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Side-blotched lizards ( Uta stansburiana Baird and Girard, 1852) use sagebrush desert habitat above cliffs and typically flee over and down the nearest cliff when disturbed. We tested antipredator escape tactics of lizards to a common local snake, the western yellowbelly racer ( Coluber mormon Baird and Girard, 1852). Our goal was to determine if lizards use cliffs as a refuge from snakes, which cannot climb the sheer rock face, and whether distance to refuge affects escape behavior. We located undisturbed lizards above a cliff and approached them from a random direction with a realistic rubber snake model. When the snake model approached, lizards fled nonrandomly toward the nearest cliff refuge, indicating considerable spatial awareness. Lizards fled more directly toward the cliff the farther from the cliff they were found. However, when beyond ~15 m from the cliff the escape behavior of lizards changed to one of flight in circles (nondirectional) without hiding. Performance capacity (endurance) of the lizards is much greater than 15 m, indicating that lizards have the physiological capacity to reach the cliff. We suggest that the costs of potential intraspecific interactions (i.e., escape into unfamiliar or a competitor’s territory) are greater than the immediate risks of predation by snakes.

Reproduction can impose “costs” associated with the burden of carrying developing embryos or eggs. Numerous studies using squamate reptiles have documented a reduction in locomotor performance related to reproduction. Recently several experimental studies have attempted to determine whether the reduction in locomotor performance is physical or physiological. However, no consensus has been reached, and there is evidence that effects are species specific. In addition, no previous studies have documented whether the reduction in performance is consistent from year to year. For this study, we measured the endurance performance on a motorized treadmill of Side-Blotched Lizards (Uta stansburiana) during their natural breeding season over two years. Our goals for this study were to determine (1) to what degree reproductive state reduces endurance, (2) whether females recover endurance capacity quickly (evidence of physical burden only), and (3) whether the change in performance associated with reproduct...

Previous studies have shown a heterogeneous expression of LH receptors in various structures of the porcine ovary. Specially striking was the existence in the preovulatory follicle of inner layers of theca interna cells devoid of LH receptor and the confinement in the corpus luteum of the LH receptor to the external cellular layers. In the present study, we have compared the steroidogenic capabilities of LH receptor-positive and -negative cells using immunocytochemistry for side-chain cleavage P450, 3β-hydroxysteroid-dehydrogenase, 17α-hydroxylase P450 and aromatase P450. We have also examined, using the same methods, the evolution of the various cell types after ovulation and during the development of the corpus luteum. In preovulatory follicles the inner layers of theca cells which were not labelled with anti-LH receptor antibodies appeared to express the steroidogenic enzymes in a way similar to that of the outer LH receptor-positive cell layers. Ovulation per se did not change the distribution of LH receptors (present in the outer luteal cells and in the granulosa) or of steroidogenic enzymes. However, 48 h after follicular rupture there was a marked decrease in overall labelling with anti-LH receptor antibody, and especially a disappearance of immunostaining in the luteal cells of granulosa origin. In the mid-luteal phase (6 days after ovulation), the receptor content seemed to increase in the peripheral luteal cells derived from the theca but the receptor did not reappear in the granulosa-derived luteal cells. Thus the down-regulation of LH receptor appeared to be reversible in the external thecal layers but irreversible in the granulosa cells. Furthermore, the distribution of the various steroidogenic enzymes in the corpora lutea delineated granulosa-derived from theca-derived cells and showed that only the external layers of the latter expressed the LH receptor. These results showed the existence in the preovulatory follicle of two theca interna regions expressing the same steroidogenic enzymes but possibly submitted to a different hormonal control. Furthermore, the cells derived from these two regions as well as the cells of granulosa origin showed a distinct pattern of variation of LH receptivity during the development of the corpus luteum. During these studies we also observed that, in the interstitial tissue, only a minority of cells which derived from remnants of atretic follicles expressed both the LH receptor and the steroidogenic enzymes. Journal of Endocrinology (1996) 148, 435–446

PML (promyelocytic leukemia) is a protein involved in the t (15;17) translocation of promyelocytic leukemia and is mainly localized in nuclear bodies. Here we show that PML exerts a very powerful enhancing activity (up to 20-fold) on the transactivating properties of the progesterone receptor (PR) and has a similar effect on several other steroid hormone receptors. There is probably a direct or indirect interaction between PR and PML, because when the latter was expressed at high concentrations it shifted PR into the nuclear bodies. The use of deletion mutants showed that both activation functions (AF1 and AF2) of PR as well as the coiled coil and His-Cys-rich domains of PML were required for transcriptional enhancement. The fusion protein PML-RAR which is not localized in nuclear bodies, also enhanced the transactivating activity of PR, but this effect was totally suppressed by the administration of retinoic acid. PML, which is ubiquitously expressed, may thus be involved in the transactivation properties of steroid hormone receptors. This mechanism may also play a role in the oncogenic properties of PML-RAR and in their suppression by retinoic acid.

Patients with familial male-limited precocious puberty present with early onset of puberty. Several missense mutations in the LH receptor gene that cause amino acid substitutions in the sixth transmembrane segment of the receptor protein have been shown to be a cause of the disorder. We have identified a novel LH receptor gene mutation in a patient with familial male-limited precocious puberty that results in a threonine for methionine substitution at position 398 in the second transmembrane segment of the receptor protein. In vitro expression in human embryonic kidney 293 cells of this LH receptor mutant and two previously described LH receptor mutants showed that cAMP production in the absence of hormone was elevated up to 25-fold compared to the basal level of the wild-type receptor. The ED50 values of hormone-induced cAMP production were within the same range for wild-type and mutant receptors, but maximal hormone-induced cAMP production was relatively low for mutant receptors. We also produced receptors containing amino acid substitutions in both the second and sixth transmembrane segments. For these double mutants, basal receptor activities were similar to the basal activities observed in single mutants, whereas hormone-induced receptor activation was almost completely abolished.

From 1,211 breast cancers, 15 oestrogen receptor (ER) negative-progesterone receptor (PgR) positive breast cancers by conventional dextran coated charcoal steroid binding assays in cytosol were reassessed using Elisa techniques with monoclonal antireceptors antibodies in the cytosolic and nuclear fractions, and immunocytochemistry on cryostat sections. Three categories of results were found in this series. Two tumours were false negative ER due to receptor sites occupancy by hormonal contraceptive treatment. A second group of ten tumours, with high PgR concentrations and immunoreactive ER, corresponds to non ER-binding forms of receptors. One PgR positive tumour was found to be devoid of PgR by using monoclonal antiPgR antibodies might contain a progesterone binding cyst protein. Only two tumours were found to be true ER negative-PgR positive by all methods. This rare phenotype deserves further study of the regulation of the PgR gene.

Mutations of receptor genes are involved in various aspects of thyroid and gonadal pathology. Activating mutations of TSH and LH receptors are associated with hyperthyroidism and premature puberty. These mutations are dominant and lead to the synthesis of a constitutive receptor, i.e. a receptor active even in the absence of hormone. Inactivating mutations of TSH, gonadotropin and GnRH receptors are recessive. They determine either a hypothyroidism or a hypogonadism. In the case of alterations of gonadotropin receptors the hypogonadism is hypergonadotrophic. It is hypogonadotrophic in the case of mutations of the GnRH receptor.

The association of hypogonadotropic hypogonadism with anosmia defines Kallmann's syndrome. The gene of the X-linked form of this syndrome has been cloned and several mutations described. However, the relatively small number of hypogonadotropic hypogonadic patients with Kallmann's gene defects supports the hypothesis that other genes may be involved. Idiopathic hypogonadotropic hypogonadism (IHH) is not associated with anosmia. The GnRH gene was excluded as a candidate gene in IHH since no abnormality was found in several patients. The action of the GnRH is mediated through a G-protein coupled receptor present in the cell membrane of gonadotropes. The GnRH receptor was thus another candidate gene. Recently, we described the first patient with partial hypogonadotropic hypogonadism without anosmia caused by loss of function mutations of the GnRH receptor. We compare this first family with a new family presenting complete hypogonadotropic hypogonadism and a variable degree of gonadotrope deficiency in the affected kindred, and discuss genotype-phenotype correlation.

Most of the enzymes involved in steroidogenesis belong to the family of cytochrome P 450. Most of the corresponding genes have been cloned. The key enzyme for estradiol biosynthesis is P 450 arom. Several germline mutations have been described. These observations have lead to reconsider the role of estradiol. Estradiol plays a key role in bone growth and mineralisation and in gonadotrope regulation in male. Moreover, the recent discovery that an additional estrogen receptor (ER beta) is present in various tissues has advanced our understanding of the mechanisms underlying estrogen signalling. It suggests the existence of two previously unrecognized pathways of estrogen signalling: via the ER beta subtype in tissues exclusively expressing this subtype and via the formation of heterodimers in tissues expressing both ER subtypes. Various models have been suggested as explanations for the stricking cell and promoter-specific effects of estrogens and antiestrogens, all on the basis of the assumption that only a single ER exists. These models have to be reconsidered. Moreover, new antiestrogens with improved therapeutic profiles could be designed.

Two different monoclonal antibodies recognizing different epitopes were used to study the localization of luteinizing hormone/human chorionic gonadotrophin (LH/hCG) receptors in human skin. Immunolabelling was observed only in the epidermis and derived structures but not in the dermis. The basal, spinal and granular layers were stained, whereas no receptors were detected in the non-nucleated horny cells. In the growing (anagen) hair, immunostaining was found in the inner root sheath below the level of the sebaceous glands and in the outer root sheath above this level. In the resting (telogen) hair, only the latter staining was observed. In the sebaceous glands, only the thin cells close to the walls of the ducts were immunolabelled. In the eccrine sweat glands, the external clear cells were stained in the secretory portion of the gland, whereas only the cells close to the lumen were labelled in the ducts. The distribution of LH/hCG receptors was compared with that of steroidogenic enzymes (side chain cleavage cytochrome P450, adrenodoxin, 3-beta-hydroxy-5-ene steroid dehydrogenase Delta5-Delta4 isomerase, 17-hydroxylase cytochrome P450 and cytochrome P450 aromatase). Only partial overlaps were observed. The presence of LH receptor mRNA in the skin was confirmed by reverse transcription-polymerase chain reaction. Monoclonal antibodies raised against the human follicle-stimulating hormone receptor failed to detect the latter in the epidermal structures and in the dermis. The role of LH and hCG in skin modifications occurring during pregnancy and after the menopause is unknown. These hormones may possibly act by regulating steroidogenic enzymes or by modulating cell growth and differentiation.

Native thyrotropin receptor (TSHR) was purified by immunoaffinity chromatography from membrane extracts of stably transfected L cells. An ELISA test was devised to study anti-TSHR autoantibodies directly. Comparison of native TSHR with bacterially expressed, denatured TSHR showed that the latter was not recognized by the autoantibodies, suggesting that they bind to conformational epitopes only present on the native receptor. The use of deglycosylated TSHR and of purified receptor ectodomain (alpha-subunit) showed that the autoantibodies recognized only the protein backbone moiety of the receptor and that their epitopes were localized entirely in its ectodomain. Autoantibodies were detected in 45 of 48 subjects with untreated Graves' disease and in 26 of 47 healthy volunteers. The affinity for the receptor was similar in the two groups (Kd = 0.25-1 x 10-10 M) and the autoantibodies belonged to the IgG class in all cases. Although the concentration of autoantibodies was higher in Graves' disease patients (3.50 +/- 0.36 mg.L-1) than in control subjects (1.76 +/- 0.21) (mean +/- SEM), there was an overlap between the groups. Receptor-stimulating autoantibodies (TSAb) were studied by measuring cAMP synthesis in stably transfected HEK 293 cells. Their characteristics (recognition of alpha-subunit, of deglycosylated TSHR, nonrecognition of bacterially expressed denatured receptor) were similar to those of the antibodies detected by the ELISA test. TSAb were only found in individuals with Graves' disease. The ELISA test measures total anti-TSHR antibodies, whereas the test using adenylate cyclase stimulation measures antibodies that recognize specific epitopes involved in receptor activation. Our observations thus disprove the hypothesis according to which Graves' disease is related to the appearance of anti-TSHR antibodies not present in normal subjects. Actually, anti-TSHR antibodies exist in many euthyroid subjects, in some cases even at concentrations higher than those found in patients with Graves' disease. What distinguishes the latter from normal subjects is the existence of subpopulation(s) of antibodies directed against specific epitope(s) of the receptor involved in its activation.

Nuclear receptors form a superfamily of transcriptional regulators including both classical hormone receptors and orphan receptors. The functional anatomy of the receptors is described as well as their interaction with coactivators, cointegrators and corepressors. Nuclear receptors activate and inhibit gene transcription by regulating histone acetylases and histone deacetylases. Receptor isoforms (oestrogen receptors alpha and beta), ligands exerting differential effects on the same receptor and the mechanism of action of antihormones are discussed.

Aryl hydrocarbon receptor (AhR) ligands such as dioxin and benzo[a]pyrene are environmental contaminants with many adverse health effects, including immunosuppression, carcinogenesis, and endothelial cell damage. We show here that a wine component, resveratrol (3,5,4'-trihydroxystilbene), is a competitive antagonist of dioxin and other AhR ligands. Resveratrol promotes AhR translocation to the nucleus and binding to DNA at dioxin-responsive elements but subsequent transactivation does not take place. Resveratrol inhibits the transactivation of several dioxin-inducible genes including cytochrome P-450 1A1 and interleukin-1beta, both ex vivo and in vivo. Resveratrol has adequate potency and nontoxicity to warrant clinical testing as a prophylactic agent against aryl hydrocarbon-induced pathology.

Detailed endocrinological studies were performed in the three affected kindred of a family carrying mutations of the GnRH receptor gene. All three were compound heterozygotes carrying on one allele the Arg262Gln mutation and on the other allele two mutations (Gln106Arg and Ser217Arg). When expressed in heterologous cells, both Gln106Arg and Ser217Arg mutations altered hormone binding, whereas the Arg262Gln mutation altered activation of phospholipase C. The propositus, a 30-yr-old man, displayed complete idiopathic hypogonadotropic hypogonadism with extremely low plasma levels of gonadotropins, absence of pulsatility of endogenous LH and alpha-subunit, absence of response to GnRH and GnRH agonist (triptorelin), and absence of effect of pulsatile administration of GnRH. The two sisters, 24 and 18 yr old, of the propositus displayed, on the contrary, only partial idiopathic hypogonadotropic hypogonadism. They both had primary amenorrhea, and the younger sister displayed retarded bone maturation and uterus development, but both sisters had normal breast development. Gonadotropin concentrations were normal or low, but in both cases were restored to normal levels by a single injection of GnRH. In the two sisters, there were no spontaneous pulses of LH, but pulsatile administration of GnRH provoked a pulsatile secretion of LH in the younger sister. The same mutations of the GnRH receptor gene may thus determine different degrees of alteration of gonadotropin function in affected kindred of the same family.

One of the most abundant protein products of human secretory endometrium is glycodelin, a glycoprotein previously referred to as PP14. Although the precise function of this protein is unknown, its unique glycosylation pattern is believed to affect immunomodulatory activity during human embryonic implantation and inhibition of sperm-egg binding after ovulation. Having confirmed the expression of glycodelin in secretory endometrial glands, we used purified endometrial epithelial cell cultures to demonstrate the hormonal regulation of glycodelin synthesis and secretion. The findings were corroborated by transiently transfecting glycodelin gene promoter-reporter constructs into human epithelioid HeLa and Ishikawa cells. Our results indicate that glycodelin protein production by endometrial epithelial cells is directly up-regulated 4- to 9-fold by progestins and antiprogestins in vitro. Transcriptional regulation of the glycodelin gene promoter expressed in HeLa cells is progesterone receptor-dependent. As observed in the primary endometrial cells, progestins and antiprogestins both act as agonists on the in vitro expression of this endometrial gene. Our findings provide insight into the regulation of this abundant endometrial protein and raise interesting questions about the physical nature of the interaction of agonist- and antagonist-bound progesterone receptors with the glycodelin gene promoter.

We report the case of an infant who presented at birth with a hypoplastic phallus associated with hypospadias. Low testosterone production, normal serum levels of steroid precursors, and increased LH in response to LH-releasing hormone supported a defect in Leydig cell differentiation or function. Conventional microscopic study of the testes showed fibroblastic cells in the interstitium. However immunocytochemical analysis using anti-LH receptor and anti-P450c17 antibodies demonstrated that about one third of these cells were Leydig cells or precursors of Leydig cells. No histological feature could distinguish the latter cells from fibroblasts. A homozygous substitution of cysteine 133 for arginine was found in the extracellular domain of the receptor. This is the first naturally occurring missense mutation found in the extracellular domain of the LH receptor. COS-7 cells transfected with the mutant receptor exhibited a marked impairment of hCG binding, whereas some cAMP production could be observed at high hCG concentrations. We propose that the partial impairment of LH receptor function, as reflected by the presence of Leydig cells, was responsible for the incomplete male pseudohermaphroditism observed in our patient.

Recent studies have suggested that human choriogonadotropin (hCG), in addition to its function in regulating steroidogenesis, may also play a role as a growth factor. Immunocytochemistry using two different monoclonal antibodies (LHR29 and LHR1055) raised against the human luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor allowed us to detect this receptor in breast cancer cell lines (T47D, MCF7, and ZR75) in individual cancer biopsies and in benign breast lesions. The receptor was also present in epithelial cells of normal human and sow breast. In the latter, its concentration increased after ovulation. The presence of LH/hCG receptor mRNA was confirmed by reverse transcription-PCR using primers extending over exons 2-4, 5-11, and 9-11. The proportion of LH/hCG-receptor positive cells and the intensity of the immunolabeling varied in individual biopsies, but there was no obvious correlation with the histological type of the cancer. These results are compatible with previous studies suggesting that during pregnancy, hCG is involved in the differentiation of breast glandular epithelium and that this hormone may play an inhibitory role in mammary carcinogenesis and in the growth of breast tumors.

The thyroid-stimulating hormone (TSH) receptor (TSHR) plays a key role in the control of thyroid growth and function. This G-protein-coupled receptor essentially activates adenylate cyclase and to a lesser extent phospholipase C. The role of the cAMP cascade in the production of thyroid hormones and in the mitogenic effect of TSH has been thoroughly studied (reviewed in Dumont et al. 1989).

The thyrotropin (TSH) receptor in human thyroid glands has been shown to be cleaved into an extracellular alpha subunit and a transmembrane beta subunit held together by disulfide bridges. An excess of the latter component relative to the former suggested the shedding of the ectodomain. Indeed we observed such a shedding in cultures of human thyrocytes and permanently transfected L or Chinese hamster ovary cells. The shedding was increased by inhibitors of endocytosis, recycling, and lysosomal degradation, suggesting that it was dependent on receptor residency at the cell surface. It was slightly increased by TSH and phorbol esters, whereas forskolin and 8-bromo-cyclic AMP were without effect. Decreasing the serum concentration in cell culture medium enhanced the shedding by an unknown mechanism. The shedding of the TSH receptor alpha domain is the consequence of two events: cleavage of the receptor into alpha and beta subunits and reduction of the disulfide bridge(s). The complete inhibition of soluble TSH receptor shedding by the specific inhibitor BB-2116 indicated that the cleavage reaction is catalyzed probably at the cell surface by a matrix metalloprotease. This shedding mechanism may be responsible for the presence of soluble TSH receptor alpha subunit in human circulation.

Leydig cell hypoplasia is a rare autosomal recessive condition that interferes with normal development of male external genitalia in 46,XY individuals. We have studied two Leydig cell hypoplasia patients (siblings born to consanguineous parents), and found them to be homozygous for a missense mutation (Ala593Pro) in the sixth transmembrane domain of the luteinizing hormone (LH) receptor gene. In vitro expression studies showed that this mutated receptor binds human choriogonadotropin with a normal KD, but the ligand binding does not result in increased production of cAMP. We conclude that a homozygous LH receptor gene mutation underlies the syndrome of autosomal recessive congenital Leydig cell hypoplasia in this family. These results have implications for the understanding of the development of the male genitalia.

Monoclonal antibodies have been raised against the porcine LH receptor and have allowed to clone the corresponding messenger RNA from testicular cells. The stricture of the LH receptor has been determined. It shows similarities but also differences with other G protein coupled receptors. Specially a large extracellular domain is specific of that new family of receptors. Variant forms of the LH receptor generated by alternative splicing and lacking transmembrane domain have been isolated. Immunochemical and immunocytochemical studies have been performed. Three different forms of the LH receptor are physiologically expressed: a mature 85kDa transmembrane species, a 68 kDa high mannose containing species corresponding to a precursor which accumulates inside the cells, and truncated 45-48kDa molecular weight species corresponding to the variant messenger RNAs identified during the cloning of the receptor. A novel zonation of the ovary has been described by immunocytochemical studies. Cross hybridization with the LH receptor clone allowed to isolate the related TSH receptor from human thyroid tissue. The human LH and FSH receptor genes have been localized to chromosome 2p21 and the TSH receptor gene to chromosome 14q31. The genes are very large (60 kbp) and have introns only within the 5' part encoding the extracellular domain of the receptor. Immunoelectron microscopic studies performed in Leydig cells and in stably transfected L cells have allowed to study intracellular traffic of the LH receptor. The same approach was used to study the transendothelial transfer of hCG in testicular microvasculature.

The progesterone receptor displays the typical three-domains structure of the steroid-thyroid receptor family. The central domain contains two 'zinc finger' structures responsible for the specific recognition of the cognate DNA sequences. The carboxy-terminal domain contains the hormone and anti-hormone binding site. Progesterone and synthetic progestins (R5020, Org 2058) activate the receptor, provoke its phosphorylation and DNA-binding ability and induce its regulatory activities. The antagonist RU38486 elicits the same sequence of events but leads to an abortive conclusion without specific gene transactivation. The progesterone receptor is down-regulated by its own ligand at the transcriptional level through inhibition of oestrogen receptor-mediated induction through protein-protein interactions. This mechanism is also inhibited by RU38486.

Hormonal receptors are rare and fragile molecules. Their study was difficult and often yielded contradictory results until immunological and molecular probes became available. Sex steroid receptor cDNAs were cloned in 1986–1987. Monoclonal antibodies had previously been prepared and for this reason the understanding of their structure and function is already very advanced to date. In contrast, gonadotropin receptors were not cloned until 1989–1990 and well-characterized antibodies still remain a problem in many cases and thus our knowledge of their mere basic structural and functional features is still preliminary in many cases.

Monoclonal antibodies have been raised against porcine LH receptor and allowed to clone the corresponding messenger RNA from testicular cells. The structure of the LH receptor have been determined. It shows similarities but also differences to other G protein coupled receptors. In particular a large extracellular domain is specific for that family of receptors. Variants forms of the LH receptor generated by alternative splicing and lacking transmembrane domains have been isolated. Immunochemical and immunocytochemical studies have been performed. Three different forms of the LH receptor are physiologically expressed: a mature 85 kDa transmembrane species, a 68 kDa high mannose containing species corresponding to a precursor which accumulate inside the cells, and truncated 45-48 kDa molecular weight species corresponding to the variant messenger RNAs identified during the cloning of the receptor. A novel zonation of the ovary has been described by immunocytochemical studies. Cross hybridisation with the LH receptor clone allowed to isolate the related human TSH receptor from thyroïds. The human LH and FSH receptor genes have been localized to chromosome 2p21 and the TSH receptor gene to chromosome 14q31. The genes are very large and have introns only within their 5' part corresponding to the extracellular domain of the receptor.

An enzyme-linked immunosorbent assay using monoclonal and polyclonal antibodies against recombinant pS2 was devised. It was used to measure pS2 concentration in the cytosol of 339 breast cancer, 15 fibroadenomas, 16 cases of benign breast disease, and 6 normal breast tissues. The mean value of pS2 concentration was higher in cancer, but the protein could be detected readily in benign tumors and even in normal breast. The concentration of pS2 was significantly lower in postmenopausal women and tumors of differentiation Grade 3. The pS2 concentration was correlated strongly with the presence of estrogen receptors (ER) and progesterone receptors (PR). No correlation was observed with the size, histologic type of the tumor, and lymph node status. The prognostic value of pS2 appeared relatively limited. It was clear cut only for a relatively small group of patients (approximately 15%), who had low concentrations of pS2 (less than or equal to 0.32 ng/mg of protein). These patients had a shorter disease-free interval and overall survival time. The most striking correlation was observed with the outcome of adjuvant hormone therapy. pS2 concentration was shown to be the most potent prognostic factor, preceding even ER.

The nuclear localization of the progesterone receptor is mediated by two signal sequences: one is constitutive and lies in the hinge region (between the DNA and steroid binding domains), the other is hormone-dependent and is localized in the second zinc finger of the DNA binding domain. The use of various inhibitors of energy synthesis in cells expressing permanently or transiently the wild-type receptor or a receptor mutated within the nuclear localization signals, demonstrated that the nuclear residency of the receptor reflects a dynamic situation: the receptor diffusing into the cytoplasm and being constantly and actively transported back into the nucleus. The existence of this nucleo-cytoplasmic shuttle mechanism was confirmed by receptor transfer from one nucleus to the other in heterokaryons. Preliminary evidence was obtained, using oestrogen receptor, that this phenomenon may be of general significance for steroid receptors.

Experimental conditions are described for the detection of steroid receptors in tissue sections or cells at the light microscope level. Current knowledge about the ultrastructural distribution of these receptors is summarized; the mechanisms of their nuclear localization are described. Karyophilic signals involved in nuclear translocation are characterized by means of in vitro mutagenesis of steroid receptor cDNAs. Studies analysing the subcellular distribution of various transfected receptor mutants in energy depleted cells together with fusion experiments provide evidence for nucleoplasmic shuttling of progesterone receptors. We conclude that the "nuclear" location of the wild type progesterone receptor reflects a dynamic equilibrium between active nuclear import and outward diffusion. We also describe the use of immunocytochemistry in pathology, especially for the detection of steroid receptors in hormone dependent tumours.

Monoclonal antibodies have been raised against porcine LH receptor and allowed to clone the corresponding messenger RNA from testicular cells. Cross hybridisation with the LH receptor clone allowed to isolate a clone corresponding to the human TSH receptor from thyroids. The structure of both receptors have been determined. They show similarities but also differences to other G protein coupled receptors. In particular a large extracellular domain is specific of that new family of receptors. Variant forms of the LH receptor lacking transmembrane domains have been isolated. The obtention of monoclonal antibodies against both receptors allowed immunochemical and immunocytochemical studies to be performed. The human LH receptor gene have been localized to chromosome 2p21 and TSH receptor gene to chromosome 14q31. The complete organisation of the human TSH receptor gene has been determined.

Several vectors were used to express the complementary DNA for breast cancer estrogen-induced protein BCEI (also called pS2) in Escherichia coli. The best results were obtained by using the pUR 290 expression vector after deletion of the sequence encoding the signal peptide of the protein. In these conditions, beta-galactosidase-BCEI/pS2 fusion protein accounted for approximately 20% of total proteins in bacterial extracts. It was purified by chromatography on DEAE-Trisacryl or by gel electrophoresis and electroelution. Polyclonal antibodies were obtained by immunization of rabbits and goats, and monoclonal antibodies were raised in mice. Two types of monoclonal antibodies were obtained: one class recognized the native protein and was very efficient for the immunoprecipitation and immunopurification of the protein from breast cancer cells; a second class recognized the denatured protein and was especially effective for immunoblot studies. BCEI/pS2 could be detected by immunocytochemistry in breast cancer biopsies using monoclonal antibodies on frozen or paraffin-embedded sections. One of the antibodies (mBCEI11) exhibited high affinity for the protein and could be used at 1.9 micrograms/ml concentration for immunolabeling of histological sections. The mBCEI11 antibody was used in immunoaffinity chromatography to purify the peptide in a single step from culture media of estrogen-treated MCF-7 cells.

Probes corresponding to human and porcine LH (luteinizing hormone) receptor cDNA were used for in situ hybridization to human chromosomes. This allowed us to assign the LH receptor gene to chromosome 2p21.

Interest in steroid hormone receptors in breast cancer stems from both theoretical and practical considerations. The malignant transformation and subsequent growth of breast cancer cells are hormonally regulated, and elucidation of the mechanisms of these processes requires an understanding of the structure and function of hormonal receptors. Moreover, receptor determination in tumour biopsies has now been used for many years as a means of predicting response to hormonal therapy and as prognostic factors in early breast cancer. Recent cloning of most of these receptors has allowed researchers to obtain a considerable wealth of new information and has provided new tools with which further questions become amenable to experimental analysis (reviews in [1–3]).

Unbound cortisol was assayed in maternal and cord plasma. A method was used in which experimental conditions are carefully selected to approach as closely as possible the in vivo situation. ∗ ∗Robin, P., Predine, J., and Milgrom, E.: J. Clin. Endocrinol. Metab. 46:277, 1978. In maternal plasma at term, the proportion of unbound cortisol (9.7 ± 1.5%) (mean ± SE) was identical to that of normal nonpregnant women (9.7 ± 0.4%). However, the concentration of unbound cortisol was 3-fold higher at term (46.0 ± 5.9 ng/ml) than that in nonpregnant women (14.2 ± 1.0 ng/ml). During labor an increase was observed, and at birth the proportion (and concentration) of unbound cortisol was extremely high varying between 17.0 ± 0.8% (92.3 ± 7.4 ng/ml) and 20.9 ± 1.4% (130.4 ± 17.9 ng/ml) (spontaneous and induced vaginal deliveries, respectively). In cord plasma at term and before labor (fetuses delivered by elective cesarean section), the proportion of unbound cortisol was high (29.1 ± 1.3 %) but its concentration (9.6 ± 1.1 ng/ml) was only slightly lower than that observed in normal adults. Labor had an important stimulatory effect as observed in cases of vaginal delivery after spontaneous labor (proportion of unbound cortisol 35.1 ± 1.2%, concentration of unbound cortisol 15.4 ± 1.35 ng/ml, respectively). No difference was observed between spontaneous and provoked labor since similarly high values of unbound cortisol were found in cord plasma after oxytocin-induced labor followed by vaginal delivery (36.2 ± 1.4% and 23.0 ± 5.2 ng/ml).

Radioautograms were made of uterine horns, cervix and vagina from guinea pigs injected with[3H]progesterone at various periods of the estrous cycle. The concentration of silver grains peaked at proestrus and estrus and then fell during metestrus, attaining a minimum at diestrus. These variations were not due to masking of binding sites by endogenous hormone since ovariectomy of guinea pigs at diestrus did not change this pattern. During proestrus and estrus, radioactivity was found in all parts of the endometrium and myometrium of the uterine horns. However, the muscle fibers and the cells of the stroma contained more silver grains than did the luminal and glandular epithelium. In the vagina, radioactivity was concentrated mostly in the nuclei of the basal layer cells of the stratified epithelium. During metestrus and diestrus, less radioactivity was present in these regions. These differences in labelling were observed simultaneously in all the uterine and vaginal cell types, suggesting that similar hormonal mechansims may control receptor variations in these different target cells.