BackgroundThe RNase III endonuclease Dicer is an important regulator of gene expression that processes microRNAs (miRNAs) and small interfering RNAs (siRNAs). The best-characterized function of miRNAs is gene repression at the post-transcriptional level through the pairing with mRNAs of protein-encoding genes. Small RNAs can also act at the transcriptional level by controlling the epigenetic status of chromatin. Dicer and other mediators of small RNA pathways are present in mouse male germ cells, and several miRNAs and endogenous siRNAs are expressed in the testis, suggesting that Dicer-dependent small RNAs are involved in the control of the precisely timed and highly organised process of spermatogenesis.Principal FindingsBeing interested in the Dicer-mediated functions during spermatogenesis, we have analysed here a male germ cell-specific Dicer1 knockout mouse model, in which the deletion of Dicer1 takes place during early postnatal development in spermatogonia. We found that Dicer1 knockout testes were reduced in size and spermatogenesis within the seminiferous tubules was disrupted. Dicer1 knockout epididymides contained very low number of mature sperm with pronounced morphological abnormalities. Spermatogonial differentiation appeared unaffected. However, the number of haploid cells was decreased in knockout testes, and an increased number of apoptotic spermatocytes was observed. The most prominent defects were found during late haploid differentiation, and Dicer was demonstrated to be critical for the normal organization of chromatin and nuclear shaping of elongating spermatids.Conclusions/SignificanceWe demonstrate that Dicer and Dicer-dependent small RNAs are imperative regulators of haploid spermatid differentiation and essential for male fertility.

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Constitutive heterochromatin at the pericentric regions of chromosomes undergoes dynamic changes in its epigenetic and spatial organization during spermatogenesis. Accurate control of pericentric heterochromatin is required for meiotic cell divisions and production of fertile and epigenetically intact spermatozoa. In this study, we demonstrate that pericentric heterochromatin is expressed during mouse spermatogenesis to produce major satellite repeat (MSR) transcripts. We show that the endonuclease DICER localizes to the pericentric heterochromatin in the testis. Furthermore, DICER forms complexes with MSR transcripts, and their processing into small RNAs is compromised in Dicer1 knockout mice leading to an elevated level of MSR transcripts in meiotic cells. We also show that defective MSR forward transcript processing in Dicer1 cKO germ cells is accompanied with reduced recruitment of SUV39H2 and H3K9me3 to the pericentric heterochromatin and meiotic chromosome missegregation. Altogether, our results indicate that the physiological role of DICER in maintenance of male fertility extends to the regulation of pericentric heterochromatin through direct targeting of MSR transcripts.

The endonuclease DICER that processes micro-RNAs and small interfering RNAs is essential for normal spermatogenesis and male fertility. We previously showed that the deletion of Dicer1 gene in postnatal spermatogonia in mice using Ngn3 promoter-driven Cre expression caused severe defects in the morphogenesis of haploid spermatid to mature spermatozoon, including problems in cell polarization and nuclear elongation. In this study, we further analyzed the same mouse model and revealed that absence of functional DICER in differentiating male germ cells induces disorganization of the cell-cell junctions in the seminiferous epithelium. We detected discontinuous and irregular apical ectoplasmic specializations between elongating spermatids and Sertoli cells. The defective anchoring of spermatids to Sertoli cells caused a premature release of spermatids into the lumen. Our findings may help also explain the abnormal elongation process of remaining spermatids because these junctions and the correct positioning of germ cells in the epithelium are critically important for the progression of spermiogenesis. Interestingly, cell adhesion-related genes were generally upregulated in Dicer1 knockout germ cells. Claudin 5 ( Cldn5 ) was among the most upregulated genes and we show that the polarized localization of CLAUDIN5 in the apical ectoplasmic specializations was lost in Dicer1 knockout spermatids. Our results suggest that DICER-dependent pathways control the formation and organization of cell-cell junctions in the seminiferous epithelium via the regulation of cell adhesion-related genes.

In basic and translational cancer research, the majority of biopsies are stored in formalin‐fixed paraffin‐embedded (FFPE) samples. Chromatin accessibility reflects the degree to which nuclear macromolecules can physically interact with chromatinized DNA and plays a key role in gene regulation in different physiological conditions. As such, the profiling of chromatin accessibility in archived FFPE tissue can be critical to understanding gene regulation in health and disease. Due to the high degree of DNA damage in FFPE samples, accurate mapping of chromatin accessibility in these specimens is extremely difficult. To address this issue, we recently established FFPE‐ATAC, a highly sensitive method based on T7‐Tn5‐mediated transposition followed by in vitro transcription (IVT), to generate high‐quality chromatin accessibility profiles with 500–50,000 nuclei from a single FFPE tissue section. In FFPE‐ATAC, which we describe here, the T7‐Tn5 adaptors are inserted into the genome after FFPE sample preparation and are unlikely to sustain the DNA breakage that occurs during reverse cross‐linking of these samples. It should, therefore, remain at the ends of broken accessible chromatin sites after reverse cross‐linking. IVT is then used to convert the two ends of the broken DNA fragments to RNA molecules before making sequencing libraries from the IVT RNAs and further decoding Tn5 adaptor insertion sites in the genome. Through this strategy, users can decode the flanking sequences of the accessible chromatin even if there are breaks between adjacent pairs of T7‐T5 adaptor insertion sites. This method is applicable to dissecting chromatin profiles of a small section of the tissue sample, characterizing stage and region‐specific gene regulation and disease‐associated chromatin regulation in FFPE tissues. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Nuclei isolation from FFPE tissue samples Basic Protocol 2: T7‐Tn5 transposase tagmentation, reverse‐crosslinking, and in vitro transcription Basic Protocol 3: Preparation of libraries for high‐throughput sequencing