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Luteinising hormone (LH) is a crucial regulator of ovarian follicle maturation, ovulation and luteinisation. Development of healthy follicles and fertile ovulation can only occur within a specific range of circulating LH concentrations, with differing upper and lower limits depending on the stage of the oestrous cycle. The objective of the three studies in this thesis was to investigate the effects of both physiological and non-physiological circulating LH levels on equine follicular maturity by examining ovulatory and steroidogenic capacity, gene expression profiles and miRNA expression in ovulatory-size follicles at various stages of the oestrous cycle and/or in response to supplementation with LH. The aim of the first study was to investigate the hypothesis that deficient circulating LH is a primary cause for the inability of equine follicles to ovulate during the physiological anovulatory season. A LH-rich equine pituitary fraction (eLH) given twice daily to early transitional mares did not restore steroidogenic capacity of the ovulatory-size follicle or advance the onset of the natural breeding season; however, it significantly stimulated follicular growth to a level similar to that occurring during the normal oestrous cycle. The results demonstrated that a deficiency in LH is critically involved in reduced follicle growth during the anovulatory season. The second study examined the effects of elevated circulating LH levels early during follicle development on follicle maturation and ovulatory ability in cycling mares, with the hypothesis that excessive LH would disrupt ovulation and produce haemorrhagic anovulatory follicles (HAFs). Treatment with eLH or a luteolytic dose of prostaglandin F2α (to stimulate an increase in endogenous levels of LH) did not have any effects on follicle growth or ovulation, but did impair follicular production of androstenedione and insulin-like growth factor 1 (IGF1), suggesting a deleterious effect of high LH on follicle and oocyte maturation. The third study examined the expression of different follicular factors associated with follicle maturation as well as microRNAs (miRNAs) in ovulatory-size follicles naturally developing under different LH milieus (oestrus, dioestrus and spring transitional period). Progesterone and IGF1 were significantly reduced in follicles developing in a low LH environment (dioestrus and transition). All four miRNAs measured, miR-378, miR-542, miR-202 and miR-21 were found at higher levels in subordinate follicles than in preovulatory follicles during oestrus. In addition miR- 202 and miR-21 were significantly increased in transitional follicles relative to oestrous follicles. The results of this study indicate that follicles developing during both the spring transitional and dioestrous periods are developmentally immature and suggested potential important roles of miRNAs in follicle maturation in the horse. In summary, although LH is a key factor promoting follicular growth, it is by itself not sufficient to restore steroidogenic activity in transitional follicles. Elevated LH levels during follicle development do not disrupt ovulation, but induce changes in follicular fluid factors related to follicle maturation and oocyte quality. Follicles developing under different LH milieus show altered miRNA expression, suggesting an important role of miRNAs in follicle maturation.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-10-10T16:40:48Z No. of bitstreams: 1 2011 - Raquel Rodrigues Costa Mello.pdf: 1289647 bytes, checksum: f0d06ec4ba47cab82e19761e6bf670ec (MD5) / Made available in DSpace on 2016-10-10T16:40:48Z (GMT). No. of bitstreams: 1 2011 - Raquel Rodrigues Costa Mello.pdf: 1289647 bytes, checksum: f0d06ec4ba47cab82e19761e6bf670ec (MD5) Previous issue date: 2011-08-05 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The Sindhi breed has excellent adaptability to adverse weather conditions and management, and good ability to milk production. Although many researches will be developed with Sindhi breed animals, such experiments are focused mainly on productive aspects, and researches involving the reproductive tract are still scarce in the Sindhi Zebu breed. Thus, the aim of this study was to evaluate the reproductive parameters of Sindhi cows, aiming to improve the efficiency of FTAI protocols in this breed. To this end, sixteen Sindhi cows with body condition score between 3,0 and 4,0 (scale from 1 to 5) were divided into two evaluation groups: In group I (BE protocol, n=8) animals received on day 0 an intravaginal P4 device and an application of 2mg of estradiol benzoate. On day 8 the device was removed and applied 10mg of Dinoprost (PGF2?), and on day 9, applied 1 mg of estradiol benzoate, and were fixed-time artificial inseminated 36 h later; in group II (BE + eCG protocol, n=8) animals were submitted through same treatment, and on day 8 were applied 400 IU of eCG. From day 0 for both treatments, the animals were examined daily by trans-rectal ultrasound device equipped with a 7,5MHz linear transducer (CHISON? D600 VET). This evaluation was performed up to ovulation. At each assessment, the largest follicles of each day (?4 mm) were measured. It used analysis of variance (ANOVA) to evaluate the results, and the means determined by Tukey test, with significance level of 5%. For data analysis, it was observed that there was no statistical difference (P>0.05) for groups I (Treatment I: no eCG) and II (Treatment II: 400 IU eCG) concerning to emergence of follicular wave, 4.5 ? 1.4 and 3.8 ? 0.3 days; interval between the emergence of the wave and ovulation, 5.8 ? 1.4 and 7.1 ? 0.3 days; interval between the emergence of the wave and implant removal, 3,5 ? 1,4 and 4,1 ? 0,3; interval between implant removal and ovulation, 74.0 ? 11.8 and 82.5 ? 4.2 hours; diameter of the dominant follicle (DF) at the time of implant removal, 7.5 ? 2.5 and 6.6 ? 1.5 mm; diameter maximum of DF, 10.47 ? 8.85 and 3.7 ? 1.2 mm; growth rate of the DF, 0.90 ? 0.83 and 0.7 ? 0.6 mm/day and ovulation rate, 75 % (6/8) and 100% (8/8), respectively. Regarding to pregnancy rate, it was observed that there was statistical difference (P<0.05) between protocols I and II (0% and 50%, respectively). Therefore, we conclude that the use of eCG in Sindhi breed cows was not effective in improving the reproductive parameters examined, perhaps because the cows are cycling at the beginning of treatment and had good body condition score during the experiment. On the other hand, there was an increase in pregnancy rate with the use of eCG, indicating that this gonadotropin may be a useful tool in FTAI protocols in this breed. / A ra?a Sindi apresenta excelente adaptabilidade ?s condi??es adversas de manejo e clima, e boa capacidade de produ??o de leite. Embora muitas pesquisas venham sendo desenvolvidas com animais desta ra?a, tais experimentos se concentram predominantemente em aspectos produtivos, pesquisas envolvendo a ?rea reprodutiva ainda s?o escassas na ra?a Sindi. Portanto, o objetivo deste trabalho foi avaliar os par?metros reprodutivos de vacas Sindi, buscando melhorar a efici?ncia dos protocolos de IATF nesta ra?a. Para tanto, 16 vacas da ra?a Sindi, com escore de condi??o corporal entre 3,0 e 4,0 (escala de 1,0 a 5,0) foram divididas em dois grupos de avalia??o: no grupo I (esquema BE, n=8), os animais receberam no dia 0 um implante intravaginal impregnado com 1,9g de progest?geno e uma aplica??o de 2mg de benzoato de estradiol. No dia 8 o implante vaginal foi removido, sendo aplicados 10mg de Dinoprost (PGF2?) e, no dia 9, aplicado 1mg de benzoato de estradiol, sendo realizada a IATF 36 horas ap?s; no grupo II (esquema BE + eCG, n=8), os animais passaram pelo mesmo tratamento, sendo que no dia 8 foram aplicadas 400UI de gonadotrofina cori?nica eq?ina (eCG). A partir do dia 0, para os dois tratamentos, os animais foram examinados diariamente por via trans-retal com aparelho de ultrassom (CHISON? D600 VET) equipado com um transdutor linear de 7,5 MHz. Esta avalia??o foi realizada at? o momento da ovula??o. Em cada avalia??o, os maiores fol?culos de cada dia (?4 mm) foram mensurados. Foi utilizada a an?lise de vari?ncia (ANOVA) para avalia??o dos resultados, sendo as m?dias determinadas pelo teste de Tukey, com n?vel de signific?ncia de 5%. Pela an?lise dos dados, observou-se que n?o houve diferen?a estat?stica (P>0,05) para os grupos I e II na emerg?ncia da onda folicular, 4,5 ? 1,4 e 3,8 ? 0,3 dias; no intervalo entre a emerg?ncia da onda e a ovula??o, 5,8 ? 1,4 e 7,1 ? 0,3 dias; no intervalo entre a emerg?ncia da onda e a retirada do implante, 3,5 ? 1,4 e 4,1 ? 0,3; no intervalo entre a retirada do implante e a ovula??o, 74,0 ? 11,8 e 82,5 ? 4,2 horas; no di?metro do fol?culo dominante (FD) na retirada do implante, 7,5 ? 2,5 e 6,6 ? 1,5 mm; no di?metro m?ximo do FD, 10,47 ? 3,7 e 8,85 ? 1,2 mm; na taxa de crescimento do FD, 0,90 ? 0,7 e 0,83 ? 0,6 mm/dia e na taxa de ovula??o, 75% (6/8) e 100% (8/8), respectivamente. Com rela??o ? taxa de prenhez, foi observada diferen?a estat?stica (P<0,05) entre os grupos I e II, sendo 0% (0/8) e 50% (4/8), respectivamente. Portanto, conclui-se que o uso do eCG em f?meas bovinas da ra?a Sindi n?o foi efetivo em melhorar os par?metros reprodutivos analisados, talvez pelo fato das vacas apresentarem bom escore de condi??o corporal durante a realiza??o do experimento. Por outro lado, houve um aumento na taxa de prenhez com o uso do eCG, indicando que este pode ser uma ferramenta ?til em protocolos de IATF nesta ra?a.

L’hormone anti-Müllerienne (AMH), une glycopréotéine exclusivement produite par les cellules de la granulosa (CG) des follicules ovariens de la femme, est un marqueur unique de du statut folliculaire ovarien. Contrairement à l’inhibine B, l’estradiol et la FSH, l’AMH est produite par un large éventail de follicules allant des follicules primaires aux follicules à petit antrum. Cependant, les mécanismes précis régulant la production d’AMH par les CG restent mal connus. Nous avons montré que la sélection folliculaire précoce au cours de la phase de transition lutéo-folliculaire, un phénomène fréquemment retrouvé chez les femmes ayant un vieillissement ovarien, caractérisé par la présence d’au moins un follicule surdéveloppé au cours de la phase folliculaire précoce, n’altérait pas la puissance de la relation entre le compte folliculaire antral et les concentrations sériques d’AMH. En revanche, cette situation perturbait significativement celle entre le nombre de follicules antraux et les taux sériques de FSH, d’inhibine B et d’estradiol. Nous avons par la suite mis en évidence, en utilisant un nouvel outil, nommé Follicular Output RaTe (FORT), que le pourcentage de follicules qui répondent effectivement à la FSH exogène en atteignant la maturation pré-ovulatoire, était négativement et indépendamment lié aux taux sériques d’AMH, ce qui va dans le sens de l’hypothèse d’un effet inhibiteur de l’AMH sur la sensibilité des follicules à la FSH. Ensuite, nous avons regardé si la production d’AMH par ovaire et par follicule était altérée chez les femmes n’ayant plus qu’un seul ovaire suite à une ovariecomie unilatérale. En effet, tout indique que chez ces femmes, des réarrangements majeurs de la folliculogenèse sont mis en place pour maintenir une fonction ovarienne malgré la perte brutale d’une partie du pool folliculaire. Ainsi, par une analyse extensive et comparative de la folliculogenèse utilisant des marqueurs hormono-folliculiares, nous n’avons pu mettre aucune modification significative chez les femmes avec un ovaire unique, comparativement aux contrôles. A l’aide du modèle précédemment utilisé, nous avons constaté une augmentation de la sensibilité des follicules antraux à la FSH exogène, évaluée par le FORT, chez des femmes avec un seul ovaire, comparativement aux femmes avec 2 ovaires. Ces résultats supportent l’hypothèse d’une augmentation de la sensibilité folliculaire à la FSH, qui pourrait faire partie des possibles mécanismes compensatoires en jeu dans le maintien d’une folliculogenèse efficace chez les femmes ayant eu une ovariectomie unilatérale.Finalement, à l’aide de 2 approches complémentaires, in vitro and in vivo, nous avons montré que la FSH et l’AMPc stimulaient la transcription de l’AMH, et que la LH avait un effet additif. Nous avons montré que les gonadotrophines et l’AMPc agissaient à travers la protéine kinase A et la P38 MAP Kinase, impliquant notamment les facteurs de transcription GATA binding factor-4 et le steroidogenic factor-1. Par ailleurs, nous avons également mis en évidence que l’expression d’AMH pouvait être régulée de manière différentielle par l’estradiol, en fonction du type de récepteur aux estrogènes exprimés par les CG. Ainsi, la chute d’expression de l’AMH au sein des CG des follicules matures, qui expriment essentiellement ERβ, est probablement liée à un effet de l’estradiol. En résumé, ces travaux de thèse ont permis d’apporter de nouvelles données sur la régulation de la croissance folliculaire et sur la production d’AMH chez la femme. / Anti-Müllerian hormone (AMH), a glycoprotein that is exclusively produced by the granulosa cells (GC) of ovarian follicles in the adult female, is a unique biomarker of ovarian follicular status. In contrast with inhibin B, estradiol and FSH, AMH is produced in a wide range of follicles that goes from the primary to the small antral stages of folliculogenesis. However, the precise mechanisms that drive AMH expression by GC remain poorly understood.We showed that untimely and/or accelerated antral follicle growth during the luteal–follicular transition, a phenomenon that is frequent in ovarian-aged women and that is characterized by the presence of at least one overdeveloped antral follicle during the first days of the follicular phase does not alter the strength of the relationship between antral follicle count and serum AMH levels but does affect the relationship between serum FSH, inhibin B and estradiol levels and the number of antral follicles. The heftiness of AMH in relation to advanced antral follicle growth provides a further explanation for the reported stronger association between serum AMH levels and antral follicle counts as compared with the other hormonal markers of the ovarian fertility status. We subsequently demonstrated, using an innovative tool, the Follicular Output RaTe (FORT), that the percentage of follicles that effectively respond to exogenous FSH by reaching pre-ovulatory maturation is negatively and independently related to serum AMH levels, which is in keeping with the hypothesis that AMH inhibits follicle sensitivity to FSH. Given this hypothesis, we wondered if per-ovary and per-follicle AMH production could be altered in patients having a single ovary as a result of unilateral oophorectomy. Indeed, all indicate that major rearrangements of folliculogenesis occur to preserve and maintain ovarian function despite the abrupt halving of follicular stockpile in these patients. We performed an extensive and comparative evaluation of the folliculogenesis using homono-follicular markers failing to show major changes in unilaterally oophorectomized when compared with control women. Using the same model, we demonstrated an increased antral follicle responsiveness to exogenous FSH, as assessed by FORT, in normo-ovulating unilaterally oophorectomized women undergoing controlled ovarian hyperstimulation. These results support the hypothesis that increased FSH sensitivity ranks among the possible compensating mechanisms at stake in the maintenance of successful folliculogenesis after unilateral oophorectomy.Finally, using complementary approaches, in vitro and in vivo, we showed that FSH and cAMP enhance AMH transcription, and LH has an additive effect. Gonadotropins and cAMP act through protein kinase A and p38 MAPK signaling pathways and involve the GATA binding factor-4 and steroidogenic factor-1 transcription factors, among others. The expression profile of AMH and the dynamics of serum AMH after gonadotropin stimulation have been interpreted as a down-regulating effect of FSH upon AMH production by GC. We also demonstrated that AMH expression can be differentially regulated by estradiol depending on the estradiol receptors by GC. Therefore the decrease in AMH expression by GC of mature follicles, which mainly express ERβ, is likely due to the effect of estradiol.In short, this Ph.D. work offers new insight into the regulation of the follicular growth and AMH production in woman.

De nos jours, la cryoconservation est une technique très largement utilisée dans les protocoles d’assistance à la reproduction ou comme outil pour la sauvegarde des ressources génétiques. Toutefois, la chienne est un modèle animal complexe pour l’application des biotechnologies de la reproduction du fait de ses nombreuses singularités anatomiques et physiologiques. L’objectif de notre travail était d’étudier et de développer une méthode de cryoconservation des ressources génétiques chez la chienne par le biais de deux types de ressources : les embryons et le tissu ovarien. Après avoir mis au point une méthode de collecte d’embryons, nous nous sommes appliqués à la constitution d’un stock d’embryons cryoconservés en prévision d’un transfert embryonnaire. L’étude et le développement d’un protocole de cryoconservation du tissu ovarien ont été abordés après avoir adapté et validé nos méthodes d’analyses in vitro. L’utilisation de plans d’expériences factoriels fractionnaires a permis de mettre en évidence les facteurs les plus influents sur la qualité de la réserve folliculaire (nature du cryoprotecteur pénétrant, cinétique de congélation, étapes d’équilibration) et de proposer un protocole de cryoconservation. La combinaison du DMSO incorporé en un seul bain d’équilibration avec une vitesse de congélation de 0,3°C/min est apparue comme la combinaison la plus appropriée à la cryoconservation de tissu ovarien chez la chienne et a permis d’observer, après xénogreffe de tissu ovarien cryoconservé, une reprise de la croissance folliculaire et de l’activité hormonale du tissu greffé / Nowadays, cryopreservation is widely used in animal assisted reproduction or safeguarding of genetic resources. Nevertheless, the bitch is a complex animal model concerning the use of this biotechnology, due to numerous anatomical and physiological peculiarities. The aim of our research work was to investigate and develop a method of cryopreservation of genetic resources in the bitch by exploring two kinds of resources: embryos and ovarian tissue. After the setting up of a method for embryo collection, we have built up a stock of cryopreserved embryo for subsequent embryo transfer. After a preliminary validation of our in vitro assessment methods, the investigation and development of a cryopreservation protocol has been conducted. The use of fractional experimental design allowed us to highlight the main factors affecting the follicular pool quality (CPA nature, freezing rate and equilibration steps). The combination of DMSO incorporated in a unique equilibration bath with a freezing rate of 0.3°C/min appeared to be suitable for the cryopreservation of bitch ovarian tissue. Finally, Follicular growth and hormonal activity resumption have been observed after xenotransplantation of cryopreserved bitch ovarian tissue

Made available in DSpace on 2016-01-26T18:55:27Z (GMT). No. of bitstreams: 1 dissertacao Bianca.pdf: 325135 bytes, checksum: 8bbf94808bc25e72a05aba3d84bca76a (MD5) Previous issue date: 2008-11-14 / The aim of the study was to evaluate the effect of bovine somatotropin in follicular deviation of sixteen Tabapuã cows. The animals received an ear implant of progesterone and 1 mg of estradiol benzoate, IM (day 0). On day 5, the females were divided into 2 groups: GI (control, n=8) and GII (treated with 500 mg bST, n=8). On day 10, the implants were removed and injected 500 µg of PGF2&#945; in all cows. Only cows with follicles bigger 9 mm received 300 µg of GnRH. Ultrasound examinations were performed each 12 hours. There was no statistical difference between the follicular deviation in GI (2.4 days) and GII (2,1 days). At the divergence moment, FD and FS of GI were 6.28±0.42 and 6.26±0.41 mm, respectively, and FD and FS of GII were 6.08±0.72 and 6.12±0.39 mm. The mean maximum diameter of FD after ovulation was at 110.0±8.43 hours in GI and 115.2±8.98 hours in GII. FS reached the maximum diameter at 55.0±20.0 hours in GI and 76.8±10.46 hours in GII. The mean maximum diameter reached by FD and FS in GI was 8.85±0.41 and 6.5±0.42 mm, respectively, and GII 9.83±0.63 and 6.87±0.35 mm. There was no significant difference (p> 0.05) in diameter of FD and FS, neither in growth rates (mm/12 hours) of the FD before and after the deviation, neither in the moment of follicle deviation / O objetivo do presente estudo foi avaliar o efeito da somatotrofina bovina na divergência folicular em vacas da raça Tabapuã. Foram utilizadas 16 vacas da raça Tabapuã, inicialmente receberam implante de progestágeno auricular concomitante à aplicação IM de 1mg de benzoato de estradiol (dia 0). No dia 5, dividiram-se as fêmeas em 2 grupos: G-I (controle, n=8) e as vacas do G-II foram tratadas com 500 mg bST (n=8). No dia 10 foi feita a retirada do crestar concomitante a aplicação de 500 &#61549;g de PGF2&#61537; e apenas nas vacas com folículos> 9 mm aplicação de 300 &#61549;g de GnRH. Foram realizados exames ultrassonográficos a cada 12 horas por 5 dias. Não houve diferença estatística entre os momentos de divergência folicular, no G-I foi de 2,4 dias e no G-II 2,1 dias. Nesse momento o FD e FS mediram 6,28 &#61617; 0,42 e 6,26 &#61617; 0,41 mm no G-I e 6,08 &#61617; 0,72 e 6,12 &#61617; 0,39 mm no G-II. O FD atingiu diâmetro máximo após a ovulação em média 110,00 ± 8,43 horas no GI e 115,20 ± 8,98 horas no GII. Já o FS atingiu o diâmetro máximo às 55,00 ± 20,00 e 76,80 ± 10,46 horas, respectivamente. A média do diâmetro máximo atingido pelo FD e FS no GI foi respectivamente 8,85 ± 0,41 e 6,50 ± 0,42 mm e no GII foi 9,83 ± 0,63 e 6,87 ± 0,35 mm. Não houve diferença significativa (p>0,05) no diâmetro do FD e FS e nem nas taxas de crescimento (mm/12h) do FD antes e após a divergência folicular. Concluímos que a aplicação de bST não afetou o diâmetro folicular, a taxa de crescimento do FD e FS antes e após a divergência, nem tampouco, o momento da divergência folicular

Made available in DSpace on 2016-07-18T17:53:05Z (GMT). No. of bitstreams: 1 dissertacao Bianca.pdf: 325135 bytes, checksum: 8bbf94808bc25e72a05aba3d84bca76a (MD5) Previous issue date: 2008-11-14 / The aim of the study was to evaluate the effect of bovine somatotropin in follicular deviation of sixteen Tabapuã cows. The animals received an ear implant of progesterone and 1 mg of estradiol benzoate, IM (day 0). On day 5, the females were divided into 2 groups: GI (control, n=8) and GII (treated with 500 mg bST, n=8). On day 10, the implants were removed and injected 500 µg of PGF2&#945; in all cows. Only cows with follicles bigger 9 mm received 300 µg of GnRH. Ultrasound examinations were performed each 12 hours. There was no statistical difference between the follicular deviation in GI (2.4 days) and GII (2,1 days). At the divergence moment, FD and FS of GI were 6.28±0.42 and 6.26±0.41 mm, respectively, and FD and FS of GII were 6.08±0.72 and 6.12±0.39 mm. The mean maximum diameter of FD after ovulation was at 110.0±8.43 hours in GI and 115.2±8.98 hours in GII. FS reached the maximum diameter at 55.0±20.0 hours in GI and 76.8±10.46 hours in GII. The mean maximum diameter reached by FD and FS in GI was 8.85±0.41 and 6.5±0.42 mm, respectively, and GII 9.83±0.63 and 6.87±0.35 mm. There was no significant difference (p> 0.05) in diameter of FD and FS, neither in growth rates (mm/12 hours) of the FD before and after the deviation, neither in the moment of follicle deviation / O objetivo do presente estudo foi avaliar o efeito da somatotrofina bovina na divergência folicular em vacas da raça Tabapuã. Foram utilizadas 16 vacas da raça Tabapuã, inicialmente receberam implante de progestágeno auricular concomitante à aplicação IM de 1mg de benzoato de estradiol (dia 0). No dia 5, dividiram-se as fêmeas em 2 grupos: G-I (controle, n=8) e as vacas do G-II foram tratadas com 500 mg bST (n=8). No dia 10 foi feita a retirada do crestar concomitante a aplicação de 500 &#61549;g de PGF2&#61537; e apenas nas vacas com folículos> 9 mm aplicação de 300 &#61549;g de GnRH. Foram realizados exames ultrassonográficos a cada 12 horas por 5 dias. Não houve diferença estatística entre os momentos de divergência folicular, no G-I foi de 2,4 dias e no G-II 2,1 dias. Nesse momento o FD e FS mediram 6,28 &#61617; 0,42 e 6,26 &#61617; 0,41 mm no G-I e 6,08 &#61617; 0,72 e 6,12 &#61617; 0,39 mm no G-II. O FD atingiu diâmetro máximo após a ovulação em média 110,00 ± 8,43 horas no GI e 115,20 ± 8,98 horas no GII. Já o FS atingiu o diâmetro máximo às 55,00 ± 20,00 e 76,80 ± 10,46 horas, respectivamente. A média do diâmetro máximo atingido pelo FD e FS no GI foi respectivamente 8,85 ± 0,41 e 6,50 ± 0,42 mm e no GII foi 9,83 ± 0,63 e 6,87 ± 0,35 mm. Não houve diferença significativa (p>0,05) no diâmetro do FD e FS e nem nas taxas de crescimento (mm/12h) do FD antes e após a divergência folicular. Concluímos que a aplicação de bST não afetou o diâmetro folicular, a taxa de crescimento do FD e FS antes e após a divergência, nem tampouco, o momento da divergência folicular

Au cours des processus de croissance folliculaire et d'ovulation, les cellules stéroïdogéniques, y compris les cellules de granulosa (CG), jouent un rôle crucial dans la maturation et la libération de l'ovocyte. Notre laboratoire a identifié et caractérisé pour la première fois tribbles pseudokinase 2 (TRIB2) dans les CG de follicules ovariens. Des études ont démontré que TRIB2 joue un rôle dans la coordination de la mitose et la morphogenèse chez la drosophile alors que chez la souris, son expression dans les cellules du cumulus serait liée à la maturation ovocytaire. Cependant, le rôle exact de TRIB2 dans la fonction des CG et ses effets sur les voies de signalisation impliquées dans la croissance folliculaire restait à être défini. La présente étude de doctorat avait donc pour but d’étudier la fonction et les partenaires de liaison de TRIB2 dans les CG de follicules ovariens bovins. À l’aide d’un modèle d’étude in vivo consistant en des CG obtenues à partir de follicules à différents stades de développement, nous avons démontré une régulation à la baisse de TRIB2 par l’hormone lutéinisante (LH) et la gonadotrophine chorionique humaine (hCG) aussi bien au niveau du messager que de la protéine. De plus, les analyses utilisant un modèle in vitro de CG en culture ont montré que la FSH stimule l'expression de TRIB2 tandis que l'inhibition de TRIB2 via CRISPR/Cas9 entraîne une réduction significative de la prolifération des CG (P<0,05). Les analyses Western blot ont montré une augmentation des niveaux de phosphorylation d’ERK1/2 (MAPK3/1) et p38MAPK (MAPK14) suite à la surexpression de TRIB2. Ces résultats suggèrent un rôle de TRIB2 dans la croissance folliculaire et la modulation des voies MAPK. Avec l’approche double hybride chez la levure, nous avons identifié CALM1, INHBA, INPPL1, NT5E, SCD, SDHB et RAB14 comme partenaires de liaison de TRIB2. Les analyses RT-PCR ont montré que ces partenaires sont régulés différemment au cours du développement folliculaire et les manipulations de l’expression de TRIB2 (inhibition ou surexpression) résulte en une régulation différente (augmentation ou diminution) de l’expression des partenaires dans les CG. Ces résultats suggèrent un rôle de TRIB2 dans la régulation de cibles effectrices en lien avec la fonction des CG et le développement folliculaire. Enfin, un modèle de CG provenant de vaches en période post-partum a été utilisé pour mieux comprendre le contrôle de l’activité des CG. Des analyses complémentaires avec ce modèle ont révélé une réduction significative de TRIB2 chez les vaches ayant un taux élevé de BHB (>1.4mmol/L) comparé à celles ayant un faible taux de BHB (<1.2mmol/L). Cette réduction était concomitante à une augmentation d’interleukines pro-inflammatoires et une réduction d’interleukines anti-inflammatoires dans les CG. L’ensemble de ces résultats supporte un rôle de TRIB2 dans la modulation de la signalisation MAPK dans les CG, apporte une preuve solide que TRIB2 pourrait agir comme un régulateur de la prolifération et de la fonction des CG et de l’expression de gènes cibles, et suggère que TRIB2 pourrait affecter la stéroïdogenèse au cours du développement folliculaire et lors de la période post-partum. / During the processes of follicular growth and ovulation, steroidogenic cells, including granulosa cells, play a crucial role in the maturation and release of the oocyte. Our lab identified and characterized for the first time tribble pseudokinase 2 (TRIB2) in GC of ovarian follicles. Previous studies have shown that TRIB2 plays a role in the coordination of mitosis and morphogenesis in Drosophila while in mice its expression in cumulus cells is linked to oocyte maturation. However, the exact mechanism of action of TRIB2 as well as its function and effects on signaling pathways in GC during follicular growth remained to be defined. This study aimed therefore to further investigate TRIB2 function and identify its binding partners in GC. TRIB2 inhibition and overexpression experiments were conducted using, respectively, CRISPR/Cas9 technology and the pQE1 system. Using an in vivo model consisting of GC obtained from follicles at different stages of development, we demonstrated a downregulation of TRIB2 by the endogenous luteinizing hormone (LH) and human Chorionic Gonadotropin (hCG) at both the messenger and protein levels. In addition, analyzes using an in vitro model of cultured GC, we showed that FSH stimulates the expression of TRIB2 while inhibition of TRIB2 via CRISPR-Cas9 resulted in a significant reduction in GC proliferation (P<0.05). Western blot analyzes showed an increase in the phosphorylation levels of ERK1/2 (MAPK3/1) and p38MAPK (MAPK14) following TRIB2 overexpression. These results suggested a role of TRIB2 in follicular growth and modulation of MAPK pathways. In the second part of the thesis, we identified CALM1, INHBA, INPPL1, NT5E, SCD, SDHB and RAB14 as binding partners of TRIB2 in GC using the yeast two-hybrid approach. RT-qPCR analyzes showed that all of these partners are present in the dominant follicles but are differently regulated during follicular development. Moreover, TRIB2 manipulation (inhibition or overexpression) results in a different regulation (up- or down-regulation) of these partners expression in GC. These results suggest a role of TRIB2 in the regulation of effector targets genes related to follicular development and GC activity. In the third part of the thesis, a GC model from postpartum cows was also used to better understand the control of GC activity. Further analyses using this model revealed a significant decrease of TRIB2 in cows with high level of BHB (>1.4 mmol/L) as compared to those with a low BHB levels (<1.2 mmol/L). This reduction of TRIB2 was concomitant with an increase in pro-inflammatory interleukins and a reduction in anti-inflammatory interleukins in GC. Overall, these results support a role of TRIB2 in the modulation of MAPK signaling in GC, provide strong evidence that TRIB2 could act as a regulator of GC proliferation and function as well as expression of target genes in GC, and suggests that TRIB2 might affect steroidogenesis during follicular development and during the post-partum period.