Treatment Of Spermatocytes Research Articles

Chromosome Condensation

During meiosis, the replicated chromatids remain condensed, which is important to prevent inappropriate DNA replication and to allow proper progression through the second meiotic cell cycle. Di Agostino et al. examined meiosis stimulated by the phosphatase inhibitor okadaic acid in mouse spermatocytes. Entry into meiosis as detected by chromatin condensation was stimulated by OA, and this could be inhibited by pretreatment of the spermatocytes with the mitogen-activated protein kinase (MAPK) inhibitor U0126. One of the downstream effectors of MAPK is the kinase p90Rsk. Spermatocytes, which are meiosis competent, expressed the p90Rsk2 isoform, whereas mitotic spermatids expressed p90Rsk3. Various assays demonstrated that p90Rsk2 was phosphorylated and activated by OA treatment of spermatocytes and that activation was blocked by exposure to the MAPK inhibitor. Furthermore, like the MAPKs, ERK1 and ERK2, p90Rsk2 was physically associated with the condensed chromatin in the meiotic cells. Histone 3 is phosphorylated in condensed chromatin, and although p90Rsk2 can phosphorylate histone 3 in vitro, the exposure of the spermatocytes to U0126 did not prevent OA-stimulated histone 3 phosphorylation. Thus, histone 3 phosphorylation correlates with chromatin condensation, but is not sufficient to cause condensation. Di Agonstino et al. found that p90Rsk2 activated the NIMA (never in mitosis in Aspergillus nidulan s)-like kinase Nek2 and the stimulation of Nek2 was inhibited in OA treated spermatocyes pretreated with U0126. If histone phosphorylation by p90Rsk2 is not the relevant target for triggering chromatin condensation, then possibly Nek2 represents the important downstream effector of p90Rsk2. The authors propose that a kinase complex forms on the chromosomes that include ERK1 and ERK2, p90Rsk2, and Nek2 and that this complex is responsible for triggering chromatin condensation in response to extracellular signals. S. Di Agostino, P. Rossi, R. Geremia, C. Sette, The MAPK pathway triggers activation of Nek2 during chromosome condensation in mouse spermatocytes. Development 129 , 1715-1727 (2002). [Online Journal]

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

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.

Regulation of the Ascaris major sperm protein (MSP) cytoskeleton by intracellular pH.

The development and locomotion of the amoeboid sperm of the nematode, Ascaris suum, depend on precise control of the assembly of their unique major sperm protein (MSP) filament system. We used fluorescence ratio imaging of cells loaded with BCECF to show that intracellular pH (pHi) is involved in controlling MSP polymerization in vivo. Spermatogenesis is marked by a cycle of MSP assembly-disassembly-reassembly that coincides with changes in pHi. In spermatocytes, which contain MSP in paracrystalline fibrous bodies, pHi was 6.8, 0.6 units higher than in spermatids, which disassemble the fibrous bodies and contain no assemblies of MSP filaments. Activation of spermatids to complete development resulted in rapid increase in pHi to 6.4 and reappearance of filaments. Treatment of spermatocytes with weak acids caused the fibrous bodies to disassemble whereas incubation of spermatids in weak bases induced MSP assembly. The MSP filaments in spermatozoa are organized into fiber complexes that flow continuously rearward from the leading edge of the pseudopod. These cells established a pseudopodial pH gradient with pHi 0.15 units higher at the leading edge, where fiber complexes assemble, than at the base of the pseudopod, where disassembly occurs. Acidification of these cells caused the MSP cytoskeleton to disassemble and abolished the pH gradient. Acid removal resulted in reassembly of the cytoskeleton, re-establishment of the pH gradient, and re-initiation of motility. MSP assembly in sperm undergoing normal development and motility and in cells responding to chemical manipulation of pHi occurs preferentially at membranes. Thus, we propose that filament assembly in sperm is controlled by pH-sensitive MSP-membrane interaction.

Detection of aneuploidy in male germ cells of mice by means of a meiotic micronucleus assay

The possibility of using a micronucleus (MN) assay in mouse germ cells for the identification of aneuploidogenic agents was evaluated by comparing the pattern of effects induced by 4 chemicals with different mechanisms of action (adriamycin, ADM; mitomycin C, MMC; chloral hydrate, CH; ethylenedinirrilotetraacetic acid, EDTA). The results obtained after treatment of spermatocytes at the premeiotic S-phase (preleptotene) indicated that only clastogenic agents (ADM and MMC) were able to induce MN at this cell stage. Previous data obtained with the saine compounds demonstrated by contrast that the micronucleus spermatid assay may detect both clastogenic and aneuploidogenic effects after treatment of diakinesis/MI/MII cells. Analysis of MN size distributions, in the present and previous spermatid samples, revealed that the clastogens ADM and MMC produced relatively more small MN than CH and EDTA. These data are in agreement with the proposed mechanism of action of the chemicals tested.

A marked ring-Y-chromosome in Drosophila hydei with a new type of white variegation

A ring-Y chromosome, R(Y)wm, of D. hydei is described which carries a complete set of fertility genes, a NOR region and a small X-chromosomal insertion (wm), which may be used as a marker. The ring has been characterized by various staining techniques. It was derived from a wmCo Y chromosome by X-ray treatment of spermatocytes. Its mode of origin allows to fix the gene order in the distal region of the long arm of the wmCoY chromosome. The white+ gene included in the ring shows a new type of position-effect variegation which is described and discussed in the context of an earlier hypothesis on a dual function of the white locus.