bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of glucose‐phosphate isomerase in relation to growth of the mouse oocyte in vivo and in vitro
Authors: Buehr Mia , McLaren Anne
Published: February 2005

The activity of the enzyme glucose‐phosphate isomerase (GPI‐1) in mouse oocytes is subject to regulation by the cis‐acting gene Gpi‐lta. Electrophoretic analysis of oocytes from 9‐ and 10‐day‐old mice showed that oocyte‐specific regulation of GPI‐1 is not observed in germ cells that have not started to grow (20 μm diameter) but appears as soon as oocyte growth begins (30 μm or larger). Three in vitro culture systems were used to examine the relation of GPI‐1 expression to oocyte growth: culture of intact neonatal ovaries, and co‐culture of dissociated oocytes and somatic cells from neonatal and from 13‐day foetal ovaries. In all three systems modification of GPI‐1 expression always occurred when oocyte growth began, showing that the presence of a normal follicle is not necessary for the expression of the gene Gpi‐lta.
Distribution of calmodulin and calmodulin‐binding proteins in membranes from bovine epididymal spermatozoa
Authors: Noland Thomas D. , Van Eldik Linda J. , Garbers David L. , Burgess Wilson H.
Published: February 2005

Reproducible concentrations of calmodulin representing approximately 0.1% of the membrane protein were detected in purified plasma membranes from bovine epididymal spermatozoa. When membranes were isolated in the presence of 1 mM EGTA, the amount of calmodulin associated with the plasma membranes was not reduced. Calmodulin‐binding proteins were detected in both purified plasma membranes and in a mixed membrane fraction containing both plasma membranes and cytoplasmic droplet membranes. A calcium‐dependent, calmodulin‐binding protein of apparent molecular weight 123,000 was detected in both fractions. In the presence of 1 mM EDTA, putative calcium‐independent calmodulin‐binding proteins of apparent molecular weights 93,000, 32,000, 18,000, and 15,000 were detected in the plasma membrane fraction. The 15,000 Mr polypeptide was also present in the mixed membrane fraction but the three proteins of higher molecular weight were reduced or absent in this fraction.
Role of the vitellus in the block to polyspermy in golden hamster eggs
Authors: Menezes J. , Peter J.
Published: February 2005

The block to polyspermy in golden hamster eggs is believed to operate only at the zona pellucida. However, changes in the egg vitellus also prevent further entry of capacitated sperm. When zona‐free hamster eggs spontaneously activated in vitro, and in vivo fertilized eggs at pronuclear stage were inseminated with capacitated human sperm, penetration did not occur. In the case of a homologous system using hamster sperm and in vivo fertilized hamster eggs, slight attachment of sperm was observed but no penetration. The cortical granules were found to be released in spontaneously activated and in fertilized eggs as observed by phase contrast microscopy. These observations suggest that the egg vitellus plays a role in the block to poiyspermy in addition to that of the zona block.
Changes in cortical granules during porcine oocyte maturation
Authors: Cran D. G. , Cheng W. T. K.
Published: February 2005

The structure, number, and distribution of cortical granules in porcine oocytes during maturation induced by human chorionic gonadotrophin (HCG) are reported.

The number of granules remained constant for 30 hr following HCG. Thereafter, there was an approximate doubling by 50 hr. At all times examined, a dark and light form were present. Up to 40 hr the latter predominated while at 50 hr there was a marked increase in the number of the former. Movement of cortical granules to the plasma membrane took place between 20 and 30 hr post‐HCG. The changes in cortical granules are correlated with the capacity of the oocytes to undergo a block to polyspermy.
The acrosomal region of the spermatozoon of Ciona intestinalis: Its relationship with the binding to the vitelline coat of the egg
Authors: Rosati F. , Pinto M. R. , Casazza G.
Published: February 2005

This paper describes a study of the apical region of the spermatozoon of Ciona intestinalis before and during its binding to the vitelline coat of the egg. A combination of the techniques of thin sectioning, negative staining, and freeze fracture has revealed that in the apical‐most region, where a small acrosomal vesicle lies on the flat tip of the nucleus, there is a cap‐like region almost completely free of particles on the P face of the plasma membrane. The particle‐free area is surrounded by two circlets of orderly arranged particles. Upon binding to the vitelline coat the particles of the distal circlet show a partial displacement, while the particles of the apical circlet remain unaltered. The relationship between the apical circlet and the binding process is discussed. The final step of the acrosome reaction, which occurs in only a few of the bound spermatozoa, consists in the fusion of the plasma membrane with the acrosomal membrane, in the dehiscence of the acrosomal contents and finally in the formation of membrane tubules.
A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa
Authors: Langlais Jean , Roberts Kenneth D.
Published: February 2005

The abundance of data pertaining to the metabolism of lipids in relation to mammalian fertilization has warranted an effort to assemble a molecular membrane model for the comprehensive visualization of the biochemical events involved in sperm capacitation and the acrosome reaction. Derived both from earlier models as well as from current concepts, our membrane model depicts a lipid bilayer assembly of space‐filling molecular models of sterols and phospholipids in dynamic equilibrium with peripheral and integral membrane proteins. A novel feature is the possibility of visualizing individual lipid molecules such as phosphatidylcholine, phosphatidylethanolamine, lysophospholipids, fatty acids, and free or esterified cholesterol. The model illustrates enzymatic reactions which are believed to regulate the permeability and integrity of the plasma membrane overlying the acrosome during interactions between the male gamete and capacitation factors present in fluids of the female genital tract. The use of radioactive lipids as molecular probes for monitoring the metabolism of cholesterol and phosphatidylcholine revealed the presence of (1) steroid sulfatase in hamster cumulus cells, (2) lecithin: cholesterol acyltransferase in human follicular fluid, (3) phospholipase A2, and (4) lysophospholipase in human spermatozoa. These enzymatic reactions can be integrated into a pathway that provides a link between the concepts of lysophospholipid accumulation in the sperm membranes and alteration of the cholesterol/phospholipid ratio as factors involved in the preparation of the membranes for the acrosome reaction. Capacitation is viewed as a reversible phenomenon which, upon completion, results in a decrease in negative surface charge, an efflux of membrane cholesterol, and an influx of calcium between the plasma and outer acrosomal membranes. Triggered by the entry of calcium, the acrosome reaction involves phospholipase A2 activation followed by a transient accumulation of unsaturated fatty acids and lysophospholipids implicated in membrane fusion which occurs during the formation of membrane vesicles in spermatozoa undergoing the acrosome reaction.
Developmental capability of rat oocytes matured in vitro in defined medium
Authors: Fleming Alan D. , Evans Gareth , Walton Elizabeth A. , Armstrong David T.
Published: February 2005

The normality of in vitro matured oocytes was compared to that of in vivo matured (ovulated) oocytes at the following stages of development: germinal‐vesicle breakdown, first polar body formation, fertilization (two polar bodies and two pronuclei with a sperm tail or first cleavage), and fetal development (day 20 fetuses). At all points, the in vitro oocytes exhibited a reduced ability, with oocytes matured cumulus‐free having the poorest. The exposure of oocytes to human chorionic gonadotropin (hCG) for 2 hr before collection or during incubation improved their rates of maturation and development to day 20 fetuses but not their ability to undergo fertilization. While beneficial, the exposure to gonadotropins before or during maturation was not essential, as evidenced by the production of two day 20 fetuses matured and fertilized in vitro without any gonadotropin (luteinizing hormone or hCG) treatment in vivo or in vitro. These data demonstrate that in the population of in vitro matured oocytes there exist individuals wholly competent of complete normal development, albeit in a reduced proportion in comparison to normally matured and ovulated oocytes. That the in vitro handling, treatment, and culture of the oocytes may be responsible for some of the reduced developmental ability observed is suggested by the developmental abilities of ovulated oocytes under different conditions. Ovulated oocytes fertilized in the donor had the highest rates of development (46%), followed by those fertilized after transfer into mated recipients' oviducts (20%). The lowest rate was achieved with in vitro fertilized oocytes (7%), which represented the group subject to the greatest degree of manipulation and distinction from the normal in vivo process.
Biochemical changes in cervical mucus‐factor infertility
Authors: Lutjen P. J. , Handley C. J. , de Witt M. T. , Trounson A. O. , McBain J. C.
Published: February 2005

Cervical‐factor infertility has generally been attributed to the presence of antispermatozoal antibodies in the secretions of the uterine cervix, despite the fact that the incidence of sperm‐specific antibodies in these women is generally low. We report here a modification in the structure of the cervical mucus of patients with a diagnosed cervical factor. Cervical mucus from patients with a cervical factor of nonimmunological origin, collected during the periovulatory phase of the menstrual cycle, had (1) a significant decrease in the content of glycosidically bound sialic acid and (2) an increased ability to act as an acceptor for sialic acid from cytidine‐5′‐monophospho‐N‐acetylneuraminic acid (CMP‐sialic acid) when incubated with an exogenous sialyltransferase; in comparison to mucus from normal fertile women. Both siaiyltransferase and fucosyltransferase activities were detected in cervical mucus, but there was no difference between fertile normal and cervical‐factor patients using the assays described. These results reinforce a possible role for sialic acid residues and their associated glycosyltransferases in the regulation of spermatozoal–cervical mucus interaction.
Lectin‐induced blockage of developmental processes in preimplantation mouse embryos in vitro
Authors: Zuzack John S. , Tasca Richard J.
Published: February 2005

This study attempts to assess the developmental importance of cell surface glycoconjugates of preimplantation mouse embryos. This was done by incubating early embryos in various lectins and analyzing subsequent development. If specific cell surface glycoconjugates (lectin receptors) are linked to specific developmental processes, such as cell division, compaction, and blastocyst formation, then different lectins should block these different developmental processes. The results show that wheat‐germ agglutinin (WGA; N‐acetyl‐D‐glucosamine‐specific) at 50 μg/ml prevents the cell division of four‐cell embryos. However, this effect of WGA occurs only in embryos with intact zonae pellucidae. Concanavalin A (Con A; α‐D‐glucose and α‐D‐mannose‐specific) treatment, 20 μg/ml, of four‐cell or early eight‐cell embryos prevents compaction, the first major change in cell shape in early mouse embryogenesis. Divalent succinly Con A does not affect development, suggesting that the Con A effect is due to crosslinking of cell surface glycoconjugates. Exposure of four‐cell or early eight‐cell embryos to 10 μg/ml Lotus Tetragonolobus puprureas agglutinin (LTA; α‐L‐fucose‐specific) or 25 μg/ml Limulus polyphemus agglutinin (LPA; sialic acid‐specific) allows compaction or development to the morula stage, but blocks blastocyst formation. All lectins tested retard cell division to some extent. Late morulae and early blastocysts are more resistant than earlier stages to all of the lectins studied. This study demonstrates that very low concentrations of these lectins affect different developmental processes, presumably based upon their sugar specificities.
A qualitative comparison of protein synthesis in the preimplantation embryos of four rodent species (mouse, rat, hamster, gerbil)
Authors: Norris Michael L. , Barton Sheila C. , Surani M. Azim H.
Published: February 2005

An examination of the protein profiles obtained from preimplantation embryos of mouse, rat, hamster, and gerbil revealed marked species differences in the one‐cell fertilized eggs but a striking similarity in the major proteins produced by blastocysts. The early change in protein profile observed in all four species suggests that activation of the embryonic genome before the second cleavage division is a rodent characteristic. The initial marked species differences resulting from putative maternal transcripts could be important in the establishment and maintenance of species integrity.
Interactions of seminal plasma and glycosaminoglycans on acrosome reactions in bovine spermatozoa in vitro
Authors: Lee Chin N. , Handrow Richard R. , Lenz Richard W. , Ax Roy L.
Published: February 2005

Previous reports indicate that glycosaminoglycans (GAGs) would enhance the occurrence of acrosome reactions in sperm in vitro, but continuous exposure of those sperm to seminal plasma prevented a significant incidence of acrosome reactions. This study was designed to evaluate the interaction of GAGs and seminal plasma to promote acrosome reactions in bull sperm in vitro. Epididymal sperm required 22 hr to exhibit acrosome reactions in response to GAGs whereas only 9 hr were needed to achieve the same effect with washed ejaculated sperm. Exposure of epididymal sperm to seminal plasma for 20 min shortened the time for induction of the acrosome reaction to 9 hr. Scatchard analyses of displacement data suggested an alteration in the binding affinity of 3H‐heparin to epididymal sperm membrane following the short‐term exposure to seminal plasma. High doses (250 and 500 μg/ml) of heparin, heparan sulfate, and chondroitin‐4‐sulfate were without effect, but doses <100 μg/ml were stimulatory in terms of enhancing acrosome reactions. Compositional studies with seminal plasma revealed a total GAG content of 1.6 mg/ml, proportioned as 61.6% chondroitin sulfates, 17.6% heparin‐like material, 0.3% hyaluronic acid, and 20.5% undetermined GAG. It is proposed that seminal plasma can alter the ability of sperm to respond to GAGs, and the high concentrations of GAGs endogenous to seminal plasma may prevent premature initiation of the membrane perturbations necessary for the acrosome reaction.
Alteration of sperm thiol‐disulfide status and capacitation in the guinea pig
Authors: Fleming Alan D. , Kosower Nechama S. , Yanagimachi Ryuzo
Published: February 2005

Epididymal spermatozoa of the guinea pig were incubated under conditions known to promote a rapid synchronous capacitation in a large proportion of the spermatozoa (Ca2+‐free medium with lysophosphatidylcholine, LC) or in Ca 2+‐free medium without LC. To study the effects of altered thiol‐disulfide status and content, incubations were conducted with reagents that maintain and increase thiol groups (DTT, GSH), maintain and increase disulfide groups (diamide, GSSG), or which irreversibly block thiol groups by alkylation (NEM). The permeable DTT inhibited LC‐induced capacitation and at high concentrations diminished the percentage of acrosome reactions in capacitated spermatozoa. The permeable diamide exhibited a stimulatory effect upon capacitation. The largely impermeable GSH and GSSG exhibited effects similar to their respective permeable counterparts but their effects were moderate and required extremely high concentrations. The DTT inhibition of LC‐induced capacitation was reversible by washing and a further 1 hr incubation. In this final incubation after removal of DTT by washing, LC was absent too so its stimulatory effect must have been accomplished prior to washing and in the presence of DTT. NEM‐alkylation of the existing thiol population did not affect LC‐induced capacitation but alkylation of the increased thiol population after prior DTT treatment was inhibitory of capacitation. These results suggest that the maintenance and/or formation of disulfide groups on enzymes or structural proteins may be a component of the capacitation process. In contrast, the formation and maintenance by alkylation of increased thiol groups but not the maintenance of existing thiol groups, is inhibitory of capacitation. The relevance of these findings to a role for a thiol‐sensitive proteinase in capacitation is discussed.
Mouse oocyte maturation modulated by a granulosa cell factor and by heparin and heparan sulfate
Authors: Sato Eimei , Ueno Hiroshi , Koide S. S.
Published: February 2005

Oocyte maturation‐preventing factor (OMPF) was extracted from bovine granulosa cells with a buffer containing 1 M urea and 5 mM EDTA. OMPF was partially purified by gel filtration on Sephadex G25 and by reversed‐phase high performance liquid chromatography. The maturation‐preventing activity of purified fractions was determined by measuring their capacity to block the spontaneous dissolution of the germinal vesicle (GVBD) of isolated cumulus‐enclosed mouse oocytes. Hyaluronic acid, chondroitin sulfate, heparin, heparan sulfate, keratan sulfate, and dextran sulfate at concentrations of 500 μg/ml did not affect the frequency of GVBD of isolated mouse oocytes. Heparin and heparan sulfate, however, blocked the inhibitory effect of OMPF, whereas the inhibition of GVBD induced with dibutyryl cAMP, forskolin, W7 (calmodulin antagonist), and 3‐isobutyl‐l‐methylxanthine (phosphodiesterase inhibitor) was not blocked. OMPF was eluted in the adsorbed fraction when chromatographed on heparin‐Agarose, showing interaction of OMPF with heparin. The present results suggest that the glycosaminoglycan matrix may influence OMPF action on oocytes.
The cortical reaction in pig oocytes during in vivo and in vitro fertilization
Authors: Cran D. G. , Cheng W. T. K.
Published: February 2005

The structure of pig oocytes after in vivo and in vitro fertilization and following treatment with the ionophore A23187 with differing levels of calcium are described, with particular reference to the cortical granules.

Fertilization in vivo and in vitro resulted in cortical granule exocytosis. Sperm penetration in vivo was more rapid than in vitro and resulted in the dispersal of the cortical granules' contents in the perivitelline space following exocytosis. The contents of the granules remained adjacent to the plasmalemma after in vitro fertilization and suboolemmar vesicles were less numerous than after in vivo fertilization. High calcium levels were necessary to induce the dispersal of the cortical granule contents following treatment with ionophore. The observations are discussed regarding their relevance to the blockage to polyspermy.
Cytochemical localization of membrane‐bound Mg2+‐dependent ATPase activity in guinea pig sperm head before and during the acrosome reaction
Authors: Usui Noriko , Yanagimachi Ryuzo
Published: February 2005

The distribution of ATPase activity in the heads of uncapacitated, capacitated, and acrosome‐reacting guinea‐pig spermatozoa was examined cytochemically using the Wachstein‐Meisel's technique. In uncapacitated spermatozoa, the reaction products of the enzyme activity were localized on both the inner surface of the plasma membrane and the outer surface of the outer acrosomal membrane. The activity was Mg2+‐dependent and inhibited by both Ca2+ and SH‐blocking agents. This Mg2+‐dependent ATPase activity was also demonstrated at the same sites in capacitated spermatozoa, whereas it was completely absent in acrosome‐reacting spermatozoa. Although we did not determine the exact time of inactivation of the enzyme, it appeared to occur before the plasma membrane fused with the underlying outer acrosomal membrane. The abrupt loss of the Mg2+‐dependent ATPase activity in the plasma and outer acrosomal membranes immediately before the onset of the acrosome reaction seems to suggest that inactivation of this enzyme by Ca2+ is one of the important biochemical events involved in the acrosome reaction.
An in vitro technique to study penetration of hamster oocyte‐cumulus complexes by using physiological numbers of sperm
Authors: Corselli J. , Talbot P.
Published: February 2005

We have developed an inexpensive in vitro system for studying cumulus penetration and fertilization by using physiological numbers of sperm. This system simulates conditions believed to exist in vivo more closely than any in current usage. In this system, 1–100 hamster sperm are used to challenge fresh hamster oocyte‐cumulus complexes (OCC). Only fresh (nonoviducal) OCC are used, as they present the most stringent challenge to sperm. Because sperm numbers are low, OCC do not disperse, and sperm can be studied microscopically during penetration of the cumulus oophorus and corona radiata. These conditions permit microscopic assessment of the sperm acrosome. Video tapes of experiments allow easy review and analysis of experiments. Results obtained employing this technique show that, in vitro, (1) capacitated, acrosome‐intact hamster sperm can penetrate the extracellular matrix between cumulus cells and bind to the zona pellucida; (2) the “figure‐eight motility” characteristic of hyperactivated hamster sperm swimming in culture medium is suppressed when sperm swim in the extracellular matrix between cumulus cells; and (3) fertilization occurs in capillary tubes when low numbers of sperm are used. The in vitro system that we have described will be useful in analyzing the mechanisms used by sperm to penetrate the cumulus and corona radiata and to clarify the role of the acrosomal enzymes in fertilization.
Localization of antigens on the inner‐acrosomal membrane of the rabbit sperm by immunofluorescence
Authors: Sheikhnejad Reza G. , Srivastava P. N.
Published: February 2005

The acrosome‐reacted spermatozoa interact with the zona pellucida through their limiting inner acrosomal membrane (IAM). The antigenic properties of IAM were determined using the antibody to IAM raised in a male guinea pig. The antisera was incubated with the acetone powder of rabbit lung, liver, kidney, heart, muscle, and blood cells. The unabsorbed antibodies specifically interacted with antigens on the IAM as determined by the immunofluorescence technique.
Cationic probes: Specificity of distribution of metal‐binding sites in bovine sperm
Authors: Nelson Leonard , Gardner Mary E.
Published: February 2005

Salts of transition elements that alter the rate of sperm cell movement act at or near calcium‐binding sites. After living bull sperm cells had been preincubated in VO43−, Ni2+, Zn2+, Mn2+, and also La3+, they were then fixed. Crisply defined organelles and the absence of particulate deposits in the morphological controls contrasted sharply with the treated specimens; the latter contained regions of increased electron density, the nature and distribution of which depended on the test substance, reflecting the differential affinities of the specific ions. La3+ formed fine dense areas, mainly at the exocytic surface of the plasma membrane. VO43− marks the cell surface but also left particulate densities within the cell. Ni2+ caused a nearly uniformly dense deposit at the surface and on the satellite fibers and axonemal microtubules. Zn2+ formed less uniform but coarser deposits, while in Mn2+ the distribution was similar to that in Zn2+ but much denser in the axonemal matrix and on the satellite fibers. Verapamil restricted the size and number of the opacities, while procaine permitted a similar distribution of slightly larger size reaction product. The differences in size and distribution of the enhanced densities were consistent and replicable for the individual assay substances. Vanadate, which specifically inhibits Na, K‐ATPase, bound to ouabain‐sensitive enzyme loci, however, completely disrupting the axonemal complex. This suggests that an important role of dynein in flagellar motion may relate to intracellular transport of Ca2+.
Effects of extracellular cations and energy substrates on the acrosome reaction of precapacitated guinea pig spermatozoa
Authors: Murphy S. J. , Roldan E. R. S. , Yanagimachi R.
Published: February 2005

It is well established that extracellular Ca2+ and an alkaline pH are essential for the acrosome reaction of precapacitated guinea pig spermatozoa. In the present study, we investigated how other extracellular cations (Na+, K+, and Mg2+) and energy substrates (glucose, lactate, and pyruvate) affect the acrosome reaction of these spermatozoa. The effect of osmolality of the medium on the reaction was also examined.

It was found that precapacitated spermatozoa could undergo the acrosome reaction in a medium with an osmolality of as low as 160 mOsmol or as high as 500 mOsmol. However, the osmolality range within which large proportions of spermatozoa underwent the acrosome reaction while retaining strong motility was 220 to 380 mOsmol. Extracellular Na+ was essential for the reaction to occur. A few spermatozoa could acrosome react in a medium with a very low concentration of Na+ (5 mM or less), but a higher concentration of Na+ (more than 60 mM) was needed to ensure the acrosome reaction in the majority of spermatozoa. High concentrations of extracellular K+ were not essential for the reaction, but the presence of K+ at physiological level ensures that the reaction will occur consistently at a high rate. Extracellular Mg2+ was not required for the acrosome reaction. The high concentrations of Mg2+ (up to 20 mM) and K+ (up to 60 mM) that were tested did not affect the incidence of the acrosome reaction. Exogenous substrates were not essential for the reaction. Although the majority of precapacitated spermatozoa could acrosome react in substrate‐free medium, the presence of substrates in the medium, particularly that of pyruvate, seemed to be beneficial to the reaction.
The decrease of Xenopus egg membrane capacity during activation might be due to endocytosis
Authors: Bernardini Giovanni , Ferraguti Marco , Peres Antonio
Published: February 2005

An endocytotic process in Xenopus eggs was studied by direct visualization of the fluorescent tracer lucifer yellow CH in 1‐μm‐thick sections. Eggs fixed 3 min after activation show small fluorescent vesicles in the cortex; those fixed after 30 min show mainly large vesicles. This process is related to membrane recycling after cortical granule exocytosis and can account for the observed decrease of the effective membrane capacity.
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

Membrane 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

Nicotinamide 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

We 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

In 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

Murine 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

Rhodaminyl‐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

Flagella 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

The 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

In 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

Methods 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

Fertilized 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

The 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

The 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

Sea 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

The 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.