epidermal-growth-factor and Azoospermia

To investigate the effects of collagen and growth factors on in vitro proliferation of human spermatogonial stem cells obtained from patients with non-obstructive azoospermia.. The experimental cross-sectional study was conducted from February 2013 to April 2015 after obtaining approval from the ethics committee of Ahvaz Jundishapur University of Medical Sciences, Iran. Testicular sperm extractions of non-obstructive azoospermic patients were obtained from the Clinical Urology and Embryology, In Vitro Fertilization Department of Imam Khomeini Hospital. Spermatogonial stem cells and Sertoli cells, obtained from human testis biopsies by a two-step enzymatic digestion method, were purified using fluorescence- activated cell-sorting and daturastramonium-lectin, and were cultured separately. To investigate a more direct influential factor on colony formation, one control and two experimental groups were formed. Group 1 acted as the control in which spermatogonial stem cells were co-cultured with Sertoli cells alone. In group 2 they were co-cultured with Sertoli cells and growth factors such as leukaemia inhibitory factor, epidermal growth factor and glial cell-derived neurotrophic factor, and in group 3 with Sertoli cells along with growth factors in the presence of collagen-coated dishes. Number and diameter of the colonies were evaluated after 7 weeks.. Specimens obtained related to 21 patients. Number and diameter of the colonies in group 3 (18±2.6 and 276.6±45.5) were significantly more than both groups 1 (3.5±1 and D1:81.6±12) and group 2(11±2.2 and 165.2±32.5) (p<0.05 each). Also, the number and diameter of colony in group 2 were significantly better than the control group (p<0.05).Expression profile of the VASA, promyelocytic leukaemia zinc-finger (PLZF), Octamer-binding transcription factor 4 (OCT4) and integrin a6 (INTGa6) were detected in all groups. Based on cytochemical findings, OCT4 was expressed in the colonies of all three groups.. According to positive effects of collagen and growth factors on the colonisation of spermatogonial stem cells, it seems that using the cells may lead to better colonisation of this type of stem cells.

Topics: Adult Germline Stem Cells; Azoospermia; Cell Culture Techniques; Cell Proliferation; Coculture Techniques; Collagen; Cross-Sectional Studies; DEAD-box RNA Helicases; Epidermal Growth Factor; Flow Cytometry; Glial Cell Line-Derived Neurotrophic Factor; Humans; Integrin alpha6; Kruppel-Like Transcription Factors; Leukemia Inhibitory Factor; Male; Octamer Transcription Factor-3; Promyelocytic Leukemia Zinc Finger Protein; Sertoli Cells

Gap junctions (GJs) allow for direct communication between adjacent cells. They are composed of connexons consisting of transmembrane proteins, connexins (Cxs). The objectives of this study were to determine if GJ proteins GJA1 (Cx43), GJB1 (Cx32) and GJB2 (Cx26) are present in the epididymis of men with a normal epididymis, to assess whether or not Cx expression and localization are altered in azoospermic patients, and to determine if epidermal growth factor (EGF) regulates GJA1 expression.. Epididymides were obtained from men with localized testis cancer with active spermatogenesis and histologically normal epididymal tubule (group 1), men with non-obstructive azoospermia secondary to Sertoli-cell only syndrome (group 2) and from azoospermic men with normal spermatogenesis and epididymal obstruction (group 3). Epididymides were subdivided into three segments: caput, corpus and cauda. Quantitative real-time RT-PCR was performed to assess GJA1, GJB1, GJB2 and EGF receptor (EGFR) mRNA levels in epididymides from patients from each group (all n=3, except n=1 for caput blockage). A human caput epididymal cell line was then used to determine the role of EGFR signaling on the regulation of human epididymal GJA1.. Real-time RT-PCR analysis revealed that GJA1, GJB1, GJB2 and EGFR were expressed along the human epididymis. In the cauda epididymidis of group 2 and 3 men, we observed a significant decrease in GJA1 (P=0.0456 and P=0.0465, respectively) and GJB1 (P=0.0450 and P=0.0497, respectively) mRNA levels when compared with group 1 men. We also observed a decrease in EGFR mRNA levels (P=0.0358) in the cauda epididymidis of group 3 men when compared with group 1. Immunocytochemistry revealed that in the epididymis, GJA1 and EGFR were localized between basal and principal cells and between adjacent principal cells. In group 2 and 3 patients, however, we noted a dramatic increase in cytosolic immunostaining for both GJA1 and EGFR in both principal and basal cells. Using a human caput epididymal cell line derived from fertile men, we demonstrated that changes in GJA1 phosphorylation could be regulated by EGF (P=0.015) and the extracellular regulated kinase 1/2 signaling pathway (P=0.03). Furthermore, while the phosphoinositide-3-kinase (PI3K)/AKT signaling pathway did not alter GJA1 phosphorylation, treatment with PI3K/AKT inhibitor LY294002 significantly (P=0.024) inhibited the EGF-stimulated increase in GJA1 total protein levels at 24 h. Immunolocalization indicated that loss of PI3K/AKT signaling was associated with increased cytosolic localization of Cx43 in this cell line.. Together, these data suggest that in azoospermic men decreased expression of EGFR may be responsible for decreasing GJA1 levels and increasing its cytosolic localization via the PI3K/AKT signaling pathway.

Topics: Adult; Apoptosis; Azoospermia; Connexin 26; Connexin 43; Connexins; Epidermal Growth Factor; Epididymis; ErbB Receptors; Gap Junction beta-1 Protein; Gap Junctions; Gene Expression Regulation; Humans; Male; Models, Biological; RNA, Messenger; Sertoli Cells; Spermatogenesis; Testis

This study aims to proliferate spermatogonial stem cells (SSCs) and compare the in-vitro effects of laminin and growth factors on the proliferation of adult human SSC.. Isolated testicular cells were cultured in DMEM supplemented with 5 % fetal calf serum (FCS). During the culture, enriched spermatogonial cells were treated with a combination of glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and mouse leukemia inhibitory factor (LIF) in the presence or absence of human placental laminin-coated dishes. Cluster assay was performed during culture. Presence of spermatogonia was determined by an ultrastructural study of the cell clusters, reverse transcription polymerase chain reaction (RT-PCR) for spermatogonial markers and xenotransplantation to the testes of busulfan-treated recipient mice. Statistical significance between mean values was determined using statistical ANOVA tests.. The findings indicated that the addition of GDNF, bFGF, EGF and LIF on laminin-coated dishes significantly increased in-vitro spermatogonial cell cluster formation in comparison with the control group (p ≤ 0.001). The expression of spermatogonial markers was maintained throughout the culture period. Furthermore, a transplantation experiment showed the presence of SSC among the cultured cells. In addition, a transmission electron microscopy (TEM) study suggested the presence of spermatogonial cells of typical morphology among the cluster cells.. It can be concluded that human SSCs obtained from non-obstructive azoospermic (NOA) patients had the ability to self-renew in the culture system. This system can be used for the propagation of a small number of these cells from small biopsies.

Topics: Analysis of Variance; Animals; Azoospermia; Biomarkers; Biopsy; Busulfan; Cell Count; Cell Culture Techniques; Cell Proliferation; Cells, Cultured; Culture Media; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glial Cell Line-Derived Neurotrophic Factor; Humans; Infertility, Male; Laminin; Leukemia Inhibitory Factor; Male; Mice; Microscopy, Electron, Transmission; Reverse Transcriptase Polymerase Chain Reaction; Serum; Spermatogonia; Stem Cell Transplantation; Stem Cells; Testis; Transplantation, Heterologous

To investigate the levels and changes of sexual hormone in serum and seminal plasma, and the epidermal growth factor (EGF), alpha-glucosidase, and fructose in the seminal plasma of non-obstructive azoospermic patients, and to assess the significance of the analysis of their chromosomes.. Thirty-six non-obstructive azoospermic males and 26 normal males were chosen. The results in reproductive endocrine and genetics obtained by radioimmunoassay, colorimetry, and chromosomes G and C banding assay were compared and analyzed to explore into the causes of infertility.. The levels of FSH, LH, PRL and E2 in the serum of the non-obstructive azoospermic patients were higher than those of the control group, but the T level had no significant difference between the two groups. There were no obvious differences in the sexual hormones except in FSH of the seminal plasma. There were 2 cases of abnormal sex chromosomes, 2 cases of big Y chromosomes and 7 cases of small Y chromosomes in the non-obstructive azoospermic patients. No correlation was found between EGF and fructose in the seminal plasma.. The level of sexual hormone in serum, alpha-glucosidase in seminal plasma and abnormal chromosomes might be closely related to non-obstructive azoospermia.

Topics: Adult; alpha-Glucosidases; Azoospermia; Case-Control Studies; Chromosome Banding; Chromosomes, Human, Y; Epidermal Growth Factor; Gonadal Steroid Hormones; Humans; Male; Semen