The Role Of Progesterone Adiponectin Receptor 7 In Mouse Reproduction

Background:Progestin-adiponectin Q receptor 7?PAQR7?is an important member of the progesterone receptor subgroup,a newly named family of membrane receptors.PAQR7 was initially cloned from the ovary of sea trout.It has a typical 7 times transmembrane structure of G-protein-coupled receptor.And it can couple with inhibitory G-protein,regulate adenylate cyclase activity,and participate in progesterone mediated oocyte maturation as progesterone membrane receptor.PAQR7 is also present in the sperm of sea trout.It can couple with an olfactory related excitatory G protein(G_olf),which mediates the sperm hyperactivity induced by progesterone.These results suggest that PAQR7 plays an important role in the reproduction of fish.In addition,PAQR7 is highly expressed in mouse and human reproductive tissues,but its role in mammalian reproduction is still unclear.Objective:The purpose of this research is to reveal the role of PAQR7 in mouse reproduction.Methods:1.Detecting the expression of PAQR7 in various tissues of mice by Real-time fluorescence quantitative PCR.2.Detecting the expression of PAQR7 in mouse sperm,testis and ovary by Western blot.3.The effects of PAQR7 antibody on mouse sperm function and the success rate of in vitro fertilization were detected.4.The?Clustered regularly interspaced short palindromic repeats/cas9,CRISPR/cas9?gene editing technology was used to construct PAQR7 systemic knockout mice,and the knockout results were verified by genotype identification,Real-time fluorescent quantitative PCR,Western blot,and immunofluorescence.5.Phenotype identification of PAQR7 knockout mice:?1?Male mice of different genotypes were mating with female mice of different genotypes and then their fertility phenotype were recorded;?2?Fertility phenotype of PAQR7 knockout mice were detected by in vitro fertilization technology;?3?Sperm motility-related parameters of mice were detected by computer assisted analysis system;?4?Observing the morphology of testis?spermatozoa and ovaries and weighing the testis and ovaries with a balance;?5?The physiological state of reproductive tissues were evaluated by Hematoxylin-eosin staining;?6?The changes of hormone levels in the peripheral blood of female mice were detected by Enzyme-Linked Immunosorbent Assay.6.Real-time quantitative PCR was used to detect the expression of progesterone receptor subunits and progesterone receptor family of PAQR7 in female and male mice.7.The expression of reproductive-related genes in different genotypes of female and male mice were detected by Real-time quantitative PCR.Results:1.The results of Real-time quantitative PCR showed that PAQR7 was highly expressed in the testis and ovary of mice.2.PAQR7 can be detected in the total and membrane protein of mouse testis,ovary and sperm by Western blot.3.PAQR7 specific antibody can significantly inhibit the sperm progressivity?PR??P<0.05?,and reduce the success rate of in vitro fertilization?IVF??P<0.05?.4.The systemic PAQR7 knockout mice were successfully constructed by CRISPR/cas9 gene editing technique.The deletion of 812bp located in the third exon of PAQR7 in the homozygous knockout mice(PAQR7^-/-)resulted in the premature termination of translation of the frameshift mutation and then the deletion of 39-345 amino acid sequence of translation protein?wild-type PAQR7 has 345 amino acid?was predicted.5.?1?The results of in vitro fertilization showed that the fertility of PAQR7^-/-male mice was not affected,but the average litters and the average litter size of PAQR7^-/-female mice and wild-type male mice or PAQR7^-/-male mice significantly reduced?P<0.05?;?2?The results of in vitro fertilization showed that the fertilization rate of PAQR7^-/-female mice and wild-type male mice or PAQR7^-/-male mice significantly reduced?P<0.05?.The results of computer-assisted analysis showed that the sperm concentration,total motility,progressivity,movement rate and head side swing of PAQR7^-/-male mice decreased significantly?P<0.05?.The results of morphology of testis,ovaries and spermatozoa showed that the quality of testis of PAQR7^-/-male mice decreased significantly?P<0.05?,the index of testis decreased?P<0.05?,the ovaries became smaller,the abnormality of spermatozoa tail was obvious,and the rate of abnormality was significantly higher than that of wild-type mice?P<0.05?.?5?HE staining showed that there were cavities in part of seminiferous tubules of testis of PAQR7^-/-male mice.The number of antral follicles in ovaries of PAQR7^-/-female mice decreased,the number of primordial follicles decreased significantly,the number of corpus luteum increased,and most of follicles showed atresia and apoptosis.?6?The levels of anti mullerian tube hormone and estradiol hormone in serum of PAQR7^-/-female mice were significantly lower than those of wild-type mice,and the levels of progesterone and follicle stimulating hormone showed no statistical differences?P>0.05?.6.The relative expression levels of PGRMC1?PGRMC2?NENF in the testis of PAQR7^-/-male mice significantly increased?P<0.05?,the expression of CYB5D2 significantly decreased?P<0.05?,the expression of PAQR5?PAQR6?PAQR8?PAQR9 which belong to PAQR subfamily decreased significantly?P<0.05?,but there was no significant difference in the expression of PGR;The relative expression levels of PGR?NENF?PAQR5?PAQR6?PAQR8?PAQR9 in the ovary of PAQR7^-/-female mice were significantly higher than that of wild type mice?P<0.05?,but the expression of PGRMC1?PGRMC2 and CYB5D2 showed no statistical differences.7.The expression of Spata25?Tex19.2?Znrf4?Tssk6?InsL3 which are closely related to spermatogenesis,motility and morphogenesis decreased significantly?P<0.05?.The expression of DMC1?POU5F1?GDF-9?ZP3?SYCP3?NOBOX which are closely related to ovarian follicle growth,ovarian development and normal fertilization significantly decreased?P<0.05?.Conclusions:In conclusion,we believe that PAQR7 plays a very important role in the reproductive process of mice,and plays an important role in the maintenance of sperm morphology,motility and ovarian function.