L-carnitine enhances oocyte maturation and development of parthenogenetic embryos in pigs

The objective was to determine whether adding L-carnitine in IVM/IVC medium enhanced maturation and developmental competence of porcine oocytes in vitro. Oocyte maturation rates did not differ significantly among groups supplemented with 0, 0.25, 0.5, or 1 mg/mL of L-carnitine added during IVM (although 2 mg/mL of L-carnitine reduced maturation rate). Compared with control oocytes, those treated with 0.5 mg/mL of L-carnitine during IVM had greater (P < 0.05) rates of blastocyst formation after parthenogenetic activation, and these blastocysts had less (P < 0.05) apoptosis. Adding 0.5 mg/mL of L-carnitine during IVM also significantly reduced intracellular reactive oxygen species (ROS), and increased glutathione (GSH) concentrations. With or without glucose supplementation, 0.5 mg/mL of L-carnitine in the IVM medium significantly hastened nuclear maturation of oocytes. Moreover, supplementing the IVM medium with either glucose or L-carnitine increased (P < 0.05) percentages of oocytes that reached the metaphase II (MII) stage, relative to a control group. Final maturation rates in IVM medium containing either glucose or L-carnitine were not significantly different. Adding L-carnitine (0 to 2 mg/mL) to IVC medium for activated porcine oocytes did not significantly affect development. However, 0.5 mg/mL of L-carnitine in IVC medium significantly reduced reactive oxygen species levels and apoptosis in activated blastocysts, although glutathione concentrations were not significantly altered. In conclusion, adding L-carnitine during IVM/IVC improved developmental potential of porcine oocytes, and also the quality of parthenogenetic embryos, probably by accelerating nuclear maturation, and preventing oxidative damage and apoptosis.     

The effects of resveratrol on porcine oocyte in vitro maturation and subsequent embryonic development after parthenogenetic activation and in vitro fertilization

We investigated the effects of resveratrol, a phytoalexin with various pharmacologic activities, on in vitro maturation (IVM) of porcine oocytes. We investigated intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, as well as gene expression in mature oocytes, cumulus cells, and in vitro fertilization (IVF)-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and IVF. After 44 h of IVM, no significant difference was observed in maturation of the 0.1, 0.5, and 2.0 μM resveratrol groups (83.0%, 84.1%, and 88.3%, respectively) compared with the control (84.1%), but the 10.0 μM resveratrol group showed significantly decreased nuclear maturation (75.0%) (P < 0.05). The 0.5- and 2.0-μm groups showed a significant (P < 0.05) increase in intracellular GSH levels compared with the control and 10.0 μM group. Intracellular ROS levels in oocytes matured with 2.0 μM resveratrol decreased significantly (P < 0.05) compared with those in the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rates and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) than the control group. Cumulus-oocytes complex treated with 2.0 μM resveratrol showed lower expression of apoptosis-related genes compared with mature oocytes and cumulus cells. Cumulus cells treated with 2.0 μM resveratrol showed higher (P < 0.05) expression of proliferating cell nuclear antigen than the control group. IVF-derived blastocysts derived from 2.0 μM resveratrol-treated oocytes also had less (P < 0.05) Bak expression than control IVF-derived blastocysts. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating gene expression during oocyte maturation.     

The new system of shorter porcine oocyte in vitro maturation (18 hours) using ≥8 mm follicles derived from cumulus-oocyte complexes

Despite recent efforts to improve in vitro maturation (IVM) systems for porcine oocytes, developmental competence of in vitro-matured oocytes is still suboptimal compared with those matured in vivo. In this study, we compared oocytes obtained from large (≥8 mm; LF) and medium (3–7 mm; MF) sized follicles in terms of nuclear maturation, intracellular glutathione and reactive oxygen species levels, gene expression, and embryo developmental competence after IVM. In the control group, cumulus-oocyte complexes (COCs) were aspirated from MF and matured for 22 hours with hormones and subsequently matured for 18 to 20 hours without hormones at 39 °C, 5% CO₂in vitro. In the LF group, COCs were obtained from follicles larger than 8 mm and were subjected to IVM for only 18 hours. The ovaries have LF were averagely obtained with 1.7% per day during 2012 and it was significantly higher in the winter season. The results of the nuclear stage assessment of the COCs from the LFs are as follows: before IVM (0 hours); germinal vesicle stage (15.2%), metaphase I (MI) stage (55.4%), anaphase and telophase I stages (15.8%), and metaphase II (MII) stage (13.6%). After 6 hours IVM; germinal vesicle (4.2%), MI (43.6%), anaphase and telophase I (9.4%), and MII (42.8%). After 18-hour IVM; MI (9.7%) and MII (90.3%). Oocytes from LF showed a significant (P < 0.001) increase in intracellular glutathione (1.41 vs. 1.00) and decrease in reactive oxygen species (0.8 vs. 1.0) levels compared with the control. The cumulus cells derived from LFs showed lower (P < 0.1) mRNA expression of COX-2 and TNFAIP6, and higher (P < 0.1) mRNA expression of PCNA and Nrf2 compared with the control group-derived cumulus cells. After parthenogenetic activation, in vitro fertilization and somatic cell nuclear transfer (SCNT) using matured oocytes from LFs, the embryo development was significantly improved (greater blastocyst formation rates and total cell numbers in blastocysts) compared with the control group. In conclusion, oocytes from LFs require only 18 hours to complete oocyte maturation in vitro and their developmental competence is significantly greater than those obtained from MFs. Although their numbers are limited, oocytes from LFs might offer an alternative source for the efficient production of transgenic pigs using SCNT.     

Vitamin C enhances porcine cloned embryo development and improves the derivation of embryonic stem-like cells

Porcine cloning through somatic cell nuclear transfer (SCNT) has been widely used in biotechnology for generating animal disease models and genetically modified animals for xenotransplantation. Vitamin C is a multifunctional factor that reacts with several enzymes. In this study, we used porcine oocytes to investigate the effects of different concentrations of vitamin C on in vitro maturation (IVM), in vitro culture (IVC), and the derivation of nuclear transfer embryonic stem-like cells (NT-ESCs). We demonstrated that vitamin C promoted the cleavage and blastocyst rate of genetically modified cloned porcine embryos and improved the derivation of NT-ESCs. Vitamin C integrated into IVM and IVC enhanced cleavage and blastocyst formation (P < 0.05) in SCNT embryos. Glutathione level was increased, and reactive oxygen species levels were decreased (P < 0.05) due to vitamin C treatment. Vitamin C decreased the gene expression of apoptosis (BAX) and increased the expression of genes associated with nuclear reprogramming (NANOG, POU5F1, SOX2, c-Myc, Klf4, and TEAD4), antioxidation (SOD1), anti-apoptotic (Bcl2), and trophectoderm (CDX2). Moreover, vitamin C improved the attachment, derivation, and passaging of NT-ESCs, while the control group showed no outgrowths beyond the primary culture. In conclusion, supplementation of vitamin C at a dose of 50 µg/ml to the IVM and IVC culture media was appropriate to improve the outcomes of porcine IVM and IVC and for the derivation of NT-ESCs as a model to study the pre- and post-implantation embryonic development in cloned transgenic embryos. Therefore, we recommend the inclusion of vitamin C as a supplementary factor to IVM and IVC to improve porcine in vitro embryonic development.     

Production of viable cloned miniature pig embryos using oocytes derived from domestic pig ovaries

For production of viable somatic cell nuclear transferred (SCNT) miniature pig embryos, in vitro condition for controlling the quality of recipient oocytes derived from domestic pig ovaries should be evaluated. In the present study, to get information on optimal in vitro maturation (IVM) condition of oocytes, we investigated the effect of IVM duration of recipient oocytes on subsequent development of SCNT miniature pig embryos, the maturation-promoting factor (MPF) activity in recipient oocytes before and after SCNT, and the occurrence of premature chromosome condensation (PCC) and spindle morphologies of donor nuclei following SCNT. The optimal window of the IVM period in terms of in vitro developmental ability of SCNT embryos was determined to be 36-40 h after the start of IVM. The use of recipient oocytes matured for 36 and 40 h resulted in a high level of MPF activity before and after SCNT, and increased the occurrence of PCC in transferred nuclei compared to the use of oocytes matured for 44 and 52 h. The proportion of abnormal spindle-like structures increased as the IVM period was prolonged. In addition, SCNT embryos constructed from recipient cytoplasts obtained after 40 h of maturation by using fetal fibroblasts of miniature pigs were transferred to surrogate miniature pigs, and developed to full term. These results suggest that recipient oocytes matured for 36 h and 40 h effectively induce PCC with a normal cytoskeletal structure because of a high level of MPF activity; furthermore, the 40-h IVM period improves in vitro development of SCNT embryos to the blastocyst stage, resulting in the production of viable cloned miniature pigs.     

The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development effect of beta-mercaptoethanol, cysteine and cystine

Supplementation of IVM medium with cysteamine, beta-mercaptoethanol, cysteine and cystine induced bovine oocyte glutathione (GSH) synthesis, but only the effect of cysteamine on the developmental competence of these oocytes was tested. During IVM of sheep oocytes, cysteamine but not beta-mercaptoethanol increased embryo development. However, it is not known how long the high intracellular oocyte GSH levels obtained after IVM with thiol compounds, can be maintained. Thus, the present study was carried out to evaluate the effects of supplementing maturation medium with 100 microM beta-mercaptoethanol, 0.6 mM cysteine and 0.6 mM cystine on 1) intracellular GSH level after IVM, 2) after IVF, 3) in 6 to 8-cell embryos and 4) on embryo development. In oocytes after IVM and in presumptive zygotes after IVF, intracellular GSH levels were significantly higher in the treated groups (P < 0.05). While, GSH content in 6 to 8-cell embryos was similar among treatment groups (P > 0.05). Differences in cleavage rates and the percentage of embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) for treated oocytes than for those matured in the control medium. We conclude from the results that the high intracellular GSH levels after induction of GSH synthesis in bovine IVM by thiol compounds remain during IVF and are still present at the beginning of IVC, improving developmental rates. Moreover, the results indicate that this metabolic pathway is an important component of the cytoplasmic maturation process that affects the subsequent steps of in vitro embryo production.     

Effects of Zinc Supplementation During In Vitro Maturation on Meiotic Maturation of Oocytes and Developmental Capacity in Yak

Zinc (Zn) is an essential trace element that is required during mammalian developmental processes. The objective of this study was to investigate the effects of Zn supplementation during in vitro maturation (IVM) on the developmental capacity of yak (Bos grunniens) oocytes. Cumulus expansion, nuclear maturation, intracellular glutathione (GSH), reactive oxygen species (ROS) levels, superoxide dismutase (SOD) activity, subsequent embryonic development, and the expression of Zn transporters (ZnTs) and Zrt and Irt-like proteins (ZiPs) were evaluated. The Zn concentrations in yak plasma and follicular fluid were 0.740?±?0.012 and 0.382?±?0.009 μg/mL, respectively. The cumulus expansion did not show significant differences in COCs after matured with or without Zn supplementation (P?>?0.05). The intracellular GSH was higher in oocytes matured with 1 or 2 mg/L Zn than in control group (0 mg/L) (P?<?0.05). However, ROS levels of oocytes matured with 1 or 2 mg/L Zn were reduced significantly compared with the control and 0.5 mg/L groups (P?<?0.05). The SOD activity was increased significantly after Zn supplementation. The cleavage rate was not significantly different after Zn supplementation (P?>?0.05). Percentages of matured oocytes that developed into the blastocyst stage after IVF were 47.9, 50.5, 60.4, and 58.9% for 0, 0.5, 1, and 2 mg/L Zn groups, respectively. Gene expression analysis revealed that the expression patterns associated with Zn were changed after Zn supplementation. In conclusion, Zn supplementation to IVM improved yak oocyte maturation and subsequent development by increasing GSH and SOD activity, decreasing ROS in oocytes.     

Scriptaid Treatment Decreases DNA Methyltransferase 1 Expression by Induction of MicroRNA-152 Expression in Porcine Somatic Cell Nuclear Transfer Embryos

Abnormal epigenetic reprogramming of donor nuclei after somatic cell nuclear transfer (SCNT) is thought to be the main cause of low cloning efficiencies. A growing body of evidence has demonstrated a positive role of Scriptaid, a histone deacetylase inhibitor (HDACi) that belongs to an existing class of hydroxamic acid-containing HDACis, on the development competence of cloned embryos in many species. The present study investigated the effects of Scriptaid on the development of porcine SCNT embryos in vitro and its mechanism. Treatment with 300 or 500 nM Scriptaid for 20 h after activation significantly increased the percentage of SCNT embryos that developed to the blastocyst stage and the total number of cells per blastocyst and significantly decreased the percentage of apoptotic cells in blastocysts. Scriptaid treatment significantly increased the level of histone H3 acetylated at K9 and the conversion of 5-methylcytosine into 5-hydroxymethylcytosine and significantly decreased the level of histone H3 trimethylated at K9 at the pronuclear stage. As a potential mechanism for the DNA methylation changes, our results showed that the expression of DNA methyltransferase 1 was frequently down-regulated in Scriptaid-treated embryos in comparison with untreated embryos and was inversely correlated to endogenous microRNA-152 (miR-152). Taken together, these findings illustrated a crucial functional crosstalk between miR-152 and DNMT1. Meanwhile, mRNA and protein levels of POU5F1 and CDX2 were increased in Scriptaid-treated embryos. mRNA levels of Caspase3, and Bax were significantly decreased and that of Bcl-xL was significantly increased in Scriptaid-treated embryos. In conclusion, these observations would contribute to uncover the nuclear reprogramming mechanisms underlying the effects of Scriptaid on the improvement of porcine SCNT embryos.     

Leptin improves in-vitro maturation of goat oocytes through MAPK and JAK2/STAT3 pathways and affects gene expression of cumulus cells

We investigated whether the recombinant leptin (1, 10, 100 ng/mL) influences the meiotic maturation of goat oocytes, whether the MAPK and JAK2/STAT3 pathways mediate the effects of leptin during in-vitro maturation, and whether leptin differently affects the abundance of mRNAs relevant to leptin signal transduction and apoptosis in oocytes and cumulus cells. The addition of leptin to the maturation medium positively affected the number of oocytes that completed nuclear maturation. Nuclear oocyte maturation stimulated by leptin was significantly impaired when we added the specific inhibitors of MAPK (U0126) and JAK2/STAT3 (AG490) to the maturation medium. The addition of leptin (10 ng/mL) during maturation did not affect the expression of AMPKα1, PPARα, Caspase 3, and BCL2 genes in oocytes or cumulus cells. The PPARγ and BAX mRNA abundances were significantly reduced in cumulus cells in the leptin group compared to the control group. Our results demonstrate that supplementation of the in-vitro maturation medium with leptin significantly improves nuclear maturation and reveal the important role of the MAPK and JAK2/STAT3 signaling pathways in establishing the leptin-mediated nuclear maturation of goat oocytes. Moreover, leptin treatment affects PPARγ and BAX gene expression in cumulus cells.     

Coculturing denuded oocytes during the in vitro maturation of bovine cumulus oocyte complexes exerts a synergistic effect on embryo development

The present study examined the effect of coculturing cumulus oocyte complexes (COCs) and denuded oocytes (DOs) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation, zona pellucida (ZP) hardening, the pattern of fertilization and glutathione peroxidase 1 (GPX1) gene expression in the oocyte. Furthermore, the rate of embryonic development and the quality of blastocysts were examined for both COCs and DOs. Three IVM conditions were studied: 1) the coculture of 12 COCs and 60 DOs, 2) COC control with 12 COCs, and 3) DO control with 60 DOs. The IVM was performed in a 120-μl droplet of TCM199-based IVM medium. Following IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were conducted separately for the COCs and DOs (DO coculture) from the IVM coculture group. Coculturing COCs and DOs increased the percentage of oocytes reaching the blastocyst stage and the total number of cells per blastocyst in both the COC coculture (44.4 ± 8.6 vs 26.7 ± 9.7%, P < 0.01, and 137.9 ± 24.9 vs 121.7 ± 21.1, P < 0.05) and the DO coculture (20.5 ± 5.0 vs 11.1 ± 2.5%, P < 0.01, and 121.9 ± 27.5 vs 112.3 ± 33.2, P < 0.05) compared to their respective control groups. The synergistic effects of coculturing were detected as increased nuclear and cytoplasmic maturation, the prevention of ZP hardening, increased monospermic fertilization and increased expression of GPX1 in the oocytes in response to endogenous oocyte-secreted factors. In conclusion, coculturing COCs and DOs may be an effective culture system for both intact COCs and immature DOs.     

Quality of vitrified porcine immature oocytes is improved by coculture with fresh oocytes during in vitro maturation

It is essential to enhance the in vitro maturation (IVM) condition for immature oocytes after cryopreservation, particularly if limited numbers of oocytes collected from specific donors. The objective of this study was to determine if quality of vitrified porcine immature oocytes was enhanced by coculturing with fresh oocytes during IVM. To distinguish fresh versus vitrified oocytes, we used two types of coculture systems: (a) transwell two‐chamber coculture; (b) labeling and tracing fresh oocytes with CellTracker? Green CMFDA during conventional culture. Coculture systems significantly accelerated meiotic progression of vitrified oocytes and significantly increased blastocyst formation rates following parthenogenetic activation and somatic cell nuclear transfer. Reactive oxygen species generation in vitrified oocytes was ameliorated by the coculture conditions, with no significant difference between fresh and vitrified oocytes for intracellular glutathione level. Both coculture systems significantly increased rate of normal mitochondrial distribution in vitrified oocytes, but did not affect fluorescence intensity of mitochondria. The percentage of oocytes with normal endoplasmic reticulum (ER) distribution and ER fluorescence intensity were significantly higher in vitrified oocytes cocultured with fresh oocytes. After 20 hr of IVM, mRNA expression of COX2, HAS2, PTX3, and TNFAIP6 remained significantly higher in cumulus cells derived from vitrified oocytes and coculture systems significantly decreased the expression of these genes. Additionally, coculture methods prevented the reduction of mRNA expression for BMP15, ZAR1, POU5F1, and DNMT3A in vitrified oocytes. In conclusion, oocyte quality and subsequent embryo development of vitrified porcine immature oocytes were significantly improved by fresh oocyte coculture during IVM.     

Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey

Somatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 degrees C) without CO(2) supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p < 0.05). There were no significant differences in the maturation rate (70.0 vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p < 0.05) and 8-cell (0 vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo development; however, different donor cell types (cumulus and fibroblast) resulted in different developmental potentials of SCNT embryos.     

In vitro maturation of porcine oocytes using a defined medium and developmental capacity after intracytoplasmic sperm injection

To establish a defined in vitro maturation culture system for porcine oocytes, we examined the effects of adding cysteine (Cys) and epidermal growth factor (EGF) to the maturation medium. Furthermore, to evaluate cytoplasmic maturation, we investigated GSH concentrations and embryo development after intracytoplasmic sperm injection (ICSI). The basic media for IVM were modified TCM199 containing 10% newborn calf serum (NBCS) or 0.1% polyvinyl alcohol (PVA), supplemented with amino acids. Adding EGF (10 ng/ml) or EGF + Cys (0.57 mM) to the defined medium (0.1% PVA + amino acids) increased (P < 0.05) the rate of nuclear maturation relative to the defined medium (without these additives). After ICSI, oocytes matured in a medium supplemented with NBCS, Cys and EGF had a higher (P < 0.05) rate of pronuclear formation rate than oocytes matured in the defined IVM medium. Although there was no significant difference in cleavage rates between NBCS- and PVA-containing media supplemented with both Cys and EGF, the rate of blastocyst development was lower (P < 0.05) in the defined medium than in the NBCS-containing medium. Intracellular GSH concentrations of oocytes matured in the NBCS- and PVA-containing media supplemented with both Cys and EGF were higher (P < 0.05) than in oocytes matured in PVA alone or in oocytes before maturation. Adding Cys and EGF to a defined medium for porcine IVM improved rates of nuclear maturation and cleaved oocytes following ICSI, probably due to increased GSH concentrations. Also, embryos derived from oocytes matured in the defined medium (with the addition of Cys and EGF) developed into blastocysts after ICSI.