Percoll Density Gradient Centrifugation Research Articles

Immunopurification of B lymphocytes from sea bass Dicentrarchus labrax (L.).

The monoclonal antibody DLIg3, specific for immunoglobulins and B cells of the teleost fish Dicentrarchus labrax (Mediterranean sea bass), was used to enrich immunoreactive cells from peripheral blood, spleen, and head kidney leukocytes. The purification was performed by immunomagnetic sorting of leukocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by immunofluorescence and cytofluorimetric analysis. Following a single immunopurification step, the percentages of DLlg3-purified cells were 61% +/- 6% from peripheral blood leukocytes, 66% +/- 5% from splenocytes, and 77% +/- 9% from head kidney cells. DLIg3-immunopurified cells, from the head kidney of antigen-primed fish displayed an enhanced proliferation index when incubated with the immunization antigen. DLIg3-purified cells from head kidney lymphoid tissue were employed for RNA extraction and complementary DNA synthesis, and in reverse transcriptase polymerase chain reaction experiments using specific primers corresponding to the sequences of the sea bass immunoglobulin light chain, and of T-cell receptor. DLIg3-purified cells displayed enhanced expression of the immunoglobulin gene, and lower expression of T-cell receptor.

Binding of Zona Binding Inhibitory Factor-1 (ZIF-1) from Human Follicular Fluid on Spermatozoa

Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.

Identification of cytokeratins in bovine sperm outer dense fibre fractions.

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)-insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll(R) density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB-insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS-insoluble material was collected and quantitatively dissolved in 8 M urea. SDS-gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti-cytokeratin antibodies detected two urea-soluble, SDS-insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45-kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1-18 and CK 20), KL1, was the only antibody that reacted with the 66-kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope-like structures, we suggest that these intermediate filaments play an important structural or tension-bearing role in sperm flagella.

In vivo activation of pro-form Bombyx cysteine protease (BCP) in silkmoth eggs: localization of yolk proteins and BCP, and acidification of yolk granules

The present study was designed to investigate the process of acidification of yolk granules during embryogenesis. In oocytes of mature Bombyx mori silkmoth, yolk proteins and a cysteine protease (pro-form BCP) were found in yolk granules. BCP was localized in small sized yolk granules (SYG, 3–6 μm in diameter) and yolk proteins in large sized granules (LYG, 6–11 μm in diameter), which might result in a spatial separation of protease and its substrates to avoid unnecessary hydrolysis. The granules were isolated on Percoll density gradient centrifugation. Although separation of LYG and SYG was incomplete, the granules sedimented in different fractions when using unfertilized egg extract, in which LYG was recovered from heavier fractions and BCP from lighter fractions. Acid phosphatase, as well as other lysosomal marker enzymes tested, was recovered from LYG-containing fractions. When extracts were prepared from developing eggs (day 3), some BCP-containing granules co-sedimented with LYG. The inactive pro-form BCP was activated in vivo, in parallel with yolk protein degradation, and as demonstrated previously in vitro under acidic conditions ( Takahashi et al., 1993). These results suggest that acidification occurs in yolk granules during embryogenesis. This was also confirmed using acridine orange fluorescent dye. In early development, most yolk granules were neutral, but became acidic during embryonic development. SYG were progressively recovered in heavier density fractions, displaying acidic interior. In this fraction, BCP-containing granules seem to be associated with larger granules (6–11 μm in size). In addition, SYG (BCP containing granules) were likely to be acidified earlier than LYG. Our results suggest that acidification initiates yolk degradation through activation of pro-form BCP.

Metabolic and ultrastructural responses of lupine embryo axes to sugar starvation

Embryo axes isolated from germinating lupine seeds were cultivated in vitro for 24-96 h over media containing either 60 mmol/L sucrose or no sucrose. Ultrastructural studies showed that large vacuoles were accumulating in a central region of primary parenchyma cells in sucrose starved lupine embryo axes, whereas cytoplasm along with organelles were forced to a periphery of the cells. We suggest that the autolysis of cytoplasmic proteins contributes to the accumulation of the vacuoles and this suggestion is consistent with the results of the characterisation of protein content. The level of cytosolic proteins was reduced by 50% and the activity of cytosolic marker enzyme, PEP carboxylase, was reduced by 46% in starved embryos as compared to control. The mitochondria from starved tissues were not degraded. The level of mitochondrial proteins was reduced by only 10% and the activity of mitochondrial NAD-isocitrate dehydrogenase decreased by 8% as a result of starvation. As demonstrated by the results of Percoll density gradient centrifugation, sucrose starvation caused an increase of 49% in many of the higher density mitochondria fractions, whereas many of the lower density mitochondria fractions were decreased by 33%. The samples of mitochondria from starved embryo axes were determined to have higher respiration activity in the presence of glutamate and malate as compared to control samples. EPR-based analyses of free radicals showed the presence of free radicals with a signal at g = 2.0060 in embryo axes. The level of the radical was two times higher in sucrose-starved embryo axes than in control (the level of this radical increased in senescing plant tissues as well). The results of EPR-based quantitation of Mn2+ ions revealed that the level was a few times higher in starved material than in control. Starved embryo axes, however, do possess a number of adaptive mechanisms protecting them from oxidative damage. Densitometric analyses of gels revealed an increase in the activity of SOD in sugar-starved embryos, whereas CAT and POX activities were lower in axes grown without sucrose as compared to control. Superoxide dismutase, catalase and peroxidase zymogram analyses showed that synthesis of new isoforms was not induced by sugar starvation. An accumulation of phytoferritin was found in plastids of sucrose starved embryos. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.

Removal of T and B lymphocytes by in-line filtration: evaluation of the efficiency of a polyester filter type (Pall WBF-2) by flow cytometric counting.

The aim of this study was to investigate whether in-line filtration, using a polyester filter for the preparation of red cell concentrates (RCC) and plasma (PL), leads to an altered proportion of T and B lymphocytes in the fraction of residual white blood cells (WBC). The capacity of Pall WBF-2 in-line filters to reduce the numbers of T and B lymphocytes from red blood cell concentrates (RCC) and plasma (PL) of 22 donations was investigated by three-colour flow cytometry (FC) using the Tritest-Trucount kit. T and B lymphocytes were identified using monoclonal antibodies (mAbs) against CD3, CD19 and CD45, conjugated with fluorescein isothiocyanate, phycoerythrin or peridinin chlorophyll protein-A, respectively. As the number of B cells was below the detection limit of the FC method, WBC of the respective blood components of healthy donors were concentrated 25-fold by Percoll density-gradient centrifugation. In this fraction the absolute numbers of T and B cells, as well as their ratio, were determined using the Attractor software, which provides a discrimination of rare cell counts from FC in relation to debris. The mean numbers, as well as minima and maxima of T and B lymphocytes per unit, were as follows. T cells in RCC: 4.51 x 10(3) (1.68 x 10(2)-4.09 x 10(4)) and in PL: 1.35 x 10(3) (2.21-1.78 x 10(4)); B cells in RCC: 2.33 x 10(3) (7.10 x 10(1)-9.15 x 10(3)) and in PL: 2.33 x 102 (7.5 x 10(2)-2.8 x 10(3)). T cells were retained, on average, at a higher level than B cells: 3.01 times higher in RCC and 1.01 times higher in PL. After filtration, the ratio of T and B lymphocytes changed in RCC (1.95 : 1) compared with unfiltered blood, where it was 5.83 : 1. In PL the ratio did not change notably compared to unfiltered blood. The results of this research show that cell concentration (using gradient centrifugation) in combination with an appropriate FC acquisition and analysis procedure, allows both residual T and B lymphocytes (being under the detection limit without cell concentration) to be determined.

Sputum processing: a new method to improve cytospin quality

Sputum examination is being increasingly used as a non-invasive method for studying airway inflammation. However, the application of sputum still presents some methodological problems and the results of sputum analysis may be substantially flawed by salivary contamination, cell and mucus debris. In addition, much work is needed to deepen the possibility of extensive application of cell and molecular biology techniques to sputum analysis. In an attempt to improve the technique of sputum processing, we investigated the effect of: (i) 20 and 11 microm filtration in addition to 40 microm on salivary contamination; (ii) Percoll density gradient centrifugation on sputum slides quality; (iii) a culture medium (Minimum Essential Medium containing HEPES 22 mm, pH 7.4: MEM) as washing and suspension solution compared to PBS on cell viability. Induced sputum samples were obtained in 37 asthmatics. 21 samples were processed as selected sputum and 16 samples as entire expectorates. After dithiotreitol (DTT) homogenization, each specimen was aliquoted in two parts of equal volume. One portion was processed with the usual method, the other using a modified method: cell pellet was suspended in sterile MEM, filtered through 40, 20 and 11 microm net filters and separated from the residual debris by Percoll gradient centrifugation. As compared to the current sputum processing this method resulted in: (i) no selective bronchial cellular loss; (ii) a significant decrease of salivary contamination, particularly in entire expectorates in which squamous cells were reduced from 47 (36) to 15.5% (20) as median values and interquartile range; (iii) a higher proportion of good quality cytospins; (iv) maintenance of cell viability over the time (88% vs. 81% in MEM and PBS, respectively) 1 h after sample collection. In the present study we demonstrated that the proposed method is feasible and makes it possible to overcome most of the technical limits met with the commonly used method, pointing to a potential extension of induced sputum application for more sophisticated techniques.

Rat small hepatocytes for liver support: growth and metabolic activities in culture

It has been reported that small hepatocytes need to be cocultivated with nonparenchymal cells for proliferation in vitro. The purpose of this study was to investigate whether purified small hepatocytes would proliferate in the absence of nonparenchymal cells and to determine the their metabolic activities in culture. Purified small hepatocytes were obtained by centrifugation in Percoll density gradients. Small hepatocytes were seeded on collagen-coated dishes at a cell density of 4 × 104 cells/cm2 and were cultured in a hormonally modified Williams' medium E with 10 mM nicotinamide (WE-HN: group I). Comparision of 1 mM dimethyl sulfoxide (DMSO) (group II) or 5 mM retinoic acid (group III) in WE-HN was performed. Cell proliferation, as indicated by DNA content, and metabolic function, as assessed by urea synthesis, gluconeogenesis, and albumin production, were measured for 14 days. In each group, DNA content increased significantly during 14 days in culture. An increase of approximately 400% was observed in group II. The cell proliferation of group II was confirmed by microscopic observations. Urea synthesis and gluconeogenesis in each group declined gradually from days 1 to 14. Albumin production also fell, but to a less extent. We found that the purified small hepatocytes could proliferate without cocultivation of nonparenchymal cells. Further, they expressed metabolic activities of mature hepatocytes.

Rapid and intense induction of apoptosis in human eosinophils by anti-CD30 antibody treatment in vitro

79 Rapid and Intense Induction of Apoptosis in Human Eosinophils by Anti-CD30 Antibody Treatment In Vitro Maki Terakawa*, Kenji Matsumoto§, Kenju Miura*, Hirohisa Saito¥ *Suntory Biomed Research Limited, Osaka, Japan §National Children's Medical Research Center, Tokyo, Japan ~National Children's Hospital, Tokyo, Japan Apoptosis is an important cellular mechanism for controlling cell viability and proliferation. With respect to eosinophils, cytokines prolong their survival while corticosteroids reduce their survival in vitro. CD30, a member of the TNF/NGF receptor family, is reported to be expressed on the surface of many cell types including Hodgkin's lymphoma cells. CD30 is capable of inducing apoptosis after antibody treatment on some cell lines but not all. To determine whether this surface structure is involved in apoptosis of eosinophils, we examined the effect of anti-CD30 antibody treatment on the viability of human eosinophil. Eosinophils were purified from human peripheral blood with percoll density gradient centrifugation and negative selection with immunomagnetic beads. Purified eosinophils (> 99%) were suspended in RPMI-1640 medium supplemented with 10% FCS and 1 ng/ml IL-5 at a cell density of 5x105/ml. Expression of CD30 on eosinophils was examined by flow cytometry. Eosinophils were cultured in 24-well culture plate previously coated with various concentrations of anti-CD30 antibodies (1 to 1,000 ng/ml) or control antibody (irrelevant IgG 1 control mAb, 100 ng/ml), and cell viability was examined by FITC-conjugated Annexin V and propium iodide after 4 hrs of culture. Resting eosinophils expressed low but consistent amounts of CD30 (Mean Fluorescent Intensity: 8.9_+ 1.4 vs 2.6 _+ 0.2 for anti-CD30 (AC10) and irrelevant IgG1 mAb, respectively, n = 5, p 1 ng/ml anti-CD30 antibody in a concentrationand time-dependent manner. %Annexin V ÷ cells at 4 hrs was: 5.3 +0.9, 58.7 -+ 6.7, 64.2 _+ 4.6, or 3.8 _+ 0.7, for IgG1 control mAb, anti-CD30 (HRS-4), anti-CD30 (Bet-H8), or anti-CD30 (Ber-H2), respectively (Mean -+ SEM, n=6-8, * p < 0.001). By DNA fragmentation assay, there was evidence of ladder formation in anti-CD30 (HRS-4 or Ber-H8)-treated eosinophils, consistent with apoptosis. We conclude that eosinophil express low level of CD30, and that treatment with some anti-CD30 antibody shortened eosinophil survival. This suggests that CD30 on eosinophil regulates their survival, perhaps by inducing apoptosis.

Increased Release of Free Fe Ions in Human Erythrocytes During Aging in the Circulation

We investigated whether free Fe ions were released in erythrocytes during aging process in the circulation. Young and senescent erythrocytes were separated from freshly drawn human blood by Percoll density gradient centrifugation. Two different methods were employed for determination of free Fe ions in erythrocytes, desferrioxamine (DFO) method and bleomycin method. DFO-chelatable Fe ions were detected in whole erythrocytes from 2 donors, and the DFO-chelatable free Fe ion levels in senescent erythrocytes were higher than those in young erythrocytes. Bleomycin-sensitive Fe ions, which was rather lower than DFO-chelatable Fe ions, were also detected in whole erythrocytes from 5 donors, and the free Fe ion levels in senescent erythrocytes were also higher than those in young erythrocytes. Free Fe ions may be derived from oxidative damage of hemoglobin, because treatment of whole erythrocytes or purified oxyhemoglobin with hydrogen peroxide gave increased free Fe ions. The results indicated that free Fe ions were released from erythrocytes during aging process in the circulation. Released free Fe ions would promote oxidative damages of the cells during aging process.

Stimulatory effect of lactate on testosterone production by rat Leydig cells

Previously we found that the increased plasma testosterone levels in male rats during exercise partially resulted from a direct and luteinizing hormone (LH)-independent stimulatory effect of lactate on the secretion of testosterone. In the present study, the acute and direct effects of lactate on testosterone production by rat Leydig cells were investigated. Leydig cells from rats were purified by Percoll density gradient centrifugation subsequent to enzymatic isolation of testicular interstitial cells. Purified rat Leydig cells (1 x 10(5) cells/ml) were in vitro incubated with human chorionic gonadotropin (hCG, 0.05 IU/ml), forskolin (an adenylyl cyclase activator, 10(-5) M), or 8-bromo-adenosine-3':5'-cyclic monophosphate (8-Br-cAMP, 10(-4) M), SQ22536 (an adenylyl cyclase inhibitor, 10(-6)-10(-5) M), steroidogenic precursors (25-hydroxy-cholesterol, pregnenolone, progesterone, and androstenedione, 10(-5) M each), nifedipine (a L-type Ca(2+) channel blocker, 10(-5)-10(-4) M), or nimodipine (a potent L-type Ca(2+) channel antagonist, 10(-5)-10(-4) M) in the presence or absence of lactate at 34 degrees C for 1 h. The concentration of medium testosterone was measured by radioimmunoassay. Administration of lactate at 5-20 mM dose-dependently increased the basal testosterone production by 63-187% but did not alter forskolin- and 8-Br-cAMP-stimulated testosterone release in rat Leydig cells. Lactate at 10 mM enhanced the stimulation of testosterone production induced by 25-hydroxy-cholesterol in rat Leydig cells but not other steroidogenic precursors. Lactate (10 mM) affected neither 30- nor 60-min expressions of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein. The lactate-stimulated testosterone production was decreased by administration of nifedipine or nimodipine. These results suggested that the physiological level of lactate stimulated testosterone production in rat Leydig cells through a mechanism involving the increased activities of adenylyl cyclase, cytochrome P450scc, and L-type Ca(2+) channel.

Comparisons of oocyte maturation times and of three methods of sperm preparation for their effects on the production of goat embryos in vitro

Various times of in vitro maturation of oocytes, and three methods of separating spermatozoa from frozen-thawed semen (Percoll density-gradient centrifugation, swim-up, and glass-wool filtration), were compared for their effects on goat embryo production in vitro. Cumulus-oocyte-complexes (COCs) from abattoir ovaries were matured in M199 supplemented with 10% fetal calf serum and hormones. In Experiment 1, COCs were fixed at 4 h intervals from 0 to 27 h of culture to assess oocyte nuclear maturation. A higher proportion cultured for 27 h than for 24 h were in Metaphase II (27/37, 73% vs. 18/33, 55%, P < 0.05). In Experiment 2, the effects of separation methods on total numbers and numbers of membrane-intact spermatozoa, and the acrosome reaction were compared. Total numbers after Percoll density-gradient centrifugation were ∼4 times higher than after swim-up and ∼2 times higher than after glass-wool filtration (P < 0.001). Progression of the acrosome reaction was not affected differentially. In Experiments 3 and 4, after 27 h of culture the COCs were inseminated with sperm isolated by the three methods. In Experiment 3, presumptive zygotes were examined for pronucleus (PN) formation at 6, 12, 18 and 24 h post-insemination. At 12 h, male PN formation rate from Percoll-treated spermatozoa was higher than from sperm subjected to swim-up and glass-wool treatments (20/37, 54% vs. 6/37, 16% and 6/38, 16%, respectively; P < 0.001). In Experiment 4, embryos were compared for cleavage at 48 h and development into blastocysts, hatching rates and cell number at 192 h. The rates of cleavage and blastocyst formation in the Percoll-treated group were higher (P < 0.05) than in the swim-up and glass-wool groups (62% and 18% vs. 50% and 11%, and 45% and 8%, respectively). Similarly, the mean cell number in the Percoll group was higher (P < 0.05) than in the swim-up and glass-wool groups (167 ± 5 vs. 149 ± 4 and 126 ± 4, respectively). We conclude that Percoll density-gradient centrifugation is superior to the other two methods for separating goat spermatozoa from frozen-thawed semen in preparation for IVF.

Glucocorticoids induce basophil apoptosis

Background: Induction of apoptosis represents an important mechanism by which glucocorticoids (GCCs) exert their anti-inflammatory properties. The effects of GCCs on apoptosis have been determined in various immune cells and found to vary among different cell types. On the other hand, the effects of GCCs on apoptosis of basophils, active participants in allergic inflammation, have remained obscure. Objective: The objective of this study was to investigate the effects of GCCs on basophil apoptosis. Methods: Basophils were highly purified (purity, >97%) by Percoll density gradient centrifugation followed by negative selection. Cell status was determined by their ability to bind annexin V and exclude propidium iodide. DNA fragmentation was determined by flow cytometry. Results: Dexamethasone (DEX) significantly accelerated the decrease in live cells and increased the number of apoptotic cells in a time-dependent fashion. Light microscopy as well as DNA fragmentation assay confirmed the induction of apoptosis by DEX. A half-maximal effect was observed in a DEX concentration range from 10–9 to 10–8 mol/L. Sex steroids did not induce basophil apoptosis at all. DEX also induced basophil apoptosis in the presence of low doses of IL-3. Conclusion: GCCs exert potent apoptogenic effects on basophils. GCC-mediated apoptogenic effects on basophils might have implications with respect to the mechanism of action of this class of drugs in allergic disorders. (J Allergy Clin Immunol 2001;108:215-20.)

Murine hypodense eosinophils induce tumour cell apoptosis by a granzyme B-dependent mechanism.

Eosinophils have been shown to potentiate anti-tumour cytotoxicity in both clinical and animal studies. The mechanism by which eosinophils induce tumour cell damage, however, has largely been speculative. The purpose of this study was to identify the mechanisms involved in eosinophil-induced tumour cell cytotoxicity. To investigate eosinophil cytotoxicity, eosinophils were isolated from the peritoneal cavity of Mesocestoides corti-infected BALB/c mice, and were separated into normodense (ND) and hypodense (HD) populations using discontinuous Percoll density gradient centrifugation. The tumoricidal activity of ND and HD eosinophils was assessed using the [51Cr]-release cytotoxicity assay (a measure of cytolytic activity) and the JAM assay (a measure of apoptotic activity). Investigation of apoptosis-inducing molecules in HD eosinophils was undertaken by RT-PCR. The calcium chelator EGTA, serine protease inhibitor aprotinin and a competitive substrate for granzyme B were used to assess the role of perforin and granzyme B in HD eosinophil killing. Cytotoxic activity induced by HD eosinophils was significantly greater than that of ND eosinophils, and apoptosis was the principal killing mechanism. RT-PCR analysis revealed that HD eosinophils express mRNA for perforin, granzyme B and Fas ligand. Furthermore, HD eosinophil killing was markedly inhibited by EGTA, intracellular aprotinin and the granzyme B competitive substrate. These data are consistent with a hypothesis that murine HD eosinophils elicit tumoricidal activity via a granzyme B-dependent mechanism.

Flow Cytometric Measurement of Intracellular Th1 and Th2 Cytokine Production by Human Villous and Extravillous Cytotrophoblast

A wide variety of cytokines are present at the maternal–fetal interface, but the extreme cellular complexity of the placenta has made it difficult to determine which cytokines are produced by which cells. Hence novel flow cytometric methods have been applied to determine intracellular cytokine production by specific cell-types in placental cell suspensions. Cell suspensions were prepared from first and third trimester chorionic villi and third trimester amniochorion by enzymatic digestion and Percoll density gradient centrifugation. After overnight incubation in the presence of monensin, cells were fixed, permeabilized and labelled with antibodies for villous cytotrophoblast (cytokeratin+, MHC class I-), extravillous cytotrophoblast (cytokeratin+, MHC class 1+) and leucocytes (CD45+). These cell types were further characterized by their expression of EGFR (proliferative cytotrophoblast) and c-erbB2 (invasive cytotrophoblast). Production of IL-4, IL-10, TNF-α, IFN-γ and IL-12 was determined by simultaneous labelling with the appropriate monoclonal antibodies. Only IL-4 was detected consistently in all samples of cytotrophoblast. IL-10 was not detected but IL-10 mRNA was demonstrated in third trimester chorionic villus digests by RT-PCR. Although IL-4 secretion has not been demonstrated, these data suggest that, in vivo there may be a ‘Th2 type cytokine bias’ orchestrated by the trophoblast. It is proposed that other cytokines (including IL-10 and TNF-α) are produced by decidual leukocytes, and not cytotrophoblast, at the maternal–fetal interface.

Mechanism of folate transport across the human colonic basolateral membrane.

Previous studies from our laboratory have demonstrated the existence of a folate transporter in the human colonic apical membranes. The current studies were undertaken to examine the possible presence and function of a folate carrier in the human colonic basolateral membrane vesicles (BLMV). BLMV were purified from mucosal scrapings of colons of organ donors by a Percoll-density gradient centrifugation technique, and uptake studies were performed using a rapid filtration technique. Our results on [(3)H]Pte-Glu uptake are summarized as follows: 1) uptake was sensitive to osmolarity of the incubation medium; 2) Na(+) removal from the incubation medium did not affect folate uptake into BLMV; 3) uptake was significantly increased with decreasing incubation buffer pH from 8 to 4; 4) uptake demonstrated saturation kinetics with an apparent Michaelis constant of 9.6 +/- 0.48 microM and a maximal velocity of 8.10 +/- 0.36 pmol x mg protein(-1) x 10 s(-1); 5) uptake was markedly inhibited by the structural analog methotrexate (inhibitory constant = 8.28 +/- 1.0 microM); 6) uptake into BLMV demonstrated a trans-stimulation phenomenon; 7) anion exchange inhibitors DIDS and SITS significantly inhibited folate uptake; and 8) uptake was potential-insensitive, as voltage clamping of vesicles or making them inside positive with K(+)/valinomycin failed to influence folate uptake. Western blot analysis using purified human colonic basolateral membrane preparations and specific polyclonal antibodies against the human reduced folate carrier (hRFC) has shown expression of the hRFC protein at this membrane domain. These data demonstrate the existence of a pH-dependent, DIDS-sensitive, electroneutral, carrier-mediated mechanism for folate transport across the human colonic basolateral membranes.

Increased Superoxide Generation is Associated with Decreased Superoxide Dismutase Activity and mRNA Expression in Placental Trophoblast Cells in Pre-eclampsia

Pre-eclampsia is a multi-system disorder unique to human pregnancy. Although the aetiology of pre-eclampsia is still unknown, increased placental oxidative stress contributes to the pathophysiology of this pregnancy disorder. The goal of this study was to determine if placental trophoblast cells generate superoxide, and if there was a difference in superoxide generation and superoxide dismutase (SOD) activity between trophoblast cells isolated from pre-eclamptic placentae versus normal placentae. Placentae were obtained from nine normal and 10 pre-eclamptic pregnancies immediately after delivery. Trophoblast cells were isolated and purified by Percoll density gradient centrifugation. Superoxide generation and SOD activity were determined by spectrophotometric assays. Localization of CuZn-SOD protein within the placenta was examined by immunohistochemical staining. mRNA expression of CuZn-SOD was determined in trophoblast cells isolated from five normal and five pre-eclamptic pregnancies by Northern blot analysis. 18S ribosomal mRNA expression was used as an internal standard. We found: (1) trophoblast cells from pre-eclamptic placentae generated significantly more superoxide than trophoblast cells from normal placentae: 17.62±3.19 versus 4.70±0.76nmol/5×106cells (mean±s.e.),P< 0.01; (2) protein and mRNA expression for CuZn-SOD was mainly localized in the trophoblast cells within the placenta; and (3) SOD activity and relative mRNA expression for CuZn-SOD were significantly decreased in trophoblast cells from pre-eclamptic placentae as compared to trophoblast cells from normal placentae, SOD activity: 6.46±1.76 versus 13.01±1.67units/mg protein, P< 0.05; relative mRNA expression for CuZn-SOD: 0.25±0.09 versus 0.73±0.07, P< 0.01. We conclude that increased superoxide generation was associated with decreased SOD activity and mRNA expression for CuZn-SOD in trophoblast cells isolated from pre-eclamptic placentae. These findings support the notion of increased oxidative stress in the pre-eclamptic placenta, which may contribute to the pathophysiology of this pregnancy disorder.

Expression of placental lactogen and cytokeratin in bovine placental binucleate cells in culture.

Binucleate cells are present in ruminant placenta and play an endocrine role in the production of many hormones during pregnancy. We isolated and cultured binucleate cells from bovine placenta at middle to late gestation and characterized these cells using immunofluorescence techniques. Enriched preparations of binucleate cells were obtained using Percoll density gradient centrifugation following collagenase digestion. Binucleate cells in culture preferentially attached to collagen-coated dishes rather than to noncoated plastic dishes. The cells gradually extended their edges on collagen substrata, and finally assumed a flattened morphology. Antibodies to placental lactogen (PL) and pregnancy-associated glycoprotein-1 (PAG-1) specifically stained the majority of round binucleate cells, but not the flat cells. We found that PL-positive binucleate cells were consistently devoid of cytokeratin. In contrast, cytokeratin was expressed in PL-negative binucleate cells as well as mononuclear epithelial cells. Furthermore, the PL-negative flat binucleate cells also developed intense cytokeratin networks in the cytoplasm. These results indicate that cytokeratin expression is inversely proportionate to that of PL in cultured binucleate cells. We conclude that downregulation of cytokeratin in binucleate cells is a function of the state of cellular differentiation.

Elevated Seminal (SP) Plasma Lead (Pb2+) Levels Result in Significant Impairment of Human Sperm Function and Reduced Artificial Insemination (AI) Cycle Fecundity Rates ( f)

Objectives: Under an IRB approved protocol, 2-3 blood plasma (BP) and 2-9 SP specimens were collected over a six month period from AI semen donors (n=14) and from an unselected population (n=13) answering an advertisement for research participation. The Pb2+ levels in these specimens had low (<9.5%) intra-male variation but varied between men over a wide range (e.g., <10 μg/L to ∼1200 μg/L). Pb2+ in BP and SP were positively correlated (Spearman correlation [SC], r=0.9, p<0.0001). Comparable BP/SP Pb2+ levels were previously seen in IVF patients, whose increased Pb2+ levels: [1] were negatively correlated with conventional insemination fertilization rates (SC, n=89, r=−0.6, P<0.0001), and [2] were unrelated to serum FSH, LH or testosterone levels. Preliminary findings also suggested that Pb2+ levels and f were negatively related (n=3, SP Pb2+ <250 μg/L, f >16% vs. n=2, SP Pb2+ >700 μg/L, f <6%; t-test, P<0.004). Therefore, the purpose of this study was to determine if increased Pb2+ levels were related to levels of other toxic metals, to decreased expression of sperm biomarkers for high IVF fertilization rates, and to f. Design: A double-blind study of cadmium (Cd2+) levels, sperm function and f (in 341 AI attempts) in specimens previously examined for Pb2+ content. Metal ion effects were modeled in vitro by exposure of fertile donor sperm (n=24) under capacitating conditions to increasing doses of exogenous Pb2+ (0–5180 μg/L). Materials and Methods: Cd2+ in BP and SP was assayed by graphite furnace atomic absorption spectroscopy. WHO (1992) semen parameters were recorded for fresh semen. Motile sperm populations were obtained from frozen/thawed semen by Percoll density gradient centrifugation and were examined for surface expression of mannose (Man) receptors (MR) and non-nuclear progesterone (P) receptors (NNPR), spontaneous and agonist (free Man and P)-stimulated acrosome reactions (AR). Results were compared using SC and repeated measures analysis of variance (RMANOVA) models. Results: BP and SP Cd2+ levels were more tightly clustered than Pb2+ (range, 0.081 μg/L to 0.161 μg/L). Pb2+ and Cd2+ in BP and SP were positively correlated (SC, respectively, n=11, r=0.7, P<0.01 and n=13, r=0.7, P<0.007). SP Pb2+ levels and sperm count, motility, percentage normal morphology and NNPR were not related. In contrast, significant inverse relationships existed between SP Pb2+ levels and MR (SC, n=12, r=−0.5, P<0.007), Man AR (SC, n=15, r=−0.5, P<0.04) and P AR (SC, n=26, r=−0.4, P<0.03). SP Pb2+ and spontaneous AR were positively correlated (SC, n=16, r=0.6, P<0.01). SP Pb2+ levels and f showed a strong inverse relationship (SC, n=24, r=−0.6, P<0.02). In vitro exposure of fertile donor sperm to exogenous Pb2+ resulted in a dose-dependent decrease in MR (RMANOVA, P<0.01), Man AR (RMANOVA, P<0.0008) and a dose-dependent increase in spontaneous AR (RMANOVA, P<0.0007). Conclusions: SP Pb2+ in subjects studied are representative of the general population. Within the AI group, selected for normal WHO semen parameters, donors with high SP Pb2+ had significantly lower fertility. The correlation between Cd2+ and Pb2+ suggests other metal toxicants may play a role. But in vitro modeling shows Pb2+ alone is sufficient to induce the same characteristics (increased spontaneous AR, decreased MR, decreased agonist-induced AR) in fertile sperm as seen in vivo in subjects with high SP Pb2+. We suggest that potential donors be screened for Pb2+ levels and sperm biomarker expression prior to acceptance into an AI program. These methods may also prove useful in directing ART patients to conventional IVF or to ICSI. (Support: NIH Grant No. ES 06100 to S.B.)

Isolation of thecal cells: an assessment of purity and steroidogenic potential

In the present investigation, the responsiveness of rat thecal cells, prepared by means of an optimised discontinuous Percoll density gradient centrifugation procedure and cultured under serum-free cell culture conditions, to different concentrations of follitropin (FSH), basic fibroblast growth factor (FGF-2 or bFGF), and lutropin (LH) has been examined. The estradiol (E 2) and progesterone (P 4) contents of the cell culture medium were simultaneously determined with aliquots collected after different times of exposure to these regulatory proteins, either individually or in combination. The results confirm that no E 2 could be detected in the cell culture medium of the rat thecal cells prepared and cultured in this manner following all of these different treatments, and hence no contamination of the thecal cell preparations by granulosa cells was evident. The effects of FGF-2 and LH on the steroidogenic and cytodifferentiational properties of these rat thecal cells under serum-free cell culture procedures were also examined. The production of P 4 in the Percoll-purified rat thecal cell cultures receiving different treatments of FSH, and/or FGF-2 did not differ from the basal cell cultures, and no E 2 was detected from the same culture media. In contrast, LH (20 or 50 ng/ml) was found to enhance the production of P 4 ( P<0.05) in the serum-free cell culture media. The stimulation of P 4 production was greater at higher LH concentration (50 ng/ml) ( P<0.05). Concurrent treatment of LH (20 or 50 ng/ml) and FGF-2 (1–100 ng/ml) showed that FGF-2 inhibited the production of P 4 by LH-stimulated thecal cell cultures ( P<0.05). The inhibition by FGF-2 was greater when LH was at a lower concentration (EC 50<1 ng/ml at LH-20 ng/ml vs. EC 50>1 ng/ml at LH-50 ng/ml). The results of the present study thus indicate that rat thecal cells isolated by this optimised Percoll density centrifugation procedure maintain a very high steroidogenic potential and specificity. Consistent with the absence of contaminating granulosa cells, these rat theca cell preparations do not respond to FSH treatment in terms of E 2 production. However, these rat theca cell preparations can be stimulated by LH to express their differentiated status in serum-free medium and respond to growth factors such as FGF-2.