bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro
Authors: Naito K. , Fukuda Y. , Toyoda Y. 
Published: February 2005

AbstractPorcine follicular oocytes, collected from antral follicles (2–5 mm in diameter) of gilt ovaries, were matured in vitro with or without porcine follicular fluid (pFF), gonadotrophins (GTH) or fetal calf serum (FCS) for 48 hours at 37°C under 5% CO2 in air, and their ability of male pronucleus (mPN) formation was examined after in vitro fertilization. Formation of mPN was observed in 38.6% of penetrated oocytes matured in modified Krebs‐Ringer bicarbonate solution (TYH) 18 hours after insemination. The addition of GTH into the maturation medium did not improve the proportion of mPN‐formed oocytes (20–30%). In contrast, the mPN formation rate elevated significantly (59.5%) when the oocytes were cultured with pFF, and the addition of follicle‐stimulating hormone (FSH) enhanced this pFF action (the rate became 81.0%). In the presence of FSH, significant pFF effect was observable at the concentration of 5%, and its efficiency was elevated with the increase of pFF concentration. When the oocytes were matured with FCS, the mPN formation rate was unchanged or decreased rather than improved (0–25%). These results suggest that pFF, but not FCS, have substance(s) stimulating the ability of mPN formation in porcine oocytes.
Time‐lapse cinematography study of the germinal vesicle behaviour in mouse primary oocytes treated with activators of protein kinases A and C
Authors: Alexandre Henri , Mulnard Jacques 
Published: February 2005

AbstractA passive erratic movement of the germinal vesicle (GV), already visible in small incompetent oocytes, is followed by an active scalloping of the nuclear membrane soon before GV breakdown (GVBD) in cultured competent oocytes. Maturation can be inhibited by activators of protein kinase A (PK‐A) and protein kinase C (PK‐C). Our time‐lapse cinematography analysis allowed us to describe an unexpected behaviour of the GV when PK‐C, but not PK‐A, is activated: GV undergoes a displacement toward the cortex according to the same biological clock which triggers the programmed translocation of the spindle in control oocytes. It is concluded that, when oocytes become committed to undergo maturation, the cytoplasm acquires a PK‐A‐controlled “centrifugal displacement property” which is not restricted to the spindle.
The effects of ultrarapid freezing on meiotic and mitotic spindles of mouse oocytes and embryos
Authors: Sathananthan A. H. , Ng S. C. , Trounson A. O. , Bongso A. , Ratnam S. S. , Ho J. , Mok H. , Lee M. N. 
Published: February 2005

AbstractPreovulatory mouse oocytes and 2‐cell embryos were frozen with dimethyl sulfoxide and propanediol by an ultrarapid method. The survival of frozen oocytes was low (33–34%) compared to that of 2‐cell embryos (78–79%) with either cryoprotectant. Development to blastocysts after postthaw culture was about 7–15% for oocytes and 79–80% for the embryos.Ultrarapid freezing preserves cell structure quite well as revealed by electron microscopy, but meiotic oocytes and late 2‐cell embryos undergoing mitosis showed evidence of spindle disorganization involving loss or clumping of microtubules resulting in some scattering of chromosomes. Embryos developed from frozen eggs showed clear evidence of micronuclear formation and incomplete incorporation of chromosomal material into main nuclei. These experiments confirm our observations on freezing of human oocytes and show that spindle microtubules are sensitive to freeze‐thawing and that cryopreservation could cause chromosomal aberrations during early development. A cautious approach to the introduction of oocyte freezing in human in vitro fertilization (IVF) programs is advocated.
Endocytic origin for periaxonemal vesicles along the flagellum during mouse spermiogenesis
Authors: Segretain D. , Roussel C. 
Published: February 2005

AbstractIn mouse spermatogenesis, formation of the flagellum is associated with the presence of numerous periaxonemal vesicles. These are present in the cytoplasmic portion, limited by the deep invagination of the plasma membrane surrounding the axoneme; the number and size of these vesicles varies during spermiogenesis. The vesicles appear at step 10 in young spermatids and increase in number and size until step 14; they then rapidly decrease and disappear at step 16. Cationic ferritin (CF), an endocytic marker, directly injected in the lumen of the seminiferous tubules, labels periaxonemal vesicles, 1 hour after the injection, showing their endocytic origin. Some vesicles are membrane invaginations, still in continuity with the extracellular space, whereas others probably come from a phagocytic mechanism. The CF also shows that some vesicles flow along the axoneme and they accumulate in small cytoplasmic extensions before disappearing. All these complex endocytic phenomena go on to form certain components of the flagellum.
Staining procedure to detect viability and the true acrosome reaction in spermatozoa of various species
Authors: Didion B. A. , Dobrinsky J. R. , Giles J. R. , Graves C. N. 
Published: February 2005

AbstractA simple dual stain procedure (DS) for simultaneously determining sperm viability and acrosomal status is described. The DS includes the use of the vital stain trypan blue to detect live and dead spermatozoa and Giemsa to detect the presence or absence of an acrosome. For staining, spermatozoa are washed, incubated with trypan blue, washed, dried onto slides, and subjected to Giemsa. Dead spermatozoa stain blue in the postacrosomal region while live spermatozoa remain unstained. The acrosome stains light purple–dark pink while acrosome‐free sperm remain unstained. This staining pattern enables differentiation of spermatozoa which have undergone a true acrosome reaction (TAR) from those which have undergone a false acrosome reaction (FAR). Incubation of bull, boar, ram, and stallion spermatozoa for 60 minutes at 37°C in the presence of calcium ionophore A23187 increased the proportion of spermatozoa undergoing a TAR in all species except the stallion. Incubation of bull spermatozoa for up to 24 hours at 37°C resulted in a decrease over time in the percentage of live acrosome‐intact spermatozoa and a simultaneous increase in the percentage of spermatozoa categorized as having undergone a TAR and FAR. The DS could be a useful technique in evaluating sperm viability and acrosomal status in fertilization and clinical studies.
Incidence of chromosomal anomalies in early bovine embryos derived from in vitro fertilization
Authors: Iwasaki S. , Shioya Y. , Masuda H. , Hanada A. , Nakahara T. 
Published: February 2005

AbstractThe incidence of chromosomal anomalies in early bovine embryos derived from follicular oocytes fertilized in vitro using sperm separated by Percoll density gradient centrifugation was investigated. Overall, chromosomal anomalies were observed in 13.7% (138/1005) of embryos. There were 14 haploids (1.4%), 2 hypodiploids (0.2%), 6 hyperdiploids (0.6%), 101 triploids (10.0%), 12 tetraploids (1.2%), 2 diploid/triploid mosaics (0.2%), and 1 diploid/tetraploid mosaic (0.1%). The frequency of triploidy was caused mainly by polyspermy. There was a significant difference in the frequency of embryos with abnormal chromosomes between the two bulls used (P < 0.005), but Percoll centrifugation did not affect the observed incidence of anomalies. The frequency of chromosomal anomalies in embryos at each stage increased with delay or arrest of development. These results suggest that the incidence of chromosomal anomalies depended on the conditions of in vitro fertilization and the arrest of development.
Relationship between calcium, cyclic AMP, ATP, and intracellular pH and the capacity of hamster spermatozoa to express hyperactivated motility
Authors: White David R. , Aitken R. John 
Published: February 2005

AbstractCapacitation of hamster caudal spermatozoa at a density of 1 × 106/ml is associated with a progressive rise in cAMP levels that precedes the onset of hyperactivated motility. This increase is not expressed by caput spermatozoa incubated under identical conditions. Both the incidence of hyperactivation and the rise in cAMP levels are severely attenuated in the absence of exogenous calcium. Neither factor is restored to control levels by the addition of the phosphodiesterase inhibitor IBMX, although in the presence of exogenous calcium, this reagent increased cAMP levels, stimulated percentage motility and advanced the appearance of hyperactivation.Treatment of spermatozoa at a density of 1 × 106/ml with the calmodulin antagonist, calmidazolium (CZ), caused severe disruption of sperm motility and abolished hyperactivation, while causing only a slight reduction in cAMP content. Addition of IBMX in the presence of CZ elevated cAMP content to levels higher than normally observed during capacitation but did not restore either coordinated or hyperactivated motility.To determine both the mechanisms responsible for this elevation of cAMP content and the changes that occur during epididymal maturation to facilitate the expression of this increase, the free cytosolic calcium concentration, ATP levels, and intracellular pH of caput and caudal cells were compared. The calcium content of caudal spermatozoa rose significantly at a time when cAMP levels were increasing, while ATP content and intracellular pH fell. However, the inability of caput spermatozoa to express a rise in cAMP content was not due to deficiencies in any of these factors.These results indicate a positive role for the cAMP rise in the expression of hyperactivated motility and that the fundamental control mechanism governing both these events may be the influx of calcium that accompanies capacitation in this species.
Capacitation of bovine spermatozoa by lysophospholipids and trypsin
Authors: Wheeler Matthew B. , Seidel George E. 
Published: February 2005

AbstractBovine spermatozoa were incubated in vitro with lysophosphatidylserine (LPS), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), or trypsin. Capacitation of sperm was evaluated by penetration of the zonae pellucidae of dead bovine oocytes. Capacitation times could be shortened to 3 h or less by treatment of spermatozoa with each of these lysophospholipids (LPLs) (P < .05). The maximum oocyte penetration percentages for individual LPLs were 40% for 10 μM LPS, 24% for 160 μM LPC, 31% for 320 μM LPE, and 19% for 320 μM LPI. Capacitation also was facilitated (P < .01) by trypsin treatment of spermatozoa. Spermatozoa treated with 250 or 2,500 units/ml of trypsin penetrated more oocytes (17 and 18%) than spermatozoa treated with 0 or 25 units/ml of trypsin (0 and 3%).Spermatozoa treated with increasing concentrations of LPL showed a decrease in both the percentage of intact acrosomes and of progressively motile spermatozoa. Increasing levels of trypsin in the incubation medium also led to a decrease (P < .05) in the percentages of intact acrosomes and a decrease (P < .01) in the percentages of progressively motile spermatozoa.Percentages of live, ovulated oocytes fertilized by spermatozoa incubated for 1 h in LPS (86%, 6/7) were not different from those incubated for 24 h in control medium (71%, 5/7). Percentages of oocytes fertilized with both of these capacitation treatments were higher (P < .05) than for oocytes exposed or killed or uncapacitated sperm.Rapid induction of capacitation and the acrosome reaction can be accomplished by exogenous treatment of bovine sperm with lysophospholipids or trypsin.
Crater defect in human spermatozoa
Authors: Baccetti B. , Burrini A. G. , Collodel G. , Magnano A. R. , Piomboni P. , Renieri T. , Sensini C. 
Published: February 2005

AbstractThis report describes the “crater defect” in human spermatozoa, a malformation that consists of a nuclear and acrosomal invagination present in 100% of the cells, whereas tail structure and motility are fairly normal. The defect occurs during spermiogenesis. A possible concomitance with abnormalities in the microtubular apparatus involved in the sperm molding is discussed.
Expression and topographical localization of cell surface fucosyltransferase activity during epididymal sperm maturation in the mouse
Authors: Ram Prabha Agrawal , Cardullo Richard A. , Millette Clarke F. 
Published: February 2005

AbstractWe have demonstrated previously that spermatogenic cells in the mouse testis have high levels of fucosyltransferase activity. Furthermore, a significant portion of this activity has been localized to the cell surface (Millette et al.: Cell Biology of the Testis and Epididymis, 1987). Differential expression of fucosyltransferases and their function as ecto‐enzymes may be important in the processes of sperm maturation and fertilization in mammals. Accordingly, here we report the activity levels of fucosyltransferase (FT) in spermatozoa isolated from the mouse caput and cauda epididymides. Calculated on a per cell basis, spermatozoa from the caput epididymis have significantly more FT activity than do spermatozoa from the cauda epididymis (18.07 ± 2.2 pmol/million cells compared with 2.8 ± 0.09 pmol/million cells). Furthermore, caput sperm exhibit a more significant increase in FT activity when assayed in the presence of Nonidet P‐40. Calculated on the basis of cell surface area, however, FT activity remains constant on the head portion of spermatozoa isolated from all portions of the male reproductive tract and from capacitated spermatozoa. Measurements of FT activity in extracts of isolated sperm tails from cells at different stages of maturation indicate a greatly diminished activity in tails from sperm in the cauda epididymis. The total sperm surface area is composed predominantly of the plasma membrane surrounding the flagellar apparatus. Therefore, our data demonstrate that FT activity is retained selectively on the different topological regions of sperm, with losses during sperm maturation in the epididymis being restricted to the tail segment. Maintenance of high levels of FT activity of the plasma membranes of the mouse sperm head raise the possibility that FT is indeed involved in some aspects of sperm‐egg recognition.
Failure of spermatozoa from T/t mice to fertilize in vitro is overcome by zona drilling
Authors: Ahmad Tariq , Conover Joanne C. , Quigley Martin M. , Collins Robert L. , Thomas Anthony J. , Gwatkin Ralph B. L. 
Published: February 2005

AbstractFailure of epididymal spermatozoa from T/t mutant mice, but not from t/t individuals, to fertilize oocytes in vitro was partially overcome by opening a small aperture in the zona pellucida with acidified Tyrode's solution to permit direct access of the spermatozoon to the vitellus. This study provides a model system to evaluate requirements for successful zona drilling in the treatment of human infertility and further insights into the effects of the t complex on sperm fertility.
Microsurgical bisection of porcine morulae and blastocysts to produce monozygotic twin pregnancy
Authors: Nagashima Hiroshi , Kato Yukio , Ogawa Shyoso 
Published: February 2005

AbstractPorcine morulae were bisected by a glass needle after softening of zonae pcllucidae by pronase followed by a treatment with 0.05% trypsin/0,02% EDTA for decreasing intercel lular junction of blastomeres. Transfer of 49 half‐morulae to three recipients resulted in one pregnancy. The blastocysts were bisected symmetrically so as to leave a cellular bridge between the sister half‐embryos after the softening of zonae followed by or without the trypsin/EDTA treatment. Transfer of 47 monozygotic (MZ) pairs of half‐blastocysts to nine recipients resulted in four pregnancies. A litter of nine fetuses was obtained from seven MZ pairs of half‐blastocysts, demonstrating that at least two pairs of MZ twin fetuses were produced. It is thought that the procedure for bisecting blastocysts developed in this study is one of the potential methods of producing porcine MZ twins.
Heparin and glutathione: Physiological decondensing agents of human sperm nuclei
Authors: Reyes Rosalina , Rosado Adolfo , Hernández Omar , Delgado Néstor M. 
Published: February 2005

AbstractIt has been proposed that reduced glutathione (GSH) or other thiol reagents may partici pate in the basic mechanism by which sperm‐decondensing activity is accomplished. How ever, in vitro, these reagents seem to be inactive and require the presence of other chemi cals, usually detergents. Heparin binds specifically to the sperm membrane and provokes the decondensation of human sperm and the activation of DNA transcription and synthe sis. However, the concentrations at which these effects occur seem to be higher than those expected under physiological conditions. In the present study, thiol reagents at 10 mM concentration, either alone or combined, were completely ineffective in inducing any sig nificant nuclear decondensation after prolonged exposures (24 hr) of incubation. Heparin, 153.8 μM, was capable of inducing only a small increase in nuclear swelling. However, GSH at concentrations as low as 0.1 mM in combination with heparin induces deconden sation of human sperm nuclei in vitro. When GSH concentration was kept constant at 5 mM, nuclear decondensation was induced with heparin at concentrations as low as 11.6 μM, and a maximal decondensation (90%) was obtained with only 21.6 μM of heparin. The latter is more than ten times less than the minimal active concentration of heparin used alone.
Calcium‐binding proteins of boar spermatozoan plasma membranes: Identification and partial characterization
Authors: Peterson R. N. , Chaudhry Prem , Tibbs Brian 
Published: February 2005

AbstractCalcium‐binding proteins (CBPs) of boar spermatozoa and boar seminal plasma were identified by using a 45Ca overlay technique to detect these proteins on transblots of PAGE‐separated proteins. A single CBP (Mr ∼ 300 kDa) was detected in seminal plasma. This protein binds specifically to the plasma membrane overlying the principal segment and is removed from sperm during capacitation. The protein was purified for further charac terization by anion exchange chromatography and gel filtration. In addition, six major proteins (30, 35, 38, 42, 52, and 66 kDa) which do not originate from accessory gland secretions were found to be strongly associated with the plasma membrane. Most of these proteins are not integral to the membrane and appear to develop an association with the plasma membrane during cpididymai maturation. Similarly, calmodulin‐binding proteins appear to develop strong associations with the plasma membrane during epididymal transit.
Direct contact is required between serum albumin and hamster spermatozoa for capacitation in vitro
Authors: Dow Mark P. D. , Bavister Barry D. 
Published: February 2005

AbstractA dialysis unit was used to test whether direct physical contact between serum albumin and hamster spermatozoa is required for capacitation and/or the acrosome reaction. Sperm and bovine serum albumin (BSA) were incubated cither together (direct incubation) or separated by a dialysis membrane (indirect incubation). Sperm viability was supported with “sperm motility factors” (hypotaurine and epinephrine) and polyvinylalcohol (PVA). Spermatozoa became capacitated and underwent acrosome reactions when directly incubated in medium containing BSA (TALP‐PVA), but did not undergo acrosome reactions when indirectly incubated with BSA (medium TLP‐PVA). When sperm were first incubated for 4 hr indirectly with BSA, followed by 4 hr direct incubation with BSA, capacitation did not occur during indirect incubation. These findings indicate that an “intimate association” is necessary between serum albumin and spermatozoa to support capacitation under in vitro incubation conditions. The data are consistent with the concept of direct transfer of compounds from sperm to albumin and/or vice versa during sperm capacitation.
Gamete membrane fusion in hamster spermatozoa with reacted equatorial segment
Authors: Vigil Pilar 
Published: February 2005

AbstractMammalian spermatozoa must undergo many changes to be able to fertilize the oocyte. One of these changes, the acrosome reaction, has been established as a requisite for gamete membrane fusion to occur; it consists of the fusion and vesiculation of the sperm plasma membrane with the outer acrosomal membrane of the principal segment of the acrosome. Reaction of the equatorial segment has occasionally been observed. The objective of the present work was to determine whether the presence of the sperm plasma membrane over the equatorial segment is necessary for gamete membrane fusion to occur.Golden hamster spermatozoa were capacitated in vitro in TAPL 10K, and the maximum possible percentage of acrosome reaction was determined at 82.79% + 1.69% SD (P = 0.27; r = 0.21). Ultrastructural studies showed that 93.6% of the reacted spermatozoa in this population had their principal and equatorial segments reacted. The fertilizing ability of these spermatozoa was assayed using zona‐free hamster oocytes. The percentage of fertilized ova obtained was 98.8% (308/312). Ultrastructural studies snowed the presence of spermatozoa with reacted equatorial segment inside the cytoplasm of immature oocytes. The evidence presented in this work demonstrates that the plasma membrane of spermatozoa with reacted equatorial segment retains its ability to fuse with the oocyte.
Activation of Ca2+channels during the acrosome reaction of sea urchin sperm is inhibited by inhibitors of chymotrypsin‐like proteases
Authors: Matsumura Kiyotaka , Aketa Kenji 
Published: February 2005

AbstractProbable participation of sperm protease in the acrosome reaction was investigated using several inhibitors and substrates. Among those examined, L‐l‐tosylamide‐2‐phenylethyl chloromethyl ketone (TPCK) and chymostatin, chymotrypsin inhibitors, p‐nitrophenyl‐p′‐guanidinobenzoate (NPGB), a serine protease inhibitor, and N‐benzoyl‐L‐tyrosine ethyl ester (BTEE), a chymotrypsin substrate, inhibited the egg jelly‐induced acrosome reaction of Strongylocentrotus intermedius. TPCK and BTEE, however, did not inhibit the reaction caused by ionophores, A23187, or nigericin. To know the mechanism of inhibition by chymotrypsin inhibitors and substrates of the egg jelly‐induced acrosome reaction, intraccllular Ca2+ concentration ([Ca2+]i) and pH (pHi) were measured with fura‐2 and 2′,7′‐bis (carboxy‐ethyl)carboxyfluorescein (BCECF), respectively. Egg jelly caused increase of [Ca2+]i which was depressed by BTEE. Egg jelly also caused a transient rise of pHi, which was not depressed by BTEE. In the presence of verapamil, the acrosome reaction by egg jelly was significantly inhibited concomitant with depressed increase of [Ca2+]i. The rise of pHj was not depressed by verapamil. Thus, modes of action of BTEE and of verapamil are similar to each other. Bringing these findings together, the authors present a view that a chymotrypsin‐like protease of sea urchin sperm activates verapamil‐sensitive Ca2+ channels, which take part in the acrosome reaction.