Localization Of Estrogen Receptor Alpha Research Articles

Abstract 4372: Metformin inhibits nuclear localization of estrogen receptor alpha in breast cancer cells, in part, by inhibition of CYP-mediated epoxyeicosatrienoic acid (EET) biosynthesis

Abstract Introduction: Cancer cell-intrinsic CYP monooxygenases promote tumor progression. In ER+HER2-, breast cancer cells, CYP3A4 promotes tumor growth, in part, through epoxyeicosatrienoic acid (EET) biosynthesis (1). The biguanide diabetes drug metformin is currently being studied in breast cancer clinical trials. We have discovered that metformin binds to the active site heme of CYP3A4, thereby inhibiting EET biosynthesis (1). Structure-based design led to the discovery of the 100-fold more potent metformin analog N1-hexyl-N5-benzyl-biguanide (HBB). HBB specifically inhibits CYP3A4 arachidonic acid (AA) epoxygenase activity in breast cancer cells, suppresses the growth of breast cancer cell lines (IC50=3-30 uM), and inhibits growth of the MCF-7 ER+ mammary tumor model, similar to CYP3A4 gene silencing. Although CYP3A4 synthesizes EETs, how EETs may promote tumor growth is unknown. Recently, we found that HBB inhibits nuclear transit of estrogen receptor alpha (ERα) and 70kD FITC-dextran, but the mechanism was unknown (2). Metformin was discovered to inhibit nuclear pore complex (NPC) function, but the mechanism was also unknown (3). We therefore hypothesized that CYP-derived EETs function as second messengers that regulate nuclear translocation of ERα in breast cancer by promoting permeability of the NPC. This hypothesis implies that biguanide drugs suppress nuclear translocation of the ERα through inhibition of CYP-derived EETs that serve as second messengers to open the NPC. Methods: Breast cancer cells were serum-starved for 16 hours and then treated with metformin, HBB, the (±)-14,15-EET regioisomer, or EET agonists, for varying times (30 min to 6 hours). Cells were fixed, permeabilized, and incubated with antibodies specific for ERα. After incubation with fluorescent secondary antibodies and Hoechst 33342 dye, cell images were acquired with a confocal microscope and the nuclear ERα fluorescence signal was quantified relative to the Hoechst signal. Quantitative PCR was used to measure the expression of ERα regulated genes. Results: (±) 14,15-EET (5 uM) treatment for 1 hour increased nuclear ERα by 66 ± 26% (n=9, p=0.031) in MCF-7 cells and 77 ± 24% (n=7, p=0.015) in ZR-75 cells. Treatment with EET agonists C22 (5 uM) and EET-A (5 uM) for 1 hour increased nuclear ERα by 32 ± 13% (n=7, p=0.047) and 39 ± 14% (n=7, p=0.02) in MCF-7 cells. Metformin (5 mM) treatment for 6 hours reduced nuclear ERα in MCF-7 by 35 ± 4.9 % (mean ± SEM, n=8, p=0.003). HBB (20 uM) treatment for 2 hours reduced the expression of three ERα regulated genes in MCF-7 cells: estrogen receptor alpha (ESR1) was reduced by 15 ± 1.7% (n=3, p=0.001), progesterone receptor (PGR) by 21 ± 0.6% (n=3, p=0.02) and L-type amino acid transporter 1 (SLC7A5) by 14 ± 0.2% (n=3, p=0.002). Conclusion: CYP epoxygenase activity in ER+HER2- breast cancer cells produces EETs, which function as second messengers that promote nuclear transit of ERα. Metformin and HBB inhibit nuclear ERα translocation, in part, through inhibition of CYP3A4 AA epoxygenase activity and this inhibition reduces ERα mediated gene expression, providing a novel mechanism for ERα regulation. 1. Cell Chem Biol. 2017 Oct 19; 24(10) 1259 - 1275. 2. Abstract of 109th AACR meeting (#LB-023), April 2019 3. Cell. 2016 Dec 15;167(7):1705-1718 Citation Format: Zhijun Guo, Jianxun Lei, Shaoping Wu, Julissa Molina-Vega, Paloma Cervantes, Aline R. Rom'Mand, Dylan C. Castillejo Mijangos, John R. Falck, Carol Lange, Jinhua Wang, David A. Potter. Metformin inhibits nuclear localization of estrogen receptor alpha in breast cancer cells, in part, by inhibition of CYP-mediated epoxyeicosatrienoic acid (EET) biosynthesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4372.

Immunohistochemical localization of estrogen receptor alpha (ERα) in the oviduct of Indian buffalo during follicular and luteal phases of estrous cycle.

The localization and distribution of estrogen receptor alpha (ERα) in different segments of oviduct of buffalo during follicular and luteal phases of estrous cycle were investigated using immunohistochemistry. Tissue samples from the different segments of oviduct from 12 buffaloes (six each during follicular and luteal phases of estrous cycle) were collected from slaughter house after assessing the gross morphology of ovaries. In addition, blood samples were collected from the animals before slaughter to estimate levels of estrogen and progesterone hormones. The tissue distribution of estrogen receptor was determined by immunohistochemical technique using one-step polymer HRPO staining system. The estrogen receptor was localized in the lamina epithelialis, propria submucosa, tunica muscularis, and tunica serosa. The maximum localization was observed in the lamina epithelialis, where both ciliated and secretory cell types were positive for ERα. Percentage of positive cells varied during the follicular and luteal phases of estrous cycle. The lining epithelium of oviductal glands was also intensely positive for ERα. No immunostaining was observed in any tunic of the oviduct when the primary antibody was replaced by antibody diluent or buffer, and it served as negative control. The data showed that highest immune positive cells were observed in the ampulla region of the oviduct and these cells were lowest in the utero-tubal junction (p < 0.05). Infundibulum, ampulla, and isthmus showed a higher percentage of ERα-positive cells during follicular phase of estrous cycle as compared with those of the luteal phase of estrous cycle (p < 0.05). There was no significant difference in the percentage positive cells during the two phases of estrous cycle in the utero-tubal junction. Immunogold labeling with anti-ERα antibody confirmed the findings of immunohistochemical study at subcellular level. The higher expression during the follicular phase was directly correlated with the level of estrogen hormone.

Changes in the immunohistochemical localization of estrogen receptor alpha and in the stereological parameters of the testes of mature and aged chickens (Gallus domesticus)

The aim of this study was to detect through immunohistochemistry the presence of the estrogen receptor alpha (ER-α) in different cell subpopulations, as well as to evaluate the histological and stereological changes of the testes of mature and aged chickens. Histological results demonstrated several changes in the tubular compartment and interstitial tissue in aged chicken testis as compared to mature chicken testis. Stereological results revealed that the mature chicken testis increases in volume as well as the total volume of the tubular compartment, whereas the total volume of the interstitial tissue and the total volume of Leydig cells diminishes as compared to aged chicken testis. The increase in the total volume of Leydig cells shown in aged chicken testis is due to the increase of cellular volume of Leydig cells, and not in their number, which decreases in aged chicken testis. Results also revealed positive ER-α immunostaining in mature and aged chicken testes, but cellular distribution of ER-α immunostaining changed according to the animal's age. In mature chicken testis, the ER-α was localized in the nuclei of germ and somatic cells. In contrast, in the aged chicken testis, only scarce spermatogonia presented ER-α immunoreactivity. This differential expression of ER-α may contribute to regulate the reproductive function in the mature chicken or the cessation of reproductive function in aged chickens.

Dynamic subcellular localization of estrogen receptor alpha during the first two cleavages of mouse preimplantation embryos

Zygotic gene activation (ZGA) is one of the most important events after mouse fertilization, but the mechanisms underpinning it are still unclear. Estrogen receptor alpha (ERα) is a versatile player in animal development. Our preliminary studies showed that ERα-specific antagonists blocked mouse 2-cell development and inhibited ZGA related gene expression, indicating an indispensable role of maternal ERα in early mouse preimplantation embryo development (PED). Here, we performed immunostaining detection to investigate the cell cycle specific subcellular localization of ERα, and serine 118 phosphorylated ERα (pERα-S118), during the first two cleavages of mouse PED. Our results showed that ERα nuclear localization appeared at 1-cell S-phase, disappeared at metaphase, and reappeared since the G1 phase of 2-cell embryos. Nuclear expression of pERα-S118 started at the 1-cell S-phase, but was absent at the G1 phase of 2-cell embryos, and reappeared since the 2-cell S-phase. Interestingly, pERα-S118 showed a dynamic expression pattern among nuclear, nuclear surface, cytoplasm and cell membrane. These results indicate that maternal derived ERα might still function in the first two cleavages of mouse PED, during which ZGA occurs.

Nonylphenol affects activity of human reproductive cells

Nonylphenol (NP) is an industrial compound belonging to Endocrine Disrupting Chemicals (EDCs) with xenoestrogenic activity abundantly present in the environment. Its xenoestrogenic activity was demonstrated both in vitro and in vivo. Estrogens play an important role in development and growth of human endometrium and prostate. Since NP mimics endogenous estrogens, it could have a negative influence on normal physiology of both organs. In this study we examined the effects of NP and 17-β-estradiol (E2) on human prostate adenocarcinoma epithelial cell line (LNCaP) and endometrial adenocarcinoma cell line Ishikawa. Firstly, we performed a MTT assay in order to see cell proliferation. We found that both NP and E2 stimulated prostate and endometrial cell proliferation but in different manner. In fact, on human prostate cells NP induced a non-monotonic increase of cell proliferation whereas on endometrial cells its effect was dose-dependent. Secondly, we studied expression and localization of estrogen receptor alpha (ERα) in both LNCaP and Ishikawa cells. Immunofluorescence and western blot analyses revealed that both NP and E2 induced cytoplasm-nucleus translocation of ERα even if E2 was able to induce an early nuclear translocation respect to NP. Finally through qPCR analysis we studied changing in gene expression of genes involved in proliferation pathways. We observed that in prostate cells NP and E2 induced upregulation of cyclin D1, ki-67, cytochine IL-8 and IL-1β together with a downregulation of cyclin E. On the contrary, in human endometrial cells gene upregulation was present only after E2 treatment whereas NP seem have no effects on these specific gene targets. These results showed that NP has estrogenic activity on human reproductive cells both in male and female system, but probably it may also interfere with other molecular pathways to induce a deregulation of normal cell physiology as seen in human endometrial cells.

Changes in the cellular localization of estrogen receptor alpha in the growing and regressing ovaries of Gallus domesticus during development

In this work, the presence of estrogen receptor alpha (ER-α) was determined in different cell subpopulations in the left growing and right regressing ovaries of Gallus domesticus from 13-day-old chicken embryos to one-month-old chickens by immunohistochemistry. Results revealed positive ER-α immunostaining in both ovaries during development, but the percentage, staining intensity, and cellular distribution of ER-α immunostaining changes according to whether it is the left or right ovary and with the animal’s age. In the left ovary, the ER-α was localized in the nuclei of the germinal epithelium and in germ cells of the ovarian cortex, as well as in the interstitial cells, undifferentiated cells, and epithelial cells of the lacunar channels of the ovarian medulla in all ages. In contrast, in the right ovary from 13-day-old chicken embryos to one-week-old chickens, only the epithelial cells of lacunar channels were ER-α immunoreactive, but in the right ovary of one-month-old chickens both the epithelial cells of lacunar channels and the interstitial cells presented ER-α. These results demonstrate differential expression of ER-α in both chicken ovaries during development in a cell type-specific distribution, suggesting that these differences may be regarded as an important cause in the process of asymmetric ovarian development in the chicken.

Estrogen receptor alpha localization in the testes of men with normal spermatogenesis

It is known that estrogens act on the male reproductive tract by binding to estrogen receptors (ER) α and β. However, studies on ER localization in the human testis are discordant. The aim of this study was to investigate the localization of ERα in the testes of adult men with normal spermatogenesis. Semen analysis of ten adult men revealed azoospermia. FSH, LH and testosterone serum concentrations were within normal values, and the volume of the testes was normal, hence obstructive azoospermia was suspected. The tissues from testicular surgical biopsies were fixed in Bouin's fluid and embedded in paraffin. Assessments of the seminiferous epithelium (scoring 10 to -1), the number of Leydig cells (scoring 1 to 5), the areal fraction of intertubular space (IS), measurements of seminiferous tubule diameter, and the thickness of the tubular wall, were performed on microscopic sections. Immunohistochemical staining was applied with monoclonal antibodies against ERα. The mean spermatogenesis score was 10 points; IS - 30.6 ± 8.1%; seminiferous tubule diameter - 193.9 ± 19.4 μm; thickness of tubular wall - 7.44 ± 1.1 μm; number of Leydig cells - 1.6 ± 1.1 points. Immunohistochemical staining showed the localization of ERα to be in the Sertoli and Leydig cell cytoplasm, while ERα was absent in germ cells. The results of testicular tissue analysis confirmed its normal structure and normal, full spermatogenesis. The presence of ERα in Sertoli and Leydig cells in normal human testis demonstrated in this study suggests that estrogens may affect testicular function.

Estrogen receptor alpha localization in the testes of men with normal spermatogenesis

It is known that estrogens act on the male reproductive tract by binding to estrogen receptors (ER) a and b. However, studies on ER localization in the human testis are discordant. The aim of this study was to investigate the localization of ERa in the testes of adult men with normal spermatogenesis. Semen analysis of ten adult men revealed azoospermia. FSH, LH and testosterone serum concentrations were within normal values, and the volume of the testes was normal, hence obstructive azoospermia was suspected. The tissues from testicular surgical biopsies were fixed in Bouin’s fluid and embedded in paraffin. Assessments of the seminiferous epithelium (scoring 10 to –1), the number of Leydig cells (scoring 1 to 5), the areal fraction of intertubular space (IS), measurements of seminiferous tubule diameter, and the thickness of the tubular wall, were performed on microscopic sections. Immunohistochemical staining was applied with monoclonal antibodies against ERa. The mean spermatogenesis score was 10 points; IS — 30.6 ± 8.1%; seminiferous tubule diameter — 193.9 ± 19.4 μm; thickness of tubular wall — 7.44 ± 1.1 μm; number of Leydig cells — 1.6 ± 1.1 points. Immunohistochemical staining showed the localization of ERa to be in the Sertoli and Leydig cell cytoplasm, while ERa was absent in germ cells. The results of testicular tissue analysis confirmed its normal structure and normal, full spermatogenesis. The presence of ERa in Sertoli and Leydig cells in normal human testis demonstrated in this study suggests that estrogens may affect testicular function.

Localization of Estrogen Receptor Alpha and Progesterone Receptor B in the Bovine Ovary During the Follicular and Luteal Phase of the Sexual Cycle

Summary Ovarian steroid hormones, mainly estrogen and progesterone, play a central role in the regulation of ovarian functions. The biological effects of progesterone (PR) and estrogen (ER) are dependent on the activation of both PR and ER isoforms. In the present study, we studied the cellular distribution and localization of progesterone receptor B (PR-B) and estrogen receptor alpha (ERα) in the bovine ovaries during the follicular and luteal phases of the sexual cycle using immunohistochemical methods. The estrous cycle stage of 23 Holstein bovines was evaluated by gross and histological appearance of ovaries and blood steroid hormone values. Tissue samples from ovaries were fixed in 10% formaldehyde for routine histological processing. ERα immunoreactivity was observed in nuclei of granulosa cells of growing follicles at all stages from primary to mature follicles, epithelial cells of the germinal epithelium, stroma cells, theca cells, corpora lutea cells and cells of the corpora albicantia. Nuclear staining for PR-B was not detected in the granulosa cells in all sizes of follicles, but was positively stained in germinal epithelium cells, stroma cells, corpora albicantia cells and corpora lutea cells. In conclusion, ERα and PR-B immunoreactivity in bovine ovary were not indicate too much difference between the phases of the sexual cycle but have important differences in cell-spesific localization.

Estrogen Receptor Alpha Localization in Thai Swamp Buffalo Oviduct during the Follicular and Luteal Phases

Estrogen Receptor Alpha Localization in Thai Swamp Buffalo Oviduct during the Follicular and Luteal Phases

Characterization of estrogen receptors alpha and beta in uterine leiomyoma cells

Cellular and subcellular localization of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in uterine leiomyomas. Retrospective study. University of La Laguna (ULL) and Canary University Hospital (HUC). Premenopausal and postmenopausal women with uterine leiomyomas. Hysterectomy and myomectomy. Estrogen receptor alpha was only present in smooth muscle cells with variation in the subcellular location in different leiomyomas. Estrogen receptor beta was widely distributed in smooth muscle, endothelial, and connective tissue cells with nuclear location in all cases studied; variations were only found in the muscle cells for this receptor. Estrogens operate in leiomyoma smooth muscle cells through different receptors, alpha and beta. However they only act through the ERbeta in endothelial and connective cells.

Cellular localization of estrogen receptor-alpha (ERα) and -beta (ERβ) mRNA in the boar testis

Boar testes synthesize high amounts of estrogens which are known to stimulate several male sexual functions in a variety of extragonadal target tissues. Possible effects within the testis depend on the existence of the estrogen receptor subtypes alpha and beta (ERalpha, ERbeta). The precise cellular localization of these subtypes within the testis was, so far, based mainly on protein expression studies using different antibodies in several species including boars shows contradictory results. Therefore, we investigated the ERalpha and ERbeta gene expression using RT-PCR of testis homogenates and RT-PCR after UV-single cell microdissection combined with in-situ hybridization of four fertile boars with an average age of 32 weeks. Both ERalpha and ERbeta mRNA were found in testis homogenates. Using in-situ hybridization and UV-single cell microdissection ERalpha mRNA was present in type A and type B spermatogonia up to mid-pachytene primary spermatocytes in stage V-VIII and stage I of the seminiferous epithelial cycle, but not in other cells. ERbeta mRNA was found only in Sertoli cells. Interstitial Leydig cells revealed neither ERalpha nor ERbeta mRNA. The data suggest a direct impact of estrogen in the boar on Sertoli cell function via ERbeta and germ cell formation via ERalpha.

3P269 海馬神経細胞での遠心分離法によるERαの分布解析(神経回路・脳の情報処理))

3P269 海馬神経細胞での遠心分離法によるERαの分布解析(神経回路・脳の情報処理))

Estrogen receptor (ER)alpha and ER beta are both expressed in human ejaculated spermatozoa: evidence of their direct interaction with phosphatidylinositol-3-OH kinase/Akt pathway.

Human and animal models have evidenced how estrogen insufficiency is associated with abnormal spermatogenesis and male infertility. We previously demonstrated that estradiol is able to influence both capacitation and acrosome reaction in human ejaculated spermatozoa. It remains to be elucidated whether the biochemical changes induced by estradiol, in a rapid nongenomic way, are mediated by a single estrogen receptor (ER) or by the two ER subtypes, ER alpha and ER beta. In the present study, we have first demonstrated the concomitant expression of ER beta and ER alpha in human ejaculated spermatozoa. By RT-PCR and Southern blot, transcripts of both ERs were detected. Western blot analysis showed ER alpha and ER beta proteins at the same size as the "classical" ERs. The localization of ER alpha and ER beta with the immunocytochemistry shows a differential distribution of the two ER subtypes, the former being prevalently located in the midpiece, but the latter being in the tail. Estradiol has been associated with sperm longevity; however, the mechanism through which estradiol acts in sperm survival was never investigated. Upon estradiol exposure, we observed an enhanced phosphorylation of the proteins involved in the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway like PDK1, Akt, GSK-3, Bcl-2, together with ERK1/2, which was also involved in cell survival signals. Moreover, such phosphorylations were reduced in the presence of ICI 182, 780, addressing the role of estradiol and ERs in sperm survival. For instance we have provided, for the first time, a different interaction of the two ERs with the PI3K/Akt pathway, because ER alpha interacts with the p55 regulatory subunit of PI3K, whereas ER beta interacts with Akt1. However, it still remains to be elucidated whether the functional role of each of the ER subtypes in sperm survival signaling is redundant or distinct.

ラット海馬におけるエストロゲン受容体ERαの分布の解析

ラット海馬におけるエストロゲン受容体ERαの分布の解析

Subcellular distribution of native estrogen receptor alpha and beta isoforms in rabbit uterus and ovary.

The association of estrogen receptors with non-nuclear/cytoplasmic compartments in target tissues has been documented. However, limited information is available on the distribution of estrogen receptor isoforms, specially with regard to the newly described beta isotype. The subcellular localization of estrogen receptor alpha and beta isoforms was investigated in rabbit uterus and ovary. Native alpha and beta subtypes were immunodetected using specific antibodies after subjecting the tissue to fractionation by differential centrifugation. The ovary expressed alpha and beta estrogen receptors in predominant association to cytosolic components. However, in the uterus, a substantial proportion of the total estrogen binding capacity and coexpression of the two isoforms was detected in mitochondria and microsomes. The mitochondrial-enriched subfraction represented an important source of 17beta-estradiol binding, where the steroid was recognized in a stereospecific and high affinity manner. The existence of mitochondrial and membrane estrogen binding sites correlated with the presence of estrogen receptor alpha but mainly with estrogen receptor beta proteins. Using macromolecular 17beta-estradiol derivatives in Ligand Blot studies, we could confirm that both alpha and beta isoforms were expressed as the major estrogen binding proteins in the uterus, while estrogen receptor alpha was clearly the dominant isoform in the ovary. Other low molecular weight estrogen receptor alpha-like proteins were found to represent an independent subpopulation of uterine binding sites, expressed to a lesser extent. This differential cellular partitioning of estrogen receptor alpha and beta forms may contribute to the known diversity of 17beta-estradiol effects in target organs. Both estrogen receptor alpha and beta expression levels and cellular localization patterns among tissues, add complexity to the whole estrogen signaling system, in which membrane and mitochondrial events could also be implicated.

Localization of estrogen receptor alpha (ERα) in pyramidal neurons of the developing rat hippocampus

During development, estrogen has a variety of effects on morphological, biochemical and electrophysiological properties of hippocampal neurons. Correspondingly, estrogen receptor (ER) binding and mRNA increase transiently in the developing hippocampus. In this study, we used immunocytochemistry to determine the localization of the ERα subtype in the developing rat hippocampus. Nuclear staining was present in pyramidal cells and some interneurons of the CA1 and CA3 regions of the developing rat hippocampus. Little or no immunoreactivity was observed in postnatal day (P)0 animals (day of birth=P0), however, beginning on P4, ERα-immunoreactivity (ERα-ir) was visible and reached maximal levels by P10. These levels subsequently declined to low levels so that by P15, levels approximated those of adult females. Western blot analysis confirmed that this antibody recognized a 67 kDa protein, characteristic of the full-length ERα protein, in the hippocampus and pituitary. Furthermore, most of the ERα-immunopositive cells in the hippocampus were located in the pyramidal cell layer, and did not co-localize appreciably with γ-aminobutyric acid (GABA) at any age examined. We conclude, based on the immunocytochemical localization of ERα, that the effects of estrogen on biochemistry and morphology of the developing hippocampus may be direct through the ERα subtype in hippocampal pyramidal cells.

A hormone pulse induces transient changes in the subcellular distribution and leads to a lysosomal accumulation of the estradiol receptor α in target tissues

An intrauterine pulse-stimulation with estradiol induced changes in the subcellular localization of estrogen receptor alpha in porcine endometrium, as detected with F(ab') fragments of various anti-receptor antibodies covalently linked to nanogold. The low-sterically hindered immunoreagents--recognizing different epitopes within the hormone binding domain--allowed for an efficient immunolabeling of estradiol receptor alpha, detecting it both in the cytoplasm and the nucleus of nonstimulated epithelium cells. In the cytoplasm, the receptor often seemed to be associated with actin filaments and the endoplasmatic reticulum. After the stimulation with estradiol, a predominantly nuclear localization and a labeling of nucleoli was observed. Our immunoelectron microscopy study demonstrates a localization of the receptor in cytoplasmic organelles that increased after the hormone pulse. These organelles exhibited the morphological properties of lysosomes and relocated to the perinuclear area. In analogous cytoplasmic organelles, the presence of cathepsin D was detected via indirect immunogold labeling, justifying their classification as lysosomes. Quantitative examinations revealed that not only the number of lysosomes in the proximity of the nucleus but also their immunostaining for estradiol receptor alpha increased significantly after the hormone pulse. Thus, estradiol induces both the rapid shift of receptor into the nucleus, a slower perinuclear accumulation of lysosomes and an increase of lysosomal ERalpha-immunoreactivity. These results suggest a role for lysosomes in the degradation of receptor shuttling out of the nucleus. This could serve as termination of the estradiol receptor alpha-dependent activation of target cells. This hypothesis is strengthened by the fact that the receptor content in uterine tissue declined drastically few hours after the hormone pulse.