Melatonin Alleviates the Toxicity of High Nicotinamide Concentrations in Oocytes: Potential Interaction with Nicotinamide Methylation Signaling

Abstract

Despite the numerous studies on melatonin and nicotinamide (NAM, the active form of vitamin B3), the linkage between these two biomolecules in the context of signaling pathways regulating preimplantation embryo development has not yet been investigated. In this study, we used bovine oocyte model to elucidate the effect of melatonin on the developmental competence of oocytes under the stress of high NAM concentrations. Results showed that NAM (20 mM) administration during in vitro maturation (IVM) significantly reduced oocyte maturation and actin distribution, while induced reactive oxygen species (ROS) accumulation and mitochondrial dysfunction, the multiple deleterious effects that were alleviated by melatonin (10−7 M). The RT-qPCR and/or immunofluorescence showed upregulation of the apoptosis (Caspase-3, Caspase-9, and BAX), autophagy (Beclin-1, LC3A, LC3B, ATG7, LAMP1, and LAMP2), cell cycle (P21, P27, and P53), and DNA damage (COX2 and 8-OxoG) specific markers in oocytes matured under NAM treatment, compared to NAM-melatonin dual-treated and the untreated ones. In addition, the total cleavage and blastocyst development rate, as well as the total number of cells and the inner cell mass (ICM) per blastocyst, were reduced, while DNA fragmentation was induced, in the group of NAM sole treatment than NAM-melatonin cotreatment and control. Inspecting the underlying mechanisms behind NAM-associated toxicity revealed an increase in transcription pattern of NAM methylation (NNMT and AHCY) genes in NAM-treated oocytes while the opposite profile was observed upon melatonin supplementation. In conclusion, to our knowledge, this is the first study reporting that melatonin can protect oocytes and embryos from NAM-induced injury through its ROS-scavenging activity together with potential interaction with NAM methylation signaling.

Attenuation of Oxidative Stress and Regulation of AKT Signaling by Vanillic Acid during Bovine Pre-Implantation Embryo Development

“…Counting the blastocyst inner cell mass (ICM) and trophectoderm (TE) cells have been used as indicators of cultured blastocyst quality, known as the CDX2-based differential staining assessment [ 21 , 22 ]. As shown in Figure 3 , the numbers of the total cells and TE cells per blastocyst were significantly higher in the VA-supplemented group (171.32 ± 5.97 and 123.42 ± 3.93, respectively) than the untreated control (157.47 ± 5.38 and 108.68 ± 4.59, respectively).…”

“…Differential staining of TE and ICM cells was performed as previously stated [ 21 ]. In brief, fixed blastocysts (20 per group) were washed in DPBS and permeabilized with 0.25% Triton X-100 for 20 min at room temperature.…”

Exogenous Melatonin Directly and Indirectly Influences Sheep Oocytes

“…MT-adjusted Nrf2 signaling decreased ROS in COCs and promoted swine ( 63 ) and mouse ( 64 ) oocyte IVM ( Figure 4B ). MT inhibited nicotinamide methylation signaling, promoting bovine oocyte IVM ( 65 ). In addition, MT is also involved in the Notch signaling pathway ( 66 ), the H4K16 deacetylation pathway ( 43 ), and the mTOR pathway ( 67 ).…”

Effects of nicotinamide on follicular development and the quality of oocytes

“…It was found that 10 mM NAM treatment in the IVF medium of bovine-fertilized embryos for 3 h reduced ROS in embryonic cells and significantly increased sirtuin1 expression and the blastocyst formation rate, indicating that NAM enhanced the developmental competence of bovine embryos in vitro via antioxidant activity [ 47 ]. However, a study showed that 20 mM NAM caused ROS accumulation and mitochondrial dysfunction in bovine oocytes, impairing oocyte maturation and embryonic developmental potential [ 48 ] Furthermore, in a review, investigators have pointed out that 5 mM NAM has a positive effect on the viability and replication of cells, but NAM exceeding 20 mM causes cell apoptosis [ 49 ]. The results of our study suggested that 5 mM may be an appropriate concentration for NAM supplementation in vitro, which improved oocyte maturation and fertilization in mice.…”