Morphology of the testis and the epididymis in rats with dihydrotestosterone (DHT) deficiency.

The aim of the study was to estimate morphology in the testis and epididymis of adult rats, treated with finasteride for 28 days (the time period of two seminiferous epithelium cycles) and 56 days (the time period of one spermatogenesis). A 28 days long DHT deficiency did not significantly influence the structure of seminiferous epithelium. After 56 days of treatment, finasteride induced sloughing of immature genninal cells (spermatids and rarely pachytene spermatocytes) into the lumen of the seminiferous tubules. A reduced content of spermatozoa was observed in the lumen of rat epididymis in rats with 56-day-long deficiency. The results indicated that 5alpha-reductase 2 activity is important for the maintenance of spermatogenesis. The decreased content of spermatozoa in the epididymal lumen of rats, treated with finasteride during one course of spermatogenesis, could reflect seminiferous epithelium condition.

Evaluation of mouse preimplantation embryos cultured in media enriched with insulin-like growth factors I and II, epidermal growth factor and tumor necrosis factor alpha.

Culture of preimplantation embryos is complex and requires strictly defined culture media to sustain their viability and quality. In the current study, an effort was made to evaluate comprehensively the quality of mice embryos, grown in media enriched with IGF I, IGF II, EGF and TNFalpha. For that purpose, critically chosen and thoroughly described, complex morphological methods based on contrast-phase, fluorescent and confocal microscopy were used. The study evaluated blastulation and hatching rates, total blastocyst cells, inner cell mass cell numbers (differential staining) as well as identified embryo cells with positive reactions for necrosis or apoptosis (TUNEL). The critical evaluation of the effects of the studied cytokines allowed for simultaneous, meticulous assessment of the applied study methods. Significantly more blastocysts were found in culture media enriched with IGF-I, IGF II and EGF. Significantly more hatched blastocysts were found in media with IGF-I and IGF II. Additionally, IGF I and II increased inner cell mass and total blastocyst cell numbers. Very few cells with necrosis and apoptosis were found in the culture media enriched with IGF I, IGF II and EGF. TNFalpha produced negative effects. The observed effects were dose-dependent.

Computerized analysis of cytochemical reactions for dehydrogenases and oxygraphic studies as methods to evaluate the function of the mitochondrial sheath in rat spermatozoa.

Cytochemical reactions for mitochondrial NADH-dependent dehydrogenases (diaphorase/NADH which is related to flavoprotein), NAD-dependent dehydrogenases (isocitrate, malate) and succinate dehydrogenase were carried out in rat spermatozoa. In addition to a morphological evaluation, the intensity of the reactions was assessed using a computer image analysing system (Quantimet 600 S). The intensity of the reactions was examined in sperm midpieces by measuring integrated optical density (IOD) and mean optical density (MOD). The activity of mitochondrial respiratory chain complexes was also analysed using the polarographic method. In the population of spermatozoa studied, all whole spermatozoa midpieces were completely filled with formazans, the product of the cytochemical reaction. These morphological findings corresponded to the values obtained for IOD and MOD for the given enzymes. In the oxygraphic studies, the spermatozoa demonstrated consumption of oxygen in the presence of substrates for I, II and IV complexes and their mitochondria revealed normal integrity and sensitivity to the substrates and inhibitors. However, the oxygraphic studies revealed differences between the sperm and somatic cells. These differences concerned the stimulation of pyruvate oxidation by malate, the lack of an effect of malonic acid on phenazine methosulphate (an acceptor of electrons) oxidation and the lack of an effect of cytochrome c on ascorbate oxidation. The cytochemical method, together with densitometric measurements, enables: (1) the reaction intensity to be determined objectively; (2) subtle and dramatic differences in reaction intensity to be revealed between spermatozoa that do not differ under morphological evaluation of the intensity; (3) possible defects within the mitochondrial sheath to be located and assessed in a large number of spermatozoa. This method can be used as a screening method alongside the routine morphological examination of spermatozoa. On the other hand, the oxygraphic method in the inner membrane of mitochondria can reveal functional changes which are related to the action of respiratory chain complexes and display characteristic features of mitochondria energy metabolism. The methods used are complementary and allow the complex evaluation of mitochondria in spermatozoa. Both methods can be used in experimental and clinical studies.

Pharmacokinetics of intravenously administered digoxin and histopathological picture in rabbits with experimental bile duct obstruction.

This study aimed to examine the effect of obstructive cholestasis on the pharmacokinetics of digoxin. Eighteen male rabbits were randomly ascribed to the two study groups: the sham-operated control group and the examined group - with common and cystic bile duct ligations. Digoxin was administered intravenously as a single dose of 0.02 mg/kg, and blood samples were withdrawn for up to 24 h. Digoxin concentrations were determined by the FPIA method. The pharmacokinetic parameters were calculated using a noncompartmental analysis. During the whole observation period the blood serum concentrations of digoxin were statistically higher in animals with obstructive cholestasis versus the controls. A significant increase in the area under the plasma concentration-time curve, decrease in the total body clearance and in the volume of distribution on the 6th day after the bile ducts ligation as compared to the sham-operated controls, were observed. The obtained results suggest an impaired elimination of digoxin in obstructive cholestasis in rabbits.

[Internal standardization of sperm morphology evaluated according to strict morphological criteria]. / Próba standaryzacji badan morfologicznych plemników w oparciu o material wlasny pracowni seminologicznej.

OBJECTIVE: To evaluate sperm morphology and find cut-off values for local andrology lab based on morphological strict criteria. To compare the results to WHO guidelines. MATERIAL AND METHODS: Strict morphological criteria were applied to 300 sperm smears stained according to the Papanicolaou method. Specific sperm defects were described in details. The results were analyzed statistically. RESULTS: The normal sperm morphology was found in 18.54% of cases, which is less than the cut-off value suggested in the WHO guidelines. The lab cut-off value aimed by the 25th percentile was 8%. CONCLUSION: Sperm morphology requires broader multi-center standardization to enable compatible exchange of morphological data and find predictive factors of sperm fertilizing potential.

[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.

Phospholipid content and lamellar structures in the epididymal epithelial cells of rats treated chronically with lead acetate [Pb(II)].

The electron-microscopic observations accomplished covered epididymal epithelial cells of rats receiving lead acetate for five times longer than the duration of one spermatogenesis. These cells were found to possess a large number of vacuoles and conglomerates containing plicated membranes or tightly packed myelin-like lamellar formations. Further observations also revealed the formation of lamellar structures in mitochondria, dilatation of cisternae in the Golgi apparatus, and increased phagocytosis of spermatozoa by epithelial cells. The presence of a large amount of membranous material correlated with the increased content of phospholipids in epididymal epithelial cells. It may be suggested that the presence of such a great quantity of lamellar structures in epididymal epithelial cells of rats treated chronically with lead is the result of several processes, including the augmented synthesis of membranes associated with encircling the deposits of lead, autophagy in the cells, as well as intensified phagocytosis of spermatozoa.

In vivo and in vitro studies on the toxicity of Hoechst 33342 (Ho342). Implications for employing Ho342 for the isolation of haematopoietic stem cells.

The fluorescence dye Hoechst 33342 (Ho342) is employed for isolating early haematopoietic cells and the aim of this study was to evaluate the in vivo and in vitro toxicity of this compound. First, by employing a murine model we studied the influence of this dye on the morphology of the different organs of animals that have been injected intravenously with increasing doses of Ho342. Accordingly, we found that Ho342 at relatively low doses (0,3 M) caused morphological changes in the spleen and lungs and at higher doses (1,5 & 6 M) damaged also the liver. In contrast, kidneys appear to be relatively resistant to this dye. Next, since Ho342 is employed for isolating early haematopoietic cells by FACS, we have been looking for potential toxicity of this dye against normal human haematopoietic progenitors. Accordingly, CD34+ cells isolated from cord blood (CB) samples were exposed to increasing doses of Ho342 (0-50 microM). We found that the low concentration of Ho342 (10 microM) recommended for isolating HSC significantly inhibited the clonogenecity of human erythroid progenitors (BFU-E). The higher doses of Ho342 have also been toxic against normal human myeloid progenitors (CFU-GM). This study shows that Ho342 could potentially damage human cells. This fact should be considered whenever Ho342 has to be employed for isolating living cells.

Pharmacokinetics of intragastrically administered digoxin in rabbits with experimental bile duct obstruction.

A change in the functioning of the liver as a result of experimental cholestasis could result in a change in the biotransformation of drugs. The aim of this study was to evaluate the effect of extrahepatic cholestasis on the pharmacokinetics of digoxin. The investigation was performed on male rabbits randomly divided into two groups: sham-operated and animals with bile-duct ligation. Digoxin (0.02 mg kg-1) was administered intragastrically as a single dose. Biomedical and anatomo-pathological tests and pharmacokinetic assays were performed before the operation and on the 6th day after surgery. A significant increase in area under the serum concentration-time curve and in mean residence time, a decrease in total body clearance, a reduction in the volume of distribution and increases in maximum concentration and the time to reach maximum concentration were observed in animals with the bile-duct ligation. These results suggest reduced elimination of digoxin in animals with obstructive cholestasis.

[The effect of uropolinum on phagocytosis of sperm by cultured peritoneal macrophages in rats]. / Wplyw uropoliny na fagocytoze plemników w hodowlach makrofagów otrzewnowych u szczurów.

Peritoneal macrophages and sperm were cultured with and without uropolinum. The macrophages were isolated from female rat peritoneal cavities and sperm from male rat cauda epididymis. Sperm phagocytosis index was estimated in cultures with increasing concentrations of uropolinum. Authors concluded that uropolinum inhibited sperm phagocytosis by peritoneal macrophages. The results were verified by electron microscopic examinations. The latter additionally revealed that uropolinum enhanced an adhesion between the macrophages.

The ultrastructure of the testis in rats after long-term treatment with lead acetate.

Studies were performed to investigate the influence of long-term lead acetate treatment on morphology of rat testis. No marked changes were observed by means of light microscopy. At all stages (I-XIV) of the seminiferous epithelium cycle, all generations and layers of spermatogenic cells were present. Electron-microscopic studies did not reveal any ultrastructural changes neither in seminiferous epithelium nor in Sertoli cells. In Leydig cells also, no ultra-structural abnormalities were visible. Macrophages of testicular interstitial tissue contained electron-dense inclusions, usually located inside phagolisosome-like vacuoles. X-ray micro-analysis revealed that the inclusions contained lead.

Pharmacokinetics of phenazone (antipyrine) in rabbits with experimental common bile duct obstruction.

1. An altered functional state of liver due to experimental cholestasis could result in a change in the biotransformation of drugs. The aim of this study was to evaluate an influence of obstructive cholestasis on the pharmacokinetics of phenazone (antipyrine). 2. The investigation was carried out on male rabbits, randomly allocated into two groups: shamoperated and animals with biliary ducts ligation. Phenazone was administered intragastrically as a probe of drug metabolism. 3. Measurements, i.e. laboratory and pharmacodynamic tests, as well as pharmacokinetic assays, were performed before the operation as well as 10-12 days after the bile duct ligation. At the end of the study livers were examined macro- and microscopically and biochemical analysis of the liver microsomes was performed. 4. The measured pharmacokinetic parameters suggested an impaired biotransformation of phenazone in animals with obstructive cholestasis, leading to a slower drug elimination.

Ultrastructure of spermatozoa from the cauda epididymis in rat chronically treated with lead acetate [Pb(II)].

Electron microscopic studies were performed on spermatozoa from the lumen of cauda epididymis in rats receiving lead acetate (II) for a period 5-fold longer than one spermatogenesis. There were numerous ultrastructural changes of spermatozoa. Mitochondrial spiral, outer dense fibers and axoneme were affected. Based on the present results it may be suggested that lead can damage spermatozoa in the epididymis.

Effect of lecithin on the development of experimental atherosclerosis in rabbits.

Thirty male mongrel rabbits were divided into 3 equal groups: (1) controls, (2) animals receiving a high-fat diet (HFD) containing cholesterol and coconut oil, and (3) HFD + lecithin. The experiment lasted 12 weeks. In animals receiving a HFD (group II) the QRS interval (ECG) was significantly prolonged, and the concentration of lipid fractions (except for HDL-cholesterol) and malondialdehyde (MDA) in the blood was markedly elevated. Distinctly lower concentrations of zinc in the serum and cytochrome P-450 in the liver microsomes were observed. When administered together with the HFD, lecithin normalized QRS interval duration, inhibited the diet-induced lipid metabolism disturbances, reduced MDA concentrations and increased zinc concentrations in the serum. Elevated under the influence of lecithin, the content of cytochrome P-450 in the liver microsomes was shown. Surface area of the lipid deposits at 12 weeks, measured planimetrically averaged 24.43% in HFD-fed animals vs 9.28% in lecithin-treated rabbits. We conclude that lecithin is responsible, not only for the hypolipidaemic properties, but also for the antiatherosclerotic action in rabbits.

Extractum Fagopyri reduces atherosclerosis in high-fat diet fed rabbits.

A group of 30 male mongrel rabbits was divided into 3 subgroups: controls, animals receiving a high-fat diet (HFD) containing cholesterol and coconut oil, HFD + Extr. Fagopyri (EF) were treated for 12 weeks. Surface areas of lipid deposites after 12 weeks of treatment measured planimetrically in the intima of the aorta, averaged 86.5% in HFD-fed animals, but 68.6% in EF treated rabbits. The positive effect of EF was confirmed histopathologically. The finding of this study is that the EF administration results in the reduction of atherosclerotic plaque formation.

Pharmacokinetics of lidocaine and its major metabolite- monoethylglycinexylidide (MEGX) in rabbits with experimental common bile duct obstruction.

The aim of this study was to evaluate, using an experimental model, the effect of obstructive cholestasis on the pharmacokinetics of lidocaine and the formation rate of its major metabolite- monoethylglycinexylidide (MEGX)-in rabbits. The investigation was carried out on 20 rabbits, randomly divided into two groups: a control one sham-operated and an experimental one-animals with biliary duct ligation. The measurements, i.e. laboratory and pharmacodynamic tests, as well as pharmacokinetic assays were performed prior to the operation as well as 10-12 days after the bile duct ligation. At the end stage of the study, livers were examined macro- and microscopically and biochemical analysis of the liver microsomes were performed. Lidocaine was given intravenously, as a bolus of 6 mg/kg. Blood for pharmacokinetic assay was sampled within 6 h following the drug administration, and MEGX concentration was evaluated 15 min after lidocaine had been administered. The immunofluorescence polarization method was employed for determination of lidocaine and MEGX concentrations. The one-compartment open model was used for calculations.

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.

Morphology of in vitro sperm phagocytosis by rat peritoneal macrophages under influence of oily contrast medium (Lipiodol).

Morphology of sperm phagocytosing rat peritoneal macrophages (PM) in control and Lipiodol added cultures was evaluated under light as well as scanning electron and transmission electron microscopes. The spermiophagic index (SPI) was assessed to estimate an inhibitory effect of Lipiodol on sperm phagocytosis to be compared with the outcome of morphological studies. Lipiodol inhibits in vitro sperm phagocytosis as indicated by SPI and evaluation under light, scanning electron and transmission electron microscopes. The highest SPI was seen in control cultures (14.44), declined in cultures with 0.25% and 0.5% Lipiodol (3.3 and 3.02 respectively) to reach the lowest value with 1% Lipiodol (1.60). The macrophages in cultures with Lipiodol were covered by a thin layer of oily contrast medium. The layer produced changes in the macrophages' shape and the cytoplasmic surface configuration. Lipiodol hinders both sperm phagocytosis and movement of PM by altering their shape and impairing pseudopodia and protrusions formation. Morphological studies of sperm phagocytosis by PM can help explain therapeutic effects of oily contrast media used in hysterosalpingography. Number of studies have suggested therapeutic effects of hysterosalpingography (HSG). These studies have demonstrated an increase in pregnancy rates after HSG in previously infertile women (Fullenlove, 1969; Palmer, 1960). Pregnancies were even more numerous after applying oil, than water based contrast media (DeCharney et al., 1980; Schwabe, 1983). Sawatari et al., 1993 have lately shown, that Lipiodol significantly increases pregnancy rates in animals. Hypothesis has been put forward as to the possibility of modulating activity of these agents exerted upon the peritoneal and oviductal macrophages (Boyer et al., 1986; Schwabe et al., 1983).(ABSTRACT TRUNCATED AT 250 WORDS)