Piasecka M

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.

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.

Administration of flutamide alters sperm ultrastructure, sperm plasma membrane integrity and its stability, and sperm mitochondrial oxidative capability in the boar: in vivo and in vitro approach.

Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 μg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p < 0.05) in flutamide-treated boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p < 0.05). No further decrease in the membrane integrity was found when the effect of anti-androgen lasted for 24 h. On the other hand, a decrease in sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p < 0.05). Characterization of sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal.

Sperm mitochondria diaphorase activity – a gene mapping study of recombinant inbred strains of mice

In order to study the genetic control of semen quality parameters, we derived a set of recombinant inbred (RI) mice from crosses between two inbred strains, KE and CBA/Kw, which differ significantly in gamete quality and fertility parameters. In this work, we used male mice from the two parental strains and from ten RI strains to map genes controlling quantitative traits such as sperm mitochondrial diaphorase activity, and assessed the correlation between this trait, sperm motility and in vivo fertilization efficiency. We analyzed sperm mitochondrial dehydrogenase (diaphorase) activity (NADH-dependent NBT assay) cytochemically by means of computerized image densitometry and obtained values for four parameters: 1) integrated optical density (IOD) for all pixels of the midpiece, 2) mean optical density (MOD) for the midpiece pixels, 3) length of sperm midpiece and 4) area of sperm midpiece. Polymorphic microsatellite marker profiles were prepared for 20 mouse chromosomes in the ten RI strains. We used Map Manager QTX software to correlate the strain distribution patterns (SDPs) of the four measured parameters with the SDPs of the analyzed markers. Hypothetical genes modifying diaphorase activity were mapped to chromosomal region 19q43-19q47, containing, for example, Poll, Sfxn2, Cyp17a1 and Usmg5 genes. Chromosomal regions 18q44 and 18q49-18q80 also showed correlation with the SDPs of diaphorase activity. Katnal2, Me2 and StARD6 candidate genes were proposed from this region. Diaphorase activity in the mouse sperm midpiece did not correlate with in vivo fertilization efficiency, but was negatively correlated with the linearity and straightness of sperm movement.

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

Summary. 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 ultrastructural abnormalities were visible. Macrophages of testicular interstitial tissue contained electron‐dense inclusions, usually located inside phagoliso‐some‐like vacuoles. X‐ray micro‐analysis revealed that the inclusions contained lead.

Novel Morphological Findings of Human Sperm Removal by Leukocytes in In Vivo and In Vitro Conditions: Preliminary Study

Biological significance of increased number of leukocytes in ejaculate remains a subject of controversy. The aim of this research was to describe the morphological characteristics of the sperm elimination by leukocytes in in vivo and in vitro conditions using natural stimulator of the immune system—uropathogenic Escherichia coli, O75:HNT, isolated from semen.

Evaluation of spermatozoa of the rat in hyperprolactinaemia induced by Metoclopramide

Summary. Metoclopramide (MCP) in doses of 2.2 mg kg−1 body weight, being administered intraperitoneally to rats for 14 d, provokes a premature release of germ cells in the testes. The number of spermatozoa is reduced. In addition the spermatozoa exhibit defects of head and tail structures. After the administration of MCP for 14 d, followed by a 14‐d break in applying the drug, the number of spermatozoa in the lumen of the epididymal duct continues to be low. Many spermatozoa are damaged. Only after a 66‐d‐break in MCP administration is the morphology of the seminiferous epithelium restored, and a large number of spermatozoa appear in the lumen of the epididymal duct.

Bacterial Contamination of Boar Semen and its Relationship to Sperm Quality Preserved in Commercial Extender Containing Gentamicin Sulfate.

This study was designed to determine the degree and type of bacterial contamination in boar semen (79 ejaculates from Large White and Landrace boars) and its consequences for sperm quality during storage (27 extended semen samples, 16°C for five days) under practical conditions of artificial insemination (AI). The results revealed the presence of aerobic bacteria in 99% of the ejaculates (from 80 to 370 ×106 colony-forming units/mL). Most of the ejaculates contained two or three bacterial contaminants, while the Staphylococcus, Streptococcus, and Pseudomonas bacterial genera were most frequently isolated. Also detected were Enterobacter spp., Bacillus spp., Proteus spp., Escherichia coli, P. fluorescens, and P. aeruginosa. In general, the growth of certain bacterial types isolated prior to semen processing (Enterobacter spp., E. coli, P. fluorescens, and P. aeruginosa) was not discovered on different days of storage, but fluctuations (with a tendency towards increases) were found in the frequencies of Bacillus spp., Pseudomonas spp., and Staphylococcus spp. isolates up to the end of storage. Semen preserved for five days exhibited decreases in sperm motility and increases in the average number of total aerobic bacteria; this was associated with sperm agglutination, plasma membrane disruption, and acrosome damage. We inferred that, due to the different degrees and types of bacterial contaminants in the boar ejaculates, the inhibitory activity of some antimicrobial agents used in swine extenders (such as gentamicin sulfate) may be limited. Because such agents can contribute to the overgrowth of certain aerobic bacteria and a reduction in the quality of stored semen, procedures with high standards of hygiene and microbiological control should be used when processing boar semen.

The effect of bacteriospermia and leukocytospermia on conventional and nonconventional semen parameters in healthy young normozoospermic males

Bacterial semen inflammation/infection is an important diagnostic and therapeutic problem in contemporary andrology. The molecular mechanism by which inflammatory mediators compromise the fertilizing potential of germ cells is complex and multifactorial, and it remains unclear. To improve the understanding of the pathophysiology of human subfertility/infertility caused or complicated by reproductive tract inflammation/infection, we simultaneously evaluated a set of conventional (standard semen analysis) and nonconventional sperm parameters, including subcellular changes in sperm membranes (phospholipid scrambling, peroxidative damage, and phosphatidylserine (PS) externalization), mitochondria (mitochondrial transmembrane potential, ΔYm, and oxidoreductive capability), and DNA fragmentation in healthy young normozoospermic males with asymptomatic bacteriospermia and leukocytospermia. Both bacteriospermia and leukocytospermia had a deleterious effect on standard sperm parameters, including sperm concentration, motility and morphology. Bacteriospermia was associated with a simultaneous decrease in mitochondrial transmembrane potential and an increase in PS externalization, and with DNA fragmentation in both live and dead sperm. The highest MDA concentrations in sperm lysates were observed in the presence of leukocytes. This study demonstrates for the first time that bacteriospermia and leukocytospermia compromise sperm quality in healthy young normozoospermic males. Bacteria mainly participate in intrinsic mitochondria-dependent apoptotic cell death mechanisms. Oxidative stress plays a relevant role in decreasing routine sperm parameters during leukocytospermia. The value of these observations may be significant and may support the development of a new diagnostic platform (biomarkers) for infertile males with infections in the reproductive tract.

The presence and role of progesterone receptor in the ovaries of postmenopausal women who have not applied hormone replacement therapy.

At present, not much is known about progesterone receptor (PR) expression and localization in postmenopausal women ovaries. In the ovaries of reproductive age women, PR is localized in internal theca and granulosa cells, corpus luteum, ovary surface epithelium (OSE) and in stroma. PR expression depends on the serum concentration of progesterone, estrogen, gonadotropin and androgen. The goal of the conducted studies was to examine PR localization and expression in the ovaries of postmenopausal women who have not applied hormone replacement therapy so far. Also, the correlation was examined between PR expression and localization in the ovaries, steroid and gonadotropin hormone serum concentrations, and influence of the time from the last menstruation. The material came from 50 postmenopausal women who had their ovaries removed due to non-neoplastic diseases. The women were divided into 3 groups (A, B, C) depending on the time from the last menstruation. The follitropin (FSH), luteotropin (LH), estradiol (E2), testosterone (T), androstendione (A) and dehydroepiandrosterone sulphate (DHEAS) concentrations in blood plasma were measured. Monoclonal mouse anti-human PR antibody was used for immunohistochemical detection (examination involved 50 postmenopausal ovaries). Between particular groups, E2 serum concentrations did not differ, but FSH, LH, T, A, DHEAS serum concentrations were significantly different. Immunohistochemical nuclear localization of PR in postmenopausal women ovaries was observed. PR expression was similar in all three groups (A, B, C). PR expression was observed in OSE nuclei and invaginations cysts deriving from the isolation of invaginated epithelium and metaplastic columnar epithelium and in stroma. In the ovaries of postmenopausal women who have not applied hormone replacement therapy so far, PR was detected in all three groups. Its expression did not depend on the time from menopause and was similar in all examined groups. FSH, LH, T, A, DHEAS serum concentrations did not influence PR expression.