bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructural studies of spermatogenesis in the anthocerotales. III. Gamete morphogenesis: From spermatogenous cell through midstage spermatid
Authors: Duckett Jeffrey G. , Carothers Zane B. , Moser John W.
Published: February 2005

An ultrastructural examination of spermatogenesis in Phaeoceros has shown nucleoli to be present in spermatogenous cells and to persist until the centrioles become associated with nuclei of young spermatids. At the onset of multilayered structure (MLS) formation, well‐defined aggregations of osmiophilic strands begin to form in the nuclei of young spermatids and disappear shortly after chromatin condensation starts in the midstage spermatids. When the centrioles in the young spermatids are orientated perpendicular to the nuclear envelope, the nucleoplasm immediately in front of them is densely stained. Where the spline tubules of the MLS extend over the nucleus, the nuclear envelope is devoid of pores, and the inner nuclear membrane is contacted internally by the local deposition of dense staining nucleoplasm. Chromatin condensation begins with strands extending perpendicularly from the dense staining nucleoplasm beneath the spline and continues with the nuclear beak becoming filled with condensed chromatin. As the MLS lamellae disappear acropetally, the rear portion of the anterior mitochondrion (AM) extends back under the nuclear beak which now narrows to a size that approximates the anterior end of the nucleus of a spermatozoid. By the end of the mid‐spermatid stage, the nucleus has coiled approximately one gyre of a helix and the five or six central slpine tubules extend over the plastid which is now located beneath the front end of the AM. Several profiles of endoplasmic reticulum confluent with the nuclear envelope are present. Possible factors which might play a role in determining the morphology of the mid‐spermatids are discussed.
Rabbit testis proacrosin: Immunological similarities between testis, sperm proacrosins and the initially formed testis acrosin
Authors: Mukherji Sudhir K. , Scibienski Robert J. , Meizel Stanley
Published: February 2005

Anti‐rabbit proacrosin IgG was prepared from goat serum following immunization with a homogeneous preparation of rabbit testis proacrosin. The “auto‐activation” products of purified testis proacrosin were separated into 68,000 and 34,000 molecular weight (mol wt) acrosins by Sephadex G‐100 column chromatography. Immunodiffusion analysis of testis and epididymal sperm proacrosins and acrosins on agarose gel against goat anti‐rabbit testis proacrosin showed immunological identity between rabbit testis and sperm proacrosins and the initial testis acrosin (mol wt 68,000). However, the 34,000 mol wt form of testis acrosin showed weaker reaction with the antibody and only partial identity with the proacrosin and the 68,000 mol wt form of acrosin. These results suggest that there is no major structural difference between testis and sperm proacrosins and between proacrosin and the 68,000 mol wt acrosin, but such a structual change occurs when the 34,000 mol wt acrosin is formed.
Initiation, prolongation, and reactivation of the motility of salmonid spermatozoa
Authors: Benau Danny , Terner Charles
Published: February 2005

The motility of salmonid spermatozoa initiated by dilution of the milt with ovarian fluid or isotonic saline is brief duration; it was believed that it can be activated only once in the life of the spermatozoon. Dilution of the milt with an equal volume of isotonic saline (0.12 M‐NaCl) containing 5 mM‐3‐isobutyl‐1‐methylxanthine (MIX) prolonged and intensified sperm motiliy. When motility had stopped after initial mobilization with saline or ovarian fluid, it could be reactivated by addition of MIX; reactivated spermatozoa fertilized eggs. Dilution with saline containing K+ (24 mEq/liter) did not initiate sperm motility even in the presence of MIX. The spermatozoa were mobilized by subsequent with 0.12 M‐NaCl. The concentration of adenosine triphosphate (ATP) in sperm suspensions dropped on dilution with saline and rose as motility ceased, but declined without subsequent recovery following dilution with MIX‐saline. The concentration of cyclic adenosine monophosphate (cAMP) rose and fell sharply on initiation of motility and rose again after motility had declined. While salmonid spermatozoa can be mobilized by dilition with saline alone, the effectiveness of MIX in reactivating “spent” spermatozoa supports the assumption that cAMP plays a role in the initiation of sperm motility.
Ovoperoxidase activity in ionophore treated mouse eggs. I. Electron microscopic localization
Authors: Gulyas Bela J. , Schmell Eli D.
Published: February 2005

A mammalian ovoperoxiadase activity has been detected in ionophore activated mouse eggs. The peroxidase activity was demonstrated at the electron microscopic level using the 3,3′‐diaminobenzidine (DAB) histochemical method. A positive DAB reaction was detected in a portion of the intact cortical granules of untreated or DMSO treated control eggs. In the ionophore activated eggs, the DAB reaction product was routinely detected by electron microscopy, predominantly on the cell surface, that is on the zona pellucida, in the perivitelline space, and in association with the cortical granule exudates. Furthermore, the peroxidase inhibitors phenylhydrazine and sodium sulfite prevented DAB staining in ionophore activated oocytes. These results indicate the presence of an ovoperoxidase, possibly of cortical granule origin, on the surface of activated mammalian eggs, detectable by histochemical means.
Membrane potential, pH and the activation of surf clam oocytes
Authors: Finkel Toren , Wolf Don P.
Published: February 2005

Unfertilized oocytes of the surf clam, Spisula solidissima, have resting membrane potentials of −18 ± 7 mV (n = 20). Within five seconds of sperm addition, an electrophysiologically detectable response was apparent, which was characterized by a rapid and prolonged depolarrization depolarization followed four to five minutes post‐insemination by the beginning of the beginning of a steady hyperpolarization to approximatelv −70 mV. This final hyperpolarization was completed within ten minutes of sperm addition. The initial rapid depolarization following insemination may result from a transient increase in sodium conductance, and it may be crucial in preventing polyspermy, since the degree of polyspermy in Spisula oocytes was sensitive to external sodium ion concentrations. Evidence was obtained that changes in intracellular pH are essential for oocvte activation. Using germinal vesical breakdown (GVB) as a marker for activation, it was shown that agents that raise intracellular pH (ammonia and procaine) induced GVB, whereas agents that lower intracellular pH pH (Na‐acetate or Na‐propionate seawater) inhibited GVB.
Partial activation of sea‐urchin eggs by bonellin
Authors: Cariello Lucio , de Nicola Giudici Marina , Zanetti Laura
Published: February 2005

We here describe further studies on the action of bonellin on sea‐urchin eggs. Bonellin brings about Some of the changes that are known to occur in the egg upon fertilization. In particular, it appears to cause the increased rate of incorporation of amino acids into proteins, the increase of the voltage noise, and the exocytosis of some of the cortical granules. A comparison with the effect of ammonia is discussed.
Membrane differentiations in spermatozoa of the squid, Loligo pealeii
Authors: Olson Gary E. , Linck Richard W.
Published: February 2005

Regional differences in the structure of the plasma membrane and acrosome membrane of squid spermatozoa were studied by freeze‐fracture and thin section electron microscopy. In regions of close apposition the plasma membrane and acrosome membrane are adjoined to one another by regularly spaced linkages. These linkage sites, overlie a set of fibers located at the inner face of the acrosomal membrane. The acrosomal fibers terminate in a layer of granular material located at the base of the acrosome. Detergent treatment of sperm releases the fibers and granular material as an interconnected complex. Freeze‐fracture replicas reveal a random arrangement of intramembranous particles in the plasma membrane over the sperm head and linear aggregates of intramembranous particles in the acrosomal membrane. Several regional differences in the structure of the flagellar plasma membrane are present. The thickness of the glycocalyx is progressively reduced distally along the flagellum. Freeze‐fracture replicas show evenly spaced linear arrays of intramembranous particles which extend parallel t o the flagellar long axis. Examination of spermatozoa extracted to disrupt flagellar geometry suggest that the dense fiber‐doublet microtubule complexes are attached to the plasma membrane. The possible functional role of these membrane differentiations and their relationship t o membrane structures in mammalian spermatozoa are discussed.
Optimization of culture conditions for the formation of sperm cells in pollen tubes of tradescantia
Authors: Lafleur Gloria J. , Gross Amy E. , Mascarenhas Joseph P.
Published: February 2005

A culture medium and culture conditions are described that enable generative cell division and sperm formation to occur in a large proportion (greater than 70%) of the pollen tubes of Tradescantia paludosa within six to eight hours of culture of pollen. The nature of the nitrogen source, speed of shaking, and ratio of pollen to medium are important parameters in determining the extent of sperm formation. Addition of the plant hormones indole acetic acid, gibberellic acid, and kinetin to the growth medium does not influence generative cell division.
Kinematics of human spermatozoa
Authors: David G. , Serres C. , Jouannet P.
Published: February 2005

A microcinematographic (50 f/s) study was performed on motile human spermatozoa. Eighty percent were found to have a linear trajectory and a pseudo‐sinusoidal head displacement pattern. Throughout their progression, the spermatozoa periodically rotated on their longitudinal axis at a frequency equal to that of flagellar wave formation. These waves were found always to begin on the same side of the cell and to propagate in the flattened plane of the head until the moment of rotation. At this time the wave had reached a point near the middle of the flagellum. Beyond this point, the flagellum moves out of the plane of the head. Different variables used to characterize the movement of spermatozoa included the velocity of progression, amplitude and velocity of head displacement, frequency of rotation, wave amplitude, and propagation velocity of the flagellar wave. Among these variables, it was the propagation velocity of the wave that was found to be best correlated with the velocity of spermatozoan progression. This flagellar wave exhibited two stages, one of initiation and one of propagation.
The assessment of human sperm morphology in surface replica preparations for transmission electron microscopy
Authors: Mortimer David
Published: February 2005

Human spermatozoa were treated with the detergents Hyamine 2389, Triton X‐100, or sodium dodecyl sulphate (SDS) and surface replica preparations and Papanicolaou‐stained smears made. From the ultrastructural studies it was found that Hyamine caused a dissolution of the plasmalemma to reveal the outer acrosomal membrane that was often absent over the anterior cap region; whereas Triton, in addition, caused severe damage to the entire acrosome. Treatment with SDS rapidly cleaved the tails from the majority of the spermatozoa, stripped the plasmalemma from both the head and tail regions of the cells, and also removed the whole of the outer acrosomal membrane plus most of the acrosomal contents. However, when viewed in the Papanicolaou‐stained preparations, the spermatozoa were all of apparently normal morphology.

These studies make it clear that some caution must be used when interpreting human sperm morphology assessments made under the light microscope. While it is not suggested that the more involved technique of surface replication should be used in routine semen analyses, it does represent a valuable technique for use with research material.
Inhibition of fertilization in cattle by passive immunization with anti‐zona pellucida serum
Authors: Tsunoda Y. , Soma T. , Sugie T.
Published: February 2005

Passive immunization with antiserum prepared against isolated bovine zonae pellucidae inhibited fertilization in the cow. The minimum dosage of antiserum (titer 27 by immunofluorescence) required for complete inhibition was 2 ml/kg of body weight.
Vitelline layer lytic activity in sperm extracts of sea urchin, Hemicentrotus pulcherrimus
Authors: Yamada Yuji , Aketa Kenji
Published: February 2005

A factor which dissolves the vitelline layer was extracted from sperm of the sea urchin, Hemicentrotus pulcherrimus. Turbidity of the suspension was reduced when isolated vitelline layers were mixed with this sperm factor. When the mixture was subjected to SDS polyacrylamide gel electrophoresis, some of the protein bands of the vitelline layer were seen to be missing. The lytic activity of the factor was heat labile, completely inhibited by L‐1‐tosyl‐amide‐2‐phenyl‐ethylchloromethyl ketone and partially inhibited by soybean trypsin inhibitor. Chymotrypsin activity was detected, but not trypsin, arylsulfatase, or glycosidase. These results suggest that a chymotrypsin‐like enzyme participates in lysis of the vitelline layer by the fertilizing spermatozoon.
The effects of carnitine, glycerylphosphorylcholine, caffeine, and egg yolk on the motility of rat epididymal spermatozoa
Authors: Turner T. T. , Giles R. D.
Published: February 2005

Experiments were performed to further the understanding of epididymal processes involved in the acquisition of sperm motility. Samples of luminal contents were collected by micropuncture from four regions of the rat epididymis. These samples were incubated in various diluents to observe the effects of the diluents on sperm motility. Consonant with previous reports, 40 mM glycerylphosphorylcholine (GPC) and 60 mM DL‐carnitine reduced overall motility scores of cauda epididymidal spermatozoa but did not prevent normal initiation of motility. Additionally, control sperm cells and cells treated with carnitine could reinitiate full motility after becoming immotile. Spermatozoa treated with GPC could not reinitiate motility. The sperm cells in our system thus react to GPC and carnitine in fundamentally different ways, the exact nature of which remains to be determined.

Spermatozoa from the distal caput epididymidis evidenced high motility scores when diluted in a 5% egg yolk + 10 mM caffeine diluent. It was demonstrated, however, that the subjective appearance of full motility in these immature cells was not supported by actual progressive motility as measured in an assay of linear distance traveled. It was concluded that neither 10 mM caffeine, 5% egg yolk, nor their combination was sufficient to induce progressive motility in immature rat spermatozoa.
Acid‐extracted nuclear proteins and ultrastructure of human sperm chromatin as revealed by differential extraction with urea, mercaptoethanol, and salt
Authors: Tanphaichitr Nongnuj , Sobhon Prasert , Chalermisarachai Panpaka , Patilantakarnkool Malinee
Published: February 2005

Basic proteins were differentially extracted from the purified heads of ejaculated human sperm by successive treatment with (1) 1% Triton X‐100, 1% mercaptoethanol (ME); (2) 8 M urea, 1% ME; (3) 8 M urea, 1% ME, 0.2 M NaCl; and (4) 8 M urea, 1% ME, 0.6 M NaCl. Nonhistones were extracted in the first, second, and third treatments; histones, in the second and third; and most of the protamines, in the fourth, together with DNA. Corresponding electro microscopic studies of the sperm pellets after each treatment suggest that the nucleoprotamines are discrete portions of chromatin which are organized in the form of long thick cords and oval bodies of about 400–550 Å in diameter interlinked with very thin strands of fibers about 20–50 Å thick, which could represent the naked DNA depleted of its constituent histones.
Radio‐iodination of plasma membrane polypeptides from isolated mouse spermatogenic cells
Authors: Millette Clarke F. , Moulding Christopher T.
Published: February 2005

Cell surface polypeptides of mouse pachytene spermatocytes and round spermatids (steps 1–8) have been iodinated using 1,2,3,6,tetracholoro‐3α, 6α‐diphenylglycouril (IODOGEN). Labeled proteins have been assayed using two‐dimensional polyacrylamide electrophoresis and radioautography. Purified plasma membranes, prepared from both spermatocytes and spermatids after the iodination of intact cells, exhibit 25–30 polypeptides which label reproducibly. No significant qualitative differences are noted in the labeled polypeptide map obtained from each of the purified cell types. Iodinated proteins range in molecular weight from greater than 100k daltons to approximately 40k daltons. The isoelectric points of labeled constituents range from pI 5.7 to 7.2. Three polypeptides represent the major iodinated species: p 94/5.8, p 75/5.9, and p 53/7.1. Comparison with total plasma membrane constituents assayed using Coomassie brilliant blue indicates that many of the radioactively labeled proteins are not present in quantities sufficient to allow ready detection without isotopic techniques. As a result, many of the proteins identified autoradiographically represent newly described surface components of mouse pachytene spermatocytes and round spermatids. The preparation of purified plasma membrane fractions prior to electrophoresis ensures that all iodinated species are in fact cell surface components. Furthermore, experiments designed to assess the vectorial nature of the IODOGEN‐catalyzed labeling procedure suggest that most, if not all, of the iodinated species are exposed on the external side of the cell plasma membrane. Therefore, these studies have (1) identified hitherto unrecognized plasma membrane components of mouse pachytene spermatocytes and round spermatids and (2) provided the first available biochemical data concerning the molecular orientation of particular proteins in the surface membranes of developing mouse spermatogenic cells.
Reaction of sperm with egg‐derived hydrogen peroxide helps prevent polyspermy during fertilization in the sea urchin
Authors: Boldt Jeffrey , Schuel Herbert , Schuel Regina , Dandekar Pramila V. , Troll Walter
Published: February 2005

Recent evidence suggests roles for egg derived hydrogen peroxide (H2O2) and ovoperoxidase (secreted by cortical granules) in both fertilization envelope hardening and the block to polyspermy in sea urchins. Strongylocentrotus purpuratus eggs were found to release H2O2 during the cortical reaction at fertilization. Treatment of sperm with equivalent concentrations of H2O2 resulted in a rapid loss of sperm fertilizing ability. Attempts were made to induce polyspermy by utilizing ovoperoxidase inhibitors at concentrations known to inhibit fertilization envelope hardening. Eggs fertilized in phenylhydrazine became polyspermic, while 3‐amino‐1,2,4‐triazole‐treated eggs did not. These data suggested that a sperm peroxidase might be involved in preventing polyspermy. This hypothesis was tested by the addition of phenylhydrazine or 3‐amino‐1,2,4‐trizaole to H2O2‐treated sperm. Phenylhydrazine acted to protect sperm fertility from H2O2, while 3‐amino‐1,2,4‐triazole increased the adverse effect of H2O2. Simultaneous addition of both inhibitors to sperm incubated in H2O2 gave an intermediate value of sperm fertility. These data indicate that (1) H2O2 generated by sea urchin eggs during the cortical reaction at fertilization is used for two separate processes, fertilization envelope hardening and the prevention of polyspermy; (2) ovoperoxidase is probably not involved in preventing polyspermy; and (3) egg‐derived H2O2 reacts directly with sperm enzymes to prevent polyspermy. The phenylhydrazine‐sensitive enzyme in the sperm is probably a peroxidase that acts to inactivate sperm, while the 3‐amino‐1,2,4‐triazolesensitive enzyme is probably a catalase which protects sperm from H2O2. This hypothesis is consistent with model experiments on horseradish peroxidase and bovine liver catalase.
Production, characterization, and use of ionophore‐induced, calcium‐dependent acrosome reaction in ram spermatozoa
Authors: Shams‐Borhan Gabriele , Harrison R. A. P.
Published: February 2005

A system has been developed for inducing a calcium‐dependent acrosome reaction in ram spermatozoa in vitro using the calcium ionophore A23187. The resultant reaction is accompanied by release of the acrosomal enzymes hyaluronidase and acrosin, but there is no release of the cytoplasmic enzyme glucose 6‐phosphate isomerase. In any given cell, the visible acrosome reaction apparently takes place rapidly, but there is a variable delay before the reaction occurs. Under optimum conditions, about 90% of treated spermatozoa show an acrosome reaction within one hour.

Preincubation of the spermatozoa with the proteinase inhibitors p‐amino‐benzamidine or p‐nitrophenylguanidinobenzoate allows two stages of the reaction to be distinguished ultrastructurally, a membrane fusion stage followed by a dispersal of the acrosomal matrix. In the presence of the inhibitors, the first stage is delayed but is completed within 1 hour, whereas the second remains largely incomplete.

In the presence of calcium, ionophore concentrations which induce an acrosome reaction abolish sperm motility rapidly and completely. However, by adding serum albumin shortly after addition of ionophore, motility can be preserved while the acrosome reaction occurs as usual; the motility pattern observed under these conditions is of the “whip‐lash” or “activated” type.

Although the motile ionophore‐treated spermatozoa were unsuccessful at penetrating normal mature sheep oocytes in vitro, they were able to penetrate zona‐free oocytes, after which swelling and decondensation of the sperm head took place.
Enrichment of primary pachytene spermatocytes from the human testis
Authors: Shepherd Robert W. , Millette Clarke F. , DeWolf William C.
Published: February 2005

Normal adult human testis has been separated using a combination of mechanical and enzymatic procedures to yield a suspension of viable single cells. The predominant cell types comprising this suspension are as follows: primary pachytene spermatocytes (7% of total cells), round spermatids (17%), residual bodies and condensing spermatids (31%), and Leydig cells (15%). Separated human germ cells viewed by Nomarski differential interference microscopy closely resemble mouse spermatogenic cells in relative size and appearance. Isolation of an enriched population of human pachytene spermatocytes has been achieved using unit gravity sedimentation (STA‐PUT) according to protocols originally developed for murine tissue. Pachytene cells are enriched to 75% and are contaminated only with Leydig cells and binucleated spermatid symplasts. Ultrastructural examination of isolated human pachytene spermatocytes indicates that these cells, as well as isolated round spermatids, exhibit a normal in situ morphology. Spermatocytes, for example, show numerous synaptonemal complexes, nuclear pores, annulate lamellae, and dictyosome‐like saccules. Round spermatids after isolation exhibit peripheral mitochondria, annulate lamellae, developing acrosomes, and other morphological features characteristic of early spermiogenesis. Therefore, enriched populations of human spermatogenic cells seem suitable for analysis using immunofluorescent, autoradiographic, or serological methods. In particular, isolated human spermatocytes should be useful for the analysis of molecular events involved in meiosis and should facilitate investigations concerning the pathophysiology of certain human infertility conditions.
Evidence for nonmuscle myosin in bovine ejaculated spermatozoa
Authors: Tamblyn Toby M.
Published: February 2005

Mammalian spermatozoa contain nonmuscle actin and many of the components of regulatory systems thought to be involved in nonmuscle actin‐myosin function. An actin‐stimulated adenosine triphosphate hydrolase (ATPase) activity has been fractionated from bovine ejaculated spermatozoa by immobilized‐actin affinity chromatography. The actinstimulated ATPase activity has a specific activity (0.04 ± 0.02 mM phosphate released/min/mg protein) similar to nonmuscle myosins from other mammalian cells and tissues, but it does not have appreciable K+‐EDTA ATPase activity. The sperm actin‐myosin may function in sperm morphogenesis in the seminiferous tubule, in capacitated spermatozoa undergoing an acrosome reaction, or in decondensation of the sperm nucleus after fertilization.
Stimulation of hamster sperm capacitation and acrosome reaction in vitro by glucose and lactate and inhibition by the glycolytic inhibitor α‐chlorohydrin
Authors: Dravland Eric , Meizel Stanley
Published: February 2005

Studies were made of the effects of D(+)‐glucose, L‐lactate and pyruvate on in vitro capacitation and acrosome reactions (AR) of hamster sperm using a more “defined” medium that that used in previous similar studies. In the absence of glucose or lactate, sperm underwent very few AR and activation (whiplash‐like motility characteristic of capacitated hamster sperm) was reduced compared to those events in sperm preincubated in the presence of glucose plus lactate plus pyruvate. Glucose and pyruvate supported more AR than glucose alone, but less than glucose, lactate, and pyruvate. The glycolytic inhibitor α‐chlorohydrin (10 μm) inhibited AR by 50% and reduced activation by less. When glucose was added to sperm incubated 2 hr with pyruvate and lactate, the number of AR observed after 4 hr was the same as that obtained when glucose was present throughout the incubation. When glucose was added after 3.5 hr, AR were delayed for 1 hr and lower numbers of sperm underwent AR. In the presence of lactate and pyruvate, 0.38 mM glucose was able to support activation and AR as well as 3.24 mM glucose. These results indicate that exogenous glucose and lactate are necessary for in vitro capacitation and AR of hamster sperm; only low levels of exogenous glucose are required; exogenous glucose is not required during the first 2 hr of capacitation; and glycolytic activity is necessary for capacitation and the AR.
Cross fertilization between sea urchin eggs and oyster spermatozoa
Authors: Osanai Kenzi , Kyozuka Kei‐ichiro
Published: February 2005

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.
Stimulation of endogenous protein phosphorylation in oocytes of Sabellaria alveolata (polychaete annelid) at meiosis reinitiation induced by protease, fertilization, or ionophore A 23187
Authors: Peaucellier Gérard , Doree Marcel , Demaille Jacques G.
Published: February 2005

Incorporation of [32P]‐phosphate into proteins was enhanced when Sabellaria oocytes were stimulated with specific protease to continue from prophase I block to metaphase I block. The rate of incorporation was increased 50 fold between onset of treatment and germinal vesicle breakdown (GVB). The same result was obtained when release from prophase block involved fertilization, or activation with ionophore A 23187. In all cases, meiosis was associated with phosphorylation of an 18,000 dalton protein, which is perhaps not labeled in prophase‐blocked oocytes. Phosphorylation of a 38,000–40,000 dalton doublet of membrane proteins, which are among the main phosphorylated proteins in intact oocytes, was also strongly enhanced in vitro in homogenates prepared from oocytes following release from prophase block.
Changes in the stainability and sulfhydryl level in the sperm nucleus during sperm‐oocyte interaction in mice
Authors: Miller Margaret A. , Masui Yoshio
Published: February 2005

During maturation in the epididymis, mouse sperm nuclei become difficult to stain with Giemsa and its component basic dyes. Mature sperm from the cauda epididymis can be stained only after DTT treatment. Stainable sperm such as those from the testis accumulate 3H NEM when examined by autoradiography, while unstainable sperm do not, indicating a close correlation between the basic dye binding capacity and SH levels in the sperm nuclei. During insemination of zonafree ovarian oocytes with a germinal vesicle (GV), mature sperm nuclei become stainable and capable of binding with 3H NEM. At the same time, sperm have established pronase‐resistant contact with the oocyte. Similarly, sperm nuclei become stainable during fertilization when the sperm attachment to the egg becomes pronase resistant. However, these changes occur before sperm chromatin decondensation begins. Therefore, it is suggested that S‐S bonds in sperm nucleoproteins are reduced when the sperm establish a stable contact with the egg plasma membrane, thus reversing sperm maturational changes. The reduction of S‐S bonds may be a prerequisite for sperm chromatin decondensation.
Chromatin transitions in the fertilizing sperm nucleus of the sea urchin, strongylocentrotus purpuratus
Authors: Kunkle Mel
Published: February 2005

Electron microscopic analysis of fertilization in the sea urchin, Strongylocentrotus purpuratus, has been carried out in an effort to establish the sequence of events involving dispersion of the paternal chromatin. Subsequent to loss of the nuclear envelope the condensed sperm chromatin begins to disperse under the influence of egg cytoplasmic factors. However, this process does not proceed at a uniform rate as is observed in other species examined to date. Portions of the paternal genome rapidly transform into dispersed chromatin while other adjacent regions disperse at a reduced rate. This variation in the time sequence of dissociation of the paternally derived chromosomes results in a reticulum of electron lucent and electron dense chromatin within the developing male pronucleus. As the paternally derived chromatin is dispersing and migrating centrad, membranous vesicles of maternal origin become aligned along the peripheral aspect of the chromatin. Deposition of a continuous bilaminar nuclear envelope around the dispersing sperm chromatin results in the formation of the definitive male pronucleus. At the time the male pronucleus is formed the paternally derived chromosomes have not completely dispersed and are visualized as a reticulum of condensed and dispersed chromatin. These results indicate that not all the paternally derived chromatin is modified in the same manner during pronuclear development.
The structure and formation of rolled and crested bull spermatozoa
Authors: Cran D. G. , Dott H. M. , Wilmington J. W.
Published: February 2005

The ejaculate of a Friesian bull contained three abnormalities: rolled, crested, and knobbed spermatozoa. The rolled form had marked lateral curvature of the head, occasionally forming a complete tube. All three forms were of testicular origin and it is likely that the first two were developmentally related.
Bioelectric responses at fertilization: Separation of the events associated with insemination from those due to the cortical reaction in sea urchin, Lytechinus variegatus
Authors: Hülser Dieter , Schatten Gerald
Published: February 2005

The bioelectric responses at fertilization of the sea urchin Lytechinus variegatus are a complex series of membrane potential and resistance changes that occur concomitant with gamete fusion, ionic fluxes, and the cortical granule discharge. This work attempts to separate the electrical effects of sperm‐egg interactions from those of the cortical reactions. Two approaches were taken to discern the electrical events associated with insemination, distinct from cortical granule discharge: (1) fertilization of eggs treated with 3% urethane, 10 mM procaine, or 10 mM nicotine, to prevent the cortical reaction and (2) refertilization of fertilized eggs (denuded with 1 mM aminotriazole containing 1 mg/ml soybean trypsin inhibitor). Cortical granule discharge in the absence of sperm incorporation was investigated by artificial activation with 5 μM A23187 or by fertilization in the presence of 10 μM cytochalasin D, which prevents incorporation.

These results are consistent with a model in which the sperm‐egg interaction triggers both a rapid (50–400 msec), but minor (≅10 mV), electrical transient that leads to an action potential and then both the Na+‐dependent fast block to polyspermy and the late block resulting from the secretion of the cortical granules.
Synthetic peptides are not chemoattractants for bull sperm
Authors: Miller Richard L.
Published: February 2005

Recent claims that bull sperm display chemotaxis to synthetic peptides known to be chemotactically active for neutrophils are not based on direct observation of sperm behavior. When these observations are made, no change in sperm motility or direction is seen. The supposed chemoattractant effect is probably based on disruption by the peptides of the sperm acrosome, resulting in increased sperm adhesion to the glass surface inside the pipette containing the supposed chemoattractive peptide.
Acquisition of zona binding by ram spermatozoa during epididymal passage, as revealed by interaction with rat oocytes
Authors: Fournier‐Delpech S. , Courtens J. L. , Pisselet C. L. , Delaleu B. , Courot M.
Published: February 2005

To assess the ability of ram spermatozoa to bind to oocytes, spermatozoa (2.5–200 × 106/500μ1) taken from the rete testis, or from various regions of the epididymis (head, body, and tail) were mixed with cumulus‐free heterologous oocytes obtained from immature superovulated rats. After incubation for 30–45 min in Parker 199 Hepes medium at 35°C, testicular spermatozoa were unable to bind to the zona at any of the concentrations used. However, spermatozoa from the middle body of the epididymus were able to bind to the zona and this binding reached a maximum in the distal body and in the tail of the epididymis. The spermatozoa were bound by their heads. Electron microscopy showed that the plasma membrane and the acrosome of the bound sperm remained intact, without any sign of an acrosome reaction.
Preimplantation losses of zygotes in superovulated immature rats
Authors: Sherman D. , Nelken Michal , Kraicer P. F.
Published: February 2005

The development of zygotes in immature, superovulated, and adult female rats was followed by flushing their oviducts and uteri on each of the days L1–L4 (LO is day of sperm finding). Superovulated rats showed a 3.6 times greater rate of preimplantation losses. The loss occurred between L1 and L2 in immatures and was restricted to some of the females; in matures the loss was between L3 and L4. In the immatures some zygotes were already in the uterus on L2. The loss of zygotes in the immature rats is attributed to accelerated transport leading to expulsion as well as lysis of some zygotes within the tract. These latter may be the zygotes identified as morphologicaly abnormal. It is speculated that PMSG may force the ovulation of unfit oocytes.
Postovulatory aging of human ova: I. Light microscopic observations
Authors: Ortiz M. E. , Salvatierra A. M. , López J. , Fernandéz E. , Croxatto H. B.
Published: February 2005

The morphologic changes undergone by the human secondary oocyte following ovulation were assessed by light microscopy in 57 specimens recovered from the Fallopian tube and endometrial cavity between 24 and 144 hr after the luteinizing hormone peak in plasma. Ovarecovered shortly after ovulation were surrounded by a large cumulus mass comprising approximately 20,000 follicular cells. Whenever it was possible to perform a detailed observation of the perivitelline space in these ova, the presence of a polar body was recognized. The oocyte usually occupied an excentric position within the cumulus. Ovum denudation appeared to proceed by breakdown of the cumulus into fragments and release of the oocyte with a small number of cells attached to the zona. As a consequence of this process the oocyte surrounded by a few layers of cells frequently coexisted with large fragments of the cumulus. Progress of ovum denudation was time dependent and proceeded at a relatively slow pace. Some uterine ova still had cells attached to the zona. At 96 hr after the LH peak 40% of the ova underwent fragmentation of the cytoplasm giving rise to anucleated pieces of varying sizes. The dimensions of the zona pellucida and ooplasm presented wide individual variations as well as some time related changes. The mean external diameter of the zona ± SD of 43 ova was 161.6 ± 14.6 μm.

The occurrence of denudation and cytoplasmic fragmentation were more clearly related to the postovulatory age of the ovum than to the site of recovery. The rate of denudation of human oocytes seems to proceed at a much lower speed in comparison with small mammals currently used as laboratory animals.
Insulin induction of meiosis of rana pipiens oocytes: Relation to endogenous progesterone
Authors: Lessman Charles A. , Schuetz Allen W.
Published: February 2005

The follicle wall was previously shown to be involved in insulin induction of oocyte maturation in Rana pipiens ovarian follicles. Steroidogenic involvement in insulin induction of maturation was investigated following development of a radioimmunoassay (RIA) for progesterone to measure endogenous progesterone associated with in vitro incubates. Insulin and frog pituitary homogenate (FPH) were both found to elevate progesterone levels significantly in these incubates. FPH was more effective in elevating progesterone levels than insulin and caused progesterone increase of about 2 orders of magnitude greater than insulin. Removal of the follicle wall eliminated the steroidogenic effects of insulin. Considerable interanimal variation was observed in the ability of insulin to induce oocyte germinal vesicle breakdown (GVBD) in intact follicles. The hypothesis was proposed that differences in endogenous progesterone might explain this variation. To test this hypothesis, an experiment was carried out in which hormone production and follicular sensitivity to insulin were simultaneously determined in follicles obtained from the same animals. Results of the experiment show that the ability of insulin to induce GVBD, as indicated by the effective concentration needed for 50% response (ED50), was strongly correlated with the levels of endogenous progesterone as measured by RIA. The results provide direct evidence that insulin's action on the follicle wall involves steroid production. It was thus concluded that increased endogenous progesterone facilitates GVBD induction by insulin. It is unclear how the two hormones interact to produce an enhanced effect, but interactions at the receptor or postreceptor level may be involved. This follicle system may provide important insights into the mode of action and interaction of these two important hormones.
Preimplantation embryo development in the mouse: Role of histidine decarboxylase
Authors: Hudgins Louanne , Mukerjee S. , Dey S. K.
Published: February 2005

The present study determines the effect of a specific and an irreversible inhibitor of histidine decarboxylase (HDC), α‐fluoromethylhistidine (α‐FMH) on the mouse preimplantation embryo development in vitro. The embryo culture technique was used to assess the effect of α‐FMH. Embryos recovered at 0800–0900 hr (AM) on day 3 of pregnancy were 4–8 cells, whereas those recovered at 1600–1630 hr were mostly 8‐cell compacted embryos. Of the day 3‐AM embryos, 81.3 ± 4.3% developed to blastocysts within 48 hr when cultured in the medium alone, but addition of α‐FMH (0.19 or 0.38 mM) drastically reduced the blastocyst formation to 26.6 ± 7 or 16.8 ± 4.3%. Most of them were arrested before the compaction stage. Addition of L‐histidine, the substrate for HDC, did not alter the inhibition of blastocyst formation in the presence of α‐FMH (37.2 ± 10.9%). Of the day 3‐PM embryos, 99.3 ± 0.7% developed to blastocyst stage when cultured in the medium alone and addition of α‐FMH (0.19 or 0.38 mM) did not affect the embryo development (92.1 ± 4.3 or 81.9 ± 9.9% developed to blastocysts). The birth of healthy young following transfer of these blastocysts into pseudopregnant mice indicates normal development of the embryos under this condition. The results suggest that histamine synthesis may be required for the process of compaction and thus the formation of blastocyst.
The mechanism of cumulus cell‐oocyte uncoupling: Evidence for the participation of both cumulus cells and oocytes
Authors: Eppig John J. , Ward‐Bailey Patricia F.
Published: February 2005

Cumulus cells are metabolically coupled to oocytes via heterologous gap junctions. This coupling terminates near the time of ovulation, and the termination appears to be correlated with the mucification of the cumulus cells lying immediately adjacent to the oocytes. The first objective of this project was to determine whether follicle stimulating hormone (FSH) induction of cumulus cell‐oocyte uncoupling could occur independently of FSH‐stimulated cumulus mucification (expansion). Intercellular coupling was measured as a percentage of radiolabeled choline (or its metabolites) that was incorporated into the oocyte relative to the total amount of radiolabel incorporated into the entire cumulus cell‐oocyte complex. It was found that the complete suppression of FSH‐stimulated cumulus expansion with chondroitin sulfate B had no suppressive effect on FSH‐stimulated cumulus cell‐oocyte uncoupling. This finding showed that FSH‐stimulated cumulus expansion was not required for cumulus cell‐oocyte uncoupling. Since 17β‐estradiol, testosterone, or progesterone could not induce maximal cumulus cell uncoupling, it was concluded that the uncoupling‐promoting action of FSH was probably not mediated by steroid hormones.

A partial uncoupling of cumulus cells and oocytes was found when spontaneous oocyte maturation had occurred in the absence of FSH. This partial uncoupling was prevented by incubation of cumulus cell‐oocyte complexes in concentrations of dibutyryl cyclic adenosine monophosphate (dbcAMP) or 3‐isobutyl‐1‐methyl xanthine (IBMX) (0.25 and 0.10 mM respectively) that suppressed spontaneous oocyte maturation without inducing cumulus expansion. These inhibitors also prevented the maximal induction of uncoupling that would have been provoked by biological grade preparations of either FSH or luteinizing hormone (LH). It was concluded that two factors were required to bring about maximal cumulus cell‐oocyte uncoupling: one factor was dependent upon the action of gonadotropins on cumulus cell function, the other factor appeared to be a function of the oocytes, since maximal uncoupling could occur only after the germinal vesicles had broken down.
Structural characterization of isolated rat epididymal epithelial cells
Authors: Olson G. E. , Jonas‐Davies J. , Hoffman L. H. , Orgebin‐Crist M.‐C.
Published: February 2005

An enriched suspension of rat epididymal epithelial cells was prepared by sequential enzymatic removal of connective tissue and peritubular cells from the epididymal tubule. The viability, structural characteristics, and pattern of polypeptides synthesized by the isolated cells were determined. Electron microscopic analysis revealed that the isolated principal cells were intact and retained their polarized morphology. Several light microscopic protocols were employed to evaluate the percentage of epithelial cells in the suspensions. These included (1) the visualization of the pattern of FITC‐lectin binding in which the principal cells could be identified by their polarized fluorescence; (2) the visualization of prominent autofluorescent granules in the cell cytoplasm which appeared to be characteristic of only epithelial cells; and (3) immunochemical staining with an antikeratin antibody which was reactive only with cells of epithelial origin. These structural probes indicated that between 80% and 90% of the isolated cells were epithelial in nature. Two‐dimensional gel electrophoresis revealed a complex pattern of polypeptides synthesized by the epithelial cells; these results are compared to those of earlier studies utilizing minced whole epididymis.
Analysis of preimplantation golden hamster conceptuses resulting from spermatozoa aged in utero
Authors: Bell Cindy L. , Shaver Evelyn L.
Published: February 2005

The effects of aging golden hamster spermatozoa in the female reproductive tract on the percentage of ova fertilized, the stage of development, and the chromosome complement of the resulting zygotes were studied. Females were inseminated artificially with cauda epididymal sperm at 6 h (control), 10, 15,18, or 21 h before the estimated time of ovulation. A decrease in the percentage of ova fertilized was found as the time spermatozoa were aged in utero prior to ovulation increased. The zygotes collected at 56 h postovulation from females inseminated 15 or 18 h prior to ovulation were delayed in development, as judged by the number of blastomeres. Although an increase of chromosomally abnormal zygotes was not found, a possibility exists that mosaicism may have been present, as evidenced by zygotes with unequal sized blastomeres, and went undetected.
Sea urchin cleavage is inhibited by ozone
Authors: Acevedo Mildred E. , Carroll Edward J. , Heath Robert L.
Published: February 2005

The first cell division of Strongylocentrotus purpuratus embryos which normally occurs at 84 min postinsemination (at 17°C) was specifically blocked by exposure to small amounts of ozone at 30 min postinsemination. Exposure of embryos to the same ozone level at 60 min postinsemination was teratogenic but did not block cell division. Previous work [Sweet et al, Science 209:931–933, 1980] using normal and several cancerous human cell lines showed that ozone blocked growth and proliferation; the present work shows that ozone specifically inhibits cleavage of sea urchin embryo cells that do not grow during the period of early development.
Detection and kinetics of the normal acrosome reaction of mouse sperm
Authors: Dudenhausen Elizabeth , Talbot Prudence
Published: February 2005

A procedure is described for quantifying the percentage of normal acrosome reactions in fixed mouse sperm. This technique, which is a modification of the triple stain technique (TST) devised for human sperm [Talbot and Chacon, 1981a, b], utilizes trypan blue exclusion to identify the percentage of dead sperm in culture and subsequent staining of fixed sperm to differentiate the acrosome. Four classes of mouse sperm can be distinguished with the TST: (1) “live,” unreacted sperm, (2) “live,” acrosome‐reacted sperm (normal reactions), (3) dead, unreacted sperm, and (4) dead, acrosome‐reacted sperm (degenerative acrosome reactions). We have used this technique to study the time course of the normal acrosome reaction of mouse sperm in vitro. Our results with TST show that a low percentage of mouse sperm undergo normal acrosome reactions during the first 30 min of incubation; a maximum level of reactions is reached by 2 hr. Calcium was required in the medium for occurrence of normal reactions.