分子佐剂C3d增强草原兔尾鼠卵透明带3 DNA疫苗的免疫应答

为增强免疫不育的效果,探讨分子佐剂C3d对草原兔尾鼠透明带3(LaguruslagurusZonaPellucida3,LZP3)基因免疫的调节作用。构建了含3个拷贝C3d的lzp3基因真核细胞表达质粒pcDNA3-LZP3-C3d3(pcD-LC),以不含C3d的质粒pcDNA3-LZP3(pcD-L)为对照,体外转染HeLa细胞,RT-PCR和Westernblot分别检测到lzp3mRNA和蛋白的表达。对NIH雌性小鼠肌肉进行基因免疫注射,ELISA结果表明pcD-LC免疫诱导的特异性抗LZP3-IgG水平明显高于pcD-L对照组(P<0.05),且有长期免疫应答效应;MTT结果显示pcD-LC能够诱导淋巴细胞增殖活性的提高。抗生育实验表明pcD-LC免疫组显著降低窝产仔数(P<0.05),且卵巢切片正常。研究结果证实C3d能够增强lzp3DNA疫苗的特异性免疫应答并达到抗生育效果,为DNA疫苗控制草原兔尾鼠的深入研究提供了基础。     

Entamoeba dispar: Cultivation with Sterilized Crithidia fasciculata1

Four isolates of Entamoeba dispar identified by their hexokinase and phosphoglucomutase isoenzyme profile and by their failure to react with Entamoeba histolytica-specific monoclonal antibody (4G6) could be grown in either Diamond's BI-S-33 medium, newly developed BCSI-S (Biosate cysteine starch iron-serum) medium, or casein-free YI-S medium in the presence of Crithidia fasciculata (ReF-1:PRR) sterilized by heating 56° C for 30 min and subsequent incubation with 1% hydrogen peroxide for 24 hours at 4° C. After the cultures were maintained for over 50 passages, the amebae were identified as E. dispar by isoenzyme analysis, polymerase chain reaction with E. histolytica- and E. dispar-specific primers, i.e. p11 plus p12 and p13 plus p14, respectively, and by negative reactivity with monoclonal antibody 4G6. The flagellates added to the culture were judged to be metabolically inactive based on the results of nuclear magnetic resonance spectroscopy, electron microscopy, and polarographic analysis. All of these findings suggest that E. dispar can grow in vitro with metabolically inactive C. fasciculata as a culture associate.     

Cortactin is implicated in murine zygotic development

Cortactin is a cortex-enriched protein implicated in Arp2/3 complex-mediated actin polymerization. However, the physiological role of cortactin remains unknown. We have generated a mouse strain in which the allele of murine cortactin was disrupted by a gene trapping vector. The resulting heterozygous mice developed normally and were fertile, but embryonic fibroblasts derived from heterozygous animals displayed partial impairment in PDGF-induced membrane ruffling. No homozygous offspring or early embryos even at the two-cell stage were detected. Analysis of oocytes revealed a gradual decrease in the detection of homozygous zygotes after fertilization. In normal oocytes arrested at meiotic metaphase II (MII), cortactin immunoreactivity was detected in an apical layer that overlies the maternal chromosome and overlaps with a polarized cortex enriched with actin. The formation of the polarized cortactin layer was diminished upon treatment with latrunculin B, an actin polymerization inhibitor. After resumption of meiosis II, the majority of cortactin protein was accumulated into the second polar body. Microinjection of MII-arrested eggs with either cortactin antibody or RNA encoding a cortactin mutant deficient in Arp2/3 complex binding disrupted the integrity of the actin cap and inhibited emission of the second polar body triggered by parthenogenesis. Our data suggest that cortactin plays an important role in the mechanics of asymmetric division in oocytes.     

Mouse granulosa cells contribute more to the mRNA synthesis of mZP2 than oocyte does

In order to elucidate the biogenesis of mouse zona pellucida 2 (mZP2) protein, RT-PCR, and in situ hybridization were carried out to localize the expression of mouse ZP2 mRNA. Cumulus cells of the OCC (Oocyte-Cumulus cell Complex) were isolated from the oocytes after superovulation for the RNA extraction. The frozen sections of ovaries from adolescent and aged mice were prepared to hybridize with RNA probe of mouse ZP2. mRNA of ZP2 was detected in isolated cumulus cells by RT-PCR. Results of in situ hybridization showed that the mRNA of ZP2 was synthesized in both oocyte and granulosa cells at different folliculogenesis stages; and the expression of ZP2 mRNA in granulosa cells was stronger than that in oocyte; much weaker expression of mZP2 was detected in the follicles of aged mouse. These suggest that the entire amount of ZP2 mRNA generated in the granulosa cells layer should be much more than that in oocyte. Therefore, we think that the granulosa cells contribute more to the mZP2 mRNA synthesis than oocyte does.     

Cx37 and Cx43 Localize to Zona Pellucida in Mouse Ovarian Follicles

In the ovarian follicle, granulosa cells adjacent to the oocyte extend processes through the zona pellucida matrix, and these projections establish gap junctions both with the oocyte and with neighboring transzonal projections. The identity of connexins contributing to gap junctions between transzonal projections has not been extensively studied. Here, we examined the expression pattern of Cx37 and Cx43 in mouse zona pellucida using multiple connexin-specific antibodies. Immunofluorescence staining revealed abundant Cx37 and Cx43 puncta within the zona pellucida of both preantral and antral follicles. Cx37 persisted in the zona pellucida of mature follicles up to 5 h after an ovulatory stimulus whereas Cx43 was reduced in the zona pellucida by 3 h after an ovulatory stimulus. We suggest that in addition to its role in oocyte-granulosa cell communication, Cx37 could enable a distinct communication pathway between those granulosa cells that are in direct contact with the oocyte.     

Dog Zona Pellucida Glycoprotein-3 (ZP3): Expression inEscherichia coliand Immunological Characterization

An internal fragment (978 bp) corresponding to the dog zona pellucida glycoprotein-3 (DZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was amplified by polymerase chain reaction from a full-length cDNA clone. The amplifiedSacI andPstI restricted fragment was cloned in-frame downstream of the T5 promoter underlacoperator control for expression in the pQE-30 vector. Recombinant DZP3 (rec-DZP3) was expressed as a polyhistidine fusion protein inEscherichia coli.Optimum expression of rec-DZP3 was observed at 1.0 mM isopropyl-β-d-thiogalactopyronoside. Immunoblots with a murine monoclonal antibody, MA-451 (raised against porcine ZP3β-a homologue of DZP3 and cross-reactive with dog zona pellucida), revealed a major band of 42 kDa. Localization studies revealed that the recombinant protein was present only in an insoluble intracellular fraction. Further optimization studies revealed that the level of expression of rec-DZP3 was significantly higher in Luria broth medium containing glycerol rather than glucose and maximum expression was observed when cultures were induced during the mid-log phase of growth. Batch fermentation with glycerol as the carbon source yielded 30 mg/L of rec-DZP3 compared to 4 mg/L from a shake flask culture. Immunization of two male rabbits with Ni-NTA-purified rec-DZP3 and two female dogs with the rec-DZP3 conjugated to diphtheria toxoid generated high antibody titers against rec-DZP3 as determined by enzyme-linked immunosorbent assay. Rabbit immune serum reacted with porcine ZP3β but failed to react with porcine ZP3α in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on dog ovarian sections showed positive fluorescence with zona pellucida. The availability of rec-DZP3 will help in evaluating its efficacy for fertility regulation in stray dogs.     

The Biological and Functional Significance of the Sperm Acrosome and Acrosomal Enzymes in Mammalian Fertilization

The mammalian spermatozoon undergoes continuous modifications during spermatogenesis, maturation in the epididymis, and capacitation in the female reproductive tract. Only the capacitated spermatozoa are capable of binding the zona-intact egg and undergoing the acrosome reaction. The fertilization process is a net result of multiple molecular events which enable ejaculated spermatozoa to recognize and bind to the egg's extracellular coat, the zona pellucida (ZP). Sperm–egg interaction is a species-specific event which is initiated by the recognition and binding of complementary molecule(s) present on sperm plasma membrane (receptor) and the surface of the ZP (ligand). This is a carbohydrate-mediated event which initiates a signal transduction cascade resulting in the exocytosis of acrosomal contents. This step is believed to be a prerequisite which enables the acrosome reacted spermatozoa to penetrate the ZP and fertilize the egg. This review focuses on the formation and contents of the sperm acrosome as well as the mechanisms underlying the induction of the acrosome reaction. Special emphasis has been laid on the synthesis, processing, substrate specificity, and mechanism of action of the acid glycohydrolases present within the acrosome. The hydrolytic action of glycohydrolases and proteases released at the site of sperm-zona binding, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoon, are important factors regulating the penetration of ZP. We have discussed the most recent studies which have attempted to explain signal transduction pathways leading to the acrosomal exocytosis.     

Relationship between the length of sperm preincubation and zona penetration in the golden hamster: A scanning electron microscopy study

Hamster spermatozoa are able to fertilize a high percentage of zona-intact hamster oocytes when they are preincubated for 2 hr in a chemically defined medium. From this time on, the longer the preincubation time the lower the percentage penetration. Spermatozoa preincubated for 6 or more hr are unable to cross the zona pellucida, retaining however their ability to fuse with zona-free hamster oocytes. Zona-intact hamster oocytes, as described above, were observed with the scanning electron microscope. When the oocytes were inseminated with spermatozoa preincubated for 1 to 5 hr the outer surface of the zona showed the penetrating spermatozoa and the sperm tracks made by those that failed to cross it. With longer preincubation times no penetrating spermatozoa were observed, and very few sperm tracks were present on the outer surface of the zona. Control experiments showed that neither eggs, spermatozoa, nor fertilization were affected by the medium recovered after long preincubations. These results show that care should be taken regarding the preincubation time when using the in-vitro fertilization technique.     

Effect of Colchicine on Cell Membrane and on Biopterin Transport in Crithidia fasciculata*

SYNOPSIS. Incorporation of ¹⁴C-labeled biopterin into Crithidia fasciculata was inhibited by 1 mM colchicine or lumicolchicine. These substances do not penetrate the cell membrane, hence they cannot interact with the subpellicular microtubules. In view of this, interference of colchicine with biopterin transport must occur on the outer surface of the cell membrane. Binding of colchicine to Crithidia was not temperature-dependent and did not exhibit saturation kinetics. These facts exclude a binding as in the case of tubulin, or similar proteins which may be present in the membrane. The results suggest an inhibition reflecting steric hindrance of the biopterin carrier system.     

Light microscopic observations of alterations in staining of the zona pellucida of porcine follicular oocytes: Possible early indication of atresia

Serial sections of porcine ovaries were examined in an attempt to detect early signs of oocyte degeneration/atresia using special staining. Porcine ovaries were fixed in Bouin's fixative and embedded in paraffin using routine techniques. Serial sections (8 μm) were mounted on glass slides and stained with Shorr's S3 and hematoxylin stain. Several criteria were used for examining general histology of the antral follicles: condition of the granulosa layer, antral cavity, the oocyte and its surrounding zona pellucida, and the cumulus layers. A change in the staining characteristic of the zona pellucida was the single most striking observation in all ovaries examined. In presumably healthy follicles, the zona pellucida was uniformly stained green, the granulosa layer was intact with fewer than three pyknotic cells per section, and a uniform basement membrane (stained green) separated the intact theca layers from the remainder of the follicle. In those follicles showing some degree of degenerative changes in the follicular wall, the zona pellucida was stained a bright orange. In the last stages of degeneration, follicles exhibited many pyknotic nuclei throughout the granulosa layers, the layer of granulosa cells was in many cases separated from the basement membrane, and the antrum was infiltrated with lymphocytes. In these follicles, the zona pellucida was always stained orange. Frequently, the zona pellucida was partially stained orange before any detectable changes could be seen in other elements of the follicular wall. Additionally, many non-antral (primary) follicles exhibited oocytes with orange-stained zonae pellucidae. In terminal stages of follicular degeneration, collapsed follicles were infiltrated by connective tissue elements stained bright orange and green. These structures very often contained dying oocytes always with bright orange-stained zonae pellucidae. Scattered throughout the ovarian stroma were many orange-stained remnants of zonae pellucidae. It is thought that perhaps the characteristic staining of the zona pellucida with Shorr's S3 stain may give an early, previously undetectable indication of follicular atresia.