The long non-coding RNA, MHM, plays a role in chicken embryonic development, including gonadogenesis

MHM is a chicken Z chromosome-linked locus that is methylated and transcriptionally silent in male cells, but is hypomethylated and transcribed into a long non-coding RNA in female cells. MHM has been implicated in both localised dosage compensation and sex determination in the chicken embryo, but direct evidence is lacking. We investigated the potential role of MHM in chicken embryonic development, using expression analysis and retroviral-mediated mis-expression. At embryonic stages, MHM is only expressed in females. Northern blotting showed that both sense and antisense strands of the MHM locus are transcribed, with the sense strand being more abundant. Whole mount in situ hybridization confirmed that the sense RNA is present in developing female embryos, notably in gonads, limbs, heart, branchial arch and brain. Within these cells, the MHM RNA is localized to the nucleus. The antisense transcript is lowly expressed and has a cytoplasmic localization in cells. Mis-expression of MHM sense and antisense sequences results in overgrowth of tissues in which transcripts are predominantly expressed. This includes altered asymmetric ovarian development in females. In males, MHM mis-expression impairs gonadal expression of the testis gene, DMRT1. Both MHM sense and antisense mis-expression cause brain abnormalities, while MHM sense causes an increase in male-biased embryo mortality. These results indicate that MHM has a role in chicken normal embryonic development, including gonadal sex differentiation

TGFβ superfamily signaling regulators are differentially expressed in the developing and adult mouse testis

Transforming growth factor-beta (TGFβ) superfamily ligands are produced by and act upon testicular cells to control testis morphogenesis and adult fertility. Ligand production changes during testis development and dysregulated signaling affects the number of cells comprising each lineage and their development, with several components of this diverse signaling pathway linked to male infertility. To test the hypothesis that TGFβ superfamily signaling regulators are differentially expressed during mouse testis development, we surveyed expression of Hgs, Zfyve9, Smurf1 and Net25 by northern blot and in situ hybridization and SMURF2 and MAN1 by western blot and immunohistochemistry. Expression of these genes is highly regulated and differs between the first spermatogenic wave and adult spermatogenesis. Zfyve9 transcripts were first detected in Sertoli cells and spermatogonia at 5 days post partum (dpp) whereas Hgs mRNA was first detected in pachytene spermatocytes at 15 dpp. Smurf1 mRNA was broadly expressed at 0 and 5 dpp but restricted to spermatogonia and early spermatocytes at 15 dpp and spermatogonia, spermatocytes and round spermatids in adults. SMURF2 was limited to gonocyte nuclei at birth but was nuclear in all cells at 5 dpp. SMURF2 was absent from 15 dpp differentiating spermatogonia and early spermatocytes but readily detected in adult pachytene spermatocytes and round spermatids. MAN1 and Net25 also had different expression profiles, with MAN1 undetectable at 5 dpp. Differential synthesis of signaling modulators explains how Sertoli cells and spermatogenic cells, which all possess TGFβ superfamily signaling machinery and reside within the same microenvironment, respond differently to the same ligand

Smad3 dosage determines androgen responsiveness and sets the pace of postnatal testis development

The establishment and maturation of the testicular Sertoli cell population underpins adult male fertility. These events are influenced by hormones and endocrine factors, including FSH, testosterone and activin. Activin A has developmentally regulated effects on Sertoli cells, enhancing proliferation of immature cells and later promoting postmitotic maturation. These differential responses correlate with altered mothers against decapentaplegic (SMAD)-2/ 3 signaling: immature cells signal via SMAD3, whereas postmitotic cells use both SMAD2 and SMAD3. This study examined the contribution of SMAD3 to postnatal mouse testis development. We show that SMAD3 production and subcellular localization are highly regulated and, through histological and molecular analyses, identify effects of altered Smad3 dosage on Sertoli and germ cell development. Smad3(-/-) and Smad3(-/-) mice had smaller testes at 7 d postpartum, but this was not sustained into adulthood. Juvenile and adult serum FSH levels were unaffected by genotype. Smad3-null mice displayed delayed Sertoli cell maturation and had reduced expression of androgen receptor (AR), androgen-regulated transcripts, and Smad2, whereas germ cell and Leydig cell development were essentially normal. This contrasted remarkably with advanced Sertoli and germ cell maturation and increased expression of AR and androgen-regulated transcripts in Smad3(-/-) mice. In addition, SMAD3 was down regulated during test is development and testosterone up-regulated Smad2, but not Smad3, in the TM4 Sertoli cell line. Collectively these data reveal that appropriate SMAD3-mediated signaling drives normal Sertoli cell proliferation, androgen responsiveness, and maturation and influences the pace of the first wave of spermatogenesis, providing new clues to causes of altered pubertal development in boy

Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis

Germ cell proliferation, migration and survival during all stages of spermatogenesis are affected by stem cell factor signalling through the c-Kit receptor, the expression and function of which are vital for normal male reproductive function. The present study comprehensively describes the c-Kit mRNA and protein cellular expression profiles in germ cells of the postnatal and adult rodent testis, revealing their significant elevation in synthesis at the onset of spermatogenesis. Real-time PCR analysis for both mice and rats matched the cellular mRNA expression profile where examined. Localization studies in normal mouse testes indicated that both c-Kit mRNA and protein are first detectable in differentiating spermatogonia. In addition, all spermatogonia isolated from 8-day-old mice displayed detectable c-Kit mRNA, but 30-50% of these lacked protein expression. The c-Kit mRNA and protein profile in normal rat testes indicated expression in gonocytes, in addition to differentiating spermatogonia. However, in the irradiated adult rat testes, in which undifferentiated spermatogonia are the only germ cell type, mRNA was also detected in the absence of protein. This persisted at 3 days and 1 and 2 weeks following treatment with gonadotrophin-releasing hormone (GnRH) antagonist to stimulate spermatogenesis recovery. By 4 weeks of GnRH antagonist treatment, accompanying the emergence of differentiating spermatogonia, both mRNA and protein were detected. Based on these observations, we propose that c-Kit mRNA and protein synthesis are regulated separately, possibly by influences linked to testis maturation and circulating hormone levels. © 2006 Society for Reproduction and Fertility

Thrombin stimulation of proteoglycan synthesis in vascular smooth muscle is mediated by protease-activated receptor-1 transactivation of the transforming growth factor βtype I receptor

Growth factors modify the structure of the glycosaminoglycan (GAG) chains on biglycan leading to enhanced LDL binding. G-protein receptor-coupled agonists such as thrombin, signal changes the structure of proteoglycans produced by vascular smooth muscle cells (VSMCs). One component of classical G-protein-coupled receptor (GPCR) signaling invokes transactivation of protein tyrosine kinase receptors such as the epidermal growth factor receptor. Serine/threonine receptor growth factors such as transforming growth factor-(TGF)-β are potent activators of proteoglycan synthesis. We have used the model of proteoglycan synthesis to demonstrate that the signaling paradigm of GPCR signaling can be extended to include the transactivation of serine/threonine receptor, specifically the TGF-β type I receptor (TβRI) also known as activin-like kinase (ALK) V. Thrombin stimulated elongation of GAG chains and increased proteoglycan core protein expression and these responses were blocked by the TβRI antagonist, SB431542 and TβRI siRNA knockdown, as well as several protease-activated receptor (PAR)-1 antagonists. The canonical downstream response to TGF-βis increased C-terminal phosphorylation of the transcription factor Smad2 generating phospho-Smad2C (phosphorylation of Smad2 C-terminal region). Thrombin stimulated increased phospho-Smad2C levels, and the response was blocked by SB431542 and JNJ5177094. The proteolytically inactive thrombin mimetic thrombin-receptor activating peptide also stimulated an increase in cytosolic phospho-Smad2C. Signaling pathways for growth factor regulated proteoglycan synthesis represent therapeutic targets for the prevention of atherosclerosis, but the novel finding of a GPCR-mediated transactivation of a serine/threonine growth factor receptor almost certainly has implications well beyond the synthesis of proteoglycans

CRISPRi enables isoform-specific loss-of-function screens and identification of gastric cancer-specific isoform dependencies

10.1186/s13059-021-02266-6Genome Biology2214

Author Correction: CRISPRi enables isoform-specific loss-of-function screens and identification of gastric cancer-specific isoform dependencies (Genome Biology, (2021), 22, 1, (47), 10.1186/s13059-021-02266-6)

10.1186/s13059-021-02314-1Genome Biology2218