Knockdown of Y-box binding protein 1 induces autophagy in early porcine embryos

Y-box binding protein 1 (YBX1) plays important roles in RNA stabilization, translation, transcriptional regulation, and mitophagy. However, its effects on porcine preimplantation embryos remain unclear. In this study, we knocked down YBX1 in the one-cell (1C) stage embryo via small interfering RNA microinjection to determine its function in porcine embryo development. The mRNA level of YBX1 was found to be highly expressed at the four-cell (4C) stage in porcine embryos compared with one-cell (1C) and two-cell (2C) stages. The number of blastocysts was reduced following YBX1 knockdown. Notably, YBX1 knockdown decreased the phosphatase and tensin homolog-induced kinase 1 (PINK1) and parkin RBR E3 ubiquitin protein ligase (PRKN) mRNA levels. YBX1 knockdown also decreased PINK1, active mitochondria, and sirtuin 1 levels, indicating reduced mitophagy and mitochondrial biogenesis. Furthermore, YBX1 knockdown increased the levels of glucose-regulated protein 78 (GRP78) and calnexin, leading to endoplasmic reticulum (ER) stress. Additionally, YBX1 knockdown increased autophagy and apoptosis. In conclusion, knockdown of YBX1 decreases mitochondrial function, while increasing ER stress and autophagy during embryonic development.


Introduction
A mitochondrion is an essential organelle that controls energy conversion and ATP production (Matsumoto et al., 2012).Mitochondrial fission and fusion maintain the mitochondrial morphology, homeostasis, and inheritance via mitochondrial biogenesis and mitophagy (Ma et al., 2020).Mitophagy is crucial for mitochondrial quality control (Dlamini et al., 2021).Dysfunctional mitochondria can generate apoptotic signals to induce cell death (Zhang, 2013).Endoplasmic reticulum (ER) is another important organelle which coordinates stress-related signaling pathways critical for maintaining the crosstalk between intracellular and extracellular environments (Lin et al., 2008;Ron and Hubbard, 2008;Kuo et al., 2022).Excess unfolded or misfolded proteins accumulate in the ER lumen leading to ER stress, disrupting ER function, thereby activating the unfolded protein response (UPR) (Senft and Ronai, 2015).ER and mitochondria together regulate various cellular processes, for example, lipid biosynthesis, apoptosis, and mitophagy (Dlamini et al., 2021).
In this study, we hypothesized that YBX1 is crucial for porcine embryonic development.To evaluate this hypothesis, we reduced the expression of YBX1 by microinjection of YBX1 siRNA to explore the role of YBX1 during the early development of porcine embryos.Additionally, we examined mitochondrial function, GRP78, calnexin, LC3, and caspase 3 following YBX1 knockdown.The results suggest that YBX1 affects embryonic development by influencing mitochondrial function, ER stress, autophagy, and apoptosis.

Material and methods
Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States of America).

Parthenogenetic activation and in vitro culture
Briefly, 1 mg/mL hyaluronidase was used to detach the cumulus cells.Oocyte with a polar body was selected for activation.Denuded oocytes were parthenogenetically activated using two direct-current pulses of 110 V for 60 µs in 280 mM mannitol containing 0.1 mM CaCl 2 , 0.05 mM MgSO 4 , 0.01% polyvinyl alcohol (PVA, w/v), and 0.5 mM 4-(2-hydroxyethyl) piperazine-l-ethanesulfonic acid.After that, activated oocytes were cultured with 0.4% bovine serum albumin (BSA) and 7.5 μg/mL cytochalasin B in PZM-5 for 3 h to inhibit pseudo-second polar body extrusion.Activated oocytes were washed 3 times and cultured in PZM-5 with 0.4% BSA in a 4well plate under the same condition as IVM.Subsequently, embryos were cultured for 7 days, and then examined the blastocyst development rate (blastocyst development rate = number of blastocysts/number of embryos).

Microinjection
For the knockdown groups, the sequences of the small interfering RNAs (siRNAs) was the same with previous study (Jiang et al., 2023b).YBX1 siRNA (50 μM) was microinjected into the cytoplasm of a parthenogenetically activated oocyte via an Eppendorf Femto-Jet (Eppendorf, Hamburg, Germany) and Nikon Diaphot Eclipse TE300 inverted microscope (Nikon, Tokyo, Japan) equipped with the Narishige MM0-202N hydraulic 3-dimensional micromanipulator (Narishige, Amityville, NY, United States).As a control, the company provided negative siRNA (sense: UUCUCCGAACGU GUCACGUTT, antisense: ACGUGACACGUUCGGAGAATT) was microinjected into the cytoplasm of a parthenogenetically activated oocyte under the same conditions.Embryos were cultured in PZM-5 medium for 1 or 2 or 7 d after microinjection.

Colocalization assay of mitochondria and TOM20
For the colocalization of mitochondria and TOM20, 4-cell stage embryos were incubated with 500 nM MitoTracker Red CMXRos (M7512; Thermo Fisher Scientific) at 38.5 °C for 30 min.After 3 washes with PZM-5, the staining of TOM20 was the same as in the immunofluorescence staining.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
RNA was extracted from a pool of 30 embryos per group (control group and YBX1 knockdown group) using the Dynabead mRNA DIRECT kit (61,012; Thermo Fisher Scientific).cDNA was synthesized using a cDNA synthesis kit (Thermo Fisher Scientific), according to the manufacturer's instructions.Quantitative reverse transcription-PCR was performed using a fast real-time PCR system (ABI StepOnePlus).Real-time quantitative PCR (qPCR) was performed according to previous article (Jiang et al., 2023a).18S rRNA was used as the reference gene.All primer sequences used in this study are listed in Table 1.Relative genes expression levels were determined using the 2 −ΔΔCT method.

Statistical analysis
Each experiment was repeated at least three times and results were presented as the mean ± standard error of the mean.The GraphPad Prism 5 software (GraphPad, San Diego, CA, United States of America) was used for statistical analysis.A t-test was used to compare the results between groups.p < 0.05 was considered statistically significant.3 Results

Subcellular distribution and expression of YBX1 during embryo development
To investigate its subcellular localization during embryonic development, we performed immunofluorescence staining to determine the location of YBX1 in two-cell (2C; n = 6), four-cell (4C; n = 6), morula (MO; n = 5) and blastocyst (BL; n = 6) stage embryos.As shown in Figures 1A,B, YBX1 localized in the cytoplasm and the immunofluorescence (IF) intensity of YBX1 was gradually increased.Next, we examined the mRNA levels of YBX1 during embryonic development.We observed that the mRNA levels of YBX1 were increased from the 4C to BL stage compared with the 1C and 2C stages (Figure 1C), indicating that YBX1 is a zygotic gene.Moreover, the results of Western blotting were similar to those of IF and real-time quantitative PCR.Taken together, these data indicate the presence of YBX1 in porcine embryos, which may play a key role in embryonic development.

Discussion
In this study, we found that the loss of YBX1 decreased PINK1 and PRKN expression levels, thereby decreasing mitochondrial function and increasing ER stress autophagy as well as apoptosis during embryonic development (Figure 6).
Mitophagy is the selective degradation of mitochondria via autophagy (Dlamini et al., 2021).It usually occurs in mitochondria that are defective after injury or stress.
Mitophagy promotes the renewal of mitochondria and prevents the accumulation of dysfunctional mitochondria, which may lead to cellular degeneration (Wang et al., 2022).Mitophagy is activated in mammalian cells by PINK1/Parkinmediated mitophagy (Iorio et al., 2021).PINK1 affects the health of mitochondrial (Nguyen et al., 2016).PINK1 activates Parkin via phosphorylation, which is an E3 ubiquitin ligase located in the cytoplasm (Bingol and Sheng, 2016;Wang et al., 2022).YBX1, a positive regulator of mitophagy, by enhancing PINK1/PRKNmediating mitophagy in brown adipocyte (Wu et al., 2022).In this study, YBX1 knockdown decreased PINK1 and PRKN mRNA levels and reduced PINK1 protein levels, indicating that YBX1 may regulate their mRNA stability and protein expression to influence mitophagy.SIRT1 is a marker of mitochondrial biogenesis (Majeed et al., 2021).Previous studies have shown that SIRT1 has emerged as an important regulator of mitochondrial function (Jian et al., 2012;Li et al., 2017;Niu et al., 2020).MitoTracker Red CMXRos was used to detect mitochondrial activity.It has been shown that decreased mitochondrial activity decreases mitochondrial function (Niu et al., 2019;Niu et al., 2020).Additionally, in aged oocytes, decreased MitoTracker Red were accompanied by decreased PINK1 and PRKN (Niu et al., 2020).Here, knockdown of YBX1 significantly reduced SIRT1 expression and mitochondrial activity, indicating that YBX1 is important for mitochondrial biogenesis.Therefore, YBX1 is crucial for mitophagy and mitochondrial biogenesis.
ER serves as a crucial organelle involved in the biosynthesis of lipids, proteins, and secreted proteins as well as an important site of calcium homeostasis (Lin et al., 2019).ER stress is triggered by the accumulation of unfolded or misfolded proteins in the ER that induce UPR (Feldman et al., 2005).UPR is mainly regulated by three sensors, including BiP (also known as GRP78) (Ghemrawi et al., 2018;Song et al., 2018).ER-mitochondrial play important roles in regulating the mitochondrial dynamics, inflammation, autophagy, and apoptosis.Therefore, we examined ER function in porcine embryos.The results indicated that YBX1 knockdown increased the expression levels of calnexin and GRP78, inducing ER stress.Subsequently, increased ER stress induced UPR.Previous article indicated that YBX1 depletion induces UPR (Sun et al., 2018), which is consistent with our finding.
Autophagy is a crucial cellular response to stress that degrades and removes unfolded proteins and damaged organelles to protect the cells (Yu et al., 2004;Dikic and  Elazar, 2018).As mitophagy and ER stress induce autophagy and apoptosis, we determined the expression levels of their markers, LC3 and caspase 3 during embryonic development.We found that YBX1 knockdown increased LC3 protein level.In cancer cells and preadipocytes, YBX1 knockdown reduces LC3 protein expression (Wu et al., 2022;Gong and Zhang, 2023;Wu et al., 2023).In this study, LC3 mRNA levels were decreased but protein expression was increased, contrary to the results, which may be due to the uniqueness of different cells or tissues.Additionally, YBX1 has been identified as an RNA-binding protein and a DNAbinding protein, mainly involved in translational repression, RNA stabilization, and transcriptional regulation.One study showed that overall translation levels were increased in YBX1depleted embryos (Sun et al., 2018), which may lead to increased LC3 protein levels.GRP78 is important for both ER stress and autophagy (Li et al., 2008).PINK1 knockdown increases autophagy and apoptosis (Niu et al., 2019).Here, YBX1 knockdown increased GRP78 level and decreased PINK1 level, thereby increasing autophagy.In addition, YBX1 knockdown has been reported to upregulate the levels of apoptosis-related genes, such as FAS, tumor necrosis factor, and caspase (Kloetgen et al., 2020).One study reported that oxygen-glucose deprivation/ reoxygenation downregulates YBX1 expression, whereas YBX1 overexpression attenuates growth inhibition and apoptosis in PC12 cells (Tuerxun et al., 2021).However, the mRNA levels of BCL2 were elevated after YBX1 knockdown, contrary to the study by Feng et al. (Feng et al., 2021).The elevation of BCL2 may be due to cellular self-protection, which inhibits apoptosis and autophagy.BCL-x1 is a member of the Bcl-2 family of proteins, which are anti-apoptotic proteins.A previous study showed that apoptosis was increased but the mRNA levels of BCL-x1, BAX, and caspase-3 were decreased (Zhang et al., 2020).In addition, it was found that Bcl-2 and Bcl-xL enhance autophagy under certain conditions, such as in response to treatment with etoposide and staurosporine (Fan and Zong, 2013).Moreover, YBX1 is a DNA/RNA-binding protein that affects transcription and translation.The levels of transcription and translation are not exactly the same; therefore, an increase in BCL2 mRNA level does not mean that the protein is also elevated.Therefore, it is possible that BCL2 is increased in YBX1 knockdown embryos.These findings indicate that YBX1 knockdown induces autophagy and apoptosis.Taken together, our results indicate that YBX1 decreases PINK1 and PRKN expression levels and mitochondrial function and induces ER stress, thereby causing autophagy and apoptosis during embryonic development.

FIGURE 2
FIGURE 2 YBX1 knockdown impairs embryo development.(A) Real-time quantitative PCR results of YBX1 mRNA expression levels in the control and YBX1 KD groups.Compared with the control group, the expression level of YBX1 mRNA was significantly lower in the YBX1 KD group.(B) YBX1 knockdown was confirmed via Western blotting.(C) Relative YBX1 protein intensity after YBX1 knockdown.(D) Immunofluorescence staining of YBX1 in the control and YBX1 KD groups at the four-cell stage.Blue, DNA; green, YBX1.Scale bar, 20 µm.(E) Relative fluorescence intensity of YBX1 at the four-cell stage.The relative fluorescence intensity of YBX1 was significantly lower in the YBX1 KD group at the 4C stage compared to the control group.(F) Blastocyst development rate after YBX1 knockdown.(G) Total cell number/blastocyst after YBX1 knockdown.*p < 0.05, **p < 0.01 indicate significant differences between groups.

FIGURE 3
FIGURE 3 YBX1 knockdown impairs mitochondrial function.(A) Immunofluorescence staining of PINK1 in the control and YBX1 KD groups at the four-cell and blastocyst stages.Blue, DNA; red, PINK1.Scale bar, 20 µm.(B) Relative fluorescence intensity of PINK1 at the four-cell stage.The relative fluorescence intensity of PINK1 was significantly lower in the YBX1 KD group at the 4C stage compared to the control group.(C) Relative fluorescence intensity of PINK1 at the blastocyst stage.The relative fluorescence intensity of YBX1 was significantly lower in the YBX1 KD group at the BL stage compared to the control group.(D) Relative mRNA expression of PINK1 at the blastocyst stage.(E) Relative mRNA expression of PRKN at the blastocyst stage.(F) Protein levels of SIRT1 in the control and YBX1 KD groups.(G) Immunofluorescence staining of TOM20, Mito Tracker in the control and YBX1 KD groups at the four-cell stage.Blue, DNA; Green, TOM20; Red, Mito Tracker; Scale bar, 20 µm.(H) Relative fluorescence intensity of Mito Tracker at four-cell stage.Compared with the control group, the relative fluorescence intensity of Mito Tracker in 4C stage in the YBX1 KD group was significantly lower.*p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences between groups.

Figure 5I )
Figure5I) were significantly increased.According to the above results, YBX1 knockdown induces autophagy and apoptosis.

FIGURE 4
FIGURE 4 YBX1 knockdown induces ER stress.(A) Immunofluorescence staining of GRP78 in the control and YBX1 KD groups at the four-cell stage.Blue, DNA; red, GRP78.Scale bar, 20 µm.(B) Relative fluorescence intensity of GRP78 at the four-cell stage.Compared with the control group, the relative fluorescence intensity of GRP78 in 4C stage in the YBX1 KD group was significantly higher.(C) Immunofluorescence staining of calnexin in the control and YBX1 KD groups at the four-cell stage.Blue, DNA; red, calnexin.Scale bar, 20 µm.(D) Relative fluorescence intensity of calnexin at the four-cell stage.Compared with the control group, the relative fluorescence intensity of calnexin in 4C stage in the YBX1 KD group was significantly higher.(E) Protein levels of calnexin and GRP78 in the control and YBX1 KD groups.(F) Relative protein levels of calnexin and GRP78.*p < 0.05 and ***p < 0.001 indicate significant differences between groups.

FIGURE 5
FIGURE 5 YBX1 knockdown induces autophagy and apoptosis.(A) Immunofluorescence staining of LC3 in the control and YBX1 KD groups at the four-cell stage.Blue, DNA; red, LC3.Scale bar, 20 µm.(B) Relative fluorescence intensity of LC3 at the four-cell stage.Compared with the control group, the relative fluorescence intensity of LC3 in 4C stage in the YBX1 KD group was significantly higher.(C) Immunofluorescence staining of LC3 in the control and YBX1 KD groups at the blastocyst stage.Blue, DNA; red, LC3.Scale bar, 20 µm.(D) Relative fluorescence intensity of LC3 at the blastocyst stage.Compared with the control group, the relative fluorescence intensity of LC3 in BL stage in the YBX1 KD group was significantly higher.(E) Relative mRNA expression of LC3 at the four-cell stage.(F) Relative protein levels of LC3 in the control and YBX1 KD groups.(G) Immunofluorescence staining of caspase 3 in the control and YBX1 KD groups at the blastocyst stage.Blue, DNA; red, caspase 3. Scale bar, 20 µm.(H) Relative fluorescence intensity of caspase 3. Compared with the control group, the relative fluorescence intensity of Caspase 3 in blastocyst stage in the YBX1 KD group was significantly higher.(I) Relative mRNA expression of Caspase3, BAX and BCL2 at the four-cell stage.*p < 0.05, **p < 0.01 and ***p < 0.001 indicate significant differences between groups.

FIGURE 6
FIGURE 6YBX1 is important for embryonic development in pigs.YBX1 knockdown decreases PINK1 and PRKN levels and affects mitochondrial function, thereby inducing ER stress, autophagy, and apoptosis.

TABLE 1
Information of primers used for RT-PCR.
Note: The annealing temperature for all reactions was 60 °C.F: forward primer; R: reverse primer.