bluuebunny/crossref_metadata_2025_split huggingface dataset:
Freeze‐fracture observations of unfertilized and fertilized hamster oocytes with special reference to the use of lipid probes
Authors: Clark Judy M. , Koehler James K. , Smith W. Dianne 
Published: February 2005

AbstractMembrane and cytoplasmic changes were observed after in vitro fertilization of hamster oocytes by examining freeze‐fracture replicas. The density of intramembranous particles on areas of membrane between microvilli increased following fertilization. Although the intramembranous particle density of microvilli is higher than that on the intermicrovillar membrane of unfertilized eggs, it did not change significantly after fertilization. Cytoplasmic changes in the Golgi complex and mitochondria upon fertilization indicate a change in cellular activity. Lipid binding probes were applied to the oocyte membranes in order to study the distribution of specific lipids before and after fertilization. Probes included the B‐hydroxy‐steroid complexing molecules, filipin and tomatin, and an anionic lipid binding antibiotic, polymyxin B. Both tomatin and filipin complex with steroids in the P and E faces of the plasma membrane (including the polar bodies), cortical granules and vesicles deeper in the cytoplasm, and the Golgi complex, leaving mitochondria, pronuclei, endoplasmic reticulum, and the majority of vesicles unlabeled. Polymyxin B binding is dependent on its application before or after fixation or in association with EGTA. With its application we detected both minor membrane perturbations of wrinkles and particle redistributions and major perturbations of vesicle fusions, the formation of blebs, and the loss of membrane morphology. Neither the distribution nor apparent quantity of these probes changed overall following fertilization, but this impression does not include specific sites of sperm‐egg fusion.
Nicotinamide inhibition of Xenopus laevis oocyte maturation
Authors: Godeau Francois , Sahni Mukesh K. , Boquet Patrice , Koide S. S. 
Published: February 2005

AbstractNicotinamide at a concentration of 20 mM prevented progesterone‐induced germinal vesicle breakdown (GVBD) of Xenopus laevis oocytes in vitro in a reversible manner, whereas nicotinic acid did not. The vitamin was inhibitory even when applied 30 min after the addition of progesterone and blocked the induction of GVBD by microinjected maturation‐promoting factor (MPF). Nicotinamide also prevented the burst of protein phos‐phorylation associated with MPF expression and increased the oocyte cAMP level, whereas intracellular NAD content and protein synthesis were not affected. The present results suggest that nicotinamide acts by increasing the intracellular level of cAMP and by blocking MPF action.
Technique for the long‐term storage of lobster (Homarus) spermatophores
Authors: Ishida Tamako , Talbot P. , Kooda‐Cisco M. 
Published: February 2005

AbstractWe have developed a procedure for the long‐term storage of lobster (Homarus) sperm. Sperm are collected from males by electrically stimulating the area around the gonopore. They are transferred with a bamboo stick to a plastic test tube containing paraffin oil and are stored for variable periods of time at 4–7°C. Sperm stored up to 289 days appear morphologically normal (equivalent to unstored sperm) when examined by phase contrast microscopy and electron microscopy, and morphologically normal sperm are able to undergo acrosome reactions. After longer periods of storage, degenerative changes begin to develop in sperm. These include loss of the nuclear spikes, condensation of the subacrosomal material, and distortion of the acrosome. Sperm stored better in spermatophores that had thick walls than in those with thin walls. In some spermatophores, bacteria were found in the sperm mass after storage; in general, sperm in these spermatophores were morphologically abnormal. This technique provides a means for saving lobster sperm for subsequent use in experiments or for artificial insemination of female lobsters. It may be adaptable to other invertebrate species that produce spermatophores.
Electron microscopic study of the in vivo zona pellucida binding ability of epididymal ram spermatozoa
Authors: Fournier‐Delpech S. , Crozet N. , Courot M. 
Published: February 2005

AbstractIn the ram, spermatozoa develop the ability to initiate pregnancy only after reaching the body of the epididymis. To determine the zona pellucida binding ability of ram spermatozoa collected from different levels of the epididymis, sufficient numbers of motile sperm cells of different epididymal origin were inseminated surgically below the uterotubal junction of ewes at the time of ovulation. Intratubal ova were recovered 24 hr later, and those having spermatozoa attached to the zona were examined by transmission electron microscopy to assess the characteristics of the bound spermatozoa. Data indicate that the ability of the capacitated spermatozoa to adhere to the zona pellucida depends on sperm egg binding sites that develop on the acrosomal membranes from the apex to equatorial segment during epididymal transit.
Evidence for a binding site on the sperm plasma membrane which recognizes the murine zona pellucida: A binding site on the sperm plasma membrane
Authors: Poirier Gary R. , Robinson Roderick , Richardson Richard , Hinds Kathy , Clayton Deborah 
Published: February 2005

AbstractMurine cauda epididymal sperm preincubated in either a modified Krebs‐Ringer or M 199 solution bind to cumulus‐free, zona pellucida‐intact eggs. Pretreatment of such eggs with an affinity purified preparation of the seminal inhibitor binding component (acceptor), isolated from epididymal sperm, reduces in a concentration dependent manner, the number of sperm that bind. Treatment of cauda sperm, preincubated in either of the above two media, with the seminal inhibitor, also reduces the number of sperm able to bind. Incubation of cauda sperm in the Krebs‐Ringer solution for up to 4 h does not affect their ability to bind the seminal inhibitor. Omission of bovine serum albumin from the preincubation medium results in a significant reduction in sperm binding. These data are interpreted to mean that the seminal inhibitor acceptor sites on the sperm surface of incubated sperm function in the in vitro binding to the zona pellucida.
Microfilaments during sea urchin fertilization: Fluorescence detection with rhodaminyl phalloidin
Authors: Cline C. A. , Schatten G. 
Published: February 2005

AbstractRhodaminyl‐labeled phalloidin is used to demonstrate the distribution of microfilaments during fertilization and early development in eggs of the sea urchins Arbacia punctulata and Lytechinus variegatus. The surface of unfertilized eggs have numerous punctate fluorescence sites at which rhodaminyl phalloidin binds, indicating the presence of actin oligomers or polymers. During fertilization this punctate pattern of fluorescence begins to change. Within thirty seconds of insemination, the fertilization cone is first detectable with this technique as an erect structure on the surface of the egg. The fertilization cone grows to a maximum size by 8–9 minutes, and is resorbed by 16 minutes after insemination. The surface of the fertilized egg displays numerous fluorescent fibers by 10 minutes after insemination rather than the punctate fluorescence observed in unfertilized eggs, indicative of the burst of microfilament assembly resulting in microvillar elongation. The elongated microfilaments persist through cytokinesis. Staining is also detected throughout the cortices of unfertilized, fertilized, and cleaving eggs. Cytochalasin E (10 μM, 30 min) prevents microfilament elongation and cytokinesis and reduces the cortical staining intensity after fertilization. At cleavage, contractile rings, appearing as narrow equatorial bundles of fibers, have been detected in Lytechinus variegatus as transient structures.
Acetylcholinesterase in sea urchin spermatozoa
Authors: Cariello Lucio , Romano Giovanna , Nelson Leonard 
Published: February 2005

AbstractFlagella contain the bulk of spermatozoan acetylcholinesterase. Brief sonication of sea urchin sperm suspended in Tris‐buffered (pH 8.0), Ca, Mg‐free artificial sea water (F‐ASW) containing 10 mM ethylene diaminetetracetic acid, (EDTA) doubled the specific activity over that of the intact spermatozoa. Lipids were removed from the solubilized supernatant of the tail membrane fraction by ether extraction. Hydrolysis of acetylthiocholine in the presence of dithiobisnitrobenzoic acid (DTNB) was monitored spectrophotometrically at 412 nm by the Ellman procedure. The enzyme was purified by affinity chromatography on a Sepharose cyanogen bromide gel to which the cholinesterase inhibitor trimethyl (para‐aminophenyl) ammonium chloride was coupled. The enzyme was eluted from the column with a discontinous NaCl gradient (0.1–0.5 M). The active fraction recovered at 0.35 M NaCl contained 0.007% of the initial total sperm cell protein with a 500‐fold increase in specific activity.Twenty‐four hr centrifugation on a 5–20% sucrose density gradient at 50,000g in a Beckman L5‐75 centrifuge yielded peaks at 14.7 S and 9.1 S. In the presence of 1% Triton X‐100, three peaks appeared: 23.3 S, 13.7 S, and 9.1 S. These sedimentation coefficients resemble those of the electroplax acetylcholinesterase (AChE) forms A8 and A4.Eserine completely inhibited the activity of the purified enzyme, which exhibits a substrate optimum at 4 mM acetylcholine. The activity is depressed by 75% at 10 mM ACh and by 90% at 25 mM. The Km was 2.1 × 10−4 M. In the sperm cell the enzyme that terminates the action of intracellularly synthesized ACh may be involved in controlling ionophoric channels that regulate transmembrane transport of calcium.
Ionophore‐induced efflux of sodium and potassium ions from sea urchin eggs
Authors: Carroll Edward J. , Heath Robert L. 
Published: February 2005

AbstractThe efflux of K+ and Na+ from sea urchin eggs during Ca2+ ionophore A23187‐induced parthenogenesis was studied in a K+ and Na+‐free artificial seawater using extracellular ion‐specific electrodes. We have probed this model system with monovalent cation‐specific ionophores to determine if they affect K+ efflux in the unfertilized egg and whether any changes in ionophore sensitivity are observed during egg activation. In 500 mM choline chloride, 10 mM CaCl2, 50 mM MgCl2, 10 mM Tris‐Cl pH 8.0, A23187 induced a rapid efflux of K+ and Na+ from the eggs after a short lag time (10–15 seconds). After the burst, the rate of K+ efflux remained higher than the pre‐activation rate, but was lower than during the burst phase, while the rate of Na+ efflux became nearly zero. Monovalent cation‐specific ionophores (valinomycin, gramicidin and nigericin) had no effect on K+ efflux from the unfertilized eggs in our model system. However, once the egg was activated by A23187, each of the above ionophores caused a prolongation of the burst phase for many minutes. These results show that the unfertilized egg plasma membrane (using our artificial conditions) is not susceptible to the monovalent cation‐specific antibiotics and suggest that either the inserted cortical granule membrane or the developing fertilization envelope interacts with these ionophores to cause the change in rate‐limiting step for K+ efflux observed egg activation.
Formation of the acrosome and basal body during spermiogenesis in a marine snail, Nerita picea (Mollusca: Archaeogastropoda)
Authors: Buckland‐Nicks John , Chia Fu‐Shiang 
Published: February 2005

AbstractIn Nerita picea the proacrosomal granule is formed basally in the early spermatid from one large cisterna of the Golgi body, with which the other Golgi‐derived vesicles fuse. After the proacrosomal granule has attached to the plasma membrane and invaginated to form a cup shape, one cisterna of endoplasmic reticulum inserts into the open end and deposits a granular secretion on the inner surface. Subsequently, the proacrosome migrates along the plasma membrane to the apex of the nucleus, but the Golgi body remains basal, as occurs in other archaeogastropods and also many polychaete annelids. However, the final shape and structure of the acrosome is similar to that of mesogastropods. The annulus attaches the distal centriole to the plasma membrane early in spermiogenesis. The production of the flagellum by the distal centriole not only expands the plasma membrane posteriorly but moves the centriolar complex to the nucleus, causing an invagination of the plasma membrane where it is bound by the annulus. During proacrosome migration, the Golgi body secretes a dense tube around the flagellum, and the mitochondria fuse into two spheres at the base of the nucleus. The nuclear plug that closes off the intranuclear canal until this stage rapidly reorganizes itself into two tubes of material inside the canal. The centrioles continue flagellar production, break away from the annulus, and move deep into the intranuclear canal where they fuse together to form the basal body of the sperm. In the maturing spermatid, the two mitochondria fuse into a single sheath that spirals around the flagellum. The annulus does not migrate posteriorly but remains anterior to the midpiece, which is unusual for a filiform sperm. Spermiogenesis in Nerita picea has features in common with both archaeogastropods and mesogastropods but also has some unique features. These observations lend credence to the idea that the Neritidae are a transitional group between Archaeogastropoda and Mesogastropoda.
Measurement of intracellular calcium in human spermatozoa
Authors: Irvine D. Stewart , Aitken R. John 
Published: February 2005

AbstractMethods for the investigation of cell‐associated calcium and intracellular calcium were studied in washed ejaculated human spermatozoa. Experiments using 45Ca2+ indicated that human spermatozoa were permeant to calcium and that a significant proportion of the cellassociated calcium (approximately 50%) was accumulated in the mitochondrion. This necessitated the use of alternative procedures to measure cytoplasmic free calcium. The ability of human spermatozoa to accumulate and de‐esterify the intracellular fluorescent calcium indicator Quin‐2 was established. Using this technique, the resting level of free intracellular calcium in human spermatozoa was found to be 146.0 ± 19.9 nM, and was significantly elevated upon addition of the divalent cation ionophore ionomycin. In further experiments designed to illustrate the applications of the Quin technique, data was obtained suggesting that the mechanisms controlling intracellular calcium in human spermatozoa are temperature dependent but do not involve voltage‐sensitive calcium channels.
Effect of centrifugation of mouse eggs on their development in vitro and in vivo
Authors: Nakamura K. , Tsunoda Y. , Nagai T. , Sugie T. 
Published: February 2005

AbstractFertilized mouse eggs were centrifuged at 5,000g, 10,000g, 15,000g, and 20,000g for 3 and 10 min or at 25,000g for 10 min. The pronuclei of centrifuged eggs became more distinctive than those of uncentrifuged eggs. The proportion of centrifuged and uncentrifuged eggs that developed to blastocysts was not significantly different. There was also no significant difference in the proportion of live fetuses obtained following the transfer of blastocysts developed from centrifuged and uncentrifuged eggs. This study demonstrated that there is no effect of centrifugation on the subsequent development of mouse eggs.
Comparison of some properties of oocytes segregating in F2 hybrids between KE and CBA/Kw inbred strains of mice
Authors: Wabik‐Sliz Barbara 
Published: February 2005

AbstractThe time taken to dissolve the zona pellucida was compared with fertilizability as well as the meiotic maturation rate of the oocytes from the same (KE × CBA) F2 females. The presence of granular material in oocyte cytoplasm was also examined. It was found that for F2 females in which the zona pellucida digestion was fast, the number of fertilized oocytes was high; for F2 females with zonae pellucidae more resistant to enzyme, the number of fertilized oocytes was low. The correlation between the two characters was significant, indicating their common genetic and/or physiological control. The low or high solubility of zona pellucida did not correlate with the rate of meiotic maturation of the oocyte. This suggests separate factors controlling these two characters. A separate factor seems to control the appearance of granules in cytoplasm since their presence interfered neither with zona pellucida solubility nor with maturation rate of the oocyte.
Serum maintains the fertilizability of mouse oocytes matured in vitro by preventing hardening of the zona pellucida
Authors: Downs Stephen M. , Schroeder Allen C. , Eppig John J. 
Published: February 2005

AbstractThe effects of serum and cumulus cells during oocyte maturation in vitro on subsequent oocyte fertilizability and zona pellucida digestability have been examined. Cumulus cell‐enclosed oocytes were cultured 15–16 hours in medium containing bovine serum albumin plus varying concentrations of fetal bovine serum (FBS). When the serum concentration was decreased incrementally from 5% to 0%, resistance to chymotrypsin digestion increased accordingly in a dose‐dependent manner. The zona pellucida digestion time for oocytes matured in serum‐free medium was increased nearly 500% above that for control oocytes matured in 5% FBS. Also, fertilization was decreased as serum concentration was lowered; the fertilization percentage for oocytes matured in serum‐free serum was reduced by over 90%. This loss of fertilizability was correlated with an absence of sperm within the vitellus following insemination of matured ova. Increasing the serum concentration from 5% to 10% had no effect on fertilization or zona pellucida digestion time. Serum deprivation, even for a short period of time, at the onset of culture significantly reduced the fertilizability of cumulus cell‐enclosed oocytes and increased the zona pellucida digestion time. Removal of serum after oocyte maturation in vitro had little effect. The presence of an intact cumulus oophorus during maturation in vitro was important in the maintenance of fertilizability and zona digestability. These data support the idea that serum deprivation and/or removal of the cumulus cells during oocyte maturation in vitro results in an alteration of the zona pellucida that is manifested as an increased resistance to proteolytic digestion and sperm penetration.
Sea urchin egg‐cortical granule protease has arginyl proteolytic specificity
Authors: Green Jeffrey D. 
Published: February 2005

AbstractSea urchin eggs secrete esteroproteolytic activity at fertilization. This enzyme has been shown to be proteolytic toward embryo protein and casein, but a systematic study of its substrate specificity has not been done. In this communication we present data that demonstrates for the first time that the cortical granule protease from Strongylocentrotus purpuratus eggs cleaves arginyl residues in a protein substrate, lysozyme. We have developed a sensitive reverse‐phase high‐performance liquid chromatography (RP‐HPLC) assay that detects femtomole levels of trypsin and chymotrypsin protease activity [Green, 1986: Anal Biochem 152:83–88]. In the sea urchin system, we have detected protease activity from as few as 50 eggs. Correlating the RP‐HPLC analysis with a spectrophotometric Nα‐benzoyl‐L‐arginine ethyl ester assay, we have found that each egg secretes approximately 40 attomoles of trypsin‐like activity. This general method should be quite useful in investigations into the natural substrate of the egg protease.
Requirement of monovalent cations in the acrosome reaction of guinea pig spermatozoa
Authors: Bhattacharyya Amitabha , Roldan Eduardo R. S. , Yanagimachi Ryuzo 
Published: February 2005

AbstractThe majority of the spermatozoa precapacitated in Ca2+‐free medium underwent the acrosome raction rapidly when they were transferred to Ca2+‐containing medium. The presence of Na+ and Ca2+ in the medium was essential for the acrosome reaction. The vast majority of spermatozoa failed to undergo the reaction in Ca2+ medium lacking monovalent ions, although they remained motile. At the concentration of 140 mM, Na+, K+, Rb+, and Cs+ all supported the reaction at the maximum level, but at 50 mM the latter three ions were not as effective as Na+. Li+ was least effective in supporting the reaction. Virtually no acrosome reactions took place when precapacitated spermatozoa were first exposed to Na+ medium (no Ca2+) and then to Ca2+ medium (no Na+). On the other hand, a considerably higher proportion of spermatozoa acrosome reacted when they were exposed to these media in the reverse order. The most efficient acrosome reactions took place when the medium contained both a monovalent ion (Na+) and Ca2+ simultaneously. Possible mechanisms by which monovalent and divalent cations participate in the acrosome reaction are discussed.
Human sperm‐egg interaction in vitro
Authors: Sathananthan A. Henry , Ng S. C. , Edirisinghe R. , Ratnam S. S. , Wong P. C. 
Published: February 2005

AbstractThe early events of gamete membrane fusion and sperm incorporation are portrayed, which are possibly the earliest pictures of human conception on record.Oocytes recovered at laparoscopy from an in vitro fertilization programme using clomiphene and human menopausal and chorionic gonadotrophin stimulation were zona‐punctured and examined for polyspermic penetration 3–6 hours after insemination. Three eggs were penetrated, and one donor became pregnant in the same cycle.Fusion occurred between the oolemma and the midsegment of the sperm cell membrane extending from the equatorial vestige of the acrosome to the anterior region of the postacrosomal segment. Only acrosome‐reacted sperm were capable of fusing with the egg. Fusion was followed by incorporation of the spermhead into the ooplasm by a process akin to phagocytosis. Sperm chromatin decondensation occurred by progressive inflation and disorganization of the original nuclear envelope.The mechanism of gamete fusion and sperm incorporation resembles that observed during human monospermic fertilization and generally conforms to that reported for eutherian mammals.
Effect of triton X‐100 on ultrastructure, reactivation, and motility characteristics of ram spermatozoa
Authors: Vishwanath R. , Swan M. A. , White I. G. 
Published: February 2005

AbstractFor optimal extraction and reactivation of ram sperm, glutamate, dithiothreitol, magnesium, and cyclic AMP were required in a medium of pH 7.9. On extraction with 0.01 % Triton X‐100, ram sperm were only partially demembranated, and extensive areas of plasma membrane remained intact especially in the midpiece region. Treatment with 0.1% Triton X‐100 removed all plasma membranes and extracted the mitochondrial membrane and matrix. In the absence of ATP, 16.6% ± 0.4 of the partially demembranated sperm were motile, but sperm extracted with 0.1% Triton X‐100 were completely immotile. On adding ATP partially demembranated sperm reactivated better (81.6% ± 2.8) than sperm completely demembranated in 0.1 % Triton X‐100 (39.5% ± 4.6). The release of intracellular LDH rose linearly with increasing concentrations of the detergent from 0.01 to 0.05%, at which it plateaued. There was a significant increase in beat frequency and forward velocity of partially demembranated sperm when treated with ATP. Partially demembranated sperm had intact mitochondria that presumably were still able to produce ATP, although the spermatozoan movement was stimulated by exogenous ATP.