Differential Effects of Bisphenol A and Diethylstilbestrol on Human,Rat and Mouse Fetal Leydig Cell Function

Endocrine disruptors (ED) have been incriminated in the current increase of male reproductive alterations. Bisphenol A (BPA) is a widely used weak estrogenic environmental ED and it is debated whether BPA concentrations within the average internal exposure are toxic. In the present study we investigated the effects of 10⁻¹² to 10⁻⁵ M BPA concentrations on fetal Leydig cell function, as fetal life is a critical period of sensitivity to ED effects on male reproductive function. To this aim, fetal testes from human at 6.5–10.5 gestational weeks (GW) or from rat and mouse at a comparable critical period of development (14.5 days post-coitum (dpc) for rat and 12.5 dpc for mouse) were explanted and cultured using our validated organotypic culture system in the presence or absence of BPA for 1–3 days. BPA concentrations as low as 10⁻⁸ M reduced testosterone secretion by human testes from day 1 of culture onwards, but not by mouse and rat testes where concentrations equal to 10⁻⁵ M BPA were required. Similarly, 10⁻⁸ M BPA reduced INSL3 mRNA levels only in human cultured testes. On the contrary, 10⁻⁵ and 10⁻⁶ M diethylstilbestrol (DES), a classical estrogenic compound, affected testosterone secretion only in rat and mouse testis cultures, but not in human testis cultures. Lastly, contrarily to the DES effect, the negative effect of BPA on testosterone produced by the mouse fetal testis was maintained after invalidation of estrogen receptor α (ERα). In conclusion, these results evidenced i) a deleterious effect of BPA on fetal Leydig cells function in human for concentrations from 10⁻⁸ M upwards, ii) species-specific differences raising concerns about extrapolation of data from rodent studies to human risk assessment, iii) a specific signaling pathway for BPA which differs from the DES one and which does not involve ERα.     

Bisphenol A Alters n-6 Fatty Acid Composition and Decreases Antioxidant Enzyme Levels in Rat Testes: A LC-QTOF-Based Metabolomics Study

Background

Male reproductive toxicity induced by exposure to bisphenol A (BPA) has been widely reported. The testes have proven to be a major target organ of BPA toxicity, so studying testicular metabolite variation holds promise for the discovery of mechanisms linked to the toxic effects of BPA on reproduction.

Methodology/Principal Findings

Male Sprague-Dawley rats were orally administered doses of BPA at the levels of 0, 50 mg/kg/d for 8 weeks. We used an unbiased liquid chromatography-quadrupole time-of-flight (LC-QTOF)-based metabolomics approach to discover, identify, and analyze the variation of testicular metabolites. Two n-6 fatty acids, linoleic acid (LA) and arachidonic acid (AA) were identified as potential testicular biomarkers. Decreased levels of LA and increased levels of AA as well as AA/LA ratio were observed in the testes of the exposed group. According to these suggestions, testicular antioxidant enzyme levels were detected. Testicular superoxide dismutase (SOD) declined significantly in the exposed group compared with that in the non-exposed group, and the glutathione peroxidase (GSH-Px) as well as catalase (CAT) also showed a decreasing trend in BPA treated group.

Conclusions/Significance

BPA caused testicular n-6 fatty acid composition variation and decreased antioxidant enzyme levels. This study emphasizes that metabolomics brings the promise of biomarkers identification for the discovery of mechanisms underlying reproductive toxicity.     

Cadmium-Induced Oxidative Stress in the Rat Testes: Protective Effects of Betaine

The aim of the present study was to evaluate the antioxidant effects of betaine against oxidative stress and pathological changes mediated by cadmium in the testes of rats. The adult male Wistar rats were allocated into three experimental groups as follows: the cadmium group received cadmium chloride at the dosage of 2 mg/kg intraperitoneally thereafter, the rats treated by physiological saline for 10 consecutive days. The betaine plus cadmium group received betaine at the dosage of 1.5 % w/w of the total diet orally for 10 consecutive days and cadmium chloride injected at the 2nd day of the betaine treatment. The control rats were injected physiological saline. Both testes of rats were removed for antioxidant assay and pathological changes evaluation on days 5 and 10 after cadmium toxicity. TBARS concentration (as a lipid peroxidation marker) was significantly higher in the cadmium group by day 10 compared to control and betaine plus cadmium groups, and it was significantly higher in cadmium group by day 5 in comparison with the controls. Catalase (CAT) and glutathione peroxidase activities decreased significantly by day 10 in cadmium group when compared to the controls. In contrast, CAT and superoxide dismutase activities increased significantly by day 10 in betaine plus cadmium group when compared to the cadmium group. In addition, the antioxidant effects of betaine could prevent testicular pathological changes in betaine plus cadmium group. The present data allow us to exploit the advantages of this nutrient agent in future studies.     

Protective Effects of Antioxidants Against Cadmium-induced Oxidative Damage in Rat Testes

The protective effects of melatonin, vitamin E, and selenium alone or in combination were tested against cadmium-induced oxidative damage in rat testes. A total of 60 male rats were equally divided into five study groups, one of which acted as control receiving subcutaneous injections of physiological saline. The remaining four groups were treated with subcutaneous injections of cadmium chloride at a dose of 1 mg/kg weight. The first study group received no treatment. The second group was treated with a combination of 60 mg/kg vitamin E and 1 mg/kg sodium selenite. Group 3 was treated with 10 mg/kg melatonin, and the fourth group received a combination of vitamin E, sodium selenite, and melatonin at the doses mentioned above. After 1 month, the animals were killed, and the testes were excised for histological inspection and determination of tissue malondialdehyde and the activity of superoxide dismutase. The animals receiving no treatment showed significantly higher malondialdehyde levels and reduced activity of the enzyme (p < 0.05). Treatment with antioxidants resulted in a significant reduction in malondialdehyde when compared to the nontreated animals (p < 0.05) and an increase in the superoxide dismutase activity that was almost the same as the controls. The combination of melatonin, vitamin E, and selenium appears to have the more profound effect against cadmium-induced testicular injury.     

Effects of Fluoride on Expression of P450, CREM and ACT Proteins in Rat Testes

Fluoride (F) is an essential trace element that humans and animals ingest from water, air, and fluoride-containing products; however, excessive fluoride absorption can damage a variety of organs and tissues, including the male reproductive system. Our previous studies found that fluoride exposure lowered sperm quality and interfered with spermatogenesis; however, the exact mechanism remained unclear. Proteins cytochrome P450 (P450), cAMP-responsive element modulator (CREM), and activator of CREM in testis (ACT) play the key roles in spermatogenesis and sperm motility. To investigate whether fluoride affects the expression of P450, CREM, and ACT, we used immunohistochemical techniques to determine expression levels of these proteins in testes of rats administered 100 mg NaF/L for 2 weeks via drinking water. The results showed that P450 expression was decreased while CREM and ACT expression was increased in the fluoride group, compared to the control. These data suggest that fluoride can impair male reproduction by affecting expression of P450, CREM, and ACT in the testes.     

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.     

Characterization of Rat Testes Mitochondrial Retinoylating System and Its Partial Purification

Retinoylation (retinoic acid acylation), a posttranslational modification of proteins occurring in a variety of eukariotic cell lines both in vivo and in vitro, was studied in rat testes mitochondria. all-trans-Retinoic acid, a highly active form of vitamin A in inducing cellular differentiation, is incorporated covalently into proteins of rat testes mitochondria. The maximum retinoylation activity of rat testes mitochondrial proteins was 21.6 pmoles mg protein(-1) 90 min(-1) at 37 degrees C. The activation energy was 44 kJ mol(-1) from 5 to 37 degrees C. The retinoylation activity had a pH optimum of 7.5. The retinoylation process was specific for the presence of ATP, ADP, and GTP (even if only 30% of the control). The half saturation constant (Km) was 0.69 microM for all-trans-retinoic acid, while the inhibition constant (Ki) was 1.5 microM for 13-cis-retinoic acid. Retinoylation was not inhibited by high concentrations of myristic acid (MA) and palmitic acid (PA), indicating that retinoylation and acylation reactions involved different rat testes mitochondrial proteins. The ATP or CoASH saturation curves of retinoylation reaction showed sigmoidal behavior with apparent half saturation constants (K0.5) of 6.5 mM ATP and 40.6 microM CoASH. On SDS-gel electrophoresis, the hydroxylapaptite/celite eluate showed various protein bands between 25 and 80 kDa. This retinoylated protein was purified 17-fold with respect to the mitochondrial extract.     

Bisphenol A Modulates Calcium Currents and Intracellular Calcium Concentration in Rat Dorsal Root Ganglion Neurons

The endocrine-disrupting chemical bisphenol A (BPA) is used to manufacture plastics including food containers, and it may leach into these containers. Consumption of BPA that has leached out of plastics may be harmful as recent research highlighted that BPA can induce alterations in the nervous system. In the present work, we studied the effects of BPA on Ca²⁺ channels in dorsal root ganglion (DRG) neurons. Using whole-cell patch-clamp recordings, we found that I _Ca could be reduced by BPA in a concentration-dependent manner. Additionally, BPA shifted the activation curve of calcium currents toward a depolarizing direction and increased the slope factor of the curve. The inactivation curve for the currents was also assessed, and the curve shifted toward the depolarizing direction, although it was not significant. Moreover, inhibitory effects of BPA on the increments of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) induced by 50 mM KCl were observed in DRG neurons using a laser scanning confocal microscopy assay. Further work revealed that the PKA and PKC pathways may be involved in the inhibitory effects of BPA since the PKA antagonist GÖ-6983 and the PKC antagonist H-89 significantly alleviated the inhibitory effects of BPA on I _Ca. As such, the results of the present study provide direct evidence that BPA decreases I _Ca and impairs calcium homeostasis, which may be involved in any toxic effects of BPA on DRG neurons.     

Comparative Investigation of Mesenchymal Stem Cells Isolated from the Bone Marrow and Fetal Liver of Mouse and Rat

We studied the properties of cells forming fibroblast colonies from the bone marrow and fetal liver of mouse and rat. Bone marrow and fetal liver cells formed colonies in vitro including fibroblasts as well as a considerable proportion of macrophages. The colonies formed from bone marrow and hepatic cells of rat differed from the murine ones by a higher proportion of fibroblasts. Most colonies derived from the bone marrow of both mouse and rat included a fraction of cells expressing alkaline phosphatase, and hence, capable of osteogenic differentiation; the colonies derived from the fetal liver included low proportions of such cells. The cell layers derived from the colony-forming fibroblasts of both bone marrow and fetal liver of mouse maintained hematopoiesis in the peritoneal cavity of irradiated mice, which indicated that these progenitor cells can form hematopoietic microenvironment.     

Antioxidant Defense of Betaine Against Oxidative Stress Induced by Ethanol in the Rat Testes

Oxidative stress is one of the factors associated with decline in fertility and betaine has been shown to bear antioxidant and methyl donor properties in our recent studies. Thus, we designed the present study to examine antioxidant and methyl donor abilities of betaine in oxidative stress induced by ethanol in the rat testes. The adult male Sprague-Dawley rats were divided into four experimental groups and treated daily for 2?months as follows: control, ethanol (4?g/kg, orally), betaine (1.5?% of total diet, orally), and betaine plus ethanol (betaine, 1.5?% of total diet and after 120?min, ethanol 4?g/kg). Sperm motility and concentration significantly increased in betaine group when compared to the ethanol?Ctreated rats. The main antioxidant enzyme (GPx) activity significantly increased (in order compensatory) in ethanol-treated rats when compared to betaine group while, antiperoxidative enzyme (CAT) activity significantly increased in betaine plus ethanol group as compared to ethanol-treated rats. Total homocysteine (tHcy) and TBARS concentration (as a lipid peroxidation marker) also significantly decreased in betaine and betaine plus ethanol groups as compared to ethanol-treated rats. Overall, methyl donor and antioxidant properties of betaine are promising and reduce the elevated tHcy and TBARS concentrations in betaine plus ethanol group. Therefore, betaine might be used as a potential therapy in hyperhomocysteinemia and oxidative stress induced by ethanol in alcoholism.     

Effects of Fetal Methylazoxymethanol Acetate Lesion on the Synaptic Neurochemistry of the Adult Rat Striatum

Abstract We used the cytotoxic properties of methylazoxymethanol acetate (MAM), which ablates mitotically active neuroblasts, to eliminate neurons in the fetal striatum to define the factors that regulate the development of the synaptic circuitry of this region. Adult rats whose mothers received a single intraperitoneal injection of 20 mg/kg of MAM on gestational days (DG) 14-17 were used in this study. MAM treatment at 14 DG caused a 49% decrease in striatal mass whereas treatment at 17 DG reduced the striatal weight by only 16%; MAM treatment on 15 or 16 DG gave intermediate results. Histologic analysis of Nissl-stained sections did not reveal an obvious disruption of striatal organization, although the region was clearly hypoplastic. The hypoplasia was associated with significant increases in the specific activities of choline acetyltransferase and tyrosine hydroxylase, although total activities of these enzymes per striatum were significantly depressed with the 14 or 15 DG treatments. In contrast, the specific activity of glutamate decarboxylase was unaffected by MAM treatment whereas the total activity of this enzyme was reduced commensurate with the degree of striatal hypoplasia. In rats lesioned at 15 DG, there was a similar 30% increase in the specific activities of all presynaptic dopaminergic markers studied. In contrast, the specific activity of the synaptosomal uptake process for [³H]choline was elevated by 60%, the specific activity of choline acetyltransferase was increased by only 30%, and the concentration of acetylcholine in the striatum was unchanged. Whereas the specific activities of glutamate decarboxylase and of the synaptosomal uptake process for [³H]γ-aminobutyric acid ([³H]GABA) were unaffected by the 15 DG MAM treatment, the concentration of GABA was increased significantly by 20%. The specific binding of [³H]spiroperidol, [³H]quinuclidinyl benzilate ([³H]QNB). and [³H] muscimol to, respectively, dopamine, muscarinic, and GABA receptors was unchanged by the 15 DG MAM lesion. The nigral dopaminergic perikarya appeared unaffected by the 15 DG MAM lesion in that the tyrosine hydroxylase activity remained normal. Consistent with the loss of striatal GABAergic perikarya, the specific activities of glutamate decarboxylase and of the synaptosomal uptake process for [³H]GABA were significantly reduced in the substantia nigra; however, the concentration of endogenous GABA was twofold greater than in control in this terminal region. The results of these studies indicate that the nigro-striatal dopaminergic pathway only partially compensates for the loss of neurons in its terminal field within the hypoplastic striatum. Striatal cholinergic and GABAergic neurons differ considerably in their responses to the MAM lesion, suggesting that they are derived from different neuroblast pools. Finally, the altered synaptic relationships induced by the fetal lesion may affect neurotransmitter turnover as evidenced by disparities in GABA and acetylcholine levels when compared with other presynaptic markers for the GABAergic and cholinergic neurons.     

Simvastatin Treatment Ameliorates Injury of Rat Testes Induced by Cadmium Toxicity

Cadmium-induced testicular toxicity is mediated through oxidative stress and inflammation which eventually lead to cell death. Simvastatin, the antihyperlipidemic agent, exhibits additional antioxidant and anti-inflammatory activities. The aim of the present work was to investigate the protective effect of simvastatin against cadmium-induced testicular toxicity in rats. The rats received a single intraperitoneal (i.p.) injection of cadmium chloride (2 mg/kg). Simvastatin treatment (5 mg/kg/day, i.p.) was applied for three consecutive days, starting 1 day before cadmium administration. Cadmium significantly decreased serum testosterone, and testicular reduced glutathione and catalase activity, and significantly increased testicular malondialdehyde, nitric oxide, and cadmium ion levels. Simvastatin significantly ameliorated the biochemical changes induced by cadmium. Cadmium-induced testicular tissue injury observed by histopathological examination was attenuated by simvastatin. In addition, simvastatin significantly decreased the expression of inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α, nuclear factor-κB, and caspase-3, and increased heme oxygenase-1 expression in testicular tissue of rats exposed to cadmium toxicity. It was concluded that simvastatin, through its antioxidant and anti-inflammatory activities, provided a significant protective effect against cadmium-induced testicular toxicity in rats. However, starting treatment with simvastatin before cadmium exposure, as done in the present work, is not clinically applicable. Therefore, other investigations are needed to assess the protective effect of simvastatin treatment following induction of cadmium testicular toxicity.     

Fetal Rat Pancreas in Organ Culture: Effects of Serum on the Development of the Endocrine Cells

Eighteen-day-old rat fetal pancreas was grown in organ culture for four days on medium consisting of tissue culture Medium 199 and varying concentrations of chicken serum. The glucagon and somatostatin concentration of the explants was decreased when the serum concentration of the medium was reduced from 50 to 10%. There was a further reduction in these hormones when the explants were cultured on Medium 199 alone. Explant insulin content was reduced only when serum was omitted from the medium. A serum factor tripeptide was not able to substitute for this serum requirement. Heat-inactivation of the serum resulted in a significant increase in medium insulin content and an increase in both the insulin and glucagon contents of the explants. This increase in hormone content was directly correlated with increases in the beta and alpha cell volumes of the explants. There was no change in the somatostatin content or delta cell volume of the explants grown on heat-inactivated medium. It is suggested that the serum is an important component of the culture media and is apparently required in high concentration for the development of the islet cells in vitro. The islet cell types differ in their requirement for serum. The alpha and delta require a higher concentration than do the beta cells.     

A Rapid Two-Step Purification of Rat Liver Fetal Thymidine Kinase

The fetal isoenzyme of thymidine kinase was purified to apparent homogeneity from cytosols of rat fetuses liver. A two-step purification including anion exchange chromatography and affinity chromatography was developed. The purified enzyme appears as oligomeric with a relative molecular weight of 71 kDa. In denaturing media its molecular weight was 24 kDa, and its pHi 8.3.     

Gangliosides in Human Fetal Brain

The ganglioside concentration and composition were determined in 42 human fetal brains from gestational week 10 to 22, a period that is morphologically characterized by rapid neuroblast proliferation and migration. The ganglioside concentration was constant during this period, approximately 1 mumol of ganglioside sialic acid/g of fresh tissue weight. At gestational week 10 the ganglioside pattern was dominated by gangliosides of the ganglio b series, with the major ganglioside being GT1b, contributing 40% of total ganglioside sialic acid, whereas GD1b and GD3 contributed only 15 and 10%, respectively. The proportion of b series ganglioside decreased to gestational week 22, with the most pronounced relative reduction affecting GD3, but also GT1b and GD1b to a lesser extent. The ganglioside GQ1b increased in content from gestational week 10 and peaked around week 16. The proportion of GD1a increased markedly between gestational week 12 and 14 and slowly between week 14 and 18 and then increased rapidly from week 20. Ganglioside GM1 underwent a similar change. Gangliosides of the lacto series contributed 6-10% of ganglioside sialic acid between gestational week 10 and 15, and thereafter the proportion slowly decreased. 3'-isoLM1 decreased rapidly in content from gestational week 10 (20 nmol/g of fresh weight) to week 22 (less than 0.5 nmol/g of fresh weight), whereas the gangliosides of the neolacto series (3'-LM1 and 3',8'-LD1) showed a slower and less marked decline in level. The biological significance of the ganglioside changes is discussed.