bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation, composition, and biological activity of sugar chains of porcine oocyte zona pellucida 55K glycoproteins
Authors: Yurewicz Edward C. , Pack Beverley A. , Sacco Anthony G. 
Published: February 2005

AbstractZP3, a preparation of the 55K families of porcine oocyte zona pellucida, possesses carbohydrate‐dependent ligand activity for boar sperm. The aim of the present study was to analyze ZP3 N‐ and O‐linked oligosaccharides with respect to size distribution, composition, and role in sperm‐zona recognition events. Digestion of denatured ZP3 with peptide N‐glycosidase F (PNGaseF) released the majority of N‐glycans which fractionated on Sephadex G‐75 resin as a polydisperse population with apparent molecular masses ranging from 1,900–8,200 Da. The higher molecular weight N‐glycans were characterized by the presence of strongly anionic sulfated/sialylated polylactosamine structures. Alkaline‐borohydride treatment of the PNGaseF‐digested core proteins liberated O‐glycans as a heterogeneous population of oligosaccharide alcohols, which were fractionated on a Sephadex G‐50 column. Compositional analyses indicated sulfated polylactosamine units associated with the higher molecular weight O‐glycans. Preincubation of boar sperm with ZP3 or purified O‐glycans, but not N‐glycans, inhibited subsequent attachment to zona‐encased oocytes. Purified O‐glycans were, however, 2 to 3 orders of magnitude less effective than ZP3 as competitive ligands. The results document the extreme heterogeneity of the ZP3 carbohydrate moiety, in large part attributable to a board spectrum of variably sized N‐ and O‐linked sulfated polylactosamines. Ligand competition bioassays suggest that O‐glycans mediate, at least in part, the sperm adhesive properties of ZP3 and strongly imply that high‐affinity interaction of ZP3 sugar chains with complementary sperm receptors is dependent upon their covalent association with core proteins.
Preparation of spermatogenic cell populations at specific stages of differentiation in the human
Authors: Blanchard Y. , Lavault M. T. , Quernee D. , Le Lannou D. , Lobel B. , Lescoat D. 
Published: February 2005

AbstractStudying biochemical events in human spermatogenesis requires separated populations of spermatogenic cells. Dissociation of these cells was performed by a Trypsin‐DNAse method adapted from the technique used for rodents. Cell separation was performed by centrifugal elutriation. Seven populations were collected, one further purified by Percoll gradient centrifugation, giving nine different cell populations. The efficiency of the cell separation was evaluated by phase contrast microscopy, flow cytometric DNA analysis, and electron microscopy. Five populations were enriched in spermatids: two in round spermatids (87% and 73%), another in round (52%) and elongating (44%) spermatids, another constituted by 80% elongating spermatids, and the last by 90% elongated spermatids. Two of the four remaining populations were enrichied in primary spermatocytes (74% and 54%); another population was the upper part of the Percoll gradient and constituted cytoplasmic lobes and residual bodies (89%); the last population was made up of various cells, with no specific enrichment. Electron microscopic observations revealed good preservation of the separated cells; only the flagella from elongated spermatids were lost. Furthermore, an unusual pattern of nucleoplasm distribution during stages 2–4 of spermatid differentiation was observed and its signification is discussed with regard to the shape of the human spermatozoon.
Developmental expression of cathepsin L and c‐rasHa in the mouse placenta
Authors: Hamilton Richard T. , Bruns Kerry A. , Delgado Michael A. , Shim Jae‐Kyoung , Fang Yu , Denhardt David T. , Nilsen‐Hamilton Marit 
Published: February 2005

AbstractAn investigation is described of the expression of the cysteine proteinase cathepsin L during placental development. In addition, whether cathepsin L expression is linked to c‐rasHa expression in development, as it is in metastatic cells, is examined. Large amounts of cathepsin L and its transcript are present in the mouse placenta, more than six times more than in adult kidney and liver. Throughout gestation, cathepsin L and its transcript are located in the giant cells and spongiotrophoblasts of the placenta. Several forms of different mobility on denaturing gels are found in the placenta. Their apparent molecular weights, as determined from the gels, are 43,000, 39,000, 29,000, and 20,000. The 39‐kDa form is procathepsin L. The 29‐kDa and 20‐kDa forms are lysosomal cathepsin Ls. The 39‐kDa procathepsin L and the 20‐kDa mature cathepsin L are the most abundant species in the placenta and are present in about equal amounts throughout gestation. At any time during gestation, placental minces synthesize and secrete only procathepsin L. The amniotic fluid of the fetus contains the 43‐kDa form of cathepsin L and procathepsin L, but no detectable amounts of mature cathepsin L. By contrast, serum from nonpregnant or pregnant mice contains three forms of cathepsin L (i.e., the 43‐kDa form, procathepsin L, and mature cathepsin L). Cathepsin L and the rasHa oncogene are expressed in two coincident waves corresponding to periods during which the placenta is invasive and just before parturition. The presence of large amounts of cathepsin L in the placenta suggests that the proteinase has a significant function there. Expression of cathepsin L in the placenta is potentially under the control of the ras gene product p21; both are under developmental control.
Sperm selection capacity of the human zona pellucida
Authors: Menkveld Roelof , Franken Daniel R. , Kruger Thinus F. , Oehninger Sergio , Hodgen Gary D. 
Published: February 2005

AbstractPrevious hemizona assay (HZA) results have illustrated a positive and significant correlation between the percentage of morphologically normal spermatozoa in the semen and the number of spermatozoa tightly bound to the zona pellucida. The present study was designed to evaluate the morphologic features using strict criteria of spermatozoa tightly bound to the zona pellucida. Semen samples of 4 normozoospermic and 11 teratozo‐ospermic men were used to compare the percentage of normal spermatozoa in the semen with that found (1) after swim‐up separation and (2) bound to the zona under HZA conditions. The mean (± SEM) % normal forms for normozoospermic men in semen, after swim‐up and zona‐bound spermatozoa were 21.5 ± 1.6, 27.5 ± 2.9, and 44.8 ± 3.4, respectively. A significantly higher % of normal forms were found among zona‐bound sperm compared to swim‐up forms (p = 0.02) and seminal sperm (p = 0.02). The mean % of normal sperm forms present in semen, after swim‐up and zona pellucida‐binding for teratozoospermic men, were 3.7 ± 0.9, 5.8 ± 1.6 and 15.6 ± 3.1, respectively. Significant differences existed between the % of normal sperm forms found in the swim‐up and zona‐bound spermatozoa (P = <0.01 and P = <0.0003, respectively) compared to the original ejaculates. Results indicate that a selective process against abnormal spermatozoa occurs at the site of the zona pellucida. Spermatozoa classified as normal or slightly abnormal are selectively bound to the zona in favor of abnormal (bizarre) spermatozoa; particularly those sperm cells with acrosomal abnormalities are either actively excluded or simply cannot bind to the zona or do it with a low efficiency due to inherent defects. This newly identified human zona property of sperm selectivity points out another potential use of the HZA, i.e., selection of sperm to be used in assisted fertilization.
Early development and X‐chromosome inactivation in mouse parthenogenetic embryos
Authors: Tada Takashi , Takagi Nobuo 
Published: February 2005

AbstractEarly development and X‐chromosome inactivation were studied in ethanol‐induced mouse parthenogenones. About 24% of oocytes transferred to 0.5‐day pseudopregnat recipients successfully implanted. However, only 49%, 20%, and 16% of implanted parthenogenones survived 5, 6, and 7 days later, respectively. Abnormal development was evident in every parthenogenone as early as 5 days after activation with the degenerating polar trophectoderm. These embryos were destined to become either small disorganized embryos or embryonic ectoderm vesicles bounded by the visceral endoderm. Only 2 of 51 representative 6‐ to 8‐day parthenogenones sectioned had morphology of the normal egg cylinder, although growth retardation was evident. Spontaneous LT/Sv parthenogenones shared similar morphological features. In late blastocysts, the frequency of cells with an apparently inactivated X chromosome was lower in parthenogenones than in fertilized embryos. The failure of X‐inactivation in the trophectoderm seems to contribute to the defective development of parthenogenones.
Purification and characterization of a sperm motility‐dynein ATPase inhibitor from boar seminal plasma
Authors: Iwamoto Teruaki , Tsang Angus , Luterman Maynard , Dickson John , de Lamirande Eve , Gagnon Claude , Okuno Makoto , Mohri Hideo 
Published: February 2005

AbstractA sperm motility inhibitor from boar seminal plasma was purified. The purification procedure included dialysis against 0.1 M Tris‐HCl containing 0.1 mM DTT and chromatographies on SP‐Sephadex C‐25 and Phenyl‐Sepharose CL‐4B. With this procedure, the seminal plasma motility inhibitor (SPMI) preparation was highly purified with a 18% recovery of inhibitory activity. The molecular weight of SPMI in native conditions has been estimated at 50,000 by molecular sieving, but 3 polypeptides with molecular weights of 14,000, 16,000 and 18,000 were observed following polyacrylamide gel electrophoresis in denaturing conditions. SPMI is a thermolabile basic protein that is stable between pH 6 and pH 11. The observations that SPMI effects on motility of demembranated spermatozoa are reversed by Mg.ATP and that SPMI inhibited bull dynein ATPase in a concentration‐dependent manner suggest that this protein blocks the motility of demembranated spermatozoa by interfering with dynein arm function.
Enhanced in vitro growth of murine fibroblast cells and preimplantation embryos cultured in medium supplemented with an amphipathic peptide
Authors: Reed W. A. , White K. L. , Enright F. M. , Holck J. , Jeffers G. W. , Jaynes J. M. 
Published: February 2005

AbstractPreliminary studies on the proliferative effects of lytic peptides were carried out using NIH 3T3 murine fibroblast cells and human lymphocytes. Cells were cultured in various concentrations of three different amphipathic peptides (SB‐37, Shiva‐1, and Vishnu), and enhanced proliferation was determined by uptake of 3Hthymidine with treated cells compared with control cultures. Enhanced proliferation of 3T3 cells was observed in cultures containing 50 μM or less SB‐37. The primary study consisted of 263 four‐cell‐ to eight‐cell‐stage mouse embryos from naturally bred mice and incubated in Whitten's medium containing 0.2, 1, or 10 μM of the amino terminus of an amphipathic cecropin B analog (Vishnu) or in Whitten's medium alone. Embryos were cultured to the hatched blastocyst stage, and effect of treatment was determined by the rate of growth to that stage of development. Statistical analysis revealed that culture in all three levels of Vishnu significantly accelerated in vitro growth of these stages of preimplantation embryos compared with controls. These results indicate that Vishnu promotes increased cleavage rates of embryos in vitro. A growth factor receptor clustering mechanism of action is proposed. This peptide may have some potential as an embryo culture medium additive to enhance in vitro growth rate.
Importance of mammalian sperm metalloendoprotease activity during the acrosome reaction to subsequent sperm‐egg fusion: Inhibitor studies with human sperm and zona‐free hamster eggs
Authors: Díaz‐Pérez Eneida , Meizel Stanley 
Published: February 2005

AbstractWe have previously shown that each of the metalloendoprotease (MEP) inhibitors phosphoramidon, diethylenetriaminepentaacetic acid, and carbobenzoxy‐L‐phenlalanine, when present only during the human sperm acrosome reaction (AR), will not inhibit the AR or sperm motility but will decrease the number of sperm that penetrate zona‐free hamster eggs. The present study was designed to investigate whether this inhibition of penetration is due to an effect on sperm binding to the egg plasma membrane and/or to an effect on the actual membrane fusion event. In these studies we used ionomycin to initiate the AR and assayed binding in a Ca2+‐free medium and fusion in Ca2+‐containing medium in the same experiment. Eggs were loaded with the fluorescent dye Hoechst 33342, and the appearance of fluorescence in a sperm head indicated that fusion had occurred. The three MEP inhibitors reduced binding only slightly but inhibited the actual fusion step by 50–60% (determined with an equation that corrected for any inhibition of fusion due to inhibition of binding). MEP inhibitors present only during gamete interactions had little or no effect on fusion. We also found that phosphoramidon‐inhibitable MEP activity was released during the ionomycin‐initiated AR. Incubation of AR supernatant containing MEP activity with previously acrosome‐reacted, phosphoramidon‐treated sperm resulted in a large reversal of the phosphoramidon‐inhibitory effect on sperm‐egg fusion. These results support the hypothesis that the acrosomal phosphoramidon‐inhibitable MEP released during the AR acts directly or indirectly during that event to increase the fusibility of the sperm plasma membrane region required for subsequent sperm‐egg fusion.
Insulin‐like growth factor‐1 stimulates growth of mouse preimplantation embryos in vitro
Authors: Harvey Mark B. , Kaye Peter L. 
Published: February 2005

AbstractBecause recent studies have particularly implicated the insulin growth factor family in early development, the effects of insulin‐like growth factor (IGF‐1) on the development of mouse embryos in vitro were investigated in detail. When added to the medium for culture of two‐cell embryos, IGF‐1 stimulated the number of cells in the resultant blastocysts after 54 hr, entirely by increasing the number of cells in the inner cell mass (ICM) (16.0 ± 0.5 vs. 12.6 ± 0.5 cells/ICM). This stimulation was also achieved when ICMs were isolated from blastocysts prior to culture for 24 hr with IGF‐1 (22.3 ± 1.0 vs. 17.5 ± 0.8 cells/ICM). There was no effect of IGF‐1 on trophectoderm (TE) cell proliferation. In morphology studies, IGF‐1 also increased the proportion of blastocysts (62% ± 3% vs. 49% ± 4%) while decreasing the number of embryos remaining as morulae (32% ± 3% vs. 38% ± 2%) or in the early cleavage stages (7% ± 3% vs. 13% ± 3%) after 54 hr culture from the two‐cell stage. All these effects were achieved with EC50s of approximately 60 pM IGF‐1, which is in the range for IGF‐1 receptor mediation; however, cross reaction with insulin, IGF‐2, or other unknown receptors is not excluded. Nonetheless, the results show that physiological concentrations of IGF‐1 (17–170 pM, 0.1‐1 ng/ml), which have been observed in the reproductive tract, affect the early embryo, suggesting a normal role for this factor in the regulation of growth of the developing conceptus before implantation.
Structural aspects of bovine oocyte maturation in vitro
Authors: de Loos F. , van Beneden T. , Kruip T. A. M. , van Maurik P. 
Published: February 2005

AbstractBovine cumulus‐oocyte complexes (COCS) were collected from 4–8 mm follicles and graded inot four categories on their morphological characteristics. These four categories were matured in vitro and processed for transmission electron microscopy at 24 h after the onset of culture. The morphology of the four groups of oocytes was analysed and compared with that of occytes that had matured in vivi and were collected 20–23 h after the preovulatory luteinizing hormone peak. After in vivo maturation, oocytes formed a homogeneous group with respect to their morphologicalo characteristics. After in vitro muturation, the oocytes formed a heterogeneous group with respect to their morphology between as well as within the four categories of oocytes. Oocytes from the first three categories showed the same morphology after in vitro maturation. The fourth category showed some specific characteristics: (1) vacuolization, (2) flattening of cumulus cells, and (3) almost complete lack of cortical granules in some category 4 oocytes. These characteristics are interpreted as signs of degeneration. Besides these aspects of degeneration, other deviations from normal development were seen: (1) retraction of cumulus cell process endings from the oocyte without the breaking down of these processes, (2) retardation of some aspects of the cytoplasmic maturation, and (3) incomplete cumulus expansion. It is concluded that oocytes capable of development in vitro show a large morphological variability before the onset of culture. In vitro maturation systems can support normal development, but many oocytes show signs of degeneration and deviant development after in vitro maturation.
Requirement for glucose during in vitro culture of sheep preimplantation embryos
Authors: Thompson J. G. , Simpson A. C. , Pugh P. A. , Tervit H. R. 
Published: February 2005

AbstractGlucose utilization by sheep embryos was examined in 8‐cell (N = 36) and blastocyst (N = 36) stages, by measuring conversion of [5−3H]glucose to 3H2O. Fifty percent glucose utilization occurred at 0.79 ± 0.69 mM for 8‐cell embryos and ∼0.06 ± 0.15 mM for blastocysts. Development of 1‐ and 2‐cell sheep embryos (N = 264) was examined under different glucose concentrations (0, 1.5, 3, or 6 mM) and in the presence or absence of 0.33 mM pyruvate and 3.3 mM lactate (PL). Overall, the presence of glucose was detrimental (P < 0.001) to embryonic development. By contrast, the presence of pyruvate and lactate was beneficial (P<0.001) to development. An interaction was observed between the concentration of glucose and presence or absence of PL (P <0.05). An optimum level of glucose occurs at 0–3 mM in the presence of PL (P ±0.1). Development to the blastocyst stage was observed in medium when supplemented with amino acids and albumin alone. Thus, glucose metabolism is not critical for embryonic development, but beneficial at low concentrations. High concentrations can inhibit development, possibly by inhibiting the tricarboxylic acid (TCA) cycle. Sheep embryos may also be using amino acids as an energy source for development.
Cleavage rate of haploid and diploid parthenogenetic mouse embryos during the preimplantation period
Authors: Henery C. C. , Kaufman M. H. 
Published: February 2005

AbstractThe lack of a paternal genome in parthenogenetic embryos clearly limits their postimplantation development, but apparently not their preimplantation development, since morphologically normal blastocysts can be formed. The cleavage rate of these embryos during the preimplantation period gives a better indication of the influence of their genetic constitution than blastocyst formation. Conflicting results from previous studies prompted us to use a more suitable method of following the development of haploid and diploid parthenogenetic embryos during this period.Two classes of parthenogenetic embryos were analysed following the activation of oocytes in vitro with 7% ethanol: (1) single pronuclear (haploid) embryos and (2) two pronuclear (diploid) embryos. Each group was then transferred separately during the afternoon to the oviducts of recipients on the 1st day of pseudopregnancy. Control (diploid) 1‐cell fertilised embryos were isolated in the morning of finding a vaginal plug, and transferred to pseudopregnant recipients at approximately the same time of the day as the parthenogenones. Embryos were isolated at various times after the HCG injection to induce ovulation, from each of the three groups studied. Total cell counts were made of each embryo, and the log mean values were plotted against time. The gradient of the lines indicated that (1) the cell doubling time of the diploid parthenogenones was 12.25 ± 0.34 h, and was not significantly different from the value obtained for the control group (12.74 ± 1.17 h), and that (2) the cell doubling time of the haploid parthenogenones (15.25 ± 0.99 h) was slower than that of the diploid parthenogenones and the control diploid group. It appears from these findings that the ability of diploid parthenogenetic embryos to divide normally during the preimplantation period does not require the presence of a paternal genome. Diploid parthenogenones would, therefore, appear to be at a developmental advantage over haploid parthenogenones at the time of implantation, as their cell number is greater, and it is likely that this is related to the total number of cells that contribute to their inner cell mass.
Hemizona assay for measuring zona binding in the lowland gorilla
Authors: Lanzendorf S. E. , Holmgren W. J. , Johnson D. E. , Scobey M. J. , Jeyendran R. S. 
Published: February 2005

AbstractThe hemizona assay is a diagnostic test used to evaluate the binding potential of spermatozoa to zonae pellucida and has been used to predict fertilization potential in the human. In this study, frozen‐thawed gorilla spermatozoa were coincubated with human hemizonae to evaluate tight binding and to assess the use of human zonae in evaluating sperm fertility. Matching hemizonae were incubated with human sperm to serve as a control. For evaluation of binding studies in a homologous system, matching halves of gorilla hemizonae were coincubated with both gorilla and human sperm. Whole, intact zonae of both human and gorilla oocytes were also coincubated with heterologous sperm to determine of penetration into the perivitelline space could occur. This study found that gorilla sperm bound well to both gorilla and human hemizonae, with a mean of 112.5 and 81.0 tightly bound sperm, respectively. Human sperm also bound to gorilla (mean 229.5) and human (mean 236.5) hemizonae. Following incubation with intact gorilla zonae, motile human sperm were found within the perivitelline space. However, gorilla sperm were not visible within the perivitelline space of nonviable human oocytes. These findings demonstrate that the zonae of nonviable human oocytes can be used to assess sperm binding of gorilla sperm. Studies continue for optimizing assay condition and correlation of findings with the fertility potential of gorilla sperm.
Inhibitory effect of purines in meiotic maturation of denuded mouse oocytes
Authors: Shim Chanseob , Lee Dae Kee , Lee Chung Choo , Cho Wan Kyoo , Kim Kyungjin 
Published: February 2005

AbstractThe potential action of purines, such as hypoxanthine and adenosine, in meiotic arrest was examined using denuded mouse oocytes. The spontaneous meiotic maturation of denuded oocytes was significantly inhibited by hypoxanthine and/or adenosine in a dose‐dependent manner. Germinal vesicle breakdown (GVBD) was inhibited even at a low concentration (1 nM) of hypoxanthine, when hypoxanthine was microinjected into the cytoplasm of denuded oocytes. This inhibitory action was potentiated by co‐injection with allopurinol, a metabolic blocker of hypoxanthine that can block a metabolic pathway to uric acid. By contrast, a microinjection of adenosine was no longer effective in inhibiting GVBD. Inhibitory action of purines in meiotic maturation was correlated with sustaining intracellular cAMP levels. GVBD was resumed by econazole, one of the nitroimidazole derivatives which act as inhibitors of catalytic subunit of adenylate cyclase. This compound was effective in counteracting the effect of adenosine, but not the action of 3‐isobutyl‐1‐methylxanthine (IBMX) on GVBD, indicating that adenosine is probably exerted at the level of oocyte plasmalemma. These data suggest that the inhibitory action of hypoxanthine and adenosine in oocyte meiotic maturation may be involved in the regulation of cAMP metabolism in a differential manner.