Uterine Luminal Epithelium Research Articles

Progesterone regulation of glutathione S-transferase Mu2 expression in mouse uterine luminal epithelium during preimplantation period

To investigate the differential expression and regulation of Gstm2 in mouse uterus during early pregnancy. Experimental animal study. University research laboratory. Sexually mature female Kunming white strain mice. Delayed and activated implantation, pseudopregnancy, hormonal treatment. The expression of Gstm2 mRNA was detected by in situ hybridization and reverse-transcription polymerase chain reaction (RT-PCR). By in situ hybridization, there were a low level of Gstm2 expression in luminal epithelium on day 3 and a strong level in the luminal epithelium on day 4 during early pregnancy. The expression pattern of Gstm2 in the pseudopregnant uterus was similar to that during early pregnancy. By RT-PCR, Gstm2 was strongly detected in the uteri on days 3 and 4 of pregnancy and pseudopregnancy. Gstm2 expression was strongly detected in the luminal epithelium under delayed implantation, but not seen after delayed implantation was activated by estrogen. In the ovariectomized mouse uterus, Gstm2 expression was strongly up-regulated by progesterone via progesterone receptor. The results showed that Gstm2 was highly expressed in the uterine luminal epithelium during preimplantation period and up-regulated by progesterone.

Effects of early gestation GH administration on placental and fetal development in sheep

Ovine GH (oGH) is synthesized in placental tissue during maximal placental growth and development. Our objectives were to localize oGH mRNA in the placenta, and study the impact of exogenous GH on twin pregnancies during the normal window (35-55 days of gestational age; dGA) of placental expression. In situ hybridization localized oGH mRNA in uterine luminal epithelium but not in tissues of fetal origin. While maternal GH and IGF-I concentrations were increased (P<0.001) approximately tenfold, uterine, uterine fluid, placental, and fetal weights were unaffected by treatment at either 55 or 135 dGA. Fetal length, liver weight, and liver weight per kg of body weight were unaffected by maternal GH treatment. However, in the cotyledon, IGF-binding protein (BP)-1 and IGFBP-4 mRNA concentrations were increased (P<0.05), while IGFBP-2 mRNA was decreased (P<0.05). The concentration of mRNA for IGFBP-3 was unaffected by treatment. Within the caruncle, IGFBP-1 mRNA was decreased (P<0.05), while IGFBP-3 and IGFBP-4 mRNA were increased (P<0.05), and IGFBP-2 mRNA was unchanged due to GH treatment. While our data indicate that elevated maternal GH and IGF-I concentrations during early and mid-gestation do not enhance placental and fetal growth in twin pregnancies, localization of GH mRNA in uterine luminal epithelium could explain GHs transitory expression from 35 to 55 dGA, since by the end of this period the majority of the uterine luminal epithelium has fused with chorionic binucleate cells forming the placental syncytium.

Interleukin 1 Signaling Is Regulated by Leukemia Inhibitory Factor (LIF) and Is Aberrant in Lif−/− Mouse Uterus1

This study addresses the regulation of the interleukin 1 (IL1) system in the murine uterine luminal epithelium (LE) and stroma by leukemia inhibitory factor (LIF). Using RT-PCR we compared expression of Il1a, Il1b, Il1rn, Il1r1, and Il1r2 during the pre- and peri-implantation periods of pregnancy in wild-type (WT) and LIF-null LE and stroma. In WT LE, Il1a transcripts were down-regulated on Day 4 of pregnancy (D4), with renewed expression by the evening of D4 (D4 pm). In Lif(-/-) LE there was a gradual decrease in expression on D2, and expression became undetectable by D6. Il1b and Il1r1 expression were similar in WT and null mice, but Il1rn expression was almost completely lost during the peri-implantation period in Lif(-/-) LE. In the stroma, Il1a was sharply down-regulated on D4 and reappeared on D4 pm but was only expressed from D3 to D5 in the null mice. Stromal Il1r1 and Il1r2 were also misregulated. Il1rn showed constitutive expression in null stroma in contrast to the loss of expression on D4 in the WT mouse. In Lif-deficient mice, immunostaining indicated a reduction of endometrial IL1A at the time of implantation and of IL1B in stroma. LE-stromal coculture revealed that LIF stimulated the apical secretion of both IL1A and PTGES2 by LE cells without affecting basal secretion of IL1A and with only a small effect on basal PTGES2 secretion. We conclude that Il1a and Il1rn in LE and Il1a, Il1rn, and Il1r1 in stroma are regulated by LIF, which stimulates apical secretion of IL1A by LE.

Actin binding protein expression is altered in uterine luminal epithelium by clomiphene citrate, a synthetic estrogen receptor modulator

Clomiphene citrate (CC), a synthetic oestrogen, is often prescribed as a superovulator in treating infertility. Although CC works efficiently, pregnancy rates following CC treatment are approximately 10 times lower than "natural" rates. This study investigates how a dose of 1.25 mg CC given to ovariectomized rats before the implantation priming hormones (a single dose of progesterone for 3 days and a dose of estradiol-17beta on d3, P-P-PE), alters the expression and distribution of alpha-actinin, gelsolin and vinculin. Actin binding proteins show a specific distribution within the uterine epithelium during implantation, linking the actin cytoskeleton to integrin expression on the uterine surface and in this way aiding "adhesiveness" for blastocyst apposition to the uterine epithelium. In this study, immunocytochemistry on frozen uterine sections using mouse monoclonal antibodies against alpha-actinin, gelsolin and vinculin and peroxidase-conjugated secondary antibodies, show that CC, administered before the P-P-PE regimen, down-regulates the expression of vinculin, does not alter the expression of gelsolin and up-regulates alpha-actinin on the uterine apical surface, when compared to P-P-PE treated animals. All three proteins are down-regulated on the apical surface of the luminal epithelium and glands in all groups when compared to pregnant controls. Vinculin was only localized in the basolateral compartment of the uterine epithelial cells in the CC treated groups. By down-regulating these proteins on the uterine surface and up-regulating vinculin on the basolateral membrane of the epithelium, CC may impede adhesion and invasion of blastocysts at implantation. These results may aid the exogenous manipulation of uterine tissue to control fertility and improve assisted reproductive out-comes.

414. The contraceptive potential of a long-acting IL-11 inhibitor

Interleukin 11 (IL-11) signalling is essential for the establishment of pregnancy in mice, through its action on the differentiation of uterine endometrial stromal cells (decidualisation), a critical process during embryo implantation. IL-11Rα deficient mice are infertile due to defective decidualisation1. IL-11 expression peaks between days (D) 4.5–9.5 of pregnancy (D0: day of plug) in mouse decidua. We examined the effect of administering (intraperitoneal [IP] injection or vaginal gel) a PEGylated IL-11 antagonist (PEGIL-11A) on decidualisation and pregnancy outcome in mice. The sera half-life of PEGIL-11A (IC50 2.8nM) following IP injection was 24h, compared with &lt;1 h for the non-PEGylated antagonist (IC50 0.26nM). Following IP injection, PEGIL-11A localised to decidual cells and blocked the IL-11 decidual target protein, cyclin D3. IP injection of 600µg/application PEGIL-11A (or PEG control) at 1000 h and 1600 h on D3 and 1000 h on D4 (n = 4/group), resulted in smaller implantation sites than controls on D6 due to retarded mesometrial decidual formation. On D10, severe decidual destruction was visible: implantation sites contained regions of haemorrhage and the uterine luminal epithelium had reformed, suggesting a return to oestrous cycling. Following vaginal application in aqueous placebo gel, PEGIL-11A localised to decidual cells. Vaginal application of 200µg/application PEGIL-11A (or control) twice daily from D2 to D5 (n = 4/group), resulted in smaller implantation sites than controls on D6 due to partial inhibition of mesometrial decidual formation. This study demonstrates that PEGIL-11A blocked IL-11 action in the uterus, resulting in total pregnancy loss, equivalent to the IL-11Rα deficient mouse. In women, IL-11 and its receptor are produced by the uterine luminal and glandular epithelium during the period of uterine receptivity2, suggesting that IL-11 may act during initial blastocyst attachment to the luminal epithelium as well as stromal decidualisation. This study provides proof-of-principle for the development of a novel, non-hormonal contraceptive for women. (1) Robb L et al. Nature Medicine 1998; 4: 303–308. (2) Dimitriadis E et al. Molecular Human Reproduction 2000; 6: 907–914.

Upregulation of Indian Hedgehog Gene in the Uterine Epithelium by Leukemia Inhibitory Factor During Mouse Implantation

Leukemia inhibitory factor (LIF) and Indian hedgehog (Ihh) are essential for embryo implantation in mice and are regulated by the actions of 17beta-estradiol (E2) and progesterone, respectively. The present study examined the effect of LIF on Ihh and Ihh-related factors in the uterine luminal epithelium during the implantation period using a DNA microarray. Expression of Ihh mRNA reached its peak on the forth day of pregnancy, and progesterone receptor (Pgr) mRNA decreased on the fifth day of pregnancy in wildtype mice. On the other hand, these changes in expression were not seen in LIF-/- mice. Ihh and Pgr mRNA were upregulated by LIF injection in delayed implantation mice. This up-regulation of Pgr was transient and preceded an increase of Ihh mRNA. Ihh mRNA also increased after E2 injection in delayed implantation mice of the LIF-/- genotype. E2 did not affect transcription of Pgr mRNA in the uterine luminal epithelium of delayed implantation LIF-/- mice. Using an antibody against the C-terminal epitope of Ihh, unprocessed Ihh proteins, but not C-terminal peptides, by autoproteolytic cleavage of Ihh were detected by western blot analysis. Unprocessed Ihh did not show quantitative changes between the wildtype and LIF-/- mice during the implantation period. Transcription of hedgehog acyltransferase was not influenced by LIF and E2 injection. In conclusion, LIF, which has a crucial role in E2 action for initiation of implantation, caused transient induction of Pgr mRNA and subsequent upregulation of Ihh mRNA, which mediates progesterone-Pgr actions for successful implantation.

Blocking LIF action in the uterus by using a PEGylated antagonist prevents implantation: A nonhormonal contraceptive strategy

Blastocyst implantation is a critical stage in the establishment of pregnancy. Leukemia inhibitory factor (LIF) is essential for mouse blastocyst implantation and also plays a role in human pregnancy. We examined the effect of a potent LIF antagonist (LA) on mouse implantation. In mice, LIF expression peaks on day 3.5 of pregnancy (D3.5) (D0.5 = day of mating plug detection) in the uterine glandular epithelium. LA (7 mg/kg per day) administered from D2.5 to D4.5 via four hourly i.p. injections plus continuous administration via miniosmotic pump resulted in complete implantation failure. To improve its pharmacokinetic properties, we conjugated LA to polyethylene glycol (PEG), achieving a significant increase in serum levels. PEGylated LA (PEGLA) (37.5 mg/kg per day) administered via three i.p. injections between D2.5 and D3.5 also resulted in complete implantation failure. PEGLA immunolocalized to the uterine luminal epithelium at the time of blastocyst implantation. Both LA and PEGLA reduced phosphorylation of the downstream signaling molecule STAT3 in luminal epithelial cells on D3.5. The effects of PEGLA were found to be endometrial, with no embryo-lethal effects observed. These data demonstrate that administration of a PEGylated LIF antagonist is an effective method of targeting LIF signaling in the endometrium and a promising novel approach in the development of nonhormonal contraceptives for women.

Effects of leukemia inhibitory factor on lectin-binding patterns in the uterine stromal vessels of mice

Lectin histochemistry was performed on mouse uteri to determine what effects leukemia inhibitory factor (LIF) has on carbohydrate epitope expressions at the time of implantation. Twenty-two biotinylated lectins were used in this study. Following injection of LIF, specific binding to the apical surface of the uterine glandular epithelium (GE) was recognized by six lectins. Particularly, binding of the lectin from Griffonia ( Bandeiraea) simplicifolia was specific to the glandular epithelium close to the luminal epithelium. Succinylated wheat germ agglutinin (WGA), which has specificity for oligosaccharides recognized by WGA without sialic acid residues, showed weaker binding to the uterine luminal epithelium (LE) and the stroma than WGA, suggesting that terminal residues of glyco-conjugates on these tissues may be modified by sialic acids. Lectin binding to the glandular and luminal epithelium was not influenced by LIF. However, three lectins including a lectin from Dolichos biflorus showed specificity for stromal vessels 6 h after LIF injection. Since the lectin from D. biflorus binds to neo-vascular vessels, LIF may play a role in regulating maternal angiogenesis directly and/or indirectly during implantation.

Up-regulation of Per1 expression by estradiol and progesterone in the rat uterus

It has been established that estrogen can alter circadian rhythms in behavior and endocrine physiology in rodents. The uterus is a reproductive organ that is critically dependent on regulation by ovarian steroids. Here, we examined the expression of Per1 in different compartments of the uterus, and explored whether the ovarian steroids could regulate Per1 expression employing ovariectomized rat uterus. RT-PCR analysis showed that Per1 was cyclically expressed in the uterus. As revealed by in situ hybridization, the staining intensity of Per1 mRNA was stronger at ZT 8 than at ZT 0 in the uterine luminal epithelium (LE), stroma (S), and myometrium (M) compartments, but was not changed in the glandular epithelium (GE). Both in situ hybridization and immunofluorescence analyses revealed that estradiol (E(2)) administration induced high expression of Per1 in the LE, GE, and M, and less expression in the S compartment. Progesterone (P(4)) treatment resulted in an obvious enhancement of Per1 expression in the LE, GE, and S, but unchanged in the M compartment. Furthermore, the E(2)- and P(4)-activated Per1 expression was significantly repressed by their respective antagonists, ICI182 780 and RU486. These findings were further supported by RT-PCR analysis of Per1 expression in cultured uterine stromal cells. Collectively, the present data indicate that E(2) and P(4) might be involved in modification of circadian rhythm via direct regulation of the expression of clock genes.

KRUPPEL-LIKE FACTOR 9 REGULATES CELL PROLIFERATION AND COMPARTMENTAL EXPRESSION OF ESTROGEN AND PROGESTERONE RECEPTORS IN THE MOUSE UTERINE ENDOMETRIUM

The uterine endometrium undergoes dynamic changes in proliferation and differentiation in response to estrogen (E) and progesterone (P) during the estrous cycle and pregnancy. E and P exert their functions through their respective nuclear receptors, estrogen receptor (ESR) and progesterone receptor (PGR), which interact with specific nuclear proteins to influence transcription. Our laboratory has identified Krüppel-like factor 9 (KLF9), a member of specificity protein (SP)/KLF family of transcription factors as a PGR-interacting protein that affects PGR transcriptional activity. Female mice null for the Klf9 gene have fewer numbers of successfully implanting embryos and decreased litter size, due partly to delayed onset of proliferation in uterine luminal epithelium (LE), resulting in asynchronous development between the embryo and the uterus. We hypothesize that the delayed initiation of endometrial cell proliferation in Klf9-knockout (KO) mice may be due to reduced uterine sensitivity to E and P mediated by KLF9. To address this, we compared endometrial cell proliferation in ovariectomized wild type (WT) and KO mice treated with E or P+EP following a regimen that mimicked the hormonal milieu of the uterine endometrium at early pregnancy. Endometrial cell proliferation was evaluated by immunostaining for Proliferating Cell Nuclear Antigen (PCNA). Acute E treatment (18h) increased PCNA immunoreactivity in endometrial LE, stroma (ST), and glandular epithelium (GE) in WT but not KO mice. Transcript levels of the proliferation-associated genes insulin-like growth factor I (Igf1) and CCAAT/enhancer binding protein, beta (Cebpb), measured by quantitative RT-PCR, were similar in uteri of WT and KO mice after acute E treatment. Chronic E treatment (32h) resulted in comparable PCNA immunoreactivity among cell types in both WT and KO mice. Proliferation status of LE, GE, and ST did not differ as a function of genotype with P+EP-treatment. Since ESR and PGR expression dictate cellular responsiveness to E and P, we determined if KLF9 influenced expression levels of ESR-α (ESR1) and PGR in LE, ST, and GE by immunohistochemistry. The absence of KLF9 in ST cells of KO mice resulted in subsequent loss of: (a) P+EP-mediated increase in LE cell ESR1 expression; (b) E-inhibition of LE cell PGR expression; and (c) P-mediated reversal of E-inhibition of LE cell PGR expression, relative to WT counterparts. Moreover, P+EP-induced increase in PGR-positive ST cells was higher in WT than in KO mice. Given that LE cells lack endogenous KLF9, results suggest that the timely proliferation of LE, which is requisite for its subsequent differentiation and receptivity to embryo implantation, is regulated by E/ESR1-mediated signaling from ST to LE, possibly via an ST-derived factor(s). Thus, KLF9 is an essential mediator of E and P action in the uterus via its distinct regulation of ESR1 and PGR expression levels in specific cell types. We suggest that ST expression of KLF9 is critical for communication between uterine LE and ST required for successful embryo implantation and pregnancy (NIH HD21961). (poster)

INTRAUTERINE INFUSION OF RECOMBINANT TRANSFORMING GROWTH FACTOR BETA LATENCY-ASSOCIATED PEPTIDE DISRUPTS PORCINE CONCEPTUS DEVELOPMENT

The beta transforming growth factors (TGFbeta) and their receptors (TGFbeta-R) are present at the conceptus-maternal interface in pigs and other mammals. TGFbeta is released as latent precursors which cannot interact with TGFbeta-R prior to activation. In pigs, amounts of active TGFbeta increase during conceptus elongation in preparation for attachment to the endometrium; active TGFbeta may play roles in conceptus-uterine interactions at this time. The latency-associated peptide of TGFbeta (LAP) is part of a complex that comprises latent TGFbeta and contains an arg-gly-asp (RGD) amino acid sequence which can bind to and activate integrins. We hypothesize that both active TGFbeta and LAP contribute to normal conceptus development and implantation. Objectives of these studies are to: 1) determine temporal and spatial distribution of LAP at the conceptus-maternal interface of pigs during the implantation process, and 2) determine effects of intrauterine infusion of LAP on conceptus development during the peri-implantation phase of pregnancy. In study one, cross-bred gilts were ovariohysterectomized on Days 10, 12, 16, 20, and 24 of gestation (n=3/day). LAP distribution at implantation and nonimplantation sites was determined using immunofluorescence microscopy. On Days 10 and 12 of gestation, LAP was present at the apical and basal aspects of uterine luminal epithelia, apical surfaces of glandular epithelia and within the lumens of uterine glands. Epithelial expression decreased between Days 16 and 20, suggesting either decreased TGFbeta production or increased TGFbeta activation. On Day 24, when conceptus attachment is nearly complete, LAP was nearly undetectable at non-implantation sites, however aggregrates of LAP were present at sites of adhesion between conceptus and uterine tissues. Thus, collective temporal and spatial localization suggests a role for LAP as an extracellular matrix protein available for interaction with conceptus and/or maternal integrins that maintains conceptus attachment. In study 2, surgically-implanted mini-osmotic pumps were used to continually infuse uterine horns with molar excesses of recombinant LAP in a porcine serum albumin (PSA) vehicle or PSA alone beginning on Day 9 of gestation; others received no infusion. Gilts were ovariohysterectomized on Day 13 of gestation and conceptuses examined. Conceptuses from LAP-infused gilts (n=3 gilts) grossly appeared retarded in their development and some appeared to be degenerating compared to those recovered from gilts which received PSA or no infusion. In two litters, conceptuses were spherical or oval in contrast to the filamentous morphology of conceptuses in litters receiving PSA and no infusion. Histological evaluation confirmed degeneration of the trophoblast in LAP-infused gilts, while integrity of the uterine epithelium was maintained. Effects most likely resulted from inactivation of TGFbeta by the infused LAP, but effects of integrin activation by the RGD sequence of LAP are also possible and will be further investigated through infusion of an LAP mutant protein containing an RGE sequence in place of the RGD. This is the first report of targeted in vivo disruption of pre-implantation conceptus development by direct infusion of a recombinant protein into the uterus of pregnant pigs. Results suggest critical roles for TGFbeta in conceptus survival and elongation. Supported by NRICG #2005-35203-15798 from USDA CSREES. (platform)

SECRETED PHOSPHOPROTEIN 1 ISOLATED FROM UTERINE SECRETIONS PROMOTES INTEGRIN-MEDIATED ADHESION OF PORCINE TROPHECTODERM CELLS

In domestic pigs, over two-thirds of reproductive losses occur during the first 25 days of gestation, a period during which rapid conceptus development, trophoblast adhesion and implantation are initiated. Successful conceptus implantation requires pregnancy-specific alterations in extracellular matrix (ECM) at the conceptus-maternal interface. The pig luminal epithelium (LE) immediately adjacent to the implanting conceptus secretes SPP1 (also known as osteopontin), an ECM molecule involved in cell-cell and cell-ECM communication that is believed to mediate conceptus-uterus interactions. We hypothesized that SPP1 expressed by the uterine LE bridges to integrin receptors on conceptus trophectoderm to stimulate adhesion at the conceptus-maternal interface. The objective of this study was to determine if SPP1 isolated from uterine secretions (uterine milk) supports trophectoderm cell adhesion in vitro. To accomplish this, we employed a unilaterally pregnant sheep model, where the ovary ipsilateral to the right uterine horn was removed and a double ligature placed at the base of the right uterine horn at the bifurcation. At the following estrus, sheep were mated to generate gravid and nongravid uterine horns, hysterectomized on day 120 and the uterine milk within nongravid horns collected. SPP1 was purified from uterine milk using a DEAE-Sephacel column (ion exchange chromatography) followed by dual phenyl-Sepharose columns (hydrophobic chromatography). A 45 kDa fragment, previously identified at the apical surface of the sheep LE during pregnancy, and a 25 kDa fragment were isolated. The spontaneously immortalized pig trophectoderm cell line pTr2 was allowed to attach to serial dilutions (20 μg/ml to 40 ng/ml) of the following proteins: bovine milk SPP1 (bSPP1; purified in the manner described above), ovine uterine milk SPP1 (oSPP1), recombinant rat SPP1 containing the RGD cellbinding sequence (rSPP1-RGD), recombinant rat SPP1 containing an RAD sequence in place of the RGD sequence (rSPP1-RAD), full-length human fibronectin (FN; positive control) and BSA (negative control). Dose-dependent cell adhesion was observed for bSPP1, oSPP1 and rSPP1- RGD, all of which supported higher levels of adhesion than FN at concentrations ranging from 0.15 and 1.25 μg/ml. Only at concentrations of 2.5 μg/ml and higher did FN support greater pTr2 adhesion than the various SPP1 proteins. Cell adhesion was not supported by rSPP1-RAD over BSA controls at any concentration, strongly suggesting that adhesion to the various SPP1 proteins and FN occurred through RGD-binding integrin receptors. Integrins require the presence of divalent cations to allow ligand binding. This property was used to further confirm that pTr2 cell adhesion to SPP1 was integrin-mediated. Cells were allowed to attach in the presence of varying levels of divalent cations: 1) no cations; 2) 1 mM Ca2+ + 2 mM Mg2+; 3) 2 mM Ca2+ + 1 mM Mg2+; or 4) 0.1 mM Mn2+. No attachment was observed in the absence of divalent cations, confirming that porcine trophectoderm attachment to SPP1 is integrin-mediated. This study is the first to demonstrate that a native form of uterine SPP1, secreted by the uterine glandular epithelium and present at the conceptus-maternal interface, can promote dose- and cation-dependent integrin-mediated adhesion of pig trophectoderm cells. (Supported by NRI USDA Grant 2006-35203-17199). (platform)

Effect of postnatal exposure to benzo[ a]pyrene on the uterus of immature rats

The objective of this study was to investigate the morphological effects of postnatal exposure to benzo[ a]pyrene (B[ a]P) on the development of the uterus, uterine estrogen receptor (ER α) expression, and the uterine response to estrogen stimulation using the uterotrophic bioassay in rats. Neonates were injected on each postnatal day (PND) 1–14 with B[ a]P (0.1, 1.0 and 10.0 mg/kg), ethynylestradiol (EE; 1.0 μg/kg) or vehicle (control group). All animals were killed on PND 23. Postnatal administration of B[ a]P with doses of 1.0 and 10.0 mg/kg induced significant ( P<0.01) reduction of uterine weight and significantly lowered ( P<0.05) ER α expression in the luminal epithelium. The increase in uterine weight and luminal epithelium heights after EE stimulation (1.0 μg/kg) on PND 20–22 was significantly higher ( P<0.01) in all groups in comparison with corresponding non-stimulated groups. However, the uterotrophic response in rats postnatally exposed to EE and B[ a]P was significantly lower ( P<0.01) than in controls. In the control and EE groups, EE stimulation on PND 20–22 induced a significant ( P<0.01) decrease in ER α immunoreactivity of the luminal epithelium. In contrast, rats postnatally treated with B[ a]P showed no change in the density of ER α immunostaining when detected after estrogenic stimulation. The present study showed that postnatal exposure to B[ a]P caused pathological changes in constitution and maturation of uterine ER α resulting in disturbed morphological development and uterine dysfunction in immature rats.

Heparanase Expression and Function During Early Pregnancy in Mice1

Embryo implantation is a complex process that involves interactions between cell-surface and extracellular components of the blastocyst and the uterus, including blastocyst adhesion to the uterine luminal epithelium, epithelial basement membrane penetration and stromal extracellular matrix remodeling, angiogenesis, and decidualization. These processes all involve interactions with heparan sulfate (HS) proteoglycans, which harbor various growth factors and cytokines and support cell adhesion. Heparanase (HPSE) is an endo-beta-glucuronidase that cleaves HS at specific sites. HPSE also can act as an adhesion molecule independent of its catalytic activity. Thus, HPSE is a multifunctional molecule contributing to and modulating HS-dependent processes. Exogenously added HPSE improves embryo implantation in mice; however, no information is available regarding the normal pattern of HPSE expression and activity during the implantation process in any system. Using several approaches, including real-time RT-PCR, in situ hybridization, and immunohistochemistry, we determined that uterine HPSE expression increases dramatically during early pregnancy in mice. Heparanase mRNA and protein were primarily expressed in decidua and were rapidly induced at the implantation site. Uterine HPSE activity was characterized and demonstrated to increase >40-fold during early pregnancy. Finally, we demonstrate that the HPSE inhibitor PI-88 severely inhibits embryo implantation in vivo. Collectively, these results indicate that HPSE plays a role in blastocyst implantation and complements previous studies showing a role for HS-dependent interactions in this process.

Lipin1 Regulation by Estrogen in Uterus and Liver: Implications for Diabetes and Fertility

Estrogens are essential for fertility and also have important effects on regulation of adiposity and the euglycemic state. We report here that lipin1, a candidate gene for lipodystrophy and obesity that is a phosphatidic acid phosphatase critical in regulation of cellular levels of diacylglycerol and triacylglycerol and a key regulator of lipid utilization, is rapidly and robustly down-regulated in the uterus by estradiol via the estrogen receptor. Lipin1 is expressed predominantly in the uterine luminal and glandular epithelium, and during the estrous cycle, lipin1 is lowest when blood levels of estrogen are highest. Lipin1 is expressed throughout all cells in the liver of ovariectomized female mice, and a sustained down-regulation is observed at the mRNA, protein and immunohistochemical levels after estrogen administration. Because the coupling of proper energy use and availability is central to reproduction, we also investigated expression of lipin1 in the uterus and liver of several mouse models of diabetes. Nonobese diabetic (NOD) mice, which have high blood levels of estrogen and impaired fertility, were severely deficient in lipin1 in the uterus and liver, which, interestingly, could be restored by insulin treatment. By contrast, nonobese diabetic/severe combined immunodeficient (NOD-SCID) mice, which do not develop diabetes, showed normal levels of lipin1. Our findings of lipin1 regulation by estrogen in two key target organs suggest a new role for this lipid-regulating phosphatase not only in central metabolic regulation but also in uterine function and reproductive biology. Estrogen regulation of lipin1 may provide a mechanistic link between estrogens, lipid metabolism, and lipid signaling.

Regulation of Expression of Fibroblast Growth Factor 7 in the Pig Uterus by Progesterone and Estradiol1

Fibroblast growth factor 7 (FGF7) stimulates cell proliferation, differentiation, migration and angiogenesis. The consensus is that FGF7, expressed by mesenchymal cells, binds FGF receptor 2IIIb (FGFR2) on epithelia, thereby mediating epithelial-mesenchymal interactions. The pig uterus is unique in that FGF7 is expressed by the luminal epithelium (LE) and FGFR2 is expressed by the LE, glandular epithelium (GE), and trophectoderm to effect proliferation and differentiated cell functions during conceptus development and implantation. FGF7 expression by the uterine LE of pigs increases between Days 9 and 12 of the estrus cycle and pregnancy, as circulating concentrations of progesterone increase, progesterone receptors (PGR) in the uterine epithelia decrease, and the conceptuses secrete estradiol-17beta (E(2)), for pregnancy recognition. Furthermore, E(2) increases the expression of FGF7 in pig uterine explants. The present study investigates the relationships between progesterone, E(2), and their receptors and the expression of FGF7 in the pig uterus in vivo. Pigs were ovariectomized on Day 4 of the estrus cycle and injected i.m. daily from Day 4 to Day 12 with either corn oil (CO), progesterone (P4), P4 and ZK317,316 (PZK), E(2), P4 and E(2) (PE), or P4 and ZK and E(2) (PZKE). All gilts (n = 5/treatment) were hysterectomized on Day 12. The results suggest that: 1) P4 is permissive to FGF7 expression by down-regulating PGR in LE; 2) P4 stimulates PGR-positive uterine stromal cells to release an unidentified progestamedin that induces FGF7 expression by LE; 3) E(2) and P4 can induce FGF7 when PGR are rendered nonfunctional by ZK; and 4) E(2) from conceptuses interacts via estrogen receptor alpha, but not estrogen receptor beta in LE to induce maximal expression of FGF7 in LE on Day 12 of pregnancy in pigs.

Immunolocalization of insulin-like growth factor I (IGF-I) during preimplantation in rat uterus

Objective The aim of this study was to determine the insulin-like growth factor (IGF-I) immunolocalization in rat uterus at preimplantation period. Design The tissue samples were examined from pregnant animals between gestational day 1 and 5 using immunocytochemistry. Results While both the uterine luminal epithelium and the glandular epithelium were stained strongly from gestational day 1--3 by IGF-I antibody, the IGF-I immunoreactivity was moderate in myometrium and capillary endothelium. At this period, the IGF-I immunoreactivity was weakly present in a few endometrial stromal cells. At day 4, while IGF-I immunostaining intensity was particularly decreased in the basal domains of uterine luminal epithelium and glandular epithelium, it was similar to the first 3 days of gestation in myometrium and capillary endothelium. The endometrial vascularization and the number of stromal cells immunoreacting with IGF-I increased. The differentiation of stromal cells into decidual cells was seen at gestational day 5 and IGF-I was strongly expressed in the decidualizing cells. The IGF-I immunoreactivity in uterine luminal epithelium and glandular epithelium was similar to gestational day 4. IGF-I immunoreactivity was strongly detected in all of endometrial stromal cells. Conclusion These results indicate that IGF-I expression in the rat uterus changes in the early pregnancy process and increase toward to day when implantation will be initiated.

Immunodetection of Thyroid Hormone Receptor (Alpha1/Alpha2) in the Rat Uterus and Oviduct

The aim of this study was to investigate the immunolocalization and the existence of thyroid hormone receptors (THR) (alpha1/alpha2) in rat uterus and oviduct. For this purpose 6 female Wistar albino rats found in estrous period were used. Tissue samples fixed in 10% neutral formalin were examined immunohistochemically. Sections were incubated with primary mouse-monoclonal THR (alpha1/alpha2) antibody. In uterus, THR (alpha1/alpha2) immunoreacted strongly with uterine luminal epithelium, endometrial gland epithelium and endometrial stromal cells and, moderately with myometrial smooth muscle. In oviduct, they were observed moderately in the epithelium of the tube and the smooth muscle cells of the muscular layer.In conclusion, the presence of THR in uterus and oviduct suggests that these organs are an active site of thyroid hormones.

Relative transcript abundance of oxytocin receptor gene in porcine uterus during luteolysis and early pregnancy

The aim of the present study was to investigate transcript localization of the oxytocin receptor (OTR) gene in different cells of the porcine uterus during luteolysis and early pregnancy (days 14-16) using in situ hybridization (ISH). OTR mRNA was localized in the uterine luminal epithelium (LEC), glandular epithelium (GEC), stromal cells (SC) of the endometrium, in the longitudinal muscle layer (LM) and circular muscle layer (CM) of the myometrium. The OTR transcript was quantified by optical density units of silver grains. The OTR transcript levels in the endometrium and myometrium were statistically higher during luteolysis than during early pregnancy (P<0.05). Besides, during luteolysis, the mRNA level was higher in the LEC, GEC of the endometrium and LM of the myometrium compared to that observed in the SC of endometrium and CM of the myometrium, respectively (P<0.05). In summary: 1) the level of OTR mRNA in uterine tissues is higher during luteolysis compared to early pregnancy, 2) the OTR transcript level in endometrial cells did not correspond to the sensitivity of these cells to oxytocin (OT), 3) the myometrial expression of the OTR gene is appropriate to control contractile activity and secretion of PG during luteolysis.

Neonatal porcine endometrial development and epithelial proliferation affected by age and exposure to estrogen and relaxin

In the pig, temporospatially regulated proliferation of uterine luminal (LE) and glandular (GE) epithelium between birth (postnatal day = PND 0) and PND 15 is essential for success of endometrial development. Exposure of gilts to estrogen (E) or relaxin (RLX) during this period disrupts uterine development, and neonatal E exposure can compromise adult uterine function. Neonatal uterotrophic effects of E and RLX, administered for 2 days beginning on PND 12, can be inhibited with the antiestrogen ICI 182,780 (ICI) indicating crosstalk between RLX and E signaling systems. Here, objectives were to determine effects of: (study 1) neonatal age and (study 2) exposure to E, RLX, and ICI on porcine neonatal uterine histoarchitecture and patterns of epithelial cell proliferation as reflected by proliferating cell nuclear antigen labeling index. In study 1, uteri were obtained on PND 0, 3, 6, 9, 12 and 15. Glandular epithelium, absent at birth, was observed by PND 3. Overall, epithelial labeling index increased from birth to PND 3, declined from PND 6–9 in LE and GE, and increased to PND 15 in GE. In study 2, uteri were collected on PND 14 after administration of vehicle, E, or RLX for 2 days, or following pretreatment with ICI. Alone, E was uterotrophic and adenogenic and increased labeling index in both LE and GE. Both RLX and ICI increased proliferation in GE. Effects of E and RLX were attenuated by ICI, providing further support for crosstalk between these signaling systems in the developing neonatal porcine endometrium.