Characterization of in vivo autophagy during avian spermatogenesis

Spermatogenesis is a complex cellular process that includes many subcellular events that are essential for the production of healthy spermatozoa. Autophagy is a physiological process that plays a significant role in the process of spermatogenesis; however, autophagy during avian spermatogenesis has not yet been reported. In the current study, we characterized in vivo autophagy throughout the process of domestic fowl spermatogenesis. Autophagy-specific markers, including microtubule-associated protein light chain 3 (LC3), sequestosome 1 (p62), and autophagy-related 7 (Atg7), were used to confirm the occurrence of autophagy in testicular germ cells. The protein expression of Atg7, LC3, and p62 in domestic fowl testes was confirmed by Western blotting. The immunohistochemical staining indicated a strong localization of LC3 and Atg7 within spermiogenic cells (intermediate and late spermatids) and primary spermatocytes. However, poorly expressed in cells (spermatogonia) that were located near the basement membrane. The immunofluorescence staining results showed the opposite tendency for LC3 and p62. LC3 was more strongly localized within the elongated spermatids, while p62 was strongly localized within the early spermatids. Moreover, the ultrastructural components of autophagy were revealed by transmission electron microscopy. Well-developed autophagosomes and multivesicular bodies were found to be prominent in primary spermatocytes (zygotene and pachytene) and spermiogenic cells. Furthermore, other vesicular structures, such as early endosomes and amphisomes, were also observed during spermatogenesis. The above findings collectively suggest that autophagy is active during spermatogenesis and that the level of autophagy increases from the basal to the luminal regions of the seminiferous tubules of domestic fowl testes. We propose that autophagic pathways may be involved in multiple functions to sustain spermatogenesis.