The effect of paternal folate status on folate metabolism and development in pre- and postnatal rat brain

Abstract

The impact of paternal folate status on folate metabolism and the development of pre- and postnatal rat brains were examined by studying the effects of combining paternal folate sufficiency (PS) or deficiency (PD) with maternal folate sufficiency (MS) or deficiency (MD) on folate content, the expression of three folate transporters, global DNA methylation and histological observations. Rats were mated after 4 weeks of being fed a folic acid-deficient (without folic acid) or folic acid-supplemented (8.0 mg of folic acid/kg diet) diet. Fetuses were killed on day 20 of gestation for prenatal experiment, and pups were killed on day 21 of lactation for postnatal experiment. In the fetal whole brain, the mean folate content was decreased in the PD × MD and PD × MS groups (P < 0.05), and the proton-coupled folate transporter (PCFT) expression were increased in the PD × MD and PD × MS groups (P < 0.0001). The reduced folate carrier 1 (RFC1) expression was highest in the PD × MD group than other three groups (P < 0.05). In the 3-week-old pups, the cortical mean folate content was decreased in the PS × MD and PD × MD groups (P < 0.0001), and the expression of PCFT and RFC1 did not differ among the four groups. The hippocampal mean folate content was decreased in the PS × MD, PD × MS and PD × MD groups (P < 0.0001), and the PCFT expression was lower in the PD × MD (P < 0.05) group than in the PS × MS and PS × MD groups, whereas the expression of RFC1 was not different among the four groups. In the fetal whole brain, global DNA methylation was decreased in the PS × MD, PD × MS and PD × MD groups (P < 0.0001). In the 3-week-old pups, cortical global DNA methylation did not differ among the four groups. The hippocampal global DNA methylation was decreased in the PS × MD, PD × MS and PD × MD groups (P < 0.0001). In the cortex, the number of brain-derived neurotrophic factor (BDNF)-immunopositive cells was decreased in the PS × MD, PD × MS and PD × MD groups (P < 0.001). The number of glial fibrillary acidic protein (GFAP)-immunopositive cells was increased in the PS × MD, PD × MS and PD × MD groups (P < 0.0001). The myelin basic protein (MBP) expression did not differ among the four groups. In the hippocampus, the number of BDNF-immunopositive cells was decreased in the PS × MD, PD × MS and PD × MD groups (P < 0.0001). The number of GFAP-immunopositive cells in the hippocampus was increased in the PS × MD, PD × MS and PD × MD groups (P < 0.01). The MBP expression level was decreased in the PD × MD group. In conclusion, the impact of paternal folate status might be as important to the normal growth and development of the brains of offspring as maternal folate status.;본 연구는 임신 전 남성의 엽산 영양상태에 따른 자손 뇌의 엽산 대사와 발달에 미치는 후생유전체학적 영향을 동물 실험을 통하여 알아보고자 하였다. 5주령 Sprague-Dawley종 수컷 흰쥐와 암컷 흰쥐을 두 그룹 (엽산 결핍군, D; 엽산 보충군, S) 으로 나누어 4주간 실험 식이를 공급하였으며 (D, 0 mg; S, 8.0 mg of folic acid/kg diet), 5주째에 교배를 시켜 각각 아빠 엽산 보충군 × 엄마 엽산 보충군 (PS × MS), 아빠 엽산 보충군 × 엄마 엽산 결핍군 (PS × MD), 아빠 엽산 결핍군 × 엄마 엽산 보충군 (PD × MS), 아빠 엽산 결핍군 × 엄마 엽산 결핍군 (PD × MD)으로 나누었다. 재태 연령 20일째의 fetal whole brain과 생후 3주령 새끼 쥐의 cortex, hippocampus를 이용하여 엽산 함량, folate transporter의 발현, global DNA methylation, 그리고 neural differentiation marker의 발현을 측정하였다. Fetal whole brain에서의 엽산 함량의 경우 PD × MS군이 PS × MS군보다 낮았다 (P < 0.05). 또한, proton-coupled folate carrier (PCFT)의 발현은 PD × MS군이 PS × MD군 만큼 증가 하였으며 (P < 0.0001), reduced folate carrier 1 (RFC1)의 발현은 PD × MD군에서 가장 많이 증가하였다 (P < 0.05). 생후 3주령 새끼 cortex의 엽산 함량은 PD × MS 군과 PS × MS군 간에 차이가 없었으나, hippocampus에서는 PD × MS군의 엽산 함량이 PS × MD군, PD × MD군의 엽산 함량 수준으로 감소되었다 (P < 0.0001). 생후 3주령 새끼 cortex의 PCFT와 RFC1 발현에는 네 군간에 차이가 없었다. Hippocampus의 RFC1 발현은 네 군간에 차이가 없었으나 PCFT 발현은 PD × MS군이 PD × MD군 만큼 감소하였다 (P < 0.01). Fetal whole brain (P < 0.0001)과 생후 3주령 새끼의 hippocampus (P < 0.0001)에서의 5-methylcytosine (5-mC)은 PD × MS군에서 감소하였다. 반면 cortex에서는 네 군간에 차이를 보이지 않았다. PS × MS군과 비교했을 때, 생후 3주령 새끼 cortex에서의 brain-derived neurotrophic factor (BDNF)의 발현은 나머지 세 군 모두에서 감소하였으며 (P < 0.001), glial fibrillary acidic protein (GFAP)의 발현은 모두 증가하였다 (P < 0.0001). 하지만 MBP의 발현은 네 군간에서 차이를 보이지 않았다. Hippocampus의 BDNF 발현도 cortex와 마찬가지로 나머지 세 군 모두에서 정상군에 비해 발현이 감소하였으며 (P < 0.001), GFAP 발현은 이 세 군에서 모두 증가하였다 (P < 0.01). 하지만 MBP 발현의 경우 PD × MD군에서만 감소하였다. 결론적으로, 임신 전 아빠 엽산 결핍은 엄마 엽산 결핍 못지않게 자손 뇌의 성장 및 발달에 중요하게 작용하는 것으로 보이며, 이는 아빠의 후성유전체학적인 영향을 받은 것으로 사료된다. 이에 자손 뇌의 정상적인 성장 및 발달을 위해 가임 기간 동안 여성뿐만 아니라 가임 남성의 엽산 영양 상태를 양호하게 유지하여야 할 것이다.