Dissection of the autophagic route in oocytes from atretic follicles

Autophagy is a conserved process that functions as a cytoprotective mechanism; it may function as a cell death process called programmed cell death type II. There is considerable evidence for the presence of autophagic cell death during oocyte elimination in prepubertal rats. However, the mechanisms involved in this process have not been deciphered.


INTRODUCTION
Autophagy is a crucial process for eliminating and recycling intracellular contents. It is an evolutionarily conserved method that involves degradative lysosomal enzymes (Ryter et al., 2013;Zhang & Wang, 2018). In addition to the cytoprotective effects of the degradation of damaged organelles and unused proteins, it is now recognized that elevated and sustained autophagy levels can induce cell death (reviewed in Galluzzi et al., 2012). Several cell death processes have been deciphered in multicellular organisms, of which apoptosis and autophagy are the best characterized (Matsuda et al., 2012;Nottola et al., 2006;Shen et al., 2014). Autophagic cell death is morphologically identified by the large-scale production of autophagic vesicles (Clarke & Puyal, 2012), involving the participation of several members of the ATG family and their mammalian orthologs (Nakatogawa et al., 2009;Yang & Klionsky, 2009). Cell death plays a key role during sexual maturation in the gonads of female mammals through a process called follicular atresia. During atresia, the germinal cells (oocytes) that are not selected for ovulation are degraded. Oocytes in prepubertal Wistar rats are eliminated in multiple ways, including cell death, in which autophagy is implicated (Escobar et al., 2008;Escobar et al., 2010;Escobar et al., 2012;Escobar et al., 2013;Escobar et al., 2019;Ortiz et al., 2006).
The serine/threonine kinase mammalian target of rapamycin (mTOR) is associated with autophagy regulation (Reddy et al., 2020). mTOR forms two complexes: mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2). The mTORC1 pathway controls several processes, including protein and lipid synthesis and autophagy (Wullschleger et al., 2006). In a nutrient-rich environment, mTORC1 inhibits autophagy by phosphorylating the complex furthest upstream of autophagy, UNC-51-like kinase 1 (ULK1). ULK1 dephosphorylation is initiated under starvation conditions to induce autophagy (reviewed in Chen et al., 2021;Davis et al., 2017). During the autophagic process, autophagosome formation involves the participation of autophagy-related proteins (ATG proteins), many of which have mammalian orthologs (Nakatogawa et al., 2009;Yang & Klionsky, 2009). Increased levels of ATG proteins are associated with elevated autophagic activation (Liang et al., 1999). mTORC1 negatively regulates autophagy by the canonical class I phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB or AKT)/mTOR signaling pathway (Li et al., 2012). Activated AKT phosphorylates and thus inhibits tuberous sclerosis proteins 1 and 2 (TSC1/2) (Saucedo et al., 2003;Stocker et al., 2003), resulting in mTORC1 activation (Sancak et al., 2007;Thedieck et al., 2007;Vander Haar et al., 2007;Wang et al., 2007). Cellular stressors, such as low cellular energy levels and hypoxia, activate TSC1/2 to inhibit mTORC1 (Gwinn et al., 2008;Inoki et al., 2006), resulting in decreased mTORC1 activity. Although mTOR has been demonstrated to play various roles in the mammalian ovary, a detailed analysis of the relationship of mTOR and autophagy to oocyte cell death has not been carried out. Although mTOR has been demonstrated to play various roles in the mammalian ovary, a detailed analysis of the relationship of mTOR and autophagy to oocyte cell death has not been carried out. We have previously reported the presence of cell death type autophagic to eliminate the oocyte in atretic follicles from prepubertal rats' ovaries. Although our evidence has shown that autophagy is implied in follicular atresia in the ovary from the beginning of post-natal life (Ortiz et al., 2006), an important frequency of autophagic oocytes has been identified in 19-and 28-day old rats (Escobar et al., 2008;Escobar et al., 2010).
In this work, we analyzed the distribution and behavior of proteins associated with the autophagy process, both upstream and downstream of mTOR.

Histological procedure
Ovaries from five rats of each age were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at pH 7.2 for 24 h at room temperature, embedded in paraffin, and sectioned. Five-micrometer-thick sections from each ovary were deposited onto glass microscope slides covered with poly-L-lysine (Sigma, St. Louis, MO, USA). Deparaffinized tissue sections were incubated for 10 min in Harris hematoxylin, rinsed with water for 10 min, dipped in acid alcohol for 20 s, submerged in an ammonia solution (1% NH 3 OH), placed in 50% and then 60% ethanol for 1 min each, immersed in alcoholic eosin for 5 min, dipped twice in 95% ethanol for 2 min each and then in 100% ethanol for 1 min, and submerged in xylol for 3 min. Finally, the slides were covered with Permount. The slides were observed under a Nikon Eclipse E600 microscope (Nikon Corporation, Japan). Images were recorded with a Nikon DXM1200F digital camera (Nikon Corporation). All animals were handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Ethical Committee under the protocol number PI_2019_02_009.

Immunolocalizations
Antigen unmasking of deparaffinized tissue sections was performed by microwaving the tissue sections in 0.1 M citrate buffer at pH 6 (BioGenex, Fremont, CA, USA) in a Panasonic microwave oven for 3 min at 1300 W and then for 6 min at 780 W. After cooling, the sections were washed with PBS and incubated with the primary antibodies diluted in PBS for 18 h at 4 • C (Table 1). After washing, the slides were incubated for 2 h in the dark at room temperature with a secondary antibody coupled to a fluorochrome (Table 1). Next, the preparations were washed and counterstained with DAPI (Sigma) to evaluate the DNA distribution. The slides were covered with a mounting medium for fluorescence microscopy (Vectashield Mounting Medium; Vector Labs, Burlingame, CA, USA). The slides were observed under fluorescence using a Nikon Eclipse E600 microscope. The specificity of the antibody was determined by Western blotting. At least five series of ovaries from five rats of each age were used in each immunolocalization. All the oocytes from each series were recorded to conduct the quantitative analysis.

Simultaneous immunolocalizations
Lamp1 and p-mTOR (S2448) Antigens in deparaffinized tissue sections were unmasked by microwaving the tissue sections in 0.1 M citrate buffer at pH 6 (BioGenex) in a Panasonic microwave oven for 3 min at 1300 W and then for 6 min at 780 W. After cooling, the sections were washed with PBS and incubated with the primary antibody rabbit anti-Lamp1 for 18 h at 4 • C. After washing, the slides were incubated with anti-immunoglobulin biotinylated Super Sensitive MultiLink (BioGenex) for 20 min. Subsequently, the slides were washed again and incubated for 1 h in the dark at room temperature with the secondary antibody streptavidin coupled to Texas Red (Life Science Products Inc., Chestertown, MD, USA). At least five series of ovaries from five rats of each age were used in each immunolocalization. All the oocytes from each series were recorded to make the quantitative analysis.
LC3A and p-mTOR (S2448) Antigens in deparaffinized tissue sections were unmasked by microwaving the tissue sections in 0.1 M citrate buffer at pH 6 (BioGenex) in a Panasonic microwave oven for 3 min at 1300 W and then for 6 min at 780 W. After cooling, the sections were washed with PBS and incubated with a mixture of the primary antibodies anti-LC3 A and phosphorylated-mTOR (p-mTOR) (S21448) for 18 h at 4 • C. After another washing step, the slides were incubated with the secondary antibodies for 1 h in the dark at room temperature. Next, the preparations were washed and counterstained with DAPI (Sigma) to evaluate the DNA distribution. The slides were covered with a mounting medium for fluorescence microscopy (Vectashield Mounting Medium; Vector Labs). The slides were observed under fluorescence using a Nikon Eclipse E600. At least five series of ovaries from five rats of each age were used in each immunolocalization. All the oocytes from each series were recorded to conduct the quantitative analysis.
Beclin 1 and Bak, or Bax, or active caspase-3 Seriated sections of ovaries from five rats of each age (19 and 28 days old) were used. Antigens in deparaffinized tissue sections were unmasked by microwaving the tissue sections in 0.1 M citrate buffer at pH 6 (Bio-Genex) in a Panasonic microwave oven for 3 min at 1300 W and then for 6 min at 780 W. After cooling, the sections were washed with PBS and incubated with a mixture of the primary antibodies anti-Beclin 1 and Bak, Bax, or active caspase-3 for 18 h at 4 • C. After another washing step, the slides were incubated with the secondary antibodies for 1 h in the dark at room temperature. The preparations were washed and counterstained with DAPI (Sigma) to evaluate the DNA distribution. Finally, the slides were covered with Vectashield Mounting Medium (Vector Labs) for fluorescence microscopy. The sections were observed under fluorescence using a Nikon Eclipse E600. All the oocytes from the five seriated sections were recorded to make the quantitative analysis.

EPON resin embedding
The ovaries from five rats of each age were sectioned into approximately 1 mm 3 slices and fixed by immersion in 2.5% glutaraldehyde-4% formaldehyde in PBS at pH 7.2 for 90 min at room temperature. The tissues were then dehydrated using a graded ethanol series and embedded in EPON (Embed-812; Electron Microscopy Science, Hatfield, PA, USA). Semi-thin sections were stained with toluidine blue. Selected areas were thinsectioned, and seriated sections were mounted on single-slot copper grids. Conventional staining was performed with uranyl acetate and lead citrate. Sections were examined under a JEOL 1010 electron microscope operated at 80 kV. Digital images were taken with a CCD-300RT MT 1 Hamamatsu camera (Hamamatsu Photonics K.K., Japan).

Western blotting
Eight ovaries from 19-and 28-day-old rats were used to oocyte isolation, three repetitions were made. The oocytes were placed in RIPA lysis buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 0.1% SDS, 1 mM PMSF, 0.5% sodium deoxycholate, and 1% Nonidet P-40) supplemented with a complete protease inhibitor cocktail (Roche, Mannheim, Germany) for 15 min. Fifty micrograms of total protein were loaded onto a 12% SDS-PAGE gel. The proteins were transferred to polyvinylidene fluoride membranes and incubated for 1 h at room temperature in blocking buffer. Subsequently, the membranes were incubated with antibodies (Table 1). Next, the proteins were tagged by incubation with peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) at a 1:10,000 dilution in blocking buffer for 1 h at room temperature (see Table 1). Horseradish peroxidase was used as the substrate (Immobilon Western; Millipore Corporation, Billerica, MA, USA). Specific labeling was detected by chemiluminescence. The film (Hyperfilm; Amersham Biosciences, Piscataway, NJ, USA) was exposed to the membranes to detect chemiluminescence.

Rapamycin treatment
Isolated oocytes from eight ovaries from 19-and 28-dayold rats were incubated with 5 nM (Feldman et al., 2009) for 24 h. Afterwards, the oocytes were lysed as described above to extract total proteins to develop a Western Blot technique. Then, 50 μm of total protein were loaded onto a 12% SDS-PAGE gel. Proteins transferred into membranes were used to identify mTORS2448, LC3-I, and LC3-II.

Statistical analysis
At least 120 oocytes from each age were classified into two groups: normal and altered. Immunodetected proteins were quantified using the Analyze tool of the ImageJ software. The measured area of each cell was limited to compare the relative fluorescence intensities in normal and altered oocytes. At least five seriated slices from ovaries from five rats were recorded with a digital camera to quantify the fluorescence intensity; all the oocytes in each slide were photographed. ANOVA followed by Tukey's multiple comparison test was used to compare the differences between the two groups. Significance was set at p < 0.05.

Follicular atresia in ovaries from 19-and 28-day-old rats
The morphology of the rats' ovaries was characterized using a histological technique. Our observations revealed the presence of follicles at different stages of development in the ovaries of both 19and 28-day-old rats (Figure 1a,b). Normal follicles have a regular shape, and the cellular junctions of the granulosa cells are conserved (Figure 1ai,bi). The atretic follicles were easily identified by their altered shape and the detachment of granulosa cells between them; additionally, the morphology acquired by the oocytes revealed changes corresponding to different cell death processes (Figure 1aii,bii). Specifically, compacted and/or fragmented oocytes suggest apoptosis, whereas oocytes with a highly vesiculated cytoplasm implied an autophagic event, indicating autophagic cell death (Figure 1aiii,biii). Autophagic oocytes from atretic follicles exhibited an important cytoplasmatic region undergoing degradation ( Figure 1c); in some follicles, only cytoplasmatic debris was observed.

Increased levels of autophagic process in oocytes from atretic follicles indicate autophagic cell death
To support evidence for the role of autophagy in oocyte elimination in prepubertal ovaries, the autophagic proteins mTOR, p-mTOR (S2448), ULK1, Beclin 1, light chain 3 A (LC3 A), and lysosomal-associated membrane protein 1 (Lamp1) were identified in cells with a large quantity of clear vesicles. The proteins were identified using seriated histological sections from ovaries.
First, the autophagic cells with clear cytoplasmic vesicles were identified by phase contrast illumination. Additionally, non-altered follicles were identified and were considered normal follicles. Highly altered vesiculated oocytes in diverse growth phases were identified in the follicles (Figure 2). Once the clear vesicles were confirmed, the distribution of the different proteins was evaluated. The labeling corresponding to the pro-autophagic proteins Beclin 1, LC3 A, and Lamp1 was significantly higher in oocytes with abundant clear vesicles than in normal oocytes. In 19-day-old oocytes, autophagic oocytes were 176.9% more labeled than normal; LC3 A was increased in autophagic oocytes by 220%, and Lamp1 was increased in the same type of oocytes by 147% (Figure 2a). The same pattern was observed in oocytes from 28-day-old rats ( Figure 2b). In this first step, the objective was to corroborate that the morphology of abundant clear vesicles corresponds to increased autophagy. Then, non-seriated slides were used to identify autophagic proteins. It is important to note that immunodetections were made in different oocytes sharing similar morphology with abundant clear vesicles (see phase contrasts images [CF] in Figure 2), and there is a correspondence between the phase-contrast images and immunolocalization of each identified protein, but not between the images of different immunolocalizations.
Along with the identification of autophagic proteins downstream from mTOR, the presence of mTOR and p-mTOR (S2448) was detected (Figure 3a,c). Our observations demonstrated that both labeling to mTOR were higher in normal oocytes than in highly altered vesiculated oocytes. Labeling to mTOR was increased by 48% and 45% in autophagic oocytes from the ages of 19 and 28 days, respectively. Similar results were observed regarding p-mTOR (S2448): 70% and 66%, respectively (Figure 3b,d). Together, these results demonstrate the high rate of autophagy occurring in altered oocytes for cell elimination.
To ensure that selected oocytes diagnosed as autophagic were not undergoing apoptosis, a simultaneous immunodetection of the pro-apoptotic proteins active caspase-3, Bax, and Bak combined with autophagic protein Beclin 1 were made. Both morphology and positive labeling to Beclin 1, at least four times more than apoptotic markers in 19-day-old and two times more than in 28-day-old specimens, as well as the negative labeling corresponding to active caspase-3, allowed us to corroborate that the studied oocytes were undergoing autophagy (Supplementary Figures S2  and S3). Moreover, oocytes with a compacted shape and non-evident clear vesicles were positive to apoptotic proteins and evidenced a low label to Beclin 1 compared with the autophagic oocytes ( Supplementary Figures S2  and S3).

Antagonistic presence of downregulators of autophagy and pro-autophagic proteins in oocytes undergoing cell elimination
To define the relationship between proteins that downregulate autophagy and those that promote autophagy, all the proteins were identified in the same oocyte. This was possible because serial sections of the same oocyte were used to identify each protein; additionally, double immunolocalizations of p-mTOR (S2448) were performed with two decisive proteins for autophagy of LC3 A and Lamp1.
The results showed that the levels of mTOR and p-mTOR (S2448) were negatively correlated with those of Beclin 1, LC3 A, and Lamp1. In normal oocytes, levels of mTOR and p-mTOR (S2448) were higher than those of the other proteins. Beclin 1 was four and three times higher in autophagic than in normal oocytes from 19-and 28-day-old rats, respectively. LC3 A reached a double quantity of labeling in autophagic oocytes. Lamp1 showed the same tendency, reaching an eightfold increase in 19-day-old oocytes and doubling in 28-day-old ones. Contrarily, mTOR was 60% higher in normal oocytes than in autophagic ones (Figure 4). The positive labeling to p-mTOR (S2448) was increased three times and ten times in 19-and 28-day-old oocytes, Labeling corresponding to Beclin 1, LC3 A, and Lamp1 proteins was significantly higher in altered oocytes than in normal ones. Representative images from observations in ovaries from five rats of each age group. Scale bars: 10 μm. (c) Quantitative fluorescence intensity data indicating the presence of the different proteins identified in normal and altered oocytes. Data are mean ± standard error of the mean. n = 120 to each group of age; p < 0.05. Lamp1, lysosomal-associated membrane protein 1; LC3 A, light chain 3 A; PC, phase contrast respectively ( Figure 5). All these data evidenced that in altered vesiculated oocytes, the labeling corresponding to both mTOR proteins was decreased, and the proautophagic proteins were increased (Figures 4 and 5), indicating increased autophagic activity.
Double-immunolocalization of p-mTOR (S2448, which downregulates autophagy) and LC3 A and Lamp1 (involved in autophagic vesicle elongation and autophagosome degradation, respectively) showed antagonistic labeling: p-mTOR (S2448) labeling was increased in healthy oocytes, whereas LC3 A and Lamp1 labeling was decreased. Conversely, in highly altered vesiculated oocytes, p-mTOR (S2448) labeling was low, and LC3 A and Lamp1 labeling was high. These results corroborate our findings that mTOR FIGURE 3 Detection of mTOR and p-mTOR (S2448). (a) An oocyte from a 19-day-old rat. Altered oocyte has an elevated presence of clear vesicles compared with normal oocyte. Labeling corresponding to mTOR and p-mTOR (S2448) was reduced in altered oocyte, indicating autophagic activation. (b) Quantitative fluorescence intensity data evidence at a low-level labelling in autophagic cells. (c) An oocyte from a 28-day-old rat. Similar to 19-day old oocytes, an increased labeling corresponding to mTOR and p-mTOR (S2448) is observed in non-autophagic oocyte. (d) Quantitative fluorescence intensity to mTOR and p-mTOR (S2448). Scale bars: 20 μm. Data are mean ± standard error of the mean. n = 180 to each group of age; p < 0.05. mTOR, mammalian target of rapamycin; PC, phase contrast; p-mTOR, phosphorylated-mTOR proteins regulate autophagy in oocytes undergoing autophagic cell death.

mTOR activity is inhibited via PI3K/AKT/mTOR and MAPK/ERK signaling
To define the pathway by which mTOR is downregulated to allow increased autophagy, the upstream mTOR signaling pathways, such as PI3K/AKT and mitogenactivated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), were analyzed using isolated enriched cell oocyte fractions. The results showed that FIGURE 4 Simultaneous immunolocalization of mTOR with Beclin 1, LC3 A, and Lamp1. mTOR showed increased labeling in normal oocytes compared with altered oocytes. The other proteins had low levels of labeling in normal oocytes and high levels in altered oocytes. Immunodetection was carried out in the same oocytes. Scale bars: 10 μm. Data are mean ± standard error of the mean. n = 60 to each group of age; p < 0.05. Lamp1, lysosomal-associated membrane protein 1; LC3 A, light chain 3 A; mTOR, mammalian target of rapamycin; PC, phase contrast FIGURE 5 Double immunolocalization of p-mTOR (S2448) and Beclin 1, LC3 A, and Lamp1 proteins. In normal oocytes, p-mTOR (S2448) was increased compared with altered oocytes. By contrast, levels of Beclin 1, LC3 A, and Lamp1 were higher in normal oocytes than in altered ones. Scale bars: 10 μm. Data are mean ± standard error of the mean. n = 60 to each group of age; p < 0.05. Lamp1, lysosomal-associated membrane protein 1; LC3 A, light chain 3 A; mTOR, mammalian target of rapamycin; PC, phase contrast; p-mTOR, phosphorylated-mTOR p90RSD, ERK 1/2, ribosome protein S6 (RPS6), and RAB11 were significantly expressed in oocytes from rats at both ages (Figure 6), suggesting that MAPK signaling pathways were involved in the increased autophagy in prepubertal oocytes. Additionally, the expression of p-AKT1 (S473) and p-RPS6 (S235/236) was practically absent, maintaining a correlation with low levels of mTOR and high rates of autophagy. These results are in line with the results of the immunodetection. Moreover, the absence of AKT indicates the downregulation of mTOR, promoting autophagy activated by growth factor deprivation. Interestingly, the low levels of the two mTOR proteins immunodetected in autophagic oocytes coincided with these results, indicating that the limitation of growth factors plays a role in activating autophagy.
In the present study, phosphorylated ERK 1/2 was strongly expressed, but conversely, RPS6 (pS235/236) was not expressed, indicating that ERK 1/2 does not promote cell proliferation. According to these results, we proposed that this protein could indirectly activate autophagy during the atretic process. To clarify this supposition, we identified the presence of TSC 1 and 2, which are associated with ERK and mTOR. The TSC 1/2 complex downregulates mTOR to promote autophagy under low-energy conditions. However, this complex is inhibited by AKT and ERK, which are absent and overexpressed, respectively, in oocytes. Our results revealed an increase in TSC1 and TSC2 in autophagic FIGURE 7 TSC1 and TSC2 immunodetection in normal and altered oocytes from ovaries of 19-and 28-day-old rats. (a) Cytoplasmic localization of TSC1 and TSC2 in normal oocytes was lower than that observed in altered oocytes. This distribution and behavior were present in oocytes from rats of both ages studied. (b) Relative fluorescence quantitation of TSC1 and TSC2 labeling. Scale bars: 10 μm. Data are mean ± standard error of the mean. n = 60 to each group of age; p < 0.05. PC, phase contrast; TSC, tuberous sclerosis protein oocytes, indicating the participation of these proteins in autophagic cell death in oocytes of 19-and 28-day-old rats ( Figure 7). Notably, the levels of positive labeling corresponding to TSC2 were two and three times higher in altered oocytes than in normal oocytes of 19-and 28-day old rats, respectively (Figure 7).

mTOR inhibition via Rapamycin does not improve native autophagy process
To corroborate the idea that the energy limitation is the major player in inducing the autophagic process in atretic oocytes, mTOR was inhibited via Rapamycin. After treatment with Rapamycin, p-mTOR (S2448) was decreased in treated oocytes from 19-and 28-day-old rats by 10% and 48%, respectively. Additionally, expression of LC3-II was conserved, evidencing progression of the autophagic process (Figure 8a). Meanwhile, the patterns of expression of via PI3K/AKT/mTOR and MAPK/ERK signaling were unaltered (Figure 8b).

Activation and inactivation of ULK1 in oocytes undergoing cell elimination
To confirm the low activity of mTOR by the downregulation of autophagy, the phosphorylation sites of ULK1 were analyzed. A reduction by half in the level of p-ULK (S757) was observed in autophagic oocytes compared with normal oocytes. Conversely, the level of p-ULK1 (S555) labeling was four times greater in autophagic oocytes than in normal ones (Figure 9). These results indicate that autophagy is activated not only by growth factor limitation but also by a low energy level, inducing AMPK phosphorylation in ULK1 on S555.
Considering that mitochondria are a crucial supply of energy, we analyzed the autophagosome content at the ultrastructural level. In oocytes undergoing autophagic cell death, autophagic vesicles were observed, and these vesicles often contained mitochondria or mitochondrial debris (Figure 10), suggesting selective intracellular degradation directed toward mitochondria. To define the specificity of the autophagic process in oocyte cell death, the p62 protein was immunodetected. Decreased labeling was identified in autophagic oocytes; by contrast, the labeling intensity of p62 was elevated in normal oocytes (10% and 100% to 19-and 28-day old, respectively) ( Figure 11).

DISCUSSION
Autophagy is a cytoprotective mechanism for cells in adverse environments. However, sustained increased levels of autophagy induce cell death. Supporting this, autophagic cell death has been reported during the elimination of oocytes in prepubertal Wistar rats (Escobar et al., 2008;Escobar et al., 2010;Escobar et al., 2012;Escobar et al., 2013;Escobar et al., 2019;Ortiz et al., 2006). In mammalian ovaries, the germinal cells are inside the follicles, which have a complex structure of theca and granulosa (somatic) cells surrounding each oocyte. The granulosa cells play several roles in supporting the oocyte, including supplying nutrients and growth factors. During the elimination of follicles not selected for ovulation, the follicles undergo several morphological and biochemical changes, such as the detachment of granulosa cells from the oocyte, contributing to oocyte cell death. Focusing on this pathway, we hypothesized that the dissociation of somatic and germinal cells induces intracellular protein activation that favors low levels of nutrients and growth factors. Our results clearly demonstrate the elimination of oocytes by autophagic cell death: a significant difference in the labeling of autophagic proteins between normal and altered oocytes was observed. The proteins analyzed in this work included Beclin 1, LC3 A, and Lamp1, which were increased in altered oocytes compared with normal oocytes, demonstrating increased autophagic activity (Akazawa et al., 2004;Carloni et al., 2008;Klionsky et al., 2012;Ma and Blenis, 2009;Zhu et al., 2009). Additionally, we demonstrated that low levels of mTOR and p-mTOR (S2448) were correlated with an increased level of autophagy. It has been amply documented that FIGURE 11 Immunodetection of p62 protein in normal and altered oocytes. Labeling corresponding to the presence of p62 was different in the two types of follicles. In normal oocytes, the labeling was elevated compared with that in atretic ones. These oocytes were from rats at the two ages studied. Scale bars: 10 μm. Data are mean ± standard error of the mean. n = 120 to each group of age; p < 0.05.l. PC, phase contrast mTORC1 acts as an autophagic regulator by inhibiting the ULK1 complex and thus autophagy (Weikel et al., 2015); specifically, p-mTOR (S2448) is indicative of autophagic repression (Reynolds et al., 2002), as it is attenuated with amino acid starvation (reviewed in Cheng et al. 2004). These data provided the first key signal to evaluate the transduction pathways related to mTOR inactivation and the beginning of autophagy. mTOR is part of the PI3K/AKT pathway, which is a classic upstream pathway of mTORC1 signaling (Lauring et al., 2013). The expression of p-AKT (S473) was not present in our study system, and this reinforced mTOR downregulation, which increased the rate of autophagy in the oocytes undergoing cell elimination. Upregulation of p-ERK1/2 (Y204/197) was observed during oocyte elimination; this activation could be regulated via the absence of AKT expression because AKT negatively regulates ERK activation by phosphorylating inhibitory sites in the Raf N-terminus (Cheung et al., 2008;Dhillon et al., 2002;Guan, 2000;Zimmermann & Moelling, 1999). Additionally, AKT phosphorylates diverse substrates, including TSC2; this phosphorylation inactivates Ras homolog enriched in brain (RHEB), which in turn potentiates mTORC1, resulting in inhibition of autophagy (Huang & Manning, 2009;Manning & Toker, 2017). Interestingly, the absence of AKT during oocyte cell death could indirectly promote the downregulation of mTORC1 via the TSC2 pathway. This hypothesis is supported in the present work by the increased presence of TSC2 in oocytes undergoing autophagic cell death.
In our study model -oocyte elimination through a normal physiological process -the upregulation of ERK 1/2 could be explained by the low levels of p-AKT because ATK negatively regulates ERK activation (Cheung et al., 2008;Dhillon et al., 2002;Guan, 2000;Zimmermann & Moelling, 1999), thus increasing ERK 1/2 levels. The complex role of autophagy in cell physiology implies the participation of several activation pathways in mTOR regulation. The antagonistic functions of ERK in the cell physiology have been documented (reviewed in Cagnol & Chambard, 2010); several studies have associated ERK activity with neuron autophagic cell death (Chu et al., 2004;Subramaniam & Unsicker, 2010), and thus the upregulation of ERK may be related to mTOR downregulation, promoting autophagic cell death in oocytes.
An important finding obtained in this study was the presence of TSC1/2 in altered oocytes. The increased labeling of TSC1/2 in autophagic oocytes suggests low energy, implying a decrease in basal high-energy phosphate levels and a reduction in the oxidative capacity of the mitochondria. This statement is supported by the observations of this work: first, the low staining of p62 protein in autophagic cells, indicating selective mitochondrial phagocytosis; second, a high quantity of autophagic vesicles with diverse content in different degree of degradation. Some ultrastructural images of vesicles of single membrane point to autophagolysosomes containing membranous debris; in fact, some conserve the multimembrane rearrangement like mitochondrial membranes. Finally, our statement that autophagy in oocytes on atretic follicles from 19-and 28-day-old rats is triggered by means energy limitation is supported by the Rapamycin treatment since, although the mTOR expression was reduced, the autophagic process was not significantly increased. mTOR is a pharmacological target of Rapamycin (reviewed in Sabatini, 2017): treatment with Rapamycin inhibits the mTOR protein and reduces protein synthesis and growth by inhibiting cell proliferation (Terada et al., 1995). In fact, mTOR has been stablished as a central regulator of anabolic cellular metabolism (Beretta et al., 1996;Chung et al., 1992;Jefferies et al., 1994;Singh et al., 1979;Terada et al., 1995). Considering these important roles of mTOR as metabolic censor, the fact mTOR inhibition did not exert effect in treating the oocytes supports the importance of the energy influence in inducing autophagy in a normal-physiological germinal cell elimination process.
Taken together, these findings indicate that autophagy is triggered by the combined stimuli of starvation conditions and energy depletion, inducing the participation of PI3K/AKT/mTOR and MAPK/ERK signaling to synergize and promote autophagic cell death of the oocytes in prepubertal Wistar rats.
The present findings are new evidence to aid the comprehension of the complex via of cell elimination or geminal line in female organisms. Our findings could be a precedent to considering different aspects during the follicular atresia, particularly as regards oocyte cell death, where depletion directly impacts reproductive capacity. Our results suggest the importance of energy limitation for inducing programmed cell elimination in a physiological system, that is, for cells that are eliminated in a normal process of selection. We identified that although the conditions were changed to induce a different via of autophagy activation by means the pharmacological inhibition of the mTOR protein, the germinal cells continued their self-programmed cell elimination. These findings open the possibility to use the pathway of energy limitation to stimulate cell elimination of not desired cells such as cancer cells as an alternative to the actual treatments.