Gynecol. Endocrinol. 4 (1990) 205-213
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Detection of estrogen receptors in the trigonum and urinary bladder with an immunohistochemical tech nique G. Bussolati, A. Tizzani*, G. Casetta*, P. Cassonit, D. Pacchioni, P. Piana*, A. Revellit and M. Massobriot Department of Biomedical Sciences and Human Oncology, Division of Pathological Anatomy, “Institute of Nephrourology and fFaculty of Clinical Obstetrics and G y n e cology ‘ D ’ , University of Turin, Ospedale Mauriziano ‘Umberto I’, Turin, Italy Abstract
In order to detect estrogen receptors (ER) in the female bladder, 5 premenopausal and 10 postmenopausal women affected by gynecological diseases were submitted to cystoscopy, during which both the trigonum and the bladder lateral wall were biopsied. A new, simple, cost-effective immunohistochemical technique was employed to stain the estrogen specific binding sites. ER were found in the trigonum of 3 premenopausal and 4 postmenopausal subjects, while the bladder lateral wall was always ER-negative. A comparison with previously used ER detection methods and a discussion of further hypothetical applications of the immunohistochemical technique in the study of the lower female urogenital tract are reported. Introduction
The existence of specific binding sites for estrogen in the human female lower urinary tract was first demonstrated, in 1981, by Iosif et al.’, who used a selfprepared method2 employing 3H-estradiol to stain the receptor and dextrancoated charcoal in order to remove the unbound tritiated steroid. In the same year the presence of estrogen receptors (ER) in the human female urethra and bladder was confirmed by Ingelman-Sundberg et al.3, who used the isoelectric focusing technique previously described by Gustafsson et a1.4. Later on, ER Correspondence to: Prof. M. Massobrio, Reparto 5C, Ospedale Mauriziano ‘Umberto I’, Largo Turati 62, 10128 Turin, Italy
205
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
206 Bussolati, T i z z a n i , Casetta, Cassoni, Pacchioni, Piana, Revelli and Massobrio were also found with a method using3H-estradiol and a dextran-coated charcoal separation technique in both the cytoplasmic and nuclear fractions prepared from female rabbit urethra and bladder5. More recently, a very similar method employing 3H-progesterone and dextran charcoal was used to demonstrate the existence of progestin receptors (PR) in female rabbit urethra and bladder after estrogen premedication6. The techniques employed in these studies are quite complicated, expensive, and require a relatively large amount of tissue, which is not easy to obtain by endoscopic biopsy. Moreover, they dose the receptor content and measure the dissociation constant (KD), but they cannot show the distribution of receptors in different cell layers of the tissue specimen. The present study describes a new, simple, cost-effective approach to ER detection and its application to the study of the female lower urinary tract.
Materials and methods Five premenopausal and 10 postmenopausal women affected by gynecological diseases were included in the study. Some clinical data are summarized in Tables 1 and2. The pharmacological history was negative, as far as hormonal therapies were concerned, in 14 subjects. O n the contrary, a 67-year-old woman, who had previously undergone hystero-ovariectomy for endometrial adenocarcinoma, had been treated with radiotherapy (40 Gy) followed by MAP therapy (1 g daily) for 10 days. All the subjects underwent a cystoscopy, during which 2 biopsies were performed, one on the trigonum, the other on the bladder lateral wall. tmmediately after the excision, the specimens were embedded in OCT compound, frozen at -30 "C and stored at -80 "C until use. Serial sections (5 pm) were cut on a cryostat a t -20 "C and applied on gelatinized slides. A routine histological examination was performed in all cases. The slides were fixed in 3.7% formaldehyde in phosphate buffered saline pH 7.6 (PBS) for 10 minutes at room temperature, then washed in PBS for 10 minutes. Sections were subsequently transferred into cold methanol (-20 "C) for 5 minutes, then into cold acetone (-20 "C) for 3 minutes, and washed twice in PBS for 10 minutes before the immunostaining procedure. This method employs a monoclonal antibody against estrogen receptor (Abbott, Wiesbaden, West Germany), followed by a sensitive peroxidaseantiperoxidase technique (PAP). Briefly, following the ER-ICA procedure the specimens were treated with normal goat serum to prevent non-specific binding of subsequent reagents. They were then incubated with the monoclonal (rat) antibody to human estrogen receptor for 60 minutes at room temperature in a humidified chamber. Sections were then incubated with the bridging antibody, a goat anti-rat IgG for 30 minutes at room temperature. Finally, the slides were treated with a rat PAP complex for 30 minutes at room temperature. All the reagents were used at the kit dilution. A chromogen-substrate solution containing 202 and 3.3'diaminobenzidine (DAB) in PBS pH 7.6 was added
Normal mucosa Normal mucosa
K cervix Ib
K cervix IIb
40
41
44
A. R.
V. G.
R . M.
Stress incontinence
K cervix Ib Metaplasia
Metaplasia
Stress incontinence
K cervix Ib
38
Normal mucosa
K cervix Ib
M. L.
~~
34
Cystoscopy
D. C.
Admittance diagnosis
Age
Patients
Table 1 Clinical data of the premenopausal women studied
Squamous metaplasia
Normal mucosa
Normal mucosa
Squamous metaplasia
Normal mucosa
Histology
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
-
-
+++ +++
Bladder
-
-
Trigonum
ER staining
3
-_
65 (13)
67 (17)
68 (20)
71 (22)
74 (31)
M. A.
P. c.t
F. L.
T. E.
S. D. _
_
Ovarian cyst Recurrent cystitis
K cervix Stress incontinence
Ovarian cyst
Stress incontinence
K endometrium
K endometrium
K ovary
~
~
Normal mucosa
Normal mucosa
Normal mucosa
Normal mucosa
Normal mucosa
Trigonal hyperemia
*Figures in parentheses represent years of menopause tPrevious hystero-ovariectomy followed by radiotherapy (40 Gy) and MAP (1 g daily)
_
64 (19)
Trigonal hyperemia
K cervix
A. L.
P. v.
Metaplasia
K endometrium Stress incontinence
T. T.
Stress incontinence
Squamous metaplasia
~
Normal mucosa
Normal mucosa
Normal mucosa
Normal mucosa
Normal mucosa
Epithelial hyperplasia
Normal mucosa
Squamous metaplasia
Normal mucosa
Normal mucosa Metaplasia
Histology
K endometrium Stress incontinence
Cystoscopy
K cervix 111
Admittance diagnosis
G. M. R.
Age*
L. L.
Patients
Table 2 Clinical data of the postmenopausal women studied
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
+
+++
-
+++
Trigonum
Bladder
ER staining
a
3
h =t
?J
3.
0
9 2
Detection of estrogen receptors in (he-fernalelower urinary tract
209
to each specimen for 5-10 minutes a t room temperature. Sections were then counterstained with methyl green in PBS for 5 minutes, dehydrated, cleared and mounted in balsam. Known ER-positive and ER-negative breast carcinomas were used as controls. Nuclear immunostain was graded as negative (-) or slightly (+), moderately (+ +) or strongly (+ +) positive.
+
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
Results ER were identified in fresh frozen tissue sections of the trigonum of 7 out of 15 patients, 3 out of 5 premenopausal and 4 out of 10 postmenopausal subjects. A strong ER positivity was found in the trigonum of 2 premenopausal and 2 postmenopausal patients. Moreover, 3 further subjects (1 pre- and 2 postmenopausal) showed a slight ER positivity in the trigonum. Trigonal ER positivity was present in 6 cases of cervical carcinoma and in 1 case of endometrial cancer. However, we could not find any correlation between ER positivity, underlying pathology, age, cystoscopic o r histological findings. Moreover, ER distribution was homogeneous in the different cell layers of the trigonal specimen. O n the contrary, negative immunohistochemical ER stains were always obtained with tissue specimens taken from the bladder wall. Cystoscopic and histological findings, as well as ER stain results, can be seen in Tables 1 and 2. Discussion The presence of estrogen specific binding sites in the human female lower urinary tract is strongly suggested by the subjective relief of symptom^'^^ and by the objective improvement of urethral function' in stress-incontinent postmenopausal women after estrogen treatment. Also, several data"'*" showing a marked effect of estrogen therapy on both blood flow and density of al-adrenergic receptors in the human urethra provide evidence that the urethra is a target for estrogen action. The existence of ER in the urethra and bladder has been investigated by several authors in different experimental conditions and by means of different methods. Iosif et al.' in 1981 first demonstrated the presence of ER in cytosolic and nuclear fractions prepared from the human urethra, trigonum and urinary bladder. They studied 3 postmenopausal women who underwent urethrocystectomy for cancer of the bladder, and 1 35-year-old woman who was urethrocystectomized for chronic non-specific cystitis and neurogenic bladder. Before the operation, the 3 older patients received radiation therapy and were pretreated with polyestradiol phosphate, 40 m g i.m. in a single dose. Cytosolic and nuclear ER content was determined by a procedure using 3H-estradiol to stain the receptor and dextran-coated charcoal to separate the unbound aspecific radioactivity2. ER were found in the urethra of all the patients, both in the cytoplasmic and nuclear fractions, whereas they were found in the bladder of
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
210 Bussolati, T i z z a n i , Casetta, Cassoni, Pacchioni, Piana, Revelli and Massobrio
Figure 1 Tissue ofthe trigonum with a mixture of ER-positive (dark) and ER-negative (pale) cells. The positive reaction is located in the nuclei. ERICA, frozen sections. (Top: x 19; bottom: x 24)
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
Detection of estrogen receptors in the female lower urinary tract
21 1
only 2 subjects and exclusively in the nuclear fraction. The trigonum contained ER in the cytosolic fraction of 1 patient, and in the nuclear fraction of 3. The apparent dissociation constants for cytosol and nuclear ER were respectively 0.65 X M and 0.74 X lop9M, similar to those reported for ER in the uterus and vagina". Another study, by Ingelman-Sundberg et al.3, confirmed the existence of ER in the human female lower urinary tissues. These authors studied 7 women (2 premenopausal and 5 postmenopausal) admitted for urinary stress-incontinence and subjected to an operation with pubococcygeal repairI3. All the patients underwent hormonal treatments with estrogens or estroprogestins before the operation. Cytosol ER were determined by isoelectric focusing4. Significant amounts of ER were found in the urethra (in 5 out of 7 patients), in the bladder (in 3 out of 4 patients in which the tissue specimen was adequate) and in the pubococcygeal muscle (in 3 out of 6 patients). Later on, ER in the urethra and bladder were demonstrated also in the ovariectomized rabbit, premedicated with a single injection of 4 mg polyestradiol phosphate5. In this study, performed with the tritiated estradiol-dextran charcoal technique, ER in the urethral cytosolic and nuclear fractions were found to be 10 times lower than in the uterus, whereas ER in the bladder were as low as 1/20 of those in the uterine tissue. The K D of urinary tract ER was approximately 1 X lop9 M. In the present study, 15 women who underwent cystoscopy for gynecological pathology (mainly gynecological cancers needing preoperatory endoscopic stadiation) were examined for the presence of ER in the trigonum and bladder. A new immunohistochemical procedure, employing a rat monoclonal antibody anti-ER, was used. This method has proved to be effective for ER detection in several other fresh frozen tissue^'^, whereas the results in fixed tissue specimens are by n o w considered unsati~factory'~. O u r data confirm the effectiveness of this technique in identifying ER in the lower female genital tract. The bladder wall was ER-negative in all the subjects. The discrepancy in ER content between the trigonum and the bladder is probably due to the different embriological origin of these tissues. The hypothesis of trigonal ER positivity due to neoplastic invasion of the trigonum was excluded by accurate histological examination. The advantages of the immunohistochemical method in comparison with the techniques using tritiated steroids and dextran-coated charcoal are as follows: (a) less work and time (approximately 4 hours) are needed by the pathologist; (b) only a small amount of tissue (a few milligrams) is necessary; (c) no radioactive material is used; (d) the results are easily comprehensible, even to an inexperienced operator. Furthermore, automated analyzing procedures are available for a precise quantification of ER positivity''. Further hypothetical applications of the immunohistochemical technique for ER detection could be: (1) the study of trigonal responsiveness to steroids in pathological conditions likely to be related to hormonal influences (especially the urethral syndrome, climacteric atrophic trigonitis, etc.); (2) ER identifica-
212 Bussolati, Tizrani, Casetta, Cassoni, Pacchioni, Piana, Revelli and Massobrio tion on urinary cytologic material; (3) the study of ER prognostic meaning in vescical and urethral malignancies.
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
References 1. Iosif, C. S . , Batra, S., Ek, A. and Astedt, €3. (1981). Estrogen receptors in the human female lower urinary tract. A m . J . Obrtet. Gynecol., 141, 817 2. Batra, S. (1979). Subcellular distribution and cytosolic receptors of progesterone and of estradiol-17beta in the rabbit myometrium: effect of progesterone treatment. Biol. Reprod., 21,483 3. Ingelman-Sundberg, A., Rosen, J., Gustafsson, S. A. and Carlstrom, K. (1981). Cytosol estrogen receptors in the urogenital tissues in stressincontinent women. Acta Obstet. Gynecol. Scand., 60, 585 4. Gustafsson, J. A., Gustafsson, S. A., Nordenskjold, B., Okret, S., Silfversward, C. and Wrange, 0. (1978). Estradiol receptor analysis in human breast cancer tissue by isoelectric focusing in polyacrilamide gel. Cancer Res., 38, 4225 5. Batra, S. and Iosif, C. S. (1983). Femaleurethra: a target for estrogenaction. J . Urol., 129, 418 6. Batra, S. and Iosif, C. S. (1987). Progesterone receptors in the female lower urinary tract.]. Urol., 138, 1301 7. Salmon, U. J., Walter, R. I. and Geist, S. H. (1941). The use of estrogens in the treatment of dysuria and incontinence in postmenopausal women. Am. J . Obstet. Gynecol., 42, 845 8. Brown, A. D. G. (1977). Postmenopausal urinary problems. Clin. Obstet. Gynecol., 4, 181 9. Rud, T . (1980). The effects of estrogens and gestagens on the urethral pressure profile in urinary continent and stress incontinent women. Acta Obstet. Gynecol. Scand., 59, 265 10. Batra, S . , Bjellin, L., Sjogren, C., Ioseif, C. S. and Widmark, E. (1986). Increases in blood flow of the female rabbit urethra following low dose estrogens. J . Urol., 136, 1360 11. Larsson, B., Anderson, K. E., Batra, A . , Matiasson, A. and Sjogren, C. (1984). Effect of estradiol on norepinephrine-induced contraction, alpha adrenoceptor number and norepinephrine content in the female rabbit urethra. J . Pharmacol. Exp. Ther., 229, 557 12. Bayard, F., Damilano, S . , Robel, P. and Baulieu, E. E. (1978). Cytoplasmic and nuclear estradiol and progesterone receptors in human endometrium. J . Clin. Endocrinol. Metab., 46, 635 13. Ingelman-Sundberg, A. (1947). Extra-vaginal plastic repair of the pelvic floor for prolapse of the bladder neck; a new method to operate for stress incontinence. Gynecologia, 123, 242 14. McCarty, K. S., Jr, Miller, L. S., Cox, E. B., Konrath, J. and McCarty, K. S., Sr (1985). Estrogen receptor analyses. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies. Arch. Pathol. Lab. Med., 109, 716
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
15. Cohen, C., Unger, E. R . , Sgoutas, I]., Bradley, N . and Chenngis, M . (1989). Automated imniunohistochemical estrogen receptor in fixed embedded breast carcinonias. h i . J . CIirr. Patlid., 92, 669 16. Bacus, S . , Flowers, J. L., Press, M. F., Bacus, J. W. and McCarty, K. S . , J r (1988). The evaluation of estrogen receptor in primary breast carcinoma by computer-assisted image analysis. Am. J . Cliri. Pnthol., 90, 233
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
Detection of estrogen receptors in the trigonum and urinary bladder with an immunohistochemical tech nique G. Bussolati, A. Tizzani*, G. Casetta*, P. Cassonit, D. Pacchioni, P. Piana*, A. Revellit and M. Massobriot Department of Biomedical Sciences and Human Oncology, Division of Pathological Anatomy, “Institute of Nephrourology and fFaculty of Clinical Obstetrics and G y n e cology ‘ D ’ , University of Turin, Ospedale Mauriziano ‘Umberto I’, Turin, Italy Abstract
In order to detect estrogen receptors (ER) in the female bladder, 5 premenopausal and 10 postmenopausal women affected by gynecological diseases were submitted to cystoscopy, during which both the trigonum and the bladder lateral wall were biopsied. A new, simple, cost-effective immunohistochemical technique was employed to stain the estrogen specific binding sites. ER were found in the trigonum of 3 premenopausal and 4 postmenopausal subjects, while the bladder lateral wall was always ER-negative. A comparison with previously used ER detection methods and a discussion of further hypothetical applications of the immunohistochemical technique in the study of the lower female urogenital tract are reported. Introduction
The existence of specific binding sites for estrogen in the human female lower urinary tract was first demonstrated, in 1981, by Iosif et al.’, who used a selfprepared method2 employing 3H-estradiol to stain the receptor and dextrancoated charcoal in order to remove the unbound tritiated steroid. In the same year the presence of estrogen receptors (ER) in the human female urethra and bladder was confirmed by Ingelman-Sundberg et al.3, who used the isoelectric focusing technique previously described by Gustafsson et a1.4. Later on, ER Correspondence to: Prof. M. Massobrio, Reparto 5C, Ospedale Mauriziano ‘Umberto I’, Largo Turati 62, 10128 Turin, Italy
205
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
206 Bussolati, T i z z a n i , Casetta, Cassoni, Pacchioni, Piana, Revelli and Massobrio were also found with a method using3H-estradiol and a dextran-coated charcoal separation technique in both the cytoplasmic and nuclear fractions prepared from female rabbit urethra and bladder5. More recently, a very similar method employing 3H-progesterone and dextran charcoal was used to demonstrate the existence of progestin receptors (PR) in female rabbit urethra and bladder after estrogen premedication6. The techniques employed in these studies are quite complicated, expensive, and require a relatively large amount of tissue, which is not easy to obtain by endoscopic biopsy. Moreover, they dose the receptor content and measure the dissociation constant (KD), but they cannot show the distribution of receptors in different cell layers of the tissue specimen. The present study describes a new, simple, cost-effective approach to ER detection and its application to the study of the female lower urinary tract.
Materials and methods Five premenopausal and 10 postmenopausal women affected by gynecological diseases were included in the study. Some clinical data are summarized in Tables 1 and2. The pharmacological history was negative, as far as hormonal therapies were concerned, in 14 subjects. O n the contrary, a 67-year-old woman, who had previously undergone hystero-ovariectomy for endometrial adenocarcinoma, had been treated with radiotherapy (40 Gy) followed by MAP therapy (1 g daily) for 10 days. All the subjects underwent a cystoscopy, during which 2 biopsies were performed, one on the trigonum, the other on the bladder lateral wall. tmmediately after the excision, the specimens were embedded in OCT compound, frozen at -30 "C and stored at -80 "C until use. Serial sections (5 pm) were cut on a cryostat a t -20 "C and applied on gelatinized slides. A routine histological examination was performed in all cases. The slides were fixed in 3.7% formaldehyde in phosphate buffered saline pH 7.6 (PBS) for 10 minutes at room temperature, then washed in PBS for 10 minutes. Sections were subsequently transferred into cold methanol (-20 "C) for 5 minutes, then into cold acetone (-20 "C) for 3 minutes, and washed twice in PBS for 10 minutes before the immunostaining procedure. This method employs a monoclonal antibody against estrogen receptor (Abbott, Wiesbaden, West Germany), followed by a sensitive peroxidaseantiperoxidase technique (PAP). Briefly, following the ER-ICA procedure the specimens were treated with normal goat serum to prevent non-specific binding of subsequent reagents. They were then incubated with the monoclonal (rat) antibody to human estrogen receptor for 60 minutes at room temperature in a humidified chamber. Sections were then incubated with the bridging antibody, a goat anti-rat IgG for 30 minutes at room temperature. Finally, the slides were treated with a rat PAP complex for 30 minutes at room temperature. All the reagents were used at the kit dilution. A chromogen-substrate solution containing 202 and 3.3'diaminobenzidine (DAB) in PBS pH 7.6 was added
Normal mucosa Normal mucosa
K cervix Ib
K cervix IIb
40
41
44
A. R.
V. G.
R . M.
Stress incontinence
K cervix Ib Metaplasia
Metaplasia
Stress incontinence
K cervix Ib
38
Normal mucosa
K cervix Ib
M. L.
~~
34
Cystoscopy
D. C.
Admittance diagnosis
Age
Patients
Table 1 Clinical data of the premenopausal women studied
Squamous metaplasia
Normal mucosa
Normal mucosa
Squamous metaplasia
Normal mucosa
Histology
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
-
-
+++ +++
Bladder
-
-
Trigonum
ER staining
3
-_
65 (13)
67 (17)
68 (20)
71 (22)
74 (31)
M. A.
P. c.t
F. L.
T. E.
S. D. _
_
Ovarian cyst Recurrent cystitis
K cervix Stress incontinence
Ovarian cyst
Stress incontinence
K endometrium
K endometrium
K ovary
~
~
Normal mucosa
Normal mucosa
Normal mucosa
Normal mucosa
Normal mucosa
Trigonal hyperemia
*Figures in parentheses represent years of menopause tPrevious hystero-ovariectomy followed by radiotherapy (40 Gy) and MAP (1 g daily)
_
64 (19)
Trigonal hyperemia
K cervix
A. L.
P. v.
Metaplasia
K endometrium Stress incontinence
T. T.
Stress incontinence
Squamous metaplasia
~
Normal mucosa
Normal mucosa
Normal mucosa
Normal mucosa
Normal mucosa
Epithelial hyperplasia
Normal mucosa
Squamous metaplasia
Normal mucosa
Normal mucosa Metaplasia
Histology
K endometrium Stress incontinence
Cystoscopy
K cervix 111
Admittance diagnosis
G. M. R.
Age*
L. L.
Patients
Table 2 Clinical data of the postmenopausal women studied
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
+
+++
-
+++
Trigonum
Bladder
ER staining
a
3
h =t
?J
3.
0
9 2
Detection of estrogen receptors in (he-fernalelower urinary tract
209
to each specimen for 5-10 minutes a t room temperature. Sections were then counterstained with methyl green in PBS for 5 minutes, dehydrated, cleared and mounted in balsam. Known ER-positive and ER-negative breast carcinomas were used as controls. Nuclear immunostain was graded as negative (-) or slightly (+), moderately (+ +) or strongly (+ +) positive.
+
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
Results ER were identified in fresh frozen tissue sections of the trigonum of 7 out of 15 patients, 3 out of 5 premenopausal and 4 out of 10 postmenopausal subjects. A strong ER positivity was found in the trigonum of 2 premenopausal and 2 postmenopausal patients. Moreover, 3 further subjects (1 pre- and 2 postmenopausal) showed a slight ER positivity in the trigonum. Trigonal ER positivity was present in 6 cases of cervical carcinoma and in 1 case of endometrial cancer. However, we could not find any correlation between ER positivity, underlying pathology, age, cystoscopic o r histological findings. Moreover, ER distribution was homogeneous in the different cell layers of the trigonal specimen. O n the contrary, negative immunohistochemical ER stains were always obtained with tissue specimens taken from the bladder wall. Cystoscopic and histological findings, as well as ER stain results, can be seen in Tables 1 and 2. Discussion The presence of estrogen specific binding sites in the human female lower urinary tract is strongly suggested by the subjective relief of symptom^'^^ and by the objective improvement of urethral function' in stress-incontinent postmenopausal women after estrogen treatment. Also, several data"'*" showing a marked effect of estrogen therapy on both blood flow and density of al-adrenergic receptors in the human urethra provide evidence that the urethra is a target for estrogen action. The existence of ER in the urethra and bladder has been investigated by several authors in different experimental conditions and by means of different methods. Iosif et al.' in 1981 first demonstrated the presence of ER in cytosolic and nuclear fractions prepared from the human urethra, trigonum and urinary bladder. They studied 3 postmenopausal women who underwent urethrocystectomy for cancer of the bladder, and 1 35-year-old woman who was urethrocystectomized for chronic non-specific cystitis and neurogenic bladder. Before the operation, the 3 older patients received radiation therapy and were pretreated with polyestradiol phosphate, 40 m g i.m. in a single dose. Cytosolic and nuclear ER content was determined by a procedure using 3H-estradiol to stain the receptor and dextran-coated charcoal to separate the unbound aspecific radioactivity2. ER were found in the urethra of all the patients, both in the cytoplasmic and nuclear fractions, whereas they were found in the bladder of
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
210 Bussolati, T i z z a n i , Casetta, Cassoni, Pacchioni, Piana, Revelli and Massobrio
Figure 1 Tissue ofthe trigonum with a mixture of ER-positive (dark) and ER-negative (pale) cells. The positive reaction is located in the nuclei. ERICA, frozen sections. (Top: x 19; bottom: x 24)
Gynecol Endocrinol Downloaded from informahealthcare.com by Nyu Medical Center on 01/06/15 For personal use only.
Detection of estrogen receptors in the female lower urinary tract
21 1
only 2 subjects and exclusively in the nuclear fraction. The trigonum contained ER in the cytosolic fraction of 1 patient, and in the nuclear fraction of 3. The apparent dissociation constants for cytosol and nuclear ER were respectively 0.65 X M and 0.74 X lop9M, similar to those reported for ER in the uterus and vagina". Another study, by Ingelman-Sundberg et al.3, confirmed the existence of ER in the human female lower urinary tissues. These authors studied 7 women (2 premenopausal and 5 postmenopausal) admitted for urinary stress-incontinence and subjected to an operation with pubococcygeal repairI3. All the patients underwent hormonal treatments with estrogens or estroprogestins before the operation. Cytosol ER were determined by isoelectric focusing4. Significant amounts of ER were found in the urethra (in 5 out of 7 patients), in the bladder (in 3 out of 4 patients in which the tissue specimen was adequate) and in the pubococcygeal muscle (in 3 out of 6 patients). Later on, ER in the urethra and bladder were demonstrated also in the ovariectomized rabbit, premedicated with a single injection of 4 mg polyestradiol phosphate5. In this study, performed with the tritiated estradiol-dextran charcoal technique, ER in the urethral cytosolic and nuclear fractions were found to be 10 times lower than in the uterus, whereas ER in the bladder were as low as 1/20 of those in the uterine tissue. The K D of urinary tract ER was approximately 1 X lop9 M. In the present study, 15 women who underwent cystoscopy for gynecological pathology (mainly gynecological cancers needing preoperatory endoscopic stadiation) were examined for the presence of ER in the trigonum and bladder. A new immunohistochemical procedure, employing a rat monoclonal antibody anti-ER, was used. This method has proved to be effective for ER detection in several other fresh frozen tissue^'^, whereas the results in fixed tissue specimens are by n o w considered unsati~factory'~. O u r data confirm the effectiveness of this technique in identifying ER in the lower female genital tract. The bladder wall was ER-negative in all the subjects. The discrepancy in ER content between the trigonum and the bladder is probably due to the different embriological origin of these tissues. The hypothesis of trigonal ER positivity due to neoplastic invasion of the trigonum was excluded by accurate histological examination. The advantages of the immunohistochemical method in comparison with the techniques using tritiated steroids and dextran-coated charcoal are as follows: (a) less work and time (approximately 4 hours) are needed by the pathologist; (b) only a small amount of tissue (a few milligrams) is necessary; (c) no radioactive material is used; (d) the results are easily comprehensible, even to an inexperienced operator. Furthermore, automated analyzing procedures are available for a precise quantification of ER positivity''. Further hypothetical applications of the immunohistochemical technique for ER detection could be: (1) the study of trigonal responsiveness to steroids in pathological conditions likely to be related to hormonal influences (especially the urethral syndrome, climacteric atrophic trigonitis, etc.); (2) ER identifica-
212 Bussolati, Tizrani, Casetta, Cassoni, Pacchioni, Piana, Revelli and Massobrio tion on urinary cytologic material; (3) the study of ER prognostic meaning in vescical and urethral malignancies.
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