Modulation of the IgE immune response to BSA by fragments of the antigen. I. Suppression by free fragments and by fragments conjugated to homologous gamma-globulin

Nonimmunogenic peptic fragments of bovine serum albumin (BSA), Fraction Ia, suppressed immune response to BSA in mice. Splenic T lymphocytes from mice treated with these fragments suppressed the anti-DNP response in irradiated mice reconstituted with DNP-BSA-primed cells, indicating carrier-specific suppression. The conjugate of Fraction Ia with mouse γ-globulin (MGG) was found to be an effective suppressive substance but it did not induce suppressor T cells. B cells from mice given Ia-MGG were unresponsive to BSA when transferred to irradiated recipients along with either normal or BSA-primed T cells. Thus, unresponsiveness to BSA was mediated by either T or B lymphocytes, depending whether the inducing substance was a free fragment of the antigen or fragments conjugated to homologous γ-globulin.     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk     

Albumin inhibits cytotoxic activity of lysophosphatidylcholine by direct binding

Fetal bovine serum (FBS) was found to protect Jurkat T cells from LPC-induced cytotoxicity. Twenty micromolar LPC-induced cytotoxicity of 80-90% of the cells in media without FBS for 3 h, whereas 50-70% in media with 0.5% FBS. However, LPC-induced cytotoxicity was not observed in the presence of 5% FBS in media. The cytotoxicity was specific for LPC among lysophospholipids tested and significantly observed with palmitoyl (C16:0) LPC, stearoyl (C18:0) LPC, and oleoyl (C18:1) LPC among 11 synthetic LPCs. Furthermore, the cytoprotective effect of FBS was observed only when it was added before the treatment, but not after the treatment of LPC, and premixing of FBS and LPC before addition to the cells ameliorated LPC-induced cytotoxicity. Finally, albumin, a major constituent of FBS, prevented completely LPC-induced cytotoxicity even at as low as 3 microM concentration. We also found that five molecules of LPC could sequentially bind to one BSA using isothermal titration calorimetry. The above results suggest that the cytotoxic activity of LPC could be attenuated by albumin in blood. Finally, it should be cautioned that, when experiments are conducted with LPC dissolved in assay buffers containing albumin, the albumin in the buffer could influence the results.     

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.     

A novel serum-free medium for the cultivation of Vero cells on microcarriers

Summary A novel serum-free medium for the cultivation of Vero cells on microcarriers was developed,which composed of the 1:1 mixture of Dubecco's Modified Eagle Medium: Nutrient Mixture F12, bovine serum albumin(BSA) or human serum albumin(HSA), epidermal growth factor(EGF), gelatin and Dbiotin. Both BSA and EGF were effective on cell growth, adhesion and spreading. Further addition of gelatin and biotin led to the enhanced cell adhesion and spreading without growth promoting activity. The serum-free medium was suitable for the cultivation of vero cells on several different microcarriers with cell density reached over 3×l0⁶cells/ml.     

Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: cloning efficiency, growth in suspension, and selection of drug-resistant mutant phenotypes

Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, we measured cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured in reduced serum with or without additives (1 microgram/ml insulin, 3 X 10(-7) M linoleic acid, 1 X 10(-8) M H2SeO3) or bovine serum albumin (BSA, 1% wt/vol). With the additives and less than or equal to 0.5% fetal bovine serum (FBS), Ham's F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle's minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. Acceptable cloning efficiencies were obtained with 2% FBS plus BSA without additives in either medium; the addition of BSA resulted in improved colony size and more compact colony morphology. In alpha MEM, satisfactory growth rates and maximum saturation densities in suspension culture were obtained only with 5% FBS; in Ham's F12, 1% FBS + deoxycytidine + BSA yielded satisfactory suspension growth. Spontaneous mutant frequencies were compared for each medium containing 10% dialyzed FBS (DFBS), 1% FBS plus BSA, or 2% FBS plus BSA. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at the aprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxyuridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethylnitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels.     

Serum and alpha 2-macroglobulin induce transient hyperpolarizations in the membrane potential of an osteoblastlike clone

Using microelectrode techniques, we have observed that the application of serum or alpha 2-macroglobulin (alpha 2M) induces transient hyperpolarizations in the membrane potential of a rat osteosarcoma clone (ROS 17/2). Hyperpolarizations arose from activation of Ca2+-dependent K+ channels by transient increases in the concentration of intracellular free Ca2+. Hyperpolarizing spikes were observed for several h following the addition of fetal bovine serum (FBS) to cell cultures. Application of small volumes of FBS or alpha 2M rapidly induced synchronized bursts of hyperpolarizing spikes. No response was elicited by serum-free medium, latex beads, or bovine serum albumin (BSA). Immunofluorescence labeling patterns were consistent with the receptor-mediated endocytosis of alpha 2M but not BSA. The ligand specificity and kinetics of these hyperpolarizations suggest that they are associated with a receptor-mediated event, possibly an early stage of receptor-mediated endocytosis.     

Mechanical agitation of hybridoma suspension cultures: Metabolic effects of serum, pluronic F68, and albumin supplements

Metabolic effects of the medium supplements, fetal bovine serum (FBS), Pluronic F68, and bovine serum albumin (BSA) were compared for agitated bioreactor cultures of hybridoma cells. Agitation speeds up to 600 rpm, without entrainment of gas bubbles by sparging or vortex formation, allowed examination of cell interactions with turbulent fluid forces. For cultures in FBS-supplemented RPMI media, there was no significant effect of intense turbulent fluid shear on cell growth, metabolism, or antibody, production. Serum-free cultures (Pluronic F68 or BSA supplements) at 600 rpm demonstrated greatly increased glycolysis rates during exponential growth relative to controls. Nutrient limitations caused increased rates of decline of the viable cell concentrations and a reduction in final antibody titers by around 70%. The Pluronic F68 and BSA supplements did not lead to cell protection by modifying metabolism under conditions of intense turbulent fluid shear. Supplementing the protein-free medium with FBS reduced glycolysis rates in exponential growth phase, but this did not prevent a high rate of viable cell decline and low antibody titers. We concluded that FBS does not have a metabolic effect on cells subjected to intense turbulent fluid shear. Although the agitation conditions employed in this study were more intense than generally required for agitated bioreactor culture of hybridomas, we have demonstrated the importance of considering metabolic effects of turbulent fluid forces on cultures using nutrient-rich basal media, in addition to the considerations of gas bubble effects described by other workers. (c) 1992 John Wiley & Sons, Inc.     

A serum-free medium for use in a cumulus cell co-culture system for bovine embryos derived from in vitro maturation and in vitro fertilization

The objective of this study was to develop a serum-free medium for the co-culture of bovine embryos that would yield a percentage of blastocysts equal to that obtained with fetal bovine serum (FBS)-supplemented medium. Cumulus cell-enclosed oocytes (CEO) matured and inseminated in vitro were cultured in a tissue culture medium (TCM)-199 or in a serum-free medium (bovine embryo culture medium; BECM) until 240 h post insemination. Replacement of 10% (v/v) FBS with either 3 mg crystallized bovine serum albumin (BSA)/ml or 3 mg fatty acid-free BSA/ml in TCM-199 had no effect (P > 0.14) on embryo development to the >or= 2-cell (51 to 60%), >or= 8-cell (24 to 33%), blastocyst (16 to 19%) and hatched-blastocyst (7 to 10%) stages at 48, 96, 192 and 240 h post insemination, respectively. Oocyte-enclosing cumulus cells in BSA-supplemented medium grew in clusters rather than in layers, as was noted in FBS-supplemented medium. When CEO were cultured in fatty acid-free BSA-supplemented media (TCM-199 and BECM), a significantly (P < 0.001) higher percentage of oocytes developed to blastocysts after culture with (22%) or without (18%) a cumulus cell monolayer than after denuding the oocytes (7%). Glucose in concentrations of 0 to 5.56 mM added for periods of 18 and 120 h post-insemination had neither a stimulatory nor a deleterious effect on preimplantation development. In conclusion, a serum-free medium supplemented with BSA can be successfully used in a cumulus cell co-culture system for bovine embryos.     

A comparison of bovine serum albumin and chicken ovalbumin as supplements for the serum-free growth of chinook salmon embryo cells,CHSE-214.

Chicken ovalbumin and bovine serum albumin (BSA) were compared as supplements to the basal medium, L-15, for the serum-free cultivation of the Chinook Salmon Embryo cell line, CHSE-214. Unlike L-15 alone, ovalbumin and some commercial BSA preparations allowed cell proliferation and development of confluent monolayer cultures. However, only a fatty acid-free BSA (2%) supported continuous proliferation for two years through approximately 15 subcultivations. For this, subcultivation was achieved with non enzymatic cell dissociation solutions. Also, new serum-free subcultivation techniques were developed that utilized avian egg white trypsin inhibitors to terminate the action of either bovine or cod trypsin. Finally, CHSE-214 were successfully cryopreserved in 2% BSA, allowing all cell cultivation steps to be performed in the absence of FBS.     

The meiotic response of cumulus cell-enclosed mouse oocytes to follicle-stimulating hormone in the presence of different macromolecules.

Experiments were carried out to determine the effect of different macromolecules on the follicle-stimulating hormone (FSH)-induced maturation of mouse oocytes in culture. Cumulus cell-enclosed oocytes (CEO) were isolated from gonadotropin-primed mice and maintained in meiotic arrest for 17-18 h with the cAMP analogue, dibutyryl cAMP (dbcAMP). Germinal vesicle breakdown (GVB) was stimulated by the addition of FSH. Medium was supplemented with either no macromolecule or with varying concentrations of polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), crystallized bovine serum albumin (BSA), or fetal bovine serum (FBS). Oocyte maturation in all FSH-free cultures occurred at a frequency of about 30% or below. High frequencies of maturation were achieved when FSH was added to macromolecule-free medium or to cultures containing PVP, PVA, or BSA. Crystallized BSA was the most effective of these in supporting stimulation of maturation (94% GVB at 3 mg/ml, compared with 72-74% with synthetic polymer-supplemented or macromolecule-free media). The BSA effect was not due to contaminating fatty acids, and a less pure fraction V BSA was not as effective in supporting FSH-induced maturation. FBS suppressed FSH stimulation of maturation in a dose-dependent fashion. Sera from pigs, goats, horses, and rats were also inhibitory, but bovine calf serum (BCS) permitted a high maturation frequency (80% GVB). When added to medium containing either FBS or BCS, crystallized BSA had no effect on FSH-stimulated maturation, but fraction V BSA suppressed maturation in both serum-supplemented media. Under no conditions did FSH stimulate maturation in cumulus cell-free oocytes. These results demonstrate that hormone-induced oocyte maturation is supported in vitro by nonprotein polymers as well as BSA and that the behavior of the oocyte-cumulus cell complex depends on the purity of the BSA sample. In addition, serum contains inhibitory factors that suppress the positive response to FSH. Thus, the choice of macromolecular supplement is of critical importance when testing the hormone responsiveness of isolated cumulus cell-enclosed oocytes in culture.     

Experimental pitfalls in evaluating vectorial protein secretion in vitro; Sertoli cell secretion of androgen-binding protein and transferrin in two-compartment culture chambers

Summary We examined the influence of various Millipore filter pretreatments on the amounts of androgen-binding protein (ABP) and transferrin (Trf) found in the outer (OC) and inner (IC) compartment of two-compartment Sertoli cell (Sc) cultures. When Sc were cultured on untreated Millipore filters, less than 10% of ABP was found in OC during 3 initial culture days compared to similar cultures on pretreated filters. Most of the glycoprotein was shown to be bound by the filters. Pretreatment of Millipore filters with 5% bovine serum albumin (BSA) or 2% fetal bovine serum (FBS) maximally saturated the nonspecific protein-binding sites resulting in OC:IC ratio of ABP similar to that found in cultures on polycarbonate membranes, which exhibit very low protein-binding capacity. In contrast to ABP, about 40% of Trf was bound by the Millipore filter on Day 1, with only trace amounts bound thereafer. These differences were due to much higher secretion rate of Trf than ABP, resulting in a relatively smaller fraction of Trf bound to the filter. Again, the nonspecific binding of Trf was greatly reduced by filter pretreatment with 5% BSA or 2% FBS. It is concluded that complete saturation of protein-binding sites of cellulose ester supports is necessary for reliable evaluation of vectorial protein secretion by Sc and other polarized epithelial cells maintained in this type of culture. The implications of partial saturation of protein-binding sites of culture support in interpreting experimental results are discussed. This work was supported in part by grant HD-17802 (A.S.) from the National Institutes of Health, Bethesda, MD.     

Influence of albumin on granulocyte and macrophage colony-forming efficiency

Mouse granulocyte and macrophage precursors were assayed in plasma clot and fibrin clot cultures, and the effect of bovine serum albumin (BSA) on colony formation was investigated. The number of granulocyte colonies (CFU-g) and clusters increased as the albumin concentration was increased and the number of macrophage colonies (CFU-m) and clusters concomitantly decreased. The albumin-mediated suppression of macrophage colony formation was overcome by the addition of more than 10% fetal bovine serum (FBS) to the plasma clot culture. The effect of BSA and fatty-acid-free BSA on colony-forming efficiency was also tested in fibrin clot cultures containing 10% FBS. Both BSA and fatty-acid-free BSA at a final concentration of 0.5-2% enhanced CFU-g colony formation, while both forms of BSA reduced the number of CFU-m colonies. However, neither BSA nor fatty-acid-free BSA had any effect on colony formation in FBS-free fibrin clot cultures, and only BSA enhanced colony formation when transferrin, linoleic acid, alpha-thioglycerol and dextran were added to the culture. The number of CFU-g (15.6 +/- 3.1) was higher in cultures containing BSA, transferrin, etc., than the number (9.8 +/- 2.5) in cultures without BSA and including transferrin, etc., than the number (9.8 +/- 2.5) in cultures without BSA and including transferrin, etc. (p less than 0.01). The number of CFU-m (32.0 +/- 6.8) in cultures containing BSA and the other four factors was lower than the number (72.2 +/- 5.6) in the culture without BSA (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of serum on calcium mobilization in the submandibular cell line A253

The effects of serum on inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ mobilization in the human submandibular cell line A253 were studied. Exposure of A253 cells to fetal bovine serum (FBS) elicited a 3.3-fold increase in IP3 formation and a concentration-dependent transient increase in cytosolic free Ca2+ concentration ([Ca2+]i), which was similar in Ca2+-containing and Ca2+-free media. Newborn bovine serum (NBS), but not bovine serum albumin (BSA), induced a similar response. The Ca2+ release triggered by FBS was significantly (88%) reduced by the phospholipase C inhibitor U73122, indicating that Ca2+ release induced by FBS is through the PLC pathway. Pretreatment with the tyrosine kinase inhibitor genistein abolished the FBS- and NBS-induced Ca2+ release, suggesting that tyrosine kinase plays an important role in mediating the Ca2+ release. Pre-exposure to ATP or thapsigargin (TG) significantly reduced the FBS-induced [Ca2+]i increase, indicating that Ca2+ release caused by FBS is from the TG- or ATP-sensitive Ca2+ store. While FBS exposure elicited a large Ca2+ release, it reduced Ca2+ influx. Furthermore, FBS significantly inhibited the Ca2+ influx activated by the depletion of intracellular stores by ATP or TG. These results suggest that (1) serum elicits Ca2+ release from ATP- and TG-sensitive stores, which is mediated by IP3; (2) the serum-induced Ca2+ release may be modulated by a tyrosine kinase-associated process; and (3) serum strongly inhibits Ca2+ influxes including the store depletion-activated Ca2+ influx.     

Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: Cloning efficiency,growth in suspension,and selection of drug-resistant mutant phenotypes

Summary Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, we measured cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured in reduced serum with or without additives (1 μg/ml insulin, 3×10⁻⁷ M linoleic acid, 1×10⁻⁸ M H₂SeO₃) or bovine serum albumin (BSA, 1% wt/vol). With the additives and ≤0.5% fetal bovine serum (FBS), Ham’s F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle’s minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. Acceptable cloning efficiencies were obtained with 2% FBS plus BSA without additives in either medium; the addition of BSA resulted in improved colony size and more compact colony morphology. In alpha MEM, satisfactory growth rates and maximum saturation densities in suspension culture were obtained only with 5% FBS; in Ham’s F12, 1% FBS+deoxycytidine+BSA yielded satisfactory suspension growth. Spontaneous mutant frequencies were compared for each medium containing 10% dialyzed FBS (DFBS), 1% FBS plus BSA, or 2% FBS plus BSA. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at theaprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxyuridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethyl-nitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels. This work was performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under Contract W-7405-ENG-48 and supported by the Environmental Protection Agency under Interagency Agreements IAG-D5-E681-AN and AO.     

Characterization of a serum factor that decreases albumin mRNA in cultured hepatocytes

Summary When primary cultures of hepatocytes are exposed to media containing fetal bovine serum (FBS) there is a rapid decrease in levels of tissue-specific mRNAs such as albumin mRNA. We used Northern blot analysis to examine mRNA levels in cultured hepatocytes, and characterized the factor in FBS that significantly reduces the steady state albumin mRNA level. Neonatal bovine serum or serum derived from platelet-poor calf plasma proved as potent as did FBS, but commercial bovine serum albumin did not exhibit this inhibitory activity. Inhibitory activity of FBS was not removed by moderate heat treatment, dialysis, or extraction with organic solvents. However, incubation of FBS with a highly anionic detergent such as 0.1% sodium dodecyl sulfate orN-lauroyl sarcosine, followed by extensive dialysis, resulted in sera that did not inhibit expression of albumin mRNA. These sera supported cell attachment and seemed non-toxic toward the cells. Ammonium sulfate fractionation of FBS showed the activity was present in the 45 to 70% fraction, and trypsin digestion destroyed the inhibitory activity. Gel exclusion chromatography gave a molecular weight 60 000 to 70 000. Fractionation of serum proteins by DEAE-Sephacel or Cibacron blue-agarose showed enrichment for albumin in the most active fractions. Interestingly, metabolic labeling of secreted and cellular proteins with³⁵S-methionine and cysteine showed no significant difference between hepatocytes maintained for 2 days beforehand in serum-free or serum-supplemented media, and no difference between detergent-treated FBS and control FBS. Therefore, FBS contains a factor that causes a significant decrease in steady state levels of mRNA for albumin and other mRNAs of tissue specific function, but under these conditions albumin mRNA levels are not paralleled by synthesis of albumin or other proteins.     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

Hierarchy of T cell dependency in antibody response among different antigens.

T cell dependency of antibody response to polyvinylpyrrolidone (PVP), sheep red blood cells (SRBC), bovine gamma globulin (BGG), and bovine serum albumin (BSA) was examined. PVP and the other three are known as a T cell-independent antigen and T cell-dependent antigens respectively. Adult mice were thymectomized, X-irradiated, reconstituted with syngeneic bone marrow cells (TxXB mice), with bone marrow cells plus thymus cells (TxXBT mice), or with bone marrow cells treated with anti-Thy-1.2 serum and complement (TxXB-theta mice) and used as experimental animals. The anti-PVP response of TxXBT mice was significantly lower than that of TxXB mice, suggesting that T cells exerted a suppressive effect on the response to PVP. Both IgM and IgG responses to SRBC and BGG occurred even in TxXB-theta mice with the aid of bacterial lipopolysaccharide (LPS). However, a significant response to BSA was not observed in TxXB mice even in the presence of LPS or several other adjuvants. These results indicate that the T cell dependency of antigens is different among so called thymus-dependent antigens, that antibody response less dependent on the helper action of T cells can be supported by LPS in the absence of T cells, and that anti-BSA response seems to be extremely T cell dependent.