Enhanced proapoptotic gene expression of XAF1, CASP8 and TNFSF10 in the bovine endometrium during early pregnancy is not correlated with augmented apoptosis

Transcript abundances of extrinsic apoptosis factors. Messenger RNA expression of extrinsic TNF family members FASLG (C), TNFSF10 (D) and TNF (E) and their death receptors FAS (F), TNFRSF10B (G) and TNFRSF1B (H) in endometria and conceptuses are shown as means ΔCq±SEM. Normalized Cq (ΔCq) values were subtracted from the arbitrary value 30. The effect of the day within the status is shown by different superscript letters (x–z in cyclic animals and a–c in pregnant animals). Asterisks indicate significant differences (p<0.05). nd indicates non-determinable values.

Messenger RNA expression of caspases as well as inhibitor of apoptosis (IAPs) and their antagonists. Messenger RNA expression of initiator CASP8 (A) and effector CASP3 (B) as well as inhibitor of apoptosis (IAPs) BIRC4 (E) and BIRC5 (F) in addition to their antagonists XAF1 (G) and DIABLO (H) in endometria and corresponding conceptuses are shown. Messenger RNA expression is shown as means ΔCq±SEM. Caspase activity of CASP8 (D) and CASP3/7 (E) in endometrial homogenates is presented as means±SEM relative light units per μg total protein (RLU/μg TP). The effect of the day within the status is shown by different superscript letters (x–z in cyclic animals and a–c in pregnant animals). Asterisks indicate significant differences (p<0.05). nd indicates non-determinable values.

Bioactivity of IFNT in uterine flushing fluids (A) and messenger RNA expression of IFN type I receptor subunits IFNAR1 (B) and IFNAR2 (C). Results are shown as means±SEM. Asterisks indicate significant differences (p<0.05).

Localisation of apoptotic cells by TUNEL in bovine endometrium and immunohistochemical localisation of FASLG in bovine tissue sections. TUNEL experiments: The negative control was incubated without rTdT enzyme (A) and the positive control with 10 Units DNase/ml (insert). Light microphotograph of an endometrial section showing TUNEL-positive cells (B). Arrows point to apoptotic cells. FASLG immunohistochemistry: Pictures show representative sections of the superficial endometrium (C), the deep endometrium (D), endothelial cells (E) and day 18 trophoblasts (F). Inserts represent negative controls.

Apoptosis can be either elicited by the intrinsic pathway through proapoptotic Bcl-2-family factors (BAX) or the extrinsic pathway via death receptor ligands (FASLG, TNFSF10 and TNF) by binding to respective receptors (FAS, TNFRSF10B, TNFRSF1A,-B). Both pathways cause activation of initiator caspases (CASP8) and effector caspases (CASP3, 6, 7). Subsequent activation of caspase activated DNases result in DNA fragmentation and apoptosis. Proapoptotic components (XAF1, DIABLO) may diminish the effects of antiapoptotic factors (BCL2L1, CFLAR, BIRC4, 5). Although expression of proapoptotic genes XAF1, CASP8 and TNFSF10 was elevated in pregnant animals in the presence of trophoblast IFNT the activity of caspases and the incidence of apoptotic cells did not increase. Inhibitory mechanisms preventing endometrial cells from apoptosis are presumed.