Development of sheep embryos in vitro in a medium supplemented with different serum fractions

Sheep embryos will generally develop into expanded blastocysts in vitro only in culture media supplemented with serum or serum components. In order to better understand how serum supports embryo development, a batch of ovine serum was fractionated by (a) ultrafiltration into two components containing substances with molecular weights greater and less than 10 Kd (kilodaltons), and (b) gel filtration into protein fractions 1, 2 and 3, containing groups of proteins with mean molecular weights of about 500, 150 and 65 Kd, respectively. The principal protein in fraction 3 was albumin. Day 6 sheep morulae were cultured in vitro for 48 hours in a bicarbonate-buffered salt solution supplemented with various concentrations of ovine serum or of these components or protein fractions of serum. Morulae could develop to fully expanded blastocysts in medium supplemented with whole serum or with the greater than 10 Kd component or protein fraction 3 only, but could not develop in medium supplemented with the less than 10 Kd component only or with the less than 10 Kd component and protein fractions 1 or 2. However, the proportion of embryos that developed fully in medium supplemented with the greater than 10 Kd component or protein fraction 3 was increased by adding the less than 10 Kd component of serum to the medium. The addition of protein fraction 2 decreased the proportion of embryos that developed to expanded blastocysts in medium containing protein fraction 3 and the less than 10 Kd component, but not in medium containing whole serum. Since the compositions of different sera may vary markedly, these results suggest (a) reasons why different sera vary in their ability to support embryo development in vitro, and (b) factors which may influence development of the sheep embryo in the uterus, where plasma proteins comprise nearly all the protein in the fluid bathing the embryo.     

In vitro development of bovine morulae in bovine serum albumin, normal steer serum and uterine flushings

One hundred fifty-three excellent and good bovine morulae were cultured in Ham's F-10, supplemented with 10 % steer serum, bovine serum albumin, or uterine flushings (64 mg protein/ml) to compare embryo development. Embryos were observed every 12 h in culture. Treatment differences were evaluated by assigning a numerical value of 0 to 4 to each embryo representing the stage of development reached in vitro. The final morphological developmental score of the embryos was comparable for steer serum (2.66) and bovine serum albumin (2.50), but it differed significantly for uterine flushings obtained from ovariectomized, steroid-supplemented cows (< 0.1) or heat-treated uterine flushings (0.07). Since albumin alone was able to support development, it suggests that the albumin component of steer serum may be responsible for the observed development. Uterine fluids were unable to support growth of embryos, suggesting that incompatibility may be due to asynchrony between the early bovine embryo and uterine constituents, or a concentration of uterine components may exacerbate actions of inhibitory substances.     

Rete testis fluid (RTF) proteins: purification and characterization of RTF albumin

A major 68-kDa protein in ram rete testis fluid (RTF) is shown to be chemically and immunologically indistinguishable from albumin in ovine serum. Data obtained with two-dimensional gel electrophoresis of RTF demonstrate the presence of additional proteins with a molecular mass of 68 kDa that do not react with antisera against sheep serum albumin. Biochemical characteristics of albumin preparations isolated by immunoaffinity chromatography from ovine serum and from RTF were compared. Albumin from both sources had the same apparent molecular mass of 68 kDa, the same isoelectric point of approximately 4.2, and neither bound specifically to Concanavalin A. Analysis of tryptic peptide maps, obtained with reverse-phase high-pressure liquid chromatography, indicated no significant differences between digests of the two purified albumin preparations. Results indicate that RTF albumin and serum albumin are the same protein, which implies that RTF albumin may originate from serum. Albumin levels in RTF, collected from different rams and measured by radioimmunoassay, varied between 46 and 164 micrograms/ml, constituting between 11 and 17% of total RTF protein, while albumin levels in sheep plasma were 40,000 micrograms/ml. The protein composition of RTF is discussed in relation to the relative amounts of various components contributed by testis cells and the amounts derived from serum.     

Use of a protein-free medium for the in vitro development of sheep embryos]

The employment of protein-free medium for the culture of ovine embryos collected at the 1-2 cell stage from superovulated ewes was investigated. For this purpose sheep zygotes were randomly allocated in four treatment groups: T1) CZB medium + bovine serum albumin (BSA) on sheep oviductal monolayer (SOM), T2) CZB + polyvinyl alcohol (PVA) + SOM, T3) CZB + PVA + SOM supplemented with inositol (I) and serine (S), T4) TCM 199 + 10% fetal calf serum + SOM. Standard culture conditions were 2 ml of medium in 35 mm Petri dishes, under 5% CO2 in air at 39 degrees C. The percentages of morulae and blastocysts were recorded after 4 and 7 days of culture. After 4 days of culture there was no significant difference (P > 0.05) in the percentage of morulae between embryos cultured in T1 (86%), T2 (85%), T3 (88.8%), and T4 (87.5%). After 7 days the percentages of blastocysts were T1 (70%), T2 (50%), T3 (55.5%) and T4 (46.8%). These data suggest that a protein-free medium, CZB + PVA and CZB + PVA + I + S, can support ovine preimplantation embryo development in vitro; however CZB medium supplemented with BSA enhances development to blastocyst.     

A low molecular weight extract of bovine serum albumin stimulates rabbit blastocyst cell division and expansion in vitro

Fraction V bovine serum albumin (BSA) was dissolved in 5% formic acid and filtered through a molecular filter with a cutoff of Mr 10 000. The freeze-dried filtrate stimulated increased cell division of cultured rabbit morulae to blastocysts (up to 8-fold increase in cell number) and increased blastocyst expansion (greater than 2-fold). These results show that some samples of commercial BSA contain an embryonic growth factor of a low molecular weight.     

Variability in different lots of commercial bovine serum albumin affects cell multiplication and hatching of rabbit blastocysts in culture

Rabbit morulae were cultured in vitro for 4 days in a synthetic culture medium supplemented with two different lots of commercial bovine serum albumin (BSA) and two different amino acid formulations in a factorial 2 x 2 arrangement. One lot of BSA caused complete hatching of a proportion of blastocysts and formation of more than twice as many cells per blastocyst (hatched and unhatched) as that of the second BSA lot which did not cause complete hatching of any blastocysts. The mean cell numbers of hatched blastocysts were more than twice those of non-hatched blastocysts. There was no significant effect of amino acid formulation.     

Detection of intracellular forms of secretory aspartic proteinase in Candida albicans.

The extracellular proteinase (EPR) of Candida albicans was induced in a medium containing bovine serum albumin as sole nitrogen source. There were two intracellular forms in cells induced to produce EPR, a 43 kDa protein (EPR) and a 45 kDa protein (cross-reacting material of EPR; CRM-EPR); these were detected by immunoblotting using anti-EPR antiserum. The 43 kDa protein (EPR) may be the same as the extracellular form judging by molecular mass, and the 45 kDa protein (CRM-EPR) may be a precursor form of EPR. Many dense granules were observed by electron microscopy near the plasma membrane of the mother cells in EPR-producing cells. Both the 43 and 45 kDa proteins were recovered in a membrane fraction and were solubilized by Triton X-100. When the membrane fraction was further fractionated by sucrose density gradient centrifugation, the 43 and 45 kDa proteins were differentially fractionated. This suggests that they were located in different membrane-bound structures and is consistent with an assumption that the 45 kDa protein is a precursor for EPR.     

Alteration in biological properties of human albumin during storage

The effects of human albumin preparations on oxidative energy metabolism and lipid svnthesis were investigated in rat liver slices incubated with sodium [1-¹⁴C]acetate as precursor. Labeled CO₂ production and incorporation of precursor into the major lipid classes was increased 2 to 3-fold by fresh preparations of albumin (fraction V), and by defatted fraction V, whereas highly purified cystalline albumin was less active. Albumin preparations from various commercial suppliers varied widely in activity. Activity of fraction V was preserved during storage at ?20°C, and gradually lost at +3°C in the course of 1 year. In contrast, defatted fractions rapidly lost activity in storage at both temperatures. After 1 year in storage at +3°C, albumin preparations became inhibitory to CO₂ production and lipid synthesis. The results suggest that commercial albumin used in metabolic studies, and in clinical situations may have unpredictable or undesirable effects related to state of purity and storage conditions of the protein.     

Effect of fractions of horse and fetal bovine serum on neoplastic conversion of C3H mouse cells in tissue culture

Summary Cultures derived from C3H/He mouse embryos were grown in medium NCTC 135 supplemented with horse serum, fetal bovine serum, or various combinations of large and small molecule fractions of horse and fetal bovine serum. Cultures in medium NCTC 135 alone or in medium 135 supplemented with the small molecule fraction of either horse or fetal bovine serum did not grow as continuous long term lines. The best growth was obtained when the cultures were in medium containing the large molecule fraction of fetal bovine serum either alone or in combination with a small molecule fraction. Cells grown in the presence of the low molecular weight fraction of horse serum invariably produced tumors on injection into syngeneic animals. Cells in the small molecular weight fraction of fetal bovine serum combined with the large molecular weight fraction of horse serum produced tumors after a prolonged period in vitro. *** DIRECT SUPPORT *** A00S8010 00003     

Effect of fetal and adult bovine albumins on human lymphocyte proliferation in vitro

Fetal and adult bovine albumins were isolated and compared with the commercially obtained fraction V of Cohn, in terms of their ability to support cell proliferation, when added as a supplement to serum-free lymphocyte cultures activated with phytohemagglutinin. It was found that fetal albumin is the least efficient of the three proteins in permitting lymphoproliferation. A difference between the kinetics of proliferation of lymphocyte cultures supplemented with albumin and with serum was also observed.     

Cryo-survival and development of bovine blastocysts are enhanced by culture with recombinant albumin and hyaluronan

Recombinant albumin can be used to supplement culture medium for the maturation and fertilization of bovine oocytes and subsequent embryo development to the blastocyst stage. Recombinant albumin was able to support blastocyst development at rates equivalent to that of bovine serum albumin (BSA) supplemented media. Supplementation of media containing recombinant albumin and citrate stimulated blastocyst expansion. Culture with recombinant albumin and citrate significantly increased the ability of the resultant blastocysts to re-expand and hatch following cryopreservation. The further addition of the glycosaminoglycan hyaluronan to the culture medium containing either BSA or recombinant albumin also increased the ability of blastocysts to survive cryopreservation. Inclusion of recombinant albumin and hyaluronan in culture media facilitates the development of physiological defined culture conditions. For bovine embryos this has implications for both research and commercial applications where defined reproducible conditions are desirable.     

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.     

The time-course development of the effects of ethanol ingestion on choline oxidase

Male rats were fed a low-fat diet containing 36% of calories as ethanol, and the time-course development of the effects of ethanol on liver mitochondrial oxidation of choline was determined. Ethanol induced an increase in choline oxidase at days 2, 5 and 7 after being introduced into the diet. Due to an observed 32% increase in total fatty acids in the whole liver, defatted bovine serum albumin was added to the buffer used to homogenize the liver. The presence of bovine serum albumim resulted in a significant decrease in choline oxidase activity at days 2 and 5; however, ethanol still induced an increase in choline oxidase activity in these mitochondria. The total fatty acid concentration of mitochondria prepared in the absence of bovine serum albumin increased steadily until day 5; however, by day 7 the fatty acid concentration had returned to control levels. The addition of bovine serum albumin to the homogenization medium prevented the increase in the total amount of fatty acids. The fatty acid composition of the bovine serum albumin-treated mitochondria, however, was not different from the mitochondria is isolated in the absence of bovine serum albumin. Further, the addition of a free fatty acid to isolated mitochondrial preparations caused about a 100% increase in choline oxidase. These data are consistent with the idea that choline oxidase may be regulated to some extent by an influx or an increase in free fatty acids in the liver as a result of ethanol ingestion. Thus, a second mechanism has been described which contributes to the increase in choline oxidase after ethanol ingestion.     

Role of bovine albumin in a serum-free suspension cell culture medium.

A serum-free culture medium, supplemented with 1% bovine serum albumin, supported the growth of both primary and continuous suspension-type cultures of various mammalian tumor cells. The role of albumin added to the medium was also studied. Defatted albumin failed to support cell growth, unless reconstituted with its lipid extract. Similarly, defatted albumin when combined with oleic and linoleic acids, also supported cell growth. Therefore, albumin-bound fatty acids play an important growth-promoting role in serum-free medium.     

The nature of the nutritional importance of serum albumin to Glossina morsitans

The nutritional significance of albumin protein and its constituent amino acids or associated impurities to Glossina morsitans was evaluated. Flies fed serum-free or albumin-free diets or diets containing delipidated serum or delipidated albumin failed to reproduce. The sizes of offspring produced by flies fed on diets containing different commercial albumins varied in proportion to the amount of bound lipid present in the albumin. FLies fed on albumin-containing diets supplemented with serum lipoproteins produced heavier offspring than flies fed on unsupplemented diets or on diets supplemented by other serum proteins. Delipidation of serum lipoproteins abolished this supplementary effect suggesting a possible similarity between lipoprotein-associated and albumin-bound lipid in terms of their importance to the nutrition and reproduction of tsetse. It is concluded that the observed nutritional importance of albumin to tsetse flies may derive from albumin-associated substances rather than albumin per se.     

Rates of pinocytic capture of simple proteins by rat yolk sacs incubated in vitro.

The rates of pinocytic uptake of a number of small 125I-labelled simple proteins (insulin, ribonuclease A and lysozyme) by rat yolk sacs incubated in vitro were determined both before and after treating these proteins with reagents that are known to increase the rate of capture of 125I-labelled bovine serum albumin. Uptake of the untreated forms of all three proteins was extremely rapid, indicating that adsorptive pinocytosis is the principal mechanism by which yolk-sac cells capture these simple proteins, but these rates show no simple correlation with molecular charge. In contrast with albumin, the rates of uptake of treated proteins were either unchanged or lower than that of the corresponding untreated protein preparations; polymeric forms of 125I-labelled lysozyme larger than dimers were ingested at rates significantly lower than that of the monomer.     

Cholesterol as a limiting factor in the growth of Mycoplasma pneumoniae.

Ultracentrifugation was used to separate three commercial lots of bovine serum fraction (BSF) into components designed to contain lipoproteins. Each BSF lot and component was tested for ability to support the growth of tree strains of Mycoplasma pneumoniae. In general, the level of growth-promoting activity corresponded to the amount of cholesterol present in the BSF or BSF components rather than to the amount or type of lipoprotein Cholesterol was the limiting nutritional factor of BSF with low growth-promoting activity. The addition of cholesterol and bovine serum albumin to BSF with low activity resulted in growth equal to or greater than that observed for BSF with high growth-promoting activity. When cholesterol was added to agar medium containing BSF of low activity, mycoplasma colonies were greater in number, possessed larger mean diameters, and had centers that were more distinct than those observed when this BSF was used alone. Variability in growth-promoting actions of commercial lots of BSF was eliminated by increasing their cholesterol content to an optimum level. An adjustment of the cholesterol and albumin levels of any serum product used in culture media may provide a simple convenient method to improve growth and isolation of mycoplasmas.     

Identification of N-acetylglucosamine-binding immunoglobulins in chicken egg yolk and serum distinct from the major mannose-binding immunoglobulins.

An N-acetyl-D-glucosamine (GlcNAc)-binding protein of 170 kDa has been isolated from hen serum and egg yolk. Another GlcNAc-binding protein of higher molecular mass was present only in the serum. The 170 kDa protein co-electrophoresed and co-chromatographed in gel filtration with a chicken IgG, and behaved identical to chicken IgG in double immunodiffusion with goat anti-chicken gamma chain antiserum. The sugar-binding hierarchy for the serum and yolk binding proteins, determined with bovine serum albumin neoglycoproteins, was GlcNAc greater than N-acetyl-D-galactosamine greater than glucose = galactose = L-fucose greater than mannose. This hierarchy was unlike any previously reported GlcNAc-binding proteins. The larger serum binding protein component was shown to be an IgM by double immunodiffusion with goat anti-chicken mu chain antiserum. The serum and yolk GlcNAc-binding proteins comprise a unique set of sugar-binding immunoglobulins distinct from the previously reported hen serum and yolk mannose-binding proteins (Wang et al., 1986).     

The effect of soluble rat liver proteins on the activity of microsomal stearoyl-CoA and linoleoyl-CoA desaturase.

1. The influence of bovine serum albumin and soluble rat liver proteins on the activity of rat liver microsomal delta9 and delta6 desaturases has been studied. 2. In the absence of bovine serum albumin, the delta9 desaturase which converts stearoyl-CoA into oleoyl-CoA, shows a non-linear correlation between enzyme activity and protein concentration. 3. Optimum concentrations of bovine serum albumin have three main effects on the enzyme activity: (i) establishes a linear relationship between enzyme activity and protein concentration, (ii) stimulates the enzyme activity 2--3-fold and (iii) raises the optimum substrate concentration from 10 to 100 muM. 4. A highly purified soluble liver protein of molecular weight 24 000 also stimulated the enzyme activity and brought about a linear relationship between enzyme activity and protein concentration. 5. It was concluded that the non-linear kinetics were due to limiting amounts of substrate binding protein in the microsomal preparations. 6. The delta6 desaturase which converts linoleoyl-CoA into gamma-linolenoyl-CoA was also stimulated by bovine serum albumin and soluble liver proteins. 7. The significance of the fatty acid-binding proteins is discussed.     

Derivation and characterization of a zebrafish liver cell line

ZF-L cells were derived from normal adult zebrafish liver, and have been growing in culture for more than 100 generations. The cells were derived in basal nutrient medium supplemented with fetal bovine serum (FBS), trout serum, trout embryo extract, bovine insulin and mouse epidermal growth factor. After 50 generations in culture, optimal growth of the cells was achieved in medium supplemented with FBS (5%) and trout serum (0.5%). ZF-L cells were hypodiploid (modal chromosome number= 46) and exhibited an epithelial morphology. ZF-L cell homogenates exhibited alanine and aspartate aminotransferase, glucose-6-phosphatase and alkaline phosphatase enzyme activities. The cells synthesized and released several proteins into the culture medium, including a 70 kDa protein recognized by anti-bovine serum albumin IgG.Abbreviations NF -naphthoflavone - EGF epidermal growth factor - EROD 7-ethoxyresorufinO-deethylase - FBS fetal bovine serum - PBS phosphate-buffered saline - PMSF phenylmethylsulfonyl fluoride - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin