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, 50% humidity, and a 14/10-hour 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 Univeristy of Yamanashi (Ethics Approval Number: A6-19). Animal handling and experimental procedures adhered to the 3Rs principles and were conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan). All mice used in this study were euthanized by cervical dislocation.

Female ICR (Institute of Cancer Research) mice were injected intraperitoneally with 7.5 IU of equine chorionic gonadotropin (eCG), followed 48 hours later by 7.5 IU of human chorionic gonadotropin (hCG) (ASKA Pharmaceutical, Tokyo, Japan) to induce superovulation. After hCG injection the superovulated females were mated with ICR males, and about 16 hours later, a vaginal plug check was performed. Approximately 6 or 30 hours after confirmation of a vaginal plug (for collection of 1-cell or 2-cell embryos, respectively), pluged female mice were then euthanized by cervical dislocation and the fallopian tubes were collected in microcentrifuge tube. The collected fallopian tubes were placed in HEPES-supplemented mCZB medium (HEPES-mCZB) (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) [40]. The oviduct was flushed from the fimbrial end with HEPES-mCZB under a stereomicroscope. The flushed 1-cell or 2-cell stage embryos were collected and transferred into drops of mCZB medium (5.56 mM glucose-containing) or CZB medium (glucose-free) [14, 15]. Embryos were maintained at 37 °C under 5% CO₂ in air until the appropriate developmental stage was achieved. The experimental procedures were conducted according to previously published protocols with minor modifications [41].

IVF was performed as previously described [42]. Briefly, male ICR mice aged 10 weeks or older were euthanized by cervical dislocation, and the cauda epididymides were collected. The cauda epididymides were punctured to release sperms, which were then placed in droplets of human tubal fluid (HTF) medium and incubated at 37 °C in 5% CO₂ for at least 30 minutes to allow capacitation. HTF medium was also used for insemination during IVF [43]. Female ICR mice aged 8 weeks or older were superovulated as described above. 16 h after hCG injection, the cumulus-oocyte-complexes (COCs) were collected from the oviducts and transferred into HTF medium droplets. Aliquots of sperm were added to the medium containing COCs to achieve a final concentration of 1.0 $\times$ 10⁶ sperm/mL, and the mixture was incubated for 6 hours. Additionally, to investigate the effects of glucose exposure, embryos were transferred to mCZB or CZB after a shortened incubation period of 2 hours, in addition to the standard 6-hour incubation protocol. Six hours after insemination, fertilized zygotes were identified by observing the extrusion of the second polar body and the presence of two pronuclei under an inverted microscope (IX71; Olympus, Tokyo, Japan). After confirmation of fertilization, unfertilized, damaged, or fragmented embryos were discarded.

CQ (C6628; Sigma-Aldrich, St. Louis, MO) was dissolved in H₂O to a concentration of 5 mM and aliquoted into in 1-µL portions. A 5 mM CQ stock solution was diluted in CZB medium to final concentrations of 4 µM, 2 µM, and 1 µM. For CQ treatment, the collected embryos were transferred to CQ-containing medium droplets and cultured at 37 °C in a humidified atmosphere of 5% CO₂.

Rotenone powder (R0090; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 2 mM stock solution. The stock solution was aliquoted in 1-µL portions and stored at –30 °C. Before use, it was diluted in CZB medium to obtain final concentrations of 0–4 µM.

OSMI-1 powder (AB235455; Abcam, Cambridge, UK) was dissolved in DMSO to prepare a 10 mM stock solution. A 10 mM stock solution of OSMI-1 was diluted in CZB medium to final concentrations of 15 µM and 30 µM. For OSMI-1 treatment, the collected embryos were transferred to OSMI-1-containing medium droplets and cultured at 37 °C in a humidified atmosphere of 5% CO₂.

Immunostaining was performed as previously described with slight modifications [44]. To further assess embryo quality, blastocysts cultured with or without CQ supplementation were subjected to immunostaining for caudal type homeobox 2 (Cdx2) and Nanog, markers of the trophectoderm (TE) and inner cell mass (ICM), respectively. Briefly, embryos were washed twice in phosphate-buffered saline (PBS) containing 1% polyvinyl alcohol (PBS-PVA) and fixed in 4% paraformaldehyde (PFA) in PBS for 30 minutes at room temperature. After washing in PBS-PVA, embryos were incubated overnight at 4 °C in blocking buffer (0.1% Triton X-100 and 1% bovine serum albumin [BSA] in PBS). The embryos were then incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. The following primary antibodies were used: anti-Nanog (1:500, ab80892, Abcam, Cambridge, UK) , anti-Cdx2 (1:500, MU392-UC, BioGenex, Fremont, CA, USA), anti-LC3 pAb (1:1000, PM036, MBL, Nagoya, Japan), and mouse anti-O-GlcNAc RL2 (1:500, NB300-524, Novus Biologicals, Centennial, CO, USA). After washing in PBS-PVA, embryos were subsequently incubated with secondary antibodies, Alexa Fluor 488-conjugates anti-rabbit IgG (1:500) and Alexa Flour anti-mouse IgG (1:500), for 2 h at room temperature. After washing with PBS, embryos were mounted on glass slides in Vectashield (Vector Laboratories, Burlingame, CA, USA) supplemented with 1 µg/mL 4^′,6-diamidino-2 phenylindole (DAPI). Fluorescence images were acquired using a fluorescence microscope (BZ-800; Keyence, Osaka, Japan) with identical laser settings applied to all samples.

To examine the changes in autophagy activity induced by low-concentration CQ treatment, DAPGreen (Dojindo, Kumamoto, Japan) staining was performed on CQ-treated embryos, as well as untreated and naturally mated embryos at the morula embryo stage. DAPGreen was dissolved in DMSO to prepare a 0.02 mM stock solution, which was then diluted 200-fold in mCZB medium to a final concentration of 1 µM. Embryos were incubated in this solution for 30 min at 37 °C, washed with mCZB medium, and incubated for an additional 30 min. Fluorescence signals were observed using a fluorescence microscope (BZ-800; Keyence, Osaka, Japan) under identical exposure settings.

0.5 mM Mito Tracker (Thermo M7513) stock was aliquoted into 1 µL portions and stored at –30 °C. Upon use, it was diluted 1000-fold in mCZB medium, and embryos were stained for 30 minutes.

Statistical analyses were conducted using JMP Pro software version 17.0 (SAS Institute Inc., Cary, NC, USA). Data were analyzed using one-way ANOVA followed by Tukey’s or Fisher’s LSD multiple comparison tests when appropriate. p-values less than 0.05 were categorized as statistically significant. Graph figures were generated by GraphPad Prism 10 Version 10.3.1 (2024) (GraphPad Software, LLC, Boston, MA, USA). Blastocyst formation rates were analyzed using the Tukey–Kramer test for pairwise comparisons, with each independent experiment regarded as one biological replicate.

In our previous study, we evaluated the concentration-dependent effects of CQ on preimplantation development using 2-cell embryos (2-CF) obtained by oviductal flushing after in vivo fertilization. When examining implanted embryos at progressively lower concentrations, the lowest concentration at which embryo development was possible was 2.0 µM CQ. In this experiment, embryos obtained via in vitro fertilization (IVF) and 1-cell flush (1-CF) after in vivo fertilization were subjected to low-concentration CQ treatment from the 2-cell stage for 48 hours to investigate effects on development and cell differentiation. Treatment groups consisted of 0, 1.0, 2.0, and 4.0 µM CQ (Fig. 1A). Unexpectedly, the IVF-derived development rate showed a tendency to decrease in a CQ concentration-dependent manner. Notably, embryos treated with 2.0 µM CQ exhibited a significant decline in developmental rate compared with the 2-CF group, resulting in blastocyst formation rates below 30% (Fig. 1B). Next, to examine the effects on cell differentiation, immunostaining was performed using antibodies against Cdx2 and Nanog, molecular markers of the trophectoderm (TE) and inner cell mass (ICM), respectively (Fig. 1C). The total cell number in each group decreased in a CQ concentration-dependent manner, with 2-CF embryos exhibiting the highest cell number (Fig. 1D). Similarly, the number of Cdx2-positive cells (Fig. 1E) and Nanog-positive cells (Fig. 1F) was highest in the 2-CF. Regardless of treatment with 0, 1.0, or 2.0 µM CQ, the number of cells in the 1-CF showed a significant decrease compared to the 2-CF. Next, we performed DAPGreen staining of 2-CF and IVF embryos at the morula-stage as well as morula flush embryos (MF) obtained by flushing the morula stage, to examine their autophagy activity levels (Fig. 1G). At the morula stage, DAPGreen relative fluorescence intensity was highest in IVF embryos. Conversely, the lowest fluorescence intensity was observed in embryos that developed in vivo up to the morula stage (Fig. 1H). These results suggest that IVF embryos exhibit the highest autophagic activity, reflecting an autophagy-dependent mode of development that is consistent with their sensitivity to CQ.

Fig. 1.

Effects of CQ treatment on the development and cell differentiation of IVF and in vivo-fertilized embryos. (A) Representative images of the blastocyst stage of IVF embryos and the blastocyst stage of in vivo fertilized embryos (1-CF, 2-CF) at each CQ concentration. Embryos cultured from the 2-cell stage in CZB medium were cultured at concentrations of 0 µM, 1.0 µM, 2.0 µM, and 4.0 µM CQ. Scale bar = 100 µm. (B) Blastocyst formation rate in the CQ-treated groups (1-CF, 2-CF and IVF embryos): 0 µM, 1.0 µM, 2.0 µM, and 4.0 µM CQ (p 0.05). (C) Representative images of DAPI (blue), Cdx2 (red), and Nanog (green) immunostaining in 1-CF, 2-CF and IVF embryos with 2.0 µM CQ treatment which shows DAPI, Cdx2, Nanog, and Merge immunostaining images. Scale bar = 100 µm. Comparison among the three groups after cell counting: 2-CF, 1-CF, and IVF embryos. Total cell number (D), Cdx2-positive cell number (E) and Nanog-positive cell number (F) of these embryos has been analyzed. (G) Representative DAPGreen fluorescence images of morula-stage embryos derived from in vivo–fertilized embryos and IVF embryos. Scale bar = 100 µm. (H) Relative DAPGreen fluorescence intensity. Data represent the mean $\pm$ SEM from at least three independent replicates per treatment group. Different letters indicate significant differences (p 0.05). 2-CF, 2-Cell Flush; 1-CF, 1-Cell Flush; MF, Morula Flush; IVF, In Vitro Fertilizaion.

In this experiment, we further investigated the dependence of preimplantation embryos on mitochondrial respiratory chain function by employing rotenone (Ro), a mitochondrial complex I inhibitor, to examine energy metabolism mediated by mitochondria in detail. First, we used 2-CF cultured in vivo up to the 2-cell stage, 1-CF cultured in vivo up to the 1-cell stage, IVF embryos were treated with Ro at concentrations of 0 µM, 1.0 µM, 2.0 µM, and 4.0 µM starting from the 2-cell stage. These embryos were then cultured in vitro to examine Ro sensitivity differences influenced by fertilization conditions and in vitro culture duration (Fig. 2A). In 2-CF embryos, the development rate decreased in a Ro concentration-dependent manner, dropping to a blastocyst formation rate of 16% in the 2.0 µM Ro group. Furthermore, in the 4.0 µM Ro group, only IVF embryos developed to the blastocyst stage, but the blastocyst rate was a mere 11%. However, both IVF embryos and 1-CF embryos showed similar blastocyst development rates at 0 µM Ro, 1.0 µM Ro, and 2.0 µM Ro (Fig. 2A). These results suggest that 24 hours of in vitro culture starting from the 1-cell stage may reduce Ro sensitivity, regardless of the fertilization environment. Furthermore, based on previous results, glucose exposure up to the two-cell stage—which differs from the in vivo environment—is considered a factor altering Ro sensitivity depending on culture conditions. Therefore, focusing on glucose exposure time in IVF embryos, we cultured embryos in vitro up to the 2-cell stage using mCZB (glucose-containing) and CZB medium (glucose-free) after IVF, under conditions of either standard 6-hour insemination or 2-hour insemination, followed by Ro treatment. Under standard 6-hour IVF conditions, no difference in Ro sensitivity was observed between mCZB and CZB culture conditions, regardless of Ro concentration (Fig. 2B). However, when the IVF duration was shortened to 2 hours to minimize glucose effects, culture in CZB medium until the 2-cell stage resulted in a Ro concentration-dependent decrease in blastocyst formation (Fig. 2C). To further clarify the effects of shortened fertilization time and avoidance of glucose exposure until the 2-cell stage on embryo development and blastocyst quality, immunofluorescence staining was performed using antibodies against Cdx2 and Nanog (Fig. 2D–F). Analysis results showed a tendency for total cell count, TE cell count, and ICM cell count to decrease in a Ro concentration-dependent manner. However, the difference in effects between mCZB and CZB medium resulted in only a slight decrease in cell count at all cells counts, with no significant effect observed. These results suggest that shortening the medium exposure time to 2 hours and culturing cells in CZB medium until the two-cell stage increases subsequent rotenone sensitivity. To determine whether mitochondrial metabolism was increased, mitochondrial membrane potential at the 2-cell stage was compared using Mito Tracker (Fig. 2G). The results showed that Mito Tracker fluorescence intensity was significantly increased by both the 2-hour medium treatment and culture in CZB medium until the two-cell stage (Fig. 2H). These results suggest that minimizing glucose exposure until the 2-cell stage increases mitochondrial activity at that stage and enhances dependence on mitochondrial function during subsequent development.

Fig. 2.

Comparison of blastocyst formation rates in IVF embryos treated with various concentrations of rotenone (Ro) from the 2-cell stage onward. (A) Blastocyst formation rates of IVF and 2-CF, 1-CF, and 0 µM, 1.0 µM, 2.0 µM, 4.0 µM Ro. Comparison of blastocysts cultured in CZB medium from 6 hours post-fertilization until the 2-cell stage. (B) Blastocyst formation rates after 0 µM, 1.0 µM, 2.0 µM Ro treatment. Comparison of in vitro fertilized blastocysts cultured in mCZB and CZB medium from 6 hours post-fertilization until the 2-cell stage. (C) Blastocyst formation rates after 0 µM, 1.0 µM, 2.0 µM Ro treatment. Comparison of in vitro fertilized blastocysts cultured in mCZB and CZB medium from 2 hours post-fertilization until the 2-cell stage. (D) Comparison of total cell numbers after DAPI, Cdx2, and Nanog immunostaining in IVF blastocysts cultured starting at 2 hours after insemination to the 2-cell stage in mCZB and CZB media with 0 µM, 1.0 µM, 2.0 µM and 4.0 µM Ro. (E) Comparison of Cdx2-positive cell numbers. (F) Comparison of Nanog-positive cell numbers. (G) MitoTracker fluorescence images of embryos cultured in mCZB and CZB medium from 2 hours post-fertilization to the 2-cell stage, with or without glucose. Scale bar = 25 µm. (H) Plot of MitoTracker fluorescence intensity in mCZB and CZB medium. Data represent the mean $\pm$ standard error of the mean (SEM) from at least three independent replicates per treatment group. Different letters indicate significant differences (p 0.05).

In this experiment, to investigate whether glucose exposure time up to the 2-cell stage could cause differences in CQ susceptibility during preimplantation embryo development, we shortened the insemination time from 6 hours to 2 hours in conventional IVF embryos and considered the glucose concentration (2.25 mM) in the HTF medium used for insemination. Furthermore, we used CZB medium, which is mCZB medium with glucose removed, for in vitro embryo culture and examined the effects of glucose exposure from fertilization to the 2-cell stage. CQ concentrations were 0, 1.0, 2.0, and 4.0 µM CQ, as previously used, and low-concentration CQ treatment was performed for 48 hours from the 2-cell stage to the morula stage after IVF. Embryo development rates decreased in the 2.0 and 4.0 µM CQ groups after the morula stage (Fig. 3A). Like previous experiments, a CQ concentration-dependent decrease in development rate was observed. However, the blastocyst formation rate was 91% in the 1.0 µM CQ group, 71% in the 2.0 µM CQ group, and 20% in the 4.0 µM CQ group (Fig. 3B). Surprisingly, these results demonstrated that the development rate of IVF using mCZB medium could be restored in all treatment groups by reducing glucose exposure time (Fig. 3A,B). Furthermore, as previously, cell counts for total cells, TE, and ICM cells were measured and compared using immunostaining. Regarding total cell count, a CQ concentration-dependent decrease in cell number was observed as in previous studies. However, in this experiment using CZB medium, cell numbers increased in all experimental groups compared to conventional IVF using mCZB medium. Notably, the 2.0 µM CQ-treated group showed a marked tendency toward increased cell numbers (Fig. 3C). The cell count in the 0 µM CQ-treated group was 53, in the 1.0 µM CQ-treated group it was 40, in the 2.0 µM CQ-treated group it was 33, and in the 4.0 µM CQ-treated group it was 21. Furthermore, a CQ concentration-dependent decrease in the number of Cdx2- and Nanog-positive cells was also observed (Fig. 3D). Next, DAPGreen staining was performed at the 2-cell stage and 4–8-cell stage to examine changes in autophagy activity with and without glucose. The experimental groups consisted of embryos cultured in vitro in mCZB medium until the 2-cell stage or 4–8-cell stage (mCZB) and embryos cultured in vitro in CZB medium (Fig. 3E). At the 2-cell stage, DAPGreen fluorescence intensity was significantly higher in CZB embryos. Conversely, at the 4–8-cell stage, mCZB embryos showed a significantly higher trend (Fig. 3F,G). These results indicate that autophagy activity differs at least depending on glucose availability. To confirm these differences in autophagy activity, LC3 immunostaining was performed. Consistently, the relative LC3 fluorescence intensity was lower in the CZB group than in the mCZB group at the 2-cell stage, whereas this pattern was reversed at the 4–8-cell stage, suggesting enhanced LC3 degradation via autophagy (Fig. 3H–J).

Fig. 3.

Effects of CQ treatment on the development and cell differentiation of IVF embryos cultured from 2 hours post-insemination to the 2-cell stage in glucose-free medium. (A) Representative images of IVF embryos cultured to the 2-cell stage in mCZB and CZB medium, then treated with 0 µM CQ, 1.0 µM CQ, 2.0 µM CQ, or 4.0 µM CQ. Scale bar = 100 µm. (B) Blastocyst formation rate after CQ treatment in IVF embryos cultured in vitro with mCZB and CZB medium with 0 µM CQ, 1.0 µM CQ, 2.0 µM CQ, 4.0 µM CQ (p 0.05). (C) Representative images of DAPI (blue), Cdx2 (red), and Nanog (green) immunofluorescence staining in IVF embryos treated with 2.0 µM CQ after culture in mCZB and CZB medium. Scale bar = 25 µm; shows DAPI, Cdx2, Nanog, and Merge immunofluorescence images. (D) Comparison of total cell numbers, Cdx2 and Nanog-positive cell numbers in CQ-treated mCZB and CZB IVF embryos after cell counting. (E) DAPGreen fluorescence images of 2-cell stage and 4–8-cell stage embryos cultured in vitro in mCZB and CZB medium. Scale bars = 125 µm (top) and 25 µm (bottom). (F) Relative fluorescence intensity of mCZB and CZB cultures at the 2-cell stage. (G) Relative fluorescence intensity of mCZB and CZB cultures at the 4–8 cell stage. (H) LC3 fluorescence images for each mCZB and CZB experimental group at the 2-cell stage and 4–8-cell stage. Scale bar = 25 µm. (I) Relative LC3 fluorescence intensity during the 2-cell stage. (J) Relative LC3 fluorescence intensity during the 4–8 cell stage. No statistical significance (n.s.) was found between the two groups. Data represent the mean $\pm$ SEM from at least three independent replicates per treatment group. Different letters indicate significant differences (p 0.05).

In this study, we examined the effects of the OGT inhibitor OSMI-1 (OS) on embryonic sensitivity to CQ by treating embryos with OSMI-1 at concentrations of 15 or 30 µM. IVF embryos were cultured in mCZB medium with OS from 2 hours post-insemination until the 2-cell stage. These 2-cell stage IVF embryos were then cultured in mCZB and the blastocyst rate was examined. In the absence of CQ, embryos treated with 15 µM OS showed a blastocyst rate of 96%, equivalent to the control group. In contrast, embryos treated with 30 µM OS had a blastocyst rate of 82%, showing a slight tendency toward reduced development compared to the control (Fig. 4A). Furthermore, when MitoTracker staining was performed at the 2-cell stage, fluorescence intensity was significantly increased by 30 µM OS treatment compared to the control group (Fig. 4B,C). Furthermore, it was experimentally confirmed that OS treatment with 15 µM and 30 µM significantly reduced O-GlcNAc modification levels compared to the untreated control group (Fig. 4D,E). Subsequently, CQ treatment was applied to each of these embryos after the 2-cell stage to investigate the effect on CQ sensitivity. Unexpectedly, in the presence of 1.0 µM CQ, the developmental rate was significantly reduced by treatment with either 15 or 30 µM OS. Similarly, in the 2.0 µM CQ group, the blastocyst rate was greatly reduced by OS treatment. In the 4.0 µM CQ-treated group, development to the blastocyst stage did not occur after 15 µM OS treatment (Fig. 4F). These results indicate that OS treatment up to the 2-cell stage caused a decrease in the development rate of CQ-treated embryos compared to the control group. Additionally, autophagy activity was measured by DAPGreen staining in the 2-cell stage of each OS-treated group (Fig. 4G). The results showed a significant increase in fluorescence intensity in the 15 µM OS group compared to the 0 µM OS group, and a similar trend was also observed in the 30 µM OS group (Fig. 4H). Collectively, these data suggest that glucose-exposure after fertilization reduces mitochondrial function and autophagy activity via O-GlcNAc modification.

Fig. 4.

Comparison of development rates following OS treatment up to the 2-cell stage and CQ treatment from the two-cell stage onward. (A) Comparison of blastocyst formation rates between embryos cultured from 2 hours post-insemination until the 2-cell stage in mCZB (0 µM OS) versus embryos treated with 15 µM OS or 30 µM OS. (B) Images stained with MitoTracker fluorescence at the 2-cell stage in OS-treated embryos (0 µM OS, 15 µM OS, 30 µM OS). Scale bar = 25 µm. (C) Plots of MitoTracker fluorescence intensity. (D) Fluorescence images of O-GlcNAc modification levels during the 2-cell stage. Scale bar = 25 µm. (E) Plots of O-GlcNAcylation intensity. (F) Blastocyst formation rate of embryos treated with 0 µM, 15 µM, or 30 µM OS until the 2-cell stage, followed by 48-hour treatment with various concentrations of CQ (0 µM, 1.0 µM, 2.0 µM, 4.0 µM). (G) Representative DAPGreen staining images of each OS-treated embryo (0 µM OS, 15 µM OS, 30 µM OS) at the 2-cell stage. Scale bars = 125 µm (top) and 25 µm (bottom). (H) Comparison of DAPGreen fluorescence intensity after OS treatment. DAPGreen fluorescence intensity was normalized to the 0 µM OS group, which was set to 1. Data represent the mean $\pm$ SEM from at least three independent replicates per treatment group. Different letters indicate significant differences (p 0.05). 0 µM OS, 0 µM OSMI-1; 15 µM OS, 15 µM OSMI-1; 30 µM OS, 30 µM OSMI-1.