The Functional Roles And The Underlying Mechanism Of HPAQR7 In Mediating Human Sperm To Respond To Progesterone

Background:After undergoing the development process in the testis and the mature process in the epididymis,mammalian sperm are still incapable of fertilizing the egg.When sperm enter into the female reproductive tract,the changes of physicochemical environment stimulate the important physiological functions of sperm,including capacitation,hyperactivation,chemotaxis,acrosome reaction,which facilitates sperm fertilizing the egg.Studies have shown that follicular fluid,cumulus cell secretion,and zona pellucida were involved in the activation of these important physiological functions of mammalian sperm.As an active ingredient in follicular fluid and cumulus cell secretion,progesterone stimulates calcium influx and chloride outflow in a short period.Meanwhile,it improves the concentration of intracellular cyclic adenonucleotide,which will in turn affect sperm capacitation,acrosome reaction,hyperactivation and chemotaxis,etc.Since the effects of progesterone on human sperm are unable to be achieved by regulating gene expression,they are called the non-genomic effects of progesterone.In recent years,the prevailing viewpoint is that progesterone regulates various physiological functions of human sperm by activating the calcium signal,which mainly be regulated by sperm-specific calcium channel(CatSper).However,there is still several controversy about whether progesterone directly affects CatSper.Recently,Miller et al found that progesterone activated ABHD2,the hydrolase existed in the plasma membrane of human sperm.Subsequently,ABHD2 would hydrolyze cannabinoid 2-AG(Cat Sper channel inhibitor)in human sperm membranes and eventually led to CatSper channel activation.These observations suggested that progesterone didn't activate the calcium current of human sperm by acting on CatSper channels directly.However,there are not enough solid evidences to prove that ABHD2 is the progesterone receptor in human sperm at present.Progestin–adiponectin Q receptor(PAQR),a newly named family of membrane protein receptors,has seven transmembrane domains,which is similar to G protein-coupled receptors.To date,11 members of PAQRs could be detected in human,including two subpopulations of progesterone receptors and adiponectin receptors.Among these,human progesterone-adiponectin receptor 7(hPAQR7,also known as mPR?)belongs to the progesterone receptor subpopulation in the PAQR family,which is observed mainly in the reproductive tissues,such as ovary and testis.The results of isotope-labeled progesterone binding experiments showed that progesterone bound to hPAQR7 receptor with characteristics of high affinity,saturation,specificity and reversibility,which were consistent with the progesterone receptor features.More importantly,hPAQR7 protein can be detected in the membrane of mature human sperm and is mainly located in the sperm tail.Thus,hPAQR7 seems to be related to the regulation of sperm motility.However,as the ideal candidate receptor for progesterone in human sperm,the functional roles and the underlying mechanism of hPAQR7 in mediating human sperm to respond to progesterone are still unclear.Objective:This article aims to reveal the functional roles and the underlying mechanism of hPAQR7 in mediating human sperm to respond to progesterone,enrich and improve the existing activation mechanisms of progesterone on CatSper in human sperm,and provide a brandly new molecular target for male infertility diagnosis and treatment.Methods:1)The expression and distribution of hPAQR7 in the testis of the young and the elderly were detected by immunohistochemistry;2)The presence of hPAQR7 transcripts and proteins in mature human spermatozoa was detected by reverse transcription PCR and western blotting;Affinity chromatography and ultra-high resolution microscopy were performed to validate progesterone will bind to hPAQR7 in human sperm and further determined the binding sites of progesterone in human sperm;4)Sperm-penetrating methylcellulose test was used to evaluate the ability of human sperm hyperactivation;5)Chromatin staining(CTC)was performed to assess human sperm acrosome reaction;6)Single-cell calcium imaging system was utilized to detect the changing of intracellular calcium concentration;7)Western blotting was carried out to quantify hPAQR7 content in human sperm;8)Super-resolution microscopy was used to verify the correlation of progesterone and hPAQR7;9)Immunofluorescence was employed to investigate the colocalization of hPAQR7,CatSper and ABHD2 in human sperm;10)Sperm patch clamping was carried out to determine the effect of hPAQR7 antibody on progesterone-activated sperm CatSper channel currents;11)Co-immunoprecipitation was performed to verify the interaction between hPAQR7,CatSper,and ABHD2.Results:1)hPAQR7 was mainly expressed in human testicular germ cells,and the expression level of hPAQR7 was higher in germ cells that located near the luminal.Notably,the hPAQR7 expression was general down-regulated in the testis of the elderly,suggested that hPAQR7 might be related to sperm function regulation;2)Both hPAQR7 transcripts and proteins could be detected in human mature sperm,and hPAQR7 was a membrane protein which mainly located in the sperm tail;3)Progesterone would bind to the head and tail of human sperm,hPAQR7 could also combine with progesterone and hPAQR7 in combination with progesterone was mainly distributed in the sperm tail;4)The hPAQR7 monoclonal antibody obtained from the extracellular amino acid sequence of hPAQR7 could greatly inhibit the activation of intracellular calcium concentration and CatSper currents by progesterone in human sperm,suggested that hPAQR7 would mediate the activation effect of progesterone on CatSper channel,then the following changes in calcium signal would ultimately affect the sperm function regulation;5)Results of co-immunoprecipitation showed that hPAQR7 antibody could form one protein complex with CatSper subunit3 and ABHD2 receptor,indicated the interaction was existing between hPAQR7 and CatSper3,ABHD2 receptor.These observations further implyed that hPAQR7-CatSper-ABHD2 formed one structure complex in modulating progesterone non-genomic effects on human sperm;6)The clinical sample screening results showed that in 42% of male infertility patients,the ability of sperm to respond to progesterone decreased(calcium signal and CatSper current).Additionally,hPAQR7 expression in more than 50% of these male infertility cases were down-regulated,and this indicated that the decrease of hPAQR7 content contributed to the decline response to progesterone in male infertility patients.Conclusion:In summary,we speculated that hPAQR7 would mediate human mature sperm response to progesterone via intracellular signaling pathway of progesterone-hPAQR7-ABHD2-CatSper.