MAP kinase activity is downregulated by phorbol ester during mouse oocyte maturation and egg activation in vitro

Perfluorononanoic acid (PFNA), a member of PFAS, is frequently detected in human blood and tissues, even in follicular fluid of women. The exposure of PFNA, but not PFOA and PFOS, is positively correlated with miscarriage and increased time to pregnancy. Toxicological studies indicated that PFNA exposure is associated with immunotoxicity, hepatotoxicity, developmental toxicity, and reproductive toxicity in animals. However, there is little information regarding the toxic effects of PFNA on oocyte maturation. In this study, we investigated the toxic effects of PFNA exposure on mouse oocyte maturation in vitro. Our results showed that 600 μM PFNA significantly inhibited germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) in mouse oocytes. Our further study revealed that PFNA induced abnormal metaphase I (MI) spindle assembly, evidenced by malformed spindles and mislocalization of p-ERK1/2 in PFNA-treated oocytes. We also found that PFNA induced abnormal mitochondrial distribution and increased mitochondrial membrane potential. Consequently, PFNA increased reactive oxygen species (ROS) levels, leading to oxidative stress, DNA damage, and eventually early-stage apoptosis in oocytes. In addition, after 14 h culture, PFNA disrupted the formation of metaphase II (MII) spindle in most PFNA-treated oocytes with polar bodies. Collectively, our results indicate that PFNA interferes with oocyte maturation in vitro via disrupting spindle assembly, damaging mitochondrial functions, and inducing oxidative stress, DNA damage, and early-stage apoptosis.

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Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation

Blood ◽

10.1182/blood.v87.12.5287.bloodjournal87125287 ◽

1996 ◽

Vol 87 (12) ◽

pp. 5287-5296 ◽

Cited By ~ 45

Author(s):

YL Zu ◽

Y Ai ◽

A Gilchrist ◽

ME Labadia ◽

RI Sha'afi ◽

...

Keyword(s):

Protein Kinase ◽

Enzymatic Activity ◽

Map Kinase ◽

Phorbol Ester ◽

Map Kinases ◽

Phosphorylation Site ◽

Human Neutrophils ◽

Inhibitory Peptide

In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen- activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase- activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP- stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.

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Effects of steroids on mouse oocyte maturation in vitro

Reproduction ◽

10.1530/jrf.0.0600331 ◽

1980 ◽

Vol 60 (2) ◽

pp. 331-338 ◽

Cited By ~ 45

Author(s):

D. M. Smith ◽

D. Y. Tenney

Keyword(s):

Oocyte Maturation ◽

Mouse Oocyte ◽

Maturation In Vitro

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Co-culture with pig membrana granulosa cells modulates the activity of cdc2 and MAP kinase in maturing cattle oocytes

Zygote ◽

10.1017/s0967199400003166 ◽

1996 ◽

Vol 4 (3) ◽

pp. 247-256 ◽

Cited By ~ 18

Author(s):

Jan Motlík ◽

Peter Šutovský ◽

Jaroslav Kalous ◽

Michal Kubelka ◽

Jiří Moos ◽

...

Keyword(s):

Map Kinase ◽

Granulosa Cells ◽

Kinase Activity ◽

Histone H1 ◽

Meiotic Spindle ◽

Nuclear Envelope Breakdown ◽

Transmission Electron ◽

Initial Culture ◽

Metaphase I

SummaryBovine cumulus-enclosed oocytes, initially cultured up to diakinesis (8h of initial culture) or metaphase I (12h of initial culture), were subsequently co-cultured for 6 h in contact with pig membrana granulosa (PMG) cells and then assayed for histone H1 and MAP kinase activities. In addition, the phosphorylation state of ERK 1,2 proteins was determined by Western blotting. The alterations in nuclear envelope breakdown, meiotic spindle formation and the patterns of chromosome condensation were analysed by immunofluorescence and transmission electron microscopy. The diakinesis-stage oocytes (initially cultured for 8h) already possessed high histone H1 kinase and MAP kinase activities that were correlated with condensed and partially individualised chromosomes. The ERK 1 and most ERK 2 proteins were partly phosphorylated. Following the 6h co-culture of these oocytes with PMG a rapid decrease in MAP kinase activity and a slower decrease in histone H1 kinase occurred, as well as ERK 1 and ERK 2 dephosphorylation. Both kinase activities and ERK 1,2 phosphorylation were fully restored following the release of the oocytes from co-culture and a subsequent culture in the absence of PMG. Moreover, the clumped bivalents were reindividualised and 56% of these oocytes reached metaphase II after 20 h of culture without PMG. The metaphase I oocytes, initially cultured for 12 h, displayed a fusiform meiotic spindle and a metaphase array of chromosomal bivalents, accompanied by high levels of both histone H1 and MAP kinase activity. Co-culture of MI oocytes with PMG abolished the activity of both kinases and caused the dephosphorylation of ERK 1 and ERK 2. Furthermore, the spindle microtubules were depolymerised and the chromosomal bivalents clumped into a single mass. Neither of the protein kinase activities nor the meiotic spindle were restored following subsequent culture in the absence of PMG for up to 20 h. These observations indicate that under in vitro conditions membrana granulosa cells can cause a prompt decrease in histone H1 and MAP kinase activities, and metaphase I oocytes. While these events are fully reversible in late diakinesis oocytes, metaphase I oocytes did not complete maturation after release from co-culture.

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Tyrosine phosphorylation and activation of homologous protein kinases during oocyte maturation and mitogenic activation of fibroblasts.

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2517 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2517-2528 ◽

Cited By ~ 118

Author(s):

J Posada ◽

J Sanghera ◽

S Pelech ◽

R Aebersold ◽

J A Cooper

Keyword(s):

Cell Cycle ◽

Map Kinase ◽

Tyrosine Phosphorylation ◽

Oocyte Maturation ◽

Mammalian Cells ◽

Map Kinases ◽

Kinase Activity ◽

Mitotic Cell ◽

Meiotic Maturation ◽

Sea Star

Meiotic maturation of Xenopus and sea star oocytes involves the activation of a number of protein-serine/threonine kinase activities, including a myelin basic protein (MBP) kinase. A 44-kDa MBP kinase (p44mpk) purified from mature sea star oocytes is shown here to be phosphorylated at tyrosine. Antiserum to purified sea star p44mpk was used to identify antigenically related proteins in Xenopus oocytes. Two tyrosine-phosphorylated 42-kDa proteins (p42) were detected with this antiserum in Xenopus eggs. Xenopus p42 chromatographs with MBP kinase activity on a Mono Q ion-exchange column. Tyrosine phosphorylation of Xenopus p42 approximately parallels MBP kinase activity during meiotic maturation. These results suggest that related MBP kinases are activated during meiotic maturation of Xenopus and sea star oocytes. Previous studies have suggested that Xenopus p42 is related to the mitogen-activated protein (MAP) kinases of culture mammalian cells. We have cloned a MAP kinase relative from a Xenopus ovary cDNA library and demonstrate that this clone encodes the Xenopus p42 that is tyrosine phosphorylated during oocyte maturation. Comparison of the sequences of Xenopus p42 and a rat MAP kinase (ERK1) and peptide sequences from sea star p44mpk indicates that these proteins are close relatives. The family members appear to be tyrosine phosphorylated, and activated, in different contexts, with the murine MAP kinase active during the transition from quiescence to the G1 stage of the mitotic cell cycle and the sea star and Xenopus kinases being active during M phase of the meiotic cell cycle.

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