Background
Successful implantation is dependent on the intricate genetic and molecular signalling dialogue between the receptive uterus and active embryo [
1]. A better understanding of the molecular events underlying the regulation of embryo implantation may improve the ability to treat infertility. To date, although many molecular modulators have been identified during the implantation period, the precise molecular mechanism underlying embryo implantation is still unknown.
CREBZF is as a member of the CREB (cAMP-response-element-binding protein)/ATF (activating transcription factor) family of basic region-leucine zipper (b-ZIP) transcription factors [
2]. Previously studies have found that CREBZF gene expression produces two isoforms, CREBZF-L (long isoform of CREBZF, also known as SMILE) and CREBZF-S (short isoform of CREBZF, which was previously designated as Zhangfei or ZF) [
3]. However, unlike other b-ZIP proteins CREBZF lacks the ability to bind any of the consensus recognition elements for b-ZIP proteins and cannot activate promoters containing these motifs [
2,
4]. Instead, CREBZF requires heterodimerisation with other factors to bind target promoters and to regulate downstream genes, which may include VP16 [
5], Luman (or CREB3, L-ZIP) [
6], Activating transcription factor 4 (ATF4, or CREB2) [
7], X-box-binding protein-1 (XBP1) [
8] and tropomyosin-related kinase A (trkA) [
9,
10]. A potential reason for the inability of CREBZF to recognise these promoters may be the absence of a critical asparagine residue in its basic domain, which forms the DNA-recognition motif, NxxAAxxCR, in all b-ZIP proteins [
2,
4].
Zhangfei was first identified via its interaction with the Herpes Simplex Virus-1 (HSV-1)-related cellular protein Host Cell Factor 1 (HCF-1) and has been proposed to play a role in inhibiting the replication of the herpes simplex virus [
2,
5]. Recent studies have shown that Zhangfei could potentially play an important role in the mammalian unfolded protein response (UPR) [
6‐
8]. Zhangfei might activate apoptosis in ONS-76 medulloblastoma cells via the NGF/TrkA pathway [
10,
11]. Currently, the precise physiological function of SMILE remains unclear. Recently, SMILE has been reported as a novel transcriptional co-regulator of a variety of nuclear receptors, including oestrogen receptors, glucocorticoid receptor, constitutive androstane receptor, hepatocyte nuclear factor 4α and oestrogen receptor-related receptor γ [
3,
12,
13]. In another study, SMILE played an important role in the development of beta cell dysfunction induced by glucolipotoxicity [
14]. Zhangfei exhibits a liver- and cell-type-specific expression pattern; however, SMILE is expressed ubiquitously in mouse tissues and tumour-derived cells [
3]. Thus, it is certainly possible that the two isoforms exhibit different regulatory functions in specific cellular contexts. However, until recently, the physiological function of CREBZF in mammalian reproduction has not been reported. To explore the potential function of CREBZF during pregnancy, we sought to investigate in detail the expression and regulation of CREBZF mRNA and protein in the mouse uterus during the oestrous cycle and peri-implantation period.
Methods
Animals and treatments
The experimental use of mouse for this study was performed according to the Committee for the Ethics on Animal Care and Experiments in Northwest A&F University. Mature mice (Kunming White outbred strain, 8-to 10-wk-old) were purchased from the laboratory animal centre of Xi’An JiaoTong University and housed at a temperature- (24+/−2°C) in a light-controlled room (12 h light: 12 h darkness) with free access to food and water.
Oestrous cycles were tracked by performing daily vaginal smears, and only those mice that demonstrated a regular 4-day oestrous cycle were used for these experiments. Mouse models of early pregnancy, pseudopregnancy, delayed implantation and activation, artificial decidualisation and hormonal treatment were produced as described in our previous reports [
15,
16]. Eight mice were used in each stage or treatment in this study. To confirm the reproducibility of the results, each sample was analysed in triplicate. The entire uterus was collected immediately following sacrifice by cervical dislocation, and half of each uterus was immediately processed for immunohistochemistry, whereas the remaining half was frozen in liquid nitrogen for further extraction of total RNA and protein.
Embryo collection
Female mice were superovulated via an intraperitoneal injection of 5 IU of pregnant mare’s serum gonadotrophin (PMSG, Ningbo Sansheng Pharmaceutical Co., Ltd. China) at 4:00 pm followed by human chorionic gonadotrophin (hCG, Ningbo Sansheng Pharmaceutical Co., Ltd. China) 48 hr later. Treated female mice were mated with fertile males of the same strain to induce natural pregnancy. Embryos at the zygote, 2-cell and 4-cell stage were collected from oviducts at 24 hr, 46 hr and 54 hr, respectively. However, the 8-cell stage embryo, morula and blastula were collected from the uterine horns at 68 hr, 72 hr and 96 hr, respectively. A total of 160 mice were used for embryo collection and 35–40 pooled embryos per developmental stage were analyzed in each group. Three independent experiments were performed.
Total RNA was extracted from the uterine horns and embryo using Trizol (Invitrogen, Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. DNase (TaKaRa Bio, Inc., Dalian, China) was used to remove genomic DNA contamination prior to RT. Extracted RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water, and the RNA concentration and purity were estimated by reading the absorbance at 260 and 280 nm on a spectrophotometer (Eppendorf, Inc., Hamburg, Germany). The cDNAs were synthesised using PrimeScriptTM RT reagent Kit (TaKaRa Bio, Inc., Dalian, China) according to the manufacturer’s instructions. The final volume of the reaction was 20 μl, including 800 ng of total RNA. The reverse transcription product was stored at −20°C.
The GenBank accession number of the mRNA, primer sequences and annealing temperatures are listed in Table
1. Real-time PCR was performed using three biological replicates and technical triplicates/duplicates of each cDNA sample in the LightCycler system (iQ5, Bio-Rad Laboratories, Inc., Hercules, USA) using SYBR® Premix Ex Taq
TM II Kit (TaKaRa Bio, Inc., Dalian, China), according to the manufacturer’s protocol. Each PCR reaction (total volume of 20 μl) consisted of 2 μl reverse transcription product, 0.8 μl of each 10 μM forward and reverse primer, 10 μl SYBR® Premix Ex Taq
TM II, and 6.4 μl RNase-free water. The cycling conditions included a denaturation step at 95°C for 30 sec, followed by 45 PCR cycles of 95°C for 5 sec and 60°C for 20 sec. A melting curve analysis was performed at the end of each PCR program to exclude the formation of nonspecific products. Gene mRNA quantifications were performed using the 2
-△△Ct method and the amount of transcripts in each sample was normalised using RPLP0 and GAPDH as the internal control gene to correct for differences in the amount of cDNA used [
17,
18].
Table 1
The primer sequences used for real-time quantitative PCR
SMILE
| NM_145151.2 | AF: 5'-TAATCGGCTCAAGAAGAAGG-3' | 144 | 60 |
AR: 5'-CGTAGGTAGCGACTCTCC-3' |
RPLP0
| NM_007475 | AF:5'- GGACCCGAGAAGACCTCCTT-3' | 85 | 60 |
AR:5'- GCACATCACTCAGAATTTCAATGG-3' |
GAPDH
| NM_008084 | AF:5'- TCACTGCCACCCAGAAGA-3' | 186 | 60 |
| | AR:5'- GACGGACACATTGGGGGTAG-3' | | |
Immunohistochemistry
Uterine tissues were fixed in 4% (v/v) paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) in phosphate-buffered saline (PBS; pH 7.4) for 24 hr, dehydrated through a graded ethanol series, and embedded in paraffin. Five μm-thick sections were mounted onto glass slides that were precoated with poly-L-Lysine solution (Sigma, St. Louis, MO, USA) and incubated overnight at 37°C. After dehydration, the samples were placed in citrate buffer (pH 6.0), and antigen retrieval was performed by treating the samples (twice) in a microwave oven at 750 W for 5 min. The slides were then washed in PBS. Sections were pretreated with 0.3% (v/v) H2O2 in methanol to quench endogenous peroxidase activity. After several washes with PBS, the sections were incubated with 10% rabbit serum for 30 min at 37°C. Following blocking, the sections were incubated with goat anti-CREBZF polyclonal antibody (Santa Cruz, sc-49328; diluted 1:200 with PBS) for 12 hr at 4°C, washed with PBS, and incubated with biotinylated anti-goat IgG antibody (MaiXin-Bio Technology Co., Ltd., Fuzhou, China) for 1 hr at 37°C. Sections were washed three times with PBS, and then incubated with HRP-labelled streptavidin (SA-HRP) for 30 min at 37°C. Next, positive reactions were visualised using a diaminobenzidine (DAB)-peroxidase substrate (Sigma-Aldrich Co. LLC, Louis, MO, USA) and 30 sec counterstaining with haematoxylin. Negative control slides without the addition of primary antibody or substitution with an appropriate dilution of normal goat IgG was performed in parallel. Slides were imaged using a digital microscope (BA400, Motic, Wetzlar, Germany).
Western blotting analyses
Proteins obtained from uterine tissues were extracted using the Total Protein Extraction Kit (Nanjing Keygen Biotech Co., Ltd., Nanjing, China) according to the supplier’s instructions. The total protein per sample was separated using 12% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by electrotransfer to polyvinylidene fluoride (PVDF) membranes (Millipore; Bedford, MA). The membrane was treated with blocking buffer (5% nonfat dried milk in Tris-buffered saline [TBS] containing 0.1% Tween 20) for 1 hr at room temperature and incubated overnight at 4°C with antibodies against CREBZF (Santa Cruz, sc-49328; 1:500 dilution) or β-actin (1:1,000; Beijing CWBIO Co., Ltd., Beijing, China) as a loading control. The membranes were washed three times with TBS, and then incubated with biotinylated anti-goat IgG antibody or biotinylated anti-mouse IgG antibody (1:5,000; MaiXin-Bio Technology Co., Ltd., Fuzhou, China) for 1 hr at room temperature. After washing, the membranes were incubated with SA-HRP for 30 min at room temperature. Finally, the immunoreactive bands were visualised by DAB staining (Sigma) at room temperature for 5 min, and the immunoreactive bands were imaged using a digital microscope (Tanon-4100, Tanon Science & Technology Co., Ltd., Shanghai, China) and densitometric analyses were processed with Quantity one v4.62 (Bio-Rad).
Immunofluorescence
Embryos were fixed in 4% paraformaldehyde for 30 min at room temperature followed by permeabilising in 0.5% Triton X-100 in PBS. After washing in PBS, the embryos were treated with blocking solution of 1% bovine serum albumin in PBS for 1 hr, followed by incubation with goat anti-CREBZF polyclonal antibody (Santa Cruz, sc-49328; 1:500 dilution) at 4°C overnight. Embryos were then washed three times with PBS, and incubated for 1 hr at room temperature in a 1:500 dilution of Cy3-labelled donkey anti-goat IgG (Beyotime Institute of Biotechnology, Jiangsu, China). The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Embryos were viewed under a laser-scanning confocal microscope (A1R, NiKon). Additionally, negative control embryos per developmental stage were treated in the same manner as described above, but omitting the primary antibody or primary and secondary antibodies, respectively.
Statistical analysis
All experiments were replicated at least three times for each group and the data were presented as the mean ± S.E.M. Data were analysed using ANOVA, followed by Fisher’s Least Significant Different Test (Fisher LSD) and Independent-Samples T test with SPSS software (Version 13.0; SPSS, Inc., Chicago, IL). Differences were considered significant when P < 0.05.
Discussion
This study mainly reported on the changes in the expression of two isoforms of CREBZF, as well as their cellular localisation throughout the oestrous cycle and peri-implantation period in the mouse uterus. These results indicated that SMILE was the predominant form and Zhangfei was not detected in the uterus, which was consistent with the expression pattern of SMILE in the male mouse prostate and testis [
3]. Moreover, our results suggested that SMILE had a cycle-dependent expression in mouse uterus and was clearly localised in both luminal and glandular epithelial cells. A relatively higher expression of SMILE mRNA and protein at the oestrus phase accompanies the female as she reaches optimal sexual receptivity and ovulating time. Thus, we speculated that the elevated levels of SMILE might contribute to the induction of embryo earlier during development and implantation. Indeed, SMILE was localised in the cytoplasm and nucleus of mouse pre-implantation embryos, and the level of SMILE mRNA was gradually increased with embryo development. In addition, SMILE was expressed at high levels in the luminal and glandular epithelium at the proestrus and oestrus phase, and at basal levels in the metestrus and diestrus phase. These data also suggested that SMILE might be under cell-specific sex hormonal control in the mouse uterine epithelium and appeared to be important factors for implantation.
During pregnancy the uterus undergoes a series of programmed morphological changes, resulting in an extensive tissue reorganisation to accommodate the growing embryo [
19]. The time from days 1 to 5 of pregnancy is critical for embryo implantation in mice, and days 4 to 5 of pregnancy is regarded as the implantation window. The embryo apposes and attaches to the uterine luminal epithelium, which is the initial step for a successful pregnancy [
20]. The expression levels of SMILE mRNA and protein were gradually decreased in the mouse uterus from days 1 to 3, but were sharply up-regulated on day 4. Furthermore, the uterine expression of the SMILE protein in the luminal epithelium was predominantly localised at the implantation site on days 5 of pregnancy, demonstrating temporal and spatial specificity during the period of mouse embryo implantation. Taken together, these results suggested that SMILE might play an essential role in the initial attachment of a blastocyst to the uterine epithelia in the mouse. However, the up-regulation of SMILE was restricted to day 5 and was not expressed in decidualised cells on days 6–7 of early pregnancy or under artificial decidualisation, although SMILE was also highly expressed in glandular epithelial cells, which might not be important for decidualisation.
In the present study, SMILE was lowly expressed on days 1–2 in the pseudopregnant uteri and was not observed in the uterus from days 3–6 of pseudopregnancy, indicating that SMILE expression was dependent on the presence of the embryo. Moreover, SMILE mRNA and protein were highly expressed in the uterus when the delayed implantation was terminated by via treatment compared with the expression under conditions of delayed implantation. In addition, the localisation of SMILE protein in the trophectoderm and increased expression of SMILE mRNA and protein at the blastocyst stage might be a prerequisite for establishing contact between the blastocyst and uterine epithelium. These results suggested that SMILE expression in the mouse uterus required the presence of an active blastocyst during the peri-implantation period.
Implantation has long been known to be a steroid hormone-dependent process. The coordinated action of both E
2 and P
4 is necessary for the preparation and process of implantation in the mouse endometrium [
21]. Because both E
2 and P
4 are essential for the induction of implantation in the mouse, we examined the hormonal regulation of SMILE expression. It has been reported that CREBZF isoforms regulated the inhibition of the transactivation of oestrogen receptors via a small heterodimer partner in a cell-type-specific manner, although CREBZF isoforms did not interact directly with oestrogen receptors [
3]. CREBZF isoforms as a novel corepressor of nuclear receptors may interact with SIRT1 to regulate the transactivation of oestrogen receptor-related receptor γ [
13]. SIRT1 plays an important role in the development of human uterine receptivity via inducing E-cadherin expression [
22]. Our studies found that E
2 could significantly up-regulate the expression and localisation of SMILE in the luminal epithelium and glandular epithelial cells in ovariectomised mice. In addition, P
4 had no significant effects on SMILE expression in the ovariectomised mouse uterus. Similar hormonal regulation was also consistent with the spatiotemporal expression of SMILE mRNA and protein in the mouse uterus during the oestrous cycle in this study. High levels of E
2 were observed in mice during proestrus and oestrus, decreasing to low levels during metestrus and diestrus [
23]. During normal mouse pregnancy, higher levels of E
2 were detected on day 1 and day 4, a surge in E
2 on day 1 stimulated uterine epithelial cell proliferation and induced the expression of progesterone receptors. An increase in E
2 levels on day 4 combined with the high P
4, which further stimulated uterine stromal proliferation and induced the endometrial receptivity for the blastocyst to implant [
21,
24]. The results in ovariectomised mice, oestrous cyclic mouse in pregnant mice confirmed our supposition that the expression of SMILE in the peri-implantation uterus may be mainly modulated by E
2.
Competing interests
The authors declare that they have no competing interests with respect to the authorship and/or publication of this article.
Authors’ contributions
PL and AW designed the experiments and drafted the manuscript. NW and JZ collected the uterine samples and embryos. FC, XW, and XL performed the experiments. YJ organised and supervised the project. All authors read and approved the final manuscript.