bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of adenosine‐metabolizing enzymes modulates mouse sperm fertilizing ability: A changing role for endogenously generated adenosine during capacitation
Authors: Monks Nicola J. , Fraser Lynn R.
Published: February 2005

The effect of inhibiting adenosine‐metabolizing enzymes on sperm fertilizing ability was studied to investigate a possible role for endogenously generated adenosine in the regulation of capacitation. The compounds used have been shown to be effective inhibitors of the relevant enzymes in similarly incubated mouse sperm suspensions. Inhibition of 5′‐nucleotidase activity with α,β‐methylene adenosine 5′‐diphosphate (AMPCP), to reduce available endogenous adenosine, caused a dose‐dependent inhibition of the fertilizing ability of partially capacitated spermatozoa, which was significant with 100 and 250 μM AMPCP. Conversely, inhibition of adenosine deaminase with 100 nM coformycin, to increase available endogenous adenosine, promoted the fertilizing ability of partially capacitated spermatozoa when the fertilization rate of control suspensions was low. However, coformycin had no effect on sperm suspensions with moderate fertilizing ability, and it inhibited fertilizing ability when added to capacitated spermatozoa. These data are consistent with a promotion of the early stages of capacitation by endogenously generated adenosine and suggest that sensitivity to adenosine changes as capacitation proceeds. Because the majority of adenosine‐metabolizing enzyme activity resides in or is directed toward the extracellular compartment in such suspensions, these effects of adenosine may be mediated at the outer surface of the cell. By interacting with receptors on adenylate cyclase, externally produced adenosine could modulate intracellular levels of cyclic adenosine monophosphate (cAMP), thereby influencing fertilizing ability.
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

Porcine 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

A 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

Preovulatory 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

In 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

A 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

The 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

Capacitation 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

Bovine 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

This 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

We 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

Failure 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

Porcine 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

It 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

Calcium‐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

A 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

Mammalian 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

Probable 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.
Effects of follicle‐stimulating hormone and ovarian steroids during vitro meiotic maturation on fertilization of rat oocytes
Authors: Zhang X. , Armstrong D. T.
Published: February 2005

The present study examined the effects of gonadotropins and ovarian steroids during in vitro meiotic maturation of rat oocytes on their ability to undergo in vitro fertilization. Fully grown oocytes were isolated from antral follicles of immature rats and cultured as oocyte‐cumulus cell complexes (OCC) under conditions in which completion of meiotic maturation occurs spontaneously. They were then exposed to spermatozoa under conditions in which oocytes matured in vivo exhibit high fertilization rates. Compared with oocytes from pregnant mare's serum gonadotropin (PMSG) or follicle‐stimulating hormone (FSH)‐treated rats, a simiiar proportion of the oocytes (>80%) from untreated rats underwent germinal vesicle breakdown, but such oocytes had a lower rate of fertilization (70% vs. 20%). The presence of FSH during in vitro maturation restored the fertilization rate for oocytes from untreated rats, while a cytochrome P450 inhibitor, aminoglutethimide phosphate abolished this beneficial effect of FSH. The addition of progesterone during the in vitro maturation period duplicated the beneficial effect of FSH on fertilization rate of oocytes from untreated rats; oestradiol‐17β was less effective in this regard, and 5α‐dihydrotestosterone was ineffective. These findings indicate that FSH and progesterone, although having no apparent effect on nuclear maturation of the oocyte, play an important role during oocyte maturation in enabling normal fertilization to occur.
Microtubule assembly is required for the formation of the pronuclei, nuclear lamin acquisition, and DNA synthesis during mouse, but not sea urchin, fertillization
Authors: Schatten Heide , Simerly Calvin , Maul Gerd , Schatten Gerald
Published: February 2005

Microtubule assembly is required for the formation of the male and female pronuclei during mouse, but not sea urchin, fertilization. In mouse oocytes, 50 μM colcemid prevents the decondensation of the maternal meiotic chromosomes and of the incorporated sperm nucleus during in vitro fertilization. Nuclear lamins do not associate with either of the parental chromatin sets although peripherin, the PI nuclear peripheral antigen, appears on both. DN A synthesis docs not occur in these fertilized, colcemid‐arrested oocytes. This effect is limited to the first hours after ovulation, since colcemid added 4–6 hours later no longer prevents pronuclear development, lamin acquisition, or DNA synthesis. Neither microtubule stabilization with 10 μM taxol nor microfilament inhibition with 10 μM cytochalasin D or 2.2 μg/ml lalrunculin A prevent these pronuclear events; these drugs will inhibit the apposition of the pronuclei at the egg center. In sea urchin eggs, colcemid or griseofulvin treatment doe? not result in the same effect and the male pronucleus forms with the attendant accumulation of the nuclear lamins. The differences in the requirement for microtubule assembly during pronucleus formation may be related to the cell cycle: In mice the sperm enters a meiotic cytoplasm, whereas in sea urchin eggs it enters an interphase cytoplasm. Refertilization of mitotic sea urchin eggs was performed to test the possibility that this phenomenon is related to whether the sperm enters a meiotic/mitotic cytoplasm or one at interphase; during refertilization at first mitosis, the incorporated sperm nucleus is unable to decondense and acquire lamins. These results indicate a requirement for microtubule assembly for the progression from meiosis to first interphase during mouse fertilization and suggest that the cytoskeleton is required for changes in nuclear architecture necessary during fertilization and the cell cycle.
Changes in the distribution of intramembranous particles and filipin‐reactive membrane sterols during in vitro capacitation of golden hamster spermatozoa
Authors: Suzuki Fumie , Yanagimachi Ryuzo
Published: February 2005

Membrane alterations accompanying in vitro capacitation of hamster spermatozoa were examined using the freeze‐fracture technique with or without use of filipin, a sterol‐binding probe. In the spermatozoa prior to or at 10 min after start of incubation in capacitating medium, large (about 11 nm) and small (8–9 nm) intramembranous particles (IMPs) were present in the periacrosomal region of the sperm plasma membrane (PAPM). Filipin sterol complexes (FSCs) were densely (about 500/μ2) distributed in the PAPM prior to incubation. The density of FSCs in the PAPM was reduced by 70–80% of the original density by 2 hr of incubation. At the same time, small patches of IMP‐free areas appeared in the plasma membrane above the equatorial and middle segments of the acrosome. By the end of 3 hr of incubation, the majority of small IMPs had disappeared from the PAPM. Remaining large and small IMPs tended to aggregate in the PAPM. During incubation in capacitation medium, “cords,” or linear arrangements of closely packed IMPs, appeared near the posterior ring of the sperm head. These observations strongly suggest that the acrosome reaction of the hamster spermatozoa is preceded by the removal (deletion) of filipin‐reactive sterols (FRSs) and the disappearance of small IMPs from the lipid bilayer of PAPM.
Effect of follicle‐stimulating hormone on metabolism and maturation in Bufo arenarum oocytes
Authors: de Atenor Maria S. Budeguer , de Legname Hortensia Salomón , Legname Arnaldo H.
Published: February 2005

In the fully grown Bufo arenarum oocyte, carbohydrate breakdown during the autumn‐winter season is accomplished mainly through the glycolytic pathway followed by the krebs cycle. During the breeding season (spring‐summer), carbohydrates are used mainly through the pentose phosphate cycle and through the variant of the Krebs cycle known as the glutamic aspartic cycle.

The metabolism operating in the oocytes was determined using the following paramenters: 1) the capacity of isolated mitochondria to oxidize citrate and fumarate; 2) the enzymatic activities of phosphofructokinase (PEK) and glucose‐6‐phosphate dehydrogenase (G‐6‐PDH); and 3) cirate and ATP compartmentalization.

The present paper shows that follicle‐stimulating hormone (FSH) would be one of the factors responsible for summer metabolism, since ovary fractios obtained from winter specimens treated with the hormone acquired the metabolic characteristics corresponding to oocytes obtained from breeding‐season animals from dose‐response, and response in the function of time curves, it could be assumed that the optimum doses and times were 0.1 μg FSH/ml of incubation medium and 30 min treatment, respecitively.

The metabolic effect of FSH upon oocytes could be mediaated by the adenylate cyclase cAMP system, since treatment of ovric fractions with cAMP 10‐3 M reproduced the effects obtained with the hormone. In addition, 0.02 mg/ml tetracyline proved to block the effect of FSH. A direct metabolic action of FSH on body cavity oocytes (without follicle cells) was observed when submitting these oocytes to the same hormonal treatment.
X‐Y chromosome dissociation in mouse strains difering in efficiency of spermatogenesis: Elevated frequency of univalents in pubertal males
Authors: Krzanowska Halina
Published: February 2005

Dissociation of the X‐Y chromosome bivalent in diakinesis‐metaphase I spermatocytes of adult mice was significantly more frequent in the CBA strain (29%) than in C57, KP, or KE strains (7–11%). Autosome dissociatio (1–5%) involved only the smallest chromosome pairs. Eleyatedfrequency of X‐Y dissociation in the CBA strain correlates with significantly lower testes weight and lower yield of spermatogenesis, which suggests that sex bivalent dissociation man be responsible for some loss of spermatogenic cells. However, sperm quality is not affected, the percentage of normal spermatozoa and their fertlizing capacity being higher in CBA thatn in the remaining strains. Two congenic strains, KE and KE. CBA (the latter with the Y chromosome introduced from CBA), had the same level of X‐Y dissociatios, suggesting that the Y chromosome plays no rle in the determination of this character. In comparison with adult males pubertal (27–29 day‐old) males had twice as hig a frequency of X‐Y dissociation in KE an KP strains, and combined frequeicies of dissociated sex and autosome bivalents were significantly higher in pubertal males of all tested strains. Although te level of chromosome dissociation is not sufficient to explain increased mortality of germ cells observed in pubertal males, it could be one of the contributing factors.
Effects of glutathione (GSH and GSSG) and glutathione reductase (GR) on zona‐free hamster oocyte ability to decondense human sperm
Authors: Lassalle B. , Testart J.
Published: February 2005

Effects of reduced glutathione (GSH), oxidized glutathione (GSSG), or glutathione reductasc (GR) supply were studied on the ability of hamster oocytes to be fertilized by human sperm. Zona‐free oocytes were pretreated with these compounds prior to sperm insemination. Oocyte pretreatment with high concentrations of GSH or GSSG (50 or 100 mM. 30 min) significantly increased the penetrated oocyte rate (PR). Polyspermy was not increased except when high concentrations of GSH (100 mM) were used. Incubation of oocytes with GR (1 or 10IU/ml) prior to sperm insemination induced increasing dose‐dependent PR. Polyspermy increased significantly with 10 mM GR in oocyte incubation medium. Oocyte incubation for 30 min with the sulfhydryl blocking agent iodoacetamide (1 mM) led to a drastic decrease in oocyte penetration and in polyspermy.

Our results demonstrate an original way to increase the efficacy of human‐hamster heterospecific fertilization. Various hypotheses are discussed explaining these observations which open new investigations for heterospecific and homospecific in vitro fertilization.
Motility and fertilizing ability of rat epididymal spermatozoa washed by a continuous gradient of percoll
Authors: Miyake M. , Coney P. , Iritani A. , Kling O. R.
Published: February 2005

Removal of epididymal fluids from epididymal sperm suspension is an important step for the study of sperm motility, capacitation, and the acrosome reaction. The technique of washing should minimize damage to viable spermatozoa but at the same time efficiently remove debris, non‐sperm cells, and biological fluids. We examined sperm motility and fertilizability in vitro of rat epididymal spermatozoa after washing with Percoll continuous gradient. Nine milliliters (ml) of 50%N‐2‐hydroxyethylpiperazine‐N1‐2‐ethanesulfonicacid (HEPES) buffered Percoll solution was centrifuged at 20,000g for 45 minutes to form a continuous gradient. One hundred to 300μ of sperm suspension was loaded onto the surface of the gradient and centrifuged at 150 g and 1,500 g for 10 minutes. Two main layers of spermatozoa were formed, one of high (lower layer) and one of low (upper layer) motility. At centrifugation 1,500g, the sperm density and motility in the lower layer were greater than at 150g.Spermatozoa from both layers at 150g and l,500g were diluted with modified Krebs‐Ringer's bicarbonate solution (mKRB) and preincubated for 5 hours. Superovulated eggs collected from 21–25‐day‐old Wistar strain immature rats were introduced into the preincubated sperm suspension for insemination and fixed 5‐5.5 hours later for observation of fertilization. Spermatozoa from both layers, 150g and 1,500 g, showed the same fertilizability in vitro as control spermatozoa. From these results we conclude that Percoll gradients can be used for washing rat epididymal sperm for the study of sperm physiology including fertilization.
Lipid peroxidation in human spermatozoa as relatd to midpiece abnormalities and motility
Authors: Rao B. , Soufir J. C. , Martin M. , David G.
Published: February 2005

The formation of malondialdehyde (MDA), a product of lipid peroxidation (LPO), was measured in human spermatozoa from 27 subjects with normal sperm characteristics. Peroxidation of lipids in washed spermatozoa was induced by catalytic amounts of ferrous ions and ascorbate and malondiaidehyde dctermint‐d by thiobarbituric method. MDA formation varied considerably from one sample to another. The studied population showed a strong correlation between lipid peroxidation potential (amount of MDA formed by 108 spermatozoa after 1 hour of incubation) and 1) initial motility r = −0.623, P = 0.001; and 2) morphologic abnormalities of the midpiece r = 0.405, P = 0.05. These results suggest that poor motility is linked with membrane fragility and that spermatozoa with midpiece abnormalities probably have membrane and/or cytoplasmic antiperoxidant system defects. Because LPO potential is related to the two most important characteristics of fertility, it seems possible that it has the potential to become a good biochemical index of semen quality.
Effect of the degree of maturation of mouse oocytes at fertilization: A source of chromosome imbalance
Authors: Badenas J. , Santaló J. , Calafell J. M. , Estop A. M. , Egozcue J.
Published: February 2005

It has been suggested that the abnormal maturation of the human oocyte during fertilization in vitro may result in chromosome imbalance and, induce embryonic loss. Using a mouse model, we have studied the influence of the degree of oocyte maturation (either immaturity or overmaturity) on the chromosome characteristics of embryos at the first‐cleavage division. Immature oocytes were obtained 2–3 h or 3–4 h before the expected ovulation time (b.o.). Overmaturation was induced by aging the newly ovulated oocytes in vitro for 3,6, and 12 h. Our results show a significant decrease in the fertilization rate in the immature groups (65.53% at 2–3 h b.o. and 16.59% at 3–4 h b.o. vs. 78.22% at control) and after 12 h of in vitro aging (69.39%), while a significant increase of this parameter was found at 3 h of aging (82.59%) as compared to the other groups. No significant differences were found in the occurrence of aneup'.oidy or hypcrhaploidy in embryos obtained from immature, newly ovulated, and overmature oocytes. Finally, an increased incidence of polyploidy was detected in immature, 2–3 h b.o. (31.20%), and overmature, 3 h (23.04%) and 6 h (31.61%), groups as compared to the control group (14.59%).
The plasama membrane of Xenopus laevis spermatozoon
Authors: Bernardini Giovanni , Zanmarchi Gemma , Belgiojoso Pio
Published: February 2005

Xenopus laevis sperm plasma membrane ultrastructure has been studied by means of freeze‐fracture, deep‐etching, and lectin‐gold binding. Xenopus spermatozoa differ from those of other species in that their plasma membrane does not exhibit topographical domains. In fact, no geometric arrangement or characteristic array of particles is present on fractured plasma membrane. Fractures rarely occur in acrosomal or nuclear membranes. Wheat germ agglutinin receptors are distributed homogeneously, on the plasma membrane of the sperm head and tail.
Complex spermatozoon of the live‐bearing half‐beak, Hemirhamphodon pogonognathus (Bleeker): Ultrastructural description (Euteleostei, Atherinomorpha, Beloniformes)
Authors: Jamieson B. G. M.
Published: February 2005

The spermatozoon of Hemirhamphodon pogonognalhus shows modifications that are frequent though not obligate in internally fertilizing sperm, notably elongation of the nucleus and extension of the mitochondria of the midpiece as an elongate sheath around the proximal region of the axoneme. These similarities to poecilid and jenynsid sperm are considered homoplasic. As in the mature sperm of all but one investigated teleost, an acrosome is absent. The elongate, blade‐shaped, electron‐dense nucleus has a mean length of 3.2 μm; its basal implantation fossa, less than one‐tenth of the length of the nucleus, houses the anterior half of the distal and only centriole (of triplet construction with satellite rays), a centriolar plug, and a mass connecting the centriole to the wall of the fossa. A unilateral putative centriole adjunct is present. The anterior region of the axoneme is surrounded by a mitochondrial sleeve, and internal to this, separated by a cisterna, by a submitochondrial sleeve. The mitochondrial sleeve unites posteriorly with the submitochondrial sleeve. Between the submitochondrial sleeve and the axoneme is a space, the cytoplasmic canal, that is open to the exterior posteriorly. The discrete, cristate mitochondria, in their sleeve, are unique in investigated atherinomorph sperm in being bilateral, grouped on only two opposing sides of the axoneme, with an arc‐shaped ‘intermitochondrial link’ between. The 9 + 2 flagellum is unique for the Animalia in having 23 radial subplasmalemmal rods, repeated longitudinally (periodicity 0.025 pm) in a quasicrystalline array. Internal fertilization is deduced to have arisen in the Exocoetoidei independently of that in the Cyprinidcntiformes.
Ultrasound‐guided transfundal uterine sperm recovery from Macaca fascicularis
Authors: VandeVoort Catherine A. , Tollner Theodore L. , Tarantal Alice F. , Overstreet James W.
Published: February 2005

Previous studies from this center have indicated that the cynomolgus macaque (Macaca fascicularis) may serve as a model for human sperm interaction with the cervix and uterus. In some macaque species, transcervical aspiration of the uterine contents carries a significant risk of disturbing the cervical milieu due to the serpentine nature of the cervix. The only alternatives have been surgical procedures such as laparotomy or laparoscopy. In this paper, we report our experience with a new technique for ultrasound‐guided sampling of spermatozoa in the macaque uterus. Twenty adult female cynomolgus macaques were monitored for menses (first day of menses = day l), and one mating per cycle was allowed on day 10, 11, or 12. In one group of ten animals, cervical mucus was sampled at 3 or 18 hr postcoitus (pc) and ultrasound‐guided uterine aspiration was performed at 24 hours pc. In a second group of ten monkeys, uterine aspiration was at six hr pc and sperm numbers and motility were counted in the uterine fluid. Uterine fluid was obtained from fourteen of twenty monkeys. Pregnancy occurred in ten of the twenty experimental cycles. Ultrasound‐guided uterine aspiration appears to be a reliable method for the evaluation of sperm transport in female macaques. The correlations between uterine sperm recovery and cervical mucus sperm populations arc discussed. The high conception rate in treatment cycles indicates that this procedure can be performed without apparent risk to pregnancy.
Fabrication of Nanoscale Rings, Dots, and Rods by Combining Shadow Nanosphere Lithography and Annealed Polystyrene Nanosphere Masks
Authors: Kosiorek Adam , Kandulski Witold , Glaczynska Hanna , Giersig Michael
Published: February 2005

The application of shadow nanosphere lithography for the preparation of large‐area, two‐dimensional, metallic nanostructures of different shape is described. Through changing the mask morphology by temperature processing and varying the evaporation conditions, particles with morphologies such as rings, rods, and dots have been produced. This process allows outstanding control of the size and morphology of the particles. The efficient technique is shown to scale down the size of metallic nanoparticles from 200 to 30 nm, while preserving the original nanosphere spacing and order. The 150‐nm‐diameter Fe rings produced by this method show ferromagnetic behavior, which was predicted by theoretical simulation. All the experimental results were confirmed by computer simulations, which also showed the possibility of creating periodic arrays of any other geometrical shape.
Increased nuchal translucency and distended jugular lymphatic sacs on first‐trimester ultrasound
Authors: Bekker M. N. , Haak M. C. , Rekoert‐Hollander M. , Twisk J. , Van Vugt J. M. G.
Published: February 2005

Objective

To investigate the presence and volume of jugular lymphatic sacs (JLS) in first‐trimester fetuses with normal nuchal translucency thickness (NT) and in those with increased NT.

Methods

This was a prospective study of 26 fetuses with NT > 95th percentile, which were compared with 137 fetuses with normal NT. Following crown–rump length (CRL) and NT measurement the neck region of the fetus was studied by transvaginal ultrasound. The JLS presented as spheroidal translucencies in the anterolateral region of the neck.

Results

The prevalence of JLS differed significantly between fetuses with enlarged NT and the control group (P < 0.0001). In the group of 26 fetuses with increased NT, 22 had clearly visible JLS. Chorionic villus sampling revealed aneuploidy in 10 and euploidy in 16 fetuses. In the control group two fetuses, with NT values of 2.8 mm and 2.9 mm, had JLS; pregnancy outcome was normal in both cases. Logistic regression analysis in the total study group showed that an increase in NT was associated with a greater probability of JLS being present (for NT = 3–3.5 mm, probability = 0.67; for NT > 3.5 mm, probability = 0.93). In fetuses with JLS, an increase in CRL was associated with a significant increase in right JLS volume (r = 0.51; P‐value = 0.01) and a non‐significant increase in left sac volume (r = 0.40; P‐value = 0.09). Increase in NT was not associated with a significant increase in JLS volume.

Conclusion

There is a significant association between increased NT and the presence of JLS on first‐trimester ultrasound. In our opinion, the pathophysiological explanation for increased NT lies in a disturbance in lymphangiogenesis. Copyright © 2005 ISUOG. Published by John Wiley & Sons, Ltd.
Late signals are required for the stimulation of DNA synthesis in rat mammary fibroblasts by growth factors
Authors: Chen Hai-Lan , Rudland Philip S. , Smith John A. , Fernig David G.
Published: February 2005

Maximal stimulation of DNA synthesis in quiescent rat mammary (Rama) 27 fibroblasts is elicited by epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) 18 h after the initial addition of the growth factors-the ‘lag’ period. At maximally-stimulating concentrations, EGF and bFGF are interchangeable 9 h after their initial addition. When the initial concentration of growth factor is below that required to elicit a maximal response, it is possible to increase the level of DNA synthesis by increasing the concentration of growth factor 9 h after its initial addition. When the initial concentration of growth factor is high, substitution by a lower concentration of growth factor after 9 h allows a greater proportion of cells to synthesize DNA than would be expected from a continuous low dose of growth factor. Similar results are obtained when both the growth factor and its concentration are changed 9 h after the initial addition of growth factor. However, when EGF at a low concentration is substituted for a high concentration of EGF or bFGF the resulting increase in the levels of DNA synthesis is greater when EGF rather than bFGF is added for a second time. The half-life of the growth-stimulatory signals delivered by EGF and by bFGF 9 h after their initial addition is 1–2 h. These results suggest that to stimulate DNA synthesis: (i) EGF or bFGF must deliver a signal(s) continuously; (ii) the initial signals produced by EGF and bFGF are equivalent; (iii) the signals produced between 9–18 h by EGF may be different to those produced by bFGF.