Reduced Amino Acids in the Bovine Uterine Lumen of Cloned versus In Vitro Fertilized Pregnancies Prior to Implantation

Abstract

Fetal overgrowth and placental abnormalities frequently occur in pregnancies following somatic cell nuclear transfer (SCNT). An optimal intrauterine supply of amino acids (AA) is of specific importance for the development of the bovine preimplantation embryo, and a defective regulation of AA supply might contribute to pregnancy failures. Thus, we analyzed 41 AA and derivatives by liquid chromatography-tandem mass spectrometry in uterine flushings of day 18 pregnant heifers carrying in vitro fertilized (IVF) or SCNT embryos, which were cultured under identical conditions until transfer to recipients. The concentrations of several AA were reduced in samples from SCNT pregnancies: L-leucine (1.8-fold), L-valine (1.6-fold), L-isoleucine (1.9-fold), L-phenylalanine (1.5-fold), L-glutamic acid (3.9-fold), L-aspartic acid (4.0-fold), L-proline (2.6-fold), L-alanine (2.0-fold), L-arginine (2.5-fold), and L-lysine (1.9-fold). The endometrial transcript abundance for the AA transporter solute carrier family 7 (amino acid transporter, L-type), member 8 (SLC7A8) was also 2.4-fold lower in SCNT pregnancies. O-phosphoethanolamine (PetN) was 11-fold (p=0.0001) reduced in the uterine fluid of animals carrying an SCNT conceptus, pointing toward changes of the phospholipid metabolism. We provide evidence for disturbed embryo–maternal interactions in the preimplantation period after transfer of SCNT embryos, which may contribute to developmental abnormalities. These are unlikely related to the major embryonic pregnancy recognition signal interferon-tau, because similar activities were detected in uterine flushings of the SCNT and IVF groups.

Introduction

Since the first cloning of a ruminant from cultured cells in the late nineties of the last century (Campbell et al., 1996; Wilmut et al., 1997), embryos produced by somatic cell nuclear transfer (SCNT) have frequently sustained a number of abnormalities leading to embryonic losses from early embryonic stages. During pregnancy, placental abnormalities occur, comprising hydroallantois and placental dysfunction, enlarged and fewer placentomes, enlarged umbilical vessels, and preterm abortion (Young et al., 1998). Newborns frequently suffer from respiratory failures, reluctance to suckle, and perinatal death (Constant et al., 2006; Young et al., 1998). Manipulations of the early blastocyst and inadequate in vitro culture conditions are supposed to cause primarily neonatal oversize. For instance, culture with serum, nonprotein nitrogen diet, asynchronous transfer, but in particular cloning by SCNT, entail the large offspring syndrome (Young et al., 1998) most likely due to aberrations in epigenetic reprogramming of somatic nuclei (Dean et al., 2001; Niemann et al., 2008). Anomalies in endometrial gene expression can be detected as early as day 18 of pregnancy by microarray approaches, suggesting aberrant signaling of the SCNT conceptus in the embryo–maternal interface (Bauersachs et al., 2009).

Bovine trophoblasts initiate the first apposition to the endometrium at day 17 postinsemination (Wathes and Wooding, 1980). During the prolonged preimplantation phase, the “uterine milk” is of importance for the supply of nutrients to permit the tremendous trophoblast elongation prior to attachment to the endometrium and subsequent formation of a placenta. Amino acids (AA) in the uterine fluid are of utmost importance for nutrient supply and protein synthesis (Gao et al., 2009c). As components of enzymes, cytokines, and hormones, AA contribute to multiple functions in biosynthesis, metabolism, and development. AA are required for the synthesis of nucleotides and may act as antioxidant agents preventing cellular damage caused by reactive oxygen species. The presence of both essential and nonessential AA is crucial to meet the requirements of a developing bovine blastocyst in vitro (Liu and Foote, 1995). Further, the amounts of AA increase during the preimplantation phase in the ewe (Gao et al., 2009c) and the cow in vivo (Groebner et al., 2011).

Ruminant preimplantation blastocysts fail to elongate in vitro. A lack of knowledge exists regarding the basic needs of a developing blastocyst after day 7. Transporter systems expressed in the membranes of diverse cell types of the ovine endometrium (Gao et al., 2000a, 2009b) mediate the uptake from the maternal blood and the directional transfer through the endometrial tissue into the uterine lumen. The selective accumulation of cationic, anionic, aromatic, or aliphatic AA is tightly regulated by either sodium-dependent or -independent membrane spanning transporter systems with different affinities for their substrates and with overlapping substrate specificity.

As altered trophoblast differentiation has been found in day 17 bovine SCNT conceptuses in addition to abnormal placental development present at early stages postimplantation (Arnold et al., 2006), we hypothesized that abnormalities in bovine cloned pregnancies may originate from disturbed embryo–maternal communication during the preimplantation period (Bauersachs et al., 2009). IVF-derived embryos were used as reference to SCNT conceptuses because both IVF and cloned embryos underwent a comparable in vitro cultivation prior to the transfer (n=2 per recipient) into the surrogate dam. Pregnancy rates were 77% for the IVF and 59% for the SCNT group. In both groups, about 40% twin pregnancies were obtained. The genetic variability was comparable in both groups as the donor fibroblast cell cultures were generated from different fetuses as previously described (Bauersachs et al., 2009). In the present study, we asked if AA abundances in the uterine lumen differ between pregnant heifers carrying IVF versus SCNT conceptuses. AA concentrations were determined by highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS). Furthermore, the mRNA levels of endometrial AA transporters were determined as a first step to clarify if the presence of cloned embryos may affect maternal AA transfer into the uterine lumen.

Material and Methods

Production of IVF and SCNT embryos, embryo transfer, and collection of samples

SCNT and IVF procedures were performed as previously described (Hiendleder et al., 2004). Embryos were cultured under identical conditions to the blastocyst stage (day 7). Briefly, putative embryos were cultured in 400-μL droplets of synthetic oviduct fluid medium supplemented with 5% estrous cow serum, 40 μL/mL of 50×BME Amino Acids Solution (#B6766, Sigma Aldrich, St. Louis, MO), and 10 μL/mL of 100×MEM Nonessential Amino Acids Solution (#M7145, Sigma Aldrich) covered with mineral oil. The culture atmosphere was 5% CO₂, 5% O₂, and 90% N₂ at 39°C and maximum humidity. Two SCNT or IVF blastocysts (grade 1) were transferred per estrus-synchronized Simmental recipient heifer (day 7 of the estrous cycle). Preparation of recipient animals and embryo transfer were done as described by Klein et al. (2006). The recipients (n=8 per group) were slaughtered 11 days later, the uteri were recovered, and flushed with 100 mL phosphate-buffered saline (PBS; pH 7.4) for the recovery of uterine fluids and conceptuses (Bauersachs et al., 2005). The flushing fluid was centrifuged at 800×g for 10 min and the supernatant was stored at −20°C until further usage (Ulbrich et al., 2009). Animals were termed pregnant if filamentous trophoblast tubes and at least 1 embryonic disc were observed. A twin pregnancy was diagnosed if two embryonic discs were detected. Endometrial tissue samples were collected, preserved, and processed for isolation of RNA as described previously (Bauersachs et al., 2009).

Antiviral activity of interferon-tau (IFNT) in uterine flushings

IFN production was measured in uterine flushings by using a bioassay based on the inhibition of the cytopathic effect of vesicular stomatitis virus (Indiana strain) on Madin–Darby bovine kidney (MDBK) cells (1.5×10⁶ cells/mL) (Rubinstein et al., 1981; Stojkovic et al., 1999). The NIH recombinant human IFN-alpha2 preparation (No. Gxa 01-901-535, NIH-Research Reference Reagent Note No. 31, 1984) was included for standardization in each assay. The standard dilution series covered a range from 1.7×10² pg/mL to 6.6×10⁻¹ pg/mL IFN-alpha2 with a 50% inhibition of the cytopathic effects at a concentration of 2.4 pg/mL IFN-alpha2. The antiviral activity was shown to be mediated by IFNT, as the effects of supernatant and an appropriate control IFNT preparation were blocked by specific anti-IFN sera (kindly provided by Dr. R.M. Roberts, University of Missouri, Columbia, MO) (Klemann et al., 1990). Recombinant bovine IFNT (PBL Biomedical Laboratories, Surrey, BC, Canada) inhibited the cytophathic effects to 50% at a concentration of 0.44 U/mL (9.4 pg/mL).

Analysis of AA in the uterine lumen

In 40 μL of uterine flushings 41 AA and derivatives were labeled by the isobaric tagging for relative and absolute quantification (iTRAQ) methodology using the AA45/32 Starter Kit according to the manufacturer's instructions (Applied Biosystems, Carlsbad, CA) and examined via LC-MS/MS (3200QTRAP LC/MS/MS, Applied Biosystems) as previously described (Kaspar et al., 2009). The data were analyzed using the Analyst^®61666; 1.5 Software. Total protein (TP) content in uterine flushings was determined by a commercially available Bicinchoninic Acid Assay (Sigma-Aldrich). To circumvent discrepancies with flushing procedures, data are shown as mean nmol AA/mg TP±SEM.

Reverse transcriptase-quantitative PCR (RT-qPCR)

Total RNA from endometrial intercaruncular tissue samples was isolated using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA) according to manufacturer's instructions. Quality of RNA was determined by the Agilent 2100 Bioanalyzer (Eukaryote total RNA Nano Assay, Agilent Technology, Palo Alto, CA). RNA integrity numbers were between 7 and 10. Quantity was spectroscopically determined at 260 nm by the Nanodrop 1000 ND-1000 (peqLab Biotechnologie GmbH). A two-step real-time RT-qPCR was conducted as described recently (Ulbrich et al., 2009). The RT-qPCR experiments were performed in agreement with the MIQE guidelines (Bustin et al., 2009). One microgram of total RNA was reverse transcribed in a volume of 60 μL by using random hexamer primers. Quantitative PCR reactions using the LightCycler DNA Master SYBR Green I protocol (Roche Diagnostics, Indianapolis, IN) were carried out. The primers listed in Table 1 were used to amplify specific fragments referring to the selected transcripts. The cycle number required to achieve a definite SYBR green fluorescence signal (Cq) was calculated by the second derivative maximum method (LightCycler software version 4.05, Roche). The Cq is correlated inversely with the logarithm of the initial template. Because polyubiquitine was not differentially expressed between the experimental groups, the transcript abundances of target genes were normalized against this reference gene. To avoid negative values while allowing an evaluation of a relative comparison between two genes, data are presented as means±SEM subtracted from the arbitrary value 30 (ΔCq). Thus, a high ΔCq resembles high gene expression and an increase of one ΔCq corresponds to a twofold increase of mRNA transcripts.

Table 1.

Sequences of Primer Pairs, Corresponding References, as Well as the Resulting Fragment Lengths Are Shown

Transporter/enzyme (alternative names)	High-affinity substrate	Primer sequence	Sequence accession ID	Product length (bp)
SLC1A1 (EAAT-3)	acidic amino acids (Glu/Asp)	AGT GTC ACT GCC ACC GCC G ACC GGT CCA GGA GCC AGT CA	U72534	145
SLC1A5 (ASCT-2)	small neutral amino acids (Ala /Ser/Cys)	CTG GGG GCG AGG TTG AGG GT TCC CAC AGG GGC GTA CCA CA	AY039236, BC123803, EF551339	176
SLC6A6 (TAUT)	β-amino acids (Tau/bAla)	CCT GGC CTG GCC TTC ATC GC GCA GGT CAA CCA AGG ACG TGA TCT G	AF260239	121
SLC7A1 (CAT-1)	basic amino acids (Arg/Lys)	AAC CTC GGG TGC CAT TGC CG TGG GCT GCT CGG GCT GGT AT	DQ399522	143
SLC7A5 (LAT-1)	branched chain, aromatic, large neutral amino acids	CCC GTG TTC GTG GGC CTG TC TGA GGG TAC GGG CGT CAG CA	AF174615, BC126651	147
SLC7A8 (LAT-2)	branched chain, aromatic, large neutral amino acids	GGC CAC CCG GGT TCA AGA CG AAG CCA GTG CGA TGA GGC CG	EU118967	167
SLC15A3 (PHT2)	di- and tripeptides, His	CCC GAC TGT GGC ACC GAC AC GGC GAT GTC CTC TTG CGG GG	BC149465	104

Statistical analysis

AA concentration and AA transporter expression data were statistically evaluated using the general linear models (GLM) procedure (SAS program package release 9.1.3; SAS Institute, Inc., Cary, NC). The model included the Group (IVF vs. SCNT) as fixed effect and IFNT concentration in the flushing medium as covariate. The effect of Group was considered significant at p<0.05. Graphs were plotted using Sigma-Plot 8.0 (SPSS Inc., Chicago, IL).

Results

Antiviral activity of IFNT in uterine flushings of heifers carrying in vitro fertilized versus cloned conceptuses

The IFNT bioactivity was similar in the uterine flushings of both groups and corresponded to 1.1×10⁴±0.2×10⁴ and 1.5×10⁴±0.3×10⁴ U/mL in IVF and SCNT pregnancies, respectively (p>0.05).

Neutral nonessential amino acids in the uterine fluid

Glycine (Gly) was the most abundant AA in uterine flushings of both groups with 232.3 versus 371.0 nmol Gly/mg TP in the uterine flushings of animals carrying SCNT versus IVF-derived conceptuses (group p=0.01) (Table 2). Luminal L-serine (Ser) was not different between the groups (p>0.05). The concentration of L-proline (Pro) was 2.6-fold less abundant in the uterine lumen of heifers pregnant with a cloned conceptuses (26.2 vs. 66.8 nmol Pro/mg TP) (group p=0.003). L-alanine (Ala) was 2.0-fold (group p=0.005) reduced in the uterine flushings of animals carrying conceptuses generated by SCNT (Ala: 93.1 vs. 184.9 nmol Ala/mg TP). L-glutamine (Gln) and L-asparagine (Asn) concentrations were 141.0 vs. 283.4 nmol Gln/mg TP (group p=0.003) and 17.1 versus 27.8 nmol Asn/mg TP (group p=0.01) in the uterine flushings of heifers carrying conceptuses generated by SCNT versus IVF.

Table 2.

Intrauterine Amino Acid (AA) Abundances of Neutral and Acidic and Cationic AA as Well as AA Derivatives Presented as Mean [nmol aa/mg Total Protein (TP)] ± SEM

			Statistical Analysis
Amino acid	IVF mean ± SEM (nmol/mg TP)	SCNT mean ± SEM (nmol/mg TP)	Group (p-value)	IFNT (p-value)
Neutral non-essential AA
Gly	371 ± 45.7	232.3 ± 25.3	0.01	0.3
Ser	131.4 ± 26.6	141.9 ± 19.6	1.0	0.07
Pro	66.7 ± 6.5	26.2 ± 5.2	0.003	0.4
Ala	184.9 ± 20.1	93.1 ± 18.3	0.005	0.5
Gln	283.4 ± 35.0	141 ± 23.2	0.003	0.3
Asn	27.8 ± 2.7	17.1 ± 2.6	0.01	0.4
Neutral essential AA
Thr	48.7 ± 7.2	39.3 ± 8.1	0.2	0.06
Phe	26.8 ± 2.9	17.3 ± 3.2	0.03	0.2
Tyr	21 ± 2.5	16.9 ± 3.5	0.3	0.2
Trp	5.7 ± 0.6	4.7 ± 0.9	0.3	0.1
Leu	41.5 ± 4.3	23.4 ± 4.2	0.007	0.3
Val	49.2 ± 4.5	29.9 ± 4.8	0.008	0.3
Ile	22.8 ± 2.4	11.8 ± 2.0	0.003	0.4
Acidic AA
Glu	247.9 ± 28.0	62.8 ± 14.8	<0.0001	0.6
Asp	46 ± 8.2	11.2 ± 2.7	0.007	0.8
Cationic AA
Lys	77 ± 6.9	40.6 ± 10.5	0.008	0.006
His	31.3 ± 3.5	25.5 ± 4.9	0.2	0.09
Arg	74.2 ± 6.0	30.2 ± 6.2	0.001	0.3
Components of the urea cycle
Orn	13.1 ± 0.9	7.5 ± 2.0	0.004	0.006
Cit	4.1 ± 0.4	3 ± 0.4	0.1	0.7
Asa	2.4 ± 0.8	nd	nd	nd
AA derivatives
Tau	79.2 ± 4.4	100 ± 9.2	0.03	0.1
PEtN	62.7 ± 9.7	5.7 ± 1.2	0.0001	0.4
Hyp	4.5 ± 0.4	3.1 ± 0.4	0.02	0.6
Car	1.6 ± 0.2	0.4 ± 0.1	0.04	0.1
M3His	3.3 ± 0.3	1.3 ± 0.2	0.0003	1.0

The statistic model included the Group (IVF vs. SCNT) as fixed effect and IFNT concentration in the uterine flushings as a covariate. The effects were considered significant at p<0.05.

Acidic amino acids in uterine fluid

Both L-glutamic acid (Glu) and L-aspartic acid (Asp) (Table 2) were 3.9-fold (group p<0.0001) and 4.1-fold (group p=0.007) less abundant in the uterine flushing of SCNT pregnancies (Glu: 62.8 vs. 247.9 nmol Glu/mg TP; Asp: 11.2 vs. 46.0 nmol Asp/mg TP).

Neutral essential amino acids in the uterine fluid

The essential AA L-threonine (Thr) (Table 2) did not differ between the two groups (44.0 nmol Thr/mg TP on average). Although the aromatic AA L-phenylalanine (Phe) was lower (group p=0.03) in the uterine flushings of heifers carrying a cloned animal (17.3 vs. 26.8 nmol Phe/mg TP), L-tyrosine (Tyr) and L-tryptophan (Trp) were not different between the groups (Tyr: 19.0 nmol Tyr/mg TP and Trp: 5.2 nmol Trp/mg TP on average). The branched chain AA L-leucine (Leu), L-valine (Val) and L-isoleucine (Ile) were 1.8-fold (23.4 vs. 41.5 nmol Leu/mg TP; group p=0.007), 1.6-fold (29.9 vs. 49.2 nmol Val/mg TP; group p=0.008) and 1.9-fold (11.8 vs. 22.8 nmol Ile/mg TP; p=0.003) less abundant in the uterine fluid derived from SCNT vs. IVF pregnancies.

Cationic amino acids in the uterine fluid

The cationic AA L-lysine (Lys) and L-arginine (Arg) in uterine flushings from SCNT pregnancies were 1.9-fold (40.6 vs. 77.1 nmol Lys/mg TP; group p=0.008, IFNT p=0.006) and 2.5-fold (30.2 vs. 74.2 nmol Arg/mg TP; group p=0.001) less abundant than in flushings from IVF pregnancies (Table 2). In contrast, the concentration of L-histidine (His) did not differ between the two groups (28.4 nmol His/mg TP on average).

Intrauterine components of the urea cycle

L-ornithine (Orn) (Table 2) was 1.8-fold less abundant in the uterine flushings of animals pregnant with a SCNT conceptus (7.5 vs. 13.1 nmol Orn/mg TP, group p=0.004, IFNT p=0.006). However, L-citrulline (Cit) (3.5 nmol Cit/mg TP on average) was not significantly different between the two groups. Argininosuccinic acid (Asa) was only detectable in the uterine lumen of heifers carrying an IVF-derived conceptus (2.4 nmol Asa/mg TP), but was below the detection limit in SCNT pregnancies.

Further AA derivatives in the uterine fluid

The uterine luminal concentration of the nonproteinogenic sulfonic acid taurine (Tau) was higher in the SCNT than IVF group (100.0 vs. 79.2 nmol Tau/mg TP, group p=0.03; Table 2). The strongest reduction in SCNT pregnancies was found for O-phosphoethanolamine (PEtN) (11-fold, 5.7 vs. 62.7 nmol PEtN/mg TP, group p=0.0001). Concentrations of hydroxyproline (Hyp) were lower in the SCNT group (3.1 vs. 4.5 nmol Hyp/mg TP on average, group p=0.02). L-carnosine (Car) and 3-methyl-L-histidine (M3His) were reduced [0.4 vs. 1.6 nmol Car/mg (group p=0.04), and 1.3 vs. 3.2 nmol M3His/mg TP (group p=0.0003), respectively] in uterine flushings from SCNT vs. IVF pregnancies. Ethanolamine (EtN average, 22.7 nmol EtN/mg TP), sarcosine (Sar average, 1.4 nmol Sar/mg TP) 1-methyl-L-histidine (M1His average, 3.0 nmol M1His/mg TP), L-alpha-amino-n-butyricacid (Abu average, 3.0 nmol Abu/mg TP) L-alpha-aminoadipicacid (Aad average, 6.3 nmol Aad/mg TP) delta-hydroxylysin (Hyl average, 2.2 nmol Hyl/mg TP), cystathione (Cth average, 0.5 nmol Cth/mg TP), and L-cystine (Cys average, 6.3 nmol Cys/mg TP) in the uterine flushings of pregnant heifers at day 18 (data not shown) were not different between the two groups (p>0.05).

Gene expression of endometrial AA transporter

The expression of the large neutral AA transporter SLC7A8 (also known as LAT2) was 2.4-fold lower in the endometrium of heifers carrying a cloned conceptus vs. an IVF-derived (mean ΔCq 23.4 vs. 24.6 SCNT; group p=0.004). The mRNA expression of further endometrial transporters was not affected by the type of conceptus (Table 3).

Table 3.

Gene Expression of Endometrial AA Transporter

	mRNA expession (log₂)		Statistical analysis
AA transporter	IVF mean ΔCq ± SEM	SCNT mean ΔCq ± SEM	Group (p-value)	IFNT (p-value)
SLC1A1	25.0 ± 0.1	25.1 ± 0.2	0.6	0.7
SLC1A5	21.7 ± 0.1	22.2 ± 0.3	0.3	0.7
SLC6A6	23.5 ± 0.1	23.2 ± 0.4	1.0	0.6
SLC7A1	24.4 ± 0.1	24.5 ± 0.3	0.8	0.2
SLC7A5	22.5 ± 0.2	21.5 ± 0.5	0.09	0.2
SLC7A8	24.6 ± 0.2	23.4 ± 0.2	0.003	0.8
SLC15A3	25.8 ± 0.1	26.0 ± 0.3	0.7	0.7

Values are shown as mean ΔCq ± SEM and were regarded as significantly different if p<0.05. The statistic model included the Group (IVF vs. SCNT) as fixed effect and IFNT concentration in the uterine flushings as a covariate. The effects were considered significant at p<0.05.

Discussion

Inadequate composition of culture media unveil the early environmental sensitivity of in vitro-derived blastocysts, frequently leading to placental abnormalities in later stages of pregnancy (Sinclair et al., 1999). Specifically, the overgrowth phenotype and the disproportionate placentomes indicate a severe metabolic issue in clone pregnancies in ruminants. An optimal temporal and spatial supply with AA is essential for the development of the conceptus (Gardner, 1998). During the prolonged ruminant preimplantation phase, AA increase considerably in the ovine (Gao et al., 2009c) and bovine (Groebner et al., 2011) uterine fluid to nourish the rapidly elongating conceptus. Insufficient supply of AA during early stages of ruminant pregnancy might cause or at least contribute to developmental deficiencies.

Alterations in endometrial gene expression during preimplantation demonstrated that the maternal endometrium responds inappropriately in case of inadequate embryonic signaling (Bauersachs et al., 2009; Mansouri-Attia et al., 2009). In the present report we demonstrate for the first time that next to a peculiar endometrial transcriptome, the uterine luminal histotroph encompassing the embryo is altered in SCNT pregnancies as well. The histotroph is particularly important for the preimplantation phase of the embryo, and alterations thereof may imply metabolic challenges affecting the development at later stages of pregnancy and adult life (Waterland and Jirtle, 2004).

The finding of altered AA concentrations in uterine flushings from clone pregnancies, specifically essential AA which cannot be synthesized by the embryo, confirm the importance of the reciprocal embryo–maternal interactions prior to implantation required for an adequate embryonic development. The histotroph consists of endometrial and embryonic secretions as well as maternal transudate, and for many signaling substances, either contribution is difficult to disentangle. But clearly, essential AA must be derived by maternal supply. Thus in our model, inadequate embryonic signaling, likely being caused by epigenetic abnormalities of SCNT embryos (Bauersachs et al., 2009; Dean et al., 2001; Santos et al., 2003; Shi et al., 2003), must have led to the inadequate maternal transport of essential AA into the uterine histotroph, which may in turn impact embryonic development. Interestingly, neither the size of the trophoblast nor the intrauterine bioactivity of IFNT were found to differ between cloned and IVF-derived conceptuses (Stojkovic et al., 1999), which is in line with our observations. A significant effect of intrauterine IFNT concentrations was only found for the cationic AA Lys and Orn. Thus, the differences in intrauterine AA concentrations are mainly independent of the predominant ruminant pregnancy recognition signal IFNT. Further investigation is needed to elucidate other specific signals involved.

Apart from the concentrations of essential AA such as Phe and Arg and branched chain AA, nonessential neutral and acidic AA were additionally reduced in the uterine fluid of SCNT pregnancies. The small neutral AA Pro and Ala were 1.6- to 2.6-fold less abundant in the uterine fluids of heifers carrying an SCNT conceptus, while intrauterine acidic AA Glu and Asp were found 75% less abundant. Enzymatic facilities for the synthesis of nonessential AA are entirely available in mammals, wherefore these AA can be easily converted and therefore serve as additional energy substrate as well as anaplerotic molecules for replenishing intermediates of the citrate cycle, allowing the development of the fast growing conceptus. In particular, the acidic AA Asp can be converted into oxalacetate, whereas Glu enters the citrate cycle upon conversion into alpha-ketogluterate. Nonessential AA such as Ala, and less likely Gln and Arg, are additionally required for the fixation of excessive amounts of ammonium to avoid an accumulation of free ions due to their toxic effects (Orsi and Leese, 2004). The reduced intrauterine concentrations of small neutral and acidic AA in the presence of a conceptus derived by SCNT may thus implicate difficulties in mobilization of adequate amounts of nutrients for the rapid elongation process.

Cationic AA regulate a number of metabolic processes, and the reduced availability of the latter may imply developmental failures of the early embryo. In particular, Arg is required for the synthesis of multiple signaling molecules such as nitric oxide acting on vasodilation, angiogenesis, tissue remodeling, and regulation of apoptosis. Low Arg bioavailability in human placentas causes severe damages due to insufficient nitric oxide dependent vasodilatation and excess formation of reactive oxygen species associated with preeclampsia (Noris et al., 2004). Ornithine, derived from Arg, is a substrate for the synthesis of polyamines regulating protein synthesis and angiogenesis during ovine placentation (Kwon et al., 2003). Lys in its posttranslational modified form hydroxylysine (Hyl) is a main component of collagen in the extracellular matrix. In preparation of implantation and placentation, impaired induction of tissue remodeling and vasculogenesis due to insufficient supply of required AA might be involved in abnormal placentome formation, cuboidal chorionic epithelium structure, and decreased allantoic blood vessel development often occurring in pregnancies with SCNT-derived embryos (Hill et al. 2000, 2001). Increased placentomes and fetal oversize in later pregnancy stages might be entailed by compensatory mechanisms due to the reduced availability of numerous proteinogenic AA at preimplantation.

Endometrial SLC7A8 mRNA was less expressed in the endometrium of SCNT pregnancies, while the expression of SLC7A5 (also known as LAT1) was not affected. Covalently associated with the noncatalytic subunit 4F2hc/CD98, both transporters mediate the transmembrane transfer of essential large neutral aromatic and branched chain AA independent of sodium ions (Pineda et al., 1999). SLC7A5 and SLC7A8 act as obligatory exchanger with a 1:1 stoichiometry. In contrast to SLC7A5, mainly expressed in growing cells, SLC7A8 rather mediates the directed basolateral transport in differentiated cells (Verrey, 2003). However, due to its broader substrate range, SLC7A8 also transports smaller neutral AA. In the endometrium of pregnant ewes, SLC7A8 is most abundant in the luminal epithelial and subepithelial stroma with highest expression during the elongation phase between days 16 and 20 (Gao et al., 2009b). The decreased supply of intrauterine essential AA in the presence of a SCNT conceptus might lead to reduced protein synthesis of the elongating trophoblast. As each branched chain AA was approximately twofold less abundant, the SCNT conceptuses possibly displayed a reduced ability to direct the maternal transport into the uterine fluid, potentially mediated via SLC7A8. Defective signaling mechanisms provided by the conceptus might account for decreased activity of the maternal transport systems independent of IFNT resulting in a defective implantation and placentation with increased placentomes in order to compensate early nutrient deficiency during the early stages.

As components of biological membranes, phospholipids are required for various regulatory processes and act as precursor molecules for many bioactive substances, such as eicosanoids and lysophospholipids. In the mouse, the distribution of distinct phospholipids is known to change during implantation and phospholipids were shown to be involved in cell death and angiogenic events at implantation sites (Burnum et al., 2009). Disturbances in sphingolipid metabolism were demonstrated to cause pregnancy loss by inadequate blood vessel formation and excessive cell death (Mizugishi et al., 2007). O-phosphoethanolamine (PEtN), dramatically reduced in SCNT pregnancies, is a degradation product of phospholipids. Due to the differences in implantation and placentation between species it is of question whether phospholipids act in the same manner during implantation in ruminants, but a contribution of phospholipids in bovine pregnancy might be assumed. Prostaglandins (PG) are central eicosanoid lipid molecules of major importance during numerous reproductive events. Phospholipase A2 mediates the release of arachidonic acid from membranal phospholipids and cyclooxygenase subsequently catalyzes the reaction from arachidonic acid to PGH2, the precursor molecule of functional PG. Increases of PG in the uterine lumen appear to be essential for successful embryonic development in the uterine fluid prior to implantation (Ulbrich et al., 2009). If PEtN were a byproduct of PG synthesis, further clarification would thus be needed to evidence whether the 11-fold reduced PEtN in SCNT pregnancies might be a sign for a modified capacity of cloned conceptuses to affect maternal PG synthesis.

Due to the termination of pregnancy at peri-implantation, it is not possible to further predict pregnancy outcome and offspring viability of the embryos studied herein. However, severe failures are known to be associated with pregnancies of cloned embryos (Dean et al., 2001). Our results suggest that SCNT embryos appear to be less capable of inducing the endometrial transport of AA into the uterine lumen. This not only emphasizes the importance of a well-orchestrated embryo–maternal crosstalk prior to implantation, but specifically draws attention to the signaling capacity of the embryo to drive the maternal environment allocating optimal growth conditions. The decreased supply of numerous AA and derivatives in SCNT pregnancies might affect the development of the fast growing preattachment conceptus and entail placental abnormalities in later stages of pregnancy. As a consequence, overcompensation in later stages of implantation and placentation may result in enlarged placentomes and fetal oversize. This hypothesis is in line with observations of repeated ultrasound monitoring of pregnancies, that is, initially reduced growth of cloned bovine fetuses compared to fetuses derived by fertilization (Chavatte-Palmer et al., 2006). The data evidence the orchestrated signaling beyond the predominant pregnancy recognition signal IFNT required for the adequate accommodation of the preimplantation embryo. Our animal model thus may likely be used to further identify signaling mechanisms possibly disturbed during epigenetic reprogramming in SCNT embryos, but of principal importance during early preimplantation development.

Footnotes

Acknowledgments

We greatly acknowledge the support of the German Research Foundation (UL 350/1-2, FOR 478) and, in part, by the Federal Ministry of Education and Research (FUGATOplus- REMEDY) in addition to the generous financial support by the Society for Reproduction and Fertility and the Gender Issue Incentive Fund (Technische Universitaet Muenchen). We sincerely thank Angela Sachsenhauser for excellent technical assistance, and Gabriele Schmidt and Ronny Scheundel for conducting the LC-MS/MS.

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

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