The Relationship Between TRNA Cleavage And DNMT2 In Mature Mouse Sperms

tRNA plays a key role in protein synthesis. In addition to its canonical function in protein biosynthesis, tRNA also participates in reverse transcription, protein degradation and cell communication. Under stress condtion, tRNA can be cleaved. tRNA derived fragments were found enriched in mature mouse sperms. Nevertheless, it is unclear why these fragments were produced in sperms under a normal physiological condition.Bioinformatic analysis done in our group revealed that these fragments are mostly derived from tRNAs which were known as the substrates of DNMT2. DNMT2 is a member of DNA methyltransferase family, which mainly methylates tRNA and prevents it from cleavage under stress condition. This study aims to address the correlation between tRNA cleavage and DNMT2 in sperms using mouse models.The main results are as follows:1. tRNA cleavage in mature mouse sperms by northern blotTotal tRNAs were extracted from mouse liver, testis, and mature sperms. The stability of tRNAAsp, tRNAGly and tRNAGlu was examined by northern blot. The results showed that above three tRNAs exerted a higher degradation level in mature mouse sperms than that in mouse liver and testis. This is consistent with tRNA cleavage in mature mouse sperms in previous report.2. Location and expression of DNMT2 proteinVarious mouse and porcine organs and sperms were collected and used to prepare sections or smear. DNMT2 immunostaining was performed on these materials. The results demonstrated that DNMT2 was expressed in mouse and porcine hearts, livers, spleens, lungs and kidneys, but not in the sperms from testis or epididymis. Total protein was extracted from above mouse organs and mature sperms, and subjected to western blot analysis using DNMT2 antibody. The results also indicated that DNMT2 was expressed in these organs, but was depleted from mature mouse sperms.3. The detection of DNMT2 mRNATotal RNAs were extracted from mouse livers and mature sperms and were reverse transcribed into cDNA. The c DNA was used as template to amplify mouse β-actin and DNMT2 genes by PCR, where β-actin served as an internal control. While β-actin was successfully amplified from mouse livers and mature sperms, DNMT2 was only amplified from livers but not detectable in the sperm amplification. To further confirm the depletion of DNMT2 mRNA in mature mouse sperms, in situ hybridization was conducted on mouse liver and testis sections using biotinylated probe for DNMT2 mRNA. While the in situ hybridization revealed the presence of DNMT2 mRNA in mouse liver, it showed the absence of the DNMT2 mRNA in mature mouse sperms, supporting the depletion of DNMT2 in mature mouse sperms at transcription level.4. DNA m~5C modification in mature mouse spermsImmunostaining on mouse testis was performed using m~5C antibody to detect m~5C modification in mouse sperm DNA. While m~5C modification was detected in earlier stages of mouse spermatogenesis, a gradual decreasing trend in DNA m~5C level was observed in the process, and this modification disappeared completely in mature mouse sperms. This is in line with the expression pattern of DNMT2.Conclusions: In mature mouse sperms, tRNA is more prone to cleavage than in other mouse tissues. DNMT2 was depleted in mature mouse sperms at both RNA and protein levels. As DNMT2 protects tRNA against stress-induced cleavage by m~5C methlyation at position 38, our data demonstrated that the lack of DNMT2 in mature mouse sperms contribulted to tRNA cleavage, thus leading to the accumulation of tRNA fragments in mature mouse sperms. More details are needed to address the mechanism. Our data also revealed more details for the underlying mechannism of spermatogenesis.