Reproductive Dysfunction In Non-Obese Diabetes Female Mouse

BackgroundType 1 diabetes mellitus（T1DM）is an organ specific autoimmune disease characterized by the destruction of isletβcells mediated by T cells,resulting in a decrease in insulin production,leading to hyperglycemia.The pathogenesis of T1DM remains unclear,which may be caused by genetic or environmental factors,such as virus infection or exposure to toxic chemicals.In the past decade,the incidence rate of T1DM has a significant increase worldwide and exhibit a trend of early onset.Female T1DM patients with poor blood glucose management will seriously impact their reproductive health,mainly including low fertility,hypogonadism caused by damage to hypothalamic pituitary ovarian（HPO）axis,abortion and congenital malformations.It is reported that up to 40%of female T1DM patients exhibit symptoms such as menstrual disorders,hyperandrogenemia or premature menopause.Non obese diabetic（NOD）mice are recognized a classical T1DM animal model.The autoimmune and genetic characteristics of NOD mice are similar to that of human T1DM,which provide solid basement for using NOD mice as suitable animal model to study reproductive dysfunction of female T1DM patients.ObjectiveThis study takes NOD mice,a classical animal model of spontaneous T1DM,as the research object,focus on the reproductive system of NOD female mice,analyze the abnormalities in morphology and function of reproductive organs,and further discuss the potential mechanism of reproductive dysfunction in female patients caused by T1DM.The aim of this study is to provide support for clinical prevention and treatment of reproductive dysfunction in female patients with T1DM.Method1.NOD female mice were caged with ICR male mice to examine the fertility of NOD female mice.2.Immunofluorescence staining was used to detect the expression of CD4⁺T lymphocytes,CD8⁺T lymphocytes and insulin⁺isletβcells in the pancreas of NOD female mice at different ages.3.HE staining,immunohistochemistry and immunofluorescence staining were used to detect the morphological and functional changes of ovary and uterus in ICR and NOD female mice at different age.4.Western blot was used to determine the expression of targeted proteins related to ovarian reserve capacity,uterine receptivity,apoptosis and angiogenesis in ovary and uterus of NOD female mice.5.Protein assay was used to quantify the expression of related inflammatory cytokines in the serum of NOD female mice at different ages.6.RNA-seq was performed to screen the differentially expressed genes in ovarian and uterine tissues between ICR and NOD female mice.Result1.The fertility result showed that the fertility of NOD female mice was significantly impaired than that of ICR female mice.2.The immunofluorescence staining results of insulin,CD4 and CD8 in pancreatic tissue found that with the age increase of NOD female mice,the normal function of pancreas was gradually impaired,and the degree of inflammatory cells infiltration in islets were significantly increased.3.The results of HE and Masson staining,immunohistochemistry of CD31 and Western blot showed that with the age increase,the ovarian follicle development of NOD female mice was abnormal,the morphology and function were impaired,and the angiogenesis was damaged.Simultaneously,the expression of apoptotic protein Caspase-3 was significantly up-regulated and anti-apoptotic protein Bcl-2down-regulated.4.The results of HE,Masson and PAS staining,immunohistochemistry of CD31and Ki67,immunofluorescence of CD3 and Western blot demonstrated that with the age increase,the morphology and function of uterine tissue in NOD female mice were disordered,along with the decrease of angiogenesis and the increase of fibrosis level and T lymphocytes infiltration.5.The results of protein assay determined that the level of serum inflammatory cytokines in NOD female mice were significantly increased with the age increase.6.The RNA-seq results of ovary and uterus founded that the differentially expressed genes were mainly enriched in the signaling pathways,such as metabolism,cytokine receptor and chemokine.Conclusion1.The reproductive function of NOD female mice have been impaired before the onset of T1DM.2.The reproductive capacity was significantly affected with the age increase of female NOD mice,and the morphology and function of ovary and uterus in mice were abnormal,which was mainly characterized by the abnormal expression of inflammatory cytokines,excessive immunocytes infiltration in main reproductive organs and the disorder of energy metabolism.