Immunohistochemical and stereological study on spermatogonial cell populations of rats treated with etoposide and carnitine in the prepuberty

Abstract

Chemotherapeutic agents, although efficient in the treatment of various types of cancers, lead to undesirable side effects. One of these effects is infertility. Etoposide, a potent chemotherapeutic drug used in the treatment of child and adolescent cancer, affects both cancer and healthy cells and causes severe damages to spermatogenesis, especially when administered during prepubertal phase. In a previous study, we observed that carnitine, an amino acid present in the epididymal fluid, reduces the testicular damages caused by etoposide when administerd during the prepuberty. The aim of this study is to investigate whether the administration of carnitine (250mg/Kg, intraperitonially) to prepubertal rats (25 to 32dpp), previously to etoposide (40mg/Kg/8 days, ip; total dose fragmented in daily equal doses), protects the stem/progenitor spermatogonia, reducing the damage to the future reproductive capacity of the animals (at 64 and 127dpp). Undifferentiated and differentiated spermatogonia will be analyzed by in situ immunostaining using specific markers (anti-GFR±1, anti-Plzf e anti-UTF1) and subsequently quantified. To evaluate the effects of etoposide and carnitine on the proliferation of undifferentiated spermatogonia, these cells will be isolated from control and treated animals, maintained in vitro and quantified. The final impact of the treatment with etoposide and carnitine will be evaluated through the analysis of: 1) concentration of step 19 spermatids and daily sperm production; 2) sperm concentration in the different epididymis regions and spermatic transit in the epididymis. The integrity of the sperm DNA will be analyzed using the Alkaline Comet Assay. The parameter will be analyzed in the pubertal and adult animals of both "sham" control (treated with 0.9% saline solution) and etoposide-treated groups, which received these treatments during prepuberty. The global data analysis can provide novel information about: 1) the effects of the treatment with etoposide and carnitine on spermatogenesis and male fertility, focusing its impact in the proliferation of stem/progenitor spermatogonia, concerning the critical balance between renewing and differentiation; 2) the potential protective action of carnitine to the germ cell lineage of rats treated during the prepuberty and its repercussions on the reproductive and spermatic parameters, including the study of sperm genotoxicity caused by etoposide; these parameters will be scrutinized in later stages of development and sexual maturity. (AU)