Thesis
Reference
Functions of small RNAs and Dicer1 in mammalian spermatogenesis
ROMERO, Yannick
Abstract
Sexual reproduction in mammals requires production and subsequent fusion of functional gametes originating from individuals of opposite sex, in order to perpetuate the species. In male mammals, production of spermatozoa occurs in the testis throughout the biological process named spermatogenesis. MicroRNAs and endogenous small-interfering RNAs inhibit the protein synthesis by silencing both the translation initiation and elongation or by activating the mRNA degradation. Their biogenesis depends on DICER1. We developed a transgenic mouse model in which the Dicer1 gene was inactivated in a specific and fully penetrant manner in the male germ cell lineage. Our results indicate that Dicer1 is required for completion of normal spermatogenesis, since its deletion leads to cumulative defects in meiosis and spermiogenesis resulting in the absence of functional spermatozoa and thus to complete infertility. In this perspective, we opened a new path in germ cells biology that might unravel certain issues of male infertility.
ROMERO, Yannick. Functions of small RNAs and Dicer1 in mammalian spermatogenesis. Thèse de doctorat : Univ. Genève, 2011, no. Sc. 4367
URN : urn:nbn:ch:unige-180246
DOI : 10.13097/archive-ouverte/unige:18024
Available at:
http://archive-ouverte.unige.ch/unige:18024
Disclaimer: layout of this document may differ from the published version.
1 / 1
UNIVERSITÉ DE GENÈVE
Département de Génétique FACULTÉ DES SCIENCES
et Évolution Professeur Ivan Rodriguez
Département de Médecine Génétique FACULTÉ DE MÉDECINE
et Développement Professeur Jean-Dominique Vassalli
Functions of Small RNAs and Dicer1 in Mammalian Spermatogenesis
THÈSE
présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Yannick Romero de
Grenoble (France)
Thèse n°4367
GENÈVE 2011
Atelier d’impression ReproMail, Uni Mail
UN IVE RSITÉ DE GENÈVE
FACULTÉ DES SCIENCES
Doctorat ès sciences Mention biologie
Thèse de
Monsieur Yannick ROM EPO
intitulée
“Functions of SmaII RNAs and Diceri in Mommalian Spermotogenesis”
La Faculté des sciences, sur le préavis de Messieurs J.-D. VASSALLI, professeur ordinaire et directeur de thèse (Faculté de médecine, Département de médecine génétique et développement), I. RODRIGUEZ, professeur ordinaire et codirecteur de thèse (Département de génétique et évolution), S. NEF, docteur (Faculté de médecine, Département de médecine génétique et développement) et de Madame N. KOTAJA, docteure (Institute of Biomedicine, University of Turku, Finland), autorise l’impression de la présente thèse, sans exprimer d’opinion sur les propositions qui y sont énoncées.
Genève, le 4 novembre 2011
Thèse
-4367
-Le Doye Jean-Marc TRISCONE
N.B.- La thèse doit porter la déclaration précédente et remplir les conditions énumérées dans les ‘Informations relatives aux thèses de doctorat à l’université de Genève’.
1 CONTENTS
SUMMARY ... 4
RESUME EN FRANÇAIS ... 6
ABBREVIATIONS ... 8
I. INTRODUCTION ... 10
A. STRUCTURE AND FUNCTION OF THE MAMMALIAN TESTIS ... 10
A.1. Testicular development ... 10
A.2. Structure of the adult testis ... 13
A.2.a. The interstitial compartment ... 13
A.2.b. The seminiferous tubule ... 15
A.3. Spermatogenesis ... 17
A.3.a. The proliferative phase of spermatogenesis ... 17
A.3.b. The meiotic phase ... 20
A.3.b.1. Preleptotene and leptotene stages ... 20
A.3.b.2. Zygotene stage ... 22
A.3.b.3. Pachytene stage ... 23
A.3.b.4. Diplotene stage, Meiosis I and Meiosis II divisions ... 25
A.3.b.5. Histone dynamics during Meiosis I ... 27
A.3.c. The post-meiotic maturation phase ... 28
A.3.c.1. Formation of the flagellum ... 30
A.3.c.2. Development of the acrosome structure ... 31
A.3.c.3. Nuclear condensation and DNA compaction ... 33
A.3.c.4. Cytoplasm elimination and residual body formation ... 34
A.3.c.5. Histone modifications ... 34
A.3.d. Maturation of the spermatozoon ... 35
A.3.e. The spermatogenic and seminiferous epithelium cycles ... 37
A.3.f. Hormonal regulation of spermatogenesis ... 39
A.4. Phenotypes of spermatogenic failures... 42
A.5. Post-transcriptional gene control during spermatogenesis ... 43
A.5.a. Alternative splicing ... 43
A.5.b. General translational control of gene expression ... 44
A.5.c. 3’UTR mediated translational control ... 45
B. POST-TRANSCRIPTIONAL REGULATION MEDIATED BY NON-CODING RNAs ... 47
B.1. microRNA mediated post-transcriptional control ... 47
B.1.a. microRNA genes ... 48
B.1.b. miRNA biogenesis ... 49
B.1.c. Function and role of microRNAs ... 52
B.1.c.1.mRNA degradation ... 53
B.1.c.2. Regulation at the initiation step of protein synthesis ... 54
B.1.c.3. Regulation at the elongation step of protein synthesis ... 54
B.1.c.4. miRNAs are genetic fine-tuners ... 56
B.1.d. Storage of repressed mRNAs ... 56
B.2. siRNAs mediated post-transcriptional control ... 58
B.3. PiRNAs ... 60
2
B.4. Transposable elements processing ... 60
B.4.a. SINE transposons ... 62
B.4.b. Regulation of transposable RNAs activity in organisms ... 63
B.4.c. Function and role of SINE RNA in transcriptional inhibition ... 64
B.5. The pivotal role of DICER1 ... 66
B.5.a. Structure and molecular function of DICER1 ... 66
B.5.b. Role of Dicer1 in mammals: Dicer1 Knockouts ... 68
C. DICER1 FUNCTIONS IN REPRODUCTION ... 71
C.1. DICER1 function in ovarian cells ... 71
C.1.a. Depletion of Dicer1 in female somatic lineage ... 71
C.1.b. Dicer1 ablation in the female germ cell lineage ... 72
C.2. DICER1 function in the testis ... 73
C.2.a. Essential role for Dicer1 in somatic cells of the testis ... 73
C.2.b. Role of Dicer1 and miRNAs in male germ cells ... 74
AIM OF THE THESIS ... 77
II. RESULTS ... 78
A. Assessing the role of Dicer1 in germ cells during mammalian spermatogenesis (Research article) ... 78
B. Depletion of Dicer1 in mammalian embryonic gonads ... 95
B.1. The HS:Cre; Dicer1lox mice ... 96
B.1.a. Material and methods ... 97
B.1.b. Results ... 99
B.1.c. Conclusions ... 103
B.2. The Ck19:Cre; Dicer1lox mice ... 104
B.2.a. Material and methods ... 104
B.2.b. Results ... 105
B.2.c. Conclusions ... 108
B.3. The Wt1-GFP:Cre; Dicer1lox mice ... 108
B.3.a. Material and methods ... 108
B.3.b. Results ... 109
B.3.c. Conclusions ... 111
B.4. The inducible Wt1:CreERT2; Dicer1lox mice ... 111
B.4.a. Material and methods ... 112
B.4.b. Results ... 112
B.4.c. Conclusions ... 114
B.5. General Conclusions ... 114
III. ADDITIONAL RESULTS AND PUBLICATIONS ... 118
A. Research article: “Dicer Is Required for Haploid Male Germ Cell Differentiation in Mice.” ... 118
B. Review article in preparation: “ARN non-codants et Spermatogenèse” ... 131
C. Manuscript in preparation: “The Glucocorticoid-induced leucine zipper (GILZ) is essential for spermatogonial survival in mice” ... 142
D. Manuscript in preparation: Role of IR and IGF1R in adreno-gonadal primordium development. ... 161
3
IV. DISCUSSION AND PERSPECTIVES ... 197
V. REFERENCES ... 207
VI. ACKNOWLEDGMENTS ... 222
Illustration table Figure 1. Schematic representation of sex determination and differentiation in mice. ... 11
Figure 2. Anatomy of the mammalian testis. ... 14
Figure 3. Schematic representation of the seminiferous epithelium. ... 16
Figure 4. Potential number of germ cells. ... 19
Figure 5. The meiotic cycle of male germ cells. ... 21
Figure 6. Schematic representation of chromosomal structure during meiotic prophase I in germ cells. ... 21
Figure 7. Spermatocytes at different meiotic prophase I stages. ... 24
Figure 8. Caspase-dependent apoptotic pathways involved in germ cell development. ... 26
Figure 9. Developmental stages and expression of associated genes of haploid germ cells during spermiogenesis. ... 29
Figure 10. Electron microscopy of mouse spermatozoa. ... 36
Figure 11. Murine seminiferous epithelium cycle. ... 38
Figure 12. Hormonal regulation of spermatogenesis. ... 41
Figure 13. Canonical and non-canonical miRNA biogenesis. ... 51
Figure 14. Exogenous and endogenous-siRNA biogenesis. ... 59
Figure 15. Biogenesis of male germ cells Piwi-interacting RNAs. ... 61
Figure 16. Structure of SINE B2 RNA and its role on RNA pol II activation. ... 65
Figure 17. DICER1 is at the centre of multiple molecular pathways. ... 70
4
SUMMARY
Sexual reproduction in mammals requires production and subsequent fusion of functional gametes originating from individuals of opposite sex, in order to perpetuate the species. In mammals, the testis is the male reproductive organ that allows the production of spermatozoa, the male gametes, throughout the biological process named spermatogenesis. The development and differentiation of diploid germ cells into haploid mature spermatozoa is a complex process involving the nutritional, structural, and hormonal support of multiple testis
‘cell types, as well as intrinsic genetic regulation. Post-transcriptional gene regulation is one of the main genetic events occurring in the germinal compartment. For instance, during the haploid phase of spermatogenesis, namely spermiogenesis, the high DNA compaction prevents gene transcription. However, essential proteins are required for differentiation from round to elongated spermatids. Thus, transcripts are stored before undergoing translation.
Since a decade, a novel mechanism of post-transcriptional and translational regulation mediated by small non-coding RNAs was discovered. Small non-coding RNAs, namely microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs) are able to bind the 3’UTR of target transcripts and inhibit the protein synthesis by silencing both the translation initiation and elongation or by activating the mRNA degradation. The biogenesis of this novel class of genetic regulators is dependent on multiple enzymes, including the RNAse III endonuclease named DICER1. In the testis, the specific depletion of Dicer1 in Sertoli cells leads to infertility, and the partial absence of DICER1 in embryonic germ cells leads to sub-fertility. These evidences suggest that small RNA machinery is essential for spermatogenesis. To confirm this hypothesis, we developed a transgenic mouse model in which the Dicer1 gene was inactivated in a specific and fully penetrant manner in the male germ cell lineage. We conducted an in-depth analysis of the phenotype of Dicer1 mutant
5 throughout the spermatogenesis process. It allowed us to better characterize the role of small RNA biogenesis machinery in the germ cells biology.
Our results indicate that Dicer1 is required for completion of normal spermatogenesis, since the deletion of Dicer1 in male germ cells leads to multiple cumulative defects in meiosis and spermiogenesis resulting in the absence of functional spermatozoa and thus to complete infertility. We found that meiotic prophase I progression was delayed, and apoptosis was hugely increased at the pachytene stage. Moreover, late prophase I stage was altered with an increased number of metaphasic cells, which presents abnormal metaphasic plates. The remaining cells that could undergo meiotic division displayed abnormal developmental features during spermiogenesis such as acrosome formation and nuclear condensation defects accompanied with mitochondria hyperplasia. Analysis of the very few remaining non-motile epididymal sperm (representing 1% compared to control individuals) revealed tremendous defects of the head and flagellum shape. Moreover, transposable elements such as SINE RNAs are up-regulated in mutant germ cells, suggesting that they might contribute to such phenotype. It is obvious that further analyses are required for a better comprehension of the roles and impact of the small non-coding RNA machinery in the completion of spermatogenesis. In this perspective, we opened a new path in germ cells biology that might unravel certain issues of male infertility.
6
RESUME EN FRANÇAIS
La reproduction sexuelle chez les mammifères nécessite la production et la fusion ultérieure de gamètes fonctionnels provenant d’individus de sexes opposés, ceci afin de perpétuer l’espèce. Chez les mammifères, le testicule est l’organe reproducteur mâle qui permet la production de spermatozoïdes, les gamètes mâles, grâce au processus biologique appelé spermatogenèse. Le développement et la différenciation de cellules germinales diploïdes en spermatozoïdes haploïdes matures est un processus complexe impliquant le support nutritionnel, structurel et hormonal de plusieurs types cellulaires du testicule ainsi qu’une régulation génétique intrinsèque. La régulation génétique post-transcriptionnelle est l’un des évènements génétiques principaux qui survient dans le compartiment germinal. Pendant la phase haploïde de la spermatogenèse, c’est-à-dire la spermiogenèse, la forte compaction de l’ADN empêche la transcription génétique. Cependant, des facteurs essentiels sont requis pour la différenciation de spermatides rondes en spermatides allongées. Donc les transcrits sont stockés avant d’être traduits. Depuis une décennie, un nouveau mécanisme de régulation post-transcriptionnelle et traductionnelle s’effectuant par l’intermédiaire de petits ARN non- codants a été découvert. Les petits ARN non-codants, c’est-à-dire les microRNAs (miRNAs) et les petits ARN interférents endogènes (endo-siRNAs) sont capables de se lier à la partie 3’UTR d’ARN messagers cibles et d’inhiber leur synthèse protéique en les réprimant soit au niveau de l’initiation et l’élongation de la traduction soit en activant la dégradation de l’ARNm lui-même. La biogénèse de cette nouvelle classe de régulateurs génétiques est dépendante de plusieurs enzymes, incluant l’endonucléase Ribonucléase III nommé DICER1.
Dans le testicule, la déplétion spécifique de Dicer1 dans les cellules de Sertoli aboutit à l’infertilité, et l’absence partielle de DICER1 dans les cellules germinales embryonnaires mène à une sous-fertilité. Ces preuves suggèrent que la machinerie des petits ARNs est essentielle pour la spermatogenèse. Afin de confirmer cette hypothèse, nous avons développé
7 un modèle murin transgénique dans lequel le gène Dicer1 est inactivé d’une manière spécifique et complètement efficace dans les cellules germinales mâles. Nous avons mené une analyse en profondeur du phénotype des mutants Dicer1 tout au long du processus de spermatogenèse. Cela nous a permis de mieux caractériser le rôle de la machinerie de biogénèse des petits ARNs dans la biologie des cellules germinales. Nos résultats indiquent que Dicer1 est requis pour la réalisation d’une spermatogenèse normale, puisque la suppression de Dicer1 dans les cellules germinales aboutit à de multiples défauts cumulatifs de méiose et de spermiogenèse, résultant en une absence de spermatozoïdes fonctionnels et une infertilité complète. Nous avons montré que la progression de la prophase I de la méiose était différée, et que l’apoptose augmentait de manière importante au stade pachytène. De plus, la phase tardive de prophase I est altérée accompagnée d’une augmentation de cellules en métaphase qui présentent une plaque métaphasique anormale. Les cellules restantes qui ont pu atteindre la division méiotique présentent des traits développementaux anormaux tels que des défauts de formation de l’acrosome et de condensation nucléaire accompagnés d’une hyperplasie des mitochondries. L’analyse du peu de spermatozoïdes non-motiles restants (représentant 1%, comparé aux individus contrôle) a révélé des défauts de la forme de la tête et du flagelle. De plus les éléments transposables tels que les ARN SINE sont surexprimés dans les cellules germinales mutantes, ce qui suggère qu’ils puissent contribuer à un tel phénotype. Il est évident que des analyses plus poussées sont requises pour une meilleure compréhension du rôle et de l’impact de la machinerie des petits ARNs non-codants dans la réalisation de la spermatogenèse. Dans cette perspective, nous avons ouvert une nouvelle voie dans la biologie des cellules germinales qui pourrait élucider certaines questions sur l’infertilité masculine.
8
ABBREVIATIONS
Ace : Acetylation AE : Axial Element BTB : Blood-Testis Barrier
cDNA: complementary DNA
CSF1 : Colony Stimulating Factor 1
D : Diplotene
DCR1 : DICER1 protein Dcr1: Dicer1 gene
DNA : DeoxyriboNucleic Acid DS : Double Strand
DSB : Double Strand Breaks
E: Embryonic day
EM: Electron Microscopy
endo-siRNA: endogenous small interfering RNA FSH: Follicle-Stimulating Hormone
GC: Germ Cell
GnRH: Gonadotropin-Releasing Hormone
H3: Histone H3
HS: Heat-Shock
IAP: Intracisternal-A-Particle
IF: ImmunoFluorescence
IHC: ImmunoHistoChemistry Int: Intermediary
K: Lysine
K.O.: Knock-Out
Kb: Kilobase
KDa: KiloDalton
L: Leptotene
LE: Lateral Element LH: Luteinizing Hormone
LINE: Long Interspersed Nuclear Element
Me : Methylation
MI : Meiosis I
MII : Meiosis II
miRNA.: microRNA
MSCI: Meiotic Sex Chromatin Inactivation
Nt: Nucleotides
ORF: Open Reading Frame
P: Pachytene
P5: Post-natal day 5
PCR: Polymerase Chain Reaction PGC: Primordial Germ Cell
PGD2: Prostaglandin
pi-RNA: PIWI- interacting RNA
Pl: Pre-leptotene
PMSR: Post-Meiotic Sex Chromosome Repression
Pol: Polymerase
9 PTGS: Post-Transcriptional Gene Silencing
RA: Retinoic Acid
RNA : RiboNucleic Acid RNP: RiboNucleoParticle SC: Synaptonemal Complex
SCP: Synaptonemal Complex Protein SINE: Short Interspersed Nuclear Element SPCTES: Spermatocytes
SPGA: Spermatogonia A
SPGB: Spermatogonia B
SPTDS: Spermatids
SPZ: Spermatozoa
SS: Single Strand
SSC: Spermatogonial Stem Cell UTR: Untranslated Region
Z : Zygotene
10
I. INTRODUCTION
A. STRUCTURE AND FUNCTION OF THE MAMMALIAN TESTIS
Sexual reproduction is the biological process by which new generations of individuals arise from the coupling of two begetters from opposite sex. The testis, in mammals, is the main component of the male reproductive system and is devoted to the production of the male gametes, the spermatozoon. The precise homeostasis of the testis is thus required for reproduction, since functional spermatozoa are essential for the fertilization of the female gamete, namely the oocyte, allowing the perpetuation of the species.
In this chapter, I will first describe how the testis develops during foetal and post-natal life. I will next dedicate part of this chapter to the description of spermatogenesis and its alterations.
Finally, this chapter will summarize regulation of spermatogenesis at the post-transcriptional level.
A.1. Testicular development
In mammals, sex is defined by the nature of sex chromosomes, XX in females that trigger the development of ovaries and XY in males that trigger the development of testes. Both organs originate from a common and undifferentiated embryonic structure, the bipotential gonad, which develops from the intermediate mesoderm as a paired structure on the ventromedial surface of the mesonephros. Around embryonic day 10 (E10) in mouse, the bipotential gonad starts to differentiate and will further give rise to a testis or an ovary from E11.5 onward.
11 Figure 1. Schematic representation of sex determination and differentiation in mice.
The bipotential gonad develops as either a testis or an ovary during the sex determination period according to the Y-encoded gene Sry, which triggers the male genetic program (mediated by Sox9, FGF9 and PGD2). In the absence of Sry, the development of an ovary is dependent of RspoI, Wnt4 and FoxL2 (and BMP2, not shown here (Kashimada, Pelosi et al.
2011)) genes. Finally, either Leydig (males) or theca (females) cells produced hormones that allow the masculinization or feminization of the rest of the embryo. Adapted from(Kashimada and Koopman 2010).
12 The bipotential gonad is composed of somatic cells, which originate from the embryonic mesoderm and primordial germ cells (PGCs). Between E7.5 until E9.5-E10, germ cells proliferate and migrate from the basis of the allantois along the hindgut in order to populate the genital ridges; at this time they are called gonocytes (Tam and Snow 1981; Ginsburg, Snow et al. 1990). The differentiation of the bipotential gonad into a testis depends on the expression of the Y-encoded gene Sry (Sex-determining Region of Y chromosome). Actually, a subset of Sertoli cells expresses Sry transiently and specifically between E10.5 to E12.5.
SRY triggers the activation of the male genetic program via the up-regulation of Sox9.
Subsequently, SOX9 triggers the up-regulation of Fgf9 and increases PGD2 expression. FGF9 and PGD2 also control Sox9 expression in an autocrine/paracrine regulatory loop, leading to the differentiation of Sertoli cells. Moreover, paracrine signals mediated by PGD2 allow the recruitment of supplemental Sertoli cells in the male differentiation process (Koopman, Munsterberg et al. 1990; Brennan and Capel 2004). It is noteworthy that in the absence of Sry, in XX gonads, antagonistic signals such as Rspo1/Wnt4/β-catenin and FoxL2 pathways repress male commitment and promote the female differentiation process (Parma, Radi et al.
2006; Chassot, Ranc et al. 2008) and (Figure 1). In XY embryos, around E12, committed Sertoli cells surround gonocytes in order to form testicular cords, themselves surrounded by peritubular cells, which will ultimately give rise to the seminiferous tubules in adult. In the meantime, the Leydig cells start to differentiate within the interstitial tissue and secrete the androgens necessary for the masculinization of the Wolffian derivatives and the urogenital system. Germ cells and somatic cells then enter a proliferation phase leading to an increase in size of the developing testis. A quiescent step follows this germ cells proliferation phase until the perinatal period where they will enter a differentiation phase to give rise to spermatogonia (Boulogne, Olaso et al. 1999).
13 A.2. Structure of the adult testis
The mammalian adult testes are ovoid organs found in different locations depending on species. In mice and humans, they lie outside the abdominal cavity in a peritoneal invagination, named the scrotum (descended and scrotal). In mouse and human, the testicular descent occurs in two sequential phases (the transabdominal and the inguino-scrotal descent) and is under the control of Leydig cells-produced hormones such as INSL3 (Insulin-like 3) (Nef and Parada 1999; Werdelin and Nilsonne 1999).
The testicular structure is relatively conserved during evolution. A fibrous and muscular connective tissue, the Tunica albuginea surrounds the testis. The testis is composed of two nested compartments, the interstitial tissue and the seminiferous tubules. The testis fulfils both an endocrine function since it secretes androgens and an exocrine function since it secretes and releases mature spermatozoa (Figure 2).
A.2.a. The interstitial compartment
The interstitial tissue (representing approximately 8% of the testis in rat) is a connective tissue composed of multiple structures and cell types, mainly Leydig cells, macrophages and lymphocytes (Russell and de Franca 1995). The major role of Leydig cells is to produce and secrete hormones in particular testosterone. The hypothalamic-pituitary-testicular axis hormonal control will take part into the development of the testis after birth in mouse (see section A.3.f.), and, androgens are essential for the maintenance of spermatogenesis. Immune cells are widely represented in the interstitial compartment of the normal, unaffected testis in order to defend it against infection. Indeed, inflammation and infection of the testis can disrupt spermatogenesis and lead to infertility (Schuppe and Meinhardt 2005).
14 Figure 2. Anatomy of the mammalian testis.
The mammalian testis is ovoid and surrounded by a fibrous and muscular connective tissue:
the Tunica albuginea (Albuginea). Two nested compartments are present: the seminiferous tubules and the interstitial tissue located between tubules. Germ cells differentiate within the tubules and give rise to spermatozoa, which pass through the rete testis and the epididymis, where they acquire their motility. Mature spermatozoa accumulate in the Vas deferens and/or are finally ejaculated. For details, see section (A.2). Adapted from (Calvel 2010).
15 A.2.b. The seminiferous tubule
The seminiferous tubule is the place where spermatogenesis occurs (Figure 3). It allows the production of haploid gametes from diploid spermatogonial stem cells (SSCs), following a series of proliferation, differentiation and maturation steps. The tubule is surrounded by peritubular myoid cells that are contractile, allowing the flowing of sperm through the tubules (Kormano and Hovatta 1972). Tubules have at their periphery a basal lamina, secreted by both Sertoli and peritubular cells, on which rests the seminiferous epithelium. Sertoli cells also provide a structural and nutritional support for germ cells, allowing them to differentiate and migrate in a centripetal manner until they reach the lumen as spermatozoa. The polarity of Sertoli cells allows the organization of the epithelium. Indeed, Sertoli cells established tight junctions between them that split the epithelium into a basal compartment, in contact with the basal lamina, and an adluminal (apical) compartment, containing the most differentiated germ cells. The selective barrier, which appears before germ cell meiosis, is a gatekeeper of the seminiferous epithelium’s integrity. Indeed, expression of specific genes such as occludin and claudin 3, 5 and 11 at the Sertoli cells barrier protects germ cells against chemicals and drugs (Mruk and Cheng 2004) and against the immune system (Morrow, Mruk et al. 2010). The role of the tight junctions is not only protective; it allows selective sorting of nutrients and provides a microenvironment for SSCs where multiple factors, including diffusible GDNF (glial-derived neurotrophic factor) are essential for the germ cells maintenance (see section A.3.a.). Indeed, depletion of SSCs is observed in GDNF-null allele mouse whereas overexpression of GDNF in Sertoli cells lead to an accumulation of non-differentiated spermatogonia, favouring their self-renewal (Meng, Lindahl et al. 2000; Chen, Ouyang et al.
2005).
16 Figure 3. Schematic representation of the seminiferous epithelium.
Interstitial cells (only Leydig cells are represented) are depicted at the top of the scheme. The interstitial tissue ensures the endocrine function of the testis by producing and secreting androgens. The place where germ cells differentiate into male gametes is the seminiferous epithelium. Sertoli cells support nutritionally and structurally germ cells that differentiate in a centripetal manner. Spermatozoa are released in the lumen. For details, see section (A.2.a and A.2.b). Adapted from (Calvel 2010).
17 A.3. Spermatogenesis
The spermatogenic process refers to the differentiation and maturation of diploid spermatogonial cells to generate haploid spermatozoon. Spermatogenesis is composed of three main phases. First, the proliferation step whereby spermatogonia undergo a series of mitotic events, followed by a meiotic phase during which germ cells become haploid and, finally spermiogenesis which corresponds to the maturation of haploid round spermatids into spermatozoa, that will be released into the lumen after spermiation.
A.3.a. The proliferative phase of spermatogenesis
The daily and continuous production of millions of gametes throughout adult male life relies on the presence of spermatogonia that reside at the base of the seminiferous epithelium. They are flattened in basal pole and have a rounded surface in contact with Sertoli cells. There are three different types of spermatogonia; the spermatogonial stem cell (SSC), the proliferative spermatogonia and the differentiating spermatogonia.
The self-renewal ability of SSCs is necessary for the maintenance of the spermatogenic process. Interestingly, SSCs were shown to be highly resistant to radiation and to survive more easily compared to other germ cell types (Dym and Clermont 1970). The maintenance of SSCs is dependent on the homing of these cells in a protective microenvironment, provided by somatic surrounding cells, as well as on their intrinsic molecular characteristics. Multiple Sertoli’s specific factors such as GDNF, ERM, Kit-L (through the activation of the PI3- Kinase pathway) and ID4 were necessary for their proliferation, survival and differentiation (Blume-Jensen, Jiang et al. 2000; Kissel, Timokhina et al. 2000; Meng, Lindahl et al. 2000;
Chen, Ouyang et al. 2005; Oatley, Kaucher et al. 2011).
More recently, the work of Oatley and co-workers on purified primary Thy1+ germ cells (known to be a SSC marker) showed that SSCs express a specific growth factor receptor, CSF1-R, and that Thy1+ cells responded, in vitro, to CSF1. Interestingly, addition of
18 recombinant CSF1 in SSCs cultures, supplemented with GDNF and FGF2, enhanced their self-renewal (Oatley, Oatley et al. 2009). The expression of CSF1 was found principally in Leydig cells, and to a lower extent in peritubular myoid cells in pre-pubertal and young adult mice. Taken together, these results show that the proliferation phase depends not only on the spermatogonia themselves but also on somatic cells (i.e. Sertoli, Leydig and peritubular myoid cells); they could explain why the niche is close to the interstitial tissue and blood vessels.
The mechanism of differentiation and proliferation of SSCs is still not clear, but the model of Huckins and Oakberg described below is now currently accepted. In mouse, SSCs (also called Aisolated or Asingle) give rise to multiple spermatogonial cell types recognizable by their morphology. Type A spermatogonia display no heterochromatin features, differentiating intermediate (In) spermatogonia contain moderate amounts of heterochromatin and type B spermatogonia display large amounts of heterochromatin. Actually, Aisolated cells give rise to either one Aisolated or two proliferative type A spermatogonia (Apr: Apaired). Apr spermatogonia undergo a series of cell divisions and differentiate into a cohort of 4, 8 then 16 Aal (Aaligned or A1, Figure 4) spermatogonia linked by cytoplasmic bridges and developing as a syncytium, which allows them to share molecular factors. Apr and Aal are referred as transient amplifying progenitors giving rise to A1 spermatogonia by maturing without further dividing. These then undergo a series of cell divisions proliferating into spermatogonia A2, A3 and A4. Finally, type A spermatogonia differentiate after cell divisions into Intermediate and subsequently type B spermatogonia (Huckins 1971; Oakberg 1971; de Rooij and Russell 2000; Oatley and Brinster 2008). At the end of the differentiation of B spermatogonia, they enter the crucial first meiotic phase by dividing into primary spermatocytes. In mice, by post-natal day (P)8, all spermatogonial cell types are present in the tubule. Transition between type B spermatogonia and spermatocytes is due to retinoic acid induced by Sertoli cells, its binding to
19 Figure 4. Potential number of germ cells.
16 A1 spermatogonial cells joined by cytoplasmic bridges can give rise to 4096 spermatids at the end of spermatogenesis along the multiple steps of differentiation. After mitotic proliferation (A1 to B spermatogonia), meiosis I (primary spermatocytes) and meiosis II (secondary spermatocytes) germ cells develop as haploid spermatids. Numbers on the right represent the number of cells at each step. From (Russell 1990).
20 spermatogonia RARγ (Retinoic Acid Receptor) leads to the synthesis of STRA8 (Stimulated by Retinoic Acid 8) allowing germ cells to enter into meiosis (Anderson, Baltus et al. 2008;
Mark, Jacobs et al. 2008).
A.3.b. The meiotic phase
At the end of the proliferative phase, after a last mitotic division, type B spermatogonia give rise to spermatocytes (or meiocytes), namely meiotic phase cells. These cells duplicate their DNA, becoming tetraploid, then undergo multiple cis and trans homologous recombination events between paternal and maternal chromosomes, followed by two divisions -the reduction division (Meiosis I: chromosomal segregation) and equation division (Meiosis II: chromatid separation) - in order to finally form haploid spermatids.
The first meiotic division is subdivided into the Prophase I (which is the longest step of the meiotic phase); the Metaphase I, the Anaphase I and the Telophase I. During ProphaseI, spermatocytes progress through preleptotene, leptotene (chromosomes begin to condense), zygotene (homologous chromosome pairing), pachytene (synapsis and recombination) and diplotene (chromosomal separation) steps (Figures 5, 6 &7).
A.3.b.1. Preleptotene and leptotene stages
Between P8 and P10, type B spermatogonia give rise to preleptotene spermatocytes, which directly enter the Synthetic (S) phase of the cell cycle, by immediately duplicating their DNA content. At the morphological level, preleptotene cells are very similar to type B spermatogonia except that they are smaller with less condensed perinuclear heterochromatin areas (Russell and Frank 1978).
Cells migrate from the basal side of the tubule and they become rounded during the preleptotene to leptotene transition. Moreover, heterochromatin areas gradually faint to form thin linear chromatin threads, indicating that chromosomes are condensing but not paired.
21 Figure 5. The meiotic cycle of male germ cells.
In i (grey highlighting) is depicted the first meiotic division (Meiosis I). During Interphase (a), DNA is replicated, then cells enter Prophase I (b to f, for details see section A.3.b).
Alignment and subsequent separation of chromosomes occurs on the equatorial plate during Metaphase (g). Cells divide at the Anaphase step (h) giving rise to two daughters cells at Telophase (i). Stretching of each sister chromatids to each pole of daughter cell occurs in metaphase (j) of Meiosis II (ii) and then separation at Anaphase (k). Finally, four haploid gametes by the end of Meiosis II are produced (l). (Ruwanpura, McLachlan et al. 2010).
Figure 6. Schematic representation of chromosomal structure during meiotic prophase I in germ cells.
Chromosomes structure and synapsis elements are represented according to spermatocytes developmental stages (bottom time-line). For details, see section A.3.b. (RN, Recombination Nodules). From (Baker, Plug et al. 1996).
22 The first SCs protein factors (Synaptonemal Complex Protein 2 and 3; SYCP2 and 3) position along the chromosome’s axis (AE: axial element) associated with the two sister chromatids (Moens, Heyting et al. 1987; Schalk, Dietrich et al. 1998). The analysis of Rec8 mutant mice, in which synapsis is affected and spermatocytes fail to undergo complete meiosis showed the importance of chromosomal pairing for meiosis. Rec8 is required for normal SC formation by preventing the pairing of sister chromatid instead of that of homologous chromosomes (Xu, Beasley et al. 2005). At the same time, generation of programmed double strand breaks (DSBs) occurs in order to prepare future homologous chromosomal recombination. It was shown that the trans-esterase SPO11 is necessary for the catalysis of such DSBs (Baudat, Manova et al. 2000; Romanienko and Camerini-Otero 2000). In response to SPO11-generated DSBs, the H2AX histone variant is phosphorylated by ATM (Burma, Chen et al. 2001) at serine-139 residue to become γ-H2AX. During leptotene, γ-H2AX is abundant and spreads along a large proportion of the developing axial element (Mahadevaiah, Turner et al. 2001;
Fernandez-Capetillo, Lee et al. 2004) allowing the recruitment of recombination enzymes (Baarends and Grootegoed 2003). Deletion of γ-H2AX leads to impaired spermatogenesis, arrested at the pachytene step and associated with chromosomal segregation and recombination defects (Celeste, Petersen et al. 2002) (see below).
A.3.b.2. Zygotene stage
At the zygotene stage, homologous chromosomes have paired, SCs can be observed by electron microscopy as a tripartite structure composed by the two lateral elements (LEs), originating from the AEs, and a central element linked together by transverse filaments. SC proteins localize alongside homologous chromosomes which appear thicker, γ-H2AX is present in all chromosomes and so as recombination proteins such as DMC1/RAD51 complexes. DMC1, a recombinase, which is associated with Rad51 recombinase allows the strand exchange at pachytene, localises to specific recombination foci on the AE and SC. In
23 Spo11 mutant mice, the recruitment of this complex is impaired and no foci are observed, showing the crucial role of DSBs for meiotic progression (Baudat, Manova et al. 2000).
A.3.b.3. Pachytene stage
By pachytene, the longest prophase I stage, pairing of the chromosomes occurs.
Chromosomal recombination (or crossovers, CRs) occurs during this period, allowing a rearrangement of the genetic material that leads to a genome completely different from the rest of the organism. Two main mismatch repair factors, namely MLH1 and MLH3 (MutL Homolog 1 and 3) localise to chiasmata (the chromosomal location where connections are established between sister chromatids). They are necessary for meiotic progression since their disruption leads to impaired spermatogenesis, increased apoptosis and infertility (Baker, Plug et al. 1996; Edelmann, Cohen et al. 1996; Lipkin, Moens et al. 2002).
Morphologically, pachytene spermatocytes are characterized by an increase in size compared to zygotene spermatocytes, and by the appearance of a specific structure in the nucleus, the sex body (or XY body) which contains the sex chromatin. Indeed, X and Y chromosome pairing is limited and occurs via their transcriptionally active pseudo-autosomal regions (PAR), along their short arm. Silencing of transcription in the rest of the X and Y chromosomes occurs, this transcriptional repression being termed Meiotic Sex Chromatin Inactivation (MSCI). MSCI is indispensable for spermatogenesis since pairing of X and Y chromosome and transcriptional inactivation were shown to be essential for fertility (Burgoyne, Mahadevaiah et al. 1992; Royo, Polikiewicz et al. 2010). Interestingly, 87% of X- linked miRNAs (67/77 X-linked miRNAs) escape MSCI, which suggests that they may play a crucial function, perhaps, in post-transcriptional regulation occurring at later stages (Song, Ro et al. 2009).
Several factors, including γ-H2AX (see Figure 7), localize to the sex body in a SPO11- and ATM-independent manner (Mahadevaiah, Turner et al. 2001; Barchi, Roig et al. 2008).
24 Figure 7. Spermatocytes at different meiotic prophase I stages.
(A-D) P12 (post-natal day 12) spermatocytes stained by immunofluorescence with γ-H2AX (Blue), SCP3 (Red) and DMC1 (Green) antibodies.
(A) Pre-leptotene spermatocyte. SCP3 starts to localise on the axial element and phosphorylation of H2AX histone variant into γ-H2AX begins. (B) Leptotene spermatocyte.
SCP3 is positioned along sister chromatids, γ-H2AX is widely expressed in the nucleus and DMC1 starts to be expressed in the nucleus. (C) Zygotene spermatocyte. Sister chromatids are synapsed as depicted by SCP3 staining, γ-H2AX is present in the whole nucleus and DMC1 proteins positioned at recombination sites (crossing-overs). (D) Pachytene spermatocyte. γ-H2AX is mainly present in the sex body, DMC1 is widely expressed in the sex body, sister chromatids are fully synapsed (SCP3 staining).
C D
B A
25 Consequently, γ-H2AX staining disappears from autosomal regions in conjunction with synapsis (Mahadevaiah, Turner et al. 2001). Fernandez-Capetillo and co-workers partially unravelled its role in the sex body biology. Indeed, in H2AX-deficient spermatocytes, the recruitment of sex body specific markers such as MacroH2A1.2 and XMR was impaired, and the transcriptional silencing (MSCI) did not occur (Fernandez-Capetillo, Mahadevaiah et al.
2003). This clearly demonstrated the importance of γ-H2AX in the heterochromatinization (transcriptional silencing) and synapsis of sex chromosomes.
Another specific characteristic of the pachytene stage is the increase of apoptosis, which regulates the number of germ cells per Sertoli cell and avoids the propagation of chromosomal abnormalities due to defects in synapsis or in MSCI. For review, spermatocytes unable to complete synapsis were found to be deleted by apoptosis in a p53-independent pathway (Odorisio, Rodriguez et al. 1998; Shaha, Tripathi et al. 2010). Pachytene spermatocyte’s apoptosis, mediated by BAX (B-cell leukemia/lymphoma 2-associated X protein) and BCLXL (BCL2-L1, B-cell leukemia/lymphoma 2-like 1), occurs in wild-type testis, and is required for spermatogenesis in order to maintain a suitable number of germ cells for Sertoli cell’s supporting function (Rodriguez, Ody et al. 1997). Moreover, FAS, a type I transmembrane protein, which is involved in caspase-dependent apoptosis in rat spermatocytes(Moreno, Lizama et al. 2006), is required for normal spermatogenesis mediated by Fas-L(Fas Ligand) secreted by Sertoli cells (Lizama, Alfaro et al. 2007) and (Figure 8). In summary, the pachytene stage of meiosis prophaseI is one of the most sensitive stages of spermatogenesis since multiple molecular and cellular processes occur in a coordinate manner in order to avoid the propagation of chromosomal abnormalities.
A.3.b.4. Diplotene stage, Meiosis I and Meiosis II divisions
By the diplotene stage, dysynapsis takes place. Homologous chromosomes begin to condense, preparing for separation at the Meiosis I division, and are attached by their telomeric ends and
26 A
B
Figure 8. Caspase-dependent apoptotic pathways involved in germ cell development.
(A) Three different pathways are described, the intrinsic (mitochondrial), the extrinsic (death receptor) and the endoplasmic reticulum (ER) pathways. In the mitochondrial pathway, BAX is translocated and allow the release of cytochrome C, which in turn binds to APAF1. This activates procaspase 9,by cleaving it, and in turn triggers the activation of caspases 3, 6 and 7 leading to apoptosis. The activation of death receptor is dependent on the ligation of FAS ligand (FASL). FADD (FAS-associated death domain) binds to the cytosolic part of the receptor, recruiting procaspase 8, which when activated in turn activates caspases 3, 6 and 7.
A link between these two pathways might involve the activation of BID via caspase 8: BID can cause the release of cytochrome C mediated by BAX. In the ER pathway, caspase 12 is overexpressed and activates executioner caspases 3, 6 and 7. From(Ruwanpura, McLachlan et al. 2010).
(B) Apoptosis is required for correct completion of spermatogenesis. Multiple actors of the apoptotic pathways are differentially (spatially and temporally) expressed in germ cells in order to regulate the number of germ cells, or to eliminate cells with chromosomal abnormalities or morphological defects. From (Shaha, Tripathi et al. 2010)
27 chiasmata. Diplotene spermatocytes, which are the largest germ cells, undergo, successively, the metaphase, anaphase and telophase of meiosis I, giving rise to secondary spermatocytes.
The second division is also brief, consisting in the separation of sister chromatids. Finally, secondary spermatocytes give rise to haploid round spermatids.
A.3.b.5. Histone dynamics during Meiosis I
Chromatin is composed of DNA and histones, and the way histones are modified affects the way they interact with each other. There are four main types of histones: H2A, H2B, H3 and H4, but multiple variants found in the genome (one example is H2AX; see section A.3.b.1).
The heterochromatic state (transcriptionally repressed) or euchromatic state (transcriptionally active) of chromatin can be modulated by the post-translational modification of the basic N- terminal end of histones (Lachner and Jenuwein 2002). Some arginine residues can be methylated, some lysine residues can be acetylated, methylated or ubiquitylated and some serine residues can be phosphorylated (for review see Zamudio, Chong et al. 2008). Such epigenetic modifications are involved in crucial steps of the SC formation, recombination, MSCI and sex body formation (Khalil and Wahlestedt 2008; Zamudio, Chong et al. 2008).
There are multiple examples of histone modifications necessary for the SC formation during the meiotic prophase I. For instance, histone H3 is tri-methylated at lysine residue 9 (H3K9me3) by SUV39H (a histone methyl-transferase) until the end of the zygotene step, and depletion of Suv39h in germ cells leads to impaired synapsis (Peters, O'Carroll et al. 2001).
Recombination is also dependent on the epigenetic modification of histone H2B, which is ubiquitylated (uH2B) by RAD6 allowing methylation of lysine residue 4 (K4) of histone H3 (H3K4me) by SET1. This leads to chromatin relaxation at recombination hot spots where SPO11 induces DSBs, (for review see Hernandez-Hernandez, Vazquez-Nin et al. 2009).
Dynamics of histone modifications are crucial for accessibility to key enzymes for DNA recombination and repair. H3K4me3 prevents the precocious incorporation of the DIDO3
28 nuclear protein in the SC; DIDO3 is required for the complete formation of the SC, Dido mutant mice display mild meiotic defects and are sterile because few spermatids are produced (Prieto, Kouznetsova et al. 2009)
Additionally, post-translational modification of histones is essential for MSCI. For instance, the tri-methylation of lysine 9 of histone 3 (H3K9me3) by SUV39H leads to the binding of proteins such as HP1, which is necessary for the propagation of heterochromatin domains (required for transcriptional silencing), and further post-meiotic differentiation (Lachner, O'Carroll et al. 2001; Peters, O'Carroll et al. 2001). Acetylation and methylation of histone 3 and histone 4 on X and Y-chromosomes during meiotic phases (and in round spermatids) are concordant with their inactive transcriptional state (Khalil, Boyar et al. 2004); thus, specific gene silencing associated histone modifications, such as H3K9me2, are present in the sex body (Khalil, Boyar et al. 2004). G9a (a H3K9 methyl transferase) or Meisetz (a H3K4 methyl transferase, specific for tri-methylation) are involved in sex body formation; defects in these leads to deficiency of the SC formation, impairment of XY body formation and loss of spermatocytes and an for the latter (Hayashi, Yoshida et al. 2005; Tachibana, Nozaki et al.
2007).
Conversely, local histone acetylation allows the relaxation of chromatin structure and enables transcription (Jenuwein and Allis 2001).
In summary meiosis I, principally, is tightly regulated at chromatin level in order to produce functional haploid spermatids containing the genetic material required for the generation of viable spermatozoa.
A.3.c. The post-meiotic maturation phase
The post-meiotic phase, also called spermiogenesis, corresponds to the morphological and functional differentiation of the spermatids rising from secondary spermatocytes. During
29 Figure 9. Developmental stages and expression of associated genes of haploid germ cells during spermiogenesis.
Spermiogenesis is composed of 16 spermatids stages, the associated roman numbers (I-XII) represent the seminiferous epithelium cycle. Annotated genes are essential for different biological processes revealed by mouse Knockouts. From (Yan 2009).
30 spermiogenesis, no cell divisions take place but major molecular and cellular changes occur which affect the chromatin state, the nuclear form as well as the distribution of organelles and also generate novel structures such as the acrosome and the flagellum (Figure 9).
A.3.c.1. Formation of the flagellum
One of the main characteristics of spermatid differentiation is their progressive elongation.
Indeed, “young” spermatids are round, but they rapidly develop a flagellum, which will be necessary for late motility of the sperm (although acquirement of motility will occur only in the epididymis and during fertilization). The centrioles that were necessary for spindle assembly and chromosomal segregation during meiosis I migrate towards the cell surface.
One of the two centrioles nucleates the axoneme, which consists of 9+2 microtubules doublets and causes the formation of a protrusion of the plasma membrane. These centrioles move towards the nucleus to the opposite position of the acrosome (see next paragraph) to form the connective piece of the sperm. Addition of multiple components occurs to the developing flagellum to form the middle, principal and end pieces (Figure 9). Recruitment of mitochondria originates from the whole cytoplasm and these associate around the middle piece to provide the energy necessary for sperm motility, the structure proteins ODF1 and ODF2 (Outer Dense Fibre) accumulate around the middle and principal piece to form the outer dense fibres which will confer elasticity to the flagellum (Russell 1990).
Another important structure participating in spermatid elongation is the manchette. It is a transient structure composed of α-tubulin and β-tubulin heterodimers, whereas γ-tubulin localises to the perinuclear ring (Mochida, Tres et al. 1998; Mochida, Rivkin et al. 2000).
Kinesins and dynein molecular motors are also associated with the manchette, linking it with the nucleus and allowing transport of molecules and vesicles along microtubules, for example KIFC1 protein (Yang and Sperry 2003). Several mutations linked with abnormal manchette structure leads to sperm differentiation defects. For instance, infertile azh mutant mice express
31 a truncated form of HOOK1 protein, which is essential for connection between microtubules and cellular structures (Mendoza-Lujambio, Burfeind et al. 2002). Intronic insertion of a neo resistance cassette, in the keratin9 gene, leads to ectopic manchette and defects in tails shape (Rivkin, Eddy et al. 2005). Mutations of specific genes such as E-MAP-115, a microtubule associated protein(Penttila, Parvinen et al. 2003), or LIS1, an acetylhydrolase (Nayernia, Vauti et al. 2003) which acts on PAF, a phospholipase (Benoff 1998; Roudebush 2001;
Cheminade, Gautier et al. 2002) also affect the formation and the shape of the manchette, or of the whole flagellum, as well as the head shape, including acrosomal formation. Thus, regulation of molecule and organelle transport by the manchette is crucial for the establishment of flagellar structure for the formation of the acrosome.
A.3.c.2. Development of the acrosome structure
The acrosome is a germ-cell specific feature of male gametes. It is an exocytotic vesicle necessary for the fertilization of the oocyte since it will release proteins such as protease zymogens, protease inhibitors and zona pellucida-binding proteins allowing the penetration of the sperm and thus the transmission of the paternal genetic material (Baba, Kashiwabara et al.
1989; Williams and Jones 1993; Baba, Niida et al. 1994; Kohno, Yamagata et al. 1998).
Formation of the acrosome originates in the early steps of spermiogenesis, when the Golgi apparatus differentiates and migrates towards the plasma membrane, concomitantly with the chromatoid body. In early spermatids, the acrosome is round (also called the acrosomal vesicle at this stage), then the nucleus moves toward the plasma membrane and the acrosome flattens and appears denser. By the end of spermiogenesis, the polarity of spermatids is obvious, with the head (nucleus and acrosome) and the tail (flagellum).
The acrosome is essential for fertility, its absence leads to sterility (Sotomayor and Handel 1986; Baccetti, Burrini et al. 1991). Multiple acrosomal defects have been reported, all demonstrate the importance of correct assembly of this structure in spermatids. Components
32 of the acrosome includes soluble proteins, insoluble matrix proteins and membrane proteins, it is generally hypothesized that different proteins are sequentially involved in the acrosomal reaction at fertilization (Olson, Winfrey et al. 1988; Olson and Winfrey 1994; Tanii, Araki et al. 1994; Westbrook-Case, Winfrey et al. 1994). Multiple soluble proteins are specific of the acrosome such as dipeptidyl peptidase II and cysteine-rich secretory protein 2 (Hardy, Oda et al. 1991). The insoluble acrosomal matrix contains apexin (Noland, Friday et al. 1994;
Westbrook-Case, Winfrey et al. 1995; Kim, Foster et al. 2001), SP56 (Kim, Cha et al. 2001), the calcium-dependent cysteine proteases, Calpain 1 and 2 (Ben-Aharon, Brown et al. 2005), serine protease acrosin and acrosin-binding protein (Baba, Niida et al. 1994). Some components, such as SP56, undergo post-translational modifications during acrosomal exocytosis (Kim, Cha et al. 2001). Male mice with a disruption of the acrosin (acr) gene are sub-fertile due to a reduced penetration in the egg (Nayernia, Adham et al. 2002). Moreover, acrosomal matrix components are necessary for the binding to the zona pellucida of the oocytes during fertilization (Buffone, Foster et al. 2008). Multiple specific transmembrane proteins such as Cyritestin, a testis specific protein belonging to the ADAM family (a disintegrin and metalloprotease), also constitute the membrane of the acrosome; its mutation in male mice leads to infertility due to impaired zona pellucida binding (Nishimura, Cho et al.
2001).
It is obvious that correct acrosome formation as well as a precise compartmentalization of its component are required for efficient penetration of the sperm into the oocyte during fertilization and thus for the paternal genetic material to finally reach the maternal pronucleus.
Concomitantly with acrosomal development, the nucleus of young spermatids and the genetic information it contains undergo multiple changes.
33 A.3.c.3. Nuclear condensation and DNA compaction
During spermiogenesis, the nucleus condenses dramatically; this reflects the extensive DNA compaction, which results from the replacement of somatic histones by testis’s specific proteins, namely the protamines. At the end of spermiogenesis, the compaction of DNA is 6- fold higher than in a mitotic cell, because the overall basicity of protamines is higher than that of somatic histones, thus leading to a stronger link with the acidic DNA. It seems that compaction process is necessary for the protection of paternal DNA against exogenous damages (Lewis, Song et al. 2003; O'Brien and Zini 2005). The intensive replacement of somatic histones by testis specific histones (beginning at meiosis) is followed by their replacement by transition proteins (Tnp1 and Tnp2). Later on, transition proteins are replaced by protamines (Prm1 and Prm2). Depletion of these factors leads to chromatin remodelling and condensation defects. When the Tnp1 and Tnp2 genes are disrupted, sperm nucleus condensation is impaired and DSBs generated (Zhao, Shirley et al. 2004). PRM1 and PRM2 are necessary for correct head morphology and chromatin integrity (Cho, Jung-Ha et al.
2003). Depletion of CamK4, which phosphorylates protamines, leads to a loss of elongating spermatids (Wu, Ribar et al. 2000). Impairment of Jhdm2a, involved in chromatin demethylation in spermatids, leads to condensation defects as well as to a decrease of Tnp1 and Prm1 genes transcription (Okada, Scott et al. 2007).
These changes in chromatin-associated proteins lead to transcriptional inactivity during spermatid elongation. Nevertheless, the differentiation of spermatids requires the synthesis of specific proteins, meaning that transcription and translation are uncoupled. This implies storage and regulation of mRNAs transcribed in round spermatids and their correct expression in elongating spermatids. It is noteworthy that translational regulation affects 70% of poly- adenylated mRNAs in the adult testis ((see Cataldo, Mastrangelo et al. 1999) and section I.A.5.).
34 A.3.c.4. Cytoplasm elimination and residual body formation
Finally, the differentiation of spermatids involves the elimination of much of their cytoplasm, since 75% of spermatid’s volume is lost during elongation. Two mechanisms play a part in this process. First, phagocytosis mediated by the tubulobulbar complexes established between Sertoli cells and spermatids, and, secondly, the release of a cytoplasmic vesicle called residual body. This structure contains the molecules (RNA, proteins) and the organelles (Golgi and endoplasmic reticulum) unnecessary for sperm survival once it is released from the seminiferous epithelium (for review see Manandhar, Schatten et al. 2005). Despite this drastic reduction of cytoplasm volume, a cytoplasmic droplet remains around the neck of the sperm and was found to be the site for water entry allowing swelling capacity (Yeung, Sonnenberg- Riethmacher et al. 1999; Yeung, Wagenfeld et al. 2000).
By the end of spermiogenesis, elongated spermatids display an acrosome, a condensed nucleus with a thin tail and a cytoplasmic droplet (figure 10) and are released into the lumen during the spermiation step.
A.3.c.5. Histone modifications
As mentioned in section A.3.b.5, the histone acetylation allows active transcription. In spermatogonia, preleptotene and elongating spermatids H2A, H2B and H4 are hyper- acetylated. Furthermore, it was demonstrated that the replacement of histones in condensing spermatids, by transition proteins and further protamines, was dependent on histone hyperacetylation (Hazzouri, Pivot-Pajot et al. 2000; Govin, Caron et al. 2004).
Moreover, it is known that sex chromatin is also transcriptionally silenced after meiosis (known as Post-Meiotic Sex chromosome Repression; PMSR). Namekawa and co-workers have shown that 87% of the X-linked genes are suppressed post-meiotically (Namekawa, Park et al. 2006). Interestingly, among the 67 X-linked miRNA genes that escape MSCI during pachytene, 49 also escape PMSR ((Song, Ro et al. 2009) and see section A.3.b.3).
35 Furthermore, the study of mice with deletions of the Y chromosome long arm (MSYq-) showed an increase of X and Y genes expression. This increase is accompanied by a decrease of H3K9me3 (mainly located in the XY body in wild-type mice) and H4K8Ac showing the importance of chromatin remodelling marks in the transcription state in spermatids (Reynard and Turner 2009).
These data show the importance of transcriptional and post-transcriptional regulation of either autosomal and sex chromatin during the spermiogenic phase in mammals. A crucial aspect of this type of regulation is mainly dependent on the epigenetic context, which in turn might allow early spermatids reaching last differentiation steps of spermiogenesis.
A.3.d. Maturation of the spermatozoon
Once released into the lumen of the seminiferous tubule, spermatozoa are not yet fully mature. The passive transport of sperm cells outside the testis occurs thanks to the seminiferous fluid produced by Sertoli cells. The contraction of both tunica albuginea and peritubular myoid cells (under the control of prostaglandin F2α and vasopressin (Howl, Rudge et al. 1995; Tripiciano, Filippini et al. 1998) allow the evacuation of this non-motile sperm. Spermatozoa migrate towards the rete testis to which the seminiferous tubules are connected, then pass through the efferent ducts, which connect the rete testis and the caput epididymis. The spermatozoon will acquire its motility and mobility in the body and the tail of the epididymis (corpus and cauda, respectively). Indeed, motility is acquired according to specific conditions, found within the distal parts of the epididymis, such as low pH, increased concentration of K+ and low concentration of Na+ ions as well as the presence of specific epididymal proteins (for review see Cooper 2011). The mouse epididymis expresses 75 specific genes (Johnston, Jelinsky et al. 2005), transcriptome analysis helped to identify novel epididymal genes and showed that 27% of the epididymal expressed genes are under the
36 Figure 10. Electron microscopy of mouse spermatozoa.
(A) shows the overall morphology of a murine spermatozoa composed of four major parts: the head, mid-piece, principal piece and end piece. In (B) is a longitudinal section of the head, showing principally the acrosome and the nucleus, and the neck showing partly the mitochondria alignment and outer dense fibres of the mid-piece. The picture in (C) shows the transverse section of the mid-pieces with mitochondria sheath, the cytoplasmic droplet is present in (D). (E&F) are micrograph of the principal piece architecture at two different locations and (G) shows the axoneme structure of the end piece. From (Yan 2009).
37 control of testicular factors present in the seminal fluid (Jalkanen, Shariatmadari et al. 2006;
Sipila, Pujianto et al. 2006). Interestingly, in man, nuclear condensation, which started during spermiogenesis, continues in the epididymis since formation of disulphide bonds in protamines is increased, and nuclear shape is modified (Auger and Dadoune 1993). Finally, the sperm is released in the vas deferens, then into the ejaculatory ducts to be ejaculated.
A.3.e. The spermatogenic and seminiferous epithelium cycles
Spermatogenesis is a well-coordinated process, in time and space. The differentiation of germ cells is unidirectional from the base of the seminiferous epithelium to the lumen. As shown previously, mitotic spermatogonia as well as preleptotene spermatocytes localise at the basal lamina, whereas leptotene and zygotene cells move away from the base of the seminiferous tubule and pass through the Blood-Testis-Barrier (BTB). From this point, they become more round and progress to the adluminal side of the tubule. In summary, adult seminiferous tubules contain several types of germ cells, and each cell type is layered within the tubule in a centripetal manner. Moreover, arrangement of germ cells from successive generations happens in typical cellular associations within the seminiferous tubules known as spermatogenic stages. In mice, we can observe twelve spermatogenetic cellular associations in the seminiferous tubule. These associations are called stages of the seminiferous epithelium cycle and are annotated from I to XII ( see (Oakberg 1956) and figure 11). The essential base of this arbitrary classification of the stages originates from the analysis of the acrosome’s shape of spermatids and the structure of the chromatin of spermatogonia and spermatocytes (Oakberg 1956). The sequence of these twelve stages in mouse is named the seminiferous epithelium cycle and is repeating all along the reproductive lifetime of a given individual in a wave-like manner. Since germ cells are only migrating in a centripetal manner and not laterally, the different stages follow each other according to the progression of germ cells
