Dicer1 And Pp2ac¦Á Meiosis Pre-function Studies In Spermatogenesis

Objective:spermatogenesis is a multiple process consist of several component elements which is strictly regulated at both the transcriptional and the posttranscriptional levels. Meiosis is a germ cell specific form and also be regulated at different levels. It is anticipated that unraveling meiosis in the testis will further our understanding of the dysfunction of mammalian spermatogenesis. To examine the function of Dicer1 which is a key enzyme of miRNA process and protein phosphatase PP2A specifically in pre-meiosis and early meiosis,we used a conditional knock-out mouse model.Methods: In this study, we first used two mouse models that expresses Cre recombinase from the Stra8 locus which is specifically in spermatogonial cells and preleptotene spermatocyte and a floxed Dicer1 or PP2ACαconditional allele. After marking, genome DNA extraction and identification of genotype using PCR, we obtained homozygous mutants of Dicer1 or PP2ACαin spermatogonial cells and preleptotene spermatocyte. In all figures, control refers to heterozygous mutants or wild type. Dicer1(or PP2ACα)-deficient testis weights were compared to age-matched littermates at a series of age. For analysis of the seminiferous tubules and spermatogenesis, testis from mutant and control mice of different weeks were dissected and fixed in Bouin fixative. Sections were stained for hematoxylin-eosin and also for Tunel analysis to detect the apotosis. Testis RNA was extracted and subjected to reverse transcription (RT), and the cDNA product was used as template for real-time PCR to quantify the efficiency of knockout. Single cell suspensions were prepared from testis of adult mice. The percentage of haploid cells were estimated by FACS. To test for fertility, male germline mutant mice were crossed to wildtype female mice, and the number of offspring was added up.Results: MircroRNAs(miRNAs) are a kind of small noncoding RNA which has~22 nucleotide that posttranscriptionally regulate gene expression. The miRNAs are crucial for a devial of cellular funcitons include survival, proliferation and apoptosis. Dicer1 is an evolutionarily conserved ribonuclease III that is necessary for for synthesis ofmature functionalmicroRNAs (miRNAs). To examine the function of Dicer1 specifically in the germline and spermatogenesis, a conditional knock-out mouse model of Dicer1 and a male germline special Stra8-cre mouse that express cre only in early-stage spermatogonia have been used. We have successfully obtained homozygous Dicer1(PP2ACα)-deleted mice. At P14 when the meiosis was in the preleptotene stage, the testis from homozygous mutant mice were normal just as the control. But after the complete of the first spermatogenic wave, Dicer1-deficient mice showed a defects in testis development. The testis from adult deficient mice were nearly equal to half of the wild-type. Results of hematoxylin-eosin revealed defects in spermatogenesis, spermatid and mature sperm was nearly absenct in the seminiferous tubules. Some of the tubules are extremely dysplasia due to absent of almost all of the germ cells. Meanwhile, some tubules were nearly normal and a small quantity of mature spermatozoa were present in epididymis. But in those nearly normal tubules we also noticed some anachromasis cells which we considered as karyopyknotic spermatogonial cells or spermatocyte, and these may be a signal of apoptosis. In the deficient epididymis, there were also many exfoliated cells. Males carrying only one mutant allele of Dicer1 exhibited normal phenotype. The results of real-time PCR revealed that the expression quantity of Dicer1 and pluripotent gene Oct4 was~80% less than those of control littermates, but no statistically significant decrease of Prm1, Prm2, Tnp1, Tnp2 which were markers of advanced stage of spermatogenesis were obtained. Tunel analysis had indicated a notable increase of apoptosis in mutant testis and epididymis since P14. The percentage of haploid cells in mutant testes was ~13% lower than wild-type according to FACS analysis. On the contrary, the percentage of diploid and tetraploid cells were higher than that of wild-types. Mutant males exhibited a significant reduction in fertility compared to wild-types.The phenotype of PP2ACα-deficient mice was slightly different from that of Dicer1-deficient mice. The decrement of testes weight of PP2AC mutants was less than Dicer1 mutants, and seldom sertoli cell-only tubules were obtained, while most tubules still contained spermatogonial cells and spermatocytes. But elongating spermatids were rarely observed in mutant tubules. Additionally, a large number of exfoliated cells were present in PP2ACαmutant adult epididymis which were usually absent in wild-types. The protein expression level of PP2ACαwas dramatically degraded in homozygous mutant mice. Tunel analysis indicating a notable increase of apoptosis in mutant testis and epididymis since P15, but no apoptosis were observed in adult mutant mice. Tunel analysis indicating a notable increase of apoptosis in mutant testis and epididymis since P14. The percentage of haploid cells in mutant testes was ~25% lower than wild-type, on the contrary, the percentage of diploid and tetraploid cells were much higher than that of wild-type, even higher than the percentage of haploid cells. Mutant males were infertility according to initial results. Overall, our results reveal critical and diverse roles for Dicer1 and PP2ACαin the spermatogenesis and meiosis.Conclusion: Meiosis is an important phase where many posttranscriptional regulation occurred. The abnormality of meiosis will lead to defects of spermatogenesis. Dicer1 and miRNAs are essential in meiosis and spermatogenesis, delete of Dicer1 in pre-meiosis or early-stage spermatogonia will lead to spermatogenesis defects. PP2A- deficient which plays essential roles in mitosis and meiosis also causes spermatogenesis arrest.