bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructure of cortical granule release and zona interaction in monospermic and polyspermic human ova fertilized in vitro
Authors: Sathananthan A. H. , Trounson A. O.
Published: February 2005

Cortical granule release and interaction with the zona pellucida are reported in monospermic and polyspermic fertilized ova and early human embryos cultured in vitro.

Twenty‐seven preovulatory oocytes from women with tubal or idiopathic infertility were recovered by laparoscopy, after induction of follicular maturation with clomid and human chorionic gonadotropin. These were then inseminated with husband's or donor sperm, cultured for 3–72 hr, routinely fixed in glutaraldehyde/osmium and examined ultrastructurally.

Evidence of cortical granule release was observed in all ova and embryos investigated and their contents were identified either at the egg surface or in the perivitelline space or interacting with the inner zona, apparently reinforcing its structure. The latter is very likely the morphological expression of the zona reaction.

Delayed release was seen in certain regions of normally fertilized ova and particularly in polyspermic ova, where massive “explosions” of granules occurred. This was attributed to delayed cortical maturation. The mechanics of release were similar in both monospermic and polyspermic ova. Spontaneous dehiscence was also described in one injured unfertilized oocyte.

The significance of the cortical and zona reactions as an effective block to polyspermy at the level of the inner zona, which becomes more impenetrable to supplementary sperm, is discussed.
Evidence against a (2)n synchronous increase of spermatogonia to produce spermatocytes in drosophila hydei
Authors: Hanna Peter J. , Liebrich Winfrid , Hess Oswald
Published: February 2005

The numbers of primary spermatocytes within cysts as well as numbers of postmeiotic spermatids in bundles in Drosophila hydei were determined. Within the contents of a single testis the cysts of primary spermatocytes are found to contain 5–11 germ cells. Furthermore, the number of spermatocytes per cyst is age‐dependent, in that pupae have a mean of 8.1 cells whereas fertile adult males have a mean of 7.1 cells. Counts of spermatids in section of testes add further support to the view that the primary spermatocytes, from which the spermatids originated, were not formed in a strict geometric progression.
Metabolism of lipid and carbohydrate in sea urchin spermatozoa
Authors: Mita Masatoshi , Yasumasu Ikuo
Published: February 2005

When the dry sperm of the sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial sea water at 0°C and at 20°C, they became motile and the levels of ATP and creatine phosphate decreased rapidly. The level of ADP hardly changed, and the AMP level increased after the dilution. After the dilution, the respiratory rate at 2°C was almost one fifth that of 20°C. Both phospholipid and glycogen were used for the energy sources in sea urchin sperm. The level of phospholipid was 10‐fold higher than that of glycogen in the dry sperm. The phospholipid level decreased after dilution at 20°C, though the level hardly changed at 0°C, suggesting that phospholipid was hardly metabolized the lower temperature. The level of α ‐glycerophosphate increased at 20°C after the dilution but did not change at 0°C. The level of glycogen decreased after the dilution, regardless of the temperature. The glycolysis was also activated after the dilution. Of the intermediates of the tricarboxylic acid cycle, the citrate concentration increased at 0°C and the malate concentration also increased at 0°C and especially strongly at 20°C.
Changes in cAMP‐dependent protein kinase activity in the 10,000g sediment of sea urchin embryos during early development
Authors: Fujino Yukio , Yasumasu Ikuo
Published: February 2005

cAMP‐dependent protein kinase was found in the sediment obtained by centrifuging a homogenate of sea urchin embryos at 10,000g for 20 min, and was solubilized with 1% Triton X‐100. This enzyme was eluted at 0.16 M NaCl in a linear concentration gradient on a DEAE‐cellulose column, at which cAMP‐dependent protein kinase found in the supernatant was also eluted. The enzyme activity was enhanced about 1.5‐fold in the presence of 1 μM cAMP, and increased somewhat by adding cGMP or cIMP. The activation by cAMP of protein kinase in the sedimentable fraction was lower than in the supernatant fraction. The properties of the enzyme found in the 10,000g sediment and in the supernatant differ somewhat. The activity of the cAMP‐dependent protein kinase in the 10,000g sediment was high in the embryos at the blastula, the swimming blastula, and the mesenchyme blastula stages. On the other hand, the activity was undetectable in unfertilized eggs and in embryos at the morula, the gastrula, and the pluteus stages.
Progressive defects observed in mouse sperm during the course of three generations of selenium deficiency
Authors: Wallace Edith , Calvin Harold I. , Cooper George W.
Published: February 2005

Three successive generations of mice were fed a Torula yeast based Se‐deficient diet with or without 0.1 ppm Se in the drinking water. The Se‐deficient mice, in the course of three generations, showed a decrease in body weight, testis weight, epididymal weight, and sperm production. The percentage of morphologically abnormal sperm increased in successive generations. The majority of sperm defects were found in the midpiece region of the tail. Many of these aberrant sperm were motile. A progressive decrease in fertility was noted during the first two generations of Se deficiency. This system confirms the essential role of Se in spermatogenesis and provides a model for the evaluation of the primary effect of Se deprivation on the structural development of sperm.
Nuclear and chromatin structure in rat spermatozoa
Authors: Koehler James K. , Würschmidt Utta , Larsen Mary Pat
Published: February 2005

The nuclei of mature mammalian spermatozoa contain a highly ordered, lamellar substructure, presumably constituting the nucleoprotein of the haploid chromosomal complement. With a view toward constructing a plausible model of chromatin packing in sperm, we have determined some of the quantitative parameters associated with these “nuclear lamellae” in rat spermatozoa. Epididymal sperm from white, Sprague‐Dawley rats were examined by conventional sectioning methods, freeze fracture of fixed and unfixed specimens, and by whole mount replica techniques. Fixation and glycerolation did not significantly alter nuclear structure as seen by freeze fracture. Numerical data obtained from cross fractures of sperm heads indicate that the number of lamellae are quite constant at 10.4 ± 1.8 and that the linear measure of the lamellae is 7.2 ± 2.3 μm per cross fracture. The total area of cross fracture, assuming an elliptical profile is 2.3 k 0.7 μm2 and the thickness of the lamellae is 18.2 ± 3.5 nm with a range of 13.5 to 25.5 nm. An estimate of the total surface area of the nuclear lamellae could be made from measurements of projected nuclear area (from replicas and sections) as 173 ± 15 μm2. From these data and the known amount of DNA in the rat sperm nucleus, a model can be proposed for the organization of the nucleoprotein in these lamellar sheets. It is suggested that the chromatin is arranged in a coiled‐coil configuration closely associated together in a side‐by‐side fashion and continuous in extent. Approximate calculations based on this simple model are within a factor of 2 or 3 of predicting the correct amount of DNA in the sperm nucleus.
Hormonal regulation of zona‐binding ability and fertilizing ability of rat epididymal spermatozoa
Authors: Fournier‐Delpech S. , Hamamah S. , Tananis‐Anthony C. , Courot M. , Orgebin‐Crist M.‐C.
Published: February 2005

Rat spermatozoa from the proximal caput, the proximal corpus, the middle corpus, and the distal cauda epididymidis were examined for their ability to bind to the zona pellucida after a 1‐, 2.5‐, or 4.5‐h incubation at 34°C with rat eggs in cumulus. Caput spermatozoa did not bind to the zona after 1, 2.5, or 4.5 h of incubation. Corpus spermatozoa did bind to the zona, but the percentage of eggs with bound spermatozoa and number of bound spermatozoa per egg increased with the length of incubation. Cauda spermatozoa bound readily to the zona pellucida, and their zona binding ability did not change with longer incubations. It thus appears that rat spermatozoa gradually acquire the ability to bind to the zona pellucida in the corpus epididymidis.

The zona‐binding capacity of cold immobilized cauda spermatozoa, defined as the percentage of eggs with bound spermatozoa, increased with the number of spermatozoa incubated and reached a plateau characteristic of the endocrine status of the animal. After castration, zona‐binding ability is progressively lost from day 3 until day 10 where it is nil. Testosterone supplementation maintains zona‐binding ability to control levels. Similarly, fertilizing ability declines from day 5 after castration until day 10. Testosterone prevents this loss of fertilizing ability. It thus appears that the development of zona‐binding ability during epididymal transit is, like the development of fertilizing ability, under androgen regulation.

The close correlation between the onset of fertilizing ability and zona‐binding ability during maturation, the loss of fertilizing ability and zona‐binding ability after castration, and the recovery of both fertilizing ability and zona‐binding ability with testosterone treatment suggests that the androgen‐dependent development of zona‐binding ability is an important component of the acquisition of sperm fertilizing ability during epididymal transit.
Factors influencing the penetration of human spermatozoa into cervical mucus in vitro
Authors: Katz D. F. , Overstreet J. W. , Tom R. A. , Hanson F. W.
Published: February 2005

Experiments were conducted to determine some of the biological variables that may affect sperm penetration of human cervical mucus in vitro. Quantitative tests of cervical mucus penetration were carried out to determine the percentage of successful collisons (PSC) between seminal spermatozoa and the semen‐mucus interface. Fifteen duplicate comparisons and 15 triplicate comparisons of PSC values were made, each using individual samples of semen and mucus. In most cases the difference between any two comparisons was less than 10%, and there was no correlation between the magnitude of the difference between tests and the absolute value of the PSC. The triplicate comparison showed no correlation between the PSC values and the location of the mucus in the collection catheter (proximal, middle, distal). In 15 experiments the semen was serially diluted with an aliquot of its own plasma to determine the effect of sperm concentration on the PSC. No effects were observed until the sperm concentration fell below 10 × 106 sperm/ml, when the PSC appeared to increase. These results indicate that the tests should be applicable to all but the most severely oligospermic semen samples.
Influence of the flagellar wave development and propagation on the human sperm movement in seminal plasma
Authors: Serres C. , Feneux D. , Jouannet P. , David G.
Published: February 2005

Microcinematographic analysis (50 frames/sec) of the movement of 57 ejaculated human spermatozoa showed a heterogeneity of the flagellar parameters (velocity of the wave development (Vd), velocity of the wave propagation (Vc), beat frequency) corresponding to that of head trajectories (progressive velocity and amplitude of the lateral head displacement). According to our results the interdoublet sliding velocity might control the progressive velocity of the spermatozoa through the wave propagation velocity. The presence of the principal bend on the proximal part of the flagellum (20 μm) might inhibit the initiation of a new wave. The complex relation existing between Vc and Vd seems to indicate that the two velocities are partly independent of one another.
Anti‐inflammatory drugs promote polyspermic fertilization in sea urchins
Authors: Schuel Herbert , Traeger Eveline , Schuel Regina , Boldt Jeffrey
Published: February 2005

Sea urchin eggs are known to release H2O2 during the cortical reaction at fertilization to help prevent polyspermy by inactivating excess sperm in the vicinity. This process resembles the peroxidatic killing of bacteria by phagocytic leukocytes during inflammation. Associated with these reactions in leukocytes, arachidonic acid is released from phospholipids and can be oxidized via the cyclooxygenase pathway to produce prostaglandins. Nonsteroidal anti‐inflammatory drugs (NSAID) that are cyclooxygenase inhibitors in somatic cells were used to determine whether Arbacia punctulata and Strongylocentrotus purpuratus eggs use these processes to help prevent polyspermy. The potent cyclooxygenase inhibitor indomethacin causes a dose (10–100 μM) and sperm density dependent induction of polyspermy if added before the egg completes the cortical reaction. It does not retard elevation of the fertilization envelope and does not promote polyspermy by protecting sperm from peroxidatic inactivation by egg‐derived H2O2. Other potent cyclooxygenase inhibitors, flufenamate and meclofenamate, also induce polyspermy at 10–60 μM. Aspirin, a weak cyclooxygenase inhibitor in somatic cells, does not cause polyspermy at 5 mM. These findings provide evidence that prostaglandins or other cyclooxygenase‐derived metabolites may help assure monospermic fertilization in sea urchins.
The site of the acrosome reaction during in vivo penetration of the sheep oocyte
Authors: Crozet N. , Dumont M.
Published: February 2005

In vivo fertilization of sheep eggs has been studied by electron microscopy. Remnants of the acrosome reaction were present at the zona surface of every penetrated egg, indicating that the acrosome reaction in sheep occurs at the surface of the zona pellucida.

To determine whether follicular oocytes could specifically bind spermatozoa, oocytes isolated from different size classes of antral follicles were transferred into the oviducts of mated ewes, recovered 4 hr 30 min later, and analyzed by electron microscopy. Oocytes from follicles up to 1 mm in diameter failed to bind spermatozoa and were not penetrated. In contrast, the zona of oocytes from follicles ⩾ 2 mm in diameter induced the acrosome reaction. These oocytes were penetrated but failed to achieve cortical granule exocytosis and so to mount a block to polyspermy. Moreover, sperm nuclei incorporated into the ooplasm did not decondense although the sperm nuclear envelope was dispersed.
Optimization of procedures for separation of motile and nonmotile bull and rabbit spermatozoa with bovine serum albumin gradients
Authors: Wall R. J. , Jerrard D. A. , Parrish J. J. , Foote R. H.
Published: February 2005

The effect of equipment design, separatory media, and time and temperature of separation were studied. Discontinuous 4%/10% bovine serum albumin (BSA) gradients were used to isolate highly motile spermatozoa in rabbit and bull semen. For all conditions tested, motility of spermatozoa collected from the 4% BSA gradient layer (top) was less than or equal to the motility of the unseparated controls. Fractions collected from the 10% BSA gradient layer contained highly motile spermatozoa. In experiment 1, washed bull spermatozoa were diluted with phosphate‐buffered saline (PBS) containing 2% BSA or 4% BSA before being fractionated on BSA columns contained in test tubes. Inclusion of BSA in PBS tended to reduce loss of motility during washing, but the proportion of sperm recovered was highest in PBS. In experiment 2, motility and recovery of buck spermatozoa collected from the 10% BSA gradient region tended to be higher when fractionation temperature was 30°C as compared to 35°C, and motility was significantly higher when incubation time was 30 min as compared to 1 hr. The proportion of sperm recovered was unaffected by incubation time. In experiments 1 and 2, 41 of 48 separations resulted in at least one fraction containing spermatozoa with motility greater than or equal to 90%. In the third experiment, the surface area on which bull and buck spermatozoa were layered was increased by forming the 4%/10% BSA gradients in conical supports. Separation of sperm on conically shaped columns was not as effective as on cylinders. The use of cylinders to support the BSA gradients and a separation time of 30 min at 30°C is recommended.
Changes in motility that accompany the acrosome reaction in hyperactivated hamster spermatozoa
Authors: Suarez Susan S. , Katz David F. , Meizel Stanley
Published: February 2005

High‐speed videomicrography was used to compare the movement characteristics of hamster epididymal sperm which completed the acrosome reaction in vitro with those of unreacted sperm in the same sample. More than 90% of the motile sperm incubated for 4.25 hr in a modified Tyrode's medium containing bovine serum albumin, taurine, and epinephrine were hyperactivated and about half were acrosome reacted. The flagella of reacted sperm beat with significantly lower frequency and bent into more acute curves than those of unreacted sperm. Lowered beat frequencies were not attributable to aging, because sperm induced to react synchronously at 3.5 hr using lysophosphatidyl choline beat with similar lowered frequencies. Both acrosome‐reacted and unreacted hyperactivated sperm swam in circular trajectories resulting from asymmetrical flagellar beating. The flagellar beating of unreacted sperm was more symmetrical; consequently, they swam in larger circles and had the potential to cover space more rapidly. Some unreacted sperm, perhaps in transition towards hyperactivation, swam in helical trajectories.

When preincubated sperm were added to slides containing oocytes in cumulus, some unreacted sperm initiated cumulus penetration. All reacted sperm failed to do so, adhering instead to the cumulus at its boundary. Reacted sperm attached to the zonae pellucidae of cumulus‐free oocytes via the region of the inner acrosomal membrane. Unreacted sperm attached via the equatorial region, but pivoted about the point of attachment, thus failing to generate sustained thrust against the zona. In conclusion, unreacted hyperactivated sperm have a different potential than reacted sperm for movement and interaction with egg vestments.
A preliminary report on the establishment of pregnancies in an in vitro fertilization (IVF) programme at the University of British Columbia (UBC)
Authors: Moon Young S. , Yuen Basil Ho , Pride Sheila M. , Rowe Timothy C. , Poland Betty J. , McComb Peter F. , Gomel Victor
Published: February 2005

Between July 1982 and November 1983, two pregnancies were established using in vitro fertilization and embryo transfer (IVF and ET) procedures with three different schedules to induce follicular maturation. All women were cycling normally and had inoperable or absent fallopian tubes. Of 83 oocytes aspirated from 24 patients (31 cycles), 75% were considered mature and 25% immature by the morphological characteristics of the oocytes and cumulus cells. Oocytes were preincubated for 6–24 hours, and after insemination, 60% cleaved to the two‐to‐four‐cell stage. The superovulation induction schedule employing hMG administered according to the individually adjusted treatment scheme established two pregnancies. This schedule was considered the superior regimen, as it gave the highest proportion of mature oocytes (89%) which cleaved (78%). The pregnancy‐attaining follicle showed a high progesterone:estradiol‐17β ratio (P4/E2) in its microenvironment of aspirated follicular fluid, culture media of granulosa cells, and oocyte‐cumulus complex. Our observations indicate a high P4/E2 ratio in the pregnancy‐attaining follicle, and thereby reflect a further parameter in influencing maturation of the oocytes most likely to implant.
Solubilization and enrichment of boar sperm membrane proteins
Authors: Klint Marianne , Sege Karin , Curman Bengt , Plöen Leif , Peterson Per A.
Published: February 2005

Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X‐100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X‐100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out.

SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined.

The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins.
The morphological and ultrastructural appearance of the crater defect in stallion spermatozoa
Authors: Johnson L. A. , Hurtgen J. P.
Published: February 2005

The appearance of the nuclear abnormality “crater defect” is described in spermatozoa from the domestic horse (Equadae). Under scanning electron microscopy, the defect appears as a severe depression at any area of the sperm nucleus or as a blister or swelling at some point on the sperm nucleus. Ultrastructurally, the crater appears as a nuclear vacuole containing amorphous material similar to that described in bull and boar sperm. The craters ranged in size from 0.5 to 2 μm in diameter. Within ejaculates of stallions having this defect, the percentages of sperm with crater defects varied widely over time, being as high as 60% and as low as 15%.
Interaction of isolated components from mammalian sperm and egg
Authors: Sullivan Robert , Bleau Gilles
Published: February 2005

In order to identify those proteins from the plasma membrane of hamster spermatozoa that exhibit an affinity for components of the zona pellucida we have used the Western blot technique. Zonae pellucidae from postovulatory hamster oocytes were solubilized by exposure to an acidic pH and then radiolabeled using the Bolton‐Hunter reagent. These 125I‐zona pellucida proteins retain their immunoreactivity and migrate in three heterogeneous bands when submitted to SDS‐PAGE electrophoresis. Membrane proteins from epididymal spermatozoa of mature hamsters were extracted by treatment with Nonidet P‐40 and then submitted to electrophoresis (SDS‐PAGE). The proteins in the gel were electrophoretically transferred to a nitrocellulose membrane and then probed with the radiolabelled zone pellucida proteins. 125I‐zona pellucida proteins bind preferentially to a sperm protein with a molecular weight of 26,400 ± 1,400 daltons (n = 9). Using a similar procedure it was shown that this protein also binds 125I‐Concanavalin A. The interaction between the sperm protein and the 125I‐zona pellucida proteins shows species specificity as demonstrated by the fact that the hamster 125I‐zona pellucida proteins do not bind to proteins extracted from ram, bull, and stallion spermatozoa. Whether this sperm protein could be implicated be implicated in the process of sperm‐egg interaction is under investigation.
Sperm aging in the male after sexual rest: Contribution to chromosome anomalies
Authors: Martin‐DeLeon Patricia A. , Boice Melinda L.
Published: February 2005

The chromosome complement was studied in first‐cleavage metaphases of mouse zygotes resulting from sperm aged in the male physiologically, after sexual rest. Females were inseminated by control males mating at 3‐day intervals while experimentals mated to males that had had a sexual rest of 14 or more days. A total of 1954 eggs were collected 33–35 h post‐HCG from 101 superovulated females mated to 42 controls and 43 experimental males. The fertilization rate was similar in both groups, being 84% and 85%, respectively. G‐banded or Q‐banded chromosomes were analyzed in 301 (68.3%) controls and 392 (49%) experimental first‐cleavage metaphases. The overall rate of chromosome anomalies in controls was 4.45% as compared to 10.94% in experimentals, a highly significant difference. In the experimental group compared to controls, the frequency of trisomy, triploidy, structural rearrangements, and tetraploidy increased from 3.9% to 6.9%, 0% to 1.6%, 0.8% to 2.8%, and 0% to 1.3%, respectively. The genomic source of origin of the abnormalities was determined on the basis of differential condensation of the genomes. In the experimentals, grossly unbalanced sperm (diploids, disomics, double disomics, and those with large fragments) fertilized significantly more oocytes compared to controls. Our results implicate an advantage either in numbers or fertilizing capability for chromosomally abnormal sperm in a physiologically aged population.
7‐aminoactinomycin D binding and the final stages of sperm chromatin processing in the mouse
Authors: Balhorn Rod , Kellaris Kennan , Corzett Michele , Clancy Colin
Published: February 2005

Quantitative fluorescence microscopy is used to compare the binding of the fluorescent, 7‐amino derivative of actinomycin D to a sonication resistant fraction of late‐step mouse spermatids and sperm isolated from three regions of the epididymis. Staining conditions are described that optimize 7‐AMD binding to air‐dried smears of these cells and permit quantitative analyses of 7‐AMD binding to sperm chromatin at the single cell level. These studies show that the step 12–16 spermatid undergoes an 8–10‐fold reduction in 7‐AMD binding as it completes differentiation in the testis and progresses through the epididymis. The first 2–3‐fold reduction in binding, associated with biochemical changes in chromatin structure brought about during spermatid differentiation, occurs gradually and is complete by the time the sperm reaches the caput epididymis. An additional four‐fold reduction is observed as the sperm passes through the corpus. This final alteration, which occurs in a single concerted step, may be reversed by dithiothreitol treatment and appears to be effected through the formation of a final, small group of sperm nuclear protein disulfide crosslinks.
The development of meiotic competence in the rat: Role of hormones and of the stage of follicular development
Authors: Bar‐Ami S. , Tsafriri A.
Published: February 2005

Hypophysectomy of 15‐day‐old rats (hypox) markedly reduced the normal development of meiotic competence and abolished the development of antral follicles between days 21 and 31 postpartum (pp). Here the correlation among age of the rats, stage of follicular development, and meiotic competence was examined. Administration of pregnant mare's serum gonadotropin (PMSG‐3IU) or insertion of an estradiol‐17β (E2) capsule to hypox rats induced the development of meiotic competence provided the treatment started after day 20 pp. Hormonal treatments at an earlier age were not effective in inducing meiotic competence in hypox rats. The induction of meiotic competence by PMSG or E2 was associated with an increase in the number of granulosa cells and formation of follicular antrum. The finding that PMSG and E2 failed to induce meiotic competence when administered prior to day 21 pp suggests that the development of meiotic competence is an age‐dependent process. When the hormonal treatments commenced after day 21, both follicular development and meiotic competence were induced.
Flagellar movement of human spermatozoa
Authors: Ishijima Sumio , Oshio Shigeru , Mohri Hideo
Published: February 2005

Flagellar movement of human spermatozoa held by their heads with a micropipette was recorded by means of a video‐strobe system. Spermatozoa were studied in normal Hanks' solution, Hanks' solution with increased viscosity, cervical mucus, and hyaluronic acid.

When flagellar movement in normal Hanks' solution was observed from the direction parallel to the beating plane, segments of the flagellum in focus did not lie on a straight line but on two diverging dashed lines. The distance between the two dashed lines was about 20% of the bend amplitude in the major beating plane. These observations indicate that flagellar beating of human spermatozoa in normal Hanks' solution is not planar. In contrast, segments of the flagellum in focus lay on a straight line when the spermatozoa were observed in Hanks' solution with increased viscosity, cervical mucus, or hyaluronic acid. In normal Hanks' solution, free swimming spermatozoa rotated constantly around their longitudinal axes with a frequency similar to the beat frequency, whereas little or no rotation of spermatozoa occurred in Hanks' solution with increased viscosity, in cervical mucus, or in hyaluronic acid.

We conclude that human spermatozoa in normal Hanks' solution beat with a conical helical waveform having an elliptical cross section, the semiaxes of which have a ratio of 0.2. The three‐dimensional geometry of the flagellar movement is responsible for the rotation of the sperm around their longitudinal axes.
Ultrastructural and cytochemical changes of the head components of human spermatids and spermatozoa
Authors: Dadoune J. P. , Alfonsi M. F.
Published: February 2005

The ultrastructural study of chromatin condensation simultaneously with the evolution of the perinuclear organelles was conducted in the spermatids and epididymal and ejaculated spermatozoa of man with the aid of the “en bloc” alcoholic PTA staining and the EDTA regressive method.

The round nuclei of young spermatids (steps 1, 2) were characterized by the persistence of nucleoli that were PTA positive, and the presence of a subacrosomal layer of well‐stained peripheral chromatin. In the beginning of the phase of nuclear elongation (step 3), the central chromatin also became dense, like the peripheral chromatin, while the nuclear ring and the associated manchette and the two anlages of the postacrosomal dense lamina and the posterior ring appeared. During steps 4 and 5, the sliding of the nuclear ring and the manchette, the growth of the postacrosomal dense lamina, and the progression of the posterior ring towards the base of the nucleus were seen along with structural and cytochemical modifications of the chromatin. In the flattened nuclei of step 4 spermatids, coinciding with the loss of the nucleolar components, the chromatin achieved maximum compactness in the entire nucleus and was PTA positive. In the spermatids of step 5, the disappearance of peripheral dense chromatin and the specific staining of the chromatin granules marked the beginning of the second stage of transformation of the basic nucleo‐proteins. The condensed nuclei of the mature spermatids were partially stained by PTA in step 6 and totally unstained in step 7. The PTA staining revealed the persistence of PTA‐positive chromatin areas in the nuclei of certain spermatids otherwise mature. The morphological aspect of the chromatin then remained the same in the nuclei of epididymal and ejaculated spermatozoa.

These observations suggest that in man, as in other mammals studied, new proteins accumulate in the elongating nuclei of spermatids and are replaced at the phase of maturation by sperm‐specific nucleoproteins. The defects in condensation of the chromatin that occur during spermiogenesis could be related to the modalities of accumulation of intermediate nucleoproteins.
Oviducal pars recta‐induced degradation of vitelline coat proteins in relation to acquisition of fertilizability of toad eggs
Authors: Takamune Kazufumi , Yoshizaki Norio , Katagiri Chiaki
Published: February 2005

In the toad Bufo bufo japonicus the vitelline coat (VC) of the uterine egg (UEVC) is more readily lysed by the sperm lysin than the VC of coelomic egg (CEVC). Fluorometric determinations of released proteins after incubation of the VC with the sperm lysin in vitro revealed that the CEVC is not completely refractory to the lysin but increases in susceptibility after treatment with a pars recta extract (PRE). Experiments employing isolated pars recta granules showed that both this increase of VC susceptibility and the acquisition of egg fertilizability are ascribable to the contents of the granules. SDS‐PAGE analyses of VC proteins revealed that in comparison with CEVC, UEVC lacks 40–52K proteins concomitant with the increased stainability of 39K protein and the appearance of 36K protein. These changes in SDS‐PAGE profiles were observed either in oviducal eggs after passage through the pars recta or in coelomic eggs treated with PRE but were inhibited when coelomic eggs were treated with PRE containing soybean trypsin inhibitor (SBTI) and leupeptin. Likewise, the acquisition of fertilizability by treatment of coelomic egg with PRE was inhibited by SBTI. Dejellied uterine eggs were successfully fertilized when pretreated with trypsin inhibitors before insemination but were not fertilized when pre‐treated with concanavalin A. We propose that the hydrolytic degradation of certain VC proteins due to the tryptic activity of pars recta granules renders the VC susceptible to the sperm lysin, so that the eggs are made receptive to a fertilizing sperm.
The ovulation and activation of primary and secondary oocytes in LT/Sv strain mice
Authors: Kaufman M. H. , Howlett S. K.
Published: February 2005

Eggs were isolated from the oviducts or ovaries of LT/Sv strain mice in order to investigate the pathways taken by them following spontaneous or induced parthenogenetic activation. The chromosome preparations from the ovarian oocytes that matured in vitro to metaphase I were all morphologically normal. Of 42 recently ovulated eggs that failed to activate parthenogenetically in culture, 57% on nuclear densitometric analysis were found to have the normal 2C amount of DNA, and 1N (haploid) number of chromosomes present, and were arrested at metaphase II. Somewhat unexpectedly, 43% had a 4C amount of DNA, and 2N (diploid) number of chromosomes present, had been arrested at metaphase I, and were evidently ovulated as primary oocytes. Following parthenogenetic activation, the majority of oocytes extruded a polar body and developed a single pronucleus. The activated eggs could be divided into two sub‐populations according to the diameter (and therefore volume) of the pronucleus—in one group this was about one‐third greater than in the other. The chromosome constitution of the two groups was determined separately at the first cleavage mitosis. Those with a normal‐sized pronucleus were invariably haploid, while those with an enlarged pronuclear volume were invariably found to be diploid. The chromosomes in the diploid spreads often appeared to be associated in homologous pairs. We conclude that almost uniquely in LT/Sv strain females eggs may be activated parthenogenetically at either stage of meiotic maturation giving rise to diploid or haploid embryos, respectively.
Development of ability to penetrate the cumulus oophorus by hamster spermatozoa capacitated in vitro, in relation to the timing of the acrosome reaction
Authors: Cummins J. M. , Yanagimachi R.
Published: February 2005

Hamster spermatozoa were tested for their ability to penetrate the intact cumulus matrix at low sperm:egg ratios (approximately 3:1). Uncapacitated spermatozoa attached to the surface of the cumulus and could not penetrate. Spermatozoa capacitated in vitro began to be able to penetrate after about 2 hr of preincubation, coincidentally with the first appearance of hyperactivation and spontaneous acrosome reactions. In all, 628 in vitro incubated spermatozoa were evaluated on and in cumuli: 270 could penetrate, but only ten of these were judged to have intact, “unmodified” acrosomes. Almost all spermatozoa capable of penetrating showed optically “modified” and swelling acrosomal caps, and this confirms previous observations on cumulus penetration in vivo. Penetration appeared limited to a phase in capacitation prior to completion of the acrosome reaction, as spermatozoa that had lost the acrosomal cap penetrated poorly and showed reduced viability. Penetration of the cumulus was inhibited by the hyaluronidase inhibitor sodium aurothiomalate. Cumulus penetrating ability could result either from a change in surface properties of the sperm at capacitation, which renders them less “sticky” to the matrix, or from release or activation of a “cumulus lysin.” We conclude that the ability to enter the cumulus matrix coincides with physiological changes in spermatozoa that occur during a terminal phase of capacitation preceding complete loss of the acrosomal cap, and that initiation of this process in vivo must precede sperm‐zona contact.
In vitro fertilization with normal development in the sheep
Authors: Crozet Nicole , Huneau D. , Desmedt Véronique , Théron Marie‐Claire , Szöllösi D. , Torrès Suzanne , Sévellec Claude
Published: February 2005

Ovine tubal (n = 87) and ovarian in vitro matured oocytes (n = 99) were fertilized in vitro with ejaculated spermatozoa capacitated for 8 h in modified defined medium buffered with Hepes. High levels of fertilization were obtained as assessed by development to two‐to six‐cell stage within 40 h (75. 8% for ovulated and 62. 6% for in vitro matured oocytes). Electron microscope analysis of oocytes 20–22 h after insemination indicated that in vitro fertilization approximated the in vivo events. Embryos (two‐ to six‐cell) were transferred surgically to the oviducts of pseudopregnant rabbits. Three days later, 42 (from ovulated oocytes) and 15 (from in vitro matured oocytes) embryos were recovered; 26 (61. 9%) and 10 (66. 6%), respectively, had cleaved at least once. Embryos incubated in vivo (n = 20 from ovulated oocytes; n = 9 from in vitro matured oocytes) were transferred surgically to the uteri of seven and four recipient ewes resulting in four and two pregnancies, respectively, from which three and one, respectively, have been maintained ( > 3 months). The first lamb resulting from the in vitro fertilization of an ovulated oocyte was born. In addition, six embryos (two‐ to four‐cell) from tubal oocytes and ten embryos (two‐ to six‐cell) from in vitro matured oocytes were directly transferred to the oviducts of two and three ewes, respectively. Two pregnancies resulting from in vitro matured fertilized oocytes are in progress ( > 3 months).
Analysis of the biological properties of antibodies raised against intact and deglycosylated porcine zonae pellucidae
Authors: Henderson Colin J. , Hulme Martin J. , Aitken R. John
Published: February 2005

Zonae pellucidae (ZP) were isolated from 1,500 porcine ovaries and heat solubilized to generate approximately 15 mg ZP glycoprotein. Analysis of this material by isoelectric focusing, one‐dimensional electrophoresis, and gas chromatography indicated the presence of a major glycoprotein species that exhibited considerable microheterogeneity with respect to its charge (pI 7.5–3.5) and molecular mass (45–85 kDa) and that contained 39.6% carbohydrate, predominantly N‐acetylglucosamine.

Chemical deglycosylation of porcine ZP using trifluoromethanesulphonic acid (TFMS) resulted in the production of five discrete protein bands on one‐dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) with molecular masses of 66, 52, 36, 32, and 16 kDa.

Antisera raised in rabbits and marmosets to ZP and/or deglycosylated ZP (DGZP) were used in immunoblotting experiments to demonstrate the retention of immunogenicity by DGZP and the cross‐reactivity of the antisera with their heterologous antigen. These studies indicated that antisera that were capable of inhibiting the fertility of primates in vivo and the penetration of the human ZP in vitro reacted preferentially with 3 of the 5 products of deglycosylation, with molecular masses of 66, 52, and 36 kDa. Anti‐DGZP antibodies were also shown to interact with intact porcine and human ZP and, with the latter, to block the ability of human spermatozoa to both bind to and penetrate this structure.
Use of salt‐stored zonae pellucidae for assessing rabbit sperm capacitation for in vitro fertilization
Authors: Fayrer‐Hosken R. A. , Brackett B. G.
Published: February 2005

Zonae pellucidae of tubal and follicular oocytes were collected and prepared for salt storage. Cumulus and corona radiata cells were removed from oocytes with hyaluronidase and a small bore pipette. The oocytes (referred to as zonae since vitelli were rendered nonfunctional) were stored in a salt solution at 4°C. Using in utero capacitated sperm, the penetrability of zonae from tubal and follicular oocytes stored immediately after collection was compared to controls, i.e., in vitro development of tubal ova to the 4‐cell stage within 24 hr. The penetration rates were 100% (8 penetrated/ 8 inseminated), 77.8% (7 penetrated/ 9 inseminated), and 100% (10 fertilized/10 inseminated), respectively, and these were not statistically different. The mean (x) numbers of sperm able to penetrate the zonae, into the perivitelline space (PVS) for tubal (34.0) and follicular (1.1) oocytes were significantly different (P < 0.01). However, following maturational incubation before salt storage, zonae of tubal and follicular origin showed no significant differences in penetrability of in utero capacitated sperm when assessed by percent penetration, or mean numbers of sperm cells reaching the PVS: tubal zonae, 100% (15/15), and follicular zonae, 100% (18/18), and mean number of sperm in the PVS (x [tubal zonae] = 12.4, and x [follicular zonae] = 11.8). The penetrability of tubal zonae with and without maturational incubation was compared, and no significant differences in penetrability by in utero capacitated sperm were present when assessed by percent penetration nonmatured 92.6% (25/27) and matured 93.3% (28/30) and mean number of sperm in the PVS (x [nonmatured] = 3.33 and x [matured] = 2.41).

In vitro capacitation of ejaculated rabbit sperm by serum treatment was assessed by the penetration of salt‐stored zonae, zonae‐free hamster oocytes (ZFHO), and in vitro fertilization of freshly collected tubal oocytes. None of 60 salt‐stored zonae and none of 31 tubal oocytes were penetrated, and these values were significantly (P < 0.005) smaller than the 9 of 78 (12%) zona‐free hamster ova that were penetrated by sperm cells from the same sample.

In vitro capacitation of ejaculated rabbit sperm by washing and preincubation was assessed by the penetration of salt‐stored zonae, zona‐free hamster oocytes (ZFHO), and fertilization of freshly collected tubal oocytes. Seventy‐six of 80 salt‐stored zonae were penetrated, and this was significantly (P < 0.005) greater than the 67 of 87 tubal oocytes fertilized and 30 of 35 ZFHO penetrated, which were not significantly different.

The salt‐stored zonae were more readily penetrated by capacitated sperm when compared to tubal oocytes. However, the ZFHO are more penetrable than salt‐stored zonae and tubal oocytes when incompletely capacitated sperm is used. A useful role for this approach in studies dealing with sperm fertilizing ability is anticipated.
Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody
Authors: Moore H. D. M. , Smith C. A. , Hartman T. D. , Bye A. P.
Published: February 2005

A monoclonal antibody generated against hamster epididymal spermatozoa and recognizing an antigen within the acrosome was used in conjunction with FITC‐antimouse immunoglobulin as a marker of the human acrosome during sperm development, capacitation, and the acrosome reaction. The specificity of binding of the monoclonal antibody was assessed using immunolocalization by epi‐fluorescence and electron microscopy. Immunofluorescence revealed that antibody bound over the entire anterior acrosome in hamster and human spermatozoa. Ultrastructural localization indicated that antigen was predominantly present on the inner face of the outer acrosomal membrane and within the acrosomal content. Qualitative specificity was studied using a highly purified preparation of hamster acrosomes in an enzyme‐linked immunosorbent assay. Since the antibody rapidly visualized human acrosomes, it was used to detect abnormal acrosome morphology of mature spermatozoa and to mark spermatids present in the ejaculate. During incubation in capacitating medium, changes in the immunofluorescence of live or methanol fixed spermatozoa were correlated with incubation interval and the ability of spermatozoa to fuse with zona‐free hamster oocytes. Spermatozoa bound to zona‐free hamster oocytes displayed no fluorescence, confirming that acrosome loss occurred before spermatozoa attached to the vitellus.
Homospermic versus heterospermic insemination of zona‐free hamster eggs to assess fertility of fluorochrome‐labeled acrosome‐reacted bull spermatozoa
Authors: Davis A. P. , Graham J. K. , Foote R. H.
Published: February 2005

Fresh spermatozoa from six bulls, with fertility ranging from 64% to 78%, (based upon 59‐day nonreturn rates for 159,448 cows inseminated) were mixed with zona‐free hamster eggs in 15 heterospermic pair inseminations. Five of the bulls were used in homospermic insemination studies. Prior to incubation, spermatozoa from each bull were labeled with contrasting fluorescent stains pretested for effects on spermatozoa. Equal numbers of spermatozoa were mixed and treated with liposomes of dilauroylphosphatidylcholine to induce the acrosome reaction. Spermatozoa from split ejaculates within a male competed against each other equally in the hamster egg test, indicating that the staining procedure did not affect egg penetration rates. Bulls differed in their egg penetration rates when their sperm were inseminated either homospermically or heterospermically, but the differences in the homospermic inseminations were not significantly correlated with sire fertility. The number and percentage of sperm which penetrated eggs, and the number of eggs penetrated in the heterospermic competitive tests were highly correlated with fertility (r ≥ 0.86). Therefore, egg penetration rates from heterospermic inseminations appear to be valuable indicators of fertility and much more sensitive predictors than results from homospermic inseminations.