Early pregnancy-induced transcripts in peripheral blood immune cells in Bos indicus heifers

Abstract

Immune cells play a central role in early pregnancy establishment in cattle. We aimed to: (1) discover novel early-pregnancy-induced genes in peripheral blood mononuclear cells (PBMC); and (2) characterize the temporal pattern of early-pregnancy-induced transcription of select genes in pBMc and peripheral blood polymorphonuclear cells (PMN). Beef heifers were artificially inseminated on D0 and pregnancies were diagnosed on D28. On D10, 14, 16, 18, and 20, blood was collected for isolation of PBMC and PMN from heifers that were retrospectively classified as pregnant (P) or non-pregnant (NP). PBMC samples from D18 were submitted to RNAseq and 220 genes were differentially expressed between pregnant (P) and non-pregnant (NP) heifers. The temporal abundance of 20 transcripts was compared between P and NP, both in PBMC and PMN. In PBMC, pregnancy stimulated transcription of IFI6, RSAD2, IFI44, IFITM2, CLEC3B, OAS2, TNFSF13B, DMKN and LGALS3BP as early as D18. Expression of IFI44, RSAD2, OAS2, LGALS3BP, IFI6 and C1R in PMN was stimulated in the P group from D18. The novel early-pregnancy induced genes discovered in beef heifers will allow both the understanding of the role of immune cells during the pre-attachment period and the development of technologies to detect early pregnancies in beef cattle. In cattle, pregnancy success depends on the maintenance of a functional corpus luteum (CL) beyond the time of luteolysis, which normally occurs between days 15 and 18 of the estrous cycle 1,2. The CL maintenance in ruminants occurs in response to interferon-τ (IFN-τ) secreted by the conceptus 3. The INF-τ glycoprotein binds to its receptors and consequently inhibits the pulsatile secretion of prostaglandin F2α (PGF2α) from the endometrium, preventing luteolysis 4. Consequently, the CL remains active and secretes progesterone (P4) in concentrations sufficient for the establishment of pregnancy 1. Success in this sequence of events determines the outcome of pregnancy; however, as many as 50% of pregnancies fail until day 17 after artificial insemination (AI) 5. The mechanisms that mediate embryo survival and death are incompletely understood, but the maternal immune system plays an important role in embryo development during the pre-attachment period 6. A functional connection between the maternal immune system and the developing embryo is IFN-τ. The day-4 bovine embryo is already capable of synthesizing IFN-τ, which regulates the local immune environment in the oviduct 7. In addition, the in vitro development of bovine embryos from morula (day-5) to blastocyst stage (day-9), starts signaling the uterine epithelial and immune cells in co-culture to modulate the anti-inflammatory response mediated by IFN-τ 8. These changes contribute to conceptus growth and maternal immune modulation to prevent conceptus rejection 9. Therefore, an immunological crosstalk between embryo and immune cells exist locally in the uterus, however, how those signals influence the immune cells is currently unclear. Studies in ruminants during the last d...

Feasibility and accuracy of using different methods to detect pregnancy by conceptus-stimulated genes in dairy cattle

“…The potential use of LGALS3BP expression as a pregnancy marker before d 20 post-TAI was recently indicated by our group because its expression is elevated in PBMC from d 10 to 20 of pregnancy in beef heifers (Rocha et al, 2020). Intriguingly, the present results were distinct, indicating that the presence of a viable bovine embryo did not stimulate LGALS3BP expression in immune cells from blood or milk but did stimulate it in CC samples.…”

“…Recently, other studies have used total immune cells from whole blood (Yoshino et al, 2018) or milk (Schanzenbach et al, 2017) or cervical and vaginal mucosa cells (Kunii et al, 2018) for accessing gene expression and predicting pregnancy status. In addition, novel potential pregnancy markers were recently reported by our group in beef heifers (Rocha et al, 2020) and need to be tested for early pregnancy diagnosis in dairy cattle. From these potential markers, LGALS3BP expression in immune cells was always 50% greater in pregnant heifers from d 10 to 20 of pregnancy.…”

“…The genes GAPDH, cyclophilin A (PPIA), ribosomal RNA (18S), and β-actin (ACTB) were tested as reference genes. Primers were previously described for cattle Rocha et al, 2020).…”

Comparison of the Ability of High and Low Virulence Strains of Non-cytopathic Bovine Viral Diarrhea Virus-1 to Modulate Expression of Interferon Tau Stimulated Genes in Bovine Endometrium

“…They then showed that measurement of ISG15 mRNA could provide a reliable indicator of early pregnancy on day 18 of gestation in heifers but not in mature cows (81). These findings have since been confirmed in beef breeds using both polymorphonuclear neutrophils and peripheral blood mononuclear cells (82,83) and extending to a number of other ISGs (84). Although the results of ISG15 up-regulation in early pregnancy were highly repeatable between studies, de Melo et al (85) found that ISG15 measurement was less reliable for confirmation of pregnancy than the use of circulating pregnancy-associated glycoprotein or Doppler ultrasound.…”

Conceptus-modulated innate immune function during early pregnancy in ruminants: a review

“…Our interest in the pattern recognition receptors (PRR) started when we performed an enrichment analysis (unpublished) with our transcriptomic data in PBMC of pregnant and nonpregnant heifers (Rocha et al, 2020). The PPR pathway came up as the most significant in PBMC of pregnant heifers on day 18 post-AI, suggesting that PRR is the main pathway affected by pregnancy during this period in immune cells.…”