Cytokine-Like Protein 1(Cytl1): A Potential Molecular Mediator in Embryo Implantation

Abstract

Cytokine-like protein 1 (Cytl1), originally described as a protein expressed in CD34+ cells, was recently identified as a functional secreted protein involved in chondrogenesis and cartilage development. However, our knowledge of Cytl1 is still limited. Here, we determined the Cytl1 expression pattern regulated by ovarian hormones at both the mRNA and protein levels. We found that the endometrial expression of Cytl1 in mice was low before or on the first day of gestation, significantly increased during embryo implantation, and then decreased at the end of implantation. We investigated the effects of Cytl1 on endometrial cell proliferation, and the effects on the secretion of leukemia inhibitory factor (LIF) and heparin-binding epidermal growth factor (HB-EGF). We also explored the effect of Cytl1 on endometrial adhesion properties in cell-cell adhesion assays. Our findings demonstrated that Cytl1 is an ovarian hormone-dependent protein expressed in the endometrium that enhances the proliferation of HEC-1-A and RL95-2 cells, stimulates endometrial secretion of LIF and HB-EGF, and enhances the adhesion of HEC-1-A and RL95-2 cells to JAR spheroids. This study suggests that Cytl1 plays an active role in the regulation of embryo implantation.

Fig 1. Expression of Cytl1 in mouse endometrial tissues and human endometrial cells.

(A) Reverse-transcription PCR analysis of Cytl1 mRNA expression in the endometrium at different stages of early pregnancy; representative images of tissues are shown. (B) Greyscale analysis of Cytl1 expression relative to β-actin expression. (C) Real-time PCR analysis of Cytl1 mRNA expression in human endometrial cell lines (HEC-1-A, RL95-2) and a human trophoblastic cancer cell line (JAR). Data represent mean±SEM. *P < 0.05; **P < 0.01.

Fig 2. The effects of ovarian hormones on endometrial Cytl1 expression.

(A) Reverse-transcription PCR analysis of Cytl1 mRNA expression in vitro after treatment with ovarian hormones or control (B). Greyscale analysis of Cytl1 expression relative to β-actin expression. Cytl1 protein expression regulated by ovarian hormones or control were analyzed by ELISA in human endometrial cell lines HEC-1-A (C), RL95-2 (D) and the human trophoblastic cell line JAR. (E) Cytl1 expression levels are represented by OD₄₅₀ values, and (F) the positive control represents the result of standard sample detected by ELISA. Data shown for each sample are average of results from three array wells. P, progesterone; E, estradiol. Data represent mean±SEM. *P < 0.05; **P < 0.01.

Fig 3. Effects of Cytl1 on endometrial cell proliferation.

Proliferation of HEC-1-A (A) and RL95-2 (B) cells after treatment with different concentrations of Cytl1 or control were determined by CCK-8 assay. Proliferation is represented by optical density (OD) values. P, progesterone. Data represent mean±SEM. *P < 0.05; **P < 0.01.

Fig 4. The effect of Cytl1 on the endometrial expression of LIF and HB-EGF.

(A) Reverse -transcription PCR analysis of LIF and HB-EGF mRNA expressions in human endometrial cell lines (HEC-1-A, RL95-2) after treatment with different concentrations of Cytl1. LIF expression in the culture supernatant of HEC-1-A (Ba) and RL95-2 (Bc), and HB-EGF expression in HEC-1-A (Bb) and RL95-2 (Bd) were detected by ELISA. The standard curve prepared according to manufacturer’s instruction was used to determine sample concentrations. P, progesterone. Data represent mean±SEM. *P < 0.05; **P < 0.01.

Fig 5. The effect of Cytl1 on JAR spheroid adhesion to endometrial cells.

The percentages of JAR spheroid adhesion to HEC-1-A (A) and RL95-2 (B) cells were determined using cell-cell adhesion assays; JAR spheroid adhesion was quantified using CCK8 assays; results are presented as optical density (OD) (C). P, progesterone. Data represent mean±SEM of three independent experiments. *P < 0.05; **P < 0.01.