LIN28 lets BLIMP1 Take the Right Course

The transcription factor BLIMP1 is a master regulator of primordial germ cell (PGC) specification and is suppressed by the microRNA let-7. In a recent issue of Nature, West and colleagues use a unique in vitro ES cell differentiation strategy to show that LIN28 is an essential regulator of PGC formation through inhibition of let-7 maturation and consequential induction of BLIMP1

Loss of inhibin alpha uncouples oocyte-granulosa cell dynamics and disrupts postnatal folliculogenesis

AbstractTargeted disruption of the inhibin α gene (Inha–/–) in mice results in an ovarian phenotype of granulosa cell tumors that renders the animals infertile. Little is known about the reproductive defects prior to tumor development. Here, we report novel data on early follicle dynamics in Inha–/– mice, which demonstrate that inhibin α has important consequences upon follicle development. Morphological changes in both germ and somatic cells were evident in postnatal day 12 ovaries, with Inha−/− mice exhibiting numerous multilayered follicles that were far more advanced than those observed in age-matched controls. These changes were accompanied by alterations in follicle dynamics such that Inha−/− ovaries had fewer follicles in the resting pool and more committed in the growth phase. Absence of inhibin α resulted in advanced follicular maturation as marked by premature loss of anti-Müllerian hormone (AMH) in secondary follicles. Additionally, gene expression analysis revealed changes in factors known to be vital for oocyte and follicle development. Together, these data provide key evidence to suggest that regulation of the inhibin/activin system is essential for early folliculogenesis in the prepubertal mouse ovary

Spermatozoa lacking Fertilization Influencing Membrane Protein (FIMP) fail to fuse with oocytes in mice

Fujihara, Y., Lu, Y., Noda, T., Oji, A., Larasati, T., Kojima-Kita, K., . . . Ikawa, M. (2020). Spermatozoa lacking fertilization influencing membrane protein (FIMP) fail to fuse with oocytes in mice. Proceedings of the National Academy of Sciences of the United States of America, 117(17), 9393-9400. doi:10.1073/pnas.191706011

Metabolism of JQ1, an Inhibitor of Bromodomain and Extra Terminal Bromodomain Proteins, in Human and Mouse Liver Microsomes†

JQ1 is a small-molecule inhibitor of the bromodomain and extra terminal (BET) protein family that potently inhibits the bromodomain testis-specific protein (BRDT), which is essential for spermatogenesis. JQ1 treatment produces a reversible contraceptive effect by targeting the activity of BRDT in mouse male germ cells, validating BRDT as a male contraceptive target. Although JQ1 possesses favourable physical properties, it exhibits a short half-life. Because the details of xenobiotic metabolism play important roles in the optimization of drug candidates and in determining the role of metabolism in drug efficacy, we investigated the metabolism of JQ1 in human and mouse liver microsomes. We present the first comprehensive view of JQ1 metabolism in liver microsomes, distinguishing nine JQ1 metabolites, including three monohydroxylated, one de-tert-butylated, two dihydroxylated, one monohydroxylated/dehydrogenated, one monohydroxylated-de-tert-butylated and one dihydroxylated/dehydrogenated variant of JQ1. The dominant metabolite (M1) in both human and mouse liver microsomes is monohydroxylated on the fused three-ring core. Using recombinant cytochrome P450 (CYP) enzymes, chemical inhibitors and the liver S9 fraction of Cyp3a-null mice, we identify enzymes that contribute to the formation of these metabolites. Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) is the main contributor to the production of JQ1 metabolites in vitro, and the CYP3A4/5 inhibitor ketoconazole strongly inhibits JQ1 metabolism in both human and mouse liver microsomes. Our findings suggest that JQ1 half-life and efficacy might be improved in vivo by co-administration of a selective CYP inhibitor, thereby impacting the use of JQ1 as a probe for BRDT activity in spermatogenesis and as a probe or therapeutic in other systems

Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm-oocyte fusion in mice

Noda, T., Lu, Y., Fujihara, Y., Oura, S., Koyano, T., Kobayashi, S., . . . Ikawa, M. (2020). Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm-oocyte fusion in mice. Proceedings of the National Academy of Sciences of the United States of America, 117(21) doi:10.1073/pnas.192265011

Disruption of Gastrulation and Heparan Sulfate Biosynthesis in EXT1-Deficient Mice

AbstractMutations in the EXT1 gene are responsible for human hereditary multiple exostosis type 1. The Drosophila EXT1 homologue, tout-velu, regulates Hedgehog diffusion and signaling, which play an important role in tissue patterning during both invertebrate and vertebrate development. The EXT1 protein is also required for the biosynthesis of heparan sulfate glycosaminoglycans that bind Hedgehog. In this study, we generated EXT1-deficient mice by gene targeting. EXT1 homozygous mutants fail to gastrulate and generally lack organized mesoderm and extraembryonic tissues, resulting in smaller embryos compared to normal littermates. RT-PCR analysis of markers for visceral endoderm and mesoderm development indicates the delayed and abnormal development of both of these tissues. Immunohistochemical staining revealed a visceral endoderm pattern of Indian hedgehog (Ihh) in wild-type E6.5 embryos. However, in both EXT1-deficient embryos and wild-type embryos treated with heparitinase I, Ihh failed to associate with the cells. The effect of the EXT1 deletion on heparan sulfate formation was tested by HPLC and cellular glycosyltransferase activity assays. Heparan sulfate synthesis was abolished in EXT1 −/− ES cells and decreased to less than 50% in +/− cell lines. These results indicate that EXT1 is essential for both gastrulation and heparan sulfate biosynthesis in early embryonic development

Homogeneous and Functional Group Tolerant Ring-Closing Metathesis for DNA-Encoded Chemical Libraries

Reaction heterogeneity, poor pH control, and catalyst decomposition in the ring-closing metathesis (RCM) of DNA–chemical conjugates lead to poor yields of the cyclized products. Herein we address these issues with a RCM reaction system that includes a novel aqueous solvent combination to enable reaction homogeneity, an acidic buffer system which masks traditionally problematic functional groups, and a decomposition-resistant catalyst which maximizes conversion to the cyclized product. Additionally, we provide a systematic study of the substrate scope of the on-DNA RCM reaction, a demonstration of its applicability to a single-substrate DNA-encoded chemical library that includes sequencing analysis, and the first successful stapling of an unprotected on-DNA [i, i+4] peptide

Genetic evidence that SMAD2 is not required for gonadal tumor development in inhibin-deficient mice

<p>Abstract</p> <p>Background</p> <p>Inhibin is a tumor-suppressor and activin antagonist. Inhibin-deficient mice develop gonadal tumors and a cachexia wasting syndrome due to enhanced activin signaling. Because activins signal through SMAD2 and SMAD3 in vitro and loss of SMAD3 attenuates ovarian tumor development in inhibin-deficient females, we sought to determine the role of SMAD2 in the development of ovarian tumors originating from the granulosa cell lineage.</p> <p>Methods</p> <p>Using an inhibin α null mouse model and a conditional knockout strategy, double conditional knockout mice of Smad2 and inhibin alpha were generated in the current study. The survival rate and development of gonadal tumors and the accompanying cachexia wasting syndrome were monitored.</p> <p>Results</p> <p>Nearly identical to the controls, the Smad2 and inhibin alpha double knockout mice succumbed to weight loss, aggressive tumor progression, and death. Furthermore, elevated activin levels and activin-induced pathologies in the liver and stomach characteristic of inhibin deficiency were also observed in these mice. Our results indicate that SMAD2 ablation does not protect inhibin-deficient females from the development of ovarian tumors or the cachexia wasting syndrome.</p> <p>Conclusions</p> <p>SMAD2 is not required for mediating tumorigenic signals of activin in ovarian tumor development caused by loss of inhibin.</p

Molecular Dissection and Testing of PRSS37 Function Through Lc-Ms/Ms and the Generation of a PRSS37 Humanized Mouse Model

The quest for a non-hormonal male contraceptive pill for men still exists. Serine protease 37 (PRSS37) is a sperm-specific protein that when ablated in mice renders them sterile. In this study we sought to examine the molecular sequelae of PRSS37 loss to better understand its molecular function, and to determine whether human PRSS37 could rescue the sterility phenotype of knockout (KO) mice, allowing for a more appropriate model for drug molecule testing. To this end, we used CRISPR-EZ to create mice lacking the entire coding region of Prss37, used pronuclear injection to create transgenic mice expressing human PRSS37, intercrossed these lines to generate humanized mice, and performed LC-MS/MS of KO and control tissues to identify proteomic perturbances that could attribute a molecular function to PRSS37. We found that our newly generated Prss37 KO mouse line is sterile, our human transgene rescues the sterility phenotype of KO mice, and our proteomics data not only yields novel insight into the proteome as it evolves along the male reproductive tract, but also demonstrates the proteins significantly influenced by PRSS37 loss. In summary, we report vast biological insight including insight into PRSS37 function and the generation of a novel tool for contraceptive evaluation

GASZ promotes germ cell derivation from embryonic stem cells

AbstractPrimordial germ cells (PGCs) are the first germ-line population that forms from the proximal epiblast of the developing embryo. Despite their biological importance, the regulatory networks whereby PGCs arise, migrate, and differentiate into gametes during embryonic development remains elusive, largely due to the limited number of germ cells in the early embryo. To elucidate the molecular mechanisms that govern early germ cell development, we utilized an in vitro differentiation model of embryonic stem cells (ESCs) and screened a series of candidate genes with specific expression in the adult reproductive organs. We discovered that gain of function of Gasz, a gene previously reported to participate in meiosis of postnatal spermatocytes, led to the most robust upregulation of PGC formation from both human and murine ESCs. In contrast, Gasz deficiency resulted in pronounced reduction of germ cells during ESC differentiation and decreased expression of MVH and DAZL in genital ridges during early embryonic development. Further analyses demonstrated that GASZ interacted with DAZL, a key germ cell regulator, to synergistically promote germ cell derivation from ESCs. Thus, our data reveal a potential role of GASZ during embryonic germ cell development and provide a powerful in vitro system for dissecting the molecular pathways in early germ cell formation during embryogenesis