Estrogen Response System Research Articles

Proliferative and Apoptotic Pathways in the Testis of Quail Coturnix coturnix during the Seasonal Reproductive Cycle.

Simple SummaryThe quail Coturnix coturnix exhibits an annual cycle of testis size, sexual steroid production, and spermatogenesis. The testicular levels of both 17β-estradiol (E2) and androgens are higher during the reproductive period compared to the non-reproductive period, suggesting that estrogens act in synergy with the androgens for the initiation of spermatogenesis. Therefore, the present study aimed to investigate the estrogen responsive system in quail testis in relation to the reproduction seasons, with a focus on the molecular pathways activated in both active and regressive quail testes. The results indicated that estrogens participated in the activation of mitotic and meiotic events during the reproductive period by activating the ERK1/2 and Akt-1 pathways. In the non-reproductive period, when the E2/ERα levels are low, ERK1/2 and Akt-1 pathways remain inactive and apoptotic events occur. Our results suggest that the activation or inhibition of these molecular pathways plays a crucial role in the physiological switch “on/off” of the testicular activity in male quail during the seasonal reproductive cycle.The quail Coturnix coturnix is a seasonal breeding species, with the annual reproductive cycle of its testes comprising an activation phase and a regression phase. Our previous results have proven that the testicular levels of both 17β-estradiol (E2) and androgens are higher during the reproductive period compared to the non-reproductive period, which led us to hypothesize that estrogens and androgens may act synergistically to initiate spermatogenesis. The present study was, therefore, aimed to investigate the estrogen responsive system in quail testis in relation to the reproduction seasonality, with a focus on the molecular pathways elicited in both active and regressive quail testes. Western blotting and immunohistochemistry analysis revealed that the expression of ERα, which is the predominant form of estrogen receptors in quail testis, was correlated with E2 concentration, suggesting that increased levels of E2-induced ERα could play a key role in the resumption of spermatogenesis during the reproductive period, when both PCNA and SYCP3, the mitotic and meiotic markers, respectively, were also increased. In the reproductive period we also found the activation of the ERK1/2 and Akt-1 kinase pathways and an increase in second messengers cAMP and cGMP levels. In the non-reproductive phase, when the E2/ERα levels were low, the inactivation of ERK1/2 and Akt-1 pathways favored apoptotic events due to an increase in the levels of Bax and cytochrome C, with a consequent regression of the gonad.

Seasonal variation of cell proliferation and apoptosis in the efferent ductules and epididymis of the Neotropical bat Artibeus lituratus (Chiroptera, Phyllostomidae)

The balance between cell proliferation and apoptosis is important for maintenance of male fertility, being influenced by a variety of stimuli including androgens and estrogens. However, studies concerning regulation of these processes along the male reproductive tract under physiological conditions are scarce. Therefore, in this study, we investigated the profile of cell proliferation and apoptosis in the efferent ductules and epididymis of the Neotropical bat Artibeus lituratus, a seasonal breeder that presents natural variation in components of the androgen and estrogen responsive systems along the circannual cycle. Low rates of cell proliferation and apoptosis were found in the efferent ductules and epididymis of A. lituratus during the reproductive period, as few epithelial cells were positive for MCM7 (proliferation marker) and cleaved caspase-3 or TUNEL (apoptosis markers). In contrast, during the regressive period, the rate of both proliferating and apoptotic cells was significantly higher in the epithelium lining the efferent ductules as well as throughout the epididymis. The increased proliferative activity at this phase was positively correlated with the expression of estrogen receptor alpha (ERα), whereas the variation in apoptosis appears to be unrelated to the local expression of androgen and estrogen receptors. Together, these data suggest that cell proliferation and apoptosis are differentially modulated in the efferent ductules and epididymis of A. lituratus during the annual reproductive cycle, and support the hypothesis that ERα may be important in preparing the male reproductive tract for sexual recrudescence.

Estradiol concentration and the expression of estrogen receptors in the testes of the domestic goose (Anser anser f. domestica) during the annual reproductive cycle

Seasonal fluctuations in the activity of bird testes are regulated by a complex mechanism where androgens play a key role. Until recently, the role played by estrogens in males has been significantly underestimated. However, there is growing evidence that the proper functioning of the testes is associated with optimal estradiol (E2) concentration in both the plasma and testes of many mammalian species. Estrogens are gradually emerging as very important players in hormonal regulation of reproductive processes in male mammals. Despite the previously mentioned, it should be noted that estrogenic action is limited by the availability of specific receptors—estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Interestingly, there is a general scarcity of information concerning the estrogen responsive system in the testes of male birds, which is of particular interest in exploring the phenomenon of seasonality of reproduction. To address this question, we have investigated for the first time the simultaneous expression of testicular ERα and ERβ genes and proteins with the accompanying plasma and testicular E2 concentrations during the annual reproductive cycle of male bird. The research model was the domestic goose (Anser anser f. domestica), a species whose annual reproductive cycle can be divided into 3 distinct phases characterized by changes in testicular activity. It has been revealed that the stable plasma E2 profile did not correspond to changing intratesticular E2 profile throughout the experiment. The expression of ERα and ERβ genes and proteins was detected in gander testes and it fluctuated on a seasonal basis with lower level in breeding and sexual reactivation stages and higher level during the nonbreeding stage. Our results demonstrated changes in testicular sensitivity to estrogens in male domestic goose during the annual reproductive cycle. The seasonal pattern of estrogen receptors (ERs) expression was analyzed against the hormonal background and a potential mechanism of ERs regulation in bird testes was proposed. The present study revealed seasonal variations in the estrogen responsive system, but further research is needed to fully explore the role of estrogens in the reproductive tract of male birds.

Estrogen prevents cardiac and vascular failure in the ‘listless’ zebrafish (Danio rerio) developmental model

The presence of a robust estrogen (E2) response system throughout heart and blood vessel tissues of vertebrates, including humans, has led to the speculation that this ubiquitous hormone may play a prominent role in the health and maintenance of the adult cardiovascular system (CVS). We previously established an embryonic zebrafish model called ‘listless’, which results from the inhibition of E2 synthesis by treatment with aromatase enzyme inhibitors (AI). These fish have outward symptoms similar to the human condition of congestive heart failure and tamponade. E2 replacement therapy (1) reduced the severity of cardiac sac abnormalities, (2) protected heart function, (3) prevented reduction in heart size, and (4) reduced blood vessel deterioration. Nitric oxide may be a critical downstream mediator of these events. We also demonstrate that removal of fluid around the heart increases survival of AI-treated fish. These results strongly indicate the importance of E2 in the developing CVS of the zebrafish and offer a potential model for the study of its role in CVS development, maintenance, and disease conditions.

Differential expression and seasonal variation on aquaporins 1 and 9 in the male genital system of big fruit-eating bat Artibeus lituratus

Efferent ductules and epididymis are involved in water and solute transport, which is indispensable for storage and maintenance of the sperm viability. The reabsorption process involves proteins such as aquaporins (AQP), which has been described in the male genital system of limited species, including primate, rodents, cats and dogs. To contribute with information about AQPs in the male system, here we investigated the distribution of AQP1 and AQP9 in the tropical bat Artibeus lituratus, along the annual reproductive cycle. A. lituratus is a seasonal breeder with natural variation in components of the androgen and estrogen responsive system, thus being a good model for exploring the AQPs modulation. AQP1 was found restricted to differentiating spermatids, efferent ductules epithelium and venular endothelia along the male tract. AQP9 was detected throughout the epididymis being more abundant in the cauda and ductus deferens, but was not found in testis, rete testis and efferent ductules. Contrasting with AQP1 which appear to be constitutively expressed, there was seasonal variation in AQP9 expression, which was reduced in regressed epididymis. The AQP9 does not appear to be modulated by estradiol or androgens, but possibly by other factor related to luminal sperm. The establishment of specific function for aquaporins in the male tract remains undetermined; however, the cellular distribution presently found are compatible with the main function of AQP1, as a selective water channel, and AQP9, which is a conduct for water and a plethora of neutral solutes present in the epididymis milieu such as glycerol and urea.

Seasonal variation in estrogen receptor ERα, but not ERβ, androgen receptor and aromatase, in the efferent ductules and epididymis of the big fruit-eating bat Artibeus lituratus

The efferent ductules (ED) are a major target for estrogens, which act via the estrogen receptors ERα (ESR1) and ERβ (ESR2). ERα has been found in the ED of all species studied so far. However, in the epididymis (EP), the expression of ERα is controversial, as is data about the occurrence of aromatase in the epithelium lining the excurrent ducts. Therefore, to further investigate this estrogen-responsive system, we used a seasonal breeder, the Neotropical bat, Artibeus lituratus, in which testicular expression of androgen (AR) and estrogen (ER) receptors vary with reproductive phase. The localization of aromatase, ERα, ERβ and AR in the ED and EP of A. lituratus was investigated. The results showed that aromatase, AR and ERβ were distributed throughout the excurrent ducts and did not vary during the annual reproductive cycle. Conversely, ERα was detected primarily in the ED epithelium, had marked seasonal variation and was increased during regression, especially in the EP epithelium. The results suggest that ERα may be involved in preparing the male genital tract for recrudescence. Together, the data obtained under natural conditions emphasize that specific segments of the excurrent ducts downstream of the testis are the primary targets for estrogen action via ERα, which is similar to previous findings in animals lacking functional ERα.

Cloning of three estrogen receptors (ER) from killifish ( Fundulus heteroclitus): Differences in populations from polluted and reference environments

Epidemiological, wildlife, and laboratory studies support the hypothesis that chemicals released into the environment through anthropogenic activities are responsible for abnormalities of reproduction and development. Although the New Bedford Harbor (NBH) killifish population has survived and reproduced successfully for >50 yr (∼20 generations), fish have high body burdens of the major NBH contaminants (polychlorinated biphenyls); elevated levels of P450 aromatase B and vitellogenin mRNA (markers of estrogen effect); and evidence of endocrine disruption. To investigate possible adaptive changes in the estrogen response system of NBH killifish, we cloned the estrogen receptors (ER) from killifish populations resident in NBH and a relatively unpolluted reference site (Scorton Creek MA, SC). ERα, -βa, and -βb cDNAs encoding full-length polypeptides of 620, 543, and 672 amino acids, respectively, were identified. Each ER subtype had multiple splice variants, single nucleotide polymorphisms (SNPs), and a characteristic tissue distribution and developmental profile. As measured by real-time quantitative RT-PCR analysis, the overall tissue distribution of each ER was similar in NBH and SC fish, implying that the regulatory pathways which maintain tissue-specific expression are largely unchanged by long term pollutant exposure. Nonetheless, a striking difference was seen in the quantity of mRNA of the estrogen-inducible gene ERα, which was significantly lower in brain, liver and ovaries of reproductively active NBH as compared to SC females. Paradoxically, despite the “estrogenic” NBH environment, ERα mRNA levels did not differ in reproductively inactive NBH and SC females, or in males at the two sites at any time of year. We interpret results in NBH fish as due in part to a deficit of circulating estrogen, and in part to pollutant-mediated hyporesponsiveness of the ERα gene. In marked contrast to adult fish, ERα was elevated ∼5-fold in NBH as compared to SC embryos/larvae, perhaps indicative of estrogenic chemicals in yolk. We conclude that contaminants in the NBH environment impact molecular components of the estrogen signaling pathways in resident killifish populations. Whether these changes are transient or heritable requires further study.

Estrogen response system in the reproductive tract of the male turtle: An immunocytochemical study

Portions of the reproductive tract of the male ( Trachemys scripta) turtle were examined by immunocytochemistry for evidence of the capacity to produce and respond to estrogen hormones (via the expression of P450 aromatase and estrogen receptors). Aromatase was detected in both the Sertoli and Leydig cells of the testis and was expressed at different levels during the spermatogenic cycle, being highest in the quiescent testis and lowest during germ cell meiosis. ERα was found in the Leydig cells surrounding the seminiferous tubules as well as in the epithelial cells of the excurrent canals (rete testis, efferent ductule, and epididymis). ERβ immunoreactivity was found in both the spermatogonia and Sertoli cells in the testis, and in the epithelial cells of excurrent canals.

Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: Gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation

In teleost fish, the predominant brain form of cytochrome P450 aromatase (P450aromB) is a neural marker of estrogen effect, and an entry point for studying the role of hormonal and environmental estrogens on neurodevelopment and neuroplasticity. As part of a project using zebrafish to investigate these issues, we developed and validated a rapid, sensitive, and reproducible real-time polymerase chain reaction (PCR) assay for quantifying and comparing P450aromB and P450aromA expression in unfertilized eggs, embryos/larvae, and dissected tissues of adult fish. Results confirm that P450aromB and -A predominate in brain and ovary, respectively, and further show that the degree of overlapping expression (ratio, B:A) is 100:1 in brain, 1:50 in ovary, 1:1 in eye, and 2:1 in testis. Sex differences were observed in eye only (female > male). When compared to whole ovaries, unfertilized eggs had similar levels of P450aromA but enrichment of P450aromB, which suggests preferential synthesis or accumulation in mature oocytes. Both of the maternally derived aromatase isoforms were rapidly degraded post-fertilization, but the onset of embryonic P450aromB expression (5 hpf) was much earlier than P450aromA (48 hpf), and reached higher maximum levels (e.g., 10-fold at 72 hpf). Consistent with earlier reports, P450aromB but not -A was estrogen-inducible, but the estrogen response system in embryos was far more robust than in adults (>100- vs. <4-fold maximal induction, respectively). Application of this real-time PCR assay to measurement of P450aromB and -A in zebrafish embryos has utility for routine screening of chemicals and environmental samples for estrogen-like bioactivity and neural effects.

Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish ( Carassius auratus) and zebrafish ( Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci ( cyp19a and cyp19b) are differentially expressed in brain (P450aromB⪢A) and ovary (P450aromA⪢B) and have a different developmental program (B⪢A) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24–48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (α, β, and γ). The 5′-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (a⪢b) are opposite to fish pituitary (b⪢a). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30–48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.

Estrogen agonist and antagonist action on the human estrogen receptor in Drosophila.

The estrogen receptor (ER) regulates the expression of genes involved in the growth, proliferation and differentiation of skeletal, cardiovascular, neural and reproductive tissues. A basic scheme for the mechanism for ER action has been developed, but precise details on the interactions between ER and the cellular signaling and transcription machinery required for receptor-mediated regulation of specific target genes are still lacking. We have developed a genetic approach to explore the functional interactions of ER. In this work, we describe the development of an estrogen responsive system in the fruit fly, Drosophila melanogaster. Transgenic flies carrying the human ER alpha and an estrogen responsive green fluorescent protein (GFP) reporter gene were constructed. In vivo expression of the GFP reporter gene was observed when larvae were grown on a food source containing steroidal or nonsteroidal estrogens. The induction of the reporter gene by estrogens was blocked upon treatment with tamoxifen, an estrogen antagonist. However, we failed to recapitulate ligand-independent activation of the receptor in vivo or in cultured Drosophila cells. An estrogen responsive Drosophila system could be used to identify and characterize the complex functional interactions between ER and the other components of the cellular transcriptional apparatus.

Regulation of protein kinase C δ by estrogen in the MCF-7 human breast cancer cell line

We have previously shown that estrogen up-regulates expression of protein kinase C (PKC) δ in the rat and rabbit corpus luteum as well as in luteinized rat granulosa primary cell cultures. To determine whether a similar regulation of the PKC δ isoform by estrogen occurred in another estrogen responsive system, we investigated the estrogen receptor positive MCF-7 human breast cancer cells. In a characterization of PKC isoforms in MCF-7 cells we determined that PKC δ was the predominant PKC isoform. However in contrast to the effect of estrogen on PKC δ expression in ovarian cells, estrogen treatment of MCF-7 cells resulted in a significant decrease in PKC δ protein and mRNA expression in a time and dose dependent manner. Treatment of MCF-7 cells with 10 −10–10 −8 M estrogen for 7 days down-regulated specifically PKC δ mRNA and protein while expression of other PKC isoforms was unchanged. The opposite regulation of PKC δ expression in ovarian and breast cancer cells prompted us to evaluate the type of estrogen receptor present in both cell types. Results showed that luteinized rat granulosa cells expressed predominantly estrogen receptor β while the MCF-7 cells expressed predominantly estrogen receptor α and barely detectable levels of estrogen receptor β. These results suggest that the differential ability of estrogen to regulate PKC δ expression could potentially be a result of differential signaling through the two estrogen receptor subtypes.

Estrogen regulation of transforming growth factor- α in ovarian cancer

Transforming growth factor alpha (TGF α) may be induced by estrogen in estrogen responsive systems and can contribute to the growth-modulatory effects of this hormone. To test whether TGF α contributes to estrogen-regulated growth in ovarian cancers, we have compared the effects of 17 β-estradiol (E 2) and TGF α in a range of ovarian carcinoma cell lines. Addition of E 2 to the estrogen receptor (ER)-positive cell lines (PE01, PE04 and PE01 CDDP) produced a 2–4 fold increase in TGF α protein concentrations in media conditioned by the cells. Both E 2 and TGF α stimulated the growth of the PE01 and PE04 lines and inhibited the growth of the PE01 CDDP line. Furthermore, the E 2-mediated growth effects could be reversed by an epidermal growth factor (EGF) receptor-targeted antibody. E 2 also down-regulated EGF receptor expression in ER-positive cell lines. In a series of primary ovarian tumors, higher concentrations of ER were associated with an increased percentage of tumors expressing TGF α mRNA and a decreased percentage expressing EGF receptor protein. All these data are consistent with E 2 increasing production of TGF α in ER-positive ovarian cancer and this in turn acting through the EGF receptor to modulate growth in an autocrine manner.

The growth inhibitory effect of conjugated linoleic acid on MCF-7 cells is related to estrogen response system.

Conjugated linoleic acid (CLA) has been shown to have a direct oncostatic action on MCF-7 human breast cancer cells in culture. However, the mechanism involved is not fully elucidated. In this study we have examined whether the inhibitor action is related to the estrogen responsiveness of MCF-7 cells. Our results demonstrate that CLA selectively inhibits proliferation of ER positive MCF-7 cells as compared with ER negative MDA-MB-231 cells. Cell cycle studies indicated that a higher percentage of CLA treated MCF-7 cells remained in the G0/G1 phase as compared to control and those treated with linoleic acid (LA). CLA also inhibited expression of c-myc in MCF-7 cells. These results demonstrate that CLA may inhibit MCF-7 cell growth by interfering with the hormone regulated mitogenic pathway. We are reporting for the first time the involvement of CLA, a dietary factor, in the regulation of hormone mediated mitogenic pathways in ER positive breast cancer cell proliferation in vitro.

In vitro and in vivo interactions between nuclear receptors at estrogen response elements.

To study mechanisms involved in the antiestrogenic effect of retinoic acid (RA), previously described in mammalian cells, we used in vitro and in vivo approaches. One hypothesis was direct competition between nuclear receptors (ER, RAR and RXR) at the DNA level. We first showed in vitro that the RAR/RXR heterodimer could weakly bind an ERE and that retinoid receptors reduced binding of ER to an ERE. We next checked whether, in yeast, direct competition between receptors that recognize the same responsive element could be monitored in a reconstituted heterologous estrogen-responsive system, by determining the expression of a reporter gene. We then co-transformed RAR and RXR in an estrogenic responsive strain. This model demonstrated that, even though RAR/RXR was able to bind an ERE, the addition of retinoic acid had no inhibitory effect on estrogen-induced responses in this yeast system, unlike in mammalian cells. Interference between these receptors should require other factors than interactions at the ERE level. This model could be used to identify mammalian factors interacting with estrogen and retinoic acid receptors which could play a role in crosstalk between these receptors.

Estrogen receptor, progesterone receptor, and HER-2/neu protein in breast cancers from pregnant patients.

Because the occurrence of breast cancer during pregnancy is uncommon and because the high levels of estrogens and progestins associated with pregnancy could cause false-negative results from ligand binding assays (LBA), the actual incidence of steroid hormone receptor positivity in tumors from this subset of women is unclear. Estrogen receptor (ER) and progesterone receptor (PgR) were determined using LBA methods in 15 tumors from 15 pregnant patients with breast cancer. In addition, immunohistochemistry was done for ER, PgR, pS2, heat shock protein 27 (hsp27), and HER-2/neu on 12 of the 15 tumors. Five of 15 (33%) tumors were positive for ER by LBA, compared with 52% of tumors from age-matched nonpregnant patients. Six of 12 (50%) were ER-positive by immunohistochemistry. For PgR, 7 of 15 (47%) tumors were positive by LBA, compared with 42% of tumors from nonpregnant patients. Ten of 12 (83%) stained positive for PgR. By LBA, 67% of tumors studied were positive for ER or PgR or both, as opposed to 57% of tumors from the nonpregnant comparison group. Two other estrogen receptor-mediated proteins, pS2 and hsp27, were present by staining in 8 of 12 (67%) and 10 of 12 (83%) of tumors, respectively. Seven of 12 tumors (58%) had positive staining for HER-2/neu, whereas only 16% of age-matched nonpregnant patients had positive-staining tumors. By LBA, the incidence of ER and PgR in breast tumors from pregnant women was not significantly different from that of tumors from nonpregnant age-matched patients. Some ER-negative tumors were PgR, pS2, or hsp27 positive, indicating that an intact estrogen response system was operative although ER was not detectable by standard LBA.

The growth inhibitory action of melatonin on human breast cancer cells is linked to the estrogen response system

The pineal hormone, melatonin, was examined for its capacity to modulate the proliferation of a panel of human breast cancer cell lines. Melatonin inhibited, to a varying extent, the proliferation of all three estrogen-responsive cell lines, but had no effect on estrogen-insensitive breast tumor cell lines. Melatonin was also able to specifically block estrogen-induced proliferation in MCF-7 breast cancer cells. However, this action was abolished in the presence of tamoxifen. Therefore, it appears that the antiproliferative effects of melatonin are mediated through the estrogen-response pathway.

Bipotential actions of estrogen on progesterone biosynthesis by ovarian cells. II. Relation of estradiol's stimulatory actions to cholesterol and progestin metabolism in cultured swine granulosa cells.

Although both inhibitory and stimulatory actions of estradiol on swine granulosa cells have been described, the bases for these inconsistent effects are not clear. We have tested properties of ovarian follicles and in vitro culture conditions that result in consistently stimulatory effects of estradiol on progesterone biosynthesis. Stimulatory actions of estradiol (in contrast to inhibitory effects) were critically dependent upon the density of granulosa cells in culture and the size and maturational status of the parent Graafian follicles. Granulosa cells isolated from small, rather than medium or large sized, swine follicles exhibited the greatest peak response to estradiol, although half-maximally stimulatory concentrations (ED50) of estradiol were similar (mean, 81 ng/ml). Granulosa cells from atretic follicles also secreted increased quantities of progesterone in response to estradiol, but the ED50 for estrogen stimulation was significantly higher (ED50 = 322 ng/ml estradiol) than that of comparable healthy follicles (ED50 = 109 ng/ml). This estrogen-responsive system was used to test the mechanisms subserving estrogen's trophic actions on granulosa cells. Estradiol significantly enhanced the activity of 3 beta-hydroxysteroid dehydrogenase with consequently increased production of progesterone and 20 alpha-hydroxypregn-4-en-3-one. Estrogen also augmented functional cholesterol side-chain cleavage activity in a dose- and time-dependent fashion with a resultant increase in pregnenolone biosynthesis. Moreover, parallel observations documented concordant dose responses for the synthesis of all three major progestins by pig granulosa cells. The trophic actions of estrogen on the steroidogenic pathway were associated with enhanced hydrolysis of endogenous cholesteryl ester stores but were not significantly antagonized by inhibition of de novo cholesterol biosynthesis. We conclude that suitable follicle selection and appropriate in vitro culture conditions provide a consistently estrogen-responsive granulosa-cell system, in which estradiol modulates certain key aspects of progestin and cholesterol metabolism. These trophic actions of estrogen are likely to prepare granulosa cells for the increased rates of progesterone biosynthesis ultimately required by fully differentiated luteal cells.

Facile geometric isomerization of phenolic non-steroidal estrogens and antiestrogens: Limitations to the interpretation of experiments characterizing the activity of individual isomers

In many estrogen responsive systems the isomers of tamoxifen are known to have different biological character-the trans isomer is generally an antagonist and the cis isomer an agonist. Attempts to similarly characterize the isomers of hydroxytamoxifen (which differ greatly in their affinity for the estrogen receptor) are shown to be complicated by their facile isomerization. This isomerization was studied in cultures of estrogen receptor positive MCF-7 human breast cancer cells and monitored by HPLC under reversed phase conditions. Hydroxytamoxifen isomers that are initially 99% pure, undergo a time and temperature dependent isomerization, so that after 2 days in tissue culture medium at 37°C they have isomerized to the extent of 20%. This isomerization occurs in the cell-free medium alone and cannot be attributed to a metabolic conversion by the cells. The isomerization occurs much more slowly at 4 than at 37° C and can be reduced considerably by various antioxidants (butylated hydroxytoluene, ascorbate, α-tocopherol, retinoic acid and retinal); however, at concentrations that block isomerization, these antioxidants are toxic to the cells. Although the medium contains both the cis and trans isomers of hydroxytamoxifen, the MCF-7 cells preferentially accumulate the trans isomer and the material associated with the nuclear estrogen receptor is, in all cases, mainly the higher affinity trans isomer. A similar preference of the estrogen receptor for the trans isomer is seen with diethylstilbestrol, resulting again in almost exclusive accumulation of the trans isomer in the receptor binding site. These experiments indicate the importance of verifying the isomer compositions of easily isomerizable non-steroidal estrogens and antiestrogens, such as diethylstilbestrol and hydroxytamoxifen, both in stock solutions and in experimental samples (especially those derived from receptor-associated material), so as to ascertain that the activity of the individual isomers is being correctly assigned.

Nuclear translocation and interactions of the estrogen receptor in uterus and mammary tumors

The mechanism of the nuclear translocation of the estrogen receptor (ER) and the nature of the acceptor sites interacting with the ER-estrogen complex are unknown. We report here results of our laboratory aiming to solve the following problems in the uterus and mammary tumors systems. 1. Mechanism of the activation of the cytosol ER leading to its nuclear translocation: The nuclear translocation of ER is triggered by all ligands tested thus far either agonist or antagonist. From the physico chemical characteristics of the nuclear ER extracted by micrococcal nuclease digestion, we propose that the mechanism of the activation of ER is not the Ca 2+-dependent proteolysis of the receptor and might be a simple conformational change. 2. Is DN A a biological acceptor of ER? our results are in agreement with, but do not prove such an hypothesis, (a) The native nuclear ER is extracted after micrococcal nuclease digestion in a form which is still able to bind DNA. (b) Intercalating drugs inhibit 50% of translocation of the ER to the nucleus, (c) Preliminary experiments with 5-bromodeoxyuridine aiming to cross-link in vino the nuclear ER to DNA in MCF 7 cells are also consistent with this hypothesis. 3. Acceptor and Effector sites: The relationship between the nuclear components interacting with the receptor and possibly responsible for its nuclear retention (acceptor sites) and the nuclear components interacting with the receptors to give a response (effector sites) is discussed on the basis of results obtained in estrogen responsive and unresponsive systems. Results obtained in independent mammary tumors of C3H mice and dimethylbenzanthracene treated rats, and with the antiestrogens in MCF, cells strongly suggest that the nuclear translocation step is required but not sufficient to induce the estrogenic response.