Aim： To investigate the expression of Spindlin 1 （Spin First, reverse-transcription polymerase chain reaction present in mouse testis. Then the expression patterns 1） isoform2 and assess its function in mouse testi...Aim： To investigate the expression of Spindlin 1 （Spin First, reverse-transcription polymerase chain reaction present in mouse testis. Then the expression patterns 1） isoform2 and assess its function in mouse testis. Methods： （RT-PCR） was used to determine whether Spinl isoform2 is of the isoform between newborn and adult mice testes were compared by immunoblot analysis. Finally, the diversity of its localization in mice testes at different ages （days 0, 7, 14, 21, 28 and 60） was observed by immunohistochemistry. The localization of the protein in mouse sperm was also investigated by immunofluorescence. Results： The RT-PCR results show that Spinl isoform2 is present in mouse testis. As shown by immunoblot analysis, the isoform was more highly expressed in adult testes compared with newborn testes. Interestingly, Spinl isoform2 did not show up in the cytoplasm of primary spermatocytes until day 14. Also, the protein exists at the tail of the mouse sperm. Conclusion： Spinl isoform2 is a protein expressed highly in adult testis, which might be involved in spermatogenesis and could be necessary for normal sperm motility.展开更多
Mammalian testis development is a complex and highly sophisticated process. To study the dynamic change of normal testis development at the transcriptional level, we investigated mouse testes at three postnatal ages: ...Mammalian testis development is a complex and highly sophisticated process. To study the dynamic change of normal testis development at the transcriptional level, we investigated mouse testes at three postnatal ages: 6 days postnatal, 4 weeks old, and 10 weeks old, representing infant (PN1), juvenile (PN2), and adult (PN3) stages, respectively. Using ultra high-throughput RNA sequencing (RNA-seq) technology, we obtained 211 million reads with a length of 35 bp. We identified 18837 genes that were expressed in mouse testes, and found that genes expressed at the highest level were involved in spermatogenesis. The gene expression pattern in PN1 was distinct from that in PN2 and PN3, which indicates that spermatogenesis has commenced in PN2. We analyzed a large number of genes related to spermatogenesis and somatic development of the testis, which play important roles at different developmental stages. We also found that the MAPK, Hedgehog, and Wnt signaling pathways were significantly involved at different developmental stages. These findings further our understanding of the molecular mechanisms that regulate testis development. Our study also demonstrates significant advantages of RNA-seq technology for studying transcriptome during development.展开更多
Sperm development is critical for male reproductive capability;any disruption during the process of spermatogenesis will result in male infertility. In this research, we used the C-Nap1 encoded by the gene of Cep250 k...Sperm development is critical for male reproductive capability;any disruption during the process of spermatogenesis will result in male infertility. In this research, we used the C-Nap1 encoded by the gene of Cep250 knockout mouse line as the model to evaluate the impact of absent C-Nap1 on spermatogenesis. To investigate the interaction between C-Nap1 and spermatogenesis, we utilized single-cell RNA sequencing to analyze 10,332 C-Nap1+/+and 13,308 C-Nap1-/-testicular cells. We identified five main cell types within seminiferous tubules, including spermatogonia, Sertoli cells, spermatogonia stem cells, Leydig cells, and spermatocytes. We found a critical reduction in testicular spermatogonia and spermatocytes in C-Nap1-null testes,compared to its C-Nap1+/+controls. By combining uniform manifold approximation and projection clustering and psedotime ordering, we distinguished five spermatogonial stages/subtypes, demonstrating that type B spermatogonia differentiation and meiotic initiation are impaired during C-Nap1-null spermatogenesis. Following gene ontology enrichment analysis,meiosis-specific genes downregulated in the C-Nap1-/-testicular cells were further verified by reverse transcription polymerase chain reaction(RT-PCR).Based on the differential gene expression, certain downregulated genes such as Ctnnb1 and Aurka encoding C-Nap1-binding potential β-Catenin and Aurka are encountered, which may account for defective type B spermatogonia differentiation and meiotic entry in C-Nap1-null testes.展开更多
基金This research was supported by grants from the 973 Program （2006CB504002, 2006CB944002） and the National Natural Science Foundation of China （30425006）.
文摘Aim： To investigate the expression of Spindlin 1 （Spin First, reverse-transcription polymerase chain reaction present in mouse testis. Then the expression patterns 1） isoform2 and assess its function in mouse testis. Methods： （RT-PCR） was used to determine whether Spinl isoform2 is of the isoform between newborn and adult mice testes were compared by immunoblot analysis. Finally, the diversity of its localization in mice testes at different ages （days 0, 7, 14, 21, 28 and 60） was observed by immunohistochemistry. The localization of the protein in mouse sperm was also investigated by immunofluorescence. Results： The RT-PCR results show that Spinl isoform2 is present in mouse testis. As shown by immunoblot analysis, the isoform was more highly expressed in adult testes compared with newborn testes. Interestingly, Spinl isoform2 did not show up in the cytoplasm of primary spermatocytes until day 14. Also, the protein exists at the tail of the mouse sperm. Conclusion： Spinl isoform2 is a protein expressed highly in adult testis, which might be involved in spermatogenesis and could be necessary for normal sperm motility.
基金supported by the National Basic Research Program of China(2011CB944100,2011CB944101)the National Natural Science Foundation of China (90919024)+1 种基金the Special Foundation Work Program of Ministry of Science and Technology (2009FY120100)the National High Technology Research and Development Program of Ministry of Science and Technology of China (2012AA020409)
文摘Mammalian testis development is a complex and highly sophisticated process. To study the dynamic change of normal testis development at the transcriptional level, we investigated mouse testes at three postnatal ages: 6 days postnatal, 4 weeks old, and 10 weeks old, representing infant (PN1), juvenile (PN2), and adult (PN3) stages, respectively. Using ultra high-throughput RNA sequencing (RNA-seq) technology, we obtained 211 million reads with a length of 35 bp. We identified 18837 genes that were expressed in mouse testes, and found that genes expressed at the highest level were involved in spermatogenesis. The gene expression pattern in PN1 was distinct from that in PN2 and PN3, which indicates that spermatogenesis has commenced in PN2. We analyzed a large number of genes related to spermatogenesis and somatic development of the testis, which play important roles at different developmental stages. We also found that the MAPK, Hedgehog, and Wnt signaling pathways were significantly involved at different developmental stages. These findings further our understanding of the molecular mechanisms that regulate testis development. Our study also demonstrates significant advantages of RNA-seq technology for studying transcriptome during development.
基金The Fundamental Research Funds for the Central Universities,Grant/Award Number:E1E43201X2The Beijing Municipal Natural Science Foundation,Grant/Award Number:Z200021+2 种基金The CAS Interdisciplinary Innovation Team,Grant/Award Number:JCTD-2020-04The National Key Research and Development Program of China,Grant/Award Numbers:2021YFE0201100,2022YFA1103401The National Natural Science Foundation of China,Grant/Award Number:31872839。
文摘Sperm development is critical for male reproductive capability;any disruption during the process of spermatogenesis will result in male infertility. In this research, we used the C-Nap1 encoded by the gene of Cep250 knockout mouse line as the model to evaluate the impact of absent C-Nap1 on spermatogenesis. To investigate the interaction between C-Nap1 and spermatogenesis, we utilized single-cell RNA sequencing to analyze 10,332 C-Nap1+/+and 13,308 C-Nap1-/-testicular cells. We identified five main cell types within seminiferous tubules, including spermatogonia, Sertoli cells, spermatogonia stem cells, Leydig cells, and spermatocytes. We found a critical reduction in testicular spermatogonia and spermatocytes in C-Nap1-null testes,compared to its C-Nap1+/+controls. By combining uniform manifold approximation and projection clustering and psedotime ordering, we distinguished five spermatogonial stages/subtypes, demonstrating that type B spermatogonia differentiation and meiotic initiation are impaired during C-Nap1-null spermatogenesis. Following gene ontology enrichment analysis,meiosis-specific genes downregulated in the C-Nap1-/-testicular cells were further verified by reverse transcription polymerase chain reaction(RT-PCR).Based on the differential gene expression, certain downregulated genes such as Ctnnb1 and Aurka encoding C-Nap1-binding potential β-Catenin and Aurka are encountered, which may account for defective type B spermatogonia differentiation and meiotic entry in C-Nap1-null testes.
