Analysis of mouse oocyte mechanics shows that effective tension drops 6-fold from prophase I to metaphase II; the metaphase II egg has a 2.5-fold tension differential between the cortex over the spindle and the opposite cortex. Manipulation of actin, myosin-II, or ERMs alters tension levels and induces spindle abnormalities during meiosis II.

In eukaryotic organisms, the largely cytosolic copper and zinc containing superoxide dismutase enzyme (Cu/Zn SOD) represents a key defense against reactive oxygen toxicity. Although much is known about the biology of this enzyme under aerobic conditions, less is understood regarding the effects of low oxygen on Cu/Zn SOD from diverse organisms. We show here that like bakers’ yeast (Saccharomyces cerevisiae), adaptation of the multicellular Caenorhabditis elegans to growth in low oxygen involves strong down-regulation of its Cu/Zn SOD. Much of this regulation occurs at the post-translational level where CCS-independent activation of Cu/Zn SOD is inhibited. Hypoxia inactivates the endogenous Cu/Zn SOD of C. elegans Cu/Zn SOD as well as a P144 mutant of S. cerevisiae Cu/Zn SOD (herein denoted as Sod1p) that is independent of CCS. In our studies of S. cerevisiae Sod1p we noted a post-translational modification to the inactive enzyme during hypoxia. Analysis of this modification by mass spectrometry revealed phosphorylation on serine 38. Serine 38 represents a putative proline-directed kinase target site located on a solvent exposed loop that is positioned at one end of the Sod1p beta-barrel, a region immediately adjacent to residues previously shown to influence CCS-dependent activation. Although phosphorylation of serine 38 is minimal when the Sod1p is abundantly active (e.g., high oxygen), up to 50% of Sod1p can be phosphorylated when CCS-activation of the enzyme is blocked, e.g., by hypoxia or low copper conditions. Serine 38 phosphorylation can be a marker for inactive pools of Sod1p.

Mammalian oocytes in ovarian follicles are arrested in meiosis at prophase I. This arrest is maintained until ovulation, upon which the oocyte exits from this arrest, progresses through meiosis I and to metaphase of meiosis II. The progression from prophase I to metaphase II, known as meiotic maturation, is mediated by signals that coordinate these transitions in the life of the oocyte. ENSA (α-endosulfine) and ARPP19 (cAMP-regulated phosphoprotein-19) have emerged as regulators of M-phase, with function in inhibition of protein phosphatase 2A (PP2A) activity. Inhibition of PP2A maintains the phosphorylated state of CDK1 substrates, thus allowing progression into and/or maintenance of an M-phase state. We show here ENSA in mouse oocytes plays a key role in the progression from prophase I arrest into M-phase of meiosis I. The majority of ENSA-deficient oocytes fail to exit from prophase I arrest. This function of ENSA in oocytes is dependent on PP2A, and specifically on the regulatory subunit PPP2R2D (also known as B55δ). Treatment of ENSA-deficient oocytes with Okadaic acid to inhibit PP2A rescues the defect in meiotic progression, with Okadaic acid-treated, ENSA-deficient oocytes being able to exit from prophase I arrest. Similarly, oocytes deficient in both ENSA and PPP2R2D are able to exit from prophase I arrest to an extent similar to wild-type oocytes. These data are evidence of a role for ENSA in regulating meiotic maturation in mammalian oocytes, and also have potential relevance to human oocyte biology, as mouse and human have genes encoding both Arpp19 and Ensa.

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