bluuebunny/crossref_metadata_2025 huggingface dataset:
Analytical subcellular distribution of cAMP‐dependent protein kinase activity in bull sperm
Authors: Pariset Claude C. , Weinman Jacqueline S. , Escaig Francoise T. , Guyot Michele Y. , Iftode Francine C. , Weinman Serge J. , Demaille Jacques G.
Published: February 2005

The distribution of the cAMP‐dependent protein kinase activity in bull ejaculated sperm has been investigated. This activity proved to be mainly present in a soluble form inside the cell. Sperm fractionation into heads and flagellar fragments, using differential centrifugation or centrifugal elutriation, has shown that the particulate cAMP‐dependent protein kinase activity was mainly associated with the flagellar structures. A much activity was shown to be associated with the head fraction. Some activity could also be detected in the purified plasma membrane fraction.
The effects of short−term incubation (aging) of mouse oocytes on in vitro fertilization, zona solubility, and embryonic development
Authors: Gianfortoni Joseph G. , Gulyas Bela J.
Published: February 2005

The effects of in vitro aging of cumulus−intact versus cumulus−free metaphase II mouse oocytes were studied with respect to zona solubility and fertilization rates. Furthermore, zygotes from the in vitro fertilization studies were incubated and their developmental progress was recorded. The zona pellucida showed a gradual increase in resistance to dissolution by α−chymotrypsin with in vitro aging over a period of 6 hr. This effect was greater in cumulus−free as compared to cumulus−intact ova, but it was not nearly as profound as that seen in the control in vivo fertilized eggs. The fertilization rate of in vitro aging cumulus−intact ova compared favorably with the control in vivo aging group over a 6−hr time period. This was in sharp contrast to the decreased fertilization rate of in vitro aging cumulus−free ova over the same period of time. Lastly, development of zygotes to the blastocyst stage was also evaluated. The rate of first cleavage was similar in all experimental groups and compared favorably with the in vivo controls. However, further development to blastocysts of in vitro aged cumulus−free ova showed a marked decrease when compared to the cumulus−intact group and the in vivo fertilized controls. Thus we established a direct relationship between zona digestion time of in vitro aged cumulus−free oocytes and a decrease of fertilization rates in the mouse.
Scanning electron microscope study of in vitro prepenetration gamete interactions
Authors: Jedlicki A. , Barros C.
Published: February 2005

Hamster and mouse capacitated spermatozoa were interacted in vitro with hamster and mouse eggs in homologous and heterologous combinations. Also, fertilized and trypsin treated unfertilized hamster eggs, and unfertilized rat eggs were made to interact with capacitated hamster spermatozoa. The surface of the zona pellucida was then examined with the scanning electron microscope. It was found that sperm attachment, followed by sperm binding and penetration through the zona pellucida, was observed only when homologous gamete combinations were used. Binding of the spermatozoa to the zona was evidenced by the lytic effect of the acrosomal enzymes on the zona substance. When fertilized eggs and trypsin‐treated unfertilized hamster eggs were mixed with capacitated hamster spermatozoa as well as in the heterologous gamete combinations, we found that the spermatozoa were able to establish attachment but not binding. Under these conditions the outer surface of the zona pellucida was never found to have penetration tracks made by the spermatozoa. Failure of heterologous spermatozoa to cross the foreign zona pellucida is believed to be associated with the inability of the foreign spermatozoa to establish binding and to the inability of their acrosomal enzymes to digest the zona. A similar mechanism is believed to work in zona‐reacted and in trypsin‐treated hamster eggs inseminated in vitro with homologous spermatozoa.
Estradiol synergism and inhibition of insulin‐induced meiotic maturation in Rana pipiens oocytes in vitro
Authors: Lessman Charles A. , Schuetz Allen W.
Published: February 2005

Estradiol 17‐β (E2) was found to either inhibit or synergize Na‐insulin (Ins)‐induced meiotic maturation of Rana oocytes. Inhibition of Ins activity occurred in the presence of the follicular investments of the oocyte; synergism with Ins occurred in oocytes denuded of the follicle wall. Similarly, co‐incubation of E2 with frog pituitary homogenate (FPH) or pregnenolone (Pe) significantly decreased meiotic reinitiation as determined by germinal vesicle dissolution (GVD) in follicle‐enclosed oocytes. By contrast, E2 had no consistently significant effect on progesterone (P)‐induced meiosis in follicle‐enclosed oocytes. Furthermore, E2 had no significant effect, either inhibitory or synergistic, on Pe‐ or P‐induced GVD of denuded oocytes. Thus, of the meiotogens tested (Ins, P, Pe, FPH), all but P were consistently inhibited by E2 in the presence of the follicle wall. Na‐insulin was the only meiotogen tested (Ins, P, Pe) which was potentiated by E2 in denuded oocytes, However, when E2 and Ins were spatially separated on the surface of individual intact follicles, the result was synergism of Ins‐induced GVD rather than inhibition. These results suggest that Ins acts to induce GVD in the denuded oocyte through a mechanism distinct from that used by P (ie, Ins mechanism allows E2 synergism while the P mechanism does not). The E2 inhibitory effect on Ins‐induced GVD appears to be dependent upon simultaneous exposure of follicle wall tissue to mixtures of E2 and Ins. The synergistic effect of E2 on Ins‐induced GVD is dependent upon the simultaneous exposure of the oocyte surface to Ins and E2, either as a homogenous mixture in the case of denuded oocytes or as single substances at independent sites, for follicle‐enclosed oocytes.
Immunological approach to the characterization of the outer acrosomal membrane of boar spermatozoa
Authors: Hinrichsen Adriana C. , Töpfer‐Petersen Edda , Dietl Thomas , Schmoeckel Christian , Schill Wolf‐B.
Published: February 2005

Antiserum to purified boar spermatozoan outer acrosomal membrane (OAM) was raised in rabbits and adsorbed with boar liver and serum glutaraldehyde cross‐linked immunoadsorbents. The IgG fraction of the antiserum was purified by (NH4)2SO4 precipitation followed by ion‐exchange chromatography. Indirect immunofluorescence showed bright fluorescent staining of the acrosomal cap of boar spermatozoa and to a lesser extent of the acrosomes of bull and goat spermatozoa after incubation with anti‐OAM‐IgG. Immuno‐electron microscopy further confirmed the specificity of the antibody for the OAM. Preincubation of the anti‐OAM‐IgG with isolated OAM, completely abolished its reactivity. When tested by ELISA, anti‐OAM‐IgG reacted with boar, bull, goat, and human spermatozoa; however, its binding activity to boar spermatozoa was significantly greater as compared to spermatozoa from the other species tested.

In an effort to identify OAM antigens recognized by this antiserum, the isolated boar OAM was labeled either with 3H or with 125I and solubilized by mild detergent treatment. The extracted components were immunoprecipitated with anti‐OAM‐IgG and protein A‐bearing S. aureus and the thus isolated antigens were analysed on SDS‐PAGE. The results suggest that anti‐OAM‐IgG recognized one high molecular 3H‐labeled glycoprotein (270 kd), and four 125I‐labeled polypeptides of lower molecular weight of the boar OAM.
Exocytosis of glycogen during maturation of amphibian oocytes
Authors: Taghy‐Sadak Zoubida , Vilain Jean‐Pierre
Published: February 2005

The repartition and fate of glycogen β has been followed during progesterone‐induced maturation of amphibian oocytes. The use of specific staining, both at the cytological and ultrastructural level, demonstrates that glycogen tends to be extruded from the oocyte during maturation of the urodeles Pleurodeles waltlii and Ambystoma mexicanum. No such effect of the hormone is observed in Xenopus laevis, where only a slight centrifuge migration of the glycogen could be recorded.

Stacks of annulate lamellae increase during the early phase of in vitro progesterone‐induced maturation (2 to 9 hours after progesterone application). After germinal vesicle breakdown (about 12 hours after beginning the progesterone treatment) annulate lamellae have disappeared and numerous masses of vesicles are present in the cytoplasm of Pleurodeles and Ambystoma matured oocytes. We never observed any close relation between the annulate lamellae and these vesicles.
Human sperm chromosomes obtained from hamster eggs after sperm capacitation in TEST‐yolk buffer
Authors: Brandriff B. , Gordon L. , Watchmaker G.
Published: February 2005

Human sperm chromosomes were obtained after capacitation with TES‐Tris (TEST) yolk buffer and fusion with Syrian hamster eggs. Semen samples could be stored at 4°C for 3 days and remain functional in the assay system. The efficiency of TEST yoik buffer for obtaining karyotypes was as good as, or greater than, the efficiency of standard BWW medium containing human serum albumin.
Influence of uterine secretions on the chromatin of ram spermatozoa at different stages of maturation: Cytophotometric study of Feulgen‐DNA after in vitro incubation
Authors: Nicolle J. C. , Fournier‐Delpech S. , Courot M.
Published: February 2005

Ram spermatozoa taken from the epididymal head, body, or tail or from the ejaculate were examined by microspectrometry after incubation in vitro with ewe uterine fluids at 37°C for 20 hours. Compared with incubation in Ringer's solution, uterine fluid incubation resulted in a decrease in nuclear Feulgen‐DNA content. This decrease was greater for more immature spermatozoa (29.0 and 47.3% for spermatozoa from head and body, respectively) than for more mature spermatozoa (17.7 and 4.0% for spermatozoa from the tail and the ejaculate, respectively). In parallel with this decrease, there was a condensation of the chromatin which resulted in a decreased nuclear surface area, especially in spermatozoa taken from the epididymal body. Therefore, it would appear that, during epididymal maturation, changes in the ability of spermatozoa to maintain embryonic development as the spermatozoa mature are due to changes in chromatin structure.
The retention of 125I‐labeled plasma membrane proteins in bovine spermatozoa cryopreserved in egg‐yolk citrate extender
Authors: Zafian P. T. , Amann R. P.
Published: February 2005

Cryopreservation of bovine sperm in egg‐yolk citrate extender (EYC) usually maintains fertility. Since plasma membrane proteins are important for the fertilizing potential of sperm, the possible loss of membrane proteins from sperm subjected to cryopreservation in EYC was evaluated. Sperm were washed and labeled with 125I without significantly reducing motility. Radiolabeled sperm were a) held for 2 hr at 22°C in N‐2‐hydroxyethylpiperazine‐N'‐ethanesulfonic acid (HEPES)‐buffered saline containing 1% polyvinyl alcohol, b) cooled to 5 °C in glycerol‐free EYC and held for 3 hr, or c) frozen‐thawed in EYC containing 7% glycerol. Sperm were solubilized and proteins were separated by electrophoresis under denaturing conditions. Freeze‐thawing dislodged most egg‐yolk proteins from the spermatozoal plasma membrane that were bound to and retained by sperm that only were cooled to 5 °C. Autoradiography resolved 11‐18 bands of 125I polypeptides. There was no difference (P > 0.05) in the amount of 125I protein retained by frozenthawed and cooled sperm. However, the radioactivity in two polypeptide bands (MW = 105 K and 24.2 K) was less (P < 0.05) for sperm held at 22 °C in HEPES‐buffered saline. Thus, holding sperm in buffered saline at 22 °C resulted in a greater loss of 125I proteins from the plasma membrane than did cryopreservation of sperm in EYC. Cryopreservation did not induce greater loss of 125I proteins from the plasma membrane than simply cooling sperm to 5 °C in EYC.
The change in the nucleus pattern during chromatin condensation in Eurygaster integriceps spermatids
Authors: Danilova L. V. , Gaber E. S. , Shinyaeva L. I.
Published: February 2005

When spermiogenesis of Eurygaster integriceps occurs, the pattern of the round nucleus of the spermaiid changes under the influence of a manchette consisting of two large groups of microtubules (up to 130–140 in each). The pericentriolar matter was found to be involved in the changes in the nuclear pattern; it takes part in the formation of the asymmetric intranuclear channel, open at one side. Two zones appear in the nucleus that differ greatly in their rates of chromatin condensation. The two main parts of the future nucleus are thereby established: the apical one with a greater rate of chromatin condensation, which gives origin to the two‐walled cylinder, and the outer part, which grows for a long time and extends caudally to the middle piece. At the later stages of nucleus formation, when its larger part is separated from the surface of the middle piece, the vesicles of the endoplasmic reticulum that penetrate between the Nebenkern and the larger part of the nucleus, play a certain role.
Interaction of human spermatozoa with the human zona pellucida and zona‐free hamster oocyte following capacitation by exposure to human cervical mucus
Authors: Gould John E. , Overstreet James W. , Hanson Frederick W.
Published: February 2005

The functional capacity for sperm interaction with the human zona pellucida and zona‐free hamster oocyte was tested after human spermatozoa were capacitated by passage through a column of human cervical mucus. The results were compared with those obtained when spermatozoa from an aliquot of the same semen sample were capacitated by the standard laboratory methods involving sequential washing by dilution and centrifugation of the semen. Washed‐capacitated sperm suspensions were more successful than mucus‐capacitated sperm in attaching to the zona‐free hamster oocyte and in fusing with its oolemma. However, the ability of mucus‐capacitated sperm to penetrate the human zona pellucida was equal to washed capacitated sperm. These experiments demonstrate an approach that may be useful in comparative studies of human sperm capacitation in vivo and in vitro.
Ultrastructural organization of heterochromatin within sea urchin sperm nuclei
Authors: Aboukarsh Nanma , Kunkle Mel
Published: February 2005

Chromatin within swollen or lysed isolated sperm nuclei of the sea urchin, Strongylocentrotus purpuratus, was examined by electron microscopy. Spread preparations of lysed sperm nuclei demonstrated dense aggregates of nondispersed material and beaded filaments radiating from these aggregates. These beaded fibers are similar in size and appearance to the “beads‐on‐a‐string” seen as characteristic of chromatin spreads from numerous interphase nuclei. The beads are nucleosomes that have an average diameter of 130 Å. The interconnecting string is 40 Å indiameter and corresponds to the spacer DNA. In thin sections of swollen nuclei the sperm chromatin appears to be composed of 400 Å superbeads that are closely apposed to form 400 Å fibers. As the chromatin disperses, the superbeads are seen to be attached to one another by chromatin fibers of 110 Å diameter. In thin sections, the 400 Å superbeads appear to disperse directly into the 110 Å fibers with no intervening structures. This work demonstrates that the heterochromatin in Strongylocentrotus purpuratus sperm nuclei is composed of nucleosomes that form 100 Å filaments that are compacted into 400 Å superbeads. The superbeads coalesce to give the morphological appearance of 400 Å fibers.
Influence of hormones on the inhibitory activity of oocyte maturation present in conditioned media of porcine granulosa cells
Authors: Anderson Larry D. , Stone Sarah Lipford , Channing Cornelia P.
Published: February 2005

This study examines the influence of follicular maturation as well as the role of various hormones upon the secretion of an oocyte maturation inhibitor (OMI) from porcine granulosa cells incubated in vitro. The results demonstrate that the OMI substance, secreted into the media by granulosa cells, is present in a low molecular‐weight fraction (< 10,000 daltons) similar to that found in follicular fluid of porcine antral follicles. Also, as follicular development progresses, the granulosa cells lose their ability to secrete OMI. More importantly, hormones appear to regulate OMI secretion: FSH stimulates OMI secretion and androgens inhibit OMI secretion. These data provide evidence for the proposal of the following hypothesis concerning hormonal regulation of oocyte, meiosis by OMI in the porcine follicle: Whether the oocyte resumes meiosis, either during atresia or ovulation, is dependent upon the proper milieu of gonadotropins, cyclic‐AMP, and steroids within the microenvironment of the follicular compartment. The cellular interactions of these hormones, particularly FSH and androgens, control the amount of OMI (and possibly other intrafollicular factors) secreted in the follicle, which may be involved in either maintaining the immature state or permitting meiotic maturation.
Development of membrane sensitivity to the parthenogenetic agent calcium ionophore A23187 during meiotic maturation in the hamster oocyte
Authors: Eusebi Fabrizio , Salustri Antonietta
Published: February 2005

The response to the parthenogenetic agent Ca‐ionophore A23187 was studied in hamster oocytes undergoing meiotic maturation, by using electrophysiological techniques. Following germinal vesicle breakdown, the activating agent induces a long‐lasting hyperpolarization accompanied by an increased membrane conductance. The duration of the response progressively shortens during the long metaphase I stage. Terminal metaphase I oocytes respond to A23187 by a hyperpolarization that is very similar to that seen in metaphase II oocytes. The ionic mechanism of the change in the membrane sensitivity to A23187 during meiotic maturation is discussed.
Intracellular localization and surface expression of a stage‐specific embryonic glycoprotein
Authors: Polak‐Charcon Sylvie , Calarco‐Gillam Patricia , Johnson Lincoln V.
Published: February 2005

The intracellular and cell surface localization of an embryonic glycoprotein antigen (BL) has been investigated in preimplantation mouse embryos using ultrastructural immunocytochemistry. Several interesting points have emerged: (1) BL antigens are exclusively localized subjacent to the plasma membrane in the cortical region of cells, whereas antigens detected by a control antibody against mouse L cells are distributed throughout the embryo. (2) The distribution of BL antigens is polarized beginning with the first cleavage, with expression confined to the cortex underlying the free or apical portions of cells. No antigen is present underlying regions of cell contact. (3) Although embryonic synthesis of BL antigens does not begin until the two‐cell stage, BL antigens are observed in unfertilized eggs, a fact verified by immunoblotting.
Transmission electron microscopy of impaired spermatogenesis in an avian hybrid
Authors: Swan M. A.
Published: February 2005

The mechanisms involved in the impaired spermatogenesis of male goldfinch x canary hybrids were investigated by transmission electron microscopy and compared with spermatogenesis in the testes of the parent species.

In the parent species the testes were of normal structure, with the only unusual observation being that the Sertoli cells were variable in cytoplasmic electron density. In hybrid birds the Sertoli cells were either electron dense or electron lucent with respect to both nucleus and cytoplasm.

In the hybrids examined in this study, no spermatozoa were produced. Spermatogenic stages were arrested without formation of synaptonemal complexes. Centrioles were abnormally arranged in both somatic and germ cells. When they moved away from the basement lamina the germ cells degenerated and were phagocytosed. No focal tight junctions were present between Sertoli cells overlying what would normally have been the basal compartment of the tubule. The basement lamina was unusually thickened, peritubular cells were abnormal in structure, and numerous plasma cells were present in the interstitial tissue.

The observations reported here suggest that there was an immunological basis for degeneration of germ cells in the hybrid testis.
Characterization of paracrystalline inclusions in chinese hamster oocytes and early embryos
Authors: Parkening T. A. , Payer A. F. , Given R. L.
Published: February 2005

Paracrystalline inclusions, readily visible with light microscopy, were found to be present in ovarian oocytes (beginning with unilaminar follicles), ovulated ova, and preimplantation embryos of the Chinese hamster. These inclusions appeared to be aggregates of finer filamentous structures visible only with electron microscopy. The use of various histochemical techniques suggested that the paracrystals were composed of protein with little or no lipid associated with them. Autoradiographic studies using 3H‐uridine and enzymatic studies did not support the hypothesis that the paracrystalline inclusions were composed of RNA masked by a crystalline protein lattice.
The involvement of methyl transfer reactions and S‐adenosylhomocysteine in the regulation of bovine sperm motility
Authors: Goh Pauline , Hoskins Dale D.
Published: February 2005

The methylation of proteins and phospholipids has been demonstrated in mammalian sperm. The role of these methylation reactions in the regulation of sperm motility is, however, unclear. A combination of agents [adenosine, L‐homocysteine and erythro‐9‐(2‐hydroxyl‐3‐nonyl) adenine (EHNA)] known to increase the level of S‐adenosylhomocysteine (AdoHcy), a competitive inhibitor of S‐adenosylmethionine (AdoMet)‐mediated methylations, has been shown to inhibit rabbit sperm motility. The level of AdoHcy in response to these agents has, however, not been measured. Therefore, enzymatic protein carboxylmethylation (PCM) and the consequences of its inhibition by elevated intrasperm AdoHcy levels on motility were studied using bovine sperm. Incubation of bovine cauda epididymal sperm with L‐[methyl‐3H]methionine resulted in a linear increase in PCM for up to 2 h. Treatment of sperm with adenosine (0.5 mM), L‐homocysteine (0.5 mM), and EHNA (0.25 mM) produced a dramatic increase in AdoHcy levels after 15 min and a 97% inhibition of PCM after 0.5 h, followed by an inhibition of sperm motility (58% and 97%) after 1 and 2 h. Similar but less marked results were obtained with L‐homocysteine (0.5 mM) alone. Individually, cycloleucine (20 mM), an inhibitor of AdoMet synthetase, and adenosine (0.5 mM) also inhibited PCM (87% and 70%, respectively) after 2 h but had no effect on sperm motility or AdoHcy levels. These studies show for the first time that agents that elevate AdoHcy levels inhibit sperm motility. This, however, leaves open the question of whether protein carboxylmethylation or other methyl transfer reactions, eg, involving phospholipid, might be critical to motility inhibition by AdoHcy. Thus, the mechanism by which AdoHcy inhibits sperm motility remains unclear.
A rat oocyte differentiation antigen (OA‐1) detected by a monoclonal antibody
Authors: Banka Carole L. , Unger Michael W. , Dulbecco Renato , Erickson Gregory F.
Published: February 2005

Cell surface antigenic changes associated with differentiation of the rat oocyte and early embryo have been demonstrated with a monoclonal antibody (anti‐OA‐1). Antigen is first detectable coincident with initiation of oocyte growth, is a constant feature of all growing oocytes and displays a redistribution during meiotic maturation. Following fertilization, antigen is detectable on the surface of the embryo through the four‐cell stage. This first monospecific marker for the rat oocyte and embryo should prove useful in probing structure/function relationships in oocyte growth, meiotic maturation fertilization, and/or early embryonic development.
Initiation of cleavage in starfish eggs by the injection of triton‐treated spermatozoa
Authors: Yamada Hisafumi , Hirai Setsuro
Published: February 2005

Triton X‐100‐treated spermatozoa were injected into immature (fully grown, germinal vesicle stage) or mature (pronuclear stage) oocytes of the starfish, Asterina pectinifera, to study relation between initiation of cleavage and cortical reaction. Immature oocytes into which Triton X‐100‐treated spermatozoa were injected were treated with 1‐methyladenine. Such immature oocytes initiated cleavage after completion of meiosis without formation of the fertilization membrane. The same results were obtained when Triton X‐100‐treated spermatozoa were injected into mature oocytes. Control oocytes into which only calcium‐free sea water was injected did not cleave. These results indicate that the initiation of cleavage is independent of the cortical reaction but dependent on the existence of spermatozoa (spermatozoon) in the egg cytoplasm.
An improved method for isolation of fertile zona‐free mouse eggs
Authors: Boldt Jeffrey , Wolf Don P.
Published: February 2005

Studies of sperm‐egg fusion using zona‐free mouse eggs are impaired by the procedures used for removal of the zona pellucida. Methods involving proteolytic digestion or mechanical aspiration through micropipettes are limited in that proteases can adversely affect fertility and mechanical removal often results in low egg yields. An efficient procedure for preparation of zona‐free mouse eggs was developed using a combined enzymatic (chymotrypsin) mechanical approach (CT‐M procedure). Zona‐intact eggs, obtained after hyaluronidase treatment, were exposed to 0.001% α‐chymotrypsin in medium containing 0.5% bovine serum albumin (BSA). Brief (2 minute) exposure to chymotrypsin under these conditions caused pronounced zona distention in a majority (80‐90%) of the eggs, facilitating mechanical removal and resulting in a high yield of zona‐free eggs. Eggs prepared by the CT‐M method displayed identical penetration levels relative to mechanically denuded eggs. CT‐M prepared eggs also showed sperm concentration dependent penetration levels and demonstrated a plasma membrane block to polyspermy, qualities previously observed in mechanically prepared eggs [Wolf DP, 1978, Dev Biol 64:1–10]. Eggs could be exposed to 0.001% CT for zona distention over a 2‐10‐minute time period with no detrimental effects on fertility. The effect of chymotrypsin was also studied by treating zona‐free eggs for 30 minutes over a 1‐1,000–μg/ml range of enzyme, and a concentration‐dependent reduction in penetration levels was observed. These results indicate that the CT‐M method is a useful procedure for the isolation of large numbers of zona‐free mouse eggs.
Cytoplasmic deletion processes during spermatogenesis in mosses
Authors: Miller C. C. J. , Duckett J. G.
Published: February 2005

Comparative ultrastructural observations reveal that cytoplasmic deletion during spermatogenesis inSphagnumand other mosses (Bryopsida) has two distinct phases. In young spermatids, Golgi‐derived vesicles produce the mucopolysaccharide sheaths in which the gametes are liberated. Golgi bodies, however, play no part in removal of cytoplasm during gamete maturation. Rounding off of the cells during this process results in a 50% reduction in volume.

Mid‐spermatid stages inSphagnumare characterised by the sequential loss of Golgi bodies and endoplasmic reticulum (ER) but no further diminution of the cytoplasm. The final stages of nuclear metamorphosis and chromatin condensation, in late spermatids, are marked by the sudden appearance, in the otherwise featureless central cytoplasm, of a membrane vesicle complex (MVC) comprising cisternae, tubules, and smooth and coated vesicles. Following repositioning of the MVC beneath the plasma membrane, rapid shrinkage of the cytoplasm is associated with the presence of vesicle fusion profiles at the cell surface. The MVC is considered to be intimately involved in cytoplasmic breakdown and loss. Acid phosphatase activity can be detected throughout spermatogenesis. Spermatogenous cells and young spermatids possess relatively low levels of the enzyme, restricted to the ER and perinuclear space, but particularly high levels occur in the MVC region of late spermatids ofSphagnum.

The deletion process in Bryopsida is much more gradual than that ofSphagnum. Mid‐spermatids contain sheets of ER, Golgi with small vesicles, and irregular cisternae associated with coated vesicles. Vacuoles derived either from dilation of the ER or the coated vesicle complexes gradually increase in size and number at the expense of the cytoplasm. During the early stages of chromatin condensation, a large central vacuole opens onto the anterior face of the gametes. Further discharge of vesicles continues throughout gamete maturation.

A comparative survey of spermatogenesis in land plants indicates that cytoplasmic deletion is achieved in different ways in different groups. We speculate that the spermatozoids of the common ancestor of archegoniate plants probably possessed large amounts of cytoplasm. The deletion mechanisms may have originated from a contractile vacuole apparatus.
Effect of Ca2+ channel antagonists on the acrosome reaction of guinea pig and golden hamster spermatozoa
Authors: Roldan E. R. S. , Shibata S. , Yanagimachi R.
Published: February 2005

The effects of Ca2+ channel antagonists on the motility and acrosome reaction of guinea pig spermatozoa were examined by incubating the spermatozoa continuously in Ca2+‐containing capacitating media with 10−6 M to 10−4 M antagonist. Antagonists tested were four voltage‐gated Ca2+ channel antagonists (verapamil, nifedipine, nimodipine, and FR–34235) and two ligand‐gated channel antagonists (NaNO2 and Na‐nitroprusside). None of these antagonists could block the acrosome reaction. Instead, three antagonists (verapamil, nimodipine, and FR‐34235, each at 10−4 M) accelerated the onset of the acrosome reaction with a subsequent decrease in sperm motility. Nifedipine and Na‐nitroprusside at the same concentration caused a complete loss of sperm motility by 4 hr of incubation with no substantial effect on the rate of acrosome reaction. The detrimental effect of antagonists on the motility of spermatozoa appears to be due to a direct, Ca2+‐independent, membrane‐perturbing action of the reagents.

The acrosome reaction was not inhibited when guinea pig spermatozoa were precapacitated in Ca2+‐free medium (with a low concentration of lysolecithin) in the continuous presence of antagonists. An acceleration of the onset of the acrosome reaction by verapamil (10−4 M) was also demonstrated in the golden hamster.

These results may be interpreted as indicating that the entry of extracellular Ca2+ into spermatozoa, which triggers the acrosome reaction of guinea pig and hamster spermatozoa, is not mediated by Ca2+ channels. This is in marked contrast with the case reported in invertebrate spermatozoa. Possible mechanisms by which some of the antagonists stimulate the acrosome reaction and affect the motility of mammalian spermatozoa are discussed.
Movement and dissolution of the nucleus (germinal vesicle) during Rana oocyte meiosis: Effect of demecolcine (Colcemid) and centrifugation
Authors: Lessman Charles A. , Marshall William S. , Habibi Hamid R.
Published: February 2005

Demecolcine (Colcemid; DE), a colchicine derivative, augmented meiosis reinitiation by progesterone in the follicle‐enclosed oocyte of the frog, Rana pipiens. Whereas DE treatment alone had a minor stimulatory effect on germinal vesicle dissolution (GVD), this treatment elicited significant germinal vesicle movement (GVM) as evidenced by translocation of the GV to the oocyte surface. The effects of DE on GVM and progesterone‐induced GVD were also elicited in oocytes lacking follicle cells or other follicle wall components (type IV follicles), indicating that DE has a direct action on the oocyte itself. DE alone did not alter oocyte membrane voltage (Vm), resistance (Rm), or current (Im) and did not interfere with the changes in these parameters usually elicited by progesterone. After 5 hr incubation of follicle‐enclosed oocytes with either DE or progesterone, or combinations of both, the GV could be moved to the animal pole surface with less centrifugal force compared to control follicles. This result suggests that a decrease in ooplasmic viscoelasticity is induced by progesterone, which is mimicked by DE before GVM or GVD normally begins. The results presented here support the idea that DE‐sensitive oocyte components such as microtubules are involved in the process of steroid‐induced meiosis. These findings provide a physiological basis for future studies of cytoskeletal involvement in the events of meiosis.
Osmotic swelling of maturing rat spermatozoa and Lysis of caput spermatozoa by acylcarnitines and acylcholines
Authors: Cooper T. G.
Published: February 2005

The form that rat spermatozoa assume when swelling in hyposmotic media depends on the position of the cytoplasmic droplet, previous exposure to hypertonic media, and the stiffness of the flagellum. Bending at the end of the midpiece occurs when the swelling droplet is situated at this site; this occurs in midcaput cells, but sperm from more proximal sites do not bend in this fashion. Stiffening of caput sperm stored in vitro reduces the incidence of such midpiece bending but looping at the tip of the tail still occurs, and previous exposure of caput spermatozoa to hypertonic media also prevents hairpin bend formation. Mature sperm from the cauda are too stiff to form hairpin loops when placed in hypotonic media unless first treated with a penetrating disulphydryl‐reducing agent, after which swollen spherical vesicles can result from very flexible flagella confined within an intact membrane. Long‐chain acylcarnitines are more potent lytic agents than acylcholines, but, for both, chain lengths of 16 carbon atoms is optimal for preventing the swelling of rat caput sperm.
Nucleolar fine structure and RNA synthesis in bovine oocytes from antral follicles
Authors: Crozet Nicole , Kanka J. , Motlik J. , Fulka J.
Published: February 2005

Nucleolar ultrastructural changes occurring in vivo in bovine oocytes during follicular growth were analyzed by electron microscopy. The rates of in vitro incorporation of 3H‐uridine by oocytes of the same size class were evaluated by autoradiography.

One to two large fibrillogranular, vacuolated nucleoli were present in oocytes from small to medium antral follicles 0.5–3 mm in diameter. These oocytes showed intense hnRNA and rRNA synthesis. The homogeneous, agranular nucleoli in oocytes from follicles 3–4 mm in diameter were composed of a compact fibrillar material. This morphological change was accompanied by an impairment of nucleolar transcriptional activity as well as by a decrease in hnRNA synthesis.
Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid
Authors: Suarez Susan S. , Wolf Don P. , Meizel Stanley
Published: February 2005

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G‐75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G‐75 fraction containing the peak of acrosome reaction‐inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona‐free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.
Induction of the acrosome reaction in guinea pig spermatozoa by calmodulin antagonist W‐7
Authors: Nagae T. , Srivastava P. N.
Published: February 2005

The effect of the calmodulin antagonist W‐7 on the capacitation and the acrosome reaction of guinea pig spermatozoa was examined. The characteristic features of the acrosome reaction induced by W‐7 were the dependence on the composition and pH of the medium and on the presence of sodium bicarbonate. The most effective concentration of W‐7 for inducing the acrosome reaction was approximately 5 μM, which is far less than the Kd for calmodulin. Moreover, W‐7 enhanced the ability of spermatozoa to acquire capacitation in a Ca2+‐free medium. The spermatozoa induced to undergo the acrosome reaction by W‐7 were capable of penetrating the zona‐free hamster eggs. W‐5, which has a lower affinity for calmodulin than W‐7, also induced the acrosome reaction in the same manner as W‐7. These results suggest that the naphthalenesulfonamide derivatives W‐7 and W‐5 can induce the acrosome reaction in guinea pig spermatozoa via capacitation in a pH‐dependent, Ca2+‐calmodulin‐independent manner.
Difference of fertilizing capacity between testicular sperm and vas deferens sperm in Cynops pyrrhogaster
Authors: Matsuda Motoko
Published: February 2005

The fertilizing capacity was compared between testicular and vas deferens sperm in Cynops pyrrhogaster. The testicular sperm was not capable of fertilizing jelly eggs. In contrast, the vas deferens sperm was already capable of fertilizing the newt jelly eggs. There was no inhibitory factor for fertilizing jelly eggs in the testis. These results suggest that the testicular sperm is immature as to the fertilizing capacity. The testicular sperm gained the fertilizing capacity for the jelly eggs by treatment with Holtfreter's solution or 1/20 strength Holtfreter's solution. The treatment may promote the step of maturation to achieve the fertilizing capacity. The treated testicular sperm did not fertilize dejellied eggs, although vas deferens sperm fertilized dejellied eggs. Therefore, the maturation state of the treated testicular sperm is different from that of vas deferens sperm. Newt sperm may be matured within the vas deferens, as the newt does not have an organ like the mammalian epididymis.
The kinetics of the first wave of spermatogenesis in the newborn male mouse
Authors: Sung Weng Kong , Komatsu Masanobu , Jagiello Georgiana M.
Published: February 2005

A combination of autoradiography and air‐dried techniques was used to calculate the duration of the major meiotic stages in the first wave of spermatogenesis in the newborn mouse. The data indicated that the entry into meiosis occurred asynchronously over 2 days, and the time required for each stage and the total cycle was constant. These time intervals were nearly identical with those estimated for adult animals in the present study and by other authors.
Amino acid transport and exchange in unfertilized mouse eggs
Authors: Miller J. G. O. , Schultz G. A.
Published: February 2005

The mouse egg is an ideal model for the kinetic study of neutral amino acid exchange transport in development in that it possesses strong exchange‐mediated uptake/efflux with little other efflux. Exchange transport was examined by preloading eggs in unlabeled or labeled amino acid and then measuring subsequent amino acid fluxes. The existence of two L‐like (leucine‐preferring) systems, a low‐affinity exchange system, and a high‐affinity weakly exchanging system is suggested by the differences between preloaded and control eggs in uptake specificity and in the kinetic parameters of methionine uptake (respectively, Km = 220 and 69 μM, Vmax = 98 and 32 fmol/min/egg). The Vmax of exchange‐mediated efflux of methionine is similar to that for uptake into preloaded eggs, but the Km is about an order of magnitude higher. Preloading in alanine and serine (preferred substrates, along with cysteine, of system ASC) decreased the subsequent uptake of radiolabeled methionine, suggesting some ASC activity in the unfertilized mouse egg. The plateau in accumulation of leucine and phenylalanine, characteristic of simple exchange (mediated by system L), is not shared by methionine, suggesting the existence of another system (possibly an A, or alanine‐preferring, system) for the transport of methionine. Amino acid transport in the mouse egg is more complex than previously thought, the egg possessing four interacting systems for the transport of the neutral amino acids.
Effect of sperm motility on human embryo quality in in vitro fertilization
Authors: Chye Ng Soon , Edirisinghe W. R. , Wong P. C. , Ratnam S. S.
Published: February 2005

Sperm motility is important for penetration of the zona pellucida, and this parameter has been reported to be the single most important factor determining fertilization rates. As there was no report on the relationship between sperm motility and embryo quality, we investigated the influence of sperm motility on embryo quality in 41 patients with tubal disease and/or obstruction. The patients were either unstimulated or stimulated with clomiphene or clomiphene and human menopausal gonadotrophin. Of 116 oocytes collected, 86 (74.1%) fertilised and cleaved; of these only 44 embryos had clear equal blastomeres without fragmentation (grade 3). Grade 3 embryos were equally distributed through all initial sperm motility categories, and through all categories of sperm concentration after swim‐up. The ratio of motile sperm concentration in the initial semen sample to the final sperm concentration after swim‐up varied from 0.5 to 67, and grade 3 embryos were distributed randomly from low to high ratios. The pregnancy rate in this series was only 14.6% per replacement. The rate of gestational sacs per embryo replaced was 7.0% (6/86); if “poor” embryos were excluded, the rate was 9.1% (6/66). The absence of correlation between sperm motility and embryo quality is discussed on morphological grounds.
Action of phosphatidylcholine in protecting ram sperm from cold shock
Authors: Simpson Ann M. , Swan M. A. , White I. G.
Published: February 2005

Rapid cooling (cold shocking) of washed ejaculated ram sperm to 0°C irreversibly reduced motility, tail beat frequency, and respiration and increased the uptake of 45Ca2+. The plasma membranes were removed from the sperm head, and the acrosomes were detached from the nuclei. The plasma membranes of the middle piece were removed, and the mitochondria contained pale and expanded cristae, similar in appearance to ATP‐deprived mitochondria in the “condensed” configuration. The presence of 2.0 mg/ml phosphatidylcholine (lecithin) in the medium prevented ultrastructural damage on cold shock, and the motility, tail beat frequency, respiratory rate, and calcium uptake were maintained at levels similar to washed sperm. As the “protective” effect of phosphatidylcholine against cold shock was maintained to a certain extent after rewashing and centrifuging the sperm prior to cold shock, the interaction of phosphatidylcholine with ram sperm membranes may be fairly “tight” and not easily disrupted.
Vital staining and acrosomal evaluation of bovine sperm
Authors: Aalseth E. P. , Saacke R. G.
Published: February 2005

Dried smears prepared from vitally stained sperm were evaluated as a method of simultaneously determining sperm viability and acrosomal morphology. A combination Fast Green FCF‐Eosin B stain was used. The stained smears were examined at × 1, 250 using differential interference contrast microscopy (DIC). For comparison, the percentage of sperm with intact acrosomes was also determined from wet smears using DIC. Acrosomal morphology was not altered by the staining procedure, as the percentage of intact acrosomes was similar whether quantitated from wet or stained smears. Absence of eosinophilic staining in the acrosome was used as an indication of sperm viability. The percentage of sperm with unstained acrosomes was highly correlated with the percentage of intact acrosomes quantitated from stained smears. Thus, vital staining provided an indication of sperm viability comparable to acrosomal integrity, a highly reliable technique. The major advantages of using dried stained smears were more thorough examination of individual sperm without sperm activity interference, simultaneous evidence of sperm viability and morphology, and the opportunity to delay evaluation. In addition, diluting spermatozoa in complex or simple media with or without egg yolk or follicular fluid did not interfere with subsequent staining or acrosomal evaluation.
Hormonal dissociation of ovulation and maturation of oocytes: Ovulation of immature amphibian oocytes by prostaglandin
Authors: Schuetz Allen W.
Published: February 2005

Prostaglandin involvement in ovulation and maturation of amphibian (Rana pipiens) ovarian follicular oocytes was investigated using in vitro‐cultured ovarian follicles. Exposure of follicles to PGF2α during culture stimulated variable but generally low levels of ovulation without concomitant induction of maturation. Addition of PGF2α to cultured follicles markedly enhanced the incidence of ovulation in follicles exposed to progesterone or frog pituitary homogenate (FPH). Onset of the ovulatory process was further accelerated following addition of PGF2α to FPH‐treated follicles. PGE, in contrast to PGF2α, exhibited no stimulatory effects on ovulation and consistently inhibited ovulation induction by FPH and progesterone. Cytological analysis of follicles undergoing ovulation revealed that ovulation of immature oocytes induced by PGF2α varied markedly from that seen following FPH or progesterone stimulation of follicles in vivo or in vitro. Immature oocytes in contrast to maturing oocytes were typically ovlulated with follicle cells still attached to the vitelline membrane. The observations indicate that PGF2α effected follicle rupture and contraction of the follicular epithelium and theca without prior separation of the follicle cells from the oocyte. Selective inhibitors of steroid synthesis (cyanoketone) and protein synthesis (cycloheximide) inhibited FPH‐induced ovulation and maturation. PGF2α reversed the inhibitory effects of cyanoketone and cycloheximide on FPH‐induced ovulation but not maturation of oocytes. Neither prostaglandins alone or in combination with progesterone or FPH induced ovulation of oocytes following removal of the follicular epithelium. Ovulatory effects of PGF2α appear to be mediated through the follicular epithelium. Results indicate that ovulation and maturation of amphibian oocytes can be induced independently of each other by separate classes of hormones. Normal synchronization of ovulation and maturation of oocytes may require the combined action of prostaglandins and steroids acting within different follicular compartments.