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Tight control of oocyte maturation and of gamete release is essential for successful sexual reproduction in the animal kingdom. These processes are precisely coordinated by endocrine and/or environmental cues, depending on the species, but much remains to be learned about their regulation. Within the Cnidaria, many hydrozoan jellyfish are known to spawn mature gametes following dark/light transitions. To characterise the cellular and molecular machinery linking light reception and oocyte maturation initiation, I have studied the hydrozoan jellyfish Clytia hemisphaerica. My thesis work had three parts, each involving the identification of a key molecular component of this process.My initial study was part of a collaboration with N. Takeda (Asamushi) and R. Deguchi (Sendai), who identified the endogenous oocyte Maturation-Inducing Hormones (MIH) in Clytia as WPRPamide-related tetrapeptides, generated by cleavage of two neuropeptide precursors. I showed by in situ hybridization and immunofluorescence that Clytia MIH is produced by neurosecretory cells of the gonad ectoderm that co-express the two precursor genes, and that it is secreted upon light stimulation. This study paved the way for identification of regulators acting upstream and downstream of MIH release in the gonads, specifically the ones involved in photoreception in the gonad ectoderm, and in MIH reception by the oocytes. To identify the Clytia MIH receptor (CheMIHR) in the oocytes, I compiled a shortlist of 16 candidate G protein-coupled receptors (GPCRs) from gonad transcriptome data. I cloned all 16 cDNAs and, using a cell culture-based "GPCR deorphanization" assay (collaboration with P. Bauknecht and G. Jékély; MPI, Tübingen), identified one GPCR that was activated by synthetic MIH peptides. Its in vivo function as the essential MIH receptor was confirmed by CRISPR/Cas9 gene editing. Introduction of a frame-shift mutation in the CheMIHR gene impaired growth of Clytia polyp colonies and also the spawning behaviour of mature medusae. Confirming the function of CheMIHR, oocyte maturation in CheMIHR mutants could not be triggered by light or by synthetic MIH, but could be restored using cell-permeable analogues of cAMP, known to act downstream of MIH reception in hydrozoan oocytes. Phylogenetic analyses showed that Clytia MIHR is related to a subset of bilaterian neuropeptide hormone receptor families involved in diverse physiological processes, including regulation of reproduction. Accordingly, in situ hybridization showed the expression of Clytia MIH precursors and MIHR in non-gonadal neural cells, suggesting a wider role of Clytia MIH-MIHR besides oocyte maturation initiation.To address gonad photoreception, I showed that Clytia spawning is selectively induced by blue-cyan light, and then identified using gonad transcriptome data an opsin photopigment (Opsin9) highly expressed in the ectoderm. Strikingly, in situ hybridization showed that Opsin9 is expressed in the MIH-secreting cells. Introduction of a frame-shift mutation into the Opsin9 gene via CRISPR/Cas9 prevented oocyte maturation and spawning of mutant jellyfish in response to light. Anti-MIH immunofluorescence and rescue experiments with synthetic MIH showed that the essential function of Opsin9 is upstream of MIH release. Spawning in Clytia thus appears to be regulated by a dual function photosensory-neurosecretory cell type, perhaps retained from a distant metazoan ancestor...; Un contrôle précis de la maturation ovocytaire et de la ponte sont essentiels au succès de la reproduction sexuée au sein le règne animal. Ces processus sont coordonnés précisément par des signaux endocriniens et/ou environnementaux, selon les espèces, mais beaucoup reste à apprendre sur leurs régulations. Chez les cnidaires, de nombreuses méduses du groupe des hydrozoaires sont connues pour produire des gamètes en réponse à la transition nuit/jour. Pour caractériser les machineries cellulaires et moléculaires liant la réception de la lumière à l'initiation de la maturation ovocytaire, j'ai étudié la méduse hydrozoaire Clytia hemisphaerica. Mon travail de thèse s’est découpé en trois parties, chacune impliquant l'identification d'un composant moléculaire clé de ce processus.Mon étude initiale faisait partie d'une collaboration avec N. Takeda (Asamushi) et R. Deguchi (Sendai), chercheurs qui avaient, avant le début de ma thèse, identifié chez Clytia les Hormones d'Incitation de Maturation ovocytaire endogènes (MIH) comme étant des tétrapeptides de type WPRPamide, produit par clivage de deux précurseurs à neuropeptides. J'ai montré par hybridation in situ et immunofluorescence que les deux gènes précurseurs du MIH sont exprimés par un type de cellules neurosécrétrices localisées au niveau de l’ectoderme de la gonade, et que les peptides MIH sont sécrétés par ces mêmes cellules suite à une stimulation lumineuse. Cette étude a posé les bases permettant l'identification des régulateurs agissant en amont et en aval du MIH, et plus spécifiquement ceux impliqués dans la photoréception de l’ectoderme de la gonade et la réception du MIH par les ovocytes.Pour identifier le récepteur du MIH de Clytia (CheMIHR) dans les ovocytes, j'ai compilé à partir de données transcriptomiques issues de tissus de gonades, une liste de 16 protéines candidates de la famille des Récepteurs Couplés aux Protéines G (GPCR). J'ai cloné les 16 cDNAs et, utilisant une méthode de « deorphelinisation » de GPCR basée sur de la culture cellulaire (collaboration avec P. Bauknecht et G. Jékély; MPI, Tübingen), j’ai pu identifier un GPCR activée par des peptides MIH synthétiques. Sa fonction in vivo comme récepteur essentiel du MIH a été confirmée par la méthode d'édition génétique CRISPR/CAS9. La délétion ainsi produite, entraînant un déplacement du cadre de lecture au sein du gène CheMIHR, a détérioré la croissance des colonies de polypes et le comportement de ponte des méduses matures. Confirmant la fonction de CheMIHR, la maturation ovocytaire chez des mutants CheMIHR ne pouvait pas être déclenchée par la lumière ou par addition de MIH synthétiques, mais pouvait être rétablie en utilisant des analogues au cAMP, molécule connue pour agir en aval de la réception du MIH dans les ovocytes d’hydrozoaires. Des analyses phylogénétiques ont montré que Clytia MIHR est affilié à un sous-ensemble de familles de neuropeptides de bilaterians impliqués dans divers processus physiologiques, notamment la régulation de la reproduction. Des hybridations in situ sur les méduses Clytia, ont en outre montré l'expression des précurseurs de CheMIH et de CheMIHR dans des cellules neurales hors de la gonade, suggérant un rôle plus large du couple CheMIH-MIHR que la seule initiation de la maturation ovocytaire.Pour mieux comprendre la photoréception des gonades chez Clyita, j'ai montré que la ponte est sélectivement incitée par la lumière bleu-cyan, et mis en évidence, grâce à l’analyse de données de transcriptome de gonade, qu’un photopigment de la famille des Opsin (Opsin9) est hautement exprimé dans l'ectoderme. De façon saisissante, les hybridations in situ ont montré que le gène Opsin9 est exprimé dans les mêmes cellules sécrétant le MIH. L'introduction d'une mutation de changement de cadre de lecture dans le gène Opsin9 via la technologie CRISPR/Cas9 a empêché la maturation ovocytaire et la ponte des méduses mutantes en réponse à la lumière...

Tight control of oocyte maturation and of gamete release is essential for successful sexual reproduction in the animal kingdom. These processes are precisely coordinated by endocrine and/or environmental cues, depending on the species, but much remains to be learned about their regulation. Within the Cnidaria, many hydrozoan jellyfish are known to spawn mature gametes following dark/light transitions. To characterise the cellular and molecular machinery linking light reception and oocyte maturation initiation, I have studied the hydrozoan jellyfish Clytia hemisphaerica. My thesis work had three parts, each involving the identification of a key molecular component of this process.My initial study was part of a collaboration with N. Takeda (Asamushi) and R. Deguchi (Sendai), who identified the endogenous oocyte Maturation-Inducing Hormones (MIH) in Clytia as WPRPamide-related tetrapeptides, generated by cleavage of two neuropeptide precursors. I showed by in situ hybridization and immunofluorescence that Clytia MIH is produced by neurosecretory cells of the gonad ectoderm that co-express the two precursor genes, and that it is secreted upon light stimulation. This study paved the way for identification of regulators acting upstream and downstream of MIH release in the gonads, specifically the ones involved in photoreception in the gonad ectoderm, and in MIH reception by the oocytes. To identify the Clytia MIH receptor (CheMIHR) in the oocytes, I compiled a shortlist of 16 candidate G protein-coupled receptors (GPCRs) from gonad transcriptome data. I cloned all 16 cDNAs and, using a cell culture-based "GPCR deorphanization" assay (collaboration with P. Bauknecht and G. Jékély; MPI, Tübingen), identified one GPCR that was activated by synthetic MIH peptides. Its in vivo function as the essential MIH receptor was confirmed by CRISPR/Cas9 gene editing. Introduction of a frame-shift mutation in the CheMIHR gene impaired growth of Clytia polyp colonies and also the spawning behaviour of mature medusae. Confirming the function of CheMIHR, oocyte maturation in CheMIHR mutants could not be triggered by light or by synthetic MIH, but could be restored using cell-permeable analogues of cAMP, known to act downstream of MIH reception in hydrozoan oocytes. Phylogenetic analyses showed that Clytia MIHR is related to a subset of bilaterian neuropeptide hormone receptor families involved in diverse physiological processes, including regulation of reproduction. Accordingly, in situ hybridization showed the expression of Clytia MIH precursors and MIHR in non-gonadal neural cells, suggesting a wider role of Clytia MIH-MIHR besides oocyte maturation initiation.To address gonad photoreception, I showed that Clytia spawning is selectively induced by blue-cyan light, and then identified using gonad transcriptome data an opsin photopigment (Opsin9) highly expressed in the ectoderm. Strikingly, in situ hybridization showed that Opsin9 is expressed in the MIH-secreting cells. Introduction of a frame-shift mutation into the Opsin9 gene via CRISPR/Cas9 prevented oocyte maturation and spawning of mutant jellyfish in response to light. Anti-MIH immunofluorescence and rescue experiments with synthetic MIH showed that the essential function of Opsin9 is upstream of MIH release. Spawning in Clytia thus appears to be regulated by a dual function photosensory-neurosecretory cell type, perhaps retained from a distant metazoan ancestor...; Un contrôle précis de la maturation ovocytaire et de la ponte sont essentiels au succès de la reproduction sexuée au sein le règne animal. Ces processus sont coordonnés précisément par des signaux endocriniens et/ou environnementaux, selon les espèces, mais beaucoup reste à apprendre sur leurs régulations. Chez les cnidaires, de nombreuses méduses du groupe des hydrozoaires sont connues pour produire des gamètes en réponse à la transition nuit/jour. Pour caractériser les machineries cellulaires et moléculaires liant la réception de la lumière à l'initiation de la maturation ovocytaire, j'ai étudié la méduse hydrozoaire Clytia hemisphaerica. Mon travail de thèse s’est découpé en trois parties, chacune impliquant l'identification d'un composant moléculaire clé de ce processus.Mon étude initiale faisait partie d'une collaboration avec N. Takeda (Asamushi) et R. Deguchi (Sendai), chercheurs qui avaient, avant le début de ma thèse, identifié chez Clytia les Hormones d'Incitation de Maturation ovocytaire endogènes (MIH) comme étant des tétrapeptides de type WPRPamide, produit par clivage de deux précurseurs à neuropeptides. J'ai montré par hybridation in situ et immunofluorescence que les deux gènes précurseurs du MIH sont exprimés par un type de cellules neurosécrétrices localisées au niveau de l’ectoderme de la gonade, et que les peptides MIH sont sécrétés par ces mêmes cellules suite à une stimulation lumineuse. Cette étude a posé les bases permettant l'identification des régulateurs agissant en amont et en aval du MIH, et plus spécifiquement ceux impliqués dans la photoréception de l’ectoderme de la gonade et la réception du MIH par les ovocytes.Pour identifier le récepteur du MIH de Clytia (CheMIHR) dans les ovocytes, j'ai compilé à partir de données transcriptomiques issues de tissus de gonades, une liste de 16 protéines candidates de la famille des Récepteurs Couplés aux Protéines G (GPCR). J'ai cloné les 16 cDNAs et, utilisant une méthode de « deorphelinisation » de GPCR basée sur de la culture cellulaire (collaboration avec P. Bauknecht et G. Jékély; MPI, Tübingen), j’ai pu identifier un GPCR activée par des peptides MIH synthétiques. Sa fonction in vivo comme récepteur essentiel du MIH a été confirmée par la méthode d'édition génétique CRISPR/CAS9. La délétion ainsi produite, entraînant un déplacement du cadre de lecture au sein du gène CheMIHR, a détérioré la croissance des colonies de polypes et le comportement de ponte des méduses matures. Confirmant la fonction de CheMIHR, la maturation ovocytaire chez des mutants CheMIHR ne pouvait pas être déclenchée par la lumière ou par addition de MIH synthétiques, mais pouvait être rétablie en utilisant des analogues au cAMP, molécule connue pour agir en aval de la réception du MIH dans les ovocytes d’hydrozoaires. Des analyses phylogénétiques ont montré que Clytia MIHR est affilié à un sous-ensemble de familles de neuropeptides de bilaterians impliqués dans divers processus physiologiques, notamment la régulation de la reproduction. Des hybridations in situ sur les méduses Clytia, ont en outre montré l'expression des précurseurs de CheMIH et de CheMIHR dans des cellules neurales hors de la gonade, suggérant un rôle plus large du couple CheMIH-MIHR que la seule initiation de la maturation ovocytaire.Pour mieux comprendre la photoréception des gonades chez Clyita, j'ai montré que la ponte est sélectivement incitée par la lumière bleu-cyan, et mis en évidence, grâce à l’analyse de données de transcriptome de gonade, qu’un photopigment de la famille des Opsin (Opsin9) est hautement exprimé dans l'ectoderme. De façon saisissante, les hybridations in situ ont montré que le gène Opsin9 est exprimé dans les mêmes cellules sécrétant le MIH. L'introduction d'une mutation de changement de cadre de lecture dans le gène Opsin9 via la technologie CRISPR/Cas9 a empêché la maturation ovocytaire et la ponte des méduses mutantes en réponse à la lumière...

International audience; The production of fertile eggs with the capacity to develop into larvae and subsequently into marketable fish is centrally important to the aquaculture industry. This entails not only the programmed production of large numbers of eggs, but also high quality eggs with the potential to support normal development and high survival of offspring to juvenile and later stages of development and growth. Numerous studies highlight the maternal contributions to the development of embryos, including transcripts that regulate cell division and determine oocyte polarity, pattern development during early and late embryonic stages and the transition from maternal to zygotic gene expression and translation. Since most fish embryos develop independently within an enclosed egg envelope, they rely on compounds deposited within the oocytes during their various stages of development. In addition to regulatory nucleic acids (maternal DNA and RNA), these include proteins and other compounds that contribute to the structure and function of the egg envelope and the bulk molecular cargo that will be used as a source of cellular energy and structural components for formation of embryos and larvae. These latter components notably include yolk lipids and proteins deposited during oocyte growth and water acquired at the same time and during cytoplasmic maturation. In this review we highlight recent advances made in revealing the transcripts deposited within the oocyte that contribute to the structural and morphological development of the embryo, and to the regulation of gene expression and translation during oocyte development. Significant advances have been made in revealing the molecular mechanisms of lipid accumulation and metabolism within the oocyte, the intricacies of yolk protein formation via endocytosis of multiple yolk precursor proteins by multiple oocyte receptors, and the complex machinery supporting massive accumulation of water by maturing oocytes of many species. Additionally, many advances have been made in our understanding of the endocrine regulation of all of these processes during oogenesis. We provide here an overview of recent advances in our knowledge on these various aspects of oogenesis and identify several gaps in our knowledge for future studies.

The endogenous peptides and small proteins extracted from bovine ovarian follicular cells (oocytes, cumulus and granulosa cells) were identified by Top-down High Resolution Mass Spectrometry (TD-HR-MS/MS) in order to annotate peptido- and proteoforms detected using qualitative and quantitative profiling method based on ICM-MS (Intact Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry). The description and analysis of these Top-down MS data in the context of oocyte quality biomarkers research are available in the original research article of Labas et al. (2017) http://dx.doi.org/10.1016/j.jprot.2017.03.027 [1]. Raw data derived from this peptidomic/proteomic analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD004892). Here, we described the inventory of all identified peptido- and proteoforms including their biochemical and structural features, and functional annotation of correspondent proteins. This peptide/protein inventory revealed that TD-HR-MS/MS was appropriate method for both global and targeted proteomic analysis of ovarian tissues, and it can be further employed as a reference for other studies on follicular cells including single oocytes. Keywords: Top-down proteomics, Bovine, Ovary, Follicular cells

International audience; BACKGROUND:We previously showed that the homeodomain transcription factor HOXB9 is expressed in mammalian oocytes and early embryos. However, a systematic and exhaustive study of the localization of the HOXB9 protein, and HOX proteins in general, during mammalian early embryonic development has so far never been performed.RESULTS:The distribution of HOXB9 proteins in oocytes and the early embryo was characterized by immunofluorescence from the immature oocyte stage to the peri-gastrulation period in both the mouse and the bovine. HOXB9 was detected at all studied stages with a dynamic expression pattern. Its distribution was well conserved between the two species until the blastocyst stage and was mainly nuclear. From that stage on, trophoblastic cells always showed a strong nuclear staining, while the inner cell mass and the derived cell lines showed important dynamic variations both in staining intensity and in intra-cellular localization. Indeed, HOXB9 appeared to be progressively downregulated in epiblast cells and only reappeared after gastrulation had well progressed. The protein was also detected in the primitive endoderm and its derivatives with a distinctive presence in apical vacuoles of mouse visceral endoderm cells.CONCLUSIONS:Together, these results could suggest the existence of unsuspected functions for HOXB9 during early embryonic development in mammals.

Three complete vitellogenin (Vtg) polypeptides of European sea bass (Dicentrarchus labrax), an acanthomorph teleost spawning pelagic eggs in seawater, were deduced from cDNA and identified as VtgAa, VtgAb and VtgC based on current Vtg nomenclature and phylogeny. Label free quantitative mass spectrometry verified the presence of the three sea bass Vtgs or their product yolk proteins (YPs) in liver, plasma and ovary of postvitellogenic females. As evidenced by normalized spectral counts, VtgAb-derived protein was 2- to 5-fold more abundant, depending on sample type, than for VtgAa, while VtgC-derived protein was less abundant, albeit only 3-fold lower than for VtgAb in the ovary. Western blotting with Vtg type-specific antisera raised against corresponding gray mullet (Mugil cephalus) lipovitellins (Lvs) detected all three types of sea bass Vtg in the blood plasma of gravid females and/or estrogenized males and showed that all three forms of sea bass Lv undergo limited partial degradation during oocyte maturation. The comparatively high levels of VtgC-derived YPs in fully-grown oocytes and the maturational proteolysis of all three types of Lv differ from what has been reported for other teleosts spawning pelagic eggs in seawater but are similar to recent findings for two species of North American Moronidae, the striped bass (Morone saxatilis) and white perch (Morone americana), which spawn pelagic and demersal eggs, respectively in fresh water. Together with the high Vtg sequence homologies and virtually identical structural features of each type of Vtg between species, these findings indicate that the moronid multiple Vtg systems do not substantially vary with reproductive environment., This research was supported by awards to Ozlem Yilmaz from the International Fulbright Visiting Student Researcher Program for doctoral research in the United States, and to Craig V. Sullivan from the North Carolina Agricultural Foundation, Inc.

L’infertilité touche environ 15% des couples en âge de procréer. Depuis 1978, la fécondation in vitro (FIV) a révolutionné la médecine reproductive en permettant la naissance de plus de 5 millions d’enfants dans le monde. Malgré de nombreux progrès, l’enjeu actuel reste l’augmentation des taux de grossesse en FIV, puisque 3 cycles de FIV sur 4 sont soldés par un échec avec plus de 80% des embryons transférés qui ne s’implantent pas. Récemment, la prokinéticine 1 (PROK1) a été proposée comme nouveau biomarqueur prédictif de l’implantation embryonnaire, suggérant un rôle pour ce facteur dans la folliculogenèse, et plus particulièrement dans l’acquisition par l’ovocyte de la compétence à donner un embryon apte au développement et à l’implantation dans la cavité utérine. Mon projet de thèse a porté sur l’étude du rôle de PROK1 dans l’acquisition de la compétence ovocytaire, avec pour objectifs d’évaluer la concentration de PROK1 en fonction de la maturité ovocytaire, ainsi que de caractériser les régulations hormonales de l’expression de PROK1 et de ses récepteurs par les deux hormones clés de la folliculogenèse, la FSH et l’hCG, dans des cultures primaires de cellules folliculaires collectées dans le cadre de FIV-ICSI. Mes résultats ont démontré l’augmentation significative de l’expression de PROK1 in vivo et in vitro au cours de la maturation folliculaire. Si ces résultats préliminaires se confirment, le dosage de PROK1 pourrait être considéré comme un nouveau biomarqueur de routine pour le choix de l’embryon à réimplanter, permettant ainsi d’envisager l’augmentation des taux de grossesse en FIV.

Pregnancy‐Associated Glycoproteins belong to a multigenic family of aspartic proteases synthetized in the trophoblast of ruminants during gestation. In the Bos taurus specie, there are 21 PAG segregated in two phylogenetic groups (ancient and modern). Except that modern PAG are produced in binucleate cells and reach the maternal blood circulation where their assays are used as gestation diagnosis, data concerning PAG are fragmentary. In this study, we interested in the characterization of PAG during gestation to understand the placental differentiation and to discover data that could improve the current gestation diagnosis in the cow. We showed that PAG are differentially regulated according to their phylogenetic origin and trophoblastic areas. Modern PAG are essentially synthetized in cotyledons whereas ancient PAG are restricted to the intercotyledonary trophoblast, except for PAG2. Moreover, each PAG (or type of PAG) are produced in distinct and specific cellular populations including binucleate cells synthetizing ancient PAG. Before the implantation, only ancient PAG are expressed and the cellular population synthetizing PAG11 change completely 17 days post‐insemination. Besides, PAG11 is secreted in the uterus lumen and would be an active aspartic protease even as a zymogen. Finally, we demonstrated that the somatic cells of the cumulus‐oocyte complex expressed PAG11 and that this synthesis is correlated to the oocyte maturation.; Les PAG forment une famille multigénique de protéases aspartiques exprimées dans le trophoblaste des ruminants au cours de la gestation. Chez Bos taurus, cette famille comprend 21 membres divisés en deux groupes phylogénétiques (ancien et moderne). Excepté le fait que les PAG modernes sont synthétisées par les cellules binucléées et atteignent la circulation maternelle où leur dosage sert de diagnostic de gestation, les informations relatives aux PAG restent fragmentaires. Dans cette étude, nous nous sommes intéressés à différents aspects relatifs à ces protéines au cours de la gestation afin de mieux comprendre la différentiation placentaire et de découvrir des informations pouvant permettre l’amélioration des techniques actuelles de diagnostic de gestation. Nous avons montré que les PAG sont différemment régulées en fonction de leur origine phylogénétique et des compartiments trophoblastiques. Les PAG modernes sont essentiellement produites par les cotylédons tandis que les anciennes sont restreintes au trophoblaste intercotylédonaire à l’exception de la PAG2. De plus, il existe des populations cellulaires distinctes et spécifiques de chaque PAG (ou type de PAG) dont des cellules binucléées exprimant des PAG anciennes. Avant l’implantation, seules les PAG anciennes sont exprimées et la population cellulaire responsable de la synthèse de PAG11 change radicalement vers 17 jours post‐insémination. La PAG11 est d’autre part sécrétée dans la lumière utérine et serait une protéase active sous sa forme proenzyme. Enfin, nous avons montré l’existence de la synthèse d PAG11 dan les cellules somatiques du complexe ovocyte‐cumulus et la corrélation de son expression avec la maturation ovocytaire.

Poster P-088 - Session : Protéomique; absent

In vitro maturation of oocytes is a crucial step in assisted reproductive technologies in cattle; however, the molecular mechanisms of cumulus contribution to oocyte developmental potential require more investigation. Based on transcriptomic data, we studied by using real-time RT-PCR and western blot in bovine cumulus cells, the kinetics of expression of several candidate genes involved in oxidative stress response, apoptosis, steroid metabolism and signal transmission throughout IVM. Phosphorylations of the components of the main signaling pathways were also analyzed. In addition, IVM was performed in different maturation mediums which influenced the cumulus apoptosis, progesterone secretion and oocyte developmental competence. Glutathione-S-transferase A1 (GSTA1) transcript and protein abundance significantly decreased throughout IVM progression. Similarly, transcript levels of FSH receptor and aromatase (CYP19A1) and protein levels of three steroidogenic enzymes (steroidogenic acute regulatory protein, cytochrome P450scc and 3-beta-hydroxysteroid dehydrogenase) decreased along with progression of maturation and especially since 10 hours of IVM. Expression of progesterone receptor (PGR) and clusterin (CLU) mRNA and phosphorylations of protein kinases AKT, MAPK P38 and SMAD2 were particularly increased at 10 hours of IVM. This expression pattern supposed the role of these factors during oocyte metaphase-I check point of meiosis. Levels of CLU, GSTA1 and FSHR transcripts were higher in 199 basic hormone-free medium as compared to the medium 199EM, enriched in gonadotropins and growth factors, in which we recorded the higher developmental rate and progesterone secretion. Higher phosphorylation levels of SMAD2, AKT and MAP kinase JNK1, but not of MAP kinases ERK1/ERK2 or P38, was positively correlated with oocyte developmental competence and progesterone secretion and negatively correlated with cumulus apoptosis rate. These factors and signaling pathways in cumulus cells are potentially involved in controlling different stages of oocyte nuclear maturation and acquirement of its developmental potential.

Diplôme : Dr. d'Universite

Diplôme : Dr. d'Universite

BACKGROUND: Dialogue between the oocyte and cumulus cells is essential for oocyte maturation. A prospective laboratory research project was designed to evaluate transcription of specific genes in cumulus cells harvested before intracytoplasmic sperm injection from pre-ovulatory follicles, according to individual oocyte nuclear maturity and developmental competence. Genes were chosen because their expression was induced by the LH peak [Steroidogenic Acute Regulatory protein (STAR), Cyclooxygenase 2 (COX2 or PTGS2), Amphiregulin (AREG)] or because they were involved in oocyte lipidic metabolism [Stearoyl-Coenzyme A Desaturase 1 and 5 (SCD1 and SCD5)] or in gap-junctions [Connexin 43 (CX43 or GJA1)]. METHODS: mRNA levels in cumulus cells were assessed by real-time PCR. RESULTS: Expression levels of all genes investigated, except Cx43, were increased after resumption of meiosis. Nuclear maturation was thus associated with increased expression of STAR, COX2, AREG, SCD1 and SCD5 by cumulus cells. When considering only cumulus associated with metaphase II oocytes, gene expression was independent of morphological status at Day 2. In contrast, transcript levels were lower and distributed over a narrower range in cumulus enclosing oocytes achieving blastocyst development at Day 5/6 than in cumulus enclosing oocytes unable to develop beyond the embryo stage. CONCLUSION: Further developmental potential from embryo to blastocyst stage was associated with lower expression in a narrow range for these genes.

BackgroundIn lower vertebrates, steroid-induced oocyte maturation is considered to involve membrane-bound progestin receptors. Two totally distinct classes of putative membrane-bound progestin receptors have been reported in vertebrates. A first class of receptors, now termed progesterone membrane receptor component (PGMRC; subtypes 1 and 2) has been studied since 1996 but never studied in a fish species nor in the oocyte of any animal species. A second class of receptors, termed membrane progestin receptors (mPR; subtypes alpha, beta and gamma), was recently described in vertebrates and implicated in the progestin-initiated induction of oocyte maturation in fish.MethodsIn the present study, we report the characterization of the full coding sequence of rainbow trout PGMRC1 and mPR beta cDNAs, their tissue distribution, their ovarian expression profiles during oogenesis, their hormonal regulation in the full grown ovary and the in situ localization of PGMRC1 mRNA in the ovary.ResultsOur results clearly show, for the first time in any animal species, that rainbow trout PGMRC1 mRNA is present in the oocyte and has a strong expression in ovarian tissue. In addition, we show that both mPR beta and PGMRC1, two members of distinct membrane-bound progestin receptor classes, exhibit highly similar ovarian expression profiles during the reproductive cycle with maximum levels during vitellogenesis and a down-expression during late vitellogenesis. In addition, the mRNA abundance of both genes is not increased after in vitro hormonal stimulation of full grown follicles by maturation inducing hormones.ConclusionTogether, our findings suggest that PGMRC1 is a new possible participant in the progestin-induced oocyte maturation in fish. However, its participation in the process of oocyte maturation, which remains to be confirmed, would occur at post-transcriptional levels.

Background The hormonal control of oocyte maturation and ovulation as well as the molecular mechanisms of nuclear maturation have been thoroughly studied in fish. In contrast, the other molecular events occurring in the ovary during post-vitellogenesis have received far less attention. Methods Nylon microarrays displaying 9152 rainbow trout cDNAs were hybridized using RNA samples originating from ovarian tissue collected during late vitellogenesis, post-vitellogenesis and oocyte maturation. Differentially expressed genes were identified using a statistical analysis. A supervised clustering analysis was performed using only differentially expressed genes in order to identify gene clusters exhibiting similar expression profiles. In addition, specific genes were selected and their preovulatory ovarian expression was analyzed using real-time PCR. Results From the statistical analysis, 310 differentially expressed genes were identified. Among those genes, 90 were up-regulated at the time of oocyte maturation while 220 exhibited an opposite pattern. After clustering analysis, 90 clones belonging to 3 gene clusters exhibiting the most remarkable expression patterns were kept for further analysis. Using real-time PCR analysis, we observed a strong up-regulation of ion and water transport genes such as aquaporin 4 (aqp4) and pendrin (slc26). In addition, a dramatic up-regulation of vasotocin (avt) gene was observed. Furthermore, angiotensin-converting-enzyme 2 (ace2), coagulation factor V (cf5), adam 22, and the chemokine cxcl14 genes exhibited a sharp up-regulation at the time of oocyte maturation. Finally, ovarian aromatase (cyp19a1) exhibited a dramatic down-regulation over the post-vitellogenic period while a down-regulation of Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) was observed at the time of oocyte maturation. Conclusion We showed the over or under expression of more that 300 genes, most of them being previously unstudied or unknown in the fish preovulatory ovary. Our data confirmed the down-regulation of estrogen synthesis genes during the preovulatory period. In addition, the strong up-regulation of aqp4 and slc26 genes prior to ovulation suggests their participation in the oocyte hydration process occurring at that time. Furthermore, among the most up-regulated clones, several genes such as cxcl14, ace2, adam22, cf5 have pro-inflammatory, vasodilatory, proteolytics and coagulatory functions. The identity and expression patterns of those genes support the theory comparing ovulation to an inflammatory-like reaction.

Holothuria scabra est l'holothurie comestible à haute valeur marchande la plus distribuée et la plus exploitée dans l'Indo-Pacifique tropical. Dès lors, et à cause de la demande croissante des pays importateurs et consommateurs, les stocks naturels de l'espèce sont confrontés actuellement un problème de surexploitation. A Madagascar, les situations économique, sociale et politique difficiles ainsi que l'insuffisance d'un plan efficace pour l'aménagement de cette exploitation, rendent la situation plus délicate encore. La surexploitation se traduit par la diminution de la production, la raréfaction de certaines espèces, la dégradation de la qualité du produit et entraîne une forte concurrence entre les exploitants et le non respect de la législation existante. Un plan d'aménagement de cette exploitation est proposé dans ce travail. Celui-ci concerne la gestion de la pêche, la restauration et la pérennisation des stocks naturels, et la valorisation des produits. L'holothuriculture est considérée comme la solution d'avenir pour pallier une demande sans cesse croissante en trépang, restaurer les stocks naturels et sauver les espèces d'holothuries menacées d'extinction. \, Doctorat en sciences, Spécialisation biologie animale, info:eu-repo/semantics/nonPublished

The aim of this study was to describe and compare the gametogenic activity of Ruditapes decussatus in situ and in hatchery. Criteria of maturity were characterized and the positive role of conditioning in the success of artificial reproduction was underlined. Specimens sampled in the South of Tunisia were conditioned at the experimental station of Monastir (Tunisia). The parameters studied were the variation of the condition index and the frequency distribution of oocyte diameters. Results showed that the reproductive cycle was continuous in situ. Gametogenesis started in January and February. Gonadic activity increased from March to November – December. Spawning events occurred irregularly from June to December. In winter conditioning, mature oocytes were produced from March and spawning began in April. The increase in temperature and food abundance shortened the gametogenesis duration; ripe gametes were produced two months earlier compared to natural environment. In summer conditioning, the reproductive cycle was the same as that in the natural site, maturation and spawning occurred simultaneously. In bivalves, the blockage of oocytes at prophase I is released only during the egg-laying period. The second blockage occurs during metaphase I and is released by fertilization. In the case of the venerid Ruditapes decussatus, when we dissect the gonad we obtain oocytes which are blocked at prophase I and so cannot be fertilized. In order to release this blockage, we used serotonin (5-HT). This study is the first to scrutinise the maturation of oocytes induced by serotonin in R. decussatus. We have thus demonstrated that adding 20 µM of serotonin in an external application allows the recovery of meiosis and the passage of oocytes from germinal vesicle (G.V.) to germinal vesicle break down (GVBD) in approximately 90 minutes at a temperature of 20°C. The highest percentage of oocytes obtained in GVBD was 67%. We have also evaluated the competence of oocytes in GVBD during fertilization, and found the greater the degree of reproductive condition, the higher is the percentage of oocytes that can undergo GVBD. From an estimated threshold of 53% of oocytes capable of GVBD, broodstock placed in a controlled environment are liable to spawn when they undergo thermal shocks. This allows us to consider this percentage as a criterion for the evaluation of oocytes during fertilization, Cette étude nous a permis tout d'abord de décrire et de comparer la gamétogenèse chez R. decussatus, en milieu naturel (sud tunisien) et en milieu contrôlé, de définir les critères d'évaluation de leur maturité et de mettre en évidence le rôle positif du conditionnement dans la réussite de la reproduction artificielle. Afin de répondre à ces objectifs, des approches qualitatives (analyse histologique) et semi-quantitatives (suivi d'un indice de condition et analyse d'images) sont utilisées. Les résultats obtenus montrent que le cycle sexuel est continu. En janvier et février, les individus débutent leur gamétogenèse. De mars à novembre – décembre, l'activité gonadique est importante. La période d'émissions gamétiques s'étale de juin à décembre avec un intervalle de temps irrégulier entre les émissions. En conditionnement d'hiver, les palourdes produisent des ovocytes matures à partir de mars et les émissions gamétiques commencent en avril. Le cycle est alors accéléré grâce à l'élévation de température et l'abondance de nourriture, les reproducteurs produisent alors des gamètes matures plutôt dans l'année, en avance de trois mois par rapport au milieu naturel. Quant au conditionnement d'été, l'évolution du cycle sexuel est la même qu'en milieu naturel et les périodes de maturation et d'émissions gamétiques coïncident parfaitement. Ainsi, cette étude nous a permis de fournir des résultats essentiels pour la maîtrise du conditionnement de la palourde R. decussatus, permettant aux écloseurs d'évaluer l'état de maturité des géniteurs, de concentrer leurs efforts sur le conditionnement uniquement à certaines périodes de l'année allant de février à mai, ce qui leur permet de gagner autant en temps qu'en coût de production. Par ailleurs, afin de maîtriser d'avantage l'élevage de la palourde et perfectionner les techniques de production en écloserie, nous avons tenté de trouver une technique alternative aux chocs thermiques qui nous permet d'obtenir plus facilement et plus rapidement des gamètes matures pour la conduite d'un élevage larvaire. La méthode proposée est celle de la dissection de la gonade (stripping). Cette étude étant la première à s'intéresser à la maturation ovocytaire pouvant être induite par la sérotonine chez cette espèce de bivalve en milieu contrôlé. Nous avons ainsi montré que l'ajout de 20 µM de sérotonine au milieu extérieur permet la reprise de la méiose des ovocytes strippés en environ 90 minutes à 20°C. Le maximum de réussite étant de 67%. Par ailleurs, nous avons aussi pu évaluer la compétence des ovocytes débloqués à la fécondation. En effet, plus les géniteurs entrent en maturation plus le pourcentage des ovocytes fécondables augmente. A partir d'un seuil estimé à 53%, les géniteurs placés en milieu contrôlé, répondent positivement aux chocs thermiques subis. Ceci nous permet d'estimer ce pourcentage comme un critère d'évaluation de la compétence des ovocytes à la fécondation et ainsi de choisir le moment propice pour la réalisation d'un élevage larvaire. En 3ème et dernière partie, nous avons recherché si des variations des paramètres immunitaires (cellulaires et biochimiques de l'hémolymphe) pouvaient être associées à des changements physiologiques liés à la reproduction, ceci dans le but d'approfondir notre étude, de mieux comprendre le comportement hémocytaire pendant le cycle de reproduction et de chercher de nouveaux critères de maturation en moyennant de nouvelles approches autres que celles couramment utilisées (histologie et analyse d'image) dans la littérature. Ainsi les mécanismes physiologiques liés à la reproduction entraînent des changements profonds dans les paramètres de défense de R. decussatus en milieu naturel ou en milieu contrôlé. En période de reproduction, on assiste à une migration des hémocytes des tissus vers le compartiment circulatoire pour le transport de métabolites pour l'approvisionnement des gonades d'où l'augmentation de la concentration hémocytaire observée pendant cette période. En période d'émissions gamétiques, on assiste à une diminution de la concentration hémocytaire, liée à la mobilisation des hémocytes vers la gonade. Dans le cas de la résorption complète de la gonade, une forte augmentation de la concentration hémocytaire est signalée, ceci nous permet d'attribuer aux hémocytes un rôle potentiel de nettoyage après atrésie. Outre les paramètres cellulaires, les paramètres biochimiques de l'hémolymphe peuvent aussi témoigner des changements cycliques liés à la reproduction, en particulier en période de maturation gamétique où une forte synthèse d'enzymes lysosomales dans le sérum est observée, liée à leur rôle dans le transport de réserve. Ainsi afin de préciser le rôle des hémocytes et approfondir les liens entre la reproduction et l'immunité chez les bivalves, des dosages plus précis des activités fonctionnelles des hémocytes sont préconisés ; d'une part des tests fonctionnels tels que l'évaluation de la phagocytose pourraient être inclus dans le protocole, en association avec les activités microbicides afin de mieux préciser le rôle des hémocytes dans la défense, d'autre part des activités biochimiques tel que des mesures de métabolites comme les lipides dans l'hémolymphe afin d'évaluer l'activité hémocytaire dans la reproduction.

Carps form the major inland fishery resource inthe lndian peninsula. The economic and growthpotential offered by the carps is instrumental in thedevelopment of the inland capture fisheries. With a vastextent of lndia's landscape tucked away from the sea,inland fishery has often proved to be a greatersubstitute for the seafood. Among the total inlandfishery catch, the carps form the major part. Theirpotential to withstand a wide range of salinity (500-3000ppm), comparatively faster growth rate andincreased fecundity have made them a viable speciesfor culture. Relatively larger size attained by the carpsas well as their increased commercial value havepaved way for further research in the development ofthese fishes.

Recent data showing the occurrence of IGF in fish, mainly teleostean fish, and their biological activity in gonads are reviewed. IGF-I does occur in all vertebrates and first evidence of IGF-II occurrence in fish has been reported. IGF-I is expressed in the testis and the ovary, and receptors for the peptide have been demonstrated in both gonads. Besides, IGF-I seems implied in spermatogonial proliferation and in oocyte maturation. Both germinal cells and somatic cells could be responsive to IGF-I stimulation., Cet article présente une courte revue des résultats montrant l'existence des IGF chez quelques espèces de poisson, essentiellement des téléostéens, et leurs rôles potentiels dans les gonades. Il est maintenant acquis que l'IGF-I existe chez tous les vertébrés, y compris les plus primitifs. L'existence de l'IGf-II semble également se confirmer. La synthèse du premier de ces peptides est possible dans le testicule et l'ovaire où ses récepteurs ont été mis en évidence. L'IGF-I paraît impliqué dans la régulation des multiplications goniales chez le mâle et celle de la maturation meïotique de l'ovocyte chez la femelle. A la fois les cellules somatiques et les cellules germinales pourraient être sensibles à l'IGF-I.

In carp determination was made of the gonadotropin (GtH2) level and oocyte maturity state after stimulation with LHRH analogue (D-Trp6 LH-RH) in pure form or as the DECAPEPTYL preparation characterized by prolonged action. Two thermal regimes were applied prior to hormonal injections : 1) fish where kept in water of a constant temperature of 20°C, and 2) water temperature was elevated from 15 to 20°C by 1°C daily. Fish exposed to the 2nd treatment, as compared with those subjected to the 1st one, displayed a significantly higher (p

International audience

International audience

Diverses observations et résultats d'expériences réalisées à la Salmoniculture du Conseil Supérieur de la Pêche à Augerolles sont présentés, concernant les techniques de reproduction artificielle adaptées à la physiologie de l'Ombre commun. Les caractéristiques physiologiques mises en évidence sont proches de celles de la Truite avec cependant une sensibilité accrue aux basses températures , qui peuvent ralentir très fortement la fin de la maturation ovocytaire. Ces observations et expériences conduisent les auteurs à formuler un certain nombre de conseils pratiques., Various observations and results of experiments carried out in the hatchery of the "Conseil Supérieur de la Pêche" in Augerolles are presented, concerning the technics of artificials pawning adapted to the physiology of grayling . It is shown that th e physiological characteristics are nearly t he same as those of brown trout . Nevertheless highs ensitivity to low temperatures reduces the speed of the finaloocyte maturation. These observations and experiments lead the authors to give practical recommendations.

Trout ovaries were processed in vitro to determine relationships between the following parameters — oocyte sensitivity to the maturational steroid 17α-hydroxy-20β-dihydroprogesterone (17α,20β-OH-P); follicular sensitivity to the maturational gonadotropin s-GtH; inhibitory potency of exogenous oestradiol-17β (E2) during s-GtH-induced maturation; and level of E2 in the plasma. The sensitivity to hormones was estimated by the median efficient dose (MED) for oocyte maturation in vitro. The peripheral migration of the germinal vesicle coincided with an increase in oocyte sensitivity to 17α,20β-OH-P. A significant correlation was observed between plasma E2 level and the follicular sensitivity to s-GtH, but not between plasma E2 and the oocyte sensitivity to 17α,20β-OH-P. It was concluded that the peripheral migration of the GV is a morphological event which coincides with an increase in oocyte sensitivity, and that E2 is a physiological regulator of follicular sensitivity to GtH.

The changes in cAMP were followed in trout oocytes incubated in vitro after defolliculation performed by either enzymatic or manual dissection. Both defolliculation methods induced a highly significant rise in oocyte cAMP level (4.5 times the basal level of control [follicle-enclosed oocytes], after 6 h). Treatment of defolliculated oocytes with 17 alpha-hydroxy, 20 beta-dihydroprogesterone (17 alpha, 20 beta-OH-P) (10(-6) M), which induced oocyte maturation (germinal vesicle breakdown [GVBD]) was able, first, to interrupt the increase of oocyte cAMP level promoted by defolliculation and then to lower this level significantly down to values that still remained higher than folliculated controls. Very low concentrations of 17 alpha, 20 beta-OH-P (1.38-55.6 10(-9) M), or physiological doses of testosterone (0.35 10(-6) M, in the range found in vivo before ovulation) were able to induce a similar decrease of oocyte cAMP level without inducing GVBD. Under the same experimental conditions estradiol (0.35 10(-6) M) exhibited no action. These results suggest that some factor(s) originating in the follicle (FIF), inhibit the oocytes' tendency to accumulate cAMP before the final surge of 17 alpha, 20 beta-OH-P. This factor might be a follicular steroid such as testosterone or nonmaturing concentrations of 17 alpha, 20 beta-OH-P. Moreover our data favour the hypothesis that the final surge of 17 alpha, 20 beta-OH-P could induce distinct intraoocyte mechanisms: the first induces an irreversible blockage of cAMP level before the inhibitory action of the FIF is suppressed by ovulation, and the second mechanism leads to GVBD.

International audience; A dual cannulation of free-swimming rainbow trout is used to estimate the metabolic clearance rate (MCR) of Estradiol-17β (E2-17β) by both single injection and constant infusion methods. It is shown that E2-17β MCR changes significantly during the progress of oogenesis, mainly at the end of the sexual cycle. The same changes in MCR and very similar values are found with both single injection and constant infusion methods: MCR is stable (28.8 ml/hr/kg) from the postovulation period (throughout endogenous vitellogenesis) to early exogenous vitellogenesis. It decreases significantly during advanced exogenous vitellogenesis (18.7 ml/hr/kg) and increases clearly at the onset of oocyte maturation (40.9 ml/hr/ kg). A direct relationship between MCR and plasma E2-17β occurs: Plasma E2-17β levels increase (advanced exogenous vitellogenesis) when MCR decreases. Then estradiol decline takes place at the same time that MCR reaches its highest values (oocyte maturation). An increase in MCR is probably one event required to allow the establishment of an appropriate hormonal environment for oocyte maturation.

Ovulation induced by a single injection of 17 α -hydroxy-20 β -dihydroprogesterone (17 α –20 β P) in rainbow trout at the subperipheral germinal vesicle stage was compared with spontaneous ovulation. Three criteria were considered: the time course of the maturation-ovulation sequence, the embryo and fry viability, and the rate of aging of ova retained in the body cavity after ovulation. Following treatment with 17 α –20 β P at 13°C, the various stages of oocyte maturation took place between day 1 and day 4 and ovulation occurred on days 3, 4, and 5 with a peak on day 4. The same timing was observed in the case of spontaneous ovulation. Treatment with 17 α –20 β P did not affect embryo or fry viability until the end of yolk sac resorption. Lastly, in both natural and steroid-induced ovulation, ova fertility had decreased significantly by the 9th day of retention in the female body cavity, at 13°C. The overall rate of ova aging was similar in both groups. These data demonstrate that ovulation induced by 17 α –20 β P at the subperipheral germinal vesicle stage is close to the natural, spontaneous, process.

Le cycle reproducteur des poissons est sous la dépendance de l'hypophyse qui élabore et secrète les hormones gonadotropes. Ces hormones interviennent lors du déroulement de la gamétogenèse et sont produites activement pendant la maturation ovocytaire puis l'ovulation. La technique d'induction de ponte dite technique d'hypophysation consiste en un apport exogène d'hormone gonadotrope par injection d'hypophyses pour provoquer le déroulement de phases du cycle sexuel qui ne se produisent pas spontanément en condition de captivité comme l'ovulation.

The endocrine processes which control oocyte maturation (resumption of meiosis) and ovulation have been studied in vitro in the trout Salmo gairdneri. Follicular maturation is ultimately under the control of a pituitary gonadotropin which induces the follicle to synthesize specific steroids; these steroids act in turn directly on the oocyte to promote maturation. The systematic study of the in vitro efficiency of various steroids have shown that 17alpha-hydroxy- 20beta-dihydroprogesterone plays a preferential role in initiating maturation; this steroid has a high affinity for a plasma protein system. The efficiency of this steroid, similarly to the efficiency of the gonadotropin, can be modulated by other circulating steroids. The precise chronology of some events of follicle maturation have been defined using inhibitors of protein and RNA synthesis. The ovulatory process (sensu stricto: expulsion of matured oocyte from the follicular envelopes) has been experimentally dissociated from oocyte maturation, and some mediators likely to act on ovulation have been identified. These data permit the consideration of novel means of intervention at the ovarian level to synchronize maturation and ovulation in fish, in order to give new tools for progress in aquaculture., Nous avons étudié in vitro les processus endocrines qui contrôlent la maturation des ovocytes (reprise de la meiose) et l'ovulation chez la truite arc-en-ciel, Salmo gairdneri. La maturation folliculaire est en dernier lieu sous le contrôle d'une gonadotropine hypophysaire qui déclenche dans le follicule la synthèse de stéroides spécifiques; ces stéroides, à leur tour, agissent directement sur l'ovocyte et activent la maturation. L'étude systématique de l'efficacité in vitro de divers stéroides démontre que la 17alpha hydroxy-20beta dihydroprogesterone joue un rôle préférentiel dans l'amorcage de la maturation; ce stéroide à une haute affinité pour un système de protéines plasmiques. L'efficacité de ce stéroide, tout comme celle de la gonadotropine, peut être modulée par d'autres stéroïdes en circulation. Nous avons défini, à l'aide d'inhibiteurs de protéines et synthèse de la RNA, la chronologie précise de certaines étapes de maturation des follicules. Le processus d'ovulation (sensu stricto: expulsion de l'ovocyte mûr des enveloppes folliculaires) a été dissocié expérimentalement de la maturation de l'ovocyte, et certains médiateurs susceptibles d'activer l'ovulation ont été identifiées. Grâce à ces données, on peut considérer de nouveaux moyens d'intervenir au niveau ovarien pour synchroniser la maturation et l'ovulation chez les poissons, fournissant ainsi de nouveaux outils pour le progrès de l'aquaculture.

Daily changes in carp gonadotropin levels in adult female carp and daily changes in carp oocyte sensitivity to carp hypophysial homogenate, in vitro and in vivo, were investigated. A total of three series of experiments were carried out. Gonadotropin levels were radioimmunologically determined. The results of series 1 and 2 experiments were subjected to statistical analysis with the use of cosinors circle and ellipse of errors. It has been found that in the mature female carp in the pre-spawning period with the light periods being long (L:D = 16:8) the apogee for gonadotropin occurs 10 hr after the onset of the light period. The sensitivity of the oocytes, in terms of the percentage of mature oocytes (after GVBD) following a 24-hr incubation of ovarian fragments with the hypophysial homogenate, reached the highest value at 1300, i.e. 9 hr after the onset of the light period. It was also found that the injections of carp hypophysial homogenate made at 0900 were much more efficient in inducing ovulation than those at 2100.

Un dosage radioimmunologique spécifique a été utilisé pour suivre la libération de 17 alpha hydroxy - 20 beta dihydroprogestérone par des follicules ovariens entiers de 2 truites arc-en-ciel, au cours de la maturation ovocytaire, induite in vitro par la gonadotropine glycoproteique maturante de salmonidé (s-GtH). Sans stimulation gonadotrope, la production de ce progestagène reste très faible. Elle s'accroît fortement avec l'augmentation des niveaux de s-GtH maintenus dans le milieu. A chacun de ces niveaux, elle est plus importante pour des follicules provenant de la femelle donneuse la plus proche de sa date d'ovulation supposée. Par ailleurs les fortes quantités de progestagène produites sont en accord avec les fortes teneurs mesurées dans le plasma d'animaux prêts à ovuler., A specific radioimmunoassay has been used to follow the synthesis of the 17 alpha hydroxy-20 beta dihydroprogesterone in vitro by ovarian follicles of 2 Rainbow Trout, during the oocyte maturation stimulated with a pure salmon glycoproteic maturational gonadotropin. Very low levels have been measured without stimulation. The progestagen production was increasing with the level of GtH dose used. It was much higher with ovarian follicles coming from the female sacrified near the period of ovulation. The high quantities, produced were in good agreement with the high levels found in plasma of ovulating females.

L'efficacité de la 17 alpha-hydroxy-20 beta-dihydroprogestérone (17 alpha-20 beta Pg) ou d'un extrait gonadotrope hypophysaire de Truite sur la maturation intrafolliculaire in vitro des ovocytes de Truite peut être modulée par des stéroïdes qui ne présentent pas d'action maturante directe: l'efficacité de l'extrait gonadotrope est abaissée par l'oestradiol et l'oestrone et augmentée par la testostérone. Comme ces mêmes stéroïdes n'ont aucun effet significatif sur la maturation induite par la 17 alpha-20 beta Pg, leur site d'action se situe probablement au niveau des enveloppes folliculaires. Les corticostéroïdes et en particulier le cortisol et la cortisone augmentent l'efficacité de l'extrait gonadotrope, mais stimulent encore plus fortement l'efficacité de la 17 alpha-20 beta Pg, suggérant un effet direct sur la sensibilité ovocytaire à ce «progestagène»., The efficiency of 17 alpha-hydroxy-20 beta-dihydroprogesterone (17 alpha-20 beta Pg) or of a Trout pituitary gonadotropic extract to induce intrafollicular maturation of trout oocytes can be modulated by some steroids which do not present any direct maturing action: gonadotropic extract efficiency is lowered by estradiol and estrone, and enhanced by testosteron. As these steroids do not present a significant effect on 17 alpha-20 beta Pg induced maturation, their site of action may be located in the follicular tissues. The corticosteroids, particularly cortisol and cortisone enhance the maturing efficiency of gonadotropin and stimulate much more strongly the efficiency of 17 alpha-20 beta Pg. This suggests a direct effect on oocyte sensitivity to 17 alpha-20 beta Pg.

Une expérimentation a été conduite en vue de mettre au point une technique d'induction de la maturation ovocytaire et de l'ovulation par traitement hormonal chez le Brochet Esox lucius. Les résultats obtenus montrent : 1) La possibilité d'induire la maturation ovocytaire et l'ovulation par une seule injection intrapéritonéale d'hormone gonadotrope de saumon partiellement purifiée (0,1 mg/kg) effectuée dans les 24 heures qui suivent la capture. Le taux d'ovulation est élevé : 89 % en moyenne, et les ovules obtenus sont de bonne qualité (taux de fécondation estimé à 40 degrés-jours : 83 % en moyenne). A 12 °C, l'ovulation intervient 4 jours après l'injection hormonale. L'insémination artificielle peut alors s'opérer sur les ovules des différentes femelles que le traitement a synchronisées. Les ovules doivent être inséminés dans les 24 heures, sous peine de vieillissement et de chute rapide de la fécondabilité. Une bonne estimation du bilan final de l'incubation peut être obtenue par simple dénombrement des « oeufs blancs » à 40 degrés-jours. 2) La dégénérescence rapide d'une grande proportion d'ovocytes consécutive au transport des femelles et à leur maintien en captivité : le taux moyen d'ovulation, de 96 % si le traitement est appliqué immédiatement après la capture, chute à 40 % si l'animal est traité après 3 jours de captivité., The efficiency of various hormonal treatments (partially purified salmon gonadotropin - PPSG and HCG administered alone or in association with 17a- hydroxy-20β-dihydroprogesterone - 17a-20β P) on oocyte maturation and ovulation was investigated in submature northern pike (Esox lucius) females (at the initial oocyte stage, the germinal vesicle was in peripheral position). A dose of 0.1 mg PPSG/kg body weight gave satisfactory ovulation (89%) and fertilization (83 %) rates. Ovulation occured 4 days after treatment. A dose of 0.03 mg/kg induced oocyte maturation but not ovulation. A dose of 0.03 mg PPSG/kg followed 24 hours later by 3 mg 17a-20β P/kg induced full maturation and partial ovulation. A single dose of 3 mg 17a-20β P caused oocyte maturation in all animals and partial ovulation or no ovulation. In all treatments including 0.03 mg PPSG/kg or less, fertilization rates were low (20 to 40%). A three day captivity prior to treatment led to ovarian atresia and to a significant decrease in ovulation rates. When eggs were left in the ovaries after ovulation, a significant drop of fertility due to aging occured within 2 to 3 days. A good estimate of the overall incubation success can be provided by a simple count of dead eggs at 40 degree-days.

17α-Hydroxy-20β-dihydroprogesterone and 17α-hydroxyprogesterone were found in higher concentrations in serum from female Salmo gairdneri undergoing final oocyte maturation immediately before ovulation than in serum from spermiating male trout. Other steroids (11-deoxycorticosterone, 11-deoxycortisol and progesterone) which have been implicated in oocyte maturation and/or ovulation in lower vertebrates were not identified at such high concentrations and the differences between the serum of both sexes were not so great. These results confirm that 17α-hydroxy-20β-dihydroprogesterone and 17α-hydroxyprogesterone, the most potent inducers of trout oocyte maturation in vitro, are present in the blood when oocyte maturation occurs. The concentration of testosterone was found to be higher in serum from female than from male trout indicating that testosterone is unlikely to be the principal androgen in trout. High concentrations of 11-oxotestosterone in male and barely detectable levels in female fish support the hypothesis that 11-oxotestosterone is an important androgen in the regulation of testicular activity.

Le cycle reproducteur des poissons est sous la dependance de l'hypophyse qui elabore et secrete les hormones gonadotropes. Ces hormones interviennent lors du deroulement de la gametogenese et sont produites activement pendant la maturation ovocytaire puis l'ovulation. La technique d'induction de ponte dite technique d'hypophysation consiste en un apport exogene d'hormone gonadotrope par injection d'hypophyses pour provoquer le deroulement de phases du cycle sexuel qui ne se produisent pas spontanement en condition de captivite comme l'ovulation.

The plasma concentrations of the glycoproteic gonadotropin (GTH) and of the 17 alpha hydroxy-20 beta dihydroprogesterone (17 alpha 20 beta OHP) have been followed in the course of ovulation in 6 rainbow trout, using radioimmunoassays on samples taken every two days. A first slow and limited GTH increase (10 to 20 ng/ml) is detected before and during the ovulation, while the 17 alpha 20 beta OHP rises sharply (up to three to five hundreds nanogrammes per millilitre). Then the progestagen levels drop back to their previous values, for about three weeks after ovulation. At the same time, a second higher GTH increase (25 to 20 ng/ml) is taking place. The relations between these two hormones, maturation and ovulation are discussed

Experiments were carried out to determine if some environmental factors usually present in spawning ponds at the time of ovulation could potentiate the effects of LHRH or LHRH-A on the plasma level of GtH, oocyte maturation or ovulation in the common carp, Cyprinus carpio . In one experiment females were held in the absence of a male in cages placed in rearing ponds containing a dense population of unicellular algae; the cages touched the bottom but there was no vegetation inside the cages. When LHRH-A was given in two injections (the first 5 μg/kg, the second, 6 h later, 50 μg/kg) plasma GtH was increased and followed oocyte maturation. Ovulation occurred in 50% (3 of 6) of the females. In another experiment, using a similar LHRH-A treatment schedule, the females were held in aquaria with added vegetation to serve as a spawning substrate; in such a simplified environment the effect of LHRH-A was not potentiated. Similarly, the natural environment of spawning ponds including the presence of males was of no benefit to the response to low doses of LHRH or LHRH-A when the water temperature was low (12°C). These results suggest that some undetermined environmental factors besides vegetation present in a pond situation may potentiate the effect of LHRH-A, possibly via abatement of the action of an endogenous gonadotropin release-inhibitory factor.

International audience; Coho salmon (Oncorhynchus kisutch) were induced to ovulate 3-5 wk ahead of the normal time using partially purified salmon gonadotropin (SG-G100) alone or in combination with 17 alpha hydroxy 20 beta dihydroprogesterone (17 alpha -20 beta P). Treatment with 17 alpha -20 beta P alone induced final oocyte maturation but not ovulation and was associated with a negative feedback upon endogenous gonadotropin. The most successful treatments consisted of a 'priming' injection of 0.1 mg/kg SG-G100 followed within 2-3 days by an injection of either 1 mg/kg SG-G100 or 3 mg/kg 17 alpha -20 beta P. Ova from the ovulated fish were successfully fertilized. This is the first occasion in which the time of spawning has been controlled in adult migrant Pacific salmon.

La 17 alpha hydroxy-20 beta dihydroprogesterone injectée (2 mg/kg) a des femelles dont les ovocytes sont au stade de la < vésicule germinative semi-periphérique > permet d'obtenir un groupement des ovulations entre 5 et 9 jours après le traitement. Au cours des premiers stades de la maturation ainsi déclenchée, l'augmentation de la t-GtH est inférieure à celle des témoins, accreditant l'hypothèse d'une action directe sur les ovocytes. L'action de la progestrone est moins efficace.

Induction de l'ovulation et stimulation de la spermiation par le LH-RH ou un analogue de LH-RH associé ou non au pimozide chez quelques espèces de poissons téléostéens. Bases biologiques de l'aquaculture

International audience; In fish there are few data on the gonadotropin-releasing hormone (Gn-RH) neurosecretory activity, which could explain long- and short-term variations of the gonadotropin secretion. There is no biological species specificity between mammal and fish Gn-RH; although there is a structural difference, they are, on the contrary, characterized by a high immunological specificity which does not allow measurement of fish Gn-RH using radioimmunoassay for LH-RH. We have synthesized salmon Gn-RH according to the formula recently proposed by Sherwood (N. Sherwood, L. Eiden, M. Brownstein, J. Spies, J. Rivier, and W. Vale, 1983. Proc. Natl. Acad. Sci. USA 80, 2794-2798). Its activity has been tested by its ability to stimulate the gonadotropin hormone (GtH) secretion in vivo in testosterone-implanted juvenile rainbow trout, and for the recognition of synthesized Gn-RH (s-Gn-RH) perykaria by a specific antibody raised against the s-Gn-RH in regions of the brain described as containing LH-RH immunoreactive-like material. A radioimmunoassay has been developed for the salmon Gn-RH, and its specificity to measure trout Gn-RH has been tested. Using this assay, the brain and pituitary Gn-RH contents have been measured throughout the final phases of maturation and ovulation. Brain Gn-RH increases from the end of vitellogenesis (8.9 +/- 0.76 ng/brain) to ovulation (more than 15 ng/brain). Pituitary Gn-RH is lower (1.58 +/- 0.69 ng/pituitary) at the end of vitellogenesis and follows a similar profile as in the brain, except for a significant decrease just prior the beginning of oocyte maturation. The correlations between Gn-RH levels and GtH pituitary and plasma levels show that total brain Gn-RH is never correlated to the GtH, suggesting that the increase in the brain Gn-RH content is related to a Gn-RH system closely related to maturation and ovulation, which remains to be investigated. On the contrary, pituitary Gn-RH levels are well correlated with pituitary and plasma GtH levels, indicating that pituitary Gn-RH levels might represent a good index of the Gn-RH neurosecretory activity in the fish hypothalamohypophysial complex, given the absence of a portal system in teleost.

Chez de nombreuses espèces de poissons élevés en captivité, la phase finale de maturation ovocytaire et l'ovulation ne s'accomplissent qu'après apport exogène d'hormone hypophysaire., In many species of fish raised in captitivty, the final phase of oocyte maturation and ovulation only occurs after exogenous pituitary hormone is supplied.