Seminal plasma N-glycome as a new biomarker of environmental exposure associated with semen quality.

Male infertility, a condition that has during the last decade raised significant concern, is a diagnostically demanding and socially sensitive topic. The number of unsolved issues on infertility etiology, especially potential environmental causes, in couples demonstrates the need for further investigations into infertility biomarkers. Semen parameters are often insufficient for reliable profiling of male infertility. Thus, this study aims to evaluate for the first time seminal plasma N-glycosylation as a biomarker of environmental exposure in semen samples from 82 normozoospermic men and 84 men with abnormal semen parameters and compare it with genome damage measured by DNA fragmentation. We obtained information about chronic exposure to environmental factors from the self-reported questionnaire, and determined sperm DNA fragmentation by sperm chromatin dispersion, while N-glycans were characterized with liquid chromatography-mass spectrometry (LC-MS). Based on previously published results, ten N-glycans were selected. Results show that the selected seminal plasma N-glycans were significantly associated with smoking, exposure to pesticides, air pollution, agents emitted during photocopying, alcohol consumption, and obesity. Some N-glycans showed a simultaneous association with DNA fragmentation, semen parameters, and environmental stressors. These subgroups of N-glycans are new potential candidates for biomonitoring of exposure to different environmental factors in men with semen abnormalities.

TSH prevents bone resorption and with calcitriol synergistically stimulates bone formation in rats with low levels of calciotropic hormones.

Thyroid-stimulating hormone exerts both antiresorptive and anabolic effects on bone remodeling in aged ovariectomized rats and thyroid stimulating hormone-receptor null mice, supported by clinical results demonstrating that low thyroid-stimulating hormone level is associated with increased bone loss. To further explore the effect of thyroid-stimulating hormone on bone metabolism we introduced here a rat model with removed thyroid and parathyroid glands to obtain low serum concentrations of thyroid and parathyroid hormone, calcitonin and 1,25(OH)2D3. Surgery resulted in hypocalcemia, low parathyroid and thyroid hormone, 1,25(OH)2D3, C-telopeptide, and osteocalcin serum level. Intermittent administration of thyroid-stimulating hormone resulted in a further decrease of serum calcium and decreased level of serum C-telopeptide due to the suppression of bone resorption, while in the same animals osteocalcin in serum was higher indicating an increased bone formation rate. A combination of thyroid-stimulating hormone and 1,25(OH)2D3 significantly increased the serum Ca2+, C-telopeptide and serum osteocalcin values. MicroCT analyses of the distal femur and proximal tibia showed that rats treated with 1,25(OH)2D3 alone or in a combination with thyroid-stimulating hormone had an increased trabecular bone volume, and enhanced trabecular bone quality. Biomechanical testing of the trabecular bone showed an increased maximal load for 105% and 235%, respectively, in rats treated with 1,25(OH)2D3 alone, or in a combination with thyroid-stimulating hormone. We suggest that thyroid-stimulating hormone independently of calciotropic hormones suppressed bone resorption and stimulated bone formation, while in combination with 1,25(OH)2D3 acted synergistically on bone formation resulting in an increased bone volume.

Initiation of steroidogenesis precedes expression of cholesterologenic enzymes in the fetal mouse testes.

Sexual differentiation is a carefully regulated process that ultimately results in a development of the male or female phenotype. Proper development of the male phenotype is dependent upon the action of testosterone and anti-mullerian hormone. Leydig cells start to produce testosterone around day 12.5 in the fetal mouse testis, and continue to produce high levels of this hormone throughout gestation. In the present study, we examined whether expression of lanosterol 14alpha-demethylase (cyp51) and cytochrome P450 NADPH reductase, both involved in the cholesterol production, occurs simultaneously with proteins required for the production of steroid hormones. Immunocytochemical staining with the antibodies against cyp51, cytochrome P450 NADPH reductase, steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydrogenase I (3beta-HSD I) was used to determine the ontogeny of expression of these four proteins. As expected, 3beta-HSD I and StAR proteins were detected on day 12.5 p.c., while expression of cyp51 and NADPH cytochrome P450 reductase appeared 1 day later, on day 13.5. Thereafter, the expression of all four proteins remained strong throughout gestation. Results of this study suggest that initial steps of steroid hormone production in murine Leydig cells are mostly dependent on exogenously derived cholesterol, while from day 13.5 onwards, mouse Leydig cells are able to synthesize cholesterol and are therefore not dependent on exogenous cholesterol resources.

Genomic damage in children accidentally exposed to ionizing radiation: a review of the literature.

During the last decade, our knowledge of the mechanisms by which children respond to exposures to physical and chemical agents present in the environment, has significantly increased. Results of recent projects and programmes focused on children's health underline a specific vulnerability of children to environmental genotoxicants. Environmental research on children predominantly investigates the health effects of air pollution while effects from radiation exposure deserve more attention. The main sources of knowledge on genome damage of children exposed to radiation are studies performed after the Chernobyl nuclear plant accident in 1986. The present review presents and discusses data collected from papers analyzing genome damage in children environmentally exposed to ionizing radiation. Overall, the evidence from the studies conducted following the Chernobyl accident, nuclear tests, environmental radiation pollution and indoor accidental contamination reveals consistently increased chromosome aberration and micronuclei frequency in exposed than in referent children. Future research in this area should be focused on studies providing information on: (a) effects on children caused by low doses of radiation; (b) effects on children from combined exposure to low doses of radiation and chemical agents from food, water and air; and (c) specific effects from exposure during early childhood (radioisotopes from water, radon in homes). Special consideration should also be given to a possible impact of a radiochemical environment to the development of an adaptive response for genomic damage. Interactive databases should be developed to provide integration of cytogenetic data, childhood cancer registry data and information on environmental contamination. The overall aim is to introduce timely and efficient preventive measures, by means of a better knowledge of the early and delayed health effects in children resulting from radiation exposure.

Investigation of spermatozoa and seminal plasma by fourier transform infrared spectroscopy.

Fourier transform infrared (FT-IR) spectra of human spermatozoa and seminal plasma were recorded and analyzed. The procedure that was established for sample preparation enabled acquisition of reproducible spectra. The parameter I(1087)/I(966) for controlling spectra reproducibility was defined. The assignment of bands was carried out using an empirical approach and the origin of the "sperm specific doublet", the bands at 968 cm(-1) and 981 cm(-1), was determined. The principal component regression (PCR) algorithm was used to define the specific spectral regions correlating to characteristics of spermatozoa, such as concentration, straight-line velocity (VSL), and beat cross frequency (BCF). Then, simple spectral parameters, such as band intensities and band ratios, were tested to determine which one best correlates to characteristics of spermatozoa. The region of the amide I band, between 1700 cm(-1) and 1590 cm(-1), was defined as a specific spectral region that correlates to the concentration of spermatozoa. The parameter that gave the linear dependence to the concentration of spermatozoa was the intensity of the amide I band. For VSL, the bands between 1119 cm(-1) and 943 cm(-1) were defined as the specific spectral region. The relative amount of nucleic acids with respect to proteins showed linear dependence on the straight-line velocity of spermatozoa. BCF showed the best correlation to the bands between 3678 cm(-1) and 2749 cm(-1), which largely represent lipids and proteins. These results suggest that FT-IR spectroscopy can serve as an adjunct to conventional histopathology studies.

Expression of the proliferating cell nuclear antigen and protein products of tumour suppressor genes in the human foetal testis.

Tumour suppressor genes retinoblastoma (Rb1) and adenomatous polyposis coli (Apc) as well as the proliferating cell nuclear antigen (PCNA) are involved in embryonic development. The purpose of the present study was to investigate the expression of Rb1 protein, APC protein and PCNA during development of the human foetal testis. Qualitative analysis of their expression at the single-cell level was performed using immunohistochemistry on archive samples of the foetal testis (18-37 gestation week). Stereological parameters (volume density, absolute volume, numerical density, absolute number) were calculated for quantification of the overall expression of those proteins that were expressed frequently enough for such an analysis. PCNA was frequently expressed in nuclei of immature Sertoli cells and prospermatogonia and less frequently in surrounding peritubular (myoid) and interstitial cells. The pRb1 protein was present in nuclei of prospermatogonia and Sertoli cells but was absent from the interstitial tissue. APC protein was expressed in the cytoplasm of a very small number of prospermatogonia and interstitial (Leydig) cells. The overall expression of PCNA in all stages of development was higher than pRb1 expression.

From testicular biopsy to human embryo.

The aim of the study was to investigate the role of a testicular biopsy in the diagnosis and therapy of infertile men with a non-obstructive azoospermia. Overall, 70 testicular biopsies from infertile men were analysed. Samples were obtained by the "open testicular biopsy" method. After dissection, several pieces of the tissue were immediately immersed into the Sperm Prep Medium (Medi-Cult) and fixative (5.5% buffered glutaraldehyde). Tissue samples transported in Sperm Prep Medium were plunged into Sperm Freezing Medium (Medi-Cult) and were stored in liquid nitrogen for potential in vitro fertilization procedures. The tissue was also processed for semithin sections and transmission electron microscopy. Semithin sections from 8 infertile patients demonstrated regular testis structure and fully preserved spermatogenesis (control biopsies). In the remaining 62 cases, spermatogenesis was impaired and a variety of pathological changes could be seen: disorganization and desquamation of spermatogenic cells, spermatid or spermatocyte "stop", spermatogonia only, "Sertoli cells only" or tubular fibrosis. However, in 65% of cases (despite the above mentioned changes of seminiferous epithelium) foci of preserved spermatogenesis could be detected. These cases were classified as "mixed atrophy" of seminiferous tubules. In 63% of infertile patients, a successful extraction of sperm from the biopsy could be performed. In azoospermic patients, histological analysis of testicular biopsy proved to be very useful in terms of diagnosis as well as therapy, i.e. for further in vitro fertilization procedures.

A functional cytochrome P450 lanosterol 14 alpha-demethylase CYP51 enzyme in the acrosome: transport through the Golgi and synthesis of meiosis-activating sterols.

Mammalian lanosterol 14 alpha-demethylase (CYP51) is a microsomal cytochrome P450 that demethylates lanosterol to FF-MAS, an oocyte meiosis-activating sterol and late intermediate of cholesterol biosynthesis. Herein we report CYP51 unequivocally localized to acrosomal membranes of male germ cells in mouse, bull, and ram, in which it synthesizes FF-MAS in the presence of the acrosomal form of nicotinamide adenine dinucleotide phosphate reduced-P450 reductase. In the mouse, CYP51 (53 kDa) resides in endoplasmic reticulum (ER) and Golgi during all phases of acrosome development, indicating an intracellular transport from ERs through the Golgi to the acrosome. CYP51 (50 kDa) also resides on acrosomal membranes of bull- and ram-ejaculated sperm. In mouse liver, a 53-kDa CYP51 is no longer detected in trans Golgi, suggesting retrieval back to the ER and no further transport to other organelles. Glycosylated high-molecular-mass CYP51-immunoreactive proteins in acrosomal membranes of bull and ram and Golgi-enriched fractions of mouse liver indicate that mammalian CYP51s are subjected to posttranslational modifications in the Golgi. In conclusion, CYP51 is the first cytochrome P450 enzyme to be detected on acrosomal membranes. It exhibits a unique, cell-type-specific intracellular transport that is in agreement with its cell-type-specific physiological role: production of cholesterol in the liver and sterols with signaling properties in sperm. Demethylation of lanosterol to FF-MAS by the acrosomal lanosterol 14 alpha-demethylase enzyme complex demonstrates for the first time the ability of ejaculate sperm to synthesize meiosis-activating sterols.

CNS angiitis in graft vs host disease.

Graft-vs-host disease (GVHD) is a potentially treatable cause of progressive neurologic decline after bone marrow transplantation (BMT). The authors present histologic confirmation of CNS granulomatous angiitis in a child with chronic GVHD after BMT. Since cranial MRI showed only nonspecific findings, CNS vasculitis associated with GVHD after BMT may be underdiagnosed.

Glial cell line-derived neurotrophic factor (GDNF) and its receptors GFRalpha-1 and GFRalpha-2 in the human testis.

Glial cell line-derived neurotrophic factor (GDNF) and its receptors GFRalpha-1 and GFRalpha-2 were found in the human testis during fetal development (15-34 weeks of gestation) and in adult men (51-86 years of age) by means of RT-PCR, immunohistochemistry and Western blot techniques. Gene expression of GDNF could be established in the human testis and immunoreactivity (IR) for GDNF was detectable in Leydig cells, Sertoli cells, some spermatocytes and round spermatids as well as in smooth muscle cells of the wall of arterioles and small arteries. In the adult human testis, Sertoli and Leydig cells showed GFRalpha-1-IR, whereas GFRalpha-2-IR was located exclusively in Leydig cells. Different to man, in the rat GDNF-IR in Sertoli cells was detectable only until postnatal day10, providing evidence for species related variability in the expression of GDNF. These findings suggest a critical role for GDNF during the differentiation of testicular structures and provide evidence for an additional important function in the adult human and rodent testis.

Effects of various cryopreservation media and freezing-thawing on the morphology of rat testicular biopsies.

Currently, testicular sperm extraction is successfully combined with intracytoplasmic sperm injection into the oocyte (ICSI). Several pieces of a testicular biopsy can be frozen and thawed until the ICSI attempt. In this study, the effects of freezing-thawing on the morphology of rat testicular biopsies stored in different cryopreservation media were analysed. Each cryopreservation medium contained glycerol and/or dimethyl sulfoxide (DMSO) as cryoprotectants. In general, both glycerol and DMSO, when applied at moderate concentrations (6-25%), preserved the structure of the seminiferous epithelium. The freezing-thawing procedure had no significant effect on tubular diameter; however, it caused a 'folding' of the lamina propria and notable damage to Sertoli cells, spermatogonia and spermatocytes. Round and elongated spermatids and spermatozoa displayed occasional nuclear damage, vacuolization, and shrinkage/swelling of the cytoplasm. However, the vast majority of these cells maintained their normal structure in nearly all the applied cryomedia. It is concluded that freezing-thawing of testicular biopsies, and the cryopreservation medium, have a significant impact on the structure of the seminiferous epithelium, particularly on its basal compartment.

Differentiation of rat neural tissue in a serum-free embryo culture model followed by in vivo transplantation.

AIM: To analyze neural tissue differentiation in a unique, chemically-defined in vitro culture model of gastrulating rat embryo proper by use of transmission electron microscopy (TEM), proliferating cell nuclear antigen (PCNA) expression, and in vivo transplantation after a 2-week culture in serum-free or serum-supplemented media. Influence of protein-free medium unfavorable for differentiation of neural tissue in vitro was compared with favorable serum-free media enriched with transferrin or albumin. Differentiation of mesodermal derivatives in transplants was also investigated. METHODS: We cultivated 9.5-day-old Fischer rat embryos on the gas-liquid interface in the protein-free Eagle's Minimum Essential Medium (MEM), in MEM with either iron-saturated holotransferrin (50 microg/mL) or iron-free apotransferrin (50 microg/mL), and in medium saturated with either bovine serum albumin (BSA) (4 mg/mL or 400 microg/mL) or rat serum (50%). After the two-week culture period, light microscopy, TEM, and immunohistochemical method for detection of PCNA were done. Some explants were transplanted under the kidney capsule of adult male rats to be cultured in vivo for additional two weeks. Chi-square test or Fisher exact test were used to compare the proportion of tissues developed. RESULTS: Proportion of differentiated neural tissue was similar in explants cultivated in apotransferrin- and holotransferrin-supplemented media (13/33 and 9/20, respectively), but higher than in explants cultivated in protein-free medium (1/13). Neurons and glia cells produced a neuropil structure. Myelinization occurred only in serum-supplemented medium. PCNA expression was detected in a small number of neural tissue cells, even in serum-free cultivated embryos. Differentiation of brain-like tissue, cerebrospinal, and vegetative ganglionic cells occurred in all groups of transplants. However, in the transplants derived from protein-free medium, the proportion of neural tissue, cartilage, bone, skeletal and smooth muscle was significantly lower than in transferrin-supplemented media (p<0.01). Albumin seemed to promote differentiation of all tissues except vegetative ganglionic cells. CONCLUSION: Nerve tissue differentiated to a rather high degree in a two-week in vitro postimplantation embryo culture. Transferrin or albumin, as the only proteins used for serum-free precultivation, significantly improved subsequent differentiation of nerve tissue and mesodermal derivatives in transplants in vivo.

Localisation of lanosterol 14alpha-demethylase in round and elongated spermatids of the mouse testis: an immunoelectron microscopic and stereological study.

Lanosterol 14alpha-demethylase (CYP51) is a microsomal cytochrome P450 enzyme involved in the postsqualene cholesterol biosynthetic pathway. CYP51 removes 14alpha-methyl group from lanosterol], forming FF-MAS (folicular fluid meiosis activating sterol) which accumulates in gonads. The goal of our study is to determine the expression of CYP51 protein in the male gonad. Using electron microscopic immunogold techniques, CYP51 is localised on inner and outer acrosomal membranes of male germ cells, the round and elongated spermatids. Significance of CYP51 localization on the acrosome which is a Golgi-derived organelle is not known, but we propose that CYP51-formed FF-MAS can function as a signalling sterol during fertilisation.

Report of a new DRB1*13 allele: DRB1*1336.

This brief communication describes the characterization of a new allele, DRB1*1336.

Chemically defined protein-free in vitro culture of mammalian embryo does not restrict its developmental potential for differentiation of skin appendages.

In a unique serum- and protein-free chemically defined in vitro culture model of postimplantation mammalian development the epidermis differentiates regularly, although the differentiation of other tissues is impaired due to the lack of the serum. The present study in that model was done to estimate more carefully the degree of epidermal differentiation in defined media supplemented with some growth- or differentiation-stimulating substances. The main objective was to discover by grafting in vivo to the richer environment whether simple protein-free culture conditions restrict an inherent embryonic potential for differentiation of skin appendages. Embryonic parts of E9.5 gastrulating Fischer rat embryos were cultivated for 2 weeks in the protein-free Eagle's minimum essential medium supplemented with holotransferrin, apotransferrin, insulin and/or Na(2)SeO(3) and in controls cultivated in protein-free medium or in serum-supplemented medium. In all experiments there was a high incidence of differentiation of the epidermis. A high level of epidermal differentiation was confirmed for the first time at the ultrastructural level. A well-differentiated cornified layer and cells connected with desmosomes containing keratohyaline masses and cytokeratin filaments were found. A strong immunohistochemical signal for the proliferating cell nuclear antigen was always detected in the basal layer of the epidermis showing that those cells were still able to proliferate. Finally, embryos precultivated for 1 or 2 weeks in the protein-free medium and media supplemented with apotransferrin or serum were grafted under the kidney capsule for an additional 2 weeks. It was discovered that even after spending 2 weeks in the simple protein-free medium in vitro, embryos retained their developmental potential for differentiation of skin appendages (hair and sebaceous glands).

The development of blood and lymph vessels of human parathyroid glands in embryonal, fetal and postnatal period.

The aim of the article is to investigate the development of blood and lymph systems in human parathyroid glands in prenatal and postnatal periods. The first capillaries are observed in these glands already in the lunar month 2. At the middle of pregnancy blood supply is increased, being extremely abundant in lunar months 9 and 10, as well as during the first year of life. As parts of the lymph system, intercellular lymph spaces are noticed in the parathyroid glands already in the lunar month 2, and also later, when lymph vessels are situated along the gland or in its connective capsule and within the gland parenchyma respectively. All these findings could be connected with the early function of these glands, as well as with the possibility that parathyroid hormone (PTH) is not transferred by blood only but by lymph as well.

Mast cells in testicular biopsies of infertile men with 'mixed atrophy' of seminiferous tubules.

Mast cells in the bilateral testicular biopsies of 30 patients with a 'mixed atrophy' of seminiferous tubules were analysed. Seven biopsies from vasectomized patients served as controls. With regard to their characteristic location within testicular tissue, two groups of mast cells could be distinguished, in both control and infertile patients: 'interstitial' mast cells (located between Leydig and other interstitial cells as well as in the vicinity of blood vessels) and 'peritubular' mast cells (located in the close proximity of the tubular lamina propria or incorporated in the lamina propria itself). Morphometric data indicated a significant increase in the number and volume of mast cells in infertile patients when compared with controls. In the biopsies of infertile patients that were analysed both 'interstitial' and 'peritubular' mast cells showed a significant increase in their number and volume, although it appeared that 'peritubular' mast cells increased at a higher rate than 'interstitial' mast cells. A significant negative correlation was found between the following variables: volume and number of mast cells, testis volume and the status of spermatogenesis evaluated by Johnsen's scoring. It was concluded that the increased presence of mast cells is closely associated with an impairment of spermatogenesis.

Anthropological measurements of the skeleton and parathyroid glands growth during fetal development.

The article presents the investigation of histomorphological differentiation and growth of parathyroid glands in human fetus from the second to the ninth lunar month. The longer and the shorter diameter of these glands were measured. The obtained values are compared with the development and the growth intensity of the skeleton (biparietal head diameter and femoral length of a fetus from lunar months 3 to 9) and with the role of the placenta in the mentioned processes. The results of our investigation show the concordance of the skeletal growth with the development and histomorphological differentiation of these glands. The factors involved in these processes point out the complex relationship between the mother and fetus during osteogenesis, expressed on the hormonal level, in mineral metabolism and placental activity.

Successful testicular sperm extraction (TESE) in spite of high serum follicle stimulating hormone and azoospermia: correlation between testicular morphology, TESE results, semen analysis and serum hormone values in 103 infertile men.

Spermatozoa recovered from testicular biopsies can be used through intracytoplasmic sperm injection (ICSI) to achieve a pregnancy. To assess the likelihood of successful testicular sperm extraction (TESE) in men suffering from severe oligo- or azoospermia, bilateral biopsy specimens were obtained. Following semi-thin sectioning, the morphology of testicular samples was graded according to a modified Johnsen score. TESE was performed in parallel to this histological examination. The number of isolated spermatozoa was assessed in a semiquantitative way. From 103 patients investigated, 64 (62.1%) showed azoospermia in a preceding semen analysis and 29 (28.2%) patients had sperm concentrations between 0.1 and 1 x 10(6)/ml. In 10 patients who had higher sperm counts, most spermatozoa were non-motile. Spermatozoa could be detected after TESE in the testicular tissue of 49 (77%) azoospermic men. When follicle stimulating hormone (FSH) concentration was normal, most patients had detectable spermatozoa after TESE. Nearly one-third of patients with mildly elevated FSH had no spermatozoa. Thirty-nine percent of patients in whom FSH was elevated to more than twice normal and 50% of patients with grossly elevated FSH had no detectable spermatozoa. In all, 82.8% of men with sperm concentrations between 0.1 and 1x10(6)/ml in their ejaculate showed spermatozoa in the tissue sample after TESE. Our data demonstrate that, contrary to previous recommendations, infertile men with azoospermia and high FSH values should be reconsidered for testicular biopsy, provided that tissue samples can be cryopreserved for later TESE/ICSI treatment.