Effect of intermittent treatment with tamoxifen on reproduction in male rats

M. K. Gill-Sharma, N. Balasinor, P. Parte

Department of Neuroendocrinology, Institute for Research in Reproduction (ICMR), Parel, Mumbai  400012, India

Asian J Androl  2001 Jun; 3:  115-119

Keywords: tamoxifen; estrogen; intermittent treatment; fertility

Abstract

Aim: To identify the antifertility effect of intermittent oral administration of tamoxifen in male rat. Methods: Tamoxifen was administered orally at a dose of 0.4 mgkg^-1d^-1 with an intermittent regime for 120 days. Treated and control rats were mated with cycling female rats on days 60, 90 and 120 of treatment. The mated males were sacrificed and the weights of reproductive organs were recorded, and the serum levels of LH, FSH, testosterone and estradiol estimated by radioimmunoassay. In the female rats, the numbers of implantation sites, corpora lutea, and numbers of normal and resorbed foetuses were recorded on d 21 of gestation. The potency, fecundity, fertility index, litter size and post-implantation loss were then calculated. Results: The fecundity of male rats was completely suppressed by tamoxifen while the potency was maintained at the control level. The fertility index was significantly decreased. No viable litters were sired. Post implantation loss, indicative of nonviable embryos, was observed but was not significantly increased above the control level. The weights of the testes, epididymides, ventral prostate and seminal vesicles were significantly reduced. The blood LH and testosterone levels were significantly decreased, but not FSH and estradiol. Conclusion: Intermittent oral tamoxifen administration completely suppressed the fecundity of adult male rats with reserved potency.

1 Introduction

2 Materials and methods

2.1 Animals and grouping

Adult male and virgin female rats of Holtzman strain, 75-90 days of age, were housed at a temperature of 22-23, humidity of 50-55% and lighting cycle of 14 h light/10 h dark.  Commercial rat pellets and tap water were available ad libitum. Female rats with normal estrus cycles were used in mating studies. Male rats were divided at random to 4 groups of 6 rats each: the continuous schedule group, the intermittent schedule group, and two relevant control groups.

2.1.1 Continuous  group

Male rats were admnistered tamoxifen citrate (Lyka, Bombay) uniformly suspended in water 0.4 mgkg^-1d^-1 per os via a rat feeding tube for 90 days. Rats were mated on d 60 and d 90 (1 male2 females).  The presence of sperm in the vaginal smear or persistent diestrous was taken as the proof of mating.   Mated female rats were sacrificed on d 21 of pregnancy and the litter size and the numbers of implantation sites, resorbed fetuses and corpora lutea were recorded.  Male rats were sacrificed by decapitation on d 60 and d 90.  Blood was allowed to clot at 4 overnight.  Serum was collected by centrifugation at 800g for 20 min and stored at -30 until radioimmunoassay of LH, FSH, testosterone and estradiol.  Testes and accessory sex organs were collected and weighed on an electronic balance.

2.1.2 Intermittent group

Male rats were daily administered 0.4 mgkg^-1d^-1 tamoxifen citrate per os as described in the previous section, for 60 days. After 10 days of rest, tamoxifen was re-administered for two 20-day periods intervened with another 10-day rest. At the end of the course, the treated male rats were mated (1 male2 females). After that the rats were treated as described in the previous section.

2.1.3 Continuous Control

The vacant vehicle was administered for 60 days. Other treatments were similar to the Continuous group.

2.1.4 Intermittent Control

The vacant vehicle was administered at a similar schedule as the Intermittent group. Other treatments were also similar to the Intermittent group.

2.2 Fertility observation

The following parameters were determined: potency, fecundity, fertility index, litter size and post implantation loss as described earlier^[6].

2.2.1 Potency

The ability of the male rat to mate was expressed as the ratio of female rats mated (inseminated or in persistent dioestrus) to the number of female rats exposed for mating100.

2.2.2 Fertility Index

Expressed as the ratio of the number of implantation sites to the number of corpora lutea.

2.2.3 Fecundity

Expressed as the ratio of the number of male rats siring at least one viable fetus to the total number of male rats exposed for mating100.

2.2.4 Post Implantation loss^[15]

Expressed as the ratio of the difference between the implantation sites and viable fetuses to the implantation sites per rat100.

2.3 Hormonal assay

LH and FSH were assayed according to Balasinor et al^[16].  The standard curve for LH (NIADDK-Rat-LH-RP-2) and FSH (NIADDK-Rat-FSH-RP-2) ranged from 10 pg to 12.5 ng per assay tube.  The inter- and intra-assay coefficient of variation were 9% and 6% for LH and 10% and 6% for FSH, respectively. Testosterone and estradiol were assayed as described by Juneja et al^[2].  The standard curves ranged 3.9-500 pg for testosterone and 5-200 pg for estradiol.  Inter- and intra-assay coefficients of variation were 11% and 11.5% for testosterone and 10% and 6% for estradiol, respectively.

2.4 Statistical analysis 

Hormone concentrations and tissue weights were subjected to analysis of variance.  Significance in differences between groups were analyzed using Duncan's multiple range test. Data relating to potency, fecundity, fertility index, litter size and post implantation loss were subjected to non-parametric Kruskal Wallis ANOVA and Mann Whitney UWilcoxon test. Level of significance was set at P<0.05.

3 Results

3.1 Effect on potency

The potency of the male rats chronically treated with tamoxifen over 90 days was significantly lower than that of the age matched controls.  Incorporation of 10-day non-treatment intervals subsequent to 60 day treatment was effective in preventing the decline in potency.  A discontinuous feeding schedule adopted after the rats are rendered sterile after 60 days of chronic treatment restored the potency to control values (Table 1).

Table 1.  Effect of tamoxifen on fertility of male rats. MeanSEM. ^bP<0.05, compared with controls.

3.2 Effect on fecundity  

The fecundity of the male rats chronically treated with tamoxifen over 60 days was reduced to zero.  An insignificant rise in fecundity was observed when the chronic treatment was prolonged to 90 days.  Incorporation of 10-day non-treatment intervals subsequent to 60-day chronic treatment could suppress the spurt in fecundity.  Although, the number of viable fetuses was drastically reduced, a marginal increase in the fertility index observed in treated groups reflected an insignificant shift to the production of non-viable litters as can be inferred from the PIL (post implantation loss) values (Table 1).

3.3 Effect on hormonal profile   

The plasma LH and testosterone levels were significantly reduced after 90 days of chronic and 120 days of discontinuous treatment with tamoxifen.  No significant effect was seen on estradiol after either of the two schedules.  However, a significant increase in plasma FSH was observed after 90 days of chronic tamoxifen treatment (Table 2).

Table 2.  Effect of tamoxifen on serum hormone profile in male rats. MeanSEM. ^cP<0.01, compared with controls.

3.4 Effect on tissue weights    

Table 3.  Effect of tamoxifen on organ weights in male rats. MeanSEM. ^cP<0.01, compared with controls.

4 Discussion

In the present study, it was shown that in rats treated with tamoxifen for 90 days continuously, there were a tendency of reduction in potency and a brief but significant spurt in fecundity. However, when the drug was administered in a discontinuous manner, the decline in potency and spurt in fecundity were no longer observed. Testosterone deprivation is known to impair the reproductive behaviourand fertilizing ability of the male^[17,18]. However, low levels of testosterone alone or in combination with physiological levels of estradiol do suffice to maintain the ability of the male rat to mate^[18-20].  The observed tendency of the decrease in potency may therefore not be the consequence of low testosterone levels. Since the effects of tamoxifen and its metabolites on reproductive behaviour are not so well documented, tentative parallels can be drawn from studies with other anti-estrogens. Based on studies with synthetic estrogens (RU-2858) and antiestrogens (MER 25) in gonadectomised rats of both sexes, a hypothesis was advanced for their mechanism of action pertaining to reproductive behaviour.  According to this hypothesis, an intact phenolic group confers estrogenicity and maintains lordosis whereas a substituted phenol confers antiestrogenecity and maintains mounting behaviour^[21].  Incidentally, tamoxifen, bearing a substituted phenol group, is metabolized in the rat to 4-hydroxy tamoxifen bearing an intact phenolic group. It can be speculated that tamoxifen and its hydroxylated metabolites have the potential to produce conflicting effects on the reproductive behaviour of male rats. Recent reports have also implicated synthetic compounds with inherent estrogenicity in the decline of male fertility^[22]. Tamoxifen and its metabolites may also have the potential to produce antagonistic effects on fertility. In that case any measure devised to minimize the levels of physiologically active metabolites could modify the tamoxifen effects, both on the reproductive behaviour and the fertility.

Since the incorporation of drug-free intervals improved potency without restoring testosterone levels to control value, it could also be inferred that tamoxifen metabolites interfered with mating behaviour at the level of receptors. 

In conclusion, our data suggest that incorporation of drug-free intervals limits the availability of the estrogenic and antiestrogenic metabolites of tamoxifen, with an improvement in the reproductive behaviour and reversal of fecundity in male rats chronically fed tamoxifen. The observed antifertility effects of oral tamoxifen reflect a cumulative effect of both estrogenic and antiestrogenic properties, perhaps involving the effects of metabolites.

Acknowledgements

References

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Correspondence to: Dr. M.K. Gill-Sharma, Institute for Research in Reproduction (ICMR), J.M.Street, Parel, Mumbai 400012, INDIA.
Tel: +91-22-413 2111 ext. 267   Fax: +91-22413 9412 
E-mail: dirirr@vsnl.com
Received 2000-08-22     Accepted 2000-11-21