| 摘要: | 本研究之目的，乃在探討調控家禽性別分化之基因，在雞胚孵化初期之基因表現，及孵化期間尿囊液內雌性素之濃度。目前家禽的性別決定基因仍然有許多存疑，位於家禽的Z性染色體上之DMRT1基因，被認為是雄性胚胎發育的重要基因，其表現受到溫度的影響，然而不只有DMRT1基因會影響到家禽性別分化，還有其他許多相關的家禽性別分化基因，均會對家禽性別分化的過程造成影響。除了DMRT1基因以外， SOX9和FOXL2以及CYP19A1基因都會影響家禽性別的分化。本研究之目的，在探討家禽胚胎發育早期調控性別分化相關基因之表現，並探討孵化溫度對性內泌素分泌之影響。實驗分為兩組，A組為控制組(溫度100℉/濕度58%)，B組為升溫組(溫度102℉/濕度54%)，家禽受精蛋均於控制組孵化環境下入孵，升溫組於孵化第3(E3)日將孵化條件調成溫度102℉，濕度54%，於孵化第5日(E5)再將孵化條件調整為和控制組一樣，於E3至E6，每天打開24個胚，取其胚體並直接收集血線，另自胚體萃取RNA，進行Real Time RT-PCR以分析DMRT1、SOX9、FOXL2和CYP19A1等基因之表現，自血液萃取DNA以分析雞胚性別。另於E7至E11每日自24個雞胚取尿囊液分析雌性素。根據實驗結果顯示，升溫孵化改變DMRT1表現的強度，但SOX9、FOXL2、CYP19A1不會受到升溫孵化的影響。雌性素於雌性胚胎發育第8日起才達可偵測範圍，雄性胚胎則需到胚胎發育第10日起才達可偵測範圍。無關性別，尿囊液之雌性素分泌濃度皆隨胚胎發育階段遞增，雌性遞增幅度明顯較快且較大。進一步觀察控制組和升溫組的雌性胚胎estrone濃度後發現，升溫孵化使得雌性胚胎estrone濃度降低，推測可能有部分雌性胚胎轉為雄性胚胎，影響整體的平均值。然而本試驗需要一一檢視個別胚胎之基因型性別與表現型性別，再與雌性素濃度比對，才能證實此一假設，但因目前試驗並未取得胚胎性腺，以致無法驗證此一假設，未來應進一步設計可同時偵測轉性個體之試驗方法予以求證。
The purpose of this research is to study the gene regulation and the allantoic estrogen levels during embryo development. Currently, poultry sex-determining genes are still not completely explored. DMRT1 gene is located on poultry Z chromosome and is considered to be an important gene responsible for male embryonic development, and also known to be thermosensitive. In addition, many other relevant genes are found to be related to gender development, such as SOX9, FOXL2 and CYP19A1 genes. This study was designed to investigate the gene expression during early embryonic development, also to investigate the effect of incubation temperature on the steroid hormones secretion. Fertilized eggs were divided into two groups, control group was hatched at temperature 100℉, 58% humidity, thermoregulation group was hatched at the same condition except E3-E5, during that time, temperature was raised to 102℉, and the relative humidity was reduced to 54%. From E3 to E6, 24 embryos were removed from the eggs each day, the blood vessels were collected for DNA extraction and for gender determination, and the RNA was extracted from the embryo for DMRT1, SOX9, FOXL2 and CYP19A1 gene expression analysis using Real Time RT-PCR. From E7 to E11, allantoic fluid was drawn from 24 chick embryos for analysis of estrogen. The results show that DMRT1 and SOX9 gene expression at any stage from E3-E5 were stronger in male embryo than that in female embryos. Raising incubation temperature strengthen the expression of DMRT1, but have no effect on SOX9, FOXL2 and CYP19A1 expression. Estrogen can be detected in the female embryo from the E8, yet in male, only until E10 can reach the detectable level. Independent of gender, the estrone levels increased as embryo development, and the rate of increase is more significant in female rather than that in male. Further observation found that raising incubation temperature reduced the estrone levels in female embryos suggesting that there may be some reversed sex embryos occurred that some female embryos converted into male embryos. This study has confirmed some poultry sexual differentiation associated gene expression and estrogen hormone profile. Further efforts need to be done as to refine the experimental design, and to increase the sample size and to study more of the sex differentiation related genes. |
