从临床活检样本中富集人类早期精母细胞的新型分选方法。

Meghan Robinson B.Sc. , Kevin Zhou B.Sc. , Sonia H.Y. Kung M.Sc. , Fatih Karaoğlanoğlu M.Sc. , Andrew Golin M.D. , Armita Safa M.Sc. , Charley Cai B.Sc. , Luke Witherspoon M.D. , Faraz Hach Ph.D. , Ryan Flannigan M.D.
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引用次数: 0

摘要

目的确定能否利用荧光激活细胞分拣技术(FACS)从人类睾丸活检组织中筛选出早期精母细胞:设计:通过对单细胞 RNA 测序(scRNAseq)人类睾丸组织进行生物信息学分析,确定早期精母细胞的潜在表面标志物。将 3 名精子发生正常的参与者的睾丸精子提取(TESE)样本消化成单细胞悬浮液并冷冻保存。从每个样本中提取 200-400 万个细胞,并使用针对已确定表面标记物的抗体进行 FACS 分选,作为单独的生物重复。每份活检样本中仍有一部分细胞未分选,作为对照。然后对分选的细胞进行表征,以富集早期精母细胞:实验室研究:干预措施:无:干预措施:无:主要结果测量:对分选的细胞进行特征描述,以确定精子发生各阶段标记物的 RNA 表达。通过对人类睾丸福尔马林固定石蜡包埋(FFPE)组织的反应验证分选标记物:结果:丝氨酸蛋白酶50(TSP50)和SWI5依赖性同源重组修复蛋白1(SFR1)被鉴定为早期精母细胞的潜在特异性表面蛋白。经 FACS 分选后,TSP50 分选群体占总群体的 1.6-8.9%,其减数分裂前标记物维甲酸刺激(STRA8)的 RNA 表达平均增加了 23 倍。免疫组化显示,TSP50在减数分裂前期未分化胚胎细胞转录因子1(UTF1)-/双性和Mab-3相关转录因子1(DMRT1)-/STRA8+精原细胞以及SYCP3+/Protamine 2(PRM2)-精母细胞中的染色模式很强:这项研究表明,TSP50可用于从人类睾丸活检组织中富集早期表达STRA8的精母细胞,为减数分裂开始阶段的定向scRNAseq分析和生殖细胞体外功能检测提供了一种方法。这将有助于研究精子发生这一关键阶段的调控通路细节,以前很难从整个组织样本中进行富集。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A novel sorting method for the enrichment of early human spermatocytes from clinical biopsies

Objective

To determine if early spermatocytes can be enriched from a human testis biopsy using fluorescence-activated cell sorting (FACS).

Design

Potential surface markers for early spermatocytes were identified using bioinformatics analysis of single-cell RNA–sequenced human testis tissue. Testicular sperm extraction samples from three participants with normal spermatogenesis were digested into single-cell suspensions and cryopreserved. Two to four million cells were obtained from each and sorted by FACS as separate biologic replicates using antibodies for the identified surface markers. A portion from each biopsy remained unsorted to serve as controls. The sorted cells were then characterized for enrichment of early spermatocytes.

Setting

A laboratory study.

Patients

Three men with a diagnosis of obstructive azoospermia (age range, 30–40 years).

Intervention

None.

Main Outcome Measures

Sorted cells were characterized for RNA expression of markers encompassing the stages of spermatogenesis. Sorting markers were validated by their reactivity on human testis formalin-fixed paraffin-embedded tissue.

Results

Serine protease 50 (TSP50) and SWI5-dependent homologous recombination repair protein 1 were identified as potential surface proteins specific for early spermatocytes. After FACS sorting, the TSP50-sorted populations accounted for 1.6%–8.9% of total populations and exhibited the greatest average-fold increases in RNA expression for the premeiotic marker stimulated by retinoic acid (STRA8), by 23-fold. Immunohistochemistry showed the staining pattern for TSP50 to be strong in premeiotic undifferentiated embryonic cell transcription factor 1/doublesex and Mab-3 related transcription factor 1/STRA8+ spermatogonia as well as SYCP3+/protamine 2 spermatocytes.

Conclusion

This work shows that TSP50 can be used to enrich early STRA8-expressing spermatocytes from human testicular biopsies, providing a means for targeted single-cell RNA sequencing analysis and in vitro functional interrogation of germ cells during the onset of meiosis. This could enable investigation into details of the regulatory pathways underlying this critical stage of spermatogenesis, previously difficult to enrich from whole tissue samples.

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来源期刊
F&S science
F&S science Endocrinology, Diabetes and Metabolism, Obstetrics, Gynecology and Women's Health, Urology
CiteScore
2.00
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审稿时长
51 days
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