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This dissertation focused on two classes of molecules implicated in processes of implantation and placentation in sheep and pigs. Study one examined the temporal/spatial distribution of several Small Integrin-Binding Ligand, N-Linked Glycoprotein (SIBLING) family members in cyclic and pregnant ovine uterus. Studies two and three evaluated the relationships between progesterone (P4) and estrogen (E2) and their receptors (PGR and ESR1, respectively) on FGF7 mRNA expression within the endometrium and placenta of pigs. Study one showed that dentin sialophosphoprotein (DSPP) was first detected in luminal epithelium (LE) of Day 15 cyclic and pregnant sheep. Stromal expression of DSPP was first detected on Day 20 of pregnancy in stratum compactum and remained prominent in stroma through Day 120. Stromal DSPP protein was positively influenced by the conceptus based upon analysis of a unilaterally pregnant ewe model system. Immunoreactive dentin matrix protein 1 (DMP1), matrix extracellular phospoglycoprotein (MEPE) were localized to the stroma of cyclic and pregnant sheep, however, these proteins appeared to be constitutively expressed. BSP was not detected in ovine endometrium. Study two determined the effects of E2, P4, P4+E2, P4+the PGR antagonist (ZK137, 316), and P4+E2+ZK on FGF7 mRNA expression in uterine LE of ovariectomized pigs. Results indicate that P4 is permissive to FGF7 mRNA expression by down-regulating PGR in LE; P4 stimulates PGR-positive uterine stromal cells to release an as yet unidentified progestamedin that induces FGF7 mRNA expression by LE; E2 and P4 can induce FGF7 mRNA in the absence of PGR rendered nonfunctional by ZK. Study three showed the expression of ESR1, PGR and FGF7 in the uterine and placental tissue of pregnant pigs from Day 20 through 85. Results reveal a positive correlation between stromal cell expression of PGR and FGF7 mRNA which suggests that P4 is permissive to FGF7 mRNA expression by down-regulating PGR in LE. FGF7 mRNA in later pregnancy is maintained by the release of progestamedin from PGR-positive stromal cells. A novel finding was the presence of ESR1 in porcine placenta on Days 20 through Day 85 of pregnancy suggesting that E2 may play important roles in the placental biology of the pig.

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Le rôle des progestatifs et du récepteur de la progestérone (PR) dans la carcinogénèse mammaire est maintenant établi. Le PR est exprimé sous deux isoformes PRA et PRB dont l'expression est équimolaire mais qui différent par leur activité transcriptionnelle des gènes cibles. La surexpression de PRA est associée à un pronostic défavorable du cancer mammaire, et est observée chez les femmes à haut risque génétique de cancer du sein. L'utilisation d'antagonistes pour inhiber l'activité de PR pourrait constituer une stratégie thérapeutique potentielle. L'objectif de cette thèse a été d'évaluer les effets de l'ulipristal acétate (UPA), un anti-progestatif, sur la tumorigenèse mammaire dans deux modèles d'études complémentaires. In vitro, dans la lignée cancéreuse mammaire bi-inductible (MDA-iPRAB) développée au laboratoire. Nous avons démontré qu'UPA inhibe la prolifération cellulaire induite par la progestérone en présence de PRA. L'expression de certains gènes clés de la tumorigenèse mammaire, cibles de PRA a été étudiée. Une corrélation entre avec la prolifération cellulaire et l'expression du facteur anti-apoptotique BCL2-L1 (messager et protéine) a été démontrée. L'activation transcriptionnelle, dépendante de la progestérone et inhibée par l'UPA, s'accompagne d'un recrutement spécifique et sélectif de PRA sur des séquences régulatrices du gène BCL2-L1, comme le démontrent les expériences de ChIP. In vivo, nous avons évalué l'efficacité anti-tumorale de l'UPA ainsi qu'une nouvelle classe d'antagoniste sélective et passive de PR, les « APRns » dans un modèle murin de xénogreffe de tumeur mammaire humaine HBCx34. UPA ralentit la croissance tumorale, diminue la prolifération cellulaire (Ki67, PCNA) et inhibe l'expression de BCL2-L1. L'ensemble de nos résultats démontrent une action antiproliférative et apoptotique de l'UPA ce qui suggère une utilisation potentielle d'antagonistes de PR dans la prise en charge du cancer du sein. / The role of progestins and the progesterone receptor (PR) in breast tumorigenesis has now been elucidated. PR is expressed as two isoforms PRA and PRB, differing by their structure and their transcriptional activity. They are often co-expressed in normal breast tissue but the predominance of PRA expression is associated with mammary carcinogenesis, a bad prognosis and an endocrine-resistance. Antiprogestins inhibit mammary tumor growth in several experimental models. The aim of this thesis was to investigate the effect of a well-known selective PR modulator (SPRM), Ulipristal acetate (UPA) in two complementary breast cancer models. In vitro, we used the newly established bi-inducible breast cancer cell line “MDA-iPRAB”, and demonstrated an anti-proliferative activity of UPA in progesterone-dependent, PRA-specific MDA-iPRAB cell proliferation. We also studied the expression of PRA-target genes involved in mammary tumorigenesis, and showed a correlation between cell proliferation and the expression of the anti-apoptotic factor BCL2-L1 (transcript and protein). The transcriptional activation of BCL2-L1 was stimulated by the progesterone in MDA-iPRA cell line, inhibited by UPA and was associated with a specific and selective recruitment of PRA to BCL2-L1 regulatory regions (ChIP assays). In vivo, we evaluated the anti-tumoral activity of UPA and a new class of selective and passive progesterone receptor antagonist ‘APRns' in patient-derived breast cancer xenograft in nude mice. UPA slowed down mammary tumor growth, decreased cell proliferation (Ki67, PCNA) and inhibited the BCL2-L1 expression. Further investigation are needed to determine the actions of APRns. In summary, UPA has an antiproliferative and a pro-apoptotic activities, which suggests a potential interest of UPA in breast cancer endocrine therapy.

Neutrophils infiltrate myometrium and decidual tissue prior to parturition. Activated neutrophils release reactive oxygen species (ROS) and tumor necrosis factor α (TNFα), which might increase expression of pro-labor genes such as matrix metalloproteinase-1 (MMP-1), progesterone receptor (PR) A/B ratio, and cause demethylation of DNA. These changes might cause labor. Decidual tissue was obtained from consented, healthy women at term (37+ weeks of gestation) not in labor (no contractions, without cervical effacement), term labor and preterm labor (under 37 weeks of pregnancy). Decidual and myometrial cells in culture were treated with (1) ROS, (2) TNFα, or (3) 5-aza-2’-deoxycytidine. Total RNA was extracted, converted to cDNA and evaluated by qRT-PCR for MMP-1, PR-A+B and PR-B. TNFα increased MMP-1 by 17 fold in decidual cells and more than 12 fold in myometrial cells. PR-A/B was increased by 5.6 fold in decidua. ROS up-regulated MMP-1 by 6 fold and elevated the PR-A/B ratio by 4.5 fold in decidual tissue. DNA demethylation increased MMP-1 by about 4 and 11 fold in decidual and myometrium, respectively. The PR-A/B ratio was increased by 4 fold in decidua and the PR-B was decreased by 40% in the myometrium due to DNA demethylation. Decidual tissue in preterm labor showed a 7-fold increase in MMP-1 over term laboring and over a 15-fold increase over term not in labor tissue. In conclusion, MMP-1 expression and PR-A/B ratio was increased by neutrophil products possibly through a mechanism of DNA methylation in decidua and myometrium. Preterm decidua showed a dramatic increase in MMP-1 over normal labor tissue. TNFα and ROS increased expression of MMP-1 to possibly initiate parturition. These data might help explain mechanisms responsible for preterm labor unrelated to infection or premature rupture of membranes.

Made available in DSpace on 2016-08-10T10:39:22Z (GMT). No. of bitstreams: 1 IASMIM RIBEIRO DA COSTA.pdf: 1307730 bytes, checksum: f0fcc7daee903a60a0822fcd55c47c64 (MD5) Previous issue date: 2010-06-09 / Endometriosis is defined by the appearance of foci of endometrial tissue with glandular features and / or stromal identical to the uterine cavity at locations other than the endometrium, showing a strong genetic component. It focuses primarily on women of reproductive age and may be related to infertility in 30% to 40% of the cases. It's a histological diagnosis of pathology, which must be confirmed the clinical suspicion and the macroscopic findings found in surgery. It can affect several organs, and because of it is called multi-systemic. The exaggerated expression of estrogen receptors and progesterone receptor defects are consequences of the abnormality of the progestational effect on the endometrium and ectopic topic. Changes in its function may thus facilitate the emergence of the disease because, in the broad sense, fails to antagonize the proliferative effects of estrogens. Recently, several polymorphisms of the progesterone receptor have been described. Among them stands out PROGINS polymorphism Alu consisting of a insertion 306 bp in the intron G between the exon 7 and 8 of progesterone receptor gene in humans. This study aims to determine the possible correlation between endometriosis and polymorphisms of the Progesterone Receptor Gene (PROGINS). The endometriosis group consisted of 54 patients from a referral center for infertility laparoscopy-video and infertility of Goiania (FÉRTILE). The control group included 45 women without history of endometriosis by anaminesis. Genotypes for PROGINS polymorphism (A1/A1, A1/A2 and A2/A2) were determined by PCR. The frequency of polymorphic genotypes (A1/A2 and A2/A2) is two times higher in patients with endometriosis (33,3%) than in the control group (15,5%). Statistical significance was found indicating an association between the polymorphism PROGINS and pathology endometriosis (P = 0,0426). / A endometriose é definida como aparecimento de focos de tecido endometrial com características glandulares e/ou estromais idênticos aos da cavidade uterina em outras localizações, que não o endométrio, apresentando um forte componente genético. Incide principalmente em mulheres em idade reprodutiva, podendo estar relacionada com infertilidade em 30% a 40% dos casos. É uma patologia de diagnóstico histológico, devendo o mesmo, confirmar as suspeitas clínicas e os achados macroscópicos encontrados em cirurgias. Pode afetar vários órgãos, sendo por isso mesmo denominada atualmente de multi-sistêmica. A expressão exagerada de receptores de estrogênio e defeitos no receptor de progesterona são consequências da anormalidade do efeito progestacional sobre o endométrio tópico e ectópico. Alterações da sua função podem, portanto, facilitar o aparecimento da moléstia porque, no sentido amplo, deixa de antagonizar os efeitos proliferativos dos estrogênios. Recentemente, vários polimorfismos do receptor de progesterona têm sido descritos. Dentre eles destaca-se o polimorfismo PROGINS que consiste em uma inserção Alu de 306 pb no íntron G entre o exon 7 e 8 do gene do receptor de progesterona humano. Este estudo tem como objetivo verificar a possível correlação entre a endometriose e o Polimorfismo do Gene do Receptor de Progesterona (PROGINS). O grupo endometriose foi composto por 54 pacientes de um centro de referência em videolaparoscopia e infertilidade de Goiânia (FÉRTILE). O grupo controle incluiu 45 mulheres sem diagnóstico de endometriose por anamnese. Os genótipos para o polimorfismo PROGINS (A1/A1, A1/A2 e A2/A2) foram determinados por PCR. A frequência dos genótipos polimórficos (A1/A2 e A2/A2) é duas vezes maior nas pacientes com endometriose (33,3%) do que no grupo controle (15,5%). Houve significância estatística que indicasse a associação entre o polimorfismo PROGINS e a patologia endometriose (P = 0,0426).

Les hormones ovariennes sont impliquées dans le développement du cancer du sein des femmes porteuses de mutations du gène BRCA1. L’étude du tissu mammaire normal de femmes mutées et non mutées nous a permis de montrer des altérations de la signalisation de l’estradiol et la progestérone dans un modèle de greffes chez la souris. Ces dérégulations concernent l’activité proliférative et l’expression des récepteurs hormonaux, et sont hétérogènes d’une patiente mutée à l’autre. De plus, une diminution de l’expression de la forme phosphorylée en sérine 211 du récepteur des glucocorticoïdes a été mise en évidence dans le tissu muté BRCA1. Ces observations posent la question de la place d’unantiprogestatif/anti-glucocorticoïde tel que l’ulipristal acétate (UPA) dans le traitement préventif des femmes mutées pour BRCA1. Les effets de l’UPA ont été caractérisés in vitro dans les cellules mammaires normales pour préciser les conséquences de son utilisation sur le sein. Son action a ensuite été examinée sur les xénogreffes de sein de patientes mutées et non mutées. Nous montrons que l’UPA est capable d’inhiber la prolifération du tissu muté lorsqu’elle est induite par la progestérone. Ce résultat encourage son utilisation dans la prévention tumorale mais indique une spécificité d’action en fonction des dérégulations hormonales associées au tissu muté. / Ovarian hormones, estradiol and progesterone, are known to promote breast carcinogenesis in BRCA1 mutated women. Using normal mammary gland xenografted in mice, we found that hormonal responses were deregulated in a heterogeneous fashion within BRCA1 mutation carriers. These observations raise the question of a potential use ofantiprogestin treatment as ulipristal acetate (UPA) to prevent breast tumorigenesis in BRCA1mutated women. Studies with this experimental model and epithelial normal breast cells in vitro, revealed that UPA alone had no proliferative activity on breast tissue but was efficient to inhibit proliferative activity when induced by progesterone treatment in mutated tissue. UPAcould be a promising treatment to prevent breast tumorigenesis for some mutated women. In addition, we observed a severe decrease of the phosphorylated Serine 211 glucocorticoid receptor isoform in BRCA1 carriers compared to non mutated tissue. This observation suggests that the glucocorticoid receptor expression and activation should be taken intoaccount in BRCA1 carriers.