bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of the normal XY male and sex‐reversed XXSxr pseudomale mouse epididymis
Authors: Lebarr Deborah K. , Blecher Stan R. , Moger William H. 
Published: February 2005

AbstractXXSxr pseudomale mice (chromosomally XX animals “sex‐reversed” by the Sxr factor) develop testes and produce sufficient androgens for masculinization as assessed at the macroscopic level. However, adult XXSxr pseudomales lack the epididymal initial segment (I.S.) In this study prenatal and postnatal epididymal development was examined histologically and biochemically, and it was found that XXSxr pseudomales are indistinguishable from normal XY males up to day 21 of postnatal life. By 25 days postnatally, before the onset of the pubertal androgen surge, the I.S. precursor is evident in normal animals but absent in XXSxr mutants. No major abnormalities were seen in other segments of the XXSxr epididymis. Our data suggest that androgen levels in testis and epididymis are not higher in normal XY males than in XXSxr pseudomale mice of the same age. Inadequate availability of androgens at the target site is unlikely to be the cause of the epididymal abnormality in XXSxr pseudomale mice.
Dynamics of paternal chromatin changes in live one‐cell mouse embryo after natural fertilization
Authors: Adenot Pierre G. , Szöllösi Maria S. , Geze Marc , Renard Jean‐Paul , Debey Pascale 
Published: February 2005

AbstractUsing video‐enhanced fluorescence microscopy, we describe in live mouse zygotes the paternal chromatin changes undergone after fertilization. We focus on the sperm recondensation process and the formation of the paternal pronucleus, in relationship with the progression of maternal chromatin. Chromatin is labeled with the vital fluorophore Hoechst 33342. Our conditions of dye concentration and irradiation allow a continuous following of the dynamics of changes without major perturbation. We combine these observations with ultrastructural analysis performed by electron microscopy of the same eggs fixed at chosen stages. We show that the highly recondensed state corresponds to the appearance of the nuclear envelop and therefore the beginning of the pronuclear stage.
The Binding Characteristics of Cholinergic Sites in Rabbit Spermatozoa
Authors: Young R. J. , Laing J. C. 
Published: February 2005

AbstractBinding of neurotrophic ligands to rabbit spermatozoa was studied. Nicotinic cholinergic antagonists, [3H]α‐bungarotoxin and [3H]dihydro‐β‐erythroidine (DE), bound with high affinity to different sites in the tails of rabbit spermatozoa with the former binding to 10,207 sites/cell and the latter to 562 sites/cell. α‐Bungarotoxin and DE sites resemble nicotinic sites in brain in binding affinity and specificity. [3H]Quinuclidinyl benzilate (QNB), a muscarinic cholinergic antagonist, also bound with high affinity to a single class of sites located in the heads and tails of rabbit spermatozoa. The binding characteristics of the sperm muscarinic site are similar to muscarinic sites in both innervated and noninnervated cells. Rabbit spermatozoa incubated for 16–18 h in a medium which supported motility for an extended period possessed fewer binding sites than nonincubated spermatozoa for [3H]α‐bungarotoxin and [3H]QNB and the KD for the latter ligand was also lower. Ligands specific for the kappa and delta opiate receptors showed no affinity for rabbit spermatozoa.
Galactosyltransferase activity is restricted to the plasma membranes of equine and bovine sperm
Authors: Fayrer‐Hosken Richard A. , Caudle A. B. , Shur Barry D. 
Published: February 2005

Abstractβ1, 4‐Galactosyltransferase (GalTase) is localized to the plasma membrane of mouse sperm, in which it mediates the binding of sperm to glycoconjugate residues in the egg zona pellucida. In this study, the presence and subcellular distribution of sperm GalTase were determined in two other mammalian species that yield sufficient sperm for subecellular fractionation. Equine and bovine semen were collected, and the plasma membranes (PM), outer acrosomal membranes (OAM), and inner acrosomal membranes (IAM) were sequentially removed. The purities of the isolated membrane preparations were determined by transmission electron microscopy and found to be ≥90%, 96%, and 98% for equine PM, OAM, and IAM, respectively, and ≥80%, 94%, and 97% for bovine PM, OAM, and IAM, respectively. GalTase activity was assayed under optimal conditions in all membrane preparations and was preferentially localized to the isolated PM both in equine and in bovine spermatozoa. The selective localization of GalTase to the sperm PM in two other species suggest that it may serve as a generalized gamete receptor during initial sperm–egg binding in mammals.
Nuclear transfer in the bovine embryo: A comparison of 5‐day, 6‐day, frozen‐thawed, and nuclear transfer donor embryos
Authors: Westhusin M. E. , Pryor J. H. , Bondioli K. R. 
Published: February 2005

AbstractMicromanipulation and electrofusion were utilized for nuclear transfer in bovine embryos. Embryonic blastomeres from 5‐day (estrus = day 0), 6‐day, frozen‐thawed 5‐day, and first‐generation nuclear transfer embryos (embryos were themselves a product of nuclear transfer with the original donor being a 5‐day embryo) were transferred into bisected bovine oocytes by electrofusion. The percentage of donor cells fusing with the recipient oocytes was compared between different types of donor embryos. The percentage of embryos developing normally into morula or blastocysts following 6 days culture in the sheep oviduct was also recorded and compared between different donor embryo types. No significant differences were found between donor blastomeres for the percent successfully fused to oocytes: 5‐day, 294 of 513 (57.3%); 6‐day, 252 of 405 (62.2%); frozen‐thawed 5‐day, 111 of 144 (77.1%); nuclear transfer, 142 of 223 (63.7%); or the percent developing normally following nuclear transfer: 5‐day, 92 of 444 (20.7%); 6‐day, 84 of 357 (23.5%); frozen‐thawed 5‐day, 32 of 127 (25.2%); nuclear transfer, 31 of 199 (15.6%). These data suggest that a variety of donor embryos can successfully be utilized for bovine embryo cloning. Also, development of blastomeres from frozen‐thawed 5‐day donors and from donors that are themselves the product of nuclear transfer suggest that the production of multiple identical offspring is possible by frozen storage of seed stock and serial recloning.
Two major acrosomal proteins act on different parts of the oocyte vitelline coat in the abalone, Haliotis discus
Authors: Usui Noriko , Haino‐Fukushima Kazu 
Published: February 2005

AbstractTwo proteins of molecular weights 20,000 (20K) and 15,500 (15.5K) are the major soluble substances released from the acrosomal vesicle of the abalone, Haliotis discus, spermatozoon. A crude preparation of them has been shown to possess lytic activity on the oocyte vitelline coat (VC). To elucidate the role(s) of each acrosomal protein (AP) in VC lysis, oocytes were examined after treatment with various AP preparations. The VC, which is about 1 μm thick, is composed of thin outer and inner electron‐dense layers and a thick main layer of a fine filamentous feltwork. When oocytes were treated with a crude preparation containing both APs, the outer layer disappeared and the feltwork of the main layer loosened extensively. A preparation containing predominantly the 20K AP dissolved the outer layer completely and the main layer to some extent, whereas another preparation containing predominantly the 15.5K AP caused loosening of the main layer without alteration of the outer layer, suggesting that the 20K AP acts on the outer layer, whereas the 15.5K AP acts on the main layer. However, when purified, each AP by itself failed to dissolve the VC, although lysis occurred in a 1:1 mixture of these preparations. Moreover, when the oocytes were pretreated with the 20K AP and thoroughly washed, the 15.5K AP alone could induce lysis. These results suggest that the lysis of the outer layer requires both APs but not simulataneously. The 15.5K AP, which is located posteriorly in the acrosomal vesicle, must be released to act on the VC following the action of the 20K AP.
The effects of α‐amanitin and cycloheximide on nuclear progression, protein synthesis, and phosphorylation during bovine oocyte maturation in vitro
Authors: Kastrop P. M. M. , Hulshof S. C. J. , Bevers M. M. , Destrée O. H. J. , Kruip Th. A. M. 
Published: February 2005

AbstractBovine cumulus oocyte complexes were cultured for various periods and either denuded and orcein stained or radiolabeled with 35S‐methionine or 32P‐orthophosphate. Specific inhibitors were added to the culture medium to investigate mRNA and protein synthesis requirements for both nuclear and cytoplasmic changes during maturation in vitro. Inhibition of mRNA synthesis by α‐amanitin during the first 2 h of culture prevented the phosphorylation of some specific proteins preceding GVBD and decreased the occurrence of GVBD from 97% to 27%. In addition, in oocytes that had undergone GVBD, only part of the changes in protein synthesis after GVBD were observed. Addition of α‐amanitin after 3 h of culture had no effect on meiotic maturation. When cumulus oocyte complexes were cultured in the presence of cycloheximide, the phosphorylation of specific proteins was also blocked and only 5% of the oocytes underwent GVBD. Addition of cycloheximide after 4, 6, or 8 h of culture resulted in an increasing percentage of GVBD, but the oocytes became arrested in metaphase I. When cycloheximide was added from 12 h of culture onwards, nuclear progression to metaphase II was increasingly restored.It is concluded that after the onset of culture, both mRNA and protein synthesis are necessary for the phosphorylation of specific proteins and for GVBD. Further‐more, transcription during the first hours of culture is needed for the synthesis of new proteins after GVBD.
Two‐dimensional polyacrylamide gel electrophoresis characterization of apz, a sperm protein involved in zona binding in the pig and evidence for its binding to specific zona glycoproteins
Authors: Peterson R. N. , Campbell P. , Hunt W. P. , Bozzola John J. 
Published: February 2005

AbstractA boar sperm integral plasma membrane protein (APz) involved in the adhesion of uncapacitated and capacitated sperm to the porcine zona pellucida (ZP) has been characterized by two‐dimensional polyacrylamide gel electrophoresis (PAGE) and tested for its ability to bind to various zona glycopeptides. APz shows microheterogeneity and focuses over a wide pH range, with predominant forms focusing above pH 7. The protein, when excised from nonreducing polyacrylamide gels, inhibited sperm‐egg binding and bound heat‐solubilized zonae preventing these zonae from blocking sperm binding to eggs. In an indirect assay, a polyclonal monovalent antibody, which blocks sperm‐egg binding and which is absorbed by APz, was used to determine the ability of zona glycopeptides to prevent the sperm‐egg blocking activity of the antibody from being absorbed by intact sperm. When whole heat‐solubilized ZP was added to sperm at doses that block sperm‐egg binding and the excess ZP was removed, the sperm‐egg blocking activity of the antibody was not absorbed by these sperm, and antibody‐containing supernatants blocked the binding of untreated sperm to eggs as effectively as antibody that was not mixed with fresh sperm. When alpha ZP3 was used in the same manner, sperm‐egg blocking activity again was not absorbed by antibody‐treated cells. Beta ZP3, however, failed to block sperm‐egg binding and failed to absorb the sperm‐egg blocking activity of the antibody. These findings support the argument that the action of APz is physiologically significant and involves specific binding sites on the ZP3 component of the ZP.
Expression of ras‐like proteins in embryonic and adult cells of Xenopus laevis
Authors: Hanocq‐quertier Jacqueline , Hanocq Françoise 
Published: February 2005

AbstractA polyclonal antibody raised against v‐Ha‐ras p21 was purified and its specificity was checked on Ha‐ras transformed cell lines. It was used to immunoprecipitate p21 from different Xenopus laevis cell types: brain cells, blood cells, and embryonic material. By one‐dimensional Western blot analysis, we show that ras p21 is synthesized very early in oogenesis and accumulates throughout vitellogenesis. The ras p21 content, estimated to be 1.1 ng in the full‐grown oocyte, remains constant during oocyte maturation and egg cleavage. Increase in the amount of ras p21 occurs at the beginning of neurulation. Two‐dimensional Western blot patterns reveal the presence of multiple molecular forms of p21 in all Xenopus cell types studied. The numerous resolved polypeptides were ascribed to the expression of at least two different ras genes. Furthermore, specific charge modification of the ras polypeptides are observed in brain, blood, and embryonic cells. During oogenesis and early embryonic development, differences in two‐dimensional patterns mainly concern variations in the relative amounts of the different polypeptides. The results are discussed in relation to the well documented synthesis activities of the growing oocyte and of the early developing embryo.
Involvement of superoxide radicals in the mouse two‐cell block
Authors: Noda Yoichi , Matsumoto Hisashi , Umaoka Yoh , Tatsumi Kenichi , Kishi Junji , Mori Takahide 
Published: February 2005

AbstractThe effect of oxygen toxicity on the development of mammalian embryos was asssessed by the use of superoxide dismutase (SOD), a potent scavenger of superoxide radicals. Mouse pronuclear embryos recovered 17 h after human chorionic gonadotropin (hCG) were cultured in medium BWW at 37°C under an atmosphere of 5% CO2 in air. Culture of mouse pronuclear embryos in the presence of Cu ∣ Zn‐SOD (500 μg/ml) significantly increased the blastulation rate (44.6%) when compared with the control culture system (4.2%). Essentially the same effects were observed in SOD containing either Mn or Fe in the catalytic center. Heat treatment of the SOD preparation, and the addition of anti‐SOD antibodies to the culture medium, significantly reduced the attenuation of the two‐cell block by SOD, indicating that this effect is SOD dependent. SOD activity was detected in rabbit oviduct fluid (3,675 ± 3,084 mlU/mg protein) by electron spin resonance. These results suggest that active oxygen is involved in the two‐cell block phenomenon in mouse embryos exposed to air and that SOD in the oviduct may play an important role in the protection of embryos from superoxide radicals.
Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment
Authors: Modlinski Jacek A. , Smora̧ Zdzisław 
Published: February 2005

AbstractThe development of nuclear‐transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight‐ and 16‐cell stages (oocytes and zygotes) or with pronuclei‐containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight‐cell stages were also transferred into pseudopregnant does. Development of nuclear‐transfer oocytes was distinctly better than that of nuclear‐transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight‐ and 16‐cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four‐cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four‐cell embryos developed from enucleated zygotes receiving eight‐cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16‐cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte‐ and zygote‐derived nuclear‐transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16‐cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight‐cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight‐cell nucleus in the development of reconstituted zygotes.
In vitro and in vivo association of an oviduct estrus‐associated protein with bovine zona pellucida
Authors: Wegner Carole C. , Killian Gary J. 
Published: February 2005

AbstractThe interaction between the bovine egg zona pellucida and a 97 kDa estrus‐associated protein produced by the oviduct was examined in vitro and in vivo. In vitro matured bovine eggs were incubated with oviduct fluid recovered throughout the estrous cycle from separate indwelling cannulae placed in the ampulla and isthmus of the same oviduct. Immunofluorescence techniques and a polyclonal antiserum against the 97 kDa protein were used to localize this protein on washed eggs previously incubated with oviduct fluid. Intensity and distribution of immunofluorescence varied with stage of cycle and to a lesser degree with region of oviduct from which the oviduct fluid was obtained. The most intense fluorescence was observed on the zonae pellucidae of eggs incubated with oviduct fluid pooled from days near estrus and ovulation compared to fluid pooled from luteal stage days. The immunofluorescence of isthmus‐derived oviduct fluid was more intense than was ampulla‐derived oviduct fluid collected near estrus. The zonae pellucidae of 7‐day‐old embryos flushed from the uterus displayed immunofluorescence comparable to that observed on the zonae pellucidae of eggs incubated in vitro with peri‐estrus oviduct fluid. No immunofluorescence was observed associated with the perivitelline space, egg cytoplasm, or blastomeres. The apparent uptake of a 97 kDa estrus‐associated protein by the zonae pellucidae of eggs in vitro and embryos in vivo may indicate that this protein functions in fertilization and/or early embryo development.
Proteasome (multicatalytic proteinase) of sea urchin sperm and its possible participation in the acrosome reaction
Authors: Matsumura Kiyotaka , Aketa Kenji 
Published: February 2005

AbstractThe egg jelly‐induced acrosome reaction of the sea urchin, Strongylocentrotus intermedius, was inhibited by succinyl‐Leu‐Leu‐Val‐Tyr‐4‐methylcoumaryl‐7‐amide (Suc‐Leu‐Leu‐Val‐Tyr‐MCA), but not by Suc‐Ala‐Ala‐Pro‐Phe‐MCA. The proteases with hydrolytic activity toward the former were purified from sperm extract by DEAE‐Sephacel and hydroxylapatite chromatographies, Sephacryl S‐300 gel filtration, and heparin‐Sepharose CL‐6B chromatography. Two types of protease were separated, and the molecular weights were estimated to be 65 and 700 kDa, respectively, by gel filtration. The former was accompanied by hydrolytic activity toward Suc‐Ala‐Ala‐Pro‐Phe‐MCA, which was not hydrolyzed by the latter. Polyacrylamide gel electrophoresis of 700 kDa protease gave a single protein band under nondenaturing conditions and at least eight bands in the range of 22–33 kDa in the presence of sodium dodecyl sulfate (SDS). The substrate specificity and the inhibitor sensitivity of 700 kDa protease indicate that it contains two types of the activity, one is chymotrypsin‐type and the other trypsin‐type. The former activity was enhanced by poly‐L‐lysine or SDS. These properties of 700 kDa protease are similar to those of proteasomes (multicatalytic proteinases) isolated from various eukaryotic sources. We had previously shown that inhibitors of chymotrypsin‐like proteases inhibit the increase of intracellular Ca2+ concentration by egg jelly, resulting in the inhibition of the acrosome reaction of St. intermedius (Matsumura and Aketa, Gamete Res 23:255–266, 1989). Bringing these findings together, we suggest that the chymotrypsin‐like activity of sperm proteasome participates in the onset of the acrosome reaction of St. intermedius.
Maturation of guinea pig sperm in the epididymis involves the modification of proacrosin oligosaccharide side chains
Authors: Anakwe Onyeama O. , Sharma Sunita , Hoff Henry B. , Hardy Daniel M. , Gerton George L. 
Published: February 2005

AbstractProacrosin from guinea pig cauda epididymal sperm has a lower molecular weight compared with the testicular zymogen. In this study, we have examined the structural basis of this change and where the conversion in proacrosin molecular weight occurs during sperm maturation. Immunoblotting of trifluoromethane‐sulfonic acid‐deglycosylated testicular and cauda epididymal sperm extracts with antibody to guinea pig testicular proacrosin demonstrated that the polypeptide backbones of proacrosins from the testis and cauda epididymal sperm had the same molecular weights (∽44,000). Keratanase, an endo‐β‐galactosidase specific for lactosaminoglycans, partially digested testicular proacrosin but had no effect on proacrosin from cauda epididymal sperm. In extracts of testis, caput epididymis, and corpus epididymis analyzed by immunoblotting, anti‐proacrosin recognized a major antigen with an apparent molecular weight (Mr) of 55,000, although a 50,000‐Mr minor antigen began to appear in the corpus epididymis. By contrast, extracts of cauda epididymis, vas deferens, and cauda epididymal sperm had the 50,000 Mr protein as the only immunoreactive antigen. By enzymography following electrophoresis, the major bands of proteolytic activity in extracts of testis, caput epididymis, and corpus epididymis had 55,000 Mr. A band of protease activity with 55,000 Mr also appeared in extracts of the corpus epididymis. However, the most prominent bands of proteolytic activity in cauda epididymis, vas deferens, and cauda epididymal sperm had 50,000 Mr. In addition, two other major protease activities were detected with 32,000 and 34,000 Mr; the relationships of these proteases to proacrosin are unclear. From these results, we conclude that the oligosaccharides of proacrosin are altered during epididymal transit and that this modification occurs in the corpus epididymis. Such structural alterations may be functionally significant, since sperm acquire the ability to fertilize eggs during epididymal maturation.