Single-nucleus and spatial transcriptomics of paediatric ovary: Molecular insights into the dysregulated signalling pathways underlying premature ovarian insufficiency in classic galactosemia

IF 7.9 1区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Clinical and Translational Medicine Pub Date : 2024-10-23 DOI:10.1002/ctm2.70043

Raghuveer Kavarthapu, Hong Lou, Thang Pham, Han Do, Mary E. Soliman, Taylor Badger, Ramya Balasubramanian, Victoria Huyhn, Maria De La Luz Sierra, Jacqueline C. Yano Maher, Veronica Gomez-Lobo

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Abstract

Background

Classic galactosemia (CG) is an inborn error of galactose metabolism caused by mutations in the GALT gene. Premature ovarian insufficiency (POI) is a later complication that affects 80% of women with CG due to a significant decline in ovarian reserve (primordial follicle pool). The definite mechanisms underlying the early onset of POI in CG patients are not fully understood.

Methods

In this study, we performed single-nucleus RNA sequencing (snRNA-seq) and spatial transcriptomics on ovary tissue biopsies from prepubertal girls diagnosed with CG to investigate dynamic changes in gene expression and altered signalling pathways in granulosa cells, oocytes, and stromal cells.

Results

We generated single-nucleus and spatial transcriptomics atlas of human ovaries from prepubertal girls diagnosed with and without CG. snRNA-seq profiling of the paediatric ovary revealed a diverse ovarian microenvironment with seven distinct major cell types. Our transcriptomic analysis revealed an increase in the expression of several endoplasmic reticulum stress and oxidative stress associated genes, which can promote apoptosis of granulosa cells in CG. PTEN/PI3K/AKT signalling, which is crucial for primordial follicle activation and survival was dysregulated as supported by upregulated PTEN transcripts and a significant reduction in phospho-AKT levels in the granulosa cells and oocytes. We also found a marked increase in expression of phospho-H2A.X, LC3A/B and CASP9 in the primordial follicles of CG ovaries suggesting DNA damage, autophagy, and accelerated follicular atresia. Furthermore, we noticed genes participating in extracellular matrix organisation, integrin and gap junction signalling, essential for structural support of the ovarian stroma were profoundly altered.

Conclusions

Our findings provide molecular insights into the dysregulated cellular signalling pathways essential for primordial follicle growth and survival that can explain the etiology of POI in CG patients. This study has implications in the development of future therapeutic interventions to preserve ovarian function and promote female reproductive health.

Highlights

Created a comprehensive single-nucleus transcriptomic atlas and spatial landscape of paediatric ovary tissue from prepubertal girls diagnosed with classic galactosemia (CG).
Our transcriptomic analysis revealed activation of genes associated with ER-stress signalling, oxidative stress response and ATM signalling/DNA damage response as shown by significant increase in expression of p-EIF2A, p-H2A.X and LC3A/B in the primordial follicles of CG ovary.
PTEN/PI3K/AKT signalling pathways was dysregulated evidenced by a significant reduction in phospho-AKT expression in the primordial follicles of CG ovary, suggesting impaired follicle activation and survival.

Abstract Image

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小儿卵巢单核和空间转录组学：经典半乳糖血症患者卵巢早衰信号通路失调的分子研究。

背景：典型半乳糖血症（CG）是一种由 GALT 基因突变引起的先天性半乳糖代谢错误。由于卵巢储备功能（原始卵泡池）显著下降，卵巢早衰（POI）是一种晚期并发症，影响到80%的CG女性患者。CG患者早期出现卵巢功能不全的确切机制尚不完全清楚：在这项研究中，我们对确诊为 CG 的青春期前少女的卵巢组织活检物进行了单核 RNA 测序（snRNA-seq）和空间转录组学研究，以探讨颗粒细胞、卵母细胞和基质细胞中基因表达的动态变化和信号通路的改变：我们从诊断出患有和未患有CG的青春期前少女的卵巢中生成了单核和空间转录组图谱。我们的转录组分析表明，一些内质网应激和氧化应激相关基因的表达量增加，这些基因可促进 CG 中颗粒细胞的凋亡。PTEN/PI3K/AKT信号对原始卵泡的活化和存活至关重要，而颗粒细胞和卵母细胞中上调的PTEN转录本和显著降低的磷酸-AKT水平证明了这一点。我们还发现，在CG卵巢的原始卵泡中，phospho-H2A.X、LC3A/B和CASP9的表达明显增加，这表明DNA损伤、自噬和卵泡闭锁加速。此外，我们还注意到参与细胞外基质组织、整合素和缝隙连接信号传导的基因发生了深刻变化，这些基因对卵巢基质的结构支撑至关重要：我们的研究结果从分子角度揭示了原始卵泡生长和存活所必需的细胞信号通路失调，这可以解释 CG 患者 POI 的病因。这项研究对开发未来的治疗干预措施以保护卵巢功能和促进女性生殖健康具有重要意义：创建了一个全面的单核转录组图谱和小儿卵巢组织空间图谱，这些小儿卵巢组织来自被诊断为典型半乳糖血症（CG）的青春期前女孩。我们的转录组分析显示，CG卵巢原始卵泡中p-EIF2A、p-H2A.X和LC3A/B的表达显著增加，这表明与ER应激信号、氧化应激反应和ATM信号/DNA损伤反应相关的基因被激活。PTEN/PI3K/AKT信号通路失调，表现为CG卵巢原始卵泡中磷酸-AKT的表达明显减少，这表明卵泡的活化和存活能力受损。

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来源期刊

Clinical and Translational Medicine Multiple-

CiteScore

15.90

自引率

1.90%

发文量

450

审稿时长

4 weeks

期刊介绍： Clinical and Translational Medicine (CTM) is an international, peer-reviewed, open-access journal dedicated to accelerating the translation of preclinical research into clinical applications and fostering communication between basic and clinical scientists. It highlights the clinical potential and application of various fields including biotechnologies, biomaterials, bioengineering, biomarkers, molecular medicine, omics science, bioinformatics, immunology, molecular imaging, drug discovery, regulation, and health policy. With a focus on the bench-to-bedside approach, CTM prioritizes studies and clinical observations that generate hypotheses relevant to patients and diseases, guiding investigations in cellular and molecular medicine. The journal encourages submissions from clinicians, researchers, policymakers, and industry professionals.