This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. PTPN11 (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.

Melatonin, a nighttime-secreted antioxidant hormone produced by the pineal gland, and AKT, a serine/threonine-specific protein kinase, have been identified as regulators for several cellular processes essential for reproduction. The current study aimed to investigate the potential interplay between melatonin and AKT in bovine oocytes in the context of embryo development. Results showed that the inclusion of SH6, a specific AKT inhibitor, during in vitro maturation (IVM) significantly reduced oocyte maturation, cumulus cell expansion, cleavage, and blastocyst development that were rescued upon addition of melatonin. Oocytes treated with SH6 in the presence of melatonin showed lower levels of reactive oxygen species (ROS) and blastocysts developed exhibited low apoptosis while the mitochondrial profile was significantly improved compared to the SH6-treated group. The RT-qPCR results showed up-regulation of the mRNA of maturation-, mitochondrial-, and cumulus expansion-related genes including GDF-9, BMP-15, MARF1, ATPase, ATP5F1E, POLG2, HAS2, TNFAIP6, and PTGS2 and down-regulation of Bcl-2 associated X apoptosis regulator (BAX), caspase 3, and p21 involved in apoptosis and cell cycle arrest in melatonin-SH6 co-treated group compared to SH6 sole treatment. The immunofluorescence showed high levels of caspase 3 and caspase 9, and low AKT phosphorylation in the SH6-treated group compared to the control and melatonin-SH6 co-treatment. Taken together, our results showed the importance of both melatonin and AKT for overall embryonic developmental processes and, for the first time, we report that melatonin could neutralize the deleterious consequences of AKT inhibition, suggesting a potential role in regulation of AKT signaling in bovine oocytes.

Nicotinamide (NAM), the amide form of vitamin B3, plays pivotal roles in regulating various cellular processes including energy production and maintenance of genomic stability. The current study aimed at deciphering the effect of NAM, when administered during in vitro maturation (IVM), on the developmental competence of bovine preimplantation embryos. Our results showed that low NAM concentrations reduced the oxidative stress and improved mitochondrial profile, total cleavage and 8–16 cell stage embryo development whereas the opposite profile was observed upon exposure to high NAM concentrations (10 mM onward). Remarkably, the hatching rates of day-7 and day-8 blastocysts were significantly improved under 0.1 mM NAM treatment. Using RT-qPCR and immunofluorescence, the autophagy-related (Beclin-1 (BECN1), LC3B, and ATG5) and the apoptotic (Caspases; CASP3 and 9) markers were upregulated in oocytes exposed to high NAM concentration (40 mM), whereas only CASP3 was affected, downregulated, following 0.1 mM treatment. Additionally, the number of cells per blastocyst and the levels of SIRT1, PI3K, AKT, and mTOR were higher, while the inner cell mass-specific transcription factors GATA6, SOX2, and OCT4 were more abundant, in day-8 embryos of NAM-treated group. Taken together, to our knowledge, this is the first study reporting that administration of low NAM concentrations during IVM can ameliorate the developmental competence of embryos through the potential regulation of oxidative stress, apoptosis, and SIRT1/AKT signaling.