Introduction

Autophagy is a system that degrades unnecessary intracellular components, including misfolded and aggregated proteins as well as damaged organelles1,2. This process plays a crucial role in maintaining cytoplasmic homeostasis and supplying nutrients by recycling degradation products under various metabolic conditions, including starvation. Upon autophagy induction, an isolation membrane forms and engulfs the target components, leading to the formation of an autophagosome. The autophagosome subsequently fuses with a lysosome to form an autolysosome, where the contents are degraded by hydrolytic enzymes3,4. The resulting degradation products are then released into the cytoplasm and utilized for new protein synthesis and other cellular functions5. Autophagy is generally regulated according to cellular nutrient levels through the mechanistic target of rapamycin (mTOR)6. Under nutrient-rich conditions, mTOR is activated and phosphorylates the ULK1 complex, an initiator of autophagy, thereby inhibiting autophagy. In contrast, under nutrient-deprived conditions, mTOR becomes inactive, allowing the ULK1 complex to be phosphorylated by AMPK, which triggers autophagy. The activated ULK1 complex subsequently activates the Class III PI3K complex, promoting the elongation of the isolation membrane and facilitating the formation of autophagosomes7.

Autophagy is essential for preimplantation development in mice8,9. Autophagy is deeply involved in this process, as its deficiency has been reported to inhibit protein synthesis, leading to developmental arrest at the 4/8-cell stages, which is partially rescued by supply amino acids (AAs)8. Interestingly, autophagy is suppressed in oocytes by an unknown mechanism but is activated after fertilization9.These findings suggest that the primary role of autophagy in preimplantation embryos is to supply AAs for protein synthesis. Moreover, the mRNA levels of autophagy-related genes are high immediately after fertilization and during early development but gradually decrease as development progresses, a pattern observed in multiple species10,11,12.

At the 8-cell stage, blastomeres transition from a loosely assembled structure of spherical cells to a tightly connected form through a process called “compaction”, leading to the formation of the morula stage embryo13. Following division into 16 cells, two distinct cell populations emerge: outer cells with polarity and inner cells lacking polarity14. This polarity regulates Hippo signaling, which determines cell fate toward either the trophectoderm (TE) or inner cell mass (ICM)15. In outer cells, cell polarity inhibits Hippo signaling, allowing nuclear translocation of YAP and subsequent expression of Cdx2, leading to differentiation into TE. Conversely, in inner cells lacking cell polarity, Hippo signaling is active, leading to phosphorylation of YAP, which prevents its nuclear translocation and promotes differentiation into the ICM.

Previous studies on autophagy at later stages of preimplantation after the 8-cell stage have employed chemical inhibitors such as rapamycin (an autophagy activator through mTOR inhibition) and 3-methyladenine (3-MA, an autophagy inhibitor)10. The addition of these compounds to culture media has been reported to affect blastocyst formation rates and cell differentiation into the TE and ICM and. Furthermore, multiple studies have shown that autophagy activity reflects embryo quality, and autophagy regulation through various pathways improves developmental rates and other outcomes16,17,18,19,20. Studies using embryoid body models lacking Atg5 or Beclin1 have demonstrated that autophagy is necessary for the clearance of apoptotic cells and that its inhibition impairs embryoid body cavitation21. However, despite these studies, the detailed roles and regulation of autophagy in the late stages of preimplantation development remains largely unclear.

The aim of this study is to elucidate the role of autophagy during preimplantation development. Chloroquine (CQ) is an FDA-approved drug widely used in research and clinical trials22,23,24. In autophagy, CQ is known to block the fusion of autophagosomes with lysosomes25,26. In this study, we elucidated the developmental stages at which autophagy is required and its role during preimplantation development using CQ as well as the dynamics of autophagy and its regulatory mechanisms in the late stages of preimplantation development.

Results

Stage-specific effects of chloroquine-mediated autophagy inhibition on embryonic development

First, we observed changes in autophagic activity during mouse preimplantation development using the autophagy detection probe DAPGreen. DAPGreen is incorporated into the double membrane during autophagosome formation and fluoresces in response to the lipid environment, thereby labeling autophagosomes and autolysosomes27. From the zygote to the 4-cell stages after IVF, DAPGreen fluorescence was detected throughout the cytoplasm (Fig. 1a), consistent with previous studies8. After compaction, autophagic activity varied among individual cells, and larger fluorescent granules were observed compared to earlier stages. Fluorescence signals were observed in the outer cells at the morula stage and only in the TE at the blastocyst stage.

Fig. 1
figure 1

Stage-specific effects of CQ-induced autophagy inhibition on embryonic development. (a) DAPGreen staining detected autophagosomes and autolysosomes from 2cell to blastocyst stage. Top: DAPGreen fluorescence images. Bottom: Merged bright-field and DAPGreen fluorescence images. White circles indicate ICM. Scale bars represent 30 μm. (b) The thick bars in the left panel indicate CQ treatment at various stages, while the right graph shows the corresponding blastocyst rates. Tukey–Kramer’s HSD test, significance level P < 0.05. (c) Developmental results are shown at 96 h post-IVF are shown at various CQ treatment time points, as depicted in (b).

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Next, to clarify the role of autophagy in mouse preimplantation development, we treated IVF embryos with 0 to 50 µM CQ and assessed the blastocyst formation rate. The rate of blastocyst formation decreased in a dose-dependent manner. Under CQ treatment at 10 µM, a concentration equivalent to genetic autophagy inhibition in cancer cells28, all embryos arrested at the 4 and 8 (4/8)-cell stages, similar to Atg5-null embryos8, suggesting that CQ treated embryos could mimic autophagy-deficient embryos (Table 1). To confirm that CQ indeed inhibited autophagy in our system, we quantitatively analyzed the size of autophagosomes in CQ-treated embryos. We observed a significant enlargement of autophagosomes compared to untreated controls, consistent with effective inhibition of autophagic flux by CQ (Fig. S1). We then exposed embryos to 10 µM CQ at different developmental stages to evaluate its stage-specific effects as shown in Fig. 1b. Treatment from the zygote to the 2-cell stage did not significantly affect development; however, treatment from the zygote to the 4/8-cell stages, as well as from the 2-cell to the 4/8-cell stages, resulted in a significant reduction in developmental rates. Additionally, CQ treatment beyond the 4/8-cell stages led to a significant decrease in blastocyst formation at all time points, with embryos exhibiting lethality when treated from the 4/8-cell to the morula stage (Fig. 1b,c; Table 2). Importantly, in the "zygote to 4/8-cell" treatment group, CQ was removed at the 4/8-cell stage, allowing embryos to recover from autophagy inhibition and subsequently develop to the blastocyst stage. These findings suggest that the role of autophagy in preimplantation development is not limited to and is also crucial beyond the 4 and 8-cell stages.

Table 1 Developmental rates following 0–50 µM CQ treatment from 6 to 96 h after IVF.
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Table 2 Developmental rates following various time points of 10 µM CQ treatment.
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To further investigate the role of autophagy from the 4/8-cell to the morula stage, we treated embryos from the 4/8-cell stage onward with lower-dose CQ (2.5 µM), which still allows embryos to develop to the blastocyst stage, and performed immunostaining for Cdx2 (a TE marker) and Nanog (an ICM marker) to analyze cell numbers. At the 8 to 16-cell stage of the morula, total cell numbers remained unchanged across all treatment groups. However, in the 2.5 µM CQ treatment group, the proportion of Cdx2-positive cells was significantly reduced (0 µM: 68.75%, 2.5 µM: 47.61%) (Fig. S2). Furthermore, in the 5 µM treatment group, the expression levels of both Cdx2 and Nanog were significantly decreased with the reduced total cell number (Fig. S2b,c). At the blastocyst stage, the 2.5 µM treatment group showed no significant change in cell number, while the treated embryos showed a tendency toward reduced Cdx2-positive cell numbers but increased Nanog-positive cell numbers, even though the difference was not significant (Fig. S3b,c). On the other hand, the 5 µM treatment group exhibited a significant reduction in both Cdx2-positive and Nanog-positive cells, as well as in the total cell number (Fig. S3a–c). These results suggest that autophagy during the 4/8-cell to morula stage is involved in cell differentiation, particularly differentiation into TE cells.

Rescue of developmental defects by AA supplementation under autophagy inhibition

Since the primary function of autophagy is considered to be AA supply, we examined whether the developmental arrest caused by 10 µM CQ treatment from the 4/8-cell to morula stage could be rescued by AA supplementation in the culture medium. It should be noted that although CZB medium originally contains 0.5 mM glutamine (Table S1), this alone may not be sufficient to alleviate the potential amino acid deficiency associated with autophagy inhibition. Although the blastocyst formation rate was slightly reduced in CZB + AA, embryos cultured in 10 µM CQ + AA progressed to the blastocyst stage (Fig. 2a and b). These results indicate that AA supply through autophagy is essential not only during early preimplantation development but also in later stages following compaction.

Fig. 2
figure 2figure 2figure 2

Rescue of autophagy inhibition by AA supplementation. Developmental rates (a) and developmental results (b) following culture with 10 μM CQ treatment from the 4/8-cell stages to the blastocyst stage with/without AA supplementation. Scale bars represent 20 μm. (c) The embryos at the morula stage, cultured with CQ and AA as shown in panel ‘a,’ were immunostained for Cdx2 and Nanog, as well as with DAPI. Scale bars represent 50 μm. (d) The graph shows the numbers of DAPI-positive cells counted in the embryos, as shown in (c). Tukey–Kramer’s HSD test, CZB vs. CZB + AA + CQ p = 0.007, CZB + CQ vs. CZB + AA + CQ p = 0.0015. (e) The graph shows the proportion of Cdx2- and Nanog-positive cells per total cell number counted in the embryos, as shown in (c). Tukey–Kramer’s HSD test, Cdx2: CZB + CQ vs. CZB + AA + CQ p = 0.0012. Nanog: CZB vs. CZ + AA + CQ p = 0.0418, CZB + AA vs. CZB + CQ p = 0.0286, CZB + CQ vs. CZB + AA + CQ p = 0.0005. (f) The proportion of positive/negative cells in each embryo, as shown in (c). The cells are classified into four types: Nanog-positive (Nanog + , green), Cdx2-positive (Cdx2 + , red), double positive for Nanog and Cdx2 (Nanog + /Cdx2 + , yellow), and double negative (Nanog-/Cdx2-, gray). Tukey–Kramer’s HSD test, Cdx2-/Nanog-: CZB vs. CZB + CQ p = 0.0034, CZB + CQ vs. CZB + AA + CQ p = 0.0045. Cdx2 + /Nanog + : CZB vs. CZB + CQ p = 0.0035. (g) The embryos at the morula stage, cultured with CQ and AA as shown in (a), were immunostained for TFAP2C and Nanog, as well as with DAPI. Scale bars represent 50 μm. (h) The graph shows the proportion of TFAP2C- and Nanog-positive cells per total cell number counted in the embryos, as shown in (g). Tukey–Kramer’s HSD test, TFAP2C: no significant differences were observed. Nanog: CZB + CQ vs. CZB + AA + CQ p = 0.0322. (i) Relative fluorescence intensity of Nanog (green) and TFAP2C (red) in embryos shown in (g). Calibration was performed using DAPI fluorescence intensity.

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Next, to investigate the effects on cell differentiation, embryos at the 4/8-cell stage were treated with 2.5 µM CQ and AA for 24 h, and the resulting morulae were subjected to immunostaining. We used 2.5 µM CQ instead of 10 µM because embryos treated with 10 µM CQ showed severe morphological abnormalities, making it unsuitable for detailed analysis of cell differentiation. Immunostaining for Nanog and Cdx2 confirmed that, consistent with previous results (Fig. S2a,c), CQ treatment reduced the number of Cdx2-positive cells, but this reduction was rescued to levels comparable to CZB medium upon AA supplementation (Fig. 2c–e). Furthermore, CQ treatment increased the proportion of cells that did not express either Nanog or Cdx2, whereas AA supplementation reduced this proportion (CZB: 1.0%, CZB + CQ: 6.9%, CZB + CQ + AA: 1.2%) (Fig. 2f).

Additionally, we analyzed the expression of TFAP2C29, a transcription factor upstream of Cdx2, by immunostaining. TFAP2C was highly expressed in all experimental groups (Fig. 2g–i), but only in the CQ-treated group was a decrease in fluorescence intensity observed. This reduction was rescued by AA supplementation (Fig. 2j). These data suggest that autophagy is involved in differentiation into TE cells through AA supply, likely via protein degradation.

Spatial regulation of autophagy following embryo compaction

Since our findings suggested a role for autophagy in the morula stage and beyond, we analyzed autophagy dynamics from the 4-cell stage to the morula stage using a "dual fluorescence staining" method, which we developed in this study to distinguish new and old autophagosomes/autolysosomes in the same cells. DAPGreen and DAPRed share the same properties in marking autophagosomes with different fluorescent colors27. The accurate evaluation of autophagic activity requires the analysis of autophagic flux. This method enables its visualization and distinguishes new and old autophagosomes/autolysosomes, allowing the capture of the entire process from formation to degradation30.

First, we used the fluorescent probe DAPRed to stain autophagosomes induced at the 4-cell stage. DAPRed originally appears red which was converted to magenta in this study for higher contrast. Subsequently, embryos that had developed into the morula stage after 24 h were stained with DAPGreen to label newly formed autophagosomes. Confocal microscopy revealed autophagosomes distinguishably marked by magenta which was formed at the 4-cell stage, green which was formed at the morula stage, and white (overlapping signals from both stages), indicating the presence of both old and newly formed autophagosomes. DAPRed fluorescence from the 4-cell stage was distributed throughout the embryo at the morula stage, whereas DAPGreen fluorescence from the early morula stage was localized only to the outer cells of the embryo (Fig. 3a). Furthermore, particularly strong autophagy signals were observed in some outer cells, suggesting autophagosome accumulation in specific parts of embryos without degradation (Fig. 3a, arrow). Next, we performed dual staining from the 4-cell stage to late morula stages with more than 16 cells. The results showed autophagy activity in all cells regardless of their position, but fluorescence accumulation was observed mainly in outer cells, consistent with early morula-stage findings (Fig. 3b). We then extended the dual staining analysis to examine differences in autophagy activity from the morula to blastocyst stage. Similar to the earlier findings, TE and ICM exhibited distinct autophagy signals. Both DAPRed and DAPGreen fluorescence were detected in TE, whereas only DAPRed fluorescence was observed in ICM, suggesting that autophagy activity in ICM returned extremely low again (Fig. 3c).

Fig. 3
figure 3

Cell position-dependent autophagy activity at the morula and blastocyst stages. Confocal microscopy images of dual fluorescence staining with DAPGreen and DAPRed at different time points. First, DAPRed (magenta) stained embryos at the earlier developmental stage, and second, DAPGreen (green) stained the embryos at the later developmental stage. Cyan corresponds to DAPI. Scale bars represent 30 μm. (a) First, embryos were stained at the 4-cell stage with DAPRed, and second, with DAPGreen at the morula stage. Arrows indicate strong co-localization of DAPRed and DAPGreen. White circles indicate inner cells, and no fluorescence of DAPGreen was observed. (b) First, embryos were stained at the 4-cell stage with DAPRed, and second, with DAPGreen at the late morula stage more than 16 cells. DAPGreen fluorescence was also observed in inner cells. (c) First, embryos were stained at the morula stage with DAPRed, and second, with DAPGreen at the blastocyst stage. Top photos (TE) focused on TE cells and bottom photos (ICM) focused on ICM. White circles indicate ICM.

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These findings reveal that throughout late preimplantation development, autophagy dynamics differ between cells destined for TE and ICM. TE cells consistently exhibited high autophagy activity, while ICM cells maintained low autophagy activity, with a transient increase in the late morula stage.

Regulation of autophagy in the morula stage by Hippo signaling

Our previous findings revealed that autophagy activity during the morula stage differs depending on cell position. Additionally, prior studies have reported that Hippo signaling suppresses autophagy31. Based on this, we investigated whether Hippo signaling regulates autophagy during the morula stage. The key Hippo signaling proteins MST1/2 can be inhibited by the small-molecule inhibitor XMU-MP-1. When Hippo signaling is active, MST1/2 phosphorylates the downstream kinases LATS1/2, which in turn phosphorylates YAP, thereby regulating the expression of target genes. Treatment with XMU-MP-1 from the morula stage (24 h) onward resulted in a dose-dependent inhibition of blastocyst development and significantly suppressed differentiation into the ICM (Fig. S4). We then performed dual fluorescence staining on XMU-MP-1-treated morula-stage embryos, which had been treated 12 h prior to observation. The results showed that inner cells exhibited autophagy fluorescence comparable to that of outer cells (Fig. 4a). Furthermore, when embryos were treated with XMU-MP-1 from the morula to blastocyst stage and subjected to dual staining, no autophagy activity was detected in the ICM (Fig. 4b). These findings suggest that autophagy dynamics in the morula stage are spatially regulated by Hippo signaling. However, in the blastocyst stage, autophagy appears to be controlled by mechanisms independent of Hippo signaling.

Fig. 4
figure 4

Regulation of autophagy at the morula stage by Hippo signaling. Confocal microscopy images reveal dual fluorescence staining with DAPGreen (green) and DAPRed (magenta) in embryos treated with 10 µM XMU-MP-1. Scale bars represent 30 μm. (a) First, embryos were stained at the 4-cell stage with DAPRed, then treated with XMU-MP-1 for 12 h, and second, stained with DAPGreen at the morula stage. White circles indicate inner cells at the morula stage. DAPGreen fluorescence was observed in inner cells upon XMU-MP-1 treatment. (b) First, embryos were stained at the morula stage with DAPRed, then treated with XMU-MP-1 for 12 h, and second, stained with DAPGreen at the blastocyst stage. Photos shown as ‘TE’ focused on TE cells and photos show as ‘ICM’ focused on ICM. White circles indicate ICM at the blastocyst stage. Autophagy was not detected in the ICM of embryo with XMU-MP-1 treatment.

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Discussion

In this study, we elucidated the role of autophagy and its regulatory mechanisms during the late stages of preimplantation mouse embryo development. Atg5 KO embryos reportedly exhibit complete developmental arrest at the 4/8-cell stages8, preventing direct assessment of the essentiality of autophagy during the 4 and 8-cell to morula stage. Consistently, we found that autophagy inhibition by CQ completely halted development from the 4/8-cell stages to the morula stage. CQ inhibits the fusion of autophagosomes with lysosomes32 and also disrupts lysosomal function by increasing the intralysosomal pH. As a result, in addition to autophagy, other lysosome-dependent intracellular processes such as endocytosis are also likely to be impaired33,34. In fact, inhibition of lysosomal function has been reported to cause developmental delays in embryos35, and thus, we cannot completely exclude the possibility that such effects may have contributed to the phenotypes observed in this study.

We analyzed the impact of CQ exposure on cell differentiation during the 4/8-cell to morula stage. CQ treatment resulted in fewer TE and ICM cells at the blastocyst stage. At the morula stage, treatment with 5.0 µM CQ significantly reduced the total cell count, as well as the number of Cdx2- and Nanog-positive cells, while 2.5 µM CQ selectively reduced the number of Cdx2-positive cells. These findings suggest that autophagy plays a role in cell differentiation. Previous studies have shown that autophagy inhibition by 3-MA increases POU5F1 (OCT4) protein expression at the blastocyst stage in mouse embryos10, and that autophagy induction by rapamycin increases TE cell numbers in bovine embryos11. Taken together, the reduction in Cdx2 expression at the morula stage due to autophagy inhibition could have disrupted the balance of cell differentiation between the ICM and TE36,37, as evidenced by a decrease in TE cells.

The developmental arrest and differentiation defects caused by CQ treatment were partially rescued by AA supplementation. We then examined TFAP2C, an upstream transcription factor of Cdx2, and found that CQ treatment did not alter the number of TFAP2C-positive cells, but significantly reduced fluorescence intensity. However, this reduction was also restored by AA supplementation. mTOR complex 1 (mTORC1) is known to be activated in AA rich environments38,39,40. TFAP2C is translationally regulated by mTOR and regulates the ICM–TE bipotency program by promoting the co-expression of ICM and TE lineage genes at early stages of development41,42, and subsequently forms a complex with YAP and TEAD4 in the nucleus to activate Cdx2 transcription specifically in outer cells following lineage segregation43. These results suggest that autophagy-mediated AA supply is also important even at the late stages of preimplantation development.

We developed a dual fluorescence staining methods to characterize autophagy dynamics at the morula and blastocyst stages. In the morula, newly induced autophagy was observed only in the outer cells, while the inner cells retained autophagy induced at the 4-cell stage. Subsequently, autophagy was activated in all the cells, initiating cavitation and blastocyst formation. In the blastocyst, new autophagy was observed exclusively only in the TE, consistent with the higher energy demands of TE cells compared to ICM cells44,45.

Inhibition of MST1/2, a core kinases of Hippo signaling, disrupted autophagy dynamics at the morula stage, leading to the induction of autophagy even in inner cells. However, Hippo signaling inhibition did not induce autophagy in the ICM of blastocysts. During the transition from the morula to the blastocyst stage, Hippo signaling gradually diminishes within the ICM, promoting the expression of pluripotency factors46,47. This suggests that autophagy in the blastocyst stage is regulated by pathways independent of Hippo signaling. Our findings indicate that Hippo signaling regulates autophagy during the morula stage. The Hippo signaling inhibitor, XMU-MP-1 suppresses MST1/2 phosphorylation. MST1/2 phosphorylates Beclin1, a component of the class III PI3K complex, thereby inhibiting its recruitment to the complex and suppressing autophagy31. In Hippo signaling inhibition experiments, suppression of MST1/2-mediated Beclin1 phosphorylation may have promoted its recruitment to the class III PI3K complex, leading to autophagy induction in inner cells. Integrating these findings, Hippo signaling is active in the inner cells of the morula48, where it suppresses autophagy. In contrast, Hippo signaling is inactive in outer cells, allowing autophagy induction and AA supply, which in turn activates mTOR to enhance the translation of TFAP2C41. This facilitates YAP nuclear translocation and Cdx2 transcription, thereby promoting TE differentiation. (Fig. 5). Taken together, our study illustrates a new mechanism underlying cell differentiation into the TE, in which Hippo signaling regulates autophagy depending on cell position to supply AAs, resulting in expression TFAP2C through mTOR at the morula stage. Interestingly, despite predicted mTOR activation in the outer cells, autophagy was not suppressed. While mTOR plays a central role in autophagy regulation in many cell types49, preimplantation embryos may regulate autophagy independently of mTOR by activating the class III PI3K complex9. Thus, autophagy regulation in the morula stage may be primarily governed by class III PI3K rather than mTOR.

Fig. 5
figure 5

A model of autophagy’s role in cell differentiation at the morula stage. In inner cells, autophagy is suppressed due to the activation of the Hippo signaling pathway47. Specifically, MST1/2, the core kinases of this pathway, presumably inhibit autophagy, by phosphorylating Beclin131. In addition, phosphorylated YAP by LATS1/2 cannot translocate to the nucleus, leading cells to differentiate into the ICM. In contrast, in outer cells, autophagy is not inhibited by Hippo signaling, enabling AA supply, activation of mTOR, and upregulation of TFAP2C, which supports differentiation into the TE lineage41.

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This study elucidated the role of autophagy at the morula stages, demonstrating that autophagy-mediated AA supply promotes TE differentiation. Additionally, we found that autophagy is spatially regulated by Hippo signaling. On the other hand, inhibiting Hippo signaling did not induce autophagy in the ICM, suggesting that a different regulatory pathway may govern autophagy at the blastocyst stage. As limitations of this study, it should be noted that we used in vitro fertilized (IVF) embryos and relied primarily on immunostaining-based analyses. Autophagy dynamics in mouse preimplantation embryos may differ between IVF and in vivo fertilized embryos, and this should be taken into consideration when interpreting the results. In addition, the analysis was largely qualitative; therefore, combining immunostaining with live imaging and more quantitative approaches in future studies would provide a more comprehensive understanding. Future studies should focus on identifying the pathway responsible for autophagy reactivation at the late morula stage and the regulatory mechanisms of autophagy in the ICM. A deeper understanding of these processes will provide a more comprehensive view of the role of autophagy in preimplantation development.

Methods

Animals

ICR strain female and male mice, aged 8–12 weeks, were purchased from Shizuoka Laboratory Animal Center (SLC) Inc. (Hamamatsu, Japan). The mice were maintained in a SPF room (25 °C, a relative humidity of 50%, and a 14/10-h light–dark cycle). Mice were fed ab libitum with a standard pelleted diet and allowed free access to distilled water. All the animal experiments were approved by the Animal Experimentation Committee at the University of Yamanashi, Japan, (protocol number A4-10) and conducted in accordance with the ethical guidelines. The animal research was carried out in accordance with the ARRIVE guidelines.

In vitro fertilization (IVF) and embryo culture

For IVF, sperm were collected from the cauda epididymis of ICR male mice and capacitated in human tubal fluid (HTF) medium for 30–60 min. After capacitation, sperm concentration was measured, and IVF was conducted at a concentration of 1 × 10⁶ sperm/ml. Cumulus-oocyte complexes (COCs) were collected from ICR female mice, which had been superovulated by an injection of 5 IU pregnant mare serum gonadotropin (PMSG) followed by 5 IU human chorionic gonadotropin (hCG) 48 h later (ASKA Pharmaceutical, Tokyo, Japan).

After IVF, presumptive zygotes were washed several times and cultured in droplets of Chatot-Ziomek-Bavister (CZB) medium at 37 °C in a humidified atmosphere containing 5% CO₂. The embryos were cultured in CZB medium supplemented with CQ (Sigma-Aldrich, MO, USA), amino acids (AAs) (MEM Amino Acids Solution (50X); Thermo Fisher Scientific Inc., Waltham, MA), or XMU-MP-1 (Selleck Chemicals, TX, USA) at designated time points.

Immunostaining

Embryos were washed twice in PBS containing 1% polyvinyl alcohol (PBS-PVA) and fixed in 4% PFA in PBS for 30 min at room temperature. Subsequently, embryos were washed in PBS-PVA and incubated overnight at 4 °C with blocking buffer (0.1% Triton X-100 and 1% BSA in PBS). The anti-CDX2 monoclonal antibody( 1:500; BioGenex, San Ramon, CA, USA, MU392A-UC) to detect TE cells, anti-Nanog rabbit polyclonal antibody (1:500; Abcam, Cambridge, UK, ab80892) to detect the ICM cells and anti-TFAP2C(1:500; AP-2γ antibody; Santa Cruz Biotechnology, TX, USA, sc-12762) were the primary antibodies used. Embryos were incubated for 3 h at 4 °C with the primary antibody diluted in blocking buffer. After washing in PBS-PVA, embryos were subsequently incubated with Alexa Fluor 568-labelled goat anti-mouse IgG secondary antibody (Invitrogen/Molecular Probes, Eugene, OR) at a 1:500 dilution for 2 h at room temperature. Finally, embryos were mounted on glass slides in Vecta-shield (Vector Laboratories Inc., Burlingame, CA) supplemented with 1 μg/ml 4’,6-diamidino-2-phenylindole (DAPI). The images were obtained with an All-in-one Fluorescence Microscope (BZ-X800; KEYENCE, Osaka, Japan).

Autophagy staining

Autophagosomes were stained by incubating the embryos with 0.5 µM DAPGreen (Dojindo, japan) in CZB for 30 min. The embryos were then transferred to Hepes-CZB drops on a glass-bottom dish (35 mm dish;MatTek corp., MA, USA) for observation. Confocal microscopy (Olympus FV1200; Olympus, Tokyo, Japan) was used to capture images of each embryo along the Z-axis in 10 sections.

Dual fluorescence staining

At the initial developmental stage to be observed, autophagosomes were stained by incubating the embryos with DAPRed 0.5 µM (Dojindo, Japan) in CZB for 30 min, followed by washing with CZB. After 24 h, embryos at a more advanced stage were incubated with DAPGreen 0.5 µM (Dojindo, Japan) in CZB for 30 min to stain newly formed autophagosomes. Embryos were then placed in Hepes-CZB drops on a glass-bottom dish and imaged using the same confocal microscopy conditions as those used for autophagy staining (Olympus FV1200; Olympus, Tokyo, Japan; Z-stack, 10 sections). We used ImageJ Fiji (22743772) to convert the DAPRed fluorescence images to magenta.

Statistical analysis

Statistical analyses were accomplished using JMP Pro software version 16.0 (SAS Institute Inc., Cary, NC). P-values less than 0.05 were regarded as statistically significant.