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.

[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.

[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.

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.

DNA content in the mouse spermatogonia B and spermatid nuclei during treatment with insecticides.

The DNA content in nuclei of germ cells: spermatogonia B and spermatides in the maturation phase of male mice receiving commercial insecticide preparations commonly used in this country was cytophotometrically measured. The following insecticides were tested: 1% Metox-30 solution, containing 30% of methoxychlorine (p,p-dimethoxydiphenyltrichlorethane), 0,3% solution of Sadofos-30 containing 30% of malathione (1,2-dicarboethoxyethyl-0,0-dimethyldithiophosphate) and 0,3% solution of Foschlor-50 containing about 50% trichlorfon (2,2,2-trichloro-1-hydroxyethyl-0,0-dimethyl-diphosphonate). It was found that a statistically significant number of spermatogonium B and spermatide nuclei in the phase of maturation of the animals treated with the insecticides exhibited an abnormal DNA content as compared with the nuclei of control animals. The chromatin of these nuclei is also more sensitive to acid hydrolysis in Feulgen reaction.