Amand Chesnel

Evidence for a conserved role of retinoic acid in urodele amphibian meiosis onset

Pleurodeles waltl is a urodele amphibian displaying a ZZ/ZW genetic mode of sex determination. Gonad differentiation can later be modulated by hormone treatment. To investigate germ cell differentiation, we analyzed the expression of the meiosis marker PwDmc1 and show that germ cells enter meiosis in late larval life in females, and 2 months after metamorphosis in males. Organotypic cultures of gonad–mesonephros complexes demonstrated that retinoic acid triggers meiosis entry in P. waltl. In vivo analyses of both PwRaldh2 and PwCyp26b1 expressions, the enzymes required for RA synthesis and degradation respectively, indicate that meiosis onset depends on PwCyp26b1 repression in the gonad during normal or steroid‐induced sex‐reversed development. Taken together, our results show that RA‐dependent meiosis entry could be a conserved mechanism of germ cell differentiation in vertebrates and provide evidence for crosstalk between steroid and RA signaling in the course of sex differentiation. Developmental Dynamics 238:1389–1398, 2009.

Estrogens promote proliferation of the seminoma-like TCam-2 cell line through a GPER-dependent ERα36 induction.

Seminoma, originated from carcinoma in situ cells (CIS), is one of the main causes of cancer in young men. Postpubertal development of these testicular germ cell tumors suggests a hormone-sensitive way of CIS cell proliferation induction. Using the unique seminoma TCam-2 cell line, we demonstrate that both estradiol and testosterone can stimulate TCam-2 cell proliferation in the absence of the estradiol receptor ERα. We establish that estradiol can activate GPER-cAMP/PKA signalling pathway. TCam-2 cells express ERα36, a truncated isoform of the canonical ERα receptor, the expression of which is rapidly induced after estrogen treatment in a GPER-dependent manner. ERα36 knockdown indicates that ERα36 is (i) a downstream target of E(2)-activated GPER/PKA/CREB pathway, (ii) required for estradiol-dependent EGFR expression, (iii) necessary for cell proliferation. Colocalization of ERα36 with cytoskeleton microfilaments suggests a role of estrogens in cell motility. Our results highlight the functional role of ERα36 in context of seminoma cell proliferation and the importance of testing ERα36 in vivo as a possible future prognostic marker.

Sexual Development of the Urodele Amphibian Pleurodeles waltl

Pleurodeles waltl is a urodele amphibian that displays a ZZ/ZW genetic mode of sex determination involving a putative W-borne dominant determinant. This determining pathway can be environmentally inhibited since heat treated ZW larvae undergo a functional female to male sex reversal. Moreover, both genetic sexes can be reversed by treatment of larvae with steroid hormones suggesting they are the major players in the differentiation process. Indeed we demonstrated that i) aromatase expression and activity increase just before ovarian differentiation, ii) aromatase inhibitors induce a female to male sex reversal, iii) estrogens induce male to female sex reversal whereas the opposite is obtained with non-aromatizable androgens, iv) steroidogenic factor 1 and estrogen receptor alpha both display a female-enriched expression following the increase in aromatase activity. The role of endogenous hormones was investigated in a parabiosis model. Surprisingly, in ZW/ZZ associations, the ZW gonad could not differentiate suggesting that the ZZ parabiont produces an inhibiting factor, prior to ovarian differentiation. The role of AMH in this process is discussed, keeping in mind that Müllerian ducts are maintained in males. The development of antibodies and new molecular tools in the near future should help us to better understand the sexual development of this vertebrate.

Temporal and spatial SOX9 expression patterns in the course of gonad development of the caudate amphibian Pleurodeles waltl

The SOX family of transcription factors is thought to regulate gene expression in a wide variety of developmental processes. Namely, SOX9 expression is conserved in vertebrate sex determination or differentiation. Nevertheless, information about caudate amphibians is lacking. In this study, we provide data on Pleurodeles waltl, a species that displays a ZZ/ZW genetic mode of sex determination and a temperature-dependent mechanism of female-to-male sex reversal. Phylogenetic analysis of SOX9 P. waltl ortholog reveals that the deduced protein segregates from the group of anuran and could be more closely related to amniote than to anamniote. However, SOX9 lacks the PQA-rich domain present in amniotes. In larvae, SOX9 is expressed in both sexes in gonad-mesonephros complexes as soon as stage 42, before gonad differentiation. At stage 54(60d) at which testis differentiation is already in progress, analyses of isolated gonads reveal a male-enriched expression of SOX9, which was quantified by real-time PCR. At the end of metamorphosis (stage 56), SOX9 shows a nuclear localization only in the testis. In adults, SOX9 is still expressed in testes and ovaries. In the ovary, SOX9 is found in oocytes from stage I to stage VI but it is never detected in the nucleus. Our results suggest that in P. waltl, like in non mammalian vertebrates, SOX9 could play a role during the late phase of gonad differentiation rather than in sex determination. Its role in germ cells of the adult ovary has still to be elucidated.

Lifelong testicular differentiation in Pleurodeles waltl(Amphibia, Caudata)

BackgroundIn numerous Caudata, the testis is known to differentiate new lobes at adulthood, leading to a multiple testis. The Iberian ribbed newt Pleurodeles waltl has been studied extensively as a model for sex determination and differentiation. However, the evolution of its testis after metamorphosis is poorly documented.MethodsTestes were obtained from Pleurodeles waltl of different ages reared in our laboratory. Testis evolution was studied by several approaches: morphology, histology, immunohistochemistry and RT-PCR. Surgery was also employed to study testis regeneration.ResultsIn this species, the testis is linked to the lung. This association consists of connective tissue derived from the mesorchium and the coelomic epithelium surrounding the lung and takes place at the end of larval life. This tissue contains lobules including primordial germ cells with a typical large and polylobular nucleus. The anterior part of the testis remains thin and undifferentiated while the posterior part differentiates in a large first testis lobe where spermatogenesis occurs during the first year of life. The undifferentiated status of the anterior part is attested by the lack of expression of the testis marker Dmrt1 and the meiosis entry marker Dmc1. Three-year-old Pleurodeles waltl possess multiple testes made up of two lobes. The second lobe appears at the caudal extremity of the first one from residual primordial germ cells located near or even inside efferent ducts in the glandular tissue that usually appears following spermatozoa extrusion. Surprisingly, in the case of surgical elimination of the anterior part of the testis, de novo spermatogenesis is stopped in the first lobe which becomes restricted to the glandular tissue. Following first testis lobe removal, the anterior part of the testis regenerates a new testis lobe, a process stimulated in the presence of DHT.ConclusionPleurodeles waltl constitute an original gonochoristic vertebrate model in which testis differentiation is observed up to adulthood.

Analysis of the effects of different alcohols on MCF-7 human breast cancer cells

Abstract: Alcohol consumption is known to be an increased risk factor for breast cancer, but the underlying molecular mechanisms are not well understood. We have recently shown that the exposure of MCF‐7 breast cancer cells to 0.1% ethanol enhanced their proliferation and increased their content in both estrogen receptor‐α (ERα) and aromatase. The aim of the present work was to determine if the effects of ethanol could be mimicked by other short‐chain aliphatic alcohols such as methanol and 1‐butanol. Our results show that these compounds do not stimulate MCF‐7 cell proliferation. An increase in ERα content was observed by Western blot in methanol‐treated cells, but this parameter was not affected in butanol‐treated cells. Neither of these two alcohols induced an increase in aromatase mRNA level. So despite a similarity in molecular structure, these primary alcohols do not exert the same effects. Taken together, these results suggest that the increase in aromatase expression might be a key event required for the enhanced proliferation observed in the presence of ethanol.

Sex Determination and Sexual Differentiation in Amphibians

Publisher Summary This chapter reviews sex determination and differentiation in amphibians. The class amphibia is composed of three orders: Anura, Caudata, and Gymnophiona. The order Anura (more than 4500 species) includes frogs and toads; caudate order comprises salamanders, newts, sirens, amphiuma, waterdogs, and mudpuppies; and gymnophiona is the least studied and consists of caecilians that resemble giant earthworms rather than typical amphibians. The chapter describes the genetic sex determination and a wide variety of mechanisms regulate sex determination/differentiation in amphibians, which is associated with sex chromosomes that display various patterns, from homomorphism to heteromorphism. Gonadal differentiation is also discussed. The general features leading the undifferentiated gonad to differentiate as an ovary or a testis appear to be similar in anurans and caudates, but the origin of germ cells is very different. It also focuses on sensitivity to temperature, a factor that can induce sex reversal in several species by modulating steroid hormone synthesis. These molecules are indeed major players in the sex differentiation process.

Mammary epithelial cell phenotype disruption in vitro and in vivo through ERalpha36 overexpression

Estrogen receptor alpha 36 (ERα36) is a variant of the canonical estrogen receptor alpha (ERα66), widely expressed in hormone sensitive cancer cells and whose high expression level correlates with a poor survival prognosis for breast cancer patients. While ERα36 activity have been related to breast cancer progression or acquired resistance to treatment, expression level and location of ERα36 are poorly documented in the normal mammary gland. Therefore, we explored the consequences of a ERα36 overexpression in vitro in MCF-10A normal mammary epithelial cells and in vivo in a unique model of MMTV-ERα36 transgenic mouse strain wherein ERα36 mRNA was specifically expressed in the mammary gland. By a combination of bioinformatics and computational analyses of microarray data, we identified hierarchical gene networks, downstream of ERα36 and modulated by the JAK2/STAT3 signaling pathway. Concomitantly, ERα36 overexpression lowered proliferation rate but enhanced migration potential and resistance to staurosporin-induced apoptosis of the MCF-10A cell line. In vivo, ERα36 expression led to duct epithelium thinning and disruption in adult but not in prepubescent mouse mammary gland. These phenotypes correlated with a loss of E-cadherin expression. Here, we show that an enhanced expression of ERα36 is sufficient, by itself, to disrupt normal breast epithelial phenotype in vivo and in vitro through a dominant-positive effect on nongenomic estrogen signaling pathways. These results also suggest that, in the presence of adult endogenous steroid levels, ERα36 overexpression in vivo contributes to alter mammary gland architecture which may support pre-neoplastic lesion and augment breast cancer risk.

286 Estrogens and Alkylphenols Promote Proliferation of the Seminoma-like TCam-2 Cell Line Through ERa36-dependent Pathways

Background: Seminoma, originated from carcinoma in situ cells (CIS), is one of the main causes of cancer in young men. Postpubertal developmentof these testicular germ cell tumors suggests a hormone-sensitive way of CIS cell proliferation induction probably stimulated by lifelong exposure to endocrine disruptors. In a first step to understand the mechanisms underlying the deleterious effects of endocrine disrupting compounds on germ cells, we aimed to decipher the estrogen-dependent transduction pathways in TCam2 cells. Then, we began to assess the effects of a [4tert-octyl + 4-nonylphenol] mix on testicular germ cell tumors in vitro and in vivo. Material and Methods: In this study, we used the unique seminoma TCam-2 cell line which do not express the canonical ERa66 estrogen receptor but Era36, a truncated isoform retaining the DNA-binding, partial dimerisation and ligand-binding domains and a specific C-term 27 aa sequence. Cells were exposed to either estradiol at concentrations in the range of those detected in an adult human testis or to a [4tert-octyl + 4-nonylphenol] mix used at low doses − i.e. those found in food and drinking water. In vitro, we performed cell proliferation assays, siRNA- or shRNA-directed knockdown, microarray- directed gene targeting and signaling pathways identification after short term (1h) or mid-term (24h or 48h) treatment. We also addressed the question of TCam-2 derived tumor growth in xenografted Nude mice treated with the [4tert-octyl + 4-nonylphenol] mix. Results: We demonstrate in vitro that estradiol and the alkylphenol mix trigger TCam-2 cell proliferation through ERa36-dependent pathways. We establish that estradiol can activate GPER-cAMP/PKA signaling pathway. Stable ERa36 knockdown indicates that ERa36 is (i) necessary for cell proliferation (ii) a downstream target of estradiol-activated GPER/PKA/CREB pathway, (iii) required for estradiol-dependent EGFR expression. The [4tert-octyl + 4-nonylphenol] mix signaling pathway is clearly ERa36 dependent but seems to be partially non-estrogenic. Finally, we show that the [4tert-octyl+ 4-nonylphenol] mix stimulates tumor growth in TCam-2 xenografted Nude mice. Conclusions: Our results highlight the functional role of ERa36 in context of seminoma cell proliferation and the importance of testing ERa36 in vivo as apossible marker for endocrine disruptor susceptibility