During pregnancy in several species including humans and rodents the endometrium undergoes decidualization. This process of differentiation from endometrial to decidual tissue occurs only after the onset of implantation in mice. It can also be artificially-induced causing the formation of deciduomal tissue. The purpose of this study was to compare the gene expression profile of the developing decidua in pregnant mice with the deciduoma formed after artificial induction in an effort to identify conceptus-influenced changes in uterine gene expression during decidualization. We induced decidualization artificially by transferring blastocyst-sized ConA-coated agarose beads into the uterus on Day 2.5 of pseudopregnancy. Recently published work has found this model to be more “physiological” than other methods. Total RNA was isolated from blastocyst and bead induced “implantation” sites of the uteri of Day 7.5 pregnant (decidua) and pseudopregnant (deciduoma) mice, respectively. This RNA was then used for microarray analysis using Mouse Illumina Beadarray chips. This analysis revealed potential differential mRNA levels of only 45 genes between the decidua and bead-induced deciduoma tissues. We confirmed the differential mRNA levels of 31 of these genes using quantitative RT-PCR. Finally, the level and localization of some of the mRNA’s for select genes (Aldh3a1, Bcmo1, Guca2b, and Inhbb) identified by our microarray analysis were examined in more detail. This study provides the identity of a small set of genes whose expression in the uterus during decidualization may be influenced by molecular signals from the conceptus.

During decidualization, uterine natural killer cells are the most abundant immune cell types found in the uterus. Although it is well known that they play key roles in spiral arteriole modification and the maintenance of decidual integrity seen after mid-pregnancy, their roles in the differentiation of decidual cells and accompanying angiogenesis during the process of decidualization is less well-characterized. To address this, we used whole-genome Illumina BeadChip analysis to compare the gene expression profiles in implantation segments of the uterus during decidualization on Day 7.5 of pregnancy between wild-type and uterine natural killer cell-deficient (interleukin-15-knockout) mice. We found almost 300 differentially-expressed genes and verified the differential expression of approximately 60 using quantitative RT-PCR. Notably there was a lack of differential expression of genes involved in decidualization and angiogenesis and this was also verified by quantitative RT-PCR. Similar endothelial cell densities and proliferation indices were also found in the endometrium between the implantation site tissues of wild-type and knockout mice undergoing decidualization. Overall, the results of this study reveal that uterine natural killer cells likely do not play a major role in decidualization and accompanying angiogenesis during implantation. In addition, the study identifies a large number of genes whose expression in implantation-site uterine tissue during decidualization depends on interleukin-15 expression in mice.

Tnfrsf9 gene expression has previously been characterized in the late pregnant mouse uterus and placenta. However, little is known about its expression in the uterus during the implantation phase of early pregnancy. Since TNFRSF9 is known to play a potentially important role in immune function in general, the purpose of this study was to assess the levels and localization of Tnfrsf9 expression in the mouse uterus and conceptus during implantation. Relative Tnfrsf9 mRNA levels were significantly increased in implantation compared to non-implantation site tissue on Days 6.5- 8.5 of pregnancy. This increase did not depend on the presence of the conceptus, as mRNA levels were not significantly different between pregnant implantation sites and artificially-induced deciduomas. Localization using in situ hybridization revealed that a subpopulation of endothelial as well as uterine natural killer cells expressed Tnfrsf9 in the endometrium during implantation. In the developing conceptus, primary trophoblast giant and ectoplacental cells expressed Tnfrsf9 on Days 6.5-8.5. This was followed by expression in the trophoblast giant cell layers surrounding the conceptus on Day 9.5 of pregnancy. Since 2 main splice forms of Tnfrsf9 mRNA exist and encode proteins which have distinct biological functions, we also determined their presence in these tissues. Both mRNA splice forms were present in uterine and conceptus tissues as determined by RT-PCR. In conclusion, both membrane and soluble forms of Tnfrsf9 are expressed in specific cell types of the uterus and conceptus during the progression of implantation in mice and thus may play an important role in this process.

Critical windows of development are often more sensitive to endocrine disruption. The murine pituitary gland has two critical windows of development: embryonic gland establishment and neonatal hormone cell expansion. During embryonic development, one environmentally ubiquitous endocrine-disrupting chemical, bisphenol A (BPA), has been shown to alter pituitary development by increasing proliferation and gonadotrope number in females but not males. However, the effects of exposure during the neonatal period have not been examined. Therefore, we dosed pups from postnatal day (PND)0 to PND7 with 0.05, 0.5, and 50 μg/kg/d BPA, environmentally relevant doses, or 50 μg/kg/d estradiol (E2). Mice were collected after dosing at PND7 and at 5 weeks. Dosing mice neonatally with BPA caused sex-specific gene expression changes distinct from those observed with embryonic exposure. At PND7, pituitary Pit1 messenger RNA (mRNA) expression was decreased with BPA 0.05 and 0.5 μg/kg/d in males only. Expression of Pomc mRNA was decreased at 0.5 μg/kg/d BPA in males and at 0.5 and 50 μg/kg/d BPA in females. Similarly, E2 decreased Pomc mRNA in both males and females. However, no noticeable corresponding changes were found in protein expression. Both E2 and BPA suppressed Pomc mRNA in pituitary organ cultures; this repression appeared to be mediated by estrogen receptor-α and estrogen receptor-β in females and G protein-coupled estrogen receptor in males, as determined by estrogen receptor subtype-selective agonists. These data demonstrated that BPA exposure during neonatal pituitary development has unique sex-specific effects on gene expression and that Pomc repression in males and females can occur through different mechanisms.

Endocrine-disrupting chemicals are prevalent in the environment and can impair reproductive success by affecting the hypothalamic-pituitary-gonadal axis. The developing pituitary gland is sensitive to exposure to endocrine-disrupting chemicals, such as bisphenol A (BPA), and sex-specific effects can occur. However, effects on the critical window of neonatal pituitary gland development in mice have not been explored. Therefore, this study determined baseline gene expression in male and female pituitaries and consequences of environmental exposure to 17β-estradiol (E2) and BPA on transcription of genes exhibiting sex differences during the neonatal period. Through microarray and quantitative RT-PCR analysis of pituitaries at postnatal day (PND)1, 3 genes were differentially expressed between males and females: Lhb, Fshb, and intracellular adhesion molecule-5 (Icam5). To see whether E2 and BPA exposure regulates these genes, pituitaries were cultured at PND1 with 10(-8) M E2 or 4.4 × 10(-6) M BPA. E2 decreased expression of Lhb, Fshb, and Icam5 mRNA in females but only significantly decreased expression of Icam5 in males. BPA decreased expression of Icam5 similarly to E2, but it did not affect Lhb or Fshb. Importantly, in vivo exposure to 50-μg/kg · d E2 from PND0 to PND7 decreased expression of Lhb, Fshb, and Icam5 mRNA in both males and females, whereas 50-mg/kg · d BPA exposure during the same time frame decreased expression of Icam5 in females only. Overall, we have uncovered that genes differentially expressed between the sexes can be regulated in part by hormonal and chemical signals in vivo and directly at the pituitary and can be regulated in a sex-specific manner.