bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flagellar movement of human spermatozoa
Authors: Ishijima Sumio , Oshio Shigeru , Mohri Hideo 
Published: February 2005

AbstractFlagellar movement of human spermatozoa held by their heads with a micropipette was recorded by means of a video‐strobe system. Spermatozoa were studied in normal Hanks' solution, Hanks' solution with increased viscosity, cervical mucus, and hyaluronic acid.When flagellar movement in normal Hanks' solution was observed from the direction parallel to the beating plane, segments of the flagellum in focus did not lie on a straight line but on two diverging dashed lines. The distance between the two dashed lines was about 20% of the bend amplitude in the major beating plane. These observations indicate that flagellar beating of human spermatozoa in normal Hanks' solution is not planar. In contrast, segments of the flagellum in focus lay on a straight line when the spermatozoa were observed in Hanks' solution with increased viscosity, cervical mucus, or hyaluronic acid. In normal Hanks' solution, free swimming spermatozoa rotated constantly around their longitudinal axes with a frequency similar to the beat frequency, whereas little or no rotation of spermatozoa occurred in Hanks' solution with increased viscosity, in cervical mucus, or in hyaluronic acid.We conclude that human spermatozoa in normal Hanks' solution beat with a conical helical waveform having an elliptical cross section, the semiaxes of which have a ratio of 0.2. The three‐dimensional geometry of the flagellar movement is responsible for the rotation of the sperm around their longitudinal axes.
Ultrastructural and cytochemical changes of the head components of human spermatids and spermatozoa
Authors: Dadoune J. P. , Alfonsi M. F. 
Published: February 2005

AbstractThe ultrastructural study of chromatin condensation simultaneously with the evolution of the perinuclear organelles was conducted in the spermatids and epididymal and ejaculated spermatozoa of man with the aid of the “en bloc” alcoholic PTA staining and the EDTA regressive method.The round nuclei of young spermatids (steps 1, 2) were characterized by the persistence of nucleoli that were PTA positive, and the presence of a subacrosomal layer of well‐stained peripheral chromatin. In the beginning of the phase of nuclear elongation (step 3), the central chromatin also became dense, like the peripheral chromatin, while the nuclear ring and the associated manchette and the two anlages of the postacrosomal dense lamina and the posterior ring appeared. During steps 4 and 5, the sliding of the nuclear ring and the manchette, the growth of the postacrosomal dense lamina, and the progression of the posterior ring towards the base of the nucleus were seen along with structural and cytochemical modifications of the chromatin. In the flattened nuclei of step 4 spermatids, coinciding with the loss of the nucleolar components, the chromatin achieved maximum compactness in the entire nucleus and was PTA positive. In the spermatids of step 5, the disappearance of peripheral dense chromatin and the specific staining of the chromatin granules marked the beginning of the second stage of transformation of the basic nucleo‐proteins. The condensed nuclei of the mature spermatids were partially stained by PTA in step 6 and totally unstained in step 7. The PTA staining revealed the persistence of PTA‐positive chromatin areas in the nuclei of certain spermatids otherwise mature. The morphological aspect of the chromatin then remained the same in the nuclei of epididymal and ejaculated spermatozoa.These observations suggest that in man, as in other mammals studied, new proteins accumulate in the elongating nuclei of spermatids and are replaced at the phase of maturation by sperm‐specific nucleoproteins. The defects in condensation of the chromatin that occur during spermiogenesis could be related to the modalities of accumulation of intermediate nucleoproteins.
Oviducal pars recta‐induced degradation of vitelline coat proteins in relation to acquisition of fertilizability of toad eggs
Authors: Takamune Kazufumi , Yoshizaki Norio , Katagiri Chiaki 
Published: February 2005

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

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

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

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

AbstractZonae pellucidae (ZP) were isolated from 1,500 porcine ovaries and heat solubilized to generate approximately 15 mg ZP glycoprotein. Analysis of this material by isoelectric focusing, one‐dimensional electrophoresis, and gas chromatography indicated the presence of a major glycoprotein species that exhibited considerable microheterogeneity with respect to its charge (pI 7.5–3.5) and molecular mass (45–85 kDa) and that contained 39.6% carbohydrate, predominantly N‐acetylglucosamine.Chemical deglycosylation of porcine ZP using trifluoromethanesulphonic acid (TFMS) resulted in the production of five discrete protein bands on one‐dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) with molecular masses of 66, 52, 36, 32, and 16 kDa.Antisera raised in rabbits and marmosets to ZP and/or deglycosylated ZP (DGZP) were used in immunoblotting experiments to demonstrate the retention of immunogenicity by DGZP and the cross‐reactivity of the antisera with their heterologous antigen. These studies indicated that antisera that were capable of inhibiting the fertility of primates in vivo and the penetration of the human ZP in vitro reacted preferentially with 3 of the 5 products of deglycosylation, with molecular masses of 66, 52, and 36 kDa. Anti‐DGZP antibodies were also shown to interact with intact porcine and human ZP and, with the latter, to block the ability of human spermatozoa to both bind to and penetrate this structure.
Use of salt‐stored zonae pellucidae for assessing rabbit sperm capacitation for in vitro fertilization
Authors: Fayrer‐Hosken R. A. , Brackett B. G. 
Published: February 2005

AbstractZonae pellucidae of tubal and follicular oocytes were collected and prepared for salt storage. Cumulus and corona radiata cells were removed from oocytes with hyaluronidase and a small bore pipette. The oocytes (referred to as zonae since vitelli were rendered nonfunctional) were stored in a salt solution at 4°C. Using in utero capacitated sperm, the penetrability of zonae from tubal and follicular oocytes stored immediately after collection was compared to controls, i.e., in vitro development of tubal ova to the 4‐cell stage within 24 hr. The penetration rates were 100% (8 penetrated/ 8 inseminated), 77.8% (7 penetrated/ 9 inseminated), and 100% (10 fertilized/10 inseminated), respectively, and these were not statistically different. The mean (x) numbers of sperm able to penetrate the zonae, into the perivitelline space (PVS) for tubal (34.0) and follicular (1.1) oocytes were significantly different (P < 0.01). However, following maturational incubation before salt storage, zonae of tubal and follicular origin showed no significant differences in penetrability of in utero capacitated sperm when assessed by percent penetration, or mean numbers of sperm cells reaching the PVS: tubal zonae, 100% (15/15), and follicular zonae, 100% (18/18), and mean number of sperm in the PVS (x [tubal zonae] = 12.4, and x [follicular zonae] = 11.8). The penetrability of tubal zonae with and without maturational incubation was compared, and no significant differences in penetrability by in utero capacitated sperm were present when assessed by percent penetration nonmatured 92.6% (25/27) and matured 93.3% (28/30) and mean number of sperm in the PVS (x [nonmatured] = 3.33 and x [matured] = 2.41).In vitro capacitation of ejaculated rabbit sperm by serum treatment was assessed by the penetration of salt‐stored zonae, zonae‐free hamster oocytes (ZFHO), and in vitro fertilization of freshly collected tubal oocytes. None of 60 salt‐stored zonae and none of 31 tubal oocytes were penetrated, and these values were significantly (P < 0.005) smaller than the 9 of 78 (12%) zona‐free hamster ova that were penetrated by sperm cells from the same sample.In vitro capacitation of ejaculated rabbit sperm by washing and preincubation was assessed by the penetration of salt‐stored zonae, zona‐free hamster oocytes (ZFHO), and fertilization of freshly collected tubal oocytes. Seventy‐six of 80 salt‐stored zonae were penetrated, and this was significantly (P < 0.005) greater than the 67 of 87 tubal oocytes fertilized and 30 of 35 ZFHO penetrated, which were not significantly different.The salt‐stored zonae were more readily penetrated by capacitated sperm when compared to tubal oocytes. However, the ZFHO are more penetrable than salt‐stored zonae and tubal oocytes when incompletely capacitated sperm is used. A useful role for this approach in studies dealing with sperm fertilizing ability is anticipated.
Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody
Authors: Moore H. D. M. , Smith C. A. , Hartman T. D. , Bye A. P. 
Published: February 2005

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

AbstractFresh spermatozoa from six bulls, with fertility ranging from 64% to 78%, (based upon 59‐day nonreturn rates for 159,448 cows inseminated) were mixed with zona‐free hamster eggs in 15 heterospermic pair inseminations. Five of the bulls were used in homospermic insemination studies. Prior to incubation, spermatozoa from each bull were labeled with contrasting fluorescent stains pretested for effects on spermatozoa. Equal numbers of spermatozoa were mixed and treated with liposomes of dilauroylphosphatidylcholine to induce the acrosome reaction. Spermatozoa from split ejaculates within a male competed against each other equally in the hamster egg test, indicating that the staining procedure did not affect egg penetration rates. Bulls differed in their egg penetration rates when their sperm were inseminated either homospermically or heterospermically, but the differences in the homospermic inseminations were not significantly correlated with sire fertility. The number and percentage of sperm which penetrated eggs, and the number of eggs penetrated in the heterospermic competitive tests were highly correlated with fertility (r ≥ 0.86). Therefore, egg penetration rates from heterospermic inseminations appear to be valuable indicators of fertility and much more sensitive predictors than results from homospermic inseminations.
Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs
Authors: Fraser Lynn R. 
Published: February 2005

AbstractExtracellular Ca2+ is required for capacitation and fertilization in the mouse, but very little is known about the ability of other divalent cations to substitute for Ca2+. In this study, Sr2+, Ba2+, and Mg2+ were evaluated for their ability to support capacitation, the acrosome reaction, hyperactivated motility, and fertilization. Ba2+ proved to be ineffective, but Mg2+‐containing medium was able to support capacitation to a greater extent than unsupplemented Ca2+‐deficient media; despite this, Ca2+ was required for fertilization. In contrast, Sr2+ proved capable of substituting for Ca2+ in all events. Furthermore, Sr2+‐induced responses were indistinguishable from the corresponding Ca2+‐induced ones: Sperm capacitated at the same rate and underwent the acrosome reaction to the same extent. However, demonstration of sperm:egg fusion in Sr2+ required the use of zona‐free eggs. This was due not to the inability of the sperm to penetrate the zona but to the very rapid activation and cortical granule release by eggs in response to Sr2+. When zona‐intact eggs were used, the block to polyspermy had been mounted by the time sperm had penetrated the zona. A 15 min exposure to Sr2+ was sufficient to block sperm fusion, but a longer exposure was required to ensure the resumption of meiosis in eggs; such a response was surprising in that the eggs were freshly ovulated and not susceptible to activation by many different treatments. Thus Sr2+ can profoundly affect both gametes in the mouse: It substitutes completely for Ca2+ in sperm responses and rapidly activates eggs, possibly by displacing Ca2+ from intracellular stores into the cytoplasm, where the Ca2+ can then trigger the various events of activation.
A complex cortical reaction leads to formation of the fertilization envelope in the lobster, Homarus
Authors: Talbot P. , Goudeau Marie 
Published: February 2005

AbstractWe have examined the formation of the fertilization envelope in the lobsters Homarus americanus and H gammarus. Oocytes were fixed for electron microscopy either in the ovary or following extrusion from the gonopore. Mature ovarian oocytes are surrounded by a coat (envelope 1), which is comprised of small electron‐dense granules and structures resembling “bottlebrushes.” At least part of this coat is synthesized by the follicle cells of the ovary. The cortex of ovarian oocytes contains four types of vesicles that we refer to as high‐density vesicles (HDV), low‐density vesicles (LDV), moderately dense vesicles (MDV), and ring vesicles (RV). Oocytes that were electrically extruded from the gonopore and fixed immediately had an envelope identical to that of ovarian oocytes. The cortex of gonopore oocytes contained the four types of vesicles found in ovarian oocytes. When unfertilized gonopore oocytes were allowed to incubate in sea water, the oocyte cortex appeared unaltered, but envelope 1 swelled and the bottlebrushes dispersed. When recently fertilized oocytes were fixed during natural spawning or following in‐vitro fertilization, each type of vesicle was released in sequence from the cortex of the oocyte. The contents of the HDV and LDV appeared first in the perivitelline space, but their fate could not be determined at later times. The ring‐shaped elements of the RV and the moderately electron‐dense material of the MDV were released exocytotically somewhat later; these materials coalesced in the perivitelline space to form a new coat (envelope 2). Envelope 1 subsequently condensed to its original thickness and appeared firmly attached to envelope 2. Our results show that the fertilized lobster egg is surrounded by two discrete coats. The outer coat, which is formed in the ovary, undergoes a swelling/condensation cycle at spawning. The inner coat originates from a complex cortical reaction. Together these coats comprise the fertilization envelope of the lobster egg.
Rhesus monkey sperm penetration into zona‐free hamster ova: Comparison of preparation and culture conditions
Authors: Binor Zvi , Rawlins Richard G. , Van der Ven Hans , Paul W. Dmowski 
Published: February 2005

AbstractA major problem in development of nonhuman primate in vitro fertilization is the selection of donor males and repeated collection of consistent sperm samples. In practice, collection of a viable semen sample is highly dependent on operator technique and the type of animal restraint. We report an updated method for semen collection from the rhesus monkey (Macaca mulatta), use of TES‐Tris (TEST) Yolk Buffer (TYB) for prolonged sperm storage and improved results of hamster ovum penetration assay. Semen was obtained from adult males restrained with 2.0 mg/kg IM ketamine hydrochloride prior to direct penile stimulation (Grass SD‐9, frequency 150, delay 9, duration 7, volts 12–18, repeat mode, twin pulse). Liquified semen was washed and centrifuged twice at 100 × g for 5 min in BWW, Ham's F‐10 and TALP and allowed to swim‐up 60 min at 37° in 5% CO2 and air. Alternatively, semen was mixed 1:1 with TYB, refrigerated 20 h at 4°C, centrifuged at 100 × g for 5 min, and the pellet resuspended in 1.0 ml of TALP or BWW prior to use. Hamster ova penetration was achieved with capacitated macaque sperm. Penetration was significantly improved (P < .001) with preincubation in TYB followed by resuspension in TALP (79%).