| Chicken embryonic gonad is an important model to study animal organogenesis. The sex determination genes have not been ascertained yet and the genes related with gonad differentiation still need study further. To use chicken embryonic gonad models efficiently and establish practical sex transformation technique, it is urgent to do more researches about sex determination and differentiation genes. Using busulfan to treat chicken embryos is an effective method to get chimera chick. The affect of different dosages of busulfan to the survival rate of chick embryos and the depletion efficiency of primary germ cells in the acceptor chick embryo need study further. This study focused on the expression of genes related to gonadal development and the work on busulfan treating the embryos. Firstly, whole mount in situ hybridization was introduced and optimized in the study. Then the temporal and spatial expression pattern of gene Cdk2apl related with chick embryo gonad differentiation was detected using WISH combining with reverse transcript-polymerase chain reaction and immunohistochemistry staining. The gonad differentiation related gene Smarcel was cloned. The expression of Smarcel after over-expression was compared with the control groups, and the histological changes of testes and ovaries were observed. The affect of different dosages of busulfan to the survival rate of chick embryos, the numbers of primary germ cell in gonads and the histological changes of testes and ovaries were studied. The specific work is as follows:Introduce the technology of whole mount in situ hybridization in chick embryo study, based on Cdk2apl gene in chick, a pair of primers was designed to amplify the Cdk2apl fragment by reverse transcript-polymerase chain reaction and subsequently the fragment was cloned into plasmids pGEM-T. By using Sp6and T7RNA polymerase, the sense and anti-sense probes labeled by digoxigenin were transcripcd in vitro, respectively and were used to examine the expression of Cdk2apl in chick embryo of different sexes by whole-mount in situ hybridization, obtained low background and stain limpid hybridization result by control the pepsic density of protease K, the hybridization dense of probe and the washing temperature after the hybridization. In order to study the expression and regulatary functions of Cdk2apl in the development of chicken embryo, the established method whole mount in situ hybridization was used to invest the expression of Cdk2apl in both male and female chick embryo. The results indicated that at both sexes, Cdk2apl is expressed in the head mesenchyme, rhombencephalon, otic vesicles, spinal neural tube and forelimb bud of4.0day embryos and the expression level in males is significantly higher than that of females. In addition, in genital ridge and hind limb bud of the4.0d chicken embryo, Cdk2apl is obviously expressed in the males but no in the the females. After that, reverse transcript-polymerase chain reaction, whole mount in situ hybridization and immunohistochemistry staining were used to invest the expression of Cdk2apl in chick gonads at6.5-10.5d. The result showed that Cdk2apl was down-regulated with the development of embryo, the expression in female gonad is higher than that in male gonad. The alternative higher expression of Cdk2apl between male and female embryos needs further research. It is supposed that Cdk2a.pl may play a role in the development of gonad and sexual differentiation of chicken embryo based on its differential expression.To understand the potential role of Smarcel in avian sex determination and differentiation, over-expression of SMARCE1was performed in chicken embryos using the RCASBP.B retroviral vector. The results showed that Smarcel expression was up-regulated in the infected embryo gonads at the investigated stages (E6.5-E12.5) assessed by whole mount in situ hybridization and immunohistochemistry staining. The HE staining indicated that the ovarian cortex of the infected female embryos was thicker than the control, and both the testes seminiferous cord and the interstitial cell of infected male embryo were more than the control. It is supposed that SMARCE1is able to facilitate both the testes and ovaries development in embryonic chicken.Present study examined the survival rate of chicken embryos after treated with different dosages of busulfan emulsion (0μg,120μ.g,240μg,360μg and480μg). Then the numbers of primary germ cells in chicken embryonic gonads were checked and analyzed using immunohistochemistry staining of stage specific embryonic antigen-1(SSEA-1). At8.5d,11.5d and14.5d, the histological changes of testes and ovaries after treated with different dosages of busulfan were observed using HE staining. The results showed that low dosages of busulfan (<120μg) did not influence significantly on the survival rate of embryos compared with blank control (P>0.05). Medium dosages of busulfan (240μg and360μg) significantly reduced the survival rate (P<0.05), and high dosage of busulfan (480μg) furtherly reduced the survival rate (P<0.05). The testes and ovaries responsed differently to the treatment of busulfan. There existed auto regulation of primary germ cells in testes, but the primary germ cells in ovaries almost didn’t response to the treatment of busulfan. Low dosages of busulfan did not affect the morphological structure of chick embryo gonads. The amount of seminiferous cord didn’t change. The thickness of ovary cortex and the number and arrangement of cells in medulla were the same as control ones. The morphological structure of chicken embryo goands began to be abnormal with the increasing dosages of busulfan. After treated with high dosage of busulfan, the seminiferous cord in testes reduced obviously and arranged in disorder. The ovary cortex became thinner and cells in medulla reduced and scattered. These results showed that the increasing dosage of busulfan was positively correlated with both the death rate of embryos and the structural abnormalities of testes and ovaries. |
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