黄颡鱼精巢结构、精子发生及Sox基因扩增
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摘要
性腺是鱼类进行繁殖活动的基础,为了完全掌握鱼类的繁殖规律,首先要对性别决定机制、性腺分化机理和性腺的组织学、组织化学等方面进行深入的研究。只有充分了解了性腺、的分化和发育的机理,认识他们的繁殖习性才能更好人工调控鱼类的繁殖过程,为鱼类养殖生产服务。由于鱼类精巢的结构比较复杂也比较原始,不同种鱼类其精巢的外部形态以及内部结构存在很大差异,所以受到国内外学者的普遍重视。黄颡鱼Pelteobagrus fulvidraco(Richardson)属鲶形目、鳞科、黄颡鱼属,俗称嘎牙子,是江河、湖泊、水库常见的小型经济鱼类,在我国南北各大水域均有产出。黄颡鱼体色艳丽,肉质细嫩鲜美,营养丰富,无肌间刺,可食部分大,不易死亡,适温范围广,是一种可开发的新型养殖对象。由于黄颡鱼雌雄性在性别决定以后表现出明显的生长差异,但关于这个特异性状的分子调控机制还是一个未知,尤其是与性别决定有关的Sox基因的研究目前还没有报道。因此,本试验深入地分析了精巢的组织学及超微结构,以期对黄颡鱼的精巢结构,精子发生等生殖生物学内容,同时初步的探讨了Sox基因表达的差异,以期进一步了解黄桑鱼的性别决定机制及性腺发育特征,为应用相应的调控手段进行性别控制,实现全雄性养殖提供理论基础。
本研究以性成熟的黄颡鱼为试验材料,采用光学显微技术、透射电镜技术以及分子生物学中基因克隆技术对黄颡鱼精巢的组织学、超微结构以及黄颡鱼Sox基因进行研究探讨。得到结论如下:
1.通过对黄颡鱼精巢的组织学研究,进一步证实黄颡鱼的精巢为典型的小叶型精巢,但其精小叶和贮精囊结构比较复杂,故在繁殖季节不能用外力挤出精子。
2.黄颡鱼精原细胞分为两种,即A型和B型。但A型精原细胞又存在两种不同的形态,分别是A1型和A2型,两者的区别在于A1型不具有拟染色体,并且其线粒体内无嵴或有极少的嵴山现。
3.黄颡鱼除A1型精原细胞外的各期生殖细胞都存在拟染色体,并且随着细胞的不断发育拟染色体逐渐变小分散,最后在精子内完全消失。
4.黄颡鱼初级精母细胞的前期出现联会复合体,应是同源染色体之间进行同源基因交换的标志。
5.黄颡鱼精子形成过程分为三个时期,即早期、中期和晚期。
6.在扫描电镜下,黄颡鱼精子头部椭圆形,其长径约为1.70μm,短径约为1.50μm;尾部鞭毛长度大约为25～30μm,从植入窝正中伸出,植入窝口直径约为0.40μm,鞭毛直径约为0.18μm。
7.本研究采用四种引物,以黄颡鱼DNA为模板进行扩增,结果显示:黄颡鱼在220bp、400bp、450bp、750bp和900bp左右都出现谱带,无性别差异。对220bp的谱带进行测序分析,证明该片段长度为221bp,且雌雄个体间仅有一个碱基不同,为同源基因。与人的SRY基因比较,同源性为41.67%。由于其同源性较低,故不能确定该基因是否参与性别分化。
Gonad was substructure that fishes carries out breeding activity. To completely master the breeding regularity of fishes, we should carry out penetrating investigation on mechanism of sex determination, mechanism of gonadal differentiation, histochemistry and histology of gonad and so on. Only we had understood the mechanism of gonadal differentiation and development and known the breeding habit of fishes, so we would have controlled the breeding activity of fishes and applied to production of fish farming. As testicular structure is relatively complexity and originality, and there is conspicuous difference with outer morphous and interior structure for different fishes, so it has been focused by at home and abroad scholars. Pelteobagrus fulvidraco (Richardson) belongs to the Order of Siluriformes (catfish) .family of Bagridae (Bagrid catfishes). It is now becoming an important economical fish for aquaculture in China. As female and male individuality of Pelteobagrus fulvidraco ( Richardson ) had a conspicuous difference on growth after sex determination, but the report about molecular control mechanism of this specificity didn't appear at present. So this investigation analysised structure of histology and uitrastructure of testis in depth, in order to understand the content of reproductive biology about structure of testis and spermatogenesis. Simultaneously, this investigation approached about Sox gene, in order to understand mechanism of sex determination and characteristic of gonadal development. To know the propagating regularity of the fish could greatly benefit the cultivation in pond.This investigation used sexually matured Pelteobagrus fulvidraco (Richardson) to make materials of experiment. The histology, uitrastructure of testis and sox gene were investigated by light microscope, transmission electron microscope and gene clone technology of molecule biology.1. In the histology research, the result indicated that the testis was lobular type, which was typical of most teleosts. But the structures of seminiferous lobule and seminal vesicle were relatively complexity.2. The spermatogonium consisted of A type and B type, and the spermatogonium of A type had two different type, namely A l type and A2 type, which the A1 type did not have chromatoid body. There was no formation of sleeves or few sleeves occurring in the mitochondria of Al type.3. Each germ cell had chromatoid body except for the spermatogonium of Al type, and the chromatoid body became small and dispersed with the cell developing constantly, disappearing completely in the sperm.4. The prophase of primary spermatocyte would appear synaptonemal complex, which should been the symbol that the homologous chromosome carried through the change of homologous gene.
5. The formation of spermatozoa included three periods: prophase. metaphase and anaphase.6. According to the observation in the scan electron microscope, the head of sperm was ellipse, the long diameter being 1.70 μm and the short diameter being 1.50 urn: the length of flagellum of tail was about 25~30μm, the diameter being 0.18μm.7. In addition, the templates from DNA of Pelteobagrus fulvidraco( Richardson) were PCR amplified with the four primer. The results indicated that the 220 bp、 400 bp、 450 bp、750 bp and 900 bp bands were appeared, which showed that there were no sexual difference. Then the 200 bp bands were sequenced, the result proved that the length of the segment was 221 bp, and there was only a different basic group between male and female, which was homologous gene. The identity of the sequence with SRY gene of human was 41.67%. As the homology to the SRY gene was lower, whether this gene was concerned with sexual differentiation was uncertain.
本研究以性成熟的黄颡鱼为试验材料,采用光学显微技术、透射电镜技术以及分子生物学中基因克隆技术对黄颡鱼精巢的组织学、超微结构以及黄颡鱼Sox基因进行研究探讨。得到结论如下:
1.通过对黄颡鱼精巢的组织学研究,进一步证实黄颡鱼的精巢为典型的小叶型精巢,但其精小叶和贮精囊结构比较复杂,故在繁殖季节不能用外力挤出精子。
2.黄颡鱼精原细胞分为两种,即A型和B型。但A型精原细胞又存在两种不同的形态,分别是A1型和A2型,两者的区别在于A1型不具有拟染色体,并且其线粒体内无嵴或有极少的嵴山现。
3.黄颡鱼除A1型精原细胞外的各期生殖细胞都存在拟染色体,并且随着细胞的不断发育拟染色体逐渐变小分散,最后在精子内完全消失。
4.黄颡鱼初级精母细胞的前期出现联会复合体,应是同源染色体之间进行同源基因交换的标志。
5.黄颡鱼精子形成过程分为三个时期,即早期、中期和晚期。
6.在扫描电镜下,黄颡鱼精子头部椭圆形,其长径约为1.70μm,短径约为1.50μm;尾部鞭毛长度大约为25～30μm,从植入窝正中伸出,植入窝口直径约为0.40μm,鞭毛直径约为0.18μm。
7.本研究采用四种引物,以黄颡鱼DNA为模板进行扩增,结果显示:黄颡鱼在220bp、400bp、450bp、750bp和900bp左右都出现谱带,无性别差异。对220bp的谱带进行测序分析,证明该片段长度为221bp,且雌雄个体间仅有一个碱基不同,为同源基因。与人的SRY基因比较,同源性为41.67%。由于其同源性较低,故不能确定该基因是否参与性别分化。
Gonad was substructure that fishes carries out breeding activity. To completely master the breeding regularity of fishes, we should carry out penetrating investigation on mechanism of sex determination, mechanism of gonadal differentiation, histochemistry and histology of gonad and so on. Only we had understood the mechanism of gonadal differentiation and development and known the breeding habit of fishes, so we would have controlled the breeding activity of fishes and applied to production of fish farming. As testicular structure is relatively complexity and originality, and there is conspicuous difference with outer morphous and interior structure for different fishes, so it has been focused by at home and abroad scholars. Pelteobagrus fulvidraco (Richardson) belongs to the Order of Siluriformes (catfish) .family of Bagridae (Bagrid catfishes). It is now becoming an important economical fish for aquaculture in China. As female and male individuality of Pelteobagrus fulvidraco ( Richardson ) had a conspicuous difference on growth after sex determination, but the report about molecular control mechanism of this specificity didn't appear at present. So this investigation analysised structure of histology and uitrastructure of testis in depth, in order to understand the content of reproductive biology about structure of testis and spermatogenesis. Simultaneously, this investigation approached about Sox gene, in order to understand mechanism of sex determination and characteristic of gonadal development. To know the propagating regularity of the fish could greatly benefit the cultivation in pond.This investigation used sexually matured Pelteobagrus fulvidraco (Richardson) to make materials of experiment. The histology, uitrastructure of testis and sox gene were investigated by light microscope, transmission electron microscope and gene clone technology of molecule biology.1. In the histology research, the result indicated that the testis was lobular type, which was typical of most teleosts. But the structures of seminiferous lobule and seminal vesicle were relatively complexity.2. The spermatogonium consisted of A type and B type, and the spermatogonium of A type had two different type, namely A l type and A2 type, which the A1 type did not have chromatoid body. There was no formation of sleeves or few sleeves occurring in the mitochondria of Al type.3. Each germ cell had chromatoid body except for the spermatogonium of Al type, and the chromatoid body became small and dispersed with the cell developing constantly, disappearing completely in the sperm.4. The prophase of primary spermatocyte would appear synaptonemal complex, which should been the symbol that the homologous chromosome carried through the change of homologous gene.
5. The formation of spermatozoa included three periods: prophase. metaphase and anaphase.6. According to the observation in the scan electron microscope, the head of sperm was ellipse, the long diameter being 1.70 μm and the short diameter being 1.50 urn: the length of flagellum of tail was about 25~30μm, the diameter being 0.18μm.7. In addition, the templates from DNA of Pelteobagrus fulvidraco( Richardson) were PCR amplified with the four primer. The results indicated that the 220 bp、 400 bp、 450 bp、750 bp and 900 bp bands were appeared, which showed that there were no sexual difference. Then the 200 bp bands were sequenced, the result proved that the length of the segment was 221 bp, and there was only a different basic group between male and female, which was homologous gene. The identity of the sequence with SRY gene of human was 41.67%. As the homology to the SRY gene was lower, whether this gene was concerned with sexual differentiation was uncertain.
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