Guoshi Liu

Melatonin promotes the in vitro development of pronuclear embryos and increases the efficiency of blastocyst implantation in murine.

When a defect occurs in the in vitro development of a pronuclear embryo, the interruption of the subsequent implantation limits the success of assisted conception. This common problem remains to be solved. In this study, we observed that melatonin at its physiological concentration (10−7 m) significantly promoted the in vitro development of murine pronuclear embryos. This was indicated by the increased blastocyst rate, hatching blastocyst rate, and blastocyst cell number with melatonin treatment. In addition, when these blastocysts were implanted into female recipient mice, the pregnancy rates (95.0% versus control 67.8%), litter sizes (4.1 pups/litter versus control 2.7 pups/litter), and postnatal survival rates of offspring (96.84% versus control 81.24%) were significantly improved compared with their non‐melatonin‐treated counterparts. Mechanistic studies revealed that melatonin treatment upregulates gene expression of the antioxidant enzyme, superoxide dismutase (SOD), and the anti‐apoptotic factor bcl‐2 while downregulating the expression of pro‐apoptotic genes p53 and caspase‐3. Due to these changes, melatonin treatment reduces ROS production and cellular apoptosis during in vitro embryo development and improves the quality of blastocysts. The implantation of blastocysts with higher quality leads to more healthy offspring and increased pup survival.

Melatonin exists in porcine follicular fluid and improves in vitro maturation and parthenogenetic development of porcine oocytes.

Abstract:  This study focused on the effect of melatonin on in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Melatonin was measured in porcine follicular fluid of follicles of different sizes in the same ovary. Melatonin exists in follicular fluid, and the concentration is approximately 10−11 m. Its concentration decreased as the diameter of follicle increased, which suggests an effect of melatonin on oocyte maturation. Therefore, immature oocytes were cultured in vitro in maturation medium supplemented with melatonin (10−11, 10−9, 10−7, 10−5 and 10−3 m) or without melatonin. The oocytes at maturation stage were collected and activated. The parthenogenetic embryos were cultured and observed in medium supplemented with or without melatonin. Fresh immature oocytes without melatonin treatment were used as control. When only maturation medium was supplemented with 10−9 m melatonin, the cleavage rate, blastocyst rate and the cell number of blastocyst (70 ± 4.5%, 28 ± 2.4% and 50 ± 6.5%) were significantly higher (P < 0.05) than that of controls; when only culture medium was supplemented with melatonin, the highest cleavage rate, blastocyst rate and the cell number of blastocyst was observed at 10−7 m melatonin, which were significantly higher than that of controls (P < 0.05). The best results (cleavage rates 79 ± 8.4%, blastocyst rates 35 ± 6.7%) were obtained when both the maturation and culture medium were supplemented with 10−9 m melatonin respectively (P < 0.05). In conclusion, exogenous melatonin at the proper concentration may improve the in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Further research is needed to identify the effect of melatonin on in vitro and in vivo oocyte maturation and embryo development in porcine.

Changes in melatonin levels in transgenic ‘Micro-Tom’ tomato overexpressing ovine AANAT and ovine HIOMT genes

In animals, the melatonin biosynthesis pathway has been well defined after the isolation and identification of the four key genes that are involved in the conversion of tryptophan to melatonin. In plants, there are special alternative catalyzing steps, and plant genes share very low homology with the animal genes. It was of interest to examine the phenotype of transgenic Micro‐Tom tomato plants overexpressing the homologous sheep oAANAT and oHIOMT genes responsible for the last two steps of melatonin synthesis. The oAANAT transgenic plants have higher melatonin levels and lower indoleacetic acid (IAA) contents than control due to the competition for tryptophan, the same precursor for both melatonin and IAA. Therefore, the oAANAT lines lose the ‘apical dominance’ inferring that melatonin likely lacks auxin activity. The significantly higher melatonin content in oHIOMT lines than oAANAT lines provides new proof for the important role of ASMT in plant melatonin synthesis. In addition, the enhanced drought tolerance of oHIOMT lines will also be an important contribution for plant engineering.

Beneficial effect of resveratrol on bovine oocyte maturation and subsequent embryonic development after in vitro fertilization

OBJECTIVE To analyze the potential beneficial effects and mechanisms of action of resveratrol on the maturation of bovine oocytes that were incubated in different concentrations of resveratrol (0.1, 1.0, or 10.0 μM) as germinal vesicle-stage oocytes. DESIGN In vitro prospective study. SETTING University research laboratory. ANIMAL(S) Animal models for human studies. INTERVENTION(S) In vitro culture in the presence of various concentrations of the antioxidant resveratrol. MAIN OUTCOME MEASURE(S) Parameters of hormone levels, oocyte nuclear maturation, cumulus expansion, levels of intracellular glutathione and reactive oxygen species, embryonic cleavage, blastocyst formation, gene expression associated with mature bovine oocytes and cumulus cells, and level of sirtuin 1 gene expression. RESULT(S) Resveratrol statistically significantly increased progesterone secretion and decreased estradiol-17β secretion by cumulus cells. The elevated levels of progesterone activated the Mos/MEK/p42 mitogen-activated protein kinase (MAPK) cascade in the oocytes. At a concentration of 1.0 μM, resveratrol statistically significantly improved cumulus expansion, polar body formation, the (hatched) blastocyst rate, and the mean number of cells/blastocysts. Meanwhile, resveratrol statistically significantly reduced the level of reactive oxygen species (ROS) and increased the level of glutathione (GSH). For the first time, the expression of the sirtuin-1 gene was identified in granulosa cells, cumulus cells, oocytes, and blastocysts. Further studies revealed that resveratrol promoted sirtuin-1 gene expression. CONCLUSION(S) Resveratrol promoted bovine oocyte maturation and subsequent post-in vitro fertilization embryonic development by inducing progesterone secretion and an antioxidant effect, probably in a manner dependent on sirtuin-1.

Beneficial effects of melatonin on bovine oocytes maturation: a mechanistic approach

This study was performed to investigate the effect of melatonin on bovine oocyte maturation and subsequent embryonic development in vitro. The endogenous melatonin concentration in bovine follicular fluid is approximately 10−11 m. To examine the potential beneficial effects of melatonin on bovine oocyte maturation in vitro, germinal vesicle (GV) oocytes were incubated with different concentrations of melatonin (10−11, 10−9, 10−7, 10−5, 10−3 m). Melatonin supplementation at suitable concentrations significantly promoted oocyte maturation. The development of embryos and the mean cell number/blastocyst produced after in vitro fertilization were remarkably improved. The most effective melatonin concentrations obtained from the studies ranged from 10−9 to 10−7 m. The expression of melatonin receptor MT1 and MT2 genes was identified in cumulus cells, granulosa cells, and oocytes using reverse transcription PCR, immunofluorescence, and Western blot. The mechanistic studies show that the beneficial effects of melatonin on bovine oocyte maturation are mediated via melatonin membrane receptors as the melatonin receptor agonist (IIK7) promotes this effect while the melatonin receptor antagonist (luzindole) blocks this action. Mechanistic explorations revealed that melatonin supplementation during bovine oocyte maturation significantly up‐regulated the expressions of oocyte maturation‐associated genes (GDF9, MARF1, and DNMT1a) and cumulus cells expansion‐related gene (PTX3, HAS1/2) and that LHR1/2, EGFR are involved in signal transduction and epigenetic reprogramming. The results obtained from the studies provide new information regarding the mechanisms by which melatonin promotes bovine oocyte maturation in vitro and provide an important reference for in vitro embryo production of bovine and the human‐assisted reproductive technology.

Beneficial effects of melatonin on in vitro bovine embryonic development are mediated by melatonin receptor 1.

In the current study, a fundamental question, that is, the mechanisms related to the beneficial effects of melatonin on mammalian embryonic development, was addressed. To examine the potential beneficial effects of melatonin on bovine embryonic development, different concentrations of melatonin (10−11, 10−9, 10−7, 10−5, 10−3 m) were incubated with fertilized embryos. Melatonin in the range of 10−11 to 10−5 m significantly promoted embryonic development both in early culture medium (CR1aa +3 mg/mL BSA) and in later culture medium (CR1aa + 6%FBS). The most effective concentrations applied in the current studies were 10−9 and 10−7 m. Using quantitative real‐time PCR with immunofluorescence and Western blot assays, the expression of melatonin receptor MT1 and MT2 genes was identified in bovine embryos. Further studies indicate that the beneficial effects of melatonin on bovine embryo development were mediated by the MT1 receptor. This is based on the facts that luzindole, a nonselective MT1 and MT2 antagonist, blocked the effect on melatonin‐induced embryo development, while 4‐P‐PDOT, a selective MT2 antagonist, had little effect. Mechanistic explorations uncovered that melatonin application during bovine embryonic development significantly up‐regulated the expression of antioxidative (Gpx4, SOD1, bcl‐2) and developmentally important genes (SLC2A1, DNMT1A, and DSC2) while down‐regulating expression of pro‐apoptotic genes (P53, BAX, and Caspase‐3). The results obtained from the current studies provide new information regarding the mechanisms by which melatonin promotes bovine embryonic development under both in vitro and in vivo conditions.

Mitochondria Synthesize Melatonin to Ameliorate Its Function and Improve Mice Oocyte’s Quality under in Vitro Conditions

The physiology of oocyte in vitro maturation remains elusive. Generally, the oocytes have a very low maturation rate under in vitro conditions. In the current study, we found that melatonin promotes the maturation of oocytes in which mitochondria play a pivotal role. It was identified that; (1) mitochondria are the major sites for melatonin synthesis in oocytes and they synthesize large amounts of melatonin during their maturation; (2) melatonin improves mitochondrial function by increased mtDNA copy, mitochondrial membrane potential (ΔΨm) and mitochondrial distribution and ATP production in oocytes; (3) the meiotic spindle assembly is enhanced; (4) melatonin reduces ROS production and inhibits 8-oxodG formation, thereby protecting potential DNA mutation from oxidative damage. As a result, melatonin improves the quality of oocytes, significantly accelerates the developmental ability of IVF embryo. The results provide novel knowledge on the physiology of oocyte’s maturation, especially under in vitro conditions.

Microstructural percolation assisted breakthrough of trade-off between strength and ductility in CuZr-based metallic glass composites

As two important mechanical properties, strength and ductility generally tend to be muturally exclusive in conventional engineering materials. The breakthrough of such a trade-off has been potentiated by the recently developed CuZr-based bulk metallic glass (BMG) composites ductilized by a shape memory CuZr(B2) phase. Here the microstructural dependences of tensile properties for the CuZr-based BMG composites were elucidated qualitatively and modeled quantitatively, and the underlying mechanisms were unraveled. Through the microstructural percolation induced by matching the length scales of particle size and interparticle spacing, a notable breakthrough was achieved in the composites that the general conflicts between strength and ductility can be defeated. This study is expected to greatly aid in the microstructural design and tailoring for improved properties of BMG composites. It also has implications for the development of strong and ductile materials in the future.

Melatonin protects porcine oocyte in vitro maturation from heat stress.

Melatonin is a pleiotropic molecule which plays an important role in animal reproductive activities. Because of the increased global warming, the impact of heat stress (HS) on stockbreeding has become an inevitable issue to be solved. To investigate the potential effects of melatonin on the in vitro maturation of porcine oocyte under the HS, a HS model for porcine oocyte maturation has been used in this study and the different concentrations of melatonin (10−6–10−9 m) were also tested for their protective effects on oocytes. The polar body rate, the index of the nuclear maturation of the oocytes, and the cleavage rate as well as the blastocyst rate were measured to evaluate the developmental competence of the oocytes after parthenogenetic activation (PA). The results showed that HS [in vitro maturation (IVM) 20–24 hr, 42°C] significantly reduced the polar body rate of oocytes and the blastocyte rate of porcine PA embryos, while melatonin (10−7 m) application not only improved polar body rate and blastocyte rate, but also preserved the normal levels of steroid hormone which is disrupted by HS. The presence of melatonin (10−7 m) during the oocyte maturation under the HS reduced reactive oxygen species (ROS) formation, enhanced glutathione (GSH) production, inhibited cell apoptosis, and increased the gene expressions of SIRT1, AKT2, and Polg2. Importantly, the endogenously occurring melatonin of cumulus–oocyte complexes was significantly induced by HS. The results indicated that melatonin application effectively protected the oocytes from HS. These observations warranted the further studies in vivo regarding to improve the reproductive activities of animals under the global warming environment.

Effects of Melatonin on In Vitro Development of Mouse Two-Cell Embryos Cultured in HTF Medium

Melatonin is capable of improving the developmental capacity of ovine, porcine and bovine embryos in vitro. However, whether melatonin possesses similar benefits to the in vitro mouse embryonic development has yet to be determined. In this study, we assessed the effects of various concentrations of melatonin (10–13 to 10–3 M) on the in-vitro development of mouse embryos cultured in HTF medium for 96 hr; embryos cultured without melatonin were used as control. The in vitro development of mouse two-cell embryos significantly benefited from treatment with melatonin in a concentration-dependent manner. The effects of melatonin on the rates of blastocyst formation, hatching/hatched blastocysts and cell number per blastocyst were bi-phasic; all significantly increased by melatonin at 10–13 to 10–5 M and decreased by melatonin at 10–3 M. Maximal benefit of melatonin on in vitro mouse 2-cell embryo development was achieved at a concentration of 10–9 M. In comparison to control, 10–9 M melatonin increased blastocyst formation rate from 48.08 ± 5.25% to 82.08 ± 2.34% (p < 0.05), hatched blastocyst rate from 25.65 ± 11.79% to 66.47 ± 4.94% (p < 0.05), and cell number per blastocyst 62.71 ± 5.97 to 77.91 ± 10.63 (p < 0.05). Thus, our datas demonstrated firstly that melatonin has beneficial effects on the in vitro development of 2-cell mouse embryos cultured in HTF medium.