In vivo oocytes grow and mature in ovarian follicles whereas oocytes are matured in vitro in plastic culture dishes with a hard surface. In vivo oocytes show a superior developmental ability to in vitro counterparts, indicating suboptimal environments of in vitro culture. This study aimed to evaluate the influence of an agarose matrix as a culture substrate during in vitro maturation (IVM) on the development of pig oocytes derived from small antral follicles (SAFs). Cumulusoocyte complexes (COCs) retrieved from SAFs were grown in a plastic culture dish without an agarose matrix and then cultured for maturation in a plastic dish coated without (control) or with a 1% or 2% (w/v) agarose hydrogel. Then, the effect of the soft agarose matrix on oocyte maturation and embryonic development was assessed by analyzing intra-oocyte contents of glutathione (GSH) and reactive oxygen species (ROS), expression of VEGFA, HIF1A , and PFKP genes, and blastocyst formation after parthenogenesis. IVM of pig COCs on a 1% (w/v) agarose matrix showed a significantly higher blastocyst formation, intra-oocyte GSH contents, and transcript abundance of VEGFA. Moreover, a significantly lower intra-oocyte ROS content was detected in oocytes matured on the 1% and 2% (w/v) agarose matrices than in control. Our results demonstrated that IVM of SAFs-derived pig oocytes on a soft agarose matrix enhanced developmental ability by improving the cytoplasmic maturation of oocytes through redox balancing and regulation of gene expression.

The osmolarity of a medium that is commonly used for in vitro culture (IVC) of oocytes and embryos is lower than that of oviductal fluid in pigs. In vivo oocytes and embryos can resist high osmolarities to some extent due to the presence of organic osmolytes such as glycine and alanine. These amino acids act as a protective shield to maintain the shape and viability in high osmotic environments. The aim of this study was to determine the effects of glycine or/and alanine in medium with two different osmolarities (280 and 320 mOsm) during IVC on embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. To this end, IVC was divided into two stages; the 0-2 and 3-7 days of IVC. In each stage, embryos were cultured in medium with 280, 320, or 360 mOsm and their combinations with or without glycine or/and alanine according to the experimental design. Treatment groups were termed as, for example, "T(osmolarity of a medium used in 0-2 days of IVC)-(osmolarity of a medium used in 3-7 days of IVC)" T280-280 was served as control. When PA embryos were cultured in medium with various osmolarities, T320-280 showed a significantly higher blastocyst formation (29.0%) than control (22.2%) and T360-360 groups (6.9%). Glycine treatment in T320-280 significantly increased blastocyst formation (50.4%) compared to T320-280 only (36.5%) while no synergistic was observed after treatment with glycine and alanine together in T320-280 (45.7%). In contrast to PA embryonic development, the stimulating effect by the culture in T320-280 was not observed in SCNT blastocyst development (27.6% and 23.7% in T280-280 and T320-280, respectively) whereas the number of inner cell mass cells was significantly increased in T320-280 (6.1 cells vs. 9.6 cells). Glycine treatment significantly improved blastocyst formation of SCNT embryos in both T280-280 (27.6% vs. 38.0%) and T320-280 (23.7% vs. 35.3%). Our results demonstrate that IVC in T320-280 and treatment with glycine improves blastocyst formation of PA and SCNT embryos in pigs.

Nitric oxide (NO) has an important role in oocyte maturation and embryonic development in mammals. This study examined the effect of exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP) in a maturation medium on meiotic progression and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. When oocytes were exposed to 0.1 μM SNAP for first 22 h of in vitro maturation (IVM) in Experiment 1, SNAP significantly improved blastocyst development in both defined and standard follicular fluid-supplemented media compared to untreated control (48.4 vs. 31.7-42.5%). SNAP treatment significantly arrested meiotic progression of oocytes at the germinal vesicle stage at 11 h of IVM (61.2 vs. 38.7%). However, there was no effect on meiotic progression at 22 h of IVM (Experiment 2). In Experiment 3, when oocytes were treated with SNAP at 0.001, 0.1 and 10 μM during the first 22 h of IVM to determine a suitable concentration, 0.1 μM SNAP (54.2%) exhibited a higher blastocyst formation than 0 and 10 μM SNAP (36.6 and 36.6%, respectively). Time-dependent effect of SNAP treatment was evaluated in Experiment 4. It was observed that SNAP treatment for the first 22 h of IVM significantly increased blastocyst formation compared to no treatment (57.1% vs. 46.2%). Antioxidant effect of SNAP was compared with that of cysteine. SNAP treatment significantly improved embryonic development to the blastocyst stage (49.1-51.5% vs. 34.4-37.5%) irrespective of the presence or absence of cysteine (Experiment 5). Moreover, SNAP significantly increased glutathione (GSH) content and inversely decreased the reactive oxygen species (ROS) level and mitochondrial oxidative activity in IVM oocytes. SNAP treatment during IVM showed a stimulating effect on in vitro development of SCNT embryos (Experiment 7). These results demonstrates that SNAP improves developmental competence of PA and SCNT embryos probably by maintaining the redox homeostasis through increasing GSH content and mitochondrial quality and decreasing ROS in IVM oocytes.

Poor embryo quality and low blastocyst formation have been major limitations in establishment of cloned embryonic stem cells and production of cloned animals through somatic cell nuclear transfer (SCNT). Aggregation of embryos is a promising method for improving developmental competence of blastocysts. The aim of this study was to improve the blastocyst formation and the quality of parthenogenetic (PA) pig embryos by the aggregation of blastomeres at the 4-cell stage that were cultured in various type of culture dishes with or without phytohemagglutinin (PHA). The PA embryos were produced by the general method of our laboratory. On Day 2 after PA, the zona pellucida of 4 cell-stage embryos were removed by treatment with 0.5% (wt/vol) pronase solution. The 3x zona-free blastomere (ZFB) were randomly distributed in each of the following treatments for aggregation. ZFB were cultured for 5 days at 39℃ in an atmosphere 5% CO2, 5% O2, and 90% N2. In Experiment 1, effect of culture dishes on the aggregation efficiency and developmental competence of PA embryos were investigated. ZFB were cultured on non-coated (control) culture dish or dishes coated with 1% (wt/vol) agarose substrate (AS) or Well of the Well in dishes coated with 1% (wt/vol) agarose substrate (WAS). The ZFB cultured in WAS showed significantly higher (P<0.05) aggregation (81.2%) than AS and control (21.6-45.5%). The mean cell number in blastocysts derived from AS and WAS (81.4-89.3 cells/blastocyst) was significantly higher (P<0.05) than that of control (63.8 cells/blastocyst). In Experiment 2, effects of 150 ug/ml PHA treatment on the aggregation efficiency and developmental competence of embryos were investigated. The ZFB cultured in AS with PHA showed a higher (P<0.05) aggregation rate (90.0%) than that in AS without PHA, control with PHA, and control (39.2%, 57.9% and 17.5%, respectively). In conclusion, aggregation of porcine ZFB treated with PHA and agarose substrate could be a useful technique for producing improving blastocyst development with increased mean cell number of blastocysts in pigs.

In most mammals, metaphase II (MII) oocytes having high maturation promoting factor (MPF) activity have been considered as good oocytes and then used for assisted reproductive technologies including somatic cell nuclear transfer (SCNT). Caffeine increases MPF activity in mammalian oocytes by inhibiting p34cdc2 phosphorylation. The objective of this study was to investigate the effects of caffeine treatment during in Vitro maturation (IVM) on oocyte maturation and embryonic development after SCNT in pigs. To this end, morphologically good (MGCOCs) and poor oocytes (MPCOCs) based on the thickness of cumulus cell layer were untreated or treated with 2.5 mM caffeine during 22-42, 34-42, or 38-42 h of IVM according to the experimental design. Caffeine treatment for 20 h during 22-42 h of IVM significantly inhibited nuclear maturation compared to no treatment. Blastocyst formation of SCNT embryos was not influenced by the caffeine treatment during 38-42 h of IVM in MGCOCs (41.1-42.1%) but was significantly improved in MPCOCs compared to no treatment (43.4 vs. 30.1%, P<0.05). No significant effects of caffeine treatment was observed in embryo cleavage (78.7-88.0%) and mean cell number in blastocyst (38.7-43.5 cells). The MPF activity of MII oocytes in terms of p34cdc2 kinase activity was not influenced by the caffeine treatment in MGCOCs (160.4 vs. 194.3 pg/ml) but significantly increased in MPCOCs (133.9 vs. 204.8 pg/ml). Our results demonstrate that caffeine treatment during 38-42 h of IVM improves developmental competence of SCNT embryos derived from MPCOCs by influencing cytoplasmic maturation including increased MPF activity in IVM oocytes in pigs.

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.