Endogenous regulators of protein phosphatase-1 during mouse oocyte development and meiosis.
Reversible phosphorylation, involving protein kinases and phosphatases (PP), is important in regulating oocyte meiosis. Okadaic acid (OA) inhibition of PP1 and/or PP2A stimulates oocyte germinal vesicle breakdown (GVB). In oocytes, PP1 is localized in the cytoplasm and nucleus, yet endogenous regulation of oocyte PP1 has not been investigated. The objectives of the study were to identify intra-oocyte mechanisms regulating PP1 during acquisition of OA-sensitive meiotic competence and meiotic resumption. Immunohistochemical studies revealed that GVB-incompetent oocytes contained equivalent cytoplasmic and nuclear PP1. Upon development of OA-sensitive meiotic competence, PP1 displayed differential intracellular localization with significantly greater nuclear staining with distinct nucleolar rimming compared with cytoplasmic staining. Germinal vesicle-intact oocytes contained neither nuclear inhibitor of PP1, nor PP1 cytoplasmic inhibitor-1 transcripts or proteins. Reverse transcription-PCR with PP1 cytoplasmic inhibitor-2 (I2) primers and oocyte RNA amplified a predicted 330-bp product with the identical sequence to mouse liver I2. Oocytes contained a heat-stable PP1 inhibitor with biochemical properties of I2. Phosphorylation of PP1 at Thr320 by cyclin dependent kinase-1 (CDK1) causes PP1 inactivation. Germinal vesicle-intact oocytes did not contain phospho-Thr320-PP1. Upon GVB, PP1 became phosphorylated at Thr320 and this phosphorylation did not occur if GVB was blocked with the CDK1 inhibitor, roscovitine (ROSC). Inhibition of oocyte GVB with ROSC was reversible and coincided with PP1 phosphorylation at Thr320. Increased oocyte staining of nuclear PP1 compared with cytoplasmic staining at a chronological stage when oocytes gain meiotic competence, and phosphorylation and inhibition of PP1 by CDK1 at or around GVB appear to be important mechanisms in regulating oocyte PP1 activity and meiosis. In addition, these studies provide further support for PP1 being the OA-sensitive PP important in the regulation of the acquisition of meiotic competence, nuclear events during meiotic arrest, and GVB.
Wang
Xia
X
Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, Michigan 48109-0617, USA.
Swain
Jason E
JE
Bollen
Mathieu
M
Liu
Xiao-Tie
XT
Ohl
Dana A
DA
Smith
Gary D
GD
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors; D005260:Female; D007150:Immunohistochemistry / Q000379:methods; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000378:metabolism*; D008540:Meiosis / Q000502:physiology*; D051379:Mice; D008815:Mice, Inbred Strains; D019319:Okadaic Acid / Q000378:metabolism; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism; D009866:Oogenesis / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D011687:Purines / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077546:Roscovitine
D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011687:Purines; C021042:protein phosphatase inhibitor-1; D000077546:Roscovitine; D019319:Okadaic Acid; D016203:CDC2 Protein Kinase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1