Nuclear lamin expression in normal testis and testicular germ cell tumours of adolescents and adults.

Nuclear A- and B-type lamins are differentially expressed in tissues, depending on the degree of cellular differentiation and proliferative status. By studying lamin expression in testis parenchyma and testicular germ cell tumours, further insight may be gained into the degree of cellular differentiation in normal testis and into the whole spectrum of differentiation lineages found in testicular germ cell tumours. Frozen tissue sections of normal testis and the different types of testicular germ cell tumours were immunostained with monoclonal antibodies to distinct lamin subtypes. Lamin reactivity was evaluated in relation to the lineage and degree of cellular differentiation and the reactivity patterns were compared with each other and with those in normal testis. In normal testis, both A- and B-type lamins were expressed in Sertoli, Leydig, and peritubular cells, while in spermatogonia only B-type lamins were found and spermatocytes showed weak reactivity with the A-type lamin antibodies. Carcinoma in situ was most often positive for both of the B-type lamins and negative for the A-type lamins (lamins A and C). In testicular germ cell tumours, B-type lamins were always expressed, while A-type lamins were differentially expressed. Differentiated non-seminomas were positive for both of the A-type lamins, whereas embryonal carcinomas were positive for lamin C and negative for lamin A. Seminomas were negative for both of the A-type lamins, with the exception of seminomas containing a Ras mutation. Spermatogonia and seminoma cells, which follow a differentiation pathway along the spermatogenic lineage and show characteristics of germ cells, do not express A-type lamins. Non-seminomas, showing embryonal or extraembryonal differentiation, express A-type lamins to varying degrees, distinguishing embryonal carcinoma cells from other non-seminomatous components. This may aid in the evaluation of the percentage of embryonal carcinoma in non-seminomatous testicular germ cell tumours as a prognostic parameter.

Organization of non-muscle myosin during early murine embryonic differentiation.

A monoclonal antibody (3D10) recognizing myosin heavy chain was isolated following immunization with a synthetic peptide sequence of eight amino acids. The antibody reacted with purified rabbit skeletal myosin and light mero-myosin in enzyme-linked immunosorbent assays and Western immunoblotting. A band of approximately 200 kDa was detected in cell extracts of an embryonal carcinoma (EC) cell line (P19EC) and one of its cloned differentiated derivatives, suggesting reactivity against non-muscle myosin. By indirect immunofluorescence, typical myosin banding patterns were observed in cryostat sections of human skeletal and cardiac muscle tissue. In undifferentiated P19EC cells, speckled immunofluorescent staining was observed in the cytoplasm that became organized in cortical rings where the cells made direct contact with each other. These rings consisted of circular bundles of F-actin decorated by myosin. Undifferentiated embryonic stem (ES) cells derived directly from mouse embryos shared the same features, although the pattern was less pronounced. Human testicular primary germ cell tumours showed cortical staining in the embryonal carcinoma component reminiscent of the staining of EC cells in vitro while cytoplasmic staining was observed in tumour cells with a differentiated morphology. In preimplantation embryos, the immunofluorescent staining was observed at cell apices of blastomeres of morula stage embryos. In blastocysts, staining of inner cell mass cells was not detectable. By contrast, various differentiated derivatives of P19EC contained extensive F-actin microfilament bundles throughout the cytoplasm decorated with myosin. Thick stress fibers in filopodious extensions of cells were particularly highly decorated by myosin. Over the nucleus, linear arrays of myosin containing speckled patterns of immunofluorescence were observed that were not associated with F-actin. The same pattern of staining could be observed in trophectoderm cells of the blastocyst. We conclude that embryonic non-muscle myosin is organized in specific patterns depending on the state of differentiation. As the myosin is primarily associated with F-actin we suspect that it forms part of a contractile apparatus that may have significance during embryonic development.