engleski

Cell cycle regulation by RB1 protein in the testicular development of the rat

Introduction. Developing germ and Sertoli cells are under the control of cell cycle regulators like the tumor suppressor retinoblastoma gene. Deviations from the normal regulation could cause testicular disruption. Knowing the normal developmental windows in the cell cycle regulation is crucial for better understanding the sensitive periods of testicular development. Aim. This study investigated the expression of Rb1 and its phosphorylated form in normal fetal rat testicular development. Material and Methods. 16.5 – 20.5 dpc (days post coitum) fetal rat testes were immunohistochemically stained with antibodies against Rb1 (1:100, PA5-99502, Thermo Fischer) and phosphorylated Rb1 on serine 780 (phosphoRbSer780) (1:100, ab47763, Abcam). Antigen retrieval and serum blocking were performed before overnight incubation at 4°C. After H2O2 application a secondary antibody (1:1000, ab97051, Abcam) was kept for one hour. 3, 3’-diaminobenzidine- tetrahydrochloride was used for staining, and hematoxylin for counterstaining. Positive signals were counted on a minimum of ten transversely sectioned tubules and normalized to the tubule diameter. Results. Nuclear expression was characteristic for both Rb1 and phosphoRbSer780 found in germ, Sertoli, and Leydig cells. Rb1 expression was reduced from 16.5 to 20.5 dpc (p<0, 01), with the steepest fall at 18.5 dpc (p<0, 001). PhosphoRbSer780 expression was not changed significantly through the rat pregnancy. Conclusion. The dynamical change of Rb1 expression reduction is in concordance with the quiescence period starting from 18.5 in the rat testis. No change in phosphoRbSer780 expression indicates that the quiescence period is not regulated by Rb1 phosphorylation on serine 780.

testis ; development ; retinoblastoma ; cell cycle

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