Regulatory Mechanism Of ADGRG2 On Fluid Reabsorption In Efferent Ductules And Male Fertility

Male infertility is transforming from a personal issue to a public health problem because approximately 15%of reproductive-age couples are infertile,and male infertility accounts for approximately 50%of this sterility.Among male reproductive system,the efferent ductules play important roles during sperm transportation and maturation by reabsorbing the fluid of the rete testis and maintaining the homeostasis of water and ion metabolism.Whereas a dysfunction of the efferent ductules reabsorption capacity caused by a developmental defect that produces improper signaling results in epididymal obstructions and abnormal spermiostasis,which ultimately lead to infertility in both humans and other mammals.Knockout of an orphan G protein-coupled receptor,ADGRG2（adhesion G-protein coupled receptor G2）,results in male infertility due to dysregulated fluid reabsorption in the efferent ductules,suggesting an active role for this cell surface receptor in regulating these processes.However,how ADGRG2 regulates water-ion homeostasis and fluid reabsorption remains elusive.ADGRG2 belongs to the 7 transmembrane receptor superfamily,which regulates approximately 80%of signal transduction across the plasma membrane.Different types of G proteins and arrestins act as signaling hubs downstream of these GPCRs（G protein-coupled receptors）,mediating most of their functions.In the efferent ductules,it remains unclear how G proteins and their parallel signaling molecules,the arrestins,regulate reabsorption as well as fertility.Objective1.How ADGRG2 regulates water-ion homeostasis and fluid reabsorptionin the male efferent ductules;2.How G proteins regulate reabsorption as well as fertilityin the efferent ductules;3.The role of β-arrestins in regulating fluid reabsorption and male fertility in the efferent ductules;4.Effects of specific amino acid residues of ADGRG2on the coupling of ADGRG2 to the G protein subtypes.5.Whether the conditional expression of wild-type ADGRG2 was able to rescue the reproductive deficient phenotype of Adgrg2-/Y mice.MethodsThe main research methods are summarized as follows.1.Preparation of the membrane fraction of the epididymis and efferent ductulesfor Western blot and Co-immunoprecipitation Assays.2.Isolation and ligation of efferent ductuleswas used to detect the change of the diameter of the efferent ductules under different experimental conditions.3.Recombinant adenovirus and lentivirus construction was used to infect the micein vivo or infect the isolated efferent ductules.4.Measurement of intracellular pH（pHi）with a pH-sensitive fluorophore carboxy-SNARF（?）-1.5.qRT-PCR analysis of mRNA transcription profiles of G proteins and potential osmotic drivers including selective ion channels and transporters.6.Immunofluorescence staining to detect the expression and localization of the related proteins in the efferent ductules.7.Culture of mouse epididymal efferent duct epitheliumfor qRT-PCR analysis and electrophysiological related experiments.8.Co-immunoprecipitation was used to detect the interaction between related proteins and proteins in the efferent ductules.9.Whole-cell patch-clamp recordingfor electrophysiological detection of isolated epididymal efferent duct epitheliumand HEK293 cells in vitro.10.cAMP and IP1 ELISA for the detection of cAMP and IP1 levels in the tissues.11.GloSensor cAMP detects cAMP levels in HEK293 cells.12.NFAT dual-luciferase reporter assaydetectsCa2+ levels in HEK293 cells.13.Tissue HE staining for detection of related morphological indicators.14.Treatment of mice efferent ductules withCFTR siRNA dicerwas used to examine the effect of CFTR knockdown on efferent ductules cells.15.Statistics,All of the data are presented as the mean±SD from at least three independent experiments.Statistical comparisons were performed using an ANOVA with GraphPad Prism5.Significant differences were accepted atp<0₀5.Results1.Gq activity is required for fluid reabsorption and male fertility.ADGRG2 localizes in cells devoid of acetylated-tubulin staining,suggesting that it is specifically expressed in non-ciliated cells.ADGRG2-expressing non-ciliated cells have expression levels of Golf,Gi2,Gq,G11,and G13 that are higher than those in brain tissue and expression levels of Gs,G12 and Gz that are similar to those in brain tissue.We next investigated the contribution of different G protein subtype signaling pathways to fluid reabsorption in the efferent ductules using specific pharmacological interventions and knockout models.The wild-type（WT）mice did not show size alterations due to the normal reabsorption of luminal fluid,but the ligated efferent ductules derived from the ADGRG2 knockout mice displayed a 40%increase in luminal area after 72 h of in vitro culture.Consistently,a 50%reduction in Gq protein levels in Gnaq+/-mice or the application of the protein kinase C（PKC）inhibitor Ro 31-8220 significantly impaired fluid reabsorption in the efferent ductules,which mimicked the phenotype of the ductules derived from Adgrg2-/Y mice.This indicates that Gq-PKC signaling was required for efficient fluid reabsorption in the efferent ductules.The efferent ductules of the Gnaq+/-animals consistently showed the accumulation of obstructed spermatozoa compared with those of WT mice,whereas the lumen of the initial segment and caput region in Gnaq+/-mice contained significantly reduced sperm levels.Sperm numbers prepared from the caudal epididymis and the birth rate of the Gnaq+/-mice were also significantly decreased compared with their WT littermates.Taken together,these data demonstrated that among different G protein subtypes,Gq activity is required for fluid reabsorption and male fertility.2.ADGRG2 and CFTR coupling in the efferent ductules and its function in fluid reabsorptionWe examined the expression levels of these membrane proteins in the efferent ductules and ADGRG2 promoter-labeled ductule cells.Specifically,NKCC,DRA,CFTR,SLC26a9,NHE3 and Cavl.3 levels were readily measured in ADGRG2 promoter-labeled non-ciliated ductule cells.We next used a panel of pharmacological blockers to examine whether the inappropriate regulation of these membrane protein functions was involved in the ADGRG2-or Gq-mediated regulation of fluid reabsorption in the efferent ductules.Blocking CFTR activity either with GlyH-101 or CFTRinh-172 had significant effects on fluid reabsorption in the efferent ductules and pheno-copied the Adgrg2-/Y mice.Collectively,the phenotype caused by inactivating ADGRG2 and administering a CFTR channel blocker in WT mice suggested that CFTR and ADGRG2 may be functionally connected to the regulation of fluid reabsorption.CFTR is the key regulator of pH homeostasis and chloride in the reproductive and renal systems and has important functions in fluid reabsorption.The pH homeostasis was impaired in Adgrg2-/Y mice,with a pH value of 7.6 for the inner solution in the efferent ductules,compared to a pH of 7.2 in WT littermates.Moreover,application of the CFTR inhibitor CFTRinh-172 increased the pH value of the efferent ductules in WT mice by approximately 0.3 but did not have a significant effect in Adgrg2-/Y mice,suggesting that CFTR dysfunction in Adgrg2-/Y mice influences pH homeostasis.In particular,unambiguous co-localization of ADGRG2 and CFTR on the apical membrane was detectedand ADGRG2 was associated with CFTR in co-immunoprecipitation assays.Taken together,these results suggest a complex formation and functional coupling of ADGRG2 and CFTR in the non-ciliated cells of the efferent ductules.3.The outwardly rectifying whole-cell CL-current（IASGRG2-ED）of ADGRG2 promoter-labeled efferent ductule cellsWe then performed whole-cell Cl-recording of primary ADGRG2 promoter-labeled efferent ductule cells derived from WT and Adgrg2-/Y mice.Patch-clamp recording on ADGRG2 promoter-labeled non-ciliated cells derived from WT mice revealed a reversible whole-cell Cl-current（IADGRG2-ED）,which was significantly diminished in response to substitution of the bath Cl-solution with Gluc-（148.5 mM Cl-was replaced by 48.5 mM Cl-and 100 mM Gluc-）.This whole-cell Cl-current（IADGRG2-ED）was recovered once Gluc-was substituted with Cl-solution.In contrast,the IADGRG2-ED ofAdgrg2-/Y mice was substantially lower than the IADGRG2-ED of their WT littermates,which showed no significant changes in response to substitution of the bath Cl-solution with Gluc-.These results suggested that ADGRG2 deficiency in the efferent ductules significantly reduced the whole-cell Cl-current of ADGRG2 promoter-labeled non-ciliated cells.4.CFTR mediates the whole-cell Cl-current of ADGRG2 promoter-labeled efferent ductule cellsWe next examined the effects of different Cl-channel and transporter inhibitors on the IADGRG2-ED of efferent ductule cells derived from Adgrg2-/Y mice and their WT littermates.The specific CFTR inhibitor CFTRinh-172 significantly reduced the IADGRG2-ED current.Moreover,the difference in the IADGRG2-ED between Adgrg2-/Y mice and their WT littermates was eliminated by the application of CFTRinh-172.Consistently,when we knocked down CFTR expression in efferent ductules,the whole-cell Cl-current（IADGRG2-ED）of WT mice was significantly reduced.These results suggested that CFTR is essentially activated in ADGRG2 promoter-labeled efferent ductule cells,which mediate the observed outwardly rectifying whole-cell Cl-current,and ADGRG2 is required for the basic activation of CFTR in these cells.5.Gq activity is required for ADGRG2/CFTR coupling in the efferent ductulesSimilar to Adgrg2-/Y mice,the efferent ductules derived from Gnaq+/-mice exhibited imbalances in pH homeostasis.Consistently,we observed a significantly decreased whole-cell Cl-IADGRG2-ED current of the ADGRG2 promoter-RFP-labeled primary non-ciliated cells in Gnaq+/-mice compared with that observed in their WT littermates.The application of Ro 31-8220,an inhibitor of the Gq downstream effector PKC,further inhibited the observed IADGRG2-ED.These results indicated that the Gq-PKC pathway plays critical roles in basic CFTR activation in the efferent ductules,which controls Cl-and pH homeostasis for efficient fluid reabsorption.We next investigated whether Gq activation by ADGRG2 is required for CFTR function.In the efferent ductules,the Gq is localized in ADGRG2-expressing cells but not acetylated tubulin-labeled cells.Consistently,Gq was readily detected in ADGRG2 antibody immuno-precipitated complexes,suggesting a physical interaction of ADGRG2 with Gq in the efferent ductules.Moreover,the endogenous resting IP1 and cAMP levels of the ligated efferent ductules derived from the Adgrg2-/Y mice were significantly lower than those of their WT littermates.Taken together,these data indicate that Gq regulates fluid reabsorption by mediating ADGRG2/CFTR coupling,and both the Gq-IP3-PKC pathway and the Gs-cAMP pathway were activated in ADGRG2 promoter-labeled efferent ductule cells.6.ADGRG2/CFTR complex formation mediated by β-arrestin-1 but not P-arrestin-2 is essential for fluid reabsorption in the efferent ductulesWe examined the fluid reabsorption in Arrbl-/-and Arrb2-/-knockout mice.Whereas the efferent ductules derived from Arrb2-/-knockout mice showed normal fluid reabsorption as well as pH homeostasis compared to their WT littermates,these functions of the efferent ductules derived from Arrbl-/-knockout mice were significantly impaired.Moreover,whereas ADGRG2 and CFTR co-localized in the apical membrane regions of the non-ciliated cells of the efferent ductules derived from Arrb2-/-or WT mice,they were separated in Arrbl-/-mice.In P-arrestin-1-deficient efferent ductules,CFTR localized away from ezrin,an apical membrane marker,suggesting that β-arrestin-1 is required for the correct localization of CFTR.Consistently,whereas CFTR was co-immunoprecipitated with ADGRG2 in WT and Arrb2-/-mice,it was not found in ADGRG2-immunoprecipitated complexes from the efferent ductules derived from Arrbl-/-mice,further suggesting thatβ-arrestin-1 is an essential component in a signaling complex encompassing ADGRG2 and CFTR in the efferent ductules.7.Molecular determinants of ADGRG2 coupling with G protein subtypes and their contribution to the regulation of CFTR activity in vitroIn vitro,the overexpression of ADGRG2 causes constitutive Gs and Gq coupling activity;a stronger effect is observed with ADGRG2tβ.Whole-cell recordings were performed to examine the effects of ADGRG2 and CFTR co-expression on membrane currents by using an I-V analysis.The co-expression of ADGRG2 and CFTR significantly increased the amplitude and slope of the current responses,which were significantly reduced by the CFTR inhibitor CFTRinh-172,compared with cells transfected with CFTR alone,indicating that CFTR channels are activated by ADGRG2 in a recombinant system.Importantly,increased CFTR activity induced by ADGRG2 was significantly diminished by the PKC inhibitor Ro 31-8220.Taken together,these data demonstrate that ADGRG2 increases CFTR Cl-currents through the activation of Gq-PLC-PKC signaling.We selected mutations in intracellular loops 2 and 3 of ADGRG2,the coupling of these ADGRG2 mutants to CFTR activity was then examined using the whole-cell recording technique.Voltage clamps were used to generate the I-V relationships of the CFTR currents in cells co-transfected with CFTR and ADGRG2.Interestingly,although the mutant with a specific Gs signaling defect showed decreased coupling of ADGRG2 to CFTR,the Gq-dysfunctional mutant and the H696A/M697A double Gs/Gq signaling-defective mutant did not demonstrate coupling between ADGRG2 and CFTR.Taken together,these results demonstrate that specific residues in intracellular loops 2 and 3 are determinants of the G protein subtype coupling of ADGRG2.Furthermore,downstream of ADGRG2,Gq signaling is essential for CFTR activation in recombinant in vitro systems.8.Effects of the conditional expression of WT-ADGRG2 or its selective G-subtype signaling mutants on the rescue of reproductive defects in Adgrg2-/Y miceWe next examined how the molecular determinants of ADGRG2/G protein subtype interactions contribute to the function of ADGRG2 infertility in vivo.The efferent ductules of Adgrg2-/Y animals frequently exhibited the accumulation of obstructed spermatozoa and significantly reduced sperm numbers and presented morphologically abnormal spermcompared with observations in WT mice.The conditional expression of ADGRG2 in non-ciliated cells in Adgrg2-/Y mice significantly reduced the accumulation of spermatozoa and restored sperm numbers in the caudal epididymis by more than half,whereas the expression of G protein signaling-deficient mutants of ADGRG2,including Y698A,F705A,Y708A,RK803EE and HM696AA,significantly reduced this rescue effect.Specifically,conditional infection of the Gs/Gq double signaling-deficient mutant ADGRG2-HM696AA or the Gq signaling-deficient mutants Y708A and RK803EE did not result in differing levels of accumulation in the efferent ductules compared with those inAdgrg2-/Y mice infected with a control virus.To investigate whether the sperm production phenotype was related to fluid reabsorption,we isolated the efferent ductules after virus infection with the WT ADGRG2 or one of the mutants and measured the luminal area after ligation.Interestingly,conditional expression of the Gs-deficient mutations Y698A and F705A marginally reduced the inflation of the efferent ductules of Adgrg2-/Y mice,whereas the Gq signaling mutants did not exert significant effects on the luminal volume.Taken together,our results demonstrate that Gq activity is required downstream of ADGRG2,and contributes to fluid reabsorption in the efferent ductules and sperm transportation.Conclusion1.Gq activity is required for fluid reabsorption and male fertility.2.ADGRG2 and CFTR coupling in the efferent ductules are required for fluid reabsorption.3.CFTR mediates the whole-cell Cl-current of ADGRG2 promoter-labeled efferent ductule cells.4.Gq activity is required for ADGRG2/CFTR coupling in the efferent ductules.5.ADGRG2/CFTR complex formation mediated by(3-arrestin-1 but not β-arrestin-2 is essential for fluid reabsorption in the efferent ductules.6.Specific amino acid residues in intracellular loops 2 and 3 of ADGRG2 are molecular determinants of the coupling of ADGRG2 to the G protein subtypes.7.Conditional expression of wild-type ADGRG2 was able to rescue the reproductive deficient phenotype of Adgrg2-/Y mice.