Identification of 9 uterine genes that are regulated during mouse pregnancy and exhibit abnormal levels in the cyclooxygenase-1 knockout mouse

Abstract

Background: Preterm birth is the leading cause of all infant mortality. In 2004, 12.5% of all births were preterm. In order to understand preterm labor, we must first understand normal labor. Since many of the myometrial changes that occur during pregnancy are similar in mice and humans and mouse gestation is short, we have studied the uterine genes that change in the mouse during pregnancy. Here, we used microarray analysis to identify uterine genes in the gravid mouse that are differentially regulated in the cyclooxygenase-1 knockout mouse model of delayed parturition.

Methods: Gestational d18.0 uteri (n = 4) were collected from pregnant wild-type and cyclooxygenase-1 knockout mice. Part of the uterus was used for frozen sections and RNA was isolated from the remainder. Microarray analysis was performed at the Indiana University School of Medicine Genomic Core and analyzed using the Microarray Data Portal. Northern analysis was performed to confirm microarray data and the genes localized in the gravid uterus by in situ hybridization.

Results: We identified 277 genes that are abnormally expressed in the gravid d18.0 cyclooxygenase-1 knockout mouse. Nine of these genes are also regulated in the normal murine uterus during the last half of gestation. Many of these genes are involved in the immune response, consistent with an important role of the immune system in parturition. Expression of 4 of these genes; arginase I, IgJ, Tnfrsf9 and troponin; was confirmed by Northern analysis to be mis-regulated during pregnancy in the knockout mouse. In situ hybridization of these genes demonstrated a similar location in the gravid wild-type and Cox-1 knockout mouse uteri.

Conclusion: To our knowledge, this is the first work to demonstrate the uterine location of these 4 genes in the mouse during late pregnancy. There are several putative transcription factor binding sites that are shared by many of the 9 genes identified here including; estrogen and progesterone response elements and Ets binding sites. In summary, this work identifies 9 uterine murine genes that may play a role in parturition. The function of these genes is consistent with an important role of the immune system in parturition.

Figure 1

Blot and graph of Northern analysis of arginase, immunoglobulin J chain, Tnfrsf9 and troponin levels from gravid d18.0 wild-type (WT) and Cox-1 KO uteri (n = 5). Error bars show the standard error.

Figure 2

In situ hybridization for arginase, Tnfrsf9, and Tnni2 in the non-gravid and gravid d13.5 wild-type and Cox-1 KO mouse uteri. In situ hybridization was performed on (n = 4) each gravid time point and (n = 2) non-gravid mice. Shown are representative light-field images (left) and dark-field images (right) from non-gravid (NG) (50×) or gravid d13.5 uteri (25×). The slides were counterstained with hematoxylin and eosin. Areas of hybridization appear white, demonstrating deposition of silver granules.

Figure 3

In situ hybridization for arginase, Tnfrsf9, and Tnni2 in the gravid d16.5 and d19.0 wild-type and Cox-1 KO mouse uteri. In situ hybridization was performed (n = 4) from each gravid time point. Shown are representative light-field images (left) and dark-field images (right) from gravid d16.5 and d19.0 uteri (25×). The slides were counterstained with hematoxylin and eosin. Areas of hybridization appear white, demonstrating deposition of silver granules.

Figure 4

In situ hybridization for IgJ in the gravid d13.5, d16.5, d18.0 and d19.0 wild-type and Cox-1 KO mouse uteri. In situ hybridization was performed (n = 4) from each gravid time point and non-gravid mice (n = 2). Shown are representative light-field images (left) and dark-field images (right) (25×). The slides were counterstained with hematoxylin and eosin. Areas of hybridization appear white, demonstrating deposition of silver granules.

Figure 5

Bar graphs for Northern analysis of arginase, immunoglobulin J chain, Tnfrsf9 and troponin. Ten μg of RNA isolated from gravid d13.5, d16.5 and d19.0 wild-type (n = 3) and d13.5, d16.5, d19.0, d20.0 and d21.0 Cox-1 KO (n = 3) uteri were run on the gel. Error bars show the standard error.