Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation

The objective was to evaluate mitochondrial distribution, and its relationship to meiotic development, in canine oocytes during in vitro maturation (IVM) at 48, 72, and 96 h, compared to those that were non-matured or in vivo matured (ovulated). The distribution of active mitochondria during canine oocyte maturation (both in vitro and in vivo) was assessed with fluorescence and confocal microscopy using MitoTracker Red (MT-Red), whereas chromatin configuration was concurrently evaluated with fluorescence microscopy and DAPI staining. During IVM, oocytes exhibited changes in mitochondrial organization, ranging from a fine uniform distribution (pattern A), to increasing clustering spread throughout the cytoplasm (pattern B), and to a more perinuclear and cortical distribution (pattern C). Pattern A was mainly observed in germinal vesicle (GV) oocytes (96.4%), primarily in the non-matured group (P < 0.05). Pattern B was seen in all ovulated oocytes which were fully in second metaphase (MII), whereas in IVM oocytes, ∼64% were pattern B, irrespective of duration of culture or stage of nuclear development (P > 0.05). Pattern C was detected in a minor percentage (P < 0.05) of oocytes (mainly those in first metaphase, MI) cultured for 72 or 96 h. In vitro matured oocytes had a minor percentage of pattern B (P < 0.05) and smaller mitochondrial clusters in IVM oocytes than ovulated oocytes, reaching only 4, 11, and 17% of MII at 48, 72, and 96 h, respectively. Thus, although IVM canine oocytes rearranged mitochondria, which could be related to nuclear maturation, they did not consistently proceed to MII, perhaps due to incomplete IVM, confirming that oocytes matured in vitro were less likely to be competent than those matured in vivo.     

DNA fragmentation in canine oocytes after in vitro maturation in TCM-199 medium supplemented with different proteins

In the present study, the effect of different protein supplementation on meiotic nuclear configuration, DNA fragmentation (TUNEL assay) and metabolic parameters of dog oocytes cultured in vitro for 72 h was investigated. TCM-199 medium was supplemented either with 0.3% bovine serum albumin (BSA) or with 10% bitch heat inactivated plasma (OBP) collected before the LH peak or with OBP collected between the LH peak and ovulation or OBP collected after ovulation. After culture, more than 70% of the cumulus-oocyte complexes cultured in plasma groups presented extensive cell expansion, while none of those cultured in BSA showed extensive expansion of the cumulus (P < 0.05). Glucose consumption and lactate production was lower (P < 0.05) in the BSA-supplemented medium than in plasma-supplemented groups. In all groups, high amounts of alanine were produced. A higher number of oocytes with DNA fragmentation were observed in the BSA group, while in the plasma-supplemented groups more oocytes presented undistinguishable nuclear material. Only a small percentage of the oocytes (7.4-12.7%) had intact DNA after culture and within these, no differences were observed between groups in number of oocytes at each chromatin configuration stage. No differences in the percentage of oocytes reaching metaphase II (MII) were observed between experimental groups. Still, only 2% of cultured oocytes reached MII, but 85.7% of these had intact DNA. Conversely, all other chromatin configurations presented a high proportion of fragmented DNA (germinal vesicle 79.8%; meiosis resumption 73.3%; unclassified 95.2%). In conclusion, a high percentage of canine oocytes that do not complete meiotic maturation to MII are degenerated, whereas a high proportion of MII oocytes have intact DNA, independently of the protein supplement used.     

Expression of growth differentiation factor 9 (GDF-9) during in vitro maturation in canine oocytes

The aim of this study was to characterize in canine oocytes and cumulus cells the dynamic expression of growth differentiation factor 9 (GDF-9) in relation to meiotic development and cumulus expansion throughout in vitro maturation (IVM). Cumulus oocytes complexes (COCs) from ovaries of adult bitches were cultured intact for IVM during 0, 48, 72, and 96 hours. At 0 hours or after IVM, COCs were divided into two groups: one group remained with their cumulus cells and in the other group the cumulus cells were extracted. The expression levels of GDF-9 were determined in both groups using indirect immunofluorescence and Western blot analysis. For immunofluorescence assay, in vivo-matured oocytes collected from oviducts were also used as a positive control. The nuclear stage was analyzed in parallel with 4′-6-diamidino-2-phenylindole staining in denuded oocytes from all maturing groups. The intensity of fluorescence, indicative of GDF-9 expression level, decreased with time (P < 0.05). High expression was observed only in germinal vesicle nonmature oocytes; in contrast, second metaphase oocytes showed only low expression. Western blot analysis showed bands of approximately 56 kd and a split band of approximately 20 kd representing the proprotein and possibly two mature protein forms of GDF-9, respectively. The proprotein was detected in all samples, and it was highly expressed before IVM and in a lesser degree, during the first 48 hours, declining thereafter in coincidence with the expansion of the cumulus cell (P < 0.05). There was a negative correlation (r = −0.97; P < 0.05) between the expression level of GDF-9 and mucification. Mature forms were evident only in COCs, before culture and up to 48 hours of IVM. It was concluded that GDF-9 is expressed in canine oocytes and cumulus cells, mainly in the early developmental states, with low levels in mature oocytes in vitro and in vivo, representing the first approach of GDF-9 dynamic in dog oocyte maturation.     

Oocyte nucleus controls progression through meiotic maturation

We analyzed progression through the meiotic maturation in oocytes manipulated to replace the prophase oocyte nucleus with the nucleus from a cumulus cell, a pachytene spermatocyte or the pronucleus from a fertilized egg. Removal of the oocyte nucleus led to a significant reduction in histone H1 kinase activity. Replacement of the oocyte nucleus by a pronucleus followed by culture resulted in premature pseudomeiotic division and occasional abnormal cytokinesis; however, histone H1 kinase activity was rescued, microtubules formed a bipolar spindle, and chromosomes were condensed. In addition to the anomalies observed after pronuclear transfer, those after transfer of the nucleus from a cumulus cell or spermatocyte included a dramatically impaired ability to form the bipolar spindle or to condense chromosomes, and histone H1 kinase activity was not rescued. Expression of a cyclin B-YFP in enucleated oocytes receiving the cumulus cell nucleus rescued histone H1 kinase activity, but spindle formation and chromosome condensation remained impaired, indicating a pleiotropic effect of oocyte nucleus removal. However, when the cumulus cell nucleus was first transformed into pronuclei (transfer into a metaphase II oocyte followed by activation), such pronuclei supported maturation after transfer into the oocyte in a manner similar to that of normal pronuclei. These results show that the oocyte nucleus contains specific components required for the control of progression through the meiotic maturation and that some of these components are also present in pronuclei.     

The binding of okadaic acid analogs to recombinant OABP2.1 originally isolated from the marine sponge Halichondria okadai

The binding between [24-³H]okadaic acid (OA) and a recombinant OA binding protein OABP2.1 was examined using various OA analog, including methyl okadaate, norokadanone, 7-deoxy OA, and 14,15-dihydro OA, 7-O-palmitoyl DTX1, to investigate the structure activity relationship. Among them, 7-O-palmitoyl DTX1, which is one of the diarrhetic shellfish poisoning (DSP) toxins identified in shellfish, displayed an IC₅₀ for [24-³H]OA binding at 51 ± 6.3 nM (Mean ± SD). In addition, a synthetic compound, N-pyrenylmethyl okadamide, exhibited its IC₅₀ at 10 ± 2.9 nM (Mean ± SD). These results suggested that the recombinant OABP2.1 and the N-pyrenylmethyl okadamide might be core substances in a novel assay for the DSP toxins.     

Induction of meiotic maturation in mouse oocytes by adenosine analogs

In this study we have examined the meiosis-inducing influence of adenosine analogs in mouse oocytes. When a varied group of nucleosides and nucleotides were tested on overnight cultures of hypoxanthine-arrested, cumulus cell-enclosed oocytes (CEO), halogenated adenosine nucleosides, but not native adenosine, exhibited a significant meiosis-inducing capability. When tested under a variety of conditions, meiotic induction by 8-bromo-adenosine (8-Br-Ado) and a second adenosine analog, methylmercaptopurine riboside (MMPR), was especially potent in denuded oocytes (DO) compared to CEO and was not dependent on the type of inhibitor chosen to maintain meiotic arrest. Germinal vesicle breakdown (GVB) was stimulated with rapid kinetics and was preceded by an increase in AMP-activated protein kinase (AMPK) activity. Moreover, compound C, an inhibitor of AMPK, blocked the meiosis-inducing activities of both adenosine analogs. When tested for an effect on meiotic progression to metaphase II (MII) in spontaneously maturing CEO, 8-Br-Ado and the AMPK activator, 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR), increased the percentage of MII-stage oocytes, but MMPR decreased this number. Adenosine and inhibitors of de novo purine synthesis had no effect on the completion of maturation, while compound C suppressed this process. These results support the proposition that oocyte AMPK mediates the positive influence of AICAR and 8-Br-Ado on both the initiation and completion of meiotic maturation. The role of AMPK in MMPR action is less clear.     

Ultrastructure of canine oocytes during in vivo maturation

The aim of the present study was to describe the canine oocyte ultrastructural modifications during in vivo maturation, with precise reference to the timing of the LH surge and of ovulation. Twenty-five bitches were ovariectomized at specific stages between the onset of proestrus and the fifth day post-ovulation: 65 oocytes were observed by transmission electron microscopy (TEM), either before the LH surge (n = 10), between the LH surge and ovulation (n = 12) or after ovulation (n = 43). Prior to the LH surge, the oocyte nucleus had already begun its displacement to the vicinity of the oolemma and reticulated nucleoli were infrequent. The cytoplasm showed signs of immaturity (few organelles preferentially located in the cortical zone, "mitochondrial cloud", scarce cortical granules). The LH surge was immediately followed by cumulus expansion but the ovulation occurred 2 days later. Retraction of the transzonal projections and the meiotic resumption occurred after another 3 days (5 days after the LH peak). The ovulation was then followed by gradual cytoplasmic modifications. Nucleoli re-assumed a reticulated aspect around 24 hr post-ovulation. From 48 hr post-ovulation mitochondria and SER were very numerous and evenly distributed. In conclusion canine oocyte maturation began prior to the LH surge and no cytoplasmic or nuclear modifications followed immediately the LH surge and ovulation. This study suggests that two distinct signals are needed for the final in vivo maturation: one prior to the LH surge (to induce maturation) and another one, around 3 days post-ovulation (to induce meiotic resumption).     

Lysophosphatidylserine inhibits completion of meiotic maturation of rat oocytes in vitro

Follicular oocytes collected prior to the expected time of the LH surge from PMSG-treated immature rats were incubated cummulus-intact (with or without LH) or cumulus-free (CF). Oocytes were incubated in the presence or absence of lysophosphatidlylserine (LS), a naturally occurring membrane phospholipid that has been previously shown to block sperm-related membrane fusion events. Fusion events occurring during oocyte maturation that might be affected by LS include maintenance of the intact germinal vesicle (GVI) and prevention of GV breakdown (GVBD) and first polar body formation (PBI). LS had only a slight effect upon GVI. The incidence of GVI was significantly increased in only one of the three oocyte culture conditions employed (CF). Exposure to LS from the outset of collection and washing did not increase the incidence of GVI, indicating the lack of effect by LS was not owing to the passage of a sensitive period during oocyte collection. In contrast, LS was not owing to the passage of a sensitive period during oocyte colection. In contrast, LS almost completely abolished PBI in all oocyte culture conditions at 100 μ in PBI and those sperm-related fusion processes previously found to be sensitive to LS. Finally, LS or similar agents may be responsible for the block to maturation (often at anaphase I) and even the retarded cleavage observed in vitro during oocyte maturation or embryo culture in some species.     

Regulation of histone acetylation during meiotic maturation in mouse oocytes

Histone acetylation is an important epigenetic modification implicated in the regulation of chromatin structure and, subsequently, gene expression. Global histone deacetylation was reported in mouse oocytes during meiosis but not mitosis. The regulation of this meiosis-specific deacetylation has not been elucidated. Here, we demonstrate that p34(cdc2) kinase activity and protein synthesis are responsible for the activation of histone deacetylases and the inhibition of histone acetyltransferases (HATs), respectively, resulting in deacetylation of histone H4 at lysine-12 (H4K12) during mouse oocyte meiosis. Temporal changes in the acetylation state of H4K12 were examined immunocytochemically during meiotic maturation using an antibody specific for acetylated H4K12. H4K12 was deacetylated during the first meiosis, temporarily acetylated around the time of the first polar body (PB1) extrusion, and then deacetylated again during the second meiosis. Because these changes coincided with the known oscillation pattern of p34(cdc2) kinase activity, we investigated the involvement of the kinase in H4K12 deacetylation. Roscovitine, an inhibitor of cyclin-dependent kinase activity, prevented H4K12 deacetylation during both the first and second meiosis, suggesting that p34(cdc2) kinase activity is required for deacetylation during meiosis. In addition, cycloheximide, a protein synthesis inhibitor, also prevented deacetylation. After PB1 extrusion, at which time H4K12 had been deacetylated, H4K12 was re-acetylated in the condensed chromosomes by treatment with cycloheximide but not with roscovitine. These results demonstrate that HATs are present but inactivated by newly synthesized protein(s) that is (are) not involved in p34(cdc2) kinase activity. Our results suggest that p34(cdc2) kinase activity induces the deacetylation of H4K12 and that the deacetylated state is maintained by newly synthesized protein(s) that inhibits HAT activity during meiosis.     

Transforming growth factor-alpha augments meiotic maturation of cumulus cell-enclosed mouse oocytes

Growth factors have been shown to play an important role in the regulation of ovarian function. In this study, we examined the effects of transforming growth factor-alpha (TGF-alpha) on the meiotic maturation of immature mouse oocytes in vitro. Cumulus cell-enclosed oocytes were exposed to TGF-alpha with or without the meiotic inhibitor hypoxanthine (HX), and oocyte maturation was assessed by germinal vesicle breakdown (GVBD). Likewise, mechanically denuded oocytes were examined for GVBD following exposure to HX and TGF-alpha. When cumulus cell-enclosed oocytes were exposed to TGF-alpha (1 microgram/ml) in the presence of HX (4 mM), an increase in GVBD was observed first after 5 hours of culture. Maximal stimulation was reached at 24 hours when 70% of the oocytes underwent maturation in the presence of TGF-alpha and HX as compared to 33% with HX only. Concentrations of TGF-alpha as low as 0.1 ng/ml produced a similar stimulatory response after 24 hours of culture. Spontaneous maturation in the presence of TGF-alpha, but without HX, was also enhanced. The stimulation of GVBD by TGF-alpha showed an increase over time both with and without HX. When denuded oocytes were exposed to TGF-alpha in the presence of HX, no effect was observed. Our results suggest that TGF-alpha is a potent stimulator of mouse oocyte maturation in vitro and that its effect is mediated by the surrounding cumulus cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The signaling pathways linking to lysophosphatidic acid-promoted meiotic maturation in mice

The signaling pathways linking to lysophosphatidic acid-promoted meiotic maturation in mice were studied. When mouse oocyte-cumulus cells complexes were cultured with 10(-5) M lysophosphatidic acid (the LPA group), the rate of oocyte nuclear maturation was significantly increased. Additions of pertussis toxin, genistein, U73122, Ro320432, PD98059 or SB203580 significantly suppressed the increase in lysophosphatidic acid-stimulated nuclear maturation rate. These results suggested that Gi/o-coupled lysophosphatidic acid receptors activate phosphatidylinositol-specific phospholipase C, and result in ERK and MAP kinase activation, which is triggered by diacylglycerol-dependent protein kinase C. When intracellular cAMP concentrations of oocytes in the LPA and control groups were measured using the acetylation assay, the intracellular cAMP concentration of an oocyte in the LPA group was significantly lower than the control oocyte (0.117+/-0.04 fmol/oocyte vs. 0.176+/-0.036 fmol/oocyte, p<0.05). In conclusion, our results suggested that lysophosphatidic acid stimulates phospholipase C through a Gi-protein linked receptor on the surface of mouse cumulus cells and stimulates both extracellular signal-regulated kinase and p38 mitogen-activated kinase, resulting in the closure or loose of gap junctions between cumulus cells and the oocyte. The resultant early decrease of oocyte cAMP levels may promote nuclear maturation of mouse oocytes in vitro.     

Effect of vitrification on meiotic maturation and expression of genes in immature goat cumulus oocyte complexes

The aim of the study was to evaluate meiotic maturation, and expression of genes coding for oocyte secreted factors (GDF9, BMP15, TGFBR1, and BPR2) and apoptosis (BCL2, BAX and P53) after vitrification of immature goat cumulus oocyte complexes (COCs) and in vitro maturation. COCs were vitrified in a solution containing ethylene glycol, dimethyl sulfoxide and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). Freshly collected COCs (Control), COCs exposed to vitrification and dilution solutions without cryopreservation (EC) and vitrified-warmed COCs were matured in vitro for 27h. The viability of vitrified-warmed COCs 2 h post warming and in vitro maturation was similar for CL, HS and CT. The proportion of oocytes that extruded a 1st polar body and reached TI/MII was significantly higher with CT and HS followed by CL, OPS and CS. Gene expression of GDF9, BMP15, BMPR2, BAX and P53 were comparable to control levels for OPS, CL, HS and CT. The gene expression pattern in CS vitrified COCs was by contrast changed in that GDF9, BMP15, TGFBR1 and BAX were up regulated and BMPR2, BCL2 and P53 down regulated. In conclusion immature goat COCs vitrified using CT and HS showed that viability, maturation rates and expression of genes coding for oocyte secreted factors and apoptosis were similar to non-vitrified controls.     

In vitro maturation of canine oocytes co-cultured with bovine and canine granulosa cell monolayers

The present study investigated the effects of bovine granulosa cell monolayers (BGML) and canine granulosa cell monolayers (CGML) on nuclear maturation of canine oocytes with and without cumulus cells. Cumulus-oocyte complexes (COCs) or cumulus-free oocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM, control group), DMEM with BGML (BGML group), or DMEM with CGML (CGML group) for 72 h at 38.5 °C in 5% CO₂, 5% O_2, and 90% N₂. All media were supplemented with 10% of FCS, 50 ng/mL of EGF, 2 μg/mL of estradiol-17β, 0.1 IU/mL of hCG, 0.1 IU/mL of FSH, 0.25 mM of pyruvic acid, 100 μM of β-mercaptoethanol, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin. In cumulus-enclosed oocytes retrieved from ovaries at estrus and/or diestrus, the highest percentage of M-II oocytes (P < 0.05) was present in the BGML group (27.0%) compared with the CGML group (7.9%) and the control group (3.5%). In cumulus-free oocytes collected from ovaries at estrus and/or diestrus, the proportions of M-II oocytes co-cultured with the CGML were low (3.0%) and similar (P > 0.05) to proportions achieved with control (3.0%). However, the presence of BGML improved (P < 0.05) the ability of denuded oocytes to develop into M-II (10.2%). The BGML group had the highest overall meiotic resumption (P < 0.05), and least oocyte degeneration (P < 0.05) among experimental groups. In conclusion, BGML had a positive impact on the in vitro maturation system, as well as meiotic resumption of canine oocytes.     

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.     

Protein serine/threonine phosphatases as binding proteins for okadaic acid

Recently, many potent inhibitors of protein serine/threonine phosphatases (PPs) have been found. Some of them have proven to be tumor promoters in mouse skin two-step carcinogenesis and rat liver medium-term tests. Among these inhibitors, okadaic acid (OA) selectively inhibits PP2A, and its use has therefore been proposed to facilitate analysis of biological roles of this phosphatase. OA shows bimodal effects on in vitro transformation and, in addition to such epigenetic changes, also induces marked genetic changes. OA treatment for more than 1 week flattened NIH 3T3 transformants irreversibly, with loss of the transfected genes. It is also known to induce diphtheria toxin-resistant mutations in Chinese hamster lung cells and sister chromatid exchanges (SCEs) in Chinese hamster ovary cells and human lymphocytes. To analyze roles of protein phosphatases in gene stability, we isolated OA-resistant mutants. They were proven to have a mutation in the PP2A catalytic subunit, in which cysteine 269 had beensubstituted for glycine; and it was demonstrated that this region interacts with OA. The recombinant mutant protein was 4 9-fold more resistant to OA than the wild type. Although the OA resistant mutants of CHO cells expressed high levels of P-glycoprotein, inhibition of PP2A itself was suggested to lead to SCE induction. However, the number of molecular species of PP which are known to be sensitive to OA continues to increase, and we have isolated cDNA for a novel type of OA sensitive PP. Our studies indicate that the fact that the roles of PP2A cannot be elucidated using only OA is of crucial importance.     

HDAC8 drives spindle organization during meiotic maturation of porcine oocytes

ObjectivesHistone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood.Materials and methodsHDAC8‐targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI‐34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ‐tubulin dynamics and acetylation level of α‐tubulin.ResultsWe observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ‐tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI‐34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α‐tubulin.ConclusionsCollectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation.     

Pleiotropic effect of okadaic acid on maturing mouse oocytes.

Okadaic acid (OA), a potent inhibitor of types 1 and 2A protein phosphatases, was shown recently to induce chromatin condensation and germinal vesicle breakdown (GVBD) in mouse oocytes arrested at the dictyate stage by dibutyryl cAMP (dbcAMP), isobutyl methylxanthine (IBMX) and 12,13-phorbol dibutyrate (PDBu). We confirm these results using IBMX and another phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and show that OA also bypasses the inhibitory effect of 6-dimethylaminopurine (6-DMAP). It has been concluded that protein phosphatases 1 and/or 2A (PP1, 2A), involved in the negative control of MPF activation, are thus operating downstream from both the protein kinase A and protein kinase C catalysed phosphorylation steps that prevent the breakdown of GV. Similar enzymatic activities are also able to counteract the general inhibition of protein phosphorylation. However, PP1 and/or PP2A are positively involved in the activation of pericentriolar material (PCM) into microtubule organizing centres (MTOCs). This explains the inhibitory effect of OA on spindle assembly. Finally, OA interferes with the integrity and/or function of actomyosin filaments. This results in a dramatic ruffling of the plasma membrane leading to the internalization of large vacuoles, the inhibition of chromosome centrifugal displacement and, consequently, the prevention of polar body extrusion.