Suppression of Sertoli cell tumour development during the first wave of spermatogenesis in inhibin α-deficient mice.

A dynamic partnership between follicle-stimulating hormone (FSH) and activin is required for normal Sertoli cell development and fertility. Disruptions to this partnership trigger Sertoli cells to deviate from their normal developmental pathway, as observed in inhibin α-knockout (Inha-KO) mice, which feature Sertoli cell tumours in adulthood. Here, we identified the developmental windows by which adult Sertoli cell tumourigenesis is most FSH sensitive. FSH was suppressed for 7 days in Inha-KO mice and wild-type littermates during the 1st, 2nd or 4th week after birth and culled in the 5th week to assess the effect on adult Sertoli cell development. Tumour growth was profoundly reduced in adult Inha-KO mice in response to FSH suppression during Weeks 1 and 2, but not Week 4. Proliferative Sertoli cells were markedly reduced in adult Inha-KO mice following FSH suppression during Weeks 1, 2 or 4, resulting in levels similar to those in wild-type mice, with greatest effect observed at the 2 week time point. Apoptotic Sertoli cells increased in adult Inha-KO mice after FSH suppression during Week 4. In conclusion, acute FSH suppression during the 1st or 2nd week after birth in Inha-KO mice profoundly suppresses Sertoli cell tumour progression, probably by inhibiting proliferation in the adult, with early postnatal Sertoli cells being most sensitive to FSH action.

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.

Smads and cell fate: distinct roles in specification, development, and tumorigenesis in the testis.

According to the World Health Organization, a fertile man typically has a sperm count of 15 million per milliliter of semen. This spermatogenic capacity is determined by appropriate specification, proliferation, differentiation, and maturation of somatic and germ cells, events that begin during fetal development and continue throughout adulthood. These processes are orchestrated by the integration of signaling inputs from hormones and growth factors, including those of several transforming growth factor beta (TGFß) superfamily ligands. This review summarizes current knowledge of the Smad proteins, which serve functions central to fertility by transducing TGFß superfamily ligand signals in the testis. The importance of regulated Smad expression and differential utilization in signal transduction for fine-tuning cellular responses to ligands is discussed. We evaluate how primary cell culture studies and analyses of genetically modified mice have revealed distinct roles for specific Smads in primordial germ cell lineage specification, in determining the pace of testicular development and in controlling testicular tumorigenesis. This review also addresses the new insights gained from examining heterozygous mice that exhibit intriguing gene-dosage effects, outcomes that provide a new understanding of how TGFß superfamily ligands influence testis development and function. Finally, we consider the growing understanding that Smads mediate cross-talk with hormones to play a central role in determining male fertility and reproductive health.

Thrombin stimulation of proteoglycan synthesis in vascular smooth muscle is mediated by protease-activated receptor-1 transactivation of the transforming growth factor beta type 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)-beta 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-beta type I receptor (TbetaRI) 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 TbetaRI antagonist, SB431542 and TbetaRI siRNA knockdown, as well as several protease-activated receptor (PAR)-1 antagonists. The canonical downstream response to TGF-beta is 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.

INTRODUCTION: Genes contain multiple promoters that can drive the expression of various transcript isoforms. Although transcript isoforms from the same gene could have diverse and non-overlapping functions, current loss-of-function methodologies are not able to differentiate between isoform-specific phenotypes. RESULTS: Here, we show that CRISPR interference (CRISPRi) can be adopted for targeting specific promoters within a gene, enabling isoform-specific loss-of-function genetic screens. We use this strategy to test functional dependencies of 820 transcript isoforms that are gained in gastric cancer (GC). We identify a subset of GC-gained transcript isoform dependencies, and of these, we validate CIT kinase as a novel GC dependency. We further show that some genes express isoforms with opposite functions. Specifically, we find that the tumour suppressor ZFHX3 expresses an isoform that has a paradoxical oncogenic role that correlates with poor patient outcome. CONCLUSIONS: Our work finds isoform-specific phenotypes that would not be identified using current loss-of-function approaches that are not designed to target specific transcript isoforms.

Prepubertal Di-n-Butyl Phthalate Exposure Alters Sertoli and Leydig Cell Function and Lowers Bone Density in Adult Male Mice.

Phthalate exposure impairs testis development and function; however, whether phthalates affect nonreproductive functions is not well understood. To investigate this, C57BL/6J mice were fed 1-500 mg di-n-butyl phthalate (DBP) in corn oil, or vehicle only, daily from 4 to 14 days, after which tissues were collected (prepubertal study). Another group was fed 1-500 mg/kg·d DBP from 4 to 21 days and then maintained untreated until 8 weeks for determination of adult consequences of prepubertal exposure. Bones were assessed by microcomputed tomography and dual-energy X-ray absorptiometry and T by RIA. DBP exposure decreased prepubertal femur length, marrow volume, and mean moment of inertia. Adult animals exposed prepubertally to low DBP doses had lower bone mineral content and bone mineral density and less lean tissue mass than vehicle-treated animals. Altered dynamics of the emerging Leydig population were found in 14-day-old animals fed 100-500 mg/kg·d DBP. Adult mice had variable testicular T and serum T and LH concentrations after prepubertal exposure and a dose-dependent reduction in cytochrome p450, family 11, subfamily A, polypeptide 1. Insulin-like 3 was detected in Sertoli cells of adult mice administered the highest dose of 500 mg/kg·d DBP prepubertally, a finding supported by the induction of insulin-like 3 expression in TM4 cells exposed to 50 µM, but not 5 µM, DBP. We propose that low-dose DBP exposure is detrimental to bone but that normal bone mineral density/bone mineral content after high-dose DBP exposure reflects changes in testicular somatic cells that confer protection to bones. These findings will fuel concerns that low-dose DBP exposure impacts health beyond the reproductive axis.

Murine Inhibin α-Subunit Haploinsufficiency Causes Transient Abnormalities in Prepubertal Testis Development Followed by Adult Testicular Decline.

Activin production and signaling must be strictly regulated for normal testis development and function. Inhibins are potent activin inhibitors; mice lacking the inhibin-α gene (Inha-/- mice) cannot make inhibin and consequently have highly elevated activin and FSH serum concentrations and excessive activin signaling, resulting in somatic gonadal tumors and infertility. Dose-dependent effects of activin in testicular biology have been widely reported; hence, we hypothesized that male mice lacking one copy of the Inha gene would produce less inhibin and have an abnormal reproductive phenotype. To test this, we compared hormone concentrations, testis development, and sperm production in Inha+/+ and Inha+/- mice. Serum and testicular inhibin-α concentrations in adult Inha+/- mice were approximately 33% lower than wild type, whereas activin A, activin B, FSH, LH, and T were normal. Sixteen-day-old Inha+/- mice had a mixed phenotype, with tubules containing extensive germ cell depletion juxtaposed to tubules with advanced Sertoli and germ cell development. This abnormal phenotype resolved by day 28. By 8 weeks, Inha+/- testes were 11% larger than wild type and supported 44% greater daily sperm production. By 26 weeks of age, Inha+/- testes had distinct abnormalities. Although still fertile, Inha+/- mice had a 27% reduction in spermatogenic efficiency, a greater proportion of S-phase Sertoli cells and lower Leydig cell CYP11A1 expression. This study is the first to identify an intratesticular role for inhibin/inhibin-α subunit, demonstrating that a threshold level of this protein is required for normal testis development and to sustain adult somatic testicular cell function.

Prepubertal mouse testis growth and maturation and androgen production are acutely sensitive to di-n-butyl phthalate.

Phthalates are plasticizers with widespread industrial, domestic, and medical applications. Epidemiological data indicating increased incidence of testicular dysgenesis in boys exposed to phthalates in utero are reinforced by studies demonstrating that phthalates impair fetal rodent testis development. Because humans are exposed to phthalates continuously from gestation through adulthood, it is imperative to understand what threat phthalates pose at other life stages. To determine the impact during prepuberty, we assessed the consequences of oral administration of 1 to 500 mg di-n-butyl phthalate (DBP)/kg/d in corn oil to wild-type (C57BL/6J) male mice from 4 to 14 days of age. Dose-dependent effects on testis growth correlated with reduced Sertoli cell proliferation. Histological and immunohistochemical analyses identified delayed spermatogenesis and impaired Sertoli cell maturation after exposure to 10 to 500 mg DBP/kg/d. Interference with the hypothalamic-pituitary-gonadal axis was indicated in mice fed 500 mg DBP/kg/d, which had elevated circulating inhibin but no change in serum FSH. Increased immunohistochemical staining for inhibin-α was apparent at doses of 10 to 500 mg DBP/kg/d. Serum testosterone and testicular androgen activity were lower in the 500 mg DBP/kg/d group; however, reduced anogenital distance in all DBP-treated mice suggested impaired androgen action at earlier time points. Long-term effects were evident, with smaller anogenital distance and indications of disrupted spermatogenesis in adult mice exposed prepubertally to doses from 1 mg DBP/kg/d. These data demonstrate the acute sensitivity of the prepubertal mouse testis to DBP at doses 50- to 500-fold lower than those used in rat and identify the upregulation of inhibin as a potential mechanism of DBP action.

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

Transforming growth factor-beta (TGFß) 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 TGFß 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 TGFß 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 testis 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 boys.

Regulated production of SnoN2 is a feature of testicular differentiation.

Transforming growth factor betas (TGF beta s) and activins are key regulators of male fertility, affecting somatic and germ cell proliferation and differentiation in the developing and adult testis. Several studies have shown that these ligands influence discrete developmental stages, suggesting that temporal expression of modifying factors may determine their specific signaling outcomes. Upon binding to cell surface receptors, TGFbeta and activin signals are transduced intracellularly by the phosphorylation and nuclear accumulation of SMAD2 and SMAD3 transcription factors. The objective of this study was to determine the cellular localization of phosphorylated SMAD2/3 and the transcriptional repressor SnoN (Ski-like), a modifier of SMAD2/3 transcriptional activity, in mouse testes. Western blot established that only the smaller SnoN isoform, SnoN2, is produced in the testis. By immunohistochemistry, widespread phospho-SMAD2/3 distribution was observed in somatic and germ cells at all ages. In contrast, SnoN2 production was highly regulated, being detected only in gonocytes and interstitial cells at birth and in pachytene spermatocytes at puberty. In the adult, SnoN2 expression differed to that during the first wave, being ubiquitously expressed but exhibiting regulated nuclear localization. In another model of spermatogenic differentiation, the irradiated rat testis, widespread phospho-SMAD2/3 contrasted with restricted SnoN2 expression. SnoN2 was limited to interstitial cells, with reduced staining intensity observed associated with the timing of spermatogenesis resumption. We conclude that somatic and germ cells at all differentiation stages are actively transducing TGFbeta superfamily signals but that responses to these ligands may be selectively modulated by controlled production and nuclear localization of SnoN2.

Developmentally regulated SMAD2 and SMAD3 utilization directs activin signaling outcomes.

Activin is required for testis development. Activin signals via phosphorylation and nuclear accumulation of SMAD2 and SMAD3. We present novel findings of developmentally regulated activin signaling leading to specific transcriptional outcomes in testicular Sertoli cells. In immature, proliferating, Sertoli cells, activin A induces nuclear accumulation of SMAD3, but not SMAD2, although both proteins become phosphorylated. In postmitotic differentiating cells, both SMAD proteins accumulate in the nucleus. Furthermore, immature Sertoli cells are sensitive to activin dosage; higher concentrations induce maximal SMAD3 nuclear accumulation and a small increase in nuclear SMAD2. Microarray analysis identified distinct transcriptional outcomes correlating with differential SMAD utilization and new activin target genes, including Gja1 and Serpina5, which are essential for Sertoli cell development and male fertility. In transgenic mice with altered activin bioactivity that display fertility phenotypes, Gja1 and Serpina5 are significantly altered. Thus, differential SMAD utilization in response to activin features during Sertoli cell maturation.

SMAD expression in the testis: an insight into BMP regulation of spermatogenesis.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta superfamily, extensively influence events that establish male fertility, affecting germ cells and somatic cells throughout fetal and postnatal life. BMP signals are relayed by SMAD proteins, transcription factors that translocate to the nucleus upon ligand stimulation. We show that BMP signaling in the testis may be regulated by selective expression of BMP-responsive and inhibitory SMADs, with expression differing between the first wave and adult spermatogenesis. Smad1, Smad5, Smad8, Smad4, Smad6, and Smad7 expression is ubiquitous during testis development but becomes cell-specific in the adult. Furthermore, regulated SMAD6 protein expression at the onset of spermatogenesis suggests differential responsiveness of spermatogonial subpopulations to ligands. In vitro, immature Sertoli cells and spermatogonia transduce BMP2 and BMP4 signals by means of SMAD1, SMAD5, and SMAD8. Based on these findings, we extrapolate these data to interpret BMP mutant testis phenotypes in terms of SMAD availability for signal transduction.

All in the family: TGF-beta family action in testis development.

To achieve and maintain fertility, the adult mammalian testis produces many generations of sperm. While testicular integrity is established in the fetus and develops further in juvenile life, sperm production does not ensue until much later in life, following the onset of puberty. Signals from the transforming growth factor-beta superfamily of proteins are vital for governance of testis development and spermatogenesis, and this review discusses our current understanding of the mechanisms and processes in which they have been implicated with a focus on the fetal and juvenile testis.

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.

Regulated nucleocytoplasmic transport in spermatogenesis: a driver of cellular differentiation?

This review explores the hypothesis that regulation of nucleocytoplasmic shuttling is a means of driving differentiation, using spermatogenesis as a model. The transition from undifferentiated spermatogonial stem cell to terminally differentiated spermatozoon is, at its most basic, a change in the repertoire of expressed genes. To effect this, the complement of nuclear proteins, such as transcription factors and chromatin remodelling components must change. Current knowledge of the nuclear proteins and nucleocytoplasmic transport machinery relevant to spermatogenesis is consolidated in this review, and their functional linkages are highlighted not only as a means of regulating nuclear protein composition, but also as a key mechanism regulating gene transcription and hence cell fate. Through this, we hypothesize that male germ cell differentiation is mediated through regulation of nuclear transport machinery components, and thereby of the access of critical factors to the nucleus. The importance of nucleocytoplasmic trafficking to male germ cell differentiation is discussed, using the sex-determining factors Sry and SOX9, cell cycle regulators, CREM and cofactors and the Smads as specific examples, together with the roles in gametogenesis for particular nuclear transport factors in Caenorhabditis elegans and Drosophila.