During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

Epigenetic mechanisms precisely regulate sex chromosome inactivation as well as genes that escape the silencing process. In male germ cells, DNA damage response factor RNF8 establishes active epigenetic modifications on the silent sex chromosomes during meiosis, and activates escape genes during a state of sex chromosome-wide silencing in postmeiotic spermatids. During the course of evolution, the gene content of escape genes in postmeiotic spermatids recently diverged on the sex chromosomes. This evolutionary feature mirrors the epigenetic processes of sex chromosomes in germ cells. In this article, we describe how epigenetic processes have helped to shape the evolution of sex chromosome-linked genes. Furthermore, we compare features of escape genes on sex chromosomes in male germ cells to escape genes located on the single X chromosome silenced during X-inactivation in females, clarifying the distinct evolutionary implications between male and female escape genes.

Mammalian sex chromosomes arose from an ordinary pair of autosomes. Over hundreds of millions of years, they have evolved into highly divergent X and Y chromosomes and have become increasingly specialized for male reproduction. Both sex chromosomes have acquired and amplified testis-specific genes, suggestive of roles in spermatogenesis. To understand how the sex chromosome genes participate in the regulation of spermatogenesis, we review genes, including single-copy, multi-copy, and ampliconic genes, whose spermatogenic functions have been demonstrated in mouse genetic studies. Sex chromosomes are subject to chromosome-wide transcriptional silencing in meiotic and postmeiotic stages of spermatogenesis. We also discuss particular sex-linked genes that escape postmeiotic silencing and their evolutionary implications. The unique gene contents and genomic structures of the sex chromosomes reflect their strategies to express genes at various stages of spermatogenesis and reveal the driving forces that shape their evolution.Free Chinese abstract: A Chinese translation of this abstract is freely available at http://www.reproduction-online.org/content/149/6/R265/suppl/DC1.Free Japanese abstract: A Japanese translation of this abstract is freely available at http://www.reproduction-online.org/content/149/6/R265/suppl/DC2.

Coordination of stem cell fate is regulated by extrinsic niche signals and stem cell intrinsic factors. In mammalian testes, spermatogonial stem cells maintain constant production of abundant spermatozoa by alternating between self-renewal and differentiation at regular intervals according to a periodical program known as the seminiferous epithelial cycle. Although retinoic acid (RA) signaling has been suggested to direct the cyclical differentiation of spermatogonial stem cells, it remains largely unclear how their cycle-dependent self-renewal/proliferation is regulated. Here, we show that MEK/ERK signaling contributes to the cyclical activity of spermatogonial stem cells. We found that ERK1/2 is periodically activated in Sertoli cells during the stem cell self-renewal/proliferation phase, and that MEK/ERK signaling is required for the stage-related expression of the critical niche factor GDNF. In addition, ERK1/2 is activated in GFRα1-positive spermatogonial stem cells under the control of GDNF and prevent them from being differentiated. These results suggest that MEK/ERK signaling directly and indirectly maintains spermatogonial stem cells by mediating a signal that promotes their periodical self-renewal/proliferation. Conversely, RA signaling directly and indirectly induces differentiation of spermatogonial stem cells. We propose that temporally regulated activations of RA signaling and a signal regulating MEK/ERK antagonistically coordinates the cycle-related activity of spermatogonial stem cells.

To induce cell type-specific forms of gene regulation, pioneer factors open tightly packed, inaccessible chromatin sites, enabling the molecular machinery to act on functionally significant information encoded in DNA. While previous studies of pioneer factors have revealed their functions in transcriptional regulation, pioneer factors that open chromatin for other physiological events remain undetermined. In this issue of Genes & Development, Spruce and colleagues (pp. 398-412) report the functional significance of a "pioneer complex" in mouse meiotic recombination. This complex, comprised of the zinc finger DNA-binding protein PRDM9 and the SNF2 family chromatin remodeler HELLS, exposes nucleosomal DNA to designate the sites of DNA double-strand breaks that initiate meiotic recombination. Both HELLS and PRDM9 are required for the determination of these recombination hot spots. Through the identification of a pioneer complex for meiotic recombination, this study broadens the conceptual scope of pioneer factors, indicating their functional significance in biological processes beyond transcriptional regulation.

The ovarian reserve defines female reproductive lifespan, which in humans spans decades. The ovarian reserve consists of oocytes residing in primordial follicles arrested in meiotic prophase I and is maintained independent of DNA replication and cell proliferation, thereby lacking stem cell-based maintenance. Largely unknown is how cellular states of the ovarian reserve are established and maintained for decades. Our recent study revealed that a distinct chromatin state is established during ovarian reserve formation in mice, uncovering a novel window of epigenetic programming in female germline development. We showed that an epigenetic regulator, Polycomb Repressive Complex 1 (PRC1), establishes a repressive chromatin state in perinatal mouse oocytes that is essential for prophase I-arrested oocytes to form the ovarian reserve. Here we discuss the biological roles and mechanisms underlying epigenetic programming in ovarian reserve formation, highlighting current knowledge gaps and emerging research areas in female reproductive biology.

Cancer testis antigens or genes (CTA, CTG) are predominantly expressed in adult testes while silenced in most or all somatic tissues with sporadic expression in many human cancers. Concerted misexpression of numerous CTA/CTGs is rarely observed. This finding argues against the germ cell theory of cancer. A surprising number of CTA/CTGs are involved in meiotic chromosome metabolism and specifically in meiotic recombination. Recent discoveries with a group of CTGs established that their misexpression in somatic cells results in genomic instability by interfering with homologous recombination (HR), a DNA repair pathway for complex DNA damage such as DNA double-stranded breaks, interstrand crosslinks, and single-stranded DNA gaps. HR-deficient tumors have specific vulnerabilities and show synthetic lethality with inhibition of polyADP-ribose polymerase, opening the possibility that expression of CTA/CTGs that result in an HR-defect could be used as an additional biomarker for HR status. Here, we review the repertoire of CTA/CTGs focusing on a cohort that functions in meiotic chromosome metabolism by interrogating relevant cancer databases and discussing recent discoveries.

The Fanconi anemia (FA) DNA damage response (DDR) pathway regulate important cellular processes such as DNA replication, cell cycle control and DNA damage repair. Here we show that FANCD2, a key member of the FA DDR pathway, interacts with several important components of the germ-cell-specific Prmt5/piRNA pathways that orchestrate the repression of transposable elements (TEs). By using the Pou5f1-eGFP reporter mice, which marks pure populations of primordial germ cells (PGCs), we demonstrate that FA deficiency results in de-repression of TEs, depletion of PGCs, and defective spermatogenesis and oogenesis. Fancd2–KO PGCs exhibited excessive DNA damage and exacerbated apoptosis. Mechanistically, we observed a significant reduction of PRMT5-catalyzed H2A/H4R3me2s marks on the LINE1 TEs in E10.5 PGCs of Fancd2-KO; Pou5f1-eGFP and Fanca-KO;Pou5f1-eGFP embryos. Furthermore, we utilized the Fancd2-KI model to show that Fancd2 and Prmt5 co-occupied the promoter of LINE1 in WT PGCs, and that this co-occupancy was lost in FA-deficient (Fanca-KO) PGCs. These results suggest that the FA pathway takes part in TE repression in early PGCs, likely through a mechanism involving Fancd2-facilitated, Prmt5-catalyzed repressive H2A/H4R3me2s marks on TEs.

Meiosis is a key step during germ cell differentiation, accompanied by the activation of thousands of genes through germline-specific chromatin reorganization. The chromatin remodeling mechanisms underpinning early meiotic stages remain poorly understood. Here we focus on the function of one of the major autism genes, CHD8, in spermatogenesis, based on the epidemiological association between autism and low fertility rates. Specific ablation of Chd8 in germ cells results in gradual depletion of undifferentiated spermatogonia and the failure of meiotic double-strand break (DSB) formation, leading to meiotic prophase I arrest and cell death. Transcriptional analyses demonstrate that CHD8 is required for extensive activation of spermatogenic genes in spermatogonia, necessary for spermatogonial proliferation and meiosis. CHD8 directly binds and regulates genes crucial for meiosis, including H3K4me3 histone methyltransferase genes, meiotic cohesin genes, HORMA domain-containing genes, synaptonemal complex genes, and DNA damage response genes. We infer that CHD8 contributes to meiotic DSB formation and subsequent meiotic progression through combined regulation of these meiosis-related genes. Our study uncovers an essential role of CHD8 in the proliferation of undifferentiated spermatogonia and the successful progression of meiotic prophase I.

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.