Developmental Biology 281 (2005) 1–21 www.elsevier.com/locate/ydbio

Characterization of the uterine phenotype during the peri-implantation period for LIF-null, MF1 strain mice

A.A Fouladi-Nashtaa,1, C.J.P. Jonesb, N. Nijjar a, L. Mohamet a, A. Smithc, I. Chambersc, S.J. Kimber a,T

aFaculty of Life Sciences, University of Manchester, 3.239 Stopford Building, Oxford Road, Manchester M13 9PT, UK bFaculty of Medical and Human Sciences, University of Manchester, Manchester M13 9PT, UK cCentre for Development in Stem Biology, Institute for Stem Cell Research, University of Edinburgh, Edinburgh EH9 3QJ, UK

Received for publication 9 August 2004, revised 20 January 2005, accepted 21 January 2005 Available online 9 March 2005

Abstract

Leukemia inhibitory factor plays a major role in the and in its absence fail to implant. Our knowledge of the targets for LIF and the consequences of its absence is still very incomplete. In this study, we have examined the ultrastructure of the potential implantation site in LIF-null MF1 female mice compared to that of wild type animals. We also compared expression of proteins associated with implantation in luminal and stroma. Luminal epithelial cells (LE) of null animals failed to develop apical pinopods, had increased glycocalyx, and retained a columnar shape during the peri-implantation period. Stromal cells of LIF-null animals showed no evidence of decidual giant cell formation even by day 6 of pregnancy. A number of proteins normally expressed in decidualizing stroma did not increase in abundance in the LIF-null animals including desmin, tenascin, Cox-2, bone morphogenetic protein (BMP)-2 and -7, and Hoxa-10. In wild type animals, the IL-6 family member Oncostatin M (OSM) was found to be transiently expressed in the luminal epithelium on late day 4 and then in the stroma at the attachment site on days 5–6 of pregnancy, with a similar but not identical pattern to that of Cox-2. In the LIF-null animals, no OSM protein was detected in either LE or stroma adjacent to the , indicating that expression requires uterine LIF in addition to a blastocyst signal. Fucosylated epitopes: the H-type-1 antigen and those recognized by lectins from Ulex europaeus-1 and Tetragonolobus purpureus were enhanced on apical LE on day 4 of pregnancy. H-type-1 antigen remained higher on day 5, and was not reduced even by day 6 in contrast to wild type uterus. These data point to a profound disturbance of normal luminal epithelial and stromal differentiation during early pregnancy in LIF-nulls. On this background, we also obtained less than a Mendelian ratio of null offspring suggesting developmental failure. D 2005 Elsevier Inc. All rights reserved.

Keywords: LIF; Uterus; Mouse; Decidualization; Ultrastructure; Protein expression

Introduction does not decidualize and blastocysts fail to implant. However, pregnancies do result in LIF-deficient females Leukemia inhibitory factor (LIF) is a pleiotrophic following infusion or injection of exogenous LIF. LIF-null member of the interleukin 6 (IL-6) cytokine family (Haines embryos can implant after transfer into wild type pseudo- et al., 2000; Pennica et al., 1995). Considerable evidence pregnant recipient uteri, showing that embryonic LIF is not links LIF to implantation of the embryo, which occurs late essential, but wild type embryos cannot implant in null uteri on day 4 of pregnancy in mice. In LIF-null mice, the uterus (Chen et al., 2000; Stewart et al., 1992). Administration of LIF to pregnant LIF-null mice reverses their lack of decidualization as well as resulting in implantation. This T Corresponding author. Fax: +44 161 275 3915. E-mail address: [email protected] (S.J. Kimber). points to a vital role for LIF in the initiation of events 1 Present address: The University of Nottingham, School of Biosciences, preparing the uterus for an implanting embryo. In keeping Sutton Bonington Campus, LE12 5RD, UK. with this, expression of LIF mRNA peaks first on day 1 of

0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.01.033 2 A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21 pregnancy in luminal epithelium (LE) and then on day 4 of appearance of latero-basal markers in the apical membrane pregnancy in the glandular epithelium. The latter occurs (Thie et al., 1996). In luminal epithelium, a number of following the transient increase in nidatory and just molecules are downregulated or disappear at, or just after, prior to embryo attachment to the uterine epithelium for implantation, including the H-type-1 glycan (Kimber et al., implantation (Bhatt et al., 1991; Shen and Leder, 1992). 1988; Kimber and Spanswick, 2000), Muc-1 (Braga and However, LIF transcripts are also expressed in stromal cells Gendler, 1993), desmosomes (Illingworth et al., 2000), and (Fouladi Nashta et al., 2004) and by in situ hybridization tight junction proteins (Orchard and Murphy, 2002). have been localized to stroma around the implanting embryo There are no reports on the electron microscopy of the at day 5 of pregnancy (Song et al., 2000). LIF protein is also LIF-null mouse uterus and the relationship of this to detected in decidualizing stroma from day 5 (Nie et al., changes in protein expression. Therefore, we investigated 2000). It binds to its specific receptor LIF-R and by the ultrastructural characteristics of the LIF-null uterus interaction with the common IL-6 signal transduction during early pregnancy. A number of molecules have been receptor, gp-130, activates signaling in the uterus via the suggested to be regulated by LIF on day 4 of pregnancy but Jak-Stat pathway. Predominant expression of LIF-R is found this work has been at the transcript level, and there may be in the luminal epithelium where LIF activation of Stat-3 is poor correlation between mRNA and protein expression in temporally restricted to day 4 of pregnancy (Cheng et al., tissues. Little is understood about the misregulation of 2001). Current data suggest that LIF triggers changes in the proteins in LIF-null mice. Therefore, we have examined the uterus which facilitate embryo attachment and implantation. expression of proteins suggested to be direct or indirect The molecular mechanism and down stream targets by targets of LIF as well as others whose expression patterns which LIF exerts its effects and regulates the implantation suggest they are potential candidates for LIF regulation. A process is only just beginning to be understood, as is its comparison of protein expression in LE and stroma at the interaction with other factors involved in decidualization potential embryo–uterus attachment site of LIF-null females and implantation. with the implantation site of wild type animals will help to Differentiation of both luminal epithelium and under- elucidate the factors regulated by LIF and their role in lying stromal cells occurs during the pre- and peri- implantation. These studies have been undertaken on implantation period (Carson et al., 2000; Kimber and outbred MF1 mice carrying a deletion of the entire LIF Spanswick, 2000). Following appropriate ovarian steroid- open reading frame (Dani et al., 1998). Lack of implantation induced changes, together with signals from the blastocyst, and rescue by injected LIF has been confirmed. Our work stromal fibroblasts differentiate to form decidual cells that shows that a number of protein and glycosylation changes enlarge, accumulate intracellular glycogen, and become are misregulated in the LIF-null female during early polygonal and multinucleated (Abrahamsohn and Zorn, pregnancy, and the normal ultrastructural changes, which 1993). There is also an increase in vascular permeability occur between the pre- and the peri-implantation stage of together with changes in the extracellular matrix. A number pregnancy, fail to materialize. of molecular changes occur in the stroma around the implantation site during decidualization, including increased expression of Cox-2 (Lim et al., 1997; Paria et al., 2002), a Materials and methods key enzyme in prostaglandin/prostacyclin synthesis, bone morphogenetic protein-2 (BMP2) and BMP7 (Ying and Animals Zhao, 2000)membersoftheTGF-h superfamily, the homeodomain transcription factor Hoxa-10 (Lim et al., The LIF-null MF1 founder mice were provided by Dr. 1999), the intermediate filament protein desmin (Glasser Andrew Sharkey (The University of Cambridge, UK) from et al., 1987; Oliveira et al., 2000), and the enzyme an original colony generated at the Institute for Stem Cell glutathione S-transferase (GST; Murakoshi et al., 1990)as Research, University of Edinburgh, UK, from ES cells with well as extracellular matrix proteins such as laminin a targeted gene deletion (Dani et al., 1998). Since LIF-null (Glasser et al., 1987) and Tenascin-C subjacent to the female mice are infertile, propagation of LIF-null mice was luminal epithelium (Julian et al., 1994; Noda et al., 2000). achieved by mating of null males with heterozygote Luminal epithelial cells are prepared for implantation females. Female mice were placed with males overnight which takes place in the antimesometrial segment of the for mating and pregnancy was determined by the presence uterus. They become more flattened and microvilli are of a vaginal plug (day 1 of pregnancy). Wild type mice were replaced by bulbous protrusions called pinopods (Lopata produced by mating of wild type or heterozygote pairs. The et al., 2002; Murphy, 2000; Nilsson, 1966) which increase in stud LIF-null males were periodically crossed with MFI number up to day 5 postcoitus (Bansode et al., 1998). In females (Harlan, UK) to maintain the outbred stock. All rodents, these apical modifications of the uterine luminal offspring were tail tipped for genotyping. Mice were epithelium appear to mediate uptake of fluid (Enders and maintained under a controlled photoperiod of 12 h L: 12 h Nelson, 1973) and macromolecules (Parr and Parr, 1974). D with food and water supplied ad libitum. Only LIF-null Luminal epithelial cell polarity becomes less marked with the and wild type females were used for experiments and mated A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21 3 with the same genotype or occasionally heterozygous males ately either in 4% paraformaldehyde (PFA) for 4 h, or to generate pregnant animals. No differences in uterine Carnoy’s fixative (60% ethanol + 30% chloroform + 10% expression were detected with use of null or +/À males. glacial acetic acid) for 30 min–1 h and then dehydrated and Animals were killed by cervical dislocation on the required processed for wax embedding using an automatic embed- day of pregnancy and uterine tissues processed as below. ding machine. Sections (5 Am) were cut, deparaffinized, Uteri were fixed on different days of pregnancy in the rehydrated in a descending ethanol series and used for morning between 9 and 10 a.m., or on days 4 to 6 also in the immunohistochemistry. In addition, some pieces were evening between 6 and 8 p.m. All experiments and breeding frozen in OCT embedding compound cooled in liquid took place under a Home Office animal license as required nitrogen, after which 7-Am sections were cut and fixed in by the UK Home Office. 4% PFA before staining. For immuno- and lectin-staining uteri from 3 to 4 different mice were fixed and stained at Chemicals each stage for both wild type and null animals. For LIF staining, uterine pieces were fixed in 4% PFA in the All of the chemicals were from Sigma unless otherwise presence of 20% sucrose before OCT embedding for stated. Primary antibodies were used as follows: polyclonal cryosectioning. rabbit anti-mouse desmin (1:100). Goat anti-mouse COX-2 (Santa Cruz Biotechnology, California, USA) (1:50), goat Immunofluorescence staining of uterine sections anti-mouse OSM (R&D Systems, Abingdon, UK) (1:50), goat anti-murine LIF (R&D Systems and Santa Cruz) (both All deparaffinized sections were microwave treated for 1:100), rabbit anti-HOX-A10 (BABCO, Richmond, Cal- antigen retrieval with TEG buffer (1.2 g/l Tris, 0.190 g/l ifornia, USA) (1:25), affinity-purified goat anti-BMP2 EGTA in distilled water, pH: 9) followed by 30 min (N-14) (Santa Cruz Biotechnology) (1:20), rabbit anti- incubation in 50 mM NH4Cl in PBS and a further 30 min BMP7 (Biogenesis, Poole, UK) (1:20), monoclonal mouse blocking in a solution containing 1% BSA, 0.2% gelatine, anti-glutathione S-transferase, GST (Abcam, Cambridge, 0.05% saponin in PBS. The sections were incubated with UK) (1:50), affinity-purified goat anti-Tenascin C (Santa primary antibody diluted in 0.1% BSA, 0.3% Triton X-100 Cruz Biotechnology) (1:50), monoclonal antibody 11-5F in PBS overnight at 48C. After washing with 0.1% BSA, against desmoplakin (gift of Prof. D. Garrod, University of 0.2% gelatine, 0.05% saponin in PBS, they were incubated Manchester) (1:10), rabbit anti-claudin-1 and ZO-1 (Zymed, with an appropriate affinity-purified FITC-conjugated sec- Cambridge BioScience, Cambridge, UK), mouse monoclo- ondary antibody (1:250 donkey anti-goat or -rabbit anti- nal 667/9E9 against H-type-1 antigen (Monocarb Lund bodies) (Jackson Immunoresearch Laboratories), washed, Sweden) (1:20), rabbit anti-Muc-1 (a gift of Dr. Sandra and incubated with 5 Ag/ml Hoescht (33342) for 5 min. Gendler Mayo clinic Scottsdale) (1:50). Fluorescein iso- They were mounted using hydrophilic mounting media thiocyanate (FITC) conjugated donkey anti-goat, rat, or containing anti-fading reagent, Gelvatol. rabbit IgGs (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used as secondary antibodies at a Isolation and immunocytochemical staining of endometrial dilution of 1 in 125. FITC anti-digoxigenin (Boehringer epithelial cell sheets Mannheim, Lewes, UK) was used at a dilution of 1:250. Normal goat serum (NGS Sigma) was used at a dilution of Uterine horns were trimmed free of connective tissues, 1:20 for blocking non-specific binding. Texas Red-X cut in half and incubated in 0.5% dispase solution for 2 h phalloidin was purchased from Molecular Probes, Leiden, at RT. Luminal epithelium (LE) sheets were squeezed out The Netherlands (1:50). of horns using an L-shaped glass rod as described previously (Illingworth et al., 2000). Isolated LE sheets Injection of trypan blue were cut in pieces and mounted on 13-mm round cover- slips, air-dried, fixed in a solution of acetone–methanol In order to identify implantation sites (if present) in (1:1) for 10 min at À208C, rehydrated with PBS for 5 min uterine horns, all animals from day 4 p.m. onwards were and incubated in 1 in 20 normal goat serum before injected with 100 Al of 0.4% (w/v) trypan blue dye into the incubating overnight at 48C with primary antibodies tail vein (Psychoyos, 1993). After 10 min, these animals against tight junction proteins ZO-1, claudin-1, and were killed by cervical dislocation and the intensity of the desmoplakin (11.5F). The coverslips were then washed blue bands around the implantation sites was observed and with PBS and incubated for 2 h at RT with an appropriate recorded before fixation. affinity-purified FITC-conjugated secondary antibody (1:250 donkey anti-goat or -mouse antibodies) (Jackson Fixation Immunoresearch Laboratories) containing 10 Ag/ml Phal- loidin, washed, and incubated with 5 Ag/ml Hoescht For immunohistochemical staining, pieces of uterine (33342) for 5 min. They were mounted using hydrophilic horn containing the implantation site were fixed immedi- mounting media containing anti-fading reagent, Gelvatol. 4 A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21

Lectin binding KFeCn in 0.1 M sodium cacodylate buffer (pH 7.3, 270 mosM) containing 2 mM calcium chloride. Fixed pieces of Semi-thin sections 0.75 Am thick were cut on a Reichert uterine horn were postfixed in 1% osmium tetroxide and OMUIII ultramicrotome and mounted on APES-coated embedded in Taab epoxy resin (Taab Laboratories Equip- slides. Resin was removed with saturated sodium ethoxide ment Ltd., Aldermaston, UK). Three to four implantation diluted 1:1 with absolute ethanol, and sections were stained sites from different animals were examined at each stage for with a panel of 15 biotinylated lectins (Table 1)as wild type and null. Semi-thin sections 0.5 Am thick were previously described (Jones et al., 1998). Briefly, endoge- cut on a Reichert OMUIII ultramicrotome and stained with nous peroxidase was blocked with 10% (v/v) hydrogen toluidine blue and only those specimens containing the peroxide (BDH, Pool, UK) before exposure to 0.03% embryo were selected for ultra-thin sectioning. Pale gold trypsin (w/v) (Type II crude) in 0.05 M Tris buffered saline sections were cut and mounted on copper grids, contrasted (TBS) pH 7.6 for 4 min at 378C. After washing, sections with uranyl acetate and lead citrate and examined in a were incubated with 10 Ag/ml biotinylated lectin (MAA and Philips 301 electron microscope at an accelerating voltage SNA from Boehringer Mannheim, Lewes, UK; LFA and of 60 kV. AAA from EY Laboratories, San Mateo, CA, USA, and all others from Sigma) in 0.05 M TBS containing 1 mM calcium chloride (1 h at 378C), washed and treated with 5 Results Ag/ml avidin peroxidase (Sigma) in 0.125 M TBS pH 7.6 with 0.347 M sodium chloride (1 h at 378C) (Jones et al., Phenotype evaluation and histological assessment of the 1987). Sections were washed and sites of lectin binding uterus revealed with 0.05% (w/v) diaminobenzidine tetrahydro- chloride dihydrate (Aldrich Chemical Co., Gillingham, UK) The number of offspring obtained from heterozygous in 0.05 M TBS pH 7.6 and 0.015% (v/v) hydrogen peroxide crossing or crossing heterozygote females with LIF-null (30 volumes) for 5 min at 188C. Sections were rinsed, air- males was recorded (Table 2). From heterozygous matings, dried, and mounted in neutral synthetic mounting medium an average of only 17% null progeny was obtained, (BDH). Controls were as previously described (Jones et al., irrespective of sex, instead of the expected 25%, and 1997) and sections stained with WGA, SNA, MAA, and similarly null males mated with heterozygous females gave LFA were also exposed to neuraminidase [0.1 U/ml, Type only 27% null progeny instead of 50% for a Mendelian VI from Clostridium perfringens (see Jones et al., 1992)] to ratio. The number of heterozygous offspring was also cleave off terminal sialic acid before lectin incubation. reduced from the expected 50% after +/À Â +/À crossings, Sections were examined and staining intensity graded on a but no obvious differences were found in the sex ratio of numerical scale from 0 (negative) to 4 (intense). offspring of any genotype. Following trypan blue injection, blue staining at the Transmission electron microscopy implantation site indicated the degree of vascular perme- abilization, an indicator of the decidualization response. The Uterine horns were cut into segments and fixed for 1 h blue reaction was compared in uterine horns of LIF-null and using 2.5% glutaraldehyde, 1% paraformaldehyde 0, 0.1% wild type mice on the evening of day 4 through to day 7 after mating. A total of 22 LIF-null and 15 wild type mice Table 1 was injected. The blue reaction was observed in all of the Lectins used in this study and their major sugar specificities wild type mice. The intensity of the blue band was faint on Acronym Source Major specificity the evening of day 4, increased on day 5 and was strongest AAA Anguilla H type 1 antigen (Fuca1,2Galh1, on day 6; by day 5, the color had diffused slightly away anguilla Eel 3GlcNAch1), and Lewisa from the implantation point. In contrast, no blue reaction UEA-1 Ulex europaeus-1 H type 2 antigen (Fuca1,2Galh1, was observed in LIF-null mice on the evening of day 4 and Gorse 4GlcNAch1-) and Lewisy also day 7. Only 1 out of 7 mice inspected on both days 5 l LTA Tetragonolobus -fucosyl terminals (especially where and 6 showed a very faint blue reaction at presumptive purpureus Lotus clustered), Fuca1,6GlcNAc NFuca1, 2Galh1,4(Fuca1,3)GlcNAch implantation sites. WFA Wisteria floribunda GalNAca1,6Galh1- NGalNAca1,3Galh1- A comparison of the histological features of the mouse Wisteria uterus on days 5–6 after mating is shown for wild type and SNA Sambucus nigra NeuNAca2,6Gal/GalNAc- LIF-null mice in Fig. 1. In the wild type mice, there was Elderberry bark marked evidence that proliferation and differentiation of MAA Maackia amurensis NeuNAca2,3Galh1- LFA Limas flavus Certain sialyl residues stromal cells had occurred. Cell shape changed from Yellow slug fibroblastic to epithelioid and both closure of the uterine WGA Triticum vulgaris Di-N-acetyl chitobiose, N-acetyl lumen and close apposition of the implanting embryo were Wheatgerm lactosamine (especially if clustered) apparent. In contrast, the uteri of LIF-null mice did not show and some sialyl residues these changes and appeared similar to those of mice in the A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21 5

Table 2 Number of offspring born from crosses of female mice heterozygous for deletion of the LIF gene and homozygous and heterozygous male mice Mating Offspring Null (%) Hetero (%) Wild type (%) Total (%) Hetero h  hetero U Male 21 (14.9) 43 (30.5) 77 (54.6) 141 (47.3) Female 29 (18.5) 36 (22.9) 92 (58.6) 157 (52.7) Total 50 (16.8) 79 (26.5) 169 (56.7) 298

Null h  hetero U Male 33 (23.1) 110 (76.9) 0 143 (52.4) Female 40 (30.8) 90 (69.2) 0 130 (47.6) Total 73 (27) 200 (73) 0 273 first few days of pregnancy, although uterine closure was type (Figs. 2C, D). In this strain, even by 7 p.m. on day 4, sometimes observed. apical microvilli have only partially given way to pinopods on apical wild type LE, but by the morning of Transmission electron microscopy day 5, pinopods were present over the entire apical LE surface and in close association with the embryonic The ultrastructure of the implantation site and changes trophectoderm at the implantation site (Figs. 3A–D). The in the uterine luminal epithelium and underlying stromal stromal cells were decidualized and enlarged, showing cells are presented in Figs. 2–4. No differences were occasional binucleate cells and deposition of glycogen in observed between wild type and LIF-null mice in the LE the cytoplasm (Fig. 3E). In the LIF-null mice, no pinopods from uteri fixed on the morning of day 4 of pregnancy were formed on the apical surface of the LE on day 5 and (Fig. 2) except for a rather more pronounced columnar there was an absence of intimate contacts indenting the TE shape in the nulls (Fig. 2B). However, in the wild type surface (Figs. 3C, D). In 2 of the implantation sites, some uterus, stromal cell shape had changed from long contact was seen but only between the microvilla tips and fibroblastic profiles to a round or polygonal form by the the TE surface (Fig. 3D).The luminal epithelial cells evening of day 4 (Fig. 2E). On the morning of day 4, no retained their columnar shape (Fig. 3F). By day 6, the pinopods were yet observed on the apical surface of LE wild type LE layer had degenerated and trophoblast cells in MF1 wild type mice or in the LIF-null animals. penetration was underway (Figs. 4A, C). By this time, However, the abundance of glycocalyx on apical LE the stromal cells were markedly enlarged (Fig. 4E) with microvilli was noticeably greater in the null than the wild loss of extracellular matrix and the development of

Fig. 1. Histological features of uterus in LIF-null and wild type mice. Semi-thin resin sections from days 5 and 6 of pregnancy were stained with toluidine blue. The insets show high magnification of differentiated polygonal stromal cells in the wild type and undifferentiated fibroblast like stroma cells in the LIF-null mice on day 6 of pregnancy. E = embryo, scale bar = 50 Am. 6 A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21

Fig. 2. Ultrastructure of uterus in LIF À/À and wild type mice on day 4 a.m. of pregnancy. On day 4, both the wild type (A) and null mouse (B) have columnar luminal uterine epithelium (LE) bearing microvilli (arrowheads), though more lipid (Li) is apparent in the basal areas of the null cells. Scale bars: 10 Am. The microvilli of the wild type mouse (C) have less glycocalyx than the null animal (D) which has a prominent filamentous covering over many microvillous processes (arrows). Scale bars: 0.5 Am. Apart from a slight enlargement of the nuclei in the wild type (E) compared with null (F) mice, there is little difference in the ultrastructure of stromal cells underlying the luminal epithelium (LE) at this stage. Scale bars: 10 Am. intercellular contacts. The LIF-null mice, however, showed type mice, H-type-1 was expressed at the highest level on only the features of a day 1–3 uterus, with the embryo day 3 and then had decreased by day 5, becoming remaining in the uterine lumen (Fig. 4B), an absence of undetectable in the day 6 uterus, while in LIF-null mice, changes in LE such as development of pinopods and none it remained at a high level during this period (Fig. 5). of the ultrastructural stromal changes such as binucleate, Indeed, in the LIF-null uterus, H-type-1 staining was enlarged, glycogen rich cells, and development of close higher on day 5 of pregnancy than in wild type or null contact indicating decidualization (Figs. 4D, F). The LE animals at day 3 of pregnancy. The intensity of staining for microvilli did contact the trophectoderm surface in one Sialy-Lex was high on days 3–4 of pregnancy and then day 6 embryo, but only at their tips with none of the decreased in wild type uterus, as previously reported intimate contact that was observed for day 5 wild type (Kimber et al., 2001). In LIF-null animals on days 3–4 embryos. of pregnancy, staining was moderately bright but it had become negligible by day 5 after mating (Fig. 5). Some Changes in glycosylation staining was evident on day 6. No differences were found for Muc-1 glycoprotein Wild type and null LE cells showed marked differences between null and wild type animals (Fig. 6) This molecule in expression of the fucosylated H-type-1 antigen. In wild was reduced in both wild type and null animals at the time A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21 7

Fig. 3. Ultrastructure of implantation site and the stroma layer in LIF À/À and wild type mouse uterus on day 5 a.m. of pregnancy. Blastocysts (E) in wild type (A) and null (B) mice abut the luminal uterine epithelium (LE). Scale bars: (A) 10 Am, (B) 5 Am. At higher power, it can be seen that in the wild type (C), pinopods (Ud) of the luminal epithelium make close contact, sometimes indenting the surface of the blastocyst (arrows) whereas in the null mouse (D), the blastocyst is only contacted by the tips of untransformed microvilli and no intimate contact is made. Scale bars: (C) 1 Am, (D) 0.5 Am. In the stroma (St) immediately underlying the luminal epithelium (LE), wild type fibroblasts are admixed with leucocytes (Le) and their nuclei are more euchromatic than those of the null mouse (F) which contain a greater proportion of heterochromatin. Scale bars: 10 Am. of implantation as previously reported in C57BL6/J (Chen staining was also stronger in LIF-null mice throughout the et al., 2000). period studied, while SNA had a similar pattern in the two genotypes until day 5 after which staining was reduced in Lectin binding wild type but remained intense in LIF-null mice. There were no notable differences between wild type and null animals The results of lectin staining are presented in Table 3. on any day of pregnancy for any of the other lectins tested in The intensity of staining for a panel of 8 different lectins this study. plus 4 controls was compared between LIF-null and wild type mice on days 3 to 6 after mating. LTA binding was Immunostaining of junctional proteins in the consistently higher in LE and GE on days 3, and 4 a.m. and luminal epithelium p.m. in LIF-null than in wild type mice. There were no differences on days 5–6 in these animals (Fig. 7). UEA-1 Luminal epithelial sheets were immunostained for binding was also consistently more intense on day 3 and day junctional proteins ZO-1, claudin, and desmoplakin. The 4 a.m. and p.m. in the LIF-null mice while being weak and intensity of staining for each protein and the depth below patchy in the wild types. Binding was then reduced on day 5 the apical surface from which signal was obtained in the and disappeared on day 6 in both genotypes (Fig. 7). WFA cell layer as measured in optically sectioned images by 8 A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21

Fig. 4. Ultrastructure of implantation site and the stroma layer in LIF À/À and wild type mouse uterus on day 6 a.m. after mating. In the wild type mouse (A), the luminal epithelium has apoptosed and the trophectoderm (TE) now overlies compacted stromal cells (St), while in the null animal (B), the blastocyst (E) has detached from the luminal epithelium (LE) and has not developed further. PE = Parietal endoderm. Scale bars: 10 Am. Underneath the TE, close contacts between stromal fibroblasts are evident (C, arrow); in the null mouse (D), it can be seen that the luminal epithelium (LE) still retains its microvillous surface (arrows). Scale bars: (C) 5 Am, (D) 1 Am. In the deep stroma of the wild type mouse (E), intercellular contacts are well developed (arrows) and deposits of glycogen (Gly) can be seen (F). There is little change in the stroma (St) of the null animal (G). Scale bars: (E) 5 m, (F) 2 Am, (G) 10 Am. confocal microscopy, was recorded (Fig. 8, Table 4). The p.m.) or day 5 morning (Fig. 9). No staining was seen in results indicated no apparent differences between the LIF- the uterus of the LIF-null mice at any time (data not null and wild type uteri in the expression and distribution shown). of junctional proteins in the luminal epithelium. Weak Cox-2 staining was observed in both the GE and LE on day 4 in both LIF-null and wild type mice. On Immunohistochemical examination of stromal components day 5, in wild type females, it was only expressed with high intensity in both the luminal epithelium and the Sections of uterine horns were stained with antibodies stromal cells immediately around the embryo at the against several known proteins expressed in the implanta- implantation site. The area of expression and intensity tion site. The results are presented in Figs. 9–13. of staining in the stromal cells had expanded by days 6 LIF protein, as expected, was expressed in GE of wild and 7. In LIF-null animals, expression of Cox-2 was type mice on day 4 morning and to a lesser extent on day 4 detected on day 5 and was restricted to LE cells. It was evening. It was undetectable by the morning of day 5 of greatly reduced by day 6 when it was seen in a few pregnancy. No staining was seen in the stroma or LE stromal cells near the embryo which was still free in the surrounding the implantation site on day 4 evening (10 uterine lumen (Fig. 10). A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21 9

Fig. 5. Immunofluorescence staining for glycosylation changes in LIF À/À and wild type mouse uterus. Frozen sections from days 3 to 6 after mating were stained for H-type-1 and Sialy-Lex which is expressed in the luminal epithelium. Note the retention of H-type-1 staining on days 5 and 6. Scale bar = 50 Am.

In wild type mice, the IL-6 family cytokine Oncostatin 7. In contrast, the null animals showed a reduction in desmin M followed a similar but not identical pattern of staining from day 5 and only patches of desmin-positive expression to Cox-2. It was first expressed in luminal stromal cells were observed on day 6 (Fig. 11) although epithelial cells adjacent to the embryo on day 5 of staining intensity for desmin was increased in stroma pregnancy with a few adjacent positive stromal cells. immediately adjacent to the embryo. Later, by day 6, lower intensity staining was seen in the The homeodomain transcription factor, Hoxa-10, a mesometrial stroma adjacent to the embryo. In LIF-null marker for decidualization of stromal cells, showed the mice, Oncostatin M was not detected on any days of expected increased expression in the stromal layer of wild pregnancy (Fig. 10). type mice from days 5 to 7 of pregnancy. Staining was There was little difference between wild type and LIF- particularly strong in the decidualized stroma around the null mice in staining for the intermediate filament protein, embryo on day 6. However, in the LIF-nulls, although desmin, on day 4 of pregnancy. On day 5, in the wild type, Hoxa-10 protein showed some increase on day 5 adjacent to desmin was highly expressed in the primary decidual zone the embryo, compared to day 4, it remained generally at a and it spread to the secondary decidual zone on days 6 and low level in LIF-null uterus on day 6 (Fig. 11). 10 A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21

Fig. 6. Immunofluorescence staining for MUC-1 in LIF À/À and wild type mouse uterus. Frozen sections from days 3 to 5 after mating were stained with rabbit antibody to MUC-1. The strongest expression was observed in luminal epithelium on day 3 and this decreased by day 5 in both LIF-null and wild type mice. Scale bar = 50 Am.

In wild type mice, BMP2 staining was very low in the was seen on day 6 at which time a gradient of protein stroma on day 4 although marked glandular staining was expression was seen with highest levels adjacent to the evident. On days 5 and 6, it was highly expressed around the embryo. Much reduced staining was seen in null stroma, implanting embryo in the primary decidual zone and also although weak fluorescence could be seen in day 6 stroma within the myometrium. In contrast, in LIF-null mice, BMP2 together with punctate staining beneath the myometrium. staining was low or negligible on day 4 and continued at a The embryo, particularly the trophectoderm and primitive low level on later days except for some moderate epithelial endoderm, was also strongly stained on all 3 days in the wild staining seen on all days. BMP2 was also highly expressed type, and days 4 and 5 in the null mice (Fig. 12). in day 4 blastocysts in both wild type and LIF-null mice Glutathione S-transferase (GST) was expressed in both (Fig. 12). BMP-7 staining was evident on day 4 and strong LIF-null and wild type uteri in the stroma layer and in both in the wild type uterus immediately adjacent to the embryos LE and GE cells. On day 3, it was mainly expressed in the and in the primary decidual zone on day 5. Similar staining myometrium and very weakly in the LE cells. Levels

Table 3 Lectin-binding patterns in LIF À/À and wild type murine uterus Lectin Day 3 Day 4 a.m. Day 4 p.m. Day 5 Day 6 +/+ À/À +/+ À/À +/+ À/À +/+ À/À +/+ À/À LTA 1–2 3–4 0–1 2–3 1 2–3 1–2 1–2 3 2 UEA-1 1–2 3–4 0–1 2–3 0–1 3–4 0–1 0–1 0 0 WFA 1–2 3–4 1 3 2 3–4 2–3 3 1–2 2–3 AAA 0 1–2 0 0–1 0 1 0 0 0 0 SNA44 44 44 44 1–24 SNA + NA 4 4 3 3–4 4 4 3–4 3–4 1–2 4 MAA 1–2 1–2 2 1–2 2 1–3 1–2 2–3 1–2 2 MAA+NA0000000000 LFA 3 1–3 0–3 0–3 2–3 0–3 0–3 0–3 0–1 1–3 LFA+NA0000 00 00 00 WGA 3 3 1–3 1–3 1–3 1–3 0–3 0–3 0–2 0–3 WGA + NA 2–3 2–3 2–3 2–3 2–3 1–3 1–3 2–3 0–3 0–2 0: Negative, 1: weak, 2: moderate, 3: strong, 4: intense staining. A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21 11

Fig. 7. Lectin histochemistry of LIF À/À and wild type mouse uterus on days 3, 4 (morning) and 4 (evening) using Tetragonolobus purpureus (LTA) and Ulex europaeus-1 (UEA-1) agglutinins. There was stronger staining for both LTA and UEA-1 in the LIF-null apical LE compared with the equivalent wild type apical LE. Arrows delineate the faint outline of the uterine lumen in the wild type specimens. Scale bar = 100 Am. increased in the stroma and become stronger in the LE during from both heterozygous matings and null males  hetero- days 4 to 6 after pregnancy. In the wild type, there was zygous females. Analysis indicated that the number of nulls intense staining in the secondary decidual zone (Fig. 13) was 54–67% that expected for a Mendelian ratio, for both which is absent in the LIF-null animals. However, GST males and females. The lower proportion of LIF-null staining was observed in the stroma both near and away from offspring indicates some embryo loss in the MF1 strain of the embryo outside the decidual zone. In both wild type and mouse. It has been reported that, on an inbred C57BL6/J LIF-null mice, specific tenascin-C staining was negligible on background, there is no loss of null offspring in utero and day 4 although some low-level non-specific reactivity with Mendelian frequencies are obtained (Juriloff et al., 2000; the luminal surface was observed on days 4 and 5 but Stewart et al., 1992). LIF has a variety of effects on different disappeared on day 6. In wild type mice, staining was present cell types in vitro, inhibiting the differentiation of embryonic in the stromal cells adjacent to the LE at the attachment site stem cells and promoting the survival and/or proliferation of and also in a few cells in the primary decidual zone on days 5 neurons, primitive hematopoietic precursors, and primordial and 6 of pregnancy. In contrast, no staining was observed in germ cells (Hilton, 1992). LIF-deficient mice derived by the stromal cells of LIF-null mice (Fig. 13). Other proteins gene targeting techniques have dramatically decreased examined, vitronectin, cardiotrophin-1, and thrombospon- numbers of stem cells in spleen and bone marrow, while din-3 were all detected at low levels in the stroma but heterozygous animals are intermediate in phenotype, imply- showed no difference in staining between wild type and null ing that LIF has a dosage effect; defects in stem cell number (data not shown). can be compensated for by the introduction of exogenous LIF (Escary et al., 1993). Recently, it has been reported that mice doubly deficient in LIF and IGF-I all died at birth of Discussion apparent respiratory failure (Pichel et al., 2003). It is also notable that mice in which the LIF-R is deleted show In order to build up a better picture of the cellular multiple fetal and placental anomalies and die at or before aberrations which contribute to lack of implantation in LIF- birth (Ware et al., 1995) while deletion of Stat-3 leads to null mice, we have assessed the histological and ultra- embryo degeneration after day 6 (Takeda et al., 1997) and structural features of the LIF-null uterus over the implanta- embryos with a deletion in gp-130 die between day 12.5 and tion period. We have also compared expression of proteins term (Yoshida et al., 1996). On the MF1 background, LIF- and glycans which change during, or are associated with, nulls appeared slightly smaller than wild type mice, but no implantation between LIF deficient and wild type mouse apparent abnormalities were observed. Thus, the multi- uteri. Once the breeding colony was set up, one of our first functionality of LIF and its important role in neuron and stem observations was the small numbers of LIF-null offspring cell development may account for the reduced number of 12 A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21

Fig. 8. Confocal microscopic analysis of junctional proteins in the luminal epithelium of LIF À/À and wild type mouse uterus. Luminal epithelial sheets were isolated from uterine horns and stained for tight junction proteins ZO-1 and claudin-1, desmoplakin protein (green) and also the nuclear stain Hoescht (blue) and Texas red-conjugated phalloidin (red). A Z series of vertical images were captured using multiphoton microscopy to compare any changes in the depth of protein staining down the lateral membranes (Illingworth et al., 2000) and intensity of staining for these proteins on different days of pregnancy in LIF-null and the equivalent wild type mice. Scale bar = 20 Am.

LIF-null offspring. The timing of embryonic loss still epithelial and stromal cells do not occur in the LIF-null requires investigation. animals. The LE cells remain columnar (rather than becoming cuboidal) and retain microvilli with an abundant Ultrastructure and glycosylation at the apical LE surface glycocalyx on their apical surface. The trophectoderm was sometimes seen to develop close but not intimate contact Examination of the ultrastructure at the potential implan- with these microvilli on days 5–6 of pregnancy. The null tation site indicated that the normal changes in both luminal epithelium did not develop the bulbous pinopods characteristic of the wild type uterus as it moves into the Table 4 receptive period (Lopata et al., 2002; Murphy, 2000). Semi-quantitative analysis in confocal microscope images of junctional Pinopod formation has been shown to be stimulated by proteins staining in LE cells of LIF À/À and wild type murine uterus and and inhibited by estrogen (Murphy, 2000; Parr, each 1-Am section scored for staining 1983) which at first site seems at odds with the known Claudin ZO-1 Desmoplakin stimulation of LIF expression by estrogen in the mouse +/+ À/À +/+ À/À +/+ À/À (Chen et al., 2000; Stewart et al., 1992). However, a recent Day1554444 study suggests that LIF regulates a number of progesterone Day 4 6 5–7 6 5–6 4–5 4 responsive molecules in the uterus. This may suggest that Day 5 3 3 2–3 3 3 3 some component of the LIF signaling pathway is stimulated Numbers equal the number of optical sections carrying staining. by progesterone. The human equivalent of pinopods, A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21 13

Fig. 9. Staining of transverse sections of wild type uterus on day 4 morning (A,D), day 4 evening (B,E) and day 5 of pregnancy (C,F) for LIF. (A–C) Stained with Goat anti murine LIF; (D–E) Dapi nuclear stain. Note staining of glandular epithelium only on day 4 morning and evening (inset). E = embryo, GE = glandular epithelium, LE = Luminal epithelium, St = Stroma. Scale bar =100 Am. uterodomes, are not pinocytotic but have been associated seem to occur on schedule in the LIF-null uterus. The with receptivity (Nikas et al., 1995) and it has been reduction in fucosylated structures in wild type mice around suggested that they carry potential adhesion molecules the time of implantation may indicate a function to protect (Bentin-Ley et al., 1999). It is possible that the failure of the mucosal layer and as a barrier against the implanting embryos to develop intimate association with the LE of LIF- embryo. If this is true, it would seem that this barrier is null mice relates to the lack of phenotypic pinopod retained for several days in the absence of LIF. The formation by these cells. Hoxa-10 is also required for H-type-1 antigen, which our previous data have implicated pinopod formation and after Hoxa-10-antisense treatment in implantation (Kimber et al., 2001; Lindenberg et al., pinopods do not appear on the luminal epithelium at the start 1988), showed a dramatic enhancement adjacent to the of the period of receptivity (Bagot et al., 2001). Thus, both embryo in the null uterus on day 5 of pregnancy and was not Hoxa-10 and LIF appear to drive pinopod formation on the downregulated on day 6 of pregnancy when, in the wild LE. Pinopods are present on LE in experimentally delayed type, the uterus becomes refractory to embryo-implantation. implantation, after ovariectomy and progesterone delivery to H-type-1 antigen present on the LE is thought to interact pregnant rats (Psychoyos and Mandon, 1971) indicating that with components of the trophectoderm cell surface facilitat- the uterine phenotype of the LIF-null uterus is not ing embryo attachment (Poirier and Kimber, 1997). Con- equivalent to that in delay of implantation. tinued presence of H-type-1 on day 6 LE cells may indicate The failure to develop pinopods in LIF-null mice is also that LIF, either directly or indirectly, induces its down- associated with retention of glycosyl residues on the surface regulation. In previous work, we showed that H-type-1 of the LE, as shown by the continued presence of H-type-1 antigen expression requires estrogen and is inhibited by and fucosylated molecules bound by UEA-1 and LTA progesterone and that in the wild type it is downregulated lectins. In wild type animals, these fucosylated molecules more or less on schedule in pseudopregnant animals in the decrease greatly just before (UEA-1 LTA), or after (H-type-1) absence of an embryo (Kimber and Lindenberg, 1990; implantation. A number of changes in LE glycosylation are White and Kimber, 1994). Thus, its upregulation in peri- known to occur in the pre- and peri-implantation period implantation null females may reflect lack of LIF-stimula- (Kimber et al., 1988; Kimber et al., 2001). Change in tion of certain progesterone-regulated molecules. Since the glycosylation is under maternal control (Jones et al., 1993; embryo makes only superficial contact with the LE in the White and Kimber, 1994) and may also occur in the LIF-null uterus, it is clear that the increase in this epitope is remodeling of the extracellular matrix (Kleinfeld et al., insufficient to drive normal intimate attachment to LE 1976; Mulholland and Jones, 1993). A reduction in the LE without other changes taking place. It is possible that it has a glycocalyx, which is rich in various glycosylated molecules, dual function: as a component of a barrier against embryo at implantation has previously been reported (Chavez and attachment, or forming part of an attachment molecule Anderson, 1985) and our EM data show that this does not depending on the molecular context, e.g., whether ligands 14 A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21

Fig. 10. Immunofluorescence staining of COX-2 and Oncostatin-M in LIF À/À and wild type mouse uterus. There was very weak staining for COX-2 in both luminal and glandular epithelium on day 4 after mating in both null and wild type animals. COX-2 is strongly expressed at the luminal epithelium and underlying stromal cells at the implantation site on day 5 and expression extended deeper into the stroma by day 6. In LIF-null mice, expression was limited to the LE cells and only a few stromal cells expressed COX-2 in the day 6 uterus. The pattern of expression for Oncostatin M in wild type mice was similar to COX-2. In LIF- null animals, Oncostatin M was absent around the embryo on days 5 and 6 of pregnancy. E = embryo, St = stroma layer, LE = luminal epithelium. which bind H-type-1 are also expressed on the LE and make inhibited either directly or indirectly by LIF. In any case, we it unavailable for engagement with the embryo. Sialyl-Lex is have revealed an increase in fucosylation of the apical LE also regulated over the implantation period (Kimber et al., surface at days 4–5 of pregnancy in the LIF-null animals by 2001), and in LIF-null mice, this epitope showed similar two independent approaches. changes to those seen in the wild type although staining was Downregulation of the anti-adhesive, mucin of the weaker. However, the fucose-binding lectins showed similar glycocalyx, Muc-1, correlates with LE receptivity in mice differences from the wild type to those seen with antibody to (Braga and Gendler, 1993; Surveyor et al., 1995). This the a1–2 fucosylated H-type-1antigen (Kimber et al., 1988), protein disappeared on schedule from LE in LIF-null mice although in wild type animals H-type 1 is downregulated at as has already been reported for MUC-1 transcripts (Chen least a day later than the lectin-binding epitopes. This et al., 2000). The expression of Muc-1 is controlled by indicates that reduction of H-type-1 antigen may be ovarian steroids and changes during the normal estrus cycle regulated differently from these other fucosylated structures (Braga and Gendler, 1993; Surveyor et al., 1995) but it is recognized by LTA and UEA-1 but expression of all is clearly regulated independently from the pathway involving A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21 15

Fig. 11. Immunofluorescence staining of the decidual intermediate filament marker desmin and Hoxa-10 in LIF À/À and wild type mouse uterus. Desmin was expressed in the primary decidual zone of wild type uterus on day 5 and then expanded to the secondary decidual zone. In the LIF-null animals, limited numbers of uterine stromal cells in the stroma expressed desmin on day 6. Hoxa-10 was also expressed strongly in wild type primary decidual zone on days 5–6 but staining in the equivalent region of the LIF-null mice was weak. E = embryo, St = stroma layer, LE = luminal epithelium.

LIF. Although Muc-1 has been thought of as an important between wild type and LIF-null animals. It is still possible barrier to implantation in mice, the absence of Muc-1 alone that a more detailed quantitative ultrastructural examination is insufficient to allow normal interaction of the trophecto- may reveal more subtle differences. derm with the LE in LIF-null uterus, since our ultra- structural examination revealed that normal trophectoderm– Proteins associated with decidualization LE interaction does not take place. Since changes in junctional components in LE have been Although aberrations in the expression of a number of documented at the time of implantation, the distribution of mRNA species associated with decidualization have been desmosomes and tight junction proteins (Illingworth et al., reported in recent years (Daikoku et al., 2004; Rodriguez et 2000; Murphy et al., 1982) was examined in epithelial al., 2004; Song et al., 2000), relatively little information is sheets from the two sets of animals. By confocal micro- available on protein expression. We therefore compared scopy, we were unable to detect any differences in the expression of a number of proteins associated with decidu- distribution of components of either type of junction alization between LIF-null and wild type mice. A number of 16 A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21

Fig. 12. Immunofluorescence staining of BMP-2 and BMP-7 proteins in LIF À/À and wild type mouse uterus. Both BMP-2 and BMP-7 were strongly expressed in the primary decidual zone (PDZ) of wild type mice on days 5 and 6 uterus. In the LIF-null uterus, BMP-2 expression was low or negligible and BMP7 was weakly expressed in the stroma adjacent to the embryo. E = embryo, GE = glandular epithelium, LE = luminal epithelium, St = stroma layer. Scale bar = 100 Am. these molecules such as desmin, Hoxa-10, BMP-2 BMP-7 and later, at day 7, in the secondary decidual zone (Paria were either absent or weakly expressed in the LIF-null et al., 2001) especially at the mesometrial side (Ying and stroma around the embryo at days 5 to 6 after mating. Zhao, 2000). On days 5 and 6 of pregnancy, we found Desmin is classically used as a marker of decidualization in BMP2 protein expression in the primary decidual zone and rodents. Desmin-containing intermediate filaments appear in the myometrium of wild type mice in agreement with the during decidualization in uterine stromal cells from day 5 mRNA distribution. However, on the morning of day 4 of and are reduced after final differentiation on day 8 post- pregnancy, marked glandular staining was noticed. In the conception (Glasser et al., 1987; Oliveira et al., 2000). Our LIF-null uterus, BMP-2 signal was only weak and general observations are in agreement with the published pattern of on days 4, 5, and 6. BMP7 mRNA was reported in the desmin expression in wild type mice, while in the LIF-null decidualizing wild type stromal cells surrounding the females, only low expression was observed. blastocyst, with highest levels near the uterine epithelium Bone morphogenetic proteins (BMPs) are pleiotropic at 4.5 days postcoitus. With the progression of decidualiza- growth and differentiation factors belonging to the TGF-h tion, BMP7 signals in the decidua at the antimesometrial superfamily. Transcripts for BMP2 have been detected first side decreased, but strong signals were retained in the in stroma around the attaching embryo then in the primary decidual area at the mesometrial side on day 7 (Ying and A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21 17

Fig. 13. Immunofluorescence staining of glutathione S-transferase and tenascin-C in LIF À/À and wild type mice uterus. GST was expressed in both LIF-null and wild type uteri in the stroma layer and in both LE and GE cells. In the wild type, there was intense staining in the secondary decidual zone. Tenascin-C was expressed in the stroma cells adjacent to the implanting embryo on day 5. Expression can be seen in further scattered stromal cells immediately subjacent to the embryo on day 6. No expression in this region was detected in the LIF-null mice uterus, although there was some non-specific staining of the luminal epithelium. Scale bar = 100 Am, E = embryo, St = stroma layer, LE = luminal epithelium. GE = glandular epithelium.

Zhao, 2000). Our study supports these findings, showing a implantation embryo is required for differentiation of similar pattern for BMP7 protein in wild type uterus, but in visceral endoderm and cavitation of embryoid bodies LIF-null mice, there was no increase in staining in the (Coucouvanis and Martin, 1999) but neither BMP2 nor stroma adjacent to the embryo at the expected time of BMP4 induce a decidualization response in the mouse implantation. BMP2 and 7 protein expression is therefore uterus, unlike HB-EGF and IGF-1 (Paria et al., 2000). affected by the absence of LIF but this may be an indirect Glutathione S-transferases (GST) are believed to be effect, secondary to the absence of induction of the involved in the protection mechanisms against lipid decidualized stromal phenotype, and does not necessarily peroxidation (Awasthi et al., 1980) and the detoxification suggest a direct regulation of BMP expression by LIF. of xenobiotics (Alin et al., 1985; Datta et al., 1994). Interestingly, both the BMP proteins were strongly Although GST expression was detected in the rat endome- expressed by day 4 blastocyst in both wild type and null trium at proestrus and estrus (Murakoshi et al., 1990), the animals but protein expression in the embryo had decreased relative abundance of GST 8-8 increases threefold in the to lower levels by day 6. Expression of BMPs in the peri- gravid rat uterus (Singhal et al., 1996). In mouse, we found 18 A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21

GST was expressed in the uterine epithelium and stroma delayed-implantation mice, Cox-2 transcripts are not and expression was somewhat enhanced in the decidual observed after progesterone treatment but only when estro- zone in wild type but not LIF-null animals but again the gen is given to activate implantation, although blastocyst pattern suggests a secondary effect of LIF correlating with signals are also required (Chakraborty et al., 1996). That a the induction of the decidual phenotype. What role GST minimal early Cox-2 response is seen in the LIF-null plays in protecting the embryo after implantation is still animals at the potential implantation site again indicates that unclear. the uterine phenotype is not identical to that in delayed Maternal expression of Hoxa-10 is necessary for both implantation. The decidualization response has been con- pinopod formation and successful implantation (Bagot et al., sidered to rely on the induction of Cox-2 at the implantation 2001). In Hoxa-10 null mice as with LIF-null animals, site (Lim et al., 1997) which would be in keeping with the receptivity, decidualization, and implantation are defective above observations. However, more recently, it has been (Benson et al., 1996). Transfection of the uterus with shown that in the Cox-2-null uterus, decidualization is antisense to Hoxa-10 reduced uterine Hoxa-10 expression delayed but not abolished and implantation can occur and affected litter size (Bagot et al., 2000). In our study, (Cheng et al., 2002). Thus, the defect in decidualization Hoxa-10 protein expression was misregulated in the stroma and implantation in the LIF-null animals cannot result solely of LIF-null peri-implantation uterus, though this has not from the incomplete expression of Cox-2 at the implantation been shown for transcripts (Song et al., 2000). Interestingly site. The much-reduced level of Cox-2 presumably leads to Hoxa-10, which itself is regulated by progesterone and a reduction in prostaglandin and prostacyclin levels which estrogen (Ma et al., 1998; Taylor et al., 1998), regulates may still occasionally reach a threshold for slight and some progesterone responsive molecules in stroma, such as transient increased vascular permeability, as we occasion- the prostaglandin receptors EP3 and EP4, epithelial calci- ally observed an extremely faint blueing reaction in the tonin and prolactin (Daftary and Taylor, 2004), as well as uterus after Pontamine blue injection. However, these progesterone-induced uterine stromal proliferation (Lim et molecules remain below the threshold (or are increased al., 1999). Thus, although previous studies have concluded too transiently) for induction of the phenotypic changes that LIF and Hoxa-10 act to regulate implantation along characteristic of full stromal decidualization. Maintenance different pathways (Song et al., 2000), we suggest that there of the decidualization response may require other factors is a feed-back loop between LIF and Hoxa-10 protein including those from the embryo which is clearly not able to expression. This may be indirect and the lack of change in participate in the normal sequence of interactions with the Hoxa-10 mRNA in LIF-null animals suggests it acts at the LE in the absence of LIF. These data strongly confirm post-transcriptional level. previous reports on the essential role of LIF for decidual- Since it has been reported that LIF transcripts can be ization of uterine stromal cells in mice (Chen et al., 2000) detected in the subluminal stroma around the implantation but also provide strong evidence that aberrant interaction of site on day 5 of pregnancy (Song et al., 2000), we examined trophectoderm with the LE may be the primary cause of whether the protein was also expressed at that site in our this. wild type mice. Although LIF protein was detected in In this study, we showed that Oncostatin M, OSM, glands on the morning and late evening of day 4 of another member of the IL-6 cytokine family, is expressed at pregnancy, LIF protein was not detected either in glands or the implantation site in the presence of LIF. In wild type around the implantation site on day 5. The discrepancy uterus, OSM showed a pattern of expression almost between our study and that of Song et al. (2000) may relate identical to Cox-2 from days 5 to 7 of pregnancy. However, to the different strains of mice used, although we cannot rule in the null uterus, OSM was not expressed at all either in LE out levels of protein below the level of detection by the two adjacent to the embryo on day 5 or in the surrounding antibodies used. stroma on later days. The expression of OSM at the Cox-2 is a key enzyme in prostaglandin/prostacyclin implantation site and its total absence in the uterus of the synthesis, and its mRNA has been reported to be expressed LIF-null animals suggests that OSM is an unexpected direct first in the LE from the evening of day 4 and later in the or indirect target of LIF. It is conceivable that the lack of stroma immediately around the implanting embryo through OSM in the LIF-null uterus is a direct result of the impaired day 6 of pregnancy. Cox-2 mRNA is aberrantly expressed in expression of Cox-2 in the absence of LIF. However, the LIF-null mice (Song et al., 2000), signal being detected in mechanism by which LIF affects the expression of OSM the LE but not in the stroma at the implantation site. We and the role that OSM plays in the murine implantation observed a Cox-2 protein expression profile similar to that process requires further investigation. In human pregnancy, reported for mRNA in wild type animals. In the null mice, OSM is produced by decidual and glandular cells and the Cox-2 protein appeared in the LE late on day 4 and in a stimulates the release of hCG (Ogata et al., 2000). It is few cells of the stroma adjacent to the embryo on early day upregulated in the mid of the 5, but did not extend into the deeper stroma. Thus, and reported to induce stromal differentiation, enhancing expression of Cox-2 protein was similar to mRNA but a Cox-2 expression and prolactin secretion (Ohata et al., few stromal cells did express protein. In ovariectomized 2001). However, in another study, it was reported to inhibit A.A. Fouladi-Nashta et al. / Developmental Biology 281 (2005) 1–21 19 prolactin secretion from 8-Br-CAMP-induced stromal cells, This work was funded by a grant from the Biotechnology although the menstrual cycle origin of these cells is not clear and Biological Research Council, UK. (Tanaka and Umesaki, 2003). OSM has been demonstrated to induce the expression of vascular endothelial growth factor (VEGF) in different cell types (Repovic et al., 2003; References Weiss et al., 2003) suggesting an indirect role in angio- genesis. In human, two types of OSM receptor complexes Abrahamsohn, P.A., Zorn, T.M., 1993. Implantation and decidualization in have been identified, one of which is identical to the high- rodents. J. Exp. Zool. 266, 603–628. Alin, P., Danielson, U.H., Mannervik, B., 1985. 4-Hydroxyalk-2-enals are affinity LIF receptor (Gearing and Bruce, 1992). The second substrates for glutathione transferase. FEBS Lett. 179, 267–270. type is specific for OSM and shows no affinity for LIF (Liu Awasthi, Y.C., Saneto, R.P., Srivastava, S.K., 1980. Purification and et al., 1992). However, in mice, OSM does not interact with properties of bovine lens glutathione S-transferase. Exp. Eye Res. 30, the LIF-R/gp-130 complex but signals only through its 29–39. specific receptor (Ichihara et al., 1997). Bagot, C.N., Troy, P.J., Taylor, H.S., 2000. Alteration of maternal Hoxa10 expression by in vivo gene transfection affects implantation. Gene Ther. The extracellular matrix glycoprotein tenascin has been 7, 1378–1384. suggested to facilitate embryo penetration by disrupting Bagot, C.N., Kliman, H.J., Taylor, H.S., 2001. Maternal Hoxa10 is required uterine epithelial cell adhesion to the underlying basal for pinopod formation in the development of mouse uterine receptivity lamina (Julian et al., 1994) or by loosening stromal cell to embryo implantation. Dev. Dyn. 222, 538–544. contacts with ECM components such as fibronectin Bansode, F.W., Chauhan, S.C., Makker, A., Singh, M.M., 1998. Uterine luminal epithelial alkaline phosphatase activity and pinopod develop- (Chiquet-Ehrismann et al., 1995). 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