Studies On Function And Related Molecular Mechanism Of BAG6 Exon 24 Skipping Affecting Mammalian Testicular Cell Growth And Blood-testis Barrier Assembly

Spermatogenesis is a complicated physiological process,which requires specific genes to perform certain regulation functions.Alternative splicing is a process that generates different m RNA isoforms from pre-m RNA in various ways,and the final protein products may show different structures or functions,thus playing an important role in animal physiological processes,such as spermatogenesis.Transcriptome sequencing of the sexually mature and immature testes from Large White pigs had been done in this research,and the differentially expressed genes were detected.BAG6 gene was differentially expressed between the sexually mature and immature testes and enriched in spermatogenesis.The alternative splicing event analysis of the sequencing data was performed using SOAPsplice and MISO software,respectively,and two splice variants of BAG6 gene were screened out: Full-length variant（BAG6-FL）and Exon 24 skipped variant（BAG6-Δ24）.The functions of BAG6 two splice variants in testis Sertoli cells were explored.The main results are as follows:1.Transcriptome sequencing and data analysis of the sexually mature and immature testes from Large White pigs（1）We selected the testes of 60-d and 180-d Large White pigs as experimental smaples to do the transcriptome sequencing（completed by BGI Company）.Taking an FDR of 0.001 and a|log2Ratio|of 1 as cutoffs,we detected 10,095 DEGs,of which 5,199 genes were up-regulated in 180-d testes and 4,896 genes were up-regulated in 60-d testes.We verified nine DEGs（PIWIL4,BAG6,DCTN,INHA,INHBA,SP1,TESK2,PRKAR1 A and PRKACA）by q RT-PCR.The results showed that expression patterns of the eight genes were consistent with those of the RNA sequencing results,except DCTN(Pearson correlation coefficient=0.96,P=3.31×10^-5).（2）The significant enrichment analysis of GO terms in DEGs showed that 5,995 genes were classified to 34 GO terms（P<0.05）,of which 242 DEGs were significantly enriched in spermatogenesis（GO: 0007283）,which contained PIWIL family,SPATA family and BAG6.The significant enrichment analysis of KEGG pathways in DEGs showed that 8,657 genes were classified to 57 KEGG classic metabolic pathways（P<0.05）including focal adhesion（ko04510）,ECM-receptor interaction（ko04512）.BAG6 was significantly categorized into regulation of actin cytoskeleton（ko04810）and protein processing in endoplasmic reticulum（ko04141）.（3）Compared with the reference genome,we detected 19,996 and 15,964 single nucleotide polymorphisms（SNPs）in the testes of 60-d and 180-d Large White pigs,respectively.Among the 242 spermatogenesis-enriched differentially expressed genes,1,224 SNPs of 106 genes and 1,622 SNPs of 174 genes were detected in testes of 60-d and 180-d Large White pigs,respectively.Two SNPs of the differentially expressed PIWIL4 gene were validated by PCR-RFLPs.PIWIL4 g.572 C>T and g.561 G>A were significantly associated with boar semen quality traits（P<0.05）.（4）We analyzed alternative splicing events in testes of 60-d and 180-d Large White pigs by SOAPsplice software.The results showed that 30,243 alternative splicing events with 7,588 genes and 29,204 alternative splicing events with 7,466 genes were detected in 60-d and 180-d testes,respectively.In 60-d testes,the proportion of intron retention was the highest;in 180-d testes,the proportion of exon skipping,intron retention,alternative 5’ splice site and alternative 3’ splice site events were relatively high.2.The study of BAG6 gene alternative splicing on Sertoli cell growth and blood-testis barrier（1）Differentially expressed BAG6 gene splice variants were detected by MISO software（|ΔPSI|≥10%）.The different splice variants were verified by q RT-PCR.BAG6-FL splice variant was highly expressed in 180-d testes,while BAG6-Δ24 splice variant was highly expressed in 60-d testes.The subcellular localization of BAG6 different splice variants in Swine Testis（ST）cell line was detected by immunofluorescence.The results showed that both splice variants were expressed in the nucleus and cytoplasm,indicating that BAG6 exon 24 skipping had no effect on its subcellular localization.（2）MTT,flow cytometry and Western blot analysis showed that BAG6-FL splice variant promoted ST cell proliferation and inhibited ST cell apoptosis,while BAG6-Δ24 splice variant inhibited ST cell proliferation and promoted ST cell apoptosis.Co-IP experiments verified that BAG6-FL splice variant could interact with HSP70 and RAF1,while the interactions between HSP70,RAF1 and BAG6-Δ24 splice variant disappeared due to deletion of exon 24.In mouse Sertoli cells,immunofluorescence results showed that BAG6-FL splice variant could maintain the bundle structure of F-actin and the integrity of the blood-testis barrier,while BAG6-Δ24 splice variant resulted in a disordered F-actin arrangement and a destroyed blood-testis barrier.（3）q RT-PCR and Western blot results demonstrated that the splicing factor SRSF1 regulated BAG6 alternative splicing via its second RRM domain,thus down-regulating the expression of BAG6-Δ24 splice variant.By constructing the BAG6 minigene eukaryotic expression vectors with different deletion fragments and co-transfecting with SRSF1,it was found that there were two binding sites（35 bp-44 bp,45 bp-51 bp）of SRSF1 at the position of 18 bp-51 bp on the BAG6 exon 24,and point mutation experiment showed that these two binding sites were essential for SRSF1 to regulate BAG6 alternative splicing.（4）MTT,flow cytometry and Western blot analysis showed that SRSF1 promoted ST cell proliferation and inhibited ST cell apoptosis.In mouse Sertoli cells,immunofluorescence revealed that SRSF1 could maintain the bundle structure of F-actin and maintain the integrity of the blood-testis barrier.Plasmids co-transfection experiments showed that SRSF1 mediated the alternative splicing of BAG6 gene,thereby regulating Sertoli cell proliferation,apoptosis and maintaining the integrity of the blood-testis barrier.3.BAG6 gene exon 24 knockout mice exhibited damaged spermatogenesis（1）The BAG6 gene exon 24 knockout mice model was successfully constructed by CRISPR/Cas9 technology,and the reproductive organs were weighed.BAG6^exon24-/-mice had smaller testes and seminal vesicles（P<0.01）,but had similar epididymes compared with wild type mice at 8 weeks of age.By H&E staining,a great number of shedding of germ cells was observed in the testes and epididymes in 8-week-old BAG6^exon24-/-mice,and partial elongating spermatids moved towards lumen base due to the change of their cell polarity.Giemsa staining results showed that there were 75.5% abnormalities in the sperm head,neck and midpiece in BAG6^exon24-/-mice.Statistical results of litter size showed a significantly decrease in BAG6^exon24-/-mice.ELISA showed the level of testosterone in serum of 8-week-old BAG6^exon24-/-mice was significantly increased compared with wild type mice（P<0.001）,while FSH and LH levels were not significantly different.These results indicated that BAG6^exon24-/-mice exhibited damaged spermatogenesis and declined fertility.（2）Mass spectrometry（MS）-based proteomics were carried out between 8-week-old BAG6exon24+/+ and BAG6^exon24-/-mice.The results showed 96 proteins were upregulated and 31 proteins were downregulated in BAG6^exon24-/-mice.These downregulated proteins were significantly enriched in sperm flagellum “9+2” structure（cellular component）,HSP70 protein binding（molecular function）and programmed cell death（biological process）.The ultrastructures of sperm flagellum were observed by transmission electron microscopy and the results showed that the outer dense fibers of sperm midpiece in BAG6^exon24-/-mice were partially delected.（3）Western blot showed that BAX expression was significantly increased in BAG6^exon24-/-mice at 4 and 8 weeks of age compared with wild type mice（P<0.05）.TUNEL staining indicated that the number of TUNEL-positive seminiferous tubules was significantly increased（P<0.05）,and the number of TUNEL-positive cells in TUNEL-positive seminiferous tubules was also significantly increased（P<0.01）.（4）Western blot and immunofluorescence indicated that the expression levels of blood-testis barrier related proteins ZO-1,N-cadherin and β-catenin but not OCLN,were significantly decreased in BAG6^exon24-/-mice at 4 and 8 weeks of age compared with wild type mice（P< 0.05）.Subcellular locations of four proteins were changed,scattering throughout the seminiferous tubules.The cytoskeletal protein α-tubulin and F-actin were disordered in 8-week-old BAG6^exon24-/-mice.The m RNA levels of AMH,AR,SOX9 and GDNF in BAG6^exon24-/-mice were significantly decreased by q RT-PCR（P<0.05,P<0.01）.These results indicated that the integrity of the blood-testis barrier was destroyed in BAG6^exon24-/-mice.