Changjiu He

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.

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.

Melatonin-related genes expressed in the mouse uterus during early gestation promote embryo implantation

Melatonin, a superior antioxidant, is an important molecule which regulates female reproduction due to its receptor‐mediated and receptor‐independent antioxidant actions. In this study, we investigated the effect of melatonin on early gestation in a mouse model. During early gestation, the expression of the melatonins rate‐limiting enzyme, AANAT, gradually increased – in the uterus while the MT2 melatonin receptor was only expressed at day 2 of gestation and no MT1 was detected. Based on these findings, we conducted a melatonin injection experiment which demonstrated that 15 mg/kg melatonin significantly improved the number of implantation sites and the litter size. Also, the blastocyst and uterus were collected to identify the local action of melatonin. In the melatonin‐treated mice, the endometrium was thicker than in the control mice; melatonin also caused an increase in density of uterine glands, and the uterine gland index (UGI) was significantly elevated over that of the control. Serum steroid hormone measurements revealed that at day 6 of gestation (postimplantation), melatonin significantly downregulated the E2 level, with no obvious effects on progesterone. Gene expression assay revealed that melatonin significantly upregulated expression of HB‐EGF, a crucial gene involved in implantation as well as its receptor ErbB1 in the blastocyst. In addition, PRA, an important gene which influences the decidual response and luminal cell differentiation, p53, which regulates uterine through leukaemia inhibitory factor (LIF), were both increased after melatonin treatment. These data suggest that melatonin and its MT2 receptor influence early gestation. Exogenous melatonin treatment can improve mouse embryo implantation and litter size, which may have important applications in human reproductive health and animal husbandry.

Melatonin and its receptor MT1 are involved in the downstream reaction to luteinizing hormone and participate in the regulation of luteinization in different species.

The functions of melatonin in preovulatory fluid remain elusive. In the current study, we observed that the extremely high level of expression of MT1 in mice granulosa cells was rapidly induced by hCG (equivalent LH) within 2 hours and this was referred as MT1 surge. In cumulus cells, serotonin N‐acetyltransferase (SNAT) was also upregulated by hCG and led to elevated melatonin levels in ovarian follicle fluid. Melatonin application before MT1 surge significantly promoted embryo implantation, and this was probably attributed to a rise in progesterone levels in the serum. The mechanistic studies indicated that melatonin/MT1 (MLT/MT1) signaling remarkably improved the expression of corpus luteum marker genes, that is, Akr1c18 and Cyp11a1. High‐throughput sequencing results suggested that extracellular matrix (ECM) receptor interaction, focal adhesion, and activation of PI3K/Akt pathway which are involved in granulosa cell luteinization might mediate the actions of MLT/MT1 signal. In addition, this effect on luteinization was compared in different species. It was verified that high melatonin levels exist in serum at estrum of cows and help to improve the first estrus fecundation rate. These results suggested that both melatonin and MT1 are involved in the downstream reaction of hCG (LH) and they play important roles in luteinization. These findings provide the novel information on the physiology of melatonin in animal reproduction.

Beneficial Effects of Melatonin on the In Vitro Maturation of Sheep Oocytes and Its Relation to Melatonin Receptors

(1) Background: The binding sites of melatonin, as a multifunctional molecule, have been identified in human, porcine, and bovine samples. However, the binding sites and mechanisms of melatonin have not been reported in sheep; (2) Methods: Cumulus–oocyte complexes (COCs) were cultured in TCM-199 supplemented with melatonin at concentrations of 0, 10−3, 10−5, 10−7, 10−9, and 10−11 M. Melatonin receptors (MT1 and MT2) were evaluated via immunofluorescence and Western blot. The effects of melatonin on cumulus cell expansion, nuclear maturation, embryo development, and related gene (GDF9, DNMT1, PTX3, HAS2, and EGFR) expression were investigated. The level of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were evaluated in oocytes and cumulus, respectively; (3) Results: Both MT1 and MT2 were expressed in oocytes, cumulus cells, and granulosa cells. Melatonin with a concentration of 10−7 M significantly enhanced the rates of nuclear maturation, cumulus cells expansion, cleavage, and blastocyst. Melatonin enhanced the expression of BMP15 in oocytes and of PTX3, HAS2, and EGFR in cumulus cells. Melatonin decreased the cAMP level of oocytes but enhanced the cGMP level in oocytes and cumulus cells; (4) Conclusion: The higher presence of MT1 in GV cumulus cells and the beneficial effects of melatonin indicated that its roles in regulating sheep oocyte maturation may be mediated mainly by the MT1 receptor.

An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving as the mammary gland bioreactor to produce melatonin-enriched milk in sheep

Melatonin as a potent antioxidant exhibits important nutritional and medicinal values. To produce melatonin‐enriched milk will benefit the consumers. In this study, a sheep bioreactor which generates melatonin‐enriched milk has been successfully developed by the technology that combined CRISPR/Cas9 system and microinjection. The AANAT and ASMT were cloned from pineal gland of Dorper sheep (Ovis aries). The in vitro studies found that AANAT and ASMT were successfully transferred to the mammary epithelial cell lines and significantly increased melatonin production in the culture medium compared to the nontransgenic cell lines. In addition, the Cas9 mRNA, sgRNA, and the linearized vectors pBC1‐AANAT and pBC1‐ASMT were co‐injected into the cytoplasm of pronuclear embryos which were implanted into ewes by oviducts transferring. Thirty‐four transgenic sheep were generated with the transgenic positive rate being roughly 35% which were identified by Southern blot and sequencing. Seven carried transgenic AANAT, two carried ASMT, and 25 carried both of AANAT and ASMT genes. RT‐PCR and Western blot demonstrated that the lambs expressed these genes in their mammary epithelial cells and these animals produced melatonin‐enriched milk. This is the first report to show a functional AANAT and ASMT transgenic animal model which produce significantly high levels of melatonin milk compared to their wild‐type counterparts. The advanced technologies used in the study laid a foundation for generating large transgenic livestock, for example, the cows, which can produce high level of melatonin milk.