The control of reproductive cycles in higher primates is largely dependent on negative and positive feedback mechanisms by both steroidal and non-steroidal substances of the ovaries which regulate the function of hypothalamo-pituitary system. To gain insights into the role of INH A, the non steroidal ovarian hormone in the feedback control of pituitary FSH secretion, studies were conducted to examine the interrelationships of hormones throughout the menstrual cycle of the bonnet macaque. The findings of chapter II provide a detailed description of endocrine hormone profile during the menstrual cycle of the bonnet macaques with special attention to the feedback role of INH A on pituitary FSH secretion. To characterize the endocrine profile of different hormones, both ovarian (E2, P4, INH A) and pituitary (FSH, LH) hormones were measured daily for more than 40 days. To further examine the site of secretion of INH A and its relationship with pituitary FSH dynamics, surgical lutectomy and pharmacological induction of luteolysis employing the third generation GnRH R antagonist, Cetrorelix (CET) studies were carried out in the subsequent experiments. The results obtained from these studies suggest that INH A and P4 secreted from the CL during luteal phase regulate pituitary FSH secretion. The selective rise in FSH observed during the late menstrual cycle and during menstruation (referred to as luteo-follicular transition), as has been reported previously in higher primates, considered necessary for initiation of follicular growth and recruitment of follicles for ensuing menstrual cycle was characterized in the monkey. Surgical lutectomy and induction of luteolysis by CET experiments suggested that increased GnRH secretion is essential for this selective rise in FSH following withdrawal of inhibition by P4 and INH A. In clinical cases of reproductive ageing, the shortened follicular phase in human females has been identified to be the result of occurrence of early onset of FSH rise during the luteal-follicular transition period. The cause(s) of declining fertility with age in women who still have regular menstrual cycles are not clear, but issues of relationship between dysregulation of selective FSH rise in the late luteal phase and associated infertility could be examined using bonnet monkey as a model system. INH A is secreted in significant quantities by CL in higher primates and the feto placental unit suggesting its importance during fertility and pregnancy. Apart from the negative feedback regulation of pituitary FSH secretion, the complete repertoire of actions of this hormone during pregnancy is yet to be fully understood. The data presented in this thesis is the first comprehensive report showing the endocrine hormone profile of gonadotropins and ovarian hormones including INH A throughout the menstrual cycle of the bonnet macaque. The characterization of INH A profile in bonnet monkey will be of significant value for studies examining the role of INH A in higher primates. Dimeric inhibin has been suggested to be important for regulation of fertility and reproductive functions. Also, inhibin-α (one of the subunits of the dimeric protein) knock out mice model has provided convincing evidence that it acts as a tumour suppressor. A great deal of new information has been generated in recent years regarding the potential clinical usefulness of monitoring inhibin levels in blood and biological fluids in gynaecological diseases, pathological pregnancies and other disorders. Emerging clinical roles of inhibin have made INH A an important candidate molecule to study its molecular regulation. The results presented in chapter II suggested that LH regulates luteal INH A secretion (induction of luteolysis by CET administration experiment). As a first step towards understanding molecular regulation of inhibin-α expression in the macaque CL, in silico promoter analysis of macaque inhibin-α was performed and it revealed several transcriptional factor binding sites that were conserved across species. In rats FSH up regulates while preovulatory LH surge suppresses inhibin-α mRNA expression in the ovary and this suppression has been suggested to be necessary for occurrence of secondary FSH surge during metestrus. To address differential regulation of inhibin-α by LH and FSH in rat ovary during the periovulatory period, studies employing immature rats were carried out and the results are presented in chapter III. The results suggest that immature rat ovaries respond to exogenous gonadotropins in terms of LH signaling (cAMP production), luteinization (P4 production) and as well induction of ICER expression required for repression of inhibin-α subunit expression. PDE4 inhibitor (rolipram) treatment enhanced the ovarian cAMP concentrations suggesting that PDE4 play a major role in controlling intraovarian cAMP concentrations in rat ovaries. However increased cAMP concentrations did not appear to up regulate the ICER expression at the time point examined in this study. In higher primates time course of second FSH surge and continued synthesis and secretion of INH A in the CL are different from non primate species. In the monkey, the second FSH rise occurs during the late luteal phase and experiments have been carried out to examine the regulation of inhibin-α subunit expression by ICER. Expressions of ICER (mRNA/protein) and INH A were examined during different stages of CL and the results indicated no clear inverse relationship between the ICER and inhibin-α mRNAs. With no conclusive role for the ICER in regulating luteal inhibin-α observed in the study, the role of transcriptional activators in the regulation of inhibin-α like GATA4, SF-1, β-catenin were further examined. Since luteal INH A secretion was dependent on pituitary LH as determined earlier in chapter II, expressions of transcriptional activators were examined in CL of different stages and also during induced luteolysis and the results are described in chapter IV. In conclusion, our results indicate cross talk between WNT, cAMP and P38 MAP kinase signaling pathways in the regulation of luteal INH A secretion. The pituitary gonadotropin, LH, is the primary luteotropin in primate species acting to maintain the structure and function of the CL during the menstrual cycle. However whether the actions of LH are direct or mediated by local factors such as P4 remain unknown. Moreover, P4 secretion which is dominant during luteal phase has any role in regulating CL structure and function is not clearly defined. To address these and issues concerning P4 actions, initially, experiments were performed in the rat model to study the importance of P4 in the regulation of ovarian functions. An antiprogestin, RU486, was employed as a tool to uncover the PR regulated pathways during ovulation in rats and the findings are presented in the chapter V. The results indicated that blockade of PR action by RU486 during gonadotropin-induced superovulation resulted in inhibition of follicular rupture and ovulation in immature rats. Further to understand the downstream effectors of PR action, and to identify the candidate target genes of PR activation, semi-quantitative RT-PCR and western blot analyses were performed. The results obtained indicated that betacellulin, a member of EGF family and MMP-9 a proteolytic enzyme, were markedly repressed in response to RU486 treatment in rat ovaries. Also, the down stream pathway of EGF signaling leading to activation of ERK was markedly repressed in RU486 treated ovaries. It was next examined what role the P4/PR system has in the regulation of CL structure and function. Surprisingly, PR expression is absent in CL of rats, while it is present in higher primates. Experiments were carried out to examine intracrine actions of P4 in the regulation of CL structure and function in monkeys. The recently reported model system of induced luteolysis yet capable of responsive to trophic support from the laboratory provided an ideal opportunity to examine direct effects of P4 on structure and function of CL in the monkey. A series of pilot experiments were carried out in monkeys experiencing summer amenorrhea, to determine dose and mode of administration of exogeneous P4 to simulate mid luteal phase circulating P4 concentrations in monkeys subjected to induced luteolysis. Based on the results of pilot experiments, implantation of Alzet pumps containing 97.5mg of P4 was selected for maintaining mid luteal phase P4 concentrations. The microarray data of induced luteolysis previously deposited by the laboratory in NGBI’s gene expression omnibus were mined for identification and validation of differentially expressed genes of PR and its target genes following LH depletion and LH replacement experiments. Expressions of PR, PR cofactors and expressions of PR downstream target genes through out the luteal phase and in CL from day1 of menses were also examined. Analysis of expressions of genes revealed that of the 45 genes identified to be regulated by LH treatment, 4 genes were found to be responsive to P4, and 14 were identified to be responsive to both P4 and LH. Morphology of CL tissue sections revealed that P4 treatment appeared to have reversed the induced-luteolysis changes. In another experiment, implantation of P4 during late luteal phase (i.e., the period of declining P4 concentrations) for 24h caused changes in expressions of genes associated with tissue remodeling and morphology of luteal cells. Taken together, the results suggest that induced luteolysis plus P4 replacement model is suitable for assessing the effects of P4 on CL function. The results also suggest that CL could serve as target tissue for examining the genomic and non genomic actions of P4. In summary, studies carried out in the present thesis provides a comprehensive endocrine hormone profile throughout the menstrual cycle of the bonnet monkey with special emphasis on time course of INH A and FSH secretion which is very useful for future investigations. Studies have been carried out in rats and monkeys with different experimental model systems to address molecular mechanisms underlying inhibin-α regulation in the ovary in general and CL in particular. Experimental findings in monkeys could help elucidate the underlying molecular nature of CL functionality and extrapolate to understand luteal insufficiency and infertility producing conditions in humans. Also different model systems have been validated to examine the actions of P4/PR system in rats and monkeys and more importantly to address the direct effects of P4 upon monkey CL structure and function were established. Future investigations based on findings of these studies should help clarify relative roles for LH and P4 during maintenance of CL function and luteolysis.

Esta tesis se interesa por distinguir entre los efectos directos de los receptores nucleares y aquellos mediados por las rutas de transducción de señales en la transcripción de genes en respuesta a hormona y proliferación celular. Para esto, nosotros hemos expresado establemente en una línea celular T47Dy desprovista de PR, formas variantes marcadas de la isoforma B del PR en regiones involucradas bien en la unión al DNA(PRB-DBD), en su habilidad para interaccionar con ER y activar la cascada c-Src/Erk (PRB-ERID), o la incapacidad de reclutar coactivadores. La expresión génica en respuesta a progesterona en líneas celulares expresando los PRB salvaje y mutantes ha sido estudiada un microarray con 750 genes de cáncer de mama. Los resultados definen conjuntos de genes regulados en respuesta a hormona por los diferentes modos de acción del PRB, también genes dónde las rutas nucleares y no genómicas cooperan. Por último, se ha centrado la atención en la participación del gen Ciclina D1 (CCND1) en proliferación celular por hormona, el modo de acción del PR en su activación y el análisis de las regiones promotoras dónde PR se une. / This these is interested on distinguishing between direct effects of nuclear receptors and those mediated by signal transduction pathways on transcription of hormone-responsive genes and cell proliferation. For this, it stablies expressed in the PR-negative T47Dy breast cancer cell line, tagged forms of the PRB mutated at regions involved either in DNA binding, in its ability to interact with ER and activate the c-Src/Erk cascade, or the recruitment of coactivators. Gene expression in response to progestins in cell lines expressing wild type or mutant PRB has been studied by a 750 genes-containing breast cancer customized cDNA microarray. Our results define the subsets of hormoneresponsive genes regulated by the different modes of action of PRB, as well as genes where the nuclear and nongenomic pathways of PRB cooperate. Finally, it has focused the attention on the involvement of Cyclin D1 gene (CCND1) activation by hormone on cell proliferation, the mode of action of PR on its activation and the analysis of promoter regions where PR binds.

Hormone-dependent cancers (HDCs), such as those of the prostate, ovary, breast and endometrium, share characteristics that indicate similar underlying mechanisms of carcinogenesis. Through steroid hormone signalling on "down-stream" target genes, the growth, development and progression of HDCs are regulated. One such family of target genes, highly expressed in HDCs and regulated by steroid hormones, are the tissue kallikreins (KLKs). The KLKs are a multigene family of serine proteases involved in physiological processes such as blood pressure regulation, inflammation, and tumour development and progression via the hydrolysis of specific substrates. Although the KLK gene family is clearly implicated in tumourigenesis, the precise roles played by these genes are largely unknown. Additionally, except for the androgen-responsive genes, KLK2 and KLK3, the mechanisms underlying their hormonal regulation in HDCs are yet to be identified. The initial focus of this thesis was to examine the regulation of the kallikreins, KLK1 and KLK4, by estradiol and progesterone in endometrial and breast cancer cell lines. From these studies, progesterone clearly regulated KLK4 expression in T47D cells and therefore, the focus of the remaining studies was to further examine this regulation at the transcriptional level. An overview of the results obtained is detailed below. Human K1 and hK4 protein levels were increased by 10 nmol/L estradiol benzoate, progesterone, or a combination of the two, over 48 hours in the endometrial cancer cell line, KLE. However, these same treatments resulted in no change in KLK1 gene or hK1 protein levels in the endometrial cancer cell lines, HEC1A or HEC1B (only hK1 analysed). Progesterone treatment (0-100 nmol/L) over 24 hours resulted in a clear increase in KLK4 mRNA at the 10 nmol/L dose in the breast cancer cell line, T47D. Additionally, treatment of T47D cells with 10 nmol/L progesterone over 0-48 hr, resulted in the rapid expression of the hK4 protein at 2 hr which was sustained for 24 hr. Further analysis of this latter progesterone regulation with the antiprogesterone, RU486, over 24 hours, resulted in an observable decrease in hK4 levels at 1 µmol/L RU486. Although the estrogen and progesterone regulation of the hK1 protein was not further analysed, the data obtained for hK4 regulation in T47D cell lines, supported the premise that this gene was progesterone-responsive. The rapid expression of hK4 protein by progesterone at two hours suggests that KLK4 transcription is directly coupled to progesterone regulation, perhaps through progesterone receptor (PR) binding to progesterone-responsive regions within the KLK4 promoter or far "up-stream" regions. Thus, the following further studies were performed. To test this hypothesis, the transcription initiation site (TIS) and 5' flanking regions of the KLK4 gene in T47D cells were interrogated. Primer extension and 5' RACE identified the TIS 78 bp 5' of the putative ATG site for translation as identified by Korkmaz et al. (2001). This KLK4 gene transcript consists of only four exons, and thus excludes the pre/pro signal peptide. Although a TATA-box is not present within -25 to -30 bp 5' of the identified TIS, a number of consensus binding motifs for Sp1 and estrogen receptor half-sites were identified. It is possible that the Sp1 sites are involved in the basal levels of transcription for this gene. Additionally, a putative progesterone response element (PRE) was identified in the far "up-stream" regions of the KLK4 gene. Basal levels of transcription were observed within the KLK4 proximal promoter region when coupled to a luciferase reporter gene and transfected into T47D cell lines. Additionally, the KLK4 proximal promoter region did not induce the luciferase reporter gene expression when progesterone was added to the system, however, estradiol was inhibitory for luciferase gene expression. This suggests that the proximal promoter region of the KLK4 gene could contain functional EREs but not PREs. In keeping with this hypothesis, some ER half-sites were identified, but PR sites were not obvious within this region. The identified PRE in the far "up-stream" region of the KLK4 gene assembled the progesterone receptor in vitro, and in vivo, as assessed by electromobility shift assays and chromatin immunoprecipitation assays (EMSAs and ChIPs), respectively. The binding of the PR to the KLK4 PRE was successfully competed out by a PR antibody and not by an androgen receptor antibody, and thus confirms the specificity of the KLK4 PRE-PR complex. Additionally, the PR was recruited and assembled onto and off the progesterone-responsive KLK4 region in a cyclic fashion. Thus, these data strongly suggest that the PR represents one of the core components of a transcription complex for the KLK4 gene, and presumably also contributes to the expression of this gene. Moreover, these data suggest a functional coordination between the PR and the KLK4 progesterone-responsive region in T47D cells, and thus, provide a model system to further study these events in vivo.

Made available in DSpace on 2015-07-22T20:50:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-26. Added 1 bitstream(s) on 2015-08-11T03:25:58Z : No. of bitstreams: 1 Publico-11853.pdf: 1847638 bytes, checksum: 58d741297f37fd108b68cf301ea6653b (MD5) / Objetivo: Avaliar a presença do polimorfismo genético do receptor da progesterona (PROGINS) bem como as variáveis clínicas e epidemiológicas de risco para câncer de endométrio em mulheres com pólipos endometriais na pós-menopausa. Casuística e Métodos: Comparou-se em estudo caso-controle 154 mulheres menopausadas com pólipos endometriais benignos e 400 controles normais na pós-menopausa, quanto à presença do PROGINS, por meio da Reação em Cadeia da Polimerase (PCR). O grupo de pólipos endometriais foi comparado a 118 pacientes do grupo controle no tocante às variáveis clínicas e epidemiológicas de risco para câncer de endométrio. Estas variáveis foram também comparadas entre os pólipos benignos e malignos. Resultados: A comparação entre o grupo de pólipos benignos e o grupo controle mostrou significância estatística (p<0,05) para as varáveis: idade (média de 61,7 x 57,5 anos), raça não-branca (44,8% x 22,9%), anos da menopausa (média de 12,9 x 9,2 anos), paridade (média de 4,5 x 3,4 filhos), uso de tamoxifeno (5,2% x 0%), hipertensão arterial (54,5% x 29,7%) e antecedente de câncer de mama (10,4% x 0,8%) respectivamente. Após o ajuste para a idade, permaneceram com significância estatística, apenas a paridade (OR=1,13), a hipertensão arterial (OR=2,19) e o antecedente de câncer de mama (OR=14,44). Seis casos (3,75%), foram diagnosticados como pólipos malignos, nestes casos, sangramento na pós-menopausa e o tamanho grande do pólipo estiveram sempre presentes, enquando que nos pólipos benignos esta frequência foi de 23,4% para sangramento e 54,5% para pólipo grande. A hipertensão arterial foi bem mais frequente no grupo de pólipos malignos, 83,3% x 54,5% nos pólipos benignos. Não houve diferença estatisticamente significante entre os grupos quanto à presença do PROGINS, sendo no grupo de pólipos benignos a distribuição entre homozigoto selvagem, heterozigoto e homozigoto mutado de 79,9%, 19,5% e 0,6% respectivamente. No grupo controle (N=400) esta distribuição foi de 78,8%, 20,8% e 0,5% respectivamente. Conclusões: A presença do PROGINS não mostrou associação significativa com pólipos endometriais. As variáveis epidemiológicas significantemente associadas à presença de pólipos endometriais, após o ajuste para idade, foram a paridade, hipertensão arterial e o antecedente de câncer de mama (implícito o uso de tamoxifeno), além da idade mais avançada. Em nosso estudo, pólipos endometriais malignos estiveram sempre associados à presença de sangramento na pós-menopausa e tamanho grande do pólipo, sendo a hipertensão arterial achado bastante frequente. / Purpose: To evaluate the genetic polymorphism of the progesterone receptor (PROGINS), as well as clinical and epidemiological risk factors for endometrial cancer in postmenopausal women with endometrial polyps. Methods: A case control study was designed with 154 postmenopausal women with endometrial polyps, compared to a normal control group of 400 postmenopausal women. The genotyping of PROGINS polymorphism was determined by polymerase chain reaction. The group of polyps was compared to 118 normal postmenopausal controls regarding clinical and epidemiological variables. These variables were also compared between benign and malignant endometrial polyps. Results: The epidemiological variables among the group of endometrial polyps and normal control, showed statistical significance (p<0,05) for age: media of 61,7 and 57,5 years, ethnicity non-white 44,8% and 22,9%, time since menopause media of 12,9 and 9,2 years, parity media of 4,5 and 3,4 sons, tamoxifen use 5,2% and 0%, hypertension 54,5% and 29,7% and history of breast cancer 10,4% and 0,8% respectively. After age adjust, statistical significance, remained only for parity (OR=1,13), hypertension (OR=2,19) and history of breast cancer (OR=14,44). Postmenopausal bleeding and large polyps were present in all cases of malignancy. Hypertension was also very frequent in malignant polyps (83,3% and 54,5% respectively). The presence of PROGINS had no statistical significance between the group of polyps and the normal control (N=400). The presence of wild homozygosis genotype, heterozygosis and mutant homozygosis was 79,9%, 19,5% and 0,6% respectively for the polyp group, and 78,8%, 20,8% and 0,5% for the control group (p=0,208). Conclusions: There was no significant association between the presence of PROGINS and endometrial polyps. After age adjust, epidemiological variables significantly associated to endometrial polyps were elderly age, parity, hypertension, and history of breast cancer (implicit tamoxifen use). Malignant polyps in this study were always associated to postmenopausal bleeding, large polyps and frequently associated to hypertension. / TEDE / BV UNIFESP: Teses e dissertações