Decidual Prolactin-related Protein Research Articles

Genome-Wide Association Mapping for Female Infertility in Inbred Mice.

The genetic factors underlying female infertility in humans are only partially understood. Here, we performed a genome-wide association study of female infertility in 25 inbred mouse strains by using publicly available SNP data. As a result, a total of four SNPs were identified after chromosome-wise multiple test correction. The first SNP rs29972765 is located in a gene desert on chromosome 18, about 72 kb upstream of Skor2 (SKI family transcriptional corepressor 2). The second SNP rs30415957 resides in the intron of Plce1 (phospholipase C epsilon 1). The remaining two SNPs (rs30768258 and rs31216810) are close to each other on chromosome 19, in the vicinity of Sorbs1 (sorbin and SH3 domain containing 1). Using quantitative RT-PCR, we found that Sorbs1 is highly expressed in the mouse uterus during embryo implantation. Knockdown of Sorbs1 by siRNA attenuates the induction of differentiation marker gene Prl8a2 (decidual prolactin-related protein) in an in vitro model of decidualization using mouse endometrial stromal cells, suggesting that Sorbs1 may be a potential candidate gene for female infertility in mice. Our results may represent an opportunity to further understand female infertility in humans.

Luman recruiting factor is involved in stromal cell proliferation during decidualization in mice.

Decidualization is crucial for successful pregnancy in mice and humans. Although many essential molecular modulators have been identified during decidualization, the precise molecular mechanism of uterine decidualization remains largely unknown. Our previous research indicates that luman recruiting factor (LRF) is strongly expressed in decidual uteri of mice on days 6-8 of pregnancy. In this study, our aim is to determine the biological functions of LRF during decidualization in mice. We used the shLRF lentivirus to attenuate the expression of LRF, which significantly reduced the weight and size of implantation sites on days 7-8 of pregnancy. In a stromal cell culture model, LRF mRNA and protein levels increased significantly during stromal cell decidualization induced by estrogen and progesterone. LRF silencing resulted in the decidual markers decidual prolactin-related protein, insulin-like growth factor-binding protein 1 and progesterone receptor being dramatically reduced, and the decidual process was significantly inhibited. Cell-cycle analysis and cell apoptosis analysis revealed that, although no obvious apoptosis occurred in shLRF-lentivirus-infected stromal cells during decidualization, proliferation was inhibited via S-phase cell-cycle arrest, and the mitotic activity of uterine stromal cells was inhibited. An examination of cell-cycle regulatory factors indicated that the mRNA expression levels of cyclin A and cyclin B1 were significantly down-regulated after treatment with shLRF lentivirus. Thus, LRF seems to be involved in the regulation of decidualization during pregnancy by modulating the expression of the key cell-cycle regulatory factors cyclin A and cyclin B1.

Molecular cues to the anti-implantation effect of nano-puerarin in rats.

Puerarin, a selective oestrogen receptor modulator, intercepts implantation in rats, albeit at unacceptably higher doses. We developed poly lactic-co-glycolic acid-encapsulated nano-puerarin (PN) and mapped the molecular pathway underlying its anti-implantation effects. Smooth-surfaced and spherical PN having a mean diameter of ∼147nm was obtained with good yield, efficient encapsulation, and optimum drug loading. In culture, PN slowly and steadily released puerarin, which was readily taken up by the decidual cells. PN exerted a dose-dependent anti-implantation effect. As marked by attenuated expression of stromal cell desmin, alkaline phosphatase, IGFBP1, and decidual prolactin-related protein, the anti-implantation effect of PN seemed secondary to compromised decidualization. Using in vivo (pregnant and pseudopregnant rats) and in vitro (endometrial stromal cell culture) treatment models, we document that PN enforced inhibition of uterine expression of Hbegf and Hoxa10 and their downstream signalling molecules, Cyclin D3 (CCND3)/CDK4. PN also efficiently ablated the Ihh-Nr2f2-Bmp2 signalling pathway and invited the loss of uterine potential for decidualization. There was a dose-dependent up-regulation of RHOA and its effector protein kinase, ROCK1, leading to the promotion of MLC phosphorylation and actin-myosin interaction. PN also down-regulated the stromal cell activation of ERK½ and expression of MMP9. These effects acting together stabilized the stroma and inhibited the stromal cell migration. Central to this array of events was the adversely altered endometrial expression of oestrogen receptor subtypes and repression of progesterone receptor that indulged endless proliferation of luminal epithelia and distorted the precisely choreographed stroma-epithelia crosstalk. Thus, PN dismantles the endometrial bed preparation and prevents implantation.

Identification of target genes for a prolactin family paralog in mouse decidua.

Prolactin family 8, subfamily a, member 2 (PRL8A2; also called decidual prolactin-related protein; dPRP) is a member of the expanded prolactin family. PRL8A2 is expressed in the uterine decidua and contributes to pregnancy-dependent adaptations to hypoxia. The purpose of this study was to identify gene targets for PRL8A2 action within the uteroplacental compartment. Affymetrix DNA microarray analysis was performed for RNA samples from WT and Prl8a2 null tissues. Validation of the DNA microarray was performed using quantitative RT-PCR. Nine genes were confirmed with decreased expression in Prl8a2 null tissues (e.g., Klk7, Rimklb, Arhgef6, Calm4, Sprr2h, Prl4a1, Ccl27, Lipg, and Htra3). These include potential decidual, endothelial and trophoblast cell targets positively regulated by PRL8A2. A significant upregulation of Derl3, Herpud1, Creld2, Hsp90b1, Ddit3 and Hspa5 was identified in Prl8a2 null tissues, reflecting an increased endoplasmic reticulum (ER) stress response. ER stress genes were prominently expressed in the uterine decidua. We propose that PRL8A2 is a mediator of progesterone-dependent modulation of intrauterine responses to physiological stressors.

Evidence of placental endoplasmic reticulum stress in the pathophysiology of gestational diabetes mellitus

Evidence of placental endoplasmic reticulum stress in the pathophysiology of gestational diabetes mellitus

Placental antiangiogenic prolactin fragments are increased in human and rat maternal diabetes

Introduction/objectivesThe role of the placenta in diabetic mothers on fetal development and programming is unknown. Prolactin (PRL) produced by decidual endometrial cells may have an impact. Although full-length PRL is angiogenic, the processed form by bone morphogenetic protein-1 (BMP-1) and/or cathepsin D (CTSD) is antiangiogenic.The objectives were to investigate the involvement of decidual PRL and its antiangiogenic fragments in placentas from type-1 diabetic women (T1D) and from pregnant diabetic rats with lower offspring weights than controls. MethodsPRL, BMP-1, and CTSD gene expressions and PRL protein level were assessed in T1D placentas (n=8) at delivery and compared to controls (n=5). Wistar rats received, at day 7 of pregnancy, streptozotocin (STZ) (n=5) or nicotinamide (NCT) plus STZ (n=9) or vehicle (n=9). Placental whole-genome gene expression and PRL western blots were performed at birth. ResultsIn human placentas, PRL (p<0.05) and BMP-1 (p<0.01) gene expressions were increased with a higher amount of cleaved PRL (p<0.05) in T1D than controls. In rats, diabetes was more pronounced in STZ than in NCT–STZ group with intra-uterine growth restriction. Decidual prolactin-related protein (Dprp) (p<0.01) and Bmp-1 (p<0.001) genes were up-regulated in both diabetic groups, with an increased cleaved PRL amount in the STZ (p<0.05) and NCT–STZ (p<0.05) groups compared to controls. No difference in CTSD gene expression was observed in rats or women. ConclusionsAlterations in the levels of the PRL family are associated with maternal diabetes in both rats and T1D women suggesting that placental changes in these hormones impact on fetal development.

Maternal Smad3 deficiency compromises decidualization in mice

Transforming growth factor (TGF)-β and activin, members of TGF-β superfamily, are abundantly expressed in the endometrium and regulate decidualization of endometrial stroma. Smad2 and Smad3 are receptor-regulated Smads (R-Smads) that transduce extracellular TGF-β/activin/Nodal signaling. In situ hybridization results showed that Smad3 was highly expressed in the decidual zone during the peri-implantation period in mice. By using artificial decidualization, we found that Smad3 null mice showed partially compromised decidualization. We therefore hypothesized that Smad2 might compensate for the function of Smad3 during the process of decidualization. Smad2 was also highly expressed in the decidual zone and phosphorylated Smad2 was much more abundantly increased in the deciduoma of Smad3 null mice than for wild-type (WT) mice. We further employed an in vitro uterine stromal cell decidualization model, and found that decidual prolactin-related protein (dPRP) and cyclin D3, which are well-known markers for decidual cells, were significantly down-regulated in Smad3 null decidual cells, and were much more significantly reduced when the expression of Smad2 was simultaneously silenced by its siRNA (P < 0.05). However, the expression levels of dPRP and cyclin D3 remained the same when Smad2 was silenced in WT decidual cells. Collectively, these findings provide evidence for an important role of Smad3 in decidualization and suggest that Smad2 and Smad3 may have redundant roles in decidualization.

Distribution and Spatiotemporal Relationship of Activin A and Follistatin in Mouse Decidual and Placental Tissue

Problem Cells responsible for the synthesis of follistatin and activin A in the pregnant mouse endometrium have not been characterized. Method of study Immunocytochemistry was used to determine the distribution of follistatin and activin A in the pregnant mouse uterus. Results Follistatin was detected in the endometrium prior to decidualization and embryo implantation. Follistatin was not seen in fully decidualized cells, being restricted to non-decidualized fibroblasts and cells in the process of decidualization. In contrast, activin A was detected exclusively in mature antimesometrial decidual cells during involution. After day eleven of pregnancy, both substances were identified in the extracellular matrix of the spongiotrophoblast. Conclusion As previously described for decidual prolactin-related protein and the proteoglycan perlecan, follistatin and activin A were detected in the extracellular matrix of the spongiotrophoblast, suggesting that this region acts as reservoir for these growth factors in the mouse placenta.

Granulated and Non‐Granulated Decidual Prolactin‐Related Protein‐Positive Decidual Cells in the Pregnant Mouse Endometrium

Identification of the cell types responsible for the synthesis of decidual prolactin-related protein (dPRP) in the pregnant mouse endometrium. Histochemistry and immunocytochemistry were used to determine peri-implantation dPRP and perlecan distribution in the mouse uterus. We identified dPRP in pre-decidual and mature decidual cells from days 5 to 12 of pregnancy. On day 8, dPRP immunoreactivity was detected within cytoplasmic granules of a specific population of granulated decidual cells (GDCs). In mesometrial decidual cells, weak immunoreactivity was seen from days 7 to 14. Between days 11 and 14, dPRP was found in cytoplasm and in the extracellular matrix surrounding islands of spongiotrophoblast. Perlecan, a heparan sulfate proteoglycan, was co-localized with dPRP. GDCs are a putative source of dPRP in pregnant mice. Co-localization of perlecan with dPRP suggests that the former acts as a dPRP reservoir and facilitates its paracrine effect in developing placental tissues.

A uterine decidual cell cytokine ensures pregnancy-dependent adaptations to a physiological stressor

In the mouse, decidual cells differentiate from uterine stromal cells in response to steroid hormones and signals arising from the embryo. Decidual cells are crucially involved in creating the intrauterine environment conducive to embryonic development. Among their many functions is the production of cytokines related to prolactin (PRL), including decidual prolactin-related protein (DPRP). DPRP is a heparin-binding cytokine, which is abundantly expressed in uterine decidua. In this investigation, we have isolated the mouse Dprp gene, characterized its structure and evaluated its biological role. Dprp-null mice were made by replacing exons 2 to 6 of the Dprp gene with an in-frame enhanced green fluorescent protein (EGFP) gene and a neomycin (neo) resistance cassette. Heterozygous intercross breeding of the mutant mice yielded the expected mendelian ratio. Pregnant heterozygote females expressed EGFP within decidual tissue in locations identical to endogenous Dprp mRNA and protein expression. Homozygous Dprp-null mutant male and female mice were viable, exhibited normal postnatal growth rates, were fertile and produced normal litter sizes. A prominent phenotype was observed when pregnant Dprp-null mice were exposed to a physiological stressor. DPRP deficiency interfered with pregnancy-dependent adaptations to hypoxia resulting in pregnancy failure. Termination of pregnancy was associated with aberrations in mesometrial decidual cells, mesometrial vascular integrity, and disruptions in chorioallantoic placenta morphogenesis. The observations suggest that DPRP participates in pregnancy-dependent adaptations to a physiological stressor.

Stimulation of a rat uterine stromal cell line in culture reveals a molecular switch for endocrine-dependent differentiation

Differentiation of uterine stromal cells is critical for the establishment of pregnancy. This study had two purposes: (i) to validate the use of the UIII rat uterine stromal cell model for investigating mechanisms underlying decidual cell differentiation, and (ii) to use this cell model to identify a molecular switch for cellular entry into the decidual cell differentiation pathway. Quiescent rat uterine stromal cells were transfected with a 500 bp segment of the decidual prolactin-related protein (dPRP) promoter ligated to a luciferase reporter gene. Cells were incubated in low-serum medium, or in low-serum medium containing progesterone (1 microM), estradiol 17-beta (10 nM), cholera toxin (10 ng/ml) and interleukin-11 (10 ng/ml). Protein extracts were collected 48 h later and luciferase was measured in the cellular lysates. Cholera toxin and interleukin-11 stimulated luciferase expression (P< 0.05) and addition of sex steroids further increased (P< 0.05) dPRP promoter activity. Stromal cells did not proliferate (P< 0.05) under differentiation conditions. Deletion analysis of the dPRP promoter revealed maximal luciferase expression between -250 and -500 bp relative to the transcription start site. Comparison of cyclin E/Cdk2 activity between proliferating and differentiating cells showed a 3-fold increase (P< 0.05) at 12 h in differentiating cells. The results suggest that cyclin E/Cdk2 serves as a molecular switch for uterine stromal cell entry into the decidual cell differentiation pathway.

Interleukin‐11 signaling is required for the differentiation of natural killer cells at the maternal–fetal interface

Interleukin-11 (IL-11) is a multifunctional hematopoietic growth factor that has been implicated in the control of reproduction. Studies on IL-11 receptor-alpha (IL-11R alpha)-deficient mice showed that female mice are infertile due to defective decidualization. In this report, we evaluated the development of decidual cells, immune cells, and the vasculature associated with the implantation site of IL-11R alpha-deficient mice; with the aim of better understanding the nature of the fertility defect. Messenger RNAs for decidual differentiation, such as decidual prolactin-related protein and prolactin-like protein-J are expressed in the IL-11R alpha mutant. However, the number of decidual cells expressing these genes is decreased in the mutant compared with the wild-type control. Although, trophoblast cells differentiate and express placental lactogen-I in the IL-11R alpha-deficient uterine environment, they fail to progress and expand in number. Defects in the organization of the decidual vasculature were also apparent in the IL-11R alpha mutant uterus. The most dramatic effect of IL-11 signaling was on the hematopoietic environment of the uterine decidua. Differentiated/perforin-expressing uterine natural killer (NK) cells were virtually absent from implantation sites of IL-11R alpha mutant mice. NK cell precursors were capable of homing to the IL-11R alpha-deficient uterus and a known regulator of NK cell differentiation; IL-15 was expressed in the IL-11R alpha mutant uterus. Splenic NK cells from IL-11R alpha mutant mice were also able to respond to IL-15 in vitro. Thus, the defect in NK precursor cell maturation was not intrinsic to the NK precursor cells but was dependent upon the tissue environment. In summary, IL-11 signaling is required for decidual-specific maturation of NK cells.

Temporal relationships among uterine pituitary adenylate cyclase-activating polypeptide, decidual prolactin-related protein and progesterone receptor mRNAs expressions during decidualization and gestation in rats

Pituitary adenylate cyclase-activating peptide (PACAP), a novel compound with vasoactive intestinal polypeptide-like activity, was recently shown to be localized in the neuronal endings of the human uterus. The purpose of the present study was to assess the functional presence of PACAP mRNA in the decidual endometrium and its relationship to the expression levels of decidual prolactin-related protein (dPRP) and the progesterone receptor mRNAs during decidualization and pregnancy in Sprague–Dawley rats. PACAP was constitutively and temporally expressed in the decidual endometrium and gravid uterus. The time-dependent correlated expression levels of PACAP, dPRP and the progesterone receptor were induced by the neurogenic reproductive signals, i.e. the vagino-cervical/deciduogenic stimuli of decidualization and by the normal equivalent stimuli of mating/blastocyst implantation of gestation. Correlation among the mRNA expression levels of PACAP, dPRP and the progesterone receptor and the coordinated inhibitory actions of the anti-progesterone (RU-486) suggest that there is also correlated time-dependent steroid regulation of the mRNA levels of PACAP, dPRP and the progesterone receptor in the decidual and pregnant uteri. One possible functional meaning for the time-related localization of endometrial/uterine PACAP could be to facilitate endometrial blood flow and increase the availability of metabolic substrates to the developing deciduoma or embryo. The study demonstrates the potential importance of PACAP expression in the regulation of the maternal feto-placental component and suggests a prominent reproductive role for the neuropeptide in mammalian pregnancy.

Hydroxyurea inhibition of cellular and developmental activities in the decidualized and pregnant uteri of rats.

The cytotoxicity of hydroxyurea (HU), currently used to combat various cancers, sickle cell anemia and human immunodeficiency infection, was assessed by exposing decidualized and pregnant uteri of Sprague-Dawley rats to this drug. Consecutive daily doses of HU (500 mg/kg(-1)) for 4 days were injected subcutaneously during decidualization when proliferation of the deciduoma was biochemically analyzed on pseudopregnancy day 9, or injected intraperitoneally during pregnancy when uterine developmental processes were evaluated on gestation day 16. Hydroxyurea displayed prominent antiproliferative effects on decidual growth. These actions were comparable to significantly impaired (P<0.001) developmental responses (increases in post-implantation losses, in resorbed fetuses and in reduced fetal and placental weights) during pregnancy. The cellular components inhibited by HU were DNA, protein, nitric oxide synthase, a matrix metalloproteinase and decidual prolactin-related protein mRNA (P<0.05). Steroid-related endocrine events (serum progesterone concentrations, estrogen receptor and mRNA levels) were unaffected by HU, implying direct cellular action by the drug. Interestingly, endometrial alkaline phosphatase bioactivity was enhanced by HU (P<0.05). Subsequently, the reproductive toxicity of HU was apparently related to mitogenic and differentiation-induced endometrial cellular activities.

Dual expression of prolactin-related protein in decidua and trophoblast tissues during pregnancy in rats.

Decidual prolactin-related protein (dPRP) is a member of the prolactin gene family and is abundantly expressed in the rat deciduum. Previously, dPRP was shown to associate with heparin-containing molecules and was found to reside, at least in part, within the decidual extracellular matrix, where it was postulated to influence decidual cells and other cell types. The purpose of this investigation was to identify the cellular origin and the temporal and regional characteristics of dPRP expression in the rat uterus during pregnancy. Protein expression was evaluated by Western blot analysis, immunoprecipitation, and immunocytochemistry; dPRP mRNA expression was assessed by reverse transcriptase polymerase chain reaction and in situ hybridization. Decidual PRP was first detected at Day 6 of pregnancy or pseudopregnancy. Expression increased with the growth of the deciduum and then declined coincident with regression of decidual tissue. Throughout the first half of pregnancy or pseudo-pregnancy, dPRP and mRNA were predominantly localized to the antimesometrial deciduum of the developing conceptus. During the second half of gestation, expression also appeared in the chorioallantoic placenta. Trophoblast giant cells and spongiotrophoblast cells within the junctional zone of the chorioallantoic placenta expressed dPRP, as did the Rcho-1 trophoblast cell line. In conclusion, dPRP production is elevated from implantation until parturition through the participation of decidual (early pregnancy) and trophoblastic (late pregnancy) tissues.

Decidual prolactin-related protein: heterologous expression and characterization.

As a first step in understanding the role of decidual PRL-related protein (dPRP) during pregnancy, we have generated recombinant dPRP protein. In this report, we present data on the generation, purification, and characterization of recombinant dPRP protein. The dPRP complementary DNA was subcloned into the pMSXND vector, and the vector was transfected into Chinese hamster ovary (CHO) cells by electroporation. After appropriate selection, amplification, and induction procedures, recombinant dPRP was purified from conditioned medium of the CHO-dPRP cells using ultrafiltration, size-exclusion chromatography, and reverse phase HPLC. Recombinant dPRP was found to possess electrophoretic mobility, immunoreactivity, and N-terminal amino acid sequence identical to those of dPRP isolated from decidual tissue. Polyclonal antibodies were generated to the recombinant dPRP and used for Western blot analysis. dPRP is capable of binding heparin, and a significant fraction of synthesized dPRP resides within the decidual extracellular matrix. Recombinant dPRP failed to bind to PRL receptors and showed no stimulatory activity in the PRL-dependent rat Nb2 lymphoma cell proliferation assay. Additional studies have shown that heterologous expression of dPRP in CHO cells significantly increased the ability of CHO cells to form tumors in athymic mice. In conclusion, recombinant dPRP possesses characteristics similar to those of dPRP of decidual origin and is a heparin-binding protein that may facilitate the establishment of pregnancy.

Decidual prolactin-related protein: heterologous expression and characterization

As a first step in understanding the role of decidual PRL-related protein (dPRP) during pregnancy, we have generated recombinant dPRP protein. In this report, we present data on the generation, purification, and characterization of recombinant dPRP protein. The dPRP complementary DNA was subcloned into the pMSXND vector, and the vector was transfected into Chinese hamster ovary (CHO) cells by electroporation. After appropriate selection, amplification, and induction procedures, recombinant dPRP was purified from conditioned medium of the CHO-dPRP cells using ultrafiltration, size-exclusion chromatography, and reverse phase HPLC. Recombinant dPRP was found to possess electrophoretic mobility, immunoreactivity, and N-terminal amino acid sequence identical to those of dPRP isolated from decidual tissue. Polyclonal antibodies were generated to the recombinant dPRP and used for Western blot analysis. dPRP is capable of binding heparin, and a significant fraction of synthesized dPRP resides within the decidual extracellular matrix. Recombinant dPRP failed to bind to PRL receptors and showed no stimulatory activity in the PRL-dependent rat Nb2 lymphoma cell proliferation assay. Additional studies have shown that heterologous expression of dPRP in CHO cells significantly increased the ability of CHO cells to form tumors in athymic mice. In conclusion, recombinant dPRP possesses characteristics similar to those of dPRP of decidual origin and is a heparin-binding protein that may facilitate the establishment of pregnancy.

Isolation, culture, and characterization of the two cell subpopulations forming the rat decidua: differential gene expression for activin, follistatin, and decidual prolactin-related protein.

The decidual tissue of the pregnant rat is formed primarily by two markedly different decidual cell populations located in the mesometrial and antimesometrial sites of the uterus. The antimesometrial decidua functions as an endocrine organ, secreting PRL-like hormones and expressing activin and follistatin. In contrast, the mesometrial decidua has no apparent endocrine activity, but secretes abundant alpha 2-macroglobulin. To determine whether the profound difference in morphology and gene expression is inherent to each cell population or whether it results from the position of these cells in the uterus and their subjection to regionally restricted information, we isolated the giant antimesometrial decidual cells from the small mesometrial decidual cells by elutriation. We examined, using Northern and reverse transcription-polymerase chain reaction analysis, the expression of PRL-related protein (DPRP), follistatin, activin, and alpha 2-macroglobulin in the two cell populations just after cell separation and after primary culture. We also examined, using coculture, the possible cross-talk between the two cell populations. In culture, the cells maintained the morphological appearance seen in the decidua; the mesometrial cells remained less differentiated, whereas the antimesometrial cells formed giant cells. In addition, the antimesometrial cells, which have been shown previously to be the only site of DPRP messenger RNA (mRNA) expression, remained, after several days of culture, the only cells able to express this gene. The coculture experiment suggested that the absence of DPRP mRNA in the mesometrial cells is not due to inhibitory signals from the antimesometrial cells, but, rather, to the inherent inability of the mesometrial decidual cells to express the DPRP gene. The cellular expression pattern of follistatin was also not changed by cell separation. However, in sharp contrast to that of DPRP and follistatin, the cell-specific expression of activin-beta A and alpha 2-macroglobulin was markedly affected by cell separation. A remarkable switch in activin-beta A cell expression was observed when mesometrial cells were separated from antimesometrial cells and maintained in culture. Activin-beta A mRNA, which was barely detectable in these cells in situ, became expressed in higher levels than in antimesometrial cells. The isolated antimesometrial cells also acquired the ability to express the alpha 2-macroglobulin gene, whose expression is highly restricted to the mesometrial cells in vivo. No down-regulation of antimesometrial alpha 2-macroglobulin mRNA could be induced by mesometrial cells in a coculture experiment. However, alpha 2-macroglobulin mRNA levels in mesometrial cells were clearly up-regulated by antimesometrial cell signal(s).(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation, culture, and characterization of the two cell subpopulations forming the rat decidua: differential gene expression for activin, follistatin, and decidual prolactin-related protein

The decidual tissue of the pregnant rat is formed primarily by two markedly different decidual cell populations located in the mesometrial and antimesometrial sites of the uterus. The antimesometrial decidua functions as an endocrine organ, secreting PRL-like hormones and expressing activin and follistatin. In contrast, the mesometrial decidua has no apparent endocrine activity, but secretes abundant alpha 2-macroglobulin. To determine whether the profound difference in morphology and gene expression is inherent to each cell population or whether it results from the position of these cells in the uterus and their subjection to regionally restricted information, we isolated the giant antimesometrial decidual cells from the small mesometrial decidual cells by elutriation. We examined, using Northern and reverse transcription-polymerase chain reaction analysis, the expression of PRL-related protein (DPRP), follistatin, activin, and alpha 2-macroglobulin in the two cell populations just after cell separation and after primary culture. We also examined, using coculture, the possible cross-talk between the two cell populations. In culture, the cells maintained the morphological appearance seen in the decidua; the mesometrial cells remained less differentiated, whereas the antimesometrial cells formed giant cells. In addition, the antimesometrial cells, which have been shown previously to be the only site of DPRP messenger RNA (mRNA) expression, remained, after several days of culture, the only cells able to express this gene. The coculture experiment suggested that the absence of DPRP mRNA in the mesometrial cells is not due to inhibitory signals from the antimesometrial cells, but, rather, to the inherent inability of the mesometrial decidual cells to express the DPRP gene. The cellular expression pattern of follistatin was also not changed by cell separation. However, in sharp contrast to that of DPRP and follistatin, the cell-specific expression of activin-beta A and alpha 2-macroglobulin was markedly affected by cell separation. A remarkable switch in activin-beta A cell expression was observed when mesometrial cells were separated from antimesometrial cells and maintained in culture. Activin-beta A mRNA, which was barely detectable in these cells in situ, became expressed in higher levels than in antimesometrial cells. The isolated antimesometrial cells also acquired the ability to express the alpha 2-macroglobulin gene, whose expression is highly restricted to the mesometrial cells in vivo. No down-regulation of antimesometrial alpha 2-macroglobulin mRNA could be induced by mesometrial cells in a coculture experiment. However, alpha 2-macroglobulin mRNA levels in mesometrial cells were clearly up-regulated by antimesometrial cell signal(s).

Expression of decidual prolactin-related protein in the rat decidua.

It is well established that rat decidual tissue produces a PRL-like hormone(s) that binds to the PRL receptor on both the corpus luteum and the decidual cells and initiates profound changes in the endocrine milieu required for the establishment of pregnancy. The recent cloning of a decidual PRL-related protein (dPRP) prompted us 1) to determine whether the expression of this gene is triggered by decidualization of the endometrial stromal cells, 2) to examine the temporal and cell-specific pattern of its expression, and 3) to examine the role of both decidual signals and PRL on levels of its messenger RNA (mRNA). Total RNA was isolated from uteri of either nonpseudopregnant rats or pseudopregnant rats with or without decidual tissue. A 1-kilobase mRNA species hybridizing strongly with the dPRP probe was present in decidualized uteri. No dPRP mRNA could be detected in uteri not subjected to decidualization. Developmental studies indicated a constant high level of dPRP mRNA in the decidual tissue until day 12 of pseudopregnancy, followed by a marked decline at a time when extensive cell death occurs in the decidua, suggesting that dPRP is constitutively expressed in this tissue. To examine the cell-specific expression of dPRP, antimesometrial decidua was separated from mesometrial decidua, and the large antimesometrial cell population was separated from the small mesometrial cells by elutriation. The results of Northern analysis revealed clearly that dPRP is abundantly and solely expressed in the large antimesometrial cells. No dPRP mRNA could be detected in the mesometrial cells and in numerous other endocrine and nonendocrine tissues. A faint signal was observed, however, in the trophoblast. Despite the very strong paracrine regulation between the antimesometrial and mesometrial cells and the high levels of PRL receptor expression in these cells, both in vivo and coculture experiments revealed no regulation of dPRP gene expression by either PRL or mesometrial cell signal, adding further support to the possibility that once induced, dPRP remains constitutively expressed. In summary, the results of this investigation revealed that the expression of dPRP in endometrial stromal cells is triggered by the induction of decidualization and that this gene is selectively and abundantly expressed in a defined cell population located in the anti-mesometrial region of the uterus. Thus, dPRP is not only a useful indicator of decidualization, but is also an excellent marker for the differentiated antimesometrial cells.