Search

This review focuses on the role of the dialog between the oocyte and its companion somatic cells in driving folliculogenesis from the primordial to the preovulatory follicle stage. Mouse and sheep genetic models have brought complementary evidence of these cell interactions and their consequences for ovarian function. In mouse, the deletion of genes encoding connexins has shown that functional gap junction channels between oocytes and granulosa cells and between granulosa cells themselves maintain the follicle in a functionally integrated state. Targeted deletions in oocytes or granulosa cells have revealed the cell- and stage-specific role of ubiquist factors belonging to the phosphatidylinositol 3 kinase signaling pathway in primordial follicle activation, oocyte growth and follicle survival. Various models of transgenic mice and sheep carrying natural loss-of-function mutations associated with sterility have established that the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor 9 orchestrate follicle development, support cumulus metabolism and maturation and participate in oocyte meiosis arrest. Unexpectedly in sheep, mutations resulting in the attenuation of BMP signaling lead to enhanced ovulation rate, likely resulting from a lowered follicular atresia rate and the enhancement of FSH-regulated follicular maturation. Both the activation level of BMP signaling and an adequate equilibrium between BMP15 and growth differentiation factor 9 determine follicle survival, maturation, and development toward ovulation. The physiological approaches which were implemented on genetic animal models during the last 20 years have opened up new perspectives for female fertility by identifying the main signaling pathways of the oocyte-somatic cell dialog.

Reproduction in Birds is characterized by internal fertilization and external development of an embryo in a telolecithal egg containing all nutrients, biological activities and protective systems required for the proper growth of the embryo, until hatch. This article will give an overview on the chicken ( Gallus gallus ) egg yolk proteins and vitellogenesis, knowing that the formation and structure of the egg (including the yolk) is quite conserved, regardless of the Bird species, although some differences exist in the timing and number of clutches of eggs produced, and in some specific egg traits (egg weight, yolk/white proportion, biochemical composition, etc.).

La différenciation de l’ovaire a été considérée pendant plusieurs décennies comme un processus passif, se mettant en place par défaut. Le développement d’outils génétiques permettant l’invalidation de gènes chez la souris, d’approches transcriptomiques haut-débit ainsi que l’étude de modèles animaux pertinents a montré que la différenciation des gonades est un processus dynamique dans les deux sexes et a permis d’identifier des acteurs clés de la différenciation ovarienne. La détermination des cellules somatiques de l’ovaire est sous le contrôle de deux voies majeures, l’une régulée par le facteur de transcription FOXL2 avec ou sans les œstrogènes selon les espèces, et celle de la voie β-caténine (Rspo1, Wnt4, β-caténine). Ces deux cascades géniques agissent simultanément sur l’activation de la voie femelle et l’inactivation de la voie mâle. À l’inverse, la différenciation sexuelle des cellules germinales ne résulte pas uniquement du contenu chromosomique de la cellule (XX femelle et XY mâle). Elle est le résultat de l’environnement des gonocytes dans l’ébauche gonadique. Très tôt, les gonocytes femelles vont se multiplier par mitose puis s’engager dans la méiose (prophase I). Suivra la formation des follicules primordiaux, étape cruciale de la vie reproductive femelle car elle constitue la réserve ovarienne. Les connaissances sur les facteurs qui participent à ces processus moléculaires ont beaucoup progressé au cours des dix dernières années et seront présentées dans cette revue., Early ovarian development has long been thought of as a default pathway switched on passively by the absence of SRY gene. Recent genetic and transcriptomic studies challenge this view and show that two master pathways simultaneously repress male-specific genes and activate female-specific genetic cascades. This antagonistic action is maintained from embryonic stages to adulthood. The differentiation of the ovarian somatic component is regulated by both the forkhead transcription factor FOXL2 (alone or in combination with oestrogens according to the species) and β-catenin pathway activated by Wnt4 and Rspo1. The sex-specific change in the fate of primordial germ cells depends on the gonad environment. Female gonocytes actively proliferate by mitosis then enter meiosis I until the diplotene stage. Primordial follicle formation occurs when oocytes are individually surrounded with pre-granulosa cells. In mammals, the population of primordial follicles serves as a resting and finite pool of oocytes available during the female reproductive life span. Recent data on factors controlling these molecular processes will be presented in this review.

Cette revue présente l’état des connaissances sur la folliculogenèse et l’ovulation chez les mammifères. La folliculogenèse basale est une phase de croissance folliculaire lente, au cours de laquelle l’ovocyte acquiert la compétence méiotique. La folliculogenèse terminale est une phase de développement rapide, au cours de laquelle le follicule ovulatoire est sélectionné et termine sa maturation, tandis que l’ovocyte acquiert la compétence au développement. La revue décrit les différents changements fonctionnels qui s’opèrent dans le follicule et l’ovocyte au cours de ces deux phases, ainsi que les mécanismes qui les régulent et qui déterminent le quota ovulatoire caractéristique de chaque espèce ou race. Les facteurs-clés identifiés de la folliculogenèse sont les BMP, le KITLG et l’AMH pour le démarrage de croissance folliculaire, l’IGF et l’insuline pour la transition entre folliculogenèse basale et terminale, la FSH pour le début de la folliculogenèse terminale et la sélection des follicules ovulatoires, et la LH pour le développement folliculaire final, la maturation ovocytaire et l’ovulation. L’ovulation est déclenchée par une décharge préovulatoire de LH qui induit la maturation du complexe cumulus-ovocyte dans le follicule ovulatoire, suivie de la rupture de la paroi folliculaire et de la formation du corps jaune. La folliculogenèse ovarienne est contrôlée par des facteurs externes, tels que la photopériode et la nutrition, et c’est essentiellement le système hypothalamo-hypophysaire qui intègre les signaux endocriniens et environnementaux. Des modifications de l’apport alimentaire, relayées par des variations de signaux hormonaux et métaboliques, modulent l’activité de l’axe hypothalamo-hypophyso-ovarien à ses différents étages., This review is a state of the art on follicullogenesis and ovulation in mammals. Basal folliculogenesis is a phase of slow follicular growth, during which the oocyte acquires meiotic competence. Terminal folliculogenesis is a phase of rapid development, during which the ovulatory follicle is selected and completes its maturation while the oocyte acquires developmental competence. This review describes the different functional changes occurring within follicles and oocytes throughout these two phases, the underlying regulatory mechanisms and the mechanisms that determine natural ovulation rate in different species and breeds. Known key factors for folliculogenesis are BNTP, KITLG and AMH for the initiation of follicular growth, IGF and insulin for transition between basal and terminal folliculogenesis, FSH for the outset of terminal folliculogenesis and the selection of ovulatory follicles, and LH for final follicular development, oocyte maturation and ovulation. Ovulation is triggered by a preovulatory LH surge that induces maturation of the cumulus oocyte complex within the ovulatory follicle, rupture of the follicular wall and corpus luteum formation. Ovarian folliculogenesis is controlled by external factors such as photoperiod and nutrition, and the hypothalamo-pituitary system integrates endocrine and environmental signals. Nutritional supply, relayed by hormonal and metabolic signals, can modulate both the hypothalamo-pituitary and ovarian activity.

FOXL2 is a transcription factor which is crucial for the ovary. In humans, heterozygous mutations are responsible for the BPES syndrome characterized by eyelid anomalies and premature ovarian failure. Similarly in mice, Foxl2 deletion leads to complete folliculogenesis disruption and female infertility. In the goat, the Polled Intersex Syndrome mutation is responsible for the transcriptional silencing of FOXL2 in XX PIS-/- gonads that leads to female-to-male sex reversal, thus to the differentiation of testes instead of ovaries in genetically female animals homozygous for the mutation. So, FOXL2 is determining for ovarian differentiation early during development in goats, whereas it is involved in fertility tardily in mice and women. In order to understand these species specific differences, we searched for the genes and pathways regulated by FOXL2 in early goat ovaries. Thanks to RNA-sequencing of goat XY testes, XX ovaries and XX PIS-/- gonads (lacking FOXL2) at the beginning of their differentiation, we were able to (i) better characterize the role of FOXL2 in goat ovaries and show that it acts mainly as an anti-testis factor, and (ii) highlight new highly-expressed ovarian genes like DMXL2, and study its role during ovarian development using functional experiments in the mouse.; FOXL2 est un facteur de transcription crucial pour la fonction ovarienne. Dans l’espèce humaine, des mutations hétérozygotes de ce gène sont responsables de la survenue d’un syndrome associant des malformations des paupières à une insuffisance ovarienne prématurée. De même chez la souris, l’invalidation totale de Foxl2 conduit à un blocage de la folliculogenèse et donc à une infertilité femelle. Chez la chèvre, la mutation Polled Intersex Syndrome (PIS) engendre le silence transcriptionnel de FOXL2 dans les gonades XX PIS-/- ce qui conduit à une inversion sexuelle, et à la différenciation de testicules à la place d’ovaires chez les animaux génétiquement femelles homozygotes pour la mutation (inversion sexuelle de type mâle XX). FOXL2 est donc déterminant pour la différenciation ovarienne très précocement au cours du développement dans l’espèce caprine, alors qu’il ne semble impliqué que plus tardivement dans l’établissement de la fertilité chez la souris et la femme. Afin de comprendre ces différences entre espèces, nous avons recherché quels étaient les gènes et les voies de signalisation régulés par FOXL2 dans l’ovaire de chèvre au début de sa différenciation. Grâce à un séquençage à haut-débit des transcrits présents dans trois types de gonades caprines (testicules XY, ovaires XX et gonades XX PIS-/- (qui n’expriment pas FOXL2)) au début de leur différenciation, nous avons pu (i) mieux caractériser le rôle de FOXL2 dans l’ovaire caprin et montrer qu’il y agit avant tout comme un facteur anti-testiculaire, et (ii) mettre en évidence de nouveaux gènes fortement exprimés dans l'ovaire au début de sa différenciation, comme DMXL2, et nous avons étudié son rôle dans la fonction ovarienne grâce à des expériences fonctionnelles chez la souris.

We identified DMXL2 from RNA-sequencing data as a gene preferentially expressed in differentiating-ovaries in the goat species. In order to determine its role during ovarian morphogenesis, we investigated functional studies in mice. Dmxl2 invalidation (KO) is lethal within the few hours following birth. The analysis of its expression in several tissues let us show that the heart, olfactory mucosa and brain expressed Dmxl2 during development. Our results further suggest that the lethality may be due to an alteration of the synaptic transmission.Besides, both male and female gonads express Dmxl2. Notably in the ovary, its expression increases few days before follicle formation. We show that DMXL2 protein is dynamically expressed in germ cells and granulosa cells; DMXL2 is strongly detected in the oocytes whereas its expression in the granulosa cells fluctuates depending on the stage of folliculogenesis. To study Dmxl2 implication during follicle formation, and to bypass the issue of lethality at birth, we performed allograft of KO ovaries on nude recipient mice. KO grafts show an alteration in follicle formation, followed by ovarian dysgenesis.To conclude, our work shows that Dmxl2 is involved in follicle formation in mice, and makes it a new candidate to explain some cases of premature ovarian failure. The conditional knock-out of Dmxl2 in germ cells or in granulosa cells is in progress in the laboratory (please refer to Clara Gobé’s poster) and will permit to precise its specific role in each cell types.

Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK), an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO) female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK) involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues). Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed.

L’objectif principal de ce travail consiste à acquérir la cinétique de l’hormone anti-Müllérienne (AMH) de la naissance à la première mise à la reproduction chez la chevrette de contre-saison. Cette étude préliminaire ne montre pas de corrélation significative entre la concentration plasmatique en AMH, les caractéristiques de croissance et le passé in utero. En revanche, il y a une corrélation moyenne positive entre certaines caractéristiques ovariennes avant la mise à la reproduction et les taux en AMH après 3 mois. De plus, les chevrettes gestantes à la première mise à la reproduction ont un taux plus faible en AMH que les non gestantes. La valeur seuil de concentration en AMH dosée à 3 mois utilisée pour prédire la réussite de la mise à la reproduction est très fiable pour prédire les chevrettes gestantes mais inefficace pour prédire les vides., The main objective of this study is to elaborate the kinetic of anti-Müllerian hormon (AMH) from birth to first reproduction on deseasoned roe deers. This preliminary study shows that there is no significant correlation between AMH plasmatic levels, growth characteristics (weight, chest measurement) and past life in utero. However, there is a positive correlation between some ovarian characteristics before reproduction and AMH level at 3 months old. Moreover, pregnant goats have an 3 months AMH level higher than non pregnant goats. An AMH threshold value at 3 months old can be used for predicting the success of the first mate. The interest of this threshold value is very important for predicting pregnancy on the first mate but not for predicting the fail with the first mate.

Le bon déroulement de la folliculogenèse ovarienne basale, est une étape clé qui conditionne la fertilité des femelles à l'âge adulte. Pourtant, les mécanismes moléculaires contrôlant ce processus sont encore mal connus et les études d’expression limitées par la difficulté d'obtention du matériel biologique. Les données actuelles proviennent essentiellement d’études réalisées chez les rongeurs (espèces poly-ovulantes). L’objectif de ce travail est de décrire, pour les deux compartiments folliculaires (ovocytes et cellules de granulosa), les spécificités d’expression et les gènes impliqués dans le dialogue moléculaire entre ces compartiments ainsi qu'aux différents stades de développement folliculaire basal « in vivo » chez une espèce mono-ovulante : la brebis. Nous avons développé, dans un premier temps, une méthode d'analyse du transcriptome pour les stades précoces qui combine microdissection à capture laser, amplification des ARN et microarrays. Puis, nous avons présenté, par la technique RNA-seq, et pour la première fois, un répertoire de 15349 gènes décrivant le profil d’expression de ces gènes dans chaque compartiment folliculaire et en fonction de leur stage de développement. L’analyse statistique révèle une différence d’expression entre compartiments folliculaires (5130 gènes) ainsi qu’une dynamique d’expression au cours du développement (3015 gènes). Nous avons également identifié 161 et 55 gènes présentant une expression préférentiellement enrichie dans l’ovocyte et dans les cellules de granulosa, respectivement. L’analyse fonctionnelle « in silico » combinée avec les données d’expression mettent en évidence des voies de signalisation (IGF1, VEGF, FGF, NOTCH) susceptibles d’être impliquées dans le dialogue moléculaire entre les deux compartiments folliculaires. Enfin, dans nos conditions expérimentales, nous montrons d’importantes variations d’expression au cours du développement folliculaire basal, lors de la transition du follicule primaire en secondaire chez la brebis. La cinétique d’expression de gènes impliqués dans des voies de signalisation tels que BMP et WNT a été précisément décrite. Un jeu de 25 gènes a été sélectionné en tant que biomarqueurs expressionnels des différentes classes folliculaires. Il pourra être utilisé pour évaluer la croissance folliculaire basale.

National audience

National audience; The process of folliculogenesis is a complex and dynamic process that is regulated by positive and negative factors. During folliculogenesis in the mammalian ovary, the primordial follicle pool is gradually reduced through successive waves of follicle growth. It is important that this pool is carefully regulated, otherwise premature ovarian failure will occur. This process results in the ovulation of an ovocyte or more commonly, the loss of ovocytes through atresia. The majority of research has concentrated on the positive regulators of follicle growth with very little attention on the negative regulators. Nonetheless, there are several factors that have been observed to inhibit follicle activation and development. In this review, we summarize those here.; La folliculogenèse est un processus complexe et dynamique régulé par des facteurs stimulateurs et inhibiteurs. Chez les mammifères, au cours de ce processus, la réserve de follicules primordiaux est progressivement réduite au fur et à mesure des vagues de croissance folliculaire. L’ovulation résulte d’un long processus de sélection au cours duquel de nombreux follicules dégénèrent par atrésie. Les principales recherches portant sur la régulation de la folliculogenèse se sont concentrées sur les facteurs ayant une action positive sur la croissance folliculaire, et peu sur les régulateurs négatifs qui participent pourtant à la différentiation harmonieuse des follicules. Néanmoins, il existe plusieurs facteurs décrits comme inhibiteurs de l’activation et du développement folliculaire. Nous en détaillerons certains dans cette revue.

National audience; The strong between-animal variability in the number of ovulations and embryos produced after ovarian stimulation by gonadotropins is a major limit to the development of embryo biotechnologies in cattle. In reproductive medicine, anti-mullerian hormone (AMH) is now widely used as an endocrine marker of the ovarian follicular reserve. In the cow, as in the woman, AMH is secreted by the granulosa cells of growing follicles. We have shown recently that in the cow, AMH is a very good endocrine marker of the population of small antral follicles that constitute the direct target of ovarian stimulatory treatments. AMH concentration measured in plasma before treatment varies between animals and is positively correlated to the number of ovulations and transferable embryos produced after an ovarian stimulatory treatment. Interestingly, AMH concentrations can remain stable over several months for each animal. Moreover, the number of embryos produced after ovarian stimulation is highly repeatable and has a relatively good heritability. From these observations, we propose the determination of AMH concentration in the plasma of a potential donor cow as a simple predictive method to evaluate both its level of ovarian activity and its capacity to produce high or low numbers of embryos. Optimal conditions for implementing this diagnostic test in cattle remain to be defined considering the age, the breed, the physiological status and the environmental factors related to breeding conditions for each animal.; Chez les bovins, l'existence d'une forte variabilité entre animaux du nombre d'ovulations et du nombre d'embryons produits après stimulation ovarienne constitue une limite majeure au développement des biotechnologies de l'embryon. Nous avons montré récemment que chez la vache, comme chez la femme, l'hormone antimüllérienne (AMH), sécrétée par les cellules de granulosa des follicules en croissance, est un très bon marqueur endocrinien de la folliculogenèse basale et, en particulier de la population de petits follicules à antrum qui est la principale cible des traitements de stimulation ovarienne utilisant des hormones gonadotropes. Les concentrations plasmatiques d'AMH mesurées avant traitement sont variables entre animaux et positivement corrélées aux nombres d'ovulations et d'embryons transférables produits après traitement de stimulation ovarienne. En revanche, pour un même animal, les concentrations plasmatiques d'AMH apparaissent stables sur une durée de plusieurs mois. En parallèle, il existe une bonne répétabilité individuelle et une relativement bonne héritabilité du nombre d'embryons produits après stimulation ovarienne. Ces résultats permettent de proposer la mesure de la concentration plasmatique d'AMH comme une méthode prédictive simple d'évaluation du niveau d'activité ovarienne et de la capacité de production d'embryons par une vache donneuse potentielle. Les conditions optimales pour la réalisation de ce diagnostic, prenant en compte l'âge de l'animal, sa race, son état physiologique et les facteurs environnementaux liés à ses conditions d'élevage, restent encore à définir.

Diplôme : Dr. d'Université

Les mammifères femelles naissent avec un très grand nombre de follicules ovariens primordiaux (104-106); par contre, la grande majorité (99%) de ces follicules n’atteignent jamais la maturité et subissent l’atrésie, principalement par l’apoptose des cellules de la granulosa. Notre laboratoire a démontré que les hyaluronidases des mammifères induisent l’apoptose des cellules de la granulosa et sont impliquées dans l’atrésie des follicules mais que cet effet apoptotique ne serait pas dû à leur activité enzymatique. Notre modèle propose que les hyaluronidases aient un rôle dans les follicules non destinés à ovuler. Le but de la présente étude est d’évaluer la folliculogénèse et la fertilité des souris déficientes de ces enzymes. Les résultats montrent que la délétion de Hyal-3 ne semble pas affecter la fonction ovarienne des souris mais qu’il pourrait y avoir un effet compensatoire par Hyal-1 chez les souris déficientes de Hyal-3 étant donné que son expression est augmentée chez ces souris. La délétion de Hyal-1 a pour effet d’augmenter le nombre des follicules primordiaux, primaires et secondaires, particulièrement chez les souris de bas âge, et de diminuer le niveau d’apoptose des cellules de la granulosa. Afin d’évaluer la fonction de Hyal-1, -2 et -3 sans effet compensatoire entre elles, nous avons voulu créer une souris déficiente des ces 3 hyaluronidases spécifiquement dans les gonades en utilisant le système Cre/loxP. Un vecteur contenant la séquence Cre sous le contrôle du promoteur de Inhibin-α, qui conduit l’expression des gènes en aval chez les cellules somatiques des gonades, a été construit avec succès. En conclusion, cette étude nous révèle que Hyal-3 ne semble pas affecter la fonction ovarienne mais que la délétion de Hyal-1 augmente la folliculogénèse et diminue l’apoptose des cellules de la granulosa., Female mammals are born with a large number of ovarian primordial follicles, though the vast majority of these never reach the preovulatory stage and undergo atresia, mainly through granulosa cell apoptosis. Our laboratory has established that mammalian hyaluronidases induce apoptosis of ovarian granulosa cells and that they are involved in follicular atresia but that their apoptotic effect is not due to their enzymatic activity. Our model suggests that mammalian hyaluronidases might have a role in follicles not destined to ovulate. The aim of this study was to evaluate the folliculogenesis and fertility of mice devoid of these enzymes. Our results showed that Hyal-3 KO mice have normal folliculogenesis, which could be explained by a compensatory effect of Hyal-1 since its expression is upregulated in these mice. In contrast, Hyal-1 KO mice had increased numbers of primordial, primary and secondary follicles, particularly in young mice, and lower levels of granulosa cell apoptosis. In order to investigate the effect of the three hyaluronidases, Hyal-1, -2 and -3, without a compensatory effect by one another, we decided to create a transgenic mouse deficient in all these three hyaluronidases but only in the gonads by using the Cre/loxP system. We successfully created a plasmid containing the Cre sequence under the control of Inhibin-α promoter, which conducts gene expression in somatic cells of the gonads. In conclusion, the present work demonstrates that Hyal-3 does not have any effect on ovarian function, but that deletion of Hyal-1 in mice promotes increased folliculogenesis and lowers granulosa cell apoptosis.

Ecole Doctorale: Des génomes aux organismes Présentée et soutenue le 11 juin 2009 Diplôme : Dr. d'Université; La production d’ovocytes fécondables dépend de la réalisation de multiples étapes parmi lesquelles deux semblent particulièrement importantes dans l’obtention de gamètes femelles: l’entrée en méiose des cellules germinales et la formation des follicules. Chez les mammifères, les gènes impliqués dans le contrôle de ces deux étapes sont peu connus. L’objectif de notre étude a été d’identifier chez la brebis les gènes impliqués dans ces deux étapes qui se déroulent pendant la période foetale. Pour cela nous avons utilisé la technique SSH qui nous a permis d’obtenir deux banques d’ADNc soustraites. L’une contenant des transcrits exprimés préférentiellement pendant la méiose et l’autre contenant des transcrits exprimés préférentiellement pendant la formation des follicules. Parmi ces transcrits, 6080 ont été séquencés et regroupés sous forme de 2101 assemblages (contigs). L’annotation de ces 2101 assemblages montre que 99% d’entre eux possèdent une homologie de séquences avec les bases de données humaines, bovines et ovines. Les 1% restant sont considérés comme des séquences inconnues car ils ne présentent pas d’homologie avec les bases de données interrogées. Dans nos banques nous avons isolé et étudié une dizaine de transcrits dont l’expression n’avait pas été étudiée dans l’ovaire. Parmi ces transcrits, Topaz1 (Testis Ovary-specific PAZ domain gene1) est exprimé uniquement dans les gonades mâles et femelles chez l’ovin et la souris. Chez les femelles de ces deux espèces, ce transcrit est exprimé dans l’ovaire foetal avec une expression plus importante pendant la période qui coïncide avec la méiose des cellules germinales. Nous avons recherché la totalité de la séquence nucléique du transcrit Topaz1 ovin in silico et par la technique 5’RACE. Les séquences orthologues du gène Topaz1 ont été retrouvées dans le génome de plusieurs mammifères (souris, chien, vache, homme) ainsi qu’une partie de la séquence chez le poulet. La séquence nucléique de Topaz1 est très conservée entre ces différentes espèces. Au niveau protéique un domaine nommé PAZ a été identifié. Ce domaine est trouvé dans les protéines Argonaute et PIWI qui ont un rôle dans la différenciation des cellules germinales. Cette étude nous a permis d’une part d’isoler et de caractériser de nouveaux gènes impliqués dans la différenciation ovarienne précoce et d’autre part de constituer une collection de transcrits dédiée à la différenciation de l’ovaire chez la brebis qui servira à évaluer et comparer le transcriptome d’ovaires dans différentes conditions physiologiques.

Lymphatic vessels exist in nearly all tissues, yet, despite their omnipresence, there remains a large knowledge gap between the described fundamental roles of lymphatic capillaries and our understanding of their functional biology, adaptive ability, and pathological response. This thesis addressed these shortcomings by utilizing an integrative biomedical engineering approach to examine molecular and mechanical regulators of lymphatic capillaries using in vivo models of lymphatic capillary biology, function, and adaptation. Using a model of skin regeneration in the mouse tail, we demonstrated that slow interstitial flow created by lymphatic drainage was necessary for lymphatic capillary organization. This novel model permitted the identification of spatial, temporal and chemical factors governing lymphangiogenesis. In contrast to the sprouting mechanism of blood angiogenesis, lymphatic endothelial cells (LECs) were demonstrated to organize in a vasculogenesis-like manner, migrating in the direction of interstitial flow and then organizing into functional lymphatic capillaries. Lymphangiogenesis was inhibited by blocking vascular endothelial growth factor (VEGF)-C signaling from day 0, but initiation of receptor blockade once LECs had already migrated did not prevent vessel organization. This uniquely demonstrated the need for a biochemical mediator (VEGF-C) to initiate lymphangiogenesis, but that an important biomechanical force, interstitial flow, was necessary for functional capillary organization. Further insight into the necessity of interstitial flow in LEC biology was found in the response of lymphatic capillary to induced lymphedema, wherein lymphatic drainage is significantly reduced. In a mouse tail model of secondary lymphedema, we demonstrated that the edematous environment – characterized by extracellular matrix breakdown, lipid accumulation, and reduced interstitial flow – resulted in hyperplasia of LECs but concurrent poor function due to the lack of interstitial flow as an organizational guiding cue. Similar dermal matrix adaptations to dysfunctional lymphatic drainage were also noted in two mouse models of congenital lymphedema, the Chy and VEGFR-3-Ig mice, further demonstrating the intimate connection of lymphatic capillary function with tissue maintenance and remodeling. To quantitatively demonstrate the changes in lymphatic capillary uptake and tissue hydraulic conductivity found in these and other transgenic mouse models, we developed a poroelastic model of interstitial transport. Tissue hydraulic conductivity was also calculated in tissues lacking lymphatics using an unsteady-state solution, demonstrating that lymphedema causes a significant increase in tissue conductivity. This model was then utilized to assess the effects of a high fat diet, metabolic disorders, and lymphatic dysfunction on the tissue and on lymphatic capillary function. We discovered that lymphatic capillary uptake function was significantly reduced with dyslipidemia, suggesting a novel interplay between lymphatic function and lipid metabolism. Additionally, we uncovered a new and critical role for lymphangiogenesis and lymphatic transport in reproduction. We demonstrated that lymphangiogenesis is a regular, non-pathological event during folliculogenesis in the ovary. These new lymphatic capillaries are seemingly necessary for hormone transport from the ovary – an essential feedback mechanism during pregnancy. Blockade of lymphangiogenesis resulted in decreased systemic progesterone and estradiol levels and resulted in failed fetal development. In conclusion, this work highlights the critical roles of the lymphatic circulation and demonstrates the interplay between lymphatic biology and the biochemical and biophysical environment in which lymphatic capillaries reside. Interstitial flow and the interstitium modulate lymphatic behavior, and lymphatic function, in turn, controls the tissue microenvironment.

Development of follicular cysts is a frequent ovarian dysfunction in cattle. Functional changes that precede cyst formation are unknown, but a role for anti-Mullerian hormone (AMH) in the development of follicular cysts has been suggested in humans. This study aimed to characterize intrafollicular steroids and AMH during follicular growth in a strain of beef cows exhibiting a high incidence of occurrence of follicular cysts. Normal follicular growth and cyst development were assessed by ovarian ultrasonography scanning during the 8 days before slaughtering. Experimental regression of cysts was followed by rapid growth of follicles that reached the size of cysts within 3-5 days. These young cysts exhibited higher intrafollicular concentrations of testosterone, estradiol-17beta, and progesterone than large early dominant follicles did in normal ovaries, but they exhibited similar concentrations of AMH. Later-stage cysts were characterized by hypertrophy of theca interna cells, high intrafollicular progesterone concentration, and high steroidogenic acute regulatory protein mRNA expression in granulosa cells. Progesterone and AMH concentrations in the largest follicles (>= 10 mm) and cysts were negatively correlated (r = -0.45, P < 0.01). Smaller follicles (< 10 mm) exhibited higher intrafollicular testosterone and estradiol-17beta concentrations in ovaries with cysts compared to normal ovaries. During follicular growth, AMH concentration dropped in follicles larger than 5 mm in diameter and in a similar way in ovaries with and without cysts. In conclusion, enhanced growth and steroidogenesis in antral follicles < 10 mm preceded cyst formation in cow ovaries. Intrafollicular AMH was not a marker of cystic development in the cow, but low AMH concentrations in cysts were associated with luteinization.

BackgroundThe key steps in germ cell survival during ovarian development are the entry into meiosis of oogonies and the formation of primordial follicles, which then determine the reproductive lifespan of the ovary. In sheep, these steps occur during fetal life, between 55 and 80 days of gestation, respectively. The aim of this study was to identify differentially expressed ovarian genes during prophase I meiosis and early folliculogenesis in sheep.ResultsIn order to elucidate the molecular events associated with early ovarian differentiation, we generated two ovary stage-specific subtracted cDNA libraries using SSH. Large-scale sequencing of these SSH libraries identified 6,080 ESTs representing 2,535 contigs. Clustering and assembly of these ESTs resulted in a total of 2,101 unique sequences depicted in 1,305 singleton (62.11%) and 796 contigs (37.9%) ESTs (clusters). BLASTX evaluation indicated that 99% of the ESTs were homologous to various known genes/proteins in a broad range of organisms, especially ovine, bovine and human species. The remaining 1% which exhibited any homology to known gene sequences was considered as novel. Detailed study of the expression patterns of some of these genes using RT-PCR revealed new promising candidates for ovary differentiation genes in sheep.ConclusionWe showed that the SSH approach was relevant to determining new mammalian genes which might be involved in oogenesis and early follicle development, and enabled the discovery of new potential oocyte and granulosa cell markers for future studies. These genes may have significant implications regarding our understanding of ovarian function in molecular terms, and for the development of innovative strategies to both promote and control fertility.

Resume Les systemes de culture folliculaire ont ete developpes dans le but de permettre la fecondation in vitro d’ovocytes issus de follicules preleves immatures. Les techniques de folliculogenese in vitro pourraient en particulier etre appliquees en medecine de la reproduction, pour restaurer la fertilite des patientes ayant beneficie d’une cryoconservation ovarienne. Plusieurs systemes de culture, permettant la croissance in vitro de follicules de stades de developpement precoces, ont ete mis au point chez la souris et valides par des naissances de souriceaux viables. Certaines caracteristiques des systemes de culture se sont revelees essentielles pour la reussite de la folliculogenese in vitro : isolement des follicules a un stade determine, maintien de la morphologie folliculaire et apport de facteurs de croissances ou d’hormones. Ces methodes de culture folliculaire ont permis une meilleure comprehension de la physiologie ovarienne, celle, notamment de la relation entre les facteurs endocrines ou paracrines et le developpement du follicule. La technique de croissance folliculaire in vitro chez la souris est devenue un veritable outil biologique pour l’amelioration des techniques en reproduction comme pour les etudes toxicologiques.

Ovarian oogenesis and folliculogenesis are complex and coordinated biological processes which require a series of events that induce morphological and functional changes within the follicle, leading to cell differentiation and oocyte development. In this context, the challenge of the researchers is to describe the dynamics of gene expression in the different compartments and their interactions during the follicular programme. In recent years, high-throughput arrays have become a powerful tool with which to compare the whole population of transcripts in a single experiment. Here, we review the challenges of applying genomics to this model in farm animal species. The first limitation lies in limited the availability of biological material, which makes the study of the follicle compartments (oocyte, granulosa cells and thecal cells) or early embryo much more difficult. The concept of observing all transcripts at once is very attractive but despite progress in sequencing, the genome annotation remains very incomplete in non-model species. Particularly, oogenesis and early embryo development relate to the high proportion of unknown expressed sequence tags. Then, it is important to consider post-transcriptional and translational regulation to understand the role of these genes. Ultimately, these new inferred insights will still have to be validated by functional approaches. In addition toin vitroorex vivofunctional approaches, both ‘natural mutant’ ewe models and RNA interference represent, at the moment, the best hope for functional genomics. Advances in our understanding of reproductive physiology should be facilitated by gene expression data exchange and translation into a better understanding of the underlying biological phenomena.

Ovarian folliculogenesis in mammals from the constitution of primordial follicles up to ovulation is a reasonably well understood mechanism. Nevertheless, underlying mechanisms that determine the number of ovulating follicles were enigmatic until the identification of the fecundity genes affecting ovulation rate in sheep, bone morphogenetic protein-15 (BMP-15), growth and differentiation factor-9 (GDF-9) and BMP receptor-1B (BMPR-1B). In this review, we focus on the use of these sheep genetic models for understanding the role of the BMP system as an intra-ovarian regulator of follicular growth and maturation, and finally, ovulation rate.

Twenty-two Serrana goats were studied through two successive estrous cycles in order to characterize their follicular dynamics during the breeding season. The ovaries of the goats were scanned daily by real-time ultrasonography and all folliclesor=3mm were measured and classified. The data were classified by the number of follicular waves per goat to test the hypothesis that temporal and morphological differences between the last follicular wave of an ovary, irrespective of ovulation, will affect the selection of the next ovulatory wave. The mean interovulatory interval was 20.7+/-1.0 days (mean+/-S.D.). Three to five waves per estrous cycle were observed and 61.3% (19/31) of cycles had four waves. In estrous cycles with four waves, the day of onset of the first, second, third and fourth wave was 1.4+/-1.0, 6.9+/-1.4, 11.6+/-1.8 and 16.8+/-1.6, respectively. No differences (P0.05) were found between the day of onset of the first and second waves for estrous cycles with three, four or five waves. However, the day of onset of the third and fourth waves occurred later when the number of waves per estrous cycle increased (P0.001). The duration of the interwave interval (time between the day of onset of two consecutive waves) was longer when the second wave was ovulatory. The length of the growth phase (2.4+/-0.9 days) and size (5.9+/-0.7 mm) of the dominant follicle in the second wave were lower (P0.01) than for the first wave (3.3+/-1.2 days and 6.6+/-0.9 mm, respectively) and the fifth wave (4.1+/-1.2 days and 7.5+/-1.0mm, respectively). Within pairs of ovaries, the onset of the last wave occurred later (P0.05) and was less variable in ovulatory ovaries (day 16.8+/-1.4, n=20) than in anovulatory ovaries (day 15.1+/-3.7, n=20). The length of the growing phase was longer (P0.001) in the last waves of ovulatory ovaries (3.1+/-0.9 days) than in the last waves of anovulatory ovaries (1.7+/-0.8 days). These results support the hypothesis that the day of onset of the ovulatory wave is related to or, at least, conditioned by the luteolysis and the decrease in plasma progesterone. In summary, the estrous cycle of Serrana goats is characterized by sequential follicular wave growth with a great variability in their onset and duration, with the exception of the ovulatory wave. The temporal and morphological differences observed in the last wave of estrous cycle provide strong evidence for the role of progesterone in their regulation.

International audience

National audience

National audience; Cet article fait partie du dossier : Actualités en reproduction équine

Cet article fait partie du dossier : L’augmentation de la prolificité des truies : acquis, progrès envisageables et conséquences; National audience

The development of in vitro culture systems, comparable to the in vivo microenvironment in terms of effect on the oocyte growth and development could provide a valuable experimental tool for studying the mechanisms governing folliculogenesis. This tool might serve as well for practical clinical, agricultural, zoological, or biotechnological applications. This thesis reports on the importance of the microenvironment for the ovarian folliculogenesis process. The complexity of such a microenvironment was approached with a strategy based on functionalized PEG-hydrogels. The PEG matrix not only served as a scaffold, but it was also used a "reservoir" of immobilized cues. Tethered integrin-binding peptides in combination with other signaling factors aimed at better understanding the interactions of the oocyte and its surrounding granulosa cells that, most probably, determine the efficiency of the actual and the future in vitro mature oocyte production strategies. This work led also to investigating the lymph angiogenesis and the lymphatic transport in the context of oocyte maturation and their impact on mice fertility. In a first step, the mechanical properties of PEG-hydrogel were optimized for culturing secondary follicles. This report shows that the growth of the follicles was highly dependent on the mechanical properties of the surrounding environment. The optimal elastic modulus was found to be close to 900 Pa. In a second step, the effect of Arg-Gly-Asp (RGD) peptide, the minimal integrin-binding sequence, was studied. RGD presence did not influence the follicle growth rate but it significantly improved the quality of the produced oocytes. These findings demonstrated that approaching, biochemically and mechanically, the complexity of the ovarian extracellular matrix could be a winning strategy. The effect of key soluble factors was also investigated in order 1- to confirm their compatibility with the established 3-D culture system and 2- to further improve qualitatively and quantitatively the produced mature oocytes. Various combinations of gonadotropins such as the follicle-stimulating hormone (FSH) or the luteinizing hormone (LH) were tested. Interestingly, the effects of the gonadotropins in the 3-D PEG system were close to their known effects in vivo. Here the aberrant effects of these hormones in the used 2-D systems appeared clearly. c-Kit ligand (KL) is suspected to be one of the most important factors for the activation of primordial follicles and thus for controlling the exit from the resting pool. Previously, studying the effects of the two forms of KL (soluble and membrane-attached) had to cope with the lack of biologically-relevant immobilization strategies. For overcoming this problem, KL constructs were designed to include a substrate sequence for Factor XIIIa (NQEQVSPL or NQEQVSPLRCG). Thus the produced recombinant KL proteins could be enzymatically crosslinked to the PEG matrix. The different constructs of KL, including a wild type extracellular domain, were successfully cloned and produced from mammalian HEK-293 cells. The identity and the activity of the produced proteins were confirmed. Ovarian tissues from four days-old mice were cultured in PEG-hydrogels functionalized with KL. The results showed that the primordial follicles grew and were activated in the PEG-hydrogels where KL was immobilized but not when the soluble form of KL was preset in the medium. This experiment showed that the membrane-attached and the soluble form of KL play a different role in the rodent folliculogenesis. In parallel it was observed that blocking vascular endothelial growth factor receptor-3 (VEGFR-3) signaling had a critical but still unsuspected role in reproduction. This study demonstrates that variation in lymphangiogenesis is a regular, non-pathological event during folliculogenesis in the ovary; blocking lymphangiogenesis, might have an effect on hormone transport and thus on pregnancy. The reported results demonstrates that the blockade of lymphangiogenesis decreases the progesterone and estradioal levels during pregnancy and in fine results in failed fetal development. In conclusion, this study demonstrates the efficiency and the flexibility of a novel 3-D culture system. Circumventing problems inherent to the "on-plastic" standard culture, such as the loss of the granulosa-oocyte interactions, allowed the emergence of a culture system tailored for investigating fundamental folliculogenesis-related questions. Furthermore, the reported culture system might serve as a platform for developing clinical and biotechnology applications.

Chantier qualité GA; Background: Successful early folliculogenesis is crucial for female reproductive function. It requires appropriate gene specific expression of the different types of ovarian cells at different developmental stages. To date, most gene expression studies on the ovary were conducted in rodents and did not distinguish the type of cell. In mono-ovulating species, few studies have addressed gene expression profiles and mainly concerned human oocytes. Results: We used a laser capture microdissection method combined with RNA-seq technology to explore the transcriptome in oocytes and granulosa cells (GCs) during development of the sheep ovarian follicle. We first documented the expression profile of 15 349 genes, then focused on the 5 129 genes showing differential expression between oocytes and GCs. Enriched functional categories such as oocyte meiotic arrest and GC steroid synthesis reflect two distinct cell fates. We identified the implication of GC signal transduction pathways such as SHH, WNT and RHO GTPase. In addition, signaling pathways (VEGF, NOTCH, IGF1, etc.) and GC transzonal projections suggest the existence of complex cell-cell interactions. Finally, we highlighted several transcription regulators and specifically expressed genes that likely play an important role in early folliculogenesis. Conclusions: To our knowledge, this is the first comprehensive exploration of transcriptomes derived from in vivo oocytes and GCs at key stages in early follicular development in sheep. Collectively, our data advance our understanding of early folliculogenesis in mono-ovulating species and will be a valuable resource for unraveling human ovarian dysfunction such as premature ovarian failure (POF).