Activation of the Mitogen-activated Protein Kinase ERK1 during Meiotic Progression of Mouse Pachytene Spermatocytes

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 OAstimulated 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.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. The homologous chromosomes are segregated in the two daughter cells (secondary spermatocytes) during the first meiotic division, whereas the sister chromatids, through a process resembling mitosis, are segregated during the second division to generate haploid cells. The prophase of male first meiotic division is a lengthy process which ensures the correct pairing and the crossing over between homologous chromosomes. In the mouse, this process lasts approximately 10 days, during which the homologous chromosomes become partially condensed, anneal to each other, and are maintained in proximity by a structure called the synaptonemal complex. After genetic exchange (crossing over) has occurred, the synaptonemal complex disappears and chromosome condensation allows the sites of DNA exchange to become visible as chiasmata. As for mitotic division, the nuclear envelope breaks down and microtubules are assembled into a spindle. The chromosomes become aligned to the equator of the meiotic spindle and the two sets of homologous chromosomes are separated during the anaphase. While the meiotic prophase is considerably long, metaphase and anaphase transitions ...

Analysis of the gene expression profile of mouse male meiotic germ cells

“…Insights into the molecular mechanisms of the progression to the metaphase of the first meiotic division have been obtained by treatment of cultured spermatocytes with okadaic acid (OA), which overcomes normal checkpoints that ensure in vivo the slow progression of the meiotic prophase. OA triggers the sequential activation of ERK1, p90Rsk2 and Nek2, thus leading to chromosome condensation and progression to metaphase with the concurrent activation of the cyclin B/cdk1 complex (Sette et al, 1999;Di Agostino et al, 2002). The spermatids that result at the end of meiosis will then undergo spermiogenesis with the final production of mature spermatozoa.…”

“…Spermatocytes obtained after elutriation from 18-day-old mice are in the middle -late pachytene stage of the first meiotic prophase (85%) and in the leptotene -zygotene stage (10%). Contamination of spermatogonia and somatic cells in the meiotic cell population is less than 5%, whereas round spermatids, which are a common contaminant of pachytene fractions when using adult testis (Sette et al, 1999;Di Agostino et al, 2002), are absent, not being present in the immature testis used. The cRNAs prepared from the two different cell populations have been hybridized with commercially available MG-U74Av2 GeneChip probe arrays (Affymetrix Inc.), containing , 12,500 known mouse genes or EST sequences, and thus spanning approximately 1/3rd of the mouse genome.…”

“…Homogeneous populations (purity . 90%) of spermatocytes and round spermatids were obtained from testes of either 18-day-old or 36-day-old mice, respectively, by differential elutriation as previously described (Sette et al, 1999;Di Agostino et al, 2002). Spermatocyte populations from 18-day-old mice (10% at the leptotene -zygotene and 85% at the middle-late pachytene stage of the meiotic prophase) are devoid of round spermatids, which contaminate elutriation fractions from adult animals, and their purity was assessed through morphological criteria (namely, cell size and the characteristic aspect of partially condensed meiotic chromatin).…”

The Nuclear RNA-binding Protein Sam68 Translocates to the Cytoplasm and Associates with the Polysomes in Mouse Spermatocytes

“…Testes from 23-to 28-d-old CD1 mice (Charles River Italia, Calco, Italy) were used to obtain pachytene spermatocytes, secondary spermatocytes, and round spermatids by elutriation technique as described previously (Sette et al, 1999). Spermatogonia were obtained from mice 8-d-old as described previously (Rossi et al, 1993).…”

“…Secondary spermatocytes were fixed for 10 min in 4% paraformaldehyde and processed for immunofluorescence analysis using the rabbit anti-Sam68 antibody (1:500) as previously reported for other antigens (Sette et al, 1999;Di Agostino et al, 2002).…”