Estudo da inter-relação das vias autofágicas e apoptóticas durante a espermatogênese e regressão ovariana da tilápia do Nilo Oreochromis niloticus em condições de cultivo

Abstract

Apoptosis and autophagy are highly conserved signaling pathways between species, essential in several physiological. Despite recent advances, there are still many gaps in the knowledge of their interactions, and especially in non­mammalian vertebrates. In this sense, the aim of the present study was to investigate the interaction between autophagy and apoptosis in follicular atresia and spermatogenesis of Nile tilapia Oreochromis niloticus under cultivation conditions, and, for this, the project was developed in three stages. In the first stage, to investigate the interaction of autophagic and apoptotic pathways in follicular atresia, freshly spawned animals were kept in culture condition and ovarian samples were collected for morphological analysis, TUNEL technique and immunohistochemistry. The follicular atresia in the ovaries of O. niloticus was analyzed in 3 stages of regression: early (AFE), advanced (AFA) and late (AFL) atretic follicles. Early and advanced atretic follicles presented autophagy in follicular cells by the presence of large lysosomes and autophagosomes with degenerating mitochondria, organelles debris and electron­dense granules. Moreover, it were observed significant increases of immunoreactivity for Bcl­2, Cathepsin­D, Beclin­1 and LC3. In late atretic follicles, the follicular cells presented a markedly electron­lucid cytoplasm and increased apoptosis, with immunoreactivity for Bax and many TUNEL positive cells. In addition, the colocalization between LC3 and Caspase­3 in AFL and LAMP­1 and Cathepsin­D in AFE and AFA showed points of interaction between autophagy and apoptosis during follicular atresia of Nile tilapia. In the second and third stage of this work, we evaluated the performance of the autophagic and apoptotic pathways during spermatogenesis in animals submitted to system of refeeding and total food restriction for 7, 14, 21 and 28 days. Analysis of the effects of starvation on biochemical, hormonal and morphological parameters showed that, glycemic and lipid levels were significantly reduced, followed by plasma reduction of testosterone (T) and 11­ ketotestosterone (11­KT). In addition, reduced proliferation of spermatogonia and increased apoptosis of spermatocytes, spermatids and spermatozoa were observed.In the refeeding groups, the sex steroids and the proportion of germ cells had no significant alterations compared to the control group, except for reduction of spermatozoa. The performance of the autophagic pathway during spermatogenesis was evidenced by immunostaining of LC3 and Beclin­1 and by the presence of autophagic structures, such as autophagosomes, autolysomes and multilamellar bodies in secondary spermatocytes, spermatids and Sertoli and Leydig cells. However, after 21 days of starvation apoptosis increased mainly in spermatocytes and