| Ovary is one of the most important organs in female reproductive system, its function is mainly divided into two aspects. First, ovaries as the place of oocyte development and mature, differentiate into normal oocyte with fertilization ability. Secondly, ovarian steroid hormones can ragulate follicular development and reproductive cycle, and so on. Many factors regulate ovary development, such as gonadotropins, cytokines, and small nucleic acids. Lnc RNA regulate gene expression level in epigenetic regulation, transcriptional regulation and transcription regulation level. A large number of lnc RNA have been reported implicating in the individual development and the occurrence of diseases. Ovary is one of the most active organ in animal body, while the study about the function of lnc RNA in ovarian development rarely be reported. To study the function of lnc RNA in ovarian reproductive activity, we designed four consecutive experiments. The specific design of experiment, the research content and the experimental results are as follows.Experiment one: Explore specifically expression of lnc RNA in ovary. First, we analysed the gene bank of lnc RNA and obtained 16 lnc RNAs. We found 9 lnc RNAs relatively high expressed in various tissues by q PCR and lnc RNA647, lnc RNA147 and lnc RNA274 specifically high expressed in ovary.Experiment two: The expression levels of lnc RNA647, lnc RNA147 and lnc RNA274 under different state of ovary development. We detected the expression levels of these three lnc RNAs in different development stages(at age of five days, third week, fifth week, eighth week) and estrus cycle(proestrus, estrus, metaestrus, dioestrus) of ovary respectively using q PCR. Results showed that the highest expression levels of lnc RNA274 and lnc RNA147 were in avary at age of 8 weeks, from high to low the expression levels in the other three developmental stages of ovary were at age of 5 days, 5 weeks and 3 weeks. The highest expression level of lnc RNA647 was ovary at 5 weeks and 8 weeks, from high to low the expression level of lnc RNA647 in the other two stages of ovary were at age of 5 days and 3 weeks. In the different estrous stages of estrus cycle, the expression levels of the three lnc RNAs raised gradually according to the dioestrus, proestrus, estrus and metaestrusin.The expression level in metaestrusin is significantly higher among the other three stages of estrus.Experiment three: In order to study the influence on ovarian function of these three lnc RNAs, we first constructed the overexpression vector of the three lnc RNAs(ZP3 promoter-lnc RNA) successfully. We injected the plasmids of lnc RNA647, lnc RNA147,lnc RNA274 into the right ovary respectively, injected ZP3 plasmid(without target gene) into the left ovary by the method of in vivo transfection. Harvesting the ovary after 72 hours of injection, shearing part of both sides of the ovary to extract total RNA and reverse transcription for c DNA were performed. Then we detected the expression level of the three lnc RNAs and the related genes of follicular development by q PCR. The rest of ovarian tissues were made of paraffin section and then were stained with HE, and observed and counted the number of preantral follicles, antral follicles and corpus luteum. After overexpressing lnc RNA274, the expression level of FSHR increased by about 7 times, LHR increased about 18 times, GDF-9 increased by about 11 times, BMP-15 increased about 7.5 times, EGFR increased about 7.5 times compared with the control group. Corpus luteum has significant increase compared with the control group(13.3±4.9 to 4.3±0.6), while the number of preantral follicles and antral follicles has no significant differences compared with the control group. After overexpressing lnc RNA147, the expression level of FSHR, GDF-9 and BMP-15 have no significant differences, LHR increased about 4 times, EGFR increased about 5 times compared with the control group. The number of corpus luteum, preantral follicles and antral follicles has no significant differences compared with the control group. After overexpressing lnc RNA647, the expression level of FSHR, LHR, GDF-9, BMP-15 and EGFR have no significant differences. The number of corpus luteum, preantral follicles and antral follicles has no significant differences compared with the control group.Experiment four: Further to validate affection of lnc RNA274 on ovulation. We constructed transgenic mice of lnc RNA274 in one-cell embryo and obtained F0 and F1 generation of transgenic mice and wild type mice of littermates respectively. Then we detected the fertility of transgenic mice of F0 and F1 generation and wild type mice of littermates respectively. The results showed that the number of offsprings of 10#-F0 transgenic mice(10.5±0.6) was significant greater compared with wild type mice(7.75±1.7) of littermates. The number of offsprings of 1#-F1 and 10#-F1 transgenic mice(11±1.4,10±1.4) was significant greater compared with wild type mice(7.5±0.7, 6.5±0.7) of littermates. The weight and body length of offsprings were no significant differences between F0 and F1 generation of transgenic mice.In conclusion, we got three lnc RNAs which were specifically high expressed in ovary(lnc RNA647、lnc RNA147 and lnc RNA274) and they were expressed high in ovaries at age of 8 weeks and metaestrus. We obtained overexpression level of lnc RNA274, and the expression levels of related genes of follicular development were significantly elevated and the number of ovulation was significant increased through in vivo transfection experiment. Transgenic mice with target gene of lnc RNA274 were constructed and following the analysis of its phenotype, the numbers of offspring were significantly increased. The experimental results demonstrated that lnc RNA274 can promote ovulation. |
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