Mitogen-activated protein kinase (MAPK) blockade of bovine preimplantation embryogenesis requires inhibition of both p38 and extracellular signal-regulated kinase (ERK) pathways.
Blastocyst formation, as a critical period during development, is an effective indicator of embryonic health and reproductive efficiency. Out of a number of mechanisms underlying blastocyst formation, highly conserved mitogen-activated protein kinase (MAPK) signaling has emerged as a major mechanism involved in regulating murine preimplantation embryo development. The objective of our study was to ascertain the role of MAPK signaling in regulating bovine development to the blastocyst stage. Using reverse transcriptase PCR and immunohistochemical staining procedures we have demonstrated that mRNA transcripts and polypeptides encoding p38 MAPK pathway constituents are detectable in preimplantation bovine embryos from the one-cell to the blastocyst stage. Further, the effects on bovine embryo development following inhibition of p38 alpha/beta and extracellular signal-regulated kinase (ERK) signaling by treatment with SB220025 and U0126, respectively, were investigated. Eight-cell bovine embryos (50 per group; three replicates) were placed into treatments consisting of synthetic oviductal fluid (SOF) medium: SOF + SB202474 (inactive analogue), SOF + SB220025, SOF + U0124 (inactive analogue), SOF + U0126, and SOF + SB220025 + U0126. Inhibition of p38 MAPK or ERK signaling individually did not affect development to the blastocyst stage. However, when both pathways were blocked simultaneously there was a significant reduction (P < 0.05) in blastocyst formation, cell number and immunofluorescence of phosphorylated downstream pathway constituents. We have determined that, in variance to what was observed during murine preimplantation development, bovine early embryos progress at normal frequencies to the blastocyst stage in the presence of p38 MAPK inhibitors.
Department of Physiology and Pharmacology, University of Western Ontario, Child Health Research Institute, 5th Floor Victoria Research Laboratories, 800 Commissioners Road East, London, Ontario, Canada, N6A 4G5.
Pavneesh
P
Madan
Michele D
MD
Calder
Andrew J
AJ
Watson
D000199:Actins / Q000378:metabolism; D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D002070:Butadienes / Q000494:pharmacology; D002417:Cattle; D046149:Embryo Culture Techniques; D047108:Embryonic Development / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors*; D019084:Fluorescent Antibody Technique, Indirect; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D020935:MAP Kinase Signaling System / Q000502:physiology*; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011743:Pyrimidines / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors*; D000099190:MAP-Kinase-Activated Kinase 2
D000199:Actins; D002070:Butadienes; D004791:Enzyme Inhibitors; D048049:Extracellular Signal-Regulated MAP Kinases; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007093:Imidazoles; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D009570:Nitriles; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011743:Pyrimidines; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2; C057725:HSPB1 protein, human; C110631:SB 220025; C113580:U 0126