Chinese Medical Sciences Journal ›› 2020, Vol. 35 ›› Issue (1): 20-30.doi: 10.24920/003680

• Original Article • Previous Articles     Next Articles

Lrrc34 Is Highly Expressed in SSCs and Is Necessary for SSC Expansion In Vitro

Ou Jinhuan, Li Yiran, Wang Zhipeng, Jin Cheng, Li Kai, Lu Yan, Zou Dingfeng, Li Pengyu, Li Mengzhen, Miao Shiying, Wang Linfang, Song Wei()   

  1. Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences & School of Basic Medicine Peking Union Medical College, Beijing 100005, China
  • Received:2019-11-07 Online:2020-03-31 Published:2020-01-20
  • Contact: Song Wei E-mail:songwei@ibms.pumc.edu.cn
  • Supported by:
    This work was supported by grants from the National Key Research and Development Program of China(2018YFC1003500);CAMS Innovation Fund for Medical Sciences(2017-I2M-3-009);CAMS Innovation Fund for Medical Sciences(2016-12M-1-001);CAMS Innovation Fund for Medical Sciences(2017-I2M-1-004);National Natural Science Foundation of China(81672472);National Natural Science Foundation of China(31970794);State Key Laboratory Special Fund from the Ministry of Science of China(2060204)
Spermatogonial stem cells (SSCs), a subpopulation of undifferentiated spermatogonia, are the sole stem cell pool in the germline that maintains male fertility by ensuring a balance between self-renewal and differentiation via the continuous production of sperm capable of fertilization. In this article, the authors identified Lrrc34 as a candidate gene that shows differential expression among ID4-EGFP+ spermatogonia subsets by using bioinformatic analysis. Based on the important role of fibroblast growth factor 2 in SSC self-renewal and the expression of Lrrc34 in pluripotent stem cells, they speculated that Lrrc34 might act as a regulator of SSC expansion or maintenance. Verification experiments confirmed that Lrrc34 is particularly highly expressed in SSCs and is required for SSC expansion.

Abstract:

Objective To discover critical genes contributing to the stemness and maintenance of spermatogonial stem cells (SSCs) and provide new insights into the function of the leucine-rich repeat (LRR) family member Lrrc34 (leucine-rich repeat-containing 34) in SSCs from mice.
Methods Bioinformatic methods, including differentially expressed gene (DEG), gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, were used to uncover latent pluripotency-related genes. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence analyses were utilized to verify the mRNA and protein expression levels, respectively. RNA interference of Lrrc34 using siRNA was performed to detect its transient impact on SSCs.
Results Eight DEGs between ID4-EGFP+ (G) and ID4-EGFP+/TSPAN8High (TH), eight DEGs between G and ID4-EGFP+/TSPAN8Low (TL) and eleven DEGs between TH and TL were discovered, and eleven protein-protein interaction (PPI) modules were found to be significant in the PPI network of DEGs. One of the DEGs, Lrrc34, was selected as a potential pluripotency-related gene due to its differential expression among ID4-EGFP+ spermatogonia subsets and its interaction with fibroblast growth factor 2 in the fifth module. Immunofluorescence experiments exhibited specific expression of Lrrc34 in a subpopulation of undifferentiated spermatogonia marked by LIN28A, and RT-PCR experiments confirmed the high expression of Lrrc34 in SSCs from P7 and adult mice. The transient knockdown of Lrrc34 in SSCs resulted in reduced colony sizes and significant changes in the transcriptome and apoptotic pathways.
Conclusion Lrrc34 is highly expressed in mouse SSCs and is required for SSC proliferation in vitro through effects on transcriptome and signaling transduction pathways.

Key words: spermatogonial stem cells, Lrrc34, high expression, apoptosis

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