alpha-Oligodeoxynucleotide stability in serum, subcellular extracts and culture media

Degradation of a synthetic alpha-oligodeoxynucleotide was studied in order to compare its survival with naturally occurring beta-oligodeoxynucleotides in five systems used for antisense hybridization arrest experiments. In contrast to beta-oligodeoxynucleotides, alpha-oligodeoxynucleotides were not detectably degraded over 24 h at 37 degrees C in HeLa cell postmitochondrial cytoplasmic extract or RPMI 1640 with 10% fetal bovine serum, and showed significant survival after 24 h at 37 degrees C in rabbit reticulocyte lysate, fetal bovine serum and human serum.     

In vitro production of embryos alters levels of insulin-like growth factor-II messenger ribonucleic acid in bovine fetuses 63 days after transfer

The objective of this study was to determine the effect of embryo production systems on the expression of insulin-like growth factor (IGF)-II mRNA in fetal bovine tissues at Day 70 of gestation (63 days after transfer). Oocytes aspirated from ovaries of Holstein cows were matured and fertilized in vitro. Zygotes were cultured in either tissue culture medium (TCM)-199 + 10% estrous cow serum (ECS; in vitro-produced with serum [IVPS]) or TCM-199 + 1% BSA (in vitro-produced with serum restriction [IVPSR]). At 72 h postinsemination, IVPSR embryos were transferred into fresh TCM-199 + 10% ECS whereas IVPS embryos had fresh medium replaced. All embryos were cultured for an additional 96 h. In vivo-produced embryos were harvested from superovulated Holstein cows (multiple ovulations [MO]). Grade 1 blastocysts from all groups were transferred singly into Angus heifers. At Day 70 of gestation, fetuses (n = 14, 13, and 11 for MO, IVPS, and IVPSR, respectively) were collected; liver and skeletal muscle samples were snap frozen, and whole-cell RNA (wcRNA) was extracted. Levels of IGF-II mRNA were determined by RNase protection assay and quantified relative to 18S rRNA (mean arbitrary units +/- SEM). WcRNA from adult and Day 90 fetal bovine liver were used as controls. Adult liver contained 9-fold less IGF-II mRNA than liver from Day 90 fetuses (P < 0.05). Fetal livers of males originating from IVPS and IVPSR groups possessed approximately 2-fold greater levels of mRNA for IGF-II than those from MO males (0.25 +/- 0.07, 0.33 +/- 0.04, and 0.14 +/- 0.03, respectively; P < 0.05). Levels of mRNA for IGF-II tended to be lower (P = 0.07) in skeletal muscle of fetuses originating from the IVPSR group (0.043 +/- 0.005) compared to MO controls (0.070 +/- 0.008). In conclusion, at Day 70 of gestation, fetuses originating from in vitro production systems possessed altered levels of IGF-II mRNA in both liver and skeletal muscle.     

Effects of prolactin on intracellular stored calcium in the course of bovine oocyte maturation in vitro

At present there are divergent opinions as to the role of prolactin (PRL) in the mechanisms of meiotic regulation in mammals. We investigated the effects of bovine PRL (bPRL) on bovine oocyte maturation in different culture systems and varying levels of intracellular stored calcium ([Ca2+]is) in the oocytes. Cumulus-oocyte complexes (COC) were incubated in TCM 199 containing either 10% fetal calf serum (FCS) in the absence (System 1) or presence (System 2) of FSH and estradiol, or 6 mg/mL bovine serum albumin (BSA) in the presence of FSH and estradiol (System 3). Levels of [Ca2+]is in oocytes were determined by using the fluorophore chlortetracycline. The addition of 50 ng/mL bPRL to different culture media increased the percentage of oocytes at telophase I and metaphase II stages (Systems 1 and 2) and/or decreased the percentage of oocytes with degenerated chromosomes (Systems 1 and 3). Compared with the control, lower levels of [Ca2+]is were observed in oocytes cultured for 2.5 h in those systems in which bPRL decreased the rate of oocytes with degenerated chromosomes (1.27+/-0.11 vs. 1.67+/-0.09 arbitrary units (AU) in System 1, P<0.001 and 1.27+/-0.12 vs. 1.52+/-0.04 AU in System 3, P<0.001). These findings show that the effects of bPRL on bovine oocyte maturation depend on the composition of the culture system and that the decline in the rate of oocytes with degenerated chromosomes in response to bPRL may be the result of the decrease in [Ca2+ ]is levels at early stages of oocyte maturation.     

Influence of hormones and growth factors on viability,DNA, and protein content of adult hepatocytes in primary culture

Summary The survival of adult rat hepatocytes in monolayer culture was studied in the presence of different hormones (neurotensin, oxytocin, thyrotropin releasing hormone, luteinizing hormone releasing hormone, cholecalciferol, bradykinin, substance P, aldosterone, melanocyte stimulating hormone, 3,3′,5-triiodo-1-thyronine, corticosterone, human growth hormone, glucagon, insulin, progesterone, testosterone, estradiol, and dexamethasone phosphate) or growth factors (fetal bovine serum). For this purpose trypan blue exclusion, lactate dehydrogenase, and DNA and protein content were measured at 24 and 72 h of culture. 10⁻⁷ M Dexamethasone, a mixture of eight hormones, 10% fetal bovine serum, and a combination of the latter two supplements caused a more than 64% higher DNA content at 72 h when compared to control cultures. A striking agreement of these results with changes of lactate dehydrogenase leakage was observed, whereas trypan blue exclusion gave erratic results. Considerable changes of cell arrangement apparently specific for each supplement were ovserved by low magnification microscopy. It is concluded that glucocorticoids and fetal bovine serum have an outstanding effect on cell viability and that DNA or protein content or both are reliable indicators of cell viability in amitotic cultures.     

Regenerated bovine fetal fibroblasts support high blastocyst development following nuclear transfer

Regenerated bovine fetal fibroblast cells were derived from a fetus cloned from an adult cow and passaged every 2-3 days. Serum starvation was performed by culturing cells in DMEM/F-12 supplemented with 0.5% FCS for 1-3 days. In vitro matured bovine oocytes were enucleated by removing the first polar body and a small portion of cytoplasm containing the metaphase II spindle. Cloned embryos were constructed by electrofusion of fetal fibroblast cells with enucleated bovine oocytes, electrically activated followed by 5 h culture in 10 microg/mL cycloheximide + 5 microg/mL cytochalasin B, and then cultured in a B2 + vero-cell co-culture system. A significantly higher proportion of fused embryos developed to blastocysts by day 7 when nuclei were exposed to oocyte cytoplasm prior to activation for 120 min (41.2%) compared to 0-30 min (28.2%, p < 0.01). Grade 1 blastocyst rates were 85.1% and 73.3%, respectively. The mean number of nuclei per grade 1 blastocyst was significantly greater for 120 min exposure (110.63 +/- 7.19) compared to 0-30 min exposure (98.67 +/- 7.94, p < 0.05). No significant differences were observed in both blastocyst development (37.4% and 30.6%) and mean number of nuclei per blastocyst (103.59 +/- 6.6 and 107.00 +/- 7.12) when serum starved or nonstarved donor cells were used for nuclear transfer (p > 0.05). Respectively, 38.7%, 29.4%, and 19.9% of the embryos reconstructed using donor cells at passage 5-10, 11-20 and 21-36 developed to the blastocyst stage. Of total blastocysts, the percentage judged to be grade 1 were 80.9%, 79.2%, and 54.1%, and mean number of nuclei per grade 1 blastocysts, were 113.18 +/- 9.06, 100.04 +/- 6.64, and 89.25 +/- 6.19, respectively. The proportion of blastocyst percentage of grade 1 blastocysts, and mean number of nuclei per grade 1 blastocyst decreased with increasing passage number of donor cells (p < 0.05). These data suggest that regenerated fetal fibroblast cells support high blastocyst development and embryo quality following nuclear transfer. Remodeling and reprogramming of the regenerated fetal fibroblast nuclei may be facilitated by the prolonged exposure of the nuclei to the enucleated oocyte cytoplasm prior to activation. Serum starvation of regenerated fetal cells is not beneficial for embryo development to blastocyst stage. Regenerated fetal fibroblast cells can be maintained up to at least passage 36 and still support development of nuclear transfer embryos to the blastocyst stage.     

Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos

Bovine blastocysts were produced using 6 different systems: 5 commonly used in vitro culture systems (synthetic oviduct fluid medium - SOF- without fetal calf serum, SOF supplemented with 10% serum for the entire culture period, SOF supplemented with 10% serum from Day 4 of culture, M199 coculture with bovine oviduct epithelial cells, M199 coculture with granulosa cell monolayer) and 1 in vivo culture system involving collection of blastocysts from superovulated bovine donors at Day 7. Zygotes obtained from IVM/IVF were assigned randomly to 1 of the 5 systems tested and were cultured for 9 d (Day 0= day of insemination). Cleavage, development to the blastocyst stage and blastocyst sex ratio were assessed in all treatments. In addition, the effect of the IVC system on the kinetics of blastocyst development and sex ratio was assessed on Days 6, 7, 8, and 9. The presence of fetal calf serum in SOF not only resulted in faster development (19.1% of blastocysts in SOF supplemented with serum vs 7.1% in absence of serum at Day 6; P < 0.05) and increased blastocyst production (47.5% of blastocysts in SOF supplemented with serum vs 34.4% in absence of serum; P < 0.05) but it also enhanced overall male survival. The coculture systems produced fewer blastocysts than culture in SOF (27.6 to 28.3% in coculture vs 47.5% in SOF supplemented with serum; P < 0.05), but similar to SOF without fetal calf serum, they had no effect on blastocyst sex ratio.     

Effects of fetal bovine serum, FSH and 17beta-estradiol on the culture of bovine preantral follicles

We describe a 7-d culture in droplets of collagen gel of isolated small bovine preantral follicles in medium with or without 10% fetal bovine serum (FBS). In addition, the effect of human recombinant FSH and 17beta-estradiol on the morphology and growth of the preantral follicles was investigated in medium without FBS. After culture in medium with 10% FBS, the increase in follicle diameter was 13.1 +/- 8.4 microm, the percentage of BrdU-labeled cells was 49.9 +/- 11.3 and the number of cells per area granulosa was 11.1 +/- 1.8. Omission of serum from the culture medium had no effect on the percentage of labeled cells, but the diameter increase was lower and the cells were smaller. Apparently, serum affects the size of the granulosa cells from small preantral follicles rather than the stimulation of cell proliferation. Addition of human recombinant FSH and/or 17beta-estradiol to serum-free medium resulted in a larger diameter increase during culture compared with that of the control. With FSH, this was due to an increase in cell proliferation, while with estradiol this was caused by an increase in granulosa cell size. The effects of simultaneous treatment with FSH and estradiol was simply the combination of their individual effects. In conclusion, small bovine preantral follicles can be cultured for 7 d in the absence of serum and hormones. The follicles increase in diameter and react to FSH with enhanced cell proliferation and to estradiol with an increase in cell size.     

Oligodeoxynucleotide stability in subcellular extracts and culture media

Oligodeoxynucleotide degradation was studied in four systems in order to assess the importance of degradation in hybridization arrest experiments dependent on oligodeoxynucleotides complementary to mRNA sequences. Oligodeoxynucleotides were not detectably degraded over 2 h at 37 degrees C in rabbit reticulocyte lysate or Dulbecco's modified essential medium with 5% fetal calf serum, but were degraded over 2 h in HeLa cell postmitochondrial cytoplasmic extract, and were degraded within 15 min in bovine calf serum.     

Serum regulates Na+/H+ exchange in Caco-2 cells by a mechanism which is dependent on F-actin.

Regulation of Na+/H+ exchange by fetal bovine serum was studied in Caco-2 cells, an established cell line derived from a human colon carcinoma. Cells were grown as polarized monolayers on collagen-coated filters and intracellular pH measured fluorometrically with 2',7'-bis(2-carboxymethyl)-5,6-carboxyfluorescein. Na+/H+ exchange was reduced 64% when cells were deprived of serum for 4 h. In contrast to other cell types, readdition of serum for 10 min did not activate Na+/H+ exchange; however, readdition of serum for 4 h restored Na+/H+ exchange to control values. This long-term effect of serum on Na+/H+ exchange activity could not be explained by changes in intracellular buffering capacity or intracellular [Na+]. 4-h serum deprivation reduced the K(t) of the exchanger for external Na+ from 21 to 6 mM, and reduced the V(max) by 57%, but did not alter the IC50 for amiloride in the presence of 140 mM Na+. Inhibition of protein synthesis with cycloheximide (5 microM) did not alter the effect of serum removal or readdition on Na+/H+ exchange. Low temperature (13 degrees C) completely prevented the inhibition of Na+/H+ exchange caused by the removal of serum. In addition, once Na+/H+ exchange was inhibited by serum removal at 37 degrees C, maintaining cells at 13 degrees C also blocked the recovery of Na+/H+ exchange caused by serum readdition. Conversely, cytochalasin D (0.1-20 microM) blocked the reduction of Na+/H+ exchange which occurred due to 4-h serum deprivation, but did not block the restoration of Na+/H+ exchange when the cells were re-exposed to serum for a further 4 h. Colchicine (20 microM) did not alter the effect of serum removal or readdition. These data suggest that serum regulates Na+/H+ exchange activity by a posttranslational mechanism which is dependent on F-actin.     

In vitro production of bovine embryos in medium supplemented with a serum replacer: effects on blastocyst development, cryotolerance and survival to term

In this study, we evaluated a serum replacer (SR; Knockout SR, Invitrogen) in our in vitro culture systems. We hypothesized that SR would benefit bovine embryo development, since SR supported survival of embryonic stem cells (which originate from embryos). Experiment 1 compared oocyte maturation with SR versus fetal bovine serum (FBS). Following fertilization, blastocyst development was lower for oocytes matured with SR (21.5 versus 34.1, P<0.05). Experiment 2 evaluated SR for culturing embryos. Following fertilization, embryos were cultured for 3 days in KSOM, and then assigned to treatments: (1) KSOM static culture (KNM); (2) fresh KSOM (KD3); (3) KSOM+SR or (4) KSOM+FBS and cultured to Day 7 (fertilization=Day 0). Blastocyst development in FBS or SR was higher than either KNM or KD3 (48.2, 47.2, 32.7, and 35.5, respectively, P<0.05). Experiment 3 evaluated cryosurvival of embryos cultured in the same manner as Experiment 2. On Day 7, embryos were vitrified and upon warming, embryos cultured in SR had greater 24h survival rates (70.6%) than all other treatments (P<0.05). Finally, Experiment 4 evaluated effects of SR on pregnancy rate and development to term. Culture in SR was not detrimental to pregnancy or calving rates (50 and 50%, respectively), and SR calves had normal birth weights (mean=38.8 kg+/-1.5). In conclusion, the use of SR for maturation of oocytes was not beneficial; however, SR enhanced embryo culture by improving development in vitro, cryotolerance and survival, effectively replacing serum in culture.     

Nutrition and fetal brain maturation : II. Impact of maternal starvation on changing levels of acetylcholinesterase and enolase in vitro

The impact of maternal starvation during Days 17-20 of gestation was examined in 20-day fetal rat brain tissue cultured for 6 days in MEM and 10% adult rat serum. Acetylcholinesterase (AChE) activities were consistently greater in fetal brain cell cultures from starved mothers. When fetal tissues from starved mothers were continuously exposed to 72-h fasted serum, AChE activities increased from 1.03 +/- 0.14 to 1.59 +/- 0.21 mumol/h/mg protein (P less than 0.001). In fetal tissues from fed mothers, lower AChE activities were increased from 0.78 +/- 0.09 to 1.04 +/- 0.07 mumol/h/mg protein (P less than 0.05) when 72-h fasted serum was used to replace the fed serum during incubation. When fetal brain cell cultures from fed mothers were exposed for 6 days to graded concentrations of fed serum (2.5-15%), the activities of AChE fell reciprocally from 1.34 +/- 0.10 to 0.82 +/- 0.12 mumol/h/mg protein (P less than 0.05). The levels of AChE activity in tissues exposed to fasted serum were consistently greater, but fell similarly from 1.62 +/- 0.10 to 0.97 +/- 14 mumol/h/mg protein (P less than 0.01), when serum concentrations were increased from 2.5 to 15%. AChE activities were 30% higher in tissues incubated with cycloheximide 10(-3) M (P less than 0.02). Unlike AChE, fetal brain enolase activities were unaffected by maternal starvation. In fetal brain cell cultures from fed mothers, enolase fell from 1.85 +/- 0.10 to 1.37 +/- 0.12 mumol/min/mg protein following exposure to fasted instead of fed serum (P less than 0.02). In fetal cultures from starved mothers, enolase activities were depressed similarly from 1.76 +/- 0.08 to 1.41 +/- 0.09 mumol/min/mg protein when fasted replaced fed serum (P less than 0.02). Thus, the fetal brain cell cultures appear to maintain enzymatic realignments imposed by maternal starvation for at least 6 days. In addition, serum from fasted animals has significant growth inhibiting properties manifested by heightened activities of AChE and lower activities of enolase.     

Somatic cell cloning in Buffalo (Bubalus bubalis): effects of interspecies cytoplasmic recipients and activation procedures

Successful nuclear transfer (NT) of somatic cell nuclei from various mammalian species to enucleated bovine oocytes provides a universal cytoplast for NT in endangered or extinct species. Buffalo fetal fibroblasts were isolated from a day 40 fetus and were synchronized in presumptive G(0) by serum deprivation. Buffalo and bovine oocytes from abattoir ovaries were matured in vitro and enucleated at 22 h. In the first experiment, we compared the ability of buffalo and bovine oocyte cytoplasm to support in vitro development of NT embryos produced by buffalo fetal fibroblasts as donor nuclei. There were no significant differences (p > 0.05) between the NT embryos derived from buffalo and bovine oocytes, in fusion (74% versus 71%) and cleavage (77% versus 75%) rates, respectively. No significant differences were also observed in blastocyst development (39% versus 33%) and the mean cell numbers of day 7 cloned blastocysts (88.5 +/- 25.7 versus 51.7 +/- 5.4). In the second experiment, we evaluated the effects of activation with calcium ionophore A23187 on development of NT embryos after electrical fusion. A significantly higher (p < 0.05) percentage of blastocyst development was observed in the NT embryos activated by calcium ionophore and 6-DMAP when compared with 6-DMAP alone (33% versus 17%). The results indicate that the somatic nuclei from buffalo can be reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts similar to those transferred into buffalo oocytes. Calcium ionophore used in conjunction with 6-DMAP effectively induces NT embryo development.     

Growth of Chinese hamster ovary (CHO) cells in the presence of MMC in low-serum medium.

Chinese hamster ovary (CHO-WBLT) cells growing in McCoy's 5a with 10% fetal bovine serum (FBS) were adapted to 0.5% FBS in CHO-1 Complete Media System, a serum-free medium from Ventrex. Cells in these two media were exposed to 10(-7) M and 10(-8) M mitomycin C (MMC) for 24 h. Comparison of cell growth over 10 days showed that cells in 0.5% serum proliferate, though at a slower rate than cells in 10% serum. Treatment with MMC revealed that at 10(-7) M, MMC is cytotoxic to cells to both the media; at 10(-8) M, MMC is non-cytotoxic to cells in both media.     

Angiotensin-(1--7) in the ovine fetus

In the adult animal, ANG-(1-7) may counterbalance some effects of ANG II. Its effects in the fetus are unknown. Basal ANG-(1-7), ANG I, ANG II, and renin concentrations were measured in plasma from ovine fetuses and their mothers (n = 10) at 111 days of gestation. In the fetus, concentrations of ANG I, ANG-(1-7), and ANG II were 86 +/- 21, 13 +/- 2, and 14 +/- 2 fmol/ml, respectively. In the ewe, concentrations of ANG I were significantly lower (20 +/- 4 fmol/ml, P < 0.05) as were concentrations of ANG-(1-7) (2.9 +/- 0.6 fmol/ml), whereas ANG II concentrations were not different (10 +/- 1 fmol/ml). Plasma renin concentrations were higher in the fetus (4.8 +/- 1.1 pmol ANG I x ml(-1) x h(-1)) than in the ewe (0.9 +/- 0.2 pmol x ml(-1) x h(-1), P < 0.05). Infusion of ANG-(1-7) (approximately 9 microg/h) for a 3-day period caused a significant increase in plasma concentrations of ANG-(1-7) reaching a maximum of 448 +/- 146 fmol/ml on day 3 of infusion. Plasma levels of ANG I and II as well as renin were unchanged by the infusion. Urine flow rate, glomerular filtration rate, and fetal arterial blood pressure did not change and were not different than values in fetuses receiving a saline infusion for 3 days (n = 5). However, the osmolality of amniotic and allantoic fluid was significantly higher in fetuses that received ANG-(1-7). Also, compared with the saline-infused animals, mRNA expression levels of renin, the AT(1) receptor, and AT(2) receptor were elevated in kidneys of fetuses that received infusions of ANG-(1-7). Infusion of an ANG-(1-7) antagonist ([D-Ala(7)]-ANG-(1-7), 20 microg/h) for 3 days had no effect on fetal blood pressure or renal function. In conclusion, although infusion of ANG-(1-7) did not affect fetal urine flow rate, glomerular filtration rate, or blood pressure, changes in fetal fluids and gene expression indicate that ANG-(1-7) may play a role in the fetal kidney.     

Factors affecting bovine embryo development in synthetic oviduct fluid following oocyte maturation and fertilization in vitro

Employing a total of 3465 bovine oocytes this study was aimed at improving the efficiency of bovine embryo production under defined and undefined conditions. Following in vitro maturation (IVM) and in vitro fertilization (IVF), oocytes were allocated to various culture treatments using synthetic oviduct fluid (SOF). In our 3 experiments we showed that: 1) the addition of fetal calf serum (FCS 10% v/v) to SOF droplets after 20 to 24 h significantly improved blastocyst yields on Day 6 (21 vs 12%; P < 0.01), but not at later stages and resulted in significantly higher Day-8 blastocyst cell numbers (148 +/- 61 vs 92 +/- 35; P < 0.05); 2) the removal of bovine serum albumin (BSA) from the standard SOF medium resulted in significantly reduced blastocyst yields on Days 6, 7 and 8, respectively (17 vs 8%; 28 vs 18%; 31 vs 21%; P < 0.05); 3) the presence or absence of cumulus cells surrounding the presumptive zygote in culture in SOF had no effect on cleavage rate, percentage of 5-8 cell embryos or blastocyst yields (Day 6,7 or 8); 4) the culture of presumptive zygotes in SOF in an atmosphere of 5% CO2 in air (20% O2) resulted in significantly reduced development compared with culture in 5% CO2, 5% O2, 90% N2 in terms of blastocyst yield on Days 6, 7 and 8 and on Day 8 hatching rate, respectively (5 vs 22%; 9 vs 33%; 13 vs 48%; 50 vs 8%; P < 0.001) and 5) embryo density (1 embryo per 1 or 3 microl SOF) or replacing the culture medium every 48 h had no effect when SOF was supplemented with serum; however, under serum-free conditions, changing of the media resulted in a slightly improved Day-6 blastocyst yield such that renewal of serum-free medium mimicked the effect of serum addition.     

Stimulation of sodium transport by aldosterone and arginine vasotocin in A6 cells

The effects of aldosterone and arginine vasotocin (AVT) on transepithelial Na+ transport of cultured A6 cells were investigated. All experiments were performed with cells grown on Millicell TM culture-plate inserts for a period of 2-4 weeks in defined, serum-free medium. Omitting fetal bovine serum 2 days after seeding the cells on filters did not influence potential difference (PD) development or the hormonal responses tested. The cell layers were placed in an Ussing chamber for short-circuit current (ISC) and transepithelial conductance (G) measurements. Base-line values were (n = 93): PD, 51.0 +/- 0.2 mV (apical side negative); ISC, 14.55 +/- 0.06 microA/cm2; G, 0.306 +/- 0.001 mS/cm2. ISC and G were higher in cells pretreated with 10(-7) M aldosterone for 24 h in the incubator, when compared to controls (ISC, 28 +/- 2 vs. 16 +/- 2 microA/cm2, G, 0.41 +/- 0.04 vs. 0.26 +/- 0.01 mS/cm2, n = 5) and both remained stable for at least 6 h. In cells not treated with aldosterone, 10(-7) M AVT increased ISC within 1 min after addition, producing a maximum ISC within 15 min which then declined to baseline levels over the next 5 h. Addition of AVT to aldosterone-pretreated cells resulted in a significantly greater peak increase in ISC than in non-pretreated cells (change in ISC compared to controls: 8.1 +/- 0.4 vs. 4.9 +/- 0.4 microA/cm2, n = 5, P less than 0.001), indicating a synergistic effect. A dose-response curve for amiloride obtained in the presence of AVT showed that amiloride completely inhibits ISC. Pretreatment of the A6 cells with aldosterone for 24 h shifted the amiloride dose-response curve to the right, as expressed in a doubling of the apparent Ki value (from 0.17 +/- 0.02 to 0.33 +/- 0.04 microM). In conclusion, A6 cells grown in defined, serum-free medium express a greater than additive synergism between aldosterone and AVT in stimulating transepithelial Na+ transport.     

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     

Effect of angiotensin II with follicle cells and insulin-like growth factor-I or insulin on bovine oocyte maturation and embryo development

The objective was to evaluate the effects of angiotensin II (Ang II), insulin-like growth factor-I (IGF-I) and insulin on the nuclear and cytoplasmic maturation of bovine oocytes in the presence of follicular cells. Cumulus-oocyte complexes (COCs) were cultured for 22h in the presence of follicular cells (control with cells) and Ang II, IGF-I or insulin (treatments), or in the absence of follicular cells (control without cells). Using these five groups, Experiment 1 was conducted with and without the addition of gonadotrophins. Only oocytes in the Ang II group resumed meiosis at rates (88.2+/-1.8% and 90.7+/-4.3% for oocytes cultured in the absence or presence of LH/FSH, respectively) similar to those observed for oocytes cultured in the absence of follicular cells (89.7+/-0.3% and 92.6+/-2.6%; P<0.01). In Experiment 2, the effect of Ang II alone and in combination with IGF-I or insulin on oocyte maturation for 7h (germinal vesicle breakdown), 12h (metaphase I) and 22h (metaphase II) was evaluated in a design similar to that of the first experiment. Ang II plus IGF-I or insulin induced the resumption of meiosis, irrespective of the presence of gonadotrophins (P<0.01). Experiment 3 used groups similar Experiment 2 to determine the rate of subsequent embryo development, using fetal calf serum (FCS) in the culture medium. The COCs were cultured in maturation medium for 1h (1+23h), 12h (12+12h) or 24h in the presence of follicular cells and the respective treatments and for the remaining period in the absence of follicular cells to complete 24h. In Experiment 4, BSA was used in lieu of serum in the maturation medium in a 12+12h maturation system. Oocytes matured using the 12+12h system with BSA or FCS in the presence of Ang II+IGF-I had higher rates of blastocyst formation than the other treatments (P<0.05). In conclusion, Ang II reversed the inhibitory effect of follicular cells on nuclear maturation of bovine oocytes, irrespective of the presence of gonadotrophins, IGF-I and insulin. However, oocyte cytoplasmatic maturation (i.e., subsequent embryo development), was higher when Ang II and IGF-I were present in the maturation medium containing follicular cells cultured for 12+12h.