Expression of ST6GalNAc1 and sialyl-Tn antigen enhances endometrial receptivity


 BackgroundTo characterize molecular mechanism underlying the regulation of sialylated glycan expression and its roles for endometrial receptivity and embryo implantation. Here, we characterized the role of a truncated form of sialylated O-glycan, sialyl-Tn, for endometrial receptivity.MethodsThe transcriptomes of human endometrium at mid-secretory phase were analyzed by Bioinformatics. Changes in gene expression, protein, and signal pathway were measured using RT-PCR and Western blot. The cell adhesion assay was visualized using a fluorescent microscope. In peri-implantation phase of mice, the expression of leukemia inhibitory factor (LIF) and sialyl-Tn were confirmed using immunohistochemistry and immnofluorescence analysis. The effect of sialy-Tn expression on embryo implantation was estimated by in vitro fertilization and embryo transfer using mice.ResultsIn in silico analysis, expression of O-glycosylation genes, especially ST6GalNAc1, was significantly increased in the human uterus of mid-secretory phase. Overexpression of the ST6GalNAc1 gene in non-receptive human endometrial AN3CA cells enhances the attachment of trophoblastic JAr cells. In an animal study, the results clearly indicated that sialyl-Tn was expressed on the surface of the mid-secretory uterus. In addition, blockade of the receptor using free sialyl-Tn epitope diminished the implantation rates of intrauterine transferred murine embryos.ConclusionFrom these results, here we suggest that sialyl-Tn expression might be a novel factor regulating the endometrial receptivity for successful embryo implantation.

The choriocarcinoma JAr cell was obtained from the Korean Cell Line Bank (Seoul, Korea).
Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from cells using RiboEx™ (GeneAll, Seoul, Korea) and then subjected to reverse transcription with oligo-dT primers using M-MLV reverse transcriptase (Thermo Fisher Scientific). The cDNA was amplified by PCR using AccuPower®PCR PreMix (Bioneer Co., Daejeon, Korea). The primers used in this study were as follows: Adhesion assay AN3CA cells were seeded into 6 well plates and cultured to a confluent monolayer. After the pretreatment of hLA, the cells were incubated with leukemia inhibitory factor (LIF) treatment for 48 h. The JAr cells were labeled with Cell Tracker™ Green CMFDA (Invitrogen, Carlsbad, CA) and gently added onto a monolayer of AN3CA cells. After gently shaking at 40 rpm for 30 min at 37 °C, the cells were vigorously washed to remove nonbinding cells. The attached JAr cells were visualized by using a fluorescent microscope (Axio Imager M1, Carl-Zeiss, Oberkochen, Germany). Five fields of each sample were chosen randomly and the number of adhered cells was averaged after quantifying with ImageJ software (NIH; Bethesda, MD) Animals Female C57BL/6 mice (7-8 weeks old, weight 20-22 g) were purchased from Orient Bio, Co. (Seongnam, Korea). Mice were ovariectomized (OVEX) and recuperated 14 days later.
Animals were fed on a standard diet with ad libitum access to water, and kept on a 12 h light: 12 h dark cycle. All experimental procedures were examined and approved by the Animal Research Ethics Committee at the Pusan University of Korea (no. PNU-2016-1212).

Induction of peri-implantation phase and immunochemistry
Twenty-eight female mice were randomly divided into two groups: control and PE. In the prime phase, mice were injected subcutaneously (s.c.) with 100 ng β-estradiol (in 100 µl corn oil) or vehicle for 3 days. In interphase, no hormone was treated for the next two days. In the peri-implantation phase, mice were daily injected s.c. with 6.7 ng β-estradiol and 1 mg progesterone (in 100 µl corn oil) for 6 more days. Mice were respectively sacrificed on day 6, 8 and 10, and both uterine horns were excised to determine the expression of LIF and sialyl-Tn using immunohistochemistry and immunofluorescence analysis, respectively. The uterine tissues of mice were fixed in 3.7% formalin and embedded in paraffin and were then cut into 4 µm serial sections. The sections were immunostained with LIF or sialyl-Tn (FITC) antibodies, visualized with the Dako Envision kit (Dako, Jena, Germany) and fluorescent microscope (Axio Imager M1) respectively. Animals and embryo collection and Embryo transfer in utero Inbred ICR female mice (6-8 weeks old) were induced to superovulate by intraperitoneal injection of 5 IU serum gonadotropin from a pregnant mare (Sigma-Aldrich) followed by injection with 5 IU human chorionic gonadotropin (hCG; Sigma-Aldrich) 46 later. Superovulated female mice were mated with fertile male mice and euthanized by cervical dislocation 46 h after hCG injection. The day of vaginal plugging was designated as 0.5 days post coitum (dpc). Mouse 2-cell embryos were collected from oviducts on 1.5 dpc and cultured in pre-warmed the Quinn's Advantage Blastocyst Medium (SAGE/Origio, Malov, Denmark) containing 10% serum protein substitute in an incubator at 37 and 5% CO 2 .
Implantation potential of embryos from the control and sialyl-Tn epitope groups was examined via embryo transfer as described previously [22,23]. In the sialyl-Tn epitope group, embryos were transferred with a 50 ng/ml sialyl-Tn epitope. Five to six embryos from two different groups were transferred on 3.5 dpc to the contralateral uterine horn of pseudopregnant recipients provided by mating with a vasectomized male. Two days after transfer, implantation sites in uterine horns of pregnancy were detected by intravenous injection of Chicago blue dye. Clear blue bands in utero were considered implantation sites.

Statistical analysis
Statistical analysis of results was performed by a Student's t-test (for in vitro adhesion assay) or Chi-square test (for in vivo embryo transfer) using GraphPad Prism (GraphPad Software, San Diego, CA). Values are expressed as the mean ± standard deviation (SD) and the minimum significance level was set at a P value of 0.05. All experiments except for animal studies were independently performed at least 3 times.

Bioinformatic analysis
To identify the functions of protein glycosylation in human embryo implantation, we applied a bioinformatic approach with the NCBI GEO database (GSE4888) [17]. The results from GSEA using the transcriptomes of human proliferative and mid-secretory endometrium indicated that glycosylation, especially O-linked glycosylation is the most prominent and significant gene set ( Fig. 1A and B). Among the genes involved in Oglycosylation, the expression of sialyltransferases, such as ST6GalNAc1, ST6Gal1, ST3Gal6, are significantly increased in the uterus of mid-secretory phase, which is corresponding to implantation window (Fig. 1C). To identify putative targets of LIF, a major cytokine regulating endometrial receptivity [24], we performed correlation analyses using the same GEO dataset. Interestingly, a highly significant positive correlation between LIF/gp130/STAT3 signaling pathway and ST6GalNAc1 was observed ( Fig. 1D  demonstrated that non-receptive AN3CA cells did not express ST6GalNAc1 at the presence of LIF or not. Whereas, receptive Ishikawa cells expressed ST6GalNAc1 even at the absence of LIF. Thus, we exogenously overexpressed the ST6GalNAc1 gene in nonreceptive endometrial AN3CA cells. The overexpressed ST6GalNAc1 gene successfully induced both sialyl-Tn expression and receptivity to JAr cells (Fig. 2C-E). These results clearly demonstrated that the expression of ST6GalNAc1 and sialyl-Tn were sufficient for mediating the cell-cell interaction between trophoblast and endometrial cells.
Sialyl-Tn expressed at implantation window was crucial for endometrial receptivity To examine the expression of sialyl-Tn at the period of implantation window in mice endometrium, estradiol and progesterone were serially treated to ovariectomized female mice for mimicking normal hormonal cycle (Fig. 3A), according to previous studies [28,29]. The expression of LIF appeared in the uterus of estradiol and progesterone treatedmice (PE group) from day 8 and prominent at day 10 (Fig. 3B). The expression of sialyl-Tn was elevated from day 6 and day 8 in the uterus of the PE group than the control group.
However, the expressions are fuzzily distributed in the stoma region of the uterus. On day 10, the sialyl-Tn was expressed highest and prominent in the luminal region of the endometrium (Fig. 3C). To confirm the function of ST6GalNAc1 expression in embryo implantation in vivo, the genetic abrogation of its expression is required. However, there are no available knockout mice in previous research papers or mouse phenotyping centers. It is possible that knockdown of ST6GalNAc1 may be embryonic lethal. Thus, to overcome the absence of knockout mice, we confirm the in vivo effect of sialyl-Tn on endometrial receptivity by blocking its receptor using free sialyl-Tn epitopes in murine IVF model. The result clearly demonstrates that the pre-incubation of the murine embryo with free sialyl-Tn for blocking its receptor can reduce the implantation rate of the transferred murine embryo (Table 1). These results collectively suggested that the sialyl-Tn antigen expressed on the endometrium induced by LIF in the period of implantation window had a key role in the adhesion between trophoblast and endometrium. are still not reported. In this study, we blocked the receptor-ligand binding by using free sialyl-Tn epitopes, and they successfully abrogated the adhesion of trophoblast to endometrial cells in murine IVF model. However, the precise mechanism underlying the LIF/sialyl-Tn/Siglec-6 axis should be illuminated by further extensive experiments.
From our recent study, the expression of integrin αVβ3 and αVβ5 induced by LIF treatment are required for endometrial receptivity [44]. However, the pattern of integrin expression in AN3CA is very similar to receptive endometrial cells, such as HEC-1A and Ishikawa [45].
To explain the phenomena, an element added to the integrins are required. Previous studies reported that several integrins including α2, α2b, α5, β1, and β3, are Oglycosylated proteins [46,47]. In addition, other adhesive molecules such as CD44 and trophinin are also highly glycosylated proteins [48]. Thus, the precise proteins, which added by sialyl-Tn and mediating embryo implantation, should be elucidated by further extensive studies. In this study, we shed a light on the understanding of the role of protein sialylation in endometrial receptivity.