170 EXPRESSION PROFILE AND PROTEIN SECRETION OF BOVINE INTERFERON τ ON DAYS 12 TO 28 IN EARLY PREGNANCY

Abstract

Bovine interferon (bIFN) τ has been implicated as a mediator of maternal recognition of pregnancy in cattle. Ovine (o) and bIFNτ expression show temporal patterns until implantation begins (Farin et al. 1990 Biol. Reprod. 43, 210–218); therefore, the expression peaks of bIFNτ are not well understood. This study aimed to investigate the expression profile of mRNA and protein secretion of bIFNτ in conceptuses obtained from cows on Days 12 to 28 in early pregnancy. Induction of superovulation and recipient synchronization were achieved by previously reported methods (Hirayama et al. 2004 Theriogenology 62, 887–896). Embryos were recovered non-surgically on Day 8 (Day 0 = estrus). Subsequently, recovered embryos were singly transferred to recipients immediately upon evaluation. Bovine conceptuses and uterine flushings were collected non-surgically from embryo-transferred cows on Days 12 (n = 2), 14 (n = 6), 16 (n = 5), 18 (n = 4), and 20 (n = 5) with MEM. On Days 22 (n = 3), 24 (n = 5), and 28 (n = 2), bovine conceptuses were recovered within 30 min after slaughter by flushing the uterus with PBS supplemented with 0.1% BSA. Uterine flushings were also collected from 6 cows in the control group on Day 16 of the estrous cycle. Conceptuses for collection of RNA were processed individually and stored at -80°C until analysis. The flushing was centrifuged at 1800g and 4°C for 30 min to remove cellular debris, and stored at -30°C for subsequent measurement of bIFNτ. Total RNA in conceptuses were reverse transcribed for PCR. Quantification of mRNA abundance was performed by real-time PCR. The expression of each mRNA concentration was normalized to the abundance of GAPDH. BIFNτ contents of the uterine flushings were measured by RIA (Takahashi et al. 2005 Theriogenology 63, 1050–1060). Intra-assay and inter-assay CVs were 11.0 and 8.5%, respectively. Transcripts of bIFNτ were detected in all samples from Days 12 to 28; the expression of mRNA increased remarkably from Day 16 (mean ± SEM, 2.15 ± 0.66) to Day 18 (9.62 ± 4.09), and then declined on Day 20 (3.42 ± 0.86). Protein detected on Day 16 was 13.3 ± 3.02 µg. An increase of bIFNτ secretion was observed between Day 18 (39.8 ± 20.62 µg) and Day 20 (71.9 ± 17.36 µg); thereafter it decreased on Day 22 (27.2 ± 2.83 µg). BIFNτ displayed a pattern of expression similar to that of oIFNτ, with mRNA expression peaking around Day 18 (ovine, Days 12/13) and protein secretion on Day 20 (ovine, Days 15/16), thereby reflecting the comparatively later time frame of maternal recognition of pregnancy in cattle. These results indicate that the expression and secretion profiles of bIFNτ show temporal and spatial patterns in early pregnancy.