Cellular Distribution of Aquaporin 3, 7 and 9 in the Male Reproductive System: A Lesson from Bovine Study (Bos taurus).

The increasing incidence of male infertility in humans and animals creates the need to search for new factors that significantly affect the course of reproductive processes. Therefore, the aim of this study was to determine the temporospatial expression of aquaglyceroporins (AQP3, AQP7 and AQP9) in the bovine (Bos taurus) reproductive system using immunohistochemistry and Western blotting. The study also included morphological analysis and identification of GATA-4. In brief, in immature individuals, AQP3 and AQP7 were found in gonocytes. In reproductive bulls, AQP3 was observed in spermatocytes and spermatogonia, while AQP7 was visible in all germ cells and the Sertoli cells. AQP7 and AQP9 were detected in the Leydig cells. Along the entire epididymis of reproductive bulls, aquaglyceroporins were visible, among others, in basal cells (AQP3 and AQP7), in epididymal sperm (AQP7) and in the stereocilia of the principal cells (AQP9). In males of all ages, aquaglyceroporins were identified in the principal and basal cells of the vas deferens. An increase in the expression of AQP3 in the testis and cauda epididymis and a decrease in the abundance of AQP7 in the vas deferens with age were found. In conclusion, age-related changes in the expression and/or distribution patterns of AQP3, AQP7 and AQP9 indicate the involvement of these proteins in the normal development and course of male reproductive processes in cattle.

Can apoptosis and necrosis coexist in ejaculated human spermatozoa during in vitro semen bacterial infection?

PURPOSE: To evaluate whether ejaculated human spermatozoa undergo complete apoptosis or necrosis during experimental semen bacterial infection in vitro. METHODS: Apoptotic markers, including mitochondrial transmembrane potential (ΔΨm), phosphatidylserine (PS) externalization, and DNA fragmentation, have been detected simultaneously in ejaculated human sperm after their incubation with a known pathogenic (Escherichia coli), as well as with conditionally pathogenic bacterial strains (Staphylococcus haemolyticus, Bacteroides ureolyticus) and/or leukocytes. The ΔΨm and translocation of PS was evaluated using the JC-1 and Annexin V binding tests, respectively. A modified TUNEL assay with additional staining for sperm viability was used to detect the DNA fragmentation level. RESULTS: The exposure of ejaculated spermatozoa to bacterial strains was associated with a simultaneous decrease in the percentage of sperm with normal ΔΨm and an increase in the proportion of Annexin V-positive sperm. Additionally, in the presence of S. haemolyticus, B. ureolyticus and/or leukocytes, a significant increase in the percentage of live TUNEL-positive (apoptotic) as well as dead TUNEL-positive (necrotic) sperm cells was also observed. CONCLUSIONS: The cellular death observed in spermatozoa in the presence of inflammatory mediators may be due to both apoptosis and necrosis. Here, we demonstrate for the first time that direct contact of conditionally pathogenic bacteria with ejaculated human sperm may play an even greater role in the promotion of apoptosis than in case of some pathogenic bacterial strains. These findings suggest that significant bacteriospermia and leukocytospermia may be direct causes of subfertility or additional negative factors worsening the prognosis of fertility in natural and assisted procreation.

Ultrastructure of the Bovine Testis in Cattle (Bos taurus): New View.

The purpose of this study was to analyze the ultrastructure of the testes of sexually immature calves and reproductive bulls of the Polish Holstein-Friesian Black-and-White breed. Utilizing TEM, this study identified three distinct stages of seminiferous tubule development in calves, characterized by varying shapes, distributions, and arrangements of individual cells. In immature animals, early developing spermatocytes, prespermatogonia, and pre-Sertoli cells were observed within the seminiferous tubules. In sexually mature bulls, all cells of the spermatogenic series were observed, situated on a thin, multilayered basal lamina, which forms characteristic undulations. An abundant smooth endoplasmic reticulum was observed in the cytoplasm of spermatogonia in both groups of animals, forming characteristic membranous swirls. In adult bulls, spermatogonia maintain contact with each other through numerous cytoplasmic bridges and cell connections, forming small spaces with visible microvilli between them. The ultrastructural analysis facilitated the identification of morphological changes occurring during the maturation of pre-Sertoli cells, transitioning from a large euchromatic nucleus to a nucleus in which the formation of characteristic vesicles and tubules could be observed. It should also be emphasized that two types of Sertoli cells, namely dark and light electron-dense cells, can be found in cattle. These cells differ from each other, indicating that they may perform different functions. The widespread recognition of the presence of two types of Sertoli cells in cattle will undoubtedly contribute to a better understanding of the processes occurring within the testes and provide a basis for further research in this area.

Total Selenium Level and Its Distribution between Organs in Beef Cattle in Different Selenium Status.

The aim of this study was to determine the Se concentration in the main tissues of beef cattle and to evaluate the differences in tissue distribution between animals with different selenium status. Selenium concentration was determined in the serum, longissimus dorsi muscle, semitendinosus muscle, kidney, heart, liver, spleen and lungs of cows, heifers and beef bulls, using spectrofluorimetric method. Despite receiving supplementation, 55.6% animals demonstrated an optimal Se level, while 44.4% were deficient. The mean serum Se concentration was significantly higher (p < 0.05) in animals with a normal Se status than in Se-deficient animals. Differences in Se tissue distribution were observed between Se-deficient animals and those with normal Se status. The organs most susceptible to Se deficiency are the semitendinosus muscle, lungs, heart and liver. In both normal and Se-deficient animals, significantly higher Se concentrations were observed in the kidney than other organs (p < 0.05), and the lowest in the muscles. As Se deficiencies can be found among supplemented animals, the level of Se should be monitored in beef cattle in order to detect possible Se deficiencies, which may have negative health effects for animals and reduce the value of animal products as a source of Se in the human diet.

Plasma membrane changes during the liquid storage of boar spermatozoa: a comparison of methods.

Studies were performed on boar semen routinely used at the local artificial insemination (AI) centre. The semen was stored in a Safe Cell Plus commercial extender at 17 degrees C for nine days. The aim of our research was focused on changes in sperm plasma membrane integrity. The integrity of the sperm plasma membrane and acrosome as well as sperm motility decreased after dilution and during storage of the semen. The highest percentage of live sperm was identified by the eosin-nigrosin method, a lower percentage by the SYBR-14/PI test, and the lowest percentage of live cells was discovered by the hypoosmotic swelling (HOS) test (P < 0.01). There were significant differences between the results of staining methods and sperm motility (P < 0.01). No significant differences were found between the HOS test results and sperm motility. The plasma membrane integrity parameters positively correlated (P < 0.001) with each other and with sperm motility but negatively with aspartate aminotransferase activity. Our findings confirmed that the boar sperm aging changes, which increased during liquid semen preservation, were connected with the loss of function and integrity of the sperm plasma membrane. The employed complementary tests are comprehensive indicators of sperm membrane integrity during long-term semen preservation, and they can help establish the actual number of 'healthy' cells. The assays may be used in AI laboratories and should be incorporated into the routine of semen analysis.

Plasma progesterone analysis by a time-resolved fluorescent antibody test to monitor estrous cycles in goats.

The objective of the current study was to evaluate whether blood plasma progesterone (P(4)) measurements with a time-resolved fluorescent antibody test (TR-FAT) kit designed for humans was applicable for goats. The first experiment was designed to verify whether the concentrations of P(4) measured by TR-FAT can be used to monitor the estrous and ovarian activity in goats (n = 14). Blood samples (322) were collected, and the ovaries were scanned using ultrasonography. The second experiment was carried out on 4 goats (60 samples) and designed to compare the TR-FAT with radioimmunoassay (RIA). The time interval between the lowest concentrations of P(4) assayed by TR-FAT was 21 +/- 0.3 days and did not differ significantly from the length of the interestrous interval. The highest concentrations of P(4) were confirmed by detection of corpus luteum. During estrus, the mean concentration did not differ significantly between both methods. Significant differences were present during the luteal phases; however, the profiles of P(4) assayed by both methods followed a similar pattern. Regression analysis showed a correlation between the 2 methods (r = 0.98; r(2) = 0.96; P < 0.0001). The Bland-Altman plot showed that all averages were within the 95% limits of agreement; however, the differences between both methods tend to be greater as the average increases. The results demonstrated that the TR-FAT method can be applied to monitor estrous cycles in goats through measurements of plasma P(4) concentrations. Moreover, not only does the TR-FAT meet the requirements for safety, but it is also a method of high throughput, rapidity, and simplicity.

Age-related changes in human sperm DNA integrity.

Abnormal standard semen characteristics and reduced sperm chromatin maturity can appear with increasing male age. However, the influence of paternal age on semen parameters is still controversial. Therefore, this study was designed to estimate the influence of paternal age not only on conventional semen characteristics but also on sperm DNA integrity. This research was carried out on ejaculated sperm cells obtained from men (n = 1124) aged ≥40 y and <40 y. Our data revealed a decreased semen volume and an increased percentage of DFI (sperm DNA fragmentation index) in older men compared to younger men in the entire study cohort, in men with normozoospermia and in men with abnormal semen parameters. Moreover, there was a higher incidence of sperm DNA damage (>10% DFI, low fertility potential) in the groups of men aged ≥40 y than in the groups of men aged <40 y. Older men had over twice the odds ratio for high sperm DNA damage as younger men. Our findings suggest a detrimental effect of advanced paternal age on sperm chromatin integrity. The data show that the evaluation of sperm DNA has greater clinical utility than standard semen analysis in case of male fertility potential assessment.

[Influence of dihydrotestosterone deficit on treatment of prostatic diseases regarding to contemporary methods of prostatic hypertrophy treatments]. / Wplyw deficytu dihydrotestosteronu na leczenie schorze prostaty na te wspólczesnych metod farmakologicznego leczenia przerostu tego gruczolu.

In the prostate the enzyme 5alpha-reductase converts testosterone into more active form--dihydrotestosterone (DHT). This hormone strongly influences prostate function and takes part in its pathology Since the connection between androgens and pathology of this gland has been proved, pharmacological inhibition of conversion became one of the therapy strategies both for benign prostate hyperplasia and prostate cancer. Last decade has brought numerous, long term clinical trials which involved numerous men being administered finasteride and other 5alpha-reductase inhibitors in bening prostate hypertrophy (BPH) treatment and prostate cancer (PCa) prevention. The results confirmed main assumptions, however revealed complex androgens and other factors' activity overlapping with the effect of therapy on prostate disease.

The effect of human sperm chromatin maturity on ICSI outcomes.

Because sperm chromatin may play a key role in reproductive success, we verify the associations between sperm chromatin abnormalities, embryo development and the ability to achieve pregnancy. The evaluation of sperm chromatin maturity using aniline blue (AB), chromomycin A3 (CMA3) and toluidine blue (TB) staining were carried out in group of males from infertile couples that underwent ICSI. Low levels of sperm chromatin abnormalities (< 16%) were found in most subjects (> 50%). A higher percentage of TB-positive sperm cells were discovered in the men from couples who achieved ≤ 50% fertilized oocytes compared to men who achieved > 50%. No significant differences were discovered by the applied tests between the men from couples who achieved ≤ 50% and those who achieved > 50% high-quality embryos on the 3rd or 5th day after fertilization, nor between the men from couples who achieved pregnancy and those who failed. The sperm chromatin maturity did not correlate with the ICSI results. However, the ROC analysis revealed a significant predictive value of TB-positive spermatozoa only for fertilization. Therefore, the TB assay can be considered as a useful test for the prediction of fertilization. Our findings suggest that the level of sperm chromatin abnormalities of the examined men was not clinically significant. No found associations between sperm chromatin maturity and embryo development and the ability to achieve pregnancy. We could not exclude the effects of the repairing processes in the fertilized oocyte. The use of complementary tests that verify the status of the sperm chromatin seems justified.

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.

Oxidoreductive capability of boar sperm mitochondria in fresh semen and during their preservation in BTS extender.

The purpose of our study was to determine the effect of dilution and liquid-preservation of boar sperm on oxidoreductive capability of their mitochondria. The semen was diluted with BTS extender produced from water purified by destillation or by reverse osmosis. The spermatozoa were stored over a four-day period at 16-18 degrees C. The function of sperm mitochondria was assessed using the screening cytochemical test for NADH-dependent oxidoreductases (diaphorase/NADH, related to flavoprotein). Morphological assessment of cytochemical reaction was carried out using a light microscope. The intensity of the reaction was evaluated by means of a computer image analysing system (Quantimet 600S), measuring the integrated optical density (IOD) and mean optical density (MOD) of the reaction product (formazans) occurring in the sperm midpieces. In the non-diluted semen, intensive cytochemical reaction throughout the length of the sperm midpiece was observed. Furthermore, spermatozoa with the intensive reaction displayed the high optical density values. After dilution the semen with two variants of experimental extender, and as the conservation time expired, the cytochemical reaction was less intensive. Moreover, the absence of formazan deposits in various parts of the sperm midpiece was also noted. These morphological features corresponded to low values of optical density. These findings suggest that the dilution of semen and the time of sperm preservation may be critical factors that handicap energy metabolism of sperm mitochondria. The type of water used in preparing BTS extender does not have any significant effect on the oxidoreductive capability of sperm boar mitochondria.

Morphological and functional evaluation of spermatozoa from patients with asthenoteratozoospermia.

In several cases of asthenoteratozoospermia, electron microscopic investigation displayed immature sperm forms, morphological apoptotic patterns of spermatozoa and many cytoplasmic conglomerates with fragments of the sperm. In these patients, TUNEL assay showed a high percentage of spermatozoa with nuclear DNA fragmentation. Moreover, thickened and deformed midpieces were observed which contained supernumerary and redundant mitochondria with normal oxidoreductive capability and normal membrane potential. In these cases a high percentage of spermatozoa with normal DeltaPsim was detected. Nevertheless, a subpopulation of patients was found with an abnormal ultrastructure of sperm mitochondria and with a low percentage of spermatozoa with normal DeltaPsim. These findings indicate that low motility of spermatozoa may be related to abnormal morphogenesis of the midpiece containing functional mitochondria and that this may be a possible consequence of an apoptotic mechanism. Furthermore, our results show that asthenoteratozoospermia may result from dysfunction of sperm mitochondria and/or with alternations of the structures involved in sperm motility, i.e. the dense outer fibres, the fibrous sheath and the axoneme.

Fertilizing potential of ejaculated human spermatozoa during in vitro semen bacterial infection.

OBJECTIVE: To assess the in vitro effect of three bacterial isolates (Escherichia coli, serotype O75:HNT, Staphylococcus haemolyticus, and Bacteroides ureolyticus) and/or leukocytes on sperm motility, subcellular changes in sperm plasma membranes, and sperm fertilizing potential. DESIGN: An in vitro model of semen bacterial infection. SETTING: Basic research laboratory. PATIENT(S): Healthy normozoospermic volunteers and healthy blood donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm plasma membrane stability was evaluated with a LIVE/DEAD Sperm Viability Kit and with the merocyanine 540 (M540) test both performed using flow cytometry. An oxiSelect TBARS Assay Kit was used for quantitative measurement of malondialdehyde content. Functional ability of spermatozoa was assessed by hypo-osmotic swelling (HOS) test and sperm penetration assay (SPA). RESULT(S): The incubation of sperm with bacteria and/or leukocytes was associated with the reduction of their fertilizing potential demonstrated in both the HOS test and SPA, and this effect can be considered as a natural consequence of diminished motility and sperm membrane injury of lipid bilayers. Bacteroides ureolyticus demonstrated the most significant detrimental effect on sperm structure and function. CONCLUSION(S): Sperm motility and lipid sperm membrane status might be the earliest and the most sensitive indicators of sperm damage with negative consequences for male factor fertility, which can be attributed to both bacteria and leukocytes action.

Novel morphological findings of human sperm removal by leukocytes in in vivo and in vitro conditions: preliminary study.

PROBLEM: 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. METHOD OF STUDY: The study was performed on ejaculated spermatozoa from normozoospermic men with leukocytospermia (n=10, in vivo experiment) or without leukocytospermia (n=15, in vitro experiment). Morphological observations were performed using light and scanning electron microscopy. RESULTS: Sperm removal by active leukocytes mediated by traditional phagocytosis and generation of extracellular traps were observed in in vivo and in vitro experiments. CONCLUSION: Our morphological data suggest that human germ cells activate leukocytes triggering both traditional phagocytosis and a novel trapping mechanism, followed by extensive sperm elimination.