Expression of piRNAs and miRNAs in embryonic mouse ovary cells: dysregulation and apoptosis by exposure to Vinclozolin and MEHP

The early stages of embryonic development are critical for the proper formation of primordial germ cells. This process is tightly regulated by different cellular signals. Among others, small non-coding RNAs (sncRNAs) are involved in the regulation of these signals to carry out the embryonic development of germ cell linage. We analyzed the expression profiles of piRNAs and miRNAs after maternal exposure of embryos, during the first 13.5 days post-coitum, to two different endocrine disruptors (EDs) chemicals: vinclozolin and the main metabolite of phthalates (MEHP), in three successive generations after exposure only in the first generation. Our results indicate clear alterations in the expression of both piRNAs and miRNAs (including mitochondrial-related), concomitant with an increase in apoptotic cells in the female early folliculogenesis during this stage of development. However, the most drastic changes occur when the effect is generated by the exposure through both paternal and maternal pathways, rather than exclusively through the maternal exposure. Based on these findings, we hypothesize the existence of an "ovarian filtering" mechanism triggered by disruptor exposure and mediated by apoptotic mechanisms regulated by sncRNAs. This system, present only in the female germ cell lineage, could render a decrease in the follicle reserve in the adult life and would function as a defense mechanism to prevent the transmission of deleterious effects to subsequent generations. Overall design: Briefly, a first generation of mice has been crossed, which are considered generation F0. When a successful mating is detected, they are randomly assigned to Control, VZN or MEHP group. From this moment on, they are exposed during the first 13.5 days of gestation to the endocrine disruptor or control. One part of these pregnant female mice was sacrificed at 13.5 dpc to obtain the fetal ovaries by dissection and differentiation of the sexes by gonadal morphology evident at this date of development, the other part of pregnant female mice was withdrawn from exposure to ED and allowed to finish gestation and birth, to analyze the effects of exposure to ED in the following generations, obtaining embryonic ovaries also at 13.5 dpc in each subsequent generation. This process was repeated two more generations to study both the F1 generation (directly exposed during their embryonic period to DEs) and the F2 and F3 (both indirectly exposed to DEs, since only their first- and second-degree female progenitors, respectively, have been exposed). Furthermore, an additional group was made ("-Sy" group or "paternal effect -PA- group") evaluating the additive effect of the both progenitros (exposed to the same DE and dose).

胚胎发育早期对于原始生殖细胞的正常形成至关重要，这一过程受到多种细胞信号的严密调控。其中，小型非编码RNA（small non-coding RNAs, sncRNAs）参与调控上述信号通路，以保障生殖细胞谱系的胚胎发育。我们分析了仅在首代暴露于两种不同内分泌干扰物（endocrine disruptors, EDs）——烯菌酮（vinclozolin）与邻苯二甲酸酯类主要代谢产物（MEHP）后，交配后前13.5天（13.5 dpc）的母体暴露胚胎中piRNA与miRNA的表达谱。研究结果显示，piRNA与miRNA（包括线粒体相关亚型）的表达均出现显著改变，同时伴随雌性早期卵泡发生阶段凋亡细胞数量的增加。但最为剧烈的变化出现在当效应通过父本与母本双途径介导产生时，而非仅通过母本暴露途径。基于上述发现，我们提出假说：存在一种由干扰物暴露触发、并由sncRNAs调控的凋亡机制介导的“卵巢筛选”机制。这一仅存在于雌性生殖细胞谱系的系统，可能会导致成年个体的卵泡储备减少，并作为防御机制阻止有害效应传递给后代。
整体实验设计：简言之，我们首先繁育第一代小鼠（记为F0代）。当检测到成功交配后，将孕鼠随机分为对照组、烯菌酮（VZN）组与MEHP组。自此，孕鼠在妊娠前13.5天暴露于相应内分泌干扰物或对照试剂。部分孕鼠于妊娠13.5 dpc时处死，通过解剖获取胎儿卵巢，并根据该发育阶段可见的性腺形态区分性别；其余孕鼠则停止内分泌干扰物暴露，待其自然妊娠分娩，以分析内分泌干扰物暴露对后续世代的影响，并在每一代后续的13.5 dpc时获取胚胎卵巢。该实验流程在后续两代中重复进行，以分别分析F1代（胚胎发育期直接暴露于EDs）、F2代与F3代（均为间接暴露于EDs，仅其一级和二级雌性母系祖先分别接受过暴露）的效应。此外，我们额外设置了"-Sy组"或称"父本效应-PA-组"，以评估双亲均暴露于相同EDs与剂量时的叠加效应。