Kate Loveland

Prepubertal Di-n-Butyl phthalate exposure alters Sertoli and Leydig cell function and lowers bone density in adult male mice

Publisher: The Endocrine Society

Date: 08-04-2016

DOI: 10.1210/EN.2015-1936

Abstract: 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.

All in the family: TGF-β family action in testis development

Publisher: Bioscientifica

Date: 08-2006

DOI: 10.1530/REP.1.01075

Abstract: 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-β 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.

Bovine Sertoli Cells Colonize and Form Tubules in Murine Hosts Following Transplantation and Grafting Procedures

Publisher: Wiley

Date: 08-07-2008

DOI: 10.2164/JANDROL.107.004465

Abstract: The contribution of somatic cells to nonrodent male germ cell transplantation success has not been well established due to lack of cell type-specific markers to distinguish donor cells from host cells. In the present study, we first screened antibodies and a lectin to identify markers suitable for unequivocal distinction between germ cells and Sertoli cells in bovine testes compared with mouse testes. Anti-vimentin and the Dolichos biflorus agglutinin (DBA) lectin detected only bovine Sertoli cells and spermatogonia, respectively anti-NONO and anti-GCNA1 detected only mouse Sertoli and germ cells, respectively. The outcome of transplanting bovine testis cells into nude mouse testes was then studied using these markers. Our results clearly showed that immature bovine Sertoli cells survive and colonize mouse testes at 2.5 months after transplantation and that tubular structures composed of donor Sertoli cells formed adjacent to murine tubules within the host mouse testis. Bovine germ cell colonization and survival in mouse testes after transplantation were confirmed, but this was restricted to areas of bovine Sertoli cell colonization. In addition, ectopic grafts of intact bovine testis tissue and cell aggregates from hanging drop cultures were placed under the back skin and testis capsule of nude mice. Bovine Sertoli cells in ectopic grafts and aggregates were able to form tubular structures, and some bovine germ cells were observed around 2 months after implantation. This study therefore identifies a practical strategy to assess the outcome of testicular cell transplantation using different antibodies and a lectin to distinguish bovine cells from mouse cells. It identifies an approach that can readily be adapted to study other nonrodent species.

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

Publisher: Wiley

Date: 09-01-2013

DOI: 10.1002/IUB.1115

Abstract: 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 (TGFβ) superfamily ligands. This review summarizes current knowledge of the Smad proteins, which serve functions central to fertility by transducing TGFβ 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 TGFβ 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.

Regulated production of activin A and inhibin B throughout the cycle of the seminiferous epithelium in the rat

Publisher: Bioscientifica

Date: 08-2006

DOI: 10.1677/JOE.1.06706

Abstract: Production and regulation of activin A and inhibin B during the cycle of the seminiferous epithelium were investigated in adult rats. Immunohistochemistry localised the activin β A -subunit to the Sertoli cell cytoplasm, with much weaker expression in spermatocytes and spermatids. Both activin A and inhibin B, measured by ELISA were secreted by, seminiferous tubule fragments over 72 h in culture. Activin A was secreted in a cyclic manner with peak secretion from tubules isolated at stage VIII. Tubules collected during stage VI produced the least activin A. Inhibin B secretion was highest from stage IX-I tubules and lowest from stage VII tubules. Addition of interleukin-1β (IL-1β) had relatively little effect on activin A or inhibin B secretion in culture. In contrast, the peak secretion of activin A by stage VIII tubules was blocked by co-incubation with an excess of human recombinant IL-1 receptor antagonist, whereas inhibin B secretion increased slightly. Dibutyryl cAMP stimulated activin A secretion by late stage VII and VIII tubules and stimulated inhibin B across all stages. These data indicate that activin A and inhibin B are cyclically regulated within the seminiferous epithelium, with endogenous IL-1 (presumably IL-1α produced by the Sertoli cells), responsible for a peak of activin A production subsequent to sperm release at stage VIII. These data provide direct evidence that production of activin A and inhibin B by the Sertoli cell is locally modulated by IL-1α , in addition to FSH/cAMP, under the influence of the developing spermatogenic cells.

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

Publisher: Springer Science and Business Media LLC

Date: 21-06-2010

DOI: 10.1186/1477-7827-8-69

Abstract: 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. 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. 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. SMAD2 is not required for mediating tumorigenic signals of activin in ovarian tumor development caused by loss of inhibin.

Regulation of nuclear import during differentiation: The IMP alpha gene family and spermatogenesis

Publisher: Bentham Science Publishers Ltd.

Date: 08-2007

DOI: 10.2174/138920207782446151

Activins and inhibins in Sertoli cell biology

Publisher: Elsevier

Date: 2015

DOI: 10.1016/B978-0-12-417047-6.00007-7

Distinct effects of importin alpha2 and alpha4 on Oct3/4 localization and expression in mouse embryonic stem cells

Publisher: Wiley

Date: 12-08-2011

DOI: 10.1096/FJ.10-176941

Abstract: The cellular repertoire of importin (IMP) proteins that mediates nuclear import of transcription factors and chromatin remodeling agents is critical to processes such as differentiation and transformation. This study identifies for the first time independent roles for specific IMPαs in murine embryonic stem cells (mESCs), showing that mESC differentiation is accompanied by dynamic changes in the levels of transcripts encoding the IMPs, IMPα3, IMPα4, IMPβ1, and IPO5. Of these, only IMPα4 was maintained at higher levels in differentiating mESCs, correlating with the finding that IMPα4 overexpression induced a significant decrease in Oct3/4 protein levels compared to control transfections. In parallel, IMPα4 protein showed a unique and striking shift in subcellular localization from the nucleus to the cytoplasm during differentiation, which is consistent with activation of a role in nuclear import of differentiation factors. Overexpression of a dominant-negative IMPα2 isoform, when assessed against adjacent untransfected or IMPα2 transfected cells, led to both a significant reduction in endogenous Oct3/4 protein levels and inhibition of Oct3/4 nuclear localization, suggesting that IMPα2-mediated delivery of Oct3/4 to the nucleus contributes directly to maintenance of mESC pluripotency. These findings implicate IMPα2 and IMPα4 in specific but distinct roles in the fate choice between pluripotency and commitment to differentiation.

Identification of a Novel Testis-Specific Member of the Phosphatidylethanolamine Binding Protein Family, pebp-21

Publisher: Oxford University Press (OUP)

Date: 09-2002

DOI: 10.1095/BIOLREPROD.101.001446

Abstract: The phosphatidylethanolamine binding proteins (pebps) are an evolutionarily conserved family of proteins recently implicated in mitogen-activated protein (MAP) kinase pathway regulation, where they are called raf kinase inhibitory proteins. Here, we describe the cloning, cellular localization, and partial characterization of a new member, pebp-2, with potential roles in male fertility. Expression data show that pebp-2 is a testis-specific 21-kDa protein found within late meiotic and haploid germ cells in a stage-specific pattern that is temporally distinct from that of pebp-1. Sequence analyses suggest that pebp-2 forms a distinct subset of the pebp family within mammals. Database analyses revealed the existence of a third subset. Analysis suggests that the specificity/regulation of the distinct pebps subsets is likely to be determined by the amino terminal 40 amino acids or the 3' untranslated region, where the majority of sequence differences occur. Protein homology modeling suggests that pebp-2 protein is, however, topologically similar to other pebps and composed of Greek key fold motifs, a dominant beta-sheet formed from five anti-parallel beta strands forming a shallow groove associated with a putative phosphatidylethanolamine binding site. The pebp-2 gene is intronless and data suggest that it is a retrogene derived from pebp-1. Further, pebp-2 colocalizes with members of the MAP kinase pathway in late spermatocytes and spermatids and on the midpiece of epididymal sperm. These data raise the possibility that pebp-2 is a novel participant in the MAP kinase signaling pathway, with a role in spermatogenesis or posttesticular sperm maturation.

Nucleocytoplasmic transport as a driver of mammalian gametogenesis

Publisher: Elsevier BV

Date: 07-2009

DOI: 10.1016/J.SEMCDB.2009.05.002

Abstract: Adult fertility requires appropriate and coordinated instruction of somatic and germ cell activity during lineage specification, development and maturation. Driven by alterations in the complement of nuclear proteins such as transcription factors and chromatin remodelling components, these events proceed by sequential changes in gene expression in response to a myriad of signalling cues. Controlled access of proteins to the nucleus is a key driver of developmental switches. This review discusses key ex les of regulated nucleocytoplasmic transport during mammalian gametogenesis and the mechanisms underpinning these transport events, focusing on ex les critical for the establishment of fertility.

Towards delineation of a developmental α-importome in the mammalian male germline

Publisher: Elsevier BV

Date: 03-2013

DOI: 10.1016/J.BBAMCR.2012.11.005

Abstract: Nucleocytoplasmic transport mediated by importin proteins is central to many developmental processes, such as precisely regulated germ cell differentiation during spermatogenesis. Here we examine for the first time the dynamic association of importins with cargo during two successive spermatogenic stages: meiotic pachytene spermatocytes and haploid round spermatids of the adult rat testis. Immunoprecipitation followed by mass spectrometry yielded the first non-biased identification of proteins selectively interacting with importin α2, α3 and α4 in each of these cell types. Amongst the 22 novel importin binding proteins identified, 11 contain a predicted classical nuclear localization signal (cNLS) for importin α binding using a new algorithm (Kosugi et al. [22]), although only 6 of these have known nuclear functions. An importin α2-immunoprecipitated protein with a key nuclear role in meiosis, structural maintenance of chromosomes 6 (SMC6), contained a predicted bipartite NLS that was shown to be preferentially recognized by importin α together with importin β1. In contrast, the predicted cNLS of synovial sarcoma, X breakpoint 2 interacting protein (SSX2IP) was found not to confer either nuclear accumulation or direct binding to importin αs, implying that NLS prediction algorithms may identify cryptic importin binding sites or require additional refinement to increase their accuracy. Unbiased identification of importin α binding proteins in cellular differentiation represents a powerful tool to help identify the functional roles of importin αs.

Expression of nucleocytoplasmic transport machinery: Clues to regulation of spermatogenic development

Publisher: Elsevier BV

Date: 09-2011

DOI: 10.1016/J.BBAMCR.2011.03.008

Abstract: Spermatogenesis is one ex le of a developmental process which requires tight control of gene expression to achieve normal growth and sustain function. This review is based on the principle that events in spermatogenesis are controlled by changes in the distribution of proteins between the nuclear and cytoplasmic compartments. Through analysis of the regulated production of nucleocytoplasmic transport machinery in mammalian spermatogenesis, this review addresses the concept that access to the nucleus is tightly controlled to enable and prevent differentiation. A broad review of nuclear transport components is presented, outlining the different categories of machinery required for import, export and non-nuclear functions. In addition, the complexity of nomenclature is addressed by the provision of a concise yet comprehensive listing of information that will aid in comparative studies of different transport proteins and the genes which encode them. We review a suite of existing transcriptional analyses which identify common and distinct patterns of transport machinery expression, showing how these can be linked with key events in spermatogenic development. The additional importance of this for human fertility is considered, in light of data that identify which importin and nuclear transport machinery components are present in testicular cancer specimens, while also providing an indication of how their presence (and absence) may be considered as potential mediators of oncogenesis. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.

The transforming growth factor‐β superfamily in early spermatogenesis: potential relevance to testicular dysgenesis

Publisher: Wiley

Date: 08-2007

DOI: 10.1111/J.1365-2605.2007.00785.X

Abstract: Regulated transforming growth factor-beta (TGFbeta) superfamily signalling is an integral part of normal testicular development and the processes that enable the production of fertile sperm. Through shared utilization of receptors, signal transduction components and inhibitors, many ligands in this family exhibit functional overlaps this facet of their function is critical to understand because these ligands are often co-expressed and, hence, they may compete with or compensate for one another, depending on the specific cellular context. This review describes particular germ cell maturation steps governed by bone morphogenetic proteins, glial cell line-derived neurotrophic factor and activins, focusing on data predominantly from rodent studies that implicate activin and other family members in modulation of gonocyte and spermatogonial stem cell development. We also review knowledge of the TGFbeta superfamily signalling components in the human testis, exploring their potential impact on the processes associated with disrupted gonocyte development and an enhanced risk of testicular cancer.

The rise of testicular germ cell tumours: the search for causes, risk factors and novel therapeutic targets

Publisher: F1000 Research Ltd

Date: 19-02-2013

DOI: 10.12688/F1000RESEARCH.2-55.V1

Abstract: Since the beginning of the 20th century there has been a decline in the reproductive vitality of men within the Western world. The declining sperm quantity and quality has been associated with increased overt disorders of sexual development including hypospadias, undescended testes and type II testicular germ cell tumours (TGCTs). The increase in TGCTs cannot be accounted for by genetic changes in the population. Therefore exposure to environmental toxicants appears to be a major contributor to the aetiology of TGCTs and men with a genetic predisposition are particularly vulnerable. In particular, Type II TGCTs have been identified to arise from a precursor lesion Carcinoma in situ (CIS), identified as a dysfunctional gonocyte however, the exact triggers for CIS development are currently unknown. Therefore the transition from gonocytes into spermatogonia is key to those studying TGCTs. Recently we have identified seven miRNA molecules (including members of the miR-290 family and miR-136, 463* and 743a) to be significantly changed over this transition period. These miRNA molecules are predicted to have targets within the CXCR4, PTEN, DHH, RAC and PDGF pathways, all of which have important roles in germ cell migration, proliferation and homing to the spermatogonial stem cell niche. Given the plethora of potential targets affected by each miRNA molecule, subtle changes in miRNA expression could have significant consequences e.g. tumourigenesis. The role of non-traditional oncogenes and tumour suppressors such as miRNA in TGCT is highlighted by the fact that the majority of these tumours express wild type p53, a pivotal tumour suppressor usually inactivated in cancer. While treatment of TGCTs is highly successful, the impact of these treatments on fertility means that identification of exact triggers, earlier diagnosis and alternate treatments are essential. This review examines the genetic factors and possible triggers of type II TGCT to highlight target areas for potential new treatments.

Importin alpha2-interacting proteins with nuclear roles during mammalian spermatogenesis

Publisher: Oxford University Press (OUP)

Date: 12-2011

DOI: 10.1095/BIOLREPROD.111.091686

Abstract: Spermatogenesis, the process of generating haploid sperm capable of fertilizing the female gamete, requires the timely transport into the nucleus of transcription and chromatin-remodeling factors, mediated by members of the importin (IMP) superfamily. Previous IMP expression profiling implies a role for IMPalpha2 in testicular germ cells late in spermatogenesis. To identify interacting proteins of IMPalpha2 that are potential drivers of germ cell development, we performed yeast two-hybrid screening of an adult mouse testis library. IMPalpha2 interactions were verified by coimmunoprecipitation approaches, whereas immunohistochemical staining of testis sections confirmed their coexpression with IMPalpha2 in specific testicular cell types. Key interactors identified were a novel isoform of a cysteine and histidine rich protein (Chrp), a protein inhibitor of activated STAT (PIAS) family member involved in transcriptional regulation and sumoylation, Androgen receptor interacting protein 3 (Arip3), and Homologous protein 2 (Hop2), known to be involved in homologous chromosome pairing and recombination, all of which are highly expressed in the testis and show mRNA expression profiles similar to that of IMPalpha2 throughout testicular development. This is the first study to identify binding partners of IMPalpha2 in the developmental context of germ line development, and we propose that the regulated expression and timely IMPalpha2-mediated nuclear transport of these proteins may coordinate events during spermatogenesis, with IMPalpha2-mediated nuclear localization representing a potentially critical developmental switch in the testis.

The chemokine CXCL12 and its receptor CXCR4 are implicated in human seminoma metastasis

Publisher: Wiley

Date: 15-03-2013

DOI: 10.1111/J.2047-2927.2013.00081.X

Abstract: Seminoma and non-seminoma tumours increasingly occur within the western population. These tumours originate from carcinoma in situ (CIS) cells, which arise from dysfunctional gonocytes. CXCL12 and its receptors, CXCR4 and CXCR7, have been implicated in migration, proliferation and survival of gonocytes and their precursors and progeny, primordial germ cells and spermatogonial stem cells respectively. We previously found evidence that several miRNA molecules predicted to modulate CXCR4 signalling are differentially expressed during the differentiation of gonocytes into spermatogonia in mice. Bioinformatic analysis predicted these miRNA to modulate CXCR4 signalling, leading us to hypothesize that CXCL12-mediated CXCR4 signalling is involved in the disrupted differentiation of gonocytes that underpins CIS formation. Indeed, we detected CXCL12 in Sertoli cells of normal human testis, and relatively high expression in tumour stroma with concomitant weak staining in dispersed tumour cells. In contrast, CXCR4 was expressed in spermatogonial and meiotic germ cells of normal testis and in the majority of tumour cells. Quantitative RT-PCR identified elevated CXCR4 transcript levels in seminoma compared with normal testis and to non-seminoma, potentially reflecting the higher proportion of dysfunctional germ cells within seminomas. In the normal testis, expression of CXCR4 downstream signalling molecules phospho-MEK1/2 and phospho-ERK1/2 correlated with CXCR4/CXCL12 expression. Strikingly, this correlation was absent in seminoma and non-seminoma s les, suggesting that CXCL12 signalling is disrupted. Proliferation rate and cell survival were not altered by CXCL12 in either seminoma (TCam-2) or non-seminoma (833ke) cell lines. However, CXCL12 exposure induced TCam-2 cell invasion though simulated basement membrane, while in contrast, we provide the novel evidence that CXCR4-expressing non-seminoma cell lines 833ke and NTera2/D1 do not invade in response to CXCL12. These findings indicate that CXCL12 expression in the human testis may selectively influence seminoma migration and metastasis, correlating with its importance in gonocyte and spermatogonial stem cell biology.

Overlapping binding sites for importin b1 and suppressor of fused (SuFu) on glioma-associated oncogene homologue 1 (Gli1) regulate its nuclear localization

Publisher: Portland Press Ltd.

Date: 10-07-2014

DOI: 10.1042/BJ20130709

Abstract: A key factor in oncogenesis is the transport into the nucleus of oncogenic signalling molecules, such as Gli1 (glioma-associated oncogene homologue 1), the central transcriptional activator in the Hedgehog signalling pathway. Little is known, however, how factors such as Gli are transported into the nucleus and how this may be regulated by interaction with other cellular factors, such as the negative regulator suppressor of fused (SuFu). In the present study we show for the first time that nuclear entry of Gli1 is regulated by a unique mechanism through mutually exclusive binding by its nuclear import factor Impβ1 (importin β1) and SuFu. Using quantitative live mammalian cell imaging, we show that nuclear accumulation of GFP–Gli1 fusion proteins, but not of a control protein, is specifically inhibited by co-expression of SuFu. Using a direct binding assay, we show that Impβ1 exhibits a high nanomolar affinity to Gli1, with specific knockdown of Impβ1 expression being able to inhibit Gli1 nuclear accumulation, thus implicating Impβ1 as the nuclear transporter for Gli1 for the first time. SuFu also binds to Gli1 with a high nanomolar affinity, intriguingly being able to compete with Impβ1 for binding to Gli1, through the fact that the sites for SuFu and Impβ1 binding overlap at the Gli1 N-terminus. The results indicate for the first time that the relative intracellular concentrations of SuFu and Impβ1 are likely to determine the localization of Gli1, with implications for its action in cancer, as well as in developmental systems.

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

Publisher: The Endocrine Society

Date: 08-03-2011

DOI: 10.1210/EN.2010-1453

Abstract: 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.

Activin over-expression in the testis of mice lacking the inhibin α-subunit gene is associated with androgen deficiency and regression of the male reproductive tract

Publisher: Elsevier BV

Date: 07-2018

DOI: 10.1016/J.MCE.2017.10.013

Abstract: Regionalised interaction of the activins, follistatin and inhibin was investigated in the male reproductive tract of mice lacking the inhibin α-subunit (Inha

Novel scavenger receptor gene is differentially expressed in the embryonic and adult mouse testis

Publisher: Wiley

Date: 21-10-2005

DOI: 10.1002/DVDY.20594

Abstract: In an effort to understand the mechanisms that underpin gonadal differentiation at the time of sex determination, we identified a cDNA encoding a putative novel testis expressed scavenger receptor, Tesr. Based on its domain structure, we hypothesize that the function of Tesr is similar to that of other scavenger receptors that play roles in phagocytosis of apoptotic cells, cell-cell adhesion, and defense. Tesr mRNA was detected in fetal mouse gonads of both sexes at 11.5 days post coitum (dpc). From 12.0 dpc, Tesr expression rapidly decreased in the female and was maintained in the male. Expression was seen in embryonic mouse sites other than the testis, such as in brain, eye, head, heart, neural arch, and cartilage primordium. Tesr expression in the newborn testis was faint to undetectable, but it increased from 2 days postpartum (dpp) until 15 dpp and was found in a subset of interstitial cells and in germ and Sertoli cells. Tesr mRNA in the adult mouse testis was observed in Sertoli cells, spermatogonia, spermatocytes, round spermatids, and in a subset of interstitial cells. We conclude that Tesr is differentially expressed in the male vs. female embryonic gonad and is expressed in both the ovary and the testes postnatally after 2 dpp.

‘Snail factors in testicular germ cell tumours and their regulation by the BMP4 signalling pathway’

Publisher: Wiley

Date: 12-06-2020

DOI: 10.1111/ANDR.12823

Activins and inhibins in mammalian testis development: New models, new insights

Publisher: Elsevier BV

Date: 08-2012

DOI: 10.1016/J.MCE.2012.02.018

Abstract: The discovery of activin and inhibins as modulators of the hypothalamic-pituitary-gonadal axis has set the foundation for understanding their central importance to many facets of development and disease. This review contains an overview of the processes and cell types that are central to testis development and spermatogenesis and then provides an update focussed on information gathered over the past five years to address new concepts about how these proteins function to control testis development in fetal and juvenile life. Current knowledge about the interactive nature of the transforming growth factor-β (TGFβ) superfamily signalling network is applied to recent findings about activins and inhibins in the testis. Information about the regulated synthesis of signalling components and signalling regulators in the testis is integrated with new concepts that demonstrate their functional significance. The importance of activin bioactivity levels or dosage in controlling balanced growth of spermatogonial cells and their niche at different stages of testis development is highlighted.

Sex specific retinoic acid signaling is required for the initiation of urogenital sinus bud development

Publisher: Elsevier BV

Date: 11-2014

DOI: 10.1016/J.YDBIO.2014.09.016

Regulated wnt/beta-catenin signaling sustains adult spermatogenesis in mice

Publisher: Oxford University Press (OUP)

Date: 2014

DOI: 10.1095/BIOLREPROD.112.105809

Abstract: The importance of Wnt signaling for postnatal testis function has been previously studied in several mouse models, with chronic pathway disruption addressing its function in Sertoli cells and in postmeiotic germ cells. While chronic beta-catenin deletion in Sertoli cells does not profoundly affect testis development, new data indicate that Wnt signaling is required at multiple stages of spermatogenesis. We used two mouse models that allow acute disruption of Wnt signaling to explore the importance of regulated Wnt pathway activity for normal germ cell development in adult male mice. Short-term induction of mutations in Adenomatous polyposis coli (Apc) and beta-catenin (Ctnnbl), which increase and decrease Wnt signaling levels, were generated in AhCre Apc(fl/fl) and AhCre Ctnnb1(fl/fl) mice, respectively. Each exhibited a distinct phenotype of disrupted spermatogenesis that was evident within 24 h and persisted for up to 4 days. Outcomes included germ cell apoptosis and rapid loss and altered blood-testis barrier protein distribution and morphology. The functional significance of nuclear localized beta-catenin protein in spermatocytes and round spermatids, indicative of active Wnt signaling, was highlighted by the profound loss of postmitotic germ cells in both models. Developmentally regulated Wnt signaling mediators identified through transcriptional profiling of wild-type and AhCre Ctnnb1(fl/fl) mouse testes identified Wnt receptors (e.g., Fzd4) and ligands (e.g., Wnt3, Wnt3a, Wnt5b, Wnt7a, and Wnt8b). This demonstration that Wnt signaling control is essential for adult spermatogenesis supports the growing understanding that its disruption may underpin certain cases of male infertility.

A survey of Sertoli cell differentiation in men after gonadotropin suppression and in testicular cancer

Publisher: Informa UK Limited

Date: 2013

DOI: 10.4161/SPMG.24014

Hidden gems in the niche: a new approach to the study of spermatogonial stem cells

Publisher: Medknow

Date: 14-01-2013

DOI: 10.1038/AJA.2012.150

Activins in Male Reproduction

Publisher: Elsevier

Date: 2018

DOI: 10.1016/B978-0-12-801238-3.64581-6

An in vitro model demonstrates the potential of neoplastic human germ cells to influence the tumour microenvironment

Publisher: Wiley

Date: 23-05-2017

DOI: 10.1111/ANDR.12365

Abstract: Testicular germ cell tumours (TGCT) typically contain high numbers of infiltrating immune cells, yet the functional nature and consequences of interactions between GCNIS (germ cell neoplasia in situ) or seminoma cells and immune cells remain unknown. A co-culture model using the seminoma-derived TCam-2 cell line and peripheral blood mononuclear cells (PBMC, n = 7 healthy donors) was established to investigate how tumour and immune cells each contribute to the cytokine microenvironment associated with TGCT. Three different co-culture approaches were employed: direct contact during culture to simulate in situ cellular interactions occurring within seminomas (n = 9) indirect contact using well inserts to mimic GCNIS, in which a basement membrane separates the neoplastic germ cells and immune cells (n = 3) and PBMC stimulation prior to direct contact during culture to overcome the potential lack of immune cell activation (n = 3). Transcript levels for key cytokines in PBMC and TCam-2 cell fractions were determined using RT-qPCR. TCam-2 cell fractions showed an immediate increase (within 24 h) in several cytokine mRNAs after direct contact with PBMC, whereas immune cell fractions did not. The high levels of interleukin-6 (IL6) mRNA and protein associated with TCam-2 cells implicate this cytokine as important to seminoma physiology. Use of PBMCs from different donors revealed a robust, repeatable pattern of changes in TCam-2 and PBMC cytokine mRNAs, independent of potential inter-donor variation in immune cell responsiveness. This in vitro model recapitulated previous data from clinical TGCT biopsies, revealing similar cytokine expression profiles and indicating its suitability for exploring the in vivo circumstances of TGCT. Despite the limitations of using a cell line to mimic in vivo events, these results indicate how neoplastic germ cells can directly shape the surrounding tumour microenvironment, including by influencing local immune responses. IL6 production by seminoma cells may be a practical target for early diagnosis and/or treatment of TGCT.

Perturbed BMP signaling and denervation promote muscle wasting in cancer cachexia

Publisher: American Association for the Advancement of Science (AAAS)

Date: 04-08-2021

DOI: 10.1126/SCITRANSLMED.AAY9592

Abstract: Neuromuscular junction remodeling and deterioration due to impaired BMP signaling are associated with cancer-induced muscle wasting.

The histone 3 lysine 4 methyltransferase, Mll2, is only required briefly in development and spermatogenesis

Publisher: Springer Science and Business Media LLC

Date: 06-04-2009

DOI: 10.1186/1756-8935-2-5

Abstract: Histone methylation is thought to be central to the epigenetic mechanisms that maintain and confine cellular identity in multi-cellular organisms. To examine epigenetic roles in cellular homeostasis, we conditionally mutated the histone 3 lysine 4 methyltransferase, Mll2, in embryonic stem (ES) cells, during development and in adult mice using tamoxifen-induced Cre recombination. In ES cells, expression profiling unexpectedly revealed that only one gene, Magoh2 , is dependent upon Mll2 and few other genes were affected. Loss of Mll2 caused loss of H3K4me3 at the Magoh2 promoter and concomitant gain of H3K27me3 and DNA methylation. Hence Mll2, which is orthologous to Drosophila Trithorax, is required to prevent Polycomb-Group repression of the Magoh2 promoter, and repression is further accompanied by DNA methylation. Early loss of Mll2 in utero recapitulated the embryonic lethality found in Mll2 -/- embryos. However, loss of Mll2 after E11.5 produced mice without notable pathologies. Hence Mll2 is not required for late development, stem cells or homeostasis in somatic cell types. However it is required in the germ cell lineage. Spermatogenesis was lost upon removal of Mll2, although spermatogonia A persisted. These data suggest a bimodal recruit and maintain model whereby Mll2 is required to establish certain epigenetic decisions during differentiation, which are then maintained by redundant mechanisms. We also suggest that these mechanisms relate to the epigenetic maintenance of CpG island promoters.

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

Publisher: The Endocrine Society

Date: 13-06-2013

DOI: 10.1210/EN.2012-2227

Abstract: 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.

Control of KIT signalling in male germ cells: What can we learn from other systems?

Publisher: Bioscientifica

Date: 11-2009

DOI: 10.1530/REP-08-0537

Abstract: The KIT ligand (KITL)/KIT-signalling system is among several pathways known to be essential for fertility. In the postnatal testis, the KIT/KITL interaction is crucial for spermatogonial proliferation, differentiation, survival and subsequent entry into meiosis. Hence, identification of endogenous factors that regulate KIT synthesis is important for understanding the triggers driving germ cell maturation. Although limited information is available regarding local factors in the testicular microenvironment that modulate KIT synthesis at the onset of spermatogenesis, knowledge from other systems could be used as a basis for identifying how KIT function is regulated in germ cells. This review describes the known regulators of KIT, including transcription factors implicated in KIT promoter regulation. In addition, specific downstream outcomes in biological processes that KIT orchestrates are addressed. These are discussed in relationship to current knowledge of mammalian germ cell development.

The nuclear import factor importin a4 can protect against oxidative stress

Publisher: Elsevier BV

Date: 10-2013

DOI: 10.1016/J.BBAMCR.2013.06.007

Abstract: The importin (IMP) superfamily of nuclear transport proteins is essential to key developmental pathways, including in the murine testis where expression of the 6 distinct IMPα proteins is highly dynamic. Present predominantly from the spermatocyte stage onwards, IMPα4 is unique in showing a striking nuclear localization, a property we previously found to be linked to maintenance of pluripotency in embryonic stem cells and to the cellular stress response in cultured cells. Here we examine the role of IMPα4 in vivo for the first time using a novel transgenic mouse model in which we overexpress an IMPα4-EGFP fusion protein from the protamine 1 promoter to recapitulate endogenous testicular germ cell IMPα4 expression in spermatids. IMPα4 overexpression did not affect overall fertility, testis morphology/weight or spermatogenic progression under normal conditions, but conferred significantly (>30%) increased resistance to oxidative stress specifically in the spermatid subpopulation expressing the transgene. Consistent with a cell-specific role for IMPα4 in protecting against oxidative stress, haploid germ cells from IMPα4 null mice were significantly (c. 30%) less resistant to oxidative stress than wild type controls. These results from two unique and complementary mouse models demonstrate a novel protective role for IMPα4 in stress responses specifically within haploid male germline cells, with implications for male fertility and genetic integrity.

Spermatogenesis and its Regulation

Publisher: Elsevier

Date: 2007

DOI: 10.1016/B978-008055232-3.60266-1

TCam-2 seminoma cell line exhibits characteristic foetal germ cell responses to TGF-beta ligands and retinoic acid

Publisher: Wiley

Date: 13-06-2011

DOI: 10.1111/J.1365-2605.2011.01170.X

Abstract: Germ cell testicular cancer is understood to arise during embryogenesis, based on the persistence of embryonic germ cell markers in carcinoma in situ and seminoma. In this study, we examine the potential of the seminoma-derived TCam-2 cell line to be used as representative in functional analyses of seminoma. We demonstrate expression of several early germ cell markers, including BLIMP1, OCT3/4, AP2γ, NANOG and KIT. Many TGF-beta superfamily receptors and downstream transcription factors are also present in these cells including the normally foetal ACTRIIA receptor, indicating potential responsiveness to TGF-beta superfamily ligands. Treatment with BMP4 or RA induces a significant increase in ACTRIA, ACTRIIA and ACTRIIB transcripts, whereas activin A decreases ACTRIB. BMP4 and RA each support TCam-2 survival and/or proliferation. In addition, despite increased KIT mRNA levels induced by BMP4, RA and activin A, activin A does not improve survival or proliferation. The capacity for BMP4 and retinoic acid to enhance foetal germ cell survival and proliferation/self-renewal has been demonstrated in mice, but not previously tested in humans. This study is the first to demonstrate a functional response in seminoma cells, using a well-characterized cell line, consistent with their foetal germ cell-like identity.

New horizons in testis biology and men's health: a special issue of the XXVIth North American testis workshop

Publisher: Wiley

Date: 13-06-2023

DOI: 10.1111/ANDR.13462

Hanging drop cultures of human testis and testis cancer samples: a model used to investigate activin treatment effects in a preserved niche

Publisher: Springer Science and Business Media LLC

Date: 29-04-2014

DOI: 10.1038/BJC.2014.160

Drivers of germ cell maturation

Publisher: Wiley

Date: 12-2201

DOI: 10.1196/ANNALS.1336.018

Abstract: Spermatogenesis requires progression of germ line stem cells through a precisely ordered differentiation pathway to form spermatozoa. Diverse and dynamic signals from the transforming growth factor-beta (TGF-beta) superfamily influence many stages of germ cell development. For ex le, interactions between several TGF-beta superfamily ligands (bone morphogenetic proteins, activin, and glial-derived neurotrophic growth factor [GDNF]) appear to govern the onset of spermatogenesis, and we are exploring how germ cells interpret these competing signals. We examined the in vivo impact of activin on testis development using two mouse models, the inhba-/- mouse (which lacks the gene encoding the activin A subunit and dies at birth) and BK mice, with inhbb (encoding the activin betaB subunit) replacing inhba (which survive to adulthood and show delayed fertility onset in males). Distinct effects on Sertoli cell and germ cell populations during fetal and early postnatal development were measured. We recognize that specific proteins, including downstream targets of TGF-beta signals, such as Smads, must move into the nucleus to implement the gene transcription changes required for development. We hypothesized that changes at the level of cellular nuclear transport machinery may be required to mediate this. Examination of proteins involved in classical nuclear import, the importins, revealed that each importin has a developmentally regulated expression pattern in male germ cells. Because each importin binds a selected range of cargo proteins and mediates their nucleocytoplasmic passage, our findings suggest that each importin ferries cargo required for discrete stages of spermatogenesis.

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology

Publisher: Oxford University Press (OUP)

Date: 16-09-2022

DOI: 10.1093/HUMREP/DEAC189

Abstract: Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Activin A Determines Steroid Levels and Composition in the Fetal Testis

Publisher: The Endocrine Society

Date: 10-04-2020

DOI: 10.1210/ENDOCR/BQAA058

Abstract: Activin A promotes fetal mouse testis development, including driving Sertoli cell proliferation and cord morphogenesis, but its mechanisms of action are undefined. We performed ribonucleic acid sequencing (RNA-seq) on testicular somatic cells from fetal activin A-deficient mice (Inhba KO) and wildtype littermates at embryonic day (E) E13.5 and E15.5. Analysis of whole gonads provided validation, and cultures with a pathway inhibitor discerned acute from chronic effects of altered activin A bioactivity. Activin A deficiency predominantly affects the Sertoli cell transcriptome. New candidate targets include Minar1, Sel1l3, Vnn1, Sfrp4, Masp1, Nell1, Tthy1 and Prss12. Importantly, the testosterone (T) biosynthetic enzymes present in fetal Sertoli cells, Hsd17b1 and Hsd17b3, were identified as activin-responsive. Activin-deficient testes contained elevated androstenedione (A4), displayed an Inhba gene dose-dependent A4/T ratio, and contained 11-keto androgens. The remarkable accumulation of lipid droplets in both Sertoli and germ cells at E15.5 indicated impaired lipid metabolism in the absence of activin A. This demonstrated for the first time that activin A acts on Sertoli cells to determine local steroid production during fetal testis development. These outcomes reveal how compounds that perturb fetal steroidogenesis can function through cell-specific mechanisms and can indicate how altered activin levels in utero may impact testis development.

TGF-β Down Under 2011—The experts interviewed!

Publisher: Informa UK Limited

Date: 13-09-2011

DOI: 10.3109/08977194.2011.616287

Expression of hedgehog signalling components in adult mouse testis

Publisher: Wiley

Date: 12-10-2006

DOI: 10.1002/DVDY.20931

Abstract: Hedgehog (Hh) signalling is known to regulate many aspects of normal development as well as being upregulated in various cancers. Signalling is mediated by the Gli family of zinc finger transcription factors. Based on observations that deletion of one of the three Hh genes, Dhh, leads to male infertility, we hypothesized that regulated expression of Hh signalling components would be a feature of adult spermatogenesis. We used in situ hybridization to characterise Gli gene expression in juvenile and adult mouse testes. In the first wave of spermatogenesis, mRNAs encoding all three Glis are detected in spermatogonia and Sertoli cells. In adult mouse testes, these transcripts are observed in spermatogonia and spermatocytes, with reduced signal intensity in round spermatids. The mRNAs encoding key effectors of Hh signalling, Ptc2, Smo, and Fu, are also most apparent in spermatogonia, spermatocytes, and to a lower extent in round spermatids. In contrast, mRNA encoding SuFu, a negative regulator of Hh signalling, was most predominant in round spermatids and the protein is evident in round and elongating spermatids, suggesting that SuFu protein may switch off Hh signalling in haploid germ cells. Overall, the coordinated expression pattern of these genes in adult mouse testis indicates a role for Hh signalling in spermatogenesis.

Regulated nucleocytoplasmic transport during gametogenesis

Publisher: Elsevier BV

Date: 06-2012

DOI: 10.1016/J.BBAGRM.2012.01.015

Abstract: Gametogenesis is the process by which sperm or ova are produced in the gonads. It is governed by a tightly controlled series of gene expression events, with some common and others distinct for males and females. Nucleocytoplasmic transport is of central importance to the fidelity of gene regulation that is required to achieve the precisely regulated germ cell differentiation essential for fertility. In this review we discuss the physiological importance for gamete formation of the molecules involved in classical nucleocytoplasmic protein transport, including importins/karyopherins, Ran and nucleoporins. To address what functions/factors are conserved or specialized for these developmental processes between species, we compare knowledge from mice, flies and worms. The present analysis provides evidence of the necessity for and specificity of each nuclear transport factor and for nucleoporins during germ cell differentiation. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

Inhibin, activin, follistatin and FSH serum levels and testicular production are highly modulated during the first spermatogenic wave in mice

Publisher: Bioscientifica

Date: 09-2008

DOI: 10.1530/REP-08-0140

Abstract: Testicular development is governed by the combined influence of hormones and proteins, including FSH, inhibins, activins and follistatin (FST). This study documents the expression of these proteins and their corresponding mRNAs, in testes and serum from mice aged 0 through 91 days post partum (dpp), using real-time PCR, in situ hybridisation, immunohistochemistry, ELISA and RIA. Serum immunoactive total inhibin and FSH levels were negatively correlated during development, with FSH levels rising and inhibin levels falling. Activin A production changed significantly during development, with subunit mRNA and protein levels declining rapidly after 4 dpp, while simultaneously levels of the activin antagonists, FST and inhibin/activin β C , increased. Inhibin/activin β A and β B subunit mRNAs were detected in Sertoli, germ and Leydig cells throughout testis development, with the β A subunit also detected in peritubular myoid cells. The α, β A , β B and β C subunit proteins were detected in Sertoli and Leydig cells of developing and adult mouse testes. While β A and β B subunit proteins were observed in spermatogonia and spermatocytes in immature testes, β C was localised to leptotene and zygotene spermatocytes in immature and adult testes. Nuclear β A subunit protein was observed in primary spermatocytes and nuclear β C subunit in gonocytes and round spermatids. The changing spatial and temporal distributions of inhibins and activins indicate that their modulated synthesis and action are important during onset of murine spermatogenesis. This study provides a foundation for evaluation of these proteins in mice with disturbed testicular development, enabling their role in normal and perturbed spermatogenesis to be more fully understood.

Dicer1 Is Required for Differentiation of the Mouse Male Germline1

Publisher: Oxford University Press (OUP)

Date: 10-2008

DOI: 10.1095/BIOLREPROD.108.067827

Abstract: MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. Hundreds of miRNAs are expressed in mammals however, their functions are just starting to be uncovered. MicroRNAs are processed from a long hairpin mRNA transcript, down to a approximately 23-nucleotide duplex. The enzyme Dicer1 is required for miRNA processing, and mouse knockouts of Dicer1 are embryonic lethal before 7.5 days postcoitus. To examine the function of miRNAs specifically in the germline, we used a mouse model that expresses Cre recombinase from the TNAP locus and a floxed Dicer1 conditional allele. Removal of Dicer1 from germ cells resulted in male infertility. Germ cells were present in adult testes, but few tubules contained elongating spermatids. Germ cells that did differentiate to elongating spermatids exhibited abnormal morphology and motility. Rarely, sperm lacking Dicer1 could fertilize wild-type eggs to generate viable offspring. These results show that Dicer1 and miRNAs are essential for proper differentiation of the male germline.

Putting things in place for fertilization: discovering roles for importin proteins in cell fate and spermatogenesis

Publisher: Medknow

Date: 2015

DOI: 10.4103/1008-682X.154310

TheStk35locus contributes to normal gametogenesis and encodes a lncRNA responsive to oxidative stress

Publisher: The Company of Biologists

Date: 2018

DOI: 10.1242/BIO.032631

Abstract: Serine/threonine kinase 35 (STK35) is a recently identified human kinase with an autophosphorylation function, linked functionally to actin stress fibers, cell cycle progression and survival. STK35 has previously been shown as highly expressed in human testis, and we demonstrated its regulation by nuclear-localized importin α2 in HeLa cells. The present study identifies progressive expression from the Stk35 locus of 2 coding mRNA isoforms and 1 long non-coding RNA (lncRNA) in mouse testis during spermatogenesis, indicating their tightly controlled synthesis. Additionally, lncRNA transcripts are increased by exposure to oxidative stress in mouse GC-1 germ cell line. Stk35 knock out (KO) mice lacking all 3 RNAs are born at sub-Mendelian frequency, and adults manifest both male and female germline deficiency. KO males exhibit no or partial spermatogenesis in most testis tubule cross-sections KO ovaries are smaller and contain fewer follicles. Eyes of KO mice display phenotypes ranging from gross deformity to mild goniodysgenesis or iridocorneal angle malformation, to overtly normal. These findings demonstrate the tight regulation of transcription from the Stk35 locus and its central importance to fertility, eye development and cell responses to oxidative stress.

Identification of receptor tyrosine kinases in the rat testis

Publisher: Wiley

Date: 12-1993

DOI: 10.1002/MRD.1080360406

Abstract: Evidence of receptor/ligand interactions that regulate testis cell function was sought in order to broaden the current understanding of the molecular basis of testis cell function. Using reverse transcription and the polymerase chain reaction, we have obtained novel evidence for the expression of three mRNAs encoding receptor tyrosine kinases in the adult rat testis: the platelet-derived growth factor type A receptor (PDGF-RA), the basic fibroblast growth factor receptor (flg), and fetal liver kinase 1 (Flk-1). A 6.8 kb transcript encoding the PDGF-RA was observed in RNA prepared from testes of rats aged day 5 through adult, with a decline in relative abundance with increasing age after day 17. Analysis of mRNA from isolated cell preparations (day 21 Sertoli cells, adult Leydig cells, round spermatids, and primary spermatocytes) and testes depleted of specific cell types [ethane dimethane sulfonate (EDS)-treated and cryptorchid] indicated that the Leydig cell was the predominant source of this mRNA in the adult testis. The addition of PDGF-BB to cultures of highly purified adult rat Leydig cell preparations resulted in a 40% increase in LH-stimulated testosterone production, confirming a role for this growth factor in regulation of Leydig cell function. These data indicate that the Leydig cell is a principal site of action of PDGF in the testis.

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

Publisher: Informa UK Limited

Date: 2011

DOI: 10.4161/SPMG.1.1.15263

Nuclear importin α and its physiological importance

Publisher: Informa UK Limited

Date: 03-2012

DOI: 10.4161/CIB.19194

Testicular Cancer in Relation to Testicular Dysgenesis Syndrome

Publisher: Elsevier

Date: 2018

DOI: 10.1016/B978-0-12-801238-3.99197-9

Differential expression profiles of conserved Snail transcription factors in the mouse testis

Publisher: Wiley

Date: 30-01-2018

DOI: 10.1111/ANDR.12465

Abstract: Snail transcription factors are key regulators of cellular transitions during embryonic development and tumorigenesis. The closely related SNAI1 and SNAI2 proteins induce epithelial-mesenchymal transitions (EMTs), acting predominantly as transcriptional repressors, while the functions of SNAI3 are unknown. An initial examination of Snai2-deficient mice provided evidence of deficient spermatogenesis. To address the hypothesis that Snail proteins are important for male fertility, this study provides the first comprehensive cellular expression profiles of all three mammalian Snail genes in the post-natal mouse testis. To evaluate Snail transcript expression profiles, droplet digital (dd) PCR and in situ hybridization were employed. Snai1, 2 and 3 transcripts are readily detected at 7, 14, 28 days post-partum (dpp) and 7 weeks (adult). Unique cellular expression was demonstrated for each by in situ hybridization and immunohistochemistry using Western blot-validated antibodies. SNAI1 and SNAI2 are in the nucleus of the most mature germ cell types at post-natal ages 10, 15 and 26. SNAI3 is only detected from 15 dpp onwards and is localized in the Sertoli cell cytoplasm. In the adult testis, Snai1 and Snai2 transcripts are detected in spermatogonia and spermatocytes, while Snai3 is in both germ and Sertoli cells. SNAI1 protein is evident in nuclei of spermatogonia, spermatocytes, round spermatids and elongated spermatids (Stages IX-XII). SNAI2 is present in the nuclei of spermatogonia and spermatocytes, with a faint signal detected in round spermatids. SNAI3 was detected only in Sertoli cell cytoplasm, as in juvenile testes. Additionally, colocalization of SNAI1 and SNAI2 with previously identified key binding partners, LSD1 and PRC2 complex components, provides strong evidence that these important functional interactions are conserved during spermatogenesis to control gene activity. These distinct expression profiles suggest that each Snail family member has unique functions during spermatogenesis.

Expression of IFI 16 in epithelial cells and lymphoid tissues

Publisher: Springer Science and Business Media LLC

Date: 20-12-2002

DOI: 10.1007/S00418-002-0485-0

Abstract: IFI 16 is a member of the HIN-200 protein family named for their haemopoietic expression, interferon-inducibility and nuclear localisation. These proteins have been characterised as transcriptional regulators that modulate the cell cycle. IFI 16 is expressed in some haemopoietic lineages including CD34+ progenitor cells, mature lymphocytes and monocytes, but is absent from granulocytes, erythrocytes and megakaryocytes. We present a wider study of IFI 16 expression in normal human tissues using a monoclonal antibody specifically recognising the C-terminus of IFI 16. As expected, IFI 16 was detected in the nuclei of lymphocytes in the spleen, thymus, lymph node and palatine tonsil, but was also found in epithelial cells in these tissues. Interestingly, IFI 16 protein was also expressed in non-lymphoid tissues including trachea, gastrointestinal tract, skin and testis, but was absent from others including heart and brain. In each tissue, IFI 16 was predominantly expressed in surface epithelial cells and staining was strongest in basal epithelial layers. Therefore, IFI 16 expression is not restricted to cells of the immune system, but is also expressed in epithelial cells. In contrast to the perceived role of HIN-200 proteins as suppressors of cell growth, maximal expression of IFI 16 was in cells with high proliferative potential.

Defining the Impact of Estrogen on Spermatogonial Fate

Publisher: Oxford University Press (OUP)

Date: 10-2011

DOI: 10.1095/BIOLREPROD.111.095737

Developmentally regulated SMAD2 and SMAD3 utilization directs activin signaling outcomes

Publisher: Wiley

Date: 07-2009

DOI: 10.1002/DVDY.21995

Activin and follistatin interactions in the male reproductive tract: activin expression and morphological abnormalities in mice lacking follistatin 288

Publisher: Wiley

Date: 24-02-2017

DOI: 10.1111/ANDR.12337

Abstract: Activin A is an important regulator of testicular and epididymal development and function, as well as inflammation and immunity. In the adult murine reproductive tract, activin A mRNA (Inhba) expression levels are highest in the caput epididymis and decrease progressively towards the distal vas deferens. The activin-binding protein, follistatin (FST), shows the opposite expression pattern, with exceptionally high levels of the Fst288 mRNA variant in the vas deferens. This unique pattern of expression suggests that activin A and follistatin, in particular FST288, play region-specific roles in regulating the epididymis and vas deferens. The cellular distribution of activin and follistatin and structural organization of the male reproductive tract was examined in wild-type and transgenic (TghFST315) mice lacking FST288. Compared to wild-type littermates, TghFST315 mice showed a 50% reduction in serum follistatin and a significant elevation of both activin A and B. Testicular, epididymal and seminal vesicle weights were reduced, but intra-testicular testosterone was normal. A decrease in the epididymal duct diameter in the corpus and thickening of the peritubular smooth muscle in the cauda, together with increased coiling of the proximal vas deferens, were observed in TghFST315 mice. No immune cell infiltrates were detected. Immunohistochemistry indicated that epithelial cells are the main source of activins and follistatin in the epididymis and vas deferens. Activin A, but not activin B, was also localized to sperm heads in the lumen of the epididymis and vas deferens. Expression of Inhba and another immunoregulatory gene, indoleamine-2,3-dioxygenase (Ido-1), was increased approximately twofold in the TghFST315 caput epididymis, but several other genes associated with immunoregulation, inflammation or fibrosis were unaffected. Our novel data indicate that disruption of follistatin expression has significant effects on the testis and epididymis, and suggest an association between activin A and indoleamine-2,3-dioxygenase in the caput epididymis, with implications for the epididymal immunoenvironment.

Interactome of the inhibitory isoform of the nuclear transporter Importin 13

Publisher: Elsevier BV

Date: 03-2017

DOI: 10.1016/J.BBAMCR.2016.12.017

Abstract: Importin 13 (Imp13) is a bidirectional nuclear transporter of proteins involved in a range of important cellular processes, with an N-terminally truncated inhibitory isoform (tImp13) specifically expressed in testis. To gain insight into tImp13 function, we performed a yeast-2-hybrid screen from a human testis cDNA library, identifying for the first time a suite of interactors with roles in erse cellular process. We validated the interaction of tImp13 with Eukaryotic translation initiation factor 4γ2 (EIF4G2) and High mobility group containing protein 20A (HMG20A), benchmarking that with glucocorticoid receptor (GR), a known Imp13 interactor expressed in testis. Coimmunoprecipitation assays indicated association of both tImp13 and Imp13 with EIF4G2, HMG20A and GR. Quantitative confocal microscopic analysis revealed the ability of tImp13 to inhibit the nuclear localisation of EIF4G2, HMG20A and GR, as well as that of Imp13 to act as a nuclear exporter for both EIF4G2 and HMG20A, and as a nuclear importer for GR. The physiological relevance of these results was highlighted by the cytoplasmic localisation of EIF4G2, HMG20A and GR in pachytene spermatocytes/round spermatids in the murine testis where tImp13 is present at high levels, in contrast to the nuclear localisation of HMG20A and GR in spermatogonia, where tImp13 is largely absent. Interestingly, Imp13, EIF4G2, HMG20A and GR were found together in the acrosome vesicle of murine epididymal spermatozoa. Collectively, our findings show, for the first time, that tImp13 may have a functional role in the mature spermatozoa, in addition to that in the meiotic germ cells of the testis.

Deletions on mouse Yq lead to upregulation of multiple X- and Y-linked transcripts in spermatids

Publisher: Oxford University Press (OUP)

Date: 08-08-2005

DOI: 10.1093/HMG/DDI304

Abstract: Deletions on the mouse Y-chromosome long arm (MSYq) lead to teratozoospermia and in severe cases to infertility. We find that the downstream transcriptional changes in the testis resulting from the loss of MSYq-encoded transcripts involve upregulation of multiple X- and Y-linked spermatid-expressed genes, but not related autosomal genes. Therefore, this indicates that in normal males, there is a specific repression of X and Y (gonosomal) transcription in post-meiotic cells, which depends on MSYq-encoded transcripts. Together with the known sex ratio skew in favour of females in the offspring of fertile MSYqdel males, this strongly suggests the existence of an intragenomic conflict between X- and Y-linked genes. Two potential antagonists in this conflict are the X-linked multicopy gene Xmr and its multicopy MSYq-linked relative Sly, which are upregulated and downregulated, respectively, in the testes of MSYqdel males. Xmr is also expressed during meiotic sex chromosome inactivation (MSCI), indicating a link between the MSCI and the MSYq-dependent gonosomal repression in spermatids. We therefore propose that this repression and MSCI itself are evolutionary adaptations to maintain a normal sex ratio in the face of X/Y antagonism.

Interactome of the negative regulator of nuclear import BRCA1-binding protein 2

Publisher: Springer Science and Business Media LLC

Date: 30-03-2015

DOI: 10.1038/SREP09459

Abstract: Although the negative regulator of nuclear import (NRNI) BRCA1 binding protein 2 (BRAP2) is highly expressed in testis, its role is largely unknown. Here we address this question by documenting the BRAP2 interactome from human testis, using the yeast 2-hybrid system to identify BRAP2-interacting proteins with roles in erse cellular processes, including regulation of the actin cytoskeleton, ubiquitinylation, cell cycle/apoptosis and transcription. Interaction with BRAP2 in adult mouse testis with three of these, PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1), A-Kinase anchor protein (AKAP3) and DNA methyl transferase 1 (DNMT1), was confirmed by coimmunoprecipitation assays. BRAP2's ability to inhibit PHLPP1 and DNMT1 nuclear localisation was also confirmed by quantitative confocal microscopy. Importantly, the physiological relevance thereof was implied by the cytoplasmic localisation of PHLPP1, AKAP3 and DNMT1 in pachytene spermatocytes/round spermatids where BRAP2 is present at high levels and nuclear localisation of PHLPP1 and DNMT1 in spermatogonia concomitant with lower levels of BRAP2. Interestingly, BRAP2 was also present in murine spermatozoa, in part colocalised with AKAP3. Together the results indicate for the first time that BRAP2 may play an important NRNI role in germ cells of the testis, with an additional, scaffold/structural role in mature spermatozoa.

What a difference a day makes! The contribution of intrinsic FGF9 signalling to germline masculinisation

Publisher: Medknow

Date: 15-11-2010

DOI: 10.1038/AJA.2010.151

Substantial prevalence of microdeletions of the Y-chromosome in infertile men with idiopathic azoospermia and oligozoospermia detected using a sequence-tagged site-based mapping strategy

Publisher: The Endocrine Society

Date: 04-1996

DOI: 10.1210/JC.81.4.1347

Expression of Bambi Is Widespread in Juvenile and Adult Rat Tissues and Is Regulated in Male Germ Cells

Publisher: The Endocrine Society

Date: 09-2003

DOI: 10.1210/EN.2002-0124

Abstract: Members of the TGFβ superfamily may compete for receptor occupancy and intracellular signaling molecules in specific developmental circumstances. We explored the potential importance of the TGFβ family inhibitor, Bambi (Bmp and activin membrane-bound inhibitor) by examining its pattern of mRNA expression in juvenile and adult rat tissues, with a focus on reproductive organs. The 1.8-kb transcript was ubiquitous, whereas a 3-kb transcript was unique to enriched spermatocyte and spermatid cell fractions and adult testis. The full-length rat cDNA is 89% (nucleic acid) and 95% (amino acid) identical to its human homolog, hnma. Using in situ hybridization, Bambi mRNA was detected in granulosa and thecal cells of adult ovaries and in spermatogonia, spermatocytes, round spermatids, and Sertoli cells of adult testes. In addition to a persistent signal in Sertoli cells in juvenile testes, this mRNA within germ cells appeared dramatically increased as gonocytes matured into spermatogonia immediately after birth. These data indicate that TGFβ superfamily signaling within male germ cells is down-regulated at the onset of spermatogenesis. The addition of exogenous activin A to 24-h cultures of newborn rat testis fragments decreased the Bambi mRNA level. Regulated Bambi mRNA synthesis may contribute to TGFβ superfamily signaling modulation in several organs, as suggested by its discrete expression switch in male germ cells.

Changing expression and subcellular distribution of karyopherins during murine oogenesis

Publisher: Bioscientifica

Date: 12-2015

DOI: 10.1530/REP-14-0585

Abstract: Mammalian oocyte growth and development is driven by a strict program of gene expression that relies on the timely presence of transcriptional regulators via nuclear pores. By targeting specific cargos for nucleo-cytoplasmic transport, karyopherin (KPN) proteins are key to the relocation of essential transcription factors and chromatin-remodelling factors into and out of the nucleus. Using multiple complementary techniques, here we establish that KPNA genes and proteins are dynamically expressed and relocalised throughout mouse oogenesis and folliculogenesis. Of the KPNAs examined ( Kpna1 , Kpna2 , Kpna3 , Kpna4 , Kpna6 , Kpna7 , Kpnb1 , Ipo5 and Xpo1 ), all were expressed in the embryonic ovary with up-regulation of protein levels concomitant with meiotic entry for KPNA2, accompanied by the redistribution of the cellular localisation of KPNA2 and XPO1. In contrast, postnatal folliculogenesis revealed significant up-regulation of Kpna1 , Kpna2 , Kpna4 , Kpna6 and Ipo5 and down-regulation of Kpnb1 , Kpna7 and Xpo1 at the primordial to primary follicle transition. KPNAs exhibited different localisation patterns in both oocytes and granulosa cells during folliculogenesis, with three KPNAs – KPNA1, KPNA2 and IPO5 – displaying marked enrichment in the nucleus by antral follicle stage. Remarkably, varied subcellular expression profiles were also identified in isolated pre-ovulatory oocytes with KPNAs KPNA2, KPNB1 and IPO5 detected in the cytoplasm and at the nuclear rim and XPO1 in cytoplasmic aggregates. Intriguingly, meiotic spindle staining was also observed for KPNB1 and XPO1 in meiosis II eggs, implying roles for KPNAs outside of nucleo-cytoplasmic transport. Thus, we propose that KPNAs, by targeting specific cargoes, are likely to be key regulators of oocyte development.

Dynamic Hedgehog signalling pathway activity in germline stem cells

Publisher: Wiley

Date: 24-02-2014

DOI: 10.1111/J.2047-2927.2014.00187.X

Identification of novel Y chromosome encoded transcripts by testis transcriptome analysis of mice with deletions of the Y chromosome long arm

Publisher: Springer Science and Business Media LLC

Date: 12-2005

DOI: 10.1186/GB-2005-6-12-R102

Regulated production of SnoN2 is a feature of testicular differentiation

Publisher: Wiley

Date: 12-06-2009

DOI: 10.1002/JEMT.20739

Abstract: 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.

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

Publisher: Wiley

Date: 2005

DOI: 10.1002/BIES.20289

Abstract: 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 ex les, together with the roles in gametogenesis for particular nuclear transport factors in Caenorhabditis elegans and Drosophila.

Uropathogenic Escherichia coli causes fibrotic remodelling of the epididymis

Publisher: Wiley

Date: 22-08-2016

DOI: 10.1002/PATH.4748

Abstract: Despite antibiotic treatment, up to 40% of patients have impaired fertility after epididymitis due to serovars of Escherichia coli, a frequent pathogen. The reasons for infertility are unclear, but it may result from epididymal duct obstruction. To determine whether E. coli infection of the epididymis causes obstruction due to fibrosis, and to identify the key mediators, tissues from patients with epididymitis were assessed. Additionally, epididymitis was induced with uropathogenic E. coli (UPEC) or commensal serovars in wild-type and MyD88(-/-) mice, which are relatively unresponsive to bacterial pathogens. Epididymal organ cultures were treated with activin A and bacteria and their histology and levels of cytokines and fibrosis markers were analysed. Patients with epididymitis showed severe fibrosis of the epididymal duct. In mice, UPEC infection also caused fibrosis and ductal obstruction in the cauda epididymis. Levels of mRNA for fibrotic markers (α-smooth muscle actin, fibronectin) and cytokines (activin A, TNFα, IL-1α, IL-1β, IL-6) and total collagen levels were significantly elevated. This fibrotic response was blunted by the loss of MyD88. Activin A induced fibrosis in cultured epididymis, which was inhibited by the activin-binding protein follistatin. In summary, bacterial epididymitis causes fibrosis and obstruction. The milder tissue damage in Myd88(-/-) UPEC epididymitis highlights the importance of the host response to infection in causing epididymal damage. Elevated levels of activin A in vivo and fibrotic remodelling elicited by activin A in vitro indicate that this cytokine is a potential target for supplementary treatment to antibiotic therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Nuclear retention of importin α coordinates cell fate through changes in gene expression

Publisher: Wiley

Date: 30-09-2011

DOI: 10.1038/EMBOJ.2011.360

Activin and BMP Signalling in Human Testicular Cancer Cell Lines, and a Role for the Nucleocytoplasmic Transport Protein Importin-5 in Their Crosstalk

Publisher: MDPI AG

Date: 24-03-2023

DOI: 10.3390/CELLS12071000

Abstract: Testicular germ cell tumours (TGCTs) are the most common malignancy in young men. Originating from foetal testicular germ cells that fail to differentiate correctly, TGCTs appear after puberty as germ cell neoplasia in situ cells that transform through unknown mechanisms into distinct seminoma and non-seminoma tumour types. A balance between activin and BMP signalling may influence TGCT emergence and progression, and we investigated this using human cell line models of seminoma (TCam-2) and non-seminoma (NT2/D1). Activin A- and BMP4-regulated transcripts measured at 6 h post-treatment by RNA-sequencing revealed fewer altered transcripts in TCam-2 cells but a greater responsiveness to activin A, while BMP4 altered more transcripts in NT2/D1 cells. Activin significantly elevated transcripts linked to pluripotency, cancer, TGF-β, Notch, p53, and Hippo signalling in both lines, whereas BMP4 altered TGF-β, pluripotency, Hippo and Wnt signalling components. Dose-dependent antagonism of BMP4 signalling by activin A in TCam-2 cells demonstrated signalling crosstalk between these two TGF-β superfamily arms. Levels of the nuclear transport protein, IPO5, implicated in BMP4 and WNT signalling, are highly regulated in the foetal mouse germline. IPO5 knockdown in TCam-2 cells using siRNA blunted BMP4-induced transcript changes, indicating that IPO5 levels could determine TGF-β signalling pathway outcomes in TGCTs.

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

Publisher: Wiley

Date: 10-12-2007

DOI: 10.1002/DVDY.21401

Abstract: 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.

Thrombin Stimulation of Proteoglycan Synthesis in Vascular Smooth Muscle Is Mediated by Protease-activated Receptor-1 Transactivation of the Transforming Growth Factor β Type I Receptor

Publisher: Elsevier BV

Date: 08-2010

DOI: 10.1074/JBC.M109.092767

Gene expression study in the juvenile mouse testis: identification of stage-specific molecular pathways during spermatogenesis

Publisher: Springer Science and Business Media LLC

Date: 09-2006

DOI: 10.1007/S00335-006-0029-3

Abstract: A gene expression time course in the juvenile mouse testis was established using cDNA microarrays derived from a variety of isolated testis cell types. In conjunction with the use of four germ cell-deficient mouse models, a stage and cell-type classification over nine time points has been obtained and analyzed for differential expression of genes. The expression profiles have been clustered into nine groups and subjected to detailed analysis of associated gene ontology. This has allowed the correlation of particular cellular processes and functions with different expression clusters. Focused analysis of transcripts involved in cell number regulation (apoptosis and proliferation) and their spatiotemporal expression patterns are presented. The findings indicate that for genes involved in both apoptosis and proliferation, several distinct pathways regulating these processes are active in somatic and germ cell lineages.

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

Publisher: The Endocrine Society

Date: 06-2015

DOI: 10.1210/EN.2014-1555

Abstract: 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.

Concomitant loss of proapoptotic BH3-only Bcl-2 antagonists Bik and Bim arrests spermatogenesis

Publisher: Wiley

Date: 03-11-2005

DOI: 10.1038/SJ.EMBOJ.7600857

Expression of nuclear transport importins beta 1 and beta 3 is regulated during rodent spermatogenesis

Publisher: Oxford University Press (OUP)

Date: 2006

DOI: 10.1095/BIOLREPROD.105.042341

Abstract: Spermatogenic differentiation requires progressive gene expression changes, and proteins required for this must be transported into the nucleus. Many of these contain a nuclear localization signal and are likely to be transported by importin protein family members, each of which recognizes and transports distinct cargo proteins. We hypothesized that importins, as modulators of protein nuclear access, would display distinct expression profiles during spermatogenesis, indicating their potential to regulate key steps in cellular differentiation. This was tested throughout testicular development in rodents. Real-time PCR analysis of postnatal mouse testes revealed changing expression levels of Knpb1 (encoding importin beta 1) and Ranbp5 (encoding beta 3) mRNAs, with Knpb1 highest at 26 days postpartum and Ranbp5 highest in Day 26 and adult testis. Their distinctive cellular expression patterns visualized using in situ hybridization and immunohistochemistry were identical in mouse and rat testes where examined. Within the seminiferous epithelium, Knpb1 mRNA and importin beta1 protein were detected within mitotic Sertoli and germ cells during fetal and early postnatal development, becoming restricted to spermatogonia and spermatocytes in adulthood. Importin beta 3 protein in fetal germ cells displayed a striking difference in intracellular localization between male and female gonads. In adult testes, Ranbp5 mRNA was detected in round spermatids and importin beta 3 protein in elongating spermatids. This is the first comprehensive in situ demonstration of developmentally regulated synthesis of nuclear transport components. The contrasting expression patterns of importins beta 1 and 3 identify them as candidates for regulating nuclear access of factors required for developmental switches.

TGF-β superfamily signaling in testis formation and early male germline development

Publisher: Elsevier BV

Date: 09-2015

DOI: 10.1016/J.SEMCDB.2015.10.029

Abstract: The TGF-β ligand superfamily contains at least 40 members, many of which are produced and act within the mammalian testis to facilitate formation of sperm. Their progressive expression at key stages and in specific cell types determines the fertility of adult males, influencing testis development and controlling germline differentiation. BMPs are essential for the interactive instructions between multiple cell types in the early embryo that drive initial specification of gamete precursors. In the nascent foetal testis, several ligands including Nodal, TGF-βs, Activins and BMPs, serve as key masculinizing switches by regulating male germline pluripotency, somatic and germline proliferation, and testicular vascularization and architecture. In postnatal life, local production of these factors determine adult testis size by regulating Sertoli cell multiplication and differentiation, in addition to specifying germline differentiation and multiplication. Because TGF-β superfamily signaling is integral to testis formation, it affects processes that underlie testicular pathologies, including testicular cancer, and its potential to contribute to subfertility is beginning to be understood.

The changing landscape of immune cells in the fetal mouse testis

Publisher: Springer Science and Business Media LLC

Date: 12-07-2022

DOI: 10.1007/S00418-022-02129-6

Abstract: Fetal testis growth involves cell influx and extensive remodeling. Immediately after sex determination in mouse, macrophages enable normal cord formation and removal of inappropriately positioned cells. This study provides new information about macrophages and other immune cells after cord formation in fetal testes, including their density, distribution, and close cellular contacts. C57BL6J mouse testes from embryonic day (E) 13.5 to birth (post-natal day 0 PND0), were examined using immunofluorescence, immunohistochemistry, and RT-qPCR to identify macrophages (F4/80, CD206, MHCII), T cells (CD3), granulocytes/neutrophils (Ly6G), and germ cells (DDX4). F4/80 + cells were the most abundant, comprising 90% of CD45 + cells at E13.5 and declining to 65% at PND0. Changes in size, shape, and markers (CD206 and MHCII) documented during this interval align with the understanding that F4/80 + cells have different origins during embryonic life. CD3 + cells and F4/80 − /MHCII + were absent to rare until PND0. Ly6G + cells were scarce at E13.5 but increased robustly by PND0 to represent half of the CD45 + cells. These immunofluorescence data were in accord with transcript analysis, which showed that immune marker mRNAs increased with testis age. F4/80 + and Ly6G + cells were frequently inside cords adjacent to germ cells at E13.5 and E15.5. F4/80 + cells were often in clusters next to other immune cells. Macrophages inside cords at E13.5 and E15.5 (F4/80 Hi /CD206 + ) were different from macrophages at PND0 (F4/80 Dim /CD206 − ), indicating that they have distinct origins. This histological quantification coupled with transcript information identifies new cellular interactions for immune cells in fetal testis morphogenesis, and highlights new avenues for studies of their functional significance.

Changing subcellular localization of nuclear transport factors during human spermatogenesis

Publisher: Wiley

Date: 04-08-2012

DOI: 10.1111/J.1365-2605.2011.01202.X

Abstract: Spermatogenesis requires progressive changes in gene expression mediated by hormonal and local factors. Regulated macromolecular movement between nuclear and cytoplasmic compartments enables these essential responses to changing extracellular cues, and dynamic production of the nucleocytoplasmic transporters and importin proteins, throughout gametogenesis in rodents implicates them as key mediators of germline differentiation. We examined normal adult human testis expression profiles of six importins plus five additional proteins involved in nucleocytoplasmic transport. Although most were detected in the nucleus during germline differentiation, importin α4 was exclusively observed in Sertoli and germ cell cytoplasm. Many proteins were present in round spermatid nuclei (importins α1, α3, β1, β3 exportin-1, Nup62, Ran, RanBP1, RCC1), and remarkable intense nuclear and/or nuclear-associated signals were detected for importin α1, importin α3 and Nup62 in spermatocytes. This study identifies conserved aspects of nucleocytoplasmic transport during spermatogenesis and extends our knowledge of the dynamic presence of these proteins, which indicates that they contribute to germ cell-specific cargo trafficking and potentially to other functions during human spermatogenesis. We also demonstrate for the first time that importin α3 is nuclear in spermatocytes, when exportin-1 is cytoplasmic, suggesting that nuclear transport is altered during meiosis.

Testicular Cancer

Publisher: Elsevier

Date: 2010

DOI: 10.1016/B978-0-08-046884-6.01136-2

Examination of testicular lumicrine regulation of activins and immunoregulatory genes in the epididymal caput

Publisher: Wiley

Date: 31-08-2022

DOI: 10.1111/ANDR.13099

Abstract: Immunoregulatory genes encoding activin A ( Inhba ) and B ( Inhbb ), and indolamine 2,3‐dioxygenase‐1 ( Ido1 ) are highly expressed in the murine caput epididymidis, which also has a network of intraepithelial mononuclear phagocytes. This environment is postulated to promote immunological tolerance to epididymal sperm. The factors regulating the immunoregulatory agents in the epididymal caput are poorly understood. This study aimed to investigate the potential role of testicular lumicrine factors in regulating activin and other immune‐related genes in the caput epididymidis. The efferent ducts in adult C57/Bl6 mice were exposed and ligated bilaterally. Serum and tissues were collected seven days later. Animals with bilateral sham ligation and animals with no ligations (collectively referred to as the “intact” group) were used as controls. Pressure‐induced seminiferous epithelial damage due to intratubular fluid accumulation was observed in all ligated testes. Testicular inhibin was significantly increased and testosterone was elevated in some animals following bilateral ligation, but serum testosterone, serum LH, and serum inhibin were normal. Ligation caused epithelial regression in the initial segment, with similar but less severe effects in other caput segments. Activin A staining by immunohistochemistry in the epithelium was reduced in bilateral ligation, particularly in the initial segment, with moderately reduced staining intensity in the rest of the caput. Inhba expression within the caput was not significantly affected by bilateral ligation, but Inhbb was reduced by more than 60%. Transcripts encoding the macrophage‐specific receptor Cx3cr1 were significantly reduced following bilateral ligation, but other immune cell markers, Ido1 , and inflammatory genes were unaffected. These data indicate that testicular lumicrine secretion regulates several genes that are preferentially expressed in the initial segment, but has marginal effects on genes such as those encoding activin A and IDO1, which are expressed more widely in the caput.

Specific immune cell and cytokine characteristics of human testicular germ cell neoplasia

Publisher: Oxford University Press (OUP)

Date: 08-09-2016

DOI: 10.1093/HUMREP/DEW211

Abstract: Which immune cells and cytokine profiles are characteristic for testicular germ cell neoplasia and what consequences does this have for the understanding of the related testicular immunopathology? The unique immune environment of testicular germ cell neoplasia comprises B cells and dendritic cells as well as high transcript levels of IL-6 and other B cell supporting or T helper cell type 1 (Th1)-driven cytokines and thus differs profoundly from normal testis or inflammatory lesions associated with hypospermatogenesis. T cells are known to be the major component of inflammatory infiltrates associated with either hypospermatogenesis or testicular cancer. It has previously been reported that B cells are only involved within infiltrates of seminoma s les, but this has not been investigated further. Immunohistochemical characterisation (IHC) of infiltrating immune cells and RT-qPCR-based analysis of corresponding cytokine microenvironments was performed on different testicular pathologies. Testicular biopsies, obtained from men undergoing andrological work-up of infertility or taken during surgery for testicular cancer, were used in this study. S les were grouped as follows: (i) normal spermatogenesis (n = 18), (ii) hypospermatogenesis associated with lymphocytic infiltrates (n = 10), (iii) s les showing neoplasia [germ cell neoplasia in situ (GCNIS, n = 26) and seminoma, n = 18]. IHC was performed using antibodies against T cells (CD3+), B cells (CD20cy+), dendritic cells (CD11c+), macrophages (CD68+) and mast cells (mast cell tryptase+). Degree and compartmental localisation of immune cells throughout all groups analysed was evaluated semi-quantitatively. RT-qPCR on RNA extracted from cryo-preserved tissue s les was performed to analyse mRNA cytokine expression, specifically levels of IL-1β, IL-6, IL-17a, tumour necrosis factor (TNF)-α (pro-inflammatory), IL-10, transforming growth factor (TGF)-β1 (anti-inflammatory), IL-2, IL-12a, IL-12b, interferon (IFN)-γ (Th1-driven), IL-4, IL-5, IL-13, IL-23a (Th2-driven), CXCL-13, CXCL-10 and CCL-5 (chemokines). This is the first study showing a direct linkage between the distribution pattern of immune cells in hypospermatogenesis versus testicular cancer and analysis of a wide range of 17 related cyto- and chemokines. A fundamental difference between testicular inflammation patterns associated with different testicular inflammatory conditions either containing or lacking neoplastic cells was demonstrated. In hypospermatogenesis, T cells were detected, whereas B cells and dendritic cells were almost absent. Within GCNIS and seminoma, in addition to T cells, high numbers of dendritic cells and B cells were found, the latter additionally organised in cell clusters, whereas mast cells were absent. Transcripts encoding pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α), anti-inflammatory cytokines (TGF-β1), Th1-driven cytokines (IL-2 and IFN-γ) as well as chemokines (CXCL-13, CXCL-10 and CCL-5) were all significantly increased in testicular germ cell neoplasia (P ≤ 0.01), suggesting the presence of a pro-tumorigenic environment. In contrast, Th2-related cytokines (IL-5, IL-13 and IL-23a) characterised the environment within s les showing normal spermatogenesis as well as hypospermatogenesis. One of the most important outcomes is the pivotal role of IL-6 in testicular cancer that opens potential novel diagnostic and/or immune-therapeutic perspective for testis cancer. Testicular tissue composed of immune cells as well as other somatic cells and germ cells does not allow identification of specific cytokine sources or single cell types, being responsible for establishing the overall cytokine environment. In this study, laser-assisted microdissection did not reach the required efficiency for RT-qPCR analyses. Therefore, in vitro models would be suggested for addressing the above-mentioned issue. Conclusions about cytokine levels in testes with GCNIS are based on a small number of s les. The unique B cell presence and the significantly increased expression level of IL-6 in testicular germ cell neoplasia (P < 0.001) strengthen its special role in this disease. In line with current knowledge on other types of cancer, these results underline the relevance of further investigating the potential of IL-6 as early biomarker and target for therapeutic intervention in testicular germ cell neoplasia. This study (and B.K. in person) was supported by the Deutsche Forschungsgemeinschaft (DFG) as part of the International Research Training Group between Justus Liebig University of Giessen and Monash University, Melbourne (GRK 1871/1) on 'Molecular pathogenesis on male reproductive disorders'. T.H., H.-C.S. and M.B. were supported by the LOEWE focus group 'MIBIE' (male infertility during infection & inflammation)-an excellence initiative of the German state government of Hessen. From the Australian side, K.L. was supported by NHMRC grants (Fellowship, ID1079646 and Project, ID1081987) K.L., S.I. and M.H. received scholarship (S.I.) and research funding (K.L., M.H.) from Monash University. The project also drew support from the Victorian Government's Operational Infrastructure Support Program. The authors have no competing interests to declare.

Fetal Testis Dysgenesis and Compromised Leydig Cell Function in Tgfbr3 (Betaglycan) Knockout Mice1

Publisher: Oxford University Press (OUP)

Date: 2010

DOI: 10.1095/BIOLREPROD.109.078766

Abstract: Betaglycan (Tgfbr3) is a coreceptor for transforming growth factor-beta (TGFB) superfamily ligands. In the current study, a defect in seminiferous cord formation was detected in 12.5-13.5 days postcoitum (dpc) beta glycan null murine testis. Immunohistochemistry with antibodies against cell-specific markers revealed defects in somatic cell populations. To confirm these data, quantitative real-time PCR was performed to determine changes in the expression levels of genes involved in fetal testis cell differentiation and function. The expression levels of the Leydig cell markers Insl3, Cyp17a1, Cyp11a1, Star, and Hsd3b1 were reduced in knockout testis compared to wild-type testis, beginning at 12.5 dpc. Whole mount in situ hybridization confirmed that Cyp11a1 expression was reduced in the null testis, but its distribution pattern was unchanged. Apoptosis was not affected by the loss of beta glycan, but proliferation within the interstitium was reduced at 14.5 dpc. However, morphometric analysis showed no changes in Leydig cell counts between the wild-type and the knockout testes at 14.5 dpc, indicating that fetal Leydig function, rather than number, was affected by the loss of beta glycan. The expression levels of Sertoli cell markers Dhh, Sox9, and Amh were also reduced in the knockout testis at 14.5 dpc. However, the expression of fetal germ cell markers Pou5f1 and DDX4 were not changed across the genotypes at any age examined. Our data show that the presence of beta glycan is required for normal cord formation, normal fetal Leydig cell development, and the establishment of fetal testis endocrine function, thus implicating TGFB superfamily members as regulators of early fetal testis structure and function.

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

Publisher: Bioscientifica

Date: 03-2006

DOI: 10.1530/REP.1.00968

Abstract: 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.

Overlap of autism and conditions associated with atypical sex hormone levels or response: A systematic review and meta-analysis

Publisher: Elsevier BV

Date: 02-2021

DOI: 10.1016/J.RASD.2020.101693

Importin alpha mRNAs have distinct expression profiles during spermatogenesis

Publisher: Wiley

Date: 2006

DOI: 10.1002/DVDY.20569

Abstract: Importin proteins control access to the cell nucleus by mediating the nuclear transport of specific cargoes. We hypothesized that developmental regulation of gene expression may be partially effected by changes in the nuclear transport machinery complement, manifested as regulated expression of importin alpha family genes. We first clarified the identity of the five known mouse importin alpha genes relative to those for human and then determined their expression throughout postnatal rodent testis using PCR and in situ hybridization. Distinct expression patterns were observed for each. At 10 dpp, all importin alpha mRNAs were detected in spermatogonia. In the adult mouse testis, importins alpha1 and alpha3 were detected in spermatogonia and early pachytene spermatocytes. Importin alpha4 mRNA was identified in pachytene spermatocytes, alpha6 mRNA in round spermatids, and alpha2 mRNA in both of these. The distinct importin alpha expression patterns are consistent with their having specific roles and transport cargoes during spermatogenesis.

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

Publisher: CSIRO Publishing

Date: 2017

DOI: 10.1071/RD15239

Abstract: 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.

Platelet-derived growth factor ligand and receptor subunit mRNA in the Sertoli and Leydig cells of the rat testis

Publisher: Elsevier BV

Date: 02-1995

DOI: 10.1016/0303-7207(94)03471-5

Abstract: The expression of mRNAs encoding the platelet-derived growth factor (PDGF) subunits (PDGF A and PDGF B) and the PDGF receptor subunits (PDGF-R alpha and PDGF-R beta) was studied in cells of the rat testis. Leydig cells, primary spermatocytes, round spermatids, and cytoplasts were isolated from adult animals, and enriched preparations of Sertoli cells were obtained from 20-day-old animals. RNA from these cells was examined using Northern blots. Messenger RNA encoding both the PDGF-R alpha and PDGF-R beta subunits was observed in Leydig and Sertoli cell preparations but not in any of the germ cell s les. The Leydig cells contained predominantly mRNA encoding PDGF B subunits, while the Sertoli cells contained abundant mRNAs encoding both ligand subunits. Neither PDGF subunit mRNA was detected in the germ cells. This paper has not examined the expression of protein corresponding to the mRNAs detected but the data suggest that both paracrine and autocrine roles for PDGF are likely in the testis, and that PDGF may mediate communication between the interstitial compartment and the seminiferous epithelium.

The BRCA1-binding protein BRAP2 can act as a cytoplasmic retention factor for nuclear and nuclear envelope-localizing testicular proteins

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1016/J.BBAMCR.2013.05.015

Abstract: Regulation of nuclear protein import is central to many cellular processes such as development, with a key mechanism being factors that retain cargoes in the cytoplasm that normally localize in the nucleus. The breast cancer antigen BRCA1-binding protein BRAP2 has been reported as a novel negative regulator of nuclear import of various nuclear localization signal (NLS)-containing viral and cellular proteins, but although implicated in differentiation pathways and highly expressed in tissues including testis, the gamut of targets for BRAP2 action in a developmental context is unknown. As a first step towards defining the BRAP2 interactome, we performed a yeast-2-hybrid screen to identify binding partners of BRAP2 in human testis. Here we report characterization for the first time of three of these: the high mobility group (HMG)-box-domain-containing chromatin component HMG20A, nuclear mitotic apparatus protein NuMA1 and synaptic nuclear envelope protein SYNE2. Co-immunoprecipitation experiments indicate association of BRAP2 with HMG20A, NuMA1, and SYNE2 in testis, underlining the physiological relevance of the interactions, with immunohistochemistry showing that where BRAP2 is co-expressed with HMG20A and NuMA1, both are present in the cytoplasm, in contrast to their nuclear localization in other testicular cell types. Importantly, quantitative confocal microscopic analysis of cultured cells indicates that ectopic expression of BRAP2 inhibits nuclear localization of HMG20A and NuMA1, and prevents nuclear envelope accumulation of SYNE2, the first report of BRAP2 altering localization of a non-nuclear protein. These results imply for the first time that BRAP2 may have an important role in modulating subcellular localization during testicular development.

Development of a pipeline for automated, high-throughput analysis of paraspeckle proteins reveals specific roles for importin α proteins

Publisher: Springer Science and Business Media LLC

Date: 27-02-2017

DOI: 10.1038/SREP43323

Abstract: We developed a large-scale, unbiased analysis method to measure how functional variations in importin (IMP) α2, IMPα4 and IMPα6 each influence PSPC1 and SFPQ nuclear accumulation and their localization to paraspeckles. This addresses the hypothesis that in idual IMP protein activities determine cargo nuclear access to influence cell fate outcomes. We previously demonstrated that modulating IMPα2 levels alters paraspeckle protein 1 (PSPC1) nuclear accumulation and affects its localization into a subnuclear domain that affects RNA metabolism and cell survival, the paraspeckle. An automated, high throughput, image analysis pipeline with customisable outputs was created using Imaris software coupled with Python and R scripts this allowed non-subjective identification of nuclear foci, nuclei and cells. HeLa cells transfected to express exogenous full-length and transport-deficient IMPs were examined using SFPQ and PSPC1 as paraspeckle markers. Thousands of cells and ,000 nuclear foci were analysed in s les with modulated IMPα functionality. This analysis scale enabled discrimination of significant differences between s les where paraspeckles inherently display broad biological variability. The relative abundance of paraspeckle cargo protein(s) and in idual IMPs each influenced nuclear foci numbers and size. This method provides a generalizable high throughput analysis platform for investigating how regulated nuclear protein transport controls cellular activities.

The alpha-importome of mammalian germ cell maturation provides novel insights for importin biology

Publisher: Wiley

Date: 30-04-2014

DOI: 10.1096/FJ.13-244913

Abstract: Importin α proteins function as adaptors to connect a cargo protein and importin β1 in the classical nuclear import pathway. Here we measure for the first time the stoichiometry of importins α2, α3, α4, and β1 in primary cells corresponding to 2 successive stages of rat spermatogenesis: meiotic spermatocytes and haploid round spermatids. Importin α2 levels were more than 2-fold higher in spermatocytes than in spermatids, while importins α4 and β1 levels did not differ significantly. We performed a comprehensive proteomics analysis to identify binding proteins in spermatocytes and spermatids using recombinant importin α2 and α4 proteins. Among the 100 candidate partners, 42 contained a strong classical nuclear localization signal (cNLS score of>6 by cNLS Mapper), while 8 nuclear proteins lacked any cNLS. In addition, we developed a new strategy to predict which cargoes bind to importin α through the conserved C-terminal acidic domain (ARM repeats 9-10), and provided functional validation of a predicted importin α C-terminal binding segment in Senataxin and Smarca4. Evaluation of this set of candidate binding partners from spermatogenic cells using several bioinformatics approaches provides new evidence that in idual importin αs may serve unique and nonredundant roles in mediating cellular differentiation.

Analysis of activin/TGFB-signaling modulators within the normal and dysfunctional adult human testis reveals evidence of altered signaling capacity in a subset of seminomas

Publisher: Bioscientifica

Date: 11-2009

DOI: 10.1530/REP-09-0206

Abstract: Activin is a pleiotropic growth factor belonging to the transforming growth factor-β (TGFB) superfamily of signaling molecules. Regulated activin signaling is known to influence several steps in rodent male gamete differentiation. TGFB ligand isoforms, TGFB1–B3, also influence germ cell survival in the rodent testis at the onset of spermatogenesis and around the time of puberty. Given the importance of regulated activin and TGFB signaling in testis development and function, we sought to investigate the cellular production sites of activin/TGFB-signaling modulators in normal and dysfunctional adult human testes s les. Signaling transducers phosphorylated SMAD2/3, and signaling modulators SMAD6, MAN-1, inhibin α (INHA), and β-glycan were detected in Bouins fixed, paraffin–embedded adult human testis sections using immunohistochemistry. Additional s les examined were from testicular cancer patients and from normal men subjected to gonadotropin suppression with androgen-based contraceptives. Our findings identify distinct differences between normal and gonadotropin-deprived human testis in the expression and cellular localization of activin/TGFB-signaling modulators. The presence of a nuclear phosphorylated SMAD2/3 signal in all analyzed seminoma specimens indicated active activin/TGFB signaling. Moreover, a subset of seminoma specimens exhibited selective enhanced expression of β-glycan (4 out of 28 seminoma tumors), INHA (6 out of 28), and MAN-1 (6 out of 28), highlighting potential functional differences between in idual tumors in their capacity to regulate activin/TGFB signaling. Within the heterogenous nonseminomas, expression of signaling modulators was variable and reflected the degree of somatic differentiation. Thus, synthesis of activin and TGFB-signaling modulators may be affected by spermatogenic disruption and altered hormone levels in the testis.

In the Shadow of Fibrosis: Innate Immune Suppression Mediated by Transforming Growth Factor-β

Publisher: American Thoracic Society

Date: 12-2016

DOI: 10.1165/RCMB.2016-0248PS

Comparative analysis of activins A and B in the adult mouse epididymis and vas deferens

Publisher: Bioscientifica

Date: 2018

DOI: 10.1530/REP-17-0485

Abstract: Activin A regulates testicular and epididymal development, but the role of activin B in the epididymis and vas deferens is unknown. Mouse models with reduced activin A ( Inhba +/− and Inhba BK/+ ), or its complete absence ( Inhba BK/BK ), were investigated to identify specific roles of activins in the male reproductive tract. In 8-week-old Inhba +/− mice, serum activin A decreased by 70%, with a 50% reduction of gene expression and protein in the testis, epididymis and vas deferens. Activin B and the activin-binding protein, follistatin, were similar to wild-type. Testis weights were slightly reduced in Inhba +/− mice, but the epididymis and vas deferens were normal, while the mice were fertile. Activin A was decreased by 70% in the serum, testis, epididymis and vas deferens of Inhba BK/+ mice and was undetectable in Inhba BK/BK mice, but activin B and follistatin levels were similar to wild-type. In 6-week-old Inhba BK/BK mice, testis weights were 60% lower and epididymal weights were 50% lower than in either Inhba BK/+ or wild-type mice. The cauda epididymal epithelium showed infoldings and less intra-luminal sperm, similar to 3.5-week-old wild-type mice, but at 8 weeks, no structural differences in the testis or epididymis were noted between Inhba BK/BK and wild-type mice. Thus, Inhbb can compensate for Inhba in regulating epididymal morphology, although testis and epididymal maturation is delayed in mice lacking Inhba . Crucially, reduction or absence of activin A, at least in the presence of normal activin B levels, does not lead to major defects in the adult epididymis or vas deferens.

Betaglycan (TβRIII) is a Key Factor in TGF-β2 Signaling in Prepubertal Rat Sertoli Cells

Publisher: MDPI AG

Date: 09-12-2019

DOI: 10.3390/IJMS20246214

Abstract: Transforming growth factor-βs (TGF-βs) signal after binding to the TGF-β receptors TβRI and TβRII. Recently, however, betaglycan (BG) was identified as an important co-receptor, especially for TGF-β2. Both proteins are involved in several testicular functions. Thus, we analyzed the importance of BG for TGF-β1/2 signaling in Sertoli cells with ELISAs, qRT-PCR, siRNA silencing and BrdU assays. TGF-β1 as well as TGF-β2 reduced shedding of membrane-bound BG (mBG), thus reducing the amount of soluble BG (sBG), which is often an antagonist to TGF-β signaling. Treatment of Sertoli cells with GM6001, a matrix metalloproteinases (MMP) inhibitor, also counteracted BG shedding, thus suggesting MMPs to be mainly involved in shedding. Interestingly, TGF-β2 but not TGF-β1 enhanced secretion of tissue inhibitor of metalloproteinases 3 (TIMP3), a potent inhibitor of MMPs. Furthermore, recombinant TIMP3 attenuated BG shedding. Co-stimulation with TIMP3 and TGF-β1 reduced phosphorylation of Smad3, while a combination of TIMP3/TGF-β2 increased it. Silencing of BG as well as TIMP3 reduced TGF-β2-induced phosphorylation of Smad2 and Smad3 significantly, once more highlighting the importance of BG for TGF-β2 signaling. In contrast, this effect was not observed with TIMP3/TGF-β1. Silencing of BG and TIMP3 decreased significantly Sertoli cell proliferation. Taken together, BG shedding serves a major role in TGF-β2 signaling in Sertoli cells.

Specific interaction with the nuclear transporter importin alpha2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles

Publisher: American Society for Cell Biology (ASCB)

Date: 15-02-2015

DOI: 10.1091/MBC.E14-01-0678

Abstract: Importin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although in idual IMPα proteins exhibit dynamic synthesis and subcellular localization during cellular differentiation, including during spermatogenesis, little is known of how this affects cell fate. To investigate how IMPαs control cellular development, we conducted a yeast two-hybrid screen for IMPα2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMPα2 expression coincident with germ-line masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMPα2-binding partner. PSPC1-IMPα2 binding in testis was confirmed in immunoprecipitations and pull downs, and an enzyme-linked immunosorbent assay–based assay demonstrated direct, high-affinity PSPC1 binding to either IMPα2/IMPβ1 or IMPα6/IMPβ1. Coexpression of full-length PSPC1 and IMPα2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High-throughput image analysis of cells indicated IMPα2 levels can directly determine PSPC1-positive nuclear speckle numbers and size a transport-deficient IMPα2 isoform or small interfering RNA knockdown of IMPα2 each reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMPα2 nuclear import cargo in fetal testis provides novel evidence that PSPC1 delivery to paraspeckles, and consequently paraspeckle function, may be controlled by modulated synthesis of specific IMPs.

Smad7 gene delivery prevents muscle wasting associated with cancer cachexia in mice.

Publisher: American Association for the Advancement of Science (AAAS)

Date: 20-07-2016

DOI: 10.1126/SCITRANSLMED.AAC4976

Abstract: Muscle-directed Smad7 gene delivery prevents the loss of skeletal muscle mass and strength in mouse models of cachexia, an important contributor to poor prognosis in patients with advanced cancer.

Tumour necrosis factor-alpha stimulates human neutrophils to release preformed activin A

Publisher: Wiley

Date: 29-03-2011

DOI: 10.1038/ICB.2011.12

Abstract: Activin A, a member of the transforming growth factor-β superfamily, is a critical early mediator of acute inflammation. Activin A release coincides with the release of tumour necrosis factor-α (TNF-α) in models of lipopolysaccharide (LPS)-induced inflammation. The source of circulating activin A during acute inflammation has not been identified and the potential contribution of leukocyte subsets was examined in the following study. Human leukocytes from healthy volunteers were fractionated using Ficoll gradients and cultured under serum-free conditions. Freshly isolated human neutrophils contained 20-fold more activin A than blood mononuclear cells as measured by enzyme-linked immunosorbent assay (ELISA), and both dimeric and monomeric forms of activin A were detected in these cells by western blotting. Activin A was predominantly immunolocalized in the neutrophil cytoplasm. Purified neutrophils secreted activin A in culture when stimulated by TNF-α, but were unable to respond to LPS directly. Although TNF-α stimulated activin A release from neutrophils within 1 h, activin subunit mRNA expression did not increase until 12 h of culture, and the amount of activin A released following TNF-α stimulation did not change between 1 and 12 h. Specific inhibition of the p38 MAP kinase signalling pathway blocked TNF-α-induced activin release, and the secretion of activin A was not due to TNF-α-induced neutrophil apoptosis. These data provide the first evidence that neutrophils are a significant source of mature, stored activin A. Stimulation of the release of neutrophil activin A by TNF-α may contribute to the early peak in circulating activin A levels during acute inflammation.

Defining the window of germline genesis in vitro from murine embryonic stem cells.

Publisher: Oxford University Press (OUP)

Date: 02-2010

DOI: 10.1095/BIOLREPROD.109.078493

Abstract: Bone morphogenetic protein (BMP) signaling is critical for germline establishment during mouse embryogenesis. To exploit its importance for induction of germline precursors in vitro, mouse embryonic stem cells (mESCs) were cultured as embryoid body (EB) aggregates with combinations of BMP2, BMP4, and BMP8B for 3-10 days. At Day 10 of culture, well-delineated clusters of POU5F1-positive (POU5F1+) cells were visible in BMP4-treated and BMP2-treated EBs these were rarely detected in untreated and BMP8B-treated cultures. Quantitative mRNA analysis revealed that a significant elevation of markers associated with primordial germ cell development had occurred in the presence of BMP4 by Day 10, including late germline markers such as Ddx4 (Mvh). Reasoning that germline specification was established by Day 10, we surveyed earlier time points for altered levels of germline marker mRNAs. A peak of early markers, Prdm1 (Blimp1), Ifitm3 (Fragilis), and Dppa3 (Stella), was measured in Day 3 to Day 4 EBs grown in BMP4, followed by a decrease at Day 5. In contrast, other markers, Pou5f1, Nanog, Dazl, and Ddx4, progressively increased from Day 3 to Day 5. Transforming growth factor beta superfamily signaling components Acvr1 (ALK2), Smad1, and Smad5 remained relatively constant. Isolated POU5F1+ cells from BMP4-treated Day 5 EBs contained significantly elevated germline markers compared with POU5F1-negative cells, with a transcript profile differing from mESCs, verifying their unique identity. These results demonstrate that signaling by BMP2 and BMP4, but not BMP8B, enhances germline marker expression within EBs and identify Day 3 to Day 5 in EB differentiation as a window for specification of germ cells in vitro.

Monash University

Location: Australia

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Duke University

Location: United States of America

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Discovery Projects - Grant ID: DP0878102

Start Date: 2008

End Date: 12-2012

Amount: $885,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP170102379

Start Date: 2017

End Date: 12-2019

Amount: $456,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0346228

Start Date: 2003

End Date: 12-2007

Amount: $60,000.00

Funder: Australian Research Council

ARC Centres Of Excellence - Grant ID: CE0348239

Start Date: 06-2003

End Date: 12-2010

Amount: $15,878,900.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP200103463

Start Date: 01-2020

End Date: 01-2023

Amount: $510,000.00

Funder: Australian Research Council