Differentiation of human adipose stromal cells in vitro into insulin-sensitive adipocytes.

Adipose tissue-related diseases such as obesity and type 2 diabetes are worldwide epidemics. In order to develop adipose tissue cultures in vitro that mimic more faithfully the in vivo physiology, new well-characterized and publicly accepted differentiation methods of human adipose stem cells are needed. The aims of this study are (1) to improve the existing natural adipose tissue extract (ATE)-based induction method and (2) to study the effects of a differentiation method on insulin responsiveness of the resulting adipocytes. Different induction media were applied on human adipose stromal cell (hASC) monocultures to study the differentiation capacity of the induction media and the functionality of the differentiated adipocytes. Cells were differentiated for 14 days to assess triglyceride accumulation per cell and adipocyte-specific gene expression (PPARγ, adiponectin, AP2, leptin, Glut4, Prdm16, CIDEA, PGC1-α, RIP140, UCP and ADCY5). Insulin response was studied by measuring glucose uptake and inhibition of lipolysis after incubation with 100 or 500 nM insulin. The selected differentiation method included a 3-day induction with ATE, 6 days in serum-free medium supplemented with 1.15 µM insulin and 9.06 µM Troglitazone, followed by 4 days in a defined serum- and insulin-free stimulation medium. This protocol induced prominent general adipocyte gene expression, including markers for both brown and white adipocytes and triglyceride accumulation. Moreover, the cells were sensitive to insulin as observed from increased glucose uptake and inhibition of lipolysis. This differentiation protocol provides a promising approach for the induction of hASC adipogenesis to obtain functional and mature human adipocytes.

Evaluation of the selected barrier properties of retinal pigment epithelial cell line ARPE-19 for an in-vitro blood-brain barrier model.

In-vitro models that maintain complex transport mechanisms and structural properties associated with the blood-brain barrier in vivo would be useful in drug permeability and neurotoxicological studies. To evaluate the suitability of a human retinal pigment epithelial cell line for a blood-brain barrier model, we have compared the barrier properties of the human retinal pigment epithelial cell line ARPE-19, the human colonic adenocarcinoma cell line Caco-2, and primary porcine microvessel endothelial cells. The tight junction proteins occludin and ZO-1 were stained immunocytochemically. The paracellular ionic permeability was evaluated by measuring the trans-epithelial or trans-endothelial electric resistance. To evaluate the active transport mechanisms, the existence and the activity of the efflux transporters, P-glycoprotein and multidrug resistance-associated proteins, were studied. All the cell types in this study stained positively for occludin and ZO-1. However, the trans-endothelial electric resistance of ARPE-19 cells was low compared with that of primary porcine microvessel endothelial cell and Caco-2 cells. In addition, both the P-glycoprotein expression and its activity in ARPE-19 cells were low. In conclusion, the barrier properties of the human ARPE-19 cell line were not satisfactory for a blood-brain barrier model. For future studies, it is important to develop a human brain endothelial cell line with expression of the complex in-vivo properties of the blood-brain barrier.

Intra-laboratory validated human cell-based in vitro vasculogenesis/angiogenesis test with serum-free medium.

Vasculogenesis and angiogenesis are the processes by which new blood vessels are formed. We have developed a serum-free human adipose stromal cell and umbilical cord vein endothelial cell based vasculogenesis/angiogenesis test. In this study, the test was validated in our GLP laboratory following the OECD Guidance Document 34 [1] using erlotinib, acetylic salicylic acid, levamisole, 2-methoxyestradiol, anti-VEGF, methimazole, and D-mannitol to show its reproducibility, repeatability, and predictivity for humans. The results were obtained from immunostained tubule structures and cytotoxicity assessment. The performance of the test was evaluated using 26 suspected teratogens and non-teratogens. The positive predictive value was 71.4% and the negative predictive value was 50.0%, indicating that inhibition of vasculogenesis is a significant mechanism behind teratogenesis. In conclusion, this test has great potential to be a screening test for prioritization purposes of chemicals and to be a test in a battery to predict developmental hazards in a regulatory context.

Vitamin D fortification as public health policy: significant improvement in vitamin D status in young Finnish men.

BACKGROUND: Vitamin D insufficiency is common in northern countries during wintertime. In Finland, after the recommendation by the Ministry of Social Affairs and Health, vitamin D has been added to liquid milk products and margarines from February 2003. OBJECTIVE: We determined the effects of national policy on vitamin D fortification on vitamin D status among young Finnish men. DESIGN: A comparison before and after intervention with study population of 196 young Finnish men (18-28 years) was carried out. Serum 25-hydroxyvitamin D3 (25-OHD3) concentrations were determined with the OCTEIA enzymeimmunoassay by IDS (Immunodiagnostic Systems Limited, Bolden, UK) in January 2003 (n = 96) and in January 2004 (n = 100), nearly 1 year after national vitamin D fortification had started. RESULTS: The mean serum 25-OHD3 concentrations during the wintertime increased by 50% after implementation of the vitamin D fortification of dairy products. Correspondingly, the prevalence of vitamin D insufficiency (serum 25-OHD3 < 40 nmol/l) was decreased by 50% from 78% in January 2003 to 35% in January 2004. CONCLUSIONS: Our results demonstrate that national vitamin D fortification substantially improved the vitamin D status of young Finnish men. Still, a third remained vitamin D insufficient.

Sex steroid sensitivity of developing bursa of Fabricius.

Sex steroid sensitivity of the bursa of Fabricius (BF) was studied from the early embryonic time until its regression. Expression of progesterone receptor (PR) served as a dual marker: first, as a marker for progesterone sensitivity and second, as a marker for estrogen action, since it is an estrogen-induced protein. The progesterone binding molecule in the bursa was characterized by different chromatography methods and by steroid binding studies. We showed that it fulfils the criteria of a progesterone receptor by binding, structural and immunological properties. With immunohistochemistry and with the combined techniques of immunohistochemistry and autoradiography we demonstrated two cell types which express the PR: smooth muscle cells surrounding the BF and stromal cells located under the bursal epithelium and between the lymphoid follicles. The epithelium and the cells inside the lymphoid follicles were negative. Using immunoelectron microscopy the PR-expressing stromal cells were shown to be fibroblasts. The cloacal mesenchyme, from which the BF develops, was shown to be sensitive to exogenous estrogen very early during the embryonic time. The mesenchyme around and inside the developing BF reached estrogen sensitivity a few days later. The estrogen-sensitive mesenchymal cells were first seen surrounding the bursal primordium and later in the center of the plicae. During a natural sexual maturation without exogenous estradiol an expression of the PR was detected much later, at the age of 10-12 weeks after hatching. This expression correlates with the onset of the bursal regression and with the increase of the sex steroid levels in the blood. In the oviduct stroma PR was undetectable before the onset of sexual maturation. In the oviduct stroma PR becomes detectable a few weeks earlier than in the bursa.

Development of progestin-specific response in the chicken oviduct.

Avidin is a host acute defense protein induced by progestins and by inflammation caused by injurious factors such as microbes, viruses, toxic factors or tissue trauma. In the reproductive tract of egg-laying vertebrates avidin has evolved into a progestin-dependent secretory protein involved in anti-microbial action through its biotin avidity. For "progestin-dependent avidin" production, cellular differentiation by estrogen is necessary. In contrast, the expression of "progestin-independent or inflammation-induced avidin" does not require differentiation. Many cell types such as macrophages, heterophils and fibroblasts can produce avidin after non-specific cellular injuries. The wide distribution of avidin in avian, reptilian and amphibian species could be explained on the basis of its vital functions such as antimicrobial or antifungal, metabolic and immunomodulatory actions. The ontogeny of the progestin-dependent avidin synthesis is a complex event involving oviductal differentiation by steroid hormones leading to a specific gene expression. The first phase in oviductal differentiation by estrogens is characterized by a new chromatin organization and by an infiltration of progesterone receptor (PR)-containing mesenchymal cells into the subepithelial mucosa leading to epithelial cell differentiation ("mesenchymal and epithelial cell interaction"). The second phase in the differentiation of progestin-induced response is dependent on the presence of PR in the secretory cells. Two kinds of PR expression occur in the oviduct. The first is a "constitutive PR" and is found in the epithelial, submucosal and peritoneal cells of the immature chick oviduct without steroid treatment, and the second is an "inducible PR" found especially in the mucosal mesenchymal and smooth muscle cells. Avidin production requires PR in the target cells, but not all PR-containing cells can produce avidin. Therefore, in addition to PR, other transcription factors are needed to define the target cell specificity of the response to progestins. Earlier biochemical studies suggested that cytosolic and/or nuclear unoccupied PR was complexed as an 8 S form with the heat shock protein 90 (hsp90). Our immunohistochemical results, however, indicate that PR in vivo is not bound to hsp90, which is located entirely in the cytoplasm, whereas PR is an entirely nuclear protein in both ligand-occupied and unoccupied forms. Therefore, we assume that PR is a monomeric (4S) or homodimeric (5S) (chromatin?) protein associated to DNA. Ligand binding to PR appears to lead to a conformational change, dimer formation, tighter binding to PRE (progesterone responsive element) and to transcription factors, phosphorylation and proteolysis of PR as well as a chromatin change.(ABSTRACT TRUNCATED AT 400 WORDS)

Location of androgen receptor in human skin.

The distribution of androgen receptor (AR) in human skin was studied by an immunohistochemical method using a polyclonal antibody against the human AR. Skin samples of preputial skin and male and female nongenital skin were examined. The possible correlation of AR location to acne was studied in skin biopsies from skin areas affected or unaffected by acne. In preputial skin, AR was expressed in epidermal cells as well as in fibroblasts, smooth muscle cells, and endothelial cells of blood vessels in the dermal area. AR was found located also in the flat fibroblast-like cells of Pacinian corpuscles. In nongenital skin, AR was also expressed in the basal cells and glandular cells of sebaceous glands, in the outer root sheath of hair follicles, and in eccrine sweat glands. The presence of AR in different cell types in the skin reflects the numerous direct effects androgens may have on this target tissue. The distribution of AR was similar in male and female skin.

Progesterone receptor in the chick bursa of fabricius: characterization and immunohistochemical localization.

A high affinity progesterone-binding site was studied in the chick bursa of Fabricius. The dissociation constant for progesterone was 1.4 nM, and the concentration of progesterone-binding sites increased with estradiol treatment. In estradiol-treated bursas, the receptor concentration was about 240 fmol/mg protein. The binding site was specific for progestins, with the following order of affinities: ORG 2058 greater than progesterone greater than promegestone. Androgens, dexamethasone, and estradiol were weak competitors for progesterone binding in the bursa cytosols from estradiol-treated chicks. Immunoglobulin G fraction of antiserum (immunoglobulin G-RB) raised in rabbit against the B-subunit of chick oviduct progesterone receptor (PR) was used for an immunohistochemical study. The PR was found only in the interfollicular cells, which were most probably nonlymphoid cells. Staining was localized exclusively in the elongated nuclei of these cells. No staining was seen in the bursal epithelium or inside the lymphoid follicles. The results indicate that the interfollicular cells of the bursa contain specific PRs which are under estrogen regulation as in the oviduct. Thus, these cells might be under direct progesterone regulation.

Progesterone-induced avidin as a marker of cytodifferentiation in the oviduct: comparison to ovalbumin.

Immunohistochemical analysis of avidin and ovalbumin expression in the normally developing chick oviduct was compared to those changes induced by exogenous estrogen. Oviduct maturation was found to occur in two consecutive phases: slow proliferation and rapid differentiation. Mitosis was induced in the epithelium by estrogen, whereas it was inhibited by progesterone. Endogenous progesterone may retard the proliferation and prevent the differentiation, an effect that is overridden by increased estrogen concentration at the beginning of differentiation. Short secondary stimulation was shown to closely mimic normal maturation. When chicks treated with diethylstilbestrol (DES) for 1 month were allowed to mature, there were marked alterations in oviduct histology and laying behavior. The tubular glands were found to form from the surface epithelium as budlike invaginations, and these cells also contained avidin and ovalbumin. Ovalbumin production was stable in tubular glands. In contrast, the intensity of avidin staining was variable between gland cells even in the same sections. It was conspicuous that the number of avidin-expressing gland cells diminished markedly when estrogen treatment was prolonged over 1 week. After 2-week stimulation with DES, avidin was expressed predominantly by cells of the basal layer of pseudostratified surface epithelium, and ovalbumin mainly by tubular glands and cells of the luminal layer of surface epithelium. Neither of these proteins was expressed by goblet cells. Expression of progesterone receptor, characterized by two antibodies (polyclonal IgG-RB and monoclonal PR6), did not explain the heterogeneity of expression of avidin and ovalbumin, but probably reflects various differentiation stages of epithelial cells.

Distribution of progesterone receptor in chicken: novel target organs for progesterone and estrogen action.

Expression of progesterone receptor (PR) in various organs of sexually immature chickens and after estrogen treatment was studied by immunohistochemical and Western blotting analyses. Constitutive PR expression was observed in the mesothelium and stroma of the esophagus, proventriculus, liver, spleen, pancreas, heart and lung. In the urogenital tract, PR was expressed in the mesothelial and stromal cells and smooth muscle of blood vessels. Estrogen treatment induced PR expression in the stroma and smooth muscle of the gall bladder and in the epithelium and stroma of the trachea. In the ovary of immature chickens PR was localized in the epithelium, stroma and smooth muscle and was induced in the granulosal cells by estrogen. In most tissues there was more PR-B than PR-A expression and this PR-B dominance remained after estrogen treatment. These results suggest that progesterone and estrogen may have physiological effects on many organs outside the genital tract not previously known as steroid-target tissues.

Characterization of the estrogen-sensitive cells expressing progesterone receptor in the bursa of Fabricius.

Cells expressing the progesterone receptor (PR) in the bursa of Fabricius (BF) were studied with immunohistochemistry at light-microscopic level, with immunoelectron microscopy (immuno-EM) and with non-specific esterase histochemistry. The antibody (IgG-RB) directed to the B component of the chick oviduct progesterone receptor was shown by immunoblotting to be specific for the PR and to recognize the PR also in the bursa. Two cell types in the BF contain the PR: stromal cells in the interfollicular-subepithelial area and smooth muscle cells lining the BF. The PR was localized in the nuclei of these cells. The bursal epithelium and the cells inside the follicles were not stained for PR. Electron microscopically the immunoreaction precipitate was localized on condensed heterochromatin and on dispersed euchromatin. The cells expressing the PR resembled electron microscopically fibroblasts. Their cytoplasm was rich in rough endoplasmic reticulum indicating active protein synthesis. By non-specific esterase histochemistry we showed that the PR-containing cells were not macrophages, which are morphologically indistinguishable from stromal cells. In the bursae of young untreated chicks the PR was not seen, but was inducible by estradiol treatment and was spontaneously expressed after the onset of sexual maturation. It is concluded that both the stromal fibroblasts and the smooth muscle cells in the BF are estrogen and progesterone sensitive. The expression of PR after the onset of sexual maturation indicates that the BF is directly affected by sexual maturation-associated factors. We suggest that estrogen and progesterone participate in tissue remodelling during bursal involution via the stromal cells and may affect bursal functions via the smooth muscle cells.

Progesterone receptor in chicken bursa of Fabricius and thymus: evidence for expression in B-lymphocytes.

In the present work constitutive progesterone receptor (PR) expression in the chicken bursa of Fabricius was detected in the stromal, smooth muscle and follicular medullary cells and smooth muscle cells of blood vessels. PR expression was increased during sexual maturation and after estrogen treatment. Bursal medullary PR-positive cells were further characterized to be B-lymphocytes by flow cytometric analysis. In addition, estrogen induced expression of PR in the bursal FAE-cells (follicle-associated epithelial cells). In the thymus PR was expressed constitutively in the connective tissue cells of the capsule and interfollicular septa, in a few medullary cells and in vascular smooth muscle. The PR-positive medullary cells consisted of epithelial cells, large polygonal cells resembling macrophages and plasma cells. T-lymphocytes were PR-negative. Estrogen up-regulated PR expression in the thymus. Immunoblotting studies revealed that both isoforms of PR, i.e. PR-A and PR-B, were expressed in the bursa of Fabricius and thymus with PR-B dominance. These results suggest that the chicken primary lymphoid organs bursa and thymus are under regulation of estrogen and progesterone. Expression of PR in B-lymphocytes, macrophages and plasma cells in the chicken is documented for the first time and suggests evidence for direct action of progesterone on immune responses.

Heat shock protein 90 and the nuclear transport of progesterone receptor.

Steroid receptors exist as large oligomeric complexes in hypotonic cell extracts. In the present work, we studied the nuclear transport of the 2 major components of the oligomeric complex, the receptor itself and the heat shock protein 90 (Hsp90), by using different in vitro transport systems: digitonin permeabilized cells and purified nuclei. We demonstrate that the stabilized oligomeric complex of progesterone receptor (PR) cannot be transported into the nucleus and that unliganded PR salt dissociated from Hsp90 is transported into the nucleus. When nonstabilized PR oligomer was introduced into the nuclear transport system, the complex dissociated and the PR but not the Hsp90 was transported into the nucleus. If PR exists as an oligomeric form after synthesis, as suggested by the experiments with reticulocyte lysate, the present results suggest that the complex is short-lived and is dissociated before or during nuclear transport. Thus, the role of Hsp90 in PR action is likely to reside in the Hsp90-assisted chaperoning process of PR preceding nuclear transport of the receptor.

Localization of glucocorticoid receptor interacting protein 1 in murine tissues using two novel polyclonal antibodies.

OBJECTIVE: Glucocorticoid receptor interacting protein 1 (GRIP1) is a coactivator that binds to the nuclear hormone receptors in a ligand-dependent manner and mediates transcriptional activation of the target genes. The aim of this study was to investigate GRIP1 expression in various murine tissues and whether the protein is nuclear, cytoplasmic, or both. DESIGN: Two novel polyclonal antibodies against amino acids 34-47 and 468-481 of GRIP1 were raised and characterized in order to study the GRIP1 expression with immunohistochemistry. RESULTS: Transient transfection studies with COS cells showed a clearly nuclear staining pattern and also immunohistochemical localization of GRIP1 was mainly nuclear, but cytoplasmic expression was seen as well. GRIP1 was expressed in epithelial cells of the submandibular gland, gastrointestinal tract, pancreas, kidney, uterus, mammary gland, testis, prostate, trachea, lungs and adrenal gland. GRIP1 was also detected in stromal cells of colon, rectum, urinary bladder, vagina, uterus, mammary gland and trachea, and to a lesser extent in esophagus, ureter, urethra, thymus and spleen. Smooth muscle cells of the gastrointestinal and urinary tract, uterus, epididymis, prostrate and bronchioles expressed GRIP1. Blood vessels of many organs, capsule of the kidney and prostate, mesovarium, adipocytes of the mammary gland, pericardium and cartilage of the trachea were also GRIP1-positive. Liver, thyroid gland and striated muscle did not express GRIP1. CONCLUSIONS: GRIP1 was expressed in a wide variety of murine organs, and expression varied between cell types and organs. In addition to mainly nuclear localization of endogenous GRIP1, cytoplasmic expression was seen as well.

Progesterone receptor and hsp90 are not complexed in intact nuclei.

In hypotonic cell extract (cytosol), unliganded progesterone receptor (PR) is known to form an oligomeric complex with heat shock protein 90 (hsp90), and this complex does not bind to DNA. Since ligand binding has been shown to render the complex less stable in vitro, it has been proposed that ligand binding regulates DNA binding and receptor activity in vivo by altering the stability of the oligomeric complex. However, there is no direct evidence as to whether this oligomeric complex is present in vivo. The present study addressed this problem. First, we used an immunoelectron-microscopic technique and monoclonal antibodies to ascertain the location of PR and hsp90 in chick oviduct cells. Hsp90 was found in the cytoplasm and PR in the nucleus. To study the relative affinities of the PR and hsp90 antibodies, we then constructed a chimeric protein (PR-hsp90), which was expressed in the HeLa cells. Both hsp90 and PR antigens of the chimera were detected in the nuclei with the same intensity, which indicates that the antibodies have equal sensitivities in detecting their antigens. This suggests that if significant amounts of nuclear hsp90 were present in intact cells, it should have been detected by our method. Our results indicate that the PR does not exist in vivo as an oligomeric, nonDNA-binding form in the cell nuclei and that the oligomeric form found in tissue extracts is possibly formed during tissue processing.

Immunolocalization of retinoic acid receptors in rat, mouse and human ovary and uterus.

We raised an antibody against a synthetic peptide corresponding to amino acids 155-174 of human retinoic acid receptor alpha (RAR-alpha). The sequence is highly homologous in all RARs and their isoforms. When mouse and human RARs (alpha, beta and gamma) expressed in Cos cell were analysed with immunoblot, all receptors gave a specific 51 K signal. Mouse RAR-gamma gave an additional signal corresponding to 58 K. In human teratocarcinoma cells (F9) both 51 and 58K molecule sizes were detected. The RAR expression in F9 cells was slightly down-regulated in charcoal-stripped culture medium and returned to normal level after retinoic acid treatment. The 51 K protein was found in all ovarian and uterine samples, but the quantity of the 58 K protein varied in different species and organs, being highest in the mouse uterus and the rat and human ovary. Using immunohistochemistry the RARs were found in the nuclear compartment. In the rat uterus, positive immunoreaction was found mainly in the nuclei of epithelial, uterine glandular and stromal cells. In the rat ovary, positive reaction was found in the nuclei of germinal epithelial, follicular and stromal cells.

Spermatogenesis in the vitamin A-deficient rat: possible interplay between retinoic acid receptors, androgen receptor and inhibin alpha-subunit.

In order to understand the mechanisms of retinol action on the testis, testicular retinoic acid receptor alpha, beta(RAR alpha and beta), androgen receptor (AR) and inhibin alpha-subunit were studied in normal, vitamin A-deficient (VAD) and vitamin A-supplemented rats by immunohistochemistry and immunoblotting. Compared to the normal testis, expression of 110 K AR was up-regulated by vitamin A withdrawal, whereas 51 K RAR alpha remained unchanged. An additional 55 K RAR alpha signal was observed. Readministration of retinol caused a marked decrease of AR in the VAD testis. By 24 h, AR declined to below the normal level. Although the 51 K RAR alpha signal remained unchanged, the 55 K band was slightly up-regulated at 6 h after retinol administration. A 51 K RAR beta protein was seen in the VAD but in not the normal testis. The intensity of the 51 K RAR beta band remained constant before and after the administration of retinol, but it had a slight up-shift at 6 h after retinol injection, suggesting post-translational modification of the receptor. The inhibin alpha-subunit of 18 K protein was undetectable in the VAD testis and increased to above normal level at 24 h after retinol administration. Immunohistochemically, nuclear AR immunostaining was more intense in the VAD testis than in the normal testis. The intensity of immunostaining declined in all AR-positive cells after the injection of retinol, but the decrease was more evident in Sertoli than in other cells. At 24 h after retinol the immunostaining was undetectable in most Sertoli cells. The regulation of the inhibin alpha-subunit by retinol in the cytoplasm of Sertoli cells detected by immunohistochemistry was correlated to the results in immunoblotting. These results suggest a possible interplay between retinoids, androgen and inhibin signalling systems in Sertoli cells in the regulation of spermatogenesis during retinol action.

Type II antagonists impair the DNA binding of steroid hormone receptors without affecting dimerization.

Two types of steroid antagonists exert their activity by different mechanisms when bound to the cognate receptor. Type I anti-progestins, such as RU486, induce DNA binding of the human progesterone receptor (hPR), while no hPR/DNA complexes were seen in gel shift assays in the presence of the type II anti-progestin ZK98,299 or RU50,331. ZK98,299-liganded hPR exerted significantly less tight nuclear binding than receptor complexes formed with RU486. Also a type II anti-glucocorticoid (RU43,044) was detected which completely abrogated DNA binding of its cognate receptor in vivo. In keeping with the existence of two different classes of anti-progestins, agonist- or RU486-induced hyperphosphorylation of the two hPR isoforms present in the T47D breast cancer cells was not induced by ZK98,299. This lack of hyperphosphorylation was, however, not the cause but most likely the consequence, of the reduced ability of the hPR/ZK98,299 complex to interact with DNA. No "mixed-ligand" heterodimers were formed in vitro between hPR isoform A bound to ZK98,299 and R5020-liganded isoform B, but nuclear co-translocation studies indicated that ZK98,299 efficiently induced hPR dimerization in vivo.

In situ hybridization of ovalbumin mRNA in the chick oviduct reveals target cell specificity for estrogen and progesterone.

An in situ hybridization method using paraffin-embedded sections was used to characterize the chicken oviduct cells synthesizing ovalbumin mRNA due to the action of estrogen and progesterone. The cytodifferentiation of the oviduct cells was induced by 17 beta-estradiol administration to newly hatched female chicks. To avoid possible effect of estrogen on the action of progesterone the chicks were withdrawn from the estrogen by six days withdrawal period without hormone treatment. Ovalbumin mRNA was not synthesized after a period of estrogen withdrawal. Administration of estrogen induced ovalbumin mRNA in the tubular gland cells. Administration of progesterone induced the expression of ovalbumin mRNA in the surface epithelial cells. It was also found that progesterone induced mucus producing goblet cells in the surface epithelium. Estrogen did not have an effect on the mucus production, which suggests that progesterone could induce the terminal differentiation of the goblet cells. We conclude that the expression of ovalbumin in the surface epithelial cells and in the tubular gland cells is specific for progesterone and estrogen, respectively.

Subcellular location of unoccupied and occupied glucocorticoid receptor by a new immunohistochemical technique.

Recent immunohistochemical studies suggest that the unoccupied glucocorticoid receptor (GR) is cytoplasmic and that the ligand causes its translocation into the target cell nucleus. The subcellular location of GR is especially interesting in that other members of the steroid receptor superfamily appear to be nuclear. The intracellular distribution of GR was studied immunohistochemically using a new freeze-drying and vapor fixation method which eliminates the protein diffusion and redistribution possibly caused by liquid fixation techniques. We used two monoclonal antibodies against rat liver GR. Dried samples of the adrenalectomized rat brain and uterus were fixed in p-benzoquinone vapor for 3 h at 60 degrees C and embedded in paraffin. Sections were stained with a biotinylated mouse monoclonal GR antibody using the avidin-biotin-peroxidase complex. Both unoccupied and occupied GR were found in the nucleus of the target cells, fibroblasts in the uterus and nerve cells in the cortex of the brain. The staining was saturated with the cytosol of cos cells transfected with GR. No cytoplasmic staining was seen even 2 days after adrenalectomy. In conclusion we propose that GR is also located in the nucleus independently of occupation.