bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein synthesis inhibitors prevent both spontaneous and hormone‐dependent maturation of isolated mouse oocytes
Authors: Downs Stephen M. 
Published: February 2005

AbstractThe present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor‐free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3‐isobutyl‐1‐methyl‐xanthine (IBMX), or delayed by follicle‐stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone‐induced maturation. CEO were maintained in meiotic arrest for 21–22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle‐stimulating hormone (FSH). Three different protein synthesis inhibitors [CX, emetine (EM), and puromycin (PUR)] each prevented the stimulatory action of FSH on GVB in a dose‐dependent fashion. This was accompanied by a dose‐dependent suppression of 3H‐leucine incorporation by oocyte‐cumulus cell complexes. The action of these inhibitors on FSH‐ and epidermal growth factor (EGF)‐induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH‐treated groups, below even that of the control (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF‐treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP. In a time course experiment, it was determined that CX could prevent FSH‐induced maturation if added as late as 3 h after the onset of culture, but the drug was less effective in EGF‐treated cultures. It is concluded that a protein with a rapid turnover rate is involved in the spontaneous maturation of mouse oocytes and that de novo protein synthesis is a requirement for hormone induction of GVB in vitro.
Decondensation of mouse sperm chromatin in cell‐free extracts: A micromethod
Authors: Maleszewski Marek 
Published: February 2005

AbstractA micromethod is presented which makes possible the analysis of mouse sperm nucleus decondensation in vitro using very small volumes of cytoplasmic preparations, even smaller than 1μl. We show that cell‐free extracts obtained from interphase HeLa cells as well as lysates from mouse eggs and embryos can sustain early stages of mouse sperm nucleus transformation, provided the sperm nuclear envelope is damaged or removed.
Characterization of human alpha fetoprotein charge microheterogeneity during fetal development
Authors: Keel Brooks A. , Harms Robin L. , Leal Juan A. , Cho Sechin 
Published: February 2005

AbstractAliquotes of human amniotic fluid (AF), fetal serum (FS), and cord blood (CB) were obtained as by‐products of routine clinical diagnostic procedures at term or in the second trimester of pregnancy. When samples of CB were applied to a pH 5.5‐4 chromatofocusing gradient, three isoforms of AFP could be resolved; a pl 4.57 form (isoform IA, 52% AFP), a pl 4.27 form (isoform IB, 43% AFP), and one species that was bound to the column but could be eluted with 1.0 M NaCl (isoform II, pl<4.00, 5% AFP). Term AF displayed a profile similar to that observed in term CB. When samples of 15–20‐week gestation AF were chromatofocused, the immunoreactive AFP recovered was distributed between isoform IA and IB (60%) and isoform II (40%). FS and AF obtained from same pregnancy (23–26 weeks) displayed an identical chromatofocusing profile. Aliquotes of AF subjected to conA revealed 83% reactive variants compared with >95% reactive variants for CB. FS displayed a conA profile identical to CB. When individual CB charge isoforms were isolated and subjected to conA analysis, >97% of the AFP bound to conA. In contrast, when AFP isoforms IA and IB were isolated from midgestation AF, approximately 22% of the AFP did not bind to the lectin while 100% of isolated AFP isoform II eluted as the reactive variant. These data suggest that human AFP exists as at least three charge and two lectin variants and that the charge profile may change during fetal development.
Culturing two‐ to eight‐cell caprine embryos using domestic chicken eggs
Authors: Blakewood E. G. , Pool S. H. , Prichard J. F. , Godke R. A. 
Published: February 2005

AbstractEarly‐stage caprine embryos were placed in the chick embryo amnion to determine if this culture method would support the development of embryos from a farm animal species. Following superovulation and natural mating, two‐ to eight‐cell embryos were surgically collected from crossbred donor goats. Embryos were allotted to in vitro culture treatments across two different experiments (EXP). In EXP‐I, embryos allotted to Treatment A (control) were cultured in Ham's F‐10 with 10% fetal calf serum and 1% antibiotic‐antimycotic (HF‐10). Embryos in Treatment B were placed on a bovine fetal uterine fibroblast monolayer in HF‐10, embryos allotted to Treatment C were agarose embedded and injected into the amniotic cavity of a day‐4 chick embryo and those placed in Treatment D were co‐cultured in HF‐10 with day‐15 caprine trophoblastic vesicles. In EXP II Treatments A, B, and C were the same; however Treatment D was omitted. EXP‐I and EXP‐II also differed in that chick embryo co‐culture was for 72 hr in EXP‐I but was extended to 96 hours in EXP‐II. Additionally, the monolayer co‐culture was limited to 96 hr in EXP‐II; whereas, embryos in EXP‐I remained on monolayer culture for 96 hr plus an additional 72 hr for subsequent embryo evaluation. Results indicate that the amniotic cavity of the developing chick embryo enhanced the development of two‐ to eight‐cell caprine embryos through to hatching blastocysts when compared with that of the control medium alone. In addition, the beneficial effects of monolayer co‐culture appeared to be lost when the caprine embryos were removed from the monolayer, whereas, the in vitro viability of these embryos cultured in the chick embryo amnion was maintained after removal from the amniotic cavity.
Production of chimaeric bovine embryos and calves by aggregation of inner cell masses with morulae
Authors: Picard L. , Chartrain I. , King W. A. , Betteridge K. J. 
Published: February 2005

AbstractBovine inner cell masses (ICMs) were isolated by immunosurgery at day 8 or 10, or by dissection at day 14, and combined with day‐5.5 morulae. Aggregation was obtained between 89%, 62%, and 0% of the day‐5:day‐8, day‐5:day‐10, day‐5:day‐14 composites, respectively. Chromosome analysis of composites potentially carrying the 1/29 translocation as a chromosome marker and temporarily transferred to the bovine uterus for 8 days showed that chimaeric day‐14 embryos can be obtained from day‐5:day‐8 aggregation. The definitive transfer of eight day‐5:day‐8 and 11 day‐5:day‐10 composites resulted in the birth of six and four calves, respectively; five of the six, but none of the four, were chimaeric. The five chimaeras showed mostly the ICM phenotype. The morphological differences between ICMs at different stages of development were examined by electron microscopy and related to the success of the aggregation technique. It is concluded that bovine embryonic cells can regulate for at least 3 days difference in development but not 5 days even though aggregation is still possible.
Zona drilling enhances fertilization by mouse caput epididymal sperm
Authors: Wazzan Wassim C. , Gwatkin Ralph B. L. , Thomas Jr. Anthony J. 
Published: February 2005

AbstractSpermatozoa from the caput epididymis are known to be much less capable of fertilization when compared to sperm from more distal segments of the epididymis. The purpose of this study was to determine if two micromanipulative techniques, zona drilling (ZD) and a modification of partial zona dissection (PZD), could be used to enhance fertilization with caput epididymal sperm. A mouse in vitro fertilization model was used. Inseminating oocytes with 500–1,000 sperm/oocyte from the cauda epididymis as a control resulted in fertilization of 98 of 300 (32.6%) oocytes. Of those fertilized, 47 developed to the blastocyst stage (47.9%). Caput sperm fertilized 13 of 116 (11.2%) nonmanipulated oocytes. Only 1 of 13 developed into a blastocyst, while with oocyte ZD, caput sperm fertilized 24 of 144 (16.7%) oocytes, 50% of those fertilized developing to blastocyst (P=0.0129). When modified PZD was performed on oocytes, only one of 23 was fertilized, with no blastocyst development. These results indicate that acid Tyrode ZD enhances both fertilization and early embryonal development when caput epididymal sperm are used for insemination. These mouse studies suggest that ZD or other micromanipulation techniques may prove clinically useful in men with proximal epididymal obstruction where only caput sperm are available.
Observations of hamster sperm‐egg fusion in freeze‐fracture replicas including the use of filipin as a sterol marker
Authors: Clark Judy M. , Koehler James K. 
Published: February 2005

AbstractWe have extended the observations of previous transmission electron microscopy studies of sperm‐egg fusion to include those of freeze‐fracture replicas showing sperm‐egg interactions before, during, and following sperm head fusion with the egg membrane. Hamster eggs were incubated with hamster sperm under polyspermic conditions and were observed after a period of 5–30 minutes. After fixation, the eggs and sperm were exposed to filipin, which binds β‐OH‐sterols to form visible complexes in freeze‐fracture replicas. Filipin can act as a marker for egg plasma membrane wherein it is abundant, while filipin is relatively scarce in the acrosome‐reacted hamster sperm membrane, found only in the plasma membrane of the equatorial segment. The earliest sperm‐egg interactions are observed between the egg microvilli and the perforatorium and the equatorial segment of the sperm, and the initial fusion between egg and sperm occurs in the vicinity of the equatorial segment. At later stages of fusion involving the postacrosomal segment, a clear line of demarcation is observed between the filipin‐rich egg membrane and the filipin‐poor sperm postacrosomal segment, suggesting that filipin binding lipids from the egg intercalate into the sperm membrane following membrane fusion. The anterior segment of the sperm does not fuse with the egg but is instead incorporated into a cytoplasmic vesicle derived from both sperm and egg membranes. In this latter step, filipin‐sterol complexes are not found in sperm‐derived membranes suggesting that there may be barriers to the movement of filipin binding lipids from the egg into these sperm membranes.
Thermolability of mouse oocytes is due to the lack of expression and/or inducibility of Hsp70
Authors: Hendrey Jaqueline , Kola Ismail 
Published: February 2005

AbstractEukaryotic and prokaryotic cells have been shown to respond to physical and chemical stress by the induction of proteins called heat shock proteins. Heat shock protein 70 (Hsp70), is the most ubiquitous of these proteins. Although heat shock proteins are generally thought to protect cells from physiologically stressful stimuli, it cannot be assumed that this is so, because several cases exist in which thermotolerance is acquired without the production of heat shock proteins, and in several other cases the hyperproduction of these heat shock proteins does not produce thermotolerance. In this study we show that unfertilized mouse oocytes are sensitive to elevated temperatures, and that the synthesis of Hsp70 cannot be induced in these oocytes. Furthermore, our data demonstrate that the expression of Hsp70 in mouse oocytes is sufficient for the acquisition of thermotolerance. Mouse oocytes were injected with mRNA for Hsp70, and the viability of these oocytes was determined after heating. The number of viable oocytes was significantly higher in the group injected with Hsp70 mRNA and then heated compared with oocytes injected with Hsp70 antisense mRNA and sham‐injected controls treated in an identical manner. No significant differences in the number of viable oocytes were found between the group that had been injected with Hsp70 mRNA, heated, and then allowed to recover for 3 hr and the group maintained at 37°C throughout. This study demonstrates that the injection of Hsp70 mRNA into unfertilized mouse oocytes significantly enhances their thermotolerance, indicating that the thermolability of mouse oocytes is due to the lack of expression of Hsp70. This study provides direct evidence for the role of Hsp70 in the enhancement of thermotolerance of mammalian cells.
The spermicidal effect of ethylene dibromide in bulls and rams
Authors: Amir David 
Published: February 2005

AbstractThe spermicidal effect of ethylene dibromide (EDB) in bulls and rams is reviewed. Following oral or parenteral administration EDB was found to cause, by its alkylating effect in the testes of treated bulls and rams, lysis of the chromatin of the elogating spermatids during the time of somatin histones replacement by the sperm protamines. However, while in the bulls the abnormal spermatozoa issued from the affected spermatids were also collected in the ejaculates, this was not the case with treated rams. In the latter animals the abnormal spermatids seem to be phagocytized in the epididymis before their arrival in the ejaculate. In addition, whereas the alkylating effect of EDB occurred also in the upper parts of the epididymis of the bulls, causing tail and acrosome defects to the spermatozoa, in the rams such an effect seems to occur all along the epididymal duct. These differences between bulls and rams in the sites of the genital tract where the drug takes effect, and in the mechanism of this effect, reveal probable differences in the physiology of the reproductive tract between these species.
Structure and expression of the polyubiquitin gene in sea urchin embryos
Authors: Gong Zhiyuan , Cserjesi Peter , Wessel Gary M. , Brandhorst Bruce P. 
Published: February 2005

AbstractA cloned Lytechinus pictus cDNA has been identified, which includes seven direct repeats of a 228 bp sequence encoding ubiquitin and about 450 bp of 3′ noncoding sequence. The deduced amino acid sequence is identical to that of ubiquitins of other animals (though repeats 3 and 5 each have single amino acid substitutions at different positions). Southern blot analysis revealed that the sea urchin genome contains a single copy of the polyubiquitin gene, and the number of 228 bp repeat units appears to vary from seven to ten among different alleles; no other ubiquitin coding sequences were detected. The size distribution of polyubiquitin mRNA is polymorphic among different individuals, probably corresponding to the differences in copy number of the repetitive coding sequence. The abundance of cytoplasmic polyubiquitin RNA is constant throughout embryogenesis and is similar in ectoderm, endoderm, and mesoderm cells. The constant prevalence of polyubiquitin mRNA apparently results from a balance between ontogenetic changes in its rate of synthesis and its stability in the presence of actinomycin D. Accumulation of polyubiquitin RNA was not heat shock‐inducible during embryogenesis.
Water insoluble fraction of egg yolk maintains porcine sperm motility by activating adenylate cyclase
Authors: Okamura N. , Onoe S. , Sugita Y. , Paquignon M. , Dacheux F. , Dacheux J.‐L. 
Published: February 2005

AbstractThe decrease in motility of porcine cauda epididymal sperm was less than that of caput epididymal sperm in the medium containing bicarbonate. This may be due to the difference of sensitivity of adenylate cyclase to bicarbonate between mature and immature sperm; activation of mature sperm enzyme by bicarbonate was higher than that of immature sperm. Nondialysable fraction of egg yolk prevented the decrease in motility of immature sperm in the presence of bicarbonate, but it was not effective for the motility of mature sperm under the same condition, because only bicarbonate is sufficient for the maintenance of its motility. In the absence of bicarbonate, both mature and immature sperm required egg yolk to maintain motility.The favorable effect of egg yolk on the motility is ascribed to the enhancement of intracellular cAMP level. Partial fractionation of egg yolk showed that water‐insoluble lipoprotein fraction contains factor(s) which activates adenylate cyclase in sperm plasma membrane. This is the first report in which high molecular weight activator of the sperm enzyme was demonstrated.
The vitelline coat spikes: A new peculiar structure of Mytilus galloprovincialis eggs with a role in sperm‐egg interaction
Authors: Focarelli Riccardo , Rosa Domenico , Rosati Floriana 
Published: February 2005

AbstractIn the course of this study we found that in Mytilus galloprovincialis eggs long filamentous protrusions never described before, which we have termed “vitelline coat spikes,” could be clearly detected using the lectin from Dolichos biflorus, which recognizes the GalNAc residues. The spikes could be also observed by transmission electron microscope but only in some fortuitous sections could their origin in the vitelline coat be clearly observed. The spikes were also clearly visible using the scanning electron microscope. Observations of the sperm‐egg interaction very few seconds after insemination or using fixed eggs suggested that the spikes could play a role in a primary binding to the unreacted sperm. Experiments have been done to test the effect of GalNAc on the sperm‐egg binding and on the fertilization process which seem to confirm this hypothesis.
Does in vitro capacitation alter chromatin stability of ejaculated human spermatozoa? cytochemical studies
Authors: Royere D. , Hamamah S. , Nicolle J. C. , Lansac J. 
Published: February 2005

AbstractIn vitro capacitation of human spermatozoa is commonly evaluated by the progressive motility percent. However its effects on sperm chromatin have hardly been studied. Our aim was to determine the extent to which in vitro capacitation with two treatments (B2 or human follicular fluid) alters the chromatin of human spermatozoa, by using two analytical methods, acridine orange staining and Feulgen‐DNA cytophotometric measures. Ejaculates were obtained from 23 men participating in our in vitro fertilization program, and several measurements were made on the same ejaculate for each subject. No alteration was observed for the percent of native DNA after capacitation in B2, but spermatozoa incubation during the same time in human follicular fluid was followed by a significant decrease of the percent of native DNA (P < 0.01). Feulgen‐DNA content significantly increased after capacitation in either B2 or follicular fluid (P < 0.05, P < 0.001 respectively), and so did sperm nuclear surface area (P < 0.001). In this study we observed a negative correlation between Feulgen‐DNA content and fertilization rate (P < 0.02). Moreover, the greater effects on Feulgen‐DNA content were observed in men with abnormal sperm, whose spontaneous percent of native DNA was lower (P < 0.05) and Feulgen‐DNA content higher (P < 0.05) than in men with normal sperm. These results indicate that capacitation in B2 as well as in human follicular fluid may alter the chromatin stability of human spermatozoa. Such results suggest a partial decondensation state of human spermatozoa during in vitro capacitation. However, beyond some level of decondensation, the fertilizing ability could be altered.
Stage and lineage‐regulated expression of two HSP90 transcripts during mouse germ cell differentiation and embryogenesis
Authors: Gruppi Carol M. , Zakeri Zahra F. , Wolgemuth Debra J. 
Published: February 2005

AbstractThe expression of members of the heat shock protein 90 (hsp90) gene family during testicular and embryonic development was investigated. Two different hsp90 transcripts were detected in RNA from mouse testis, approximately 3.2 kb and 2.9 kb in size, and were shown to exhibit cellular and developmental stage specificity of expression. The larger, more abundant transcript was expressed at high levels in the germinal compartment of the testis, particularly in germ cells in meiotic prophase. The smaller hsp90 transcript was expressed predominantly in the somatic compartment of the testis. Expression of the two hsp90 transcripts was observed in testes of other species, suggesting an important role for hsp90 in mammalian testicular function. In addition, expression of both hsp90 transcripts was detected in the embryonic and extra‐embryonic compartments of mid‐gestation embryos.
Developmental appearance of proteins identified by two‐dimensional gel electrophoresis in mouse gonadal tissue
Authors: Durbin E. J. , Erickson R. P. , van Keuren M. L. , Iacob R. A. , Kurnit D. M. 
Published: February 2005

AbstractGonadal protein patterns of the mouse were studied during fetal development by two‐dimensional gel electrophoresis. Fetal mice at days 8.5, 10.5, 12.5, and 14.5 post‐coitum were analyzed for male or female specific proteins. Although no sex specific proteins were found, several proteins were found which were expressed in significantly different amounts in the two sexes at about the time of gonadal differentiation. Hence, quantitative differences, rather than qualitative ones, could be related to the initiation of testis or ovary development.
Requirement of an extracellular energy substrate for the guinea pig sperm acrosome reaction induced by calcium ionophore
Authors: Bhattacharyya Amitabha , Pakrashi Anita 
Published: February 2005

AbstractIt is well established that calcium ionophore A 23187 induces acrosome reaction (AcR) of uncapacitated spermatozoa in the presence of extracellular Ca2+ ions. In the present study, we have investigated how extracellular energy substrates (glucose, pyruvate, and lactate) affect the ionophore‐induced AcR of guinea pig spermatozoa. It was found that 0.3 μM concentration of A 23187 had the maximum effect to initiate AcR of guinea pig spermatozoa. Virtually no spermatozoa underwent their AcR when incubated in substrate‐free modified Tyrode's medium containing 0.3 μM A 23187 and 2 mM Ca2+. At least one exogenous substrate is essential for the ionophore‐induced AcR of spermatozoa. As for efficacy of the substrates, lactate was more effective than pyruvate and glucose. However, a better result was observed when lactate was added along with pyruvate. Malonate inhibited the ionophore‐induced AcR but not the hyperactivated motility of spermatozoa. The mitochondrial electron transport chain blockers rotenone, antimycin, and oligomycin failed to inhibit AcR, although in the presence of these blockers spermatozoa were unable to show hyperactivated motility. These results suggest that the mitochondrial citric acid cycle, not the electron transport chain, is probably the energy source for ionophore‐induced AcR of guinea pig spermatozoa.
xl21: A localized maternal transcript in Xenopus laevis
Authors: Kloc Malgorzata , Reddy Bramham A. , Miller Mill , Eastman Eric , Etkin Laurence D. 
Published: February 2005

AbstractWe describe the cloning and characterization of a partial cDNA, xl21, that represents an RNA, which is localized in the animal hemisphere of Xenopus oocytes. This RNA is also detected in an animal to vegetal gradient during early cleavage stages. The xl21 RNA titer decreases from fertilization through the gastrula stage, after which it is not detectable on northern blots. The amino acid composition of the xl21 conceptual protein derived from cDNA sequencing reveals a large number of acidic residues similar in distribution to proteins that function as transcriptional activators.