Endocrine disruptors and the fetal origin of diseases

Thesis

Reference

Endocrine disruptors and the fetal origin of diseases

CEDERROTH, Christopher

Abstract

Les perturbateurs endocriniens sont des composés qui induisent des effets néfastes sur le système endocrine. Cette thèse vise à étudier les effets et mécanismes sous-jacents en utilisant deux perturbateurs endocriniens: le diethylstilbestrol (DES) et les phytoestrogènes.

D'une part, nous avons montré "in vivo" que les actions du DES "in utero" pour induire la maldescente testiculaire (cryptorchidie) nécessitent la présence du récepteur aux oestrogènes alpha. D'autre part, les phytoestrogènes induisent des effets bénéfiques sur le métabolisme du glucose et des lipides et retardent le développement du diabète. Les effets bénéfiques sur le métabolisme du glucose sont restreints à une exposition durant la vie foetale. Cette thèse souligne l'importance de l'exposition aux perturbateurs endocriniens durant la périodes foetale, indiquant que le mode de vie de la mère durant sa grossesse, ainsi que son environnement, pourraient affecter des aspects reproducteurs et métaboliques de sa descendance.

CEDERROTH, Christopher. Endocrine disruptors and the fetal origin of diseases. Thèse de doctorat : Univ. Genève, 2009, no. Sc. 4083

URN : urn:nbn:ch:unige-19959

DOI : 10.13097/archive-ouverte/unige:1995

Available at:

http://archive-ouverte.unige.ch/unige:1995

Disclaimer: layout of this document may differ from the published version.

1 / 1

UNIVERSITÉ DE GENÈVE

Département de Zoologie et de Biologie Animale

Département de Médecine Génétique et Développement

FACULTÉ DES SCIENCES Professeur Ivan Rodriguez

FACULTÉ DE MÉDECINE Professeur Jean-Dominique Vassalli Dr. Serge Nef

Endocrine Disruptors and

the Fetal Origin of Diseases

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade Docteur ès sciences, mention biologie

par

Christopher R. CEDERROTH de

Stockholm (Suède)

THÈSE N° 4083

GENÈVE

Atelier d’impression ReproMail, Uni Mail 2009

UNIVERSITE DE GTNEVE

F A C U L T Ë D Ë S S C I E I I d C T S

Doctorqt ès sciences mention bîologîe

Thèse de /l/lonsieur Christopher CEDERPOTH

i n t i t u l é e :

" Endocrine Disrupfors ond lhe Fefol Origin of Diseqses "

Lo Foculté des sciences, sur le préovis de Messieurs J.-D. VASSALLI, professeur ordinoire et d i r e c t e u r d e t h è s e ( f o c u l t é d e M é d e c i n e - D é p o r t e m e n t d e m é d e c i n e g é n é t i q u e e t d é v e l o p p e m e n t ) , l . R O D R I G U E Z , p r o f e s s e u r o d j o i n t e t c o - d i r e c t e u r d e t h è s e ( D é p o r t e m e n t de zoologie et biologie onimole), S. NEF, docteur, privot docent et c o - d i r e c t e u r d e t h è s e ( F o c u l t é d e m é d e c i n e - D é p o r t e m e n t d e m é e c i n e g é n é t i q u e e t d é v e l o p p e m e n t ) e t A . S O T O , p r o f e s s e u r (T u f t s U n i v e r s i t y , S c h o o l o f M e d i c i n e , B o s t o n , U S A ) , o u t o r i s e I' i m p r e s s i o n d e ' l o p r é s e n t e t h è s e , s o n s e x p r i m e r d ' o p i n i o n s u r l e s p r o p o s i t i o n s q u i y sont énoncées.

G e n è v e , l e I 7 o v r i l 2 0 0 9

Thèse - 4083 (r'

L e D é c q n o l

N . B . - L o t h è s e d o i t p o r t e r l o d é c l o r o t i o n p r é c é d e n t e e t r e m p l i r l e s c o n d i t i o n s é n u m é r é e s d o n s l e s " l n f o r m o t i o n s r e l o t i v e s à u x t h è s e s d e d o c t o r o t ù I ' U n i v e r s i t é d e G e n è v e " .

Nombre d'exemploires à livrer por colis séporé à lq Fqculté : - 4

1

Remerciements

Tout d’abord, je tiens `a remercier le Dr. Serge Nef pour m’avoir donn´e la libert´e d’aborder des sujets d’une telle diversit´e, tout en m’orientant sur les priorit´es `a suivre. Il m’a appris `a ˆetre ind´ependant et `a prendre des initiatives. Sa reconnaissance et son soutien m’ont ´enorm´ement encourag´e `a initier un nouveau projet au sein de son laboratoire.

Un grand merci au Professeur Jean-Dominique Vassalli pour avoir partag´e sa vision pertinente de la science. Il a su m’encourager `a sortir des sentiers battus et `a croire en mes intuitions. Il a fortement contribu´e `a renforcer ma confiance.

Je souhaiterais remercier le Professeur Ivan Rodriguez, mon co-directeur `a la Facult´e des Sci- ences, qui m’a suivi depuis mon diplˆome et qui m’a toujours soutenu.

Merci `a la Professeure Ana Soto, mon examinatrice externe, qui m’a introduit dans le monde des perturbateurs endocriniens, et qui a partag´e sa passion `a chaque meeting du Programme National de Recherche 50.

Un grand merci `a la Professeure Fran¸coise Rohner-Jeanrenaud, la “marraine” de ma th`ese, que je souhaite particuli`erement remercier pour avoir toujours ´et´e disponible, autant profession- nellement que personnellement, ainsi qu’aux Professeurs Michelangelo Foti, Paolo Meda, Isabelle Bolon, Pedro Herrera, Benoˆıt Gauthier, Dominique Belin, Walter Reith, Thierry Pedrazzini et Fred Vom Saal pour leurs discussions scientifiques pertinentes.

Je souhaiterais remercier le Professeur Stylianos Antonorakis, pour ses discussions enrichissantes.

Son ´education scientifique, au sein de son d´epartement, m’a beaucoup stimul´e durant ma forma- tion.

Tous mes remerciements au soutien financier du Programme National de Recherche 50 sur les perturbateurs endocriniens; de la Fondation Gertrude von Meissner; de la Sir Jules Thorn Charitable Overseas Trust Reg., Schaan; de la Fondation Ernst & Lucie Schmidheiny et de la Fondation Endocrinologie.

Je suis reconnaissant envers les coll`egues du CMU et du NCCR sans qui ce travail de th`ese n’aurait pas pu aboutir, Fran¸coise K¨uhne, Laurence Tropia, Marilena Papaioannou, Jean-Luc Pitetti, Yannick Romero, Rime Madani, Rime Abla, C´eline Zimmermann ainsi qu’`a tous les membres des laboratoires des Professeurs Vassalli, Herrera, Meda et Foti.

A ma famille, m`ere, p`ere et soeur; ma belle-famille et Pyrrha; Doyen, Larry et Hiapo; pour avoir toujours ´et´e pr´esents et encourageants; et finalement, `a celle‘s que j’aime plus que tout au monde: ma femme, Manon, et ma fille, Syrane, `a qui je d´edie cette th`ese.

CONTENTS 2

Contents

1 Summary 4

2 R´esum´e 6

3 Abbreviations 8

I

Introduction 10

4 Environmental Endocrine Disruptors 10

4.1 Definition . . . 10

4.2 Effects at low doses - the BPA example . . . 11

4.3 Additive and synergistic effects . . . 12

4.4 Epigenetic alterations and transgenerational effects . . . 12

4.5 Xenoestrogens and Estrogen Receptors . . . 13

5 Male Sexual Development and Diethylstilbestrol 15 5.1 Human male reproductive disorders . . . 15

5.2 Mammalian sexual differentiation . . . 15

5.2.1 Choosing between the male or female pathway: a balance between antago- nistic signals . . . 15

5.3 Male sexual development . . . 17

5.3.1 Testicular descent occurs in two hormonally-controlled phases . . . 17

5.3.2 Insulin like-3, an essential hormone for gonadal positioning . . . 17

5.4 Xeno-estrogens and cryptorchidism . . . 19

6 Soy-Derived Phytoestrogens and Metabolism 20 6.1 Metabolic diseases and therapeutic alternatives . . . 20

6.2 Soybean composition . . . 21

6.3 Absorption and metabolism of isoflavones . . . 21

6.4 Soy consumption and phytoestrogen levels . . . 22

6.5 Phytoestrogens: complex hormetic compounds . . . 23

6.6 Role of estrogens in metabolism . . . 23

6.7 Effects of soy protein and phytoestrogens on human metabolism . . . 25

6.8 Actions of soy on metabolism in rodents . . . 26

7 Objectives 28 II

Results 29

8.2 Study 1b: Potential Detrimental Effects of a Phytoestrogen-Rich Diet on Male Fertility . . 45

9 Study 2 61 9.1 Study 2a: A Phytoestrogen-Rich Diet Increases Energy Expenditure and Decreases Adi- posity in Mice . . . 61

9.2 Study 2b: Dietary Phytoestrogens Activate AMP-Activated Protein Kinase With Improve- ment in Lipid and Glucose Metabolism. . . 69

CONTENTS 3 9.3 Study 2c: Fetal Hormonal Environment Determines Adult Glucose Homeostasis in Mice . 81

9.4 Study 2d: Health Improvements in Diabetic Mice Fed With a Phytoestrogen-Rich Diet . . 91

III

Discussion 101

10.2 Genomic or non-genomic actions of Endocrine Disruptors? . . . 101

10.3 Endocrine Disruptors and the Testis Dysgenesis Syndrome . . . 102

10.4 Beneficial effects of phytoestrogens on metabolism . . . 103

10.5 Soy proteins versus phytoestrogens . . . 104

10.6 Central actions of phytoestrogens? . . . 104

10.7 Phytoestrogens’ effects on adipose and glucose metabolism are time-dissociated . . 105

10.8 Natural molecules for the prevention of metabolic diseases and aging? . . . 106

10.9 Potentially adverse effects in consuming soy and soy-derived phytoestrogens . . . . 106

IV

Conclusions and Perspectives 108

References 109

Appendix 124

11.2 Pancreatic Insulin Content Regulation by the Estrogen Receptor ERα. . . 143

11.3 Genetic Programs that Regulate Testicular and Ovarian Development . . . 155

11.4 Of Epigenetics and Development . . . 163

11.5 Diethylstilbestrol Action on Leydig Cell Function and Testicular Descent . . . 168

11.6 Soy, Phytoestrogens and Metabolism: a Review . . . 174

1 SUMMARY 4

1 Summary

Endocrine disruptors (EDs) are man-made or environmental compounds that have potential ad- verse effects on endocrine functions including male reproductive functions and metabolism. Fetal life is a sensitive period during which the embryo is vulnerable to exogenous compounds, in partic- ular those with estrogenic activity (xenoestrogens). Indeed, gestational exposure to xenoestrogens disrupts male sexual development and leads to reproductive disorders and cancer in adulthood, in addition to its recently-discovered potential for inducing obesity. The exact mechanisms by which EDs act on the developing fetus, however, remain to be elucidated.

This thesis aimed to unravel these mechanisms through the use of two EDs that share es- trogenic properties: (i) diethylstilbestrol (DES), a potent synthetic estrogen that was given to pregnant women in the 1950’s; and (ii) phytoestrogens, natural plant-derived endocrine disruptors of weaker activity mostly abundant in soy-derived nutritional products.

In male humans and rodents, gestational exposure to DES is associated with failure of the testis to descend into the scrotum (cryptorchidism) and feminization of the urogenital tract. Our group has previously shown that in mice,in utero exposure to the natural estrogen (E2) or DES induces cryptorchidism together with a severe downregulation of Insulin like-3 (Insl3) transcripts.

Insl3 is an essential hormone for testicular descent and is secreted by the Leydig cells. Here, we tested the hypothesis that E2- or DES-induced cryptorchidism is mediated by estrogen recep- tors. Using genetic tools, we demonstrated that the estrogen receptor (ER)α is required for the disruptive effects of DES on the endocrine functions of fetal Leydig cells. Male embryos lacking ERα, but not those lacking ERβ, have normal testicular descent andInsl3 transcription remains unaffected despite E2- or DES-exposure (Study 1a).

In contrast with DES, dietary phytoestrogens (at doses relevant to human exposure) did not induce cryptorchidism. In addition, we show that dietary phytoestrogens have negligible effects on the transcriptome of fetal testes and slightly affect the fertility of male mice exposed to phy- toestrogens throughout life. We found that phytoestrogen exposure decreases fertility and sperm abundance but does not affect semen quality or erectile ability (Study 1b). Our findings suggest a weak but significant adverse effect of phytoestrogens on male reproductive functions.

While studying the effects of soy-derived phytoestrogens on male fertility, we observed that the epidydimal adipose tissue was markedly reduced in size and weight, suggesting that phy- toestrogens could modulate the energy balance. We thus decided to initiate a project aimed at understanding how dietary phytoestrogens modulate lipid and glucose metabolism. We found that, despite an increased food intake, mice exposed to dietary phytoestrogens are leaner, prob- ably because of a central effect of dietary phytoestrogens leading to increased locomotor activity coupled with an increased energy expenditure and lipid oxidation (Study 2a). In peripheral tis- sues of these mice, key energy sensors such as the AMP-activated protein kinase (AMPK) are activated and the transcription of peroxisomal oxidation genes and mitochondrial biogenesis is upregulated, strongly suggesting that dietary phytoestrogens stimulate fatty acid oxidation. In addition, we found that dietary phytoestrogens increase glucose tolerance and insulin sensitivity.

These improvements are probably the result of an improvement in the insulin pathway in skeletal muscles, which increases glucose uptake in response to insulin (Study 2b). Overall, lifelong expo- sure to dietary phytoestrogens improves lipid and glucose metabolism.

We then determined the period of life (fetal, postnatal or adult) during which such pheno- types are acquired. A reduction in adiposity is only progressively observed with an exposure to phytoestrogens occurring after birth whereas, in contrast, the improvements in glucose tolerance

1 SUMMARY 5 derive only from in utero exposure. These observations suggest that small changes in endocrine signals during fetal life may affect adult metabolic features. To test this hypothesis, we used a model known as the intrauterine position (IUP) model, according to which small variations of steroid levels in a fetus influenced by the sex of its neighboring siblings can lead to changes in adult phenotypes such as behavior, sexual performance and agressiveness. We found that IUP, in the presence and absence of phytoestrogens, influences adult glucose homeostasis, blood pressure and bone mass density (Study 2c). These results strongly indicate that some adult metabolic features are determined during fetal life through the combination of endogenous and exogenous endocrine cues.

In addition to the metabolic improvements observed in healthy mice, we found that dietary phytoestrogens are also beneficial in the context of type 2 diabetes (T2D). The development of T2D, in a genetic mouse model of diabetes (db/db), is delayed upon exposure to dietary phy- toestrogens during adulthood. Within one week of treatment, clinical symptoms of T2D such as polyurea and polydipsia are markedly reduced. Dietary phytoestrogens rapidly decrease hy- perglycemia and increase circulating insulin levels, in part, by increasing β-cell mass, ultimately prolonging lifespan of diabetic mice (Study 2d). These results suggest that dietary phytoestrogens could be used to prevent the development of T2D by reducing hyperglycemia and maintaining β-cell function.

The present thesis presents various contributions in the field of endocrine disruption. First, we show that DES inhibits the endocrine functions of fetal Leydig cells (i.e. testicular descent) through an ERα-dependent mechanism. Second, it provides strong evidence that dietary phy- toestrogens improve lipid and glucose metabolism in healthy mice and decrease the severity of the symptoms in the context of T2D. Most importantly, some metabolic parameters such as glu- cose tolerance, blood pressure and bone mass density appear to be hormonally pre-determined (programmed) during fetal life. This work underlines the importance of ED exposure during the gestational period, during which the mother’s lifestyle and environment could affect some adult reproductive and metabolic features of her progeny.

2 R ´ESUM ´E 6

2 R´ esum´ e

Les perturbateurs endocriniens sont des compos´es pr´esents dans l’environnement, qui ont le poten- tiel d’induire des effets ind´esirables sur des fonctions endocrines essentielles comme la reproduction et le m´etabolisme. Une p´eriode sensible durant laquelle un individu est vuln´erable aux perturba- teurs endocriniens, en particulier ceux qui poss`edent une activit´e œstrog´enique (x´enoestrog`enes), est la p´eriode embryonnaire et fœtale. En effet, l’exposition gestationnelle aux x´enoestrog`enes m`ene `a des probl`emes reproducteurs ainsi qu’`a l’ob´esit´e chez l’adulte. N´eanmoins, les m´ecanismes mol´eculaires qui induisent ces effets restent `a ˆetre identifi´es.

Le but de cette th`ese consiste `a identifier ces m´ecanismes en utilisant deux perturbateurs endocriniens de type x´enoestrog`ene : (i) le diethylstilbestrol (DES), un puissant œstrog`ene synth´etique prescrit `a des femmes enceintes durant les annes 50 et (ii) les phytoestrog`enes, un perturbateur endocrinien naturel avec une activit´e œstrog´enique plus faible, pr´esent en grande quantit´e dans les produits alimentaires d´eriv´es du soja.

Chez les mˆales, humains ou rongeurs, l’exposition au DES durant la gestation est associ´ee

`a la cryptorchidie (maldescente testiculaire). Notre groupe a d´ej`a montr´e que chez la souris, l’expositionin utero `a l’œstrog`ene naturel (E2) ou au DES induit une cryptorchidie, caus´ee par l’inhibition de la transcription du g`ene Insulin like-3 (Insl3) dans le testicule du fœtus. Insl3 est une hormone essentielle secr´et´ee par les cellules de Leydig et r´egulant la descente testicu- laire durant le d´eveloppement embryonnaire. Dans le cadre de cette th`ese, nous avons ´etudi´e les m´ecanismes mol´eculaires qui r´egulent les actions du DES sur l’inhibition de la descente tes- ticulaire. En utilisant la g´enomique fonctionnelle, nous avons d´emontr´e que le r´ecepteur aux œstrog`enes α (ERα), est essentiel pour permettre au DES d’exercer ses effets perturbateurs sur la fonction endocrine des cellules de Leydig fœtales. Des embryons avec une invalidation g´enique d’ERα ont une descente testiculaire normale et la transcription d’Insl3 n’est pas alt´er´ee, malgr´e le traitement au DES (Study 1a).

En revanche, les phytoestrog`enes alimentaires (`a des doses ´equivalentes aux doses auxquelles les humains sont expos´es) n’induisent pas de cryptorchidie chez la souris. De plus, les phytoe- strog`enes ont des effets n´egligeables sur le transcriptome du testicule fœtal et une exposition continue aux phytoestrog`enes affecte sensiblement la fertilit´e mˆale. En effet, nous avons observ´e chez l’adulte une l´eg`ere r´eduction de la taille des port´ees et de l’abondance de sperme. La qualit´e du liquide s´eminal et la capacit´e ´erectile, quant `a elles, ne sont pas affect´ees (Study 1b). Ces r´esultats sugg`erent que les phytoestrog`enes ont des effets faibles, n´eanmoins significatifs, sur la reproduction mˆale.

Au cours de cette ´etude, nous avons observ´e une forte r´eduction de la taille des tissus adipeux chez ces souris, sugg´erant que les phytoestrog`enes peuvent influencer la balance ´energ´etique. Nous avons donc initi´e un projet sur l’´etude des effets des phytoestrog`enes sur le m´etabolisme des lipides et du glucose. Malgr´e une augmentation de la prise alimentaire, la maigreur des souris expos´ees aux phytoestrog`enes est probablement due `a un effet sur le syst`eme nerveux central menant `a une augmentation de l’activit´e locomotrice et de la d´epense ´energ´etique (Study 2a). Dans les tissus p´eriph´eriques de ces souris, des mol´ecules sensibles aux variations ´energ´etiques telles que l’AMP- activated protein kinase (AMPK) sont activ´ees et des g`enes r´egulant l’oxydation peroxisomale et la biogen`ese mitochondriale sont sur-r´egul´es. Ces r´esultats indiquent que les phytoestrog`enes stimulent l’oxydation des acides gras. De plus, nous avons observ´e que les phytoestrog`enes aug- mentent la tol´erance au glucose et la sensibilit´e `a l’insuline. Ces am´eliorations sont probablement li´ees `a une stimulation de la voie de signalisation de l’insuline dans le muscle squelettique, ce qui augmente l’import de glucose en r´eponse `a l’insuline (Study 2b). En bref, l’exposition durant toute

2 R ´ESUM ´E 7 la vie `a une di`ete enrichie en phytoestrog`enes am´eliore le m´etabolisme lipidique et glucidique chez la souris.

Nous avons ensuite d´etermin´e si ces effets b´en´efiques sont la r´esultante d’une exposition fœtale, post-natale ou adulte. La r´eduction de l’adiposit´e n’est obtenue qu’avec une exposition apr`es la naissance, alors que les am´eliorations de la tol´erance au glucose ne proviennent que de l’exposition durant la vie fœtale. Ces observations sugg`erent que des variations fines de signaux endocriniens (endog`enes ou exog`enes) durant la vie fœtale peuvent affecter des param`etres m´etaboliques chez l’adulte. Afin de tester cette hypoth`ese, nous nous sommes pench´es sur un ph´enom`ene connu sous le nom de la position intra-ut´erine, o`u de petites variations dans les niveaux de st´ero¨ıdes du fœtus, influenc´ees par le sexe des embryons voisins, peuvent mener `a des changements de ph´enotypes adultes tels que le comportement, la performance sexuelle et l’agressivit´e. Nous avons trouv´e que la position intra-ut´erine, avec ou sans phytoestrog`enes, influence l’hom´eostasie du glucose, la ten- sion sanguine et la densit´e osseuse chez l’adulte (Study 2c). Ces r´esultats indiquent que certains param`etres m´etaboliques adultes sont d´etermin´es durant la vie fœtale, par une combinaison de signaux endocriniens environnementaux et endog`enes.

En plus des effets b´en´efiques observ´es chez des animaux sains, les phytoestrog`enes alimen- taires se sont av´er´es ´egalement b´en´efiques dans le contexte pathologique du diab`ete de type 2.

En effet, le d´eveloppement du diab`ete est retard´e chez un mod`ele de souris diab´etiques (db/db) expos´ees aux phytoestrog`enes alimentaires durant la vie adulte. En une semaine, les symptˆomes cliniques du diab`ete de type 2, tels que la polyurie et la polydipsie, sont fortement r´eduits. Les phytoestrog`enes diminuent rapidement l’hyperglyc´emie et augmentent les niveaux d’insuline cir- culante, en partie grˆace `a la pr´eservation et `a l’augmentation de la masse de cellulesβ, menant `a une augmentation de la survie des souris diab´etiques (Study 2d). Ces r´esultats indiquent que les phytoestrog`enes alimentaires peuvent avoir un potentiel pr´eventif dans le contexte du diab`ete de type 2 en diminuant l’hyperglyc´emie et en am´eliorant la fonction et le maintien des cellulesβ.

En r´esum´e, cette th`ese pr´esente plusieurs contributions dans le domaine des perturbateurs endocriniens. Premi`erement, elle montre que le DES inhibe les fonctions endocrines des cel- lules de Leydig fœtales, dont la descente testiculaire, par un m´ecanisme d´ependant d’ERα.

Deuxi`emement, elle d´emontre des effets b´en´efiques des phytoestrog`enes alimentaires dans un con- texte m´etabolique sain ou pathologique, comme le diab`ete de type 2. De plus, certains param`etres m´etaboliques comme la tol´erance au glucose, la tension sanguine et la densit´e osseuse semblent ˆetre pr´ed´etermin´ees (ou programm´ees) durant la vie fœtale par des signaux endocriniens endog`enes et exog`enes. Ce travail souligne l’importance de l’exposition aux perturbateurs endocriniens durant la p´eriode fœtale, indiquant que le mode de vie de la m`ere durant sa grossesse, ainsi que son environnement, pourraient affecter des aspects reproducteurs et m´etaboliques de sa descendance.

3 ABBREVIATIONS 8

3 Abbreviations Abbreviations

4-MBC 4-methylbenzylidene camphor

AGRP Agouti Related Protein

AMPK AMP-activated Protein Kinase

ArKO Aromatase knock-out

ARKO Androgen Receptor knock-out

BMI Body Mass Index

BPA Bisphenol-A

CHD Coronary Heart Disease

CNS Central Nervous System

CSL Cranial Suspensory Ligament

CVD Cardiovascular Diseases

CYP17A1 Cytochrome p450 17a-hydroxylase/17-20-lyase

DBD DNA Binding Domain

DBP Di(n-butyl)phthalate

DDT Dichloro-Diphenyl-Trichloroethane

DES Diethylstilbestrol

ED Endocrine Disruptor

EGCG Epigallocatechin-3-Gallate

ERα Estrogen receptor alpha

ERβ Estrogen receptor beta

ERE Estrogen Response Element

ERK Extracellular signal-Regulated Kinase FDA Food and Drug Administration FGF9 Fibroblast Growth Factor 9

GLUT4 Glucose transporter 4

HDL High-Density Lipoprotein

HP High Phytoestrogen

HPch HP chronic

HPG Hypothalamo-Pituitary-Gonadal HGP Hepatic Glucose Production

HPin HP in utero

HPpn HP post natal

IGF-1R Insulin like Growth Factor-1 Receptor

IR Insulin Receptor

IRS-2 Insulin Receptor Substrate-2

INSL3 Insulin-like 3

IUP Intrauterine Position

LBD Ligand Binding Domain

Ley I-L Leydig Insulin-like Hormone

LDL Low-Density Lipoprotein

LhRKO LH-receptor knock-out

LP Low Phytoestrogen

3 ABBREVIATIONS 9

Abbreviations

MAPK Mitogen Activated Protein Kinase

MCH Melanin Concentrating Hormone

MIN Mouse Insulinoma Cells

MIS M¨ullerian inhibiting substance mTOR mammalian Target of Rapamycin

MS Metabolic Syndrome

NPY Neuropeptide Y

NOD Non-Obese Diabetic

OC Octocrylene

OVX Ovariectomized

PCB Polychlorinated Biphenyls

PGC Primordial Germ Cell

PGC1-α PPAR gamma Co-Activator 1 alpha

PGD2 Prostaglandin D2

PDK-1 Pyruvate Dehydrogenase Kinase 1

PI3K Phosphoinositide-3 Kinase

PPARα Peroxisome Proliferator-Activated Receptor alpha qRT-PCR Quantitative real-time PCR

RIN Rat Insulinoma Cells

RLF Relaxin-Like Factor

RXFP2 Relaxin Family Peptide 2

SERM Selective Estrogen Receptor Modulator SFRE Steroidogenic factor 1 response element

SOX9 SRY-box 9

SPI Soy Protein isolate

SirT1 Sirtuin 1

SRY Sex determining Region Y

STAR Steroidogenic Acute Regulatory protein

T2D Type 2 Diabetes

TC Total Cholesterol

TDS Testis Dysgenesis Syndrome

TG Triglyceride

WAT White Adipose Tissue

WNT4 Wingless Type 4

10

Part I

Introduction

4 Environmental Endocrine Disruptors

4.1 Definition

Before any effects on humans were recorded, the concept of “endocrine disruption” was already known from reports that have taught us that synthetic chemicals or other compounds introduced into the environment by human activity (environmental contaminants) may disrupt the endocrine system of animals. Indeed, any chemical with the ability to interfere with, mimic or antagonize the function and/or the production of hormones may cause adverse effects by disrupting organ function and subsequent development. Such molecular compounds are calledendocrine disrup- tors (EDs) and are defined by the European Commission as “exogenous substances that alter function(s) of the endocrine system and, as a consequence, cause adverse health effects in an intact organism”. These man-made chemicals, as well as a few natural ones, include persistent, bioaccumulative, organohalogen compounds (including pesticides such as fungicides, herbicides and insecticides) and industrial chemicals, other synthetic products and some metals (for a non- exaustive list please see page 11). Endocrine disruptors can mimic the effect of natural hormones (agonists), interfere with the binding of hormones to their receptor (antagonists), block the synthe- sis of hormones or directly interact with hormones. Because of the important role that hormones play during development, the consequences of such disruption can be severe. These effects may include reproductive, endocrine, metabolic, immunological, neurological, behavioral and neoplas- tic changes. As observed in wildlife, the impacts are widespread and include thyroid dysfunction, decreased fertility, gross birth abnormalities, metabolic abnormalities, behavioral abnormalities, compromised immune system, demasculinization and feminization of males, defeminization and masculinization of females, in birds, fish and mammals. In the light of these observations, scien- tists have agreed on several points: i) these compounds have different effects on the embryo, fetus or perinatal organism than on the adult; ii) the period of exposure is crucial in determining the outcomes; iii) the manifestations may not occur until maturity.

The fact that humans can be sensitive to endocrine changes during fetal life has emerged from the polemics related to the tragic experience suffered by the children of mothers treated with DES during their pregnancy to avoid abortion or other pregnancy-related complications. Mil- lions of pregnant women were prescribed this drug before it was realized later on that both male and female offspring developed cancer later in life (Herbst et al., 1971; Barnes et al., 1980). A better understanding of how exogenous estrogens, hereafter named xenoestrogens, interfere with fetal development has been achieved through animal studies involving rodents which have shown that perinatal exposure to DES leads, in adulthood, to enlarged prostate (vom Saal et al., 1997), disrupted testicular function with incomplete phenotypic sexual maturation (Guyot et al., 2004) and, recently, obesity (Newbold et al., 2007). One important issue involved here, apart from the occurrence of birth defects, is the possible long-term effect, which in humans may not manifest until adolescence or even much later in life. Indeed, during embryonic development, steroid hor- mones coordinate cell differentiation, growth, organogenesis and metabolism through the control of gene expression. Adding a compound with estrogenic activities [foreign (exogenous) or natural (endogenous)], can irreversibly disrupt these embryonic processes by blocking or overstimulating pathways during key developmental periods. As a consequence, minute changes in natural hor- monal levels during fetal life may have severe consequences later in life.

Xenoestrogens are one particular class of EDs characterized by their estrogenic activity. These estrogenic compounds are part of our daily environment since they are found in numerous man-

4 ENVIRONMENTAL ENDOCRINE DISRUPTORS 11 made or natural products such as pesticides [e.g. Dichloro-Diphenyl-Trichloroethane (DDT) derivatives, methoxychlor, kepone], in products associated with plastics (e.g. BPA, nonylphe- nols), in pharmaceutical agents (e.g. DES, tamoxifen, raloxifen), in ordinary household products (breakdown products of detergents and surfactants), in industrial chemicals such as polychlo- rinated biphenyls (PCBs), in UV filters [e.g. 4-methylbenzylidene camphor (4-MBC) and oc- tocrylene (OC)] as well as in food (soy-derived phytoestrogens such as genistein, daidzein). The estrogenic activity of plastic stabilizers was discovered when researchers from Tufts University Medical School (Ana Soto and Carlos Sonnenschein) found that control breast cancer cells were proliferating just as much as estrogen-treated cells. They later found that the source of estrogen contamination was the tubes that contained their serum. They finally identified p-nonylphenol, which is used to stabilize polystyrene plastics such as in cans containing beverages or food. Since this finding, the concept of xenoestrogens affecting sexual development has been applied to many other compounds such as the infamous BPA, Di(n-butyl)phthalates (DBP), and ethinylestradiol found in oral contraceptives (Markey et al., 2001; Palanza et al., 2002; Timms et al., 2005; Mahood et al., 2006).

4.2 Effects at low doses - the BPA example

Environmental toxicology, which pursues the causes of death, cancer and genetic damage, differs from endocrine disruption which focuses on the roles that environmental chemicals may play in disrupting endocrine functions in living animals: in other words, at doses relevant to endocrine changes, not toxic changes where death occurs. Seen from this perspective of endocrine disruption, the findings by vom Saal’s group that fetal exposure to environmentally relevant parts-per-billion (ppb) doses of BPA affects the adult reproductive system, as does DES or estradiol, are striking (Nagel et al., 1997; vom Saal et al., 1997). These doses are, for instance, six times lower than what a patient might swallow during the application of a plastic dental sealant (that contains BPA). The dose-response curve follows an inverted-U shape, showing that at very low levels of BPA the effects are stronger that at intermediate doses. Vom Saal’s studies have shown that in rodents exposure to ppb levels of BPA during fetal development increases the prostate weight (Nagel et al., 1997), diminishes the weight of the epidydimis (vom Saal et al., 1998) and reduces daily sperm production (Safe, 2000). Indeed, hormones usually do not follow the classic linear type of dose response, but rather what is considered “non-monotonic”. Such responses can be U-shaped, with the greatest effects at low and saturating doses, U-inverted, with the greatest responses at intermediate doses, and biphasic, where the complexity is puzzling.

These results have attracted the immediate attention of the chemical industry, which started studying endocrine disruption. Because of BPA’s wide usage in the manufacturing of polycar- bonated plastics, polystyrene and epoxy resins commonly present in food and beverage cans as well as in dental sealants (Brotons et al., 1995; Sharman et al., 1995; Olea et al., 1996; Biles et al., 1999), the conflicting results obtained by the industry and the academics have generated significant controversy (Gross, 2007; Vandenberg et al., 2009). In contrast with DES, which was banned after the publication of the study by Herbst and colleagues (Herbst et al., 1971), the exposure of the human population to endocrine disruptors such as BPA remains important since its usage is widespread in consumer products. For instance, BPA and it’s metabolites have been measured in the blood of healthy men (1.49 ± 0.11 ng/ml) and women (0.64 ± 0.11 ng/ml), in food (4-23 µg/can) and in drinks (7-58 µg/g), and in the saliva just after the application of certain dental sealants (90-913 µg/saliva in one hour). The levels are such that no studies in humans are feasible - BPA is found in the urine of 95% of individuals (Calafat et al., 2005). The concerns of the effects of BPA are such that the Canadian Government has decided on the 17th of October 2008 to set a regulation to prohibit the imports, sales and advertizing of baby bottles in BPA-containing polycarbonates. Importantly, if BPA can act at ppb doses, other EDs may do so as well.

4 ENVIRONMENTAL ENDOCRINE DISRUPTORS 12

4.3 Additive and synergistic effects

Of major relevance is the question of whether different endocrine disruptors may act in an ad- ditive or synergistic manner which may drastically increase their adverse effects. It is known in toxicology that simultaneous exposure to environmental compounds does not have the same consequences as when each one acts alone (e.g. antagonism, additivity, etc ). These so-called

“cascading impacts” result from the interactive effect of multiple (two and more) compounds.

For instance, in amphibians it has been shown that synergism occuring between trematode infec- tion and exposure to chemical contaminants significantly increases limb deformities (Kiesecker, 2002). Alhough this study was limited to two chemicals or compounds, it is possible to envisage the contribution of three or more factors that individually have no effects but which taken to- gether may have unpredictable consequences. In support of this idea, a recent study has reported that pesticide mixtures (nine chemicals) have greater effects on the inhibition of amphibian larval growth and development (likely due to increase in stress hormones) in comparison with individual pesticides whose effects are relatively weak (Hayes et al., 2006). Despite the absence of evidence in the first study that these effects act through the endocrine system, both works are important because they raise an issue that is not necessarily brought up by the problem of antagonism or additivity, namely the emergence of a disrupting effect presented by individually non-acting chemicals in living animals.

4.4 Epigenetic alterations and transgenerational effects

Gene transcription can be also modulated by epigenetic mechanisms, which include in a large mea- sure methylation changes or post-translational modification of histones. Both epigenetic mecha- nisms may act independently or in concert to modify chromatin compaction and, in consequence, gene expression (Sadoni et al., 1999). As mentioned above, hormones during fetal life determine the transcription rate of genes to coordinate cell differentiation, growth and organogenesis during embryonic development. Since in utero exposure to endocrine disruptors correlates with adult disease formation, it is thought that epigenetic modifications are involved in this process in some manner, by permanently altering the methylation patterns on some genes during fetal life. Indeed, fetal exposure to endocrine disruptors such as BPA and genistein has been found to modulate epigenetic marks with phenotypic effects in adulthood (Dolinoy et al., 2006; Dolinoy et al., 2007).

Schematically, gene transcription can be repressed by cytosine methylation in CpG sequences (deoxycytidine - phosphate - deoxyguanosine). CpG motifs are statistically under-represented in the human genome, except in small areas named “CpG islands” located in promoters or in the first exon of more than 60% of human genes. (Bird, 2002). The more methylated the CpG island of a given regulatory region is, the weaker its level of expression will be. The various methylation profiles of CpG islands found in the different cell types are acquired during cell differentiation.

Thus, totipotent embryonic cells from a blastocyst are less methylated, whereas in CpG islands from somatic cells, methylation is elevated and correlates with the cell-specific gene expression level acquired during cell differentiation (Jaenisch, 1997). Once differentiated, the state of CpG islands methylation is transmitted during cell division, which enables the hereditary transmission to the daughter cell of certain regions of the genome in a hypermethylated state (Bird, 2002).

Different processes such as the recruitment of methyl-DNA binding proteins and histone deacety- lases, favor chromatin compaction of hypermethylated genes (Bird and Wolffe, 1999). On the other hand, transcriptionally active genes remain or become hypomethylated. Thus, changes in the methylation degree of cytosines from a CpG island, and subsequent modulation of chromatin compaction, represent a crucial epigenetic regulation which irreversibly determines cell fates dur- ing development (Wolffe and Matzke, 1999).

Some studies suggest that epigenetic modifications that permanently alter gene expression may

4 ENVIRONMENTAL ENDOCRINE DISRUPTORS 13 be transmitted through the generations (transgenerationally). It has been demonstrated through an epidemiological study that in humans the risk of developing cardiovascular complications or type 2 diabetes (T2D) is determined by the dietary trends of parents and grandparents during their teenage years (Kaati et al., 2006); nutrient excess of a grandfather just before puberty will transmit to his grandchildren a quadruple risk of developing T2D. In rodents, an example of transgenerational effects from the environment has been brought to light by a recent study in which gestating rats were exposed to vinclozolin, a fungicide introduced in the late seventies and used in viticulture, fruit culture and colza (Tomlin, 1997). In addition to its anti-fungal actions, vinclozolin possesses an anti-androgenic activity and male rats exposedin utero (F1 generation) display impaired testicular function with a severe reduction in sperm number and motility (Anway et al., 2005). More intriguingly, it was shown than this sub-fertility is transmitted through at least four generations [from F1 to F4, (Anway et al., 2005)]. This transgenerational effect of vinclozolin on testicular function is correlated with alterations in methylation states of genomic DNA, suggesting that these epigenetic modifications may be the cause of reduced fertility to be observed in future generations. These experiences underline the risks following fetal exposure to environmental EDs and strongly indicate the need for the addition of transgenerational studies to all studies evaluating the toxicological risks linked with EDs.

4.5 Xenoestrogens and Estrogen Receptors

As described above, the modus operandi of EDs is far-ranging and complex. In part, many EDs are ligands of steroid/thyroid/retinoid receptors (or nuclear receptors). Many of the studies on environmental endocrine disruptors have focused on chemicals with estrogenic activities, owing to the common estrogenic read-outs of in vitro and in vivo systems. In vitro assays rely on the proliferation of breast cancer cells (such as MCF-7), and in vivo studies are based on the measurement of uterotrophic activity (proliferation of the uterine epithelium in castrated female mice). Dr. Roy Hertz said “Notwithstanding this complex array of variability associated effects of estrogens, the sine qua non of estrogenic activity remains the mitotic stimulation of the tissues of the female urogenital tract. A substance which can elicit this response is an estrogen; one that cannot do this is not an estrogen” (Hertz, 1985).

In the case of EDs with estrogenic activity (xenoestrogens), their relation with estrogen recep- tors (ERs) appears self-evident. Similarly to the natural estrogens, xenoestrogens are thought to mediate their activity through estrogen receptorsα (ERα) and beta (ERβ). Chemicals that do not possess the phenanthrene nucleus (such as BPA and DES), the ring common to steroids, are still capable of estrogenicity. Even metals have been reported to interact with the ligand-binding domain of the ER and mimic the effects of estradiol in estrogen-responsive breast cancer cells in terms of proliferation and gene transcription (Garcia-Morales et al., 1994; Stoica et al., 2000).

Although the implication of ERs in the disruptive effects of xenoestrogens may appear obvious, very few studies have confirmed that ERs are essential in mediating these effects.

ERs are members of the nuclear receptor family regulating gene transcription. These receptors are present in many organs: for instance, ERα is present in the kidney, adrenal gland and liver, whereas ERβ is expressed in the lung, brain and heart (Kuiper et al., 1997). Both receptors are co-expressed in the reproductive organs such as the epididymis, the testis, the ovaries and the uterus as well as in metabolic organs such as the adipose tissue (Simpson et al., 1994), the skeletal muscle (Barros et al., 2006) and theβ-cell (Alonso-Magdalena et al., 2008). Thus, since the nuclear context will also influence the activity of these receptors, one cannot predict the effect of an ED in a given cell type. These receptors have a transactivation function sequence (AF1) found in the A/B N-terminal domain, the least conserved domain between both isoforms (28% homology), that modulates its transcriptional activity depending on the coactivators or corepressors bound to it.

The DNA binding domain (DBD) in the C domain recognizes estrogen response elements (EREs)

4 ENVIRONMENTAL ENDOCRINE DISRUPTORS 14 on promoters, as well as the steroidogenic factor-1 response elements (SFREs) which are ERE half sites (Vanacker et al., 1999). Finally, the E/F domain in the C-terminal part harbours the ligand- binding domain (LBD) that allows the binding of agonists or antagonists (Klinge, 2000). Upon ligand binding, the LBD suffers conformational changes that enable the recruitment of different co- factors, either co-ativators or repressors, which modulate DNA binding affinity and transcriptional responses (Dayan et al., 2006). Thus, environmental contaminants will elicit different cellular responses because of the varying structural conformations of the ER after binding to the LBD.

Both ERs have different regulatory properties despite their similar estrogen affinity and negative cross-regulatory loops modulate their transcription (Lindberg et al., 2003; Li et al., 2004). In spite of the transcriptional activity related to the nuclear property of ER, researchers have tried to explain the rapid responses (within two to five minutes) seen after estrogen stimulation, which are too fast to be transcriptional, by characterizing the so-called non-genomic pathways. Indeed, there is increasing evidence that ER are localized in the plasma membrane of estrogen target cells (Watson et al., 1995) and coupled to nitric oxide synthase to induce signaling cascades (Chambliss et al., 2000), but the precise underlying molecular mechanisms by which ERs mediate rapid non-genomic signaling remain poorly understood.

5 MALE SEXUAL DEVELOPMENT AND DIETHYLSTILBESTROL 15

5 Male Sexual Development and Diethylstilbestrol

5.1 Human male reproductive disorders

Clinical and epidemiological studies published since the 1980’s have indicated a drastic increase in the incidence of male reproductive health problems. These disorders include cryptorchidism (undescended testes) (Chilvers et al., 1984; Campbell et al., 1987), hypospadias ((Matlai and Beral, 1985), declining semen quality (Irvine et al., 1996; Andersen et al., 2000) and testicular cancer (Adami et al., 1994; Moller et al., 1998), all of which are regrouped under the termTes- ticular Dysgenesis Syndrome (TDS). The rapid increase of TDS over the past 50 years suggests that environmental or lifestyle factors and not genetic factors are most likely involved in these changes in male reproductive health. Furthermore, basic, clinical and epidemiological studies have come to a consensus in suggesting that the underlying causes of human male reproductive disorders operate in the fetus (for review see Norgil Damgaard et al., 2002). This seems logical since testicular descent, closure of the urethra along the shaft of the penis (the failure of which causes hypospadias), testicular development and differentiation, all occur during fetal life.

Among these, Diethylstilbestrol (DES), a synthetic estrogen, stands apart since it was the first and remains the most well characterized endocrine disruptor. DES was prescribed to more than 5 million pregnant women from the late 1940s to early 1970s to prevent abortion, pre-eclampsia and other pregnancy complications (for review see Jensen et al., 1995). Daughters of women treated with the drug during pregnancy have a higher risk of developing clear cell adenocarcinoma of the vagina, while male children exposed in utero to DES have a higher risk of developing the symptoms of TDS (Herbst et al., 1971; Gill et al., 1979; Herbst et al., 1979; Wilcox et al., 1995).

Herein, I describe how male and female gonads form, how male sexual development occurs and finally how testicular descent is regulated.

5.2 Mammalian sexual differentiation

In mammals, sexual differentiation occurs in three sequential steps. Genetic sex is determined at fertilization and directs the differentiation of the gonad into a testis (XY) or an ovary (XX), thus establishing the gonadal sex of the embryo. Ultimately, the phenotypic sex is established by the secretion of hormones required for the differentiation of the rest of the embryo either as a male or a female (Nef and Parada, 2000). The embryonic testis secretes three hormones: the anti- M¨ullerian hormone (AMH), produced by fetal Sertoli cells, induces regression of the M¨ullerian ducts. Testosterone, produced by Leydig cells, allows the development of Wolffian duct deriva- tives and masculinization of the external male genitalia. Finally, insulin-like 3 (Insl3) mediates transabdominal testicular descent into the scrotum. In females, the absence of AMH allows de- velopment of the M¨ullerian structures, whereas the lack of androgens induces degeneration of the Wolffian ducts. In absence ofInsl3, ovaries do not descend towards the scrotum as in males, but instead remain intra-abdominal.

5.2.1 Choosing between the male or female pathway: a balance between antagonistic signals

The mammalian gonads are derived from the intermediate mesoderm and arise as paired thick- enings of the coelomic epithelium on the ventrolateral surface of the mesonephros. In mice, the genital ridges are visible at embryonic day 10 (E10); they are composed of somatic cells and primordial germ cells (PGCs) that have migrated from the base of the allantois via the hindgut (McLaren, 2003). At this stage, the gonad is bipotential and both somatic and germ cell lineages have the capacity to differentiate into ovarian or testicular cell types. Recent findings suggest that gonadal differentiation is controlled by mutually antagonistic signals between the SRY (sex determining region Y)-box 9 (Sox9)/Fibroblast growth factor 9 (Fgf9) and wingless type 4 (Wnt4)

5 MALE SEXUAL DEVELOPMENT AND DIETHYLSTILBESTROL 16 signalling pathways (Kim et al., 2006). At 15 tail somites [∼embryonic day 11.25 (E11.5)], both Fgf9/Sox9 andWnt4 are expressed in the bipotential gonads suggesting that the undifferentiated gonad is poised between these two antagonistic signals. The differentiation of the testis or ovary is initiated when, by regulating the expression of the testis-determining factorSox9, the balance tilts towards one or the other opposite signal (Kim et al., 2006).

Testis determination In XY gonads, Sry is transiently activated (E10.5-E12.5) in the sup- porting cell lineage during the commitment to the testis pathway (Palmer et al., 1991). The onset ofSry expression will initiate the male pathway by upregulating Sox9, which in turn will upreg- ulate Fgf9 expression and increase prostaglandin D2 (PGD2) synthesis (Malki et al., 2005; Kim et al., 2006). Both Fgf9 and PGD2 will help maintain Sox9 expression by forming a feed-forward loop and ultimately directing the differentiation of the supporting lineage into Sertoli cells. Sertoli cells, in turn, will act as organizing centers to initiate cellular and morphological changes and to direct differentiation of the other cell types within the developing testis (for review see Brennan and Capel, 2004). Between E11.5-E12.5, Sertoli cells together with peritubular myoid cells will enclose the PGCs to form the testicular cords. Sertoli cells will also block the entrance of PGCs into meiosis by expressingCyp26b, a cytochrome p450 enzyme known for its capacity to metab- olize retinoic acid, promote migration of mesonephric cells and vascularization and direct Leydig cell differentiation (Brennan et al., 2004; Koubova et al., 2006).

Early ovarian development The female somatic lineage is required to initiate and orchestrate a signalling pathway that is critical for early ovarian development. In XX gonads, Wnt4 signaling antagonizes the male pathway by interfering with the upregulation of Sox9 expression. Thus, the absence of the feed-forward loop between Sox9 and Fgf9 tilts the balance towards the female path- way (Kim et al., 2006). The signalling cascade(s) orchestrating ovarian development and crosstalk between germ and somatic cells remains so far extremely sketchy, but recent findings suggest that Wnt4 and Follistatin are required for early ovarian development. Both of them are constituents of a common signaling cascade that has both anti-testis (inhibition of coelomic vessels formation) and pro-ovary (survival of meiotic germ cells) properties (Vainio et al., 1999; Jeays-Ward et al., 2003; Yao et al., 2004). Wnt4 appears to be the upstream regulator of Follistatin as it induces its expression in female somatic cells as early as E11.5 (Yao et al., 2004). These two molecules are believed to inhibit endothelial cell migration from the mesonephros, and consequently prevent coelomic vessel formation. Loss of either one of the two genes abolishes this inhibition and enables the formation of coelomic vessels on the surface of XX gonads (Jeays-Ward et al., 2003; Yao et al., 2004). Wnt4 and Follistatin are also implicated in the modulation of the apoptotic wave that controls the number and localisation of germ cells in the future ovary by creating a protective niche in the ovarian cortex that enables survival of female germ cells (Jeays-Ward et al., 2003;

Yao et al., 2004).

Although a female-specific signaling cascade is activated as early as E11.5, no marked cellular changes are observed during early ovarian development. The first cellular event occurs at around E13.5, when germ cells enter meiotic prophase as oocytes and arrest at the diplotene stage of prophase I by birth (McLaren, 2003). In contrast to XY gonads, germ cells are crucial for the for- mation and maintenance of the ovarian structure. In their absence, follicles do not assemble and ovarian dysgenesis is observed as exemplified in germ-cell deficient mice lacking the c-Kit/Steel signalling pathway (Buehr et al., 1993). An additional modification that occurs at the cellular and structural level around E13.5, is the formation of loose cordlike structures referred to as ovigerous cords; these are composed of a cluster of PGCs surrounded by somatic cells that have a mesenchymal appearance. Within the ovigerous cords, germ cells are interconnected via cyto- plasmic bridges to form a syncytium (Pepling and Spradling, 1998), and around the time of birth, certain oocytes go through an apoptotic process, while the remaining ones become surrounded by

5 MALE SEXUAL DEVELOPMENT AND DIETHYLSTILBESTROL 17 a monolayer of flattened pre-granulosa cells to form individual primordial follicles (Pepling et al., 2001).

5.3 Male sexual development

Male sexual differentiation requires the synthesis and the secretion of three testicular hormones:

testosterone, M¨ullerian inhibiting substance (MIS) and insulin-like factor 3 (Insl3) also known as Leydig Insulin-like hormone (Ley I-L), or relaxin-like factor (RLF). MIS, secreted by Sertoli cells, promotes the regression of the M¨ullerian ducts. In males, in absence of MIS, the M¨ullerian ducts would develop and ultimately give rise to the uterus, the oviduct and the upper part of the vagina. Testosterone, synthetized by Leydig cells, directs the differentiation of male accessory organs including the vas deferens, the seminal vesicles, the second phase of testicular descent and the development of the external male genitalia. Finally, Insl3, also produced by Leydig cells, mediates the first phase of testicular descent. Interference with the production or action of these three sexual hormones by EDs during gonadal development can disrupt the sexual differentiation of the male fetus.

5.3.1 Testicular descent occurs in two hormonally-controlled phases

Testicular descent is an important aspect of male sexual development. Failure of the testes to descend into the scrotum (cryptorchidism) is one of the most common birth defects in humans affecting approximately 2-3% of newborn males. Because intraabdominal temperature is toxic for male germ cells, cryptorchidism often results in infertility and increases the risk of testicular cancer (Hutson et al., 1994).

Gonadal positioning relies on the development of two ligaments that connect the gonad to the abdominal wall: the cranial ligament known as the cranial suspensory ligament (CSL) and the caudal ligament named the gubernaculum, a mesenchymal tissue that connects the developing gonad to the inguinal abdominal wall. In males, outgrowth of the gubernaculum and regression of the CSL descends the testis transabdominaly whereas in females, development of the CSL and developmental failure of the gubernaculum positions the ovaries next to the kidneys (Figure 1).

Hormonal control of testicular descent occurs in two steps: the transabdominal phase and the inguinoscrotal phase (Hutson, 1985; Hutson et al., 1994; Satokata et al., 1995). The transab- dominal phase (from E15.5 to E17.5 in mice) relocates the testes from a high abdominal position to the base of the abdomen, close to the inguinal canal. This process is mediated by androgens and Insl3. Testosterone triggers the regression of the CSL, and Insl3 acts on the gubernaculum through the binding to its receptor LGR8 to promote its outgrowth and contraction. The second phase involves the movement of the testes from the base of the abdomen to the scrotum and is mediated by androgens (for review see Nef and Parada, 2000). In females, the absence of testos- terone and Insl3 enables the CSL to develop and maintains the ovaries next to the kidneys, while the gubernaculum remains thin and elongated.

5.3.2 Insulin like-3, an essential hormone for gonadal positioning

The evidence that Insl3 plays a role in testicular descent came from the generation of mice lacking Insl3, from our group and others, or its receptor LGR8. These mice were found to exhibit bilateral cryptorchidism, abnormal gubernacular development, spermatogenesis defects, and infertility (Nef and Parada, 1999; Zimmermann et al., 1999; Gorlov et al., 2002; Bogatcheva et al., 2007). The Insl3 gene is a member of the insulin family which includes insulin, relaxin, and insulin-like growth factors I and II. As insulin, the mature Insl3 peptide is composed of A and B-chains linked by disulfide bonds (Adham et al., 1993) and binds to its receptor LGR8, also

5 MALE SEXUAL DEVELOPMENT AND DIETHYLSTILBESTROL 18

1.jpg

Figure 1: Schematic drawing describing the first phase of testicular descent in mice. In males, the presence of testosterone induces regression of the cranial suspensory ligaments (CSL), while Insl3 promotes the contraction of gubernacular cords and outgrowth of gubernacular bulbs. These morphological changes in the genital ligaments allow the testes to relocate at the base of the abdominal cavity. In females, the absence of testosterone allows the CSL to develop and retain the ovaries at their original position, while the gubernaculum elongates and does not develop because of the absence of Insl3. Legend: adrenal (a), kidney (k), coecum (c), cranial suspensory ligament (csl), ovary (o), gonad (g), bladder (b), epidydima (e), testis (t), vas deferens (vd), gubernaculum (gb - in red). From Cederroth and Nef, 2008.

known as relaxin family peptide 2 (RXFP2), which is a member of leucine-rich repeat-containing G-protein coupled receptor family (Kumagai et al., 2002). Our group and others have shown thatInsl3 expression during sex determination is sexually dimorphic (Zimmermann et al., 1997;

see appendix 1a: Nef et al., 2005). In male mice, Insl3 transcripts are first detected at E13.5 in fetal Leydig cells. Its expression remains constant up until postnatal (P) day 3-5 where a slight decrease is observed. Postnatal transcript levels of Insl3 increase again as fetal Leydig cells are being replaced by mature Leydig cells and reach the highest levels in adult testis. Insl3 gene regulation appears to be regulated differently in fetal and adult population of Leydig cells.

In fetal Leydig cells, Insl3 gene expression occurs independently of the hypothalamo-pituitary- gonadal (HPG) axis and is inhibited by estrogenic compounds such as estradiol or DES. In mature Leydig cells on the opposite,Insl3 gene expression is under the control of the HPG axis (Balvers et al., 1998; Klonisch et al., 2004; Yuan et al., 2006) and our group has shown that it remains unaffected by estrogens (Nef et al., 2000). For example, inhpg mutant mice that lack a functional HPG axis,Insl3 expression is unaffected in fetal Leydig cells whereas it is fairly absent in mature Leydig cells, suggesting that only postnatal transcription ofInsl3 requires signals from the central

5 MALE SEXUAL DEVELOPMENT AND DIETHYLSTILBESTROL 19

2.jpg

Figure 2: Schematic drawing of testicular positioning at E18.5 under normal conditions or after in utero exposure to xenoestrogens. At this stage, the transabdominal descent is completed and the testes are located at the level of the bladder neck. and the gubernaculum is contracted and well developed. Estrogen exposure induces cryptorchidism characterized by intra-abdominal testes usually located below the kidneys and a thin elongated gubernaculums. Legend: adrenal (a), kidney (k), coecum (c), cranial suspensory ligament (csl), ovary (o), gonad (g), bladder (b), epidydima (e), testis (t), vas deferens (vd), gubernaculum (gb - in red). From Cederroth and Nef, 2008.

nervous system (CNS) (Balvers et al., 1998). Confirming this hypothesis, mice lacking the LH- receptor (LhRKO) display normal transabdominal descent but postnatal bilateral cryptorchidism with impaired inguinoscrotal descent which is rescued by testosterone treatment (Klonisch et al., 2004; Yuan et al., 2006). Thus, the expression of Insl3 in fetal Leydig cells is crucial for transabominal descent and development of the gubernaculum.

5.4 Xeno-estrogens and cryptorchidism

Insl3 and LGR8 appear to play similar roles in human sexual development, although mutations in these genes are not a common cause of cryptorchidism in humans (Baker et al., 2002). Since 90%

of cryptorchidism cases are spontaneous, it would seem that environmental factors, rather than genetic factors, are the most plausible cause. Abnormal estrogen action has been hypothesized to be a possible cause for sporadic cryptorchidism in humans. For instance, treatment of preg- nant women with DES is associated with undescended testis in male offspring (Gill et al., 1979).

A clinical study showed that mothers of cryptorchid children had higher levels of free estradiol during the first trimester as compared to mothers whose offspring had normally-descended testes (Bernstein et al., 1988). Finally, Hadziselimovic and colleagues (Hadziselimovic et al., 2000) demonstrated an increased expression of estradiol in placenta of boys with cryptorchid testes.

Animal studies support the observations in humans. Indeed, for many years the main experi- mental model for intra-abdominal cryptorchidism was obtained by exposure of pregnant mice to exogenous estrogens (Hadziselimovic and Girard, 1977; Grocock et al., 1988; Perez-Martinez et al., 1996;Figure 2.). The effects of estradiol include a reduction of gubernacular outgrowth, the induction of estrogen receptors within the Wolffian ducts, and the stabilization of the M¨ullerian ducts. Our group and others have shown that in mice,in utero exposure to estradiol or DES in- duces cryptorchidism in association with a severe downregulation ofInsl3 transcription (Emmen et al., 2000; Nef et al., 2000). Similarly, gestational exposure to phthalate esters, an endocrine dis- ruptor with weak estrogenic activities, also impairsInsl3 gene expression and the transabdominal phase of testicular descent in rat embryos (Wilson et al., 2004; Shono et al., 2005).

6 SOY-DERIVED PHYTOESTROGENS AND METABOLISM 20

6 Soy-Derived Phytoestrogens and Metabolism

Because this thesis has focused most of its attention on the metabolic effects of phytoestrogens on metabolism, this section introduces phytoestrogens with an emphasis on metabolism. The relationship between phytoestrogens and reproduction is presented inStudy 1b.

6.1 Metabolic diseases and therapeutic alternatives

Obesity and its related disorders, such as type 2 diabetes (T2D), cardiovascular diseases (CVD), high blood pressure, dyslipidemia [high levels of circulating triacylglycerols and low-density lipopro- tein (LDL) cholesterol, and low levels of high-density lipoprotein (HDL) cholesterol], have recently become a major health problem reaching pandemic proportions (Engelgau et al., 2004). These diseases, commonly referred to as the Metabolic Syndrome (MS), are beginning to surpass malnu- trition and infectious diseases as the most significant contributor to ill health globally. In western societies, for the first time in modern history, life expectancy of newborns is declining as a result of these metabolic disorders (Olshansky et al., 2005). The rapid increase in obesity suggests that life-style factors such as high-calorie diets, physical inactivity and potentially environmental en- docrine disruption, rather than genetics, are the most plausible causes.

Although the source of metabolic disorders is often the diet itself, nutrition can also form part of the solution, in fact providing health benefits. Usually dietary intervention to control excess body weight, hyperglycemia and dyslipidemia has included low energy and low fat diets, but these are of limited efficacy due to the strict and long-term commitment required. However, long term health benefits can be gained from dietary proteins and bioactive non-nutrients, called phytochemicals, which could be either incorporated into the diet or be part of the food itself.

These phytochemicals are biologically active plant-derived compounds, which structurally and functionally mimic estrogens (Dixon, 2004). Phytoestrogens are found in numerous fruits and vegetables and are categorized into three classes, namely the isoflavones, lignans and coumestans.

While phytoestrogens are ubiquitous within the plant kingdom, isoflavones are mainly found the soybean the most important dietary source of phytoestrogens for humans, cattle and rodents.

Isoflavones have a non steroidal structure but possess a phenolic ring that enables them to bind the estrogen receptor (ER) and act either as estrogen agonists or antagonists (Makela et al., 1994;

Makela et al., 1995).

The fact that isoflavones have been shown to exert estrogenic effects raises the possibility that this class of phytochemicals may affect glucose and lipid metabolism. In fact, estradiol itself is a well known modulator of both obesity and glucose homeostasis. For instance, postmenopausal women develop visceral obesity and insulin resistance and are at an increased risk of diabetes but estrogen replacement therapy normalizes these abnormalities (Ahmed-Sorour and Bailey, 1980;

Bailey and Ahmed-Sorour, 1980; Gambacciani et al., 1997). From genetic studies in rodents, it has been shown that these effects are mediated by estrogen receptors (see below). This has caused researchers to focus on the identification of Selective Estrogen Receptor Modulators (SERMs) that could be of potential therapeutic interest for the treatment of metabolic disorders, without having negative effects. Studies in humans and rodents support the hypothesis that soy proteins or soy- derived phytoestrogens may be beneficial for the prevention of obesity and diabetes (Bhathena and Velasquez, 2002; Velasquez and Bhathena, 2007).

The complex interactions between soy proteins and isoflavones are fairly well understood. To understand these intricate relationships, one must assess the biological activity of soy components, both in isolation and in combination. So far, few studies have shown that pure soy-proteins or soy protein isolates (SPI) alone (in absence of isoflavones) can provide beneficial metabolic effects (Velasquez and Bhathena, 2007). The majority of the studies using SPI remain difficult

6 SOY-DERIVED PHYTOESTROGENS AND METABOLISM 21 to interpret because of the lack of clarity concerning the presence or absence of isoflavones in the diet. On the other hand, soy-derived phytoestrogens have received more attention mainly due to their benefits in decreasing age related diseases (e.g. osteoporosis, cardiovascular disease), or hormono-dependent cancers (e.g. prostate) (Setchell, 1998; Tham et al., 1998; Sacks et al., 2006).

Concerning metabolism, the American Food and Drug Administration (FDA) authorized in 1999 the labeling of health claims on food containing soy proteins, referring to the beneficial role of soy protein in reducing the risk of coronary heart disease (CHD). Attempts to show beneficial effects on metabolism in humans have been hotly debated, but studies in rodents may help in identifying the biologically relevant soy components and the intimate mechanisms involved. The purpose of this section is to examine the evidence regarding the use of soy and phytoestrogens in the prevention of obesity and diabetes mellitus in animals and humans. We also discuss the mechanisms by which soy and dietary phytoestrogens may affect glucose and lipid metabolism and improve the control of body weight and glucose homeostasis. To provide context and the requisite background information, we begin with a brief overview about soybeans, the nutritional composition of soy. We also present scientific evidence both in humans and rodents supporting or refuting the potential beneficial effects of soy and phytoestrogens on glucose and lipid metabolism.

6.2 Soybean composition

Soybean (Glycine max) is composed of macronutriments such as lipids, carbohydrates and pro- teins. Soybean lipids, which are deprived of cholesterol, contain about 15% of saturated fat, 61%

of polyunsaturated fat, and 24% of monounsaturated fat (USDA, 1979). Carbohydrates make up about 30% of the seed, with 15% being soluble carbohydrates (sucrose, raffinose, stachyose) and 15% insoluble carbohydrates (dietary fiber). The protein content of soybean varies from 36% to 46% depending on the variety (Garcia et al., 1997; Grieshop et al., 2001; Grieshop et al., 2003). Storage proteins are predominant, such as the 7S globulin (β-conglycinin) and 11S globulin (glycinin), which represent about 80% of total protein content, as well as less abundant storage proteins such as 2S, 9S, and 15S globulins (Garcia et al., 1997). Interestingly, β-conglycinin but not glycinin is capable of improving serum lipid profiles in mice and humans, in the absence of phytoestrogens (Moriyama et al., 2004; Kohno et al., 2006).

Soybean also contains micronutrients, which include isoflavones, phytates, saponins, phytos- terols, vitamins and minerals. Although beneficial effects of micronutrients such as saponins and phytosterols on cholesterol levels and absorption have been reported (Oakenfull, 2001; Lukaczer et al., 2006), there is an increasing body of literature suggesting that isoflavones may additionally have a beneficial role in lipid and glucose metabolism. Soybeans are the most abundant source of isoflavones in food. Studies have shown that there is a large variability in isoflavone content and composition in soybeans. This is function of the variety of soy grown, as well as environmental conditions (Wang and Murphy, 1994b; Caldwell et al., 2005). Abiotic and biotic stresses such as variation in temperature, drought or nutritional status, pest attack or light conditions may modify isoflavone content and composition. As a consequence, total isoflavone content may vary up to 3-fold with growth of the same soy cultivar in different geographical areas and years (Wang and Murphy, 1994a).

6.3 Absorption and metabolism of isoflavones

The metabolism of isoflavones is rather complex. The two major isoflavones, genistein and daidzein, are present in soy asβ-D-glycosides, namely genistin and daizin (seeFigure 3). These glycoside forms are biologically inactive (Setchell, 1998). Once ingested, isoflavone glycosides are hydrolyzed by bacterialβ-glucosidases in the intestinal wall, resulting in the conversion to their corresponding bioactive aglycones (genistein and daidzein). Only the aglycone forms are absorbed by the intestinal tract and are therefore biologically active. Daidzein can be further metabolized