br Results br Discussion Autophagy is

Results
Discussion Autophagy is considered as an important process in physiological and pathological ovarian function. It is related mainly to follicular atresia and CL regression (for review see [20]). However, an involvement in luteal cell survival is also proposed [21], [22]. The present study provides the first evidence of autophagy in the porcine CL throughout the estrous cycle. We have found double-membrane autophagosomes and autophagic proteins at all stages of CL development. Furthermore, the presence of autolysosomes and increased expression of autophagy-related proteins during the late luteal phase suggest that autophagy is involved in CL regression in pigs. At this time, autophagy has been detected in luteal tissue of humans [21], primates [23], rodents [14], and domestic ruminants [16], [24]. It occurs during physiological and pathological conditions, including spontaneous luteolysis [16], pregnancy [25], [26], and the irregular CL regression seen in climacteric women [21]. Since very little research has been performed on autophagy during mammalian CL development, we have now shown its presence in the porcine CL throughout the estrous cycle. We have examined CLs from the early, middle, and late luteal phases reflecting periods of luteinization, high steroidogenic activity, and luteolysis, respectively. Ultrastructural analysis of luteal Phos-tag Biotin using TEM detected the characteristic double-membrane autophagosomes at all examined developmental stages. The expression of Beclin 1, LC3-II, and Lamp 1 proteins also confirmed the presence of autophagy. Importantly, it remains unclear whether autophagy promotes cell survival and/or cell death in luteal cells. Most studies have examined autophagy only in regressing CL. However, studies on the human CL demonstrated the expression of Beclin 1 in the early and mature CL [21], which is consistent with our present findings. The current results showing the presence of autophagosomes and their marker LC3-II and expression of mRNAs and proteins for Beclin 1 and Lamp1 in the early porcine CL indicate the contribution of autophagy to luteinization. Similarly, Gaytán et al [21] have shown high Beclin 1 level in the wall of the human luteinized postovulatory follicle. Furthermore, less luteinization due to reduced expression of autophagic proteins has been found in the mouse ovary after insulin treatment [27]. In addition, dexamethasone-induced autophagy enhanced CL formation in the spiny mouse and was proposed to promote cell survival rather than cell death [22]. These authors also found autophagy to be positively correlated with lipid droplet accumulation in luteal cells, thereby underpinning steroidogenesis. In particular, autophagy-related proteins have been confirmed to have an important role in lipid droplet formation and function within nonreproductive tissues [28], [29]. In the present study, the CL at the middle of the luteal phase represents the highest steroidogenic activity, as indicated by the greatest production of P4. Therefore, at this developmental stage of the porcine CL, autophagy may influence luteal lipid metabolism. However, further work is needed to understand the interaction between lipid droplets and autophagosome components. Taken together, this evidence points to a role of autophagy in the regulation of cell survival, which can prolong CL lifespan and function during the early and middle luteal phases in pigs. Recent studies highlighted a link between autophagy and luteal regression. In rats, LC3-II–positive autophagosomes were identified during the late luteal phase and correlated with luteal cell apoptosis [14]. Furthermore, treatment of rat luteal cells with PGF2α induced autophagosome accumulation, suggesting that autophagy may be involved in luteal cell death [15]. Similarly, regressing bovine CL showed simultaneous upregulation of autophagic markers, lysosomal enzymes, and apoptotic proteins [16]. In pigs, functional luteolysis appears after day 12 of the estrous cycle that correlates with acquisition of luteolytic sensitivity in the CL [30]. The luteolytic cascade involves disruption of P4 synthesis followed by degeneration of luteal cells via apoptosis [31]. Herein, in CLs obtained from the late luteal phase, there was a decreased concentration of intraluteal P4 and accumulation of lipids indicating the cessation of steroidogenesis. In that regressing CL, we found elevated expression of autophagy-related proteins (Beclin 1 and Lamp 1) as compared with CLs from the early and middle luteal phases. The level of LC3-II protein was not increased, but the occurrence of autolysosomes exclusively at this stage of luteal development firmly suggests advanced autophagy. Its occurrence in luteal tissue undergoing spontaneous luteolysis suggests a role for autophagy in luteal cell death during regression in pigs. This hypothesis is consistent with our previous research concerning the participation of autophagy in flutamide-induced regression of the porcine CL of pregnancy [18].