Cochaperone immunophilin FKBP52 is critical to uterine receptivity for embryo implantation

Abstract

Embryo implantation in the uterus is a critical step in mammalian reproduction, requiring preparation of the uterus receptive to blastocyst implantation. Uterine receptivity, also known as the window of implantation, lasts for a limited period, and it is during this period blastocysts normally implant. Ovarian steroid hormones estrogen and progesterone (P(4)) are the primary regulators of this process. The immunophilin FKBP52 serves as a cochaperone for steroid hormone nuclear receptors to govern appropriate hormone action in target tissues. Here we show a critical role for FKBP52 in mouse implantation. This immunophilin has unique spatiotemporal expression in the uterus during implantation, and females missing the Fkbp52 gene have complete implantation failure due to lack of attainment of uterine receptivity. The overlapping uterine expression of FKBP52 with nuclear progesterone receptor (PR) in wild-type mice together with reduced P(4) binding to PR, attenuated PR transcriptional activity and down-regulation of several P(4)-regulated genes in uteri of Fkbp52(-/-) mice, establishes this cochaperone as a critical regulator of uterine P(4) function. Interestingly, ovulation, another P(4)-mediated event, remains normal. Collectively, the present investigation provides evidence for an in vivo role for this cochaperone in regulating tissue-specific hormone action and its critical role in uterine receptivity for implantation.

Fig. 1.

Female reproductive events in Fkbp52^-/- mice. (A) Normal ovulation and fertilization were examined on day 2 of pregnancy. Superovulated eggs retrieved on day 1 were subjected to in vitro fertilization. Numbers within the bars indicate number of fertilized eggs/total number of ovulated eggs. Normal and superovulation rates are not significantly different between +/+ and -/- mice (unpaired t test); however, normal or in vitro fertilization rates are significantly different between +/+ and -/- mice (χ² analysis; ^*, P = 0.01). (B) Fkbp52 and Pgr expression in preimplantation embryos as assessed by RT-PCR. (C) Representative photographs of wild-type uteri with implantation sites (IS) and Fkbp52^-/- uteri without IS on day 5 of pregnancy. Arrowheads and arrows indicate sites of ovary and implantation, respectively. (D) Representative photomicrograph of blastocysts recovered from Fkbp52^-/- mice on day 5 of pregnancy. (Bar, 50 μm.)

Fig. 2.

Uterine expression of Fkbp52, Pgr and Fkbp51. (A) In situ hybridization of Fkbp52, Pgr, and Fkbp51 in wild-type uteri on days 4 and 5 of pregnancy. (B) Comparative RT-PCR of uterine Fkbp51 in Fkbp52^+/+ (+/+), Fkbp52^+/- (+/-), and Fkbp52^-/- (-/-) mice on day 4 of pregnancy. The data are presented as fold changes (mean ± SEM) of three to four independent RNA samples. Fold changes are not significantly different between +/+, +/-, or -/- mice (unpaired t test). (C) In situ hybridization of uterine Pgr in wild-type (+/+) and Fkbp52^-/- (-/-) mice on day 4. (D) Northern blot analysis of uterine Pgr in +/+, +/-, and -/- mice on day 4. rPL7 is a housekeeping gene. Arrows in A indicate the location of blastocysts. le, luminal epithelium; ge, glandular epithelium; s, stroma. (Bar, 400 μm.)

Fig. 3.

Differential modulation of PR transactivation by FKBP52 and FKBP51. Fibroblast cell lines prepared from wild-type (WT), Fkbp52^-/-, or Fkbp51^-/- mouse embryos were cotransfected with four plasmids as described in Materials and Methods. Cells were treated with or without 0.5 nM P₄ for 16 h. The bars indicate the activity (mean ± SD; n = 3) normalized to uninduced levels of luciferase activity in each cell background. In WT cells expressing endogenous FKBP52 and FKBP51, overexpression of FKBP52 show marginal increases in the reporter activity, whereas overexpression of FKBP51 decreases the reporter activity (^*, P = 0.05). In Fkbp52^-/- cells, P₄-induced reporter activity is minimal but restored by exogenous FKBP52 (^*, P = 0.05). In Fkbp51^-/- cells retaining endogenous FKBP52, maximal reporter activity is induced; however, induced expression of FKBP51 decreases the activity. Statistical analysis was performed by using one-way ANOVA followed by paired t test.

Fig. 4.

Uterine progesterone (P₄) binding and PR activity. (A) P₄ binding in uterine cytosol of wild-type (+/+) and Fkbp52^-/- (-/-) mice. Both +/+ and -/- female mice were ovariectomized and treated with estrogen to increase uterine PR levels. Uterine cytosolic samples from +/+ and -/- mice were used for binding assays for comparison. The data shown are representative of four independent experiments. (B) FKBP52 effects on reporter gene activation in embryonic fibroblasts isolated from Fkbp52^-/- mice cotransfected with plasmid expressing human PR-B, a luciferase reporter plasmid, and an empty vector or plasmid expressing FKBP52 (p52). Each value represents the mean ± SD for three replicate samples. (C and D) In situ hybridization of Hoxa-10 and Ihh (C) and Areg and Hdc (D) in +/+ and -/- mice on day 4 of pregnancy. (E) Northern blot analysis of uterine Hoxa-10, Ihh, and Areg in +/+, +/-, and -/- mice on day 4. rPL7 is a housekeeping gene. le, luminal epithelium; ge, glandular epithelium; s, stroma. (Bar, 400 μm.) (F) Comparative RT-PCR of uterine Hdc in +/+, +/-, and -/- mice on day 4. The data are presented as fold changes (mean ± SEM) of three to four independent samples. Fold changes in Fkbp52^-/- mice are significantly different compared to combined fold changes in +/+ and +/- mice (P = 0.05, unpaired t test).

Fig. 5.

Differential expression of an estrogen-responsive gene lactoferrin (Ltf) and cell proliferation. (A) In situ hybridization of Ltf in uteri of +/+ and -/- mice on days 1 and 4 of pregnancy. (Bar, 400 μm.) (B) Histological examination of +/+ and -/- uteri on day 4 of pregnancy. (Bar, 200 μm.) (C) Immunostaining of Ki67, a marker of cell proliferation, in uteri of +/+ and -/- mice on day 4. Note stromal cell proliferation in wild-type uterus versus luminal epithelial proliferation in Fkbp52^-/- mice. Arrow points to luminal epithelial layer. le, luminal epithelium; s, stroma; myo, myometrium. (Bar, 100 μm.)