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Regulation of M-phase promoting factor activity during development of mouse male germ cells
1Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Abstract:
While the role of M-phase promoting factor (MPF) in controlling meiosis in oocytes has been well documented, very little is known about its function in male germ cells. Previous studies have localized transcripts for cyclin B1, the regulatory subunit of MPF, in male germ cells, with highest levels in postmeiotic, early round spermatids and much reduced levels in the meiotically dividing pachytene spermatocytes. The present study describes the localization of the regulatory and the catalytic subunits of MPF, CycB1 and Cdc2, respectively, to specific cell types within the testis. Immunoblotting revealed that both CycB1 and Cdc2 were present at highest levels in pachytene spermatocytes, with lower levels observed in the postmeiotic compartment. To assay for MPF activity, kinase complexes were isolated from lysates of testicular cells using p13suc1 agarose and antibodies directed against Cdc2 and CycB1. Activity of these kinase complexes was analyzed using histone H1 as an exogenous substrate. Cdc2 and CycB1-associated kinase activities were localized to the meiotically dividing pachytene spermatocytes, but not to postmeiotic spermatids.
Insights
M-phase promoting factor (MPF) activity, crucial for oocyte meiosis, is now found in male germ cells. MPF
Area of Science:
- Reproductive Biology
- Cell Cycle Regulation
- Molecular Endocrinology
Background:
- M-phase promoting factor (MPF) is essential for oocyte meiosis, but its role in male germ cells is largely unknown.
- Previous studies indicated cyclin B1 (a regulatory subunit of MPF) transcripts are highest in postmeiotic spermatids, not meiotic cells.
Purpose of the Study:
- To investigate the cellular localization and activity of MPF subunits, cyclin B1 (CycB1) and Cdc2, in male germ cells.
- To determine if MPF is active during male meiosis.
Main Methods:
- Immunoblotting to detect CycB1 and Cdc2 protein levels in testicular cell types.
- Isolation of kinase complexes using p13suc1 agarose and antibodies against Cdc2 and CycB1.
- Assay of isolated kinase complex activity using histone H1 as a substrate.
Main Results:
- Both CycB1 and Cdc2 proteins were found at highest levels in pachytene spermatocytes (meiotically dividing cells).
- Lower levels of CycB1 and Cdc2 were detected in postmeiotic spermatids.
- MPF kinase activity was localized specifically to pachytene spermatocytes, not postmeiotic spermatids.
Conclusions:
- Contrary to transcript localization, MPF protein subunits and activity are highest in meiotically active pachytene spermatocytes.
- This study demonstrates MPF plays a role in male meiosis, specifically during the pachytene stage.
- Findings highlight a discrepancy between transcript and protein/activity localization for MPF in male germ cells.