| Traditionally, sperm is considered as the key role in delivering paternal genome and then activating the embryos in fertilization, while the oocyte is responsible to regulate the development of embryos. So it tends to exist the problem that people pay attention to studying on the oocyte instead of sperm in the long run. In fertilization, protein, small non-coding RNA, mRNA could be carried into the oocyte, and then play the major role in some aspects, such as the development of the male and female gametes, the cleavage stage of development, the transformation of zygotes, the development of the embryos, even the phenotype of the offspring. Besides, it means that sperm is involved in regulating the development and the reprogramming of the nuclear, etc. Therefore, it contributes to unraveling the developmental biological riddles such as the contents brought from sperm into oocyte. This study is focused on the construction of small RNA library of sperm, oocyte and somatic cell in bovine, and screening the sperm-specific miRNA.For this reason, a high-efficient protocol of extracting the sperm RNA is the basis for studying the contents of sperm and then constructing the small RNA library. Sperm is highly condensed without too much RNA, it is rather difficult to extract the abundant RNA from sperm, especially in view that testis is lack of mature sperm, fresh semen is expensive and frozen semen has inferior motility. Moreover, more precise protocol of extracting sperm RNA and quality control are required to guarantee the qualified level of RNA. In addition, RT-PCR could be used to detect the RNA quality. By Solexa high-sequencing technique, the sperm-specific miRNA can be screened and then identified. Through injecting the external-synthesis mimic of miRNA into oocyte, the expressed level of miRNA in oocyte could be induced as that in sperm, and further we could analyze their affects on the early development.(1)The more innovated and reasonable protocol of isolating RNA from epididymis of bovine has been enacted. Further, through altering the reaction conditions on isolation of RNA and qRT-PCR confirmation of specific genes and integrity of sperm, it has been concluded that swim-up and 60°C TRIzol are more appropriate.(2)Via Illumina high-sequencing technique, the small RNA library and relevant target genes and network are analyzed and predicated. In miBRase2.0, we blasted 18-26 nt Unique sequences to specific pre-miRNA. The result is that 951 known mi RNA and 8 novel candidates are detected, as well as their regulatory networks. The sperm-borne miRNAs are very basic but important to further analysis on the embryo development, epigenetics and molecular mechanism.(3)The specific-expressed miRNAs are regarded as the candidates after confirmation of qRT-PCR,along with the analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes network. Such as miR-202, it could enhance the ability of embryo development and nuclear reprogramming. In specific, we could inject the mimic into oocyte with the similar level of that in sperm, and then detect the cleavage rate and acetylation level.(4)Constructing of miR-449 b 3G Tet-On induced expression vector, followed by screening the positive cloned cells which are used as donor cells in SCNT. Doxycycline(Doxycycline, Dox)-induced experimental group could be identified to improve the cleavage rate, nuclear reprogramming, and the quality of blastula. |
PDF Full Text Request