Preliminary Studies On The Reproductive Physiology And CDNA Cloning Of Gonadotropin Hormone Genes Of Paralichthys Lethostigma

The physiological and endocrine characteristics of reproductive cycle of southernflounder were investigated in the present study. The contents include physiologicalcharacteristics of gonadal maturation cycle, sex steroids expression pattern,gonadotropic hormones expression pattern, exogenous hormone induction, structure andfunction of gonadotropin hormone genes, etc. Multiple methods including histologicalobservation, ELISA, RACE, semi-quantitative real time PCR and exogenous hormoneinduction etc. were combined and employed in the present study. Results from thisstudy would be helpful to better understand the physiological and endocrinemechanisms underlying the annual reproductive cycle of southern flounder, furthermore,it will be facilitate the artificial induced spawning of southern flounder under farmingconditions.1. The physiological characteristics of annual ovary maturation of southern flounderThe histological observation revealed that southern flounder features asynchronousoocyte development and batch spawnings, the ovary matured once per year during thereproductive cycle. The oocyte growth could be divided into six phases in its life history,whereas in sexually matured females, only five phases of the oocyte growth could beobserved and ovary maturation cycle was divided into five stages accordingly. Thegonadosomatic index （GSI）, condition factor （CF）, hepatosomatic index （HSI） valueswere all found to increase prior to, or during the peak phase of vitellogenic growthcorresponding to the gonad development. The GSI peaked at December （20.87） andreached the lowest level of1.06at June. The HSI achieved the maximum value of4.92at November and the minimum value of1.14at December. The CF value peaked atNovember.2. Annual expression pattern of serum sex steroids levels and gonadotropic hormones of female southern flounderThe annual expression pattern of serum sex steroids including17β-Estradiol （E₂）,testosterone （T）, progesterone （P） and gonadotropic hormones including folliclestimulating hormone （FSH）,luteinizing hormone （LH） during ovary maturation cyclewere determined by enzyme-linked immunosorbent assay （ELISA）. A close relationshipbetween sex steroids levels, gonadotropic hormones and gonadal development wasobserved during annual maturation cycle of female southern flounder. Two peaks of sexsteroids levels were observed during annual ovary maturation of southern flounder inthe present study. The estradiol expression levels peaked at June （7.43pg/mL） andDecember （6.67pg/mL） respectively, wherein the first peak value of estradiolexpression level is higher than that of second peak value. The serum estradiolexpression levels maintained relative low in other months, and the minimum valueappeared at February when reached the end of spawning season with the expressionlevel was4.34pg/mL. The temperature exerted significant effect on the expression ofserum sex steroids. No significant fluctuation of serum sex steroids of testosteronelevels was found through the year in this study, it peaked at November （377.84pg/ml）and dropped to the lowest level （307.24pg/ml） at March. The testosterone expressionlevel showed significant relationship with water temperature, and its maximum valueappeared one month before the estradiol expression level reached the maximum value.The expression level of serum progesterone showed obvious annual fluctuation, and itpeaked at July （23.76ng/mL） and January （23.54ng/mL）, and the lowest level wasfound at March.The serum FSH level peaked at July and January respectively, and the first peakvalue was higher than that of the second. As for serum LH level, it peaked at June andDecember respectively. The exogenous salmon gonadotropin releasing hormone wasused to induce final maturation of oocytes of southern flounder in the present study.Both the serum FSH level and LH level peaked at96h post hormonal treatment,meanwhile, the serum progesterone level increased, but the serum estradiol level peakedat144h post hormonal treatment while testosterone level dropped to the lowest level at144h. These results provided direct endocrine index to evaluate the reproductioncondition of southern flounder broodstocks and would be helpful to better understandthe reproductive biology of southern flounder.3. Molecular cloning and expression characteristics of gonadotropic hormone genes The present study cloned and sequenced the gonadotropin hormone （FSHβ andLHβ subunit） genes from southern flounder by isolating their cDNAs. The FSHβsubunit consists of120amino acid residues, and the FSHβ cDNA is625bp in length,excluding a poly （A） tail, and consists of a107-bp5`UTR, a363-bp open reading frame（ORF）, a stop codon （TGA）, and a155-bp3`UTR. The predicted molecular weight was12918.88m.w. and the isoelectric point was5.52. Two N-glycosylation sites were foundin the cDNA sequence. The LHβsubunit consists of145amino acid residues, and theLHβ cDNA is597bp in length, excluding a poly （A） tail, and consists of a72-bp5`UTR, a438-bp open reading frame （ORF）, a stop codon （TGA）, and a87-bp3`UTR.The predicted molecular weight was16383.31m.w. and the isoelectric point was5.58.A N-glycosylation site was found in the cDNA sequence.The spatial expression pattern of these two genes were determined. The FSHβsubunit was exclusively detected in pituitary, whereas the LHβ subunit exhibiteduniversal tissue expression pattern, it could be detected in pituitary, brain, gonad, liver,heart, spleen, stomach, intestine and gill, which indicated it may bear multiplephysiological functions besides reproduction regulation in southern flounder.4. The seasonal changes in expression levels of gonadotropic hormone genes duringannual ovary maturation cycle of southern flounderThe temporal expression patterns of these two genes during ovary maturation cyclewere determined using semi-quantitative real time PCR. The pituitary mRNA level ofFSHβ gene was low at the early stage of gonadal maturation, and increased with theovarian development and peaked at December （pre-spawning stage）. The pituitary, brain,liver and gonadal LHβ mRNA level were examined during the ovarian maturation cycleof southern flounder. Wherein, the pituitary LHβ mRNA level maintained relativelyhigh level during the ovarian maturation cycle, and peaked at January （spawning stage）.The brain LHβ mRNA level increased with gonadal development and peaked atDecember. Liver LHβ mRNA level reached the maximum value at July and maintainedrelatively low level at other sample months. The gonadal LHβ mRNA level showed twoobvious peaks, and peaked at June and December respectively, which were concomitantwith that of serum estradiol level. The temporal expression patterns of FSHβ mRNAand β mRNA during ovarian maturation cycle were in accordance with morphologicalcharacteristics of oocytes maturation and expression patterns of serum sex hormones.These results provide more knowledge on reproductive regulation mechanism of southern flounder from the view of gene level.