Glutamine supplementation enhances development of in vitro-produced porcine embryos and increases leucine consumption from the medium

Abstract

Improper composition of culture medium contributes to reduced viability of in vitro-produced embryos. Glutamine (Gln) is a crucial amino acid for preimplantation embryos as it supports proliferation and is involved in many different biosynthetic pathways. Previous transcriptional profiling revealed several upregulated genes related to Gln transport and metabolism in in vitro-produced porcine blastocysts compared to in vivo-produced counterparts, indicating a potential deficiency in the culture medium. Therefore, the objective of this study was to determine the effects of Gln supplementation on in vitro-produced porcine embryo development, gene expression, and metabolism. Cleaved embryos were selected and cultured in MU2 medium supplemented with 1 mM Gln (control), 3.75 mM Gln (+Gln), 3.75 mM GlutaMAX (+Max), or 3.75 mM alanine (+Ala) until day 6. Embryos cultured with +Gln or +Max had increased development to the blastocyst stage and total number of nuclei compared to the control (P < 0.05). Moreover, expression of misregulated transcripts involved in glutamine and glutamate transport and metabolism was corrected when embryos were cultured with +Gln or +Max. Metabolomics analysis revealed increased production of glutamine and glutamate into the medium by embryos cultured with +Max and increased consumption of leucine by embryos cultured with +Gln or +Max. As an indicator of cellular health, mitochondrial membrane potential was increased when embryos were cultured with +Max which was coincident with decreased apoptosis in these blastocysts. Lastly, two embryo transfers by using embryos cultured with +Max resulted in viable piglets, confirming that this treatment is consistent with in vivo developmental competence.

Supplemental glutamine in porcine embryo culture medium modulates gene expression and enhances metabolic activity of in vitro-produced blastocysts.

Introduction

The complex environments of mammalian oviducts and uteri are difficult to mimic in vitro. Recent advances in the porcine oocyte maturation system have resulted in about 40–45% of in vitro-matured and fertilized oocytes developing to the blastocyst stage [1], but culture conditions after fertilization are still suboptimal as many embryos undergo developmental arrest or fragmentation. Furthermore, in vivo-derived porcine blastocysts have at least twofold more cells compared to in vitro-produced blastocysts [2]. Previous deep sequencing endeavors have revealed differences in transcriptional profiles between in vivo and in vitro-matured porcine oocytes [3] and in vivo and in vitro-produced porcine blastocysts [4]. These differences may partially explain the reduced viability of in vitro-produced porcine embryos, and altering the composition of the culture medium can refine expression of transcripts towards an in vivo state [5, 6]. Continuing to optimize the porcine embryo culture system is warranted as genetically modified pigs are increasingly being used as agricultural [7] and biomedical models [8–11].

Rapidly proliferating cells demonstrate metabolic alterations compared to most normal somatic cells. Several types of cancers are characterized by increased rates of aerobic glycolysis with a concomitant decrease in use of glucose for the tricarboxylic acid (TCA) cycle, known as the Warburg effect [12]. Preimplantation embryos also exhibit hallmarks of the Warburg effect, such as shuttling glycolytic intermediates to the pentose phosphate pathway for nucleotide synthesis and redox balance [13]. Furthermore, expression of glycolytic gene variants, hexokinase 2 and pyruvate kinase M2, is upregulated in preimplantation embryos, similar to certain cancer cells [14]. Therefore, harnessing information gained from cancer cell metabolism studies may provide insight for understanding metabolic pathways in preimplantation embryos and improving the current culture system.

Glutamine is a conditionally essential amino acid that is used for several processes aside from protein synthesis, including maintenance of cellular energy through TCA cycle anaplerosis, acid–base balance, and synthesis of nonessential amino acids, lipids, and nucleotides. Collectively, these processes support rapid proliferation as many types of cancers consume large amounts of glutamine and are considered “glutamine addicted” [15]. Oncogenic transformation resulting in overexpression of MYC proto-oncogene (MYC) leads to dependency on glutamine catabolism, also known as glutaminolysis, for sustaining mitochondrial metabolism instead of glucose [16]. Regarding preimplantation embryos, glutamine addition to mouse embryo culture medium allowed for progression past the two-cell block and significantly improved blastocyst development [17, 18]. Additionally, glutamine was shown to successfully replace glucose as the carbon source for the TCA cycle in porcine preimplantation embryos [19]. However, glutamine utilization for the TCA cycle by in vitro-produced porcine embryos was significantly reduced compared to in vivo-derived counterparts, which may suggest a deficiency in the culture medium [20]. Furthermore, transcript abundances of several genes involved in glutamine and glutamate transport and metabolism were upregulated in in vitro-produced porcine blastocysts compared to in vivo-derived blastocysts, indicating that culture conditions alter preimplantation embryo homeostasis [4].

Based on this information, we hypothesized that glutamine supplementation to the current porcine embryo culture medium would enhance development, correct misregulated gene expression, and improve metabolism of in vitro-produced porcine embryos. To test this, blastocyst developmental parameters were characterized, and expression of previously misregulated transcripts was assessed. Additionally, effects of glutamine supplementation on metabolic profiles, mitochondrial function, and apoptosis were determined. Finally, embryo transfers after culture with supplemental glutamine were performed to confirm developmental competence in vivo.

Materials and methods

Chemical components

Unless stated otherwise, all chemicals were purchased from Sigma Chemical Company (St. Louis, MO).

Ethics statement

Collection of ovaries from prepubertal gilts and use of live animals were in accordance with approved protocol and standard operating procedures by the Animal Care and Use Committee of the University of Missouri.

Production of porcine embryos in vitro

Cumulus–oocyte complexes were aspirated from follicles 3–6 mm in diameter by using an 18-gauge needle attached to a 10-mL disposable syringe. Cumulus–oocyte complexes with uniform cytoplasm and at least three layers of cumulus cells were placed in maturation medium (TCM-199 medium supplemented with 0.1% polyvinyl alcohol (PVA), 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 10 μg/mL of gentamicin, 0.57 mM cysteine, 10 ng/mL of EGF, 0.5 μg/mL of FSH, 0.5 μg/mL of LH, 40 ng/mL FGF2, 20 ng/mL LIF, and 20 ng/mL IGF1) and matured for 42–44 h in a humidified incubator with an atmosphere of 5% CO₂ in air at 38.5°C [1, 21]. Cumulus cells were removed from matured oocytes by vortexing for 3 min in 0.1% (w/v) hyaluronidase in Tyrode's Lactate 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (TL-HEPES)-buffered saline with 0.1% PVA. Then, metaphase-II oocytes with extrusion of the first polar body were selected for in vitro fertilization (IVF).

Thirty oocytes were washed and placed into 50 μL droplets of IVF medium (modified Tris-buffered medium containing 2  mg/mL bovine serum albumin (BSA) and 2  mM caffeine) in a mineral oil overlay and maintained at 38.5°C until sperm were added. The sperm used for IVF in all experiments were from a single collection of a Landrace boar and stored as frozen pellets in liquid nitrogen. A 0.1-mL frozen semen pellet was thawed in 3 mL of sperm washing medium (Dulbecco's phosphate-buffered saline (Gibco, Gaithersburg, MD) supplemented with 0.1% FAF-BSA and 10 μg/mL gentamicin). Sperm were washed by centrifugation in 45% Percoll solution and then in modified Tris-buffered medium. The spermatozoa pellet was resuspended in IVF medium to 0.5 × 10⁶ cells/mL. Then, 50 μL of the sperm suspension was added to the oocyte droplets to obtain a final concentration of 0.25 × 10⁶ cells/mL. Gametes were incubated together in a humidified incubator with an atmosphere of 5% CO₂ at 38.5°C for 4 h.

Embryo culture

After IVF, presumptive zygotes were removed from the droplets, washed, and transferred in groups of 50–500 μL of porcine zygote medium 3 plus 1.69 mM arginine and 5 μM PS48 (MU2) in a four-well dish at 38.5°C in a humidified atmosphere of 5% CO₂ in air [5, 22, 23]. At 28–30 h after fertilization, cleaved embryos were randomly selected and moved in equal numbers to 500 μL of MU2 with 0, 1 (control), 2.5, 5, or 10 mM L-glutamine in a four-well dish at 38.5°C in a humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂ until day 6 post-fertilization. To determine the optimal concentration of glutamine, percentage developed to the blastocyst stage was recorded at day 6 for each treatment. Blastocysts were fixed in 2% paraformaldehyde for 30 min at room temperature, stained with Hoechst 33342 (10 μg/mL) for 15 min, and total number of nuclei was recorded after visualization with a UV filter attached to a Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan).

For all subsequent experiments, cleaved embryos were cultured in 500 μL of MU2 with 1 mM L-glutamine (control), 3.75 mM L-glutamine (+Gln), 3.75 mM GlutaMAX without 1 mM L-glutamine (+Max; Gibco), or 3.75 mM L-alanine with 1 mM L-glutamine (+Ala) to day 6 at 38.5°C in a humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂. GlutaMAX is an L-alanyl-L-glutamine dipeptide, which is used in several cell culture systems because it is more stable in solution and mitigates the rise in ammonia due to spontaneous glutaminolysis. Percentage to the blastocyst stage and total number of nuclei were recorded.

RNA extraction and cDNA synthesis

Day 6 blastocysts cultured in MU2, MU2+Gln, MU2+Max, or MU2+Ala were washed with diethyl pyrocarbonate-treated phosphate-buffered saline, collected in pools of 10 in 0.6-mL tubes, and snap-frozen in liquid nitrogen for storage at –80°C. Four biological replicates were collected. Total RNA was extracted from each pool by using an RNeasy Micro Kit (Qiagen, Germantown, MD) and eluted in 12 μL of nuclease-free water. Exactly 4 μL of total RNA was used for cDNA synthesis and amplification with the Ovation Pico WTA System V2 according to the manufacturer's instructions (NuGEN Technologies, Inc., San Carlos, CA). Following amplification, cDNA samples were purified with the DNA Clean & Concentrator-25 (Zymo Research, Irvine, CA).

Relative quantitative PCR

Relative quantitative PCR was performed with each cDNA sample as template for genes involved in amino acid transport, glutaminolysis, amino acid synthesis, nucleotide synthesis, mitochondrial function, and apoptosis by using IQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA). Primers were designed by using Integrated DNA Technology software (Idtdna.com; Coralville, IA), and all primers used for this study were listed in Supplementary Table S1. Efficiency tests were conducted for each primer set by generating a standard curve of 10 ng dilutions from a 50 ng/μL pooled cDNA reference sample. Quantitative PCR was conducted in triplicate on the CFX Connect Real-Time System (Bio-Rad Laboratories) for each concentration (50, 5, 0.5, 0.05, and 0.005 ng/μL) for validation of each set. Accepted primer sets had standard curve R² values of ≥0.99 with efficiencies of 95–105%. Samples from every biological replicate were diluted to 5 ng/μL, and quantitative PCR was run in triplicate to determine differential expression of the selected transcripts with the conditions: 95°C for 3 min, and 40 cycles of 95°C for 10 s, 55°C for 10 s, and 72°C for 30 s. A dissociation curve was generated after amplification to ensure that a single product was amplified.

Abundance of each mRNA transcript was calculated relative to the housekeeping gene, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma polypeptide (YWHAG) and a reference sample, which consisted of pooled cDNA from four biological replicates of in vivo-fertilized/in vivo-derived blastocysts and in vivo-fertilized/in vitro-cultured blastocysts [4]. YWHAG was selected as the housekeeping gene because stable expression of this transcript has been observed during porcine preimplantation development [24]. The comparative quantification cycle (Cq) method was used to determine relative mRNA expression for each treatment.

Evaluation of blastocyst metabolism

After 28–30 h of culture, cleaved embryos were individually moved into 13 μL droplets of MU2, MU2+Gln, MU2+Max, or MU2+Ala in 75-mm petri dishes and covered with light mineral oil. Embryos were cultured at 38.5°C in a humidified incubator with an atmosphere of 5% CO₂, 5% O₂, and 90% N₂ until the blastocyst stage at day 6. Fully expanded or hatching blastocysts were removed from the droplets, and spent media (10 μL; n = 4 per treatment) were snap-frozen in liquid nitrogen and stored at –80°C. Unspent media (10 μL; n = 3 per treatment) that did not contain embryos during the culture period were snap-frozen in liquid nitrogen for each group as a control and stored at –80°C. Four biological replicates were collected.

Metabolite consumption and production were analyzed by using gas chromatography (GC) and mass spectrometry (MS) at the University of Missouri Metabolomics Center. Media samples (10 μL) were thawed and added to a 2-mL glass vial containing 3 μg/mL umbelliferone in 80% methanol before being dried under nitrogen at 45°C by using a RapidVap Vertex Evaporator (Labconco, Kansas City, MO). Dried samples were methoximated by incubating with 25 μL of methoxyamine HCl (15 mg/mL) in pyridine for 60 min at 50°C. After centrifuging at 3000 × g for 5 min at room temperature, 25 μL of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS, Thermo Fisher Scientific, Waltham, MA) was added, vortexed, and incubated for 60 min at 50°C for derivatization. Samples were centrifuged at 3000 × g for 5 min at room temperature, and the supernatant was transferred to a 150-μL glass insert in a GC-MS autosampler vial for analysis by using an Agilent 6890N GC coupled to a 5973N mass selective detector (MSD; Agilent Technologies, Santa Clara, CA). Exactly 1 μL of each sample was injected in a 15:1 split ratio with the inlet and transfer line held at 280°C. Separation occurred through a 60-m DB-5MS column (0.25 mm in diameter and 0.25 μm film thickness; Agilent Technologies) with a 1.0 mL/min flow rate of helium gas. The first program consisted of a start temperature of 80°C, a ramp of 8°C/min to 315°C, and a 12 min hold. The second program started at 315°C and ramped 10°C/min to 325°C with a 5 min hold. Masses between 50 and 650 m/z were scanned at 2.46 scans per second after electron impact ionization.

By using MSD ChemStation Enhanced Data Analysis (Agilent), raw data files were converted to computable document format for relative quantification. Peak detection and deconvolution was conducted for each sample by using the Automated Mass spectral Deconvolution and Identification System (AMDIS; National Institute of Standards and Technology, Gaithersburg, MD). Retention index calibration was performed by using an alkane mix ranging from 8 to 34 carbons. Metabolites were annotated by the corresponding retention index and mass spectra by using Noble Foundation, Golm, and Adams metabolite databases. Data extraction was conducted by using Metabolomics Ion-based Data Extraction Algorithm (MET-IDEA) [25], and the resulting data were normalized to the internal standard, umbelliferone. Preliminary experiments demonstrated that fractions of glutamate and glutamine converted to pyroglutamate during each run on the GC-MS. Due to the similar patterns of consumption or production across treatment groups, mean areas of chromatographic peaks for glutamine, glutamate, and pyroglutamate were added together. Mean areas of the chromatographic peaks of metabolites in the unspent medium for each treatment and replicate were subtracted from the mean areas of the chromatographic peaks of metabolites in the respective spent medium to calculate relative changes in terms of consumption or production.

Mitochondrial membrane potential analysis

For mitochondrial membrane potential (ΔΨm) staining, day 6 blastocysts from each treatment group were collected and cultured in MU2, MU2+Gln, MU2+Max, or MU2+Ala along with 2.5 μM JC-10 (Enzo Life Sciences Inc., Farmingdale, NY) for 30 min at 38.5°C in 5% CO₂, 5% O₂, and 90% N₂. Additionally, two embryos were incubated with 20 μM valinomycin (Thermo Fisher Scientific) and 2.5 μM JC-10 as depolarized controls, and two embryos from each treatment were unstained as negative controls (Supplementary Figure S1). All groups were subsequently stained with 10 μg/mL Hoechst 33342 for 15 min at room temperature. Embryos were washed three times in TL-HEPES with 0.1% PVA and mounted on slides in their respective culture medium. Four biological replicates were imaged by using epifluorescence illumination linked to a Nikon Eclipse E600 microscope with a 10 objective under a FITC filter for detection of J-monomers (low mitochondrial membrane polarization), a Texas Red filter for J-aggregates (high mitochondrial membrane polarization), and a UV filter for DNA. Each embryo was analyzed by using Fiji for signal intensity of red and green fluorescence [26]. Relative mitochondrial membrane potential was determined as the ratio of J-aggregate to J-monomer staining intensity.

ATP assay

Adenosine 5΄-triphosphate (ATP) contents in single blastocysts were determined by using the ATP bioluminescent somatic cell assay kit. Day 6 blastocysts from each treatment group were individually transferred to 0.2-mL tubes in 12.5 μL of TL-HEPES and stored at –80°C. After thawing, 12.5 μL of Milli Q H₂O and 25 μL of somatic cell ATP releasing reagent were added to each tube and kept on ice for 5 min. Negative controls (TL-HEPES only) and standards (0.10–10.0 pmol ATP/12.5 μL Milli Q H₂O) were prepared in the same manner. To a white 96-well plate, 50 μL of assay mix (diluted 1:25 with dilution buffer) containing luciferin, luciferase, and MgSO₄ were added to appropriate wells and equilibrated for 3 min in the dark. Entire volumes within tubes were added to individual wells, and luminescence was immediately measured by using an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, MA). ATP (pmol/blastocyst) was calculated by using the seven-point standard curve of log₁₀ luminescence vs log₁₀ ATP standard.

Terminal deoxynucleotidyl transferase dUTP nick end labeling assay

Day 6 blastocysts from each treatment group were fixed in 2% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 for 1 h at room temperature. Nuclei with DNA damage in the blastocysts as an early indicator of apoptosis were labeled by incubating in 25 μL of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) solution (fluorescein-conjugated dUTP and terminal deoxynucleotidyltransferase) for 1 h at 38°C (In Situ Cell Death Detection Kit, Roche Diagnostics, Mannheim, Germany). Negative control groups were incubated with only the “label” solution containing fluorescein-conjugated dUTP. Positive control groups were incubated with 16 Kunitz units of DNase I in 25 μL of TUNEL solution. Blastocysts were washed three times in TL-HEPES, and all nuclei were stained with Hoechst 33342 (10 μg/mL) for 30 min at room temperature. Then, blastocysts were mounted on glass slides and imaged by using epifluorescence illumination. The apoptotic index of each blastocyst was calculated as the percentage of TUNEL-positive nuclei out of the total number of nuclei.

Embryo transfer

Day 6 blastocysts cultured in MU2+Max were placed in 3 mL of manipulation medium (9.50 g TCM-199, 0.05 g NaHCO₃, 0.75 g HEPES, 1.76 g NaCl, 3.00 g BSA, 1 mL gentamicin, 1000 mL Milli Q H₂O) with 5 μM PS48 in polystyrene tubes (BD Biosciences, San Jose, CA). After being transported at 37°C to the University of Missouri Swine Research Complex, 40 blastocysts were loaded into a tomcat catheter and surgically transferred into the ampullary-isthmic junction of a cycling gilt on day 3 of her estrous cycle. Pregnancy was determined by heat checking and monitoring by ultrasound after day 25. Surrogates were checked weekly until farrowing. Sexes and birth weights were recorded.

Statistical analysis

All experiments were repeated at least four times so that replicate variation could be assessed. The percentage data used to quantify the development to the blastocyst stage were analyzed by a generalized linear mixed model (PROC GENMOD). Total number of nuclei, gene expression (2^−ΔΔCq), mitochondrial membrane potential, ATP contents, and apoptotic indexes were analyzed by linear mixed models (PROC MIXED). Shapiro-Wilk test was used for assessing the normality assumption for each experiment. For data with severe deviation from the normality assumption, we applied a log transformation, after which data complies with the normality assumption. Treatment was modeled as a fixed factor, and biological replicate was modeled as a random factor. For blastocyst metabolism, outliers were determined based on total signal and the first principal component from principal component analysis by using MetaboAnalyst 3.0. Differences in consumption or production of individual metabolites were analyzed by using a linear mixed model with a fixed treatment effects and random biological replicate effects. Significance was discovered by testing hypotheses using least square estimates. Type I error and family-wise error rate were controlled at level of 0.05. All of these analyses were conducted using SAS version 9.4 (SAS Institute, Cary, NC).

Results

Glutamine supplementation improves porcine embryo development

Supplementation of glutamine (Gln) to our current MU2 culture medium was investigated to determine the effects on porcine preimplantation development. Presumptive zygotes in MU2 cleaved at a rate of 58.6 ± 2.8% at 28–30 h post-fertilization and were placed in equal numbers into the treatments for each replicate. Compared to the control MU2 medium (1 mM Gln), addition of 2.5 and 5 mM Gln significantly increased development to the blastocyst stage and total number of nuclei in the blastocysts (Figure 1A and B). Glutamine deprivation (0 mM) resulted in significantly lower development to the blastocyst stage but did not decrease total number of nuclei compared to the control. Moreover, development to the blastocyst stage and total number of nuclei in the blastocysts after supplementation with 10 mM Gln were not different from the control, indicating possible toxicity at this concentration.

Figure 1.

Blastocyst development after culture with different concentrations of glutamine. (A) Percentage of embryos developed to the blastocyst stage at day 6. Values were determined from six replicates (n = 40–50 cleaved embryos per treatment per replicate). (B) Total number of nuclei in day 6 blastocysts. Values were determined from five replicates (n = 15 per treatment per replicate). Data are presented as mean ± standard error. Different lowercase letters indicate statistical differences (P < 0.05).

The average value of 3.75 mM Gln (+Gln) was chosen as the supplemental concentration for subsequent experiments because 2.5 and 5 mM Gln enhanced development to the blastocyst stage but no differences were observed between these two concentrations. Supplementation of 3.75 mM GlutaMAX (+Max), a L-alanyl-L-glutamine dipeptide, to MU2 without glutamine was investigated for effects on blastocyst development and metabolism due to its increased stability during culture. To account for alanine in GlutaMAX, 3.75 mM alanine (+Ala) was also supplemented separately to MU2. Embryos cultured with +Gln or +Max had significantly increased development to the blastocyst stage compared to the control (68.9 ± 2.7% or 70.7 ± 3.5%, respectively, vs 58.9 ± 2.6%), while +Ala did not improve blastocyst development (Figure 2A). Moreover, embryos cultured with +Gln or +Max had increased total number of nuclei compared to the control and embryos cultured with +Ala (Figure 2B). Applicability of these treatments to other breeds or stains of pigs will need to be determined by future experiments.

Figure 2.

Blastocyst development after culture with glutamine, GlutaMAX, or alanine. (A) Percentage of embryos developed to the blastocyst stage at day 6. Values were determined from five replicates (n = 40–50 cleaved embryos per treatment per replicate). (B) Total number of nuclei in day 6 blastocysts. Values were determined from five replicates (n = 15 per treatment per replicate). Data are presented as mean ± standard error. Different lowercase letters indicate statistical differences (P < 0.05).

Expression of transporters and metabolism-related genes are altered after glutamine supplementation

Quantitative PCR was used to determine the effects of glutamine supplementation on genes involved in amino acid transport, glutaminolysis, amino acid synthesis, and nucleotide biosynthesis. With respect to transporters, expression of solute carrier family 1 member 1 (SLC1A1), a high-affinity glutamate transporter, was significantly decreased in all groups compared to the control. Expression of solute carrier family 7 member 5 (SLC7A5), a glutamine and large neutral amino acid bidirectional transporter, was significantly decreased in embryos cultured with +Gln or +Max. Expression of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, was not different between any groups (Figure 3A). Expression of glutaminase (GLS) was significantly increased in embryos cultured with +Gln compared to the control, and embryos cultured with +Max tended (P = 0.06) to have increased expression of GLS. In addition, transcript abundance of an enzyme involved in asparagine synthesis, asparagine synthetase (ASNS), was significantly decreased in embryos cultured with +Gln or +Max, and pyrroline-5-carboxylate reductase 1 (PYCR1), which is involved in proline synthesis, was decreased in embryos cultured with +Max. Expression of glutamate dehydrogenase 1 (GLUD1), glutamic-pyruvic transaminase 2 (GPT2), and phosphoserine aminotransferase 1 (PSAT1) did not differ between groups (Figure 3B). Additionally, expression of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), phosphoribosyl pyrophosphate amidotransferase (PPAT), and phosphoribosyl pyrophosphate synthetase 1 (PRPS1), which are involved in nucleotide biosynthesis, did not differ between groups (Figure 3C).

Figure 3.

Relative abundance of transcripts involved in (A) amino acid transport, (B) glutaminolysis and amino acid synthesis, and (C) nucleotide synthesis from embryos cultured in MU2, MU2+Gln, MU2+Max, or MU2+Ala determined by qPCR. Data are presented as mean ± standard error. Different lowercase letters indicate statistical differences (P < 0.05).

Effects of glutamine supplementation on blastocyst metabolism

Gas chromatography and mass spectrometry were used to analyze spent medium on day 6 from each treatment group for 22 metabolites. The following metabolites did not differ based on consumption or production by the embryos between treatments: arginine, aspartate, glycerol, glycine, isoleucine, lactate, lysine, methionine, ornithine, phenylalanine, proline, pyruvate, serine, succinate, threonine, tryptophan, tyrosine, and valine. As shown in Figure 4, alanine was significantly consumed by embryos cultured with +Ala compared to all other groups. Glutamine, glutamate, and pyroglutamate exhibited similar patterns of production across the treatment groups. The normalized values of the chromatographic peaks were added together because the amount of glutamine and glutamate conversion to pyroglutamate was unable to be determined. Embryos cultured with +Max had significantly increased production of glutamine and glutamate in the media compared to all other treatments. Furthermore, embryos cultured with +Gln had significantly increased production of glutamine and glutamate compared to embryos cultured with +Ala but not the control. Embryos cultured with +Gln or +Max had significantly increased consumption of leucine compared to the control (Figure 4).

Figure 4.

Differentially produced (above 0) or consumed (below 0) metabolites in the culture medium from embryos cultured in MU2, MU2+Gln, MU2+Max, or MU2+Ala. Chromatographic peak values for spent culture medium which contained one day 6 blastocyst were normalized to unspent culture medium which did not contain an embryo during the culture period. Values for glutamine, glutamate, and pyroglutamate were added together and represented as Gln+Glu. Values were determined from four replicates (n = 16 per treatment). Data are presented as mean ± standard error. Different lowercase letters indicate statistical differences (P < 0.05).

Mitochondrial gene expression and membrane potential increases in response to glutamine supplementation

Glutamine enhances mitochondrial function through its catabolism; thus, we evaluated effects of glutamine supplementation on mitochondrial gene expression, membrane potential, and ATP concentrations in day 6 blastocysts. Compared to the control, embryos cultured with +Gln had significantly increased expression of presequence translocase associated motor 16 (PAM16) and single-stranded DNA binding protein 1 (SSBP1), and embryos cultured with +Max had significantly increased expression of inner membrane mitochondrial protein (IMMT), PAM16, and SSBP1. Expression of PTEN induced putative kinase 1 (PINK1) was not different between any groups (Figure 5A).

Figure 5.

Effects of glutamine supplementation on mitochondrial activity. (A) Relative abundance of transcripts involved in mitochondrial function from embryos cultured in MU2, MU2+Gln, MU2+Max, or MU2+Ala determined by qPCR. (B) Representative images of J-aggregate and J-monomer staining in day 6 blastocysts from each treatment. Nuclei are stained with Hoechst 33342. Scale bars = 50 μm. (C) Ratios of J-aggregate to J-monomer fluorescence intensities. Values were determined from four replicates (n = 40 per treatment). (D) ATP contents of day 6 blastocysts assessed by a luminescence assay. Values were determined from four replicates (n = 20 per treatment). Data are presented as mean ± standard error. Different lowercase letters indicate statistical differences (P < 0.05).

Mitochondrial membrane potential (ΔΨm) was determined in blastocysts from each treatment by measuring red to green fluorescence emission ratios after staining with JC-10. When embryos were cultured with +Max, mitochondrial ΔΨm was significantly increased compared to control embryos (0.83 ± 0.1 vs 0.61 ± 0.1; Figure 5B and C). However, there were no significant differences in ATP concentrations (pmol/embryo) between any of the treatments (Figure 5D).

Decreased expression of apoptosis markers and apoptotic index after glutamine supplementation

As glutamine deficiency can result in apoptosis [15], we hypothesized that glutamine supplementation would decrease apoptosis by maintaining appropriate energy within the blastomeres. Transcript abundance of apoptotic marker, BCL2 associated agonist of cell death (BAD), was decreased in the embryos cultured with +Max or +Ala, while tumor protein p53 (TP53) expression was decreased in embryos cultured with +Gln, +Max, or +Ala compared to the control. Expression of BCL2 associated X, apoptosis regulator (BAX) was not different between any of the groups (Figure 6A).

Figure 6.

Effects of glutamine supplementation on apoptosis. (A) Relative abundance of transcripts involved in apoptosis from embryos cultured in MU2, MU2+Gln, MU2+Max, or MU2+Ala determined by qPCR. (B) Apoptotic indexes determined by TUNEL staining. Values were determined from four replicates (n = 40 per treatment). Data are presented as mean ± standard error. Different lowercase letters indicate statistical differences (P < 0.05).

The apoptotic indexes of day 6 blastocysts were determined by a TUNEL assay. Control embryos had a significantly increased apoptotic index than embryos cultured with +Gln or +Max (8.2 ± 0.5% vs 6.5 ± 0.8% and 5.5 ± 0.6%, respectively). Additionally, embryos cultured with +Ala had a significantly increased apoptotic index compared to embryos cultured with +Max (7.4 ± 0.5% vs 5.5 ± 0.6%; Figure 6B).

Determining in vivo competence through embryo transfer

Two embryo transfers were each performed with 40 in vitro-produced day 6 blastocysts cultured in MU2+Max to ensure this treatment could produce viable offspring. MU2+Max was chosen because this treatment demonstrated the most improvements in blastocyst quality from the parameters measured compared to the control. Both litters were farrowed on time. The first litter had 10 piglets with one stillborn, no mummies, seven males, and three females, and an average birthweight of 1.3 kg with a standard deviation of 0.03 kg. The second litter had 12 piglets with one stillborn, no mummies, eight males, and four females, and an average birthweight of 1.1 kg with a standard deviation of 0.17 kg. This information confirms that MU2+Max can produce live offspring after embryo transfer.

Discussion

Highly proliferative cells utilize glutamine for a variety of biochemical functions. Most notably, several types of cancer cells have increased glutamine uptake and catabolism to drive the TCA cycle and for synthesis of nonessential amino acids, lipids, and nucleotides [27]. Activated T cells are another class of highly proliferative cells that exhibit increased glutamine uptake, and glutamine deprivation impairs proliferation and cytokine secretion [28]. When activated T cells were subjected to oxidative stress, glutamine supplementation was shown to decrease the number of apoptotic cells by suppressing caspases 3 and 8 as well as increasing intracellular concentrations of glutathione, demonstrating additional antioxidant properties of glutamine [29]. Glutamine has also been established as a crucial energy source for preimplantation embryos of several species, including mice, pigs, and humans [19, 30, 31]. However, embryo culture can adversely affect metabolism as evidenced by a 50% decrease in glutamine utilization in the TCA cycle by in vitro-produced porcine blastocysts compared to their in vivo-derived counterparts [20]. This discrepancy and the low rate of porcine embryo development to the blastocyst stage in culture may partially be attributed to glutamine deficiency or deficiency of other amino acids in the medium.

Glutamine is liable to degradation during in vitro culture, resulting in increased ammonia concentrations that may be toxic to preimplantation embryos [32]; therefore, the more stable L-alanyl-L-glutamine dipeptide, GlutaMAX, was investigated as an alternative to preserve blastocyst quality. L-alanyl-L-glutamine is mainly transported into cells as the intact dipeptide through solute carrier family 15 member 1 (SLC15A1) which is also expressed in porcine blastocysts [4]. Within the cells, glutamine and alanine are liberated to contribute to the intracellular pools of these amino acids for potential use in various metabolic pathways. In the current study, supplementation of glutamine as a single amino acid (+Gln) or in GlutaMAX (+Max) to MU2 medium improved blastocyst development and total number of nuclei as predicted, reaffirming the similarity between preimplantation embryos and other highly proliferative cell types in terms of glutamine requirements. Decreased blastomere proliferation observed in in vitro-produced porcine blastocysts appears to be the result of the culture period after fertilization because in vitro-matured and fertilized porcine oocytes that developed to blastocysts in vivo had significantly higher numbers of cells than those that developed in vitro [33]. Therefore, the increase in total number of nuclei after culture with +Gln or +Max suggests an improvement in the medium composition to promote proliferation. Although GlutaMAX is more stable in culture, no differences in development were observed between embryos cultured with +Gln or +Max, which is consistent with a recent report in porcine parthenogenotes [34].

In vitro-produced embryos are developmentally delayed compared to their in vivo counterparts, which has been shown to be partially attributed to metabolic alterations [20, 35]. A deep sequencing endeavor by Bauer et al. revealed vast differences in the transcriptional profiles of in vivo-derived versus in vitro-produced porcine blastocysts [4]. Of these differences, several transcripts related to glutamine and glutamate transport and metabolism were differentially regulated between the two groups. Decreased expression of SLC1A1, which encodes a high-affinity glutamate transporter, in the embryos cultured with +Gln or +Max concurs with the idea that MU2 control embryos upregulate expression of this transporter due to a deficiency of intracellular glutamate. Increased production of glutamine and glutamate into the medium by +Max embryos is consistent with previous reports in pigs and other species [36–38] and suggests that the intracellular requirements of these two amino acids are satisfied. However, Booth et al. observed glutamine consumption by porcine embryos, but the concentration of glutamine was reduced compared to conventional NCSU-23 medium [39]. SLC7A5 heterodimerizes with SLC3A2 to initiate glutamine efflux and large neutral amino acid influx in a wide variety of cells. Leucine is one of the primary amino acids transported into the cell through this complex [40]. Embryos cultured with +Gln or +Max had increased consumption of leucine, which may have corrected an intracellular deficiency as evidenced by the decrease in expression of SLC7A5 in both of these groups. Leucine can activate mammalian target of rapamycin complex 1 (mTORC1) in murine trophoblast, allowing for increased protein synthesis [41]; thus, utilization of leucine in preimplantation porcine embryos must be clarified to determine if the same mechanism is occurring.

Expression of several transcripts related to glutaminolysis, nonessential amino acid synthesis, and nucleotide synthesis were measured to determine if glutamine supplementation had effects on these pathways. Increased glutamine catabolism in cancer cells is often the result of overexpression of MYC, an oncogenic transcription factor, which upregulates expression of GLS mRNA and protein [16, 42]. GLS is the first enzyme in the glutaminolysis pathway which catalyzes hydrolysis of glutamine to glutamate and ammonium within the mitochondria. Supplementation of glutamine enhanced proliferation of ovarian cancer cells in a dose-dependent manner and increased activity of GLS within the cells [43]. Thus, several anticancer therapeutics target glutamine transport into the cell and subsequent metabolism. For example, CB-839, an allosteric inhibitor of GLS, has moved into clinical trials for breast and certain types of blood cancers [44]. GLS expression was significantly increased in embryos cultured with +Gln and tended to be increased in embryos cultured with +Max, indicating the potential for increased glutaminolysis. This is also consistent with decreased expression of SLC1A1 and increased production of glutamine and glutamate into the culture medium. Moreover, KRAS proto-oncogene, GTPase (KRAS) transformed cancer cells exhibit increased TCA cycle activity driven by glutamine-derived carbon and synthesis of nonessential amino acids by using glutamine-derived nitrogen compared to untransformed cells [45]. Embryos cultured with +Gln or +Max had decreased expression of ASNS for asparagine synthesis, and embryos cultured with +Max had decreased expression of PYCR1 for proline synthesis. These observations are in agreement with the ability of cells to endogenously synthesize non-essential amino acids from glutamine [44], and deficiencies of asparagine and proline may have been relieved with glutamine supplementation. Lastly, no difference in expression was observed for CAD, PPAT, or PRPS1, which encode enzymes for nucleotide biosynthesis, suggesting that glutamine supplementation may not affect these pathways at the blastocyst stage.

Within the mitochondria, glutaminolysis is a major pathway which promotes the TCA cycle for energy production. Although mitochondria are thought to be relatively quiescent during preimplantation development, pharmacological inhibition of mitochondrial function in mouse precompaction embryos has been shown to hinder blastocyst development and reduce cell numbers [46]. Mitochondria-related transcripts were upregulated in embryos supplemented with +Gln (PAM16, SSBP1) or +Max (IMMT, PAM16, SSBP1), indicating glutamine supplementation may enhance mitochondrial function and preserve structural integrity. IMMT maintains crista junctions, PAM16 is involved in nuclear-encoded mitochondrial protein transport, and SSBP1 is involved in mitochondrial biogenesis. At compaction, metabolic activity increases as evidenced by increases in oxygen consumption to provide energy for sodium pumping into the blastocoel cavity and macromolecule synthesis [47, 48]. Additionally, as a measure of cellular health, mitochondrial membrane potential (ΔΨm) indicates pumping of protons across the inner membrane during oxidative phosphorylation and can be measured by staining with cationic, lipophilic dyes, such as JC-1 [49]. JC-10 was used for the current study because it is more water soluble than JC-1, but both dyes function in a similar manner. At low ΔΨm (<100 mV), JC-1 remains in monomeric form and emits green fluorescence, while high ΔΨm (>140 mV) causes JC-1 to aggregate inside the mitochondria, known as J-aggregates, and emits red fluorescence [49]. Embryos cultured with +Max had increased ΔΨm compared to the control; however, ATP content between all of the groups did not differ. While paradoxical, this discrepancy might be due to higher polarization of mitochondria in embryos cultured with +Max but not increased ATP synthesis or increased consumption of ATP by embryos cultured with +Max. Consistent with improved mitochondrial profiles, expression of apoptosis initiator, BAD, was downregulated in embryos cultured with +Max and cellular stress indicator, TP53, was downregulated in embryos cultured with +Gln or +Max. Furthermore, embryos cultured with +Gln or +Max had lower percentages of nuclei with DNA damage as an early indicator of apoptosis after the TUNEL assays, indicating development of better quality blastocysts. Suzuki et al. observed that glutamine supplementation (2 mM) reduced intracellular H₂O₂ concentrations and DNA damage independent of glutathione concentrations in porcine preimplantation embryos [50]. Decreased apoptosis observed in embryos cultured with +Gln or +Max could be due to corrections in gene expression and improved mitochondrial activity.

Two embryo transfers were performed by using embryos cultured with +Max to assess their in vivo developmental competence. The purpose of the embryo transfers was simply to demonstrate that the culture system is compatible with development to term. A previous study of ours showed great improvements in the quality of blastocysts, but those blastocysts failed to establish a pregnancy [6]. Here, the apparent increase in embryo quality was confirmed to be compatible with full-term development. The first litter contained 10 piglets with one stillborn, and the second litter contained 12 piglets with one stillborn. Importantly, this study was conducted by using FLI-matured oocytes which have increased maturation to the metaphase-II stage and cumulus cell expansion, and litters by using these oocytes had nine piglets on average [1]. Therefore, studies focusing on enhancing preimplantation development before transfer, such as the current study, are crucial to continue improving the entire in vitro-produced porcine embryo system. Future studies will use MU2 + Max and will refer to this new medium formulation as MU3.

Due to the fact that embryos supplemented with +Max may have been increasing intracellular concentrations of alanine through uptake of the dipeptide, supplementation of alanine (+Ala) was a necessary control. Supplementation of alanine along with glycine has been shown to improve bovine preimplantation development, but these effects were not observed with only alanine supplementation [51, 52]. Interestingly, embryos cultured with +Ala in the present study demonstrated increased consumption of alanine from the culture medium compared to all other groups. A transamination reaction catalyzed by GPT2 can convert alanine to pyruvate and alpha-ketoglutarate to glutamate which could increase intracellular levels of glutamate and explain decreased expression of SLC1A1 in embryos cultured with +Ala. Expression of BAD and TP53 were decreased in embryos cultured with +Ala compared to the control, suggesting that this treatment may have anti-apoptotic effects during embryo development. Further investigation is required to clarify the role of alanine in preimplantation embryos.

Extensive research has revealed that glutamine is an important amino acid for in vitro production of preimplantation embryos, but there is a deficit of information regarding effects of glutamine on gene expression and metabolism in the embryos. In the current study, supplemental concentrations of glutamine as a single amino acid or in a dipeptide, GlutaMAX, resulted in enhanced blastocyst development and modulation of genes involved in glutamine and glutamate transport and metabolism. Furthermore, these blastocysts exhibited increased consumption of leucine from the culture medium with concurrent decreased expression of SLC7A5. Apoptotic indexes were decreased after glutamine supplementation which is consistent with improved mitochondrial integrity and activity, and viable piglets were obtained after culturing embryos in GlutaMAX. However, the effects of glutamine and leucine on molecular signaling pathways within blastomeres need to be further elucidated to understand the roles of these amino acids during all aspects of preimplantation development.

Supplementary data

Supplementary Figure S1. Representative images of depolarized and negative controls for JC-10 staining. Nuclei are stained with Hoechst 33342. Scale bars = 50 μm.

Supplementary Table S1. Transcripts selected for quantitative PCR.

Notes

Edited by Dr. Peter J. Hansen, PhD, University of Florida

Conflict of Interest: The authors have declared that no conflict of interest exists.