Levels of sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) messenger ribonucleic acid (mRNAs) in ovarian endometriosis

The proliferation, differentiation, and devel- opment of normal uterine endometrium is regulated by sex steroids. In some cases, when endometrial tissue exhibits aberrant growth, which occurs ectopically in various locations in the pelvic cavity (endometrio- sis), it exhibits the cyclic functional respon- siveness of normal endometrium. In other cases an immature or unripe variety devel- ops, which is only responsive to the estro- genic stimulus and not to progesterone (Diz- erega et al, 1980). Estrogen-induced growth of endometrio- sis might be partly explained by the fol- lowing evidence. Most endometriosis is less responsive to progestogens, due to the low level of progesterone receptor rel- ative to that of estrogen receptor (Tamaya et al, 1979), while progestogen has an anti- estrogenic effect (Dorfman ef al, 1961 ). Recently, a great deal of evidence has indicated that sex hormone-binding globu- lin (SHBG) and corticosteroid-binding glob- ulin (CBG) play a role in the intracellular action of sex steroid hormones in target cells. The expressions of SHBG and CBG have been demonstrated immunohisto- chemically in human endometrium, prostate (Mercier-Bodard et al, 1987) and breast tissue (Sinnecker et al, 1990), and in pitu- itary (Perrot-Applanat etal, 1984) and thy- roid glands (Kuhn et al, 1986). Moreover, mRNA expressions of SHBG and CBG have been analyzed in human endometrial cancer cell lines (Mercier-Bodard et al, 1991) and rhesus monkey testis (Ham- mond et al, 1987a) by Northern blot hybridization. These results prompted us to investi- gate the expression of SHBG and CBG mRNA in order to know the mechanism of estrogen-induced growth of endometrio- sis.

Materials Human uterine endometria were obtained by endometrial biopsy from 35 patients (aged from 25 to 39 years) with a regular menstrual cycle at the Department of Obstetrics and Gynecology, Gifu University School of Medicine from July 1990 to June 1993. Part of the specimen was submit- ted for histological dating (Noyes et al, 1950). Agreements for the study were obtained from patients and from the Research Committee on Human Subject of Gifu University School of Medicine. Pelvic endometriosis tissues were obtained from 6 patients who underwent conser- vative or definitive surgery for ovarian endometrio- sis. These specimens were immediately frozen in liquid nitrogen and prepared for the following procedures, such as RNA isolation, Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). Poly(A)+RNA isolation Frozen tissue (100 mg) was ground, transferred to a Teflon-glass homogenizer containing 1 ml lysis/binding buffer (100 mM Tris-HCI pH 8.0, 500 mM LiCl, 10 mM EDTA (ethylenediamine- tetraacetic acid) pH 8.01% SDS (sodium dodecyl sulfate), 5 mM DTT (dithiothreitol) and homoge- nized manually at 4°C. After spinning at 20 000 g for 30 s, the supernatant was transferred to an Eppendorf tube. Polyadenylated mRNA (poly(A)+RNA) was isolated from tissue with mag- netic beads, Dynabeads Oligo (dT)25 (Dynal AS, Oslo, Norway) (Jacobsen et al, 1990). The poly(A)+RNA concentration was determined by UV absorption at 260 and 280 nm. Preparation of labeled probe SHBG cDNA (1 143 bp) and CBG cDNA (1 215 bp) were synthesized from human liver poly(A)+RNA (Clontech Laboratories, Palo Alto, CA) using the PCR (as detailed below for the PCR). The primers used for SHBG cDNA synthesis using PCR were 5’-CAGCACACCCGCCAGGGATGGGCC-3’ (SHBG-5’:1-24, Exon I) and 3’-CCGTTACCGT- GACTGCGAAGGGTA-5’ (SHBG-3’:1120-1143, Exon VIII) (Hammond et al, 1987b). The primers used for CBG cDNA synthesis were 5’-ATGC- CACTCCTCCTGTACAC-3’ (CBG-5’: 1-20, Exon II) and 3’-TGAACCCAGTGTAAGAGACC-5’ (CBG-3’:1205-1224, Exon V) (Hammond et al, 1987a). The DNA probe was labeled with biotiny- lated dUTP using a Polar Plex Random Primer Biotin Labelling Kit (Millipore, Burlington, MA). Northern blot analysis Poly(A)+RNA (45 !g) was denatured at 65°C for 15 min, size-fractionated by electrophoresis through 1 % agarose-formaldehyde gel, and blot- ted onto a nylon membrane (immobilon-S; Milli- pore) by capillary transfer for 20 h using 10 x x standard saline citrate (SSC: 1.5 M NaCI, 0.15 M sodium citrate, pH 7.0). After blotting, the mem- brane was dried at 75°C for 20 min and then cross- linked by ultraviolet irradiation (33 000 ¡W/cm2 at 254 nm). Prehybridization was performed at 42°C for 4 h in a mixture containing 5 x SSC, 50% for- mamide, 2 x Denhardt’s reagent, 0.1 % SDS, and 100 pg/ml denatured salmon sperm DNA. The same solution was used for the hybridization but included the biotin-labeled probe (26 ng/ml). After hybridization at 42°C for 24 h, the membrane was washed for 20 min at room temperature and then finally washed twice with 0.1 x SSC for 30 min at 65°C. The detection reaction using a Plex Chemiluminescent Kit (New England BioLabs, Beverly, MA). The membrane was exposed to Kodak XAR-5 films (Eastman Kodak, Rochester, NY) for 15 min. Reverse transcription Poly(A)+RNA (10 ng) was reverse transcribed for 1 h at 42°C with a mixture of 600 units of M-MLV reverse transcriptase (Gibco BRL, Gaithersburg, MD) and the following reagents: 50 mM Tris-HCI buffer pH 8.3; 75 mM KCI; 15 mM MgCl2; 40 units of RNAsin (Promega, Madison, WI); 10 mM DTT; 0.5 mM dNTP mix; 1.5 wg oligo d(T)!2_!e (Phar- macia, Uppsala, Sweden); and 3 wg acetylated bovine serum albumin in 50 wl volume. The reac- tion mixture was incubated for 5 min at 95°C to inactivate M-MLV reverse transcriptase. Polymerase chain reaction The primers used to amplify SHBG DNA frag- ments were: 5’-TGTAGAATCAAATCCCGGGA- 3’ (SHBG-5’; 591-610, Exon V) and 3’-TTCCAC- CACAAGAGAAGACC-5’ (SHBG-3’; 790-809, Exon Vil) (Hammond et al, 1987b). The size of PCR products for SHBG mRNA was 219 bp (syn- thesized by Ricaken Co Ltd). The primers for CBG DNA fragments were synthesized: 5’- ATGACCTTGGAGATGTGCTG-3’ (CBG-5; 929- 948, Exon IV) and 3’-TGAACCCAGTGTAAGA- GAAC-5’ (CBG-3’; 1205-1224, Exon V) (Hammond et al, 1987a). The size of PCR prod- ucts for CBG mRNA was 296 bp. The primers to amplify glyceraldehyde-3-phosphate dehydro- genase (G3PDH) were 5’-TGAAGGTCGGAGT- CAACGGATTTGGT-3’ (G3PDH-5’; 71-96, Exon I) and 3’-CACCACCTG GAGTACCGGGTGTAC- 5’ (G3PDH-3’; 1053-1030, Exon VIII) (Arcali et al, 1984) (Clontech Laboratories, Palo Alto, CA). The size of the PCR product for G3PDH mRNA was 983 bp. PCR with reverse transcribed poly(A)+RNAs as templates (1 Ill) and 5 pmol of each specifc primer was carried out using a DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk, CT) with 0.5 units of Amplitaq DNA polymerase (Perkin- Elmer Cetus) in a buffer containing 50 mM KCI, 10 mM Tris-HCI buffer pH 8.3, 1.5 mM MgCIZ and 0.2 mM dNTPs in 20 wl volume. Each PCR cycle consisted of 1 min at 94°C for denaturation, 2 min at 60°C for annealing and 3 min at 72°C for extension with a DNA Thermal Cycler (Perkin-Elmer Cetus). Thirty-eight cycles of PCR for SHBG mRNA, 31 cycles for CBG mRNA and 23 cycles for G3PDH were performed after a serial dilution of cDNAs reverse transcribed to obtain the appropriate range of linear amplifi- cation of each PCR product. Gel electrophoresis An aliquot of amplified PCR products (8 Ill) after addition of 2 wl of loading dye mix (0.25% bro- mophenol blue and 30% glycerol in distilled water) was electrophoresed on 2% NuSieve 3:1 agarose (FMC BioProducts, Rockland, ME) gels in Tris- borate/EDTA buffer (45 mM Tris-borate, pH 8.0, 1 mM EDTA) in a 100 V constant-voltage field for 50 min. The strength of photographed ethidium bromide-staining PCR products was analyzed densitometrically by calculating the area with total integrated optical density (IOD) using Bio Image (Millipore Corporation, Bedfold, MA). IOD shows arbitrary units calculated by Bio Image. DNA sequence Amplified PCR products were electrophoresed on 2% agarose gels. The SHBG and CBG cDNA fragments were isolated from excised gel slices using a OIAEX agarose gel extraction kit (Qia- gen, Hilden, Germany) and inserted in pT7 Blue T-vector (Novergen, Madison, WI). After trans- formation of pT7 Blue T-vector with insertion into Nova Blue competent cells (Novergen) and ampli- fication of the cells, double-stranded plasmid DNA was isolated. Both strands of PCR fragments were treated with a Circum Vent Thermal Cycle Deoxy DNA-sequencing Kit (New England Bio Labs, Beverly, MA) with biotinylated M13/pUC reverse sequencing primer and biotinylated T7 promoter primer, and were sequenced by elec- trophoresis on denaturing polyacrylamide gels (5% Hydrolink Long Ranger gel; AT Biochem, Malvern PA) at a constant power of 75 W for 3 h. After transfer of sequencing DNA fragments to a nylon membrane (immobilon-S; Millipore), the membrane was dried, and UV cross-linked. The sequencing DNA bands were detected with a Plex 5 Chemiluminescent Subkit (New England BioLabs). Statistics The levels of mRNA was compared by a Stu- dent’s t test. Correlation coefficients were deter- mined by Spearman’s rank test. Differences were considered to be significant at P < 0.05. All data were expressed as mean ± SD.

SHBG mRNA and CBG mRNA in uterine endometrium A single dominant form of SHBG mRNA of 1.6 kb was detected by the biotinylated SHBG DNA probe in human endometrium and was the same in size as SHBG mRNA in the endometrial carcinoma cells (Mercier- Bodard et al, 1991) (fig 1 A). A single domi- nant form of CBG mRNA of approximately 1.6 kb was also detected in human endometrium and was the same size as CBG mRNA in rhesus monkey liver (Ham- mond et al, 1987a) (fig 1 B). Lower copies of SHBG and CBG mRNAs in endometrium were present at too low concentrations for evaluation by Northern blot analysis, so we performed RT-PCR. Amplified SHBG and CBG mRNAs were detected with the expected size in the uterine endometrium and the pelvic endometriosis in all samples (fig 2), and DNA sequences of both PCR products were identical to those of SHBG and CBG cDNAs (fig 3). In other words, SHBG and CBG mRNA were detected in endometriotic tissues in addition to normal endometrium. SHBG mRNA and CBG mRNA levels in endometriosis IOD for CBG, SHBG and G3PDH mRNAs levels obtained by RT-PCR were plotted on a log-log scale against the serial dilution of SHBG, CBG and G3PDH CDNA. There is a good linear relationship between the amount of input template and the output measurement (fig 4). Relative quantification of mRNA was obtained from the IOD on the graph of SHBG and CBG, based on the indi- vidual dilution of CDNA, giving an IOD equiv- alent to the IOD 0.5 on G3PDH graph. After standardization, the mRNA level was assigned as a corrected IOD value. The rel- ative amount of SHBG mRNA after stan- dardization to the G3PDH mRNA level indi- cated that the endometrial SHBG mRNA level was significantly higher in the secretory phase (1.46 ± 0.37 corrected IOD) than (P < 0.02) in the proliferative phase (0.38 ± 0.26 corrected IOD) of the mentrual cycle (fig 5). The relative amount of CBG mRNA was investigated in the same way as described above (fig 4) and the endometrial CBG mRNA level was also significantly higher in the secretory phase (0.45 ± 0.15 corrected IOD) than (P < 0.05) in the proliferative phase (1.26 ± 0.81 corrected IOD) of the menstrual cycle (fig 5). The level of SHBG mRNA in endometri- otic tissue (4.07 ± 2.40 corrected IOD) was higher than that in normal secretory phase endometrium. On the other hand, the level of CBG mRNA in the endometriotic tissues (0.27 ± 0.17 corrected IOD) was lower than that in secretory phase endometrium (fig 5). Moreover, the ratio of SHBG mRNA level to CBG mRNA level was derived as the average of the individual ratio from each tis- sue. The ratio of SHBG mRNA/CBG mRNA in endometriosis was approximately 20 times higher than that in the normal endometrium (fig 6).

Estrogen-induced growth of endometriotic lesions has been documented even under the influence of progesterone during the regular menstrual cycle. In the female repro- ductive tract, the cooperative interaction of estrogen and progesterone (progestogen) plays an important role in biological events. In particular, progestogen has antiestro- genic effects, inhibiting cellular proliferation and bringing about cellular differentiation. Therefore, the substances binding estrogen and progestogen should be investigated in the study of steroid action mechanisms in reproduction. SHBG and CBG are plasma glycopro- teins that bind steroid hormones such as estrogen and androgen (SHBG, Mercier- Bodard et al, 1970) and corticosteroid and progesterone (CBG, Seal and Doe, 1966) with relatively high affinity. It has been assumed that the steroid-glycoprotein complex is inactive and only the free steroids are biologically active at the tar- get cells (Hoffmann et al, 1969; Vermeulen and Ando, 1979). However, in recent stud- ies, SHBG and CBG coupling to mem- brane-binding sites, which activate adeny- late cyclase and lead to cellular accumulation of cAMP (Nakhla etal, 1988; Nakhla et al, 1990), has been demon- strated in human decidual endometrium (Strel’chyonok et al, 1984) and prostate (Hryb etal, 1985) for SHBG, and in human liver (Strel’chyonok and Awakumov, 1983), prostate (Hryb et al, 1986) and decidual endometrium (Avvakumov et al, 1988) for CBG indicating that protein-bound steroid may also be available (Siiteri ef al, 1982; Selby, 1990). Moreover, the expression of SHBG and CBG, and their mRNAs, has been detected in the target cells (as described in the Introduction) immunohis- tochemically (Perrot-Applanat et al, 1984; Kuhn et al, 1986; Mercier-Bodard et al, 1987; Sinnecker et al, 1990) and by North- ern blot analysis (Mercier-Bodard et al, 1991; Hammond et al, 1987a). This sug- gests that SHBG and CBG might be involved in the regulatory system of some steroid actions as an intracellular reservoir or buffer which regulates the free fraction of steroid hormones in their target cells (Mercier-Bodard etal, 1991). In addition, the synthesis of endometrial SHBG and CBG might be complexly regulated by steroid hormones such as estrogen and progesterone, in a manner different from that in the liver (Misao et al, 1994a, b). The expression of SHBG and CBG mRNAs has been documented in the normal endometrium and the endometriosis, indi- cating that SHBG and CBG are synthesized in target tissue cells. The expression of SHBG mRNA appears to be higher in endometriosis than in nor- mal endometrium, indicating that SHBG is synthesized more in the endometriosis than CBG. In addition, it is assumed that intra- cellular SHBG is more involved as a store of estrogen in the regulatory system of steroidal action in the endometriotic cells, while the ratio of the SHBG mRNA level to that of CBG mRNA is much higher in endometriosis than in the endometrium. Therefore, estrogen might be more stored intracellularly due to the abundance of SHBG, thus providing the cellular estrogen- predominant milieu.

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