Recent 2 decades, including in vitro maturation (IVM), assisted reproductive technologies (ARTs) achieved noteworthy development. However the efficiency of ARTs with in vitro matured oocytes is still lower than that with in vivo oocytes. To overcome those limitations, many researchers attempted to adapt co-culture system during IVM and consequently maturation efficiency has been increased. The beneficial effects of applying co-culture system is contemplated base on communication and interaction between various somatic cells and oocytes, achievement of paracrine factors, and spatial effects of extracellular matrix (ECM) from somatic cell surface. The understanding of co-culture system can provide some information to narrow the gap between in vitro and in vivo. Here we will review current studies about issues for understanding cu-culture system with various somatic cells to improve in vitro maturation microenvironment and provide bird view and strategies for further studies.

Inflammation mainly mediated by innate immune cells as the first line of host defense against pathogens is an acute response that limits tissue damage and eliminates pathogens in the body. In triggering inflammation, several pattern recognition receptors work together; membrane-associated Toll-like receptors, c-type lectin receptors, retinoic acid-inducible gene-like helicase receptors, absent in melanoma-like receptors, and cytosolic nucleotide-binding domain and leucine-rich repeat receptors. Among them, inflammasome is a newly trigger of inflammation in response to exogenous and endogenous stimuli and its activation leads to the assembly of multiprotein platforms composed of NLRP3 (NOD-like receptor family, pyrin domain containing 3), ASC (apoptosis associated speck-like protein containing a CARD), and procaspase 1. Thus, the activated inflammasome activates caspase 1, resulting in processing and secretion of interleukin (IL)-1β. Recent emerging data suggest that dysregulated metabolites, i.e., amyloids, ceramides, and cholesterol crystals, have been classified as inflammasome activators. In addition, IL-1β may play a critical role in the pathogenesis of chronic inflammation-induced disorders such as Alzheimer’s diseases, type 2 diabetes, and atheriosclerosis. This review introduces the basic concept of inflammasome activation and auto-inflammatory diseases. In addition, it discusses the updated signaling models of inflammasome activation that link metabolic dysfunction in order to outline future therapeutic approaches to inflammasome-mediating diseases.

The present study assessed the effect of FSH and LH on oocyte meiotic, cytoplasmic maturation and on the expression level and polyadenylation status of several maternal genes. Cumulus-oocyte complexes were cultured in the presence of FSH, LH, or the combination of FSH and LH. Significant cumulus expansion and nuclear maturation was observed upon exposure to FSH alone and to the combination of FSH and LH. The combination of FSH and LH during entire IVM increased the mRNA level of four maternal genes, C-mos, Cyclin B1, Gdf9 and Bmp15, at 28 h. Supplemented with FSH or LH significantly enhanced the polyadenylation of Gdf9 and Bmp15; and altered the expression level of Gdf9 and Bmp15. Following parthenogenesis, the exposure of oocytes to combination of FSH and LH during IVM significantly increased cleavage rate, blastocyst formation rate and total cell number, and decreased apoptosis. In addition, FSH and LH down-regulated the autophagy gene Atg6 and upregulated the apoptosis gene Bcl-xL at the mRNA level in blastocysts. These data suggest that the FSH and LH enhance meiotic and cytoplasmic maturation, possibly through the regulation of maternal gene expression and polyadenylation. Overall, we show here that FSH and LH inhibit apoptosis and autophagy and improve parthenogenetic embryo competence and development.

The objective of this study was to examine the effect of in vitro maturation (IVM) medium, cytochalasin B (CB) treatment during intracytoplasmic sperm injection (ICSI), and electric activation on in vitro development ICSI-derived embryos in pigs. Immature pig oocytes were matured in vitro in medium 199 (M199) or porcine zygote medium (PZM)-3 that were supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 21～22 h. ICSI embryos were produced by injecting single sperm directly into the cytoplasm of IVM oocytes. The oocytes matured in PZM-3 with 61.6 mM NaCl (low-NaCl PZM-3) tended to decrease (0.05<P<0.1) nuclear maturation when compared with oocytes matured in M199 (76.9% vs. 83.8%) but no significant differences were found in embryo cleavage, blastocyst formation, and mean number of cells in blastocyst (73.8% vs. 74.6%, 11.1% vs. 12.1%, and 28.4 cells vs. 30.1 cells, respectively). The oocyte degeneration was not reduced by CB treatment during ICSI (11.9%) when compared with no treatment control (11.3%) while the treatment showed detrimental effects (P<0.05) on embryonic cleavage (40.0%) and blastocyst formation (1.8%) rates when compared with control (60.0% and 11.5%, respectively). For activation of ICSI oocytes, additional electric stimulus has no positive or negative effect on in vitro development of preimplantation stage ICSI porcine embryos. Our results demonstrate that CB treatment during ICSI inhibits embryonic development of ICSI oocytes and additional electric activation after ICSI has no effect in improving ICSI embryonic development in pigs. Further studies are needed to improve ICSI efficiency by investigating factors influencing embryonic development after ICSI in pigs.

In canine, oocytes are ovulated at the GV (germinal vesicle) stage and they have to fulfill maturation phase before reaching metaphase II stage. The efficiency of in vitro maturation is still very low. Therefore, the aim of this study was to investigate the effect of in vitro maturation on nuclear changes of immature canine oocytes recovered from different reproductive stages ovaries and different culture conditions. The oocytes were cultured in TCM-199 with supplement at 5% and for 72 h. The nuclear maturation of canine oocytes was evaluated with Hoechst 33342 stain under fluorescence microscope (Fig. 1). The results of this study detected differences in in vitro maturation rate between oocytes recovered from follicle status and non-follicle status ovaries. However, these differences were not significant as indicated in Table 1 and Fig. 2. In regard to the effect of culture condition with supplements, we did not found significant differences compared with control group (Table 2, Table 3). One of the reasons for this data could be the conditions that ovaries were exposed during slaughtering process or the long distant transportation of the ovaries. Although these data have not shown clearly significant differences results compared with control, furthermore the different reproductive status ovaries was beneficial for maturation of oocytes in vitro and can be a basic part of knowledge to improve in vitro maturation of canine oocytes.

The technique of SCNT is now well established but still remains inefficient. The in vitro development of SCNT embryos is dependent upon numerous factors including the recipient cytoplast and karyoplast. Above all, the metaphase of the second meiotic division (MII)　oocytes have typically become the recipient of choice. Generally high level of MPF present in MII oocytes induces the transferred nucleus to enter mitotic division precociously and causes NEBD and PCC, which may be the critical role for nuclear reprogramming. In the present study we investigated the in vitro development and pregnancy of White-Hanwoo SCNT embryos treated with caffeine (a protein kinase phosphatase inhibitor). As results, the treatment of 10 mM caffeine for 6 h significantly increased MPF activity in bovine oocytes but does not affect the developmental competence to the blastocyst stage in bovine SCNT embryos. However, a significant increase in the mean cell number of blastocysts and the frequency of pregnant on 150 days of White-Hanwoo SCNT embryos produced using caffeine treated cytoplasts was observed. These results indicated that the recipient cytoplast treated with caffeine for a short period prior to reconstruction of SCNT embryos is able to increase the frequency of pregnancy in cow.

Sphingosine-1-phosphate (S1P) has a many function involved proliferation, differentiation and survival of many cells. In this study, to investigate whether S1P improve the developmental competence of porcine embryos, 50 nM of S1P were supplemented during in vitro maturation (with EGF or without EGF) medium and/or in vitro culture (IVC) medium. Addition of S1P was significantly increased the rate of oocytes reaching metaphase II (MII) compared to the control (83.5 vs. 64.1%) in without EGF medium, but not with EGF medium (89.5 vs. 84.6%). When treated with 1 μM of N1N-dimethylsphingosine (DMS), a sphingosine kinase inhibitor which is blocked endogenous generation of S1P, the meiotic progression rates to MII stage (without EGF: 45.2 and with EGF: 66.7%) were significantly decreased and degeneration rates (without EGF: 51.2 and with EGF: 30.1%) were increased in both medium compared to control group during IVM periods. Also, the rates of blastocyst formation was significantly increased in the S1P treated group compared to control group (29.0 vs. 19.2%) of EGF supplemented medium, whereas there were no effect in the EGF free medium (9.0 vs. 10.5%). After 12 h IVM, the phosphorylation of ERK1 and ERK2, which is major signaling pathway of MAP kinase, were increased in the S1P group than that of control or DMS group. When supplemented of S1P during IVC, the rates of blastocyst formation and total cell number (30.2% and 40.6) were significantly increased in S1P-treated group compared with control (20.1% and 32.5), DMS (12.3% and 25.1), and S1P plus DMS group (24.7% and 33.6). The percentage of apoptosis nuclei in the S1P group was significantly decreased than other groups. Also, the rates of blastocyst formation (26.7 vs. 14%) and total cell number (42.8 vs. 32.5) were significantly increased in the S1P group than those of control group when S1P added during the entire IVM and IVC periods. Taken together, our results indicate that S1P supplementation in IVM and/or IVC medium affects beneficial effect of meiotic maturation and subsequent developmental competence of porcine embryos.

The 3-isobutyl-1-methylxanthine (IBMX) is non-selective phosphodiesterase and is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte. The present study was conducted to analyze: (1) nuclear maturation (examined by the Hoechst staining), (2) whether cytoplasmic maturation (examined by the intracellular glutathione (GSH) concentration) of porcine oocytes is improved during meiotic arrest after prematuration (22 h) with IBMX. Before in vitro maturation (IVM), oocytes were treated with 1 mM IBMX for 22 h. After 22 h of pre-maturation, the higher rate of IBMX treated group oocytes were arrested at the germinal vesicle (GV) stage (42.3%) than control IVM oocytes (10.1%). It appears that the effect of IBMX on the resumption of meiosis has shown clearly. In the end of IVM, the reversibility of the IBMX effect on the nuclear maturation has been corroborated in this study by the high proportions of MII stage oocytes (72.5%) reached after 44 h of IVM following the 22 h of inhibition. However, intracellular GSH concentrations were lower in the oocytes treated with IBMX than the control oocytes (6.78 and 12.94 pmol/oocyte, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pre-treated with IBMX for 22 h did not equal that of control oocytes in the current IVM system. These results indicate that pre-maturation with IBMX for 22 h may not be beneficial in porcine IVM system.

The present study was conducted to develop a simple method for porcine oocyte maturation without CO2 regulation. In experiment 1, we evaluated that the effect of CO2 non-supplement on porcine oocyte maturation. Cumulusoocyte complexes (COCs) were collected from 2～6 mm follicles and divided into three groups (Control, tube-CO2, and tube-non-CO2). For control, COCs were cultured in 4-well multidish in a CO2 incubator. For tube-CO2, COCs were cultured in a round-bottom tube in a CO2 incubator, and for tube-non-CO2, COCs were cultured in a round-bottom tube sealed tightly without CO2 supplement in a dry incubator. The proportion of oocytes reached to metaphase II (M-II) was not significantly different among three groups (87.9% to 91.4%). In experiment 2, we evaluated the effect of CO2 non-supplement during oocyte maturation on development of embryos. Oocytes with a polar body were divided into two groups (Control and tube-non-CO2) and applied 1.1 kV/cm or 1.2 kV/cm voltages for parthenogenetic activation. After activation, embryos were cultured for 6 days and examined the development. The proportion of embryos cleaved was not significantly different among treatment (86.3% to 91.5%). The proportion of embryo reached to blastocyst stage was not significantly different among treatment (13.9% to 25.2%). The cell number of blastocysts was not significantly different among treatment (29.0 to 32.4). In conclusion, oocytes cultured in a dry incubator without CO2 supplement have enough competence to development after parthenogenetic activation. These results would be useful for transporting oocytes or embryos a long distance.

Biotechnologies for cloning animals and in vitro embryo production have the potential to produce biomedical models for various researches. Especially, pigs are a suitable model for xenotransplantation, transgenic production and various areas of reproductive research due to its physiological similarities to human. However, utilization of in vitro-produced embryos for transfer remains limited. Despite improvement over past few decades, obstacles associated with the production of good quality embryos in vitro still exist which limit the efficiency of cloning. One of major problems includes improper in vitro maturation (IVM) and culture (IVC). Oxidative stress caused from in vitro culture conditions contributes to inadequate IVM and IVC which leads to poor developmental competence of oocytes, failure of fertilization and embryo development. To reduce the oxidative stress, various antioxidants have been used to IVM and IVC system. However, limited information is available on the effects of resveratrol on livestock reproductions. Resveratrol is a polyphenolic natural product and well known as an antioxidant in foods and beverages (e.g. in grapes and red wine). Resveratrol is known to be cardioprotective, anticarcinogenic, anti-inflammatory, antioxidant and antiapoptotic. This paper will review the effects of resveratrol on in vitro maturation of oocytes and embryo development.

It is well established that mammalian cumulus cell (CC) expansion requires BMP15 (bone morphogenetic protein bone morphogenetic protein 15) and GDF9 (growth differentiation factor 9). However, the mechanisms of the factors in CC expansion are largely unclear. This study was conducted to examine the two paracrine factors and their receptor SMAD intracellular signaling mechanism of mediating porcine CC expansion and oocyte maturation, and to compare COCs (Cumulus–oocyte complexes) maturation to DOs (Denuded oocytes). COCs and DOs were in vitro matured in medium with FSH, LH and TGFB superfamily antagonists. Our results showed that the expansion of COCs was unaffected by addition of GDF9 and BMP15 recombinant protein, but cumulus cell proliferation and DOs maturation rate were enhanced. The mRNA expressions of SMAD receptor confirmed that oocytes secreted factors that activate SMAD3,4 and SMAD1 in granulosa cells and oocytes, but unaffected SMAD2. Treatment of COCs with a SMAD2/3 phosphorylation inhibitor (SB431542) inhibited CC expansion and expression of TNFAIP6. SB431542 also was revealed to inhibit DOs maturation. The activation of CC SMAD signaling by oocytes, and the requirement of SMAD2/3 signaling for expansion and oocyte maturation were studied in pig. Nonetheless, porcine oocyte maturation without SMAD2/3 signaling is likely to be needed for optimal matrix formation, but also BMP15 and GDF9 is likely to be needed in oocyte.

Although evidences showed that histone deacetylation plays an important role in the mitotic and meiotic cell cycle, but the mechanisms are still unclear. Level of histone acetylation can be easily changed by deacetylase inhibitors (HDACi) i.e trichostatin A (TSA) and valporic acid. In this study, we determined whether the inhibition of histone deacetylation by TSA could affect porcine oocyte maturation and aging process. Our results showed that treated COCs with 100 nM TSA significantly increase the GVBD in each time group than 0, 5, 50 nM but no significantly different from that of higher concentration (200 nm or 300 nM). No significant differences on maturation, blastocyst development, MAPK pattern and expressions of apoptosis gene when treated oocytes with 100 nM TSA for the first 24h of IVM compared with control and 5, 50 nM TSA. However, in the oocytes treated with 200 nM and 300 nM TSA for first 24 h, MAPK significantly decreased and abnormal spindle were observed. But, in prolonged (64 h) of TSA treated group has no significantly different in control. Another data observed that after 24h TSA-treat to prolonged group were significantly decreased of MAPK activation and normal spindle than the other group. We concluded that TSA played a critical role in meiotic progression in porcine oocytes through the regulation of arrest GVBD, which prolonging the in vitro maturation time, but unaffected the subsequent pre-implantation embryo developmental potential and embryonic qualities. Moreover, the histone deacetylase inhibitor TSA may artificially control porcine oocyte maturation time and delay porcine oocyte aging process.

In the present study, we investigated the effect of porcine follicular fluid (PFF) concentration (10% vs. 1%) and protein-free media (PFF 0%) on maturation of porcine oocytes in vitro and analysed difference in gene expression in resulting blastocysts following parthenogenetic activation. Three groups were tested; 1) 10% PFF: Tissue culture medium (TCM) 199+10% PFF; 2) 1% PFF: TCM 199+1% PFF; and 3) 0.1% PVA: TCM 199+0.1 PVA. Cumulus-oocyte-complexes were cultured in the respective media containing gonadotrophin (1 ug/ml), epidermal growth factor (10 ng/ml), cystein (0.57 mM), sodium pyruvate (0.91 mM), insulin (5 ug/ml), 9-cis retinoic acid (5 nM) for 20～22 h and then without hormonal supplements for an additional 20-22 h. Data was analyzed using statistical analysis system(SAS) program. There was no significant difference in oocyte maturation rate. However, significantly higher (p<0.05) proportions of embryos developed to the blastocyst stage when oocytes were matured in 10% PFF group (45%) than in the 1% PFF group (31.1%). The total cell numbers were not significantly different among groups (52 ± 1.3 vs. 54.6±3.1 vs. 54.4±2.5, respectively). The relative abundance (ratio to beta-actin mRNA) of gene transcripts related to apoptosis in blastocysts was measured by real- time PCR. The expression of anti-apoptotic gene (BclxL) was up-regulated and the expression of pro-apoptotic gene (Bax) was down-regulated in 10% PFF group than in the other groups. Therefore, it can be concluded that supplementation of 10% PFF during in vitro maturation improves embryo development by reduction of apoptosis. * This study was supported by IPET (#311011-05-1-SB010), RNL Bio (#550-20120006), MKE (#10033839-2011-13), Institute for Veterinary Science, the BK21 program and TS Corporation.

The present study examined the expression of porcine sirtuin 1–3 (Sirt1–3) genes in immature (germinal vesicle; GV stage), mature (metaphase II; MII stage) oocytes, preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the role of sirtuins in oocyte nuclear and cytoplasmic maturation, and embryonic development of PA and IVF embryos using sirtuin inhibitor [5 mM nicotinamide (NAM) and 100 μM sirtinol]. The expression of Sirt1–3 mRNA was significantly (p<0.05) up-regulated during IVM. The expression patterns of Sirt1–3 mRNA in preimplantation embryos of PA, IVF and SCNT were gradually (p<0.05) decreased from MII stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1 and Sirt3 in SCNT blastocysts were significantly lower than IVF blastocysts. Treatment with nicotinamide (NAM) during IVM resulted in significantly decreased nuclear maturation but it was restored when NAM treated with 2 μM resveratrol (RES; known as antioxidant and sirtuin activator) compared to the control (control: 88.9%, NAM: 67.9% and NAM+RES: 86.4% respectively). Intracellular reactive oxygen species (ROS) level of oocytes matured with NAM was significantly increased and with NAM+RES was significantly decreased compared to the control. Treatment with sirtuin inhibitors during IVC resulted in significantly decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate both PA and IVF embryos. Oocytes treated with NAM during IVM showed significantly lower expression of PCNA, Bax, Bcl-2, POU5F1 and Sirt1–3 compared to the control. Oocytes treated with NAM+RES during IVM restored gene expression except POU5F1. Similarly, PA derived blastocysts treated with NAM during IVM showed down-regulation of PCNA, Bax, Bcl–2, POU5F1 and Sirt1–2. The blastocysts derived from PA embryos treated with sirtuin inhibitors during IVC showed lower (p<0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly de -creased in both NAM and sirtinol treated groups compared to the control. These findings indicate that Sirt1–3 which are transcribed and stored during oocyte maturation may have physiological and important roles in porcine oocyte maturation and preimplantation embryonic development by regulating gene expressions. * This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121), Rural Development Administration, Republic of Korea.

The objective of the present study was to investigate the effects of different concentrations of sorbitol supplementation for in vitro maturation medium and in vitro culture medium, on porcine cumulus oocyte complexe(COC) maturation and subsequent developmental capacity after parthenogenetic activation. Porcine COC were cultured for 44 h(0～ 22 h termed MI stage and 22～44 h termed MII stage) in TCM199 without(— ) or with(+) sorbitol (20 μM, 100 μM, 200 μM), and the resultant metaphase II oocytes cultured in PZM-3 for 7 days following activation. Our results showed that supplementation with appropriate concentrations of sorbitol (20 μM) during full term maturation culture(MI+/MII+) significantly(p<0.05) improved blastocyst formation rates and total cell number. When the concentration of sorbitol were increased to 100 μM and 200 μM during maturation culture, the maturation rate of COC were significantly reduced compared with 20 μΜ or control groups. Also blastocyst formation rates significantly(p<0.05) reduced with increasing concentration of sorbitol(200 μM). Supplementation with sorbitol(20 μM, 50 μM, 100 μM) into PZM-3 for in vitro culture significantly(p<0.05) inhibited blastocyst formation compared with control group. However, the blastocyst formation rates start to rise again when 50 μ M sorbitol was used for the first 48 hours and then cultured in PZM-3 without sorbitol. There was no significant difference in cell number between control and sorbitol treated groups. When the activated oocytes were cultured in PZM-3 for 48h and then cultured in PZM-3 with sorbitol, interestingly, the blastocyst formation rate was similar to that of PZM-3 with sorbitol for in vitro culture and significantly lower than control group. These results suggest that addition of low concentrations of sorbitol(20 μM) during oocyte maturation is beneficial for subsequent blastocyst development and improved embryo quality. However, treatment with sorbitol supplementation during in vitro culture medium is negative effect to blastocyst formation.