bluuebunny/crossref_metadata_2025 huggingface dataset:
An improved method to visualize human sperm chromosomes using zona‐free hamster eggs
Authors: Chaudhuri J. P. , Yanagimachi R.
Published: February 2005

An improved method for human sperm chromosome preparation is described. Improvements include (1) the use of a sperm population with high motility and normal morphology for insemination, (2) the insemination and postinsemination culture of zona‐free eggs in Holmes medium, which does not require serum supplementation, (3) control of sperm concentration at insemination to avoid heavy polyspermy, (4) reduction of the occurrence of egg agglutination by placing the medium for postinsemination culture in a ring or crescent shape instead of a droplet, and (5) application of a two‐step fixation method to augment the efficiency of chromosome preparation.
Fish germinal cells. I. Comparative spermatology of seven Cyprinid species
Authors: Baccetti B. , Burrini A. G. , Gibertini G. , Mazzini M. , Zerunian S.
Published: February 2005

The spermatozoa of seven fishes belonging to Cyprinid family are examined. They have no acrosome, like all Teleost fishes, a spheroidal or slightly elliptic nucleus, always eccentrically placed on the tail, two variously oriented centrioles, and a postnuclear cytoplasmic region of various size that contains some mitochondria (2 to 10) and surrounds a periaxonemal postnuclear canal. The tail is of moderate length (from 36 to 60 μm) and contains a “9 + 2” axoneme: both dynein arms are present. Comparative examination of the spermatozoa in the seven species shows that significant differences occur among them, even when they belong to the same genus. These concern the tail length; the position of the centrioles, the proximal with respect to the central one and with respect to the nucleus; the number of mitochondria, which is in relationship to the depth of the postnuclear canal. In the uniform general pattern of the ultrastructure of the Cyprinid spermatozoa, each species is characterized by a particular organization of the sperm organelles; in this respect, the two species examined by us, Leuciscus cephalus and souffia, are more closely related, even if easily recognizable one from the other. From a phylogenetic point of view, the comparative spermatology of the Cyprinid fishes suggests that the mitochondrial number is a good character, which enables us to order them in a phylogenetic arrangement.
Influence of the concentration of sperm on the percentage of eggs fertilized for three strains of mice
Authors: Robl James M. , Dziuk Philip J.
Published: February 2005

This study was conducted to determine the optimal concentration of sperm to use for the insemination of females to detect differences among strains of mice in the percentage of eggs fertilized. Female ICR mice were inseminated with sperm of concentrations ranging from 0.25 to 8 × 106/50 μl from males of either DBA/2N, CF1, or C57BL/6N strains. Differences among strains were detected only when approximately 50% of the eggs were fertilized but not when each of the strains fertilized either a high or low percentage of eggs. The optimal concentration of sperm therefore was the concentration that gave approximately 50% fertilized eggs.
A further study of lysolecithin−mediated acrosome rection of guinea pig spermatozoa
Authors: Yanagimachi Ryuzo , Suzuki Fumie
Published: February 2005

When guinea pig spermatozoa are preincubated for 1 hr in Ca2+−free medium containing a low concentration of lysolecithin (LC, 85 μg/ml) and then exposed to 2 mM Ca2+ by diluting the preincubation medium with an equal volume of LC−free, 4 mM Ca2+−containing medium, the majority of the spermatozoa undergo acrosome reaction promptly. On the other hand, when the preincubated spermatozoa are exposed to 2 mM Ca2+ without reducing the original concentration of LC in the medium, none of them undergo acrosome reaction. These spermatoza can acrosome−react if they are transferred to an LC−free medium. These results and those of some other experiments suggest that in the presistent presence of a high concentration of LC in the medium, exogenous Ca2+ essential for the acrosome reaction either does not penetrate the sperm plasma membrane or, if it does, it cannot alter the membrane for the acrosome reaction, at least under the experimental conditions employed. Freeze−fracture examination of the sperm plasma membrane has revealed that small areas or patches free of intramembranous paarticles (IMPs) appear in the membrance during sperm preincubation, and these IMP−free areas expand drastically in response to Ca2+ when the LC conccentration in the medium is reduced at the time Ca2+ is added to the medium. In contrast, IMP−free areas remain unchanged even after exposure of spermatozoa to Ca2+ if the concentration of LC remains at its original level of 85 μg/ml.
The influence of postovulatory age on the rate of cleavage in in‐vitro fertilized rat oocytes
Authors: Shalgi R. , Kaplan R. , Kraicer P. F.
Published: February 2005

The purpose of this study was to analyze the effect of postovulatory ‘aging’ in the oviduct on the rate of zygotic development. Two ovulatory ages were tested: oocytes collected from the oviducal ampullae 1) soon after ovulation (denoted freshly ovulated) or 2) 7‐hour postovulation. All the oocytes were from superovulated immature rats. By manipulation of the timing of the ovulatory hormone treatment, it was possible to place both types of oocytes into sperm suspension from the same pool and at the same time. The oocytes and spermatozoa were coincubated overnight. Cleavage was established by interference contrast microscopy. The time of the first cleavage of ova from the 7‐hour postovulation group was clearly advanced. Because the cleavage time curves were not parallel, no reliable estimate of the time difference could be made, but it was clearly in the range of 2 hr. This shift could not be related to any difference in the time of sperm penetration. Both groups of oocytes underwent penetration by spermatozoa at the same time. The time interval between maximal sperm penetration (94% of oocytes in both groups) and maximal cleavage (50% in both groups) was 23 hr in the freshly ovulated and 21 hr in the 7‐hour postovulatory eggs. Nor was the difference related to polyspermy, which was approximately 14% in both groups. These results support the hypothesis that developmental processes are under way in the oocyte before fertilization, but at a much slower rate than after fertilization.
Maturation of pig and rat oocytes transplanted into surrogate pig follicles in vitro
Authors: Fleming Alan D. , Kuehl Thomas J. , Armstrong David T.
Published: February 2005

Pig oocytes obtained from slaughterhouse material and rat oocytes obtained from PMSG‐treated immature females were incubated as isolated oocytes or injected into explanted pig follicles (5–8 mm). Free oocytes of both species, with or without their cumulus investment or gonadotropins during culture, matured at high rates after 30 hr or 9–10 hr of culture, respectively. Gonadotropic stimulation was necessary for maturation of both the native and injected cumulus‐intact pig oocytes in follicle culture. Cumulus‐free pig oocytes injected into follicle failed to mature in response to gonadotropic stimulation, suggesting an inability to perceive or respond to stimulation. Injected rat oocytes, however, matured irrespective of cumulus investment or gonadotropic stimulation. Their maturation was delayed and reduced at 9 hr. These results in the rat suggest that the pig follicular environment is incapable of regulating rat oocyte maturation but rather presents a permissive or supportive environment for their maturation. The explanted surrogate follicles from the pig or other species may provide a useful model for the study of oocyte‐follicle interactions in oocyte maturation within or between species.
A mouse embryo culture system for quality control testing of human in vitro fertilization and embryo transfer media and fetal cord sera
Authors: Cheung Sau W. , Strickler Ronald C. , Yang Victoria C. , de Vera Michelle , Spitznagal Edward L.
Published: February 2005

Swiss white mice were superovulated, mated, and sacrificed to recover two‐cell embryos that were cultured in Ham's F‐10 supplemented with 15% fetal serum. In 16 experiments, media enriched with fetal bovine serum (FBS) supported blastocyst development from 80% ± 19% (mean ± S.D.) of two‐cell embryos. Culture media + FBS was the positive control when 74 batches of heat‐inactivated human fetal cord serum (hFCS) were tested. Statistical analyses indicated two distinct populations: 49 hFCS promoted blastocyst formation and 25 hFCS grew fewer blastocysts. In five studies, 35/47 two‐cell embryos recovered from mice oviducts in media + FBS and immediately incubated formed blastocysts (75% ± 10%). In six comparison studies where the recovered embryos stood at room temperature for 30 minutes before incubation, only 18/57 (29% ± 21%) became blastocysts. When the colony was housed for 1 week in rooms with Shell No Pest Strips as treatment for mites, only 11/125 two‐cell embryos became blastocysts (9%). In contrast, animals housed in quarters decontaminated with chlorine bleach had reduced breeding efficiency and produced fewer two‐cell embryos. We conclude that (1) Ham's F‐10 + FBS is an excellent positive control to test new batches of hFCS; (2) hFCS that supports blastocyst formation from ≥75% of two‐cell embryos is adequate for human use; (3) pesticide treatment of breeding colonies and cooling of murine embryos during harvest both impaired in vitro blastocyst development; and (4) chlorine bleach cleansing of animal quarters reduced the number of successful matings.
Alteration of the human sperm surface during in vitro capacitation as assessed by lectin‐induced agglutination
Authors: Singer Sherry L. , Lambert Hovey , Cross Nicholas L. , Overstreet James W.
Published: February 2005

Human sperm incubated in vitro in BWW medium containing 35 mg/ml human serum albumin acquire the capacity to penetrate the human zona pellucida and to fuse with the zona‐free hamster oocyte. We have studied changes in lectin‐induced agglutination of human sperm during incubation in this medium to detect alterations in the sperm surface which may be correlated with the acquisition of these functions. Sperm incubated for 1, 6, or 24 hr were combined with two‐fold dilutions of lectins for 30 min at 37°C, in 5% CO2, balance air. When pooled data from five donors were analyzed, the average sperm agglutination titer of wheat germ agglutinin (WGA), phytohemagglutinin‐E (PHA), Lens culinaris agglutinin (LCA), peanut agglutinin (PNA), and Pisum sativum agglutinin (PSA) was found to increase significantly (P ≤ 0.06) with incubation in vitro, although there was considerable variation between ejaculates. Ulex europaeus and Dolichos biflorus agglutinins did not agglutinate human sperm (≤250 μg/ml). Results of this screening demonstrate the alteration of sperm surface components during in vitro incubation and suggest that WGA, PHA, LCA, and PSA may prove useful in efforts to correlate changes in the sperm surface with the ability of the sperm to fertilize the egg.
Immunohistochemical localization of the transferrin receptor in the seminiferous epithelium of the rat
Authors: Brown William R. A.
Published: February 2005

The transferrin receptor has been immunohistochemically localized in the seminiferous epithelium of the rat with a monoclonal antibody, MRC OX26, which recognizes the transferrin receptor glycoprotein. The receptor was detectable on mitotically and meiotically dividing germ cells and, less abundantly, on round spermatids. It was lost from germ cells during spermatid elongation and was undetectable on immature spermatozoa. The transferrin receptor was also present on Sertoli cells in the testes of immature animals and on Sertoli cells in the testes of aspermatogenic animals that had been irradiated in utero. It was not detectable on Sertoli cells in the testes of cryptorchid animals. These studies demonstrate that the transferrin receptor is abundant on dividing germ cells as well as dividing somatic cells.
An examination of the method of sizing the spermatozoa of the domestic fowl and duck with an electronic counter
Authors: Hashimoto H. , Terada T. , Tsutsumi Y.
Published: February 2005

Although many estimations have been made electronically of mammalian sperm volume, detailed investigations have not been reported for avian spermatozoa with an electronic counter. In the present study, sizing of spermatozoa of fowls and Muscovy and Pekin drakes was examined using a Coulter counter (model ZB). In our preliminary work on fowl sperm volumes, we found mono‐ or di‐morphic distribution displays that were modified depending on the combination of amplification (AMP) and aperture current (APC). Therefore, methodology to estimate the fowl and drake sperm volume was examined. Dilution of semen had no effect on the dimorphic distribution pattern of the sperm volume. Density‐gradient centrifugation did not separate two kind of particles in the semen in either continuous or discontinuous Percoll gradients; therefore, we varied settings of AMP and APC, and found that the most suitable settings for measuring sperm volumes of these birds are 1 for AMP and 8 for APC. With these settings, mean volumes of spermatozoa were 5.1 μm3 for fowls, 5.7 μm3 for Muscovy drakes, and 5.6 μm3 for Pekin drakes.
The response of rhesus monkey sperm motility to cervical mucus and solid surfaces
Authors: Katz D. F. , Phillips D. M.
Published: February 2005

Movement characteristics of rhesus monkey spermatozoa were analyzed using high‐speed cinemicrography. In the first experiment, spermatozoa were studied at 100 frames/sec in diluted semen near a surface, and after entering ovulatory cervical mucus from a bonnet monkey. In mucus, the spermatozoa swam more slowly, with reduced flagellar beat frequencies. The beat shape was altered, and there was less lateral yawing of the sperm head. In the second experiment, spermatozoa in diluted semen were studied at 500 frames/sec in deep preparations, while swimming near a surface or when in the midplane of these preparations. Those sperm in the midplane swam faster, but with lower beat frequencies than those near the surface, and exhibited much more pronounced yawing motions. Such distinctions in sperm motion are probably hydromechanical in origin and may be significant during transport in the female.
Artificial insemination of the American lobster (Homarus)
Authors: Talbot P. , Thaler C. , Wilson P.
Published: February 2005

A technique has been developed for artificially inseminating freshly molted female lobsters (Homarus). Using the criterion that eggs were fertilized if they showed development of normal cleavage patterns, at least 49.5% of the inseminated females extruded fertilized eggs. Sixty‐two percent of the fertilized eggs developed to the eyespot stage, and 26.6% of these subsequently hatched. Eggs were not fertilized in 7.7% of the extrusions. In 42% of the samples, it was not possible to establish whether fertilization had occurred because either samples were collected prior to initiation of cleavage or eggs were too poorly preserved to assess cleavage with confidence. It is probable that many of the eggs that were evaluated as equivocal were also fertilized. This technique should be valuable in the experimental and commercial aquaculture of lobsters and might be directly applicable to other crustaceans.
The evolution of hamster sperm motility during capacitation and interaction with the ovum vestments in vitro
Authors: Katz David F. , Cherr Gary N. , Lambert Hovey
Published: February 2005

Movement characteristics of golden hamster spermatozoa were studied upon collection from the cauda epididymis, during an incubation which capacitates the spermatozoa in vitro, during penetration of the cumulus, and during attachment to and penetration of the zona pellucida. High‐speed videomicrography was employed to quantitate flagellar beat frequency and shape. The status of the acrosome was also assessed. During capacitation, hamster spermatozoa become increasingly invigorated before the onset of hyperactivated motility. Within the cumulus, beat frequency and curvature are reduced, apparently in response to the physical resistive properties of the matrix material. These properties appear to vary within the cumulus. Initial attachment to the zona precedes completion of the acrosome reaction, is non‐rigid, and is accompanied by increased beat frequency and curvature. Subsequently, the onset of rigid binding to the zona, completion of the acrosome reaction, and increased flagellar beat frequency are very closely associated in time. The latter produces an increase in thrust against the zona. Preliminary results indicate that ensuing zona penetration requires not more than five minutes, is at oblique angles, and is associated with a continuation of vigorous flagellar beating.
Ultrastructure of the sperm and spermatogenesis of three species of Mytilidae (mollusca, bivalvia)
Authors: Hodgson A. N. , Bernard R. T. F.
Published: February 2005

The mature sperm of three species of the subfamily Mytilinae (Choromytilus meridionalis, Mytilus galloprovincialis, Aulacomya ater) are of the primitive type. The sperm are 50–55 μm long, with a distinct head, midpiece of four to six mitochondria, and tail. In all three species the acrosome is in the form of an elongated hollow cone; yet each has its own characteristic morphology, indicating that sperm ultrastructure can be used as an aid to bivalve identification. A reexamination and comparison of the acrosomes from five subclasses of bivalve show that each subclass has an acrosome with a unique form. The process of spermatogenesis is similar in all three species; early spermatogonia are located close to the wall of the germinal follicle, and successive stages are displaced toward the center. Observations support the theories of acrosome formation by coalescence of numerous proacrosomal vesicles and of reduction in mitochondrial number by mitochondrial fusion.
Lower temperature of the cauda epididymidis facilitates the storage of sperm by enhancing oxygen availability
Authors: Djakiew D. , Cardullo R.
Published: February 2005

The temperature coefficients for the oxygen consumption rate of sperm from the rat, mouse, bull, and sea urchin were measured to be within a range of 2.7 to 3.0. For a reduction in temperature from 37°C to 30°C, the respiration rate of mammalian sperm declined by a factor of approximately 2.1. Hence, the availability of oxygen to sustain these sperm substantially increased over this temperature range. In addition, the solubility of oxygen in cauda epididymidal fluid increased by 7.6% for a decline in temperature from 37°C to 30°C. The increased availability of oxygen to sperm at the lower temperature is related to the increased storage capacity of the cauda epididymidis in scrotal mammals. In this context, it is suggested that the evolutionary migration of the cauda epididymidis to a cooler, extra‐abdominal location may have been influenced by the increased availability of oxygen to sperm and hence resulted in an increased capacity to sustain and thereby store more sperm per unit volume of duct.
Immunochemical localization of epididymal protein DE on rat spermatozoa: Its fate after induced acrosome reaction
Authors: Cameo Mónica S. , Echeverria Fernanda Gonzalez , Blaquier J. A. , Burgos M. H.
Published: February 2005

The localization of rat epididymal protein DE on cauda epididymis spermatozoa was studied with a specific antibody and the peroxidase antiperoxidase (PAP) immunocyto‐chemical reaction. At the light microscopic level, all spermatozoa appeared to be labeled over the dorsal portion of the head, whereas tails were negative. This observation was confirmed using scanning electron microscopy. A large number of particles were seen on the external surface of the plasma membrane covering the acrosomal region and a smaller number on the postacrosomal portion. Flagella appeared free of particles. Sperm suspensions were incubated in conditions that induce capacitation and the acrosome reaction, and, in this instance, the permanence of protein DE on the vesicles and the postacrosomal region of the membrane were observed. The localization of this epididymal protein on the sperm surface is compatible with a role in the gamete interaction process.
Developmental capacity of mouse oocytes following maintenance of meiotic arrest in vitro
Authors: Downs Stephen M. , Schroeder Allen C. , Eppig John J.
Published: February 2005

It was shown previously that the frequencies of fertilization and pre‐ and post‐implantation embryonic development of mouse oocytes matured in vitro were similar to those of oocytes matured in vivo (Schroeder and Eppig, Dev Biol 102:493–497, 1984). The present study determined the developmental capacity of mouse oocytes after they had been maintained in meiotic arrest in vitro by substances thought to be important regulators of meiosis in vivo. Oocytes were maintained in meiotic arrest for 12 or 24 h in medium containing maturation inhibitor(s), washed free of inhibitor, and cultured 16 h in inhibitor‐free (control) medium to permit meiotic maturation. Four different medium supplements were used to maintain meiotic arrest: (1) 100 μM dibutyryl cAMP plus 1 mM hypoxanthine; (2) 4 mM hypoxanthine plus 0.75 mM adenosine (H + AR); (3) 300 μM dibutyryl cAMP; and (4) 50 μM IBMX. Parallel groups of oocytes were treated to the same experimental protocol except that no inhibitory compounds were used; eg, oocytes were cultured a total of 28 or 40 h in control medium that permitted the resumption of maturation. These latter groups tested the effect of extended culture of mature oocytes on subsequent development. Control oocytes were cultured 16 h in control medium. Oocytes were inseminated and subsequently assessed for development to two‐cell and blastocyst stages. When oocytes were first cultured 12 or 24 h in medium that maintained meiotic arrest, development to two‐cells in all groups but one were within 10% of controls (70%). The 24 h H + AR group was the one exception (47% two‐cells). By contrast, culturing oocytes for 28 or 40 h in inhibitor‐free medium resulted in a precipitous decrease in development to two cells (27% and 7%, respectively). Blastocyst development followed the same pattern. When uridine (U) was added to H + AR medium, development to two cells was increased significantly. Also, the addition of FSH to the maturation medium significantly increased both two‐cell and blastocyst development in the H + AR and H + AR + U groups. Transfer of compacted morulae from the H + AR + U/FSH group into pseudopregnant hosts produced live young 19 days postinsemination.

These data demonstrate that prolonged culture of oocytes matured in vitro decreased their capacity to undergo normal development following insemination, but if oocytes were maintained in meiotic arrest during prolonged culture and then allowed to mature spontaneously, their developmental potential was significantly preserved. These results also lend support for a physiological role of cAMP and purines in the maintenance of meiotic arrest in vivo.
Phosphoproteins are structural components of bull sperm outer dense fiber
Authors: Brito Mónica , Figueroa Jaime , Vera Juan C. , Cortés Patricia , Hott René , Burzio Luis O.
Published: February 2005

To isolate the bull sperm outer dense fibers, the cells were treated at room temperature with 0.05% cetyltrimethylammonium bromide and 7 mM 2‐mercaptoethanol. This treatment solubilized most of the sperm structure except for the sperm head and the fibrillar complex. The latter was purified by discontinuous sucrose gradient centrifugation. Electron microscopy showed that in the isolated complex each fiber remains attached to the corresponding segmented column of the connecting piece.

Analysis of the fibrillar complex by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed three major components with molecular weights of 85,000, 33,000, and 11,000. These were purified, and their amino acid composition was found to be similar to that of the polypeptides of rat sperm outer dense fibers. Furthermore the components of 85,000 and 33,000 daltons contain about 2 mol of phosphoserine per mole of polypeptide, which indicates that they are phosphoproteins.
Flow sorting of X and Y chromosome‐bearing spermatozoa into two populations
Authors: Johnson L. A. , Flook J. P. , Look M. V. , Pinkel D.
Published: February 2005

The only established difference on which to base the separation of X and Y chromosome‐bearing spermatozoa is chromosomal constitution. This difference is quantifiable both from chromosome morphology (karyotype) and from DNA content. Flow cytometric techniques were used to measure relative DNA content of the X and Y populations and to flow‐sort spermatozoa from Chinchilla laniger. Epididymal spermatozoa were recovered in PBS, fixed in 80% ethanol, treated with papain and dithioerythritol, and stained for DNA with Hoechst 33342. Sperm nuclei were analyzed and sorted on an EPICS V flow cytometer/cell sorter, modified specifically for spermatozoa. Two clearly resolved peaks (coefficient of variation < 1.5%) with approximately 7.5% difference in DNA content between X and Y chromosome‐bearing spermatozoa were evident. Sperm nuclei were sorted from a portion of the X and Y peaks at a rate of 55 nuclei/sec for each population. Purities of individual X and Y populations averaged 95% as determined by reanalysis of the sorted populations. Successful sorting of Chinchilla X and Y chromosome‐bearing spermatozoa into separate populations may aid in the identification of a biochemical marker that could be used to discriminate between the two sperm populations and lead to a practical procedure for sexing spermatozoa.
Reproduction in mice: Protein kinase mimics the sperm effect on preimplantation embryo development
Authors: Chaykin Sterling , Davé Sheela
Published: February 2005

The creation of an environment in mouse fallopian tubes that is sufficient to sustain preimplantation embryo development is known to require the participation of spermatozoa in excess of those involved in the process of fertilization. We have now found that highly purified cAMP‐dependent protein kinase can substitute for spermatozoa in the facilitation of the first cleavage of mouse embryos. Both spermatozoa and purified protein kinase induce increases in fallopian phosphoproteins. It is suggested that nonfertilizing spermatozoa could exert their effects on preimplantation embryo development through the provision of protein kinase.
Incidence of chromosome anomalies in first‐cleavage mouse embryos obtained from frozen‐thawed oocytes fertilized in vitro
Authors: Glenister P. H. , Wood Maureen J. , Kirby Carol , Whittingham D. G.
Published: February 2005

To assess the effect of low temperature storage on mouse oocytes we (1) examined the capacity for normal development of embryos derived from frozen oocytes fertilized in vitro after transfer to pseudopregnant foster mothers and (2) analyzed the chromosome complement at the first cleavage division. Fewer frozen than control oocytes were fertilized (36% vs 66%), but after embryo transfer the proportion of fertilized eggs that implanted (67–68%) and formed normal foetuses (50–53%) was similar in the two groups. Freezing did not affect the observed incidence of aneuploidy (1.5–3.3%). The frequency of polyploid embryos derived from frozen oocytes was almost double that of controls (15.8% vs 8.5%), but it is unclear whether this is a real effect of freezing or is an artifact produced by the chromosome preparation technique.
Features of a seminal proteinase inhibitor‐ zona pellucida‐binding component on murine spermatozoa
Authors: Robinson Roderick , Richardson Richard , Hinds Kathy , Clayton Debra , Poirier Gary R.
Published: February 2005

Murine cauda epididymal sperm contain sites on the plasma membrane over the apical portion of the acrosome that recognize proteinase inhibitors and the homologous zona pellucida. Ten times more of the component can be extracted from cauda and ductus sperm than from equal numbers of caput and corpus sperm. Likewise, few sperm from the upper epididymal regions are able to bind seminal inhibitor, while the majority of sperm from the cauda and ductus do bind. Cauda epididymal and ductus sperm lose little of their ability to bind inhibitor after a 4‐hour in vitro incubation in either a capacitating or a noncapacitating medium. The percentage of naturally inseminated sperm with the seminal inhibitor bound to their surface decreases to about 10 after 4 hours in utero. Approximately 80% of these sperm show positive fluorescence when given the opportunity to rebind the inhibitor, and these sperm do have an intact plasma membrane over the apical portion of the acrosome. Furthermore, after 4 hours in utero, the inhibitor bound in the same region of the sperm head as it did on freshly ejaculated sperm. The seminal inhibitor inhibits the binding of sperm to the zona if added during the first 15 minutes of incubation but has no effect on attachment.

The data indicate that sperm gain the ability to bind the seminal inhibitor during the epididymal sojourn. Furthermore, this binding capacity is not lost during in vitro or in utero incubation. The site is not involved in sperm‐zona attachment but does participate in the binding of sperm to the zona.
Inhibition of in vitro fertilization by mouse anti‐mouse sperm sera and preliminary antigen identification
Authors: Hamilton Marilyn S. , Vernon Robert B.
Published: February 2005

Antisperm antibodies are implicated as one causative factor of infertility, but the target antigens have not been identified. Immune responses to sperm antigens are qualitatively variable even within a single mouse strain. We took advantage of this variability and immunized individual female mice to allogeneic sperm to reflect their natural exposure during mating. We determined the ability of the individual sera to inhibit in vitro fertilization and to bind to sperm antigens separated by electrophoresis. Compared to preimmune sera, four of five immune sera significantly inhibited in vitro fertilization. The serum from individual mice bound variable panels of sperm antigens. By comparing the panels, we identified two polypeptides with molecular weights of 40,000 and 44,000 that were bound by all sera. We propose that these molecules may be good candidates for further investigation of the immunoprophylaxis of pregnancy.
Absorption of compounds in medium by the oil covering microdrop cultures
Authors: Miller K. F. , Pursel V. G.
Published: February 2005

Microdrops of medium under either paraffin or silicone oil are commonly used for culture of early mammalian embryos. Radiolabeled estradiol, progesterone, and androstenedione in drops of medium under oil decreased by 51, 89, and 77%, respectively, after 24‐hr incubation. Up to 14% of labeled estradiol moved to another drop of medium by passing through the oil. Several other substances tested (FSH, leucine, glucose, lactate, sodium ion, PGE2, PGF2α) did not pass into the oil. Both paraffin and silicone oils can alter the composition of culture medium by absorbing and transferring certain types of compounds.
Boar sperm surface glycoproteins: Isolation, localization, and temporal expression during spermatogenesis
Authors: Klint Marianne , Fridberger Anita , Menge Alan , Sällström Jan , Plöen Leif
Published: February 2005

Boar sperm glycoprotein fractions were isolated by Lens culinaris hemagglutinin affinity chromatography of detergent‐solubilized ejaculated spermatozoa, followed by preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In order to develop methods for further investigations of the sperm proteins, we proceeded with two of the isolated glycoproteins.

Antibodies were raised in female rabbits against each of the two sperm glycoproteins. By a combination of immunosorbent chromatography, using the antibodies obtained, and preparative SDS polyacrylamide gel electrophoresis, highly purified sperm proteins were isolated. The sperm proteins were immobilized on Sepharose gel columns and specific immunoglobulin Fab fragments were enriched by affinity chromatography. The specificity of the Fab fragments was ascertained by immunoprecipitation analysis. The Fab fragments were used in indirect immunofluorescence analysis to localize the corresponding antigens on the surface of boar spermatozoa. Both antigens were exclusively confined to the postacrosomal region.

Immunohistochemical staining of boar testis sections revealed that both antigens are expressed from the spermatid stage. This technique also revealed that one of the antigens congregated at the Golgi complex‐acrosome region during spermatogenesis.
Flow cytometry of X and Y chromosome‐bearing sperm for DNA using an improved preparation method and staining with Hoechst 33342
Authors: Johnson L. A. , Flook J. P. , Look M. V.
Published: February 2005

A new and improved method of preparing mammalian spermatozoa for high resolution flow cytometric DNA analysis and flow sorting is described. Ejaculated or cryopreserved sperm were briefly sonicated to remove tails and then stained with Hoechst 33342. This simple procedure was found superior to more severe treatments of dimethylsulfoxide washes, fixation in 80% ethanol, and protease digestion of the sperm membranes and tails by papain. Flow cytometric DNA analyses of sperm samples subjected to varying sonication times indicated that X and Y chromosome‐bearing sperm populations could be well resolved with as little as 15‐sec sonication. In addition, a comparison of sonicated samples stained with four concentrations of bisbenzimide (Hoechst 33342) or 4′,6‐diamidino‐2‐phenylindole (DAPI) indicated that 2.5 or 5.0 μg/ml of Hoechst was sufficient to resolve the X and Y sperm populations. In order to quantitatively describe the flow cytometric data, several indices (sample quality, orientation and splitting) were developed.
Receiver‐operating characteristics curves for semen analysis variables: Methods for evaluating diagnostic tests of male gamete function
Authors: Peng H‐Q. , Collins J. A. , Wilson E. H. , Wrixon W.
Published: February 2005

This study draws on decision analysis to estimate the utility of seminal variables in predicting pregnancy. The infertile couples selected (n = 709) excluded those who had donor insemination or known female factors (history of pelvic infection, ovulatory disorders, endometriosis, and tubal disease). Diagnostic test properties were calculated with respect to pregnancy for two variables derived from the semen analysis report: sperm density, and the total number of motile sperm in the ejaculate (TMS). Receiver‐operating characteristic (ROC) curves based on sensitivity and specificity revealed that none of the proposed thresholds for sperm density or TMS is clearly a best choice. The highest positive predictive values for sperm density (75%) and TMS (75%) were associated with cutoff points at five million sperm per milliliter and five million motile sperm per ejaculate, respectively. The false positive rates when these cutoff points were used were 4.3% (sperm density) and 5.6% (TMS). ROC analysis provides visual and numeric vidence of the quality of a diagnostic test and constitutes a set of procedures for incorporating information from new tests of male gamete function into the diagnostic assessment of infertility.
Formation of the rupture site in preovulatory hamster and mouse follicles: Loss of the surface epithelium
Authors: Talbot Prudence , Martin Gary G. , Ashby Hansel
Published: February 2005

Changes in the morphology of epithelial cells covering the sides and apex of ovarian follicles were examined in mice and hamsters during the final 13 hr before rupture using light and electron microscopy. At the time of the surge of luteinizing hormone, approximately 13 hr before follicle rupture, epithelial cells along the follicle sides are spherical, covered with microvilli, and remain so throughout the entire cycle. As ovulation approaches, cells at the apex become progressively flatter, increase in diameter, and undergo a reduction in the number and length of microvilli. By 2 hr before ovulation, the microvilli are present only along the boundary between adjacent cells and the cells are in different stages of degeneration. In some cells, the cytoplasm is electron dense and the nuclei are pyknotic. Other cells become electron lucent and cytoplasmic elements are leached from the cell. The apical plasma membrane is lost first over the center of the cell and later over the periphery. Epithelial cells detach from the apex individually until a large patch devoid of cells is formed. This includes the site of eventual rupture. The loss of epithelial cells from the apex of ovarian follicles of other species is compared with our results, and the processes involved in stretching, degeneration, and sloughing of the epithelial cells are discussed.
The distribution of organelles in mammalian oocytes following centrifugation prior to injection of foreign DNA
Authors: Cran D. G.
Published: February 2005

The centrifugation of oocytes from the domestic species is a necessary prerequisite to allow visualization of nuclei for the introduction of foreign DNA. This improvement in visibility which has allowed the production of transgenic animals is accompanied by a clear stratification of the organelles. In immature oocytes from the sheep, pig, and cow, four distinct zones are formed. These comprise, lipid, membrane‐bound vesicles, organelle‐free cytoplasm, and mitochondria. In mature ovine oocytes, a fifth zone of smooth endoplasmic reticulum (SER) beneath that of the vesicles is formed. This SER is produced at discrete locations within the untreated cell. The potential for removal of these fractions has implications for relating patterns of protein synthesis with particular structural components.

In intact oocytes, the cortical granules are located in a peripheral position beneath the plasma membrane. However, even after subjection to high centrifugal force (65,000g for 60 min), they maintain their original location. However, treatment with the cytoskeletal inhibitors, nocodozole and cytochalasin, results in rapid exocytosis after centrifugation. It is concluded that the maintenance of the spatial relationships of this organelle is mediated through the peripheral cytoskeleton.
DNA synthesis following microinjection of heterologous sperm and somatic cell nuclei into hamster oocytes
Authors: Naish Sally J. , Perreault Sally D. , Zirkin Barry R.
Published: February 2005

We have investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in 3H‐thymidine after being parthenogenetically activated by sham microinjection, or microinjected with hamster, mouse, rabbit, or fish sperm nuclei, or hamster hepatocyte nuclei. Within 6 hr of sham or nucleus microinjection, nuclei of each type underwent transformation into pronuclei and synthesized DNA. These results demonstrated that the hamster egg can access and utilize its own and each type of template provided, whether homologous or heterologous. However, pronuclei derived from hamster sperm nuclei were more likely to be synthesizing DNA at 6 hr than pronuclei derived from sperm nuclei of other species. We conclude that the mechanisms employed by the hamster oocyte to transform hamster sperm nuclei into pronuclei and to effect DNA synthesis in these nuclei are not specific for the hamster sperm nucleus. Nevertheless, these mechanisms apparently operate more efficiently when the hamster sperm nucleus, rather than a heterologous sperm nucleus, is present.
Effects of in utero and in vitro incubation on the lipid‐bound fatty acids and sterols of porcine spermatozoa
Authors: Evans R. W. , Weaver D. E. , Clegg E. D.
Published: February 2005

Sperm‐rich semen and washed porcine spermatozoa were incubated for up to 2 hr either in utero in the presence of oviduct fluid or in vitro at 37°C. Sperm lipids were extracted and separated into phospholid and neutral lipid fractions. Eleven phospholipid and five neutral lipid fatty acids were identified and quantified using GC and GC‐MS. The percentage of 22:5n6, the major phospholipid fatty acid, decreased slightly but significantly during 1.5 hr of in utero incubation (41.2–38.0%), but after 2.0 hr of in utero incubation no significant difference was observed (40.0%). None of the phospholipid fatty acids changed in concentration during in vitro incubation. The mole ratio of phospholipid to phospholipid fatty acid (1.00:1.27) did not change during incubation. The levels of neutral lipid‐bound 14:0 decreased (43.5% to 31.8%) and that of 18:0 increased (11.1% to 18.2%) during in utero incubation. Similar but less pronounced changes were observed during in vitro incubation. (43.5% to 36.0%; and 11.1% to 15.8%, respectively).

Two major sterols, cholestrol (73%) and desmosterol (27%) were identified by gas chromatography–mass spectrometry. The mole ratio of phospholipid to sterol (2.47:1:00) did not change during incubation.
Zona pellucida penetration assay for capacitation of bovine sperm
Authors: Wheeler Matthew B. , Seidel George E.
Published: February 2005

In vitro capacitation and in vitro zona penetration systems are described for bovine spermatozoa. The criterion for capacitation was penetration by sperm of either 1) the zona pellucida of dead bovine oocytes, or 2) live, intact oocytes in which subsequent development was observed. Sperm were washed free of seminal plasma by centrifugation and incubated in a modified Tyrode's medium (TALP) in a 5% CO2‐air atmosphere at 38°C. Dead bovine oocytes were obtained from > 1 mm follicles from ovaries stored at −20°C. Spermatozoa at a final concentration of 107 ml were coincubated with the oocytes for 3 hr after capacitation treatments.

Initial experiments established that: 1) dead bovine oocytes can be penetrated by bovine sperm capacitated in the rabbit reproductive tract; 2) oocytes should be used for the penetration test within 24 hr of thawing; 3) oocytes could be stored for 24 hr at 4°C after penetration before evaluation; and 4) penetration of zonae pellucidae of dead bovine oocytes by sperm is species specific.

The percentages of dead oocytes penetrated after incubating frozen‐thawed sperm for 0, 6, 12, 24, and 30 hr prior to incubation with oocytes were 17, 24, 34, 10, and 0% respectively; the corresponding percentages for fresh semen were 1, 2, 5, 42, and 25%. Addition of 1% bovine seminal plasma to suspensions of washed, capacitated sperm decreased oocyte penetration percentages from 48% for the control (no seminal plasma) to 23%.

Spermatozoa capacitated in vitro for 24 hr fertilized 15 of 22 ovulated oocytes (68%) from superovulated cows. Three embryos developed to the morula stage, but no pregnancy resulted after transfer of these embryos to bovine recipients.

The present results provide the basis for a reliable (interassay C.V. = 13.4%) in vitro assay system for capacitation based on sperm penetration of the zona pellucida of dead bovine oocytes.
Membrane molecules involved in adhesion properties of cultured sertoli cells
Authors: Zani Bianca M. , Ziparo Elio , Russo Mario A. , Filippini Antonio , Stefanini Mario
Published: February 2005

Membrane components involved in adhesion properties of cultured Sertoli cells have been studied by a combination of immunological and biochemical methods. An antiserum prepared against Sertoli cells induced reversible rounding and detachment of the cells from the culture dishes. The cell surface morphology during detachment was studied by scanning electron microscopy and indirect immunofluorescence. A Triton soluble fraction of crude membrane preparations inhibited the antibody‐induced detachment. The antibodies recognized a restricted number of membrane glycoproteins [detectable as prominent bands on Sodium dodecylsulphate polyacrilamide gel electrophoresis (SDS‐PAGE), Mr 170, 140, 80, and 48K] both in the Triton soluble fraction of crude membrane preparation and on intact Sertoli cells. The data suggest that the molecules involved in adhesion properties of cultured Sertoli cells are integral membrane glycoproteins exposing antigenic determinants at the cell surface.
A cortical granule‐specific enzyme, B‐1,3‐glucanase, in sea urchin eggs
Authors: Wessel Gary M. , Truschel Margaret R. , Chambers Scott A. , McClay David R.
Published: February 2005

The ultrastructural localization of B‐1,3‐glucanase in three species of sea urchin eggs was determined using a monospecific antibody in an electronmicroscopic immunogold procedure. In all three species, Lytechinus variegatus, Strongylocentrotus purpuratus, and Arbacia punctulata, B‐1,3‐glucanase was localized specifically to the cortical granules. No other organelle within the egg contained significant label. During the fertilization reaction, B‐1,3‐glucanase was released from cortical granules into the perivitelline space and became associated with the hyaline layer. No significant label was found in association with the fertilization envelope.
Immunological approach to characterize proacrosin and various acrosin forms in boar and man by monoclonal antibodies
Authors: Kaufmann M. , Schönwald D. , Mansouri A. , Günther E.
Published: February 2005

Three different monoclonal rat antibodies, Acr1, Acr2, and Acr3, have been established against boar proacrosin. They are shown by enzyme‐linked immunosorbent and immunoblot assays to react with boar proacrosin and several different acrosin molecules derived therefrom during activation. The epitopes detected by the three antibodies are different from each other, one being highly sensitive to reduction and periodate treatment. The antibodies crossreact with various proacrosin and acrosin molecules derived from human sperm extract; they also show indirect immunofluorescent staining of the acrosomal region of ejaculated sperm from normal men but fail to react with round‐headed spermatozoa.
Correlation between sperm creatine phosphokinase activity and sperm concentrations in normospermic and oligospermic men
Authors: Huszar Gabor , Corrales Marcelia , Vigue Lynne
Published: February 2005

Toward the development of biochemical probes for the assessment of sperm function we have measured the activities of sperm creatine‐N‐phosphotransferase (CPK). There was a highly significant inverse correlation (P < 0.001 in all comparisons) between sperm CPK activities and sperm concentrations in specimens of normospermic and oligospermic men with > 30 million sperm/ml (0.106 ± 0.01 SEM, Nequals;90, expressed as CPK U/100 million sperm), 20–30 million sperm/ml (0.333 ± 0.07 SEM, Nequals;30) and 10–20 million sperm/ml (0.583 ± 0.12 SEM, Nequals;30) when compared with the CPK values of the < 10 million/ml specimens (2.242 ± 0.46 SEM, Nequals;30). Furthermore, the distribution of CPK activities within these four groups showed that 96%, 67%, 43%, and 4% of the samples, respectively, were in the < 0.250 CPK U/100 million sperm normal range (mean + 2 SD of the > 30 million sperm/ml group). However, there was no relationship between sperm CPK activities and the values of sperm motility (P > 0.15) or morphology (Pequals;0.38) in the samples. The migrated sperm fractions (significantly improved in motility and velocity parameters) showed CPK activities lower than the initial semen specimens (P < 0.01, Nequals;150). In fact, in some oligospermic men the CPK activities of the migrated sperm fractions were within the range of normospermic samples. The data suggest that sperm CPK values in the initial specimens and the degree of improvement in the migrated sperm fractions reflect the relative concentrations of a “normal” sperm subpopulation. We propose that CPK activities and similar objective biochemical parameters may be important in predicting sperm quality and the fertilizing potential of oligospermic men.