Progression Of Nuclear Maturation Research Articles

Modulation of phosphatidylinositol 3-kinase activity during in vitro oocyte maturation increases the production of bovine blastocysts.

This study aimed to evaluate the effect of regulating phosphatidylinositol 3-kinase (PI3K) activity on the kinetics of oocyte nuclear maturation and the blastocyst rate. To evaluate oocyte viability, nuclear maturation rate and in vitro embryo production, cumulus-oocyte complexes (COCs) were maintained for 0, 10 min, 6 h or 22 h in TCM 199 medium supplemented with 20 nM wortmannin, an inhibitor of PI3K. After each period, COCs were transferred to the same medium without wortmannin and kept under the same conditions until completion of 22 h of in vitro maturation (IVM). To evaluate the effect of time on progression of nuclear maturation, COCs cultivated with 20 nM wortmannin was maintained for 22, 28 or 34 h of IVM. To determine the effect of wortmannin on the activity of maturation-promoting factor (MPF), COCs were kept under IVM conditions in the presence of the inhibitor for 0, 1, 3, 6, or 8 h. Exposure of COCs to wortmannin decreased (P < 0.05) the percentage of oocytes that reached metaphase II (MII) up to 22 h, MPF activity and reduced PI3K activity by 30%. However, after 28 and 34 h, 70% of oocytes reached the MII stage in the presence of inhibitor Moreover, COCs matured in the presence of wortmannin showed an increase (P < 0.05) in the blastocyst rate. These findings suggested that the regulation of the PI3K activity during IVM of bovine COCs interfered with the meiotic progression due to control of MPF activity, positively affecting the blastocyst rate.

Effect of Gaseous Atmosphere on Different Supplementation Medium for In Vitro Canine Oocyte Maturation

The aim of the present research was to verify the influence of two different atmosphere gases (A: 5%CO 2 , B: 5%CO 2 , 5%O 2 and 90%N 2 ) on three different maturation media on in vitro maturation (IVM) rates for oocytes from anoestrous and diestrous bitches that were cultured in vitro for 48h. Oocytes harvested from bitches were selected and allocated into three maturation media groups, cultured in atmosphere A or B. Group 1, oestrous bitch serum; Group 2, bovine serum albumin (BSA), glutamine, cisteamine; Group 3, BSA, estrogen (E 2 ), epidermal growth factor (EGF). Progression of nuclear maturation was evaluated under fluorescent microscopy after staining with Hoechst 33342 solution. The results demonstrated that different maturation media was not influenced by the atmosphere in any case. Group 1 showed the lowest maturation rates in both cases: 4.3±1.3 vs 7.3±2.1 and 7.9±2.5 for groups 1, 2 and 3 respectively in atmosphere A (p≤ 0.5), 0 vs 7.2± 2.9 and 9.3± 4.0 in atmosphere B (p= 0.33). Maturation rates were similar among treatments 2 and 3 in both atmospheres, were slightly higher for treatment 3. Moreover, group 1 obtained the highest degeneration rates: 65.5±7.3 vs 39.63±7.1, 28.19±6.4 (p= 0.018) and 71.43±12.2 vs 27.71±10, 25.33± 10.20 (p= 0.010) for mediums 1, 2, and 3 in atmospheres A and B respectively. Our findings indicate that atmosphere B seems to be more favorable habitat for canine oocytes and independently of gassed, maturation with not controlled supplementation, with estrous bitch serum, is not a good option.

Maternal Liver Damage Delays Meiotic Resumption in Bovine Oocytes through Impairment of Signalling Cascades Originated from Low p38MAPK Activity in Cumulus Cells

The main objective of the present study is to investigate the molecular mechanism underlying the delay in progression of nuclear maturation in oocytes derived from cows with damaged livers (DL cows), which was previously reported. In present study, delayed progression of nuclear maturation of oocytes derived from DL cows relative to oocytes derived from cows with healthy livers (HL cows) was accompanied by low maturation promoting factor (MPF) activity (0.43 fold, p<0.05). When cumulus cells were removed from cumulus-oocyte complexes and the denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two liver conditions. In addition, gap junctional communication (GJC) between the oocyte and cumulus cells was higher in DL cows than in HL cows at 3 and 7h of in vitro maturation (IVM) (p<0.05). Supplementation of IVM medium with epidermal growth factor (EGF) increased the ratio of germinal vesicle breakdown (GVBD) of oocytes derived from DL cows to the level seen in oocytes derived from HL cows. Additionally, the level of p38MAPK phosphorylation at 0h of IVM was significantly lower in cumulus cells derived from DL cows than in cumulus cells derived from HL cows (HL cows, 53.5%; DL cows, 28.9%; p<0.05). Thus, a low level of p38MAPK phosphorylation in cumulus cells induced slow GJC closure between oocyte and cumulus cells, which resulted in slow meiotic maturation of oocytes derived from DL cows.

Effect of maternal age on the developmental competence and progression of nuclear maturation in bovine oocytes

Progression of meiotic division in oocytes and early embryonic development are affected by oocytes quality. In most mammals, oocyte quality declines with increase in maternal age. The main aim of the present study is to investigate the effect of maternal age on developmental competence, progression of meiotic division, and associated kinetics of maturation promoting factor (MPF) activity in bovine oocytes. Oocytes were collected from the ovaries of young and old cows (here after referred to as young cow oocytes and old cow oocytes, respectively). When old cow oocytes were matured and fertilized in vitro, the rate of abnormal fertilization was greater than that in young cow oocytes. Moreover, progression of nuclear maturation and activation of MPF during oocyte maturation (or inactivation of MPF and formation of pronucleus after insemination) were faster in old cow oocytes than in young cow oocytes. Relative expression of cyclin B, cyclin-dependent kinase 1 and MAD2 transcripts in either immature or mature oocytes did not differ between the two groups. When cumulus cells (CC) were removed and denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two age groups. Moreover gap junctions between oocytes and CC disappeared more rapidly during maturation of old cow oocytes than of young cow oocytes. These results suggest that the fertilization ability of old cow oocytes is low and that premature progression of meiotic division in these oocytes is partly due to impaired oocyte-CC gap junctions communication.

Effect of follicular fluid collected from various diameter follicles on the progression of nuclear maturation and developmental competence of pig oocytes

Supplementing in vitro maturation medium with porcine follicular fluid (FF) improves maturation rate, male pronucleus formation, and monospermic fertilization of pig oocytes. This study examined, (1) if there are differences in FF derived from large follicles (LF, 5–6 mm in diameter) and small follicles (SF, 3–4 mm in diameter) on the effect of supplementing the maturation medium with FF on the progression of nuclear maturation, fertilization rate, and developmental competence of porcine oocytes; (2) whether the FF source influences the effect of the FF on the maturation medium on the survival rate and proliferation rate of cumulus cells (CCs) and the expansion of cumulus-oocyte-complexes (COCs); (3) whether the oocyte source (oocytes collected from LFs or SFs) influences the effect of FF on the progression of the nuclear maturation of oocytes; (4) whether the factors in the FF that affect the kinetics of nuclear maturation are proteins, and the range of the molecular weight of the FF factors. In experiment 1, adding FF from LFs (LFF) significantly accelerated nuclear maturation and improved the fertilization rate; the developmental ratio was comparable with those of adding FF from SFs (SFF). In experiment 2, adding LFF, but not SFF, improved the CC survival rate, although the FF source did not affect the proliferation rate. Expansion of COCs was greater with SFF than LFF. In experiment 3, LFF promoted nuclear maturation of oocytes collected from only LFs. There was a significant interaction between the FF source and the oocyte source in the effect on nuclear maturation stages at 36 h of maturation. In experiment 4, treatment of FF with heat or trypsin diminished the difference between the effect of LFF and SFF on the progression of nuclear maturation. In addition, the predominant effect of LFF compared to that of SFF on nuclear maturation was not affected by ultrafiltration of the FF with a 30-kDa filter, but was diminished by ultrafiltration with a 100-kDa filter. The present study suggests that some proteins present in LFF that range in molecular weight from 30 to 100 kDa improve the developmental competence of oocytes probably via progression of nuclear maturation and cumulus cells viability.

344 FOLLICULAR FLUIDS COLLECTED FROM LARGE FOLLICLES IMPROVED THE PROGRESSION OF IN VITRO NUCLEAR MATURATION OF PIG OOCYTES

The present study examined the effects of supplementation of in vitro maturation medium with porcine follicular fluids (pFFs) on the progression of nuclear maturation, subsequent fertilization, and development of pig oocytes. The pFFs were collected from follicles of different diameters: small follicles (SFs, 3–4 mm in diameter) and large follicles (LFs, 5-6 mm). In experiment 1, oocytes were collected from follicles 3–6 mm in diameter. The oocytes were cultured in maturation medium (NCSU-37) supplemented (10% v/v) with pFF from SFs (SpFF) or LFs (LpFF); the kinetics of nuclear maturation, fertilization rate, and developmental competence of oocytes were evaluated. In experiment 2, oocytes were collected from SFs or LFs, and the effects of supplementation of SpFF and LpFF on the nuclear maturation of these oocytes were examined. In experiment 3, each pFF was denaturated by heat treatment (60�C for 30 min) or trypsin treatment (200 �g mL-1), and the effects of these pFFs on the nuclear maturation of pig oocytes were examined. In experiment 4, pFFs were ultrafiltered, and separated into fractions greater and less than 30 kDa; the effects of the separated pFFs on nuclear maturation were examined. We used Student&amp;apos;s t-test to examine the differences among the results; a P value less than 0.05 was considered to be significant. In experiment 1, the progression of nuclear maturation was significantly faster when the maturation medium was supplemented with LpFF than with SpFF (MI rates at 36 h after culture: LpFF, 20.0%, and SpFF, 35.0%; MII rates at 36 h after culture: LpFF, 33.3%, and SpFF, 20.0%). The rates of polyspermic fertilization were significantly lower in the maturation medium with LpFF than in that with SpFF (LpFF, 31.7%; SpFF, 66.7%). Moreover, the rates of monospermic fertilization (LpFF, 45.0%; SpFF, 21.7%) and blastocyst formation (LpFF, 15.2%; SpFF, 3.0%) were significantly greater in the medium supplemented with LpFF than with SpFF. In experiment 2, we observed that LpFF accelerated the progression of nuclear maturation of pig oocytes collected from LFs (MII rates at 36 h after culture: LpFF, 61.7%; SpFF, 35.5%), but not those collected from SFs (MII rates at 36 h after culture: LFs, 15.0%; SFs, 11.7%). In experiment 3, the accelerating effect of pFF on nuclear maturation was eliminated by heat (MII rates at 36 h after culture: LpFF, 30.0%; SpFF, 25.0%) or trypsin treatment (MII rates at 36 h after culture: LpFF, 20.0%; SpFF, 21.7%). In experiment 4, only LpFF with a molecular weight greater than 30 kDa had positive effects on the progression of nuclear maturation (MII rates at 36 h after culture with LpFF: &amp;gt;30 kDa, 30.0%; &amp;lt;30 kDa, 11.7%). The present study suggests that LpFF contains some proteins that improve the progression of nuclear maturation and the developmental competence of oocytes.

Progression of Nuclear Maturation and p34cdc2 Kinase Activity in Porcine Oocytes during In Vitro Culture in Different Media.

In the present study, to elucidate the cause of different developmental potential in porcine oocytes matured in two media, mTCM199 and mNCSU37, we examined the time course of changes in the nuclear states and p34cdc2 kinase activity of oocytes during meiotic maturation. The p34cdc2 kinase activity of oocytes cultured in mTCM199 gradually increased from earlier in the culture time periods than that of oocytes in mNCSU37, and that of oocytes in mNCSU37 rapidly increased from the middle of the culture period, but the oocytes in both media had similar levels of p34cdc2 kinase activity at the end of the culture period. Oocytes cultured in mTCM199 underwent GVBD and reached the MII stage earlier in the culture period than those in mNCSU37. Moreover, MII oocytes cultured in either mTCM199 or mNCSU37 had a similar level of p34cdc2 kinase activity at the end of the culture period. It was therefore concluded that the difference in developmental competence of oocytes matured in different media seems to be independent of p34cdc2 kinase activity.

Behavior of hamster sperm nuclei incorporated into eggs at various stages of maturation, fertilization, and early development. The appearance and disappearance of factors involved in sperm chromatin decondensation in egg cytoplasm

Golden hamster eggs at various stages of maturation, fertilization, and early development were freed from the zona pellucida and inseminated in vitro with capacitated, acrosome-reacted spermatozoa. Electron microscopic observations showed that the spermatozoa were capable of fusing with eggs in all stages from the germinal vesicle stage through the four-cell stage. The ability of the eggs to fuse with the spermatozoa was high throughout all stages of maturation, decreased slightly following fertilization, and was markedly reduced after the first cleavage division. Eggs beyond the four-cell stage were incapable of fusing with spermatozoa. The ability of the egg cytoplasm to decondense sperm chromatin first appeared about the time of the breakdown of the germinal vesicle, increased with the progression of nuclear maturation, reached a maximum at the time of ovulation, diminished following fertilization, reappeared during the first cleavage division, and disappeared again after the completion of the division. It appears that the factors of conditions which cause the decondensation of sperm chromatin appear (or become active) only during a certain period of the cell cycle. The possible chemical nature and origin of the factors responsible for the decondensation of sperm chromatin within the egg cytoplasm were discussed.