We describe the discovery and preclinical characterization of a potent and selective lysophosphatidic acid receptor 1 (LPAR1) antagonist with a direct-acting antifibrotic mechanism. 18a was initially identified as a potent non-carboxylic acid LPAR1 antagonist in an LPA-induced myocardin-related transcription factor A (MRTF-A) nuclear translocation assay. Modifications to the aromatic elements in the structure allowed for improvements in metabolic stability and the mitigation of GSH adduct formation, but in vitro to in vivo clearance disconnects were observed with several potent sulfonamides (e.g., 27b) across preclinical species. Through modification of the sulfonamide, 42 (GS-2278) emerged as a potent LPAR1 antagonist with a suitable in vitro profile and desirable pharmacokinetic properties for oral QD dosing. GS-2278 dose-dependently blocked LPA-induced histamine release and demonstrated efficacy in an interventional model of bleomycin-induced lung fibrosis. However, CNS-related toxicity was observed in dogs, and based on these findings, the clinical development of GS-2278 for IPF was halted.

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H/ACA small nucleolar RNAs (snoRNAs) guide pseudouridylation as part of a small nucleolar ribonucleoprotein complex (snoRNP). Disruption of H/ACA snoRNA levels in stem cells impairs pluripotency, yet it remains unclear how H/ACA snoRNAs contribute to differentiation. To determine if H/ACA snoRNA levels are dynamic during differentiation, we comprehensively profiled H/ACA snoRNA abundance in multiple murine cell types and during differentiation in three cellular models, including mouse embryonic stem cells and mouse myoblasts. We determined that the profiles of H/ACA snoRNA abundance are cell-type specific, and we identified a subset of snoRNAs that are specifically regulated during differentiation. Additionally, we demonstrated that a decrease in Snora27 abundance upon differentiation corresponds to a decrease in pseudouridylation of its target site within the E-site transfer RNA (tRNA) binding region of the 28S ribosomal RNA (rRNA) in the large ribosomal subunit. Together, these data point toward a potential model in which H/ACA snoRNAs are specifically regulated during differentiation to alter pseudouridylation and fine tune ribosome function.

Spermatogenesis is a differentiation process that requires dramatic changes to DNA architecture, a process governed in part by Transition Nuclear Proteins 1 and 2 (TNP1 and TNP2). Translation of Tnp1 and Tnp2 mRNAs is temporally disengaged from their transcription. We hypothesized that RNA regulatory proteins associate specifically with Tnp mRNAs to control the delayed timing of their translation. To identify potential regulatory proteins, we isolated endogenous mRNA/protein complexes from testis extract and identified by mass spectrometry proteins that associated with one or both Tnp transcripts. Five proteins showed strong association with Tnp transcripts but had low signal when Actin mRNA was isolated. We visualized the expression patterns in testis sections of the five proteins and found that each of the proteins was detected in germ cells at the appropriate stages to regulate Tnp RNA expression.

Phosphoinositide 3-kinase (PI3K) β signaling is required to sustain cancer cell growth in which the tumor suppressor phosphatase and tensin homolog (PTEN) has been deactivated. This manuscript describes the discovery, optimization, and in vivo evaluation of a novel series of PI3Kβ/δ inhibitors in which PI3Kβ potency was built in a PI3Kδ-selective template. This work led to the discovery of a highly selective PI3Kβ/δ inhibitor displaying excellent pharmacokinetic profile and efficacy in a human PTEN-deficient LNCaP prostate carcinoma xenograft tumor model.

Improved therapies for cancer and other conditions have resulted in a growing population of long-term survivors. Infertility is an unfortunate side effect of some cancer therapies that impacts the quality of life of survivors who are in their reproductive or prereproductive years. Some of these patients have the opportunity to preserve their fertility using standard technologies that include sperm, egg, or embryo banking, followed by IVF and/or ET. However, these options are not available to all patients, especially the prepubertal patients who are not yet producing mature gametes. For these patients, there are several stem cell technologies in the research pipeline that may give rise to new fertility options and allow infertile patients to have their own biological children. We will review the role of stem cells in normal spermatogenesis as well as experimental stem cell-based techniques that may have potential to generate or regenerate spermatogenesis and sperm. We will present these technologies in the context of the fertility preservation paradigm, but we anticipate that they will have broad implications for the assisted reproduction field.

SHP2 is a widely expressed protein tyrosine phosphatase required for signal transduction from multiple cell surface receptors. Gain and loss of function SHP2 mutations in humans are known to cause Noonan and LEOPARD syndromes, respectively, that are characterized by numerous pathological conditions including male infertility. Using conditional gene targeting in the mouse, we found that SHP2 is required for maintaining spermatogonial stem cells (SSCs) and the production of germ cells required for male fertility. After deleting SHP2, spermatogenesis was halted at the initial step during which transit-amplifying undifferentiated spermatogonia are produced from SSCs. In the absence of SHP2, proliferation of SSCs and undifferentiated spermatogonia was inhibited, thus germ cells cannot be replenished and SSCs cannot undergo renewal. However, germ cells beyond the undifferentiated spermatogonia stage of development at the time of SHP2 knockout were able to complete their maturation to become sperm. In cultures of SSCs and their progeny, inhibition of SHP2 activity reduced growth factor-mediated intracellular signaling that regulates SSC proliferation and cell fate. Inhibition of SHP2 also decreased the number of SSCs present in culture and caused SSCs to detach from supporting cells. Injection of mice with an SHP2 inhibitor blocked the production of germ cells from SSCs. Together, our studies show that SHP2 is essential for SSCs to maintain fertility and indicates that the pathogenesis of infertility in humans with SHP2 mutations is due to compromised SSC functions that block spermatogenesis.

Spermatogenesis is a highly organized and proliferative system. The spermatogonial stem cell (SSC) maintains spermatogenesis throughout adult life by balancing self renewal with differentiation. The growth factor GDNF (glial cell line-derived neurotrophic factor) is necessary for maintenance of mouse SSCs in vitro and in vivo, but the downstream effects of GDNF in SSCs are not well understood. Here we describe the role of GDNF in regulating RNA binding protein YBX1 in the mouse testis. Using immunohistochemistry, we show that YBX1 is expressed uniquely by cells on the basement membrane of seminiferous tubules, including stem and progenitor spermatogonia. Additionally, we show that YBX1 is phosphorylated in germ cells in vitro and in vivo, and we show through growth factor withdrawal in SSC cultures, that GDNF signaling modulates YBX1 phosphorylation. Specifically inhibiting either phosphatidylinositol-3-kinase (PI3K) or AKT in SSC cultures also causes a loss of YBX1 phosphorylation, suggesting that GDNF works through the PI3K/AKT pathway to phosphorylate YBX1. We next wanted to identify mRNA targets of YBX1, and whether the targets change in response to growth factor stimuli. We immunoprecipitated YBX1 from SSC cultures grown in the presence or absence of GDNF, and isolated co-immunoprecipitated mRNAs. In normal growth conditions (containing GDNF), YBX1 associated with mRNA for genes expressed by all germ cells (Dazl and Ddx4), but not those expressed specifically by more undifferentiated germ cells (GFRa1, Ngn3 and Plzf). Furthermore, association of YBX1 with a specific mRNA correlates with an increase in protein levels for that gene. These data collectively lead us to conclude that YBX1 mediates growth factor-dependent translational regulation of transcripts important for coordinating SSC maintenance, renewal and/or differentiation. Supported by Magee-Womens Research Institute and Foundation and NIH grants HD08160, RR018500 and HD055475.

With the recent publication of 'Direct Differentiation of Human Pluripotent Stem Cells into Haploid Spermatogenic Cells' in CELL REPORTS, several fundamental and clinical avenues for future research are now available. In this article, we review the discoveries reported and also consider the implications for the management of male infertility as well as new contraceptive designs.

Commentary on Oatley et al., “Inhibitor of DNA binding 4 is expressed selectively by single spermatogonia in the male germline and regulates the self-renewal of spermatogonial stem cells.”

A role for RNA regulation in male germ cell development is well established and evolutionarily conserved. The significance of RNA regulation in germ cell biology is enhanced by recent identification of unique small RNAs (piRNAs), cellular components (nuage) and proteins (such as DAZL, NANOS3 and PIWIL2) with specific and necessary functions in spermatogenesis. Furthermore, previous microarray results from our lab indicate that RNA regulatory proteins are over-represented by mouse spermatogonial stem cells (SSCs). The Ybx1 gene identified in that study encodes the protein YBX1 which has varied cellular functions including translational regulation. RT-PCR indicated that Ybx1 mRNA was expressed in all mouse tissues analyzed, including the testis, though its specific role in the testis is unclear. In the testis, we show that YBX1 is expressed specifically by cells on the basement membrane of mouse seminiferous tubules, consistent with the location of undifferentiated spermatogonia. Additionally, in vitro (immunocytochemistry in SSC culture) and in vivo (immunohistochemistry in histological sections of the testis) studies show that YBX1 is co-expressed with the pan germ cell marker DAZL as well as the stem and progenitor spermatogonia marker, PLZF. To begin investigating the regulation of YBX1, we looked at its phosphorylation status and found that YBX1 is phosphorylated in the adult mouse testis. Interestingly previous in vitro studies show that AKT directly phosphorylates YBX1 and that this phosphorylation regulates the ability of YBX1 to bind mRNA. In SSCs the AKT pathway is activated by GDNF (glial cell derived neurotrophic factor) signaling. GDNF is the factor necessary for long-term renewal of mouse SSCs in vitro. Thus, we hypothesize that GDNF-activated AKT signaling is mediated, at least in part, by YBX1 regulation of target mRNAs. This work was supported by NIH grant RR18500 and Magee-Womens Research Institute and Foundation. (poster)

This article will provide an updated review of spermatogonial stem cells and their role in maintaining the spermatogenic lineage. Experimental tools used to study spermatogonial stem cells (SSCs) will be described, along with research using these tools to enhance our understanding of stem cell biology and spermatogenesis. Increased knowledge about the biology of SSCs improves our capacity to manipulate these cells for practical application. The chapter concludes with a discussion of future directions for fundamental investigation and practical applications of SSCs.

Gene expression and consequent biological activity of adult tissue stem cells are regulated by signals emanating from the local microenvironment (niche). To gain insights into the molecular regulation of spermatogonial stem cells (SSCs), gene expression was characterized from SSCs isolated from their cognate niches of cryptorchid (stem cell-enriched), wild-type, and busulfan-treated (stem cell-depleted) mouse testes. Quantitative assessment of stem cell activity in each testis model was determined using an in vivo functional assay and correlated with gene expression using Affymetrix MGU74Av2 microarrays and the ChipStat algorithm optimized to detect gene expression from rare cells in complex tissues. We identified 389 stem/progenitor spermatogonia candidate genes, which exhibited significant overlap with genes expressed by embryonic, hematopoietic, and neural stem cells; enriched spermatogonia; and cultured SSCs identified in previous studies. Candidate cell surface markers identified by the microarray may facilitate the isolation and enrichment of stem and/or progenitor spermatogonia. Flow cytometric analyses confirmed the expression of chemokine receptor 2 (Ccr2) and Cd14 on a subpopulation cryptorchid testis cells (alpha 6-integrin(+), side scatter(10)) enriched for SSCs. These cell surface molecules may mark progenitor spermatogonia but not SSCs because Ccr2(+) and Cd14(+) fractions failed to produce spermatogenesis upon transplantation to recipient testes. Functional annotation of candidate genes and subsequent immunohistochemistry revealed that proteins involved in post-transcriptional regulation are overrepresented in cryptorchid testes that are enriched for SSCs. Comparative analyses indicated that this is a recurrent biological theme among stem cells.