| Spermatogonia developed into mature sperm undergoing meiosis and spermiogenesis. During these series of differentiation, the cytoplasm lost, chromatin condensed and transcriptional activity reduced gradually, which led to RNA quantity in sperm extremely low. At present, the fact that sperm contained kinds of RNA had been confirmed, but whether there were transcriptional activity still be debated. The study on the trend of transcriptional expression during spermiogenesis becomes necessary. The laboratory had studied the transcriptional expression of mouse during spermiogenesis by high-through sequence, found a large number of different expression genes among the round, elongated spermatids and mature sperm, and validated the reliability of results by quantitative polymerase chain reaction(Q-PCR). Whether there was the same trend for the expression of the transcription process in Holstein bull sperm, it still needed to be further studied.The study collected testists and epididymis of Holstein bull and captured 90 576 round spermatids, 122 651 elongated spermatids and 67 818 spermatogonial stem cells(three biological replicates) by adjusting and optimizating frozen sections details, prolonging the staining time effectively and groping each parameter of laser capture microdissection based on preliminary experiments with mice. At the same time, we obtained 107 orders epididymal spermatozoa by epididymids flotation. The above experiments provided reliable experiment materials.In this study, we obtained a better method — RNeasy Kit to extract sperm RNA(0.023~0.025 pg RNA/sperm) by comparing two extraction kits and trizol method, which laid technology foundation for the following high-through sequencing. We extracted the RNA of round spermatids, elongated spermatids and spermatogonial stem cells using PicoPureTM RNA Isolation Kit. We selected CD45 and CDH1 genes which expressed specificly in white blood cells and epithelial cells to detect the contamination of somatic cells, and housekeeping gene GAPDH(design primers by cross-intron) to check the genomic contamination by reverse transcription polymerase chain reaction(RT-PCR). These provided high-quality samples for high-through sequencing. In addition, the study analyzed the difference of RNA quantity in single cell among spermatogenic cells with one-way analysis of variance and found that the RNA quantity in round spermatids, elongated spermatids, epididymal sperm and spermatogonial stem cells were different significantly(P< 0.05). The RNA quantity decreased with the loss of cytoplasmic during spermatogenesis and spermiogenesis, and RNA quantity in the round and elongated spermatids were 752-fold and 413-fold higher than epididymal sperm. The spermatogenic cells RNA was amplified using Smart-seq2 and the cDNA integrity was tested by Aligent 2100. The amount of each cDNA sample was higher than 20 ug, and the products size were between 1-2kb, which met the requirements of building RNA-seq libraries. These studys provided the reliable samples for RNA-seq in sampling, RNA preparation, quality test and trace amplification. It also will provide an important reference for exploring the mechanism of spermatogenesis and spermiogenesis. This study established stable frozen section method and cell sampling techniques systerm based on bovine testicular tissue characteristics, and captured large number of round spermatids, elongated spermatids and spermatogonial stem cells. At the same time, the study got a better method to preparate sperm RNA, and the RNA content and trends of single cell during spermiogenesis were also abtained. Besides, the enough cDNA were acquired using Smart-seq2. These studies laid the foundation for high-throughput sequencing, as well as the mechanism of spermatogenesis and provide an important reference. |
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