engleski

Immunogold electron microscopic study on enzymes involved in the production of meiosis activating sterols within the testis

Lanosterol 14alfa-demethylase (CYP51) is a cytochrome P450 enzyme involved in the cholesterol biosynthesis. In the presence of NADPH-cytochrome P450 reductase, CYP51 converts lanosterol to FF-MAS, an intermediate of the cholesterol biosynthesis. FF-MAS could significantly affect spermatogenesis at the final stages of spermatid maturation. Our previous reports demonstrated a high expression of CYP51 mRNA in the rat testis and P450 at the ultrastructural level, using the mouse testis as an experimental model. Mature CD1 mice, housed in groups of three, were subjected to a cervical dislocation. Their testes were immediately dissected, mildly fixed in a mixture of 2% paraformaldehyde and 0.1% glutaraldehyde and processed for electron microscopy. Ultrathin sections were placed on nickel grids and incubated with rabbit polyclonal antibodies to CYP51 and P450 reductase. The appropriate secondary antibody (goat-anti rabbit) bearing 10nm gold particles was used in order to visualise the location of the antigen - primary antibody complex. The sections were examined by the Zeiss 902A electron microscope. EM figures were evaluated using a non-biased system grid (with 42 test points) according to Weibel and the number (Nv) of gold particles for the two different enzymes was determined. Within the mouse testis, a high expression of CYP51 and P450 reductase was found on the membranes of the smooth endoplasmic reticulum of Leydig cells. CYP51 and P450 reductase were co-localised on the inner and outer membranes of the acrosome of round and elongated spermatids. A number of gold particles were found in the apical part of Sertoli cells (surrounding the acrosome) and on the cisternae of the smooth endoplasmic reticulum (in the proximity of lipid droplets and residual bodies) of these cells. Our stereological data demonstrated the colocalisation of the investigated enzymes. High number of gold particles was found within round and a moderate number of these particles in elongated spermatids. In addition, small number of gold particles was found in the cytoplasm of Sertoli cells.

lanosterol-14alfa-demetilaze; P450 reductase; meiosis activating sterols; immunogold electron microscopy; testis

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