High Plasma Estradiol-17Β Levels in Dogs with Benign Prostatic Hyperplasia and Azoospermia

FULL PAPER Theriogenology

High Plasma Estradiol-17β Levels in Dogs with Benign Prostatic Hyperplasia and Azoospermia

Eiichi KAWAKAMI1), Eri AMEMIYA1), Kenji NAMIKAWA1), Chikako KASHIWAGI1), Tatsuya HORI1) and Toshihiko TSUTSUI1)

1)Department of Reproduction, Nippon Veterinary and Animal Science University, 1–7–1 Kyonan-cho, Musashino-shi, Tokyo 180–8602, Japan

(Received 15 June 2000/Accepted 25 December 2000)

ABSTRACT. The semen quality of 22 dogs (4 to 7 years old) with benign prostatic hyperplasia (BPH) was examined at the hospital of our university, and 4 of the 22 BPH dogs were diagnosed as azoospermic. The mean peripheral plasma estradiol-17β (E2) level (17.3 pg/ ml) of the 18 BPH dogs with spermatogenic function was higher than that of 5 normal male dogs and their mean T level (1.7 ng/ml) was lower. The mean E2 level (27.3 pg/ml) of the 4 BPH dogs with azoospermia was significantly higher than the value in the BPH dogs with spermatogenic function (P<0.01), and the mean T level (1.1 ng/ml) was significantly lower (P<0.05). Five normal male dogs were given 10 intramuscular injections of estradiol benzoate (E2B) 5 µg/kg, at 3-day intervals to investigate the relationship between high plasma E2 levels and the cause of the BPH and azoospermia. Their testes and prostates were measured and biopsied both before and 30 days after the start of E2B injections. At 30 days after the start of the E2B injections, the mean peripheral plasma T levels had decreased by half, and the mean testicular volume had decreased to 88% of original volume. The numbers of spermatocytes, spermatids, and spermatozoa in the seminiferous tubules of all of the dogs were significantly lower (P<0.05, 0.01). In addition, the mean prostatic volume increased to 130%, the mean height of the glandular epithelium decreased, and the glandular lumen became increased in diam- eter. These findings indicate that both BPH and serious spermatogenic dysfunction may be simultaneously induced by protracted high plasma E2 levels in dogs. KEY WORDS: azoospermia, benign prostatic hyperplasia, canine, estradiol-17β. J. Vet. Med. Sci. 63(4): 407–412, 2001

Canine benign prostatic hyperplasia (BPH) in dogs is azoospermia in dogs. known to occur chiefly in animals more than 6 years old [6], and its occurrence is presumed to be associated with abnor- MATERIALS AND METHODS mal estradiol-17β (E2) and testosterone (T) secretion by the testes associated with advanced age [5, 19]. T is converted Animals: The subjects of this study were 22 dogs (4 to 7 to 5α-dihydrotestosterone (DHT) in the canine prostate, and years old) diagnosed with BPH at the teaching hospital of prostatic function is maintained by the androgen effects of our university between April 1992 and March 1995. Blood DHT [10, 11]. DHT concentration in the prostates of dogs samples were collected from them to determine their plasma with BPH has been reported to be higher than in normal hormone levels, and semen samples were collected 2 or 3 dogs [8, 14]. Accumulation of DHT in the prostate is accel- times weekly to evaluate semen quality. Each semen sam- erated by high plasma E2 levels and is thought to cause BPH ple was examined for the volume and pH, and then sperm [8, 14]. concentration in the semen was determined by hematocy- Testicular and plasma E2 are produced by both Leydig tometer counts. The percentage of motile sperm was deter- cells and Sertoli cells [23]. Infertility in a few human mined by counting actively motile sperm on a warmed glass patients has been attributed to elevated testicular E2 concen- slide. Four (4 to 6 years old; one Shetland Sheepdog, two trations [13], and the plasma E2 levels of some azoospermic Beagles, and one Maltese) of the 22 dogs with BPH were men have been found to be higher than those of normal men diagnosed as having azoospermia by reason of no there [9, 29]. Long term E2 administration has been shown to being sperm in their semen. The reproductive tracts in the 4 inhibit T secretion by the testis in rats [28], to cause the dis- BPH dogs were found to be not obturated because of obser- appearance of spermatids and spermatozoa from the semin- vation of a few germ cells in the semen. The semen quality iferous tubules of bull testes [21], and to induce the of the other 18 BPH dogs was almost normal. The diagnosis testicular atrophy of dogs [15]. However, in many cases the of BPH was based on prostatic size measured by radiogra- cause of spermatogenic arrest in dogs is unknown [7, 24]. In phy and ultrasonography and clinical signs, e.g., hematuria, the present study, the peripheral plasma E2 and T levels of dysuria, or rectal obstruction [22]. BPH dogs with azoospermia and of BPH dogs with sper- Five normal male beagles (3 to 5 years old) cared for in matogenic function were examined together with the our university were used to assess changes in the volume of changes in the volume and histological findings of the testes the testes and prostates and their histological appearance. and the prostates of normal dogs after estradiol benzoate (E2 Another 3 normal male beagles were used to assess the B) administration to determine the cause of both BPH and changes in plasma E2 levels after a single E2B injection. 408 E. KAWAKAMI ET AL.

Hormone assays: Plasma E2 and T levels in the blood were higher than the values in the 5 normal dogs (Table 1), samples were measured by radioimmunoassay [20, 30]. and there was a significant difference between the mean Rabbit antisera to E2 -6-CMO-BSA and T-11α-succinate- plasma E2 level of the BPH dogs with azoospermia and the BSA were used. The intra-assay and inter-assay coeffi- normal dogs (P<0.01). cients of variation were 7.3% and 12.6% for E2, respec- The mean plasma T levels in both the BPH dogs with tively, and 3.0% and 9.6% for T, respectively. azoopsermia and the BPH dogs with spermatogenesis were E2B injection: The 5 normal beagles were given 10 intra- lower than the values in the normal dogs (Table 1). There muscular injections of E2B (Gynandol; Sankyo Zoki, Inc., was a significant difference between the mean plasma T Tokyo), 5 µg/kg, at 3-day intervals. The other 3 normal level of the BPH dogs with azoospermia and the normal male beagles were given a single 5 µg/kg intramuscular dogs (P<0.05). injection of E2B. Plasma E2 levels in normal dogs after a single E2B injec- Peripheral blood collection to measure plasma T levels: tion: The mean (± S. E.) peripheral plasma E2 levels of the 3 Peripheral blood samples were collected from the 5 normal normal dogs peaked (50 ± 3 pg/ml) 2 hr after the single intra beagles before and 30 days after the start of E2B injection to muscular injection of 5 µg/kg E2B (Fig. 1). The mean E2 examine their plasma T levels. level (17 ± 3 pg/ml) 72 hr after E2B injection was still higher Testicular and prostatic biopsies and histological exami- than the pretreatment value (11 ± 1 pg/ml). nation: Before and 30 days after the start of E2B injection, Plasma T levels in normal dogs after E2B injection: The under halothane inhalation anesthesia, the sizes of the right mean plasma T level (0.9 ± 0.2 ng/ml) in the 5 normal dogs and left testes were measured in the scrotum, and the right 30 days after the start of the E2B injections was significantly testis was biopsied with a scalpel. Laparotomy was per- lower than the pretreatment value (2.1 ± 0.3 ng/ml). formed to measure the exact size of the prostate and to per- Volume of the testes and prostates of normal dogs after form a prostatic biopsy. E2B injections: The mean testicular volume of the normal Testicular and prostatic volume were calculated accord- dogs 30 days after the start of the E2B injections decreased ing to the formula (4/3π⋅abc⋅1/8: a, length; b, width; c, thick- significantly to 88% of the pretreatment value (P<0.01) ness) [27]. The biopsied testicular tissue was embedded in (Table 2). The mean prostatic volume, however, increased paraffin, sectioned at 3 µm, and stained with PAS-hematox- significantly to 130% of the pretreatment value (P<0.05) ylin. Round transverse sections of 5 seminiferous tubules of (Table 2). the testis and 5 glandular alveoli of the prostate were exam- Histology of the testes and prostates of normal dogs after ined histologically. The diameter of the seminiferous E2B injections: Large numbers of the various types of germ tubules and the height of the epithelium of the alveoli were cells and spermatozoa were seen in all seminiferous tubules measured with a micrometer. The numbers of type A sper- in the testes before treatment (Fig. 2-a). The well-developed matogonia, pachytene primary spermatocytes, round sper- secretory epithelium of the glandular alveoli was composed matids, and spermatozoa according to the classification of of tall columnar cells, and the shape of the glandular lumen Ibach et al. [12] were counted in 5 seminiferous tubules at was irregular because of the marked projection of the glan- stage No. 9 of the seminiferous epithelial cycle by the dular epithelium into the lumen (Fig. 3-a). The mean diam- method described previously [18]. The crude germ cell eter of the glandular epithelium into the lumen (Fig. 3-a). counts were corrected by the method of Abercrombie [1]. The mean diameter of the seminiferous tubules 30 days after Statistical analysis: Statistical significance was tested by the start of the E2B injections, however, was significantly the unpaired Student’s t test. The data are summarized as lower than the pretreatment value (P<0.01) (Table 3), and mean values ± standard error (S. E.). the numbers of primary spermatocytes, round spermatids and spermatozoa were significantly lower than before the RESULTS injections (P<0.05, 0.01) (Fig. 2-b and Table 3). The mean height of the glandular epithelium 30 days after the start of Plasma E2 and T levels in dogs with BPH: The mean (± the E2B injections was significantly lower than the pretreat- S. E.) plasma E2 levels in both the 4 BPH dogs with ment value (P<0.01) (Table 3), and the glandular lumen azoospermia and the 18 BPH dogs with spermatogenesis became wider because of reduction of the epithelial projec-

Table 1. Mean (± S.E.) peripheral plasma estradiol-17β (E2) and testosterone (T) levels in 4 dogs with benign prostatic hyperplasia (BPH) and azoospermia (AZ dogs), 18 dogs with BPH and spermatogenic function (SP dogs) and 5 normal dogs (NO dogs)

E2 (pg/ml)T (ng/ml) AZ dogs SP dogs NO dogs AZ dogs SP dogs NO dogs 27.3 ± 3.3** 17.3 ± 0.8 14.8 ± 1.2 1.1 ± 0.1* 1.7 ± 0.1 2.1 ± 0.3 ** P<0.01 and * P<0.05, in comparison with both SP dogs and NO dogs. A CAUSE OF BOTH BPH AND AZOOSPERMIA IN DOGS 409

Fig. 1. Changes in the mean (± S. E.) peripheral plasma E2 levels of 3 normal dogs after a single intramuscular injection of E2B, 5 µg/kg.

Table 2. GGMean (± S.E.) volume (cm3) of the testes and tions into the lumen (Fig. 3-b). Squamous metaplasia of the prostates of 5 normal dogs before and 30 days after the glandular epithelium after E2B injections was not observed. start of estradiol benzoate injections DISCUSSION Testes Prostates

Before 7.0 ± 1.8 (100%) 6.2 ± 1.7 (100%) Abnormally elevated E2 and reduced T secretion by the After 6.2 ± 0.1** ( 88%) 8.1 ± 0.6* (130%) testes has been reported to be a cause of both human [26] ** P<0.01 and P<0.05*, in comparison with before. and canine [5, 19] BPH, and it is necessary to administer E2 for a long period as well as androgen to artificially induce

Fig. 2. Histological changes in the testes of a normal dog after E2B injections (PAS-hematoxylin stain, × 200). (a) Testis before E2B injection. Normal spermatogenesis is observed in all of the seminiferous tubules. (b) Testis 30 days after the start of E2B injection. Seminiferous tubules diameter and the num- ber of germ cells in the tubules have decreased markedly. 410 E. KAWAKAMI ET AL.

Fig. 3. Histological changes in the prostate of a normal dog after E2B injections (PAS-Hematoxylin stain, × 200). (a) Prostate before E2B injection. Tall columnar epithelial cells and marked projections of the epithelium into the glandular lumen are observed. (b) Prostate 30 days after the start of E2B injection. Low epithelial cell height and reduction of epithelial projections into the lumen are observed.

Table 3. Mean (± S.E.) diameter of the seminiferous tubules, number of germ cells in the testes and epithelial cell height of the prostatic alveoli of 5 normal dogs before and 30 days after the start of estradiol benzoate injections Testesa) Prostates Seminiferous tubule Type A Pachytene primary Round Spermatozoa Epithelial cell diameter (µm) spermatogonia spermatocytes spermatids height (µm) Before 195 ± 30.7 ± 0.1 7.2 ± 0.9 18.7 ± 1.2 15.3 ± 1.2 15.8 ± 1.4 After 166 ± 8** 0.6 ± 0.1 4.1 ± 0.5* 11.7 ± 0.9** 6.4 ± 0.8** 11.0 ± 0.4* a) Germ cells were counted in stage 9 of the seminiferous epithelial cycle by the classification of Ibach et al. [12]. ** P<0.01 and * P<0.05, in comparison with before.

BPH in orchidectomized dogs [2, 6]. The peripheral plasma prostate and consequently cause BPH [8, 14]. Therefore, E2 levels of normal dogs 1–3 days after an intramuscular elevated plasma E2 levels are thought to be more important injection of 5 µg/kg of E2B were similar to the values (20– than reduced plasma T levels for the occurrence of BPH. 40 pg/ml) reported by us in azoospermic dogs with elevated Some cases of azoospermic human patients have been plasma E2 levels [17], and thus the dose of E2B administered reported in which the spermatogenic function in the testes to the normal dogs in the present study was concluded to was affected by increased plasma E2 levels and decreased T have been appropriate. In this study, the mean plasma E2 levels [9, 29]. Increased E2 in the blood plasma of some level in the dogs with spontaneous BPH was higher than the azoospermic men has been found to be due to increased value in normal dogs, and E2 treatment induced expansion secretion by the testes [9], and E2 treatment to male animals of the glandular lumen by reducing epithelial projections for long periods is well known to inhibit androgen produc- into the lumen. The volume of the stromal tissue in the tion [16] and spermatogenesis in the testes [16, 21], and to canine prostate has been shown to be increased by E2 treat- cause testicular atrophy [15]. In the present study, the mean ment [4, 25], and thus it is assumed that spontaneous and plasma E2 level of the BPH dogs with azoospermia was artificial BPH in dogs is the result of both expansion of the higher than in both the BPH dogs with spermatogenic func- glandular lumen and an increase in stromal tissue caused by tion and the normal dogs. Therefore, BPH dogs with exces- high plasma E2 levels. Protracted high plasma E2 levels in sive plasma E2 levels are assumed to have a tendency to the dog were found to induce accumulation of DHT in the suffer from azoospermia. Decreases in the numbers of the A CAUSE OF BOTH BPH AND AZOOSPERMIA IN DOGS 411 various type of germ cells were observed in all the seminif- and peripheral oestradiol levels in patients affected by erous tubules of the normal dogs after E2B treatment. The azoospermia due to seminiferous tubular damage. Int. J. reductions of seminiferous tubule diameter and testicular Androl. 4: 161–171. volume in the normal dogs were presumably caused by a 10. Giorgi, E.P., Grant, J. K., Stewart, J.C. and Reid, J. 1972. decrease in the number of germ cells after E B treatment. Androgen dynamics in vitro in the canine prostate gland. J. 2 Endocrinol. 55: 421–430. Greater numbers of E2B injections may induce the disap- 11. Haltmeyer, G.C. and Eik-Nes, K.B. 1972. Production and pearance of spermatozoa in the seminiferous tubules of nor- secretion of 5α-dihydrotestosterone by the canine prostate. mal dogs. Acta Endocrinol. 69: 394–402. As BPH chiefly occurs in dogs more than 6 years old [3], 12. Ibach, B., Weissbach, L. and Hilscher, B. 1976. Stages of the aging is a factor in the abnormal steroid hormone secretion cycle of the seminiferous epithelium in the dog. Andrologia 8: by the testes of dogs with BPH. However, not only the age 297–307. of the dog, but genetic defects, nutrition and environment 13. Ichikawa, T. 1995. A study of intratesticular aromatase activity are also presumably related to the development of BPH and in male infertility. Jpn. J. Urol. 86: 940–948. azoospermia, and it seems necessary to thoroughly investi- 14. Issacs, J. T. and Coffey, D.S. 1981. Changes in dihydrotesto- gate the other possible causes of abnormal hormone secre- sterone metabolism associated with the development canine benign prostatic hyperplasia. Endocrinology 108: 445–453. tion by canine testes. 15. Jabara, A. G. 1962. Some tissue changes in the dog following In conclusion, the results of this study demonstrated that stilboestrol administration. Austral. J. Exp. Biol. 40: 293–308. an elevated E2 secretory function of the testes may simulta- 16. Kalla, N.R., Nisula, B.C., Menard, R. and Loriaux, D. L. 1980. neously induce both BPH and serious spermatogenic dys- The effect of estradiol on testicular testosterone biosynthesis. function in dogs. Especially, BPH dogs with high plasma E2 Endocrinology 106: 35–39. levels are assumed to have a tendency to suffer from 17. Kawakami, E., Hori, T. and Tsutsui, T. 1997. Changes in β azoospermia. This finding suggests that inhibitors of E2 plasma luteinizing hormone, testosterone and estradiol-17 synthesis in the testes may be useful in treating canine BPH levels and semen quality after injections of gonadotropin and spermatogenic dysfunction. releasing hormone agonist and human chorionic gonadotropin in three dogs with oligozoospermia and two dogs with azoospermia. Anim. Reprod. Sci. 47: 157–167. REFERENCES 18. Kawakami, E., Tsutsui, T., Yamada, Y., Ogasa, A. and Yamau- chi, M. 1988. Testicular function of scrotal testes after the 1. Abercrombie, M. 1946. Estimation of nuclear population from cryptorchidectomy in dogs with unilateral cryptorchidism. microtome sections. Anat. Rec. 94: 239–247. Jpn. J. Vet. Sci. 50: 1239–1244. 2. Bartsch, G., Brungger, A., Deklerk, D.P., Coffey, D.S. and 19. Kawakami, E., Tsutsui, T., Shimizu, M., Orima, H., Makanae, Role, H.P. 1987. Light-microscopic stereologic analysis of Y., Yajima, K. and Ogasa, A. 1993. Effects of administration spontaneous and steroid-induced canine prostatic hyperplasia. of chlormadinone acetate on canine prostatic hypertrophy. J. J. Urol. 137: 552–558. Vet. Med. Sci. 55: 631–635. 3. Brooks, J. R., Busch, R.D., Patanelli, D.J. and Steelman, S.L. 20. Makino, T., Inano, K., Yoshida, T., Den, N., Takagi, S. and 1973. A study of effects of a new anti-androgen on the hyper- Kanbegawa, A. 1973. Radioimmunoassay of sex steroid hor- plastic dog prostate. Proc. Soc. Exp. Biol. Med. 143: 647–655. mones. (4) Radioimmunoassay of testosterone. Clin. Endo- 4. Chaisiri, N. and Pierrepoint, C.G. 1980. Examination of the crinol. (Tokyo) 21: 867–873 (in Japanese). distribution of oestrogen receptor between the stromal and epi- 21. Ogasa, A., Sugawa, Y. and Matsuyama, S. 1963. Relationship thelial compartments of the canine prostate. The Prostate 1: between male reproductive function and estrogen. II. Effect of 357–366. injected estrogen on the testicular tissue of bull and rat. Jpn. J. 5. Cochran, R.C., Edwing, L.L. and Niswender, G.D. 1981. Anim. Reprod. 9: 12–16 (in Japanese with English summary). Serum levels of follicle stimulating hormone, luteinizing hor- 22. O’Shea, J. D. 1962. Studies on the canine prostate gland. I. α α mone, prolactin, testosterone, 5 -dihydrotestosterone, 5 - Factors influencing its size and weight. J. Comp. Pathol. 72: α β β androstane-3 , 17 -diol, and 17 -estradiol from male beagles 321–331. with spontaneous or induced benign prostatic hyperplasia. 23. Pomerantz, D.K. 1979. Effects of in vitro gonadotropin treat- Invest. Urol. 19: 142–147. ment on estrogen levels in the testis of the immature rat. Biol. 6. Deklerk, D.P., Coffey, D. S., Ewing, L.L., McDermott, I.R., Reprod. 21: 1247–1255. Reiner, W.G., Robinson, C.H., Scott, W.W., Strandberg, J.D., 24. Renton, J.R. and Aughey, E. 1971. Certain interesting aspects Talalay, P., Walsh, P.C., Wheaton, L.G. and Zirkin, B.R. 1979. of infertility in two shetland collies. Vet. Rec. 88: 408–411. Comparison of spontaneous and experimentally induced canine 25. Rohr, H. P., Naef, H. R., Holliger, O., Oberholzer, M., Ibach, prostatic hyperplasia. J. Clin. Invest. 64: 842–849. B., Weissbach, L. and Bartsch, G. 1981. The effect of oestro- 7. Evans, J. and Renton, J.R. 1973. A case of azoospermia in a gen on stromal growth of the dog prostate: a quantitative ultra- previously fertile dog with subsequent recovery. Vet. Rec. 92: structual study. Urol Res. 9: 201–207. 198–199. 26. Takezawa, Y., Fukabori, Y., Suzuki, T., Imai, K. and 8. Ewing, L.L., Berry, S.J. and Higginbottom, E.G. 1983. Dihy- Yamanaka, H. 1992. Endocrine environment of benign pros- drotestosterone concentration of beagle prostatic tissue: effect tatic hyperplasia: relationships of sex steroid hormone levels of age and hyperplasia. Endocrinology 113: 2004–2009. with age and the size of the prostate. Jpn. J. Urol. 83: 664–671. 9. Forti, G., Giusti, G., Pazzagli, M., Fiorelli, R., Borrelli, D., 27. Tunn, U., Serge, Y., Schenck, B. and Neumann, F. 1979. Bio- Cicchi, P., Guazzelli, R., Conti, C., Scarselli, G., Franchini, M., chemical and histological studies on prostates in castrated dogs Boninsegni, R., Mannelli, M. and Serio, M. 1981. Spermatic after treatment with androstandiol, oestradiol and cyptoterone 412 E. KAWAKAMI ET AL.

acetate. Acta Endocrinol. 91: 373–384. Clin. Endocrinol. 16: 39–47. 28. Van der Molen, H. J., Brinkmann, A. O., Jong, F.H. and Rom- 30. Yoshida, T., Den, N., Ozaki, H., Takagi, S., Makino, T. and merts, F. F. 1981. Testicular estrogens. J. Endocrinol. 89: 33– Kanbegawa, A. 1973. Radioimmunoassay of sex hormones. 45. (2) Radioimmunoassay of estradiol. Clin. Endocrinol. (Tokyo) 29. Wu, F.C.W., Swanston, I.A. and Baird, D. T. 1982. Raised 21: 651–656 (in Japanese). plasma oestrogens in infertile men with elevated levels of FSH.