Immunohistochemistry and in situ hybridization of P-45017 alpha (17 alpha-hydroxylase/17,20-lyase).

Cytochrome P-45017 alpha catalyzes both 17 alpha-hydroxylation and 17,20-side-chain cleavage in steroidogenesis and lies at a key branch point in the pathways of steroid hormone biosynthesis. To obtain information on the precise localization of P-45017 alpha in swine testis, ovary, and adrenal, we undertook the simultaneous detection of P-45017 alpha mRNA and protein by combining immunohistochemistry with in situ hybridization. In situ hybridization was performed on 4% paraformaldehyde-fixed, paraffin-embedded sections by employing either a 39-base oligomer or a cDNA insert (1.7 KB) of porcine testis P-45017 alpha as DNA probe. Immunohistochemical study was performed by employing anti-P-45017 alpha. Hybridization signals were obtained in Leydig cells of the testis, theca interna of the ovarian follicle, and zona fasciculata reticularis cells of the adrenal cortex. Oligonucleotide probing yielded lower background signal than the cDNA probe. No specific signals were obtained in seminiferous tubules of the testis, medulla, and zona glomerulosa of the adrenal, and in membrana granulosa and interstitial cells of the ovary. Hybridization signals were obtained in the cells where immunoreactivity of the enzyme was observed by immunohistochemistry, except for some Leydig cells of the testis and theca interna cells of the ovary in which only immunoreactivity but not hybridization signal was observed. The present study provided detailed information about the precise cellular localization of P-45017 alpha expression at both the protein and mRNA levels in swine adrenal glands and gonads. This approach of simultaneous immunohistochemistry and in situ hybridization analysis of steroidogenic enzymes can be applied in the future to tissues exhibiting abnormal steroid metabolism and should contribute to a better understanding of steroidogenesis.     

Alleviation of lindane induced toxicity in testis of Swiss mice (Mus musculus) by combined treatment with vitamin C, vitamin E and alpha-lipoic acid

Mitigation of lindane induced toxicity in testis of Swiss mice by combined treatment with vitamin C, vitamin E and alpha-lipoic acid has been evaluated. Male healthy mice (40), 8-10 weeks old were randomly selected and divided into 4 groups, control (C); lindane (L); antioxidant (A) and antioxidant plus lindane (A+L). Group C animals were administered only the vehicle (olive oil); in group L lindane was administered orally at a dose of 40 mg/kg body wt.; in group A combination of antioxidants at a dose of 125 mg/kg body wt.(vitamin C: 50 mg/kg body wt., vitamin E: 50 mg/kg body wt. and alpha-lipoic acid: 25 mg/kg body wt.) was administered orally; in group A+L both antioxidants (125 mg/kg body wt.) and lindane (40 mg/kg body wt.) were administered at their respective doses. In group A+L antioxidants were administered 1 h prior to lindane administration. All treatments were continuously given for 60 days. Histopathological changes due to lindane intoxication indicated shrunken and distorted seminiferous tubules, sparse Leydig cells and blood vessels and atrophy in the tissue. The testis weight also decreased significantly. Lindane treated group showed increased lipid peroxidation, whereas glutathione, glutathione peroxidase, superoxide dismutase, catalase and protein were significantly decreased compared to control. Lindane induced damage was minimized by administration of antioxidants. Results suggest that combined pretreatment with antioxidants can alleviate the damage caused to testis by lindane.     

R 76713, a new specific non-steroidal aromatase inhibitor

The effects of R 76713, a new triazole derivative, on rat ovarian, testicular and adrenal steroidogenesis were investigated both in vitro and in vivo. In vitro R 76713 is a very potent inhibitor of the aromatase enzyme in rat granulosa cells, showing an IC50-value of 3.0 +/- 0.2 nM. The compound is about 1000 times more active than aminoglutethimide which shows an IC50-value of 3900 +/- 2800 nM in the same system. R 76713 is also a highly selective aromatase inhibitor. In cultures of ovarian, testicular and adrenal cells, formation of progesterone, androgens and glucocorticoids was only affected by drug concentrations higher than 1 microM. In vivo, single oral drug doses of 0.05 mg/kg lowered plasma estradiol levels of PMSG-primed female rats by more than 90%. An ED50-value of 0.005 mg/kg could be calculated. A single oral dose of 1 mg/kg suppressed plasma estradiol levels almost completely for 24 h. A dose of 0.1 mg/kg lowered plasma estradiol by more than 90% for 8 h. In vivo, R 76713 also showed a highly selective profile. In LHRH/ACTH-injected rats, plasma levels of testicular and adrenal steroids remained unchanged after administration of a drug dose of 20 mg/kg. R 76713 at drug concentrations of 10 microM, showed no interaction in vitro with estrogen-, progestin-, androgen- and glucocorticoid-receptors. Given orally at 20 mg/kg for 3 days the compound also showed no estrogen or androgen agonistic or antagonistic effects.     

Evidence of adrenal 18-hydroxylase inhibition by metyrapone in man.

The influence of metyrapone (M) on the adrenal 18-hydroxylation was studied in two groups of healthy young men. In group I, serum concentrations of 18-OH-11-deoxycorticosterone (18-OH-DOC) fell significantly after a single oral dose of 40 mg/kg of M at 8.00 h, while those of 11-deoxycorticosterone (DOC) increased by a factor of about 500 within 4 hours after drug administration. Serum concentrations of 18-OH-DOC remained suppressed up to 14,00 h and tended to increase up to 16.00 h with a concomitant increase of plasma ACTH. In group II, serum concentrations of 18-OH-DOC and corticosterone (B) were slightly lowered eight hours after oral administration of 30 mg/kg of M at midnight in comparison with measurement of the previous day. Serum concentrations of 11-deoxycortisol (S) and DOC were markedly increased after drug administration. These findings indicate an inhibitory effect of M on adrenal 18-hydroxylation in addition to 11-hydroxylation under in vivo conditions. The slight increase of 18-OH-DOC at 16.00 h in group I and the only slight decrease of this steroid 8 hrs after drug administration in group II may be explained by declining enzyme blockade and a superimposed ACTH stimulation of the adrenal cortex at this time.     

Morphological and functional changes in several endocrine glands induced by hypothyroidism in the rat

The effect of hypothyroidism upon the morphology and the function of several endocrine glands was studied in radiothyroidectomized male rats. It was found that T3, T4, insulin, prolactin and corticosterone levels were significantly lower in hypothyroid rats. TSH levels were significantly higher in these animals while no changes were depicted in testosterone levels. The administration of T4 drew back to normal range the above-mentioned altered serum hormone levels. The studies performed with light microscopy revealed alterations only in the TSH secretory cells of the adenohypophysis. Conversely, when using the electron microscope to study the different endocrine glands, clear alterations were depicted in the TSH and prolactin secretory cells of the adenohypophysis, as well as in the pancreatic B cells and the cells of the zona fasciculata of the adrenal cortex. No abnormal changes were demonstrable at the level of the seminiferous tubules of the testis. All the above morphological changes were corrected by the administration of T4 to hypothyroid rats. These results suggest that the hypothyroid state is a complex hormonal dysfunction rather than a single hormonal defect. The secretory alterations are accompanied by fine cellular alterations in the corresponding glands.     

Investigation of N-acylethanolamines on adrenal cortex function

The possibility of NAE to take part in the regulation of the function of adrenal glands was studied. It was shown that two times NAE (18:0) injection in a dose 5 mg/kg of weight increased the content of 11-hydroxysteroids in blood of intact male rats. NAE caused the raise of the blood hormone content in 4 fold under the immobilization stress. It is apparent that augment of stress response under the influence of NAE in vivo is explained by the activation of hypophysis-adrenal cortex system. In vitro NAE lowered steroidogenesis by near 40%. One can suggest that this decrease is caused by membranotropic properties of NAE.     

Effect of N-acylethanolamines on adrenal cortex function

The possibility of NAE to take part in the regulation of the function of adrenal glands was studied. It was shown that two times NAE (18:0) injection in a dose 5 mg/kg of weight increased the content of 11-hydroxysteroids in blood of intact male rats. NAE caused the raise of the blood hormone content by 4 times under the immobilization stress. It is apparent that augment of stress response under the influence of NAE in vivo is explained by the activation of hypophysis-adrenal cortex system. In vitro NAE lowered steroidogenesis by near 40%. One can suggest that this decrease is caused by membranotropic properties of NAE.     

The adrenal gland and progesterone stimulates testicular steroidogenesis in the rat in vivo

Administration of pharmacological doses of glucocorticoid to male rats in vivo suppresses adrenal steroidogenesis and inhibits testicular steroidogenesis by inhibiting the anterior pituitary secretion of LH. In contrast, administration of ACTH to these pharmacologically-suppressed rats stimulates the adrenal secretion of progesterone and testicular steroidogenesis. The mechanism by which ACTH increases testicular steroidogenesis is dependent on the presence of the adrenal gland and is reproduced by the administration of progesterone. The conclusion from these data is that the adrenal gland has an important role in generating external signals that modulate the hypothalamic-pituitary-gonadal axis in male rats. The adrenal secretion of glucocorticoid acts as a negative signal to testicular steroidogenesis whereas progesterone acts as a positive signal. The adrenal secretion of progesterone and its conversion to testosterone by steroidogenic enzymes in the cytoplasm of the Leydig cell may provide an alternative pathway for testosterone biosynthesis and may account for the increased plasma testosterone levels during the acute phase of stress and mating.     

Effects of dietary equol on the pituitary of the ovariectomized rats.

The aim of our study was to evaluate the effects of dietary equol, metabolite of a phytoestrogen daidzein, on the secretion of prolactin (PRL) and lutenizing hormone (LH), as well as the expression of estrogen receptors (ERalpha, ERbeta and truncated estrogen receptor-1 (TERP-1) in the pituitary gland of ovariectomized (ovx) female Sprague-Dawley rats. Two doses of equol (50 mg/kg of chow and 400 mg/kg of chow) were used and the results were compared with the effects of estradiol 3-benzoate (E2B), also given at two doses (4.3 mg/kg of chow and 17.3 mg/kg of chow). Treatment period was 3 months. Dietary equol administration at the high dose increased significantly serum PRL levels. This effect was also observed in the E2B group but this difference did not reach statistical significance. Surprisingly, high dose dietary equol treatment also significantly increased serum LH levels, which was in contrast to E2B treatment where serum LH levels were significantly decreased at both doses. Serum LH levels in the equol low group were unaffected. Equol treatment had no effects on pituitary ERalpha or ERbeta gene expression. In contrast, high dose E2B treatment increased significantly pituitary ERalpha mRNA levels but decreased those of ERbeta. Both doses of E2B also increased significantly pituitary TERP-1 mRNA levels. This effect was also observed in the equol high group but at a much smaller magnitude. In conclusion, high dose dietary equol administration to ovx rats exerts estrogenic like effects on the lactotropes and anti-estrogenic on the gonadotropes.     

Some aspects of corticosterone metabolism in lithium treated rats

The effect of lithium administration on adrenal steroidogenesis has been studied in rats. An increase in plasma corticosterone (CS) levels was observed and this appears to be due to increased steroidogenesis. This effect on the adrenal is mediated via ACTH, suggesting an involvement of the pituitary adrenal axis.     

Effect of biseptol on the adrenal cortex of the white rat in postoperative shock

The study aimed to find out the effect of sulfonamide combined with Trimetaprim-Biseptol 480 on the adrenal cortex in post-operative shock after removal of SPIGELian lobe (lobectomy of the lobus caudatus and unilaterally of one kidney with its suprarenal gland. The study was performed on a material of white rats which were post-operatively administered Biseptol 480 in doses 5 times bigger than those given to men. It was attempted to determine histochemically the intensity of the adrenal cortex' function by testing the number of lipid droplets, activity of the main enzyme of steroidogenesis (beta-hydroxy steroid dehydrogenase) and the level of alpha-ketols (as the final stage of steroidogenesis). Pathomorphologic examinations were also performe. On the basis of the present study's results, it was observed that - in the case of liver-lobectomy - the zona fasciculata and zona reticularis are functionally stimulated but the zona glomerulosa becomes insufficient. In the case of nephrectomy plus suprarenal gland's removal, all the adrenal cortex becomes insufficient. Administration of Biseptol in the 1st case contributed to hormonal inactivation of the zone glomerulosa cells, but in the 2nd case, it caused an increased activity of steroid dehydrogenase and an increase of the alpha-ketol level in the zona fasciculata.     

Zona glomerulosa of the adrenal gland in a transgenic strain of rat: a morphologic and functional study

Transgenic rats for the murine Ren-2 gene display high blood pressure, low circulating levels of angiotensin II, and high renin content in the adrenal glands. Moreover, transgenic rats possess and increased aldosterone secretion (maximal from 6 to 18 weeks of age), paralleling the development of hypertension. To investigate further the cytophysiology of the adrenal glands of this strain of rats, we performed a combined morphometric and functional study of the zona glomerulosa of 10-week-old female transgenic rats. Morphometry did not reveal notable differences between zona glomerulosa cells of transgenic and age- and sex-matched Sprague-Dawley rats, with the exception of a marked accumulation of lipid droplets, in which cholesterol and cholesterol esters are stored. The volume of the lipid-droplet compartment underwent a significant decrease when transgenic rats were previously injected with angiotensin II or ACTH. Dispersed zona glomerulosa cells of transgenic rats showed a significantly higher basal aldosterone secretion, but their response to angiotensin II and ACTH was similar to that of Sprague-Dawley animals. Angiotensin II-receptor number and affinity were not dissimilar in zona glomerulosa cells of transgenic and Sprague-Dawley rats. These data suggest that the sustained stimulation of the adrenal renin-angiotensin system in transgenic animals causes an increase in the accumulation in zona glomerulosa cells of cholesterol available for steroidogenesis, as indicated by the expanded volume of the lipid-droplet compartment and the elevated basal steroidogenesis. However, the basal hyperfunction of the zona glomerulosa in transgenic animals does not appear to be coupled with an enhanced responsivity to its main secretagogues, at least in terms of aldosterone secretion.     

Investigations on the effects of long-term administration of a methionine-enkephalin analogue on the adrenal zona fasciculata of rats treated with dexamethasone or dexamethasone and ACTH

Prolonged (7 days) methionine-enkephalin (DALA) treatment provoked a dose-dependent increase in the volume of zona fasciculata cells of dexamethasone-administered rats, along with a notable rise in the plasma concentration of corticosterone and the activity of 11 beta-hydroxylase. Comparable dose-dependent effects were observed after chronic administration of ACTH to dexamethasone-suppressed rats. The chronic administration of the maximum dose of DALA (500 micrograms/kg/day) was found to significantly further the trophic action of ACTH on the zona fasciculata of dexamethasone-treated animals. It is suggested that enkephalins act independently of and synergistically with ACTH in stimulating the growth and steroidogenic capacity of the rat adrenal zona fasciculata.     

The effect of leptin on adrenal glands in rats with streptozotocin-induced diabetes: an experimental study

OBJECTIVE: To determine the cross-section area of adrenal medulla and the percentage of Ki-67 (a proliferation marker) of the adrenal gland in diabetic rats after leptin injection. STUDY DESIGN: Twenty-nine male Wistar rats were randomly divided into 3 groups: control (C) group (n = 9), diabetes mellitus (DM) group (n = 10) and leptin-injected diabetes mellitus (DM+L) group (n = 10). Experimental DM was induced by a single intraperitoneal dose of streptozotocin (40 mg/kg). After this, leptin (100 microg/kg) was injected subcutaneously for a period of 2 weeks in the diabetic group. RESULTS: An atrophy of adrenal medulla in the DM group was observed, and this atrophy returned to normal morphology after injection of leptin. In addition, an increase in the Ki-67 percentage was demonstrated in the zona reticularis layers in the DM+L group. CONCLUSION: Our study indicated that leptin stimulates the sympathoadrenal system and the androgen producing adrenal cortex in DM rats.     

Mechanism of action of tetracycline on the functional state of the adrenal cortex]

The mechanism of stimulation of the adrenal cortex function by tetracycline was studied on albino rats. It was shown that tetracycline administered orally in a dose of 200 mg/kg regularly induced an increase in the corticosterone levels in the peripheral blood of the animals by the 15th day of the antibiotic use. It was shown on the animals with an experimentally suppressed function of the hypophysis by prolonged administration of hydrocortisone acetate that tetracycline primarily stimulated the hypophysis function resulting in production and excretion of increased amounts of the adrenocorticotropic hormone into the blood. The hormone increased the production of corticosterone in the adrenal glands which resulted in its higher levels in the peripheral blood.     

Beneficial effects of DL-alpha-lipoic acid on cyclophosphamide-induced oxidative stress in mitochondrial fractions of rat testis

The present study investigated the protective efficacy of dl-alpha-lipoic acid on the peroxidative damage and abnormal antioxidant levels in the mitochondrial fraction of testis in cyclophosphamide (CP) administered rats. Male Wistar rats of 140+/-20 g were categorized into four groups. Two groups were administered CP (15 mg/kg body weight once a week for 10 weeks by oral gavage) to induce testicular toxicity; one of these groups received lipoic acid treatment (35 mg/kg body weight intraperitoneally once a week for 10 weeks, 24 h prior to CP administration). A vehicle-treated control group and a lipoic acid drug control group were also included. The mitochondrial fraction of untreated CP-exposed testis showed 1.84-fold increase in lipid peroxidation, along with a significant (P<0.001) increase in hydrogen peroxide levels. In CP-exposed rats, we observed abnormal changes in the activities/levels of mitochondrial enzymic (superoxide dismutase, glutathione peroxidase and glutathione reductase) and non-enzymic (reduced glutathione, ascorbate and alpha-tocopherol) antioxidants. CP-treated rats also showed decline in the activities of mitochondrial enzymes such as succinate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase. In contrast, rats pretreated with lipoic acid showed normal lipid peroxidation and antioxidant defenses, thereby showing the protection rendered by lipoic acid.     

Vitamin E prevents nonylphenol-induced oxidative stress in testis of rats

In the present study we have investigated if administration of nonylphenol-induced oxidative stress in various subcellular fractions of adult rat testis and the effect of vitamin E on reactive oxygen species mediated nonylphenol toxicity. Male rats were administered orally with nonylphenol at 1, 10 and 100 microg/kg body weight per day for 45 days with and without supplementation of vitamin E (20 mg/kg body weight). In nonylphenol-treated rats the activities of antioxidant enzymes superoxide dismutase and glutathione reductase decreased significantly while the levels of lipid peroxidation increased significantly in the crude homogenate and in the mitochondrial and microsome-rich fractions of testis. Co-administration of nonylphenol and vitamin E did not cause changes in the activities of antioxidant enzymes in various subcellular fractions of rat testis. The results suggest that graded doses of nonylphenol elicit depletion of antioxidant defence system in rat testis, indicating nonylphenol induced oxidative stress in the testis of rats which could be reversed by the administration of vitamin E.     

环磷酰胺对雄性大鼠生殖免疫功能的影响

目的观察环磷酰胺对大鼠睾丸及其细胞免疫的影响,探讨抗肿瘤药物在生殖免疫功能中的机制。方法选用16只15周龄SD大鼠,随机分为对照组和实验组,每组8只;实验组腹腔注射环磷酰胺20mg/kg/d,连续5天,用药两个月后,应用HE染色法研究大鼠睾丸远期组织学变化,用原位缺口末端标记法（TUNEL方法）检测生精小管中生殖细胞凋亡,放射免疫法检测血清睾酮（T）、卵泡刺激素（FSH）、黄体生成素（LH）,流式细胞术进行血液T淋巴细胞亚群分析。结果实验组睾丸生精小管直径缩小、间距增宽、生精上皮变薄、生殖细胞层次和数量减少、生精小管腔多未见精子形成,实验组睾丸生精小管直径、面积、生殖细胞数均显著低于对照组（P〈0.01）;实验组与对照组比较生殖细胞凋亡增多,差异显著（P〈0.01）;实验组与对照组比较血清T明显降低,差异显著（P〈0.01）,血清FSH、LH水平两组间差异无显著性;血液T淋巴细胞亚群分析,实验组与对照组比较CD3＋CD4＋、CD4＋/CD8＋明显降低（P〈0.01）,CD3＋CD8＋明显升高（P〈0.01）。结论环磷酰胺对大鼠睾丸远期损害明显,促进生殖细胞凋亡,降低睾酮的分泌,并抑制T淋巴细胞的免疫功能。     

Tissue-selective inhibition of cholesterol synthesis in vivo by pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor

Tissue selectivity of pravastatin sodium (pravastatin) in inhibition of cholesterol synthesis was investigated and its effect was compared with other 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, such as lovastatin, simvastatin and ML-236B. Inhibition of cholesterol synthesis in vivo was measured by incorporation of radioactivity into the sterol fraction 1 h after intraperitoneal injection of [14C]acetate to mice. The drugs were orally administered to mice 2 h before the acetate injection. When pravastatin at a dose of 20 mg/kg was administered to mice, about 90% inhibition of cholesterol synthesis was observed in liver and ileum, but the inhibition was less than 14% in kidney, spleen, adrenal, testis, prostate and brain. This tissue selectivity of pravastatin was also demonstrated even in varying doses (5-100 mg/kg) and time (75-180 min) after drug administration. Other 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors did not show such a tissue-selective inhibition of sterol synthesis under the same conditions. These results obtained with the in vivo study were confirmed in vitro by the inhibition of sterol synthesis in various cultured cells and rats lenses, as well as by cellular uptake of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.