In Silico and In Vitro Investigation of the Piperine's Male Contraceptive Effect: Docking and Molecular Dynamics Simulation Studies in Androgen-Binding Protein and Androgen Receptor.

Understanding the molecular mechanism of action of traditional medicines is an important step towards developing marketable drugs from them. Piperine, an active constituent present in the Piper species, is used extensively in Ayurvedic medicines (practiced on the Indian subcontinent). Among others, piperine is known to possess a male contraceptive effect; however, the molecular mechanism of action for this effect is not very clear. In this regard, detailed docking and molecular dynamics simulation studies of piperine with the androgen-binding protein and androgen receptors were carried out. Androgen receptors control male sexual behavior and fertility, while the androgen-binding protein binds testosterone and maintains its concentration at optimal levels to stimulate spermatogenesis in the testis. It was found that piperine docks to the androgen-binding protein, similar to dihydrotestosterone, and to androgen receptors, similar to cyproterone acetate (antagonist). Also, the piperine-androgen-binding protein and piperine-androgen receptors interactions were found to be stable throughout 30 ns of molecular dynamics simulation. Further, two independent simulations for 10 ns each also confirmed the stability of these interactions. Detailed analysis of the piperine-androgen-binding protein interactions shows that piperine interacts with Ser42 of the androgen-binding protein and could block the binding with its natural ligands dihydrotestosterone/testosterone. Moreover, piperine interacts with Thr577 of the androgen receptors in a manner similar to the antagonist cyproterone acetate. Based on the in silico results, piperine was tested in the MDA-kb2 cell line using the luciferase reporter gene assay and was found to antagonize the effect of dihydrotestosterone at nanomolar concentrations. Further detailed biochemical experiments could help to develop piperine as an effective male contraceptive agent in the future.

A Chinese herbal formula, Wuzi Yanzong pill, improves spermatogenesis by modulating the secretory function of Sertoli cells.

OBJECTIVE: To evaluate the effects of the Chinese herbal formula Wuzi Yanzong Pill (, WYP) on the spermatogenesis and specific secretory functions of Sertoli cells in rat model and to investigate the underlying mechanism. METHODS: Five groups of male Sprague-Dawley rats including the control group, the model group, the low-dose WYP group, the medium-dose WYP group and the high-dose WYP group (5 in each group) were treated daily with vehicle, multiglycosides of Tripterygium wilfordii Hook f (GTW) either alone (20 mg/kg) or followed by WYP (0.5, 1.0, or 2.0 g/kg daily), respectively for 30 days. Serum levels of follicle-stimulating hormone (FSH), inhibin B (INHB) and testosterone (T) were evaluated using enzyme-linked immunosorbent assay. Androgen-binding protein (ABP) gene expression and transferrin (TF) protein expression in testis tissue specimens of all rats were determined using real-time reverse transcriptase polymerase chain reaction and Western blotting analysis, respectively. Histopathological alterations in the testis were determined using Johnsen's score. RESULTS: The toxicity of GTW towards Sertoli cell secretory functions and spermatogenesis was accompanied by increased serum FSH concentrations and decreased INHB and T concentrations. Upregulated ABP mRNA levels, and decreased TF protein expression and Johnsen's scores were detected in the model group compared with the control group P<0.05 or P<0.01). Oral high-dose WYP administrations to GTW-treated rats effectively alleviated all of the GTW-induced changes in specific secretory functions of Sertoli cells (ABP, INHB and TF). Furthermore, serum T level and Johnsen's score of the testis increased greatly compared with the model group (P<0.01). CONCLUSION: WYP has the ability to improve the spermatogenesis, possibly through modulating the secretory proteins expression of Sertoli cells.

Gibberellic acid acts as an agonist of steroidogenesis in male rats.

Testicular steroidogenesis has significant implication in male reproductive function. Although the effects of various signalling molecules on testicular functions have been studied earlier, the influence of the plant hormone gibberellic acid (GA3 ) on steroidogenesis has not been investigated. Acute (4 h) and subacute (15 days) studies using this compound through oral administration (150 µg day(-1) ) to groups of normal and diabetic Wistar male rats were therefore carried out. Results indicate that (i) enhanced activity of steroidogenic markers 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), elevated tissue testosterone (T) content, increased steroidogenic acute regulatory protein (StAR) and androgen binding protein (ABP) levels with reduced lipid peroxidation and improved antioxidant defence in this treatment group of normal and diabetic rat testis, and (ii) elevated lipid peroxidation and diminished antioxidant defence, with insignificant change in 3ß-HSD and 17ß-HSD activity and testosterone level in acute treatment group of normal and diabetic rats testis, were noted. The observed increase in the activity of testicular 3ß-HSD and 17ß-HSD along with elevated testosterone content established GA3 as an inducer of steroidogenesis in rat.

Kolaviron prevents carbendazim-induced steroidogenic dysfunction and apoptosis in testes of rats.

The study evaluated the protective role of kolaviron (an isolated biflavonoid from the seed of Garcinia kola) and vitamin E in carbendazim-induced reproductive dysfunction in male rats. Adult male Wistar rats were orally exposed to carbendazim (200mg/kg) singly or in combination with kolaviron (100 and 200mg/kg). Exposure to carbendazim significantly decreased the activities of superoxide dismutase and catalase but markedly increased sialic acid concentration and lipid peroxidation in the testes of rats. Western blot analysis revealed that carbendazim treatment decreased the expression of steroid acute regulatory (StAR) protein and androgen binding protein (ABP) with concomitant decrease in activities of steroidogenic enzymes. Germ cell apoptosis in carbendazim-treated rats was confirmed by TUNEL assay. However, pretreatment with kolaviron and vitamin E restored the testicular antioxidant status and steroidogenesis and decreased apoptotic nuclei to near control level in carbendazim-treated rats. Kolaviron may prove useful in combating carbendazim-induced reproductive toxicity.

Co-expression of vasopressin and androgen-binding protein in the rat hypothalamus.

In previous studies we have observed the expression of androgen binding protein (ABP) in the rat hypothalamo-neurohypophysial system. With immunocytochemical double staining we found partial co-localization with oxytocin. In the present study we used antibodies to the anti-diuretic hormone arginine vasopressin (AVP) for co-localization with ABP in the rat hypothalamus. Both antigens were seen in the magnocellular paraventricular and supraoptic nuclei. Dense fiber networks with varicosities containing both AVP and ABP immunoreactivity were visible throughout the hypothalamus, the median eminence and in the posterior pituitary lobe. Double immunostaining revealed also co-existence in the parvocellular portion of the paraventricular nucleus and in the suprachiasmatic nucleus. ABP immunoreactive neurons in the preoptic region were devoid of AVP staining, AVP neurons in the bed nucleus of the stria terminalis stained only occasionally for ABP. We conclude that both the magnocellular and the parvocellular hypothalamic vasopressin systems are capable of expressing the steroid binding globulin, which is probably subject to axonal transport, along with the peptide hormone. Intrahypothalamic expression of ABP may be among the mechanisms necessary for rapid actions of steroids on hypothalamic neuroendocrine systems.

Distribution of androgen-binding protein in the rat hypothalamo-neurohypophyseal system, co-localization with oxytocin.

Androgen-binding protein (ABP) is known to be expressed in the male and female rat hypothalamus. In the present study, we observed immunocytochemically ABP in neurons of the magnocellular hypothalamic nuclei, in the preoptic region and in the lateral hypothalamus. Dense fiber networks with varicosities, containing ABP immunofluorescence, were visible throughout the hypothalamus, the median eminence and in the posterior pituitary lobe. Double immunostaining revealed a partial coexistence of ABP-and oxytocin immunoreactivity in a portion of the magnocellular perikarya. ABP was isolated by affinity chromatography from hypothalamus homogenates. Western blots resulted in immunoreactive (IR) bands with an approximate molecular weight of 35 and 50 kDa. Mass spectrometry of these preparations confirmed the presence of ABP, which was almost identical to ABP isolated from rat testis. It is likely that ABP, expressed in magnocellular oxytocinergic neurons, is subject to axonal transport and release in the hypothalamo-neurohypophyseal system.

Genistein alters growth but is not toxic to the rat prostate.

The mortality of clinical prostate cancer is lower in Asian populations than in American or European men. Asian men typically consume more soy than their Western counterparts, leading to the investigation of individual components, particularly phytoestrogens, as protective factors against prostate cancer. Genistein, the predominant isoflavone in soy, has been reported to reduce the incidence of prostate cancer in animal models, but the underlying biological action remains to be elucidated. The purpose of this investigation was to identify the effects of the phytoestrogen, genistein and the synthetic estrogen diethylstilbestrol (DES), as a control, on development and function of the rat dorsolateral prostate (DLP) when given in the diet. The effects of testosterone and dihydrotestosterone (DHT) injections were also tested. Analysis of individual lobes of the DLP revealed that 1000 mg/kg, but not 250 mg/kg, of a genistein AIN-76A diet slightly reduced lateral prostate type 1 (LP1) bud perimeter. However, expression of the secretory dorsal protein 1 (DP1) and 5alpha-reductase type II activity were not altered in the prostate. This suggested that prostate differentiation, and not toxicity, had occurred. DES in the diet reduced and testosterone injections elevated relative prostate weights and perimeters of the dorsal, LP1, lateral prostate type 2 and DP1 expression. DHT increased relative prostate weights but did not significantly increase individual lobe perimeter. Unlike DES, maximally tolerated doses of genistein in the diet were not toxic to the rat prostate.

Non-steroidal L-245,976 acts as a classical antiandrogen in vitro.

Non-steroidal antiandrogens have been employed in the management of prostate cancer, but the mechanism of action is unclear due to a lack of good tissue culture models. The growth of a hamster ductus deferens cell line (DDT1) is highly dependent upon the addition of 10 nM testosterone to synthetic serum-free media. We describe a non-steroidal compound N-(4-chlorophenyl)-(Z,Z)-2,3-bis(-cyclopropylmethylene) cyclopentanecarboxamide (L-245976) which antagonizes the action of testosterone on DDT1 cells at 10 microM but exhibits little or no effect on cell growth by itself. This compound also blocks the binding of 3H-dihydrotestosterone (DHT) to the human androgen receptor (AR) with an IC50 of approximately 28 microM. In addition, L-245976 was found to antagonize DHT-dependent transactivation of the AR via the probasin gene promoter at comparable doses with no agonist activity.

The androgen-specific probasin response element 2 interacts differentially with androgen and glucocorticoid receptors.

The nuclear receptors constitute a large family of transcription factors characterized by a well conserved DNA-binding domain. The receptors for glucocorticoids, progestins, mineralocorticoids, and androgens constitute a subgroup because they bind in vitro with high affinity to DNA elements containing a partial palindrome of the core sequence 5'-TGTTCT-3'. In vivo, however, the corresponding steroids differentially regulate the expression of their target genes, even when more than one receptor type is present in a particular cell. The DNA-binding domains of the androgen and of the glucocorticoid receptors bind most androgen response elements with similar relative affinities. In contrast, one element (5'-GGTTCTTGGAGTACT-3') which was recently described in the promoter region of the probasin gene selectively interacts with the DNA-binding domain of the androgen receptor and not with that of the glucocorticoid receptor. From studies with chimeric elements, it can be deduced that it is the left subsequence 5'-GGTTCT-3' which excludes the glucocorticoid receptor domain from binding. In co-transfection experiments where the ARE of the C3(1) gene is responsive to both androgens and glucocorticoids, the probasin element is induced only by androgens and not by glucocorticoids. The existence of response elements which are recognized preferentially by the androgen receptor provides yet another possible mechanism to explain the differences of the in vivo effects between androgens and other steroids of the subgroup.

Impairment of testicular endocrine function after lead intoxication in the adult rat.

To clarify the mechanism of the action of lead on male reproductive function, adult male rats were injected intraperitoneally (i.p.) with lead acetate (8 mg/kg/day of lead), 5 days a week for 35 days. Despite this high dose, germ cells and Sertoli cells did not appear to be major targets of lead. However, lead determination in the reproductive organs showed that the accessory sex glands are such a target. Epididymal function was unchanged. In lead-exposed rats, plasma and testicular testosterone dropped by about 80%, but plasma luteinizing hormone (LH) only dropped by 32%. After luteinizing hormone releasing hormone (LHRH) stimulation of the pituitary, the plasma LH level reached the control one, but plasma testosterone remained significantly reduced by 37%. The sharp decrease in the testosterone:LH ratio in lead-exposed rats, combined with the significant reduction of intertubular tissue volume in the testes, indicate impaired Leydig cell function.

Steroidogenic and morphogenic characteristics of human peritubular cells in culture.

We have explored the morphogenic and functional characteristics of human peritubular cells originating from seminiferous tubule (ST) fragments isolated from the testes of two prepubertal patients with the androgen insensitivity syndrome. These ST were cultured in Dulbecco's Modified Eagle's Medium-Ham's F-12 supplemented with antibiotics, transferrin, hydrocortisone, vitamin E, and 3% fetal bovine serum. A centrifugal growth of elongated fibroblast-like cells peripheral to the ST explants was observed. Muscle-specific actin and 3 beta-hydroxysteroid dehydrogenase were evident in the peritubular area and in the elongated cells growing from the tubules. Histochemistry was negative in the tubules themselves, revealing the mixed nature of these cultures. The ST fragments were lost after subculturing, leaving a homogeneous monolayer of fibroblast-like cells. The steroidogenic potential of these cells was demonstrated by the secretion of testosterone (T) to the culture medium. T secretion was stimulated by hCG in a time-dependent fashion (patient 1: Day 11, 84% and Day 15, 200%; patient 2: Day 8, 73% and Day 11, 32% over basal). FSH also stimulated T secretion (patient 1: Day 5, 136% and Day 8, 89%; patient 2: Day 8, 117% and Day 11, 129% over basal). Furthermore, T secretion by these cultures was 100% higher than that observed in mesenchymal cells obtained from the testicular intertubular space in the same patient. Spontaneous T secretion and hormone responses declined progressively to cease by 25 days in culture. These results suggested the involvement of Sertoli cell (SC)-secreted factor(s) in the regulation of T secretion by peritubular cells. In order to further explore possible paracrine interactions between peritubular and Sertoli cells, we carried out heterologous cocultures with rat SC. After 72 h a striking redistribution of both cell types was observed with the formation of cord-like structures. Ultrastructural examination of these cords showed the formation of a basement membrane between epithelial (Sertoli) and mesenchymal cells of peritubular origin. No resumption of T secretion was observed, but an increase in androgen-binding protein (ABP) production by rat SC under basal (37%) and FSH-stimulated (52%) conditions was evident. Our results show that in the human peritubular compartment, cells exist that can alternatively express steroidogenic functions, associate with SC in a specific mesenchymal-epithelial interaction, and exert regulatory influences on ABP secretion by SC. In addition they indicate that communicating events in the testis are preserved throughout evolution.

Surface-associated glycosyltransferase activities in rat Sertoli cells in vitro.

We have previously demonstrated fucosyltransferase (FT) activity on mouse germ cell surfaces at different stages of spermatogenesis. To complement these findings, here we report FT activity on the Sertoli cell (SC) surface. SC isolated and cultured from 20-day-old rat testes displayed FT activity with a Vmax of 12.5 pmoles/mg protein/min and a Km of 22 microM, while purified Sertoli cell plasma membranes (SCPM) showed FT activity with a Vmax of 10 pmoles/mg protein/min and a Km of 18.2 microM for GDP-[14C]-L-fucose. Fucosyltransferase activities were 16.7 and 2.6 pmoles/mg protein/min in SC and SCPM, respectively; approximately 16% of FT activity is, therefore, on the cell surface. To test whether the expression of FT activity in SC was regulated by hormones and growth factors, SC were cultured in serum-free medium supplemented with insulin, transferrin, sodium selenite, and epidermal growth factor (medium 4F) or in 4F plus follicle-stimulating hormone, testosterone, hydrocortisone, and vitamin E (medium 8F). We found that FT activity in SC is not modulated by these hormones or growth factors (4F or 8F). For comparison with FT, galactosyltransferase (GalTase) activities in SC and SCPM were also determined. SC displayed GalTase activity with a Vmax of 50 pmoles/mg protein/min and a Km of 38.5 microM, while SCPM showed GalTase activity with a Vmax of 25 pmoles/mg protein/min and a Km of 20.8 microM for UDP-[3H]-galactose. Galactosyl-transferase activities were 29.2 and 9.6 pmoles/mg protein/min in SC and SCPM, respectively. Therefore, approximately 33% of the total cell GalTase activity was detected on the surface membranes of rat Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The 56 kDa androgen-binding protein in human genital skin fibroblasts: its relation to the human androgen receptor.

We have recently described in genital skin fibroblasts (GSF) a relatively abundant 56 kDa protein with androgen-binding activity. This protein is missing in GSF of most patients with complete androgen insensitivity syndrome (CAI). The protein has many characteristics compatible with the androgen receptor; it has in fact been tentatively considered as a precursor or degradation form of the prototypic (approximately 100 kDa) human androgen receptor. We have prepared an antiserum to this protein, which allowed us to detect it as a direct product by in vitro translation of mRNA from GSF. It is thus very unlikely to be a degradation product of a larger precursor. Furthermore, covalent photolytic labeling of this protein with the androgen analogue [3H]mibolerone revealed a much lower affinity for this protein than is known for the androgen receptor. Finally, the GSF of two exceptional patients with complete androgen insensitivity syndrome due to negligible androgen receptor-binding activity express this protein normally, as determined on two-dimensional gels by Western blot analysis with the antiserum and by photolytic covalent labeling with androgen analogues. These data indicate that the protein is not a precursor or a degradation product of the receptor; nor is it androgen-induced. They are more compatible with the idea that the protein is another member of the steroid/thyroid/retinoic acid receptor supergene family, perhaps as an unorthodox product of the human androgen receptor gene.

Bipolar secretion of androgen-binding protein and transferrin by Sertoli cells cultured in a two-compartment culture chamber.

The influence of various medium supplements on Sertoli cell (Sc) monolayer permeability to [3H]inulin and polarized secretion of transferrin (Trf) and androgen-binding protein (ABP) was investigated in a two-compartment culture chamber. Sc from 14- and 18-day-old rats were maintained for 13 days in one of the following media: Modified Eagle's Medium-Ham's F-12 (DFM), DFM plus insulin, epidermal growth factor, progesterone, hydrocortisone, and vitamins A and E (6F), 6F plus testosterone (7F), 7F plus FSH (8F), 6F plus 2% fetal bovine serum (6F2S), 6F plus 5% fetal bovine serum (6F5S). The monolayer permeability to [3H]inulin decreased rapidly during the initial 3-5 days of culture for the Sc isolated from 18- and 14-day old animals, then remained stable in all media except DFM. Morphological examination revealed the presence of tight junctions between neighbouring Sc in both age groups, indicating their de novo formation. Secretion of Trf was lowest in DFM and steadily declined. In all other media, Trf secretion peaked on day 5 and remained relatively constant after day 7. Medium 7F only slightly and inconsistently increased the secretion, whereas 8F was always highly stimulatory compared to 6F. Supplementation of 6F with serum resulted in the greatest Trf secretion. In the case of ABP, three different secretion patterns were noted depending on the medium composition; secretion was greatest in the presence of 5% fetal bovine serum. The medium supplements also differentially affected the polarity of Trf and ABP secretion. The ratio of Trf secreted to the outer and inner compartments (OC/IC) was approximately 2.0 in DFM and was not influenced by supplements in 6F, 7F, and 8F. However, in serum-containing media, the OC/IC ratio gradually increased with time to about 5.0 on day 13. The average OC/IC ratio of ABP was 1.7 in DFM and, in contrast to Trf, declined to 0.7 in other serum-free media. Serum supplementation reversed and increased the ABP ratio to about 6.0 on day 13. These data indicate that Sc grown on permeable supports form confluent monolayers that limit the diffusion of macromolecules, most likely due to the formation of tight junctions. The monolayer permeability as well as the total and polarized secretion of Trf and ABP are differentially regulated by hormones and serum factors.

Androgen binding protein levels and FSH binding to testicular membranes in vitamin A deficient rats and during subsequent replenishment with vitamin A.

ABP levels in the testes and epididymides of vitamin A deficient-retinoic acid maintained rats were only 20 and 6% respectively as compared with those in normal rats. The number of FSH receptors in the testes of vitamin A deficient rats, as measured by [125I]ovine FSH binding to isolated testicular membranes, was only 40% of that in the testes of normal rats. Supplementation of retinyl acetate to these vitamin A deficient rats for 16 days resulted in restoration of the number of FSH receptors to normal levels. On the other hand, ABP levels were restored to 32 and 34% only in the testes and epididymides respectively.

Differential regulation of testicular transferrin and androgen-binding protein secretion in primary cultures of rat Sertoli cells.

Specific RIAs for rat transferrin (rTF) and androgen-binding protein (rABP) were used to determine whether the secretion of these proteins was coordinately regulated in the Sertoli cell under a variety of conditions. Sertoli cell-enriched primary cultures were prepared from the testes of 20-day-old rats, and rTF and rABP were assayed in medium from the same culture. There was a strong effect of cell density on both rABP and rTF secretion per cell, with increased secretion per cell at high densities. Human TF (hTF), FeSO4, and desferrioxamine had little or no effect on rTF secretion. The age of the animal at the time of preparation of cells for culture had a strong effect on the pattern of rTF and rABP secretion in vitro; however, the effects of animal age, time in culture, and medium supplementation differed for the two proteins. In cultures prepared from 20-day-old animals, insulin, epidermal growth factor, and testosterone stimulated both rTF and rABP secretion, although to different extents. Retinoic acid was required for the stimulation and maintenance of rTF secretion, but had no effect on rABP secretion in the presence of insulin, hTF, and epidermal growth factor. Conversely, FSH and isoproterenol stimulated rABP, but not rTF, secretion. These data suggest that the secretion of rABP and rTF by Sertoli cells is under differential control.

Age-dependent pattern of androgen-binding protein secretion from rat Sertoli cells in primary culture.

The pattern and hormonal control of rat androgen-binding protein (rABP) secretion in vitro was investigated using animals of different ages to initiate primary cultures of Sertoli cells. Sertoli cells were isolated from testes of 7- to 31-day-old rats and cultured for periods of up to 30 days in serum-free medium, medium supplemented with insulin, transferrin, and epidermal growth factor (3F), or 3F plus FSH, testosterone, progesterone, hydrocortisone, and vitamin E (8F). The amount of rABP secreted by Sertoli cells during the first 24 h in culture (initial rate) exhibited an age-dependent pattern which reflected the apparent in vivo activity of these cells. Between 7 and 25 days of age, the initial rate of rABP secretion per Sertoli cell increased 20-fold; a further 2-fold increase occurred between 25 and 35 days of age. The pattern of rABP secretion exhibited by Sertoli cells cultured for several weeks was dependent not only on added factors (3F or 8F), but also on Sertoli cell age, expressed as the total of animal age plus time in culture (total age). In cultures of Sertoli cells isolated from very young animals (7-10 days old), the rate of rABP secretion increased until 20 days (total age), but declined thereafter. This early increase in rABP secretion was augmented by, but not dependent on, hormone additions. In contrast, Sertoli cells isolated from older animals always showed decreasing rates of secretion with time in culture. Furthermore, Sertoli cells from very young animals retained the capacity to respond to hormones in vitro with increased secretion of rABP and maintenance of cell viability. This responsiveness decreased with age, similar to the loss of hormone response seen in vivo after puberty. In conclusion, culture conditions were established which permitted the study of FSH-dependent and independent regulation of rABP secretion and of the acquisition of hormone resistance at the time of puberty. The initial rate of rABP secretion in culture (first 24 h) correlates with the age of the animal from which the cultures are obtained. The pattern of rABP secretion during subsequent long term culture is determined by the total age (animal and culture age), with increasing rates of secretion up to 20 days and decreasing rates thereafter. This inherent pattern of rABP secretion as well as loss of responsiveness of the Sertoli cell to hormonal stimulation appear to be programmed early in development.