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Tetraspanins play critical roles in various physiological processes, ranging from cell adhesion to virus infection. The members of the tetraspanin family have four membrane-spanning domains and short and large extracellular loops, and associate with a broad range of other functional proteins to exert cellular functions. Here we report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membrane-spanning partner protein, EWI-2. The reversed cone-like molecular shape of CD9 generates membrane curvature in the crystalline lipid layers, which explains the CD9 localization in regions with high membrane curvature and its implications in membrane remodeling. The molecular interaction between CD9 and EWI-2 is mainly mediated through the small residues in the transmembrane region and protein/lipid interactions, whereas the fertilization assay revealed the critical involvement of the LEL region in the sperm-egg fusion, indicating the different dependency of each binding domain for other partner proteins. Tetraspanins play critical roles in various physiological processes, ranging from cell adhesion to virus infection. Here authors report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membrane-spanning partner protein, EWI-2.

The mammalian epididymis is the organ for functional sperm maturation. In rodents, the initial segment, the most proximal region of the epididymis, plays a critical role in sperm maturation. The luminal epithelial differentiation and the following gene expression of the initial segment are regulated by the lumicrine signaling, a testis-epididymis transluminal secreted signaling. Adhesion G protein-coupled receptor G2 (ADGRG2) is expressed in the efferent duct and the initial segment epididymis. In the preceding study, Adgrg2 ablation decreased the expression of several genes expressed in the initial segment. Such downregulated genes include those known to be regulated by lumicrine signaling, suggesting the involvement of ADGRG2 in lumicrine signaling. The present study examined whether ADGRG2 is associated with the lumicrine signaling regulating epididymal initial segment differentiation and gene expression. Adgrg2-null mice were generated by CRISPR/CAS9-mediated genome editing. The postnatal differentiation of the Adgrg2-null male epididymal initial segment was histologically comparable with that of control wild-type animals. The RNA-seq of Adgrg2-null mice was performed together with those of efferent duct-ligated and W/Wv mice in both of which lumicrine signaling is defective. The comparative transcriptome analyses clarified that the expressions of genes expressed in the initial segment and regulated by lumicrine signaling were decreased by Adgrg2 nullification. However, the extent of such downregulations observed in Adgrg2null epididymis was not so prominent compared with those of lumicrine signaling deficient Nell2–/–, efferent duct-ligated, or W/Wv mice. Collectively, these findings indicate that ADGRG2 is dispensable for the lumicrine regulation of epididymal initial segment differentiation. Summary Sentence The association of ADGRG2 with the lumicrine-mediated epididymal initial segment differentiation and gene expression was examined by generating Adgrg2-null mice and concluded to be dispensable. Graphical Abstract

CRISPR-Cas9 associates with a guide RNA to target and cleave a specific DNA site next to a protospacer adjacent motif (PAM). Streptococcus pyogenes Cas9 (SpCas9), the one most often used for genome editing, only recognizes the NGG sequence (where N is any nucleobase) as the PAM, which restricts regions in the genome that can be targeted. To address this limitation, Nishimasu et al. created a SpCas9 variant that recognizes NG rather than NGG. The SpCas9-NG variant increased the targeting range, had a specificity similar to that of the wild-type enzyme, and could be used with a base editor. Thus, SpCas9-NG is a powerful addition to the CRISPR-Cas9 genome engineering toolbox and will be useful in a broad range of applications, from basic research to clinical therapeutics. Science , this issue p. 1259 An engineered CRISPR-Cas9 nuclease increases the range of genomic sequences that can be targeted in Cas9-mediated genome engineering. The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non–base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.

Astrocytes play a key role in brain homeostasis and functions such as memory. Specifically, astrocytes express multiple receptors that transduce signals via the second messenger cAMP. However, the involvement of astrocytic cAMP in animal behavior and the underlying glial–neuronal interactions remains largely unknown. Here, we show that an increase in astrocytic cAMP is sufficient to induce synaptic plasticity and modulate memory. We developed a method to increase astrocytic cAMP levels in vivo using photoactivated adenylyl cyclase and found that increased cAMP in hippocampal astrocytes at different time points facilitated memory formation but interrupted memory retention via NMDA receptor–dependent plasticity. Furthermore, we found that the cAMP-induced modulation of memory was mediated by the astrocyte–neuron lactate shuttle. Thus, our study unveils a role of astrocytic cAMP in brain function by providing a tool to modulate astrocytic cAMP in vivo.

Ovulation in the mouse and other mammals is controlled by hormones secreted by the hypothalamo-pituitary-ovarian axis. We describe anovulation and infertility in female mice lacking the microRNAs miR-200b and miR-429. Both miRNAs are strongly expressed in the pituitary gland, where they suppress expression of the transcriptional repressor ZEB1. Eliminating these miRNAs, in turn, inhibits luteinizing hormone (LH) synthesis by repressing transcription of its β-subunit gene, which leads to lowered serum LH concentration, an impaired LH surge, and failure to ovulate. Our results reveal roles for miR-200b and miR-429, and their target the Zebl gene, in the regulation of mammalian reproduction. Thus, the hypothalamo-pituitary-ovarian axis was shown to require miR-200b and miR-429 to support ovulation.

During early pregnancy in the mouse, nidatory estrogen (E2) stimulates endometrial receptivity by activating a network of signaling pathways that is not yet fully characterized. Here, we report that bone morphogenetic proteins (BMPs) control endometrial receptivity via a conserved activin receptor type 2 A (ACVR2A) and SMAD1/5 signaling pathway. Mice were generated to contain single or double conditional deletion of SMAD1/5 and ACVR2A/ACVR2B receptors using progesterone receptor (PR)-cre. Female mice with SMAD1/5 deletion display endometrial defects that result in the development of cystic endometrial glands, a hyperproliferative endometrial epithelium during the window of implantation, and impaired apicobasal transformation that prevents embryo implantation and leads to infertility. Analysis of Acvr2a- PRcre and Acvr2b -PRcre pregnant mice determined that BMP signaling occurs via ACVR2A and that ACVR2B is dispensable during embryo implantation. Therefore, BMPs signal through a conserved endometrial ACVR2A/SMAD1/5 pathway that promotes endometrial receptivity during embryo implantation. Building on the known role of BMP signalling in implantation, the authors define the role of uterine ACVR2A and ALK3 (via SMAD1/5) in vivo in regulating murine endometrial receptivity and embryo implantation.

Background A mixture of spermatozoa and accessory gland secretions (from seminal vesicles, prostates, and coagulating glands) is ejaculated into the female reproductive tract at copulation. However, the physiological function of accessory glands on male fecundity remains unclear. Methods Publications regarding the physiological functions of male accessory glands were summarized. Main findings (Results) The functions of accessory glands have been studied using male rodents surgically removed coagulating glands (CG), prostates (PR), or seminal vesicles (SV). CG‐removed males are fertile or subfertile, while the fecundity of PR‐removed males is controversial. SV‐removed males show copulatory plug defects, leading to fewer sperm in the uterus and severe subfertility. TGM4, SVS2, and PATE4 were identified as essential factors for copulatory plug formation. When the sufficient number of epididymal spermatozoa was artificially injected into a uterus (AI method), they could efficiently fertilize oocytes, implicating that accessory gland secretions are not essential. Seminal vesicle secretions (SVSs) improved fertilization rates only when low numbers of spermatozoa were used for AI. The changes of uterine environment by SVSs could not improve the pregnancy rate. Conclusion Accessory gland factors are critical for copulatory plug formation and support sperm fertilizing ability.

The mammalian sperm midpiece has a unique double-helical structure called the mitochondrial sheath that wraps tightly around the axoneme. Despite the remarkable organization of the mitochondrial sheath, the molecular mechanisms involved in mitochondrial sheath formation are unclear. In the process of screening testisenriched genes for functions in mice, we identified armadillo repeat-containing 12 (ARMC12) as an essential protein for mitochondrial sheath formation. Here, we engineered Armc12-null mice, FLAG-tagged Armc12 knock-in mice, and TBC1 domain family member 21 (Tbc1d21)-null mice to define the functions of ARMC12 in mitochondrial sheath formation in vivo. We discovered that absence of ARMC12 causes abnormal mitochondrial coiling along the flagellum, resulting in reduced sperm motility and male sterility. During spermiogenesis, sperm mitochondria in Armc12-null mice cannot elongate properly at the mitochondrial interlocking step which disrupts abnormal mitochondrial coiling. ARMC12 is a mitochondrial peripheral membrane protein and functions as an adherence factor between mitochondria in cultured cells. ARMC12 in testicular germ cells interacts with mitochondrial proteins MIC60, VDAC2, and VDAC3 as well as TBC1D21 and GK2, which are required for mitochondrial sheath formation. We also observed that TBC1D21 is essential for the interaction between ARMC12 and VDAC proteins in vivo. These results indicate that ARMC12 uses integral mitochondrial membrane proteins VDAC2 and VDAC3 as scaffolds to link mitochondria and works cooperatively with TBC1D21. Thus, our studies have revealed that ARMC12 regulates spatiotemporal mitochondrial dynamics to form the mitochondrial sheath through cooperative interactions with several proteins on the sperm mitochondrial surface.

Flagella and cilia are evolutionarily conserved cellular organelles. Abnormal formation or motility of these organelles in humans causes several syndromic diseases termed ciliopathies. The central component of flagella and cilia is the axoneme that is composed of the '9+2' microtubule arrangement, dynein arms, radial spokes, and the Nexin-Dynein Regulatory Complex (N-DRC). The N-DRC is localized between doublet microtubules and has been extensively studied in the unicellular flagellate Chlamydomonas. Recently, it has been reported that TCTE1 (DRC5), a component of the N-DRC, is essential for proper sperm motility and male fertility in mice. Further, TCTE1 has been shown to interact with FBXL13 (DRC6) and DRC7; however, functional roles of FBXL13 and DRC7 in mammals have not been elucidated. Here we show that Fbxl13 and Drc7 expression are testes-enriched in mice. Although Fbxl13 knockout (KO) mice did not show any obvious phenotypes, Drc7 KO male mice were infertile due to their short immotile spermatozoa. In Drc7 KO spermatids, the axoneme is disorganized and the '9+2' microtubule arrangement was difficult to detect. Further, other N-DRC components fail to incorporate into the flagellum without DRC7. These results indicate that Drc7, but not Fbxl13, is essential for the correct assembly of the N-DRC and flagella.

Endometrial decidualization, a prerequisite for successful pregnancies, relies on transcriptional reprogramming driven by progesterone receptor (PR) and bone morphogenetic protein (BMP)-SMAD1/SMAD5 signaling pathways. Despite their critical roles in early pregnancy, how these pathways intersect in reprogramming the endometrium into a receptive state remains unclear. To define how SMAD1 and/or SMAD5 integrate BMP signaling in the uterus during early pregnancy, we generated two novel transgenic mouse lines with affinity tags inserted into the endogenous SMAD1 and SMAD5 loci ( Smad1 HA/HA and Smad5 PA/PA ). By profiling the genome-wide distribution of SMAD1, SMAD5, and PR in the mouse uterus, we demonstrated the unique and shared roles of SMAD1 and SMAD5 during the window of implantation. We also showed the presence of a conserved SMAD1, SMAD5, and PR genomic binding signature in the uterus during early pregnancy. To functionally characterize the translational aspects of our findings, we demonstrated that SMAD1/5 knockdown in human endometrial stromal cells suppressed expressions of canonical decidual markers ( IGFBP1, PRL, FOXO1 ) and PR-responsive genes ( RORB , KLF15 ). Here, our studies provide novel tools to study BMP signaling pathways and highlight the fundamental roles of SMAD1/5 in mediating both BMP signaling pathways and the transcriptional response to progesterone (P4) during early pregnancy.