Cloning And Characterization Of Duck Leptin Receptor CDNA And Effect Of Exogenous Leptin On Duck Reproductive Function

Leptin is a protein secreted primarily by white adipose tissue, in addition to its effects on food intake and energy expenditure balance, leptin has been implicated as a mediator of diverse physiological processes, such as growth, development and reproduction. There are numerous studies about leptin in mammals, however, little is known about biological role of leptin in avian. In the present study, two isoforms of duck leptin receptor (LEPR) were cloned and the tissue and organ distribution of leptin receptors in duck was determined. The effect of the exogenous leptin on the recovery of regressed ovary in fasted ducks was studied, the possible mechanism was discussed, and the possible mechanism was dis ussed. Finally, the prokaryotic highly expression vector for mouse leptin was constructed and the fusion protein was purified. The main results are as follow:(1) By the method of reverse transcription-PCR (RT-PCR) and Rapid Amplification of cDNA Ends (RACE), two isforms of duck LEPR from duck ovary cDNA were cloned. The two isoforms of LEPR were named LEPR-a and LEPR-b, and both complete sequences of coding region were obtained. By sequence analysis, the 3468bp cDNA of LEPR-b coding region encodes a protein of 1155 amino acid. The deduced amino acid sequence of duck lepr-b showed 81.7% identity to the chicken LEPR-b, showed 51. 9% and 50. 1% identity to the human and mouse LEPR-b respectively. Duck LEPR-b contained all functionanl domains to activate JAK-STAT pathway as mammals' LEPR-b. Duck LEPR-a was a isoform of LEPR which was firstly found in avian. The 2670 bp cDNA of duck LEPR-a encoded a protein of 889 amino acid. Dcuk LEPR-a contained only a JAK2 binding box in the cytoplasm region, so duck LEPR-a which like mammals' LEPR-a may not activate the JAK-STAT pathway(2) The semi-quantitative RT-PCR was performed to determine the expression pattern of the duck LEPR-a and LEPR-b in fourteen tissue and organ samples of adult ducks. The results showed that the duck LEPR-b mRNA was expressed in all tissues and organs , expecially highly in pituitary, gonad, lung, kidney, muscle and adipose; the expression of duck LEPR-a in most tissues and organs was less than LEPR-b, duck LEPR-a was only expressed highly in testis> cerebellum and pituitary.(3) 2 days of fasting caused the regression of the duck ovaries, 3 days of refeeding could not restore the regressed ovaries according to the morphological and histologic characteristics of ovaries. And the regression of ovaries caused by fasting might be related to the decrease of plasma E₂ and P₄ , and the decrease of the expression of ovarian ER-β,FSHR,LHR,GnRH-I,GHR and LEPR-b mRNA. A morderate dose of exogenous leptin (250μg/kg body weight/day) could promote the recovery of regressed ovary in fasted ducks , and it may be related to the increase of plasma E₂ caused by the leptin treatment, and it also may related to the increase of the expression of ovarian ER-β,FSHR,LHR,GnRH-1,GHR and LEPR-b mRNA. At the meantime, it was also found that high dose leptin treatment (1000μg/kg body weight/day) may inhibit the recovery of regressed ovary, it might be related to the decrease of plasma E₂ , and the decrease of the expression of ovarian ER-β,FSHR,LHR,GnRH-I,GHR and LEPR-b mRNA.(4) Fasting significantly decreased the expression of ovarian LEPR-b mRNA, and leptin can stimulate the expression of ovarian LEPR-b mRNA. It might be related to the leptin role in the recovery of ovary. It was also found that ovarian LEPR-a mRNA expression increase in regressed ovary, so it is supposed that the LEPR-a may be the mediator in the leptin to LEPR-b pathway.