卤虫（Artemia salina）孵化腺细胞分化时相的免疫细胞化学研究（简报）

动物孵化酶(hatching enzyme HE)是早期胚胎在特定发育阶段由孵化腺细胞产生和分泌的,在动物早期胚胎孵化中具有关键性作用^[4]。孵化腺细胞(hatching gland cell,HGC)一般为单细胞腺体,是从胚胎发育到特定阶段(孵化前)出现、至胚胎孵出后的特定时期消失的一时性细胞(transient type of     

Amphibian hatching gland cells: pattern and distribution in anurans

The hatching gland (HG) is a transient organ, found in most anuran embryos and early larvae, and located on the dorsal side of the head. The enzymes secreted by hatching gland cells (HGCs) aid the embryos to escape from their enveloping coats. Analysis of HG morphology and distribution in 20 anuran species from six families using scanning electron microscopy revealed small differences in the shape and pattern of the gland particularly in the length and width of the posterior mid-dorsal extension of the gland. The four species of foam-nest making leptodactylids examined had HGs of a somewhat different shape to the others, but otherwise, there was little sign of a relationship between HG shape and taxonomic position. In the single Eleutherodactylus species examined, cells with the appearance and location of HGCs were transiently present long before the active stage of hatching. No sign of HGCs was seen on the head surface of one species, Phyllomedusa trinitatis. It seems possible that in this species, hatching is achieved by a mechanical rather than an enzymatic mechanism. The microvilli characteristic of the surfaces of HGCs were quite variable in density and length from species to species, and at different stages. HGCs remained at the surface of the embryo for some time after hatching and the possibility of a post-hatching function is briefly discussed.     

卤虫(Artemia salina)孵化腺细胞分化时相的免疫细胞化学研究(简报)

动物孵化酶(hatching enzyme,HE)是早期胚胎在特定发育阶段由孵化腺细胞产生和分泌的,在动物早期胚胎孵化中具有关键性作用。孵化腺细胞(hatching gland cell,HGC)一般为单细胞腺体,是从胚胎发育到特定阶段(孵化前)出现、至胚胎孵出后的特定时期消失的一时性细胞(transient type ofcells)。完全分化的HGC内充满了低电子密度的酶原颗粒(孵化酶原颗粒),在鱼胚中的分布因物种而异。在大多数鱼中,HGC分布在胚体的外表面和/或卵黄囊中,一般为外胚层来源。如在虹蹲鱼HGC分布在胚体的前表面、卵黄囊、咽部、鳃的内表面及外表面,属于外胚层来源。而日本鳉鱼HGC     

鲤胚胎孵化腺细胞

鲤胚胎孵化腺为单细胞腺体,发生于外胚层,可特异地被PAS染色。最早可在眼色素期检验出孵化腺细胞(Hatching gland cell,HGC)它们主要分布在头部腹面及头部与卵黄囊连接处。开始,HGC位于表皮细胞下面,随发育迁移到胚胎表面。根据扫描和透射电镜观察,在分泌孵化酶的前后,HGC区表面细胞呈鸡冠花状和疣状两种突起。前者系HGC处于分泌孵化酶期间;后者系HGC业已完成分泌作用,由于相邻的表皮细胞活动而形成的。HGC内富有粗面内质网、线粒体、核糖体和高尔基体,并由后者合成酶原颗粒。HGC在完成分泌作用后,仍留在表皮中,以后逐渐退化,但在孵化后30h仍可见残留的HGC。     

Ultrastructural changes in hatching-gland cells of pike embryos (Esox lucius L.) and evidence for their degeneration by apoptosis

Summary Around hatching, when the pike embryo sheds its acellular egg envelope, marked changes occur in the cellular covering of the embryo. This cellular covering consists of a peridermal layer and a mono-layered presumptive epidermis. The periderm begins to disintegrate shortly before hatching and is sloughed off in the first posthatching period. The cellular covering produces hatching enzyme, the protease that partly dissolves the zona radiata interna of the acellular envelope. By means of the peroxidase-anti-peroxidase staining method with antibodies against hatching enzyme the cells producing this enzyme (hatching gland cells, HGCs) could be identified ultrastructurally. They are interspersed as single cells between the periderm and the presumptive epidermis. The secretory cycle of the HGC was studied. Hatching enzyme is released by an exocytotic secretory process in which multiple secretion into a secretion vacuole predominates. Exocytosis into surrounding intercellular spaces also occurs. These results show that the HGCs are merocrine glands. The HGC also has some holocrine nature, however, in that only a single, massive release of its secretory product occurs. The death of the transitory HGCs in posthatching stages is characterized by condensation of the cell, formation of surface protuberances and splitting up into globular cell fragments. Eventually these fragments are ingested by epidermal cells and digested. These results lead to the conclusion that the pike HGCs degenerate by apoptosis, unlike true holocrine cells.     

草鱼孵化腺超微结构及孵化酶形成与释放的研究

草鱼胚胎孵化腺为单细胞腺体,发生于外胚层,主要分布在胚胎头部及头部与卵黄囊连接处,尤以眼睛腹下方最多且典型。其形成、释放酶的过程具有一定的规律性,可划分为5个时期:形成前期、迁移期、分泌期、衰退期和消失期。畸形胚胎头部表皮细胞中很少有HGC的分化或HGC分化不完全,其形态结构也呈现畸形。       

INDUCTION OF THE FROG HATCHING GLAND CELL FROM EXPLANTED PRESUMPTIVE ECTODERMAL TISSUE BY LICL

When cells of the superficial layer explanted from the presumptive ectoderm of a Rana japonica early gastrula embryo at stage 10 were cultured in standard salt solution for 4–7 days, they differentiated into cement gland cells (CGCs), cilia cells (CCs) and common epidermal cells (CECs). When, however, these explants were treated with LiCl and transferred to Barth's solution, hatching gland cells (HGCs) and pigment cells were induced.
The optimum condition for inducing differentiation of HGC was treatment with 70 mM LiCl for 6–8 hr at 18°C. The best ability to react to the HGC-inducing stimuli resided in the superficial layer of the dorsal presumptive epidermis of the embryo at stage 10. Upon repeated stimulation, explants from stage 8 embryos underwent differentiation into nerve and pigment cells, whereas those from stage 11 embryos differentiated into CCs and CECs. Under optimum conditions, the total volume of HGCs induced amounted to about 70% of the explanted tissue. The culture media from LiCl-induced HGCs showed an apparent jelly-digesting activity, strongly indicating that the cells were functionally identical with those differentiated in situ .     

Ultrastructural Studies on Development of the Tail Gland of a Cuttlefish, Sepiella japonica

Hatching glands in embryos of teleosts and amphibians have been reported to be indispensable for hatching of the embryos. The cephalopod has capsuled eggs, so we expected to find some exocrine organ in the embryos that functioned as a hatching gland. The tail gland (Hoyle's organ) has been suspected to be a hatching gland in the cephalopod, and therefore we examined it during the course of development of cuttlefish embryos. Cells in the tail gland appeared similar to the hatching gland cells (HGCs) of teleosts and amphibians, and contained a number of secretion granules that also resembled the hatching enzyme granules (HEGs) in HGCs of teleosts and amphibians in size, electron density and distribution in the cells. However, a few of these granules were discharged one after another from an early stages, whereas most of them were retained up to the stage just before hatching, and then discharged all at once. The former process of trickling discharge was similar to that in amphibians and the latter process of abrupt discharge resembled that in teleosts.     

Factors influencing the in vitro hatching of mouse blastocysts

The influence of bovine serum albumin (BSA) concentration on embryo hatching and the number of embryos cultured per drop of culture medium was examined in F1 (C57BL/6J × DBA/2J), C3HeB/FeJ strain and Line E mice. Embryos collected from F1 and Line E mice exhibited uniform hatching rates at BSA concentrations between 1 and 10 mg/ml, and embryo numbers ranging from 1 to 10 per 3 μ1 of culture medium. The hatching of C3HeB/FeJ blastocysts was greater at the higher concentrations of BSA and higher embryo densities. When the C3HeB/FeJ embryos were grown at high densities until morula and then cultured singly in fresh media they hatched at a low rate. However, when allowed to develop until the blastocyst stage before replotting, the embryos hatched at a high rate. C3HeB/FeJ embryos cultured singly until morula and then placed in groups of 10 hatched at a high rate. Single C3HeB/FeJ embryos, cultured in medium conditioned by the prior presence of embryos at high densities, hatched at a slightly higher frequency than those cultured in fresh medium. There was no tendency of embryos developing from the two-cell to the eight-cell stages to hatch when cultured in the presence of high densities of hatching blastocysts.     

Analysis of the origin and development of hatching gland cells by transplantation of the embryonic shield in the fish, Oryzias latipes

Hatching gland cells of the medaka, Oryzias latipes, have been observed to differentiate from the anterior end of the hypoblast, which seems to first involute at the onset of gastrulation. These results suggest that the hatching gland cells of medaka originate from the embryonic shield, the putative organizer of this fish. The present study investigated whether hatching gland cells really originate from the embryonic shield in the medaka. Transplantation experiments with embryonic shield and in situ hybridization detection of hatching enzyme gene expression as a sign of terminal differentiation of the gland cells were carried out. The analysis was performed according to the following processes. First, identification and functional characterization of the embryonic shield region were made by determining the expression of medaka goosecoid gene and its organizer activity. Second, it was confirmed that the embryonic shield had an organizer activity, inducing a secondary embryo, and that the developmental patterns of hatching gland cells in primary and secondary embryos were identical. Finally, the hatching gland cells as identified by hatching enzyme gene expression were found to coincide with the dye-labeled progeny cells of the transplanted embryonic shield. In conclusion, it was determined that hatching gland cells were derived from the embryonic shield that functioned as the organizer in medaka.     

Two constituent proteases of a teleostean hatching enzyme: concurrent syntheses and packaging in the same secretory granules in discrete arrangement.

Formation, accumulation, and storage of two components of the Oryzias latipes hatching enzyme, high and low choriolytic enzymes (HCE and LCE), were examined by immunocytochemical and immunoblotting methods. Both of the enzymes were found to be formed specifically in the hatching gland cells at the stages of lens formation to eye pigmentation and their accumulation proceeded markedly and concurrently up to Day 5.5 embryos (the stage just before hatching). The amount of HCE formed was more abundant than that of LCE. In the hatching gland cells, HCE and LCE were found to be packaged in the same secretory granules but in distinct arrangement; HCE is localized to the inside of granules whereas LCE is situated at the periphery of the same granules. Their segregated arrangement is compatible with their relative quantities formed per embryo. The results provide not only the cellular and developmental basis for a view that this hatching enzyme is an enzyme system composed of HCE and LCE but also a clue to the regulatory mechanism of concurrent syntheses of two different specific proteins in the same embryonic cell.     

Properties of the hatching enzyme from Xenopus laevis.

Using an anti-(glutathione S-transferase-UVS.2 cDNA) Ig and uterine egg vitelline envelope (UEVE) protein of Xenopus laevis as probes, the hatching enzyme (HE) from Xenopus was solubilized in hatching medium and purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular mass and enzymatic properties. The hatching medium solubilized the UEVE and contained molecules reactive to the anti-(GST UVS.2) Ig against Xenopus HE. It was found that the HE had a molecular mass of 60 kDa, and often preparations also contained a 40-kDa form. The 60-kDa HE had a high hydrolytic and UEVE-solubilizing activity, and its activities against Boc-Leu-Gly-Arg-7-amino-4-methylcoumarin (-NH-Mec) and UEVE were inhibited by anti-(GST UVS.2) Ig in a dose-dependent manner. The 60-kDa form was easily autodigested into a 40-kDa form. The 40-kDa molecule alone had no detectable UEVE-solubilizing activity, even it still had high hydrolytic activity. It probably represents the main protease domain of the 60-kDa form after loss of two CUB repeats during autodigestion or digestion. The autodigestion of the 60-kDa molecule into 40-kDa molecule is probably a congenital behavior for successfully dissolving the embryo envelope during the hatching process. The two molecules may play different roles at different stages of the hatching process, during which they co-ordinate with each other to achieve complete solubilization of the embryo envelope, similar to the high and low choriolytic enzymes in medaka (Oryzias latipes). Their hydrolytic activity against Boc-Leu-Gly-Arg-NH-Mec was optimal at pH of 7.4, and with an apparent Km value of 200 micromol.L-1 at 30 degrees C. The HE is very sensitive to trypsin-specific inhibitors such as leupeptin, (4-amidino-phenyl)methane sulfonyl fluoride, diisopropyl fluorophosphate (DFP) and N-alpha-tosyl-L-lysylchloromethane (Tos-Lys-CH2Cl), indicates that it is a trypsin-type protease. The results on EDTA and some metal ions, combined with the occurrence of a astacin family metalloprotease-specific 'HExHxxGFxHE' sequence in the deduced HE amino-acid sequence, indicates that this HE is a Zn2+ metalloprotease.     

Selective Degradation of Specific Components of Fertilization Coat and Differentiation of Hatching Gland Cells during the Two Phase Hatching of Bufo japonicus Embryos

The embryonic hatching process in the toad, Bufo japonicus , consists of two phases: rupture of the outer jelly strings at stage 20 (neural tube) and an escape from the inner jelly layers and fertilization coat (FC) of individual embryos at stage 23 (tailbud). SDS-PAGE analyses of FCs revealed that, of the eight major protein bands, two components with 58 K and 62 K in molecular weight gradually decreased from stage 18–19 on and totally disappeared at stage 22. When the FCs were treated with a hatching medium prepared by culturing denuded prehatching embryos, both 58 K and 62 K components of the FCs were solubilized, and in the solubilized materials 18 K and 31 K components appeared. Electron microscopy showed that a meshwork of filament bundles present in the FCs before stage 17 became dissociated at stage 19–20, and completely disappeared at stage 23, just before the hatching of embryos. Hatching gland cells (HGCs), an epidermal cell with numerous secretory granules, were first identified at stage 19, and underwent active secretion of the granules during stage 19–23. These results indicate that the hydrolytic degradation of 58K and 62 K components in FCs effected by the hatching enzyme constitutes the basic mechanism of embryonic hatching during both the first and second phases.     

EFFECT OF ACTINOMYCIN D ON THE DIFFERENTIATION OF HATCHING GLAND CELL AND CILIA CELL IN THE FROG EMBRYO

The forehead epidermis of the stage 18–20 R. japonica embryo includes the hatching gland cell (HGC) which contains cell-specific secretory granules. The cilia cell (CC) and common epidermal cell (CEC) constitute the epidermis of the entire body surface, in addition to the forehead region.
Culture of superficial epidermal explants from various embryonic portions at various developmental stages revealed that HGCs are derived from cells localized on the neural crest in the stage 13a (early neural plate) embryo. When explants from the presumptive HGC area were treated with 1 ug/ml actinomycin D (AMD), the formation of secretory granules in HGCs was inhibited either by continuous treatment from stage 13 or by an 8-hr treatment at stage 13b. Similarly, the ciliogenesis in CCs was inhibited. The differentiation of CECs was entirely unaffected by any of the AMD treatment. After release from AMD, mucous vesicles, characteristic of the CEC, were formed in cells whose differentiation into HGC and CC had been suppressed by the antibiotic. Thread complexes and clumps of coiled strings were found in the nuclei of AMD-affected cells.
It is concluded that the DNA-dependent RNA syntheses which direct secretory granule formation in the HGC and ciliogenesis in the CC occur during a limited period at stage 13b, viz. , 20 hr before their cytodifferentiation becomes appreciable.     

Changes in the second stage juveniles of Globodeva rostochiensis prior to hatching in response to potato root diffusate

cAMP levels in eggs of G. rostochiensis and the diameter of the nucleolus of the nucleus within the dorsal pharyngeal gland cell of the second stage juvenile have been measured as indicators of the response of the nematode to the hatching stimulus in potato root diffusate. The nucleolus increased from 2.72 ± 0.103 μm for unhatched individuals to 3.28 ± 0.14 μm and 3.88 ± 0.15 μm after soaking eggs in potato root diffusate for 3 and 4 days respectively. Juveniles expressed from unstimulated eggs in water to potato root diffusate for 4–5 days showed a similar increase in size of the nucleolus to 3.94 ±0.15 μm but those released into water for this time had smaller nucleoli of 3.20 ± 0.98 μm. The change in diameter of the nucleolus is probably related to the accumulation of secretions in this gland cell before hatching. Preliminary results with dibutyryl analogues of CAMP and cGMP showed some inhibition of hatch in 10% potato root diffusate. Theophylline had a similar effect but NaF was dissimilar in that the effect of this inhibitor was not reversible. A standard radioimmunoassay showed that significant changes in cAMP levels occurred in the unhatched juveniles within cysts after treatment with potato root diffusate for 2.5 or 8 h compared with values for cysts kept in water. This change occurs before other known responses of the juveniles to potato root diffusate and it defines the period of interest for future work on the initial action of hatching factor.     

Homoiogenetic regulation through the ectoderm on localized expression of the hatching gland phenotype in the head area of Xenopus embryos

Ectoderm pieces explanted from embryos of Xenopus laevis were cultured and examined for differentiation of hatching gland cells, using immunoreactivity against anti-XHE (Xenopus hatching enzyme) as a marker. The anterio-dorsal ectoderm excised from stage 12-13 (mid-late gastrula) embryos developed hatching gland cells. Meanwhile, the posterio-, but not the anterio-dorsal ectoderm from stage 11 (early gastrula) embryos developed these cells, although it is not fated to do so during normogenesis. This hatching gland cell differentiation from stage 11 posterior ectoderm was not affected by conjugated sandwich culture with the mesoderm but was suppressed when explants contained an anterior portion of the ectoderm. Conjugated cultures of anterior and posterior portions of the ectoderm in various combinations indicated that differentiation of hatching gland cells from stage 11 posterior and stage 12 anterior portions was suppressed specifically by stage 11 anterior ectoderm. Northern blot analyses of cultured explants showed that XHE was expressed in association with XA-1, suggesting its dependence on the anteriorized state. These results indicate that the planar signal(s) emanating from stage 11 anterior ectoderm participates in suppression of the expression of the anteriorized phenotype so that an ordered differentiation along the anteroposterior axis of the surface ectoderm is accomplished.