bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of epiblast‐specific cDNA clones by differential hybridization with polymerase chain reaction‐amplified probes derived from single embryos
Authors: Varmuza Susannah , Tate Peri 
Published: February 2005

AbstractA mouse day 7.5 embryonic ectoderm cDNA library containing 2 × 106 clones was screened by differential hybridization with polymerase chain reaction (PCR)‐amplified probes derived from a single embryo. Day 7.5 ectoplacental cone and embryonic ectoderm served as the source of mRNA to make minus and plus probes, respectively. In a limited screen of fewer than 2,000 clones, 23 up‐regulated clones were identified by the difference in hybridization singal with the two probes. DNA sequence analysis revealed the nature of some, but not all, of the clones. Northern blot and in situ hybridization with a subset of the clones confirmed the utility of the approach, since the differential signal was also observed in these experiments. This approach may prove useful for identifying genes that play a role during development.
Prefertilization treatment of mice with platelet activating factor affects pregnancy
Authors: Podsiadly B. T. , Adamson L. M. , Stanger J. D. , Smart Y. C. , Roberts T. K. 
Published: February 2005

AbstractEmbryo‐derived platelet activating factor (EPAF) is thought to be either biologically similar to platelet activating factor (PAF) or responsible for PAF liberation in vivo. We have previously shown that premating PAF treatment in the mouse renders the platelets nonresponsive to EPAF, leading to a reduced implantation rate in these animals. In this study, we have shown that females, injected with PAF before mating, show altered embryo development in vivo on day 4 postfertilization. This is manifested as an interruption of compaction, a reduced cell number per embryo, and reduced embryo number per mouse. Results suggest that EPAF represents an early pregnancy signal that supports embryo development. The most likely mechanism is via platelet activation, since only those mice that showed thrombocytopenia after fertilization were found to have normal embryos on day 4 postmating.
Purification and characterization of vitellogenin and lipovitellins of the sand crab Emerita asiatica: Molecular aspects of crab yolk proteins
Authors: Tirumalai R. , Subramoniam T. 
Published: February 2005

AbstractIn the mole crab Emerita asiatica, the main yolk proteins consist of two slow moving lipovitellins (Lv I and Lv II) of glycolipoprotein nature. Lv I cleaves into subunits (MW: 109,000 and 105,000) and Lv II gives rise to six subunits (MW: 65,000, 54,000, 50,000, 47,000, 44,000, and 42,000) in SDS‐PAGE (with β‐mercaptoethanol). In order to observe the stability of Lv II as well as to achieve better resolution of the proteins, two different buffer systems (Phosphate buffered saline and tris‐buffered saline), 40% sucrose, and glass distilled water were used as homogenizing media. Among them, better resolution was achieved with tris‐buffered saline and 40% sucrose, and tris‐buffered saline seems to be the ideal medium for elution of Lv II. The analysis of biochemical constituents of the major Lv II reveals a percentage composition of 69.325, 27.927, and 2.753 respectively for protein, lipid, and bound sugars. In the I stage embryo, protein comprises about 67.276%, lipid 29.65%, and bound sugars 3.015%. Vitellogenin (Vg) electrophoretically corresponding to the Lv I and Lv II was present in the female haernolymph during the entire period of embryogenesis. The number of subunits(8) of Vg in all stages remained unaltered and their approximate molecular weights were Vg1, 91,000; Vg2, 87,000; Vg3, 83,000; Vg4, 61,000; Vg5, 58,000; Vg6, 45,000; Vg7, 42,000; and Vg8, 38,000. Different proteins present in the embryos (I and IV stage) and the serum obtained from the animal carrying the I stage embryo were separated by gel‐filtration in high performance liquid chromatography (HPLC). Sephadex (G‐200) gel filtration chromatography was used to purify the Lv II in large quantity.Total lipid extracted from Lv II as well as the embryos belonging to different stages of development were separated into their constituent neutral, glycolipids, and phospholipids, using silicic acid column chromatography. Thin layer chromatography (TLC) was used to isolate the different phospholipids purified from various stages of embryos and Lv II. As many as seven different phospholipids were separated from Lv II and I and IX stage embryos; and whereas thin layer chromatogram of V and VI stage embryos showed six different phospholipids, embryos of VII and VIII stage contained four phospholipid species. Cholesterol, glycolipids, and individual phospholipids isolated from the Lv II and I stage embryo were quantified spectrophotometrically and the results were discussed. © 1992 Wiley‐Liss, Inc.
Recovery of penetration ability in protease‐treated zona‐free mouse eggs occurs coincident with recovery of a cell surface 94 kD protein
Authors: Kellom Theresa , Vick Angela , Boldt Jeffrey 
Published: February 2005

AbstractPrevious studies have demonstrated that protease treatment of zona‐free mouse eggs impairs sperm‐egg interaction (Boldt et al.: Biol Reprod 39:19–27. 1988) and causes modification of a 94 kD egg plasma membrane protein (Boldt et al., Gamete Res 23:91–101, 1989), In this report, the ability of eggs to recover penetration ability following protease treatment was examined. Zona‐free mouse eggs were isolated and treated with either trypsin or chymotrypsin (1 mg/ml, 20 min), then cultured for 0, 3, or 6 hr before insemination. Eggs cultured for 3 or 6 hr displayed significantly higher penetration levels than eggs inseminated immediately after protease treatment, indicating a recovery of penetration ability during the 3 or 6 hr incubation period. The recovery of penetration ability was not blocked by inclusion of cyclohexamide (50 μg/ml) during the 3 or 6 hr culture period, indicating that protein synthesis was not required for recovery of fusion ability. Cell surface radiolabeling studies with 125I revealed that a 94 kD cell surface protein was lost immediately following trypsin or chymotrypsin treatment but was found on the egg surface after the 3 or 6 hr recovery period. Recovery of the 94 kD egg surface protein occurred in the presence of cyclohexamide, and metabolic radiolabeling studies with 35S‐methionine confirmed that synthesis of a 94 kD protein was blocked by cyclohexamide. These results suggest that the recovery of penetration ability after protease treatment of zona‐free eggs is due to recovery of the 94 kD cell surface protein, providing further evidence for the involvement of the 94 kD protein in sperm‐egg interaction. © 1992 Wiley‐Liss, Inc.
Subzonal insemination of a single mouse spermatozoon with a personal computer‐controlled micromanipulation system
Authors: Kobayashi Kazuhiko , Kato Keisuke , Saga Masahiko , Yamane Masami , Rothman Cappy M. , Ogawa Shyoso 
Published: February 2005

AbstractA personal computer‐controlled micromanipulation system was developed for automatic injection of spermatozoa into the perivitelline space of mouse ova. A pair of three‐dimensional hydraulic micromanipulators driven by pulse motors was used for this automatic system. The pulse signals that regulate the motors are initiated by the computer program, and these signals cause the micromanipulator to move the microtool precisely. The computer program was designed to perform the most effective movements of the sperm injection needle used during manual micromanipulation. Prior to the manipulation, the computer locates the tip of the injection needle and the end of the egg‐holding pipette in the microscope field using image processing. The trajectory of the injection needle is determined according to these initial positions. Using this robotic system, subzonal insemination with a single mouse spermatozoon was attempted in a total of 143 ova. The sperm insertion was successfully completed in all cases without damaging any of the ova. Spermatozoa treated with ionophore A23187 and those without the treatment were used. The fertilization rate (68.8%) of the ova inseminated with treated sperm was significantly higher than that (37.5%) obtained with the nontreated sperm (P < 0.05). These findings suggest the feasibility and potential for further applications of a robotic microinsemination system and, in addition, that a higher fertility rate in the subzonal insemination of mouse ova can be achieved with the ionophore treatment of spermatozoa. © 1992 Wiley‐Liss, Inc.
Characterization of low Mr Zona Pellucida binding proteins from boar spermatozoa and seminal plasma
Authors: Parry R. V. , Barker P. J. , Jones R. 
Published: February 2005

AbstractA group of low Mr (16 kDa ‐ 23 kDa) glycoproteins on ejaculated boar spermatozoa have been shown to have high affinity for homologous zona pellucida glycoproteins (ZPGPs). These ZPGP binding proteins are derived from seminal plasma as shown by their absence from epididymal spermatozoa and their presence in seminal plasma as identified by N‐terminal amino acid sequence analysis. They bind to ZPGPs by a polysulphate recognition mechanism similar to that found for proacrosin‐ZPGP interactions. The haemagglutination activity of boar seminal plasma is also associated with these low Mr glycoproteins. It is suggested that they play a role in regulating the rate of sperm capacitation and survival in the female reproductive tract. © 1992 Wiley‐Liss, Inc.
Developmental regulation ofan snRNP core protein epitope during pig embryogenesis and after nuclear transfer for cloning
Authors: Prather Randall S. , Rickords Lee F. 
Published: February 2005

AbstractThe appearance and stabilization of a core protein epitope of the snRNP is developmentally regulated during pig embryogenesis. The epitope recognized by the monoclonal antibody Y12 is present in the germinal vesicle of mature ooocytes and interphase nuclei of late 4‐cell stage (24 to 30 hours post cleavage to the 4‐cell stage) to blastocyst stage embryos. There was no antibody iocalization within pronuclei, or nuclei of 2‐cell or early 4‐cell stage embryos. Zygotes or 2‐cell stage embryos cultured in the presence of α‐amanitin to the late 4‐cell stage showed no immunoreactivity, whereas control embryos had immunoreactivity. Thus antibody localization was correlated with RNA synthesis and RNA processing that begins by 24 hours post cleavage to the 4‐cell stage. A final experiment showed no detectable immunoreactivity in 16‐cell stage nuclei that had been transferred to enucleated activated meiotic metaphase II oocytes. Since immunoreactivity is associated with active RNA synthesis and RNA processing, it suggests that the 16‐cell stage nucleus, which is RNA synthetically active, does not process RNA after nuclear transfer to an enucleated activated meiotic metaphase II oocyte. © 1992 Wiley‐Liss, Inc.
Purification and partial characterization of the 17 kDa sperm coating protein from boar seminal plasma
Authors: Moos JiřRí , Veselskýa Leopold , Pěknicov´ Jana , Drahoréd Josef 
Published: February 2005

AbstractSperm coating proteins of 16, 17, and 19 kDa have been purified from boar seminal plasma. The 17 kDa protein has been identified as an antigen recognized by monoclonal antibody ACR.3 and is thus identical to low molecular mass zona pellucida binding protein from boar spermatozoa (Moos et al., 1990). The 17 and 19 kDa proteins are glycosylated and tend to form hetero‐complexes. The 17 kDa ACR.3 antigen is sequentially released from the sperm cell surface during capacitation and, after induction of the acrosome reaction, the 16 kDa form was also observed. Immunocytochemical studies on boar reproductive tissues have suggested that the seminal vesicle epithelium may be the source of these proteins. © 1992 Wiley‐Liss, Inc.
Increasing carbon dioxide from five percent to ten percent improves rabbit blastocyst development from cultured zygotes
Authors: Hallden Kristine , Li Jianming , Carney Edward W. , Foote Robert H. 
Published: February 2005

AbstractOne‐cell rabbit zygotes were cultured at 39°C in basal synthetic medium II (BSM‐II) with 5%, 10%, or 15% CO2 and humidified air to determine the effect of CO2 concentration on development in vitro. After 4 days in culture, 37% of the embryos grown in 10% or 15% CO2 had reached the hatching blastocyst stage, but only 10% of the embryos were hatching when cultured under 5% CO2 (P = 0.01). Over all blastocysts, cell numbers were 207, 246, and 205 for the 5%, 10%, and 15% CO2 treatments, respectiveiy. In a second experiment to determine if there was a beneficial effect, particularly at the blastocyst stage, of a higher concentration of CO2, embryos were cultured 4 days in either 5% or 10% CO2 or for 2 days in 5% CO2 followed by 2 days in 10% CO2. The numbers of blastomeres per embryo and embryo diameter were greater (P > 0.05) in embryos cultured continuously in 10% CO2 or in 10% CO2 only during days 3 and 4 of culture than in embryos cultured continuously in 5% CO2. In a third experiment, one‐cell rabbit zygotes were cultured with 5% or 10% CO2 in a defined, protein‐free medium consisting of 1:1 RPMI 1640 and Dulbecco's modified Eagle's medium. The proportion of embryos hatching and cell counts were significantly greater (P > 0.01) when cultured in the presence of 10% CO2. These data indicate that a 10% CO2 atmosphere exerts a beneficial effect on the development of zygotes into expanding and hatching rabbit blastocysts in vitro. © 1992 Wiley‐Liss, Inc.
Adsorption of oviductal fluid proteins by the bovine sperm membrane during in vitro capacitation
Authors: McNutt Tamara , Rogowski Lee , Vasilatos‐Younken Regina , Killian Gary 
Published: February 2005

Abstract125I‐labeled oviductal fluid (ODF) proteins and antiserum to ODF were used to determine whether ODF proteins associate with the sperm membrane during in vitro capacitation. Luteal and nonluteal pools of ODF were obtained from oviduct catheters during the estrous cycle. Washed sperm (50 × 106 sperm/ml) were incubated up to 4 h in a protein‐free modified Tyrode's medium (MTM), or MTM supplemented with 40% ODF, or 0.5 ng 125I‐labeled ODF proteins. Solubilized sperm membrane proteins and incubatin media containing ODF proteins were separated by gel electrophoresis. Membranes isolated from bovine sperm previously incubated with ODF, absorbed five 125I‐proteins: A doublet at 85–95 kDa, and others at 24, 34, 53, and 66 kDa. The amount of 66 kDA 125I‐protein associated with the sperm decreased during the incubation, whereas the amount of 85 to 95‐kDa protein did not. Western blot analyses also detected the presence of ODF proteins (53, 66, 85–95, and 116 kDa) in solubilized membranes from sperm incubated in ODF. The 85 to 95‐kDa protein in ODF decreased in ODF. The 85 to 95kDa protein in ODF decreased in apparent molecular weight by 5 kDa when associated with the sperm membrane. At 53 kDa ODF proteins which associated with sperm were transformed from two to three separate proteins. These studies indicate that the surface of sperm is modified by adsorption of ODF proteins ot the membrane during in vitro capacitation. (c) 1992 Wiley‐Liss, Inc.
Automated analysis of rabbit sperm motility and the effect of chemicals on sperm motion parameters
Authors: Young J. , Bodt B. A. , Iturralde T. G. , Starke W. C. 
Published: February 2005

AbstractAppropriate software settings and optimum procedures were determined for the measurement of the motion parameters of rabbit spermatozoa by the CellSoft (Cryo Resources Ltd., Montgomery, NY) computer‐assisted digital image analysis system. The system was used to follow motion parameter changes occurring in spermatozoa incubated for 6 hr with or without exposure to chemicals. Mean amplitude of lateral head displacement (AALH) increased over the 6 hr period, while curvilinear velocity (Vc) first increased and then decreased. Values for linearity (Lin), or beat cross frequency (BCF), were unchanged. The majority of spermatozoa progressed linearly, with rapid rotation of the sperm head, but subpopulations of spermatozoa with different swimming patterns appeared after 1–3 hr of incubation. Percentage motile sperm and Vc were most sensitive to the action of the compounds (pyrogallol, hydroquinone, ammonium oxalate, triethyl phosphite, and pinacolyl alcohol), while BCF was least affected. The decline in percentage of motile sperm was dependent on duration of exposure and chemical concentration. Mean Vc of the sperm population decreased rapidly upon chemical exposure and remained at a low value until motility ceased. The initial decrease in Vc was dependent on the concentration of the added compound. Motion‐based indices—motility concentration (MCl50), motility time (MTl50), and velocity (VI)—were defined and used as toxicological endpoints. The rank order of these indices, the end point of the neutral red in vitro assay for cytotoxicity, and LD50 values for the five compounds were the same, suggesting that chemical inhibition of sperm motility may be useful as a method for the in vitro assessment of chemical cytotoxicity. © 1992 Wiley‐Liss, Inc.
Rat sertoli and spermatogenic cells express a similar gene, and its product is antigenically related to an outer dense fiber‐associated protein
Authors: Smith Frank F. , Mertz James R. , Krebs Ingeborg , Tres Laura L. , Chae Chi‐Bom , Zakeri Zahra , Engelhardt Jeff , Hoover Dennis , Tenniswood Martin , Kierszenbaum Abraham L. 
Published: February 2005

AbstractWe have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfidelinked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5‐min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis. © 1992 Wiley‐Liss, Inc.
Developmental expression of the S35‐S45/SGP‐2/TRPM‐2 gene in rat testis and epididymis
Authors: Zakeri Zahra , Curto Marcello , Hoover Dennis , Wightman Karen , Engelhardt Jeff , Smith Frank F. , Kierszenbaum Abraham L. , Gleeson Timothy , Tenniswood Martin 
Published: February 2005

AbstractTestosterone‐repressed prostate message‐2 (TRPM‐2) was originally isolated and cloned from the regressing ventral prostate of the rat. In this tissue, and in other hormone‐dependent tissues such as the mammary gland, this gene is induced in the absence of the appropriate trophic hormone. Sequence analysis of the cDNA and genomic clones of TRPM‐2 have demonstrated that the coding sequence of this gene is identical to S35‐S45 (also known as SGP‐2 and clusterin), which is constitutively expressed by the Sertoli cells of the adult testis. Using Northern, slot blot, S1‐nuclease analysis, and in situ hybridization, we have investigated the regulation of TRPM‐2 expression in the testis and epididymis during development. Slot blot analysis of RNA extracted from the testis and epididymis of 7‐, 14‐, 28‐, 35‐, and 91‐day‐old rats demonstrates that the gene is induced to detectable levels between days 7 and 14 and that the relative level of expression does not change significantly after day 14. In situ hybridization using frozen sections of testis from day 2‐, 7‐, 14‐, 28‐, 35‐, and 91‐day‐old rats confirms that there is little expression of TPRM‐2 in the seminiferous epithelium of 7‐day‐old rats, but this increases considerably after 14 days, primarily in Sertoli cells but also in association with meiotic developing spermatogenic cells. However, TRPM‐2 mRNA is expressed in the rete testis at 2 days of age, reaches a peak at 35 days of age, and continues to be expressed in the adult. Slot blot analysis demonstrates that TRPM‐2 is also induced in the epididymis between 7 and 14 days of age, although, as has been demonstrated by in situ hybridization, TRPM‐2 mRNA is detectable in the epithelial cells in the head of the epididymis but is barely detectable in the midportion or tail regions. Northern analysis suggests that the size of the TRPM‐2 transcript in the testis also changes during development. In the early stages of testicular development, the TRPM‐2 transcript appears to be a broad band of approximately 1.5 kb, while the transcript in the adult appears to be approximately 1.8 kb in length. S1‐nuclease protection assays suggest that this increase in size is not due to differential splicing of the first exon of TRPM‐2/SGP‐2 and most probably reflects a difference in the polyadenylation of the mRNA in the testis at different times during development. © 1992 Wiley‐Liss, Inc.
Monoclonal antibody AG7 inhibits fertilization post sperm‐zona binding
Authors: Brucker Cosima , Sandow Bruce A. , Blackmore Peter F. , Lipford Grayson B. , Hodgen Gary D. 
Published: February 2005

AbstractMonoclonal antibodies (mAbs) against sperm cells are currently being used in an effort to define spermatozoal antigens involved in the fertilization process. We have produced a number of anti‐human sperm mAbs by immunization of female mice with the 100,000 × g supernatant of octylglycoside‐solubilized washed human sperm. From a panel of mAbs, 1 antibody, AG7, was selected and characterized due to its fertilization‐inhibiting characteristics. MAb AG7 defines a sperm acrosome antigen‐1 (SAA‐1) located in the acrosomal region of human sperm as evaluated by indirect immunofluorescence. Staining of life sperm cells indicated that the antigen is present on the sperm surface. SAA‐1 was also found on sperm of several other mammalian species, implying evolutionary conservation of the antigen. SAA‐1 was first observed on testicular sperm and can be followed through epididymal transit, ejaculation, and capacitation. When applied in a mouse in vitro fertilization assay, mAb AG7 inhibits fertilization by greater than 95%, and inhibition is dose dependent, with half‐maximal inhibition at 0.8 μg/ml. The block to fertilization could not be attributed to sperm agglutination, inhibition of motility, interference with adhesion to the zona pellucida, or inhibition of fusion with the oocyte membrane. MAb AG7 was demonstrated to inhibit calcium influx in spermatozoa in vitro (measured using the fluorescent indicator fura 2), a prerequisite for the acrosome reaction. Initial biochemical characterization of the antigen suggests it is proteinlike in nature, with a molecular weight of approximately 220 kD. The results suggest that SAA‐1, identified by mAb AG7, is a sperm antigen crucially involved in the fertilization process, possibly an atypical steriod receptor or ion channel located within the sperm plasma membrane. © 1992 Wiley‐Liss, Inc.