Free amino acids are present in the natural environment of the preimplantation embryo, and their availability can influence early embryo development. Glutamic acid is one of the amino acids with highest concentrations in female reproductive fluids, and we investigated whether glutamic acid/glutamate can affect preimplantation embryo development by acting through cell membrane receptors. Using RT-PCR, we detected 15 ionotropic glutamate receptor transcripts and 8 metabotropic glutamate receptor transcripts in mouse ovulated oocytes and/or in vivo developed blastocysts. Using immunohistochemistry, we detected expression of two AMPA receptor subunits, three kainate receptor subunits and member 5 metabotropic glutamate receptor protein in blastocysts. Extracellular concentrations of glutamic acid starting at 5 mM impaired mouse blastocyst development, and this fact may be of great practical importance since glutamic acid and its salts (mainly monosodium glutamate) are widely used as food additives. Experiments with glutamate receptor agonists (in combination with gene expression analysis) revealed that specific AMPA receptors (formed from GRIA3 and/or GRIA4 subunits), kainate receptors (formed from GRIK 3 and GRIK 4 or GRIK 5 subunits) and GRM5 glutamate receptor were involved in this effect. The glutamic acid-induced effects were prevented or reduced by pre-treatment of blastocysts with AMPA, kainate and GRM5 receptor antagonists, further confirming the involvement of these receptor types. Our results show that glutamic acid can act as a signaling molecule in preimplantation embryos, exerting its effects through activation of cell membrane receptors.

The aim of the present study was to test the hypothesis that leptin can directly mediate the negative effect of maternal obesity on preimplantation embryos. As previously shown, maternal obesity retards early embryonic development in vivo and increases the incidence of apoptosis in blastocysts. When two-cell embryos isolated from control and obese mice were transferred to identical (leptin free) conditions in vitro, no differences in any growth or quality parameters were recorded, including apoptosis incidence in blastocysts. Embryos isolated from control mice responded to transfer to environments with a high concentration of leptin (10 ng/mL) with a significant increase in arrest at the first or subsequent cell cycle. However, the majority of non-arrested embryos developed into blastocysts, showing morphology comparable to those cultured in the leptin-free group. On the other hand, the exposure of embryos isolated from obese mice to high leptin concentration in vitro did not retard their development. Furthermore, these embryos developed into blastocysts, showing a lower incidence of apoptosis. In vivo-developed blastocysts recovered from obese mice showed elevated expression levels of the proapoptotic gene BAX and the insulin-responsive glucose transporter gene SLC2A4. In conclusion, elevated leptin levels have both positive and negative effects on preimplantation embryo development in vitro, a response that likely depends on the body condition of the embryo donor. Moreover, these results suggest that leptin acts as a survival factor rather than an apoptotic inductor in embryonic cells. Since no elevations in the expression of the leptin receptor gene (LEPR) or fat metabolism-associated genes (PLIN2, SLC27A4) were recorded in blastocysts recovered from obese mice, the role of leptin in mediating the effects of obesity on embryos at the peripheral level is likely lower than expected.

The aim of the present work was to investigate the impact of maternal obesity on DNA methylation in 3 ovulated oocytes, and to compare the response of in vitro-developing preimplantation embryos originating 4 from control and obese mice to insulin. An intergenerational, diet-induced obesity model was used to 5 produce outbred mice with an increased body weight and body fat. Two-cell and eight-cell embryos 6 recovered from obese and control mice were cultured in a medium supplemented with 1 or 10 ng/mL 7 insulin until blastocyst formation. In the derived blastocysts, cell proliferation, differentiation, and death 8 rates were determined. The results of immunochemical visualization of 5-methylcytosine indicated a 9 slightly higher DNA methylation in ovulated metaphase II oocytes recovered from obese females; 10 however, the difference between groups did not reach statistical significance. Expanded blastocysts 11 developed from embryos provided by control dams showed increased mean cell numbers (two and eight-12 cell embryos exposed to 10 ng/mL), an increased inner-cell-mass/trophectoderm ratio (two-cell embryos 13 exposed to 1 ng/mL and eight-cell embryos exposed to 10 ng/mL), and a reduced level of apoptosis (two 14 and eight-cell embryos exposed to 10 ng/mL). In contrast, embryos originating from obese mice were 15 significantly less sensitive to insulin; indeed, no difference was recorded in any tested variable between 16 the embryos exposed to insulin and those cultured in insulin-free medium. Real-time RT-PCR analysis 17 showed a significant increase in the amount of insulin receptor transcripts in blastocysts recovered from 18 obese dams. These results suggest that maternal obesity might modulate the mitogenic and antiapoptotic 19 responses of preimplantation embryos to insulin.