0021-972x/92/7504-1140$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 75, No. 4 Prmted in U.S.A.
The Expression of Human Chorionic Gonadotropin/ Human Luteinizing Hormone Receptors in Ectopic Human Endometrial Implants S. R. LINCOLN,
Z. M. LEI,
CH.
V. RAO,
Department of Obstetrics and Gynecology, University Louisville, Kentucky 40292
AND of
M. A. YUSSMAN Louisville
School of Medicine,
ABSTRACT
contain receptor mRNA and receptor protein. The glands contain more receptor mRNA and receptor protein than stromal cells in implants similar to uterine endometrium from patients with or without endometriosis. However, there is no consistent difference in the expression of receptors in implants compared to uterine endometria from patients with or without endometriosis. Contrary to ectopic endometrial implants, unaffected or normal peritoneum contain neither receptor mRNA nor receptor protein. In summary, we conclude that ectopic endometrial implants contain hCG/LH receptor mRNA and receptor protein, which suggests new possibilities in the medical treatment of endometriosis. (J Clin Endocrinol Metab 75: 1140-1144,1992)
Our laboratory previously demonstrated that normal human endometrium contains hCG/human LH receptors. Since ectopic endometrial implants in endometriosis arise directly at least in part from uterine endometrium, we investigated whether the implants continue the expression of these receptors. The presence of hCG/LH receptor mRNA and/or immunoreactive receptor protein in ectopic endometrial implants on pelvic peritoneum, uterine endometrium, and unaffected or normal peritoneum from patients with (n = 12) and without (n = 14) clinically apparent endometriosis was examined by in situ hybridization and immunocytochemistry analyses. The results showed that the peritoneal biopsies with visible or microscopic endometrial implants
H
CG AND human LH are glycoprotein hormones of similar structure and function (1). They are primarily secretedby the placenta and anterior pituitary, respectively. These two hormones bind to the same receptors in target gonadal cells to stimulate steroidogenesis(l-3). Recent studies from our laboratory have shown that several nongonadal human reproductive tissuesalso contain hCG/LH receptors. The tissuesinclude endometrium (4), myometrium (4), Fallopian tubes (5), placenta (4, 6), fetal membranes(4), decidua (4), human gestational trophoblastic neoplasms (7), and blood vesselsin someof these tissues(8). Endometriosisis a benign gynecological diseasecharacterized by the presence of endometrium in ectopic locations throughout the body. The pelvic peritoneum and/or ovaries are the common sitesof implantation. This diseaseis seenin 25-35s of the infertile patient population and is characterized in part by dysmenorrhea, pelvic pain, dyspareunia, and infertility (9). Since normal human endometrium expresses the hCG/LH receptor gene, we investigated the possibility of whether the ectopic endometrial implants on pelvic peritoneum from endometriosisalso expressthis gene. The present study demonstrates that ectopic endometrial implants also contain hCG/LH receptor mRNA and receptor protein. Materials and Methods The sources of all the materials, reagents, riboprobes, performance of in situ hybridization,
etc., used for transcribing and immunocytochem-
Received December 23, 199 1. Address all correspondence and requests for reprints to: Dr. Ch.V. Rao, Department of Obstetrics and Gynecology, 438 MDR Building, University of Louisville, Louisville, Kentucky 40292.
istry have previously been described (4, 7, 8). Biopsy specimens were taken from 26 patients undergoing laparotomy (n = 1) or total abdominal hysterectomy/bilateral salpingooophorectomy (n = 1) or diagnostic laparoscopy (n = 24) for chronic pelvic pain, unexplained infertility, or both at the University of Louisville affiliated hospitals. Twelve patients (age range, 22-40 yr) had clinically observable endometriosis, and 14 patients (age range, 21-40 yr) did not. Eight of the 12 endometriosis patients and 11 of the 14 nonendometriosis patients were in the proliferative phase of the cycle. According to American Fertility Society classification criteria, 6 endometriosis patients had stage 1 disease, and 2 each had stage 2 through stage 4 disease. Neither group of women had been on any hormonal treatment for infertility before the surgical procedures. Biopsies of uterine endometrium and normal appearing peritoneum were taken from all patients. In addition, peritoneal biopsies were taken from the visible sites of ectopic endometrial implants from the 12 patients with clinical endometriosis. These peritoneal biopsies were taken predominantly from the anterior cul-de-sac or posterior cul-de-sac near the uterosacral ligaments. The specimens were frozen in liquid nitrogen until processing for in situ hybridization and immunocytochemistry. The preparation of 32P-labeled antisense and sense riboprobes from porcine hCG/LH receptor cDNA (nucleotides -11 to 2089) in pBluescript SK(+), kindly provided by Dr. Hugues Loosfelt (Paris, France), and performance of in situ hybridization were previously described (7, 8). Hybridization with the sense probe carried out at the same time under identical conditions served as a negative control. Two polyclonal antibodies to synthetic fragments of rat luteal hCG/ LH receptors (anti-PCR II, l-11 and anti-LHR 1%38), kindly provided by Dr. Patrick Roche, Mayo Clinic (Rochester, MN), and a monoclonal antibody to rat luteal hCG/LH receptors, kindly provided by Dr. Judith Luborsky, TSI Mason Research Institute (Worcester, MA), were used for immunocytochemical detection of hCG/LH receptor protein. The details of the procedure were previously described (4, 7, 8). The receptor antibodies were used at 1:500 (polyclonal) or 1:lOO (monoclonal) dilution. Three types of controls were performed: 1) polyclonal receptor antibodies preabsorbed with corresponding synthetic receptor peptides, also kindly provided by Dr. P. Roche, and the monoclonal receptor antibody preabsorbed with bovine luteal microsomes prepared in our laboratory (lo), 2) omission of the unabsorbed receptor antibodies, and
1140
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hCG/LH
RECEPTORS
3) substitution of the unabsorbed receptorantibodies with nonspecific immunoglobulin G during the immunostaining procedure. The immunostaining of all specimens including controls was performed at the same time using identical conditions. Three of the authors independently evaluated the degree and pattern of immunostaining by examining the slides under an Olympus microscope (New Hyde Park, NY). The representative photographs presented in this manuscript were processed at the same time by a commercial vendor using computerized equipment.
Results In situ hybridization analysis with 32P-labeledantisense riboprobe shows that endometrial glands and stromal cells in ectopic endometrial implant (Fig. 1A) and uterine endometrium from patients with (Fig. 1B) and without (Fig. 1C) apparent endometriosis contain silver grains. These grains, which reflect the presenceof hCG/LH receptor mRNA, are absentwhen 32P-labeledsenseprobe, which cannot basepair with receptor mRNA, was used (Fig. 1, D, E, and F). The glands contain more hybridization signals due to antisense probe than stromal cells in ectopic endometrial implants similar to uterine endometrium (Fig. 1, A, B, and C). The grain distribution in ectopic endometrial implants is similar to that in uterine endometrium (Fig. 1, A VS.B and C). Figure 2 shows that the immunostaining for hCG/LH receptor protein is present in ectopic endometrial implant (A) and uterine endometrium from patients with (B) or without (C) apparent endometriosis. In ectopic endometrial implants, as in uterine endometria, the glands contain more receptor immunostaining than stromal cells (Fig. 2, A-C). There are no obvious differences in the receptor immunostaining between ectopic endometrial implant and uterine endometria (Fig. 2, A VS.B and C). Some biopsies of normal appearing peritoneum from patients with (2 of 12) and without (4 of 14) clinically observable endometriosis contained microscopicendometrial glands, and they all were immunostained for hCG/LH receptors (Fig. 2, H-M).
IN ENDOMETRIOSIS
1141
The receptor immunostaining was absent in unaffected adjacent peritoneum from endometriosis patients (Fig. 2A) and in peritoneum (Fig. 2G) of patients without endometriosis.The receptor immunostaining was also absent when the receptor antibody preabsorbed with excessreceptor peptide was used for immunostaining ectopic endometrial implants (Fig. 2D) and uterine endometria (Fig. 2, H and J) from patients with or without endometriosis. Similar results were obtained when unabsorbed receptor antibody was omitted or substituted with nonspecific immunoglobulin G during the immunostaining procedure (data not shown). Another polyclonal receptor antibody (anti-PCRII, l-l 1) and a monoclonal receptor antibody gave results similar to those presented in Fig. 2 using polyclonal receptor antibody, LHR 1538. Discussion The present studies were prompted by the recent findings that human uterus contains a major 4.3-kilobase and a minor 2.6-kb hCG/LH receptor mRNA transcript (8). These transcripts along with the immunoreactive receptor protein of 100 kilodaltons are present in endometrial glands, stromal cells, myometrial smooth muscle, and uterine blood vessels (4, 8). Among these uterine cells, glands contain more receptor transcripts and receptor protein than the other cells (4, 8). All of the uterine cells show an increasein receptors from the proliferative to secretory phase (4, 8). These findings suggestedthat hCG/LH may directly regulate human uterine functions. Consistent with this possibility, we have recently demonstrated that hCG can increase both the number and size of cultured human myometrial smooth muscle cells (11). Since ectopic human endometrial implants originate from uterine endometria, it was of interest to determine whether the implants also express hCG/LH receptors. The results clearly demonstrate that the endometrial implants, but not unaffected adjacent peritoneum or peritoneum from nonen-
FIG. 1. In situ hybridization for hCG/ LH receptor mRNA in ectopic (A and D) and uterine endometrium (B and E) from endometriosis patients and uterine endometrium (C and F) from patients without endometriosis. 32P-Labeled antisense riboprobe was used in A-C, and sense probe was used in D-F. Glandular epithelium is indicated by arrows, and stromal cells by arrowheads. Magnification, X384.
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11
A IF---
_-:
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hCG/LH
RECEPTORS
dometriosis patients, contain hCG/LH receptor mRNA and receptor protein. Although endometrial implants in other anatomical locations in the body were not examined, it is quite likely that they may also contain these receptors because of the common origin and similarity of implants from different locations (12). The overall receptor expression and the glands containing more receptors than stromal cells are similar in both ectopic endometrial implants and uterine endometria. These findings are consistent with the previous observations that glands and stroma in implants are active as those in uterine endometria (12). Contrary to the lower estrogen and progesterone receptor concentrations in implants compared to those in uterine endometrium (13, 14), hCG/LH receptor gene expression appears to be similar. Compared to bovine corpus luteum, the specific binding of [‘251]hCG to human uterine tissue is very low (unpublished data from our laboratory). Nevertheless, the ligand binding in ectopic endometrial implants and uterine endometrium was not determined because the amount of tissues from the biopsies was not adequate. In the present study we did find higher uterine endometrial expression of receptors in the secretory compared to the proliferative phase as previously reported (4, 8). However, we could not find a similar consistent difference in ectopic endometrial implants. This is probably due to two reasons. First, the ectopic endometrial implants may not always be in phase with uterine endometrium. Second, the amount of endometrium is often too small in implants to determine the differences in immunostaining. We had too few samples to determine whether receptor expression was dependent on the stage of endometriosis. Because of sample procurement problems, we have not been able to study receptor expression during or after hormonal therapy. This study does not attempt to determine the functional significance of hCG/LH receptors in ectopic endometrial implants. Since these implants originate from the endometrium or an analog shared with the endometrium, the function in implants is probably similar to that in uterine endometrium. This is currently being investigated. The treatment of endometriosis patients with GnRH analogs (GnRHa) results in regression of implants and relief from the symptoms (15). These therapeutic benefits have been attributed to decreased estradiol levels due to desensitization of the hypothalamic-hypophyseal axis resulting in decreased LH levels (16-18). Although the role of estradiol in the growth of endometriosis is convincing, it is not universally accepted. The direct result of decreased LH secretion due to chronic GnRH-a treatment of the endometrial implants is unknown. This was not previously suspected because the existence of hCG/LH receptors in ectopic endometrial implants was not known until the present work. Endometriosis subsides during pregnancy, and in normal or FIG. 2. Light microscope
immunocytochemistry for immunoreactive and E) from endometriosis patients and uterine endometrium immunostaining for the receptors in microscopic implants. The M, and the receptor antibody preabsorbed with excess receptor and unaffected adjacent peritoneum from endometriosis patient
IN ENDOMETRIOSIS
1143
pseudomenopause induced by surgical or medical castration (17-22), the conditions in which the hCG and hLH levels are elevated. This does not necessarily rule out the possible direct effect of decreased hLH levels achieved by GnRHa in the treatment of endometriosis. The beneficial effects of pregnancy and menopause on endometriosis could be related to various hormonal and biophysical alterations that occur. In summary, the present study demonstrates for the first time that ectopic human endometrial implants, but not unaffected or normal peritoneum, contain hCG/LH receptor mRNA and receptor protein. This finding suggests new possibilities in the medical treatment of endometriosis. References 1.
Pierce JG, Parsons TF. 1981 Glycoprotein function.
Annu
Rev Biochem.
hormones:
structure
2. Catt KJ, Harwood JP, Clayton RN, et al. 1980 Regulation receptors
and
gonadal
and
50:466-495.
steroidogenesis.
Recent
Frog
of peptide Horm Res.
361557-622. 3. Rao ChV. 1982 Receptors
for gonadotropins in human ovaries. In: Muldoon TH, Mahesh VB, Presz-Ballester B, eds. Recent advances in fertility research, developments in reproductive endocrinology, part A. New York: Liss; pp 123-135.
4. Reshef E, Lei ZM, Rao ChV, Pridham DD, Chegini N, Luborsky JL. 1990 The presence of gonadotropin receptors in nonpregnant human uterus, Clin Endocrinol
5. Bailey-Pridham
6.
7.
8. 9. 10.
11.
12. 13.
human Metab.
placenta, fetal 70:421-430.
membranes,
and decidua.
J
DD, Lei ZM, Rao ChV, Reshef E, Luborsky J.
The presence of gonadotropin receptors in human fallopian tubes [Abstract O-0261. Proc of the 45th Annual Meet of the American Fertility Society. 1989. Lei ZM, Rao ChV. 1992 Gonadotropin receptors in human fetoplacental unit: implications for hCG as an intracrine, paracrine and endocrine regulator of human fetoplacental function, In: Cedard L, Firth JA, eds. Trophoblast research. New York: Plenum Press; vol 6, In Press. Lei ZM, Rao ChV, Ackerman DM, Day TG. 1992 The expression of human chorionic gonadotropin/human luteinizing hormone receptors in human gestational trophoblastic neoplasms. J Clin Endocrinol Metab. 74:1236-1241 Lei ZM, Reshef E, Rao ChV. The expression of human chorionic gonadotropin/luteinizing hormone receptors in human endometrial and myometrial blood vessels. J Clin Endocrinol Metab. In Press. Cramer DW. 1987 Epidemiology of endometriosis. In: Wilson EA, ed. Endometriosis. New York: Liss; pp 5-22. Mitra S, Rao ChV. 1978 Gonadotropin and prostaglandin binding sites in rough endoplasmic reticulum and Golgi fractions of bovine corpora lutea. Arch Biochem Biophys. 191:331-340. Kornyei J, Lei ZM, Rao ChV. Human myometrial smooth muscle cells are novel targets of direct action of hCG [Abstract 4451. Proc of the Annual Meet of the Sot for Gynecol Invest. 1991. Bergqvist A, Ljungberg 0, Myhre E. 1984 Human endometrium and endometriotic tissue obtained simultaneously: a comparative histological study. Int J Gynecol Pathol. 3:135-145. Bergqvist A, Rannevik G, Thorell J. 1981 Estrogen and progesterone cytosol receptor concentration in endometriotic tissue and intrauterine endometrium. Acta Obstet Gynecol Stand. lOl(Suppl):53-
58. 14. Janne 0, Kauppila
A, Kokko E, Lantto T, Ronnberg L, Vihko R.
1981 Estrogen and progestin receptors in endometriosis comparison with endometrial tissue. Am J Obstet Gynecol.
lesions: 141:562-
hCG/LH receptor protein in ectopic (A and D) and uterine endometrium (B (C and F) and peritoneum (G) from patients without endometriosis. H-M show unabsorbed polyclonal receptor antibody (LHR 15-38) was used in A-C and Gpeptide was used in D-F. Glands are indicated by arrows, stroma by arrowheads, by a star in A. Magnification of A-M, x150.
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1144
LINCOLN
566. 15. Dlugi AM, Miller JD, Knittle J. 1990 Lupron depot (leuprolide acetate for depot suspension) in the treatment of endometriosis: a randomized, placebo-controlled, double-blind study. Fertil Steril. 54:419-427. 16. Barbieri RL, Gordon A-MC. 1991 Hormonal therapy of endometriosis: the estradiol target. Fertil Steril. 56:820-822. 17. Fraser HM, Sandow J, Cowen GM, Lumsden MA, Haining R, Smith SK. 1990 Long term supression of ovarian function by a luteinizing-hormone releasing hormone antagonist implant in patients with endometriosis. Fertil Steril. 53:61-68. 18. Venturini PL, Fasce V, Constantini S, Anserine P, Cucuccio S,
ET AL.
19.
20.
21. 22.
JCE & M .1992 Voll5.No4
deCecco L. 1990 Treatment of endometriosis with Goserelin depot, a long acting gonadotropin-releasing hormone agonist analog: endocrine and clinical results. Fertil Steril. 54:1021-1027. Kistner RW. 1975 Endometriosis and infertility. In: Kistner RW, Behrman SJ, eds. Progress of infertility, 2nd ed. Boston: Little, Brown; p 345. Marrs RP, Vargyas JM. 1986 Pelvic endometriosis. In: Mishell DR, Davajan V eds. In: Infertility, contraception and reproductive endocrinology. Oradell: Medical Economics; pp 499-519. Hammond CB, Haney AF. 1978 Conservative treatment of endometriosis. Fertil Steril. 30:497-509. Vernon MW, Wilson EA. 1985 Studies on the surgical induction of endometriosis in the rat. Fertil Steril. 44:684-694.
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Vol. 75, No. 4 Prmted in U.S.A.
The Expression of Human Chorionic Gonadotropin/ Human Luteinizing Hormone Receptors in Ectopic Human Endometrial Implants S. R. LINCOLN,
Z. M. LEI,
CH.
V. RAO,
Department of Obstetrics and Gynecology, University Louisville, Kentucky 40292
AND of
M. A. YUSSMAN Louisville
School of Medicine,
ABSTRACT
contain receptor mRNA and receptor protein. The glands contain more receptor mRNA and receptor protein than stromal cells in implants similar to uterine endometrium from patients with or without endometriosis. However, there is no consistent difference in the expression of receptors in implants compared to uterine endometria from patients with or without endometriosis. Contrary to ectopic endometrial implants, unaffected or normal peritoneum contain neither receptor mRNA nor receptor protein. In summary, we conclude that ectopic endometrial implants contain hCG/LH receptor mRNA and receptor protein, which suggests new possibilities in the medical treatment of endometriosis. (J Clin Endocrinol Metab 75: 1140-1144,1992)
Our laboratory previously demonstrated that normal human endometrium contains hCG/human LH receptors. Since ectopic endometrial implants in endometriosis arise directly at least in part from uterine endometrium, we investigated whether the implants continue the expression of these receptors. The presence of hCG/LH receptor mRNA and/or immunoreactive receptor protein in ectopic endometrial implants on pelvic peritoneum, uterine endometrium, and unaffected or normal peritoneum from patients with (n = 12) and without (n = 14) clinically apparent endometriosis was examined by in situ hybridization and immunocytochemistry analyses. The results showed that the peritoneal biopsies with visible or microscopic endometrial implants
H
CG AND human LH are glycoprotein hormones of similar structure and function (1). They are primarily secretedby the placenta and anterior pituitary, respectively. These two hormones bind to the same receptors in target gonadal cells to stimulate steroidogenesis(l-3). Recent studies from our laboratory have shown that several nongonadal human reproductive tissuesalso contain hCG/LH receptors. The tissuesinclude endometrium (4), myometrium (4), Fallopian tubes (5), placenta (4, 6), fetal membranes(4), decidua (4), human gestational trophoblastic neoplasms (7), and blood vesselsin someof these tissues(8). Endometriosisis a benign gynecological diseasecharacterized by the presence of endometrium in ectopic locations throughout the body. The pelvic peritoneum and/or ovaries are the common sitesof implantation. This diseaseis seenin 25-35s of the infertile patient population and is characterized in part by dysmenorrhea, pelvic pain, dyspareunia, and infertility (9). Since normal human endometrium expresses the hCG/LH receptor gene, we investigated the possibility of whether the ectopic endometrial implants on pelvic peritoneum from endometriosisalso expressthis gene. The present study demonstrates that ectopic endometrial implants also contain hCG/LH receptor mRNA and receptor protein. Materials and Methods The sources of all the materials, reagents, riboprobes, performance of in situ hybridization,
etc., used for transcribing and immunocytochem-
Received December 23, 199 1. Address all correspondence and requests for reprints to: Dr. Ch.V. Rao, Department of Obstetrics and Gynecology, 438 MDR Building, University of Louisville, Louisville, Kentucky 40292.
istry have previously been described (4, 7, 8). Biopsy specimens were taken from 26 patients undergoing laparotomy (n = 1) or total abdominal hysterectomy/bilateral salpingooophorectomy (n = 1) or diagnostic laparoscopy (n = 24) for chronic pelvic pain, unexplained infertility, or both at the University of Louisville affiliated hospitals. Twelve patients (age range, 22-40 yr) had clinically observable endometriosis, and 14 patients (age range, 21-40 yr) did not. Eight of the 12 endometriosis patients and 11 of the 14 nonendometriosis patients were in the proliferative phase of the cycle. According to American Fertility Society classification criteria, 6 endometriosis patients had stage 1 disease, and 2 each had stage 2 through stage 4 disease. Neither group of women had been on any hormonal treatment for infertility before the surgical procedures. Biopsies of uterine endometrium and normal appearing peritoneum were taken from all patients. In addition, peritoneal biopsies were taken from the visible sites of ectopic endometrial implants from the 12 patients with clinical endometriosis. These peritoneal biopsies were taken predominantly from the anterior cul-de-sac or posterior cul-de-sac near the uterosacral ligaments. The specimens were frozen in liquid nitrogen until processing for in situ hybridization and immunocytochemistry. The preparation of 32P-labeled antisense and sense riboprobes from porcine hCG/LH receptor cDNA (nucleotides -11 to 2089) in pBluescript SK(+), kindly provided by Dr. Hugues Loosfelt (Paris, France), and performance of in situ hybridization were previously described (7, 8). Hybridization with the sense probe carried out at the same time under identical conditions served as a negative control. Two polyclonal antibodies to synthetic fragments of rat luteal hCG/ LH receptors (anti-PCR II, l-11 and anti-LHR 1%38), kindly provided by Dr. Patrick Roche, Mayo Clinic (Rochester, MN), and a monoclonal antibody to rat luteal hCG/LH receptors, kindly provided by Dr. Judith Luborsky, TSI Mason Research Institute (Worcester, MA), were used for immunocytochemical detection of hCG/LH receptor protein. The details of the procedure were previously described (4, 7, 8). The receptor antibodies were used at 1:500 (polyclonal) or 1:lOO (monoclonal) dilution. Three types of controls were performed: 1) polyclonal receptor antibodies preabsorbed with corresponding synthetic receptor peptides, also kindly provided by Dr. P. Roche, and the monoclonal receptor antibody preabsorbed with bovine luteal microsomes prepared in our laboratory (lo), 2) omission of the unabsorbed receptor antibodies, and
1140
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 22 November 2015. at 13:42 For personal use only. No other uses without permission. . All rights reserved.
hCG/LH
RECEPTORS
3) substitution of the unabsorbed receptorantibodies with nonspecific immunoglobulin G during the immunostaining procedure. The immunostaining of all specimens including controls was performed at the same time using identical conditions. Three of the authors independently evaluated the degree and pattern of immunostaining by examining the slides under an Olympus microscope (New Hyde Park, NY). The representative photographs presented in this manuscript were processed at the same time by a commercial vendor using computerized equipment.
Results In situ hybridization analysis with 32P-labeledantisense riboprobe shows that endometrial glands and stromal cells in ectopic endometrial implant (Fig. 1A) and uterine endometrium from patients with (Fig. 1B) and without (Fig. 1C) apparent endometriosis contain silver grains. These grains, which reflect the presenceof hCG/LH receptor mRNA, are absentwhen 32P-labeledsenseprobe, which cannot basepair with receptor mRNA, was used (Fig. 1, D, E, and F). The glands contain more hybridization signals due to antisense probe than stromal cells in ectopic endometrial implants similar to uterine endometrium (Fig. 1, A, B, and C). The grain distribution in ectopic endometrial implants is similar to that in uterine endometrium (Fig. 1, A VS.B and C). Figure 2 shows that the immunostaining for hCG/LH receptor protein is present in ectopic endometrial implant (A) and uterine endometrium from patients with (B) or without (C) apparent endometriosis. In ectopic endometrial implants, as in uterine endometria, the glands contain more receptor immunostaining than stromal cells (Fig. 2, A-C). There are no obvious differences in the receptor immunostaining between ectopic endometrial implant and uterine endometria (Fig. 2, A VS.B and C). Some biopsies of normal appearing peritoneum from patients with (2 of 12) and without (4 of 14) clinically observable endometriosis contained microscopicendometrial glands, and they all were immunostained for hCG/LH receptors (Fig. 2, H-M).
IN ENDOMETRIOSIS
1141
The receptor immunostaining was absent in unaffected adjacent peritoneum from endometriosis patients (Fig. 2A) and in peritoneum (Fig. 2G) of patients without endometriosis.The receptor immunostaining was also absent when the receptor antibody preabsorbed with excessreceptor peptide was used for immunostaining ectopic endometrial implants (Fig. 2D) and uterine endometria (Fig. 2, H and J) from patients with or without endometriosis. Similar results were obtained when unabsorbed receptor antibody was omitted or substituted with nonspecific immunoglobulin G during the immunostaining procedure (data not shown). Another polyclonal receptor antibody (anti-PCRII, l-l 1) and a monoclonal receptor antibody gave results similar to those presented in Fig. 2 using polyclonal receptor antibody, LHR 1538. Discussion The present studies were prompted by the recent findings that human uterus contains a major 4.3-kilobase and a minor 2.6-kb hCG/LH receptor mRNA transcript (8). These transcripts along with the immunoreactive receptor protein of 100 kilodaltons are present in endometrial glands, stromal cells, myometrial smooth muscle, and uterine blood vessels (4, 8). Among these uterine cells, glands contain more receptor transcripts and receptor protein than the other cells (4, 8). All of the uterine cells show an increasein receptors from the proliferative to secretory phase (4, 8). These findings suggestedthat hCG/LH may directly regulate human uterine functions. Consistent with this possibility, we have recently demonstrated that hCG can increase both the number and size of cultured human myometrial smooth muscle cells (11). Since ectopic human endometrial implants originate from uterine endometria, it was of interest to determine whether the implants also express hCG/LH receptors. The results clearly demonstrate that the endometrial implants, but not unaffected adjacent peritoneum or peritoneum from nonen-
FIG. 1. In situ hybridization for hCG/ LH receptor mRNA in ectopic (A and D) and uterine endometrium (B and E) from endometriosis patients and uterine endometrium (C and F) from patients without endometriosis. 32P-Labeled antisense riboprobe was used in A-C, and sense probe was used in D-F. Glandular epithelium is indicated by arrows, and stromal cells by arrowheads. Magnification, X384.
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11
A IF---
_-:
_.
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hCG/LH
RECEPTORS
dometriosis patients, contain hCG/LH receptor mRNA and receptor protein. Although endometrial implants in other anatomical locations in the body were not examined, it is quite likely that they may also contain these receptors because of the common origin and similarity of implants from different locations (12). The overall receptor expression and the glands containing more receptors than stromal cells are similar in both ectopic endometrial implants and uterine endometria. These findings are consistent with the previous observations that glands and stroma in implants are active as those in uterine endometria (12). Contrary to the lower estrogen and progesterone receptor concentrations in implants compared to those in uterine endometrium (13, 14), hCG/LH receptor gene expression appears to be similar. Compared to bovine corpus luteum, the specific binding of [‘251]hCG to human uterine tissue is very low (unpublished data from our laboratory). Nevertheless, the ligand binding in ectopic endometrial implants and uterine endometrium was not determined because the amount of tissues from the biopsies was not adequate. In the present study we did find higher uterine endometrial expression of receptors in the secretory compared to the proliferative phase as previously reported (4, 8). However, we could not find a similar consistent difference in ectopic endometrial implants. This is probably due to two reasons. First, the ectopic endometrial implants may not always be in phase with uterine endometrium. Second, the amount of endometrium is often too small in implants to determine the differences in immunostaining. We had too few samples to determine whether receptor expression was dependent on the stage of endometriosis. Because of sample procurement problems, we have not been able to study receptor expression during or after hormonal therapy. This study does not attempt to determine the functional significance of hCG/LH receptors in ectopic endometrial implants. Since these implants originate from the endometrium or an analog shared with the endometrium, the function in implants is probably similar to that in uterine endometrium. This is currently being investigated. The treatment of endometriosis patients with GnRH analogs (GnRHa) results in regression of implants and relief from the symptoms (15). These therapeutic benefits have been attributed to decreased estradiol levels due to desensitization of the hypothalamic-hypophyseal axis resulting in decreased LH levels (16-18). Although the role of estradiol in the growth of endometriosis is convincing, it is not universally accepted. The direct result of decreased LH secretion due to chronic GnRH-a treatment of the endometrial implants is unknown. This was not previously suspected because the existence of hCG/LH receptors in ectopic endometrial implants was not known until the present work. Endometriosis subsides during pregnancy, and in normal or FIG. 2. Light microscope
immunocytochemistry for immunoreactive and E) from endometriosis patients and uterine endometrium immunostaining for the receptors in microscopic implants. The M, and the receptor antibody preabsorbed with excess receptor and unaffected adjacent peritoneum from endometriosis patient
IN ENDOMETRIOSIS
1143
pseudomenopause induced by surgical or medical castration (17-22), the conditions in which the hCG and hLH levels are elevated. This does not necessarily rule out the possible direct effect of decreased hLH levels achieved by GnRHa in the treatment of endometriosis. The beneficial effects of pregnancy and menopause on endometriosis could be related to various hormonal and biophysical alterations that occur. In summary, the present study demonstrates for the first time that ectopic human endometrial implants, but not unaffected or normal peritoneum, contain hCG/LH receptor mRNA and receptor protein. This finding suggests new possibilities in the medical treatment of endometriosis. References 1.
Pierce JG, Parsons TF. 1981 Glycoprotein function.
Annu
Rev Biochem.
hormones:
structure
2. Catt KJ, Harwood JP, Clayton RN, et al. 1980 Regulation receptors
and
gonadal
and
50:466-495.
steroidogenesis.
Recent
Frog
of peptide Horm Res.
361557-622. 3. Rao ChV. 1982 Receptors
for gonadotropins in human ovaries. In: Muldoon TH, Mahesh VB, Presz-Ballester B, eds. Recent advances in fertility research, developments in reproductive endocrinology, part A. New York: Liss; pp 123-135.
4. Reshef E, Lei ZM, Rao ChV, Pridham DD, Chegini N, Luborsky JL. 1990 The presence of gonadotropin receptors in nonpregnant human uterus, Clin Endocrinol
5. Bailey-Pridham
6.
7.
8. 9. 10.
11.
12. 13.
human Metab.
placenta, fetal 70:421-430.
membranes,
and decidua.
J
DD, Lei ZM, Rao ChV, Reshef E, Luborsky J.
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hCG/LH receptor protein in ectopic (A and D) and uterine endometrium (B (C and F) and peritoneum (G) from patients without endometriosis. H-M show unabsorbed polyclonal receptor antibody (LHR 15-38) was used in A-C and Gpeptide was used in D-F. Glands are indicated by arrows, stroma by arrowheads, by a star in A. Magnification of A-M, x150.
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