Adiponectin Deficiency Promotes Endometrial Cancer Development In Vivo

Background Adiponectin(APN)is an endogenous collagen-like protein that is abundantly expressed and secreted by adipocytes.Clinical studies have indicated that there is a significant correlation between low serum concentration of adiponectin and the incidence as well as progression of endometrial carcinoma.Our previous cellular studies have shown that adiponectin directly inhibits human endometrial cell proliferation and induced their apoptosis.However,the direct in vivo evidence that adiponectin deficiency promotes the development of endometrial carcinoma is still sorely needed.Objectives To study the mechanism of adiponectin deficiency on endometrial cancer development in vivo.Methods 1.Using single Pten gene knockout mice as a model of endometrial hyperplasia and carcinoma.Breeding and genotyping the six group mice including Pten+/+(APN+/+,APN+/-,APN-/-)and Pten+/-(APN+/+,APN+/-,APN-/-)by crossing Pten+/-and APN+/-mice.2.We observed and recored the times,sizes and positions of endometrial cancer occurred in each group of mice.3.To study some key proteins that related to signaling pathway of endometrial cancer development and the mechanism by immunohistochemistry and western blotting.4.WT,APN+/-,and APN-/-mice were treated with DES to investigated histological changes of endomtrium.Results 1.Breeding APN-/-Pten+/-and APN+/-Pten+/-primary endometrial cancer mice First we crossed Ptenloxp/loxp to ZP3-cre mice to obtain Pten+/-mice;and then crossed Pten+/-to APN+/-to get F1 generation mice;Finally crossed mice between F1 generation mice to obtain six group mice Pten+/+(APN+/+,APN+/-,APN-/-)and Pten+/-(APN+/+,APN+/-,APN-/-).Expression of Pten and APN were respectively decreased in Pten+/-and APN+/-mice,which were examined by genotyping PCR,western blotting and ELISA.Serum adiponectin was absent in APN-/-mice.2.Adiponectin deficiency increased the incidence of endometrial cancer in Pten+/-mice(1)At 30 week-old,the wet weight of uterus was increased and there were several nodules on the uterine body in APN-/-Pten+/-mice.The incidence of endometrial cancer in APN-/-Pten+/-mice was 66.7%(6/9),which in Pten+/-mice was only 28.6(2/7)%.(2)The endometrial histology of mice had no changes at12 week-old.(3)The incidence of CAH in APN-/-Pten+/-mice were 40%(2/5),however,20%(1/5)in Pten+/-mice at 22 week-old.3.Adiponectin deficiency induced the up-regulated expression of cyclin D1 in endometrial cancer(1)Immunohistochemical analysis showed that adiponectin deficiency reduced the expression of PAX2 or even loss,with the increased expression of Ki 67.(2)Western blotting analysis showed that single Pten deficiency had no changes in the expression of cyclin D1,However,adiponectin deficiency promoted the expression of cyclin D1(P<0.01,vs WT;P<0.05,vs Pten+/-),Immunohistochemical analysis was also consistent with this.However,the expression of cyclin E2 had any change in adiponectin deficiency mice.4.Adiponectin deficiency induced the increased expression of p-Erk in endometrial cancer(1)Western blotting analysis showed that the expression of p-Erk was increased in APN-/-mice(P<0.05,vs WT).No changes occurred in Pten+/-mice,however adiponectin deficiency induced the increased expression of p-Erk(P<0.001,vs Pten+/-).Similarly,immunohistochemical analysis was also consistent with this result.(2)Pten deficiency induced the increased expression of p-Akt(P<0.001,vs WT).However,no changes were founded in adiponectin deficiency mice.5.DES induced the complexity of atypical hyperplasia in APN deficient mice After DES treatment:(1)At 6 month-old,the uterus were showed cystic change and solid areas in APN-/-mice on gross.(2)The histological analysis of endometrium showed simple hyperplasia or CAH.(3)The expression of Ki67 was increased in the endometrium of APN-/-mice.Conclusion Adiponectin deficiency induced the endometrial cancer development and progression,with the increased incidence of endometrial cancer by activating the Erk1/2/Cyclin D1 signaling pathway.Background Adiponectin,secreted by adipocytes,is negatively correlated with adipocyte mass.Clinical investigations and epidemiological studies have shown that low serum adiponectin is associated with infertility in obesity women and PCOS patients.A recent research has found that non-obese women in normal fertility group have significantly higher adiponectin level in follicular fluid than the group with low fertility.Consistent with this,the abundance of all adiponectin isoforms is significantly reduced in circulation in the female pigs with subfertility.However,the in vitro studies showed the conflicting effects of adiponectin on female reproductive functions.In this study,we deployed adiponectin knockout mouse to study the effects of adiponectin deficiency on ovarian function and fertility as well as the underlying molecule mechanisms.Objectives To study the effects of adiponectin deficiency on ovarian function and female fertility as well as the related mechanism.Methods 1.To investigate the female mice fertility in Adiponectin heterozygotic(APN+/-)and homozygotic deletion(APN-/-)mice.2.To study the ability of ovarian response to gonadotropin in adiponectin deficient mice by injecting of PMSG/h CG.3.All follicles composed of primordial、primary、preantral、antral and atretic follicles were counted in every twelfth section.All these follicle types were expressed as percentages of the total number of follicles..4.Different stages of estrous cycle(proestrus、estrus、metestrus、and diestrus)were determined by Toluidine blue stain.The serum levels of Gn RH,E2,FSH,LH and progesterone were analyzed with ELISA.5.To detect the expression of Bax,IGFBP-4,LHR and CYP11A1 genes by Real-time PCR and immunohistochemistry.Results 1.The effects of female fertility in adiponectin deficient mice Homozygotic deletion of adiponectin gene in female mice led to significant reduction in litter sizes compared with the control group respectively(17.8 vs 30.3,p<0.05).2.Ovulation defects in adiponectin deficient mice Adiponectin KO mice led to significant reduction in the number of oocytes following ovulation induction.The ability of ovarian response to gonadotropin was declined.3.Abnormal follicle development in adiponectin deficient mice at 16 week-old Adiponectin deficient female mice clearly showed a substantial increase in atretic follicles and markedly reduced number of preantral and antral follicles,particularly in adiponectin null mice.The percentages of atretic follicles in the heterozygotic and homozygotic mutant mice were 12.9% and 19.3%,respectively,which was in sharp contrast to that in the wild type mice(8.5%,in both cases,p<0.001).4.Abnormal estrous cycles and sex hormones in adiponectin deficient mice APN-/-mice presented significant changes in four stages of estrus cycle which characterized by extended periods of diestrus and shortened time of estrus.Serum FSH and progesterone levels were significantly decreased in adiponectin deficient mice,however,serum LH level was increased in APN-/-mice.5.Dysregulation of gene expression in adiponectin deficent mice The expression of Bax and IGFBP-4 was markedly increased in the ovaries of adiponectin null mice.However,LH receptor and CYP11A1 were significantly lower in adiponectin deficient mice than in wild-type mice.Conclusion Adiponectin deficiency impaired female fertility,folliculogenesis,and steroidogenesis through upregulation of apoptosis-related genes(BAX,IGFBP-4)and downregultion of LH receptor and CYP11A1.