bluuebunny/crossref_metadata_2025 huggingface dataset:
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

The 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

Gonadal 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

It 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

We 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.
Zona pellucida modifications in the mouse in the absence of oocyte activation
Authors: Vincent C. , Turner K. , Pickering S. J. , Johnson M. H.
Published: February 2005

Mouse oocytes arrested in metaphase II exhibit zona hardening and a reduced fertilization rate after exposure to the cryoprotectant dimethylsulfoxide (Johnson J, In Vitro Fertil Embryo Transfer 6:168–175, 1989) but do not undergo parthenogenetic activation (Johnson and Pickering, Development 100:313–324, 1987). This paper shows that dimethylsulfoxide cause proteolytic modification of the zona pellucida glycoprotein ZP2 and inhibition of sperm binding. These effects of dimethylsulfoxide are caused by premature exocytosis of the cortical granules, a process that is initiated usually on fertilization. A model for the mechanism of action of dimethylsulfoxide is proposed based on the combined effects of cytoskeletal modification and osmotic shock. The presence of serum before and during the exposure to dimethylsulfoxide was found to reduce significantly these deleterious effects on the mouse zona pellucida without inhibiting the cortical granule release. These results highlight the suitability of dimethylsulfoxide as a tool to study the mechanisms leading to cortical granule release. Use of dimethylsulfoxide allows the separation of oocyte parthenogenetic activation from cortical granule release, and addition of serum allows separation of cortical granule release from the action of the cortical granule contents. Their use allows a dissection of the mechanisms underlying each of these three related events.
Preimplantation embryo biopsy: Detection of trisomy in a single cell biopsied from a four‐cell mouse embryo
Authors: Kola Ismail , Wilton Leeanda
Published: February 2005

This paper describes an efficient technique for the production of metaphase spreads from single blastomeres biopsied from four‐cell preimplantation mouse embryos. The karyotype obtained by chromosomal analysis of single biopsied cells is shown to be fully predictive of subsequent fetal karyotype. The data in this study also demonstrate that the entire process of embryo biopsy and karyotypic analysis of biopsied blastomeres does not adversely affect the ability of biopsied embryos to form fetuses after transfer into pseudopregnant recipients. This study has potential clinical relevance in that it demonstrates that chromosomally defective embryos can be accurately identified before implantation. In addition, the techniques developed in this study may facilitate more efficient procedures for the genesis of animal models for human disorders such as Down syndrome and Alzheimers disease.
Follicle somatic cells influence pig oocyte penetrability and cortical granule distribution
Authors: Galeati G. , Modina S. , Lauria A. , Mattioli M.
Published: February 2005

The influence of somatic cells on oocyte penetrability was studied during in vitro maturation. Four experiments were carried out. In the first, pig oocytes were fertilized in vitro immediately after collection (immature oocytes) or after being cultured for 44 hr with cumulus cells connected to the whole wall of the extroverted follicle (follicle oocytes) or without cumulus cells (denuded oocytes) (Mattioli et al.: Gamete Res 20:177–183, 1988a). In follicle and immature oocytes, 12 hr after insemination, the sperm were equally distributed between zona and ooplasm; in denuded oocytes, the majority (90.5%) of the sperm were located in the zona. In the second experiment, zonae pellucidae of follicle and denuded oocytes, obtained after 44 hr of culture, were incubated with a sperm suspension for 2 hr at 39°C to evaluate the zona binding. The different number of sperm found on the zona pellucida of follicle or denuded oocytes (49.3 ± 2.97 vs. 37.8 ± 1.77) did not achieve statistical significance (P > 0.05). In the third experiment, follicle and denuded oocytes were denuded of their zonae after maturation and then fertilized in vitro. The number of sperm recorded in the ooplasm of zona‐free follicle oocytes was significantly higher than that recorded in zona‐free denuded oocytes (9.2 ± 0.93 vs 1.19 ± 0.28) (P<0.01). In the last experiment the influence of somatic cells on the distribution of cortical granules was evaluated. Pig oocytes were denuded at different stages of culture and, after completion of maturation, processed for electron microscopy. The removal of somatic cells at 5–20 hr of culture resulted in a premature migration of cortical granules underneath the oolemma. An increase of exocytotic events was also recorded. These data demonstrate that the presence of cumulus cells during maturation is fundamental for the pig oocyte to maintain its penetrability. The persistence of the interaction between cumulus cells and oocyte stabilizes the distribution of cortical granules, and the interruption of this coupling induces cortical granule exocytosis and may be responsible for the reduced penetrability of oocytes matured without cumulus cells.
Nucleocytoplasmic translocation of ribosomal RNA in the rabbit blastocyst: Participation of sulfhydryl groups
Authors: Manes Cole
Published: February 2005

The release of ribosomes from the nucleus in the rabbit blastocyst was investigated by pulse‐labeling embryos to within 5 min of the earliest appearance of radiolabeled ribosomal RNA (rRNA) in the cytoplasmic fraction. The accumulation of radiolabeled 4.7 and 1.9 kilobase mature rRNA species in the cytoplasm was then followed during a 2 hour chase period, using polyacrylamide gel electrophoresis to identify the rRNAs. Colchicine, cytochalasin B, KCN, and EDTA were found to have no effect on the release of radiolabeled rRNA from the blastocyst nucleus during the 2 hour chase. Oligomycin, a known inhibitor of the nuclear envelope nucleoside triphosphatase, and the protein synthesis inhibitors puromycin and cycloheximide blocked rRNA release after a short delay. In contrast, actinomycin D and the sulfhydryl‐reactive agents N‐ethylmaleimide and diamide produced an abrupt and complete block to further rRNA release. The results indicate that ribosomes leave the nuclear compartment by an energy‐dependent process. They further underscore the importance of reduced sulfhydryl groups in a rapidly growing blastocyst with a high level of oxidative metabolism.
Analysis of proteins expressed at the time of murine organogenesis
Authors: Van Keuren Margaret L. , Iacob Ruxandra A. , Kurnit David M.
Published: February 2005

Two‐dimensional electrophoretograms were prepared from wild‐type C57BL/6J embryos from day 7.5 through day 9.0 of development. This time period encompasses a critical window of development as the embryo traverses from an egg cylinder through major organogenesis. Consequently, we term this resource MOPED (for mouse organogenesis protein electrophoresis database). By resolving and analyzing the behavior of approximately 1,000 polypeptides per time point, we were able to track many of these polypeptides through this time period in development. Of special note was a burst of induced protein synthesis that was observed on day 8.5 of development. Polypeptides observed in mouse embryos that match those identified previously in mouse fibroblasts were noted. Two of them (the intermediate filament‐associated protein and tropomyosin‐4) were significantly altered in 8.5 day embryos. As more polypeptides are designated, it will be possible to expand the known proteins in the database. MOPED establishes the patterns of synthesis of a large number of polypeptides during a crucial period of development. Thus MOPED is designed to analyze proteins relevant to mouse embryogenesis in the future.
Guinea pig proacrosin is synthesized principally by round spermatids and contains O‐linked as well as N‐linked oligosaccharide side chains
Authors: Anakwe Onyeama O. , Sharma Sunita , Hardy Daniel M. , Gerton George L.
Published: February 2005

Proacrosin is the zymogen precursor of acrosin, a sperm protease believed to play an essential role in fertilization. In this study, we used primary cultures of guinea pig spermatogenic cells to examine the temporal appearance and mechanisms of synthesis and processing of proacrosin during acrosome development. Following [35S]methionine incorporation and immunoprecipitation, cultured spermatogenic cells were found to synthesize two forms of proacrosin (Mr 54,000 and 57,000). Proacrosin was synthesized mainly by round spermatids. By immunoblotting, proacrosin became very prominent in round spermatids and persisted throughout spermiogenesis. Pluse‐chase experiments demonstrated that the Mr 54,000 form of proacrosin was converted to the Mr 57,000 form, presumably reflecting posttranslational processing of carbohydrate side chains. When spermatogenic cells were cultured in the presence of tunicamycin, the synthesized proacrosin had an Mr of 54,000. However, in vitro translation of mRNA extracted from guinea pig testis followed by immunoprecipitation indicated that the core polypeptide of procrosin has an Mr of 44,000. Guinea pig spermatogenic cells incorporated glucosamine and fucose into the oligosaccharides of proacrosin. Treatment of guinea pig testis proacrosin with N‐glycosidase or O‐glycosidase reduced the Mr by 3–7%. These results indicate that proacrosin is synthesized by postmeiotic cells and the enzyme contains N‐ and O‐linked oligosaccharides.
Complement component C1q and its receptor are involved in the interaction of human sperm with zona‐free hamster eggs
Authors: Fusi F. , Bronson R. A. , Hong Y. , Ghebrehiwet B.
Published: February 2005

C1q is a component of the classical complement pathway that can react with the Fc‐fragment of immunoglobulins and with other proteins, such as fibronectin, laminin, and a specific C1q receptor present on several cell types. Given its role in many adhesion systems, mainly related to phagocytosis, we tested the effects of C1q on the interaction between human spermatozoa and zona‐free hamster eggs. The presence of C1q in the medium used for gamete coincubation resulted in promotion of sperm‐oolemma adhesion and an inhibition of penetration. The number of adherent sperm per egg at 5 μg/ml concentration was 90 ± 35 vs. 29 ± 7 for the control (P < 0.001). At 1 μg/ml, the lower concentration at which C1q had an effect, the number of penetrating sperm/egg was 0.6 vs. 1.7 for the control without C1q (P < 0.01), and the percent of penetrated eggs was 28% vs. 85%. At 50 μg/ml, the percent of penetrated eggs was 7%, with a penetration index of 0.07. The addition of C1q to the medium resulted in sperm agglutination, which varied between sperm donors. The presence of C1q receptors, as detected by anti‐C1qR monoclonal antibodies (Mabs), was demonstrated both on zona‐free hamster eggs by immunobead rosetting and on human spermatozoa by immunobead binding and indirect immunofluorescence. Mabs directed against different epitopes of C1qR had different effects on gamete interaction, with a partial inhibition of penetration mediated by some of them. The binding of C1q to antibody‐free human spermatozoa was also demonstrated both by means of indirect immunofluorescence and utilizing 125I‐C1q. Cur results suggest that C1q and its receptor could be involved in fertilization and provide additional support for the hypothesis that sperm incorporation by the oocyte utilized a family of cell membrane receptors or binding sites similar to those involved in phagocytosis.
Immunoelectron microscopic localization of small nuclear ribonucleoproteins during bovine early embryogenesis
Authors: Kopečný V. , Fakan S. , Pavlok A. , Pivko J. , Grafenau P. , Biggiogera M. , Leser G. , Martin T. E.
Published: February 2005

Small nuclear ribonucleoproteins (snRNPs) were localized using human autoimmune or monoclonal anti‐snRNP antibodies and ultrastructural immunocytochemistry in early preimplantation bovine embryos before and at transcription onset. Bovine cleavage stages up to 16‐cell embryos were obtained either by culture of in vitro‐fertilized ovarian oocytes or by isolation at slaughter of embryos fertilized and developed in vivo. Before transcription onset, up to the four‐cell stage, diffuse labeling of nucleoplasm was detected, whereas cytoplasm labeling remained low. At the transcription onset, labeling of all eight‐cell embryo nuclei was markedly concentrated at the borderline of already formed, condensed chromatin aggregates, where it was associated mainly with perichromatin fibrils. The condensed chromatin blocks revealed by several different staining methods were more prominent than is the case in most somatic cells. The degree of chromatin condensation and the pattern of its distribution differed between in vivo‐ and in vitro‐produced eight‐cell embryos: the in vitro embryos showed a higher degree of chromatin condensation and a peripheral distribution of chromatin blocks. A relation of this observation to the developmental potential of cow embryos is suggested. In two‐ and four‐cell embryos, intensive labeling was seen in interchromatin granules, which, in their turn, were often seen in close proximity to the nucleous precursor bodies, or in the four‐cell stage were interconnected to them. No labeling was ever seen, using antibodies specific for the snRNP Sm antigen, in nucleolar precursor bodies during embryonic nucleologenesis nor in the resulting nucleoli. There was some incidental labeling of the large central vacuole appearing at the beginning of the nucleolus precursor body transformation, testifying the nucleoplasmic origin of this entity.
Cyclic appearance of cytoplasmic NOR–silver‐stained particles in sea urchin embryos
Authors: Amikura Reiko M. , Ihnuma Morio , Aikawa Eizo , Yamada Hisafumi , Nagano Hiroshi
Published: February 2005

When sea urchin embryos were subjected to nucleolar organizer region (NOR)‐silver staining, densely stained particles were observed in the cytoplasm. The appearance of these cytoplasmic particles (CPs) was cell‐cycle dependent. During early development, the CPs were detected at interphase, but not during mitosis; they disappeared at metaphase and reappeared at telophase. The CPs appeared periodically even when embryos were treated with cytochalasin B or aphidicolin, which inhibits the progression of cytokinesis and nuclear division, respectively. By contrast, CPs were not detected in the colchicine‐treated embryos in which both cytokinesis and nuclear divisions were prevented. The CPs were observed only in the embryos whose stage was early blastula (about 6th to 7th cleavage) or earlier; no CPs were detected even at interphase in the embryos at late blastula (about 8th to 9th cleavage) or later. Electron microscopic evaluation showed CPs to be granular structures, similar to heavy bodies. Also, sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) showed that 95‐kDa and 38‐kDa proteins were the NOR–silver‐staining proteins in sea urchin embroys. These proteins existed during the course of the cell cycles. These results suggest that (1) the cyclic appearance of the CPs or heavy bodies is closely related to the cell cycle as well as the programming of the embryogenesis, but independent of the cycle of cytokinesis and nuclear division; (2) 95‐kDa and 38‐kDa proteins are the major NOR–silver‐staining proteins in sea urchin embryos.
Mouse blastocysts respond metabolically to short‐term stimulation by insulin and IGF‐1 through the insulin receptor
Authors: Harvey Mark B. , Kaye Peter L.
Published: February 2005

Insulin specifically stimulates protein synthesis in compacted mouse embryos on days 3 and 4 after fertilization, with an EC50 of 0.5 pM (Harvey and Kaye, 1988). The identity of the receptor mediating this short‐term effect of insulin was further examined by dose–response studies with IFG‐1 and by using a specific anti‐insulin receptor antiserum that has no appreciable cross‐reaction with IGF‐1 receptors. IGF‐1 caused a maximum 40% stimulation of protein synthesis after 4 h exposure (similar to the response to insulin) with an EC50 of 150 pM IGF‐1. The insulin receptor‐specific antiserum, or IgGs isolated from it, also stimulated protein synthesis at dilutions as high as 1:1,000 to the same degree as insulin (∼40%). This agonistic action of the insulin receptor antiserum, the EC50 of 150 pM for IGF‐1, and the previously established EC50 of 0.5 pM for insulin, all with similar maximal stimulation, strongly support the conclusion that the short‐term metabolic stimulation of mouse blastocysts by insulin is mediated by insulin receptors. Immunosurgical isolation of inner cell masses before and after exposure to 1.7 pM insulin (sufficient to stimulate only the insulin receptor) showed that insulin stimulates protein synthesis in these cells as well as in the trophectoderm cells of the blastocyst. This finding suggests that in intact blastocysts, insulin may travel across the trophectoderm to the inner cell mass, acting anabolically on both tissues. Analysis of the agonistic effect of the B‐10 antiserum showed there was no evidence of an unresponsive subpopulation of embryos.
Sperm analysis by flow cytometry using the fluorescent thiol labeling agent monobromobimane
Authors: Seligman J. , Shalgi R. , Oschry Y. , Kosower N. S.
Published: February 2005

The fluorescent labeling agent monobromobimane (mBBr) was used to label thiols and disulfides (after reduction of sperm disulfides by dithiothreitol) in intact spermatozoa. Bimane‐labeled sperm of several mammalian species were analyzed by flow cytometry (FCM) and examined by fluorescent microscopy. FCM analysis showed sperm thiol oxidation to disulfides during epididymal maturation. FCM of labeled mature spermatozoa showed differences among species in the sperm thiol content. Heterogeneity in thiol content of sperm within individual samples was also observed. In addition, FCM patterns showed heterogeneity among and within samples in the content of disulfides and their resistance to reduction. FCM analysis reflected the microscopic appearance of the labeled spermatozoa. FCM analysis of bimanelabeled spermatozoa offers a convenient method for the study of sperm thiol–disulfide status and permits detection of sperm subpopulations within an individual sample. FCM analysis of mBBr‐labeled spermatozoa may serve as a test to evaluate sperm quality.
Characterisation of 24‐kD proteins from rat testes using polyclonal sera reactive to human sperm antigens
Authors: Shaha Chandrima , Seshadri T. , Suri A. , Talwar G. P.
Published: February 2005

A group of antigens of 24‐kD Mr from rat testes were characterised biochemically. These antigens were part of a larger molecule of ∼200 kD. On treatment with disulfide bond reducing agent, the 200‐kD molecule was reduced to subunits. Immunoreactivity was confined to a doublet of ∼24 kD and a single band of ∼50 kD Mr after the reduction. Glycoprotein in nature, this antigen shared immunoreactive epitopes with a 40‐kD antigen on human spermatozoa. Antiserum raised in rabbits against the 24‐kD antigen from rat testes reacted with antigens on the acrosome of human spermatozoa. Agglutination of sperm could be induced by the antiserum. The carbohydrate residue could be removed by mannosidase digestion. Chemical deglycosylation studies showed a slight decrease in molecular weight. Immunoreactivity was however not completely lost after chemical deglycosylation. Isoelectric focusing of the antigen identified nine isoelectric species. Two relatively minor species showed immunoreactivity. Acrosome‐reacted spermatozoa showed loss of antigens from acrosome.
Developmental regulation of a serum response element binding activity in amphibian embryos
Authors: Varley Joel , Brennan Sean
Published: February 2005

As part of our studies of transcriptional control during early development in vertebrates, we have examined embryos of the amphibian Xenopus laevis for the presence of sequence‐specific DNA‐binding proteins, using gel electrophoresis mobility‐shift assays. Our analysis has focused on sequence elements in the cytoskeletal actin gene, whose embryonic transcription is initially activated at the gastrula stage, approximately 16 hours after fertilization. We detect activites capable of specific binding to two known transcriptional regulatory elements, the serum response element and the GC‐box, located in the 5′‐flanking region of the cytoskeletal actin gene. Binding activity specific for a region downstream of the transcriptional startsite is also detected, in a region which may be involved in controlling developmental activation of this gene. Serum response element‐binding activity, as well as the downstream binding activity, is enriched in extracts from gastrula and neurula stage embryos, compared to egg extracts, suggesting that increased levels of one or both of these activities might play a role in developmentally timed transcriptional activation of the cytoskeletal actin gene in the embryo.
Vitrification of mouse oocytes: Improved rates of survival, fertilization, and development to blastocysts
Authors: Shaw P. W. , Fuller B. J. , Bernard A. , Shaw R. W.
Published: February 2005

Rall and Fahy's (1985) vitrification procedure for the cryopreservation of 8‐cell embryos was applied to unfertilized mouse oocytes. Unchanged, this method resulted in a mean of 24.1% of vitrified oocytes fertilizing and developing to blastocysts in vitro. Exposure of oocytes to the cryoprotectant media, but without the vitrification, resulted in 30.8% developing to blastocysts. Modifications to the durations of and media used in the dilution and equilibration steps of the procedure produced a final protocol giving a mean of 55.4% of vitrified oocytes and 72.4% of nonvitrified VS1‐exposed oocytes developing to blastocysts; 85.7% of control oocytes develop to blastocysts. Osmotically induced damage was found to be the most important cause of loss of viability in these methods. Cooling of oocytes to 5–8°C during the procedure had no significant effect on their viability. No parthenogenetic activation of oocytes occurred as a result of exposure to the procedure.
Quantification of Y chromosome bearing spermatozoa of cattle using in situ hybridization
Authors: Schwerin Manfred , Blottner Steffen , Thomsen Preben D. , Roschlau Daniela , Brockmann Gudrun
Published: February 2005

An easy assay for quantification of Y chromosome‐bearing sperm (Y‐sperm) is needed, especially to monitor sperm separation techniques. In the present study a tritiated bovine male‐specific DNA fragment was tested for identification of Y‐sperm by in situ hybridization. A protocol for in situ hybridization to bovine sperm was developed and used to study the proportion of Y‐sperm of 12 bulls. The usefulness of the method in optimization of sperm separation procedures is illustrated through analysis of fractions of sperm separated by Percoll density gradient centrifugation.
Effects of actinomycin D and cycloheximide on transcript levels of IGF‐I, actin, and albumin in hepatocyte primary cultures treated with growth hormone and insulin
Authors: Johnson Thomas R. , Trojan Jerzy , Rudin Susan D. , Blossey Betty K. , Ilan Judith , Ilan Joseph
Published: February 2005

The stability of several RNA transcripts in cultured hepatocytes is known to increase when serum is omitted from the culture medium. In order to investigate possible mechanisms for this phenomenon, we examined the effects of actinomycin D and cycloheximide on the levels of actin, albumin, and insulin‐like growth factor I transcripts in primary cultures incubated in serumfree medium. The levels of IGF‐I and albumin transcripts per culture increased for the first 4 hours following addition of actinomycin D and then declined. The levels of actin transcripts and total RNA per culture declined immediately following actinomycin D addition in a manner consistent with exponential decay. IGF‐I and albumin transcript levels were relatively unaffected by cycloheximide, while actin transcript levels increased 7‐fold over 7 hours. The half‐lives of actin transcripts and total RNA were calculated to be 4.6 to 7.7 hours and 11 to 19 hours, respectively, with no statistically significant correlation with hormone treatment. The data suggest that the stability of albumin and IGF‐I transcripts, but not actin transcripts, is controlled in part by an actinomycin D‐sensitive process.
Glucose and phosphate inhibit respiration and oxidative metabolism in cultured hamster eight‐cell embryos: Evidence for the “Crabtree effect”
Authors: Seshagiri Polani B. , Bavister Barry D.
Published: February 2005

The development of hamster eight‐cell embryos is inhibited by glucose in culture medium containing inorganic phosphate (Pi). This is hypothetically attributed to the “Crabtree effect,” in which enhanced glycolysis inhibits respiratory activity and oxidative metabolism. To examine this hypothesis, oxygen consumption of hamster eight‐cell embryos was measured using a microelectrode. A two‐ to three‐fold decrease in oxygen consumption was observed in embryos cultured with glucose and Pi. Oxidizable substrates and intermediates of the Krebs cycle supported embryo development only in the absence of glucose and Pi; Krebs cycle inhibitors (fluoroacetate and arsenite) arrested embryo development. Under anaerobic conditions, pyruvate and lactate did not support embryo development. Inhibition of both respiration and oxidative activity in cultured hamster embryos by glucose and Pi is consistent with the existence of a Crabtree effect and indicates that the metabolic properties of preimplantation embryonic cells differ markedly from those of most somatic cells and resemble some cancer cells.
Changes in maturation‐promoting activity in the cytoplasm of pig oocytes throughout maturation
Authors: Mattioli M. , Galeati G. , Bacci M. L. , Barboni B.
Published: February 2005

Maturation‐promoting factor (MPF) was examined in maturing pig oocytes by electrofusing them with germinal vesicle (GV) oocytes. Oocytes containing high levels of MPF (MI or MII stages) induced the breakdown of the GV introduced by fusion and the formation of the metaphase plate in 1 hr. A similar effect was seen when two or three GV oocytes were fused with a MII oocyte and then incubated for 1 hr in the presence of cycloheximide (a specific protein synthesis inhibitor), indicating that high levels of preformed MPF are present at the metaphase stage. During the maturation in vitro of cumulus‐enclosed oocytes, a first sharp rise in MPF was seen between 26 and 29 hr of culture (MI stage); MPF declined after 2 hr (AI‐TI stages) and again reached high levels at 35 hr, where it remained for the rest of maturation. Denuded oocytes showed a similar behavior, but MPF appeared 9 hr earlier and the rise, due to the asynchronous maturation of these oocytes, was not as sharp as in cumulus enclosed oocytes. Cycloheximide was used to study protein synthesis requirements for oocyte maturation. Intact GV were observed after 44 hr of culture when cycloheximide was added at 26 hr or earlier, and chromosome decondensation and pronuclear formation were observed when the drug was added at 32 hr. Transcriptional requirements were investigated by treating the oocytes with α‐amanitin, an RNA polymerase inhibitor. This drug could completely inhibit the maturation of cumulus‐enclosed oocytes, but this was a somatic cell‐mediated effect since denuded oocytes were insensitive to this treatment. Enucleated oocytes exhibited an increase in MPF after 24 hr of culture, and low levels of this factor were seen after 40 h of maturation. These data indicate that both rises in MPF require active protein synthesis, whereas transcription is not necessary for the resumption of meiotic cycle. Nuclear activity may be required for the second rise in MPF.
Ion channels in boar sperm plasma membranes: Characterization of a cation selective channel
Authors: Cox Thomas , Campbell P. , Peterson R. N.
Published: February 2005

Plasma membranes isolated from cauda epididymal and ejaculated boar sperm were inserted into planar lipid bilayers and examined for the presence of ion channels. Channel fusion was frequently observed; the most prominent was a nonselective cation channel which conducted K, Na, Cs, Ca, and Ba. Channel opening did not show a strict dependence on voltage but was partially blocked by verapamil, nitrendipine, and ruthenium red. A channel with these characteristics was observed when plasma membranes were isolated by high‐pressure nitrogen cavitation (650 psi, 78% sperm head plasma membranes) or at very low nitrogen pressures (50 psi, 90% sperm head plasma membranes), suggesting that this channel may be present in the plasma membrane overlying the sperm head.
Effect of cryopreservation on the frequency of chromosomal abnormalities and sex ratio in human sperm
Authors: Martin Renee H. , Chernos Judy E. , Rademaker Alfred W.
Published: February 2005

The effects of cryopreservation on the frequency and type of chromosomal abnormalities in human sperm were investigated. Employing a technique that enables direct visualization of human sperm chromosomes following in vitro penetration of hamster oocytes, sperm samples from 10 normal men were examined before and after freezing in liquid nitrogen. A total of 1,960 sperm karyotypes were analyzed, 1,132 before freezing and 828 after freezing. There was no significant difference in the frequency of structural chromosomal anomalies (10.5% prefreeze vs. 8.5% postfreeze), but there was a significant decrease in the frequency of numerical abnormalities (5.2% prefreeze vs. 3.0% postfreeze). However, there was a large excess of hypohaploid complements compared with hyperhaploid complements, suggesting that the hypohaploid complements were caused by technical artefact. A conservative estimate of aneuploidy, derived by doubling the hyperhaploid frequencies, did not differ before (0.4%) and after (0.4%) freezing. There was no evidence for interdonor variability in response to sperm cryopreservation for total chromosomal abnormalities, structural abnormalities, and sex ratios. The sex ratios were also not affected by cryopreservation and did not differ significantly from the theoretical 50%. It is concluded that cryopreservation does not affect the frequencies of chromosomal abnormalities or alter the sex ratio in human sperm, provided that an adequate cryoprotective buffer and freezing system is employed.
Analysis of a murine male germ cell‐specific transcript that encodes a putative zinc finger protein
Authors: Höög Christer , Schalling Martin , Grunder‐Brundell Eva , Daneholt Bertil
Published: February 2005

A cDNA species, corresponding to a gene with testis‐specific expression (TSGA), was isolated from a testis cDNA library. The temporal and spatial expression of TSGA was studied by in situ hybridization as well as RNA filter hybridization. In tissue sections, the TSGA sequence was confined to cells within the seminiferous tubules. For filter hybridization, RNA was isolated from testis of prepubertal rats of different ages as well as from enriched populations of various germ cell types. It was found that TSGA is expressed only in male germ cells and that the steady‐state level of TSGA transcripts reaches a maximum during the meiotic and the postmeiotic stages of germ cell development, suggesting a meiotic or postmeiotic function for the encoded protein. TSGA encodes a putative protein having 1,214 amino acids and contains a zinc finger, a structure that previously has been shown to mediate binding to nucleic acids.
Activation of a two‐cell stage‐specific gene following transfer of heterologous nuclei into enucleated mouse embryos
Authors: Latham Keith E. , Solter Davor , Schultz Richard M.
Published: February 2005

Zygotic gene activation occurs at the two‐cell stage in the mouse embryo, resulting in the appearance of many new proteins, including a stage‐specific family of related proteins of Mr 70,000. The mechanisms that regulate the stage‐specific expression of these proteins were examined by transplanting nuclei from oocytes, four‐cell‐stage blastomeres, inner cell mass cells, cultured embryonic stem cells, or differentiated endoderm‐like PYS2 cells to enucleated one‐cell embryos. Although none of these cell types synthesizes the 70 kDa complex, all were able to direct the synthesis of the 70 kDa complex following transplantation and overnight culture to the two‐cell stage. These results suggest that the embryonic cytoplasm can exert a dominant, positive regulatory influence on a variety of heterologous nuclei that results in the transcription of a stage‐specific gene. In addition, these results indicate that activation of the gene(s) coding for the 70 kDa complex is not dependent on prior programming during oogenesis and oocyte maturation, and that repression of the gene(s) coding for this complex after the two‐cell stage does not involve irreversible gene inactivation.
Liposome‐Mediated DNA uptake by sperm cells
Authors: Bachiller Daniel , Schellander Karl , Peli Janos , Rüther Ulrich
Published: February 2005

To investigate the potential use of sperm cells as vectors to transfer exogenous DNA via the fertilization of oocytes into the germ line of mice, we have used liposomes to transfect DNA into the sperm head. Although the DNA transfer into sperm mediated by liposomes was very efficient and no obvious reduction in the fertilization frequency of oocytes could be detected, we were unable to generate transgenic mice by this method.
Patterns of transgene inheritance in rainbow trout (Oncorhynchus mykiss)
Authors: Penman D. J. , Iyengar A. , Beeching A. J. , Rahman A. , Sulaiman Z. , Maclean N.
Published: February 2005

There have been very few studies of the inheritance of introduced genes (transgenes) in fish. We have followed the inheritance of the mammalian fusion gene MTrGH from founder generation transgenics (originating from eggs microinjected with the MTrGH DNA) to offspring in crosses with control fish.

Initial screening of the founder generation transgenics was by analysing DNA from blood samples. Only three out of six fish which carried the novel gene in blood DNA transmitted it to their offspring, despite the presence of the gene in DNA extracted from the sperm of all four male fish in this group. The frequency of transgenics in the progeny groups from the three fish which transmitted the gene varied widely: in one of these groups more than one type of MTrGH restriction pattern was found. These results suggest widespread mosaicism in founder generation transgenics.
Cryopreservation of mouse half‐morulae and chimeric embryos by vitrification
Authors: Nagashima Hiroshi , Kobayashi Kazuhiko , Yamakawa Hirohito , Ogawa Shyoso
Published: February 2005

Mouse half‐morulae were cryopreserved ≤1, 3, 6, and 12 hr after bisection by the vitrification method using 25% glycerol and 25% 1,2‐propanediol as cryoprotectant. The developmental rates of the frozen‐thawed half‐embryos to blastocysts in vitro were 77.8% (63/81), 82.0% (41/50), 92.1% (117/127), and 0% (0/37), respectively. Sixty‐one of the half‐embryos that had been vitrified 6 hr after the bisection followed by transfer to five recipients resulted in a total of ten (16.4%) normal fetuses. Chimeric mouse embryos constructed by two half‐morulae were also vitrified 6 and 16 hr after aggregation. Survivors were obtained from the former case: 40 (80.0%) of 50 frozen‐thawed embryos developed in vitro to blastocysts, and, after transfer, six chimeric offspring were obtained from the 34 vitrified chimeric embryos. These results showed that mouse half‐morulae and chimeric embryos could be cryopreserved by the vitrification method. It seems possible to manufacture a chimeric mouse embryo of defined genotypic composition that can be analyzed during its frozen state using the identical half‐embryos of the components.
Effect of ethanol and methanol on the motility of Saccostrea commercialis sperm and sperm models
Authors: Molinia F. C. , Swan M. A.
Published: February 2005

The organic solvents methanol and ethanol at concentrations of 2.5% and 5% (v/v), respectively, were found to significantly (P < 0.001) decrease the radius of curvature and track velocity of S. commercialis sperm. To observe the effects of the solvent directly on the axoneme, S. commercialis sperm models were prepared by extraction with Triton X‐100 and reactivation with ATP in media containing acetate anions, DTT, magnesium, and cAMP. Concentrations of 0.1% Triton X‐100 demembranated sperm while 0.01% and 0.05% Triton X‐100 permeabilized sperm. Sperm models were successfully produced after reactivation with 1 mM ATP. At pH 8.25, 1% (v/v) ethanol or methanol was observed to increase waveform asymmetry and significantly (P < 0.001) decrease track velocity of 0.1% Triton X‐100 demembranated sperm models. Similarly 1% (v/v) ethanol increased tailwave asymmetry and decreased track velocity of 0.01% and 0.05% Triton X‐100 permeabilized sperm models. Reactivated motility of 0.05% Triton X‐100 permeabilized sperm models prepared at pH 7.8 were poor and improved after treatment with 7% (v/v) ethanol, which increased waveform asymmetry and doubled the track velocity of sperm. This stimulatory effect of ethanol was unchanged in the presence of the alcohol dehydrogenase inhibitor pyrazole. Concerning the precise mechanism of action of ethanol on the axoneme, we conclude that a stimulatory or inhibitory effect of ethanol is dependent on the pH of the sperm model system used.
Improvement of flow cytometry analysis and sorting of bull spermatozoa by optical monitoring of cell orientation as evaluated by DNA specific probing
Authors: Métézeau Philippe , Cotinot Corinne , Colas Guy , Azoulay Martine , Kiefer Héléne , Goldberg Michel E. , Kirszenbaum Marek
Published: February 2005

Flow cytometry is a potential method for the separation of X and Y bearing spermatozoa, on the basis of their relative DNA content evaluated by the fluorescence emission intensity due to specific fluorochrome DNA staining. However, spermatozoa DNA is highly condensed and nuclei exhibit flat non spherical shape, which can produce artefacts impeding accurate analysis. In order to avoid these limitations, decondensation of DNA performed by enzymatic treatment and a modification of the flow cytometer that orients the spermatozoa relative to the laser beam are generally used. In this work, we describe alternative methods and materials for selection of (1) decondensed and thus dead spermatozoa without orientation, sorted on the basis of only the 10% spermatozoa containing the least DNA (expected Y) and the 10% spermatozoa containing the more DNA (expected X), or (2) native spermatozoa homogeneously oriented using a simultaneous measurement of Axial light loss (extinction) and Forward angle light scatter. For testing enrichment of each selected fraction we have worked out a molecular hybridization procedure using X and Y specific DNA probes. We analyse and sort bull spermatozoa on these basis: the purity obtained for these fractions is 80% without orientation after enzymatic treatment, and 70% on live spermatozoa “optically” oriented.
Growth and DNA replication in rabbit blastocysts
Authors: Lawitts Joel A. , Butler James E. , Kiessling Ann A. , Biggers John D.
Published: February 2005

DNA content and DNA polymerase activity were measured on rabbit blastocysts removed from the uterus at 24‐hr intervals over the period of days 4–7 postcoitum (pc). Median DNA content increased 53 times over the 72‐hr period, from 25.3 ng on day 4 to 1,360 ng on day 7. Median DNA polymerase activity (fmole of radiolabeled nucleotide incorporated in 30 min at 37°C) increased 393‐fold from day 4 to day 7: 32.8 to 12,900. These embryos also increased in surface area and volume by34‐fold and 6,078‐fold, respectively. Litters containing individuals with high DNA content also tended to have similar individuals with high DNA polymerase activity. Therefore, DNA polymerase activity may be a useful measure of the potential for the next cell division. A large amount of variation existed between blastocysts in all parameters measured. An analysis of variance, conducted to partition variation between litters and within litters, determined that within‐litter variation was actually greater than that between litters, resulting in intraclass correlation coefficients less than 0.5. There was also a positive regression of DNA content and DNA polymerase activity on surface area in 6‐ and 7‐day‐old blastocysts after eliminating variation attributable to litters. The developmental pattern of DNA polymerase activity in the rabbit may be quantitatively different from that described in the mouse. The pattern in mammals is very different from that described in several nonmammalian species.
Distribution of filamentous actin in and around spermatids and in spermatozoa of Australian conilurine rodents
Authors: Breed W. G. , Leigh C. M.
Published: February 2005

The distribution of filamentous actin around the maturing sperm head and in spermatozoa of four species of Australian conilurine rodents was investigated at the light and electron microscopic levels. Similar results were obtained for all the species studied. Mechanically isolated spermatids had NBD‐phallacidin‐positive longitudinal bands of fluorescence over the dorsolateral surface and, in late spermatids, bands of bright fluorescence passed perpendicularly from the dorsal convex to ventral concave surface. TEM observations indicated that these regions corresponded to filaments of ectoplasmic specializations and granular filamentous material around the tubulobulbar complexes, respectively. In testicular and cauda spermatozoa NBD‐phallacidin fluorescent material was present in the two ventral processes that extended from the upper concave surface of the sperm head; also fainter material occurred along the concave border and as a dorsocaudal spur. Its distribution was identical for testicular and cauda spermatozoa. TEM of late spermatids showed that in the ventral process closest to the apical hook there were between 170 and 245 filaments, which attached to the inner surface of the postacrosomal dense lamina; in the more caudal ventral process about 70 filaments occurred. No filaments were, however, visible in the mature spermatozoon but, after immunocytochemical labelling for actin, deposition of gold particles was evident over ventral processes of both late spermatids and cauda spermatozoa. Within the female tract these ventral processes made contact with the zona matrix and were taken into the egg cytoplasm unchanged in morphology. The possible functional significance of the filamentous actin in these structures is discussed.
Expression of a gene encoding a rabbit sperm membrane protein in mammalian cells
Authors: Liu Qiangyuan , Wang Linfang , Miao Shiying , Zhao Min , Zong Shudong , Yan Yuan‐Chang , Koide S. S.
Published: February 2005

A general mammalian expression vector designated pSV2‐EP was reconstructed by inserting an oligonucleotide fragment into pSV2‐dhfr. This vector allowed insertion of cDNAs with EcoRI cohesive ends. The pSV2‐EP contains a simian virus 40 (SV40) early promoter, origin for DNA replication, SV40 poly‐A site, splicing site, an initiator ATG downstream from the promoter and an EcoRI site for the insertion of cDNA fragment screened from Àgt11 expression libraries. A recombinant plasmid (pSVRS‐1) was constructed by inserting RSD‐1, a cDNA encoding a rabbit sperm tail protein, into the EcoRI site of the pSV2‐EP vector. Chinese hamster ovarian (CHO) dhfr‐negative cells were contransformed with pSV2‐dhfr and pSVRS‐1 by the calcium phosphate method. In selective culture medium without thymidine and hypoxanthine, several cell lines were obtained containing mRNA and DNA that hybridized with RSD‐1. One of these transformed cell lines stained intensely with anti‐rSMP‐B antibodies, demonstrating that the RSD‐1 was expressed in the transformed CHO cells.