Reduced HSP70 expression under mild hypothermia is involved in bovine embryo growth suppression

Abstract

Bovine blastocysts cultured under mild hypothermia (MH) can be maintained with non-hatching viable embryos compared to normothermic controls (38.5°C). However, the mechanism by which mildly hypothermic culture delays embryonic growth has not yet been elucidated. This study evaluated the number of cells in embryos cultured under MH conditions and the expression of genes involved in embryonic differentiation. Bovine blastocysts cultured under MH conditions exhibited reduced cell numbers and interferon-tau mRNA expression. Both forkhead box O3 (FOXO3) mRNA expression and FOXO3 protein level in blastocysts cultured under MH conditions were higher than those in normothermic controls (P < 0.05). On the phosphorylated FOXO3 protein level, there was no significant difference between blastocysts cultured under MH and normothermic conditions. In contrast, no significant difference was observed in the ATP content of blastocysts between the MH and normothermic groups. In blastocysts cultured under MH conditions, cold-inducible RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) mRNA expression increased, and heat shock protein 70 (HSP70) mRNA expression decreased compared to that in normothermic controls (P < 0.05). Considering that HSP70 is involved in preventing apoptosis, these results suggest that MH retards embryonic development via apoptosis induced by HSP70 downregulation during the culture period.

When cryopreserving bovine blastocysts, in vitro-produced (IVP) embryos are more susceptible to freezing-induced damage than in vivo-produced embryos, which can reduce embryo survival [1]. Many strategies have been developed to avoid cryodamage during the freeze-thaw processes, including the addition of cryoprotectants [2] and antioxidants [3]. Recently, a method was developed that enables IVP bovine blastocysts to be maintained at supra-zero temperatures [4]. Ideta et al. [5] demonstrated that the hypothermic storage of bovine embryos using an anti-freezing protein-containing medium maintained the high viability of bovine blastocysts. Belinsky and Antic [6] demonstrated that the culture of pluripotent human stem cells at 35°C strikingly reduced unwanted spontaneous differentiation during hESC and iPSC maintenance compared with 37°C. Focusing on the maintenance of pluripotency in embryos, we previously revealed that bovine blastocysts cultured under mild hypothermia (MH, 33°C) could be held with non-hatching viable embryos compared with those cultured under normothermia (38.5°C) [7]. However, the mechanism by which blastocysts are maintained longer in the blastocyst state by 33°C culture has not been clarified yet.

Our previous study demonstrated that the transcription of RNA-binding motif protein 3 (Rbm3), a cold-inducible transcription factor, was upregulated in bovine blastocysts at MH temperatures [7]. Heat- and cold-shock proteins modulate cell proliferation and cause cell cycle arrest under temperature stress [8,9,10]. For example, heat shock protein 70 (HSP70) is elicited by stress at certain stages of development and protects against damage caused by various stressors during embryonic development [11]. In addition, heat shock proteins (HSPs) are important modulators of apoptosis and prevent programmed cell death during embryogenesis [12]. HSP70 is synthesized at a specific embryonic stage, and the presence of antibodies to heat shock protein not only significantly decreases development to the hatched blastocyst stage, but also increases the frequency of TUNEL-stained cells (i.e., undergoing apoptosis) in murine embryos [13]. Therefore, we hypothesized that heat and shock proteins are involved in embryo maintenance during the blastocyst stage under MH conditions.

In the present study, we examined the number of cells in embryos cultured under MH conditions and the expression of genes involved in embryonic differentiation. We further investigated whether HSP affected the developmental capacity of bovine blastocysts.

Materials and Methods

Materials and ethical considerations

Bovine ovaries were collected from a commercial slaughterhouse (Mie Matsusaka Meat Corporation, Mie, Japan). Therefore, no prior ethical approval was required for this study. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.

In vitro embryo production

Embryo production was performed in vitro as previously described [7]. Briefly, groups of 30–50 cumulus-oocyte complexes (COCs) were cultured in 500-μl droplets of IVM medium and incubated for 21 h at 38.5°C in 5% CO₂ and saturated humidity. Up to 30 COCs were incubated in 100-µl droplets of IVF medium containing sperm from Black Japanese bulls for 6 h at 38.5°C in 5% CO₂ and saturated humidity. After IVF, up to 50 zygotes were cultured in 500-μl droplets of IVC medium for 7 days at 38.5°C in 5% CO₂, 5% O₂, and 90% N₂ with saturated humidity.

The IVM medium consisted of Medium 199, HEPES supplemented with 10% newborn calf serum (NBCS, heat-inactivated; Thermo Fisher Scientific, Waltham, MA, USA), 0.02 AU/ml follicle-stimulating hormone (Antrin-R10; Kyoritsu Seiyaku, Tokyo, Japan), 50 ng/ml epidermal growth factor, 5.0 μg/ml dibutyryl cAMP, and 10 μg/ml gentamicin sulfate. The IVF culture medium was IVF100 (Research Institute for Functional Peptides, Yamagata, Japan). Charles Rosenkrans 1 medium [14] with amino acids containing 5% NBCS and 10 μg/ml gentamicin sulfate was used as the IVC medium.

Experimental procedure

Blastocysts obtained 7 days after IVF were collected and classified into codes 1–2 according to the International Embryo Transfer Society guidelines [15]. After washing, groups of three blastocysts were transferred to wells of ultra-low attachment U-bottomed 96-well plates (PrimeSurface; Sumitomo Bakelite, Tokyo, Japan) containing 100 μl of a blastocyst culture medium (potassium simplex optimization medium (KSOM) [16] with 10% NBCS and 10 µg/ml gentamicin sulfate) and cultured at 33°C or 38.5°C in 5% CO₂ with saturated humidity. Embryos were collected on days 7, 8, 9, and 10, and the number of cells in the blastocyst stage and hatched blastocyst stage embryos was counted on each day after staining the nuclei with 10 μg/ml Hoechst 33342 (Thermo Fisher Scientific). The expression levels of pou class 5 homeobox 1 (POU5F1), caudal type homeobox 2 (CDX2), and interferon tau (IFNT) were quantified by qRT-PCR. In addition, blastocysts were collected on day 8 and the ATP levels and mRNA expression of bcl-2-associated X protein (BAX), b-cell leukemia/lymphoma 2 protein (BCL2), forkhead box O3 (FOXO3), P53, fas cell surface death receptor (FAS),HSP60, HSP70, HSP90, cold-inducible RNA-binding protein (CIRP), and RBM3 were measured. Protein levels of FOXO3 and phosphorylated FOXO3 were measured by immunofluorescence staining of blastocysts collected on day 8.

Total cell count

Blastocysts and hatched blastocysts classified as code 1 or 2 were collected and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 25°C for 20 min. After washing three times with PBS containing 0.3% polyvinylpyrrolidone, the fixed embryos were treated with Hoechst 33342, mounted on slides with an antifade reagent (Vector Laboratories, Burlingame, CA, USA), and staining signals were recorded via fluorescence microscopy (IX71; Olympus, Tokyo, Japan).

RNA extraction and quantitative real-time reverse transcription PCR

Ten blastocysts were pooled in approximately 15 µl of the blastocyst culture medium, placed in a 1.5 ml microtube, and immediately immersed in liquid nitrogen. RNA was extracted from pooled blastocysts using a Nucleospin RNA XS Kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. The isolated RNA was reverse-transcribed into cDNA using ReverTra Ace qPCR RT Master Mix with Genomic DNA Remover (TOYOBO, Osaka, Japan), following the manufacturer’s instructions. cDNA samples were stored at –20°C until use. Quantitative real-time PCR was performed using an Applied Biosystems QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific) in a 20 µl reaction mixture containing 10 pmol of each primer, diluted cDNA as a template, and THUNDERBIRD SYBR qPCR Mix (TOYOBO). Each primer pair was designed based on a similarity-based search using BLAST, and the sequences for each gene are shown in Supplementary Table 1. cDNA sequences were amplified after 40 cycles of denaturation (15 sec, 95°C) and annealing/extension (60 sec, 58°C). The formation of a single product was determined by melting curve analysis. mRNA was quantified using the comparative Ct method, and β-actin was used as an endogenous reference, as in previous experiments under mild hypothermic conditions [17]. Fold increases above control levels were determined using the ^2ΔΔCt method, where ΔΔCt = ΔCt treatment – ΔCt control.

ATP assay

The ATP content in blastocysts was measured using an ATP assay kit (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer’s instructions. Blastocysts obtained 7 days after IVF were collected and cultured at 33°C or 38.5°C in a blastocyst culture medium. Expanded blastocysts were collected on day 8 and washed three times with Dulbecco’s phosphate-buffered saline (DPBS) before incubation. The embryos were then incubated with 100 μl working solution on a microplate mixer and incubated in the dark for 10 min at 25°C. Luminescence signals were detected using a luminometer (Infinite 200 PRO microplate reader; Tecan Group, Zurich, Switzerland), and their intensities were calculated using a calibration curve generated from serial dilutions of ATP standard solutions in PBS.

Immunofluorescence staining

Blastocysts were collected on day 8 and classified as codes 1–2, according to IETS guidelines [7]. Cells were fixed in 4% paraformaldehyde in PBS at 25°C for 20 min. After three washes with PBS containing 0.3% polyvinylpyrrolidone, the embryos were treated with 0.5% Triton X-100 in PBS at 25°C for 60 min, blocked in PBS supplemented with 5 mg/ml bovine serum albumin and 0.02% Tween-20 (PBST) at 4°C overnight, and then stained with the following antibodies: anti-FOXO3 (Polyclonal; 10176-2-AP; 1:100; Proteintech, Rosemont, IL, USA) and anti-Phospho-FOXO3 (Ser315) (Polyclonal; 28755-1-AP; 1:100; Proteintech). Following three washes with PBST, the embryos were incubated with a secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit IgG; 1:1000; Thermo Fisher Scientific) in PBST at 25°C for 1 h. Embryos were mounted on slides in an anti-fade reagent (VECTASHIELD Mounting Medium; Vector Laboratories), and staining signals were recorded using a fluorescence microscope (IX71; Olympus). The fluorescence intensity of the entire embryo was quantified using ImageJ v. 1.53 (National Institutes of Health).

Statistical analysis

The results are expressed as the mean ± standard error of the mean. The normality of total cell number, mRNA expression, ATP concentration, and fluorescence intensity of FOXO3 and phosphorylated FOXO3 in embryos cultured under hypothermic and normothermic conditions was verified, and statistical analysis was performed using Student’s t-tests when the population was normally distributed and the Mann-Whitney U test when the population was not normally distributed. Statistical significance was set at P < 0.05.

Results

The total cell number in the hatched blastocysts on day 10 cultured at 33°C was significantly lower than that of those cultured at 38.5°C (P < 0.05, Fig. 1). On day 9, the expression of IFNT mRNA in the hatched blastocysts cultured at 33°C was significantly lower than that in those cultured at 38.5°C (P < 0.05, Fig. 2A). The expression of POU5F1 mRNA in blastocysts cultured at 33°C tended to be higher than in those cultured at 38.5°C (P < 0.1, Fig. 2B). In contrast, the expression of CDX2 mRNA in hatched blastocysts cultured at 33°C tended to be lower than in those cultured at 38.5°C (P < 0.1, Fig. 2C). On day 8, the expression of FOXO3 mRNA in blastocysts cultured at 33°C was significantly higher than in those cultured at 38.5°C (P < 0.05, Fig. 3). There were no differences in BAX, BCL2, p53, or FAS mRNA expression in the blastocysts cultured at either temperature. The intensity of FOXO3 on day 8 blastocysts cultured at 33°C was significantly higher than in those cultured at 38.5°C (P < 0.05, Figs. 4A and 4B), whereas no significant difference in the intensity of phosphorylated FOXO3 was observed between the two conditions (Figs. 4C and 4D). There were no significant differences in ATP concentration in blastocysts on day 8 cultured at 33°C or 38.5°C (Fig. 5). HSP70 mRNA expression in blastocysts cultured at 33°C on day 8 was significantly lower than that in blastocysts cultured at 38.5°C (P < 0.05, Fig. 6A), but there was no difference in their HSP60 and HSP90 mRNA expression. The expression of RBM3 and CIRP mRNA in blastocysts cultured at 33°C on day 8 was significantly higher than those cultured at 38.5 °C (P < 0.05, Fig. 6B).

Fig. 1.

Total cell number in bovine blastocysts cultured in vitro from days 7 to 10 at different temperatures (33 and 38.5°C). Morphological categories: blastocyst (Bl) and hatched blastocyst (Hatch-Bl). Error bar means the standard error of the mean. * P < 0.05.

Fig. 2.

mRNA levels of differentiation-associated genes in bovine blastocysts cultured at different temperatures (33 and 38.5 °C) from days 7 to 10. Relative mRNA levels of IFNT (A), POU5F1(B), and CDX2 (C). The mRNA level of each gene was normalized to that of β-actin. Morphological categories: blastocyst (Bl) and hatched blastocyst (Hatch-Bl). The experiments were repeated thrice. Error bar means the standard error of the mean. * P < 0.05.

Fig. 3.

mRNA levels of apoptosis-related genes in bovine blastocysts cultured at different temperatures (33 and 38.5°C) on day 8. Relative mRNA levels of BAX, BCL2, FOXO3, P53, and FAS. The mRNA level of each gene was normalized to that of β-actin. The experiments were repeated thrice. Error bar means the standard error of the mean. * P < 0.05.

Fig. 4.

Protein levels of FOXO3 and phosphorylated FOXO3 in bovine blastocysts cultured at different temperatures (33 and 38.5°C) on day 8. (A) Representative images of bovine blastocysts cultured at 38.5°C (left) and 33°C (right) groups with anti-FOXO3 antibody staining. (B) Mean pixel intensities of FOXO3. Individual mean pixel intensity per embryo was quantified using ImageJ software. Error bar means the standard error of the mean. * P < 0.05. (C) Representative images of bovine blastocysts cultured at 38.5°C (left) and 33°C (right) groups with anti-phosphorylated FOXO3 antibody staining. (D) Mean pixel intensities of phosphorylated FOXO3. Individual mean pixel intensity per embryo was quantified using ImageJ software. Error bar means the standard error of the mean.

Fig. 5.

Effect of mild hypothermia on embryonic metabolic functions on day 8. ATP concentrations in bovine blastocysts cultured at different temperatures (33 and 38.5°C). Error bar means the standard error of the mean. * P < 0.05.

Fig. 6.

mRNA levels of heat shock protein and cold shock protein-related genes in bovine blastocysts cultured at different temperatures (33 and 38.5°C) on day 8. Relative mRNA levels of the heat shock proteins HSP60, HSP70, and HSP90 (A) and cold shock proteins CIRP and RBM3 (B). The expression level of each gene was normalized to that of β-actin. The experiments were repeated thrice. Error bar means the standard error of the mean. * P < 0.05.

Discussion

MH culture (33°C) decreased the total number of hatched bovine blastocysts on day 10 and IFNT mRNA expression on day 9. Both FOXO3 mRNA expression and FOXO3 protein levels in blastocysts cultured under MH conditions were higher than those in the normothermic controls (P < 0.05). Regarding phosphorylated FOXO3 protein levels, there was no significant difference between blastocysts cultured under MH and normothermic conditions. These results indicated that MH culture inhibited cell proliferation and differentiation by inducing apoptosis. This study further demonstrated that MH culture conditions decreased HSP70 mRNA expression and increased RBM3 and CIRP mRNA expression in blastocysts. Taken together, these results suggest that MH suppresses embryonic development via apoptosis induced by HSP70 downregulation.

The present study showed that on day 10, the cell number in hatched blastocysts cultured at 33°C was lower than in blastocysts cultured at 38.5°C. These results indicated that MH culture conditions suppressed cell proliferation in bovine embryos. On day 9, IFNT mRNA expression was significantly lower in hatched blastocysts cultured at 33°C compared with those cultured at 38.5°C. IFNT is a trophectoderm (TE)-derived cytokine responsible for maternal recognition of pregnancy in ruminants [18]. The secretion of IFNT protein by bovine embryos increases from day 4 after IVF, reaching a maximum during intimate contact of the embryos with the uterus on day 16 after IVF [19]. Furthermore, on day 9, blastocysts exhibited an increasing trend in POU5F1 mRNA expression, a pluripotency marker, whereas hatched blastocysts exhibited a decreasing trend in CDX2 mRNA expression, a TE marker. These findings are consistent with the suppression of embryonic cell differentiation under MH conditions. Thus, we suppose that the downregulation of IFNT mRNA expression in day 9-hatched blastocysts under MH conditions may be caused by the suppression of differentiation in TE.

In this study, FOXO3 mRNA expression and FOXO3 protein levels were higher in bovine blastocysts cultured in MH than in the normothermic controls. FOXO3 is a pivotal transcription factor that regulates the transcription of several genes important for cell cycle control [20] and apoptosis in human epithelial kidney cells [21], human breast carcinoma cells [22, 23], and mouse embryonic fibroblasts [23]. The FOXO protein family is phosphorylated by Akt following activation of the PI3K-Akt pathway, leading to its translocation to the cytoplasm, inactivation, and subsequent degradation via the proteasome [24]. In this study, the FOXO3 protein levels in embryos cultured under MH conditions were higher than those in embryos cultured at 38.5°C, but the phosphorylated FOXO3 protein levels did not differ between the groups. These findings suggest that apoptosis was induced in embryos cultured under MH conditions. Furthermore, the proliferation and differentiation of bovine blastocysts cultured under MH conditions may be suppressed by apoptosis. In contrast, no significant difference was found in the ATP concentration in blastocysts cultured under MH and normothermic conditions. ATP is a molecule that serves as a major energy source in cells, and ATP production is an important factor in expanding the blastocoel cavity at the blastocyst stage [25]. These results indicate that blastocysts cultured under MH conditions may maintain metabolic conditions similar to those cultured under normothermic conditions, despite the suppression of cell proliferation and differentiation. Moreover, we also demonstrated that embryos cultured at 38.5°C until day 7 and subsequently at 33°C for 2 days were transferred nonsurgically into three Holstein milking cows, resulting in two confirmed pregnancies (Supplementary Information 1).

In this study, on day 8, the expression of HSP70 mRNA in blastocysts cultured at 33°C was significantly lower than in those cultured at 38.5°C. This result is consistent with a previous report demonstrating that HSP70 mRNA expression decreases under moderately hypothermic conditions in human liver cells [26]. HSP70 is a member of a family of molecular chaperones involved in innate immunity and protection against environmental stress [27]. It is highly expressed in proliferating mammalian cells, such as tumor cells, and its expression is cell cycle-dependent [28]. In HeLa cells, the expression of HSP70 mRNA rapidly increases upon entry into the S phase and declines in the late S and G2 phases [29]; a transient increase in HSP70 synthesis was observed during the early S phase. Downregulation or selective inhibition of HSP70 suppresses cell proliferation in cancer cells [30]. The addition of antibodies against HSP70 to early embryos cultured in vitro suppresses the blastocyst formation rate in cattle [31] and mice [32]. Taken together, the downregulation of HSP70 mRNA expression in bovine blastocysts cultured under MH conditions may have resulted in an increased proportion of non-hatching viable embryos. In addition, the downregulation of HSP70 by FOXO3 induces apoptosis after heat shock in human endothelial cells [33]. This finding led us to speculate that bovine blastocysts cultured under MH conditions undergo apoptosis because of decreased HSP70 expression (See Supplementary Fig. 1).

On day 8, CIRP and RBM3 mRNA expression levels were upregulated in bovine blastocysts cultured under MH conditions. CIRP and RBM3 belong to a group of stress-responsive proteins. In hibernating animals, these genes are transcriptionally upregulated in response to low temperatures [34, 35], thereby protecting cell viability and promoting cell function [36, 37]. Recent mechanistic investigations have revealed that CIRP and RBM3 modulate G0/G1, G1/S [38], and G2/M transitions [36, 39], respectively. Hypothermic conditions slow cell proliferation and cause cell cycle arrest. Thus, the upregulation of CIPR and RBM3 genes in bovine embryos cultured under MH conditions may be associated with embryo growth retardation. The present study has some limitations. Further studies are required to determine whether the CIPR and RBM are involved in the inhibition of cell proliferation and cell cycle arrest in bovine embryos cultured under MH conditions.

When cells are cultured at low temperatures, many pathways are activated, resulting in various physiological responses [40,41,42]. It is well-accepted that various physiological responses are also involved in the suppression of cell proliferation in embryos and the maintenance of the blastocyst state under MH culture conditions. The present study only partially clarified the changes in HSP and associated apoptosis-inducing pathways. Further studies are needed to elucidate this phenomenon.

In conclusion, our results show that MH suppressed cell proliferation and differentiation in bovine blastocysts via HSP70 downregulation-induced apoptosis. These findings help us understand the effects of MH culture conditions on bovine embryos and are expected to contribute to the development of optimal transport mechanisms and in vitro holding of bovine embryos.

Conflict of interests

The authors declare no conflict of interest.