1α,25-Dihydroxyvitamin D(3) signaling pathways on calcium uptake in 30-day-old rat Sertoli cells.

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is the active metabolite of vitamin D(3) and the major calcium regulatory hormone in tissues. The aim of this work was to investigate the mechanism of action of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells from 30-day-old rats. Results showed that 10(-9) and 10(-12) M 1,25D(3) increased the rate of (45)Ca(2+) uptake 5 and 15 min after hormone exposure and that 1α,25(OH)(2) lumisterol(3) (JN) produced a similar effect suggesting that 1,25D(3) action occurs via a putative membrane receptor. The involvement of voltage-dependent calcium channels (VDCC) in 1,25D(3) action was evidenced by using nifedipine, while the use of Bapta-AM demonstrated that intracellular calcium was not implicated. Moreover, the incubation with ouabain and digoxin increased the rate of (45)Ca(2+) uptake, indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, we demonstrated that the mechanism underlying the hormone action involved extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) activation in a phospholipase C-independent way. Furthermore, a local elevation of the level of cAMP, as demonstrated by incubating cells with dibutyryl cAMP or a phosphodiesterase inhibitor, produced an effect similar to that of 1,25D(3), and the inhibition of protein kinase A (PKA) nullified the hormone action. In conclusion, the stimulatory effect of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells occurs via VDCC, as well as PKA, PKC, and ERK activation. These protein kinases seem to act by inhibiting Na(+)/K(+)-ATPase or directly phosphorylating calcium channels. The Na(+)/K(+)-ATPase inhibition may result in Na(+)/Ca(2+) exchanger activation in reverse mode and consequently induce the uptake of calcium into the cells.

Effect of 1α,25-dihydroxyvitamin D3 in plasma membrane targets in immature rat testis: ionic channels and gamma-glutamyl transpeptidase activity.

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is critical for the maintenance of normal reproduction since reduced fertility is observed in vitamin D-deficient male rats. The aim of this study was to investigate the effect of 1,25D(3) in 30-day-old rat testicular plasma membrane targets (calcium uptake and gamma-glutamyl transpeptidase (GGTP) activity), as well as to highlight the role of protein kinases in the mechanism of action of 1,25D(3). The results demonstrated that 1,25D(3) induced a fast increase in calcium uptake in rat testis through a nongenomic mechanism of action. This effect was dependent on PKA, PKC and MEK. Moreover, ionic channels, such as ATP- and Ca(2+)-dependent K(+) channels and Ca(2+)-dependent Cl(-) channels, are involved in the mechanism of action. The use of BAPTA-AM showed that [Ca(2+)](i) was also implicated, and the incubation with digoxin produced an increase in (45)Ca(2+) uptake indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, 1,25D(3) was able to increase the GGTP activity. Considered together, our results indicate a PKA/PKC/MEK-dependent 1,25D(3) pathway as well as ionic involvement leading to (45)Ca(2+) uptake in immature rat testis. These findings demonstrate that 1,25D(3) stimulates calcium uptake and increases GGTP activity which may be involved in male reproductive functions.

Regulation of aromatase expression by 1α,25(OH)2 vitamin D3 in rat testicular cells.

It is well known that the vitamin D endocrine system is involved in physiological and biochemical events in numerous tissues, especially gut, bone and kidney but also testis. Therefore, in this study the effect and mechanisms of action of 1α,25(OH)(2) vitamin D(3) (1,25D) on aromatase gene expression in immature rat Sertoli cells were evaluated. Vitamin D receptor transcripts were present in immature Sertoli cells as well as in adult testicular germ cells and somatic cells. The treatment of immature Sertoli cells with 100 nM 1,25D increased the amount of aromatase transcript, mainly in 30-day-old rats. The protein kinase A (PKA) blocker, H89, partially inhibited the 1,25D effect. The stimulation of aromatase gene expression in 30-day-old Sertoli cells by the agonist 1α,25(OH)(2) lumisterol(3), and the suppression of the 1,25D effect by the antagonists 1ß,25(OH)(2) vitamin D(3) and (23S)-25-dehydro-1α (OH)-vitamin D(3)-26,23-lactone suggested, besides a genomic effect of 1,25D, the existence of non-genomic activation of the membrane-bound vitamin D receptor involving the PKA pathway.

Effects of the methanol extract of Basella alba L (Basellaceae) on steroid production in Leydig cells.

In this study, Leydig cells were purified from 70 day-old Sprague Dawley male rats and incubated with 10 and 100 µg/mL of methanol extract of Basella alba (MEBa) for 4 hours followed by the evaluation of cell viability, steroid (testosterone and estradiol) production, and the level of aromatase mRNA. Results showed that MEBa did not affect Leydig cell viability. At the concentration of 10 µg/mL, MEBa significantly stimulated testosterone and estradiol production (p < 0.01 and p < 0.03, respectively), and enhanced aromatase mRNA level (p < 0.04). These observations suggest that MEBa directly stimulated testosterone, estradiol and aromatase mRNA levels in isolated Leydig cells.

Aromatase gene expression in immature rat Sertoli cells: age-related changes in the FSH signalling pathway.

Aromatase, the enzyme responsible for the transformation of androgens into oestrogens, is encoded by the cyp19 gene expressed in the testis. The aim of the present study was to analyse the evolution of aromatase gene expression under FSH control in rat Sertoli cells between 10 and 30 days post partum, corresponding to the end of the proliferative period of Sertoli cells, establishment of the blood-testis barrier and acquisition of the mature phenotype. The maximum stimulatory effect of FSH on aromatase gene expression was obtained in 20-day-old rat Sertoli cells, compared with cells from 10- and 30-day-old rats, in parallel with the differentiation of Sertoli cells. Using two effectors of the protein kinase A pathway (i.e. forskolin and dibutyryl-cAMP) revealed differential effects between cells from rats aged 20 and 30 days, implying the involvement of another signalling pathway. Experiments using the specific phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 revealed that PI3-K is strongly involved in FSH-induced aromatase expression in Sertoli cells from both 20- and 30-day-old rats. In vivo, this decrease could be explained by a negative effect exerted by germ cells because, in coculture, aromatase gene expression in 20-day-old Sertoli cells is greatly diminished.

Immunomodulatory, beta-cell, and neuroprotective actions of fenugreek oil from alloxan-induced diabetes.

Immunological disorders and nephropathy are among the most frequent and serious complications of diabetes mellitus. This shows that fewer infiltrated inflammatory cells evidenced by reverted back to near the normal value of white blood cell, mean corpuscular volume, and lymphocytes counts as interleukin-6 in pancreas. Also, fenugreek oil significantly improved blood glucose levels, glucose intolerance, and insulin sensitivity compared to the diabetic group. The pancreatic islet and less beta-cells damage were observed after the administration of fenugreek oil to diabetic rats. Moreover, diabetic rats showed low activities of superoxide dismutase, catalase, glutathione peroxidase, and reduced glutathione content in kidney, which were restored to near normal levels by treatment with fenugreek oil. The increased levels of lipid peroxidation, creatinine, albumin, and urea in diabetic rats decreased significantly in diabetic rats treated with fenugreek oil. Diabetic rats treated with fenugreek oil restored almost a normal architecture of pancreas and kidney. In conclusion, this study reveals the efficacy of fenugreek oil in the amelioration of diabetes, hematological status, and renal toxicity which may be attributed to its immunomodulatory activity and insulin stimulation action along with its antioxidant potential.

Inhibitory effect of estrogens, phytoestrogens, and caloric restriction on oxidative stress and hepato-toxicity in aged rats.

OBJECTIVE: To investigate the protective effect of 17beta-estradiol (E2), peganum harmala extract (PHE) administration and calorie restriction (CR) treatment (60%) on oxidative stress and hepato-toxicity in aged rats. METHODS: Eighteen months old animals that were treated at the age of 12 months were divided into 4 groups: normal control group with free access to food, E2 treatment group, PHE treatment group and CR treatment group of the food given to control group. Six male rats at the age of 4 months were used as a reference group. RESULTS: Aging significantly decreased superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), and increased lactate deshydrogenase (LDH), gamma-glytamyl transferase (GGT), phosphatase alkalines (PAL), aspartate and lactate transaminase (AST and ALT) activities in the liver. Aging also induced an increased lipid peroxidation level, histological changes and a decreased E2 level. However, treatment with E2, PHE, and CR increased 17beta-estradiol, and decreased hepatic dysfunction parameters and lipid peroxidation as well as histological changes in the liver of aged rats. CONCLUSION: The antioxidant and hepatoprotective activity of PHE and CR is possibly attributed to its ability to increase E2 level, which as an antioxidant, acts as a scavenger of ROS. Further studies on the pharmaceutical functions of E2 in males may contribute to its clinical application.

Hyperglycaemia, stress oxidant, liver dysfunction and histological changes in diabetic male rat pancreas and liver: protective effect of 17 beta-estradiol.

Oxidative stress is thought to play a crucial role in the pathogenesis of chronic diabetic complications. We investigated the protective effects of 17 beta-estradiol (E2) on alloxan-induced stress oxidant, hepatic dysfunction and histological changes in male rats liver and pancreas. Our results showed that 17 beta-estradiol could attenuate the increase of blood glucose in plasma and normalise the hepatic glycogen level. In addition, E2 enhanced superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) (by 207, 52 and 72%, respectively, as compared to diabetic rats), reduced lipid peroxidation in the hepatic tissue (by 54%) and improved the liver dysfunction parameters by the significant decrease of gamma-glytamyl transferase (GGT), phosphatases alkalines (PAL), lactate deshydrogenase (LDH) and aspartate and lactate transaminases (AST and ALT) activities which increased in diabetic rats. Moreover, 17 beta-estradiol treatment in diabetic rats protects against alloxan-induced pancreatic beta-cells and hepatic cells damages.

Age-related decrease in aromatase and estrogen receptor (ERalpha and ERbeta) expression in rat testes: protective effect of low caloric diets.

AIM: To examine the effects on rat aging of caloric restriction (CR1) and undernutrition (CR2) on the body and on testicular weights, on two enzymatic antioxidants (superoxide dismutase and catalase), on lipid peroxidation and on the expression of testicular aromatase and estrogen receptors (ER). METHODS: CR was initiated in 1-month-old rats and carried on until the age of 18 months. RESULTS: In control and CR2 rats an age-related decrease of the aromatase and of ER (alpha and beta) gene expression was observed; in parallel a diminution of testicular weights, and of the total number and motility of epididymal spermatozoa was recorded. In addition, aging in control and CR2 rats was accompanied by a significant decrease in testicular superoxide dismutase, catalase activities, and an increase in lipid peroxidation level (thiobarbituric acid reactive substance), associated with alterations of spermatogenesis. Conversely, caloric restriction-treatment exerted a protective effect and all the parameters were less affected by aging. CONCLUSION: These results indicate that during aging, a low caloric diet (not undernutrition) is beneficial for spermatogenesis and likely improves the protection of the cells via an increase of the cellular antioxidant defense system in which aromatase/ER could play a role.

Protective effects of estrogens and caloric restriction during aging on various rat testis parameters.

AIM: To investigate the effects of 17beta-estradiol (E2), Peganum harmala extract (PHE) and caloric restriction (CR) on various testis parameters during aging. METHODS: Twelve month-old male rats were treated for 6 months with either E2 or PHE, or submitted to CR (40%). RESULTS: Our results show that estrogens and CR are able to protect the male gonad by preventing the decrease of testosterone and E2 levels as well as the decrease of aromatase and estrogen receptor gene expressions. Indeed, E2, PHE and CR treatments induced an increase in the superoxide dismutase activities and decreased the activity of testicular enzymes: gamma-glutamyl transferase, alkaline phosphatase, lactate deshydrogenase as well as the aspartate and lactate transaminases in aged animals. In addition, the testicular catalase and gluthatione peroxidase activities were enhanced in E2, PHE and CR-treated rats compared to untreated animals at 18 months of age. Moreover, the positive effects of estradiol, PHE and CR were further supported by a lower level of lipid peroxidation. Recovery of spermatogenesis was recorded in treated rats. CONCLUSION: Besides a low caloric diet which is beneficial for spermatogenesis, a protective antioxydant role of estrogens is suggested. Estrogens delay testicular cell damage, which leads to functional senescence and, therefore, estrogens are helpful in protecting the reproductive functions from the adverse effects exerted by reactive oxygen species (ROS) produced in large quantities in the aged testis.

Positive effects of green tea on hepatic dysfunction, lipid peroxidation and antioxidant defence depletion induced by cadmium.

Cadmium (Cd) is a highly toxic environmental and industrial cumulative pollutant that affects many organs, especially the liver. The present study was designed to evaluate the antioxidant effect of green tea on cadmium-induced hepatic dysfunction and oxidative stress in rats. Adult male Wistar rats were administered cadmium by injection of 20 micromoles/kg bw/every 3 days for six months. This study revealed significant (p < 0.05) liver dysfunction, lipid peroxidation and a decline in antioxidant enzyme activities in the liver of cadmium-treated rats compared to control animals. Compared to control rats, the activities of lactate dehydrogenase (LDH), gammaglutamyl transferase (GGT), acid phosphatase (PAC), phosphatase alkaline (PAL), as well as bilirubin and thiobarbituric acid-reactive substances (TBARs), were significantly (p < 0.05) increased in Cd-treated rats. Moreover, antioxidant enzyme activities, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase, were significantly (p < 0.05) decreased in the liver of cadmium-treated rats. The oral administration of 5% aqueous green tea extract, along with cadmium treatment for six months, caused a significant (p < 0.05) improvement in cadmium-induced toxicity by significantly decreasing (p < 0.05) the activities of enzymatic markers of liver dysfunction (LDH, GGT, PAC, PAL activities, as well as the bilirubin rate). Indeed, green tea extract significantly increased (p < 0.05) antioxidant enzymatic activities (SOD, Catalase, GPX) in rat liver, compared to those given cadmium alone. Thus, the oral administration of green tea, along with cadmium significantly (p < 0.05) improves cadmium-induced liver dysfunction and stress oxidant in rats' liver.

Protective effects of Peganum harmala extracts on thiourea-induced diseases in adult male rat.

Cancers and hepatoprotective prevention using traditional medicines have attracted increasing interest. The aim of our study was to characterize the putative protective effects of ethanol and chloroform extracts of Peganum harmala on thiourea-induced diseases in adult male rat. We seek to determine the effects of these plant extracts on body weight, thyroid and endocrine cancer parameters. In addition the putative hepatoprotective effect was checked by the determination of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and the bilirubin level in the blood. Our data show that ethanol and chloroform extracts of Peganum harmala protected the animal against the carcinogenic effects induced by thiourea since neuron-specific enolase (NSE) and thyroglobulin (TG) levels were back to the normal range. In addition, the observed-hepatocytotoxicity after thiourea treatment was greatly reduced (AST and ALT activities were respectively 270 IU/l and 60 IU/l and in the same order of magnitude as in the untreated rats) as well as the bilirubin levels (6 micromol/l) especially for animals receiving the choroform preparation. Therefore we may suggest that extracts of Peganum harmala are efficient to reduce the toxicity induced by thiourea in male rat as far as the above parameters are concerned.

Effects of maternal undernutrition during lactation on aromatase, estrogen, and androgen receptors expression in rat testis at weaning.

The goal of this study was to evaluate the effects of maternal malnutrition during lactation on serum levels of testosterone and estradiol, testicular testosterone concentration, aromatase, testicular androgen (AR) and estrogen alpha (ERalpha) receptors expression in the pups at weaning. From parturition until weaning, Wistar rats were separated into three groups: (C) control group, with free access to a standard laboratory diet containing 23% protein; protein-energy restricted (PER) group, with free access to an isoenergy and protein-restricted diet containing 8% protein; and energy-restricted (ER) group, receiving standard laboratory diet in restricted quantities, which were calculated according to the mean ingestion of the PER group. All pups were killed at weaning, corresponding to 21 days post partum. Compared with the C group, body weights (C=48 +/- 2.3 g; PER=20 +/- 1.3 g; ER=25.4 +/- 0.9 g; P<0.01) and testicular weights (C=0.15 +/- 0.02 g, PER=0.05 +/- 0.01 g, ER=0.06 +/- 0.02 g, P < 0.001) of both PER and ER groups were lower. However, there was no significant difference in the testicular/body weight ratio in PER and ER groups compared with the C group. The testosterone serum concentration (ng/ml) was significantly higher in the PER group compared with ER and C groups (C=0.09 +/- 0.012; PER=0.45 +/- 0.04; ER=0.15 +/- 0.03, P < 0.01). Testicular testosterone concentration (C=2.1 +/- 0.43; PER=6.5 +/- 0.7; ER=13 +/- 2.3, P < 0.01) was increased in treated groups when compared with controls. The estradiol serum concentration (pg/ml) was lower in both dietary groups (C=74 +/- 4.6; PER=49 +/- 3.2; ER=60 +/- 5.5, P < 0.01). The amounts of aromatase mRNA and ERalpha transcripts were significantly lower (P<0.05) in PER and ER groups; conversely AR (both mRNA and protein) was significantly enhanced (P<0.05) in treated animals. The nutritional state in early phases of development is important since we have demonstrated here that the maternal malnutrition during lactation leads to alterations in estradiol and testosterone serum concentrations, testicular testosterone concentration, AR and ERalpha expression together with a decrease of aromatase expression. All together, these changes of steroid status may be deleterious for future germ cell development and reproductive function of these male pups submitted to early malnutrition.

Relationship between chromatin organization, mRNAs profile and human male gamete quality.

Spermiogenesis is a complex process leading to the formation of motile spermatozoa characterized by a highly stable chromatin compaction that transfers the paternal genome into the oocyte. It is commonly held that these haploid cells are devoid of transcriptional and translational activities and that the transcripts represent remnants of stored mRNAs. Recently, the chromatin organization of mature spermatozoa has been revisited as a double nucleoprotamine-nucleohistone structure possessing less-condensed regions sensitive to nuclease activity, which could be implicated in the expression of genes involved in the early embryo development. The existence of a complex population of mRNAs in human sperm is well-documented, but their role is not yet elucidated. Evidence for a latent transcriptional capacity and/or a potential de novo translation in mature spermatozoa from fertile men are essential for understanding the last steps of sperm maturation, such as capacitation and acrosome reaction. As such, we have documented the relationship between sperm quality and the distribution of sperm RNAs by showing divergent levels of transcripts encoding for proteins involved in either nuclear condensation (protamines 1 and 2) or in capacitation (eNOS and nNOS, c-myc) or in motility and sperm survival (aromatase) between low and high motile sperm issued from the same sample. Therefore, analyzing the profile of mRNAs could be helpful either as a diagnostic tool for evaluating male fertility after spermatogenesis or for prognosis use for fertilization.

Gonadal expression of aromatase and estrogen receptor alpha genes in two races of Tunisian mice and their hypofertile hybrids.

House mice (Mus musculus domesticus) in Tunisia consists of two races, one carries the 40-acrocentric standard karyotypes and the other one is a robertsonian race (2n=22) homozygous for nine centric fusions (Rb). The F1 hybrids between the two chromosomal races showed a significant decrease in reproductive success and litter size. Such results can be related to the formation of meiotic trivalent in the hybrids leading to the production of viable aneuploid gametes and post-zygotic elimination of embryos due to chromosomal non disjunction events at meiosis. Moreover, testicular histology of F1 and backcross males showed in some cases a breakdown in spermatogenesis. In both females and males, androgens but also estrogens play an important role in gametogenesis. In this study, we have studied aromatase and estrogen receptor alpha (ERalpha) gene expression in the gonads of the two parental races and their chromosomal hybrids. The results showed that aromatase and ERalpha mRNAs are expressed in hybrid males of inter-racial crosses (female22Rb x male40Std and female40Std x male22Rb) and in hybrid females of inter-racial crosses (female22Rb x male40Std) as in the two parental races. However, in hybrid females of inter-racial crosses (female40Std x male22Rb) the amount of aromatase transcripts decreased sharply suggesting that this gene is involved in the breakdown of hybrid fertility in females, but not in males. However, in hybrid males, a putative post-translational modification of this enzyme, in terms of activity, should be verified.

Aromatase and estrogen receptors in male reproduction.

Aromatase is a terminal enzyme which transforms irreversibly androgens into estrogens and it is present in the endoplasmic reticulum of numerous tissues. We have demonstrated that mature rat germ cells express a functional aromatase with a production of estrogens equivalent to that of Leydig cells. In humans in addition to Leydig cells, we have shown the presence of aromatase in ejaculated spermatozoa and in immature germ cells. In most tissues, high affinity estrogen receptors, ERalpha and/or ERbeta, mediate the role of estrogens. Indeed, in human spermatozoa, we have successfully amplified ERbeta mRNA but the protein was not detectable. Using ERalpha antibody we have detected two proteins in human immature germ cells: one at the expected size 66 kDa and another at 46 kDa likely corresponding to the ERalpha isoform lacking exon 1. In spermatozoa only the 46 kDa isoform was present, and we suggest that it may be located on the membrane. In addition, in men genetically deficient in aromatase, it is reported that alterations of spermatogenesis occur both in terms of the number and motility of spermatozoa. All together, these observations suggest that endogenous estrogens are important in male reproduction.

Apoptotic effects of 25-hydroxycholesterol in immature rat Sertoli cells: prevention by 17beta-estradiol.

The aim of the present study was to determine whether or not apoptosis occurs in Sertoli cells in presence of 25-hydroxycholesterol, an oxysterol derived from cholesterol-containing foods or endogenous oxidation. Here, we provide evidence that 25-hydroxycholesterol can induce cultured Sertoli cells of immature rat to undergo apoptosis. The cell death was identified by analysis of fragmented DNA detected using enzyme-immunoassay. After 48 h of treatment with 50 microM of 25-hydroxycholesterol, apoptosis increased by 70% in Sertoli cells. Moreover, 50 microM of 25-hydroxycholesterol inhibited the incorporation of [14C] acetate into cholesterol by 70%. Addition of mevanolate to prevent isoprenoid deficiency do not inhibit the apoptosis generated by 25-hydroxycholesterol. In contrast, this increase of DNA fragmentation was reversed by addition of caspase-3 inhibitors as Ac-DEVD-CHO or Ac-ESMD-CHO. Bcl-2 mRNA level in the Sertoli cells decreased by 60% after 24 h exposure to 25-hydroxycholesterol. In parallel, Bax mRNA level increased by 40% in the Sertoli cells incubated in presence of 50 microM of 25-hydroxycholesterol. Physiological concentrations of 17beta-estradiol (10 or 100 nM) elicited a significant protection on apoptosis generated by 25-hydroxycholesterol in Sertoli cells. Our results show that the 25-hydroxycholesterol would control the cholesterol synthesis without toxic effect in immature rat Sertoli cells, these cells being able to protect themselves by estradiol production.

Induction of apoptosis by 25-hydroxycholesterol in adult rat Leydig cells: protective effect of 17beta-estradiol.

Testicular macrophages can convert cholesterol into 25-hydroxycholesterol which strongly stimulates Leydig cell testosterone production. We demonstrated that 25-hydroxycholesterol reduced cholesterol biosynthesis in adult rat Leydig cells. This oxysterol can also be cytotoxic. As hydroxylated cholesterol can induce apoptosis in various cells, we investigated cell death produced by 25-hydroxycholesterol. Apoptosis was characterized by TUNEL assay and by DAPI test. Addition of 25-hydroxycholesterol, during 24h, induced a dose dependent increase of apoptosis. This effect was reduced by a treatment with a caspase-3 inhibitor (Ac-DEVD-CHO). 25-Hydroxycholesterol is known to stimulate testosterone production, but an increase of intracellular or culture medium testosterone level does not modify significantly the percentage of apoptotic cells. In contrast, addition of 17beta-estradiol (2 nM) induced a decrease of apoptotic cells. These data suggested that this oxysterol can be used by rat Leydig cells in culture for sterol metabolism, but also induces apoptosis which could be inhibited by 17beta-estradiol.

Effects of extracts from Hibiscus macranthus and Basella alba mixture on testosterone production in vitro in adult rat testes slices.

AIM: To find an in vitro system for the measurement of the androgenic effects of different extracts of Hibiscus macranthus (Malvaceae) and Basella alba (Basellaceae). METHODS: The production of testosterone from testes slices incubated in two media, either Krebs-Henseleit buffer containing 0.5% Bovine serum albumin (BSA) or Dubecco's Modified Eagle's medium-F12 Ham nutrient mixture (DME/Ham F12), under a mixture of 5% CO2 in 95% air was determined either in the presence or absence of cofactors and Hibiscus macranthus plus Basella alba (HMBA) extracts. RESULTS: The testosterone production was increased in testes slices incubated in DME/Ham F12 medium in response to the cofactors (49%) and aqueous extracts (34%-60% according to dilutions). Under the same atmospheric conditions, there was no positive response of the testes slices to either cofactor or HMBA extract stimulation in Krebs-Henseleit buffer containing 0.5% BSA. In further investigations related to the effect of HMBA, the DME/Ham F12 medium was used. The results obtained from the in vitro test showed that the activity was present mainly in methylene chloride and methanol, since these extracts induced an increase in testosterone production by testes slices. CONCLUSION: The testes slice system is suitable to be used for further in vitro investigations of the isolation of androgenic bioactive components of plants.

Differential expression of steroidogenic factor-1/adrenal 4 binding protein and liver receptor homolog-1 (LRH-1)/fetoprotein transcription factor in the rat testis: LRH-1 as a potential regulator of testicular aromatase expression.

Aromatase converts testicular androgens to estrogens, which are essential for male fertility. Aromatase expression in testis occurs via transcription from promoter II, and requires the presence of a nuclear receptor half-site that binds the orphan receptor steroidogenic factor-1 [SF-1 (nuclear receptor 5A1)] to mediate basal and (in part) cAMP-induced transcription. We hypothesized that liver receptor homolog-1 (LRH-1) (nuclear receptor 5A2), a receptor closely related to SF-1, could also play a role in regulating aromatase expression in the testis. We demonstrate expression of LRH-1 in adult rat and immature mouse Leydig cells (LHR-1 > SF-1) as well as in pachytene spermatocytes and round spermatids but not in Sertoli cells, which in contrast, express high levels of SF-1. In transient transfection assays using TM3 Leydig cells and TM4 Sertoli cells, a rat promoter II luciferase reporter construct was stimulated by cotransfection of LRH-1 expression vector. Mutation analysis showed that induction by LRH-1 in TM3 and TM4 cells requires an AGGTCA motif at position -90, to which LRH-1 bound in gel shift analysis. We therefore provide evidence that LRH-1 plays an important role in the regulation of aromatase expression in Leydig cells. The colocalization of LRH-1 and aromatase to multiple testis cell types suggests that LRH-1 may have important effects on estrogen production, testis development, spermatogenesis, and testicular carcinogenesis.