Two experiments were conducted to determine whether in ewes uterine concentrations of estrogen and progesterone receptors are affected by the presence of a conceptus or by the hormonal milieu associated with extremes in photoperiod to which ewes are exposed. In Exp.1, nine mature ewes were unilaterally ovariectomized by removing an ovary bearing the corpus luteum (CL). The ipsilateral uterine horn was ligated at the external bifurcation and a portion of the anterior ipsilateral uterine horn was removed and assayed for endometrial nuclear and cytosolic concentrations of estrogen receptor (ER) and progesterone receptor (PR) by exchange assays. After a recovery estrous cycle, ewes were bred to a fertile ram. On day 18 of gestation a 10 ml jugular blood sample was collected for measurement of serum concentrations of estradiol -17β (E₂) and progesterone by radioimmunoassay. Ewes were relaparotomized on day 18 and the remaining uterine tissue was removed. Endometrium from both the pregnant and nonpregnant uterine horn was assayed for nuclear and cytosolic ER and PR concentrations. Nuclear and cytosolic ER concentrations on day 10 of the cycle were greater than in endometrium of gravid and nongravid uterine horns on day 18 of gestation (p<.01). Endometrial nuclear PR levels were also greater on day 10 of the cycle than in the pregnant (p<.05) and nonpregnant horn (p<.01) on day 18 of gestation. There were no differences in nuclear and cytosolic ER and PR concentrations between the pregnant and nonpregnant uterine horn on day 18. Serum levels of E₂ and progesterone on day 18 of gestation were 16.56 ± 2.43 pg/ml and 1.74 ± 0.57 ng/ml, respectively. These data suggest that duration of exposure of the uterus to progesterone and(or) the presence of the conceptus causes a reduction in uterine concentrations of ER and PR. Further, an effect of the conceptus, if any, is exerted via a systemic route. In Exp. 2, ten mature ewes were bilaterally ovariectomized in early October. During the onset of the winter solstice (late December), a 10 ml blood sample was collected from five ewes for analysis of serum levels of E₂ and progesterone. Ewes were then laparotomized and approximately one-third to one-half of a uterine horn was removed and assayed for endometrial nuclear and cytosolic ER. The contralateral horn was ligated at the external bifurcation and 10 μg of E₂ in 3 ml of physiological saline was injected into the uterine lumen of the ligated horn. After 48 h, a jugular blood sample was collected for steroid analysis and a section of the E₂ treated horn was removed and assayed for endometrial cytosolic and nuclear ER. This procedure was repeated on the remaining five ewes during the height of the summer solstice (late June). Endometrial nuclear and cytosolic concentrations of ER prior to and after exogenous E₂ stimulation were similar during the winter and summer solstice (p>.05). However, treatment with E₂ increased endometrial nuclear and cytosolic concentrations of ER compared with those of the nonstimulated uterine horn during the winter and summer solstice (p<.05 for each). Serum levels of E₂ prior to luminal treatment of ewes with E₂ during the winter and summer solstice did not differ (16.55 ± 4.05 vs 16.00 ± 3.0 pg/ml, respectively, p>.05). Serum levels of E₂ 48 h after administration of E₂ did not differ among ewes at the winter and summer solstice (18.75 ± 2.4 vs 18.65 ± 1.65 pg/ml, respectively, p>.05). Serum levels of progesterone were basal (<0.10 ng/ml) and did not differ in ewes prior to and after E₂ treatment at the winter and summer solstice (p>.05). These data indicate that physiological factors and(or) hormones such as prolactin and melatonin secreted in response to extremes in photoperiod do not appear to influence uterine concentrations of ER in ovariectomized ewes. / Graduation date: 1991

Both estradiol and progeterone are proposed autocrine or paracrine regulators of ovarian function in primate species. However, specific receptors for these steroids have not been localized to individual compartments of the primate ovary. Using immunocytochemical techniques, estradiol receptors were detected in the germinal epithelium, but not other structures, of ovaries obtained from rhesus or cynomolgus monkeys during the follicular and luteal phases of the menstrual cycle. In contrast, progesterone receptors were present in stromal and interstitial tissue, the thecal layers of healthy and atretic follicles, as well as the functional corpus luteum. These results are consistent with the concept of a receptor-mediated role for progesterone, but not estrogen, within the predominant gametogenic and endocrine structures, e.g., the follicle and corpus luteum, of the primate ovary. The recent discovery of distinct cell types in the corpus luteum of domestic ungulates has revised concepts on the control of luteal function in these species. Studies were designed to test the hypothesis that the primate corpus luteum consists of cell subpopulations that differ in physical characteristics, function and regulation. Cells enzymatically-dispersed from the monkey corpus luteum at mid-luteal phase of the menstrual cycle differed in size (diameter) and the presence of the steroidogenic enzyme, 3β-hydroxysteroid dehydrogenase (3β-HSD). Analysis of dispersed cells for forward and 90° light scatter properties by flow cytometry revealed two distinct continua (Cα and Cβ). These continua were isolated using the sorting capabilities of the flow cytometer. Cα contained single cells of ≤ 15 μm and cell clusters; the cells were typically 3β-HSD-negative nonsteroidogenic. Cβ consisted of single cells that increased in size up to 40 μm and were 3β-HSD-positive. Cβ was divided into two regions (R₁ and R₃) and the cells isolated. R₁ cells were ≤ 15 μm whereas R₃ cells were ≥ 20 μm. Basal progesterone and estrogen production by R₃ cells was greater than that produced by R₁ cells (as determined by radioimmunoassay of the incubation media). Relative stimulation of progesterone production by hCG, cAMP or PGE₂ was not different between R₁ and R₃ luteal cells. These results support the hypothesis that the primate corpus luteum consists of distinct cell subpopulations which differ in size and steroidogenic capacity. However, the cell types which secrete progesterone are typically responsive to gonadotropin and PGE₂, possibly via a cAMP-mediated pathway.

No description available.

Orientador: José Luiz Morais Vasconcelos / Banca: Ciro Moraes Barros / Banca: José Buratini Junior / Banca: Mario Binelli / Banca: Paula de Carvalho Papa / Resumo: O objetivo desse experimento foi avaliar a histomorfometria uterina, a expressão gênica endometrial de ER, PR, OTR, COX-1 e COX-2, e a expressão protéica de ER e PR (porcentagem de núcleos positivos e intensidade de coloração dos núcleos positivos, em unidades arbitrárias [UA], à imunohistoquímica) em vacas em anestro ao longo do desenvolvimento da onda folicular. Vacas Nelore primíparas em anestro pós-parto foram avaliadas diariamente por ultrassonografia ovariana, visando acompanhar o desenvolvimento folicular, e submetidas à histerectomia quando o folículo dominante atingiu o diâmetro de 7,0 mm (FP; n = 4), 8,5 mm (FM; n = 4) ou 10,0 mm (FG; n = 4). Um grupo adicional (PRO; n = 4) consistiu em vacas submetidas a remoção de bezerros (RB; 48 h) quando o folículo dominante atingiu o diâmetro de 9,5 mm e histerectomizadas ao final da RB. Os dados foram analisados pelo PROC GLM do programa SAS. A concentração sérica de estradiol no dia da histerectomia foi maior nas vacas do grupo PRO em relação aos demais grupos (FP: 0,7 pg/mL; FM: 0,8 pg/mL; FG: 1,1 pg/mL; PRO: 2,9 pg/mL; P < 0,05). Não houve efeito de grupo (P > 0,1) nas variáveis histomorfométricas e na expressão gênica endometrial de ER, PR, OTR, COX-1 e COX-2. As vacas do grupo PRO apresentaram maior intensidade de coloração dos núcleos positivos para ER nas células epiteliais glandulares superficiais (FP: 75,9 UA; FM: 75,2 UA; FG: 71,5 UA; PRO: 88,5 UA), intensidade de coloração dos núcleos positivos para PR nas células epiteliais glandulares profundas (FP: 70,2 UA; FM: 73,1 UA; FG: 69,2 UA; PRO: 85,7 UA) e porcentagem de núcleos positivos para PR no estroma subepitelial superficial (FP: 58,2%; FM: 58,0%; FG: 57,3%; PRO: 63,4) em relação às vacas dos demais grupos (P < 0,05). As vacas dos grupos FG e PRO apresentaram maior porcentagem de núcleos positivos para ER no estroma... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate the uterine histomorphometry, gene expression of ER, PR, OTR, COX-1 and COX-2, and protein expressions of ER and PR (percentage of positive nuclei and staining intensity of positive nuclei, in arbitrary units [AU], at immunohistochemistry) in anestrous cows throughout the development of a follicular wave. Primiparous postpartum anestrous Nelore cows were evaluated daily by ovarian ultrasonography and submitted to hysterectomy when the dominant follicle reached the diameter of 7.0 mm (FP; n = 4), 8.5 mm (FM; n = 4), or 10.0 mm (FG; n = 4). An additional group (PRO; n = 4) consisted in cows submitted to temporary weaning (TW; 48 h) when the dominant follicle reached 9.5 mm and hysterectomized at the end of TW. Data were analyzed by PROC GLM of SAS. Serum concentration of estradiol at the day of hysterectomy was greater in cows from PRO group than in cows from the other groups (FP: 0.7 pg/mL; FM: 0.8 pg/mL; FG: 1.1 pg/mL; PRO: 2.9 pg/mL; P < 0.05). There were no effects of group (P > 0.1) on histomorphometrical variables and on endometrial gene expression of ER, PR, OTR, COX-1, and COX-2. Cows from PRO group had greater staining intensity of ER-positive nuclei in shallow glandular epithelium (FP: 75.9 AU; FM: 75.2 AU; FG: 71.5 AU; PRO: 88.5 AU), staining intensity of PR-positive nuclei in deep glandular epithelium (FP: 70.2 AU; FM: 73.1 AU; FG: 69.2 AU; PRO: 85.7 AU), and greater percentage of PR-positive nuclei in shallow subepithelial stroma (FP: 58.2%; FM: 58.0%; FG: 57.3%; PRO: 63.4%) than cows from the other groups (P < 0.05). Cows from FG and PRO groups had a greater percentage of ER-positive nuclei in deep subepithelial stroma than cows from the other groups (FP: 76.0%; FM: 76.4%; FG: 83.6%; PRO: 86.1%; P < 0.05). In the other endometrial areas, the protein expressions of ER and PR were similar among groups. We concluded that in anestrous... (Complete abstract click electronic access below) / Doutor

Background: Estrogen receptor (ER)- and progesterone receptor (PR)-negative (ER-PR-) breast cancer is associated with higher grade and poorer prognosis compared with other breast cancer subtypes. High parity, coupled with lack of breastfeeding, has been associated with an increased risk of ER-PR- cancer. The mechanism of this etiology is unclear, and may be obfuscated by ER and PR correlation with each other as well as other prognostic tumor characteristics. Methods: Using population-based and clinic-based ascertained cases and controls from the Breast Cancer Family Registry, I examined reproductive risk factors, including parity, breastfeeding, and oral contraceptive (OC) use, in relation to ER and PR status, using polytomous logistic regression (for the population-based data) and the method of generalized estimating equations (GEE) (for the clinic-based data) as well as the pseudo-conditional likelihood approach, which accounts for correlated outcome variables. Results: High parity (≥ 3 live births) combined with lack of breastfeeding, was positively associated with ER-PR- tumors (odds ratio [OR]=1.57, 95% confidence interval [CI] 1.10-2.24, population-based cases vs. controls) relative to nulliparity. There was no association with ER-PR- tumors and parity in women who breastfed (OR=0.93, 95%CI 0.71-1.22) relative to nulliparous women. Associations with ER-PR- cancer were higher across all races/ethnicities among women who did not breastfeed compared with women who did. Population-based and clinic-based data were generally in agreement (OR=2.07, 95% CI 1.09-3.91, clinic-based cases vs. controls, relative to nulliparity). When adjusted for the correlation of PR-status and grade, to ER-status, the association between high parity +lack of breastfeeding and ER- status, was maintained. OC use before year 1975 was associated with an increased risk of ER-PR- tumors (OR=1.32, 95% CI 1.04-1.67, population-based data, cases vs. controls) relative to never use of OCs. For women who began OC use in 1975 or later there was no increased risk. Analysis of OC use in clinic-based data agreed with the findings of the population-based data. Conclusions: My findings support that there are modifiable factors for ER-PR- breast cancer, and that breastfeeding in particular may mitigate the increased risk of ER-PR-cancers seen from multiparity. The mechanism of both risk and risk mitigation may operate primarily through the estrogen, rather than progesterone, pathway.

RESUMO A similaridade entre o tecido da mama e o da glândula salivar está bem estabelecida. A porção das estruturas acinares e ductais destes órgãos são basicamente semelhantes. Estes aspectos, associados ao fato de que uma coexistência de carcinomas da mama e de glândula salivar, têm sido relatados em uma incidência maior do que a esperada. Guiaram estudos tentando determinar a importância dos receptores de estrógeno e progesterona em adenomas pleomórficos (AP). A neoplasia é mais freqüente nas glândulas salivares e exibe uma predileção para o sexo feminino. Recentemente a presença do receptor de estrógeno (RE) e do receptor de progesterona (RP) tem sido investigada no AP, entre outras neoplasias de glândula salivar, questionando-se a possibilidade da existência da dependência hormonal. A expressão dos receptores hormonais nos carcinomas de mama é importante para determinar o prognóstico e a probabilidade de responder à manipulação hormonal. Neoplasias que apresentam positividade para ambos os receptores, estrógeno e progesterona, exibem maior probabilidade de resposta à terapia anti-estrogênica do que as neoplasias que são negativas para estes receptores. Baseando-se na literatura científica pertinente, o presente trabalho se propõe a investigar a presença da proteína RE e da proteína RP em APs humanos, relacionando-os com a proliferação de linhagens celulares sob a influência destes hormônios. A técnica utilizada foi a imuno-histoquímica para a pesquisa dos RE e RP em 10 APs emblocados em parafina pertencentes ao arquivo do Serviço de Patologia Cirúrgica da FOUSP, e de duas linhagens de APs estabelecidas no mesmo serviço: uma derivada de um paciente do sexo masculino e a outra de um paciente do sexo feminino. No meio de cultivo onde subculturas destas células proliferavam foram diluídos 17-b-estradiol e progesterona. Através de contagens destas células em períodos pré-determinados (24 horas, 48 horas e 72 horas), pretendeu-se verificar a influência dos respectivos hormônios na multiplicação celular. Como controle positivo utilizou-se uma linhagem denominada T-47D, que foi largamente estudada na literatura. A T-47D é derivada de um carcinoma metastático de mama, reconhecidamente hormônio dependente. E como controle negativo, utilizou-se de uma linhagem de carcinoma epidermóide, denominada HN30. Encontrou-se positividade para o RE em 7 de 10 APs estudados (4 em homens e 3 em mulheres) e positividade para o RP em 8 Aps estudados (4 em mulheres e 4 em homens). Pela análise estatística, constatou-se que existe uma diferença significativa no índice proliferativo entre o controle e as células submetidas à ação do 17-b-estradiol e da progesterona. Para a linhagem derivada do paciente do sexo masculino houve diferença entre o controle e as células expostas ao 17-b-estradiol e a progesterona somente nas últimas 72 horas. Para a linhagem derivada do sexo feminino constatou-se uma diferença significativa entre o controle e as células sob a influência da progesterona, a partir de 48 horas de proliferação celular. A diferença significativa entre o controle e as células sob a ação do 17-b-estradiol ocorreu somente a partir das 72 horas, sugerindo que o AP poderia ser uma neoplasia influenciada pela ação hormonal. / SUMARY It is well established the similarity between mammary and salivary glands especially between the acinic and ductile structures. These aspects, associated to the fact of coexistence of breast carcinomas and of salivary gland tumors been described, leaded studies in attempt to determine the importance of the ERs and Pr in pleomorphic adenomas (PA), the most frequent salivary gland tumor and with predilection for the females. Lately, the presence of ERs and of the PRs has been investigated in PA and other salivary gland tumors pointing out their hormonal dependency. The expression of hormone receptors in breast carcinomas is crucial to determine a presence for both receptors. These tumors exhibit better response to anti-estrogenic therapy than the negative ones. Basing on the pertinent scientific literature, the present study proposes to investigate the presence of the RE and of the RP in humans PA and connecting them with cellular proliferation in vitro, under the influence of these hormones. Immunohistochemistry technique was used for the detection of RE and RP in paraffin embedded 10 PAs from the files of the Department of Oral Pathology, School of Dentistry, University of São Paulo, and two PA cell lines one from a male patient and other female. The culture midia was supplied with, 17-b-estradiol and progesterone. A growth curve was performed (24 hours, 48 hours and 72 hours) to verify the influence of the respective hormones in the cellular proliferation. As a positive control T-47-D cells derived from a hormone dependent metastatic breast carcinoma were used, and as negative control HN30 cells, derived from a tongue squamous cell carcinoma. 7 of 10 PAs were positive (4 in men and 3 in women) for RP and 8 of 8 PAs (4 in women and 4 in men) for RE. The statistical analysis verified a significant difference in the proliferative index between the control cells and the ones submitted to the action of the 17-b-estradiol and of the progesterone: for male derived lineage a difference was only observed in the last 72 hours. In the other hand, for the female derived lineage a significant difference was verified starting from 48 hours, suggesting that PA can be influenced by hormonal action.

RESUMO A similaridade entre o tecido da mama e o da glândula salivar está bem estabelecida. A porção das estruturas acinares e ductais destes órgãos são basicamente semelhantes. Estes aspectos, associados ao fato de que uma coexistência de carcinomas da mama e de glândula salivar, têm sido relatados em uma incidência maior do que a esperada. Guiaram estudos tentando determinar a importância dos receptores de estrógeno e progesterona em adenomas pleomórficos (AP). A neoplasia é mais freqüente nas glândulas salivares e exibe uma predileção para o sexo feminino. Recentemente a presença do receptor de estrógeno (RE) e do receptor de progesterona (RP) tem sido investigada no AP, entre outras neoplasias de glândula salivar, questionando-se a possibilidade da existência da dependência hormonal. A expressão dos receptores hormonais nos carcinomas de mama é importante para determinar o prognóstico e a probabilidade de responder à manipulação hormonal. Neoplasias que apresentam positividade para ambos os receptores, estrógeno e progesterona, exibem maior probabilidade de resposta à terapia anti-estrogênica do que as neoplasias que são negativas para estes receptores. Baseando-se na literatura científica pertinente, o presente trabalho se propõe a investigar a presença da proteína RE e da proteína RP em APs humanos, relacionando-os com a proliferação de linhagens celulares sob a influência destes hormônios. A técnica utilizada foi a imuno-histoquímica para a pesquisa dos RE e RP em 10 APs emblocados em parafina pertencentes ao arquivo do Serviço de Patologia Cirúrgica da FOUSP, e de duas linhagens de APs estabelecidas no mesmo serviço: uma derivada de um paciente do sexo masculino e a outra de um paciente do sexo feminino. No meio de cultivo onde subculturas destas células proliferavam foram diluídos 17-b-estradiol e progesterona. Através de contagens destas células em períodos pré-determinados (24 horas, 48 horas e 72 horas), pretendeu-se verificar a influência dos respectivos hormônios na multiplicação celular. Como controle positivo utilizou-se uma linhagem denominada T-47D, que foi largamente estudada na literatura. A T-47D é derivada de um carcinoma metastático de mama, reconhecidamente hormônio dependente. E como controle negativo, utilizou-se de uma linhagem de carcinoma epidermóide, denominada HN30. Encontrou-se positividade para o RE em 7 de 10 APs estudados (4 em homens e 3 em mulheres) e positividade para o RP em 8 Aps estudados (4 em mulheres e 4 em homens). Pela análise estatística, constatou-se que existe uma diferença significativa no índice proliferativo entre o controle e as células submetidas à ação do 17-b-estradiol e da progesterona. Para a linhagem derivada do paciente do sexo masculino houve diferença entre o controle e as células expostas ao 17-b-estradiol e a progesterona somente nas últimas 72 horas. Para a linhagem derivada do sexo feminino constatou-se uma diferença significativa entre o controle e as células sob a influência da progesterona, a partir de 48 horas de proliferação celular. A diferença significativa entre o controle e as células sob a ação do 17-b-estradiol ocorreu somente a partir das 72 horas, sugerindo que o AP poderia ser uma neoplasia influenciada pela ação hormonal. / SUMARY It is well established the similarity between mammary and salivary glands especially between the acinic and ductile structures. These aspects, associated to the fact of coexistence of breast carcinomas and of salivary gland tumors been described, leaded studies in attempt to determine the importance of the ERs and Pr in pleomorphic adenomas (PA), the most frequent salivary gland tumor and with predilection for the females. Lately, the presence of ERs and of the PRs has been investigated in PA and other salivary gland tumors pointing out their hormonal dependency. The expression of hormone receptors in breast carcinomas is crucial to determine a presence for both receptors. These tumors exhibit better response to anti-estrogenic therapy than the negative ones. Basing on the pertinent scientific literature, the present study proposes to investigate the presence of the RE and of the RP in humans PA and connecting them with cellular proliferation in vitro, under the influence of these hormones. Immunohistochemistry technique was used for the detection of RE and RP in paraffin embedded 10 PAs from the files of the Department of Oral Pathology, School of Dentistry, University of São Paulo, and two PA cell lines one from a male patient and other female. The culture midia was supplied with, 17-b-estradiol and progesterone. A growth curve was performed (24 hours, 48 hours and 72 hours) to verify the influence of the respective hormones in the cellular proliferation. As a positive control T-47-D cells derived from a hormone dependent metastatic breast carcinoma were used, and as negative control HN30 cells, derived from a tongue squamous cell carcinoma. 7 of 10 PAs were positive (4 in men and 3 in women) for RP and 8 of 8 PAs (4 in women and 4 in men) for RE. The statistical analysis verified a significant difference in the proliferative index between the control cells and the ones submitted to the action of the 17-b-estradiol and of the progesterone: for male derived lineage a difference was only observed in the last 72 hours. In the other hand, for the female derived lineage a significant difference was verified starting from 48 hours, suggesting that PA can be influenced by hormonal action.

The goal of the current research was to examine the expression, signaling and function of the membrane progestin receptors (mPRs) in the ovarian follicular cells of the Atlantic croaker (Micropogonias undulatus) and in human breast cancer cells. Multiple studies have examined the role of mPRs in the germ cells of several vertebrate classes, yet few studies have examined the role of the mPRs in the somatic cells of reproductive tissues. Therefore this research examines the mechanism of mPR action and its function in somatic cells of female reproductive tissues. Results from studies on the expression, localization and signaling of the mPR[alpha] in co-cultures of granulosa and theca cells from the croaker suggest that the mPR[alpha] is localized to the plasma membrane of both cell types and that the mPR[alpha] is associated with and signals via pertussis toxin-sensitive inhibitory G proteins to decrease intracellular cAMP and activate ERK. In addition, exposure of follicular co-cultures to progestins that activate the mPR[alpha] results in a decrease in serum starvation-induced cell death which is not replicated by progestins which activate the nuclear progestin receptor (nPR), indicating mPR mediation. Similar studies in two immortalized human breast cancer cell lines, MDA-MB-468 and SKBR3, suggest that the mPR[alpha] is also present in the membranes of these cells and signals in human breast cancer cell lines via activation of a pertussis toxin-sensitive G protein to significantly decrease in intracellular cAMP and activate ERK. Progesterone exposure also decreased serum starvation-induced cell death in SKBR3 cells which are nPR positive and in MDA-MB-468 cells which are nPR negative. Synthetic progestins which activate the nPR but not the mPR were ineffective in inhibiting death in either cell type suggesting that the mPR is the mediator of this progestin action. mPR[alpha], mPR[beta] and mPR[gamma] expression analysis of paired normal and malignant breast tissue biopsies from thirteen women revealed that at least one mPR isoform was upregulated in the malignant tissue of 70% of the women. In addition the expression of mPR[gamma] was positively correlated with the expression of the nPR and CK19, a breast epithelial cell marker. / text

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 89-109 and 224-228).