| The reason why the chicken-quail hybrid embryos died at an early time was probably because of thedifference on the expression of the relevant genes of the chickens and the quails in the process of sexdifferentiation. The main cause of the embryo’s death was closely related to the estrogen receptor (ER) andthe relevant genes regulated by the ER. Therefore, the research adopted the relevant genes of the sexdifferentiation of the ER and so on as the primary target.In order to meet the needs of the sampling in the follow-up experiments, we have carried out anexperimental research of improving the fertilization rate and survival rate of the chicken-quail hybridembryos. On the day after two days of insemination, we collected the mixed sperms of the roosters andinseminated the quails in the morning and evening twice. Besides, when the quails are at the peak of thelaying period, that is, when the laying rate was above80%, and with the adoption of the Grumbach rotatingincubator imported from Germany whose temperature was ranging from37.8℃to38.6℃and humidity isranging from60%to80%, and when we inseminated the quails at the superficial part of the vagina, on twoconsecutive days insemination, sooner or later each day,1times,1days interval and then operate better, sowe could achieve good results.In this study the expression profiles of3β-HSD, P-450c17, P450arom and Estrogen receptor (ER)genes were analyzed to understand the potential influence of3β-HSD, P-450c17, P450arom and Estrogenreceptor (ER) on the early embryonic development and sex differentiation in quail embryos. The sexes ofquail embryos were identified by PCR amplification of Wpkci gene, and then10female and10maleembryos at every time point (day3,4,5,6,7and8of incubation) of the early development stage weresampled. Real-time PCR was applied to analyze the expression profiles of3β-HSD, P-450c17, P450aromand Estrogen receptor (ER) genes at different time points of the early development stage (day3-8ofincubation) in quail embryos. The results showed that the expression levels of P-450c17, P450arom and ERwere all higher in female than that in male on day4of incubation: for P450arom gene, it was detected onlyin female embryos throughout the incubation period and displayed the peaking level on day4of incubation（P﹤0.01）; for ER gene, the highest expression levels were detected on day4of incubation both in femaleand male, but the expression level in female was significantly higher than that in male （P﹤0.05）; forP-450c17, the expression level was higher in female than in male, but the difference was not significant（P﹥0.05）. The expression level of3β-HSD gene was significantly higher in female than in male on day5ofincubation（P﹤0.01）. These results indicated that3β-HSD, P-450c17, P450arom and ER genes played animportant role in the process of sex differentiation during the early development stages of quail embryos,especially at the day4to day5of incubation.In order to analyze the genes that played an important role in sex differentiation of birds, this studywas designed to detect expression profiles of estrogens and its related genes in sex-reversed chickenembryos. In the present study, after injection with formestane and17β-estradiol, expression of ER,3βHSD,P450c17, and p450arom were measured at the early development stages (84h,96h,120h,144h,168hand192h incubation) in chicken embryos by real-time PCR. Embryos treated with formestane and17β-estradiol were designed as test group, while embryos non-treated but sex differential comprised controlgroup. The results showed that: expression of ER,3βHSD, P450c17and P450arom in control groupshowed normal expression trend, but when compared with test group, the difference was significant (P＜0.05or P＜0.01). For the test group, ER expression showed a downward trend, whereas, P450aromexpression increased sharply. However, there was no obvious trend for the expression trend of3βHSD andP450c17genes. It was indicated that estrogen regulation was a complex process. Once injected with exogenous hormone, the normal regulation process would be disturbed significantly.Because the steroid hormone played an important role in the gonad development of the poultry, thegene expression of the5enzyme which catalyzed the biosynthesis of the steroid hormone was particularlyimportant. In the period of the sex differentiation of the chicken-quail hybrid embryos, the expressionrelated to the ER was of great significance in sex differentiation. By making use of the PCR embryossex-identifying technology and RT-PCR technology to analyze the regular pattern of the expression of3β-HSD, P-450c17, Aromatase, ER and other genes so as to explore the regular pattern of the expression ofthe relevant genes of different genders, different development time of the embryos, the ER, etc,. Bycontrasting the expression of the genes of chickens and quails, we could make an analysis of the effects ofthe ER in the early death of the large number of the hybrid embryos and the related regulatory mechanism.According to the research, we found that the expression peak of the female hybrid embryos of3β-HSDgenes occurs at3d6h(78h),4d(96h) and5d(120h), while the expression of the male embryos is clearlylower; and the expression peak of the female hybrid embryos of P-450c17genes occurs at3d6h(78h),remarkably higher than that of the male embryos. Moreover, after that, both the female embryos and themale ones had a low level of the expression. The expression peak of the female hybrid embryos ofP450arom genes occurs at3d(72h),3d12h(84h),4d(96) and5d(120h), while the expression of the maleembryos was not in the form of P450arom genes; and the expression peak of the female hybrid embryosand that of the male hybrid embryos of ER genes both occur at3d(72h) and4d12h(108h), but theexpression of the female embryos is higher. Besides, the expressions of both the female embryos and themale ones were low at4d(96h) and5d(1200h); and after6d(144h), both expressions were low.We have adopted the RIA method to determine the levels of estradiol and testis every3h period fromthe third day to the sixth day of the chickens, quails and the hybrid embryos. The results showed that thelevel of testosterone was apparently higher than that of the early developing embryos of the chickens andquails, and the level of the estradiol showed fluctuating changes. In addition, the disorder of the hormonesynthesis of the sex differentiation was also the reason that led to the abnormality of the development of thehybrid embryos, which was also the evidence of the abnormality of the gene expression in the generatingprocess of the above sex hormones.Based on the above research results, it was in the process of the sex differentiation that the relevantgenes from the chickens and quails differed in their expressions and then led to the early death of theembryos. Thus, we could find that the main reasons of the death of the embryos was closely related to theexpressions of the ER and the relevant genes regulated by it. ER indeed played an important role in theearly death of the hybrid embryos. The abnormality occurred in the hybrids showed that the hybrids hadtheir own expression pattern of genes. Moreover, the time when the pattern occurs was consistent with thetime of death. |
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