice.