Ca2+ Cascade and Meiotic Resumption of the Caprine Primary Oocyte

In this study, we investigated the fluctuations of concentration of intracytoplasmic free Ca(2+) during in vitro maturation of caprine primary oocytes and its role in meiotic resumption. Oocytes that were extracted from caprine ovaries were cultured and allowed to mature in vitro to determine their developmental stages including germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase of the first meiotic division (MI) and metaphase of the second meiotic division (MII). Intracytoplasmic free Ca(2+) turnovers of caprine oocytes at these different developmental stages were measured using the calcium fluorescent probe Fura-2/AM (C(44)H(47)N(3)O(24)) to investigate the dynamics of cytosolic free Ca(2+) during in vitro maturation of oocytes and the role of Ca(2+) in inducing the initiation of meiotic resumption of oocytes. Moreover, the oocytes were cultured in Ca(2+) culture medium and Ca(2+)-free culture medium to examine the effect of extracellular Ca(2+) on the oocyte maturation. The results indicated that Ca(2+) concentrations at GV, GVBD, MI and MII stages were 78.06, 147.41, 126.97 and 97.73 nmol/l, respectively, and that 86.30% of oocytes remained at the GV stage and no oocyte developed to MII in Ca(2+)-free culture medium, and 1.1% of oocytes stayed at the GV stage and 83.5% of oocytes developed to MII in Ca(2+) culture medium. These results suggest that the occurrence of GVBD and cell cycle progression to MI and MII stages are closely related to Ca(2+), and that extracellular Ca(2+) performs a specific function for the initiation of meiotic resumption in caprine oocytes.     

In Vitro Compaction of Germinal Vesicle Chromatin is Beneficial to Survival of Vitrified Cat Oocytes

The immature cat oocyte contains a large-sized germinal vesicle (GV) with decondensed chromatin that is highly susceptible to cryo-damage. The aim of the study was to explore an alternative to conventional cryopreservation by examining the influence of GV chromatin compaction using resveratrol (Res) exposure (a histone deacetylase enhancer) on oocyte survival during vitrification. In Experiment 1, denuded oocytes were exposed to 0, 0.5, 1.0 or 1.5 mmol/l Res for 1.5 h and then evaluated for chromatin structure or cultured to assess oocyte meiotic and developmental competence in vitro . Exposure to 1.0 or 1.5 mmol/l Res induced complete GV chromatin deacetylation and the most significant compaction. Compared to other treatments, the 1.5 mmol/l Res concentration compromised the oocyte ability to achieve metaphase II (MII) or to form a blastocyst. In Experiment 2, denuded oocytes were exposed to Res as in Experiment 1 and cultured in vitro either directly (fresh) or after vitrification. Both oocyte types then were assessed for meiotic competence, fertilizability and ability to form embryos. Vitrification exerted an overall negative influence on oocyte meiotic and developmental competence. However, ability to reach MII, achieve early first cleavage, and develop to an advanced embryo stage (8–16 cells) was improved in vitrified oocytes previously exposed to 1.0 mmol/l Res compared to all counterpart treatments. In summary, results reveal that transient epigenetic modifications associated with GV chromatin compaction induced by Res is fully reversible and beneficial to oocyte survival during vitrification. This approach has allowed the production of the first cat embryos from vitrified immature oocytes.     

The localization and function of p38α mitogen‐activated protein kinase in rat oocytes

P38α mitogen‐activated protein kinase (MAPK), which is a member of the canonical MAPK family, is activated in response to various extracellular stresses and plays a role in multiple cellular processes. In this study, we investigated the expression, subcellular localization and functional roles of p38α MAPK during the meiotic maturation of rat oocytes. We found that p38α MAPK phosphorylation (p‐p38α MAPK, indicative of p38α MAPK activation) was low at the germinal vesicle (GV) stage, increased 3 hr after germinal vesicle breakdown (GVBD) and maintained its maximum at metaphase I (MI) or metaphase II (MII). The p‐p38α MAPK mainly accumulated in the GV and had no obvious expression in the nucleus. From GVBD to MII, p‐p38α MAPK was distributed in the cytoplasm around either the chromosomes or the spindle. We used SB203580, an inhibitor of p38α MAPK, to investigate the possible functional role of p38α MAPK during rat oocyte meiotic maturation. Treatment of GV stage oocytes with 20 μM SB203580 blocked p‐p38α MAPK activity, and the spindles appeared abnormal. Additionally, the rate of GVBD after 3 hr of culture with 20 μM SB203580 (58.8%) was significantly inhibited compared with the control (82.5%, p < .05), and the polar body extrusion rate after 12 hr of culture with SB203580 was also significantly decreased compared with the control (40.1% vs 73.3%, p < .05). Taken together, these data indicate that p38α MAPK may play a vital role in rat oocyte meiotic maturation.     

Effect of vitrification at different meiotic stages on epigenetic characteristics of bovine oocytes and subsequently developing embryos

Vitrification by the Cryotop method is frequently used for bovine oocyte cryopreservation. Nevertheless, vitrified oocytes still have reduced developmental competency compared with fresh counterparts. The objective of this study was to compare the effect of vitrification either at the germinal vesicle (GV) stage or at the metaphase II (MII) stage on epigenetic characteristics of bovine oocytes and subsequently developing embryos. Our results demonstrated that vitrification of oocytes at each meiotic stage significantly reduced blastocyst development after in vitro fertilization (IVF). However, vitrification at the GV stage resulted in higher blastocyst development than did vitrification at the MII stage. Irrespective of the meiotic stage, oocyte vitrification did not affect 5-methylcytosine (5mC) immunostaining intensity in oocyte DNA. However, at both stages, it caused a similar reduction of 5mC levels in DNA of subsequently developing blastocysts. Oocyte vitrification had no effect on the intensity of H3K9me3 and acH3K9 immunostaining in oocytes and subsequent blastocysts. The results suggest that irrespective of meiotic stage, oocyte vitrification alters global methylation in resultant embryos although such alteration in the oocytes was not detected. Oocyte vitrification might not influence histone acetylation and methylation in oocytes and resultant embryos. Vitrification at the immature stage was more advantageous for blastocyst development than at the mature stage.     

Chromatin Configurations in the Ferret Germinal Vesicle that Reflect Developmental Competence for In Vitro Maturation

In several mammalian species, the configuration of germinal vesicle (GV) chromatin correlates with the developmental competence of oocytes. Yet, no study has been published on the configuration of GV chromatin in ferret, nor is it known whether a specific configuration predicts meiotic competence in this species, in spite of the potential importance of ferret cloning to the study of human disease and to species conservation efforts. Here, we report on an analysis of the chromatin configuration in ferret GV oocytes and on how they correlate with meiotic development. Three distinct configurations were identified based on the degree of chromatin condensation: (1) fibrillar chromatin (FC), featuring strands of intertwined chromatin occupying most of the visible GV region; (2) intermediate condensed chromatin (ICC), characterized by dense, irregular chromatin masses throughout the GV; and (3) condensed chromatin (CC), which is highly compact and centered around the nucleolus. We also found that chromatin configuration was related to the extent of association with cumulus cells in cumulus–oocyte complexes; CC-configured oocytes were most often surrounded by a compact cumulus layer and also a compact corona but FC-configured oocytes were associated with neither. In addition, increasing chromatin condensation corresponded to an increase in oocyte diameter. Finally, following in vitro culture, significantly more CC-configured oocytes underwent maturation to meiotic metaphase II than did FC- or ICC-configured oocytes. We conclude that, in ferret, chromatin condensation is related to the sequential achievement of meiotic competencies during oocyte growth and differentiation, and thus can be used as a predictor of competence.     

Cryopreservation of Immature Bovine Oocytes to Reconstruct Artificial Gametes by Germinal Vesicle Transplantation

Joining immature gamete cryopreservation and germinal vesicle transplantation (GVT) technique could greatly improve assisted reproductive technologies in animal breeding and human medicine. The present work was aimed to assess the most suitable cryopreservation protocol between slow freezing and vitrification for immature denuded bovine oocytes, able to preserve both nuclear and cytoplasmic competence after thawing. In addition, the outcome of germinal vesicle transfer procedure and gamete reconstruction was tested on the most effective cryopreservation system. Oocytes, isolated from slaughterhouse ovaries, were stored after cumulus cells removal either by slow freezing or by vitrification in open pulled straws. After thawing, oocytes were matured for 24 h in co-culture with an equal number of just isolated intact cumulus enclosed oocytes, and fixed in order to evaluate the stage of meiotic progression and cytoskeleton organization. Our results showed that after warming, vitrified oocytes reached metaphase II (MII) in a percentage significantly higher than oocytes cryopreserved by slow freezing (76.2% and 36.5% respectively, p < 0.05). Moreover, vitrification process preserved the organization of cytoskeleton elements in a higher proportion of oocytes than slow freezing procedure. Therefore vitrification has been identified as the elective method for denuded immature oocytes banking and it has been applied in the second part of the study. Our results showed that 38.3% of oocytes reconstructed from vitrified gametes reached the MII of meiotic division, with efficiency not different from oocytes reconstructed with fresh gametes. We conclude that vitrification represents a suitable method of GV stage denuded oocyte banking since both nuclear and cytoplasmic components derived from cryopreserved immature oocytes can be utilized for GVT.     

Changes of HCO3-/Cl- exchanger activity during meiotic maturation in Balb/c strain mouse oocytes and zygotes

Intracellular pH-regulatory mechanisms are acquired by growing mouse oocytes with meiotic competence, and these mechanisms become fully active when the oocytes develop to the germinal vesicle (GV) stage as shown in CF1 and Balb/c strains mice. On the other hand, there is some evidence showing that intracellular pH-regulatory mechanisms are inhibited at the stages of Metaphase I (MI) and II (MII) oocytes in the CF1 strain mouse and hamster. Since it has been shown that the intracellular pH regulatory mechanism can be functionally different among mouse strains (e.g., CF1, Balb/c), the aim of this study was to investigate the activity of HCO3-/Cl- exchanger (anion exchanger, AE), which protects cells against alkalosis during the meiotic maturation process, in the GV oocyte up to the pronuclear (PN) zygote derived from the Balb/c strain mouse. Intracellular pH (pHi) was recorded using a microspectrofluorometric technique during meiotic maturation stages. KSOM-based solutions were used as culture and recording solutions. AE activity was determined using a Cl- removal assay and was reported as the change in pHi per minute. AE activity was high in GV stage oocytes but was significantly inhibited at the MI and MII stages. AE activity was higher in the PN zygote stage. This activity was significantly inhibited in all oocyte and zygote stages by 4,4'-Diisocyanatostilbene-2,2'-disulfonic acid disodium salt. After alkalosis induction, the pHi of MI and MII stage oocytes did not completely recover; however, almost complete recovery occurred in the GV stage oocytes and PN zygotes. These results suggest that AE is inhibited during the meiotic maturation process in the Balb/c strain mouse.     

Vitrification of fully grown and growing porcine oocytes using germinal vesicle transfer

The survival rate of vitrified germinal vesicle (GV) stage porcine oocytes is very low, and it is not known if the vitrification damages the nucleus, cytoplasm or both. We have evaluated the eventual GV or cytoplasmic damage in fully grown (FG) and growing vitrified oocytes. Fifty-five percent of nonvitrified FG cumulus-denuded oocytes reached the metaphase II (MII) stage in culture. When growing oocytes from preantral (PA) and early antral (EA) follicles were matured in vitro, almost all oocytes were arrested at the GV stage (GV stage: PA 88.9 and EA 79.5%, respectively). When fresh GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes and matured in vitro, some of them reached the MII stage (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). The maturation rate of vitrified FG oocytes was only 6.1% but increased dramatically when vitrified GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes (MII stage: VitFG/FG 43.9%, VitPA/FG 7.1% and VitEA/FG 26.3%, respectively). These results were not significantly different from those for the nonvitrified groups (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). We activated the reconstructed oocytes that received fresh or vitrified GVs (FG/FG, EA/FG, VitFG/FG and VitEA/FG) and examined their embryonic development. Cleaved embryos (nonvitrified groups 13.0-61.8%, vitrified groups 33.3-40.0%) and blastocysts (nonvitrified groups 0.0-18.2%, vitrified groups 0.0-2.9%) were obtained after activation. These results demonstrate that vitrified porcine GVs maintain maturational and developmental competence and that vitrification predominantly damages the cytoplasm.     

Mitochondrial Patterns in Bovine Oocytes with Different Meiotic Competence Related to Their in vitro Maturation

This study was designed to specify chromatin and mitochondrial patterns in bovine oocytes with different meiotic competence in relation to maturation progress, resumption of meiosis, MII onset and completion of maturation. Oocytes with greater or lesser meiotic competence, recovered separately from medium (MF) and small follicles (SF), were categorized according to morphology. Four oocyte categories, healthy and light‐atretic MF and healthy and light‐atretic SF oocytes were matured and collected at 0, 3, 7, 16 and 24 h of maturation. Specific differences in terms of chromatin and mitochondrial patterns were found among the maturing oocyte categories. Resumption of meiosis was accelerated in light‐atretic oocytes, as compared with healthy oocytes, regardless of their meiotic competence. More competent oocytes activated mitochondria twice during maturation, before resumption of meiosis and before completion of maturation, while less competent oocytes did it only once, before completion of maturation. Changes in mitochondrial activity differed in light‐atretic compared with healthy in both more and less competent oocytes. Healthy meiotically more competent oocytes formed clusters and produced ATP for the whole time of maturation until its completion, while light‐atretic more competent oocytes and healthy less competent oocytes reduced these activities earlier, at MII onset. Contrary to these oocyte categories, light‐atretic less competent oocytes increased cluster formation significantly before resumption of meiosis. It can be concluded that bovine oocytes with different meiotic competence and health differed in the kinetics of mitochondrial patterns during maturation.     

Localization of gamma-tubulin in mouse eggs during meiotic maturation, fertilization, and early embryonic development

Gamma-tubulin, a member of the tubulin superfamily, is a peri-centriolar component which is considered to be essential for microtubule nucleation. The dynamics of gamma-tubulin during mouse oocyte meiotic maturation, fertilization, and early cleavage as well as the co-localization of gamma-tubulin and alpha-tubulin during the formation of the meiotic I spindle were studied by confocal microscopy. We found that gamma-tubulin was evenly distributed in the germinal vesicle (GV) stage oocyte. After germinal vesicle breakdown (GVBD) gamma-tubulin dots were localized in both the cytoplasm and the vicinity of the condensed chromosomes, and aligned at both poles of the meiotic spindle at prometaphase I and metaphase I. At anaphase I and telophase I, gamma-tubulin was detected between the separating chromosomes, while it was absent in the midbody. At the MII stage, gamma-tubulin was again accumulated at the spindle poles. Alpha-tubulin had a similar distribution pattern as gamma-tubulin in the cytoplasm and radiated from gamma-tubulin foci close to the chromosomes during the meiotic spindle formation. After fertilization, gamma-tubulin was translocated from spindle poles to the area between separating chromatids and distributed around the pronuclei. It aggregated into some dots during the interphase, but was distributed on the mitotic spindle poles in early embryos. Our results suggest that gamma-tubulin is essential for microtubule nucleation and spindle formation during mouse oocyte meiosis, fertilization, and early embryo cleavage.     

Exposure to L-Ascorbic Acid or α-Tocopherol Facilitates the Development of Porcine Denuded Oocytes from Metaphase I to Metaphase II and Prevents Cumulus Cells from Fragmentation

It is known that alpha-tocopherol (vitamin E) and L-ascorbic acid (vitamin C) can modulate many biochemical processes intracellularly or extracellularly as antioxidants. The objective of the present study was to investigate the effects of alpha-tocopherol and L-ascorbic acid on porcine oocyte meiotic maturation, viability and the functions of cumulus cells. In two independent experiments, porcine oocytes with or free from cumulus cells were exposed to different levels of alpha-tocopherol (0, 10, 100 and 200 microM) or L-ascorbic acid (0, 50, 250 and 750 microM). Cumulus expansion, cumulus cell DNA fragmentation, meiotic maturation and degeneration of oocytes were assessed 48 h after in vitro culture. The results showed that: (1) neither alpha-tocopherol nor L-ascorbic acid influenced cumulus expansion but both prevented cumulus cell DNA fragmentation. (2) Alpha-tocopherol lowered the percentage of denuded oocytes (DOs) arrested at germinal vesicle stage (GV). Among the oocytes undergoing germinal vesicle breakdown (GVBD) proportion, fewer DOs treated by alpha-tocopherol were at metaphase I (MI) and more at metaphase II (MII). L-ascorbic acid caused lower percentage of DOs arrested at GV stage and higher percentage of DOs undergoing GVBD, especially at MII. The influences of alpha-tocopherol and L-ascorbic acid were not obvious in cumulus-enclosed oocytes (CEOs). (3) Both vitamins compromised the viability of CEOs and DOs. These results indicate that exposure to alpha-tocopherol or L-ascorbic acid promotes the development of porcine DOs from MI to MII and prevents cumulus cell DNA fragmentation at certain levels, especially 10 microM alpha-tocopherol or 250 microM L-ascorbic acid.     

Intermediate Filament Keratin Dynamics During Oocyte Maturation Requires Maturation/M‐Phase Promoting Factor and Mitogen‐Activated Protein Kinase Kinase Activities in the Hamster

Research related to intermediate filaments in mammalian oocytes remains poorly advanced. We investigated keratin reorganization in oocytes during meiotic maturation using immunofluorescence, and examined effects of inhibitors for cdc2 and mitogen‐activated protein kinase kinase (MAPKK) on keratin assembly. In germinal vesicle (GV) oocytes (n = 26), large and oval‐shaped aggregates of non‐fibrillar keratin were found in the cortical ooplasm (designated as a ‘cortical’ pattern). The delicate network of keratin filaments was concentrated in the GV periphery. The large keratin aggregates began to divide into small fragments at the pro‐MI/MI stage (n = 22, designated as a ‘fragmented’ pattern). Some keratin fragments were occasionally broken down into several granules at the peripheral region. In the MII oocytes (n = 24), the filament network was extended over the ooplasm and numerous keratin granules were scattered across the oocyte (designated as a ‘granular’ pattern). After 12 h of incubation with roscovitine, 66.7% of the oocytes (20/30) were at the GV stage and showed a cortical pattern of keratin. After incubation with U0126, most oocytes (83.9%, 26/31) were at the MII stage; most of them (76.9%, 20/26) showed a fragmented pattern of keratin. The increasing complexity of keratin filament network from the GV to MII stages suggests a possible role in maintaining cell integrity under physical stress after ovulation. In fact, maturation/M‐phase promoting factor is necessary for such keratin reorganization, as is meiotic nuclear progression. In addition, MAPKK is involved in keratin reorganization from a fragmented pattern to a granular pattern.     

Dynamic changes in histone acetylation during sheep oocyte maturation

The changes in histone acetylation are not always consistent in various cell types and at different developmental stages. We immunostained specific antibodies against acetylated lysine 9 of histone H3 and acetylated lysines 5 and 12 of histone H4 in an effort to understand the detailed changes in histone acetylation during sheep oocyte meiosis. We found that the acetylation fluorescence signals of H3/K9 and H4/K12 on chromatin appeared intensively in the germinal vesicle (GV), late-GV (L-GV), and germinal vesicle breakdown (GVBD) stages and became weak in metaphase I (MI); however staining reappeared in anaphase I-telophase-I (AI-TI) and metaphase II (MII). Furthermore, staining was detected in the first polar bodies. The fluorescence signals of H4/K5 first appeared in the MI stage and became intensive in the AI-TI stage; however they were barely detectable in MII stage chromosomes and first polar bodies. We conclude that the acetylation patterns of H3/K9 and H4/K12 during oocyte meiotic maturation are similar and that the pattern of H4/K5 is unique.     

Ultrastructure of immature and mature human oocytes after cryotop vitrification

In vitro maturation of vitrified immature germinal vesicle (GV) oocytes is a promising fertility preservation option. We analyzed the ultrastructure of human GV oocytes after Cryotop vitrification (GVv) and compared it with fresh GV (GVc), fresh mature metaphase II (MIIc) and Cryotop-vitrified mature (MIIv) oocytes. By phase contrast microscopy and light microscopy, the oolemmal and cytoplasmic organization of fresh and vitrified oocytes did not show significant changes. GVv oocytes showed significant ultrastructural alterations of the microvilli in 40% of the samples; small vacuoles and occasional large/isolated vacuoles were abnormally present in the ooplasm periphery of 50% of samples. The ultrastructure of nuclei and mitochondria-vesicle (MV) complexes, as well as the distribution and characteristics of cortical granules (CGs), were comparable with those of GVc oocytes. MIIv oocytes showed an abnormal ultrastructure of microvilli in 30% of the samples and isolated large vacuoles in 70% of the samples. MV complexes were normal, but mitochondria-smooth endoplasmic reticulum aggregates appeared to be of reduced size. CGs were normally located under the oolemma but presented abnormalities in distribution and matrix electron density. In conclusion, Cryotop vitrification preserved main oocyte characteristics in the GV and MII stages, even if peculiar ultrastructural alterations appeared in both stages. This study also showed that the GV stage appears more suitable for vitrification than the MII stage, as indicated by the good ultrastructural preservation of important structures that are present only in immature oocytes, like the nucleus and migrating CGs.     

Maturation Status, Protein Synthesis and Developmental Competence of Oocytes Derived from Lambs and Ewes

The efficacy of oocyte selection for in vitro embryo production depends on the abundance and diameter of follicles, cumulus layers around the oocytes and subsequent fertilization. Application of `ovum pick-up' technique allows us to utilize partially matured oocytes for embryo production even from juvenile subjects. To compare their developmental competence, oocytes derived from lambs and ewes and cultured in maturation medium for up to 26 h were assessed at 2 h intervals by confocal microscopy after chromatin and microtubulin-specific fluorochrome labelling. Lamb oocytes reached second meiotic metaphase (MII) at lower numbers at 24 h (60.0%) and 26 h (28.6%) whereas 85.7% of adult-derived oocytes attained MII status by 24 h of maturation. Radiolabelling of oocyte proteins revealed higher incorporation of [³⁵S-]-methionine and [³⁵S]-cysteine in adult-derived oocytes compared to lamb oocytes. Although the cleavage rate of lamb oocytes was similar to that of ewe oocytes, the proportion reaching blastocyst stage was significantly lower (p < 0.05) in the lamb-derived oocytes. However, blastocysts from both types of oocytes displayed similar cell lineage allocations to inner cell mass and trophectoderm.     

Distribution of protein disulfide isomerase during maturation of pig oocytes

Oocyte maturation in mammals is characterized by a dramatic reorganization of the endoplasmic reticulum (ER). In mice, the ER forms accumulations in the germinal vesicle (GV) stage and distinctive cortical clusters in metaphase II (MII) of the oocyte. Multiple evidence suggests that this ER distribution is important in preparing the oocyte for Ca²⁺ oscillations, which trigger oocyte activation at fertilization. In this study, we investigated the time course and illustrated the possible functional role of ER distribution during maturation of porcine oocytes by immunostaining with protein disulfide isomerase (PDI). PDI forms clusters in the cytoplasm of oocytes. After immunostaining, PDI clusters were identified throughout the cytoplasm from the GV to metaphase I (MI) stage; however, at the MII stage, the PDI formed large clusters (1–2 µm) in the animal pole around the first polar body. PDI distribution was prevented by bacitracin, a PDI inhibitor. Our experiments indicated that, during porcine oocyte maturation, PDI undergoes a dramatic reorganization. This characteristic distribution is different from that in the mouse oocyte. Moreover, our study suggested that formation of PDI clusters in the animal pole is a specific characteristic of matured porcine oocytes.     

Phosphorylation of histone H3 on Ser10 by auto-phosphorylated PAK1 is not essential for chromatin condensation and meiotic progression in porcine oocytes

Background

The p21-activated kinase 1 (PAK1) is essential for mitosis and plays an important role in the regulation of microtubule assembly during oocyte meiotic maturation in mice; however, little is known about its role in porcine oocytes.

Result

Total p21-activated kinase 1 (PAK1) and phosphorylated PAK1 at Thr423 (PAK1^Thr423) were consistently expressed in porcine oocytes from the germinal vesicle (GV) to the second metaphase (MII) stages, but phosphorylation of histone H3 at Ser10 (H3^Ser10) was only expressed after the GV stage. Immunofluorescence analysis revealed that PAK1^Thr423 and H3^Ser10 colocalized on chromosomes after the GV stage. Blocking of endogenous PAK1^Thr423 by injecting a specific antibody decreased the phosphorylation level of H3^Ser10; however, it had no impact on chromatin condensation, meiotic progression, cleavage rate of blastomeres or the rate of blastocyst formation.

Conclusion

Phosphorylation of PAK1^Thr423 is a spontaneous activation process and the activated PAK1^Thr423 can promote the phosphorylation of H3^Ser10; however, this pathway is not required for meiotic maturation of porcine oocytes or early embryonic development.