bluuebunny/crossref_metadata_2025_split huggingface dataset:
Changes in elemental composition of fertilized and unfertilized northern pike (Esox lucius) eggs incubated in buffer with and without dimethyl sulfoxide: An X‐ray microanalysis study
Authors: Schmehl M. K. , Graham E. F. 
Published: February 2005

AbstractFertilized and unfertilized eggs from the northern pike (Esox lucius) were incubated 2 hr in buffer with 0 and 10% (v/v) dimethyl sulfoxide and then quickly frozen in the wells of aluminum blocks submerged in liquid nitrogen. Control eggs and ovarian fluid were similarly frozen immediately after collection. The frozen eggs were sectioned, freeze dried, mounted on stubs, and carbon coated. X‐ray microanalysis was used to determine changes in element levels and dimethyl sulfoxide (Me2SO) penetration in the zona radiata, cytoplasm, cortical alveoli, and egg yolk. Unfertilized eggs incubated without Me2SO showed decreased levels of Na, Cl, and K in the zona radiata; fertilized eggs, incubated without Me2SO showed decreased levels of Na, P, and Cl in the zona radiata and increased levels of K in the cytoplasm; unfertilized eggs, incubated with 10% Me2SO showed decreased Na and Cl in the zona radiata, decreased K in the cytoplasm and increased K in the cortical alveoli; fertilized eggs incubated in buffer with 10% Me2SO showed decreased levels of Na, P, Cl, and K (zona radiata), P, Cl, and K (cytoplasm), Na (yolk), and increased Cl in the yolk (all P<.01). Me2SO (v/v) levels reached 1.5‐3.1% in the zona radiata, 0‐3.2% in cytoplasm, 2.3‐8.7% in cortical alveoli, and 0‐1.6% in the yolk. Unfertilized eggs showed more Me2SO penetration than fertilized eggs.
Hamster oocyte fertilizability after 4°C storage
Authors: Barros C. , Herrera E. , Fuenzalida I. , Argüello B. 
Published: February 2005

AbstractThe purpose of the present work was to study the feasibility of using hamster oocytes stored at 4°C in M‐2 culture medium for 24 and 48 hours in the evaluation of human sperm fertilizability. A total of 1,394 oocytes were stored for 24 hours and 1,234 were stored for 48 hours. After the storage period all the ooctyes were stained with fluorescein diacetate, proving the physical integrity of the egg plasma membrane. Twenty‐five and 22 semen samples were used to compare their ability to penetrate freshly ovulated and 24‐and 48‐hour‐stored hamster oocytes. Freshly ovulated and 24‐hour‐stored oocytes were penetrated at percentages that in more than 95% of the cases showed no significant differences. The same experiments carried out with oocytes stored for 48 hours showed that in 75% of the cases no significant differences were found. The use of oocytes preserved at 4°C when large numbers of semen samples are to be tested for fertilizability is recommended.
Protamine in the rat, its fate in vivo, and its degradation in vitro by egg homogenate
Authors: Betzalel Moshe , Shalgi Ruth , Moav Boaz 
Published: February 2005

AbstractSpermatogenesis in the male rat involves chemical and physical transformation of chromatin. There is a shift in the chemical composition of rat testicular sperm chromatin containing histones to nucleoprotamines in the mature sperm. Rat protamine contains a high percentage of arginine. The biosynthesis and kinetics of H3‐arginine incorporation into rat sperm protamine was at its peak in the testis 2 days after intratesticular injection. The specific activity of protamine in the testis was reduced to 50% 5 days after injection. Specific activity of protamine in the epididymis was maximal at 9 days after injection and remained at a similar level for 12 days. The kinetics of H3‐arginine and H3‐thymidine in ejaculated sperm were compared. It was found that maximal specific activity of H3‐arginine (H3‐Arg/No. of sperm) in the spermatozoa was obtained 20–22 days following its intratesticular injection. The peak of H3‐thymidine specific activity (H3‐thymidine/No. of sperm) was obtained 53–59 days following the injection.H3‐Arg labeled sperm were capacitated in vitro and were allowed to penetrate zona free rat eggs in vitro. The disappearance of the label from the sperm head was observed 1–2 hr after penetration. Protamine labeled in vitro with I125 incubated with extracts of freshly ovulated rat eggs shows that protamine could be degraded by the cytoplasmic extract (10–30%). The proteolytic activity is maximal at pH 7.0–8.0. Cytoplasmic extracts from cumulus cells incubated with I125 rat protamine under identical conditions did not show any significant proteolytic activity. Trypsin degradation of rat protamine shows a different pattern of degradation. It is assumed that the H3‐labeled protamine in the sperm chromatin is removed and probably degraded early after penetration, making possible the remodeling of the sperm chromatin by embryonic histones.
Partial purification and characterization of sperm receptor hydrolase, a cortical granule proteoesterase, from eggs of the sea urchin Strongylocentrotus purpuratus
Authors: Lois Augusto F. , Lackey Debora A. , Carroll Edward J. 
Published: February 2005

AbstractSperm receptor hydrolase, one of two classes of cortical granule proteoesterases (E.C.3.4.4.4) was purified approximately 30‐fold with 80% yield from Strongylocentrotus purpuratus cortical granule exudate. Sperm receptor hydrolase preparations were free of vitelline delaminase activity (the other class of cortical granule proteoesterase) and had less than 1% of the starting levels of cortical granule peroxidase and β‐1,3‐glucanohydro‐lase activities. Native polyacrylamide gel electrophoresis coupled with a protease activity stain showed that three proteases were present in the most highly purified preparations of sperm receptor hydrolase. Each of the three proteases has the same molecular weight of 60,000, but different isoelectric points of 2.4, 3.8, and 5.5. The Km value of the mixture of proteases for α‐N‐benzoyl‐L‐arginine ethyl ester as substrate was 263 μM at pH 8.4 and 30°C; the pH dependence of Vm showed a single prototrophic group with a pK of 6.7 and an enthalpy of ionization of 8.6 kcal‐mol−1. The values of these kinetic parameters are consistent with an enzyme‐active site containing histidine. Phenylmethyl sulfonyl fluoride, tosyl lysine chloromethyl ketone, several proteinaceous trypsin inhibitors, and p‐aminobenzamidine inhibited the esterase activity of the proteases. These data suggest that sperm receptor hydrolases are serine proteases.
Effect of in vivo oocyte aging on sperm chromatin decondensation in the golden hamster
Authors: Jedlicki A. , Barros C. , Salgado A. M. , Herrera E. 
Published: February 2005

AbstractAged spontaneously activated hamster oocytes recovered from adult females 18 and 24 hours after ovulation were at the pronuclear stage. These oocytes and fresh controls were inseminated in vitro with capacitated hamster spermatozoa and observed with the phase‐contrast microscope. The percentage of fertilization in fresh control oocytes was 98%, as compared to 36% and 18% when the oocytes were recovered 18 and 24 hours after ovulation, respectively. The mean number of sperm decondensations per egg in control oocytes was 10, and in the aged ones it was 0.69 and 0.12 when the oocytes were recovered 18 and 24 hours after ovulation, respectively. When similarly treated oocytes were studied with scanning and transmission electron microscopy, it was found that the degree of gamete membrane fusion was greater than that observed with the phase‐contrast microscope, but that most of the spermatozoa failed to decondense the chromatin. We suggest that parthenogenetic oocytes at the pronuclear stage are in a similar stage of the cell cycle as in fertilized eggs, in which the cytoplasm does not have the ability to decondense the sperm chromatin.
Two simple methods for detecting acrosome‐reacted human sperm
Authors: Cross Nicholas L. , Morales Patricio , Overstreet James W. , Hanson Frederick W. 
Published: February 2005

AbstractWe describe two methods for detecting acrosome reactions of human sperm at the light microscopic level. The techniques include the use of a supravital stain to detect dead sperm in order to differentiate between “physiological” and “degenerative” acrosome reactions. Sperm are incubated with the supravital stain Hoechst 33258 (a fluorescent DNA‐binding dye with limited membrane permeability), washed, suspended in 95% ethanol for fixation and permeabilization, and dried onto slides. The sperm are then labeled either by indirect immunofluorescence using rabbit anti‐human sperm antiserum or with fluoresceinated Pisum sativum agglutinin (PSA). Both probes intensely label the acrosomal region of acrosome‐intact sperm. Electron microscopy revealed the major site of PSA binding to be the acrosomal contents. Acrosome‐reacted sperm have diminished acrosomal labeling by both probes; sperm with nuclei labeled by Hoechst stain are considered nonviable, and are excluded from the assay. Both assays are rapid, give similar results, and detect an increase in acrosome reactions following exposure to the ionophore A23187.
Examination of living and fixed gametes and early embryos stained with supravital fluorochromes (Hoechst 33342 and 3,3′‐dihexyloxacarbocyanine iodide)
Authors: Luttmer Shirley J. , Longo Frank J. 
Published: February 2005

AbstractWe have examined living and fixed gametes and early embryos of surf clams, sea urchins, and hamsters stained with the supravital dyes Hoechst 33342 for DNA and 3,3′‐dihexyloxacarbocyanine iodide (DIOC6) for mitochondria and endoplasmic reticulum. Hoechst staining (10 μM) was confined exclusively to egg and sperm chromatin and, in living marine specimens, did not interfere with sperm motility, fertilization, or nuclear activity during meiosis or early embryogenesis. Although Hoechst staining did not appear to affect the motility of hamster sperm, only zonae‐free eggs inseminated. Because chromatin retained Hoechst 33342 stain during fertilization, the paternally and maternally derived chromosomes of living and fixed preparations fluoresced and their number, organization, and location within the zygote cytoplasm could be determined. Hence, polyspermy and other nuclear abnormalities were amenable to examination in these stained preparations. DIOC6 staining (8.7 μM) was restricted primarily to the mitochondria of spermatozoa. Eggs stained with DIOC6 (0.87 to 8.7 μM) were brightly fluorescent because of their size and the presence of large numbers of mitochondria and other DIOC6‐positive organelles. Sea urchin and surf clam sperm stained with DIOC6 fertilized unstained eggs and the location of the incorporated sperm mitochondrion up to first cleavage was followed. Although hamster sperm stained with DIOC6 were less motile than unstained sperm, they were capable of inseminating only zonae‐free eggs. These observations demonstrate that staining with supravital fluorochromes provides a rapid and useful method to analyze macromolecular and organelle changes in a variety of living and fixed gametes and embryos.
Characterization of cAMP‐dependent protein kinase and its endogenous substrate proteins in ram testicular, cauda epididymal, and ejaculated spermatozoa
Authors: Wooten Marie W. , Voglmayr Josef K. , Wrenn Robert W. 
Published: February 2005

AbstractMammalian spermatozoa have been shown to possess cAMP‐dependent protein kinase (A‐PK) and endogenous substrate proteins for this enzyme. A study of the kinase system was undertaken to determine changes that may be associated with sperm maturation by comparing immature testicular with mature cauda epididymal and ejaculated spermatozoa. Absolute activity levels of A‐PK, stimulated over a concentration range of 10−9 to 10−5 M, was significantly greater in testicular than ejaculated spermatozoa. At an optimal cAMP concentration (10−6M), testicular spermatozoa had significantly greater amounts of cAMP‐dependent protein kinase activity than did cauda or ejaculated spermatozoa. Electrophoretic analysis and autoradiography of NP‐40‐soluble protein extracts revealed the presence of two substrate proteins (Mr = 62,000 and 44,000) in all three types of spermatozoa. In addition, a phosphoprotein (Mr = 20,000) was detected in mature cauda and ejaculated but not immature testicular spermatozoa. The phosphorylation of these substrate proteins was both dose and time dependent. Examination of cyclic AMP phosphodiesterase activity revealed significantly higher levels in testicular than ejaculated spermatozoa. These results indicate marked alterations in cAMP‐modulated protein phosphorylation and dephosphorylation systems in ram spermatozoa during epididymal maturation.
In vitro fertilization of hamster and human oocytes by microinjection of human sperm
Authors: Lassalle B. , Courtot A. M. , Testart J. 
Published: February 2005

AbstractIn this study, swollen sperm heads were obtained after the injection of human sperm into the perivitelline space of hamster oocytes. The number of injected sperm and the sperm concentration in the preincubation medium were found to have an influcnce on the rate of penetrated hamster oocytes. The optimal injected sperm number was always between five to 12 to obtain 8, 37, and 36% penetration for donors A, B, and C, respectively. The optimal sperm concentration in preincubation medium was between 6 and 22 × 106 sperm/ ml to obtain 16, 47, and 43% penetration for donors A, B, and C, respectively. The rate of polyspermic oocytes was related to the injected sperm number (0, 55, and 100% for one to four, five to 12, and more than 12 injected sperm respectively). Ten human mature oocytes were injected with the sperm from six normal donors. Five fertilized eggs were obtained, and of these four cleaved in in vitro culture.
Distribution by sex of mouse fetuses in the intrauterine position
Authors: Endo A. , Goto T. , Sakai N. 
Published: February 2005

AbstractWe examined whether or not the sex of the fetuses of polytocous animals distributes randomly in the position along the uterine horn in 255 mouse litters. The fetal sex ratio did not differ significantly among the three intrauterine segments (ovarian, middle, and cervical). Based on the number of fetuses examined in this study, it can be stated that even if sex ratio differences exist among the segments, the ratio in individual segments would fall mostly inside the ±0.1 range, when the overall sex ratio (M/(M+F)) is around 0.5.
Spontaneous continuous release of spermatozoa and its predawn surge in male passerine birds
Authors: Quay W. B. 
Published: February 2005

AbstractSpontaneous, continuous release of morphologically normal spermatozoa occurs in males of species of passerine (order Passeriformes) birds that were examined. It was demonstrated and studied quantitatively in temporarily captive and isolated house sparrows and house finches by means of repetitive cloacal lavages and extraction of excreta. It is suggested that this phenomenon could be exploited to facilitate comparative and quantitative evaluations of release of spermatozoa in relation to diverse environmental, physiological, and social factors.
Seasonal variation in estrous cycling in the mouse: Implications for artificial insemination
Authors: Watson J. Gary , Chaykin Sterling 
Published: February 2005

AbstractArtificial insemination in the C3HeB/FeJ inbred strain of mice has been shown to be more successful at the middle and end of the calendar year. The reasons are twofold: 1) an increase in the number of normal estrous cycles exhibited by females and 2) an increase in the tightness of the phasing of ovarian and vaginal events. The latter phenomenon was found to be the key to the success of artificial insemination, since it permitted the use of vaginal smears to predict accurately the time females could be expected to ovulate and, therefore, the appropriate time for artificial insemination. Seasonal variations in the frequency of estrous cycling also have been observed in SJL/J and B6D2F1/J females.
Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona‐free hamster ova in vitro
Authors: Clarke R. N. , Johnson L. A. 
Published: February 2005

AbstractThe effect of liquid storage and cryopreservation of boar spermatozoa on sperm motility, acrosomal integrity, and the penetration of zona‐free hamster (ZFH) ova was examined. The sperm penetration assay (SPA) provides valuable information on specific events of fertilization and is a potentially useful indicator of sperm fertility. Ejaculated semen from 4 boars was subjected to 3 treatments: fresh (FRE, no storage), liquid‐stored (LIS, stored at 18°C for 3 days), and frozen (FRO, frozen by pellet method and stored at −196°C for 3 days). A highly motile sperm population was isolated by the swim‐up procedure (1 hr). FRE and LIS were incubated an additional 3 hr at 39°C in a Tris‐buffered medium to elicit capacitation and the acrosome reaction. Sperm motility and acrosomal integrity were assessed before and after incubation. For the SPA, sperm and eggs were incubated at 39°C for 3 hr in Hams F‐10 medium. Each egg was assessed for sperm penetration, sperm binding, and stage of development. Percentages of sperm motility and sperm with a normal apical ridge (NAR) prior to incubation were 78 and 78 (FRE), 75 and 69 (LIS), and 28 and 50 (FRO). After incubation, percentages of motility, NAR, and acrosome‐reacted sperm were 34, 10, and 73 (FRE); 43, 24, and 51 (LIS); and 18, 13, and 59 (FRO). A somewhat higher (P < .05) percentage of ZFH ova was penetrated by FRE (45.8) than by LIS (42.0). Penetration of ZFH ova by FRO was markedly (P < .05) reduced (30.2). Sperm penetration was not significantly correlated with motility or acrosomal integrity before or after incubation, regardless of treatment. These data suggest that the SPA can be used in conjunction with conventional measures of semen analysis in assessing the potential fertilizing capacity of boar sperm and that liquid storage is superior to frozen storage with respect to preserving sperm fertility.
Ultrastructural study of the mature egg of Tethya citrina sarà and melone (porifera, demospongiae)
Authors: Gaino Elda , Burlando Bruno , Buffa Paola , Sarà Michele 
Published: February 2005

AbstractTethya citrina is an oviparous demosponge in which eggs are distributed in clumps within the choanosome. The cytoplasm of the mature egg presents a peripheral cortex consisting of a slightly granular layer sandwiched between two densely granular, vesiculated ones. The cortex probably has a specialized, trophic function. Mesohyl bacteria are phagocyted at the egg surface, included in vacuoles, and transferred across the cortical sheath toward the inner cytoplasm. The region of the egg extending between the cortex and the nucleus shows a lacunary system mostly developed beneath the cortical envelope. The noncortical cytoplasm also contains lipid droplets, dense rodlike bodies, and phagosomelike granules. Most of the latter are probably autophagosomes, forming lacunae and supporting autosynthetic vitellogenesis. Rodlike inclusions are probably proteinaceous; they likely originate within the phagosomes and represent the actual yolk material.
Distribution of microtubules during the first meiotic cell division in the mouse oocyte: Effect of taxol
Authors: Rime Hélène , Jessus Catherine , Ozon René 
Published: February 2005

AbstractMouse oocytes were stained by antitubulin antibody and by an anti‐MAP1 antibody (JA2) during the first meiotic cell division. At germinal vesicle stage, JA2 antibody exclusively stains the nucleoplasm. When the nuclear envelope breaks down (GVBD) (2–3 h of culture), numerous foci of microtubules appear around the disrupting nuclear envelope; they are decorated by anti‐MAP1 antibody. After 10–12 h of culture, the antigen component detected with anti‐MAP1 antibody is present in the meiotic spindle.The antimitotic agent taxol (10 μm) induces microtubule formation, mainly at the periphery of the germinal vesicle. At GVBD, taxol provokes the formation of a large microtubular array stained with both antibodies, which is associated with the condensed chromosomes. Furthermore, numerous cytoplasmic asters become visible in the cytoplasm. At metaphases I and II, taxol induces an important enlargement of the metaphase spindles and increases the number of cytasters.
Structural organization of the sperm chromatin in a fern (Scolopendrium vulgare) studied by spreading methods
Authors: Khlifi Samira , Robert Daniel 
Published: February 2005

AbstractTo investigate chromatin organization, we applied the spreading techniques to nuclei isolated from Scolopendrium spermatozoids.Well‐dispersed chromatin shows three types of fibers: beaded fibers corresponding to a nucleosomal filament with adjacent nucleosomes in close contact, smooth fibers (14 nm in diameter) associated in a complex network, and knobby fibers constituted by local supercoiling of a very thin (4 nm) smooth filament. Along the knobby fibers, beads of variable size are irregularly spaced.The knobby fibers lie parallel and coalesce in thick bundles. The sperms basic proteins identified by electrophoretic analysis probably promote the supercoiling and the side‐to‐side attachment of the knobby fibers, which are all the more abundant in spread preparations. These results indicate that knobby fibers are probably located in the outer part of the sperm nucleus in which the chromatin is densely packed. As for the nucleosomal and smooth filaments, they may be situated in the inner part.
Intratesticular injection as a method to assess the potential toxicity of various agents and to study mechanisms of normal spermatogenesis
Authors: Russell Lonnie D. , Saxena Nirmal K. , Weber James E. 
Published: February 2005

AbstractTo better understand, to optimize, and to validate the technique of intratesticular (i.t.) injection, several parameters related to i.t. injection were examined. Volumes exceeding 50 μl could be injected i.t.; however, testes frequently became excessively turgid and backflow of injected fluids occurred. Thus, a volume of 50 μl or less was deemed optimal for injection. To determine the rate of distribution of substances throughout the testis, trypan blue was injected i.t. near the caudal pole of the testis, and the movement of dye was monitored. Within 2 min, the dye had spread approximately 1 cm from the site of injection, and in 5 min it had spread twice that distance. In 2 h, the dye had become distributed throughout the testis except at its extreme cranial pole. Seminiferous tubules did not take up dye, indicating that the spread of dye was via peritubular lymphatics. Seminiferous tubule histology appeared virtually unaffected by i.t. injection, even at regions adjacent to the site of injection, when a sterile 26‐gauge or smaller bore needle was utilized. To determine disappearance from the testis, radiolabeled inulin was injected i.t. Half time for absorption was achieved at 1.75 h. Potential vehicles were expolored in which compounds with a variety of physical properties could be injected. Gum tragacanth, normal saline, ethylene glycol, dimethyl sulfoxide (DMSO) mixed 1:1 with normal saline, sesame oil, and propylene glycol were found to be suitable injection vehicles, whereas ethanol, dissolved in normal saline in concentrations as low as 0.5% was found unsuitable. To assess vehicle efficiency, various vehicles were utilized with a known testicular toxin (taxol) and injected into one testis, and the histology was compared with the contralateral testis injected with vehicle alone. All vehicles, found suitable above, allowed dispersion of taxol to influence areas distant from the site of injection. Intratesticular injection assesses the potential of agents to directly affect the testis, and systemic metabolism is avoided. Their rapid spread throughout the lymphatics of the testes allows seminiferous tubules to be exposed to agents in innocuous vehicles more rapidly and in higher concentration than is often possible when using systemic injections.