【作者】 吕翠仙；

【导师】 樊廷俊；

【作者基本信息】 中国海洋大学 ， 海洋生物， 2005， 硕士

【摘要】 在中国对虾(Penaeus chinensis)的早期胚胎发育到一定阶段后必须从卵壳中孵化出来才能进一步发育,而孵化必须依赖于孵化酶(Hatching Enzyme,HE)对卵壳的降解才能完成。孵化腺细胞(Hatching Gland Cells,HGCs)是孵化酶的特异性分泌细胞,它们还是一类孵化前的特定时期出现、孵化后的特定时期消失的一时性细胞(a transient type of cells)。由于动物HE具有高度保守的氨基酸序列,因此许多动物的HGCs均可特异性地被GST-UVS.2抗体识别。为了查清甲壳类动物孵化腺细胞的分化规律,本文以中国对虾的早期胚胎为材料利用GST-UVS.2抗体和辣根过氧化物酶标记的羊抗兔第二抗体对孵化腺细胞的分化时相及其超微结构特征进行了研究。 免疫组织化学反应结果显示,典型的HGCs开始出现于受精后约17小时(孵化前15小时)的胚胎中,一般呈圆形或卵圆形,内含深染的孵化酶原颗粒,其起始分化的部位弥散分布于整个胚体;随发育进程的延续,HGCs的数量逐渐增多,细胞逐渐变大,所含酶原颗粒的浓度也越来越大,并开始向胚胎的前端和背部体表处迁移;至胚胎临近孵化时,HGCs的数量、体积及所含酶原颗粒的浓度达到顶峰,主要集中在头部和背部体表处;胚胎完成孵化后,HGCs所含酶颗粒浓度变淡,细胞也变小,在孵化后10小时的胚胎中孵化腺细胞在胚胎中呈全身弥散分布,孵化后15小时,孵化腺细胞聚缩并沿头胸部的体腔分布;在孵化后20小时孵化酶颗粒已基本消失殆尽。因此,对虾HGCs是一种在孵化前15小时开始出现、到胚胎孵化后20小时消失的一时性细胞。 通过对中国对虾受精后32小时(孵化前5小时)和受精后39小时(孵化后2小时)受精卵的透射电镜观察到:2000倍下,观察到孵化前5小时胚体内存在大量卵黄脂滴和正在发育中的肌丝结构,HGCs沿体表分布,呈不规则形状,部分HGCs有微绒毛结构,部分HGCs表面呈不规则的凸起状,孵化后2h的胚胎HGCs中含有空泡状结构。4000-6000倍下,观察到两个时期的HGCs内含有较更多还原

【Abstract】 Hatching enzyme (HE), synthesized in hatching gland cells (HGCs), plays vital roles in animal hatching. Immunocytochemical techniques employing anti-GST-UVS.2 antiserum, prepared from Xenopus HE and with specificity to Penaeus chinensis HE, were first used to investigate the differentiation and variability of hatching gland cells (HGCs) in the hatching process of embryos of marine shrimp, Penaeus chinensis, in this study. HGCs with immunoreactivity to anti-GST-UVS.2 antiserum were identified, for the first time, in shrimp embryos during hatchingprocess.Immunocytochemical staining results showed that: HE-positive immunoreactivity is really specific to Penaeus chinensis HE, and its appearance and disappearance are closely correlated with the hatching process of Penaeus chinensis; Penaeus chinensis HGCs, first appeared in embryos 15 hours before hatching and disappeared 20 hours after hatching, were also a transient type of cells, with an existence period of 35 hours; the back portion of Penaeus chinensis embryo is probably the initial position of HE secretion, and likely to be the main position of HE secretion as well; the appearance of HGCs is in a synchronous mode from places all over the embryos, and their disappearance is also in a synchronous mode; the number of HGCs increased gradually along with embryo development process and reached a maximum number at hatching. Contrarily, the number of HGCs decreased gradually after hatching, and HGCs disappeared 20 hours after hatching. However, the intensity of HE-positive reaction was almost at the same level at the period of HGCs’presence. Therefore, it can concluded that marine shrimp Penaeus chinensis HGCs, as a transient type of cells, first appeared in embryos 15 hours before hatching and disappeared in embryos 20 hours after hatching, and with distinguished patterns of appearance, disappearance and distribution in embryos. All these findings indicate that HGC is a kind of transient type of cells, which first appeared all over the body of embryo 15 hours before hatching, and disappeared 20 hours after hatching.By the ultrastructure study of the marine shrimp embryos, we find some muscle fibril and more yelk distributing the whole embryo; HGCs which form in ovum and rotundity posited in the surface of the body and some HGCs develop the micro-floccus structure; there are abnormity salient on the HGC surface before secreting zymogen granules, whereafter secreting there will develop vacuole in the HGC; the nucleolus of HGC is relatively larger than common cells and organelles such as endoplasm、 ribosome and mitochondria are very abundantly, as magnify the HGC, we find more nuclear pore complexes; there are much electron dencity unequal aymogen granules of roundish in 0.3μm ~0.5μm distributed in the HGCs.更多还原