Hostname: page-component-77f85d65b8-g98kq Total loading time: 0 Render date: 2026-04-18T03:40:59.761Z Has data issue: false hasContentIssue false
  • English
  • Français

Interferon stimulated genes as peripheral diagnostic markers of early pregnancy in sheep: a critical assessment

Published online by Cambridge University Press:  06 May 2016

V. Mauffré ,
B. Grimard ,
C. Eozenou ,
S. Inghels ,
L. Silva ,
C. Giraud-Delville ,
D. Capo ,
O. Sandra  and
F. Constant
V. Mauffré*
Affiliation:
Ecole Nationale Vétérinaire d’Alfort, UMR 1198 Biologie du Développement et Reproduction, Université Paris Est, 7 av du général de Gaulle, F-94704 Maisons-Alfort Cedex, France UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
B. Grimard
Affiliation:
Ecole Nationale Vétérinaire d’Alfort, UMR 1198 Biologie du Développement et Reproduction, Université Paris Est, 7 av du général de Gaulle, F-94704 Maisons-Alfort Cedex, France UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
C. Eozenou
Affiliation:
UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
S. Inghels
Affiliation:
Ecole Nationale Vétérinaire d’Alfort, UMR 1198 Biologie du Développement et Reproduction, Université Paris Est, 7 av du général de Gaulle, F-94704 Maisons-Alfort Cedex, France UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
L. Silva
Affiliation:
Ecole Nationale Vétérinaire d’Alfort, UMR 1198 Biologie du Développement et Reproduction, Université Paris Est, 7 av du général de Gaulle, F-94704 Maisons-Alfort Cedex, France UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
C. Giraud-Delville
Affiliation:
UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
D. Capo
Affiliation:
INRA, UE 1298 Unité Commune d’Expérimentation Animale, Domaine de Bressonvilliers, F-91630 Leudeville, France
O. Sandra
Affiliation:
UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
F. Constant
Affiliation:
Ecole Nationale Vétérinaire d’Alfort, UMR 1198 Biologie du Développement et Reproduction, Université Paris Est, 7 av du général de Gaulle, F-94704 Maisons-Alfort Cedex, France UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
*
† Email: vincent.mauffre@vet-alfort.fr
Get access

Abstract

We investigated the diagnostic reliability of pregnancy detection using changes in interferon stimulated gene (ISG) messenger RNA (mRNA) levels in circulating immune cells in ewes. Two different groups of ewes (an experimental group, experiment 1 and a farm group, experiment 2) were oestrus-synchronized and blood sampled on day 14 (D0=day of insemination in control animals, experiment 1) and day 15 (experiment 2). Real-time PCR were performed to evaluate the abundance of different ISG mRNAs. In the experimental group, peripheral blood mononuclear cells of 29 ewes born and bred in experimental facilities were isolated using a Percoll gradient method. Gene expression for Chemokine (C-X-C motif) ligand 10 (CXCL10), Myxovirus (influenza virus) resistance 1 (MX1) and Signal transducer and activator of transcription 1 (STAT1) mRNA were, respectively, 8.3-fold, 6.1-fold and 2.7-fold higher (P<0.001) in pregnant compared with non-pregnant ewes. The receiver operating characteristic (ROC) curves generated from the real-time PCR data demonstrated that a reliable cut-off could be established for CXCL10, MX1 and STAT1. In the farm group of animals, peripheral blood leucocytes of 37 cross-bred multiparous ewes bought from several herds were isolated using the PAXgene® procedure. This blood sampling procedure is achievable in farms, whereas the Percoll method is not. No significant differences (P>0.10) in CXCL10, STAT1, MX1, Myxovirus (influenza virus) resistance 2 (MX2) and ISG15 ubiquitin-like modifier (ISG15) mRNA expression were found between pregnant and non-pregnant ewes. The ROC curves and the hierarchical classification generated from the real-time PCR data failed to discriminate between pregnant and non-pregnant animals. In this group of animals, our results show a strong variability in ISG expression patterns: 17% of animals identified as non-pregnant by the five tests were in fact pregnant, only 52% of pregnant animals had at least two positive results (two genes above threshold), whereas up to five positive results (five genes above threshold) were needed to avoid misclassification. In conclusion, this study illustrates the high variability in ISG expression levels in immune circulating cells during early pregnancy and, therefore, highlights the limits of using ISG expression levels in blood samples, collected on PAXgene® tubes on farms, for early pregnancy detection in sheep.

Keywords

interferon stimulated genespregnancy diagnosisearly pregnancyimplantationsheep

Information

Type
Research Article
Information
animal , Volume 10 , Issue 11 , November 2016 , pp. 1856 - 1863
Copyright
© The Animal Consortium 2016 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Article purchase

Temporarily unavailable

References

Andronicos, N, Hunt, P and Windon, R 2010. Expression of genes in gastrointestinal and lymphatic tissues during parasite infection in sheep genetically resistant or susceptible to trichostrongylus colubriformis and haemonchus contortus. International Journal for Parasitology 40, 417–429.Google Scholar
Bromfield, JJ, Santos, JEP, Block, J, Williams, RS and Sheldon, IM 2015. Physiology and endocrinology symposium: uterine infection: linking infection and innate immunity with infertility in the high-producing dairy cow. Journal of Animal Science 93, 2021–2033.Google Scholar
Carvalho, AV, Eozenou, C, Healey, GD, Forde, N, Reinaud, P, Chebrout, M, Gall, L, Rodde, N, Padilla, AL, Delville, CG, Leveugle, M, Richard, C, Sheldon, IM, Lonergan, P, Jolivet, G and Sandra, O 2016. Analysis of STAT1 expression and biological activity reveals interferon-tau-dependent STAT1-regulated SOCS genes in the bovine endometrium. Reproduction, Fertility and Development 28, 459–474.Google Scholar
Colazo, MG and Mapletoft, RJ 2014. A review of current timed-AI (TAI) programs for beef and dairy cattle. The Canadian Veterinary Journal 55, 772–780.Google Scholar
Diskin, MG and Morris, DG 2008. Embryonic and early foetal losses in cattle and other ruminants. Reproduction in Domestic Animals 43, 260–267.CrossRefGoogle ScholarPubMed
Düvel, A, Maaß, J, Heppelmann, M, Hussen, J, Koy, M, Piechotta, M, Sandra, O, Smith, DGE, Sheldon, IM, Dieuzy-Labaye, I, Zieger, P and Schuberth, HJ 2014. Peripheral blood leukocytes of cows with subclinical endometritis show an altered cellular composition and gene expression. Theriogenology 81, 906–917.Google Scholar
Gifford, CA, Racicot, K, Clark, DS, Austin, KJ, Hansen, TR, Lucy, MC, Davies, CJ and Ott, TL 2007. Regulation of interferon-stimulated genes in peripheral blood leukocytes in pregnant and bred, nonpregnant dairy cows. Journal of Dairy Science 90, 274–280.CrossRefGoogle ScholarPubMed
Gray, CA, Abbey, CA, Beremand, PD, Choi, Y, Farmer, JL, Adelson, DL, Thomas, TL, Bazer, FW and Spencer, TE 2006. Identification of endometrial genes regulated by early pregnancy, progesterone, and interferon tau in the ovine uterus. Biology of Reproduction 74, 383–394.CrossRefGoogle ScholarPubMed
Green, JC, Okamura, CS, Poock, SE and Lucy, MC 2010a. Measurement of interferon-tau (IFN-τ) stimulated gene expression in blood leukocytes for pregnancy diagnosis within 18–20 days after insemination in dairy cattle. Animal Reproduction Science 121, 24–33.CrossRefGoogle Scholar
Green, JC, Okamura, CS, Mathew, DJ, Newsom, EM and Lucy, MC 2010b. Hot topic: successful fixed-time insemination within 21 days after first insemination by combining chemical pregnancy diagnosis on day 18 with a rapid resynchronization program. Journal of Dairy Science 93, 5668–5672.CrossRefGoogle Scholar
Greiner, M, Pfeiffer, D and Smith, RD 2000. Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. Preventive Veterinary Medicine 45, 23–41.Google Scholar
Hammerle-Fickinger, A, Riedmaier, I, Becker, C, Meyer, HD, Pfaffl, M and Ulbrich, S 2010. Validation of extraction methods for total RNA and miRNA from bovine blood prior to quantitative gene expression analyses. Biotechnology Letters 32, 35–44.Google Scholar
Han, H, Austin, KJ, Rempel, LA and Hansen, TR 2006. Low blood ISG15 mRNA and progesterone levels are predictive of non-pregnant dairy cows. Journal of Endocrinology 191, 505–512.CrossRefGoogle ScholarPubMed
Jiang, L, Sorensen, P, Rontved, C, Vels, L and Ingvartsen, K 2008. Gene expression profiling of liver from dairy cows treated intra-mammary with lipopolysaccharide. BMC Genomics 9, 443.Google Scholar
Johnson, H, Torres, CG, Carvallo, F, Duchens, M and Peralta, OA 2015. Endometrial expression of selected transcripts in postpartum of primiparous Holstein cows with clinical and subclinical endometritis. Animal Reproduction Science 156, 34–39.CrossRefGoogle ScholarPubMed
Kizaki, K, Shichijo-Kizaki, A, Furusawa, T, Takahashi, T, Hosoe, M and Hashizume, K 2013. Differential neutrophil gene expression in early bovine pregnancy. Reproductive Biology and Endocrinology 11, 6.CrossRefGoogle ScholarPubMed
Lucy, MC 2001. Reproductive loss in high-producing dairy cattle: where will it end? Journal of Dairy Science 84, 1277–1293.Google Scholar
Mitterhuemer, S, Petzl, W, Krebs, S, Mehne, D, Klanner, A, Wolf, E, Zerbe, H and Blum, H 2010. Escherichia coli infection induces distinct local and systemic transcriptome responses in the mammary gland. BMC Genomics 11, 138.CrossRefGoogle ScholarPubMed
Oliveira, JF, Henkes, LE, Ashley, RL, Purcell, SH, Smirnova, NP, Veeramachaneni, DNR, Anthony, RV and Hansen, TR 2008. Expression of interferon (IFN)-stimulated genes in extrauterine tissues during early pregnancy in sheep is the consequence of endocrine IFN-τ release from the uterine vein. Endocrinology 149, 1252–1259.CrossRefGoogle ScholarPubMed
Ott, TL and Gifford, CA 2010. Effects of early conceptus signals on circulating immune cells: lessons from domestic ruminants. American Journal of Reproductive Immunology 64, 245–254.Google Scholar
Prezeau, N, Silvy, M, Gabert, J and Picard, C 2006. Assessment of a new RNA stabilizing reagent (Tempus Blood RNA) for minimal residual disease in onco-hematology using the EAC protocol. Leukemia Research 30, 569–574.Google Scholar
Pugliesi, G, Miagawa, BT, Paiva, YN, França, MR, Silva, LA and Binelli, M 2014. Conceptus-induced changes in the gene expression of blood immune cells and the ultrasound-accessed luteal function in beef cattle: how early can we detect pregnancy? Biology of Reproduction 91, 95.Google Scholar
Rainard, P, Cunha, P, Bougarn, S, Fromageau, A, Rossignol, C, Gilbert, FB and Berthon, P 2013. T Helper 17-associated cytokines are produced during antigen-specific inflammation in the mammary gland. PLoS One 8, e63471.Google Scholar
Rodrigues Hoffmann, A, Dorniak, P, Filant, J, Dunlap, KA, Bazer, FW, de la Concha-Bermejillo, A, Welsh, CJ, Varner, P and Edwards, JF 2013. Ovine fetal immune response to cache valley virus infection. Journal of Virology 87, 5586–5592.CrossRefGoogle ScholarPubMed
Romano, JE and Christians, CJ 2008. Early pregnancy diagnosis by transrectal ultrasonography in ewes. Small Ruminant Research 77, 51–57.CrossRefGoogle Scholar
Romano, JE and Larson, JE 2010. Accuracy of pregnancy specific protein-B test for early pregnancy diagnosis in dairy cattle. Theriogenology 74, 932–939.Google Scholar
Romano, JE, Thompson, JA, Forrest, DW, Westhusin, ME, Tomaszweski, MA and Kraemer, DC 2006. Early pregnancy diagnosis by transrectal ultrasonography in dairy cattle. Theriogenology 66, 1034–1041.Google Scholar
Schalm, OW and Jain, NC 1986. Schalm’s veterinary hematology. Lea & Febiger, Philadelphia, PA, USA.Google Scholar
Scott, JL, Ketheesan, N and Summers, PM 2009. Spermatozoa and seminal plasma induce a greater inflammatory response in the ovine uterus at oestrus than dioestrus. Reproduction, Fertility and Development 21, 817–826.Google Scholar
Sheridan, MP, Browne, JA, MacHugh, DE, Costello, E and Gormley, E 2012. Impact of delayed processing of bovine peripheral blood on differential gene expression. Veterinary Immunology and Immunopathology 145, 199–205.Google Scholar
Shirasuna, K, Matsumoto, H, Kobayashi, E, Nitta, A, Haneda, S, Matsui, M, Kawashima, C, Kida, K, Shimizu, T and Miyamoto, A 2012. Upregulation of interferon-stimulated genes and interleukin-10 in peripheral blood immune cells during early pregnancy in dairy cows. Journal of Reproduction and Development 58, 84–90.Google Scholar
Spencer, TE, Sandra, O and Wolf, E 2008. Genes involved in conceptus – endometrial interactions in ruminants: insights from reductionism and thoughts on holistic approaches. Reproduction 135, 165–179.Google Scholar
Stevenson, JL, Dalton, JC, Ott, TL, Racicot, KE and Chebel, RC 2007. Correlation between reproductive status and steady-state messenger ribonucleic acid levels of the Myxovirus resistance gene, MX2, in peripheral blood leukocytes of dairy heifers. Journal of Animal Science 85, 2163–2172.CrossRefGoogle ScholarPubMed
Stevenson, JS, Cartmill, JA, Hensley, BA and El-Zarkouny, SZ 2003. Conception rates of dairy cows following early not-pregnant diagnosis by ultrasonography and subsequent treatments with shortened ovsynch protocol. Theriogenology 60, 475–483.CrossRefGoogle ScholarPubMed
Yankey, S, Hicks, B, Carnahan, K, Assiri, A, Sinor, S, Kodali, K, Stellflug, J and Ott, T 2001. Expression of the antiviral protein Mx in peripheral blood mononuclear cells of pregnant and bred, non-pregnant ewes. Journal of Endocrinology 170, R7–R11.CrossRefGoogle ScholarPubMed