記住我
ORIGINAL ARTICLE
Year : 2021 | Volume
: 5
| Issue : 4 | Page : 199-205
Leukemia inhibitory factor enhanced the developmental and implantation compatibility of mouse embryos in co-culture with human endometrial epithelial cells
Ali Hosseini1, Bahar Movaghar2, Showra Amani Abkenari3, Hassan Nazari4, Mehrdad Bakhtiyari1
1 Department of Anatomy, Faculty of Medicine; Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
2 Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
3 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
4 Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran
Correspondence Address:
Mehrdad Bakhtiyari
Department of Anatomy, Faculty of Medicine, Iran University of Medical Sciences, Tehran; Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran
Iran
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2096-2924.327881
Objective: Among the various in vitro embryo culture systems, co-culture has demonstrated remarkable effects in pre-implantation embryo development owing to the production of embryo-nourishing factors. Nevertheless, little is known about the secretion of these factors. Therefore, in this study, the effect of leukemia inhibitory factor (LIF), one of the most important nourishing factors in the early development of mouse embryos, in human endometrial epithelial cells (hEECs) was evaluated.
Methods: Two-cell stage embryos were collected from the oviducts of hyper-stimulated and mated mice and cultivated in a co-culture with an hEEC monolayer with or without LIF. The quality and developmental and attachment potential rates of cultured embryos were evaluated by determining the levels of octamer-binding transcription factor 4 (Oct4) and caudal type homeobox 2 (Cdx2) transcripts.
Results: LIF significantly increased the developmental rate (82.67% vs. 61.04%, respectively) and attachment rate (64% vs. 45.45%, respectively) of mouse embryos co-cultured with hEECs compared to those in untreated embryos. The expression levels of Oct4 and Cdx2 in blastocysts cultured in the presence of LIF were higher than those in blastocysts cultured without LIF.
Conclusions: Despite the secretion of LIF by hEECs during co-culture with embryos, the amount of this factor was insufficient, and its addition to the culture media could increase the developmental potential of embryos.
Keywords: Caudal Type Homeobox 2; Embryo Development; Leukemia Inhibitory Factor; Octamer-Binding Transcription Factor 4
Leukemia inhibitory factor (LIF) is highly expressed in the uterine endometrial glands before blastocyst implantation.[1] This cytokine is involved in the survival, differentiation, and proliferation of different cells in adults and embryos.[2],[3] LIF regulates indispensable biological activities during early embryonic development, including blastocyst formation, hatching, and implantation in embryos by inducing the receptivity of the endometrial epithelium for blastocyst attachment and preparing the stroma for implantation and placental development.[4],[5],[6] LIF also maintains the totipotency of the inner cell mass (ICM) of the blastocyst during diapause (arrested development of the blastocyst before implantation) and the ability of the trophoblasts to invade the endometrium.[7]
Several protocols have been designed to optimize the developmental rate and quality of embryos in vitro. Among these, co-culture with somatic cells has been performed to simulate the maternal environment by releasing bioactive factors and supporting cell survival and growth in order to increase the development and implantation potential of in vitro-produced embryos and improve the outcomes of infertility treatment.[8],[9]
Numerous growth factors, receptors, and binding proteins are spatially expressed in the endometrial epithelial cells of mammals. During pre-implantation development, many of these factors and their receptors are also expressed in embryos. The maintenance of mitochondrial function, decrease in apoptosis in the embryo, and increase in blastocyst formation has been shown in some experiments. Embryos co-cultured with endometrial epithelial cells could produce a combination of embryo-nourishing factors, such as LIF.[10],[11]
Although experiments have shown that the co-culture of embryos with somatic cells can improve embryonic development, culture conditions associated with changing normal embryonic development only support early in vitro development in any species.[12] This is probably due to the low effective volume of secretory growth factors in co-cultured somatic cells in vitro. Accordingly, adding some essential growth factors to the culture medium, besides using a co-culture embryo culture system, facilitates embryo development.[13],[14]
Several reports have also demonstrated the effect of LIF supplementation on the developmental rate and blastocyst formation[15],[16] and showed that LIF deficiency could significantly decrease the survivability of embryos during development to the blastocyst stage.[17] These findings strongly suggest that LIF is a critical factor in the normal development of pre-implantation embryos. Although the role of LIF during implantation has been extensively investigated, the co-administration of this factor and co-culture of embryos with endometrial cells during pre-implantation embryogenesis are yet to be experimentally considered.
The goal of this study was to explore the possible role of LIF in the early development and implantation potential of mouse embryos in co-treatment with human endometrial epithelial cells (hEECs). Although the size of blastocysts and the effective cavity expansion and hatching indicate the ICM and trophectoderm (TE), other characteristics of these cells may be compromised by hEECs and LIF. Thus, in this study, the expression levels of Oct4 and Cdx2, key transcription factors for the formation of the TE and ICM, respectively, were also investigated.
MethodsThis study was approved by the Institutional Ethical Committee of the Iran University of Medical Sciences (IR.IUMS.FMD.REC 1395.27960). Written informed consent was obtained from all participants.
Isolation, identification, and culture of human endometrial epithelial cells
Endometrial samples were collected on the day of luteinizing hormone peak +4 of the menstrual cycle (LH + 4) from healthy fertile women aged 25–35 years with a normal menstrual cycle and without any hormonal or IUD device treatment for at least 3 months before sampling. Endometrial samples were obtained using a Pipelle aspirator device (Cooper Surgical, USA) from the fundal region of the endometrial cavity.
The hEECs were obtained from the endometrium samples following three washes with phosphate-buffered saline (PBS) (Gibco, Invitrogen), division into 1 × 1 mm pieces, and 1 h of enzymatic digestion in PBS containing 1 mg/mL collagenase type IV (Sigma-Aldrich, Steinheim, Germany), 0.05 mg/mL trypsin EDTA (Gibco, Invitrogen), and 50 IU/mL DNase at 37°C. After filtration of the entire cell mixtures with a 70 μm cell strainer (BD Falcon, MA, USA) to remove debris and filtration of the remaining cells with a 40 μm cell strainer (BD Falcon, MA, USA) for separating stromal cells from epithelial fragments, the stromal cells were cultured in a culture medium consisting of DMEM/F12 (Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), insulin-transferrin-selenium (ITS) (Gibco, Invitrogen), penicillin/streptomycin (Sigma-Aldrich, Steinheim, Germany), L-glutamine (Gibco, Invitrogen), and non-essential amino acids (Gibco, Invitrogen). The collected epithelial fragments were exposed to Accutase (Millipore, USA) for 20 min to obtain single epithelial cells. Differential adhesion was used to purify epithelial cells and eliminate stromal cells in the epithelial cell mixture by the culture of epithelial cell mixtures in a culture medium containing 75% DMEM/F12 and 25% MCDB 105 (Sigma-Aldrich) with 10% FBS for 2 h. After adherence of the stromal cells to the plate, epithelial cell suspensions were collected and preserved in liquid nitrogen.
Mouse anti-cytokeratin 7 antibody was used to identify hEECs. For this purpose, the cultured cells were washed thrice with PBS, fixed with 4% paraformaldehyde for 10 min, and washed again with PBS-Tween. The cells were then treated with 0.03% Triton X-100 in PBS for 10 min for permeabilization. PBS containing 1% bovine serum albumin (BSA) was used to block non-specific binding for 30 min. The cells were incubated with primary antibodies, mouse anti-cytokeratin 7 (1:150, PDM097 IGg1, Diagnostic BioSystems, Pleasanton, CA, USA) at 4°C overnight and with secondary antibody (FITC-conjugated anti-mouse IgG; Abcam, CA, UK 1:200 in PBS) for 30 min at room temperature (22°C–25°C). After washing with PBS-Tween, 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) was applied to dye cell nuclei. Evaluations and cell counts were performed in triplicate using an Olympus fluorescence microscope (Olympus, Japan) [Figure 1].
Figure 1: Characterization and purity of isolated human epithelial endometrial cells, as revealed via immunofluorescence using cytokeratin 7.Isolated and identified hEECs were cultured in DEME-F12 containing 10% FBS at 37°C and 5% CO2 and then used for in vitro mouse embryo co-culture after the cells reached 50% confluence.
Embryo acquisition and cultivation
Animals
Experiments were performed on 20 adult female NMRI mice (6–8-week-old) obtained from Pasture Institute of Iran (Tehran, Iran). All experiments were performed in accordance with the international guidelines for the Care and Use of Laboratory Animals[18] and were approved by the Institutional Ethics Committee of Iran University of Medical Sciences.
The stimulation of ovulation was carried out by subjecting the female NMRI mice to an intraperitoneal (IP) injection of 7.5 IU pregnant mare serum gonadotropin (PMSG) followed by an IP injection of 7.5 IU human chorionic gonadotropin (hCG) 48 h later. Each female mouse was placed next to a male of the same breed. Female mice with a confirmed vaginal plaque were sacrificed by cervical dislocation 39–40 h after mating to collect 2-cell stage embryos from their oviduct by flushing with DMEM/F12 on the second day after hCG injection. Good-quality embryos were isolated and randomly cultivated in an embryo co-culture system in an embryo culture medium (DMEM-F12 containing 10% FBS) with 1,000 IU/mL or without LIF (Sigma-Aldrich) up to the blastocyst stage. Embryo growth was recorded every 24–96 h.
In order to prepare hEECs for the embryo co-culture system, one day before embryo acquisition, the hEECs were thawed and cultured in a culture medium and approximately 1 h before the co-cultures and washed three times with PBS and incubated with the embryo culture medium for acclimatization. Embryo attachment was also tested by mechanical shaking of the culture dish and washing with PBS, 96 h after embryo co-culture with hEECs.
Evaluation of caudal type homeobox 2 and octamer-binding transcription factor 4 produced in mouse blastocysts by immunohistochemistry
At 3 d post-culture (~114 h post-mating), the blastocysts were fixed in 4% paraformaldehyde for 30 min using immunocytochemistry. For this purpose, the embryos were washed three times with PBS containing 0.1% Triton X-100, incubated with 5% normal goat serum (Sigma-Aldrich, Germany) in PBS for 30 min, and then incubated with the primary antibodies (1:400; rabbit Anti-CDX2 antibody (ab227201) and rabbit Anti-Oct4 antibody (ab18976)) overnight at 4°C. The embryos were then incubated with the secondary antibodies (1; 1,000; goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) and goat anti-rabbit IgG (Alexa Fluor® 647) (ab150079) for cdx2 and oct4, to quantify differentiation and proliferation, respectively).
Cell nuclei were counterstained with 0.1% DAPI to count the total number of cells.
Evaluation of caudal type homeobox 2 and octamer-binding transcription factor 4 expression by real-time reverse transcription-polymerase chain reaction
Real-time polymerase chain reaction (PCR) was used to determine the expression of Cdx2 and Oct4 genes individually in a single blastocyst. Five blastocysts were analyzed in five replicates, and a single embryo was analyzed in each replicate. The RNA of blastocysts was extracted using the RNeasy Plus Micro Kit (Qiagen, Hilden, Germany). Briefly, samples were lysed and homogenized in 350 μL of RLT Plus buffer. The homogenized lysate samples were transferred to a gDNA Eliminator spin column placed in a 2 mL collection tube and centrifuged for 30 s at 8,000 × g. The flow-through of samples was mixed with 350 μL of 70% ethanol, transferred to an RNeasy MinElute spin column placed in a 2 mL collection tube, and centrifuged for 15 s at 8,000 × g. After washing the spin column membrane of RNeasy MinElute spin column three times with 700 μL Buffer RW1, 500 μL Buffer RPE, and then 500 μL of 80% ethanol by centrifugation for 2 min at 8,000 × g, and the RNA was eluted with 10 μL RNase-free water. The extracted RNA was translated into cDNA using a reverse transcription enzyme (Takara Bio Inc., Japan). cDNA synthesis was performed according to the manufacturer's instructions. Briefly, a mixture of buffer, enzyme reverse transcription, oligo-(dT), and random hexamer was added to the extracted RNA sample and incubated at 42°C for 20 min to synthesize cDNA, followed by incubation at 85°C for 15 s to neutralize the enzymes. The synthesized cDNA (1 μg) was amplified using PCR and evaluated. Each PCR using Cyber Green (Takara Bio Inc., Japan) was performed on a Biosystems (ABI 7500 thermocycler, Warrington, UK) StepOne and StepOnePlus real-time PCR systems, according to the manufacturer's protocol. Approximately 40 cycles were specified for each cycle of PCR, and the temperature for each cycle was 94°C for 15 s and then 60°C for 60 s. The accuracy of each amplification curve was validated by the melting curve using a melt temperature that was specific for each product of each gene. The no-template control and no-reverse transcriptase control were used to check for contamination of the PCR reagents. Data were analyzed using LinReg PCR software version 2012.0 (USA) to determine the threshold cycle number (Ct). The mean efficiency (E) values for each gene were determined from the amplification profiles of individual samples using the same software. The following formula was applied to determine the relative gene expression in embryos compared to that in the control group for treatment.[19] Five embryos were analyzed in each group individually, and GAPDH was used as an internal control. Each experiment was repeated thrice. The sequences of the primers used are listed in [Supplementary Table 1].
Statistical analysis
Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS version 22.0) for Windows (IBM Corp., Armonk, NY, USA). Continuous variables were expressed as mean ± standard error of the mean and categorical variables as frequencies (percentages). The normality of the variables was checked using the Kolmogorov–Smirnov test. Chi-square and t-test were used to evaluate the differences between groups. Statistical significance was set at P < 0.05.
ResultsEmbryo development in vitro
The co-culture of hEECs with LIF significantly increased the developmental rate of mouse embryos compared to that in the group without LIF [Table 1] and [Figure 1]; the day 3 blastocyst and hatched blastocyst rates showed a significant difference between the two groups (P < 0.05). The implantation compatibility of cultured blastocysts, determined using blastocysts attached to hEECs [Figure 2], was also significantly increased in the presence of LIF (P < 0.05) [Table 1].
Table 1: Developmental rate of 2-cell mouse embryos cultured in vitro up to the blastocyst stage and attachment to endometrial epithelial cells in the presence and absence of LIFTo quantify the proliferation (ICM cell number) and differentiation (TE cell number) rates, blastocysts were fixed on day 3 post-culture and immunostained using Oct 4 and Cdx2 antibodies, respectively [Figure 3]. The total cell number of mouse blastocysts co-cultured with hEECs was affected by LIF. The embryos that developed to the blastocyst stage in the presence of LIF had a higher total cell number (P < 0.05) than those cultured in the absence of LIF. However, the difference in ICM and TE cell percentage between the blastocysts developed in the two groups was not significant [Table 2].
Figure 2: Attachment of the mouse blastocyst to human endometrial epithelial cells in the embryo co-culture system.
Figure 3: Quantification of the proliferation and differentiation rates of in vitro-cultured mouse blastocysts using immunostaining with OCT4 and CDX2 antibodies. Oct4: Octamer-binding transcription factor 4; Cdx2: Caudal type homeobox 2.
Table 2: The cell numbers of mouse blastocysts co-cultured with human endometrial epithelial cells in the presence and absence of LIFThe relative abundance (RA) of Oct4 and Cdx2 transcripts at the blastocyst stage in the two experimental groups are shown in [Figure 4]. As shown, the expression of Oct4 in blastocysts cultured in the presence of LIF was higher than that in blastocysts without LIF (P < 0.05). Similarly, higher expression of Cdx2 was observed in blastocysts treated with LIF during in vitro co-culture with hEECs than in blastocysts without LIF (P < 0.05).
Figure 4: The abundance of Oct4 (a) and Cdx2 (b) transcripts in in vitro culture of the mouse blastocyst. mRNA from embryos was reverse-transcribed and subjected to real-time quantitative PCR. All levels were normalized to GAPDH mRNA expression. Values with superscripts “*” refer to significant (P < 0.05) differences in relative transcript abundance between groups. Oct4: Octamer-binding transcription factor 4; Cdx2: Caudal type homeobox 2; PCR: Polymerase chain reaction. Discussion The goal of this study was to investigate the likely role of LIF in the early development and implantation potential of mouse embryos subjected to co-culture with hEECs. Therefore, the expression levels of Oct4 and Cdx2, key transcription factors for the formation of the TE and ICM, respectively, were compared in the presence and absence of LIF. Mammalian IVF studies have recently shown an increased cryo-survivability and implantation rate per embryo at the blastocyst stage compared to that at earlier pre-implantation stages.[20],[21] Co-culture of embryos with feeder cells leads to an increase in the blastocyst formation rate and improved blastocyst quality because of the release of bioactive factors.[8],[9],[22] Numerous growth factors are spatially expressed in the endometrial epithelial cells of the mammals used in this study. Therefore, using these cells in co-culture with embryos can provide a combination of embryo-nourishing factors.[23]
Rather than exposing the embryos to a large number of factors present with hEECs in the co-culture medium, which may exert beneficial and inhibitory effects, this study examined the specific effects of the growth factor LIF on blastocyst formation and quality in vitro. Embryo development in co-culture with hEECs was enhanced in the presence of LIF; the blastocyst formation rate increased from 61.04% to 82.67% (P < 0.05), and the blastocyst attachment rate increased from 45.45% to 64%.
Previous studies have reported that LIF deficiency can significantly decrease the development of embryos into blastocysts and reduce the number of cells in the ICM and TE.[17],[24] The blastocyst expresses LIF receptor mRNA and LIF mRNA in the endometrium at the highest level at implantation in mice[24],[25] and many other species, such as humans.[26],[27] Thus, the presence of LIF through co-culture and LIF production by feeder cells or addition to the culture medium can be beneficial for the development of embryos. It has been shown that the co-culture of mouse embryos with monolayers that express LIF (human embryonic fibroblasts and Vero cells) resulted in higher blastocyst formation rates than that observed in non-LIF-expressing cells (human placental villous core mesenchymal cells).[28]
The effects of growth factors and cytokines on mammalian pre-implantation embryo development have been extensively studied. Interestingly, the data obtained from the use of LIF in in vitro studies are conflicting. This factor has either stimulatory[4],[5],[14],[15],[29] or no effects[13],[30],[31],[32] on blastocyst development in vitro. Although some conflicting results have been obtained, the differences in effects of LIF treatment during embryo culture and blastocyst formation may be attributed to the culture media and environment of the embryos (e.g. stress or deprivation of critical developmental conditions).[33] This difference may explain why a sufficient amount of each growth factor is needed during pre-implantation embryonic development, which is consistent with the results of our present study on LIF treatment in a co-culture system.
Barmat et al. showed the secretion of some growth factors, such as LIF, from human tubal epithelial cells and assumed that these factors were responsible for the positive effects of co-culture.[34] In addition, LIF has increasingly been shown to be involved in the blastocyst–endometrium dialog and in preparing a receptive endometrium, which is required for successful implantation.[35],[36],[37] The combined actions of the ovarian hormones E and P4 on the uterus mediate embryo implantation. In addition, LIF, a pro-inflammatory cytokine, plays a pivotal role in regulating uterine receptivity. LIF is expressed in the endometrial glands and strongly affects the luminal epithelium of the uterus. LIF mediates its function by activating the JAK-STAT pathway specifically in the uterine luminal epithelium and subsequent induction of many additional pathways, including those with many transcription factors that initiate a cascade of changes affecting the epithelium.[35],[38],[39]
The data of this study indicate that despite the secretion of some factors present during in vitro embryonic development from feeder cells in a co-culture embryo culture system, the amount of these factors is not sufficient. LIF enhanced the developmental potential of mouse embryos despite the co-culture of embryos with hEECs. Therefore, it can be argued that contentment to co-culture system in a simple culture medium without any specific supplement, which provides all essential factors, alone is insufficient for in vitro embryo development.
Supplementary information is linked to the online version of the paper on the Reprod Dev Med website.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
留言 (0)