bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cyclic nucleotide metabolism in mouse epididymal spermatozoa during capacitation in vitro
Authors: Stein D. M. , Fraser L. R. 
Published: February 2005

AbstractThe role of cyclic nucleotides in sperm capacitation is equivocal. Using conditions known to support mouse sperm capacitation after 120 min incubation in vitro, the cAMP and cGMP contents of epididymal spermatozoa were measured and the cGMP/cAMP ratio determined. The initial high cAMP content detected upon release of spermatozoa decreased within 30 min to a lower plateau, which was then maintained throughout incubation. With the cGMP content remaining approximately constant, the cGMP/cAMP ratio increased over 120 min. In the presence of 2 mM caffeine, an increased cAMP content was noted at 0 and 30 min before a fall to the plateau level. To investigate cyclic nucleotide metabolism, adenylate cyclase and phosphodiesterase activities were compared in two sperm populations, one essentially uncapacitated and the other incubated for 120 min. Adenylate cyclase activity, higher in the presence of 2 mM Mn2+ compared to Mg2+, showed increased activity at 120 min compared to 30 min incubation, while phosphodiesterase activity decreased during this period. The ability of spermatozoa to form adenosine and inosine from cAMP indicated endogenous 5′‐nucleotidase and deaminase, as well as phosphodiesterase, activities. Although the endogenous cAMP content appeared to remain constant during the time that acrosome loss, hyperactivated motility and fertilizing ability can be demonstrated, activities of the enzymes responsible for cAMP metabolism indicate an increased potential for cAMP availability and turnover. The increased cGMP/cAMP ratio may also play a role during capacitation.
Morphological and ultrastructural characterization of mammalian spermatozoa processed for flow cytometric DNA analyses
Authors: Garner D. L. , Johnson L. A. , Lake S. , Chaney N. , Stephenson D. , Pinkel D. , Gledhill B. L. 
Published: February 2005

AbstractThe morphological and ultrastructural changes that occur during preparation of porcine, bovine, and murine spermatozoa for flow cytometric quantification of the relative DNA content of the X‐ and Y‐chromosome‐bearing sperm populations were examined. Ejaculated spermatozoa from the boar and bull were washed using a series of dimethyl sulfoxide (DMSO) solutions prior to fixation, whereas the epididymal mouse spermatozoa were washed only in phosphate‐buffered saline (PBS). Spermatozoa from all three species were then fixed in ethanol and processed for fluorochrome staining by a treatment regimen consisting of sulfhydryl reduction and proteolysis. The processed sperm nuclei were stained for DNA with the fluorochrome, 4′‐6‐diamidino‐2‐phenylindole (DAPI) before quantification by flow cytometry. Scanning and transmission electron micrographs of sperm heads taken at various steps of the preparation and staining procedures show 1) that the rigorous washing procedure disrupted the plasma and outer acrosomal membranes, 2) that ethanol fixation resulted in removal of the outer membranes and disintegration of the nuclear envelope, and 3) that thiol and proteolysis treatment removed the remaining cellular organelles including the tail and rapidly induced partial decondensation of the tightly packed chromatin. Sequential micrographs showed that the nuclear matrix of all three species increased in thickness about twofold during the preparation and staining. Consequently, the harsh procedures currently used for quantitative staining of DNA for high‐resolution flow cytometric analyses destroy most cellular organelles and thereby prevent simultaneous characterization of DNA content and other sperm cell constituents.
Fate of argyrophilic proteins during sea‐urchin (Hemicentrotus pulcherrimus) oogenesis detected by NOR‐silver staining
Authors: Amikura Reiko M. , Ihnuma Morio 
Published: February 2005

AbstractNOR‐silver staining technique was applied to paraffin sections of sea urchin ovaries. The fibrillar component and the granular component of the nucleolus were stained dark brown and yellowish tan, respectively. Silver‐stained particles were also observed in germinal vesicles (GV) during oocyte growth. After the breakdown of GV, such particles appeared both in female pronucleus and egg cytoplasm, suggesting dispersion of the stained materials from the GV to the egg cytoplasm in the course of the breakdown of GV. The silver‐stained sites disappeared by the pretreatment with NaOH, trypsin, and pepsin but not with HCl, and RNase, indicating the protein derivation of silver‐stained sites. These results indicate the stained materials are kinds of protein.
Postinsemination changes in the amoeboid sperm of a nematode, nippostrongylus brasiliensis
Authors: Wright E. J. , Sommerville R. I. 
Published: February 2005

AbstractNematode sperm undergo a capacitationlike change called activation, after insemination into the female reproductive tract. In this paper we examine the morphological and functional consequences of activation of the sperm of Nippostrongylus brasiliensis by comparing the ultrastructure of sperm in the reproductive tracts of males and females and by analysing locomotion using time‐lapse cinematography. Sperm in males consist of a long tail, composed of highly condensed chromatin, and a cytoplasmic region containing numbrane‐bound secretory products of the Golgi called membranous organelles (MOs) as well as mitochondria. Following activation, the MOs fuse with the plasma membrane, release their electron‐dense contents, and remain connected to the outside by a narrow channel.A small region at the anterior of sperm from males is amoeboid and can produce small pseudopodia. In sperm from the uterus this region enlarges considerably, the groundplasm changes from a granular to a filamentous from and the sperm become fully motile. During locomotion a cycle of events occurs at the leading edge beginning with the extension of a small pseudopodium. A constriction ring forms across its base and as the sperm progresses the constriction ring moves back along the cell, but remains stationary relative to the substrate. The location of new pseudopodia at the leading edge dictates the direction of movement of the spermatozoon. Since Cytochalasin B had no effect on the translocation of sperm, the polymerization of actin appears not to play a part in locomotion.
Binding of fluorescent lectins to the surface of germ cells from fetal and early postnatal mouse gonads
Authors: Felici Massimo De 
Published: February 2005

AbstractFluorescent lectins were used to study the chemical nature of carbohydrate moieties present on the surface of female and male germ cells isolated from mouse gonads during fetal and early posnatal development. Concanavalin A (ConA), lens culinaris agglutinin (LCA), ricinus communis agglutinin (RCAI) and wheat germ agglutinin (WGA) bound intensely to the germ cell plasma membrane at all stages studied. Other lectins such as ulex europaeus agglutinin (UEAI) and agglutinin (SBA) did not bind or bound moderately (SBA to female germ cells only).Distinct developmental‐related changes were observed when female germ cells were labeled with fluorescein‐conjugated peanut agglutinin (PNA) or dolichos biflorus agglutinin (DBA). DBA and PNA binding was absent or weak in fetal female and male germ cells, but became intensely positive in oocytes in the immediate postnatal period. The percentage of oocytes stained with DBA increased during the first three days after birth, and from day 3–4 onwards all oocytes were strongly labeled.I suggest that these changes in lectin binding reflect changes in biochemical structure of the oocyte surface related to differentiative events occurring in the mouse ovary immediately after birth.
Analytical subcellular distribution of cAMP‐dependent protein kinase activity in bull sperm
Authors: Pariset Claude C. , Weinman Jacqueline S. , Escaig Francoise T. , Guyot Michele Y. , Iftode Francine C. , Weinman Serge J. , Demaille Jacques G. 
Published: February 2005

AbstractThe distribution of the cAMP‐dependent protein kinase activity in bull ejaculated sperm has been investigated. This activity proved to be mainly present in a soluble form inside the cell. Sperm fractionation into heads and flagellar fragments, using differential centrifugation or centrifugal elutriation, has shown that the particulate cAMP‐dependent protein kinase activity was mainly associated with the flagellar structures. A much activity was shown to be associated with the head fraction. Some activity could also be detected in the purified plasma membrane fraction.
The effects of short−term incubation (aging) of mouse oocytes on in vitro fertilization, zona solubility, and embryonic development
Authors: Gianfortoni Joseph G. , Gulyas Bela J. 
Published: February 2005

AbstractThe effects of in vitro aging of cumulus−intact versus cumulus−free metaphase II mouse oocytes were studied with respect to zona solubility and fertilization rates. Furthermore, zygotes from the in vitro fertilization studies were incubated and their developmental progress was recorded. The zona pellucida showed a gradual increase in resistance to dissolution by α−chymotrypsin with in vitro aging over a period of 6 hr. This effect was greater in cumulus−free as compared to cumulus−intact ova, but it was not nearly as profound as that seen in the control in vivo fertilized eggs. The fertilization rate of in vitro aging cumulus−intact ova compared favorably with the control in vivo aging group over a 6−hr time period. This was in sharp contrast to the decreased fertilization rate of in vitro aging cumulus−free ova over the same period of time. Lastly, development of zygotes to the blastocyst stage was also evaluated. The rate of first cleavage was similar in all experimental groups and compared favorably with the in vivo controls. However, further development to blastocysts of in vitro aged cumulus−free ova showed a marked decrease when compared to the cumulus−intact group and the in vivo fertilized controls. Thus we established a direct relationship between zona digestion time of in vitro aged cumulus−free oocytes and a decrease of fertilization rates in the mouse.
Scanning electron microscope study of in vitro prepenetration gamete interactions
Authors: Jedlicki A. , Barros C. 
Published: February 2005

AbstractHamster and mouse capacitated spermatozoa were interacted in vitro with hamster and mouse eggs in homologous and heterologous combinations. Also, fertilized and trypsin treated unfertilized hamster eggs, and unfertilized rat eggs were made to interact with capacitated hamster spermatozoa. The surface of the zona pellucida was then examined with the scanning electron microscope. It was found that sperm attachment, followed by sperm binding and penetration through the zona pellucida, was observed only when homologous gamete combinations were used. Binding of the spermatozoa to the zona was evidenced by the lytic effect of the acrosomal enzymes on the zona substance. When fertilized eggs and trypsin‐treated unfertilized hamster eggs were mixed with capacitated hamster spermatozoa as well as in the heterologous gamete combinations, we found that the spermatozoa were able to establish attachment but not binding. Under these conditions the outer surface of the zona pellucida was never found to have penetration tracks made by the spermatozoa. Failure of heterologous spermatozoa to cross the foreign zona pellucida is believed to be associated with the inability of the foreign spermatozoa to establish binding and to the inability of their acrosomal enzymes to digest the zona. A similar mechanism is believed to work in zona‐reacted and in trypsin‐treated hamster eggs inseminated in vitro with homologous spermatozoa.
Estradiol synergism and inhibition of insulin‐induced meiotic maturation in Rana pipiens oocytes in vitro
Authors: Lessman Charles A. , Schuetz Allen W. 
Published: February 2005

AbstractEstradiol 17‐β (E2) was found to either inhibit or synergize Na‐insulin (Ins)‐induced meiotic maturation of Rana oocytes. Inhibition of Ins activity occurred in the presence of the follicular investments of the oocyte; synergism with Ins occurred in oocytes denuded of the follicle wall. Similarly, co‐incubation of E2 with frog pituitary homogenate (FPH) or pregnenolone (Pe) significantly decreased meiotic reinitiation as determined by germinal vesicle dissolution (GVD) in follicle‐enclosed oocytes. By contrast, E2 had no consistently significant effect on progesterone (P)‐induced meiosis in follicle‐enclosed oocytes. Furthermore, E2 had no significant effect, either inhibitory or synergistic, on Pe‐ or P‐induced GVD of denuded oocytes. Thus, of the meiotogens tested (Ins, P, Pe, FPH), all but P were consistently inhibited by E2 in the presence of the follicle wall. Na‐insulin was the only meiotogen tested (Ins, P, Pe) which was potentiated by E2 in denuded oocytes, However, when E2 and Ins were spatially separated on the surface of individual intact follicles, the result was synergism of Ins‐induced GVD rather than inhibition. These results suggest that Ins acts to induce GVD in the denuded oocyte through a mechanism distinct from that used by P (ie, Ins mechanism allows E2 synergism while the P mechanism does not). The E2 inhibitory effect on Ins‐induced GVD appears to be dependent upon simultaneous exposure of follicle wall tissue to mixtures of E2 and Ins. The synergistic effect of E2 on Ins‐induced GVD is dependent upon the simultaneous exposure of the oocyte surface to Ins and E2, either as a homogenous mixture in the case of denuded oocytes or as single substances at independent sites, for follicle‐enclosed oocytes.
Immunological approach to the characterization of the outer acrosomal membrane of boar spermatozoa
Authors: Hinrichsen Adriana C. , Töpfer‐Petersen Edda , Dietl Thomas , Schmoeckel Christian , Schill Wolf‐B. 
Published: February 2005

AbstractAntiserum to purified boar spermatozoan outer acrosomal membrane (OAM) was raised in rabbits and adsorbed with boar liver and serum glutaraldehyde cross‐linked immunoadsorbents. The IgG fraction of the antiserum was purified by (NH4)2SO4 precipitation followed by ion‐exchange chromatography. Indirect immunofluorescence showed bright fluorescent staining of the acrosomal cap of boar spermatozoa and to a lesser extent of the acrosomes of bull and goat spermatozoa after incubation with anti‐OAM‐IgG. Immuno‐electron microscopy further confirmed the specificity of the antibody for the OAM. Preincubation of the anti‐OAM‐IgG with isolated OAM, completely abolished its reactivity. When tested by ELISA, anti‐OAM‐IgG reacted with boar, bull, goat, and human spermatozoa; however, its binding activity to boar spermatozoa was significantly greater as compared to spermatozoa from the other species tested.In an effort to identify OAM antigens recognized by this antiserum, the isolated boar OAM was labeled either with 3H or with 125I and solubilized by mild detergent treatment. The extracted components were immunoprecipitated with anti‐OAM‐IgG and protein A‐bearing S. aureus and the thus isolated antigens were analysed on SDS‐PAGE. The results suggest that anti‐OAM‐IgG recognized one high molecular 3H‐labeled glycoprotein (270 kd), and four 125I‐labeled polypeptides of lower molecular weight of the boar OAM.
Exocytosis of glycogen during maturation of amphibian oocytes
Authors: Taghy‐Sadak Zoubida , Vilain Jean‐Pierre 
Published: February 2005

AbstractThe repartition and fate of glycogen β has been followed during progesterone‐induced maturation of amphibian oocytes. The use of specific staining, both at the cytological and ultrastructural level, demonstrates that glycogen tends to be extruded from the oocyte during maturation of the urodeles Pleurodeles waltlii and Ambystoma mexicanum. No such effect of the hormone is observed in Xenopus laevis, where only a slight centrifuge migration of the glycogen could be recorded.Stacks of annulate lamellae increase during the early phase of in vitro progesterone‐induced maturation (2 to 9 hours after progesterone application). After germinal vesicle breakdown (about 12 hours after beginning the progesterone treatment) annulate lamellae have disappeared and numerous masses of vesicles are present in the cytoplasm of Pleurodeles and Ambystoma matured oocytes. We never observed any close relation between the annulate lamellae and these vesicles.
Human sperm chromosomes obtained from hamster eggs after sperm capacitation in TEST‐yolk buffer
Authors: Brandriff B. , Gordon L. , Watchmaker G. 
Published: February 2005

AbstractHuman sperm chromosomes were obtained after capacitation with TES‐Tris (TEST) yolk buffer and fusion with Syrian hamster eggs. Semen samples could be stored at 4°C for 3 days and remain functional in the assay system. The efficiency of TEST yoik buffer for obtaining karyotypes was as good as, or greater than, the efficiency of standard BWW medium containing human serum albumin.
Influence of uterine secretions on the chromatin of ram spermatozoa at different stages of maturation: Cytophotometric study of Feulgen‐DNA after in vitro incubation
Authors: Nicolle J. C. , Fournier‐Delpech S. , Courot M. 
Published: February 2005

AbstractRam spermatozoa taken from the epididymal head, body, or tail or from the ejaculate were examined by microspectrometry after incubation in vitro with ewe uterine fluids at 37°C for 20 hours. Compared with incubation in Ringer's solution, uterine fluid incubation resulted in a decrease in nuclear Feulgen‐DNA content. This decrease was greater for more immature spermatozoa (29.0 and 47.3% for spermatozoa from head and body, respectively) than for more mature spermatozoa (17.7 and 4.0% for spermatozoa from the tail and the ejaculate, respectively). In parallel with this decrease, there was a condensation of the chromatin which resulted in a decreased nuclear surface area, especially in spermatozoa taken from the epididymal body. Therefore, it would appear that, during epididymal maturation, changes in the ability of spermatozoa to maintain embryonic development as the spermatozoa mature are due to changes in chromatin structure.
The retention of 125I‐labeled plasma membrane proteins in bovine spermatozoa cryopreserved in egg‐yolk citrate extender
Authors: Zafian P. T. , Amann R. P. 
Published: February 2005

AbstractCryopreservation of bovine sperm in egg‐yolk citrate extender (EYC) usually maintains fertility. Since plasma membrane proteins are important for the fertilizing potential of sperm, the possible loss of membrane proteins from sperm subjected to cryopreservation in EYC was evaluated. Sperm were washed and labeled with 125I without significantly reducing motility. Radiolabeled sperm were a) held for 2 hr at 22°C in N‐2‐hydroxyethylpiperazine‐N'‐ethanesulfonic acid (HEPES)‐buffered saline containing 1% polyvinyl alcohol, b) cooled to 5 °C in glycerol‐free EYC and held for 3 hr, or c) frozen‐thawed in EYC containing 7% glycerol. Sperm were solubilized and proteins were separated by electrophoresis under denaturing conditions. Freeze‐thawing dislodged most egg‐yolk proteins from the spermatozoal plasma membrane that were bound to and retained by sperm that only were cooled to 5 °C. Autoradiography resolved 11‐18 bands of 125I polypeptides. There was no difference (P > 0.05) in the amount of 125I protein retained by frozenthawed and cooled sperm. However, the radioactivity in two polypeptide bands (MW = 105 K and 24.2 K) was less (P < 0.05) for sperm held at 22 °C in HEPES‐buffered saline. Thus, holding sperm in buffered saline at 22 °C resulted in a greater loss of 125I proteins from the plasma membrane than did cryopreservation of sperm in EYC. Cryopreservation did not induce greater loss of 125I proteins from the plasma membrane than simply cooling sperm to 5 °C in EYC.
The change in the nucleus pattern during chromatin condensation in Eurygaster integriceps spermatids
Authors: Danilova L. V. , Gaber E. S. , Shinyaeva L. I. 
Published: February 2005

AbstractWhen spermiogenesis of Eurygaster integriceps occurs, the pattern of the round nucleus of the spermaiid changes under the influence of a manchette consisting of two large groups of microtubules (up to 130–140 in each). The pericentriolar matter was found to be involved in the changes in the nuclear pattern; it takes part in the formation of the asymmetric intranuclear channel, open at one side. Two zones appear in the nucleus that differ greatly in their rates of chromatin condensation. The two main parts of the future nucleus are thereby established: the apical one with a greater rate of chromatin condensation, which gives origin to the two‐walled cylinder, and the outer part, which grows for a long time and extends caudally to the middle piece. At the later stages of nucleus formation, when its larger part is separated from the surface of the middle piece, the vesicles of the endoplasmic reticulum that penetrate between the Nebenkern and the larger part of the nucleus, play a certain role.
Interaction of human spermatozoa with the human zona pellucida and zona‐free hamster oocyte following capacitation by exposure to human cervical mucus
Authors: Gould John E. , Overstreet James W. , Hanson Frederick W. 
Published: February 2005

AbstractThe functional capacity for sperm interaction with the human zona pellucida and zona‐free hamster oocyte was tested after human spermatozoa were capacitated by passage through a column of human cervical mucus. The results were compared with those obtained when spermatozoa from an aliquot of the same semen sample were capacitated by the standard laboratory methods involving sequential washing by dilution and centrifugation of the semen. Washed‐capacitated sperm suspensions were more successful than mucus‐capacitated sperm in attaching to the zona‐free hamster oocyte and in fusing with its oolemma. However, the ability of mucus‐capacitated sperm to penetrate the human zona pellucida was equal to washed capacitated sperm. These experiments demonstrate an approach that may be useful in comparative studies of human sperm capacitation in vivo and in vitro.
Ultrastructural organization of heterochromatin within sea urchin sperm nuclei
Authors: Aboukarsh Nanma , Kunkle Mel 
Published: February 2005

AbstractChromatin within swollen or lysed isolated sperm nuclei of the sea urchin, Strongylocentrotus purpuratus, was examined by electron microscopy. Spread preparations of lysed sperm nuclei demonstrated dense aggregates of nondispersed material and beaded filaments radiating from these aggregates. These beaded fibers are similar in size and appearance to the “beads‐on‐a‐string” seen as characteristic of chromatin spreads from numerous interphase nuclei. The beads are nucleosomes that have an average diameter of 130 Å. The interconnecting string is 40 Å indiameter and corresponds to the spacer DNA. In thin sections of swollen nuclei the sperm chromatin appears to be composed of 400 Å superbeads that are closely apposed to form 400 Å fibers. As the chromatin disperses, the superbeads are seen to be attached to one another by chromatin fibers of 110 Å diameter. In thin sections, the 400 Å superbeads appear to disperse directly into the 110 Å fibers with no intervening structures. This work demonstrates that the heterochromatin in Strongylocentrotus purpuratus sperm nuclei is composed of nucleosomes that form 100 Å filaments that are compacted into 400 Å superbeads. The superbeads coalesce to give the morphological appearance of 400 Å fibers.