Trophoblast Major Histocompatibility Complex Class I Expression Is Associated with Immune-Mediated Rejection of Bovine Fetuses Produced by Cloning

Abstract

Trophoblast cells from bovine somatic cell nuclear transfer (SCNT) conceptuses express major histocompatibility complex class I (MHC-I) proteins early in gestation, and this may be one cause of the significant first-trimester embryonic mortality observed in these pregnancies. MHC-I homozygous-compatible (n = 9), homozygous-incompatible (n = 8), and heterozygous-incompatible (n = 5) SCNT pregnancies were established. The control group consisted of eight pregnancies produced by artificial insemination. Uterine and placental samples were collected on Day 35 ± 1 of pregnancy, and expression of MHC-I, leukocyte markers, and cytokines were examined by immunohistochemistry. Trophoblast cells from all SCNT pregnancies expressed MHC-I, while trophoblast cells from age-matched control pregnancies were negative for MHC-I expression. Expression of MHC-I antigens by trophoblast cells from SCNT pregnancies was associated with lymphocytic infiltration in the endometrium. Furthermore, MHC-I-incompatible conceptuses, particularly the heterozygous-incompatible ones, induced a more pronounced lymphocytic infiltration than MHC-I-compatible conceptuses. Cells expressing cluster of differentiation (CD) 3, gamma/deltaTCR, and MHC-II were increased in the endometrium of SCNT pregnancies compared to the control group. CD4(+) lymphocytes were increased in MHC-I-incompatible pregnancies compared to MHC-I-compatible and control pregnancies. CD8(+), FOXP3(+), and natural killer cells were increased in MHC-I heterozygous-incompatible SCNT pregnancies compared to homozygous SCNT and control pregnancies.

Keywords: cattle; immunohistochemistry; miscarriage; pregnancy; somatic cell nuclear transfer.

FIG. 1

Crown-to-rump length (cm) and weight of embryos at Day 35 ± 1 of gestation. SCNT pregnancies were either MHC-I homozygous-compatible (n = 9), homozygous-incompatible (n = 8), or heterozygous-incompatible (n = 5). Age-matched control pregnancies (n = 8) were established by AI. There was no significant difference between any of the groups.

FIG. 2

Percent area of MHC-I⁺ trophoblast cells in the placenta, and dispersed CD3⁺ and CD4⁺ T cells in the endometrium in control (n = 8), MHC-I homozygous-compatible SCNT (n = 9), MHC-I homozygous-incompatible SCNT (n = 8), and MHC-I heterozygous-incompatible SCNT (n = 5) pregnancies at Day 35 ± 1 of pregnancy. Age-matched control pregnancies were established by AI. Groups with different superscripts were significantly different (P < 0.05).

FIG. 3

Immunohistochemical labeling of trophoblast for MHC-I (A–D), and endometrial tissue for CD3 (E–H) and CD4 (I–L) of cows at Day 35 ± 1 of pregnancy. A, E, and I) Pregnancies established by AI (control). B, F, and J) MHC-I homozygous-compatible SCNT pregnancies. C, G, and K) Homozygous-incompatible SCNT pregnancies. D, H, and L) Heterozygous-incompatible SCNT pregnancies. Bar = 50 μm.

FIG. 4

Percent area of dispersed CD8⁺, FOXP3⁺, and γ/δTCR⁺ lymphocytes in the endometrium of Day 35 ± 1 pregnancies established by SCNT, which were MHC-I homozygous compatible (n = 9), MHC-I homozygous incompatible (n = 8), or MHC-I heterozygous incompatible (n = 5). Age-matched control pregnancies were established by AI (n = 8). Groups with different superscripts were significantly different (P < 0.05).

FIG. 5

Percent area of dispersed cells positive for CR2, MHC-II, and NCR1 in the Day 35 ± 1 pregnant endometrium. Pregnancies were established by either AI (control, n = 8) or SCNT. SCNT pregnancies were MHC-I homozygous compatible (n = 9), MHC-I homozygous incompatible (n = 8), or MHC-I heterozygous incompatible (n = 5). Groups with different superscripts were significantly different (P < 0.05).

FIG. 6

Percent area of CD68⁺ cells in the endometrium and placenta of pregnancies at Day 35 ± 1 of gestation. SCNT pregnancies were MHC-I homozygous compatible (n = 9), MHC-I homozygous incompatible (n = 8), or MHC-I heterozygous incompatible (n = 5). Age-matched control pregnancies were established by AI (n = 8). Groups with different superscripts were significantly different (P < 0.05).

FIG. 7

Immunohistochemical labeling of cells for TNF (A–D), IL12 (E–H), and CSF2 (I–L) in the endometrium of cows at Day 35 ± 1 of pregnancy. A, E, and I) Pregnancies established by AI (control). B, F, and J) MHC-I homozygous-compatible SCNT pregnancies. C, G, and K) Homozygous-incompatible SCNT pregnancies. D, H, and L) Heterozygous-incompatible SCNT pregnancies. Bar = 50 μm.