Theriogenology 81 (2014) 225–229
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Theriogenology journal homepage: www.theriojournal.com
Amino acid profiles in first trimester amniotic fluids of healthy bovine cloned pregnancies are similar to those of IVF pregnancies, but not nonviable cloned pregnancies Wenli Zhou a, *, Grant Gosch b, Trina Guerra a, Diane Broek b, Guoyao Wu c, Shawn Walker a, Irina Polejaeva a,1 a
ViaGen, Austin, Texas, USA Trans Ova Genetics, Sioux Center, Iowa, USA c Department of Animal Science, Texas A&M University, College Station, Texas, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2013 Received in revised form 4 September 2013 Accepted 12 September 2013
Somatic cell nuclear transfer (SCNT), or cloning, is one of the assisted reproductive technologies currently used in agriculture. Commercial applications of SCNT are presently limited to the production of animals of high genetic merit or the production of the most elite show cattle owing to its relatively low efficiency. In current practice, 20% to 40% of SCNT pregnancies do not result in viable offspring. In an effort to better understand some of the anomalies associated with SCNT pregnancies, we investigated amino acid compositions of first trimester amniotic fluid. In this retrospective study, amniotic fluids were collected from SCNT and control IVF pregnancies at Day 75 of gestation and grouped according to the pregnancy results: control IVF (IVF), viable SCNT pregnancies that resulted in live healthy calves (SCNT-HL), nonviable SCNT pregnancies that were aborted before Day 150 (SCNT-ED), and nonviable SCNT pregnancies that were aborted after Day 150 or produced deceased calves (SCNT-LD). High-performance liquid chromatography (HPLC) was used to analyze the concentrations of 22 amino acids (AAs) in the amniotic fluid samples. There were no differences in average AA concentrations between IVF and SCNTHL groups, whereas SCNT-LD and SCNT-ED had higher levels of total AA concentrations. Concentrations of asparagine, citruline, arginine, and valine were significantly higher in the SCNT-LD group. Both SCNT-LD and SCNT-ED groups had relatively large intragroup variances in AA concentrations. Urea concentration was also measured in the SCNT amniotic fluid samples. No correlations between urea concentrations and arginine concentrations or pregnancy outcomes were found. The findings in this study not only deepen the understanding on SCNT pregnancy anomalies, but also provide a potentially useful screening tool for assessing viable and nonviable SCNT pregnancies. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Cloning Amniotic fluid Amino acids Cattle IVF
1. Introduction Suboptimal reproductive efficiency of somatic cell nuclear transfer (SCNT) impedes its broader application [1]. * Corresponding author. Tel.: þ1 512 461 6317; fax: þ1 512 266 4980. E-mail address: [email protected] (W. Zhou). 1 Present address: Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322-4815. 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.09.012
Although SCNT in vitro embryo development and pregnancy establishment rates are very similar to those of IVF embryos, the loss of SCNT pregnancies at later gestational stages is significantly higher, accounting for 20% to 40% of total embryos transferred [2]. Abnormal placentation is often observed in SCNT pregnancies, and is believed to be one of the major common defects identified in unsuccessful SCNT pregnancies [3,4]. Aberrant molecular profiles in endometria and extra-embryonic tissues of SCNT transfers
226
W. Zhou et al. / Theriogenology 81 (2014) 225–229
[5,6] suggest that the placentation problem could begin as soon as embryos enter uteri. Changes in chemical compositions of amniotic fluid in SCNT pregnancies were also observed [7], indicating the alteration in the relative contribution of the fetal and placental tissues to the amniotic compartments. Thus, chemical compositions of amniotic fluid in as early as the first trimester could be potential markers for detecting early onset placentation defects and fetal anomalies. Amniotic fluid provides an environment in which the fetus and its metabolic function develop. The volume and composition of amniotic fluid are therefore strictly regulated during development, reflecting the dynamic balance maintained through exchange between maternal circulation and fetal environment [8–10]. Amniotic fluid also provides the fetus with amino acids (AAs), which are the building blocks for biomolecules involved in physiologic growth and development, and precursors of nitrogenous substances with varied regulatory functions [11]. Any aberrations may elicit fetal stress or compromise fetal development. To the best of our knowledge, there has been no report on the study of AA profiles in amniotic fluids bearing SCNT fetuses. Thus, the objective of this study was to determine if the AA profiles in first trimester amniotic fluid reflected the competency of SCNT pregnancies. In this study, we compared AA profiles of amniotic fluids from viable SCNT pregnancies that resulted in live healthy calves (SCNT-HL), nonviable SCNT pregnancies that were aborted or produced deceased calves (SCNT-ED), and IVF pregnancies with over 95% healthy calf delivery rate (SCNTLD). Amniotic fluid was drawn at Day 75 of gestation. Day 75 of gestation was selected based on the practical consideration should AA profile be used as a screening marker for SCNT pregnancy competency. At this stage, the amniotic fluid volume is greatly increased and pregnancy termination can be performed if an AA profile indicates a nonviable pregnancy. Our results show that the AA profiles between viable SCNT pregnancies and IVF pregnancies are very similar; whereas, significant differences in the AA profiles exist between nonviable SCNT pregnancies and viable SCNT or IVF pregnancies. 2. Materials and methods 2.1. Pregnancy preparation and amniotic fluid collection We produced IVF and SCNT embryos using the protocols described previously [12] with the modification that synthetic oviductal fluid medium containing 0.4% BSA was used as an embryo culture medium. On Day 7, the embryos were examined morphologically and graded using the International Embryo Transfer Society grading system [13]. Stages 4 to 8 and grades 1 to 2 were transferred to synchronized Angus recipients. Pregnancies were confirmed by ultrasonography and pregnant recipients were monitored every 30 days by ultrasonography or palpation throughout the gestation. Pregnancy outcomes including birth weight of SCNT calves were recorded. Pregnant recipients at Day 75 of gestation stage were subjected to amniocentesis. The procedure was performed as previously described [14] with the following modification: animals
were not raised in the anterior end, they remained standing horizontal in the chute and a 60 cm long 17-ga needle was used for aspiration. Approximately 10 mL of clear, bloodfree amniotic fluid was carefully drawn under ultrasound guidance. The fluid was aliquoted into Eppendorf tubes, snap frozen in liquid nitrogen, and stored at 80 C until analysis. Sixty SCNT samples were collected: 15 SCNT-ED, 21 SCNT-LD, and 14 SCNT-HL. Amniotic fluids were also drawn from 10 IVF pregnancies at the same gestational stage. Both SCNT and IVF pregnancies represented both genders and comprised various commercial breeds. 2.2. Amino acid analysis We performed AA analysis as previously described [15]. Upon arrival, amniotic fluid samples were thawed and centrifuged to remove cells and debris, followed by deproteinization with an equal volume of 1.5 mol/L HClO4. The neutralized extracts were then treated with o-phthaldialdehyde, and the AA concentrations were determined by fluorometric HPLC as described by Wu and Knabe [16]. All 20 protein-coding AAs, except for cysteine and proline, along with four other important nonprotein AAs (b-alanine, citrulline, taurine and ornithine) were analyzed. The AAs in samples were quantified using Waters Millenium-32 software [17]. Total AA concentration was determined as the sum of all measured. For each AA, both absolute concentration and relative concentration normalized to the total concentration were used in different analyses as stated. The AA concentrations were compared among the four groups (SCNT-ED, SCNT-LD, SCNT-HL, and IVF) as described. 2.3. Urea analysis Urea concentration in each amniotic fluid sample was determined using QuantiChrom Urea Assay kit (BioAsssay Systems, Haywood, CA) following the manufacturer’s protocol. 2.4. Statistical analysis The analysis was conducted using R-software version 2.13. We performed ANOVA to compare AA concentration differences among groups. The F-test was performed to compare AA concentration variances between groups. 3. Results Average individual concentration of the majority of AAs and consequently the total AA concentration were elevated in nonviable SCNT pregnancies, compared with those in the viable SCNT and control IVF pregnancies (Table 1). Asparagine (Asn), citrulline (Cit), arginine (Arg), and valine (Val) were significantly higher (P < 0.03) in SCNT-LD compared with IVF and SCNT-HL. Concentrations of serine (Ser), glutamine (Glu), threonine (Thr), taurine (Tau), tyrosine (Tyr), isoleucine (Ile), and lysine (Lys) were also elevated in the nonviable pregnancies but not significantly higher (P < 0.1). There were no differences in any of the AA concentrations between the SCNT-HL and IVF groups. Intragroup variances of AA concentrations were similar between
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Table 1 Mean value and standard deviation (SD) of amino acid concentrations in four groups of samples. AA (nmol/mL)
ASP GLU ASN SER GLN HIS GLY THR CIT ARG b-ALA TAU ALA TYR TRP MET VAL PHE ILE LEU ORN LYS Total
IVF (n ¼ 10)
SCNT-HL (n ¼ 14)
SCNT-LD (n ¼ 21)
SCNT-ED (n ¼ 15)
Mean
SD
Mean
SD
Mean
SD
Mean
SD
5.98 54.3 39.5 101 148 15.3 153 73.6 9.78 62.7 3.06 47.5 362 104 5.81 20.4 52.1 73.7 13.7 34.1 47.6 101 1527
1.94 25.8 10.1 27.1 45.9 7.14 57.1 21.2 3.52 22.0 2.68 25.1 107 21.0 10.1 16.8 16.6 27.4 2.60 10.4 25.2 43.5 377
6.35 54.1 50.2 108 159 20.5 171 78.7 11.2 78.0 3.63 46.2 376 116 7.32 25.5 68.0 89.1 17.5 38.1 48.5 114 1687
2.13 15.7 12.3 29.0 42.0 10.2 48.0 19.0 4.20 20.3 2.98 22.9 69.0 29.0 10.7 10.7 23.0 37.7 5.20 10.0 24.3 41.6 344
6.74 56.0 56.7a 125 216 30.8 207 96.6 19.4a 94.0a 5.27 74.9 405 123 8.02 30.2 93.9a 95.8 25.4 55.2 56.7 135 2016
3.05 16.3 19.2 42.0 79.0 26.8b 89.0 38.5 14.2b 23.6 4.37 53.6b 94.0 27.9 10.3 14.4 46.1b 32.1 16.2b 38.2b 30.5 56.5 646
8.60 108 53.2 135 247 35.1 288 120 18.8 68.6 9.52 53.9 612 123 12.3 57.1 124 112 41.4 87.2 67.6 153 2536
2.31 11.5 20.3b 36.4 171b 485b 121b 36.6 11.0b 27.2 3.49 24.3 229b 25.5 12.3 50.3b 72.7b 46.0 27.2b 53.6b 17.3 58.4 989
Abbreviations: ALA, Alanine; ARG, Arginine; ASN, Asparagine; ASP, Aspartic acid; b-ALA, beta-Alanine; CIT, Citrulline; GLN, Glutamine; GLU, Glutamic acid; GLY, Glycine; HIS, Histidine; ILE, Isoleucine; LEU, Leucine; LYS, Lysine; MET, Methionine; ORN, Ornithine; PHE, Phenylalanine; SER, Serine; TAU, Taurine; THR, Threonine; TRP, Tryptophan; TYR, Tyrosine; VAL, Valine. a Significantly higher mean compared with IVF (P < 0.05). b Significantly higher variance compared with IVF (P < 0.05).
SCNT-HL and IVF (Table 1). It was in the nonviable SCNT groups that greater variations of AA concentrations were observed. Compared with IVF, Cit, Val, Ile, leucine (Leu), and histidine (His) had significantly higher variances in both SCNT-LD and SCNT-ED (F-test, P < 0.05; Fig. 1). Relative AA concentrations were compared among groups. There was no difference between SCNT-HL and IVF in the relative AA concentration (Fig. 2). However, the relative concentrations of Glu, His, Cit, and all three branched-chain AA (Val, Ile, and Leu) were significantly higher in SCNT-LD. In addition, Cit and Val were elevated in SCNT-ED, whereas the
percentage of alanine (Ala) was significantly reduced in SCNT-LD. Greater Arg concentrations were found in some nonviable SCNT pregnancies, which raises the possibility that the urea level in amniotic fluid might also be elevated because Arg could be directly hydrolyzed by arginase to form urea in multiple cell types [18]. The urea concentration was then determined. There was no correlation between urea concentration in amniotic fluid and the concentration of Arg (correlation coefficient R2 ¼ 0.0132; Fig. 3).
Fig. 1. Boxplots of amino acids that had a significantly greater variance in nonviable somatic cell nuclear transfer (SCNT) pregnancies. In each panel from left to right: IVF, SCNT in healthy calves, SCNT in deceased calves, and nonviable SCNT pregnancies that were aborted before Day 150.
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Fig. 2. Average relative amount of amino acids in four groups of samples. * P < 0.05 compared with IVF.
4. Discussion Amino acids have important roles in embryonic survival, growth, and development [19]. Until now, little was known about changes in amniotic fluid AAs of compromised SCNT pregnancies. Our results show that AA concentration profiles are similar in IVF and viable SCNT pregnancies, whereas the concentrations are generally higher in amniotic fluids of nonviable SCNT pregnancies. Amniotic fluid AAs mainly come from maternal and fetal circulation through the placenta during the first trimester [10,20]. Transport can occur via a simple diffusion with glucogenic AAs such as Arg, Asn, and Val being preferably transported [6]. The fact that the concentrations of the majority of AAs listed in Table 1 were elevated in the nonviable SCNT pregnancies suggests that it was probably not the transport of any specific AA alone that was impaired in these pregnancies, but rather through broader biochemical mechanisms. One potential cause may be the abnormal SCNT placenta structure and function, which could facilitate more active transportation and metabolism of AAs. Indeed, the overall surface area and branching of the capillary networks were found to be increased in cattle SCNT placentomes [21,22]. In human patients with nonimmune hydrops fetalis, disordered capillary permeability and edema were assumed to be responsible for increased levels of AA concentrations in the patient’s amniotic fluid [23]. Hydrallantois is among the major abnormalities observed in cattle SCNT [3]. In our studies, pregnancies with a high level of AA concentration also manifested various degrees of hydrops during later gestational stages. Another potential cause of the AA accumulation in nonviable pregnancies could be owing to an increased rate of fetal apoptosis. Fetal cell apoptosis results in protein degradation and the release of AAs into amniotic fluid. In support of this view, pregnancies with very high levels of AA were usually aborted soon after the timing of amniocentesis. Greater AA concentrations could also be the result of a lesser water volume in amniotic fluid owing to impaired transport of water and ions across the placental and amniotic membranes. Amniotic fluid is rapidly accumulated during the first trimester either through fetal skin or through the mother across uterine decidua and/or placenta surface [24,25]. The area and permeability of the
membrane surface can also affect the water flow. However, as observed in an earlier study, there was no difference in amniotic fluid volume among SCNT, artificial insemination, and IVF pregnancies at Day 50 or 100 of gestation, although the biochemical composition in the SCNT amniotic fluid was noticeably changed [7]. Similarly, in this study we did not observe any volume differences during amniocentesis. Amniotic fluid is also a nutrient reservoir for the fetus, which constantly swallows amniotic fluid in the first half of pregnancy [10,20]. The composition of AA in the fetal stomach was found to be highly correlated with the composition in the amniotic fluid at Day 190 of gestation [10]. The AAs are essential building blocks of proteins and play important roles in biosynthesis, metabolism, growth, and health. Excess AA can result in severe adverse effects owing to AA imbalance and antagonism as well as production of ammonia and its toxicity to embryos [11,26]. It is possible that abnormal placenta development at an early gestational stage causes greater accumulations of AAs in amniotic fluid and, in turn, feeds the fetus and affects development at later fetal stages. We observed no correlations between AA concentrations and fetal sex, breeds, or the birth weight of live and deceased SCNT calves (SCNT-HL and SCNT-LD groups; data not shown). The SCNT pregnancies with amniocentesis were 20% more likely to be aborted by Day 150 of gestation than those without the procedure, whereas IVF pregnancies were minimally affected by the procedure (less than 5% of an increase in the abortion rate after the procedure; TransOva, unpublished data). To separate the procedure-induced
Fig. 3. Correlation between Arg and urea concentrations in amniotic fluid samples.
W. Zhou et al. / Theriogenology 81 (2014) 225–229
abortion from the nonviable pregnancies, we divided the unsuccessful SCNT pregnancies into two groups: one with abortion before Day 150 (SCNT-ED) and another with abortion after Day 150 or death at birth (SCNT-LD). Thus, the SCNT-ED group included natural abortions and abortions caused by the amniocentesis procedure itself. In another words, some of the otherwise viable pregnancies would be aborted and included in this group. As expected, the AA concentrations varied the most among SCNT-ED samples (Table 1; Fig. 1). Separating SCNT-LD from SCNT-ED allowed us to focus on the developmental defects caused by SCNT and to identify potential AA markers for nonviable SCNT pregnancies that manifest at later gestational stages. Late-stage pregnancy loss is an important factor limiting the broader adaptation of SCNT technology in livestock reproduction. Current SCNT technology imposes significant financial loss to breeders and causes various degrees of physical and emotional stress to recipients. By applying the knowledge gained from this study, one can potentially use AAs in amniotic fluid as biomarkers to identify and thereby terminate problematic pregnancies at an earlier stage of gestation. Acknowledgments The authors thank Dr. Thomas Bunch for helpful discussions and critical reading of the manuscript. References [1] Faber DC, Ferre LB, Metzger J, Robl JM, Kasinathan P. Agro-economic impact of cattle cloning. Cloning Stem Cells 2004;6:198–207. [2] Panarace M, Aguero JI, Garrote M, Jauregui G, Segovia A, Cane L, et al. How healthy are clones and their progeny: 5 years of field experience. Theriogenology 2007;67:142–51. [3] Chavatte-Palmer P, Camous S, Jammes H, Le Cleac’h N, Guillomot M, Lee RS. Review: placental perturbations induce the developmental abnormalities often observed in bovine somatic cell nuclear transfer. Placenta 2012;33(Suppl):S99–104. [4] Palmieri C, Loi P, Ptak G, Della Salda L. Review paper: a review of the pathology of abnormal placentae of somatic cell nuclear transfer clone pregnancies in cattle, sheep, and mice. Vet Pathol 2008;45: 865–80. [5] Bauersachs S, Mitko K, Ulbrich SE, Blum H, Wolf E. Transcriptome studies of bovine endometrium reveal molecular profiles characteristic for specific stages of estrous cycle and early pregnancy. Exp Clin Endocrinol Diabetes 2008;116:371–84. [6] Degrelle SA, Jaffrezic F, Campion E, Le Cao KA, Le Bourhis D, Richard C, et al. Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer. PLoS One 2012;7:e38309.
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[7] Li N, Wells DN, Peterson AJ, Lee RS. Perturbations in the biochemical composition of fetal fluids are apparent in surviving bovine somatic cell nuclear transfer pregnancies in the first half of gestation. Biol Reprod 2005;73:139–48. [8] Kwon H, Spencer TE, Bazer FW, Wu G. Developmental changes of amino acids in ovine fetal fluids. Biol Reprod 2003;68: 1813–20. [9] Wu G, Bazer FW, Tou W. Developmental changes of free amino acid concentrations in fetal fluids of pigs. J Nutr 1995;125:2859–68. [10] Baetz AL, Hubbert WT, Graham CK. Developmental changes of free amino acids in bovine fetal fluids with gestational age and the interrelationships between the amino acid concentrations in the fluid compartments. J Reprod Fertil 1975;44:437–77. [11] Wu G. Amino acids: metabolism, functions, and nutrition. Amino Acids 2009;37:1–17. [12] Zhou W, Xiang T, Walker S, Farrar V, Hwang E, Findeisen B, et al. Global gene expression analysis of bovine blastocysts produced by multiple methods. Mol Reprod Dev 2008;75:744–58. [13] Robertson I, Nelson R. Certification and identification of the embryos. In: Stringfellow DA, Seidel SM, editors. Manual of the International Embryo Transfer Society. Savoy, IL: International Embryo Transfer Society; 1998. p. 103–34. [14] Kamimura S, Nishiyama N, Ookutsu S, Goto K, Hamana K. Determination of bovine fetal sex by PCR using fetal fluid aspirated by transvaginal ultrasound-guided amniocentesis. Theriogenology 1997;47:1563–9. [15] Gao H, Wu G, Spencer TE, Johnson GA, Li X, Bazer FW. Select nutrients in the ovine uterine lumen. I. Amino acids, glucose, and ions in uterine lumenal flushings of cyclic and pregnant ewes. Biol Reprod 2009;80:86–93. [16] Wu G, Knabe DA. Free and protein-bound amino acids in sow’s colostrum and milk. J Nutr 1994;124:415–24. [17] Li X, Rezaei R, Li P, Wu G. Composition of amino acids in feed ingredients for animal diets. Amino Acids 2011;40:1159–68. [18] Wu G, Morris Jr SM. Arginine metabolism: nitric oxide and beyond. Biochem J 1998;336:1–17. [19] Bazer FW, Wu G, Johnson GA, Kim J, Song G. Uterine histotroph and conceptus development: select nutrients and secreted phosphoprotein 1 affect mechanistic target of rapamycin cell signaling in ewes. Biol Reprod 2011;85:1094–107. [20] Underwood MA, Gilbert WM, Sherman MP. Amniotic fluid: not just fetal urine anymore. J Perinatol 2005;25:341–8. [21] Lee RS, Peterson AJ, Donnison MJ, Ravelich S, Ledgard AM, Li N, et al. Cloned cattle fetuses with the same nuclear genetics are more variable than contemporary half-siblings resulting from artificial insemination and exhibit fetal and placental growth deregulation even in the first trimester. Biol Reprod 2004;70:1–11. [22] Miglino MA, Pereira FT, Visintin JA, Garcia JM, Meirelles FV, Rumpf R, et al. Placentation in cloned cattle: structure and microvascular architecture. Theriogenology 2007;68:604–17. lu M, Kuyumcuoglu U, Guzel AI, Celik Y, Kale E. Amniotic [23] Erdemog fluid amino acid levels in non-immune hydrops fetalis: a casecontrol study. Braz J Med Biol Res 2011;44:725–8. [24] Beall MH, van den Wijngaard JP, van Gemert MJ, Ross MG. Regulation of amniotic fluid volume. Placenta 2007;28:824–32. [25] Beall MH, van den Wijngaard JP, van Gemert MJ, Ross MG. Amniotic fluid water dynamics. Placenta 2007;28:816–23. [26] Wu G. Functional amino acids in growth, reproduction, and health. Adv Nutr 2010;1:31–7.
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Theriogenology journal homepage: www.theriojournal.com
Amino acid profiles in first trimester amniotic fluids of healthy bovine cloned pregnancies are similar to those of IVF pregnancies, but not nonviable cloned pregnancies Wenli Zhou a, *, Grant Gosch b, Trina Guerra a, Diane Broek b, Guoyao Wu c, Shawn Walker a, Irina Polejaeva a,1 a
ViaGen, Austin, Texas, USA Trans Ova Genetics, Sioux Center, Iowa, USA c Department of Animal Science, Texas A&M University, College Station, Texas, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2013 Received in revised form 4 September 2013 Accepted 12 September 2013
Somatic cell nuclear transfer (SCNT), or cloning, is one of the assisted reproductive technologies currently used in agriculture. Commercial applications of SCNT are presently limited to the production of animals of high genetic merit or the production of the most elite show cattle owing to its relatively low efficiency. In current practice, 20% to 40% of SCNT pregnancies do not result in viable offspring. In an effort to better understand some of the anomalies associated with SCNT pregnancies, we investigated amino acid compositions of first trimester amniotic fluid. In this retrospective study, amniotic fluids were collected from SCNT and control IVF pregnancies at Day 75 of gestation and grouped according to the pregnancy results: control IVF (IVF), viable SCNT pregnancies that resulted in live healthy calves (SCNT-HL), nonviable SCNT pregnancies that were aborted before Day 150 (SCNT-ED), and nonviable SCNT pregnancies that were aborted after Day 150 or produced deceased calves (SCNT-LD). High-performance liquid chromatography (HPLC) was used to analyze the concentrations of 22 amino acids (AAs) in the amniotic fluid samples. There were no differences in average AA concentrations between IVF and SCNTHL groups, whereas SCNT-LD and SCNT-ED had higher levels of total AA concentrations. Concentrations of asparagine, citruline, arginine, and valine were significantly higher in the SCNT-LD group. Both SCNT-LD and SCNT-ED groups had relatively large intragroup variances in AA concentrations. Urea concentration was also measured in the SCNT amniotic fluid samples. No correlations between urea concentrations and arginine concentrations or pregnancy outcomes were found. The findings in this study not only deepen the understanding on SCNT pregnancy anomalies, but also provide a potentially useful screening tool for assessing viable and nonviable SCNT pregnancies. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Cloning Amniotic fluid Amino acids Cattle IVF
1. Introduction Suboptimal reproductive efficiency of somatic cell nuclear transfer (SCNT) impedes its broader application [1]. * Corresponding author. Tel.: þ1 512 461 6317; fax: þ1 512 266 4980. E-mail address: [email protected] (W. Zhou). 1 Present address: Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322-4815. 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.09.012
Although SCNT in vitro embryo development and pregnancy establishment rates are very similar to those of IVF embryos, the loss of SCNT pregnancies at later gestational stages is significantly higher, accounting for 20% to 40% of total embryos transferred [2]. Abnormal placentation is often observed in SCNT pregnancies, and is believed to be one of the major common defects identified in unsuccessful SCNT pregnancies [3,4]. Aberrant molecular profiles in endometria and extra-embryonic tissues of SCNT transfers
226
W. Zhou et al. / Theriogenology 81 (2014) 225–229
[5,6] suggest that the placentation problem could begin as soon as embryos enter uteri. Changes in chemical compositions of amniotic fluid in SCNT pregnancies were also observed [7], indicating the alteration in the relative contribution of the fetal and placental tissues to the amniotic compartments. Thus, chemical compositions of amniotic fluid in as early as the first trimester could be potential markers for detecting early onset placentation defects and fetal anomalies. Amniotic fluid provides an environment in which the fetus and its metabolic function develop. The volume and composition of amniotic fluid are therefore strictly regulated during development, reflecting the dynamic balance maintained through exchange between maternal circulation and fetal environment [8–10]. Amniotic fluid also provides the fetus with amino acids (AAs), which are the building blocks for biomolecules involved in physiologic growth and development, and precursors of nitrogenous substances with varied regulatory functions [11]. Any aberrations may elicit fetal stress or compromise fetal development. To the best of our knowledge, there has been no report on the study of AA profiles in amniotic fluids bearing SCNT fetuses. Thus, the objective of this study was to determine if the AA profiles in first trimester amniotic fluid reflected the competency of SCNT pregnancies. In this study, we compared AA profiles of amniotic fluids from viable SCNT pregnancies that resulted in live healthy calves (SCNT-HL), nonviable SCNT pregnancies that were aborted or produced deceased calves (SCNT-ED), and IVF pregnancies with over 95% healthy calf delivery rate (SCNTLD). Amniotic fluid was drawn at Day 75 of gestation. Day 75 of gestation was selected based on the practical consideration should AA profile be used as a screening marker for SCNT pregnancy competency. At this stage, the amniotic fluid volume is greatly increased and pregnancy termination can be performed if an AA profile indicates a nonviable pregnancy. Our results show that the AA profiles between viable SCNT pregnancies and IVF pregnancies are very similar; whereas, significant differences in the AA profiles exist between nonviable SCNT pregnancies and viable SCNT or IVF pregnancies. 2. Materials and methods 2.1. Pregnancy preparation and amniotic fluid collection We produced IVF and SCNT embryos using the protocols described previously [12] with the modification that synthetic oviductal fluid medium containing 0.4% BSA was used as an embryo culture medium. On Day 7, the embryos were examined morphologically and graded using the International Embryo Transfer Society grading system [13]. Stages 4 to 8 and grades 1 to 2 were transferred to synchronized Angus recipients. Pregnancies were confirmed by ultrasonography and pregnant recipients were monitored every 30 days by ultrasonography or palpation throughout the gestation. Pregnancy outcomes including birth weight of SCNT calves were recorded. Pregnant recipients at Day 75 of gestation stage were subjected to amniocentesis. The procedure was performed as previously described [14] with the following modification: animals
were not raised in the anterior end, they remained standing horizontal in the chute and a 60 cm long 17-ga needle was used for aspiration. Approximately 10 mL of clear, bloodfree amniotic fluid was carefully drawn under ultrasound guidance. The fluid was aliquoted into Eppendorf tubes, snap frozen in liquid nitrogen, and stored at 80 C until analysis. Sixty SCNT samples were collected: 15 SCNT-ED, 21 SCNT-LD, and 14 SCNT-HL. Amniotic fluids were also drawn from 10 IVF pregnancies at the same gestational stage. Both SCNT and IVF pregnancies represented both genders and comprised various commercial breeds. 2.2. Amino acid analysis We performed AA analysis as previously described [15]. Upon arrival, amniotic fluid samples were thawed and centrifuged to remove cells and debris, followed by deproteinization with an equal volume of 1.5 mol/L HClO4. The neutralized extracts were then treated with o-phthaldialdehyde, and the AA concentrations were determined by fluorometric HPLC as described by Wu and Knabe [16]. All 20 protein-coding AAs, except for cysteine and proline, along with four other important nonprotein AAs (b-alanine, citrulline, taurine and ornithine) were analyzed. The AAs in samples were quantified using Waters Millenium-32 software [17]. Total AA concentration was determined as the sum of all measured. For each AA, both absolute concentration and relative concentration normalized to the total concentration were used in different analyses as stated. The AA concentrations were compared among the four groups (SCNT-ED, SCNT-LD, SCNT-HL, and IVF) as described. 2.3. Urea analysis Urea concentration in each amniotic fluid sample was determined using QuantiChrom Urea Assay kit (BioAsssay Systems, Haywood, CA) following the manufacturer’s protocol. 2.4. Statistical analysis The analysis was conducted using R-software version 2.13. We performed ANOVA to compare AA concentration differences among groups. The F-test was performed to compare AA concentration variances between groups. 3. Results Average individual concentration of the majority of AAs and consequently the total AA concentration were elevated in nonviable SCNT pregnancies, compared with those in the viable SCNT and control IVF pregnancies (Table 1). Asparagine (Asn), citrulline (Cit), arginine (Arg), and valine (Val) were significantly higher (P < 0.03) in SCNT-LD compared with IVF and SCNT-HL. Concentrations of serine (Ser), glutamine (Glu), threonine (Thr), taurine (Tau), tyrosine (Tyr), isoleucine (Ile), and lysine (Lys) were also elevated in the nonviable pregnancies but not significantly higher (P < 0.1). There were no differences in any of the AA concentrations between the SCNT-HL and IVF groups. Intragroup variances of AA concentrations were similar between
W. Zhou et al. / Theriogenology 81 (2014) 225–229
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Table 1 Mean value and standard deviation (SD) of amino acid concentrations in four groups of samples. AA (nmol/mL)
ASP GLU ASN SER GLN HIS GLY THR CIT ARG b-ALA TAU ALA TYR TRP MET VAL PHE ILE LEU ORN LYS Total
IVF (n ¼ 10)
SCNT-HL (n ¼ 14)
SCNT-LD (n ¼ 21)
SCNT-ED (n ¼ 15)
Mean
SD
Mean
SD
Mean
SD
Mean
SD
5.98 54.3 39.5 101 148 15.3 153 73.6 9.78 62.7 3.06 47.5 362 104 5.81 20.4 52.1 73.7 13.7 34.1 47.6 101 1527
1.94 25.8 10.1 27.1 45.9 7.14 57.1 21.2 3.52 22.0 2.68 25.1 107 21.0 10.1 16.8 16.6 27.4 2.60 10.4 25.2 43.5 377
6.35 54.1 50.2 108 159 20.5 171 78.7 11.2 78.0 3.63 46.2 376 116 7.32 25.5 68.0 89.1 17.5 38.1 48.5 114 1687
2.13 15.7 12.3 29.0 42.0 10.2 48.0 19.0 4.20 20.3 2.98 22.9 69.0 29.0 10.7 10.7 23.0 37.7 5.20 10.0 24.3 41.6 344
6.74 56.0 56.7a 125 216 30.8 207 96.6 19.4a 94.0a 5.27 74.9 405 123 8.02 30.2 93.9a 95.8 25.4 55.2 56.7 135 2016
3.05 16.3 19.2 42.0 79.0 26.8b 89.0 38.5 14.2b 23.6 4.37 53.6b 94.0 27.9 10.3 14.4 46.1b 32.1 16.2b 38.2b 30.5 56.5 646
8.60 108 53.2 135 247 35.1 288 120 18.8 68.6 9.52 53.9 612 123 12.3 57.1 124 112 41.4 87.2 67.6 153 2536
2.31 11.5 20.3b 36.4 171b 485b 121b 36.6 11.0b 27.2 3.49 24.3 229b 25.5 12.3 50.3b 72.7b 46.0 27.2b 53.6b 17.3 58.4 989
Abbreviations: ALA, Alanine; ARG, Arginine; ASN, Asparagine; ASP, Aspartic acid; b-ALA, beta-Alanine; CIT, Citrulline; GLN, Glutamine; GLU, Glutamic acid; GLY, Glycine; HIS, Histidine; ILE, Isoleucine; LEU, Leucine; LYS, Lysine; MET, Methionine; ORN, Ornithine; PHE, Phenylalanine; SER, Serine; TAU, Taurine; THR, Threonine; TRP, Tryptophan; TYR, Tyrosine; VAL, Valine. a Significantly higher mean compared with IVF (P < 0.05). b Significantly higher variance compared with IVF (P < 0.05).
SCNT-HL and IVF (Table 1). It was in the nonviable SCNT groups that greater variations of AA concentrations were observed. Compared with IVF, Cit, Val, Ile, leucine (Leu), and histidine (His) had significantly higher variances in both SCNT-LD and SCNT-ED (F-test, P < 0.05; Fig. 1). Relative AA concentrations were compared among groups. There was no difference between SCNT-HL and IVF in the relative AA concentration (Fig. 2). However, the relative concentrations of Glu, His, Cit, and all three branched-chain AA (Val, Ile, and Leu) were significantly higher in SCNT-LD. In addition, Cit and Val were elevated in SCNT-ED, whereas the
percentage of alanine (Ala) was significantly reduced in SCNT-LD. Greater Arg concentrations were found in some nonviable SCNT pregnancies, which raises the possibility that the urea level in amniotic fluid might also be elevated because Arg could be directly hydrolyzed by arginase to form urea in multiple cell types [18]. The urea concentration was then determined. There was no correlation between urea concentration in amniotic fluid and the concentration of Arg (correlation coefficient R2 ¼ 0.0132; Fig. 3).
Fig. 1. Boxplots of amino acids that had a significantly greater variance in nonviable somatic cell nuclear transfer (SCNT) pregnancies. In each panel from left to right: IVF, SCNT in healthy calves, SCNT in deceased calves, and nonviable SCNT pregnancies that were aborted before Day 150.
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Fig. 2. Average relative amount of amino acids in four groups of samples. * P < 0.05 compared with IVF.
4. Discussion Amino acids have important roles in embryonic survival, growth, and development [19]. Until now, little was known about changes in amniotic fluid AAs of compromised SCNT pregnancies. Our results show that AA concentration profiles are similar in IVF and viable SCNT pregnancies, whereas the concentrations are generally higher in amniotic fluids of nonviable SCNT pregnancies. Amniotic fluid AAs mainly come from maternal and fetal circulation through the placenta during the first trimester [10,20]. Transport can occur via a simple diffusion with glucogenic AAs such as Arg, Asn, and Val being preferably transported [6]. The fact that the concentrations of the majority of AAs listed in Table 1 were elevated in the nonviable SCNT pregnancies suggests that it was probably not the transport of any specific AA alone that was impaired in these pregnancies, but rather through broader biochemical mechanisms. One potential cause may be the abnormal SCNT placenta structure and function, which could facilitate more active transportation and metabolism of AAs. Indeed, the overall surface area and branching of the capillary networks were found to be increased in cattle SCNT placentomes [21,22]. In human patients with nonimmune hydrops fetalis, disordered capillary permeability and edema were assumed to be responsible for increased levels of AA concentrations in the patient’s amniotic fluid [23]. Hydrallantois is among the major abnormalities observed in cattle SCNT [3]. In our studies, pregnancies with a high level of AA concentration also manifested various degrees of hydrops during later gestational stages. Another potential cause of the AA accumulation in nonviable pregnancies could be owing to an increased rate of fetal apoptosis. Fetal cell apoptosis results in protein degradation and the release of AAs into amniotic fluid. In support of this view, pregnancies with very high levels of AA were usually aborted soon after the timing of amniocentesis. Greater AA concentrations could also be the result of a lesser water volume in amniotic fluid owing to impaired transport of water and ions across the placental and amniotic membranes. Amniotic fluid is rapidly accumulated during the first trimester either through fetal skin or through the mother across uterine decidua and/or placenta surface [24,25]. The area and permeability of the
membrane surface can also affect the water flow. However, as observed in an earlier study, there was no difference in amniotic fluid volume among SCNT, artificial insemination, and IVF pregnancies at Day 50 or 100 of gestation, although the biochemical composition in the SCNT amniotic fluid was noticeably changed [7]. Similarly, in this study we did not observe any volume differences during amniocentesis. Amniotic fluid is also a nutrient reservoir for the fetus, which constantly swallows amniotic fluid in the first half of pregnancy [10,20]. The composition of AA in the fetal stomach was found to be highly correlated with the composition in the amniotic fluid at Day 190 of gestation [10]. The AAs are essential building blocks of proteins and play important roles in biosynthesis, metabolism, growth, and health. Excess AA can result in severe adverse effects owing to AA imbalance and antagonism as well as production of ammonia and its toxicity to embryos [11,26]. It is possible that abnormal placenta development at an early gestational stage causes greater accumulations of AAs in amniotic fluid and, in turn, feeds the fetus and affects development at later fetal stages. We observed no correlations between AA concentrations and fetal sex, breeds, or the birth weight of live and deceased SCNT calves (SCNT-HL and SCNT-LD groups; data not shown). The SCNT pregnancies with amniocentesis were 20% more likely to be aborted by Day 150 of gestation than those without the procedure, whereas IVF pregnancies were minimally affected by the procedure (less than 5% of an increase in the abortion rate after the procedure; TransOva, unpublished data). To separate the procedure-induced
Fig. 3. Correlation between Arg and urea concentrations in amniotic fluid samples.
W. Zhou et al. / Theriogenology 81 (2014) 225–229
abortion from the nonviable pregnancies, we divided the unsuccessful SCNT pregnancies into two groups: one with abortion before Day 150 (SCNT-ED) and another with abortion after Day 150 or death at birth (SCNT-LD). Thus, the SCNT-ED group included natural abortions and abortions caused by the amniocentesis procedure itself. In another words, some of the otherwise viable pregnancies would be aborted and included in this group. As expected, the AA concentrations varied the most among SCNT-ED samples (Table 1; Fig. 1). Separating SCNT-LD from SCNT-ED allowed us to focus on the developmental defects caused by SCNT and to identify potential AA markers for nonviable SCNT pregnancies that manifest at later gestational stages. Late-stage pregnancy loss is an important factor limiting the broader adaptation of SCNT technology in livestock reproduction. Current SCNT technology imposes significant financial loss to breeders and causes various degrees of physical and emotional stress to recipients. By applying the knowledge gained from this study, one can potentially use AAs in amniotic fluid as biomarkers to identify and thereby terminate problematic pregnancies at an earlier stage of gestation. Acknowledgments The authors thank Dr. Thomas Bunch for helpful discussions and critical reading of the manuscript. References [1] Faber DC, Ferre LB, Metzger J, Robl JM, Kasinathan P. Agro-economic impact of cattle cloning. Cloning Stem Cells 2004;6:198–207. [2] Panarace M, Aguero JI, Garrote M, Jauregui G, Segovia A, Cane L, et al. How healthy are clones and their progeny: 5 years of field experience. Theriogenology 2007;67:142–51. [3] Chavatte-Palmer P, Camous S, Jammes H, Le Cleac’h N, Guillomot M, Lee RS. Review: placental perturbations induce the developmental abnormalities often observed in bovine somatic cell nuclear transfer. Placenta 2012;33(Suppl):S99–104. [4] Palmieri C, Loi P, Ptak G, Della Salda L. Review paper: a review of the pathology of abnormal placentae of somatic cell nuclear transfer clone pregnancies in cattle, sheep, and mice. Vet Pathol 2008;45: 865–80. [5] Bauersachs S, Mitko K, Ulbrich SE, Blum H, Wolf E. Transcriptome studies of bovine endometrium reveal molecular profiles characteristic for specific stages of estrous cycle and early pregnancy. Exp Clin Endocrinol Diabetes 2008;116:371–84. [6] Degrelle SA, Jaffrezic F, Campion E, Le Cao KA, Le Bourhis D, Richard C, et al. Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer. PLoS One 2012;7:e38309.
229
[7] Li N, Wells DN, Peterson AJ, Lee RS. Perturbations in the biochemical composition of fetal fluids are apparent in surviving bovine somatic cell nuclear transfer pregnancies in the first half of gestation. Biol Reprod 2005;73:139–48. [8] Kwon H, Spencer TE, Bazer FW, Wu G. Developmental changes of amino acids in ovine fetal fluids. Biol Reprod 2003;68: 1813–20. [9] Wu G, Bazer FW, Tou W. Developmental changes of free amino acid concentrations in fetal fluids of pigs. J Nutr 1995;125:2859–68. [10] Baetz AL, Hubbert WT, Graham CK. Developmental changes of free amino acids in bovine fetal fluids with gestational age and the interrelationships between the amino acid concentrations in the fluid compartments. J Reprod Fertil 1975;44:437–77. [11] Wu G. Amino acids: metabolism, functions, and nutrition. Amino Acids 2009;37:1–17. [12] Zhou W, Xiang T, Walker S, Farrar V, Hwang E, Findeisen B, et al. Global gene expression analysis of bovine blastocysts produced by multiple methods. Mol Reprod Dev 2008;75:744–58. [13] Robertson I, Nelson R. Certification and identification of the embryos. In: Stringfellow DA, Seidel SM, editors. Manual of the International Embryo Transfer Society. Savoy, IL: International Embryo Transfer Society; 1998. p. 103–34. [14] Kamimura S, Nishiyama N, Ookutsu S, Goto K, Hamana K. Determination of bovine fetal sex by PCR using fetal fluid aspirated by transvaginal ultrasound-guided amniocentesis. Theriogenology 1997;47:1563–9. [15] Gao H, Wu G, Spencer TE, Johnson GA, Li X, Bazer FW. Select nutrients in the ovine uterine lumen. I. Amino acids, glucose, and ions in uterine lumenal flushings of cyclic and pregnant ewes. Biol Reprod 2009;80:86–93. [16] Wu G, Knabe DA. Free and protein-bound amino acids in sow’s colostrum and milk. J Nutr 1994;124:415–24. [17] Li X, Rezaei R, Li P, Wu G. Composition of amino acids in feed ingredients for animal diets. Amino Acids 2011;40:1159–68. [18] Wu G, Morris Jr SM. Arginine metabolism: nitric oxide and beyond. Biochem J 1998;336:1–17. [19] Bazer FW, Wu G, Johnson GA, Kim J, Song G. Uterine histotroph and conceptus development: select nutrients and secreted phosphoprotein 1 affect mechanistic target of rapamycin cell signaling in ewes. Biol Reprod 2011;85:1094–107. [20] Underwood MA, Gilbert WM, Sherman MP. Amniotic fluid: not just fetal urine anymore. J Perinatol 2005;25:341–8. [21] Lee RS, Peterson AJ, Donnison MJ, Ravelich S, Ledgard AM, Li N, et al. Cloned cattle fetuses with the same nuclear genetics are more variable than contemporary half-siblings resulting from artificial insemination and exhibit fetal and placental growth deregulation even in the first trimester. Biol Reprod 2004;70:1–11. [22] Miglino MA, Pereira FT, Visintin JA, Garcia JM, Meirelles FV, Rumpf R, et al. Placentation in cloned cattle: structure and microvascular architecture. Theriogenology 2007;68:604–17. lu M, Kuyumcuoglu U, Guzel AI, Celik Y, Kale E. Amniotic [23] Erdemog fluid amino acid levels in non-immune hydrops fetalis: a casecontrol study. Braz J Med Biol Res 2011;44:725–8. [24] Beall MH, van den Wijngaard JP, van Gemert MJ, Ross MG. Regulation of amniotic fluid volume. Placenta 2007;28:824–32. [25] Beall MH, van den Wijngaard JP, van Gemert MJ, Ross MG. Amniotic fluid water dynamics. Placenta 2007;28:816–23. [26] Wu G. Functional amino acids in growth, reproduction, and health. Adv Nutr 2010;1:31–7.
