Isolation of motile spermatozoa: Comparison of Percoll centrifugation, SpermPrep filtration, and swim-up techniques

1993 ◽  
Vol 10 (7) ◽  
pp. 485-487 ◽  
Author(s):  
S�ren Ziebe ◽  
Claus Yding Andersen
Keyword(s):  
Percoll Centrifugation

Role of diacylglycerol in adrenergic-stimulated 86Rb uptake by proximal tubules

1990 ◽  
Vol 258 (5) ◽  
pp. F1133-F1138 ◽  
Author(s):  
A. D. Baines ◽  
R. Drangova ◽  
P. Ho
Keyword(s):  
Protein Kinase ◽  
Proximal Tubule ◽  
Proximal Tubules ◽  
K Channel ◽  
Rat Proximal Tubule ◽  
Percoll Centrifugation

We used rat proximal tubule fragments purified by Percoll centrifugation to examine the role of diacylglycerol (DAG) in noradrenergic-stimulated Na+ reabsorption. Tubular DAG concentration and ouabain-inhibitable 86Rb uptake increased within 30 s after adding norepinephrine (NE) and remained elevated for at least 5 min. NE (1 microM) increased DAG content 17% and ouabain-inhibitable 86Rb uptake 23%. Cirazoline-stimulated 86Rb uptake was not inhibited by BaCl, quinidine, or bumetanide (1-10 microM) or by the omission of HCO3- or Cl- from the medium, but it was completely inhibited by ouabain and furosemide. Oleoyl-acetyl glycerol, L-alpha-1,2-dioctanoylglycerol, and L-alpha-1,2-dioleoylglycerol (DOG) increased total 86Rb uptake 8-11%. 12-O-tetradecanoylphorbol-13-acetate (TPA) (5 nM) increased uptake by only 4%. Staurosporine at 5 nM inhibited DOG stimulation completely, whereas 50 nM staurosporine was required to inhibit NE stimulation completely. Sphingosine inhibited DOG stimulation by 66% but did not inhibit NE stimulation. Amiloride (1 mM) completely blocked DOG stimulation. Monensin increased 86Rb uptake 31% and completely blocked the DOG effect but reduced the NE effect by only 26% (P = 0.08). In tubules from salt-loaded rats, NE did not increase DAG concentration, but NE-stimulated 86Rb uptake was reduced by only 23% (P = 0.15). Thus DAG released by NE may stimulate Na+ entry through Na(+)-H+ exchange. NE predominantly stimulates Na(+)-K(+)-adenosinetriphosphatase (ATPase) by activating a protein kinase that is insensitive to DAG and TPA and is inhibited by staurosporine but not by sphingosine. NE may also stimulate K+ efflux through a BaCl-insensitive K+ channel that is inhibited by millimolar furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Gastric estrogen directly induces ghrelin expression and production in the rat stomach

2006 ◽  
Vol 190 (3) ◽  
pp. 749-757 ◽  
Author(s):  
Ichiro Sakata ◽  
Toru Tanaka ◽  
Mami Yamazaki ◽  
Takashi Tanizaki ◽  
Zhao Zheng ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Male Rat ◽  
Pcr Analysis ◽  
Regulation Mechanism ◽  
Female Rat ◽  
Rat Stomach ◽  
Ghrelin Gene ◽  
Cell Enrichment ◽  
Percoll Centrifugation

Ghrelin, an endogenous ligand for the GH secretagogue receptor, is predominantly produced in the stomach. Little is known about the regulation mechanism of gastric ghrelin. Here, we report that estrogen synthesized in the stomach induces rat gastric ghrelin gene expression and production. We established a gastric ghrelin cell enrichment method using Percoll centrifugation and then studied the effect of estrogen and/or its antagonist on ghrelin expression and production. Treatment with estrogen for 8 h significantly increased the level of ghrelin expression, and ICI-182 780, an estrogen receptor (ER) antagonist, completely reversed this effect. Reverse transcriptase-PCR analysis clearly showed that ERα and aromatase are expressed in the female rat stomach. Moreover, treatment with an aromatase inhibitor, 4-hydro-xyandrostenedione (formestane), significantly decreased the level of ghrelin mRNA expression in minced stomach tissue. In vivo studies revealed that the ghrelin mRNA expression and production did not change in gonadectomized rat 3 weeks after surgery. These results strongly suggest that estrogen produced in the stomach directly induces ghrelin expression and production in both female and male rat stomachs.

222 EFFECT OF THE PRESENCE OR ABSENCE OF PERCOLL CENTRIFUGATION; PENICILLAMINE, HYPOTAURINE, AND EPINEPHRINE; AND HEPARIN ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 259 ◽  
Author(s):  
J. F. Hasler ◽  
J. E. Stokes
Keyword(s):  
Percoll Gradient ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Rat Liver Cells ◽  
Lactate Medium ◽  
Bovine Oocytes ◽  
Percoll Centrifugation

Protocols for the production of bovine embryos in vitro routinely include Percoll centrifugation of semen, usually include heparin, and often include penicillamine, hypotaurine, and epinephrine (PHE) in the fertilization media. This study examined the contribution of each of these components to the success of in vitro fertilization of bovine oocytes and subsequent blastocyst development. Bovine oocytes were aspirated from 2- to 10-mm follicles within 5 h after slaughter of cattle at a local abattoir. Groups of 30 to 40 cumulus–oocyte complexes (COC) were matured in 0.5 mL of TCM-199 with 10% FCS, 4 µg mL–1 of FSH, and 6 µg mL–1 of LH (NOBL Laboratories, Sioux Center, IA, USA) for 24 h (39°C, 4% CO2 in air). The COC were then washed and placed in 0.5 mL of modified Tyrode-lactate medium for IVF with various combinations of 2 µg mL–1 of heparin, 20 µM penicillamine, 10 µM hypotaurine, and 1 µM epinephrine. Each group of COC was inseminated with 0.25 × 106 frozen–thawed sperm from a single bull after 30 min of centrifugation with (Exp. 1) or without (Exp. 2) a 45/90% Percoll gradient with sperm TALP. Oocytes were vortexed to remove the cumulus after 18 h and placed in co-culture wells containing a monolayer of buffalo rat liver cells and 0.5 mL of Menezo’s B2 medium supplemented with 10% FCS. On the fourth day of in vitro culture, cleavage was defined as 2 cells or greater and embryos were transferred to fresh co-culture wells. There were 4 replicates in the first experiment and 6 in the second. Data were analysed by ANOVA. In the first experiment, the use of a Percoll gradient during centrifugation for separation of viable sperm from seminal plasma and cryprotectants resulted in significantly higher cleavage and Day 8 blastocyst rates than did the absence of Percoll when PHE and heparin were used together, and both cleavage and blastocyst rates were lower when only PHE or heparin was used separately compared with when both were used together (Table 1). The absence of Percoll, PHE, and heparin resulted in the lowest rates of cleavage and development. In the second experiment, the absence of either PHE or heparin resulted in lower cleavage rates, but not blastocyst rates, compared with the use of both, and the absence of both resulted in the lowest cleavage and blastocyst rates in spite of the use of Percoll. Table 1.Effects of Percoll; penicillamine, hypotaurine, and epinephrine (PHE); and heparin on cleavage and subsequent embryo development per oocyte

Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations

1986 ◽  
Vol 22 (4) ◽  
pp. 201-211 ◽  
Author(s):  
Bill L. Kreamer ◽  
Jeffrey L. Staecker ◽  
Norimasa Sawada ◽  
Gerald L. Sattler ◽  
M. T. Stephen Hsia ◽  
...  
Keyword(s):  
Rat Hepatocyte ◽  
Low Speed ◽  
Centrifugation Method ◽  
Percoll Centrifugation ◽  
Isolated Rat

Mechanisms of extracellular hydrogen peroxide clearance by alveolar type II pneumocytes

1990 ◽  
Vol 69 (6) ◽  
pp. 2078-2084 ◽  
Author(s):  
P. C. Engstrom ◽  
L. Easterling ◽  
R. R. Baker ◽  
S. Matalon
Keyword(s):  
Hydrogen Peroxide ◽  
Trypan Blue ◽  
Enzymatic Digestion ◽  
Alveolar Type ◽  
Type Ii ◽  
Oxidant Injury ◽  
Minimum Essential Medium ◽  
Atii Cell ◽  
Type Ii Pneumocytes ◽  
Percoll Centrifugation

This study quantified the ability of freshly isolated alveolar type II (ATII) pneumocytes to reduce extracellularly produced hydrogen peroxide (H2O2) and identify the mechanisms involved. ATII cells were isolated to high purity (greater than 85%) from rabbit lungs by enzymatic digestion and Percoll centrifugation and suspended in Eagle's minimum essential medium (MEM). They were then coincubated with either 500 microM xanthine and 10 mU/ml xanthine oxidase (XO; pH 7.4; 25 degrees C) or 300 microM H2O2. The extracellular H2O2 concentration [H2O2] was measured in the following conditions over a 60-min period: 1) MEM alone, 2) untreated (control), 3) 3-amino-1,2,4-triazole (ATZ)-treated, or 4) 1-chloro-2,4-dinitrobenzene-treated ATII cells. Addition of xanthine and XO to MEM alone resulted in a time-dependent increase in [H2O2], reaching a plateau value of approximately 300 microM after 45 min. In the presence of control ATII cells (1 x 10(6) cells/ml), [H2O2] remained at control levels. When coincubated with 300 microM H2O2, ATII cells cleared H2O2 at a higher rate than an equivalent amount of free catalase. Incubation with ATZ decreased ATII cell catalase activity by 89% and significantly impaired their ability to clear H2O2 (half-life = 18.1 +/- 2.7 vs. 1.3 +/- 0.1 min, P less than 0.01). ATZ-treated cells were more susceptible to oxidant injury, as shown by their decreased ability to exclude trypan blue after 60 min of H2O2 exposure. On the other hand, glutathione-depleted cells scavenged H2O2 at the same rate as controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Percoll centrifugation eliminates mold contaminants from cell cultures

1991 ◽  
Vol 27 (4) ◽  
pp. 273-276 ◽  
Author(s):  
Patricia A. Kruk ◽  
Nelly Auersperg
Keyword(s):  
Cell Cultures ◽  
Percoll Centrifugation

Bulk isolation of renal PCT and PST. I. Glucose-dependent metabolic differences

1990 ◽  
Vol 259 (1) ◽  
pp. F164-F175 ◽  
Author(s):  
C. E. Ruegg ◽  
L. J. Mandel
Keyword(s):  
Oxygen Consumption ◽  
Oxidative Metabolism ◽  
Metabolic Regulation ◽  
Glucose Utilization ◽  
Leucine Aminopeptidase ◽  
Glycolytic Enzyme ◽  
Metabolic Substrate ◽  
Distal Marker ◽  
Proximal Tubular ◽  
Percoll Centrifugation

A new procedure for separately isolating milligram quantities of rabbit renal proximal straight (PST) or convoluted (PCT) tubules is described, and the differential abilities of these segments to utilize glucose as a metabolic substrate are investigated. Separate dissection of the cortical cortices and the outer medullary stripe, followed by collagenase digestion and discontinuous Percoll centrifugation, provide enriched populations (greater than 98% pure) of PCT (37 mg) and PST (14 mg), respectively, per rabbit. The purity of PCT and PST fractions was quantitated morphologically and by comparing the enriched activity of the proximal tubular marker leucine aminopeptidase and deenriched activity of the distal marker hexokinase to previously published values reported from microdissection studies. To investigate glucose-dependent metabolic differences, PCT and PST suspensions (1 mg/ml) were preincubated in Dulbecco's modified Eagle's-Ham's F-12 medium for 1 h before being incubated for 30 min in buffer with or without glucose as the only available metabolic substrate. In glucose-containing buffer, PST segments maintained their oxygen consumption and ATP contents at levels significantly higher than PCT segments. These differential responses between PST and PCT were glucose-dependent because they were abolished when segments were incubated under glucose-free conditions. Because responses in PCT were glucose-independent, these results suggest that PCT cannot utilize glucose to support oxidative metabolism, whereas PST segments can oxidatively metabolize this substrate. These differences in glucose utilization do not correlate with the distribution of glycolytic enzyme activities, suggesting that differential metabolic regulation of these enzymes may determine the ability of each segment to utilize glucose.

Functional capacity and fertilizing longevity of frozen-thawed scimitar-horned oryx (Oryx dammah) spermatozoa in a heterologous in vitro fertilization system

2000 ◽  
Vol 12 (8) ◽  
pp. 413 ◽  
Author(s):  
Justine K. O'Brien ◽  
Terri L. Roth
Keyword(s):  
In Vitro Fertilization ◽  
Functional Capacity ◽  
Fertilization Success ◽  
Transport Mechanisms ◽  
Vitro Fertilization ◽  
Sperm Characteristics ◽  
Fertilization System ◽  
Percoll Centrifugation

This study was conducted to determine if cryopreservation and thawing reduces the quality of scimitar-horned oryx spermatozoa and thus might be responsible for sub-optimal artificial insemination (AI) efficiency. Functional capacity of frozen–thawed oryx spermatozoa was compared in a heterologous bovine in vitro fertilization (IVF) system after being prepared by four methods. Fertilizing longevity was also assessed after thawing and pre-incubating spermatozoa for 12 or 24 h before IVF. Sperm characteristics (viability, morphology, acrosomal and capacitation status) were superior for samples prepared by Percoll centrifugation and standard swim-up compared with microdrop swim-up and wash methods. Regardless of variation in sperm characteristics over time, fertilization success and embryo development were high and did not differ among treatments. Fertilization and cleavage success for spermatozoa pre-incubated for 12 h before IVF were comparable with that achieved with non-incubated spermatozoa. Even 24 h after thawing, spermatozoa were capable of fertilizing oocytes, but percentage fertilization and embryo cleavage were significantly lower than for spermatozoa pre-incubated for 12 h. Overall, functional capacity of oryx spermatozoa after thawing appears comparable with that of domestic bull spermatozoa. When used for AI, frozen—thawed oryx spermatozoa should be capable of fertilizing oocytes in females ovulating 12 or even 24 h after insemination, providing sperm transport mechanisms are adequate. The functional capacity and fertilizing longevity of oryx sperm after thawing is high, and therefore unlikely to be responsible for decreased AI efficiency in the scimitar-horned oryx.

Sign in / Sign up

Export Citation Format

Share Document