Biomedical subjects

D R Armant

Publications and source records attributed to D R Armant.

At least 37 records · Page 2Linked to original sources

PubMed1995

Beta 1- and beta 3-class integrins mediate fibronectin binding activity at the surface of developing mouse peri-implantation blastocysts. Regulation by ligand-induced mobilization of stored receptor.

Implanting mouse blastocysts adhere through their abembryonic surfaces to the endometrial extracellular matrix. Because blastocysts cultured on fibronectin in vitro dissociate to form trophoblast outgrowths, it is unclear whether this adhesion is initially mediated by fibronectin receptors on the apical or basolateral surface of the trophectoderm. Intact blastocysts were examined in a ligand binding assay utilizing the fibronectin cell binding domain attached to fluorescent microspheres. Fibronectin binding activity on the apical surface of the trophectoderm was confined to the abembryonic pole of the blastocyst, where trophoblast differentiation initiates, and was regulated temporally in accordance with blastocyst outgrowth. Soluble fibronectin (IC50 = 0.2 microM) or Gly-Arg-Gly-Asp-Ser-Pro, but not laminin, competitively inhibited fibronectin binding activity. Addition of antibodies against the alpha v, alpha 5, beta 1, or beta 3 integrin subunits also inhibited binding activity. Blastocysts cultured in the absence of an adhesive substratum exhibited fibronectin binding activity only after exposure to immobilized or soluble ligand. Potentiation of binding activity by ligand was unaffected by cycloheximide but was sensitive to brefeldin A inhibition of protein trafficking. These findings suggest that the interaction of fibronectin with the trophectoderm induces a translocation event that up-regulates fibronectin binding beta 1- and beta 3-class integrins on the apical surface.

Amino Acid Sequence↗

PubMed1995

Molecular interactions between fibronectin and integrins during mouse blastocyst outgrowth.

To investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum-free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin-binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell-binding domain (FN-120) and the Arg-Gly-Asp (RGD) recognition sequence. A 40 kD COOH-terminal chymotryptic fragment of fibronectin containing both a heparin-binding region and an alternate (non-RGD) cell-binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin-binding domain, including the CS1 alternate cell-binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits alpha 3, alpha 5, alpha IIb, alpha v, beta 1 and beta 3, but not alpha 4, all bound to trophoblast cells. Antibodies raised against either the beta 1 or beta 3 integrin subunits significantly inhibited fibronectin-mediated outgrowth. These findings demonstrate the key role of the central cell-binding domain of fibronectin in trophoblast adhesion, and suggest four RGD-binding integrins, alpha 3 beta 1, alpha 5 beta 1, alpha IIb beta 3, and alpha v beta 3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation.

Amino Acid Sequence↗

PubMed1995

Rapid inhibition of mRNA synthesis during preimplantation embryo development: vital permeabilization by lysolecithin potentiates the action of alpha-amanitin.

Lysolecithin was used to permeabilize embryonic cells to impermeant compounds without compromising embryo viability. Within 2 min of exposure to 0.05% lysolecithin, mouse morulae became permeable to inositol 1,4,5-trisphosphate, leading to the release of calcium from intracellular stores. Although these morulae remained permeable for at least 2 h, they developed normally to the blastocyst stage during subsequent culture in vitro. Lysolecithin permeabilization was then used to potentiate the internalization of alpha-amanitin. Pretreatment with lysolecithin for 2 min markedly accelerated the onset of transcriptional inhibition from 3 h to 10 min after alpha-amanitin addition. The rapid inhibition by alpha-amanitin of mRNA synthesis in lysolecithin-treated embryos provided a precise method for delineating developmentally important transcriptional periods. Using this protocol, we found that mRNAs required for embryonic cavitation were synthesized between 87 and 91 h post-hCG, shortly before blastocoel formation commenced.

Amanitins↗

PubMed1995

Improved accuracy of sperm motility assessment using a modified Micro-Cell sperm counting chamber.

OBJECTIVE: To assess the effect of modifications made in the Micro-Cell sperm counting chamber on the motility of washed human spermatozoa. DESIGN: In a 2 x 2 experimental design, human sperm samples were washed with or without protein supplementation, loaded into modified or unmodified Micro-Cell chambers, and assessed by automated semen analysis for changes associated with sperm adhesion to the glass chamber surfaces. PARTICIPANTS: Twenty-one men who were undergoing semen analysis and presented with > or = 50% motile sperm, or normal donors. MAIN OUTCOME MEASURES: Reduced motility and elevated lateral head displacement associated with sperm adhesion to glass surfaces were compared using a doubly repeated measures analysis of variance. RESULTS: Addition of protein to the sperm washing solution partially reversed the adhesion of spermatozoa to the glass of unmodified Micro-Cell chambers. Chambers manufactured to reduce cell adhesion to glass surfaces yielded the highest motility and lowest lateral head displacement, whether the sperm were washed in a solution supplemented with or without protein. CONCLUSION: These findings indicate that, as in raw semen, kinematics of washed sperm can be measured reliably in the modified Micro-Cell chamber.

Cell Adhesion↗

PubMed1994

Mouse trophoblast cell invasion of extracellular matrix purified from endometrial tissue: a model for peri-implantation development.

We have investigated the invasive activity of mouse trophoblast cells during embryo implantation in vitro by culturing blastocysts with extracellular matrix (ECM) purified from mouse endometrium obtained on day 4 of pregnancy. Endometrium was dissected from lyophilized mouse uteri, and intact ECM was isolated by sequential precipitation in nonionic detergent and high salt. Electron microscopic examination of the ECM revealed typical collagen fibers plus an amorphous material resembling basement membrane. Electrophoretic analysis of the ECM revealed an enrichment of high molecular weight proteins, and immunoblotting indicated the presence of fibronectin, laminin, entactin, and type IV collagen, but not the intracellular proteins 2',3'-cyclic nucleotide-3'-phosphodiesterase or vimentin. Mouse blastocysts cultured with this ECM attached to it within 3 days, and the trophoblast cells began to migrate through the matrix in a manner resembling trophoblast invasion in utero. Unlike blastocysts cultured on plastic surfaces, the trophoblast did not flatten and become disorganized, but retained a polarized, spherical structure. Fluorescent microscopy with fluorescein isothiocyanate-labeled phalloidin revealed a high degree of microfilament organization and established that actin was absent from the ECM preparation. In the presence of a serum substitute, differentiation continued through yolk sac formation. Without serum components, yolk sac did not form; however, light and electron microscopic examination indicated that the invasive behavior of trophoblast cells persisted and was comparable to that of trophoblasts cultured in the presence of the serum substitute. A three-dimensional model for investigating trophoblast behavior in ECM from the endometrium should be of great value in elucidating the cellular and molecular events surrounding the process of blastocyst implantation.

Animals↗

PubMed1994

The effects of in vitro cocaine exposure on human sperm motility, intracellular calcium, and oocyte penetration.

OBJECTIVE: To determine if cocaine exposure affects human sperm motility, intracellular calcium level, and fertilizing capability. DESIGN AND METHODS: Human semen samples were treated with 1 to 1,000 microM cocaine hydrochloride for up to 2 hours in vitro. Sperm motion kinematics were measured by computer-assisted semen analysis (CASA). Spermatozoan intracellular calcium was determined by laser cytometry. The sperm fertilizing capability was assessed using the zona-free hamster oocyte penetration test. RESULTS: After a short exposure (15 minutes) to cocaine, the sperm motion kinematic parameters, straight line velocity and linearity, were decreased in the high concentration groups. However, after a longer exposure (2 hours) to cocaine, the differences were no longer significant. Cocaine treatment did not alter spermatozoa intracellular calcium levels. Most importantly, human sperm treated with cocaine at a high concentration were fully capable of penetrating zona-free hamster oocytes. CONCLUSION: Human spermatozoa acutely exposed to high concentrations of cocaine initially demonstrate a decrease in two motion kinematics, straight line velocity and linearity. However, overall, cocaine exposure had no significant effects on sperm motility and fertilizing capability.

Calcium↗

PubMed1994

Blastocyst cavitation is accelerated by ethanol- or ionophore-induced elevation of intracellular calcium.

To extend our previous finding that ethanol stimulates murine preimplantation development, we focused in the current study on the cavitation and expansion of the blastocyst. Ethanol stimulation of blastocyst cavitation and expansion was optimal at a concentration of 0.1% and required only a 5-min exposure. Because intracellular levels of calcium were transiently increased in the ethanol-exposed embryos, we determined the effect of directly increasing calcium on the rates of blastocyst cavitation and expansion. Treatment with the calcium ionophore, A23187, altered development much as did ethanol, indicating that ethanol may stimulate preimplantation development by increasing the intracellular calcium concentration. The relationship between intracellular calcium levels and blastocoel volume suggests that morphogenetic events during preimplantation development, particularly cavitation and blastocyst formation, may be regulated through modulation of intracellular calcium levels.

Aniline Compounds↗

PubMed1994

Mouse blastocyst outgrowth and implantation rates following exposure to ethanol or A23187 during culture in vitro.

This study focused on the effects of ethanol on blastocyst outgrowth and implantation in mice. Blastocysts were exposed to ethanol in Ham's F10 medium and then cultured free of ethanol on fibronectin-coated Petri dishes to assess trophoblast cell adhesion and migration. The time necessary for half of the embryos to outgrow was significantly less (P < 0.05) following treatment with 0.1%, 0.2%, 0.4% or 1.0% (w/v) ethanol for either 5 min or 24 h compared with controls. The rate of trophoblast cell migration was determined by measuring the mean area of outgrowing embryos using an image analysis system. Blastocysts exposed to ethanol for 5 min produced a greater (P < 0.05) average outgrowth area than did stage-matched controls. Acceleration of blastocyst cavitation by ethanol is known to be associated with an increase in the intracellular concentration of calcium. Here, treatment with the calcium ionophore A23187 stimulated (P < 0.05) trophoblast outgrowth and accelerated (P < 0.05) the rate of cell migration. In an attempt to correlate the effect of ethanol on outgrowth in vitro with implantation in utero, cultured blastocysts were either not exposed to ethanol or exposed to 0.1% ethanol for 5 min and transferred 24 h later to the uteri of pseudopregnant dams. The implantation rate (39.4%, n = 376) and the rate of development to term (2.45 pups per mouse, n = 20) were higher (P < 0.05) in mice receiving ethanol-treated embryos compared with those receiving control embryos (20.8%, n = 331; 1.16 pups per mouse, n = 18, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Porcine zona pellucida ZP3 alpha glycoprotein mediates binding of the biotin-labeled M(r) 55,000 family (ZP3) to boar sperm membrane vesicles.

The two M(r) 55,000 glycoproteins, ZP3 alpha and ZP3 beta, of porcine zona pellucida copurify as a preparation designated ZP3. Gamete binding assays have implicated ZP3 alpha, but not ZP3 beta, as participating in sperm-zona recognition events. We now report that boar sperm contain membrane-associated binding sites with specificity for ZP3 alpha. Biotin-labeled (b-) preparations of ZP3 bind to intact boar sperm in a saturable manner, with localization on the anterior head region. Membrane vesicles obtained from capacitated sperm by nitrogen cavitation retain b-ZP3 binding sites as determined by an enzyme-linked method employing alkaline phosphatase-conjugated strepavidin. In competitive binding assays using b-ZP3 (0.1 microgram/ml) as probe, heat-solubilized zonae and ZP3 were effective competitors, whereas the nonzona molecules fetuin and fucoidin were not. Digestion of ZP3 with endo-beta-galactosidase, an enzyme that trims polylactosamines, enhanced its affinity for membrane receptors. In contrast treatments such as chemical deglycosylation, pronase digestion, or disruption of disulfide bonds abolished the ligand activity of ZP3. Finally, purified ZP3 alpha was an at least 100-fold better antagonist than purified ZP3 beta. The results demonstrate that binding of b-ZP3 to isolated boar sperm membranes is mediated by sperm receptors with specificity for the ZP3 alpha macromolecular component and reveal a complex contribution of both carbohydrate and protein moieties toward the ligand activity of this sperm adhesive zona molecule.

Animals↗

PubMed1993

Development of in vitro fertilized mouse embryos exposed to ethanol during the preimplantation period: accelerated embryogenesis at subtoxic levels.

This study examined the effects of ethanol (EtOH) on mouse preimplantation development using an in vitro culture method. Embryos at the 1-cell, 2-cell, or 4-cell stage were exposed for 24 h to medium containing EtOH, then further cultured without EtOH to determine their ability to form blastocysts and to eventually hatch from the zona pellucida. EtOH exposure either arrested or enhanced normal development, depending on dose and embryonic stage of exposure. Exposure of 1-cell and 2-cell embryos to 1.6% (w/v) EtOH decreased blastocyst formation and hatching, and exposure of 1-cell embryos to 0.4% EtOH inhibited their development. At 0.1%, EtOH had an opposite effect, causing an increase in the percent blastocyst formation of treated 1-cell and 2-cell embryos. Neither inhibition nor stimulation of blastocyst formation occurred in 4-cell embryos exposed to 0.1-1.6% EtOH. Using an in vitro outgrowth model of implantation, embryos that reached the blastocyst stage were further tested for their ability to produce differentiated trophoblast cells. Blastocysts previously exposed to 0.1% EtOH during the 1-cell stage appeared to form adhesive trophoblasts earlier than control embryos, indicating that EtOH exposure can induce precocious differentiation of the trophoblast cells. The EtOH treated blastocysts contained significantly more cells than control blastocysts. These results indicate that EtOH can alter preimplantation development by either inhibiting or accelerating cell growth and differentiation.

Animals↗

PubMed1993

Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential.

A thermodynamic model was used to evaluate and optimize a rapid three-step nonequilibrium freezing protocol for one-cell mouse embryos in the absence of cryoprotectants (CPAs) that avoided lethal intracellular ice formation (IIF). Biophysical parameters of one-cell mouse embryos were determined at subzero temperatures using cryomicroscopic investigations (i.e., the water permeability of the plasma membrane, its temperature dependence, and the parameters for heterogeneous IIF). The parameters were then incorporated into the thermodynamic model, which predicted the likelihood of IIF. Model predictions showed that IIF could be prevented at a cooling rate of 120 degrees C/min when a 5-min holding period was inserted at -10 degrees C to assure cellular dehydration. This predicted freezing protocol, which avoided IIF in the absence of CPAs, was two orders of magnitude faster than conventional embryo cryopreservation cooling rates of between 0.5 and 1 degree C/min. At slow cooling rates, embryos predominantly follow the equilibrium phase diagram and do not undergo IIF, but mechanisms other than IIF (e.g., high electrolyte concentrations, mechanical effects, and others) cause cellular damage. We tested the predictions of our thermodynamic model using a programmable freezer and confirmed the theoretical predictions. The membrane integrity of one-cell mouse embryos, as assessed by fluorescein diacetate retention, was approximately 80% after freezing down to -45 degrees C by the rapid nonequilibrium protocol derived from our model. The fact that embryos could be rapidly frozen in the absence of CPAs without damage to the plasma membrane as assessed by fluorescein diacetate retention is a new and exciting finding. Further refinements of this protocol is necessary to retain the developmental competence of the embryos.

Animals↗

PubMed1993

Recombinant entactin promotes mouse primary trophoblast cell adhesion and migration through the Arg-Gly-Asp (RGD) recognition sequence.

In vitro culture of mouse blastocysts during the period coinciding with implantation has revealed that primary trophoblast cells can adhere and migrate in serum-free medium when provided with certain extracellular matrix components, including fibronectin and laminin. Tightly associated with laminin is the glycoprotein, entactin, that may play an important role in basement membrane assembly and cell attachment. Mouse blastocysts were studied using this in vitro model to determine whether entactin was capable of mediating trophoblast invasive activity. Although entactin has never been shown to promote cell migration, we report here that recombinant entactin supported blastocyst outgrowth in a dose-dependent manner, with a maximal effect at 20-50 micrograms/ml. The ability of trophoblast cells to adhere and migrate on entactin was specifically inhibited by anti-entactin antibody, but not by antibodies raised against laminin. The synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, that contains the Arg-Gly-Asp (RGD) integrin recognition site, reversibly inhibited entactin-mediated blastocyst outgrowth in a dose-dependent manner, but had no effect on laminin-mediated outgrowth. The synthetic peptide, Gly-Phe-Arg-Gly-Asp-Gly-Gln, that comprises the actual RGD-containing sequence within entactin, promoted trophoblast outgrowth when immobilized on the substratum. Furthermore, a mutated recombinant entactin, altered to contain a Glu in place of Asp at the RGD site, provided no trophoblast cell adhesive activity. We conclude that entactin promotes trophoblast outgrowth through a mechanism mediated by the RGD recognition site, and that it may play an important role during invasion of the endometrial basement membrane at implantation.

Amino Acid Sequence↗

PubMed1992

Purification of a tumor-specific PNA-binding glycoprotein, gp200, from a human embryonal carcinoma cell line.

A 200-kDa peanut agglutinin (PNA)-binding glycoprotein, gp200, has been purified and partially characterized from the human embryonal carcinoma cell line, HT-E (833k). Tissue distribution analysis of this molecule by lectin blotting with PNA of detergent-extracted proteins from human cell lines and tissues demonstrated expression limited to nonseminomatous germ cell tumors. The 200-kDa protein was purified with lectin affinity and gel filtration chromatography. Purification to apparent homogeneity was demonstrated by one- and two-dimensional gel electrophoresis. Characterization of gp200 revealed it to be a surface integral membrane glycoprotein; however, gp200 could also be purified from the culture media of EC cells, suggesting gp200 has an extracellular role. The carbohydrate groups of gp200 are N-linked and partially sialylated and contain terminal galactose residues. These initial studies suggest that the PNA-defined glycoprotein, gp200, is a candidate for a nonseminomatous germ cell tumor marker.

Cell Line↗

PubMed1992

In vitro exposure of preimplantation mouse embryos to cocaine and benzoylecgonine inhibits subsequent development.

Cocaine is used by over 20% of women of reproductive age. Although there have been numerous studies focusing on its effects on reproductive processes, none has evaluated its direct effect on preimplantation development. We have investigated the effect of cocaine and its major metabolite, benzoylecgonine, on in vitro preimplantation mouse embryogenesis. One-cell embryos were exposed at the one-, two-, four-, or eight-cell stage for 24 hr to medium containing 0-400 micrograms/ml cocaine or benzoylecgonine and then cultured to the blastocyst stage. Cocaine had its strongest inhibitory effect at the earliest stages of development. At the one- and two-cell stages, there was a significant inhibition of blastocyst formation following exposure to cocaine concentrations of 25-400 micrograms/ml, and at the four-cell stage there was an inhibitory effect at 100 and 400 micrograms/ml cocaine. Benzoylecgonine inhibited the development of embryos to blastocyst only at the one- and two-cell stages, at concentrations of 100-400 micrograms/ml. These findings suggest that cocaine is capable of blocking preimplantation embryogenesis, particularly following exposure at the earliest stages, and that this toxicity may abate as cocaine is biotransformed to benzoylecgonine.

Animals↗

PubMed1991

Cryomicroscopic analysis of intracellular ice formation during freezing of mouse oocytes without cryoadditives.

Kinetics of intracellular ice formation (IIF) under various freezing conditions was investigated for mouse oocytes at metaphase II obtained from B6D2F1 mice. A new cryostage with improved optical performance and "isothermal" temperature field was used for nucleation experiments. The maximum thermal gradient across the window was less than 0.1 degrees C/10 mm at sample temperatures near 0 degrees C. The dependence of IIF on the initial concentration of the suspending medium was found to be pronounced. The mean IIF temperatures were found to be -9.56, -12.49, -17.63, -22.20 degrees C for freezing at 120 degrees C/min in 200, 285, 510, and 735 mosm phosphate-buffered saline, respectively. For concentrations higher than 735 mosm, the kinetics of IIF showed a break point at approximately -31 degrees C. Below -31 degrees C, all the remaining unfrozen oocytes underwent IIF almost immediately over a temperature range of less than 3 degrees C. This dramatic shift in the kinetics of IIF suggests that there were two distinct mechanisms responsible for IIF during freezing. The effect of the cooling rate on the kinetics of IIF was also investigated in isotonic PBS. At 1 degrees C/min none of the oocytes contained ice, whereas, at 5 degrees C/min all the oocytes contained ice. The mean IIF temperatures for cooling rates between 1 and 120 degrees C/min were almost constant with an average of -12.82 +/- 0.6 degrees C (SEM). In addition, constant temperature experiments were conducted in isotonic PBS. The percentages of oocytes with IIF were 0, 50, 60, and 95% for -3.8, -6.4, -7.72, and -8.85 degrees C. In undercooling experiments, IIF was not observed until approximately -20 degrees C (at which temperature the whole suspension was frozen spontaneously), suggesting the involvement of the external ice in the initiation of IIF between approximately -5 and -31 degrees C during freezing of oocytes.

Animals↗

PubMed1991

Cell interactions with laminin and its proteolytic fragments during outgrowth of mouse primary trophoblast cells.

Mouse blastocysts in serum-free culture for 24-48 h become attachment-competent, adhere to fibronectin- or laminin-coated surfaces, and subsequently form trophoblast outgrowths. The blastocyst laminin receptor was characterized in outgrowth studies using modified laminin. Trophoblast cells interacted with the peptide portion of laminin, but not the oligosaccharide moiety since its adhesive activity was reduced by boiling or trypsin treatment, but not by treatments that removed or modified its carbohydrate. Laminin outgrowth-promoting activity was further localized within its structural domains by use of the well-characterized proteolytic fragments of laminin, E1-4, and E8, and a synthetic peptide, CDPGYIGSR. The E1-4 fragment of laminin did not promote embryo outgrowth. However, the E8 fragment, which contains a heparin-binding domain as well as sites recognized during cell adhesion and neurite outgrowth, vigorously promoted outgrowth in both the presence and absence of heparin, heparan sulfate, or heparinase. Consistent with these results, outgrowth on intact laminin was not inhibited by CDPGYIGSR, a sequence within the E1-4 fragment that is known to mediate the adhesion of some cell types. It is concluded from these results that early trophoblast cells adhere to peptide in the E8 domain of laminin using a mechanism that is independent of the one used for adhesion to fibronectin.

Amino Acid Sequence↗

PubMed1990

Water transport and estimated transmembrane potential during freezing of mouse oocytes.

The kinetics of water transport and the changes in transmembrane potential during freezing of mouse oocytes in isotonic phosphate buffered saline (PBS) were simulated using thermodynamic models. The permeability to water at 0 degree C, Lpg, and the activation energy, ELp, of metaphase II mouse oocytes from B6D2F1 mice were determined to be 0.044 +/- 0.008 micron/min-atm and 13.3 +/- 2.5 kcal/mol during freezing at 2 degrees C/min. The inactive cell volume was determined to be 0.214 with a correlation coefficient of 0.995, indicating that the oocytes closely follow the ideal Boyle-van't Hoff relation. The mean value of the oocyte diameter was 79.41 +/- 4.62 microns. These results were used to predict the behavior of mouse oocytes under various freezing conditions. The effect of the cooling rate on the cell volume and cytoplasm undercooling was investigated. The changes in transmembrane potential were also investigated during freezing of mouse oocytes. The computer simulations showed that at the beginning of the freezing process (-1 degrees C), the fast growth of ice in the extracellular solution causes a sharp increase of the membrane potential. It is predicted that the change in membrane potential is substantial for almost all cooling rates. Estimations show that values as high as -90 mV may be reached during freezing. The hyperpolarization of the membrane may cause orientation of the dipoles within the membrane. For membrane proteins with 300 debye dipole moment, the theoretical prediction suggests that the percentage of dipoles aligned with the membrane potential increases from 16% at 0 degrees C prior to freezing to 58% at -8 degrees C after seeding of the external ice followed with a cooling at 120 degrees C/min.

Animals↗

PubMed1990

The influence of chamber characteristics on the reliability of sperm concentration and movement measurements obtained by manual and videomicrographic analysis.

To assess the influence of chamber design and depth on the accuracy and precision of sperm measurements, manual counting of a standardized latex bead solution and automated sperm motility measurements were made using a Makler chamber (Sefi Medical Industries, Haifa, Israel), Neubauer hemocytometer (American Optical Company, Buffalo, NY), and a new, disposable device (Micro-Cell; Cyto Fluidics, Inc., Silver Spring, MD). Bead counts obtained with the Micro-Cell chamber or hemocytometer were not statistically different from those determined by electronic particle counting, whereas Makler chamber counts were 62% higher. Makler counts had a significantly higher standard deviation, suggesting that counts made with this device are less reproducible. Analyzing live sperm samples, the percentage of motile sperm determined using Micro-Cell and Makler chambers were similar. However, significant differences in sperm concentration and mean velocity were found. The Micro-Cell disposable chamber provided consistent and accurate data on sperm concentration, percent motility, and mean velocity. These differences in sperm measurements emphasize the importance of sampling chamber characteristics on data reliability.

Cell Survival↗