| As sex is one of the greatest advances in the history of biological evolution, sex determination has been attracting people’s attention all this time. With the progress of science and technology, genetic determination of mammalian gender was accepted by the discovery of Sex-determining Region on the Y Chromosome(SRY). However there is large difference between avian sex determination and mammals. Although several genes which associated with the sex-determining have been found on Z and W chromosome of bird, there is no key gene like SRY gene in mammalian. It is interesting that sex reversal phenomena the hen is crowing under natural was recorded in the history. Researchers also discovered that sex determination and differentiation process are vulnerable to influenced by sex hormones. Because of sex reversal chicken is the ideal model for study of avian sex determination and differentiation, as well as the potential economic value, researchers tried various methods to research sex reversal chicken in recent years.Our laboratory has a wealth of experience on obtaining male phenotypic sex reversal chicken via drugs induction and gonads removed. At present we come across two problems: the revers of male phenotypic is unstable in sex reversal chicken and the incomplete development of vas deferens. In this study, eggs were dealing with exemestane oil drug and aromatase mixed with testosterone oil drug. Right gonad of the chicks was removed after hatching 3~5d. Cross-group induced by drug-tracking in chicken was start at the first day. Observing the changes in apparent characters of sex reversal chicken and verified their genetic sex by PCR when grow up to aged 1 months. Measured the expression level of six sex determining genes in gonadal by real-time fluorescence quantitative PCR at aged 1, 2, 3, 4 months respectively. At the age of 6 months compared the development of vas deferens and sperm. In addition, explored the fertility of male phenotypic sex reversal chicken by artificial insemination in vitro. Results are as follows:Either 0.7mg exemestane oil drug or 0.7 exemestane mixed 0.5mg testosterone oil drug injected in per egg, hatchability was reduced significantly. Two death peaks occurred in the early incubation and hatching period respectively, leaded to the final hatching rates is lower than 40%. It illustrated that exemestane is toxic to embryonic. Compared the rate of weak chicken between and the exemestane group and the mixed drugs group, the latter was significantly higher than the former, indicate excess testosterone is bad for embryogenesis.Compared group A, B, C and D with control group: at 1 month old, comb of experimental group was more prominent developed. Tail shape and combative personality of A and C were more similar to the control group. Testis of A and C group were slightly smaller on volume, but shape, color are similar to control group. Testis of group B and D were similar to control group on shape at one month age and two months age and control group similar, but appearance turned partly rough, color nonuniform. At three months age the surface of gonad became completely rough, color turned pink white, and stop growing. At four months age, it has turn light white, sheet, rougher, edge blur. Indicated that female chicken use alone exemestane can’t maintain male phenotypic stability, but the drug mixed testosterone can make stable male phenotypic sex reversal.DMRT1, SF1, DAX1, AMH, SOX9 and P450 arom gene were expressed differently in gonad between the groups of male phenotypic sex reversal chicken. Levels of DMRT1, AMH and SOX9 gene expression were higher in the gonads of A, C and the control group than B, D groups. Levels of SF1、DAX1 and P450 arom gene expression were higher in B, D group than those in A, C and the control group. Testosterone increased DMRT1, SOX9, AMH gene and decreased SF1, DAX1, P450 arom gene expression in gonads of male phenotypic reversal chicken. Consequently, DMRT1, SOX9 and AMH gene closely related with the formation of the testes.At six months age of A group, adult male phenotype chickens presented male secondary sex characteristics with crowing loudly. Yellowish-white testicles looks like broad bean(3.06 cm vertical axis, 1.22 cm horizontal axis, and 3.83 g weight), firming and vas deferens well developed. Spermatozoon movements were observed in spillage from the right gonads. Support cells, spermatogenesis cells of various stages and spermatozoa were observed on tissue slices of the right gonads. The section of vas deferens which close to epididymis was filled with sperms. Tube wall of the vas deferens in midpiece increased thickness and lumen decreased with seldom sperms. The lumen of vas deferens at the far-end was filled with parenchymal tissue. Indicates that group A which were dealing with exemestane oil drug added testosterone could maintain male sexual characteristics stable, produce sperm, form complete vas deferens, but could not obtain generation by natural mating.Conclusion:(1)phenotypic stability male phenotypic reversal chicken could obtain inducing by exemestane combined testosterone. Sexualmaturity chicken with completely front end of vas deferens, but the end of the lumen was filled by parenchyma.(2)Testosterone increased DMRT1, SOX9, AMH gene and decreased SF1, DAX1, P450 arom gene expression in gonads of male phenotypic reversal chicken. Consequently, DMRT1, SOX9 and AMH gene closely related with the formation of the testes. |
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