Porcine In Vitro Maturation Research Articles

Effects of the addition of insulin-transferrin-selenium (ITS) and/or metformin to the in vitro maturation of porcine oocytes on cytoplasmic maturation and embryo development.

One of the main problems of porcine in vitro maturation (IVM) is incomplete cytoplasmatic maturation. Nuclear and cytoplasmic maturation will determine the future success of fertilisation and embryo development. Insulin-transferrin-selenium (ITS) has insulin-like and antioxidant effects, and metformin (M) is an insulin-sensitiser and antioxidant drug. To assess the effects of adding ITS and/or M in porcine IVM media on cytoplasmic maturation and early embryo development. Cumulus -oocyte complexes (COC) were IVM with M (10-4 M), ITS (0.1% v/v), M+ITS or no adding (Control). ITS increased glucose consumption compared to Control and M (P <0.01), and M+ITS did not differ from ITS or Control. Redox balance: M, ITS and M+ITS increased glutathione (P <0.01) and decreased lipid peroxidation (P <0.005). The viability of cumulus cells by flow cytometry increased with M (P <0.005) and decreased with ITS (P <0.001); M+ITS did not differ from Control. After IVF, M increased penetration and decreased male pronucleus (P <0.05). Embryo development: cleavage increased with M (P <0.05), and blastocysts increased with ITS and M+ITS (P <0.05). The number of blastocyst cells increased with ITS (P <0.05). Adding ITS and M+ITS to porcine IVM media benefits embryo development to blastocysts, but ITS alone has better effects than M+ITS. ITS is an excellent tool to improve IVM and embryo development after IVF in pigs.

Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

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 50µg/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.

Effects of Stem Cell Factor/c-Kit Signaling on In Vitro Maturation of Porcine Oocytes and Subsequent Developmental Competence After Fertilization.

Stem cell factor (SCF), also known as c-Kit ligand, plays an important role in the proliferation of primordial germ cells and the survival of oocytes during follicular development. The aim of this study was to investigate the effect of SCF/c-Kit signaling on in vitro maturation (IVM) of porcine oocytes by analyzing nuclear and cytoplasmic maturation, oocyte size, cumulus cell expansion, and developmental competence to the blastocyst stage. Moreover, mRNA expression patterns of porcine cumulus cells and oocytes were evaluated using qRT-PCR. Following 42 h of IVM, 10 and 50 ng/mL SCF-treated groups exhibited significantly (P < 0.05) increased polar body extrusion rates and intracellular glutathione levels compared with the control group. The cumulus expansion index significantly (P < 0.05) increased in all SCF-treated groups compared with the control samples. mRNA levels of the proapoptotic gene Bax and apoptosis-related cysteine peptidase Caspase3 were lower in SCF-treated cumulus cells than in the control group. Notably, the diameter of oocytes after IVM, the mRNA expression of well-known oocyte-secreted factors (GDF9 and BMP15), and an oocyte-specific protein essential for ovulation and oocyte health (YBX2) were significantly (P < 0.05) higher in SCF-treated than in non-treated oocytes. Inhibition of c-Kit during porcine IVM using ACK2, an antagonistic blocker of c-Kit, significantly (P < 0.05) decreased the polar body extrusion rate compared with the control, as well as blastocyst formation rate compared with the 10 ng/mL SCF-treated group. In conclusion, the effect of SCF/c-Kit-mediated signaling during porcine IVM could be ascribed to the reduced expression of apoptosis-related genes and higher expression of oocyte-specific/secreted factors.

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.

Effect of Interleukin-7 on In Vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Developmental Potential after Parthenogenetic Activation.

Simple SummaryOocyte-secreted factors play an essential role in oogenesis and fertility through bidirectional crosstalk between oocytes and somatic cells. Interleukin-7, known as an oocyte-secreted factor, has recently been shown to improve oocyte developmental competence through interaction with cumulus cells around the oocytes. This study aimed to investigate the effects of interleukin-7 on porcine cumulus-oocyte complexes during in vitro maturation. Our results showed that supplementation with interleukin-7 during in vitro maturation exerted beneficial effects on porcine oocyte meiotic maturation by upregulating antioxidant-related genes and enhanced the subsequent developmental potential of porcine embryos after parthenogenetic activation.Interleukin-7 (IL-7) is a cytokine essential for cell development, proliferation and survival. However, its role in oocyte maturation is largely unknown. To investigate the effects of IL-7 on the in vitro maturation (IVM) of porcine oocytes, we analyzed nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, and subsequent embryonic developmental competence after parthenogenetic activation (PA) under several concentrations of IL-7. After IVM, IL-7 treated groups showed significantly higher nuclear maturation and significantly decreased intracellular ROS levels compared with the control group. All IL-7 treatment groups exhibited significantly increased intracellular GSH levels compared with the control group. All oocytes matured with IL-7 treatment during IVM exhibited significantly higher cleavage and blastocyst formation rates after PA than the non-treatment group. Furthermore, significantly higher mRNA expression levels of developmental-related genes (PCNA, Filia, and NPM2) and antioxidant-related genes (GSR and PRDX1) were observed in the IL-7-supplemented oocytes than in the control group. IL-7-supplemented cumulus cells showed significantly higher mRNA expression of the anti-apoptotic gene BCL2L1 and mitochondria-related genes (TFAM and NOX4), and lower transcript levels of the apoptosis related-gene, Caspase3, than the control group. Collectively, the present study suggests that IL-7 supplementation during porcine IVM improves oocyte maturation and the developmental potential of porcine embryos after PA.

140 Effect of addition of different concentrations of L-carnitine during porcine in vitro maturation on embryo quality and development

l-Carnitine (LC) plays an important role in the catabolism of lipids and protects cells from the damage caused by reactive oxygen species due to its antioxidant activity. The aim of this study was to evaluate the effect of adding different concentrations of LC during porcine invitro maturation on embryo quality and development. The cumulus–oocyte complexes were obtained by follicular aspiration from ovaries of slaughtered sows and matured invitro for 44h without LC (control) or with different concentrations of LC (0.6 or 1.25mg mL−1) (Sigma-Aldrich) in TCM-199 supplemented with human menopausal gonadotrophin and cyclic AMP (cAMP) during the first 22h. Invitro fertilization was performed with fresh boar semen for 4h in 100-µL drops of TCM-199 with caffeine, bovine serum albumin, sodium lactate, and pyruvate (20 denuded oocytes per drop, 1×106 spermatozoa mL−1). Presumptive zygotes were washed and cultured in NCSU 23 at 39°C, 7% O2, 5% CO2, and humidity. The cleavage rate was registered on Day 2 and the blastocyst rate on Day 7. Embryo quality was assessed by counting the number of cells per blastocyst (Hoescht 33342) and late apoptosis index (TUNEL-positive cells/total cells). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was performed according to the kit protocol (Roche). LC significantly decreased the cleavage rate (control: 46.2%; LC0.6: 32.1%; LC1.25: 37.9%; P&amp;lt;0.05, Chi-squared test). No significant differences were detected in the blastocyst rate (control: 19.2%; LC0.6: 17%; LC1.25: 10,2%, Chi-squared test) or in number of cells per blastocyst (control: 51.97±3; LC0.6: 56.11±4; LC1.25: 45.62±4, ANOVA). There was embryo hatching in LC treatments but not in the control (control: 0%, LC0.6: 11%; LC1.25: 7.6%). The apoptosis index decreased in LC1.25 compared with LC0.6 (Control: 7,6±1.3%; LC0.6: 10±1.1%; LC1.25: 5,5±0.8%; P&amp;lt;0.05, ANOVA) but there was no significant difference in the apoptosis index between control and LC treatments. In conclusion, LC treatments decreased the cleavage rate but did not modify the blastocyst rate and allowed embryo hatching.

Maternal Cytokines CXCL12, VEGFA, and WNT5A Promote Porcine Oocyte Maturation via MAPK Activation and Canonical WNT Inhibition.

Maternal regulatory factors endow the oocyte with developmental competence in vivo, which might be absent in current in vitro maturation (IVM) systems, thereby compromising oocyte quality. In the present study, by employing RNA sequencing data analysis, we expect to identify potential contributing factors to support porcine oocyte maturation through binding to their receptors on the oolemma. Here, C-X-C motif chemokine ligand 12 (CXCL12), vascular endothelial growth factor A (VEGFA), and Wingless-type MMTV integration site family member 5A (WNT5A), termed CVW, are selected and confirmed to be important maternal cytokines for porcine oocyte maturation. Combined supplementation of CVW promotes the nuclear maturation percentage from 57.2% in controls to 75.9%. More importantly, these maternal cytokines improve the developmental potential of matured oocytes by parthenogenesis, fertilization, and cloning, as their blastocyst formation efficiencies and total cell numbers are increased. CVW supplementation also enlarges perivitelline space and promotes cumulus expansion, which results in a more complete transzonal projection retraction on the zona pellucida, and a reduced incidence of polyspermy in fertilized oocytes. Meanwhile, inhibiting the CVW receptor-mediated signaling pathways severely impairs oocyte meiotic resumption and cumulus expansion during IVM. We further determine that maturation improvement by CVW is achieved through activating the MAPK pathway in advance and inhibiting the canonical WNT pathway at the end of the IVM period. These findings provide a new combination of three cytokines to promote the porcine IVM process, which also holds potential to be used in human assisted reproduction technologies as well as in other species.

Transient meiotic arrest maintained by DON (6-diazo-5-oxo-l-norleucine) enhances nuclear/cytoplasmic maturation of porcine oocytes.

The developmental competence of in vitro-matured oocytes is still lower than that of the in vivo-matured oocytes due to precocious meiotic resumption and inappropriate cytoplasmic maturation. Although numerous efforts have been attempted to accomplish better in vitro maturation (IVM) condition, only limited progress has been achieved. Thus, a current study was conducted to examine the effects of 6-diazo-5-oxo-l-norleucine (DON, an inhibitor of hyaluronan synthesis) during the first half period of IVM on nuclear/cytoplasmic maturation of porcine oocytes and subsequent embryonic development. Based on the observation of the nucleus pattern, metaphase II (MII) oocyte production rate in 1 µM DON group was significantly higher than other groups at 44 h of IVM. The 1 µM of DON was suggested to be optimal for porcine IVM and was therefore used for further investigation. Meiotic arrest effect of DON was maximal at 6 h of IVM, which was supported by the maintenance of significantly higher intra-oocyte cAMP level. In addition, increased pERK1/2 levels and clear rearrangement of cortical granules in membrane of MII oocytes matured with DON provided the evidence for balanced meiosis progression between nuclear and cytoplasmic maturation. Subsequently, DON significantly improved blastocyst formation rate, total cell numbers, and cellular survival in blastocysts after parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Altogether, our results showed for the first time that 1 µM DON can be used to increase the yield of developmentally competent MII oocytes by synchronizing nuclear/cytoplasmic maturation, and it subsequently improves embryo developmental competence.

Growth differentiation factor 8 regulates SMAD2/3 signaling and improves oocyte quality during porcine oocyte maturation in vitro†.

Growth differentiation factor 8 (GDF8), also known as myostatin, is a member of the transforming growth factor-β (TGF-β) family and has been identified as a strong physiological regulator of muscle differentiation. Recently, the functional role of GDF8 in reproductive organs has received increased interest following its detection in the human placenta and uterus. To investigate the effects of GDF8 during porcine oocyte in vitro maturation (IVM), we assessed the quality of matured oocytes. Furthermore, we investigated the specific gene transcription and protein activation levels in oocytes and cumulus cells after IVM and subsequent embryonic development after in vitro fertilization and parthenogenetic activation. Prior to these experiments, the concentration of GDF8 in porcine follicular fluid was determined. During the entire IVM period, 1.3 ng/mL GDF8 and its signaling inhibitor SB431542 (SB) at 5 μM were added as control, SB, SB+GDF8, and GDF8 groups, respectively. Our results demonstrate that supplementation with GDF8 during porcine oocyte IVM enhanced both meiotic and cytoplasmic maturation, with altered transcriptional patterns, via activation of Sma- and Mad-related protein 2/3 (SMAD2/3). Using the pharmacological inhibitor SB431542, we demonstrated that inhibition of GDF8-induced Smad2/3 signaling reduces matured oocyte quality. In conclusion, for the first time, we demonstrated paracrine factor GDF8 in porcine follicular fluid in vivo. Furthermore, we showed that GDF8 supplementation improved mature oocyte quality by regulating p38 mitogen-activated protein kinase phosphorylation and intracellular glutathione and reactive oxygen species levels during porcine IVM.

GDF8 activates p38 MAPK signaling during porcine oocyte maturation in vitro

Growth Differentiation Factor 8 (GDF8) is a member of the transforming growth factor-β (TGF-β) family and has been identified as a strong physiological regulator. This factor is expressed as a paracrine factor in mural granulosa cells. To investigate the effects of GDF8 on the in vitro maturation (IVM) of porcine oocytes, we assessed the quality of matured oocytes as well as the specific gene transcription and protein activation levels in oocytes and cumulus cells (CCs) after IVM and subsequent embryonic development after in vitro fertilization (IVF) and parthenogenetic activation (PA). Supplemental concentrations (0, 1, 10, and 100 ng/ml) of GDF8 were provided in IVM medium. Supplementation with GDF8 during IVM induced transcription of specific TGF-β receptor genes, such as ActRIIb and Alk4/5, and the recognition of the GDF8 by these receptors induced phosphorylation of p38 MAPK. Activated p38 MAPK signaling changed oocyte maturation and cumulus expansion-related gene transcription: Nrf2 and Bcl-2 in oocytes and PCNA, Nrf2, Has2, Ptx3, and TNFAIP6 in CCs. The altered gene expression pattern during IVM resulted in a 10% lower level of intracellular ROS in mature oocytes. The improved cytoplasmic maturation led to an increase in the fertilization efficiency and subsequent embryonic developmental competence. The embryonic development showed increases in the blastocyst formation rate and higher transcription levels of POU5F1 and BCL-2 in the blastocysts. The present study suggests that supplementation of GDF8 during IVM synergistically improved the developmental potential of IVF- and PA-derived porcine embryos by reducing the intracellular ROS level in oocytes by altering the transcription of specific genes and increasing the phosphorylation of p38 MAPK during IVM. In conclusion, for the first time, our results demonstrate that GDF8 can act as a paracrine factor to modulate oocyte maturation by regulating p38 MAPK phosphorylation and intracellular ROS level during porcine IVM.

The effect of poly(ADP-ribosyl)ation inhibition on the porcine cumulus-oocyte complex during in vitro maturation

Poly(ADP-ribosyl)ation (PARylation) plays important roles in DNA repair, apoptosis, transcriptional regulation, and cell death, and occurs via the activity of poly(ADP-ribose) polymerases (PARPs). Previous studies have shown that PARylation affects mouse and porcine pre-implantation development and participates in mechanisms of autophagy. However, there have not yet been reported the role of PARylation during in vitro maturation (IVM) of porcine oocytes. Thus, we investigated the effect of PARylation inhibition on this process; cumulus-oocyte complexes (COCs) were cultured with 3-aminobenzamide (3-ABA, PARP inhibitor) during porcine IVM. Full cumulus expansion was significantly reduced (10.34 ± 1.23 [3-ABA] vs. 48.17 ± 2.03% [control]), but nuclear maturation rates were not changed in the 3-ABA treatment group. Especially, we observed that cumulus cells were little expanded after 22 h in 3-ABA treated COCs. The mRNA expression levels of oocyte maturation- and cumulus expansion-related genes were evaluated at 22 and 44 h. GDF9, BMP15, COX-2, and PTX3 expression were upregulated at 44 h, whereas the levels of HAS2 and TNFAIP6 were downregulated in the 3-ABA treated group. Furthermore, 3-ABA treatment significantly decreased the developmental rate (28.24 ± 1.06 vs. 40.24 ± 3.03%) and total cell number (41.12 ± 2.10 vs. 50.38 ± 2.27), but increased the total apoptotic index (6.44 ± 0.81 vs. 3.08 ± 0.51) in parthenogenetically activated embryos. In conclusion, these results showed that PARylation regulates cumulus expansion through the regulation of gene expression and affects developmental competence and quality in parthenogenetic embryos.

Nitric oxide synthase (NOS) inhibition during porcine in vitro maturation modifies oocyte protein S-nitrosylation and in vitro fertilization.

Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization.

New elements in the C-type natriuretic peptide signaling pathway inhibiting swine in vitro oocyte meiotic resumption.

C-type natriuretic peptide (CNP) and its cognate receptor, natriuretic peptide receptor (NPR) B, have been shown to promote cGMP production in granulosa/cumulus cells. Once transferred to the oocyte through the gap junctions, the cGMP inhibits oocyte meiotic resumption. CNP has been shown to bind another natriuretic receptor, NPR-C. NPR-C is known to interact with and degrade bound CNP, and has been reported to possess signaling functions. Therefore, NPR-C could participate in the control of oocyte maturation during swine in vitro maturation (IVM). Here, we examine the effect of CNP signaling on meiotic resumption, the amount of cGMP and gap junctional communication (GJC) regulation during swine IVM. The results show an inhibitory effect of CNP in inhibiting oocyte meiotic resumption in follicle-stimulating hormone (FSH)-stimulated IVM. We also found that an NPR-C-specific agonist (cANP([4-23])) is likely to play a role in maintaining meiotic arrest during porcine IVM when in the presence of a suboptimal dose of CNP. Moreover, we show that, even if CNP can increase intracellular concentration of cGMP in cumulus-oocyte complexes, cANP((4-23)) had no impact on cGMP concentration, suggesting a potential cGMP-independent signaling pathway related to NPR-C activation. These data support a potential involvement of cANP((4-23)) through NPR-C in inhibiting oocyte meiotic resumption while in the presence of a suboptimal dose of CNP. The regulation of GJC was not altered by CNP, cANP((4-23)), or the combination of CNP and cANP((4-23)), supporting their potential contribution in sending signals to the oocytes. These findings offer promising insights in to new elements of the signaling pathways that may be involved in inhibiting resumption of meiosis during FSH-stimulated swine IVM.

Replacement of glutamine with the dipeptide derivative alanyl-glutamine enhances in vitro maturation of porcine oocytes and development of embryos

The presence of glutamine (Gln) in in vitro maturation (IVM) and in vitro culture (IVC) medium is a more potent factor for improving porcine oocyte and embryo development than other amino acids. However Gln is inherently unstable and spontaneously breaks down into ammonia, and therefore interferes with proper development. To avoid this adverse effect, Gln was replaced in the present study with its stable dipeptide derivative alanyl-glutamine (Ala-Gln) and the effects of this replacement on porcine IVM and IVC were evaluated. Replacement of Gln with Ala-Gln during IVM did not improve nuclear maturation, however numbers of early cleaved embryos were significantly increased after activation. Blastocyst formation rates were also significantly improved by using Ala-Gln during IVM. Replacement of Gln with Ala-Gln during IVC significantly increased total cell numbers in blastocysts. Blastocyst formation rate was also significantly higher when Ala-Gln was used in both IVM and IVC. In conclusion, the use of Ala-Gln rather than Gln gives better results for development in both porcine IVM and IVC.

Development ofin vitroproduced porcine embryos according to serum types as macromolecule

This study was conducted to establish an in vitro maturation (IVM) system by selection of efficient porcine serum during porcine in vitro production. To investigate the efficient porcine serum (PS), different types of PS [newborn pig serum, prepubertal gilt serum (PGS), estrus sow serum, and pregnancy sow serum] were used to supplement IVM media with or without gonadotrophin (GTH) and development rates of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos were then compared. The maturation rates of the PGS group was significantly higher when GTH was not added. Additionally, during development of PA embryos without GTH, the PGS group showed significantly higher cleavage and blastocyst formation rates. Moreover, the cleavage rates of IVF embryos were significantly higher in the PGS group, with no significant differences in the blastocyst formation. However, when GTH was supplemented into the IVM media, there were no significant differences among the four groups in the cleavage rates, development rates of the blastocyst, and cell number of the blastocyst after PA and IVF. In conclusion, PGS is an efficient macromolecule in porcine IVM, and GTH supplementation of the IVM media is beneficial when PS is used as macromolecule, regardless of its origin.

Effect of Trans-ε-Viniferin on &lt;i&gt;In Vitro&lt;/i&gt; Porcine Oocyte Maturation and Subsequent Developmental Competence in Preimplantation Embryos

ABSTRACTTrans-ε-viniferin is a naturally occurring polyphenol belonging to the stilbenoid family that has been isolated from Vitis amurensis, one of the most common wild grapes in Asia. We investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and developmental competence after in vitro fertilization (IVF) or parthenogenesis (PA). We observed that trans-ε-viniferin treatment during IVM did not improve nuclear maturation rates of oocytes in any group, but significantly increased (P<0.05) intracellular glutathione (GSH) levels and reduced reactive oxygen species (ROS) levels in the 0.5 µM treatment group. Trans-ε-viniferin treatment during IVM of recipient oocytes promoted higher (P<0.05) expression of DNA methyltransferase-1 (DNMT1) mRNA in the 0.5 µM treatment group as compared with the control group. However, the expression of essential transcriptional and apoptosis-related genes did not significantly differ from that of the control. In cumulus cells, pro-apoptosis gene expressions were changed as apoptosis decreased. Oocytes treated with trans-ε-viniferin during IVM did not have significantly different cleavage rates or blastocyst formation rates after PA, but total cell numbers were significantly higher (P<0.05) in the 0.5 and 5.0 µM treatment groups compared with those in the control group. IVF embryos showed similar results. In conclusion, these results indicate that trans-ε-viniferin treatment during porcine IVM increased the total cell number of blastocysts, possibly by increasing intracellular GSH synthesis, reducing ROS levels, increasing DNMT1 gene expression of oocytes and decreasing pro-apoptosis gene expressions of cumulus cells.

Regulation of Gap-Junctional Communication Between Cumulus Cells During In Vitro Maturation in Swine, a Gap-FRAP Study1

Intercellular gap-junctional communication (GJC) plays an important role in ovarian cell physiology. Closure of GJC has been proposed to be involved in oocyte maturation, particularly in the resumption of meiosis, both in vivo and in vitro, by controlling the flow of meiosis inhibitors, such as cAMP and cGMP. Understanding how GJC dynamics are regulated during in vitro maturation (IVM) could provide a powerful tool for controlling meiotic resumption and oocyte maturation in vitro. Since little is known about the GJC dynamic regulation between cumulus cells, we have developed an assay based on recovery of calcein fluorescence in photobleached cumulus cells, a gap-FRAP assay. The GJC profile has been characterized during the first hours of porcine IVM. We showed that equine chorionic gonadotropin (eCG) and epidermal growth factor (EGF) down-regulated GJC effectiveness between cumulus cells. However, human chorionic gonadotropin was not down-regulating GJC effectiveness. We also showed that the GJC network expanded during this period and that this effect was not regulated by gonadotropins. Porcine follicular fluid present in the maturation medium also had an impact on GJC regulation, increasing GJC network establishment and the effectiveness of calcein transfer rate between cumulus cells. These results show that both eCG and EGF are regulating the decrease in GJC effectiveness after 4.5 h of IVM, while the network extension is gonadotropin independent. Regulation of GJC between cumulus cells would then be specifically regulated during in vitro IVM.

190 EFFECT OF TRANS-ε-VINIFERIN ON IN VITRO PORCINE OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENTAL COMPETENCE IN PRE-IMPLANTATION EMBRYOS

Trans-ε-viniferin is a naturally occurring polyphenol belonging to the stilbenoids family. Trans-ε-viniferin is isolated from Vitis amurensis, 1 of the most common wild grapes in Korea, Japan and China. We investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and developmental competence after IVF or parthenogenesis (PA). At the laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Chungbuk National University, trans-ε-viniferin was purified from the leaves and stems of Vitis amurensis. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. First, in total, 594 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of trans-ε-viniferin (0, 0.1, 0.5, 1.0 and 5.0 μM) with 10% porcine follicular fluid, 10 IU mL–1 of eCG and 10 IU mL–1 of hCG. After 22 h in maturation culture, the COC were cultured in hormone-free medium supplemented with various concentrations of trans-ε-viniferin for an additional 22 h and then nuclear maturation was evaluated. Second, in total, 300 matured oocytes were used to examine the effects of different trans-ε-viniferin concentrations (0, 0.5 and 5.0 μM) on porcine oocyte intracellular glutathione (GSH) and reactive oxygen species (ROS) levels. Lastly, the developmental competence of oocytes matured with different concentrations of trans-ε-viniferin (0, 0.5 and 5.0 μM) was evaluated after IVF or PA. In total, 711 embryos were evaluated. As results, we observed that trans-ε-viniferin treatment during IVM did not improve the nuclear maturation of oocytes in any group (84.2, 86.6, 85.5, 83.3 and 79.2%, respectively), but significantly increased (P &lt; 0.05) intracellular GSH levels in the 0.5 μM group (0 μM vs 0.5 μM; 14.6 vs 16.8 pmol oocyte–1) and reduced ROS levels (0 μM vs 0.5 μM and 50 μM; 174.6 vs 25.7 and 23.8 pixel oocyte–1). Oocytes treated with trans-ε-viniferin during IVM did not have significantly different cleavage rates or blastocyst formation rates after IVF, but total cell numbers were significantly higher (P &lt; 0.05) in the 0.5 and 5.0 μM treatment groups (53.6 ± 4.0 and 47.9 ± 3.1) compared to the control group (36.4 ± 2.2). The PA embryos showed similar results; there were no significant differences in cleavage rates and blastocyst formation rates, but the total cell number significantly increased in the 0.5 and 5.0 μM treatment groups (59.6 ± 4.2 and 60.8 ± 4.6) compared to the control group (43.1 ± 2.1). In conclusion, these results indicate that trans-ε-viniferin treatment during porcine IVM increased total cell number of blastocysts, possibly through increasing intracellular GSH synthesis and reducing ROS levels. This work was supported by a grant from the Korea institute of Planning &amp; Evaluation for Technology in Food, Agriculture, Forestry &amp; Fisheries, Republic of Korea.

Effects of melatonin on in vitro maturation of porcine oocyte and expression of melatonin receptor RNA in cumulus and granulosa cells

Melatonin is a multifunctional molecule that mediates several circadian and seasonal processes in animal reproduction. Melatonin and its metabolites are antioxidants and free radical scavengers. We investigated the effects of melatonin on porcine oocyte maturation and embryo development. We then investigated the local expression of the melatonin receptor 1 (MT1) gene in cumulus cells, granulosa cells, and the oocytes with the reverse transcription-polymerase chain reaction (RT-PCR) method. We further evaluated the antioxidant effects [reactive oxygen species (ROS) levels in cumulus-oocytes complexes] of melatonin supplementation during in vitro maturation (IVM). Compared with control, melatonin supplementation (10 ng/mL) during IVM resulted in a greater proportion of oocytes extruding the polar body (75.6% versus 84.6%). Significantly greater proportion of parthenogenetically activated oocytes developed to blastocysts when the in vitro medium was supplemented with melatonin; however, cleavage frequency and blastocyst cell number were not affected by the treatment. RT-PCR analysis revealed the expression of MT1 gene in cumulus and granulosa cells but not in oocytes. Melatonin-treated oocytes had significantly lower levels of ROS than did control (untreated) oocytes. We conclude that exogenous melatonin has beneficial effects on nuclear and cytoplasmic maturation during porcine IVM. Some of the observed effects may be mediated by receptor binding and while others may have been receptor independent, e.g., direct free radical scavenging.