Effect of bovine oocyte transportation system on embryonic quality

Abstract

The conventional ovum pick-up method requires oocytes to be transported from local farms to the laboratory, where they undergo nuclear maturation. However, atmospheric conditions for oocyte transportation differ from those for normal oocyte maturation in vitro. In this study, we examined the effects of conventional and modified oocyte transport conditions on oocyte quality and subsequent embryonic development. Cumulus-oocyte complexes were collected from slaughterhouse-derived bovine ovaries and cultured in few drops of medium on plastic plates in a CO₂-incubator (Control), in plastic tubes containing medium (C-T) in air, or in tubes containing gellan gum and medium (MC-T) in air. C-T conditions reduced mitochondrial functionality (mitochondrial membrane potential and adenosine triphosphate), lipid content, and DNA methylation but increased mitochondrial DNA copy number and phosphorylated AMP-activated protein kinase (P-AMPK) levels compared to those in control oocytes. Furthermore, RNA sequencing analysis of blastocysts derived from these oocytes revealed that C-T conditions affected mitophagy- and AMPK-signaling-related genes. However, MC-T conditions attenuated these C-T-associated changes. In conclusion, conventional C-T conditions affect oocyte metabolism and alter embryo quality, whereas the use of gellan gum as a substrate ameliorates such adverse effects. The oocyte transportation system is inadequate for embryonic production and can induce epigenetic changes. Modifying these conditions with gellan gum is a useful counter-measure.

In recent decades, in vitro embryo production methods have been evaluated based on the developmental rate of blastocysts. However, it is known that in vitro culture processes induce epigenetic alterations in embryos, resulting in abnormalities, including large offspring syndrome [1]. Therefore, we investigated different methods for in vitro embryo production to minimize the potential risk to calves. Embryo transfer (ET) is a crucial method for increasing the number of genetically superior cows produced, with more than two million cases reported globally [2]. In recent years, ovum pick-up (OPU)-in vitro fertilization (IVF) has become mainstream, accounting for about 80% of bovine embryo production globally [3,4,5,6]. In general, OPU is performed by local farmers, and the collected oocytes are transported to a laboratory for in vitro maturation (IVM) in air. In contrast, in the genera, laboratory-based IVM method, oocytes are cultured in an incubator (38.5°C, 5% CO₂). However, the effects of oocyte transportation mechanism on embryo quality have not yet been fully examined.

Compared to culture conditions in the laboratory where Earls’ salts medium is used under 5% CO₂ in air, oocyte transportation systems use Hanks’ salt medium, which maintains a pH of 7.2–7.6, even in air. The major differences in these culture conditions were the atmospheric conditions and the bicarbonate concentration in the medium. It has been reported that the incubation of ovarian cells in air causes mitochondrial dysfunction and metabolic abnormalities in cells [7]. In addition, high oxygen levels inhibit embryonic development and induce the production of reactive oxygen species (ROS) [8, 9], whereas CO₂ inhibits the formation of ROS in cells [10]. Furthermore, oocyte transportation methods differ from conventional culture conditions in the laboratory, such as culture substrate, oil coverage of the medium, and volume of the culture medium. In line with this, culture substrate and medium volume affect oocyte and embryo quality, including parameters such as lipid content, mitochondrial function, and DNA methylation [11,12,13]. Furthermore, metabolites derived from the tricarboxylic acid (TCA) cycle in the mitochondria influence DNA methylation and chromatin configuration in bovine embryos [14]. Thus, we hypothesized that conventional oocyte transportation systems could potentially affect the energy status, mitochondrial function, ROS content, and DNA methylation. In addition, we explored a method that ameliorates the alterations caused by the conventional oocyte transportation system. They are simple to produce, have transparent characteristics that enable easy observation, and form a soft gel substrate [15]. In our previous study, a gel substrate composed of polysaccharides (gellan gum) was shown to reduce reactive oxygen species (ROS) levels, increase adenosine triphosphate (ATP) content in oocytes, and improve embryonic development [16]. Therefore, gel substrates can be used for oocyte transportation to reduce the damage caused by conventional oocyte transportation systems.

In this study, we compared three oocyte culture conditions: general IVM in a 5% CO₂ incubator, IVM in tubes in air that mimic the oocyte transportation system, and IVM in tubes containing gellan gum in air. We found that the conventional oocyte transportation system was insufficient for embryo production and that modifying the conditions with gellan gum is a useful counter-measure.

Materials and Methods

Chemicals and media

All the chemicals were purchased from Nacalai Tesque (Kyoto, Japan) unless otherwise stated. IVM medium (IVM-I) used for the general condition (Control) was TCM199 with Earle’s salt solution (Gibco, Grand Island, NY, USA) supplemented with NaHCO₃ (2.2 g/l), 10 mM taurine, 10 ng/ml EGF (Sigma-Aldrich, St. Louis, MO, USA), 100 IU/ml penicillin, 0.1 µg/ml streptomycin, and 10% fetal calf serum (FCS; 5703H; ICN Pharmaceuticals, Costa, Mesa, CA, USA). IVM medium (IVM-A) used for the transportation conditions (C-T and MC-T) was TCM199 with Hanks’ salt solution (Sigma-Aldrich) supplemented with NaHCO₃ (0.35 g/l) and the same contents as IVM-I. The IVF medium was synthetic oviduct fluid (SOF) [17] supplemented with 4 mg/ml fatty-acid-free bovine serum albumin (BSA) and 10 IU/ml heparin. The medium used for in vitro culture (IVC) of embryos was SOF supplemented with essential and non-essential amino acids (Sigma-Aldrich) and FCS (1% for 18–48 h post-insemination, 5% for 48 h to 7 days).

Bovine ovaries and collected oocytes

Bovine ovaries (from crossbred male Japanese Black and female Holstein cattle) were collected from a slaughterhouse. Ovaries were transported to the laboratory in phosphate-buffered saline (PBS) containing 100 IU/ml penicillin and 0.1 µg/ml streptomycin at 25°C within 4 h. Cumulus cell-oocyte complexes (COCs) were collected from antral follicles (3–5 mm in diameter) using an 18 G needle (Terumo, Tokyo, Japan) connected to a 10 ml syringe (Terumo).

Gel preparation

The gellan gum gel used for the MC-T cells was prepared using a previously described method [9]. A polysaccharide gel consisting of gellan gum (Sansho Co., Ltd., Osaka, Japan) was dissolved in PBS and heated in an autoclave (125 °C, 20 min; Tomy Co., Ltd., Tokyo, Japan). Dissolved gellan gum (300 µl) was added to a 2.0 ml tube (Ina-optika corporation, Osaka, Japan) and then cooled to form a gel. The gel was equilibrated with IVM-A medium overnight and the medium was replaced with fresh IVM-A medium before the experiment.

IVM

The collected COCs were randomly allocated to one of three conditions. In the Control condition, COCs (10 COCs/100 µl) were matured under general conditions (CO₂ incubator: 5% CO₂ in air; ASTEC Co., Ltd., Ibaraki, Japan) covered with oil. In the Condition for transportation (C-T), COCs (50 COCs/500 µl) were matured in a 2.0 ml tube (Ina-optika corporation) containing IVM-A in air (block incubator: Taitec-online, Nagoya, Japan) covered without oil. In the modulated conditions for transportation (MC-T), COCs (50 COCs/500 µl) were matured in a 2.0 ml tube containing IVM-A in air (block incubator: Taitec-online) covered without oil. During transportation, the oil was mixed with the medium. Therefore, the oil was not used under these conditions. In addition, differences in the culture plate and the presence of oil did not affect the experimental parameters of mitochondrial membrane potential (MMP) and mitochondrial DNA copy number (Mt-cn) (Supplementary Fig. 1). The polysaccharide gel was placed at the bottom of the tube, and the culture medium was placed on it (Fig. 1). All incubations were conducted at 38.5°C for 21 h. The nuclear maturation rate was determined by applying 5 µg/ml Hoechst 33342 (Sigma) stain under a Leica DMI 6000 B microscope using LAS AF software (Leica, Wetzlar, Germany), and oocytes at the metaphase II stage (M2) with the first polar body and clear spindle were considered mature oocytes.

Fig. 1.

Submatic design of oocytes maturation used for transportation. Control condition (Control): COCs were incubated in a droplet made of the IVM medium (Earle’s salt, IVM-I) (10 COCs/100 µl) on a plastic plate in a CO₂ incubator. Condition for oocyte transportation (C-T) condition: COCs were incubated in 2 ml tube containing IVM medium (Hanks’ salt, IVM-A) (50 COCs/500 µl) in air. Modified condition for oocyte transportation (MC-T) condition: COCs were incubated in 2 ml tube in which IVM medium (Hanks’ salt, IVM-A) (50 COCs / 500 µl) was put on 0.5% gellan gum substrate (300 µl).

IVF and IVC

IVM oocytes were fertilized with frozen and thawed semen derived from Japanese Black bulls. The sperm samples were washed with a discontinuous gradient Percoll solution (Cytiva Tokyo, Japan, 30 and 60% in IVF medium) and co-incubated with COCs at a concentration of 1 × 10⁶ sperm/ml for 6 h. Then, COCs were transferred to the IVC medium and cultured for 42 h. Cleaved embryos were denuded from the cumulus cells using a fine-pulled pipette and cultured in IVC medium (50 µl) for 5 days. To determine fertilization rate, presumptive zygotes (18 h post-insemination) were incubated in acetic ethanol (1:3) for 15 min. Zygotes with two clear pronuclei were considered normally fertilized oocytes, as determined using a stereomicroscope (Olympus Corporation, Tokyo, Japan). The total number of blastocysts was determined using Hoechst 33342 staining under a fluorescence microscope (Keyence, Osaka, Japan).

Measurement of MMP

IVM oocytes were stained using 0.5 µM Mitotracker Orange CMTMRos (Molecular Probes, Eugene, OR, USA) and 0.5 µM Mitotracker Green FM (Molecular Probes) for 30 min. The oocytes were then washed thrice with 0.2% polyvinyl alcohol (PVA)-PBS and mounted on glass slides. Fluorescence images of whole oocytes were obtained under a Leica DMI 6000 B microscope using the LAS AF software (Leica), and the fluorescence intensities of the equatorial regions of the oocytes were quantified using ImageJ software (ver. 1.54p, NIH, Bethesda, MD, USA). Orange values (active mitochondria) were normalized to green values (whole mitochondria).

Measurement of ATP content

Denuded IVM oocytes after IVM and blastocysts (7 days post-insemination) were individually transferred to a tube containing distilled water (50 µl). The ATP content of individual oocytes was measured using an ATP assay kit (Toyo Inc., Tokyo, Japan). Luminescence generated by the ATP-dependent luciferin-luciferase reaction was measured using a luminometer (Spark 10 M; Tecan Japan Co., Ltd., Kanagawa, Japan).

Mitochondrial DNA copy number

The mitochondrial DNA copy number (Mt-cn) in IVM oocytes and blastocysts was measured by quantitative polymerase chain reaction (PCR) using serial dilutions of standard DNA, as described previously [18]. DNA of each oocyte was extracted by heating at 55°C for 30 min and at 98°C for 10 min in the extraction buffer (Tris-HCl containing 20 mM Nonidet P-40, 0.9% Tween 20, and 0.4 mg/ml proteinase K). PCR was conducted using the CFX ConnectTM Real-Time PCR system (Bio-Rad, Hercules, CA, USA), the KAPA SYBR FAST qPCR Kit (Roche, Indianapolis, IN, USA), and specific primers. The primer sets used to determine bovine Mt-cn were 5′-ACCCTTGTACCTTTGCAT-3′ and 5′-TCTGGTTTCGGGCTCGTTAG-3′ (product size: 81 bp, NC_006853.1) [19,20,21]. The PCR program was 95°C for 3 min, followed by 40 cycles of 98°C for 5 sec and 60°C for 10 sec. The standard DNA used was the plasmid vector from which the PCR product was cloned. The copy number of the standard was calculated using Avogadro’s constant and the vector DNA concentration. All PCR efficiencies were greater than 1.99.

Immunostaining

IVM oocytes, 8-cell stage embryos (48 h post-insemination), and blastocyst-stage embryos were fixed in 4% paraformaldehyde in PBS overnight and permeabilized in 0.25% Triton X-100 for 30 min. The oocytes and embryos were blocked in PBS containing 5% BSA for 1 h. Subsequently, these samples were incubated with a primary antibody rabbit polyclonal anti-AMP-activated protein kinase (AMPKalpha; 1:200; Cell Signaling, Danvers, MA, USA; Cat# 5831S), phosphorylated AMPKalpha (P-AMPKalpha; Cell Signaling; Cat# 2531S), fatty acid synthase (FASN; Santa Cruz Biotechnology, Santa Cruz, CA, USA; Cat# sc-20140) and 5-methylcytosine (5mC: D3S2Z, Cell Signaling; Cat# 28692S) overnight, followed by treatment with a secondary Alexa-Flor-555-conjugated anti-Rabbit IgG Fab2 antibody (Cell Signaling; Cat# 4413S) for AMPKalpha, P-AMPKalpha and 5mC immunostaining and an Alexa-Fluor-488 anti-Rabbit IgG Fab2 antibody (Cell Signaling; Cat# 4412S) for FASN immunostaining. For 5mC staining, oocytes and embryos were treated with 1 N HCl for 1 h before blocking. Oocytes and embryos were mounted and observed under a Leica DMI 6000 B microscope using the LAS AF software (Leica). Fluorescence intensities were quantified using the ImageJ software (NIH, Bethesda, MD, USA). To examine FASN, the fluorescence intensity of the equatorial region of oocytes was evaluated. The fluorescence intensity of the nuclear region was evaluated for AMPK, P-AMPK, and 5mC (NIH).

Measurement of reactive oxygen species (ROS)

IVM oocytes and blastocysts were stained using 5 µM CMH2DCFDA (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min and then washed three times with PVA-PBS. The oocytes and blastocysts were mounted on a depression slide and observed under a Leica DMI 6000 B microscope using the LAS AF software (Leica). The fluorescence intensities of the equatorial regions of the oocytes were quantified using ImageJ software (NIH).

Measurement of lipid content

IVM oocytes and blastocysts were fixed overnight in 4% paraformaldehyde in PBS. Then, samples were washed with PVA-PBS, and lipid content was determined by staining with 1 µg/ml Nile red (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 10 min. The samples were mounted on glass slides and observed under a Leica DMI 6000 B microscope using the LAS AF software (Leica). The fluorescence intensities of the equatorial regions of oocytes or blastocysts were quantified using ImageJ software (NIH).

RNA sequencing analysis of the blastocyst stage

Twenty-five blastocysts from the control, C-T, and MC-T groups were used for RNA extraction. Three samples were prepared from each experimental group using a differential ovary series. RNA extraction was conducted using RNAqueous™-Micro (Thermo Fisher Scientific), and the quality and concentration of the total RNA were determined using a Bioanalyzer RNA Pico kit and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The RNA quality index was 9.8 ± 0.1. cDNA libraries of the RNA from blastocysts were prepared using the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). The concentrations of the cDNA libraries were determined using an Agilent High-Sensitivity DNA kit and Bioanalyzer 2100 (Agilent Technologies). The concentration of the cDNA libraries was reassessed using a Kapa Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA). The multiplexed samples were sequenced in single-read 100 bp reads using the NextSeq 1000 system (Illumina, San Diego, CA, USA).

Raw data were generated using bcl2fastq2 v2.20.0.422. software (Illumina), according to the manufacturer’s instructions. Sequence preparation, reference genome mapping, and differential gene expression analyses were performed using the CLC Genomics Workbench ver. 23.0.2 (Qiagen, Hilden, Germany). To prepare the sequencing data, the adapter sequences, ambiguous nucleotides, and low-quality sequences were removed. The remaining sequence data were aligned to the Bos taurus genome sequence (ARS-UCD1.2/bosTau9) to count sequence reads. Gene expression was evaluated as transcripts per million (TPM). Differentially expressed genes (DEGs) were determined using transcriptomic tools from the CLC Genomics Workbench with a threshold of P < 0.05. Raw RNA sequencing (RNA-seq) data for blastocysts were registered in the DDBJ database under BioProject number PRJDB20374.

Analysis of RNA-seq data

Volcano plots of the DEGs between the C-T/control and MC-T/control conditions were created using the CLC Genomics Workbench (Qiagen). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in the DEGs (P < 0.05, fold change 2.0, TPM > 0.1) were predicted using a functional annotation tool (DAVID, https://david.ncifcrf.gov, accessed on February 7, 2023), using Bos taurus as a background species. The distribution of the three conditions was analyzed by principal component analysis (PCA) using the CLC Genomics Workbench.

K-medoids analysis

DEGs between the Control and C-T conditions (P < 0.05, fold change 2.0, TPM > 0.1) were clustered into six groups based on the differences between the C-T and MC-T conditions using the K-medoids method (CLC Genomics Workbench). K-medoids analysis assigns DEGs to clusters using an algorithm [22]. In the K-medoids analysis, the DEGs were clustered into K separate clusters. These procedures assign DEGs to clusters such that the distance between DEGs within the same cluster is small, whereas the distances between clusters are large. A cluster of DEGs with the greatest differential directional changes between the C-T/control and MC-T/C-T conditions was selected and used to predict Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. These genes were affected by C-T conditions, but their expression was ameliorated by MC-T conditions.

Experimental design

Experiment 1: The effects of IVM culture conditions (Control, C-T, and MC-T conditions) on embryonic development (M2, normal fertilization, cleavage, blastulation rates, and total cell number of the blastocysts) were examined. Approximately 20 oocytes were used as replicates, and the experiment was performed six times for maturation and normal fertilization rates and eight times for cleavage and blastulation rates.

Experiment 2: The effects of IVM culture conditions on mitochondrial function and p-AMPK and AMPK levels in oocytes and blastocysts were examined. Approximately 30 oocytes were used for each experiment.

The effects of IVM culture conditions on ROS levels, lipid content, and FASN expression in oocytes and blastocysts were examined. Approximately 30 oocytes and blastocysts were used in each experiment. For each analysis, oocytes in the metaphase 2 stage were selected for measurement (Supplementary Fig. 2).

Experiment 3: The effects of IVM culture conditions on 5-mC levels, which indicate global DNA methylation levels in oocytes, 8-cell stage embryos, and blastocysts, were examined. Approximately 20 oocytes and blastocysts or 30 8-cell stage embryos were used in the experiment.

Experiment 4: RNA-seq was performed on blastocysts derived from Control, C-T, and MC-T oocytes. Data were analyzed using a functional annotation tool and K-medoid clustering.

Statistical analysis

Data were analyzed using the Shapiro–Wilk test for normality. Parametric data were analyzed using a one-way ANOVA followed by the Tukey–Kramer post-hoc test. Nonparametric data were analyzed using the Kruskal–Wallis test followed by the Steel–Dwass test for multiple comparisons. M2 normal fertilization, cleavage, and blastulation rates were arcsine-transformed before analysis. All data are presented as mean ± standard error of the mean (SEM). Statistical significance was set at P < 0.05.

Results

Experiment 1

The maturation rate did not differ among the experimental groups (Control, C-T, and MC-T). The C-T condition reduced the normal fertilization rate compared with the Control and MC-T conditions. The cleavage rate in the control condition was the lowest, whereas the blastulation rate and total cell number did not differ between the conditions (Table 1).

Table 1. Effect of culture conditions on oocyte maturation and subsequent embryonic development.

Groups	IVM and IVF				IVC
	No. of		Rate of M2 (%)	Rate of Normal (%)	No. of		Rate of		Blastocysts
	Trials	Oocytes			Trials	Oocytes	≥ 8-cells	Blast	No.	TCN
Control	6	247	71.5 ± 4.5	86.8 ± 1.1 a	8	157	50.4 ± 1.0 a	20.3 ± 1.5	17	117.1 ± 13.3
C-T	6	233	68.4 ± 3.4	81.2 ± 2.3 b	8	162	55.6 ± 0.8 b	24.7 ± 1.3	26	99.4 ± 5.8
MC-T	6	237	77.6 ± 5.1	89.1 ± 1.3 a	8	159	57.9 ± 0.9 b	25.2 ± 1.6	28	114 ± 10.3

Data is presented as average ± standard error mean. Rate of oocytes at the metaphase II stage (M2), rate of normal fertilization (Normal), rate of embryos derived over 8-cell stage (≥ 8-cells), rate of embryos development to do blastocyst stage (Blast), total cell number of the blastocysts (TCN) and number of the oocytes or embryos (No.). ^a–b P < 0.05.

Experiment 2

The C-T condition significantly reduced the MMP (Fig. 2A) and ATP content in oocytes compared with the other conditions (Fig. 2B), whereas the ATP content in the resultant blastocysts did not differ among the conditions (Fig. 2C). Similarly, the Mt-cn of oocytes was highest in the C-T condition, and the high Mt-cn remained at the blastocyst stage, with a significant difference between the Control and MC-T conditions (Fig. 2D). Because the energy status of oocytes in the C-T condition was low (Fig. 2B), we examined the expression levels of the AMPK energy sensor. Phosphorylated AMPK levels were significantly higher in the C-T condition than in the control condition (Fig. 2F), whereas AMPK expression levels were comparable among the conditions (Fig. 2G).

Fig. 2.

Effect of culture conditions on mitochondrial functions in oocytes and embryos. The mitochondrial membrane potential (MMP) was determined by MitoTracker staining using the ratio of orange (active mitochondria) to green (whole mitochondria) in oocytes (A). Average content of adenosine triphosphate (ATP) in individual oocytes (B) and blastocysts (C). Mitochondria DNA copy number (Mt-cn) in individual oocytes (D) and blastocysts (E). The expression levels of p-AMPK (F) and AMPK (G) in oocytes. Representative images of (H) MMP (H), (I) nuclear p-AMPK, and (J) AMPK staining. Arrow, nuclear. Data on MMP, ATP content, AMPK, and P-AMPK were analyzed using one-way ANOVA followed by the Tukey–Kramer post-hoc test, while data on Mt-cn were analyzed using the Kruskal–Wallis test followed by the Steel-Dwass test. Data are presented as the mean ± SEM, * P < 0.05. The number of samples (N.) is indicated in each figure.

The ROS content in the oocytes tended to be higher under C-T conditions, but the difference was not significant (Fig. 3A). However, ROS levels in the resultant blastocysts were higher in the C-T condition than in the control condition (Fig. 3B). Lipid content was the lowest in oocytes under the C-T condition (Fig. 3E), and this difference persisted in blastocyst-stage embryos (Fig. 3F). The expression levels of FASN were significantly lower in the C-T condition than in the control condition (Fig. 3G).

Fig. 3.

Effect of culture conditions on ROS content, lipid content, the expression levels of FASN and 5mC expression levels in oocytes and embryos. Relative ROS levels in the oocytes (A) and blastocysts (B). Representative images of the ROS content in oocytes (C) and blastocysts (D). Relative lipid contents of oocytes (E) and blastocysts (F). Relative FASN expression levels in oocytes (G). Representative images of lipid content (oocytes, H; blastocysts, I) and FASN (J) in oocytes. Relative expression levels of 5mC in oocytes (K), 8-cell stage embryos (L), and blastocysts (M). Representative images of 5mC in oocytes, 8-cell stage embryos, and blastocysts (N). FASN and 5mC levels in 8-cell stage embryos were analyzed using one-way ANOVA followed by the Tukey–Kramer post-hoc test, whereas lipid, ROS content, and 5mC levels in oocytes and blastocyst stage embryos were analyzed using the Kruskal–Wallis test followed by the Steel-Dwass test. Data are presented as the mean ± SEM, * P < 0.05. The number of samples (N.) is indicated in each figure. The expression level of the control was set to 1.0.

Experiment 3

The levels of 5mC in oocytes and blastocysts were similar among the conditions (Figs. 3K, M), whereas in 8-cell embryos, they were significantly higher under the C-T condition than under the Control and MC-T conditions (Fig. 3L).

Experiment 4

In the RNA-Seq analysis, 957 DEGs were detected between the C-T and Control conditions, whereas 490 DEGs were identified between the MC-T and Control conditions. A volcano plot of these DEGs showed that a greater number of genes were downregulated in blastocyst-stage embryos under C-T or MC-T conditions than under control conditions (Figs. 4A, B). As shown in Fig. 4C, 364 DEGs overlapped between the C-T and MC-T conditions. The overlapping DEGs were associated with focal adhesions and tight junctions. In addition, unique DEGs under C-T conditions (593 genes) were associated with mitophagy and AMPK signaling, whereas unique DEGs under MC-T conditions (126 genes) were associated with endocytosis (the top five pathways in Fig. 4D).

Fig. 4.

Results of RNA-seq of blastocysts derived from Control, C-T or MC-T oocytes. Volcano plots of differential expression genes (DEGs) of the C-T vs. control (A) and MC-T vs. control (B) groups. Venn diagram of DEGs to determine overlapping or unique DEGs for the C-T and MC-T conditions (C) compared with the control condition. KEGG pathways associated with overlapping or unique DEGs (D).

Using the DEGs between the Control and C-T conditions, six clusters were created via K-medoid analysis (Fig. 5A). Two clusters (clusters 3 and 6) showing large adverse directional changes (C-T/control vs. MC-T/C-T conditions) were selected (Fig. 5B). These two clusters, which included 255 DEGs, were subjected to a pathway analysis. Autophagy and mitophagy were the significantly enriched pathways (Fig. 5C). In addition, DEGs associated with the mitophagy pathway were mostly reduced in the C-T condition compared with those in the control condition (Supplementary Fig. 3).

Fig. 5.

K-medoids analysis of DEGs. Images of the six clusters of DEGs (A). The DEGs between Control and C-T conditions were divided into six clusters using k-medoid analysis. Clusters 3 and 6 showed large adverse directional changes between the C-T and MC-T conditions compared with the control (B). Pathways enriched in genes associated with clusters 3 and 6 (C).

Discussion

This study showed that C-T conditions affected oocyte metabolism, including mitochondrial function, lipid synthesis, and DNA methylation in the resultant embryos. Furthermore, RNA-seq analysis of blastocysts revealed that C-T conditions affect the expression of mitophagy-related genes. However, the MC-T conditions attenuated the changes induced by the C-T conditions.

Mitochondrial quantity and quality are important for oocytes. It has been reported that oocytes with higher MMP, ATP content, and MT-cn have higher developmental competence than their counterparts with lower values of these parameters [23,24,25]. Atmospheric conditions, including CO₂ concentrations and NaHCO₃ content, differed between the C-T and conventional IVM methods. In hamster ovarian cells, low CO₂ levels induce mitochondrial dysfunction and metabolic abnormalities [7]. In this context, we found that C-T conditions reduced MMP and ATP content in oocytes. This bicarbonate concentration is a potential cause of alterations in mitochondrial function; however, a precise evaluation needs to be conducted in further experiments. AMPK is a conserved sensor of cellular energy changes that is activated by increased AMP/ATP and/or ADP/ATP ratios [26]. In addition, P-AMPK levels were high in oocytes cultured under C-T conditions, indicating that the oocyte energy status was low under these conditions. Additionally, RNA-seq analysis of blastocysts showed that AMPK signaling was a major pathway for the DEGs, which is reminiscent of the low-energy status of oocytes in the C-T condition. As mitochondrial dysfunction is accompanied by ROS production in human oocytes [27], high ROS content was observed in blastocyst-stage embryos derived from C-T oocytes. Notably, C-T conditions increase the number of mitochondria in oocytes, even in blastocyst-stage embryos. Mitochondrial number increases during oocyte maturation [28], remains constant after fertilization, and increases further in blastocyst-stage embryos [29]. The number of mitochondria is maintained through well-orchestrated mitochondrial biosynthesis and degradation. When degradation is inhibited, low-quality mitochondria accumulate in porcine oocytes [30, 31]. Mitophagy plays an important role in the removal of defective mitochondria [32]. Using RNA-seq, we found that the DEGs induced by C-T conditions were associated with mitophagy, and that the expression of mitophagy-related genes was reduced (Supplementary Fig. 3). Together, these results suggest that CT oocytes have impaired mitophagy, which increases Mt-cn and ROS levels.

Oocyte lipids are used for ATP production in mitochondria and are important for subsequent embryonic development. In line with this notion, it has been reported that highly competent oocytes have higher lipid contents [33, 34]. Mitochondrial dysfunction reduces lipid content in mouse oocytes [35]. In this study, C-T conditions reduced the lipid content of oocytes, and this reduction was observed in blastocyst stage embryos. Therefore, metabolic changes in the oocytes may persist for long periods and extend to the blastocyst stage. However, it is unclear whether this phenomenon was due to increased lipid metabolism or decreased lipid synthesis. The C-T condition reduced FASN protein expression levels in oocytes and blastocysts (fold change: –2.81, P < 0.05). FASN is a key enzyme in lipid synthesis [36]; therefore, it has been suggested that C-T conditions reduce lipid synthesis in oocytes and blastocysts.

Interestingly, C-T conditions increase the levels of DNA methylation in 8-cell-stage embryos, indicating that the C-T system has a long-term impact on embryo development because the 8-cell embryo stage is a crucial time point for active demethylation and zygotic activation in cows [37]. Mitochondrial dysfunction reduces ATP production in oocytes and induces high levels of DNA methylation in subsequent embryos [38]. Therefore, it has been suggested that mitochondrial dysfunction induced by C-T conditions results in high DNA methylation in 8-cell stage embryos. Furthermore, this is noteworthy because our previous study showed that IVM conditions using xanthan gum and locust bean gum gel substrates significantly decreased the 5mC at the 8-cell stage. In the present study, we demonstrated that TGFB1, derived from increased F-actin levels, plays a role in embryo demethylation [13]. Consistent with this, we previously reported that gellan gum substrate for IVM increases F-actin levels in porcine oocytes [16].

We examined whether MC-T conditions could recover oocytes from the damage caused by C-T conditions. MC-T conditions attenuate C-T condition-induced alterations in mitochondrial function, mitochondrial number, and lipid metabolism. We have previously shown that gel substrates used in MC-T reduce ROS levels in porcine oocytes [16]. Various studies have shown that antioxidants are effective for oocyte maturation [39,40,41]. We suggest that the beneficial effects of the gel substrate used under MC-T conditions were partly due to its antioxidant properties. RNA-seq and K-medoid analyses showed that MC-T conditions ameliorated differentially expressed mitophagy-related genes that were severely affected by C-T conditions. Furthermore, gellan gum used under MC-T conditions activates autophagy in chondrocytes [42]. Mitophagy is important for embryonic development [43, 44]. Therefore, MC-T cells may improve mitochondrial quality by regulating mitophagy, which removes dysfunctional mitochondria. However, PCA using PC1 and PC2 or PC1 and PC3 showed that the C-T and MC-T conditions had distinct effects on gene expression compared to the control condition, indicating that even the MC-T condition was still insufficient. Analysis of DEGs that overlapped between the C-T and MC-T conditions showed that tight junctions and focal adhesions in blastocysts were affected. Tight junctions and focal adhesions are essential in early embryos [45,46,47]. Other factors, including the culture tube, presence of oil, and atmospheric conditions, may affect the expression of genes related to tight junctions and focal adhesions. Consistently, PCA analysis of the DEGs revealed that the MC-T and C-T conditions overlapped compared to the control condition (Supplementary Fig. 4). These issues need to be addressed in future studies.

In this study, we first showed that the conventional oocyte transportation method, which mimics an oocyte transportation system using OPU in the field, affected mitochondrial function and metabolism in oocytes, and DNA methylation in subsequent embryos compared to the laboratory method. However, modifying these conditions with gellan gum attenuates these adverse effects and may be useful for embryo production using OPU.

Conflicts of interests

The authors declare no conflicts of interest.