Endometrial caspase 1 and interleukin-18 expression during the estrous cycle and peri-implantation period of porcine pregnancy and response to early exogenous estrogen administration

Abstract

Background: The role for endometrial secretion of cytokines during the establishment of pregnancy in a number of mammals is well established. The current study determined endometrial expression of caspase 1 (CASP1) and interleukin-18 (IL18) during the estrous cycle and early pregnancy, and following early estrogen administration, which induces conceptus loss during early development in pigs.

Methods: Gilts were hysterectomized on either D 0, 5, 10, 12, 15 and 18 of the estrous cycle, or D 10, 12, 15 or 18 of pregnancy. The abundance of endometrial CASP1 mRNA was unaffected by day of the estrous cycle, however there was a 6 and 10-fold increase in expression on D 15 and 18 of pregnancy. Endometrial expression of IL18 mRNA increased 5-fold between D 10 to 18 in cyclic and pregnant gilts. Total recoverable IL18 in uterine flushings was greater in pregnant compared to cyclic gilts on D 15 and 18.In the second experiment, mated gilts were treated with either corn oil (CO) or estrogen (E) on D 9 and 10 and hysterectomized on either D 10, 12, 13, 15 or 17 of pregnancy. The current study localizes the presence of CASP1 to the epithelial layer of the endometrium for the first time. Further, a day x treatment interaction was detected for endometrial CASP1 mRNA and protein abundance as E stimulated an earlier increase on D 13 compared to CO gilts. Although IL18 mRNA expression remained unaltered from the E treatment, protein abundance was significantly attenuated on D 15 and 18 in response to E treatment.

Conclusions: Endometrial expression of CASP1 and IL18 is associated with establishment of pregnancy in pigs. Alteration of CASP1 and IL18 following premature exposure of the uterus to estrogen during early pregnancy may contribute to conceptus loss between Days 15 to 18 of pregnancy.

Figure 1

Relative mRNA abundance in endometrial IL18 mRNA expression from gilts (n = 4/day/status) on days 0, 5, 10, 12, 15, and 18 of the estrous cycle (open bars) and days 10, 12, 15 and 18 of pregnancy (black bars). Abundance of mRNA was calculated from the real-time PCR analysis as described in Methods and Materials. A significant day effect on relative mRNA units (mean ± SEM) was identified for endometrial IL18 (P < 0.05). Days without a common superscript represent a statistical difference.

Figure 2

Relative content of total IL18 (pg) in uterine flushing collected from gilts (n = 4/day/status) on days 0, 5, 10, 12, 15 and 18 of the estrous cycle (open bars) and days 10, 12, 15 and 18 of pregnancy (black bars). A day × status interaction (P < 0.05) was detected for total IL18. Bars without a common superscript represent a statistical difference.

Figure 3

Relative mRNA adundance in endometrial caspase-1 mRNA expression from gilts (n = 4/day/status) on days 0, 5, 10, 12, 15, and 18 of the estrous cycle (open bars) and days 10, 12, 15 and 18 of pregnancy (black bars). Abundance of mRNA was calculated from the real-time PCR analysis as described in Methods and Materials. A significant day × status interaction on relative mRNA units (mean ± SEM) was identified for endometrial caspase-1 (P < 0.05). Bars without a common superscript represent a statistical difference.

Figure 4

In situ hybridization analysis of caspase-1 mRNA expression in porcine endometrium. Protected transcripts in endometrium from day 15 of pregnancy was visualized by liquid emulsion autoradiography and imaged under bright-field and dark-field illumination. Endometrial sections from E treated section on Day 12 of pregnancy was hybridized with radiolabeled sense cRNA probe to serve as a negative control. Caspase-1 mRNA expression is abundant in the luminal (L) and glandular (G) epithelium but low to absent in the stroma (S). 4× Objective and 10× eyepiece.

Figure 5

Relative content of total caspase-1 protein (pg) in uterine flushings from gilts (n = 4/day/status) on days 10, 12, 15 and 18 of the estrous cycle (open bars) and pregnancy (black bars). Content of caspase-1 in the uterine luminal flushings of cyclic and pregnant gilts exhibited a day by status interaction (P < 0.05). Bars without a common superscript represent a statistical difference.

Figure 6

Relative mRNA abundance in endometrial IL18 mRNA expression of control (open bars) and estrogen (black bars) treated gilts (n = 4/day/treatment) on days 10, 12, 13, 15 and 17 of pregnancy. Abundance of mRNA was calculated from the real-time PCR analysis as described in Methods and Materials. No significant main effects or interaction were observed on relative mRNA units (mean ± SEM) for endometrial IL18 (P > 0.05).

Figure 7

Relative content of total IL18 (pg) in uterine flushing collected from control (open bars) and estrogen (black bars) treated gilts (n = 4/day/treatment) on days 10, 12, 13, 15 and 17 of pregnancy. A day × treatment interaction (P < 0.05) was detected. Bars without a common superscript represent a statistical difference.

Figure 8

Relative mRNA abundance in endometrial caspase-1 mRNA expression of control (open bars) and estrogen treated (black bars) gilts (n = 4/day/treatment) on days 10, 12, 13, 15 and 17 of pregnancy. Abundance of mRNA was calculated from the real-time PCR analysis as described in Methods and Materials. A significant day by treatment interaction was observed on relative mRNA units (mean ± SEM) was identified for endometrial caspase-1 (P < 0.05). Bars without a common superscript represent a statistical difference.

Figure 9

Relative content of total caspase-1 protein (pg) in uterine flushing collected from control (open bars) and estrogen treated (black bars) gilts (n = 4/day/treatment) on days 10, 12, 13, 15 and 17 of pregnancy. A day × treatment interaction (P < 0.05) was detected. Bars without a common superscript represent a statistical difference.