Protein Phosphatase 2A Affects MiR-138-5p To Promote Apoptosis Of Spermatocytes

Background:Spermatogenesis disorder is the most common cause of male infertility and is idiopathic in about 20%of azoospermia patients.Non-obstructive azoospermia（NOA）is mainly explained by congenital factors leading to failure of spermatogenesis.Due to the complexity of spermatogenesis and testicular function,the lack of unrelated family cases and confirmatory studies,and the high genetic heterogeneity pose a challenge to the field.Current understanding of monogenic causes of NOA is very limited.Protein Phosphatase 2A（PP2A）is a member of the highly conserved serine/threonine protein phosphatase family.The latest family reports that homozygous mutation of PP2A regulatory subunit can cause 46,XY gonadal dysgenesis syndrome,but the specific regulation mechanism of spermatogenesis is still unclear.Purpose:Our research group constructed a germline conditional knockout of PP2A catalytic subunit alpha gene Ppp2ca(Ppp2ca^{c KO})mice.The purpose of high-throughput miRNA sequencing in testis tissue was to analyze the differentially expressed miRNA and differentially expressed genes in testis of Ppp2ca^{c KO} mice by bioinformatics method to elucidate the regulation of PP2A-mediated miRNA and its target genes on spermatogenesis.Since miR-138-5p is highly conserved between humans and mice and has complete sequence identity,miR-138-5p was selected for this study.The expression of miR-138-5p was significantly reduced in the testis of Ppp2ca^{c KO} mice.Meanwhile,the atypical GATA transcription repressor Trps1 was highly expressed.To investigate the effect of miR-138-5p on the proliferation and apoptosis of spermatocytes and the targeting relationship between miR-138-5p and Trps1.Methods:Germline conditional Ppp2ca knockout mice were constructed based on the principle of Cre-lox P recombinase principle.Epididymal sperm smears and testicular and epididymal hematoxylin-eosin staining were performed to determine the testicular development and spermatogenesis of the knockout mice.The expression of PP2Ac protein in testicular tissue was determined by Western blot（WB）.Double immunofluorescence staining was used to determine the expression and localization of PP2Ac protein in germ cells.TUNEL staining was used to determine the apoptosis of seminiferous tubules in Ppp2ca^{c KO} mice.High-throughput sequencing was used to screen the differentially expressed miRNAs in the testis of Ppp2ca^{c KO} mice.The expression of miR-138-5p in GC2 cells was detected after PP2A enzyme was inhibited by Okadaic acid.TUNEL staining and flow cytometry were used to detect the effect of miR-138-5p on GC2 cell apoptosis.RT-q PCR and WB were used to detect the effect of miR-138-5p on Trps1 and apoptosis-related molecules in GC2 cells.Bioinformatics predicted the targeting relationship between miR-138-5p and Trps1 and its binding targets.Luciferase gene reporter assay was used to explore the targeted binding between miR-138-5p and Trps1.Results:The testis weight of the Ppp2ca^{c KO} mice was significantly reduced,and no spermiocytes and mature sperm were observed in the sperm smear.In Ppp2ca^{c KO} mice,the number and layers of germ cells were significantly reduced,and the chromatin condensation was abnormal and chromatin was scattered in the nucleus with a large number of vacuolated structures in spermatocytes.TUNEL staining showed obvious apoptosis of spermatocytes.Mi RNA high-throughput sequencing identified 36significantly differentially expressed miRNAs.The expression of miR-138-5p was significantly decreased in the testis of Ppp2ca^{c KO} mice and the expression of miR-138-5p was significantly decreased in GC2 cells after inhibiting PP2A enzyme activity.TUNEL and flow cytometry showed that overexpression of miR-138-5p inhibited the apoptosis of GC2 cells.Inhibition of miR-138-5p promoted apoptosis of GC2 cells.Luciferase reporter assay confirmed that miR-138-5p targeted Trps1 and negatively regulated the expression level of Trps1.Overexpression of miR-138-5p decreased the expression of Trps1,increased the expression of apoptosis-related factor Bcl2,and decreased the expression of Bax,Caspase3 and Caspase9.When the expression of miR-138-5p was inhibited,the expression of Trps1 and apoptosis-related factor Bcl2 decreased,and the expression of Bax,Caspase3 and Caspase9 increased.Conclusions:Ppp2ca^{c KO} mice have a significant reduction in testicular weight and failure of spermatogenesis.Ppp2ca^{c KO} mice showed significantly reduced layers of germ cells in seminiferous tubules,aberrant chromatin and vacuolated structures in spermatocytes.TUNEL staining showed that the spermatocytes had obvious apoptosis signals.In the testis tissue of Ppp2ca^{c KO} mice,miR-138-5p was significantly down-regulated and Trps1 was significantly up-regulated.Mi R-138-5p targeted Trps1and negatively regulated Trps1 expression.Overexpression of miR-138-5p inhibited apoptosis,and inhibition of miR-138-5p promoted apoptosis of GC2 cells.In conclusion,miR-138-5p may promote spermatocyte apoptosis by targeting Trps1 after PP2A enzyme dysfunction.