Failure to maintain full-term pregnancies in pig carrying klotho monoallelic knockout fetuses.

BACKGROUND: Small animals that show a deficiency in klotho exhibit extremely shortened life span with multiple aging-like phenotypes. However, limited information is available on the function of klotho in large animals such as pigs. RESULTS: In an attempt to produce klotho knockout pigs, an sgRNA specific for klotho (targeting exon 3) was designed and Cas9-sgRNA ribonucleoproteins were transfected into porcine fibroblasts. Transfected fibroblasts were cultured for one to 2 days and then directly used for nuclear transfer without selection. The cloned embryos were cultured in vitro for 7 days and analyzed to detect modifications of the klotho gene by both T7E1 and deep sequencing analysis. Modification succeeded in 13 of 20 blastocysts (65%), 8 of which (40.0%) were monoallelic modifications and 5 (25.0%) were biallelic modifications. Based on high mutation rates in blastocysts, we transferred the cloned embryos to 5 recipient pigs; 1 recipient was pregnant and 16 fetuses were recovered at Day 28 post transfer. Of the 16 fetuses, 9 were resorbing and 7 were viable. Four of 9 (44.4%) resorbing fetuses and 3 of the 7 (42.9%) viable fetuses had monoallelic modifications. Thus, 3 klotho monoallelic knockout cell lines were established by primary culture. A total of 2088 cloned embryos reconstructed with 2 frame-shifted cell lines were transferred to 11 synchronized recipients. Of the recipients, 7 of 11 eleven (63.6%) became pregnant. However, none of the pregnancies was maintained to term. To discover why klotho monoallelic knockout fetuses were aborted, expression of aging- and apoptosis-related genes and klotho protein in placentas from klotho monoallelic knockout and wild-type fetuses was investigated. Placentas from klotho monoallelic knockout fetuses showed negatively changed expression of aging- and apoptosis-related genes with lower relative expression of klotho protein. These results indicated that the reason why klotho monoallelic knockout fetuses were not maintained to term was possibly due to decreased klotho expression in placentas, negatively affecting aging- and apoptosis-related genes. CONCLUSIONS: Klotho monoallelic knockout porcine fetal fibroblasts were successfully established. However, pigs carrying klotho monoallelic knockout fetuses failed to maintain full-term pregnancy and a decrease in klotho expression in placenta likely leads to pregnancy loss.

Enhancement of epigenetic reprogramming status of porcine cloned embryos with zebularine, a DNA methyltransferase inhibitor.

Aberrant epigenetic reprogramming is known to be a major cause of inefficient somatic cell nuclear transfer (SCNT) in pigs, and use of epigenetic modification agents, such as DNA methyltransferase inhibitors (DNMTis), is a promising approach for enhancing SCNT efficacy. Here, we attempted to find the optimal condition of zebularine (Zb), a DNMTi, treatment on porcine SCNT embryos during in vitro culture (IVC). As results, treatment with 5 nM Zb for 24 hr showed the highest rate of embryo development to blastocyst compared to other groups (p < .05). Also, the relative intensities of global DNA methylation levels of anti-5-methylcytosine in pseudo-pronuclear (PNC), 2-cell and 4-cell stages were significantly lower in the Zb-treated group (p < .05), however, changes in methylation levels of centromeric satellite repeat were noted only in PNC and blastocyst stages. In addition, significant positive alterations in the relative expression of genes related to pluripotency (OCT4 and SOX2), histone acetylation (HAT1, HDAC1, HDAC2, and HDAC3) and DNA methylation (DNMT1 and DNMT3a) were observed compared to the control (p < .05). In conclusion, we found that Zb could modify DNA methylation levels in the early stages of porcine SCNT embryos and promote their developmental competence.

Effects of manganese on maturation of porcine oocytes in vitro and their subsequent embryo development after parthenogenetic activation and somatic cell nuclear transfer.

This study was carried out to examine the effects of manganese (Mn) on the developmental competence of porcine oocytes during in vitro maturation (IVM) after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). Upon treatment of porcine oocytes with different concentrations (0, 3, 6, and 12 ng/ml) of Mn during IVM, PA was performed to determine the optimum concentration. Following PA, the rate of blastocyst formation was higher significantly in treated porcine oocytes at 6 ng/ml of Mn than in other groups (P < 0.05). However, there was no substantial difference in the cleavage rate and total blastocyst cell numbers among all groups. SCNT was performed using the optimal concentration of Mn from PA, which showed an improved blastocyst formation rate in treated oocytes compared to that in control group (P < 0.05). However, the cleavage rate and total cell numbers per blastocyst were not different between the control and the Mn treated groups after SCNT. Additionally, oocyte nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels were assessed. There was no significant difference observed in nuclear maturation among all the groups. However, enhanced intracellular GSH levels while lower levels of ROS were seen in the Mn treated group compared to the control group (P < 0.05). Thus, these results indicate that Mn supplementation can improve the developmental competence of porcine PA and SCNT embryos by increasing GSH and decreasing ROS levels.

Improved early development of porcine cloned embryos by treatment with quisinostat, a potent histone deacetylase inhibitor.

Recently, the modification of the epigenetic status of somatic cell nuclear transfer (SCNT) embryos by treatment with histone deacetylase inhibitors (HDACis) has made it possible to alter epigenetic traits and improve the developmental competence of these embryos. In the current study, we examined the effects of an HDACi, quisinostat (JNJ), on the in vitro development of porcine cloned embryos and their epigenetic nuclear reprogramming status. SCNT embryos were cultured under various conditions, and we found that treatment with 100 nM JNJ for 24 h post activation could improve blastocyst formation rates compared to the control (P < 0.05). Therefore, this was chosen as the optimal condition and used for further investigations. To explore the effects of JNJ on the nuclear reprogramming of early stage embryos and how it improved cloning efficiency, immunofluorescence staining and quantitative real-time PCR were performed. From the pseudo-pronuclear to 2-cell stages, the levels of acetylation of histone 3 at lysine 9 (AcH3K9) and acetylation of histone 4 at lysine 12 (AcH4K12) increased, and global DNA methylation levels revealed by anti-5-methylcytosine (5-mC) antibody staining were decreased in the JNJ-treated group compared to the control (P < 0.05). However, JNJ treatment failed to alter AcH3K9, AcH4K12, or 5-mC levels at the 4-cell embryo stage. Moreover, JNJ treatment significantly upregulated the expression of the development-related genes OCT4, SOX2, and NANOG, and reduced the expression of genes related to DNA methylation (DNMT1, DNMT3a, and DNMT3b) and histone acetylation (HDAC1, HDAC2, and HDAC3). Together, these results suggest that treatment of SCNT embryos with JNJ could promote their developmental competence by altering epigenetic nuclear reprogramming events.

Sonic hedgehog signaling mediates resveratrol to improve maturation of pig oocytes in vitro and subsequent preimplantation embryo development.

The beneficial effects of resveratrol on in vitro maturation (IVM) have been explained mainly by indirect antioxidant effects and limited information is available on the underlying mechanism by which resveratrol acts directly on porcine cumulus oocyte complexes (COCs). Recently, several studies reported that sonic hedgehog (SHH) signaling mediates resveratrol to exert its biological activities. Furthermore, SHH is an important signaling molecule for follicle development, oocyte maturation, and embryo development. Therefore, to elucidate the relationship between resveratrol and SHH signaling, we designed three groups: (i) control; (ii) resveratrol; and (iii) resveratrol with cyclopamine (SHH signaling inhibitor). We evaluated the effects of these agents on cumulus expansion, oocyte maturation, embryo development after parthenogenetic activation, expression levels of mRNAs in cumulus cells, oocytes and blastocysts, and protein expression in COCs. Resveratrol significantly increased the proportion of COCs exhibiting complete cumulus expansion (degree 4), oocyte nuclear maturation, cleavage and blastocyst formation rates and total cell numbers, which were blocked in the presence of cyclopamine. At the same time, a significant increase in the expression levels of mRNAs related to cumulus expansion, oocyte maturation and SHH signaling-related mRNAs and proteins from the resveratrol treatment group was also inhibited by simultaneous addition of cyclopamine. In conclusion, our results indicate that SHH signaling mediates resveratrol to improve porcine cumulus expansion, oocyte maturation, and subsequent embryo development.

A potential role of knockout serum replacement as a porcine follicular fluid substitute for in vitro maturation: Lipid metabolism approach.

The use of supplements, such as porcine follicular fluid (pFF), fetal bovine serum and human serum albumin are widely used during in vitro maturation (IVM) in different species but these supplements contain undefined components that cause technical difficulties in standardization and influence the efficiency of IVM. Knockout serum replacement (KSR) is a synthetic protein source, without any undefined growth factors or differentiation-promoting factors. Therefore, it is feasible to use KSR as a defined component for avoiding effects of unknown molecules in an IVM system. In this study, the rates of oocyte maturation and blastocyst formation after parthenogenetic activation (PA), somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) were significantly higher in the 5% KSR supplemented group than in the unsupplemented control group and more similar to those of the 10% pFF supplemented group. Moreover, the intensity of GDF9, BMP15, ROS, GSH, BODIPY-LD, BODIPY-FA, and BODIPY-ATP staining showed similar values between 5% KSR and 10% pFF, which have significant difference with control group. Most of the gene expression related to lipid metabolism with both supplements exhibited similar patterns. In conclusion, 5% KSR upregulated lipid metabolism and thereby provides an essential energy source to sustain and improve oocyte quality and subsequent embryo development after PA, SCNT, and IVF. These indications support the idea that KSR used as a defined serum supplement for oocyte IVM might be universally used in other species.

Stimulatory Effects of Melatonin on Porcine In Vitro Maturation Are Mediated by MT2 Receptor.

Melatonin is a multifunctional molecule with numerous biological activities. The fact that melatonin modulates the functions of porcine granulosa cells via the MT2 receptor suggests the possibility of MT2 receptor-mediation for melatonin to promote cumulus expansion of porcine cumulus-oocyte complexes (COCs). Therefore, we investigated the presence of MT2 in porcine COCs, and the effects of melatonin with or without selective MT2 antagonists (luzindole and 4-P-PDOT) on this process; COCs underwent in vitro maturation culturing with six different conditions (control, melatonin, luzindole, 4-P-PDOT, melatonin + luzindole or melatonin + 4-P-PDOT). Cumulus expansion, oocyte nuclear maturation, and subsequent embryo development after parthenogenetic activation (PA) were evaluated. In experiment 1, MT2 was expressed in both oocytes and cumulus cells. In experiment 2, melatonin significantly increased the proportion of complete cumulus expansion (degree 4), which was inhibited by simultaneous addition of either luzindole or 4-P-PDOT. A similar pattern was observed in the expression of genes related to cumulus expansion, apoptosis, and MT2. In experiment 3, no significant difference was observed in immature, degenerate, and MII oocyte rates among the groups. In experiment 4, melatonin significantly increased blastocyst formation rates and total blastocyst cell numbers after PA, but these effects were abolished when either luzindole or 4-P-PDOT was added concomitantly. In conclusion, our results indicate that the MT2 receptor mediated the stimulatory effects of melatonin on porcine cumulus expansion and subsequent embryo development.

The HDAC Inhibitor LAQ824 Enhances Epigenetic Reprogramming and In Vitro Development of Porcine SCNT Embryos.

BACKGROUND/AIMS: Hypoacetylation caused by aberrant epigenetic nuclear reprogramming results in low efficiency of mammalian somatic cell nuclear transfer (SCNT). Many epigenetic remodeling drugs have been used in attempts to improve in vitro development of porcine SCNT embryos. In this study, we examined the effects of LAQ824, a structurally novel histone acetylase inhibitor, on the nuclear reprogramming and in vitro development of porcine SCNT embryos. METHODS: LAQ824 treatment was supplemented during the culture of SCNT embryos. The reprogramming levels were measured by immunofluorescence and quantified by image J software. Relative expression levels of 18 genes were analyzed by quantitative real-time PCR. RESULTS: 100 nM LAQ824 treatment of post-activation SCNT embryos for 24 h significantly improved the subsequent blastocyst formation rate. The LAQ824 treatment enhanced histone 3 lysine 9 (H3K9) levels, histone 4 lysine 12 (H4K12) levels, and reduced global DNA methylation levels as well as anti-5-methylcytosine (5-mC) at the pseudo-pronuclear and 2-cell stages. Furthermore, LAQ824 treatment positively regulated the mRNA expression of genes for histone acetylation (HAT1, HDAC1, 2, 3, and 6), DNA methylation (DNMT1, 3a and 3b), development (Pou5f1, Nanog, Sox2, and GLUT1) and apoptosis (Bax, Bcl2, Caspase 3 and Bak) in blastocysts. CONCLUSION: Optimum exposure (100 nM for 24 h) to LAQ824 post-activation improved the in vitro development of porcine SCNT embryos by enhancing levels of H3K9 and H4K12, reducing 5-mC, and regulating gene expression.

Generation of CMAHKO/GTKO/shTNFRI-Fc/HO-1 quadruple gene modified pigs.

As an alternative source of organs for transplantation into humans, attention has been directed to pigs due to their similarities in biological features and organ size. However, severe immune rejection has prevented successful xenotransplantation using pig organs and tissues. To overcome immune rejection, recently developed genetic engineering systems such as TALEN coupled with somatic cell nuclear transfer (SCNT) to make embryos could be used to produce pigs compatible with xenotransplantation. We used the TALEN system to target the non-Gal antigen cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in pigs that is naturally deleted in humans. Gal-deleted cells expressing both soluble human tumor necrosis factor receptor I IgG1-Fc (shTNFRI-Fc) and human hemagglutinin -tagged-human heme oxygenase-1 (hHO-1) were transfected with a TALEN target for CMAH. Cells lacking CMAH were negatively selected using N-glyconeuraminic acid (Neu5Gc)/magnetic beads and the level of Neu5Gc expression of isolated cells were analyzed by FACS and DNA sequencing. Cloned embryos using 3 different genetically modified cell clones were respectively transferred into 3 recipients, with 55.6% (5/9) becoming pregnant and three cloned pigs were produced. Successful genetic disruption of the CMAH gene was confirmed by sequencing, showing lack of expression of CMAH in tail-derived fibroblasts of the cloned piglets. Besides decreased expression of Neu5Gc in piglets produced by SCNT, antibody-mediated complement-dependent cytotoxicity assays and natural antibody binding for examining immuno-reactivity of the quadruple gene modified pigs derived from endothelial cells and fibroblasts were reduced significantly compared to those of wild type animals. We conclude that by combining the TALEN system and transgenic cells, targeting of multiple genes could be useful for generating organs for xenotransplantation. We produced miniature pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc/hHO-1 that will be suitable for xenotransplantation by overcoming hyperacute, acute and anti-inflammatory rejection.

Melatonin regulates lipid metabolism in porcine oocytes.

It is being increasingly recognized that the processes of lipogenesis and lipolysis are important for providing an essential energy source during oocyte maturation and embryo development. Recent studies demonstrated that melatonin has a role in lipid metabolism regulation, including lipogenesis, lipolysis, and mitochondrial biogenesis. In this study, we attempted to investigate the effects of melatonin on lipid metabolism during porcine oocyte in vitro maturation. Melatonin treatment significantly enhanced the number of lipid droplets (LDs) and upregulated gene expression related to lipogenesis (ACACA, FASN, PPARγ, and SREBF1). Oocytes treated with melatonin formed smaller LDs and abundantly expressed several genes associated with lipolysis, including ATGL, CGI-58, HSL, and PLIN2. Moreover, melatonin significantly increased the content of fatty acids, mitochondria, and ATP, as indicated by fluorescent staining. Concomitantly, melatonin treatment upregulated gene expression related to fatty acid ß-oxidation (CPT1a, CPT1b, CPT2, and ACADS) and mitochondrial biogenesis (PGC-1α, TFAM, and PRDX2). Overall, melatonin treatment not only altered both the morphology and amount of LDs, but also increased the content of fatty acids, mitochondria, and ATP. In addition, melatonin upregulated mRNA expression levels of lipogenesis, lipolysis, ß-oxidation, and mitochondrial biogenesis-related genes in porcine oocytes. These results indicated that melatonin promoted lipid metabolism and thereby provided an essential energy source for oocyte maturation and subsequent embryonic development.

Melatonin influences the sonic hedgehog signaling pathway in porcine cumulus oocyte complexes.

Melatonin, which is synthesized in the pineal gland and peripheral reproductive organs, has antioxidant properties and regulates physiological processes. It is well known that melatonin affects in vitro maturation (IVM) of oocytes and embryonic development in many species. However, beneficial effects of melatonin on IVM have been explained mainly by indirect antioxidant effects and little information is available on the underlying mechanism by which melatonin directly acts on porcine cumulus oocyte complexes (COCs). Sonic hedgehog (Shh) signaling is important for follicle development, oocyte maturation, and embryo development, and there may be a relationship between melatonin and Shh signaling. To examine this, we designed three groups: (i) control, (ii) melatonin (10-9  mol/L), and (iii) melatonin with cyclopamine (2 µmol/L; Shh signaling inhibitor). The aim of this study was to investigate the effects of these agents on cumulus expansion, oocyte maturation, embryo development after parthenogenetic activation (PA), gene expression in cumulus cells, oocytes and blastocysts, and protein expression in COCs. Melatonin significantly increased the proportion of COCs exhibiting complete cumulus expansion (degree 4), PA blastocyst formation rates, and total cell numbers, which were inhibited by addition of cyclopamine. Simultaneously, the expression of cumulus expansion-related genes (Ptgs1, Ptgs2, and Has2) and Shh signaling-related genes (Shh, Pthc1, Smo, and Gli1) and proteins (Ptch1, Smo, and Gli1) in cumulus cells was upregulated in the melatonin-treated group, and these effects were also inhibited by cyclopamine. In conclusion, our results suggest that Shh signaling mediates effects of melatonin to improve porcine cumulus expansion and subsequent embryo development.

PXD101 significantly improves nuclear reprogramming and the in vitro developmental competence of porcine SCNT embryos.

In this study, we investigated the effects of the histone deacetylase inhibitor PXD101 (belinostat) on the preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos and their expression of the epigenetic markers histone H3 acetylated at lysine 9 (AcH3K9). We compared the in vitro developmental competence of SCNT embryos treated with various concentrations of PXD101 for 24h. Treatment with 0.5 µM PXD101 significantly increased the proportion of SCNT embryos that reached the blastocyst stage, in comparison to the control group (23.3% vs. 11.5%, P<0.05). We tested the in vitro developmental competence of SCNT embryos treated with 0.5 µM PXD101 for various amounts of times following activation. Treatment for 24h significantly improved the development of porcine SCNT embryos, with a significantly higher proportion of embryos reaching the blastocyst stage in comparison to the control group (25.7% vs. 10.6%, P<0.05). PXD101-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and four fetuses developed. PXD101 treatment significantly increased the fluorescence intensity of immunostaining for AcH3K9 in embryos at the pseudo-pronuclear and 2-cell stages. At these stages, the fluorescence intensities of immunostaining for AcH3K9 were significantly higher in PXD101-treated embryos than in control untreated embryos. In conclusion, this study demonstrates that PXD101 can significantly improve the in vitro and in vivo developmental competence of porcine SCNT embryos and can enhance their nuclear reprogramming.

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer.

Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P>0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P<0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2mM valproic acid for 24h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

SINE-Associated LncRNA SAWPA Regulates Porcine Zygotic Genome Activation.

In mice, retrotransposon-associated long noncoding RNAs (lncRNA) play important regulatory roles in pre-implantation development; however, it is largely unknown whether they function in the pre-implantation development in pigs. The current study aims to screen for retrotransposon-associated lncRNA in porcine early embryos and identifies a porcine 8-cell embryo-specific SINE-associated nuclear long noncoding RNA named SAWPA. SAWPA is essential for porcine embryonic development as depletion of SAWPA results in a developmental arrest at the 8-cell stage, accompanied by the inhibition of the JNK-MAPK signaling pathway. Mechanistically, SAWPA works in trans as a transcription factor for JNK through the formation of an RNA-protein complex with HNRNPA1 and MED8 binding the SINE elements upstream of JNK. Therefore, as the first functional SINE-associated long noncoding RNAs in pigs, SAWPA provides novel insights for the mechanism research on retrotransposons in mammalian pre-implantation development.

Generation of transgenic Wuzhishan miniature pigs expressing monomeric red fluorescent protein by somatic cell nuclear transfer.

Red fluorescent protein and its variants enable researchers to study gene expression, localization, and protein-protein interactions in vitro in real-time. Fluorophores with higher wavelengths are usually preferred since they efficiently penetrate tissues and produce less toxic emissions. A recently developed fluorescent protein marker, monomeric red fluorescent protein (mRFP1), is particularly useful because of its rapid maturation and minimal interference with green fluorescent protein (GFP) and GFP-derived markers. We generated a pCX-mRFP1-pgk-neoR construct and evaluated the ability of mRFP1 to function as a fluorescent marker in transgenic Wuzhishan miniature pigs. Transgenic embryos were generated by somatic cell nuclear transfer (SCNT) of nuclei isolated from ear fibroblasts expressing mRFP1. Embryos generated by SCNT developed into blastocysts in vitro (11.65%; 31/266). Thereafter, a total of 685 transgenic embryos were transferred into the oviducts of three recipients, two of which became pregnant. Of these, one recipient had six aborted fetuses, whereas the other recipient gave birth to four offspring. All offspring expressed the pCX-mRFP1-pgk-neoR gene as shown by PCR and fluorescence in situ hybridization analysis. The transgenic pigs expressed mRFP1 in all organs and tissues at high levels. These results demonstrate that Wuzhishan miniature pigs can express mRFP1. To conclude, this transgenic animal represents an excellent model with widespread applications in medicine and agriculture.

Melatonin Regulates Lipid Metabolism in Porcine Cumulus-Oocyte Complexes via the Melatonin Receptor 2.

Previous studies suggest that the inclusion of melatonin (MTn) in in vitro maturation protocols improves the developmental competence of oocytes by scavenging reactive oxygen species (ROS). However, the molecular mechanisms integrating melatonin receptor (MT)-mediated lipid metabolism and redox signaling during in vitro cumulus-oocyte complex (COC) development still remain unclear. Here, we aimed to elucidate the potential role of MTn receptors in lipid metabolic adjustments during in vitro porcine COC development. We observed that MTn-mediated Gsα-cAMP/PKA signaling facilitated lipolysis primarily through the MT2 receptor and subsequently increased fatty acid (FA) release by hydrolyzing intracellular triglycerides (TGs) in cumulus cells. Furthermore, CD36 was a critical FA transporter that transported available FAs from cumulus cells to oocytes and promoted de novo TG synthesis in the latter. In addition, MTn regulated lipogenesis and intracellular lipolysis to maintain lipid homeostasis and limit ROS production, thereby supporting oocyte cytoplasmic maturation and the subsequent embryo development. Taken together, these findings provide insight into the possible mechanism integrating MT2-mediated lipid homeostasis and redox signaling, which limits ROS production during in vitro COC development. Therefore, understanding the dynamics of the interactions between lipid homeostasis and redox signaling driven by MT2 is necessary in order to predict drug targets and the effects of therapeutics used to improve female reproductive health.

Tannin Reduces the Incidence of Polyspermic Penetration in Porcine Oocytes.

Tannin (TA) improves porcine oocyte cytoplasmic maturation and subsequent embryonic development after in vitro fertilization (IVF). However, the mechanism through which TA blocks polyspermy after IVF remains unclear. Hence, the biological function of organelles (cortical granule [CG], Golgi apparatus, endoplasmic reticulum [ER], and mitochondria) and the incidence of polyspermic penetration were examined. We found no significant difference in oocyte nuclear maturation among the 1 µg/mL, 10 µg/mL TA, and control groups. Moreover, 100 µg/mL TA significantly reduced 1st polar body formation rate compared to the other groups. Additionally, 1 and 10 µg/mL TA significantly increased the protein levels of GDF9, BMP15, and CDK1 compared to the control and 100 µg/mL TA groups. Interestingly, 1 and 10 µg/mL TA improved the normal distribution of CGs, Golgi, ER, and mitochondria by upregulating organelle-related gene expression and downregulating ER stress (CHOP) gene expression. Simultaneously, 1 and 10 µg/mL TA significantly increased the proportion of normal fertilized oocytes (2 pronuclei; 2 PN) and blastocyst formation rate compared to the control, as well as that of 100 µg/mL TA after IVF by upregulating polyspermy-related genes. In conclusion, TA during IVM enhances 2PN and blastocyst formation rates by regulating organelles' functions and activities.

Ramelteon Reduces Oxidative Stress by Maintenance of Lipid Homeostasis in Porcine Oocytes.

This study aimed to determine the underlying mechanism of ramelteon on the competence of oocyte and subsequent embryo development in pigs during in vitro maturation (IVM). Our results showed that the cumulus expansion index was significantly lower in the control group compared to the ramelteon groups (p < 0.05). Moreover, supplementation of 10−11 and 10−9 M ramelteon significantly increased the cumulus expansion and development-related genes expression, and reduced apoptosis in cumulus cells (p < 0.05). In oocytes, the nuclear maturation rate was significantly improved in 10−11, 10−9, and 10−7 M ramelteon groups compared to the control (p < 0.05). Additionally, the level of intracellular GSH was significantly increased and ROS was significantly decreased in ramelteon-supplemented groups, and the gene expression of oocyte development and apoptosis were significantly up- and down-regulated by 10−11 and 10−9 M ramelteon (p < 0.05), respectively. The immunofluorescence results showed that the protein levels of GDF9, BMP15, SOD1, CDK1, and PGC1α were significantly increased by 10−11 M ramelteon compared to the control (p < 0.05). Although there was no significant difference in cleavage rate, the blastocyst formation rate, total cell numbers, and hatching/-ed rate were significantly improved in 10−11 M ramelteon group compared to the control (p < 0.05). Furthermore, embryo development, hatching, and mitochondrial biogenesis-related genes were dramatically up-regulated by 10−11 M ramelteon (p < 0.05). In addition, the activities of lipogenesis and lipolysis in oocytes were dramatically increased by 10−11 M ramelteon compared to the control (p < 0.05). In conclusion, supplementation of 10−11 M ramelteon during IVM improved the oocyte maturation and subsequent embryo development by reducing oxidative stress and maintenance of lipid homeostasis.

Tannin Supplementation Improves Oocyte Cytoplasmic Maturation and Subsequent Embryo Development in Pigs.

To investigate the effects of tannins (TA) on porcine oocyte in vitro maturation (IVM), different concentrations of TA (0, 1, 10 and 100 µg/mL) were supplemented with a maturation medium and the COCs and subsequent embryonic development were examined. The results showed that 10 µg/mL TA significantly improved the cumulus expansion index (CEI), cumulus-expansion-related genes (PTGS1, PTGS2, PTX-3, TNFAIP6 and HAS2) expression and blastocyst formation rates after parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) compared to the control groups, but not oocyte nuclear maturation. Nevertheless, 10 µg/mL TA dramatically enhanced the mRNA expression of oocyte-development-related genes (BMP15, GDF9, CDC2 and CYCLIN B1), GSH, ATP, SOD1, PGC1α, BMP15, GDF9 and CDC2 levels and reduced intracellular ROS level in porcine oocytes. These results indicated that porcine oocyte cytoplasmic maturation was improved by 10 µg/mL TA treatment during IVM. In contrast, a high concentration of TA (100 µg/mL) significantly decreased the CEI and PTGS1, PTGS2, PTX-3 and HAS2 mRNA expressions in cumulus cells, and reduced oocyte nuclear maturation and the total cell numbers/blastocyst. In general, these data showed that 10 µg/mL TA supplementation has beneficial effects on oocyte cytoplasmic maturation and subsequent embryonic development in pigs.