Leukemia inhibitory factor and its receptor: expression and regulation in the porcine endometrium throughout the estrous cycle and pregnancy

Objective Leukemia inhibitory factor (LIF) binds to a heterodimeric receptor composed of LIF receptor (LIFR) and glycoprotein 130 (GP130) to transmit signals into the cell. LIF plays an important role in reproduction by regulating immune response, decidualization, and implantation in several species. However, the expression of LIF and LIFR in the endometrium throughout the estrous cycle and pregnancy in pigs is not fully understood. Methods We analyzed the expression of LIF and LIFR in the endometrium on days 0 (estrus), 3, 6, 9, 12, 15, and 18 of the estrous cycle, and days 12, 15, 30, 60, 90, and 114 of pregnancy, in conceptuses on days 12 and 15, and in chorioallantoic tissues on days 30, 60, 90, and 114 of pregnancy in pigs. We also determined the effects of estrogen and progesterone on the expression of LIF and LIFR in endometrial tissues. Results The expression of LIF increased in the endometrium during the late diestrus phase of the estrous cycle and during mid- to late- pregnancy, while the expression of LIFR increased during early pregnancy. The expression of LIF was induced by increasing doses of estrogen, whereas the expression of LIFR was induced by increasing doses of progesterone. Conclusion These results indicate that the expression of LIF and its receptor LIFR in the endometrium is regulated in a stage-specific manner during the estrous cycle and pregnancy, suggesting that LIF and its receptor signaling system may play critical roles in regulating endometrial function in pigs.


INTRODUCTION
Leukemia inhibitory factor (LIF) is a pleiotropic member of the interleukin6 (IL6) family of cytokines [1,2]. LIF regulates cellular functions by binding to a membranebound heterodi meric receptor, LIF receptor (LIFR) and glycoprotein 130 (GP130) [3]. Binding of LIF to LIFR and GP130 forms a high affinity functional receptor complex and activates the Janus kinase signal transducer and activator of the transcription signaling pathway [3,4]. In addition to the wellknown function of LIF in inhibiting differentiation of mouse embryonic stem cells to maintain pluripotency and selfrenewal [5], LIF is involved in a variety of biological pro cesses such as cell differentiation, bone metabolism, inflammation, vascularization, and embryogenesis [3].
In the female reproductive tract, LIF plays important roles in regulation of immune re sponse, decidualization, and implantation in several animal species [1,3,6]. In humans, LIF is expressed in the endometrial glands during the luteal phase of the menstrual cycle, and LIFR and GP130 are expressed in endometrial luminal epithelial cells during the cycle and in decidual stroma during early pregnancy [7,8]. LIF affects endometrial receptivity by inducing decidualization of stromal cells and increasing IL6 and IL15 production in decidual cells during the implantation period [9]. In mice, Lif is expressed in endometrial glands at the highest levels on day 4 of pregnancy [10], while Lifr and Gp130 are expressed in luminal epithelial cells on days 3 to 5 of pregnancy [11]. Lifnull mice are infer tile due to defects in implantation and decidualization [2,12], and LIF induces recruitment of various leukocytes into the site of implantation in mice [1,6]. In pigs, the expression of LIF and its receptor has been reported in the endometrium. LIF is expressed in the endometrium between days 10 and 18 of the estrous cycle and early pregnancy [13,14], and LIFR and GP130 are expressed in the endometrium during the estrous cycle and pregnancy and in the placenta [1518]. However, the expression of LIF and LIFR in the endometrium throughout all stages of the estrous cycle and pregnancy has not been de termined.
The expression of LIF is increased by leptin, IL1β, tumor necrosis factorα, and transforming growth factorβ in endo metrial tissues, cultured endometrial cells, and decidua in humans [1]. In mice, endometrial expression of Lif is induced by a nidatory surge of estrogen at implantation [10,19]. In golden hamsters, Lif expression is induced by estrogen while the expression of Lifr and Gp130 is induced by progesterone [20]. In pigs, the expression of GP130 is not affected by estro gen and progesterone [18]. However, the regulatory mechanism of LIF and LIFR expression in the endometrium is not fully understood in pigs. Therefore, to clarify the role of LIF in the endometrium during the estrous cycle and pregnancy in pigs, we examined the temporal and cell typespecific expression of LIF and LIFR in the endometrium throughout the estrous cycle and pregnancy and the regulation of LIF and LIFR by steroid hormones, estrogen and progesterone, in endometrial tissues.

Animals and tissue preparation
All experimental procedures involving animals were conduct ed in accordance with the Guide for Care and Use of Research Animals in Teaching and Research and approved by the Insti tutional Animal Care and Use Committee of Yonsei University and the National Institute of Animal Science. Sexually mature crossbred female gilts were assigned randomly to either cyclic or pregnant status. The reproductive tracts of gilts were ob tained immediately after slaughter at a local slaughterhouse on either days 0, 3, 6, 9, 12, 15, or 18 of the estrous cycle or days 12, 15, 30, 60, 90, or 114 of pregnancy (n = 36/d/status). Pre gnancy was confirmed by the presence of apparently normal filamentous conceptuses in uterine flushings on days 12 and 15 and the presence of embryos and placenta on later days of pregnancy. Conceptus tissues were obtained from uterine flushings on days 12 and 15 of pregnancy, and chorioallantoic tissues were obtained on days 30, 60, 90, and 114 of pregnancy (n = 34/d).
Endometrium, dissected free of the myometrium, was col lected from the middle portion of each uterine horn, snap frozen in liquid nitrogen, and stored at -80°C prior to RNA extraction. For in situ hybridization, crosssections of endo metrium were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) (pH 7.4) for 24 h and then embedded in paraffin as previously described [21]. For endometrial ex plant tissue cultures, endometrial tissues were obtained from sexually immature crossbred female gilts (n = 8) with no evi dence of ovulation at a local slaughterhouse immediately after pigs were slaughtered.
Total RNA extraction and reverse transcription polymerase chain reaction for LIF and LIFR cDNAs Total RNA was extracted from endometrial, chorioallantoic, and conceptus tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer' s recommendations. The quantity of RNA was assessed spectrophotometrically, and the integrity of RNA was validated following electropho resis in 1% agarose gel.
Four micrograms of total RNA from endometrial, chorio allantoic, and conceptus tissues were treated with DNase I (Promega, Madison, WI, USA) and reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen, USA) to obtain cDNAs. The cDNA templates were then diluted 1:4 with nucleasefree water and amplified by polymerase chain reaction (PCR) using Taq polymerase (Takara Bio, Shiga, Ja pan). The PCR conditions, sequences of primer pairs for LIF and LIFR, and expected product sizes are listed in Table 1. The PCR products were separated on 2% agarose gel and visualized by ethidium bromide staining. The identity of each amplified PCR product was verified by sequence analysis after cloning into the pCRII vector (Invitrogen, USA).

Quantitative real-time reverse transcription polymerase chain reaction
To analyze levels of LIF and LIFR mRNAs in endometrial and chorioallantoic tissues, realtime reverse transcription (RT) PCR was performed using the SYBR Green method with the Applied Biosystems StepOnePlus System (Applied Biosys tems, Foster City, CA, USA). Complementary DNAs were synthesized from 4 μg total RNA isolated from different uter ine endometrial tissues, and newly synthesized cDNAs (total volume of 21 μL) were diluted 1:4 with nucleasefree water and then used for PCR. Power SYBR Green PCR Master Mix (Applied Biosystems, USA) was used for PCR reactions. The final reaction volume of 20 μL included 2 μL of cDNA, 10 μL of 2X Master mix, 2 μL of each primer (100 nM), and 4 μL of dH 2 O. PCR conditions and sequences of primer pairs used for LIF and LIFR are listed in Table 1. The results are reported as expression relative to the level detected on day 0 of the es trous cycle after normalization of the transcript amount to the endogenous porcine ribosomal protein L 7 (RPL7) con trol by the 2 -ΔΔCT method [22].

Non-radioactive in situ hybridization
We performed nonradioactive in situ hybridization as pre viously described [23,24] with minor modifications. Sections (5 μm thick) were rehydrated through successive baths of xylene, 100% ethanol, 95% ethanol, diethylpyrocarbonate (DEPC)treated water, and DEPCtreated PBS. Tissue sec tions were boiled in citrate buffer (pH 6.0) for 10 min. After washing in DEPCtreated PBS, sections were digested using 5 μg/mL Proteinase K (Sigma, St. Louis, MO, USA) in TE (100 mM TrisHCl, 50 mM ethylenediaminetetraacetic acid, pH 7.5) at 37°C. After postfixation in 4% paraformaldehyde, sec tions were incubated twice for 15 min each in PBS containing 0.1% active DEPC and equilibrated for 15 min in 5× saline sodium citrate (SSC). The sections were prehybridized for 2 h at 68°C in hybridization mix (50% formamide, 5× SSC, 500 μg/mL herring sperm DNA, 250 μg/mL yeast tRNA; 200 μL on each section). Sense and antisense LIF and LIFR riboprobes were generated using partial cDNAs cloned into pCRII vec tors by linearizing with appropriate restriction enzymes and labeling with digoxigenin (DIG)UTP using a DIG RNA La beling kit (Roche, Indianapolis, IN, USA). The probes were denatured for 5 min at 80°C and added to the hybridization mix. The hybridization reaction was carried out at 68°C over night. Prehybridization and hybridization reactions were performed in a box saturated with a 5× SSC50% formamide solution to avoid evaporation, and no coverslips were used. After hybridization, sections were washed for 30 min in 2× SSC at room temperature, 1 h in 2× SSC at 65°C, and 1 h in 0.1× SSC at 65°C. Probes bound to the section were detected immunologically using sheep antiDIG Fab fragments cova lently coupled to alkaline phosphatase and nitroblue tetrazolium chloride/5bromo4chloro3indolyl phosphate (toluidine salt) as chromogenic substrate, according to the manufacturer' s protocol (Roche, USA).

Explant cultures
Endometrial tissues from immature gilts were dissected from the myometrium and placed into warm phenol redfree Dul becco's modified Eagle's medium/F12 (DMEM/F12) culture medium (Sigma, USA) containing penicillin G (100 IU/mL) and streptomycin (0.1 mg/mL) as described previously [21] with some modifications. The endometrium was minced with scalpel blades into small pieces (2 to 3 mm 3 ), and aliquots of 500 mg were placed into T25 flasks with serumfree modified DMEM/F12 containing 10 μg/mL insulin (Sigma, USA), 10 ng/mL transferrin (Sigma, USA), and 10 ng/mL hydro cortisone (Sigma, USA). Endometrial explants were cultured immediately after mincing in the presence of increasing doses of estradiol17β (E 2 ; 0, 5, 50, 500, or 5,000 pg/mL; Sigma, USA) and progesterone (P 4 ; 0, 0.3, 3, 30, or 300 ng/mL; Sigma, USA) for 24 h with rocking in an atmosphere of 5% CO 2 in air at 37°C. Explant tissues were then harvested and total RNA was extracted for realtime RTPCR analysis to determine the effects of E 2 and P 4 on expression of LIF and LIFR mRNAs. These experiments were conducted using endometrial tissues from eight immature gilts.

Statistical analysis
Data from realtime RTPCR for LIF and LIFR expression during the estrous cycle and pregnancy were subjected to analysis of variance using the general linear models proce dures of SAS (Cary, NC, USA). As sources of variation, the model included day, pregnancy status (cyclic or pregnant, days  30, 60, 90, and 114), and effects of dose in explant culture for data from realtime RTPCR for LIF and LIFR expression were analyzed by least squares regression anal ysis. Data are presented as means with standard error of the mean. A pvalues less than 0.05 were considered significant.

Expression of LIF and LIFR mRNAs in the endometrium during the estrous cycle and pregnancy in pigs
We performed realtime RTPCR analysis to determine whe ther LIF and LIFR mRNAs were expressed in the endometrium in pigs. As shown in Figure 1, realtime RTPCR analysis showed that the expression of LIF in the endometrium in creased at late diestrus and proestrus phases during the estrous cycle (linear effect of day, p<0.05), while the expression of LIFR did not change during the cycle. On days 12 and 15 postestrus, LIF expression was affected by day (p<0.05), but not by pre gnancy status and day×status, and LIFR expression was affected by pregnancy status (p<0.05) and day×status (p<0.05), but not by day. The expression of LIFR mRNA was greater on day 15 of pregnancy than day 15 of the estrous cycle (p<0.05). Dur ing pregnancy, the expression of LIF increased towards term pregnancy with the greatest expression on day 90 of pregnancy (linear effect of day, p<0.01), and whereas the expression of LIFR was greatest on day 15 and decreased thereafter (linear effect of day, p<0.01).

Expression of LIF and LIFR mRNA in conceptuses during early pregnancy and chorioallantoic tissue during later stage of pregnancy
We performed RTPCR using cDNA from conceptuses from days 12 and 15 to determine whether conceptuses express LIF and LIFR during early pregnancy. LIF and LIFR mRNAs were detected in conceptus tissues on both days of pregnancy (Figure 2A). We performed realtime RTPCR analysis to determine if the expression of LIF and LIFR mRNAs changed in chorioallantoic tissues during mid to late pregnancy. The expression of LIF mRNA increased toward term pregnancy (linear effect of day, p<0.01), whereas the expression of LIFR mRNA decreased after day 30 and remained low until term (linear effect of day, p<0.05) ( Figure 2B).

Localization of LIF and LIFR mRNAs in the endometrium during the estrous cycle and pregnancy in pigs
Next, we performed in situ hybridization analysis to determine which cell type(s) express LIF and LIFR mRNAs in the endo metrium. The expression of LIF mRNA was mainly localized to endometrial luminal and glandular epithelial cells during the estrous cycle and pregnancy and to the chorioallantoic membrane during mid to late pregnancy. LIFR mRNA was localized primarily to endometrial epithelial cells and stromal cells during the estrous cycle and pregnancy, with strong sig nal intensity in stromal cells on day 15 of pregnancy ( Figure 3).

Effects of E 2 and P 4 on LIF and LIFR mRNA expression in the endometrium
Because LIF mRNAs increased significantly at late diestrus and proestrus phases in this study and the expression of Lif and Lifr is induced by the steroid hormones estrogen and pro gesterone in rodent endometrium [19,20], we determined whether E 2 and P 4 affected the expression of LIF and LIFR Figure 1. Expression of leukemia inhibitory factor (LIF; A) and leukemia inhibitory factor receptor (LIFR; B) mRNAs in the endometrium during the estrous cycle and pregnancy in pigs. Endometrial tissue samples from cyclic and pregnant gilts were analyzed by real-time reverse transcription polymerase chain reaction, and data are reported as expression relative to that detected on day 12 of the estrous cycle after normalization of the transcript amount to the endogenous ribosomal protein L7 control. Data are presented as means with standard errors. mRNAs in endometrial explant cultures from immature gilts, that were not exposed to cyclical ovarian hormones. We treated endometrial explant tissues with 0, 5, 50, 500, or 5,000 pg/mL E 2 , and found that the expression of LIF, but not LIFR, was  increased by increasing doses of E 2 (linear effect of dose, p< 0.01) (Figure 4). Furthermore, we treated endometrial explant tissues with 0, 0.3, 3, 30, or 300 ng/mL P 4 and found that in creasing doses of P 4 induced the expression of LIFR mRNA (linear effect of dose, p<0.01), but not LIF mRNA ( Figure 5).

DISCUSSION
The novel findings of this study in pigs are as follows: i) LIF and LIFR mRNAs are expressed in the endometrium through out the estrous cycle and pregnancy in a stagespecific manner; ii) conceptus tissues on days 12 and 15 and chorioallantoic tissues from day 30 to term express LIF and LIFR mRNAs; iii) LIF and LIFR mRNAs are localized to epithelial and stromal cells in the endometrium; and iv) the expression of LIF in the endometrium is increased by E 2 , while LIFR is increased by P 4 . To our knowledge, this is the first report to determine the expression of LIF and LIFR throughout the estrous cycle and pregnancy in pigs.
LIF and LIFR are expressed in the endometrium, and their expression changes depending on the stage of the reproduc tive cycle and pregnancy, in humans and rodents. In human endometrium, LIF expression increases during the secretory phase of the menstrual cycle [7,8], and LIFR and GP130 are expressed in epithelial cells during the cycle [8]. In mice, Lif expression in the endometrium is greatest on day 4 during pregnancy, which coincides with the onset of blastocyst im plantation, and remains low after implantation [10]. In the Figure 4. Effects of E 2 on leukemia inhibitory factor (LIF) and leukemia inhibitory factor receptor (LIFR) mRNA levels in endometrial explant cultures. Endometrial explants from immature gilts were cultured in the presence of increasing doses of E 2 (0, 5, 50, 500, or 5,000 pg/mL) at 37°C for 24 h. For each treatment, experiments were performed with endometria from eight gilts. Abundance of mRNA expression determined by real-time reverse transcription polymerase chain reaction analyses is relative to that for LIF and LIFR mRNAs in the control group of endometrial explants (0 pg/mL) after normalization of transcript amounts to ribosomal protein L7 mRNA. Data are presented as least squares means with standard errors. E 2 (pg/mL) E 2 (pg/mL) Figure 5. Effects of P 4 on leukemia inhibitory factor (LIF) and leukemia inhibitory factor receptor (LIFR) mRNA levels in endometrial explant cultures. Endometrial explants from immature gilts were cultured in the presence of increasing doses of P 4 (0, 0.3, 3, 30, or 300 ng/mL) at 37°C for 24 h. For each treatment, experiments were performed with endometria from eight gilts. Abundance of mRNA expression determined by real-time RT-PCR analyses is relative to that for LIF and LIFR mRNAs in the control group of endometrial explants (0 pg/mL) after normalization of transcript amounts to ribosomal protein L7 mRNA. Data are presented as least squares means with standard errors.

P 4 (ng/mL) P 4 (ng/mL)
present study, we found that LIF was expressed in the endo metrium throughout the estrous cycle and pregnancy in pigs. During the estrous cycle, LIF expression was greatest at late diestrus and proestrus phases. These phases correspond to the period of luteolysis and follicular development, which, in turn, causes decline of progesterone and increase of estrogen. Because endometrial prostaglandin (PG) F 2α production is critical for the induction of luteolysis [25], we hypothesize that LIF is involved in endometrial function for the produc tion of luteolytic PGs. In addition, because the proportion of macrophages in the endometrium significantly decreases at the time of implantation in Lifnull mice [26] and immune cell recruitment into the endometrium increases during the pro estrus phase after luteolysis in pigs [27], LIF may be involved in regulation of immune cell recruitment into the endome trium during the estrous cycle. LIF plays a critical role in implantation and decidualization in the uterus to increase receptivity to the developing embryo during early pregnancy in humans and rodents [1]. In pigs, the expression of LIF in the endometrium during pregnancy was greatest during mid to late stages of pregnancy. Because LIF increases cell attachment and viability of porcine tropho blast cells in vitro [14] and was most expressed during mid to late pregnancy, it is likely that LIF plays an important role in placental development and cell adhesion between the tro phoblast and endometrial epithelial cells during mid to late stages of pregnancy. The endometrial expression of GP130, which forms a heterodimeric LIF receptor with LIFR, also increases during midpregnancy with the greatest expression on day 60 of pregnancy in pigs [18]. Based on the expression pattern of endometrial LIF during pregnancy, it is likely that the function of LIF for conceptus implantation in pigs is not as critical as in humans and rodents. This may be due to dif ferences in implantation and placentation in pigs, which forms a noninvasive true epitheliochorial type placenta and does not require a decidualization process during the implantation period, whereas in humans and rodents the endometrium is decidualized for implantation and an invasive hemochorial type placenta is formed when the embryo is implanted.
The expression of LIFR in the endometrium has been shown in humans [7,8], rodents [20], and pigs [16,17], but the tem poral pattern of LIFR expression throughout the menstrual or estrous cycle and pregnancy has not been studied in any species so far. In this study, we found that the expression of LIFR in the endometrium was constitutive throughout the estrous cycle in pigs, but changed during pregnancy with the greatest abundance on day 15. These findings suggest that LIFR and GP130 are both expressed in the endometrium, but their endometrial expression during pregnancy is differ entially regulated in pigs.
Porcine conceptus tissues expressed LIF and LIFR during early pregnancy, and chorioallantoic tissues also expressed LIF and LIFR during mid to late pregnancy. These data co incide with previous reports that LIF and LIFR are expressed in the conceptus and fetal membrane tissues [14,15]. We fur ther determined that LIF expression in chorioallantoic tissues increased towards term pregnancy in pigs, while LIFR expres sion decreased, suggesting a role of LIF in placental function. Based on localization of LIF and LIFR in the endometrium and conceptus tissues, it is likely that LIF acts on endometrial epithelial cells and conceptus/chorioallantoic tissues in an autocrine and/or paracrine manner to affect endometrial and conceptus function during pregnancy in pigs.
LIF is expressed in endometrial epithelial cells during the menstrual cycle and endometrial epithelial cells, decidual cells, and decidual leukocytes during early pregnancy in humans [8,28]. LIFR is expressed in only endometrial luminal and glandular epithelial cells during the cycle, whereas it is ex pressed in endometrial epithelial cells, endothelial cells, and villous and extravillous trophoblasts during pregnancy in humans [8,28]. In this study, LIF expression was detected primarily in endometrial epithelial cells during the estrous cycle and pregnancy and in chorioallantoic membrane during mid to late pregnancy in pigs. LIFR expression was also de tected in endometrial epithelial cells in the endometrium, and endometrial stromal cells expressed LIFR at increased levels on day 15 of pregnancy, which is the time when many immune cells such as T cells and NK cells are recruited into the endo metrium during pregnancy in pigs [29]. These data suggest that LIF and LIFR act on endometrial epithelial cells in an autocrine manner and on stromal cells in a paracrine manner.
The expression of LIF is increased by cytokines, leptin, and estrogen in endometrial cells in humans and rodents [1,10,19], and the expression of Lifr is induced by P 4 , but not by E 2 , in the endometrium of golden hamsters [20]. Based on the pat terns of LIF and LIFR expression in the endometrium during the estrous cycle and pregnancy observed in this study and reports in other species [1,10,19,20], we hypothesized that ovarian steroid hormones affect the expression of LIF and LIFR in the porcine endometrium. Indeed, our results in this study indicate that estrogen increased the expression of LIF and progesterone increased the expression of LIFR in endome trial tissues. These data suggest that estrogen and progesterone play critical roles in regulating the expression of LIF and LIFR in the endometrium during the estrous cycle and pregnancy, and that the regulation of endometrial LIF and LIFR expres sion by steroid hormones is similar among species. In mice, the expression of Lif dramatically increases in the endome trium at the time of implantation, and estrogen is responsible for the increase of endometrial Lif expression [10,19]. In pigs, conceptus implantation initiates on day 12 of pregnancy and the implanting conceptus produces estrogen, which acts as a maternal pregnancy recognition signal [25]. Thus, we postu lated that endometrial expression of LIF increased in response to conceptusderived estrogen in pigs. However, our results showed that LIF expression was not different between day 12 of the estrous cycle and pregnancy. This suggests that concep tusderived estrogen may not be responsible for endometrial expression of LIF in pigs, and that the regulatory mechanism underlying endometrial LIF expression is different between rodents and pigs because of differences in implantation and placentation.
We also found that the endometrial expression of LIFR was greatest on day 15 of pregnancy, when the implanting concep tus produces the large amounts of cytokines such as interferons (IFNs), IFNγ, and IFNδ [25], and the endometrium pro duces cytokines and chemokines such as cysteineXcysteine motif chemokine ligand (CXCL) 9, CXCL10, and CXCL11 [29]. Thus, it is possible that these cytokines derived from the conceptus and the endometrium affect the expression of endometrial LIFR at the time of implantation in pigs, but further study of the factors regulating endometrial LIFR expression on day 15 of pregnancy are necessary to elucidate this hypothesis.
In conclusion, in this study we showed that LIF and LIFR are expressed in the endometrium in a stagespecific manner during the estrous cycle and pregnancy and in conceptus and chorioallantoic tissues during pregnancy in pigs. Estrogen and progesterone regulate the expression of LIF and LIFR in the endometrium, respectively. Although the role of LIF in the endometrium during the estrous cycle and pregnancy is not fully understood, our results indicate that LIF and its receptor signaling system play important roles in regulating endome trial function during the estrous cycle and at the maternal conceptus interface during mid to latepregnancy in pigs.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.