Introduction
Embryo implantation, endometrial stromal cell decidualization and formation of a functional placenta are critical processes in the establishment and maintenance of pregnancy. The molecular events of early implantation in humans are difficult to study due to the lack of tissues available. However, gene targeting studies have identified a small number of cytokines that are unequivocally required for implantation in mice, including interleukin 11 (IL-11) and leukemia inhibitory factor (LIF) [
1,
2].
IL-11 was initially described as a growth factor synergising with other factors in the regulation of hematopoiesis. More recently it has been demonstrated to have pleiotropic actions, including an anti-inflammatory role, in various cell types [
3,
4]. IL-11 belongs to a family of cytokines that includes oncostatin M, IL-6, LIF, cardiotropin 1 and ciliary neurotropic growth factor. Activities of this family of cytokines are mediated via high affinity receptor complexes composed of a ligand-specific alpha (α) subunit and a common signalling subunit gp130. IL-11 binds to a complex of IL-11 receptor (R) α and gp130 [
5]. Female mice with a null mutation of the gene encoding IL-11Rα are infertile due to a defective uterine decidualization response to the implanting blastocyst [
1,
6]. In both rats and mice, IL-11 mRNA is expressed in the primary decidual zone while mRNA for both receptor components, IL-11Rα and gp130, are expressed throughout the peri-implantation period [
1,
7]. Furthermore, comparison of the predicted IL-11 amino acid sequences of mouse, rat, non-human primate and human reveals that IL-11 is highly conserved among species [
7].
Although there are many similarities in the processes of implantation and placentation between the rodent and human, important differences exist. In the human, implantation is initiated in the secretory phase of the menstrual cycle during which time the endometrium is extensively remodelled in preparation for the implanting blastocyst. In particular, the terminal differentiation or decidualization of endometrial stromal cells occurs spontaneously at this time (even in the absence of a blastocyst) and continues throughout pregnancy [
8‐
11]. However, in the mouse and rat, decidualization occurs post-implantation in response to the blastocyst and continues throughout pregnancy [
12].
IL-11 and IL-11Rα mRNA are expressed in human endometrium throughout the menstrual cycle [
13‐
15]. Immunoreactive IL-11 increases in luminal epithelium and glands in the mid-late secretory phase but is most intense in the decidualized stromal cells late in the cycle [
13,
16]. IL-11Rα protein has also been detected in the endometrium throughout the menstrual cycle [
16]. During the first trimester of pregnancy, IL-11Rα mRNA and IL-11 protein are present in decidua and in chorionic villi [
15] but reduced in anembryonic compared to normal pregnancies, identifying a possible critical role for IL-11 signalling in pregnancy [
15]. Functions of IL-11 in the human endometrium have been examined using
in vitro cell culture models; IL-11 progresses endometrial stromal cell decidualization and also increases decidualized and non-decidualized stromal cell survival [
17,
18].
In the human, early stages of implantation are difficult to study due to the lack of availability of tissue. As a prelude to examining the function of IL-11 in human implantation and placentation, the aim of this study was to determine the localisation of IL-11 and IL-11Rα at implantation sites of rhesus and cynomolgus macaque and in first trimester human decidua and placenta.
Materials and Methods
Tissue collection
Adult cynomolgus monkeys (
Macaca fascicularis) were housed at the California Regional Primate Centre. All animal procedures conformed to the requirements of the Animal Welfare Act, and the protocols were approved by the Institutional Animal Use and Care Administrative Committee at the University of California, Davis, USA. The timing of pregnancies has been described previously [
19]. Briefly, animals were bred twice during the ovulation stage of the cycle and the second mating was designated day 0 of pregnancy. Pregnancy was verified by ultrasound at day 12 of pregnancy. Intact uterus was removed at days 12 (n = 2), 14 (n = 1), 17 (n = 1), 27 (n = 1), 31 (n = 1), 49 (n = 1), 74 (n = 1), 150 (n = 1) of pregnancy. Tissues were fixed with 4% paraformaldehyde in phosphate buffer for 4 hrs and processed to paraffin wax blocks.
Rhesus monkeys (Macaca mulatta) were maintained in the Fu-Zhou Primate Research Centre, China. All experimental work was approved by the Animal Ethics Committee at the Institute of Zoology, Chinese Academy of Sciences, Beijing. Estrous cycles of female monkeys were monitored for two to three cycles before sampling. Uterine tissue was collected 1 day before ovulation (ov-1, n = 3) and at 5 (ov+5, n = 3), 10 (ov+10, n = 3) and 15 (ov+15, n = 2) days after ovulation. For the tissue from pregnant monkeys, animals were permitted to mate over a period of 3 days at the anticipated time of ovulation. The second day of mating was designated day 0 of pregnancy. The presence of a conceptus was confirmed by ultrasound diagnosis and rectal examination. Samples of uterus were collected from pregnant monkey implantation sites between day 24 and day 35 (n = 8). At the appropriate time of either the estrous cycle or pregnancy, the monkeys were sacrificed, the uterus was removed (with or without placenta) and appropriately selected wedges of full thickness uteri (myometrium, endometrium, placenta of < 0.5 mm in diameter) were fixed in buffered formalin overnight at 4°C, washed in Tris-buffered saline (pH 7.4) and processed to paraffin wax blocks.
Human decidua and placenta were collected from healthy women undergoing elective termination. All were singleton pregnancies at 8–9 weeks of gestation without any known fetal abnormality. Tissue from term pregnancy was collected after spontaneous vaginal delivery. All tissue collection followed informed consent and was under approval from Monash Medical Centre Human Research and Ethics Committee, Clayton, Vic., Australia. All tissues were fixed in 10% neutral buffered formalin for 12–16 hrs at 4°C and processed to paraffin wax blocks.
Immunohistochemistry
Immunohistochemistry for IL-11 was conducted using a monoclonal anti-huIL-11 antibody (5E3) produced in our laboratory raised against recombinant human IL-11 (a kind gift of Genetics Institute). A second monoclonal antibody (MAB618; R&D Systems Inc., Minneapolis, USA) was also used to verify the specificity of immunostaining in the macaque tissues as previously described [
13]. Immunostaining for IL-11Rα was performed using a monoclonal anti-huIL-11Rα (4D12) as previously described [
18]. A commercially available antibody (sc-993, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) confirmed specificity of the IL-11Rα antibody in macaque uterus.
Paraffin sections (5 μm) of endometrium from all stages of the rhesus monkey menstrual cycle and implantation sites, cynomolgus monkey implantation sites, human placenta and decidua were dewaxed in histosol and rehydrated through descending grades of ethanol. Endogenous peroxidase activity was quenched by immersion in 3% H2O2 in methanol for 10 min. Sections were then incubated with blocking solution containing 10% horse serum (H0146; Sigma Aldrich Inc. Missouri, USA), 5% human serum and TBS (pH 7.4) for 30 min. Primary antibodies were applied diluted to 5 μg/ml (IL-11) and 2.5 μg/ml (IL-11Rα) in blocking solution for 18 hrs at 4°C. Antibody localisation was detected by sequential application of biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) in blocking solution, and an avidin-biotin-complex conjugated to HRP (Vectastain ABC Elite kit; Vector Laboratories). The substrate used was diaminobenzidine (DAB) (Zymed, San Francisco, USA), which forms an insoluble brown precipitate, and nuclei were counterstained blue with Harris hematoxylin (Sigma Diagnostics, St. Louis, USA). Human term placenta was used as a positive control and a section from a single block was included in each staining run for quality control. An isotype matched negative control at the same concentration as the primary antibody was included for each tissue.
Immunohistochemistry for cytokeratin in rhesus monkey implantation sites was conducted using anti-human cytokeratin (CAM 5.2; Becton Dickinson Immunocytochemistry Systems, San Jose, California, USA) using a technique similar to that above except that the primary antibody, pre-diluted by the manufacturer, was applied for 18 hrs at 4°C. Immunohistochemistry for cytokeratin in cynomolgus monkey tissues was performed similarly but tissue sections were digested with 0.1% pepsin (Sigma Aldrich, St Louis, USA) prior to incubation with antibody.
Assessment of immunostaining
Immunostaining in individual cellular compartments within each section was scored blind by two independent observers from 0 (negative) to +++ (maximal staining intensity) relative to the positive and negative controls.
Discussion
IL-11 signalling has been identified as critical for female fertility in the mouse [
1,
6]. This is the first study to establish that both IL-11 and IL-11Rα protein are present in the cycling rhesus monkey endometrium and in early implantation sites of cynomolgus and rhesus monkeys. Both proteins were also detected in human first trimester placenta and decidua.
In the cycling rhesus monkey endometrium, staining for IL-11 and IL-11Rα increased during the secretory phase of the oestrous cycle compared to the proliferative phase. Similarly, studies in the human identified IL-11 immunostaining to be maximal in the late secretory phase of the menstrual cycle [
13,
14,
20]. However, a third study reported very little staining for IL-11 in cycling human endometrium, probably reflecting differences in antibody or immunohistochemical staining techniques, although a significant increase in IL-11 gene expression was found in secretory phase endometrium compared to proliferative phase endometrium [
15]. Maximal IL-11 and IL-11Rα immunostaining in rhesus monkey endometrium was found during the mid-late secretory phase. Maximal IL-11Rα staining was found in the mid-secretory phase, the time when implantation is most likely to occur. Furthermore, all the major cellular compartments stained positively for IL-11 although the glandular epithelial cells in the basalis were the most highly stained cell type. Staining for IL-11Rα was maximal in the luminal epithelium and glands. This is in accordance with previous findings that showed strong staining for IL-11Rα in glandular and luminal epithelium in cycling human endometrium, although with little cyclical variation [
16,
20]. In the human, in the late secretory phase, strongest IL-11 staining is seen in the decidualizing stromal cells, but this process does not occur until later in the rhesus monkey.
There are very few functional studies in the human identifying a role for IL-11 in the endometrium. IL-11 has been shown to be involved in stromal cell decidualization and decidualized stromal cell survival in vitro [
17,
18]., but whether such actions in vivo result from IL-11 of epithelial or other cellular origin is not yet known. In addition, the cyclical variation in staining for IL-11 and IL-11Rα indicates a role for IL-11 signalling in uterine receptivity. It is likely that epithelial IL-11 has additional, as yet unidentified, functions perhaps on the implanting blastocyst.
As it is not possible to examine early implantation sites in the human, samples were collected from pregnant cynomolgus and rhesus monkeys. During both the early and late lacunar stage of implantation in the cynomolgus monkey, very little staining was seen for IL-11, and this was in the epithelial plaques, cytotrophoblast in the trophoblast shell, glands, decidual and vascular smooth muscle cells. Staining in the trophoblast shell was variable likely reflecting the different populations of trophoblast cells present. In contrast, virtually no staining for IL-11Rα was present either in the trophoblast or the glands, and only pale staining was present in the stroma, endothelial and smooth muscle cells. It therefore seems that IL-11 has a minimal, if any, role in early migration of trophoblast within the arterioles, since very little IL-11 is detectable during the lacunar stage.
In contrast there was a dramatic increase in staining for both IL-11 and IL-11Rα at day 17 of pregnancy compared to the earlier time points. At this time, large numbers of cytotrophoblast cells invade the arterial wall most likely from the lumen [
21]. Sub-populations of trophoblast cells that invaded arteries, along with smooth muscle and endothelial cells, and many cells in the trophoblast shell showed staining for IL-11 and IL-11Rα. This suggests IL-11 may facilitate trophoblast invasion into the spiral arteries, a critical step in placentation.
Later in gestation, as seen in both the cynomolgus and rhesus monkey implantation sites, the most intriguing finding was the absence, or very little staining for both IL-11 and IL-11Rα from the trophoblast shell. There appears to be a switching off of IL-11 production in the trophoblast shell and a switching on in the villi by day 24 of pregnancy in the rhesus monkey. By the third week of pregnancy, the cytotrophoblast cells within the anchoring villi and trophoblast shell are heterogeneous and respond to adjacent constituents [
22]. Heterogeneity also exists in cytotrophoblast cells in proximal, mid and distal regions (adjacent to the trophoblast shell) of the cell columns [
22]. Thus, the observation that sub-populations of trophoblast cells stained for IL-11 and IL-11Rα likely reflects differences in IL-11 function during trophoblast migration.
The most consistent finding was the high IL-11 and IL-11Rα in the decidua in all three primate species. This likely reflects an involvement in the continuing process of decidualization of endometrial stromal cells, as demonstrated in vitro for the human [
17]. The intense staining for IL-11 and IL-11Rα in human first trimester decidua was not surprising since stromal differentiation continues to provide decidual cells well into pregnancy. This confirms previous data showing immunoreactive IL-11 and IL-11Rα mRNA in such cells [
15]. Both IL-11 and IL-11 Rα protein were also present in syncytio- and cyto-trophoblast of the chorionic villi in first trimester pregnancy, in accord with the documented IL-11Rα mRNA expression in these cells [
15]. Thus, very similar expression patterns are present in the rhesus monkey and human implantation sites.
The most striking difference in IL-11 and IL-11Rα staining between species was the lack of staining in villous trophoblast in the cynomolgus monkey compared to the rhesus monkey and the human. Inconsistencies may be related to differences in tissue fixation but this is unlikely since staining was similar in the decidual cells, and trophoblast cells that invaded arteries in all three species. The results indicate that IL-11 has multiple roles in implantation and early placentation. The potential importance of IL-11 signalling in early placentation is emphasised by the defective production of IL-11 and IL-11Rα in decidua and placenta of women with anembryonic pregnancies [
15].
The data presented in this paper thus indicate a complex and critical role for IL-11 signalling in preparing the endometrium for implantation, early trophoblast invasion and stromal cell decidualization in the primate. IL-11 and IL-11Rα production by cycling endometrium is upregulated at the time an embryo is likely to implant. In addition, the precise location of IL-11 and IL-11Rα during early implantation and placentation suggests that aberrant IL-11 signalling or blocking IL-11 action may lead to reduced fertility. Furthermore, reduced IL-11 signalling in primate endometrium or early pregnant decidua and placenta may be a therapeutic target for treatment of infertility.
Authors' contributions
ED conceived, designed and coordinated the study, carried out and scored the immunohistochemistry studies, and drafted the manuscript. LR prepared antigens, raised and tested the antibodies used for immunohistochemistry. YXL obtained ethics approval for rhesus monkey studies, obtained and prepared rhesus monkey tissues for immunohistochemistry. ACE obtained ethics approval for cynomolgus monkey studies, obtained and prepared cynomolgus monkey tissues for immunohistochemistry, conducted the cynomolgus monkey cytokeratin staining. CS participated in performing the immunohistochemistry studies. EW obtained ethics approval for collection of human placenta and decidua, and prepared tissues for the immunohistochemistry studies. LAS conceived the study and participated in drafting the manuscript.