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Endocrinology Vol. 141, No. 5 1872-1881
Copyright © 2000 by The Endocrine Society

ARTICLES

Implantation and Decidualization Defects in Prolactin Receptor (PRLR)-Deficient Mice Are Mediated by Ovarian But Not Uterine PRLR1

Jeff Reese, Nadine Binart, Naoko Brown, Wen-ge Ma, B. C. Paria, S. K. Das, Paul A. Kelly and S. K. Dey

Departments of Pediatrics (J.R., N.Br., B.C.P.), Molecular and Integrative Physiology (W.-g.M., S.K.De.) and Obstetrics and Gynecology (S.K.Da.), Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160-7338; and INSERM U344 Molecular Endocrinology (N.Bi., P.A.K.), Paris, France

Address all correspondence and requests for reprints to: Jeff Reese, Department of Pediatrics, 3043 Wescoe Building, 3901 Rainbow Boulevard, University of Kansas Medical Center, Kansas City, Kansas 66160-7338. E-mail: jreese{at}kumc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PRL and its homologs accomplish their biological effects through the PRL receptor (PRLR). We evaluated the expression and function of PRLR in the embryo and uterus during the periimplantation period because PRLR deficiency results in implantation failure. In wild-type mice, PRLR expression was localized to undecidualized stromal cells in the antimesometrial border on days 6–8 of pregnancy. A small population of PRLR-expressing cells was observed adjacent to the ectoplacental cone in the mesometrial stroma. Low levels of PRLR expression were also detected in the developing embryo on days 6–8. To determine the significance of PRLR expression in this distribution, we examined implantation and decidualization in PRLR-/- mice. Progesterone (P4) administration rescued infertility in PRLR-/- mice from the periimplantation period to midgestation. Artificially induced decidualization was absent in pseudopregnant PRLR-/- mice but was identical to wild-type in P4-treated PRLR-/- mice. Furthermore, wild-type and P4-treated PRLR-/- mice had similar expression of the implantation-specific genes, LIF, amphiregulin, HB-EGF, COX-1, COX-2, PPAR{delta}, Hoxa-10, cyclin-D3, VEGF, and its receptors, Flk-1 and neuropilin-1. Together, these results show that luteal P4 production via ovarian PRLR signaling is required for implantation and early pregnancy. The function of uterine PRLR remains unclear. However, the eventual loss of pregnancy in P4-treated PRLR-/- mice suggests that uterine PRLR may be essential for the support of late gestation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PRL, SYNTHESIZED BY the anterior pituitary and to a lesser extent by numerous extrapituitary tissues (1), has a diverse array of functions. These include regulation of fluid balance, growth and development, endocrine and metabolic activity, behavior, immunity, and reproduction (2). In addition, PRL regulates mammary gland development, stimulates lactation, and affects ovarian functions (3). PRL and PRL-like proteins are produced by decidual cells in the uterus (4, 5, 6, 7, 8), but their function in this tissue is unknown.

In mice and other rodents, PRL and related lactogens mediate their actions via a single receptor (PRLR). This receptor is a single-pass transmembrane protein and belongs to the class I cytokine receptor superfamily (9, 10). Alternative splicing of the PRLR gene yields one long and several short forms with sequence variation in the cytoplasmic tail domain (11, 12, 13, 14, 15). PRLR activation leads to numerous responses including the development of the corpus luteum and the mammary gland ducts and alveoli. Although PRLR is widely expressed (16, 17, 18), there is little information on its cell-specific expression in the embryo and uterus during early pregnancy. The significance of PRL signaling during this period is illustrated by PRLR-deficient female mice where reproductive failure is characterized by reduced ovulation and abnormal cyclicity, impaired fertilization, and the inability of wild-type embryos to implant in PRLR-/- uteri after blastocyst transfer (19). These mice also display abnormal mammary gland development and altered maternal behavior (19, 20). One cause of infertility in PRLR-/- mice is defective corpus luteum formation, thereby limiting progesterone (P4) support for implantation and placental development. P4 administration rescues the periimplantation deficits in PRLR-/- mice, although pregnancy losses often occur during and after midgestation (21). Similarly, mice deficient for PRL are also infertile and have abnormal mammary gland development (22).

The uterine production of PRL and PRL-like proteins and the infertility of PRL-/- and PRLR-/- mice suggest a critical role for PRLR signaling in the establishment of pregnancy. However, the expression and functions of PRLR during the periimplantation period in the mouse are unknown. Thus, we examined the expression of PRLR in the mouse uterus and embryo from the onset of pregnancy to midgestation using in situ hybridization. PRLR messenger RNA (mRNA) accumulation occurred in undecidualized stromal cells in the antimesometrial border, suggesting that these cells are the site of PRL signaling during early pregnancy. P4 administration to PRLR-/- mice rescued implantation and decidualization failures. Furthermore, there was no difference in the expression pattern of several implantation-specific genes between wild-type and P4-treated PRLR-/- mice. These results suggest that PRLR-specific uterine and/or embryonic functions during the periimplantation period are primarily influenced by ovarian PRLR, but that PRLR expression in the uterus may be required to maintain pregnancy during late gestation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and tissue preparation
Homologous recombination and gene targeting techniques were used to create mice with a premature termination signal in exon 5, resulting in failure of PRLR mRNA and protein expression (19). Wild-type and PRLR-/- mice on a 129/Bl6 genetic background were generated by heterozygous crossbreeding. PCR analysis of tail DNA determined the genotypes of the offspring. Animals were housed in the animal care facility at the University of Kansas Medical Center according to NIH and institutional guidelines for the care of laboratory animals. Adult females were mated with fertile males of the same strain. The morning of finding a vaginal plug was designated day 1 of pregnancy. A subgroup of PRLR-/- females received daily P4 supplementation from day 2–19 (2 mg/mouse, sc, in sesame oil). For in situ hybridization, wild-type and P4-treated PRLR-/- uteri were collected at 0830–0930 h on days 1–8 of pregnancy and flash frozen. The weight of individual implantation sites was determined at the time of tissue sectioning. On days 1–3, pregnancy was confirmed by recovery of embryos from oviducts or from the uterine lumen of one horn on day 4. On day 5 (0830–0930 h), implantation sites were visualized by iv injections of 0.1 ml Chicago Blue dye solution (1% in saline). The animals were killed 3 min later to identify the blue bands (implantation sites) along the uterus (23).

Induction of decidualization
To induce the decidual cell reaction (deciduoma), sesame oil (25 µl) was infused intraluminally in one uterine horn on day 4 of pseudopregnancy. Uterine weights of the infused and noninfused (control) horns were recorded on day 8 in wild-type and PRLR-/- mice. The decidual cell reaction was confirmed by histological examination. Mechanical trauma, a more potent stimulus of the decidual response, was also used since oil infusion may result in nonuniform uterine swelling. The effects of ovarian steroids on artificially induced decidual response were evaluated in response to mechanical trauma by silk suture. Wild-type and PRLR-/- mice were ovariectomized and allowed to recover for 2 weeks. Ovariectomized mice were treated with P4 and E2 to sensitize the uterus for optimal decidualization (24). The treatment schedule was the following: E2 (100 ng) for 3 days (days 1–3), no treatment on days 4 and 5, P4 (1 mg) + E2 (10 ng) on days 6–8, and P4 on days 9–12. The uterine lumen was traumatized on day 8 by passage of a 3–0 silk suture through the length of one uterine horn, without disturbing the contralateral horn. A segment of suture was left in place for the remainder of the hormone treatment period. Mice were killed 4 days later to record the weights of treated and untreated horns.

Hybridization probes
A partial complementary DNA (cDNA) containing the mouse extracellular coding region for the PRLR was subcloned into a pGEM-2 vector. This region is common to long and short forms of PRLR and served as a template for generating cRNA probes (13). Mouse-specific cDNAs to HB-EGF, amphiregulin, COX-1, COX-2, PPAR{delta}, LIF, Flk-1, neuropilin-1, Hoxa-10, and cyclin D3 were used to generate cRNA probes using the appropriate polymerases (23, 25, 26, 27, 28, 29, 30, 31). A human-specific VEGF probe that cross hybridizes with mouse VEGF mRNA was also used (25). 35S-labeled sense or antisense cRNA probes used for in situ hybridization had specific activities of approximately 2 x 109 dpm/µg.

In situ hybridization
Frozen sections of uteri from wild-type and PRLR-/- mice were mounted onto the same glass slides. Sections of ovary from wild-type animals served as positive controls for PRLR mRNA localization. In situ hybridization was performed as described previously (23, 32). Frozen sections (11 µm) from days 1–8 of pregnancy were mounted onto poly-L-lysine-coated slides. When required, uterine sections were cut serially to detect the sites of blastocysts. Sections were fixed in 4% paraformaldehyde solution in PBS for 15 min at 4 C. After prehybridization, sections were hybridized to 35S-labeled antisense cRNA probes at 45 C for 4 h in 50% formamide hybridization buffer. As negative controls, sections were hybridized to 35S-labeled sense probes. After hybridization and washing, the sections were incubated with RNase A (20 µg/ml) at 37 C for 20 min. RNase A-resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak Co., Rochester, NY). The slides were poststained with hematoxylin and eosin.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of PRLR mRNA in the periimplantation mouse uterus
To analyze the pattern of PRLR expression in the embryo and uterus during early pregnancy, we examined the distribution of PRLR mRNA by in situ hybridization with a cRNA probe that recognizes all forms of PRLR (13) (Fig. 1Go). The results demonstrate that uterine PRLR expression was low to undetectable on days 1 and 2. On days 3 and 4, low levels of accumulation were noted in the subepithelial stroma (data not shown). There was no significant PRLR expression on day 5. On days 6–8, distinct accumulation of PRLR mRNA was noted in subepithelial stromal cells at the mesometrial pole and undifferentiated stromal cells at the antimesometrial pole. On days 6–7, low levels of signals were also present in the epiblast region of the embryo. On day 8, a weak signal for PRLR mRNA also began to appear in the embryo, mostly in the distal endoderm or Reichert’s membrane. No specific autoradiographic signals were detected when uterine sections were hybridized with the sense probe (data not shown).



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  		Figure 1. In situ hybridization of PRLR mRNA in the periimplantation mouse embryo and uterus. Brightfield and corresponding darkfield photomicrographs of representative sections of implantation sites on day 5 (a, b), day 6 (c, d), day 7 (e, f), and day 8 (g, h) are shown at 20x. bl, Blastocyst, em, embryo; sdz, secondary decidual zone; m, mesometrial, am, antimesometrial.

 
Decidualization is defective in PRLR-deficient mice
After blastocyst attachment to the uterine luminal epithelium, subepithelial stromal cells undergo extensive proliferation and differentiation into decidual cells. The attachment reaction and ensuing decidual response coincide with increased endometrial vascular permeability. During pregnancy in the mouse, the stimulus for decidualization is the implanting blastocyst. However, this process can be experimentally induced in pseudopregnant or steroid hormonally prepared uteri by intraluminal oil infusion or mechanical stimulation (33). Although implantation fails to occur in PRLR-/- mice, whether this receptor is also critical for decidualization is not known. To address this question, we examined this process in PRLR-/- mice by intraluminal oil infusion on day 4 of pseudopregnancy. The results demonstrate that while all (7/7) of the wild-type mice showed an average of 4-fold increase in uterine weights with decidualization, none of the PRLR-/- females showed this response (Fig. 2AGo). To circumvent the steroid hormone deficiency resulting from defective corpus luteum development and function (21), we examined the decidualization process in ovariectomized mice treated with an established steroid regimen that optimizes uterine responses to decidualization. All of the wild-type (6/6) and PRLR-/- (7/7) mice showed decidualization response (Fig. 2BGo), demonstrating the rescue of this event by P4 despite the absence of the PRLR. Collectively, these results show that the initial attachment reaction and the subsequent decidualization process could be supported by the administration of exogenous ovarian steroids in the absence of PRLR.



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  		Figure 2. Experimentally induced decidual cell reaction in wild-type and PRLR-/- mice. A, Response to intraluminal oil injection on day 4 of pseudopregnancy, with uterine weights recorded on day 8. In these experiments, PRLR-/- mice did not receive P4 supplementation. B, Response to mechanical trauma in ovariectomized, steroid-treated females. Uterine weights recorded 4 days after insertion of silk sutures into the uterine lumen as deciduogenic stimuli. Data represented as mean ± SEM, * P < 0.05 (Student’s t test).

 
Implantation sites in P4 treated PRLR-/- females
In the mouse, secretion of P4 from newly formed corpora lutea accompanied by preimplantation ovarian estrogen secretion on the morning of day 4 is critical for the establishment of uterine receptivity for implantation. In PRLR-/- females, P4 administration rescues preimplantation embryo development (21). We examined the extent to which implantation failure in these mice is rescued by P4 in regard to the weight of individual implantation sites. As shown in Fig. 3Go, implantation sites in wild-type and P4-treated PRLR-/- mice are similar on days 5–10 of pregnancy, although a decreasing trend is noted in PRLR-/- mice after day 8. There was no difference in the number of wild-type and PRLR-/- implantation sites during this period. However, P4-treated PRLR-/- mice (n = 9) had significantly fewer pups than wild-type mice (n = 5) at the time of birth (2.3 ± 3.1 vs. 7.6 ± 1.9, P < 0.01) and an increase in the number of resorption sites.



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  		Figure 3. Weight of implantation sites in wild-type and P4-treated PRLR-/- females on days 5–10 of pregnancy. Implantation sites were identified by blue dye injection on day 5. Implantation sites from day 6 onward are visually distinct and do not require any special manipulation. Weights of implantation sites at each time point averaged from three to seven mice. Data represented as mean ± SEM. *, P < 0.01 (Student’s t test).

 
Expression of implantation-specific genes in the uterus of P4 treated PRLR-/- mice
Our results demonstrate that impaired implantation and decidualization in PRLR-/- mice are rescued by P4 replacement. To determine whether this is related to the correct expression of implantation-specific genes, we examined the expression pattern of several genes that are associated with the implantation and decidualization process. The regulated expression of many growth factors and their receptors in the uterus and embryo during the periimplantation period suggests their importance in implantation (34). Thus, we examined the expression of leukemia inhibitory factor (LIF) and amphiregulin (Ar) on days 4 and 5, and heparin-binding EGF-like growth factor (HB-EGF) on day 5 of pregnancy in the wild-type and P4-treated PRLR-/- mice. LIF is expressed in uterine glandular epithelium on day 4 and disruption of this gene causes implantation failure (35). Ar is a P4-regulated gene in the mouse uterus that is expressed in the epithelium on the morning of day 4 and becomes highly localized in the luminal epithelium surrounding the blastocyst with the initiation of implantation (27). Among the EGF family of growth factors, HB-EGF appears to be more relevant to implantation. In contrast to Ar, HB-EGF is localized in the luminal epithelium solely at the site of blastocyst apposition 6–7 h before the attachment reaction occurs at midnight on day 4 (23). As shown in Fig. 4Go, the distribution of LIF and Ar mRNA in P4-treated PRLR-/- uteri on days 4 and 5 is similar to wild-type mice. Furthermore, HB-EGF is correctly expressed in the luminal epithelium at the sites of implantation in P4-treated PRLR-/- mice (Fig. 4Go).



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  		Figure 4. In situ hybridization of LIF, Ar and HB-EGF mRNAs in periimplantation uteri of wild-type and P4-treated PRLR-/- mice. Dark-field photomicrographs for LIF mRNA on day 4 (a and b) and day 5 (c and d), Ar mRNA on day 4 (e and f) and day 5 (g and h) and HB-EGF mRNA (i and j) on day 5 are shown at 80x (a and b), 40x (e and f) and 20x (c and d, g–j). ge, Glandular epithelium; le, luminal epithelium; s, stroma; myo, myometrium; bl, blastocyst; m, mesometrial; and am, antimesometrial.

 
Hoxa-10, an abdominal B class homeobox gene, is expressed in mouse uterine stromal cells under the stimulation of P4, and is up-regulated in the stroma during decidualization. The absence of this gene causes implantation and decidualization failures in mice (29, 36). Cyclin D3, a cell-cycle regulatory gene, is also up-regulated in uterine stromal cells at the sites of implantation with the onset of the decidual cell reaction (28). The expression of this gene is down-regulated in Hoxa-10-/- uteri, implying a relationship between Hoxa-10 and cyclin D3 in cell cycle regulation (28). The present investigation did not reveal any aberrant spatiotemporal expression of these genes in P4-treated PRLR-/- uteri (Fig. 5Go), again suggesting that the rescue of implantation and decidualization by P4 is normal with respect to these genes.



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  		Figure 5. In situ hybridization of Hoxa-10 and cyclin D3 mRNAs at implantation sites on days 5 and 6 of pregnancy in wild-type and P4-treated PRLR-/- mice. Darkfield photomicrographs of mRNA localization for Hoxa-10 on day 5 (a and b) and day 6 (c and d), and cyclin D3 on day 5 (e and f) and day 6 (g and h) are shown at 20x. bl, Blastocyst; em, embryo; pdz, primary decidual zone; sdz, secondary decidual zone; m, mesometrial pole; and am, antimesometrial pole.

 
We also examined the expression of cyclooxygenase isoforms (COX-1 and COX-2), which are rate-limiting enzymes in the synthesis of prostaglandins (PGs). These genes have a restricted cellular expression pattern in the mouse uterus during the periimplantation period (25). Using COX-1 and COX-2 null mice and selective COX inhibitors, we have recently established that COX-2 is essential for ovulation, fertilization, implantation, and decidualization (32). Further, COX-2 derived prostacyclin is the major PG that is produced at the site of implantation in the mouse and mediates its effects in implantation via PPAR{delta}, a member of the steroid hormone nuclear receptor superfamily (30). We have observed that both COX isoforms (Fig. 6Go) and PPAR{delta} (data not shown) are correctly expressed in P4-treated pregnant PRLR-/- uteri.



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  		Figure 6. In situ hybridization of COX-1 and COX-2 in periimplantation uteri of wild-type and P4-treated PRLR-/- mice. Dark-field photomicrographs of COX-1 mRNA on days 4 (a and b) and 5 (c and d), and of COX-2 on days 5 (e and f) and 8 (g and h) are shown at 40x (a and b) and 20x (c–h). le, Luminal epithelium; s, stroma; bl, blastocyst; em, embryo; sdz, secondary decidual zone; m, mesometrial pole; and am, antimesometrial pole.

 
Uterine vascular permeability and angiogenesis are hallmarks of the implantation process. Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells and an inducer of angiogenesis. We have previously demonstrated that VEGF and its receptors have a specific spatiotemporal expression pattern in the mouse uterus that suggests their involvement in uterine vascular permeability and angiogenesis during implantation (25, 31). In P4-treated PRLR-/- uteri, these genes were correctly expressed, revealing their presence in both the mesometrial and antimesometrial decidual beds (Fig. 7Go). Overall, the results obtained by in situ hybridization cannot determine quantitative differences in gene expression. However, the cell-specific hybridization for each of these genes suggests that P4-treated PRLR-/- uteri have similar patterns of gene expression to those of wild-type.



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  		Figure 7. In situ hybridization of VEGF and Flk-1 mRNAs at the implantation sites on day 6 of pregnancy in wild-type and P4-treated PRLR-/- mice. Darkfield photomicrographs of VEGF mRNA (a and b) and of Flk-1 (c and d) are shown at 20x. em, Embryo; pdz, primary decidual zone; sdz, secondary decidual zone; m, mesometrial pole; and am, antimesometrial pole.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The highlights of the present investigation are the demonstration of temporal and cell-specific localization of the PRL receptor in the uterus during early pregnancy, and the restoration of implantation and decidualization failures in PRLR-/- mice with correct cell-specific expression of several implantation-specific genes after P4 replacement. These results imply that aberrant preimplantation embryo development and implantation in PRLR-/- mice are due to P4 deficiency resulting from abnormal corpus luteum function.

PRL mediates over 300 different biologic functions (2). Normally, PRL secreted by the pituitary acts in an endocrine manner on target tissues. However, it is also produced by a number of extrapituitary tissues and has autocrine and paracrine functions (1). The placenta and decidua also generate several PRL-like proteins during pregnancy (8). The biologic activity of PRL and certain lactogenic hormones is regulated by the PRLR. However, the receptors for many PRL-like ligands produced by the deciduum have not yet been identified. Although various forms of the PRLR are expressed in many tissues, its expression in the gravid mouse uterus has not been characterized. Our results show very little expression of PRLR in the embryo and uterus during the preimplantation period. While PRLR is widely expressed in later stages of fetal development (17, 18, 37), the early embryo is capable of cellular differentiation and proliferation in the absence of PRL signaling. The generation of viable PRL-/- and PRLR-/- mice also suggests that embryo-uterine interactions during the preimplantation period are not directly dependent on embryonic PRLR activation (19, 22).

In the gravid uterus, decidual cells eventually form two distinct entities, the mesometrial and antimesometrial decidua (38, 39). On days 6–8 of pregnancy, we observed increasing PRLR signals in a rim of undifferentiated stromal cells in the antimesometrial pole. The cells expressing PRLR were small and densely packed, and located outside of the secondary decidual zone in the capsule region (40). This region of PRLR-expressing cells was wider at the junction of mesometrial and antimesometrial regions. A small population of cells expressing PRLR was also consistently observed in the subepithelial stroma adjacent to the uterine crypt and superior to the ectoplacental cone. The function of PRLR expression in these cells is unknown. Recently, rat decidual cells were shown to contain receptors that bind to and are activated by pituitary and decidual PRLs (41, 42). In these studies, PRLR mRNA was initially found in both mesometrial and antimesometrial decidua. Over time, PRLR disappeared from the antimesometrial decidual cells and was primarily localized in mesometrial decidual cells. These studies were performed on size-fractionated decidual cells, assuming that the smaller cells were of mesometrial origin. In contrast, we observed that PRLR expression was limited to undifferentiated small stromal cells in the lateral and antimesometrial regions. This discrepancy may be related to passage of small undifferentiated cells with the mesometrial fraction during elutriation of dispersed decidual cells (39, 42). Conversely, it is possible that PRL ligand-receptor relationships during decidualization in the mouse may be different from the rat. The human PRLR is expressed in amnion, chorion, decidua, and placenta during late gestation, but no information is available during early pregnancy (43, 44). Until recently, PRL signaling in the reproductive tract has primarily focused on the role of ovarian PRLRs. However, the rodent antimesometrial decidua actively secretes growth factors and PRL-like hormones, suggesting that an autocrine and/or paracrine pathway exists for PRLR action within the uterus (7, 42, 45). Activation of the PRLR in this distribution may transduce a signal for stromal cell regeneration, or limit the ability of these cells to engage in the decidual response. The lack of PRLR expression in the mesometrial decidua suggests that early placental development occurs without the direct influence of PRL. This is somewhat surprising, given the extensive expression of placental lactogens during this period, and suggests that additional receptors may be available for these ligands. Alternatively, PRL-like ligands present at midgestation may not be biologically indispensable. Finally, although PRL regulates many aspects of the immune response, it seems unlikely that immunomodulation of the maternal host response to the fetus would be concentrated in the antimesometrial border, opposite to the maternal-fetal interface. Thus, a definitive role for uterine PRLRs remains speculative at this time.

PRLR-/- mice are sterile, with irregular cycles and impaired ovulation. The failure of implantation and decidualization was suspected as an additional cause of infertility because PRLR-/- mice are infertile even after the transfer of wild-type blastocysts (19). To this end, a decidual response could not be induced in PRLR-/- mice, but complete restoration was observed after ovariectomy and supplementation with estrogen and P4. These results suggest that decidualization in the mouse is dependent on ovarian rather than uterine PRLR activation, and the subsequent production of P4 by the corpus luteum. The minimal requirements for P4 in the maintenance of pregnancy have been established (46). By direct measurement and by their response to supplementation, PRLR-/- mice are known to have insufficient P4 levels (21). However, continuous P4 treatment does not appear to completely rescue pregnancy, with increased losses occurring after midgestation. The effect of P4 treatment on the formation of the deciduum was examined after supplementation with P4. The weight of implantation sites was similar in wild-type and supplemented PRLR-/- mice through day 10 of pregnancy, although a small difference was noted on day 8. Furthermore, the expression pattern of P4-dependent genes such as amphiregulin, COX-1, and Hoxa-10 was similar in wild-type and P4-supplemented PRLR-/- mice. These results suggest that the correction of reproductive deficits by P4 in PRLR-/- mice is accomplished with correct expression of P4-dependent genes that are important in early pregnancy. Finally, we did not detect alterations in expression patterns of specific uterine genes that regulate growth and differentiation, positional identity, or vascular tone and permeability. Thus, the rescue of pregnancy failure by P4 and the cause of pregnancy loss at a later stage in PRLR-/- mice cannot be ascribed to aberrant spatial expression of genes that normally contribute to the establishment of pregnancy. In conclusion, PRLR-mediated P4 production in the ovary appears to be critical for implantation and decidualization. On days 6–8 of pregnancy, uterine PRLR expression is restricted to a subpopulation of undecidualized cells adjacent to the uterine crypt and in the antimesometrial stroma. Although the function of PRLR in these cells is unknown, we cannot exclude their contribution to normal decidual function.


   Acknowledgments
 
We thank Lovella Tejada for her assistance on this project.


   Footnotes
 
1 This work was supported by NIH Grants RR-11825, HD-37677 (to J.R.), HD-35114 (to B.C.P.), ES-07814 (to S.K.Da.), HD-12304, HD-29968 (to S.K.De.), Akzo-nobel-Organon, 99D293B, and Association pour la recherche sur le cancer, 9952 (to N.Bi.). NICHD Center Grants in Reproductive Biology (HD-33994) and Mental Retardation and Developmental Disabilities (HD-02528) provided various core facilities. Back

Received December 3, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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