bluuebunny/crossref_metadata_2025_split huggingface dataset:
Outer acrosomal membrane of guinea pig spermatozoa: Isolation and structural characterization
Authors: Olson G. E. , Winfrey V. P. , Winer M. A. , Davenport G. R. 
Published: February 2005

AbstractWe describe a protocol to isolate a highly enriched fraction of outer acrosomal membrane from guinea pig spermatozoa and present new data on the ultrastructure of this membrane domain. Cauda epididymal spermatozoa were suspended into a low ionic strength buffer and subjected to brief homogenization; this stripped the plasma membrane from the spermatozoa and severed the acrosomal apical segment from the spermatozoon. The crescent‐shaped apical segments retained the outer acrosomal membrane and specific components of the acrosomal matrix. Enriched fractions of apical segments were isolated on discontinuous sucrose gradients and the outer acrosomal membrane purified by subsequent centrifugation onto Percoll density gradients. The isolated outer acrosomal membrane did not form vesicles, but instead rolled up into spiral sheets. Both thin section and negatively stained specimens revealed a paracrystalline arrangement of filaments associated with the luminal surface of the membrane. The isolated outer acrosomal membrane revealed a limited number of polypeptides by SDS‐PAGE, and the polypeptide pattern was distinct from the plasma membrane fraction. The isolated acrosomal membranes possessed no oubain sensitive Na+, K+‐ATPase activity, whereas about 20% of the ATPase activity of the plasma membrane enriched fraction was inhibited by oubain. The potential function of the structural differentiations of the outer acrosomal membrane in the membrane fusion events of the acrosome reaction is discussed.
Identification of a male‐specific histocompatibility protein on preimplantation porcine embryos
Authors: White K. L. , Anderson G. B. , Berger T. J. , Bondurant R. H. , Pashen R. L. 
Published: February 2005

AbstractAn indirect immunofluorescence assay was used to detect the presence of male‐specific protein(s) on various stages of preimplantation porcine embryos. Embryos were collected at slaughter from the reproductive tracts of day−2.5, −4, −5, −6, and −8 (day 0 = first day of estrus) sows and gilts. Embryos were placed in medium containing an anti‐male primary antibody, washed, and transferred to culture drops containing a fluorescein isothiocyanate (FITC)‐labeled secondary antibody. Embryos were classified as either fluorescent (H‐Y positive) or nonfluorescent (H‐Y negative), transferred to coded drops, and karyotyped to examine sex chromosomes. A total of 91 eight‐cell to blastocyst stage embryos were evaluated; of these, 46% were classified as fluorescent and 54% as nonfluorescent. Of readable metaphase spreads (65%) from these embryos, 81% (48 of 59, P < 0.005) were correctly sexed by immunological detection of the male‐specific antigen. Although 13 % (2/15)of four‐cell embryos evaluated were classified as fluorescent, the accuracy with which embryos at this stage were sexed by detection of H‐Y antigen was not different from 50%. Fifty percent of eight‐cell embryos were classified as H‐Y positive with 78% of embryos correctly sexed. It was concluded that the eight‐cell embryo is the earliest stage of development for which there is evidence for expression of H‐Y antigen. Detection of the male‐specific protein was difficult at the expanded blastocyst stage.
Impaired spermatogenesis in the finch hybrid L. castaneothorax by L. punctulata: Transmission electron microscopy and genetic analysis
Authors: Swan M. A. , Christidis L. 
Published: February 2005

AbstractAs part of a project to determine whether there is any correlation between the form of hybrid sterility and the genetic relatedness of the parental species, we studied a male intrageneric hybrid between two finch species (Lonchura custaneothorax × L. punctuluta), and compared the ultrastructural basis of hybrid sterility in this species with that reported by Swan [1985] for an intergeneric bird hybrid. In the latter study the sterility appeared to have an autoimmune basis, due to lack of Sertoli‐Sertoli tight junctions. In the hybrid examined in the present study, lanthanum tracing showed that the junctions were tight. There was no testicular immune reaction; the parental species were almost identical in chromosomal constitution, having only a small inversion difference on chromosome 5, and only two structural protein differences could be detected through examination of the variation at 38 protein loci. Nevertheless, the hybrid appeared sterile and had the following ultrastructural testicular features. Intercellular bridges where present were usually abnormal in structure; centrioles in a centriole pair were arranged in parallel. Many spermatocytes and spermatids degenerated and were phagocytosed by Sertoli cells. Some spermatids progressed to mature testicular spermatozoa in sperm bundles, but commonly had multiple (2–4) axonemes or disrupted doublets and accessory fibers. The multiple axonemes present in most spermatids inserted separately into the base of the nucleus and the multiple centrioles were capable of organizing separate neck structures. We conclude that these cytological abnormalities were caused by genic effects and discuss why they appeared to be restricted to the germ line.
Effects of exposing bovine sperm to bovine serum albumin, or freeze‐thawing, on sperm‐bound amidase activity
Authors: Schenk J. L. , Amann R. P. 
Published: February 2005

AbstractIsolation of a self‐selected population of motile spermatozoa is possible by using a gradient of bovine serum albumin (BSA). We determined if exposure to BSA altered the sperm or if isolated sperm differed from nonisolated cells in terms of motility or activity of sperm‐bound amidase, either before or after subsequent cryopreservation. Exposure of sperm to 6% BSA in egg yolk Tris extender induced changes in the plasma and acrosomal membranes of sperm that resulted in exposure and activation of sperm‐bound amidase (P < .01). In experiment 2, semen extended in egg yolk Tris was cooled to 5°C or layered onto a solution of 6% BSA in extender at 37°C, from which the sperm that had swum into the BSA solution were recovered 2 h later and cooled to 5°C. Sperm in both treatments were cryopreserved. The percentage of progressively motile sperm was determined visually and by track motility. Activity of sperm‐bound amidase exposed to substrate was evaluated. After recovery of sperm from the 6% BSA solution, 81% were progressively motile as compared to 59% in the starting samples (P < .01). However, the amount of exposed sperm‐bound amidase also was greater (P < .05) this was a deleterious change. Immediately after thawing, more (P < .01) sperm were motile in samples of isolated sperm than for nonisolated cells (43 vs 24%), but after incubating the thawed sperm for 1 h at 37°C there was no difference. After freezing and thawing of sperm, amidase activity was higher (P < .05) for the isolated sperm than for nonisolated cells. Thus, isolation of sperm using a 6% BSA gradient increased the proportion of progressively motile sperm, but decreased the percentage of sperm with an intact acrosome, based on measurements of amidase activity.
Sperm chromosome analysis as a new system to test human X‐ and Y‐sperm separation
Authors: Ueda Katsunori , Yanagimachi R. 
Published: February 2005

AbstractThe correlation between the percentage of F‐body‐positive spermatozoa (F‐sperm) and that of Y‐chromosome‐bearing spermatozoa (Y‐sperm) determined by human sperm chromosome analysis was examined after spermatozoa were separated by an albumin column method [Ericsson et al, Nature 246:421–424:1973]. The percentages of F‐sperm were 43.1 ± 4.8 (36.1–48.8) and 45.8 ± 8.3 (35.3–66.5) before and after separtion, respectively. A slight but significant (p < 0.05) increase in F‐sperm after the separation was observed in two semen donors. A total of 570 sperm karyotypes was analyzed and the percentage of Y‐sperm ranged from 36.0 to 59.1 with a mean of 47.3. In one semen donor, the percentage of Y‐sperm (45.6) was significantly (p< 0.05) higher than that of F‐sperm (35.3) after separation. In the remaining nine semen donors, the results obtained by chromosome analysis corresponded well with those obtained by F‐body scoring. The validity of the albumin column method in isolating a Y‐sperm‐rich sperm population is discussed.
Formation of the rupture site in preovulatory hamster follicles: Morphological and morphometric analysis of thinning of the granulosa and thecal layers
Authors: Martin G. G. , Talbot P. 
Published: February 2005

AbstractThe wall of hamster ovarian follicles is composed of the following cell layers: granulosa, theca interna, theca externa, and the surface epithelium. To determine the morphological changes occurring in the follicle during formation of the rupture site, we measured: the thickness of each layer, the number of cells in each layer, and size of cells in each layer, at the apex and base of follicles at specific times during the final 13 hr before ovulation. Changes in the wall occur in 3 stages. During stage 1 (T0–T8), which includes the first 8 hr following the surge in luteinizing hormone, the apical and basal walls thin at the same rate and the antrum increases in size. During stage 2 (T9–T12), there is no change in the thickness of the apical and basal follicle wall nor in the size of the antrum. During the third stage (T12–T13), the size of the antrum decreases slightly and there is an increase in the thickness of the basal wall, which is correlated with its constriction caused by the contraction of smooth muscle cells. The apical wall rapidly thins to the point of rupture. The morphology of cells from each layer is described. Theca interna cells form the final tissue preventing escape of the oocyte‐cumulus complex. The roles of cell migration, stretching, and death in thinning of the apical wall are described, and the mechanisms involved in follicle rupture are discussed.
Ovulation and fertilization of primary and secondary oocytes in LT/Sv strain mice
Authors: O'Neill G. T. , Kaufman M. H. 
Published: February 2005

AbstractIn this study, the chromosome constitution of both unfertilized oocytes and fertilized eggs isolated from the oviducts of LT/Sv strain mice were analyzed. Air‐dried chromosome preparations from unfertilized oocytes revealed that about one‐third of those examined were ovulated as primary oocytes. These were arrested at metaphase of the first meiotic division and exhibited the characteristic “tetrad” chromosome configuration. The remaining two‐thirds of the unfertilized oocytes were ovulated at metaphase of the second meiotic division. The fertilized eggs were isolated from the oviducts of LT/Sv females previously mated to (C57BL × CBA) F1 hybrid males. Analysis of the fertilized eggs at metaphase of their first cleavage mitosis revealed that about one‐third of the eggs examined were digynic triploids, whereas the remaining two‐thirds had the normal diploid chromsome constitution. In the triploids, the 40 female chromosomes present (mouse, n = 20) were derived from a single diploid pronucleus formed after the extrusion of a first polar body, and following the monospermic fertilization of primary oocytes. The female pronuclear‐derived chromosomes invariably exhibited “homologous pairing,” and these were associated at their centromeres. The ovulation, penetration, and subsequent fertilization of primary oocytes is an extremely unusual phenomenon in mammals and only appears to occur on a regular basis in LT/Sv mice. The premature “cytoplasmic maturation” of these oocytes is of interest, as they clearly have the same developmental capacity as secondary oocytes. The significance of these observations in relation to folliculogenesis and litter size in LT/Sv mice is discussed.
Phosphodiesterase activity of mouse sperm incubated under conditions that modulate fertilizing potential in vitro
Authors: Monks Nicola J. , Fraser Lynn R. 
Published: February 2005

AbstractCyclic AMP has been implicated as a regulator of capacitation, but the control of its metabolism in sperm remains obscure. A recent study of mouse sperm has shown capacitation‐related changes in the activities of both adenylate cyclase, which increased during incubation, and cyclic nucleotide phosphodiesterase, which decreased. The present study was conducted to extend these observations by measuring phosphodiesterase activity in sperm incubated in media with modified calcium and/or glucose content, conditions known to modulate fertilizing ability. Phosphodiesterase activity of sequential sperm samples, taken first when sperm are essentially uncapacitated and then when they are either partially or completely capacitated, decreased with time under all conditions, and in each case the greater fall in activity was seen in the medium that would support the greater change in fertilizing ability of the sperm population. Sperm washed by centrifugation to remove epididymal fluid also displayed a reduction in phosphodiesterase activity with time. The medium surrounding the sperm contained about half of the total phosphodiesterase activity, as well as 5′‐nucleotidase and adenosine deaminase. The crude enzyme preparation showed complex kinetic behavior when assayed over a range of cAMP concentrations, but the reduction in activity with time was seen at all substrate levels. The observed changes in phosphodiesterase activity, together with the increased adenylate cyclase activity seen under these sperm incubation conditions, would increase cAMP availability with time, thus providing further evidence for a fundamental role for cAMP in controlling the events of capacitation.
Protein synthesis in mature human oocytes
Authors: Gifford D. J. , Fleetham J. A. , Mahadevan M. M. , Taylor P. J. , Schultz G. A. 
Published: February 2005

AbstractAs a first step toward understanding control of gene expression in early human development, an analysis of protein synthesis and amino‐acid transport in unfertilized mature oocytes was initiated. Qualitative patterns of protein synthesis were examined in individual oocytes cultured in medium containing radiolabeled methionine. No differences in synthetic pattern of proteins, resolved by one‐dimensional electrophoresis and fluorography, were observed in oocytes analyzed from times varying from 12 to 52 hr following collection by laparoscopy. Contamination by follicular or corona radiata cells was readily distinguished on the basis of increased relative synthesis of a polypeptide with Mr = 44,000, a dominant product of synthesis in follicular cells. Based on the specific activity of the methionine precursor, the absolute rate of synthesis was measured to be about 50 pg/oocytc/hr, a value higher than in mouse unfertilized eggs. No difference in protein synthetic rate was observed in oocytes analyzed at 12 hr postcollection versus later times up to 50 hr postcollection. Competition of methionine uptake by leucine, efflux of radiolabeled methionine from preloaded oocytes into medium containing methionine and uptake of methionine in medium with low sodium ion concentration was observed. These findings are consistent with the presence of an L (leucine‐preferring) system for neutral amino acid transport, similar to that in mouse and rabbit eggs. Total protein was measured to be about 150 ng/oocyte, a value five times that of the mouse. These studies provide basic data for further analysis of oocytes and perhaps preimplantation stage embryos in the future.
A computer‐assisted assay for mouse sperm hyperactivation demonstrates that bicarbonate but not bovine serum albumin is required
Authors: Neill James M. , Olds‐Clarke Patricia 
Published: February 2005

AbstractMammalian sperm hyperactivation (HA) is a change in motility that accompanies capacitation (CAP) and is dependent on calcium (Ca) (Yanagimachi and Usui, Exp Cell Res 89:161, 1974). HA may be important for transport through the female tract and/or for fertilization. To develop an objective and quantitative assay for HA in individual mouse sperm, a computer‐assisted motion‐analysis system was used to describe sperm trnaslational movements. To determine which movements were characteristic of HA, Ca‐dependent motility was identified. This was done by incubating sperm with or without calcium (Ca+ or Ca− sperm, respectively), and determining the range of values for each motility parameter that was present only among Ca+ sperm. To do this, we compared frequency distributions of motility parameter values at the time of maximal CAP (90 min). CAP was monitored by measuring the level of in vitro fertilization and by evaluating the pattern of chlortetracycline binding to individual sperm heads [Ward and Storey, Dev Biol 104:287, 1984]. Two Ca‐dependent motility subgroups were apparent: 1) a “slow‐speed” subgroup with a curvilinear velocity (Vc) < 169 μm/sec that had none of the characteristics expected of HA sperm; and 2) a subgroup with higher speeds (Vc > 169 μm/sec) and wider‐amplitude head movements as measured by curvilinear progressiveness ratio (PRc < 0.56). The latter subgroup was selected as HA, since the frequencies and time course were similar to those for CAP in the same population. Two media components known to be important for CAP, bicarbonate and bovine serum albumin (BSA) were then tested to determine whether they were necessary for HA. Incubation of sperm without bicarbonate prevented HA, but omitting BSA did not affect HA during the first 3 hrs. These data suggest that HA is not tightly coupled with CAP.
Influence of the calcium ionophore A23187 on rat egg behavior and cortical F‐actin
Authors: Battaglia David E. , Gaddum‐Rosse Penelope 
Published: February 2005

AbstractRat eggs treated with the calcium ionophore A23187 and subjected to long‐term observation by phase microscopy were found to undergo many developmental changes that are normally associated with fertilization. These included cortical granule exocytosis and the abstriction of the second polar body. In addition, time‐lapse video microscopy revealed that, unlike untreated eggs, whose surfaces remained relatively immotile, the ionophore‐treated eggs underwent a lengthy period of surface undulatory activity. Since all of these events were remarkably similar in timing and morphology to those seen in fertilized eggs, we conclude that A23187 is capable of activating rat eggs. Using NBD‐phallacidin, the distribution of F‐actin in ionophore‐activated eggs was determined. During most of the postactivation period the eggs possessed an uninterrupted, uniform band of polymerized actin encompassing the entire cortex of the egg. However, during a discrete 1.5‐h period after the formation of the second polar body, an area adjacent to the region of polar body abstriction exhibited more intense staining than the rest of the cortex. Cytochalasin B treatment caused a dramatic reduction and/or rearrangement in cortical NBD‐phallacidin staining in activated eggs as compared to activated controls not exposed to the drug. We observed that all the developmental changes described above could be produced in the absence of exogenous calcium, suggesting that the rat egg possesses internal stores of calcium sufficient to elicit an activational response. We conclude that the ionophore‐induced release of free calcium ions into the cytosol stimulates many of the developmental changes that are normally seen during fertilization. These results indicate that calcium influx and cytoskeletal activity are correlated during the activation of this animal egg.
Changes in binding of a 27‐kilodalton chimpanzee cauda epididymal protein glycoprotein component to chimpanzee sperm
Authors: Young L. G. , Gould K. G. , Hinton B. T. 
Published: February 2005

AbstractMotility patterns of caput epididymal chimpanzee sperm, caput epididymal chimpanzee sperm incubated in vitro with chimpanzee cauda epididymal fluid, and cauda epididymal chimpanzee sperm were assessed quantitatively. Sperm recovered from the caput epididymis showed no motility, whereas sperm recovered from cauda epididymis showed progressive forward motility. After incubation in cauda fluid, approximately 25% of caput epididymal sperm showed some motile activity. Electrophoretic analysis of 125I‐labeled sperm plasma membrane preparations revealed that the surface of caput epididymal sperm, incubated in cauda fluid, was modified by the appearance of a major protein‐glycoprotein surface component with an apparent molecular weight of 27 kilodaltons (kD). THis 27‐kD component was not detected on caput epididymal sperm incubated in buffer or in caput fluid. However, it was present in cauda fluid and on cauda epididymal sperm. Binding to caput epididymal sperm was cell specific in that chimpanzee erythrocytes incubated in cauda fluid did not bind this 27‐kD cauda fluid component. Motility patterns of ejaculated chimpanzee sperm and of ejaculated chimpanzee sperm incubated in the uterus of adult female chimpanzees also were assessed quantitatively. Ejaculated sperm showed progressive forward motility, whereas in utero incubated ejaculated sperm showed hyperactivated motility typical of capacitated sperm. Electrophoretic analysis of 125I‐labeled sperm plasma membrane preparations revealed the loss of a 27‐kD component from the surface of ejaculated sperm after in utero incubation. No significant change in the 125I‐distribution pattern was detectable when ejaculated sperm were incubated in buffer. These results suggest that the lumenal fluid component, which becomes adsorbed to the surface of chimpanzee sperm during maturation in the epididymis and which is removed from the surface of mature chimpanzee sperm in the female reproductive tract, affects sperm motility.
Detection of acrosome‐reacted toad sperm based on specific lectin binding to the inner acrosomal membrane
Authors: Takamune Kazufumi 
Published: February 2005

AbstractWhen the sperm of the toad Bufo japonicus were treated with fluorescein isothiocyanate (FITC)‐conjugated peanut agglutinin (PNA), soybean agglutinin (SBA), or Dolichos biflorus agglutinin (DBA), a few sperm fluoresced at the acrosomal region. The number of sperm showing this lectin binding to the acrosome increased significantly upon mild sonication of the sperm suspension. Electron microscopy revealed that ferritin‐conjugated PNA bind not to the outer acrosomal and overlying plasma membranes, but specifically to the surface of the inner acrosomal membrane exposed by sonication. Both the percentage of FITC‐PNA‐labeled sperm and the activity of vitelline coat lysin released by sperm increased in good correlation with increasing sonication time, although the PNA‐labeled sperm decreased in number upon longer sonication. These results indicate that the binding of FITC‐PNA to the sperm provides a reliable measure of the acrosome reaction of Bufo sperm.
Sperm‐egg interaction in the mouse using live and glutaraldehyde‐fixed eggs
Authors: Soldani Patrizia , Rosati Floriana 
Published: February 2005

AbstractA study of interaction between gametes in the mouse, using live and glutaraldehyde‐fixed eggs, showed that, while in live eggs the binding is a two‐step process (“early” and “late” binding), the process is one step when the spermatozoa interact with fixed eggs. The second point that emerged from this study is that uncapacitated and capacitated spermatozoa bind the zonae pellucidae of live and fixed eggs in the same way, but only the capacitated spermatozoa bound to live eggs undergo a complete acrosome reaction and penetrate the zona pellucida. Moreover, it has been shown that binding to fixed eggs, just as to live eggs, is Ca2+‐dependent, and it can be reversed by EGTA. Fixed eggs thus are a good model to study only one step of the sperm‐egg interaction removed from all the other events of the fertilization process.