Academic literature on the topic 'Cellule de la granulosa [CG]'

Recent studies suggest that non-steroid factors, such as cytokines, may play a role in ovarian processes. The purpose of this study was to explore cellular sites of interleukin (IL)-6 biosynthesis in rabbit follicles and to investigate IL-6 modulation in granulosa and theca cell functions. In this report development of rabbit preovulatory follicles was induced by 200 mIU equine chorionic gonadotropin (eCG) daily for 2 days. Seventy-two hours after the last injection ovaries were excised and granulosa and theca cells isolated. The two types of cells were preincubated for 24 h in Minimum Essential Medium (MEM) with 5% fetal calf serum (FCS), and then incubated for 24 h in MEM-2.5% FCS with appropriate stimulants. Results showed that rabbit granulosa and theca cell culture supernatants contained IL-6 bioactivity and that its production was inhibited by FSH and human CG and stimulated by IL-1. IL-6 inhibited gonadotropin-induced progesterone production, but not basal secretion, in both cell types, without a cytotoxic effect. IL-6 affected cAMP generation and steps distal to cAMP formation, but the mechanism of IL-6 action on progesterone differed in granulosa and theca cells. Taken together our results suggest that gonadotropins, by inhibiting IL-6 production, could control, in our model, IL-6 modulation of gonadotropin action on steroidogenesis.

Abstract We have examined the expression and regulation of the two estrogen receptor (ERα and ERβ) genes in the rat ovary, using Northern blotting, RT-PCR, and in situ hybridization histochemistry. Northern blotting results show that the ovary expresses both ERα and ERβ genes as single (∼6.5-kb) and multiple (ranging from ∼1.0-kb to ∼10.0-kb) transcripts, respectively. ERα mRNA is expressed at a level lower than ERβ mRNA in immature rat ovaries. This relationship appears unchanged between sexually mature adult rats and immature rats. In sexually mature adult rats undergoing endogenous hormonal changes, whole ovarian content of ERβ mRNA, as determined by RT-PCR, remained more or less constant with the exception of the evening of proestrus when ERβ mRNA levels were decreased. Examination of ERβ mRNA expression at the cellular level, by in situ hybridization, showed that ERβ mRNA is expressed preferentially in granulosa cells of small, growing, and preovulatory follicles, although weak expression of ERβ mRNA was observed in a subset of corpora lutea, and that the decrease in ERβ mRNA during proestrous evening is attributable, at least in part, to down-regulation of ERβ mRNA in the preovulatory follicles. This type of expression and regulation was not typical for ERα mRNA in the ovary. Although whole ovarian content of ERα mRNA was clearly detected by RT-PCR, no apparent modulation of ERα mRNA levels was observed during the estrous cycle. Examination of ERα mRNA expression at the cellular level, by in situ hybridization, showed that ERα mRNA is expressed at a low level throughout the ovary with no particular cellular localization. To further examine the potential role of the preovulatory pituitary gonadotropins in regulating ERβ mRNA expression in the ovary, we used immature rats treated with gonadotropins. In rats undergoing exogenous hormonal challenges, whole ovarian content of ERβ mRNA, as determined by RT-PCR, remained more or less unchanged after an injection of PMSG. In contrast, a subsequent injection of human CG (hCG) resulted in a substantial decrease in whole ovarian content of ERβ mRNA. In situ hybridization for ERβ mRNA shows that small, growing, and preovulatory follicles express ERβ mRNA in the granulosa cells. The preovulatory follicles contain ERβ mRNA at a level lower than that observed for small and growing follicles. In addition, there is an abrupt decrease in ERβ mRNA expression in the preovulatory follicles after hCG injection. The inhibitory effect of hCG on ERβ mRNA expression was also observed in cultured granulosa cells. Moreover, agents stimulating LH/CG receptor-associated intracellular signaling pathways (forskolin and a phorbol ester) readily mimicked the effect of hCG in down-regulating ERβ mRNA in cultured granulosa cells. Taken together, our results demonstrate that 1) the ovary expresses both ERα and ERβ genes, although ERβ is the predominant form of estrogen receptor in the ovary, 2) ERβ mRNA is localized predominantly to the granulosa cells of small, growing, and preovulatory follicles, and 3) the preovulatory LH surge down-regulates ERβ mRNA. These results clearly implicate the physiological importance of ERβ in female reproductive functions.

Abstract An endocrine type of voltage-activated sodium channel (eNaCh) was identified in the human ovary and human luteinized granulosa cells (GC). Whole-cell patch-clamp studies showed that the eNaCh in GC is functional and tetrodotoxin (TTX) sensitive. The luteotrophic hormone human CG (hCG) was found to decrease the peak amplitude of the sodium current within seconds. Treatment with hCG for 24–48 h suppressed not only eNaCh mRNA levels, but also mean Na+ peak currents and resting membrane potentials. An unexpected role for eNaChs in regulating cell morphology and function was indicated after pharmacological modulation of presumed eNaCh steady-state activity in GC cultures for 24–48 h using TTX (NaCh blocker) and veratridine (NaCh activator). TTX preserved a highly differentiated cellular phenotype. Veratridine not only increased the number of secondary lysosomes but also led to a significantly reduced progesterone production. Importantly, endocrine cells of the nonhuman primate corpus luteum (CL), which represent in vivo counterparts of luteinized GC, also contain eNaCh mRNA. Although the mechanism of channel activity under physiological conditions is not clear, it may include persistent Na+ currents. As observed in GC in culture, abundant secondary lysosomes were particularly evident in the regressing CL, suggesting a functional link between eNaCh activity and this form of cellular regression in vivo. Our results identify eNaCh in ovarian endocrine cells and demonstrate that their expression is under the inhibitory control of hCG. Activation of eNaChs in luteal cells, due to loss of gonadotropin support, may initiate a cascade of events leading to decreased CL function, a process that involves lysosomal activation and autophagy. These results imply that ovarian eNaChs are involved in the physiological demise of the temporary endocrine organ CL in the primate ovary during the menstrual cycle. Because commonly used drugs, including phenytoin, target NaChs, these results may be of clinical relevance.

Abstract Granulosa cell tumors comprise approximately 10% of ovarian tumors and, although rare, are clinically important due to their potential for malignancy and recurrence. Although their morphological features have been carefully described, the global changes in gene expression associated with their formation remain undetermined. To initiate this characterization, we used a transgenic mouse model in which granulosa cell tumors occur with 100% penetrance in CF-1 mice that harbor a novel transgene encoding a chimeric LHβ subunit. When this transgene is expressed in other strains of mice, including (C57BL/6♀ × CF-1♂,Tg) F1 hybrids, luteomas develop even though levels of LH remain high. This dichotomous response permits a longitudinal comparison of global changes in transcriptomes uniquely associated with either granulosa cell tumors or luteomas. Herein we report numerous changes in the transcriptome, including a decrease in LH receptor mRNA and increases in several mRNAs that encode secreted proteins previously associated with granulosa cell tumors. Furthermore, we identified a constellation of mRNAs that encode proteins that may serve as new markers for this tumor phenotype. Additional experiments indicated that periodic treatment with human CG prevented formation of granulosa cell tumors in mice genetically predisposed to tumor development and, instead, led to the appearance of luteomas. More importantly, ovarian transcriptomes from the luteomas induced by ovulatory doses of human CG permitted refined confirmation of gene expression changes that were uniquely associated with either granulosa cell tumors in the permissive CF-1 genetic background or in luteomas in the F1 hybrids. Together, these dynamic changes in the ovarian transcriptome indict various signaling pathways potentially involved in mediating the actions of LH over time and, depending on genetic background, the formation of either a luteoma or a granulosa cell tumor.

Recently, interference RNA (RNAi), inducing inhibition of the specific gene expression, attracted a lot of attention. Many researchers have reported that the 21-mer small interference RNA (siRNA) is introduced into target cells and then siRNA can suppress the gene expression. RNAi is a useful tool for functional analysis of specific genes. However, there is little information about RNAi for the analysis of gene function in reproductive physiology in ruminants. Thus, this study was aimed at evaluating RNAi for the analysis of cyclooxygenase-2 (Cox-2) mRNA expression in bovine cumulus-granulosa (CG) cells as well as prostaglandin F2� (PGF2�) production. We investigated both the effective concentration of Cox-2 siRNA and the effect of the introduction time of siRNA on Cox-2 mRNA expression. Bovine CG cells were collected at slaughterhouse and cultured in 4-well dishes. After the cells reached confluency, two experiments were conducted. In the present study, Cox-2 siRNA was simply added to culture medium with lipofectamine" 2000 (Invitrogen Japan K. K., Tokyo, Japan) as the transfection reagent. In experiment 1, the concentration of 0, 100, 250, and 500 pM of Cox-2 siRNA was introduced into the CG cells. After 24 h of introduction, the amount of mRNA expression for Cox-2 was measured by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. In experiment 2, 250 pM siRNA for Cox-2 was introduced into CG cells for 0, 3, 6, 12, and 24 h. After culture, the amount of mRNA expression for Cox-2 was measured and the culture medium was collected to determine PGF2� concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by 100 pM siRNA introduced into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression (10% of that of the 0 pM siRNA group). Moreover, the suppressive effect of 250 pM siRNA was observed at 6 h after introduction (60% of that of the 0 pM siRNA group at 0 h) and the reduction of mRNA expression by RNAi became more obvious over 12 h (10% of that of the 0 pM siRNA group at 0 h). On the other hand, the PGF2� concentration in the culture medium was not significantly different at 12 h after siRNA introduction, however, the PGF2� concentration of the RNAi treatment group at 24 h was significantly lower than that of the 0 pM siRNA group at the same time point. These results suggest that gene silencing by Cox-2 siRNA is a means of analyzing the function and expression of specific genes in bovine CG cells. This study was supported by the Japan Society for the Promotion of Science for Young Scientists.

The P2U purinoceptor (P2UR) has been identified pharmacologically in the ovary. However, the expression and regulation of the P2UR messenger RNA (mRNA) in human ovarian cells are still poorly characterized. The present study was designed to examine the expression and regulation of the P2UR in human granulosa-luteal cells (hGLCs) by RT-PCR and Northern blot analysis. A PCR product corresponding to the expected 599-bp P2UR complementary DNA was obtained from hGLCs. Molecular cloning and sequencing of the PCR product revealed an identical sequence to the reported P2UR complementary DNA. Two mRNA transcripts of 2.0 kb and 4.6 kb were identified in hGLCs using Northern blot analysis. The expression of the P2UR mRNA was down-regulated by human CG in a dose- and time-dependent manner. Treatment with 8-bromo-cAMP and forskolin also attenuated P2UR mRNA levels. Calcium signaling following the activation of the P2UR in single hGLCs was studied using microspectrofluorimetry. It revealed that, like ATP, uridine triphosphate (UTP) also induced cytosolic calcium mobilization in a dose-dependent manner. These results demonstrate for the first time that the P2UR mRNA is expressed in hGLCs and that P2UR mRNA is regulated by human CG, cAMP, and forskolin. The P2UR expressed in hGLCs functional because activation of the P2UR by ATP or UTP resulted in rapid and transient mobilization of cytosolic calcium at the single cell level. These findings further support a potential role of this neurotransmitter receptor in the human ovary.

Abstract Although X-linked inhibitor of apoptosis protein (Xiap) is an important intracellular suppressor of apoptosis in a variety of cell types and is present in ovary, its physiological role in follicular development remains unclear. The purpose of the present studies was to examine the modulatory role of Xiap in the proapoptotic action of tumor necrosis factor-α (TNFα) in rat granulosa cells. Granulosa cells from equine CG-primed immature rats were plated in RPMI 1640 medium containing 10% FCS and subsequently cultured in serum-free RPMI in the absence or presence of TNFα (20 ng/ml), the protein synthesis inhibitor cycloheximide (10 μm), and/or adenoviral Xiap sense or antisense complementary DNA. TNFα alone failed to induce granulosa cell death, but in the presence of cycloheximide, it markedly increased the number of apoptotic granulosa cells (as assessed by in situ terminal deoxynucleotidyl transferase-mediated deox-UTPbiotin end labeling and DNA fragmentation analysis). Western analysis indicated that TNFα alone increased the Xiap protein level, a response significantly reduced by adenoviral Xiap antisense expression. Down-regulation of Xiap expression by antisense complementary DNA induced granulosa cell apoptosis, which was potentiated by the cytokine. Inhibition of nuclear factor-κB activation by N-acetyl-cysteine and SN50 suppressed Xiap protein expression and enhanced apoptosis induced by TNFα. The latter phenomenon was readily attenuated by adenoviral Xiap sense expression. In conclusion, these findings suggest that Xiap is an important intracellular modulator of the TNFα death signaling pathway in granulosa cells. Its expression is regulated by the TNFα via a nuclear factor-κB-mediated mechanism.

The meiotic competence and meiosis resumption of Blue fox (Alopex lagopus) oocytes from anoestrous animals were followed. Oocyte–cumulus complexes (OCC) were cultured in modified TC 199 medium with or without FSH, recombinant bovine somatotrophin (bST) and okadaic acid (OA). The results showed that oocytes less than 100 μm in diameter did not achieve germinal vesicle breakdown (GFBD) by 72 h of culture, which indicates their meiotic incompetence. Oocytes larger than 100 µm in diameter underwent GVBD after 48 h of culture (27%) and reached metaphase II (MII) after 72 and 96 h (20% and 27%) in control medium. Both bST and OA accelerated resumption of meiosis (bST: 55% GVBD and 42% MII after 48 h; OA: 66% GVBD after 18 h). In contrast, FSH significantly reduced meiosis resumption (only 3% GVBD and MII after 72 h) and induced changes in the shape of cumulus granulosa (CG) cells and F-actin assembly typical for cumulus expansion. However, the innermost layers of CG cells (corona radiata) remained connected with the oocyte via gap junctions until the end of culture. Cumuli of oocytes cultured in control, bST-supplemented or OA-supplemented medium did not expand (changes in cell shape and F-actin redistribution did not occur). Moreover, especially in media with bST and OA an increased detachment and rapid disconnection of their gap junctions with the oocyte were observed. These results suggest that under in vitro conditions FSH stimulates expansion of the CG cells and the attached membrana granulosa cells but in contrast it secures heterologous gap junctions between cytoplasmic processes of the corona radiata cells and oolemma during 3 days of culture. Thus, in agreement with the in vivo situation in which Canidae oocytes are ovulated in the GV stage, the cumulus, mainly corona radiata cells, controls resumption of meiosis in Blue fox oocytes under in vitro conditions also.

Abstract Angiogenesis is an essential event during the development of the ovarian follicle and ensuing formation of the corpus luteum. We investigated the presence of angiogenin, a potent inducer of angiogenesis, and the regulatory mechanisms of its production in the human ovary. Follicular fluid (FF) and granulosa cells (GCs) were collected from women undergoing in vitro fertilization and embryo transfer. The presence of angiogenin in FF and GCs was demonstrated by Western blot analysis. The production of angiogenin by cultured GCs was stimulated with the addition of human CG or cAMP or under the hypoxic milieu. Concentrations of angiogenin in FF from an individual follicle were positively correlated with those of progesterone, but not estradiol and testosterone. Given the presence of angiogenin in FF and up-regulation of its production by human CG and hypoxia, it seems logical to assume that angiogenin may play a role as a local angiogenic factor in the human ovary.

Granulosa cells in the ovulatory follicle express messenger ribonucleic acid encoding vascular endothelial growth factor (VEGF), an agent that may mediate the neovascularization of the developing corpus luteum, but it is not known whether luteinizing granulosa cells synthesize and secrete VEGF during the periovulatory interval. Studies were designed to evaluate the effects of an in vivo gonadotropin surge on VEGF production by macaque granulosa cells (study 1) and to test the hypothesis that gonadotropins act directly on granulosa cells to regulate VEGF production (study 2). Monkeys received a regimen of exogenous gonadotropins to promote the development of multiple preovulatory follicles. Nonluteinized granulosa cells (i.e. preovulatory; NLGC) and luteinized granulosa cells (i.e. periovulatory; LGC) were aspirated from follicles before and 27 h after an ovulatory gonadotropin bolus, respectively. Cells were either incubated for 24 h in medium with or without 100 ng/mL hCG (study 1) or cultured for 6 days in medium with or without 100 ng/mL hCG or 0.1, 1, 10, and 100 ng/mL of recombinant human LH (r-hLH) or r-hFSH (study 2). Culture medium was assayed for VEGF and progesterone. In study 1, LGC produced 8-fold greater levels of VEGF than NLGC (899 ± 471 vs. 111 ± 26 pg/mL, mean ± sem; P < 0.05). In vitro treatment with hCG increased (P < 0.05) VEGF production by NLGC to levels that were not different from the LGC incubated under control conditions. In vivo bolus doses of r-hCG (100 and 1000 IU) and r-hFSH (2500 IU) were equally effective in elevating granulosa cell VEGF production. In study 2, in vitro treatment with r-hFSH, r-hLH, and hCG markedly increased (P< 0.05) VEGF and progesterone production by the NLGC in a dose- and time-dependent manner. By comparison, the three gonadotropins (100 ng/mL dose) only modestly increased VEGF and progesterone production by LGC. These experiments demonstrate a novel role for the midcycle surge of gonadotropin (LH/CG or FSH) in primates to promote VEGF production by granulosa cells in the periovulatory follicle. Further, the data demonstrate that FSH-like as well as LH-like gonadotropins directly stimulate VEGF synthesis by granulosa cells.

Le Deoxynivalenol (DON) est une mycotoxine majeure retrouvée dans l’alimentation animale et celle-ci est connue pour réduire la fertilité des truies en inhibant la sécrétion de progestérone par les cellules de granulosa. Chez le bétail, DON est métabolisée en de-epoxy DON (DOM-1) dans le rumen, et DOM-1 peut atteindre des concentrations élevées dans le sang et les liquides folliculaires. Une des voies majeures de signalisation activée par DON est le ribotoxic stress response (RSR), lequel induit une auto-phosphorylation de la protéine kinase R (PKR) et réduit l’activation des MAP kinases incluant la MAPK3/1. Il n’a pas encore été démontré que ces mycotoxines affectent la reproduction chez les bovins. Les objectifs de cette thèse sont (1) de déterminer comment et à quelles doses DON affecte la fonction des cellules de granulosa et d’élucider les mécanismes d’action entrant en jeu; et (2) déterminer comment et à quelles doses la mycotoxine majeure DON et son métabolite DOM-1, affectent la fonction des cellules de la thèque chez le bétail. Les résultats sont présentés dans trois articles distincts. Dans le premier article, nous explorons les effets de DON sur les cellules de granulosa bovines; les traitements avec DON résultant en une inhibition significative de la sécrétion d’oestradiol et de progestérone (P4), et en une augmentation de la proportion de cellules apoptotiques après 4 jours de traitement. Les expériences de Western-Blot démontrent une stimulation significative de la phosphorylation de ERK1/2 et de MAPK14 entre 15 et 30 minutes après le début du traitement des cellules par DON. Par la suite, nous avons déterminé les effets de DON sur les gènes cibles de ERK1/2. En effet, les niveaux d’ARNm de EGR1 et FOS sont transitoirement augmentés avec des niveaux maximum à 1h de traitement par DON, tandis que les niveaux d’ARNm de iv COX2 et GADD45B sont augmentés mais plus de 24h après le début du traitement par DON. Dans le second article, les effets de DON et DOM-1 sur les cellules de thèque ont été étudiés. Le traitement des cellules par DOM-1 résulte en une inhibition dosedépendante de la sécrétion de P4 et de testostérone, et en une augmentation de la proportion de cellules apoptotiques, tandis que DON inhibe la sécrétion de P4 sans altérer celle de la testostérone ou bien le pourcentage de cellules mortes. Les deux mycotoxines sont effectives de manière maximale à des concentrations de 1 ng/ml (en revanche, DON affecte les cellules de granulosa à 100 ng/ml). Les résultats de Western-Blot démontrent la phosphorylation rapide de MAPK3/1, PKR et de JUN kinase après un traitement par DON ou DOM-1. En présence d’un inhibiteur spécifique de PKR, DON et DOM-1 sont incapables d’induire la phosphorylation de MAPK3/1, et l’effet inhibiteur de DON sur la phosphorylation de MAPK14 est en partie abrogé. Néanmoins, l’inhibiteur de PKR augmente davantage la phosphorylation de MAPK14 induite par DOM-1. Ensemble, ces résultats suggèrent que DON active le RSR dans les cellules de thèque et les cellules de granulosa bovines, et que les cellules de la thèque sont plus sensibles que les cellules de granulosa aux effets de DON. Ces données démontrent pour la première fois l’habilité de DOM-1 à affecter les fonctions et la survie cellulaires.
Deoxynivalenol (DON) is a major mycotoxin found in animal feed and is known to reduce fertility in pigs by inhibiting progesterone secretion from granulosa cells. In cattle, it is metabolized to de-epoxy DON (DOM-1) in the rumen, and DOM-1 can reach high concentrations in blood and follicular fluid. One of the major pathways activated by DON is the ribotoxic stress response (RSR), which involves autophosphorylation of protein kinase R (PKR) and downstream activation of MAP kinases including MAPK3/1. It is not known if these mycotoxins affect bovine reproduction. The objectives of present thesis were (1) to determine how and at what doses DON affects ovarian granulosa cell function and to elucidate its mechanism of action; and (2) to determine how and at what doses major mycotoxin DON and its metabolite DOM-1 affect theca cell function in cattle. The results are separated into three articles. In the first article the effects of DON on granulosa cells were explored; treatment with DON resulted in a significant inhibition of estradiol and progesterone (P4) secretion, and an increase in the proportion of apoptotic cells after 4 days of treatment. Western blot demonstrated significant upregulation of ERK1/2 and MAPK14 phosphorylation within 15-30 minutes of adding DON. We then determined the effect of DON on ERK1/2 target genes; EGR1 and FOS mRNA levels were transiently stimulated with maximum levels at 1 h of adding DON, whereas COX2 and GADD45B mRNA levels were upregulated but not until 24 h after DON treatment. In the second article, the effects of DON and DOM-1 on theca cells were assessed. Treatment with DOM-1 resulted in a dose-dependent inhibition of P4 and testosterone secretion, and an increase in the proportion of apoptotic cells, while DON inhibited P4 but did not alter testosterone secretion or the percentage of dead cells. Both ii DON and its metabolite were maximally effective at concentrations of 1 ng/ml (in contrast, the effects of DON on occur at 100ng/ml). Western blot demonstrated rapid phosphorylation of MAPK3/1, PKR and of JUN kinase after addition of DOM-1 or DON. Interestingly, phosphorylation of MAPK14 was significantly increased by DOM-1 but decreased by DON. The addition of a PKR inhibitor abrogated the ability of DON and DOM-1 to increase phosphorylation of MAPK3/1, and partly abrogated the inhibitory effect of DON on MAPK14 phosphorylation, however, the PKR inhibitor further increased the phosphorylation of MAPK14 caused by DOM-1. Together, these results suggest that DON activates the RSR in bovine granulosa and theca cells, and that theca cells are more sensitive than granulosa cells to the effects of DON. The data also demonstrate for the first time in any cell type the ability of DOM-1 to affect cell function and health.

Les « Facteurs de croissance des fibroblastes» (FGF) agissent comme des régulateurs locaux sur la qualité des follicules et sont connus pour promouvoir la prolifération des cellules de granulosa, réduire l’apoptose et la stéroïdogenèse. Parmi la sous-famille FGF8, FGF18 est une exception puisqu’il semblerait avoir une fonction pro-apoptotique alors que FGF8 n’a pas été jusqu’à présent rapporté comme altérant la viabilité des cellules de la granulosa. Ces deux ligands ont un mode d’activation similaire et il pourrait être proposé que toute la sous-famille FGF8 ait la même réponse. L’objectif de cette étude était de déterminer si FGF8 et FGF18 activaient la même réponse précoce de gènes dans des cultures de granulosa bovine. Pour répondre à cette question, nous avons cultivé des cellules de la granulosa dans du milieu de culture sans sérum pendant 5 jours. Le jour 5, les cellules ont été traitées avec FGF8 ou FGF18. Nous avons eu recours à une approche de « puce à ADN » afin d’identifier la réponse précoce de gènes induite par FGF8 et FGF18, et les données ont été confirmées par des PCRs en temps réel lors d’une expérience in vitro où les cellules de granulosa ont été traitées avec FGF8 et FGF18 pendant différents temps. L’analyse du puce à ADN a identifié 12 gènes surexprimés par FGF8, incluant SPRY2, NR4A1, XIRP1, BAMBI, EGR1, FOS et FOSL1. A l’inverse, FGF18 n’a régulé aucun gène de manière significative. Les analyses de PCR ont confirmé l’augmentation d’ARNm codant pour EGR1, EGR3, FOS, XIRP1, FOSL1, SPRY2, NR4A1 et BAMBI après 2 h de traitement. FGF18 a entrainé seulement une augmentation de l’expression de EGR1 après 2 h de traitement parmi tous les gènes testés. Ces résultats démontrent donc que FGF8 et FGF18, malgré leur similarité dans le mode d’activation de leurs récepteurs, agissent sur les cellules de la granulosa via différentes voies de signalisation. FGF8 et FGF18, sont donc tous les deux capables de stimuler l’expression de EGR1, mais les voies de signalisation induites par la suite divergent.
Fibroblast growth factors (FGF) act as local regulators of follicular health and are known to increase granulosa cell (GC) proliferation, reduce apoptosis and decrease steroidogenesis. One exception is FGF18, which appears to be a pro apoptotic member of the FGF8-subfamily while FGF8 has not been reported to alter GC health. These two ligands have similar activation patterns and it could be proposed that all FGF8-subfamilies would have the same response. The objective of this study was to determine if FGF8 and FGF18 activate the same early response genes in cultured bovine GC. To address this we cultured GC in serum free medium for five days. On day 5, cells were challenged with FGF8 or FGF18. We used a microarray approach to identify early response genes altered by FGF8 and FGF18, and data were confirmed by real-time PCR in an independent time-course experiment. Microarray identified 12 genes up-regulated by FGF8, including SPRY2, NR4A1, XIRP1, BAMBI, EGR1, FOS and FOSL1. In contrast FGF18 did not result in significant regulation of any gene. PCR analysis confirmed the stimulation of abundance of mRNA encoding EGR1, EGR3, FOS, XIRP1, FOSL1, SPRY2, NR4A1 and BAMBI after 2 hours of challenge. FGF18 resulted in an increase of EGR1 mRNA abundance at 2 h, but not of the other genes tested. These results demonstrate that FGF8 and FGF18, despite reportedly similar receptor activation patterns, act on granulosa cells through different intracellular pathways. Both FGF8 and FGF18 stimulate EGR1 expression, but thereafter their signaling pathways diverge.

Comprendre les événements paracriniens qui régulent la fertilité chez la vache est nécessaire non seulement en raison de l'importance agricole de cette espèce, mais aussi pour son utilisation potentielle comme modèle chez l’humain. L'oxyde nitrique (NO), un gaz de radicaux libres, a été impliqué dans la croissance folliculaire et l'ovulation chez les rongeurs et d'autres espèces, mais chez la vache c’est une énigme fascinante : le NO est produit par les cellules de la granulosa bovine et est régulé par la FSH, mais la présence et le profil d'expression des enzymes responsables de la synthèse de NO (NOS) dans les cellules de la granulosa tout au long de la croissance folliculaire ne sont pas claires. Les objectifs de cette thèse sont: (1) élucider le mécanisme de contrôle des NOS et les conséquences de la production d'oxyde nitrique pour le fonctionnement des cellules de la granulosa au cours du développement folliculaire chez la vache et (2) déterminer la régulation des NOS pendant la cascade ovulatoire induite par LH chez les cellules de la granulosa bovine et si l'activité des NOS pour l’expression des gènes critiques dans la cascade ovulatoire chez cette espèce. Les résultats sont séparés en 2 articles. Dans le premier article, la régulation de NOS2 dans les cellules de la granulosa bovine a été explorée. L'abondance des ARNm codant pour NOS2 a été stimulée par la FSH et l’IGF1 en augmentant l’estradiol, et un blocage de l'action de l’estradiol a conséquemment réduit les niveaux d'ARNm codant pour NOS2. De plus, l'inhibition de l'activité des NOS a augmenté l'apoptose dans les cellules de la granulosa in vitro. Dans le second article, il a été démontré que le pic de LH induit une activation des NOS dans les cellules de la granulosa, et que l'activité de NOS induit la production de NO, ce qui est essentiel pour l’expression des gènes critiques dans la cascade ovulatoire induite par LH comme EREG/AREG/PTGS2. Ensemble, les résultats présentés dans ces 2 articles suggèrent que les niveaux physiologiques d'activité des NOS peuvent contribuer à la croissance et la survie des cellules de la granulosa et indiquent également que NO peut être essentiel pour l'ovulation chez les bovins.
Understanding the paracrine events that regulate fertility in the cow is necessary not only because of the agricultural importance of this species, but also its potential use as a model for humans. Nitric oxide (NO), a free-radical gas, has been implicated in follicular growth and ovulation in rodents and other species, but the cow is an intriguing enigma: NO is produced by bovine granulosa cells and is regulated by FSH, but the presence and the expression pattern in granulosa cells of the enzymes responsible for NO synthesis (NOS) throughout follicular growth are unclear. The objectives of the present thesis were (1) to elucidate the mechanism of control of NOS and the consequences of nitric oxide production for granulosa cell function during follicle development in cattle; and (2) to determine the regulation of NOS during the LH-induced ovulatory cascade in bovine granulosa cells and whether NOS activity is critical for the ovulatory cascade in this species. The results are separated in 2 articles. In the first article, the regulation of NOS2 in bovine granulosa cells was explored. Abundance of mRNA encoding NOS2 was stimulated by FSH and IGF1 through increased estradiol, and a blockade of estradiol action consequently lowered NOS2 mRNA levels. Further, inhibition of NOS activity increased apoptosis in granulosa cells in vitro. In the second article, it was demonstrated that the LH surge induces NOS activation in granulosa cells, and that NOS activity induces the production of NO, which is essential for EREG/AREG/PTGS2 expression, critical genes in the LH-induced ovulatory cascade. Together, the results presented in these 2 articles suggest that physiological levels of NOS activity may contribute to growth and survival of granulosa cells, and also indicate that NO may be essential for ovulation in cattle.

Au cours des dernières années, une sélection génétique importante a été faite pour améliorer la production de lait des bovins, ceci au détriment des performances reproductives. Cette diminution de performance n’a cependant pas été rapportée chez la génisse présentant un même potentiel génétique. Cette immense production de lait et les changements métaboliques qui l’accompagnent ont donc un impact négatif sur l’efficacité reproductive des vaches laitières qui subissent un stress métabolique supérieur à celui des génisses. Le but de l’étude était d’acquérir une meilleure connaissance des différences moléculaires et métaboliques entre ces deux groupes d’animaux pour amener à une meilleure compréhension de la pathogenèse de l’infertilité chez la vache laitière. Pour ce faire, les vagues folliculaires de vaches en lactation (30-50 jours en lait; N = 12) et de génisses (N = 10) ont été synchronisées par ablation écho guidée des follicules et par traitement hormonal avec injection de prostaglandine et insertion d’un implant de progestérone. L’aspiration du liquide folliculaire et des cellules de la granulosa du follicule dominant a été faite au jour 6. Les paramètres métaboliques mesurés chez les animaux à partir de prises de sang, faites au jour 6, confirment un plus grand stress métabolique chez la vache, les niveaux de BHBA, acides biliaires et cholestérol étant plus élevés et le niveau de glucose plus bas chez celles-ci. Un total de six échantillons a été utilisé pour le séquençage d’ARN et des analyses bio-informatiques ont été effectuées. Plusieurs gènes et voies de signalisation ont présenté des différences entre les deux groupes d’animaux incluant le cycle cellulaire et la production d’hormones. Une confirmation des résultats par PCR en temps réel a été faite, mais la grande variation intragroupe a nui à l’obtention de résultats significatifs. Conjointement, une culture primaire de cellules de la granulosa a été réalisée pour évaluer l’effet des acides biliaires sur la stéroïdogenèse suite à la détection d’une plus grande quantité de ceux-ci chez la vache laitière. La présence d’acide biliaire dans la culture cellulaire cause une diminution de l’accumulation d’estradiol ainsi que de l’expression des gènes CYP19A1 et CYP11A1. Les résultats présentés dans ce mémoire indiquent une différence potentielle au niveau métabolique et moléculaire des follicules dominants entre la vache laitière et la génisse pouvant avoir une responsabilité dans la diminution de l’efficacité reproductive observée chez la vache laitière.
Over the last fifty or more years, genetic selection has been employed to improve milk production in dairy cattle. This selection was made at the expense of reproductive performance. The observed decrease in fertility does not occur in heifers with the same genetic merit. The enormous milk production and the metabolic challenge that accompany it have a negative impact on the reproductive efficiency due to the metabolic stress of lactation. The purpose of the study was to gain a better knowledge of the molecular and metabolic difference between the two groups of animals in order to better understand the pathogenesis of infertility in dairy cows. To do this, the follicular wave of twelve lactating cows (30-50 days in milk; N = 12) and ten heifers (N = 10) were synchronized by ultrasound guided follicle ablation and by hormonal treatment with injection of prostaglandin-F2α and insertion of a progesterone implant. Follicular fluid and granulosa cells of the dominant follicle were aspirated on day 6. The metabolic indicators BHBA, total bile acids, cholesterol and glucose, were measured in the animals from the blood samples also taken on day 6 confirming greater metabolic stress in the cows when compared to the heifers. A total of six samples were used for RNA sequencing and bioinformatics analyses were performed. Several genes and signaling and cellular function pathways were shown to differ between the two groups of animals, including the cell cycle signaling pathway and hormone production pathway. A confirmation of the results by real-time PCR was undertaken, but the great intragroup variation obviated significant results. In the second set of experiments, primary culture of granulosa cells was conducted to evaluate the effect of bile acids on steroidogenesis to further explore the larger amount of the bile acids in the dairy cows when compared to heifers. The results demonstrate a difference in the metabolic status of the animals; BHBA, total bile acids and cholesterol being higher and glucose being lower in the dairy cow relative to the heifer. Presence of bile acids in the granulosa cell culture caused a decrease in expression of CYP19A1, CYP11A1 and estradiol accumulation. The differences at the metabolic and molecular level of the dominant follicles between dairy cows and heifers may be implicated in the reduced reproductive efficiency of the dairy cows.