Temporal and spatial expression of leukemia inhibitory factor in rabbit uterus during early pregnancy

Leukemia inhibitory factor (LIF) has been shown to play an important role in the implantation of mouse blastocysts. The present study was designed to document the appearance of LIF in the rabbit uterus during early pregnancy and to determine whether changes just prior to implantation, similar to those in mice, occurred. LIF was localized in endometrial epithelium, myometrium, and endometrial glands. A low level of LIF was detected in the uterus of nonestrous and estrous females. LIF expression reached its highest level on day 5 of pregnancy and declined on days 6 and 7. By day 13 of pregnancy, little endometrial LIF was apparent. The expression of LIF on day 5 of pseudopregnancy was similar to that on day 5 of pregnancy. LIF expression was much higher at implantation sites than that at nonimplantation areas on day 7 of pregnancy. It is concluded that LIF may be important for the implantation of rabbit blastocysts. © 1994 Wiley-Liss, Inc.     

Leukemia inhibitory factor, leukemia inhibitory factor receptor, and glycoprotein 130 in rhesus monkey uterus during menstrual cycle and early pregnancy

This goal of this study was to examine immunohistochemical distribution of leukemia inhibitory factor (LIF), LIF receptor (LIFR), and glycoprotein (gp) 130 in rhesus monkey uterus during the menstrual cycle and early pregnancy. Pregnancy rate was significantly reduced in the control group from 66.7% (12 of 18) to 22.2% (4 of 18) with an injection of goat anti-human recombinant LIF immunoglobulin G into the uterine lumen on Day 8 of pregnancy. LIF was mainly localized in glandular and luminal epithelium. LIF immunostaining during the luteal phase was stronger than it was during the proliferative phase. LIF staining gradually increased from Day 3 of pregnancy and reached its highest level on Day 9. LIFR was mainly localized in the glandular and luminal epithelium. LIFR staining during the luteal phase was stronger than it was during the proliferative phase. LIFR staining began to increase from Day 3 of pregnancy and reached a high level on Days 9 and 11. Gp130, a signal-transducing receptor component of LIF, was mainly localized in the glandular epithelium. A high level of gp130 was found on Days 16 and 20 of menstrual cycle, and from Days 5 to 11 of pregnancy. These results suggest that LIF may play an important role in monkey implantation, as it does in mice.     

Gene expression of leukemia inhibitory factor (LIF) and macrophage colony stimulating factor (M-CSF) in bovine endometrium during early pregnancy

Leukemia inhibitory factor (LIF) and macrophage colony stimulating factor (M-CSF), members of the group of hemopoietic cytokines, play a primary role in the control of embryo development and implantation and in the growth of the placenta in humans and mice. Gene expressions of LIF and M-CSF were investigated using quantitative RT-PCR in bovine endometrial tissues during early and mid-pregnancy (Days 16-17, 20-21, 30-36, 48-49 and 74-140) and during the estrous cycle (Days 13-14). Leukemia inhibitory factor and M-CSF genes were expressed in all samples examined. Significant differences were found between the gene expression patterns of LIF and M-CSF. Leukemia inhibitory factor expression level at Days 48-49 was the highest in caruncular endometrium, however, the large variability negated any significant differences. Leukemia inhibitory factor expression levels in intercaruncular endometrium at Days 48-49 and 74-140 of pregnancy were greater than at Days 13-14 of the estrous cycle and at other days of pregnancy. No significant change was recognized in M-CSF expression levels in caruncular endometrium. Macrophage colony stimulating factor expression level in intercaruncular endometrium at Days 74-140 was greater than those of the other samples. These results suggest that LIF and M-CSF are produced in the endometrium and may play different roles in early and mid-pregnancy.     

Effect of leukemia inhibitory factor in experimental cisplatin neuropathy in mice

In this study, the effect of leukemia inhibitory factor (LIF) on cisplatin (CDDP)-induced neuropathy was evaluated. Mice were treated with CDDP, 2 mg/kg i.p. twice a week nine times. During the last week some of the mice were also injected with LIF, 2 mug/kg s.c. every other day for a total of four injections. Development of neuropathy was evaluated with changes in tail flick latency and sensory nerve conduction velocity (NCV). At the end of the treatment period dorsal root ganglia (DRG) were microscopically examined. Some of the DRGs were explanted into extracellular matrix, covered with culture medium and incubated for 3 days. During and at the end of the incubation, cellular migration and axonal outgrowth from the DRGs were quantified. LIF proved effective in reversing the increase in tail flick latency (p<0.05) and improving the reduction in NCV induced by CDDP. CDDP led to smaller nuclear and somatic size in neurons, while with LIF, the latter was restored to control values (p<0.01). No apoptotic nucleus was observed among DRG neurons while very few and moderate numbers detected among satellite and Schwann cells, respectively. With LIF, none of the cells had apoptosis. CDDP caused a decrease in the number of migrating cells and in the length of outgrowing axons while LIF treatment restored both capacities (p<0.05) In conclusion, in CDDP-induced neuropathy, LIF was found to be effective in correcting some functional and morphological deteriorations related with major involvement of Schwann cells.     

Platelet-activating factor induces the expression of early pregnancy factor activity in female mice

When synthetic platelet-activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was injected into mature female mice during dioestrus, pro-oestrus or oestrus, it induced the expression of early pregnancy factor (EPF) activity in the sera of these animals within 1 h of injection. The sera of similarly injected males, metoestrous or immature females did not display any EPF activity. The results suggest that embryo-derived PAF may be the ovum factor responsible for triggering the generation of serum EPF activity during the preimplantation stages of pregnancy.     

Platelet-activating factor in the rabbit uterus during early pregnancy

Platelet-activating factor (PAF) concentrations were low in the non-pregnant, oestrous uterus (mean +/- s.e.m.: 2.2 +/- 1.2 pmol/g, n = 3). However, uterine PAF increased dramatically during pregnancy to a maximum of 37.8 +/- 4.90 pmol/g (n = 7) on Day 5. By Day 7, PAF concentrations in the uteri of pregnant rabbits had returned to levels similar to those found at oestrus. In contrast, uterine PAF in pseudopregnant rabbits peaked at 30.6 +/- 2.8 pmol/g (n = 8) on Day 4, declined to 20.5 +/- 2.4 pmol/g (n = 8) on Day 5 and then remained at that concentration through Day 7. Uterine PAF co-migrated with synthetic PAF (1-O-hexadecyl-2-acetyl-sn-glycero-phosphocholine) in both thin-layer and normal-phase high-performance liquid chromatography. PAF activity in the uterus during pregnancy and pseudopregnancy was found almost exclusively in the endometrium; little or no PAF was found in myometrium, uterine flushings or blastocysts. While no PAF was detected in blastocysts on Days 5 and 6 of pregnancy, the presence of the embryo appears to modulate biosynthesis and/or degradation of PAF by the uterus, since PAF decreased significantly in uterine tissue apposed to the implanting embryo (but not in similar areas between such attachment sites). Increased concentrations of PAF in the preimplantation rabbit uterus followed by a dramatic decrease on the day of blastocyst attachment suggest that this potent inflammatory autacoid may play a vital role in implantation.     

GDNF-induced leukemia inhibitory factor can mediate differentiation via the MEK/ERK pathway in pheochromocytoma cells derived from nf1-heterozygous knockout mice

Glial cell line-derived neurotrophic factor (GDNF) can induce neuron-like differentiation of mouse pheochromocytoma (MPC) cell lines derived from mice with a heterozygous knockout mutation of nf1, the murine counterpart of the human gene mutated in neurofibromatosis type 1 (NF1). Here, we show that GDNF-induced differentiation in the MPC 862L cell line is mediated by the MEK/extracellular signal-regulated kinase (ERK) pathway. Neurite outgrowth, increased expression of growth-associated protein 43, and decreased incorporation of bromodeoxyuridine (BrdU) were induced by treatment with GDNF, H-RasV12, or a constitutively active MEK2. GDNF also induces leukemia inhibitory factor (LIF) via the MEK/ERK pathway, and LIF itself can elicit these differentiative changes via a cell-extrinsic autocrine/paracrine pathway. Treatment with anti-LIF neutralizing antibody depleted the differentiative activity of the conditioned medium from cells stimulated for MEK/ERK signaling, while recombinant LIF could induce differentiation in MPC cells, indicating that LIF is the sole factor with differentiative activity. LIF could activate MEK1/2 and STAT3, but LIF-induced differentiation was blocked only by the MEK1/2-specific inhibitor U0126, indicating that the MEK/ERK pathway is necessary for LIF action in MPC cells. Our findings suggest that LIF may be utilized for signaling mediated by GDNF and may be important in the pathobiology of neuroendocrine tumors.     

Heparanase expression and function during early pregnancy in mice

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.     

Identification of a putative inhibitor of early pregnancy factor in mice

Previous studies have indicated that early pregnancy factor (EPF) produced in the pre- and peri-implantation stage of pregnancy appears to consist of inactive components which combine to produce the active species. This is in contrast with EPF produced later in gestation which appears to consist of a single active species. The original studies on ammonium sulphate fractionation of mouse serum and in-vitro culture of mouse ovaries and oviducts have been repeated but tested in the bioassay for EPF, the rosette inhibition test, over an extended range of dilutions. This revealed that the two components in early pregnancy can be understood as EPF and an inhibitor(s). Once this inhibitor is removed, the active fractions in both early and late pregnancy sera exhibit similar behaviour in the above assay. It was shown also that the ovary alone is the source of activity but that this is modulated by an inhibitory substance(s) from the oviduct. Reversed-phase HPLC studies on purified 'early' EPF confirm that active and inhibitory components are present and demonstrate that the active component exhibits an identical elution pattern to 'late' EPF. Thus as pregnancy proceeds, it is not EPF that alters but rather the inhibitor(s), which disappears from the circulation soon after implantation. This substance(s) is under hormonal control, being present during oestrus as well as the early stages of pregnancy; it may be an important biological regulator of EPF. Its action in the rosette inhibition test has profound implications for further study using this bioassay.     

Alterations in epithelial glycocalyx of rabbit uteri during early pseudopregnancy and pregnancy, and following ovariectomy

Pseudopregnant, pregnant, and ovariectomized rabbits were utilized to study hormonal mediation of uterine epithelial surface negativity and glycocalyx morphology, and to seek local effects of blastocysts at sites of implantatioN. A loss of surface negativity [polycationic ferritin (PCF) binding] by day 6 of pregnancy or pseudopregnancy was noted, accompanied by alterations in epithelial glycocalyx. Uteri from estrous animals, or ovariectomized animals receiving oil or estradiol injections, bound PCF and exhibited a "globular" glycocalyx. Uteri from day 6 pseudopregnant or pregnant animals, or ovariectomized animals receiving progesterone injections, did not bind PCF or exhibit a globular glycocalyx. Both PCF binding and the globular character of the epithelial glycocalyx were sensitive to neuraminidase and trypsin treatment, suggesting sialoglycoprotein contribution to surface negativity. Implanting blastocysts had no detectable local effect on surface negativity, but did induce local reduction of epithelial glycocalyx at sites of implantation. Results of this study suggest that uterine epithelial glycocalyx alterations during the preimplantation period reflect a general response to progesterone stimulation, primarily qualitative in nature, related to the acquisition of receptivity to ovo-implantation.     

Analysis of neuronal and glial phenotypes in brains of mice deficient in leukemia inhibitory factor

Leukemia inhibitory factor (LIF) can regulate the survival and differentiation of certain neurons and glial cells in culture. To determine the role of this cytokine in the central nervous system in vivo, we examined the brains of young and adult mice in which the LIF gene was disrupted. Immunohistochemical staining of neurons for choline acetyltransferase, tyrosine hydroxylase, serotonin, parvalbumin, calbindin, neuropeptide Y, vasoactive intestinal polypeptide, and calcitonin gene-related peptide revealed no significant differences between null mutant and wild-type (WT) brains. In contrast, analysis of glial phenotypes demonstrated striking deficits in the LIF-knockout brain. Staining with several anti-glial fibrillary acidic protein (GFAP) antibodies showed that the number of GFAP-positive cells in various regions of the hippocampus in the female mutant is much lower than in the WT. The null male hippocampus also displays a significant, though less marked deficit. The number of astrocytes in the mutant hippocampus, as determined by S-100 staining, is not, however, significantly different from WT. In addition, quantification of immunohistochemical staining of female, but not male, mutants reveals a significant deficit in myelin basic protein content in three brain regions, suggesting alterations in oligodendrocytes as well. Thus, while overall brain histology appears normal, the absence of LIF in vivo leads to specific, sexually dimorphic alterations in glial phenotype. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 509–524, 1998     

Antibodies to early pregnancy factor retard embryonic development in mice in vivo

Previous work in this laboratory has shown that passive immunization of mice against early pregnancy factor (EPF) leads to failure to maintain pregnancy. The findings presented in this paper demonstrate that this treatment affects the development of the embryos very early in gestation. By Day 3, 54 and 25% of embryos in the 2 groups treated with anti-EPF immunoglobulin (Ig)G and IgM, respectively, had not developed to the 4-cell stage, compared with 12 and 1% in the control groups. None of the embryos in the mice treated with anti-EPF had developed beyond the 8-cell stage. A similar delay in development after treatment was observed on Day 4. The effect apparent during the early stages of cleavage is an indirect rather than a direct one, as 2-cell embryos (32-36 h post coitum), cultured in vitro in the presence of anti-EPF antibodies, developed uninterrupted to the morula and blastocyst stage. The delay in development did not appear to be caused by a disruption of the normal pattern of circulating progesterone, as progesterone concentrations on Day 4 were within the normal range for Quackenbush mice.     

Fetal loss in mice exposed to magnetic fields during early pregnancy

The effects of low-frequency magnetic fields (MFs) on early pregnancy were studied in CBA/S mice. The magnetic field was a 20 kHz, 15 μT sawtooth. Pregnant females were divided into four groups, two control groups and two exposed groups. One group was exposed to MFs continuously from day 1 postconception (pc) until day 5.5 pc, and the other group was exposed continuously until day 7 pc. All animals were sacrificed on day 19 pc, the day before partus, and their uterine contents were analyzed. No significant increase in the resorption (early fetal death) rate was found in the exposed animals compared to the sham controls. In the group exposed during days 1.0–5.5 pc, the body weight and length of the living fetuses were significantly decreased. Except on day 3 pc (progesterone) and day 13 pc (calcium) in the treated groups, there were no significant differences in progesterone and calcium levels in peripheral blood. Implantation occurred on the same day in MF-treated and control animals. © 1995 Wiley-Liss, Inc.     

Studies using leukemia inhibitory factor (LIF) knockout mice and a LIF adenoviral vector demonstrate a key anti-inflammatory role for this cytokine in cutaneous inflammation

Previous work has implicated the cytokine leukemia inhibitory factor (LIF) in cutaneous inflammation, although results have differed as to whether LIF is pro- or anti-inflammatory in this setting. We examined edema, inflammatory cell infiltration, and cytokine responses following CFA injection in the adult mouse footpad. Inflammatory cell infiltration and edema are significantly enhanced when CFA is injected in LIF knockout mice as compared with injection of wild-type littermates. Moreover, local injection of an adenoviral vector encoding LIF suppresses both measures of inflammation. In contrast, injection of an adenoviral vector encoding beta-galactosidase has no discernable effect on inflammation. In addition, comparison of the CFA responses in LIF knockout vs wild-type skin reveals that LIF is an important regulator of IL-1beta, IL-6, IL-7, IL-2Ralpha, and IFN-gamma in cutaneous inflammation. These and our previous data indicate that both endogenous and exogenous LIF are anti-inflammatory in the CFA model and that LIF is a key regulator of the cytokine cascade. The results also indicate that adenoviral gene delivery can be an effective therapeutic approach in this paradigm.     

Thymocyte subpopulations during early fetal development in sheep

Phenotypic analysis of thymocytes during fetal development may identify subpopulations which are either absent or difficult to detect in postnatal thymus. A panel of monoclonal antibodies specific for sheep lymphocyte antigens (SBU-T1, -T4, -T8, -T6) was used to identify thymocyte subpopulations in postnatal and fetal sheep. Thymuses were analyzed by two-color immunofluorescence and flow cytometry or by immunohistology. Two-color immunofluorescent staining of postnatal sheep thymus with anti-SBU-T4 and anti-SBU-T8 revealed four relatively distinct subpopulations with particular localizations: a) SBU-T4-T8-, predominantly outer cortex (12%); b) SBU-T4+T8+, inner cortex (74%); c) SBU-T4+T8-, medulla (10%), and d) SBU-T4-T8+, medulla (4%). One- and two-color immunofluorescent analysis of cells from early fetal thymuses demonstrated the appearance of SBU-T8+ cells well before SBU-T4+ cells. Immunohistologic staining of fetal sheep thymus at various stages of gestation (term = 150 days) revealed that lymphoid cells and MHC class II-positive dendritic cells first appeared at 35 days, at which stage the thymic epithelium was weakly positive for class I MHC antigens but negative for class II MHC antigens. The earliest lymphocyte antigens detectable on fetal sheep thymocytes were SBU-LCA and SBU-T1. By 40 days, the antigens SBU-T6, SBU-T4, and SBU-T8 were detectable on a small number of thymocytes; SBU-T8 preceded SBU-T4, and the number of SBU-T8+ thymocytes always exceeded the number of SBU-T4+ thymocytes throughout early gestation. At 50 days, a thymic medulla appeared and thereafter grew rapidly in size. Immunoperoxidase staining of serial sections of the fetal neck revealed cortical-type thymocytes outside the thymus from 40 days onward, before the appearance of a thymic medulla. However, by 60 days, only medullary-type thymocytes were observed either extrathymically or within the interlobular septa of the thymus, indicating that only thymocytes with a medullary phenotype leave the thymus from this stage of gestation.     

Differential expression of genes linked to the leukemia inhibitor factor signaling pathway during the estrus cycle and early pregnancy in the porcine endometrium

The objective of this study was to determine the expression profiles of leukemia inhibitory factor (LIF) and its receptor (LIFR), interleukin 6 receptor (IL6R), tumor protein p53 (TP53) and B-cell CLL/lymphoma 2 (BCL2) in the porcine endometrium on selected days of the estrous cycle and pregnancy. Time- and reproductive status (estrous cycle/pregnancy)-specific patterns of expression were identified for all investigated genes. The most pronounced changes were seen on Days 12 and 14 of pregnancy when maternal recognition of pregnancy and implantation, respectively, occurs in pigs.