Intermitochondrial cement (IMC) harbors piRNA biogenesis machinery and exonuclease domain-containing proteins EXD1 and EXD2 in mouse spermatocytes.

BACKGROUND: Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway. AIMS: In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis. RESULTS: The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3'-5' domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility. CONCLUSION: Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells.

Wild-type and mutant SOD1 localizes to RNA-rich structures in cells and mice but does not bind RNA.

Many of the genes whose mutation causes Amyotrophic Lateral Sclerosis (ALS) are RNA-binding proteins which localize to stress granules, while others impact the assembly, stability, and elimination of stress granules. This has led to the hypothesis that alterations in the dynamics of stress granules and RNA biology cause ALS. Genetic mutations in Superoxide Dismutase 1 (SOD1) also cause ALS. Evidence demonstrates that SOD1 harboring ALS-linked mutations is recruited to stress granules, induces changes in alternative splicing, and could be an RNA-binding protein. Whether SOD1 inclusions contain RNA in disease models and whether SOD1 directly binds RNA remains uncertain. We applied methods including cross-linking immunoprecipitation and in vitro gel shift assays to detect binding of SOD1 to RNA in vitro, in cells with and without stress granules, and in mice expressing human SOD1 G93A. We find that SOD1 localizes to RNA-rich structures including stress granules, and SOD1 inclusions in mice contain mRNA. However, we find no evidence that SOD1 directly binds RNA. This suggests that SOD1 may impact stress granules, alternative splicing and RNA biology without binding directly to RNA.

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment.

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

FYCO1 and autophagy control the integrity of the haploid male germ cell-specific RNP granules.

Ribonucleoprotein (RNP) granules play a major role in compartmentalizing cytoplasmic RNA regulation. Haploid round spermatids that have exceptionally diverse transcriptomes are characterized by a unique germ cell-specific RNP granule, the chromatoid body (CB). The CB shares many characteristics with somatic RNP granules but also has germline-specific features. The CB appears to be a central structure in PIWI-interacting RNA (piRNA)-targeted RNA regulation. Here, we identified a novel CB component, FYCO1, which is involved in the intracellular transport of autophagic vesicles in somatic cells. We demonstrated that the CB is associated with autophagic activity. Induction of autophagy leads to the recruitment of lysosomal vesicles onto the CB in a FYCO1-dependent manner as demonstrated by the analysis of a germ cell-specific Fyco1 conditional knockout mouse model. Furthermore, in the absence of FYCO1, the integrity of the CB was affected and the CB was fragmented. Our results suggest that RNP granule homeostasis is regulated by FYCO1-mediated autophagy.

DICER Regulates the Formation and Maintenance of Cell-Cell Junctions in the Mouse Seminiferous Epithelium.

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.

Retromer vesicles interact with RNA granules in haploid male germ cells.

Spermatozoa are produced during spermatogenesis as a result of mitotic proliferation, meiosis and cellular differentiation. Postmeiotic spermatids are exceptional cells given their haploid genome and remarkable sperm-specific structural transformations to compact and reshape the nucleus and to construct the flagellum and acrosome. These processes require delicate coordination and active communication between distinct cellular compartments. In this study, we elucidated the interplay between the haploid RNA regulation and the vesicular transport system. We identified a novel interaction between VPS26A/VPS35-containing retromer vesicles and the chromatoid body (CB), which is a large ribonucleoprotein (RNP) granule unique to haploid male germ cells. VPS26A/VPS35-positive vesicles were shown to be involved in the endosomal pathway, as well as in acrosomal formation that is dependent on the Golgi complex-derived vesicular trafficking. While the exact role of the retromer vesicles in the CB function remains unclear, our results suggest a direct functional link between vesicle transport and CB-mediated RNA regulation.

An atlas of chromatoid body components.

The genome of male germ cells is actively transcribed during spermatogenesis to produce phase-specific protein-coding mRNAs and a considerable amount of different noncoding RNAs. Ribonucleoprotein (RNP) granule-mediated RNA regulation provides a powerful means to secure the quality and correct expression of the requisite transcripts. Haploid spermatids are characterized by a unique, unusually large cytoplasmic granule, the chromatoid body (CB), which emerges during the switch between the meiotic and post-meiotic phases of spermatogenesis. To better understand the role of the CB in male germ cell differentiation, we isolated CBs from mouse testes and revealed its full RNA and protein composition. We showed that the CB is mainly composed of RNA-binding proteins and other proteins involved RNA regulation. The CB was loaded with RNA, including pachytene piRNAs, a diverse set of mRNAs, and a number of uncharacterized long noncoding transcripts. The CB was demonstrated to accumulate nascent RNA during all the steps of round spermatid differentiation. Our results revealed the CB as a large germ cell-specific RNP platform that is involved in the control of the highly complex transcriptome of haploid male germ cells.

Epigenetic regulation of male germ cell differentiation.

Male germ cell differentiation is a complex developmental program that produces highly specialized mature spermatozoa capable of independent movement and fertilization of an egg. Germ cells are unique in their capability to generate new organisms, and extra caution has to be taken to secure the correct inheritance of genetic and epigenetic information. Male germ cells are epigenetically distinct from somatic cells and they undergo several important epigenetic transitions. In primordial germ cells (PGCs), epigenome is reprogrammed by genome-wide resetting of epigenetic marks, including the sex-specific imprinting of certain genes. Postnatal spermatogenesis is characterized by drastic chromatin rearrangements during meiotic recombination, sex chromosome silencing, and compaction of sperm nuclei, which is accomplished by replacing near to all histones by sperm-specific protamines. Small RNAs, including microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs) and PIWI-interacting RNAs (piRNAs) are also involved in the control of male gamete production. The activities of small RNAs in male germ cells are diverse, and include miRNA- and endo-siRNA-mediated posttranscriptional mRNA regulation and piRNA-driven transposon silencing and the control of DNA methylation in PGCs. In this chapter, we give a brief review on the epigenetic processes that govern chromatin organization and germline-specific gene expression in differentiating male germ cells.

Dicer is required for haploid male germ cell differentiation in mice.

BACKGROUND: The 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 FINDINGS: Being 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/SIGNIFICANCE: We demonstrate that Dicer and Dicer-dependent small RNAs are imperative regulators of haploid spermatid differentiation and essential for male fertility.

Chromatoid body and small RNAs in male germ cells.

The chromatoid body (CB) is a germ granule in the cytoplasm of postmeiotic haploid round spermatids that is loaded with RNA and RNA-binding proteins. Following the discovery of small non-coding RNA-mediated gene regulation and the identification of PIWI-interacting RNAs (piRNAs) that have crucial roles in germ line development, the function of the CB has slowly begun to be revealed. Male germ cells utilise small RNAs to control the complex and specialised process of sperm production. Several microRNAs have been identified during spermatogenesis. In addition, a high number of piRNAs are present both in embryonic and postnatal male germ cells, with their expression being impressively induced in late meiotic cells and haploid round spermatids. At postmeiotic stage of germ cell differentiation, the CB accumulates piRNAs and proteins of piRNA machinery, as well as several other proteins involved in distinct RNA regulation pathways. All existing evidence suggests a role for the CB in mRNA regulation and small RNA-mediated gene control, but the mechanisms remain uncharacterised. In this review, we summarise the current knowledge of the CB and its association with small RNA pathways.

Accumulation of piRNAs in the chromatoid bodies purified by a novel isolation protocol.

Haploid male germ cells are featured by an intriguing cytoplasmic cloud-like structure that has been named as chromatoid body (CB) on the basis of its staining properties and appearance under a microscope. Notwithstanding its early discovery in the late 19th century, the function of the CB is still largely obscure. Emerging evidence suggests a role for the CB and other similar RNA-containing granules, such as germ plasm in lower organism and processing bodies in somatic cells, in the control and organization of RNA processing and/or storage. Despite the increasing scientific demand, the lack of CB purification protocols has still been the main obstacle in the functional characterization of this structure. We have successfully isolated CBs from mouse testis by a novel immunoaffinity purification procedure and validated by several different methods that pure CB fractions are obtained. Analysis of the CB RNA content reveals enrichment of PIWI-interacting RNAs (piRNAs), further emphasizing the role of CB as the RNA processing body.