bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of motile spermatozoa by density gradient centrifugation in Percoll®
Authors: Lessley Bruce A. , Garner Duane L. 
Published: February 2005

AbstractA procedure using centrifugation in density gradients composed of Percoll was developed for isolation of spermatozoa from mammalian semen. To evaluate the technique, rabbit, human, or bovine semen was layered over continuous Percoll gradients ranging in density from 1.02 to 1.13 gm/ml and centrifuged at 1,500g for 45 min. After centrifugation, the seminal plasma remained above the gradient, whereas the spermatozoa and seminal particles were distributed within the gradient according to their buoyant densities. Unlike most washing techniques, no sperm pellet was formed; instead, the spermatozoa were concentrated into a compact band above the most dense layer of Percoll. The spermatozoa recovered from the gradient were easily resuspended by gentle techniques. Thus, the mechanical stress to the spermatozoa was minimized. Osmotic stress to the spermatozoa was also negligible as the Percoll gradients were isotonic throughout. Spermatozoa obtained by this technique possessed motility equivalent to that of spermatozoa in the unfractionated semen. Sperm suspensions recovered from the gradients contained less than 5% of the nonspermatozoal particles present in the original samples of unfractionated semen. Soluble seminal components were also efficiently removed from the spermatozoa. Thirty billion bovine spermatozoa could be fractionated on a single gradient without loss of effectiveness. Recovery of spermatozoa from these preparative separations averaged 80%. These results demonstrated that Percoll was a superior medium for efficient density gradient isolation of motile spermatozoa free of contamination by other seminal constituents.
Adenylate cyclase activity in porcine sperm in response to female reproductive tract secretions
Authors: Berger T. , Clegg E. D. 
Published: February 2005

AbstractAdenylate cyclase activities were studied in porcine sperm in the presence and absence of Mn++ before and after incubation in vivo and in vitro. Incubation of sperm in vivo for 30 min increased the Mg++‐stimulated adenylate cyclase activity from 35.1 pmoles cyclic AMP formed per mg protein per 10 min to 50.4 pmoles. The activity stimulated by Mg++ and Mn++ increased from 392 to 729 pmoles after 30 min of in vivo incubation. Activity after incubation in vivo for 120 min was not different from activity after 30 min. In vitro incubation of porcine sperm in Ca++‐free Ringer‐fructose resulted in no change, but incubation in oviductal and uterine flushings obtained from gilts soon after ovulation increased Mg++‐stimulated activity by 24% and Mg++−+ Mn++‐stimulated activity by 49%. In vitro incubations in preovulatory flushings plus follicular fluid or in bovine serum albumin also increased adenylate cyclase activity.
Effect of Na+‐free seawater on energy metabolism in sea urchin spermatozoa with special reference to coenzyme A and carnitine derivatives
Authors: Mita Masatoshi , Yasumasu Ikuo 
Published: February 2005

AbstractWhen the dry sperm of sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial seawater, they became motile and the level of ATP decreased. However, after dilution in Na+‐free seawater, the sperm were still immotile and the ATP level remained constant. CoA, carnitine, and their derivatives as intermediates of lipid metabolism were also measured in sperm. The levels of CoA derivatives were much higher than those of carnitine derivatives. The free and acid‐soluble CoA level decreased and the long‐chain acyl‐CoA level increased after dilution in artificial seawater, However, they did not change after dilution in Na+‐free seawater. The levels of carnitine derivatives hardly changed after the dilution in both sorts of artificial seawater, The respiratory rate was extremely low in Na+‐free seawater, Furthermore, concentrations of pyruvate, lactate, citrate, and malate remained essentially constant after dilution in the Na+‐free seawater, whereas they changed markedly after dilution in the artificial seawater.
Nucleoprotein changes during male pronuclear development as determined by the ammoniacal silver reaction
Authors: Longo Frank J. 
Published: February 2005

AbstractSperm nucleoprotein changes during male pronuclear development in fertilized sea urchin (Arbacia punctulata) eggs have been examined utilizing the ammoniacal silver reaction (ASR) at the light and electronic microscopic levels of observation. Previous studies and control preparations indicated that the ASR has an affinity for basic proteins, staining intensely those rich in arginine residues. Differences in the affinity of the paternally derived chromatin to the ASR prior to, during, and following pronuclear development were observed. Relative to the female pronucleus the unincorporated sperm nucleus was densely stained. Upon its entry into the egg the sperm nucleus showed a two‐fold increase in staining, indicating an augmentation in the availability of reactive sites already present in the paternally derived chromatin or an accumulation of “new” reactive sites from the egg cytoplasm. With the dispersion of the sperm nucleus there was a progressive decrease in staining intensity of the paternally derived chromatin. Subsequent to pronuclear fusion the paternally derived chromatin, recognized by its relatively dense staining, was seen at one pole of the zygote nucleus. With time there was a gradual regression in the size and staining intensity of the paternally derived chromatin within the zygote nucleus. Changes in reactivity of sperm‐derived chromatin are discussed in reference to previous studies of chromatin transitions at fertilization.
Lysophosphatidylserine inhibits completion of meiotic maturation of rat oocytes in vitro
Authors: Fleming Alan D. , Armstrong David T. 
Published: February 2005

AbstractFollicular oocytes collected prior to the expected time of the LH surge from PMSG‐treated immature rats were incubated cummulus‐intact (with or without LH) or cumulus‐free (CF). Oocytes were incubated in the presence or absence of lysophosphatidlylserine (LS), a naturally occurring membrane phospholipid that has been previously shown to block sperm‐related membrane fusion events. Fusion events occurring during oocyte maturation that might be affected by LS include maintenance of the intact germinal vesicle (GVI) and prevention of GV breakdown (GVBD) and first polar body formation (PBI). LS had only a slight effect upon GVI. The incidence of GVI was significantly increased in only one of the three oocyte culture conditions employed (CF). Exposure to LS from the outset of collection and washing did not increase the incidence of GVI, indicating the lack of effect by LS was not owing to the passage of a sensitive period during oocyte collection. In contrast, LS was not owing to the passage of a sensitive period during oocyte colection. In contrast, LS almost completely abolished PBI in all oocyte culture conditions at 100 μ in PBI and those sperm‐related fusion processes previously found to be sensitive to LS. Finally, LS or similar agents may be responsible for the block to maturation (often at anaphase I) and even the retarded cleavage observed in vitro during oocyte maturation or embryo culture in some species.
Sperm‐aggregating factor from Spisula oocytes
Authors: Sato Eimei , Segal Sheldon J. , Koide S. S. 
Published: February 2005

AbstractA membrane protein possessing sperm‐aggregating activity was partially purified from Spisula oocyest. Spisula oocytes were incubated with three different media: A) 1 M urea, 5 mM EDTA, 10 mM Tris‐HCI, pH 7.4, B) 1 M urea, 10 mM Tris‐HCI, pH 7.4, and C) 5 mM EDTA in artificial sea water. Oocytes incubated in media A or B at 22°C were viable up to 15 min of treatment based on the trypan blue exclusion test. After this treatment period, oocyte viability gradually decreased as demonstrated by a progressive increase in the uptake of the dye. However, oocytes excluded the dye when incubated in medium C for 2 hr or longer. Oocytes incubated in medium A or B did not undergo germinal vesicle breakdown (GVBD) on exposure to sperm, while GVBD was induced on treatment with 70 mM KCI, suggesting removal or alteration of sperm receptors by the treatment.When sperm were incubated with oocyte extract prepared by treatment with medium A or B, they aggregated and formed clusters. The clusters remained unchanged for at least 1 hr at 22–24°C and sperm within the aggreates were motile. Extracts of Spisula oocytes showed species specificity by not agglutinating sperm of Arbacia punctulata, Asterias forbesi, ovalipes ocellatus, or Chaetopterus peramentaceus. The factor was puridied by ammonium sulfate fractionation (30% saturation) and by gel filtration on a Sephadex G 100 column. Four major protein peaks were eluted. Fraction comprising the second and third peaks possessed sperm‐aggregating activity at an affective does od 2.5 μg of protein per ml. The factor is a heat‐stable protein with an estimated molecular weight (mol wt) of 15 to 25 kdaltons.
Comparative morphometrics of oligochaete spermatozoa and egg‐acrosome correlation
Authors: Jamieson B. G. M. , Richards K. S. , Fleming T. P. , Erséus C. 
Published: February 2005

AbstractUltrastructural variation between spermatozoa of nine lumbricid species has examined for Prearson r correlations, by principal components and coordinates analysis, and graphically, with particular reference to the acrosmoe. Results are compared with a survey of acrosome in seven other oligochaete families. Acrosome lengths correlates highly with length of the primary acrosome vesicle (PAV) though absolute lengths of the two in an acrosome may differ greatly. High correlation ofacrosome lenghts with thickness of the zone pellucida of the oocyte in lumbricida and other families is consistent with the hypothesis that intertaxon variation in acrosome length in an adaptation to the thickness of the zona. This variation is brought about by variation in the length of the acrosome tube, which is a unique structure of the Clitellasta. Correlation with zona thickness is considered premarily to be that of the acrosome (and tube) length and not of the PVA. Other sperm components do not show high correlation with zona depth, except an enigmatic correlation re of axial rod width. Lumbricid arcosome lenght shows a moderate positive allometry relative to zona depth. The short acrosome of Tubificidae and Enchytraeidae (microdriles) correlate with short oocyte microvilli and narrow zonae. Acrosome lenght‐zona correlation is not a secondary expression of body size (which varies indiscriminately in lumbricids) or egg size; microdriles have larger eggs than megadriles. Mwgadrile features of the acrosome of the tubificidan Phreodrilus, including greater length, are attributed to postulated internal fertilization. It is suggested that the oliochatee acrosome penetrates the zone between, and possibly manipulated by, its microvilli before the acrosome reaction (which is briefly charaterized electron microscopically) occurs or progresses far.
Calmodulin intracelluar concentration and cAMP‐dependent protein kinase activity in human sperm samples in relation to sperm motphlogy and motlity
Authors: Pariset Claude C. , Roussel Claude , Weinman Serge J. , Demaille Jacques G. 
Published: February 2005

AbstractCalmodulin concentration and cAMP‐dependent protein kinase activity have been determined in human sperm samples. No significant differences have been noticed in the motility index of two groups of sperm samples differing in their intracellular concentration of calmodulin. It was however found that a low intracellular clamodulin concentration is frequently associated with particular anomalies of the head and tail region. In contrast, a positive correlation has been demonstrated between the motility index and the whole extractable cAMP‐dependent protein activity of the different sperm samples. In suspension s, When the cAMP‐dependent protein kinase activity was measured on intact sperm suspensions, a positive correlation between the activity and the motility index was also found.
Inhibition of fertilization in the rabbit by anti‐acrosin antibodies
Authors: Dudkiewicz Alan B. 
Published: February 2005

AbstractRabbit acrosin was purified from detergent extract of epididymal spermatozoa by molecular‐sieve Chormatography (Sephadex G‐75) and Priflavin‐Sepharose affinity chromatography. Affinity Purified rabbit acrosin (Specific activity = 63.7 U Per mg Protein) exhibited one major band (Mr = 36,000) on SDS‐polyacrylamide‐slab gel electrophoresis. Sheep werweantibodies, Preared against acid‐glycerol‐stabilized acrosin, were Fractionated sequentitally with 50 and 33% ammonium sulfate. Anti‐acrosin immunoglobulins were specific for the acrosome as evidenced by indirect fluorescence lebeling, and they inhibited acrosin's proteolytic activity when essayed for their effect on fertilization invivo, antiacrosin‐treated sperm produced the lowest persent fertility (7.1%) followed by preimmune y‐globulins(69%), Krebs‐ringer hosphate‐glucose‐serum(96.4%) Analysis of antibody‐treated sperm revealed no significiant chang in their motility or agglutination patterns. Antiacrosin antibodies, therefore, can effectivily neutrlize acrosin and inhibit fertilization when placed at the site of sperm‐egg interaction in vivo.
The sperm head of the plains mouse, Pseudomys australis: Ultrastructure and effects of chemical treatments
Authors: Flaherty Sean P. , Breed William G. 
Published: February 2005

AbstractThe ultrastructure of the sperm head of the plains mouse, Pseudomys australis, and the effects of chemical treatments on the sperm head components has been investigated to determine the nature of the material in the hooks on the apical margine of the sperm head. Ultrastructural studies indicated that the dorsal hook contained nuclear, subacrosomal, and acrosomal material, whereas the two ventral hooks were largely composed of an extention of the subacrosomal material with two thin acrosomal projections at their base. Acrosomal material was dispersed by mild detergent treatment, where as the bulk of the material in the ventral hooks were generally found to be similar to the subacrosomal material in the dorsal hook in their resistance to the various chemical treatments. Treatment of sperm with NaOH or guanidine‐hydrochloride and DTT revealed two layers of material in the ventral hooks.
Ribosomal preparations may induce maturation in Xenopus laevis oocytes
Authors: Baltus E. , Hanocq‐Quertier J. 
Published: February 2005

AbstractXenopus laevis oocytes undergo maturation when they are injected with large quantities of crude ribosomes from various origins: X laevis full‐grown or matured oocytes, Xenopus ovaries and embryos, Xenopus liver or mouse liver. All have the same efficiency, whatever their origin: they include 50‐90% maturation in the injected oocytes at about the same speed as progesterone treatment. The ribosomal preparations are inactive wen injected into recipient oocytes pretreated with cholera toxin or cycloheximide. After dissociation with the high salt extract, but not with the subunits. Hypotheses concernning the mode action of this ribosomal extract are disussed.
Biochemical studies of mammalian oogenesis: Synthesis of 5S and 4S RNA during growth of the mouse oocyte
Authors: Boreen Stuart M. , Gizang Elena , Schultz Richard M. 
Published: February 2005

AbstractThe relative proportion of synthesis of 5S RNA to 5S plus 4S RNA is constant during growth of the mouse oocyte. In conjunction with results of a previous study [Brower et al, 1981], the synthesis of 28S, 18S, 5S, and 4S RNA is coordinate during mouse oocyte growth.
Relationship between the length of sperm preincubation and zona penetration in the golden hamster: A scanning electron microscopy study
Authors: Barros C. , Jedlicki A. , Bize I. , Aguirre E. 
Published: February 2005

AbstractHamster spermatozoa are able to fertilize a high percentage of zona‐intact hamster oocytes when they are preincubated for 2 hr in a chemically defined medium. From this time on, the longer the preincubation time the lower the percentage penetration. Spermatozoa preincubated for 6 or more hr are unable to cross the zona pellucida, retaining however their ability to fuse with zona‐free hamster oocytes.Zona‐intact hamster oocytes, as described above, were observed with the scanning electron microscope. When the oocytes were inseminated with spermatozoa preincubated for 1 to 5 hr the outer surface of the zona showed the penetrating spermatozoa and the sperm tracks made by those that failed to cross it. With longer preincubation times no penetrating spermatozoa were observed, and very few sperm tracks were present on the outer surface of the zona.Control experiments showed that neither eggs, spermatozoa, nor fertilization were affected by the medium recovered after long preincubations.These results show that care should be taken regarding the preincubation time when using the in‐vitro fertilization technique.
Ultrastructural studies of the porcine zona pellucida during the solubilization process by Li‐3,5‐diiodosalicylate
Authors: Dietl J. , Czuppon A. B. 
Published: February 2005

AbstractIsolated porcine zonae pellucidae were investigated using transmission electron microscopy. It was found that the fine structure of the zona is not homogenous. The region near the oocyte consists of a more tightly packed micellar structure than the structure near the external surface. However, no clear boundary between the two structural features could be detected. The assumption that the molecular structure of the external and internal surface of the zona is not the same is substantiated by the finding that antibodies against the whole zona structure react apparently only with the more loosely bound structure on the external surface. This effect is attributed to differences in the spacial arrangements of the micellar structures rather than to differences in chemical composition. Solubilization of the zona results in a reorientation of the otherwise randomly interconnected structure. At lower Li‐3,5‐diiodosalicylate concentration (0.05 M) the fibrils seems to expand so that the individual fibers lie almost parallel to each other. At a higher Li‐3,5‐diiodosalicylate concentration (0.2 M) the individual micelles begin to break up from the zona surface, while at a still higher concentration (0.3 M) the rigid structure of the zona is completely solubilized. In the latter case no residual material could be detected in the sediment following high speed centrifugation of the mixture. These electron microscopic results correlated with the protein concentrations in the supernatant indicating that the maximal protein content (35 ng/zona) is obtained at 0.3 M or higher Li‐3,5‐diiodosalicylate concentrations.
The onset and maintenance of hyperactivated motility of spermatozoa from the mouse
Authors: Cooper T. G. 
Published: February 2005

AbstractEstimates were made of the proportion of freely motile mouse spermatozoa displaying hyperactivated motility by an objective photographic method employing stroboscopic illumination under dark‐field conditions and examining displacements of the sperm head and bend angles of the sperm tail. In media known to support in vitro fertilisation hyperactivation gradually appeared reaching about 40% by 6 hr incubation, and it was not promoted by 2 mM caffeine or 0.1 mM Bt2 cAMP or washing the cells free of epididymal fluid. Raising the osmolarity of the medium to 400 mOSM with electrolytes, but not nonelectrolytes, did promote hyperactivation (60% by 2 hr) suggesting that the ionic strength of the medium was important. Hyperactivation in high ionic strength media could be prevented by removing or chelating Ca2+, or replacing Ca2+ with Ba2+ or Mg2+, when nonhyperactivated motility was maintained, but Sr2+, like Ca2+, permitted hyperactivated motility. Hyperactivation in low ionic strength medium could be promoted by the ionophore A23187, suggesting that Ca2+ movement into the cells is important. Of a range of glycolytic substrates tested supporting nonhyperactivated motility in the presence of lactate, only glucose supported hyperactivation. Addition to glucose— or Ca2+ — free, high ionic strength media after 2 hr increased hyperactivation immediately (glucose) or after a lag of 2 hr (Ca2+) suggesting that glucose acts on a Ca2+ — primed system. Removal from high ionic strength medium, chelation of Ca2+ or inhibition of glucose metabolism did not prevent hyperactivation continuing once it had been initiated, indicating different requirements for initiation and maintenance of this form of motility.
Culture of mouse preimplantation embryos as a quality control assay for human in vitro fertilization
Authors: Ackerman Steven B. , Swanson R. James , Stokes Gordon K. , Veeck Lucinda L. 
Published: February 2005

AbstractTechniques for culturing preimplantation mouse embryos from the two‐cell stage to blastocyst are described, and the importance of this system to quality control assay the media and supplements used in human in vitro fertilization (IVF) procedures is discussed. Embryos from B6CBAF1mice were cultured in a commonly used mouse culture medium, modified Krebs‐Ringer‐bicarbonate (Krebs'), or in a commonly used human culture medium, Ham's F10 nutrient mixture supplemented with human fetal cord serum (FCS), and results were not significantly different. Using the mouse embryo culture system, tests on 174 preparations of FCS resulted in 24.1% producing less than 75% morula or blastocyst stages after 72 h in culture, compared to 9.5% of the Krebs' control cultures. Results of the mouse embryo culture system using 98 FCS subsequently used in human IVF were compared to results from the VIP Human In Vitro Program of Eastern Virginia Medical School of Norfolk, Virginia, from June 1982 through June 1983. The data suggest that prescreening of media and supplements using this mouse embryo culture system may indicate sources of factors potentially detrimental to the success of human IVF procedures.
A thin‐layer isoelectric focusing method for the separation of proteins in follicular fluid and seminal plasma
Authors: Nayudu P. L. , Gook D. , Lopata A. , Cornell F. N. , McLachlan R. 
Published: February 2005

AbstractA thin‐layer electrofocusing method has been developed for the separation of proteins in human follicular fluid, seminal plasma, and blood plasma/serum. The method has a number of unique features. It is simple and rapid, and it produces distinct, undistorted bands that can be readily quantitated. Any samples solubilized by standard methods can be analyzed, and it can be adapted to favor specific regions of the pH spectrum. The gels used are composed of agarose and sorbitol, and they are formed between a plastic template and a Mylar film. Sample volumes ranging from 0.5 to 5 μl can be used. Protein separation is usually completed in 30–40 min at constant power of 1 or 1.5 W. The use of low power ensures that little heat is generated during the run and hence only minimal temperature control is needed. Carbon rod electrodes are a special feature as they allow the inverted gel to rest in direct contact with the electrodes, eliminating the necessity for electrode solutions. The inverted gel position allows fluid to drain from the surface of the gel and so prevents distortion of protein patterns. This overcomes the problems inherent in conventional horizontal electrofocusing of unpurified biologic fluids such as plasma, follicular fluid, and seminal plasma. Rapid protein staining is carried out with fixed and dried gels. The excellent resolution and the lack of distortion make this method suitable for reliable quantitative analysis of differences between samples by means of laser beam densitometry. This has enabled us to assess differences in specific follicular fluid proteins between individual follicles in the human, and to correlate these differences with fertilizability and cleavage potential of oocytes derived from such follicles.