The Regulation And Mechanism Of FHL2 Gene On Spermatogenesis And Male Reproductive Performance

Spermatogenesis is the basic of maintaining normal reproductive function in male animals,which is regulated by hormones,genes,transcription factors and other factors.FHL2（Four and a half LIM domains 2）,as a transcriptional co-activator/co-repressor,participates in a variety of cellular processes such as regulator regulation of gene expression,apoptosis,cell cycle,cytoskeleton,and signal transmission.Our previous research results show that knockdown of FHL2 causes follicular development obstruction and female fecundity reduction.In addition,FHL2 is expressed in both male Sertoli cells and spermatid,and knockdown of FHL2 in Sertoli cells isolated and cultured in vitro to affect its secretion function.Therefore,it is inferred that FHL2 may play an important regulatory role in spermatogenesis and male reproductive function.In this study,FHL2 gene knockout mice were used as models to study the regulation of FHL2 on spermatogenesis and reproductive function of male animals and its mechanism.The main research findings are as follows:1 FHL2 expression profile analysis and identification of gene knockout mice（1）Analysis of FHL2 tissue expression profile and spatiotemporal expression profile:FHL2 is expressed in the testes;The expression level of FHL2 in the testes of 7（P7）,14（P14）and 21 days old（P21）mice was approximately twice that of P35,P56,and P84 mice;FHL2 immune signals were mainly located in the nuclei of all levels of spermatid in the testes of male mice before sexual maturity;（2）Identification of FHL2 knockout mice:Based on the FHL2 gene knockout(FHL2^-/-)mice constructed by the research group in the early stage,and validation of the knockout mouse model was achieved using PCR,qRT-PCR and Western Blot methods.2 FHL2 deletion leads to the delay of the first wave of spermatogenesis（1）Male gonad dysplasia in FHL2^-/-mice:compared to WT mice of the same age,the weight and volume of the testicles in P21,P35,and P56 FHL2^-/-mice were significantly reduced,the testicular organ index and diameter of the seminiferous tubules were reduced,and the thickness of the seminiferous epithelium became thinner;（2）The deletion of FHL2 leads to the delay of the first wave of spermatogenesis:It was found that FHL2 knockout led to the delay of round spermatid and elongated spermatid in seminiferous tubules;Mature sperm can be detected in the epididymal tail P42 of WT mice,while sperm can be detected in the FHL2^-/-mouse P49,and density is significantly lower than that of WT;（3）Loss of FHL2 leads to blocked meiosis initiation:Immunofluorescence results showed that the number of spermatogonium of FHL2^-/-mice was not significantly different from WT,but the number of primary spermatocyte of P10（20.76±1.01 vs.11.22±0.32）and the proportion of convoluted seminiferous tubules with primary spermatocyte（66.05±2.96%vs.24.87±6.47%）were significantly reduced;The expression of meiosis initiation marker（Stra8）in seminiferous tubules of FHL2^-/-mice（P21）was significantly lower than that of WT mice;（4）The loss of FHL2 led to a decrease in the number of spermatid:compared with WT,there was no significant difference in the number of spermatogonium in the seminiferous tubules of FHL2^-/-mice（P21,P35）.The number of primary spermatocyte,round spermatid and elongated spermatid were significantly reduced,and the total number of spermatid in the seminiferous tubules was reduced（P21,137.73±11.64 vs.93.06±2.35,p<0.01）;（5）FHL2 deficiency leads to delayed maturation of Sertoli cells:Immunofluorescence results showed that the expression of AMH in the seminiferous tubules of FHL2^-/-mice（P10,P14）was significantly higher than that of WT mice;WT mice could hardly detect AMH signals in the P14 seminiferous tubules,which indicating basic maturation of supporting cells,while FHL2^-/-mice still had about 50%positive AMH signals in the seminiferous tubules;（6）FHL2 deletion affects the biological process related to spermatogenesis:the testicular tissues of 21 days old WT and FHL2^-/-mice were collected for transcriptome sequencing,and a total of 362 differentially expressed genes were identified.GO analysis showed that differentially expressed genes were mainly enriched in the biological processes related to spermatogenesis,such as sex differentiation,sperm differentiation,sperm development,meiosis cell cycle and synaptonemal complex.GSEA is enriched to G protein coupled receptor,gap junction,chromatin condensation and other related signaling pathways.3 FHL2 deficiency leads to a decrease in sperm production and reproductive ability（1）FHL2 deficiency leads to a decrease in reproductive ability:Compared to WT mice of the same age,FHL2^-/-male mice at 12 weeks of age（12 W）showed 40%decrease in offspring（20.86±1.74 vs 12.71±3.01）,and significant decrease in thrombus detection rate and number of uterine embryo implantation compared to mating females.（2）FHL2 deficiency leads to a decrease in spermatogenic ability and semen quality:Compared to WT mice of the same age,the total number of spermatogenic cells and various spermatogenic cells in FHL2^-/-mice decreased;The epididymal semen density of FHL2^-/-mice significantly decreased by 43%,and the sperm deformity rate significantly increased.4 FHL2 deficiency leads to premature decline in reproductive function（1）Loss of FHL2 leads to early termination of reproductive function:Fecundity test results show that 20 months old FHL2^-/-mice lose reproductive capacity,but WT mice can still reproduce normally at 23 months old;The statistical results of litter size showed that after the peak reproductive period,the descendants of FHL2^-/-mice was significantly lower than that of WT mice;（2）FHL2 deficiency leads to premature atrophy of the gonads:Compared to WT mice of the same age,the testicular volume,mass,and epididymis weight of FHL2^-/-mice aged23 months significantly decreased;（3）Loss of FHL2 results in the shedding of seminiferous epithelium:HE staining results show that the convoluted seminiferous tubules of WT mice still maintain normal morphology and structure until 23 months of age,while the shedding and empty tubes of spermatid can be detected in the convoluted seminiferous tubules of FHL2^-/-mice at 12months of age;At the age of 23 months,both the epididymal body and epididymal tail of FHL2^-/-mice showed empty tubes,and the sperm density was significantly lower than that of WT mice,the sperm deformity rate significantly increased.The results of this study indicate that FHL2 plays an important role in regulating Spermatogenesis and male reproductive function.The deletion of FHL2 inhibits the meiosis initiation and spermatogenesis,leading to the delay of the first wave of spermatogenesis,the decline of spermatogenesis and reproductive capacity,and the early termination of reproductive function in male mice.This study provides a theoretical basis for further elucidating the regulatory network of male reproductive function and provides new targets for the regulation of male reproductive function.