The physiological importance of CD151 tetraspanin is known from somatic cells and its outside-in signalling through integrins was described. In male germ cells, two tetraspanins, CD9 and CD81, are involved in sperm-egg membrane fusion, and similarly to integrins, they occupy characteristic regions. We report here on a newly discovered presence of CD151 in sperm, and present its expression and distribution during spermatogenesis and sperm transition during the acrosome reaction. We traced CD151 gene and protein expression in testicular cell subpopulations, with strong enrichment in spermatogonia and spermatids. The testicular and epididymal localization pattern is designated to the sperm head primary fusion site called the equatorial segment and when compared to the acrosome vesicle status, CD151 was located into the inner acrosomal membrane overlying the nucleus. Moreover, we show CD151 interaction with α6 integrin subunit, which forms a dimer with β4 as a part of cis-protein interactions within sperm prior to gamete fusion. We used mammalian species with distinct sperm morphology and sperm maturation such as mouse and bull and compared the results with human. In conclusion, the delivered findings characterise CD151 as a novel sperm tetraspanin network member and provide knowledge on its physiology in male germ cells.

• MeSH
• antigeny CD151 chemie   genetika   metabolismus   MeSH
• exprese genu * MeSH
• fluorescenční protilátková technika MeSH
• integrin alfa6 chemie   metabolismus   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• myši MeSH
• spermie metabolismus   MeSH
• testis metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zárodečné buňky metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples.

• MeSH
• antigeny CD44 metabolismus   MeSH
• antigeny nádorové metabolismus   MeSH
• CD antigeny metabolismus   MeSH
• glykoproteiny metabolismus   MeSH
• integrin alfa6 metabolismus   MeSH
• lidé MeSH
• molekuly buněčné adheze metabolismus   MeSH
• nádorové biomarkery metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorové kmenové buňky metabolismus   patologie   MeSH
• nádory prostaty metabolismus   patologie   MeSH
• nádory tračníku metabolismus   patologie   MeSH
• peptidy metabolismus   MeSH
• proliferace buněk * MeSH
• průtoková cytometrie metody   MeSH
• techniky in vitro MeSH
• testy nádorových kmenových buněk metody   MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

PURPOSE: The purpose of this study was to investigate the expression of cytokeratin (CK) 8 in the corneoconjunctival epithelium. METHODS: In 17 cadaveric corneoscleral discs and 3 other discs, the presence of CK8 alone or CK8, together with CK3, CK15, vimentin, and integrin α6, was investigated by using indirect immunohistochemistry on radial cryosections. Four corneoscleral discs stored in organ culture were used for the preparation of tangential sections of the limbus and for the isolation of limbal epithelial cells and their subsequent cultivation. CK8 expression was examined by RT-PCR in the corneal, limbal, and conjunctival epithelium. RESULTS: Sixty percent of the cadaveric corneoscleral samples and all samples stored in organ culture revealed positivity for CK8 in the basal epithelial layer of the limbus. Positive basal cells formed a single line or separated clusters. The signal for CK8 became weaker toward the surface of the limbal epithelium. The colocalization of CK8 with vimentin and CK15 in the limbus was also found. CK3 showed only occasional positivity in some of the surface limbal cells. The expression of integrin α6 in the basal membrane was absent or decreased under the CK8-positive clusters. Cell cultures revealed strong positivity for CK8 in approximately 80% of the cultured cells, and CK8 expression in the cornea, limbus, and conjunctiva was determined by RT-PCR. CONCLUSIONS: The study demonstrates the strong expression of CK8 in limbal epithelial basal cells, which is maintained during the differentiation and migration of the limbal cells toward the central corneal epithelium.

• MeSH
• buněčné kultury MeSH
• dospělí MeSH
• epitelové buňky metabolismus   MeSH
• exprese genu MeSH
• fluorescenční mikroskopie MeSH
• fluorescenční protilátková technika nepřímá MeSH
• integrin alfa6 metabolismus   MeSH
• keratin-15 metabolismus   MeSH
• keratin-3 metabolismus   MeSH
• keratin-8 genetika   metabolismus   MeSH
• konjunktiva cytologie   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• limbus corneae cytologie   metabolismus   MeSH
• messenger RNA genetika   MeSH
• mladiství MeSH
• orgánové kultury - kultivační techniky MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• rohovkový epitel cytologie   metabolismus   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• vimentin metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH