Autophagy is an intracellular lysosome-mediated degradation process that involves the sequestration of cytoplasmic organelles and macromolecules within autophagosomes. The products of degradation are recycled, so they become available substrates for anabolic pathways and energy production. In addition to a pro-survival role, increasing evidence demonstrated that autophagy also acts as a pro- death mechanism under different conditions [1]. In several mammalian species, it has been demonstrated that autophagy is also involved in processes related to reproduction such as gametogenesis, and embryo and placenta development, while in sheep little is known about the role of autophagy in reproductive processes. It has been also demonstrated in several species includi ng rodents and pigs that autophagy is involved in the regulation of follicle destiny in the ovary, inducing a programmed cell death in granulosa cells (GCs) through a cooperative role with apoptosis [2]. As in ovine species it has never been investigated, the aim of the present study was to evaluate whether there is a correlation between autophagy and follicular development by analyzing the expression of the autophagosomal marker, LC3 protein, in GCs of sheep antral follicles. For this purpose, sheep ovaries were collected from local slaughterhouses and antral follicles were isolated and classified into three groups (healthy, early atretic, advanced atretic), based on their health status according to validated morphological parameters. Then, GCs were collect ed and pooled from each group, and then were divided in two parts to carry out assays for both apoptosis (TUNEL) and autophagy. For the latter, the amount of LC3-I protein converted to LC3-II, which is an index of autophagic induction, was assessed in GCs by western blotting, and the subcellular localization of the LC3 protein was evaluated by immunofluorescence and confocal microscope analysis. Our preliminary results confirmed the activation of apoptosis in GCs of atretic antral follicles. Moreover, we demonstrated for the first time a different abundance of the autophagic marker LC3 in sheep GCs of healthy vs atretic antral follicles, indicating a correlation between follicular health status and autophagy enhancement and suggesting that also in sheep, like in other species, the process of autophagy might play a role in follicular fate. [1] Baehrecke, Eric H. "Autophagy: dual roles in life and death?." Nature reviews Molecular cell biology 6.6 (2005): 505 -510. https://doi.org/10.1038/nrm1666 [2] Escobar, M. L., O. M. Echeverría, and G. H. Vázquez-Nin. "Role of autophagy in the ovary cell death in mammals." Autophagy-a double-edged sword-cell survival or death (2013): 423-441. http://dx.doi.org/10.5772/54777
Does autophagy play a role during follicular development in sheep's ovary?
Aurora Scudieri;Luisa Gioia
;Domenico Iuso;Pasqualino Loi
2023-01-01
Abstract
Autophagy is an intracellular lysosome-mediated degradation process that involves the sequestration of cytoplasmic organelles and macromolecules within autophagosomes. The products of degradation are recycled, so they become available substrates for anabolic pathways and energy production. In addition to a pro-survival role, increasing evidence demonstrated that autophagy also acts as a pro- death mechanism under different conditions [1]. In several mammalian species, it has been demonstrated that autophagy is also involved in processes related to reproduction such as gametogenesis, and embryo and placenta development, while in sheep little is known about the role of autophagy in reproductive processes. It has been also demonstrated in several species includi ng rodents and pigs that autophagy is involved in the regulation of follicle destiny in the ovary, inducing a programmed cell death in granulosa cells (GCs) through a cooperative role with apoptosis [2]. As in ovine species it has never been investigated, the aim of the present study was to evaluate whether there is a correlation between autophagy and follicular development by analyzing the expression of the autophagosomal marker, LC3 protein, in GCs of sheep antral follicles. For this purpose, sheep ovaries were collected from local slaughterhouses and antral follicles were isolated and classified into three groups (healthy, early atretic, advanced atretic), based on their health status according to validated morphological parameters. Then, GCs were collect ed and pooled from each group, and then were divided in two parts to carry out assays for both apoptosis (TUNEL) and autophagy. For the latter, the amount of LC3-I protein converted to LC3-II, which is an index of autophagic induction, was assessed in GCs by western blotting, and the subcellular localization of the LC3 protein was evaluated by immunofluorescence and confocal microscope analysis. Our preliminary results confirmed the activation of apoptosis in GCs of atretic antral follicles. Moreover, we demonstrated for the first time a different abundance of the autophagic marker LC3 in sheep GCs of healthy vs atretic antral follicles, indicating a correlation between follicular health status and autophagy enhancement and suggesting that also in sheep, like in other species, the process of autophagy might play a role in follicular fate. [1] Baehrecke, Eric H. "Autophagy: dual roles in life and death?." Nature reviews Molecular cell biology 6.6 (2005): 505 -510. https://doi.org/10.1038/nrm1666 [2] Escobar, M. L., O. M. Echeverría, and G. H. Vázquez-Nin. "Role of autophagy in the ovary cell death in mammals." Autophagy-a double-edged sword-cell survival or death (2013): 423-441. http://dx.doi.org/10.5772/54777I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
