bluuebunny/crossref_metadata_2025 huggingface dataset:
Ovulation and fertilization of primary and secondary oocytes in LT/Sv strain mice
Authors: O'Neill G. T. , Kaufman M. H.
Published: February 2005

In this study, the chromosome constitution of both unfertilized oocytes and fertilized eggs isolated from the oviducts of LT/Sv strain mice were analyzed. Air‐dried chromosome preparations from unfertilized oocytes revealed that about one‐third of those examined were ovulated as primary oocytes. These were arrested at metaphase of the first meiotic division and exhibited the characteristic “tetrad” chromosome configuration. The remaining two‐thirds of the unfertilized oocytes were ovulated at metaphase of the second meiotic division. The fertilized eggs were isolated from the oviducts of LT/Sv females previously mated to (C57BL × CBA) F1 hybrid males. Analysis of the fertilized eggs at metaphase of their first cleavage mitosis revealed that about one‐third of the eggs examined were digynic triploids, whereas the remaining two‐thirds had the normal diploid chromsome constitution. In the triploids, the 40 female chromosomes present (mouse, n = 20) were derived from a single diploid pronucleus formed after the extrusion of a first polar body, and following the monospermic fertilization of primary oocytes. The female pronuclear‐derived chromosomes invariably exhibited “homologous pairing,” and these were associated at their centromeres. The ovulation, penetration, and subsequent fertilization of primary oocytes is an extremely unusual phenomenon in mammals and only appears to occur on a regular basis in LT/Sv mice. The premature “cytoplasmic maturation” of these oocytes is of interest, as they clearly have the same developmental capacity as secondary oocytes. The significance of these observations in relation to folliculogenesis and litter size in LT/Sv mice is discussed.
Phosphodiesterase activity of mouse sperm incubated under conditions that modulate fertilizing potential in vitro
Authors: Monks Nicola J. , Fraser Lynn R.
Published: February 2005

Cyclic AMP has been implicated as a regulator of capacitation, but the control of its metabolism in sperm remains obscure. A recent study of mouse sperm has shown capacitation‐related changes in the activities of both adenylate cyclase, which increased during incubation, and cyclic nucleotide phosphodiesterase, which decreased. The present study was conducted to extend these observations by measuring phosphodiesterase activity in sperm incubated in media with modified calcium and/or glucose content, conditions known to modulate fertilizing ability. Phosphodiesterase activity of sequential sperm samples, taken first when sperm are essentially uncapacitated and then when they are either partially or completely capacitated, decreased with time under all conditions, and in each case the greater fall in activity was seen in the medium that would support the greater change in fertilizing ability of the sperm population. Sperm washed by centrifugation to remove epididymal fluid also displayed a reduction in phosphodiesterase activity with time. The medium surrounding the sperm contained about half of the total phosphodiesterase activity, as well as 5′‐nucleotidase and adenosine deaminase. The crude enzyme preparation showed complex kinetic behavior when assayed over a range of cAMP concentrations, but the reduction in activity with time was seen at all substrate levels. The observed changes in phosphodiesterase activity, together with the increased adenylate cyclase activity seen under these sperm incubation conditions, would increase cAMP availability with time, thus providing further evidence for a fundamental role for cAMP in controlling the events of capacitation.
Protein synthesis in mature human oocytes
Authors: Gifford D. J. , Fleetham J. A. , Mahadevan M. M. , Taylor P. J. , Schultz G. A.
Published: February 2005

As a first step toward understanding control of gene expression in early human development, an analysis of protein synthesis and amino‐acid transport in unfertilized mature oocytes was initiated. Qualitative patterns of protein synthesis were examined in individual oocytes cultured in medium containing radiolabeled methionine. No differences in synthetic pattern of proteins, resolved by one‐dimensional electrophoresis and fluorography, were observed in oocytes analyzed from times varying from 12 to 52 hr following collection by laparoscopy. Contamination by follicular or corona radiata cells was readily distinguished on the basis of increased relative synthesis of a polypeptide with Mr = 44,000, a dominant product of synthesis in follicular cells. Based on the specific activity of the methionine precursor, the absolute rate of synthesis was measured to be about 50 pg/oocytc/hr, a value higher than in mouse unfertilized eggs. No difference in protein synthetic rate was observed in oocytes analyzed at 12 hr postcollection versus later times up to 50 hr postcollection. Competition of methionine uptake by leucine, efflux of radiolabeled methionine from preloaded oocytes into medium containing methionine and uptake of methionine in medium with low sodium ion concentration was observed. These findings are consistent with the presence of an L (leucine‐preferring) system for neutral amino acid transport, similar to that in mouse and rabbit eggs. Total protein was measured to be about 150 ng/oocyte, a value five times that of the mouse. These studies provide basic data for further analysis of oocytes and perhaps preimplantation stage embryos in the future.
A computer‐assisted assay for mouse sperm hyperactivation demonstrates that bicarbonate but not bovine serum albumin is required
Authors: Neill James M. , Olds‐Clarke Patricia
Published: February 2005

Mammalian sperm hyperactivation (HA) is a change in motility that accompanies capacitation (CAP) and is dependent on calcium (Ca) (Yanagimachi and Usui, Exp Cell Res 89:161, 1974). HA may be important for transport through the female tract and/or for fertilization. To develop an objective and quantitative assay for HA in individual mouse sperm, a computer‐assisted motion‐analysis system was used to describe sperm trnaslational movements. To determine which movements were characteristic of HA, Ca‐dependent motility was identified. This was done by incubating sperm with or without calcium (Ca+ or Ca− sperm, respectively), and determining the range of values for each motility parameter that was present only among Ca+ sperm. To do this, we compared frequency distributions of motility parameter values at the time of maximal CAP (90 min). CAP was monitored by measuring the level of in vitro fertilization and by evaluating the pattern of chlortetracycline binding to individual sperm heads [Ward and Storey, Dev Biol 104:287, 1984]. Two Ca‐dependent motility subgroups were apparent: 1) a “slow‐speed” subgroup with a curvilinear velocity (Vc) < 169 μm/sec that had none of the characteristics expected of HA sperm; and 2) a subgroup with higher speeds (Vc > 169 μm/sec) and wider‐amplitude head movements as measured by curvilinear progressiveness ratio (PRc < 0.56). The latter subgroup was selected as HA, since the frequencies and time course were similar to those for CAP in the same population. Two media components known to be important for CAP, bicarbonate and bovine serum albumin (BSA) were then tested to determine whether they were necessary for HA. Incubation of sperm without bicarbonate prevented HA, but omitting BSA did not affect HA during the first 3 hrs. These data suggest that HA is not tightly coupled with CAP.
Influence of the calcium ionophore A23187 on rat egg behavior and cortical F‐actin
Authors: Battaglia David E. , Gaddum‐Rosse Penelope
Published: February 2005

Rat eggs treated with the calcium ionophore A23187 and subjected to long‐term observation by phase microscopy were found to undergo many developmental changes that are normally associated with fertilization. These included cortical granule exocytosis and the abstriction of the second polar body. In addition, time‐lapse video microscopy revealed that, unlike untreated eggs, whose surfaces remained relatively immotile, the ionophore‐treated eggs underwent a lengthy period of surface undulatory activity. Since all of these events were remarkably similar in timing and morphology to those seen in fertilized eggs, we conclude that A23187 is capable of activating rat eggs. Using NBD‐phallacidin, the distribution of F‐actin in ionophore‐activated eggs was determined. During most of the postactivation period the eggs possessed an uninterrupted, uniform band of polymerized actin encompassing the entire cortex of the egg. However, during a discrete 1.5‐h period after the formation of the second polar body, an area adjacent to the region of polar body abstriction exhibited more intense staining than the rest of the cortex. Cytochalasin B treatment caused a dramatic reduction and/or rearrangement in cortical NBD‐phallacidin staining in activated eggs as compared to activated controls not exposed to the drug. We observed that all the developmental changes described above could be produced in the absence of exogenous calcium, suggesting that the rat egg possesses internal stores of calcium sufficient to elicit an activational response. We conclude that the ionophore‐induced release of free calcium ions into the cytosol stimulates many of the developmental changes that are normally seen during fertilization. These results indicate that calcium influx and cytoskeletal activity are correlated during the activation of this animal egg.
Changes in binding of a 27‐kilodalton chimpanzee cauda epididymal protein glycoprotein component to chimpanzee sperm
Authors: Young L. G. , Gould K. G. , Hinton B. T.
Published: February 2005

Motility patterns of caput epididymal chimpanzee sperm, caput epididymal chimpanzee sperm incubated in vitro with chimpanzee cauda epididymal fluid, and cauda epididymal chimpanzee sperm were assessed quantitatively. Sperm recovered from the caput epididymis showed no motility, whereas sperm recovered from cauda epididymis showed progressive forward motility. After incubation in cauda fluid, approximately 25% of caput epididymal sperm showed some motile activity. Electrophoretic analysis of 125I‐labeled sperm plasma membrane preparations revealed that the surface of caput epididymal sperm, incubated in cauda fluid, was modified by the appearance of a major protein‐glycoprotein surface component with an apparent molecular weight of 27 kilodaltons (kD). THis 27‐kD component was not detected on caput epididymal sperm incubated in buffer or in caput fluid. However, it was present in cauda fluid and on cauda epididymal sperm. Binding to caput epididymal sperm was cell specific in that chimpanzee erythrocytes incubated in cauda fluid did not bind this 27‐kD cauda fluid component. Motility patterns of ejaculated chimpanzee sperm and of ejaculated chimpanzee sperm incubated in the uterus of adult female chimpanzees also were assessed quantitatively. Ejaculated sperm showed progressive forward motility, whereas in utero incubated ejaculated sperm showed hyperactivated motility typical of capacitated sperm. Electrophoretic analysis of 125I‐labeled sperm plasma membrane preparations revealed the loss of a 27‐kD component from the surface of ejaculated sperm after in utero incubation. No significant change in the 125I‐distribution pattern was detectable when ejaculated sperm were incubated in buffer. These results suggest that the lumenal fluid component, which becomes adsorbed to the surface of chimpanzee sperm during maturation in the epididymis and which is removed from the surface of mature chimpanzee sperm in the female reproductive tract, affects sperm motility.
Detection of acrosome‐reacted toad sperm based on specific lectin binding to the inner acrosomal membrane
Authors: Takamune Kazufumi
Published: February 2005

When the sperm of the toad Bufo japonicus were treated with fluorescein isothiocyanate (FITC)‐conjugated peanut agglutinin (PNA), soybean agglutinin (SBA), or Dolichos biflorus agglutinin (DBA), a few sperm fluoresced at the acrosomal region. The number of sperm showing this lectin binding to the acrosome increased significantly upon mild sonication of the sperm suspension. Electron microscopy revealed that ferritin‐conjugated PNA bind not to the outer acrosomal and overlying plasma membranes, but specifically to the surface of the inner acrosomal membrane exposed by sonication. Both the percentage of FITC‐PNA‐labeled sperm and the activity of vitelline coat lysin released by sperm increased in good correlation with increasing sonication time, although the PNA‐labeled sperm decreased in number upon longer sonication. These results indicate that the binding of FITC‐PNA to the sperm provides a reliable measure of the acrosome reaction of Bufo sperm.
Sperm‐egg interaction in the mouse using live and glutaraldehyde‐fixed eggs
Authors: Soldani Patrizia , Rosati Floriana
Published: February 2005

A study of interaction between gametes in the mouse, using live and glutaraldehyde‐fixed eggs, showed that, while in live eggs the binding is a two‐step process (“early” and “late” binding), the process is one step when the spermatozoa interact with fixed eggs. The second point that emerged from this study is that uncapacitated and capacitated spermatozoa bind the zonae pellucidae of live and fixed eggs in the same way, but only the capacitated spermatozoa bound to live eggs undergo a complete acrosome reaction and penetrate the zona pellucida. Moreover, it has been shown that binding to fixed eggs, just as to live eggs, is Ca2+‐dependent, and it can be reversed by EGTA. Fixed eggs thus are a good model to study only one step of the sperm‐egg interaction removed from all the other events of the fertilization process.
Polyclonal antibodies to a 32‐KDA deglycosylated polypeptide from porcine zonae pellucidae will prevent human gamete interaction in vitro
Authors: Henderson Colin J. , Braude Peter , Aitken R. John
Published: February 2005

The major deglycosylated polypeptides of the porcine zona pellucida (ZP), with molecular masses of 66, 52, 36, and 32 kDa, were purified to homogenity with one‐dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE). Immunofluorescence studies demonstrated that antibodies to the DGZP fraction, and the 66‐ and 32‐kDa polypeptides, bound predominantly to the outer ZP; however, only the first two of these antisera formed an immunoprecipitate around the outer human ZP. In immunoblotting experiments using polyclonal antisera raised to these molecules all four polypeptides exhibited cross‐reactivity with each other and their parental glycoprotein families (ZP 1–4). In addition, the antisera were tested in an in vitro human gamete bioassay to determine their contraceptive potential; antibodies to the 32‐kDa deglycosylated polypeptide inhibited human gamete interaction to the greatest extent, 5.3% (± 1.2%), relative to a control value of 100%.
Influence of proteins in rat cauda epididymidal lumen fluid on cauda sperm motility
Authors: Turner T. T. , Reich G. W.
Published: February 2005

Rat cauda epididymidal spermatozoa are kept quiescent in the cauda lumen by a protein fraction termed “immobilin”, but it has not been shown whether or not this activity is unique to the immobilin fraction. Cauda fluid was subjected to gel filtration chromatography and the eluents contributing to three peaks of absorbance at A280 were pooled, lyophylized, and reconstituted at equivalent concentrations. Their relative viscoelasticity and their effects on sperm motility were determined. Peak 1 (P1), containing proteins > 400 kd, retained the greatest sperm‐immobilizing activity, but P2 and P3 also had sperm‐immobilizing activity related to their viscoelasticity. P1 and an immobilin fraction obtained by ultracentrifugation of rat cauda fluid were generally similar in their sperm‐immobilizing activity, viscoelasticity index, electrophoretic patterns, and binding characteristics to Concanavalin A; thus, the sperm‐immobilizing factor obtained by gel filtration chromatography and the immobilin fraction obtained by centrifugation are believed to be the same products. Further, it was shown that intraluminal testicular fluids did not inhibit cauda sperm motility even at epididymal‐like protein concentrations; thus, it is believed that the sperm‐immobilizing factor(s) is of epididymal, not testicular, origin.
Toxicity potential of residual ethylene oxide on fresh or frozen embryos maintained in plastic straws
Authors: Schiewe Mitchel C. , Schmidt P. M. , Pontbriand D. , Wildt D. E.
Published: February 2005

The toxic effects of residual ethylene oxide (EtO), a frequently used gas‐sterilant, on embryos either frozen for long‐term purposes or stored acutely for 30 min to 9 hr in a fresh condition in 0.25‐ml straw containers were evaluated. In Experiment 1, fresh embryos were frozen (using conventional technology) in straws previously aerated for 0 hr to 8 mo after EtO sterilization. With the exception of the 8‐mo group in which survival and quality ratings were depressed, embryo viability was not affected significantly by short‐term prefreeze and post‐thaw exposure to EtO residues. Experiment 2 was conducted to analyze the influence of prefreeze exposure to EtO residues on embryo development in vitro for embryos temporarily stored in previously sterilized straws aerated for different intervals. Compared to non‐EtO‐sterilized control straws, the development, quality, and viability of embryos exposed to EtO‐treated straws were compromised (p < 0.05) as the aeration interval decreased and the exposure interval increased. The combined results of both experiments indicate that EtO‐treated straws can be used to cryopreserve gametes efficiently, but only if the aeration interval is ≥72 hr and the prefreeze duration of exposure is ⩽3 hr.
Hamster sperm motility transformation during development of hyperactivation in vitro and epididymal maturation
Authors: Suarez Susan S.
Published: February 2005

The transformation of hamster sperm motility during capacitation in vitro and during maturation in the caudal epididymis was analyzed and compared using videomicrography. Sperm recovered from the distal portion of the caudal epididymis, as well as ejaculated sperm recovered from the uterus exhibited low amplitude, planar flagellar beating. By 3 hr of incubation under capacitating conditions, the caudal epididymal sperm were swimming in helical patterns apparently produced by significantly increased acuteness of flagellar bending and by torsion seen as abrupt, periodic turning of the head. By 4 hr, most sperm were hyperactivated, swimming in circles resulting from asymmetrical, planar flagellar bending that was significantly more acute than the preceding patterns. When motility parameters of fresh sperm were compared with those of sperm swimming in the transitional helical pattern and with hyperactivated sperm, transitional sperm had significantly higher net and average path velocities than the others, indicating that they covered space at the greatest rate. This suggests that the transitional phase plays an important role in sperm transport. Sperm recovered from the proximal region of the caudal epididymis, near the corpus, swam in either the helical or hyperactivated patterns, or a mixture of the two. The means of their flagellar curvature ratios and linear indices were intermediate between helical and hyperactivated mean values. Thus, sperm undergoing final maturation in the caudal epididymis reverse the pattern of development of hyperactivation. Also, the development of hyperactivated motility must therefore entail induction of a preexisting potential for flagellar movement, rather than a maturational process.
The role of membrane‐active lipids in the protection of ram spermatozoa during cooling and storage
Authors: Holt W. V. , North R. D.
Published: February 2005

The interactions of ram spermatozoa with exogenous liposomes of varying composition were studied, with the aim of examining the mechanisms by which some lipids protect against cold‐induced damage during cryostorage. Liposomes containing various preparations of phosphatidylcholine and cholesterol enhanced sperm survival during storage at 5°C, both in terms of motility and acrosomal integrity. A membrane‐fluidizing agent, A2C, was slightly deleterious in this respect. Cholesterol‐containing liposomes were not superior in their effects to those prepared without cholesterol. Thus stabilization of the plasma membrane by cholesterol loading may be unimportant.

When sodium vanadate was used as a functional probe of membrane integrity, the cryoprotective effects of lipids were apparent despite increased plasma membrane permeability. Incubation of spermatozoa with positively charged liposomes, containing stearyl‐amine, caused considerable loss of motility and acrosomal damage, coupled with cellular aggregation. There was also some evidence that the presence of calcium lessened the effectiveness of liposomes in protecting spermatozoa against damage during cooling.
Differential distribution of the P1 and P2 protamine gene sequences in eutherian and marsupial mammals and a monotreme
Authors: Johnson Paula A. , Yelick Pamela C. , Liem Hetty , Hecht Norman B.
Published: February 2005

At the protein level, the P1 protamine is the predominant form of mammalian protamine, present in all mammalian spermatozoa analyzed to date. An additional variant, the P2 protamine, has been detected only in spermatozoa of the mouse, hamster and human. Southern blot analysis of a group of restriction enzyme‐digested mammalian DNAs has revealed the presence of sequences homologous to the P1 and the P2 mouse protamine genes in diverse species. In agreement with protein studies, nucleotide sequences homologous to the mouse P1 protamine cDNA are widespread, being present in the genomic DNAs of human, rat, dog, ram, horse, bull, hamster, baboon, flying fox (megabat), microbat, boar, North American opossum, and wallaby. Although we detect genomic sequences with strong homology to the mouse protamine 2 cDNA in rat and hamster, we also find weaker but reproducible hybridization to the genomic DNA of human, boar, dog, bull, microbat, wallaby, and platypus. With the exception of the human, the P2 protamine has not been detected in the spermatozoa of these latter species.
Ultrastructure of spermatogenesis in the sea star, Asterina minor
Authors: Yamagata Atsuko
Published: February 2005

The ultrastructural features of spermatogenesis were investigated in the hermaphroditic sea star Asterina minor. The primordial germ cells in the genital rachis contain small clusters of electron‐dense material (nuage material) and a stack of annulate lamellae. They also have a flagellum and basal body complex situated close to the Golgi complex. After the development of the genital rachis into the ovotestis, spermatogenic cells increase in number and differentiation begins. Nuage material is observed in spermatogonia, but it gradually disappears in spermatocytes. The annulate lamellae do not exist beyond the early spermatogonial stage. By contrast, a flagellum and basal body complex are found throughout spermatogenesis. The Golgi‐derived proacrosomal vesicles appear in the spermatocyte and coalesce to form an acrosomal vesicle in the early spermatid. The process of acrosome formation is as follows: (1) a lamella of endoplasmic reticulum (ER) continuous with the outer nuclear membrane encloses the posterior portion of the acrosomal vesicle; (2) the vesicle attaches to the cell membrane with its anterior portion; (3) periacrosomal material accumulates in the space between the acrosomal vesicle and the ER; (4) the nucleus proper changes its features to surround the acrosome; (5) amorphous, electron‐dense material is deposited under the electron‐dense disk; and (6) the nucleus forms a hollow opposite the electron‐dense material.
Quantitative comparison of the passage of homologous and heterologous spermatozoa through the uterotubal junction of the golden hamster
Authors: Smith T. Timothy , Koyanagi F. , Yanagimachi R.
Published: February 2005

A quantitative method was used to determine whether the spermatozoa of foreign species could pass through the uterotubal junction (UTJ) of the hamster as efficiently as homologous (hamster) spermatozoa. Estrous female hamsters were artificially inseminated with epididymal spermatozoa of homologous and heterologous (foreign) species. The number and distribution of spermatozoa in the oviduct were determined several hours after insemination (shortly before ovulation). The passage of immotile (dead) hamster spermatozoa through the UTJ was also examined.

It was found that the spermatozoa of all foreign species tested (rat, mouse, guinea pig, and rabbit), as well as immotile hamster spermatozoa, could pass through the UTJ but did so in much smaller numbers compared to live hamster spermatozoa. This was not specifically due to poor survival of foreign spermatozoa in the hamster uterus, as the viability of all inseminated spermatozoa (including hamster spermatozoa) was considerably reduced by 1 h after insemination. While a large number of live hamster spermatozoa were distributed throughout the caudal isthmus at the time of examination, none or only a very few foreign spermatozoa had advanced this far. The few foreign and immotile spermatozoa that reached the caudal isthmus were confined to the first ascending loop of this segment. Some possible causes for the small number and retarded advance of foreign spermatozoa in the hamster oviduct were discussed.
Fertilizing potential of acrosome‐defective sperm following microsurgical injection into eggs
Authors: Lanzendorf S. , Maloney M. , Ackerman S. , Acosta A. , Hodgen G.
Published: February 2005

Ejaculates from three infertile men were examined ultrastructurally and found to include a high number of amorphous acrosomeless spermatozoa. Two of the patient's spermatozoa exhibited the typical characteristics of round‐head syndrome—spherical‐shaped heads completely absent of acrosome and postacrosomal sheath. The semen of the third patient was found to contain a mixture of round‐headed and irregularly shaped acrosomeless sperm and a small percentage of normal acrosome‐intact sperm. Previous studies have shown that acrosomeless sperm do not have the ability to bind or penetrate zona‐free hamster eggs (Weissenberg et al., Syms et al.). In an attempt to determine if such amorphous sperm are capable of decondensation and pronuclear formation, sperm of all three men were microsurgically injected into zona‐intact hamster eggs. All of the sperm injected were found to be capable of decondensation or pronuclear formation, suggesting that if the inability to penetrate an egg is bypassed, the sperm of these infertile men are capable of participating in the early events of fertilization.
Chimerization of highly asynchronous murine blastomeres: Developmental alteration?
Authors: Prather R. S. , First N. L.
Published: February 2005

Previous studies have shown that early embryos contain information that can alter the developmental fate of adjacent cells and transferred nuclei. In this report we show that a specific combination of cells from early murine embryos, a single blastomere from an eight‐cell embryo placed under the zona pellucida with a two‐cell embryo, results in a difference in incorporation of 3H‐uridine and expression of two protein bands between the chimeric treatment group and the nonchimeric controls, a single blastomere from an eight‐cell embryo in a separate zona pellucida and a two‐cell embryo. The incorporation of 3H‐uridine in the chimeric group and nonchimeric control group was significantly different at 45 hours after chimerization (P < .02). A stage‐specific protein band (52k) on a polyacrylamide gel detected with fluorography was found to be qualitatively different (present more often; P < .01) and another stage‐specific protein band (48k) was found to be quantitatively different (more protein; P equals; .07) in the chimeric treatment vs. the nonchimeric controls at 45 hours after chimerization. These results suggest communication between the cells resulting in a change in their incorporation of uridine and protein synthetic profiles.
DNA content of spermatozoa in different genetic lines of turkeys
Authors: Lamošová D. , Baumgartner J.
Published: February 2005

The variability of DNA content in turkey spermatozoa of four different lines and its correlation with body weight and sperm concentration were studied. In lines selected for lower body weight the DNA content was 2.034 and 2.036 pg per spermatozoon. In lines selected for higher body weight the DNA content was 2.267 and 2.370 pg per spermatozoon. Sperm concentration in 1 mm3 of semen, however, was higher in lines with a lower body weight (6.08–6.21 million) in comparison with lines selected for higher body weight (5.46–5.67 million). The correlations between the DNA content and sperm concentration were negative (r ranged from −.457 to −.860).
Actin synthesis is not regulated by granulosa cells in mouse growing and preovulatory oocytes
Authors: Canipari R. , Bevilacqua A. , Colonna R. , De Felici M. , Mangia F.
Published: February 2005

The synthesis and intracellular distribution of actin were studied in isolated dictyate and metaphase II mouse oocytes by (1) sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of newly synthetized oocyte protein and (2) cytochemical F‐actin labeling by fluorescent phalloidin. Unpermeabilized, fully grown oocytes bound phalloidin intensely at the level of the zona pellucida (ZP), such ZP‐associated actin representing a significant portion of total actin found in these cells. In contrast, phalloidin binding to ZP was very low in growing oocytes and was undetectable in ovulated, metaphase II eggs. When ZP‐associated actin of fully grown oocytes was removed by prolongedly exposing oocytes to α‐chymotrypsin, the amount of newly synthesized actin displayed by cumulus‐enclosed oocytes was reduced to a level comparable to that shown by oocytes isolated from granulosa cells. We demonstrate that ZP‐associated actin belongs to granulosa cell processes that remain within the ZP as a consequence of oocyte isolation procedures. We conclude that actin synthesis of mouse oocytes is not regulated by granulosa cells.
Viability of mouse half‐embryos in vitro and in vivo
Authors: Lawitts Joel A. , Graves Charles N.
Published: February 2005

Mouse embryos at the 2‐, 4‐, 8‐cell, and morula stage were divided in half by using microsurgical procedures and were either grown in vitro up to the blastocyst stage or transferred at the late morula stage into the uteri of pseudopregnant recipients. A relatively high percentage of the half embryos from 2‐cell (70%), 4‐cell (75%), 8‐cell (93%), or morula stage embryos (75%) developed into blastocysts in vitro. However, the overall development in vivo of half embryos was low, as 3%, 13%, 8%, and 1% of half embryos from the 2‐cell, 4‐cell, 8‐cell, and morula stages, respectively, developed into live fetuses.

Embryos which were divided in half at different stages developed at different rates in vitro. This determined the stage of embryonic development at the time of transfer, which might have interacted with the stage of pseudopregnancy of the recipients to influence embryo survival in vivo.
Culture of in vitro fertilized rabbit ova
Authors: Keefer C. L. , Fayrer‐Hosken R. A. , Brown L. M. , Brackett B. G.
Published: February 2005

Embryonic development of in‐vitro fertilized rabbit ova was assessed following in‐vitro culture in four different serum supplemented media. A mixture of Basal Medium Eagle (BME) and Ham's F10 medium (1:1) provided better support for in‐vitro development than Ham's F10, BME, or regular acidic saline (RAS). In‐vitro embryonic development in the BME/Ham's F10 mixture was synchronous with in‐vivo development through at least 55 hr of culture. After 54 hr of culture, embryos transferred to the oviduct of a synchronous pseudopregnant recipient were able to implant at the same rate as simultaneously transferred embryos grown in vivo. BME/Ham's F10 supplemented with 10% newborn calf serum was highly supportive of rabbit embryo development following in‐vitro fertilization.
Refertilization in eggs of the sea urchin Strongylocentrotus purpuratus
Authors: Kubota Lynda F. , Carroll Edward J.
Published: February 2005

To determine the role of the sea urchin egg plasma membrane in the species‐specificity of fertilization, the ability of denuded activated eggs to be heterospecifically refertilized was determined. Our initial studies included evaluating the effectiveness of three commonly used methods of vitelline envelope (VE) removal using indirect immunofluorescence microscopy with antibodies directed against the VE. Unfertilized Strongylocentrotus purpuratus eggs were extracted with 0.01 M dithiothreitol (DTT) for 3 min or digested with 1.0 mg/ml pronase for 1 hr. Eggs were also fertilized, then diluted into a divalent‐free medium to produce thin, elevated envelopes (VE*s) that were mechanically removed by sieving the eggs through nylon mesh. We found that both DTT extraction and pronase digestion were not completely effective in VE removal, and mechanical removal methods gave rise to a mixed population of eggs, those that had their VEs removed and those with a collapsed envelope that was not detectable at the light microscope level. Therefore, a new method of VE removal was developed. Eggs with VE*s were prepared followed by treatment with 0.01 M DTT to solubilize the envelopes. Nearly 100% of the denuded activated eggs incorporated one or more homologous and heterologous sperm, suggesting that the egg plasma membrane does not function in determining the species‐specificity of fertilization.
Effect of cholesterol and phosphatidylcholine of different chain length on acrosome breakdown and fertilizing capacity of amphibian spermatozoa
Authors: Fontdevila Marina F. Diaz , Bloj Bernabe , Cabada Marcelo O.
Published: February 2005

The effect of different lipids on the fertilizing capacity of Bufo arenarum spermatozoa and on acrosome breakdown of Leptodactylus chaquensis spermatozoa was studied. Sonicated vesicles of egg yolk phosphatidylcholine (1 mM) were as effective as vesicles of egg yolk phosphatidylcholine:cholesterol (molar ratio 1:0.9) in inhibiting the fertilizing capacity of Bufo arenarum spermatozoa. This suggests that cholesterol depletion from the spermatozoa was not the cause of the fertility loss. Bufo arenarum spermatozoa were incubated with phosphatidylcholines with even chain length from 6 to 18 carbons. At a concentration of 0.01 mM, didecanoyl‐phosphatidylcholine reduced fertilizing capacity to 10% in a few minutes and to 0% within 60 minutes. Didodecanoyl‐phosphatidylcholine required 2 hours to reduce fertility to 10% and 4 hours to cause a 100% loss of fertilizing capacity. A concentration of didecanoyl‐phosphatidylcholine as low as 5 × 10−4 mM caused a more than 95% fertility loss in less than five minutes. At a concentration of 0.1 mM, didecanoyl‐phosphatidylcholine induced complete acrosome breakdown in Leptodactylus chaquensis spermatozoa in 15 minutes, whereas didodecyl‐phospatidylcholine required 2 hours. At a concentration 100‐fold lower didecanoyl‐phosphatidylcholine induced complete acrosome breakdown in 2 hours. Electron microscopic observations in both species showed loss of acrosome caused by the action of the didecanoyl‐phosphatidylcholine. Longer chain phosphatidylcholines exerted an inhibitory effect on Bufo arenarum spermatozoa fertilizing capacity at a higher concentration when in a vesicular form.
A major secretory protein from rat seminal vesicle binds ejaculated spermatozoa
Authors: Manco G. , Abrescia P.
Published: February 2005

The rat seminal vesicle produces large amounts of a protein‐rich fluid that greatly contributes to semen volume. RSV IV, a protein abundantly secreted from this gland, binds in vitro to rat epididymal spermatozoa. However, there is no evidence that this protein may have an in vivo role as a sperm‐coating antigen. We report in this paper that high‐molecular‐weight RSV IV immunologically related proteins can be detected on ejaculated spermatozoa, but not on epididymal spermatozoa. After incubation of purified RSV IV with ejaculated spermatozoa in freshly recovered semen or with epididymal spermatozoa in a medium containing the coagulating gland secretion, sperm‐bound proteins with analogous properties were detected. These results support the hypothesis that RSV IV is modified at ejaculation to an high‐molecular‐weight, sperm‐coating antigen.
Phagocytosis of sperm heads lacking the acrosomal process by unfertilized starfish oocytes
Authors: Kyozuka Keiichiro , Osanai Kenzi
Published: February 2005

In sperm of the starfish Asterina pectinifera, the acrosomal process and the flagellum were mechanically separated from the sperm head with a disperser. The sperm head fraction was then used to examine the direct interaction between the sperm head and the egg surface. Sperm heads lacking the acrosomal process and the flagellum did not fertilize oocytes, even after removal of the vitelline coat. Transmission electron microscopy showed that each denuded oocyte engulfed the sperm head without gamete membrane fusion. The sperm‐engulfing response, similar to phagocytosis, was induced without the mediation of the acrosomal process. The present results suggest that the process of sperm incorporation consists of two independent events, acrosomal process‐egg surface fusion and the phagocytotic movement of the egg surface.
Fertilization in a chiton: Acrosome‐mediated sperm‐egg fusion
Authors: Buckland‐Nicks John , Koss Ron , Chia Fu‐Shiang
Published: February 2005

Contrary to the widely accepted view that chiton sperm lack acrosomes and that fertilization in this group occurs via a micropyle, we demonstrate here that fertilization in Tonicella lineata occurs by acrosome‐mediated sperm‐egg fusion. The acrosome is a small vesicle containing two granules located at the tip of the sperm. The eggs have an elaborate hull (=chorion), which is formed into cupules that remain covered by follicle cells until maturity. When dissected ripe eggs were exposed to sperm in vitro, the sperm were attracted only to open cupules, inside which they swam through one of seven channels to the base where they penetrated the hull. The acrosome fired on contact with, or in, the hull, and during passage through it the apical granule was exhausted while the basal granule was exposed. If sperm contacted follicle cells between the cupules the acrosome did not react. The vitelline layer beneath the hull contains pores arranged in a regular pattern. Embedded in the base of each pore is an egg microvillus. Having penetrated the hull the sperm anterior filament located a pore and fused with the tip of the egg microvillus projecting into it. This created a membranous tube, through which the sperm nucleus was injected into the egg. The egg membrane appeared to be raised up into a small fertilization cone around the penetrating sperm, the vitelline layer became slightly elevated, and some cortical granules were released by exocytosis.
Follicular factors influence oocyte fertilizability by modulating the intercellular cooperation between cumulus cells and oocyte
Authors: Mattioli M. , Galeati G. , Bacci M. L. , Seren E.
Published: February 2005

In order to investigate whether the follicular tissue influences cumulus‐oocyte interaction and, consequently, the fertilizability of the egg, four experiments were carried out. In the first, cumulus‐enclosed pig oocytes were cultured for 44 h in control medium (modified TCM‐199) or in follicle‐conditioned medium, and the intercellular coupling was studied by measuring 3H‐uridine uptake. In control medium the intercellular cooperation started to decline immediately, and at 24–32 h the uncoupling was almost complete. By contrast, in follicle, conditioned medium, it remained at high levels until 24‒32 h. In the second experiment protein synthesis patterns of oocytes were studied. Oocytes cultured in conditioned medium were characterized by a 45‐kD protein band, while those maturing in control medium were identifiable by a marked 56‐kD band. In the third experiment mature oocytes were fertilized in vitro. The percentage of penetrated egg was higher in oocytes matured in conditioned medium than in control medium. In addition, only oocytes matured in conditioned medium could consistently decondense spermatozoa and form male pronuclei. Metabolic cooperation, protein synthesis patterns, and fertilizability were also studied in oocytes matured in control medium supplemented with either 17β‐estradiol or progesterone or testosterone or dihydrotestosterone or androstenedione or ether extract of conditioned medium. Only ether extract and progesterone stimulated cumulus oocyte interaction and sperm decondensation. In the last experiment oocytes denuded at different stage of their maturation in conditioned medium were fertilized in vitro. The longer the eggs were cultured with the cumulus, the higher was their penetrability. Moreover, only oocytes denuded after 40 h of culture could, once fertilized, promote the formation of male pronuclei.

These data demonstrate that follicular secretions are fundamental for the maintenance in vitro of a functional intercellular coupling between cumulus and oocyte, which is necessary for the egg to become penetrable by spermatozoa and to acquire the conditions required for the formation of male pronuclei.
Evidence for sequential deployment of secretory enzymes during the normal acrosome reaction of guinea pig sperm in vitro
Authors: DiCarlantonio Gayle , Talbot P.
Published: February 2005

Experiments were conducted to determine if acrosomal enzymes are released simultaneously or in sequence during the normal acrosome reaction. Epididymal guinea pig sperm were incubated in a chemically defined, calcium‐containing medium which supports normal acrosome reactions within 4–5 hours at 37°C. The sperm suspensions were monitored for motility, normal acrosome reactions, and false acrosome reactions during in vitro incubation. At specified time intervals, the sperm were separated from the incubation medium by centrifugation, and the distribution of dipeptidyl peptidase (DPP II) and acrosin activity was determined by biochemically assaying the hydrolysis of trialanine and N‐benzoyl‐L‐arginine ethyl ester (BAEE), respectively. When calcium was present, there was a significant increase in DPP II activity in the supernatants by 1 hour of incubation and a slight decline at later time points. This release was not correlated with false or normal acrosome reactions (loss of the acrosomal cap) monitored by phase‐contrast microscopy but probably represents a very early stage in the normal acrosome reaction. This early stage is difficult to detect at the light microscope level because sperm are still in rouleaux and because membrane fusion is not directly observable. In contrast, acrosin activity, which was assayed in the same supernatants, increased at later times when sperm were observed to have completed normal acrosome reactions. The ultrastructural distribution of DPP II was determined in sperm pellets collected during in vitro incubation by using the DPP II substrate lysyl‐alanyl‐4‐methoxy‐2‐naphthyamide. In freshly isolated cauda epidiymal sperm, reaction product is confined to the light‐staining area in the dorsal bulge of the acrosome. However, by 1 hour of incubation, the light‐staining area of many sperm was partially or completely dispersed, while other regions of the acrosome were unchanged. Our data are consistent with the conclusions that DPP II is a highly soluble component of the guinea pig sperm acrosome and that its release occurs during the initial phase of the acrosome reaction while sperm are still in rouleaux. Structural changes in the acrosome associated with DPP II release were detectable by electron microscopy but not by light microscopy. Acrosin, which is less soluble than DPP II, is released at a later time during the acrosome reaction. Both DPP II and acrosin appear to be partially inhibited following their release from sperm. A complete understanding of the sequential release and extracellular activities of the acrosomal enzymes will be necessary to fully define their functions in fertilization.
A comparison of sperm morphology and silver nitrate staining characteristics in the domestic ferret and the black‐footed ferret
Authors: Curry Patrick T. , Ziemer Terry , Van der Horst Gerhard , Burgess Warren , Straley Monte , Atherton Robert W. , Kitchin Robert M.
Published: February 2005

Ejaculated sperm from the domestic ferret (Mustela putorius furo) and the black‐footed ferret (Mustela nigripes) were compared for differences in morphological abnormalities and argentophilic protein distribution. Thawed domestic ferret sperm was also compared to fresh sperm to determine whether there were any effects on cell morphology due to cryopreservation. There were statistically significant differences between the two species of ferret in two of the categories scored. The domestic ferret had a higher frequency of cells that were bent in the midpiece and in the principal piece, and a higher frequency of headless and tailless cells when compared to the black‐footed ferret. There were no statistically significant differences in cell morphology between the fresh and cryopreserved ejaculates of the domestic ferret employing a standard egg yolk cryoextender. Silver nitrate staining distribution was different between the two species in both the head and tail region.
Production of H‐Y antibody in the ascites fluid of mouse and localization of the antigen on cells and tissues
Authors: Iyer Subramanian V. , Nandedkar Tarala D. , Hegde Umashashi C.
Published: February 2005

A procedure is described for the production of large amounts of ascites fluid containing specific H‐Y antibody. The distribution of H‐Y antigen on mouse epididymal spermatozoa, thymocytes, and splenocytes was carried out using this specific antibody in the microcytotoxicity test and ELISA. Employing the indirect immunofluorescent technique, the H‐Y antigen was localized on the acrosomal membrane of mouse epididymal and washed ejaculated human spermatozoa and on the entire membrane of mouse splenocytes and thymocytes. Immunohistochemical localization of the antigen in the testicular section indicated its presence in the cytoplasm of Leydig cells and on the membrane of Sertoli cells and sperm heads.
Fertilization by sperm injection in the rabbit
Authors: Keefer Carol L.
Published: February 2005

Whole rabbit spermatozoa and isolated sperm nuclei were microinjected directly into the ooplasm of hamster and rabbit ova. These injected sperm decondensed and formed male pronuclei during subsequent in‐vitro culture. Injection of whole spermatozoa and sperm nuclei prepared by a protocol known to allow in‐vitro capacitation of ejaculated spermatozoa yielded a significantly higher (P < 0.01) number of activated rabbit ova containing male pronuclei than did injection of uncapacitated epididymal sperm nuclei or ejaculated sperm nuclei. Rabbit ova fertilized by sperm injection were capable of undergoing normal‐appearing cleavage division during 22 h of culture.
Induction of cross‐fertilization between sea urchin eggs and starfish sperm by polyethylene glycol treatment
Authors: Kyozuka Keiichiro , Osanai Kenzi
Published: February 2005

Cross‐fertilization between sea urchin eggs (Strongylocentrotus nudus) and starfish sperm (Asterina pectinifera) was induced by treatment with polyethylene glycol (PEG). Without treatment with PEG, the denuded egg surface (jelly coat‐ and vitelline coat‐free) engulfed the head of acrosome‐reacted sperm; however, sperm penetration did not occur [Kyozuka and Osanai, 1988]. When these eggs were exposed briefly to PEG (molecular weight 3,000) in seawater, the sperm entered the egg by membrane fusion. Cortical granules were discharged, and embryogenesis began following sperm penetration. PEG did not induce parthenogenesis in Strongylocentrotus eggs. Egg activation is thus closely linked with gamete membrane fusion.