Activin A Determines Steroid Levels and Composition in the Fetal Testis

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.

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Testicular Somatic Cells, not Gonocytes, Are the Major Source of Functional Activin A during Testis Morphogenesis

Endocrinology ◽

10.1210/en.2011-1288 ◽

2011 ◽

Vol 152 (11) ◽

pp. 4358-4367 ◽

Cited By ~ 8

Author(s):

Denise R. Archambeault ◽

Jessica Tomaszewski ◽

Andrew J. Childs ◽

Richard A. Anderson ◽

Humphrey Hung-Chang Yao

Keyword(s):

Cell Proliferation ◽

Sertoli Cell ◽

Somatic Cells ◽

Activin A ◽

Protein Product ◽

Conditional Knockout ◽

Seminiferous Tubules ◽

Testis Size ◽

Testis Development ◽

Testis Cord

Proper development of the seminiferous tubules (or testis cords in embryos) is critical for male fertility. Sertoli cells, somatic components of the seminiferous tubules, serve as nurse cells to the male germline, and thus their numbers decide the quantity of sperm output in adulthood. We previously identified activin A, the protein product of the activin βA (Inhba) gene, as a key regulator of murine Sertoli cell proliferation and testis cord expansion during embryogenesis. Although our genetic studies implicated fetal Leydig cells as the primary producers of testicular activin A, gonocytes are another potential source. To investigate the relative contribution of gonocyte-derived activin A to testis morphogenesis, we compared testis development in the Inhba global knockout mouse, which lacks activin A production in all cells (including the gonocytes), and a steroidogenic factor 1 (Sf1)-specific conditional knockout model in which activin A expression in testicular somatic cells is disrupted but gonocyte expression of activin A remains intact. Surprisingly, testis development was comparable in these two models of activin A insufficiency, with similar reductions in Sertoli cell proliferation and minor differences in testis histology. Thus, our findings suggest activin A from male gonocytes is insufficient to promote Sertoli cell proliferation and testis cord expansion in the absence of somatic cell-derived activin A. Evaluation of adult male mice with fetal disruption of activin A revealed reduced testis size, lowered sperm production, altered testicular histology, and elevated plasma FSH levels, defects reminiscent of human cases of androgen-sufficient idiopathic oligozoospermia.

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CORTICOSTEROIDS SUPPRESS PLASMINOGEN ACTIVATION IN THE BOVINE SERTOLI CELL

Journal of Endocrinology ◽

10.1677/joe.0.108r001 ◽

1986 ◽

Vol 108 (1) ◽

pp. R1-R3 ◽

Cited By ~ 13

Author(s):

N. Jenkins ◽

J.D. Ellison

Keyword(s):

Plasminogen Activator ◽

Sertoli Cell ◽

Sertoli Cells ◽

Rna Synthesis ◽

Fold Increase ◽

Maximum Response ◽

Plasminogen Activation ◽

Half Maximum ◽

Direct Effects ◽

Dose Dependent

ABSTRACT Bovine FSH stimulated a six fold increase in secretion of plasminogen activator by immature bovine Sertoli cells with half-maximum response (ED50) at 145ng/ml. Treatment with FSH and either dexamethasone, cortisol or corticosterone produced a dose-dependent suppression of PA activity, with ED50 values of 35, 320 and >650nmol/l respectively. Effects of dexamethasone required over 6 h incubation to become significant (P < 0.001), and were blocked by inhibitors of RNA synthesis and translation. These data demonstrate direct effects of corticosteroids on Sertoli cells, resulting in the synthesis of antiprotease factors which antagonize the actions of FSH.

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Two distinct Sertoli cell states are regulated via germ cell crosstalk†

Biology of Reproduction ◽

10.1093/biolre/ioab160 ◽

2021 ◽

Author(s):

Rachel L Gewiss ◽

Nathan C Law ◽

Aileen R Helsel ◽

Eric A Shelden ◽

Michael D Griswold

Keyword(s):

Germ Cell ◽

Sertoli Cell ◽

Germ Cells ◽

Sertoli Cells ◽

Seminiferous Tubule ◽

Seminiferous Epithelium ◽

Critical Component ◽

Cell Transcriptome ◽

Cell Crosstalk ◽

Blood Testis Barrier

Abstract Sertoli cells are a critical component of the testis environment for their role in maintaining seminiferous tubule structure, establishing the blood-testis barrier, and nourishing maturing germ cells in a specialized niche. This study sought to uncover how Sertoli cells are regulated in the testis environment via germ cell crosstalk in the mouse. We found two major clusters of Sertoli cells as defined by their transcriptomes in Stages VII–VIII of the seminiferous epithelium and a cluster for all other stages. Additionally, we examined transcriptomes of germ cell-deficient testes and found that these existed in a state independent of either of the germ cell-sufficient clusters. Altogether, we highlight two main transcriptional states of Sertoli cells in an unperturbed testis environment, and a germ cell-deficient environment does not allow normal Sertoli cell transcriptome cycling and results in a state unique from either of those seen in Sertoli cells from a germ cell-sufficient environment.

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Role of Nodal signalling in testis development and initiation of testicular cancer

Reproduction ◽

10.1530/rep-18-0641 ◽

2019 ◽

Vol 158 (2) ◽

pp. R67-R77 ◽

Cited By ~ 2

Author(s):

Katrine Harpelunde Poulsen ◽

Anne Jørgensen

Keyword(s):

Testicular Cancer ◽

Germ Cell ◽

Sertoli Cell ◽

Cell Function ◽

Testicular Development ◽

Fetal Life ◽

Cell Specification ◽

Testis Development ◽

Fetal Testis ◽

Cancer Pathogenesis

Testicular development from the initially bipotential gonad is a tightly regulated process involving a complex signalling cascade to ensure proper sequential expression of signalling factors and secretion of steroid hormones. Initially, Sertoli cell specification facilitates differentiation of the steroidogenic fetal Leydig cells and establishment of the somatic niche, which is critical in supporting the germ cell population. Impairment of the somatic niche during fetal life may lead to development of male reproductive disorders, including arrest of gonocyte differentiation, which is considered the first step in the testicular cancer pathogenesis. In this review, we will outline the signalling pathways involved in fetal testis development focusing on the Nodal pathway, which has recently been implicated in several aspects of testicular differentiation in both mouse and human studies. Nodal signalling plays important roles in germ cell development, including regulation of pluripotency factor expression, proliferation and survival. Moreover, the Nodal pathway is involved in establishment of the somatic niche, including formation of seminiferous cords, steroidogenesis and Sertoli cell function. In our outline of fetal testis development, important differences between human and mouse models will be highlighted to emphasise that information obtained from mouse studies cannot always be directly translated to humans. Finally, the implications of dysregulated Nodal signalling in development of the testicular cancer precursor, germ cell neoplasia in situ, and testicular dysgenesis will be discussed – none of which arise in rodents, emphasising the importance of human models in the effort to increase our understanding of origin and early development of these disorders.

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Proliferative Phase Sertoli Cells Display a Developmentally Regulated Response to Activin in Vitro

Endocrinology ◽

10.1210/en.2002-220595 ◽

2003 ◽

Vol 144 (2) ◽

pp. 474-483 ◽

Cited By ~ 58

Author(s):

Jeremy J. Buzzard ◽

Paul G. Farnworth ◽

David M. de Kretser ◽

Anne E. O’Connor ◽

Nigel G. Wreford ◽

...

Keyword(s):

Sertoli Cell ◽

Sertoli Cells ◽

Activin A ◽

Paracrine Factor ◽

Binding Studies ◽

Developmentally Regulated ◽

Rat Pups ◽

Peritubular Cells

We have used cultures of highly purified, proliferating rat Sertoli cells collected from d 3, 6, and 9 rat pups to investigate the role of activin A on Sertoli cell division. These studies demonstrate that activin A acts directly on d 6 and 9, but not d 3, Sertoli cells to induce proliferation, both alone and synergistically with FSH. In addition to stimulating proliferation, activin A induces secretion of inhibins A and B as determined by specific ELISAs. We demonstrate that the synergy between activin A and FSH is not due to local actions of secreted inhibin or follistatin. We have used real-time fluorometric RT-PCR to demonstrate that activin regulates expression of activin receptor and follistatin mRNA by Sertoli cells. Saturation binding studies using 125I-activin A indicate that synergy between activin and FSH may be due to increased numbers of activin receptors on the Sertoli cell. Finally, we show that activin A was secreted at high levels by cultured peritubular cells but was undetectable in high purity proliferating Sertoli cell cultures, suggesting that activin A functions as a paracrine factor during postnatal testis development.

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232. Developmentally regulated activin A signal transduction by Sertoli cells is required for normal mouse testis development

Reproduction Fertility and Development ◽

10.1071/srb08abs232 ◽

2008 ◽

Vol 20 (9) ◽

pp. 32

Author(s):

C. M. Itman ◽

C. Small ◽

M. Griswold ◽

A. K. Nagaraja ◽

M. M. Matzuk ◽

...

Keyword(s):

Signal Transduction ◽

Sertoli Cell ◽

Sertoli Cells ◽

Normal Mouse ◽

Mouse Testis ◽

Nuclear Accumulation ◽

Developmentally Regulated ◽

Testis Development ◽

Differentiated Cells ◽

Immature Cells

Activin A, a TGF-β superfamily ligand, is critical for normal mouse testis development and quantitatively normal sperm production. Testicular activin production changes during development, being substantially higher in the immature testis relative to the adult [1, 2]. Activin influences the Sertoli cell, the nurse cell to developing sperm, enhancing proliferation during its immature phase, but not following terminal differentiation [3]. In the Inha−/− mouse, chronic excessive activin production results in Sertoli cell-derived tumours [4] whereas reduced activin bioactivity, in the InhbaBK/BK mouse, delays fertility [5]. Activin signals are transduced by the phosphorylation and nuclear accumulation of the transcription factors SMAD2 and SMAD3. By comparing activin signal transduction in immature v. terminally differentiated Sertoli cells, using quantitative confocal microscopy and western blot analysis of total and phosphorylated SMAD2 and SMAD3, we discovered that mouse Sertoli cells exhibit developmentally regulated activin responses. Activin induces nuclear accumulation of SMAD3, but not SMAD2, in immature cells, although both proteins are phosphorylated. In contrast, terminally differentiated cells exhibit nuclear accumulation of both SMAD2 and SMAD3. We observed that this shift coincides with decreased SMAD3 production at puberty and changes in FSH-induced Smad transcription, which favours Smad3 in immature cells but promotes Smad2 synthesis in terminally differentiated cells. Furthermore, whereas removal of SMAD3 from the Inha−/− mouse rescues the tumour phenotype [6], we demonstrated that insufficient SMAD3 production impairs testis growth. We hypothesised that this developmentally regulated SMAD utilisation drives specific transcriptional outcomes. Using microarray and quantitative PCR, we identified novel activin target genes displaying developmental stage-specific expression patterns coinciding with differential SMAD usage, including Gja1 and Serpina5 which are required for male fertility. These mRNAs are also modulated in vivo, increased 1.5–2 fold in Inha−/− testes and decreased by half in InhbaBK/BK testes, confirming that normal testis development requires carefully regulated activin production and responsiveness. (1) Buzzard J et al. 2004. Endocrinology 145(7): 3532–3541 (2) Barakat et al. 2008. Reproduction 2008 Epub ahead of print (3) Boitani C et al. 1995. Endocrinology 136(12): 4538–4544 (4) Matzuk M et al. 1992. Nature 360: 313–319 (5) Brown C et al. 2000. Nature Genetics 25(4): 453–457 (6) Li Q et al. 2007. Molecular Endocrinology 21(10: 2472–2486

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149. Smad3 DOSAGE INFLUENCES TESTICULAR MATURATION

Reproduction Fertility and Development ◽

10.1071/srb09abs149 ◽

2009 ◽

Vol 21 (9) ◽

pp. 67

Author(s):

C. Itman ◽

C. Wong ◽

D. A. Jans ◽

M. Ernst ◽

K. L. Loveland

Keyword(s):

Cell Proliferation ◽

Sertoli Cell ◽

Sertoli Cells ◽

Post Partum ◽

Cell Maturation ◽

Physiological Status ◽

Testis Weight ◽

Testis Development ◽

Smad3 Expression ◽

Cell Mitogen

Activin A, a TGF-beta superfamily ligand which signals via Smad2 and Smad3, is critical for normal mouse testis development and quantitatively normal sperm production. Whereas activin enhances immature Sertoli cell proliferation (1), excessive activin production causes Sertoli cell tumours (2); this is alleviated when mice lack Smad3 (3). Sertoli cells exhibit developmentally regulated Smad utilization in activin signalling. Immature Sertoli cells signal via Smad3 while the onset of Smad2-mediated signal transduction correlates with Sertoli cell maturation (4). This change coincides with decreased testicular Smad3 production at puberty and a shift in follicle stimulating hormone (FSH)-induced Smad transcription, from Smad3 in 6 dpp (days post partum) Sertoli cells to Smad2 in 15 dpp cells. These findings suggest that Smad3 is more important for testis development than adult spermatogenesis. To test this hypothesis, we examined testis development in Smad3+/– and Smad3–/– mice. At 7 dpp, testis weight and cord diameter were reduced in Smad3–/–mice, indicating impaired Sertoli cell proliferation. Levels of FSH, a potent Sertoli cell mitogen, were unaltered. Histological analysis revealed advanced spermatogenesis in heterozygous mice, with round spermatids already present at 16 dpp. Quantitative PCR also identified advanced Sertoli and germ cell maturation in Smad3+/– mice, while Leydig cell maturation appeared unaltered. Adult Smad3+/– and Smad3–/– mice were fertile, but had smaller testes. This is the first study relating Smad3 levels to puberty onset and identifies the Smad3+/– mouse as a model of peripheral precocious puberty with otherwise normal physiological status, i.e. no gonadal tumours and normal FSH levels. These results demonstrate that FSH influences testis growth and maturation by regulating Smad3 expression and highlights the importance of testing whether environmental factors, toxicants and endocrine disruptors affect Smad3 expression, thereby leading to altered testis development.

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The use of Y-chromosome-specific repeated DNA sequences in the analysis of testis development in an XX/XY mouse

Development ◽

10.1242/dev.101.supplement.143 ◽

1987 ◽

Vol 101 (Supplement) ◽

pp. 143-149

Author(s):

Lalji Singh ◽

Shoichi Matsukuma ◽

K. W. Jones

Keyword(s):

Y Chromosome ◽

Sertoli Cell ◽

Dna Sequences ◽

Sertoli Cells ◽

Dna Probes ◽

Cellular Composition ◽

Repeated Dna ◽

Repeated Dna Sequences ◽

Testis Development

A study, by means of Y-chromosome-specific repeated DNA probes, of mouse (ST) with small testes is reviewed. Mouse ST was shown to be a somatic mosaic of 10 % XY and 90 % XX cells. The cellular composition of the azoospermic testis reflected the overall proportions of XX and XY cells but it was found that XY cells predominated in the Sertoli cells of the testis tubules. These findings have been interpreted to indicate a fundamental role for the Sertoli cell in inducing testis organization in the indifferent gonadal rudiment, involving the expression of the Y chromosome.

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Regulation of inhibin secretion by Sertoli cell-enriched cultures

Acta Endocrinologica ◽

10.1530/acta.0.1020136 ◽

1983 ◽

Vol 102 (1) ◽

pp. 136-143 ◽

Cited By ~ 27

Author(s):

G. Verhoeven ◽

P. Franchimont

Keyword(s):

Sertoli Cell ◽

Sertoli Cells ◽

Cyproterone Acetate ◽

Aromatase Activity ◽

Pituitary Cells ◽

Immature Rats ◽

Enriched Cultures ◽

Dose Dependent ◽

Pregnant Mare Serum Gonadotrophin ◽

Foetal Bovine Serum

Abstract. Sertoli cells secrete a factor which has the same bioactivity as ovine testicular lymph inhibin: it selectively suppresses the secretion of FSH by cultured pituitary cells. We investigated the factors that acutely modulate the secretion of this inhibin by cultured Sertoli cells derived from immature rats. The secretion of inhibin was studied on day 7 of culture after a 24 h period of incubation in the presence or absence of steroids, gonadotrophins and foetal bovine serum, added alone or in various combinations. It could be demonstrated that aromatisable as well as non-aromatisable natural and synthetic androgens promote the secretion of inhibin in a dose-dependent way. FSH and pregnant mare serum gonadotrophin — at concentrations that clearly stimulate Sertoli cell aromatase activity — did not affect basal or androgen-stimulated production of inhibin. hCG was equally uneffective. The effect of androgens was not modified by the addition of an aromatase inhibitor but it was neutralized by the antiandrogen cyproterone acetate. Oestradiol-17β did not influence the secretion of inhibin whereas progesterone decreased it. Serum enhanced basal as well as androgen stimulated secretion of inhibin. It is concluded that androgens are the major factor which acutely stimulates the production of Sertoli cell inhibin.

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Regulation of activin A and inhibin B secretion by inflammatory mediators in adult rat Sertoli cell cultures

Journal of Endocrinology ◽

10.1677/joe.1.06266 ◽

2005 ◽

Vol 187 (1) ◽

pp. 125-134 ◽

Cited By ~ 33

Author(s):

Y Okuma ◽

A E O’Connor ◽

J A Muir ◽

P G Stanton ◽

D M de Kretser ◽

...

Keyword(s):

Sertoli Cell ◽

Inflammatory Mediators ◽

Sertoli Cells ◽

Reproductive Function ◽

Inhibitory Effect ◽

Activin A ◽

Inhibin B ◽

Hormonal Stimulation ◽

Adult Rat

The regulation of Sertoli cell activin A and inhibin B secretion during inflammation was investigated in vitro. Adult rat Sertoli cells were incubated with the inflammatory mediators, lipopolysaccharide (LPS), interleukin-1β (IL-1β), IL-6 and the IL-1 receptor antagonist (IL-1ra) over 48 h in culture. Activin A, inhibin B and IL-1α were measured in the culture medium by specific two-site ELISAs. Both IL-1β- and LPS-stimulated activin A and inhibited inhibin B secretion. LPS also stimulated the production of IL-1α in the cultures. In contrast to IL-1β, IL-6 had no effect on activin A, although it did have a significant inhibitory effect on inhibin B secretion. Ovine follicle-stimulating hormone (FSH) and the cAMP analogue dibutyryl cAMP opposed the actions of IL-1 and LPS by suppressing activin A and IL-1α secretion and by stimulating inhibin B. Blocking IL-1 activity in the cultures by addition of an excess of IL-1ra completely prevented the response of activin A to exogenous IL-1β, and reduced the response to LPS by 50%. In the presence of IL-1ra, basal secretion of inhibin B was increased, but IL-1ra was unable to reverse the suppression of inhibin B by LPS. These data indicate the importance of both IL-1 isoforms in regulating secretion of activin A and inhibin B by mature Sertoli cells during inflammation. The data also establish that inflammation exerts its effects on activin A and inhibin B secretion via other pathways in addition to those mediated by IL-1, and that hormonal stimulation by FSH and cAMP moderates the Sertoli cell response to inflammation. Interference with the complex interactions between these cytokines and hormones may contribute to the disruption of reproductive function that can accompany infection and illness in men.

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