哺乳动物胚胎透明带脱落机制的研究进展

胚胎着床是一个连续的动态过程,其中胚泡从透明带中准时孵出是着床的关键.透明带脱落的机制主要是子宫或(和)胚泡分泌物部分或全部溶解透明带后,胚泡在细胞数量增加及细胞运动的机械压力作用下通过透明带的某一位点孵出.     

Trypsin-like hatching protease from mouse embryos: evidence for the presence in culture medium and its enzymatic properties

Enzymatic properties of a protease involved in hatching of mouse embryos were examined. A trypsin-like protease, which most efficiently hydrolyzed t-butoxycarbonyl-Leu-Ser-Thr-Arg-4-methylcoumaryl-7-amide, was demonstrated in culture medium of mouse hatching embryos. The enzyme was strongly inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, leupeptin, antipain, N alpha-tosyl-L-lysyl-chloromethane, soybean trypsin inhibitor, and Trasylol, but not or weakly inhibited by p-chloromercuribenzoic acid, EDTA, E-64, pepstatin, chymostatin, and bestatin, suggesting a trypsin-like serine proteinase. The protease activity in the medium gradually elevated during the course of hatching, whereas the embryo-associated activity showed no significant change. Furthermore, pyroglutamyl-Leu-argininal, the strongest inhibitor for the enzyme among peptidyl argininals, all of which are potent trypsin inhibitors, showed the strongest inhibition toward hatching. Thus, a trypsin-like protease secreted from hatching embryos into the culture medium may participate in mouse hatching, probably as a hatching enzyme.     

Evidence that protease action is not specifically involved in the hatching of rabbit blastocysts caused by commercial bovine serum albumin in culture

Commercial samples of bovine serum albumin (BSA) in a complex medium caused growth of 1-cell rabbit embryos to completely hatched blastocysts. Heat treatment of the BSA at 65 or 80 degrees C significantly decreased blastocyst formation and expansion and destroyed the ability to cause blastocyst hatching. Addition of trypsin at levels down to 20 ng/ml caused the formation of hatched blastocysts which degenerated rapidly. The effects of 5 protease inhibitors (ovomucoid trypsin inhibitor, alpha-1-antitrypsin, TAME, TLCK and soybean) were tested. Ovomucoid trypsin inhibitor, TAME and TLCK significantly inhibited blastocyst hatching but only at the highest concentration used. These inhibitors also reduced blastocyst formation and expansion, indicating that their effect was not specifically on blastocyst hatching in vitro. It is concluded that hatching of rabbit blastocysts is probably not dependent on protease action.     

EFFECTS OF TWO PROTEASE INHIBITORS ON SEA URCHIN AND AMPHIBIAN EGG DEVELOPMENT*

After a review of our present knowledge about the effects of proteases and protease inhibitors on cell growth and egg fertilization, the results of experiments where sea urchin and amphibian eggs were treated with two protease inhibitors (TPCK, TLCK) are described. Cleavage was hardly affected, but gastrulation quickly stopped or was incomplete. The morphogenetic abnormalities which follow can be explained by abnormal gastrulation and other factors: persistence of remnants of the fertilization membrane in sea urchin larvae and dissociation of the ectoderm cells in Xenopus embryos.     

Inhibition of Mouse Blastocyst Hatching by Subsite-Specific Trypsin Inhibitors,Peptidyl Argininals1

To explore the substrate or subsite specificity of a mouse hatching enzyme, effects of leupeptin [acetyl(P₄)-Leu(P₃)-Leu(P₂)-argininal(P₁)] and its analogs (peptidyl argininals) on mouse blastocyst hatching were investigated. The compounds containing benzyloxycarbonyl group (Z) in the P₄ position inhibited the hatching more strongly than those containing acetyl group or unprotected N-terminal amino acid. Among five Z-Leu-P₂-argininals, a derivative containing a P₂ Ser residue was the most potent inhibitor, and the derivatives containing Leu, Thr, Pro, and Gly in the P₂ position followed in this order. Then, we synthesized four Z-P₃-Ser-argininals and tested their effects on hatching. The result indicated that the compound with Phe residue in the P₃ position was the strongest inhibitor, and the Leu-, Pro-, and Ala-containing derivatives were ranked in this order. Thus, among Z-dipeptidyl-argininals tested, Z-Phe-Ser-argininal most potently inhibited the mouse embryonic hatching, suggesting the preference of the mouse hatching enzyme for Phe(P₃)-Ser(P₂)-Arg(P₁) sequence as a substrate.     

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.     

Developmental potential of biopsied mouse blastocysts

The health of a preimplantation embryo can be diagnosed in one or more cells biopsied from the conceptus. Here, we tried to evaluate the impact of biopsy of some trophectoderm cells from hatching mouse blastocysts on their further in vitro implantation and early egg cylinder formation. Of 374 blastocysts evaluated 112 hours after hCG, 34% initiated hatching with a small number of mural, polar, or intermediate trophectoderm cells. Half of 59 embryos that underwent induction of hatching by zona puncturing herniated some cells through this opening. After removal of cells with a glass microneedle from spontaneously hatching blastocysts, viability assessed by vital FDA staining was impaired, as well as the in vitro zona pellucida shedding and implantation. When polar trophectoderm cells were biopsied, a significantly lower number of embryos reached the egg cylinder stage.     

Protease Inhibitors Cause Necrotic Cell Death in Chlamydomonas reinhardtii by Inducing the Generation of Reactive Oxygen Species

Protease inhibitors affecting the activity of the proteasome were reported to induce programmed cell death (apoptosis) in some mammalian cell lines. Proteasome activity can be suppressed by specific peptide derivatives and by N‐tosyl‐lysine‐chloromethyl‐ketone (TLCK) and N‐tosyl‐phenylalanine‐chloromethyl‐ketone (TPCK), which affect the trypsine‐ and chymotrypsine‐like activities of the proteasome, respectively. Particularly TLCK and TPCK caused necrotic cell death in the unicellular green alga Chlamydomonas reinhardtii. As a control, the effects of these protease inhibitors on the survival of human WISH cells were also studied. Bleaching of the Chlamydomonas cells after addition of TLCK or TPCK indicated that reactive oxygen species (ROS) were involved in this process. Indeed, increased levels of ROS were detected in Chlamydomonas cells treated with TLCK or TPCK. Furthermore, cell death induced by these protease inhibitors was accelerated by illumination and prevented or slowed down by scavengers of ROS.     

Properties of Intracellular Hatching enzyme in the Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

The intracellular hatching enzyme was confirmed to be particulate-bound in the sea urchin, Hemicentrotus pulcherrimus. The enzyme was solubilized most effectively by sonication in buffer containing 12.5 mM CaCl₂, and 0.5 M KCl. The intracellular hatching enzyme is suggested to be activated by an antipain- or elastatinal-susceptible protease(s) on its solubilization. Since the intracellular hatching enzyme solubilized in the absence of protease inhibitors was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, the active hatching enzyme is concluded to be a chymostatin-sensitive serine protease. The enzyme required CaCl₂, and KCl or NaCl for both stability and activity. The preference of the enzyme of anions as sodium salts was as follows: Cl⁻ > NO₃⁻ > I⁻ > SCN⁻. The apparent molecular weights of the intracellular hatching enzyme (IHE) and the hatching enzyme secreted from the blastula with or without the fertilization envelope (SHE or dSHE) were estimated as 89,000, 135,000, 80,000, respectively. On incubations with isolated fertilization envelopes as an enzyme substrate, the apparent molecular weights of dSHE and IHE increased to 128,000 and 105,000, respectively.     

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 plasticity in the tropical gastropod Nerita scabricosta

Hatching plasticity has been documented in diverse terrestrial and freshwater taxa, but in few marine invertebrates. Anecdotal observations over the last 80 years have suggested that intertidal neritid snails may produce encapsulated embryos able to significantly delay hatching. The cause for delays and the cues that trigger hatching are unknown, but temperature, salinity, and wave action have been suggested to play a role. We followed individual egg capsules of Nerita scabricosta in 16 tide pools to document the variation in natural time to hatching and to determine if large delays in hatching occur in the field. Hatching occurred after about 30 d and varied significantly among tide pools in the field. Average time to hatching in each pool was not correlated with presence of potential predators, temperature, salinity, or pool size. We also compared hatching time between egg capsules in the field to those kept in the laboratory at a constant temperature in motionless water, and to those kept in the laboratory with sudden daily water motion and temperature changes. There was no significant difference in the hatching rate between the two laboratory treatments, but capsules took, on average, twice as long to hatch in the laboratory as in the field. Observations of developing embryos showed that embryos in the field develop slowly and continuously until hatching, but embryos in the laboratory reach the hatching stage during the first month of development and remain in stasis after that. Instances of hatching plasticity in benthic marine invertebrates, like the one in N. scabricosta, could greatly enhance our ability to investigate the costs and benefits of benthic versus planktonic development, a long‐standing area of interest for invertebrate larval biologists.     

Isolation and in vitro synthesis of trypsin from the biting fly,Stomoxys calcitrans

The synthesis of [3H]trypsinlike enzyme by the fat body was followed in Stomoxys calcitransin vitro using a radioimmunoassay (RIA) developed against mammalian trypsin. Using high specific activity [3H]valine, trypsinlike activity was followed in midgut epithelial cells, thoracic muscle, and fat body removed from sugar-fed flies. Excreta protease of S. calcitrans was partially purified using charge and hydroxylapatite gel chromatography. Seventy-five percent of the enzyme eluted from these gels was inhibited by tosyl-L-lysine chloromethyl ketone HCI (TLCK) and was classified as trypsinlike. Electrophoresis of the trypsinlike enzyme indicated that it was only 50% pure. Trypsinlike activity from S. calcitrans bound to α₁-globulin IV-I and formed a complex that was dissociated on a P-100 Bio-Gel column. Binding between the protease and the α₁-gobulin IV-I caused a 1.4-fold increase in the apparent molecular weight of the protease on the P-100 Bio-Gel column. Trypsinlike activity was characterized in the midgut and excreta by affinity binding to covalently linked TLCK and tosyl-L-lysine chloromethyl ketone HCI (TAME)Sepharose 4B gels. Between 50% and 55% of the excreta protease and 5669% of the midgut protease bound to the affinity gels and was trypsinlike. Protease activity that did not bind to the gels was not inhibited by TLCK and did not have the esterolytic activity of trypsin.     

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.     

Significance of the number of embryonic cells and the state of the zona pellucida for hatching of mouse blastocysts in vitro versus in vivo

We investigated the course of mouse blastocyst hatching in vitro after experimental modulation of the hatching process by growth hormone or by laser treatment and compared it to embryos grown in vivo. When embryos were grown in vitro, successful hatching was dependent on blastocyst expansion and was based on a minimum number of embryonic cells. Embryos grown in the presence of growth hormone were more advanced in their development and hatched earlier. When an artificial opening was laser-drilled into the zona pellucida, hatching occurred at lower numbers of embryonic cells. In vivo, escape from the zona pellucida occurred earlier and independent of blastocyst expansion. However, when we isolated in vivo-grown blastocysts with intact zonae that had developed in vivo and then cultured them in vitro, blastocysts started to expand and hatched the following day when a sufficiently high number of embryonic cells was present. Our data show that successful hatching in vitro is dependent on a sufficiently high number of embryonic cells, which enables blastocyst expansion and zona shedding. In vivo, the lower number of embryonic cells detected in zona-free blastocysts indicates that the underlying mechanism of zona escape is different, does not depend on blastocyst expansion, and presumably involves lytic factors from the uterus.     

Hatching mechanism of the Chinese soft-shelled turtle Pelodiscus sinensis

The mechanism by which the embryo hatches out of the egg envelope, the vitelline membrane and egg white, was studied in the Chinese soft-shelled turtle Pelodiscus sinensis. The cDNA of the turtle hatching enzyme (HE) was 1555 bp-long and a mature enzyme of 321 amino acids. The mature HE was composed of an astacin protease domain of 200 amino acids and a CUB domain of 121 amino acids, and the estimated molecular size was 35,311. The protease domain contained two active site consensus sequences, HExxHxxGFxHExxRxDR and MHY. An immunoblotting test of an extract of allanto-chorions revealed a 40-kDa band by cross-reaction with the anti-Xenopus HE antiserum. The first change in the envelopes was the appearance of a hole, 1 mm in diameter, at the location around the animal pole of day 8 incubation eggs. A cluster of tall cells, forming a circle in the avascular chorion of day 8 embryos and facing the edge of the hole, had various sizes of inclusion bodies and secretory granules that were labeled by immuno-electron microscopic staining with the antiserum. The egg envelopes were degraded gradually from the animal pole side towards the vegetal pole side in accordance with translocation of the avascular site of the chorion in the same direction. Labeled cells degenerated, presumably when the chorion was underlain by allantois in succeeding developmental stages. The vitelline membrane and egg white were totally digested, presumably by secreted HE, during the hatching period and were consumed for embryonic growth.     

Potential role in development of the major cysteine protease in larvae of the brine shrimp Artemia franciscana

Encysted embryos and larvae of the brine shrimp Artemia franciscana contain a cysteine protease which represents over 90% of the protease activity in these organisms. We have used immunocytochemical methods to determine the localization and potential role of the cysteine protease in development of young larvae. In prenauplius larvae, there is intense staining for the protease on the basal side of the epidermal layer in the posterior region and diffuse staining for the protease throughout the embryo. In first instar larvae, cysteine-protease staining becomes intense in the midgut-forming area where a reticulum-like pattern emerges in cells with an abundance of yolk platelets. Cysteine-protease staining in second instar larvae becomes intense in the apical side of epidermal cells and in the basal and apical zones of midgut cells. Subcellular localization of the protease in the epidermis and midgut of young larvae using immunogold electron microscopy suggests that most is located in the cytosol and extracellular matrix adiacent to these cells. Addition of cysteine-protease inhibitors to the growth medium, especially the fluoromethyl ketone Z-Phe-Ala-CH₂F, inhibits growth and segmentation of the thorax. Collectively, these observations suggest that the major cysteine protease in embryos and larvae functions in yolk utilization, as a hatching enzyme, in apolysis during the molt cycle, and as a digestive enzyme when the swimming larvae begin to feed.     

Hatching enzyme immunolocalization during embryonic development of the ascidians Ciona intestinalis and Phallusia mammillata

Summary

Different stages of the embryonic development of the ascidians, Ciona intestinalis and Phallusia mammillata, were observed by confocal microscopy after treating embryos with polyclonal antibody raised against C. intestinalis hatching enzyme and after staining with FITC-conjugated second antibody. In both species fluorescence is localized, at the gastrula stage, in the ectoderm. At the subsequent neurula and tail bud stages, in C. intestinalis, the enzyme is localized in the anterior region and tail region, while in P. mammillata it is only present in the anterior region.     

Effect of Insect Growth Inhibitors Upon Hatching of the Nematodes Nippostrongylus brasiliensis and Nematospiroides dubius

Summary

The effect of certain insect growth inhibitors upon hatching of eggs of the nematodes Nippostrongylus brasiliensis and Nematospiroides dubius is described. Two azasteroids did not affect hatching while a nonsteroidal amine and amide inhibited hatching, apparently by blocking embryogenesis. Possible modes of action by the hatching inhibitors and their potential use as novel nematicides are discussed.     

Cathepsins B, H and L in Peritoneal Macrophages and Hepatopancreas of Carp Cyprinus carpio

Cathepsin B was purified from the crude extract of carp hepatopancreas by a modified method, and the specific activity increased about 3,400-fold with a 17% recovery. The purified enzyme gave a single protein band on Native-PAGE, whereas two bands with molecular weights of 30,000 (single chain) and 26,000 (heavy chain) migrated on SDS-PAGE. The enzyme potently hydrolyzed Z-Arg-Arg-MCA and Z-Phe-Arg-MCA but lacked the ability to hydrolyze most of the other MCA substrates. The kinetic constants of the enzyme with two Z-blocked substrates revealed that V_max and K_cat values of Z-Phe-Arg-MCA were much higher than Z-Arg-Arg-MCA, while their K_m values were approximate. The optimum hydrolysis pH and temperature of the enzyme for these two substrates were determined to be pH 6.0 and 45°C, respectively. A variety of protease inhibitors such as E-64, DTNB, antipain, leupeptin, chymostatin, TLCK and TPCK and metal compounds such as CuCl₂, CdCl₂, HgCl₂, and Zn(CH₃COO)₂ were able to significantly inactivate the enzyme. In contrast, cysteine and 2-mercaptoethanol activated the enzyme, with cysteine being more effective for activation than 2-mercaptoethanol over the tested concentrations.     

Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus

Two novel extracellular serine proteases were purified to homogeneity from the cell-free culture filtrate of an obligate alkalophilic Bacillus sphaericus by a combination of ultrafiltration, ammonium sulfate precipitation and chromatographic methods. The enzymes showed similar substrate specificities, but differed in hydrophobicity and molecular mass. Protease A was a monomeric protease with a relative molecular mass (M _r) of 28.7 kDa, whereas protease B, with a M _r of 68.0 kDa, apparently consisted of smaller subunits. The purified protease A had a specific activity on hemoglobin of 5.1 U/mg protein compared to 40.9 U/mg protein in the case of protease B. Both proteases were most active on SAAPF-pNa, a substrate for chymotrypsin-like serine proteases. However, the K _m values of these two proteases on SAAPF-pNa were higher than that for α-chymotrypsin, indicating a lower affinity of proteases A and B for this substrate compared to chymotrypsin. Unlike other Bacillus serine proteases, neither protease A nor B stained with Coomasie blue R-250, even with loading of a large amount of protein, and they stained poorly with the silver staining method. However, NH₂-terminal amino acid sequencing of protease B revealed a high similarity with subtilisin Carlsberg (67% homology). Almost total inhibition of both proteases by PMSF, but very little/no inhibition by trypsin and chymotrypsin inhibitors (TPCK and TLCK) or thiol reagents (PCMB and iodoacetic acid), further supported the view that the enzyme belonged to the serine protease family. Journal of Industrial Microbiology & Biotechnology (2001) 26, 387–393. Received 05 November 2000/ Accepted in revised form 23 April 2001