Exogenous retroelement integration in sperm and embryos affects preimplantation development.

Retroelement transcripts are present in male and female gametes, where they are typically regulated by methylation, noncoding RNAs and transcription factors. Such transcripts are required for occurrence of retrotransposition events, while failure of retrotransposition control may exert negative effects on cellular function and proliferation. In order to investigate the occurrence of retrotransposition events in mouse epididymal spermatozoa and to address the impact of uncontrolled retroelement RNA expression in early preimplantation embryos, we performed in vitro fertilization experiments using spermatozoa preincubated with plasmid vectors containing the human retroelements LINE-1, HERVK-10 or the mouse retroelement VL30, tagged with an enhanced green fluorescence (EGFP) gene-based cassette. Retrotransposition events in mouse spermatozoa and embryos were detected using PCR, FACS analysis and confocal microscopy. Our findings show that: (i) sperm cell incorporates exogenous retroelements and favors retrotransposition events, (ii) the inhibition of spermatozoa reverse transcriptase can decrease the retrotransposition frequency in sperm cells, (iii) spermatozoa can transfer exogenous human or mouse retroelements to the oocyte during fertilization and (iv) retroelement RNA overexpression affects embryo morphology and impairs preimplantation development. These findings suggest that the integration of exogenous retroelements in the sperm genome, as well as their transfer into the mouse oocyte, could give rise to new retrotransposition events and genetic alterations in mouse spermatozoa and embryos.

Synergistic effect of follicle-stimulating hormone receptor and androgen receptor gene variants on semen quality.

Follicle-stimulating hormone (FSH), interacting with its receptor (FSHR), participates in the production of spermatozoa and androgens. Androgens exert their effects on male sex determination, development and sperm production by binding to androgen receptor (AR). In the present study, we sought to explore the potential synergistic effects of FSHR and AR gene variants on sperm quality. 200 oligozoospermic and 250 normozoospermic men were examined. DNA was extracted from spermatozoa, and the FSHR 307 (T/A), FSHR 680 (N/S) and AR (CAG)n polymorphisms were genotyped. Their parallel analysis revealed six combined genotypes. A gradual reduction of sperm motility, from long AR allele-Thr307Thr/Asn680Asn carriers to long AR allele-Ala307Ala/Ser680Ser carriers and from short AR allele-Thr307Thr/Asn680Asn carriers to short AR allele-Ala307Ala/Ser680Ser carriers was revealed in normozoospermic men (P < 0.001). Similar associations were observed in oligozoospermic men (P < 0.001). In our series, the synergism of the long AR alleles with the FSHRThr307/Asn680 allelic variant was associated with increased sperm motility, while the synergism of the short AR alleles with the FSHRAla307/Ser680 allelic variant was associated with decreased motility, supporting the significance of these genes in semen quality.

Semen quality is influenced by androgen receptor and aromatase gene synergism.

STUDY QUESTION: Does synergism between AR(CAG)(n) and CYP19(TTTA)(n) polymorphisms influence the quality of sperm? SUMMARY ANSWER: AR(CAG)(n) and CYP19(TTTA)(n) polymorphisms had a synergistic effect on sperm concentration and motility. WHAT IS KNOWN ALREADY: Androgens exert their action in the testicular tissue by binding to androgen receptor (AR), while their action is mediated by the aromatase P450 enzyme (CYP19). AR(CAG)(n) alleles are associated with sperm motility and CYP19(TTTA)(n) allelic variants have implications for sperm concentration and motility. STUDY DESIGN, SIZE, DURATION: Two hundred oligozoospermic and 250 normozoospermic men who presented for infertility investigation were examined during a period of 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS: Conventional semen analysis was performed. DNA was extracted from spermatozoa and both polymorphisms were genotyped by polymerase chain reaction. Serum hormone levels were determined. MAIN RESULTS AND THE ROLE OF CHANCE: Six combined genotypes were identified between the 18 AR(CAG)(n) alleles with 12-32 repeats and the 6 CYP19(TTTA)(n) alleles with 7-12 repeats. A gradual reduction in the sperm concentration (10(6)/ml) and motility (%) from long AR allele-non-CYP19(TTTA)(7) allele carriers to long AR allele-CYP19(TTTA)(7) homozygotes and from short AR allele-non-CYP19(TTTA)(7) carriers to short AR allele-CYP19(TTTA)(7) homozygotes was observed in normozoospermic men (means ± SD; concentration: 93 ± 53.1 versus 65 ± 48.6 and 85 ± 60.1 versus 37 ± 17.2l, P < 0.002; motility: 63 ± 10.3 versus 55 ± 14.5 and 52 ± 19.6 versus 41 ± 13.7, P < 0.001, respectively). Similar associations were observed in oligozoospermic men (concentration: 10 ± 4.2 versus 9 ± 5.9 and 10 ± 6.3 versus 6 ± 3.1, P < 0.03; motility: 47 ± 17.1 versus 39 ± 6.2 and 39 ± 22 versus 27 ± 18.3, P < 0.003, respectively). The above associations of the combined genotypes with sperm concentration and motility were confirmed in the total study population (P < 0.006 and P < 0.001, respectively). LIMITATIONS, REASONS FOR CAUTION: Our study population was limited to Greek Caucasian adult males, residents of Northwest Greece. WIDER IMPLICATIONS OF THE FINDINGS: The confirmation of our findings in other populations would verify the significance of AR and CYP19 genes for spermatogenesis. STUDY FUNDING/COMPETING INTERESTS: This study did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. The authors declare no conflicts of interest.

Dramatic reduction in sperm parameters following bariatric surgery: report of two cases.

Severe obesity constitutes the main public health crisis of the industrialised world. Bariatric surgery has been proposed as the most efficient treatment of obesity. In this study, we report the potential effects of bariatric surgery on semen parameters in male partners of couples undergoing assisted reproduction. These patients had been tested in the context of infertility treatment in two consecutive cycles before and after bariatric surgery. A marked reduction in sperm parameters was observed in a period of twelve to eighteen months after surgery. This unfavourable effect had also remarkable effects on the assisted reproduction outcome, necessitating the counselling of patients before bariatric surgery.

The N-myc oncogene in human neuroblastoma cells: down-regulation of an angiogenesis inhibitor identified as activin A.

Angiogenesis, the formation of new blood vessels, is seen during embryonic development and tumor progression, but the mechanisms have remained unclear. Recent data indicate that developmental and tumor angiogenesis can be induced by cellular oncogenes, leading to the enhanced activity of molecules stimulating angiogenesis. However, activated oncogenes might also facilitate angiogenesis by down-regulating endogenous inhibitors of angiogenesis. We report here that enhanced expression of the N-myc oncogene in human neuroblastoma cells down-regulates an inhibitor of endothelial cell proliferation, identified by amino acid sequencing as being identical with activin A, a developmentally regulated protein. Down-regulation appears to involve interaction of the N-Myc protein with the activin A promoter. In addition, activin A inhibits both endothelial cell proliferation in vitro and angiogenesis in vivo, and it induces hemorrhage in vivo. We suggest that the N-myc-induced down-regulation of activin A could contribute to developmental and tumor angiogenesis.

Secretion of ribonucleases by normal and immortalized cells grown in serum-free culture conditions.

The requirement of serum in cell culture is a major limitation for studies on secreted ribonucleases (RNases) because serum contains a high amount of ribonucleolytic activity. Defined culture condition is thus of interest to improve our knowledge of the RNase biology. We report here that cells from three different types and origins, Chinese hamster lung fibroblasts, bovine smooth muscle cells, and human endothelium-derived EA.hy926 cells, proliferate consistently in the presence of a basal medium supplemented with bovine serum albumin, high-density lipoproteins, basic fibroblast growth factor, insulin, and transferrin. Using a new quantitative radio-RNase inhibitor assay, two distinct ribonucleolytic assays, and a radioimmunoassay against angiogenin, it is shown that RNases became apparent in media conditioned by cell monolayers. Both the hamster lung fibroblast and the EA.hy926 cell lines secreted larger amounts of RNase inhibitor-interacting factors and RNase activity than normal smooth muscle cells. The serum-free medium represents an alternative way to grow these cells and allows investigation of biosynthesis and functions of RNases in culture. It should be useful to identify and quantitate unambiguously specific members of the RNase family secreted by normal versus tumor cells in culture.

MYCN oncogene and angiogenesis: down-regulation of endothelial growth inhibitors in human neuroblastoma cells. Purification, structural, and functional characterization.

Angiogenesis, the formation of new blood vessels, is seen during embryonic development and tumor progression, but the mechanisms have remained unclear. Recent data indicate that tumor angiogenesis can be induced by cellular oncogenes, leading to the enhanced activity of molecules stimulating angiogenesis. However, activated oncogenes might also facilitate angiogenesis by down-regulating endogenous inhibitors of angiogenesis. We report here that enhanced expression of the N-myc oncogene in human neuroblastoma cells down-regulates three inhibitors of endothelial cell proliferation. One of them was identified by amino acid sequencing as being identical with activin A, a developmentally-regulated protein. Down-regulation involves interaction of the N-myc protein with the activin A promoter. Work is ongoing to characterize the other two endothelial cell inhibitors. We suggest that the N-myc induced down-regulation of angiogenesis inhibitors could contribute to tumor angiogenesis.

Expression of receptors for human angiogenin in vascular smooth muscle cells.

Human angiogenin is a plasma protein with angiogenic and ribonucleolytic activities. Angiogenin inhibited both DNA replication and proliferation of aortic smooth muscle cells. Binding of 125I-angiogenin to bovine aortic smooth muscle cells at 4 degrees C was specific, saturable, reversible and involved two families of interactions. High-affinity binding sites with an apparent dissociation constant of 0.2 nm bound 1 x 104 molecules per cell grown at a density of 3 x 104.cm-2. Low-affinity binding sites with an apparent dissociation constant of 0.1 micrometer bound 4 x 106 molecules.cell-1. High-affinity binding sites decreased as cell density increased and were not detected at confluence. 125I-angiogenin bound specifically to cells routinely grown in serum-free conditions, indicating that the angiogenin-binding components were cell-derived. Affinity labelling of sparse bovine smooth muscle cells yielded seven major specific complexes of 45, 52, 70, 87, 98, 210 and 250-260 kDa. The same pattern was obtained with human cells. Potential modulators of angiogenesis such as protamine, heparin and the placental ribonuclease inhibitor competed for angiogenin binding to the cells. Together these data suggest that cultured bovine and human aortic smooth muscle cells express specific receptors for human angiogenin.

Internalization and processing of human angiogenin by cultured aortic smooth muscle cells.

Human angiogenin is a 14-kDa plasma protein with angiogenic and ribonucleolytic activities. Angiogenin binds specifically to aortic smooth muscle cells, activates second messenger pathways, and inhibits their proliferation. Human and bovine aortic smooth muscle cells were used to study the internalization and intracellular fate of human angiogenin at 37 degrees C. Using a specific antibody against angiogenin, we found that the internalized native protein was localized in the perinuclear region at 30 min and then dispersed throughout the cytoplasm. In conditions favoring receptor-mediated endocytosis, internalization of iodinated angiogenin showed a first peak at 5 min and then further increased for up to 24 h. The half-life of the molecule, calculated as 12 h in chase experiments, could contribute to its intracellular accumulation. In cell extracts, in addition to the 14-kDa protein, a 8.7-kDa fragment was observed at 24 h, and three fragments with molecular mass of 10.5, 8.7, and 6. 1 kDa were detected at 48 h. Our data point to a specific internalization and processing of human angiogenin by aortic smooth muscle cells.

The widespread expression of angiogenin in different human cells suggests a biological function not only related to angiogenesis.

Angiogenin is a secreted polypeptide that induces neovascularization in vivo. The expression of angiogenin by human cells in culture was investigated by using a specific radioimmunoassay and by cDNA hybridization. Angiogenin immunoreactivity was widely but differentially produced by anchorage-dependent growing cells including vascular endothelial cells from saphenous and umbilical veins, aortic smooth muscle cells, fibroblasts (from embryos, new-borns and adults), and tumour cells. Endothelial cells from saphenous veins and the endothelium-derived EA.hy926 cell line released immunoreactivity whatever the stage of the culture, including release at the lag phase, during exponential growth and at the confluent phase. However, the rate of accumulation of angiogenin varied as a function of EA.hy926 cell density. As compared to anchored cells, normal peripheral blood cells and tumour cells of myelomonocytic and megakaryocytic origin did not noticeably secrete angiogenin except at low levels. A myeloma cell line supernatant contained as much angiogenin cross-reactivity as did anchored cells, while four tumour T-cell lines expressed the cross-reactivity at different levels, i.e. from undetectable levels to a high level. A 0.9-kb angiogenin messenger RNA was detected by Northern-blot analyses in a variety of representative cells correlating with the presence of immunoreactivity in the cell-culture media. The widespread expression pattern of angiogenin suggests a physiological function that is not restricted to the neovascularization process.

Controlled release systems based on poly(lactic acid). An in vitro and in vivo study.

A new biodegradable delivery system based on poly(lactic acid) has been formulated, with potential applications in sustained antibiotic release against bone infection. The in vitro release of a new quinolone (pefloxacin) from low molecular weight poly(D,L-lactic acid) Mw = 2 x 10(3) lasted for 56 d whereas the in vivo delivery lasted 33 d. In both cases, the release rate is controlled by the drug diffusion and the polymer degradation, which seems to be the predominant factor. For the release experiments, discs were prepared from poly (D,L-lactide) Mw = 2 x 10(4) with drug loadings of 2% and 10% w/w. It was concluded that pefloxacin concentration remains higher than the Minimum Inhibitory Concentration (MIC) against the major causative bacteria of bone infection. The results indicate that the two different types of poly(lactic acid) can be used effectively in an implantable antibiotic release system.

Controlled release of salicylic acid from poly(D,L-Lactide).

Poly(D,L-Lactide) of high molecular weight (Mv) was prepared by ring-opening bulk polymerization of D,L-Lactide and characterized in terms of Mv, melting point and swelling behavior in buffer solution. Samples of the polymers with low and high Mv (2000 and 22 000 respectively) loaded with various amounts of salicylic acid (SA) were immersed in a buffer solution and the release of SA was recorded. The results obtained showed that swelling of the poly(D,L-Lactide) samples obeyed Fick's law, especially for those with high molecular weight, where biodegradation proceeds slowly. The release of SA seemed to follow a simplified relationship which is linear with time, at least for the early stages of delivery. The extent of linearity is dependent on the content of the acidic SA, which probably accelerates decomposition of the high molecular weight products.

Synthesis and properties of poly(lactic acid).

Poly(D,L lactic acid) was prepared by bulk polymerization of D,L lactide, both under atmospheric pressure and in vacuum. The obtained polymeric products were characterized in terms of molecular weight, Mw, melting point, calorimetric response and swelling behaviour. All products were amorphous. Their molecular weights were determined by viscosimetry and ranged from 2x10(3) to 9x10(4). Similarly, the melting points ranged from 90 to 210 degrees C. Swelling experiments, with specimens immersed in buffer solutions, showed that hydrolytic degradation started in a few days for the low Mw material, whereas for the higher molecular weight products it took much longer and probably followed a two-stage mechanism. This study suggests that the high molecular weight material could be an interesting carrier for the preparation of controlled release products, in cases where prolonged delivery is necessary.

Down-regulation of endothelial cell growth inhibitors by enhanced MYCN oncogene expression in human neuroblastoma cells.

Recent evidence indicates that the genetic alterations of the multistage process of malignant transformation appear to activate tumor neovascularization by altering the balance between stimulators and inhibitors of angiogenesis. In the present study, we have attempted to define the effect of enhanced MYCN oncogene expression on the profile of endothelial cell growth modulators in neuroblastoma cells. We report here that conditioned medium of human neuroblastoma cells with normal MYCN expression contains three inhibitors of endothelial cell proliferation, which appear to be novel proteins as judged by their physicochemical, immunological and biological properties. All three inhibitors are diminished or become undetectable upon experimental increase of MYCN expression. Our results suggest that enhanced MYCN expression in human neuroblastoma cells alters the angiogenic balance by down-regulating endothelial cell growth inhibitors but leaving the expression of the stimulators unaffected. These data shed light on the molecular mechanisms linking the genetic changes of malignant transformation with initiation of tumor angiogenesis. Moreover, our observations might explain the poor prognosis of human neuroblastomas following MYCN oncogene amplification through initiation of angiogenesis and subsequent tumor growth and spread.

Distribution and frequency of beta-thalassemia mutations in northwestern and central Greece.

OBJECTIVES: Beta-Thalassemia is a common autosomal recessive disorder resulting from over 200 different mutations of the beta-globin genes. The spectrum of beta-thalassemia mutations in Greece has been previously described in the population of the capital city of Athens, or in beta-thalassemia patients having transfusion therapy. The aim of the present study was to identify the distribution of the most common beta-thalassemia mutations in the population of northwestern and central Greece. METHODS: The data for this study were derived from a total of 1,130 unrelated subjects including 46 beta-thalassemia major, three beta -thalassemia intermedia and 1,081 carriers identified in our antenatal screening program. beta-Thalassemia mutations were identified by ARMS, DGGE and Reverse Dot Blot. RESULTS: The most common mutation, IVS-I-110, is followed, in order of frequency, by the mutations Cd-39, IVS-I-1, IVS-II-1, Cd-6, IVS-I-6, IVS-I-5, IVS-II-745, Cd-5 and 44 bp del. IVS-I-110 and Cd-39 frequencies are similar with those found in other Balkan countries. Significant differences in regional distribution were observed. The results showed a clear drift of the distribution of the most frequent IVS-I-110 mutation in the south-north (29.4, 40.0, 44.6 and 61.7%) and the east-west axis (31.8 and 44.6%). CONCLUSIONS: Population screening and prenatal diagnosis are significantly facilitated by these data. Furthermore, the detailed distribution tables of beta-thalassemia mutations are essential for counseling and extraction of genetic diversity estimates for population genetic studies in other inherited disorders.