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Induced premature G2/M-phase transition in pachytene spermatocytes includes events unique to meiosis
T Wiltshire1, C Park, K A Caldwell
1Department of Zoology, University of Tennessee, Knoxville 37996, USA.
Abstract:
Little is known about the control of events ending the lengthy prophase of meiosis I and leading to the G2/M-phase transition in mammalian spermatocytes, primarily because the relevant late pachytene, diplotene, and MI cells are present in low numbers in the testis and it is not possible to isolate them in significant numbers. We have utilized short-term cultures of pachytene spermatocytes from the mouse to study events of the G2/M cell-cycle transition induced by the protein phosphatase inhibitor okadaic acid (OA). Treatment of cultured pachytene spermatocytes with OA induced a rapid and premature onset of events leading to the M phase, visualized cytologically by nuclear envelope breakdown and chromosome condensation. After OA treatment, condensed chromosomes were seen as bivalents, not as univalents. Treatment with OA induced disassembly of synaptonemal complexes and resolution of crossovers as cytologically visible chiasmata. Chiasmata counts were similar in treated cells and control cells. Thus, surprisingly, even though the treated cells were in the pachytene substage of meiotic prophase, events of recombination were apparently completed to the point of chiasma formation in the majority of these cells. The sex chromosomes, forming the sex body of the pachytene spermatocyte, lagged behind the autosomal chromosomes in their condensation and progression toward the M phase. Treatment with OA induced an increase in histone H1 kinase activity, generally used as an indicator of metaphase-promoting factor (MPF) activity; furthermore, the OA-induced cell-cycle transition does not require new protein synthesis. These results suggest that OA treatment overrides a cell-cycle checkpoint control that normally keeps pachytene spermatocytes in a lengthy prophase and that this control may be exerted by regulation of protein phosphorylation status.
Insights
Protein phosphatase inhibitor okadaic acid (OA) prematurely triggers meiosis I progression in mouse spermatocytes. This suggests OA overrides a cell-cycle checkpoint controlling the G2/M transition by regulating protein phosphorylation.
Area of Science:
- Cell Biology
- Reproductive Biology
- Molecular Biology
Background:
- Control of the G2/M transition in meiosis I of mammalian spermatocytes is poorly understood.
- Late prophase I cells (pachytene, diplotene, MI) are scarce and difficult to isolate for study.
Purpose of the Study:
- To investigate the G2/M cell-cycle transition in mouse pachytene spermatocytes.
- To identify regulatory mechanisms controlling the end of meiotic prophase I.
Main Methods:
- Short-term culture of isolated mouse pachytene spermatocytes.
- Treatment with okadaic acid (OA), a protein phosphatase inhibitor.
- Cytological analysis of nuclear envelope breakdown, chromosome condensation, synaptonemal complex disassembly, and chiasma formation.
- Assay of histone H1 kinase activity as a marker for metaphase-promoting factor (MPF).
Main Results:
- OA treatment induced premature nuclear envelope breakdown and chromosome condensation.
- Synaptonemal complexes disassembled, and crossovers resolved into chiasmata, with counts similar to controls.
- Sex chromosomes lagged in condensation and progression compared to autosomes.
- OA increased histone H1 kinase activity, indicating elevated MPF activity.
- The OA-induced transition did not require new protein synthesis.
Conclusions:
- Okadaic acid overrides a cell-cycle checkpoint that normally maintains meiotic prophase I arrest in pachytene spermatocytes.
- This checkpoint control likely involves the regulation of protein phosphorylation status.
- Recombination appears to be completed to the chiasma formation stage before or during the OA-induced transition.