bluuebunny/crossref_metadata_2025 huggingface dataset:
Fertilization cones of inseminated sea urchin (Arbacia punctulata) oocytes: Development of an asymmetry in plasma membrane topography
Authors: Longo Frank J.
Published: February 2005

Electron microscopic and cytochemical investigations were carried out on inseminated Arbacia oocytes comparing structural and chemical properties of their microvillous surface and fertilization cones. Early fertilization cones (up to 4 min postinsemination) were relatively small, smooth surface projections of cytoplasm that engulfed the incorporated sperm nucleus. However, in contrast to surrounding microvillous areas of the oocyte surface, enlarged fertilization cones (8 to 15 min postinsemination) had a distinctive crenated appearance that persisted until their regression. When examined by various cytochemical techniques, membrane delimiting fertilization cones had a much lower affinity for agents that stain negatively charged and carbohydrate moieties (cationized ferritin, concanavalin A, ruthenium red, and alcian blue) than did other regions of the oocyte surface. This difference in surface properties of membrane delimiting the site of sperm‐egg fusion was not due solely to incorporated sperm plasma membrane and did not occur when inseminated oocytes were incubated with cytochalasin B. Little or no difference in the membrane of the fertilization cone versus microvillous areas was observed when inseminated eggs were freeze‐fractured or prepared with agents (filipin and polymixin B) to demonstrate β‐hydroxysterols and anionic phospholipids. These observations indicate that membrane delimiting the fertilization cone differs from the remainder of the oocyte surface and suggests that following insemination significant rearrangements of surface molecules take place within the egg plasmalemma that give rise to asymmetries in membrane topography.
X‐Y chromosome univalency in the testes of hyperthermic mice: I. Concomitant formation of multinucleated giant cells
Authors: Waldbieser G. C. , Chrisman C. L.
Published: February 2005

Exposure of mice to an environment of approximately 35°C and 65% relative humidity for 2–5 days caused dissociation of the X‐Y chromosome bivalent in diakinesis‐metaphase I (primary) spermatocytes. Dissociation of autosomal bivalents, chromosome fragmentation, aneuploidy, and polyploidy were not significantly affected by heat stress. Heat stress also caused formation of multinucleated giant cells in the mouse testis. Giant cell formation followed a distribution similar to X‐Y dissociation, reaching highest levels 5 days after heat stress and returning to control levels 50–70 days after heat stress. Spermatogonial activity was not permanently damaged by these conditions, and the germinal epithelium was able to repopulate. We believe that cell aberration or cell death resulting from either or both of these anomalies causes much of the decreased fertility found after subjecting scrotal mammals to high temperatures.
Partial purification and characterization of dipeptidyl peptidase II (DPP II) from guinea pig testes
Authors: DiCarlantonio G. , Talbot P. , Dudenhausen Elizabeth
Published: February 2005

Depeptidyl peptidase (DPP II) was partially purified from guinea pig testes by (NH4)2SO4 precipitation, Con A‐Sepharose 4B chromatography, and Sephadex G‐200 chromatography to a specific activity of 27.4 μmol Ala3 hydrolyzed min−1 mg−1 protein. Chromatography on a calibrated G‐200 column yielded a molecular weight of 135,000 daltons for the enzyme. Sodium dodecyl sulfate polyacrylamide electrophoresis showed an enrichment of a broad doublet at 64–66,000 daltons. The enzyme had optimal activity toward hydrolysis of L‐alanyl‐alanyl‐alanine at pH 4.5 and showed sensitivity to cations of increasing size with Tris producing the most inhibition of those tested. The enzyme was moderately inhibited by serine proteinase inhibitors. Thin‐layer chromatography revealed the dipeptidase nature of the enzyme's activity on tripeptides and dipeptidyl arylamides. A doublet of activity occurred when nitrocellulose electroblots of nondenaturing gel electrophoresis of the (NH4)2SO4 fraction were reacted with the specific DPP II substrate, lysyl‐alanyl‐4‐methoxy‐2‐napthylamide. Analytical isoelectric focusing of the G‐200 fraction followed by fluorescent enzyme activity detection that used cellulose triacetate overlay membranes impregnated with the specific DPP II substrate, lysyl‐alanyl‐7‐amino‐4‐trifluoromethylcou‐marin, revealed multiple isoforms focusing at pI = 4.8–5.6. Two prominent bands focused at pI = 4.9 and pI = 5.1. The properties of guinea pig testicular DPP II are compared and contrasted with similar dipeptidyl peptidases from other sources.
Sperm‐oocyte membrane fusion in the human during monospermic fertilization
Authors: Sathananthan A. Henry , Chen Christopher
Published: February 2005

Sperm‐oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro.

Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH‐surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7–14 hours in vitro and inseminated with preincubated sperm, fixed between 1–3 hours after insemination, and examined by transmission electron microscopy.

Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome‐reacted and had been partly incorporated into the ooplasm, while the anterior two‐thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized.

The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg.
X‐Y chromosome univalency in the testes of hyperthermic mice: II. Light and electron microscopic analysis of synaptonemal complexes
Authors: Waldbieser G. C. , Chrisman C. L.
Published: February 2005

Hyperthermia‐induced X‐Y dissociation has been observed in diakinesis‐metaphase I sper‐matocytes but not in pachytene spermatocytes, which have been implicated as highly susceptible to heat stress. To determine X‐Y dissociation in pachytene spermatocytes and to compare levels of dissociation between pachytene and diakinesis‐metaphase I spermatocytes male ICR mice were exposed to 35°C ± 0.07°C and 65% ± 0.14% relative humidity for 2 or 4 days. Control mice were housed at 24°C ± 0.04°C and 43% ± 0.58% relative humidity. Mice were killed 0, 3, 5, 8, or 10 days after stress and the testes processed for meiotic chromosome display at diakinesis‐metaphase I and synaptonemal complex display at pachynema. Twenty‐five to thirty cells per mouse at both stages of meiosis were observed with light microscopy, and pachytene spreads with transmission electron microscopy to determine heat‐stress effects on synaptonemal complex structure. Statistical analyses revealed significant linear increases in X‐Y dissociation with prolonged exposure to heat at pachynema and diakinesis‐metaphase I. Levels of pachytene dissociation were one‐half the levels of dissociation at diakinesis‐metaphase I. The resolvable structure of the lateral elements of the synaptonemal complex was not affected by heat stress.
Human sperm chromosomes in zona‐free hamster eggs: Pretreatment of spermatozoa with calcium ionophore facilitates later examination of chromosomes
Authors: Wramsby H. , Yanagimachi R.
Published: February 2005

Freshly ejaculated human spermatozoa exposed to 10 μM calcium ionophore A23187 for 30 minutes quickly bound to and penetrated into zona‐free hamster eggs. When these eggs were cultured for 13 hours in the presence of low concentrations of vinblastin and podophyllotoxin, then fixed and spread with the use of a cold fixation technique, haploid sets of sperm chromosomes were demonstrated. The number of spermatozoa that were attached to the egg surface at insemination influenced the success rate of chromosome demonstration. The best results (an average of about 0.7 chromosome set per egg) were obtained when insemination was controlled so that an average of seven spermatozoa attached to each egg at the end of a 15–30‐minute insemination period. This improved method for demonstrating human sperm chromosomes would be useful in the analysis of paternal genetic contributions in reproduction as well as in assessing the effects of environmental genotoxic agents or conditions on spermatozoa.
Action of gossypol on rat germinal cells. II. The acrosome
Authors: Baccetti B. , Bigliardi E. , Burrini A. G. , Renieri T. , Selmi M. G.
Published: February 2005

The action of gossypol on the acrosomal complex in rats has been investigated by microscopical and submicroscopical methods. The drug displays its morphological action only on spermatozoa during the transit through the epididymis, causing malformations and vesiculations. It appears to exert a primary action on the SS groups formation, disturbing morphological molding occurring in this period and inhibiting capacitation.
Interactions of aged gametes: In vitro fertilization using in vitro‐aged sperm and in vivo‐aged ova in the mouse
Authors: Smith A. L. , Lodge J. R.
Published: February 2005

A study of varying combinations of in vitro‐aged sperm and in vivo‐aged ova at 3 hr intervals from 0–24 hr resulted in failures at different steps of the fertilization process during in vitro fertilization of mouse ova. Significant decreases caused by sperm aging, ova aging, and sperm × ova aging interaction were found in sperm penetration. Pronuclear formation was not affected by sperm aging and was enhanced by ova aging, and there was a significant effect of sperm × ova aging interaction. Sperm aging significantly influenced the prometaphase stage of the fertilization process. Therefore, it is suggested that the detrimental fertilization effects resulting from aging gametes are due to different mechanisms in sperm and ova, that these mechanisms are affected at different times, and that they affect different steps in the fertilization process.
Cyclic nucleotide content and protein phosphorylation during maturation of Spisula oocytes
Authors: Adeyemo Oyewole , Shirai Hiroko , Koide S. S.
Published: February 2005

Cyclic nucleotide levels in the oocytes of the surf clam Spisula solidissima were measured during germinal vesicle breakdown (GVBD) induced by fertilization. The level of cAMP and cGMP in untreated oocytes was 8.23 ± 0.95 and 4.89 ± 0.39 pmol/106 oocytes. The ratio of cAMP to cGMP ranged from 1.5 to 2.0. The cAMP level in Spisula oocytes fluctuated after fertilization and before GVBD. The cGMP level showed minimal fluctuation, with a tendency to decrease initially followed by a subsequent rise to the basal level in a nonsynchronous manner. These changes were not statistically significant. There was a general increase in protein phosphorylation during the period after fertilization and before GVBD. The greatest increase occurred with proteins of estimated molecular weights of 52, 18, and 12 kD, analyzed by gel electrophoresis and autoradiography.
Superovulation in immature and mature Chinese hamsters
Authors: Roldan Eduardo R. S. , Horiuchi Toshitaka , Yanagimachi Ryuzo
Published: February 2005

Mature female Chinese hamsters ovulate an average of 8.8 ± 1.0 (mean ± SD) eggs per female in each estrous cycle. Superovulation can be induced in both immature and mature females by subcutaneous or intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) and either human chorionic gonadotropin (hCG) or pituitary luteinizing hormone (PLH). The best superovulation in immature females was induced by the administration of 15 IU of PMSG followed 72 hr later by injection of 15 IU of hCG (about 25 eggs per female) or 0.2 mg (200 IU) PLH (about 46 eggs per female). Ovulation started about 13–15 hr after administration of hCG (or PLH) and was completed during the next 5–6 hr. Superovulation in mature females could be induced by injecting PMSG any day of the estrous cycle, but the best superovulation (about 39 eggs per female) was induced by injecting 15 IU of PMSG on day 1 (day of ovulation) followed by the injection of 0.4 mg of PLH 72 hr later. When immature females treated with the best superovulatory protocol were mated on the evening of PLH injection, only 5% of the eggs were found fertilized 50 hr after PLH administration. On the other hand, about 60% of the eggs were found fertilized in mature females mated following treatment with the best superovulatory protocol. The majority (83–85%) of superovulated eggs obtained from both immature and mature females were normally fertilized in vitro.
Isolation of 125I‐concanavalin A‐labeled plasma membrane from unfertilized mouse eggs
Authors: Boldt Jeffrey , Wolf Don P.
Published: February 2005

A procedure was developed for isolation of plasma membrane (PM) preparations from unfertilized mouse eggs. Zona‐free mouse eggs prepared by the method of Boldt and Wolf (Gamete Res 13:213–222, 1986) were labeled with 125I‐concanavalin A (ConA) prior to sonication and fractionation on iso‐osmotic self‐generated Percoll density gradients. Experiments using the ConA‐specific sugar α‐methylmannoside (αMM) indicated that 125I‐ConA bound specifically to the egg PM. Greater than 95% of 125I‐ConA binding to zona‐free eggs was blocked in the presence of 0.1 M αMM, and incubation of eggs in αMM after 125I‐ConA labeling caused release of 85–90% of bound label. Fractionation of 125I‐ConA‐labeled eggs by Percoll density gradient centrifugation yielded a single radioactive peak at density = 1.025, corresponding to egg PM material. Prolonged incubation of 125I‐ConA‐labeled eggs or egg sonicates prior to fractionation did not alter the location of the radioactive peak, indicating that 125I‐ConA did not label other organelles. As a control, human erythrocytes were labeled with 125I‐ConA and fractionated under identical experimental conditions and yielded a single radioactive peak at density (1.020) comparable to that observed for 125I‐ConA‐labeled eggs. These results indicate that 125I‐ConA can be used as a specific marker to support PM isolation from small numbers of zona‐free mouse eggs.
Morphology and fertilizability of frozen human oocytes
Authors: Sathananthan A. Henry , Trounson Alan , Freeman Lesley
Published: February 2005

Human 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

The 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

The 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

Fine‐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

Extragonadal 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

We 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

Fertility 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

A 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

The 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

Using 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

This 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

The 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

The 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

Pooled 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

Free‐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

Unfertilized 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

The 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.
Fate of sperm tail components after incorporation into the hamster egg
Authors: Hiraoka Jun‐Ichi , Hirao Yuki‐Hisa
Published: February 2005

The changes in the cytoplasmic organelles of sperm tail in golden hamster eggs fertilized in vivo were observed by electron microscopy. Eggs were obtained from oviducts of hamsters that had been superovulated and inseminated by injection of cauda epididymal spermatozoa into the uteri. In the egg cytoplasm 10 hours after insemination, some of the mitochondria of the spermatozoon midpiece had begun to swell, and a number of multivesicular bodies were observed surrounding the midpiece. The fibrous sheath of the principal piece quickly disappeared prior to the first cleavage, whereas the axoneme and outer dense fibers were unaltered.

During the two‐cell stage, numerous multivesicular bodies gathered around the midpiece and fused with the mitochondria. The heterophagic vacuoles thus formed then gradually separated from the axial fibers. The outer dense fibers were disarranged and partially torn into small segments; then they seemed to dissociate into substructural granular components. The axonemal microtubules had begun to swell but were still present in the two blastomeres. It is indicated from these observations that at least the mitochondria of the tail constituents carried into the oocyte are digested into small molecular elements by the multivesicular bodies and are possibly distributed as nutrients for the blastomeres during the early stage of development.
Epididymal sperm profiles in young adult, middle‐aged, and testosterone‐supplemented old rats
Authors: Taylor George T. , Weiss Jürgen , Pitha Josef
Published: February 2005

Both ejaculated semen and epididymal contents from an individual male contain sperm that differ in various physicochemical characteristics. An experiment is reported in which epididymides from rats 5–24 months old were subjected to density gradient centrifugation to separate gametes of different stages of maturity. The research was designed to examine typical changes in “profiles” of sperm maturity during the reproductive lifetime of rats. Also, testosterone complexed with cyclodextrin that mimics the episodic release of the endogenous hormone was used to supplement the decreased circulating titers of some of the old males. Results revealed clear ontogenetic patterns of gradually decreasing reproductive competence as measured by absolute numbers of sperm, circulating levels of testosterone, and various other physiological markers of fertility. Sperm profiles also revealed age‐specific changes with a shift toward progressively more mature, perhaps senile, gametes that begins at middle age. Testosterone supplementation (400 μg/kg b.w./day for 30 days) failed to restore sperm numbers or other measures of physiology in the old males, but the steroid modified sperm profiles to approximate more closely the profiles characteristic of young adult males than either untreated middle‐aged or old males. The data were interpreted as suggesting that epididymal sperm profiles clearly identify males of different ages, and that the aging epididymis retains its capacity to respond to manipulations that modify the endocrine milieu.
Egg‐penetration ability and structural properties of human sperm prepared by Percoll‐gradient centrifugation
Authors: Tanphaichitr N. , Millette C. F. , Agulnick A. , Fitzgerald L. M.
Published: February 2005

Human sperm with a high zona‐free hamster egg‐penetration ability were obtained by centrifuging freshly ejaculated sperm through a discontinuous two‐step (47% and 90%) Percoll gradient at 600g at room temperature for 30 min. Highly motile sperm with good penetration ability were recovered in the pellet fraction (Per‐sperm), whereas those with low penetration ability were in the gradient interface. The increased penetration ability of Per‐sperm, as compared to sperm capacitated by other methods such as a single‐tube swim‐up or multiple‐tube swim‐up preparation, was not due to an increased proportion of acrosome reacted sperm. Rather, transmission electron microscopy indicated that Per‐sperm were devoid of coating envelopes, which were present around both the head and tail regions of noncapacitated and single‐tube swim‐up sperm. Changes to the surface of Per‐sperm were demonstrated by their decreased interaction with UEA I lectin, which binds specifically to fucose residues. Removal of the coating envelopes as well as other changes on the sperm surface may lead to an enhanced binding of Per‐sperm to the oocyte. In addition, 99% of Per‐sperm contained chromatin that was fully condensed. By contrast, about 15% of swim‐up sperm still possessed incompletely condensed chromatin. With a higher penetration ability, “clean” appearance, and homogeneity of condensed chromatin, Per‐sperm are recommended for insemination and studies of human sperm capacitation.
Attempted sexing of bovine spermatozoa by fractionation on a Percoll density gradient
Authors: Upreti G. C. , Riches P. C. , Johnson L. A.
Published: February 2005

Bovine spermatozoa were fractionated on Percoll density gradients into two major subpopulations of motile spermatozoa and a minor fraction containing mostly nonmotile spermatozoa with abnormal morphology. Fractionation required the addition of bovine serum albumin and a continuous Percoll gradient buffered with sodium bicarbonate. It is postulated that, under suitable ionic conditions, the binding of bovine serum albumin to spermatozoa amplifies subtle differences between subpopulations. These studies were directed toward separating Y‐ and X‐bearing spermatozoa. However, when the subpopulations were evaluated by flow cytometry, their Y:X ratios were similar to that of an unfractionated control.
Xenopus spermatozoon: Correlation between shape and motility
Authors: Bernardini Giovanni , Andrietti Francesco , Camatini Marina , Cosson Marie‐Paule
Published: February 2005

Xenopus spermatozoa have a characteristic corkscrew‐shaped head and a flagellum with a conventional “9 + 2” structure (Bernardini et al.: J Ultrastruct Md Struct Res: 94:188–194). They are motile in water and media of low osmolarities, but not in media of osmolarities higher than 200 mosm/liter, regardless of the ionic composition. External calcium and pH are not involved in the inhibition of sperm movement at high osmolarities.

The duration of sperm motion was less than 10 min, and both flagellar beat frequencies and sperm velocities declined progressively. However, in demembranated and reactivated tails, flagellar beating was sustained for longer times. Flagella propagated three‐dimensional waves that induced a spinning motion of the whole spermatozoon. This pattern of movement is not dependent on the shape of the sperm head, since isolated, reactivated flagella exhibited three‐dimensional waves.
Effect of follicle somatic cells during pig oocyte maturation on egg penetrability and male pronucleus formation
Authors: Mattioli Mauro , Galeati Giovanna , Seren Eraldo
Published: February 2005

In order to investigate the effect of the somatic cells of the follicle on the preparation of the oocyte for fertilization three experiments were carried out. In the first, pig oocytes, cultured for 46 h in the presence of extroverted follicles (follicle oocytes) or surrounded by the cumulus (cumulus oocytes), were exposed to sperm in an in vitro fertilization system. Follicle oocytes showed a higher rate of fertilization than that recorded in cumulus oocytes (80% vs. 47%). In addition, significantly more sperm penetrated into the ooplasm of follicular oocytes (3.77/egg) than into that of cumulus oocytes (1.42/egg). To investigate the reason for the observed fertilization difference zona‐free oocytes were studied in the second experiment. Significantly more spermatozoa were recorded in the ooplasm of follicle oocytes than in that of cumulus oocytes, thus suggesting that the effect of the follicle on fertilizability may partly depend on an action on the plasma membrane of the oocyte. A further effect of the follicular tissue was on cytoplasmic maturation: only follicular oocytes were capable of consistently promoting male pronucleus formation. In cumulus oocytes, sperm that entered the cytoplasm remained in a condensed form near the female pronucleus. In the third experiment cumulus oocytes and denuded oocytes were matured in medium that had been previously used to mature follicle oocytes. This conditioned medium was alone able to affect sperm penetration and male pronucleus formation in cumulus oocytes, but it did not exert any influence on denuded oocytes. This suggests that the observed effect of the follicle is mediated by soluble factors that, however, cannot influence the oocyte without some direct cell‐oocyte contact.
Studies of stallion sperm survival: Preservation of progressive motility of stallion spermatozoa by low ionic strength media
Authors: Bize Isabel , Driscoll David M.
Published: February 2005

Preservation of stallion sperm forward motility was studied using a video recording system in semen diluted with media of different ionic strength and sodium content. After 8 hr of incubation at room temperature, semen diluted in a low ionic strength media containing sucrose displayed 65±9% motility with 68±3% of the motile sperm showing forward motility (diameter of head trajectory ≥ 80 μm). In contrast, sperm populations diluted and incubated with a normal ionic strength media containing sodium had 56±7% motile sperm of which only 36±7% displayed forward progression. Replacement of the sodium ion in the normal ionic strength media by a different cation (choline) produced results similar to those of the media containing high sodium concentration. These results indicate that the preservation of stallion sperm progressive motility is highly dependent on the ionic strength of the media. Membrane depolarization caused by the replacement of ions by noncharged particles in the sperm's external medium might be responsible for the sustained coordination of the flagellar movements.