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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.

Gonadotropin receptors belong to a subgroup of G-protein coupled receptors characterized by a large extracellular domain responsible for the binding of the hormone. Soluble, hormone-binding, alternative splicing variants of the LH receptor, are present in high concentration. A mannose rich precursor form of LH and FSH receptor is accumulated inside target cells. FSH receptors are addressed to the basolateral domain of cells through specific signaling mechanisms. Gonadotropin receptors are also present in endothelial cells of target organ vessels and are involved in hormone transcytosis. Various genetic abnormalities of these receptors (and of the GnRH receptor) are discussed.

The thyrotropin (TSH) receptor in human-thyroid glands is 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 suggests shedding of the ectodomain. Indeed we observed such shedding in cultures of human thyrocytes and permanently transfected L or Chinese hamster ovary cells. 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 cAMP were without effect. 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-like enzyme. Shedding of the TSH receptor alpha domain is the consequence of two events: cleavage of the proreceptor into alpha and beta subunits and reduction of the disulfide bridge(s). The use of different specific inhibitors including monoclonal antibodies allowed us to implicate the enzyme protein disulfide isomerase in the reduction of TSHR disulfide bounds. The shed alpha subunit probably results in circulating TSH receptor ectodomain detected in human blood. This shedding mechanism might be implicated in the development of autoimmune diseases.

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.

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.

The exon/intron organization and the structure of the 5' flanking region of the human thyrotropin receptor gene (hTSH-R) were determined. The hTSH-R gene spans more than 60 kb and is split into ten exons. The extracellular domain is encoded by the first nine exons and part of the last exon, whereas the transmembrane and intracellular domains are encoded in totality by the last exon. The leucine-rich repeats of the extracellular domain are encoded as monomers or multimers by separate exons. The TSH receptor gene seems to have arisen by insertion of a DNA sequence encoding repeated leucine-rich elements between the regions encoding the extracellular and the transmembrane domains of a proto-receptor gene ressembling the intronless beta adrenergic receptor genes. Primer extension and S1 mapping experiments identified three transcription start sites. In the hTSH-R gene, the main site was located 157 bp upstream from the start of translation. The promoter region is very GC-rich and contains multiple SP1, ETF and AP2 binding site consensus sequences.

Complementary cDNA clones encoding the TSH (thyroid stimulatory hormone) receptor were isolated from a human thyroid lambda gt10 library using Iow stringency hybridization with LH/hCG (luteinizing hormone-human choriogonadotropic hormone) receptor probes. Sequencing of the clones showed a 764 amino acid open reading frame. The first 21 amino acids probably correspond to a signal peptide, the mature protein thus contains 743 amino acids (calculated molecular weight: 84,501 daltons). Its putative structure consists of a 394 amino acid extracellular domain, a 266 amino acid membrane spanning domain with 7 putative transmembrane segments and a 83 amino acid intracellular domain. A high degree of homology is observed with LH/hCG receptor suggesting the definition of a new subfamily of G-protein coupled receptors. Computer search showed the presence in the putative third intracellular loop of a motif resembling that described in the non receptor type protein tyrosine kinases (c-src, c-yes, c-fgr, etc...). RNA blots showed that the receptor messenger RNA consists of two major species of 4300 and 3900 nucleotides. The cDNA was inserted into an expression vector and after transfection into COS 7 cells it was shown to produce a functional TSH receptor.

7 alpha- and 7 beta-carboxymethyl-derivatives of 17-hydroxyprogesterone and 11-deoxycortisol have been synthesized. After coupling to bovine serum albumin, they were used to elicit antibodies in rabbits. No major difference in the steroid specificity of the antisera was observed when either 7 alpha- or 7 beta-epimers were used for immunization. In both cases, highly specific antisera were obtained which may possibly be used to assay human plasma 17-hydroxyprogesterone and 11-deoxycortisol without chromatographic purification.

Many technical problems are encountered when measuring estradiol and progesterone receptors in breast tumors biopsies. They are due to both intrinsic characteristics of the tissue, and to receptors instability. Suggestions are made to establish an internal quality control in each laboratory at the different steps: obtention and storage of biopsies, purity checking of the reagents, control of the assay protocol, and calculations. Several methods to prepare control pools have been proposed. They use either mammary tumors or animal target organs, and stable concentrations of receptors have been obtained by storage in liquid nitrogen or lyophilization. Published studies have demonstrated important interlaboratory variations. These discrepancies might be in part reduced by using the same assay protocol in the different participating laboratories.

A simplified method is described for the assay of cortisol in human plasma. It is based upon tryptic hydrolysis of cortisol binding proteins in the plasma followed by radioimmunoassay. Only 1 microliter of plasma is used, no extraction of the hormone is necessary and most of the procedure is amenable to automatization. The sensitivity of the method is 10 pg, the coefficient of variation is 8.7% intrassay and 11.5% in different assays. The accuracy and the specificity were also verified. Comparison of this simplified technique with a standard method (extraction by methylene chloride and radiocompetition with corticosteroid binding globulin) gave a correlation coefficient (r) of 0.964.

The solubilized ("cytosolic") receptor present in the rabbit uterus in the absence of hormone and the chromatin-bound ("nuclear") receptor obtained after injection of a progestin were compared. Crude cellular extracts were analyzed by immunoblotting and receptors were purified by immunoaffinity chromatography. With both methods it was observed that the electrophoretic mobility of the "nuclear" receptor was slower than that of the "cytosolic" receptor. This difference in mobility appeared to be due to the existence of variably phosphorylated forms of receptor. The phosphorylation reaction was examined in uterine slices. In the absence of hormone the cytosolic receptor was phosphorylated. When hormone was added the phosphorylation of receptor was markedly enhanced and the electrophoretic mobility of the "nuclear" receptor was decreased. These experiments thus show that the receptor in its "cytosolic" form is a phosphoprotein. Under the effect of the hormone the receptor is further phosphorylated on some supplementary site(s). This polyphosphoprotein is the chromatin-bound, putatively active, form of the receptor. In this respect the intracellular progesterone receptor is similar to various membrane receptors for hormones and growth factors which are phosphorylated upon binding of their ligand.

Monoclonal antibodies (mAB) against progesterone receptor (PR) and the peroxidase-antiperoxidase (PAP) method to visualize PR in paraffin sections from 68 human breast cancers were used. Ten mAB, which recognize human PR on frozen sections, were tested. Six could detect PR in paraffin sections, with Li 417, LET 456, and LET 126 giving the best results. LET 126 antibody was used for most further studies. The effects of fixation with picric acid-formaldehyde (PAF), buffered-formalin, or with Bouin solution were investigated; all fixation methods allowed PR immunolabeling, although PAF or buffered formalin usually gave the best results. Positive staining was seen in the nucleus of carcinoma cells. Variations in intensity and extent of immunoreactivity were observed in all sections and among different regions of the same specimen. These were probably related to the heterogeneity of the tumor cell population. Results were compared with the PR content in the respective tumor tissues, determined by steroid-binding assay, and with immunocytochemistry on frozen sections. It was shown that there were correlations between the immunocytochemical staining (positive or negative) and the steroid binding assay (80%) and between the immunocytochemical staining on paraffin sections and on frozen sections (78%). Weaker intensity and fewer number of PR-positive cells were found for paraffin-embedded tumors. Estrogen receptors were also detected on adjacent sections from the same paraffin-embedded tissues by use of monoclonal anti-ER antibodies (ERICA-kit[Abbott Laboratories, Chicago, IL]) and DNase pretreatment. In conclusion, this immunocytochemical method for detection of PR and ER on paraffin sections offers an alternative to frozen tissue. It allows histologic and immunocytochemical studies on the same sample and retrospective studies on stored tissue blocks.

The structure of uteroglobin, a progesterone binding protein from rabbit uterine fluid, was determined and refined at 1.34 A resolution to a conventional R-factor of 0.229. The accuracy of the co-ordinates is estimated to be 0.15 A. The isotropic temperature factor of individual atoms was refined and its average value is 11.9 A2 for the 548 non-hydrogen atoms of the protein monomer. A total of 83 water molecules was located in difference electron density maps and refined, first using a constant occupancy factor of 1 and then variable occupancy, the final (Q) being 0.63. The mean temperature factor of the water oxygen atoms is 26.4 A2. Uteroglobin is a dimer and its secondary structure consists of four alpha-helices per monomer that align in an anti-parallel fashion. There is one beta-turn between helix 2 and helix 3 (Lys26 to Glu29); 76% of the residues are part of the alpha-helices. In the core of the dimeric protein molecule, between the two monomers that are held together by two disulfide bridges, we have observed a closed cavity. Its length is 15.6 A and its width is 9 A; 14 water molecules could be positioned inside. In the "bottom" part of the protein, near the C terminus, we have observed a smaller cavity, occupied by two water molecules. The calculation of the molecular surface revealed four surface pockets whose possible functional implications are discussed below.

A lambda gt10 library containing DNAs complementary to messenger RNAs from human breast cancer T47-D cells was constructed and screened with a cDNA probe encoding the rabbit progesterone receptor. Four overlapping clones have been sequenced. The open reading frame corresponds to a protein of 933 amino acids with a molecular weight of 98,868 Da. The cysteine rich basic region supposed to be involved in DNA binding is completely homologous in the human and rabbit receptors, whereas the C-terminal end, where hormone binding is thought to take place, differs by a single amino acid change. The human progesterone receptor is characterized, as is the rabbit receptor, by the very high proline content of its N-terminal region. When mRNAs from either human breast cancer cell lines T47-D and MCF-7 or from normal human uterus tissue were blotted and probed with the cloned cDNA, four main bands were observed (5100, 4300, 3700, and 2900 nucleotides).