VASA mRNA (DDX4) detection is more specific than immunohistochemistry using poly- or monoclonal antibodies for germ cells in the male urogenital tract

Boellaard, Willem; Stoop, Hans; Gillis, Ad J. M.; Oosterhuis, J. Wolter; Looijenga, Leendert H. J.

doi:10.1097/md.0000000000007489

Cited by 10 publications

(7 citation statements)

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“…The germline and TGCT markers that we used in this study are considered to be widely accepted “gold standards” both at mRNA and protein levels [ 31 – 33 ]. However, the sole use of correlation of expression of the PIWI/piRNA pathway with these markers still leaves room for alternative explanations of our results (for instance, that PIWI/piRNA pathway may function to a different extent in both germ cells and GCNIS cells in testis tissues adjacent to TGCTs).…”

Section: Discussionmentioning

confidence: 99%

Assessment of piRNA biogenesis and function in testicular germ cell tumors and their precursor germ cell neoplasia in situ

et al. 2018

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BackgroundAberrant overexpression of PIWI/piRNA pathway proteins is shown for many types of tumors. Interestingly, these proteins are downregulated in testicular germ cell tumors (TGCTs) compared to normal testis tissues. Here, we used germline and TGCT markers to assess the piRNA biogenesis and function in TGCTs and their precursor germ cell neoplasia in situ (GCNIS).MethodsWe used small RNA deep sequencing, qRT-PCR, and mining public RNAseq/small RNA-seq datasets to examine PIWI/piRNA gene expression and piRNA biogenesis at four stages of TGCT development: (i) germ cells in healthy testis tissues, (ii) germ cells in testis tissues adjacent to TGCTs, (iii) GCNIS cells and (iv) TGCT cells. To this end, we studied three types of samples: (a) healthy testis, (b) testis tissues adjacent to two types of TGCTs (seminomas and nonseminomas) and containing both germ cells and GCNIS cells, as well as (c) matching TGCT samples.ResultsBased on our analyses of small RNA-seq data as well as the presence/absence of expression correlation between PIWI/piRNA pathway genes and germline or TGCT markers, we can suggest that piRNA biogenesis is intact in germ cells present in healthy adult testes, and adjacent to TGCTs. Conversely, GCNIS and TGCT cells were found to lack PIWI/piRNA pathway gene expression and germline-like piRNA biogenesis. However, using an in vitro cell line model, we revealed a possible role for a short PIWIL2/HILI isoform expressed in TGCTs in posttranscriptional regulation of the youngest members of LINE and SINE classes of transposable elements. Importantly, this regulation is also implemented without involvement of germline-like biogenesis of piRNAs.ConclusionsThough further studies are warranted, these findings suggest that the conventional germline-like PIWI/piRNA pathway is lost in transition from germ cells to GCNIS cells.Electronic supplementary materialThe online version of this article (10.1186/s12885-017-3945-6) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Assessment of piRNA biogenesis and function in testicular germ cell tumors and their precursor germ cell neoplasia in situ

et al. 2018

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“…These cells, which appear to be T1-prospermatogonia, migrate to the seminiferous tubules and produce T2 pre-spermatogonia during the first post-natal week. These cells fill the basement membrane and begin the spermatogenesis pathway throughout the post-pubertal life 35 , 36 .…”

Section: Discussionmentioning

confidence: 99%

VASA protein and gene expression analysis of human non-obstructive azoospermia and normal by immunohistochemistry, immunocytochemistry, and bioinformatics analysis

Amirian

Azizi

Karoii

et al. 2022

Sci Rep

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VASA, also known as DDX4, is a member of the DEAD-box proteins and an RNA binding protein with an ATP-dependent RNA helicase. The VASA gene expression, which is required for human germ cell development, may lead to infertility. Immunocytochemistry and immunohistochemistry were used to examine the expression of VASA protein in the human testis sections of azoospermic patients, in-vitro and in-silico models. Some studies of fertile humans showed VASA expression in the basal and adluminal compartments of seminiferous tubules. Our Immunocytochemistry and immunohistochemistry in infertile humans showed expression of VASA in the luminal compartments of the seminiferous tubule. The immunohistochemical analysis of three human cases with different levels of non-obstructive azoospermia revealed a higher expression of VASA-positive cells. For this purpose, Enrichr and Shiny Gene Ontology databases were used for pathway enrichment analysis and gene ontology. STRING and Cytoscape online evaluation were applied to predict proteins' functional and molecular interactions and performed to recognize the master genes, respectively. According to the obtained results, the main molecular functions of the up-regulated and downregulated genes include the meiotic cell cycle, RNA binding, and differentiation. STRING and Cytoscape analyses presented seven genes, i.e., DDX5, TNP2, DDX3Y, TDRD6, SOHL2, DDX31, and SYCP3, as the hub genes involved in infertility with VASA co-function and protein–protein interaction. Our findings suggest that VASA and its interacting hub proteins could help determine the pathophysiology of germ cell abnormalities and infertility.

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“…Similarly, Abbasi et al reported that in immunohistochemistry analysis of THY1 (A conserved marker of SSCs of bovine, rodents, and primates) positive cells were all positive for DDX4 ( 37 ). Several investigations have analyzed DDX4 expression in normal human and mice ( 24 , 38 ). In the present study, comparison of two different abnormal cases with different levels of tissue disruption was done which showed that the expression of spermatogonia had decreased.…”

Section: Discussionmentioning

confidence: 99%

“…Next studies demonstrated that DDX4. (A member of the DEAD (Asp-Glu-Ala-Asp) box family proteins) gene encodes an ATP-dependent RNA helicase which regulates the translation of at least two mRNAs during germ cell development, nanos and gurken ( 24 ). The DDX4 gene has a conserved sequence in invertebrates and vertebrates such as C. elegans, planarian, xenopus, zebrafish, frog, mouse, rat, cat, pig, rhesus macaques, goat, chicken, and human.…”

Section: Introductionmentioning

confidence: 99%

Characterization of DDX4 Gene Expression in Human Cases with Non-Obstructive Azoospermia and in Sterile and Fertile Mice

Azizi¹,

Tabar²,

Mohammadi³

et al. 2021

JRI

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Background: In mammals, spermatogenesis is the main process for male fertility that is initiated by spermatogonial stem cells (SSCs) proliferation. SSCs are unipotent progenitor cells accountable for transferring the genetic information to the following generation by differentiating to haploid cells during spermato-and spermiogenesis. DEAD-box helicase 4 (DDX4) is a specific germ cell marker and its expression pattern is localized to, spermatocytes, and spermatids. The expression in the SSCs on the basement membrane of the seminiferous tubules is low. Methods: Immunohistochemistry (IHC) and Fluidigm reverse transcriptase-poly-merase chain reaction (RT-PCR) were used to analyze the expression of DDX4 in testis tissue of fertile and sterile mice and human cases with non-obstructive azoospermia. Results: Our immunohistochemical findings of fertile and busulfan-treated mice showed expression of DDX4 in the basal and luminal compartment of seminiferous tubules of fertile mice whereas no expression was detected in busulfan-treated mice. The immunohistochemical analysis of two human cases with different levels of non-obstructive azoospermia revealed more luminal DDX4 positive cells. Conclusion: Our findings indicate that DDX4 might be a valuable germ cell marker for analyzing the pathology of germ cell tumors and infertility as global urological problems.

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VASA mRNA (DDX4) detection is more specific than immunohistochemistry using poly- or monoclonal antibodies for germ cells in the male urogenital tract

Abstract: Supplemental Digital Content is available in the text

Cited by 10 publications

References 20 publications

Assessment of piRNA biogenesis and function in testicular germ cell tumors and their precursor germ cell neoplasia in situ

Assessment of piRNA biogenesis and function in testicular germ cell tumors and their precursor germ cell neoplasia in situ

VASA protein and gene expression analysis of human non-obstructive azoospermia and normal by immunohistochemistry, immunocytochemistry, and bioinformatics analysis

Characterization of DDX4 Gene Expression in Human Cases with Non-Obstructive Azoospermia and in Sterile and Fertile Mice

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