Expression Of Amyloid Precursor Protein In Ovarian Granulosa Cells And Its Relationship With Ovarian Aging

Ovarian aging particularly early-onset ovarian aging (Premature Ovarian Aging, POA) associated with declined ability to produce eggs, poor quality of oocytes is one of the leading cause of female infertility, it is a great threat to women’s reproductive health. There are thousands of researches on ovarian aging mainly focused on finding out biomarkers which can predict and judge the ovarian function, however, study of precise mechanism underline diminished ovarian reserve is rare. It is generally believed that age is the most important factors influencing the ovarian reserve. But there is great individual difference among ovarian aging patients, which suggests the internal factors of ovary are more important than age to decide ovarian reserve. Follicular atresia result from follicle growth abnormalities and acceleration rate of granulose cells apoptosis is the primary cause of diminished ovarian reserve. To study genes involved in regulating follicle growth, development and atresia process to reveal the mechanism of diminished ovarian reserve has important significance. A research on Alzheimer’s disease (AD) found that the model transgenic mice of AD showed sterility, the level of Amyloid Precursor Protein (APP) and its metabolite levels which play a key role in AD are all increased. APP protein is a type I transmembrane glycoprotein, processed by β,γ-secretase and produced a APP carboxyl terminal fragment (C-terminal fragment, the CTF)---C99,which contains a complete sequence of Aβ.One of the processed APP products, β-amyloid is a major component of amyloid plaque, which is frequently detected in the brain tissues with Alzheimer’s disease.BACEl expresses in almost all the organs and tissues of human, it has play an important role in the cleaving process of APP,and the generation, aggregation and sedimentation of Aβ is considered to be the first and central part of the pathogenesis of AD. Some studies have confirmed that high expression of APP and its metabolites in genetically modified mice may increase the susceptibility to oxidative stress, reduce the anti-apoptosis ability and increase the death rate of cells, which suggests that APP and its metabolites may influence the growth and apoptosis of ovarian follicle as well as ovarian reserve by increase the susceptibility to apoptosis induced by oxidative stress and induce the cell apoptosis. The research on the role of the APP protein in the ovarian aging is still blank. This study proposed to detect APP,β-secretase and members of Bcl-2family expression level in ovarian granulosa cells of patients with ovarian aging, and Aβ42expression level in follicular fluid by molecular and biology method, analyze the correlation with quantity and quality of eggs and embryo as well as in vitro fertilization-embryo transfer (IVF-ET) clinical outcomes, and try to explore the possible molecular mechanism of diminished ovarian reserve mediated by APP and its metabolites, provide the theory basis for prevention and treatment of ovarian aging.Part I Expression of Amyloid precursor protein in ovarian granulosa cells and its relationship with IVF outcomesObjective:To study the expression condition of APP in granulosa cells and its relationship with ovarian aging, detected the protein and gene expression level of APP in granulosa cells of infertility patients who under IVF/ICSI-ET treatment in Nanfang Hospital, and analyze its expression differences in different age patients and the correlation with IVF outcome.Methods:1. Follicular fluid of192infertility patients was collected from the IVF/ICSI-ET programs in Reproductive Medical Center of Nanfang Hospital of Southern Medical University. All the patients were suffered from infertility due to tubal factor and male factor.2. Granulosa cells were purified from follicular fluid by Percoll density gradient centrifugation method and the red cell lysis buffer was used to eliminate the interference from red cells.3. Western-blot was applied to detect the APP protein expression level for67samples to detect the protein expression level of APP, all the samples were divided into three groups according to patients’ age:group A:<35years old; Group B:≥35&<40years old; Group C:≥40.QRT-PCR(quantitative real-time polymerase chain reaction) was applied to detect the expression of APPmRNA level for138samples, all the cumulus cells were divided into three groups according to patients’ age:group A1:<35years old; Group B1:≥35&<40years old; Group C1:≥40.4. The clinical data were collect,SPSS16.0statistical software was used to compare the APP expression level in different groups and analyze its correlation with age, antral follicle count (AFC), number of normal fertilized egg, number of oocyte, number of embryo.a=0.05was set as the inspection level and P＜0.05was considered significant.Results:1. Granulosa cells APP protein group:there were significant difference in age,AFC,total Gn does, number of oocyte,total number of normal fertilization, number of embryo;APP mRNA groups:there were significant difference in age,AFC,E2level on HCG day, total does of Gn,number of oocyte,total number of normal fertilization, number of embryo.(P＜0.05);2. Compared with young age group of granulosa cells, the APP protein expression level was significantly higher in the old age groups.(1.12±0.75,2.02±1.17vs3.47±1.22,P<0.05);APP mRNA level was also increased with age significantly.(1.88±1.34,3.58±2.58vs6.59±6.54, P<0.05);3. APP protein expression level in granulosa cells was positively correlated with age (r=0.646,P=0.000),and negatively with AFC, number of oocyte and embryo(r=-0.553,P=0.000;r=-0.601,P=0.000;r=-586,P=0.000).The APP gene expression level was positively correlated with age (r=0.488, P=0.000), and negatively with AFC, number of oocyte and embryo (r=0.189, P=0.026; r=0.175, P=0.040; r=0.177, P=0.037).Conclusions:1. Amyloid Precursor Protein and its mRNA are expressed in ovarian granulosa cells. With the increase of age, the accumulation of APP protein in ovarian is elevated, protein APP maybe a predictor of ovarian aging.2. Both APP gene and protein expression level in ovarian granulose cells are negatively correlated with AFC and number of oocyte.To some extent, the expression of APP may influence functional ovarian reserve. Part II Impact of Amyloid precursor protein and its metabolites on ovarian agingObjective:To study the expression condition of APP, BACE1, Bcl-2and Bax gene in granulosa cells and Aβ42in follicular fluid, analyze the correlation between each gene and the genes with IVF outcomes; explore the impact of APP and its metabolites on diminished ovarian reverse.Methods:1. Follicular fluid of80infertility patients was collected from the IVF/ICSI-ET programs in Reproductive Medical Center of Nanfang Hospital of Southern Medical University.2. Granulosa cells were purified from follicular fluid by Percoll density gradient centrifugation method and the red cell lysis buffer was used to eliminate the interference from red cells. The first glass of clear follicular fluid was cryo-stored after centrifugation.3. All the samples were divided into three groups according to patients’age and biomarkers of ovarian reserve(basal FSH,AMH):(1)early-onset ovarian aging/occult with poi (POA/OPOI) group:age<38, bFSH>10IU/L or AMH<1.1ng/ml,19cases;(2) physiological diminished ovarian reserve (DOR) group: age<40,29cases;(3) normal ovarian reserve(NOR) group:age<38, bFSH<10IU/L or AMH>1.1ng/ml,32cases.4. QRT-PCR was applied to detect APP, BACE1, Bax, Bcl-2gene expression; Elisa was applied to detect the expression of Aβ42.5. The clinical data were collect,SPSS16.0statistical software was used to compare the APP expression level in different groups and analyze its correlation with age, antral follicle count (AFC), number of normal fertilized egg, number of oocyte, number of embryo.a=0.05was set as the inspection level and P＜0.05was considered significant.Results:1. There were significant difference in age,AFC, E2level on HCG day, number of oocyte, total number of normal fertilization, number of oocyte in three groups. After further comparison:there is no difference in age between POA group and NOR group; there are no difference in AFC, E2level on HCG day, number of oocyte, total number of normal fertilization, number of oocyte between POA group and DOR group;2. Compared with NOR group, APP, B ACE1,Bax mRNA expression level were increased significantly, the Bcl-2level was decreased both in POA and DOR group(P<0.05);Compared with the POA group, APP,BACE1,Bcl-2and Bax mRNA expression were slightly lower in DOR group, but there was no statistically significance (P>0.05);3. The correlation analysis showed:APPmRNA was positively correlated with BACE1, Bax expression, and negatively correlated with the expression of Bcl-2(P<0.05);APP, BACE1gene expression were positively correlated with age, Bcl-2expression was negatively related with age;APP,BACE1,Bax gene expression were negatively related with the AFC,Bcl-2expression was positively correlated with AFC;APP and BACE1gene expression were negatively related with number of oocyte;BACEl and Bax gene expression were negatively related with the number of embryo(P<0.05);4. Compared with NOR group, Aβ42level was higher in POA and DORgroup, the difference was statistically significant (P<0.05). A042level in POA group was a little higher than DOR group.5. The correlation analysis showed:Aβ42level was positively correlated with BACElgene expression, and was negatively related with Bcl-2and number of oocyte.Conclusions:1. The increased expression of APP, BACE1and A042may be associated with diminished ovarian reserve.2. High expression of APP may reduce Bcl-2levels in ovarian granulose cells, which may play a role in the development of follicles, the growth and apoptosis of granulosa cells and mature of oocytes, thus affect ovarian reserve function.