Table_3_Linking Phospho-Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) to Histone Ubiquitination and Acetylation Essential for Spermatid Development During Spermiogenesis.XLS

GRTH/DDX25 is a testicular RNA helicase expressed in germ cells that plays a crucial role in completion of spermatogenesis. Previously, we demonstrated a missense mutation (R242H) of GRTH gene in Japanese infertile patients (5.8%) with non-obstructive azoospermia. This mutation upon expression in COS-1 cells revealed absence of the 61 kDa phosphorylated GRTH in cytoplasm and the presence of the 56 kDa non-phosphorylated GRTH in the nucleus. GRTH knock-in (KI) mice carrying the human GRTH (R242H) mutation, lack phosphorylated GRTH, and sperm due to failure of round spermatid elongation during spermiogenesis. To determine the impact of phosphorylated GRTH on molecular events/pathways participating in spermatid development during spermiogenesis, we analyzed transcriptome profiles obtained from RNA-Seq of germ cells from KI and WT mice. RNA-Seq analysis of 2624 differentially expressed genes revealed 1404 down-regulated and 1220 up-regulated genes in KI mice. Genes relevant to spermatogenesis, spermatid development and spermatid differentiation were significantly down-regulated. KEGG enrichment analysis showed genes related to ubiquitin-mediated proteolysis and protein processing in endoplasmic reticulum pathway genes were significantly down-regulated while the up-regulated genes were found to be involved in Focal adhesion and ECM-receptor interaction pathways. Real-Time PCR analysis confirmed considerable reduction in transcripts of ubiquitination related genes Ube2j1, Ube2k, Ube2w, Rnf8, Rnf133, Rnf138, Cul3 and increased expression of Ccnd2, Col1a, Lamb1, Cav1, Igf1, Itga9 mRNA’s in KI mice compared to WT. Also, marked reduction in protein expression of UBE2J1, RNF8, RNF138 (ubiquitination network), MOF (histone acetyltransferase), their modified Histone substrates (H2AUb, H2BUb) and H4Ac, H4K16Ac were observed in KI mice. GRTH-IP mRNA binding studies revealed that Rnf8 and Ube2J1 mRNAs from WT mice associated with GRTH protein and the binding is greatly impaired in the KI mice. Immunohistochemistry analysis showed significantly reduced expression of RNF8, MOF, H4Ac and H4K16Ac in round spermatids of KI mice. Absence of phosphorylated GRTH impairs UBE2J1, RNF8 and MOF-dependent histone ubiquitination and acetylation essential for histone replacement, chromatin condensation and spermatid elongation during spermiogenesis.

GRTH/DDX25是一种在生殖细胞中表达的睾丸RNA解旋酶，它在精子发生的完成过程中发挥着至关重要的作用。此前，我们已在5.8%的患有非阻塞性无精症的日本不育患者中证实了GRTH基因存在错义突变（R242H）。该突变在COS-1细胞中的表达结果显示，细胞质中缺失了61 kDa的磷酸化GRTH，而56 kDa的非磷酸化GRTH存在于细胞核中。携带人类GRTH（R242H）突变的人源GRTH敲入（KI）小鼠缺乏磷酸化GRTH和精子，这是由于在精子形成过程中精子小管的伸长失败。为了确定磷酸化GRTH对参与精子形成过程中精子小管发育的分子事件/途径的影响，我们分析了来自KI和野生型（WT）小鼠生殖细胞的RNA-Seq转录组数据。2624个差异表达基因的RNA-Seq分析揭示了在KI小鼠中，有1404个基因下调和1220个基因上调。与精子发生、精子小管发育和精子小管分化相关的基因显著下调。KEGG富集分析显示，与泛素化介导的蛋白质降解和内质网蛋白质加工途径相关的基因显著下调，而上调的基因被发现涉及焦点粘附和细胞外基质受体相互作用途径。实时定量PCR分析证实，与WT小鼠相比，KI小鼠中泛素化相关基因Ube2j1、Ube2k、Ube2w、Rnf8、Rnf133、Rnf138、Cul3的转录本显著减少，而Ccnd2、Col1a、Lamb1、Cav1、Igf1、Itga9 mRNA的表达增加。此外，在KI小鼠中观察到UBE2J1、RNF8、RNF138（泛素化网络）、MOF（组蛋白乙酰转移酶）、其修饰的组蛋白底物（H2AUb、H2BUb）和H4Ac、H4K16Ac的蛋白质表达显著减少。GRTH-IP mRNA结合研究显示，来自WT小鼠的Rnf8和Ube2J1 mRNA与GRTH蛋白相关联，而在KI小鼠中这种结合严重受损。免疫组化分析显示，KI小鼠的圆形精子小管中RNF8、MOF、H4Ac和H4K16Ac的表达显著减少。磷酸化GRTH的缺失损害了UBE2J1、RNF8和MOF依赖的组蛋白泛素化和乙酰化，这对于精子形成过程中的组蛋白替换、染色质凝聚和精子小管伸长是必不可少的。