bluuebunny/crossref_metadata_2025_split huggingface dataset:
Incubation of mouse sperm with lactate delays capacitation and hyperactivation and lowers fertilization levels in vitro
Authors: Neill James M. , Olds‐Clarke Patricia 
Published: February 2005

AbstractMouse sperm were incubated in medium with or without 24 mM lactate and assessed for (1) motility characteristics including hyperactivation—a computer‐assisted motion analysis system was used; (2) capacitation—a chlortetracycline fluorescent dye binding assay was used; and (3) ability to penetrate oocytes. Lactate affected all aspects of motility and delayed the rates of both hyperactivation and capacitation. When a concentration of 8 × 103 sperm/ml was used for insemination in vitro, sperm preincubated 60–90 minutes in medium with lactate prior to insemination in lactate‐free medium fertilized fewer oocytes than did sperm preincubated in lactate‐free medium. Use of a calcium‐sensitive electrode demonstrated that lactate chelated appreciable amounts of calcium in the medium. Capacitation was assayed in sperm incubated 60 minutes in medium with various concentrations of lactate or CaCl2. When medium containing lactate was compared to medium without lactate but having a similar level of free calcium, the level of capacitation of sperm incubated with lactate was less than half that of sperm incubated without lactate. These results demonstrate that including 24 mM lactate in the medium can have detrimental effects on mouse sperm hyperactivation and capacitation. The detrimental effects on capacitation are partly but not completely due to the chelation of calcium by lactate.
Fine structure of the neck of epididymal spermatozoa of schreiber's long‐fingered bat (Chiroptera: Mammalia)
Authors: Bernard R. T. F. , Hodgson A. N. 
Published: February 2005

AbstractThe structure of the spermatozoon of the long‐fingered bat (Miniopterus schreibersii) was studied using transmission electron microscopy. The neck is characterized by two asymmetrically arranged features that allow the orientation of sections in different planes and therefore aid the interpretation of the structure. There are six segmented columns, two major and four minor, in the anterior 79% of the neck, and it is only posterior to this that the major columns split. One major column is associated with doublets 9,1, and 2, and the other major column, with doublets 5 and 6. The proximal centriole is surrounded by the capitulum and segmented columns, and the distal centriole persists and is apparent in both longitudinal and transverse sections. The central pair of microtubules pass through the distal centriole and terminate at the proximal centriole. A line drawn through the two central microtubules divides the head into left and right halves. Development of the neck begins during early spermiogenesis at which time the two centrioles are associated with the production of the capitulum, segmented columns, and axoneme complex.
Adenosine potentiates forskolin‐induced delay of meiotic resumption by mouse denuded oocytes: Evidence for an oocyte surface site of adenosine action
Authors: Salustri A. , Petrungaro S. , Conti M. , Siracusa G. 
Published: February 2005

AbstractAdenosine is present in the mouse follicular fluid and has been shown to interfere with oocyte maturation in vitro. To clarify the mechanism of adenosine action on meiotic arrest, we have characterized the synergistic action of this purine with forskolin on the meiotic resumption of mouse denuded oocytes.Forskolin delays meiotic resumption by approximately 1 hour; adenosine at concentrations ranging between 30–750 μM has no significant effect. Conversely, adenosine treatment together with forskolin produces a further delay in the resumption of meiosis. This adenosine effect is dose‐dependent and mimicked by adenosine analogs like N6‐phenylisopropyl adenosine (PIA), 2‐chloroadensoine (2‐CLA), 5′‐N‐ethylcarboxamide (NECA). Dipyridamole, which inhibits adenosine transport, does not prevent the meiosis‐arresting synergistic effect of adenosine with forskolin. Adenosine causes a 50% increase of adenosine triphosphate (ATP) content in the oocyte. However, this increase is not directly responsible for the observed delay in the oocyte maturation for the following reasons: (1) the dose response of inhibition of meiotic resumption does not correlate with the doses of adenosine producing an increase in ATP; (2) dipyridamole blocks the increase in intracellular ATP, but it has no effect on the adenosine inhibition of maturation; (3) adenosine analogs inhibit oocyte maturation but do not affect intracellular ATP levels. These results suggest that the synergism of adenosine with forskolin on meiotic arrest does not require uptake of the nucleoside nor its conversion to ATP and that the adenosine effects are exerted at the level of the oocyte plasma membrane.
Wheat germ agglutinin treatment of follicle cell‐free mouse oocytes inhibits fertilization
Authors: Nuzzo Nancy A. , Zaneveld Lourens J. D. 
Published: February 2005

AbstractControversy exists whether treatment of follicle cell‐free oocytes with wheat germ agglutinin (WGA) prevents fertilization. Lack of inhibition in one case has led to the suggestion that acrosin may not be a zona lysin. To re‐examine the effect of the WGA, the zona pellucida of follicle cell‐free mouse oocytes was made more resistant to proteinase digestion by treatment with 10 or 50 μg/ml WGA. Such WGA‐treated oocytes showed decreased fertilizability when washed to remove excess WGA and incubated with capacitated spermatozoa. Oocyte cleavage was used as an end point, because a large number of spermatozoa adhered to the eggs after WGA treatment, making observation of sperm penetration and pronucleus formation unreliable. Resistance to proteinase digestion increased, and the fertilizability decreased with the higher amount of WGA. The action of WGA was most likely not mediated by a direct effect on sperm motility, sperm acrosin activity, sperm binding to the zona pellucida, or oocyte cleavage. WGA did not affect the acrosome reaction of guinea pig spermatozoa. These data show that WGA treatment of follicle cell‐free mouse oocytes results in decreased fertilizability, possibly by rendering the zona pellucida more resistant to sperm proteinase digestion.
Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion sperm
Authors: Blach E. L. , Amann R. P. , Bowen R. A. , Sawyer H. R. , Hermenet M. J. 
Published: February 2005

AbstractTransmission electron microscopy was used to confirm that a monoclonal antibody (F79.3E2; class IgGlK) was specifically localized to an antigen in the acrosomal ground substance of stallion sperm. This antibody was used to develop and validate an indirect immunofluorescent procedure to evaluate integrity of the plasma‐acrosomal membranes of stallion sperm. The concept was that primary monoclonal antibody would be “shielded” from its acrosomal antigen by an intact plasma membrane. Conversely, sperm with damaged plasma‐acrosomal membranes would exhibit green acrosomal fluorescence when viewed with an epifluorescence microscope. A lipophilic counterstain (red fluorescence) was used to insure that all sperm were visualized. Sperm in fresh‐extended or frozen‐thawed semen were incubated with hybridoma supernatant containing monoclonal antibody for 30 min at 37°C, then a second antibody (rabbit anti‐mouse IgG‐FITC) was added for 30 min at 37°C. Unbound antibody was removed by dilution and centrifugation. Sperm were resuspended in phosphate‐buffered saline containing Evan's blue as a counterstain. All sperm fluoresced bright red, regardless of the status of cell membranes, except that in cells with damaged plasma‐acrosomal membranes, the green fluorescence associated with antibody was overriding for the rostral portion. By counting fluorescent and nonflourescent “acrosomes”, the percentage of sperm with intact plasma‐acrosomal membranes was easily determined. Evaluation of five mixtures of undamaged and damaged sperm by this procedure gave a correlation of 0.91 between the percentage of damaged sperm in a mixture and the percentage of sperm with a fluorescent acrosome. Intra‐ and interassay coefficients of variability were < 6%.
Phospholipid composition of isolated guinea pig sperm outer acrosomal membrane and plasma membrane during capacitation in vitro
Authors: Stojanoff A. , Bourne H. , Andrews A. G. , Hyne R. V. 
Published: February 2005

AbstractAfter capacitation of guinea pig spermatozoa in vitro, the plasma membrane was mechanically separated from the spermatozoa in the presence or absence of HgCl2 and subsequently isolated by density gradient centrifugation. Examination of the spermatozoa by electron microscopy after homogenization in the presence of HgCl2 revealed that plasma membrane was removed only from the acrosomal region and remained predominately intact posterior to the equatorial segment of the sperm head, as well as the midpiece and tail. In comparison, spermatozoa homogenized under similar buffer conditions but in the absence of HgCl2 lose the large apical segment of the acrosome and the plasma membrane is removed essentially from the entire cell. If spermatozoa were homogenized in the absence of Hg2+, analysis of plasma membrane phospholipid composition revealed a complete loss of lysophosphatidylcholine (LPC) from the plasma membrane after incubation of spermatozoa in minimal capacitating medium (MCM‐PL) for 2 hours. Under these culture conditions the addition of Ca2+ (5 mM) to the capacitated spermatozoa induced approximately 78 ± 5% (n = 3) of the motile spermatozoa to undergo acrosome reactions while still maintaining sperm motility (80 ± 5%) (n = 3). If the spermatozoa were homogenized in the presence of Hg2+, a time course study revealed that plasma membrane LPC loss occurred between 60 and 90 minutes of incubation. This complete loss of LPC was evident when approximately half of the capacitated spermatozoa had undergone acrosome reactions. Incubation of the spermatozoa with the metabolic and acrosome reaction inhibitor, 2‐deoxyglucose (10 mM) for 2 hours, maintained the plasma membrane phospholipid composition similar to that in the noncapacitated state. These data provide evidence that changes in the plasma membrane phospholipid composition may be associated with guinea pig sperm capacitation.
Role of protein kinase C activation in oocyte maturation and steroidogenesis in ovarian follicles of Rana pipiens: Studies with phorbol 12‐myristate 13‐acetate
Authors: Francisco Susan Kleis‐San , Schuetz Allen W. 
Published: February 2005

AbstractIn ovarian follicles of Rana pipiens, frog pituitary homogenates (FPH) elevate intrafollicular progesterone levels which in turn is thought to induce meiotic resumption in the prophase I arrested oocytes. Calcium plays a role in FPH and steroid‐provoked responses in the somatic and gametic components of the follicle, presumably via effects exerted at the plasma membrane of their respective target cells. Many membrane active hormones which utilize Ca2+ in their intracellular transduction also provoke membrane phosphoinositide hydrolysis yielding inositol triphosphate (IP3) and diacyl glycerol (DAG), an activator of the CA2+‐dependent protein kinase C (PKC). The actions of phorbol 12‐myristate 13‐acetate (TPA), a potent synthetic activator of PKC, on progesterone production and oocyte maturation was examined in in vitro cultured ovarian follicles. TPA induced germinal vesicle breakdown (GVBD) in intact follicles and in oocytes denuded of somatic components, while the inactive compound phorbol 13‐monoacetate was ineffective. Further, TPA induction of GVBD exhibited similarities to progesterone‐induced GVBD, being inhibited by treatments which elevate cAMP or inhibit protein synthesis. TPA alone did not elevate intrafollicular or medium progesterone levels, as occurred in FPH‐treated follicles. TPA partially inhibited intrafollicular progesterone accumulation induced by FPH or treatments which elevate cAMP levels. These data suggest that activation of PKC plays a role in oocyte maturation independent of follicular progesterone production as occurs in response to FPH. Further, it appears that the somatic cells of the amphibian follicle also possess PKC which when activated, antagonizes cAMP generating pathway in these cells. Results indicate that protein kinase can influence oocyte maturation in Rana follicular oocytes by several mechanisms.
Kinematics of hamster sperm during penetration of the cumulus cell matrix
Authors: Drobnis E. Z. , Yudin A. I. , Cherr G. N. , Katz D. F. 
Published: February 2005

AbstractDuring capacitation, mammalian spermatozoa gain the ability to penetrate the cumulus cell matrix (CCM). The role of hyperactivated motility for this capacity is uncertain. In the present study, hamster sperm were observed during penetration and progression through the CCM, and flagellar beat patterns were quantitated by characterization of the underlying flagellar bends. Small numbers of sperm were added to cumulus masses slightly compressed on a slide (150 μm depth), and penetration was videorecorded using interference contrast optics. During penetration of the cumulus surface, sperm did not generate the large flagellar bends and asymmetric beats that are hallmarks of hyperactivation in low viscosity media. Instead, they entered slowly using high‐frequency, low‐amplitude sinusoidal flagellar motions. Within the CCM, sperm continued to move slowly, and they exhibited three distinct patterns of motility. The first was sinusoidal, produced by alternating, propagated bends: principal bends (PB) moved the head away from the beat midline, with the convex edge of the head leading, and reverse bends (RB) had the opposite curvature. The second pattern was asymmetric and sinusoidal: an extreme RB developed in the distal flagellum, was propagated distally, and was followed by a PB of less curvature. The third motility pattern was a hatchet‐like stroke of the sperm head which resulted when an extreme, nonpropagated PB developed slowly in the proximal midpiece, and was released rapidly. In this mode there were no reverse bends, and sperm did not progress. There were subpopulations of capacitating sperm in free‐swimming medium which had these same bend types and motility patterns, suggesting that qualitative flagellar movement may not change during CCM penetration. Sperm velocity in the CCM was not strongly correlated with flagellar beat kinematics, suggesting local heterogeneity in cumulus mechanical resistance and/or differences in interaction of the matrix with the surfaces of individual sperm. An effective viscosity of the cumulus near its border was estimated to be of the order of 1–4 P.
The major component of the rat sperm fibrous sheath is a phosphoprotein
Authors: Brito Monica , Figueroa Jaime , Maldonado Enrique U. , Vera Juan C. , Burzio Luis O. 
Published: February 2005

AbstractThe fibrous sheath from rat epididymal sperm was isolated by sequential extraction, first with Triton X‐100 and dithiothreitol, and then with 6 M urea and dithiothreitol. The latter extraction procedure solubilized most of the sperm components, leaving the head and the fibrous sheath as the only intact structures. This material was purified by sucrose gradient centrifugation. Electron microscopy confirmed the purity of the isolated material and revealed the characteristic structural features of the fibrous sheath.Polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulfate) of the fibrillar material, showed a complex polypeptide composition. The polypeptides with molecular weights of 80,000, 24,000, and 11,500 accounted for about 65% of the total protein of the fibrous sheath. Peptide map analyses indicated that the components of molecular weights of 80,000 and 24,000 are unrelated to the polypeptides of similar size of the outer dense fibers. On the other hand, it appears that the fibrous sheath and the outer dense fibers share the polypeptide of 11,500 daltons. The component of 80,000 daltons contains on the average about 3 mol of phosphoserine per mol of polypeptide, indicating that the most abundant polypeptide of the fibrous sheath is a phosphoprotein.
Parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro
Authors: Onodera M. , Tsunoda Y. 
Published: February 2005

AbstractThe present study was undertaken to determine conditions for parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro. The cumulus‐free eggs were submitted to square direct current pulses at output voltage of 1.0 to 2.5 kV/cm for 25 to 200 μsec. The best conditions for the activation of mouse eggs were 1.5 kV/cm for 100 μsec, in which 78% of eggs were activated, 32% of which developed to blastocysts in vitro. When the nonelectric solution (0.3M mannitol) was used for electric stimulation, the activation rate was quite low (16%). Optimal conditions for activation of rabbit eggs were 1.5 kV/cm for 200 μsec, in which 77% of eggs were activated, 25% of which developed to blastocysts. Unlike mouse eggs, rabbit eggs frequently had three pronuclei after electric stimulation.It is clearly shown that electric stimulus can induce parthenogenetic activation of the mouse and rabbit eggs in vitro.
Assessment of the viability and fertilizing potential of cryopreserved bovine spermatozoa using dual fluorescent staining and two‐flow cytometric systems
Authors: Ericsson S. A. , Garner D. L. , Redelman D. , Ahmad K. 
Published: February 2005

AbstractA dual fluorescent staining system utilizing 5 (and‐6)‐carboxy‐4′,5′‐dimethyl fluorescein diacetate (CDMFDA) and Hydroethidine (HED) was developed to provide quantifiable information reflective of spermatozoal viability and fertilizing potential. Cryopreserved spermatozoa from ten bulls on which there was fertilizing capacity information were incubated for 1.5, and 3 hr at 39°C prior to fluorogenic staining. Spermatozoa were analyzed using both a FACS Analyzer and an EPICS V flow cytometer to determine if a particular fluorescence pattern was due to an instrumental artifact or cellular processes. Five fluorescent cellular populations were identified by the FACS Analyzer and three populations by the EPICS V. Spermatozoa were quantified after each incubation time for red (HED) and green (CDMFDA) fluorescence. Viable spermatozoa retained the greatest amount of both green and red fluorescence. Dead or moribund spermatozoa had a decrease in over‐all fluorescence. The number of viable cells at 0 hr plus the number of dead or morbid cells at any time period were identified by the FACS Analyzer as important in estimating the potential fertility of a bull. The EPICS V identified the number of dead or moribund cells as being related to nonreturn rates. Incubation of samples decreased cellular viability, which resulted in reduced levels of both green and red fluorescence. Similarities between data obtained with both flow cytometers illustrated that cellular processes, not instrumental artifacts, were responsible for the decrease in over‐all fluorescence when viability declined, the relationship between the number of cells with specific fluorescence levels and nonreturn rates, and the incubative‐induced changes in fluorescence patterns.
Differentiation of binuclear spermatozoa in mice
Authors: Burkhart J. G. , Malling H. V. 
Published: February 2005

AbstractIn an electron microscopy study of abnormal spermatogenesis in mice, we have found that two discrete haploid nuclei may be located in a single spermatid cytoplasm after the second meiotic division. The spermatid continues to differentiate and forms a binucleate spermatozoon with both nuclei separately packaged within the sperm head. The Golgi apparatus of the double spermatid forms a single proacrosome that attaches to both nuclei. Apparently, one acrosomal structure differentiates to cover and compartmentalize the two haploid nuclei within the sperm head. Chromatin condensation appears normal. The head morphology and number of flagella vary in mature spermatozoa produced by this process. This work demonstrates one pathway by which polyploid spermatids continue to differentiate to spermatozoa after failure of cytoplasmic division or possibly cellular fusion.
Distribution and role of microfilaments during early events of sheep fertilization
Authors: Le Guen P. , Crozet N. , Huneau D. , Gall L. 
Published: February 2005

AbstractThe distribution of actin was studied during early events of sheep fertilization by fluorescence microscopy after staining with 7‐nitrobenz‐2‐oxal‐1.3 diazole (NBD)‐phallacidin and anti‐actin antibody and by electron microscopy after heavy meromyosin labelling. Unfertilized and fertilized eggs exhibited a continuous band of fluorescence with both NBD‐phallacidin and anti‐actin antibody. Unlike in mice, no high concentration of actin overlying the spindle was detected in ovulated sheep oocytes. At the site of sperm head incorporation, the fertilization cone developed above the decondensing male chromatin and was underlined by a submembranous area rich in microfilaments. A similar actin network was observed in the cortex of the second polar body.Cytochalasin D was used to investigate the role of actin during the fertilization process. This drug did not prevent sperm fusion and incorporation but inhibited polar body abstriction and fertilization cone development and retarded sperm tail incorporation. Moreover, in the presence of the drug, the anchorage of the metaphase II spindle at the surface of the egg was destroyed. The role of microfilaments in these early events is discussed.
Effects of non‐24‐hour days on reproductive efficacy and embryonic development in mice
Authors: Endo Akira , Watanabe Toshiaki 
Published: February 2005

AbstractICR female mice were exposed to either 22 (L11, D11) or 26 hour day (L13, D13) light/dark cycles for at least 2 weeks before mating and/or during pregnancy. The mating rates of these animals decreased considerably. When pregnant females were examined at gestation days 12.0 or 17.5, resorption rates were increased, the embryos weighed less, and development was retarded in the experimental groups with preconceptional exposure to non‐24‐hour days. We speculate that in mice maternal and paternal pre‐ and periconceptional environment of daily light/dark cycles is important for normal reproductive efficacy and normal embryonic development during pregnancy.
Factors regulating mammalian sperm migration through the female reproductive tract and oocyte vestments
Authors: Katz David F. , Drobnis Erma Z. , Overstreet James W. 
Published: February 2005

AbstractMechanisms of mammalian sperm migration through the female reproductive tract and ovum vestments are described. The perspective is biophysical as well as biochemical and morphological, and the focus is upon the role of sperm motility in these processes. Sperm forward progression is characterized as an interactive process between the the cell and its environment, and the mediation of flagellar bend propagation by the physical properties of its surroundings is described. These properties, together with flagellar beat kinematics, sperm morphology, and surface properties, determine the magnitude of the forces generated by sperm and their consequent rate of progression. Sperm interactions with the cervical mucus, the cumulus oophorus, and the zona pellucida are described. The poorly understood affinity of the sperm surface for the macromolecules of the mucus, cumulus, and zona is stressed, as is the viscoelastic structural mechanical resistance of these biopolymers to sperm motion. The kinematics and consequences of hyperactivated sperm motion are presented, with emphasis on objective characterization of such motion (as a biomarker), along with analysis of the mechanical advantage that such motion may confer on spermatozoa during egg–vestment interaction.