Effect of oral tamoxifen on semen characteristics and serum hormone profile in male bonnet monkeys

Contraception 67 (2003) 409 – 413

Original research article

Effect of oral tamoxifen on semen characteristics and serum hormone profile in male bonnet monkeys M.K. Gill-Sharma*, S. D’Souza, P. Parte, N. Balasinor, J. Choudhuri, D.D. Majramkar, M. Aleem, H.S. Juneja Department of Neuroendocrinology, National Institute for Research in Reproductive Health (ICMR), J.M. Street, Parel, Mumbai 400 012, India Received 2 December 2002; received in revised form 8 January 2003; accepted 13 January 2003

Abstract The effects of oral tamoxifen were studied at a dose of 0.4 mg/kg per day, on the serum hormones and semen parameters in adult male bonnet monkeys, for a period of 90 days. Honey was used as vehicle. Monkeys were treated with honey for 30 days, followed by tamoxifen from Day 30 –120 (90 days). Thereafter the treatment was withdrawn until Day 150 of schedule. Blood samples were drawn at 12 and 24 clock hours at monthly intervals for the analysis of luteinizing hormone, follicle-stimulating hormone and testosterone. Semen samples were also collected for analysis once a month, from Day 0 –150 of exposure. Tamoxifen treatment produced a transient but significant increase in circulating gonadotropins, at Day 90 of treatment schedule, corresponding to 60 days of treatment. Whilst serum testosterone levels were normal throughout treatment period, an increase was observed after 30 days of drug withdrawal. No effect of oral tamoxifen was evident on semen parameters, viz., volume, counts, morphology and motility. However, throughout the exposure period to honey, a significant increase was observed in sperm counts without any effect on testosterone levels. The present study suggests that oral tamoxifen has a transient antiestrogenic effect on the serum hormones and no effect on semen parameters of adult nonhuman primate males. It is concluded that bioefficacy of oral tamoxifen may have been reduced due to hepatic metabolism. © 2003 Elsevier Inc. All rights reserved. Keywords: Antiestrogen; Male pill; Nonhuman primate; Semenology; Gonadotropins

1. Introduction Tamoxifen, a nonsteroidal antiestrogen, produces estrogenic/antiestrogenic effects, depending upon sex, species, dose and route of administration [1]. Besides these, it also produces effects which are cell promoter as well as receptor specific [2– 4]. The complexity of tamoxifen effects are not only dependent upon the type of receptor, nature of regulated gene but also on the type of sex-specific metabolites formed in the liver of each species [5–7]. Tamoxifen has been shown to produce antifertility effects in the male rats at a dose of 0.4 mg/kg per day [8]. In the male rat, the effects observed were predominantly estrogenic, characterized by luteinizing hormone (LH) and testosterone suppression whereas absence of effect on follicle-stimulating hormone (FSH) appeared to reflect its antiestrogenicity. The tamoxifen-induced disorganization of the cytoarchitecture * Corresponding author. Tel.: ⫹91-24132111-2-6-7; fax: ⫹91-24139412. E-mail address: [email protected] (M.K. Gill-Sharma). 0010-7824/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0010-7824(03)00018-0

and increase in intercellular spaces, observed within the seminiferous tubules in rat, have yet to be resolved in terms of the regulatory hormone affected, as sloughing has been ascribed to FSH deprivation, aromatase inhibitors and antiestrogens in diverse experimental paradigms [9 –12]. The tamoxifen-induced effects on sperm motility from forward progressive to yawing, have been partly ascribed to direct inhibition of sperm dynein ATPase mediated by sperm estrogen receptors [13]. The tamoxifen-induced paternal effects underlying the reported loss of fertilized embryos are still being researched [14,15]. Some of these effects, notably on morphology, motility and fertility, have also been reported in male bonnet monkey, when the drug was chronically administered via Alzet pump at a dose of 0.05 mg/kg per day for 90 days [16]. The most interesting feature of the antifertility effects of tamoxifen is the absence of effects on sperm counts in both species, perhaps indicating subtle effects at the level of germ cell maturation either during germ cell differentiation or epididymal maturation [6,16]. Based on the available data, the present study was there-

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fore undertaken to assess the antifertility effects of oral tamoxifen in male bonnet monkey at the effective dose of 0.4 mg/kg per day.

2. Materials and methods 2.1. Animals Adult male bonnet monkeys were housed in cages under standard conditions of temperature 22–23°C, 50 –55% humidity and lighting cycle of 14 h light:10 h dark. The details of colony management have already been published [17]. The animals were checked regularly by an in-house veterinarian. The experiment was conducted in the months of August to March. The experiment was repeated in the subsequent year with the same set of animals. All experiments were carried out with the prior approval of the Ethics Committee of the Institute. 2.2. Drugs Tamoxifen citrate tablets containing 10 mg pure tamoxifen were obtained from Lyka (Mumbai, India). Tamoxifen tablets were crushed, a dose of 0.4 mg/kg per day mixed with 2 mL of honey and daily fed to each monkey, between 1000 –1200 h. Tamoxifen is not soluble in aqueous solvents and forms a paste in honey. Honey was used as vehicle to ensure compliance of the primates to orally administered tamoxifen. 2.3. Experimental protocol Prior to tamoxifen treatment, the monkeys were fed 2 mL of honey for a period of 30 days to account for the effects of honey, if any. Tamoxifen treatment was then started for a total period of 90 days. The monkeys were fed 0.4 mg/kg per day tamoxifen mixed in 2 mL of honey, once daily for a period of 90 days. At the end of the treatment period, the tamoxifen and honey treatment was withdrawn in all six animals for 30 days during reversal, for comparison. 2.3.1. Blood sampling Blood was collected by venipuncture from the forearm between 1000 –1200 h and 2300 –2400 h keeping in view the diurnal variations of testosterone [17,18]. At the outset, samples were collected on Day 0 and thereafter at 30-day intervals until Day 150. Blood was allowed to clot at room temperature and sera separated by centrifugation at 1000 rpm for 10 min and stored at ⫺20°C for hormone estimations. 2.3.2. Hormone assays Sera were assayed for LH and FSH by double antibody radioimmunoassays using radioimmunoassay kits for cynomolgus monkey LH and FSH (National Institute of Diabetes

and Digestive and Kidney Diseases, National Institutes of Health) according to the protocol used for human serum samples [19]. 2.3.2.1. Gonadotropin assay protocol. Aliquots of sera, containing unlabelled antigens, taken from the experimental animals were put in assay tubes containing 100 ␮L (30,000 cpm) of NIDDK I125-labeled antigens (diluted in 0.5% PBSA) and 200 ␮L of NIDDK LH or FSH antibodies [diluted in normal rabbit serum (NRS) and phosphate-buffered saline (PBS) in a ratio of 1:400]. The standard curves for LH (rec-moLH-RP-1) and FSH (rec-moFSH-RP-1) cynomolgus-monkey reference preparations provided by NIDDK (prepared by double dilution in 0.5% PBSA), were set up in the range of 0.01–12.5 ng. Standard curve assay tubes contained 100 ␮L aliquots of each standard antigen dilution, 200 ␮L of PBS, 100 ␮L of labeled antigen and 200 ␮L of primary antibody (diluted in PBS-NRS 1:400). Total antigen binding tubes were set up containing 100 ␮L of labeled antigen (diluted in 0.5% PBSA), 200 ␮L of PBS and 200 ␮L of primary antibodies (diluted in PBS-NRS 1:400). Nonspecific binding was accounted for by setting up separate tubes containing 100 ␮L of labeled hormones (diluted in 0.5% PBSA), 100 ␮L of 0.5% PBSA, 200 ␮L of PBS and 200 ␮L of PBS-NRS (1:400). Labeled and unlabelled antigens were allowed to compete for sites on the respective antibodies for 24 h at 30°C. The antigen-antibody complexes formed were precipitated by incubation with 200 ␮L of anti-rabbit gamma globulin (diluted in PBS) for 24 h at 30°C followed by 24 h at 4°C. The complexes were separated by cold centrifugation (4°C) at 2500 ⫻ g for 30 min and air-dried. The precipitated I125-labeled LH or FSH bound to their respective antibodies were counted in a gamma counter. The sensitivities for LH, FSH assays were 0.39 ng, 0.19 ng, respectively. The inter- and intra-assay coefficients of variation were 17% and 9% for LH, 3% and 4% for FSH, respectively. All assays were run in duplicates. 2.3.2.2. Testosterone assay protocol. Testosterone was estimated in the sera as described by Juneja et al. [19]. Briefly, testosterone was extracted twice from 500 ␮L of serum with 10 volumes of purified ether. Same volume of 0.01 M PBS buffer containing 0.1% gelatin (pH 7.4) was also extracted simultaneously with ether to construct buffer blanks. The ether extracts were allowed to evaporate overnight. The dried steroid residues were reconstituted in 1.5 mL of 0.1% PBS gelatin (PBSG) buffer (pH 7.4) and solubilized by heating in a water bath at 55°C for 1 h. Aliquots of 100 ␮L and 200 ␮L were assayed in duplicate for testosterone. The assay mixture contained 100 ␮L (10,000 cpm) of 3H-testosterone (1,2,6,7-3H testosterone, Amersham, UK), 100 ␮L of testosterone antibodies (raised in-house), and unlabeled testosterone (500 ␮L). The standard curve for testosterone (Testosterone; Sigma, St. Louis, MO, USA) was set up in the range of 3.9 –1000 pg. The standard curve assay tubes contained 500 ␮L of each standard antigen dilution, 100 ␮L

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411

Table 1 Temporal effects of tamoxifen on serum hormones in male bonnet monkey Affected parameters

Duration of treatment (days) 0

30

60

90

120

150

LH12 (ng/mL) FSH12 (ng/mL) T12 (ng/mL) T24 (ng/mL)

2.28 ⫾ 0.35 0.66 ⫾ 0.17 8.33 ⫾ 0.45 17.11 ⫾ 2.11

2.27 ⫾ 0.43 1.28 ⫾ 0.12 11.0 ⫾ 1.15 18.35 ⫾ 0.69

3.05 ⫾ 0.46 1.06 ⫾ 0.09 10.44 ⫾ 2.45 22.97 ⫾ 1.85

11.18 ⫾ 0.77* 5.90 ⫾ 0.24* 9.09 ⫾ 0.78 23.33 ⫾ 2.17

3.84 ⫾ 1.26 1.80 ⫾ 0.49 7.39 ⫾ 2.09 18.61 ⫾ 1.49

4.49 ⫾ 0.55 2.05 ⫾ 0.34 18.30 ⫾ 4.65* 32.68 ⫾ 3.56*

Values represent mean ⫾ SE of six male monkeys. Day 0 group represents control values, Day 30 group represents honey controls, Days 60 –120 represent values after tamoxifen treatment, Day 150 represents values after withdrawal of tamoxifen and honey treatment. “12, 24” subscripts refer to clock hours. * Significance at p ⱕ 0.05 as compared to all other days.

(10,000 cpm) of 3H-antigens and 100 ␮L of testosterone antibodies (both diluted in 0.1% PBSG). The assay mixture of total binding tubes contained 500 ␮L of PBSG, 100 ␮L (10,000 cpm) of 3H-testosterone and 100 ␮L of testosterone antibodies (both diluted in 0.1% PBSG). Nonspecific binding was accounted for by setting up tubes containing 600 ␮L of PBSG, 100 ␮L (10,000 cpm) of 3H-testosterone. The assay mixtures were incubated overnight at 4°C. The assay mixtures were then transferred to an ice bath where the free antigens were removed by adsorption with 500 ␮L of cold dextran-coated charcoal within 15 min. The soluble antigenantibody complexes were separated from charcoal-adsorbed free antigens by cold centrifugation (4°C) at 2500 ⫻ g for 10 min. The supernatants containing the bound 3H- testosterone were solubilized in a toluene-based scintillation fluid and counted in a beta counter. The sensitivity of the testosterone assay was 15.6 pg. Inter- and intra-assay coefficients of variation were 11% and 5.5%, respectively. 2.3.3. Semen analysis Semen samples were obtained from all monkeys concomitant with serum on Day 0 and at 30-day intervals thereafter, by electroejaculation at a frequency of 6 – 8 Hz and 10 –15 V. Semen was promptly processed for analysis of sperm motility, counts, morphology and semen volume according to the standard World Health Organization protocols [20]. Briefly, monkey ejaculates containing the coagulates were collected in sterile, graduated centrifuge tubes and incubated at 37°C in a water bath to permit liquefaction. The volumes of the liquefied ejaculates were recorded and expressed in milliliters. Aliquots of liquefied semen, diluted in Biggers, Whitten, and Whittingham medium in a proportion of 1:50 were incubated in serum tubes for 30 min at 37°C. Sperm morphology was assessed after staining sperm with PAP staining technique. Semen aliquots of 10 ␮L were then observed under bright field optics to assess sperm motility. Semen was further diluted 10⫻ for ascertaining sperm counts in the Neubaeur chamber. 2.3.4. Statistical analysis The data on hormones and semen parameters were analyzed by one-way analysis of variance. Significant differences between treatments, at Days 0, 30, 60, 90, 120 and

150 of treatment schedule in the six monkeys were computed by Duncan’s Multiple Range test. The level of significance was set at p 聿 0.05.

3. Results 3.1. Effects of tamoxifen on circulating gonadotropins and testosterone (Table 1) 3.1.1. Days 0 –30 The values of LH and FSH in the serum did not differ significantly between Days 0 and 30 at 12 h when honey was fed as vehicle. Testosterone (T) values in the serum also did not differ between Days 0 and 30 at 12 and 24 h. 3.1.2. Days 60 –120 Tamoxifen treatment induced a transient but significant increase in serum levels of LH and FSH on Day 60 of treatment, corresponding to 90 days of treatment schedule. The increase in gonadotropins observed at Day 60 of tamoxifen treatment was not sustained thereafter. However, tamoxifen treatment did not affect serum T levels at Days 60 –90 of treatment corresponding to Days 60 –120 of treatment schedule. 3.1.3. Day 150 The levels of LH and FSH observed after withdrawal of tamoxifen and honey treatment for 30 days, corresponding to Day 150 of treatment schedule, returned to those seen at Days 0 or 30. However, serum T concentrations at 12 and 24 h were significantly elevated at the end of treatment as compared to Days 0, 30, 60, 90 and 120 of schedule. 3.2. Effect of tamoxifen on semen parameters (Table 2) 3.2.1. Day 0 –30 Honey treatment produced no effect whatsoever on semen volume, sperm motility or morphology during the treatment period. However, a significant increase in sperm counts was evident on Day 30 of treatment schedule during which the animal was fed only honey as vehicle.

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Table 2 Temporal effects of tamoxifen on semen parameters in male bonnet monkey Affected parameters

Semen volume (mL) Sperm counts (mil/mL) Sperm motility (%) Sperm morphology (%)

Duration of treatment (days) 0

30

60

90

120

150

0.87 ⫾ 0.5 230 ⫾ 43.6 32.83 ⫾ 6.8 38.2 ⫾ 6.95

1.05 ⫾ 0.68 1100 ⫾ 224.3* 47.33 ⫾ 3.35 38.0 ⫾ 2.03

0.77 ⫾ 0.28 972 ⫾ 157.4* 48.00 ⫾ 55.35 40.3 ⫾ 2.46

0.45 ⫾ 0.2 1060 ⫾ 99* 44.8 ⫾ 5.4 36.7 ⫾ 4.5

0.64 ⫾ 0.26 773 ⫾ 99* 34.00 ⫾ 7.12 32.0 ⫾ 3.31

0.84 ⫾ 0.3 347 ⫾ 81 41 ⫾ 2.7 41 ⫾ 2.7

Values represent mean ⫾ SE of six male monkeys. Day 0 represents control group values, Day 30 represents honey controls, Days 60 –120 represent values after tamoxifen and honey treatments, Day 150 represents reversal values after withdrawal of tamoxifen and honey treatment. Morphology represents % of normal sperm. * Significance at p ⱕ 0.05 vs. all other days.

3.2.2. Day 60 –120 Tamoxifen fed in honey as vehicle did not produce any significant changes in semen volume, sperm motility or sperm morphology. However, a significant increase in sperm counts was continuously seen on Days 60 –120 of schedule as compared to Day 0 but not when compared to Day 30 (honey control). 3.2.3. Day 150 Interestingly, no effect was seen on sperm counts after 30 days of withdrawal of both honey and tamoxifen, on Day 150 of schedule when compared to untreated control values on Day 0. Significant reduction in counts was, however, evident when compared to honey control values on Day 30 of schedule.

4. Discussion The antiestrogen, tamoxifen, has been conventionally used to treat breast cancer, gynecomastia and male infertility by blocking tissue estrogen receptors [21]. Recently, oral or subcutaneous tamoxifen treatment has also been reported to induce infertility, without reducing sperm counts, in rat and nonhuman primate [6,16]. However, the mechanism through which tamoxifen reduces fertility in nonhuman primate remains to be deduced. Direct effect of the antiestrogen on the spermatozoa could be one of the mechanisms [22,23]. Both estrogen receptors alpha and beta are expressed in the male reproductive organs of nonhuman primate but their relative importance to male fertility awaits elucidation [24,25]. Subcutaneous tamoxifen treatment has been shown to produce sperm abnormalities and induce infertility in male bonnet monkey without reducing sperm counts [16]. In the present study, oral tamoxifen treatment, at the dose of 0.4 mg/kg per day, failed to produce an increase in sperm abnormalities. Interestingly, honey used as a vehicle, but not tamoxifen treatment, appeared to induce a significant and independent increase in sperm counts throughout the period of experiment. Honey appeared to have some factors which might have led to the observed increase in sperm

counts [26]. On the other hand, tamoxifen, administered in saline, is known to maintain and not increase sperm counts in normal rat or nonhuman primate with improvement reported only in infertile patients. Tamoxifen treatment produced a significant, though transient, increase in serum LH and FSH but not testosterone, after 60 days of treatment without a concomitant increase in diurnal testosterone levels which was elevated after 30 days of reversal, on Day 150 of schedule. No effects of treatment with honey or tamoxifen were seen on the semen volume, sperm morphology or motility. The absence of expected effects of oral tamoxifen treatment on semen parameters in particular, led to the conclusion that the drug may be sparingly effective either due to inadequate absorption through the hepatic portal circulation or inactivation to ineffective metabolites in the liver. Two metabolites of tamoxifen have recently been shown to be ineffective in inducing infertility in male rat [6]. Since the drug is known to be given by the oral route for the treatment of breast cancer, gynecomastia and male infertility, it was reasonable to assume that it would be absorbed in the nonhuman primates also [21]. However, there are reports that argue against the first assumption, which maintain that the drug is sparingly absorbed through the oral route in nonhuman primates [27,28]. Alternatively, the species- and sex-specific metabolism of tamoxifen reported in literature favors the second assumption [5]. The present study also supports the former argument since oral tamoxifen treatment did induce a transient but significant increase in circulating gonadotropins though not testosterone. Since oral tamoxifen treatment also produced a sufficiently strong, though transient, antiestrogenic effect at the hypothalamicpituitary level to increase gonadotropins, reduced bioavailability may have been due to hepatic metabolism rather than malabsorption. Reduced bioavailability may, however, have led to the absence of expected effects on sperm morphology and motility reported in primate males after administration of tamoxifen via Alzet pump. A recent report suggests that honey reduces the bioavailability of orally administered diltiazem [26]. Whether tamoxifen administered in honey may also have reduced its bioavailability is a moot point. Whatever the reason for the observed lack of effects, the present study suggests that it may not be feasible to use this

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drug as a male contraceptive pill, administered daily and at the concentrations recommended for treatment of human cancer or infertility [21]. Follow-up studies to ascertain the effect of honey, if any, on the pharmacokinetics of tamoxifen administered per os, at much higher doses than that used in the present study, would be useful in understanding the magnitude and true import of effects, if any, of honey on the bioavailability of tamoxifen and consequently on sperm morphology and motility of the male primate. The caveat here is that future investigations pertaining to mechanism underlying the antifertility effects of tamoxifen or other pure antiestrogens in nonhuman primates, be either limited to subcutaneous route or executed in tandem with oral route (after ascertaining the effects of vehicles on bioavailability) [29].

[10]

[11]

[12]

[13]

[14]

[15]

Acknowledgments The authors would like to acknowledge the gift of reagents for radioimmunoassays of cynomolgus monkey gonadotropins by National Institutes of Health, Bethesda, MD, USA. The kind gift of testosterone antiserum by Dr. M.I. Khatkhatay is acknowledged. The statistical assistance of Mr. D. Balaiah (Assistant Director) is acknowledged. The technical assistance of Mr. H.G. Pawar, S. Mandavkar, A. Hatle, Chavan in handling the monkeys and taking blood and semen samples is greatly appreciated. Supported by Indian Council of Medical Research (ICMR), New Delhi, India.

[16]

[17]

[18]

[19]

[20]

[21]

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