Changes in histone acetylation during oocyte meiotic maturation in the diabetic mouse

Although there is considerable evidence that diabetes can adversely affect meiosis in mammalian oocytes, acetylation status of oocytes in a diabetic environment remains unclear. The objective was to determine acetylation or deacetylation patterns (based on immunostaining) of H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16 sites at various stages during meiosis in murine oocytes from control and diabetic mice. According to quantitative real time polymerase chain reaction (qPCR), mean ± SEM relative expression of Gcn5 (1.70 ± 0.14 at metaphase [M]I and 1.27 ± 0.01 at MII, respectively), Ep300 (1.74 ± 0.04 at MI and 1.80 ± 0.001 at MII), and Pcaf (2.01 ± 0.03 at MI and 1.41 ± 0.18 at MII) mRNA in oocytes from diabetic mice were higher than those from controls (P < 0.05), whereas there was no difference (P > 0.05) during the germinal vesicle (GV) stage between the two groups (1.23 ± 0.04 for Gcn5, 0.82 ± 0.06 for Ep300, and 0.80 ± 0.07 for Pcaf). Conversely, relative mRNA expression concentrations of Hdac1, Hdac2, Hdac3, Sirt1 and Sirt2 during the germinal vesicle stage were lower in oocytes of diabetic mice (0.24 ± 0.03 for Hdac1, 0.11 ± 0.001 for Hdac2, 0.31 ± 0.03 for Hdac3, 0.28 ± 0.02 for Sirt1, and 0.55 ± 0.02 for Sirt2; P < 0.05). Similarly, the expression concentrations of these genes at the MI stage were lower in oocytes from diabetic mice (0.79 ± 0.12 for Hdac1, 0.72 ± 0.001 for Hdac2, 0.02 ± 0.001 for Sirt1, and 0.84 ± 0.08 for Sirt2; P < 0.05). Their expression concentrations at the MII stage were also lower in oocytes from diabetic mice (0.46 ± 0.03 for Hdac1, 0.93 ± 0.01 for Hdac2, 0.56 ± 0.01 for Hdac3, 0.01 ± 0.002 for Sirt1, and 0.84 ± 0.04 for Sirt2; P < 0.05). At the MI stage, however, there was no difference in the expression of Hdac3 between the two groups of oocytes (0.96 ± 0.03; P > 0.05). Taken together, diabetes altered the intracellular histone modification system, which may have contributed to changes in histone acetylation, and may be involved in the compromised maturation rate of oocytes in diabetic humans.     

CDC2A (CDK1)-mediated phosphorylation of MSY2 triggers maternal mRNA degradation during mouse oocyte maturation

Degradation of maternal mRNA is thought to be essential to undergo the maternal-to-embryonic transition. Messenger RNA is extremely stable during oocyte growth in mouse and MSY2, an abundant germ cell-specific RNA-binding protein, likely serves as a mediator of global mRNA stability. Oocyte maturation, however, triggers an abrupt transition in which most mRNAs are significantly degraded. We report that CDC2A (CDK1)-mediated phosphorylation of MSY2 triggers this transition. Injecting Cdc2a mRNA, which activates CDC2A, overcomes milrinone-mediated inhibition of oocyte maturation, induces MSY2 phosphorylation and the maturation-associated degradation of mRNAs. Inhibiting CDC2A following its activation with roscovitine inhibits MSY2 phosphorylation and prevents mRNA degradation. Expressing non-phosphorylatable dominant-negative forms of MSY2 inhibits the maturation-associated decrease in mRNAs, whereas expressing constitutively active forms induces mRNA degradation in the absence of maturation and phosphorylation of endogenous MSY2. A positive-feedback loop of CDK1-mediated phosphorylation of MSY2 that leads to degradation of Msy2 mRNA that in turn leads to a decrease in MSY2 protein may ensure that the transition is irreversible.     

Synthesis and modification of D7 protein during Xenopus oocyte maturation.

The Xenopus maternal mRNA D7 is translationally repressed during oogenesis, only becoming recruited into polysomes during oocyte maturation, with D7 protein being detectable for the first time prior to germinal vesicle breakdown (GVBD). The synthesis of D7 protein was found to be induced by a variety of maturation-promoting agents including cyclin, c-mos and crude preparations of MPF. D7 protein induced by all these agents is post-translationally modified and exists as a number of variants of differing molecular weight. In contrast to endogenous D7 mRNA, D7 RNA injected into the stage VI oocyte is efficiently translated, resulting in the accumulation of predominantly unmodified D7 polypeptides, which become increasingly modified during oocyte maturation to produce a pattern of polypeptides similar to those derived from endogenous D7 mRNA. Thus, the system that results in the post-translational modification of the D7 protein is itself activated during oocyte maturation. The nature of the protein modification is not known but does not appear to be phosphorylation. The translation of exogenous D7 RNA in the stage VI oocyte does not lead to translational derepression of endogenous D7 mRNA.     

Expression of estrogen receptor alpha and beta during mouse embryogenesis.

In adult mammals numerous target tissues and organs for estrogens exist. Little is known about possible target organs during embryogenesis other than the reproductive tract and the gonads. This is the first report on the expression of estrogen receptor beta (ERbeta) in comparison with ERalpha mRNA during mouse embryogenesis. We found expression of estrogen receptor mRNA in the reproductive tract, but also in the atrial wall, brain, kidney, urethra, bladder neck, mammary gland primordium, midgut, cartilage primordia and perichondria.     

SET improved oocyte maturation by serine/threonine protein phosphatase 2A and inhibited oocyte apoptosis in mouse oocytes

SET is a multifunctional protein involved in a variety of molecular processes such as cell apoptosis and cell-cycle regulation. In ovaries SET is predominantly expressed in theca cells and oocytes. In polycystic ovary syndrome (PCOS) patients the expression of SET was increased than healthy people. The current study was designed to determine whether SET plays a role in oocyte maturation and apoptosis, which may provide clues for the underlying pathological mechanism of follicular development in PCOS patients. Oocytes at germinal vesicle (GV) stage were collected from 6-week-old female ICR mice ovaries. The expression of SET was manipulated by AdCMV-SET and AdH1-SiRNA/SET adenoviruses. SET overexpression improved oocyte maturation whereas SET knockdown inhibited oocyte maturation. Moreover, SET negatively regulated serine/threonine protein phosphatase 2A (PP2A) activity in oocytes. Treatment with PP2A inhibitor okadaic acid (OA) promoted oocyte maturation. Furthermore, PP2A knockdown confirmed the role of PP2A in oocyte maturation, and OA was able to block the AdH1-SiRNA/SET-mediated inhibition on oocyte maturation. The central role of PP2A in SET-mediated regulation of oocyte maturation was confirmed by the finding that SET increased the expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) and PP2A inhibited their expressions. Besides, SET inhibited oocyte apoptosis through decreasing the expression of caspase 3 and caspases 8, while PP2A had no effect on oocyte apoptosis. SET promoted oocyte maturation by inhibiting PP2A activity and inhibited oocyte apoptosis in mouse in-vitro cultured oocytes, which may provide a pathologic pathway leading to impaired oocyte developmental competence in PCOS.     

Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos.

The objective of this study was to examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization. Oocytes were aspirated from 2-5 mm follicles of ovaries collected at a local abattoir. The oocyte-cumulus complexes (OCCs) were cultured for 23-25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 micrograms/ml luteinizing hormone (LH), 0.5 microgram/ml follicle-stimulating hormone (FSH), and 1 microgram/ml estradiol. After maturation for 23-25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum-free embryo culture medium (HECM-1/BSA) for 8 days, after which embryo development was assessed. Five media (SFRE, MEM alpha, TCM199, MEM alpha/+, RPMI:MEM alpha) better supported normal oocyte maturation as determined by embryo development to the two-cell (76-82%), morula/blastocyst (25-32%), and blastocyst (12-19%) stages. Oocytes that were matured in Waymouth's medium MB 752/l or Ham's F-12 had a significantly reduced incidence of cleavage to the two-cell stage (52% and 37%, respectively), which was not attributed to failure of fertilization. Of the eggs that did cleave to the two-cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polarization of mitochondria in the unfertilized mouse oocyte

Maturation of an immature oocyte into one capable of being fertilized involves tightly choreographed movements of chromosomes and organelles. The localizaton of mitochondria during maturation was studied in live mouse oocytes by confocal laser scanning microscopy (CLSM). Mitochondria were labeled with rhodamine 123 or Mitotracker (Molecular Probes, Eugene, OR) both of which are cell permeant and accumulate in mitochondria; acridine orange was used to mark chromatin. Prior to maturation, oocytes appeared to be radially symmetrical with no evident polarity; fully mature oocytes exhibited obvious polarity marked by the position of the metaphase II spindle in the cortex. CLSM revealed several interesting features of mitochondrial distribution: (1) A cortical clump of mitochondria was seen approximately 30-45to one side of the metaphase II spindle and marked the region of polar body I extrusion. (2) Large foci of mitochondria (7–14μM) were frequently found around the central region of the mature oocyte, while the central region often exhibited markedly fewer mitochondria. (3) Small mitochondrial foci (3μM) in the cortex and near the GV characterized several oocytes which failed to mature. (4) Non-spindle-associated mitochondria were not uniformly distributed in the mature oocyte but were concentrated in the hemisphere containing the metaphase II spindle. (5) The distal margins of this mitochondrial hemisphere were sharply demarcated at the cortex. These findings should help us understand organelle localization during mammalian oocyte maturation, and may give insights into possible causes of infertility and into early events of preimplantation development. © 1995 Wiley-Liss, Inc.     

Protein synthesis requirements during resumption of meiosis in the hamster oocyte: early nuclear and microtubule configurations.

The organization of chromatin and cytoplasmic microtubules changes abruptly at M-phase entry in both mitotic and meiotic cell cycles. To determine whether the early nuclear and cytoplasmic events associated with meiotic resumption are dependent on protein synthesis, cumulus-enclosed hamster oocytes were cultured in the presence of 100 micrograms/ml puromycin or cycloheximide for 5 hr. Both control (untreated) and treated oocytes were analyzed by fluorescence microscopy after staining with Hoechst 33258 and tubulin antibodies. Freshly isolated oocytes exhibit prominent nucleoli and diffuse chromatin within the germinal vesicle as well as an interphase network of cytoplasmic microtubules. After 4-4.5 hr in culture, most oocytes were in prometaphase I of meiosis as characterized by a prominent spindle with fully condensed chromosomes and numerous cytoplasmic asters. After 5-5.5 hr in culture, microtubule asters are no longer detected in most cells, and the spindle is the only tubulin-positive structure. Incubation for 5 hr in the presence of inhibitors does not impair germinal vesicle breakdown, chromatin condensation, kinetochore microtubule assembly, or cytoplasmic aster formation in the majority of oocytes examined; however, under these conditions, a population of oocytes retains a germinal vesicle, exhibiting variable degrees of chromatin condensation and cytoplasmic aster formation. Meiotic spindle formation is inhibited in all oocytes. These effects are fully reversible upon culture of treated oocytes in drug-free medium for 5 hr. The data indicate that meiotic spindle assembly is dependent on ongoing protein synthesis in the cumulus-enclosed hamster oocyte; in contrast, chromatin condensation and aster formation are not as sensitive to protein synthesis inhibitors during meiotic resumption.     

AMP-activated protein kinase is involved in hormone-induced mouse oocyte meiotic maturation in vitro

We have previously shown that AMP-activated protein kinase (AMPK) can induce the resumption of meiosis in mouse oocytes maintained in meiotic arrest in vitro. The present study was carried out to determine whether AMPK activation is involved in hormone-induced maturation. Follicle-stimulating hormone (FSH) and the EGF-like peptide, amphiregulin (AR), are potent inducers of maturation in cumulus cell-enclosed oocytes (CEO). Within 3 h of FSH treatment, phospho-acetyl CoA carboxylase (ACC) levels were increased in germinal vesicle (GV)-stage oocytes when compared to non-stimulated controls and remained elevated throughout 9 h of culture, indicating AMPK activation. A similar response to AR was observed after 6 h of culture. Using anti-PT172 antibody (binds only to activated AMPK), Western analysis demonstrated active AMPK in both FSH- or AR-treated GV-stage oocytes within 6 h. The AMPK inhibitors, compound C and adenine 9-beta-d-arabinofuranoside (araA), blocked FSH- or AR-induced meiotic resumption and ACC phosphorylation, further supporting a causal role for AMPK in hormone-induced meiotic resumption. Immunocytochemistry using anti-PT172-AMPK antibody showed an increased diffuse cytoplasmic staining and more intense punctate staining in the germinal vesicles of oocytes following treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or with FSH or AR, and this staining was eliminated by compound C or a blocking peptide for the anti-PT172 antibody. Staining of oocytes from hCG-stimulated mice with the anti-PT172 antibody also showed pronounced label in the germinal vesicles within 1-2 h. Furthermore, in oocytes from all groups, active AMPK was always observed in association with the condensed chromosomes of maturing oocytes. Taken together, these results support a role for AMPK in FSH and AR-induced maturation in vitro and hCG-induced maturation in vivo.     

Protein phosphorylation patterns during in vitro maturation of the goat oocyte

Protein phosphorylation patterns were studied by radiolabelling goat cumulus oocyte complexes with [³²P]orthophosphate for various periods of time. The radiolabelled denuded oocytes were assessed for nuclear status and were used individually for gel electrophoresis. This study demonstrated that specific changes in protein phosphorylations were programmed during goat oocyte maturation. One of the most prominent changes was a general increase in the phosphorylation rate at germinal vesicle breakdown (GVBD). From 8 hr of culture, dominant phosphoprotein bands with apparent molecular weights of 27, 31, 40, and 50 kD were observed; they remained at this level until the metaphase II stage. In the molecular weight range of 65–80 kD, the protein phosphorylation pattern exhibited characteristic differences, with a complex series of phosphoproteins appearing and disappearing, during maturation. Addition of 6-dimethylaminopurine (6-DMAP) at the onset of culture blocked the maturation process after GVBD and induced a dramatic condensation of chromatin. When added at different times after GVBD, 6-DMAP invariably induced chromosome condensation. This inhibition was partly reversible; i.e., after removal of the drug, oocytes were able to progress only until metaphase l. © 1993 Wiley-Liss, Inc.     

Effect of glucocorticoids on spontaneous and follicle-stimulating hormone induced oocyte maturation in mouse oocytes during culture

Several studies have indicated that glucocorticoids are involved in maturation of mammalian oocytes. Recently, maturation of porcine oocytes in culture was shown to be inhibited by glucocorticoids in a time- and dose-dependent manner. In addition, levels of cortisol available for biological action in fluid of preovulatory follicles are higher than that present in circulation. The present study evaluates the effect of cortisol and dexamethasone on mouse cumulus enclosed oocytes (CEO) undergoing spontaneous- and FSH-induced maturation during a 24 h culture period using breakdown of the germinal vesicle (GVBD) as end-point. FSH-induced oocyte maturation was studied using media containing 4.5 mM hypoxanthine to maintain levels of cAMP elevated, whereas spontaneous oocyte maturation was studied in a medium without hypoxanthine. In the presence of FSH (25 IU/l) the rate of GVBD was significantly elevated compared to the control. Dexamethasone (1–20 μg/ml) in combination with FSH resulted in a rate of GVBD similar to FSH alone. Cortisol (0.1–10 μg/ml) resulted in a significant higher rate of GVBD in combination with a physiological concentration of FSH (10 IU/l) as compared to the control but similar to that caused by FSH alone. Nearly all CEO that matured spontaneously resumed meiosis irrespective of whether or not cortisol was present. In conclusion, these results indicate that glucocorticoids have little or no influence on the regulation of oocyte maturation in the mouse. Species differences between mouse and pig oocytes may exist.