Liver Cell Monolayers Research Articles

Study of the l-Phenylalanine Ammonia-Lyase Penetration Kinetics and the Efficacy of Phenylalanine Catabolism Correction Using In Vitro Model Systems.

The kinetics of l-phenylalanine ammonia-lyase (PAL) penetration into the monolayer of liver cells after its release from capsules was studied. The studies showed the absence of the effect of the capsule shell based on plant hydrocolloids on the absorption of l-phenylalanine ammonia-lyase in systems simulating the liver surface. After 120 min of incubation, in all variants of the experiment, from 87.0 to 96.8% of the enzyme penetrates the monolayer of liver cells. The combined analysis of the results concludes that the developed encapsulated form of l-phenylalanine ammonia-lyase is characterized by high efficiency in correcting the disturbed catabolism of phenylalanine in phenylketonuria, which is confirmed by the results of experiments carried out on in vitro model systems. PAL is approved for the treatment of adult patients with phenylketonuria. The encapsulated l-phenylalanine ammonia-lyase form can find therapeutic application in the phenylketonuria treatment after additional in vitro and in vivo studies, in particular, the study of preparation safety indicators. Furthermore, it demonstrated high efficacy in tumor regression and the treatment of tyrosine-related metabolic disorders such as tyrosinemia. Several therapeutically valuable metabolites biosynthesized by PAL via its catalytic action are included in food supplements, antimicrobial peptides, drugs, amino acids, and their derivatives. PAL, with improved pharmacodynamic and pharmacokinetic properties, is a highly effective medical drug.

Hepato(Geno)Toxicity Assessment of Nanoparticles in a HepG2 Liver Spheroid Model

(1) In compliance with the 3Rs policy to reduce, refine and replace animal experiments, the development of advanced in vitro models is needed for nanotoxicity assessment. Cells cultivated in 3D resemble organ structures better than 2D cultures. This study aims to compare cytotoxic and genotoxic responses induced by titanium dioxide (TiO2), silver (Ag) and zinc oxide (ZnO) nanoparticles (NPs) in 2D monolayer and 3D spheroid cultures of HepG2 human liver cells. (2) NPs were characterized by electron microscopy, dynamic light scattering, laser Doppler anemometry, UV-vis spectroscopy and mass spectrometry. Cytotoxicity was investigated by the alamarBlue assay and confocal microscopy in HepG2 monolayer and spheroid cultures after 24 h of NP exposure. DNA damage (strand breaks and oxidized base lesions) was measured by the comet assay. (3) Ag-NPs were aggregated at 24 h, and a substantial part of the ZnO-NPs was dissolved in culture medium. Ag-NPs induced stronger cytotoxicity in 2D cultures (EC50 3.8 µg/cm2) than in 3D cultures (EC50 > 30 µg/cm2), and ZnO-NPs induced cytotoxicity to a similar extent in both models (EC50 10.1–16.2 µg/cm2). Ag- and ZnO-NPs showed a concentration-dependent genotoxic effect, but the effect was not statistically significant. TiO2-NPs showed no toxicity (EC50 > 75 µg/cm2). (4) This study shows that the HepG2 spheroid model is a promising advanced in vitro model for toxicity assessment of NPs.

222 EFFECT OF THE PRESENCE OR ABSENCE OF PERCOLL CENTRIFUGATION; PENICILLAMINE, HYPOTAURINE, AND EPINEPHRINE; AND HEPARIN ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

Protocols for the production of bovine embryos in vitro routinely include Percoll centrifugation of semen, usually include heparin, and often include penicillamine, hypotaurine, and epinephrine (PHE) in the fertilization media. This study examined the contribution of each of these components to the success of in vitro fertilization of bovine oocytes and subsequent blastocyst development. Bovine oocytes were aspirated from 2- to 10-mm follicles within 5 h after slaughter of cattle at a local abattoir. Groups of 30 to 40 cumulus–oocyte complexes (COC) were matured in 0.5 mL of TCM-199 with 10% FCS, 4 µg mL–1 of FSH, and 6 µg mL–1 of LH (NOBL Laboratories, Sioux Center, IA, USA) for 24 h (39°C, 4% CO2 in air). The COC were then washed and placed in 0.5 mL of modified Tyrode-lactate medium for IVF with various combinations of 2 µg mL–1 of heparin, 20 µM penicillamine, 10 µM hypotaurine, and 1 µM epinephrine. Each group of COC was inseminated with 0.25 × 106 frozen–thawed sperm from a single bull after 30 min of centrifugation with (Exp. 1) or without (Exp. 2) a 45/90% Percoll gradient with sperm TALP. Oocytes were vortexed to remove the cumulus after 18 h and placed in co-culture wells containing a monolayer of buffalo rat liver cells and 0.5 mL of Menezo’s B2 medium supplemented with 10% FCS. On the fourth day of in vitro culture, cleavage was defined as 2 cells or greater and embryos were transferred to fresh co-culture wells. There were 4 replicates in the first experiment and 6 in the second. Data were analysed by ANOVA. In the first experiment, the use of a Percoll gradient during centrifugation for separation of viable sperm from seminal plasma and cryprotectants resulted in significantly higher cleavage and Day 8 blastocyst rates than did the absence of Percoll when PHE and heparin were used together, and both cleavage and blastocyst rates were lower when only PHE or heparin was used separately compared with when both were used together (Table 1). The absence of Percoll, PHE, and heparin resulted in the lowest rates of cleavage and development. In the second experiment, the absence of either PHE or heparin resulted in lower cleavage rates, but not blastocyst rates, compared with the use of both, and the absence of both resulted in the lowest cleavage and blastocyst rates in spite of the use of Percoll. Table 1.Effects of Percoll; penicillamine, hypotaurine, and epinephrine (PHE); and heparin on cleavage and subsequent embryo development per oocyte

Characterization of a novel embryonic stem cell line from an ICSI-derived blastocyst in the African green monkey

Several cell types from the African green monkey (Cercopithecus aethiops), such as red blood cells, primary culture cells from kidney, and the Vero cell line, are valuable sources for biomedical research and testing. Embryonic stem (ES) cells that are established from blastocysts have pluripotency to differentiate into these and other types of cells. We examined an in vitro culture system of zygotes produced by ICSI in African green monkeys and attempted to establish ES cells. Culturing with and without a mouse embryonic fibroblast (MEF) cell monolayer resulted in the development of ICSI-derived zygotes to the blastocyst stage, while culturing with a buffalo rat liver cell monolayer yielded no development (3/14, 21.4% and 6/31, 19.4% vs 0/23, 0% respectively; P<0.05). One of the nine blastocysts, which had been one of the zygotes co-cultured with MEF cells, formed flat colonies consisting of cells with large nuclei, similar to other primate ES cell lines. The African green monkey ES (AgMES) cells expressed pluripotency markers, formed teratomas consisting of three embryonic germ layer tissues, and had a normal chromosome number. Furthermore, expression of the germ cell markers CD9 and DPPA3 (STELLA) was detected in the embryoid bodies, suggesting that AgMES cells might have the potential ability to differentiate into germ cells. The results suggested that MEF cells greatly affected the quality of the inner cell mass of the blastocysts. In addition, AgMES cells would be a precious resource for biomedical research such as other primate ES cell lines.

378 INTRACYTOPLASMIC SPERM INJECTION AND ESTABLISHMENT OF EMBRYONIC STEM CELLS IN AFRICAN GREEN MONKEYS (CERCOPITHECUS AETHIOPS)

There has been little embryological research in African green monkeys (Cercopithecus aethiops), although Vero and Cos7 cell lines derived from the kidney of the green monkey are used as valuable resources for testing viral infections and introducing foreign genes. Embryological research would not only contribute to medical science but would also help to maintain and propagate other monkeys, including those in the wild. We examined intracytoplasmic sperm injection (ICSI) and in vitro culture of green monkey embryos and attempted to establish embryonic stem (ES) cells, which would be precious resources. We collected mature oocytes from females treated with eCG + hCG or human FSH + hCG to induce follicular growth stimulation and oocyte maturation. Of 61 oocytes from eCG-treated females and of 44 oocytes from FSH-treated females, 41 (67.2%) and 27 (61.4%) fertilized oocytes with 2 pronuclei and a second polar body were produced with ICSI, respectively. We cultured fertilized oocytes from eCG-treated and FSH-treated females in CMRL-1066 medium containing 10% fetal bovine serum (FBS), with or without a buffalo rat liver (BRL) cell monolayer. Of 28 zygotes from eCG-treated females, 9 were cultured without a monolayer, and 3 (33.3%) developed into blastocysts; none of 19 cultured with a monolayer did. Of 22 zygotes from FSH-treated females, 18 were cultured without a monolayer, and 5 (27.8%) developed into blastocysts; none of 4 cultured with a monolayer did. Thus, there were no differences in the fertilization rates and development into blastocysts between the oocytes from eCG- and FSH-treated females. On the other hand, BRL cells did not support development into blastocysts [0/23 (0%) with a monolayer vs. 8/27 (29.6%) without a monolayer; P &amp;lt; 0.01]. The other embryos were used for experiments such as embryo transfer. Parts of the inner cell mass (ICM), isolated by removing trophectoderm and the zona pellucida (ZP) with a 27-gauge needle (5 blastocysts) or by dissolving the ZP with an enzyme (3 blastocysts) were transferred to DMEM/F12 (1 : 1) medium containing 20% FBS or knockout serum replacement with a mitomycin C-treated STO cell or mouse embryonic fibroblast cell monolayer. Only 3 ICMs formed extended colonies; the others, including the 3 blastocysts from which the ZP was dissolved, did not. When the 3 colonies were subcultured with collagenase or divided into small clusters with a needle, 2 flat colonies, as in primate ES cells, appeared. At the next subculture, the flat colonies disappeared. However, we confirmed the formation of ES-like cell colonies. These results show that it is possible to produce blastocysts with ICSI and that ES cells may be established in African green monkeys.

Alkaline phosphatase activity in bovine oocytes and preimplantation embryos as affected by removal of the zona pellucida and culture medium constituents.

The aims of the present study were: (1) to characterize alkaline phosphatase (AP) activity in bovine oocytes and embryos with intact or removed zona pellucida (ZP); and (2) to evaluate the effect of culture medium constituents on AP activity. Alkaline phosphatase activity in non-matured and matured oocytes was most evident nearest the plasma membrane and perivitelline space. In more than 90% of two- to 16-cell embryos, AP activity was observed in the perivitelline space and at blastomere contacts. In blastocysts, AP activity was localized to the trophectoderm. Only after immunodissection was AP activity detected in the inner cell mass. Removal of the ZP by pronase or mechanical means reduced AP activity. Alkaline phosphatase activity was detected in evacuated ZP after mechanical removal. Specific constituents comprising the embryo culture medium altered AP activity. Alkaline phosphatase activity was reduced in eight- to 16-cell embryos and evacuated ZP cultured in CR1aa + 0.4% bovine serum albumin compared with embryos cultured in CR1aa alone or embryos co-cultured on a monolayer of Buffalo rat liver cells in the presence of 10% fetal bovine serum. The presence of AP activity at blastomere contacts and in evacuated ZP limits its usefulness as a marker for the differentiation of embryonic cells comprising the early cleavage-stage embryo.

The effect of carbon tetrachloride and ultraviolet radiation on dolichol levels in liver cells isolated from 3- and 24-month-old male Sprague-Dawley rats.

Dolichol (D) is a long-chain polyprenoid broadly distributed in the cell membranes, possibly endowed with a free-radical scavenging activity, whose concentration in tissues increases with increasing age. No enzyme pathway for D degradation has been discovered. In order to test the hypothesis that D might undergo a non-enzymatic free-radical mediated decomposition the effects of a xenobiotic agent (carbon tetrachloride, CCl(4)) and ultraviolet-B (UV-B) radiation on D levels were studied in liver cells isolated from male ad libitum fed Sprague-Dawley rats aged 3 or 24 months. Liver cells (90 mg/ml) were incubated in sealed flasks (6 ml cell suspension each) for 0, 5, 10 and 20 min after the addition of 25, 50 or 200 microl CCl(4) in the central well. 50 ml of a 6 mg/ml liver cell suspension were poured in a 120 cm(2) Petri dish and the sediment liver cell monolayer was exposed to UVB radiation for 0, 5, 10, 20 and 40 min. At the given time, cells were taken and D was extracted and assayed by the HPLC procedure. D levels were remarkably higher in older than in younger cells as expected ( P < 0.001). Treatment with CCl(4) and UVB caused a highly significant decrease in D ( P < 0.001) whose percentage was larger in younger than in older cells. The conclusions are that free-radicals generated either by chemical or by physical agents cause a very rapid depletion of D in liver cells, and that the effect of the free radical attack on D decomposition may be lower percentage wise in older than in younger cells, which might account at least in part for the accumulation of D in older tissues.

Interferon-tau in bovine blastocysts following parthenogenetic activation of oocytes: pattern of secretion and polymorphism in expressed mRNA sequences.

A series of experiments were conducted to examine the pattern of production and secretion of interferon-tau (IFN-tau) by blastocysts following parthenogenetic activation of bovine oocytes. In the first experiment, 36.8, 24.1, and 33.2% of IVF-derived and parthenogenetically activated oocytes cultured in the presence or absence of a monolayer of buffalo rat liver cells, respectively, reached the blastocyst stage. Following individual culture of blastocysts, IFN-tau concentration in medium droplets was similar among the three groups, although IVF-derived blastocysts contained significantly more cells. In the second experiment, 156 IVF-derived blastocysts were sexed by PCR with 75 and 81, respectively, being male and female. IFN-tau secretion of these was compared to that of 70 parthenogenetic blastocysts. Female and parthenogenetic blastocysts produced significantly more IFN-tau than their male counterparts. In the third experiment, the ability of hatched blastocysts to form outgrowths and the pattern of their IFN-tau secretion were examined. Of the 48 IVF-derived blastocysts, 44 formed outgrowths compared to 41 of the 42 hatched parthenotes. Parthenogenetic outgrowths were significantly larger after 7 days, but this difference had disappeared after 14 days. IFN-tau secretion did not differ between the two groups. Lastly, sequence analyses of expressed mRNA from individual parthenogenetic blastocyst outgrowths showed four different transcript types which, based on their predicted amino acid sequence, belong to two subgroups, IFN-tau1 and IFN-tau3. In addition, one new transcript sequence was identified, encoding a new protein isoform.

Rhesus offspring produced by intracytoplasmic injection of testicular sperm and elongated spermatids

Objective: To establish pregnancies in rhesus monkeys using testicular sperm and elongated spermatids injected into oocytes. Design: Comparative animal study. Setting: Regional Primate Research Center. Animal(s): Prime, fertile rhesus monkeys. Intervention(s): Oocytes collected by laparoscopy from gonadotropin-stimulated female rhesus monkeys were injected with testicular sperm or elongated spermatids obtained from the testis of males. Cleavage stage embryos were transferred to surrogate females. Main Outcome Measure(s): Fertilization, embryo cleavage, and the establishment of pregnancies. Fertilization failures were fixed and processed for the detection of microtubules and chromatin configurations. Result(s): Fertilization, assessed by the presence of two pronuclei within 15 hours after injection, was 60% for intracytoplasmic sperm injection with testicular sperm and 47% for elongated spermatid injection. Fertilized zygotes co-cultured in Connaughts Medical Research Labs (CMRL) medium on a Buffalo Rat Liver cell monolayer resulted in hatched blastocysts after testicular sperm extraction–intracytoplasmic sperm injection and elongated spermatids. Embryos transferred at the 4- to 8-cell stage gave rise to three pregnancies: 2/3 from testicular sperm and 1/1 from an elongated spermatid. Three healthy infants were delivered by cesarean. Oocytes that failed to fertilize typically remained arrested in metaphase of meiosis. Conclusion(s): Testicular sperm and elongated spermatids can be used for fertilization in the rhesus monkey resulting in live births.

Preimplantation bovine embryos express mRNA of growth hormone receptor and respond to growth hormone addition during in vitro development.

In previous studies we demonstrated that bovine cumulus oocyte complexes (COCs) obtained from small and medium sized follicles express growth hormone receptor (GHR) mRNA and respond to growth hormone (GH) addition during in vitro maturation. The aim of this study was to investigate whether bovine zygotes and preimplantation embryos continue the expression of GHR gene after in vitro fertilization and during early embryo development and whether supplementation of GH during embryo culture affects embryo development. Therefore, COCs obtained from small and medium sized follicles were cultured in M199 supplemented with 10% FCS and gonadotropins for 24 hr. After in vitro fertilization the embryos were cultured: (a) on a monolayer of buffalo rat liver (BRL) cells in M199 supplemented with 10% FCS and 100 ng/ml bovine GH (NIH-GH-B18); (b) in droplets of serum-free BRL-conditioned medium supplemented with 100 ng/ml GH; (c) in droplets of synthetic oviductal fluid (SOF) supplemented with 100 ng/ml GH. Cultures without GH served as controls. Embryos were scored morphologically and the efficiency of the culture system was evaluated (a) as the percentage of cleaved embryos 4 days after IVF, (b) the percentage of blastocysts on Day 9 expressed on the basis of the number of oocytes at the onset of culture, and (c) the percentage of hatched blastocysts on Day 11 expressed on the basis of the total number of blastocysts present at Day 9. For gene expression, immature (GV) and mature (MII) oocytes (as positive control), embryos with less than 8 cells, 16-32 cells, and hatched blastocysts were prepared for reverse transcriptase polymerase chain reaction (RT-PCR) to assess the expression of mRNA of GHR. Messenger RNA for GHR was found in GV and MII oocytes and in all stages of embryo development. No mRNA for GH could be detected in early and expanded blastocysts produced in SOF medium. Immunoreactive GHR was found both in trophoblastic and embryonic cells of hatched blastocysts. Addition of 100 ng/ml GH during embryo culture on a monolayer of BRL cells in M199 supplemented with 10% FCS did not affect embryo development. However, GH (100 ng/ml) supplementation during embryo culture in droplets of serum-free BRL conditioned medium significantly (P < 0.05) enhanced the proportion of > 8-cell embryos. Similarly, culture of embryos in droplets of SOF medium in the presence of GH (100 ng/ml) significantly (P < 0.05) enhanced the number of > 8-cell embryos from 53.8% in control to 70.6% in GH-treated group. Day 9 blastocyst formation in SOF medium was also significantly (P < 0.01) increased in the presence of GH (33.9%) compared to the control (20.2%). Embryos cultured in SOF without GH rarely resulted in hatched blastocysts (0.7%). However, GH supplementation remarkably enhanced the proportion of the hatched blastocysts (13%). In conclusion, expression of GHR gene in preimplantation bovine embryos, presence of the receptor, and the beneficial effect of GH on cleavage, blastocyst formation and hatchability of the embryos point to the involvement of GH in early embryonic development.

Fertilization and embryo development to blastocysts after intracytoplasmic sperm injection in the rhesus monkey.

Notwithstanding the thousands of seemingly healthy children born after intracytoplasmic sperm injection (ICSI), it is not yet possible to conclude absolutely that the ICSI procedure might induce some altered development or that the ICSI protocol might not be improved even further. To address this in a clinically relevant system, the developmental potential of rhesus monkey embryos produced by ICSI is reported. Oocytes collected by laparoscopy from gonadotrophin-stimulated fertile females were fertilized by ICSI using spermatozoa obtained from fertile males by electro-ejaculation. Neither sperm immobilization prior to injection nor an additional chemical stimulus were necessary to achieve oocyte activation and pronuclear formation. Survival and activation of the injected oocytes were judged by the extrusion of the second polar body. Successful fertilization was confirmed by the presence of two pronuclei within 12 h post-ICSI. Some oocytes were fixed and processed for the detection of microtubules and chromatin. Fluorescent labelling revealed that by 12 h post-ICSI the male and female pronuclei were closely apposed and eccentrically positioned within a large microtubule aster. ICSI resulted in a 76.6 +/- 14.9% fertilization rate. First cleavage was completed within 24 h post-ICSI. Two-cell ICSI embryos were co-cultured in CMRL medium on a buffalo rat liver cell monolayer until the hatched blastocyst stage. Oocytes collected laparoscopically from stimulated monkeys can be fertilized by ICSI and will complete preimplantation embryo development in vitro demonstrating that the rhesus monkey is an excellent preclinical model for examining and understanding many aspects of human ICSI.

A comparison of Menezo's B2 and tissue culture Medium-199 for in vitro production of bovine blastocysts

The objectives of this study were, first, to evaluate the effectiveness of 2 culture media, Menezo's B2 (B2) and Tissue Culture Medium-199 (M-199), for the production of bovine blastocysts in a commercial embryo transfer program; and, second, to characterize the stage of development, quality grade and cell number of blastocysts produced in each medium. One-cell bovine embryos were produced using in vitro maturation and fertilization procedures. After fertilization, the embryos were co-cultured on Buffalo rat liver (BRL) cell monolayers in either B2 or M-199+1% BSA (M-199) medium. Both media were supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. Embryo cultures were continued undisturbed to either Day 7 or Day 8 post-insemination. In the Day 7 cultures, all blastocysts were removed for evaluation on Day 7, and the remaining embryos were cultured for a further 24 h. Any additional blastocysts that formed were removed for evaluation and designated as Day 8 disturbed embryos. All blastocysts were classified for stage and quality grade. Embryos were fixed and stained for determination of cell number. Overall, the proportion of blastocysts was greater (P = 0.0003) with B2 medium (46%) than with M-199 (33%). This was due to a larger (P = 0.0001) proportion of blastocysts produced in B2 medium when cultures were left undisturbed for 8 d (50 vs 28% for B2 vs M-199). The proportion of blastocysts on Day 7 of culture tended to differ (P = 0.073) between media (33 vs 24% for B2 vs M-199). In addition, there were more (P = 0.007) blastocysts at advanced stages of development in B2 medium on Day 7. There was no effect of type of medium on the distribution of embryo quality grades on any day examined. The number of cells per blastocyst did not differ between media but did vary significantly (P < .05) with both stage and grade. In conclusion, B2 medium was superior to M-199 medium when used in a co-culture system with BRL cells for the production of bovine blastocysts.

Growth factor expression by human oviduct and buffalo rat liver coculture cells

Objective: To characterize growth factor gene expression by passaged coculture cell lines demonstrated to enhance in vitro pre-embryo growth. Design: Ribonucleic acids isolated from the isthmus, ampullary, and fimbriae portions of the human oviduct, and from buffalo rat liver cell monolayers were subjected to Northern analysis using probes for growth factors. Setting: Academic tertiary care hospital. Patient(s): Two reproductive age women undergoing a hysterectomy and bilateral salpingectomy for benign gynecologic conditions consented to experimental use of their oviducts. Intervention(s): Cell cultures were established from fresh human oviduct segments and commercially purchased buffalo rat liver cells. After two passages, total RNA was isolated from these confluent monolayers, fractionated on denaturing agarose gels, transferred to nylon membranes, and analyzed by Northern hybridization using complementary DNAs from epidermal growth factor (EGF), stem cell factor, also known as Kit-ligand, colony-stimulating factor-1 (CSF), leukemia inhibitory factor, and interleukin-6 (IL-6). Radioactively labeled probes were prepared by in vitro transcription or by 5' end labeling. After hybridization, blots were washed at increasing strigencies to remove nonspecifically bound radioactivity and subjected to autoradiography. Result(s): Human oviduct coculture cells express EGF (kit-ligand), CSF, leukemia inhibitory factor, and IL-6. Buffalo rat liver cells contain the messenger RNA transcripts for kit-ligand and CSF. Conclusion(s): Human oviduct and buffalo rat liver coculture cells express specific growth factors. These results support the theory that coculture systems may enhance pre-embryo growth via the production of embryotrophic factors. The identification of these ligands may provide the rationale for selecting specific growth factors for media supplementation as well as contribute to our understanding of the general mechanisms involved in regulating early embryonic growth and development.

In vitro maturation of bovine oocytes in the presence of bovine activin A does not affect the number of embryos.

This study was carried out to investigate whether bovine recombinant activin A present during the in vitro maturation (IVM) of cumulus enclosed bovine oocytes affects the proportion of embryos that develop to the blastocyst stage. In addition, the effect of the presence of activin A during maturation and during embryo culture was studied. Therefore, bovine cumulus oocyte complexes were matured at 39 degrees C in a humidified atmosphere with 5% CO2 in air for 24 h in: (1) culture medium M199 supplemented with 10% foetal calf serum (FCS), luteinising hormone (LH) and follicle-stimulating hormone (FSH) and 10 ng ml-1 activin A; (2) M199 without FCS but supplemented with LH and FSH and 10 ng ml-1 activin A; (3) M199 without FCS, LH and FSH but supplemented with 10 ng ml-1 activin A. Cultures without activin served as controls. After IVF the embryos were cultured in M199 supplemented with 10% FCS on a monolayer of buffalo rat liver (BRL) cells. For the second part of the study, COCs were matured in vitro in M199 supplemented with LH and FSH and 10 ng ml-1 activin A, fertilized in vitro and the embryos were cultured (1) on a monolayer of BRL cells in M199 supplemented with 10% FCS and 10 ng ml-1 of activin A, and (2) in droplets of serum free BRL-conditioned medium supplemented with 10 ng ml-1 activin A. IVM in the presence of LH, FSH and 10 ng ml-1 activin A did not change the proportion of blastocysts present at Day 9 or the proportion of hatched blastocyst at Day 11. Activin present during maturation in the absence of serum and gonadotrophic hormones also did not alter the proportion of blastocysts or hatched blastocysts. In vitro culture of embryos on BRL cells or in BRL-conditioned medium in the presence of activin had no effect on embryonic development. It is concluded that IVM in the presence of bovine activin A has no effect on subsequent embryonic development.

Early Development of IVM/IVF Bovine Embryos Cultured with or without Somatic Cells in a Simple Serum-free Medium with Different Concentrations of CO2 and O2.

The effect of various O2 and CO2 mixtures on development of bovine embryos produced by in vitro maturation and in vitro fertilization (IVM/IVF) was examined in a newly formulated simple KSOM medium versus a complex Menezo B2 serum-free medium and in co-culture. Numbers of cells comprising blastocysts were also counted using Hoechst 33342 stain. Following routine IVM/IVF procedures oocytes and zygotes were cultured for 40 to 44 h in various media. Then 2-to 8-cell embryos had cumulus cells removed and were alotted randomly for continued culture to the same experimental culture media, but with various gas conditions. In Experiment 1, the percentage of embryos developing to and beyond morula stages in 5, 10 and 20% O2 with 5% CO2 in humidified air was 22, 15 and 12%, respectively, with the 22 versus 12% being different (P 0.05). In Experiment 2, the proportions of embryos developing into blastocysts in 5 and 10% CO2 were 15 and 6% (P<0.05), respectively, and in 5 and 10% O2 were 15 and 7%, respectively (P<0.05). Mean blastocyst cell number (82) was highest with 5% CO2 and 10% O2 even though more blastocysts were obtained with 5% CO2 and 5% O2 (P<0.05). In Experiment 3, embryos were co-cultured on monolayers of bovine oviduct epithelial cells (BOEC) or buffalo rat liver cells with KSOM medium in 5 and 10% O2 and 5% CO2. Treatment effects did not differ except 10% O2 was superior to 5% O2 when considering both morulae and blastocysts (41 vs 33%; P<0.05). These experiments indicate that the O2-CO2 concentrations can affect the development of bovine embryos produced by IVM/IVF when cultured in a simple serum-free medium with and without co-culture and that the simple KSOM medium can serve as a defined medium to study required supplementation for culturing IVM/IVF bovine oocytes.

Effect of gas atmosphere on the development of one-cell bovine embryos in two culture systems

The effect of two concentrations of oxygen on the development of bovine embryos was compared using two separate co-culture systems. In Experiment I, bovine oocytes were matured and fertilized in vitro and were then co-cultured for 7 days in 20 μl drops of M199 with 10% fetal calf serum containing oviduct cells. When cultures were performed in an atmosphere of 5% CO 2 in air (20% O 2) or in a mixture of 5% CO 2, 5% O 2 and 90% N 2 (5% O 2), 22 of 179 (12%) and 56 of 179 (31%) zygotes developed to or beyond the late morula stage (P<0.0001), respectively. After freezing, thawing and 48 hours of additional culture, 2 of 21 (10%) and 18 of 53 (34%) embryos were judged viable (P<0.001) within the respective treatment groups. In Experiment II, zygotes produced by the same means were co-cultured in 0.5 ml of M199 containing 10% fetal calf serum with monolayers of buffalo rat liver (BRL) cells. In 20% O 2, 51 of 177 (29%) zygotes developed into viable embryos, while in 5% O 2 only 9 of 177 (5%) were judged viable after 7 days of culture (P<0.0001). Post-freezing survival rates were 53% and 67% for embryos from the two respective oxygen concentration treatment groups. The transfer of 20 Grade 1 frozen/thawed embryos produced by co-culture with BRL cells produced six pregnancies (30%). These experiments show that the critical effect of oxygen concentration on embryo development in vitro and the ability of embryos produced by in vitro procedures to survive freezing can be influenced by the type of culture system employed.

Co-culture of in vitro fertilized bovine embryos with different cell monolayers.

The effects of different cell monolayers on in vitro development of early bovine embryos derived from in vitro maturation and fertilization were examined in this study. Early embryos (four to eight cells) were randomly allocated to bovine granulosa cell (GC), oviductal cell (OC), or uterine cell (UC) monolayers in Exp. 1 and to GC, skin cell (SC; from 10-d-old chicken embryos), testicular cell (TC; from 10-d-old mouse), and liver cell (LC; from 10-d-old chicken embryos) monolayers in Exp. 2, and cultured for 6 d at 38.6 degrees C in a humidified atmosphere of 5% CO2 in air. The culture medium was 12.5 mM HEPES TCM 199 supplemented with 1% calf serum and 1 mM sodium pyruvate. In Exp. 1, the percentage of four- to eight-cell embryos that developed to blastocysts on GC, OC, and UC monolayers was 26.9 (28/104), 37.5 (39/104), and 39.2 (40/102), respectively. In Exp. 2, the percentage of four- to eight-cell embryos that developed to blastocysts on GC, SC, TC, and LC monolayers was 53.3 (40/75), 42.9 (33/77), 49.3 (37/75), and 44.3 (35/79), respectively. There were no significant differences in development among groups in either experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of in vitro matured and fertilized bovine oocytes in conditioned media obtained from a transformed bovine fetal oviductal epithelial cell line (TOE136)

Objective: To characterize growth factor gene expression by passaged coculture cell lines demonstrated to enhance in vitro pre-embryo growth.Design: Ribonucleic acids isolated from the isthmus, ampullary, and fimbriae portions of the human oviduct, and from buffalo rat liver cell monolayers were subjected to Northern analysis using probes for growth factors.Setting: Academic tertiary care hospital.Patient(s): Two reproductive age women undergoing a hysterectomy and bilateral salpingectomy for benign gynecologic conditions consented to experimental use of their oviducts.Intervention(s): Cell cultures were established from fresh human oviduct segments and commercially purchased buffalo rat liver cells. After two passages, total RNA was isolated from these confluent monolayers, fractionated on denaturing agarose gels, transferred to nylon membranes, and analyzed by Northern hybridization using complementary DNAs from epidermal growth factor (EGF), stem cell factor, also known as Kit-ligand, colony-stimulating factor-1 (CSF), leukemia inhibitory factor, and interleukin-6 (IL-6). Radioactively labeled probes were prepared by in vitro transcription or by 5' end labeling. After hybridization, blots were washed at increasing strigencies to remove nonspecifically bound radioactivity and subjected to autoradiography.Result(s): Human oviduct coculture cells express EGF (kit-ligand), CSF, leukemia inhibitory factor, and IL-6. Buffalo rat liver cells contain the messenger RNA transcripts for kit-ligand and CSF.Conclusion(s): Human oviduct and buffalo rat liver coculture cells express specific growth factors. These results support the theory that coculture systems may enhance pre-embryo growth via the production of embryotrophic factors. The identification of these ligands may provide the rationale for selecting specific growth factors for media supplementation as well as contribute to our understanding of the general mechanisms involved in regulating early embryonic growth and development.

A trypsin‐sensitive avian reovirus: Isolation and experimental infection of poults and chicks

A reovirus (strain TR1) was isolated from the hock joint of a turkey with arthritis. It was rarely excreted in the faeces of poults after oral or intranasal inoculation and only occasionally following footpad inoculation. The virus did not localise in the hock joints or cause lesions. Cyclophosphamide-induced immunosuppression of poults did not modify the infection. Oral inoculation of chicks with a high dose of strain TR1 resulted in joint localisation, of virus and erosive arthritis. Footpad inoculation caused deaths in 13 of 15 chicks by 8 days pi due to hepatic necrosis. Intranasal inoculation of chicks resulted in a delayed and limited faecal excretion. However, joint lesions were most marked in chicks infected via this route. The infectivity of strain TR1 in chick embryo liver cell monolayers was reduced by almost 3.0 log(10) after treatment with 0.01% trypsin for 30 min, but the infectivity of strain R2 was unaffected. Bile salts had no effect on either virus. This trypsin-sensitive reovirus was much more pathogenic for chicks than for poults. The respiratory route may be important in the dissemination of trypsin-sensitive avian reoviruses such as this.

Co-culture systems for assessing the stability and genotoxicity of reactive 1, 2-dibromo-3-chloropropane (DBCP) metabolites

1,2-Dibromo-3-chloropropane (DBCP) induced DNA damage, measured by alkaline elution, at low concentrations (5-10 microM) in suspensions of hepatocytes and testicular cells isolated from rats. At higher concentrations (greater than or equal to 100 microM) DBCP caused DNA damage and increased the frequency of sister chromatid exchanges in Chinese hamster V79 cells. When DBCP (2.5-10 microM) was tested for its ability to cause unscheduled DNA synthesis (UDS) in monolayers of liver cells isolated from untreated rats, a clear positive response was obtained. No increase in UDS was detectable when liver cells isolated from PCB-pretreated rats were used. In contrast, DBCP (greater than or equal to 50 microM) was metabolized in hepatocytes from PCB-pretreated rats to products mutagenic to Salmonella typhimurium TA100 in co-culture with the hepatocytes, whereas control hepatocytes were substantially (40-fold) less active. No bacterial mutagenicity could be detected when the TA100 strain was co-incubated with isolated rat testicular cells and DBCP. In co-cultures of hepatocytes and V79 cells DBCP-induced DNA damage in V79 cells occurred at low concentrations (10 microM DBCP) compared to the concentration (100 microM) needed to induce DNA damage in the V79 cells incubated without hepatocytes. Testicular cells were not able to enhance DBCP-induced DNA damage in the V79 cells. The data indicate that the putative reactive DBCP episulphonium ion metabolite formed in the testicular cells presumably responsible for testicular cell DNA damage, is not capable of escaping the cell where it is formed. Other reactive DBCP metabolites generated in the liver cells seem to be able to interact with the DNA of neighbouring cells.