Effect Of Leptin On Yolk Sac And Chorioallantoic Membrane Gene Expression And Angiogenesis In The Chicken Embryo

The present study was initiated by a pioneer experiment which was aimed to investigate the effect of maternal protein restriction on offspring embryonic development in Langshan chickens. It was shown that maternal protein restriction significantly decreased leptin deposition in the egg, changed gene expression pattern in yolk sac membrane, and reduced hatch weight, Based on these findings, we raised a hypothesis that leptin in the egg may serve as a maternal signal to affect embryo development through its action on yolk sac and chorioallantoic membranes (CAM). To test the hypothesis, we injected leptin into both broiler and layer breeding eggs, and found that the effect of in ovo leptin injection on embryo development was associated mainly with alterations in expression of genes involved in hormone signalling and angiogenesis in CAM. In order to further study the effect of leptin on CAM angiogenesis, we administered leptin directly onto the surface of CAM through a window and treated primary cultured CAM epithelial cells with leptin to examine the changes of CAM angiogenesis or cell viability, as well as the expression of the angiogenesis-related genes at the levels of both mRNA and protein.1 Effects of maternal protein restriction on hormone deposition in eggs and yolk sac gene expression and hatch weight in Langshan chickensOne hundred twenty 44-week-old Langshan breeder hens were randomly divided into two groups fed diets containing low (10%, LP) or normal (15%) crude protein levels. Eggs were collected randomly for measuring leptin and corticosterone contents and for incubation under the same condition. On day 14 of incubation (E14), fetuses were weighed, CAM and yolk sac membranes were collected for quantitation of mRNAs with Real-time RT-PCR. The breeder eggs laid by LP hens contained significantly lower leptin (P<0.05), while corticosterone content was not altered. The hatch weight of LP chicks was significantly lower than their control counterparts (P< 0.05) although the E14 fetal weight did not differ.20-hydroxysteroid dehydrogenase (20HSD) and basic fiberoblast growth factor 2 (FGF2) mRNA levels in yolk sac membrane were significantly lower than control (P< 0.05), and vascular endothelial growth factor (VEGF) mRNA level also tended to decrease (P=0.052). No alterations were observed in yolk sac expression of leptin receptor (LepR), transthyretin (TTR), or CAM expression of 20HSD, VEGF, FGF2 or LepR mRNA. These results indicate that leptin may target the gene expression in extraembryonic membranes to affect embryonic development in the chicken.2 Effect of in ovo leptin administration on hatch weight, CAM angiogenesis and extraembryonic membrane gene expression in layer and broiler chickensLayer and broiler breeder eggs were injected with either 0.5μg of recombinant mice leptin in 100μL of phosphate buffered saline (PBS) or 100μL PBS before incubation. On E12, fetal body weight and length were recorded and the yolk sac and CAM were taken for mRNA quantitation. On E13, CAM was fixed and removed for measuring the vessel areas with professional software (Image-pro plus 4.5). In ovo injection of leptin decreased hatch weight of layer female chicks (P<0.05), despite similar fetal weight and length on E12. This growth retardation was associated with a significant decrease in CAM vessel area also in E13 female layer embyros (P< 0.05). In contrast, broiler chickens did not show changes in hatch weight or CAM vessel area regardless of gender. Leptin significantly up-regulated mRNA expression of VEGF, FGF2, LepR and 20HSD in CAM of layer females.20HSD mRNA was increased in CAM of layer males (P<0.05). Moreover, yolk sac VEGF mRNA were up-regulated (P<0.05) in yolk sac of layer females, while no difference was found in either of the genes investigated in yolk sac of layer males. Yolk sac LepR and VEGF gene expression was up-regulated in male broilers, while no alterations were found in the expression of either investigated genes in CAM of broilers. These results suggest that the effects of leptin on chicken embryo development, extraembryonic membrane gene expression, and CAM angiogenesis are breed and sex dependent. The hatch weight decrease in female layer chicks is associated with inhibition in CAM angiogenesis and modification of CAM gene expression.3 Effect of leptin on CAM angiogenesis and the mechanism involved To investigate the effect of leptin on CAM angiogenesis, we administered 2.5μL 0 ng,10 ng and 100 ng leptin in PBS directly onto the surface of CAM through a window on E8 and CAM was fixed and removed for observation 48 h thereafter. In addition, primary cultured CAM epithelial cells were divided into 4 groups treated with 0,1,10 and 100 ng/mL of leptin, respectively for 72 h. LepR,20HSD, VEGF and FGF2 mRNA levels were determined with Real-time RT-PCR, LepR and VEGF protein levesl were quantitated with Western blot analysis. The number of total and middle sized blood vessels showed tendencies of decrease (P=0.064, P=0.054), while small vessel and large vessel were not changed in layers regardless of gender. When taking gender into consideration, the female layers demonstrated significant decrease in the number of total blood vessels (P=0.044), as well as the middle and large sized vessels (P=0.011, P=0.014). The viability of cells was inhibited significantly after 72 h of leptin treatment. LepR gene expression was up-regulated in leptin-treated cells at the doses of 10 and 100 ng/mL, However, LepR protein levels were decreased either significantly or potentially (P=0.029, P=0.078). VEGF, FGF2 mRNA levels were increased in leptin-treated cells at all the doses investigated (P< 0.05), while VEGF protein level tended to decrease after treated with 10 and 100 ng/mL of leptin (P=0.065, P=0.07). These results indicate that leptin inhibits CAM angiogenesis in layer females, the decreased cell viability, as well as LepR and VEGF protein levels in CAM cells may be involved in such effect.