Varsha Padwal

Hyperprolactinemia affects spermiogenesis in adult male rats

The mechanisms underlying the antifertility effects of hyperprolactinemia have yet to be established in an appropriate experimental model. Hyperprolactinemia is a known side effect of fluphenazine, a broad spectrum, long-acting phenothiazine known to be dopamine type-D2 receptor antagonist. In our earlier study in adult male rats, we reported that fluphenazine at a dose of 3 mg/kg/day suppressed serum FSH but not testosterone (T) through increasing dopamine (DA) metabolism in the pituitary gland, within 60 days. Fluphenazine treatment affected sperm quality and male rats treated with fluphenazine sired fewer litters. The effects of fluphenazine-induced hyperprolactinemia on sperm quality appeared to be related to reduced FSH. We now report that FSH suppression enhanced the uptake of acridine orange (AO), a DNA intercalating, fluorescent dye by the fluphenazine- treated caput epididymal sperms with concomitant reduction in the uptake of thiolspecific monobromobimane (mBBr) fluorescent dye in vitro, suggesting greater accessibility of DNA intercalating dye to sperm chromatin and reduction in free sperm protein thiols. The concomitant increase in AO and decrease in mBBr fluorescence was suggestive of loose chromatin packaging in caput epididymal sperms after treatment with fluphenazine at 3 mg/kg/day for 60 days. The suppression in levels of protamine (P1) in caput epididymal sperms suggested that chromatin hypocompaction was due to reduced deposition of protamines in sperm chromatin. Reduction in testicular levels of cyclic adenosyl 3′, 5′ monophosphate response element modulator (CREMτ) and P1 further suggested that reduced deposition was indeed due to reduced synthesis. The concomitant reduction in testicular levels of transition protein 1 (TP1) and transition protein 2 (TP2) also suggested that hypoprotamination was due to reduced synthesis of these proteins crucial for facilitating P1 deposition. The effect appeared to have occurred at the level of translation of CREMτ, since its transcript levels were unaffected whereas those of TP1, TP2 and P1 and protamine were upregulated. The study led to the view that the effects of FSH suppression were manifest on the posttranscriptional modifications of CREMτ, as also on transcript repression of TP1, TP2, P1, which do the RNA-binding proteins bring about. Reduction in FSH did not decrease ABP expression in the testis, which has recently been implicated in the expression of transition protein 1 in vitro. However, a significant reduction was evident after fluphenazine treatment, in the immunoexpression of testicular cAMP, the mediator of FSH effects in the Sertoli cells and putative mediator of ABP effects in the spermatids. The study suggests that fluphenazine-induced hyperprolactinemia suppressed FSH and affected a putative cAMP-dependent mechanism underlying posttranscriptional modification of spermatidal genes involved in chromatin condensation, presumably by reducing the availability/secretion of ABP, a paracrine regulator of spermiogenesis in vitro.

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.

Estradiol affects androgen-binding protein expression and fertilizing ability of spermatozoa in adult male rats

The estrogenicity of certain environmental pollutants is being increasingly correlated to decline in sperm counts and fertility of the males. Qualitative effects, if any, of estrogen(s) on terminal differentiation of spermatids have been less reported. The present study suggests that exposure to estrogen(s) can also alter the status of condensed chromatin in testicular spermatozoa and reduce their fertilizing potential. A significant reduction was evident in the serum gonadotropins, testosterone, weights of reproductive organs, sperm counts and litters sired by male rats after 10 days of estradiol exposure to a dose of 0.1mg/kg/day. Estradiol treatment led to retardation of in vitro decondensation rates of sperm chromatin, reduction in the uptake of acridine orange dye by chromatin, reduction in susceptibility of chromatin to acid denaturation in vitro, reduced uptake of thiol reactive monobromobimane dye and reduced levels of immunoreactive protamine 1 in caput epididymal sperms. Concomitantly, testicular levels of immunoreactive protamine 1, transition proteins 1/2 and cyclic adenosyl response element modulator-tau (CREMtau) were significantly reduced whilst their mRNA levels were unaffected after estradiol treatment. A significant increase was observed in the testicular mRNA levels of androgen-binding protein (ABP) in estradiol treated sires. An inverse correlation was observed between ABP mRNA levels and uptake of acridine orange by estradiol treated caput sperm chromatin. The results suggest that estradiol-induced increase in ABP mRNA underlies the mechanism(s) involved in the reduction in levels of certain proteins involved in nuclear chromatin condensation during spermiogenesis.

Antifertility effects of fluphenazine in adult male rats.

The underlying mechanisms in human infertility associated with hyperprolactinemia have yet to be established. Hyperprolactinemia is a known side-effect of fluphenazine, a broad spectrum, long-acting phenothiazine known to be D2 dopamine receptor antagonist. Dose-related effects of fluphenazine decanoate were ascertained on the fertility of 60-day treated, adult male rats. Significant increase in the serum levels of prolactin and decrease in the levels of LH and FSH were seen at doses of 1–3 mg/kg/day. No effect was evident on the serum testosterone (T) and estradiol. The tissue levels of Inhibins were not affected. The weights of testes, epididymides, seminal vesicles, ventral prostate, adrenal and pituitary glands were not affected. Testicular histology showed sloughing indicating the sensitivity of this parameter to FSH deficiency. Mating occurred within 10 days of cohabitation in the control and 1–2 mg/kg/day treated groups but delayed in the 3 mg/kg/day treated group with a significant effect on potency. Implantation sites, litter size and fertility index were significantly reduced at 2–3 mg/kg/day doses of fluphenazine. No effects however were seen on sperm counts or motility whereas morphological changes were apparent in the acrosome. Chromatin decondensation in vitro was enhanced and sperm chromatin structure assay revealed DNA denaturation. Hypothalamic tyrosine hydroxylase levels were increased in 1–3 mg/kg/day dose range. Hyperprolactinemic males sired fewer pups as compared to controls. Hypothalamic tyrosine hydroxylase was upregulated at all the doses. The antifertility effects of fluphenazine-induced hyperprolactinemia appeared to be unrelated to testosterone (T). In addition, FSH decrease might have affected the intrinsic sperm quality and thereby reduced litter size.

Effects of an Antiprogestin Onapristone on the Endometrium of Bonnet Monkeys: Morphometric and Ultrastructural Studies

Abstract Our previous studies demonstrated the ability of low doses of antiprogestin ZK 98.299 (onapristone) to inhibit fertility in bonnet monkeys. In the present study cumulative effects of low doses of ZK 98.299 on the endometrial cytoarchitecture of bonnet monkeys were analyzed. Treatment with either the vehicle (n = 3) or onapristone at 2.5 mg (n = 4) or 5.0 mg (n = 3) was initiated on Day 5 of the first menstrual cycle and thereafter repeated every third day for four to seven consecutive cycles. The last treatment cycles were anovulatory in two animals treated with 2.5 mg and all animals treated with 5.0 mg. Endometrial biopsies were collected on Day 8 after the midcycle estradiol peak in ovulatory menstrual cycles and on Day 20 in anovulatory menstrual cycles during the last treatment cycle. Ultrathin sections of the fixed endometrium were stained with toluidine blue for morphometric analysis and uranyl acetate and lead citrate for ultrastructural analysis. The ZK 98.299-treated animals showed a dose-dependent endometrial atrophy as evident by a decrease in the height and diameter of the glands and early signs of compaction in the stroma. Ultrastructural analysis also revealed dose-dependent degenerative changes in the subcellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, lysosomes, and Golgi apparatus. This suggests that long-term treatment with low doses of ZK 98.299 leads to the suppression of estrogen-dependent endometrial proliferation. However, this blockade operates independent of estradiol receptor (ER) and progesterone receptor (PR) concentrations as the expressions of these steroid receptors did not show any significant changes even after prolonged treatment. The study demonstrated an antiestrogenic effect of ZK 98.299 on endometrium after prolonged treatment in bonnet monkeys.

Sperm protamine levels as indicators of fertilising potential in sexually mature male rats

We have earlier reported that administration of cyproterone acetate, fluphenazine decanoate, tamoxifen citrate, oestradiol valerate to adult male rats, at doses of 50, 5.77, 0.71, 0.28 μmol kg−1 body weight given for periods of 15, 60, 60, 10 days, respectively, partially suppressed/reduced availability of one or more reproductive hormones viz. LH, FSH, testosterone and reduced their siring ability. The reduction in epididymal sperm counts was not considerable after treatment with these drugs, but conventional methods of assessment of spermatozoa quality viz. sperm chromatin structure assay (SCSA), nuclear chromatin decondensation (NCD) assay, monobromobimane (mBBr) uptake, had shown quantifiable changes in caput sperm chromatin compaction and reduced the testicular levels of protamine 1. The present follow‐up study attempts to quantify changes in caudal sperm chromatin which has undergone compaction in the epididymis, in the altered hormonal microenvironment of rats treated with cyproterone acetate, tamoxifen citrate, fluphenazine decanoate, oestradiol valerate, at doses of 50, 5.77, 0.71, 0.28 μmol kg−1 body weight respectively given for periods of 15, 60, 60, 10 days, with a view to correlating these changes to reduction in their fertilising potential. During the androgen‐dependent transit of spermatozoa from caput to cauda epididymis, thiol group oxidation and tyrosine phosphorylation of protamine occurs in maturing sperms concomitant with development of fertilising ability. The results indicate that conventional methods viz. SCSA, NCD, mBBr uptake fail to detect changes induced by hormone deficits in sperm chromatin condensation, as a result of maturation during transit from caput to cauda epididymis. Absence of protamine 1 in epididymal sperm was observed in either testosterone or FSH deficient rats that correlated with reduced fertilising potential. The study suggests that changes in LH/T or FSH affect a hitherto unknown common molecular mechanism in the testis, underlying the protamination of rat spermatozoa. In conclusion, loss of P1 occurs in adult male rats deprived of T or FSH and is a reliable detectable change in epididymal sperm indicative of chromatin condensation defects associated with endocrine imbalance and poor fertility status.

Tamoxifen, protein kinase C and rat sperm mitochondria.

Tamoxifen at a dose of 400 μg/kg/day has been reported to reduce the fertility of adult male rats and alter the pattern of cauda sperm motility from forward progressive to circular yawing type. Since sperm motility is powered by mitochondria, the effect of tamoxifen on mitochondrial function was studied. Tamoxifen treatment significantly increased rhodamine 123 fluorescent dye uptake by sperm mitochondria, reflecting an altered mitochondrial membrane potential. ATP and DAG levels, activities of glycolytic enzymes, creatine kinase and PKC all remained unaffected by tamoxifen. This is also the first report describing the presence of PKC α and β in rat sperm. Morphological and biochemical integrity of sperm membranes was determined by electron microscopy and malondialdehyde levels, which were unaltered after tamoxifen treatment. This study indicates that the altered sperm motility induced by tamoxifen is accompanied by changes in mitochondrial membrane potential, but in the absence of any detectable change in membrane integrity, lipid peroxidation, ATP levels and activities of glycolytic enzymes, creatine kinase and PKC.

Genotypic characterization of HIV-1 C variants in PBMCs and cervicovaginal cells

Background HIV-1 is primarily a mucosal pathogen since more than 80% of infections occur through genital exposure. Vaginal intercourse, though an inefficient mode of transmission, contributes more new infections worldwide than any other route. Also, the female reproductive tract has been identified as a compartment that harbors variants distinct from blood. Present study aims to highlight viral compartmentalization between these compartments reflecting inadequate ART penetration. Methods In this study blood and cervicovaginal swabs were collected from 8 female subjects. CD4 counts and viral loads were determined. Translated amino acid sequences of C2 v3 region of env gene of proviral HIV-1 C in PBMCs and genital cells were analyzed using N-Glycosite and Geno2pheno [Co receptor] 1.2 programs for presence of N linked glycosylation sites and co receptor preference, respectively. Results Characterization of translated amino acid sequences of C2 v3 region of env gene of HIV-1 C shows variation in the number of N linked glycosylation (NLG) sites and uniform co receptor preference. Viral load varies in blood and genital secretions. Conclusion Genotypic characterization of viral variants in blood and female reproductive tract can provide information regarding their association with sexual transmission of HIV. Difference in the number of NLG sites observed may influence the affinity for host cell co receptor. Discrepancies in viral load of blood and genital secretions suggest that ART may not be uniformly effective in suppression of viral load in different compartments of the same individual.

Genotypic characterization of HIV variants from PBMCs and spermatozoa.

Background Several studies have shown that detectable levels of HIV are observed in semen in spite of undetectable viral load in blood following antiretroviral therapy (ART). Also, different HIV variants have been detected in blood and semen of the same individual, suggesting that drugs may not be uniformly effective to control the viral load and infectivity in different tissues and secretions, which in turn may affect sexual transmission of HIV. Hence, genotypic characterization of HIV variants in PBMCs and sperm of the same individual may provide information about the association of these variants with sexual transmission of HIV.