bluuebunny/crossref_metadata_2025_split huggingface dataset:
Morphology and fertilizability of frozen human oocytes
Authors: Sathananthan A. Henry , Trounson Alan , Freeman Lesley 
Published: February 2005

AbstractHuman oocytes were frozen and thawed by four methods previously used for cryopreser‐vation of human embryos. Most of these oocytes were inseminated after thawing to assess their capacity to fertilize and form pronuclear ova. Their morphology was assessed by phase‐contrast microscopy used in routine IVF. Twenty‐three oocytes were examined by electron microscopy to critically evaluate the effects of cooling and cryopreservation and to confirm fertilization.Morphological survival was observed in more than 60% of the oocytes examined after freeze‐thawing. The main features of cryoinjury were cracks in the zona pellucida, disruption of the plasma membrane and extensive disorganization of the ooplasm. Subtle changes in the cytosol of cumulus cells was also observed. Cooling to 0°C or −6°C had little effect on cytoplasmic structure. Spindles were damaged in two frozen oocytes.Cumulus cell activity, sperm binding to the zona, sperm penetration of the zona seem to be largely unaffected by freeze‐thawing. Fertilization was observed in eight oocytes after postthaw insemination and three embryos (8‐cell to morula stages) were developed from pronuclear ova on further culture. Both monospermic and polyspermic fertilization were confirmed by electron microscopy and micronuclei were detected in three pronuclear ova. The genetic implications of these nuclear aberrations are discussed.These preliminary studies indicate that oocyte freezing needs to be integrated cautiously with clinical IVF by further assessment of embryos developed from frozen oocytes.
Electrofusion for the pronuclear transplantation of mouse eggs
Authors: Tsunoda Y. , Kato Y. , Shioda Y. 
Published: February 2005

AbstractThe present study was undertaken to determine the efficiency of HVJ treatment and electrofusion for pronuclear transplantation in the mouse. The output voltage and duration of the pulses were fixed to 200 μsec at 10 V or to 150 μsec at 15 V for electrofusion, because the maximum rates of blastomere fusion of 2‐cell embryos and development of fused embryos in vitro were obtained under these conditions. Although the proportion of eggs with fused karyoplast (78%) and the fused eggs developed to morulae or blastocysts (67%) was significantly lower than those obtained after HVJ treatment (94% and 94%), the proportion of pregnant recipients and young obtained after treatment of fused eggs was not significantly different between these two procedures.It is advised that electrofusion can be used as a fusogenic procedure for pronuclear transplantation in the mouse in some cases where HVJ cannot be applied.
Formation of the ventral hooks on the sperm head of the plains mouse, Pseudomys australis
Authors: Flaherty Sean P. , Breed William G. 
Published: February 2005

AbstractThe sperm head of the plains mouse, Pseudomys australis, has three curved hooks projecting from its anterior margin. The two ventral hooks have previously been shown to consist largely of an extension of the subacrosomal material. To characterize further the structure and composition of the ventral hooks, we have examined their formation during spermiogenesis using transmission electron microscopy, silver staining, and actin localization with NBD‐phallacidin. The ventral hooks develop as an extension of the perinuclear space and postacrosomal dense lamina on the anteroventral margin of the sperm head. Bundles of 6‐nm‐thick filaments appear in the core of each hook; these are probably actin filaments based on staining of the hooks with NBD‐phallacidin. Just prior to spermiation, electron‐dense material condenses in the core of the ventral hooks and concurrently in the perinuclear space in the remainder of the sperm head. The two ventral hooks thus appear to consist of a core of perinuclear material and actin filaments, which is enclosed by a continuation of the postacrosomal dense lamina.
Phagocytosis of yeast by human oocytes: Fine structural observations
Authors: Koehler James K. , Smith W. Dianne , Ravnik Stuart 
Published: February 2005

AbstractFine‐structure observations have been made on the interaction between invasive yeast cells and human oocytes. The yeast appear to make their way through the zona pellucida and once in the perivitelline space are incorporated into phagocytic vacuoles by surface activity of the oocyte. The yeast attach to the vitelline membrane via fuzzy surface material on the cell wall, and incorporation appears to be aided by oocyte microvillar activity. Coated pits in the oocyte plasma membrane are incorporated into the phagosomes, but no lysosomal activity is seen, and neither oocytes nor yeast cels appear to undergo degeneration in the time frame investigated.
Effect of mating on sperm distribution in the reproductive tract of the male rat
Authors: Ratnasooriya W. D. , Wadsworth R. M. 
Published: February 2005

AbstractExtragonadal sperm reserves in male rats were measured in different regions of the genital tract before and subsequent to normal ejaculation. In sexually rested rats, the sperm count (million spermatozoa for the paired organs) in different regions was: distal vas, 18; proximal vas, 9.8; cauda epididymidis, 229; caput + corpus epididymidis, 154. Following mating, the sperm count was reduced in the proximal and distal vas deferens and in the cauda epididymidis. The reproductive tract of mated females was found to contain 29% (no copulatory plug) or 59% (with copulatory plug) of the estimated mean ejaculate, which was estimated from the difference between the sperm counts in the sexually rested rat and following ejaculation. It is concluded that in the rat the immediate source of spermatozoa for ejaculation is the cauda epididymidis, with a smaller contribution arising from the vas deferens.
Inhibition of progesterone‐mediated maturation of oocytes of Xenopus laevis by oocyte maturation inhibitor from pig follicular fluid: Development of a routine assay for the inhibitor with Xenopus oocytes
Authors: Pomerantz Seymour H. , Bilello Patricia A. 
Published: February 2005

AbstractWe describe an assay for oocyte maturation inhibitor (OMI) using the progesterone‐mediated maturation of Xenopus oocytes. The test fraction used was a partially purified fraction from pig follicular fluid, which gave consistent inhibition of maturation in the pig oocyte assay. In the toad assay, oocytes (30–50) from each toad were pretested to determine whether satisfactory maturation was achieved because widespread animal variation was observed. Toads whose oocytes showed > 60% maturation in the pretest could be used directly. Toads whose oocytes showed < 60% maturation were injected with pregnant mares serum gonadotropin (PMSG) in order to increase progesterone‐mediated maturation. The dose and time after injection of PMSG before harvesting oocytes, dose and duration of progesterone exposure, and order of exposure of oocytes to OMI and progesterone were important variables. Opposing effects of OMI and progesterone were seen in oocytes from toads receiving 60 IU PMSG. In the routine assay we use animals whose oocytes show > 60% matuaration in the pretest or animals treated with 12 IU of PMSG 4 to 7 days before use. Oocytes are exposed to the OMI fraction for 1 hr, progesterone is added, and incubations continued until controls reach maximum maturation (5 to 8 hr). The inhibition of toad oocyte maturation by OMI is reversible. The toad and mammalian oocyte assays were compared using more highly purified fractions of OMI and gave identical results.
Separation of motile human spermatozoa by means of a glass bead column
Authors: Daya Salim , Gwatkin Ralph B. L. , Bissessar Haimant 
Published: February 2005

AbstractFertility potential of semen depends upon the presence of viable and motile spermatozoa. A variety of techniques has been reported whereby motile spermatozoa are separated from semen for use in homologous insemination. Using a column of glass beads, we tested various sizes for spermatozoa‐separating efficiency. This procedure is quick and simple and results in a signficantly better recovery of motile and viable spermatozoa from poor‐quality semen. Therefore it has the potential for use in intrauterine insemination for treating male factor infertility.
Purification of the sperm‐binding factor and identification of a sperm attack molecule from the egg of the sea urchin, Hemicentrotus pulcherrimus
Authors: Yoshida Motonobu , Aketa Kenji 
Published: February 2005

AbstractA sperm‐binding factor, which seems to have a primary role in binding sperm to the egg, was isolated from the egg surface of Hemicentrotus pulcherrimus and purified by monitoring the neutralization of the fertilization inhibition exerted by Fab fragments against crude sperm‐binding factor. An improved purification for this sperm‐binding factor is described in the present paper. The preparation of purified sperm‐binding factor revealed one major protein band with an apparent molecular weight of 61,000 after sodium dodecylsulfate‐polyacrylamide gel electrophoresis.A substance with the fertilization inhibitory effect on sperm, was isolated by diethylaminoethyl (DEAE)‐Sephadex column chromatography in the course of purification of the sperm‐binding factor and termed “sperm attack molecule.” One precipitin line was formed between the sperm attack molecule and anti‐crude sperm‐binding factor serum in a double‐immunodiffusion test. Fab fragments were prepared against partially purified sperm‐binding factor or sperm attack molecule, and the effect of these Fab fragments on eggs was investigated. Anti‐sperm‐binding factor Fab fragments inhibited the fertilizability of eggs, whereas anti‐sperm attack molecule fab fragments did not. However, anti‐sperm attack molecule Fab fragments impaired elevation of the vitelline layer. It is possible that the sperm attack molecule prevents polyspermy.Sperm attack molecule contains 3.7% neutral sugars. Its inhibitors effect was cancelled by trypsin or heat.
Phosphorylation state of protamines 1 and 2 in human spermatids and spermatozoa
Authors: Pruslin Fred H. , Imesch Elisabeth , Winston Ronald , Rodman Toby C. 
Published: February 2005

AbstractThe basic nuclear proteins of a fraction of elongating spermatids from human tests and of a fraction of motile spermatozoa from the ejaculate, separated by ion‐exchange chromatography, were compared. Analysis by acetic acid‐urea polyacrylamide gel electrophoresis (PAGE) showed that, in both fractions, four proteins of lower mobility were coeluted with protamine 1 by 23% guanidinium chloride (GuCI) while protamine 2 alone was eluted by 50% GuCI. Treatment with alkaline phosphatase identified those four proteins as phosphorylated protamines, and cyanogen bromide (CNBr) treatment of the dephosphorylated protamines distinguished them as variants of protamine 2 and not of protamine 1. Thus far, phosphorylated forms of protamine 1 have not been detected in either spermatids or spermatozoa. Those observations indicate that protamine 2 functions in the cycle of phosphorylation‐dephosphorylation, which is essential to the process of sperm chromatin condensation, while the role of protamine 1 in human spermiogenesis is not yet defined.The presence of phosphorylated protamine in motile, presumably mature spermatozoa appears to be characteristic of human sperm but not of the sperm of other mammals and is probably the basis for the heterogeneity of chromatin condensation frequently observed in human spermatozoa.
Fertilizability of unovulated mature eggs following indomethacin administration in mice
Authors: Hayashi Seiji , Noda Yoichi , Matsumoto Hisashi , Mori Takahide 
Published: February 2005

AbstractUsing mice as subjects, we investigated the effects of indomethacin (ID) on follicle rupture and nuclear maturation, and studied the fertilizability of ova retained in the follicles as a result of ovulation inhibition by ID.Ten units each of PMSG and hCG were administered intraperitoneally to mice at 56‐hr intervals to induce superovulation. ID was administered 90 min after hCG injection. The ova recovered from the oviduct 17 hr after hCG injection numbered 32.2 ± 7.8, 16.9 ± 5.8, 5.6 ± 2.9, and 1.0 ± 1.3 for mice receiving 0, 0.5, 1.0, and 2.0 mg ID, respectively, demonstrating dose‐dependent inhibition of ovulation. Ten hours after hCG administration, the intrafollicular ova that had matured to metaphase second stage comprised 43% in both groups.The fertilization rate (73.7%, 56/76) for the follicle‐retained eggs in the 2 mg ID mice was similar to that for controls (72.9%, 62/85). Essentially the same results were seen with respect to efficacy of ovulation inhibition, rate of egg maturation, and fertilizability of the intrafollicular ova when ID was administered 30 min before hCG injection. These findings indicate that in the mouse, prostaglandins (PG), while required for follicle rupture, are not involved in the ovum maturation process, including fertilizability, under the experimental conditions employed.
Changes of elemental concentrations around/on the rat sperm plasma membrane during maturation in the male genital tract
Authors: Ozawa Yoko , Ashizawa Koji , Okauchi Keizo 
Published: February 2005

AbstractThis study was undertaken to analyze the changes of elemental concentrations around/on the plasma membrane of the head, midpiece, and principal piece regions of individual rat spermatozoa during maturation in the male genital tract by X‐ray microprobe analysis. Around/on the plasma membrane of the three different subcellular regions, concentrations of sodium, potassium, chlorine, and calcium decreased gradually during sperm passage through the male genital tract. Phosphorous showed almost constant concentrations. In contrast, magnesium concentration increased significantly through the process of maturation, and the concentration in the vas deferens was about three times higher than that in the testis. The Na‐to‐K ratios on the midpiece also increased gradually during maturation.
Cytochemical analysis of the anionic sites on the membrane of the stallion spermatozoa during the epididymal transit
Authors: López M. L. , de Souza W. , Bustos‐Obregón E. 
Published: February 2005

AbstractThe structure, relative density, and distribution of anionic sites on the surface of epididymal and ejaculated spermatozoa were studied using polycationic ferritin (CF), colloidal iron hydroxide (CIH), various enzymatic treatments, methylation, and de‐acetylation. Macro‐molecules containing sugar residues, probably sialic acid, are part of the sperm membrane and show a characteristic distribution and density that is dependent of the sperm region and of its origin. Unlike the spermatozoa of other eutheria examined, the exposure of the stallion spermatozoa to neuraminidase treatment did not produce significant changes in the density of the negative charge of the sperm surface. The ability of purified neuraminidase to act only after saponification suggests that sialic acid may be present in the acetylated form. When CIH was used it is seen that the density of the negative charge is rather uniform within a particular segment of the spermatozoa and abruptly changes at the junction of morphologically distinct segments (Between the acrosomal and post acrosomal region of the sperm head and between the post acrosomal region and middle piece of the flagellum). The acrosome presented more negative groups dissociated at pH 1.8 than the postacrosomal region. A greater concentration of anionic sites over the flagellum was also observed when CIH and CF were used. This assymetry probably represents different domains that may be related to specific functions.The cytochemical observations and the cellular electrophoretic mobility measurements did not show striking differences on the negative charge of sperm obtained from different regions of epididymis and ejaculates in contrast to previous results in other species. The spermatozoa collected from caput epididymidis bind CIH but not all population present equal response. In corpus and cauda region of epididymis the population displaying the capacity to bind CIH or CF significantly over the head and tail surface was the majority.This study corroborates that the distribution and density of terminal oligosaccharide residues on the sperm plasma membrane has species specific characteristics. The surface charge of the spermatozoa obtained either during the breeding or nonbreeding season, determined by measurements of cellular electrophoretic mobility and by the binding pattern of CIH and CF, does not show significant differences.
Failure of hamster eggs fertilized in vitro to extrude the second polar body correlates with high levels of polyspermy
Authors: Stewart‐Savage J. , Bavister B. D. 
Published: February 2005

AbstractThe occurrence of second polar body (PB2) retention in in‐vitro fertilized hamster eggs is reported. Of 2,872 eggs examined, 6.9% (199 eggs) failed to extrude PB2. Eggs that failed to extrude PB2 were more likely to be polyspermic than eggs with PB2 (63% and 14.6%, respectively), and the number of fusing sperm per egg was higher in the abnormal eggs. These data indicate that eggs which fail to extrude PB2 have an impaired block to polyspermy. The level of PB2 retention varied between females and ranged from all eggs extruding PB2 to 20% of the eggs failing to extrude PB2 (41% and 4% of the females, respectively). There was no correlation within a female between the percentage of eggs that failed to extrude PB2 and the level of polyspermy in the sister eggs with PB2. Therefore, regardless of the condition of their sister eggs, eggs that fail to extrude PB2 have an impaired block to polyspermy and eggs that extrude PB2 have a normal block.
Acrosomal enzymes and ultrastructure of unfrozen and cryotreated human spermatozoa
Authors: Mack Stephen R. , Zaneveld Lourens J. D. 
Published: February 2005

AbstractPooled semen judged to be normal in all parameters was divided into a number of aliquots which were either 1) kept untreated; 2) mixed with glycerol (10% v/v); 3) washed by centrifugation and resuspended to the original volume with buffer; or 4) washed and resuspended in buffer with glycerol (10% v/v). The progressive motility, viability, ultrastructure, and acrosomal enzyme activity (8 different hydrolases) were studied before and after cryotreatment.The described washing procedure effectively removed seminal plasma, and did not alter sperm motility, sperm viability, sperm ultrastructure, or the acrosomal enzymes studied. Glycerol (10%, v/v) had a deleterious effect on most parameters evaluated before cryotreatment. Cryotreatment severely altered the motility and viability of the spermatozoa and their acrosomal morphology but did not cause significant decreases in most of the acrosomal hydrolases measured. However, acrosin/proacrosin levels decreased by 50–80% and were correlated to the acrosomal damage. A simple assay for the measurement of acrosin/proacrosin enzyme levels in whole sperm is presented which could be used as a monitor for acrosomal integrity. No significant differences were seen between the samples cryotreated in the absence or presence of seminal plasma.
Separation of human X and Y spermatozoa by free‐flow electrophoresis
Authors: Engelmann Ute , Krassnigg Franz , Schatz Harald , Schill Wolf‐Bernhard 
Published: February 2005

AbstractFree‐flow electrophoresis is a fast and promising method for gamete separation. Pretreated seminal plasma‐free human spermatozoa were injected continuously as a fine stream into the buffer medium of the separation chamber flowing perpendicular to the forces of an electrical field, which separated the spermatozoa according to their differences in electrophoretic mobility. For characterization of the two classes of spermatozoa before and after separation, quinacrine mustard staining was used to identify the Y‐chromosome‐bearing spermatozoa carrying the fluorescent body (F‐body). Human spermatozoa moved toward the anode and were separated into two main peaks. The faster moving fraction contained nearly 80% Y‐bearing spermatozoa and the slower peak consisted mainly of pure X‐bearing spermatozoa. Whereas sperm viability as determined by eosin staining was nearly unchanged, sperm motility was reduced after free‐flow electrophoresis.
Sulfhydryl groups are involved in the activation of sea urchin eggs
Authors: Dale Brian , Russo Paola 
Published: February 2005

AbstractUnfertilized sea urchin eggs exposed to the sulfhydryl reagents Ag+ or N‐ethylmaleimide either elevated fertilizationlike membranes, formed surface protrusions, developed a clear cortical layer devoid of organelles, or cytolysed. The relative fraction of each modification varied from batch to batch and was also dose and time dependent. With Ag+ and higher doses of N‐EMI (10−3 M), the most common effect was the elevation of a membrane indicating cortical exocytosis, while at lower doses of N‐EMI protrusions were predominant. Glutathione (GSH) protected eggs against these reagents also in a dose‐dependent manner. Eggs exposed to equimolar amounts of N‐EMI and GSH, which otherwise formed membranes, produced protrusions, while increasing GSH tenfold afforded complete protection. We suggest there are two targets for the sulfhydryl reagents–the first, SH groups on proteins that regulate the release of Ca2+ from the intracellular sequestering mechanism which subsequently triggers cortical exocytosis; the second, SH groups on the egg surface that may regulate cortical organization.
Activation of phospholipase A2 of human spermatozoa by proteases
Authors: Guérette Pierre , Langlais Jean , Antaki Pierre , Chapdelaine Alcide , Roberts Kenneth D. 
Published: February 2005

AbstractThe effect of various proteases (kallikrein, plasmin, and trypsin) on sperm phospholipase A2 activity (PA2: EC 3.1.1.4) has been studied. The addition of trypsin to spermatozoa, isolated and washed in the presence of the protease inhibitor benzamidine, increased PA2 activity optimally with trypsin concentrations of 1.0–1.5 units/assay. In kinetic studies, all of the above proteases stimulated the deacylation of phosphatidylcholine (PC); in fresh spermatozoa, trypsin showed a higher activation potential than kallikrein or plasmin. In the presence of benzamidine, the activity remained at basal levels. Endogenous protease activity due to acrosin (control) resulted in an increase in PC deacylation compared to the basal level. The maximum activation time of PA2 activity by proteases was 30 min. Natural protease inhibitors (soybean trypsin inhibitor and aprotinin) kept the PA2 activity at basal levels and a by‐product of kallikrein, bradykinin, did not significantly affect the control level. Protein extracts of fresh spermatozoa exhibited the same pattern of PA2 activation upon the addition of proteases, thus indicating that the increase in PA2 activity was not merely due to the release of the enzyme from the acrosome. All of these findings suggest the presence of a precursor form of phospholipase A2 that can be activated by endogenous proteases (acrosin) as well by exogenous proteases present in seminal plasma and in follicular fluid (plasmin, kallikrein). Thus, this interrelationship of proteases and prophospholipase A2 could activate a dormant fusogenic system: the resulting effect would lead to membrane fusion by lysolipids, key components in the acrosome reaction.