bluuebunny/crossref_metadata_2025_split huggingface dataset:
Association of newly synthesized 3H‐arginine‐labeled proteins with chromatin structures in fertilization
Authors: Kopečný Václav , Pavlok Antonín 
Published: February 2005

AbstractZona‐free hamster eggs have been fertilized in vitro with boar spermatozoa in a medium enriched by arginine‐3H and the sites of localization of newly synthesized arginine‐3H–labeled proteins have been investigated using fine‐structure autoradiography. It was confirmed that such proteins are synthesized during fertilization and that they accumulate to a notable degree in decondensing sperm chromatin as well as in the chromatin of the female pronucleus and also of the second polar body. A similar process did evidently take place also in defective pronuclei, characterized by a core of a still condensed chromatin and by remaining nuclear membrane. In such male pronuclei the highest concentration of the label was seen just on the border of the condensed chromatin, on the expected site of nuclear protein exchange. It is supposed that, at least in this experimental system, any morphologically detectable sperm decondensation is accompanied immediately by a shift from sperm basic nuclear proteins to other nuclear proteins.
Ultrastructural localization of proacrosin and acrosin in ram spermatozoa
Authors: Huneau D. , Harrison R. A. P. , Fléchon J.‐E. 
Published: February 2005

AbstractProacrosin and acrosin were localized immunocytochemically at the electron microscope level in ram spermatozoa undergoing an ionophore‐induced acrosome reaction. Antigenicity was preserved after fixation with 0.5% w/v ethyl‐(dimethylaminopropyl)‐carbodimide, and an antibody preparation was used that reacted with all major forms of ram acrosin. All stages of the acrosome reaction could be observed in a single preparation. At the earliest stage, labeling was observed throughout the acrosomal contents, which were just beginning to disperse. As dispersal proceeded, labeling diminished, being associated only with visible remnants of the acrosomal matrix. By the time the acrosome had emptied, almost no labeling could be detected on the inner acrosomal membrane.The relationship between matrix dispersal and proacrosin activation was studied in isolated ram sperm heads. While proacrosin was prevented from activating, the acrosomal matrix remained compact; but as activation proceeded, the matrix decondensed and dispersed in close parallel. By the time proacrosin activation was complete, the acrosomal contents had almost entirely disappeared.We conclude that proacrosin is distributed throughout the acrosomal contents as an intrinsic constituent of the acrosomal matrix. During the acrosome reaction, proacrosin activation occurs, resulting directly in decondensation of the matrix. All the contents of the acrosome including acrosin disperse and, by the time the acrosome is empty and the acrosomal cap is lost, only occasional traces of acrosin remain on the inner acrosomal membrane. Since the acrosomal cap is normally lost during the earliest stages of zona penetration, acrosin's role in fertilization is unclear: it does not appear to be a zona lysin bound to the inner acrosomal membrane.
The oocyte‐cumulus complex: Ultrastructure of the extracellular components in hamsters and mice
Authors: Talbot P. , Dicarlantonio G. 
Published: February 2005

AbstractTo enhance preservation of the extracellular materials, we have fixed hamster and mouse oocyte cumulus complexes (OCC) for transmission electron microscopy in the presence of ruthenium red. Ruthenium red had four effects on the extracellular components of the freshly ovulated hamster OCC. It interacted with the surface of cumulus and corona radiata cells; it stabilized the extracellular matrix (ECM) that was comprised of granules and filaments; it produced moderate electron density and good structural definition in the zona pellucida, and it revealed occasional smalls granular depsits on the oolemma. The ECM observed between cells of the cumulus and corona radiata layers extended into the outer one third of the zona pellucida. The granule and filament matrix was removed from the cumulus layer, corona radiata, and pores of the zona pellucida by brief treatment with hyaluronidase. The extracellular components of oviducal OCC from hamsters and mice appeared similar to OCC removed from follicles of the hamster shortly before ovulation. However, oviducal OCC did show increased aggregation of granules in the ECM. In most cases where females had been mated and oocytes were fertilized, the extracellular components appeared similar to those seen in fresh OCC. Exceptions were noted in some oocytes that lacked cumulus and corona radiata cells. In these instances, the zona pellucida generally lacked the granule/filament matrix. After fertilization numerous small electrondense granules were noted in the perivitelline space. These were presumed to originate in the cortical granules and formed a new investing layer around the zygote. Our data suggest that the OCC becomes more difficult for a sperm to penetrate as it approaches the oocyte. The significance of these results is discussed with respect to sperm traffic in the OCC and the cortical reaction.
Comparative cytochemistry of the developing ovarian follicles of the dog, rabbit, and mouse: Origin of the zona pellucida
Authors: Tesoriero John V. 
Published: February 2005

AbstractThe reactivity of the glycoproteins of ovarian follicles of the dog, rabbit, and mouse were compared, using the Alcian blue (pH 0.5) and Alcian yellow (pH 2.5) technique at the light‐microscope level and the periodic acid‐chromic acid‐silver methenamine technique at the electron‐microscope level.In paraffin sections, the rabbit and mouse show the appearance of both zona pellucida and follicular fluid in the earliest growing follicles. In the dog, there is a sequence of development with the follicular fluid appearing late, after much of the zona has been secreted. The zona and follicular fluid are highly sulfated in all animals. Zonae pellucidae of atresia appear to lose all traces of sulfation and become highly acidic. At the electronmicroscope level, oocytes contain little if any reactive glycoprotein material which can be related to zona pellucida formation. The initial appearance of the zona material occurs between follicle cell membranes extending outwards and away from the oocyte. Follicle cells of all species consistently contain a variety of reactive Golgi bodies and granules, with exocytotic vesicles, suggesting active synthesis and secretion of zona material.Our observations suggest that in the early stages of oogenesis, the follicular epithelium is responsible for at least part of the synthesis of the zona pellucida. It is possible therefore that both the oocyte and its follicle cells participate, probably on the sequential basis, in the synthesis of the mammalian zona pellucida.
Intra‐acrosomally located acrosin‐inhibitors: Evolution and developmental patterns in mammals
Authors: Flörke‐Gerloff S. , Tschesche H. , Müller‐Esterl W. , Engel W. 
Published: February 2005

AbstractThe acrosome of the mammalian spermatozoon contains proacrosin that is autocatalytically activated to become the proteolytic enzyme acrosin during the process of fertilization. Tschesche et al [1982] isolated specific acrosin inhibitors and suggested that they block prematurely activated acrosin. Antibodies against acrosin inhibitors purified from boar spermatozoa were used to demonstrate the evolutionaary relationship and the developmental pattern of the inhibitors in mammals. Using immunofluorescent techniques the following results were obtained: (1) The spermatozoa of man, boar, bull, ram, rat, rabbit, beaver, and mole stained positive in the acrosomal portion. (2) The round‐headed spermatozoa of patients with globozoospermia and those in the ejaculates of fertile men lacked immunostaining for the inhibitors. (3) During spermatogenesis in all species, immunofluorescence for the acrosin inhibitors was first demonstrable in haploid spermatids and increased thereafter during spermatid differentiation. The stained area was found adjacent to the nuclear membrane, the position of the acrosome. (4) During teratogenesis of round‐headed spermatozoa, the immunofluorescent staining for the inhibitors becomes separated from the nuclear membrane of the spermatids and is lost in late spermatids. Since identical results have been described for acrosin and acrosomal membrane proteins both in spermatozoa and spermatids of mammalian species and during spermiogenesis of patients with globozoospermia, our results are consistent with the localisation of the inhibitors in the acrosome. Immunostaining of spermatozoa of species belonging to five different mammalian orders with the antibody against boar acrosin‐inhibitors is indicative of an evolutionary conservation of the inhibitors and their underlying genes.
Effects of caffeine, Ca++, capacitation time, and strain on in vitro fertilization in mice
Authors: Dodds J. W. , Seidel G. E. 
Published: February 2005

AbstractOocytes (N=2922) were collected from superovulated female C57B16/J X DBA2/J (B6D2F1) mice and distributed among 48 treatments consisting of a 2×3×2×2×2 factorial design. The factors were strain of spermatozoa, B6D2F1 or SJL/J; caffeine concentration in the fertilization medium, 0,2, or 6 mM; time oocytes were exposed to sperm, 1 or 2 hours; Ca++ concentration in the capacitation medium, 0 or 1.8 mM; and capacitation time, 1 or 2 hr. Ova were observed 400 min after they were initially exposed to 105 spermatozoa per ml. Ova with two or more pronuclei and a second polar body were considered fertilized, In vitro embryonic development was monitored for 5 days.B6D2F1 spermatozoa resulted in consistently higher rates of fertilization than SJL spermatozoa, 77.5% vs 38.7% when averaged over other treatments. Caffeine concentrations of 0,2, and 6 mM resulted in respective mean fertilization rates of 50.1%, 58.8%, and 65.4% (P<0.005) when averaged over other factors. Fertilization rates of ova exposed 1 and 2 hr to sperm were 53.0% and 63.3% (P<0.005). B6D2F1 spermatozoa capacitated in medium with 1.8 mM Ca++ fertilized more ova (P<0.01), 83.1%, than when no Ca++ was present, 71.9%; this effect was absent with SJL spermatozoa. The effect of capacitation time depended on strain. Fertilization rates with B6D2F1 spermatozoa were higher, 80.1%, with a 2‐hour capacitation time than with a 1‐hour capacitation time, 75.0%. Exactly the opposite was true for the SJL spermatozoa; 43.4% for the 1‐hour and 34.1% for 2‐hour capacitation (P<.01). Development to the blastocyst stage was significantly greater (P<0.025) for ova fertilized by B6D2F1 (26.8%) than by SJL spermatozoa (17.7%).
Enhancement of the acrosome reaction of hamster spermatozoa by the proteolytic enzymes, kallikrein, trypsin, and chymotrypsin
Authors: Shinohara Harumichi , Yanagimachi Ryuzo , Srivastava Prakash N. 
Published: February 2005

AbstractThe involvement of a kallikrein−kinin system in the motility of mammalian spermatozoa has been suggested by several investigators. We found that incorporation of kallikrein (0.1–1.0) unit/ml) in the sperm incubation medium did not enhance the motility of hamster spermatozoa that were already active. However, this enzyme significantly increased the incidence of the acrosome reaction. Trypsin (1.8–18 units/ml) and chymotrypsin (0.34–3.4 units/ml) also increased the incidence of the acrosome reaction, and accelerated its onset. Kinins (bradykinin and kallidin) added to the medium in a wide concentration range (1 ng/ml to 1 mg/ml) had no marked effects on either the motility or the acrosome reaction. A kallikrein−kinin system is apparently not of primary importance at least for the acrosome reaction. The enhancement of the acrosome reaction by exogenous proteinases may be due in part to accelerated removal or alteration of the sperm surface coat (glycoprotein) by the enzyme peior to the acrosome reaction. Exogenous proteinases may also act synergistically with endogenous (acrosomal) proteinases (and other enzymes) in altering membrane proteins and dispersing the acrosome matrix during the course of teh acrosome reaction.
Induction of polyspermic fertilization in sea urchins by the leukotriene antagonist FPL−55712 and the 5−lipoxygenase inhibitor BW755C
Authors: Schuel Herbert , Moss Roberta , Schuel Regina 
Published: February 2005

AbstractArachidonic acid can be oxidized via the cyclooxygenase pathway to produce prostaglandins and via the 5−lipoxygenase pathway to produce leukotrienes. These substances are known to be extremely potent regulators of cellular function in somatic tissues, particularly during inflammatory reactions. Recent studies have implicated cyclooxygenase−derived products in preventing polyspermy in sea urchins [Schuel et al, Gamete Res 10:9–19, 1984]. FPL−55712, a receptor antagonist for leukotrienes in somatic tissues, causes a dose−(1–10 μM) and sperm−density−dependent induction of polyspermic fertilization in sea urchins if added before the egg completes the cortical reaction (elevation of the fertilization envelope). Eggs pretreated with FPL−55712 become polyspermic upon subsequent insemination with untreated sperm in sea water. Sperm pretreated with the drug do not cause polyspermy when used to inseminate untreated eggs. The 5−lipoxygenase inhibitor BW775C also promotes polyspermy. FPL−55712 and BW755C do not retard elevation of the fertilization envelope. These findings imply that (1) leukotrienes may be produced via the 5−lipoxygenase pathway during fertilization in sea urchins, and (2) the reaction of leukotrienes with putative receptors on the egg's surface may modulate its receptivity to sperm during the cortical reaction, and thereby help prevent polyspermy.
Electron microscopy of mouse preimplantation embryos and isolated blastomeres immobilized on coverslips
Authors: Wiley Lynn M. , Takaki Kathy K. , Yamagata Maya 
Published: February 2005

AbstractIn this report we describe a new method of immobilizing intact mouse preimplantation embryos and their dissociated blastomeres on a flat substrate so that they may be processed en masse for electron microscopy. The embryos can be arranged so that they will be coplanar and grouped together closely enough to be included within the same plane of section. The substrate consists of Thermonex plastic coverslips that have been coated with a layer of fibroblasts or with fibronectin. The plant lectin phytohemaggiutinin (PHA) is used as a ligand to adhere the embryos onto the coverslips. This method is compatible with other ligands and with a variety of immunocytochemical procedures and can be used with live or fixed embryos.
Partial purification and characterization of an alkaline proteinase from the rabbit testis
Authors: Ninjoor Vyas , Srivastava Prakash N. 
Published: February 2005

AbstractA proteolytic enzyme capable of cleaving intact proteins and synthetic substrates α−N−benzoyl−DL−arginine β−naphthylamide (Bz‐Arg‐NNap), α‐N‐benzoyl‐L‐arginine p‐nitroanilde (Bz‐Arg‐NPhNO2), and α‐N‐benzoyl‐L‐arginine ethyl ester (Bz‐Arg‐OEt) was purified 92– fold from the rabbit testes. The enzyme exhibited optimal activity at pH 9.0 and 50°C. The polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis of the purified enzyme demonstrated multiple forms; the major band in the SDS‐polyacrylamide gel electrophoresis corresponded to a Mt 48,000. The same value was established by the gel filtration over Sephadex G‐75. The rabbit testicular alkaline proteinase (TAP) resembled acrosin in the hydrolysis of Bz‐Arg‐OEt. However, CaCl2, a potential stimulator of acrosin activity, inhibited the alkaline proteinase. The strong inhibitors of acrosin, eg pheny methyl sulphonyl fluoride (PMSF), tosyl lysine chloromethyl ketone (TLCK), and benzamidine did not inhibit the alkaline proteinase. TAP was activated by an acrosin inhibitor isolated from the rabbit testes. Since 0.5 M KCl was necessary for complete extraction of the enzyme and the bulk of the activity was present in 9,000g pellet of the testicular homogenate. The alkaline proteinase appeared to be associated with the membranous structures.
A follicular fluid component prevents gonadotropin reversal of cyclic adenosine monophosphate‐dependent meiotic arrest in murine oocytes
Authors: Downs Stephen M. , Eppig John J. 
Published: February 2005

AbstractThe effects of the putative maturation inhibitor in porcine follicular fluid on gonadotropinstimulated reversal of cyclic adenosine monophosphate (cAMP)‐maintained meiotic arrest in mouse oocytes in vitro were assessed in this study. When cumulus cell‐enclosed oocytes were cultured in a suboptimal inhibitory concentration of dibutyryl cAMP (dbcAMP), the effect of follicle‐stimulating hormone (FSH) on oocyte maturation was initially inhibitory at 3 hr, but stimulatory at 6 hr. Supplementation of the medium with an ultrafiltrate of porcine follicuiar fluid (PM10‐filtrate) completely suppressed FSH‐promoted reversal of inhibition at 6 hr. Charcoal extraction eliminated this effect of the PM10‐filtrate. FSH reversed the inhibition of maturation of cumulus cell‐enclosed oocytes maintained by a high concentration of dbcAMP and suboptimal concentrations of the phosphodiesterase inhibitor, 3‐isobutyl‐1‐methyl xanthine (IBMX), during a 21–22‐hr culture period. However, the effect of a completely inhibitory concentration of IBMX was not reversed by gonadotropin. A component of serum was also found to inhibit FSH reversal of dbcAMP‐maintained meiotic arrest, and this activity was removed by charcoal extraction. In addition, when oocytes were cultured in medium containing a suboptimal concentration of dbcAMP plus a low molecular weight fraction (< 1,000) of porcine follicular fluid, porcine serum, or fetal bovine serum, a synergistic inhibition of maturation was observed. Experiments with highly purified gonadotropins revealed that reversal of dbcAMP‐maintained meiotic arrest occurred only in response to FSH; neither highly purified luteinizing hormone nor human chorionic gonadotropin could mimic this action of FSH. Also, this effect was mediated by the cumulus cells, since FSH could not reverse dbcAMP‐maintained meiotic arrest in denuded oocytes. Furthermore, elevating cAMP levels in denuded oocytes augmented, rather than reversed, the inhibitory action of dbcAMP on oocyte maturation. These data therefore suggest that dbcAMP‐ or IBMX‐maintained meiotic arrest in vitro is reversed by an FSH‐stimulated, cAMP‐dependent process mediated by the cumulus cells and demonstrate that a factor present both in follicular fluid and serum prevents this action of the gonadotropin.
Characterization of a spectrophotometric technique for the estimation of fowl and turkey sperm motility
Authors: Wishart Graham J , Ross Frances H. 
Published: February 2005

AbstractAn objective method of estimating the motility of fowl and turkey spermatozoa, depending on their rheotactic and light‐scattering properties, has been developed. From a 1‐ to 2‐minute recorder trace of the optical density of diluted semen before and after stopping its passage through a flow cuvette, three independent constants may be simply and graphically determined. These are: ODm, the maximum optical density of semen flowing through the cuvette, shown to be dependent on the concentration of spermatozoa; % (ΔOD)m, the maximal change of optical density following cessation of flow, which has been correlated with the percentage of motile spermatozoa in the sample; and t½, the time taken for the change of optical density to reach % (ΔOD)m. This latter parameter has been correlated with forward motility of both fowl and turkey spermatozoa.
Transglutaminase inhibitors, calmodulin antagonists, and calcium channel blockers: Influence on Arbacia sperm motility
Authors: Cariello Lucio , Nelson Leonard 
Published: February 2005

AbstractSea urchin spermatozoa react to transglutaminase inhibitors, which may antagonize the function of calmodulin, in dose‐dependent fashion. The optimum concentration of diallyl‐amino propionyl benzothiophenc (DAPBT), a potent, noncompetitive inhibitor of transglutaminase, 0.01 mM, causes a 3 1/2‐fold increase in the forward swimming speed of Arbacia sperm. This effect apparently involves calcium‐dependent enzymes since manipulation of both extracellular and intracellular calcium by means of chelating agents, calcium‐channel blockers, and calmodulin antagonists depresses the stimulatory effect of DAPBT. These results suggest that in the spermatozoa the interaction of flagellar sliding filaments may be mediated by reversible cross‐linking of the contractile proteins catalyzed by a calcium‐ dependent transglutaminase.
Expression of glucose‐phosphate isomerase in relation to growth of the mouse oocyte in vivo and in vitro
Authors: Buehr Mia , McLaren Anne 
Published: February 2005

AbstractThe activity of the enzyme glucose‐phosphate isomerase (GPI‐1) in mouse oocytes is subject to regulation by the cis‐acting gene Gpi‐lta. Electrophoretic analysis of oocytes from 9‐ and 10‐day‐old mice showed that oocyte‐specific regulation of GPI‐1 is not observed in germ cells that have not started to grow (20 μm diameter) but appears as soon as oocyte growth begins (30 μm or larger). Three in vitro culture systems were used to examine the relation of GPI‐1 expression to oocyte growth: culture of intact neonatal ovaries, and co‐culture of dissociated oocytes and somatic cells from neonatal and from 13‐day foetal ovaries. In all three systems modification of GPI‐1 expression always occurred when oocyte growth began, showing that the presence of a normal follicle is not necessary for the expression of the gene Gpi‐lta.
Distribution of calmodulin and calmodulin‐binding proteins in membranes from bovine epididymal spermatozoa
Authors: Noland Thomas D. , Van Eldik Linda J. , Garbers David L. , Burgess Wilson H. 
Published: February 2005

AbstractReproducible concentrations of calmodulin representing approximately 0.1% of the membrane protein were detected in purified plasma membranes from bovine epididymal spermatozoa. When membranes were isolated in the presence of 1 mM EGTA, the amount of calmodulin associated with the plasma membranes was not reduced. Calmodulin‐binding proteins were detected in both purified plasma membranes and in a mixed membrane fraction containing both plasma membranes and cytoplasmic droplet membranes. A calcium‐dependent, calmodulin‐binding protein of apparent molecular weight 123,000 was detected in both fractions. In the presence of 1 mM EDTA, putative calcium‐independent calmodulin‐binding proteins of apparent molecular weights 93,000, 32,000, 18,000, and 15,000 were detected in the plasma membrane fraction. The 15,000 Mr polypeptide was also present in the mixed membrane fraction but the three proteins of higher molecular weight were reduced or absent in this fraction.
Role of the vitellus in the block to polyspermy in golden hamster eggs
Authors: Menezes J. , Peter J. 
Published: February 2005

AbstractThe block to polyspermy in golden hamster eggs is believed to operate only at the zona pellucida. However, changes in the egg vitellus also prevent further entry of capacitated sperm. When zona‐free hamster eggs spontaneously activated in vitro, and in vivo fertilized eggs at pronuclear stage were inseminated with capacitated human sperm, penetration did not occur. In the case of a homologous system using hamster sperm and in vivo fertilized hamster eggs, slight attachment of sperm was observed but no penetration. The cortical granules were found to be released in spontaneously activated and in fertilized eggs as observed by phase contrast microscopy. These observations suggest that the egg vitellus plays a role in the block to poiyspermy in addition to that of the zona block.