The topography of South Africa (SA) shows complex variations and is one the main factors that determine the daily weather patterns and climate characteristics. It affects for example temperature, winds and rainfall (intensity and distribution). Mesoscale numerical weather prediction (NWP) models are used to simulate atmospheric motions with high horizontal grid resolution using appropriate cumulus parameterisation schemes. They also allow users to investigate the effects of topography and surface heating on the development of convective systems. The Weather Research and Forecasting (WRF) model was applied over the complex terrain of SA to simulate extreme weather events and evaluate the influence of topography and grid resolution on the accuracy of weather simulations. This includes heavy precipitation event that lead to floods over Limpopo region of SA which was caused by the tropical depression Dando for the period 16 -18 January 2012; the heat wave events over Limpopo region for the period 22-26 October 2011 and also over Cape region for the period 15-18 January 2012. The Grell-Devenyi Ensemble (GDE) cumulus parameterization scheme was applied. The WRF model was run at a horizontal resolution of 9 km with 3 km nests, one over Limpopo and another over Cape region respectively. A total of 210 South African Weather Service (SAWS) synoptic stations data were used to verify the model, with 37 stations located over Limpopo and 88 over Cape region. The WRF model simulations are able to capture the spatial and temporal distribution of the heat wave over Limpopo and Cape regions respectively. The model verification with observational data showed that the performance statistics are in the expected range. The experiments without topography give unrealistic verification scores. The increase of model grid resolution from 9 to 3 km improved the spatial and temporal distribution and performance statistics. The above findings are in general similar for the two heat wave events, although the influence of topography over Cape region is not too pronounced. This can be attributed to different topographic variations over the Cape region as compared to the Limpopo region. The WRF model captured well the spatial and temporal distribution of rainfall patterns; verification statistics shows over-prediction of its intensity in simulation with topography. The simulation without topography shows unrealistic space and intensity of rain distribution. An increase in model grid resolution from 9 to 3 km shows improved spatial and temporal distribution of rainfall. The importance of high grid resolution and the use of non-hydrostatic equations are confirmed by the analysis of the vertical velocity distribution and moisture fluxes. The overall findings proved that topography plays a major role to weather and climate over SA. The high grid resolution allows for a better topography representation and capturing convective activities by the use of nonhydrostatic approximations. Therefore the WRF model proved to be useful forecasting tool for weather and climate simulations and can be used for operational weather forecasting over South Africa. / Dissertation (MSc)--University of Pretoria, 2014. / lk2014 / Geography, Geoinformatics and Meteorology / MSc / Unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22048号 / 工博第4629号 / 新制||工||1722(附属図書館) / 京都大学大学院工学研究科社会基盤工学専攻 / (主査)教授 中北 英一, 准教授 山口 弘誠, 准教授 田中 賢治 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Ovulatory follicle development and associated oocyte maturation involve complex coordinated molecular and cellular mechanisms not fully understood. This study addresses, for the first time in any species, the relationships among diameter, vascularization, follicular-fluid factors, and gene expression for follicle growth, steroidogenesis, angiogenesis and apoptosis in granulosa/cumulus cells and oocytes during different stages from the beginning of largest/ovulatory follicle to impending ovulation. Follicle dynamics was tracked and follicle-wall blood flow evaluated daily through transrectal ultrasonography in mares. The largest follicle of the ovulatory wave was distributed in six diameter groups (predeviation, deviation, postdeviation, early preovulatory, preovulatory, and impending ovulation) according to the targeted diameter for complete aspiration and cell harvesting. The most remarkable findings were (i) positive association between follicle development, blood flow, intrafollicular FSH, LH, estradiol, progesterone, and mRNA expression for FSHR and LHCGR in granulosa cells of largest/ovulatory follicle; (ii) plateau/decrease in follicle diameter, blood flow, and granulosa cell mRNA for FSHR, LHCGR, IGF1R, VEGFR2, CYP19A1, and CASP3 at the preovulatory stage; (iii) higher StAR and BCL2 and lower CASP3 mRNA in granulosa cells at impending ovulation; (iv) greater IGF1R mRNA in the granulosa cells at the predeviation stage; and (v) lower FSHR, LHCGR, IGF1R, and VEGFR2 mRNA in cumulus cells, and greater LHCGR and IGF1R mRNA in oocytes at the ovulatory stage. This study is a critical advance in the understanding of molecular mechanisms of follicle development and oocyte maturation and is expected to be vital for future studies targeting potential markers for follicle selection, development, and ovulation.

Thesis. 1975. M.S.--Massachusetts Institute of Technology. Dept. of Meteorology. / Bibliography: leaves 107-109. / M.S.

Thesis. 1975. Ph.D.--Massachusetts Institute of Technology. Dept. of Meteorology. / Vita. / Bibliography: leaves 202-206. / Ph.D.

Mes travaux explorent, chez la vache, le transfert entre les cellules du cumulus et l’ovocyte des transcrits d’ARN et d’autres larges molécules qui, selon notre hypothèse, pourraient être exportées des cellules somatiques à l’ovocyte, ceci afin de soutenir sa maturation. L’étude des projections transzonales (PTZ) reliant les cellules du cumulus à l’ovocyte a révélé qu’elles sont de morphologie irrégulière, qu’elles peuvent contenir des organelles (mitochondries) et des structures cellulaires (ribosomes) et qu’elles se retractent au cours de la maturation ovocytaire. Des microvésicules pouvant transférer de larges molécules ont été identifiées à la jonction entre les cellules. Pour déterminer si l’ARN est empaqueté préalablement à son transfert entre les cellules, nous avons analysé les transcrits présents dans les PTZ et avons constaté qu’ils se déplacent vers l’ovocyte en maturation. Parmi les transcrits retrouvés dans les PTZ, certains étaient commun à ceux dont l’abondance augmente dans l’ovocyte en cours de maturation de même qu’à ceux retrouvés dans les polyribosomes de l’ovocyte en maturation, suggérant ainsi le transfert et l’utilisation des transcrits provenant des cellules du cumulus. Un transcrit synthétique ajouté aux cellules du cumulus avant qu’elles ne s’attachent à l’ovocyte a été transféré à ce dernier, confirmant ainsi le potentiel des cellules à transférer des ARNm et possiblement leurs protéins associées à l’ovocyte. Nouse avons observé, sur une échelle de temps, que les transcrits sont accumulés dans les PTZ au cours des heures suivant l’abattage de l’animal, mais préalablement à l’aspiration de l’ovocyte. Le retrait des cellules du cumulus et de cette période d’accumulation des transcrits se traduit par un faible taux de maturation des ovocytes. La nécessité d’avoir des vésicules d’ARN transférées à l’ovocyte a été testée grâce à des inhibiteurs de la synthèse d’ARN, de son transport et de la formation des vésicules. L’inhibition de chacune de ces étapes a compromis la maturation des ovocytes. En se penchant sur les mécanismes impliqués dans le transfert des transcrits, nous avons découvert un candidat potentiel jouant un rôle dans l’insuffisance ovarienne précoce. Cette protéine liant les ARNm, Fragile X mental retardation (FMRP), a été retrouvée dans les cellules folliculaires et dans l’ovocyte tout au long de la folliculogenèse et de l’ovogenèse. Nous avons constaté que FMRP s’associe à la machinerie traductionnelle et aux protéines des granules d’entreposage dans l’ovocyte. L’inhibition de la protéine a significativement réduit le taux de blastocyste. En démontrant que des transcrits exogènes contribuent au développement de l’ovocyte et influencent sa maturation, nos recherches ajoutent un niveau de compréhension additionnel aux mécanismes de communication intercellulaire menant à la production de gamètes de bonne qualité. De futures expériences axées la caractérisation du transfert des transcrits et des mécanismes spécifiques en jeu contribuera à accroître notre compréhension de la compétence ovocytaire. / The reports in this thesis explored the potential for large molecule communication between the cumulus cell and the oocyte hypothesizing that large molecules, including RNA, could be transferred to the oocyte for support during maturation. Exploration of the transzonal projections (TZPs) connecting the cumulus cells to the oocyte revealed that they are irregular in shape, can contain large organelles (mitochondria) and small cellular structures (ribosomes), and that these connections retract during oocyte maturation. Microvesicles were identified at the intercellular articulations capable of sharing large molecules between the cells. To determine if RNA is transferring as cargo between cells, nascent as well as total transcripts were evaluated in the TZPs and found moving towards the oocyte during maturation. Of the transcripts found in the TZPs during maturation, some were common to those increasing in abundance in the oocyte during maturation, and on the polyribosomes of the maturing oocyte, thus suggesting transfer and use of cumulus cell transcripts. A synthetic transcript provided to some cumulus cells for reconstruction with, and transfer to the oocyte, confirmed the potential to transfer mRNA and possibly proteins. Temporally, RNA transcripts were found to accumulate in TZPs during the hours post slaughter but prior to oocyte aspiration. Removal of the cumulus cells and this period of accumulation resulted in poor oocyte maturation. The requirement of vesicle mediated RNA transfer to the oocyte was tested. Inhibitors against RNA synthesis, transport, and vesicle formation were explored and found to reduced oocyte maturation. Focusing on mechanisms that could mediate transference, we assessed an RNA binding protein candidate with implications in premature ovarian insufficiency. Fragile X mental retardation protein (FMRP) was found in follicular cells and the oocyte throughout folliculogenesis and oogenesis. Based on known roles in translation control, FMRP was shown associated with translational machinery and storage granule proteins in the oocyte. Knockdown of this protein resulted in compromised rates of blastocyst formation. Knowing that exogenous transcripts contribute to oocyte development, and influence the molecular aspects of oocyte maturation adds another layer to our understanding of intercellular communication in the production of a healthy gamete. Future characterization of the transferred transcripts and the mechanisms in control of this process will improve our understanding of oocyte health and competence.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24208号 / 工博第5036号 / 新制||工||1786(附属図書館) / 京都大学大学院工学研究科社会基盤工学専攻 / (主査)教授 中北 英一, 准教授 山口 弘誠, 教授 田中 賢治 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

L'objectif de mon travail de doctorat consistait à explorer l'expression génique du cumulus au regard de la compétence au développement de l'ovocyte bovin. Cette exploration tire son intérêt des nombreuses démonstrations du rôle crucial du cumulus auprès de l'ovocyte mammalien. En premier lieu, nous avons appliqué à des vaches de race laitière, un protocole de stimulation ovarienne permettant la modulation de la compétence ovocytaire, en agissant sur la différenciation folliculaire, via l'hormone folliculo-stimulante (FSH). Trois variantes de la compétence développementale de l'ovocyte ont ainsi pu être obtenues : acquisition de compétence en cours, compétence acquise et déclin de compétence. En dépit des différences modérées de compétence engendrées, le transcriptome des cellules du cumulus les reflète quand même. Ces variations bien que minimes, de l'expression génique du cumulus, appuient l'idée générale que la compétence ovocytaire serait le résultat non seulement de changements substantiels par l'environnement, mais aussi d'une régulation beaucoup plus fine des mécanismes folliculaires.Suite aux résultats de la première étude, nous avons voulu obtenir un portrait transcriptomique du cumulus plus contrasté. Pour ce faire, des biopsies de cumulus ont été réalisées sur des complexes ovocyte-cumulus (COC) d'abattoir suivis individuellement, de leur maturation à leur développement in vitro, et catégorisés selon que le développement ce soit arrêté à 2-8 cellules – i.e. avant l'activation du génome embryonnaire – ou qu'il se soit poursuivi jusqu'au stade de blastocyste. De cette manière, les cumuli associés à la formation d'un blastocyste, et donc à des ovocytes compétents, montrent une expression génique clairement différente de ceux dont le développement embryonnaire a échoué. Ces différences concernent des fonctions telles que : la modification post-traductionnelle, le repli des protéines, le métabolisme des acides aminés, la dégradation des protéines ou encore l'épuration des radicaux libres qui se trouvent surexprimées dans les cumuli compétents. Dans un troisième temps, c'est l'implication de l'hormone lutéinisante (LH) dans l'expression génique du cumulus qui a été évaluée. Le même protocole que celui de la première étude a été appliqué, en rajoutant cette fois des injections de Cétrorelix afin de supprimer la sécrétion de LH en fin de croissance folliculaire – i.e. lors de l'acquisition de la compétence au développement. Bien que l'absence de LH n'ait pas eu d'impact significatif sur la compétence ovocytaire, le transcriptome du cumulus a lui été significativement affecté. Ainsi, l'expression de fonctions comme la survie, la prolifération ou la croissance du follicule est inhibée par l'absence de LH. Par ailleurs, des fonctions de maintenance cellulaire telles que la transcription, le métabolisme des acides aminés et des protéines, ainsi que la production d'énergie semblent également pâtir de cette absence. Le soutien de la LH serait donc important pour les fonctions du cumulus lors de la maturation finale du follicule. Finalement, la présence du récepteur à la LH (R-LH) dans le cumulus bovin étant controversée dans la littérature, nous avons cherché à suivre l'expression génique de ce R-LH dans les cellules de COC en cours de maturation in vitro. Nous avons également examiné l'expression de la mévalonate kinase (MVK), dont le transcrit est reconnu en tant que régulateur de l'expression du R-LH ; et aussi surveillé l'expression de différentes enzymes stéroïdogènes ainsi que celle du récepteur à la progestérone (PGR). Le suivi d'expression génique a été réalisé à intervalles de trois heures, de 0 à 24 h de maturation, et n'a pour autant pas permis de détecter la présence de transcrits du R-LH dans le cumulus, tandis que l'expression génique de la MVK augmente au cours de la maturation. Les transcrits des enzymes stéroïdogènes STAR et HSD3β, bien que faiblement exprimés, ont été retrouvés ; et l'expression de CYP11A1 et PGR quant à elle augmente pendant la maturation. Ainsi, d'une part, la supplémentation en LH des milieux de maturation ne semble pas pertinente chez le bovin ; d'autre part, le cumulus pourrait être capable d'utiliser le cholestérol pour la stéroïdogenèse et de répondre à une stimulation par la progestérone puisqu'il en exprime le récepteur. Ces travaux participent ainsi à accroître la connaissance du transcriptome spécifique au cumulus bovin et son lien avec la compétence ovocytaire au développement. Enfin, les données acquises ici offrent de nouvelles pistes à explorer afin d'améliorer la qualité ovocytaire, tant in vitro qu'in vivo. / In this thesis report, the cumulus cell gene expression regarding oocyte developmental competence was explored in bovine. This interest is based on the proven crucial role in the differentiation of the cumulus to the mammalian oocyte. First, we applied an ovarian stimulation protocol to dairy cows which allows modulation of oocyte competence using FSH to influence follicular differentiation. Then, three oocyte competency states were obtained: increasing competence, competence acquired and decreasing competence. In spite of moderated competency fluctuations, cumulus cell transcriptome still reflects them. The small variations in cumulus gene expression reinforce the general idea that oocyte competence is the result not only of substantial changes due to the environment, but also of a much finer regulation of follicular mechanisms. Following the results of the first study, we wanted to obtain a more contrasted transcriptomic picture of the cumulus. To do so, cumulus biopsies were performed on slaughterhouse cumulus-oocyte complexes (COC) monitored individually, from their maturation to their in vitro development, and categorized according to whether the development was stopped at 2-8 cells – i.e. the embryonic genome activation – or continued until the blastocyst stage. In this way, the cumuli associated with a blastocyst formation, and therefore with competent oocytes, show a clearly different gene expression from those whose embryonic development has failed. These differences concern functions such as: posttranslational modification, protein folding, amino acid metabolism, protein degradation or free radical scavenging that are over-expressed in competent cumuli. Thirdly, we evaluated the involvement of luteinizing hormone (LH) in cumulus gene expression. The same protocol as that of the first study was applied, with the addition of Cetrorelix injections in order to suppress the LH secretion at the end of follicular growth – i.e. during developmental competence acquisition. Although the absence of LH did not impacted significantly on oocyte competence, the cumulus cell transcriptome was significantly affected. Thus, the expression of functions such as survival, proliferation or follicular growth is inhibited in the absence of LH. In addition, cell maintenance functions such as transcription, amino acid and protein metabolism, and energy production also seem to suffer from this absence. LH support would therefore be important for cumulus functions during the final maturation of the follicle. Finally, because of the controversy found in literature concerning the presence of the LH receptor (LH-R) in bovine cumulus, we sought to follow the gene expression of this LH-R in in vitro maturing COC cells. Also, we examined the expression of mevalonate kinase (MVK), whose transcript is recognized as a regulator of R-LH expression; and monitored the expression of various steroidogenic enzymes as well as that of the progesterone receptor (PGR). Gene expression observation was performed every 3 hours, from 0 to 24 h of maturation, and did not allow the detection of any R-LH transcripts in the cumulus, while MVK gene expression increases during maturation. The transcripts of the STAR and HSD3β steroidogenic enzymes were found, although weakly expressed; and the expression of CYP11A1 and PGR increases during maturation. Thus, on the one hand, LH supplementation of maturation media does not seem relevant in cattle; on the other hand, cumulus may be able to use cholesterol for steroidogenesis and to respond to a progesterone stimulation since it expresses its receptor. This work thus contributes to increase knowledge of the bovine cumulus transcriptome and its link with oocyte developmental competence. Finally, the data acquired here offer new avenues to explore in order to improve oocyte quality, both in vitro and in vivo.

Oocyte quality plays a critical role in establishment of pregnancies, embryo development, implantation and the health of offspring. The oocyte provides maternal factors necessary for the initial development of its embryo during the period of transcriptional silence. Despite the consistent increase in number of couples seeking assisted reproductive treatments, oocyte quality still remains as an obstacle in successful fertility treatments and the mechanisms governing the quality of oocyte are poorly understood. Among various factors that may potentially affect the quality of oocyte, the acquisition of oocyte developmental competence seems to mainly occur during the final stage of oocyte maturation. The correct temporal regulation of series of molecular events and the proper exchange of signals with surrounding follicular environment during this critical period will ensure the developmental competence of oocyte and its subsequent embryo. In order to identify molecular factors affecting oocyte quality, I have compared oocytes and cumulus cells of different qualities at a molecular level. I present in this thesis the pathways and molecules that may determine the developmental competence of oocyte as well as candidate molecular markers of oocyte and embryo quality. A cDNA microarray analysis was performed, comparing the transcriptomes of rhesus monkey MII oocytes of different qualities, high quality VVM oocytes and poor quality IVM oocytes. A small set of 59 Oocyte quality plays a critical role in establishment of pregnancies, embryo development, implantation and the health of offspring. The oocyte provides maternal factors necessary for the initial development of its embryo during the period of transcriptional silence. Despite the consistent increase in number of couples seeking assisted reproductive treatments, oocyte quality still remains as an obstacle in successful fertility treatments and the mechanisms governing the quality of oocyte are poorly understood. Among various factors that may potentially affect the quality of oocyte, the acquisition of oocyte developmental competence seems to mainly occur during the final stage of oocyte maturation. The correct temporal regulation of series of molecular events and the proper exchange of signals with surrounding follicular environment during this critical period will ensure the developmental competence of oocyte and its subsequent embryo. In order to identify molecular factors affecting oo was identified as differentially expressed between the two types of oocytes. These mRNAs are involved in steroid metabolism, cell-cell interactions, cellular homeostasis, cell adhesion, mRNA stability and translation. In addition, the overexpression of several imprinted genes in IVM oocytes were detected, indicating a possible loss of correct epigenetic programming during IVM. These results indicate that normal oocyte-somatic cell interactions may be disrupted during IVM and the interruptions of these interactions during the final phase of oocyte maturation may be the prime cause of reduced developmental competence of IVM oocytes. To elucidate oocyte quality factors linked to the cumulus cell phenotype, the transcriptomes of two types of rhesus monkey cumulus cells, IVM and VVM, were compared using a cDNA microarray analysis. In contrast to a relatively small difference between IVM and VVM oocytes, a large number of genes were differentially expressed between IVM and VVM rhesus cumulus cells. Moreover, a much larger number of differential mRNA expressions were observed comparing the transitions from pre-maturation cumulus cells to the IVM and VVM cumulus cells. The results from these array comparisons indicated that the cumulus cells may fail to achieve successfully normal gene regulation during IVM and thus make a remarkable amount of changes in gene expression to compensate for the loss. Numerous genes involved in lipid metabolism are incorrectly regulated during IVM, and the synthesis of sex hormones appears not suppressed during IVM. In addition, a panel of 24 cumulus cell markers of oocyte quality was identified. Genetic effects on oocyte quality were explored by comparing transcriptomes of oocytes obtained from two different inbred mouse strains, B6 and D2, and F1 hybrid. A clustering analysis and statistical tests showed that the transcriptome of F1 oocytes is more similar to the B6 transcriptome than to the D2 at both GV and MII stages. Also, comparison analyses of GV stage oocyte transcriptomes with MII oocyte transcriptomes from three different mouse strains indicated that the number of overdominance genes at the MII stage is bigger than the number of overdominance genes at the GV stage. In order to investigate how the genes gain the overdominance during the GV to MII transition, overdominance genes were categorized according to their mRNA expression patterns at GV and MII stages. The results showed that more than 80% of overdominance genes belong to one of the four major transition groups. The further evaluation of changes in array intensities from GV to MII stage transition revealed that F1 oocytes and inbred strain oocytes differentially regulate the mRNA abundance during oocyte maturation and that the differential regulation of mRNA abundance by the F1 genotype is responsible for the increase of the number of overdominance genes during maturation from GV stage to MII stage. A mRNA sequence analysis indicated that the gain of overdominant low in F1 mRNA expression pattern during maturation may be regulated by 3'UTR motif elements. The number of dominance genes also increase during GV to MII transition. At both GV and MII stages, there are more genes with B6 dominant mRNA expression pattern than those with D2 dominance pattern. Lipid metabolism, small molecule biochemistry and cell death are biofunctions overrepresented in both dominance and overdominance genes. In addition, `blebbing' was identified as a biofunction significantly downregulated in the F1 and B6 MII eggs, indicating that the cellular function may be involved in oocyte maturation. / Molecular Biology and Genetics / Accompanied by one .pdf file: YLEE_SupplementalTables.pdf

Les jonctions communicantes (JCs) sont particulièrement importantes au sein du follicule ovarien puisque qu’elles vont participer à la régulation de la maturation ovocytaire. Les JCs vont contrôler le flux de molécules tels que l’AMPc et le GMPc qui passent entre les cellules du cumulus et l’ovocyte et qui inhibent la reprise de la méiose. La fermeture de ces canaux, tant in vivo qu’in vitro, va entrainer la reprise de la méiose en empêchant le passage de ces molécules. Dans les études que nous avons réalisées, nous avons tout d’abord développé une méthode d’analyse fonctionnelle des jonctions communicantes (le FRAP : Fluorescence Recovery After Photobleaching) dans la structure tridimensionnelle du complexe ovocyte-cumulus. Cette technique nous a permis de déterminer que les gonadotrophines et le liquide folliculaire présent dans le milieu de maturation pouvaient moduler la dynamique de transfert entre les cellules du cumulus pendant les premières heures de maturation in vitro (MIV). Par la suite, nous avons étudié la régulation des connexines (Cx), qui sont les protéines composant la JC. Nous avons identifié les principales connexines présentes dans le COC porcin, la Cx43, la Cx45 et la Cx60. Nous avons déterminé que les gonadotrophines régulaient l’expression, la dégradation ainsi que la localisation de la connexine 43 (la principale connexine présente dans les cellules du cumulus porcin). Enfin, nous nous sommes intéressés à la régulation de la reprise de la méiose par le peptide natriurétique de type C (CNP). En effet, le CNP a été proposé pour inhiber la reprise de la méiose en se liant à son récepteur NPR-B sur les cellules du cumulus, ce qui stimule la production de GMPc. Il est ensuite transféré dans l’ovocyte, via les JCs, où il va inhiber la reprise de la méiose. Nous avons montré que le CNP et un analogue du GMPc avaient un impact différent sur la dynamique de transfert entre les cellules du cumulus. De plus, le CNP agirait sur un autre récepteur (le récepteur NPR-C) pour promouvoir l’arrêt méiotique. En conclusion, ces résultats ajoutent un niveau de compréhension supplémentaire des mécanismes régulant les JCs et la reprise de la méiose durant la MIV chez le porc. / Intercellular gap-junctional communication (GJC) plays an important role in ovarian cell physiology. Closure of GJC has been proposed to be involved in oocyte maturation, particularly in the resumption of meiosis, both in vivo and in vitro, by controlling the flow of meiosis inhibitors, such as cAMP and cGMP. In the present study, we have first developed an assay based on recovery of calcein fluorescence in photobleached cumulus cells (FRAP: Fluorescent Recovery After Photobleaching) in the three-dimensional cumulus-oocyte complex (COC), during the first hours of porcine in vitro maturation (IVM). Results obtained using FRAP technology provide evidence that the composition of the IVM medium in terms of hormones and follicular fluid clearly affects cumulus-cumulus cell GJC. We then turned our attention to the effect of gonadotropins on connexins regulation. Indeed, GJC are composed of connexins proteins. We indentified Cx43, Cx45 and Cx60 as the main connexins expressed in swine COC. We show that gonadotropins regulate Cx43 protein level, degradation and localisation in the COC during the first hours of IVM. We finally turned our attention on the effect of natriuretic peptide C (CNP) on GJC regulation and meiotic resumption during porcine IVM. Indeed, CNP has been proposed to bind to receptor natriuretic peptide receptor B (NPR-B) where it triggers the production of cyclic guanosine monophosphate (cGMP). The cGMP produced is then transferred to the oocyte through gap junctions where it promotes meiotic arrest. Here we show that CNP and a cGMP analogue have different effects on GJC and Cx43 regulation suggesting that cGMP signaling may not be the only signaling pathway involving CNP. Moreover, we found that CNP is likely to bind to receptor natriuretic peptide receptor C (NPR-C) to play a role in maintaining meiotic arrest during porcine IVM, when activated in addition to NPR-B. In conclusion, we describe new mechanisms involved in the complex regulation of dynamic changes in GJCs and meiotic resumption. A better understanding of GJC regulation during IVM may in turn provide a powerful tool to improve assisted reproduction technologies and their efficacy in mammals.