Uterine decidual response occurs in estrogen receptor-alpha-deficient mice

Abstract

Embryo-uterine interactions leading to the attachment reaction is followed by stromal cell proliferation and differentiation into decidual cells (decidualization) at the sites of blastocyst apposition. In rodents, decidualization is also induced by application of an artificial stimulus (intraluminal oil infusion) in a pseudopregnant uterus, or to one that has been appropriately prepared by exogenous progesterone (P4) and estrogen. The process of decidualization is under the control of these steroids in the presence of blastocysts or deciduogenic stimuli. Although it is well known that estrogen is required for the induction of progesterone receptors in the uterus, the functional importance of estrogen in the process of decidualization is poorly understood. To better understand the role of estrogenic actions in decidualization, we used wild-type and estrogen receptor-alpha knock-out (ERKO) mice for induction of decidualization employing a defined steroid hormonal treatment schedule. Our results demonstrate that P4 alone induces decidualization in ovariectomized wild-type or ERKO mice in response to intraluminal oil infusion in the absence of estrogen. A combined treatment of either estradiol-17beta (E2) or its catecholmetabolite 4-hydroxyestradiol-17beta(4-OH-E2) with P4 does not potentiate the decidual response produced by P4 treatment alone in either ovariectomized wild-type or ERKO mice. The induction of decidual response was associated with up-regulation of decidual cell marker genes, such as progesterone receptor, metallothionein-1, and cyclooxygenase-2. The results suggest that the stromal cell sensitivity to decidualization is critically dependent on P4-regulated events, and estrogenic induction of progesterone receptor via classical nuclear ER-alpha is not critical for this process.

Fig. 1

Effects of P₄ and/or estrogens on decidual response. Ovariectomized wild-type or ERKO mice were treated with P₄ (A) or P₄ plus E₂ or P₄ plus 4OH-E₂ (B), which sensitize the uterus for optimal decidualization. Intraluminal infusion of oil was made in one horn on the appropriate day of the hormone treatment, whereas the contralateral noninfused horn served as a control. Mice were killed 4 days later after induction of decidualization as described in Materials and Methods. These experiments were repeated three times with 3–4 mice in each group. Induction of decidualization was determined by the increases in wet weights of the infused horns as opposed to the noninfused horns. Results are mean ± sem. Values are statistically different (P < 0.05, Student’s t test) between the infused and the noninfused horns in each group. *, Bars marked with an asterisk are not significantly different from each other (P > 0.05).

Fig. 2

In situ hybridization of PR mRNA in wild-type and ERKO uteri. Induction of decidualization followed the protocol as described in the legend to Fig. 1. Darkfield photomicrographs of representative uterine sections of non-infused horn treated with P₄ (a) and infused horn treated with P₄ plus E₂ (b) or P₄ plus 4-OH-E₂ (c) or P₄ (d) from ERKO mice, as well as infused horn from wild-type mice treated with P₄ (e) are shown. Magnifications are shown at 20×. le, Luminal epithelium; s, stroma; myo, myometrium; AM, antimesometrial pole; M, mesometrial pole; dec, decidual cells. These experiments were repeated three times with three mice in each group.

Fig. 3

Quantitation of PR mRNA signals in the decidualized uteri of wild-type and ERKO mice treated with different hormones. Experimental designs are same as described in the legend to Fig. 2. The quantitation of hybridization signals was achieved by computer-assisted analysis (OPTIMA II program) of reddish brown grain densities under the darkfield microscopy. The grain densities of five different regions on each section showing positive signals were computed separately from four to five mice in each group. The grain density was used as an index for mRNA accumulation. Results are mean ± sem. Values are statistically different (P < 0.05, Student’s t test) between the infused and the noninfused horns in each group. *, Bars marked with an asterisk are not significantly different from each other (P > 0.05).

Fig. 4

In situ hybridization of COX-2 mRNA in wild-type and ERKO uteri. Induction of decidualization followed the protocol as described in the legend to Fig. 1. Darkfield photomicrographs of representative uterine sections of non-infused horn treated with P₄ (a) and infused horn with P₄ plus E₂ (b) or P₄ plus 4-OH-E₂ (c) or P₄(d) from ERKO mice, and infused horn from wild-type mice treated with P₄ (e) are shown. Magnifications are shown at 20×. le, Luminal epithelium; s, stroma; myo, myometrium; AM, antimesometrial pole; M, mesometrial pole; dec, decidual cells. These experiments were repeated two times with three mice in each group.

Fig. 5

In situ hybridization of MT-1 mRNA in wild-type and ERKO uteri. Induction of decidualization followed the protocol as described in the legend to Fig. 1. Dark-field photomicrographs of representative uterine sections of noninfused horn treated with P₄ (a) and infused horn treated with P₄ plus E₂ (b) or P₄ plus 4-OH-E₂ (c) or P₄ (d) from ERKO mice, and infused horn from wild-type mice treated with P₄ (e) are shown. Magnifications are shown at 20×. le, Luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium; AM, antimesometrial pole; M, mesometrial pole; dec, decidual cells. These experiments were repeated two times with three mice in each group.