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Indian Journal of Pharmacology
Medknow Publications on behalf of Indian Pharmacological Society
ISSN: 0253-7613 EISSN: 1998-3751
Vol. 42, Num. 5, 2010, pp. 312-317

Indian Journal of Pharmacology, Vol. 42, No. 5, September-October, 2010, pp. 312-317

Research Article

Possible mechanism of benign prostatic hyperplasia induced by androgen-estrogen ratios in castrated rats

Liu Xiang-Yun, Xu Ying-Wen, Xie Chen-Jing, Wang Jiu-Jiu, Pan Qi, Gui Bo, Sun Zu-Yue

Department of Pharmacology and Toxicology, Shanghai Institute of Planned Parenthood Research, National Evaluation Centre for the Toxicology of Fertility Regulation Drugs, Shanghai 200032, China

Correspondence Address: Sun Zu-Yue, Department of Pharmacology and Toxicology, Shanghai Institute of Planned Parenthood Research, National Evaluation Centre for the Toxicology of Fertility Regulation Drugs, Shanghai 200032, China, sunzy64@163.com

Date of Submission: 01-Feb-2010
Date of Decision: 13-May-2010
Date of Acceptance: 20-Jul-2010

Code Number: ph10090

DOI: 10.4103/0253-7613.70397

Abstract

Objectives : To explore the role of androgen-estrogen balance in benign prostatic hyperplasia (BPH) induced by varying doses of estradiol/testosterone propionate (E 2 /TP) in castrated rats.
Materials and Methods : A total of 222 rats were divided into 37 groups at random, including 35 groups of different E 2 /TP, one control, and one castrated group. All 37 groups except the control group were castrated, for eliminating endogenesis of testosterone in rats. The treated groups were administered testosterone propionate (TP; at the dosages of 0.15, 0.74, 3.7, 18.5, and 92.6 mg/kg), combined with estradiol (E 2 ; at the dosage of 0, 0.4, 2, 10, 50, 250, and 1250 μg/kg) diluted in vegetable oil for 30 days, respectively, whereas the control groups received only vegetable oil. All prostate specimens were removed under anesthesia, then fixed and embedded in paraffin, for measuring the organ quotient, volume, area of prostate glandular cavity, and the height of prostate epithelia.
Results : When the dosages of TP were 0.15, 3.7, 18.5, and 92.6 mg/kg, the degree of prostatic hyperplasia had no obvious dose-effect relationship with E 2 . When TP was 0.74 mg/kg, with the increase of the dosage of E 2 , the volume and quotient of prostate were increasing. However, when the dosage of E 2 exceeded 50 μg/kg, E 2 /TP was 5/74, the prostatic volume did not increase obviously.
Conclusion : The proper levels of E2/TP play an important role in the pathogenesis of BPH. In rats, the balance point of E 2 /TP is 5/74.

Keywords: Area of prostate glandular cavity, height of prostate epithelia, organ quotient

Introduction

Benign prostatic hyperplasia (BPH) is a common disease of men over 50, and its incidence goes up with advancing age. Statistics shows that BPH is hardly found in men less than 30 years old, [1] but in 88% of autopsies BPH were found in men aged above 80, [2] with compatible symptomatology reported in nearly 50% of men aged above 50 in the general population. The phenomenon maybe correlated with changes of sex hormone in serum of elderly population. One clinical study reported that there were low free testosterone concentrations with relative rise in serum estradiol levels in patients of BPH. [3] Testosterone level declines with age, but serum estrogen level remains unaltered, so estrogen may be involved in the development of BPH. [4]

BPH is histologically complex, involving glandular and stromal hyperplasia, fibrosis, and prostatitis. [5] The etiology of BPH is still poorly understood. It is thought to be related to the combination of aging and endocrine dysregulation. It is well documented that androgens are the primary factor for prostate disease, [6],[7] but the mechanism is still unclear. [8] While the prostate is considered the prototype androgen-dependent gland, there is rising evidence that estrogen is necessary to maintain the natural function of prostate. [9],[10] In addition, estrogens also play an important role in growth and differentiation of prostate gland. [11] We have tried to find a balance point of estradiol/testosterone propionate (E 2 /TP) in development of BPH. The results may provide further insight into the role of E 2 /TP in development of BPH.

Materials and Methods

Animal

All experiments were performed in Shanghai. Male Sprague-Dawley rats (120 ± 10 g) were obtained from the Shanghai SIPPR-BK (Shanghai Institute of Planned Parenthood Research-BK Laboratory Animal Limited Company, Shanghai, China). The animals were weighed and kept under the same conditions with free access to water and food. A total of 222 rats were divided in 37 groups with six animals in each group at random, including 35 groups of different E 2 /TP, one control group and another group of castrated animals which also served as sham control. Except the rats of control group, all rats were castrated under anesthesia with ketamine, for eliminating endogenesis of testosterone. The experiments were started in the first week after castration.

Study procedures

As testosterone propionate (TP) (3.7 mg/kg) is shown to result in rat BPH, [12] the treated groups were administered (s.c.) daily with different dosages of TP (five doses of 0.15, 0.74, 3.7, 18.5, and 92.6 mg/kg) combined with different dosages of estradiol (E 2 ) (seven doses of 0, 0.4, 2, 10, 50, 250, and 1250 μg/kg) diluted in vegetable oil for 30 days, respectively, whereas the control groups received only vegetable oil. All prostate specimens were removed under anesthesia, then fixed and embedded in paraffin. The paraffin-blocked section was consecutively cut at 5-μm thickness for hematoxylin-eosin (H&E) and immunohistochemical staining. After the prostate quotient (the weight of prostate/the weight of rats) and the volume were determined, the area of 200 glandular cavity and 200 height of prostatic epithelia of prostate in each tissues slide were measured by image analysis software after being shot by camera under light microscope. Furthermore, AR-labeled cells were detected using immunohistochemical staining as described. After deparaffination and rehydration of sections, slides were placed in sodium citrate solution (0.01 M, pH 6.0) and heated to 96-100ºC for 25 min. After cooling, sections were put into 5% BSA for 20 min. Then, sections were incubated for 2 h with primary anti-AR mouse monoclonal antibody (Boster Biotechnology Co. Ltd. Wuhai, China) diluted 1:100 in TBS, lastly, covered with cover slips. AR-labeled cells were observed under a light microscope.

Statistics

Data were expressed as mean ±SD. One-way ANOVA and P values were used to evaluate significant differences between the groups. Image-Pro Plus 6.0 was used to analysis image data.

Results

Changes in prostate after treatment

Effect of different dosages of E2 with TP (0.15 mg/kg)

There were seven groups, in which all rats were administered with TP of 0.15 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. Compared with the castrated control group, the organ quotient, volume and area of prostate glandular cavity, and the height of prostate epithelia were found to be statistically comparable (P > 0.05) [Table - 1] and [Figure - 1]. The prostates of the animals from all the treatment groups were found to be regressed, and the organ quotient and the volume of prostate were significantly less (P < 0.05) [Table - 1], compared to the control group. There were no significant differences in the area of prostate glandular cavity and height of prostate epithelia (P > 0.05) [Table - 1] and [Figure - 1].

Effect of different dosages of E 2 with TP (0.74 mg/kg)

There were seven groups, in which all rats were administered with TP of 0.74 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. Compared with the group which received TP alone, the prostate organ quotient and the volume of the other groups (E 2 ≥ 0.4 μg/kg) were enlarged significantly (P < 0.05) [Table - 2]. In addition, individual groups compared with each other. There was no statistically significant difference (P > 0.05) among the middle three groups (E 2 doses 0.4, 2.0, and 10 μg/kg) and the last three groups (E 2 doses 50, 250, and 1250 μg/kg), but compared with the middle three groups, each of the last three groups was enlarged significantly (P < 0.05) [Table - 2]. The height of prostate epithelia of the two groups (E 2 doses 250, 1250 μg/kg) was higher than the control groups, but the differences were insignificant (P > 0.05). Compared with the control group, the area of prostate glandular cavity of the last three groups was observed to be increased and the increase was statistically significant (P < 0.05) [Table - 2] and [Figure - 2].

Effect of different dosages of E 2 with TP (3.7 mg/kg)

There were seven groups which were all administered with TP of 3.7 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. Compared with the control groups, the organ quotient, the volume, and the area of prostate glandular cavity were all increased significantly (P < 0.01) [Table - 3] and [Figure - 3]. The prostate epithelia appeared high stylolitic with increase in glandular cavity, and the area of prostate glandular cavity was large.

Effect of different dosages of E 2 with TP (18.5 mg/kg)

There were seven groups which were all administered with TP of 18.5 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. Compared with the control groups, the organ quotient, the volume, and the area of prostate glandular cavity were all significantly different (P < 0.01) [Table - 4] and [Figure - 4]. The prostate epithelia appeared high stylolitic with increased glandular cavity, and the area of prostate glandular cavity was large.

Effect of different dosages of E 2 with TP (92.6 mg/kg)

There were seven groups which were all administered with TP of 92.6 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. Compared with the control group, the organ quotient, the volume, the area of prostate glandular cavity were all significantly different (P < 0.01) [Table - 5] and [Figure - 5]. The prostate epithelia appeared high stylolitic with increased glandular cavity, and the area of prostate glandular cavity was large. However, when the dosages of TP exceed 3.7 mg/kg, E 2 had little effect on prostate as compared to other groups (P < 0.05).

Androgen receptor-labeled cell assay

Effect of E2 on androgen receptor (AR) of prostate with TP (0.15 mg/kg)

There were seven groups which were all administered with TP of 0.15 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. A large number of cells were observed in the areas of prostate epithelial cells and stroma, but positive cells were hardly seen in all groups.

Effect of E 2 on androgen receptor (AR) of prostate with TP (0.74 mg/kg)

There were seven groups which were all administered with TP of 0.74 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. A few of AR-labeled cells were observed in the last three groups (E 2 were 50, 250, and 1250 μg/kg), whereas positive cells were hardly seen in the control and the other treatment groups.

Effect of E 2 on androgen receptor (AR) of prostate with TP (3.7 mg/kg)

There were seven groups which were all administered with TP of 3.7 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. AR-labeled cells appeared in all treatment groups, and especially, a number of AR-labeled positive cells were observed in the last three groups (E 2 were 50, 250, and 1250 μg/kg).

Effect of E 2 on androgen receptor (AR) of prostate with TP (18.5 mg/kg)

There were seven groups which were all administered with TP of 18.5 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. AR-labeled cells appeared in all treatment groups, but there were insignificant differences in them.

Effect of E 2 on androgen receptor (AR) of prostate with TP (92.6 mg/kg)

There were seven groups which were all administered with TP of 92.6 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. AR-labeled cells appeared in all treatment groups to similar extent, AR-labeled cells were not found and compared statistically.

Discussion

Androgens play an obligatory role in the embryonic development and function of prostate gland in adults. The essential role of androgens in prostatic development is clearly evident in genetic XY males with congenital abnormality in AR function or deficiency in 5-α-reductase, since in these individuals the prostate is either absent or incompletely developed. [13] The secretory epithelial cells express the AR, and they require continuous androgenic stimulation for survival and functional integrity. When the androgen level drops below a threshold, as is the case after surgical or chemical castration, the secretory cells undergo apoptosis, causing glandular involution. [14] In the study, when androgen is 0.15 mg/kg, even if the highest dosage (1250 μg/kg) of E 2 were administered, there were no obvious changes in the organ quotient, volume, area of prostate glandular cavity, and height of prostate epithelia. In addition, the AR-labeled cells were hardly seen through immunohistochemical examination. When the dosage of TP was 0.74 mg/kg, with the increasing of the dosage of E 2 , the volume and quotient of prostate increased. When the dosages of E 2 were 50, 250, and 1250 μg/kg in TP-0.74 mg/kg group, the area of prostate glandular cavity increased and a few little AR-labeled cells appeared. The results proved that when the TP dose was below 0.15 mg/kg, the prostate gland showed atrophy, whereas when TP dose was 0.74 mg/kg but the prostate gland was found to be hyperplastic when TP dose was 0.74 mg/kg. When TP was over 3.7 mg/kg, the organ quotient, volume, area of prostate glandular cavity showed further increase which was obvious and AR express markedly, which support the fact that androgen is a crucial hormone for prostate development. [15]

It was observed that when TP was 0.74 mg/kg combined with E 2 (0.4 μg/kg), i.e., E 2 /TP was 2/3700, the change in morphology of prostate was less; however when E 2 is 50 μg/kg, i.e., E 2 /TP of 5/74, there was obvious change in prostate gland structure. Estrogen does not always cause antiandrogen effects, but under specific conditions, it may be of benefit to induce prostatic hyperplasia. [16],[17],[18] Many studies reported that estrogens affect prostatic hyperplasia. This neonatal exposure to estradiol resulted in a permanent reduction in prostatic growth and activational response to androgens during adulthood, an effect mediated in part through a permanent reduction in AR expression. [19] Exposure to estradiol results in neonatal results in promoting prostate hyperplasia during adulthood. The effects of estrogens on prostate were found to be complicated. [19]

Therefore, we attempted to study the effect of E 2 /TP on prostate. Serum level of estrogen-androgen is 1/150 in adulthood. The incidence of BPH is related to age. With increasing age, serum level of estrogen-androgen is 1/120-1/80 in elderly, whereas it can reach 1/8 in prostate. [20] BPH could be induced by the change of E 2 /TP. Mark reported that dihydrotestosterone (DHT) plus E 2 treatment in animals increased the prostatic activity of 4-hydroxy estradiol synthase, whereas either E 2 or DHT treatment alone did not change this activity. [21] Our results show that in rats, balance point of E 2 /TP is 5/74. The proper E 2 /TP ratio plays an important role in the pathogenesis of BPH. If the optimum ratio is not maintained, it can lead to BPH. This knowledge of optimizing E 2 /TP in humans may help to prevent or cure BPH in future.

Acknowledgments

We would like to thank Gui-lin He, Xiu-rong Jiang, Gui-ming Liu, Shu-wu Xie, Zhi-ling Li and Li Ma for technical assistance.

References

1.Berry SJ, Coffey DS, Walsh PC, Ewing LL. The development of human benign prostatic hyperplasia with age. J Urol 1984;132:474-9.  Back to cited text no. 1  [PUBMED]  
2.Pavel N, Patrick M, Peter B. Worldwide patterns of prevalence and mortality from benign prostatic hyperplasia. J Urol 1995;46:41-6.  Back to cited text no. 2    
3.Tan MO, Karabiyik I, Uygur MC, Diker Y, Erol D. Serum concentrations of sex hormones in men with severe lower urinary tract symptoms and benign prostatic hyperplasia. Int Urol Nephrol 2003;35:357-63.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.Scarano WR, Cordeiro RS, Gσes RM, Carvalho HF, Taboga SR. Tissue remodeling in Guinea pig latral prostate at different ages after estradiol treatment. Cell Biol Int 2005;29:778-84.  Back to cited text no. 4    
5.Horsfall DJ, Mayne K, Ricciardelli C, Rao M, Skinner JM, Henderson DW, et al. Age-related changes in Guinea pig prostatic stroma. Lab Invest 1994;70:753-63.   Back to cited text no. 5  [PUBMED]  
6.Qian LH, Wang XL, Tu ZH. Inhibition of re growth of prostatic glandular cells by epristeride. Acta Pharmacol Sin 2001;22:847-50.   Back to cited text no. 6  [PUBMED]  
7.Chu JH, Sun ZY, Meng XL, Wu JH, He GL, Liu GM, et al. Differential metastasis-associated gene analysis of prostate carcinoma cells derived from primary tumor and spontaneous lymphatic metastasis in nude mice with orthotopic implantation of PC-3M cells. Cancer Lett 2006;233:79-88.   Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Meigs JB, Mohr B, Barry MJ, Collins MM, McKinlay JB. Risk factors for clinical benign prostatic hyperplasia in a community-based population of healthy aging men. J Clin Epidemiol 2001;54:935-44.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.Griffiths K. Estrogens and prostatic disease. International Prostate Health Council Study Group. Prostate 2000;45:87-100.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Pettersson K, Gustafsson JA. Role of estrogen receptor beta in estrogen action. Annu Rev Physiol 2001;63:165-92.   Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.Naslund MJ, Coffey DS. The differential effect of Neonatal androgen, estrogen and progesterone on adult rat prostate growth. J Urol 1986;136:1136-40.   Back to cited text no. 11  [PUBMED]  
12.Bureau of drug administration in the People's Republic of China. New Drug (Western Medicine) Preclinical Study Guideline (Pharmacy, Pharmacology, Toxicology) 1993. p. 102.  Back to cited text no. 12    
13.Griffin J. Androgen resistance: The clinical and molecular spectrum. N Engl J Med 1992;326:611-8.  Back to cited text no. 13    
14.Chatterjee B. The role of the androgen receptor in the development of prostatic hyperplasia and prostate cancer. Mol Cell Biochem 2003;253:89-101.   Back to cited text no. 14  [PUBMED]  [FULLTEXT]
15.Levine AC, Ren M, Huber GK, Kirschenbaum A. The effect of androgen, estrogen and growth factors on the proliferation of cultured fibroblasts derived from human fetal and adult prostates. Endocrinology 1992;130:2413-9.   Back to cited text no. 15  [PUBMED]  [FULLTEXT]
16.Gann P, Hennekens C, Longcope C, Verhoek-Oftedahl W, Grodstein F, Stampfer MJ. A prospective study of plasma hormone levels, non-hormonal factors, and development of benign prostatic hyperplasia. Prostate 1995;26:40-9.   Back to cited text no. 16    
17.Rhodes L, Ding VD, Kemp RK, Khan MS, Nakhla AM, Pikounis B, et al. Estradiol causes a dose-dependent stimulation of prostate growth in castrated beagle dogs. Prostate 2000;44:8-18.  Back to cited text no. 17  [PUBMED]  [FULLTEXT]
18.Jarred RA, Mcpherson SJ, Bianco JJ, Couse JF, Korach KS, Risbridger GP. Prostate phenotypes in estrogen-modulated transgenic mice. Trends Endocrinol Metab 2002;13:163-8.  Back to cited text no. 18  [PUBMED]  [FULLTEXT]
19.Woodham C, Birch L, Prins GS. Neonatal estrogens down regulate prostatic androgen receptor levels through a proteosome-mediated protein degradation pathway. Endocrinology 2003;144:4841-50.  Back to cited text no. 19  [PUBMED]  [FULLTEXT]
20.Griffiths K, Coffey D, Cockett A, Khoury S, Aso YM. The regulation of prostatic growth. The 3 rd international consultation on benign prostatic hyperplasia. In: Cockett A, Khoury S, Aso Y. editors. Monaco: 1995. p. 71-122.  Back to cited text no. 20    
21.Mark LW, Joachim GL. Possible mechanism of induction of benign prostatic hyperplasia by estradiol and dihydrotestosterone in dogs. Toxicol Appl Pharm 1996;136:211-9.  Back to cited text no. 21    

Copyright 2010 - Indian Journal of Pharmacology

Indian Journal of Pharmacology, Vol. 42, No. 5, September-October, 2010, pp. 312-317

Research Article

Possible mechanism of benign prostatic hyperplasia induced by androgen-estrogen ratios in castrated rats

Liu Xiang-Yun, Xu Ying-Wen, Xie Chen-Jing, Wang Jiu-Jiu, Pan Qi, Gui Bo, Sun Zu-Yue

Department of Pharmacology and Toxicology, Shanghai Institute of Planned Parenthood Research, National Evaluation Centre for the Toxicology of Fertility Regulation Drugs, Shanghai 200032, China

Correspondence Address: Sun Zu-Yue, Department of Pharmacology and Toxicology, Shanghai Institute of Planned Parenthood Research, National Evaluation Centre for the Toxicology of Fertility Regulation Drugs, Shanghai 200032, China, sunzy64@163.com

Date of Submission: 01-Feb-2010
Date of Decision: 13-May-2010
Date of Acceptance: 20-Jul-2010

Code Number: ph10090

DOI: 10.4103/0253-7613.70397

Abstract

Objectives : To explore the role of androgen-estrogen balance in benign prostatic hyperplasia (BPH) induced by varying doses of estradiol/testosterone propionate (E 2 /TP) in castrated rats.
Materials and Methods : A total of 222 rats were divided into 37 groups at random, including 35 groups of different E 2 /TP, one control, and one castrated group. All 37 groups except the control group were castrated, for eliminating endogenesis of testosterone in rats. The treated groups were administered testosterone propionate (TP; at the dosages of 0.15, 0.74, 3.7, 18.5, and 92.6 mg/kg), combined with estradiol (E 2 ; at the dosage of 0, 0.4, 2, 10, 50, 250, and 1250 μg/kg) diluted in vegetable oil for 30 days, respectively, whereas the control groups received only vegetable oil. All prostate specimens were removed under anesthesia, then fixed and embedded in paraffin, for measuring the organ quotient, volume, area of prostate glandular cavity, and the height of prostate epithelia.
Results : When the dosages of TP were 0.15, 3.7, 18.5, and 92.6 mg/kg, the degree of prostatic hyperplasia had no obvious dose-effect relationship with E 2 . When TP was 0.74 mg/kg, with the increase of the dosage of E 2 , the volume and quotient of prostate were increasing. However, when the dosage of E 2 exceeded 50 μg/kg, E 2 /TP was 5/74, the prostatic volume did not increase obviously.
Conclusion : The proper levels of E2/TP play an important role in the pathogenesis of BPH. In rats, the balance point of E 2 /TP is 5/74.

Keywords: Area of prostate glandular cavity, height of prostate epithelia, organ quotient

Introduction

Benign prostatic hyperplasia (BPH) is a common disease of men over 50, and its incidence goes up with advancing age. Statistics shows that BPH is hardly found in men less than 30 years old, [1] but in 88% of autopsies BPH were found in men aged above 80, [2] with compatible symptomatology reported in nearly 50% of men aged above 50 in the general population. The phenomenon maybe correlated with changes of sex hormone in serum of elderly population. One clinical study reported that there were low free testosterone concentrations with relative rise in serum estradiol levels in patients of BPH. [3] Testosterone level declines with age, but serum estrogen level remains unaltered, so estrogen may be involved in the development of BPH. [4]

BPH is histologically complex, involving glandular and stromal hyperplasia, fibrosis, and prostatitis. [5] The etiology of BPH is still poorly understood. It is thought to be related to the combination of aging and endocrine dysregulation. It is well documented that androgens are the primary factor for prostate disease, [6],[7] but the mechanism is still unclear. [8] While the prostate is considered the prototype androgen-dependent gland, there is rising evidence that estrogen is necessary to maintain the natural function of prostate. [9],[10] In addition, estrogens also play an important role in growth and differentiation of prostate gland. [11] We have tried to find a balance point of estradiol/testosterone propionate (E 2 /TP) in development of BPH. The results may provide further insight into the role of E 2 /TP in development of BPH.

Materials and Methods

Animal

All experiments were performed in Shanghai. Male Sprague-Dawley rats (120 ± 10 g) were obtained from the Shanghai SIPPR-BK (Shanghai Institute of Planned Parenthood Research-BK Laboratory Animal Limited Company, Shanghai, China). The animals were weighed and kept under the same conditions with free access to water and food. A total of 222 rats were divided in 37 groups with six animals in each group at random, including 35 groups of different E 2 /TP, one control group and another group of castrated animals which also served as sham control. Except the rats of control group, all rats were castrated under anesthesia with ketamine, for eliminating endogenesis of testosterone. The experiments were started in the first week after castration.

Study procedures

As testosterone propionate (TP) (3.7 mg/kg) is shown to result in rat BPH, [12] the treated groups were administered (s.c.) daily with different dosages of TP (five doses of 0.15, 0.74, 3.7, 18.5, and 92.6 mg/kg) combined with different dosages of estradiol (E 2 ) (seven doses of 0, 0.4, 2, 10, 50, 250, and 1250 μg/kg) diluted in vegetable oil for 30 days, respectively, whereas the control groups received only vegetable oil. All prostate specimens were removed under anesthesia, then fixed and embedded in paraffin. The paraffin-blocked section was consecutively cut at 5-μm thickness for hematoxylin-eosin (H&E) and immunohistochemical staining. After the prostate quotient (the weight of prostate/the weight of rats) and the volume were determined, the area of 200 glandular cavity and 200 height of prostatic epithelia of prostate in each tissues slide were measured by image analysis software after being shot by camera under light microscope. Furthermore, AR-labeled cells were detected using immunohistochemical staining as described. After deparaffination and rehydration of sections, slides were placed in sodium citrate solution (0.01 M, pH 6.0) and heated to 96-100ºC for 25 min. After cooling, sections were put into 5% BSA for 20 min. Then, sections were incubated for 2 h with primary anti-AR mouse monoclonal antibody (Boster Biotechnology Co. Ltd. Wuhai, China) diluted 1:100 in TBS, lastly, covered with cover slips. AR-labeled cells were observed under a light microscope.

Statistics

Data were expressed as mean ±SD. One-way ANOVA and P values were used to evaluate significant differences between the groups. Image-Pro Plus 6.0 was used to analysis image data.

Results

Changes in prostate after treatment

Effect of different dosages of E2 with TP (0.15 mg/kg)

There were seven groups, in which all rats were administered with TP of 0.15 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. Compared with the castrated control group, the organ quotient, volume and area of prostate glandular cavity, and the height of prostate epithelia were found to be statistically comparable (P > 0.05) [Table - 1] and [Figure - 1]. The prostates of the animals from all the treatment groups were found to be regressed, and the organ quotient and the volume of prostate were significantly less (P < 0.05) [Table - 1], compared to the control group. There were no significant differences in the area of prostate glandular cavity and height of prostate epithelia (P > 0.05) [Table - 1] and [Figure - 1].

Effect of different dosages of E 2 with TP (0.74 mg/kg)

There were seven groups, in which all rats were administered with TP of 0.74 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. Compared with the group which received TP alone, the prostate organ quotient and the volume of the other groups (E 2 ≥ 0.4 μg/kg) were enlarged significantly (P < 0.05) [Table - 2]. In addition, individual groups compared with each other. There was no statistically significant difference (P > 0.05) among the middle three groups (E 2 doses 0.4, 2.0, and 10 μg/kg) and the last three groups (E 2 doses 50, 250, and 1250 μg/kg), but compared with the middle three groups, each of the last three groups was enlarged significantly (P < 0.05) [Table - 2]. The height of prostate epithelia of the two groups (E 2 doses 250, 1250 μg/kg) was higher than the control groups, but the differences were insignificant (P > 0.05). Compared with the control group, the area of prostate glandular cavity of the last three groups was observed to be increased and the increase was statistically significant (P < 0.05) [Table - 2] and [Figure - 2].

Effect of different dosages of E 2 with TP (3.7 mg/kg)

There were seven groups which were all administered with TP of 3.7 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. Compared with the control groups, the organ quotient, the volume, and the area of prostate glandular cavity were all increased significantly (P < 0.01) [Table - 3] and [Figure - 3]. The prostate epithelia appeared high stylolitic with increase in glandular cavity, and the area of prostate glandular cavity was large.

Effect of different dosages of E 2 with TP (18.5 mg/kg)

There were seven groups which were all administered with TP of 18.5 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. Compared with the control groups, the organ quotient, the volume, and the area of prostate glandular cavity were all significantly different (P < 0.01) [Table - 4] and [Figure - 4]. The prostate epithelia appeared high stylolitic with increased glandular cavity, and the area of prostate glandular cavity was large.

Effect of different dosages of E 2 with TP (92.6 mg/kg)

There were seven groups which were all administered with TP of 92.6 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. Compared with the control group, the organ quotient, the volume, the area of prostate glandular cavity were all significantly different (P < 0.01) [Table - 5] and [Figure - 5]. The prostate epithelia appeared high stylolitic with increased glandular cavity, and the area of prostate glandular cavity was large. However, when the dosages of TP exceed 3.7 mg/kg, E 2 had little effect on prostate as compared to other groups (P < 0.05).

Androgen receptor-labeled cell assay

Effect of E2 on androgen receptor (AR) of prostate with TP (0.15 mg/kg)

There were seven groups which were all administered with TP of 0.15 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. A large number of cells were observed in the areas of prostate epithelial cells and stroma, but positive cells were hardly seen in all groups.

Effect of E 2 on androgen receptor (AR) of prostate with TP (0.74 mg/kg)

There were seven groups which were all administered with TP of 0.74 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. A few of AR-labeled cells were observed in the last three groups (E 2 were 50, 250, and 1250 μg/kg), whereas positive cells were hardly seen in the control and the other treatment groups.

Effect of E 2 on androgen receptor (AR) of prostate with TP (3.7 mg/kg)

There were seven groups which were all administered with TP of 3.7 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. AR-labeled cells appeared in all treatment groups, and especially, a number of AR-labeled positive cells were observed in the last three groups (E 2 were 50, 250, and 1250 μg/kg).

Effect of E 2 on androgen receptor (AR) of prostate with TP (18.5 mg/kg)

There were seven groups which were all administered with TP of 18.5 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. AR-labeled cells appeared in all treatment groups, but there were insignificant differences in them.

Effect of E 2 on androgen receptor (AR) of prostate with TP (92.6 mg/kg)

There were seven groups which were all administered with TP of 92.6 mg/kg, with different dosages of E 2 (0, 0.4, 2.0, 10, 50, 250, and 1250 μg/kg), respectively. AR-labeled cells appeared in all treatment groups to similar extent, AR-labeled cells were not found and compared statistically.

Discussion

Androgens play an obligatory role in the embryonic development and function of prostate gland in adults. The essential role of androgens in prostatic development is clearly evident in genetic XY males with congenital abnormality in AR function or deficiency in 5-α-reductase, since in these individuals the prostate is either absent or incompletely developed. [13] The secretory epithelial cells express the AR, and they require continuous androgenic stimulation for survival and functional integrity. When the androgen level drops below a threshold, as is the case after surgical or chemical castration, the secretory cells undergo apoptosis, causing glandular involution. [14] In the study, when androgen is 0.15 mg/kg, even if the highest dosage (1250 μg/kg) of E 2 were administered, there were no obvious changes in the organ quotient, volume, area of prostate glandular cavity, and height of prostate epithelia. In addition, the AR-labeled cells were hardly seen through immunohistochemical examination. When the dosage of TP was 0.74 mg/kg, with the increasing of the dosage of E 2 , the volume and quotient of prostate increased. When the dosages of E 2 were 50, 250, and 1250 μg/kg in TP-0.74 mg/kg group, the area of prostate glandular cavity increased and a few little AR-labeled cells appeared. The results proved that when the TP dose was below 0.15 mg/kg, the prostate gland showed atrophy, whereas when TP dose was 0.74 mg/kg but the prostate gland was found to be hyperplastic when TP dose was 0.74 mg/kg. When TP was over 3.7 mg/kg, the organ quotient, volume, area of prostate glandular cavity showed further increase which was obvious and AR express markedly, which support the fact that androgen is a crucial hormone for prostate development. [15]

It was observed that when TP was 0.74 mg/kg combined with E 2 (0.4 μg/kg), i.e., E 2 /TP was 2/3700, the change in morphology of prostate was less; however when E 2 is 50 μg/kg, i.e., E 2 /TP of 5/74, there was obvious change in prostate gland structure. Estrogen does not always cause antiandrogen effects, but under specific conditions, it may be of benefit to induce prostatic hyperplasia. [16],[17],[18] Many studies reported that estrogens affect prostatic hyperplasia. This neonatal exposure to estradiol resulted in a permanent reduction in prostatic growth and activational response to androgens during adulthood, an effect mediated in part through a permanent reduction in AR expression. [19] Exposure to estradiol results in neonatal results in promoting prostate hyperplasia during adulthood. The effects of estrogens on prostate were found to be complicated. [19]

Therefore, we attempted to study the effect of E 2 /TP on prostate. Serum level of estrogen-androgen is 1/150 in adulthood. The incidence of BPH is related to age. With increasing age, serum level of estrogen-androgen is 1/120-1/80 in elderly, whereas it can reach 1/8 in prostate. [20] BPH could be induced by the change of E 2 /TP. Mark reported that dihydrotestosterone (DHT) plus E 2 treatment in animals increased the prostatic activity of 4-hydroxy estradiol synthase, whereas either E 2 or DHT treatment alone did not change this activity. [21] Our results show that in rats, balance point of E 2 /TP is 5/74. The proper E 2 /TP ratio plays an important role in the pathogenesis of BPH. If the optimum ratio is not maintained, it can lead to BPH. This knowledge of optimizing E 2 /TP in humans may help to prevent or cure BPH in future.

Acknowledgments

We would like to thank Gui-lin He, Xiu-rong Jiang, Gui-ming Liu, Shu-wu Xie, Zhi-ling Li and Li Ma for technical assistance.

References

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