Different response of embryos originating from control and obese mice to insulin <i>in vitro</i>

Abstract

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.

Overweight and Fertility: What We Can Learn from an Intergenerational Mouse Obesity Model

“…Moreover, significantly higher rates of meiotic spindle and mitochondrial defects were found in oocytes of "diet reversal mice" exhibiting normalization of weight, glucose utilization, and cholesterol levels eight weeks after switching from a highfat diet to regular chow [9]. Finally, matured oocytes recovered from obese mice developed in an intergenerational model showed significantly lowered deposits of neutral lipids in the cytoplasm [19], higher DNA methylation in the nuclear area [16], and significantly lower expression of Glutathione Peroxidase 8 [37].…”

“…32 h after presumed ovulation [15,20]), on embryonic day 3 of pregnancy (ED3, approx. 56 h after presumed ovulation [16]), and on day 4 of pregnancy (ED4, approx. 80 h after presumed ovulation [11,15,16]).…”

“…56 h after presumed ovulation [16]), and on day 4 of pregnancy (ED4, approx. 80 h after presumed ovulation [11,15,16]). Recovery was performed by flushing oviducts and uteri using a flushing-holding medium [23] containing 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA).…”

“…Mice developed in this intergenerational model do not show the same massive fat accumulation as genetically modified obese mice or obese mice produced by means of the standard high-fat-diet model [12]; however, they are able to develop a somatic condition characterized by significantly higher body weight and body fat and increased plasma glucose, insulin, and leptin levels at the age of 35 days [13][14][15][16], i.e., at the time characterized by the highest ability to conceive in selected strain [17,18]. Furthermore, the acquired characteristics of body condition show long-lasting stability, i.e., there is no decrease in body weight which usually accompanies the removal of the high-fat diet.…”

“…So far, the intergenerational model of diet-induced obesity has been used to evaluate the effect of female overweight status on various qualitative parameters of ovarian cells, oocytes, and early embryos, such us lipid deposition, metabolic activity, hormonal activity, apoptosis incidence, gene expression, and DNA methylation [11,[14][15][16][19][20][21][22]. The results have helped to clarify some of the unknown aspects of this relationship.…”