The aim of this study was to investigate whether the damage to male offspring induced by cadmium (Cd) exposure during embryonic period leads to the apoptosis of ovarian granulosa cells (OGCs) in the next generation of female offspring, and whether this apoptosis in the offspring was due to paternal genetic effects. Pregnant Sprague–Dawley (SD) rats were exposed to CdCl2 (0, 0.5, 2.0, or 8.0 mg/kg) by gavage daily for 20 days to produce the filial 1 (F1) generation. F1 males were mated with newly purchased females to produce the F2 generation, and the F3 generation was generated in the same way. No apoptotic bodies were observed in the OGCs of either the F2 or F3 generation as shown by electron microscopy, and a reduced OGC apoptosis rate (detected by flow cytometry) was observed in F2 OGCs from the Cd‐exposed group. Moreover, the mRNA (qRT‐PCR) levels of Bax and Bcl‐2 and the protein (western blotting) level of pro‐caspase‐8 increased in the F2 generation (p < 0.05). The expression of apoptosis‐related miRNAs (qRT‐PCR) and methylation of apoptosis‐related genes (determined via bisulfite‐sequencing PCR) in OGCs were further determined. Compared with those of the controls, the expression patterns of microRNAs (miRNAs) in the F2 offspring were different in the Cd‐exposed group. The miR‐92a‐2‐5p expression levels were decreased in both the F2 and F3 generations (p < 0.05), while the average methylation level of apoptosis‐related genes did not change significantly (except for individual loci). In summary, this study showed that the paternal genetic intergenerational effect of male Cd exposure during embryonic period induced apoptosis of OGCs in the offspring was weakened, and the transgenerational effect disappeared; nevertheless, intergenerational and transgenerational changes in apoptosis‐related genes, epigenetic modifications, DNA methylation, and miRNAs were observed, and may be important for understanding the homeostatic mechanisms of the body to alleviate the intergenerational transmission of Cd‐induced damage.

中文翻译：

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雄性大鼠镉暴露导致雌性后代卵巢颗粒细胞凋亡和父系遗传效应


本研究旨在探讨胚胎期镉 （Cd） 暴露对雄性后代的损伤是否会导致下一代雌性后代卵巢颗粒细胞 （OGCs） 凋亡，以及后代的这种凋亡是否是由于父系遗传效应。怀孕的 Sprague-Dawley （SD） 大鼠每天通过管饲法暴露于 CdCl2 （0、0.5、2.0 或 8.0 mg/kg），持续 20 天，以产生子状 1 （F1） 代。F1 雄性与新购买的雌配以产生 F2 代，F3 代以相同的方式产生。如电子显微镜所示，在 F2 或 F3 代的 OGC 中均未观察到凋亡小体，并且在 Cd 暴露组的 F2 OGC 中观察到 OGC 细胞凋亡率降低（通过流式细胞术检测）。此外，Bax 和 Bcl-2 的 mRNA （qRT-PCR） 水平以及 pro-caspase-8 的蛋白质 （western blotting） 水平在 F2 代中增加 （p < 0.05）。进一步测定 OGC 中细胞凋亡相关 miRNAs （qRT-PCR） 的表达和细胞凋亡相关基因的甲基化 （通过亚硫酸氢盐测序 PCR 确定）。与对照组相比，Cd 暴露组 F2 后代 microRNAs （miRNAs） 的表达模式不同。F2 和 F3 代 miR-92a-2-5p 表达水平均降低 （p < 0.05），而细胞凋亡相关基因的平均甲基化水平没有显着变化（单个基因座除外）。 综上所述，本研究显示，胚胎期雄性 Cd 暴露诱导后代 OGCs 凋亡的父系遗传代际效应减弱，跨代效应消失;尽管如此，观察到细胞凋亡相关基因、表观遗传修饰、DNA 甲基化和 miRNA 的代际和跨代变化，这对于理解身体的稳态机制以减轻 Cd 诱导损伤的代际传递可能很重要。