| This thesis mainly studied on the PGRMC1( progestrone receptor membrane component1, PGRMC1) of the cultured half smooth tongue sole(Cynoglossus semilaevis) by using RACE, histology, fluorescence quantitative real-time, RNA in situ hybridization, immunohistochemistry and Western-blotting methods. We cloned the full-length cDNA sequence of PGRMC1 from the ovary of tongue sole and did a quantitative and qualitative analysis for the expression characteristics of PGRMC1 in the different tissues. At the same time, The exogenous hormone treatment and cultured in vitro for the oocytes in different development phase, the expression features of PGRMC1 were analyzed. The relationship between PGRMC1 knock down and oocyte maturation was analyzed. The present study could lay the foundation for the study of physiological function of the PGRMC1, and improve the flatfish of artificial reproduction technology. The results are as follows:1. Molecular cloning and tissue expression analysis of PGRMC1 from half smooth tongue soleWe cloned the full-length cDNA sequence of PGRMC1 from the ovary of C. semilaevis using homology cloning and RACE method. The full length cDNA of PGRMC1 was 1335 bp in size with ORF of 546 bp encoding a 181 amino acids preprohormone with a deduced molecular mass and isoelectric point of 20.64 KDa and 4.67, respectively. The PGRMC1 was a single transmembrane protein with a N-terminal transmembrane domain. Sequence alignment of C. semilaevis PGRMC1 protein with the corresponding sequences identified in other species reveals that the highest identity was 82.87% with Oryzias latipes, followed by 81.22% with Tetraodon nigroviridis. Phylogenetic analysis indicated that C.semilaevis PGRMC1 was clustered with the counterparts of Cyprinodontiformes and Tetraodontiformes. The mRNA expression of PGRMC1 could be detected at high levels in the ovary, to a lesser extent in the liver and brain, and at low levels in other tissues. In addition, we studied that the change characteristics for PGRMC1 mRNA expression levels in pituitary, brain and ovary at different ovarian developmental stages.The PGRMC1 mRNA in brain increased sharply at stage III and remained higher levels until stage V and then decreased at stage VI. The PGRMC1 mRNA in pituitary ascended progressively to the maximal value at stage V and then declined significantly post ovulation. Similarly, the levels of ovarian PGRMC1 mRNA increased gradually, reached the peak value at stage V and then declined markedly after spawning.2. The PGRMC1 expression analysis in different tissues of C. semilaevisWe analyed PGRMC1 protein sequence and selected epitope of the protein sequence. The corresponding immune polypeptide were synthesized. Polyclonal antibodies were generated by immunization of New Zealand rabbit with recombinant protein. The expression level of PGRMC1 protein was detected by using polyclonal antibody.The protein expression levels in different tissues were analyzed by Western-blotting. The tissue distributions of PGRMC1 were studied by RNA in situ hybridization and immunohistochemistry. Western-blotting results showed that the PGRMC1 protein expression were higher in the ovary and brain. The expression levels were relatively low in liver, kidney, head kidney and pituitary. Those results showed that PGRMC1 may play a main role in the reproductive organ. RNA in situ hybridization and immunohistochemistry results showed that the positive signals of PGRMC1 mRNA and protein were significantly expressed in the oocyte membrane of maturational ovary. Those findings further provided an evidence that PGRMC1 may be involved in membrane / cytoplasm and nucleus important intracellular processes. The positive signals of PGRMC1 were detected in liver, kidney, head kidney, brain and pituitary of C. semilaevis. These positive signals were distributed in different parts of the organ. In the liver, PGRMC1 was mainly localized in the bile duct and hepatic veins, which were connected with the outside world. In the kidney, it was observed that PGRMC1 was expressed in the vicinity of the renal tubules.The expression characteristics of PGRMC1 in different tissues indicated that it could mediate different physiological functions in C. semilaevis.3. The PGRMC1 expression analysis in the oocytes of C. semilaevisThe mRNA expression of PGRMC1 in the oogenesis of the different development period of ovary and the influence by the different concentration of HCG treatment were detected by the qRT-PCRand western-blotting analysis. The results showed that the highest transcriptional level of PGRMC1 appeared at the different developmental phase of oocyte. However, the overall trend was consistent. The PGRMC1 mRNA expression level in the oocyte of IV phase was the highest in the ovary of stage IV(P<0.05). The PGRMC1 mRNA expression level in the oocyte of V phase was the highest in the ovary of stage V(P<0.05).The variation of PGRMC1 expression by the HCG treatment in different stages of oogenesis was detected. The results showed that the regulation of 20IU/ml HCG on expression of PGRMC1 mRNA and protein was more significant than 10IU/ml HCG(P<0.05). It showed that the HCG have an effect on the expression levels of PGRMC1. The promoting effects of HCG is different at different phases of oocyte, and it was the most obvious in the oocyte of V phase. This shows that PGRMC1 play an important role in mature stage.4. The PGRMC1 gene knock-down from oocyte of C. semilaevisGene knock-down technology is an important experimental method to study gene function. In this study, in vitro oocytes as experimental materials, we design and synthesis PGRMC1-Morpholino inhibited PGRMC1 gene function in oocyte, and set up the control group, injection group, antisense group, antisense control group. After microinjection, we used the qRT-PCR and Western-blotting methods to detect the PGRMC1 mRNA and protein expression characteristics. Taken together,the PGRMC1 can regulate oocyte maturation process of C. semilaevis. Our results would shed light on further explore the PGRMC1 gene in reproduction function of C. semilaevis. |
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