The effect of the modulation of L2-hydroxyglutarate content in pachytene-diplotene cells of male mice. The effect of the modulation of L2-hydroxyglutarate content in pachytene-diplotene cells of male mice

We found that testis of male mice contain high concentrations of L2-hydroxyglutarate (L2HG) specifically in germ cells at pachytene/ diplotene (PD) stage of spermatogenesis. In order to elucidate the function of 2HG we decreased its level by incubating PD cells with 24 mM oxamate and to rescue the putative phenotype by incubation with 24 mM oxamate together with 0.3 mM cell permeable L2HG. Overall design: PD cells were isolated by sorting of enzyme-digested testicular cells from transgenic mice expressing tdTomato under germ cells specific promotor. PD cells were incubated for 48 hrs with 24 mM oxamate or with 24 mM oxamate together with 0.3 mM octyl L-2HG.

我们发现，雄性小鼠睾丸的生精过程中，处于粗线期/双线期（pachytene/ diplotene，简称PD）的生殖细胞内含有高浓度的L2-羟基戊二酸（L2-hydroxyglutarate，简称L2HG）。为阐明L2HG的生物学功能，我们通过向PD细胞中添加24 mM草氨酸盐（oxamate）以降低其内源L2HG水平，并通过同时添加24 mM草氨酸盐与0.3 mM细胞通透性L2HG来挽救潜在的表型缺陷。实验整体设计：通过分选经酶解消化的转基因小鼠睾丸细胞，分离得到PD细胞；该转基因小鼠在生殖细胞特异性启动子（germ cell-specific promoter）的调控下表达tdTomato荧光蛋白。将PD细胞分别用24 mM草氨酸盐，或24 mM草氨酸盐与0.3 mM辛基L-2HG（octyl L-2HG）共同孵育48小时。