70 Alterations in miRNA and tRNA-derived fragment expression during bovine conceptus elongation

Failure of the embryo to survive and establish pregnancy is due to paternal, maternal, or embryonic factors or a combination of these factors. Successful fertilization generally occurs following breeding in cattle, but embryonic mortality is especially high during the first month of gestation. Small noncoding RNAs play crucial roles in oocyte and embryonic development by regulating gene expression at the post-transcriptional level. Mature tRNAs are processed by Dicer-dependent or -independent cleavage pathways from which five classes of tRNA-derived fragments (tRFs) originate: 5′ tRFs, 3′ tRFs, internal tRFs (i-tRFs), 5′ halves, and 3′ halves. Despite extensive research on the mechanisms regulating conceptus elongation, the roles of microRNAs (miRNAs) and tRFs during this pivotal stage of preimplantation development remain largely unexplored. In the current study, small RNA sequencing was performed on conceptuses in different stages of development to profile the miRNA and tRF dynamics. Angus heifers (n = 8) were superovulated and bred using semen from two proven Bos indicus sires. Heifers were flushed on Days 13–14 of gestation using a standard flushing technique. Recovered conceptuses were individually photographed and snap-frozen for RNA extraction and small RNA sequencing. Recovered conceptuses (n = 39) were classified based on morphology into ovoid (OV, 0.2–5 mm; n = 6; each sample containing two to five embryos), tubular (TUB, <15 mm; n = 7), or filamentous (FIL, >16 mm; n = 7). To determine the differentially expressed (DE) miRNAs and tRFs, pairwise comparisons between distinct stages of development (OV vs. TUB, TUB vs. FIL) were conducted using DESeq2 version 1.40.2. Significant changes in miRNA profiles were observed only during the transition from OV to TUB. Principal component analysis (PCA) revealed clustering of OV samples away from the TUB and FIL groups (PCA1: 54%, PCA2: 7%). The expression of 81 miRNAs increased and 123 decreased in OV compared with TUB conceptuses. Similarly, for tRFs, PCA revealed distinct clustering of OV conceptuses from the TUB and FIL groups (PCA1: 42%, PCA2: 28%). The most pronounced changes in the expression of tRF were identified between the OV and TUB transition, with 494 DE tRFs (286 up and 208 downregulated in OV), with i-tRFs subtype representing 75.1% of the DE tRFs. The transition from TUB to FIL had only one upregulated tRF, an i-tRF (anticodon AsnGTT). During the OV stage, 58% of DE tRFs were of the i-tRF class, while for the TUB and FIL, the i-tRF class represented less than 35% of the DE tRFs. Conversely, the TUB and FIL conceptuses had increased abundance of the 5ʹ halves subtype (over 56%), while OV conceptuses had only 21% of 5ʹ halves tRFs. Because i-tRFs primarily modulate translation initiation, while 5ʹ halves are more directly involved in gene silencing and mRNA stability, our results suggest that distinct post-transcriptional mechanisms are at play between the OV and TUB stages. Our results provide new insights into the transcriptional regulation during bovine conceptus elongation. This fundamental knowledge is necessary to guide genomic selection and support the development of innovative tools to ameliorate the effects of embryonic mortality.