中华锯齿米虾卵黄发生的超微结构及免疫组化研究
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摘要
目前国内外许多学者对十足目甲壳动物的卵巢发育和卵子发生做了大量的研究,但有关中华锯齿米虾卵黄发生过程的研究还未见相关报道,对中华锯齿米虾生殖发育的调控机理仍然不是十分清楚。本文采用透射电镜和免疫组织化学技术系统研究了中华锯齿米虾卵母细胞及其内部各种细胞器在卵黄发生过程中的形态变化特征,以及在这一过程中滤泡细胞的结构变化情况。这项研究,一方面为甲壳动物卵黄发生研究提供理论基础,对甲壳动物生殖发育调控及育种具有重要的实际意义；另一方面对以卵黄蛋白原作为环境内分泌干扰物质的“生物标志物”等毒理学方面的深入研究具有细胞学理论指导意义；同时,还为甲壳动物的毒理学实验确定了一种良好的实验材料。
中华锯齿米虾的卵巢分为前叶、后叶两部分,前叶汇合在一起位于心脏下方、肝胰腺上方；后叶位于肠道上方并与肠道平行。在卵巢的发育过程中,可观察到不同时相的卵母细胞。
卵原细胞期：细胞圆形,体积较小,核质比较大,细胞质均匀,细胞之间排列相当紧密,没有发现滤泡细胞。
卵黄发生前期：细胞体积有所增大,但胞质仍较均匀。该时期滤泡细胞出现,但数目较少,分布也很不规则,且有逐渐将卵母细胞包围分割的趋势。
卵黄发生早期：卵黄蛋白合成全面启动,细胞体积由于卵黄蛋白的不断合成而增大,细胞质中开始出现卵黄球。卵母细胞被滤泡细胞全部包围,多层,但它们之间的关系很松散。
卵黄发生中期：细胞体积继续增大,形状越来越不规则,近似于三角形。核质比下降。卵母细胞被单层滤泡细胞层完全分割。滤泡细胞渐渐紧贴卵母细胞。
卵黄发生后期：卵黄物质继续填充卵母细胞质,部分卵母细胞的细胞核甚至消失。滤泡细胞开始缩小退化。
成熟期：卵母细胞中充满成熟的卵黄蛋白。此时滤泡细胞呈一薄层紧贴于卵母细胞乃至完全退化。
在此基础上进行电镜观察发现：中华锯齿米虾卵黄发生过程是双源性的。在卵黄发生过程中,卵母细胞中的线粒体、内质网、溶酶体和核糖体等多种细胞器均参与了其内源性合成。其中线粒体最早参与形成卵黄颗粒。在卵黄发生的中后期,内质网成为内源性卵黄蛋白合成最主要的细胞器,并最终演化成颗粒较大且有膜包被的卵黄球；溶酶体也在此时期发挥其特有的吞噬功能,通过与线粒体、内质网、环状片层结构组成自噬泡,相互融合使卵黄球的体积逐渐增大；另外,有些溶酶体通过融合分解线粒体、内质网等胞质细胞器形成髓样结构,最终形成一种电子密度很大的致密卵黄球。外源性卵黄则主要通过卵质膜形成的微绒毛和微吞饮小泡从卵周隙及滤泡细胞中摄取外源物质而形成。
通过免疫组织化学实验,充分表明：中华锯齿米虾内源性卵黄物质发生的主要场所是卵母细胞质。在卵黄发生前,卵母细胞中的卵黄物质较少,到了卵黄发生时期,卵母细胞中的卵黄物质快速积累。此外,实验中发现滤泡细胞中也存在卵黄物质,进一步证明了滤泡细胞是外源性卵黄物质来源的主要场所。同时也进一步证实了中华锯齿米虾卵黄发生是双源性的这一结论。
At present,many scholars had done a devil of investigations on the ovary development and oogenesis of Decapoda crustacean. Nevertheless,it hadn't seen the reports correlated vitellogenesis in the oocytes of Neocaridina denticulata sinensis.The study on the regulation mechanisms of reproductive development in N. denticulata sinensis was not very clear. Transmission electron microscope (TEM) and immunohistochemistry were employed to research the morphologic change characteristics of various kinds of organelles in oocytes and the structure changes on follicular cells during the vitellogenesis of N. denticulata sinensis systemly. This research would provid further rationale for studing the vitellogenesis of crustacean.It possessed significant practical purpose on reproductive development regulation and breeding in crustacean.It also would be the theory guide on using the vitellogenin as Environmental Endocrine-Disrupting Chemicals'biological marker in toxicology further research.This research provided a good kind of study subject for toxicology as well.
The N. denticulata sinensis's ovary is divided into the anterior lobe and posterior lobe. The anterior lobe joins together and locates between the heart and hepatopancreas. The posterior lobe locates above the intestinal tract and parallels with it. During the development of ovary, we could observe the oocytes during different phases.
The ovogonium:the cell was spherical. The volume was comparatively small. The nuclear-cytoplasmic ratio was large.The cytoplasm was well-distributed.All of cells disposed tightly fairly.
The prophase of vitellogenesis:the volume grew. The cytoplasm still remained well-distributed.The follicular cells appeared in this phase. But the amount was very low.The distribution of follicular cells was quite irregularity. They had a tendency to surrounding and dividing the oocytes by degrees.
The earlier period of vitellogenesis:the synthesis activity of vitellogenin started overall. The corpuscular volume was go on growing.The oocytes'cytoplasm began to appear yolk sphere.The oocytes were entirely surrounded by multilayer follicular cells.But the relation was loosing.
The metaphase of vitellogenesis:the corpuscular volume continued growing.The shape of oocytes was irregularity gradually and approximating a triangle. The oocytes were completely divided by unilaminar follicle cell.The follicular cells stuck to the oocytes tightly.
The anaphase of vitellogenesis:the oocytes were filled with vitellus material.A part of oocyte's nucleus even disappeared.The follicular cells started to degenerate and deflated.
The maturation phase:The oocytes were full of ripe vitellogenin. The follicular cells became a extreme tenuis wrap and even vanished.
Our observation was carried out on these base by transmission electron microscope. The results indicated a dual origin of the yolk. All kinds of organelles were involved in endogenous yolk formation during the vitellogenesis period,for example:mitochondria, endoplasmic reticula, lysosomes and ribosomes.The mitochondria were the first organelles involved in yolk granules formation. However, the endoplasmic reticula were the dominant organelles which played the most important role in the metaphase and anaphase of vitellogenic oocytes and then developed into the bigger yolk granules with membranes. Furthermore,the lysosomes played the typical function in the same phase.They engulfed and resolved the mitochondria and endoplasmic reticula to expand the yolk sphere's volume, at last formed high electron-dense yolk granules. Exogenous yolk formation was carried out primarily by microvillus and pinocytotic vesicles of the plasma membrane which uptook the exogenous materials from the perivitelline space or follicular cells.
The results of immunohistochemistry research would be an important test and supplementary to the above-mentioned resultant.The research discoverd by analyzing that the oocytes' cytoplasm were the major arena of endogenous yolk formation during the vitellogenesis period.In the prophase of vitellogenesis,the amount of vitellus material was very low.During the vitellogenesis period,the vitellus material accumulated quickly. In addition,there were vitellogenin in follicular cells.The result would be a further proof to certificate that follicular cells were the major arena of exogenous yolk formation in N. denticulata sinensis. Simultaneously,it also confirmed the conclusion of a dual origin of the yolk.
中华锯齿米虾的卵巢分为前叶、后叶两部分,前叶汇合在一起位于心脏下方、肝胰腺上方；后叶位于肠道上方并与肠道平行。在卵巢的发育过程中,可观察到不同时相的卵母细胞。
卵原细胞期：细胞圆形,体积较小,核质比较大,细胞质均匀,细胞之间排列相当紧密,没有发现滤泡细胞。
卵黄发生前期：细胞体积有所增大,但胞质仍较均匀。该时期滤泡细胞出现,但数目较少,分布也很不规则,且有逐渐将卵母细胞包围分割的趋势。
卵黄发生早期：卵黄蛋白合成全面启动,细胞体积由于卵黄蛋白的不断合成而增大,细胞质中开始出现卵黄球。卵母细胞被滤泡细胞全部包围,多层,但它们之间的关系很松散。
卵黄发生中期：细胞体积继续增大,形状越来越不规则,近似于三角形。核质比下降。卵母细胞被单层滤泡细胞层完全分割。滤泡细胞渐渐紧贴卵母细胞。
卵黄发生后期：卵黄物质继续填充卵母细胞质,部分卵母细胞的细胞核甚至消失。滤泡细胞开始缩小退化。
成熟期：卵母细胞中充满成熟的卵黄蛋白。此时滤泡细胞呈一薄层紧贴于卵母细胞乃至完全退化。
在此基础上进行电镜观察发现：中华锯齿米虾卵黄发生过程是双源性的。在卵黄发生过程中,卵母细胞中的线粒体、内质网、溶酶体和核糖体等多种细胞器均参与了其内源性合成。其中线粒体最早参与形成卵黄颗粒。在卵黄发生的中后期,内质网成为内源性卵黄蛋白合成最主要的细胞器,并最终演化成颗粒较大且有膜包被的卵黄球；溶酶体也在此时期发挥其特有的吞噬功能,通过与线粒体、内质网、环状片层结构组成自噬泡,相互融合使卵黄球的体积逐渐增大；另外,有些溶酶体通过融合分解线粒体、内质网等胞质细胞器形成髓样结构,最终形成一种电子密度很大的致密卵黄球。外源性卵黄则主要通过卵质膜形成的微绒毛和微吞饮小泡从卵周隙及滤泡细胞中摄取外源物质而形成。
通过免疫组织化学实验,充分表明：中华锯齿米虾内源性卵黄物质发生的主要场所是卵母细胞质。在卵黄发生前,卵母细胞中的卵黄物质较少,到了卵黄发生时期,卵母细胞中的卵黄物质快速积累。此外,实验中发现滤泡细胞中也存在卵黄物质,进一步证明了滤泡细胞是外源性卵黄物质来源的主要场所。同时也进一步证实了中华锯齿米虾卵黄发生是双源性的这一结论。
At present,many scholars had done a devil of investigations on the ovary development and oogenesis of Decapoda crustacean. Nevertheless,it hadn't seen the reports correlated vitellogenesis in the oocytes of Neocaridina denticulata sinensis.The study on the regulation mechanisms of reproductive development in N. denticulata sinensis was not very clear. Transmission electron microscope (TEM) and immunohistochemistry were employed to research the morphologic change characteristics of various kinds of organelles in oocytes and the structure changes on follicular cells during the vitellogenesis of N. denticulata sinensis systemly. This research would provid further rationale for studing the vitellogenesis of crustacean.It possessed significant practical purpose on reproductive development regulation and breeding in crustacean.It also would be the theory guide on using the vitellogenin as Environmental Endocrine-Disrupting Chemicals'biological marker in toxicology further research.This research provided a good kind of study subject for toxicology as well.
The N. denticulata sinensis's ovary is divided into the anterior lobe and posterior lobe. The anterior lobe joins together and locates between the heart and hepatopancreas. The posterior lobe locates above the intestinal tract and parallels with it. During the development of ovary, we could observe the oocytes during different phases.
The ovogonium:the cell was spherical. The volume was comparatively small. The nuclear-cytoplasmic ratio was large.The cytoplasm was well-distributed.All of cells disposed tightly fairly.
The prophase of vitellogenesis:the volume grew. The cytoplasm still remained well-distributed.The follicular cells appeared in this phase. But the amount was very low.The distribution of follicular cells was quite irregularity. They had a tendency to surrounding and dividing the oocytes by degrees.
The earlier period of vitellogenesis:the synthesis activity of vitellogenin started overall. The corpuscular volume was go on growing.The oocytes'cytoplasm began to appear yolk sphere.The oocytes were entirely surrounded by multilayer follicular cells.But the relation was loosing.
The metaphase of vitellogenesis:the corpuscular volume continued growing.The shape of oocytes was irregularity gradually and approximating a triangle. The oocytes were completely divided by unilaminar follicle cell.The follicular cells stuck to the oocytes tightly.
The anaphase of vitellogenesis:the oocytes were filled with vitellus material.A part of oocyte's nucleus even disappeared.The follicular cells started to degenerate and deflated.
The maturation phase:The oocytes were full of ripe vitellogenin. The follicular cells became a extreme tenuis wrap and even vanished.
Our observation was carried out on these base by transmission electron microscope. The results indicated a dual origin of the yolk. All kinds of organelles were involved in endogenous yolk formation during the vitellogenesis period,for example:mitochondria, endoplasmic reticula, lysosomes and ribosomes.The mitochondria were the first organelles involved in yolk granules formation. However, the endoplasmic reticula were the dominant organelles which played the most important role in the metaphase and anaphase of vitellogenic oocytes and then developed into the bigger yolk granules with membranes. Furthermore,the lysosomes played the typical function in the same phase.They engulfed and resolved the mitochondria and endoplasmic reticula to expand the yolk sphere's volume, at last formed high electron-dense yolk granules. Exogenous yolk formation was carried out primarily by microvillus and pinocytotic vesicles of the plasma membrane which uptook the exogenous materials from the perivitelline space or follicular cells.
The results of immunohistochemistry research would be an important test and supplementary to the above-mentioned resultant.The research discoverd by analyzing that the oocytes' cytoplasm were the major arena of endogenous yolk formation during the vitellogenesis period.In the prophase of vitellogenesis,the amount of vitellus material was very low.During the vitellogenesis period,the vitellus material accumulated quickly. In addition,there were vitellogenin in follicular cells.The result would be a further proof to certificate that follicular cells were the major arena of exogenous yolk formation in N. denticulata sinensis. Simultaneously,it also confirmed the conclusion of a dual origin of the yolk.
引文
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