{"paper_id":"2b0eb8cc-b07f-4f76-9829-4304818cccf3","body_text":"Original article\nLevels of sex hormone-binding globulin (SHBG) and\ncorticosteroid-binding globulin (CBG) messenger\nribonucleic acid (mRNAs) in ovarian endometriosis\nR Misao, M Hori, S Ichigo, J Fujimoto, T Tamaya\nDepartment of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu, Japan\n(Received 4 May 1994; accepted 10 January 1995)\nSummary &horbar; Recently, much evidence has indicated that sex hormone-binding globulin (SHBG) and\ncorticosteroid-binding globulin (CBG) play a role in the intracellular action of sex steroids in target\ncells. In the present work, expression of SHBG mRNA and CBG mRNA was demonstrated in tissues\nof human normal endometrium and pelvic endometriosis, using the reverse transcription-polymerase\nchain reaction (RT-PCR). SHBG mRNA levels were higher in pelvic endometriosis than in normal\nendometrium (P < 0.02), while CBG mRNA levels were lower than in normal endometrium (P < 0.05).\nThe SHBG mRNA/CBG mRNA ratio was significantly higher in pelvic endometriosis than in normal\nendometrium (P < 0.01 These findings suggest that overexpression of intercellular SHBG in endometri-\notic tissues results in the formation of the estrogen-predominant milieu, since SHBG-bound estrogen\nis considered to be protected from the metabolism in liver and available in endometrial cells, thereby\nassisting the development of the pelvic endometriosis.\nsex hormone-binding globulin / corticosteroid-binding globulin / mRNA I endometriosis\nRésumé &horbar; Niveaux des ARN messagers de la globuline liant la testostérone (ARNm SHBG) et\nde la transcortine (CBG) dans l’endométriose ovarienne. Récemment, plusieurs travaux ont mon-\ntré que la SHBG et la CBG jouent un rôle dans l’interaction intracellulaire de stéroïdes sexuels dans les\ncellules cibles. Dans cette étude, l’expression des ARNm de la SHBG et de la CBG a été mise en\névidence dans des biopsies d’endomètre humain normal et des prélèvements d’endométriose pel-\nvienne par transcription réverse puis amplification en chaîne par la polymérase. La concentration de\nI ARNm SHBG est plus élevée dans l’endométriose pelvienne que dans l’endomètre normal (P <\n0,02), les résultats inverses sont observés pour IARNm CBG (P < 0,05). Le rapport ARNm SHBG/ARNm\nCBG est significativement plus élevé dans l’endométriose pelvienne que dans l’endomètre normal\n(P < 0, 01). Étant donné que ¡’&oelig;strogène lié à la SHBG semble être protégé du métabolisme hépatique\net rendu disponible dans les cellules endométriales, toutes ces constatations suggèrent que, dans\nles tissus d’endométriose, l’augmentation de l’expression de la SHBG intracellulaire produisant un\nmilieu où l’cestrogène est prédominant accentuerait un développement de l’endométriose pelvienne.\nglobuline liant les hormones stéroïdes sexuels / transcortine lARNm / endométriose\n\n\nINTRODUCTION\nThe proliferation, differentiation, and devel-\nopment of normal uterine endometrium is\nregulated by sex steroids. In some cases,\nwhen endometrial tissue exhibits aberrant\ngrowth, which occurs ectopically in various\nlocations in the pelvic cavity (endometrio-\nsis), it exhibits the cyclic functional respon-\nsiveness of normal endometrium. In other\ncases an immature or unripe variety devel-\nops, which is only responsive to the estro-\ngenic stimulus and not to progesterone (Diz-\nerega et al, 1980).\nEstrogen-induced growth of endometrio-\nsis might be partly explained by the fol-\nlowing evidence. Most endometriosis is\nless responsive to progestogens, due to\nthe low level of progesterone receptor rel-\native to that of estrogen receptor (Tamaya\net al, 1979), while progestogen has an anti-\nestrogenic effect (Dorfman ef al, 1961 ).\nRecently, a great deal of evidence has\nindicated that sex hormone-binding globu-\nlin (SHBG) and corticosteroid-binding glob-\nulin (CBG) play a role in the intracellular\naction of sex steroid hormones in target\ncells. The expressions of SHBG and CBG\nhave been demonstrated immunohisto-\nchemically in human endometrium, prostate\n(Mercier-Bodard et al, 1987) and breast\ntissue (Sinnecker et al, 1990), and in pitu-\nitary (Perrot-Applanat etal, 1984) and thy-\nroid glands (Kuhn et al, 1986). Moreover,\nmRNA expressions of SHBG and CBG\nhave been analyzed in human endometrial\ncancer cell lines (Mercier-Bodard et al,\n1991) and rhesus monkey testis (Ham-\nmond et al, 1987a) by Northern blot\nhybridization.\nThese results prompted us to investi-\ngate the expression of SHBG and CBG\nmRNA in order to know the mechanism of\nestrogen-induced growth of endometrio-\nsis.\nMATERIALS AND METHODS\nMaterials\nHuman uterine endometria were obtained by\nendometrial biopsy from 35 patients (aged from\n25 to 39 years) with a regular menstrual cycle at\nthe Department of Obstetrics and Gynecology,\nGifu University School of Medicine from July 1990\nto June 1993. Part of the specimen was submit-\nted for histological dating (Noyes et al, 1950).\nAgreements for the study were obtained from\npatients and from the Research Committee on\nHuman Subject of Gifu University School of\nMedicine. Pelvic endometriosis tissues were\nobtained from 6 patients who underwent conser-\nvative or definitive surgery for ovarian endometrio-\nsis. These specimens were immediately frozen\nin liquid nitrogen and prepared for the following\nprocedures, such as RNA isolation, Northern blot\nanalysis and reverse transcription-polymerase\nchain reaction (RT-PCR).\nPoly(A)+RNA isolation\nFrozen tissue (100 mg) was ground, transferred\nto a Teflon-glass homogenizer containing 1 ml\nlysis/binding buffer (100 mM Tris-HCI pH 8.0, 500\nmM LiCl, 10 mM EDTA (ethylenediamine-\ntetraacetic acid) pH 8.01% SDS (sodium dodecyl\nsulfate), 5 mM DTT (dithiothreitol) and homoge-\nnized manually at 4°C. After spinning at 20 000 g\nfor 30 s, the supernatant was transferred to an\nEppendorf tube. Polyadenylated mRNA\n(poly(A)+RNA) was isolated from tissue with mag-\nnetic beads, Dynabeads Oligo (dT)25 (Dynal AS,\nOslo, Norway) (Jacobsen et al, 1990). The\npoly(A)+RNA concentration was determined by\nUV absorption at 260 and 280 nm.\nPreparation of labeled probe\nSHBG cDNA (1 143 bp) and CBG cDNA (1 215 bp)\nwere synthesized from human liver poly(A)+RNA\n(Clontech Laboratories, Palo Alto, CA) using the\nPCR (as detailed below for the PCR). The primers\nused for SHBG cDNA synthesis using PCR were\n\n\n5’-CAGCACACCCGCCAGGGATGGGCC-3’\n(SHBG-5’:1-24, Exon I) and 3’-CCGTTACCGT-\nGACTGCGAAGGGTA-5’ (SHBG-3’:1120-1143,\nExon VIII) (Hammond et al, 1987b). The primers\nused for CBG cDNA synthesis were 5’-ATGC-\nCACTCCTCCTGTACAC-3’ (CBG-5’: 1-20, Exon\nII) and 3’-TGAACCCAGTGTAAGAGACC-5’\n(CBG-3’:1205-1224, Exon V) (Hammond et al,\n1987a). The DNA probe was labeled with biotiny-\nlated dUTP using a Polar Plex Random Primer\nBiotin Labelling Kit (Millipore, Burlington, MA).\nNorthern blot analysis\nPoly(A)+RNA (45 !g) was denatured at 65°C for\n15 min, size-fractionated by electrophoresis\nthrough 1 % agarose-formaldehyde gel, and blot-\nted onto a nylon membrane (immobilon-S; Milli-\npore) by capillary transfer for 20 h using 10 x x\nstandard saline citrate (SSC: 1.5 M NaCI, 0.15 M\nsodium citrate, pH 7.0). After blotting, the mem-\nbrane was dried at 75°C for 20 min and then cross-\nlinked by ultraviolet irradiation (33 000 ¡W/cm2 at\n254 nm). Prehybridization was performed at 42°C\nfor 4 h in a mixture containing 5 x SSC, 50% for-\nmamide, 2 x Denhardt’s reagent, 0.1 % SDS, and\n100 pg/ml denatured salmon sperm DNA. The\nsame solution was used for the hybridization but\nincluded the biotin-labeled probe (26 ng/ml). After\nhybridization at 42°C for 24 h, the membrane was\nwashed for 20 min at room temperature and then\nfinally washed twice with 0.1 x SSC for 30 min\nat 65°C. The detection reaction using a Plex\nChemiluminescent Kit (New England BioLabs,\nBeverly, MA). The membrane was exposed to\nKodak XAR-5 films (Eastman Kodak, Rochester,\nNY) for 15 min.\nReverse transcription\nPoly(A)+RNA (10 ng) was reverse transcribed for\n1 h at 42°C with a mixture of 600 units of M-MLV\nreverse transcriptase (Gibco BRL, Gaithersburg,\nMD) and the following reagents: 50 mM Tris-HCI\nbuffer pH 8.3; 75 mM KCI; 15 mM MgCl2; 40 units\nof RNAsin (Promega, Madison, WI); 10 mM DTT;\n0.5 mM dNTP mix; 1.5 wg oligo d(T)!2_!e (Phar-\nmacia, Uppsala, Sweden); and 3 wg acetylated\nbovine serum albumin in 50 wl volume. The reac-\ntion mixture was incubated for 5 min at 95°C to\ninactivate M-MLV reverse transcriptase.\nPolymerase chain reaction\nThe primers used to amplify SHBG DNA frag-\nments were: 5’-TGTAGAATCAAATCCCGGGA-\n3’ (SHBG-5’; 591-610, Exon V) and 3’-TTCCAC-\nCACAAGAGAAGACC-5’ (SHBG-3’; 790-809,\nExon Vil) (Hammond et al, 1987b). The size of\nPCR products for SHBG mRNA was 219 bp (syn-\nthesized by Ricaken Co Ltd). The primers for\nCBG DNA fragments were synthesized: 5’-\nATGACCTTGGAGATGTGCTG-3’ (CBG-5; 929-\n948, Exon IV) and 3’-TGAACCCAGTGTAAGA-\nGAAC-5’ (CBG-3’; 1205-1224, Exon V)\n(Hammond et al, 1987a). The size of PCR prod-\nucts for CBG mRNA was 296 bp. The primers to\namplify glyceraldehyde-3-phosphate dehydro-\ngenase (G3PDH) were 5’-TGAAGGTCGGAGT-\nCAACGGATTTGGT-3’ (G3PDH-5’; 71-96, Exon I)\nand 3’-CACCACCTG GAGTACCGGGTGTAC-\n5’ (G3PDH-3’; 1053-1030, Exon VIII) (Arcali et\nal, 1984) (Clontech Laboratories, Palo Alto, CA).\nThe size of the PCR product for G3PDH mRNA\nwas 983 bp.\nPCR with reverse transcribed poly(A)+RNAs\nas templates (1 Ill) and 5 pmol of each specifc\nprimer was carried out using a DNA Thermal\nCycler (Perkin-Elmer Cetus, Norwalk, CT) with\n0.5 units of Amplitaq DNA polymerase (Perkin-\nElmer Cetus) in a buffer containing 50 mM KCI,\n10 mM Tris-HCI buffer pH 8.3, 1.5 mM MgCIZ and\n0.2 mM dNTPs in 20 wl volume.\nEach PCR cycle consisted of 1 min at 94°C for\ndenaturation, 2 min at 60°C for annealing and\n3 min at 72°C for extension with a DNA Thermal\nCycler (Perkin-Elmer Cetus). Thirty-eight cycles\nof PCR for SHBG mRNA, 31 cycles for CBG\nmRNA and 23 cycles for G3PDH were performed\nafter a serial dilution of cDNAs reverse transcribed\nto obtain the appropriate range of linear amplifi-\ncation of each PCR product.\nGel electrophoresis\nAn aliquot of amplified PCR products (8 Ill) after\naddition of 2 wl of loading dye mix (0.25% bro-\nmophenol blue and 30% glycerol in distilled water)\nwas electrophoresed on 2% NuSieve 3:1 agarose\n(FMC BioProducts, Rockland, ME) gels in Tris-\nborate/EDTA buffer (45 mM Tris-borate, pH 8.0,\n1 mM EDTA) in a 100 V constant-voltage field\nfor 50 min. The strength of photographed ethidium\nbromide-staining PCR products was analyzed\ndensitometrically by calculating the area with total\n\n\nintegrated optical density (IOD) using Bio Image\n(Millipore Corporation, Bedfold, MA). IOD shows\narbitrary units calculated by Bio Image.\nDNA sequence\nAmplified PCR products were electrophoresed\non 2% agarose gels. The SHBG and CBG cDNA\nfragments were isolated from excised gel slices\nusing a OIAEX agarose gel extraction kit (Qia-\ngen, Hilden, Germany) and inserted in pT7 Blue\nT-vector (Novergen, Madison, WI). After trans-\nformation of pT7 Blue T-vector with insertion into\nNova Blue competent cells (Novergen) and ampli-\nfication of the cells, double-stranded plasmid DNA\nwas isolated. Both strands of PCR fragments\nwere treated with a Circum Vent Thermal Cycle\nDeoxy DNA-sequencing Kit (New England Bio\nLabs, Beverly, MA) with biotinylated M13/pUC\nreverse sequencing primer and biotinylated T7\npromoter primer, and were sequenced by elec-\ntrophoresis on denaturing polyacrylamide gels\n(5% Hydrolink Long Ranger gel; AT Biochem,\nMalvern PA) at a constant power of 75 W for 3 h.\nAfter transfer of sequencing DNA fragments to a\nnylon membrane (immobilon-S; Millipore), the\nmembrane was dried, and UV cross-linked. The\nsequencing DNA bands were detected with a\nPlex 5 Chemiluminescent Subkit (New England\nBioLabs).\nStatistics\nThe levels of mRNA was compared by a Stu-\ndent’s t test. Correlation coefficients were deter-\nmined by Spearman’s rank test. Differences were\nconsidered to be significant at P < 0.05. All data\nwere expressed as mean ± SD.\nRESULTS\nSHBG mRNA and CBG mRNA\nin uterine endometrium\nA single dominant form of SHBG mRNA of\n1.6 kb was detected by the biotinylated\n\n\nSHBG DNA probe in human endometrium\nand was the same in size as SHBG mRNA\nin the endometrial carcinoma cells (Mercier-\nBodard et al, 1991) (fig 1 A). A single domi-\nnant form of CBG mRNA of approximately\n1.6 kb was also detected in human\nendometrium and was the same size as\nCBG mRNA in rhesus monkey liver (Ham-\nmond et al, 1987a) (fig 1 B). Lower copies\nof SHBG and CBG mRNAs in endometrium\nwere present at too low concentrations for\nevaluation by Northern blot analysis, so we\nperformed RT-PCR. Amplified SHBG and\nCBG mRNAs were detected with the\nexpected size in the uterine endometrium\nand the pelvic endometriosis in all samples\n(fig 2), and DNA sequences of both PCR\nproducts were identical to those of SHBG\nand CBG cDNAs (fig 3). In other words,\nSHBG and CBG mRNA were detected in\nendometriotic tissues in addition to normal\nendometrium.\nSHBG mRNA and CBG mRNA levels\nin endometriosis\nIOD for CBG, SHBG and G3PDH mRNAs\nlevels obtained by RT-PCR were plotted on\na log-log scale against the serial dilution of\nSHBG, CBG and G3PDH CDNA. There is\na good linear relationship between the\n\n\namount of input template and the output\nmeasurement (fig 4). Relative quantification\nof mRNA was obtained from the IOD on the\ngraph of SHBG and CBG, based on the indi-\nvidual dilution of CDNA, giving an IOD equiv-\nalent to the IOD 0.5 on G3PDH graph. After\nstandardization, the mRNA level was\nassigned as a corrected IOD value. The rel-\native amount of SHBG mRNA after stan-\ndardization to the G3PDH mRNA level indi-\ncated that the endometrial SHBG mRNA\nlevel was significantly higher in the secretory\nphase (1.46 ± 0.37 corrected IOD) than (P <\n0.02) in the proliferative phase (0.38 ± 0.26\ncorrected IOD) of the mentrual cycle (fig 5).\nThe relative amount of CBG mRNA was\ninvestigated in the same way as described\nabove (fig 4) and the endometrial CBG\nmRNA level was also significantly higher in\nthe secretory phase (0.45 ± 0.15 corrected\nIOD) than (P < 0.05) in the proliferative\nphase (1.26 ± 0.81 corrected IOD) of the\nmenstrual cycle (fig 5).\nThe level of SHBG mRNA in endometri-\notic tissue (4.07 ± 2.40 corrected IOD) was\nhigher than that in normal secretory phase\nendometrium. On the other hand, the level\nof CBG mRNA in the endometriotic tissues\n(0.27 ± 0.17 corrected IOD) was lower than\nthat in secretory phase endometrium (fig 5).\n\n\n\n\n\n\nMoreover, the ratio of SHBG mRNA level\nto CBG mRNA level was derived as the\naverage of the individual ratio from each tis-\nsue. The ratio of SHBG mRNA/CBG mRNA\nin endometriosis was approximately 20\ntimes higher than that in the normal\nendometrium (fig 6).\nDISCUSSION\nEstrogen-induced growth of endometriotic\nlesions has been documented even under\nthe influence of progesterone during the\nregular menstrual cycle. In the female repro-\nductive tract, the cooperative interaction of\nestrogen and progesterone (progestogen)\nplays an important role in biological events.\nIn particular, progestogen has antiestro-\ngenic effects, inhibiting cellular proliferation\nand bringing about cellular differentiation.\nTherefore, the substances binding estrogen\nand progestogen should be investigated in\nthe study of steroid action mechanisms in\nreproduction.\nSHBG and CBG are plasma glycopro-\nteins that bind steroid hormones such as\nestrogen and androgen (SHBG, Mercier-\nBodard et al, 1970) and corticosteroid and\nprogesterone (CBG, Seal and Doe, 1966)\nwith relatively high affinity. It has been\nassumed that the steroid-glycoprotein\ncomplex is inactive and only the free\nsteroids are biologically active at the tar-\nget cells (Hoffmann et al, 1969; Vermeulen\nand Ando, 1979). However, in recent stud-\nies, SHBG and CBG coupling to mem-\nbrane-binding sites, which activate adeny-\nlate cyclase and lead to cellular\naccumulation of cAMP (Nakhla etal, 1988;\nNakhla et al, 1990), has been demon-\nstrated in human decidual endometrium\n\n\n(Strel’chyonok et al, 1984) and prostate\n(Hryb etal, 1985) for SHBG, and in human\nliver (Strel’chyonok and Awakumov, 1983),\nprostate (Hryb et al, 1986) and decidual\nendometrium (Avvakumov et al, 1988) for\nCBG indicating that protein-bound steroid\nmay also be available (Siiteri ef al, 1982;\nSelby, 1990). Moreover, the expression of\nSHBG and CBG, and their mRNAs, has\nbeen detected in the target cells (as\ndescribed in the Introduction) immunohis-\ntochemically (Perrot-Applanat et al, 1984;\nKuhn et al, 1986; Mercier-Bodard et al,\n1987; Sinnecker et al, 1990) and by North-\nern blot analysis (Mercier-Bodard et al,\n1991; Hammond et al, 1987a). This sug-\ngests that SHBG and CBG might be\ninvolved in the regulatory system of some\nsteroid actions as an intracellular reservoir\nor buffer which regulates the free fraction of\nsteroid hormones in their target cells\n(Mercier-Bodard etal, 1991). In addition,\nthe synthesis of endometrial SHBG and\nCBG might be complexly regulated by\nsteroid hormones such as estrogen and\nprogesterone, in a manner different from\nthat in the liver (Misao et al, 1994a, b).\nThe expression of SHBG and CBG\nmRNAs has been documented in the normal\nendometrium and the endometriosis, indi-\ncating that SHBG and CBG are synthesized\nin target tissue cells.\nThe expression of SHBG mRNA appears\nto be higher in endometriosis than in nor-\nmal endometrium, indicating that SHBG is\nsynthesized more in the endometriosis than\nCBG. In addition, it is assumed that intra-\ncellular SHBG is more involved as a store of\nestrogen in the regulatory system of\nsteroidal action in the endometriotic cells,\nwhile the ratio of the SHBG mRNA level to\nthat of CBG mRNA is much higher in\nendometriosis than in the endometrium.\nTherefore, estrogen might be more stored\nintracellularly due to the abundance of\nSHBG, thus providing the cellular estrogen-\npredominant milieu.\nREFERENCES\nArcali P, Martinelli R, Salvatore F (1984) The complete\nsequence of a full length cDNA for human liver glyc-\neraldehyde-3-phosphate dehydrogenase: evidence\nfor multiple mRNA species. Nucleic Acids Res 153,\n1012 2\nAvvakumov GV, Krupenko SA, Dubovskaya LV,\nStrefchyonok OA (1988) Interaction of the transcortin-\nprogesterone complex with plasma membranes of\nhuman decidual endometrium cells. Biokhimiya 53,\n586-590\nDizerega GS, Barber DL, Hodgen GD (1980)\nEndometriosis: role of ovarian steroids in initiation,\nmaintenance and suppression. Fertil Steri133, 649-\n653\nDorfman Ri, Kincl FA, Ringold HJ (1961) Antiestrogen\nassay of neutral steroids administered by subcuta-\nneous injection. Endocrinology 68, 17-24\nHammond GL, Smith CL, Goping IS et al (1987a) Pri-\nmary structure of human corticosteroid binding glob-\nulin, deduced from hepatic and pulmonary cDNAs,\nexhibits homology with serine protease inhibitors.\nProc Natl Acad Sci USA 84, 5153-5157\nHammond GL, Underhill DA, Smith CL et al (1987b) The\ncDNA-deduced primary structure of human sex hor-\nmone-binding globulin and location of its steroid-\nbinding domain. J Biol Chem 260, 100-104\nHoffmann W, Forbes TR, Westphal U (1969) Biological\ninactivation of progesterone by interaction with cor-\nticosteroid-binding globulin and with albumin.\nEndocrinology 85, 778-781\nHryb DJ, Khan MS, Rosner W (1985) Testosterone-\nestradiol-binding globulin binds to human prostatic\ncell membranes. Biochem Biophys Res Comm 128,\n432-440\nHryb DJ, Khan MS, Romas NA, Rosner W (1986) Spe-\ncific binding of human corticosteroid-binding globu-\nlin to cell membranes. Proc Natl Acad Sci USA 83,\n3253-3256\nJacobsen KS, Breivold E, Homes E (1990) Purification\nof mRNA directly from crude plant tissues in 15 min-\nutes using magnetic oligo dT microspheres. Nudeic\nAcids Res 18, 3669\nKuhn R, Green A, Raymoure W, Siiteri P (1986) lmmuno-\ncytochemical localization of corticosteroid-binding\nglobulin in rat tissues. J Endocrinol 108, 31-36\nMercier-Bodard C, Alfsen A, Baulieu EE (1970) Sex\nsteroid binding plasma protein. Acta Endocrinol\n(Copenh) Suppl 147, 204-224\nMercier-Bodard C, Radanyi C, Roux C etal(1987) Cel-\nlular distribution and hormonal regulation of h-SBP.\nJ Steroid Biochem 27, 297-307\nMercier-Bodard C, Nivet V, Baulieu EE (1991) Effects\nof hormones on SBP mRNA levels in human can-\ncer cells. J Steroid Biochem 40, 777-785\n\n\nMisao R, Hori M, lchigo S, Fujimoto J, Tamaya T\n(1994a) Corticosteroid-binding globulin mRNA lev-\nels in human uterine endometrium. Steroids 59,\n603-607\nMisao R, Itoh N, Mori H, Fujimoto J, Tamaya T (1994b)\nSex hormone-binding globulin mRNA levels in human\nuterine endometrium. Eur J Endocrinol 131, 623-\n629\nNakhla AM, Khan MS, Roser W (1988) Induction of\nadenylate cyclase in a mammary carcinoma cell line\nby human corticosteroid-binding globulin. Biochem\nBiophys Res Commun 153, 1012-1018 8\nNakhla AM, Khan MS, Rosner W (1990) Biologically\nactive steroids activate receptor-bound human sex\nhormone-binding globulin to cause LNCaP cells to\naccumulate adenosine 3’5’-monophosphate. J Clin\nEndocrinol Metab 71, 398-404\nNoyes RW, Hertig AT, Rock J (1950) Dating the endome-\ntrial biopsy. Fertil Steril 1, 3-5\nPerrot-Applanat M, Racadot 0, Milgrom E (1984) Spe-\ncific localization of plasma corticosteroid-binding\nglobulin immunoreactivity in pituitary corticotrophs.\nEndocrinology 115, 559-569\nSeal US, Doe RP (1966) Corticosteroid-binding globulin:\nBiochemistry, physiology, and phylogeny. In: Proc\nSymposium on the Dynamics of Steroid Hormone\n(G Pincus, ed), Academic Press, New York, USA,\n63-90\nSelby C (1990) Sex hormone-binding globulin: origin,\nfunction and clinical significance. Ann Clin Biochem\n27, 532-541\nSiiteri PK, Murai JT, Hammond GL, Nisker JA, Ray-\nmoure WJ, Kuhn RW (1982) The serum transport\nof steroid hormones. Recent Prog Horm Res 38,\n457-510 0\nSinnecker G, Hiort 0, Kwan PWL, de Lellis RA (1990)\nImmunohistochemical localization of sex hormone-\nbinding globulin in normal and neoplastic breast tis-\nsue. Horm Metab Res 22, 47-50\nStrel’chyonok OA, Avvakumov GV (1983) Evidence for\nthe presence of specific binding sites for transcortin\nin human liver plasma membranes. Biochim Biophys\nActa 755, 514-517 7\nStrel’chyonok OA, Avvakumov GV, Survilo Li (1984) A\nrecognition system of sex hormone-binding protein-\nestradiol complex in human decidual endometrium\nplasma membranes. Biochem Biophys Acta 802,\n459-466\nTamaya T, Motoyama T, Ohno Y, lde N, Tsurusaki T,\nOkada H (1979) Steroid receptor levels and histology\nof endometriosis and adenomyosis. Fertil Steril 31,\n396-400\nVermeulen A, And6 S (1979) Metabolic clearance rate\nand interconversion of androgens and the influence\nof the free androgen fraction. J Clin Endocrinol Metab\n48, 320-326","source_license":"CC0","license_restricted":false}