Morphofunctional characteristics of proliferation and apoptosis of germinal epithelium: pathogenesis of temporary azoospermia after local electron irradiation

BACKGROUND: At present, the violation of male reproductive function caused by electron irradiation, leading to a decrease in the proliferative activity of germ cells, as well as the development of methods for its correction are relevant. The effect of leukocyte-poor platelet-rich plasma (LP-PRP) growth factors, which have a high regenerative capacity for the restoration of spermatogenesis, remains poorly understood. Objective of the study: immunohistochemical (IHC) assessment of the proliferation of germinal epithelium after electron irradiation with a dose of 2 Gy. METHODS: Wistar rats (n=60) were divided into groups (conventional names): (I) control (n=30), which were injected with saline; (II) single local irradiation of the testes with electrons at a dose of 2 Gy (n=30). Animals were gradually withdrawn from the experiment over 11 weeks: 5 animals one week after irradiation, and then 5 animals once every 2 weeks. The testes were studied by histological and IHC methods using antibodies to Ki-67, Bcl-2, and p53. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) method was used to study DNA fragmentation in germ cells, which were stained with TdT solution (Thermo Fisher, USA) and incubated for 60 minutes. The nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (blue spectrum) (Thermo Fisher), and the luminescence intensity was controlled in a fluorescent microscope with a set of fluorescein isothiocyanate (FITC) filters (green spectrum). RESULTS: IHC analysis of testes after irradiation showed a shift in the proliferative-apoptotic balance towards apoptosis of germ cells: a decrease in the expression levels of Ki-67 (16.3%±1.1%, P<0.05) and Bcl-2 (9.1%±1.1%, P<0.05), an increase in p53-positive cells (74.8%±1.2%, P<0.05) by the end of the experiment. CONCLUSIONS: In the experimental model, local irradiation of the testes with electrons at a dose of 2 Gy causes the development of focal hypospermatogenesis, affecting up to 1/8 of the sections of the tubules (in the first week) with a subsequent increase to 1/4 (in the second month), with a tendency to recovery in the third month (temporary azoospermia). The basis of focal hypospermatogenesis is the irradiation-induced proliferative-apoptotic imbalance towards the dominance of apoptosis, which is most pronounced in the pool of spermatogonia.