Early Pregnancy In Mice Research Articles

Progesterone and DNA Damage Encourage Uterine Cell Proliferation and Decidualization through Up-regulating Ribonucleotide Reductase 2 Expression during Early Pregnancy in Mice

Embryo implantation into the maternal uterus is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormone-dependent decidualization, which is characterized by stromal cell proliferation and differentiation. The mechanisms underlying steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood. Ribonucleotide reductase, consisting of two subunits (RRM1 and RRM2), is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis and plays an important role in cell proliferation and tumorgenicity. Based on our microarray analysis, Rrm2 expression was significantly higher at implantation sites compared with interimplantation sites in mouse uterus. However, the expression, regulation, and function of RRM2 in mouse uterus during embryo implantation and decidualization are still unknown. Here we show that although both RRM1 and RRM2 expression are markedly induced in mouse uterine stromal cells undergoing decidualization, only RRM2 is regulated by progesterone, a key regulator of decidualization. Further studies showed that the induction of progesterone on RRM2 expression in stromal cells is mediated by the AKT/c-MYC pathway. RRM2 can also be induced by replication stress and DNA damage during decidualization through the ATR/ATM-CHK1-E2F1 pathway. The weight of implantation sites and deciduoma was effectively reduced by specific inhibitors for RRM2. The expression of decidual/trophoblast prolactin-related protein (Dtprp), a reliable marker for decidualization in mice, was significantly reduced in deciduoma and steroid-induced decidual cells after HU treatment. Therefore, RRM2 may be an important effector of progesterone signaling to induce cell proliferation and decidualization in mouse uterus.

Regulation of Maternal Blood Pressure by the Conceptus During Early Pregnancy

Pregnancy has a profound impact on the homeostatic regulation of maternal systemic arterial pressure in humans [1, 2]. During the first trimester, prior to substantial blood flow through the placenta [3], there is a decrease in systemic vascular resistance likely caused by systemic vasodilation. This is accompanied by an increase in both plasma volume and cardiac output, but these are not sufficient to maintain maternal arterial pressure. The net result is a characteristic decrease in maternal mean arterial pressure through mid-gestation of pregnancy. Several mechanisms have been suggested for these cardiovascular changes, including modulation of the reninangiotensin system and increased nitric oxide production [1]. However, complete understanding of the mechanisms involved remains to be determined. Elucidation of these mechanisms may be crucial to understanding several important complications of human pregnancy such as preeclampsia, a condition clinically defined by elevated maternal blood pressure and proteinuria, associated with maternal arterial stiffness [4]. Many human obstetrical complications such as preeclampsia become clinically evident only later in gestation but are likely the result of abnormalities that occur early in gestation. Clearly, animal studies are critical to determine the mechanisms responsible for these cardiovascular changes, as it is impossible to adequately address causality directly in humans for obvious ethical reasons. In a fashion similar to that of humans, mice also exhibit the characteristic decrease in maternal arterial pressure after the onset of implantation through to mid-gestation [5–9]. This is the period of time when major changes occur in the uterus and the developing placenta but before there is much placental blood flow. Initial studies evaluated the potential role of uterine natural killer (uNK) cell-driven spiral artery modification in mediating the drop in mean arterial pressure in early pregnant mice [10]. This work suggested that uNK cells have the potential to take part in the decrease in maternal mean arterial pressure leading to mid-gestation. However, studies by Barrette et al. [11] demonstrated that the decline in blood pressure occurred normally in Rag2 / cc / mice; these mice lack uNK cells and, thus, the characteristic spiral artery modification of pregnancy. These findings suggested that conceptus-derived signals are required. Work published in the current issue of Biology of Reproduction by Barrette et al. [11] sought to answer the important question of whether the decrease in maternal mean arterial pressure occurs in uteri undergoing artificially induced decidualization in the absence of the conceptus. The authors used radiotelemetry and a model in which blastocyst-sized concanavalin A-coated agarose beads were used as a deciduogenic stimulus. This method of inducing a deciduoma has previously been shown to be more physiologically relevant than other generally used artificial stimuli [12]. Surprisingly, Barrette el al. [11] found that the decrease in maternal mean arterial pressure leading up to midpseudopregnancy does not occur in the deciduoma-bearing mice lacking a conceptus. This provides strong evidence supporting the hypothesis that signals from the conceptus initiate the decrease in maternal arterial pressure through midgestation in mice. The work of Barrette et al. [11] further provides an important proof-of-principle that conceptus-derived factors play a key role in driving maternal cardiovascular changes during early pregnancy. Because they are in direct contact with the mother, the source of the signals involved may be trophoblast cells. Indeed, mouse trophoblasts [13, 14], like human trophoblasts [15], are known to produce many vasoactive substances which could act systemically on maternal vessels. However, one cannot rule out the possibility that nonvasoactive factors from the conceptus may also regulate downstream maternally expressed vasoactive substances either in a paracrine or endocrine fashion. An exciting possibility brought up by Barrette et al. [11] is that microparticles and nanoparticles shed by trophoblasts may be involved. Microparticles are produced by human placentae during normal pregnancy, and production is generally thought to increase in diseases such as preeclampsia [16]. Depending upon their contents, microparticles can either augment or inhibit vascular functions [17]. Although demonstrated in humans, the presence of trophoblast microparticles in the maternal circulation or uterus have yet to be demonstrated during early pregnancy in mice. Presently, more work is required to determine the precise identity, function, source, and regulation of conceptus-derived factors involved in the decrease in maternal arterial pressure during early pregnancy.

Decidual spiral artery remodeling during early post-implantation period in mice: Investigation of associations with decidual uNK cells and invasive trophoblast

Circumferential remodeling of spiral arteries (SAs) during pregnancy is crucial for regulating maternal blood flow into the placenta and clinically important. However its mechanism is still ill defined in humans and mice. In mice, several important aspects of decidual SA remodeling (SAR) remain unexplored and were addressed here using morphometrics by examining SAs within the mesometrial half of the decidua basalis between embryonic day 6.5 (E6.5) and midgestation (E10.5). The data presented here provide evidence that supports the following novel conclusions about SAR: (a) SAs (defined by their muscular walls) appear between E6.5 and E7.5, undergo ‘outward hypertrophic’ SAR (SA lumen widening with muscular wall thickening) during the E7.5–E8.5 and E9.5–E10.5 periods, and ‘outward hypotrophic’ SAR (SA lumen widening with muscular wall thinning) during the E8.5–E9.5 interval. (b) ‘Outward hypotrophic’ SAR is associated with decreases in the overall number, but not density, of SA media nuclei, suggesting loss of SA muscular wall cells. Proximity of placenta-derived invasive trophoblast to SAs appears not be involved in SAR, as these cells were undetectable in the mesometrial region of decidua basalis throughout the E6.5–E10.5 period. Although the maternally derived lymphocytes of the decidual uterine natural killer (uNK) cell type are required for decidual SAR, the timing of this, the uNK cell parameter involved and the type of SAR they influence have not been adequately explored. Evidence is presented here that not all decidual SAR during this period is uNK cell-dependent. Rather, the data suggest that uNKs only influence ‘outward hypotrophic’ SAR during the E8.5–E9.5 period. Evidence is presented that the uNK cell parameter involved is the attainment of a certain maturation state (based on uNK cell size) by SA wall uNKs of the Dolichos biflorus agglutinin (DBA) lectin-positive uNK cell subset. This work also suggests that the previously shown loss of contractile mural cell character from the SA wall does not depend on either uNKs or trophoblast proximity. The novel implications of the present data for early mouse pregnancy are discussed.

Progesterone stimulates expression of follistatin splice variants Fst288 and Fst315 in the mouse uterus

Follistatin, an inhibitor of activin A, has key regulatory roles in the female reproductive tract. Follistatin has two splice variants: FST288, largely associated with cell surfaces, and FST315, the predominant circulating form. The mechanism regulating uterine expression of these variants is unknown. Quantitative RT-PCR was used to measure expression of follistatin splice variants (Fst288, Fst315), the activin βA subunit (Inhba) and the inhibin α subunit (Inha) in uterine tissues during early pregnancy (days 1–4, preimplantation) and in response to exogenous 17β-oestradiol (single s.c. injection) and progesterone (three daily s.c. injections) in ovariectomized mice. Uterine Fst288, Fst315 and Inhba expression increased during early pregnancy, with greater increases in Fst315 relative to Fst288 suggesting differential regulation of these variants. Fst288, Fst315, Inhba and Inha all increased in response to progesterone treatment. Fst288, but not Fst315, mRNA decreased in response to 17β-oestradiol treatment, whereas Inhba increased. A comparison of the absolute concentrations of uterine follistatin mRNA using crossing thresholds indicated that both variants were more highly expressed in early pregnancy in contrast to the hormone treatment models. It is concluded that progesterone regulates uterine expression of both follistatin variants, as well as activin A, during early pregnancy in the mouse uterus.Follistatin and activin are proteins with important functions in female reproduction. There are two forms of follistatin (FST288 and FST315), which interact with activin in different ways and subsequently may have different functions. We are interested in the function of these two forms of follistatin in the uterus. In this study, we examined how the expression of the two follistatin variants and activin changed in the uterus during early pregnancy in mice, as well as in response to the female hormones oestrogen and progesterone. We saw an increase in the expression of the genes for FST288, FST315 and activin in the uterus during early pregnancy. We also saw an increase in these proteins in the mouse uterus in response to progesterone treatment. We concluded that the hormone progesterone regulates expression of both follistatin variants, as well as activin, during early pregnancy in the mouse uterus.

The source of leptin, but not leptin depletion in response to food restriction, changes during early pregnancy in mice

Maternal food restriction during pregnancy results in adverse consequences for offspring, including obesity and cardiovascular disease. Early pregnancy is a critical period for this programming effect. Leptin is a regulator of energy homeostasis that also affects placental and fetal development. As food restriction results in decreased serum leptin levels, at least in non-pregnant animals, leptin depletion may be one mechanism by which food restriction affects development. The objective of this study was to test whether moderate food restriction affects serum leptin concentrations during the first half of pregnancy. We found that restriction to 50% of ad libitum consumption levels resulted in a significant decrease in serum leptin concentrations in both pregnant and non-pregnant female mice. There was no significant difference in serum leptin concentrations between non-pregnant females and at pregnancy day 11.5 when fed ad libitum. However, there was a difference in the source of leptin during pregnancy, with greater production in visceral fat in pregnant mice, and greater production in subcutaneous fat in non-pregnant mice. Leptin concentrations were dependent on time of day and time of sampling relative to feeding, particularly in restricted mice. There was a significant difference in serum leptin concentrations between fed and restricted mice when they were fed and sampled in afternoon, but not when they were fed and sampled in morning. We conclude that food restriction results in a significant decrease in leptin concentration during the first half of pregnancy in mice, but that detection of this relationship is subject to experimental design considerations.

Differential Changes in the Hypothalamic‐Pituitary‐Adrenal Axis and Prolactin Responses to Stress in Early Pregnant Mice

Stress can cause pregnancy failure but it is unclear how the mother's neuroendocrine system responds to stress to impair mechanisms establishing implantation. We analysed stress-evoked hypothalamic-pituitary-adrenal (HPA) axis responses in early pregnant mice. HPA axis secretory responses to immune stress in early-mid pregnancy were strong and similar to that in virgins, although activation of hypothalamic vasopressin neurones, rather than corticotrophin-releasing hormone neurones, may be more important in the stress response in pregnancy. The site and mode of detrimental glucocorticoid action in pregnancy is not established. Because circulating prolactin is important for progesterone secretion and pregnancy establishment, we also hypothesised that stress negatively impacts on prolactin and its neuroendocrine control systems in early pregnant mice. Basal prolactin secretion was profoundly inhibited by either immune or fasting stress in early pregnancy. Prolactin release is inhibited by tonic dopamine release from tuberoinfundibular (TIDA) neurones. However, immune stress did not increase TIDA neurone activity in the median eminence in pregnant mice [measured by 3,4-dihydroxyphenylacetic acid (DOPAC) content and the DOPAC:dopamine ratio]. By contrast, both immune stress and fasting caused weak induction of Fos in TIDA neurones. However, Fos induction does not always reflect dopamine secretion. Taken together, the data suggest that the stress-evoked profound reduction in prolactin secretion does not involve substantially increased dopamine activity as anticipated. In pregnancy, there was also attenuated recruitment of parvocellular paraventricular nucleus neurones and increased activation of brainstem noradrenergic nuclei after immune stress, indicating that other mechanisms may be involved in the suppression of prolactin secretion. In summary, low prolactin and increased circulating glucocorticoids together may partly explain how a mother's endocrine system mediates stress-induced pregnancy failure.

Prenatal exposure to lipopolysaccharide results in neurodevelopmental damage that is ameliorated by zinc in mice

There is converging evidence that during pregnancy a maternal immune response to infection can cause neurodevelopmental damage. Lipopolysaccharide (LPS)-mediated induction of metallothionein (MT) and subsequent hypozincaemia has been linked to fetal brain damage. Our group has demonstrated that Zn, when co-administered with LPS in early pregnancy in mice (gestation day (GD) 8), prevents fetal malformations and neurodevelopmental deficits in offspring. Others demonstrating fetal brain lesions have administered LPS much later in gestation (after GD 16), when the influence of LPS-mediated MT-induction on maternal plasma Zn levels, and the effect of Zn co-administration with LPS, are unknown. The aims of this study are firstly to examine whether LPS causes MT induction and maternal hypozincaemia in mid-to-late pregnancy, and secondly to determine if histochemical markers of inflammatory damage in fetal brain are affected by LPS and whether this damage can be alleviated with Zn treatment. Pregnant mice were injected with LPS (5 mg/kgbodywt.) or saline vehicle on GD 16 and then humanely killed at 8, 16 and 24 h for Zn and MT measurements, or concomitantly injected subcutaneously with Zn (2 mg/kgbodywt.) or saline and then killed on GD 18 and immunohistochemistry performed on fetal brain. Maternal hepatic MT was markedly induced after LPS-challenge and this was associated with a 38% reduction in maternal plasma Zn concentrations. Coincidentally, the fetuses of LPS-treated dams showed astrogliosis, extensive cell death and an increased number of cells producing TNF-α which was prevented with concomitant Zn treatment. These results support the premise that in mid-to-late pregnancy, an infection-mediated activation of a maternal immune response can cause MT induction that redistributes Zn in the mother, restricting fetal Zn supply, causing neurodevelopmental damage.

Localization of chondromodulin-I at the feto-maternal interface and its inhibitory actions on trophoblast invasion in vitro

BackgroundChondromodulin-I (ChM-I) is an anti-angiogenic glycoprotein that is specifically localized at the extracellular matrix of the avascular mesenchyme including cartilage and cardiac valves. In this study, we characterized the expression pattern of ChM-I during early pregnancy in mice in vivo and its effect on invasion of trophoblastic cells into Matrigel in vitro.ResultsNorthern blot analysis clearly indicated that ChM-I transcripts were expressed in the pregnant mouse uterus at 6.5-9.5 days post coitum. In situ hybridization and immunohistochemistry revealed that ChM-I was localized to the mature decidua surrounding the matrix metalloproteinase-9 (MMP-9)-expressing trophoblasts. Consistent with this observation, the expression of ChM-I mRNA was induced in decidualizing endometrial stromal cells in vitro, in response to estradiol and progesterone. Recombinant human ChM-I (rhChM-I) markedly inhibited the invasion through Matrigel as well as the chemotactic migration of rat Rcho-1 trophoblast cells in a manner independent of MMP activation.ConclusionsThis study demonstrates the inhibitory action of ChM-I on trophoblast migration and invasion, implying the potential role of the ChM-I expression in decidual cells for the regulated tissue remodeling and angiogenesis at feto-maternal interface.

Cervical Softening During Pregnancy: Regulated Changes in Collagen Cross-Linking and Composition of Matricellular Proteins in the Mouse1

A greater understanding of the parturition process is essential in the prevention of preterm birth, which occurs in 12.7% of infants born in the United States annually. Cervical remodeling is a critical component of this process. Beginning early in pregnancy, remodeling requires cumulative, progressive changes in the cervical extracellular matrix (ECM) that result in reorganization of collagen fibril structure with a gradual loss of tensile strength. In the current study, we undertook a detailed biochemical analysis of factors in the cervix that modulate collagen structure during early mouse pregnancy, including expression of proteins involved in processing of procollagen, assembly of collagen fibrils, cross-link formation, and deposition of collagen in the ECM. Changes in these factors correlated with changes in the types of collagen cross-links formed and packing of collagen fibrils as measured by electron microscopy. Early in pregnancy there is a decline in expression of two matricellular proteins, thrombospondin 2 and tenascin C, as well as a decline in expression of lysyl hydroxylase, which is involved in cross-link formation. These changes are accompanied by a decline in both HP and LP cross-links by gestation Days 12 and 14, respectively, as well as a progressive increase in collagen fibril diameter. In contrast, collagen abundance remains constant over the course of pregnancy. We conclude that early changes in tensile strength during cervical softening result in part from changes in the number and type of collagen cross-links and are associated with a decline in expression of two matricellular proteins thrombospondin 2 and tenascin C.

Effect of Sympathetic Nerves on Proliferation of Splenic Lymphocytes and Antioxidant Function of Maternal Spleen in Early Pregnant Mice

Sympathetic regulation plays an important role in fetal survival and development during early pregnancy. Maternal adaptations to pregnancy may involve changes of the spleen in its structure, size, and function. This study was therefore designed to investigate the effects of sympathetic nerves on these adaptations in the maternal spleen of mice during early pregnancy. The adult female mice were intraperitoneally injected with neurotoxin 6-hydroxydopamine (6-OHDA) for 5 consecutive days to selectively destroy the sympathetic nerves. The results were as follows: (1) the splenic weight was reduced in the 6-OHDA group by 10.9%-13.0% at E5-E9 (P < 0.05) when compared with the control group. (2) The splenic nodule and periarterial lymphatic sheath of the 6-OHDA-treated mice were smaller than those of control mice. The proliferating cell nuclear antigen positive cells of 6-OHDA-treated group were decreased by 10.9%-16.2% (P < 0.05) at E1-E9. (3) Lymphocyte proliferation indices in response to concanavalin A or lipopolysaccharide were significantly decreased (P < 0.05) in the 6-OHDA group. (4) When compared with control mice, the superoxide dismutase and glutathione peroxidase activities of 6-OHDA-treated mice were decreased by 14.3%-21.9% (P < 0.05) at E1-E9 and 17.4%-25.0% (P < 0.05) at E3-E9, respectively. In contrast, the malondialdehyde content of 6-OHDA group was increased by 10.6%-38.6% (P < 0.05) at E3-E9. The results demonstrated the regulation of pregnancy-dependent adaptations in the spleen through the sympathetic nerve activity.

Steroid Hormone-regulated let-7b Mediates Cell Proliferation and Basigin Expression in the Mouse Endometrium

Let-7b, one of the let-7 family members, was studied for its regulative role in endometrial cells during early pregnancy in mice. According to real-time RT-PCR analysis, the expression of let-7b in epithelial cells increased gradually from day 1 to day 4 of preimplantation stages and reached the highest level on day 4. On the other hand, the highest level of let-7b in stromal cells was observed on day 1, although the expression was decreased on day 2 and increased significantly on day 4. By in situ hybridization, let-7b was also found to express in uteri during days 6-8 of pregnancy. Endometrial cells isolated from prepubertal mice were treated with steroid hormones, progesterone (P4), estradiol (E2) and P4 plus E2. After 96 h of culture in the presence of steroid hormones, the expression levels of let-7b were increased in the endometrial cells, although significant differences were only observed after P4 treatment in stromal cells and after individual E2 and P4 treatments in the epithelial cells. In association with the increased let-7b expression, the cell proliferation slope, measured by a MTT assay, significantly decreased in the presence of P4 and P4 plus E2 compared with the nonhormone and E2 treatment groups during 72-108 h of culture. Furthermore, results from transfection of let-7b into stromal cells isolated from day 4 pregnant mice or prepubertal mice demonstrated that let-7b attenuated the proliferation during the periods of time examined. After transfection of let-7b into mouse stromal cells isolated from day 7 of pregnancy, the expression of Basigin (Bsg), a matrix metalloproteinase (MMP) inducer, was suppressed, as well as that of MMP-9. In conclusion, this study clarifies the expression pattern of let-7b in uterine epithelial and stromal cells during preimplantation stages in mice, as well as the inhibitory effect of let-7b associated with steroid hormones on stromal cell proliferation and on the expression of MMP-9.

Modulation of macrophage inflammatory profile in pregnant nonobese diabetic (NOD) mice

During normal early pregnancy circulating monocytes are recruited to the maternal–placental interface where they differentiate to macrophages expressing different functional phenotypes for the maintenance of tissue homeostasis. Pregnancy in the nonobese diabetic (NOD) mouse model presents some pathological features in the pre-diabetic stage. The aim of this work was to analyze the functional profile of peritoneal macrophages faced with inflammatory and phagocytic stimuli in early pregnant pre-diabetic NOD mice and their modulation by vasoactive intestinal peptide (VIP). Pregnant NOD mouse macrophages showed no basal NFκB activation, lower IL-12 and nitrites production compared with the macrophages from non-pregnant NOD mice. Their pro-inflammatory aberrant response to LPS and apoptotic cell challenge was reduced and VIP inhibited macrophage residual deleterious responses to apoptotic cells. A functional phenotype switch in macrophages during pregnancy in NOD mice and a promoting effect of VIP towards this regulatory phenotype would be in line with the central role of macrophages in the maternal–placental dialogue.

Death effector domain–containing protein (DEDD) is required for uterine decidualization during early pregnancy in mice

During intrauterine life, the mammalian embryo survives via its physical connection to the mother. The uterine decidua, which differentiates from stromal cells after implantation in a process known as decidualization, plays essential roles in supporting embryonic growth before establishment of the placenta. Here we show that female mice lacking death effector domain-containing protein (DEDD) are infertile owing to unsuccessful decidualization. In uteri of Dedd-/- mice, development of the decidual zone and the surrounding edema after embryonic implantation was defective. This was subsequently accompanied by disintegration of implantation site structure, leading to embryonic death before placentation. Polyploidization, a hallmark of mature decidual cells, was attenuated in DEDD-deficient cells during decidualization. Such inefficient decidualization appeared to be caused by decreased Akt levels, since polyploidization was restored in DEDD-deficient decidual cells by overexpression of Akt. In addition, we showed that DEDD associates with and stabilizes cyclin D3, an important element in polyploidization, and that overexpression of cyclin D3 in DEDD-deficient cells improved polyploidization. These results indicate that DEDD is indispensable for the establishment of an adequate uterine environment to support early pregnancy in mice.

GM-CSF Is an Essential Regulator of T Cell Activation Competence in Uterine Dendritic Cells during Early Pregnancy in Mice

Uterine dendritic cells (DCs) are critical for activating the T cell response mediating maternal immune tolerance of the semiallogeneic fetus. GM-CSF (CSF2), a known regulator of DCs, is synthesized by uterine epithelial cells during induction of tolerance in early pregnancy. To investigate the role of GM-CSF in regulating uterine DCs and macrophages, Csf2-null mutant and wild-type mice were evaluated at estrus, and in the periconceptual and peri-implantation periods. Immunohistochemistry showed no effect of GM-CSF deficiency on numbers of uterine CD11c(+) cells and F4/80(+) macrophages at estrus or on days 0.5 and 3.5 postcoitum, but MHC class II(+) and class A scavenger receptor(+) cells were fewer. Flow cytometry revealed reduced CD80 and CD86 expression by uterine CD11c(+) cells and reduced MHC class II in both CD11c(+) and F4/80(+) cells from GM-CSF-deficient mice. CD80 and CD86 were induced in Csf2(-/-) uterine CD11c(+) cells by culture with GM-CSF. Substantially reduced ability to activate both CD4(+) and CD8(+) T cells in vivo was evident after delivery of OVA Ag by mating with Act-mOVA males or transcervical administration of OVA peptides. This study shows that GM-CSF regulates the efficiency with which uterine DCs and macrophages activate T cells, and it is essential for optimal MHC class II- and class I-mediated indirect presentation of reproductive Ags. Insufficient GM-CSF may impair generation of T cell-mediated immune tolerance at the outset of pregnancy and may contribute to the altered DC profile and dysregulated T cell tolerance evident in infertility, miscarriage, and preeclampsia.

Proline-Rich Acidic Protein 1 (PRAP1) as a Marker for Established Uterine Receptivity.

The proline-rich acidic protein 1 (PRAP1) is mainly expressed in the epithelial cells and has been suggested to play an important role in maintaining normal growth homeostasis in epithelial cells. We have examined the expression and regulation of PRAP1 in mouse uterus. In situ hybridization indicates that PRAP1 mRNA is exclusively expressed in the uterine luminal epithelium (LE). In ovariectomized uterus, PRAP1 expression levels can be upregulated by 17β-estradiol treatment and downregulated by progesterone treatment. PRAP1 mRNA has unique temporal expression pattern in the early pregnant mouse uterus. In normal wild type uterus, PRAP1 mRNA is strongly detected in day 0.5 LE (mating night as day 0), disappears in pre-implantation day 3.5 LE, and then is intensively expressed in day 4.5 LE after implantation has initiated. These data indicate that the temporal expression of PRAP1 is associated with the establishment of uterine receptivity. This indication has been further demonstrated in experimentally-induced delayed implantation wild type mouse uterus and Lpar3(-/-) mouse uterus with delayed implantation. Indeed, PRAP1 mRNA is undetectable in the non-receptive uterus in both delayed implantation mouse models. These results suggest that PRAP1 may serve as a marker for the established uterine receptivity. However, the function of PRAP1 in the LE has not been established. (Supported by startup funding from The University of Georgia and funding from the Interdisciplinary Toxicology Program at The University of Georgia.) (poster)

A preliminary report on the toxicity of arecoline on early pregnancy in mice

Areca nut (Areca catechu) is chewed by roughly 10% of the world population, including India, Taiwan and parts of China. Lower mean birth-weight and higher neonatal jaundice were reported in the babies of betel chewing pregnant women in 1982. Although areca nut chewing during pregnancy has been associated with lower birth weight and premature delivery, the mechanism of such complications is not entirely understood. A possible contributor, arecoline, a major alkaloid in the areca nut, has been reported to be cytotoxic and genotoxic. To determine the influence of arecoline on reproduction, we study the effects of arecoline on embryos during peri-implantation stages in mice. Mice consuming varying dosages of arecoline were checked for their ability to successfully produce viable embryos. In addition, trophoblast outgrowth from mice blastocysts was evaluated the survival status of the embryos. Our investigation revealed that arecoline decreased the number of implanted embryos in early pregnant mice. In addition, trophoblast outgrowth expansion of blastocysts was also inhibited by arecoline. These observations suggest that arecoline is toxic to mouse embryos as early as peri-implantation. Improved understanding of the effects of arecoline during embryogenesis may help to establish public health policies and to develop potential treatments for such patients.

Arnebia preventing the expression of Muc1 protein decrease results in anti-implantation in early pregnant mice

Background The study was designed to explore the relationship between the anti-implantation activity of arnebia and the expression of Muc1 protein in the endometrium of early pregnant mice. Study design The aqueous extract of arnebia was administered to mice on Days 1–4 postcoitum, and the mice were sacrificed to asses the implantation rate on Day 8 postcoitum. On the night of Day 4 postcoitum and on the morning of Day 5 postcoitum, the treated mice were sacrificed to study the influence on endometrium. The extract of arnebia was administered to mice on Days 11–14 postcoitum, and the mice were sacrificed to asses abortion on Day 18 postcoitum. The reversible effect of arnebia on mice was also studied. Results The endometrium in the experimental group exhibited morphological changes compared with that in the control group. The expression of Muc1 protein was increased with the increasing doses of arnebia, while in control group, it increased a little. The experiments of pseudopregnancy and normal mice show the identical expression of Muc1. The anti-implantation effect of arnebia was reversible. Conclusion The arnebia may prevent embryo implantation by inhibiting the decrease of the Muc1 protein.

Uterine epithelial cell fucosylated structures are regulated by macrophages during early pregnancy in mouse

Uterine epithelial cell fucosylated structures are regulated by macrophages during early pregnancy in mouse

Differential expression of vascular endothelial growth factor-A isoforms in the mouse uterus during early pregnancy

While vascular endothelial growth factor (VEGF)-A mediates endometrial vascular remodelling during early pregnancy in mice, individual VEGF-A isoforms have not been investigated, despite their different biological properties. Using mice as a model, the expression of VEGF-A isoforms and receptors in the mouse uterus during early pregnancy was quantified. It was postulated that selected isoform expression would increase concurrent with increased endometrial endothelial cell proliferation at this time. Uteri were collected on days 1-5 of pregnancy and mRNA expression was quantified by quantitative reverse-transcription polymerase chain reaction, VEGF-A protein by Western blot and VEGF receptor (VEGFR)-2 by immunohistochemistry. The lowest expression of isomers Vegf(120) and Vegf(164) was observed on day 2 of pregnancy, increasing thereafter. Vegfr-2 mRNA expression was significantly higher on days 3-5 of pregnancy relative to days 1-2 (P<0.05). No significant changes were noted in Vegf(188), Nrp1 or Nrp2 mRNA. VEGF(188) protein expression was consistently higher than other isoforms. These data demonstrate differential regulation of VEGF-A isoforms in mouse uterus during early pregnancy.

Functional Differentiation of Uterine Stromal Cells Involves Cross-Regulation between Bone Morphogenetic Protein 2 and Krüppel-Like Factor (KLF) Family Members KLF9 and KLF13

The inability of the uterine epithelium to enter a state of receptivity for the embryo to implant is a significant underlying cause of early pregnancy loss. We previously showed that mice null for the progesterone receptor (PGR)-interacting protein Krüppel-like factor (KLF) 9 are subfertile and exhibit reduced uterine progesterone sensitivity. KLF9 expression is high in predecidual stroma, undetectable in decidua, and enhanced in uteri of mice with conditional ablation of bone morphogenetic protein 2 (BMP2). Given the individual importance of KLF9 and BMP2 for implantation success, we hypothesized that the establishment of uterine receptivity involves KLF9 and BMP2 functional cross-regulation. To address this, we used early pregnant wild-type and Klf9 null mice and KLF9 small interfering RNA-transfected human endometrial stromal cells (HESCs) induced to differentiate under standard conditions. Loss of KLF9 in mice and HESCs enhanced BMP2 expression, whereas recombinant BMP2 treatment of HESCs attenuated KLF9 mRNA levels. IGFBP1 and KLF9-related KLF13 expression were positively associated with BMP2 and inversely associated with KLF9. Prolonged, but not short-term, knockdown of KLF9 in HESCs reduced IGFBP1 expression. Mouse uterine Igfbp1 expression was similarly reduced with Klf9 ablation. PGR-A and PGR-B expression were positively associated with KLF9 in predecidual HESCs but not decidualizing HESCs. KLF13 knockdown attenuated BMP2 and PGR-B and abrogated BMP2-mediated inhibition of KLF9 expression. Results support cross-regulation among BMP2, KLF9, and KLF13 to maintain progesterone sensitivity in stromal cells undergoing differentiation and suggest that loss of this regulatory network compromises establishment of uterine receptivity and implantation success.