bluuebunny/crossref_metadata_2025 huggingface dataset:
Human sperm‐egg interaction in vitro
Authors: Sathananthan A. Henry , Ng S. C. , Edirisinghe R. , Ratnam S. S. , Wong P. C.
Published: February 2005

The early events of gamete membrane fusion and sperm incorporation are portrayed, which are possibly the earliest pictures of human conception on record.

Oocytes recovered at laparoscopy from an in vitro fertilization programme using clomiphene and human menopausal and chorionic gonadotrophin stimulation were zona‐punctured and examined for polyspermic penetration 3–6 hours after insemination. Three eggs were penetrated, and one donor became pregnant in the same cycle.

Fusion occurred between the oolemma and the midsegment of the sperm cell membrane extending from the equatorial vestige of the acrosome to the anterior region of the postacrosomal segment. Only acrosome‐reacted sperm were capable of fusing with the egg. Fusion was followed by incorporation of the spermhead into the ooplasm by a process akin to phagocytosis. Sperm chromatin decondensation occurred by progressive inflation and disorganization of the original nuclear envelope.

The mechanism of gamete fusion and sperm incorporation resembles that observed during human monospermic fertilization and generally conforms to that reported for eutherian mammals.
Effect of triton X‐100 on ultrastructure, reactivation, and motility characteristics of ram spermatozoa
Authors: Vishwanath R. , Swan M. A. , White I. G.
Published: February 2005

For optimal extraction and reactivation of ram sperm, glutamate, dithiothreitol, magnesium, and cyclic AMP were required in a medium of pH 7.9. On extraction with 0.01 % Triton X‐100, ram sperm were only partially demembranated, and extensive areas of plasma membrane remained intact especially in the midpiece region. Treatment with 0.1% Triton X‐100 removed all plasma membranes and extracted the mitochondrial membrane and matrix. In the absence of ATP, 16.6% ± 0.4 of the partially demembranated sperm were motile, but sperm extracted with 0.1% Triton X‐100 were completely immotile. On adding ATP partially demembranated sperm reactivated better (81.6% ± 2.8) than sperm completely demembranated in 0.1 % Triton X‐100 (39.5% ± 4.6). The release of intracellular LDH rose linearly with increasing concentrations of the detergent from 0.01 to 0.05%, at which it plateaued. There was a significant increase in beat frequency and forward velocity of partially demembranated sperm when treated with ATP. Partially demembranated sperm had intact mitochondria that presumably were still able to produce ATP, although the spermatozoan movement was stimulated by exogenous ATP.
Glycoconjugates of the human sperm surface: Distribution and alterations that accompany capacitation in vitro
Authors: Cross Nicholas L. , Overstreet James W.
Published: February 2005

We have studied changes in the binding of fluoresceinated lectins to human sperm during in vitro capacitation. We first determined the surface labeling pattern of viable sperm obtained by the swim‐up procedure. Sperm were labeled with 100 μg/ml FITC‐conjugated lectin at 4°C for 30 min. We simultaneously used Hoechst stain 33258 as a supravital stain to help differentiate surface from intracellular lectin labeling. Of 14 lectins studied, six (phytohemagglutinin‐E, concanavalin A, Ricinus communis agglutinin‐I, and the lectins of wheat germ, Lens culinaris, and Pisum sativum) bound to the entire surface of sperm, sometimes with minor local heterogeneity. Three lectins (from peanut, Maclura pomifera, and soybean) usually bound in a punctate manner, with more label on the tail than on the head. Five lectins (Ulex europaeus, Dolichos biflorus, Helix pomatia, and Vicia villosa lectins, and lectin II of Griffonia simplicifolia) bound very poorly or not at all to the sperm surface. Sperm were also inspected for changes in surface lectin binding patterns after 0, 5, and 23 hr of incubation in a capacitating medium. Two lectins showed reproducible changes. The labeling by Maclura pomifera agglutinin decreased by 5 hr in eight of ten experiments, and among sperm labeled with concanavalin A, the incidence of sperm with a highly fluorescent anterior margin of the sperm head increased by about 3.5‐fold between 0 and 5 hr. The labeling pattern of the other lectins did not change.
Formation of the cylindrical structure during the acrosome reaction of abalone spermatozoa
Authors: Usui Noriko
Published: February 2005

Spermatozoa of abalone Haliotis discus were examined before and during the acrosome reaction with special regard to one of the newly formed structures: a cylindrical structure surrounding a part of the elongated acrosomal process near the opening of the acrosomal vesicle. The structure, about 0.2 μm in diameter and about 1 μm in length, was revealed to be composed of a tightly coiled, fine tubular structure about 20 nm in diameter. In the course of the acrosome reaction, a triple‐spiral structure appeared in the anterior part of the acrosomal vesicle. Since this spiral structure was also composed of a tightly coiled 20 nm tubule(s), it was concluded that this structure was transformed into the single‐walled cylindrical structure by simple stretching in the direction of its longitudinal axis. In the clumps of spermatozoa that underwent acrosome reaction in suspension, the cylindrical structures were frequently found in contact with each other and/or other structures, indicating that they are very sticky.
Ethanol inhibits human and hamster sperm penetration of eggs
Authors: Rogers B. Jane , Cash Mervina K. M. , Vaughn William K.
Published: February 2005

The effect of alcohol on the fertilizing ability of both human and hamster spermatozoa was examined by an in vitro fertilization assay using hamster ova. Spermatozoa were incubated in capacitating media for 3 hr (hamster sperm) and 4 hr (human sperm). Hamster ova were inseminated with preincubated sperm and were examined after 2 to 3 hr. Ethanol was added to the capacitating media at concentrations of 25, 50, 100, 200, and 400 mg%. Fertilization of zona‐free hamster eggs by human spermatozoa was reduced from 49.6% in no alcohol to 16.7% in 400 mg% ethanol. Fertilization of hamster eggs by hamster sperm revealed a reduction from 63.6% to 33.7% in cumulus‐intact eggs and from 65.8% to 10.8% in cumulus‐free eggs in the presence of ethanol at 400 mg%. Hamster sperm acrosome reaction was reduced from 47% to 12%. When these hamster sperm with reduced acrosome reaction were placed with zona‐free hamster eggs, the 100% fertilization rate was not reduced; however, the fertilization index, which reflects the number of swelling sperm heads per egg, was reduced from 8.5 to 1.8. This suggests that as little as 12% of the sperm with an acrosome reaction is sufficient to fertilize 100% of the zona‐free eggs. If ethanol was added to the insemination media only, there was no inhibition of fertilization by human sperm or hamster sperm that had been previously capacitated in an ethanol‐free media. Removal of the ethanol from the preincubated sperm produced fertilization at control levels; thus the inhibitory effect is reversible. These results indicate that ethanol may affect fertilization by an inhibition of the capacitation and/or acrosome reaction process.
Effects of superovulatory doses of pregnant mare serum gonadotropin on oocyte quality and ovulatory and steroid hormone responses in rats
Authors: Yun Young W. , Yuen B. Ho , Moon Y. S.
Published: February 2005

Immature rats (aged 28 days) were injected with 4, 20, or 40 IU pregnant mare serum gonadotropin (PMSG) and sacrificed every 6 or 12 hr. Control rats (4 IU) ovulated between 60 and 72 hr, whereas rats given superovulatory doses of PMSG (20 and 40 IU) ovulated between 24 and 72 hr. The oocyte count from the superovulated rats increased slightly between 24 and 36 hr and markedly between 48 and 72 hr. Degenerated oocytes were recovered 48 and 36 hr after administration of 20 and 40 IU PMSG, respectively. Thereafter, the proportion of degenerated oocytes was dose dependent and reached a maximum at 72 (30.9%, 20 IU) and 60 hr (61.0%, 40 IU). 17β‐estradiol content of the superovulated ovaries increased significantly (P < 0.01) from 36 hr and was maximal at 60 (20 IU) or 54 hr (40 IU), when compared to the control regimen. Administration of 40 IU PMSG resulted in a biphasic increase of progesterone content with the peaks at 36 and 60 hr. Androgen content of the superovulated ovaries was lower than control levels during the first 36 hr but was significantly (P < 0.01) higher thereafter. The results suggest that these alterations in the steroid response (particularly androgens) from 36 hr onward following superovulation may be responsible for the coincidental occurrence of abnormal oocytes, possibly by disturbing the specific intrafollicular steroid environment essential for complete maturation. In addition, oocyte aging that is due to earlier activation by the exogenous luteinizing hormone activity may be a contributing factor.
Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona‐free hamster eggs by bull sperm: I. A fertility assay for fresh semen
Authors: Graham J. K. , Foote R. H.
Published: February 2005

Fresh sperm from five bulls having nonreturn rates ranging from 48% to 77% were treated with 15.7, 21.0, 26.2, 31.5, 36.7, and 42.0 μM dilauroylphosphatidylcholine (PC12) to induce the sperm acrosome reaction (AR). Treated sperm were incubated 3 hr with zona‐free hamster eggs at 39°C prior to fixation. The eggs were then stained and examined for sperm penetration. Differences in the percentages of motile sperm and of sperm exhibiting an AR among bulls were small when compared on a within‐liposome‐concentration basis. Increasing the PC12 concentration from 15.7 μM to 42.0 μM increased the percentage of sperm exhibiting an AR for all bulls. At the lowest lipid concentration (15.7 μM), the percentage of eggs penetrated by sperm from the five bulls was 6% to 36%, with 0% in controls. When sperm were incubated with increasing lipid concentrations, the egg penetration rate increased to over 80%, and the total number of sperm increased to over 100 per 36 eggs in each treatment for every bull. These penetration rates decreased at the highest lipid concentration. A correlation between the PC12 concentration maximizing egg penetration and the nonreturn rate of −.63 was found. The correlation between the PC12 concentration maximizing the total number of penetrated sperm per treatment and the bull nonreturn rate was −.96. It was concluded that PC 12 liposomes induce the AR in bull spermatozoa, which enables them to penetrate zona‐free hamster eggs. High fertility bulls required less lipid to induce the AR than did lower fertility bulls. Consequently, this assay of fresh semen could provide a laboratory method to estimate the fertility of a bull.
Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona‐free hamster eggs by bull sperm: II. A fertility assay for frozen‐thawed semen
Authors: Graham J. K. , Foote R. H.
Published: February 2005

Frozen‐thawed sperm from five bulls with fertility rates ranging from 48% to 77% were treated with seven concentrations of dilauroylphosphatidylcholine (PC12) liposomes to induce an acrosome reaction (AR) that enabled sperm to penetrate eggs. Treated sperm were incubated with liposomes for 7 min prior to insemination of zona‐free hamster eggs in vitro. Sperm and eggs were incubated 3 hr at 39°C prior to fixation, staining, and examination for sperm penetration and nuclear decondensation. The percentage of motile sperm immediately after thawing as well as after treatment with liposomes had a low correlation with sire fertility (r = .39 and ⩽.63, respectively). The percentage of sperm exhibiting an AR was more highly correlated with fertility (r ⩽ −.85). Similar correlations were found between fertility and the penetration rates of zona‐free hamster eggs or the total number of penetrating sperm. When data for two high and for two lower fertility buils were each grouped to increase information per data point the correlation between the PC12 concentration giving the maximum proportion of eggs penetrated and fertility was r = .92 (P ≤ .05). The correlation between the PC12 concentration producing the most total sperm penetrating the eggs and fertility r = .97 (P ≤ .05). It was concluded that PC12 liposomes induced an AR in bull sperm frozen‐thawed in egg yolk extender. Frozen‐thawed sperm from low fertility bulls require less PC12 to induce the AR and to penetrate zona‐free hamster eggs than do sperm from higher fertility bulls. These differences in lipid requirements may help to provide a quick, direct laboratory assay method to estimate the fertility of frozen bull semen.
Parthenogenetic activation of cattle follicular oocytes in vitro with ethanol
Authors: Nagai T.
Published: February 2005

Cattle follicular oocytes cultured in vitro for 24–33 h were treated with ethanol to induce artificial activation. When oocytes were cultured for 27–33 h before ethanol treatment, 60–68% of oocytes were activated and were found to have a female pronucleus(ei). In contrast, maturation culture of oocytes for 24–26 h resulted in low activation rates (25–38%). The female pronucleus was formed in the activated oocytes within 8–10 h of incubation after ethanol treatment. And it became visible under interference‐contrast microscope by centrifugation for 3 min at 15,000g and 10 min at 20,000g. These results indicate that ethanol treatment is effective for activation of cattle follicular oocytes and that the pronucleus formed in the activated oocyte can be visualized by centrifugation.
Ultrastructural localization of labeled acrosomal glycoproteins during in vivo fertilization in the rabbit
Authors: Kopečný V. , Fléchon J.‐E.
Published: February 2005

Rabbit spermatozoa were labeled predominantely in their acrosomal glycoproteins by 1‐3H‐glucosamine during spermiogenesis. Ova fertilized in vivo by spermatozoa labeled 22 days earlier were analyzed by fine‐structure autoradiography for the localization of the label. The latter was found associated with 1) the fused membranes of the acrosomal cap remaining on the zona pellucida surface, 2) the material released on the zona surface after the acrosome reaction and possibly detectable after tannic acid fixation, 3) the equatorial segment of the sperm head and the preequatorial swellings, and 4) other sperm components, eg, the sperm tail. No labeling, on the other hand, was detected on the denuded leading edge of spermatozoa found either in the penetration slit or in the perivitelline space. Our observations suggest the involvement of acrosomal glycoproteins in different mechanisms of sperm/zona pellucida interaction but are not in favor of a major role of (enzymatic) glycoproteins bound to the inner acrosomal membrane during the penetration of the zona pellucida.
Intermolecular cross‐linking of vitelline envelope polypeptides predominates in the hardened sea urchin fertilization envelope
Authors: Santiago Lyric , Carroll Edward J.
Published: February 2005

At fertilization, the sea urchin egg vitelline envelope (VE) elevates, and a subset of released cortical granule proteins, paracrystalline protein fraction (PCF), associates with the VE to form the fertilization envelope (FE). Cortical granule peroxidase cross‐links FE polypeptides by phenolic coupling of tyrosyl residues. We have used an immunological approach to determine which polypeptides are linked together in the hardened FE of Strongylocentrotus purpuratus. Soluble polypeptides were extracted from hardened FEs, and antibodies were prepared in rabbits against the insoluble envelope matrix (FE ghost). Whole immune serum and purified IgGs each reacted with FE ghosts when using an enzyme‐linked immunosorbent assay. VEs isolated by means of three published procedures cross‐reacted with the immune serum and purified IgGs. Soluble FE polypeptides also cross‐reacted with whole immune serum and IgGs owing to the presence of VE polypeptides. Hyalin, a protein not found in FEs, and PCF did not cross‐react with antiserum against FE ghosts. To determine which VE polypeptides were cross‐linked in the hardened FE, VE polypeptides were immunoblotted by using antiserum against FE ghosts. Most of the VE polypeptides that ranged from 68,000 to 283,000 molecular weight cross‐reacted with the antibody.
Actin and calmodulin coexist in the equatorial segment of ejaculated boar sperm
Authors: Camatini Marina , Casale Antonella
Published: February 2005

The direct overlay technique with iodinated calmodulin on boar sperm fractions evidenced a strong bond in the 45,000 molecular weight, which is the region recognized by anti‐actin antibodies. This result and the close codistribution of the two staining patterns for calmodulin and actin with immunofluorescence and immunoelectronmicroscopy suggest a possible interaction between calmodulin and actin in boar sperm.
Isolation of a stable apical segment of the guinea pig sperm acrosome
Authors: Stojanoff A. , Bourne H. , Andrews A. G. , Hyne R. V.
Published: February 2005

The large apical segments of guinea pig sperm acrosomes were mechanically separated from the spermatozoa and subsequently isolated by density gradient centrifugation. The isolated acrosomal caps were very stable and maintained their crescent morphology when suspended in sucrose‐based medium buffered at pH 5.6, with or without the acrosin inhibitor p‐aminobenzamidine (pAB). Examination under the electron microscope showed that the acrosomal caps were free of plasma membrane and were bound by an outer acrosomal membrane which was discontinuous. Enzymatic analysis after lysis of the caps indicated that acrosin and hyaluronidase were present with high specific activity, while only a trace amount of acid phosphatase activity and no arylsulphatase, phospholipase A2, or phospholipase C activities were present. Significant particulate acrosin activity, but only trace amounts of soluble acrosin activity, could be detected in the isolated acrosomal caps if assayed immediately after isolation in the absence of pAB. However, soluble acrosin activity of high specific activity was obtained after the acrosomal caps were extracted by 10% glycerol buffered at low pH (pH 3.0). The new procedures provide a means to isolate and purify guinea pig sperm apical acrosomal segments rapidly.
Lectin binding sites in the vitelline envelope of Bufo arenarum oocytes: Role in fertilization
Authors: del Pino Enrique J. , Cabada Marcelo O.
Published: February 2005

Incubation of dejellied spawned oocytes from Bufo arenarum with different lectins results in a decrease of oocyte fertility. Concanavalin A was the most effective lectin; phytohem‐agglutinin P and wheat germ lectin were less effective. Agglutinin from soybean was scarcely active. These lectin effects could be ascribed to a hindering of specific sites for some proteases, since the same treatment renders the oocyte vitelline envelope insensitive to spermatolysin (an essential requisite for fertilization) and to trypsin. Also in this case concanavalin A was the most effective lectin. Univalent concanavalin A was also effective in blocking the fertility of dejellied oocytes. These results indicate that the residues of α‐D‐glucose and α‐D‐mannose present in the vitelline envelope are involved in gamete interactions in Bufo arenarum. This idea is also supported by the finding that dejellied oocytes (fertilizables) have a number of binding sites for concanavalin A that is three or four orders of magnitude higher than coelomic or fertilized oocytes (both not penetrable by spermatozoa).
Follicular characteristics associated with viable pregnancy after in vitro fertilization in humans
Authors: Nayudu P. L. , Gook D. A. , Lopata A. , Sheather S. J. , Lloyd‐Smith C. W. , Cadusch P. , Johnston W. I. H.
Published: February 2005

The aim of this study has been the development of a noninvasive method of predicting the pregnancy potential of human oocytes and embryos intended for in vitro fertilization and embryo replacement. A multifactorial system which distinguishes, with a high degree of accuracy, between normal pregnancy, abnormal pregnancy, and non‐pregnancy‐producing embryos is reported. The variables included are (1) follicular fluid proteins alpha1‐antitrypsin, complement C3, immunoglobulin IgG2, and total protein, and total proteoglycan level separated by isoelectric focusing; (2) follicular volume; and (3) an embryo appearance rating. The study group consisted of (1) follicles which produced embryos of known performance after transfer (a) when the number of embryos transferred = the number of implantations and, (b) where one embryo transferred = no pregnancy; (2) follicles which produced oocytes which did not cleave after insemination; and (3) follicles from which no oocyte was aspirated. Canonical discriminant analysis of follicular fluid variables and follicular volume has been used to characterize the oocyte performance groups. Correct classification was achieved in 69% of normal pregnancy, 70% of abnormal pregnancy, 33% of no pregnancy, and 47% of no cleavage oocytes. An embryo appearance rating was included with the above variables for a separate discriminant analysis of only those oocytes which had formed embryos after insemination. Correct classification was achieved in 81% of normal‐pregnancy, 70% of abnormal‐pregnancy, and 70% of nopregnancy embryos.
Binding of morphologically abnormal sperm to mouse egg zonae pellucidae in vitro
Authors: Kot Mary C. , Handel Mary Ann
Published: February 2005

The ability of morphologically abnormal mouse sperm to bind to the zona pellucida of the egg was examined with techniques of fertilization in vitro. After incubation with sperm, cumulus‐free ova were scored by either phase microscopy or scanning electron microscopy for the number and type of sperm bound. The percentages of abnormal sperm bound to zonae were compared to the percentages of abnormal sperm in the inseminating suspension. In general, all abnormal classes (except broadly spatulate sperm) bound to zonae at a frequency significantly lower than their representation in the inseminating suspension. However, when the percentage of abnormal sperm was quite high, no significant difference existed between frequencies of abnormal sperm bound and in the inseminating solution. The percentage of abnormal sperm bound did not increase significantly over time. Scanning electron microscopy studies demonstrated that the association of abnormal sperm with the zona pellucida varied according to sperm morphology. Normal and some abnormal sperm bound at an angle perpendicular to the zona, while more grossly abnormal sperm bound to ova tangentially.
A quantitative assay for capacitation: Evaluation of multiple sperm penetration through the zona pellucida of salt‐stored hamster eggs
Authors: Boatman Dorothy E. , Andrews Jane C. , Bavister Barry D.
Published: February 2005

The endpoint for sperm capacitation occurs when spermatozoa become able to penetrate intact zonae pellucidae of unfertilized homologous eggs. Activation of eggs stimulated by sperm fusion or by gamete aging initiates changes in zonae pellucidae that bar further sperm entry. This zona block mechanism reduces the usefulness of eggs as indicators for sperm capacitation. Egg storage in concentrated salt solutions destroys the zona block mechanism while retaining the biological sperm receptor/activator functions of the zona pellucida [Yanagimachi et al., Fertil Steril 31:562–574, 1979]. We have developed techniques for the quantitative assay of capacitation using multiple sperm penetration into zonae and have evaluated the sperm‐response characteristics of hamster eggs stored in a modified salt solution (STOR). Sperm, preincubated in capacitating or noncapacitating treatments (CAP or NOCAP, respectively), were coincubated with unfertilized and fertilized STOR‐treated zonae for 4 hr. Then zonae were stripped of externally bound sperm and the sperm heads in the perivitelline space (PVS) were fluorescently labeled with Hoechst dye 33342. Entry of CAP sperm into the PVS of STOR‐treated unfertilized eggs was highly correlated with sperm concentration (Rw equals; .859) and was log linear from 1 × 103–2 × 105 CAP sperm/ml. At 2 × 105 CAP sperm/ml, the mean number of PVS sperm was 63.5 and the maximum observed was 158. NOCAP sperm very rarely penetrated unfertilized zonae (2 sperm into 75 eggs). Few CAP sperm entered the PVS of STOR‐treated fertilized eggs (two sperm into 54 eggs at 2 × 105 sperm/ml) unless the sperm concentration was raised to high levels. Differences between replicates were due to male but not female (ie, egg) differences. We conclude that (1) STOR‐treated unfertilized zonae can be used to accurately quantitate differences between sperm capacitation and/or fertility states and (2) the availability of large numbers of homogeneous “shelf‐stable” zonae will make it feasible to perform hamster sperm capacitation bioassays on a large scale.
Evaluation of assay conditions for the zona‐free hamster ova bioassay of boar sperm fertility
Authors: Berger Trish , Horton M. B.
Published: February 2005

The ability to penetrate zona‐free hamster ova may be a very useful test of fresh and frozen boar sperm fertility. These studies were designed to optimize assay conditions prior to evaluation of the accuracy of the bioassay in predicting boar sperm fertility. The ability to penetrate zona‐free hamster ova was greater in sperm washed on a Percoll gradient than in sperm washed by dilution and centrifugation. Penetrating ability was greater in sperm from the sperm‐rich fraction than from the whole ejaculate but did not differ among different aliquots of the sperm‐rich fraction and did not decrease when the prewashing interval was increased from 15 to 85 min. Frequency of collection of ejaculates (1, 3, or 5 times per week) did not affect the penetrating ability of the sperm. Penetration rate was greater when sperm were coincubated with zona‐free hamster ova at 39°C compared to 37°C. Sperm from an infertile boar had reduced penetrating ability compared to sperm from fertile boars (11% vs 93%, P < .001). These studies suggest that the zona‐free hamster ova bioassay may be a useful assessment of fresh boar sperm fertility.
Synthetic organic pH buffers can support fertilization of guinea pig eggs, but not as efficiently as bicarbonate buffer
Authors: Bhattacharyya Amitabha , Yanagimachi R.
Published: February 2005

Bicarbonate ions (HCO3−) in the medium are not absolutely essential for the fertilization of guinea pig eggs. However, fertilization takes place most efficiently in HCO3−‐buffered medium. Capacitation, the acrosome reaction, and zona penetration by spermatozoa can occur in HCO3−‐free media with synthetic organic buffers (i.e., MOPS, TES, HEPES, Tris, and TAPSO) but not as efficiently as in the HCO3−‐buffered medium. It appears that HCO3− functions as more than just a pH‐buffering molecule.
Effects of induction current and other factors on large‐scale electrofusion for pronuclear transplantation of mouse eggs
Authors: Kono T. , Tsunoda Y.
Published: February 2005

In this paper, we describe the procedure of large‐scale and efficient electrofusion for pronuclear transplantation in mouse eggs and the tolerance of the eggs for electric stimulus, assessed in vitro and in vivo development. The fusion chamber was arranged in parallel by dielectrodes (30‐mm length, 1‐mm width, and 2‐mm height), and 0.3 M mannitol in distilled water was used as a fusion solution. The agglutination cleavage of enucleated eggs with karyoplast was easily orientated in parallel with electrodes by alternating current between 100 and 500 kHz at 2 and 10 V/mm. Immediately after the orientation, a direct current of 150 V/mm was given for 200 μsec twice and repeated three times to induce fusion of the enucleated eggs with karyoplast. More than five eggs, at least, can be submitted to electrofusion at the same time. The eggs that were not fused were treated again in the same manner. The proportion of eggs fused with karyoplast was increased by preincubation in M16 medium prior to submitting them to the electrofusion. When the eggs were incubated for 60 min, 80% of them were fused with karyoplast by the first electric treatment; in contrast, only 19% of the eggs were fused if they were submitted to electrofusion directly. It was found that between the CD‐1 and F1 strains there was a difference in tolerance of the eggs to electric stimulus and that this was depend on the nuclei but not on cytoplasm. The proportion of development to blastocyst in the eggs fused with the pronuclear karyoplast derived from F1 (75 and 71%) was twice that of the eggs fused with the pronuclei derived from CD‐1 strain (25 and 37%). After transfer to recipients, live young were obtained from both the eggs fused with karyoplast following one or two electrofusion exposures.
Time sequence of germinal vesicle breakdown in pig oocytes after cycloheximide and p‐aminobenzamidine block
Authors: Kubelka M. , Motlík J. , Fulka J. , Procházka R. , Rimkevičová Zora , Fulka J.
Published: February 2005

All porcine oocytes cultured 20 hr in medium with 10 μg/ml cycloheximide rested in the germinal vesicle (GV) stage but with the highly condensed bivalents in nucleoplasm. When these oocytes were washed and cultured in the control medium for 2, 4, and 6 hr, germinal vesicle breakdown (GVBD) was completed in 0, 86, and 100% of them, respectively. When similarly inhibited oocytes cultured successively only 2.5 hr in the control medium were given again in cycloheximide enriched medium (3.5 hr), nearly all of them reached late diakinesis stage again. It means that oocytes cultured for 20 hr and washed free of this inhibitor of protein synthesis completed GVBD rapidly (4 hr) and protein synthesis crucial for nuclear membrane disintegration occurred already during the first 2 hr after washing of inhibitor. All oocytes cultured for 20 hr in medium with 1 mM p‐aminobenzamidine rested in GV with chromatin around the compact nucleolus. The successive culture in cycloheximide (20 hr) and p‐aminobenzamidine (10 hr) prevented GVBD in all oocytes, too. In contrast, when the oocytes washed after cycloheximide block (20 hr) were cultured in p‐aminobenzamidine enriched medium 2 and 3 hr and again for 6 hr in cycloheximide medium, the nuclear membrane dissolved in 62 and 68% of oocytes, respectively. These data suggest that inhibition of protein synthesis in pig oocytes does not prevent the high condensation of bivalents in GV. However, nuclear membrane breakdown requires the successive protein synthesis and proteolysis.
Partial characterization of a fraction from human follicular fluid that initiates the human sperm acrosome reaction in vitro
Authors: Siiteri Jon E. , Gottlieb Wayne , Meizel Stanley
Published: February 2005

A human follicular fluid (HFF) fraction prepared by Sephadex G‐75 column chromatography has been previously shown by this laboratory to initiate the human sperm acrosome reaction (AR) in vitro. In the present report, the apparent molecular weight (MW) of this AR activity determined by a longer G‐75 column than was used in the previous work was 50,000 ± 5,106. The G‐75 Sephadex void volume fractions of some but not all HFF samples were also found to contain some AR‐initiating activity. The occasional void volume activity was less potent than that of the 50,000 MW fraction and was not studied further. Further characterization of the 50,000 MW fraction was carried out. A time‐course study demonstrated that maximum AR were obtained within 5 min following the addition of the 50,000 MW fraction. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) followed by silver staining revealed that the 50,000 MW fraction was still a relatively crude preparation. Treatment of the 50,000 MW fraction with chloroform:methanol did not extract the AR‐initiating activity into the lipid phase. The AR‐initiating activity of the untreated 50,000 MW fraction was precipitated when it was boiled, but the activity was partially resistant to boiling after overnight incubation. Treatment of the 50,000 MW fraction with pronase E or with several glycosaminoglycan hydrolases did not destroy the activity. Pronase treatment resulted in a higher amount of boiling‐resistant AR‐initiating activity. The AR‐initiating activity of the untreated 50,000 MW fraction was partially dialyzable, but the activity of an undialyzed fraction did not pass through an ultrafiltration membrane with a 10,000 MW cut‐off. However, treatment of the 50,000 MW fraction with protease, peptide:N‐glycosidase F, and to a lesser extent chondroitinase ABC yielded an active lower MW activity which could pass through such an ultrafiltration membrane. The lower MW activity released by peptide:N‐glycosidase F eluted in the included volume (5,000–1,000) of a Sephadex G‐25 column. Neutral hexose but not protein or peptide was detected in the G‐25 peak of AR‐initiating activity. These results suggest that the AR‐initiating activity present in the 50,000 MW fraction of HFF: (1) is present either as two different AR factors (a high‐MW factor and a low‐MW, noncovalently bound factor) or as a single factor responsible for both the nondialyzable and dialyzable AR‐initiating activities (the latter being enzymatically released from the former), and (2) may be at least partially associated with N‐linked oligosaccharides of a glycoprotein or proteoglycan.
Intracellular pH change does not appear as a prerequisite for triggering activation of Barnea candida (Mollusca, Pelecypoda) oocytes
Authors: Brassard M. , Duclohier H. , Moreau M. , Guerrier P.
Published: February 2005

Barnea Candida oocytes, exposed to excess KCl, ammonia, or digitonin, exhibit germinal vesicle breakdown (GVBD) and reinitiate meiosis, at least up to first polar body extrusion. While we confirm that KCl—but not ammonia‐induced activation requires external calcium, our findings that digitonin is effective at any pH from 6 to 8, in the presence of calcium, while the phorbol ester TPA and diacylglycerol fail to reinitiate meiosis, strongly suggests calcium as the main trigger for this process. Preliminary experiments using the fluorescent probes fluorescein diacetate and Quin 2/AM show, moreover, that KCl and ammonia produce both an intracellular calcium surge (30 nM) and a slight alkalinization of the intracellular cytoplasm from 7.84 to 8.05.
Effect of in vitro storage of bovine spermatozoa on acrosomal integrity, proteolytic activity, binding, and initiation of penetration of oocytes
Authors: Miller B. J. , Graves C. N. , Lodge J. R.
Published: February 2005

During a 10‐day 5°C storage and subsequent 4–6‐hr 37°C incubation, both percent live spermatozoa and percent spermatozoa with an intact acrosome decreased, and percent spermatozoa with a late‐reacted or without an acrosome increased. When stored spermatozoa were mixed with oocytes, no decrease in percent of oocytes with spermatozoa bound or percent of oocytes with spermatozoa starting to penetrate occurred as storage time increased. A 58% decrease in acrosin gelatinolytic activity and a 56% decrease in acrosin esterolytic activity but no decrease in nonacrosin proteolytic activity were evident over the 10‐day storage. These studies show that a change in acrosomal morphology as well as a loss of acrosin may be responsible for the decreased fertility following extended in vitro storage of mammalian spermatozoa.
Sperm tail differentiation in the nudibranch mollusc Hypselodoris tricolor (gastropoda, opisthobranchia)
Authors: Medina A. , Moreno F. J. , García‐Herdugo G.
Published: February 2005

The sperm axoneme of Hypselodoris tricolor forms from a single centriole that is located initially beneath the plasma membrane and then migrates to the nuclear surface. A conspicuous centriolar adjunct‐like formation is present in the neck of midspermatids, but it becomes very reduced at the end of spermiogenesis. In spermatocyte and spermatid mitochondria, intracristal bodies originate from the accumulation of a dense material in some cristae. From our observations and foregoing reports, it may be concluded that the process of sperm tail differentiation in opisthobranchs resembles that in pulmonates, whereas it differs in many respects from that occurring in prosobranchs. The appearance of intracristal bodies in modified mitochondria seems to be a special feature of spermatogenesis in the opisthobranchs that does not occur in the two other groups of gastropod molluscs.
Binding of chemotactic peptide to the outer surface and to whole human spermatozoa with different affinity states
Authors: Ballesteros Luz Ma. , Delgado Nestor M. , Rosado Adolfo , Correa Carlos , Hernandez‐Perez Omar
Published: February 2005

Binding of N‐formyl‐methionyl‐L‐leucyl‐[3H]phenylalanine (fML[3H]Ph) to human ejaculated spermatozoa and to its isolated plasma membrane was studied. Our data confirm the presence of specific receptors for f‐MLPh in the human spermatozoa and suggest that whole spermatozoa receptors exist in two affinity states, one high‐affinity, low‐capacity specific receptor (Kd = 12.3 ± 0.5 nM, n = 22,285 ± 65,008 binding sites per sperm cell) and a second one (Kd = 700 ± 47 nM) that is not saturable, indicating a low‐affinity, high‐capacity nonspecific site. In contrast, sperm membrane showed only one class of binding site (Kd = 6.4 ± 0.12 nM), which was statistically different from that of the high‐affinity binding site of intact spermatozoa. To explain this difference we discuss the possibility that first, the two binding affinities represent two interconvertible states of a single receptor population, which, depending on the metabolic activity of spermatozoa, may change its physicochemical properties; or second, they reflect two different processes, binding and/or transport into the spermatozoa.
Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid
Authors: Borsuk Ewa , Mańka Renata
Published: February 2005

Our objective was to examine the developmental fate of sperm nuclei in oocytes fertilized under conditions of meiotic arrest. Therefore zona‐free metaphase II oocytes and oocyte fragments (nucleate and anucleate) were fertilized in the presence of colcemid. In anucleate oocyte fragments, normal male pronuclei develop. In contrast, in intact oocytes and nucleate fragments sperm nuclei after initial decondensation undergo secondary condensation. This state is maintained as long as the oocytes are treated with colcemid. When the drug is removed 3 h after insemination, the meiotic spindle(s) is reconstructed, the second polar body(ies) is extruded, and a female pronucleus (or micronuclei) forms. At the same time the sperm nucleus decondenses again and transforms into a male pronucleus. In addition oocytes fertilized in the presence of colcemid could not be refertilized. These observations suggest that oocytes and oocyte fragments fertilized in the presence of colcemid undergo activation despite the failure of pronucleus formation. The inhibitory effect of colcemid on the formation of pronuclei is expressed only in the presence of oocyte chromosomes. We suggest that colcemid stabilizes factors responsible for chromosome condensation that are associated with oocyte chromosomes but not factors (whether the same or different) present in the cytoplasm.
An influx of extracelluar calcium is required for initiation of the human sperm acrosome reaction induced by human follicular fluid
Authors: Thomas Paul , Meizel Stanley
Published: February 2005

The role of Ca2+ in the human sperm acrosome reaction was investigated using the fluorescent calcium indicator fura‐2. Previous experiments have shown that a Sephadex G‐75 column fraction of human follicular fluid can stimulate the human sperm acrosome reaction [Suarez SS, Wolf DP, Meizel S (1986): Gamete Res 14:107–121]. Using fura‐2, we demonstrated that this Sephadex G‐75 fraction also stimulates a rapid, transient increase in intracellular free Ca2+. This Ca2+ transient is blocked either by chelation of extracellular calcium or by addition of the Ca2+ antagonist La3+. We have also been able to stimulate the acrosome reaction in human sperm without significant loss of motility, using the divalent cation ionophore ionomycin. Acrosome reactions stimulated by whole follicular fluid, the G‐75 fraction, or ionomycin are all blocked by removal of extracellular Ca2+. These results strongly suggest that an influx of extracellular Ca2+ is responsible for intiating the acrosome reaction in human sperm treated with human follicular fluid. This is the first demonstration in mammalian sperm that a potentially physiological stimulus can cause an increase in intracellular Ca2+ concomitant with the acrosome reaction.
Success of fertilization in golden hamsters is a function of the relative gamete ratio
Authors: Stewart‐Savage J. , Bavister Barry D.
Published: February 2005

Gamete concentrations can be expressed as either absolute gamete ratios (sperm/egg), the concentration of the motile sperm (sperm/ml), or as a relative gamete ratio (sperm/egg × ml). We demonstrate that the success of hamster fertilization in vitro is a function of the relative gamete ratio and that any effect of the insemination medium geometry is minimal. Consistent fertilization occurs when the relative gamete ratio is above 103.5 sperm/egg × ml, but becomes variable above 105.0 sperm/egg × ml. At suboptimal relative gamete ratios, there is a strong sperm concentration effect on both the consistency and level of fertilization, whereas the absolute gamete ratio only affects the overall level of fertilization. These effects are seen when the sperm concentration is below 103.5 sperm/ml and when the absolute gamete ratio is below 102.3 sperm/egg. These influences are probably due to reduced sperm survival at low sperm concentrations and due to a sampling error that occurs when small numbers of sperm are transferred. When the absolute gamete ratios found in vivo in the hamster [Cummins and Yanagimachi, 1982, Gamete Res 5, 239–256] are converted to relative gamete ratios, they are similar to our in vitro results. Thus relative gamete ratios allow, for the first time, comparison between in vitro and in vivo data in the hamster, and between other rodents.
Analysis of calmodulin acceptor proteins and the influence of calmodulin antagonists on human spermatozoa
Authors: Aitken R. J. , Clarkson J. S. , Hulme M. J. , Henderson C. J.
Published: February 2005

The possible role of calmodulin in regulating a number of calcium‐dependent functions exhibited by human spermatozoa was investigated by using the antagonists trifluoperazine and calmidazolium. At high doses both antagonists inhibited the motility of human spermatozoa and induced a concomitant rise in [Ca2+]i and a decline in cAMP. Lower doses of these antagonists, particularly calmidazolium, suppressed the ability of human spermatozoa to generate reactive oxygen species and exhibit sperm‐oocyte fusion, without influencing [Ca2+]i, cAMP, or motility. This inhibition of sperm‐oocyte fusion was effective even if the spermatozoa were subsequently exposed to A23187, suggesting that calmodulin may regulate this aspect of human sperm function at a point downstream from calcium influx.

Both radiolabelling and affinity chromatography techniques were used to detect a number of calcium‐dependent and calcium‐independent calmodulin acceptor proteins in the human spermatozoon. The major calcium‐dependent acceptor proteins exhibited Mr values of 32,000 and 22,000–27,000, respectively, and did not appear to be associated with the sperm plasma membrane.
Development of enucleated parthenogenones that received pronuclei or nuclei from fertilized mouse eggs
Authors: Tsunoda Y. , Shioda Y.
Published: February 2005

This study compares the developmental potential of enucleated mouse parthenogenones that received pronuclei from fertilized eggs with those that received nuclei from late two‐cell embryos. The proportion of reconstituted parthenogenones, which received pronuclei at the one‐cell stage, that developed to blastocysts in vitro and to live fetuses after transfer to recipients was significantly lower than that of reconstituted control eggs. However, the in vitro and in vivo developmental potential of reconstituted parthenogenones that received nuclei at the two‐cell stage from fertilized late two‐cell embryos was not different from that of reconstituted control eggs.

These results were contrary to those reported previously by Mann and Lovell‐Badge (1984), who showed that parthenogenetic eggs receiving pronuclei from fertilized eggs developed well both in vitro and in vivo.
A gonadotropin‐releasing hormone agonist and an activator of protein kinase C improve in vitro oocyte maturation in Macaca fascicularis
Authors: Lefèvre B. , Gougeon A. , Peronny H. , Testart J.
Published: February 2005

Cynomolgus monkey oocytes were recovered from follicles > 1,000 μm in diameter at day 8 in gonadotropin‐stimulated cycles and cultured in vitro for 2 days. Germinal vesicle breakdown (GVBD) occurred in 30.3% of control oocytes (n = 76) compared with 54.0% and 55.0% of oocytes cultured in presence of a gonadotropin‐releasing hormone agonist (GnRHa), Buserelin (n = 50), or a protein kinase C activator, 1‐oleoyl‐2‐acetyl‐rac‐glycerol (OAG, n = 40), respective (P < .01). As similar results were obtained using OAG or Buserelin, we hypothesize that the action of Buserelin upon the primate oocyte is protein‐kinase‐C dependent. The possible effects of Buserelin on in vivo oocyte maturation have to be detemined.
Capacitation potential of the fallopian tube: A study involving surgical insemination and the subsequent incidence of polyspermy
Authors: Hunter R. H. F. , Nichol R.
Published: February 2005

In an attempt to demonstrate limitations in the capacitating potential of the Fallopian tube, ejaculated boar spermatozoa were introduced directly into the isthmus at varying intervals before ovulation. The incidence and degree of polyspermy subsequently observed were taken as indicators of the population of capacitated spermatozoa confronting the newly ovulated eggs: the more extensive the condition of polyspermy, the greater the number of capacitated spermatozoa presumed to have been available at the site of fertilization. Results are based on 673 eggs from 53 animals.

When suspensions containing 2.21–3.87 × 108 sperm per ml were introduced 36–40 hours and 26–30 hours before ovulation, 85% and 61% respectively of the eggs were polyspermic, such eggs exhibiting mainly dispermy and trispermy. By contrast, when comparable sperm suspensions from the same boar were instilled 17–18 hours before ovulation, 70% of the eggs were polyspermic but the degree of polyspermy had increased dramatically: most eggs contained 40 or more sperm heads in the vitellus, invariably forming swollen chromatin aggregates rather than male pronuclei. Surgical insemination at times closer to ovulation significantly reduced the incidence and degree of polyspermy, reaching a low of 2% with insemination 1–2 hours before ovulation. These results therefore support the concept of a limited capacitation potential of the Fallopian tube.

In a separate series of observations, mating animals shortly before surgical insemination with sperm suspensions from the same boar markedly reduced the incidence of polyspermy. This latter observation may be of clinical significance in procedures of laparoscopic or transcervical insemination into the tubes to alleviate human infertility. The manner whereby myosalpingeal physiology could be modified in response to coital stimulation is discussed.