| This thesis mainly studied on cultured half smooth tongue sole(Cynoglossus semilaevis)and Japanese flounder(Paralichthys olivaceus) by using histology, fluorescence quantitative real-time, RNA in situ hybridization, immunohistochemistry and western blotting method to do a quantitative and qualitative analysis for the expression characteristics of membrane progesterone receptor alpha(m PRα) and new membrane progesterone receptor-like(m PRL) in the different development stage of oocytes and tissues. At the same time, The expression patterns of m PRαand m PRL following h CG treatment in the different stages of oogenesis were investigated. This study provides a preliminary understanding of the expression characteristics and physiological function of two kinds of membrane progesterone receptor, and also provides theoretical basis for improving the technology of artificial breeding of flatfish. The results are as follows:1.The m PRα and m PRL expression analysis in oocytes of half smooth tongue soleThe m RNA expression of m PRα and m PRL in the different stages of oogenesis of the different development period of ovary and the influence by the different concentration of HCG treatment were detected by the fluorescence quantitative Real-time and western-blotting technical analysis. The results showed that the highest transcriptional level of m PRα and m PRL appeared at the different developmental phase of oocyte. However the overall trend is consistent.The m PRα and m PRL m RNA expression level of the oocyte in II phase were highest in the ovary of stage II(P<0.05). The m PRα and m PRL m RNA expression level of the oocyte in IV phase were the highest in the ovary of stage III and IV stages(P<0.05). The m PRα and m PRL m RNA expression level of the oocyte in V phase were the highest in the ovary of stage V(P<0.05).The variation of m PRα and m PRL expression by the different concentrations of HCG treatment in different stages of oogenesis were detected. The results showed that the regulation of 20IU/ml HCG on expression of m PRα and m PRL m RNA and protein is more obvious than10IU/ml HCG(P<0.05). It showed that the effects of HCG have a effect on the expression levels of m PRα and m PRL. HCG promoting effects is different at different phases of oocyte, and it was the most obvious in the the oocyte of V phase. This shows that m PRα and m PRL play an important role in mature stage.2.The m PRα and m PRL expression analysis in different tissues of half smooth tongue sole The protein expression levels in different tissues were analyzed by western-blotting technology.The tissue distributions of m PRα and m PRL were studied by RNA in situ hybridization and immunohistochemistry. Western blot results showed that the m PRα and m PRL protein expression in the gonads were the highest(P<0.05). The expression levels in liver, kidney,brain, head kidney and pituitary were relatively low. Those results showed that m PRα and m PRL may play a main role in gonadal tissue. The result of in situ hybridization and immunohistochemistry showed that the positive signals of m PRα and m PRL were significantly expressed in the oocyte membrane of maturational ovary. This finding further provided a evidence for m PRα and m PRL as the membrane receptor and take an exercise of its physiological function in membrane. The positive signal of m PRα and m PRL hybridization wasdistributed in the cell membrane of oocytes. The positive signal of two genes were detected in the peripheral part of head kidney, pituitary and brain. And the positive signal of two genes distributed around the bile canaliculi. This further showed the characteristics of m PRs m RNA expression of different tissues and demonstrated it mediates progesterone exercising different physiological role.3. The m PRα and m PRL expression analysis in oocytes of Japanese flounderThe m RNA expression of m PRα and m PRL in the different stages of oogenesis were studied by fluorescence quantitative Real-time technical analysis. The results showed that the highest expression value of m PRα and m PRL appeared in V phase oocytes in the ovary of stage V(P<0.05).4.The m PRα and m PRL expression analysis in different tissues of Japanese flounder The m PRα and m PRL protein expression levels in different tissues were analyzed by western-blotting technology. The m PRα and m PRL m RNA and protein distribution in the different tissues were studied by RNA in situ hybridization and immunohistochemistry. Western blot results showed that m PRα and m PRL protein expression levels in the gonads was the highest than the expression in liver, kidney, brain, head kidney and pituitary(P<0.05). This suggested that m PRα and m PRL play a main role in gonad than other tissues. The result of in situ hybridization and immunohistochemistry showed that the positive signals of m PRα and m PRL were significantly expressed in the oocyte membrane in the ovary of stage V. The relative results provided an evidence for m PRα and m PRL as the membrane receptor to take an exercise of their physiological function in membrane. The positive signal of m PRα and m PRL gene hybridization was distributed in the cell membrane of oocytes. The m PRα and m PRL were detected in the different parts of liver, kidney, head kidney, brain and pituitary. The positive signal were distributed in the peripheral of kidney, head kidney, pituitary and brain. And the positive signal were found around the bile canaliculi of liver. The current results firstly showed the characteristics of m PRs m RNA expression in different tissues of japanesr flounder, and demonstrated that m PRs may mediate progesterone to exercise the different physiological role. |
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