Cell and region specificity of Aryl hydrocarbon Receptor (AhR) system in the testis and the epididymis.

Aryl hydrocarbon receptor (AhR) plays multiple important functions in adaptive responses. Exposure to AhR ligands may produce an altered metabolic activity controlled by the AhR pathways, and consequently affect drug/toxin responses, hormonal status and cellular homeostasis. This research revealed species-, cell- and region-specific pattern of the AhR system expression in the rat and human testis and epididymis, complementing the existing knowledge, especially within the epididymal segments. The study showed that AhR level in the rat and human epididymis is higher than in the testis. The downregulation of AhR expression after TCDD treatment was revealed in the spermatogenic cells at different stages and the epididymal epithelial cells, but not in the Sertoli and Leydig cells. Hence, this basic research provides information about the AhR function in the testis and epididymis, which may provide an insight into deleterious effects of drugs, hormones and environmental pollutants on male fertility.

Lipid Accumulation Product (LAP) as an Index of Metabolic and Hormonal Disorders in Aging Men.

Lipid accumulation product (LAP) is an index used for evaluating lipid overaccumulation in adults. Our study aimed at assessing associations between LAP and metabolic syndrome (MetS) and its components, age-related testosterone deficiency syndrome (TDS), low-density cholesterol (LDL), as well as HOMA-IR (insulin resistance ratio), insulin level in non-diabetics and total testosterone (TT), estradiol E2, dehydroepiandrosterone sulphate (DHEAs) and sex hormone-binding globulin (SHBG) in aging men.313 men aged 50-75 were surveyed with regard to the prevalence of diabetes (T2DM) and hypertension (HT). Anthropometric measurements, including waist circumference and arterial pressure, were performed. We also determined the levels of fasting plasma glucose (FPG), total cholesterol (TC), high-density cholesterol (HDL), low-density cholesterol (LDL), triglyceride (TG), insulin, TT, SHBG, DHEAs, and E2.Patients with diagnosed MetS, T2DM, HT, obesity, overweight and TDS had a significantly higher LAP compared to those without these conditions. LAP was significantly positively correlated with serum TC, FPG, insulin, DHEAs, as well as APB-systolic concentration, and negatively correlated with HDL, TT, and SHBG.LAP may then be used as a simple and inexpensive biomarker of metabolic disorders, and in risk assessment related to testosterone deficiency in aging men.

Occurrence of climacteric symptoms in postmenopausal women after prophylactic bilateral ovariectomy.

PURPOSE OF INVESTIGATION: To analyse the quality of life in postmenopausal women after prophylactic bilateral ovariectomy depending on the time from menopause. MATERIALS AND METHODS: The study involved 252 postmenopausal women grouped according to the time from last menstruation: one to five years (group A), five to ten years (group B), and > ten years (group C). All women were ovariectomized during laparotomy performed for benign diseases of the uterus. Climacteric symptoms were measured with the Kupperman Index one day before and three months after surgery. RESULTS: Highly significant age differences and no substantial BMI differences were demonstrated among the study groups. Before and after surgery climacteric symptoms were reported by 17.06% and 57.8% of women, respectively. After surgery, group A women significantly more often had hot flushes, sweating, nervousness, and sleep disorders, the women in group B significantly more often reported sleep disorders, nervousness, and sweating, and the women in group C significantly more often complained of nervousness. CONCLUSION: In postmenopausal women, ovaries play the most important role during the first ten years from the last menstruation.

[Influence of Selected Reproductive Factors and Smoking on Age at Menopause]. / Einfluss ausgewählter Reproduktionsfaktoren sowie des Rauchens auf das Alter bei Eintreten der Menopause.

Introduction: Early menopause may be associated with serious health risks resulting from, for example, decreased oestrogen levels. This may occur despite hormone replacement therapy. Aim: The aim of this study was the determination of the effect of selected reproductive factors and smoking on age at the onset of menopause in women from Szczecin and surrounding areas. Material and Methods: 305 women after natural menopause were asked to complete a questionnaire, and blood samples were collected from them to test for the levels of follicle stimulating hormone (FSH) and oestradiol (E2). Results: Smoking women experienced menopause on average more than a year earlier than non-smokers, but this difference was not statistically significant. There was no statistically significant effect of age at menarche or first birth on age at the last menstrual period. Conclusions: Age at menarche and first birth were not related to age at menopause. In smoking women, menopause occurred earlier but the difference was not statistically significant.

Immunoexpression of intermediate filaments and morphological changes in the liver and bile duct of rats infected with Fasciola hepatica.

We investigated the immunoexpression of the intermediate filament proteins, cytokeratin and desmin, and the morphological changes in the liver of rats during experimental fasciolosis at 4, 7 and 10 weeks post-infection. Rats were infected with 30 Fasciola hepatica metacercariae. Paraffin sections of the liver were stained using H & E, PAS and azan stains. Immunohistochemical reactions were performed using antibodies against cytokeratin and desmin. The experimental F. hepatica infection led to fibrosis and cirrhosis of the liver, and to inflammation of the common bile ducts. The expression of cytokeratin was increased in the epithelial cells of both the liver bile ductules at 4, 7 and 10 weeks post-infection and in the common bile ducts at 7 and 10 weeks post-infection compared to uninfected rats; expression in the common bile ducts was more intense. The myofibroblasts of the liver and smooth myocytes of the interlobular bile ducts and common bile ducts, showed a slight increase in desmin expression compared to the uninfected rats. The increased expression of cytokeratins in the hyperplastic rat common bile duct epithelium during the biliary phase of fasciolosis at 7 and 10 weeks post-infection may be explained by mechanical irritation by the parasite and an inflammatory reaction in the bile duct epithelium and in periductal fibrous tissue.

Mapping of polar fox renal cortex proteins using two-dimensional gel electrophoresis and mass spectrometry--a preliminary study.

The aim of the present study was to establish protein map of polar fox (Alopex lagopus) renal cortex. Kidney cortex proteins of isoelectric point ranging from 3 to 10 were analysed using two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Sixteen protein spots corresponding to thirteen different gene products were identified. These proteins were divided into following groups: lipid and fatty acid metabolism, amino acid metabolism, energetic pathways, regulatory proteins, transport proteins and structural proteins. This is the first attempt to create reproducible 2-D map, of renal cortex proteins characteristic for polar foxes, used as animal model for carnivores. It is worth emphasizing that the results of this study may broaden currently available protein databases.

Immunohistochemical identification of aquaporin 2 in the kidneys of young beef cattle.

Aquaporin 2 (AQP2) is a small, integral tetrameric plasma membrane protein that is expressed in mammalian kidneys. The specific constitution of this protein and its selective permeability to water means that AQP2 plays an important role in hypertonic urine production. Immunolocalization of AQP2 has been studied in humans, monkeys, sheep, dogs, rabbits, rats, mice and adult cattle. We analyzed the expression of AQP2 in kidneys of 7-month-old Polish-Friesian var. black and white male calves. AQP2 was localized in the principal cells of collecting ducts in medullary rays penetrating the renal cortex and in the collecting ducts of renal medulla. AQP2 was expressed most strongly in the apical plasma membrane, but expression was observed also in the intracellular vesicles and basolateral plasma membrane. Our study provides new information concerning the immunolocalization of AQP2 in calf kidneys.

Membrane stability and mitochondrial activity of human-ejaculated spermatozoa during in vitro experimental infection with Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus.

The aim of the study was to examine an in vitro effect of the three bacterial strains (Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus) on ejaculated spermatozoa with reference to sperm membrane integrity and mitochondrial activity. The study was carried out on swim-up-separated spermatozoa from 12 normozoospermic volunteers. Sperm plasma membrane stability was evaluated by the LIVE/DEAD Sperm Viability Kit and by the merocyanine 540 test. Mitochondrial activity was evaluated using the JC-1 test as well as the NADH-dependent NBT assay. The percentage of dead cells was significantly higher in spermatozoa treated with B. ureolyticus as compared to that of control spermatozoa (P < 0.01). All the bacterial strains applied affected sperm plasma membrane architecture measured by M540 test (P < 0.01). Moreover, the presence of E. coli or B. ureolyticus was connected with significant decrease in both the number of cells with high mitochondrial transmembrane potential (ΔΨm) and the cells with normal oxidoreductive function of mitochondria (P < 0.05 as compared to untreated cells). To conclude, the contact of bacteria with ejaculated spermatozoa can be a reason for severe injury of sperm membrane stability and mitochondrial activity with potential consequences for male fertility.

Analysis of pituitary gonadotropin concentration in blood serum and immunolocalization and immunoexpression of follicle stimulating hormone and luteinising hormone receptors in ovaries of postmenopausal women.

The participation of gonadotropins in ovarian carcinogenesis is well known and is supported by studies with inhibition of pituitary gonadotropin secretion, which results in a diminished risk of cancer. However, there are few data on localization and expression of Follicle Stimulating Hormone and Luteinising Hormone Receptors (FSHR and LHR) in ovaries of healthy postmenopausal women, and their correlation with FSH and LH concentration in blood serum is unknown. The aim of our study was to analyze gonadotropin concentration in blood serum and the expression of FSHR and LHR in ovaries of 207 postmenopausal women. Patients included in the study were divided into three groups depending on the number of years since menopause. We analyzed the concentration of FSH and LH in blood serum and the expression of FSHR and LHR in ovaries. Ovaries of postmenopausal women showed numerous morphological changes in the cortex and medulla when compared to the structure of ovaries of women at reproductive age. In all groups of patients clefts in the surface epithelium and epithelial inclusion cysts were found. The concentration of FSH and LH in the blood serum of women studied increased significantly with time from menopause. Significant differences between analyzed menopausal groups were found. The highest FSH and LH concentration in blood serum were found in women with the longest period of time from menopause. Quantitatively similar expression of FSHR and LHR was found in ovarian surface epithelial cells, in epithelial inclusion cysts and in the connective tissue cells of ovarian stroma. The intensity of the immunohistochemical reaction decreased with time from menopause and with age.

Oxidative stress indices in rats under immunosuppression.

Immunosuppressants lead to generation of reactive oxygen species (ROS). Oxidative stress (OxS) can initiate chronic allograft nephropathy (CAN). The most active antioxidant enzymes, superoxide dysmutase (SOD) and catalase (CAT), are present in erythrocytes. Glutathione peroxidase (GPx) is produced in the proximal tubules of nephrons. Malonyldialdehyde (MDA) concentrations are a marker of OxS intensity in plasma. In vitro and animal model studies have shown increased or decreased OxS during treatment with tacrolimus (Tac) or cyclosporine (CyA). Results obtained in humans after solid organ transplantation have been contradictory, because of confounding factors such as ischemia-reperfusion injury, donor and recipient ages, endothelial injury, and comorbidity. The aim of this study was to assess the intensity of OxS among rats under chronic immunosuppression (IS) without a transplantation. We examined 49 male Wistar rats. IS started at 12 weeks of age was continued for 6 months: group I were controls (n=7); group II, Tac+sirolimus (Rapamycin [Rapa])+corticosteroids (CS; n=6); group III, CyA+Rapa+CS (n=4 of which 2 died); group IV, Rapa+mycophenolate mofetil (MMF)+CS (n=6); group V, CyA+MMF+CS (n=6); group VI, CsA+MMF+CS for 3 months followed by conversion to Rapa (n=6); group VII, Tac+MMF+CS (n=6 rats); and group VIII, Tac+MMF+CS for 3 months followed by conversion to Rapa (n=6). The drug doses were as follows: Tac 4 mg/kg/d; MMF 20 mg/kg/d; CyA 5mg/kg/d; Rapa 0.5 mg/kg/d; and CS 4 mg/kg/d. Multiple regression analysis revealed that all IS drugs decreased GPx activity (P<.001) except CS, which increased it (P<.0001). Multiple regression analysis showed that CsA and Tac decreased plasma MDA concentrations (P<.01), whereas CS increased them (P<.05). In conclusion, all IS drugs except CS damage proximal tubules of nephrons.

Immunohistochemical analysis of steroid receptors in ovaries of postmenopausal women--effects of aging and hormone status.

Current knowledge on immunolocalization and immunoexpression of steroid hormone receptors, especially estrogen receptor alpha (ER-alpha), progesterone receptor (PR) and androgen receptor (AR) in normal ovaries in postmenopausal women is not complete. The recognition of localization of these receptors in postmenopausal women is crucial, as many of these women receive estro-progestagene therapy, and its participation in the pathogenesis of ovarian cancer should be carefully studied. In our paper we present the results of immunohistochemical studies performed on samples from 100 post-menopausal women (aged: 48 to 60 years) who did not use hormonal therapy. The ovaries were removed during elective operation due to uterine leiomyoma, endometriosis and/or prolapsed uterine. PR, ER-alpha and AR were detected in the normal ovaries of postmenopausal women in stroma and in ovarian surface epithelium, as well as in its invagination and in epithelial inclusion cysts. The expression of PR and AR did not change, while the expression of ER-alpha decreased in time from menopause, and it was also detected in patients more than 10 years after menopause. Women older than 60 were not included in the study. The concentration of selected hormones was measured in the serum. The immunohistochemical expression of PR and AR were similar in all examined patients and did not correlate with FSH, LH, T, A, DHEAS concentrations in serum, while immunohistochemical expression of ER-alpha correlated with FSH, LH, T, A, DHEAS concentrations in serum. The significant correlation of decreasing expression of ER-alpha in normal ovarian tissue and decreasing concentrations of T, A and DHEAS in serum were found, as well as increasing serum concentrations of FSH and LH.

Human postmenopausal ovary--hormonally inactive fibrous connective tissue or more?

The ovary undergoes several changes after the menopause. In this period, the main structural changes in both the cortex and medulla were observed. In the cortex, they included: 1) reduction of its thickness; 2) epithelial inclusions forming cysts; 3) blurring the line between medulla and cortex; 4) reduction of follicles number; 5) tendency to fragmentation of corpora albicantia; 6) surface epithelium invaginations. Whereas the changes in the medulla included: 1) fibrosis and scars in stroma; 2) architectonical changes in blood vessels with hyalinization of walls and constriction of lumen. The loss of follicles and several changes in the ovary are due to apoptotic processes. Despite age related atrophic changes, the postmenopausal ovary is not devoid of hormonal activity. Our results are coherent with the reports of other researchers, and reveal that postmenopausal ovary produces trace quantities of steroid hormones, mainly androgens, and confirm the presence of steroid receptors and activity of main enzymes involved in steroidogenesis process.

Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia.

Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.

Evaluation of sperm genomic integrity of normozoospermic men: a prospective study.

The objective of our study was to evaluate the incidence of spermatozoa with nuclear DNA strand breaks in patients with normal routine sperm parameters (26 subjects). Sperm DNA fragmentation was measured using TUNEL test assessed in flow cytometer. Variable percentages of sperm with damaged DNA (9.42 +/- 7.68%; range: 2-36) were found. Two categories of patients were distinguished: (1) patients (8 out of 26 subjects) with < or = 4% of TUNEL-positive sperm and (2) patients (18 out of 26 subjects) with > 4% of TUNEL-positive sperm. A significantly lower percentage of normal sperm forms was found in patients with > 4% of TUNEL-positive sperm than in patients with < or = 4% of TUNEL-positive sperm. Moreover, a significant negative correlation (r(s) = -0.50) was noted only between a proportion of normal sperm forms and a proportion of TUNEL-positive spermatozoa. In electron microscope, a large number of spermatozoa with immature chromatin was observed more frequently in subjects with > 4% of TUNEL-positive cells (11 out of 18 subjects). Our results suggest that in some patients with normal routine sperm parameters, DNA fragmentation may be associated with poor sperm morphology. The diminished sperm genomic integrity may result from molecular disturbances in nuclear remodeling process during spermiogenesis. TUNEL assay is a screening tool that may help to discriminate between fertile and infertile men and may help to predict successful in vitro fertilization.

Diagnostic evaluation of oxidoreductive capability of sperm mitochondria.

In the present paper, morphological and functional features of human sperm midpiece, contributing to the assessment of sperm fertility potential, have been described. The NADH-dependent NBT screening assay was used to identify and visualise: 1/ morphological defects of sperm midpiece, 2/ immature sperm forms with extensive cytoplasmic retention, reflecting developmental failure in spermatogenic remodelling process, 3/ cytoplasmic sperm conglomerates, related to apoptotic bodies and 4/ sperm NADH-dependent oxidoreductase system at the mitochondrial level, related to the reaction intensity. The used assay is an adequate marker of sperm mitochondrial activity and sperm maturity. It can also help discover sperm defects that result in asthenozoospermia and can be used as an additional indicator in the evaluation of the sperm midpiece, as well as in routine morphological examination of spermatozoa, having a considerable predictive value for in vivo and in vitro fertilization.

Aging process of epithelial cells of the rat prostate lateral lobe in experimental hyperprolactinemia induced by haloperidol.

The aim of the study was to examine the influence of hyperprolactinemia, induced by haloperidol (HAL) on age related morphology and function changes of epithelial cells in rat prostate lateral lobe. The study was performed on sexually mature male rats. Serum concentrations of prolactin (PRL) and testosterone (T) were measured. Tissue sections were evaluated with light and electron microscopy. Immunohistochemical reactions for Anti-Proliferating Cell Nuclear Antigen (PCNA) were performed. In rats of the experimental group, the mean concentration of: PRL was more than twice higher, whereas T concentration was almost twice lower than that in the control group. Light microscopy visualized the following: hypertrophy and epithelium hyperplasia of the glandular ducts, associated with increased PCNA expression. Electron microscopy revealed changes in columnar epithelial cells, concerning organelles, engaged in protein synthesis and secretion.

Germ cells with nuclear DNA fragmentation related to apoptotic cells in rat testis in experimental hyperprolactinemia induced by metoclopramide.

The cells with nuclear DNA fragmentation related to apoptosis were detected by TUNEL technique in the seminiferous epithelium of control rats and of rats with experimental hyperprolactinemia induced by metoclopramide. The percentage of convoluted tubules with apoptotic cells and the number of apoptotic cells (predominantly spermatogonia and spermatocytes) was increased in the experimental group. The results indicated stage-specific germ cell apoptosis. In the experimental group, apoptotic cells were most evident at early (I-IV), middle (VII-VIII) and late (XII-XIV) stages of the seminiferous epithelium cycle, as revealed by light and electron microscopy. We suggest that a decreased concentration of testosterone and an increased concentration of prolactin could disturb spermatogenesis and contribute to the intensive apoptosis of germ cells in rats with hyperprolactinemia. Sertoli cells which have receptors for testosterone and prolactin and play an important role in spermatogenesis and in the initiation of apoptosis in seminiferous epithelium, could mediate such an influence of both hormones.

[Changes in sperm mitochondria in patients with asthenozoospermia]. / Zmiany we wstawkach plemników pacjentów z astenozoospermia.

OBJECTIVE: To evaluate functional and ultrastructural alterations of the spermatozoa midpieces in patients with asthenozoospermia and to find a correlation between the damage of the midpieces and loss of sperm motility. MATERIAL AND METHODS: Routine, morphological assessment of the midpieces stained according to the Papanicolaou method, cytochemical study of the mitochondrial activity using reaction for the diaphorase/ NADH according to the Piasecka method and electron-microscopic investigation of the midpiece structures were performed. RESULTS: The cytochemical reaction for diaphorase/NADH revealed disorders of the mitochondrial activity and subtle and drastic malformations in the spermatozoa midpieces. The unusually thickened midpieces contained the supernumerary mitochondria. In patients with severe asthenozoospermia, the damage of the accessory fibres and axonemal complex located in the midpiece, were obtained also. CONCLUSION: This study indicates that mitochondrial defects are one of the causes that may account for loss of sperm motility in the population of patients.

Electron-dense deposits in epididymal cells of rats chronically treated with lead acetate [Pb(II)].

Electron microscopic studies were performed to investigate the influence of chronic lead acetate treatment on morphology of rat epididymis. Dense, lead-loaded inclusions were found in the cytoplasm of epididymal principal cells, especially in the caput of epididymis. They were also present, but in smaller amounts, in smooth muscle cells. Usually, the inclusions were located in vacuoles, rarely without any surrounding membrane. Similar lead-containing structures were found in the epididymal lumen. The localization of lead deposits suggests the ability of lead to pass from blood vessels through the epithelial cells of the epididymis to its lumen. It can be therefore postulated, that lead can be eliminated from the male genital tract together with ejaculate.