Long-term artificial selection reveals a role of TCTP in autophagy in mammalian cells.

Long-Term Artificial Selection Reveals a Role of TCTP in Autophagy in Mammalian Cells Ke Chen,y,1 Chunhua Huang,y,1 Jia Yuan,1 Hanhua Cheng,*,1 and Rongjia Zhou*,1 1

Department of Genetics, College of Life Sciences, Wuhan University, Wuhan, People’s Republic of China These authors contributed equally to this work. *Corresponding author: [email protected], [email protected]. Associate editor: Joshua Akey y

Abstract

Key words: genetic improvement, evolution, selection, nucleophagy.

Introduction

Article

Mammalian ovaries produce mature oocytes for fertilization, which is essential for successful propagation of the species. Oocytes within the ovarian follicles are surrounded by somatic granulosa cells and cumulus cells to support the growth and development of oocytes in immature and adult ovarian follicles. Dysfunction in ovary, especially defective homologous recombination and aberrant signaling pathways (e.g., NOTCH and FOXM1), is known to contribute to ovarian cancer (Network 2011), which is the fifth leading cause of cancer death among women in the United States (Jemal et al. 2010). Aberrant cell signaling events in ovary, particularly those within ovarian granulosa cells, are associated with infertility in humans (Duggavathi and Murphy 2009; Fan et al. 2009). The production of ovarian oocytes is thought to cease before birth, and the total number of ovarian follicles is determined early in life (Johnson et al. 2004; Zou et al. 2009). Thus, ovarian senescence and menopause probably attribute to the exhaustion of reserved primordial follicles. However, recent studies suggested the existence of female germ stem cells (FGSCs) in neonatal ovaries for generating new oocytes (Johnson et al. 2004; Zou et al. 2009). These findings have significant implications in the genetic improvement of both economic and rare animals and in therapeutic expansion of the follicle pool to postpone normal or premature ovarian failure. Nevertheless, regulatory mechanisms controlling

FGSCs and how they are activated in the ovary remain unknown. Increasing evidence suggests that an oocyte–granulosa cell regulatory loop exists during folliculogenesis (Su et al. 2004; Zhang et al. 2010; Eppig and Handel 2011). Because oocytes are unable to take up some amino acids or synthesize cholesterol from acetate, cumulus cells may compensate by providing oocytes with the specific amino acids, cholesterol, and their metabolites in these metabolic pathways (Su et al. 2009). On the other hand, oocytes are known to regulate gene expression in granulosa cells through oocyte-derived paracrine factors, including growth differentiation factor 9 and bone morphogenetic protein 15 (Su et al. 2004). The crosstalk between oocytes and granulosa cells may thus coordinate oocyte maturation and the differentiation of both mural granulosa cells lining inside the follicle wall and cumulus cells surrounding the oocyte. From the primordial to antral follicle stages, a few oocytes gradually mature, whereas most follicles undergo atretic degeneration. Accurate regulation of these differential processes assuredly goes with a specific change in the follicle niche, probably within a hypoxic microenvironment of ovary, which is an unresolved mystery. Female fecundity (i.e., litter size) has been genetically improved in domestic animal species (e.g., pig) through longterm artificial selection. Litter size in domestic pigs has been under human control for over 9,000 years (Giuffra et al. 2000), with artificial selection leading to approximately 20 piglets/

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Mol. Biol. Evol. 31(8):2194–2211 doi:10.1093/molbev/msu181 Advance Access publication June 2, 2014

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Understanding genomic variation and detecting selection signatures in a genome under selection have been great challenges for a century. Activation, development/exhaustion of primordial follicles in mammalian ovary determines reproductive success, menopause/end of female reproductive life. However, molecular mechanisms underlying oogenesis, particularly under artificial selection, are largely unknown. We report that a proteome-wide scan for selection signatures in the genome over 9,000 years of artificial pressure on the ovary revealed a general picture of selection signatures in the genome, especially genomic variations through artificial selection were detected in promoter and intron regions. Crossbreeding between domestic and wild species results in more than half of the protein spots exhibiting heterosis. Translationally controlled tumor protein (TCTP) is upregulated by artificial selection and positively regulates autophagy through the AMP-activated protein kinase pathway. Notably, TCTP interacts with ATG16 complex. In addition to cytoplasmic autophagy, nucleophagy occurs in the nuclei of granulosa and cumulus cells in ovaries, indicating an importance of the nuclear material for degradation by nucleophagy. Our findings provide insight into cellular and molecular mechanisms relevant for improvement of ovary functions, and identify selection signatures in the genome for ovary function over long-term artificial selection pressure.

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litter, compared with approximately 8 piglets/litter in the closest wild relatives (Asian wild boar), which has been subject primarily to natural selection. Understanding the history of domestication and the dynamic changes that have occurred across the genome under selection has been a challenge since Darwin’s time. Comparative analysis between domestic pig and the closest wild relatives offers the potential in detecting selection signatures in the genome to understand female fecundity. Translationally controlled tumor protein (TCTP) is ubiquitously expressed in all eukaryotes (Hinojosa-Moya et al. 2008). TCTP plays key roles in cell proliferation and antiapoptosis through interaction with MCL1, Bax, Bcl-xL, p53, TSC-22, Rheb, VHL, Apaf-1, ATM, Cdc25C, and MDM2 (Liu et al. 2005; Yang et al. 2005; Chen et al. 2007; Hsu et al. 2007; Lee et al. 2008; Chan et al. 2011; Jung et al. 2011; Amson et al. 2012; Hong and Choi 2013; Jung et al. 2014). TCTP also acts as a transcription factor in the regulation of stem cell factors Oct4 and Nanog (Koziol et al. 2007). In mice, TCTP knockout leads to early embryonic lethality (Chen et al. 2007; Susini et al. 2008). In addition, TCTP expression is upregulated in many tumors and inhibition of TCTP expression can induce tumor reversion (Tuynder et al. 2002, 2004). Because of its complexity of functions and pathways in many tissues, molecular mechanisms and functions of TCTP in reproduction, especially in oogenesis, remain largely unknown. Herein, using a comparative and quantitative proteomics approach in combination with a selective breeding strategy, we report a proteome-wide scan for selection signatures designed to identify key genes for female fertility. Two-dimensional differential in-gel electrophoresis (2D-DIGE) analysis of proteomes revealed a general picture of selection signatures in the genome, as a result of long-term artificial selection of female fecundity, represented by a tripartite confrontation of protein expression patterns in the ovary (i.e., upselected, downselected, and unselected protein spots). Crossbreeding between domestic and wild species results in more than half of the protein spots exhibiting heterosis, which may disrupt the counterbalance of the three forces. We provide evidence that, in addition to cytoplasmic autophagy, nucleophagy also occurs in the nucleus of granulosa and cumulus cells in the ovary and show that TCTP, which is upselected during domestication, is a critical regulator of autophagy through the AMP-activated protein kinase (AMPK) pathway.

between two proteomes from ovaries of the domestic species, wild species, and their hybrid F1, approximately 1,500 protein spots were identified on a typical ovary gel (supplementary fig. S1, Supplementary Material online), of which 109 differential protein spots (P < 0.01, 1.5-fold cutoff) were determined by one-way analysis of variance (ANOVA) (fig. 2A, supplementary fig. S2, Supplementary Material online). Over one-third (36%) of the differential protein spots were upselected and nearly one-third (31%) were downselected, whereas artificial selection had no effect on the other onethird (33%) of the protein spots (fig. 2B–E and H). In addition, crosses between domestic and wild species resulted in more than half of the 109 protein spots exhibiting heterosis (58.7%), and approximately a quarter of the protein spots (26.6%) exhibiting hybrid disadvantages (fig. 2F and G). Using LCMS/MS techniques, 60 differential spot features were chosen for protein identification, of which 36 unique proteins were identified (supplementary tables S2–S4, Supplementary Material online). All the proteins identified by MS were further analyzed and annotated using PANTHER Gene Ontology classification (Thomas et al. 2003), indicating eight categories that mainly included proteins involved in the catalytic activity (33.33%), binding (26.19%), and structural molecule activity (21.43%) (fig. 3A).

Results Identification of Proteins in Female Fecundity by 2DDIGE and MS Analysis To search for key genes in female fecundity, a comparative and quantitative proteomics approach in combination with a selection breeding strategy was used as summarized in figure 1. Pairing the domestic pig and its closest wild relative (Asian wild boar) represents a typical model for long-term artificial selection (litter size ~20) versus natural selection (litter size ~8) for female fecundity for over 9,000 years before present (Giuffra et al. 2000). Using 2D-DIGE to detect and quantify differences in protein abundance

Selection-Altered Gene Expression Is Mainly Regulated through Transcription and/or Posttranscription and TCTP Is Upregulated by Artificial Selection To explore whether the expression level of the identified proteins is consistent with the MS results and one-way ANOVA, semiquantitative RT-polymerase chain reaction (PCR) was used to amplify 20 genes encoding the differential proteins, which confirmed 2D-DIGE/MS identification (fig. 3B–D). Western blot analysis using antibodies against six proteins was consistent with the quantitative proteomic results (fig. 3E). These data suggested that artificial selection of ovary functions altered gene expression, primarily through transcriptional and/or posttranscriptional regulations. In addition, western blot analysis showed that five proteins including TCTP were upregulated by artificial selection. Further sequence variation analysis showed that abundant mutations including point mutation, insertion, and deletions occurred in the promoter regions of these selection-altered genes in the domestic and wild pigs (fig. 4A). In contrast, no mutation was detected in equally expressed genes between the domestic and wild pigs. In addition, the mutations were associated with some binding sites of transcription factors in the selection-altered genes in domestic pigs (fig. 4A). In the markedly upregulated gene TCTP, single-nucleotide polymorphism (SNP) frequency was obviously reduced in its introns and 3’-region in the domestic pigs compared with wild pigs (fig. 4B). These variations also generated some binding sites of transcription factors in the domestic pigs (fig. 4C). Further function test of these binding sites showed that the TCTP intron 2 containing the binding site of transcription factor Oct-1/Pbx-1 in the domestic pigs in either directions had significant transcriptional activation effect on reporter gene 2195


Nucleophagy in Mammalian Cells . doi:10.1093/molbev/msu181

Chen et al. . doi:10.1093/molbev/msu181

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FIG. 1. Schematic representation of MS identification of differentially expressed proteins in ovary among domestic pigs, wild pigs, and their F1. Ovary samples were collected from two individuals of each strain: Domestic strain, wild strain, and their cross F1. After extracted and quantified, total proteins were labeled with dye Cy3 and Cy5, respectively. To normalize the various 2D-DIGEs, internal standard consisting of equal amount of proteins from each ovary was labeled with dye Cy2. 2D-DIGEs were performed, and images were analyzed to calculate significant differences in relative abundances of protein spot features. Finally, in-gel digestion and mass spectrometer analysis were used to identify the differentially expressed proteins. The detailed procedure was described in Materials and Methods section.

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FIG. 2. Artificial selection reveals three directions of gene expression pattern shift among the domestic (blue line) pigs, wild (yellow line) pigs, and their F1 (red line) by DIGE analysis. (A) The overall expression levels of the differentially expressed 109 spots. Y axis indicates the relative expression level of each spot normalized to internal standard Cy2. X axis represents the spot numbers. About 1.5-fold cutoff was used as a standard for significant difference. (B) Thirty-nine spots were high expressed in the domestic strain compared with the wild strain. (C) Thirty-four spots low expressed in the domestic strain compared with the wild strain. (D) Twelve spots low expressed in the F1 but equal in both domestic and wild strains. (E) Twenty-four spots high expressed in the F1, but equal in both domestic and wild strains. (F) In the F1, 64 spots were high expressed, including those significantly differential between the domestic and wild stain. (G) Twenty-nine spots low expressed in the F1, including those significantly different between the domestic and wild stain. (H) Relative percentage of 109 spots based on the difference between the domestic and wild stain. (I) Artificial selection results in three directions of gene expression changes: Upregulation, downregulation, and no change, and each of the directions has nearly one-third genes based on data in the panel H.

expression (fig. 4D), suggesting an enhancer role of the TCTP intron 2 in the domestic pigs. To demonstrate that Oct1 is associated with TCTP intron 2 in vivo, we performed a chromatin immunoprecipitation (ChIP) assay focusing on a

fragment of 94 bp around the Oct1 binding site. DNA fragments containing the Oct1 binding site were highly enriched during immunoprecipitation with anti-Oct1 in the ovary of the domestic pig but not in the wild pig (fig. 4E). These results 2197


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FIG. 3. Function classification, semiquantitative RT-PCR, and western blot analysis of differentially expressed genes identified by 2D-DIGE and MS. (A) Thirty-six proteins were identified by MS successfully. They were divided into eight categories based on their main functions, including catalytic activity, binding, structural molecule activity, enzyme regulator activity, transporter activity, nucleic acid binding transcription factor activity, receptor activity, and antioxidant activity. (B–D) RT-PCR confirmation of most of the genes encoding the proteins identified by MS in ovaries of the domestic strain, wild strain, and their F1. (B) Highly expressed in the ovary of the domestic strain compared with the wild strain. (C) Highly expressed in the ovary of the wild strain compared with the domestic strain. (D) Highly/lowly expressed in F1 but equally in both the domestic and wild strains. (E) Western blot analysis confirmed the differentially expressed proteins in ovaries among the domestic strain, wild strain, and their F1. GAPDH was employed as an internal control.

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FIG. 4. Genetic variation in differentially expressed genes between wild and domestic pigs. (A) Mutations in promoter regions of differentially expressed genes in domestic (D, n = 14; Landrace pigs) or wild (W, n = 15) pig. Background color on the mutated bases indicates mutation frequency. Predicted binding sites of transcription factors generated or lost in domestic pig because of the mutations are showed in the right panel. (B) SNP frequency in the TCTP introns and 3’-region in domestic (D) or wild (W) pig. Percentages were calculated based on valid sequencing data. (C) Predicted binding sites of transcription factors generated in the domestic pig in the TCTP intron 2 and 3’-region. L, Landrace pig. (D) Transcriptional activation effect of the TCTP intron 2 containing the binding site of transcription factor Oct-1/Pbx-1 in the domestic pigs on the minP-Luciferase. Fragments of both forward (Int2) and reverse (Int2-R) directions of the intron 2 were tested. The vector pGL4-promoter was used as a control. (E) Oct1 binds to the TCTP intron 2 of the domestic pig in vivo. ChIP analysis shows that a fragment of 94 bp containing the Oct1 binding site in the TCTP intron 2 (200400/200493) is detected in ovary tissue of domestic pig, but not in the wild pig. Bar indicates the standard error of the mean.

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suggested that abundant genetic variations exist in these selection-altered genes between the domestic and wild pigs, and some of these variations are associated with the binding sites of transcription factors.

TCTP Is Dominantly Expressed in the Cytoplasm of Granulosa and Cumulus Cells and the Liquor Folliculi Because TCTP is upregulated by artificial selection (fig. 3), cell types and subcellular localization of TCTP expression were 2200

examined by immunofluorescence using an antibody against TCTP in mouse ovary. In primary follicles, remarkable signals were observed in the cytoplasm of the granulosa cells, especially inner regions surrounding the oocytes (fig. 5A–E). TCTP was consistently expressed in the cytoplasm of granulosa cells in the secondary, mature, and ruptured follicles (fig. 5F–T). Expression was also observed in the cytoplasm of the cumulus cells of the mature and ruptured follicles (fig. 5K–T). In addition, positive signals appeared in the liquor folliculi. Western blot analysis confirmed the expression of both the


FIG. 5. Subcellular localization of TCTP in the granulosa and cumulus cells of the mouse follicles. Hematoxylin and eosin staining of the primary (A), secondary (F), mature (K), and ruptured follicles (P) in the mouse ovary. (B, G, L, Q) The nucleus was counterstained with Hoechst. (C, H, M, R) The ovary sections were stained with monoclonal antibody specific to TCTP (green), and the specific signals were examined by indirect immunofluorescence and confocal microscopy. Merged images of Hoechst and TCTP staining are shown in D, I, N, and S. Higher magnification images of the square regions in the panels (D, I, N, S) are shown on the right (E, J, O, T). Arrows indicate granulosa cells. White triangles indicate the cumulus oophorus. The black triangle points to the released oocyte. Dominant TCTP signals were observed in the cytoplasm of granulosa and cumulus cells. The signals also appeared in the liquor folliculi. Scale bar: 50 mm (A–D, F–I, K–N,) or 100 mm (P–S). (U) Western blot analysis confirmed the expression of the TCTP protein in the cytoplasm of granulosa and cumulus cells, and the supernatant of the primary culture of cumulus cells (condensed supernatant).


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cytoplasmic and secreted TCTP protein in primary cumulus cell culture (fig. 5U). A similar expression pattern of TCTP was also observed in the porcine ovary (supplementary fig. S3A–O, Supplementary Material online).

autophagy was induced under starvation (fig. 8C). Immunoelectron microscopy analysis confirmed that the LC3B puncta surrounds the nuclear membrane (fig. 8D and E). During nucleophagy, nuclear materials to be degraded are encapsulated by the nuclear membrane, probably through two modes of nucleophagosome generation: Exocytosis using the nuclear membrane and formation of an isolation membrane (fig. 8F). Only the active form of the autophagy protein LC3B, LC3-II, was observed in the nuclei, indicating that LC3-PE participates in the nucleophagy.

Nucleophagy Occurs in the Granulosa and Cumulus Cells

The Nuclear Envelope Participates in Autophagosome Formation in Nucleophagy The membrane source of autophagosomes has been the topic of much debate for many years. To determine the source of autophagosome membrane during nucleophagy, we induced autophagy under hypoxia and analyzed the autophagosome membrane using Nurim as a marker for the nuclear membrane by immunofluorescence and confocal microscopy (Chen et al. 2012). The results indicated that nucleophagy occurred from the nuclear membrane, which contains nuclear materials surrounded by the nuclear envelope (fig. 8A and B), whereas the nuclear autophagosomes were not observed in normal culture condition (fig. 8A). Similar nucleophagy phenomena were observed when

TCTP Regulates Autophagy through the AMPK Pathway Because TCTP is predominantly expressed in the cytoplasm of granulosa and cumulus cells, which is similar to the expression pattern of autophagy protein LC3, the question remains with respect to whether TCTP is involved in the regulation of autophagy. Previous studies have indicated that TCTP promotes mTORC1 activity to regulate cell proliferation (Hsu et al. 2007; Dong et al. 2009), and both mTORC1 and AMPK regulated autophagy (Egan et al. 2011; Kim et al. 2011). We further investigated a role of TCTP in autophagy regulation. As expected, stable TCTP knockdown (miR-TCTP) downregulated the phosphorylation of P70S6K (an indicator of mTORC1 activity) (fig. 9A). TCTP knockdown also decreased the transcriptional activation of HIF1 (HIF1 is a known downstream effector of TCTP) (fig. 9B and C), indicating an effectiveness of miR-TCTP. We therefore explored whether TCTP regulates AMPK activity using the miR-TCTP. TCTP depletion enhanced the AMPK activity under normoxic condition, as indicated by increased phosphorylation of AMPKa on Thr172, which is required for the activation of AMPK (fig. 9D). However, under hypoxic conditions, TCTP knockdown inhibited the phosphorylation of AMPKa on Thr172 (fig. 9E). As the activities of both AMPK and mTORC1 were regulated by TCTP, suggesting a role of TCTP in the regulation of autophagy, we measured the levels of autophagy protein LC3-II in COS-7 cells expressing miR-TCTP under normoxic or hypoxic conditions. Under normoxic conditions, LC3-II was increased when TCTP was knocked down (fig. 9E). However, after 24 h of hypoxia, LC3-II was decreased in the TCTP knockdown cells (fig. 9E) and LC3II puncta numbers were reduced (supplementary fig. S4B and E, Supplementary Material online). Similar results were also obtained under starvation conditions (supplementary fig. S4C and F, Supplementary Material online). Moreover, when the TCTP expression was rescued in the stable miR-TCTP cell line via infection with lentivirus expressing the TCTP gene, LC3-II puncta numbers increased markedly under hypoxic or starvation conditions (supplementary fig. S4G–I, Supplementary Material online). To further clarify that TCTP regulates autophagy through AMPK, two miR-AMPK constructs (two different target sites) were transfected into COS-7 cells to establish stable AMPK knockdown cell lines. Besides, double (AMPK and TCTP) knockdown cell lines stable-expressing miRAMPK and miR-TCTP were also established. These cells were cultured under normoxic or hypoxia conditions for 2201


The environment surrounding oocytes may be considered “hypoxic” from a molecular standpoint because the oxygen partial pressure (pO2) surrounding oocytes with a diameter greater than 200 mm in the follicular cavity of mature follicles reaches a diffusion distance of oxygen cutoff of 100–200 mm in a tissue, that is, 4–20 mmHg (0.5–2.5% oxygen) (Martin et al. 2008). As hypoxia triggers autophagy in many cell types (Rabinowitz and White 2010), some questions remain, including whether autophagy occurs in granulosa and cumulus cells and whether autophagy plays a role in follicle development and oogenesis. To address these questions, we first examined autophagy protein LC3 in ovary sections. Immunofluorescence showed that LC3 was expressed in a similar pattern as TCTP in the cytoplasm of granulosa and cumulus cells from primary, mature, and ruptured follicles (besides secreted expression of TCTP) (fig. 6). Moreover, accumulation of LC3 puncta surrounding the nuclei of granulosa and cumulus cells was remarkable, and in the nuclei, the signals were diffuse (fig. 6U–W). Western blot analysis revealed that the active form of the autophagy protein LC3, LC3-II (LC3-PE), existed in both the cytoplasm and nucleus, and only LC3-II, but not LC3-I, was detected in the nucleus (fig. 6X). In addition, a similar expression pattern of LC3 was also observed in the porcine ovary (supplementary fig. S3A’–O’, Supplementary Material online). Furthermore, accumulation of Red-LC3-II puncta was also detected surrounding the nuclei in some COS-7 cells when infected with a lentivirus expressing Red-LC3 and cultured under hypoxic conditions for 24 h, whereas most of the LC3-II puncta were observed in the cytoplasm (fig. 7A and B). Western blot analysis indicated that, similar to the granulosa and cumulus cells, only the active form LC3-II, but not LC3-I, was detected in the nucleus of COS-7 cells (fig. 7C). These results suggest that, in addition to cytoplasmic autophagy, nucleophagy also occurs in the nucleus.

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24 h. Hypoxia induced LC3-II accumulation in miR-lacZ treated cells (fig. 9F lanes 1 and 7), but not in the TCTP knockdown (fig. 9F lanes 2 and 8), the AMPK knockdown (fig. 9F lanes 3, 4, 9, and 10), or AMPK and TCTP double knockdown cells (fig. 9F lanes 5, 6, 11, and 12). These results suggested that TCTP regulates autophagy through the promotion of AMPK activity under hypoxic conditions. 2202

TCTP Interacts with ATG16 Complex Because the ATG5–ATG12/ATG16 complex are required for efficient promotion of LC3-I conjugation to phosphatidylethanolamine to be an active form, LC3-II (Romanov et al. 2012), we examined whether TCTP interacts with the components


FIG. 6. Expression of autophagy protein LC3B in granulosa cells from mouse follicles. Hematoxylin and eosin staining of the primary (A), secondary (F), mature (K), and ruptured follicles (P) in the mouse ovary. Nuclei were counterstained with Hoechst (B, G, L, Q). These ovary sections were stained with antibody specific to LC3B (green) and the specific signals examined by indirect immunofluorescence and confocal microscopy (C, H, M, R). Merged images of Hoechst and LC3B staining are shown in D, I, N, and S. Higher magnification of the square regions in the panels (D, I, N, S) are shown on the right (E, J, O, T). Dominant LC3B signals were observed in the cytoplasm of granulosa cells. Arrows indicate granulosa cells. White triangles indicate the cumulus oophorus. The black triangle points to the released oocyte. Scale bar: 50 mm (A–D, F–I, K–N) or 100 mm (P–S). (U–W) Higher magnification images of the square regions in red line in the panel J. Scale bar: 10 mm. (X) Western blot analysis confirmed the appearance of the active form of the autophagy protein LC3B, LC3-II, in the nuclei. The supernatant, medium from the primary granulosa cell culture (condensed supernatant, 5 times). Lamin A/C and GAPDH were used as internal controls for the nuclear and cytoplasmic extracts, respectively.


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FIG. 7. Accumulation of Red-LC3 puncta surrounding the nuclei and LC3-II presence in the nuclei. (A) In COS-7 cells, dominant signals from the LC3 puncta were observed in the cytoplasm; some signals also appeared in the nuclei. Scale bar: 20 mm. (B) The images were enlarged from the squares in the images in (A), highlighting accumulation of Red-LC3 puncta surrounding the nuclei. Arrows indicate Red-LC3-II signals in the nuclei. Scale bar: 5 mm. (C) Western blot analysis confirmed the appearance of the active form of the autophagy protein LC3B, LC3-II, in the nuclei, besides in the cytoplasm in COS-7 cells. Lamin A/C and GAPDH were used as internal controls of the nuclear and cytoplasmic extracts, respectively.

of the autophagy pathway including the complex. Coimmunoprecipitation assays of autophagy-related proteins ATG5, ATG6, ATG7, ATG8, ATG12, ATG16, and p62/ SQSTM1 with Flag-TCTP in 293T cells showed that

Flag-TCTP coprecipitated efficiently with ATG5, ATG12, and ATG16L1 but not with ATG6, ATG7, ATG8, and p62 (fig. 10A). The interaction of ATG5, ATG12, and ATG16L1 with TCTP was determined by reciprocal 2203


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FIG. 8. The nuclear envelope participates in autophagosome formation in nucleophagy. COS-7 cells were infected with both lentivirus expressing RedLC3 and EGFP-Nurim to establish a stably expressing cell line, and then cultured under normal conditions (A), hypoxic conditions (B), or in starvation medium MEM-EBSS (C) for 24 h. Images were taken by confocal microscopy. Arrows indicate nuclear autophagosomes containing nuclear materials surrounded by the nuclear membrane. Upper panels in (B) show two modes of nucleophagy (exocytosis and isolation membrane). The nuclei were stained with Hoechst33258. The nuclear membrane was labeled with a green color (expressing EGFP-Nurim). Red color indicates Red-LC3B protein. Merged images show the three colors overlapping. Scale bar: 5 mm. (D, E) Immunoelectron microscopy. COS7 cells transfected with EGFP-LC3B were cultured in MEM-EBSS for 24 h and the localization of EGFP-LC3B was examined by immunogold electron microscopy using anti-GFP antibody. Black dots indicate immunogold particles. N and C represent the nucleus and cytoplasm, respectively. (E) Higher magnification image of the square region in red line from the panel D. Arrows indicate representative gold particles. Dotted line indicates the nuclear envelope. Scale bar: 200 nm. (F) A model for (continued)

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coimmunoprecipitation experiments (fig. 10B–D). We further confirmed the interaction between endogenous TCTP and ATG16L1 complex. We observed that immunoprecipitates with anti-TCTP antibody contained endogenous ATG16L1 and ATG5-ATG12 but not LC3B/ATG8 (fig. 10E). In addition, both TCTP and ATG16L1 or TCTP and ATG5 were colocalized in the cytoplasm, especially coaggregated between TCTP and ATG16L (fig. 10F and G). These results indicated that TCTP binds to ATG16L1 complex in vivo. Thus, TCTP promotes autophagy under hypoxia and starvation conditions by binding to ATG16L complex and then promoting LC3-I conjugation to phosphatidylethanolamine to be an active form, LC3-II, during autophagosome formation.

to elucidate the mechanisms of mammalian oogenesis through nucleophagy. We provide clear evidence supporting that, in addition to cytoplasmic autophagy, nucleophagy occurs in the nuclei of granulosa and cumulus cells in ovaries. The nucleophagy phenomenon was also observed in yeast (Narita et al. 2011) and mouse cells with nuclear envelopathies (Park et al. 2009) and HSV-1 infection (English, Chemali, and Desjardins 2009; English, Chemali, Duron, et al. 2009). We observed nucleophagy in other kinds of mammalian cells under hypoxic conditions or in the presence of starvation stress. Recently, nucleophagy attracts more attention of the scientific world (Erenpreisa et al. 2012; Rello-Varona et al. 2012; Mijaljica and Devenish 2013). These data indicate a role of nucleophagy in various physiological or pathological conditions, whenever the nuclear materials are redundant or dispensable, such as in DNA misreplication, RNA redundancy, or proteins in the nucleocytoplasmic shuttle errors. A further topic of much debate for many years is the membrane source of autophagosomes. Notably, we show that the nuclear envelope participates in autophagosome formation in nucleophagy, which provides evidence supporting the membrane source of the autophagosome, at least some, from the nuclear envelope. A more important finding in this work is a new role of TCTP, an upselected gene, during folliculogenesis through the regulation of autophagy. TCTP is involved in a variety of cellular processes, including growth control, microtubule stabilization, calcium-binding activities, transcriptional regulation, apoptosis, and cancer (Bommer and Thiele 2004; Tuynder et al. 2004; Hsu et al. 2007; Koziol et al. 2007; Susini et al. 2008). We show that TCTP is upregulated in ovaries under the artificial selection of female fecundity, which may exert a role as an energy regulator for oogenesis in the special microenvironment of the ovary. On one hand, TCTP promotes energy synthesis through the upregulation of PGK1 expression via HIF1. On the other hand, TCTP positively regulates autophagy through the AMPK pathway. Autophagy is not only the major intracellular degradation system by which both cytoplasmic and nuclear materials are delivered to and degraded in the lysosome but it also engages in the renovation of cells and tissues (Mizushima and Komatsu 2011). TCTP, through at least the two modes discussed above, serves as a dynamic recycling and energy regulator for cellular renovation and homeostasis to ensure or maintain a proper hypoxic niche environment surrounding the oocytes during oogenesis, which is essential for female fertility.

Discussion Understanding the history of domestication, detecting selection signatures, and identifying genes or regions in a genome that are, or have been, under selection have been the topics of study for many years. This study provides a large-scale integrative analysis to understand the improvement of female fecundity and selection signatures in the genome over 9,000 years of artificial pressure. Overall, the work presented here provides a new perspective on selection and adaptation in female fecundity. We show that long-term artificial selection of female fecundity changes the gene expression pattern reflective of tripartite confrontation in the ovary, indicating the counterbalance of upselected, downselected, and unselected genes. Further disruption of the counterbalance of the three forces in regulation of gene expression, such as through crossbreeding or introduction of new genetic variation through genetic modifications, could continue to improve female fecundity. Notably here we provide a general picture of gene expression changes under selection. We show that artificial selection of ovarian functions alters gene expression mainly through transcriptional and/or posttranscriptional regulation, owing to genomic variations occurred in promoter and intron regions during domestication. Especially, genes upregulated by artificial selection mainly include those involved in the cytoplasmic cytoskeleton, molecular chaperones, and cell growth/ apoptosis, and downregulated genes are those in the immune response and nuclear cytoskeleton. Genetic manipulation of these genes may change ovary functions. Therefore, the work has significant clinical implications in the development of new therapeutic, diagnostic, and preventive approaches to the treatment of diseases involved in ovary functions, such as female infertility, polycystic ovarian syndrome, and ovarian cancer, in addition to therapeutic expansion of the follicle pool to ensure the proper length of reproductive life. Our study also provides new directions to explore nucleophagy in mammalian cells, especially in ovarian functions, and

Materials and Methods Ovary Sample Collection Ovary samples used for this study were collected from two domestic pigs (Chinese Meishan, adults), two wild pigs (Hubei

FIG. 8. Continued nucleophagy. Nuclear materials to be degraded will be encapsulated by the nuclear membrane. Two modes likely exist to generate nuclear autophagosomes; one is similar to exocytosis using the nuclear membrane (F, a), and another may be through the gradual formation of an isolation membrane to encapsulate the nuclear materials (F, b). Because the active form of the autophagy protein LC3B, LC3-II, was only present in the nuclei, LC3-PE participates in nuclear autophagosome formation. N, the nucleus; C, the cytoplasm.

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wild pigs, adults), and two crossbred pigs (F1, male wild female Meishan, adults). Ovary samples (300 mg) were taken and immediately stored in liquid nitrogen. All animal work has been conducted according to relevant national and international guidelines and was approved by the ethics review committee of the university.

Germany), then homogenized with a Dounce homogenizer (20 strokes). The tissue lysate was sonicated with a Sonic Dismembrator (10 s, 8 times) and centrifuged at 14,000 rpm for 1 h at 4 C. The supernatant (proteins) was transferred to another tube. All experiments were handled on ice. The protein concentration was determined using a protein assay kit (Bio-Rad DC Assay kit, Richmond, CA).

Protein Extraction Each ovary sample was cut into several small tissue fractions. The tissue fractions were washed three times with PBS, suspended in 1 ml DIGE lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS) with protease inhibitors (Roche, Mannheim, 2206

Cy-Dye Labeling and 2D-DIGE A total of 50 mg of ovary protein was minimally labeled with Cy2, Cy3, or Cy5 (400 pmol; GE Healthcare, Piscataway, NJ). A pooled sample consisting of an equal amount of each of the


FIG. 9. TCTP regulates autophagy through the AMPK pathway. (A) Stable TCTP knockdown in COS-7 cells downregulates the phosphorylation of P70S6K (an indicator of mTORC1 activity). Stable expression of miR-LacZ in COS-7 cells was used as a control. (B) TCTP knockdown decreases the transcriptional activation of HIF1 (HRE: hypoxia response element; HIF1 binds to the HRE of PGK1 promoter; HIF1 is a known downstream effector of TCTP). Luciferase activity was measured (n = 3). (C) Real-time quantitative PCR showed that TCTP knockdown inhibits PGK1 transcription in COS-7 cells expressing miR-TCTP. miR-LacZ was used as a control. (D) Stable TCTP knockdown in COS-7 cells induces phosphorylation of AMPKa at Thr172 under normoxic condition. (E) Stable TCTP knockdown in COS-7 cells inhibits autophagy and AMPK activity under hypoxic stress. (F) TCTP regulates autophagy through the AMPK pathway. COS-7 cells stably expressing miR-LacZ, miR-TCTP, miR-AMPKa or miR-TCTP, and miR-AMPKa were cultured under normoxic or hypoxic conditions for 24 h. GAPDH was employed as an internal control.

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six experimental samples was used as the pooled internal standard. The internal standard (50 mg) was minimally labeled with Cy2. Individual ovary protein samples (50 mg) were minimally labeled with Cy3 or Cy5 (fig. 1). Proteins labeled with Cy2 (pool), Cy3, and Cy5 were mixed and separated by isoelectric focusing (IEF) using 13-cm nonlinear IPG DryStrips (pH 3–10, GE Healthcare) according to the manufacturer’s instructions. Briefly, the IPG DryStrip was rehydrated and warmed at 30 V for 12 h before IEF, and IEF was performed at 500 V for 1 h, 1,000 V for 1 h, 8,000 V for 8 h, and 500 V for 4 h. Proteins were further separated according to their molecular weight on SDS-PAGE gels (12.5%) using the Hofer SE 600 system (GE Healthcare).

DIGE Analysis The gels were scanned using Typhoon 9410 Variable Mode Imager (GE Healthcare). The excitation wavelengths for Cy2, Cy3, and Cy5 are 488, 532, and 633 nm, respectively. The gel images were analyzed using the DeCyder 6.5 Differential In-gel

Analysis software (GE Healthcare). Including the Cy2-labeled pooled internal standard on every gel allowed measurements of the abundance of a protein in each sample relative to the internal standard. This approach also allowed accurate relative quantitation of protein spot features across different gels. Significantly changed protein spots (i.e., present in all analyzed gels and the standardized average spot volume ratios exceeded 1.5 times) were calculated using one-way ANOVA.

In-Gel Digestion The 2D gel containing 1 mg of unlabeled pooled standard sample was fixed in 30% methanol and 7.5% acetic acid, and then Coomassie stained (R-350; GE Healthcare). Protein spot features that were significantly increased or decreased (P < 0.01) were excised from the 2-DE gel and placed into 96well plates. The gels were washed twice with Milli-Q water for 15 min and then washed three times in 25 mM NH4HCO3 and 50% CH3CN for 30 min while vortexing. The gels were then dehydrated in 100% CH3CN for 10 min while vortexing 2207


FIG. 10. TCTP interacts with autophagy proteins ATG16L1 complex. (A) Cotransfection of Flag-TCTP into 293T cells was performed, together with Myc-ATG5, EGFP-myc-ATG6, Myc-ATG7, Myc-ATG8, Myc-ATG12, EGFP-myc-ATG16L1, EGFP-myc-p62, or an empty control plasmid pCMV-myc, respectively. At 48 h after transfection, the whole cell lysate was extracted for coimmunoprecipitation with anti-Myc, followed by probing with anti-Flag. (B–D) 293T cells were cotransfected with EGFP-myc-ATG16L1 (B) EGFP-myc-ATG12 (C) or Myc-ATG5 (D) together with Flag–TCTP or an empty control plasmid pCMV-myc, respectively. Band * indicates partial degradation of EGFP from the EGFP-fused protein. (E) TCTP interacts with ATG5 and ATG16L1 in vivo. The 293T cell lysates were immunoprecipitated with anti-TCTP antibody or anti-Flag antibody (control) followed by immunoblotting with antibodies against ATG16L1, ATG5, or LC3B. The bands corresponding to endogenous ATG16L1, ATG5, and LC3B are indicated. ATG5–ATG12 is a known complex. (F–G) Colocalization of TCTP with ATG16L1 (F) or ATG5 (G) in the cytoplasm under normal or starvation condition imaged by confocal microscopy. The nuclei were stained with Hoechst33258. Images were taken by confocal microscope. Arrows indicate the ATG16L1 spots. Scale bar: 5 mm.

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and allowed to air dry for 1 h. A total of 12 ml of 1.5 mM trypsin (Promega, Madison, WI) suspended in 25 mM NH4HCO3 was added, and the gels were placed at 37 C and allowed to digest overnight. The 96-well plates were then gently centrifuged, and the supernatant was removed for MS/MS analysis.

MALDI-TOF/TOF MS and LC-MS/MS Analyses

RT-PCR Analysis Total mRNA was isolated from ovarian tissue using TRIzol reagent (Invitrogen, Carlsbad, CA) and used to synthesize first-strand cDNA with M-MLV (Promega, Madison, WI). The amount of mRNA was measured by semiquantitative PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was the housekeeping gene used as a control. Primer sequences used in this study are provided in supplementary table S1, Supplementary Material online. All PCR reactions were performed using the cycle 30 s, 94 C; 30 s, 58 C; and 30 s, 72 C (supplementary table S1, Supplementary Material online). PCR products were run on a 1.5% agarose gel and visualized under UV light.

Gene Sequence Analysis Genomic DNA was isolated from ear tissues of domestic or wild pigs using routine method. PCR was performed using primers specific for promoters or introns of differentially expressed genes and conditions indicated in supplementary table S1, Supplementary Material online. All PCR products were sequenced. Sequences were analyzed using MEGA6 (www.megasoftware.net, last accessed June 18, 2014), and all sequences were aligned with reference sequences of the domestic pig retrieved from UCSC (http://genome.ucsc.edu/, last accessed June 18, 2014). Transcription factor binding sites were predicted using TFSEARCH (http://www.cbrc.jp/ research/db/TFSEARCH.html, last accessed June 18, 2014) (Heinemeyer et al. 1998).

Western Blot Analysis Western blotting was performed according to routine protocols. The whole extract was analyzed by glycine–SDS-PAGE or tricine–SDS-PAGE and transferred to a 0.45-mm PVDF membrane (Hybond-P; GE Healthcare). The membranes were blocked with 5% nonfat dried milk in TBST (20 mM 2208

Plasmid Constructs Mouse TCTP cDNA was PCR amplified using primers TCTP F and R, digested by EcoRI and XhoI, and ligated into pcDNA3.03xHA and pCMV-2xFlag cut with EcoRI and XhoI to create pcDNA3.0-3xHA-TCTP and pCMV-2xFlag-TCTP, respectively. pLove-Flag, which contains a lentiviral vector backbone, was constructed by subcloning the CMV promoter and Flag of pCMV-Tag2B into the XhoI and EcoRI sites of pSicoR-GFP (Addgene). CMV-Flag was PCR amplified using primers CMV F and R, digested by SalI and EcoRI, and ligated into pSicoR-GFP. The CMV-mTCTP synonymous mutant construct was generated by a two-step PCR-based mutagenesis procedure using pCMV-2xFlag-TCTP as the template. Firststep PCR was used to amplify two partially overlapping fragments using primers TCTP-m 5’-plus TCTP R and TCTP-m F plus TCTP-m 3’. Both fragments were annealed and used as the template for second-step PCR. Second-step PCR with primers TCTP-m F and TCTP R was used to amplify the full length of mutant TCTP. The resulting mutant second-step PCR product was cut by BamHI and XhoI and ligated into pLove-Flag to obtain the synonymous mutant CMV-mTCTP. pLove-Red-C1, which contains a lentiviral vector backbone, was constructed by subcloning the CMV promoter and encoding the region of RFP of pDsRed-C1 into the XhoI and EcoRI sites of pSicoR-GFP (Addgene). CMV-RFP was PCR amplified using RFP F and R, digested by SalI and EcoRI, and ligated into pSicoR-GFP. Red-LC3B recombinant was constructed by subcloning the encoding region of human L3CB into the BamHI and XhoI sites of pLove-Red-C1. Human LC3B cDNA was PCR amplified using LC3B F and R, digested by BamHI and XhoI, and then ligated into pLove-Red-C1. The Nurim-EGFP recombinant was constructed by cloning the


Peptide mixtures were analyzed by MALDI-TOF/TOF MS (Bruker-Daltonics AutoFlex TOF-TOF LIFT Mass Spectrometer, Bruker-Daltonics) or LC-ESI-MS/MS (LTQ, Thermo Finnigan, San Jose, CA). Proteins were identified using the Mascot software (http://www.matrixscience.com, last accessed June 18, 2014) utilizing the National Center for Biotechnology Information (NCBI) nr database or BioWorks software utilizing the NCBI Suina protein database. Valid identification required a protein score greater than 65 when peptide mixtures were analyzed using MALDI-TOF/TOF MS, and two or more peptides independently matching the same protein sequence when peptide mixtures were analyzed using LC-ESI-MS/MS.

Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) and incubated with the antibody for 1 h at room temperature, followed by the horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized by incubating membranes with ECL Plus detecting reagents. The following antibodies were used: Anti-DRP2 antibody (#9393), anti-LC3B antibody (#2775), and antiphospho-AMPKa (Thr172) (#2535) purchased from Cell Signaling (Pickering, ON); antiAMPKa antibody (#1596-1), and anti-TCTP antibody (#53041) purchased from Epitomics (Burlingame, CA); anti-TCTP antibody (H00007178-M01) purchased from Abnova (Abnova Corporation, Taipei); anti-hnRNPA1 antibody (11176-AP) and anti-PDIA3 antibody (15967-1-AP) purchased from Proteintech Group (PTGLAB, Chicago, IL); anti-Hsp60 antibody (sc-1052) and anti-Vimentin antibody (sc-7558) purchased from Santa Cruz Company (Santa Cruz, CA); anti-GAPDH antibody (CW0100) purchased from Beijing CWBIO (CWBIO, Peking, China); anti-Myc (11667149001; 1:5,000) from Roche (Roche Applied Science, Indianapolis, IN); anti-Flag (F3165; 1:5,000) from Sigma (St Louis, MO); and goat antimouse IgG horseradish peroxidase (HRP)-linked whole antibody (SA1-74039) and goat antirabbit IgG HRP-linked whole antibody (SA1-9510) purchased from Pierce Company (Rockford, IL).


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coding region of human Nurim cDNA into the HindIII and SalI sites of pEGFP-N1. The wild-type Nurim cDNA was PCR amplified using primers Nurim F and R. psicoR-Nurim-EGFP, which contains a lentiviral vector backbone, was constructed by subcloning the CMV promoter and Nurim-EGFP coding region of the Nurim-EGFP plasmid into the XbaI and NotI sites of pLove-Flag. Nurim-EGFP was PCR amplified using primers CMV F and EGFP-N R, digested by XbaI and NotI, and ligated into pLove-Flag. Human ATG5 cDNA was PCR amplified using ATG5 F and R, digested by BamHI and XhoI, and then ligated into pCMV-myc. Mouse ATG6 cDNA was amplified using ATG6 F and R, digested by EcoRI and XhoI, and then ligated into pEGFP-myc. Human ATG7 cDNA was amplified using ATG7 F and R, digested by EcoRI and SalI, and then ligated into pCMV-myc. Human ATG8 cDNA was amplified using ATG8 F and R, digested by BamHI and XhoI, and then ligated into pCMV-myc. Human ATG12 cDNA was amplified using ATG12 F and R, digested by BamHI and XhoI, and then ligated into pCMV-myc or pEGFP-myc. Mouse ATG16L1 cDNA was amplified using ATG16L1 F and R, digested by MunI and SalI, and then ligated into pEGFP-myc. Mouse p62 cDNA was PCR amplified using p62 F and R, digested by EcoRI and XhoI, and then ligated into pEGFP-myc. Porcine TCTP introns and 3’-region were PCR amplified using genomic DNA from the domestic or wild pigs and primers (TCTP-intron-2-F and R for forward direction cloning, TCTP-Intron-2R-F and R for reverse direction cloning of the intron 2, or TCTP-3-region-F and R for 3’-region cloning). PCR products were digested with XhoI and BamHI and ligated into the XhoI and BamHI-digested sites of pGL4-promoter vector containing a minimal TATA promoter and the luciferase reporter gene (Promega). TCTP-specific (miR-TCTP), APMKspecific (miR-AMPK), or control (miR-lacZ) miRNA target sequence was synthesized and cloned into pLenti6.4MSGW/EmGFP-miR containing EGFP (Invitrogen, Carlsbad, CA). The target sequences for miRNA TCTP, AMPK, or LacZ were as follows: miR-TCTP, 5’-CGACATCTACAAGATCCG GGA-3’; miR-AMPK-1#, 5’-GGAAGTTCTCAGCTGTCTT-3’; miR-AMPK-2#, 5’-GAAGATATGTGATGGGATC-3’; and miRLacZ, 5’-GACTACACAAATCAGCGATTT-3’. PCR conditions and primers for the creation of these constructs are provided in supplementary table S1, Supplementary Material online. All constructs were verified by sequencing.

appropriate antibodies. Antibodies used for the Co-IP are as follows: Anti-TCTP antibody (H00007178-M01; Abnova Corporation); anti-ATG16 antibody (#8089; Pickering); antiATG5 antibody (AP1812a; Abgent, San Diego, CA); anti-LC3Bantibody (#2775; Pickering); anti-Myc (11667149001; Roche Applied Science); and anti-Flag (F3165; Sigma).

To analyze protein interactions, coimmunoprecipitation assay was performed in 293T cells. Cells were lysed in NETN buffer consisting of 50 mM Tris–HCl at pH 8.0, 0.15 M NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% NP-40, and a 1 protease inhibitor cocktail (Roche). Cell lysates were incubated with specified antibody and Protein G Agarose (Roche) overnight at 4 C. The resins were collected by centrifugation and then washed four times with NETN buffer. Bound proteins were eluted by loading buffer (3% SDS, 1.5% mercaptoethanol, 8% glycerol, 0.01% Coomassie blue G-250, 150 mM Tris–HCl, pH 7.0), separated by SDS–PAGE, followed by immunoblotting with the

Mouse ovary samples were cryosectioned. The sections were immediately fixed with methanol for 20 min at 20 C and then permeabilized with 0.1% Triton X-100 in PBS for 10 min. After treating the samples with 5% BSA for 20 min at room temperature, the sections or glass coverslides were incubated with primary antibody (anti-TCTP or anti-LC3B) overnight at 4 C. After washing with PBS, the cells were subjected to indirect immunofluorescence using FITC-labeled or Cy3-labeled goat antirabbit IgG (CWBIO, Peking, China). The nuclei were stained with Hoechst33258. Images were taken with a confocal fluorescence microscope.

Luciferase Activity Assays HEK293T or COS-7 cells were grown in 24-well plates to 70– 80% confluence and transfected with various plasmid combinations. Approximately 36 h after transfection, the expression of firefly and Renilla luciferase was measured by the Dual-Luciferase Reporter Assay system (Promega).

Chromatin Immunoprecipitation Ovary samples (~40 mg) from domestic or wild pigs were cut into small pieces, which were crosslinked with 1% formaldehyde/PBS for 15 min at room temperature, then homogenized into single-cell suspension. The cell suspension was collected and lysed in 1 ml cold lysis buffer (10 mM Tris– HCl, pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40) supplemented with protease inhibitors (Roche) and incubated at 4 C for 5 min to allow the release of the nuclei. The cell nuclei were lysed with lysis buffer (10 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.1% sodium deoxycholate) and sonicated to shear crosslinked DNA. After centrifugation, the supernatant chromatin was immunoprecipitated with 10 mg of Oct1 antibody (Proteintech) or IgG. The beads were washed five times with lysis buffer. The cross-linking was reversed in lysis buffer at 65 C overnight. The ChIP DNAs were subjected to real-time PCR amplification using the primers specific for the TCTP intron 2 or exon 6 (supplementary table S1, Supplementary Material online).

Primary Culture of Granulosa Cells and Isolation of Cytosolic and Nuclear Proteins Adult female mice were injected with 10 U of PMSG and sequentially injected with HCG after 2 days. Oviducts were removed 24 h later. Granulosa cells were stripped from the follicles by incubation with hyaluronidase. The isolated granulosa cells were grown in Dulbecco’s modified Eagle’s medium containing 10% FBS. Cell culture supernatants were collected for western blotting. 2209


Coimmunoprecipitation Assay

Immunofluorescence Analysis

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Granulosa cells grown in a 4-cm2 plate were rinsed with PBS and treated with 0.25% trypsin solution. The cells were pelleted by centrifugation (1,000 g, 5 min) and resuspended in 100 ml of buffer A (10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 5 mM MgCl2, 1 mM DTT, protease inhibitor cocktail) for 20 min on ice, followed by the addition of 5 ml buffer B (10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 5 mM MgCl2, 1 mM DTT, protease inhibitor cocktail, 10% IGEPAL CA-630). The lysates were vigorously mixed and centrifuged for 5 min (500 g, 4 C). The cytoplasmic proteins were present in the supernatant. To extract the nuclear proteins, the nuclear pellet was resuspended in 300 ml of buffer A for 10 min at 4 C. After vigorous mixing, the nuclear suspension was centrifuged for 5 min at 4 C.

The miR-TCTP, miR-AMPK, miR-lacZ, psicoR-Red-LC3B (encoding Red-LC3B), pLove-Nurim-EGFP, and CMVmTCTP (TCTP synonymous mutant) constructs were transfected into HEK293T cells in combination with lentiviral packaging vectors pRSV-Rev, pMD2.G, and pCMV-VSV-G, respectively, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Viruses were collected 60 h after transfection. Viral supernatant from a 12-well plate was used to infect approximately 103 COS-7 cells for 48 h.

Cell Treatment with Hypoxia and Starvation COS-7 cells were cultured under normal condition (20% O2, DMEM plus 10% FBS), that is, normoxic condition. Hypoxic treatment was performed under 1% O2 with DMEM plus 10% FBS for 24 h or the cells were cultured in starvation medium minimum essential medium/Earle’s balanced salts (MEMEBSS) for 24 h.

Electron Microscopy COS7 cells transfected with EGFP-LC3B were cultured in MEM-EBSS for 24 h fixed with 4% paraformaldehyde for 1 h in 0.1 M sodium phosphate buffer (pH7.4) and washed for 5 min three times in PBS. Cells were permeabilized and blocked for 30 min with 0.2% saponin, 10% BSA, and 10% normal goat serum. COS-7 cells were stained with anti-GFP antibody overnight at 4 C, washed for 10 min six times in PBS containing 0.1% saponin, then stained for 2 h at room temperature with an antimouse IgG conjugated to 10-nm gold particle (G7777, Sigma), washing for 10 min five times in PBS containing 0.1% saponin and for another 10 min without saponin. The cells were fixed in 2.5% glutaraldehyde overnight at 4 C and postfixed in 0.5% osmium tetroxide for 30 min. The cells were then dehydrated, infiltrated with plastic, embedded in plastic, sectioned at a thickness of 80 nm, stained with 6% uranyl acetate and Reynolds lead citrate, and analyzed with a Tecnai G220 transmission electron microscope (FEI Company, Hillsboro, OR). 2210

Supplementary figures S1–S4 and tables S1–S4 are available at Molecular Biology and Evolution online (http://www.mbe. oxfordjournals.org/).

Acknowledgments This work was supported by the National Natural Science Foundation of China, the National Key Basic Research project of China, and the Key Transgenic New Organism Project.

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Artificial selection reveals the energetic expense of producing larger eggs.

Emerging role of mammalian autophagy in ketogenesis to overcome starvation.

Methods for the Detection of Autophagy in Mammalian Cells.

Regulation of autophagy by glucose in Mammalian cells.

Selection and coamplification of heterologous genes in mammalian cells.

Selection of mammalian cells resistant to a chloramphenicol analog.

Mmi1, the yeast homologue of mammalian TCTP, associates with stress granules in heat-shocked cells and modulates proteasome activity.

Transfer of yeast artificial chromosomes from yeast to mammalian cells.

LC3 family in autophagy: differential and compensatory roles in the spatiotemporal regulation of autophagy.

A human artificial chromosome recapitulates the metabolism of native telomeres in mammalian cells.

Molecular complementation of a collagen mutation in mammalian cells using yeast artificial chromosomes.

Selection on synonymous codons in mammalian rhodopsins: a possible role in optimizing translational processes.

Mammalian artificial chromosomes.

Ultrastructural analysis of autophagosome organization using mammalian autophagy-deficient cells.

Role of autophagy in cisplatin resistance in ovarian cancer cells.

Autophagy and mammalian development.

A novel platform for antibody library selection in mammalian cells based on a growth signalobody.

A systems biology investigation of neurodegenerative dementia reveals a pivotal role of autophagy.

Dual role of autophagy in lipopolysaccharide-induced preodontoblastic cells.

Artificial Selection of Gn1a Plays an Important role in Improving Rice Yields Across Different Ecological Regions.

Artificial selection on relative brain size reveals a positive genetic correlation between brain size and proactive personality in the guppy.

Transfer of a yeast artificial chromosome carrying human DNA from Saccharomyces cerevisiae into mammalian cells.

A novel sgRNA selection system for CRISPR-Cas9 in mammalian cells.

Selection at molecular level in mammalian myoglobins.