Study On The Physiological Function Of Membrane Progestin Receptor In The Reproductive Cycle Of Flatfish

The experiments studied on the cultured half smooth tongue soleCynoglossus semilaevis Günther and Japanese flounder Paralichthysolivaceus about ovarian and oocyte development, characteristics of sexsteroid hormones in reproductive cycle and annual changes, cloning andexpression levels of two kinds of membrane progesterone receptor gene,bymethods of electron microscope,histological method, RACE and real-timePCR, hormone radiation immunoassay measurement and so on.Understanding the structure characteristics of ovarian and oocytes indifferent development stages, physiological function of sex steroidhormone and two kinds of membrane progesterone receptors gene, canprovide theory basis for artificial breeding technology of flatfish.1. The development of ovary of sexually mature half smooth tonguesole Cynoglossus semilaevis could be divided into five stages and thecollected oocytes be divided into five phases by using histological methodand transmission electron microscope. We preliminary judged that thefollicle diameter of200-500um,500-800um,800-1000um and1000-1200um were respectively in Ⅲ phase, Ⅳ phase,Ⅴ phase and free in the ovarian cavity. For the follicle diameter of200-300um, thecapillaries were found in the outer of follicular cell. Moreover, surfaceepithelium enclosed follicular cell. And a large number of mitochondriawas found in the cytoplasm. The rough endoplasmic reticulum, golgicomplex, cortical alveoli and lysosome were found in the follicle diameterof300-400um. The yolk granules was found in the follicle diameter of400-500um. Transparent parallel stripes were found on the yolk granules inthe follicle diameter of500-600um. The length and number of pore canalsof membrane were reduced in the follicle diameter of600-700um. In thefollicle diameter of700-800um, their was full of yolk granules.2. Full-length cDNA encoding membrane progestin receptor alpha(mPRα) was firstly cloned from half smooth tongue sole Cynoglossussemilaevis by means of homology cloning and RACE PCR analyses. Thelength of complete cDNA sequence of mPRα gene was1319bp. Alignmentanalysis using tongue sole mPRα gene deduced animo acid sequence andanimo acid sequences of other species showed that there were seventransmembrance domains. The rooted phylogenetic tree was constructed bythe neighbor-joining method of MEGA4.0. And the identity of tongue solemPRα with other representative sequences was analysed by the Clustal Xmethods. The results indicated that the tongue sole mPRα was clusteredtogether with those of other fish mPRα. The identity was94%、93%and 90%when compared with Southern flounder (Paralichthys lethostigma),Atlantic croaker (Micropogonias undulates) and Japanese medaka (Oryziaslatipes), respectively. In contrast, identity among the mPRα of tongue soleand those of human (Homo sapiens) and cattle (Bos taurus) was low andwas only53%. A quantitative Real-time PCR（qRT-PCR）method wasdeveloped to measure mRNA expression levels of mPRα gene of femaletongue sole. Tissue expression analysis showed that mPRα mRNA wasexpressed widely in tongue sole, although the expression level was nothomogeneous. mPRα transcripts were highly abundant in brain, ovary,pituitary and gill. The less abundant level is in liver, muscle, stomach,intestine and kidney. The study of expression levels of brain,ovary andpituitary in reproductive cycle revealed that the highest expression levels ofbrain and pituitary were both in stage Ⅳ and ovary was in stage Ⅴ. Thisstudy will lay the foundation for further exploring mechanism of mPRαaction in the reproductive physiology of half smooth tongue sole3. Full-length cDNA encoding membrane progestin receptor like(mPRL) was firstly cloned from half smooth tongue sole Cynoglossussemilaevis by means of homology cloning and RACE PCR analyses. Thelength of complete cDNA sequence of mPRL gene was2002bp.Constructing the amino acid sequence phylogenetic tree of mPR familywith14species, including bony fishes and mammals, found that the evolutionary tree was divided into three branches, mPR alpha branch,mPRL branch, mPR beta branch,and half smooth tongue sole mPRLbelongs to mPRL branch. And the identity of tongue sole mPRL with otherrepresentative sequences was analysed by the Clustal X methods. Theidentity was68%and72%when compared with O. latipes and G.aculeayus.A quantitative Real-time PCR（qRT-PCR）method wasdeveloped to measure mRNA expression levels of mPRL gene of femaletongue sole. Tissue expression analysis showed that mPRL mRNA wasexpressed widely in tongue sole, although the expression level was nothomogeneous. mPRL transcripts were highly abundant in ovary, heart, gilland brain. The less abundant level is in liver, kidney and head-kidney. Thestudy of expression levels of brain,ovary and pituitary in reproductive cyclerevealed that the highest expression levels of brain, ovary and pituitarywere both in stage Ⅴ.4. Using real-time PCR to analysis the expression levels of mPRα andmPRL in Sexual maturity female Paralichthys olivaceus. Tissue expressionanalysis showed that mPRα and mPRL mRNA were expressed widely inParalichthys olivaceus, although the expression level were nothomogeneous. mPRα transcripts of Paralichthys olivaceus were highlyabundant in brain, pituitary, ovary and gill, less abundant in head-kidney,kidney, stomach and intestine.And mPRL transcripts of Paralichthys olivaceus were highly abundant in ovary,heart,gill and brain. The study ofexpression levels of brain,ovary and pituitary in reproductive cyclerevealed that the expression levels of mPRα in brain, pituitary and ovarywere disaffinity. The result revealed that the highest expression levels ofbrain and pituitary were both in stage Ⅳ and ovary was in stage Ⅴ. Inaddition，the expression levels of mPRL were similar, and the highestexpression levels of were both in stage Ⅴ.5. Analysis of estradiol and progesterone of Cynoglossus semilaevisand Paralichthys olivaceus in reproductive cycle with ovarian maturity andgene expression revealed that estradiol was rising with yolk formationand reached the highest peak before oocyte maturation.But progesteronereached the highest peak in oocyte maturation period. The regular patternof estradiol and progesterone of Cynoglossus semilaevis and Paralichthysolivaceus in reproductive cycle were similar to the expression levels ofmPRα and mPRL of brain,ovary and pituitary in reproductive cycle.Thissuggested that estradiol and progesterone affect ovarian development andmature through two kinds of membrane progesterone receptors in fish.