The Role Of Intraflagellar Transport Complex B(IFT-B) Related Proteins In Spermatogenesis

Objective:To explore the roles and mechanisms of three Intraflagellar transport complex B(IFT-B)related proteins:COP9 signalosome complex subunit 5(COPS5),Autophagyrelated protein 5(ATG5)and Leucine zipper transcription factor-like 1(LZTFL1)in spermatogenesis and male reproduction.Methods:Using yeast two-hybrid,cell co-localization analysis,co-immunoprecipitation experiment or luciferase protein complementation experiment to verify the interaction between COPS5 and IFT20,or LZTFL1 and IFT27.Western blot was used to detect the expression level of these three proteins in mouse tissues.Immunofluorescence staining was used to determine the location of related proteins in mouse spermatogenic cells.Crossing Cops5flox/flox or Atg5flox/flox mice with Stra8-iCre transgenic mice respectively to generate male germ cell-specific Cops5(Cops5 cKO)or A tg5(A tg5 cKO)knockout mouse models.Western blot,RT-PCR,qPCR or immunofluorescence staining were used to detect the gene knockout efficiency of Cops5 cKO,Atg5 cKO and the global Lztfl1 knockout(Lztfl1 KO)mice.Fertility test,sperm count,sperm motility analysis and sperm morphology were used to evaluate the fertility of the three knockout mice.HE staining,transmission electron microscopy(TEM)or scanning electron microscopy(SEM)were performed on the testis or epididymis to detect whether the spermatogenesis process of the three knockout mice was affected.Through RNA-seq,comparative proteomics,flow cytometry,TUNEL staining,mitochondrial staining,Western blot,RT-PCR or qPCR,the effects of these three gene knockouts on the corresponding downstream genes were evaluated.Results:1)COPS5 is a binding protein of IFT20.COPS5 is abundantly expressed in mouse testes,but not in mature sperm.Cops5 cKO mice were completely infertile,the sperm count was significantly reduced(9.83±2.75con vs.0.39±0.23cKO,106/mL,P<0.05),and germ cells underwent significant apoptosis at the premeiotic stage.Testicular expression of poly(ADP-ribose)polymerase-1,a protein that helps cells to maintain viability,was dramatically decreased,and FANK1,a key germ cell apoptosis regulator was also reduced in the mutant mice.Expression level of Caspase-3,a critical executioner of apoptosis,was increased in the knockout mice.Acrosome biogenesis was affected in the mutant mice.IFT20 expression level was significantly reduced in the Cops5 cKO mutant mice,and it was no longer present in the acrosome of round spermatids.However,in germ cell-specific conditional Ift20 gene knockout(Ift20 cKO)mice,the expression level of COPS5 and the localization of COPS5 in germ cells in the testis were not changed.Analysis of testicular morphology at different time points during the first wave of spermatogenesis showed that the spermatogenesis of Cops5 cKO mice was abnormal after day 16 of birth.Cell populations in the seminiferous tubules of the 16-day-control and Cops5 cKO mice analyzed by flow cytometry showed that the cell populations were similar between the two groups.A total of 2,978 proteins were identified in the proteomics study by using the testis of 16-day-control and Cops5 cKO mice.Among them,expression level of 399 proteins was significantly decreased and expression level of 65 proteins was significantly increased in the Cops5 cKO mice,these proteins are involved in autophagy,apoptosis,Golgi transport,vesicle protein transport,ciliary biogenesis,methylation,phosphorylation,ubiquitination,proteasome degradation,RNA transport or localization,mRNA splicing,nucleus and spermatogenesis and other biological processes.RNA-seq experiment revealed a total of 9,912 transcripts in which expression of 441 transcripts was significantly reduced,and only 11 transcripts significantly increased.Expression of 240 genes was significantly reduced in both protein and mRNA levels,and many are key regulators of spermatogenesis,including Ddx4,Tdrd1,Tdrd5,Mill and Mybl1,the also key components in the piRNA pathway.The expression of some down-regulated components of the piRNA pathway and sperm-related proteins was verified by qPCR or Western blot,and all showed varying levels of decline.2)The significantly reduced expression of Atg5 mRNA and ATG5 protein in Atg5 cKO mice testes,demonstrated the mouse model was successfully constructed.In Atg5 cKO mice,testicular expression of the autophagosome marker LC3-II was significantly reduced,and expression of autophagy receptor SQSTM1/p62 was significantly increased,indicating a decrease in testicular autophagy activity.The fertility of the Atg5 cKO mice was dramatically reduced with about 70%(12/17)being infertile.Sperm counts(13.85±1.70Con vs.4.0±2.83cKO,106/mL,P<0.05)and motility(67.23±2.61Con vs.41.37±3.12cKO,μm/s,P<0.05)were also significantly reduced compared to the controls.Histological examination of the Atg5 cKO testes revealed numerous,large residual bodies in the lumen of seminiferous tubules.The cauda epididymal lumen was filled with sloughed germ cells,large cytoplasmic bodies,and spermatozoa with abnormal heads and tails.Examination of cauda epididymal sperm by electron microscopy revealed misshapen sperm heads,a discontinuous accessory structure in the mid-piece and abnormal acrosome formation and loss of sperm individualization.Immunofluorescence staining of epididymal sperm showed abnormal mitochondria and acrosome distribution in the mutant mice.3)LZTFL1 is a binding protein of IFT27.Western blot showed that LZTFL1 protein is highly expressed in the mouse testes.During the first wave of spermatogenesis,the LZTFL1 protein is highly expressed during spermiogenesis.The results of immunofluorescence staining of isolated germ cells showed that LZTFL1 was distributed in the cytoplasm of round spermatids.In the elongating spermatids,LZTFL1 is present in the developing flagella and appears also close to the manchette.Analysis of global Lztfl1 knockout(Lztfl1 KO)mice showed that the fertility of KO male mice was significantly reduced,associated with low sperm motility(7.48± 1.33Con vs.3.15±2.41KO,106/mL,P<0.05)and a high level of abnormal sperm(2.78±2.21Con vs.34.9±3.2KO,%,P<0.05).In vitro assessment of fertility also revealed the significantly reduced fertilization of Lztfl1 KO mice(78.83±6.90Con vs.36.72±24.81KO,%,P<0.05).Testicular expression of IFT27 protein in the Lztfl1 KO mice was significantly reduced,but the levels of other IFT proteins(including IFT20,IFT81,IFT88 and IFT140)did not change significantly in the testes.Conclusion:The results of this study show that the three IFT-B related proteins,COPS5,ATG5 and LZTFL1 all play an important role in spermatogenesis.First of all,COPS5 is the upstream protein of IFT20,which regulates the expression and localization of IFT20.COPS5 maintains normal spermatogenesis through a variety of mechanisms,including apoptosis,IFT and piRNA pathways;Secondly,ATG5 induces autophagy to maintain normal developmental processes,which is essential for male fertility and participates in many aspects of spermatogenesis.Finally,LZTFL1 plays an important role in mouse spermatogenesis and fertility.LZTFL1 is a downstream protein of IFT27,and its expression in the testis are affected by IFT27.