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.     

Influence of co‐culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus‐enclosed porcine oocytes in a defined system

Co‐culture of cumulus‐oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte‐secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β‐mercaptoethanol. Cumulus‐oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co‐culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus‐enclosed porcine oocytes in a defined system.     

Effect of estradiol‐17β during in vitro growth culture on the growth,maturation, cumulus expansion and development of porcine oocytes from early antral follicles

Growing porcine oocytes from early antral follicles can acquire meiotic and developmental competence under suitable culture conditions, but at lower rates compared to full‐grown oocytes. We postulated that estradiol‐17β (E₂) supported the acquisition of meiotic and developmental competence as well as cumulus‐expansion ability during growth culture. Growing oocytes from early antral follicles (1.2 to 1.5 mm in diameter) were grown in vitro for 5 days in a medium containing 0, 10^?7, 10^?6, 10^?5 or 10^?4 mol/L E₂; after in vitro maturation, 35, 58, 47, 74 and 49% of oocytes matured to metaphase II, 25, 79, 77, 90 and 97% acquired cumulus‐expansion ability, and 23, 54, 63, 89 and 64% were fully surrounded by cumulus cells, respectively. Following maturation, electro‐stimulation was applied to the oocytes grown with 10^?5 mol/L E₂. After 6 days of culture, in vitro‐grown oocytes developed to the blastocyst stage at a rate similar to that for full‐grown oocytes (31% and 40%, respectively). Therefore, we suggest that the use of E₂ during growth culture improves the meiotic and developmental competence of oocytes, cumulus‐expansion ability, and cumulus cell attachment to the oocytes.     

The effect of M‐phase stage‐dependent kinase inhibitors on inositol 1,4,5‐trisphosphate receptor 1 (IP3R1) expression and localization in pig oocytes

At fertilization, inositol 1,4,5‐trisphosphate receptor type 1 (IP₃R1) has a crucial role in Ca²⁺ release in mammals. Expression levels, localization and phosphorylation of IP₃R1 are important for its function, but it still remains unclear which molecule(s) regulates IP₃R1 behavior in pig oocytes. We examined whether there was a difference in localization of IP₃R1 after in vitro or in vivo maturation of pig oocytes. In mouse oocytes, large clusters of IP₃R1 were formed in the cortex of the oocyte except in a ring‐shaped band of cortex adjacent to the spindle. However, no such clusters of IP₃R1 were observed in pig oocytes and there was no difference in its localization between in vitro and in vivo matured oocytes. We next tried to clarify which factor(s) regulates IP₃R1 localization, phosphorylation and expression using M‐phase stage‐dependent kinase inhibitors. Our results show that treatments with roscovitine (p34^cdc2 kinase inhibitor) or U0126 (mitogen‐activated protein kinase inhibitor) did not affect IP₃R1 expression or localization in pig oocytes, although the latter strongly inhibited phosphorylation. However, treatment with BI‐2536, an inhibitor of polo‐like kinase 1 (Plk1), dramatically decreased the expression level of IP₃R1 in pig oocytes in a dose‐dependent manner. From these results, it is suggested that Plk1 is involved in the regulation of IP₃R1 expression in pig oocytes.     

The radiosensitizing effect of the aurora kinase inhibitors,ENMD‐2076, on canine mast cell tumours in vitro

ENMD‐2076 is an aurora kinase inhibitor that also has multi‐target tyrosine kinase inhibitor properties. In this study, the mRNA and the protein expression of aurora‐A and aurora‐B were evaluated in three canine mast cell tumour cell lines. Dose‐dependent cytotoxicity was seen in the cells treated, and it affected the cell cycle with cells in the G2/M phase being selectively killed. The cells were also evaluated for radiosensitivity with/without ENMD‐2076, and radiosensitization was seen after 3 Gy and 6 Gy exposures with ENMD‐2076 for 48 h. Protein expression of caspase‐3 was gradually increased, and the expression intensity was highest at 24 h post irradiation in cells without ENMD‐2076 treatment, which indicates that radiation exposure with ENMD‐2076‐induced cell death faster than radiation treatment alone. Our study results suggest the potential usefulness of treating canine mast cell tumours with aurora kinase inhibitors alone or in conjunction with radiation therapy.     

Interactions between different media and follicle‐stimulating hormone supplementation on in vitro culture of preantral follicles enclosed in ovarian tissue derived from collared peccaries (Pecari tajacu Linneaus, 1758)

The aim was to verify the effect of follicle‐stimulating hormone (FSH) supplementation to α‐MEM+ or TCM199+ media on the in vitro development of ovarian preantral follicles (PFs) derived from collared peccaries. Ovaries (n = 5 pairs) were collected and divided into fragments destined to control group (non‐cultured) or treatments that were cultured for 7 days. The PFs morphology, growth and activation were evaluated by classical histology. The immunohistochemistry markers Ag‐NOR and PCNA were used for nuclear proliferation analysis, and the picrosirius red labelling was used for ovarian extracellular matrix (ECM) evaluation. After 7‐day culture, only the TCM199+ treatment maintained the proportion of intact PFs similar to day 1 (63.2%), but no differences were found among treatments (p > .05). In addition, a significant increase in the growing follicles proportion was verified for all the treatments, indicating follicular activation (p > .05). By the Ag‐NOR analysis, only the TCM199+/FSH maintained the nuclear proliferation similar to the first day (p > .05). The picrosirius red staining revealed that the ECM remained intact in all the treatments (p > .05). We suggest the use of TCM199+ medium supplemented of FSH for the in vitro development of peccaries PFs under 7‐day culturing conditions.