bluuebunny/crossref_metadata_2025_split huggingface dataset:
The incidence of aneuploidy after single pulse electroactivation of mouse oocytes
Authors: Henery C. C. , Kaufman M. H. 
Published: February 2005

AbstractA brief electric pulse often produces a high rate of activation of recently ovulated oocytes. Some other efficient parthenogenetic stimuli, such as alcohol, however, disrupt the spindle apparatus and increase the incidence of aneuploidy. In this paper, we have determined whether electroactivation per se increases the incidence of chromosomal segregation errors in haploid parthenogenones as evidenced at first cleavage mitosis.Superovulated F1 hybrid female mice were killed at 15.5, 18.5, 22.5, and 25 h after the HCG injection. Batches of 10–12 cumulus‐denuded oocytes were transferred to an electroactivation chamber containing mannitol which was connected to a high voltage pulse stimulator and the pulse was triggered once. A high proportion of oocytes activated following this treatment, but only the single‐pronuclear haploid parthenogenones were incubated overnight in medium containing colcemid, to determine the incidence of aneuploidy as evidenced at first cleavage mitosis.“Sham” electroactivation groups were also examined for evidence of activation and aneuploidy as described above. In these cases, cumulus‐denuded oocytes were put through the electroactivation chamber but the pulse was not triggered. A further group of oocytes was studied to determine the effect of handling and exposure to hyaluronidase on activation frequency and parthenogenetic pathways. Finally, the spontaneous rate of aneuploidy was examined in fertilised embryos of F1 hybrid female mice × Rb(1.3)1Bnr male mice at first cleavage mitosis.The results show that single pulse electroactivation does not increase the level of aneuploidy in single‐pronuclear parthenogenones compared to the “sham” group or the spontaneous rate observed in 1‐cell fertilised embryos, nor does aneuploidy appear to increase with postovulatory age. The developmental pathways observed in the electroactivation group are significantly different to those observed in the “sham” group, and the level of activation observed in both groups is increased through handling of oocytes and their exposure to hyaluronidase. © 1993 Wiley‐Liss, Inc.
Separation of bovine X and Y sperm based on surface differences
Authors: Cartwright Elizabeth J. , Harrington Pauline M. , Cowin Allison , Sharpe Paul T. 
Published: February 2005

AbstractAqueous two‐phase partition involving thin‐layer counter current distribution (TLCCD) has been used to assess surface heterogeneity of ejaculated bovine sperm. When partitioned in charge‐insensitive aqueous two‐phase systems, which detect non‐charge associated surface properties, the sperm fractionates into two distinct populations. Using a Y‐chromosome‐specific DNA marker, it has been shown that one of these populations is enriched in Y chromosome bearing sperm. However, this population is not pure—it consists of 80% Y sperm, with the other 20% being X sperm. All the sperm in the original population that had begun to undergo the acrosome reaction were separated into this same peak; the sex chromosome composition of these sperm is unknown. Since the aqueous partition of sperm is based on surface properties these results suggest that two populations of Y sperm exist that have different surface characteristics. © 1993 Wiley‐Liss, Inc.
Direct evidence that the mouse sex‐determining gene Sry is expressed in the somatic cells of male fetal gonads and in the germ cell line in the adult testis
Authors: Rossi Pellegrino , Dolci Susanna , Albanesi Cristina , Grimaldi Paola , Geremia Raffaele 
Published: February 2005

AbstractReverse transcriptase polymerase chain reaction (RT‐PCR) analysis shows that Sry mRNA is expressed in male fetal urogenital ridges from 12.5 day p.c. embryos, but not in enriched populations of primordial germ cells from the same embryos, indicating that Sry is expressed in the somatic cells of the embryonal gonad at the time of testis determination. We also show that, in the adult male mouse testis, Sry mRNA is expressed at high levels in meiotic and postmeiotic germ cells and, at much lower levels, also in Sertoli cells. Treatment with cyclic adenosine monophosphate (cAMP) analogs of cultured Sertoli cells from postnatal testis completely abolishes Sry mRNA expression. © 1993 Wiley‐Liss, Inc.
Protein secretion by the mouse blastocyst: Stimulatory effect on secretion into the blastocoel by transforming growth factor‐α
Authors: Dardik Alan , Doherty Adam S. , Schultz Richard M. 
Published: February 2005

AbstractWe have previously demonstrated that newly synthesized proteins are secreted into the mouse blastocoel [Dardik and Schultz (1991): Biol Reprod 45:328–333]. In the present study we examine the effect of transforming growth factor‐α (TGF‐α) on these proteins. We observe that TGF‐α stimulates secretion of these newly synthesized proteins into the blastocoel and apical medium, which faces the zona pellucida, by about 65%. Although one‐dimensional gel electrophoretic analysis does not reveal any marked differences in the patterns of newly synthesized proteins secreted into the blastocoel in response to TGF‐α, zymography reveals a marked stimulation in the secretion of several gelatinases into the blastocoel and apical medium. These results suggest additional functions for TGF‐α in mouse preimplantation development. © 1993 Wiley‐Liss, Inc.
Okadaic acid induces premature chromosome condensation reflecting the cell cycle progression in one‐cell stage mouse embryos
Authors: Dyban A. P. , de Sutter P. , Verlinsky Y. 
Published: February 2005

AbstractHaploid parthenogenetic embryos as well as fertilized mouse eggs were treated in vitro with 1–10 μM okadaic acid (OA) at the one‐cell stage. Cytogenetic analysis detected that OA induces nuclear envelope breakdown (NEBD) and premature condensation of interphase chromosomes in pronuclei as well as in 2nd polar body (PB) nuclei. G1‐, S‐, and G2‐type prematurely condensed chromosomes (PCC) were found in pronuclei of embryos of different age, which reflects their progression through the first cell cycle. In nuclei from 2nd PBs only G1‐ and S‐type PCC were observed. Using the types of PCC as a criterion of different phases of the cell cycle, it was possible to estimate that in haploid parthenogenetic embryos G1‐phase lasts until 5.5 hr post activation (hpa), S‐phase takes from 4.5 to 9.5 hpa, and from 8.5 hpa G2‐phase had started. Second PBs were found to be in G1‐phase until 6.5 hpa and S‐phase started in some as early as 5.5 hpa, but in most not before 7.5 hpa.Treatment with OA visualizes G1‐chromosomes in pronuclei as well as in 2nd PBs, and it is easy to count the number of these chromosomes and recognize a T6 marker chromosome. The possibility to apply cytogenetic analysis of G1‐chromosomes from 2nd PBs for a more accurate detection of maternal meiotic nondisjunction is discussed. © 1993 Wiley‐Liss, Inc.
Noninvasive chemical enucleation of mouse oocytes
Authors: Fulka Josef , Moor Robert M. 
Published: February 2005

AbstractA noninvasive method of enucleating mouse oocytes has been developed and evaluated. Strong chromosome to chromosome binding was induced by culturing early metaphase I oocytes in etoposide supplemented medium. Subsequent expulsion of the entire chromosome complex during polar body extrusion was facilitated by exposing the etoposide treated oocytes to a combination of cycloheximide and etoposide during anaphase and telophase. This simple two‐step chemical enucleation procedure yields fully enucleated mouse oocytes in 96% of cases. Chemically enucleated oocytes do not contain maturation promoting factor (MPF) at the end of etoposide‐cycloheximide enucleation. MPF levels are, however, restored during subsequent incubation in drug‐free medium and, after 15 h of post‐enucleation culture, the cytoplasts regain their full capacity for parthenogenetic activation and nuclear remodelling. We believe that this novel enucleation technique will greatly facilitate the research in nuclear transplantation. © 1993 Wiley‐Liss, Inc.
Nuclear transfer in cattle: Effect of nuclear donor cells, cytoplast age, co‐culture, and embryo transfer
Authors: Yang Xiangzhong , Jiang Shie , Farrell Paul , Foote Robert H. , McGrath Ann B. 
Published: February 2005

AbstractThere are many factors affecting the efficiency of nuclear transfer technology. Some are evaluated here using our novel approach by enucleating oocytes at 20–22 hr after in vitro maturation (IVM), culturing the enucleated oocytes (cytoplasts) for 8–10 hr or 18–20 hr to gain activation competence and then conducting nuclear transfer. In the first experiment, we demonstrated that cumulus cell (CC) monolayer can support some cloned embryos to develop into morulae or blastocysts. Co‐culture with CC and bovine oviduct epithelial cell (BOEC) monolayers resulted in no differences (P 0.05) in supporting the development of cloned embryos (Experiment 2). When in vitro matured oocytes were enucleated at 22 hr after IVM followed by nuclear transfer 18–20 hr later, cleavage and morula or blastocyst development of the cloned embryos were similar to those resulting from the enucleated oocytes which had been matured in vivo (Experiment 3). Frozen embryos as nuclear donor cells worked equally well as fresh embryos for cloning in embryo development which was superior to IVF embryos (Experiment 4). However, fresh embryos resulted in a higher proportion (P < 0.05) of blastomere recovery than did frozen or IVF ambryos. Finally, embryo transfer of cloned embryos from our procedure produced a viable calf, demonstrating the commercial value of this novel approach of the technology. © 1993 Wiley‐Liss, Inc.
Isolation and biochemical characterization of heparin‐binding proteins from boar seminal plasma: A dual role for spermadhesins in fertilization
Authors: Sanz Libia , Calvete Juan J. , Mann Karlheinz , Gabius Hans‐Joachim , Töpfer‐Petersen Edda 
Published: February 2005

AbstractSperm surface‐coated heparin‐binding proteins originating from secretions of the male sexual accessory glands, are known to play a pivotal role as extrinsic regulatory factors during sperm capacitation in many mammalian species. They interact with glycosaminoglycans present in the female genital tract and enhance the subsequent zona pellucida‐induced acrosome reaction. We have isolated heparin‐binding proteins from boar seminal plasma by affinity chromatography on heparin–Sepharose and reverse‐phase HPLC. N‐Terminal sequence analysis of these proteins identified a boar counterpart of the bovine capacitation factors BSP‐A1/2 (also called PDC‐109) and BSP‐A3. Several carbohydrate‐ and zona pellucida‐binding proteins, which belong to the newly described spermadhesin family, were also identified as heparin‐binding proteins. Our results imply that, besides other capacitation factors, members of the spermadhesin family may play a dual role in sperm capacitation and fertilization in the pig. © 1993 Wiley‐Liss, Inc.
Vitelline coat of Unio elongatulus egg: I. Isolation and biochemical characterization
Authors: Focarelli Riccardo , Rosati Floriana 
Published: February 2005

AbstractIn this study we isolated and purified the vitelline coat (vc) of Unio elongatulus eggs in order to investigate its protein and carbohydrate composition. SDS dissolved up to 80% of the vitelline coat protein content whereas 100 mM Ammonimum acetate (AA) at pH 11 and 1 mM lithium diiodosalicylate (LISH) dissolved only 40–50%. The ability of extremes of pH or LIS to solubilize the vitelline coats on eggs was then investigated. The results showed that pH from 7 to 11 progressively dissolved the vitelline coats without gross damage to the oocytes. SDS‐PAGE of the solubilized material revealed only two components corresponding to the main components revealed by SDS‐PAGE of the isolated vcs. These peptides have an apparent MW of 220 and 180 kD, are ConA positive, and seem to be connected to each other to form polycomponents. The latter feature is suggested by the electrophoretic pattern of the solubilized material under nondenaturing conditions. © 1993 Wiley‐Liss, Inc.
Capacity of rete testicular and cauda epididymal boar spermatozoa to undergo the acrosome reaction and subsequent fusion with egg plasma membrane
Authors: Harayama Hiroshi , Kusunoki Hiroshi , Kato Seishiro 
Published: February 2005

AbstractThe capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane was examined in rete testicular and cauda epididymal spermatozoa from boars. Sperm penetration assay using zona‐free hamster eggs demonstrated that the penetration rates for rete testicular spermatozoa preincubated for induction of the acrosome reaction for 2 and 3 h were 55% and 97%, respectively. However, most of the eggs (93%) were penetrated with polyspermy by cauda epididymal cells preincubated for 2 h. Results obtained by the triple‐stain technique revealed the percentages of acrosome‐reacted spermatozoa in the rete testicular and cauda epididymal samples preincubated for 3 h to be 61% and 74%, respectively. These results indicate that many rete testicular spermatozoa possess the capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane in vitro, which appears to be completely established only after sperm transit through at least the proximal part of the epididymis. © 1993 Wiley‐Liss, Inc.
Local alteration of cortical actin in Xenopus eggs by the fertilizing sperm
Authors: Chow Robert L. , Elinson Richard P. 
Published: February 2005

AbstractRhodamine phalloidin (Rph) staining was used to examine the microfilament organization of the Xenopus laevis egg cortex during the early stages of fertilization. Unactivated eggs possessed a cytochalasin B (CR)‐insensitive Rph‐stained matrix that was reorganized upon egg activation and diminished in the presence of CB. Xenopus laevis sperm caused a temporary local increase in Rph staining on the Xenopus cortex. In CB‐treated eggs, the local increases of cortical Rph staining later changed to a Rph‐free area. These temporary local increases of cortical Rph staining were also observed when Notophthalmus viridescens sperm fertilized Xenopus and Rana pipiens eggs, and were followed by the appearance of concentric rings of stained and unstained areas. Our data suggest that Xenopus and Notophthalmus sperm have activities that can both organize and disrupt the cortical filamentous actin of the Xenopus egg. © 1993 Wiley‐Liss, Inc.
Role of glutathione in the maturation and fertilization of pig oocytes in vitro
Authors: Yoshida Mitsutoshi 
Published: February 2005

AbstractThe present study examined, by treatment of buthionine sulfoximine (BSO), which is a specific inhibitor of glutathione (GSH) synthesis, the role of GSH in the maturation and fertilization of pig oocytes in vitro. Follicular oocytes collected from prepubertal gilts at a local slaughterhouse were cultured for 36 h in Waymouth MB 752/1 with or without BSO (1 mM), fertilized in vitro, and assessed for GSH concentration (before insemination), maturation, and fertilization. The addition of BSO to maturation medium immediately after culture (Group I), 12 h after culture (Group II), or 24 h after culture (Group III) significantly decreased the GSH concentration in pig oocytes compared with the control (P < 0.01), whereas the rate of cumulus mass expansion at 36 h of culture and the rates of nuclear maturation and sperm penetration following in vitro insemination did not differ. However, the rate of pig oocytes having condensed sperm heads was significantly lower and the rate of male pronucleus formation of pig oocytes was significantly higher in oocytes matured in the control and Group III than in oocytes matured in Groups I and II (P < 0.01). In experiment 2, when BSO was added to maturation media 15, 18, 21, or 24 h after culture, the rate of pig oocytes having condensed sperm heads was significantly lower and the rate of male pronucleus formation of pig oocytes was significantly higher in oocytes matured in the medium supplemented with BSO at 21 or 24 h of culture than in oocytes matured in other groups (P < 0.05 or P < 0.01). The results indicate that GSH synthesis occurs throughout in vitro maturation of pig oocytes and GSH is an important cytoplasmic factor for regulating sperm nuclear decondensation and male pronucleus formation following sperm penetration in pig oocytes. © 1993 Wiley‐Liss, Inc.
Purine control of mouse oocyte maturation: Evidence that nonmetabolized hypoxanthine maintains meiotic arrest
Authors: Downs Stephen M. 
Published: February 2005

AbstractHypoxanthine is present in preparations of follicular fluid and has been shown to suppress the spontaneous meiotic maturation of mammalian oocytes in vitro. The present experiments examined the possible role of hypoxanthine metabolism in mediating this meiotic arrest. Four putative inhibitors of the enzyme, hypoxanthine phosphoribosyltransferase (HPRT), which metabolizes hypoxanthine to inosine monophosphate, were tested on lysates of oocyte‐cumulus cell complexes. At a concentration of 1 mM, 6‐mercapto‐9‐(tetrahydro‐2‐furyl)‐purine (MPTF) and 6‐mercaptopurine (6‐MP) suppressed enzymatic activity by 86% and 98%, respectively, while 6‐azauridine and 2,6‐bis‐(hydroxyamino)‐9‐β‐D‐ribofuranosyl‐purine had no effect. MPTF and 6‐MP increased the inhibitory effect of hypoxanthine on germinal vesicle breakdown, but the other agents did not. The 2 active agents had similar effects on salvage activity and hypoxanthine‐maintained meiotic arrest in denuded oocytes. Also, oocytes from XO mice were more sensitive to the meiosis‐arresting action of hypoxanthine than oocytes from XX littermates, which have twice the HPRT activity. The actions of the HPRT inhibitors were not due to their conversion to nucleotides via HPRT and negative feedback on purine de novo synthesis, because azaserine and 6‐methylmercaptopurine riboside, which are more potent inhibitors of de novo synthesis, had a stimulatory, rather than inhibitory, effect on hypoxanthine‐arrested oocytes. Furthermore, several lines of evidence indicate that metabolism of hypoxanthine to xanthine and uric acid by xanthine oxidase does not mediate the inhibitory action of this purine base on meiotic maturation. The data therefore suggest that nonmetabolized hypoxanthine is responsible for the meiotic arrest observed, most likely through suppression of cAMP degradation. © 1993 Wiley‐Liss, Inc.
The sea urchin egg jelly coat is a three‐dimensional fibrous network as seen by intermediate voltage electron microscopy and deep etching analysis
Authors: Bonnell Barry S. , Larabell Carolyn , Chandler Douglas E. 
Published: February 2005

AbstractThe egg jelly (EJ) coat which surrounds the unfertilized sea urchin egg undergoes extensive swelling upon contact with sea water, forming a threedimensional network of interconnected fibers extending nearly 50 μm from the egg surface. Owing to its solubility, this coat has been difficult to visualize by light and electron microscopy. However, Lytechinus pictus EJ coats remain intact, if the fixation medium is maintained at pH 9. The addition of alcian blue during the final dehydration step of sample preparation stains the EJ for visualization of resin embedded eggs by both light and electron microscopy. Stereo pairs taken of thick sections prepared for intermediate voltage electron microscopy (IVEM) produce a threedimensional image of the EJ network, consisting of interconnected fibers decorated along their length by globular jelly components. Using scanning electron microscopy (SEM), we have shown that before swelling, EJ exists in a tightly bound network of jelly fibers, 50–60 nm in diameter. In contrast, swollen EJ consists of a greatly extended network whose fibrous components measure 10 to 30 nm in diameter. High resolution stereo images of hydrated jelly produced by the quick‐freeze/deep‐etch/rotary‐shadowing technique (QF/DE/RS) show nearly identical EJ networks, suggesting that dehydration does not markedly alter the structure of this extracellular matrix. © 1993 Wiley‐Liss, Inc.
Flow cytometric studies of bicarbonate‐mediated Ca2+ influx in boar sperm populations
Authors: Harrison R. A. P. , Mairet B. , Miller N. G. A. 
Published: February 2005

AbstractBoar spermatozoa loaded with the Ca2+ probe fluo‐3 were incubated in various Tyrode's‐based media similar to those used for in vitro fertilization (IVF), and samples were then analysed by two‐colour flow cytometry; propidium iodide was included in the media to detect membrane‐damaged (“dead”) cells.If media contained bicarbonate/CO2 (a component thought to promote capacitation), part of the live sperm population experienced a considerable influx of Ca2+ into both head and tail compartments. The percentage of responding cells reached a maximum after about 30 min, but both during and after this period there was also a steady increase in the number of dead cells. This bicarbonatemediated increase in cell death took place in the absence of external Ca2+. Evidence was obtained that the entry of propidium iodide was preceded by a change in permeability of the plasma membrane, detectable by leakage of carboxydichlorofluorescein, and it was therefore deduced that the Ca2+ influx detected by fluo‐3 was due to destabilization of the plasma membrane. A similar response could be produced by both caffeine and papaverine (best known as phosphodiesterase inhibitors), but neither cyclic AMP nor activators of adenylate cyclase had any effect. There was no influence of substrate on the process, but, in comparison to poly(vinyl alcohol), serum albumin enhanced it.The precise relevance of this destabilization to capacitation is not yet clear, but it seems significant that the process is mediated or enhanced by components often specifically included in IVF media, and that different individual cells respond after different times. © 1993 Wiley‐Liss, Inc.
ZNC(C)PR affects developmental changes of P46 phosphorylation in rat hippocampus
Authors: Chen Xiu‐Fang , Tang Tong , Zhang Jian‐Wei , Miao Hong‐Hua , Wang Tong‐Xi , Du Yu‐Cang 
Published: February 2005

AbstractProtein phosphorylation has been suggested to be correlated with brain development and with the molecular mechanism of behavioral effects of neuropeptides. The present study reports in vitro endogenous phosphorylation of P46, a membrane‐associated protein that is changed during development of the rat hippocampus. This study indicated that the degree of endogenous phosphorylation may be correlated with the establishment of synaptic connections. Interestingly, P46 was proved to be identical to a well‐known growth‐associated protein B‐50/GAP‐43 in its identical apparent molecular weight, isoelectric point, phosphorylation dependence, and the cross immunoreaction of monoclonal anti‐B‐50/GAP‐43 antibody and P46. Moreover, neonatal administration of neuropeptide ZNC(G)PR could facilitate the developmental progress of P46 endogenous phosphorylation. It is suggested that the changes in P46 phosphorylation could be involved in the cellular mechanism of ZNC(C)PR behavioral effects on learning. © 1993 Wiley‐Liss, Inc.