bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fertilization in a chiton: Acrosome‐mediated sperm‐egg fusion
Authors: Buckland‐Nicks John , Koss Ron , Chia Fu‐Shiang 
Published: February 2005

AbstractContrary 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

AbstractIn 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

AbstractExperiments 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

AbstractEjaculated 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

AbstractA 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

AbstractWhole 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

AbstractCross‐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.
Production of identical twins by separating two‐cell rat embryos
Authors: Matsumoto K. , Miyake M. , Utsumi K. , Iritani A. 
Published: February 2005

AbstractRat identical twins were produced from two‐cell embryos. In the presence of cytochalasin B, rat two‐cell embryos could be separated efficiently into two blastomeres by micromanipulation. Isolated blastomeres, embedded in agar cylinders and cultivated in ligated rat oviducts for 3 days, developed to the morula or blastocyst stage. After removing the agar, pairs of developed one‐half embryos were transferred into Day 1 oviducts or Day 4 uteri of pseudopregnant rats. The percentage of embryos, separated either in the presence or absence of cytochalasin B, that developed into live fetuses was higher in cases of uterine transfer than in cases of oviduct transfer (38% vs. 18%, 31% vs. 15%, respectively). Throughout the present experiment, nine pairs of identical twins were successfully produced. This is the first report of the production of identical rat twins by separating two‐cell embryos.
Age dependence of bovine oocyte activation
Authors: Ware C. B. , Barnes F. L. , Maiki‐Laurila M. , First N. L. 
Published: February 2005

AbstractThe ability of bovine oocytes to undergo parthenogenetic activation using either a Ca++‐Mg++‐H+ ionophore (A23187) or electric shock was investigated, as a prelude to understanding activation potential following nuclear transfer into ooplasm. Oocytes were collected from slaughterhouse ovaries by aspiration of 1–5‐mm follicles. The time of placement into maturation medium was noted, and maturational age (time in culture) measured from that point. After exposure to activating conditions eggs were cultured for a further 12–16 hours, fixed, and stained with aceto‐orcein. Oocytes that progressed to telophase or pronuclear formation were considered activated. Concentrations of A23187 ranging from 100 pM to 100 μM showed that 1–100 μM levels resulted in 94–100% activation at 30 hours maturation. Frequency of activation differed from controls (no ionophore) at 100 nM (49%; P < 0.05). With A23187 maximum response occurred between 26 and 30 hours of maturation (77% and 92%, respectively). A short pulse electric shock, capable of causing oocyte membrane fusion, gave similar results relative to maturational age (82% and 90% activation for 26 and 30 hours, respectively). Therefore, maximum response to the two activating stimuli occurred in oocytes at similar maturational ages. Exposure to activating conditions prior to onset of activating ability (18 hours) followed by another exposure at 26 hours showed that the oocytes were still fully able to activate upon reaching maturational activation competence. Because cytochalasin B is present in the medium used for nuclear transfer, oocytes were incubated with cytochalasin B prior to exposure to an activating stimulus. Frequency of activation was similar to the control treatment (61% and 73%). The effect of mechanical stress of cytoplasm removal and replacement by electrofusion on activation was also not significant. Overall, maturational age of the oocyte was the main determinant of activation ability.
Protein carboxyl methyltransferase activity specific for age‐modified aspartyl residues in mouse testes and ovaries: Evidence for translation during spermiogenesis
Authors: O'Connor Clare M. , Germain Bonnie J. , Guthrie Kathleen M. , Aswad Dana W. , Millette Clarke F. 
Published: February 2005

AbstractAn antiserum prepared against the purified protein carboxyl methltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age‐damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell‐deficient ovaries and testes obtained from mutant atrichosislatrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4–5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5‐fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.
Modification of the zona‐free hamster ova bioassay of boar sperm fertility and correlation with in vivo fertility
Authors: Berger Trish , Parker Kent 
Published: February 2005

AbstractThese studies were designed to evaluate the ability of the zona‐free hamster ova bioassay to detect differences in fertility of boar sperm. In the first study, sperm from two previously infertile boars were compared to sperm from seven previously fertile boars. The percentage of zona‐free hamster ova penetrated by sperm from the previously infertile boars was significantly lower than the percentage of ova penetrated by sperm from previously fertile boars (18% of ova penetrated vs. 83%, P < .001). In the 14 ejaculates from the previously infertile boars that had ejaculate motilities of 50% or greater, the percentage of zona‐free hamster ova penetrated continued to be lower than in ejaculates from the fertile boars. One of the two previously infertile boars consistently had a normal semen analysis. The only two observed manifestations of his reduced fertility were his zero conception rate and the limited ability of his sperm to penetrate zona‐free hamster ova. In the second study, females were inseminated with equal numbers of sperm from two previously fertile males and the paternity of offspring determined at birth. The experiment was replicated with four combinations of six boars. A high correlation was observed between the percentage of offspring sired and the ability to penetrate zona‐free hamster ova (R = .89). Neither morphology nor the ability of the sperm to undergo an acrosome reaction during in vitro incubation was correlated with fertility in the competitive mating situation. These results suggest the zona‐free hamster ova bioassay can improve the in vitro fertility assessment of fresh boar semen.
Pregnant mares' serum gonadotropin source influences fertilization and fresh or thawed embryo development, but the effect is genotype specific
Authors: Schmidt P. M. , Monfort S. L. , Wildt D. E. 
Published: February 2005

AbstractThe influence of the source of pregnant mares' serum gonadotropin (PMSG) on the num ber, quality, and in vitro development of mouse embryos before and after freezing was evaluated among three genotypes: N:NIH(S), C57BL/6N, and C3H/HeN‐MTV−. Immature females were given PMSG from one of five commercial sources. Following col lection ( 116 hr later), embryos were evaluated for stage of development, and four‐to eight‐cell embryos were pooled within genotype and assigned to standardized fresh or freeze‐thaw culture trials. Different PMSG sources stimulated the production of different num bers of total embryos (P < 0.05) but not necessarily more embryos suitable for freezing. Differences in embryo production among genotypes indicated that absolute embryo num bers using a single mouse genotype may not accurately reflect the potency of a specific gonadotropin source. The PMSG source also affected the ability of an embryo to survive in culture either immediately after collection or after frozen storage. The effect, however, was genotype specific, with some mouse strains being relatively insensitive to PMSG source, whereas gonadotropin source played a major role in determining in vitro viability in others. Development rates for freshly collected embryos differed, often inconsistently, from those of thawed embryos regardless of the PMSG source used, demonstrating that fresh embryo development cannot be used to estimate expected post‐thaw survival. In vitro development of thawed embryos is influenced not only by genotype, but also the source of the gonadotropin used to promote follicular development and oocyte maturation. These findings may explain, in part, the wide variation in embryo viability and culture rates reported among laboratories and intraspecies animal populations.
Gamete processing in an environmental chamber: Effect on in vitro fertilization outcome
Authors: Gwatkin Ralph B. L. , Quigley Martin M. , Collins Robert L. 
Published: February 2005

AbstractA study of mouse gamete processing for in vitro fertilization (IVF) under various conditions showed that it is necessary to control the atmosphere if the temperature is raised from 22°C to 37°C. The data suggest that maximum IVF success is attained by processing the gametes at 37°C, under an atmosphere of 5% O2 and 5% CO2, and overlaying the medium with silicone oil.
Influence of cumulus cell processes on oolemma permeability and lethality of isolated mouse oocytes cultured in Ca2+‐free medium
Authors: De Felici Massimo , Dolci Susanna , Siracusa Gregorio 
Published: February 2005

AbstractCumulus cell processes remaining in the zona pellucida of mouse occytes mechanically isolated from the ovary have been indirectly visualized by labeling their actin microfilament core with rhodaminyl‐phalloidin. If the isolation of the oocytes is performed in Ca2+‐free medium, the preisence of such processes allows the entry into the cell of low molecular weight molecules (such as 5‐6 carboxyfluorescein) and contributes to the death of the cell in such experimental conditions.Following dissolution of the zona pellucida (by enzymatic or acidic treatment) the oocyte is no longer permeable to small molecules and becomes resistant to Ca2+‐free medium, probably as a consequence of the collapse of cumulus cell processes. The role of cumulus cell processes and gap junctions in the permeability of mechanically isolated ovarian oocytes is discussed.
Structure of the zona pellucida and cumulus oophorus in three species of native Australian rodents
Authors: McGregor Lesley , Flaherty Sean P. , Breed William G. 
Published: February 2005

AbstractThe sperm head of many Australian hydromyine rodents has three curved hooks projecting from its anterior margin; the structure of the hooks has been characterized, but their function is unknown. In this study, we have investigated whether the hooks might have evolved to assist sperm penetration through more formidable egg vestments, particularly the zona pellucida. Cumulus‐oocyte complexes were obtained from two species that possess a three‐hooked sperm head (Pseudomys australis and P. nanus) and one species that does not (Notomys alexis) and examined by light and electron microscopy. After fixation in the presence of ruthenium red, the zona pellucida was found to consist of a fibrillar meshwork, but there were no interspecific structural differences. A corona radiata was absent, and the cumulus extracellular matrix was composed of filaments and electron‐dense granules in each species. Measurements of the zona thickness in freshly ovulated, unfixed oocytes revealed that it was thinnest (7.8 μm) in P. australis. Which has a three‐hooked sperm head, and thickest (11.4 μm) in N. alexis, the species in which the ventral hooks are absent. Hence, no correlation was found between the thickness of the zona pellucida or the structure of the cumulus‐oocyte complex, and the presence of three hooks on the sperm head. We conclude, therefore, that it is unlikely that the evolution of the three‐hooked sperm head is an adaptation for penetration of increased barriers around the oocyte.