p38 MAPK signaling during murine preimplantation development

Abstract

Mitogen-activated protein kinase (MAPK) pathways mediate some important cellular processes and are likely to also regulate preimplantation development. The role of p38 MAP kinase signaling during murine preimplantation development was investigated in the present study. p38 MAPK, p38-regulated or -activated kinase (PRAK; MK5), map kinase-activated protein kinase 2 (MK2), and heat shock protein 25 (hsp25) mRNAs and proteins were detected throughout preimplantation development. Two-cell stage embryos cultured in the presence of SB220025 and SB203580 (specific inhibitors of p38 MAPK alpha/beta), progressed to the eight-cell stage with the same frequency as controls; however, treated embryos halted their development at the 8- to 16-cell stage. In addition, embryos treated with p38 MAPK inhibitors displayed a complete loss of MK2 and hsp25 phosphorylation and also a complete loss of filamentous actin as indicated by the absence of rhodamine-phalloidin staining. In these inhibitor-treated groups, the embryos were composed of a mixture of compacting and noncompacting cells, and the embryos were one to two cell divisions behind controls. Treated embryos remained viable as the developmental blockade was rescued by removing embryos from the drug treatment and placing them in drug-free medium until they progressed to the blastocyst stage. This study demonstrates that p38 MAPK activity is required to support development through the murine preimplantation interval.

Mitogen-activated protein kinase (MAPK) blockade of bovine preimplantation embryogenesis requires inhibition of both p38 and extracellular signal-regulated kinase (ERK) pathways

“…p38 MAPK further regulates actin filament formation through MAPKAPK 2/3 (MK2) or MAPKAPK 5 (PRAK) and subsequently through heat-shock protein (Hsp) 25/27 (Lavoie et al 1995, Guay et al 1997, Huot et al 1998, DalleDonne et al 2001, Khurana & Dey 2003. More importantly we have discovered that inhibition of p38 MAPK activity during murine preimplantation development results in a blockade of development at the eight-to-16-cell stage that is fully reversible for at least a 48-h treatment interval (Natale et al 2004, Paliga et al 2005. These results have implicated p38 MAPK as an important regulator of filamentous actin and of preimplantation development in mice (Natale et al 2004).…”

“…The total RNA extracts were digested with DNase 1 to eliminate possible contamination from genomic DNA. The reverse transcriptase (RT) reactions were conducted using oligo-dT primers (Gibco BRL) as described previously (Barcroft et al 1998, Offenberg et al 2000, Natale et al 2004. Briefly, samples were incubated for 90 min at 42 8C in a total volume of 20 ml consisting of 50 mM Tris/HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 10 mM dithiothreitol, 0.5 mM dNTPs, and 200 units of Superscript II (Gibco BRL) followed by heating the samples to 95 8C for 5 min to terminate the reaction.…”

“…The effects of p38 MAPK inhibition on filamentous actin can be observed within 3 h of treatment with p38 MAPK inhibitors applied to eight-cell-stage murine embryos and this is accompanied by a decline in phosphorylated MAPKAPK 2/3 and Hsp25/27 (Paliga et al 2005). All of these effects of p38 MAPK inhibition were reversible upon removal of the inhibitor and development resumed in a delayed but normal fashion to the blastocyst stage (Natale et al 2004, Paliga et al 2005.…”

“…We have demonstrated recently that all principal constituents of the p38 MAPK family are expressed throughout murine preimplantation development (Natale et al 2004). This subfamily of the MAPK includes four protein isoforms, p38 a, b, g, and d, that regulate cellular processes such as inflammation and cytoskeleton re-arrangements as well as cell proliferation and apoptosis (Enslen et al 2000, Ono & Han 2000, Kyriakis & Avruch 2001.…”

“…More importantly we have discovered that inhibition of p38 MAPK activity during murine preimplantation development results in a blockade of development at the eight-to-16-cell stage that is fully reversible for at least a 48-h treatment interval (Natale et al 2004, Paliga et al 2005. These results have implicated p38 MAPK as an important regulator of filamentous actin and of preimplantation development in mice (Natale et al 2004). The effects of p38 MAPK inhibition on filamentous actin can be observed within 3 h of treatment with p38 MAPK inhibitors applied to eight-cell-stage murine embryos and this is accompanied by a decline in phosphorylated MAPKAPK 2/3 and Hsp25/27 (Paliga et al 2005).…”