| Spermatogenesis is a complicated and specific process of cell proliferation and differentiation including mitotic division of spermatogonia, meiotic division of spermatocytes and morphological transformation of spermatids.It is a complex process involving cell division, diffentiation and interactions between cells in the microenviroment of the seminiferous tubule. It is regulated by lots of genes; generally, these kinds of regulation genes are expressed under precise temporal and spatial regulation in sperm cells specifically. The separating and identifying of genes related to spermatogenesis and the study of their molecular regulating mechanism at proteome level are very important for clinical diganosis and treatment of male infertility.LM23 (AF492385) is a gene specifically expressed in the testes of Rattus norvegicus previously reported by our laboratory. A BLAST homology search against the NCBI non-redundant database and an Ambystoma EST database revealed that LM23 is a R. norvegicus homologue of Speedy A (Spdya). In this study, we clarified the majoy role and mechanism of LM23 in the proeess of spemratogenesis.Firstly, RT-PCR analysis of RNA from the nine different tissues including testis and ovary showed that LM23 RNA was only present in testis. Real-time PCR analysis showed that the expression level of LM23 was highest in spermatocytes and very low in spermatogonia and spermatids. In situ hybridization revealed a strong positive signal in the cytoplasm of spermatocytes and a weak signal in spermatids and spermatogonia. This testis-specific and stage-specific expression pattern suggested that LM23 might be involved in R. norvegicus spermatogenesis.To reveal the function of LM23 in the testis, we used lentivirus-mediated RNA interference (RNAi) to knock down LM23 expression in a tissue-specific manner in vivo. A lentiviral vector expressing a short hairpin RNA (shRNA) targeting LM23 was microinjected into the efferent ducts of R. norvegicus testes. The infectious lentivirus was microinjected into testes of 5-week-old R. norvegicus just completing the first wave of spermatogenesis. The enhanced green fluorescent protein (EGFP) signal in about 75% of whole testes of R. norvegicus at four weeks post-transfection is shown in a stereomicroscope view. Next, to examine the efficiency of LM23 RNAi, we analyzed the expression levels of LM23 mRNA in testes by real-time PCR at two weeks and four weeks post-transfection. Compared with scrambled RNAi-transfected testes, LM23 mRNA expression was significantly reduced (69% and 87%, respectively). There was no difference in LM23 mRNA level between scrambled RNAi-transfected testes and wild type testes. Western blot analysis showed LM23 protein expression was not detected in testis after LM23 RNAi. These data showed that the specific in vivo knockdown of LM23 in testes of R. norvegicus via lentivirus-mediated RNAi was effective and stable.The size and weight of LM23-shRNA testes had no significant differences from the controls. Seminiferous tubules of control testes were well organized and contained a full spectrum of spermatogenic cells, including spermatogonia, spermatocytes, spermatids and spermatozoa. In contrast, seminiferous tubules of LM23-shRNA testes appeared disorganized, disrupted, and shedding germ cells into the lumina; the germ cells exhibited complete meiotic arrest in spermatogenesis. Spermatocytes were accumulated, round spermatids were few and elongating spermatids, spermatozoa were absent in certain LM23-shRNA seminiferous tubules. Three major types of seminiferous tubules were observed in LM23-shRNA testes. Typeâ… tubules contained 3-4 layers of spermatocytes. In typeâ…¡tubules, there were more layers of spermatocytes and many heavily eosin-stained cells, which might be apoptotic cells. Typeâ…¢tubules were characterized by a few layers of spermatogenic cells/Sertoli cells and big empty lumina. The epididymal tubules of control R. norvegicus were filled with spermatozoa, whereas those of LM23-shRNA testes R. norvegicus were empty. A TUNEL assay showed the presence of many apoptotic cells in certain tubules, which were likely typeâ…¡tubules. In contrast, few apoptotic cells were present in typeâ… or typeâ…¢tubules. Few apoptotic cells were detected in tubules of control testes. One possible explanation for the presence of three types of tubules in LM23-knockdown testes might be coordinated differentiation of the germ cells in a given tubule. In LM23-knockdown testes, spermatogenesis proceeded from spermatogonia to spermatocytes, but further differentiation was blocked, resulting in the accumulation of spermatocytes in type I tubules. Subsequently, these spermatocytes failed to further differentiate and underwent apoptosis in typeâ…¡tubules. Eventually, most apoptotic spermatocytes were eliminated in typeâ…¢tubules.Microarray analyses of the transcriptomes of the LM23-shRNA and control testes were performed to screen for genes regulated by LM23. The results revealed that the expression of some genes related to spermatogenesis, meiosis, the cell cycle, and apoptosis were significantly changed after LM23 knockdown. Real-time PCR analysis confirmed that some meiotic genes involved in synapsis, recombination (Sycp1, Sycp2, Sycp3, Msh5) and meiotic sister-chromatid cohesion (Stag3, rec8Ll) had lower expression. Many pro-apoptotic genes were up regulated such as Bcl-2 family members including Bax, Bid3, Bak1 et al. And many anti-apoptotic genes were down regulated such as Fafl and Zfp9.Collectively, these studies demonstrate that LM23 is required for meiosis in spermatogenesis.
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