136 HIGH PROTEIN DIET INHIBITS INNER CELL MASS FORMATION AND INCREASES APOPTOSIS IN MOUSE BLASTOCYSTS DEVELOPED IN VIVO BY INCREASING THE LEVELS OF AMMONIUM IN THE REPRODUCTIVE TRACT

Abstract

Ammonium is known to adversely affect the development of mouse embryos in culture. Specifically, ammonium has been found to impair inner cell (ICM) mass formation, increase apoptosis, retard fetal development following embryo transfer and induce exencephaly. Significantly, high protein diets in cattle lead to reduced fecundity. This has been linked to elevated urea levels within fluid of the female tract. In this study we have determined the effects of a high protein diet for mice on the levels of ammonium within the female tract and the effects of such a diet on the development and viability of blastocysts developed in vivo. Outbred mice (CF1) were fed a diet of either 25% (high protein) or 14% (control) protein for 4 weeks. Females were superovulated and mated to males of the same strain. In 24 mice, oviduct fluid was collected at 22 h post hCG. Ammonium in the oviduct fluid was then quantitated fluorometrically. From other animals, blastocysts were flushed 92 h post hCG and analyzed. Blastocyst differentiation and apoptotic indices were determined. Values are mean ± SEM. Data were analysed using Student’s t-test. The levels of ammonium in the oviduct were significantly higher (P < 0.01) in females fed the high protein diet (356 ± 43 µM) compared to the control (68 ± 13 µM) (n = 12 in each group). Blastocysts (n = 139) from females fed the high protein diet had significantly lower total (43.4 ± 1.1; P < 0.05) and ICM cell numbers (12.7 ± 0.4; P < 0.01), compared to the control group (46.8 ± 0.9 and 15.4 ± 0.4 respectively; n = 124). Furthermore, blastocysts from animals fed a high protein diet had a significantly higher apoptotic index (8.7 ± 1.4; P < 0.01) compared to the control group (2.0 ± 0.5). These data show that consumption of a high protein diet results in the excess accumulation of ammonium in the fluid of the female reproductive tract of mice. These high levels of ammonium subsequently impair the formation of the fetal progenitor cells and increase cell death at the blastocyst stage. These data from in vivo-developed mouse blastocysts are similar to those for blastocysts developed in culture in the presence of 300 µM ammonium. Therefore, it is not advisable to maintain mice on a high protein diet. These data have significant implications for animal breeding, and for patients attempting IVF treatment.