H.S. Juneja

Antifertility effects of estradiol in adult male rats

The dose-related effects of estradiol 17-β at the doses 0.1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 1000 μg/kg/day were determined on sperm motility, potency, fertility parameters, serum levels of LH, FSH, PRL and testosterone, weights of testes and accessory sex organs, weights of pituitary and adrenal glands. The drug was administered daily via sc route for a period of 60 days. Dose-related effects on fertility parameters of the estradiol-treated male rats were ascertained by allowing them to mate with normal cycling female rats. Estradiol at 0.1 μg/kg/day dose significantly reduced sperm motility with no effects seen on potency or fecundity, serum LH, FSH, PRL or testosterone, weights of testes and accessory sex organs while pituitary weight increased. Estradiol at 10 μg/kg/day dose significantly reduced motility, serum LH, FSH, weights of testes and accessory sex organs, while pituitary weight increased with no effects seen on potency, fecundity, PRL or testosterone. Estradiol at 100–1000 μg/kg/day dose significantly reduced motility, potency and fecundity, serum LH, FSH and testosterone, weights of testes and accessory sex organs while serum PRL and the weights of pituitary and adrenal glands increased significantly. Histology of the testes revealed disorganization of the cytoarchitecture in the seminiferous tubules, vacuolation, absence of lumen and compartmentalization of spermatogenesis. Estradiol withdrawal, testosterone propionate at 100 μg/kg/day or antiestrogen (tamoxifen citrate) at 400 μg/kg/day prevented the histological changes. It is concluded that estradiol reduces sperm motility even at a low dose. Low doses (<10 μg/kg/ day) appear to maintain whilst high doses (>10 μg/kg/day) reversibly disrupt spermatogenesis. Prevention of disruption by testosterone or antiestrogen indicates crosstalk between androgen and estrogen receptors in Sertoli cells. Loss of potency and fecundity also suggests effects on crosstalk between these receptors in other male reproductive organs.

Estrogen receptor, calcium mobilization and rat sperm motility

Oral treatment with 0.4 mg/kg/day of tamoxifen citrate, an antiestrogen, has been reported to reduce the fertility of adult male rat, presumably through estrogen receptors expressed throughout the male reproductive tract. During the course of these studies, tamoxifen was observed to gradually alter the pattern of sperm motility in the cauda epididymides without reducing sperm counts. Studies were carried out to understand the mechanism involved in tamoxifen induced change in the sperm motility pattern. In order to study the direct effects of tamoxifen on motility, biochemical levels/activities of sperm calcium, cAMP, phosphodiesterase and dynein ATPase, normally implicated in sperm motility were studied In view of the fact that tamoxifen is a ligand of estrogen receptor, estrogen receptor alpha protein and transcript were localized on rat sperm membrane and the effect of tamoxifen studied. The present study demonstrated presence of estrogen receptor protein and mRNA in the rat sperm by immunofluorescence, western blotting and in situ hybridization respectively. Specificity of sperm estrogen receptors was confirmed by conventional binding studies using [3H]-estradiol. There was no effect of tamoxifen treatment on estrogen receptors in rat sperms. Biochemical analysis of the sperms from tamoxifen treated cauda epididymides revealed a significant increase in the levels of calcium and cAMP. A significant reduction was also apparent in the activity of dynein ATPase. Tamoxifen treatment did not alter phosphodiesterase activity. Estrogen receptors could be identified both in the control as well as tamoxifen treated rat sperms. It was concluded that tamoxifen treatment mobilized calcium from the intra- or extra-cellular pools with a concomitant increase in cAMP and presumably activation of PKA (protein kinase A). Tamoxifen altered the pattern of sperm motility through a calcium induced block in the activity of dynein ATPase, presumably through the activation of sperm phosphatase. The putative estrogen receptor mediated signal transduction pathway appears to be directly affected in the tamoxifen treated, sub-motile rat sperm.

Effect of paternal administration of an antiestrogen, tamoxifen on embryo development in rats

We have earlier reported that oral administration of tamoxifen causes a dose-dependent reduction in the fertility of adult male rats. The decrease in fertility was mainly due to an increase in pre-implantation loss without an effect on fertilizing ability. During the study, an increased incidence of post-implantation loss of conceptuses sired by tamoxifen-treated male rats was observed. A detailed study was undertaken to investigate dose-related changes in pre- and post-implantation loss and the stage(s) of development at which these losses occurred. The present study demonstrates that tamoxifen treatment produced few normal litters as well as significantly increased pre-implantation loss without affecting the rate of fertilization. Also a significant increase in the number of degenerating embryos at the 2-4-cell stage (days 1-2 of gestation), retrieved from the oviduct/uterus of females mated with tamoxifen-treated males was observed. Histology of the resorbed fetuses, in both control and treated groups, showed presence of trophoblast outgrowth indicative of early placenta formation, which normally occurs on days 8-9 of gestation. The present results suggest that pre-implantation loss occurred at the 2-4-cell stage and the post-implantation loss occurred around days 8-9 of gestation, i.e. around midgestation. The possible effects of paternal tamoxifen treatment on embryogenesis may be due to the reduction of androgens or by the blockage of the estrogen receptor by tamoxifen, thereby affecting germ cell maturation during spermatogenesis.

Effect of tamoxifen on sperm fertilising ability and preimplantation embryo development.

Recent evidences point to a role of estrogens in males. We have earlier reported that tamoxifen, a synthetic non-steroidal antiestrogen, when administered to adult male rats, in the dose range of 0.04-0.4 mg/kg per day, reduced fertility. The reduced fertility was measured in terms of fertility index (a measure of the efficiency of the ovulated ovas to fertilise and implant), fecundity (siring ability) and litter size. The present study was done to investigate whether the reduction in fertility index was due to reduction in fertilising ability or increase in pre-implantation embryo loss. Also a dose related effect of tamoxifen from 0.02 mg to 2 mg/kg per day on the fertility of the male rats was studied. To study the fertilising ability, control and tamoxifen (0.4 mg/kg per day, the most effective dose) treated adult male rats were mated with normal cycling females and the females sacrificed at day 0-4 of gestation. Eggs fertilised/unfertilised were flushed from the oviduct/uterus and the number and types of eggs were noted. The index of fertilisation, a measure of the fertilising ability was determined. The studies demonstrate that the reduction in fertility is not due to decreased fertilising ability but because of the increased pre-implantation embryo loss as evident from an increase in number of abnormal eggs in the treated group with no change in index of fertilisation. A dose related decrease in fertility was observed. The present study suggests that tamoxifen at 0.02-2-mg dose is predominantly estrogenic in males and paternal factor/s sensitive to tamoxifen is involved in embryogenesis.

Diurnal Variations and Temporal Coupling of Bioactive and Immunoactive Luteinizing Hormone, Prolactin, Testosterone and 17-Beta-Estradiol in Adult Men

Diurnal variations and temporal coupling in the circulating levels of immunoactive and bioactive luteinizing hormone (LH) and prolactin (PRL), testosterone (T) and 17-beta-estradiol (E2) in plasma of 6 healthy men (mean age 33 years) were studied. Each hormonal profile was analyzed for circadian amplitude, acrophase and nadir. Acrophases for immunoactive LH and T were coincident and ranged between clock hours 1 and 5. Acrophase for bioactive LH ranged between 9 and 12 h and was coincident with nadir for T. Acrophase for E2 ranged between 15 and 18 h and was coincident with nadir for immunoactive LH (15-17 h). Acrophase for bioactive PRL and immunoactive PRL ranged between 20-23 and 23-4 h, respectively. The circadian amplitude for T showed a negative correlation coefficient with circadian amplitude of bioactive LH (alpha = -0.86) and positive correlation coefficient with circadian amplitude of immunoactive LH (alpha = 0.94). It is inferred that immunoactive LH may be a sensor of T concentration while bioactive LH may be actually involved in the feedback regulation of T secretion. It is suggested that PRL may have a key role in the regulation of LH secretion.

Tamoxifen-induced light and electron microscopic changes in the rat testicular morphology and serum hormonal profile of reproductive hormones

The effects of oral administration of tamoxifen at doses of 40 and 200 micrograms/kg/day on testicular histology, testicular ultrastructure and serum hormonal profile were studied. The drug was administered to adult male rats over a period of 90 days and the effect was assessed at 10-day intervals. The morphometry, microscopic structures of the testis, including ultrastructure and daily sperm production rate, were evaluated. The hormone profiles of luteinizing hormone (LH), follice-stimulating hormone (FSH), testosterone, and estradiol were studied. The testes from treated animals showed disorganization of tubular elements with increased intercellular space. At day 50, the changes were extensive including presence of phagosomes. Morphometric studies showed a reduction in the spermatid and spermatozoan population (69.3%) with no changes in tubular diameter. The mean Leydig cell area was significantly lowered at day 50, at both doses. The daily sperm production rate was reduced as compared with controls. An array of degenerative changes were revealed by ultrastructural studies. The changes were extensive at day 50 at both doses. The characteristic features were lost in most of the cells with phagolysosomes becoming abundant. The cytoplasm of the cells was dense with poorly defined cytoplasmic organelles. Circulating LH levels were not modified at the 40 micrograms/kg/day dose but at 200 micrograms/kg/day, LH levels were significantly decreased. Initial transitory rise in FSH was seen with both doses. Both doses of tamoxifen decreased testosterone levels. Changes in the circulating estradiol levels were inconsistent, and no apparent relationship between dose and days of treatment was observed. Thus, this study supports our thesis of tamoxifen as a potential male contraceptive agent.

Effects of tamoxifen metabolites on fertility of male rat

The effects of chronic oral administration of tamoxifen citrate, at a dose of 0.4 mg/kg/day, were compared to those of subcutaneous (s.c) administration of tamoxifen citrate, 4-hydroxy tamoxifen, N-desmethyl tamoxifen and intermittent oral tamoxifen administration on the fertility of the male rat and its post reversal progeny. The fertility parameters of 120 day-treated male rat sires from all groups and post reversal male F1 progeny of tamoxifen-treated sires were assessed. Chronic tamoxifen treatment via oral or s.c. routes reduced the fertility of the male rat, weights of accessory sex glands, serum luteinizing hormone, and testosterone levels without altering potency or sperm counts. However, antifertility effects of s.c. treatment were comparatively more consistent than those of oral treatment. 4-hydroxy and N-desmethyl tamoxifen failed to produce significant antifertility effects in the male rat. The antifertility effects of intermittent oral treatment were more sustained than those of chronic oral tamoxifen treatment. It is inferred that hepatic metabolism of tamoxifen interferes with its antifertility effects via oral route and that the parameters affected by chronic oral exposure in the male sires are completely reversed in progeny ensuing after an adequate period of drug withdrawal.

Temporal effect of tamoxifen on cytochrome P450 side chain cleavage gene expression and steroid concentration in adult male rats

Adult male rats when treated with 0.4 mg tamoxifen (tam)/kg per day for 90 days show reduced circulating testosterone (T) and LH. The present study was designed to have an in depth understanding of tam induced androgen reduction in adult male rats. Adult male rats were orally administered 0.4 mg tam/kg per day for 30, 60 or 90 days and the temporal effects on intratesticular concentrations of pregnenolone (P(5)), progesterone (P(4)), T, 5 alpha-dihydrotestosterone (5 alpha-DHT) and estradiol (E(2)) were estimated. Control group rats were fed saline. Serum hormonal profile of LH, FSH, T and E(2) was also followed on these days. Testicular levels of cytochrome P450 scc mRNA transcripts on 30, 60 and 90 days of treatment with the same dose were quantitated by biplex RT-PCR using beta Actin as internal control followed by analysis using GelPro Analysis software.A significant reduction in intratesticular P(5), P(4), T, 5 alpha-DHT and E(2) was observed from day 30 of treatment. The P450 scc gene expression in the testis was reduced during treatment period from day 60 of treatment. This study demonstrates for the first time that tam reduces testicular pregnenolone biosynthesis through an effect on cholesterol transport and downregulation of P450 scc gene expression. In confirmation of the observed estrogenic effects of tam in this study, it is suggested that E(2) may have a role in cholesterol transport and testicular pregnenolone biosynthesis at the level of cytochrome P450 scc as shown by us.

Modulation of pituitary gonadotropins and prolactin secretion by testosterone in vitro

Investigations were undertaken to study the differential modulation of LH, FSH and PRL secretion by testosterone (T) using whole pituitary (PI) or pituitary - hypothalamus coincubates (PHC) as in vitro constructs. PI and PHC from intact and castrated rats were incubated with or without T thrice, for 24 h each, (24 h × 3, total incubation period 72 h). The spent media was replenished every 24 h. At the end of 72 h, a few of the pituitary glands were challenged with 10 nM LHRH for 4 h. The spent media and pituitary glands were analyzed for LH, FSH and PRL using specific RIAs. Incubation of PI or PHC from intact rats with T stimulated the release of LH and FSH but inhibited the release of PRL. T had no effect on the intrapituitary contents of LH but inhibited intrapituitary contents of FSH and PRL, as compared to controls incubated without T. Castration increased intrapituitary contens of LH and FSH with concomitant decrease in PRL levels. Incubation of PI or PHC from castrated rats with T inhibited intrapituitary contents of LH to intact pituitary levels, while PRL levels were further reduced instead of being ameliorated. It is concluded that PI or PHC can be used as convenient in vitro models to monitor the effect of castration or of T modulation of pituitary and hypothalamus functions. T does not affect the synthesis of LH at the gonadotroph level but facilitates the regulation of intracellular LH and FSH levels. It is postulated that T inhibits the synthesis of FSH/PRL at the gonadotroph/lactotroph levels.

The choice of a model for studying the hypothalamus-pituitary interactions in vitro.

Comparative studies on the release of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (Prl) by the whole pituitary, pituitary plus hypothalamus and pituitary-hypothalamus complex (PHC) were undertaken to choose an appropriate model for studying the hypothalamus-pituitary interactions in vitro and to relate the importance of the intact neural connections between pituitary and hypothalamus on hypothalamus-pituitary interactions. Also the effect of including dopamine (DA) at 1 X 10(-7) mol/1 in these different in vitro systems on the release of LH, FSH and Prl was investigated. The pituitary released increasing amounts of LH and FSH at 2, 4 and 6 h but the amount of Prl released remained unchanged. The rates of release of LH, FSH and Prl by the pituitary were different and were characteristic of each hormone. Co-incubation of pituitary with hypothalamus stimulated the release of LH and FSH but inhibited the release of Prl. Pituitary-hypothalamus complex behaved almost identical to behaved almost identical to pituitary plus hypothalamus system. Inclusion of 1 X 10(-7) M DA in the incubation medium stimulated the release of LH (80%) but inhibited the release of Prl (71%) by PHC. FSH was unaffected. DA had no significant effect on the release of LH, FSH and Prl by pituitary and pituitary plus hypothalamus systems. It is suggested that PHC is the system of choice for studying hypothalamus-pituitary interactions in vitro.