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D Rieger

Publications and source records attributed to D Rieger.

At least 19 recordsLinked to original sources
PubMed2002

Effect of 2,4-dinitrophenol on the energy metabolism of cattle embryos produced by in vitro fertilization and culture.

In cattle embryos, the proportion of ATP produced by glycolysis increases following the major activation of the embryonic genome, and development to the blastocyst stage is improved in the presence of 10 microM 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, from Day 5 to Day 7 of culture. In Experiment 1 of the present study, culture of cattle embryos in the presence of 10 microM DNP from Day 5 to Day 7 stimulated development to the blastocyst stage, but had no significant effects on oxygen, pyruvate or glucose uptake, or on lactate production. In Experiment 2, culture of cattle embryos in the presence of 10 microM DNP from Day 5 to Day 7, stimulated the metabolism of [2-14C]pyruvate (a measure of Krebs cycle activity) on all of Days 5, 6 and 7, and stimulated metabolism of [5-3H]glucose (a measure of glycolysis) on Day 7 only. The results show that 10 microM DNP stimulates oxidative and glycolytic metabolism in Day-5 to Day-7 cattle embryos, but this does not fully explain the observed increase in developmental competence. We propose that partial inhibition or uncoupling of oxidative phosphorylation may reduce the level of intracellular reactive oxygen species production, thereby facilitating development.

2,4-Dinitrophenol↗
PubMed2000

Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants.

Profilin is a ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cell physiology. By site-directed mutagenesis of profilin II from Dictyostelium discoideum the point mutations K114E and W3N were generated by PCR thus changing actin and poly-(L)-proline-binding activity respectively. W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin binding. The K114E profilin exhibited a profound decrease in its ability to interact with actin, whereas binding to poly-(L)-proline was essentially unchanged. Binding to phospholipids was indistinguishable from the wild-type profilin. The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., Cell 79, 303-314, 1994). Expression of K114E or W3N displayed a reduction in the F-actin content, normal cell morphology, and the transformants were capable of undergoing complete development. Interestingly, only cells that drastically overexpressed W3N could restore the aberrant phenotype, whereas the mutant protein K114E with its fully functional poly-(L)-proline binding and its strongly reduced actin-binding activities rescued the phenotype at low concentrations. Wild-type and both mutated profilins are enriched in phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline-binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities.

Animals↗
PubMed1999

Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization.

Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP. The intracellular levels of cAMP in cattle cumulus-oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis. High concentrations of iAC (1 or 5 microgram/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 microgram/ml) resulted in high rates of maturation to the MII stage (92.6 +/- 2.5 and 98.5 +/- 1.4% respectively). The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 +/- 4.2 and 45.1 +/- 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 +/- 2.9%). Finally, the addition of 0.1 microgram/ml iAC and 0.5 mM 3-isobutyl 1-methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 microgram/ml iAC (31.9 +/- 5.5 vs. 12.1 +/- 1.6 and 45.5 +/- 2.9 vs. 19.1 +/- 2.3% respectively). It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones. Mol. Reprod. Dev. 54:86-91,1999.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Effect of time interval from insemination to first cleavage on the developmental characteristics, sex ratio and pregnancy rate after transfer of bovine embryos.

In vitro produced bovine zygotes show substantial variation in the time required to complete the first cell cycle and in their in vitro development potential. A number of reports have highlighted the fact that the fastest developing embryos in vitro are most likely to be comparable with their in vivo counterparts. At 24 h after IVF, presumptive zygotes were cultured in droplets of synthetic oviduct fluid medium. Droplets were examined at regular intervals and all cleaved embryos at each time point were transferred into new droplets and cultured separately for the duration of the experiment. All uncleaved zygotes were returned to the incubator and re-examined at the successive time points until 48 h after insemination, at which time the remaining uncleaved oocytes were retained as a group. A representative number of day 7 blastocysts from zygotes that had cleaved by 30 or 36 h were transferred to synchronized recipients and pregnancy was diagnosed by ultrasonography at day 35. Glucose and glutamine metabolism was examined in zygotes and blastocysts and compared retrospectively with time of first cleavage. A representative number of blastocysts from each of the cleavage groups was sexed using PCR. Data were analysed by chi-squared and regression analysis. Development to the blastocyst stage decreased as the time from insemination to first cleavage increased (r = 0.97, P < 0.03). There was no difference in blastocyst hatching, number of blastocyst cells or pregnancy rate between the 30 and 36 h groups. The overall sex ratio was 62% males (n = 258, P < 0.0001) and was not different in the 30 and 36 h groups (61%, n = 155 versus 63%, n = 95, respectively). These results indicate that although time of first cleavage has a major influence on the probability of an embryo developing to the blastocyst stage, once that stage is attained, subsequent developmental characteristics are unrelated to the time of first cleavage.

Animals↗
PubMed1998

Effects of the in vitro chemical environment during early embryogenesis on subsequent development.

The development of the preimplantation embryo seems morphologically very simple, and embryologists previously assumed that an embryo that developed to the blastocyst stage was fully capable of normal development after transfer to the uterus of a recipient female. This complacency was disturbed by reports that exposure of early embryos to mutagens such as methylnitrosourea led to fetal abnormalities, decreased birth rates, and decreased life-span. Even more disturbing are recent reports that culture of early embryos in supposedly benign conditions can adversely affect their subsequent development. Techniques have been developed for the production of cattle and sheep embryos by in-vitro fertilization and by cloning. Such embryos must be cultured for several days before they can be transferred, and, in some cases, this has been related to abortion, very high birthweight, physical abnormalities and peri-natal mortality of the calves and lambs. This syndrome may result from an unbalanced development of the trophoblast relative to the inner-cell mass, possibly related to the presence of serum, glucose, or ammonium in the culture medium. An analogous phenomenon has been observed in human in-vitro fertilization where babies from single pregnancies have below-normal birth-weight. There is also evidence to suggest that the in-vitro environment of the gametes before fertilization can affect subsequent embryonal and fetal development. Exposure of mouse oocytes to vitrification solutions has been shown to lead to fetal malformations, and treatment of bull sperm with glutathione improves early embryo development. The common thread in these diverse observations is that development can be affected by events that occur long before any defect is apparent. Consequently, the production of a morphologically normal embryo is no guarantee that fetal development and post-natal life will be normal. This is of immediate concern in human reproductive medicine due to the increasing use of sperm injection for fertilization, and the emergence of in-vitro oocyte maturation. Further development and application of reproductive techniques would benefit from a toxicological evaluation of risk factors and exposure limits.

Animals↗
PubMed1998

The effects of epidermal growth factor and insulin-like growth factor I on the metabolic activity, nuclear maturation and subsequent development of cattle oocytes in vitro.

The effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the maturation and subsequent development of cattle oocytes in vitro were evaluated in three experiments. Cumulus-oocyte complexes (COC) were collected from cattle ovaries and matured for 20-24 in control medium or in medium containing 50 ng EGF ml-1, 100 ng IGF-I ml-1, EGF + IGF-I, or 10% (v/V) fetal calf serum plus 0.1 i.u. human menopausal gonadotrophin ml-1 (hMG). In Expt 1, treatment with EGF + IGF-I stimulated cumulus expansion, the metabolism of pyruvate and glutamine, and nuclear maturation. In Expt 2, only the metabolic measurements from oocytes that reached metaphase II were considered, and EGF + IGF-I stimulated pyruvate metabolism to the same extent as serum + hMG. In Expt 3, the oocytes were fertilized after maturation culture, and the resultant embryos cultured for up to 8 days. The cleavage was greater in the EGF and EGF + IGF-I groups than in the controls but less than in the serum + hMG group. Moreover, the number of blastocyst cells at 7 days after insemination and the proportion of cleaved embryos that developed to the blastocyst stage by day 8 was greater in the serum + hMG group than in the control group indicating that maturation treatment can affect early embryonic development. In conclusion, EGF + IGF-I can stimulate cumulus expansion, oxidative metabolism, nuclear maturation and cleavage after fertilization of bovine oocytes in vitro. The relative effects of the treatments on oocyte pyruvate metabolism in Expts 1 and 2 generally paralleled their effects on cleavage and subsequent development in Expt 3, suggesting that mitochondrial function is related to developmental potential. Further investigation is required to determine which component(s) of serum or gonadotrophin treatment is responsible for the effects on subsequent embryonic development.

Analysis of Variance↗
PubMed1997

Batch analysis of the ATP content of bovine sperm, oocytes, and early embryos using a scintillation counter to measure the chemiluminescence produced by the luciferin-luciferase reaction.

This report describes an approach to using a commercial luciferin-luciferase kit and a scintillation counter for batch analysis of the ATP content of bovine sperm, oocytes and embryos. After ATP extraction, the luciferin-luciferase assay mixture was added to as many as 48 samples at one time, and the tubes counted once, and then again, in reverse order. The square-root of the geometric mean of the two counts for a tube was directly proportional to the ATP content. The measured ATP content was directly proportional to the number of sperm, oocytes, or blastocysts in a sample. The assay is sensitive enough to measure the ATP content of a single oocyte or blastocyst, or approximately 1000 sperm. The assay does not require the complicated and expensive automated sampling and mixing apparatus that is required for batch analysis using luminometer counting. This approach may significantly simplify the determination of the ATP content of large numbers of samples.

Adenosine Triphosphate↗
PubMed1996

Structure/function studies on the pH-dependent actin-binding protein hisactophilin in Dictyostelium mutants.

Our previous studies have shown that the actin-binding protein hisactophilin from Dictyostelium discoideum is a candidate for organizing the actin cytoskeleton at the plasma membrane in a pH-dependent manner. To further characterize this interaction we isolated hisactophilin overexpression (hisII+) and hisactophilin minus (his-) mutants. D. discoideum contains two hisactophilin isoforms; both genes are independently transcribed and carry a short intron at the same position of the coding region. The deduced amino acid sequence of hisactophilin II showed a characteristic high content of 35 histidine residues out of a total 118 amino acids. After transformation of Dictyostelium AX2 wild-type cells with a genomic fragment designed to inactivate the hisactophilin I gene we obtained hisactophilin II overexpressing mutants (hisII+). Multiple integration of the vector led to strong overexpression of hisactophilin II which even outnumbered the actin concentration by a factor of two. Hisactophilin II protein showed the same biochemical properties as hisactophilin I during purification and in its pH-dependent binding to F-actin; as shown by mass spectrometry the hisactophilin II fraction was almost completely myristoylated despite of this high overexpression. The inactivation of both hisactophilin genes was achieved by gene replacement with a vector construct encompassing parts of gene I and gene II connected by a geneticin cassette. The properties of the hisII+ and his- cells with regard to growth in shaking culture and on Klebsiella plates, development, chemotaxis and morphology were not affected under normal conditions. However, the hisII+ transformants revealed a significant difference to wild-type cells and his- cells when the cytoplasmic pH was lowered by diethylstilbestrol (DES), a proton pump inhibitor. HisII+ cells were more resistant to the acidification; in contrast to AX2 wild-type cells and his- cells they did not form plasma membrane protrusions, showed an increase in F-actin content, and contained large clusters of F-actin. Lowering the internal pH caused an accumulation of hisactophilin below the plasma membrane. The fact that cells deficient in hisactophilin again lose resistance to acidification is in good agreement with the hypothesis that hisactophilin functions as a pH sensor at the plasma membrane by reversibly connecting the membrane with the actin cortical network upon local changes of the proton concentration.

Actins↗
PubMed1995

Comparison of the effects of oviductal cell co-culture and oviductal cell-conditioned medium on the development and metabolic activity of cattle embryos.

The objective of this study was to compare the development and metabolic activity of cattle embryos co-cultured with bovine oviductal cells or cultured in serum-free medium previously conditioned by bovine oviductal cells. Zygotes were produced by in vitro fertilization of oocytes from bovine ovaries obtained from an abattoir. Development to the four-cell stage occurred by 48 h after fertilization in both culture systems, but co-cultured embryos reached the 16-cell stage by 96 h, whereas those cultured in conditioned medium did not do so until 24 h later. Similarly, the morula and blastocyst stages were reached 24 h earlier in co-culture than in conditioned medium. There were significantly more cells in the blastocysts from co-culture (96.8 +/- 6.1 versus 56.7 +/- 3.3; P < or = 0.0001). The metabolism of glutamine did not differ between embryos cultured in the two systems, but the metabolism of glucose was significantly greater in embryos cultured in conditioned medium. The first significant increase in glucose metabolism occurred between the four-cell and the 16-cell stages in embryos cultured in conditioned medium, but occurred between the 16-cell and morula stages in the co-cultured embryos, such that the glucose metabolism was significantly greater at the 16-cell stage in embryos cultured in conditioned medium compared with co-cultured embryos (6.5 +/- 1.0 versus 1.5 +/- 0.4 pmol per embryo per 3 h, P < or = 0.0001). The concentration of glucose was significantly less, and that of lactate significantly greater, in co-culture medium than in conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Effects of an LHRH antagonist on the time of occurrence and amplitude of the preovulatory LH surge, progesterone and estradiol secretion, and ovulation in superovulated Holstein heifers.

Beginning on Day 10 or 11 of the estrous cycle, mature Holstein heifers were given a superovulatory regimen of twice-daily injections of porcine FSH, together with injections of PG with the fifth and sixth FSH injections. Every 12 h from 24 to 60 h after PG administration, the animals received im injections of different doses of the LH releasing hormone antagonist [N-Ac-D-Nal(2)(1), D-pCl-Phe(2), D-Trp(3), D-Arg(6), D-Ala(10)]-LHRH or vehicle. Follicular development was monitored by transrectal ultrasonography every 12 h from 24 to 120 h after PG administration. All animals were given hCG at 72 h after PG injection, and were artificially inseminated. At Day 7 of gestation, the corpora lutea were counted by ultrasonography, and embryos were collected by nonsurgical flushing of the uterus. Treatment with the antagonist resulted in a dose-dependent decrease in the amplitude of the LH surge and in delays in the time of occurrence of the LH surge, ovulation and the shift from estradiol to progesterone production. These results indicate that LHRH antagonists can be used to delay the LH surge and ovulation in superovulated heifers. This finding may be beneficial to studies in the superovulation of cattle.

Journal Article↗
PubMed1994

Changes in the metabolism of glucose, pyruvate, glutamine and glycine during maturation of cattle oocytes in vitro.

After maturation in vitro for 0, 6, 12, 18 or 24 h, the metabolism of radiolabelled glucose, glutamine, pyruvate and glycine by individual cattle oocytes was measured for 3 h. The metabolism of glucose through the Embden-Meyerhof (1.77-2.66 pmol per oocyte per 3 h) and pentose-phosphate (0.39-0.75 pmol per oocyte per 3 h) pathways was low and did not change over time. The oxidative metabolism of glucose carbon through the Krebs cycle was low throughout maturation, but increased significantly (P < or = 0.05) at 6 h (0.41 pmol per oocyte per 3 h) and 18 h (0.69 pmol per oocyte per 3 h). Pyruvate, glutamine and glycine metabolism in the Krebs cycle increased during culture. Pyruvate metabolism increased significantly from 0 h (17.3 pmol per oocyte per 3 h) to 6 h (23.3 pmol per oocyte per 3 h) and reached a maximum at 12 h (30.8 pmol per oocyte per 3 h). Glutamine metabolism was unchanged from 0 to 12 h (0.89 pmol per oocyte per 3 h), and then increased significantly at 18 h (2.25 pmol per oocyte per 3 h). Glycine metabolism increased significantly from 6 h (0.21 pmol per oocyte per 3 h) to 12 h (0.46 pmol per oocyte per 3 h) and reached a maximum at 18 h (0.68 pmol per oocyte per 3 h). The results suggest that oxidative metabolism increases, and is the major site of cellular energy production, during maturation of the cattle oocyte in vitro.

Amino Acids↗
PubMed1993

The effect of a depot injection of recombinant bovine somatotropin on follicular development and embryo yield in superovulated Holstein heifers.

Superovulated Holstein heifers (n = 32) were given a depot injection of 500 mg recombinant bovine somatotropin (rBST) or vehicle at Day 4 of the estrous cycle (7 days before the first FSH injection); at Day 11, coincidentally with the first FSH injection; or at Day 15, the time of artificial insemination. Embryos were collected nonsurgically, and the number of corpora lutea was counted by ultrasonography at Day 7 after insemination. Blood samples were taken every second day, from Day 2 of the superovulatory cycle until the day of embryo collection, and were analyzed for progesterone, somatotropin and insulin-like growth factor-1 (IGF-1). Somatotropin-treated heifers at Day 11 had a significantly higher mean number of corpora lutea than the controls (18.1 vs 13.4; P </= 0.05). Day 4 treatment tended to increase the mean number of corpora lutea (15.4; P <- 0.10), and significantly increased the overall percentage of transferable embryos (74.6 vs 58.6%; P </= 0.01). In the control animals, plasma IGF-1 was uncorrelated to somatotropin (P > 0.63), but it was negatively correlated with progesterone (P </= 0.01), suggesting that IGF-1 production in the superovulated heifer may be related to ovarian development.

Journal Article↗
PubMed1993

Embryo transfer and related techniques in domestic animals, and their implications for human medicine.

This report was commissioned by the Canadian government on the relevance of new techniques in animal embryology to the social, ethical and clinical applications of assisted human reproduction. It briefly describes the history of animal breeding, and the regulation of the female reproductive tract, ovulation and fertilization in laboratory and veterinary species. Embryo transfer is described in detail, including the synchronization of reproductive cycles, superovulation and embryo growth in vitro. Methods of experimental embryology, including bisection, sexing of spermatozoa and embryos, cloning and gene therapy are described. The relevance of these studies to human IVF are considered briefly.

Animals↗
PubMed1992

Cap100, a novel phosphatidylinositol 4,5-bisphosphate-regulated protein that caps actin filaments but does not nucleate actin assembly.

The fast and transient polymerization of actin in nonmuscle cells after stimulation with chemoattractants requires strong nucleation activities but also components that inhibit this process in resting cells. In this paper, we describe the purification and characterization of a new actin-binding protein from Dictyostelium discoideum that exhibited strong F-actin capping activity but did not nucleate actin assembly independently of the Ca2+ concentration. These properties led at physiological salt conditions to an inhibition of actin polymerization at a molar ratio of capping protein to actin below 1:1,000. The protein is a monomer, with a molecular mass of approximately 100 kDa, and is present in growing and in developing amoebae. Based on its F-actin capping function and its apparent molecular weight, we designated this monomeric protein cap100. As shown by dilution-induced depolymerization and by elongation assays, cap100 capped the barbed ends of actin filaments and did not sever F-actin. In agreement with its capping activity, cap100 increased the critical concentration for actin polymerization. In excitation or emission scans of pyrene-labeled G-actin, the fluorescence was increased in the presence of cap100. This suggests a G-actin binding activity for cap100. The capping activity could be completely inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and bound cap100 could be removed by PIP2. The inhibition by phosphatidylinositol and the Ca(2+)-independent down-regulation of spontaneous actin polymerization indicate that cap100 plays a role in balancing the G- and F-actin pools of a resting cell. In the cytoplasm, the equilibrium would be shifted towards G-actin, but, below the membrane where F-actin is required, this activity would be inhibited by PIP2.

Actin Cytoskeleton↗
PubMed1992

Developmentally related changes in the uptake and metabolism of glucose, glutamine and pyruvate by cattle embryos produced in vitro.

The metabolism of, and retention of radioactivity from, radiolabelled glucose, glutamine and pyruvate were measured in individual cattle embryos produced in vitro from the 2-cell to hatched blastocyst stage. Uptake was defined as the numeric sum of metabolism and retention of radiolabel. Glucose metabolism increased significantly between the 8- and 16-cell stages, but was accompanied by a much larger increase in glucose uptake. Consequently, the proportion of glucose uptake that was metabolized through the pentose-phosphate and Embden-Meyerhof pathways reached a minimum at those stages. From the compacted morula stage onward, the calculated uptake of [14C]glucose was only 25 to 33% of that calculated for [5-3H]glucose. This suggests that 66 to 75% of glucose carbon leaves the embryo, after metabolism to phosphoenolpyruvate, in some form other than CO2. Little or no glucose metabolism by the Krebs cycle could be detected at any stage. Both glutamine and pyruvate metabolism were relatively high at the 2- and 4-cell stages, declined to a minimum at the compacted morula stage and then increased with blastulation. Glutamine metabolism continued to increase with expansion and hatching of the blastocyst, but pyruvate metabolism did not. This suggest that, relative to the activity of the pathway from pyruvate to 2-oxoglutarate, the activity of the 2-oxoglutarate-to-oxaloacetate segment of the Krebs cycle is of increasing significance during expansion and hatching of the cattle blastocyst.

Analysis of Variance↗