Metabolism of pyruvate by isolated rat mesenteric lymphocytes, lymphocyte mitochondria and isolated mouse macrophages.

1. The activities of pyruvate dehydrogenase in rat lymphocytes and mouse macrophages are much lower than those of the key enzymes of glycolysis and glutaminolysis. However, the rates of utilization of pyruvate (at 2 mM), from the incubation medium, are not markedly lower than the rate of utilization of glucose by incubated lymphocytes or that of glutamine by incubated macrophages. This suggests that the low rate of oxidation of pyruvate produced from either glucose or glutamine in these cells is due to the high capacity of lactate dehydrogenase, which competes with pyruvate dehydrogenase for pyruvate. 2. Incubation of either macrophages or lymphocytes with dichloroacetate had no effect on the activity of subsequently isolated pyruvate dehydrogenase; incubation of mitochondria isolated from lymphocytes with dichloroacetate had no effect on the rate of conversion of [1-14C]pyruvate into 14CO2, and the double-reciprocal plot of [1-14C]pyruvate concentration against rate of 14CO2 production was linear. In contrast, ADP or an uncoupling agent increased the rate of 14CO2 production from [1-14C]pyruvate by isolated lymphocyte mitochondria. These data suggest either that pyruvate dehydrogenase is primarily in the a form or that pyruvate dehydrogenase in these cells is not controlled by an interconversion cycle, but by end-product inhibition by NADH and/or acetyl-CoA. 3. The rate of conversion of [3-14C]pyruvate into CO2 was about 15% of that from [1-14C]pyruvate in isolated lymphocytes, but was only 1% in isolated lymphocyte mitochondria. The inhibitor of mitochondrial pyruvate transport, alpha-cyano-4-hydroxycinnamate, inhibited both [1-14C]- and [3-14C]-pyruvate conversion into 14CO2 to the same extent, and by more than 80%. 4. Incubations of rat lymphocytes with concanavalin A had no effect on the rate of conversion of [1-14C]pyruvate into 14CO2, but increased the rate of conversion of [3-14C]pyruvate into 14CO2 by about 50%. This suggests that this mitogen causes a stimulation of the activity of pyruvate carboxylase.     

Activation of pyruvate dehydrogenase by insulin in isolated brown fat cells.

Isolated fat cells from rat brown adipose tissue in vitro respond to insulin with an increase of pyruvate dehydrogenase (EC 1.2.4.1) activity due to conversion of the inactive form of the enzyme (PDHb) to the active form (PDHa). Like in white adipocytes this effect depends on the presence of glucose or 2-deoxyglucose in the medium. The interrelationship between the steady state of the PDH-system and the phosphorylation state of the adenine nucleotides was studied in white adipose tissue. While insulin in the presence of 2-deoxyglucose caused a large fall of the tissue ATP/ADP ratio which could explain the increase of PDHa activity, the ATP/ADP ratio remained unchanged during incubations with insulin and glucose. Thus it appears that other factors than the ATP/ADP ratio are involved in the regulation of PDH activity by insulin the nature of which remains to be elucidated.     

Production of cloned goats from enucleated oocytes injected with cumulus cell nuclei or fused with cumulus cells

This study was designed to produce cloned goats from cumulus cells. Cloning donor nuclei were from cumulus cells either freshly isolated or cultured in vitro. Enucleated oocytes were either injected with cumulus cell nuclei without piezo-driven manipulator (injection method) or fused with cumulus cells (fusion method). The survival rate of cloned embryos, obtained by injection, was higher than that derived from fusion (62.7 and 45.9%, respectively). Two cloned goats were derived by fusion with in vitro cultured cumulus cells without starvation, but died shortly after natural birth, from respiratory difficulties. Their birth weights (2.23 kg and 2.03 kg) were within the normal range (2.0-2.7 kg) and postmortem analysis revealed no morphological abnormalities. The third cloned goat, derived by injection of nuclei from freshly isolated cumulus cells, weighed 3.3 kg at birth, and was 37% overweight compared with the average weight of the same species. This goat is healthy and well as this paper is being prepared. Nested PCR-RFLP analysis confirmed that all the cloned goats were derived from the donor cells.     

Production of the hyporeactivity factor by mouse cells

The mouse cells L 929 experiencing the state of hyporeactivity to repeated interferon induction with the aid of poly (I) X poly (C) secrete to the incubation medium a factor which suppresses interferon production. The factor is not neutralized by antiserum against murine interferon. Apart from hyporeactivity factor, interferon itself may inhibit its own production.     

Developmental potential of mouse follicular epithelial cells and cumulus cells after nuclear transfer.

The developmental potential after nuclear transfer of mouse follicular epithelial cells cultured in vitro was examined. Follicular epithelial cells surrounding growing oocytes (type 5, diameter of oocytes, 62.6 +/- 5.9 microm; n = 14) were obtained from ovaries of adult mice. Before nuclear transfer, cells were cultured for several passages and subjected to serum starvation for several days. When the nuclear-transfer oocytes were at the 2-cell stage, serial nuclear transfer was performed. Additionally, cumulus cells surrounding ovulated oocytes were used as nuclear donors, with or without thermal stimulation (from -25 degrees C to 60 degrees C for 10 min) before nuclear transfer. Nuclear-transfer oocytes with follicular epithelial cells developed into blastocysts (34%) after serial nuclear transfer, and 4 living fetuses on Day 10.5 (25%, 16 transferred) and 1 dead fetus on Day 19.5 of pregnancy (3%, 30 transferred) were obtained after transfer to recipients. Although blastocysts (20%) were obtained after serial nuclear transfer of cumulus cells, only one implantation site without a fetus was observed on Day 10.5 of pregnancy. Thermal stimulation of cumulus cells before nuclear transfer did not enhance the ability to develop into fetuses or blastocysts.     

Production of cumulus expansion enabling factor by mouse oocytes grown in vitro: Preliminary characterization of the factor

The objective of this study was to determine whether fully grown oocytes, obtained after isolation from preantral follicles and growth in vitro, secrete paracrine factors affecting granulosa cell development and function. If so, the relative ease in producing oocytes in this way could facilitate the identification and characterization of the factors. As a test of this idea, the ability of in vitro grown oocytes to produce a paracrine factor that is known to enable the isolated cumulus oophorus to undergo expansion in response to follicle stimulating hormone (FSH) was determined. Initial experiments compared culture systems, which differed in the orientation of the oocyte-granulosa cell complexes from preantral follicles to an extracellular matrix, for their ability to support oocyte growth and the acquisition of competence to resume meiosis. The systems for culture on the surface of the matrix produced larger oocytes and the highest percentage of oocytes having competence to resume meiosis. Oocytes grown using this system secreted active cumulus expansion enabling factor, albeit at levels about half that of oocytes grown in vivo. A preliminary characterization of the cumulus expansion enabling factor secreted by the oocytes grown in vitro showed that activity was lost upon treatment with either heat (65°C for 15 min) or proteinase K. Activity did not pass through a membrane having a nominal molecular weight limit (NMWL) of 100 kd but did pass through a membrane having a NMWL of 300 kd. It is concluded that cumulus expansion enabling factor is secreted by oocytes grown in vitro. This factor is probably a protein or depends upon a protein for its activity. The ease in obtaining relatively large numbers of GVB-competent oocytes using techniques for growth in vitro combined with the demonstration that these produce cumulus expansion enabling factor indicates that these protocols can be used to produce oocytes for the collection and characterization of oocyte secretory products some of which are paracrine regulators of granulosa cells. © 1993 Wiley-Liss, Inc.     

Inositol trisphosphate activates pyruvate dehydrogenase in isolated fat cells

Inositol trisphosphate, when added to permeabilized rat fat cells, led to a several-fold increase of pyruvate dehydrogenase activity due to conversion of the inactive phospho form (PDHb) to the active, dephospho form (PDHa). It is suggested that inositol trisphosphate, probably through intracellular Ca2+-mobilisation, acts as a physiological mediator of insulin for activation of the mitochondrial PDH-complex.     

Production of cloned goats by nuclear transfer of cumulus cells and long-term cultured fetal fibroblast cells into abattoir-derived oocytes

Dairy goats are ideal for the transgenic production of therapeutic recombinant proteins. The use of recombinant somatic cell lines for nuclear transfer (NT) allows the introduction of genes by transfection, increases the efficiency of transgenic animal production to 100%, and overcomes the problem of founder mosaicism. Although viable animals have been cloned via NT from somatic cells of 11 species, the efficiency has been extremely low. Both blastomere and somatic cell NT increased fetal loss and perinatal morbidity/mortality in cattle and sheep, but fetal loss and perinatal mortality appear to be relatively low in goats. In this study, we produced cloned goats by NT from cumulus cells and long-term cultured fetal fibroblast cells (FFCs) to abattoir-derived oocytes. NT embryos were constructed from electrofusion of cumulus cells (CCs), FFCs, or skin fibroblast cells (SFCs) with cytoplasts prepared from abattoir-derived ovaries. The NT embryos were activated with an optimized activating protocol (1 min exposure to 2.5 microM ionomycin followed by 2 hr incubation in 2mM 6-DMAP). Two viable cloned kids from CCs and one from long-term cultured FFCs (at passage 20-25) were born. Microsatellite analysis of 10 markers confirmed that all cloned offspring were derived from corresponding donor cells. To our knowledge, the production of cloned goat offspring using abattoir-derived oocytes receiving nuclei from CCs and long-term cultured FFCs has not been reported. The production of viable cloned animals after activation with reduced intensity of ionomycin and 6-DMAP treatment has also not been reported. Loss of cloned embryos was obvious after 45 and 90 days of pregnancy, and a lack of cotyledons, heart defects, and improperly closed abdominal wall were observed in the aborted fetuses and one cloned kid. The fusibility and in vitro developmental potential of embryos reconstructed from FFCs at passage 20-25 were significantly lower than those of embryos reconstructed from FFCs at passage 3-5, and the cloning efficiency of the long-term cultured cells was low (0.5%).     

The oxidation of pyruvate by isolated heart sarcosomes

• 1.1. Although heart sarcosomes are able rapidly to oxidize glutamate to aspartate for periods exceeding 1 h at 25°, the oxidation of pyruvate declines rapidly after about 1 min.
• 2.2. Pyruvate inhibits the oxidation of glutamate, whether added before or after the glutamate.
• 3.3. Acetoacetate inhibits the oxidation of both glutamate and pyruvate. The respiration is completely restored by the addition of catalytic amounts of malate or of α-oxoglutarate.
• 4.4. It is concluded that the inhibitions are caused by decarboxylation of oxaloacetate, catalysed by acetyl-CoA, produced by oxidation of pyruvate or from the acetoacetyl-CoA formed by reaction of acetoacetate with succinyl-CoA.

     

Evidence of new antigens in the mouse cumulus oophorus during preovulatory cumulus expansion

The effects of an antiserum (anti-COC) against ovulated mouse cumulus-oocyte complexes (COC) on in vitro fertilization of mouse oocytes were studied. Preincubation of ovulated COC with various concentrations of anti-COC led to dose-dependent impairment of fertilization rates as well as to a decrease in the number of spermatozoa attached to the zona pellucida. Anti-COC was used to probe Western blots of cumulus proteins. These cumuli were obtained from 2 experimental groups of mice corresponding to 2 different maturational stages (preovulatory immature COC or preovulatory mature COC). Two antigens (70 and 80 kDa) present in cumulus intercellular matrix from mature COC were only found as traces in matrix from immature COC. In addition, the protein pattern of the cumulus intercellular matrix was different from that of cumulus cells, whatever the COC maturational stage. These results indicate the appearance of new proteins in the cumulus oophorus during preovulatory expansion and are consistent with the contraceptive action of anti-COC. © 1993 Wiley-Liss, Inc.     

Production of a fibronectin-associated lymphokine by cloned mouse T cells

Azobenzenearsonate-specific cloned mouse T cells able to transfer delayed hypersensitivity reactions in vivo produced macrophage agglutination factor (MaggF) after stimulation with mitogen or antigen in vitro. Mitogen (Con A) elicited MAggF production directly from T cells. Responses to Ag were Ag-specific, required syngeneic accessory cells in addition to T cells, and were independent of T cell fine specificity for azobenzenearsonate. Mouse MAggF shared a number of biochemical and immunochemical properties with the fibronectins (FN): 1) high Mr similar to that of plasma FN; 2) binding to gelatin, heparin, and polyclonal antibodies and mAb specific for cellular and plasma FN; 3) inhibition of activity in solution by monoclonal anti-human FN directed against plasma FN gelatin-binding domain; and 4) action on peritoneal exudate macrophages mediated through a FN-receptor cross reactive with one on human monocytes. MAggF production required active protein synthesis and was associated with significant increases in gelatin-binding immunoreactive FN (Mr 440 kDa on immunoblotting) in culture supernatants and T cell lysates. Metabolically labeled peptides could be precipitated by anti-FN from culture supernatants of activated T cells. Stimulated cultures contained significantly more cells with immunohistologically demonstrable cytoplasmic FN than unstimulated control cultures. We suggest that T cell FN is a distinct species of cellular FN which may play an important role in mediating delayed hypersensitivity inflammatory reactions in vivo.     

Inhibition of pyruvate transport by fatty acids in isolated cells from rat small intestine.

1. A technique is described for the rapid separation of intestinal epithelial cells from the incubation medium by passage through a silicon-oil layer and collection in acid, in which their soluble constituents are released. 2. The inhibition by fatty acids of pyruvate oxidation is further studied. Measurement of pyruvate transport in epithelial cells at 0 degree C showed that short- and medium-chain fatty acids as well as ricinoleate inhibit this transport. Propionate inhibits pyruvate transport by another mechanism than octanoate. 3. Differences between pyruvate propionate and octanoate transport across the epithelial cell membrane were obtained in efflux studies. These studies revealed that acetate, propionate, butyrate and high concentrations of bicarbonate readily stimulate the efflux of pyruvate, probably by anionic counter-transport. No effects were seen with octanoate and hexanoate. The data obtained in these efflux studies suggest that lipophilicity and the pKa values of the monocarboxylic acids determine the contribution of non-ionic diffusion to overall transport. 4. Saturation kinetics, competitive inhibition by short-chain fatty acids and counter-transport suggest a carrier-mediated transport of pyruvate.     

Control of pyruvate kinase flux during gluconeogenesis in isolated liver cells

• 1.1. With pyruvate as the gluconeogenic substrate, pyruvate kinase flux, estimated isotopically, and lactate formation were inhibited by glucagon, but only slightly affected by epinephrine.
• 2.2. The glucagon effect was unchanged in the absence of calcium.
• 3.3. Ethanol increased lactate formation from pyruvate, but depressed pyruvate kinase flux.
• 4.4. These results support the role of pyruvate kinase m the cyclic mechanism which transfers mitochondrial reducing hydrogen to the cytosol.
• 5.5. Glucagon and, to a lesser degree, epinephrine inhibit lactate formation from fructose or dihydroxyacetone.
• 6.6. Ethanol also inhibits lactate formation from these substrates, suggesting the possibility that NADH may in some manner regulate pyruvate kinase flux.

     

Expression of heat shock proteins by isolated mouse spermatogenic cells.

Proteins of the hsp70 family are abundant in mouse spermatogenic cells. These cells also synthesize relatively large amounts of a 70,000-molecular-weight protein (P70) that appears to be a cell-specific isoform of hsp70, the major heat-inducible protein (R.L. Allen, D.A. O'Brien, and E.M. Eddy, Mol. Cell. Biol. 8:828-832, 1988). In this study, proteins of unstressed and heat-stressed spermatogenic cells consisting of purified preparations of preleptotene, leptotene-zygotene, pachytene spermatocytes, and round spermatids were analyzed by two-dimensional polyacrylamide gel electrophoresis. Unstressed preleptotene and leptotene-zygotene spermatocytes contained little P70, whereas relatively large amounts of P70 were present in pachytene spermatocytes and round spermatids. Labeling studies showed that P70 was synthesized primarily in pachytene spermatocytes and that little synthesis occurred in round spermatids or in preleptotene and leptotene-zygotene stages of spermatogenesis. Synthesis of hsp70 was not detectable in unstressed cells but was induced in all stages of isolated germ cells following heat stress. These results indicate that P70 is expressed in a stage-specific manner during cell differentiation, whereas hsp70 is synthesized in response to stress in all populations of isolated spermatogenic cells examined.