Disrupted fetal germline development underpins testicular germ cell neoplasia, which is increasing worldwide. The complex signaling milieu during normal testis development includes TGFβ superfamily ligands; this study tests the hypothesis that, activin A, a TGFβ superfamily member, can influence gonocyte development. The human seminoma-derived cell line, TCam-2, a model of fetal gonocytes, was cultured with activin A (1.25-25 ng/mL) for 48 h, or with 5 ng/mL activin A for short- (6, 24, and 48 h) and long-term (13 days) exposures, and downstream targets measured by qRT-PCR. Transcripts that exhibited significant dose-dependent responses to activin A included the early germ cell markers KIT, NODAL, and CRIPTO (NODALl co-receptor and activin inhibitor) which all increased and the differentiation marker DNMT3L which decreased. After 48 h, KIT, NODAL, and CRIPTO levels were significantly higher, while the differentiation marker NANOS2 was significantly lower. Interestingly, activin A exposure also significantly reduced both transcript and protein levels of the PIWI/piRNA pathway component DNMT3L. Because TCam-2 cells produce the activin inhibitor CRIPTO, CRIPTO was reduced using siRNA prior to activin A exposure. This selectively increased KIT in response to activin A. Other ligands present in the fetal testis (BMP4, FGF9, TGFβ1, and TGFβ2) induced distinct effects on germline marker expression. This study showed that activin A can directly modulate germline markers in this human gonocyte-like cell, promoting a less-differentiated phenotype. Additional findings indicate evidence of signaling crosstalk between activin A and NODAL, leading to target-specific effects on gonocyte differentiation.

中文翻译：

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激活素 A 直接调节早期人类男性生殖系分化状态的证据

胎儿生殖系发育中断是睾丸生殖细胞瘤形成的基础，这种瘤在世界范围内正在增加。正常睾丸发育过程中的复杂信号环境包括 TGFβ 超家族配体；本研究检验了以下假设：激活素 A（TGFβ 超家族成员）可以影响生殖细胞发育。人精原细胞来源的细胞系 TCam-2，一种胎儿生殖细胞模型，与激活素 A (1.25-25 ng/mL) 一起培养 48 小时，或与 5 ng/mL 激活素 A 一起培养短时间 - (6, 24和 48 小时）和长期（13 天）暴露，以及通过 qRT-PCR 测量的下游目标。对激活素 A 表现出显着的剂量依赖性反应的转录物包括早期生殖细胞标志物 KIT、NODAL 和 CRIPTO（NODAL1 共同受体和激活素抑制剂），它们都增加，分化标志物 DNMT3L 减少。48 小时后，KIT、NODAL、和 CRIPTO 水平显着升高，而分化标记 NANOS2 显着降低。有趣的是，激活素 A 暴露也显着降低了 PIWI/piRNA 通路组分 DNMT3L 的转录物和蛋白质水平。因为 TCam-2 细胞产生激活素抑制剂 CRIPTO，所以在激活素 A 暴露之前使用 siRNA 减少了 CRIPTO。这选择性地增加了 KIT 以响应激活素 A。胎儿睾丸中存在的其他配体（BMP4、FGF9、TGFβ1 和 TGFβ2）对生殖系标记物表达产生不同的影响。这项研究表明，激活素 A 可以直接调节这种人类生殖细胞样细胞中的生殖系标志物，促进分化程度较低的表型。其他发现表明激活素 A 和 NODAL 之间信号串扰的证据，导致对生殖细胞分化的目标特异性影响。