Nuclear Maturation Of Porcine Oocytes Research Articles

Epigallocatechin gallate improves meiosis maturation against Diazinon exposure in porcine oocytes

Diazinon (DZN) is a refractory organophosphorus pesticide (OP) in the surrounding environment due to its overuse in agriculture. The antioxidant activity of Epigallocatechin gallate (EGCG) from green tea is at least 100 times greater than that of vitamin C. This study aimed to study the effects of DZN on the meiotic maturation of porcine oocytes, as well as the protective roles of EGCG. Firstly, the effects of DZN and EGCG on meiotic nuclear maturation of porcine oocytes were detected, and then embryonic development was investigated by chemical parthenogenetic activation. Next, the spindle assembly, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), DNA damage, and finally the early apoptosis of oocytes were examined by immunofluorescence staining. The results revealed that DZN exposure significantly reduced the quality of porcine oocytes, such as failure of nuclear and cytoplasmic maturation, evidenced by abnormal spindle assembly, disordered chromosome alignment, low MMP, observably increased ROS, severe DNA damage, and early apoptosis. Appropriate EGCG could significantly reduce all these defects caused by DZN. In conclusion, EGCG can help prevent the harm that DZN exposure can do. These findings offer convincing support for enhancing the oocyte quality from EGCG through daily ordinary beverages.

Enhancing Oocyte Competence With Milrinone as a Phosphodiesterase 3A Inhibitor to Improve the Development of Porcine Cloned Embryos.

The objective of this study was to investigate the effect of milrinone supplementation as a phosphodiesterase 3A inhibitor during in vitro maturation (IVM) to coordinate the cytoplasmic and nuclear maturation of porcine oocytes and subsequent development of porcine cloned embryos. Brilliant cresyl blue (BCB)-stained (BCB +) oocytes, classified as well-developed, and BCB− oocytes were used in parthenogenesis (PA) and cloning, and their preimplantation development was compared. In PA embryos, BCB + oocytes had significantly higher rates of development than BCB− oocytes in terms of maturation (87.5 vs. 71.3%), cleavage (88.6 vs. 76.3%), and blastocyst development (34.3 vs. 25.3%) and also had higher cell numbers (46.9 vs. 38.9%), respectively (p < 0.05). In cloned embryos, the BCB + group also had a significantly higher blastocyst formation rate than the BCB− group (30.6 vs. 20.1%; p < 0.05). Supplementation with 75 μM milrinone during IVM of BCB− oocytes showed improvement in maturation and blastocyst development rates, which may be due to the coordinated maturation of the cytoplasm with the nucleus as an effect of milrinone. Moreover, the analysis of nuclear reprogramming via the examination of the expression levels of the reprogramming-related genes POU5F1, DPPA2, and NDP52IL in milrinone-supplemented BCB− oocytes showed higher expression levels than that in non-treated BCB− oocytes. These findings demonstrate that milrinone is useful in improving developmental competence in less competent oocytes during IVM and for proper nuclear reprogramming in the production of porcine cloned embryos by coordinating cytoplasmic and nucleus maturation.

Supplementation with Niacin during in vitro maturation improves the quality of porcine embryos

Niacin, also known as vitamin B3, has a pivotal role in energy metabolism, cellular signaling cascades regulating gene expression, and apoptosis. However, the effect of Niacin on porcine early embryo developmental competence remains to be elucidated. The present study aimed to assess the effects of Niacin treatment during in vitro maturation (IVM) on the nuclear maturation of porcine oocytes and subsequent development of in vitro embryos. In addition, the expression profiles of selected genes related to lipid metabolism, oxidative stress, and apoptosis were assessed. The IVM medium was supplemented with different concentrations of Niacin (0, 300, 600, and 900 μM). The results showed that a high concentration of Niacin (900 μM) significantly decreased cumulus expansion compared to the other groups (p < 0.05). No significant difference was observed among the experimental groups for nuclear maturation rate. Niacin treatments (300, 600, and 900 μM) during IVM significantly (p < 0.05) enhanced glutathione levels. Treatment with 300 and 600 μM significantly (p < 0.05) lowered the reactive oxygen species levels compared to treatment with 900 μM and the control group. Niacin supplementation to the IVM media significantly improved the cleavage and blastocyst rates compared to the control group. Supplementation with 300 and 600 μM of Niacin significantly increased the total cell number of blastocysts compared to supplementation with 900 μM or the control groups. Cytoplasmic lipid droplets were significantly reduced after 600 μM treatment. Supplementation of Niacin to IVM media positively affected the relative expression of genes related to energy and oxidative status (SIRT1), pro-apoptosis (BAX), anti-apoptosis (BCL2), and lipid metabolism (ACACA and PNPLA2) in cumulus cells and oocytes. Taken together, Niacin supplementation to porcine IVM media improved the developmental competence of early embryos mainly through protection against oxidative stress and its influence on energy metabolism and apoptosis pathways.

162 Supplementation of Fibroblast Growth Factor 2, Leukemia Inhibitory Factor, and Insulin-Like Growth Factor 1 (FLI) Enables Efficient Production of Competent Porcine Oocytes Without Gonadotropins in the Maturation Medium

In vitro maturation (IVM) of oocytes is a critical step in assisted reproductive technologies carried out in species such as cattle and swine, for generating oocytes capable of being fertilized in vitro and providing healthy offspring useful for biomedical and agricultural purposes. Cumulus-oocyte complexes (COC) collected from abattoir ovaries for IVM often respond poorly to gonadotropins, resulting in compromised oocyte competence. Our previous work demonstrated that the combination of fibroblast growth factor 2, leukemia inhibitory factor, and insulin-like growth factor 1 (FGF2, LIF, and IGF1; FLI) significantly improved nuclear maturation of porcine oocytes and their developmental competence. However, it is unclear whether the benefits of FLI are mediated through increased gonadotropin sensitivity of COC or improved downstream signalling. Here we investigated the effect of FLI supplementation of IVM medium with and without gonadotropins. The COC, collected from 2- to 6-mm follicles from prepubertal ovaries, were matured in 5% CO2/air at 38.5°C for 42 h in chemically defined TCM-199 medium (supplemented with 0.1% polyvinyl alcohol, 3.05 mM d-glucose, 0.91 mM pyruvate, 0.57 mM cysteine, 10 ng mL−1 epidermal growth factor), with or without FLI (40 ng mL−1 FGF2, 20 ng mL−1 LIF, and 20 ng mL−1 IGF1), and with or without gonadotropins (0.5 μg mL−1 LH, 0.5 μg mL−1 FSH), in a 2 × 2 factorial design experiment. After IVM, oocytes were fertilized in vitro and cultured under standard conditions until Day 6 when blastocyst formation was assessed. The experiment was replicated 4 times with a total of 792 oocytes. Percentage data were arcsin transformed and analysed by ANOVA to detect differences (significance, P &lt; 0.05). When FLI was absent from the maturation medium, oocytes matured in presence of gonadotropins demonstrated improved nuclear maturation (67.0 ± 2.2% v. 52.7 ± 5.3%) and blastocyst formation (23.7 ± 3.3% v. 10.2 ± 2.3%) relative to oocytes matured without gonadotropins, respectively. However, when FLI was present in the medium, the extent of oocyte maturation and subsequent blastocyst development was unaffected by gonadotrophin addition (maturation: 84.2 ± 1.8% with and 79.2 ± 3.4% without; blastocyst formation: 29.5 ± 4.3% with and 24.9 ± 4.3% without). In summary, these results suggest that FLI, rather than enhancing COC sensitivity to gonadotrophins, may activate certain downstream signalling pathways that are normally controlled by gonadotropins during IVM, thereby enhancing oocyte quality. Therefore, FLI appears able to substitute for gonadotropins in the maturation medium and supports porcine oocyte competence in the absence of FSH and LH. These observations will help us better understand the mechanisms whereby FLI enhances oocyte nuclear maturation and improves developmental competency. Supported by NIH R01HD69979, U42OD011140, and University of Missouri Food for the 21st Century Program.

Treatment of Epidermal Growth Factor (EGF) enhances Nuclear Maturation of Porcine Oocytes and Stimulates Expression of ER/Golgi Transport Proteins.

ABSTRACTThis study was conducted to investigate stimulatory effect of epidermal growth factor (EGF) on nuclear maturation and the expression level of EGF-receptor (EGFR), GM-130 (a marker of Golgi apparatus), transport protein Sec61 subunit beta (Sec61β), and coatomer protein complex subunit gamma 2 (COPG2) in porcine oocytes. The cumulus-oocyte complexes were collected from follicle with 3-6 mm in diameter. They were incubated in medium with/without EGF for 22 h (IVMⅠ) and subsequently incubated hormone-free medium with/without EGF for 22 h (IVMⅡ). Nuclear maturation state was checked by aceto-orcein stain. Protein expression of EGFR, GM-130, Sec61β, and COPG2 were measured by immunofluorescence. In results, nuclear maturation of oocytes in EGF non-treated oocytes were significantly lower than EGF-treated groups at IVMⅠ or IVMⅡ stage (P<0.05), whereas maturational rate in EGF treatment groups at both of IVM stage was higher in among the all treatment groups (P<0.05). EGFR, GM-130, Sec61β and COPG2 were expressed in the cytoplasm of oocytes. Especially, GM-130 and EGFR were strongly expressed, but Sec61β and COPG2 were weakly expressed in cortical area of cytoplasm. The protein level of GM-130, Sec61β, and COPG2 were significantly higher in the EGF-treated groups (P<0.05). However EGFR was no difference between non EGF-treated groups and control. In conclusion, EGF plays an important role in the systems for oocyte maturation with endoplasmic reticulum and Golgi apparatus. In addition, the protein levels of Sec61β and COPG2 could be changed by EGF in the porcine oocytes during maturation.

Effect of zeaxanthin on porcine embryonic development during in vitro maturation.

Zeaxanthin is a common carotenoid, which is a powerful antioxidant that protects against damage caused by reactive oxygen species. The aim of the present study was to investigate the effects of zeaxanthin supplementation on in vitro maturation of porcine embryo development. We investigated nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels during in vitro maturation, and subsequent embryonic development following parthenogenetic activation and in vitro fertilization (IVF). The oocytes were maturated and used at the metaphase II stage. After 42 hours of in vitro maturation, the zeaxanthin-treated group (0.5 mmol/L) showed significant increases in nuclear maturation (89.6%) than the control group (83.4%) (P<0.05). The intracellular GSH levels increased significantly (P<0.05) as zeaxanthin concentrations increased; ROS generation levels decreased with increased zeaxanthin concentrations, but there were no significant differences. There were no significant differences in subsequent embryonic development, cleavage rate, blastocyst stage rate, and total blastocyst cell numbers following parthenogenetic activation and IVF when in vitro maturation media was supplemented with zeaxanthin. These results suggest that treatment with zeaxanthin during in vitro maturation improved the nuclear maturation of porcine oocytes by increasing the intracellular GSH level, thereby slightly decreasing the intracellular ROS level.

Progesterone influences cytoplasmic maturation in porcine oocytes developing in vitro.

Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and development in vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p < 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p < 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression.

Effect of Antioxidant Flavonoids (Quercetin and Taxifolin) on &lt;i&gt;In vitro&lt;/i&gt; Maturation of Porcine Oocytes

Quercetin (QT) and taxifolin (TF) are structurally similar plant-derived flavonoids that have antioxidant properties and act as free radical scavengers. The objective of this study was to investigate effects of QT and TF on nuclear maturation of porcine oocytes. Effects of TF at 0, 1, 10, and 50 μg/mL on oocyte nuclear maturation (polar body extrusion) were investigated. After incubation for 44 h, there were no significant differences between the treatment and control groups except in the 50 μg/mL group which was significantly lower (59.2%, p<0.05) than the other groups (control: >80%). After parthenogenetic activation, further in vitro development of QT- or TF-treated vs control oocytes was investigated. A significantly higher proportion of QT-treated (1 μg/mL) oocytes developed into blastocysts compared to controls (24.3% vs 16.8%, respectively); however, cleavage rate and blastocyst cell number were not affected. The TF-treated group was not significantly different from controls. Levels of reactive oxygen species (ROS) and intracellular glutathione (GSH) in oocytes and embryos in a culture medium supplemented with QT or TF were measured. Both treatment groups had significantly lower (p<0.05) levels of ROS than controls, however GSH levels were different only in QT-treated oocytes. We conclude that exogenous flavonoids such as QT and TF reduce ROS levels in oocytes. Although at high concentration (50 μg/mL) both QT and TF appear to be toxic to oocytes.

Effect of cumulus cell removal at different timings during IVM on nuclear maturation of porcine oocytes

Effect of cumulus cell removal at different timings during IVM on nuclear maturation of porcine oocytes

The effects of extracellular ATP and its receptor antagonists on pig oocytes during in vitro maturation.

We measured the ATP concentrations in the porcine follicular fluid derived from three sizes of follicles (small: 6 mm in diameter). Then, the effects of pre-treatment (100 μM each for 30 min before maturation) with antagonists for extracellular ATP receptor P2X or P2Y on the nuclear maturation rate of cumulus-cell-enclosed (COs) or -denuded oocytes (DOs) up to the preovulatory stage in the presence or absence of 20 nM ATP (a similar concentration to that of medium-sized follicle fluid) were investigated. The antagonists used were pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) or reactive blue 2 (RB2), for extracellular ATP receptor P2X and P2Y, respectively. In addition, the embryonic development rates of COs pre-treated with RB2 were also evaluated. It was found that when the follicular sizes increased, the ATP concentrations significantly decreased (P < 0.05). No differences were observed in the nuclear maturation rates among all COs, regardless of pre-treatment with (+) or without (-) PPADS and in the presence (+) or absence (-) of ATP during maturation. In contrast, the nuclear maturation rate of the COs, but not DOs, in the ATP(-) RB2(+) group was significantly lower (P < 0.05) than that of the ATP(-) RB2(-) and ATP(+)RB2(-) groups. The pronuclear formation and blastocyst formation rates by parthenogenetic activation in the ATP(-) RB2(+) and ATP(+) RB2(+) groups were significantly lower (P < 0.05) than those in the ATP(-) RB2(-) group. In conclusion, it is suggested that the nuclear maturation of porcine oocytes may be influenced by the ATP receptor P2Y present in the cumulus cells.

Quercetin improves thein vitrodevelopment of porcine oocytes by decreasing reactive oxygen species levels

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 µg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 µg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 µg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 µg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development.

Insulin–transferrin–selenium (ITS) improves maturation of porcine oocytes in vitro

The objective of this study was to determine if insulin-transferrin-selenium (ITS) promoted a nuclear and cytoplasmic maturation of porcine oocytes that better supports subsequent embryonic development. The rate of oocyte in vitro maturation (IVM) in an experimental group treated with hormones for 42 h was significantly increased compared with that in a control group without hormone treatment (47.8% vs. 11.7%, respectively, p < 0.05). Following reduction of the hormone treatment period from 42 h to 21 h, which included both the first 21 h period of hormones treatment (45.4%) and the second 21 h period of hormone treatment (44.8%), the rate of oocyte IVM was still higher than that of the control group (p < 0.05). To improve porcine oocyte nuclear maturation, 1% ITS was added to medium supplemented with hormones. The rate of nuclear maturation in the ITS-treated group was significantly higher than in the ITS-untreated group (78.6% vs. 54.4%, respectively, p < 0.05). ITS treatment also significantly reduced the per cent of oocytes with type I and type III cortical granule (CG) distribution, respectively, and significantly increased the per cent of oocytes with type II CG distribution (85.3%). These observations indicated that the synchronization rates of nuclear and ooplasmic maturation reached 67.04% (78.56 × 85.33%). In conclusion, the combination of modified Tissue Culture Medium-199 (mM199) + 10 ng/ml epidermal growth factor (EGF) + 10 IU/ml pregnant mare serum gonadotrophin (PMSG) + 10 IU/ml human chorion gonadotrophin (hCG) + 2.5 IU/ml follicle stimulating hormone (FSH) + 1% ITS is suitable for culturing porcine oocytes in vitro, and effectively enhances porcine oocyte nuclear and cytoplasmic maturation.

Involvement of the metabolic hormones leptin, ghrelin, obestatin, IGF-I and of MAP kinase in control of porcine oocyte maturation

The general aim of our in vitro experiments was to study the role of the metabolic hormones leptin, ghrelin, obestatin and IGF-I and mitogen-activated protein kinase (MAPK)-dependent intracellular mechanisms in the control of nuclear maturation of porcine oocytes. For this purpose, porcine oocytes were isolated from the ovary and cultured in the presence of leptin, ghrelin, obestatin, IGF-I, MAPK blocker PD98059 and the combinations of hormones with PD98059. Proportions of matured oocytes (at metaphase II of meiosis, determined by DAPI staining) and of oocytes containing MAPK/ERK1-2 (determined by immunocytochemistry) were measured before and after culture. It was observed that the majority of oocytes isolated from the ovary before culture were immature and did not contain visible MAPK, but some oocytes were mature, and the majority of these oocytes contained MAPK. Incubation of oocytes resulted in a significant increase in the proportion of matured oocytes and in the percentage of oocytes containing MAPK in both the matured and not matured groups. Addition of IGF-I to the culture medium increased the proportion of matured oocytes, addition of leptin decreased it, and ghrelin and obestatin did not oocyte maturation. Addition of hormones did not affect the expression of MAPK in either immature or mature oocytes. PD98059, when given alone, suppressed the maturation and accumulation of MAPK in both mature and immature oocytes. When given together with hormones, PD98059 was able to reduce the stimulatory effect of IGF-I, to invert the inhibitory action of leptin to stimulatory and to induce the stimulatory action of ghrelin and obestatin on meiosis. IGF-I, ghrelin and obestatin, but not leptin, when given together with PD98059, increased the accumulation of MAPK in both immature and mature oocytes. Association of nuclear maturation and expression of MAPK in oocytes before, but not after culture, as well as the prevention of oocyte maturation by MAPK blocker suggests the involvement of MAPK-dependent intracellular mechanisms in the promotion of reinitiation, but not completion of meiosis. The effect of hormonal additions on meiosis of oocytes suggests that IGF-I is a stimulator, leptin can be an inhibitor, while ghrelin and obestatin probably do not control oocyte maturation. The ability of PD98059 to modify the effect of hormones on oocyte maturation and on MAPK expression suggests possible interference of hormones and MAPK-dependent intracellular mechanisms in oocytes. However, no influence of hormones on MAPK and lack of association between action of hormones and PD98059 on MAPK and meiosis suggest that MAPK is probably not a mediator of effect of IGF-I, leptin, ghrelin and obestatin on porcine oocyte nuclear maturation.

275 EFFECT OF REACTIVE OXYGEN SPECIES DURING IN VITRO MATURATION ON PORCINE OOCYTE NUCLEAR MATURATION AND DEVELOPMENTAL COMPETENCE

The generation of excessive reactive oxygen species (ROS) may contribute to the decreased competence of in vitro matured (IVM) oocytes. However, ROS are also generated in normal cellular metabolism and can be important regulators of cellular functions. The objective of this study was to examine the effect of ROS during IVM on porcine oocyte nuclear maturation and subsequent embryonic development. Oocytes were matured in different redox environments for 40 h in 7% CO2 in air at 38.7°C. The basic maturation medium was defined PPM supplemented with 1 mM hypoxanthine. Reactive oxygen species were generated by the hypoxanthine–xanthine oxidase (XOD) system at 3 different concentrations: XOD0 (0 mU), XOD1 (1 mU), and XOD10 (10 mU). In each XOD treatment, 2 different concentrations of cysteine (Cys) were added as an antioxidant: Cys1 (0.57 mM) and Cys2 (1.14 mM). This resulted in 6 experimental treatments in a 3 × 2 factorial design; XOD0-Cys1 was considered the control. For fertilization, gametes were co-incubated in modified Tween medium B with milk powder for 5 h and then cultured in NCSU-23 medium in 5% CO2, 10% O2 for 6 days, at which point cleavage, blastocyst development, and blastocyst cell number were determined (30–50 per treatment per replicate; 4 replicates). Data were analysed by two-way ANOVA, and differences were determined by Fisher’s least significant difference multiple-comparison test; percentage data were arcsin transformed (significance, P &lt; 0.05). Results are shown in Table 1. Percentage of mature oocytes was not different between any XOD0 and XOD1 treatments, but maturation was decreased in both XOD10 treatments. Embryonic cleavage was also decreased in both XOD10 treatments compared with the control. Blastocyst development was decreased in XOD0-Cys2 and XOD10-Cys1 when compared with the control. Blastocyst total cell number was not different between any treatments (P &gt; 0.05). In conclusion, 10-mU XOD during IVM resulted in decreased nuclear maturation, embryonic cleavage, and blastocyst development, possibly due to excessive ROS generated by XOD. The negative effect of high levels of XOD on blastocyst formation could be reversed by adding additional antioxidant capacity to the environment (Cys2). This result suggests that adequate ROS balance is important for oocyte quality. Interestingly, adding extra antioxidant capacity alone (XOD0-Cys2) was detrimental to blastocyst formation, possibly due to the creation of an environment that was too reduced. These results demonstrate the importance of keeping the redox environment balanced during oocyte maturation. Excessive oxidative or reducing environments both appear to be detrimental to oocyte developmental competence. Table 1.Nuclear maturation and developmental competence of oocytes matured in different redox environments

Positive effects of Forskolin (stimulator of lipolysis) treatment on cryosurvival of in vitro matured porcine oocytes

In order to examine its effect on oocyte lipid content and cryosurvival, Forskolin was added to the medium for in vitro maturation of porcine oocytes. Treatments were control (IVM without Forskolin during the 42 h incubation period), addition of 10 μM Forskolin for the entire 42 h (0–42) and addition of 10 μM Forskolin between 24 and 42 h only (24–42). In Experiment 1, treatments did not differ significantly in cleavage rate, but the blastocyst formation rate was lower in the 0–42 group than for control and 24–42 group oocytes (17, 32 and 40%, respectively; P < 0.05). It was shown in Experiment 2 that Forskolin treatment from 0–42 h and from 24–42 h significantly reduced lipid content of oocytes compared to that of control cells (65 and 99 vs. 140 μm 2 intensity of fluorescence, respectively; P < 0.05). In Experiment 3, the percentage of oocyte survival after cryopreservation and thawing was significantly higher in both Forskolin treatment groups than in control oocytes (72% for 0–42, 65% for 24–42 and 52% for control; P < 0.05). However, Forskolin treatment did not increase cleavage rates of vitrified in vitro matured porcine oocytes (Control group 28%, 0–42 h group 0%, 24–42 h group 26.67%). Addition of Forskolin affected the nuclear maturation of porcine oocytes. The percentage of PBE (polar body extrusion) were significantly reduced in the 0–42 h group (0–42 h group 42.00 ± 2.08 vs. Control group 79.70 ± 2.82 and 24–42 h group 70.60 ± 2.83; P < 0.05). The 24–42 h group showed similar nuclear status to that of the Control group. We propose that delipation engendered by incubation with 10 μM Forskolin during 24–42 hours of maturation increased cryosurvival of in vitro-maturated porcine oocytes and that attendant chemical lipolysis did not impair their further development as it may have done in oocytes incubated with Forskolin for the full 42 h.

Epidermal growth factor can be used in lieu of follicle-stimulating hormone for nuclear maturation of porcine oocytes in vitro

Epidermal growth factor (EGF) has been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes, but inconsistencies exist between earlier studies, probably due to differences in the culture conditions used. Using a serum- and hormone-free in vitro maturation (IVM) medium, this study investigated the specific contribution of EGF on IVM of porcine (Sus scrofa) oocytes and its interactive effects with follicle-stimulating hormone (FSH), porcine follicular fluid (pFF), cumulus cells, and serum. It was noteworthy that EGF functionally mimicked the action of FSH and could completely replace FSH for nuclear maturation (83.2 ± 4.4% vs. 55.9 ± 5.2%; mean ± SEM), whereas EGF had a synergistic effect with FSH on cytoplasmic maturation of porcine oocytes (P < 0.05). Specific inhibition of EGF receptor (EGFR) by tyrphostin AG 1478 inhibited both EGF- and FSH-induced meiotic resumption (17.9 ± 5.2% and 18.2 ± 4.4%, respectively), thereby suggesting that EGFR signaling pathway was essential for oocyte reentry into the meiotic cell cycle. Furthermore, it is possible that FSH action occurs via the EGFR signaling pathway to induce meiotic maturation, although alternate pathways could not be excluded. There were also individual or combined effects of cumulus cells, FSH, serum, and pFF with EGF on IVM of porcine oocytes (P < 0.05). Although FSH had a synergistic effect with EGF on cytoplasmic maturation, pFF masked the effects of EGF on both nuclear and cytoplasmic maturation of porcine oocytes (P < 0.05). Moreover, the presence of cumulus cells was essential for EGF action. In conclusion, a defined system was used to better examine the effects of EGF. We inferred that EGF functionally mimics FSH for nuclear maturation of porcine oocytes, and its exogenous supplementation into IVM medium can optimize the beneficial effects of FSH on cytoplasmic maturation of oocytes to obtain enhanced embryo development in vitro.

176. INHIBITION OF PORCINE OOCYTE NUCLEAR MATURATION IN VITRO USING A PHOSPHODIESTERASE INHIBITOR AND AN ADENYLATE CYCLASE ACTIVATOR

Asynchronous nuclear and cytoplasmic maturation is thought to contribute to poor embryo production in vitro. Nuclear arrest is mediated by cAMP and can be maintained within the oocyte using non-specific phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine ; IBMX) and the adenylate cyclase activator forskolin (FSK) (1). The aim of this study was to investigate the effect of IBMX and FSK supplementation on porcine oocyte nuclear maturation during COC recovery and IVM using a defined culture system. In all experiments, cAMP modulators were added to Hepes-buffered media held in collection tubes. COCs recovered from 3–5 mm diameter follicles of prepubertal ovaries were cultured in basic maturation media in the absence of FSH. Nuclear maturation was assessed using orcein dye. In Experiment 1, IVM media was supplemented with 0, 50 or 500 µM IBMX. In Experiment 2, IVM media was supplemented with 0, 5, 10, 50 and 100µM FSK. In Experiment 3, IVM medium was supplemented with combinations of IBMX and FSK to give the treatments; control, 50IBMX/50FSK, 50IBMX/100FSK, 500IBMX/50FSK and 500IBMX/100FSK. Nuclear maturation was assessed at 0, 2, 4 and 18 h after the onset of IVM. At 18 h of culture, there were no differences in the proportion of oocytes supplemented with 0, 50 or 500 µM IBMX reaching MII. Incubation with 10, 50 or 100 µM FSK resulted in 8-16% of oocytes at MII at 18 h compared to the other groups (25–29%; P &lt; 0.001). The combinations of IBMX and FSK resulted in greater proportions (86–98%) of oocytes remaining at the GV stage at 18 h compared to the control (16%; P &lt; 0.001). There were no differences in the proportion of oocytes remaining at the GV stage at the earlier time points (P &gt; 0.05). The results demonstrate that these cAMP modulators, in combination, are highly effective in maintaining porcine oocyte meiotic arrest in vitro for an extended period. (1) Albuz FK et al., Proceedings of the 25th Annual Meeting of ESHRE, Amsterdam, The Netherlands, 2009.

Effect of β-hydroxybutyrate Added to Maturation Medium on Nuclear Maturation of Pig Oocytes

Abstract: The effects of β-hydroxybutyrate (BHB) added to a modified NCSU37 medium during in vitro maturation (IVM) on nuclear maturation, and ATP and glutathione (GSH) contents of porcine oocytes were investigated. Nuclear maturation up to the metaphase stage of the second meiotic division (the M-II stage) of cumulus cell-enclosed oocytes (COs) matured with various concentrations (0.0, 2.3, 3.1, 3.9 and 4.7 mM) of BHB was observed. The nuclear maturation rate of the oocytes of the 3.1 mM group was significantly higher (P < 0.05) than that of the control (0.0 mM) group. Both the ATP and GSH contents of the oocytes matured with or without 3.1 mM BHB and removed from cumulus cells after IVM were not significantly different. These results indicate that 3.1 mM BHB added to the maturation medium may stimulate the nuclear maturation of porcine oocytes without changing their ATP and GSH contents.

256 LEPTIN AND GLUCOSE INFLUENCE PORCINE NUCLEAR MATURATION

Leptin (LEP), a product of the obese gene, regulates food intake and may be associated with the metabolic syndrome. In addition, leptin receptor has been identified in the luteal and granulosa cells of the pig, suggesting that this protein may play a role in fertility. Research indicates that LEP is associated with insulin sensitivity; thus, the addition of different concentrations of LEP and glucose (GLUC) in maturation media may have an impact on oocyte maturation. The objective of the current experiments was to determine the effect of LEP (Experiment 1), and LEP in combination with GLUC (Experiment 2) on the nuclear maturation of porcine oocytes. This could potentially be a good model system in which to study the effects of obesity and diabetes on oocyte quality. Cumulus–oocyte complexes were cultured in a chemically defined medium, Purdue porcine medium (PPM) for 42 h, in 7% CO2 in air and at 38.7°C. In Experiment 1, medium was supplemented with 5 different concentrations (0, 1, 10, 20, and 100 ng mL–1) of LEP, in the presence of 2 mm GLUC. Experiment 2 was designed as a 3 × 3 factorial, with LEP (0, 1, and 10 ng mL–1) and GLUC (0, 5, and 50 mm). Oocytes were fixed and stained after IVM (136 to 155/treatment, 7 replicates; and 39 to 90/treatment, 5 replicates for Experiment 1 and 2, respectively). Nuclear maturation was scored as 1 (mature; T or MII) or 0 (not mature). Data were analyzed by GLM ANOVA and chi-square. There was no difference in nuclear maturation between 0, 1, 10, 20, and 100 ng mL–1 LEP (79.4, 82.3, 74.5, 77.2, and 83.7% mature, respectively). According to these data, LEP alone did not have an effect on in vitro maturation of porcine oocytes when using a chemically defined maturation medium. Absence of GLUC in the medium had a negative effect on maturation (39.5%; P &lt; 0.01) compared with treatment with 5 mm (88.2%) and 50 mm (74.7%) GLUC, independent of LEP. In the presence of 5 mm GLUC, there was no difference between 0, 1, and 10 ng mL–1 LEP (83.9, 94.9, and 88.3%, respectively). However, LEP did affect nuclear maturation when COC were cultured in the absence of GLUC or with excessive GLUC. Leptin (10 ng mL–1) tended (P = 0.1) to promote nuclear maturation (46.2%) in the absence of GLUC (0 mm), compared with 0 ng mL–1 LEP (32.3%). In high-GLUC (50 mm) medium, 1 ng mL–1 LEP had a positive effect (P &lt; 0.05) on nuclear maturation (87.8%) compared with 0 ng mL–1 (66.7%) and 10 ng mL–1 LEP (72.3%). LEP (0 or 10 ng mL–1) inhibited (P &lt; 0.05) nuclear maturation in the presence of high (50 mm) GLUC compared with that in 5 mm GLUC. These data demonstrate that addition of 10 ng mL–1 LEP in maturation media may mitigate the negative effect of maturation in the absence of GLUC. Moreover, in the presence of excessive GLUC, 1 ng mL–1 LEP stimulates nuclear maturation. These results suggest that LEP and GLUC may interact to regulate oocyte nuclear maturation in obese or diabetic individuals, or both.

Nuclear Maturation of Porcine Oocytes in vitro: Effect of the Cumulus-Oocyte Complexes Quality

The objective of this study was to examine the effect of the cumulus-oocyte complexes (COCs) quality on the ability of porcine oocytes to mature in vitro. Porcine COCs were collected from 2-6 mm follicles of slaughterhouse ovaries. The oocytes used for IVM were classified into three categories based on the compactness and transparency of the cumulus investment and homogeneity and transparency of the ooplasm. The oocytes were then matured in vitro for 44 h. At 22 of maturation culture, most of the oocytes in all groups were identified still at germinal vesicle (GV) stage and metaphase I (M-I) stage. After 44 h of culture, a greater proportion of Category I and II oocytes completed in vitro maturation through the second meiotic as compared with that of Category III oocytes (Pl0.05). The proportion of oocytes remaining at M-I stage and the degenerative oocytes in Category III oocytes were significantly higher than those of oocytes in other groups (Pl0.05). These data indicate that porcine oocytes with high quality cytoplasm and a cumulus cell complement have a much greater chance of maturing in vitro than that lower quality oocytes. The morphological grading of immature oocytes is an appropriate selection criterion for their developmental ability.