Mode of Action of some Stimulants of the Hatching Enzyme Secretion in Fish Embryos*

Mode of action of two stimulants of the hatching enzyme secretion, electric current (AC) and potassium cyanide, was analyzed by applying them to Medaka embryos in the presence or absence of suppressants of nervous system-mediated secretion, tetrodotoxin or MS–222. Electric current (AC) stimulated the secretion of the hatching gland of the embryos that had been treated with these suppressants, while potassium cyanide did not. These results strongly suggest that electric current acts as a stimulant of hatching enzyme secretion directly on the gland cell itself, while potassium cyanide stimulates the secretion indirectly, probably through nervous system of the embryo. In the present experiments, it was also shown that Ca²⁺ and ionophore, X-537A, when applied directly to the hatching gland extracellularly, induced a marked secretion-associated morphological change of the gland cells instantaneously. However, it was found that chum salmon prolactin did not induce the secretion-associated morphological changes in the hatching gland cells when it was applied directly to the gland cells in situ or indirectly through embryonic circulation.     

Ultrastructural changes of the teleostean hatching gland cell during natural and electrically induced precocious secretion.

Ultrastructural changes of the hatching gland during electrically induced precocious secretion were compared with those during natural secretion in the medaka, Oryzias latipes. The gland cells are covered by a layer of epithelial cells, which adjoin one another just on the apical center of each gland cell. When the natural as well as the precocious secretion occurred, each gland cell was swollen upward and rounded, and separation of the epithelial joints occurred, giving rise to an exposure of the apical portion of the gland cells. There were marked differences between these two kinds of secretion process in the behavior of the secretory granules prior to secretion and in the mode of discharge of the secretory substances. The changes which occurred during both types of secretion and which, therefore, seemed to be essential to the secretory processes of this gland cell were the swelling up of the gland cells in the initiation of secretion and the reduction of the electron density of the zymogen granules. These secretion-associated ultrastructural changes are discussed in view of the difference in the maturation of the gland cells.     

NS-4 (nervous system antigen-4), a cell surface antigen of developing and adult mouse brain and sperm.

Morphological changes in the chorion of the Medaka, Oryzias latipes, brought about by the hatching enzyme were examined by transmission as well as scanning electron microscopy. The structure of the intact chorion, especially its thick multilamellar inner layer, does not change during development until about 1 hr before the onset of hatching. As choriolysis proceeds, the inner layer of the chorion is digested to yield soluble proteins of relatively high molecular weight. During this process it appears that each lamella is successively solubilized from the inner surface of the chorion. Finally, a thin outer layer with accompanying villi and attaching filaments remains.Under the experimental conditions used, the enzyme was in direct contact with both the inner and outer layers of the chorion. Because of this, the enzyme could penetrate the outer layer and act on some peripheral parts of the underlying inner layer. Based on these morphological changes, a mechanism is proposed to account for the solubilization of the chorion by the hatching enzyme.     

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.     

Hatching in the pike Esox lucius L.: Evidence for a single hatching enzyme and its immunocytochemical localization in specialized hatching gland cells

Antibodies against purified hatching enzyme (HE) from the pike, Esox lucius L., have been used to examine different aspects of the presence of the enzyme in the ontogeny of this teleostean fish. Immunochemical analysis indicates that the two proteolytic enzymes which occur in the hatching medium arise from a single protease, HE itself. The second proteolytic fraction found in gel filtration of hatching medium could be a heterogeneous population of complexes of HE with digestion fragments of its natural substrate, the zona radiata. Immunofluorescence microscopy by means of anti-HE antibodies demonstrates that HE is localized in the so-called hatching gland cells (HGCs). The HGCs in pike appear as oval to round cells 10–15 μm in diameter containing granules of 1.5–2.3 μm. They are found interspersed between the periderm and the presumptive epidermis. The number of HGCs and their granule content increase significantly until the 35-somite stage to reach about 1200 and 30, respectively. From then on these numbers do not change until hatching in the 66-somite stage. The distribution of the HGCs over the embryo also changes, probably since HGC precursors in the yolk sac differentiate to HGCs later than their counterparts in the head region. The immunocytochemical procedure further shows that HE can be detected from the 10-somite stage on. Discrete hatching gland remnant bodies, phagocytized by epidermal cells, are observed in larval stages until 3–7 days after emergence of the embryo.     

Presence of Active Hatching Enzyme in the Secretory Granule of Prehatching Medaka Embryos

Secretory granules of hatching gland were isolated from a 0.3 M sucrose homogenate of whole medaka embryos at prehatching stage by differential centrifugation, followed by a Percoll density gradient centrifugation. The obtained preparation was almost free of melanosomes and composed exclusively of the secretory granules of hatching gland (hatching enzyme granules), as judged by morphological as well as enzymological criteria.
The aqueous extracts of the purified secretory granules showed a specific choriolytic activity as high as about 40 times that of a partially purified secretory granule preparation, P_1,000, and represented a single protein band with molecular weight of about 21,000 on SDS-polyacrylamide gel electrophoresis. It was also revealed that a major component of the hatching enzyme preparation (P II–0.3 enzyme, 13) purified from the hatching liquid was identical with the 21,000 molecular weight band.
These results suggest that the hatching enzyme is present in the secretory granules of prehatching embryos in an active molecular form.     

The short life of the Hoyle organ of <Emphasis Type="Italic">Sepia officinalis</Emphasis>: formation,differentiation and degradation by programmed cell death

Cephalopods encapsulate their eggs in protective egg envelopes. To hatch from this enclosure, most cephalopod embryos release egg shell-digesting choriolytic enzymes produced by the Hoyle organ (HO). After hatching, this gland becomes inactive and rapidly degrades by programmed cell death. We aim to characterize morphologically the development, maturation and degradation of the gland throughout embryonic and first juvenile stages in Sepia officinalis. Special focus is laid on cell death mechanisms and the presence of nitric oxide synthase during gland degradation. Hatching enzyme has been examined in view of metallic contents, commonly amplifying enzyme effectiveness. HO gland cells are first visualized at embryonic stage 23; secretion is observed from stage 27 onwards. Degradation of the HO occurs after hatching within two days by the rarely observed autophagic process, recognized for the first time in cephalopods. Nitric oxide synthase immunopositivity was not found in the HO cells after hatching, suggesting a possible NO role in cell death signalling. Although the HO ‘life course’ chronology in S. officinalis is similar to other cephalopods, gland degradation occurs by autophagy instead of necrosis. Eggs that combine a large perivitelline space and multi-layered integument seem to require a more complex and large gland system.     

THE FINE STRUCTURE AND FUNCTION OF THE SALIVARY GLANDS OF NUCELLA LAPILLUS (GASTROPODA: MURICIDAE)

The fine structure of the tubular and acinous salivary glandsof Nucella lapillus (L.) has been studied and some histochemicaland enzyme tests have been carried out. The clusters of subepithelialcells of the tubular glands secrete a glycoprotein composedof chains of tubular macromolecules resembling secretions knownto have adhesive properties which may assist in boring. Thesecretion is rich in disulphide groups, as are many toxins,and is believed to be responsible for the recently demonstratedpharmacological activity of the glands. It is proposed thatflaccid paralysis is induced in prey by envenomation with thissecretion during rasping, after soft parts have been exposedby an ‘anti-predator’ reaction to secretion fromthe hypobranchial gland of Nucella. The secretory vesicles ofboth types of gland cells in the acinous glands have heterogeneouscontents indicating that their secretions are complex. The majorcomponent in those of the mucous cells is an acid mucopolysaccharidetypical of a lubricant or releasing agent. The ciliated basalcells resemble typical enzyme-secreting cells and the majorconstituent of their secretion is a finely granular glycoprotein. (Received 8 January 1990; accepted 5 June 1990)     

The Digestive Glands of Pinguicula: Structure and Cytochemistry

The digestive glands of the carnivorous genus Pinguicula havethree functional compartments, (a) a basal reservoir cell, (b)an intervening cell of endodermal character and (c) a groupof secretory head cells. The gland complex is derived from asingle epidermal initial. The reservoir cell, which is richin Cl^– ions, is highly turgid before discharge; it islinked by plasmodesmata to the surrounding epidermal cells,and is ensheathed by a pectin-rich inner wall layer. The endodermalcell is bounded by a Casparian strip to which the plasmalemmais tightly attached; it contains abundant storage lipid andnumerous mitochondria. The head cells of the developing glandhave labyrinthine radial walls of the transfer-cell type, theingrowths being composed of pectic polysaccharides. The boundingcuticle is discontinuous, although lacking well-formed pores.Mitochondria are numerous, with well-developed cristae; theplastids are large and ramifying, and invested by ribosomalendoplasmic reticulum. Dictyosomes are sparse, and where theyoccur, are associated with coated vesicles. Ribosomal endoplasmicreticulum is moderately abundant in the head cells, and so alsoare free ribosomes. Optical and electron microscopic localizationmethods indicate that the digestive enzymes are synthesizedin the head cells and transferred both into the vacuoles andinto the walls. There is no evidence of a granulocrine modeof secretion, and the transfer seems to be initially by directperfusion through the plasmalemma. During the final phase ofmaturation of the head cells they suffer a form of autolysis,vacuoles, cytoplasm and wall becoming confluent as all of themembranes of the cell undergo dissolution. The gland head isthus, in effect, simply a sac of enzymes at the time of theultimate discharge. Pinguicula, carnivorous plant, insectivorous plant, enzyme secretion, digestive gland     

Trypsin-like Hatching Enzyme of Mouse Blastocysts: Evidence for Its Participation in Hatching Process before Zona Shedding of Embryos6

Effects of twelve protease inhibitors on hatching of mouse embryos were investigated. Mouse hatching was strongly or moderately inhibited by trypsin inhibitors including p-toluenesulfonyl-Lys-CH₂Cl (TLCK) and chicken ovomucoid, while inhibitors for chymotrypsin and elastase showed weak or no inhibition. These results indicate the participation of a trypsin-like protease in the hatching of mouse embryos as a hatching enzyme., Since TLCK is the strongest and an irreversible inhibitor for the enzyme, timing of the participation of the hatching enzyme in the hatching process was examined by pulse treatment of embryos with TLCK before and during the zona shedding. The results indicated that a trypsin-like hatching enzyme functions before, but not during, the zona shedding of embryos, especially during a 15 h period immediately before the beginning of the shedding.     

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.     

The Involvement of Glucose-6-Phosphatase in Mucilage Secretion by Root Cap Cells of Zea mays

In order to determine the involvement of glucose-6-phosphatasein mucilage secretion by root cap cells, we have cytochemicallylocalized the enzyme in columella and peripheral cells of rootcaps of Zea mays. Glucose-6-phosphatase is associated with theplasmalemma and cell wall of columella cells. As columella cellsdifferentiate into peripheral cells and begin to produce andsecrete mucilage, glucose-6-phosphatase staining intensifiesand becomes associated with the mucilage and, to a lesser extent,the cell wall. Cells being sloughed from the cap are characterizedby glucose-6-phosphatase staining being associated with thevacuole and plasmalemma. These changes in enzyme localizationduring cellular differentiation in root caps suggest that glucose-6-phosphataseis involved in the production and/or secretion of mucilage byperipheral cells of Z. mays. Zea mays, corn, glucose-6-phosphatase, columella cell, peripheral cell, mucilage, secretion, cytochemistry     

Change in Hatching Enzyme Activity during Development of the Sea Urchin, Hemicentrotus Pulcherrimus

The time course of change in hatching enzyme activity during development of embryos of the sea urchin Hemicentrotus pulcherrimus was observed. The enzyme was present in the particulate fraction in embryos until the time of hatching and was maximal at the time of hatching. Cell fractionation studies suggested the existence of an inhibitor of the hatching enzyme. This possibility was subsequently substantiated by experiments in mixtures of fractions: the activity of hatching enzyme in the particulate fraction was inhibited by the supernatant of embryos. This inhibitory factor was heat-stable and non-dialyzable, but it was not characterized further. The activity of secreted hatching enzyme was not inhibited by this factor, suggesting that the molecular forms of hatching enzyme in embryos and in the culture supernatant are different. After hatching, the amount of increase in the hatching enzyme activity in the culture supernatant was 3.5 times the amount of decrease in enzyme activity in the embryos, suggesting that the enzyme was activated during its secretion.     

Hatching asynchrony, nestling competition, and the cost of interspecific brood parasitism

All parental hosts of heterospecific brood parasites must paythe cost of rearing non-kin. Previous research on nest parasitismby brown-headed cowbirds (Molothrus ater) concluded that competitivesuperiority of the typically more intensively begging and largercowbird chick leads to preferential feeding by foster parentsand causes a reduction in the hosts' own brood. The larger sizeof cowbird nestlings can be the result of at least two causes:(1) cowbirds preferentially parasitize species with smallernestlings and lower growth rates; and/or (2) cowbirds hatchearlier than hosts. I estimated the cost of cowbird parasitismfor each of 29 species by calculating the difference betweenhosts' published brood sizes in nonparasitized and parasitizednests and using clutch size to standardize values. In this analysis,greater incubation length and lower adult mass, surrogate measuresof the hatching asynchrony and size difference between parasiteand hosts, were both related to greater costs of cowbird parasitismwithout bias owing to phylogeny. To establish causality, I manipulatedclutch contents of eastern phoebes (Sayornis phoebe) and examinedwhether earlier hatching by a single cowbird or phoebe egg reducesthe size of the rest of the original host brood. As predicted,greater hatching asynchrony increased the proportion of theoriginal phoebe brood that was lost. This measure of the costof parasitism was partially owing to increased hatching failureof the original eggs in asynchronous broods but was not at allrelated to the size differences of older and younger conspecificnestmates. However, proportional brood loss owing to an earlierhatching conspecific was consistently smaller than brood lossowing to asynchronous cowbirds in both naturally and experimentallyparasitized phoebe nests. These results imply that althoughhatching asynchrony is an important cause of the reduction ofhost broods in parasitized clutches, competitive features ofcowbird nestlings remain necessary to explain the full extentof hosts' reproductive costs caused by interspecific brood parasitism.     

AN ULTRASTRUCTURAL STUDY OF THE GLAND OF LEIBLEIN OF MURICID AND NASSARIID NEOGASTROPODS IN RELATION TO FUNCTION, WITH A DISCUSSION ON ITS HOMOLOGIES IN OTHER CAENOGASTROPODS

The gland of Leiblein of the muricid Nucella lapillus and thenassariid Hinia reticulata has been examined by scanning andtransmission electron microscopy. The origin and functionalsignificance of its complex organization and its relationshipwith the rest of the mid-oesophagus in Nucella are discussed.It is absorptive as well as secretory, and a mechanism is proposedby which solute-rich fluids may enter the gland. Its epitheliumis composed of occasional mucous cells and two major cell types:ciliated cells engaged in protein metabolism and unciliatedcells responsible for uptake and storage of lipids and carbohydrates,both of which show evidence of pinocytotic uptake of solutesand intracellular digestion in lysosomes. Some enzyme activitypersists in the residual bodies they shed by apocrine secretion,but they remain intact in a mucous string until they reach thestomach. Preliminary ultrastructural examination indicates thatthe gland absorbs cadmium not only from the blood but also directlyfrom its lumen and that it may have the capacity to sequestera wide range of toxins. The same types of cell occur in Hiniain which their cyclical activity has been correlated with feeding.Similar cells have been identified in the oesophageal glandsof other prosobranchs. The foregut glands of carnivorous caenogastropodsare compared with the gland of Leiblein. There is an inversecorrelation between the role of the mid-oesophagus in digestionand absorption and the complexity of the stomach. (Received 16 August 2004; accepted 24 January 2005)     

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.     

小地老虎雄性附腺细微结构和功能及高温的影响

本文通过光镜、电镜和生化分析等方法,研究了小地老虎Agrotis ypsilon(Rottemberg)雄性附腺的细微结构和功能,结果表明：(1)雄性附腺是一对管状腺,基段粉红色,中段桔红色,端段乳白色。形态分化在蛹前期完成,分泌功能在羽化后4天进入旺盛期;(2)附腺细胞属蛋白质合成型,具有旺盛的合成蛋白质的能力,胞内含有致密的粗面内质网和游离核糖体颗粒,大量的分泌液泡均匀地分布在细胞质中;(3)顶浆分泌和局部分泌是腺体的二种主要分泌方式, 前者分泌的颗粒物为糖蛋白性质(Pas阳性),后者吩泌的网状物为非糖蛋白性质(Pas阴性),二者在腺腔内呈有规则的放射状排列“4”雄性附腺分泌物具有种的特异性,小地老虎、棉铃虫和粘虫等夜蛾科昆虫分泌物的蛋白电泳谱带存在明显的种间差异,高温(32℃)抑制了雄性附腺分泌某些蛋白质的能力,从而改变精液的成分。     

The Time-Course of Hatching Enzyme Secretion in the Sea Urchin Strongylocentrotus purpuratus

By two independent methods, we have determined approximately the time-course of hatching enzyme secretion in the sea urchin Strongylocentrotus purpuratus . A quick-kill method indicates that a significant fraction of the enzyme is secreted between 90% and 97% of the fertilization-hatching interval. A direct assay method indicates that the remainder of the enzyme is secreted on either side of the 90–97%"window". The entire period of secretion spans from 75% to 100% or more of the fertilization-hatching interval. For embryos raised at 15°C this translates to an interval of 4.8 or more hr.     

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。     

Ultrastructure of active and inhibited prothoracic glands.

Changes in the cytoplasm of prothoracic gland cells were compared in pharate adults, dauer pupae, and aminophylline inhibited pupae of H. cecropia. For the first 3 to 4 days after transfer from 4 to 22°C, a similar sequence of changes in the cytoplasmic elements was observed. At day 4 the cytoplasm of pharate adults exhibited further differentiation which was consistent with the initiation of secretion, while dauer and inhibited pupae remained at the stage achieved at day 4 and did not advance further even after a substantial lapse of time. These results are interpreted as indicating that the early changes represent a response by the cells to the temperature change, while the initiation of secretion requires the intervention of brain hormone.