Abstract
Okadaic acid (OA) causes meiotic progression and chromosome condensation in cultured pachytene spermatocytes and an increase in maturation promoting factor (cyclin B1/cdc2 kinase) activity, as evaluated by H1 phosphorylative activity in anti-cyclin B1 immunoprecipitates. OA also induces a strong increase of phosphorylative activity toward the mitogen-activated protein kinase substrate myelin basic protein (MBP). Immunoprecipitation experiments with anti-extracellular signal-regulated kinase 1 (ERK1) or anti-ERK2 antibodies followed by MBP kinase assays, and direct in-gel kinase assays for MBP, show that p44/ERK1 but not p42/ERK2 is stimulated in OA-treated spermatocytes. OA treatment stimulates phosphorylation of ERK1, but not of ERK2, on a tyrosine residue involved in activation of the enzyme. ERK1 immunoprecipitated from extracts of OA-stimulated spermatocytes induces a stimulation of H1 kinase activity in extracts from control pachytene spermatocytes, whereas immunoprecipitated ERK2 is uneffective. We also show that natural G(2)/M transition in spermatocytes is associated to intracellular redistribution of ERKs, and their association with microtubules of the metaphase spindle. Preincubation of cultured pachytene spermatocytes with PD98059 (a selective inhibitor of ERK-activating kinases MEK1/2) completely blocks the ability of OA to induce chromosome condensation and progression to meiotic metaphases. These results suggest that ERK1 is specifically activated during G(2)/M transition in mouse spermatocytes, that it contributes to the mechanisms of maturation promoting factor activation, and that it is essential for chromosome condensation associated with progression to meiotic metaphases.
Highlights
Male meiosis is a process in which a dyploid spermatocyte gives rise to four haploid round spermatids after a single round of DNA replication followed by two subsequent cell divisions
Treatment with 5 M okadaic acid (OA) in the absence of fetal calf serum induced a dramatic chromatin condensation which was observed in about 90% of the cells (Fig. 1A, right side), suggesting that serum is not required for G2/M transition induced by OA treatment
Densitometric analysis indicates that 4-h treatment of spermatocytes with OA induced a 4-fold increase in cyclin B1/cdc2 kinase activity (Fig. 1D), demonstrating that the increase in H1 kinase activity observed in the cytosolic extracts (Fig. 1B) is due to activation of MPF
Summary
Male meiosis is a process in which a dyploid spermatocyte gives rise to four haploid round spermatids after a single round of DNA replication followed by two subsequent cell divisions. Induction of G2/M progression by progesterone requires the expression of the proto-oncogene mos [2,3,4], activation of the mitogen-activated protein kinase (MAPK) cascade, and activation of the cyclin B1⁄7cdc complex, known as mitosis-promoting factor, or maturation-promoting factor (MPF, for review, see Ref. 5). The oocyte proceeds to the second meiotic division and arrests at metaphase II with the sister chromatids aligned on the spindle, due to the concerted action of Mos and MAPKs, which, at this stage, prevent cyclin destruction and consequent MPF inactivation [5]. OA overcomes the checkpoints that normally delay the progression of the meiotic cycle of mid- and late pachytene spermatocytes and induces nuclear envelope breakdown and chromosome condensation resembling that observed during G2/M transition after prophase I. The increase in H1 kinase activity during OA-induced G2/M transition in mouse spermatocytes is due to a concurrent activation of a cyclin/cdk activity detectable in precipitates using p13suc1-conjugated agarose [23], suggesting that activation of MPF occurs under these experimental conditions
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