Expression profile of interferon tau–stimulated genes in ovine peripheral blood leukocytes during embryonic death

Theriogenology 85 (2016) 1161–1166

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Expression profile of interferon tau–stimulated genes in ovine peripheral blood leukocytes during embryonic death M. Kose a, M.S. Kaya b, N. Aydilek b, I. Kucukaslan a, T. Bayril c, S. Bademkiran a, Z. Kiyma d, N. Ozyurtlu a, S.A. Kayis e, A. Guzeloglu f, M.O. Atli a, * a

Faculty of Veterinary Medicine, Department of Obstetrics and Gynaecology, Dicle University, Diyarbakir, Turkey Faculty of Veterinary Medicine, Department of Physiology, Dicle University, Diyarbakir, Turkey Faculty of Veterinary Medicine, Department of Animal Husbandry, Dicle University, Diyarbakir, Turkey d Faculty of Agriculture, Department of Animal Husbandry, Osmangazi University, Eskisehir, Turkey e Faculty of Medicine, Department of Biostatistics, Karabuk University, Karabuk, Turkey f Faculty of Veterinary Medicine, Department of Genetics, Selcuk University, Konya, Turkey b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 July 2015 Received in revised form 25 November 2015 Accepted 30 November 2015

Early and efficient detection of embryonic death (ED) has a valuable impact as important as early pregnancy diagnosis in ruminants. Among early pregnancy diagnosis methods, detection of the expression of interferon tau–stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) is well documented in cows and ewes. Therefore, we hypothesized that the expression profile of ISGs in PBLs might also be useful for detecting ED in these animals. For this purpose, pregnant ewes were used as an experimental model. Pregnancy was detected on Day 18 after mating by transrectal ultrasonography. Pregnant ewes were divided into a control group (sham injection on Day 18, n ¼ 10) and ED group (treated with 75 mg synthetic PGF2a on Day 18, n ¼ 12). PBLs and plasma were collected on Days 0 (mating day), 15, 18, 19, 20, 21, 23, and 25 by jugular venipuncture. Total RNA was isolated from PBLs. ISGs expression levels were determined by real-time polymerase chain reaction in triplicate. Electrochemiluminescence immunoassay was used to measure progesterone (P4) levels in plasma. In the ED group, the P4 level declined to less than 1 ng/mL on Day 19 and remained at a low level until the end of the study. Compared with that on Day 0, receptor transporter protein 4 (RTP4) and ISG15 expression was upregulated on Day 15 and remained high until Day 21 in both groups, and RTP4 and ISG15 mRNA levels were attenuated on Days 23 and 25 only in the ED group (P < 0.001). Myxovirus resistance 1 expression was upregulated on Day 15 and remained high until Day 23 in both groups, but was attenuated on Day 25 in the ED group (P < 0.05). The B2-microglobulin mRNA level did not change significantly during the study in either group. These results indicate that the decline in P4 concentration was an immediate response to PGF2a and that the embryo may have survived longer than the CL on the basis of the extended period of ISGs expression. This suggests that the absence of P4 could be the reason for ED rather than a direct effect of PGF2a. In conclusion, the expression of ISGs, including ISG15, RTP4, and myxovirus resistance 1, but not B2-microglobulin, in PBLs may serve as a marker of ED. Ó 2016 Elsevier Inc. All rights reserved.

Keywords: Interferon tau–stimulated genes Peripheral blood leukocytes Embryonic death Ewe

1. Introduction * Corresponding author. Tel.: þ90 412 248 8020; fax: þ90 412 248 8021. E-mail address: [email protected] (M.O. Atli). 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.11.032

The establishment and maintenance of pregnancy in ruminants including maternal recognition of pregnancy, implantation, and placentation depend on communication

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between the maternal system and the conceptus, and are regulated by many factors such as progesterone (P4) and interferon tau (IFN-tau). Communication failures most frequently result in termination of the pregnancy [1]. Despite the high fertilization success rate in ruminants, low birth rates clearly indicate the occurrence of embryonic death (ED) and fetal losses during pregnancy [2]. Most EDs occur during the periimplantation stage of pregnancy and directly affect fertility by extending the pregnancy interval and reducing offspring number in many species, including ruminants [3]. Most of the pregnancy losses (20%–30%) occur during the embryonic stage of pregnancy in ewes [4,5]. At present, one of the most effective fertility management strategies to prevent and reduce economic losses is to identify pregnant or nonpregnant animals as soon as possible after insemination, together with application of resynchronization programs in modern commercial enterprises [6,7]. Therefore, early and efficient detection of ED tends to increase reproductive profitability. Many techniques for monitoring ED have been evaluated for ruminants. Ultrasonography is used as a detection method during earlier stages of pregnancy; however, it requires a skilled veterinarian [4]. Measurement of progesterone levels has disadvantages in terms of not being specific to pregnancy, and the occurrence of falsepositive results because of extended interestrus intervals [8]. Pregnancy-associated glycoproteins are also not promising for this purpose, owing to their long half-life in blood plasma [8–11]. Apart from endometrium, increased expression profiles of genes, called interferon stimulated genes (ISGs; myxovirus resistance 1 (MX1), MX2, ISG15, receptor transporter protein 4 (RTP-4), the 20 -50 -oligoadenylate synthase 1, and so forth), are also found outside of uterine tissues, such as in the CL and peripheral blood leukocytes (PBLs), during pregnancy [12,13]. The question is whether extrauterine presence of ISGs can be used for the early, noninvasive detection of pregnancy. Initially, Yankey et al. [14] reported significant increases in MX1 and MX2 mRNA levels on Day 15 after insemination in the pregnant ewes. Subsequent studies have confirmed these results in dairy cows [15–17]. Moreover, exogenous administration of IFN-tau upregulates ISGs expression in the endometrium, CL, and PBLs in vivo [18,19]. The idea is that expression pattern of ISGs in PBLs could be a gold indicator of embryonic development as emphasized by many scientists [19,20]. A dying embryo would not produce sufficient amounts of IFN-tau to affect PBLs; therefore, monitoring ISG levels in those animals could facilitate detection of ED versus healthy pregnancies. Matsuyama et al. [19] reported that nonpregnant cows with an extended interestrus interval had variable ISG15 levels in their PBLs after embryo transfer. This variability was explained by ED. Owing to the impossibility of knowing the time of ED after embryo transfer; the ISGs expression profiles during ED are unknown. Therefore, in this study, we investigated the ISGs expression profiles in PBLs during ED. Pregnant ewes were chosen as an experimental model and were induced to undergo ED by injection of PGF2a. The mRNA levels of ISGs, including RTP4, interferon tau–stimulated gene 15 (ISG15), B2-microglobulin (B2M), and MX1, were investigated in their PBLs.

2. Materials and methods 2.1. Animal materials All animal experimental procedures were approved by the Local Ethics Committee of Dicle University (2011/66). The field study was completed during the breeding season in southeastern Turkey (August–September 2013). Ewes were kept on the pasture during the day and were placed in pens during the night. Ewes with at least one birth in the records and no health problems (n ¼ 22, aged 3–5 years) were used in this experiment. Estrous cycles were synchronized by two intramuscular injections of PGF2a (75 mg d-cloprostenol,Dalmazin, Vetas¸ Istanbul, Turkey) at 10-day intervals. Estrus was detected by a teaser ram. Ewes were mated with fertile rams on Day 0. Pregnancy was determined as the presence of an embryo and embryonic vesicle by transrectal ultrasonography on Day 18 after mating. Then, pregnant ewes were divided into a control group (sham injection on Day 18, n ¼ 10) and ED group (ED; treated with 75 -mg synthetic PGF2a on Day 18, n ¼ 12). Effect of the treatment on pregnancy was also evaluated by transrectal ultrasonography on Day 25. Blood samples were collected serially (on Days 0, 15, 18, 19, 20, 21, 23, and 25 after mating) from both groups. All blood samples were transferred to the laboratory on ice within 1 hour after collection. 2.2. PBLs isolation and total RNA extraction The PBLs were isolated using a 10 -mL tube containing Na-ethylenediaminetetraacetic acid according to Kurar et al. [21]. Briefly, a 10 -mL blood sample was centrifuged at 300  g for 20 minutes at 4  C. The buffy coat was harvested and resuspended in 1:5 (v:v) 0.87% Tris-NH4Cl lysis buffer. Samples were incubated at 37  C for 10 minutes and then centrifuged at 300  g for 10 minutes. The PBL pellet was washed with 10 -mL icecold PBS buffer and subjected to total RNA extraction. The RNA isolation, quality control, genomic DNA removal by DNase-I, and complementary DNA (cDNA) synthesis procedures were conducted as described by Atli et al. [22]. Briefly, total RNA isolation was performed using TRIzol reagent (Invitrogen, USA). RNA samples (2 mg) were cleaned of possible genomic DNA contamination by DNase-I treatment and then subjected to reverse transcription to synthesize first strand cDNA using the RevertAid First-Strand cDNA Synthesis Kit (Fermentas, USA) according to the manufacturer’s instructions. 2.3. Quantification of expression by real-time PCR Primers for ISGs (MX1, RTP4, ISG15, and B2M) were derived from published studies [12,15] or known ovine sequences using Primer3 and the NCBI database (http:// www.ncbi.nlm.nih.gov/). Gene expression profiles were evaluated on blood sampling days (i.e., Days 0, 15, 18, 19, 20, 21, 23, and 25 after mating) in both the control and ED groups. The reaction was set up as follows: 5 -mL SYBR Green Master Mix (2  ), 5 pmol of each primer, 1 -mL cDNA, and ddH2O to a final volume of 10 mL. Thermal

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cycling conditions were as follows: initial denaturation at 95  C for 10 minutes, followed by 40 cycles of denaturation, annealing, and amplification (95  C for 15 seconds, 60  C for 30 seconds, and 72  C for 30 seconds) on a StepOnePlus Real-Time PCR System (Applied Biosystems). Melting curve analysis was performed as follows: 95  C for 1 minute, followed by fluorescence measurement at 1  C increments between 55  C and 95  C. A negative control with no cDNA template was included in each run. The entire procedure from RNA extraction to real-time polymerase chain reaction (PCR) was performed twice as a technical replicate. Real-time PCR was performed in triplicate for each gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene for normalization of the real-time PCR data. We validated the consistency of steady-state GAPDH concentrations in the PBLs in preliminary experiments (unpublished results). To verify reaction specificity, amplification products were evaluated after separation on a 2% agarose gel. To determine the dynamic range and amplification efficiencies of the real-time PCR assays for each gene product, amplifications were performed using specific primers in duplicate on a serial dilution (1/2, 1/4, 1/8, 1/16) of pooled cDNA collected from PBLs. The amplification efficiency of qPCR was 95 to 105% for all genes. 2.4. Progesterone measurements To determine changes in progesterone (P4) concentrations during and after treatment, blood samples were collected from the jugular vein on Days 0, 15, 18, 19, 20, 21, 23, and 25. The samples were centrifuged at 3000 rpm for 20 minutes. The plasma was stored at 20  C until P4 assay. P4 concentrations were quantified using an electrochemiluminescence immunoassay kit from Roche Diagnostics (GmbH, D-68298 Mannheim, Germany) in duplicate. Before quantification, the kit was validated physiologically by testing plasma samples collected from rams and ewes in estrus, diestrus, and before and after prostaglandin injections in diestrus. The interassay and intraassay coefficients of variation for P4 were 10.3% and 6.4%, respectively.

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3. Results 3.1. Effects of treatments on pregnancy in ewes On the basis of transrectal ultrasonography, all ewes (n ¼ 22) in both groups were pregnant on Day 18. Effects of PGF2a or sham injection on pregnancy were evaluated after 7 days (on Day 25) by transrectal ultrasonography. Although all ewes in the control group were pregnant (10/ 10), the pregnancies in the ED group did not survive (0/12). 3.2. Plasma progesterone profiles of pregnant ewes during ED Plasma P4 concentrations in the ED and control groups are shown in Figure 1. The concentrations were similar in the groups until Day 18. However, that of the ED group declined to less than 1 ng/mL on Day 19 and remained at a low level until the final measurement. From Days 19 to 25, the concentrations were significantly different between the groups (P < 0.05). 3.3. Expression profiles of ISGs in pregnant ewes during the period of ED Relative expression levels of RTP4, ISG15, MX1, B2M mRNAs, in ovine PBLs of ED group compared with control group, are shown in Figure 2. The mRNA levels of RTP4 and ISG15 followed similar profiles. Compared with Day 0, levels of both genes were upregulated on Day 15 and remained high until Day 21 in both groups. However, the levels of both were attenuated in PBLs collected on Days 23 and 25 only in the ED group (P < 0.001). In the ED group, the levels of both genes were numerically higher on Day 20 and started to decrease on Day 21 (P > 0.05). The mRNA level of MX1 was upregulated on Day 15 and remained high until Day 23 in both groups, but was downregulated on Day 25 in the ED group

2.5. Statistical analysis Before statistical analysis, the efficiencies of amplification of the target genes and internal control GAPDH were examined using qPCR amplification of serial dilutions of cDNA. On the basis of confirmation that the amplification efficiencies of the target and reference genes are nearly the same, data normalization process was performed according to Livak and Schmittengen [23] via 0 2DC T method, in whichDC 0 T ¼ CT; target  CT; reference (where CT; target and CT; reference are threshold cycles for target and reference genes amplifications, respectively). Normalized data were subjected to repeated measures ANOVA to be able to take into account effects of treatment (ED vs. control), time, and treatment by time interaction. All statistical analyses were carried out by using R statistical software [24].

Fig. 1. Effect of PGF2a or sham treatment on circulating progesterone concentration in ewes. The data are presented as mean  standard error of the mean. * indicate significant differences within a day between groups at P < 0.05. Uppercase letters show differences among days within embryonic death group. Lowercase letters show differences among days within control group (P < 0.05).

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Fig. 2. Steady-state mRNA concentrations for ISG15 (A), RTP4 (B), MX1 (C), and B2-microglobulin (D) in peripheral blood leukocytes during embryonic death or control (pregnancy) in ewes. The data are presented as relative expression level  standard error of the mean. *,** differences within a day between groups at P < 0.05 and P < 0.001, respectively. 0, 15, 18, 19, 20, 21, 23, and 25 days are sample collection days. Uppercase letters show differences among days within embryonic death group. Lowercase letters show differences among days within control group (P < 0.05).

(P < 0.05). The level of B2M did not change significantly during the study in either group (P > 0.05). 4. Discussion This is the first report of ISGs expression profiles in ovine PBLs during ED in ewes. This study used luteolytic dose of PGF2a to regress pregnancy and to mimic the ED process. The daily blood samples collected during the experiment period allowed us to carefully monitor ISGs expressions in PBLs during the process of the ED. Clearly, there were distinct patterns for the expression of ISGs in PBL after the ED. In the ED group, a luteolytic dose of PGF2a was used to induce demise of the CL, which eventually led to death of the embryo because a suitable environment for the developing embryo is disturbed by the removal of P4. Our findings from this experimental model are summarized in Figure 3. The induction of luteolysis in pregnant ewes resulted in downregulation of ISGs expression in PBLs; this represents indirect evidence of the absence of IFN tau–stimulation on PBLs during the process of ED. The critical pregnancy-associated functions of IFN-tau in uterine tissues, including its role in prostaglandin secretion, conceptus elongation, immunological tolerance, and the development and secretional functions of the endometrial gland are well-described [25–28]. However, its role in tissues outside of the uterus, such as the CL and PBLs,

remain to be resolved. Pregnancy-associated increases in ISGs expression levels in PBLs show promise for the early detection of pregnancy. Moreover, previous studies have indicated that cows with an extended cycle without pregnancy have comparatively higher ISGs expression in PBLs (Wiltbank, Yılmazbas, Atli, unpublished observation, 19, 20). The cause of this may be ED at a variety of time points after fertilization. That is why we monitored ISGs expression profiles in PBLs during experimentally induced ED in ewes. Owing to the high incidence of embryonic mortality in high-production dairy cows [2,6], the mechanism of ED in ruminants is an important research topic. Therefore, we aimed to use ovine ED model in this present study as ruminant model. Furthermore, besides many similar features of IFN-tau including molecular structure and immune functions between ewes and cows, extension of estrus cycle in cows by ovine IFN-tau clearly indicate that establishment and maintenance of pregnancy use similar molecular pathways in both species [26,28–31]. Therefore, our results may provide important information for understanding the events that occur during ED in dairy cows. Our findings clearly indicate that the decline in P4 concentration was an immediate response to PGF2a, as a significant decrease occurred in 24 hours (Day 19). However, the embryo may have survived longer than the CL on the basis of the detection of RTP4, ISG15, and MX1 transcripts in PBLs beyond Day 19. In a previous study [19], induction of

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Fig. 3. A schematic model of changes in IFN tau–stimulated genes after PGF2a administration associated with embryonic death or control (pregnancy) in peripheral blood leukocytes (PBLs) in ewes. After PGF2a administration, although there were distinct patterns for each interferon tau–stimulated genes (ISGs), P4 levels declined to below 1 ng/mL on Day 19 and remained to low levels during embryonic death (ED) in the ED group. Compared with Day 0, the mRNA levels of RTP4 and ISG15 remained high until Day 21 in both groups. However, the levels of both were attenuated in PBLs collected on Days 23 and 25 only in the ED group. In the ED group, the levels of both genes were numerically higher on Day 20 and started to decrease on Day 21. The mRNA level of MX1 was downregulated on Day 25 in the ED group (P < 0.05). The level of B2-microglobulin (B2M) did not change significantly during the study in either group. Names of genes are defined in the text. An up arrow ([) indicates upregulation, a down arrow (Y) indicates downregulation, (4) indicates no changes, CL: corpus luteum, Emb: embryo, PBLs: peripheral blood leukocytes, P4: progesterone. (*) indicates differences at P < 0.05, (**) indicates differences at P < 0.001.

ISG15 and MX2 expression in PBLs by intrauterine administration of IFN-tau lasted for w10 hours, suggesting that the initial induction of ISG expression in PBLs lasted for less than 1 day; therefore, cessation of IFN-tau production by the dead embryo could be detected on the same day. In our study, ISGs expression remained at high levels until Day 23; however, P4 was long absent, which suggests that the embryos in the ED group were still producing IFN-tau and thus alive. However, they did not develop further in the absence of P4. We have reported earlier that embryos could survive to PGF2a injection on Day 18 by application of exogenous supplemental P4 [32]. Taken together, these results suggest that PGF2a alone is not detrimental to the embryo; instead, the embryo dies because of insufficient support from endometrial secretions because of the regressed CL, rather than a direct effect of PGF2a. The mRNA levels of ISG15, RTP4, and MX1 in PBLs were elevated on Day 15 of pregnancy and remained higher throughout the study period compared with Day 0. This is consistent with previous reports [13–15]. However, the ISGs expression patterns were distinct in the ED group. Although the mRNA levels of ISG15 and RTP4 were significantly decreased on Day 23, expression profiles of those genes were numerically increased in the ED group on Day 20. The fact that the P4 level was decreased on Day 19 in this group may suggest that dying embryos produce more IFN-tau and induce decreased endometrial secretion because of the low P4 level. This notion should be clarified by further studies. In addition, B2M mRNA in PBLs was affected by neither pregnancy nor ED. Moreover, the mRNA

level of MX1 was downregulated on Day 25. In an experimental model [19], there was a positive correlation between ISG15 and MX2 mRNA levels and the quantity of IFN-tau administered. ISG15 and MX2 mRNA levels were increased 2 hours after intrauterine administration of 1000 -mg IFN-tau. The increased levels remained at 6 and 10 hours, respectively. In the present study, the later response of MX1 mRNA level to ED (on Day 25) suggests that ISG15 and RTP4 in PBLs are more sensitive to IFN-tau than to MX1. Although the IFN-tau level in blood could not be directly determined, induction of ISG expression by IFN-tau outside of uterine tissues (including CL and PBLs) has been reported by many studies in ruminants [12–14]. Furthermore, intrauterine administration of IFN-tau also upregulates ISGs levels in PBLs in cyclic ruminants [18,19]. Although speculative, immune-regulatory functions of ISG15 and RTP4, including serving as chemokine receptor regulators and playing a role in the innate immune system, have been emphasized [12,33,34]. Moreover, a high ED rate in ISG15knockout mice has been reported [35]. In our study, decreased expression levels of ISG15 and RTP4 in PBLs were associated with ED; however, whether this decrease plays a role in ED warrants further investigation. Acknowledgments This study was supported by Dicle University Grant Commission of research numbered with 12-VF-59, 14-VF112 (to MOA).

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