Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior

Abstract

The long-term developmental and behavioral consequences of mammalian embryo culture are unknown. By altering the culture medium with the addition of FCS, we wanted to determine whether mouse embryos cultured under suboptimal conditions develop aberrant mRNA expression of imprinting genes at the blastocyst stage and whether fetal development, growth, and behavior of adult mice are affected. One-cell embryos obtained from superovulated female B6CBAF(1) mice were cultured for 4 days in K(+)-modified simplex optimized medium in the presence of either 10% FCS or 1 g/liter BSA. After embryo transfer, born animals were submitted to several developmental and behavior tests. The mRNA expression of some imprinting genes was significantly affected in blastocysts cultured in the presence of FCS. Two of the eight measures of preweaning development and some specific measures of neuromotor development, such as the walking activity, were delayed in the group originated with FCS. After 34 weeks, the weight of female mice cultured in vitro in the presence of FCS was significantly higher than controls. In addition, the locomotion activity of mice was altered at 5 and 15 months. Anatomopathological and histological analysis of animals at 20 months of age showed some large organs and an increase in pathologies. We have found that mice derived from embryos cultured with FCS exhibited specific behavioral alterations in anxiety and displayed deficiencies in implicit memories. Our data indicate that long-term programming of postnatal development, growth, and physiology can be affected irreversibly during the preimplantation period of embryo development by suboptimal in vitro culture.

Fig. 1.

Relative abundance of four growth-related imprinting genes (four replicates) in mice blastocysts cultured in the presence (white bars) or absence (black bars) of FCS. The mean of the four replicates was calculated for both groups, and the group with the higher value was assigned a value of 1. *, significant differences of P < 0.05.

Fig. 2.

Elevated plus maze paradigm. At 5 months of age, mice produced with FCS exhibited a greater anxiolysis than controls, as reflected in the percentage of time spent in the exposed arms of the elevated plus maze under novelty conditions (Top Left). Retesting 24 h after (familiarity) induced a fear response in controls, characterized by enhanced latency to enter into the exposed arms (Middle Left). Young mice of both sexes produced with FCS did not exhibit this conditioned fear, spending more time, exhibiting lower latencies, and displaying greater activity (Bottom) than controls. This pattern was completely reversed with aging. Animals at 15 months of age produced with FCS displayed higher anxiety states and lower activity in the exposed arms of the maze (Right). Again, animals exposed to FCS did not exhibit fear responses during retesting. Data are means ± SEM of at least 10 determinations per group. Within each color bar, different letters denote significant differences (a ≯ b; c ≯ d, P < 0.05). *, P < 0.05 versus –FCS group, Newman–Keul's test.

Fig. 3.

Body weights of male and female mice produced in vitro in the presence or absence of FCS (0–76 weeks old). *, P < 0.05, versus–FCS group, Student's t test.