FKBP52 deficiency–conferred uterine progesterone resistance is genetic background and pregnancy stage specific

Immunophilin FKBP52 serves as a cochaperone to govern normal progesterone (P₄) receptor (PR) function. Using Fkbp52^–/– mice, we show intriguing aspects of uterine P₄/PR signaling during pregnancy. Implantation failure is the major phenotype found in these null females, which is conserved on both C57BL6/129 and CD1 backgrounds. However, P₄ supplementation rescued implantation and subsequent decidualization in CD1, but not C57BL6/129, null females. Surprisingly, experimentally induced decidualization in the absence of blastocysts failed in Fkbp52^–/– mice on either background even with P₄ supplementation, suggesting that embryonic signals complement uterine signaling for this event. Another interesting finding was that while P₄ at higher than normal pregnancy levels conferred PR signaling sufficient for implantation in CD1 null females, these levels were inefficient in maintaining pregnancy to full term. However, elevating P₄ levels further restored PR signaling to a level optimal for successful term pregnancy with normal litter size. Collectively, the results show that the indispensability of FKBP52 in uterine P₄/PR signaling is a function of genetic disparity and is pregnancy stage specific. Since there is evidence for a correlation between P₄ supplementation and reduced risks of P₄-resistant recurrent miscarriages and remission of endometriosis, these findings have clinical implications for genetically diverse populations of women.

Progesterone (P₄) signaling is an absolute requirement for implantation and pregnancy maintenance in all eutherian mammals studied (1, 2). P₄ acts through its nuclear P₄ receptor (PR) to activate transcription of genes involved in ovulation, uterine receptivity, implantation, decidualization, and pregnancy maintenance. This is evident from the complete infertility of female mice lacking Pgr, the gene encoding PR (3). The failure of ovulation and implantation precludes using Pgr^–/– mice to study potential new aspects of P₄ function during pregnancy. In contrast, the targeted deletion of the Fkbp52 gene, which encodes FKBP52, an immunophilin cochaperone that optimizes PR signaling, has allowed us to address unique aspects of uterine P₄/PR signaling during pregnancy in mice.

Immunophilins are so named because of their ability to bind and mediate the actions of certain immunosuppressive drugs. They are grouped into 2 families, FK506-binding proteins (FKBPs) and cyclosporin A–binding proteins (cyclophilins [CyPs]). Some FKBP and CyP family members contain a tetratricopeptide repeat (TPR) domain that targets binding to the highly conserved C terminus of Hsp90. FKBP52, FKBP51, and CyP40 are 3 such TPR-containing cochaperones that have been identified in steroid receptor complexes (4). Like other steroid receptors, PR assembles in an ordered, multistep manner for hormone binding (5, 6); the final complex involves direct association of Hsp90 with PR, which stabilizes its ability to bind P₄ (7). At the mature stage, FKBPs and other TPR cochaperones dynamically exchange on Hsp90 such that there is a mixture of receptor complexes distinguished by which cochaperone is associated with Hsp90 (8, 9). Hormone binding stimulates interruption of the receptor/chaperone assembly cycle and promotes receptor activation. Recent findings show that cochaperones have unique influences on steroid receptor function. FKBP52 potentiates the responses of PR, androgen receptor, and glucocorticoid receptor to their respective ligands (10–12). In fact, only the mature PR complex bound to FKBP52 is capable of binding P₄ with high affinity and efficiency, although basal PR responsiveness to P₄ is still retained in the absence of FKBP52. Our recent study (12) revealed the previously unknown role of FKBP52/PR signaling in female reproduction.

The uterus consists of heterogeneous cell types that respond differentially to estrogen and P₄. For successful implantation, the uterus must be transiently receptive to implantation-competent blastocysts. For example, the prereceptive uterus on days 1–3 of pregnancy or pseudopregnancy becomes receptive to implantation on day 4 under the direction of P₄ and estrogen. By late day 5, the uterus becomes nonreceptive, since implantation-competent blastocysts transferred into uteri of pseudopregnant mice at this time fail to implant. Following implantation on day 4 evening (2200 to 2400 h), uterine stromal cells at the sites of blastocysts undergo extensive proliferation and differentiation, giving rise to decidual cells, a process termed decidualization (1, 13). Using proteomic analysis in Hoxa10^–/– mouse uteri, we identified FKBP52 as an important signaling molecule in stromal cell function during the periimplantation period (14). Consistent with the role of FKBP52 as a PR cochaperone, uterine expression of Fkbp52 and Pgr overlaps on days 4 and 5 of pregnancy (12). More importantly, we have shown that Fkbp52^–/– females on a C57BL6/129 mixed background have complete implantation failure, with normal ovulation and slightly reduced fertilization rates (12). This phenotype has recently been confirmed by another group in independently generated Fkbp52^–/– mice on the same background (15). The fact that the infertile phenotype of Fkbp52^–/– females is primarily due to implantation defects suggests differential sensitivity of the ovary and uterus to FKBP52/PR-mediated P₄ action. This tissue-specific differential sensitivity is not noted in Pgr^–/– females, in which severely compromised ovarian and uterine functions lead to complete female infertility (3, 16).

Although P₄ is commonly known as the “hormone of pregnancy,” various aspects of its roles throughout pregnancy are not well understood. Therefore, Fkbp52^–/–mice provide a unique opportunity for studying such roles of P₄ signaling throughout pregnancy. Using these null mice, we show in the present investigation that P₄/PR signaling is a function of genetic makeup and is pregnancy stage specific. For example, while the implantation failure phenotype was conserved in both C57BL6/129 and CD1 mice lacking Fkbp52, daily P₄ supplementation rescued implantation with subsequent decidualization in CD1 Fkbp52^–/– females but not in C57BL6/129 Fkbp52^–/– females. Surprisingly, experimentally induced decidualization failed to occur in Fkbp52^–/– mice on either background even with exogenous P₄ supplementation, suggesting that embryonic signals complement the uterine signaling network. Another interesting finding is the differential requirement for P₄/PR signaling at specific stages of pregnancy in P₄-treated CD1 Fkbp52^–/– females. For example, while P₄ at higher than normal levels conferred PR signaling sufficient for uterine receptivity and implantation in Fkbp52^–/– females, these levels did not maintain adequate PR signaling to sustain pregnancy, resulting in reduced litter sizes due to in utero fetal resorption and restricted growth. However, increasing P₄ levels even further restored PR signaling, to a degree sufficient to maintain full-term pregnancy with normal litter size. Collectively, these findings show that FKBP52 deficiency confers uterine P₄ resistance during pregnancy, since null females have normal PR and P₄ levels with reduced PR activity (12, 17). This study also shows that the requirement for FKBP52 in optimizing PR activity is genetic background dependent and that levels of P₄/PR signaling required to ensure successful pregnancy differ depending on pregnancy stage.

Implantation failure occurs in Fkbp52^–/– mice irrespective of genetic background. There is increasing evidence that mutation of a gene often results in altered phenotypes depending on the genetic background of mice (18). These varying phenotypes are thought to be due to differential expression and/or regulation of modifier genes (19), although identification of such modifiers remains largely unknown. There is also evidence that compensatory function of genes among the same family is genetic background dependent (20). We have recently shown that C57BL6/129 Fkbp52^–/– females have complete implantation failure, although ovulation is normal (12). To determine whether this phenotype is a function of genetic background, we established Fkbp52 deletion in CD1 mice (see Methods). First, we examined ovulation and fertilization in CD1 Fkbp52^–/– females after mating them with WT males, since Fkbp52^–/– males, irrespective of genetic background, are infertile (10). We found ovulation and fertilization to be comparable to those of WT females (Figure 1, A and B). We then asked whether implantation occurs in these mice. Initiation of implantation accompanies an increased endometrial vascular permeability at sites of blastocysts, which are visualized as distinct blue bands after injection of a blue dye solution (13, 21). CD1 Fkbp52^–/– females were examined for implantation sites (ISs) by this method on day 5 of pregnancy. We observed that only 2 of 14 CD1 Fkbp52^–/– females showed very faint blue bands (Figure 1C and Table 1). Unimplanted blastocysts were recovered from uterine flushings of null mice with implantation failure (Figure 1C), suggesting that although embryos developed to blastocysts, they failed to implant. Our data indicate that optimal P₄/PR signaling imparted by FKBP52 is critical to uterine receptivity and implantation in mice, a phenotype conserved across these genetic backgrounds. Because ovulation is normal in Fkbp52^–/– mice, our results suggest that uterine responsiveness to PR signaling differs from ovarian responsiveness. It is possible that relatively high local P₄ levels in the ovary (22), the site of its synthesis, enhance basal PR activity sufficient for ovulation and fertilization processes.

Figure 1

P₄ supplementation via Silastic implants rescues implantation failure in CD1 Fkbp52^–/– females. Ovulation (A) and fertilization (B) were examined on day 2 of pregnancy. The number of ovulated eggs was not significantly different in WT and Fkbp52^–/– (KO) females. Values are mean ± SEM; P > 0.05, unpaired Student’s t test. Fertilization rate was determined by counting the number of 2-cell embryos after flushing oviducts. Numbers above the bars indicate the total number of 2-cell embryos per total number of eggs recovered. The fertilization rate was comparable between WT and KO females (P > 0.05; unpaired Student’s t test). (C) Implantation fails in KO females but is rescued by P₄ as examined on day 5 of pregnancy. Implants containing P₄ were inserted s.c. in KO females (KO+P₄) on day 2 of pregnancy. Representative photographs of uteri with or without ISs as demarcated by blue bands in WT, KO, and KO+P₄ mice are shown. Representative photographs of blastocysts recovered from uteri of KO females without IS. Arrowheads and short arrows indicate the location of ovaries and ISs, respectively. The long arrow indicates the uterus from which unimplanted blastocysts were recovered. Scale bar: 50 μm.

Table 1

P₄ rescue of implantation failure in CD1 Fkbp52^–/– female mice

P₄ supplementation rescues implantation in CD1 Fkbp52^–/– females. We have shown that PR activity, but not PR or P₄ levels, is compromised in C57BL6/129 Fkbp52^–/– females (12, 17). It is possible that FKBP52 binding to the PR complex modulates the hormone responsiveness of PR not in an all-or-none fashion, but rather to fine-tune physiological responses to P₄. This would imply that while FKBP52 is necessary for optimal PR activity, PR signaling can still operate, albeit not as efficiently, in the absence of FKBP52. Since serum P₄ levels in CD1 Fkbp52^–/– females are similar to those in WT mice on day 5 of pregnancy (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI31622DS1), we speculated that exposing Fkbp52^–/– uteri to higher-than-normal P₄ levels would enhance PR activity to rescue pregnancy failure in the absence of FKBP52. This is consistent with our previous findings that PR activity in Fkbp52^–/– mouse embryonic fibroblasts (MEFs) reached levels similar to those in WT MEFs exposed to higher P₄ concentrations in culture (12). This may also explain why ovulation, a P₄-regulated event, is normal in Fkbp52^–/– females on both genetic backgrounds.

To determine whether P₄ supplementation rescues implantation, we used P₄-containing Silastic implants to maintain steady-state hormone levels (23). WT and Fkbp52^–/– females were mated with WT males, and Silastic implants containing P₄ were placed under the dorsal skin of Fkbp52^–/– females from day 2 of pregnancy until the day of sacrifice. We were surprised to see that P₄ supplementation not only rescued implantation in CD1 Fkbp52^–/– females examined on day 5, but the number of ISs was also comparable to that in WT mice (Figure 1C and Table 1). In contrast, exogenous P₄ supplementation was largely ineffective in rescuing implantation in C57BL6/129 Fkbp52^–/– females; only 3 of 11 (27%) and 2 of 8 (25%) null females showed implantation when examined on days 5 and 8, respectively. In addition, the number of ISs was also remarkably low, especially on day 8, compared with that in WT females (Table 2). To circumvent any contribution arising from lower fertilization rates in C57BL6/129 Fkbp52^–/– mice, we performed blastocyst transfer experiments. Day-4 WT blastocysts were transferred into uterine lumens of day-4 C57BL6/129 Fkbp52^–/– pseudopregnant females carrying Silastic P₄ implants from day 2. Again, we noted extremely poor implantation rates in these mice (Table 3).

Table 2

P₄ fails to rescue implantation failure in C57BL6/129 Fkbp52^–/– female mice

Table 3

P₄ fails to rescue implantation failure of transferred WT blastocysts in C57BL6/129 Fkbp52^–/– females

P₄ supplementation restores P₄- and implantation-regulated gene expression in CD1 Fkbp52^–/– uteri. P₄-regulated events combined with preimplantation estrogen secretion guide the uterus from a prereceptive to receptive state, allowing blastocyst attachment in the uterine wall on day 4 evening. To determine whether rescue of implantation by P₄ is reflected in the restoration of P₄-dependent uterine gene expression, we placed a Silastic P₄ implant on day 2 in CD1 Fkbp52^–/– females that had been mated with WT males and sacrificed them on day 4 of pregnancy. We selected P₄-regulated genes that encode Hoxa10, Indian hedgehog (Ihh), and amphiregulin (Areg) because of their participation in uterine receptivity (24–27). In situ hybridization results showed that exogenous P₄ treatment considerably restores expression of these genes (Figure 2A).

Figure 2

P₄ supplementation via Silastic implants corrects misexpression of genes in CD1 Fkbp52^–/– uteri. (A) In situ hybridization of P₄-regulated genes Hoxa10, Ihh, and Areg in WT, KO, and KO+P₄ uteri on day 4 of pregnancy. (B) In situ hybridization of estrogen-target genes Ltf and complement factor 3 (C3) in WT, KO, and KO+P₄ uteri on day 4 of pregnancy. Implants containing P₄ were inserted s.c. in KO females on day 2 of pregnancy. ge, glandular epithelium; le, luminal epithelium; myo, myometrium; s, stroma. Scale bar: 200 μm.

Lactoferrin (Ltf) and complement factor 3 (C3) are induced to a high degree in the uterus by estrogen and antagonized by P₄ (28, 29). They are abundantly expressed in the luminal epithelium on day 1 of pregnancy under the influence of a preovulatory estrogen surge but are dramatically downregulated on day 4 by rising P₄ levels from newly formed corpora lutea (1, 2). However, in CD1 Fkbp52^–/– mice, uterine expression of these genes was aberrantly elevated on day 4 but downregulated with exogenous P₄ supplementation (Figure 2B). Together, these findings provide evidence that P₄ supplementation restores the expression of P₄-regulated genes and counters the expression of estrogen target genes in CD1 Fkbp52^–/– uteri, shifting the uterus to a P₄-dominated milieu conducive to uterine receptivity as opposed to one of estrogenic dominance that is detrimental to uterine receptivity.

The fact that exogenous P₄ treatment fully rescues implantation in CD1 Fkbp52^–/– females but is largely ineffective in C57BL6/129 Fkbp52^–/– mice led us to examine whether P₄ treatment would restore the expression of P₄-regulated genes in C57BL6/129 Fkbp52^–/– uteri, as was observed in CD1 Fkbp52^–/– mice. We found that although exogenous P₄ treatment considerably restored Hoxa10 and Ihh expression, Areg expression remained low to undetectable (Supplemental Figure 2A). More interestingly, exogenous P₄, which normally inhibits estrogen-responsive Ltf expression in CD1 Fkbp52^–/– uteri on day 4, was not effective in attenuating Ltf expression in C57BL6/129 Fkbp52^–/– uteri (Supplemental Figure 2B). Overall, these results imply that while C57BL6/129 Fkbp52^–/– uteri are somewhat responsive to P₄ induction of target genes, they are less receptive to P₄’s influence in antagonizing estrogen target genes. These findings are significant, since excess estrogenic influence leads to uterine nonreceptivity (30). Because implantation fails in most C57BL6/129 Fkbp52^–/– mice even after P₄ treatment, we performed subsequent experiments on CD1 Fkbp52^–/– mice.

Although expression of several P₄-regulated genes was considerably restored with P₄ supplementation in CD1 Fkbp52^–/– uteri, the expression of leukemia inhibitory factor (Lif), normally expressed in day-4 WT pregnant uterine glands, was not restored by P₄ treatment in these null mice (Figure 3A). We next determined whether P₄ rescue of implantation on day 5 in CD1 Fkbp52^–/– mice is accompanied by correct expression of implantation-related genes, such as Lif, Ptgs2, and Bmp2 (31–33). We found that while these genes were not expressed at the site of blastocysts in CD1 Fkbp52^–/– females with failed implantation in the absence of P₄, P₄ supplementation restored implantation with correct expression of these genes (Figure 3, A and B). The ability of P₄ to rescue both implantation and Lif expression on day 5 of pregnancy in Fkbp52^–/– mice without salvaging Lif expression on day 4 agrees with previous observations that the first phase of Lif expression on day 4 is not as critical as its second phase of expression in stromal cells surrounding the implanting blastocyst (33, 34).

Figure 3

Delivery of P₄ via Silastic implants restores expression of implantation-related genes in CD1 Fkbp52^–/– females. (A) In situ hybridization of Lif in WT, KO, and KO+P₄ uteri on days 4 and 5 of pregnancy. (B) In situ hybridization of Ptgs2 and Bmp2 in WT, KO, and KO+P₄ uteri on day 5 of pregnancy. P₄ implants were inserted s.c. in KO females on day 2 of pregnancy. Arrowheads in A and B indicate the location of embryos. Scale bars: 200 μm. (C) Number of glands per uterine cross-section of WT and KO uteri on CD1 and C57BL6/129 backgrounds. For each animal, glands were counted in 9–12 uterine sections. Numbers above bars indicate number of mice evaluated. Values are mean ± SEM. *P < 0.05, unpaired Student’s t test.

Upon closer examination of WT and null uterine histology, we came across an interesting observation. A significant decrease in the number of glands was noted in null uteri on both genetic backgrounds (Figure 3C), a phenotype not rescued by P₄ supplementation (data not shown). This decrease in gland numbers, however, does not fully account for the altered expression of Areg, Ihh, or Lif in null uteri, since the glands that were still present failed to show normal expression patterns of these genes. There is clear evidence that both estrogen and P₄ participate in uterine gland formation in sheep (35, 36). Our data also implicate a potential role for FKBP52/PR signaling in mouse uterine gland formation and function. This is an exciting finding but warrants further investigation.

Postimplantation defects in P₄-treated CD1 Fkbp52^–/– females. Our observation of unimplanted blastocysts recovered from CD1 Fkbp52^–/– females examined on day 5 of pregnancy suggested deferral of implantation timing in these mice as is observed in cPLA2a^–/– or LPA3^–/– mice (37, 38). To test for this possibility, we sacrificed CD1 Fkbp52^–/– mice on days 6 and 8 of pregnancy. We found that unlike in cPLA2a^–/– or LPA3^–/– mice, implantation timing was not altered; rather implantation drastically failed in Fkbp52^–/– females (Figure 4, A and B, and Table 1). Again, blastocysts were recovered from CD1 Fkbp52^–/– uteri on these days, confirming implantation as the major defect in these mice (Figure 4, A and B, and Table 1).

Figure 4

P₄ delivery via Silastic implants rescues blastocyst-induced, but not oil-induced, decidualization in Fkbp52^–/– females. Representative photographs of WT, KO, and KO+P₄ uteri on day 6 (A), day 8 (B), and day 12 (C) of pregnancy are shown. Arrowheads and short arrows indicate the location of ovary and IS, respectively. Representative images of recovered unimplanted blastocysts from uteri (long arrow) of KO females without ISs are shown. Scale bars: 50 μm. (D) Oil-induced decidualization fails in KO females on both CD1 and C57BL6/129 backgrounds. Representative photomicrographs of WT, KO, and KO+P₄ uteri on day 8 of pseudopregnancy. On day 4 of pseudopregnancy, 25 μl of oil was infused intraluminally in one uterine horn (oil); the contralateral horn without oil infusion served as a control (con). (E) Fold changes in weight between oil-infused and noninfused (control) uterine horns. Numbers above the bars indicate the number of mice with decidual response per total number of mice examined. Fold changes are presented as mean ± SEM; *P < 0.05, unpaired Student’s t test.

We next determined whether P₄ supplementation could sustain pregnancy beyond day 5. To our surprise, we found that placing Silastic P₄ implants in null females allowed progression of pregnancy in 100% of CD1 Fkbp52^–/– females examined on days 6, 8, and 12 of pregnancy (Figure 4, A–C, and Table 1). However, ISs in P₄-treated CD1 Fkbp52^–/– uteri were smaller and weighed less than those in WT uteri (Table 1), suggesting a somewhat compromised decidual response.

Experimentally induced decidualization fails in Fkbp52^–/– mice irrespective of P₄ treatment. In pseudopregnant mice in the absence of embryos, the steroid hormonal milieu and responsiveness of the uterus on days 1 through 4 are similar to those of normal pregnancy. Various artificial stimuli, including intraluminal infusion of oil, can initiate many aspects of the decidual cell reaction in pseudopregnant mice if applied on day 4. Decidualization, characterized by stromal cell proliferation and differentiation into specialized types of cells with polyploidy, is critical to pregnancy establishment in many species (1). In fact, decidualization does not occur in Pgr^–/– mice (3, 16), demonstrating an absolute requirement for P₄/PR signaling in this process.

We asked whether experimentally induced decidualization occurs in Fkbp52^–/– females on both genetic backgrounds using the model of intraluminal oil infusion and if not, whether P₄ supplementation could rescue this phenotype. We observed severely compromised decidualization in both C57BL6/129 and CD1 Fkbp52^–/– females when compared with WT littermates (Figure 4, D and E). Interestingly, P₄ treatment could not restore decidualization in Fkbp52^–/– mice on either strain, with only a few swellings noted along the oil-infused uterine horn (Figure 4, D and E). That P₄ treatment rescues blastocyst implantation with decidualization in CD1 Fkbp52^–/– females but not experimentally induced decidualization is remarkable. This agrees with previous observations that gene expression differs in the decidual bed induced by blastocysts from that induced experimentally (39). Differences in decidualization in these 2 models (oil-induced versus blastocyst-induced) were noted even at the ultrastructural level (40).

P₄ delivery via Silastic implants partially restores full-term pregnancy in CD1 Fkbp52^–/– females. To investigate whether P₄ supplementation maintains full-term pregnancy in CD1 Fkbp52^–/– females, P₄ implants placed on day 2 were removed on day 17, since P₄ withdrawal is necessary to initiate labor (41). Although 9 of 13 Fkbp52^–/– mothers delivered pups of normal weight, litter sizes were significantly smaller (Figure 5A). This led us to determine when embryonic loss occurs in null females carrying P₄ implants. We observed that 70 of 106 ISs were resorbing in P₄ implant-treated Fkbp52^–/– uteri when examined on day 14, compared with only 9 of 194 in WT uteri (Figure 5, B and C). Resorbing ISs in P₄-treated Fkbp52^–/– females appeared dark blue and were infiltrated with a massive number of blood cells (Figure 5B). In addition, cytokeratin staining of sections of ISs with normal appearance from null females carrying P₄ implants showed placentas with less-developed and ill-defined spongiotrophoblast and labyrinth layers compared with those of WT females (Figure 5D).

Figure 5

P₄ delivery by Silastic implants fails to sustain pregnancy to full term. (A) Average litter size of WT and KO+P₄ mothers. Litter size is presented as mean ± SEM; *P < 0.05, unpaired Student’s t test. (B) Representative photomicrographs of WT and KO+P₄ uteri on day 14 of pregnancy. Arrowheads and arrows indicate resorbing and normal IS, respectively. A representative H&E-stained section of resorbing IS from KO+P₄ uterus shows massive infiltration of blood cells. Scale bar: 200 μm. (C) Percentage of resorption sites in WT and KO+P₄ mice on day 14 of pregnancy. *P < 0.05, unpaired Student’s t test. (D) Cytokeratin staining of WT and KO+P₄ IS on day 14. Scale bar: 200 μm. dec, decidua; lab, labyrinth; sp, spongiotrophoblast.

Although P₄/PR signaling is absolutely required for pregnancy maintenance in all eutherians thus far studied, uterine FKBP52 expression in later days of pregnancy has not yet been examined. We found that while FKBP52 is expressed in the mesometrial decidua with high expression in the placenta on days 10–14 of pregnancy, PR is mostly expressed in the decidua (Supplemental Figure 3, A and B, and data not shown). Embryonic signals direct normal decidual functions and development (39), which in turn govern placentation and embryonic growth (42). Therefore, the expression of FKBP52 and PR in the decidua suggests that maternally derived FKBP52-mediated PR signaling contributes to fetoplacental well-being, while placental expression of FKBP52 suggests a PR-independent role for FKBP52.

Excessive estrogenic influence or complement activation does not contribute to pregnancy failure in P₄-implanted Fkbp52^–/– females. We speculated that one possible explanation for the higher incidence of resorptions in Fkbp52^–/– females carrying P₄ implants is the tipping of the balance between estrogen and P₄ signaling toward estrogenic dominance. Since high levels of estrogen are detrimental to pregnancy success (30), we examined whether combined treatment with P₄ implants and ICI 182,780 (ICI; 25 μg or 125 μg/0.1 ml oil/mouse, s.c.), an estrogen receptor antagonist (Tocris Bioscience), injected on days 8–13 improves pregnancy maintenance. Combined treatment with P₄ and the high dose of ICI increased the resorption rate to 100% in Fkbp52^–/– mice, while treatment with a lower dose of ICI resulted in resorption rates similar to those observed in null mice receiving P₄ implants alone (Supplemental Figure 4). Our observation that treatment with a higher dose of ICI results in increased resorption rates suggests that appropriate estrogen signaling is also critical to pregnancy maintenance. This agrees with a previous study showing that pregnancy maintenance under the direction of P₄ is supported by low amounts of estrogen, especially on days 10 and 11 of pregnancy (43).

While the underlying causes of recurrent pregnancy failure are not well understood, one possibility is that the maternal immune response mistakenly recognizes the fetus. A recent study shows that complement activation causes growth restriction and subsequent fetal rejection, leading to pregnancy failure (44). We speculated that this pathway is activated due to reduced PR signaling from FKBP52 deficiency, especially since P₄ has antiinflammatory roles within and outside the uterus (17, 45). Since low doses of heparin inhibit the complement pathway (44, 46), Fkbp52^–/– mice carrying P₄ implants from day 2 were given a low dose of heparin (10 U/mouse) twice a day on days 8, 10, and 12 of pregnancy. However, this treatment also failed to rescue pregnancy maintenance in these mice (Supplemental Figure 4), suggesting that activation of the complement pathway is not a major contributing factor for pregnancy failure in P₄-treated Fkbp52^–/– mice.

Differential P₄/PR signaling is required for successful full-term pregnancy. Normal serum P₄ levels during pregnancy in CD1 WT mice on days 5 and 14 of pregnancy range between 40 and 47 ng/ml (Supplemental Figure 1 and Figure 6A). In our experiments, Silastic P₄ implants provided an increase in serum P₄ levels sufficient to induce uterine receptivity and rescue implantation in CD1 Fkbp52^–/– females but failed to maintain pregnancy to full term. We speculated that further increasing P₄ levels would rectify this failure. We injected P₄ s.c. at a dose of 2 mg/ml per mouse daily to further increase P₄ serum levels. We observed that in null mice, serum P₄ levels increased to approximately 156 ng/ml by daily injection as compared with approximately 100 ng/ml in mice treated with P₄ implants, as assessed on day 14 of pregnancy (Figure 6A). To our surprise, these elevated P₄ levels significantly improved pregnancy maintenance in null females; the number and weights of ISs on day 14 were comparable to those in WT mothers (Figure 6, B–D). Pregnancy maintenance in WT mice exposed to a similar P₄ injection regimen was normal (data not shown).

Figure 6

Daily P₄ injections restore pregnancy to full term in CD1 Fkbp52^–/– females. (A) Serum P₄ levels in WT and KO+P₄ mice on day 14 of pregnancy. KO mice were exposed daily to P₄ either via Silastic implants (Imp KO+P₄) or s.c. injection (Inj KO+P₄; 2 mg/ml) from day 2 of pregnancy. *P < 0.05, univariate ANOVA. (B) The average number of ISs was not significantly different in WT and Inj KO+P₄ mice on day 14; P > 0.1, unpaired Student’s t test. (C) Weights of ISs from WT and Inj KO+P₄ mice on day 14 of pregnancy were not significantly different; P > 0.1, unpaired Student’s t test. (D) A representative photomicrograph of WT and Inj KO+P₄ uteri on day 14 of pregnancy is shown. (E) The average litter size of WT and Inj KO+P₄ mice was not significantly different; P > 0.05, unpaired Student’s t test. All values are mean ± SEM.

Our next objective was to examine whether daily P₄ injection results in pregnancy to full term in null females. CD1 Fkbp52^–/– females mated with WT males were injected with P₄ daily from days 2 through 17 of pregnancy and monitored for term delivery on day 20. We observed that all P₄-injected null females carried pups to term, and the average litter size was comparable to that of WT mothers (Figure 6E). Pup weights from null and WT mothers at weaning and during early development were also similar (data not shown).

Although P₄ signaling via PR is critical to ovulation, fertilization, implantation, postimplantation growth, and pregnancy maintenance, it is not known whether a similar P₄/PR signaling mechanism determines these target and stage-specific functions or whether genetic disparity influences this signaling. By using Fkbp52^–/– females with compromised PR signaling as opposed to Pgr^–/– females with total infertility (3, 12), we address these issues for the first time to our knowledge. Our previous and present investigations provide clear evidence that the major reproductive phenotype in mice lacking Fkbp52 is unique to uterine deficiency in the context of implantation. The reason for the organ-specific dependence on FKBP52 for appropriate PR signaling is not clearly understood, but it is noteworthy, since ovulation that also requires P₄/PR signaling is normal in null females. It is possible that relatively higher P₄ levels locally in the ovary override the reduced PR signaling in the absence of FKBP52. We were also surprised to note that P₄/PR-regulated mating behavior appears normal, since null females mate and produce copulatory plugs. One possibility is that the uterus requires more robust P₄/PR signaling during pregnancy than other P₄ targets. Alternatively, FKBP52, in addition to its role in influencing PR signaling, may have a unique PR-independent role in the uterus not observed in other tissues.

P₄ signaling via PR plays major roles at essentially all stages of pregnancy, from ovulation through parturition. It is surprising to see that genetic makeup of a species alters such a fundamental signaling pathway. While FKBP52 is essential to support implantation in both strains of mice in the absence of exogenous P₄, FKBP52’s role becomes less significant in CD1 mice exposed to high levels of P₄, while still remaining crucial in C57BL6/129 mice under similar treatment conditions. The contrasting reproductive phenotypes of Fkbp52^–/– mice on C57BL6/129 and CD1 backgrounds with respect to P₄ rescue provides the first evidence to our knowledge that P₄/PR/FKBP52 signaling is a function of genetic makeup. Coordinated interactions of P₄ and estrogen are essential to uterine receptivity and implantation. While one aspect of P₄ signaling is to correctly orchestrate P₄-responsive genes, another is to appropriately constrain and/or synergize estrogen-responsive genes in the uterus. One cause of implantation failure in C57BL6/129, but not in CD1, Fkbp52^–/– females supplemented with P₄ could be the failure of the uterus to attain optimal receptivity arising from altered expression of Areg and/or Ltf. This inappropriate gene expression in the presence of P₄ may reflect even lower basal PR activity in C57BL6/129 Fkbp52^–/– uteri than in CD1 Fkbp52^–/– uteri. Alternatively, differential expression of modifier genes in these 2 strains of mice could contribute to differential uterine responsiveness to P₄/PR signaling in the absence of FKBP52.

There is evidence that rodent blastocysts synthesize P₄ or structurally similar steroids (47–49) and that gene expression differs in embryo-induced decidua and experimentally induced deciduoma (39). Since our previous results show that Fkbp52 and Pgr are expressed in blastocysts (12), it is possible that P₄ synthesized from blastocysts enhances PR/FKBP52 signaling locally at their sites of apposition in the uterus. This could explain the failure of oil-induced decidualization but not of that induced by implanting blastocysts in CD1 Fkbp52^–/– mice supplemented with P₄. Alternatively, signaling arising from blastocysts may influence other uterine functions. For example, the gene encoding heparin-binding epidermal growth factor–like growth factor (HB-EGF) is expressed in implantation-competent blastocysts, which can induce uterine Hbegf to initiate the implantation cascade. In fact, Affi-Gel Blue beads (Bio-Rad) presoaked in HB-EGF, when transferred into uterine lumens of pseudopregnant mice on day 4, show implantation-like responses similar to those induced by living blastocysts, including upregulation of Hbegf and Bmp2 (32, 50).

P₄/PR signaling is critical throughout pregnancy until its downregulation for the onset of parturition. Indeed, ovariectomizing mice at practically any stage of pregnancy causes resorptions and/or abortion (51). However, the magnitude of this signaling during various stages of pregnancy remains unknown. Although P₄ implants cannot rescue pregnancy in WT mice ovariectomized on day 8 unless they are given daily P₄ injections, similar P₄ injections alone maintain full-term pregnancy in mice ovariectomized on day 14 (51). The use of Fkbp52^–/– females has enabled us to show that the requirement for P₄/PR signaling is different for uterine receptivity, implantation, and postimplantation growth. This signaling appears to be tightly regulated, since blood levels of approximately 100 ng/ml of P₄ are adequate to induce uterine receptivity and implantation, but levels above 150 ng/ml are required for full complement of pregnancy success in the absence of FKBP52. This suggests that more robust P₄/PR signaling is required for pregnancy maintenance than is required for uterine receptivity, implantation, and decidualization. It is also possible that a burst of P₄ levels as provided by daily injections is more amenable to pregnancy sustenance than the relatively constant levels maintained with Silastic implants.

The observation that differential P₄ levels are required for various stages of pregnancy implies that signaling targets are different. We believe that P₄/PR signaling for uterine preparation, implantation, and decidualization is primarily targeted to uterine epithelial and stromal cells. This is consistent with P₄’s known roles in epithelial differentiation and stromal cell proliferation during the periimplantation period. On the other hand, P₄/PR signaling for pregnancy maintenance is directed more toward keeping the myometrium quiescent until parturition and providing sanctuary for the growing fetus from mother’s immunological surveillance. P₄/PR signaling is also known to regulate angiogenesis (52), a process integral to placental development and pregnancy maintenance. Since the events during the course of pregnancy are very dynamic, P₄/PR signaling at various targets could be overlapping. We consider FKBP52’s influence on P₄/PR signaling during pregnancy to be primarily of maternal origin, since both PR and FKBP52 are expressed in the decidua on days 10 and 12 of pregnancy. The high expression of FKBP52 in the placenta implies a PR-independent role for FKBP52 in placentation. This requires further investigation, but the putative PR-independent role is not critical, since pregnancies are completed to term with normal litter sizes in CD1 Fkbp52-null females receiving daily P₄ injections.

An interesting observation is the successful nursing of pups to weaning by P₄-injected CD1 Fkbp52^–/– mothers. Mammary morphogenesis during pregnancy requires P₄/PR signaling (16, 53), but the latter stages of lactogenesis and lactation correspond to withdrawal of this signaling (reviewed in ref. 54). It was recently reported that FKBP52 may not be critical for P₄/PR signaling in the mammary gland, since exogenously provided P₄ could stimulate mammary morphogenesis in Fkbp52^–/– mice (15). This is similar to the findings of our studies in which Fkbp52^–/– mothers were injected with exogenous P₄ prior to parturition. These results suggest that exogenously supplemented P₄ overcomes the P₄-resistant state in the mammary gland and uterus in the absence of FKBP52.

The implanting blastocyst is the stimulus for normal decidualization in mice. However, in humans, stromal cells undergo decidualization during the receptive phase in each menstrual cycle in the absence of blastocysts. This predecidualization is thought to be critical for blastocyst implantation in the pregnant uterus (55). Our findings that P₄ supplementation fails to rescue oil-induced decidualization in Fkbp52-null uteri may therefore have implication for predecidualization events in P₄-resistant women.

P₄ resistance is also a hallmark of endometriosis, a condition that affects an estimated 5 million women of childbearing age in the United States (National Women’s Health Information Center, National Institute of Child Health and Human Development [NICHD], NIH) (56–58). In fact, a recent study examining global gene expression profiles in endometria of women with or without endometriosis found dysregulation of many known P₄ target genes during the window of uterine receptivity (59). While the mechanism(s) of P₄ resistance remain unclear, one speculation is that downregulation of PR contributes to P₄ resistance; however, other studies refute this concept (57). Whether FKBP52 expression differs in normal and endometriotic tissues has not been examined.

The finding that human FKBP52 interacts with and potentiates human PR activity in MEFs (12) and our preliminary observation on a limited number of samples showing FKBP52 and PR expression in human endometria in proliferative and secretory phases (data not shown) suggest a potential role for uterine P₄/PR/FKBP52 signaling in women. In fact, 3 separate clinical trials found that P₄ treatment resulted in a statistically significant decrease in miscarriages in women with a history of 3 or more consecutive pregnancy losses (60). In this respect, pregnancy rescue in CD1 Fkbp52^–/– mice by daily P₄ injections is clinically relevant for women who are infertile due to P₄ resistance. These are exciting results, especially since no significant differences in adverse effects were found between P₄ treatment and control groups. These findings in humans are similar to our findings in mice that exogenous P₄ injections in WT females do not adversely affect pregnancy (data not shown). Fkbp52^–/– mice with normal PR and P₄ levels but reduced PR activity constitute a unique model for studying P₄ resistance specifically in uterine biology, and it is hoped that our findings will encourage the development of human studies to determine whether FKBP52 status influences P₄ resistance in the uterus.

View Supplemental data

We thank Fuhua Xu for help with statistical analysis. P₄ assays were performed by the University of Virginia Center for Ligand Assay and Analysis Core supported by an NICHD grant (U54 HD28934). This work was supported in part by grants from the NICHD (HD12304 and HD033994 to S.K. Dey; HD050315 to H. Wang) and NIDDK (DK48218 to D.F. Smith). S. Tranguch was supported by a National Research Service Award fellowship from the National Institute on Drug Abuse (F31 DA021062) and an NIDDK training grant (T32 DK07563).

Address correspondence to: Sudhansu K. Dey, Department of Pediatrics, Division of Reproductive and Developmental Biology, Vanderbilt University Medical Center, MCN-D4100, Nashville, Tennessee 37232-2678, USA. Phone: (615) 322-8642; Fax: (615) 322-4704; E-mail: sk.dey@vanderbilt.edu.

Nonstandard abbreviations used:Areg, amphiregulin; FKBP, FK506-binding protein; ICI, ICI 182,780; Ihh, Indian hedgehog; IS, implantation site; Lif, leukemia inhibitory factor; Ltf, lactoferrin; MEF, mouse embryonic fibroblast; P₄, progesterone; PR, P₄ receptor; TPR, tetratricopeptide repeat.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.117:1824–1834 (2007). doi:10.1172/JCI31622

1. Dey, S.K., et al. 2004. Molecular cues to implantation. Endocr. Rev. 25:341-373.
   View this article via: CrossRef PubMed Google Scholar
2. Wang, H., Dey, S.K. 2006. Roadmap to embryo implantation: clues from mouse models. Nat. Rev. Genet. 7:185-199.
   View this article via: CrossRef PubMed Google Scholar
3. Lydon, J.P., et al. 1995. Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev. 9:2266-2278.
   View this article via: CrossRef PubMed Google Scholar
4. Smith, D.F. 2004. Tetratricopeptide repeat cochaperones in steroid receptor complexes. Cell Stress Chaperones. 9:109-121.
   View this article via: CrossRef PubMed Google Scholar
5. Pratt, W.B., Toft, D.O. 1997. Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocr. Rev. 18:306-360.
   View this article via: CrossRef PubMed Google Scholar
6. Smith, D.F. 2000. Chaperones in progesterone receptor complexes. Semin. Cell Dev. Biol. 11:45-52.
   View this article via: CrossRef PubMed Google Scholar
7. Smith, D.F. 1993. Dynamics of heat shock protein 90-progesterone receptor binding and the disactivation loop model for steroid receptor complexes. Mol. Endocrinol. 7:1418-1429.
   View this article via: CrossRef PubMed Google Scholar
8. Barent, R.L., et al. 1998. Analysis of FKBP51/FKBP52 chimeras and mutants for Hsp90 binding and association with progesterone receptor complexes. Mol. Endocrinol. 12:342-354.
   View this article via: CrossRef PubMed Google Scholar
9. Riggs, D.L., et al. 2004. Functional specificity of co-chaperone interactions with Hsp90 client proteins. Crit. Rev. Biochem. Mol. Biol. 39:279-295.
   View this article via: CrossRef PubMed Google Scholar
10. Cheung-Flynn, J., et al. 2005. Physiological role for the cochaperone FKBP52 in androgen receptor signaling. Mol. Endocrinol. 19:1654-1666.
    View this article via: CrossRef PubMed Google Scholar
11. Riggs, D.L., et al. 2003. The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo. EMBO J. 22:1158-1167.
    View this article via: CrossRef PubMed Google Scholar
12. Tranguch, S., et al. 2005. Cochaperone immunophilin FKBP52 is critical to uterine receptivity for embryo implantation. Proc. Natl. Acad. Sci. U. S. A. 102:14326-14331.
    View this article via: CrossRef PubMed Google Scholar
13. Paria, B.C., Huet-Hudson, Y.M., Dey, S.K. 1993. Blastocyst’s state of activity determines the “window” of implantation in the receptive mouse uterus. Proc. Natl. Acad. Sci. U. S. A. 90:10159-10162.
    View this article via: CrossRef PubMed Google Scholar
14. Daikoku, T., et al. 2005. Proteomic analysis identifies immunophilin FK506 binding protein 4 (FKBP52) as a downstream target of Hoxa10 in the periimplantation mouse uterus. Mol. Endocrinol. 19:683-697.
    View this article via: CrossRef PubMed Google Scholar
15. Yang, Z., et al. 2006. FK506-binding protein 52 is essential to uterine reproductive physiology controlled by the progesterone receptor A isoform. Mol. Endocrinol. 20:2682-2694.
    View this article via: CrossRef PubMed Google Scholar
16. Mulac-Jericevic, B., Mullinax, R.A., DeMayo, F.J., Lydon, J.P., Conneely, O.M. 2000. Subgroup of reproductive functions of progesterone mediated by progesterone receptor-B isoform. Science. 289:1751-1754.
    View this article via: CrossRef PubMed Google Scholar
17. Tranguch, S., Smith, D.F., Dey, S.K. 2006. Progesterone receptor requires a co-chaperone for signalling in uterine biology and implantation. Reprod. Biomed. Online. 13:651-660.
    View this article via: PubMed Google Scholar
18. Threadgill, D.W., et al. 1995. Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science. 269:230-234.
    View this article via: CrossRef PubMed Google Scholar
19. Bonyadi, M., et al. 1997. Mapping of a major genetic modifier of embryonic lethality in TGF beta 1 knockout mice. Nat. Genet. 15:207-211.
    View this article via: CrossRef PubMed Google Scholar
20. Wang, H., et al. 2004. Rescue of female infertility from the loss of cyclooxygenase-2 by compensatory up-regulation of cyclooxygenase-1 is a function of genetic makeup. J. Biol. Chem. 279:10649-10658.
    View this article via: CrossRef PubMed Google Scholar
21. Psychoyos, A. 1973. Hormonal control of ovoimplantation. Vitam. Horm. 31:201-256.
    View this article via: PubMed Google Scholar
22. Pointis, G., Rao, B., Latreille, M.T., Mignot, T.M., Cedard, L. 1981. Progesterone levels in the circulating blood of the ovarian and uterine veins during gestation in the mouse. Biol. Reprod. 24:801-805.
    View this article via: CrossRef PubMed Google Scholar
23. Milligan, S.R., Cohen, P.E. 1994. Silastic implants for delivering physiological concentrations of progesterone to mice. Reprod. Fertil. Dev. 6:235-239.
    View this article via: CrossRef PubMed Google Scholar
24. Das, S.K., Chakraborty, I., Paria, B.C., Wang, X.N., Plowman, G., Dey, S.K. 1995. Amphiregulin is an implantation-specific and progesterone-regulated gene in the mouse uterus. Mol. Endocrinol. 9:691-705.
    View this article via: CrossRef PubMed Google Scholar
25. Lim, H., Ma, L., Ma, W.G., Maas, R.L., Dey, S.K. 1999. Hoxa-10 regulates uterine stromal cell responsiveness to progesterone during implantation and decidualization in the mouse. Mol. Endocrinol. 13:1005-1017.
    View this article via: CrossRef PubMed Google Scholar
26. Matsumoto, H., Zhao, X., Das, S.K., Hogan, B.L., Dey, S.K. 2002. Indian hedgehog as a progesterone-responsive factor mediating epithelial-mesenchymal interactions in the mouse uterus. Dev. Biol. 245:280-290.
    View this article via: CrossRef PubMed Google Scholar
27. Lee, K., et al. 2006. Indian hedgehog is a major mediator of progesterone signaling in the mouse uterus. Nat. Genet. 38:1204-1209.
    View this article via: CrossRef PubMed Google Scholar
28. Heikaus, S., Winterhager, E., Traub, O., Grummer, R. 2002. Responsiveness of endometrial genes Connexin26, Connexin43, C3 and clusterin to primary estrogen, selective estrogen receptor modulators, phyto- and xenoestrogens. J. Mol. Endocrinol. 29:239-249.
    View this article via: CrossRef PubMed Google Scholar
29. McMaster, M.T., Teng, C.T., Dey, S.K., Andrews, G.K. 1992. Lactoferrin in the mouse uterus: analyses of the preimplantation period and regulation by ovarian steroids. Mol. Endocrinol. 6:101-111.
    View this article via: CrossRef PubMed Google Scholar
30. Ma, W.G., Song, H., Das, S.K., Paria, B.C., Dey, S.K. 2003. Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation. Proc. Natl. Acad. Sci. U. S. A. 100:2963-2968.
    View this article via: PubMed Google Scholar
31. Lim, H., et al. 1997. Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell. 91:197-208.
    View this article via: CrossRef PubMed Google Scholar
32. Paria, B.C., et al. 2001. Cellular and molecular responses of the uterus to embryo implantation can be elicited by locally applied growth factors. Proc. Natl. Acad. Sci. U. S. A. 98:1047-1052.
    View this article via: CrossRef PubMed Google Scholar
33. Song, H., Lim, H., Das, S.K., Paria, B.C., Dey, S.K. 2000. Dysregulation of EGF family of growth factors and COX-2 in the uterus during the preattachment and attachment reactions of the blastocyst with the luminal epithelium correlates with implantation failure in LIF-deficient mice. Mol. Endocrinol. 14:1147-1161.
    View this article via: CrossRef PubMed Google Scholar
34. Song, H., Lim, H. 2006. Evidence for heterodimeric association of leukemia inhibitory factor (LIF) receptor and gp130 in the mouse uterus for LIF signaling during blastocyst implantation. Reproduction. 131:341-349.
    View this article via: CrossRef PubMed Google Scholar
35. Carpenter, K.D., Gray, C.A., Bryan, T.M., Welsh (Jr.), T.H., Spencer, T.E. 2003. Estrogen and antiestrogen effects on neonatal ovine uterine development. Biol. Reprod. 69:708-717.
    View this article via: CrossRef PubMed Google Scholar
36. Gray, C.A., Bazer, F.W., Spencer, T.E. 2001. Effects of neonatal progestin exposure on female reproductive tract structure and function in the adult ewe. Biol. Reprod. 64:797-804.
    View this article via: CrossRef PubMed Google Scholar
37. Song, H., et al. 2002. Cytosolic phospholipase A2alpha is crucial [correction of A2alpha deficiency is crucial] for ‘on-time’ embryo implantation that directs subsequent development. Development. 129:2879-2889.
    View this article via: PubMed Google Scholar
38. Ye, X., et al. 2005. LPA3-mediated lysophosphatidic acid signalling in embryo implantation and spacing. Nature. 435:104-108.
    View this article via: CrossRef PubMed Google Scholar
39. Bany, B.M., Cross, J.C. 2006. Post-implantation mouse conceptuses produce paracrine signals that regulate the uterine endometrium undergoing decidualization. Dev. Biol. 294:445-456.
    View this article via: CrossRef PubMed Google Scholar
40. Lundkvist, O., Nilsson, B.O. 1982. Endometrial ultrastructure in the early uterine response to blastocysts and artificial deciduogenic stimuli in rats. Cell Tissue Res. 225:355-364.
    View this article via: PubMed Google Scholar
41. McCormack, J.T., Greenwald, G.S. 1974. Progesterone and oestradiol-17beta concentrations in the peripheral plasma during pregnancy in the mouse. J. Endocrinol. 62:101-107.
    View this article via: PubMed Google Scholar
42. Bilinski, P., Roopenian, D., Gossler, A. 1998. Maternal IL-11Ralpha function is required for normal decidua and fetoplacental development in mice. Genes Dev. 12:2234-2243.
    View this article via: PubMed Google Scholar
43. Milligan, S.R., Finn, C.A. 1997. Minimal progesterone support required for the maintenance of pregnancy in mice. Hum. Reprod. 12:602-607.
    View this article via: CrossRef PubMed Google Scholar
44. Girardi, G., Yarilin, D., Thurman, J.M., Holers, V.M., Salmon, J.E. 2006. Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction. J. Exp. Med. 203:2165-2175.
    View this article via: CrossRef PubMed Google Scholar
45. Hardy, D.B., Janowski, B.A., Corey, D.R., Mendelson, C.R. 2006. Progesterone receptor plays a major antiinflammatory role in human myometrial cells by antagonism of nuclear factor-B activation of cyclooxygenase 2 expression. Mol. Endocrinol. 20:2724-2733.
    View this article via: CrossRef PubMed Google Scholar
46. Girardi, G., Redecha, P., Salmon, J.E. 2004. Heparin prevents antiphospholipid antibody-induced fetal loss by inhibiting complement activation. Nat. Med. 10:1222-1226.
    View this article via: CrossRef PubMed Google Scholar
47. Wu, J.T. 1987. Metabolism of progesterone by preimplantation mouse blastocysts in culture. Biol. Reprod. 36:549-556.
    View this article via: CrossRef PubMed Google Scholar
48. Wu, J.T., Liu, Z.H. 1990. Conversion of pregnenolone to progesterone by mouse morulae and blastocysts. J. Reprod. Fertil. 88:93-98.
    View this article via: PubMed Google Scholar
49. Carson, D.D., Hsu, Y.C., Lennarz, W.J. 1982. Synthesis of steroids in postimplantation mouse embryos cultured in vitro. Dev. Biol. 91:402-412.
    View this article via: CrossRef PubMed Google Scholar
50. Hamatani, T., et al. 2004. Global gene expression analysis identifies molecular pathways distinguishing blastocyst dormancy and activation. Proc. Natl. Acad. Sci. U. S. A. 101:10326-10331.
    View this article via: CrossRef PubMed Google Scholar
51. Sharma, R., Bulmer, D. 1983. The effects of ovariectomy and subsequent progesterone replacement on the uterus of the pregnant mouse. J. Anat. 137:695-703.
    View this article via: PubMed Google Scholar
52. Ma, W., et al. 2001. Adult tissue angiogenesis: evidence for negative regulation by estrogen in the uterus. Mol. Endocrinol. 15:1983-1992.
    View this article via: CrossRef PubMed Google Scholar
53. Mulac-Jericevic, B., Lydon, J.P., DeMayo, F.J., Conneely, O.M. 2003. Defective mammary gland morphogenesis in mice lacking the progesterone receptor B isoform. Proc. Natl. Acad. Sci. U. S. A. 100:9744-9749.
    View this article via: CrossRef PubMed Google Scholar
54. Neville, M.C., McFadden, T.B., Forsyth, I. 2002. Hormonal regulation of mammary differentiation and milk secretion. J. Mammary Gland Biol. Neoplasia. 7:49-66.
    View this article via: CrossRef PubMed Google Scholar
55. Chobotova, K., et al. 2005. Heparin-binding epidermal growth factor and its receptors mediate decidualization and potentiate survival of human endometrial stromal cells. J. Clin. Endocrinol. Metab. 90:913-919.
    View this article via: PubMed Google Scholar
56. Berkley, K.J., Rapkin, A.J., Papka, R.E. 2005. The pains of endometriosis. Science. 308:1587-1589.
    View this article via: CrossRef PubMed Google Scholar
57. Bulun, S.E., et al. 2006. Progesterone resistance in endometriosis: link to failure to metabolize estradiol. Mol. Cell. Endocrinol. 248:94-103.
    View this article via: CrossRef PubMed Google Scholar
58. Fang, Z., et al. 2004. Intact progesterone receptors are essential to counteract the proliferative effect of estradiol in a genetically engineered mouse model of endometriosis. Fertil. Steril. 82:673-678.
    View this article via: CrossRef PubMed Google Scholar
59. Kao, L.C., et al. 2003. Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility. Endocrinology. 144:2870-2881.
    View this article via: CrossRef PubMed Google Scholar
60. Oates-Whitehead, R.M., Haas, D.M., Carrier, J.A. 2003. Progestogen for preventing miscarriage. Cochrane Database Syst. Rev. 2003:CD003511.
    View this article via: CrossRef Google Scholar
61. Das, S.K., et al. 1994. Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation. Development. 120:1071-1083.
    View this article via: PubMed Google Scholar