Gametogenesis is a complex process subject to strict controls at both levels of transcription and translation. Members of a family of conserved RNA-binding proteins encoded by the DAZ genes are required for the translational regulation of gene expression essential for this process. Although loss of DAZ family genes is associated with infertility in several organisms including humans, the identity of the transcripts regulated in vivo is unknown. Using a combination of immunoprecipitation and microarray analysis, we have identified a number of mRNAs that are bound by the murine Dazl protein both in vivo and in vitro. Sequence analysis shows that these transcripts contain binding sites for Dazl, which have been conserved during evolution between human, rat and mouse. We have focussed on mouse vasa homologue (Mvh), a gene that is essential for male gametogenesis, and show that Dazl stimulates translation via the Mvh 3'-UTR. Finally, we show that germ cells of Dazl null mice contain reduced levels of Mvh protein, indicating that Dazl-mediated regulation of Mvh translation is crucial for mammalian spermatogenesis.

DAZ-related genes are essential for gametogenesis in diverse metazoa: in human males, a loss of DAZ genes is associated with infertility. These genes, expressed only in germ cells, regulate the translation of a yet undefined set of specific transcripts, and loss of function results in numerous defects throughout the mitotic and meiotic process of germ cell development. In a mouse model, absence of the autosomal Dazl gene results in a final block at zygotene of meiotic prophase. Sycp3 is also essential for meiosis, specifically for the formation of the synaptonemal complex lateral element with a mouse knockout model displaying a block in meiotic prophase similar to the Dazl knock out. Sycp3 was identified as a potential target for translational regulation by Dazl in male mouse germ cells. This was confirmed by both RNA binding and translation assays. In the Dazl knockout mouse model, Sycp3 protein levels were decreased, indicating that Dazl is required for efficient translation of the Sycp3 mRNA in vivo. Taken together these data support Sycp3 as a biologically relevant target of Dazl-mediated translation in mammals. This suggests that azoospermia associated with a decrease in DAZ gene function in humans may in part be a consequence of failure at synapsis caused by reduced levels of SYCP3 protein.

Background Immunohistochemical quantification of the immune response is prognostic for colorectal cancer (CRC). Here, we evaluate the suitability of alternative immune classifiers on prognosis and assess whether they relate to biological features amenable to targeted therapy. Methods Overall survival by immune (CD3, CD4, CD8, CD20 and FOXP3) and immune-checkpoint (ICOS, IDO-1 and PD-L1) biomarkers in independent CRC cohorts was evaluated. Matched mutational and transcriptomic data were interrogated to identify associated biology. Results Determination of immune-cold tumours by combined low-density cell counts of CD3, CD4 and CD8 immunohistochemistry constituted the best prognosticator across stage II–IV CRC, particularly in patients with stage IV disease (HR 1.98 [95% CI: 1.47–2.67]). These immune-cold CRCs were associated with tumour hypoxia, confirmed using CAIX immunohistochemistry (P = 0.0009), which may mediate disease progression through common biology (KRAS mutations, CRIS-B subtype and SPP1 mRNA overexpression). Conclusions Given the significantly poorer survival of immune-cold CRC patients, these data illustrate that assessment of CD4-expressing cells complements low CD3 and CD8 immunohistochemical quantification in the tumour bulk, potentially facilitating immunophenotyping of patient biopsies to predict prognosis. In addition, we found immune-cold CRCs to associate with a difficult-to-treat, poor prognosis hypoxia signature, indicating that these patients may benefit from hypoxia-targeting clinical trials.

Graphical abstract

Optimisation and validation of a multiplex immunofluorescence (mIF) workflow, from staining to digital image analysis (DIA), ensure assay robustness. Chromogenic immunohistochemistry (IHC) and fluorescent singleplexes are fundamental in this process, particularly when biomarkers are co‐expressed. We describe our experience developing two mIF panels and the various parameters of staining, scanning and DIA to consider when standardising a digital pathology workflow.