Influence of epidermal growth factor supplementation during in vitro maturation on nuclear status and gene expression of canine oocytes.

This study evaluated the effect of epidermal growth factor (EGF) supplementation during in vitro maturation on the meiotic status and the expression of EGF receptor (EGFr), luteinizing hormone receptor (LHr) and gap junction protein α 5 (GJA5) in canine cumulus-oocyte-complexes (COCs). COCs of ≥110 µm diameter, exhibiting dark pigmentation and completely surrounded by three or more layers of cumulus cells collected from anestrus stage ovaries in natural cycle were matured in TCM-199 supplemented with 10% fetal bovine serum, 0.57 mM cysteine, 10 µg/ml LH and FSH, and different concentrations of EGF (0, 10 and 30 ng/ml). Oocytes cultured for 72 h were fixed to assess the nuclear maturation. Expression of EGFr, LHr and GAJ5 was assessed by immunocytochemistry and real-time PCR. Proportion of metaphase II status of oocytes cultured in in vitro maturation (IVM) medium supplemented with 10 ng/ml EGF for 72 h was significantly (P<0.05) higher than 0 and 30 ng/ml EGF supplemented IVM medium (9.8% vs. 6.5% and 5.2%). In both cumulus cells and oocytes, EGFr protein was undetectable, LHr protein level of expression was low and a strong expression of GJA5 protein was observed. The relative abundance (RA) of EGFr transcript revealed low levels and the LHr expression decreased steadily with addition of EGF. However it did not vary among different concentrations of EGF supplementation. The RA of GJA5 transcript exhibited lower level at 10 ng/ml EGF supplementation. In conclusion, the supplementation of 10 ng/ml EGF in IVM media exerted a positive influence on the progression of maturation to MII phase and the expression level of GJA5 at 72 h, but did not demonstrate any stimulatory role on the expression of EGFr and LHr during the maturation of the canine IVM oocytes.

Expression pattern of oxygen and stress-responsive gene transcripts at various developmental stages of in vitro and in vivo preimplantation bovine embryos.

The present study examined the expression pattern of oxygen (O(2)) and stress-responsive gene transcripts at various preimplantation developmental stages of in vitro produced (IVP) and in vivo derived (IVD) bovine embryos. Embryos were produced in vitro from oocytes matured, fertilized and cultured in synthetic oviductal fluid (SOF) medium under low (5%) and high (20%) O(2) concentrations. In vivo embryos were derived from 18 superovulated and artificially inseminated cows. In IVP and IVD groups, embryos were collected at 2-, 4-, 8-, 16-cell morula and blastocyst stages at specific time points for gene expression analysis. The cleavage rates (69.8+/-4.8%) did not differ significantly, but blastocyst rates were significantly higher (28.5+/-3.7%) in low O(2) than those in high O(2) group (18.7+/-3.9%). Mean cell number in low O(2) (145+/-12) and high O(2) (121+/-73) IVP blastocyst were lower (P<0.05) than those of IVD blastocyst (223+/-25). The ICM ratio of IVD blastocyst (26+/-4) was lower (P<0.05) than that of IVP embryos under 5% O(2) (33+/-5) and 20% O(2) (34+/-4) concentrations, respectively. Using real time PCR, for the set of target transcripts (Glut1, Glut5, Sox, G6PD, MnSOD, PRDX5, NADH and Hsp 70.1) analyzed, there were differences in the mRNA expression pattern at 2-, 4-, 8-, 16-cell morula and Day 7 blastocyst stages between the two embryo sources. It can be concluded that, although in vitro bovine embryo culture in SOF medium under low (5%) O(2) concentration provided a more conducive environment in terms of blastocyst formation; differences in the total cell number and gene expression pattern between the IVP and IVD embryos reflected the effect of O(2) concentration.

Effects of confluent, roscovitine treatment and serum starvation on the cell-cycle synchronization of bovine foetal fibroblasts.

The present study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent, roscovitine treatment and serum starvation, in bovine foetal fibroblasts on synchronization accuracy at G0/G1, viability, apoptosis, necrosis and ploidy for use as a nuclei donor. The cells in 5-10 passages were randomly allocated into three treated groups. Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) until 90% confluent (group 1, confluent), in DMEM + 10% FBS + 30 microM roscovitine for 12 h (group 2, roscovitine), or in DMEM + 0.5% FBS for 5 days (group 3, serum starvation). Most of the cells (>80%) in all groups were arrested at the G0/G1 stage. Although the rates did not differ, cells in group 1 showed an increased cell population arrested at the G0/G1 phase. Significantly (p < 0.05) higher rates of apoptosis occurred in group 3 than in group 1 and 2 (10% vs 6% and 6%, respectively). No differences in chromosomal abnormality were observed among groups. However, by increasing the number of cell culture passages up to 15, significantly (p < 0.05) higher chromosomal abnormality was observed than in 5 and 10 passages (39% vs 28% and 23%, respectively) in group 1. The results clearly indicated that bovine foetal fibroblasts could be effectively synchronized at G0/G1 stages by all the three different treatments, confluent, roscovitine and serum starvation. However, cells in confluent showed reduced apoptosis and necrosis when they underwent 5-10 passages, exhibiting increased percentage of cells with stable chromosome diversity. Hence, cells in confluent merit further studies before they could be used as nuclear donors.

Efficient production of cloned bovine embryos using cdc2 kinase inhibitor.

This study was carried out to compare the effects of the combination of ionomycin with a H1-histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on the development of reconstituted bovine eggs. For this study, the enucleated bovine oocytes were injected with a presumptive primordial germ cell pre-treated with 1% sodium citrate, and randomly allocated into three activation groups: Group 1 (ionomycin 5 microm, 5 min), Group 2 (ionomycin + DMAP 1.9 mm, 3 h), and Group 3 (ionomycin + SPP 2 mm, 3 h). The reconstituted eggs were compared on the rates of cleavage and development with the blastocyst stage and the ploidy of embryos at 96 h post-activation. Cleavage rates and blastocyst development in Groups 1, 2 and 3 were 7 and 0%, 63 and 17%, and 53 and 14%, respectively. The chromosomal composition differed significantly (p < 0.05) among treatments. Although the embryos in Group 1 had significantly lower developments, 60% of embryos evaluated had diploid chromosomal sets. In contrast, approximately 60% of embryos in Group 2 had abnormal ploidy (21% polyploid and 38% mixoploid). In Group 3, the appearance of abnormal chromosome sets was reduced with the proportion of diploid embryos being increased to 86% (19 of 22), significantly higher (p < 0.05) than in Group 2. It can be concluded that the use of SPP with ionomycin reduces greatly the incidence of chromosomal abnormalities, and may be applicable for the activation of nuclear transplant bovine embryos.

Analysis of the cbbF genes from Alcaligenes eutrophus that encode fructose-1,6-/sedoheptulose-1,7-bisphosphatase.

The cbbF genes of the facultative chemoautotroph Alcaligenes eutrophus H16 are part of two highly homologous cbb operons. Both the chromosomal and the megaplasmid pHG1-borne copy of cbbF were cloned and sequenced. Subsequent analyses including comparison with known sequences from other organisms and heterologous expression in Escherichia coli revealed that each of the genes encodes fructose-1,6-bisphosphatase (FBPase). A closely related activity likewise operating in the Calvin carbon reduction cycle, sedoheptulose-1,7-bisphosphatase, was also catalyzed by the two isoenzymes which were purified from autotrophically grown cells of A. eutrophus. Two-dimensional gel electrophoresis allowed the separation of the cbbF gene products. Preliminary physical evidence by Southern hybridization with a heterologous gene probe was obtained for the existence of a third FBPase gene, fbp, on the chromosome of the organism. Its product is probably involved in the heterotrophic carbon metabolism.

Analysis of the genes forming the distal parts of the two cbb CO2 fixation operons from Alcaligenes eutrophus.

In the facultative chemoautotroph Alcaligenes eutrophus H16, most of the genes (cbb genes) encoding enzymes of the Calvin carbon reduction cycle are organized within two highly homologous cbb operons, one located on the chromosome and the other on the megaplasmid pHG1. Nucleotide sequencing of the promoter-distal part of the operons revealed three open reading frames, designated cbbG, cbbK, and cbbA. Similarity searches in databases and heterologous expressions of the subcloned genes in Escherichia coli identified them as genes encoding the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and a class II fructose-1,6-bisphosphate aldolase, respectively. The aldolase could be grouped together with the enzymes from Rhodobacter sphaeroides and Bacillus subtilis as a new subtype of class II aldolases. A phenotypic complementation analysis with a cbb operon mutant of A. eutrophus showed that the cbbG product is essential for autotrophic growth of the organism, whereas the products of cbbK and cbbA can apparently be substituted by isoenzymes encoded elsewhere on the chromosome. No or only low constitutive promoter activity was associated with cbbK and cbbA, respectively, confirming the two genes as parts of the cbb operon. Downstream of cbbA, the very high overall nucleotide sequence identity (about 94%) prevailing throughout the two cbb operons discontinues, suggesting that cbbA is the most promoter-distal gene of the operon.

The cbb operons of the facultative chemoautotroph Alcaligenes eutrophus encode phosphoglycolate phosphatase.

The two highly homologous cbb operons of Alcaligenes eutrophus H16 that are located on the chromosome and on megaplasmid pHG1 contain genes encoding several enzymes of the Calvin carbon reduction cycle. Sequence analysis of a region from the promoter-distal part revealed two open reading frames, designated cbbT and cbbZ, at equivalent positions within the operons. Comparisons with known sequences suggested cbbT to encode transketolase (TK; EC 2.2.1.1) as an additional enzyme of the cycle. No significant overall sequence similarities were observed for cbbZ. Although both regions exhibited very high nucleotide identities, 93% (cbbZ) and 96% (cbbT), only the chromosomally encoded genes were heterologously expressed to high levels in Escherichia coli. The molecular masses of the observed gene products, CbbT (74 kDa) and CbbZ (24 kDa), correlated well with the values calculated on the basis of the sequence information. TK activities were strongly elevated in E. coli clones expressing cbbT, confirming the identity of the gene. Strains of E. coli harboring the chromosomal cbbZ gene showed high levels of activity of 2-phosphoglycolate phosphatase (PGP; EC 3.1.3.18), a key enzyme of glycolate metabolism in autotrophic organisms that is not present in wild-type E. coli. Derepression of the cbb operons during autotrophic growth resulted in considerably increased levels of TK activity and the appearance of PGP activity in A. eutrophus, although the pHG1-encoded cbbZ gene was apparently not expressed. To our knowledge, this study represents the first cloning and sequencing of a PGP gene from any organism.

The Calvin cycle enzyme pentose-5-phosphate 3-epimerase is encoded within the cfx operons of the chemoautotroph Alcaligenes eutrophus.

Several genes (cfx genes) encoding Calvin cycle enzymes in Alcaligenes eutrophus are organized in two highly homologous operons comprising at least 11 kb. One cfx operon is located on the chromosome; the other is located on megaplasmid pHG1 of the organism (B. Bowien, U. Windhövel, J.-G. Yoo, R. Bednarski, and B. Kusian, FEMS Microbiol. Rev. 87:445-450, 1990). Corresponding regions of about 2.7 kb from within the operons were sequenced. Three open reading frames, designated cfxX (954 bp), cfxY (765 bp), and cfxE (726 bp), were detected at equivalent positions in the two sequences. The nucleotide identity of the sequences amounted to 94%. Heterologous expression of the subcloned pHG1-encoded open reading frames in Escherichia coli suggested that they were functional genes. The observed sizes of the gene products CfxX (35 kDa), CfxY (27 kDa), and CfxE (25.5 kDa) closely corresponded to the values calculated on the basis of the sequence information. E. coli clones harboring the cfxE gene showed up to about 19-fold-higher activities of pentose-5-phosphate 3-epimerase (PPE; EC 5.1.3.1) than did reference clones, suggesting that cfxE encodes PPE, another Calvin cycle enzyme. These data agree with the finding that in A. eutrophus, PPE activity is significantly enhanced under autotrophic growth conditions which lead to a derepression of the cfx operons. No functions could be assigned to CfxX and CfxY.