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The Effects of an Aromatase
Inhibitor (Letrozole) on Hormone and Sperm Production in the Stallion Introduction In the stallion, the testes produce unusually large amounts of estrogens and low amounts of testosterone compared to most mammalian species (Setchel and Cox 1982). Although a majority of testicular estrogens (converted by the aromatase enzyme from androgens) are sulfoconjugated, a significant amount are in the free and active form as estradiol-17b (E2) and estrone (E1) and thus available to stimulate physiological events in an endocrine, paracrine, or autocrine fashion (Semans et al. 1991) suggesting that estrogens may play a role in the reproductive development and endocrine function in the stallion. For decades it has been thought that testosterone is the local regulator of sperm production, but it has been recently demonstrated that factors such as growth factors, inhibin, activin and estrogen may play an important role in modulating spermatogenesis (Spiteri-Grach and Wieschlag 1993). Only recently has estrogen been implicated as a modulator of testicular function. Findings have included the following 1) the presence of P450 aromatase activity in germ cells (Nitta et al. 1993), 2) the presence of estrogen receptors in the epididymis (Ergii et al. 1997), 3) alteration of spermatogenesis after targeted disruption of the estrogen receptor gene (Eddy et al. 1996), 4) and alteration in spermatogenesis after in vivo treatment with an aromatase inhibitor (Shetty, et al. 1998). Letrozole has been reported to significantly inhibit estrogen production in the rat, human, primate, dog (Walker et al. 1994, Trunet et al. 1993) and sperm production in the primate (Shetty et al. 1998). Since the stallion produces so much estrogen, it was of interest to determine if changing the ratio of T to E would affect sperm production. Therefore, the effects of an aromatase enzyme inhibitor on 1) the ratio of T to E 2) hormone profiles and 3) sperm production were investigated. Materials and Methods Preliminary Study to Determine Effective Dose Nine normal, adult stallions were given a single oral dose of Letrozole [CGS 20 267; Novartis Pharmaceuticals AG, Basil, Switzerland (0.01mg/kg BW, n=2; 0.1mg/kg BW, n=2; 1mg/kg BW, n=2)] or vehicle (n=3) to determine the dosage and regime that would cause a 50% or greater decrease in estrogen production during the breeding season. On the day of Letrozole treatment jugular blood samples were collected at –30 min, 0, 30 min, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, and 24 hr. Samples were then taken at 32 hr, 72 hr, 4 days, 5 days, and 6 days to determine the duration of time that Letrozole induced a hormone response. Plasma was stored at –20°C until analyzed by radioimmunoassay (RIA) for LH, FSH, T, and E2. Semen was collected three weeks prior to treatment and three days post-treamtment. A dose between 0.1mg/kg 1.0 mg/kg BW resulted in a 50% decrease in E2 and a 3-fold increase in T within 6-8 hours after treatment. Hormone levels returned to baseline between 48-72 hours. There was a reported increase in the total number of progressively motile, morphologically normal spermatozoa per ejaculate as observed three days post-treatment. Long-Term Treatment Ten normal, adult stallions were given Letrozole (0.55mg/kg, n=7) or vehicle (n=3) orally every other day for 60 days during the breeding season (May 31-July 31, 1999). Letrozole was mixed with molasses so that treatment horses (average weight: 500 kg) received 5 mls of molasses mixture (275 mg of Letrozole per dose) via syringe, every other day between 9-10 am. Jugular blood samples (10 mls in heparinized tubes) were collected three times a week on Monday, Wednesday, and Friday, 30 days prior to treatment up to 60 days after treatment. Plasma was stored at –20°C until analyzed by RIA for LH, FSH, T, E2, EC and inhibin. Biopsy samples were obtained from the right testis of each stallion 30 days prior to and during the last week of treatment. Samples were snap frozen in liquid nitrogen and stored at –70 °C until analyzed. The tissue was homogenized into a cytosol and membrane fraction. The aromatase enzyme activity of the membrane fraction was determined by the tritiated water assay technique (Conley et al. 1996). Testicular concentrations of T, E2, and inhibin were determined by RIA using extracts of the homogenized tissue samples. Inter-assay coefficient of variation (CV) for all hormone assays were < 9.9% and intra-assay CV was < 16.4%. Semen was collected from all stallions twice one hour apart every other week to determine changes in the total number of progressively motile sperm per ejaculate. The total volume of the gel fraction and gel-free fraction were determined at the time of collection. Seminal pH was measured within 5 minutes of collection. One ml of semen extended in pre-warmed EZ-Mixin and 100ul of gel-free semen aliquotted into 900ul of Phosphate Buffered Formalin were placed on ice in a thermal insulated box and transferred to the veterinary andrology laboratory. Five mls of raw semen was maintained at 37°C and extended semen was kept at 25°C. Computer-Aided Sperm Motility Analysis (CASA) using Hamilton-Thorne Research (HTR)-CEROS was performed on the extended sample. Sperm concentrations were determined by manual count on a hemocytometer using the formalin diluted sample. Sperm motility and concentration were determined from the second of the two collections done. In addition to the bimontly collections, stallions were collected for seven consecutive days to determine daily sperm output (DSO) prior to, during, and post treatment. Gel volume, gel-free volume, and pH were measured immediately after collection by standardized methods, for the first five days of collection. Raw semen (0.25 mls) was diluted with 4.75 mls of pre-warmed EZ-Mixin semen extender and then analyzed using a light microscope. Formalin (7.6 mls) was mixed with 0.4 mls of raw semen to determine concentration using a hemocytometer. During the last two days of collection, gel volume, gel-free volume and pH were determined within five minutes of collection and then raw semen was extended in formalin and EZ-Mixin and transported as previously described. Concentration was counted using a hemacytometer and progressive motility was analyzed by CASA-HTR. Concentration, progressive motility (pm), and volume values were averaged from the last two days of collection and the product (conc x pm x vol) was used to determine total number of progressively motile sperm per ejaculate. Statistical Analysis Hormone data was averaged + SEM across weeks prior to (period 1), during (period 2), and after treatment (period 3) in the control group and Letrozole group. Repeated measures ANOVA and T-tests were used to determine level of significance between treatment and control values. Results and Discussion Long-term treatment with Letrozole caused an increase in plasma testosterone and a decrease in estrogen and inhibin production. When compared to control stallions, treated stallions showed a 3,6 fold increase (p < 0.05) in plasma T levels from period 1 to 2. Treated stallions, as compared to control stallions, showed a 9.1-fold, 5.0-fold and 7.9-fold decrease (p < 0.05) in levels of inhibin, LH, and EC, respectively from period 1 to 2. Changes in circulating levels of FSH and E2 were not significantly different from period 1 to 2. The aromatase activity of treated stallions was significantly suppressed (963 + 154 to 326 + 173 pmol/mg/2hr) as compared to control stallion (1141 + 10 to 897 + 116 pmol/mg/2hr) during treatment (p < 0.05). Letrozole treatment was effective indecreasing the ratio of T: EC by 8.4-fold (p < 0.05) between periods 1 and 2, while there was no significant change in the ratio of T: E2. Efficacy of oral treatment of the aromatase inhibitor was observed by measuring the average aromatase enzyme activity of testicular tissue obtained prior to and at the end of treatment. The aromatase activity of treated stallions (Figure 2) was significantly suppressed (963 + 154 to 326 + 173 pmol/mg/2hr) as compared to control stallions (1141 + 10 to 897 + 116 pmol/mg/2hr) during treatment (p < 0.05). Mean testicular values of T, E2, and inhibin are summarized in Table 1. There were no significant changes found between the control and treated group of stallions although absolute levels of each hormone appeared to decrease from pre to post treatment measurements. Seminal parameters, such as concentration, progressive motility, and volume were compared between control and treatment stallions for DSO and bimonthly collections. There were no significant differences in progressive motility or concentration among stallions. Treatment stallions showed a slight increase in the gel-free fraction of the ejaculate, but this was not proven to be significant. Total progressively motile sperm per ejaculate did not differ between groups (Figure 3 a,b). Conclusion A decrease in aromatase enzyme activity altered the T:E ratio in treatment stallions, proving that Letrozole is a potent aromatase inhibitor in stallions. Letrozole treatment significantly reduced levels of plasma EC and elevated plasma T without affecting sperm production. As seen in treated stallions, a decrease in inhibin production without a change in plasma FSH levels may suggest that T or E may modulate inhibin production locally. Table 1: Mean concentration of testosterone, estradiol, and inhibin in testicular tissue expressed as ng/mg + SEM of protein (normalized by individual sample protein concentration). Differences between control and treated stallions were not significant.
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