Ferreira Lucas G A, Kizys Marina M L, Gama Gabriel A C, Pachernegg Svenja, Robevska Gorjana, Sinclair Andrew H, Ayers Katie L, Dias-da-Silva Magnus R
Laboratory of Molecular and Translational Endocrinology (LEMT), Endocrinology Division, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
Murdoch Children's Research Institute, Melbourne, Australia.
Cell Biosci. 2024 Jan 4;14(1):3. doi: 10.1186/s13578-023-01182-5.
The absence of expression of the Y-chromosome linked testis-determining gene SRY in early supporting gonadal cells (ESGC) leads bipotential gonads into ovarian development. However, genetic variants in NR2F2, encoding three isoforms of the transcription factor COUP-TFII, represent a novel cause of SRY-negative 46,XX testicular/ovotesticular differences of sex development (T/OT-DSD). Thus, we hypothesized that COUP-TFII is part of the ovarian developmental network. COUP-TFII is known to be expressed in interstitial/mesenchymal cells giving rise to steroidogenic cells in fetal gonads, however its expression and function in ESGCs have yet to be explored.
By differentiating induced pluripotent stem cells into bipotential gonad-like cells in vitro and by analyzing single cell RNA-sequencing datasets of human fetal gonads, we identified that NR2F2 expression is highly upregulated during bipotential gonad development along with markers of bipotential state. NR2F2 expression was detected in early cell populations that precede the steroidogenic cell emergence and that retain a multipotent state in the undifferentiated gonad. The ESGCs differentiating into fetal Sertoli cells lost NR2F2 expression, whereas pre-granulosa cells remained NR2F2-positive. When examining the NR2F2 transcript variants individually, we demonstrated that the canonical isoform A, disrupted by frameshift variants previously reported in 46,XX T/OT-DSD patients, is nearly 1000-fold more highly expressed than other isoforms in bipotential gonad-like cells. To investigate the genetic network under COUP-TFII regulation in human gonadal cell context, we generated a NR2F2 knockout (KO) in the human granulosa-like cell line COV434 and studied NR2F2-KO COV434 cell transcriptome. NR2F2 ablation downregulated markers of ESGC and pre-granulosa cells. NR2F2-KO COV434 cells lost the enrichment for female-supporting gonadal progenitor and acquired gene signatures more similar to gonadal interstitial cells.
Our findings suggest that COUP-TFII has a role in maintaining a multipotent state necessary for commitment to the ovarian development. We propose that COUP-TFII regulates cell fate during gonad development and impairment of its function may disrupt the transcriptional plasticity of ESGCs. During early gonad development, disruption of ESGC plasticity may drive them into commitment to the testicular pathway, as observed in 46,XX OT-DSD patients with NR2F2 haploinsufficiency.
早期支持性性腺细胞（ESGC）中Y染色体连锁的睾丸决定基因SRY表达缺失会使双潜能性腺向卵巢发育。然而，编码转录因子COUP-TFII三种亚型的NR2F2基因变异是SRY阴性46,XX性发育睾丸/卵睾差异（T/OT-DSD）的新病因。因此，我们推测COUP-TFII是卵巢发育网络的一部分。已知COUP-TFII在胎儿性腺中产生类固醇生成细胞的间质/间充质细胞中表达，但其在ESGC中的表达和功能尚未得到探索。
通过在体外将诱导多能干细胞分化为双潜能性腺样细胞，并分析人类胎儿性腺的单细胞RNA测序数据集，我们发现NR2F2表达在双潜能性腺发育过程中与双潜能状态标记物一起高度上调。在类固醇生成细胞出现之前的早期细胞群体中检测到NR2F2表达，这些细胞在未分化性腺中保持多能状态。分化为胎儿支持细胞的ESGC失去NR2F2表达，而前颗粒细胞仍为NR2F2阳性。当单独检查NR2F2转录变体时，我们发现，在46,XX T/OT-DSD患者中先前报道的因移码变体而破坏的典型亚型A，在双潜能性腺样细胞中的表达比其他亚型高近1000倍。为了研究人类性腺细胞环境中COUP-TFII调控下遗传网络，我们在人类颗粒样细胞系COV434中构建了NR2F2基因敲除（KO）模型，并研究了NR2F2-KO COV434细胞转录组。NR2F2缺失下调了ESGC和前颗粒细胞的标记物。NR2F2-KO COV434细胞失去了对支持女性性腺祖细胞的富集，并获得了更类似于性腺间质细胞的基因特征。
我们的研究结果表明，COUP-TFII在维持卵巢发育所需的多能状态中起作用。我们提出，COUP-TFII在性腺发育过程中调节细胞命运，其功能受损可能会破坏ESGC的转录可塑性。在性腺发育早期，ESGC可塑性的破坏可能会使它们转向睾丸发育途径这正如在具有NR2F2单倍体不足的46,XX OT-DSD患者中所观察到的那样。
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