Investigation of Testosterone,Androstenone, and Estradiol Metabolism in HepG2 Cells and Primary Culture Pig Hepatocytes and Their Effects on 17βHSD7 Gene Expression

Steroid metabolism is important in various species. The accumulation of androgen metabolite, androstenone, in pig adipose tissue is negatively associated with pork flavor, odour and makes the meat unfit for human consumption. The 17β-hydroxysteroid dehydrogenase type 7 (17βHSD7) expressed abundantly in porcine liver, and it was previously suggested to be associated with androstenone levels. Understanding the enzymes and metabolic pathways responsible for androstenone as well as other steroids metabolism is important for improving the meat quality. At the same time, metabolism of steroids is known to be species- and tissue-specific. Therefore it is important to investigate between-species variations in the hepatic steroid metabolism and to elucidate the role of 17βHSD7 in this process. Here we used an effective methodological approach, liquid chromatography coupled with mass spectrometry, to investigate species-specific metabolism of androstenone, testosterone and beta-estradiol in HepG2 cell line, and pig cultured hepatocytes. Species- and concentration-depended effect of steroids on 17βHSD7 gene expression was also investigated. It was demonstrated that the investigated steroids can regulate the 17βHSD7 gene expression in HepG2 and primary cultured porcine hepatocytes in a concentration-dependent and species-dependent pattern. Investigation of steroid metabolites demonstrated that androstenone formed a 3′-hydroxy compound 3β-hydroxy-5α-androst-16-ene. Testosterone was metabolized to 4-androstene-3,17-dione. Estrone was found as the metabolite for β-estradiol. Inhibition study with 17βHSD inhibitor apigenin showed that apigenin didn’t affect androstenone metabolism. Apigenin at high concentration (50 µM) tends to inhibit testosterone metabolism but this inhibition effect was negligible. Beta-estradiol metabolism was notably inhibited with apigenin at high concentration. The study also established that the level of testosterone and β-estradiol metabolites was markedly increased after co-incubation with high concentration of apigenin. This study established that 17βHSD7 is not the key enzyme responsible for androstenone and testosterone metabolism in porcine liver cells.     

A single injection of 17β-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E₂) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E₂, as was the combination of E₂ and DHT. The relative efficacy of a single dose of T, DHT, and E₂ plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E₂B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E₂, and E₂B suggests that an appreciable, but not necessarily sustained, elevation of E₂ levels in the brain may be critical in the facilitation of male copulatory behavior in mice.     

Different mechanisms of regulation of nuclear reduced nicotinamide-adenine dinucleotide phosphate-dependent 3-oxo steroid 5alpha-reductase activity in rat liver, kidney and prostate

The regulatory mechanisms involved in the control of the nuclear NADPH-dependent 3-ketosteroid 5α-reductase (5α-reductase) activity were studied in liver, kidney and prostate. The substrate used was [1,2-³H]androst-4-ene-3,17-dione (androstenedione) (for liver and kidney) or [4-¹⁴C]androstenedione (for prostate). The hepatic nuclear 5α-reductase activity was greater in female than in male rats, was greater in adult than in prepubertal female rats, increased after castration of male rats, but was not affected by treatment with testosterone propionate or oestradiol benzoate. These regulatory characteristics are in part different from those previously described for the hepatic microsomal 5α-reductase. The renal nuclear metabolism of androstenedione, i.e. 5α reduction and 17β-hydroxy steroid reduction, was relatively unaffected by sex, age, castration and treatment with testosterone propionate. However, treatment of castrated male rats with oestradiol benzoate led to a significant increase in the 5α-reductase activity and a significant decrease in the 17β-hydroxy steroid reductase activity. Finally, the nuclear 5α-reductase activity in prostate was androgen-dependent, decreasing after castration and increasing after treatment with testosterone propionate. In conclusion, the nuclear 5α-reductase activities in liver, kidney and prostate seem to be under the control of distinctly different regulatory mechanisms. The hypothesis is presented that whereas the prostatic nuclear 5α-reductase participates in the formation of a physiologically active androgen, 5α-dihydrotestosterone, this may not be the true function of the nuclear 5α-reductase in liver and kidney. These enzymes might rather serve to protect the androgen target sites in the chromatin from active androgens (e.g. testosterone) by transforming them into less active androgens (e.g. 5α-androstane-3,17-dione and/or 5α-dihydrotestosterone).     

Zearalenone induces chromosome aberrations in mouse bone marrow: preventive effect of 17β-estradiol, progesterone and Vitamin E

The cytogenetic effect of zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, was evaluated in vivo, in mouse bone marrow cells, by assessing the percentage of cells bearing different chromosome aberrations. The studies included different conditions for animal treatment, as follows: (1) single intraperitoneal (ip) injection, (2) repeated ip injections, (3) pre-treatment for 24 h with Vitamin E (Vit E), and (4) pre-treatment for 4 h with 17β-estradiol (17β-Est) or progesterone (Prog). ZEN induced different types of chromosome aberrations, which was concentration-dependent (2–20 mg/kg bw). These doses corresponded to 0.4–4% of the LD₅₀ in the mouse. Interestingly, when the dose of ZEN (40 mg/kg) was fractionated into four equivalent doses (4 × 10 mg/kg bw), into three doses (15 + 10 + 15 mg/kg bw), or into two equivalent doses (2 × 20 mg/kg bw), given every 24 h, the percentage of chromosome aberrations increased significantly. This finding suggests that ZEN proceeds by reversible binding on receptors that could become saturated, and that it damages the chromosomes in a ‘hit and go’ manner. Furthermore, pre-treatment of animals with 17β-estradiol or progesterone significantly decreased the percentage of chromosome aberrations, suggesting that (i) these hormones bind to the same cytoplasmic receptors transported into the nucleus to elicit DNA damage, (ii) they may play a role in preventing chromosome aberrations induced by ZEN. Similarly, Vit E prevented these chromosome aberrations indicating that Vit E, previously reported to prevent most of the toxic effects induced by ZEN, may also bind to the same receptors.     

Sex Bias in Experimental Immune-Mediated,Drug-Induced Liver Injury in BALB/c Mice: Suggested Roles for Tregs,Estrogen, and IL-6

Background and Aims

Immune-mediated, drug-induced liver injury (DILI) triggered by drug haptens is more prevalent in women than in men. However, mechanisms responsible for this sex bias are not clear. Immune regulation by CD4+CD25+FoxP3+ regulatory T-cells (Tregs) and 17β-estradiol is crucial in the pathogenesis of sex bias in cancer and autoimmunity. Therefore, we investigated their role in a mouse model of immune-mediated DILI.

Methods

To model DILI, we immunized BALB/c, BALB/cBy, IL-6–deficient, and castrated BALB/c mice with trifluoroacetyl chloride-haptenated liver proteins. We then measured degree of hepatitis, cytokines, antibodies, and Treg and splenocyte function.

Results

BALB/c females developed more severe hepatitis (p<0.01) and produced more pro-inflammatory hepatic cytokines and antibodies (p<0.05) than did males. Castrated males developed more severe hepatitis than did intact males (p<0.001) and females (p<0.05). Splenocytes cultured from female mice exhibited fewer Tregs (p<0.01) and higher IL-1β (p<0.01) and IL-6 (p<0.05) than did those from males. However, Treg function did not differ by sex, as evidenced by absence of sex bias in programmed death receptor-1 and responses to IL-6, anti-IL-10, anti-CD3, and anti-CD28. Diminished hepatitis in IL-6-deficient, anti-IL-6 receptor α-treated, ovariectomized, or male mice; undetectable IL-6 levels in splenocyte supernatants from ovariectomized and male mice; elevated splenic IL-6 and serum estrogen levels in castrated male mice, and IL-6 induction by 17β-estradiol in splenocytes from naïve female mice (p<0.05) suggested that 17β-estradiol may enhance sex bias through IL-6 induction, which subsequently discourages Treg survival. Treg transfer from naïve female mice to those with DILI reduced hepatitis severity and hepatic IL-6.

Conclusions

17β-estradiol and IL-6 may act synergistically to promote sex bias in experimental DILI by reducing Tregs. Modulating Treg numbers may provide a therapeutic approach to DILI.     

Androgen Excess Produces Systemic Oxidative Stress and Predisposes to β-Cell Failure in Female Mice

In women, excess production of the male hormone, testosterone (T), is accompanied by insulin resistance. However, hyperandrogenemia is also associated with β-cell dysfunction and type 2 diabetes raising the possibility that androgen receptor (AR) activation predisposes to β-cell failure. Here, we tested the hypothesis that excess AR activation produces systemic oxidative stress thereby contributing to β-cell failure. We used normal female mice (CF) and mice with androgen resistance by testicular feminization (Tfm). These mice were exposed to androgen excess and a β-cell stress induced by streptozotocin (STZ). We find that following exposure to T, or the selective AR-agonist dehydrotestosterone (DHT), CF mice challenged with STZ, which are normally protected, are prone to β-cell failure and insulin-deficient diabetes. Conversely, T-induced predisposition to β-cell failure is abolished in Tfm mice. We do not observe any proapoptotic effect of DHT alone or in the presence of H₂O₂ in cultured mouse and human islets. However, we observe that exposure of CF mice to T or DHT provokes systemic oxidative stress, which is eliminated in Tfm mice. This work has significance for hyperandrogenic women; excess activation of AR by testosterone may provoke systemic oxidative stress. In the presence of a prior β-cell stress, this may predispose to β-cell failure.     

Mouse Leydig Cells with Different Androgen Production Potential Are Resistant to Estrogenic Stimuli but Responsive to Bisphenol A Which Attenuates Testosterone Metabolism

It is well known that estrogens and estrogen-like endocrine disruptors can suppress steroidogenic gene expression, attenuate androgen production and decrease differentiation of adult Leydig cell lineage. However, there is no information about the possible link between the potency of Leydig cells to produce androgens and their sensitivity to estrogenic stimuli. Thus, the present study explored the relationship between androgen production potential of Leydig cells and their responsiveness to estrogenic compounds. To investigate this relationship we selected mouse genotypes contrasting in sex hormone levels and differing in testosterone/estradiol (T/E₂) ratio. We found that two mouse genotypes, CBA/Lac and C57BL/6j have the highest and the lowest serum T/E₂ ratio associated with increased serum LH level in C57BL/6j compared to CBA/Lac. Analysis of steroidogenic gene expression demonstrated significant upregulation of Cyp19 gene expression but coordinated suppression of LHR, StAR, 3βHSDI and Cyp17a1 in Leydig cells from C57BL/6j that was associated with attenuated androgen production in basal and hCG-stimulated conditions compared to CBA/Lac mice. These genotype-dependent differences in steroidogenesis were not linked to changes in the expression of estrogen receptors ERα and Gpr30, while ERβ expression was attenuated in Leydig cells from C57BL/6j compared to CBA/Lac. No effects of estrogenic agonists on steroidogenesis in Leydig cells from both genotypes were found. In contrast, xenoestrogen bisphenol A significantly potentiated hCG-activated androgen production by Leydig cells from C57BL/6j and CBA/Lac mice by suppressing conversion of testosterone into corresponding metabolite 5α-androstane-3α,17β-diol. All together our data indicate that developing mouse Leydig cells with different androgen production potential are resistant to estrogenic stimuli, while xenoestrogen BPA facilitates hCG-induced steroidogenesis in mouse Leydig cells via attenuation of testosterone metabolism. This cellular event can cause premature maturation of Leydig cells that may create abnormal intratesticular paracrine milieu and disturb proper development of germ cells.     

Non-Genomic Estrogen Regulation of Ion Transport and Airway Surface Liquid Dynamics in Cystic Fibrosis Bronchial Epithelium

Male cystic fibrosis (CF) patients survive longer than females and lung exacerbations in CF females vary during the estrous cycle. Estrogen has been reported to reduce the height of the airway surface liquid (ASL) in female CF bronchial epithelium. Here we investigated the effect of 17β-estradiol on the airway surface liquid height and ion transport in normal (NuLi-1) and CF (CuFi-1) bronchial epithelial monolayers. Live cell imaging using confocal microscopy revealed that airway surface liquid height was significantly higher in the non-CF cells compared to the CF cells. 17β-estradiol (0.1–10 nM) reduced the airway surface liquid height in non-CF and CF cells after 30 min treatment. Treatment with the nuclear-impeded Estrogen Dendrimer Conjugate mimicked the effect of free estrogen by reducing significantly the airway surface liquid height in CF and non-CF cells. Inhibition of chloride transport or basolateral potassium recycling decreased the airway surface liquid height and 17β-estradiol had no additive effect in the presence of these ion transporter inhibitors. 17β-estradiol decreased bumetanide-sensitive transepithelial short-circuit current in non-CF cells and prevented the forskolin-induced increase in ASL height. 17β-estradiol stimulated an amiloride-sensitive transepithelial current and increased ouabain-sensitive basolateral short-circuit current in CF cells. 17β-estradiol increased PKCδ activity in CF and non-CF cells. These results demonstrate that estrogen dehydrates CF and non-CF ASL, and these responses to 17β-estradiol are non-genomic rather than involving the classical nuclear estrogen receptor pathway. 17β-estradiol acts on the airway surface liquid by inhibiting cAMP-mediated chloride secretion in non-CF cells and increasing sodium absorption via the stimulation of PKCδ, ENaC and the Na⁺/K⁺ATPase in CF cells.     

Combined 17β-Estradiol with TCDD Promotes M2 Polarization of Macrophages in the Endometriotic Milieu with Aid of the Interaction between Endometrial Stromal Cells and Macrophages

The goal of this study is to elucidate the effects of 17β-estradiol and TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) on macrophage phenotypes in the endometriotic milieu. Co-culture of endometrial stromal cells (ESCs) and U937 cells (macrophage cell line) was performed to simulate the endometriotic milieu and to determine the effects of 17β-estradiol and/or TCDD on IL10, IL12 production and HLA-DR, CD86 expression by U937 macrophages. We found that combining 17β-estradiol with TCDD has a synergistic effect on inducing M2 activation when macrophages are co-cultured with ESCs. Moreover, the combination of 17β-estradiol and TCDD significantly enhanced STAT3 and P38 phosphorylation in macrophages. Differentiation of M2 macrophages induced by 17β-estradiol and TCDD were effectively abrogated by STAT3 and P38MAPK inhibitors, but not by ERK1/2 and JNK inhibitors. In conclusion, 17β-estradiol and TCDD in the ectopic milieu may lead to the development of endometriosis by inducing M2 polarization of macrophages through activation of the STAT3 and P38MAPK pathways.     

17β-Estradiol Elevates cGMP and,via Plasma Membrane Recruitment of Protein Kinase GIα, Stimulates Ca2+ Efflux from Rat Hepatocytes

Rapid non-genomic effects of 17β-estradiol, the principal circulating estrogen, have been observed in a wide variety of cell types. Here we investigate rapid signaling effects of 17β-estradiol in rat hepatocytes. We show that, above a threshold concentration of 1 nm, 17β-estradiol, but not 17α-estradiol, stimulates particulate guanylyl cyclase to elevate cGMP, which through activation and plasma membrane recruitment of protein kinase G isoform Iα, stimulates plasma membrane Ca²⁺-ATPase-mediated Ca²⁺ efflux from rat hepatocytes. These effects are extremely rapid in onset and are mimicked by a membrane-impermeant 17β-estradiol-BSA conjugate, suggesting that 17β-estradiol acts at the extracellular face of the plasma membrane. We also show that 17β-estradiol binds specifically to the intact hepatocyte plasma membrane through an interaction that is competed by an excess of atrial natriuretic peptide but also shows many similarities to the pharmacological characteristics of the putative γ-adrenergic receptor. We, therefore, propose that the observed rapid signaling effects of 17β-estradiol are mediated either through the guanylyl cyclase A receptor for atrial natriuretic peptide or through the γ-adrenergic receptor, which is either itself a transmembrane guanylyl cyclase or activates a transmembrane guanylyl cyclase through cross-talk signaling.     

Metabolism of Androstenone, 17β-Estradiol and Dihydrotestosterone in Primary Cultured Pig Hepatocytes and the Role of 3β-Hydroxysteroid Dehydrogenase in This Process

Steroids metabolism plays an important role in mammals and contributes to quality of pig meat. The main objective of this study was to identify metabolites of androstenone, 17β-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. The role of 3β-hydroxysteroid dehydrogenase (3βHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor. Steroid glucuronide conjugated metabolites were detected by liquid chromatography time of flight mass spectrometry (LC-TOF-MS). 3βHSD enzyme was essential for metabolism of androstenone to 5α-androst-16-en-3β-ol, which then formed androstenone glucuronide conjugate. Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol. The ratio of the two metabolites was around 5∶1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.     

Fighting experience alters brain androgen receptor expression dependent on testosterone status

Contest decisions are influenced by the outcomes of recent fights (winner–loser effects). Steroid hormones and serotonin are closely associated with aggression and therefore probably also play important roles in mediating winner–loser effects. In mangrove rivulus fish, Kryptolebias marmoratus, individuals with higher testosterone (T), 11-ketotestosterone and cortisol levels are more capable of winning, but titres of these hormones do not directly mediate winner–loser effects. In this study, we investigated the effects of winning/losing experiences on brain expression levels of the receptor genes for androgen (AR), oestrogen α/β (ER_α/β), glucocorticoid (GR) and serotonin (5-HT_1AR). The effect of contest experience on AR gene expression depended on T levels: repeated losses decreased, whereas repeated wins increased AR gene expression in individuals with low T but not in individuals with medium or high T levels. These results lend strong support for AR being involved in mediating winner–loser effects, which, in previous studies, were more detectable in individuals with lower T. Furthermore, the expression levels of ER_α/β, 5-HT_1AR and GR genes were higher in individuals that initiated contests against larger opponents than in those that did not. Overall, contest experience, underlying endocrine state and hormone and serotonin receptor expression patterns interacted to modulate contest decisions jointly.     

The Olfactory Transcriptome and Progression of Sexual Maturation in Homing Chum Salmon Oncorhynchus keta

Reproductive homing migration of salmonids requires accurate interaction between the reception of external olfactory cues for navigation to the spawning grounds and the regulation of sexual maturation processes. This study aimed at providing insights into the hypothesized functional link between olfactory sensing of the spawning ground and final sexual maturation. We have therefore assessed the presence and expression levels of olfactory genes by RNA sequencing (RNAseq) of the olfactory rosettes in homing chum salmon Oncorhynchus keta Walbaum from the coastal sea to 75 km upstream the rivers at the pre-spawning ground. The progression of sexual maturation along the brain-pituitary-gonadal axis was assessed through determination of plasma steroid levels by time-resolved fluoroimmunoassays (TR-FIA), pituitary gonadotropin subunit expression and salmon gonadotropin-releasing hormone (sgnrh) expression in the brain by quantitative real-time PCR. RNAseq revealed the expression of 75 known and 27 unknown salmonid olfactory genes of which 13 genes were differentially expressed between fish from the pre-spawning area and from the coastal area, suggesting an important role of these genes in homing. A clear progression towards final maturation was characterised by higher plasma 17α,20β-dihydroxy-4-pregnen-3-one (DHP) levels, increased pituitary luteinizing hormone β subunit (lhβ) expression and sgnrh expression in the post brain, and lower plasma testosterone (T) and 17β-estradiol (E2) levels. Olfactomedins and ependymin are candidates among the differentially expressed genes that may connect olfactory reception to the expression of sgnrh to regulate final maturation.     

Regulation of Guinea Pig Detrusor Smooth Muscle Excitability by 17β-Estradiol: The Role of the Large Conductance Voltage- and Ca2+-Activated K+ Channels

Estrogen replacement therapies have been suggested to be beneficial in alleviating symptoms of overactive bladder. However, the precise regulatory mechanisms of estrogen in urinary bladder smooth muscle (UBSM) at the cellular level remain unknown. Large conductance voltage- and Ca²⁺-activated K⁺ (BK) channels, which are key regulators of UBSM function, are suggested to be non-genomic targets of estrogens. This study provides an electrophysiological investigation into the role of UBSM BK channels as direct targets for 17β-estradiol, the principle estrogen in human circulation. Single BK channel recordings on inside-out excised membrane patches and perforated whole cell patch-clamp were applied in combination with the BK channel selective inhibitor paxilline to elucidate the mechanism of regulation of BK channel activity by 17β-estradiol in freshly-isolated guinea pig UBSM cells. 17β-Estradiol (100 nM) significantly increased the amplitude of depolarization-induced whole cell steady-state BK currents and the frequency of spontaneous transient BK currents in freshly-isolated UBSM cells. The increase in whole cell BK currents by 17β-estradiol was eliminated upon blocking BK channels with paxilline. 17β-Estradiol (100 nM) significantly increased (~3-fold) the single BK channel open probability, indicating direct 17β-estradiol-BK channel interactions. 17β-Estradiol (100 nM) caused a significant hyperpolarization of the membrane potential of UBSM cells, and this hyperpolarization was reversed by blocking the BK channels with paxilline. 17β-Estradiol (100 nM) had no effects on L-type voltage-gated Ca²⁺ channel currents recorded under perforated patch-clamp conditions. This study reveals a new regulatory mechanism in the urinary bladder whereby BK channels are directly activated by 17β-estradiol to reduce UBSM cell excitability.     

Differential Ligand Binding Affinities of Human Estrogen Receptor-α Isoforms

Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17β-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17β-estradiol bound ER66 and ER46 with K_d values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17β-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17β-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.     

A single injection of 17 beta-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E₂) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E₂, as was the combination of E₂ and DHT. The relative efficacy of a single dose of T, DHT, and E₂ plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E₂B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E₂, and E₂B suggests that an appreciable, but not necessarily sustained, elevation of E₂ levels in the brain may be critical in the facilitation of male copulatory behavior in mice.     

Therapeutic Potential of Date Palm Pollen for Testicular Dysfunction Induced by Thyroid Disorders in Male Rats

Hyper- or hypothyroidism can impair testicular function leading to infertility. The present study was designed to examine the protective effect of date palm pollen (DPP) extract on thyroid disorder-induced testicular dysfunction. Rats were divided into six groups. Group I was normal control. Group II received oral DPP extract (150 mg kg^-1), group III (hyperthyroid group) received intraperitoneal injection of L-thyroxine (L-T4, 300μg kg^-1; i.p.), group IV received L-T4 plus DPP extract, group V (hypothyroid group) received propylthiouracil (PTU, 10 mg kg^-1; i.p.) and group VI received PTU plus DPP extract. All treatments were given every day for 56 days. L-T4 or PTU lowered genital sex organs weight, sperm count and motility, serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone (T), testicular function markers and activities of testicular 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD). Moreover, L-T4 or PTU increased estradiol (E2) serum level, testicular oxidative stress, DNA damage and apoptotic markers. Morphometric and histopathologic studies backed these observations. Treatment with DPP extract prevented LT4- or PTU induced changes. In addition, supplementation of DPP extract to normal rats augmented sperm count and motility, serum levels of LH, T and E2 paralleled with increased activities of 3β-HSD and 17β-HSD as well as testicular antioxidant status. These results provide evidence that DPP extract may have potential protective effects on testicular dysfunction induced by altered thyroid hormones.     

Studies on glutathione metabolism in ventral prostate and chemically induced prostatic carcinoma in rats

Glutathione content and the activity of glutathione reductase were examined in ventral prostate and chemically induced 11095 squamous-cell prostatic carcinoma in rats, Castration produced a significant reduction in the levels of reduced (GSH) and oxidized (GSSG) glutathione and glutathione reductase activity in the prostate. Replacement of testosterone (50 mg/kg) daily for 7 days to castrated animals elevated the reduced glutathione level and the activity of glutathione reductase almost to normal limits, Squamous-cell carcinoma was implanted in castrated and intact animals, Tumor growth in normal rats produced a decrease of almost 30% in the weight of the ventral prostate at 21 days post-implantation, although the glutathione levels remained unaffected. Much greater activity of glutathione reductase was detected in the tumor in comparison to the values noted for the normal tissue, The tumor also showed significantly higher values for the GSH/GSSG ratio, No apparent difference could be found in the rate of the growth of tumors whether implanted in normal or castrated animals, The levels of reduced and oxidized glutathione and glutathione reductase activity also seemed identical in tumors obtained from both groups of animals, Administration of testosterone (50 mg/kg) or β-estradiol (2 mg/kg) daily for 11 days to tumor-bearing castrated animals did not alter the levels of glutathione and glutathione reductase activity. A significantly higher level of blood reduced glutathione was found in tumor-bearing rats in comparison to that seen for the normal subjects. Our results demonstrate that androgen depletion and replacement therapy influence the metabolism of glutathione in rat ventral prostate. Squamous-cell carcinoma of the prostate appears to differ from the normal tissue with respect to the observed androgen effects, There is dissimilarity in the metabolism of glutathione in the two tissues since greater activity of glutathione reductase and lower values of reduced glutathione were seen in the tumor as compared to t h o s e of the ventral prostate. Treatment with β-estradiol, an antiprostatic agent, does not seem to influence the growth or glutathione metabolism of squamous-cell carcinoma of the prostate. The observed changes in blood glutathione levels might prove to be useful as an index of rapid growth of the neoplastic tissue.