Jun-Xue Jin

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 β‐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, β‐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.

Supplementation with spermine during in vitro maturation of porcine oocytes improves early embryonic development after parthenogenetic activation and somatic cell nuclear transfer

Spermine plays an important role in protection from reactive oxygen species (ROS) in bacteria, yeast, and mammalian cells, but there are few studies on the effects of spermine on porcine oocyte maturation and subsequent embryo development. The aim of this study was to determine the effects of spermine on in vitro maturation (IVM) of porcine oocytes and their developmental competence after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). We evaluated nuclear maturation, intracellular glutathione (GSH), and ROS levels in oocytes, and their subsequent embryonic development, as well as gene expression in mature oocytes, cumulus cells, and PA blastocysts. After treatment with various concentrations of spermine in IVM culture medium, there was no significant difference in nuclear maturation rate. However, spermine treatment groups (10- 500 µM) showed significantly increased intracellular GSH levels and decreased ROS levels compared to the control ( < 0.05). Furthermore, 10 µM spermine supported significantly higher blastocyst formation rates after PA than the control group ( < 0.05). According to the optimal condition from the PA results, we investigated the effects of 10 µM spermine on SCNT, and it also significantly improved blastocyst formation rates compared with the control group ( < 0.05). In evaluating the effects of 10 µM spermine on gene expression, there was significantly lower expression of a proapoptotic gene () and higher expression of an antiapoptotic gene () in cumulus cells ( < 0.05). was increased in spermine-treated oocytes. Levels of transcription for and were significantly increased in PA blastocysts. In conclusion, 10 µM spermine supplementation during IVM improved the development of porcine PA and SCNT embryos by increasing intracellular GSH, scavenging ROS levels, and regulating gene expression.

Lanosterol influences cytoplasmic maturation of pig oocytes in vitro and improves preimplantation development of cloned embryos

Lanosterol is a precursor of meiosis-activating sterols in the cholesterol biosynthetic pathway and induces a physiological signal that instructs the oocyte to reinitiate meiosis. In this study, we examined the effect of lanosterol on IVM of porcine oocytes, specifically on nuclear maturation, cytoplasmic maturation by investigating intracellular glutathione (GSH) levels and lipid content, embryonic development after parthenogenetic activation and somatic cell nuclear transfer (SCNT), and on gene expression in cumulus cells, oocytes, and SCNT-derived blastocysts. There was no significant difference in nuclear maturation rates between the control and treatment groups (10, 50, and 100 μM of lanosterol added to IVM culture medium). Supplementation with 50-μM lanosterol significantly increased lipid content and GSH levels and decreased reactive oxygen species levels compared with the control. In addition, oocytes treated with 50 μM of lanosterol exhibited significantly increased blastocyst formation rates and total cell numbers after parthenogenetic activation (30.3% and 63.9 vs. 21.6% and 36.5, respectively) and SCNT (18.2% and 53.7 vs. 12.6% and 37.5, respectively), when compared with the control group. Cumulus cells treated with 50 μM of lanosterol showed significantly increased 14α-demethylase, Δ14-reductase, and Δ7-reductase mRNA transcript levels. Significantly increased PPARγ, SREBF1, GPX1, and Bcl-2 and decreased Bax transcript levels were observed in mature oocytes treated with 50 μM of lanosterol compared with the control. SCNT blastocysts derived from 50-μM lanosterol-treated oocytes had significantly higher POU5F1, FGFR2, and Bcl-2 transcript levels than control SCNT-derived blastocysts. In conclusion, supplementation with 50 μM of lanosterol during IVM improves preimplantation development of SCNT embryos by elevating lipid content of oocytes, increasing GSH levels, decreasing reactive oxygen species levels, and regulating genes related to the cholesterol biosynthetic pathway in cumulus cells, to lipid metabolism and apoptosis in oocytes, and their developmental potential and apoptosis in blastocysts.

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.

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.

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.

Establishment and identification of cell lines from type O blood Korean native pigs and their efficiency in supporting embryonic development via somatic cell nuclear transfer

Due to their similarities with humans in anatomy, physiology, and genetics miniature pigs are becoming an attractive model for biomedical research. We aim to establish and evaluate blood type O cells derived from Korean native pig (KNP), a typical miniature pig breed in Korea. Ten cell lines derived from 8 KNP piglets and one adult female KNP (kidney and ear tissues) were established. To confirm the presence of blood type O, genomic DNA, fucosyltransferase (FUT) expression, and immunofluorescence staining were examined. Additionally, fluorescence-activated cell sorting and somatic cell nuclear transfer were performed to investigate the normality of the cell lines and to evaluate their effectiveness in embryo development. We found no significant bands corresponding to specific blood group A, and no increase in FUT expression in cell lines derived from piglets No. 1, No. 4, No. 5, No. 8, and the adult female KNP; moreover, they showed normal levels of expression of α 1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase. There was no significant difference in embryo development between skin and kidney fibroblasts derived from the blood type O KNPs. In conclusion, we successfully established blood type O KNP cell lines, which may serve as a useful model in xenotransplantation research.

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.

71 MELATONIN IMPROVES PORCINE IN VITRO MATURATION VIA SONIC HEDGEHOG SIGNALLING

Melatonin (N-acetyl-5-methoxytryptamine) is the hormone synthesised from the mammalian pineal gland, which has an antioxidant property and regulates physiological processes such as cellular metabolism. It is well known that melatonin affects in vitro maturation of oocytes and embryonic development in many species. However, limited information is available on the underlying beneficial effects of melatonin. Sonic Hedgehog (Shh) signalling is important for follicular development, oocyte maturation, and embryo development. To elucidate the relationship between melatonin and Shh signalling, we designed an experiment with the following three groups: (1) control, (2) melatonin, and (3) melatonin with cyclopamine (smoothened inhibitor) during porcine in vitro maturation. Porcine ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory at 28 to 32°C. The contents of follicles 3 to 6mm in diameter were recovered by aspiration with an 18G needle. Cumulus-oocyte complexes were pooled and cultured in TCM-199 medium for 44h. The aim of this study was to evaluate the effects of melatonin (10-9 M) with or without cyclopamine (2μM) on cumulus cell expansion (a total of 432 cumulus-oocyte complexes were used in 3 replicates), embryo development after parthenogenetic activation (a total of 432 oocytes were used in 4 replicates). Moreover, we detected gene expression related to cumulus expansion, oocyte maturation, and hedgehog signalling in cumulus cells and oocyte. Results indicated that melatonin treatment significantly increased cumulus expansion index (3.75±0.02v. 3.51±0.03 and 3.59±0.05, respectively; P<0.05) and blastocyst formation rates (30.4±2.4v. 21.9±2.2 and 20.0±2.2, respectively; P<0.05) compared with control and melatonin with cyclopamine. In addition, the expression of cumulus expansion-related genes (Ptgs1, Ptgs2, Has2, Ptx-3, and Tnfaip6) and hedgehog signalling-related genes (Shh, Pthc1, Smo, and Gli-1) in cumulus cells were up-regulated in melatonin treatment compared with control and melatonin with cyclopamine. Similarly, the expressions of oocyte maturation-related genes (GDF9 and BMP15) in porcine oocytes were up-regulated in melatonin treatment compared with control and melatonin with cyclopamine. In conclusion, Shh signalling mediated melatonin to improve porcine cumulus cell expansion, oocyte maturation, and subsequent embryo development. Further studies are needed to evaluate the effect of melatonin on protein levels of Shh signalling. Statistical analyses were performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). All data were tested for normality and homoscedasticity and then subjected to one-way ANOVA, followed by Duncans multiple range test (when the variances were assumed to be equal) or Dunnets T3 test (when the variances were assumed to be unequal) to determine differences among experimental groups. All results are expressed as means±SEM; P-values<0.05 were considered to be statistically significant.