Effect Of Perfluorooctanoic Acid(PFOA)on Gap Junctional Communication In Human Ovarian Granulosa Cells And The Relationship With Apoptosis

BackgroundEnvironmental endocrine disruptors(EEDs)can interfere with normal endocrine function.Perfluorinated and polyfluoroalkanoates(PFASs)are a class of EEDs that are widely used in daily commodities such as textiles,paper,aerospace and electroplating Due to its stable structure and low degradation,PFASs can remain in the environment as a new type of persistent organic pollutants(POPs)through bioaccumulation.PFASs have been shown to have broad toxic effects on aquatic organisms and mammals,including hepatotoxicity,immunotoxicity,neurotoxicity,endocrine toxicity,reproductive and developmental toxicity,potential genetic and carcinogenicity.The most typical and widely used PFASs are perfluorooctanoic acid(PFOA).Unlike traditional POPs,PFOA has a high affinity for proteins.When combined with proteins,it is widely ingested through the enterohepatic circulation.It is difficult to be metabolized and transformed in the body,mainly through urine and feces.Therefore,the half-life of PFOA in the body is very long about 3.5 years.Humans are exposed to PFOA via diet,air,occupation,etc.,and drinking water pollution is the most important route of exposure.At present,PFOA has been detected in human blood samples all over the world.So exploring the impact of PFOA on human reproduction has become a global problem that needs to be solved urgently.Gap junction intercellular communication(GJIC)is an intercellular membrane channel between adjacent cells formed by two linkers.It is a unique pattern of interaction between adjacent cells.The gap junction channel acts as a channel for small molecule exchange between cells,and hydrophilic molecules with a diameter of<1.5 nm or a molecular weight of<1000 Da,such as inorganic ions,cAMP and inositol 1,4,5-triphosphate which can be passively diffused by GJIC.Therefore,gap junctions play a crucial role in a wide range of physiological processes,including regulating cell growth and differentiation,maintaining tissue homeostasis and embryogenesis.GJIC is essential for the entire process of ovarian follicular development.During ovarian follicle growth,GJIC interconnects the growing oocytes with adjacent granulosa cells and granulosa cells to form a functional metabolic unit.Previous studies have shown that connexin 43(Cx43)and connexin 37(Cx37)are the major components of GJIC in ovarian tissue.Lately,studies have confirmed that PFOA can destroy the GJIC between oocytes and granulosa cells,thereby affecting the quality of mouse oocytes.In recent years,many studies have shown that PFOA exposure may have toxic effects on cells and induce apoptosis.For example,Eun et al.found that PFOA causes osteoblast apoptosis by disrupting mitochondrial function in osteoblasts.Chen et al found that PFOA exposure significantly inhibited luteal function in luteal cells induced by oxidative stress in pregnant mice.Kan et al.used iTRAQ and western blotting techniques to detect histological and proteomic changes in liver samples,and the results showed dose-dependent hepatocyte apoptosis and peroxisome proliferation.However,so far the research on apoptosis pathways mainly focuses on intracellular signal transduction pathways,ignoring the effects of cellular microenvironment and adjacent cells.Nevertheless,this does not rule out the possibility of some mechanical connection between GJIC and apoptosis.For instance,loss of connexin 43 in testicular support cells has been found to result in stagnant spermatogonia after birth,decrease number of germ cells,and impair spermatogenesis.Rita et al reported that the GJB2 mutation in the gene encoding connexin 26(Cx26)leads to apoptosis and oxidative damage in cochlear duct cells and sensorineural hearing loss.As an EED,PFOA can induce reproductive toxicity,but there are still no experimental reports on the cytotoxicity of PFOA on mammalian ovarian granules.And the mechanism that GJIC in PFOA-induced granulosa cell survival and apoptosis remains unclear.We hypothesized that PFOA-induced downregulation of connexin expression reduces GJIC and leads to apoptosis of ovarian granulosa cells.In this study,we investigated the effects of PFOA on the expression of GJIC and connexin in human ovarian granulosa cells,and then studied the possible effects and mechanisms of PFOA on apoptosis by human ovarian granulosa cell line(KGN).Objective.To investigate the effect of PFOA exposure on GJIC in ovarian granulosa cells,and to explore the role of GJIC and connexin in PFOA-induced ovarian granulosa cell apoptosis and related mechanisms.Materials and methods1)From September 2017 to January 2018,we collected 41 cases of infertility patients who received in vitro fertilization and embryo transfer(IVF-ET)or oocyte intracellular sperm injection(ICSI)due to tubal or male infertility at the Reproductive Medicine Center of the Women's Hospital of Zhejiang University School of Medicine,aged between 20 and 39 years old.All follicular fluids were collected,and granulosa cells were isolated by Fiecoll gradient centrifugation.2)We established an in vitro culture system for granulosa cells.After treatment with different concentrations of PFOA(0,0.3 ?M,3 ?M,30 ?M)for 24 hours,the cells were harvested to extract total RNA and protein.RT-PCR was used to detect the expression of gap junction-related genes Cx43 and Cx37 mRNA in granulosa cells,and the expression of gap junction-related proteins Cx43 and Cx37 was detected by western blot.3)We established an in vitro culture system for KGN cell lines.Different concentrations of PFOA(0,0.03 ?M,0.3?M,3 30?M,300 ?M)and gap junction regulators RA and CBX were added for 0,12,24,36 and 48 hours,respectively4)The cell viability was detected by CCK-8,the GJIC vitality was detected by SL/DT,the apoptosis of KGN cells was detected by Annexin V-FITC/PI apoptosis kit,and the expression of apoptosis-related proteins was detected by western blot.5)All data were statistically analyzed using the SPSS 16.0 software package.Results1)The results of in vitro culture treatment and detection in human granulosa cells indicated that compared with the control group,the expression of Cx43 in ovarian granulosa cells decreased in a concentration-dependent manner in mRNA and protein levels after PFOA exposure(P<0.05).However,Cx37 expression decreased at the mRNA level,but there was no significant change at the protein level(P>0.05)2)The of results of in vitro culture treatment and detection in KGN cells:? CCK-8 results showed that PFOA inhibited the activity of KGN cells in a concentration-time-dependent manner compared to the control group.It was most typical when exposed to PFOA(30 ?M)for 24 hours.Compared with the control group and the PFOA group,the effect of RA and CBX treatment on cell viability was not obvious,but the CBX+PFOA group significantly inhibited cell viability(P<0.05),and the RA+PFOA group was significantly increased cell viability(P<0.05).? SL/DT showed that PFOA exposure inhibited GJIC vitality in KGN cells compared to the control group(P<0.05).Compared with the PFOA group,GJIC vitality was down-regulated in the CBX+PFOA group(P<0.05),and was up-regulated in the RA+PFOA group(P<0.05).? Apoptosis by flow cytometry showed that PFOA exposure induced apoptosis of KGN cells compared with the control group(P<0.05).Compared with PFOA group,CBX increased the apoptosis of KGN cells induced by PFOA(P<0.05),and RA decreased PFOA-induced apoptosis of KGN cell(P<0.05).?Western blot analysis showed that PFOA exposure down-regulated the expression of apoptosis-related proteins Bcl-2,and up-regulated the expression of Bax and Cleaved-caspase3.The difference was statistically significant.Conclusion1)PFOA exposure can down-regulate the expression of GJIC-related genes and inhibit the function of GJIC in ovarian granulosa cells.2)PFOA may affect the proliferation of ovarian granulosa cells by inhibiting GJIC,ultimately leading to apoptosis.