Spermatogenesis requires intricate interactions between the germline and somatic cells. Within a given cross section of a seminiferous tubule, multiple germ and somatic cell types co-occur. This cellular heterogeneity has made it difficult to profile distinct cell types at different stages of development. To address this challenge, we collected single-cell RNA sequencing data from ∼35,000 cells from the adult mouse testis and identified all known germ and somatic cells, as well as two unexpected somatic cell types. Our analysis revealed a continuous developmental trajectory of germ cells from spermatogonia to spermatids and identified candidate transcriptional regulators at several transition points during differentiation. Focused analyses delineated four subtypes of spermatogonia and nine subtypes of Sertoli cells; the latter linked to histologically defined developmental stages over the seminiferous epithelial cycle. Overall, this high-resolution cellular atlas represents a community resource and foundation of knowledge to study germ cell development and in vivo gametogenesis.

This study examines metal binding to metallo-β-lactamase VIM-2, demonstrating the first successful preparation of a Co(II)-substituted VIM-2 analogue. Spectroscopic studies of the half- and fully metal loaded enzymes show that both Zn(II) and Co(II) bind cooperatively, where the major species present, regardless of stoichiometry, are apo- and di-Zn (or di-Co) enzymes. We determined the di-Zn VIM-2 structure to a resolution of 1.55 Å, and this structure supports results from spectroscopic studies. Kinetics, both steady-state and pre-steady-state, show that VIM-2 utilizes a mechanism that proceeds through a very short-lived anionic intermediate when chromacef is used as the substrate. Comparison with other B1 enzymes shows that those that bind Zn(II) cooperatively are better poised to protonate the intermediate on its formation, compared to those that bind Zn(II) non-cooperatively, which uniformly build up substantial amounts of the intermediate.

Polycomb Repressive Complex 2 (PRC2) methylates lysine 27 in histone H3, a modification associated with epigenetic gene silencing. This complex plays a fundamental role in regulating cellular differentiation and development, and PRC2 overexpression and mutations have been implicated in numerous cancers. In this review, we examine recent studies elucidating the first crystal structures of the PRC2 core complex, yielding seminal insights into its catalytic mechanism, substrate specificity, allosteric regulation, and inhibition by a class of small molecules that are currently undergoing cancer clinical trials. We conclude by exploring unresolved questions and future directions for inquiry regarding PRC2 structure and function.Polycomb group (PcG) proteins represent transcriptional repressors that are present in single cell eukaryotes through multicellular organisms (1,2). The genes encoding many of these proteins were initially characterized in Drosophila as key regulators of epigenetic silencing of homeotic genes (3,4). Subsequent studies demonstrated that PcG proteins function in the context of large heteromeric complexes that repress gene expression within facultative heterochromatin. These PcG complexes include Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2), as well as the more recently identified Phorepressive Complex (PhoRC) and Polycomb Repressive Deubiquitinase (PR-DUB) (1,5-7). The PRCs possess intrinsic histone modifying activities that contribute to their functions in transcriptional repression. PRC1 monoubiquitinates Lys119 in histone H2A (H2AK119ub1) and can compact chromatin by binding to nucleosomes, whereas PRC2 is a lysine methyltransferase (KMT) that trimethylates Lys27 in histone H3 (H3K27me3), a modification associated with PcG silencing (8-15). PRC1 has been shown to bind H3K27me3, whereas PRC2 can recognize H3K27me3 and H2AK119ub1, facilitating the recruitment of the PRCs to specific genomic loci (16)(17)(18)(19). The interdependence of their enzymatic activities and chromatin localization illustrates how PRC1 and PRC2 can function in concert to epigenetically silence gene expression (20).PRC2 comprises multiple subunits that facilitate its biological functions. The minimal core complex that exhibits methyltransferase activity comprises the core subunits Embryonic Ectoderm Development (EED), Suppressor of Zeste 12 (SUZ12) and the catalytic subunit Enhancer of Zeste Homolog 1 or 2 (EZH1 or EZH2) that possess a conserved catalytic SET domain found in many [21][22][23] (9)(10)(11)20,24,25). Many of these non-core subunits possess intrinsic histone or DNA binding activity and can mediate recruitment of PRC2 to chromatin and promote H3K27 methylation (20,(26)(27)(28)(29). Deletion of the genes encoding the PRC2 core subunits in mice results in morphological defects and embryonic lethality, underscoring their importance in regulating epigenetic programs that are essential to development and differentiation (22,30,31). PRC2 has also been shown to have context-dependent roles in cancer ...

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility.

Male fertility throughout life hinges on the successful production of motile sperm, a developmental process that involves three coordinated transitions: mitosis, meiosis, and spermiogenesis. Germ cells undergo both mitosis and meiosis to generate haploid round spermatids, in which histones bound to the male genome are replaced with small nuclear proteins known as protamines. During this transformation, the chromatin undergoes extensive remodeling to become highly compacted in the sperm head. Despite its central role in spermiogenesis and fertility, we lack a comprehensive understanding of the molecular mechanisms underlying the remodeling process, including which remodelers/chaperones are involved, and whether intermediate chromatin proteins function as discrete steps, or unite simultaneously to drive successful exchange. Furthermore, it remains largely unknown whether more nuanced interactions instructed by protamine post-translational modifications affect chromatin dynamics or gene expression in the early embryo. Here, we bring together past and more recent work to explore these topics and suggest future studies that will elevate our understanding of the molecular basis of the histone-to-protamine exchange and the underlying etiology of idiopathic male infertility.

Previous crystallographic and mutagenesis studies have implicated the role of a position-conserved hairpin loop in the metallo-β-lactamases in substrate binding and catalysis. In an effort to probe the motion of that loop during catalysis, rapid-freeze-quench double electron electron resonance (RFQ-DEER) spectroscopy was used to interrogate metallo-β-lactamase CcrA, which had a spin label at position 49 on the loop and spin labels (at positions 82, 126, or 233) 20–35 Å away from residue 49, during catalysis. At 10 milliseconds after mixing, the DEER spectra show distance increases of 7, 10, and 13 Å between the spin label at position 49 and the spin labels at positions 82, 126, and 233, respectively. In contrast to previous hypotheses, these data suggest that the loop moves nearly 10 Å away from the metal center during catalysis and that the loop does not clamp down on the substrate during catalysis. This study demonstrates that loop motion during catalysis can be interrogated on the millisecond time scale.

Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by passive electrostatics between DNA and the arginine-rich core of protamines. However, phylogenetic analysis reveals several non-arginine residues that are conserved within, but not across, species. The functional significance of these residues or post-translational modifications are poorly understood. Here, we investigated the functional role of K49, a rodent-specific lysine residue in mouse protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In vivo, an alanine substitution (P1 K49A) results in ectopic histone retention, decreased sperm motility, decreased male fertility, and in zygotes, premature P1 removal from paternal chromatin. In vitro, the P1 K49A substitution decreases protamine-DNA binding and alters DNA compaction/decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential to ensure reproductive fitness.

2 Highlights • Multipotent Tcf21 + MPs can differentiate into somatic testis cell types • Tcf21 + cells contribute to testis and ovary somatic cells during gonadal development • Tcf21 + cells replenish somatic cells of the aging testis and in response to tissue injury • Testis Tcf21 cells resemble resident fibroblast populations in multiple organs 3 Summary Testicular development and function relies on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity decline in aging and disease. Whether the adult testis maintains a reserve progenitor population with repair or regenerative capacity remains uncertain. Here, we characterized a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that Tcf21 + cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as reserve somatic progenitors following injury. In vitro, Tcf21 + cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, Tcf21 + cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be leveraged in development of future therapies for hypoandrogenism and/or infertility.