Molecular Human Reproduction, Vol. 6, No. 4, 337-343,
April 2000
© 2000 European Society of Human Reproduction and Embryology
Embryo development |
Leukaemia inhibitory factor in the endometrium of the common marmoset Callithrix jacchus: localization, expression and hormonal regulation
Primate Biology Division, Primate Biology Division, Cell Biology Division, Primate Biology Division, Institute for Research in Reproduction (ICMR), Jehangir Merwanji Street, Parel, Mumbai 400 012, India
Abstract
In the present study, changes in the immunohistochemical localization of leukaemia inhibitory factor (LIF) in the endometrium during various phases of ovarian cyclicity of the common marmoset have been reported. LIF was absent during the early and late follicular phases. LIF was observed mainly in the cytoplasm of the endometrial glands during the early luteal phase, reached maximum intensity during the mid-luteal phase and declined again during late luteal phase. In-situ hybridization also showed a similar cyclic pattern in the expression of LIF. Stromal cells only showed signals for LIF during the mid-luteal phase. In ovariectomized marmosets, graded dosages of oestradiol alone failed to induce the appearance of LIF protein. Progesterone treatment following oestradiol priming, however, induced distinct glandular localization of LIF, indicating that LIF is a progesterone-dependent protein. Thus endometrial LIF is under maternal control and is secreted in response to the increased progesterone concentrations in circulation. It is possible that high concentrations of LIF during mid-luteal phase may prepare the endometrium for blastocyst implantation in marmosets.
cyclicity/endometrium/LIF/localization/regulation
Introduction
Leukaemia inhibitory factor (LIF) is a glycoprotein with pleiotropic activity (Tomida et al., 1984
; Metcalf, 1991). Recent studies demonstrated that LIF might have an important role in implantation in various mammalian species. In mouse, LIF was transiently expressed in the endometrial epithelium with a distinct peak of protein as well as mRNA at the time of blastocyst implantation (Bhatt et al., 1991; Yang et al., 1995a). The uterine expression of LIF during pseudopregnancy in the mouse was identical to that of its expression during normal pregnancy, indicating maternal control of LIF (Bhatt et al., 1991). The evidence that the expression of LIF is essential for implantation has been provided by the LIF-knockout mouse model (Stewart et al., 1992). High concentrations of LIF receptor and gp 130 (the signal transducer) are present in rabbit endometrium prior to implantation (Yang et al., 1995b). In pigs, the concentration of LIF mRNA in the endometrium was highest on day 11 while the LIF protein in the luminal fluid was high on day 12 (i.e. 2 days prior to implantation), suggesting the importance of maternal LIF in preparing the endometrium for blastocyst implantation (Anegon et al., 1994). In sheep uterus, endometrial LIF mRNA expression remained relatively constant throughout the oestrous cycle with highest expression at the time of implantation and placentation (Vogiagis et al., 1997).
In the endometrium of rhesus monkeys, LIF transcripts were elevated during the secretory phase (Ace and Okulicz, 1995; Okulicz et al., 1996). In women, various workers have reported expression of endometrial LIF mRNA during the postovulatory phase of the menstrual cycle (Charnock-Jones et al., 1994; Kojima et al., 1994; Arici et al., 1995; Cullinan et al., 1996; Vogiagis et al., 1996). Immunohistochemical and in-situ hybridization studies also demonstrated the presence of LIF and LIF mRNA in the uterine glandular and luminal epithelium during the secretory phase of the cycle (Cullinan et al., 1996; Vogiagis et al., 1996). LIF has also been detected in uterine flushings during the expected time of implantation in normal fertile women, while LIF concentration was significantly reduced in women with unexplained infertility, suggesting its importance in embryo implantation (Laird et al., 1997). Human blastocysts have also been shown to express LIF receptor transcripts (Charnock-Jones et al., 1994; van Ejik et al., 1996). LIF significantly improved the quality and quantity of human blastocysts in vitro (Dunglison et al., 1996), suggesting a beneficial role of LIF in embryo development.
In the mouse model, LIF appears to be regulated by oestradiol (Bhatt et al., 1991; Yang et al., 1996) whereas in the rabbit, its expression appears to be controlled by progesterone (Yang et al., 1996). Studies carried out in rhesus monkeys and humans indicated the role of progesterone in regulating endometrial LIF (Ace and Okulicz, 1995; Cullinan et al., 1996; Okulicz et al., 1996; Vogiagis et al., 1996). Postovulatory administration of the progesterone receptor antagonist RU-486 reduced the endometrial glandular immunolocalization of LIF at the expected time of implantation (Gemzell-Danielsson et al., 1997), supporting the role of progesterone in regulation of endometrial LIF. However, in rhesus monkeys, postovulatory mifepristone treatment did not cause any changes in LIF localization (Ghosh et al., 1997), contradicting GemzellDanielsson's report.
Thus, LIF may have widespread importance during early pregnancy in various mammalian species. Studies on non-human primates are restricted to rhesus monkeys only. The common marmoset, Callithrix jacchus, showed unique features in reproduction, including lack of menstruation and presence of postpartum ovulation and subsequent pregnancy (Hearn, 1980; McNeilly et al., 1981; Kholkute, 1984). The profiles of steroid hormones during ovarian cyclicity and pregnancy were also quite different as compared to Old World monkeys (Chambers and Hearn, 1979; Kholkute, 1984). Furthermore, the marmoset embryo adhered to the uterine luminal epithelium at a later period (around day 11 after ovulation) compared to rhesus monkeys, baboons and humans (Moore et al., 1985). In view of these distinct features, we undertook to study the localization, expression and hormonal regulation of LIF in the endometrium of the common marmoset.
Materials and methods
Animals
Colony-bred adult female common marmosets (2–4 years old) weighing between 300 and 400 g were selected. The females were individually caged for this study. Full details of husbandry and management of marmosets in our Primate facility have been published (Kholkute, 1984; Puri et al., 1989).
Ovarian cyclicity
Blood samples (0.4 ml) were collected from the femoral vein on alternate days between 14:00 and 15:00 for a period of 45–60 days. Plasma was separated and kept frozen (–20°C) until further processed. Tonoferon (East India Pharmaceuticals, Calcutta, India) was given orally to replenish iron.
Ovarian cyclicity was monitored by measuring oestradiol and progesterone concentrations by radioimmunoassay (Chambers and Hearn, 1979; Kholkute, 1984). The follicular phase was considered as that part of the cycle in which the progesterone concentrations were <10 ng/ml and the luteal phase was designated by progesterone concentrations >10 ng/ml. The occurrence of ovulation was assessed by an increase in oestradiol concentrations (>1 ng/ml) followed by a rise in progesterone (>10 ng/ml) within 2 days. Decline in progesterone concentrations below 10 ng/ml suggested the beginning of the follicular phase of the next cycle.
Depending on the concentrations of plasma oestradiol and progesterone, the animals were assigned to various phases of ovarian cyclicity as: early follicular phase (plasma progesterone < 10 ng/ml, day 2 to day 4 of the next cycle), late follicular (day 7 or 8 of next cycle), early luteal (day 2 to day 4 following oestradiol peak), mid-luteal (8–10 days following oestradiol peak) and late luteal (14–16 days following oestradiol peak). The various phases of the cycles were also confirmed at the time of surgery by the morphological appearance of the ovaries (follicular development, presence or absence of corpora lutea and presence of stigma). At least three females were included in each group.
Under ketamine anaesthesia, the entire uterus was removed and cut horizontally into small tissue blocks. One portion was placed in Tissue Tek and immediately mounted on a cryostat chuk. Serial sections (5 µm) were cut on a Leitz cryostat and mounted on clean glass slides precoated with gelatin. The sections were stored at –20°C and used for localization of LIF protein within 3–4 weeks. For in-situ hybridization studies, uteri were fixed in 4% formaldehyde overnight at 4°C and processed as per routine histology and embedded in paraffin. Sections (5 µm) were cut on a microtome, put on gelatin-coated slides and stored at –20°C for up to 3 weeks.
Ovariectomy
Bilateral ovariectomy was performed in regularly cycling females (n = 12) by mid-ventral incision under ketamine anaesthesia (10 mg/kg) and sterile conditions. Antibiotic was administered i.m. on the day of operation followed by alternate days for a week. The animals were rested for at least 4 weeks after the surgery.
Simulation of artificial ovarian cyclicity
This pilot experiment was performed to standardize the dose of oestradiol and progesterone so as to mimic the endogenous concentrations of these hormones during follicular phase and luteal phase of the normal ovarian cycle. Ovariectomized animals were randomly divided into following groups (n = 3/group): group 1: olive oil; group 2: 10 µg oestradiol benzoate; group 3: 50 µg oestradiol benzoate; and group 4: 5 mg progesterone.
All the animals received s.c. injections in 0.2 ml of olive oil. Blood samples were collected at 0 h (prior to injection) 6 h and 24 h following treatment. Plasma was separated and stored at –20°C until further processed. These females were used again for studying the hormonal regulation of LIF, following a 4 week rest period.
Hormonal regulation of LIF
Ovariectomized animals were divided into following groups (n = 4 in each group): group 1: olive oil; group 2: 10 µg/day oestradiol benzoate for day 1 to day 4, + 20 µg/day from day 5 to day 7 plus 50 µg oestradiol benzoate on day 8; group 3: oestradiol benzoate treatment as per group 2 followed by 5 mg/day progesterone injection from day 9 to day 17.
Twenty-four hours following the last injection, the entire uterus was removed under ketamine anaesthesia and processed for immunohistochemical localization of LIF.
Animal handling procedures were approved by the Institute's Ethics Committee for the use and care of non-human primates in biomedical research.
Radioimmunoassays
Oestradiol and progesterone were estimated in 50 µl of plasma by radioimmunoassay using matched assay reagents supplied by the World Health Organization (Sufi et al., 1987). These assays have already been validated (Kholkute, 1984). The sensitivity of the oestradiol assay was 160 pg/ml. The inter- and intra-assay coefficients of variation were 14% and 9% respectively (n = 9). The sensitivity of progesterone assay was 400 pg/ml with inter- and intra-assay coefficients of variation 12% and 10% respectively (n = 26).
Immunohistochemistry of LIF
Immunolocalization of LIF was carried out by a published method (Cameron et al., 1996). Briefly, cryostat sections (5 µm) were fixed in cold acetone at –20°C for 10–15 min. Non-specific binding was blocked with normal sheep serum (NSS) for 1 h and the sections were incubated overnight at 4°C with goat anti-recombinant human LIF antibody (R & D systems, Minneapolis, MN, USA, diluted with PBS 1:50 dilution). Following PBS washes, sections were incubated with second antibody (biotinylated goat antihorse) followed by a complex of avidin and biotinylated alkaline phosphatase (Vectastain, ABC-AP kit) according to the manufacturer's protocol. Endogenous alkaline phosphatase activity was inhibited with levamisole (13–15 mg/l; Sigma Chemicals Co., St Louis, MO, USA).
Non-specific reaction was monitored by replacement of primary antiserum with non-immune serum and by omission of the second antibody. Negative control sections were included in each immunostaining protocol. The staining intensity was graded on a scale: no stain (–), weak (+), moderate (++) and intense (+++).
Localization of LIF mRNA in marmoset uterus by non-radioactive in-situ hybridization with digoxigenin (Dig)-labelled oligodeoxynucleotides
Chemicals
Paraformaldehyde (PFA), BSA (98–99% pure) Ficoll-400, polyvinylpyrrolidone (mol wt. 360 000), salmon testicular DNA (phenol chloroform extract), yeast transfer RNA (tRNAm type X-S), dextran sulphate (mol. wt. 500 000), deoxy-ATP (dATP), gelatin, normal sheep immunoglobulin G (IgG), heparin (grade 1, 181 USP U/mg), sodium dodecyl sulphate (SDS), RNase-A (Type IIIA, protease and DNase free), Triton X-100, Brij-35, formamide, and diethyl pyrocarbonate were purchased from Sigma Chemical Co. Dig-11-dUTP, terminal deoxynucleotidyl transferase (TdT) and anti-Dig IgG (Fab), nitroblue tetrazolium (NBT), 5 bromo-4-chloro-3-indolyl phosphate (BCIP) and nylon membranes were obtained from Boehringer Mannheim (Indianapolis, IN, USA).
Oligo DNA
The LIF antisense oligo-DNA selected was complementary to the mRNA sequence coding for amino acids of the human LIF protein (Gough et al., 1988) as follows: 5'- CAA GTA AAC TGG AGG AGA GAG GGA CGA TGG GAG GAC GAT GCA GGG GAC AAG-3'. The LIF sense oligo-DNA selected corresponded to that mRNA sequence as follows: 5'-GAA CAG GGG ACG TAG CAG GAG GGT AGC AGG GAG AGA GGA GGT CAA ATG AAC-3'.
A non-homologous oligo-DNA sequence selected for control studies was based on the mRNA sequence for the progesterone receptor (PR) protein (amino acids 806–820) of the human as follows: 5'-CAG GAG TTT GTC AAG CTT CAA GTT AGC CAA GAA GAG TTC CTC TGT-3' (Misrahi et al., 1987). All oligo-DNA were synthesized on an Automatic Applied Biosystems DNA Synthesizer (model 391 PCR-MATE EP, Foster City, CA, USA) using the phosphoromidite method and purified by polyacrylamide gel electrophoresis (obtained from Carl Roth GmbH, Karlsruhe, Germany).
Labelling of oligo-DNA by TdT
The oligo-DNA were labelled at their 3' end with Dig 11-dUTP by TdT. The labelling mixture (40 µl) contained 25 mmol/l Tris–HCl (pH 6.6, 25°C), 200 mmol/l potassium cacodylate (0.25 mg/ml), BSA, 7.5 mmol/l CaCl2, 5 µmol/l dATP, 50 µmol/l UTdT, 125 µmol/l Dig-II dUTP and 1 µg oligo-DNA. After 15 min incubation at 37°C, the labelled DNA was separated from unreacted compounds by ethanol precipitation with glycogen (40 µg) as recommended by Boehringer Mannheim. The final precipitate was suspended in 40 µl TE buffer (10 mmol/l Tris–HCl buffer, pH 7.4 containing 1 mmol/l EDTA).
Dot blot hybridization
All procedures were carried out at room temperature. Two µl drops of the sense oligo-DNA solution were pipetted on the nylon membrane that had been pretreated with 10xSSC (1xSSC = 0.15 mol/l NaCl and 0.015 mol/l sodium citrate, pH 7.0) in a series of spots at 1 pg to 10 ng/spot. After fixation with UV light, the membranes were incubated at 42°C for 2 h with prehybridization medium containing 10 mmol/l Tris–HCl (pH 7.4), 1 mmol/l EDTA, 0.6 mol/l NaCl, 1x Denhardt's solution, 500 µg/ml yeast tRNA, 250 µg/ml salmon testicular DNA and 50% deionized formamide. Then the membranes were hybridized at 42°C for 15–17 h with 0.5 mg/ml antisense or non-homologous Dig-DNA probes in the medium containing 10 mmol/l Tris–HCl (pH 7.4), 1 mmol/l EDTA, 0.6 mol/l NaCl, 1x Denhardt's solution, 250 µg/ml yeast tRNA, 125 µg/ml salmon testicular DNA, 10% dextran sulphate and 40% deionized formamide. After successive washings with 2xSSC (15 min, twice), 1xSSC (15 min), 0.5xSSC (15 min) 0.075% Brij-35 in PBS (120 mmol/l NaCl, 2.7 mmol/l KCl and 10 mmol/l phosphate buffer, pH 7.4, 15 min) and PBS (15 min), the membranes were immersed for 1 h in a blocking solution that contained 5% BSA, 500 µg/ml normal sheep IgG,100 µg/ml salmon testicular DNA and 100 µg/ml yeast tRNA in PBS. The reaction with anti-Dig polyclonal antibody conjugated to alkaline phosphatase at 1:500 dilution with the blocking solution was performed for 1 h. Then the membranes were washed with 0.075% Brij-35 in PBS (20 min three times) and with PBS alone (15 min, twice). The alkaline phosphatase activity was detected by incubating the sections with BCIP and NBT. Then the spot intensity was compared.
In-situ hybridization
The sections were dewaxed and passed through various graded alcohol concentrations and finally kept in distilled water. The sections were treated with 0.2 mol/l HCl for 5 min at room temperature and afterwards washed in TEC buffer (100 mmol/l Tris–HCl, 150 mmol/l EDTA and 2 mmol/l CaCl2, pH 7.4) for 5 min twice. Proteinase K (1 µg/ml) digestion was carried out at 37°C for 30 min in TEC buffer.
The sections were post-fixed in 4% paraformaldehyde in PBS (0.1 mol/l, pH 7.4) for 10 min followed by rinses in 2xSSC for 10 min at room temperature. Prehybridization was carried out at 37°C for 30 min using 50 µl prehybridization solution containing 1xDenhardt's salt, formamide (50%), salmon sperm DNA (500 µg/ml) and yeast transfer RNA (250 µg/ml). The sections were washed in 2xSSC for 10 min and hybridized at 42°C in 50 µl hybridizing cocktail which contained the same ingredients as the prehybridizing mix with the addition of either sense or antisense probes and dextran sulphate (10%). Post-hybridization stringency washes included three rinses of 4xSSC (twice, 10 min), 2xSSC (5 min) and 1xSSC (2 min).
The sections were washed briefly with Tris/NaCl buffer (100 mmol/l Tris–HCl, 150 mmol/l NaCl, pH 7.5). Non-specific binding of the antibody was blocked by incubating the sections with 2% normal sheep serum and 0.3% Triton X-100 in Tris/NaCl buffer at room temperature for 30 min. The sections were incubated with 100–200 µl of an anti-Dig polyclonal antibody conjugated to alkaline phosphatase at 1:500 dilution in Tris/NaCl buffer at 4°C overnight. The sections were washed briefly in Tris/NaCl buffer and then with Tris/NaCl/MgCl2 buffer (10 mmol/l MgCl2, pH 9.5). Endogenous activity was blocked by incubating the sections with 5 mmol/l levamisole in Tris/NaCl buffer for 20 min at room temperature. The alkaline phosphatase activity was detected by incubating the sections with BCIP and NBT according to Boehringer Mannheim's instruction manual. The sections were washed in distilled water and mounted in Dabco (Sigma).
Control experiments
To confirm the specificity of LIF mRNA signals, various types of control experiments were conducted. Sense probe was used as a negative control in every run. Some sections were hybridized with LIF antisense probe but without second antibody (i.e. anti-Dig antibody) to provide definitive evidence for the sequence specificity of the signal. To evaluate the degree to which commonly abundant RNA, such as ribosomal RNA and tRNA contributed to the staining, paraffin sections of LIF-negative control tissue, namely lymph node from human, was also used for in-situ hybridization studies.
Results
Immunohistochemical localization of LIF during ovarian cyclicity
Immunolocalization of LIF protein in the endometrium of marmoset is shown in Figure 1. Staining was absent in the endometrium in the negative control section (early luteal phase) treated with NSS in place of primary antiserum (Figure 1A). Immunoreactivity was also absent in endometrial glands and stroma during the late proliferative phase (Figure 1B). Moderate staining for LIF was first observed in the endometrial glands during the early luteal phase while during the mid-luteal phase intense immunoreactivity was observed in the glands (Figure 1C and D). The localization of LIF protein was mainly observed in the cytoplasm of endometrial glands at the apical region (towards lumen). In some sections weak immunostaining was also seen in the stroma. During the late secretory phase, weak localization of LIF was observed in the endometrial glands. The results are summarized in Table I.
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Dot blot hybridization
When LIF sense oligo-DNA was hybridized with Dig-labelled LIF antisense oligo-DNA or Dig-labelled unrelated oligo-DNA, the unrelated oligo-DNA detected no sense DNA. The LIF antisense probe was able to detect 1 pg sense DNA. Thus the antisense probe was of sufficient sensitivity to be used for detection of signals in in-situ hybridization.
Localization of LIF mRNA in the endometrium
During the proliferative phase, hybridized Dig-LIF antisense probe failed to detect any signal in the various endometrial compartments (Figure 2A). Similarly no staining was observed in the endometrium with the Dig-LIF sense probe (Figure 2B). In addition to this negative control, when sections of human lymph node were hybridized with the LIF antisense probe, no LIF mRNA signals were observed (not shown). However, during early secretory phase, moderate positive signals for LIF mRNA were clearly detected predominantly in the glandular epithelial cells. In stroma, these signals were weak (Figure 2C). The signals in the glandular epithelium were mainly in the perinuclear region with occasional supranuclear localization. During the mid-luteal phase, intense signals were localized in the glands while in stroma the signals were moderate (Figure 2D). The signals were enhanced in some of the stromal cells closer to the glands. During the late secretory phase of the ovarian cycle, the signals for LIF mRNA were considerably weaker in the glands and absent in the stroma (Figure 2E).
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Immunohistochemical localization of LIF during artificially simulated cycles
Studies carried out in ovariectomized marmosets showed absence of LIF localization in the endometrium of ovariectomized animals (Figure 3A
) as well as in the negative control endometrial sections. In the endometrial sections of oestradiol benzoate-treated animals, a uniform non-specific background staining was seen (Figure 3B). Administration of progesterone, following oestradiol priming, induced distinct intense immunostaining of LIF predominantly in the cytoplasm of the glands (C). Localization of immunoreactivity was observed mainly in the apical region of the endometrial glands (Figure 3D). The results are summarized in Table I
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Hormonal profile
The plasma concentrations of oestradiol and progesterone during the follicular and the luteal phase of natural ovarian cycles and following simulation of artificial cyclicity by exogenous administration of oestradiol and progesterone are shown in Table II
. Administration of 10 µg oestradiol benzoate was found to raise the plasma oestradiol concentrations to 620 ± 178 pg/ml by 6 h and to 327 ± 187 pg/ml by 24 h. These concentrations were comparable to those found during the follicular phase of the normal ovarian cycle. Administration of 50 µg oestradiol benzoate elevated plasma oestradiol concentrations comparable to those of the peak oestradiol concentrations. Similarly 5 mg progesterone injection elevated the plasma concentrations of progesterone as observed during the luteal phase of the ovarian cycle (Table II).
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Discussion
The results of the present study demonstrate a cyclic pattern of LIF localization in the endometrium of the common marmoset, a non-menstruating primate species. LIF protein was localized in the glandular epithelium during the early secretory phase, reached maximum intensity during the mid-luteal phase and declined again during the late secretory phase of the ovarian cycle. Using in-situ hybridization, a similar phase-dependent pattern of LIF mRNA was observed in the endometrium.
The expression of endometrial LIF during the secretory phase of the cycle has been demonstrated in rhesus monkeys (Ace and Okulicz, 1995; Ghosh et al., 1997). In humans, maximal LIF mRNA was observed during the mid-secretory phase (Charnock-Jones et al., 1994; Kojima et al., 1994; Arici et al., 1995). The expression and protein localization was mainly seen on the glandular and luminal epithelia (Cullinan et al., 1996; Vogiagis et al., 1996) while stromal localization has also been reported (Cameron et al., 1996).
The results of the present study are thus similar to those reported for rhesus monkeys and humans. Circulatory concentrations of steroid hormones during the ovarian cycle and early pregnancy vary substantially in these species. Chronological events of implantation in marmosets, rhesus monkeys and humans also differ considerably. Despite differences in hormonal concentrations, variability in blastocyst development and mode of implantation, endometrial LIF expression in all these species was elevated during the luteal phase of the cycle, indicating a common physiological role of LIF in diverse primate species. LIF derived from epithelial cells may act in a paracrine manner on the embryo and/or as an autocrine regulator of implantation. Furthermore, in the common marmoset, localization of LIF was not restricted to the implantation period. The presence of LIF during early and late luteal phases reflects a need for this cytokine in preparing the endometrium before implantation and during the post-implantation period. Thus LIF may have additional functions in decidualization and early pregnancy (Vogiagis et al., 1997; Vogiagis and Salamonsen, 1999).
The hormonal regulation of LIF in vivo has been studied in mice (Bhatt et al., 1991), rabbits (Yang et al., 1996) and rhesus monkeys (Ace and Okulicz, 1995). Endometrial LIF secretion in rabbits and rhesus monkeys appears to be controlled by maternal progesterone. The results of the present study utilizing normal cycling and ovariectomized marmosets also suggest that endometrial LIF is predominantly under the influence of progesterone. Our results are in agreement with studies in ovariectomized rhesus monkeys, in which up-regulation of LIF gene expression by progesterone was observed (Ace and Okulicz, 1995; Okulicz et al., 1996). A recent study (Hambartsoumian et al., 1998) in women without ovarian function, however, demonstrated an inhibitory effect of progesterone on endometrial LIF production and thus contradicts earlier reports in normally menstruating women (Charnock-Jones et al., 1994; Kojima et al., 1994; Arici et al., 1995; Hambartsoumian, 1998a). The regulation of endometrial LIF secretion in normally menstruating women is likely to be under the influence of ovarian growth factors/cytokines (Giudice et al., 1996) which are absent/altered in women without ovarian function. Therefore, it is indeed difficult to compare results obtained following limited steroid substitution under pathological conditions to those observed in normally menstruating women. Furthermore, it seems that endometrial LIF is differentially regulated in fertile women, in women with unexplained infertility and in women without ovarian function (Hambartsoumian, 1998a,b; Hambartsoumian et al., 1998). In-vitro studies utilizing human endometrial cells or explant culture or decidual cell culture also showed contradictory results (Arici et al., 1995; Laird et al., 1997; Sawai et al., 1997; Hambartsoumian, 1998b). Thus results obtained in various studies vary substantially. The effects of progesterone on endometrial LIF seem to be complex and depend on various factors including species, physiological status, cell type or tissue utilized for culture, etc. Nonetheless, these studies highlight that although steroid hormones play a major role in regulation of endometrial LIF, normal functioning of various other cytokines and growth factors, and their interactions, are also crucial for the in-vivo modulation of LIF (Kishimoto et al., 1994; Arici et al., 1995). Several cytokines and growth factors are known to be produced by or to act on the endometrium and are thought to play a central role in endometrial function (Guidice, 1994; Klentzeris, 1997).
In conclusion, the results of the present study have demonstrated for the first time localization and expression of LIF in endometrial glands during the secretory phase of the ovarian cycle in the common marmoset. Presence of LIF during mid-luteal phase suggests its important role in embryo implantation. Furthermore, LIF appeared to be a progesterone-dependent protein. Endometrial LIF expression during the secretory phase of the cycle in marmosets, rhesus monkeys and humans suggests the involvement of LIF in implantation in diverse species of primates.
Acknowledgments
The authors are grateful to Dr H.S.Juneja, Director for his encouragement and interest in this study. We thank the World Health Organization's Special Programme of Research Development and Research Training in Human Reproduction for the supply of reagents for oestradiol and progesterone assays. We also acknowledge the technical assistance provided by Mr A.S.Patankar, S.T.Ghanekar and Ms Sushma Gadkar as well as the staff of the Marmoset Colony for the care and management of animals.
Notes
1 To whom correspondence should be addressed 
References
Ace, C. and Okulicz, W. (1995) Differential gene regulation by estrogen and progesterone in the primate endometrium. Mol. Cell. Endocrinol., 115, 95–103.[Web of Science][Medline]
Anegon, I., Cuturi, M.C., Godard, A. et al. (1994) Presence of leukaemia inhibitory factor and interleukin 6 in porcine uterine secretions prior to conceptus attachment. Cytokine, 6, 493–499.[Web of Science][Medline]
Arici A., Engin, O., Atar, E. et al. (1995) Modulation of leukaemia inhibitory factor gene expression and protein biosynthesis in human endometrium. J. Clin. Endocrinol. Metab., 80, 1908–1915.[Abstract]
Bhatt, H., Brunet, J.J. and Stewart, C.L. (1991) Uterine expression of leukaemia inhibitory factor coincides with onset of the blastocyst implantation. Proc. Natl. Acad. Sci. USA, 88, 11408–11412.
Cameron, S.T., Critchley, H.O.D., Buckley, C.H. et al. (1996) The effects of postovulatory administration of onapristone on the development of a secretory endometrium. Hum. Reprod., 11, 40–49.
Chambers, P.L. and Hearn, J.P. (1979) Peripheral plasma levels of progesterone, 17ß-estradiol, testosterone, androstenedione, chorionic gonadotrophin during pregnancy in marmoset (Callithrix jacchus). J. Reprod. Fertil., 56, 23–32.
Charnock-Jones, D.S., Sharkey, A., Fenwick, P. et al. (1994) Leukaemia inhibitory factor mRNA concentration peaks in human endometrium at the time of implantation and blastocyst contains mRNA for the receptor at this time. J. Reprod. Fertil., 101, 421–426.
Cullinan, E.B., Abbondanzo, S.J., Anderson, J.S. et al. (1996) Leukaemia inhibitory factor (LIF) and LIF receptor expression in human endometrium suggests a potential autocrine/paracrine function in regulating embryo implantation. Proc. Natl. Acad. Sci. USA, 93, 3115–3120.
Dunglison, G.F., Barlow, D.H. and Sargent, I.H. (1996) Leukaemia inhibitory factor significantly enhances the blastocyst formation rates of human embryos cultured in serum-free medium. Hum. Reprod., 11, 191–196.
Gemzell-Danielsson, K., Swahn, M.I. and Bygdeman, M. (1997) The effect of various doses of mifepristone on endometrial leukaemia inhibitory factor expression in mid-luteal phase: an immunohistochemical study. Hum. Reprod., 12, 1293–1297.
Ghosh, D., Kumar, S.P.G. and Sengupta, J. (1997) Effect of early luteal phase administration of mifepristone (RU 486) on leukaemia inhibitory factor, transforming growth factor ß and vascular endothelial growth factor in the implantation stage endometrium of the rhesus monkey. J. Endocrinol., 159, 115–125.
Giudice, L.C. (1994) Growth factors and growth modulators in human endometrium: their potential relevance to reproductive medicine. Fertil. Steril., 61, 1–17.[Web of Science][Medline]
Gough, N.M., Gearing, D.P., King, J.A. et al. (1988) Molecular cloning and expression of the human homologue of the murine gene encoding myeloid leukaemia inhibitory factor. Proc. Natl. Acad. Sci. USA, 85, 2623–2627.
Guidice, I.C., Cataldo, N.A., van Dessel, H.J. et al. (1996) Growth factors in normal ovarian follicular development. Semin. Reprod. Endocrinol., 14, 179–196.[Web of Science][Medline]
Hambartsoumian, E. (1998a) Endometrial leukaemia inhibitory factor (LIF) as a possible cause of unexplained infertility. Am. J. Reprod. Immunol., 39, 137–143.
Hambartsoumian, E. (1998b) Leukaemia inhibitory factor (LIF) production in human decidua and its relationship with pregnancy hormones. Gynecol. Endocrinol., 11, 17–22.
Hambartsoumian, E., Taupin, J.l., Moreau, J.F. et al. (1998) In vivo administration of progesterone inhibits the secretion of endometrial leukaemia inhibitory factor in vitro. Mol. Hum. Reprod., 4, 1039–1044.
Hearn, J.P. (1980) Primate models for early human pregnancy. In Serio, M. and Martini, L. (eds), Animal Models for Human Reproduction. Raven Press, New York, pp. 319–332.
Kholkute, S.D. (1984) Plasma progesterone and estradiol levels during postpartum ovulation and early pregnancy in common marmoset, Callithrix jacchus. Primates, 25, 538–543.
Kishimato, T., Tetsuya, T. and Shuzuo, A. (1994) Cytokine signal transduction. Rev. Cell, 76, 253–262.
Klentzeris, L.D. (1997) The role of endometrium in implantation. Hum. Reprod., 12, 170–175.[Medline]
Kojima, K., Kanzaki, H., Iwai, M. et al. (1994) Expression of leukaemia inhibitory factor in human endometrium and placenta. Biol. Reprod., 50, 882–887.[Abstract]
Laird, S.M., Tuckerman, E.M., Dalton, C.F. et al. (1997) The production of leukaemia inhibitory factor by human endometrium: presence in uterine flushings and production by cell in culture. Hum. Reprod., 12, 569–574.
McNeilly, A.S., Abbott, D.H., Lunn, S.F. et al. (1981) Plasma prolactin concentrations during the ovarian cycle and lactation and their relationship to return of fertility post partum in the common marmoset. J. Reprod. Fertil., 62, 353–360.
Metcalf, D. (1991) The leukaemia inhibitory factor (LIF). Int. J. Cell Cloning, 9, 95–108.[Medline]
Misrahi, M., Atger, M., Auriol, L. et al. (1987) Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA. Biochem. Biophys. Res. Commun., 143, 740–748.[Web of Science][Medline]
Moore, M., Jem, S. and Hearn, J.P. (1985) Early implantation stages in the marmoset Am. J. Anat., 172, 265–276.
Okulicz, W.C., Ace, C.I., Longscope, C. et al. (1996) Analysis of differential gene regulation in adequate versus inadequate secretory phase endometrial cDNA population from rhesus monkey. Endocrinology, 137, 4844–4850.[Abstract]
Puri, C.P., Patil, R.K., Kholkute, S.D. et al. (1989) Progesterone antagonist lilopristone: a potent abortifacient in the common marmoset. Am. J. Obstet. Gynaecol., 161, 240–253.
Sawai, K., Matsuzaki, N., Okada, T. et al. (1997) Human decidual cell biosynthesis of leukaemia inhibitory factor: regulation by decidual cytokines and steroid hormones. Biol. Reprod., 56, 1274–1280.[Abstract]
Stewart, C.L., Kaspar, P., Brunet, L.J. et al. (1992) Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature, 359, 76–79.[Medline]
Sufi, S.B., Donaldson, A. and Jeffcoate, S.L. (1987) Programme for the Matched Assay Reagents for the Radioimmunoassay of Hormones in Reproductive Physiology. World Health Organization Method Manual, World Health Organization, Geneva.
Tomida, M., Yamamoto-Yamaguchi, Y. and Hozumi, M. (1984) Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L 929 cells. J. Biol. Chem., 259, 10978–10982.
Van Ejik, M.J.T., Mandelbaum, J., Salat-Baroux, J. et al. (1996) Expression of LIF receptor subunits LIFR 9B and gp 130 in human oocytes and preimplantation embryos. Mol. Hum. Reprod., 2, 355–360.
Vogiagis, D. and Salamonsen, L.A. (1999) The role of leukaemia inhibitory factor in the establishment of pregnancy. J. Endocr., 160, 181–190.[Abstract]
Vogiagis, D., Marsh, M.M., Fry, D.C. et al. (1996) Leukaemia inhibitory factor in human endometrium throughout the menstrual cycle. J. Endocr., 148, 95–102.
Vogiagis, D., Fry, R.C., Sandelman, R.M. et al. (1997) Leukaemia inhibitory factor in ovine endometrium during the oestrus cycle and early pregnancy. J. Reprod. Fertil., 109, 279–288.
Yang, Z., Le, S., Chen, D. et al. (1995a) Leukaemia inhibitory factor (LIF), LIF receptor and gp 130 in the mouse uterus during early pregnancy. Mol. Reprod. Dev., 42, 407–414.[Web of Science][Medline]
Yang, Z., Le, S., Chen, D. et al. (1995b) Expression patterns of leukaemia inhibitory factor receptor (LIFR) and gp 130 receptor components in rabbit uterus during early pregnancy. J. Reprod. Fertil., 103, 249–255.
Yang, Z., Chen, D., Le, S. et al. (1996) Differential hormonal regulation of leukaemia inhibitory factor (LIF) in rabbit and mouse uterus. Mol. Reprod. Dev., 43, 470–476.[Web of Science][Medline]
Submitted on August 16, 1999; accepted on January 13, 2000.
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