Expression And Function Of Anoctamin1Calcium-activated Chloride Channel In Mouse Ovarian Granulosa Cells

Calcium-activated chloride channels (CaCCs) are a kind of chloride channelregulated by [Ca2+]iand widely expressed in various tissues and implicated inphysiological processes. Transmembrane proteins with unknown function16A(TMEM16A, also called ANO1) has been identified as a major component of CaCCsin2008. The study of ANO1’s structure-function relationships is a hot point recently.It has been found that ANO1plays an important role in many physiological processes,such as fluid secretion in exocrine glands and intestinal tract, sensory transduction,and regulation of vessels smooth muscle contraction and so on. However, theexpression and function of ANO1in the reproductive system are little known.Follicular development was a very complicated process and under the strictcontrolling of a huge system, including gonad stimulating hormone, steroid hormonesand some local factors. Estrogens are produced mainly in granulosa cells in the ovary andplay crucial roles in female fertility by supporting normal folliculogenesis in the ovary andmodulating the structure and function of female reproductive tissues. Abnormal level ofestrogens may induce abnormal folliculogenesis, and involved in severe reproductive disordersleading to infertility and malignancies in female reproductive system such as polycystic ovariansyndrome, premature ovarian failure, ovarian cancers and granulosa cells cancer. The aim of thisstudy was to explore the expression of the Anoctamin1(ANO1) CaCC in mouse ovary and itsrole in the regulation of gonadotropin-induced estrogen production. This discovery may providenew insights into the regulation of the molecular mechanisms of folliculogenesis and ovulation.【Objectives】The aim of this study was to explore the expression of the Anoctamin1(ANO1)CaCC in mouse ovary and its role in the regulation of the molecular mechanisms offolliculogenesis and ovulation.【Methods】1. RT-PCR and Western blot were used to detect the mRNA and protein expression of ANO1in mouse ovary and granulosa cells (GCs). Byimmunohistochemistry and immunofluorecence analyses, we determined the locationof ANO1in mouse ovary.2. Patch-clamp recordings, including whole-cell patch-clamp recordings andinside-out patch-clamp recordings, were used to identify the electrophysiological properties offreshly isolated mouse GCs. At the same time, we used chloride channel inhibitor (NFA) andANO1special inhibitor (T16Ainh-A01) to analyze the electrophysiological properties ofANO1CaCC on GCs.3. ANO1mRNA in primary cultured mouse GCs was knocked down using twopairs of shRNAs targeting mouse ANO1mRNA by lentiviral transduction. ELISAanalysis was used to identify the estrogen level after ANO1mRNA knock down. Insome experiments, small molecule ANO1inhibitor (T16Ainh-A01), activator (Eact)orMEK inhibitor (U0126) were added in GCs culture medium to identify the function andmolecular mechanism of ANO1in regulation of estrogen production.4. The expression rule of ANO1was analyzed through three experiments asfollows:(1) We used vulva state observation and vaginal smears to determine thestage of the estrous cycle, real-time PCR to identify the mRNA expression variationof ANO1in the ovary during the estrous cycle.(2) We used PMSG and hCG to mimicthe process of folliculogenesis and ovulation, real-time PCR to identify the mRNAexpression variation of ANO1in the ovary GCs at different time of ovulation.(3)Different diameter of follicles were isolated under the microscope, the proteinexpression variation of ANO1in different diameter of follicles were identified byimmunohistochemistry and Western blot.5. The dehydroepiandrosterone (DHEA) induced mouse PCOS model was established.ovaries were collected for morphological studies by hematoxylin-eosinstaining (HE)staining and ANO1expression analysis by immunoblotting. Blood estradiol level wasmeasured using a Mouse estrogen ELISA Kit.【Results】1. RT-PCR products of the predicted sizes for ANO1, ANO4, ANO6and ANO10were detected in whole ovary and freshly isolated GCs. Western blot analysis detected asingle protein band at~114kDa in mouse ovary and GCs. Immunohistochemistry of ANO1 expression mainly in granulosa cells, much weaker staining was also seen in other cell typesincluding theca cells, interstitial cells, oocytes and ovarian surface epithelium.Immunofluorescence clearly demonstrated plasma membrane expression of ANO1inprimary cultured GCs.2. Whole-cell patch clamp recordings on freshly isolated single GC revealed apronounced outward rectifying property of the ANO1CaCC channel when [Ca2+]i was600nM and calcium and voltage-dependent property. These electrical properties weresimilar to the classic CaCCs’. The Ca2+-activated Cl-current was completely blockedby a small molecule ANO1inhibitor T16Ainh-A01. The electrical properties ofinside-out patch clamp recordings were as follows: the holding potential was50mV,when zero Ca2+perfusion solution was applied to the bath (intracellular side), no Cl-current appeared; but when perfusion solution was changed from zero Ca2+to1μMCa2+, CaCC channels were activated and Cl-currents were evoked remarkably. TheCa2+-activated Cl-current was completely blocked by a small molecule ANO1inhibitor T16Ainh-A01and Cl-channel blocker niflumic acid (NFA). These resultsdemonstrated the functional expression of ANO1CaCC channel in mouse GCs.3. Western blot analysis indicating about70%lower ANO1protein level in GCsafter lentivirus-mediated shRNA expression. Knockdown of ANO1mRNA orincubation with a selective inhibitor T16Ainh-A01enhanced estradiol production,whereas a selective ANO1activator Eact significantly inhibited estradiol productionin primary cultured granulosa cells. The ANO1expression and activation increasesthe phosphorylation of ERK1/2and decreases aromatase expression. This effect canbe abolished by MEK inhibitor U0126. These data determined its negative regulationon estrogen production possibly through MEK-ERK signaling cascade.4. The expression of ANO1mRNA is remarkably higher in the proestrous andestrous phases during the estrous. Immunohistochemistry and Western blot of ANO1expression in different stage ovarian follicles shows that the expression of ANO1protein is higher when the diameter is bigger. The highest expression of ANO1proteinis in large antral follicle, the lowest expression of it is in ovulated follicle.5. Compared with control group, we detected a remarkable decrease in the ANO1protein expression in DHEA-induced PCOS group. Serum estradiol level in DHEA-induced PCOS mice was significantly increased compared with control mice.【Conclusion】1. The present study identified for the first time the expression and function ofANO1Ca2+-activated chloride channel in mammalian ovarian granulosa cells.2. This Ca2+-activated Cl-current is likely mediated exclusively by ANO1inmouse ovarian GCs.3. The negative regulation of ANO1on estrogen production in granulosa cellspossibly through MEK-ERK signaling cascade regulated aromatase expression.4. Ovarian ANO1mRNA expression was stage-specific during the normal estrouscycle and regulated by gonad stimulating hormone. It may be involved in theregulation in ovulation induced by LH surge. The present study provided new insightsinto the molecular mechanisms for the regulation of folliculogenesis and ovulation.5. The expression of ANO1mRNA and protein were remarkable decreased in aDHEA-induced mouse PCOS model.【Significance】The present study identified for the first time the expression and function ofANO1Ca2+-activated chloride channel in mammalian ovarian granulosa cells. Basedon these found, we further studied the physiology role of ANO1in ovarian granulosacells. We found that ANO1activation downregulates estrogen production in granulosacells possibly through MEK-ERK signaling cascade. It may be involved infolliculogenesis and ovulation. The present study provided new insights into themolecular mechanisms for the regulation of folliculogenesis and ovulation.