Thesis
Reference
Monitoring gonadal somatic cell differentiation during sex determination using single-cell RNA sequencing
STEVANT, Isabelle
Abstract
Mammalian sex determination is a particularly interesting model to study cell fate decision.
Depending on the genetic sex, the common gonadal primordium is able to differentiate as two different organs, the testis and the ovary. This PhD project aimed to dissect with single-cell RNA-sequencing the process of sex determination in mouse by characterising the transcriptomic changes occurring in the different somatic cell lineages from the bipotential state to advanced developing testes and ovaries. For this purpose, we proceeded to single-cell RNA sequencing of the gonadal somatic cells (NR5A1 expressing cells) from XX and XY mice at key stages of the gonad development. With cell lineage predictive algorithms and pseudotime ordering, we were able to reconstruct the chronology of events driving testis and ovary development, as well as sex fate decision in both supporting and steroidogenic cell lineages.
STEVANT, Isabelle. Monitoring gonadal somatic cell differentiation during sex
determination using single-cell RNA sequencing . Thèse de doctorat : Univ. Genève, 2018, no. Sc. 5200
URN : urn:nbn:ch:unige-1058409
DOI : 10.13097/archive-ouverte/unige:105840
Available at:
http://archive-ouverte.unige.ch/unige:105840
Disclaimer: layout of this document may differ from the published version.
Département de Génétique Médecine et Développement
FACULTÉ DE MÉDECINE Prof. Emmanouil T. Dermitzakis
Département de Génétique Médecine et Développement
FACULTÉ DE MÉDECINE Prof. Serge Nef
Département d’Informatique FACULTÉ DES SCIENCES
Dr. Frédérique Lisacek
Monitoring gonadal somatic cell differentiation during sex determination
using single-cell RNA sequencing
THÈSE:
présentée à la faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention bioinformatique
par Isabelle Stévant
de Nantes (France)
Thèse no5200 Genève, 2018
Mammalian sex determination is a particularly interesting model to study cell fate decision. De- pending on the genetic sex, the common gonadal primordium is able to differentiate as two different organs, the testis and the ovary. The fate of this primordium is determined by the presence or the absence of a short gene located on the Y chromosome:Sry(1,188 base pairs only). The supporting cell lineage is the first cell type to differentiate in the gonad. Its differentiation into either Sertoli (XY) or pre-granulosa (XX) cells determines the phenotypic sex of the individual. The expression ofSryin a sensitive window of time controls the differentiation of the supporting cells as Sertoli cells, while in the absence ofSry, the supporting cells differentiate as pre-granulosa cells through the activation of the WNT/β-catenin pathway. Once committed, the supporting cells of both sex control the differentiation of the other somatic cells of the gonads, including the steroidogenic cells (theca cells in XX and Leydig cells in XY) that will secrete the hormones necessary for the development of the secondary sexual characteristics.
Numerous questions regarding sex determination remain. The cell composition of the bipotential gonad is a matter of debate. The classical model states the presence of two cell lineages, the supporting cells and the steroidogenic cells, prior to sex determination. Recent studies challenged this statement toward a common progenitor cell population but did not provide clear evidence.
How and when these the cell lineages are specified remain unclear. After lineage specification, the cells operate sex determination by adopting their respective sex specific cell type. The precursors of the steroidogenic cells in both sex are still poorly characterised. While supporting cells differentiate cell autonomously as early as E11.5 in mouse, the steroidogenic cell lineage differentiation is delayed (E12.5 in XY, after birth in XX) and occurs under the influence of the committed supporting cells.
This PhD project aimed to dissect with single-cell RNA-sequencing the process of sex determination in mouse by characterising the transcriptomic changes occurring in the different somatic cell lineages from the bipotential state to advanced developing testes and ovaries.
For this purpose, we proceeded to single-cell RNA sequencing of the gonadal somatic cells (NR5A1 expressing cells) from XX and XY mice at key stages of the gonad development. With cell lineage predictive algorithms and pseudotime ordering, we were able to reconstruct the chronology of events driving testis and ovary development, as well as sex fate decision in both supporting and steroidogenic cell lineages. We detected a single cell population prior sex determination that is able to specify into either supporting and steroidogenic fate. We characterised the dynamic of expression underlying the specification and the differentiation of these two cell lineages in both sex.
With this study we provide the most resolutive transcriptomic study of early gonadal development in both sex and revisit the classical model of sex determination.
La détermination du sexe chez les mammifères est un modèle particulièrement intéressant pour étudier la décision du destin cellulaire. Selon le sexe génétique, le primordium gonadique est capable de se différencier en deux organes différents, le testicule et l’ovaire. Le devenir de ce pri- mordium est déterminé par la présence ou l’absence d’un seul court gène situé sur le chromosome Y:Sry(1,188 paires de bases seulement). La lignée supportrice est le premier type cellulaire à se différencier dans la gonade. Sa différenciation en cellules de Sertoli ou pré-granulosa détermine le sexe phénotypique de l’individu. L’expression deSrydans une fenêtre temporelle précise contrôle la différenciation des cellules supportrices en cellules de Sertoli, tandis qu’en l’absence deSry, les cellules supportrices se différencient en pré-granulosa par l’activation de la voie de signalisation WNT/β-caténine. Une fois engagées, les cellules supportrices des deux sexes contrôlent la différen- ciation des autres cellules somatiques de la gonade, et en particulier des cellules stéroïdogéniques (cellules de la thèque chez les XX et cellules de Leydig chez les XY) qui sécrètent les hormones nécessaires au développement des caractères sexuels secondaires.
De nombreuses questions concernant la détermination du sexe demeurent. La composition cel- lulaire de la gonade bipotentielle est un sujet de débat. Le modèle classique indique la présence de deux lignées cellulaires avant la détermination du sexe: la lignée supportrice et la lignée stéroï- dogénique. Des études récentes ont remis en question ce dogme en faveur d’une population de cellules progénitrices commune, mais n’ont pas fourni de preuve claire. Comment et quand ces lignées cellulaires se spécifient restent flous, d’autant plus que les précurseurs des cellules stéroïdogéniques dans les deux sexes sont encore mal caractérisés. Alors que les cellules sup- portrices se différencient façon autonome dès E11.5 chez la souris, la différenciation de la lignée stéroïdogénique se produit plus tardivenement (E12.5 dans XY, après la naissance dans XX) et est controlée par les cellules supportrices.
Ce projet de thèse visait à étudier le processus de détermination du sexe chez la souris grâce au single-cell RNA sequencing (scRNA-seq) en caractérisant les changements transcriptomiques survenant dans les différentes lignées cellulaires somatiques depuis l’état bipotentiel jusqu’aux testicules et ovaires en développement.
Dans ce but, nous avons procédé au séquençage de l’ARN des cellules somatiques de gonades (exprimant NR5A1) de souris XX et XY aux stades clés de leur développement. Avec les algorithmes de prédiction de lignée cellulaire et en rangeant les cellules selon un pseudotemps, nous avons pu reconstituer la chronologie des événements qui déterminent le développement des testicules et des ovaires, ainsi que la décision du devenir sexuel dans les lignées cellulaires support et stéroï- dogéniques. Nous avons détecté une population de cellules uniques avant la détermination du sexe qui est capable de se spécifier soit en cellules supportrices, soit en cellules stéroïdogéniques.
Nous avons caractérisé la dynamique de l’expression des gènes qui conduit à la différenciation de
ces deux lignées cellulaires dans les deux sexes.
Cette étude transcriptomique est à ce jour la plus précise dans le domaine de la détermination du sexe et a permis de revoir le modèle classique de la différentation des cellules lors de la phase critique de différenciation des gonades.
Here we are, four years, 11 months and seven days since the beginning of my PhD. What a long journey, paved of moments of joy, deception, surprise, frustration, satisfaction, and despondency.
During these past years, I learned to cultivate my schizophrenia, as being part of a biology lab and a bioinformatics lab. In the end, I have a strange feeling of not being an accomplished biologist, nor an accomplished bioinformatician (hello, my dear impostor syndrome!). Don’t be mad at me, but I think I will never be able to be pride of what I have done, that’s part of me, that’s the curse of the perfectionists.
I have to thank a lot of persons, starting with Prof. Serge Nef, who entrusted me this project, and his lab. You pushed me in the deep end swimming pool, as you used to say, and I finally managed to swim. It was nice to have a boss like you, with whom I could speak frankly, maybe too much sometimes. We argued, a lot, but we also shared nice and fun moments, in particular during the European Testis Workshops.
I would like to thank the former members of the Nef lab, Piere Calvel, Jean-luc Pitetti, Céline Zimmermann, who warmly welcomed me when I arrived and made me comfortable; Vanessa Bianda, who regularly went to "wash her testis" with such enthusiasm. I really hope that you have now found your career path and that makes you feel happy and more self-confident, you deserve it.
Quentin Gex and Dario Sessa, for their constant good mood, their good bad jokes and their third and fourth degree humour. And finally Jessica Escoffier, for her desperate attempts to make me feel pride of my work, and her support when I was down. Even if we did not always agree, I felt like I was with my sister when I talked with you.
I would like to warmly thank Béatrice Conne and Françoise Kuhne, our lab "mums". You taught me so much, your are so important for the lab, we owe you a lot.
Rita Rahban, sweet little Rita, I am so proud of you, I saw you grow, scientifically and personally.
Keep going, never give up, and keep your smile.
And finally Yasmine Neirijnck, I have learned so much from you, the rigour, the tenacity, the patience. I was happy to find another person that cannot wake up in the morning, so I was not the latest to arrive anymore in the lab. I also really enjoyed our daily debriefing in the tram back to home.
I thank Prof. Manolis Dermitzakis and all his lab members. The demanding and time consuming mouse work prevented me to spend a lot of time in the lab so in the end we did not interact a lot. I would like to particularly thank Marco Garieri, Nikos Panousis and Julien Bryois for their support and their help in understanding the bioinformatics PhD program that is still a bit mysterious to me even now; Cédric Howald for the technical assistance on Vital-IT and video game discussions;
Ana Vinuela and Andrew Brown for your help and support after the tough lab meetings; Olivier
Delaneau for his good mood and sometimes absurd but funny discussions. A big thanks to Luciana Romano and Deborah Penet for their technical expertise in Illumina sequencing. I really enjoyed working with you and I appreciated that you took the time to show me all the steps of the sequencing run of my samples. And the last but not the least, a huge thanks to Ancilla Stephani, your smile, your dedication, your help, it is always a pleasure to come to see you and discuss and laugh about everything.
A particular thanks to Christelle Borel, who supported me in the hellish handling of the C1 single- cell protocol and made me face the level of expectation for a PhD student. You were sometimes tough but always just and right. You made me assume my choice and responsibilities.
This work could not be achieved without the assistance of the different facility platforms of the University of Geneva. In particular the flow cytometry platform with Jean-Pierre Aubry, Cécile Gamero and Grégory Schneiter, who are always so nice with us, even when we arrive in late and when we ask for a cell sorting during the midday break. You are the super-heros of cell sorting!
Thanks also to the IGE3 genomic platform for the single-cell capture, particularly Mylène Docquier, Didier Chollet and Brice Petit. No offence but I hope I will never have to do this again, I still have grievance against the C1 machine and the random cell capture success.
Thanks to all the collaborators I met, the Jabaudon’s lab for the nice collaboration that end up with a successful publication; Prof. Lee Smith from Edinburgh, who injected himself mice for a lineage tracing that didn’t work because of a defective cre, I really appreciate the effort; and the team of Bernard Jégou and Frédéric Chalmel from Rennes for the serious and less serious discussion during conferences; and I thank Prof. Andy Greenfield to have proposed to be part of my PhD jury.
Finally I would like to thank and to apologise to Arnaud, because I was horrible at home, with detestable mood all the time, and so exhausted the weekend I was staying grumpy in the sofa or in front of my computer working or distressing with video games. Sorry for that, I will do my best to not be the same during my next job and to try to be less selfish (I said "try"...).
Genève, le 7 mars 2018 I. S.
Abstract i
Résumé iii
Acknowledgements v
1 Introduction 1
1.1 Overview of the PhD Project . . . 1
1.2 Sex Determination and Gonad Differentiation in Mammals . . . 4
1.2.1 History of Sex Determination . . . 4
Tales and Mythologies of Sex . . . 4
From Tales to Science: Sex under the Microscope . . . 6
The "female testis" . . . 7
First observations of spermatozoa . . . 8
The mammalian egg and the ovary . . . 8
Leydig cells . . . 9
Sertoli cells . . . 9
Intimate matting: the fecundation . . . 9
Early Stages of Genetics: the Sex Chromosomes . . . 10
Sex and Chemistry: Hormones . . . 12
The Discovery of the Testis-determining Factor . . . 15
The Search for the Ovary-determining Factor . . . 17
1.2.2 The Current State of Mammalian Gonadal Development . . . 19
The Genital Ridge Formation and the Bipotential Gonads . . . 19
Male Sex Determination and Testis Development . . . 21
Sryswitches on maleness . . . 21
Mesonephric cell migration . . . 23
Testis cord formation . . . 24
Foetal Leydig cell differentiation . . . 25
Female Sex Determination and Early Ovarian Development . . . 28
RSPO/WNT/β-catenin signaling pathways . . . 29
Granulosa cell differentiation . . . 29
Germ cell cysts and primordial follicles . . . 30
Postnatal theca cell differentiation . . . 31
The Battle of the Sexes . . . 32
1.3 Single-cell RNA Sequencing Applied to Developmental Biology . . . 34
1.3.1 Principles of scRNA-seq Technology . . . 34
Cell Capture Strategies . . . 35
Microwell cell isolation . . . 35
Fluidigm C1 autoprep system . . . 36
Droplet-based systems . . . 37
RNA-Sequencing Strategies . . . 38
1.3.2 Technical Aspects of Performing scRNA-seq . . . 39
Tissue Preparation . . . 39
Experimental Design and Internal Controls . . . 39
Cell Number . . . 41
Sequencing Depth . . . 41
1.3.3 Computational Challenges of scRNA-seq Data Analysis . . . 42
Dealing with Sparse and High Dimensional Data . . . 42
Clustering the Cells . . . 43
Feature selection . . . 43
Dimensionality reduction . . . 44
Defining group of cells . . . 45
Identifying Transcriptional Signatures . . . 45
Prediction of the Cell Lineages and Pseudotime Ordering . . . 46
2 Objectives 47 3 Results 49 3.1 Deciphering cell lineage specification during male sex determination with single-cell RNA sequencing . . . 49
3.1.1 Contribution . . . 49
3.2 Supporting cell lineage specification and sex-specific differentiation into Sertoli or Granulosa cells occurs sequentially during gonad sex determination . . . 73
3.2.1 Manuscript in Preparation . . . 73
3.2.2 Contribution . . . 73
4 Discussion 99 4.1 The E10.5 NR5A1+Cells as Common Progenitors of the Supporting and Steroidogenic Cells . . . 100
4.1.1 The E10.5 CE Cells are not Predefined as Supporting or Interstitial/Stromal Cells100 4.1.2 The E10.5 CE Cells Differentiate as Sertoli and Foetal Leydig Cells . . . 101
4.1.3 The NR5A1+Cells do not Originate from the Migrating Mesonephric Cells . . 103
4.2 Supporting Cell Lineage Specification and Sexual Dimorphism . . . 104
4.2.1 The Supporting Cell Lineage Commits Independently of the Genetic Sex . . . 104
4.2.2 The Sertoli and Pre-Granulosa Cells Differentiate Asymmetrically . . . 105
4.3 Interstitial/Stromal Cells as a Source of Steroidogenic Cells . . . 106
4.4 Limitations and Perspectives . . . 107
4.4.1 Limited Number of Cells . . . 107
4.4.2 Robustness of the Cell Clustering and the Lineage Prediction . . . 108
4.4.3 Perspectives of the PhD Project . . . 109
5 Additional works 111 5.1 Research articles . . . 111
5.1.1 Loss of Function Mutation in the Palmitoyl-Transferase HHAT Leads to Syn- dromic 46,XY Disorder of Sex Development by Impeding Hedgehog Protein Palmitoylation and Signaling . . . 111 5.1.2 The Dynamic Transcriptional Profile of Sertoli Cells During the Progression of
Spermatogenesis . . . 124 5.1.3 Sequential transcriptional waves direct the differentiation of newborn neurons
in the mouse neocortex . . . 141 5.1.4 Insulin and IGF1 receptors are essential for the development and steroidogenic
function of Leydig cells . . . 146 5.2 Review articles . . . 162
5.2.1 Single cell transcriptome sequencing: a new approach for the study of mam- malian sex determination . . . 162
Bibliography 173
1.1 Overview of the PhD Project
Among the genetic characteristics that influence people’s physical identity throughout their lives, the most important is surely that of sex. The ultimate goal of male and female sex differentiation and development is to provide organisms with the necessary attributes for sexual reproduction. In mammals, the genetic sex of individuals is determined at fertilization with the introduction of an X or a Y chromosome from a spermatozoon to an X-bearing oocyte. Phenotypic sex is only revealed during foetal development, when the gonads start to differentiate into ovaries or testes (at around six embryonic weeks in humans, and at embryonic day (E)11.5 in mice).
Both ovaries and testes develop from a common primordium, the adrenogonadal primordium (AGP), composed of a thickening layer of multipotent cells at the surface of the mesonephroi (intermediate mesoderm) and migrating primordial germ cells (PGCs). The exact moment the gonads begin their differentiation into either ovaries or testes is called sex determination. Following sex determination, a cascade of events leads to the differentiation of the bipotential cell lineages into their respective sex specific cell types and ultimately the formation of functional reproductive organs (Figure 1.1). Subsequently to the development of the ovaries or testes, the whole embryo will adopt the secondary sexual characteristics such as male and female reproductive tracts and appendixes.
Sex determination is a robust developmental programme but sometimes glitches appear and lead to sexual ambiguity. Disorder of sex development (DSD) are defined by rare "congenital conditions in which development of the chromosomal, gonadal or anatomical sex is atypical" [Hughes et al., 2006]. In humans, it has been estimated that one in 4,500–5,500 births presents ambigous genilatia preventing a binary gender assignment [Thyen et al., 2006, Sax, 2002]. Many different causes of DSD have been identified, such as congenital adrenal hyperplasia (CAH), androgen insensitivity syndrome (AIS), Klinefelter syndrome (47,XXY), and Turner syndrome (45,XO), but in 40% of cases, the aetiology remains unaccounted.
The difficulty in identifying the genetic causes of DSD conditions partly resides in the fact that our knowledge of the genetic sex determination programme is limited. Fundamental aspects of gonadal differentiation and early development are still controversial, in particular the origin and characteristics of several somatic cells including the steroidogenic cells (Leydig and theca cells).
Figure 1.1 –Schematic representation of the developing gonads.
Current knowledge of sex determination was built on gene by gene knock-in/knock-out in mice and transcriptomic analysis of a pool of purified cells. The first method suffers from a very low throughput and produces partial knowledge of the function of one gene or a limited set of genes.
The second method’s caveat resides in the lack of specific reporters for the different cell types within the gonad, which makes the appreciation of the cell type heterogeneity difficult, especially before sex determination. Moreover, a pool of purified cells results in an average message of non synchronous differentiating cells and thus blurs the chronology of gene expressions.
The synergy between different disciplines such as physics, electronics, computer science and molecular biology enables the development of increasingly efficient research technologies, al- lowing for the push back of limitations. As a result, single-cell sequencing is now becoming a critical technology by giving access to the DNA and RNA of the inner components of tissues, cells themselves. Single-cell RNA sequencing (scRNA-seq) can be used to appreciate the diversity of cell types of a given tissue, and combined with time-course experiments, it allows the monitoring of cell differentiation at the transcriptomic level. Numerous bioinformatics programmes have been developed to study time-course single-cell experiments but they all have their own specificity. Some of them needa prioriknowledge of the data, while others support only linear change in a single precursor cell population and do not support multipotent cell lineage models. The development of new single-cell automatic systems and bioinformatics tools is still ongoing but current solutions are sufficient to produce exciting results.
This multi-disciplinary PhD project applies scRNA-seq technology in the context of sex determi- nation in mice to gain a deeper understanding of the sex fate decision and the differentiation of the somatic cells from bipotential gonads to differentiated ovaries and testes. The first part of the
project uses transgenic mice carrying a specific GFP reporter to isolate the somatic cells of the gonads at key steps of sex determination into male and female. Cells are then processed for the single-cell capture, RNA reverse-transcribed into cDNA, and prepared for sequencing. The second part consists in the bioinformatics analysis of the sequencing data to identify the different cell types, reconstruct the cell lineages and identify genetic programs driving cell fate decision.
1.2 Sex Determination and Gonad Differentiation in Mammals
How most living creatures segregate into males and females raises many questions. Ancient texts testify that the origin of sex differentiation was debated long before the establishment of science.
Most of these ancient writings are narratives void of fact and can be considered mainly as tale and myth. That said, these stories often reveal surprising insights into our present knowledge of biology.
1.2.1 History of Sex Determination
Tales and Mythologies of Sex
The most widespread story about the origin of sexual differenciation is undoubtedly the story of Adam and Eve (Genesis 2: 21-24, written between the 8thand 2ndcenturies B.C., figure 1.2). The biblical tale relates that God created Adam in his image, then created the first woman, Eve, from one of Adam’s ribs. Something we are less acquainted with is the existence of a second interpretation of this story. Indeed, the Old Testament, written in Hebrew, mentions the worldzela, which can be translated as "rib" or "side", as is the case with the Latin wordcosta[Cassuto, 1961]. This second translation was considered the correct meaning by a rabbinical tradition whick existed before the Christian era. According to this tradition, Adam was in fact a hermaphrodite, both male and female, able to procreate by himself. God then decided to create a woman from Adam’s side,i.e.
to separate the female and male sides, generating two incomplete individuals who need each other to reproduce [Krappe, 1936, Koppelman, 2006]. This interpretation of a bisected original hermaphrodite represents a more egalitarian version of the creation of the first woman than the interpretation of the creation of the woman from Adam’s ribs.
Figure 1.2 –Adam and Eveby Peter Paul Rubens, 1597-1600, oil on panel (Antwerp, Rubenshuis).
This vision of a divided individual is also described in Plato’sSymposium(around 380 B.C), where Aristophanes relates the origin of the sexes. According to this account, primeval men were round, with four hands, four feet, and two faces looking in opposite directions (figure 1.3). These men were so strong that they might represent a threat to the gods, and thus Zeus decided to cut them into two. However as soon as they were separated, they sought to re-unite and embrace [Jowett, 1970].
The stories of Adam and Eve and primeval men seem to derive from an ancient Oriental legend known in Mesopotamia, India and Persia, and may be older than 5000 years [Krappe, 1936].
Figure 1.3 –Image of an androgyne, detail of an ancient Greek amphora, 4th century BC (unknown source).
In antiquity, male children were far more welcome than female children, as they were believed to be quicker at calming the wrath of the gods. From a more pragmatic point of view, they were also considered to provide good support for the parents in old age. This preference for producing a boy was the main motivation in the search for understanding how sex is determined, in order to eventually control it [McCartney, 1922].
Plato was one of many Greek philosophers who proposed successive explanations (figure 1.4). A commonly held view at that time was that organisms are composed of the four elements: fire (hot), water (cold), air (moist), and earth (dry). Males were regarded as hotter than females, and thus were thought to be constituted from a high proportion of the superior element of fire, while females were mainly constituted from water [Lesky, 1951]. It was also commonly believed that the right side was associated with heat, and the left with cold [Lloyd, 1973]. Following these beliefs, Parmenides (6thcentury B.C) hypothesized that the sex of a child was determined by its position in the womb:
boys on the right side, and girls on the left. Anaxagoras, a contemporary of Plato, thought that males were formed from the semen of the right testis, and females from the semen of the left. Both theories were criticized by Aristotle (384-322 B.C.) who illustrated that children of both sexes can be carried on the same side of the uterus, and that men with only one testicle could have both male and female children [Mittwoch, 2000].
In a series of booksOn the Generation of Animals, Aristotle synthesized his observations on the differences between males and females and their origins (figure 1.4). Males were stronger animals, he stated, constituted with a high proportion of the fire element. With their heat, they converted food into a perfect concoction, the semen, in a limited quantity. This semen passed through the entire male body to collect all of its characteristics, and this explained why children resembled their parents. Females, because of their low proportion of the fire element, were considered weaker animals. Because they lacked heat, they were not able to produce semen. Instead, they produced an abundant quantity of blood, the catamenia (i.e.the menstrual blood), forming the material of the embryo. The male discharged his semen in the female by copulation and the embryo resulted from the mixture of both semen and catamenia. If the male mastered the production of his semen, the embryo formed as a male. If he failed to master the semen production because of a deficiency
of heat, the embryo took the opposite form, that of the female.
"The female, then, provides matter, the male the principle of motion. And as the products of art are made by means of the tools of the artist, or to put it more truly by means of their movement, and this is the activity of the art, and the art is the form of what is made in something else, so is it with the power of the nutritive soul."–Aristotle (On the Generation of AnimalsII-1)
Figure 1.4 –Plato (left) and Aristotle (right), a detail ofThe School of Athens, a fresco by Raphael.
While we may now smile at Aristotle’s account of sex determination, he was probably the closest to reality. He raised the idea that both male and female produce the materials to conceive, as well as the role of male semen in the determination of a child’s sex. He also referred to the fact that the sex of the embryo is formed during its development after a period of ambiguity by the formation of organs that differ between male and female. Finally, he associated the testes as responsible for masculinization by observing the transformation of the bodies of eunuchs from having masculine traits to exhibiting more feminine traits.
Since antiquity, each period and culture has developed its own beliefs around what influences sex determination, from bandaging women’s feet, to men’s testes, to even eating specific foods [McCartney, 1922]. The theories of heat, laterality (left or right testis and left or right side of the womb), or the implication of food in the determination of a child’s sex, despite numerous objections, have survived for nearly 2,000 years [Mittwoch, 2000].
From Tales to Science: Sex under the Microscope
Until the 19thcentury, knowledge about sex determination and reproduction was based more on intuition than on reliable facts. Even though it had been obvious for a long time that birds’ eggs are the origin of their progeny, the existence of mammalian eggs remained unclear. The common view in 1660 was that in mammals, male semen mixed in the uterus with the hypothetical female semen and this mixture turned into an egg and then a fetus.
(a) Portrait of Reiner de Graaf [de Graaf, 1671].
(b)Portrait of Antonie van Leeuwenhoek (by Jan Verkolje, around 1680).
(c)Reproduction of Van Leeuwenhoek’s mi- croscope (Deutsches Museum, Munich)
(d)Spermatozoa of rabbit and dog [Lewen- hoeck, 1677].
Figure 1.5 –Reiner de Graaf and Antonie van Leeuwenhoek.
The "female testis" The first evidence of the existence of mammalian eggs came from Reiner de Graaf (1641-1673) in Delft, the Netherlands (figure 1.5a). After studying the morphology and function of testes1, De Graaf worked on the female reproductive tract and was one of the first to perform a human ovary dissection. The female reproductive organs in chickens were called ovaries as they produce eggs (ovumin Latin). Meanwhile, for mammals, reproductive organs were called
"female testes", as people did not view them as the equivalent of chicken ovaries. De Graaf pointed out that the structures in the "female testis" were very similar to the chicken ovaries. He found small vesicles full of liquids which he thought corresponded to the albuminous fluid in a bird egg.
He confirmed the analogy with a succession of experiments and proposed that the vesicles, or eggs,
1At that time, the testis was thought to be a glandular organ containing amorphous tissue with no defined structures.
By dissecting testes, De Graaf observed that they are composed of long and fine tubules he called "vessels". These vessels join up to form a larger tube, the "efferent vessels", leading to the epididymis, itself connected to the vas deferens, thus depicting the whole path of the semen [Loriaux and Loriaux, 2016].
contained a necessary element for foetal development. Following this, De Graaf observed ovaries and female reproductive tracts in rabbits from 30 minutes to 29 days aftercoitus. He was able to track the "ova" from the ovary to the uterus, confirming his idea that the egg is produced by the ovary and travels through the uterus. What he was actually looking at were follicles full of fluid, not the real oocyte that he could not see without the use of a microscope. The conclusion of his work was that all animals originate from eggs that exist before coitus in the female testis. Hence, the female "testes" were called ovaries [Loriaux and Loriaux, 2016].
First observations of spermatozoa The use of a microscope by Antonie van Leeuwenhoek (1632- 1723) constituted an important step forward in the understanding of reproduction (figure 1.5b).
Van Leeuwenhoek worked as a draper, in his own shop in Delft, in the Netherlands. His interest in lens-making derived from his need to evaluate the thread quality of materials. He developed glass lenses that had a greater magnifying power than had ever been made before (200x–400x)(figure 1.5c).
He presented his lenses to his friend Reiner de Graaf, who was convinced that microscopy would help him considerably in his study of the reproductive system.
De Graaf contacted the Royal Society of London to share Van Leeuwenhoek’s work, and the society recognised the achievement and in 1673 published a letter with Van Leeuwenhoek’s microscopic observations. Unfortunately, De Graaf died suddenly at the age of 32 and was never able to benefit from Van Leeuwenhoek’s invention. With his powerful lenses, Van Leeuwenhoek set out to observe a variety of organisms living in liquids and discovered, among other things, the existence of bacteria, infusoria (now known as protists), and blood cells in capillaries. He was the first person to observe sperm cells in human and dog fluids and gave them the name ofanimacula(figure 1.5d). He claimed that theseanimaculesmay play an important role in the formation of the embryo, going against the theory of spontaneous generation which was popular at the time [Ruestow, 1983].
Between 1780 and 1785, Lazzaro Spallanzani experimented with fecundation in frogs and concluded that the seminal liquid was necessary for fertilizing the eggs, in accordance with Van Leeuwenhoek’s hypothesis. From the 17thcentury until the 19thcentury, the democratization of the microscope allowed many scientists to observe sperm cells and ova in mammals. In 1821, the Swiss biologist Jean-Louis Prevost and the French chemist Jean-Baptiste André Dumas described and compared the properties ofanimaculesin many species including mammals. They noticed the absence of animaculesin the spermatic liquid of young and old animals and correlated these observations with the reproductive capacity of the animals. They also observed thatanimaculestaken from a testis were immobile, but once they had passed the seminal vesicle, they became mobile. In 1824 they published the first evidence of the role ofanimaculesin fecundation [Diderot and D’Alembert, 1827].
The mammalian egg and the ovary In parallel to this work, Karl Ernst von Baer, an Estonian scientist, set out to find the mammalian egg within the ovary. Studying the dog, he found a floating body in the liquid of the follicle previously discovered by De Graaf. He realized that what he observed was actually the mammalian egg. In 1827, he published a treatise in LatinDe Ovi Mammalium et Hominis Genesiin which he described for the first time the origin of the mammalian ovum and foetal development. In the main figure of his book, we can see the terms still used today to describe the follicles, such as "membrana granulosa", "thecae stratum externum2" and "corpus luteum3"
2Thecaerefers to a sheath or a covering.
3Luteummeans the colour yellow.
(figure 1.6).
(a)Full-page, hand-coloured, engraved plate from the book. (b)Extract of the figure legend with the current terms to describe the ovary.
Figure 1.6 –Main figure of Karl Ernst von Baer’s bookDe Ovi Mammalium et Hominis Genesi.(1827)
Leydig cells In 1850, Franz Leydig, a young German scientist who helped to establish the first Microscopy Institute of the medical faculty at Würzburg, published a comparative histological study of the male reproductive system in various mammals (figure 1.7a). The manuscript includes the first description of the testicular cells that would later bear his name. Tissue preparation at that time was far removed current methods (fixation, dehydration, embedding, sectioning and staining). The common procedures consisted of teasing living tissue with needles, or manually cutting slices as thin as possible. Chemical treatment was also used to break connective tissues or to stain the tissue to enhance contrasts (carmine, cochineal, saffron, madder, or indigo). Under these conditions, he first described Leydig cells as individual multinucleated cells between the tubules with fatty granules as he could not discern sharp contours nor boundaries within the mass [Christensen, 2007].
Sertoli cells Not so long after the first description of Leydig cells, in 1862, a young student named Enrico Sertoli from the University of Pavia, Italy, purchased a brand new microscope for his research studies under Professor Oehl (figure 1.7b). He collected human testicular tissues and used different types of preparations including microdissections of seminiferous tubules, pieces of fresh tissue, and frayed sections of tubules. He observed and meticulously drew branched cells enclosing the seminiferous cells (progressing spermatic cells). He also described spermatozoa at the centre of the tubules and hypothesized that the branched cells did not produce the spermatozoa but were involved in their production. He referred to these cells as "mother cells", an intuitive perception of the true role of Sertoli cells as supporting spermatogenesis [Hess and França, 2005].
Intimate matting: the fecundation By the end of the 19thcentury, it had become clear that re- production occurs with the mating between ananimaculaformed within the testis, and an egg,
(a)Portrait of Franz Leydig. (b)Portrait of Enrico Sertoli in 1865 [França et al., 2016].
Figure 1.7 –Franz Leydig and Enrico Sertoli.
coming from the ovary. The mystery of reproduction was finally solved by Oscar Hertwig in 1878 with his work on fertilization in sea urchins. He observed a sperm cell head entering an egg and the nuclei of the two cells fused [Hertwig, 1878]. However, the question of how male and female sexes are determined remained unsolved.
Early Stages of Genetics: the Sex Chromosomes
Before the advent of the era of genetic following the discoveries of Mendel, hypotheses on sex determination were directed toward environmental factors. In the 19thcentury, food was suspected to be the most decisive factor; it was believed that the mother’s nutrition during the first three months of pregnancy could affect the sex of the child [Düsing, 1884]. Temperature was also considered to be a factor, based on the assumption that females are colder than males and so heat may tend to produce males.
The change in the basis of sex determination from the environment to genetics emerged with the discovery of string-like bodies in the nuclei of cells discovered by Walther Flemming in 1882, and named chromosomes by Heinrich Wilhelm Waldeyer in 1888, after he observed their behaviour when stained with specific dyes. In 1891, Hermann Henking, a German biologist, was using microscopy to study firebug sperm formation and noticed that some sperm had 12 chromosomes while others had only 11. While observing the stages of meiosis of the forming sperm, he found that this extra chromosome looked different from all the others. He named this chromosome the "X element" to emphasizes its mysterious nature. He then studied female grasshopper egg formation but was not able to find the X element [Henking, 1891, Brown, 2003]. Henking hypothesized that this element may be related to sex determination in insects, but he was unable to find any direct evidence.
Ten years after Henking’s discovery, American zoologist Clarence Erwin McClung began extensive studies around the idea that the X element must be implicated in sex determination. He renamed
this chromosome the "accessory chromosome", as it seemed to have a different role compared to the other chromosomes. He decided to carry out a comparative study of spermatogenesis in many different animals, not only insects. He discovered that the accessory chromosome was present in 50/50 proportions in sperm cells. He realized that only one characteristic varies in a 50/50 proportion in many species, and this characteristic is sex [McClung, 1902].
(a) Edmund Beecher Wilson (October 19, 1856 - March 3, 1939), pioneering American zoologist and geneticist
(b)Image from his textbook "The cell in Development and Inheritance", second edition, 1900.
(c)Nettie Maria Stevens (July 7, 1861 - May 4, 1912), early Amer- ican geneticist.
(d) Nettie Stevens’s microscope, Bryn Mawr College.
Figure 1.8 –Edmund B. Wilson and Nettie Stevens discovered the XX/XY sex determination system.
Subsequent to this finding, Edmund Beecher Wilson found that spermatocytes of different insect species exhibited either the presence or the absence of a chromosome, and also that there was a difference in the size of one of the chromosome pairs [Wilson, 1905] (figure 1.8a & 1.8b). The same year Nettie Maria Stevens, investigating the common mealworm, found that one chromosome was smaller than the other but only in males, not in females. She concluded that this chromosome may be responsible for male sex determination and called the short chromosome "Y" and the larger one
"X" [Stevens, 1905] (figure 1.8c & 1.8d).
Although Wilson and Stevens agreed that X and Y chromosomes were related to sex determination, they strongly disagreed about how the chromosomes influenced sex. Stevens believed that sex was
one of the traits carried by the X chromosome, as with any other chromosomes carrying multiple traits. Wilson believed there was “a whole-chromosome effect – one X kept things tilted towards maleness, while two Xs pushed the balance in favour of femaleness” [Richardson, 2013]. Both scientists dedicated their work to refuting the other but Stevens, affected by breast cancer, died in 1912 at the age of 50 before she could validate her hypothesis. Wilson was the first to call the pair of XX or XY chromosomes the "sex chromosomes" and this designation has since been adopted in the scientific literature.
The exact number of 46 chromosomes in humans, including the sex chromosomes, was established in 1956 by Charles Ford and John Hamerton. Men present an X and a Y chromosome, whereas women have two X chromosomes [Ford and Hamerton, 1956]. Since then, cytogenetics studies have pointed out aberrations in human chromosomes. The first was the discovery of Down’s syndrome by Marthe Gautier and colleagues in Paris in May 1958 [Lejeune et al., 1959]. A year later, in 1959, Patricia Jacobs reported a patient with Klinefelter syndrome, a male phenotype with an XXY chromosome set [Jacobs and Strong, 1959] (figure 1.9), and Ford and Fraccaro both reported two patients with Turner syndrome, female phenotype, presenting a single X chromosome [Ford et al., 1959, Fraccaro et al., 1959]. These findings demonstrated that, in humans, the male phenotypic sex is due to the presence of the Y chromosome, and does not depend on the number of X chromosomes. This constitutes the first evidence of the role of the Y chromosome as the male-determining factor.
Figure 1.9 –Klinefelter syndrome: 47,XXY karyotype.
Sex and Chemistry: Hormones
Even though it has been known since antiquity that castrated animals show a loss of secondary sexual characteristics, the exact role of the gonad was not elucidated until the 20thcentury.
At the end of the 19thcentury, Dr. Charles Édouard Brown-Sequard, a Mauritian physiologist and neurologist, was in the habit of performing experiments on himself (figure 1.10a). He correlated the
(a)Portrait of Charles-Édouard Brown-Séquard. (b)(A)Magazine advertisement showing extracts of animal organs, including testicular fluid. (B) An advertisement for “Sequarine,” also known as
“Brown-Séquard Elixir” [Rengachary and Colen, 2008]
Figure 1.10 –Dr. Brown-Séquard and his "elixir of life".
decrease of strength and vitality caused by old age to what he observed in eunuchs and concluded that this must be due to a loss of some unknown substance created by the testicles.
"There is no need of describing at length the great effects produced on the organization of man by castration, when it is made before the adult age. It is particularly well known that eunuchs are characterized by their general debility and their lack of intellectual and physical activity."–Dr. Brown-Sequard (The Elixir of Life, 1889)
To prove his hypothesis, he prepared concoctions of water and fluids from different fractions of dogs’
and guinea pigs’ testicles. He gave himself multiple hypodermic injections of these preparations and reported miraculous effects of rejuvenation and prolonged human life. His unprecedented discovery was later called by the scientific community "the elixir of life"(figure 1.10b). It appeared much later that the effects he had described were in fact placebo effects, as the active hormone, testosterone, is hydrophobic, and was thus absent in his preparation [Rengachary and Colen, 2008].
In the 1920s and 1930s, the scientific community was focused on research into hormones, and experiments inspired by the work of Dr. Brown-Sequard led to the discovery of testosterone. In the early 1920s, Leo Stanley, an American physicist, injected thousands of prisoners with testicular crush and observed beneficial results in general health in 80% of cases [Blue, 2009]. In 1926, Fred Koch from the University of Chicago obtained a large stock of bull testicles from stockyards and managed to extract an unidentified substance. He injected the preparation into capons (cocks
castrated at a young age). Capons are totally unable to crow and do not present secondary sexual characteristics. After receiving the injections, it appeared that the capons started to crow like normal cocks [F Gallagher and Koch, 1929].
In 1935, Ernst Laqueur and his team in Amsterdam extracted 10mg of a hormone from 100kg of bull testes and named it testosterone (a contraction of the words "testis", "sterol" and "hormone"). In August of the same year, Ruzicka and Butenandt in Basel managed to synthetize testosterone from cholesterol; their work was rewarded with a Nobel Prize in 1939 [Nieschlag and Nieschlag, 2014].
Figure 1.11 –Female free-martin, by John Hunter, Plate from "The Works of John Hunter", 1835
The hypothetical implication of hormones in sex determination received positive evidence with the parallel studies of free-martins in 1916 by Keller and Tandler, and by Frank Rattray Lillie. Free- martins are female-male dizygotic twins in cattle where the male presents phenotypically normal characteristics and the female free-martin presents asexual gonads or even sterile testes, and a general masculinized phenotype (figure 1.11). The researchers discovered that during the gestation of cattle twins, the two chorions are linked together by vascular anastomosis. In the absence of vascular anastomosis, female-male dizygotic twins develop normally. They concluded that sex determination, especially masculinization, is due to a hormone spread by blood [Lillie, 1916, Clarke, 1998, Freeman, 2007].
Following this discovery, many scientists experimented with different ways to understand how hormones influence sex determination, including gonadal grafts, castration and parabiosis4. They demonstrated that in some cases, the female or the male sex can be dominant, with a trans- differentiation of the testes into ovaries andvice versa. During the 1950s, the French endocrinologist Alfred Jost revealed the existence of a hormone controlling the sex fate of embryos, elucidating the mystery of the free-martins. He experimented with testicular grafts onto female rabbit embryos and demonstrated that the testes’ hormonal secretions caused regression of the Müller ducts (normally persistent only in females) and the persistence of the Wolffian ducts (normally persistent only in males). He replaced the testis graft with a testosterone propionate crystal but obtained a partial effect (figure 1.12).
4In physiology, parabiosis consists of joining two living organisms together surgically.
Figure 1.12 –Schema of Alfred Jost’s experiments [Claes and Ball, 2016].
Jost identified that the fetal testis influenced the development of the male tract and the external genital organs. His experiments proved that these changes were controlled by two different factors:
testosterone and a secreted "anti-Müllerian hormone" or "Müllerian-inhibiting substance" (AMH or MIS).
In addition to the AMH discovery, Jost was also the first to demonstrate that Sertoli cells control testicular organization by surrounding the germ cells to form the testis chords [Josso, 2008].
The Discovery of the Testis-determining Factor
By 1959, thanks to the work of Charles Ford and Patricia Jacobs, the Y chromosome had been identified as the initiator of male development. Ford and Jacobs showed that genes on the Y chro- mosome induce testis development, which in turn produces the hormones AMH and testosterone that masculinize the embryo. In the absence of a Y chromosome, the gonads develop as ovaries.
Over the following decades, research focused on trying to identify the testis-determining factor (TDF in humans, SRY in mice). It was hoped that its discovery would be the key to deciphering the mechanism of sex determination and testis development. By analyzing cases of sex reversal due to homologue recombination with the X and Y chromosomes, the region where the TDF is located was narrowed down to a small region on the short arm of the Y chromosome (figure 1.13).
To identify suitable candidate genes, five key criteria for favourable testis-determining candidate genes have been established. The TDF has to be conserved amongst mammals. It has to be located in the small region of the Y chromosome previously identified. It has to be expressed during sex determination, in the somatic cells of the testis. Its activity should control the activity of other genes and be cell autonomous. Finally, a mutation should induce a defect in testis development leading to XY females [Koopman et al., 2016].
In the 1980s, two teams worked intensively on the identification of the TDF. The UK team consisted
Figure 1.13 –Schema of the expected genomic location of the testis determining factor over time. ZFY: Zinc finger Y-chromosomal gene; SRY: Sex-determining Region of Y chromosome gene (adapted from [McLaren, 1990]).
of Peter Goodfellow, a human molecular geneticist, and Robin Lovell-Badge, a mouse developmen- tal biologist, who joined their complementary expertise in London. The USA team worked under David Page at the Whitehead Institute in Cambridge, Massachusetts. In 1987, Page was the first to publish a strong candidate, a gene coding for the zinc-finger protein ZFY [Page et al., 1987]. It transpired later that this was not the right gene. TheZFY gene is in fact expressed ubiquitously in humans and its mouse homologue is not expressed in the male foetal gonads, and, finally, a mutation in this gene does not cause any sex reversal in humans or mice.
In London, a cohort of four XX patients presenting masculinization with testis-like tissues were carrying in common a portion of 35kb of the Y chromosome. Goodfellow’s team extracted and fragmented this DNA region and performed Southern blots with male and female DNA from several mammals to test for male-specific binding. Only one of these fragments presented male-specific binding in several mammalian genomes. As the Y chromosome is poorly conserved in mammalian species, this fragment of the human Y chromosome was likely to be the one containing theTDF gene. Sequencing of the fragment revealed an open reading frame coding for a single exon gene they called with prudence "sex-determining region of the Y chromosome" (SRYin human,Sryin mice) [Sinclair et al., 1990, Gubbay et al., 1990].
The reasonable continuation of this work was naturally to make a transgenic XX mouse carrying Sryand this was undertaken by the team of Lovell-Badge. They injected mouse embryos with 14 kb clones containing the mouseSryopen reading frame and its flanking regions. The mouse embryos were analyzed at 14 dayspost coitum. Chromosomal sex and the presence of the transgene were verified by PCR. After several unsuccessful attempts, they obtained the first XX sex-reversed embryos.
In the following experiments, they allowed the litter to birth and grow. They again obtained an XX transgenic mouse with a complete sex reversal that was able to mate with females. They called the mouse Randy (figure 1.14). Randy had a normal male reproductive tract but had small testes and was not able to perform spermatogenesis.
Lovell-Badge et al.’s paper was published in 1991 and made front-page news in the UK. The secret to making a male was finally revealed. It was hoped that this significant discovery would open the gate
(a)Naturecover with the presentation of the complete sex reversed XX mouse carry- ing theSrytransgene
(b)Complicated photo shooting for theNaturecover. Left-to-right: Nigel Vivian, Robin Lovell-Badge, Peter Koopman and Neal Cramphorn.
Figure 1.14 –Randy, the complete sex reversed XX mouse carrying theSrytransgene [Koopman et al., 2016].
to a deeper understanding of the genetic basis of sex determination. In reality,SRY/Sryturned out to be extremely difficult to work with. The gene is small, expressed in a very small number of cells in a brief period of time, in a tissue difficult to access. Anti-bodies specific for SRY were difficult to develop because of its similarity to SOX proteins [Bradford et al., 2007]. SRY is a high-mobility group (HMG) transcription factor but its binding site is small and degenerated. Primary gonad cell cultures quickly loseSRY/Sryexpression. In summary, all the classic methods to study its function and targets were impractical.
The Search for the Ovary-determining Factor
Until recently, sex determination was described as a default female programme that is overwhelmed by the dominant male pathway in the presence of the Y chromosome. In contrast with the dramatic morphological changes taking place during early testis development, ovarian development appears to be dormant with no obvious changes until the last days before birth. Because of this, the study of ovarian development was hampered and the molecular mechanisms remained mysterious, as they do even today. However, to develop an organ as complex as the ovary, there has to be a female specific genetic programme. With the advent of the transcriptomics methods developed at the end of the 1990s, such as microarrays, it has been revealed that the ovary also shows sex specific gene expression as early as the initiation of sex determination, at the exact same moment as the sex-specific gene expression of the testis [Nef et al., 2005]. This raises the question of the existence of a female-determining factor that could act in a similar way as SRY.
As for the TDF, the female-determining factor has to fulfil certain criteria. It needs to be expressed in a female specific manner at the moment of sex determination; the presence of the function of
the gene should lead to an XY male-to-female sex reversal, and conversely, loss of the function should induce an XX female-to-male sex reversal. Three good candidates were identified: the transcription factor FOXL2, the orphan nuclear factor DAX1 (or NR0B1), and the Wnt signalling molecule WNT4. Foxl2 was identified in goats as responsible for an XX autosome female-to-male sex reversal [Pailhoux et al., 2002]. Although null mutation of this gene in mice revealed its role in granulosa cell differentiation, it did not cause XX female-to-male sex reversal. Duplication of DAX1andWNT4in humans has been identified as inducing an XY male-to-female sex reversal [Jordan et al., 2001, Zanaria et al., 1994]. However, a knock-out ofDax1in XX mice resulted in totally fertile females. Regarding theWnt4loss of function in mice, it induced a Müllerian duct regression and a partial female-to-male sex reversal. In light of all of these results, the existence of a unique female-determining factor was questioned.
In 2006, the teams of Andreas Schedl in Nice and Giovanna Camerino in Rome identified RSPO1 as having mutated in two independent families. One of the individuals was suffering from hyper- keratosis, a predisposition to squamous cell carcinoma, and, more importantly, a complete XX female-to-male sex reversal [Parma et al., 2006]. This was the first reported case of a single gene mutation causing an XX female-to-male sex reversal in humans. In their study, the researchers showed that theRspo1gene was expressed around E12.5 in mice, corresponding to early ovarian development. It has also been shown thatRspo1has a highly conserved role in ovarian development among vertebrates [Smith et al., 2008]. However, loss of the function in mice results in a partial XX female-to-male sex reversal [Chassot et al., 2008, Tomizuka et al., 2008] and gain of the function of theRspo1gene does not disturb testis development in XY males or XX sex-reversed mice [Buscara et al., 2009].
RSPO1 is a secreted protein belonging to a small family of growth factors. It is implicated in the activation of Wnt4 andβ-catenin pathways. It has now been acknowledged that the synergy of RSPO1 and WNT4 is essential for normal ovarian development.
To conclude, conversely to testis development, there is not one but several ovary determining factors and yet others may still be discovered.
1.2.2 The Current State of Mammalian Gonadal Development
Although the genetic sex of mammals is cast at fertilization, with the introduction of an X or a Y chromosome by a spermatozoa to a bearing X oocyte, embryos do not display any sexual phenotypic differences during a long period of ambiguity. The first event that triggers the development of male or female characteristics is the differentiation of the gonads as testes or ovaries. Since the discovery ofSryin 1990, gonadal development and sex determination have been studied intensively. However, major questions remain regarding different aspects covering the whole gonadal development, from the genital ridge formation in the embryo to the functional organs in the adult. The following sections summarize current knowledge concerning the embryonic development of the gonads, in both males and females.
The Genital Ridge Formation and the Bipotential Gonads
The genital ridges, or adreno-gonadal primordium (AGP), constitute the primordium from which the ovaries and testes develop, as well as the adrenal glands [Hatano et al., 1996]. These appear at between four and five weeks of gestation in humans, and around embryonic day E9.5 in mice5from a precise region of the ventral surface of the intermediate mesoderm. The genital ridges develop as paired narrow bands of proliferating cells on either sides of the dorsal mesentery (figures 1.15a and 1.15b).
The growth of the genital ridges occurs by the proliferation of the coelomic epithelium (CE) and subsequently by the fragmentation of the underlying basement membrane, allowing the delami- nation of the proliferating CE into the inner mesenchymal region of the mesonephros [Karl and Capel, 1998, Schmahl et al., 2000] (figure 1.15c). Genital ridge thickening starts at E10.3 following an anteroposterior axis, that is, the anterior part is composed of multi-layers of cells, while the posterior part is still single-layered [Hu et al., 2013]. By E10.5, the adrenal primordium separates from the genital ridge. It has been postulated that the anterior part contributes to the adrenal primordium, and the posterior part to the gonadal primordium [Ikeda et al., 1994, Hatano et al., 1996, Bandiera et al., 2013]. Concurrently with the thickening, the genital ridges are colonized by the migrating primordial germ cells (PGCs). The PGCs develop far from the gonads and have to travel a long way to reach their final destination. The PGCs appear in the epiblast of mice and migrate from the primitive streak to the endoderm around E7.5. At E8.0, they migrate along the endoderm (the future hindgut) and by E9.5 into the dorsal mesentery. Finally, the PGCs reach the genital ridges at E10.5 to settle in the gonads (figure 1.15c, for review, see [Richardson and Lehmann, 2010]).
The molecular control of the genital ridge formation remains elusive. Many important factors have been identified by studying mutant mice, providing partial but precious information about the genetic regulation of gonadogenesis (table 1.1).
One of the first expressed critical factors for gonad formation isWt1(Wilms’ tumor 1). This zinc- finger transcription factor is known to play multiple roles in the development of various organs such
5The age of mouse embryos is generally defined by counting the total somites or, more commonly, the tail somites.
However, we found discrepancies in different studies regarding the exact age when the genital ridges begin to develop.
Various studies state that the genital ridges develop from E9.0, E9.5, E10.0, or E10.5 [Hacker et al., 1995, Karl and Capel, 1995, Nef and Parada, 2000, Chen et al., 2012, Tanaka and Nishinakamura, 2014].
(a) Mouse embryo at E10.
carrying theTg(Nr5a1-GFP) transgene [Stallings et al., 2002]. The genital ridge ap- pears in green (merging of bright field and UV light).
(b)Transversal section of the E10.5Tg(Nr5a1- GFP)embryo. Immunohistochemistry against GFP reveals the genital ridges composed of a single layer of coelomic epithelial cells.
(c)Schematic representation of the genital ridge formation (adapted from [Svingen and Koopman, 2013]).
Figure 1.15 –Genital ridge formation in mouse
as the kidneys, gonads, heart and nervous system.Wt1is expressed in the whole urogenital ridge (comprising the future gonads and kidneys) from E9.5. TheWt1gene encodes 24 isoforms, one of which is essential for the genital ridge development: WT1-KTS. Disruption of the WT1-KTS isoform causes increased cell death, leading to impaired genital ridges [Kreidberg et al., 1993]. The earliest gene expressed specifically in the genital ridges is the evolutionary conserved transcription factor Gata4(GATA-binding protein 4) [Hu et al., 2013].Gata4is expressed in the CE cells of the anterior part of the genital ridge as early as E10.0, and its expression extends along the anteroposterior axis until E10.4. Mice lackingGata4fail to initiate the thickening of the genital ridge due to impared CE cell proliferation and the absence of basement membrane fragmentation. Gata4deletion also negatively impacts the expression ofNr5a1andLhx9, two other key regulators of genital ridge development (table 1.1). Nr5a1has been identified as a determinant regulator of genital ridge development, AGP separation, and later, gonadal supporting and steroidogenic cell lineage differentiation [Hatano et al., 1996, Park et al., 2005, Lasala et al., 2011, Shima et al., 2012]. The expression pattern ofNr5a1is the same asGata4but slightly delayed, suggesting thatGata4might promoteNr5a1expression [Hu et al., 2013]. In mutant mice with a heterozygous deletion ofNr5a1 (Nr5a1+/-), embryos display undeveloped adrenal glands and a reduction of cell proliferation [Bland
Table 1.1 –Critical factors involved in the genital ridge development.
Genes Full names Phenotypes References
Nr5a1 (Ad4BP, Sf1)
Nuclear receptor sub- family 5, group A, mem- ber 1
Constitutive knockout ofNr5a1causes the degen- eration of the gonadal ridge by apoptosis and com- plete absence of both the adrenal glands and the gonads.
[Luo et al., 1994, Sadovsky et al., 1995]
Wt1 Wilms’ tumor 1 Constitutive induced mutation ofWt1leads to a disruption of the urogenital development, in particular to the absence of gonads.
[Kreidberg et al., 1993]
Lhx9 LIM homeobox 9 ConstitutiveLhx9knockout mice show absence of gonads due to coelomic epithelial cell prolif- eration failure at E10.5.Lhx9knockout does not affect the separation of the adrenal primordium.
[Birk et al., 2000]
Emx2 Empty spiracles home- obox 2
ConstitutiveEmx2knockout mice display a fail- ure of the genital ridge thickening leading to the absence of gonads, but the adrenal gland develop- ment is not affected.
[Miyamoto et al., 1997]
Six1 and Six4
SIX Homeobox 1 and 4 Six1/Six4double knockout results in smaller go- nads with a male-to-female sex-reversal due to an impairedSryexpression.Six1/Six4regulates Nr5a1andZfpm2, a direct regulator ofSry.
[Fujimoto et al., 2013]
Gata4 GATA-binding protein 4 Conditonal knockout ofGata4induced at E8.75 impairs epithelial proliferation and basement membrane fragmentation. Gata4 controls the ex- pression ofNr5a1andLhx9.
[Hu et al., 2013]
Insr and Igf1r
Insulin receptor and IGF receptor 1
InsrandIgf1rdouble knockout affects the expres- sion ofNr5a1and reduces the proliferation rates of the somatic progenitor cells in both XX and XY prior sex determination. Mice present a male- to-female sex reversal and complete absence of adrenal glands.
[Nef et al., 2003, Pitetti et al., 2013]
et al., 2004]. Homozygous deletion ofNr5a1leads to the absence of adrenal glands; however, the genital ridge thickening initiation is present but reduced, and the gonads regress via apoptosis by E12.5 [Luo et al., 1994, Sadovsky et al., 1995].
Once the adrenal primordium has split from the genital ridge, the gonadal primordium constitutes the bipotential gonad at the origin of both testis and ovary. The XX and XY bipotential gonads are transcriptionally identical, except for the genes located on the Y chromosome, and the X inactivation genes [Nef et al., 2005, Munger et al., 2013]. Lineage tracing experiments have demonstrated that the gonadal somatic progenitor cells from the genital ridge contribute to multiple cell lineages of the testis and the ovary [Karl and Capel, 1998, Schmahl et al., 2000, DeFalco et al., 2011, Liu et al., 2015, Liu et al., 2016]. These lineages include the supporting cell lineage (the Sertoli and granulosa cells) and the steroidogenic cells (the foetal and adult Leydig cells, and theca cells). Whether the cell lineages are already predefined in the bipotential gonads prior to sex determination is a matter of debate.
Male Sex Determination and Testis Development
Sryswitches on maleness Male sex determination is set at between six to seven weeks of gestation in humans and around E11.0 in mice. The differentiation of the bipotential gonads as testes relies on the presence of the Y chromosome, more precisely, on the presence of theSrygene located on
