Low-Concentration Essential Amino Acids in PZM-3 Improve the Developmental Competence of Porcine Embryos Produced by Handmade Cloning

Abstract

Essential amino acids (EAA) of inappropriate concentration have been reported to compromise the development of embryo. This study aimed to investigate the effect of EAA on the developmental competence of porcine embryos produced by either handmade cloning (HMC) or parthenogenetic activation (PA). In experiment 1, we examined the in vitro developmental competence of PA embryos after culture in PZM-3 containing different concentrations (v/v) of EAA (0%, 1%, and 2%). The results indicated that reducing the concentration of EAA from 2% to 1% significantly improved the blastocyst formation (36% vs. 54%), while 0% would compromise the blastocyst formation rate (54% vs. 38%). In experiment 2, we further investigated the effect of EAA concentration (1% and 2%) on the in vitro developmental competence and gene expression of HMC embryos. Blastocyst rate significantly increased by reducing concentration of EAA (41% vs. 53%) and those genes upregulated were enriched in oxidative phosphorylation, PPAR signaling pathway, and metabolism-related pathways. In experiment 3, the in vivo developmental competence of HMC embryos cultured in the medium supplemented with 1% EAA was examined. Embryos derived from both non-gene-modified fetal fibroblasts (FFs) and gene-modified fetal fibroblasts (GMFFs) were transferred to recipients. The pregnancy rates were 83% and 78% separately. Out of the pregnancies, 5 (FFs) and 6 (GMFFs) were successfully developed to term. Our study indicates that supplementing EAA to embryo culture medium at a concentration of 1% can improve the in vitro developmental competence of porcine HMC embryos and the blastocyst obtained can successfully develop to term, which could be beneficial for the production of gene-modified piglets.

Introduction

Due to similarities in size, physiological characteristics, anatomy, metabolism, and genetic background, pigs are regarded as an ideal model for studying embryo development and human disease. Somatic cell nuclear transfer (SCNT) is one of important strategies used to produce these models. However, low efficiency of pig SCNT limits its extensive application, possibly due to the incomparable in vitro culture system with in vivo environment. Culture medium is one of the key factors that is crucial for the development of SCNT embryos, especially for the handmade cloning (HMC) embryos that lack zona pellucida (Du et al., 2007) and thus cannot be transplanted into the recipient at earlier stages before blastocyst.

Amino acids as nutrient substance play key roles at the early development of embryos (Van Winkle, 2001) and are extensively added to the mammal embryo culture medium as an important supplement. It has been proved in many species that amino acids in culture medium were beneficial for the in vitro developmental competence of embryos (Devreker et al., 2001; Lane and Gardner, 1997a, 1997b; Liu et al., 1996; Park et al., 2014; Steeves and Gardner, 1999), while their beneficial effect on the development of embryos closely depends on types and concentration. Essential amino acids (EAA) are a group of amino acids that could not be synthesized by animal organism itself, and the deletion of any EAA from in vitro culture medium would result in cell degeneration and death (Eagle, 1959), indicating their essential role in supporting cell survival and growth.

However, there were several studies reported that addition of EAA could compromise the development of bovine embryos (Liu and Foote, 1995), mice embryos (Gardner and Lane, 1993), and porcine parthenogenetic embryos (Park et al., 2014; Van Thuan et al., 2002), and decrease the blastocyst rate. Steeves and Gardner (1999) reported that the development of bovine embryos was negatively correlated with the concentration of EAA. Furthermore, Lane et al. (2001) found that the development of mouse embryos could be significantly improved by reducing the concentration of EAA to the half of Eagle's concentration (Eagle, 1959). Study in pig also showed that the blastocyst rate of porcine in vitro-fertilized (IVF) embryos could be significantly increased by reducing concentration of EAA to 1% (v/v) (Beebe et al., 2009).

Despite this, it is still not known the best concentration of EAA for the developmental capacity of porcine HMC embryos, because most of those studies were performed with IVF or pathenogenetic activation (PA) embryos (Beebe et al., 2009) or limited to the in vitro culture stage. Many reports showed that the in vitro culture conditions of SCNT embryos were different from those of parthenogenetic or fertilized embryos (Boiani et al., 2005; Chung et al., 2002; Gupta et al., 2007; Heindryckx et al., 2001; Kim et al., 2006; Lee et al., 2004), and one study suggested that amino acids have no beneficial effect on the developmental ability of horse cloned embryos (Choi et al., 2003). Thus, investigating the effect of EAA on porcine HMC embryos will help to elevate the cloning efficiency, which could benefit the production of pig model human disease and also pig breeding.

Our study aimed to investigate the effect of EAA on both in vitro and in vivo developmental competence of porcine HMC embryos. First, we compared the effect of different concentrations of EAA on in vitro parthenogenetic activated embryos. We then investigated the effect of EAA on porcine embryos produced by HMC (Du et al., 2007). The blastocyst rate was checked 6 days after culture and the quality of the embryo was estimated by counting the cell number. In the end, to investigate the effect of reducing EAA concentration on further in vivo developmental competence of porcine HMC embryos, blastocysts generated with both wide-type and genetically modified fibroblasts were cultured in medium with optimized concentration of EAA, and then subjected to transplantation.

Materials and Methods

We purchased all chemicals from Sigma Chemical (St.Louis, MO) unless otherwise stated. All media were equilibrated in 5% CO₂ at 38.5°C for at least 4 hours before use.

Ethics statement

All procedures related to animal experiment were approved by the Life Ethics and Biological Safety Review Committee of BGI-Research

Establishment of donor cell lines

The establishment of FFs was performed as described previously (Liu et al., 2015). Briefly, pregnant recipients were sacrificed 40 days after insemination and fetuses were cut into 1 m³ pieces in 0.9% sodium chloride solution with scalpel and forceps. Small pieces were digested with both trypsin and collagenase type IV solution. After centrifugation (400 g, 6 minutes), supernatant was discarded and the cell pellets were washed in PBS twice. The digested cells were transferred into culture dish filled with Dulbecco's minimum Essential medium (DMEM; Gibco) containing 10% (v/v) fetal bovine serum (FBS), 1% glutamine (Invitrogen), and 1% nonessential amino acid for further culture at 37°C in an atmosphere of 5% CO ₂ in air until fetal fibroblasts (FFs) were confluent. The cell lines were passaged at least three times before being prepared as donor cells.

Genetically modified donor cells were also prepared as reported by Liu et al. (2015). Briefly, FFs were transfected by plasmids containing the fragments of target DNA by electroporation. Subsequently, cells were selected in DMEM containing 400 μg/mL geneticin (G418; Gibco), 15% (vol/vol) FBS, 1% l-glutamine, and 1% NEAA. Positive cell colonies were screened by PCR and passaged before being used as donors for HMC.

Collection and in vitro maturation of porcine oocytes

Abattoir-derived gilt ovaries were transported to laboratory at 38.5°C and those cumulus-oocyte complexes (COCs) in 3–6 mm follicles were recovered. Then COCs with compact cumulus cells of more than two layers were further selected in microscope and cultured in 450 μL TCM-199 containing 10% (v/v) porcine follicular fluid, 10% (v/v) FBS, 1000 IU/mL PMSG (Suigonan Vet; Skovlunde, Denmark), and 1000 IU/mL hCG. All COCs were cultured at 38.5°C in an atmosphere with 5% CO₂ for 40–44 hours in an incubator (Galaxy R+, Germany) before use.

Parthenogenetic activation of oocytes

Before electronic activation, expanded cumulus cells were denuded from oocytes with 0.2% hyaluronidase by pipetting vigorously and rinsed with T2 (HEHPS buffered TCM-199 containing 2% FBS). Denuded oocytes were placed in the fusion chamber covered with activation medium (0.1 mM MgSO₄, 0.3 M mannitol, 0.1 mM CaCl₂, and 0.01% PVA) and attached to one wire at an alternating current (AC) of 0.14 kV/cm and 700 kHz. The activation was performed with a DC pulse (80 μsec, 0.63 kV/cm). Afterward, oocytes were further activated in PZM-3 medium containing 10 μg/mL cycloheximide and 5 μg/mL cytochalasin B at 38.5°C in 90% N₂, 5% O₂, and 5% CO₂ with maximum humidity for 4–6 hours, and then flushed and cultured in pre-equilibrated PZM-3.

Handmade cloning

The process of HMC was performed as Liu et al. (2015) described before. The denuded COCs were placed in T33 containing 3.3 mg/mL pronase for about 25 seconds to partially digest zonae pellucidae, and then washed in T10 and T20 drops twice. Then oocytes were lined up in T10 drops containing 3 μg/mL cytochalasin B. The polar body was located by rotating oocytes with a fire-polished glass pipette. Under a stereomicroscope, denucleation of oocytes was completed, seperating polar body and about one thirds of its nearby cytoplasm by using Ultra Sharp Splitting Blades (AB Technology, Pullman, WA). The putative cytoplast was collected, while the part with polar body was discarded.

We performed fusion in two steps and the initiation of activation was included in the second step. Donor cells were equally distributed in a 20 μL T2 drop. Cytoplasms were stored in a drop of T0 containing 1.5 mg/mL phytohemagglutinin (PHA; ICN Pharmaceuticals, Australia) for 2 seconds, and then immediately transferred to the 20 μL T2 drop to attach a single fibroblast cell individually. Then, the cytoplast fibroblast cell pair was transferred to the fusion chamber covered with fusion medium (0.01% PVA and 0.3 M mannitol) after being equilibrated in fusion chamber for 10 seconds. Pairs were attached to fusion chamber wire with donor cell furthest away from wire by using an AC of 700 kHz and 0.6 kV/cm.

The fusion was performed with a DC of 2.0 kV/cm for 9 μs. After that, we transferred pairs to T10 drops to wait for fusion and activation. About 1 hour after fusion, fused pair and another putative cytoplast were aligned to wire of the fusion chamber with the same AC as used in the first step. Activation was completed by a single DC pulse (80 μs, kV/cm). When fusion happened, the reconstructed embryos were further activated in the same way as used in PA. After that, the embryos were rinsed and cultured in fresh PZM-3 with Well of Well system (Vajta et al., 2000).

To examine the effect of the concentration of EAA (Sigma B6766) on the developmental capacity of porcine PA embryos generated by our production system, we cultured PA embryos in the media supplemented with 0%, 1%, or 2% (v/v) EAA. The morphology of blastocysts was observed under stereomicroscope and blastocyst rates were calculated on day 6.

We further investigated the effect of concentration of EAA on the development of HMC embryos. However, medium without EAA was no longer applied. HMC embryos were cultured in the PZM-3 media supplemented with 1% or 2% (v/v) EAA. The blastocyst rates were calculated on day 6. Furthermore, the quality of embryos was assessed by mean cell number of blastocysts after Hoechst staining (Fig. 1).

FIG. 1.

Porcine blastocysts produced by HMC and stained with Hoechst on Day 6 after culture in medium containing 1% (A, C) or 2% EAA (B, D) (scale bar = 100 μm). HMC, handmade cloning; EAA, essential amino acid. Color images are available online.

The further in vivo developmental competence of embryos cultured in the medium supplemented with an optimized concentration of EAA (1%) was investigated. We first investigated in vivo developmental capacity of HMC embryos derived from WT porcine fibroblast cell lines. To investigate whether the optimized culture medium would facilitate the production of genetically modified pig models, we further investigated those embryos reconstructed with gene-modified donor cells. Day 6 blastocysts were collected and transferred into recipients as described above. Pregnancy was diagnosed by ultrasonography 4 weeks after transfer and confirmed every 2 weeks until delivery (Fig. 2).

FIG. 2.

Pregnancy of one recipient confirmed in the right horn (A) and the left horn (B) of uterus by ultrasonography 4 weeks after embryo transfer.

Embryo evaluation and nuclear staining

The development of embryos was assessed 6 days after culture (∼144 hours). Blastocysts rate were calculated. Total cell number of blastocysts was counted under a fluorescence microscope (SZX7, Olympus, Tokyo, Japan) after being fixed in glycerol containing 15 μg/mL Hoechst 33258.

Embryo transfer

Day 6 blastocysts cultured in 1% EAA group were transferred into the two uterine horns of surrogates on day 5 of estrus surgically. Four weeks after surgery, recipients were screened by ultrasonography to check pregnancy for the first time and every 2 weeks afterward. All care-related procedure of animals used in this work were authorized by the Ethics Committee of BGI.

RNA sequencing and data analysis

Smart-seq2 method was applied as described previously to obtain transcriptome data of each embryo (Picelli et al., 2013). Briefly, the embryos were recovered with a pulled glass tip and transferred into lysis buffer following reverse transcription-polymerase chain reaction, sequence-specific reverse transcription, and preamplification polymerase chain reaction. Then cDNA obtained was used to construct library and then sequenced by BGI-seq T1. RNA-seq reads were mapped to the WZSP reference genome (Minipig_v3) using HISAT2 (Kim et al., 2015) (version 2.0.4) with parameters “-q—no-unal—dta—un-conc-gz.”

Then, the expression levels of each gene were calculated by the fragments per kilobase of exons per million fragments mapped (FPKM) using StringTie (Pertea et al., 2016) with parameters “-t -C -e -B -A” based on the result of HISAT2. A twofold variance in expression levels and a p < 0.05 were used as cutoffs to define differentially expressed genes. The p value was calculated using R software (DESeq2) (Love et al., 2014). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using the R software (clusterProfiler) (Yu et al., 2012). All data were obtained from at least 3 replicates. ANOVA analysis was carried out using SPSS12.0. A value of p < 0.05 was supposed to be statistically significant.

Result

Experiment 1: Effects of the concentrations of EAAs on PA embryo development in culture

The effect of different concentrations of EAA in medium on porcine PA embryos in vitro development is shown in Table 1. The blastocyst rate was significantly increased when the concentration of EAA in culture medium was reduced from 2% to 1% (36% ± 3% vs. 54% ± 2%; p < 0.05). However, further reducing the concentration of EAA from 1% to 0% would decrease the blastocyst rate (54% ± 2% vs. 38% ± 3%; p < 0.05). Supplementing EAA to culture medium at a concentration of 0% and 2% resulted in a similar blastocyst rate (38% ± 3% vs. 36% ± 3%; p > 0.05).

Table 1.

Effect of the Concentration of Essential Amino Acids on Porcine Pathenogenetic Activation Embryo Development in Culture

Concentration of EAA in culture medium (%)	NO. of oocytes activated	NO. of blastocysts on day 6	Mean blastocyst rates ± SEM
0	114	44	38 ± 3^a
1	235	128	54 ± 2^b
2	120	44	36 ± 3^a

Values with different superscripts in the same column mean significant difference (p < 0.05). The experiment was repeated five times.

PA, pathenogenetic activation; EAA, essential amino acid.

Experiment 2: Effect of reduced EAAs on porcine HMC embryo development in culture

Based on the results of experiment 1, the HMC embryos were cultured in PZM-3 medium with two different concentrations of EAA (1%, 2%) to further evaluate the effect of the EAA on the developmental capacity of HMC embryos. As shown in Table 2, the blastocyst rate of porcine HMC embryos was significantly increased when concentration of EAA was reduced from 2% to 1% (41% ± 3% vs. 53% ± 3%; p < 0.05). However, the mean cell number of blastocysts showed no significant difference between them (38 ± 1 vs. 37 ± 2; p > 0.05) (Table 2 and Fig. 1).

Table 2.

Effect of the Concentration of Essential Amino Acids on Porcine Handmade Cloning Embryo Development in Culture

Concentration of EAA in culture medium (%)	NO. of reconstructed embryos	NO. of oocytes activated	NO. of blastocysts on day 6	Mean cell NO. of blastocysts ± SEM
1	279	148	51 ± 3^c	38 ± 1^e
2	276	114	41 ± 3^d	37 ± 2^e

Values with different superscripts in the same column mean significant difference (p < 0.05). The experiment was repeated six times.

HMC, handmade cloning.

Furthermore, Smart-seq2 was applied to investigate gene expression of embryo cultured in medium supplemented with two different concentrations of EAA. A total of 6968 genes were detected in total (Supplementary Table S1), including 199 genes that were upregulated while 177 genes were downregulated when the concentration of EAA in medium was reduced from 2% to 1% (Fig. 3A and Supplementary Table S1).

FIG. 3.

Data analysis of all DEGs in pig HMC embryo cultured in the medium containing 1% (1:100) and 2% (1:50) EAA. (A) Volcano plots showing the DEGs between 1% and 2% EAA conditions. Blue: genes upregulated in 1:100 (p < 0.05 and log2 Fold Change < -1); Red: genes downregulated in 1:100 (p < 0.05 and log2 Fold Change >2); Gray: genes with no expression change. (B) Expression levels of representative genes. (C, D) Top10 enriched GO terms (C) and KEGG pathways (D) of genes upregulated in 1% and 2% EAA conditions. DEG, differentially expressed gene. Color images are available online.

Figure 3B showed that the expression of DNA repairing-related genes such as BRCA2, REV3L, FAAP24, and TMEM123, a surface receptor involved in oncotic cell death, was significantly higher in embryos cultured in medium containing 2% EAA than in those embryos cultured in medium containing 1% EAA, while fatty acid metabolism or intracellular transport-related genes including ACAT1, ACSL6, and FABP3 was found significantly upregulated in embryos when the concentration of EAA in medium was reduced to 1%.

GO term and KEGG pathway enrichment analyses further revealed that genes that function of the 199 upregulated genes were enriched in oxidative phosphorylation, PPAR signaling pathway, and metabolic processes such as fatty acid derivative metabolic process, pyruvate metabolism, and nucleotide metabolic process (Fig. 3C, D and Supplementary Table S2), whereas those of the 177 downregulated genes were enriched in mRNA metabolic process, DNA conformation change and Fanconi anemia pathway (Fig. 3C, D and Supplementary Table S2).

Experiment 3: In vivo development of cloned porcine embryos cultured in the medium supplemented with EAA at a concentration of 1%

The in vivo developmental competence of HMC embryos cultured in the medium supplemented with 1% EAA is shown in Table 3. One thousand three hundred forty-nine HMC blastocysts reconstructed with FFs were transferred into 12 recipients, resulting in 10 pregnancies (83%) (Fig. 2). Five recipients successfully went to term and delivered 15 piglets, out of which 12 were born alive (80%). One thousand thirty-six embryos reconstructed with four genetically modified fetal fibroblasts (GMFFs) were transferred into nine recipients, resulting in seven pregnancies (78%). Six recipients went to term and delivered 16 piglets, out of which 7 were born alive (44%).

Table 3.

In Vivo Development of Cloned Porcine Embryos Cultured in the Medium Supplemented with 1% Essential Amino Acids

Type of donor cells	NO. of cloned embryos transferred on day 6	NO. of recipients	NO. of pregnant recipients (%)	NO. of farrowed recipients	NO. of piglets	NO. of piglets born alive (%)
FF	1349	12	10 (83)	5	15	12 (80)
GMFF	1036	9	7 (78)	6	16	7 (44)

FF, fetal fibroblast; GMFF, gene-modified fetal fibroblast.

Discussion

The demands for amino acids vary a lot at the different stages of embryo development (Lane and Gardner, 1997a; Steeves and Gardner, 1999; Van Winkle, 2001). However, the effect of EAA on HMC porcine embryo remains largely unknown. Culture medium PZM3 used for in vitro culture of porcine embryos was designed to contain EAA at a 1:50 dilution (2%) of commercially available solution (Sigma B6766) more than 15 years ago (Yoshioka et al., 2002) and remain unchanged so far. That might compromise the development of embryos and could be optimized.

As it has been reported that decreasing the concentration of EAA in the culture medium could enhance the developmental ability of IVF and PA embryos in bovine (Liu and Foote, 1995) and mouse (Lane et al., 2001), we therefore first investigated the effect of reduced EAA on the development of porcine PA cultured in PZM-3. EAA was added to culture medium at concentration of 0%, 1%, and 2%, and we found that 1% EAA provided the best condition for the development of porcine PA with the highest blastocyst rate. Our results are identical with the study on porcine IVF embryos (Beebe et al., 2009), although different culture systems were used. PZM-3 was used for one-step culture in our study, while NCSU 23 was used in two-step culture in Beebe's study (Beebe et al., 2009), which indicated that reduced EAA in culture medium have the beneficial effect despite different culture conditions.

No difference was observed between 0% EAA and 2% EAA, indicating that 2% EAA did not benefit from the development of porcine PA embryos, which may result from inappropriate concentration, for excess amino acids could produce ammonium that is detrimental to the development of embryos (Gardner and Lane, 1993). It has been reported that nonpolar EAA could significantly hinder the development of porcine embryo from diploids to blastocyst stage (Van Thuan et al., 2002), probably due to inappropriate concentration of EAA used in their study (2%), given our results indicated that at appropriate concentration (1%), EAA that mainly composed of nonpoplar amino aicds added to culture medium still could benefit the development of porcine PA embryo from one-cell stage to blastocyst stage.

We next investigated the effect of EAA on porcine HMC embryos cultured under the same condition as experiment 1, for many reports showed that the in vitro culture conditions of SCNT embryos were different from those of parthenogenetic or fertilized embryos (Boiani et al., 2005; Chung et al., 2002; Gupta et al., 2007; Heindryckx et al., 2001; Kim et al., 2006; Lee et al., 2004). The results showed that 1% EAA provides a better condition for HMC embryos when compared to 2% EAA, which was similar with our results in porcine PA embryos, suggesting that reducing the EAA concentration has beneficial effect on the developmental competence of porcine embryo regardless of their origins.

We also assessed the quality of the HMC embryos by calculating the blastocyst cell number. In agreement with previous report in porcine IVF embryo (Beebe et al., 2009), the mean cell number of blastocysts was not elevated by reducing concentration of EAA, which suggested that, although the number of embryo developing to blastocyst stage could be increased by reducing EAA concentration, the quality of blastocysts is not improved significantly.

One of possible reasons why blastocyst rates of porcine embryos could be elevated by reducing EAA concentration could be that ammonium in the medium was decreased (Lane et al., 2001) because surplus amino acids can spontaneously degrade into ammonium as a byproduct of metabolic activities of embryos (Gardner and Lane, 1993; Kleijkers et al., 2016; Virant-Klun et al., 2006). There were studies showing that ammonium in the culture environment was detrimental to the in vitro development of mouse embryo (Lane and Gardner, 2003; Zander et al., 2006), while production of ammonium could be decreased by reducing the concentration of EAA in the medium (Lane et al., 2001).

To gain more insight into the effect of low concentration of EAA on pig embryo development, we further investigated the gene expression pattern in HMC embryos cultured in medium containing 1% or 2% EAA by single-cell RNA sequencing. Our results suggested that, when the concentration of EAA in medium was reduced from 2% to 1%, those genes involved in oxidative phosphorylation and metabolism process such as fatty acid derivative metabolic process, pyruvate metabolism, and nucleotide metabolic process were significantly upregulated, indicating that metabolic activities were enhanced. OF note is that PPAR signaling pathway was also enriched in embryo cultured in 1% EAA. PPAR signaling pathway was found to be not only involved in lipid metabolism and glucose homeostasis (Yi et al., 2016) but also plays an important critical role in the proliferation of embryonic stem cells (Lee et al., 2009).

Besides, positive regulation of G1/S transition of mitotic cell cycle pathway was also enriched. This suggested that embryo cultured in 1% EAA medium probably has higher developmental capacity than those cultured in 2% EAA medium in the subsequent developmental stage, although we did not find any significant difference in cell number of the embryos at blastocyst stage between two groups. We also analyzed those genes upregulated in embryos cultured in medium containing 2% EAA. Interestingly, those genes were enriched in mRNA metabolic process, DNA conformation change, and Fanconi anemia pathway, which is a biochemical network that assists in DNA repair.

The expression of DNA repairing-related genes such as BRCA2, REV3L, and FAAP24 was significantly higher in embryos cultured in medium containing 2% EAA than in those embryos cultured in medium containing with 1% EAA, indicating that DNA of embryos cultured in medium containing with 2% EAA may probably be damaged by ammonium from surplus amino acids by spontaneously breaking down, and resulted in lower blastocyst rate. Besides, the expression of a surface receptor involved in oncotic cell death, TMEM123, was significantly higher in embryos cultured in medium containing 2% EAA than in those embryos cultured in medium containing 1% EAA, indicating that former probably have lower developmental capacity. However, whether the ammonium could contribute to DNA damage and how it affects cell proliferation need further investigation.

It has been reported that the conditions optimized for in vitro development of embryos may not be beneficial for in vivo development and may not lead to alive offspring (Redel et al., 2016; Spate et al., 2012). Thus, we further examined the in vivo development of HMC porcine embryos produced with optimized concentration of EAA. We first transferred WT cell line-derived HMC blastocysts cultured in the PZM-3 containing 1% EAA to 12 surrogates. The pregnancy rate was 83%, which is higher than we achieved before (Yang et al., 2016). Five recipients developed to term and delivered 15 piglets, out of which 12 were born alive. These data suggested porcine embryos cultured with optimized concentration of EAA provide high developmental capacity.

In addition, we examined the effect of optimized concentration of EAA on the in vivo development of HMC embryos derived from gene-edited donor cells, considering that one of most important applications of HMC is to produce animal model of human disease. In this test, seven of nine recipients were pregnant. One recipient aborted and the remaining successfully developed to term, giving birth to 17 genetically modified piglets. Our results indicated that our improved PZM-3 could also support to produce genetically modified pig models for the research of human diseases.

In conclusion, reducing the concentration of EAA in the medium could elevate the in vitro developmental capacity of porcine HMC embryos with a higher blastocyst rate. Ammonium spontaneously degraded from high surplus EAA can probably damage DNA of embryos. The improved PZM-3 contained an optimized concentration of EAA that could support full-term development of porcine embryos, which would not only significantly improve the production efficiency of pig model of human disease but also benefit other applications such as pig breeding and xenotransplantation.

Footnotes

Acknowledgment

We would like to thank Hongsheng Li and Xinzhi Pang from BGI Ark Biotechnology (BAB) for helping embryo reconstruction.

Author Disclosure Statement

The authors declare they have no competing financial interests. All institutional and national guidelines for the care and use of laboratory animals were followed.

Funding Information

The project is supported by National Natural Science Foundation of China (No. 81903159) and Shenzhen Municipal Government of China (No. 20170731162715261).

Supplementary Material

References

Beebe

L.F.

, Vassiliev

, McIlfatrick

, and Nottle

M.B.

(2009). Adding essential amino acids at a low concentration improves the development of in vitro fertilized porcine embryos. J. Reprod. Dev. 55, 373–377.

Boiani

, Gentile

, Gambles

V.V.

, Cavaleri

, Redi

C.A.

, and Scholer

H.R.

(2005). Variable reprogramming of the pluripotent stem cell marker Oct4 in mouse clones: distinct developmental potentials in different culture environments. Stem Cells. 23, 1089–1104.

Choi

Y.H.

, Chung

Y.G.

, Walker

S.C.

, Westhusin

M.E.

, and. Hinrichs

(2003). In vitro development of equine nuclear transfer embryos: effects of oocyte maturation media and amino acid composition during embryo culture. Zygote. 11, 77–86.

Chung

Y.G.

, Mann

M.R.

, Bartolomei

M.S.

, and Latham

K.E.

(2002). Nuclear-cytoplasmic “tug of war” during cloning: effects of somatic cell nuclei on culture medium preferences of preimplantation cloned mouse embryos. Biol. Reprod. 66, 1178–1184.

Devreker

, Hardy

, Van den Bergh

, Vannin

A.S.

, Emiliani

, and Englert

(2001). Amino acids promote human blastocyst development in vitro. Hum. Reprod. 16, 749–756.

, Kragh

P.M.

, Zhang

, Li

, Schmidt

, Bogh

I.B.

, Zhang

, Purup

, Jorgensen

A.L.

, Pedersen

A.M.

, Villemoes

, Yang

, Bolund

, and Vajta

(2007). Piglets born from handmade cloning, an innovative cloning method without micromanipulation. Theriogenology. 68, 1104–1110.

Eagle

(1959). Amino acid metabolism in mammalian cell cultures. Science. 130, 432–437.

Gardner

D.K.

, and Lane

(1993). Amino acids and ammonium regulate mouse embryo development in culture. Biol. Reprod. 48, 377–385.

Gupta

M.K.

, Uhm

S.J.

, and Lee

H.T.

(2007). Differential but beneficial effect of phytohemagglutinin on efficiency of in vitro porcine embryo production by somatic cell nuclear transfer or in vitro fertilization. Mol. Reprod. Dev. 74, 1557–1567.

10.

Heindryckx

, Rybouchkin

, Van Der Elst

, and. Dhont

(2001). Effect of culture media on in vitro development of cloned mouse embryos. Cloning. 3, 41–50.

11.

Kim

, Langmead

, and Salzberg

S.L.

(2015). HISAT: a fast spliced aligner with low memory requirements. Nat. Methods. 12, 357–360.

12.

Kim

H.S.

, Lee

G.S.

, Kim

J.H.

, Kang

S.K.

, Lee

B.C.

, and Hwang

W.S.

(2006). Expression of leptin ligand and receptor and effect of exogenous leptin supplement on in vitro development of porcine embryos. Theriogenology. 65, 831–844.

13.

Kleijkers

S.H.

, van Montfoort

A.P.

, Bekers

, Coonen

, Derhaag

J.G.

, Evers

J.L.

, and Dumoulin

J.C.

(2016). Ammonium accumulation in commercially available embryo culture media and protein supplements during storage at 2–8 degrees C and during incubation at 37 degrees C. Hum. Reprod. 31, 1192–1199.

14.

Lane

, and Gardner

D.K.

(1997a). Differential regulation of mouse embryo development and viability by amino acids. J. Reprod. Fertil. 109, 153–164.

15.

Lane

, and Gardner

D.K.

(1997b). Nonessential amino acids and glutamine decrease the time of the first three cleavage divisions and increase compaction of mouse zygotes in vitro. J. Assist. Reprod. Genet. 14, 398–403.

16.

Lane

, and Gardner

D.K.

(2003). Ammonium induces aberrant blastocyst differentiation, metabolism, pH regulation, gene expression and subsequently alters fetal development in the mouse. Biol. Reprod. 69, 1109–1117.

17.

Lane

, Hooper

, and Gardner

D.K.

(2001). Effect of essential amino acids on mouse embryo viability and ammonium production. J. Assist. Reprod. Genet. 18, 519–525.

18.

Lee

M.Y.

, Lee

Y.J.

, Kim

Y.H.

, Lee

S.H.

, Park

J.H.

, Kim

M.O.

, Suh

H.N.

, Ryu

J.M.

, Yun

S.P.

, Jang

M.W.

, and Han

H.J.

(2009). Role of peroxisome proliferator-activated receptor (PPAR)delta in embryonic stem cell proliferation. Int. J. Stem Cells. 2, 28–34.

19.

Lee

S.H.

, Kim

D.Y.

, Nam

D.H.

, Hyun

S.H.

, Lee

G.S.

, Kim

H.S.

, Lee

C.K.

, Kang

S.K.

, Lee

B.C.

, and Hwang

W.S.

(2004). Role of messenger RNA expression of platelet activating factor and its receptor in porcine in vitro-fertilized and cloned embryo development. Biol. Reprod. 71, 919–925.

20.

Liu

, Dou

, Xiang

, Li

, Lin

, Pang

, Zhang

, Chen

, Luan

, Xu

, Yang

, Liu

, Li

, Wang

, Yang

, Bolund

, Vajta

, and Du

(2015). Factors determining the efficiency of porcine somatic cell nuclear transfer: data analysis with over 200,000 reconstructed embryos. Cell Reprogram. 17, 463–471.

21.

Liu

, and Foote

R.H.

(1995). Effects of amino acids on the development of in-vitro matured/in-vitro fertilization bovine embryos in a simple protein-free medium. Hum. Reprod. 10, 2985–2991.

22.

Liu

, Foote

R.H.

, and Simkin

M.E.

(1996). Effect of amino acids and alpha-amanitin on the development of rabbit embryos in modified protein-free KSOM with HEPES. Mol. Reprod. Dev. 45, 157–162.

23.

Love

M.I.

, Huber

, and Anders

(2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550.

24.

Park

C.H.

, Jeong

Y.H.

, Jeong

Y.I.

, Kwon

J.W.

, Shin

, Hyun

S.H.

, Jeung

E.B.

, Kim

N.H.

, Seo

S.K.

, Lee

C.K.

, and Hwang

W.S.

(2014). Amino acid supplementation affects imprinted gene transcription patterns in parthenogenetic porcine blastocysts. PLoS One. 9, e106549.

25.

Pertea

, Kim

, Pertea

G.M.

, Leek

J.T.

, and Salzberg

S.L.

(2016). Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat. Protoc. 11, 1650–1667.

26.

Picelli

, Bjorklund

A.K.

, Faridani

O.R.

, Sagasser

, Winberg

, and Sandberg

(2013). Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat. Methods. 10, 1096–1098.

27.

Redel

B.K.

, Spate

L.D.

, Lee

, Mao

, Whitworth

K.M.

, and Prather

R.S.

(2016). Glycine supplementation in vitro enhances porcine preimplantation embryo cell number and decreases apoptosis but does not lead to live births. Mol. Reprod. Dev. 83, 246–258.

28.

Spate

L.D.

, Redel

B.K.

, Brown

A.N.

, Murphy

C.N.

, and Prather

R.S.

(2012). Replacement of bovine serum albumin with N-methyl-D-aspartic acid and homocysteine improves development, but not live birth. Mol. Reprod. Dev. 79, 310.

29.

Steeves

T.E.

, and Gardner

D.K.

(1999). Temporal and differential effects of amino acids on bovine embryo development in culture. Biol. Reprod. 61, 731–740.

30.

Vajta

, Peura

T.T.

, Holm

, Paldi

, Greve

, Trounson

A.O.

, and Callesen

(2000). New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system. Mol. Reprod. Dev. 55, 256–264.

31.

Van Thuan

, Harayama

, and Miyake

(2002). Characteristics of preimplantational development of porcine parthenogenetic diploids relative to the existence of amino acids in vitro. Biol. Reprod. 67, 1688–1698.

32.

Van Winkle

L.J.

(2001). Amino acid transport regulation and early embryo development. Biol. Reprod. 64, 1–12.

33.

Virant-Klun

, Tomazevic

, Vrtacnik-Bokal

, Vogler

, Krsnik

, and Meden-Vrtovec

(2006). Increased ammonium in culture medium reduces the development of human embryos to the blastocyst stage. Fertil. Steril. 85, 526–528.

34.

Yang

, Vajta

, Xu

, Luan

, Lin

, Liu

, Tian

, Dou

, Li

, Liu

, Zhang

, Li

, Yang

, Bolund

, Yang

, and Du

(2016). Production of pigs by hand-made cloning using mesenchymal stem cells and fibroblasts. Cell Reprogram. 18, 256–263.

35.

, Chen

, Sa

, Zhong

, Xing

, and Zhang

(2016). High concentrations of atmospheric ammonia induce alterations of gene expression in the breast muscle of broilers (Gallus gallus) based on RNA-Seq. BMC Genomics. 17, 598.

36.

Yoshioka

, Suzuki

, Tanaka

, Anas

I.M.

, and Iwamura

(2002). Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol. Reprod. 66, 112–119.

37.

, Wang

L.G.

, Han

, and He

Q.Y.

(2012). clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 16, 284–287.

38.

Zander

D.L.

, Thompson

J.G.

, and Lane

(2006). Perturbations in mouse embryo development and viability caused by ammonium are more severe after exposure at the cleavage stages. Biol. Reprod. 74, 288–294.

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