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Endogenous retroviral gene elements (syncytin-Rum1 and BERV-K1), interferon-τ, and pregnancy associated glycoprotein-1 are differentially expressed in maternal and fetal tissues during the first 50 days of gestation in beef heifers

Endogenous retroviral gene elements (syncytin-Rum1 and BERV-K1), interferon-τ, and pregnancy... Endogenous retroviral gene elements (syncytin-Rum1 and BERV-K1), interferon-τ, and pregnancy associated glycoprotein-1 are differentially expressed in maternal and fetal tissues during the first 50 days of gestation in beef heifers K. J. McLean,* M. S. Crouse,* M. R. Crosswhite,* D. N. Black,* C. R. Dahlen,* P. P. Borowicz,* L. P. Reynolds,* A. K. Ward,* B. W. Neville,† and J. S. Caton* *Department of Animal Sciences and Center for Nutrition and Pregnancy, North Dakota State University, Fargo, 58102; and †Central Grasslands Research Extension Center, North Dakota State University, Streeter 58483 ABSTRACT: We hypothesized that the endogenous compared with NP controls or any other day of early retroviruses [ERV: syncytin-Rum1 and (BERV-K1)], pregnancy. In contrast, syncytin-Rum1 expression in and pregnancy hormones [interferon-τ (IFN-τ), and I-CAR only tended (P = 0.09) to change across days pregnancy associated glycoprotein-1 (PAG-1)] would of early pregnancy and did not differ ( P = 0.27) in FM be differentially expressed whereas progesterone and tissues. In CAR, the expression of BERV-K1 was not insulin concentrations in maternal blood would remain different ( P > 0.79) at d 16 and 22, was intermediate steady during early gestation. To test this hypothesis at d 28, 34, and 40, and was greatest on d 50 (108-fold Angus crossbred heifers (n = 46; ~15 mo of age; BW = increase compared with NP). Expression of BERV-K1 363 ± 35 kg) were fed native grass hay, supplemented in FM was increased (P < 0.01) on d 28, 34, and 50 with cracked corn to gain 0.3 kg/d, and given ad libi- compared with NP controls, but at d 40 did not differ tum access to water. All heifers were subjected to a 5-d from NP controls. The mRNA expression of IFN-τ in CO-Synch + CIDR estrous synchronization protocol FM at d 22 was greater (P < 0.01) than all other days and AI (breeding = d 0). Ovariohysterectomies were of gestation. In CAR, expression of PAG-1 increased (P performed on d 16, 22, 28, 34, 40, and 50 of gestation < 0.001) dramatically on d 40 (20,000-fold) and d 50 and at d 16 of the estrous cycle for non-pregnant (NP) (86,000-fold) compared with NP heifers (P < 0.01). In controls. Utero-placental tissues [maternal caruncle ICAR, expression of PAG-1 was greater (P < 0.05) on d (CAR); maternal intercaruncular endometrium (ICAR); 28 and 40 (fold increases of 113 and 102, respectively, and fetal membranes, (FM, chorion on d 16, chorioal- compared with NP). Insulin concentrations were not lantois on d 22 to 50)] were collected from the uterine different ( P = 0.53) but progesterone was greater (P < horn ipsilateral to the corpus luteum (CL). Tissues were 0.01) on d 16, 22, 28, 34, and 40 compared with d 50 of flash frozen and stored at –80°C. Expression of mRNA gestation. These data confirm differential ERV, IFN-τ, was evaluated using qPCR. In CAR, syncytin-Rum1 and PAG-1 gene expression during critical time points expression was greater (P < 0.01) on d 50 (81.5-fold) of early gestation in utero-placental tissues. Key words: bovine, early pregnancy, endogenous retroviruses, hormones, maternal recognition © 2017 American Society of Animal Science. This is an open access article distributed under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Transl. Anim. Sci. 2017.1:239–249 doi:10.2527/tas2017.0026 INTRODUCTION The authors would like to thank the North Dakota State Board of Agricultural Research and Education and a USDA Seed Grant (USDA-NIFA-AFRI-GRANT11890931) for research support. Placental formation during early gestation is vital Appreciation to personnel at the NDSU Animal Nutrition and to the establishment and maintenance of pregnancy. Physiology Center, the NDSU Animal Science Nutrition Laboratory, The developing conceptus requires a fully functional and the NDSU Animal Science Reproductive Physiology Laboratory placenta for exchange of nutrients, respiratory gases, is expressed for help in conducting this experiment. and metabolic wastes throughout pregnancy (Meschia, Corresponding author: joel.caton@ndsu.edu 1983; Bassil et al., 1995; Reynolds and Redmer, 1995). Received March 30, 2017. In ruminants, trophoblast stem cells fuse to form the Accepted May 23, 2017. Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 240 McLean et al. syncytial plaques, which are multinucleated cells that and the cervix were ligated, and then the uterus was can contain up to 25 nuclei in sheep (Wooding, 1984) removed. Uterine contents were held in place with a and 8 nuclei in cattle (Wooding and Wathes, 1980). In 24-cm Crafoord Coarctation Clamp (Integra-Miltex; addition to nutrient and gas exchange, the syncytio- Plainsboro, NJ), placed just cranial to the cervical liga- trophoblast produces hormones, including progester- tures, during and after removal from the body cavity. one for maintenance of gestation (Bazer et al., 1991), Following surgery, heifers were kept in individual pens interferon-τ (IFN-τ) for pregnancy recognition (Spencer during recovery and stitches were removed 14 d after et al., 2007), and pregnancy associated glycoprotein-1 surgery (McLean et al., 2016a). Heifers were randomly (PAG-1). The syncytiotrophoblast will also interact selected for ovariohysterectomy on d 16 based on the with the maternal immune system during early gesta- inability to confirm viability of pregnancy via ultra - tion and the establishment of pregnancy (reviewed in sound. Heifers without any evidence of a conceptus in Moffett and Loke, 2004, 2006). the uterus on d 16 were deemed not pregnant and re- The Bovidae genome contains 24 endogenous ret- moved from the study. Pregnancy was confirmed via roviral gene elements (ERV) depending on the spe- transrectal ultrasonography on d 22 and again on the d cies (Garcia-Etxebarria and Jugo, 2013). Five ERV are of surgery (d > 28). expressed in bovine trophoblast cells: syncytin-Rum1 (Cornelis et al., 2013), BERVE-A, BERVE-B, BERV-K1, Tissue Collecting and Processing and BERV-K2 (Koshi et al., 2012) The envelope pro- teins of syncytin-Rum1, BERVE-A and BERV-K1 may Immediately on removal from the body cavity, be involved with cell-to-cell fusion that occurs in bovine tissues were trimmed of excess broad ligament, fat, trophoblast during early gestation (Cornelis et al., 2013; and non-reproductive tissues. Utero-placental tissues Nakaya et al., 2013). In addition, Sharif et al. (2013) [maternal caruncle (CAR); maternal intercaruncular argued that ERV function as nutrient sensors during endometrium, (ICAR), fetal membranes (FM; cho- the development of the placenta, and thus may interact rioallantois, d 22 and later)] were obtained from the with insulin, which is also indicative of animal nutrient uterine horn containing the conceptus, as previously status. Thus, we hypothesized that the mRNA of en- described (Grazul-Bilska et al., 2010). There were no dogenous retroviruses (syncytin-Rum1 and BERV-K1), FM collected until d 22 due to insufficient develop - IFN- τ, and PAG-1 would be differentially expressed, ment of tissues for adequate collection, extraction, and whereas serum progesterone and insulin concentrations analysis on d 16. After collection, all tissues were snap would remain steady during early gestation. frozen in liquid nitrogen cooled isopentane (Sigma- Aldrich; St. Louis, MO) and stored at –80°C. MATERIALS AND METHODS Blood samples were taken via jugular venipuncture on d 16, 22, 28, 34, 40, and 50 of gestation until the heifer All animal procedures were conducted with ap- underwent ovariohysterectomy. Non-bred, non-pregnant proval from the Institutional Animal Care and Use control heifers were sampled on d 16 of the estrous cy- Committee at North Dakota State University (A14053). cle. Blood samples were collected in 10 mL vacutainer Commercial Angus crossbred heifers (n = 46; ~ 15 mo tubes (Becton Dickinson Healthcare; Franklin Lakes, of age; BW = 362.3 ± 34.7 kg) were transported 229 NJ), allowed to clot, and stored at 4°C until processing. km from Central Grasslands Research Extension Center Samples were centrifuged for 30 min at 3,000 rpm and (Streeter, ND) to the Animal Nutrition and Physiology 4°C after which serum was removed and stored at –20°C. Center (North Dakota State University, Fargo). Heifers Concentrations of progesterone and insulin in serum were housed in pens with 6 heifers per pen and fed daily were determined using an Immulite 1,000 (Siemens AG; at 0800 h. Heifers were maintained on an ad libitum Munich, Germany). Sensitivity of the assays was 0.2 ng/ native grass hay diet, granted ad libitum access to wa- ml and 2 µIU/ml for progesterone and insulin, respec- ter, and supplemented with cracked corn to maintain a tively. Intra-assay CV for progesterone and insulin were positive energy balance. All heifers were subject to 5-d 4.08% and 19.25%, respectively. CO-Synch + CIDR (Zoetis Inc., Parsippany, NJ) estrus synchronization protocol and AI to a single Angus sire Real-time Reverse Transcriptase Quantitative PCR (day of breeding = d 0; Bridges et al., 2008). Heifers were ovariohysterectomized on d 16, 22, 28, 34, 40, or The RNA was extracted and purified via an RNeasy 50 (n = 9, 6, 6, 7, 6, and 5 respectively) of gestation Mini Kit (Qiagen Inc., Valencia, CA). The concentra- and at d 16 of the estrus cycle for non-bred, non-preg- tion of RNA extracted was determined using Take3 nant controls (NP; n = 7). During surgery, the left and module of a Synergy H1 Microplate Reader (BioTek right uterine arteries, the left and right spiral arteries, Instruments Inc., Winooski, VT). A total of 1 µg of Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Early pregnancy gene expression in heifers 241 RNA was used for cDNA synthesis via a QuantiTect mine if coefficients for linear, quadratic, cubic, exponen - Reverse Transcription Kit (Qiagen Inc.). Primer se- tial models were significantly different than zero. When quences (Table 1) were obtained from previous litera- multiple models were found significant, best fit was ture for syncytin-Rum1 (Cornelis et al., 2013), BERV-K1 determined from the regression analyses by which had (Nakaya et al., 2013), IFN- τ (Hickman et al., 2013), the smallest P value and the greatest r . Concentrations and PAG-1 (Patel et al., 2004). Primer validation for op- of progesterone and insulin were analyzed as repeated timum cDNA concentration and primer efficiency for measures using the MIXED procedure of SAS with day each tissue type was completed before qPCR analysis. as the variable and cow as the subject for a repeated Gene expression was analyzed using a 7500 Fast Real- measure. Progesterone was analyzed with a Compound Time PCR System (Applied Biosystems, Thermo Fisher Symmetry covariance structure. Insulin was analyzed Scientific Inc., Grand Island, NY) with SYBR Green with a Toeplitz covariance structure. Means were sepa- Master Mix (Bio-Rad Laboratories, Hercules, CA). rated using the LSMEANS statement of SAS with differ - Gene expression for maternal tissues was calculated us- ences determined at a P-values ≤ 0.05. -ΔΔCT ing the 2 method (Livak and Schmittgen, 2001) with β-actin as the reference gene and the average of NP RESULTS expression as the control (set to 1) within each tissue. Fetal membrane gene expression was calculated using Endogenous Retroviruses the same methods with the exception that the average of d 22 FM expression as the control (set to 1) within Expression of syncytin-Rum1 in CAR was greater each gene. Gene expression analysis of syncytin-Rum1, (P < 0.01; Fig. 1A) by 81.5-fold on d 50 compared with BERV-K1, IFN-τ, and PAG-1 across day was performed NP controls and all other days of gestation. In ICAR, separately from analysis of syncytin-Rum1, BERV-K1, syncytin-Rum1 expression tended (P = 0.09; Fig. 1B) IFN-τ, and PAG-1 expression across tissues within a to increase until d 28 and then decrease as pregnancy given day of gestation to compare expression between progressed from d 28 to 50. The expression of syncy- tissues. Across tissue gene expression was calculated tin-Rum1 in FM during the first 50 d of gestation did -ΔΔCT using the 2 method (Livak and Schmittgen, 2001) not change over time (P = 0.27; Fig. 1C). The expres- with β-actin as the reference gene and the average of sion of BERV-K1 was not different ( P > 0.79) at d 16 ICAR expression as the control (set to 1) on each day and 22 compared with NP control heifers. The mRNA of gestation. levels of BERV-K1 in CAR were intermediate at d 28, 34, and 40 and greater (P < 0.01) on d 40 and 50 com- pared with NP, d 16, and d 22 heifers; whereas d 28 and Statistical Analysis 34 were intermediate. In addition, d 50 was greater (P Statistical analyses were conducted via the GLM < 0.01) compared with d 28 and 34 (Fig. 2A). In ICAR, procedure of SAS version 9.4 (SAS Inst. Inc., Cary, NY), BERV-K1 was less (P = 0.003) on d 16 and 22 com- with individual heifer as the experimental unit. During pared with d 40 and 28 and greatest (P = 0.003) on d 28 pregnancy, relative pattern of mRNA expression for with a 12.9-fold increase but then returned to NP levels syncytin-Rum1, BERV-K1, IFN-τ, and PAG-1 in CAR, (Fig. 2B) during the first 50 d of gestation. In FM, the ICAR, and FM was determined via the REG procedure expression of BERV-K1 increased (P = 0.001) from d of SAS. Regression analyses were conducted to deter- 22 to d 34 with a 27.4-fold increase compared with d Table 1. Primer sequences of syncytin-Rum1, bovine endogenous retrovirus-K1 (BERV-K1), interferon–τ (IFN-τ), and pregnancy associated glycoprotein-1 (PAG-1) Gene of interest Primer direction Product size (bp) Sequence GenBank accession number Syncytin-Rum1 Forward 2464 TGGTATGACTATCTTGCTGGCTTC NM_001305454 Reverse TGGGCTGTGAGTAGTTCTAAT BERV-K1 Forward 2142 GGAAATCACGATGTCCT NM_001245951 Reverse GGAGAGGAGGCGCTTACCTG IFN-τ Forward 1313 CAGGACAGAAAGACTTTGG NM_001015511 Reverse GTGCTCTGTGTAGAAGAGGTTG PAG-1 Forward 1295 TCCAGCCTGTTCTACACACGTT NM_174411 Reverse AGGTGATCCTGAAGGTCTTATTGG Primer sequences were obtained from Cornelis et al., 2013 (syncytin-Rum1), Nakaya et al., 2013 (BERV-K1), Hickman et al., 2013 (IFN-τ), and Patel et al., 2004 (PAG-1). All sequences are represented from 5’ to 3’. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 242 McLean et al. Figure 1. Expression of syncytin-Rum1 in reproductive tissues dur- Figure 2. Expression of bovine endogenous retrovirus-K1 (BERV-K1) ing the establishment of pregnancy in beef heifers: A) syncytin-Rum1 in in reproductive tissues during the establishment of pregnancy in beef heifers: maternal caruncles (CAR), B) syncytin-Rum1 in uterine endometrium A) BERV-K1 in maternal caruncles (CAR), B) BERV-K1 in uterine endome- (ICAR), and C) syncytin-Rum1 in fetal membranes (FM). Data presented trium (ICAR), and C) BERV-K1 in fetal membranes (FM). Data presented as -ΔΔCT -ΔΔCT as a 2 -fold change normalized to β-Actin and the average of non- a 2 -fold change normalized to β-Actin and the average of non-pregnant pregnant (NP; maternal tissues) or d 22 FM (fetal tissues). Expression pat- (NP; maternal tissues) or d 22 FM (fetal tissues). Expression pattern line tern line ( – – ) via regression (P < 0.05); regression analysis does not ( – – ) via regression (P < 0.05); regression analysis does not include NP include NP heifers. Means without a common superscript differ ( P < 0.05). heifers. Means without a common superscript differ ( P < 0.05). Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Early pregnancy gene expression in heifers 243 22 heifers, while d 28 was intermediate. Expression of BERV-K1 in FM decreased from d 34 to 40 and increased again at d 50. The d 50 increase in mRNA expression of BERV-K1 in FM represents a 32.3-fold increase compared with d 22 FM (P = 0.001; Fig. 2C). Pregnancy Hormones The maternal recognition signal in ruminants, IFN-τ, was not detected in maternal tissues, CAR and ICAR; thus, analysis of IFN-τ expression as pregnancy progressed was only conducted in FM and no across tissue comparisons were made. The mRNA expres- sions of IFN-τ at d 22, which was used as baseline for all FM tissues, was greater (P < 0.01) than all other days of gestation (Fig. 3). Expression levels of PAG-1 increased dramatically with d 40 and 50 being 20,000 and 86,000-fold, respectively, greater than NP heifers in CAR (Fig. 4A). Due to the magnitude of relative fold change on d 40 and 50 in CAR for PAG-1 they were removed and the same analysis was conducted to deter- mine if differences existed early in gestation (d 16, 22, 28, and 34) compared with NP heifers. Relative expres- sion of PAG-1 was increased (P < 0.001) on d 22 and 34 (3,876- and 5,368-fold, respectively) but was not differ - ent (P > 0.10) on d 16 and 28 compared with NP heifers. In ICAR, expression of PAG-1 was greater (P < 0.05) on d 28 and 40 compared with NP with fold increases of 113 and 102, respectively, and d 22, 34, and 50 were intermediate (Fig. 4B). Expression of PAG-1 in FM tis- sue was similar (P = 0.33) across all days evaluated Figure 4. Expression of pregnancy associated glycoprotein-1 (PAG- 1) in reproductive tissues during the establishment of pregnancy in beef heifers: A) PAG-1 in maternal caruncles (CAR), B) PAG-1 in uterine en- Figure 3. Expression of (IFN-τ) in fetal membranes (FM) during the es- dometrium (ICAR), and C) PAG-1 in fetal membranes (FM). Data pre- -ΔΔCT -ΔΔCT tablishment of pregnancy in beef heifers. Data presented as a 2 -fold change sented as a 2 -fold change normalized to β-Actin and the average of normalized to β-Actin and the average of d 22 FM (fetal tissues). Expression pat- non-pregnant (NP; maternal tissues) or d 22 FM (fetal tissues). Expression tern line ( – – ) via regression (P < 0.05); regression analysis does not include pattern line ( – – ) via regression (P < 0.05); regression analysis does not NP heifers. Means without a common superscript differ ( P < 0.05). include NP heifers. Means without a common superscript differ ( P < 0.05). Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 244 McLean et al. Figure 5. Concentrations of hormones in serum of beef heifers during early gestation. A) Concentrations of circulating progesterone in serum of beef heifers B) Concentrations of circulating insulin in serum of beef heifers. Hormones are reported as a pooled mean from heifers (n = 38, 30, 25, 18, 11, and 5 for d 16, 22, 28, 34, 40, and 50; respectively) on each d. Means without a common superscript differ ( P < 0.05). (Fig. 4C). Concentrations of progesterone (Fig. 5A) on Table 2. Relative expression patterns for syncytin- d 50 (1.8 ± 1.8 and 3.6 ± 1.0 ng/mL; respectively) was Rum1, bovine endogenous retrovirus-K1 (BERV-K1), decreased (P < 0.01) compared with all other days (7.4 interferon-τ (IFN-τ), and pregnancy associated glyco- ± 0.6 ng/mL). Concentrations of insulin were similar (P protein-1 (PAG-1) during early pregnancy in beef heifers = 0.53) among all days of gestation evaluated (Fig. 5B). Equation for the best fit 1 2,3 2 Gene regression model P-value R Temporal Patterns of Expression Syncytin-Rum1 0.4125x CAR (Fig. 1A) y = 1.6298e 0.002 0.47 Regression analysis for expression of syncytin-Rum1, FM (Fig. 1C) y = 0.504x + 0.61 0.03 0.92 BERV-K1, IFN-τ, and PAG-1 across days of gestation BERV-K1 0.3567x was used to determine temporal changes in expression CAR (Fig. 2A) y = 8.4859e < 0.001 0.84 0.2619x pattern during the first 50 d of pregnancy (Table 2). In ICAR (Fig. 2B) y = 0.9091e 0.009 0.23 3 2 FM (Fig. 2C) y = 3.8167x – 35.62x + 0.01 0.89 CAR, syncytin-Rum1 (P = 0.002), BERV-K1 (P < 0.001), 102.79x – 68.984 and PAG-1 (P < 0.001) all had exponential expression IFN-τ patterns during the first 50 d of gestation. Expression pat - –1.374x FM (Fig. 3) y = 3.8167e < 0.001 0.73 terns for BERV-K1 (P = 0.009), and PAG-1 (P < 0.001) PAG-1 in ICAR were also exponential; however, syncytin-Rum1 1.1099x CAR (Fig. 4A) y = 32.007e < 0.001 0.93 expression in ICAR had no (P > 0.19) pattern of expres- 0.4691x ICAR (Fig. 4B) y = 4.967e < 0.001 0.50 sion. In FM, a linear pattern (P = 0.03) was observed in Tissue expression in maternal caruncles (CAR), uterine endometrium syncytin-Rum1 expression but a cubic pattern was found (ICAR), and fetal membranes (FM). for BERV-K1 (P = 0.01). The pattern of expression for 2 Regression analysis does not include non-pregnant heifers. IFN-τ in FM was exponential (P < 0.001) but there was Equation variables are y = gene and x = day. only a tendency for an exponential pattern of expression for PAG-1 (P > 0.08; Table 2). Expression of syncytin-Rum1 mRNA was greater in FM compared with ICAR (P < 0.002) and CAR (P < 0.004) Tissue Comparisons from on d 22, 28, and 34 of gestation. At d 40 syncytin- For comparison of gene expression across tissues on Rum1 in CAR and FM tissues were similar (P = 0.34) a given day the expression of syncytin-Rum1, BERV-K1, compared with ICAR. However, at d 50 syncytin-Rum1 IFN-τ, and PAG-1 was normalized to their average ex- expression in CAR was greater (P = 0.01) compared pression in ICAR tissues. In maternal tissues, CAR and with ICAR and FM tissues (Table 3). ICAR had similar mRNA expression of syncytin-Rum1 There were no differences between tissues ( P > 0.36; from d 16 to 40 and in NP tissues (P > 0.32; Table 3). Table 4) in BERV-K1 on d 16 and 22 in pregnant heifers However, at d 50 syncytin-Rum1 mRNA expression in and NP expression. On d 28 of gestation expression of CAR was greater than ICAR (P < 0.05) and increased BERV-K1 in FM was greater (P < 0.01) than that of ma- (P < 0.0001) by 190.3-fold over NP baseline (Table 3). ternal tissues CAR and ICAR. Expression of BERV-K1 Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Early pregnancy gene expression in heifers 245 Table 3. Relative fold change of syncytin-Rum1 Table 5. Relative fold change of pregnancy associated expression in maternal caruncles (CAR), uterine glycoprotein-1 expression in maternal caruncles (CAR), endometrium (ICAR), and fetal membranes (FM) uterine endometrium (ICAR), and fetal membranes 1 1 during the first 50 d of pregnancy in beef heifers (FM) during the first 50 d of pregnancy in beef heifers Tissue type Tissue type Gestation, d CAR ICAR FM SEM P-value Gestation, d CAR ICAR FM SEM P-value 2 2 NP 5.1 1.5 – 2.8 0.67 NP 0.01 6.84 – 4.44 0.55 16 2.4 2.2 – 0.5 0.77 16 29.67 9.93 – 8.50 0.12 a a b b a a 22 1.2 0.6 4.8 0.8 < 0.01 22 10.27 3.16 1.69 2.41 0.05 a a b 28 1.9 1.5 11.0 1.4 < 0.01 28 1.47 1.45 1.11 0.46 0.83 a a b 34 14.1 6.8 128.5 11.7 < 0.01 34 4.98 1.10 10.30 3.01 0.13 40 13.9 1.5 23.6 7.5 0.10 40 11.10 2.58 2.73 2.45 0.05 b a a b a a 50 190.3 1.1 71.7 28.6 < 0.01 50 143.98 1.23 8.91 13.84 < 0.01 a,b a,b Means within rows without a common superscript differ ( P < 0.05). Means within rows without a common superscript differ ( P < 0.05). 1 1 Data normalized to β-Actin and the average for normalized ICAR for Data normalized to β-Actin and the average for normalized ICAR for -ΔΔCT -ΔΔCT 2 values. 2 values. 2 2 NP: non-pregnant controls ovariohysterectomized at d 16 of luteal cycle. NP: non-pregnant controls ovariohysterectomized at d 16 of luteal cycle. Table 4. Relative fold change of bovine endogenous 2009; Dupressoir et al., 2011; Cornelis et al., 2012, 2013, retrovirus-K1 (BERV-K1) expression in maternal 2014, 2015). To date, mammalian placentas found to caruncles (CAR), uterine endometrium (ICAR), and form syncytium exhibit 2 morphologies, syncytiotroph- fetal membranes (FM) during the first 50 d of preg - blast and syncytial plaques. The syncytiotrophoblast is a nancy in beef heifers multinucleated tissue layer of the placenta whereas syn- cytial plaques are multinucleated cells that form at the Tissue type feto-maternal interface. Formation of syncytial plaques, Gestation, d CAR ICAR FM SEM P-value which consist of both fetal and maternal cells, is unique NP 0.47 2.33 – 1.03 0.48 to ruminants among eutherian mammals. The conjoin- 16 3.06 1.32 – 0.60 0.06 22 1.90 1.44 8.37 1.10 0.36 ing of cells of fetal and maternal origin makes separation a a b 28 0.98 1.33 6.73 3.87 < 0.01 of maternal and fetal transcriptome extremely difficult a a b 34 10.06 1.44 45.01 6.27 < 0.01 and is a limitation of this data but expression of ERV a a b 40 5.63 2.29 16.04 4.27 0.04 in maternal tissues, CAR and ICAR as reported here, a a b 50 21.04 1.26 62.55 11.12 < 0.01 is understandable in the uterus. The classical functions a,b Means within rows without a common superscript differ ( P < 0.05). for ERV of immunosuppressive and cell to cell fusion Data normalized to β-Actin and the average for normalized ICAR for (Dupressoir et al., 2011; Cornelis et al., 2013) and previ- -ΔΔCT 2 values. ously established ERV expression in fetal tissues (Blond NP: non-pregnant controls ovariohysterectomized at d 16 of luteal cycle. et al., 2000; Mi et al., 2000; Dupressoir et al., 2011; Cornelis et al., 2013) and now, from this study, ERV ex- in fetal membranes remained elevated (P < 0.04) on d 34, pression in the maternal endometrium are intriguing to 40, and 50 of gestation (45.01, 16.04, and 62.55, respec- potential roles in the establishment of pregnancy such tively; Table 4) compared with maternal tissues. When as maternal recognition, uterine immunotolerance, and comparing PAG-1 expression across tissues, there were overall placental development. no differences in maternal tissues in NP or d 16 of gesta - The measurement of basal mRNA expression during tion or across CAR, ICAR, or FM on d 28, 34, or 40 of the first 50 d of gestation is entirely novel for the ERV, gestation. On d 22 and 50, however, PAG-1 expression BERV-K1. McLean et al. (2016b) reported across tissue was increased in CAR with a fold increase of 10.27 and and day of gestation expression during early gestation 143.98, respectively (Table 5) compared to NP. but did not establish general mRNA expression pat- terns. These data are necessary to begin understanding DISCUSSION the roles of ERV in pregnancy success. As stated ear- lier, we hypothesized that the ERV (syncytin-Rum1 and Endogenous retroviral gene elements contribute to BERV-K1), IFN-τ, and PAG-1 would be differentially the formation of the multinucleation within placental expressed while progesterone and insulin concentrations formation in a wide variety of mammals with many dif- would remain steady during early gestation. In keep- ferent placental morphologies (Blond et al., 2000; Mi et ing with our hypothesis, we found BERV-K1 began to al., 2000; Dupressoir et al., 2005, 2009; Heidmann et al., increase near d 28; whereas, syncytin-Rum1 expression Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 246 McLean et al. was only different at d 50 of gestation in CAR but both with peaks at d 34 and 50. These data agree with char- exhibited exponential patterns of expression from d 16 acteristic functions of BERV-K1 for cell to cell fusion to 50 of gestation. This coincides with the time period (Nakaya et al., 2013) during binucleate cell formation when Winters et al. (1942) reported the greatest amount within placental trophoblast cells and syncytial plaques of multinucleated cells and syncytial plaque formations. between fetal and maternal cells. As well as known The early increase of BERV-K1 over syncytin-Rum1 may events during early gestation such as maternal recogni- be due to the increased cell to cell fusion capabilities of tion, embryonic adhesion with the uterine endometrium, BERV-K1, which agrees with data from Nakaya et al. and placentation. Placental development is necessary (2013). Cornelis et al. (2013), Nakaya et al. (2013) and for the transfer of nutrients responsible for the rapid fe- our data presented here indicate that ruminants have at tal growth that must occur during late gestation. least 2 ERV, syncytin-Rum1 and BERV-K1. This finding The secretion of IFN-τ from the trophoblast is widely is similar to ERV in the rodent placenta, syncytin-A and accepted as the ruminant signal for pregnancy recogni- -B (Dupressoir et al., 2011) and the human placenta syn- tion and inhibition of luteolysis (Thatcher et al., 1989; cytin-1 and -2 (Fisher et al., 1989). While syncytin-A and Bazer et al., 1991; Bazer, 1992; Mann et al., 1999; -B and syncytin-1 and -2 are homologous genes it is cur- Spencer and Bazer, 2004; Spencer et al., 2007). The rently unknown if syncytin-Rum1 and BERV-K1 are also secretion of IFN-τ must occur before the initiation of homologous to the mouse and human genes. Knockout luteolysis on d 18 of the estrous cycle. After this point mice for syncytin-A exhibit abnormal embryogenesis, ul- concentrations of IFN-τ decreased dramatically back to timately terminating gestation between d 11.5 and 13.5 basal levels which is in agreement with data from the of gestation (Dupressoir et al., 2009). While termination current research where IFN-τ mRNA expression at d 22 of rodent gestation occurs later in pregnancy, compara- was greater (P < 0.01) compared with all other days of tively, than the timeframe in this study; these data may gestation and exhibited a negative exponential pattern of be taken to imply that these BERV-K1 and syncytin-Rum1 expression in FM. Interferon-τ stimulates the production are important to placentation, placentome formation, and of many other proteins such as: ubiquitin-like interferon successful pregnancy in beef cattle. However, more work stimulated gene 15, myxovirus resistance 1, and 2ꞌ-,5ꞌ- remains to be completed to determine roles for BERV-K1 oligoadenylate synthetase 1, which may be necessary for and syncytin-Rum1 during gestation. the establishment of pregnancy (Glaucia Teixeira et al., The increased mRNA expression occurred in ICAR 1997; Perry et al., 1999; Binelli et al., 2001; Bazer et al., earlier (d 28) during pregnancy than in CAR for BERV-K1. 2015). Another such protein, PSP-B, is produced in de- The BERV-K1 increase in ICAR occurred at the end of the tectable quantities as early as d 15 of gestation (Butler et adhesion phase of implantation, which further supports al., 1982; Sasser et al., 1986); however, concentrations previous data that demonstrated BERV-K1 has increased vary greatly until after d 30 (Sasser et al.,1986; Humblot expression during early gestation and fusogenic functions et al., 1988a; Sasser et al., 1991; Vasques et al., 1995). (Koshi et al., 2012; Nakaya et al., 2013). Thus, the role The limited secretion of PSP-B early in gestation agrees of BERV-K1 in placental formation is likely in cotyledon with our data in which we observed mRNA expression of formation and subsequent syncytial plaque development. PAG-1 during the first 34 d of gestation. However, the presence of syncytin-Rum1 and BERV-K1 in The exponential increase in expression of PAG-1 maternal tissues is not in agreement with previous data for may indicate a greater prevalence in placental devel- syncytin genes in cattle (Cornelis et al., 2013) or BERV-K1 opment as gestation progresses and may be stimulat- expression in trophoblast cells (Koshi et al., 2012). The ed by IFN-τ and ERV. However, secondary functions increase earlier in gestation of BERV-K1 expression com- may be to aide IFN-τ and ERV in fetal protection dur- pared with syncytin-Rum1 may indicate a greater role for ing implantation via immune suppression (Wooding BERV-K1 for cell to cell fusion not only between tropho- et al., 2005). In addition, expression pattern for PAG-1 blast cells of the fetus but also in syncytial plaque for- in CAR was exponential with a mean fold change of mation and the combination of maternal and fetal cells. 18,000 compared with NP. Pregnancy associated glyco- These data may also support previous data (Imakawa et proteins from binucleated cells seem to interact exten- al., 2015) that suggested BERV-K1 is replacing syncytin- sively with maternal connective tissue which develops Rum1as the main catalyst in placental cell to cell fusion. during placental villi formation (Wooding et al., 2005). Although means were not different from d 22 to 50 It has been speculated that PAG may possibly be in- of gestation the linear increase in mRNA expression of volved in proteolytic activation of growth factors and syncytin-Rum1 in FM could be aiding in cell to cell fu- other molecules specific to pregnancy, protection of fe - sion occurring during placental development, which is tal tissues from maternal immune response, transport of rapidly occurring during this time (Winters et al., 1942). hormones between fetal and maternal tissues, and cell Expression pattern of BERV-K1 was cubic in nature to cell fusion (Wooding et al., 2005). Our data presented Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Early pregnancy gene expression in heifers 247 here confirms expression of PAG-1 during pregnancy, creased on d 50 supporting roles in cell to cell fusion and which would support Wooding et al. (2005) suggested placental development. This research also established roles of PAG-1 in cell to cell fusion during placentation. basal expression patterns for syncytin-Rum1, BERV-K1, Combined these data may also indicate an interaction IFN-τ, and PAG-1 which can be used in future research with ERV to promote the cell to cell fusion needed for to determine the influence of treatments on pregnancy. syncytial plaques formation and placental development While these data provide evidence for differential ex - to support fetal growth throughout gestation. pression the functions and interactions between syncy- Progesterone must also be present for IFN-τ to tin-Rum1, BERV-K1, IFN- τ, and PAG-1 remain to be suppress the release of PGF stimulated by oxytocin elucidated and should be the focus in future studies to 2α (Meyer et al., 1995) to maintain pregnancy (Mann and determine the importance in fetal and placental develop- Lamming, 2001; Green et al., 2005; Mann et al., 2006; ment and the establishment of pregnancy. Bazer et al., 2015). Our data clearly demonstrates el- evated circulating progesterone concentrations (> 5 LITERATURE CITED ng/mL) in pregnant heifers on all days except for d Bassil, S., J. P. Magritte, J. Roth, M. Nisolle, J. Donnez, and S. 50. Pregnant cattle will not only maintain a functional Gordts. 1995. Uterine vascularity during stimulation and its corpus luteum (CL) but also have greater progester- correlation with implantation in in-vitro fertilization. Hum. Reprod. 10:1497–1501. doi:10.1093/HUMREP/10.6.1497 one concentrations compared with non-pregnant cattle Bazer, F. W. 1992. Mediators of maternal recognition of preg- (Henricks et al., 1971; Humblot et al., 1988b; Humblot, nancy in mammals. Proc. Soc. Exp. Biol. Med. 199:373–384. 2001). The drop in progesterone, regardless of treatment, doi:10.3181/00379727-199-43371A on d 50 is intriguing; while the placenta does become Bazer, F. W., W. W. Thatcher, P. J. Hansen, M. A. Mirando, T. L. Ott, the major source of progesterone in sheep and horses and C. Plante. 1991. Physiological mechanism of pregnancy rec- ognition in ruminants. J. Reprod. Fertil. 43:39–47. this does not occur in cattle (reviewed in Hoffmann and Bazer, F. W., W. Ying, X. Wang, K. A. Dunlap, B. Zhou, G. A. Schuler, 2002). Our data could be interpreted to mean Johnson, and G. Wu. 2015. The many faces of interferon that by d 50 the CL has begun to share progesterone tau. Invited Review. Amino Acids 47:449–460. doi:10.1007/ secretion with the placenta but progesterone synthesis s00726-014-1905-x within the placenta remains to be completely elucidated Binelli, M., P. Subramaniam, T. Diaz, G. A. Johnson, T. R. Hansen, (reviewed in Hoffmann and Schuler, 2002). and W. W. Thatcher. 2001. Bovine interferon-τ stimulates Janus kinase-signal transducer and activator of transcription The time points assessed in this study, specifical - pathway in bovine endometrial epithelial cells. Biol. Reprod. ly d 16, 34, and 50 of gestation are influential to the 64:654–665. doi:10.1095/biolreprod64.2.654 expression of syncytin-Rum1, BERV-K1, IFN-τ, and Blond, J. L., D. Lavillette, V. Cheynet, O. Bouton, G. Oriol, S. Chapel- PAG-1 and may be important for the establishment of Fernandes, B. Mandrand, F. Mallet, and F. L. Cosset. 2000. An pregnancy. On d 16 the embryo must support the main- envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells tenance of a functional CL and as such this has been expressing the type D mammalian retrovirus receptor. J. Virol. termed the period of maternal recognition. Day 34 is the 74:3321–3329. doi:10.1128/JVI.74.7.3321-3329.2000 approximate end of adhesion when the embryo has suc- Bridges, G. A., L. A. Helser, D. E. Grum, M. L. Mussard, C. L. cessfully completed implantation. Finally, d 50 is when Gasser, and M. L. Day. 2008. 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Is there a role for endog - electron microscope immunocytochemical studies of the distribu- enous retroviruses to mediate long-term adaptive phenotypic re- tion of pregnancy associated glycoproteins (PAGs) throughout sponse upon environmental inputs? Philos. Trans. R. Soc. Lond. B. pregnancy in the cow: Possible functional implications. Placenta Biol. Sci. 368:20110340–20110353. doi:10.1098/rstb.2011.0340 26:807–827. doi:10.1016/j.placenta.2004.10.014 Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Translational Animal Science Oxford University Press

Endogenous retroviral gene elements (syncytin-Rum1 and BERV-K1), interferon-τ, and pregnancy associated glycoprotein-1 are differentially expressed in maternal and fetal tissues during the first 50 days of gestation in beef heifers

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Abstract

Endogenous retroviral gene elements (syncytin-Rum1 and BERV-K1), interferon-τ, and pregnancy associated glycoprotein-1 are differentially expressed in maternal and fetal tissues during the first 50 days of gestation in beef heifers K. J. McLean,* M. S. Crouse,* M. R. Crosswhite,* D. N. Black,* C. R. Dahlen,* P. P. Borowicz,* L. P. Reynolds,* A. K. Ward,* B. W. Neville,† and J. S. Caton* *Department of Animal Sciences and Center for Nutrition and Pregnancy, North Dakota State University, Fargo, 58102; and †Central Grasslands Research Extension Center, North Dakota State University, Streeter 58483 ABSTRACT: We hypothesized that the endogenous compared with NP controls or any other day of early retroviruses [ERV: syncytin-Rum1 and (BERV-K1)], pregnancy. In contrast, syncytin-Rum1 expression in and pregnancy hormones [interferon-τ (IFN-τ), and I-CAR only tended (P = 0.09) to change across days pregnancy associated glycoprotein-1 (PAG-1)] would of early pregnancy and did not differ ( P = 0.27) in FM be differentially expressed whereas progesterone and tissues. In CAR, the expression of BERV-K1 was not insulin concentrations in maternal blood would remain different ( P > 0.79) at d 16 and 22, was intermediate steady during early gestation. To test this hypothesis at d 28, 34, and 40, and was greatest on d 50 (108-fold Angus crossbred heifers (n = 46; ~15 mo of age; BW = increase compared with NP). Expression of BERV-K1 363 ± 35 kg) were fed native grass hay, supplemented in FM was increased (P < 0.01) on d 28, 34, and 50 with cracked corn to gain 0.3 kg/d, and given ad libi- compared with NP controls, but at d 40 did not differ tum access to water. All heifers were subjected to a 5-d from NP controls. The mRNA expression of IFN-τ in CO-Synch + CIDR estrous synchronization protocol FM at d 22 was greater (P < 0.01) than all other days and AI (breeding = d 0). Ovariohysterectomies were of gestation. In CAR, expression of PAG-1 increased (P performed on d 16, 22, 28, 34, 40, and 50 of gestation < 0.001) dramatically on d 40 (20,000-fold) and d 50 and at d 16 of the estrous cycle for non-pregnant (NP) (86,000-fold) compared with NP heifers (P < 0.01). In controls. Utero-placental tissues [maternal caruncle ICAR, expression of PAG-1 was greater (P < 0.05) on d (CAR); maternal intercaruncular endometrium (ICAR); 28 and 40 (fold increases of 113 and 102, respectively, and fetal membranes, (FM, chorion on d 16, chorioal- compared with NP). Insulin concentrations were not lantois on d 22 to 50)] were collected from the uterine different ( P = 0.53) but progesterone was greater (P < horn ipsilateral to the corpus luteum (CL). Tissues were 0.01) on d 16, 22, 28, 34, and 40 compared with d 50 of flash frozen and stored at –80°C. Expression of mRNA gestation. These data confirm differential ERV, IFN-τ, was evaluated using qPCR. In CAR, syncytin-Rum1 and PAG-1 gene expression during critical time points expression was greater (P < 0.01) on d 50 (81.5-fold) of early gestation in utero-placental tissues. Key words: bovine, early pregnancy, endogenous retroviruses, hormones, maternal recognition © 2017 American Society of Animal Science. This is an open access article distributed under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Transl. Anim. Sci. 2017.1:239–249 doi:10.2527/tas2017.0026 INTRODUCTION The authors would like to thank the North Dakota State Board of Agricultural Research and Education and a USDA Seed Grant (USDA-NIFA-AFRI-GRANT11890931) for research support. Placental formation during early gestation is vital Appreciation to personnel at the NDSU Animal Nutrition and to the establishment and maintenance of pregnancy. Physiology Center, the NDSU Animal Science Nutrition Laboratory, The developing conceptus requires a fully functional and the NDSU Animal Science Reproductive Physiology Laboratory placenta for exchange of nutrients, respiratory gases, is expressed for help in conducting this experiment. and metabolic wastes throughout pregnancy (Meschia, Corresponding author: joel.caton@ndsu.edu 1983; Bassil et al., 1995; Reynolds and Redmer, 1995). Received March 30, 2017. In ruminants, trophoblast stem cells fuse to form the Accepted May 23, 2017. Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 240 McLean et al. syncytial plaques, which are multinucleated cells that and the cervix were ligated, and then the uterus was can contain up to 25 nuclei in sheep (Wooding, 1984) removed. Uterine contents were held in place with a and 8 nuclei in cattle (Wooding and Wathes, 1980). In 24-cm Crafoord Coarctation Clamp (Integra-Miltex; addition to nutrient and gas exchange, the syncytio- Plainsboro, NJ), placed just cranial to the cervical liga- trophoblast produces hormones, including progester- tures, during and after removal from the body cavity. one for maintenance of gestation (Bazer et al., 1991), Following surgery, heifers were kept in individual pens interferon-τ (IFN-τ) for pregnancy recognition (Spencer during recovery and stitches were removed 14 d after et al., 2007), and pregnancy associated glycoprotein-1 surgery (McLean et al., 2016a). Heifers were randomly (PAG-1). The syncytiotrophoblast will also interact selected for ovariohysterectomy on d 16 based on the with the maternal immune system during early gesta- inability to confirm viability of pregnancy via ultra - tion and the establishment of pregnancy (reviewed in sound. Heifers without any evidence of a conceptus in Moffett and Loke, 2004, 2006). the uterus on d 16 were deemed not pregnant and re- The Bovidae genome contains 24 endogenous ret- moved from the study. Pregnancy was confirmed via roviral gene elements (ERV) depending on the spe- transrectal ultrasonography on d 22 and again on the d cies (Garcia-Etxebarria and Jugo, 2013). Five ERV are of surgery (d > 28). expressed in bovine trophoblast cells: syncytin-Rum1 (Cornelis et al., 2013), BERVE-A, BERVE-B, BERV-K1, Tissue Collecting and Processing and BERV-K2 (Koshi et al., 2012) The envelope pro- teins of syncytin-Rum1, BERVE-A and BERV-K1 may Immediately on removal from the body cavity, be involved with cell-to-cell fusion that occurs in bovine tissues were trimmed of excess broad ligament, fat, trophoblast during early gestation (Cornelis et al., 2013; and non-reproductive tissues. Utero-placental tissues Nakaya et al., 2013). In addition, Sharif et al. (2013) [maternal caruncle (CAR); maternal intercaruncular argued that ERV function as nutrient sensors during endometrium, (ICAR), fetal membranes (FM; cho- the development of the placenta, and thus may interact rioallantois, d 22 and later)] were obtained from the with insulin, which is also indicative of animal nutrient uterine horn containing the conceptus, as previously status. Thus, we hypothesized that the mRNA of en- described (Grazul-Bilska et al., 2010). There were no dogenous retroviruses (syncytin-Rum1 and BERV-K1), FM collected until d 22 due to insufficient develop - IFN- τ, and PAG-1 would be differentially expressed, ment of tissues for adequate collection, extraction, and whereas serum progesterone and insulin concentrations analysis on d 16. After collection, all tissues were snap would remain steady during early gestation. frozen in liquid nitrogen cooled isopentane (Sigma- Aldrich; St. Louis, MO) and stored at –80°C. MATERIALS AND METHODS Blood samples were taken via jugular venipuncture on d 16, 22, 28, 34, 40, and 50 of gestation until the heifer All animal procedures were conducted with ap- underwent ovariohysterectomy. Non-bred, non-pregnant proval from the Institutional Animal Care and Use control heifers were sampled on d 16 of the estrous cy- Committee at North Dakota State University (A14053). cle. Blood samples were collected in 10 mL vacutainer Commercial Angus crossbred heifers (n = 46; ~ 15 mo tubes (Becton Dickinson Healthcare; Franklin Lakes, of age; BW = 362.3 ± 34.7 kg) were transported 229 NJ), allowed to clot, and stored at 4°C until processing. km from Central Grasslands Research Extension Center Samples were centrifuged for 30 min at 3,000 rpm and (Streeter, ND) to the Animal Nutrition and Physiology 4°C after which serum was removed and stored at –20°C. Center (North Dakota State University, Fargo). Heifers Concentrations of progesterone and insulin in serum were housed in pens with 6 heifers per pen and fed daily were determined using an Immulite 1,000 (Siemens AG; at 0800 h. Heifers were maintained on an ad libitum Munich, Germany). Sensitivity of the assays was 0.2 ng/ native grass hay diet, granted ad libitum access to wa- ml and 2 µIU/ml for progesterone and insulin, respec- ter, and supplemented with cracked corn to maintain a tively. Intra-assay CV for progesterone and insulin were positive energy balance. All heifers were subject to 5-d 4.08% and 19.25%, respectively. CO-Synch + CIDR (Zoetis Inc., Parsippany, NJ) estrus synchronization protocol and AI to a single Angus sire Real-time Reverse Transcriptase Quantitative PCR (day of breeding = d 0; Bridges et al., 2008). Heifers were ovariohysterectomized on d 16, 22, 28, 34, 40, or The RNA was extracted and purified via an RNeasy 50 (n = 9, 6, 6, 7, 6, and 5 respectively) of gestation Mini Kit (Qiagen Inc., Valencia, CA). The concentra- and at d 16 of the estrus cycle for non-bred, non-preg- tion of RNA extracted was determined using Take3 nant controls (NP; n = 7). During surgery, the left and module of a Synergy H1 Microplate Reader (BioTek right uterine arteries, the left and right spiral arteries, Instruments Inc., Winooski, VT). A total of 1 µg of Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Early pregnancy gene expression in heifers 241 RNA was used for cDNA synthesis via a QuantiTect mine if coefficients for linear, quadratic, cubic, exponen - Reverse Transcription Kit (Qiagen Inc.). Primer se- tial models were significantly different than zero. When quences (Table 1) were obtained from previous litera- multiple models were found significant, best fit was ture for syncytin-Rum1 (Cornelis et al., 2013), BERV-K1 determined from the regression analyses by which had (Nakaya et al., 2013), IFN- τ (Hickman et al., 2013), the smallest P value and the greatest r . Concentrations and PAG-1 (Patel et al., 2004). Primer validation for op- of progesterone and insulin were analyzed as repeated timum cDNA concentration and primer efficiency for measures using the MIXED procedure of SAS with day each tissue type was completed before qPCR analysis. as the variable and cow as the subject for a repeated Gene expression was analyzed using a 7500 Fast Real- measure. Progesterone was analyzed with a Compound Time PCR System (Applied Biosystems, Thermo Fisher Symmetry covariance structure. Insulin was analyzed Scientific Inc., Grand Island, NY) with SYBR Green with a Toeplitz covariance structure. Means were sepa- Master Mix (Bio-Rad Laboratories, Hercules, CA). rated using the LSMEANS statement of SAS with differ - Gene expression for maternal tissues was calculated us- ences determined at a P-values ≤ 0.05. -ΔΔCT ing the 2 method (Livak and Schmittgen, 2001) with β-actin as the reference gene and the average of NP RESULTS expression as the control (set to 1) within each tissue. Fetal membrane gene expression was calculated using Endogenous Retroviruses the same methods with the exception that the average of d 22 FM expression as the control (set to 1) within Expression of syncytin-Rum1 in CAR was greater each gene. Gene expression analysis of syncytin-Rum1, (P < 0.01; Fig. 1A) by 81.5-fold on d 50 compared with BERV-K1, IFN-τ, and PAG-1 across day was performed NP controls and all other days of gestation. In ICAR, separately from analysis of syncytin-Rum1, BERV-K1, syncytin-Rum1 expression tended (P = 0.09; Fig. 1B) IFN-τ, and PAG-1 expression across tissues within a to increase until d 28 and then decrease as pregnancy given day of gestation to compare expression between progressed from d 28 to 50. The expression of syncy- tissues. Across tissue gene expression was calculated tin-Rum1 in FM during the first 50 d of gestation did -ΔΔCT using the 2 method (Livak and Schmittgen, 2001) not change over time (P = 0.27; Fig. 1C). The expres- with β-actin as the reference gene and the average of sion of BERV-K1 was not different ( P > 0.79) at d 16 ICAR expression as the control (set to 1) on each day and 22 compared with NP control heifers. The mRNA of gestation. levels of BERV-K1 in CAR were intermediate at d 28, 34, and 40 and greater (P < 0.01) on d 40 and 50 com- pared with NP, d 16, and d 22 heifers; whereas d 28 and Statistical Analysis 34 were intermediate. In addition, d 50 was greater (P Statistical analyses were conducted via the GLM < 0.01) compared with d 28 and 34 (Fig. 2A). In ICAR, procedure of SAS version 9.4 (SAS Inst. Inc., Cary, NY), BERV-K1 was less (P = 0.003) on d 16 and 22 com- with individual heifer as the experimental unit. During pared with d 40 and 28 and greatest (P = 0.003) on d 28 pregnancy, relative pattern of mRNA expression for with a 12.9-fold increase but then returned to NP levels syncytin-Rum1, BERV-K1, IFN-τ, and PAG-1 in CAR, (Fig. 2B) during the first 50 d of gestation. In FM, the ICAR, and FM was determined via the REG procedure expression of BERV-K1 increased (P = 0.001) from d of SAS. Regression analyses were conducted to deter- 22 to d 34 with a 27.4-fold increase compared with d Table 1. Primer sequences of syncytin-Rum1, bovine endogenous retrovirus-K1 (BERV-K1), interferon–τ (IFN-τ), and pregnancy associated glycoprotein-1 (PAG-1) Gene of interest Primer direction Product size (bp) Sequence GenBank accession number Syncytin-Rum1 Forward 2464 TGGTATGACTATCTTGCTGGCTTC NM_001305454 Reverse TGGGCTGTGAGTAGTTCTAAT BERV-K1 Forward 2142 GGAAATCACGATGTCCT NM_001245951 Reverse GGAGAGGAGGCGCTTACCTG IFN-τ Forward 1313 CAGGACAGAAAGACTTTGG NM_001015511 Reverse GTGCTCTGTGTAGAAGAGGTTG PAG-1 Forward 1295 TCCAGCCTGTTCTACACACGTT NM_174411 Reverse AGGTGATCCTGAAGGTCTTATTGG Primer sequences were obtained from Cornelis et al., 2013 (syncytin-Rum1), Nakaya et al., 2013 (BERV-K1), Hickman et al., 2013 (IFN-τ), and Patel et al., 2004 (PAG-1). All sequences are represented from 5’ to 3’. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 242 McLean et al. Figure 1. Expression of syncytin-Rum1 in reproductive tissues dur- Figure 2. Expression of bovine endogenous retrovirus-K1 (BERV-K1) ing the establishment of pregnancy in beef heifers: A) syncytin-Rum1 in in reproductive tissues during the establishment of pregnancy in beef heifers: maternal caruncles (CAR), B) syncytin-Rum1 in uterine endometrium A) BERV-K1 in maternal caruncles (CAR), B) BERV-K1 in uterine endome- (ICAR), and C) syncytin-Rum1 in fetal membranes (FM). Data presented trium (ICAR), and C) BERV-K1 in fetal membranes (FM). Data presented as -ΔΔCT -ΔΔCT as a 2 -fold change normalized to β-Actin and the average of non- a 2 -fold change normalized to β-Actin and the average of non-pregnant pregnant (NP; maternal tissues) or d 22 FM (fetal tissues). Expression pat- (NP; maternal tissues) or d 22 FM (fetal tissues). Expression pattern line tern line ( – – ) via regression (P < 0.05); regression analysis does not ( – – ) via regression (P < 0.05); regression analysis does not include NP include NP heifers. Means without a common superscript differ ( P < 0.05). heifers. Means without a common superscript differ ( P < 0.05). Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Early pregnancy gene expression in heifers 243 22 heifers, while d 28 was intermediate. Expression of BERV-K1 in FM decreased from d 34 to 40 and increased again at d 50. The d 50 increase in mRNA expression of BERV-K1 in FM represents a 32.3-fold increase compared with d 22 FM (P = 0.001; Fig. 2C). Pregnancy Hormones The maternal recognition signal in ruminants, IFN-τ, was not detected in maternal tissues, CAR and ICAR; thus, analysis of IFN-τ expression as pregnancy progressed was only conducted in FM and no across tissue comparisons were made. The mRNA expres- sions of IFN-τ at d 22, which was used as baseline for all FM tissues, was greater (P < 0.01) than all other days of gestation (Fig. 3). Expression levels of PAG-1 increased dramatically with d 40 and 50 being 20,000 and 86,000-fold, respectively, greater than NP heifers in CAR (Fig. 4A). Due to the magnitude of relative fold change on d 40 and 50 in CAR for PAG-1 they were removed and the same analysis was conducted to deter- mine if differences existed early in gestation (d 16, 22, 28, and 34) compared with NP heifers. Relative expres- sion of PAG-1 was increased (P < 0.001) on d 22 and 34 (3,876- and 5,368-fold, respectively) but was not differ - ent (P > 0.10) on d 16 and 28 compared with NP heifers. In ICAR, expression of PAG-1 was greater (P < 0.05) on d 28 and 40 compared with NP with fold increases of 113 and 102, respectively, and d 22, 34, and 50 were intermediate (Fig. 4B). Expression of PAG-1 in FM tis- sue was similar (P = 0.33) across all days evaluated Figure 4. Expression of pregnancy associated glycoprotein-1 (PAG- 1) in reproductive tissues during the establishment of pregnancy in beef heifers: A) PAG-1 in maternal caruncles (CAR), B) PAG-1 in uterine en- Figure 3. Expression of (IFN-τ) in fetal membranes (FM) during the es- dometrium (ICAR), and C) PAG-1 in fetal membranes (FM). Data pre- -ΔΔCT -ΔΔCT tablishment of pregnancy in beef heifers. Data presented as a 2 -fold change sented as a 2 -fold change normalized to β-Actin and the average of normalized to β-Actin and the average of d 22 FM (fetal tissues). Expression pat- non-pregnant (NP; maternal tissues) or d 22 FM (fetal tissues). Expression tern line ( – – ) via regression (P < 0.05); regression analysis does not include pattern line ( – – ) via regression (P < 0.05); regression analysis does not NP heifers. Means without a common superscript differ ( P < 0.05). include NP heifers. Means without a common superscript differ ( P < 0.05). Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 244 McLean et al. Figure 5. Concentrations of hormones in serum of beef heifers during early gestation. A) Concentrations of circulating progesterone in serum of beef heifers B) Concentrations of circulating insulin in serum of beef heifers. Hormones are reported as a pooled mean from heifers (n = 38, 30, 25, 18, 11, and 5 for d 16, 22, 28, 34, 40, and 50; respectively) on each d. Means without a common superscript differ ( P < 0.05). (Fig. 4C). Concentrations of progesterone (Fig. 5A) on Table 2. Relative expression patterns for syncytin- d 50 (1.8 ± 1.8 and 3.6 ± 1.0 ng/mL; respectively) was Rum1, bovine endogenous retrovirus-K1 (BERV-K1), decreased (P < 0.01) compared with all other days (7.4 interferon-τ (IFN-τ), and pregnancy associated glyco- ± 0.6 ng/mL). Concentrations of insulin were similar (P protein-1 (PAG-1) during early pregnancy in beef heifers = 0.53) among all days of gestation evaluated (Fig. 5B). Equation for the best fit 1 2,3 2 Gene regression model P-value R Temporal Patterns of Expression Syncytin-Rum1 0.4125x CAR (Fig. 1A) y = 1.6298e 0.002 0.47 Regression analysis for expression of syncytin-Rum1, FM (Fig. 1C) y = 0.504x + 0.61 0.03 0.92 BERV-K1, IFN-τ, and PAG-1 across days of gestation BERV-K1 0.3567x was used to determine temporal changes in expression CAR (Fig. 2A) y = 8.4859e < 0.001 0.84 0.2619x pattern during the first 50 d of pregnancy (Table 2). In ICAR (Fig. 2B) y = 0.9091e 0.009 0.23 3 2 FM (Fig. 2C) y = 3.8167x – 35.62x + 0.01 0.89 CAR, syncytin-Rum1 (P = 0.002), BERV-K1 (P < 0.001), 102.79x – 68.984 and PAG-1 (P < 0.001) all had exponential expression IFN-τ patterns during the first 50 d of gestation. Expression pat - –1.374x FM (Fig. 3) y = 3.8167e < 0.001 0.73 terns for BERV-K1 (P = 0.009), and PAG-1 (P < 0.001) PAG-1 in ICAR were also exponential; however, syncytin-Rum1 1.1099x CAR (Fig. 4A) y = 32.007e < 0.001 0.93 expression in ICAR had no (P > 0.19) pattern of expres- 0.4691x ICAR (Fig. 4B) y = 4.967e < 0.001 0.50 sion. In FM, a linear pattern (P = 0.03) was observed in Tissue expression in maternal caruncles (CAR), uterine endometrium syncytin-Rum1 expression but a cubic pattern was found (ICAR), and fetal membranes (FM). for BERV-K1 (P = 0.01). The pattern of expression for 2 Regression analysis does not include non-pregnant heifers. IFN-τ in FM was exponential (P < 0.001) but there was Equation variables are y = gene and x = day. only a tendency for an exponential pattern of expression for PAG-1 (P > 0.08; Table 2). Expression of syncytin-Rum1 mRNA was greater in FM compared with ICAR (P < 0.002) and CAR (P < 0.004) Tissue Comparisons from on d 22, 28, and 34 of gestation. At d 40 syncytin- For comparison of gene expression across tissues on Rum1 in CAR and FM tissues were similar (P = 0.34) a given day the expression of syncytin-Rum1, BERV-K1, compared with ICAR. However, at d 50 syncytin-Rum1 IFN-τ, and PAG-1 was normalized to their average ex- expression in CAR was greater (P = 0.01) compared pression in ICAR tissues. In maternal tissues, CAR and with ICAR and FM tissues (Table 3). ICAR had similar mRNA expression of syncytin-Rum1 There were no differences between tissues ( P > 0.36; from d 16 to 40 and in NP tissues (P > 0.32; Table 3). Table 4) in BERV-K1 on d 16 and 22 in pregnant heifers However, at d 50 syncytin-Rum1 mRNA expression in and NP expression. On d 28 of gestation expression of CAR was greater than ICAR (P < 0.05) and increased BERV-K1 in FM was greater (P < 0.01) than that of ma- (P < 0.0001) by 190.3-fold over NP baseline (Table 3). ternal tissues CAR and ICAR. Expression of BERV-K1 Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Early pregnancy gene expression in heifers 245 Table 3. Relative fold change of syncytin-Rum1 Table 5. Relative fold change of pregnancy associated expression in maternal caruncles (CAR), uterine glycoprotein-1 expression in maternal caruncles (CAR), endometrium (ICAR), and fetal membranes (FM) uterine endometrium (ICAR), and fetal membranes 1 1 during the first 50 d of pregnancy in beef heifers (FM) during the first 50 d of pregnancy in beef heifers Tissue type Tissue type Gestation, d CAR ICAR FM SEM P-value Gestation, d CAR ICAR FM SEM P-value 2 2 NP 5.1 1.5 – 2.8 0.67 NP 0.01 6.84 – 4.44 0.55 16 2.4 2.2 – 0.5 0.77 16 29.67 9.93 – 8.50 0.12 a a b b a a 22 1.2 0.6 4.8 0.8 < 0.01 22 10.27 3.16 1.69 2.41 0.05 a a b 28 1.9 1.5 11.0 1.4 < 0.01 28 1.47 1.45 1.11 0.46 0.83 a a b 34 14.1 6.8 128.5 11.7 < 0.01 34 4.98 1.10 10.30 3.01 0.13 40 13.9 1.5 23.6 7.5 0.10 40 11.10 2.58 2.73 2.45 0.05 b a a b a a 50 190.3 1.1 71.7 28.6 < 0.01 50 143.98 1.23 8.91 13.84 < 0.01 a,b a,b Means within rows without a common superscript differ ( P < 0.05). Means within rows without a common superscript differ ( P < 0.05). 1 1 Data normalized to β-Actin and the average for normalized ICAR for Data normalized to β-Actin and the average for normalized ICAR for -ΔΔCT -ΔΔCT 2 values. 2 values. 2 2 NP: non-pregnant controls ovariohysterectomized at d 16 of luteal cycle. NP: non-pregnant controls ovariohysterectomized at d 16 of luteal cycle. Table 4. Relative fold change of bovine endogenous 2009; Dupressoir et al., 2011; Cornelis et al., 2012, 2013, retrovirus-K1 (BERV-K1) expression in maternal 2014, 2015). To date, mammalian placentas found to caruncles (CAR), uterine endometrium (ICAR), and form syncytium exhibit 2 morphologies, syncytiotroph- fetal membranes (FM) during the first 50 d of preg - blast and syncytial plaques. The syncytiotrophoblast is a nancy in beef heifers multinucleated tissue layer of the placenta whereas syn- cytial plaques are multinucleated cells that form at the Tissue type feto-maternal interface. Formation of syncytial plaques, Gestation, d CAR ICAR FM SEM P-value which consist of both fetal and maternal cells, is unique NP 0.47 2.33 – 1.03 0.48 to ruminants among eutherian mammals. The conjoin- 16 3.06 1.32 – 0.60 0.06 22 1.90 1.44 8.37 1.10 0.36 ing of cells of fetal and maternal origin makes separation a a b 28 0.98 1.33 6.73 3.87 < 0.01 of maternal and fetal transcriptome extremely difficult a a b 34 10.06 1.44 45.01 6.27 < 0.01 and is a limitation of this data but expression of ERV a a b 40 5.63 2.29 16.04 4.27 0.04 in maternal tissues, CAR and ICAR as reported here, a a b 50 21.04 1.26 62.55 11.12 < 0.01 is understandable in the uterus. The classical functions a,b Means within rows without a common superscript differ ( P < 0.05). for ERV of immunosuppressive and cell to cell fusion Data normalized to β-Actin and the average for normalized ICAR for (Dupressoir et al., 2011; Cornelis et al., 2013) and previ- -ΔΔCT 2 values. ously established ERV expression in fetal tissues (Blond NP: non-pregnant controls ovariohysterectomized at d 16 of luteal cycle. et al., 2000; Mi et al., 2000; Dupressoir et al., 2011; Cornelis et al., 2013) and now, from this study, ERV ex- in fetal membranes remained elevated (P < 0.04) on d 34, pression in the maternal endometrium are intriguing to 40, and 50 of gestation (45.01, 16.04, and 62.55, respec- potential roles in the establishment of pregnancy such tively; Table 4) compared with maternal tissues. When as maternal recognition, uterine immunotolerance, and comparing PAG-1 expression across tissues, there were overall placental development. no differences in maternal tissues in NP or d 16 of gesta - The measurement of basal mRNA expression during tion or across CAR, ICAR, or FM on d 28, 34, or 40 of the first 50 d of gestation is entirely novel for the ERV, gestation. On d 22 and 50, however, PAG-1 expression BERV-K1. McLean et al. (2016b) reported across tissue was increased in CAR with a fold increase of 10.27 and and day of gestation expression during early gestation 143.98, respectively (Table 5) compared to NP. but did not establish general mRNA expression pat- terns. These data are necessary to begin understanding DISCUSSION the roles of ERV in pregnancy success. As stated ear- lier, we hypothesized that the ERV (syncytin-Rum1 and Endogenous retroviral gene elements contribute to BERV-K1), IFN-τ, and PAG-1 would be differentially the formation of the multinucleation within placental expressed while progesterone and insulin concentrations formation in a wide variety of mammals with many dif- would remain steady during early gestation. In keep- ferent placental morphologies (Blond et al., 2000; Mi et ing with our hypothesis, we found BERV-K1 began to al., 2000; Dupressoir et al., 2005, 2009; Heidmann et al., increase near d 28; whereas, syncytin-Rum1 expression Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 246 McLean et al. was only different at d 50 of gestation in CAR but both with peaks at d 34 and 50. These data agree with char- exhibited exponential patterns of expression from d 16 acteristic functions of BERV-K1 for cell to cell fusion to 50 of gestation. This coincides with the time period (Nakaya et al., 2013) during binucleate cell formation when Winters et al. (1942) reported the greatest amount within placental trophoblast cells and syncytial plaques of multinucleated cells and syncytial plaque formations. between fetal and maternal cells. As well as known The early increase of BERV-K1 over syncytin-Rum1 may events during early gestation such as maternal recogni- be due to the increased cell to cell fusion capabilities of tion, embryonic adhesion with the uterine endometrium, BERV-K1, which agrees with data from Nakaya et al. and placentation. Placental development is necessary (2013). Cornelis et al. (2013), Nakaya et al. (2013) and for the transfer of nutrients responsible for the rapid fe- our data presented here indicate that ruminants have at tal growth that must occur during late gestation. least 2 ERV, syncytin-Rum1 and BERV-K1. This finding The secretion of IFN-τ from the trophoblast is widely is similar to ERV in the rodent placenta, syncytin-A and accepted as the ruminant signal for pregnancy recogni- -B (Dupressoir et al., 2011) and the human placenta syn- tion and inhibition of luteolysis (Thatcher et al., 1989; cytin-1 and -2 (Fisher et al., 1989). While syncytin-A and Bazer et al., 1991; Bazer, 1992; Mann et al., 1999; -B and syncytin-1 and -2 are homologous genes it is cur- Spencer and Bazer, 2004; Spencer et al., 2007). The rently unknown if syncytin-Rum1 and BERV-K1 are also secretion of IFN-τ must occur before the initiation of homologous to the mouse and human genes. Knockout luteolysis on d 18 of the estrous cycle. After this point mice for syncytin-A exhibit abnormal embryogenesis, ul- concentrations of IFN-τ decreased dramatically back to timately terminating gestation between d 11.5 and 13.5 basal levels which is in agreement with data from the of gestation (Dupressoir et al., 2009). While termination current research where IFN-τ mRNA expression at d 22 of rodent gestation occurs later in pregnancy, compara- was greater (P < 0.01) compared with all other days of tively, than the timeframe in this study; these data may gestation and exhibited a negative exponential pattern of be taken to imply that these BERV-K1 and syncytin-Rum1 expression in FM. Interferon-τ stimulates the production are important to placentation, placentome formation, and of many other proteins such as: ubiquitin-like interferon successful pregnancy in beef cattle. However, more work stimulated gene 15, myxovirus resistance 1, and 2ꞌ-,5ꞌ- remains to be completed to determine roles for BERV-K1 oligoadenylate synthetase 1, which may be necessary for and syncytin-Rum1 during gestation. the establishment of pregnancy (Glaucia Teixeira et al., The increased mRNA expression occurred in ICAR 1997; Perry et al., 1999; Binelli et al., 2001; Bazer et al., earlier (d 28) during pregnancy than in CAR for BERV-K1. 2015). Another such protein, PSP-B, is produced in de- The BERV-K1 increase in ICAR occurred at the end of the tectable quantities as early as d 15 of gestation (Butler et adhesion phase of implantation, which further supports al., 1982; Sasser et al., 1986); however, concentrations previous data that demonstrated BERV-K1 has increased vary greatly until after d 30 (Sasser et al.,1986; Humblot expression during early gestation and fusogenic functions et al., 1988a; Sasser et al., 1991; Vasques et al., 1995). (Koshi et al., 2012; Nakaya et al., 2013). Thus, the role The limited secretion of PSP-B early in gestation agrees of BERV-K1 in placental formation is likely in cotyledon with our data in which we observed mRNA expression of formation and subsequent syncytial plaque development. PAG-1 during the first 34 d of gestation. However, the presence of syncytin-Rum1 and BERV-K1 in The exponential increase in expression of PAG-1 maternal tissues is not in agreement with previous data for may indicate a greater prevalence in placental devel- syncytin genes in cattle (Cornelis et al., 2013) or BERV-K1 opment as gestation progresses and may be stimulat- expression in trophoblast cells (Koshi et al., 2012). The ed by IFN-τ and ERV. However, secondary functions increase earlier in gestation of BERV-K1 expression com- may be to aide IFN-τ and ERV in fetal protection dur- pared with syncytin-Rum1 may indicate a greater role for ing implantation via immune suppression (Wooding BERV-K1 for cell to cell fusion not only between tropho- et al., 2005). In addition, expression pattern for PAG-1 blast cells of the fetus but also in syncytial plaque for- in CAR was exponential with a mean fold change of mation and the combination of maternal and fetal cells. 18,000 compared with NP. Pregnancy associated glyco- These data may also support previous data (Imakawa et proteins from binucleated cells seem to interact exten- al., 2015) that suggested BERV-K1 is replacing syncytin- sively with maternal connective tissue which develops Rum1as the main catalyst in placental cell to cell fusion. during placental villi formation (Wooding et al., 2005). Although means were not different from d 22 to 50 It has been speculated that PAG may possibly be in- of gestation the linear increase in mRNA expression of volved in proteolytic activation of growth factors and syncytin-Rum1 in FM could be aiding in cell to cell fu- other molecules specific to pregnancy, protection of fe - sion occurring during placental development, which is tal tissues from maternal immune response, transport of rapidly occurring during this time (Winters et al., 1942). hormones between fetal and maternal tissues, and cell Expression pattern of BERV-K1 was cubic in nature to cell fusion (Wooding et al., 2005). Our data presented Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018 Early pregnancy gene expression in heifers 247 here confirms expression of PAG-1 during pregnancy, creased on d 50 supporting roles in cell to cell fusion and which would support Wooding et al. (2005) suggested placental development. This research also established roles of PAG-1 in cell to cell fusion during placentation. basal expression patterns for syncytin-Rum1, BERV-K1, Combined these data may also indicate an interaction IFN-τ, and PAG-1 which can be used in future research with ERV to promote the cell to cell fusion needed for to determine the influence of treatments on pregnancy. syncytial plaques formation and placental development While these data provide evidence for differential ex - to support fetal growth throughout gestation. pression the functions and interactions between syncy- Progesterone must also be present for IFN-τ to tin-Rum1, BERV-K1, IFN- τ, and PAG-1 remain to be suppress the release of PGF stimulated by oxytocin elucidated and should be the focus in future studies to 2α (Meyer et al., 1995) to maintain pregnancy (Mann and determine the importance in fetal and placental develop- Lamming, 2001; Green et al., 2005; Mann et al., 2006; ment and the establishment of pregnancy. Bazer et al., 2015). Our data clearly demonstrates el- evated circulating progesterone concentrations (> 5 LITERATURE CITED ng/mL) in pregnant heifers on all days except for d Bassil, S., J. P. Magritte, J. Roth, M. Nisolle, J. Donnez, and S. 50. Pregnant cattle will not only maintain a functional Gordts. 1995. Uterine vascularity during stimulation and its corpus luteum (CL) but also have greater progester- correlation with implantation in in-vitro fertilization. Hum. Reprod. 10:1497–1501. doi:10.1093/HUMREP/10.6.1497 one concentrations compared with non-pregnant cattle Bazer, F. W. 1992. Mediators of maternal recognition of preg- (Henricks et al., 1971; Humblot et al., 1988b; Humblot, nancy in mammals. Proc. Soc. Exp. Biol. Med. 199:373–384. 2001). The drop in progesterone, regardless of treatment, doi:10.3181/00379727-199-43371A on d 50 is intriguing; while the placenta does become Bazer, F. W., W. W. Thatcher, P. J. Hansen, M. A. Mirando, T. L. 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Is there a role for endog - electron microscope immunocytochemical studies of the distribu- enous retroviruses to mediate long-term adaptive phenotypic re- tion of pregnancy associated glycoproteins (PAGs) throughout sponse upon environmental inputs? Philos. Trans. R. Soc. Lond. B. pregnancy in the cow: Possible functional implications. Placenta Biol. Sci. 368:20110340–20110353. doi:10.1098/rstb.2011.0340 26:807–827. doi:10.1016/j.placenta.2004.10.014 Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/1/3/239/4636621 by Ed 'DeepDyve' Gillespie user on 10 April 2018

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Translational Animal ScienceOxford University Press

Published: Sep 1, 2017

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