Expression of MHC I Isoforms in Bovine Placentomes: Impact of Cloning

Abstract

Major histocompatibility complex class I (MHC-I) gene expression in the placenta is modulated to tailor the maternal immune response to fetal antigens during pregnancy. This study evaluated MHC-I expression through immunohistochemistry (IHC) using an anti-mouse preimplantation embryo development (PED) clone Qa-2 and anti-bovine leukocyte antigen I (BoLA) monoclonal antibody clone IL-A88 (n = 23), as well as RT-qPCR (n = 17) for classical and non-classical (BoLA-NC) genes in control and cloned bovine placentomes during early and near-term gestation. Control samples showed minimal Qa-2 protein expression in early gestation, with intense labeling in trophoblasts and the maternal uterine epithelium near term. In contrast, cloned samples exhibited intense Qa-2 labeling in both maternal and trophoblastic epithelia at both stages, while trophoblast giant cells (TGCs), located apposed to the maternal epithelium, showed no labeling. Control samples exhibited intense IL-A88 labeling in the maternal epithelium at both stages. In cloned samples, weak to no labeling was observed in early gestation, with intense labeling in maternal and fetal epithelium near term. RT-qPCR revealed significant upregulation of BoLA-NC3 in early gestation, with sustained elevated expression in cloned samples in the near term. These findings suggest that altered BoLA protein expression and gene regulation in cloned pregnancies may contribute to pregnancy complications and increased losses.

Keywords: BoLA; IL-A88; Qa-2; bovine placenta; chorionic villi; cloned bovine.

Figure 1

Cluster analysis of MHC I isotype and peptide sequences recognized by Qa-2 (X80933.1) and IL-A88 (NM_001201460.1) antibodies.

Figure 2

Detection of MHC by Qa-2 antibody in bovine placentomes from control and cloned pregnancies. (A) Control at 60 days of pregnancy. Absence of Qa-2 in all tissues. (B) Control at 90 days of pregnancy. Absence of Qa-2 expression. (C) Control at term. Increased Qa-2 expression restricted to trophoblast and maternal epithelium. (D) Clone at 60 days of pregnancy. Qa-2 restricted to trophoblast and maternal epithelium. (E) Clone at 90 days of pregnancy. Increased Qa-2 labeling in trophoblast and maternal epithelium and low labeling in trophoblast giant cells near maternal epithelium. (F) Clone at term. The same pattern as (E) but with low intensity. (F`) IgG control. Brown is an immune reaction product; blue is a hematoxylin counterstain. Endometrial stroma (S), maternal epithelium (E), trophoblast (T), and mesenchyme (M). Bar = 20 µm.

Figure 3

Detection of MHC by Qa-2 antibody in bovine clone placentomes at 90 days of pregnancy. Trophoblastic giant cells (TGCs—arrow) migrate into disorganized maternal epithelium (E). The deeper the TGCs were in the maternal epithelium, the lower the Qa-2 expression. Brown: immune reaction product; blue: hematoxylin counterstain. Endometrial stroma (S), maternal epithelium (E), trophoblast (T), and TGC (arrow). Bar = 20 µm.

Figure 4

Immunohistochemistry for IL-A88 in bovine placentomes from control and cloned pregnancies. (A) Control at 60 days of pregnancy, the anti-IL-A88 signal is observed in the maternal epithelium and light expression in the trophoblast. (B) Control at 90 days of pregnancy, anti-IL-A88 is absent in trophoblast. (C) Control at term, low presence of anti-IL-A88 in maternal epithelium and trophoblast. (D) Clone at 60 days of pregnancy. (E) Clone at 90 days of pregnancy. The maternal epithelium has a light IL-A88 signal in both (D,E), whereas it is absent in trophoblasts. (F) Clone at term, the presence of anti-IL-A88 in the maternal epithelium and trophoblast with more intensity than in controls (C). Brown is an immune reaction product; blue is a hematoxylin counterstain. (F`) IgG control. Endometrial stroma (S), maternal epithelium (E), trophoblast (T), and mesenchyme (M). Bar = 20 µm.

Figure 5

Expression of BoLA-NC1 (a), BoLA-NC2 (b), BoLA-NC3 (c), BoLA-NC4 (d), and JSP-1 (e) in bovine placentomes from control (dark bar) and cloned (grey bar) pregnancies. In the X axis, the pregnancy period (first or third trimester); in the Y axis, gene expression is shown in dCt. Data are least squares means ± S.E.M. Lowercase letters represent significance (p ≤ 0.05) between control placentomes. Uppercase letters represent significance (p ≤ 0.05) between cloned placentomes. Continuous bars represent significance (p ≤ 0.05) inside pregnancy age.