Calmodulin modulates the cyclic AMP level in Xenopus oocyte

Progesterone decreases the cAMP level of Xenopus oocytes which had been pretreated with cholera toxin (6 nM) and IBMX (1 mM); its action is obtained either by exposure to external hormone (1 micro M) or by microinjection of 50 nl of a 1 mM progesterone solution in paraffin oil. The cAMP content can be decreased in hormone-free oocytes by the calcium ionophore A 23187 or by microinjection of calcium-calmodulin. Conversely when endogenous calcium-calmodulin is inhibited by microinjection of either anticalmodulin antibodies or fluphenazine the cAMP content is increased. In all experimental conditions (high or low levels of intracellular calmodulin), progesterone is always capable of decreasing the oocyte cAMP concentration. Our results favor the view that the cAMP content is negatively controlled, probably via an inhibition of the adenylate cyclase activity, by two parallel mechanisms: the first involves calmodulin, the second results in an action of progesterone which does not require the intermediary formation of the calcium-calmodulin complex.     

Regulatory contribution of heterotrimeric G-proteins to oocyte maturation in the sea urchin

Regulation of animal oocyte maturation is hypothesized to involve heterotrimeric G-proteins. It is difficult to test this hypothesis though without knowing what G-proteins are present in these cells and where are they localized. We set out to test the hypothesis that G-proteins regulate maturation in the sea urchin oocyte by identifying resident G-proteins in oocytes and eggs, and then investigating their function. We find four families of G-protein alpha-subunits (Galphai, Galphaq, Galphas, and Galpha12) present in both oocytes and eggs of the sea urchin. Three of them, Galphai, Galphaq, and Galphas are present on the plasma membrane of the oocyte, while the fourth is located on cytoplasmic vesicles. Upon oocyte maturation, these proteins remain in eggs, and continue to be expressed in embryonic tissues. To test the functional contribution of the G-proteins to the regulation of oocyte maturation, we employ specific intervening reagents, including antibodies and competitor peptides to each Galpha subunit, and specific Galpha toxins. We find that Gi is a main candidate for a positive regulator of sea urchin oocyte maturation. These studies provide a foundation to further test specific hypotheses of the G-protein mediated regulation of oocyte maturation, fertilization, and early development in the sea urchin.     

XGef is a CPEB-interacting protein involved in Xenopus oocyte maturation

XGef was isolated in a screen for proteins interacting with CPEB, a regulator of mRNA translation in early Xenopus development. XGef is a Rho-family guanine nucleotide exchange factor and activates Cdc42 in mammalian cells. Endogenous XGef (58 kDa) interacts with recombinant CPEB, and recombinant XGef interacts with endogenous CPEB in Xenopus oocytes. Injection of XGef antibodies into stage VI Xenopus oocytes blocks progesterone-induced oocyte maturation and prevents the polyadenylation and translation of c-mos mRNA; injection of XGef rescues these events. Overexpression of XGef in oocytes accelerates progesterone-induced oocyte maturation and the polyadenylation and translation of c-mos mRNA. Overexpression of a nucleotide exchange deficient version of XGef, which retains the ability to interact with CPEB, no longer accelerates oocyte maturation or Mos synthesis, suggesting that XGef exchange factor activity is required for the influence of overexpressed XGef on oocyte maturation. XGef overexpression continues to accelerate c-mos polyadenylation in the absence of Mos protein, but does not stimulate MAPK phosphorylation, MPF activation, or oocyte maturation, indicating that XGef may function through the Mos pathway to influence oocyte maturation. These results suggest that XGef may be an early acting component of the progesterone-induced oocyte maturation pathway.     

XGef mediates early CPEB phosphorylation during Xenopus oocyte meiotic maturation

Polyadenylation-induced translation is an important regulatory mechanism during metazoan development. During Xenopus oocyte meiotic progression, polyadenylation-induced translation is regulated by CPEB, which is activated by phosphorylation. XGef, a guanine exchange factor, is a CPEB-interacting protein involved in the early steps of progesterone-stimulated oocyte maturation. We find that XGef influences early oocyte maturation by directly influencing CPEB function. XGef and CPEB interact during oogenesis and oocyte maturation and are present in a c-mos messenger ribonucleoprotein (mRNP). Both proteins also interact directly in vitro. XGef overexpression increases the level of CPEB phosphorylated early during oocyte maturation, and this directly correlates with increased Mos protein accumulation and acceleration of meiotic resumption. To exert this effect, XGef must retain guanine exchange activity and the interaction with CPEB. Overexpression of a guanine exchange deficient version of XGef, which interacts with CPEB, does not enhance early CPEB phosphorylation. Overexpression of a version of XGef that has significantly reduced interaction with CPEB, but retains guanine exchange activity, decreases early CPEB phosphorylation and delays oocyte maturation. Injection of XGef antibodies into oocytes blocks progesterone-induced oocyte maturation and early CPEB phosphorylation. These findings indicate that XGef is involved in early CPEB activation and implicate GTPase signaling in this process.     

Essential role of ubiquitin C-terminal hydrolases UCHL1 and UCHL3 in mammalian oocyte maturation

Ubiquitin C-terminal hydrolases (UCHs) comprise a family of deubiquitinating enzymes that play a role in the removal of multi-ubiquitin chains from proteins that are posttranslationally modified by ubiquitination to be targeted for proteolysis by the 26S proteasome. The UCH-enzymes also generate free monomeric ubiquitin from precursor multi-ubiquitin chains and, in some instances, may rescue ubiquitinated proteins from degradation. This study examined the roles of two oocyte-expressed UCHs, UCHL1, and UCHL3 in murine and rhesus monkey oocyte maturation. The Uchl1 and Uchl3 mRNAs were highly expressed in GV and MII oocytes, and were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Microinjection of the UCH-family enzyme inhibitor, ubiquitin-aldehyde (UBAL) to GV oocytes prevented oocyte meiotic progression beyond metaphase I in a majority of treated oocytes and caused spindle and first polar body anomalies. Injection of antibodies against UCHL3 disrupted oocyte maturation and caused meiotic anomalies, including abnormally long meiotic spindles. A selective, cell permeant inhibitor of UCHL3, 4, 5, 6, 7-tetrachloroidan-1, 3-dione also caused meiotic defects and chromosome misalignment. Cortical granule localization in the oocyte cortex was disrupted by UBAL injected after oocyte maturation. We conclude that the activity of oocyte UCHs contributes to oocyte maturation by regulating the oocyte cortex and meiotic spindle.     

Nuclear-cytoplasmic interactions during ovine oocyte maturation

The present studies have been undertaken to investigate the interactions that occur between the nucleus and cytoplasm of ovine oocytes at various stages during meiotic maturation. We report that the nucleus of ovine fully grown dictyate stage oocytes can be efficiently removed by a microsurgical enucleation procedure. It is demonstrated that between the initiation of maturation and germinal vesicle breakdown certain newly synthesized polypeptides are selectively sequestered in the oocyte nucleus and the major sequestered polypeptide has a relative molecular mass of 28,000, which represent at least 9% of the total labelled polypeptides transferred to the oocyte nucleus during the first 4 h of maturation. The experiments provide evidence that the removal of the oocyte nucleus at various times before germinal vesicle breakdown (GVBD) does not prevent the major series of changes in protein synthesis that occurs after entry into a metaphase. We conclude therefore that the mixing of the nucleoplasm and cytoplasm is not essential for the initiation or progression of the protein reprogramming process during maturation. In addition, the experiments show that the development of the ability to condense chromatin during ovine oocyte maturation is independent of the oocyte nucleus. The combined results strongly support the hypothesis that the extensive series of translational changes that occur in oocytes during maturation are controlled by cytoplasmic rather than nuclear factors.     

Disappearance of a novel protein component of the 26S proteasome during Xenopus oocyte maturation

We have prepared polyclonal antibodies against Xenopus 20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development of Xenopus laevis. A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.     

In vivo induction of oocyte maturation and ovulation in zebrafish

The maturation of fish oocytes is a well-characterized system induced by progestins via non-genomic actions. In a previous study, we demonstrated that diethylstilbestrol (DES), a non-steroidal estrogen, induces fish oocyte maturation via the membrane progestin receptor (mPR). Here, we attempted to evaluate the effect of DES as an environmental endocrine disrupting chemical (EDC) upon fish oocyte maturation using live zebrafish. DES triggered oocyte maturation within several hours in vivo when administrated directly into the surrounding water. The natural teleost maturation-inducing hormone, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20beta-DHP) also induced oocyte maturation in vivo. Steroids such as testosterone, progesterone or 17alpha-hydroxyprogesterone were also effective in vivo. Further studies indicated that externally applied 17,20beta-DHP even induced ovulation. In contrast to 17,20beta -DHP, DES induced maturation but not ovulation. Theoretically this assay system provides a means to distinguish pathways involved in the induction of ovulation, which are known to be induced by genomic actions from the pathway normally involved in the induction of oocyte maturation, a typical non-genomic action-dependent pathway. In summary, we have demonstrated the effect of EDCs on fish oocyte maturation in vivo. To address the effects, we have explored a conceptually new approach to distinguish between the genomic and non-genomic actions induced by steroids. The assay can be applied to screens of progestin-like effects upon oocyte maturation and ovulation for small molecules of pharmacological agents or EDCs.     

Induction of starfish oocyte maturation by disulfide-reducing agents

Oocyte maturation was found to be induced by disulfide-reducing agents such as dithiothreitol (DTT) and 2,3-dimercapto-1-propanol (BAL) in the starfish, Asterina pectinifera. The follicular envelopes around the oocytes broke and retracted into small clumps of cells on treatment with these reagents, as in the case of 1-methyladenine. Upon insemination, fertilizable eggs obtained by treatment with DTT formed a tight fertilization membrane and underwent cleavage. Such eggs developed normally to bipinnaria larvae. Cysteine and glutathione-SH had no effect in inducing oocyte maturation. On the other hand, pretreatment with sulfhydryl reagents such as p-chloromercurybenzoate (PCMB), iodoacetamide (IAM) and N-ethylmaleimide (NEM) completely suppressed 1-methyladenine-induced oocyte maturation. This inhibitory effect of sulfhydryl reagents on oocyte maturation was diminished by subsequent treatment with DTT or BAL with or without 1-methyladenine. Pretreatment with o-iodosobenzoate failed to inhibit 1-methyladenine-induced oocyte maturation.     

Proteomic profiling of murine oocyte maturation

In an effort to better understand oocyte function, we utilized two-dimensional (2D) electrophoresis and mass spectrometry to identify proteins that are differentially expressed during murine oocyte maturation. Proteins from 500 germinal vesicle (GV) and metaphase II-(MII) arrested oocytes were extracted, resolved on 2D electrophoretic gels, and stained with silver. Analysis of the gels indicated that 12 proteins appeared to be differentially expressed between the GV and MII stage. These proteins were then cored from the 2D gels and identified by mass spectrometry as: transforming acidic coiled-coil protein 3 (TACC3), heat shock protein 105 (HSP105), programmed cell death six-interacting protein (PDCD6IP), stress-inducible phosphoprotein (STI1), importin alpha2, adenylsuccinate synthase (ADDS), nudix, spindlin, lipocalin, lysozyme, translationally controlled tumor protein (TCTP), and nucleoplasmin 2 (NPM2). Interestingly, PDCD6IP, importin alpha2, spindlin, and NPM2 appear slightly larger in mass and more acidic on the MII oocyte gel compared to the GV oocyte gel, suggesting that they may be post-translationally modified during oocyte maturation. Given NPM2 is an oocyte-restricted protein, we chose to further investigate its properties during oocyte maturation and preimplantation development. Real-Time RT-PCR showed that NPM2 mRNA levels rapidly decline at fertilization. Indirect immunofluorescence analysis showed that, with the exception of cortical localization in MII-arrested oocytes, NPM2 is localized to the nucleus of both GV stage oocytes and all stages of preimplantation embryos. We then performed one-dimensional (1D) western blot analysis of mouse oocytes and preimplantation embryos and found that, as implicated by the 2D gel comparison, NPM2 undergoes a phosphatase-sensitive electrophoretic mobility shift during the GV to MII transition. The slower migrating NPM2 form is also present in pronuclear embryos but by the two-cell stage, the majority of NPM2 exists as the faster migrating form, which persists to the blastocyst stage.     

Control of oocyte maturation in cows--biological factors

Since bovine in vitro fertilization became possible in the early 80s, a lot of effort has been done to clarify the mechanisms of what seems more and more one of the crucial steps in this procedure, being oocyte maturation. Undoubtedly, many biological factors act together to prepare the immature oocyte for a successful development to a competent embryo after fertilization. Defects in oocyte maturation can possibly be caused by an inadequate nuclear or cytoplasmic maturation or even by a failure of both. There is a general agreement upon the fact that the origin of the oocyte can play an important role. Oocytes derived from very small follicles show a lower rate of maturation and lower blastocyst development with currently used maturation protocols. Parthenogenetic activation of small size follicle derived oocytes suggests that their poor development was not caused by fertilization problems but more likely by intrinsic oocyte factors. Similar developmental rates achieved through nuclear transfer and parthenogenetic activation suggests that the nucleus of the incompetent oocyte may not be the sole reason for a poor development. Another important factor appears to be the donor animal age. The younger the donor animal, the more impaired is its oocyte's developmental competence in most of the embryo IVP systems. Treatment with exogeneous gonadotropins can be beneficial in young donors on the oocyte cleavage rates but does not always increase the final blastocyst outcome. This review briefly documents some of the biological factors and their possible effects on the developmental capacities of the bovine oocyte in vitro.     

Molecular characterization of the starfish inositol 1,4,5-trisphosphate receptor and its role during oocyte maturation and fertilization.

The release of calcium ions (Ca(2+)) from their intracellular stores is essential for the fertilization of oocytes of various species. The calcium pools can be induced to release Ca(2+) via two main types of calcium channel receptor: the inositol 1,4,5-trisphosphate receptor (IP(3)R) and the ryanodine receptor. Starfish oocytes have often been used to study intracellular calcium mobilization during oocyte maturation and fertilization, but how the intracellular calcium channels contribute to intracellular calcium mobilization has never been understood fully, because these molecules have not been identified and no specific inhibitors of these channels have ever been found. In this study, we utilized a novel IP(3)R antagonist, the "IP(3) sponge," to investigate the role of IP(3) during fertilization of the starfish oocyte. The IP(3) sponge strongly and specifically competed with endogenous IP(3)R for binding to IP(3). By injecting IP(3) sponge into starfish oocyte, the increase in intracellular calcium and formation of the fertilization envelope were both dramatically blocked, although oocyte maturation was not blocked. To investigate the role of IP(3)R in the starfish oocyte more precisely, we cloned IP(3)R from the ovary of starfish, and the predicted amino acid sequence indicated that the starfish IP(3)R has 58-68% identity to mammalian IP(3)R types 1, 2, and 3. We then raised antibodies that recognize starfish IP(3)R, and use of the antibodies to perform immunoblot analysis revealed that the level of expression of IP(3)R remained unchanged throughout oocyte maturation. An immunocytochemical study, however, revealed that the distribution of starfish IP(3)R changes during oocyte maturation.     

Preovulatory endocrinology and oocyte maturation in superovulated cattle

Preovulatory follicular and oocyte nuclear maturation was studied in donor cattle induced to superovulate with a PMSG- or FSH-prostaglandin regimen. Plasma concentrations of progesterone (P4) and luteinizing hormone (LH) and follicular fluid levels of progesterone and estradiol-17β were measured and related to the oocyte nuclear maturation stages. The oocyte donors could be divided into two distinct groups. Group I had entirely normal periovulatory P4 and LH concentrations, and the majority of follicles and oocytes followed a characteristic pattern, clearly time-related to the LH peak. Group II had deviating levels of P4 and/or LH in the pre- and periovulatory period, and a majority of their follicles and oocytes had disturbed maturation, such as abnormal P4:E2 ratio in follicles and prematurely activated or meiotically arrested oocytes. It is concluded that a certain proportion of superovulated cows and heifers develop abnormal follicular/oocyte maturation and constitute poor oocyte, and probably also embryo, donors.     

New insight into the role of phosphodiesterase 3A in porcine oocyte maturation

Background  

The ovulatory surge of gonadotropins triggers oocyte maturation and rupture of the ovarian follicle. The resumption of nuclear maturation in the oocyte from the prophase stage is characterized by germinal vesicle breakdown (GVBD). It has previously been shown that specific inhibition of cAMP degradation by PDE3 prevents the resumption of oocyte meiosis. However, no report has characterized the activity of PDE3 in the porcine oocyte, or the implication of the cAMP-PDE3 pathway in the entire nuclear maturation process. In this study, PDE3 activity in the oocyte was assessed during in vitro maturation (IVM) and the possible roles of the cAMP-PDE3 pathway in the resumption and progression of meiosis were investigated in terms of different models of oocyte maturation.     

Comparative study of the molecular mechanisms of oocyte maturation in amphibians

Maturation-promoting factor (MPF), a complex of Cdc2 and cyclin B, is the final inducer of oocyte maturation. Its activity is controlled by inhibitory phosphorylation of Cdc2 on Tyr15/Thr14 and activating phosphorylation on Thr161. Full-grown immature oocytes of the African clawed frog Xenopus laevis contain inactive MPF (pre-MPF) that comprises cyclin B-bound Cdc2 phosphorylated on Tyr15/Thr14 and Thr161. The synthesis of Mos, but not cyclin B, after stimulation by the maturation-inducing steroid progesterone, is believed to be necessary for initiating Xenopus oocyte maturation through Tyr15/Thr14 dephosphorylation of pre-MPF. In contrast, amphibians other than Xenopus (and also fishes) employ a different mechanism. Full-grown immature oocytes of these species contain monomeric Cdc2 but not cyclin B. MPF is formed after hormonal stimulation by binding of the newly produced cyclin B to the pre-existing Cdc2 and is immediately activated through Thr161 phosphorylation. Mos/MAP kinase is neither necessary nor sufficient for initiating maturation in fishes and amphibians except for Xenopus. We propose a new model of MPF formation and activation during oocyte maturation that is applicable to all amphibians (as well as fishes), based on a novel concept that pre-MPF is an artificial molecule that is not essential for inducing oocyte maturation.     

Boron deficiency disables Xenopus laevis oocyte maturation events

Processes of oocyte maturation that may be affected by boron (B) deficiency were studied to potentially determine a possible biochemical role of B in the Xenopus laevis oocyte. More specifically, the Xenopus oocyte membrane progesterone receptor (OMPR) in B-deficient oocytes was characterized by evaluating progesterone affinity for the OMPR and OMPR responsiveness to progesterone stimulation. The responsiveness of B-deficient oocytes to microinjection of a purified oocyte cytoplasmic fraction (OCF) from B-adequate oocytes was also studied to evaluate which aspects of the maturation process were affected by B deficiency. Results suggested that B deficiency resulted in incomplete oocyte maturation and that maturation could not be induced by the administration of exogenous progesterone. Progesterone successfully induced germinal vesicle breakdown (GVBD) in oocytes from females fed a B-supplemented diet (+B) and females administered a traditional diet of beef liver and lung (B adequate). Addition of exogenous B to the -B oocytes increased the rate of progesterone-induced GVBD slightly. The B-deficient X. laevis oocytes were capable of undergoing GVBD when endogenously stimulated by microinjected purified B-adequate OCF. These results indicated that the inability of the B-deficient oocytes to undergo GVBD was not associated with the cytoplasmic induction process specifically, but possibly in the progesterone receptor or signal transduction pathways. Radio-binding studies found that progesterone binding to the B-deficient OPMR was greatly reduced compared to B-adequate or B-supplemented OMPR. Moreover, washout studies determined that progesterone binding to the OMPR in B-deficient oocytes was more transient than the B adequate or +B oocytes.