Insulin and branched-chain amino acid depletion during mouse preimplantation embryo culture programmes body weight gain and raised blood pressure during early postnatal life

Abstract

Mouse maternal low protein diet exclusively during preimplantation development (Emb-LPD) is sufficient to programme altered growth and cardiovascular dysfunction in offspring. Here, we use an in vitro model comprising preimplantation culture in medium depleted in insulin and branched-chain amino acids (BCAA), two proposed embryo programming inductive factors from Emb-LPD studies, to examine the consequences for blastocyst organisation and, after embryo transfer (ET), postnatal disease origin. Two-cell embryos were cultured to blastocyst stage in defined KSOM medium supplemented with four combinations of insulin and BCAA concentrations. Control medium contained serum insulin and uterine luminal fluid amino acid concentrations (including BCAA) found in control mothers from the maternal diet model (N-insulin+N-bcaa). Experimental medium (three groups) contained 50% reduction in insulin and/or BCAA (L-insulin+N-bcaa, N-insulin+L-bcaa, and L-insulin+N-bcaa). Lineage-specific cell numbers of resultant blastocysts were not affected by treatment. Following ET, a combined depletion of insulin and BCAA during embryo culture induced a non sex-specific increase in birth weight and weight gain during early postnatal life. Furthermore, male offspring displayed relative hypertension and female offspring reduced heart/body weight, both characteristics of Emb-LPD offspring. Combined depletion of metabolites also resulted in a strong positive correlation between body weight and glucose metabolism that was absent in the control group. Our results support the notion that composition of preimplantation culture medium can programme development and associate with disease origin affecting postnatal growth and cardiovascular phenotypes and implicate two important nutritional mediators in the inductive mechanism. Our data also have implications for human assisted reproductive treatment (ART) practice.

Keywords: Birth weight; Blastocyst; Branched-chain amino acids; DOHaD (developmental origins of health and disease); Insulin; Systolic blood pressure.

Fig. 1

Schematic representation of the murine experimental model used in this study. The effects of branched-chain amino acids (bcaa) and/or insulin depletion (Low, L = 50%) on in vitro blastocyst development and health of the resultant offspring following embryo transfer was studied. Two-cell embryos were in vitro cultured for 66 h until the blastocyst stage. The control medium was termed “normal” (N) and represented 100% of the serum insulin levels and uterine luminal fluid amino acid concentrations (N-Insulin + N-bcaa), including bcaa, found in pregnant (E3.5) mice fed with normal levels of protein (18% casein) taken from .

Fig. 2

Effect of branched-chain amino acids (bcaa) and/or insulin depletion on in vitro preimplantation development in mice (Logistic regression). (A) Blastocyst formation (n = number of 2-cell embryos cultured) of embryos used for ET (11–16 replicates, 2–10 females per replicate). (B) Blastocyst formation (n = number of 2-cell embryos cultured) of embryos used for cell allocation analysis (14 replicates, 2–4 females per replicate). (C) Representative in vitro-derived blastocysts from the control group (N-insulin + N-bcaa) (scale bar = 100 μm). 2-cell embryos were in vitro cultured up to the blastocyst stage in either 100% (N = Normal) or 50% (L = Low) serum insulin levels with either 100% or 50% of bcaa concentrations found in the uterine luminal fluid of well-fed mice.

Fig. 3

Branched-chain amino acids (bcaa) and/or insulin depletion did not affect cell number (A) or cell allocation variables (B) of expanded blastocysts in mice (n = number of blastocyst analysed) 0.14 replicates (2–4 females per replicate) (ANOVA). 2-cell embryos were in vitro cultured up to the blastocyst stage in either 100% (N = Normal) or 50% (L = Low) serum insulin levels with either 100% or 50% of bcaa concentrations found in the uterine luminal fluid of well-fed mice.

Fig. 4

A combined depletion of insulin and branched-chain amino acids (bcaa) during in vitro preimplantation embryo development results in the production of offspring with a non sex-specific increase in birth weight (A,C) and early postnatal growth (B,D). Murine offspring is the result of transferring blastocysts (into well-fed embryo recipients) derived from 2-cell embryos cultured in either 100% (N = Normal) or 50% (L = Low) serum insulin levels with either 100% or 50% of bcaa concentrations found in the uterine luminal fluid of well-fed mice. Compared to controls (N-insulin + N-bcaa), the L-insulin + L-bcaa group showed increased birth weight and higher body weight at weeks 5–8 and 4–6 in males and females respectively. n = offspring number derived from 8 to 9 litters. Multilevel random effect regression analysis. *P < 0.05, ♦P < 0.10 (Trend).

Fig. 5

A combined depletion of insulin and branched-chain amino acids (bcaa) during in vitro preimplantation embryo development results in the production of offspring with a sex specific increase in blood pressure but with no significant changes in glucose metabolism. Murine offspring is the result of transferring blastocysts (into well-fed embryo recipients) derived from 2-cell embryos cultured in either 100% (N = Normal) or 50% (L = Low) serum insulin levels with either 100% or 50% of bcaa concentrations found in the uterine luminal fluid of well-fed mice. “LIFE” indicates the mean value of the measurements done during postnatal life (i.e. at weeks 9, 15 and 21). Compared to controls (N-insulin + N-bcaa), L-insulin + L-bcaa males showed increased systolic blood pressure at week 9. n = offspring number derived from 8 to 9 litters. Multilevel random effect regression analysis. *P < 0.05, ♦P < 0.10 (Trend).

Fig. 6

A combined depletion of insulin and branched-chain amino acids (bcaa) during in vitro preimplantation embryo development results in the production of offspring with no significant changes in organ weight, but slight alteration in organ allometry. Murine offspring is the result of transfer of blastocysts (into well-fed embryo recipients) derived from 2-cell embryos cultured in either 100% (N = Normal) or 50% (L = Low) serum insulin levels with either 100% or 50% of bcaa concentrations found in the uterine luminal fluid of well-fed mice. Compared to controls (N-insulin + N-bcaa), L-insulin + L-bcaa females showed increased heart:body weight ratio. n = offspring number derived from 8 to 9 litters. Multilevel random effect regression analysis. *P < 0.05, ♦P < 0.10 (Trend).