Mitogen-Activated Protein Kinase Dynamics During the Meiotic G2/MI Transition of Mouse Spermatocytes

BIOLOGY OF REPRODUCTION 71, 570–578 (2004) Published online before print 14 April 2004. DOI 10.1095/biolreprod.104.027938 Mitogen-Activated Protein Kinase Dynamics During the Meiotic G2/MI Transition of Mouse Spermatocytes1 Amy Inselman3 and Mary Ann Handel2 Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840 ABSTRACT Cellular and genetic approaches were used to investigate the requirements for activation during spermatogenesis of the extracellular signal-regulated protein kinases (ERKs), more commonly known as the mitogen-activated protein kinases (MAPKs). The MAPKS and their activating kinases, the MEKs, are expressed in specific developmental patterns. The MAPKs and MEK2 are expressed in all premeiotic germ cells and spermatocytes, while MEK1 is not expressed abundantly in pachytene spermatocytes. Phosphorylated (active) variants of these kinases are diminished in pachytene spermatocytes. Treatment of pachytene spermatocytes with okadaic acid (OA), to induce transition from meiotic prophase to metaphase I (G2/MI), resulted in phosphorylation and enzymatic activation of ERK1/2. However, U0126, an inhibitor of the ERK-activating kinases, MEK1/2, did not inhibit OA-induced MAPK activation or chromosome condensation. Analysis of spermatocytes lacking MOS, a mitogenactivated protein kinase kinase kinase responsible for MEK and MAPK activation, revealed that MOS is not required for OAinduced activation of the MAPKs. OA-induced MAPK activation was inhibited by butyrolactone I, an inhibitor of cyclin-dependent kinases 1 and 2 (CDK1, CDK2); thus, these kinases may regulate MAPK activity. Additionally, spermatocytes lacking CDC25C condensed bivalent chromosomes and activated both MPF and MAPKs in response to OA treatment; therefore, there is a CDC25C-independent pathway for MPF and MAPK activation. These studies reveal that spermatocytes do not require either MOS or CDC25C for onset of the meiotic division phase or for activation of MPF and the MAPKs, thus implicating a novel pathway for activation of the ERK1/2 MAPKs in spermatocytes. kinases, meiosis, spermatogenesis INTRODUCTION Spermatogenesis is initiated by production of mitotically proliferating spermatogonia from spermatogonial stem cells, and meiosis is initiated after a precise number of mitotic divisions by the differentiated spermatogonia. During the extended meiotic prophase, nuclear events include homologous chromosome pairing, recombination, and chromatin remodeling. As spermatocytes enter the meiotic diSupported by a grant from the NIH, HD33816, to M.A.H. Correspondence and current address: Mary Ann Handel, The Jackson Laboratory, 600 Main St., Bar Harbor, ME 04609. FAX: 207 288 6073; e-mail: 3 Current address: Laboratory for Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709. 1 2 Received: 28 January 2004. First decision: 16 February 2004. Accepted: 1 April 2004. Q 2004 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org 570 vision phase, synaptonemal complex breakdown, chromosome condensation, and spindle assembly ensue. Although these events serve as hallmarks of the onset of the division phase, the regulatory mechanisms governing the processes of the meiotic G2/MI transition are not well understood. Reversible protein phosphorylation is universally involved in regulation of both mitotic and meiotic cell divisions [1]. Indeed, during meiosis, protein phosphorylation accompanies synaptonemal complex disassembly, chromosome condensation, and nuclear envelope breakdown [2– 5], and there is a dramatic increase in levels of phosphorylated proteins as spermatocytes exit prophase and enter metaphase (Cobb and Handel, unpublished results). These observations imply that reversible protein phosphorylation plays an important role in the regulation of meiotic divisions during spermatogenesis. However, although phosphorylation reactions are presumed essential, the roster of specific kinases required for the successful completion of the G2/MI transition during male meiosis is not yet known. Experimental analysis of the G2/MI transition in spermatocytes has been difficult, in part because of the low frequency of testicular spermatocytes progressing through this transition but also because of the lack of unique size characteristics that would facilitate their purification. This difficulty was partially overcome by the discovery that okadaic acid (OA) treatment of pachytene spermatocytes in vitro overrides the normal checkpoints that delay entry into metaphase I and induces a precocious MI-like state, characterized by condensation of bivalent metaphase chromosomes and synaptonemal complex breakdown [2]. OA is thought to act indirectly through activation of maturation promoting factor (MPF). MPF is a complex of two subunits, the catalytic p34cdc2 (CDK1) subunit and the CYCLIN B1 regulatory subunit. Activation of MPF occurs through a two-step process involving the phosphorylation of Thr161 by the CDK-activating kinase and the dephosphorylation of Thr14 and Tyr15 by a CDC25 phosphatase [6, 7]. MPF is suspected to be required for the meiotic G2/MI transition of spermatocytes in vitro since pachytene spermatocytes pretreated with butyrolactone I, a potent and specific inhibitor of the cyclin-dependent kinases, did not condense bivalent chromosomes in response to OA treatment [4]. These observations, however, are also consistent with a role for CDK2, which has recently been shown to be required for the meiotic division of spermatocytes [8, 9]. The role of CDC25C in MPF activation has recently come into question. Mice homozygously null for the Cdc25c gene have no abnormal phenotype and are fertile [10], suggesting that CDC25C is not required or that there are redundant phosphatases that substitute in its absence. The requirement for CDC25C in the spermatocyte’s OA-induced G2/MI transition is investigated here. MAP KINASE DYNAMICS IN SPERMATOGENESIS MATERIALS AND METHODS Animals and Germ Cell Isolation ICR mice were obtained from Harlan (Indianapolis, IN); those carrying the Mostm1Ev (hereafter designated Mos2) and Cdc25ctm1Hpw (hereafter designated Cdc25c2) targeted null alleles were generously provided by Drs. John Eppig (The Jackson Laboratory, Bar Harbor, ME) and Helen Piwnica-Worms (Washington University, St. Louis, MO), respectively. Mice were maintained under standard conditions in accordance with the National Institutes of Health and U.S. Department of Agriculture standards. Mixed germ cell preparations were obtained by enzymatic digestion of adult testes. The testes were detunicated and digested in 0.5 mg/ml collagenase in Krebs-Ringer bicarbonate (KRB) buffer at 328C for 20 min, followed by a subsequent digestion in 0.5 mg/ml trypsin at 328C for 13 min. After digestion, the germ cells were fi (...truncated)