Abstract
During its journey through the oviduct, the bovine embryo may induce transcriptomic and metabolic responses, via direct or indirect contact, from bovine oviduct epithelial cells (BOECs). An in vitro model using polyester mesh was established, allowing the study of the local contact during 48 h between a BOEC monolayer and early embryos (2- or 8-cell stage) or their respective conditioned media (CM). The transcriptomic response of BOEC to early embryos was assessed by analyzing the transcript abundance of SMAD6, TDGF1, ROCK1, ROCK2, SOCS3, PRELP and AGR3 selected from previous in vivo studies and GPX4, NFE2L2, SCN9A, EPSTI1 and IGFBP3 selected from in vitro studies. Moreover, metabolic analyses were performed on the media obtained from the co-culture. Results revealed that presence of early embryos or their CM altered the BOEC expression of NFE2L2, GPX4, SMAD6, IGFBP3, ROCK2 and SCN9A. However, the response of BOEC to two-cell embryos or their CM was different from that observed to eight-cell embryos or their CM. Analysis of energy substrates and amino acids revealed that BOEC metabolism was not affected by the presence of early embryos or by their CM. Interestingly, embryo metabolism before embryo genome activation (EGA) seems to be independent of exogenous sources of energy. In conclusion, this study confirms that early embryos affect BOEC transcriptome and BOEC response was embryo stage specific. Moreover, embryo affects BOEC via a direct contact or via its secretions. However transcriptomic response of BOEC to the embryo did not manifest as an observable metabolic response.
Introduction
During its 4-day journey through the oviduct (Hackett et al. 1993), the bovine embryo undergoes important morphological and transcriptional events such as the first mitotic cell divisions, the timing of which is reflective of developmental competence (Lonergan et al. 1999) and embryonic genome activation (EGA) involving a switch from dependence on transcripts stored in the oocyte to de novo transcription (Memili & First 2000). There is convincing evidence demonstrating a positive influence of the oviduct on embryo development in terms of embryo cryotolerance, gene expression and developmental competence (Enright et al. 2000, Lazzari et al. 2002, Rizos et al. 2002, Lonergan et al. 2003). However, the embryo effect on the oviduct is less studied. In mice, the presence of embryos in the oviduct increased the expression of specific genes such as thymosin beta-4, ribosomal protein L41 and nonmuscle myosin light chain 3 (Lee et al. 2002). In pigs, Almiñana et al. (2012) reported that the presence of embryos reduced the expression of genes related with immune function. Also, transforming growth factor-alpha, transforming growth factor-beta-binding protein 2 and atrial natriuretic factor receptor-like were upregulated in the presence of four-cell porcine embryos (Chang et al. 2000).
It is likely that the effect of any putative embryo-derived signals on the cells of the oviduct would be magnified in litter-bearing species due to the presence of multiple embryos in the oviduct. That notion is supported by a previous study from our group in cattle, a mono-ovulatory species, which failed to detect a transcriptomic response of the oviduct to the presence of a single embryo, while a response was observed after the transfer of multiple embryos (Maillo et al. 2015). In the horse, the presence of a single embryo altered the oviduct transcriptome (Smits et al. 2016), when epithelial cells were collected locally from the ampullary–isthmic junction. Combined, these findings suggest that the presence of a single embryo in the oviduct may induce a very local effect on oviduct transcriptome which is difficult to detect. Furthermore, in vivo studies remain challenging, due to the relative inaccessibility of the oviduct. BOECs have been cultured in vitro in many systems, as monolayers, in perfusion chambers, in suspension, in polarized or in 3D systems (reviewed by Maillo et al. 2016). Recent in vitro culture strategies have mimicked the morphological and functional changes occurring in the oviduct epithelium using an air–liquid interface (Chen et al. 2017, 2018) or using the state-of-the-art oviduct-on-a-chip platform (Ferraz et al. 2018). However, considering the complexity of studies aiming to understand the regulatory mechanisms controlling embryo–maternal communication, the simple BOEC in vitro, cultured as a monolayer might provide a good starting point to study embryo signals. For instance, Schmaltz-Panneau et al. (2014) identified differentially expressed genes related to the immune system and interferon signaling by co-culturing early embryos for 8 days on a 13-day-old BOEC monolayer (Schmaltz-Panneau et al. 2014). Also, in a recent study carried out by our group using the BOEC monolayer model, we reported that bovine embryo–oviduct interaction in vitro reveals an early cross-talk mediated by bone morphogenetic protein (BMP) signaling (García et al. 2017).
In order to test the hypothesis that the early embryo induces transcriptional and metabolic changes in BOEC, we established an in vitro co-culture system allowing both local and temporal contact between early bovine embryos and oviduct isthmus epithelial cells, where development occurs in vivo. The objectives were (i) to assess the gene expression response of BOEC to early embryos and to test whether it is due to a contact-dependent signal or the result of BOEC interaction with embryo secretions and (ii) to examine the metabolic changes of BOEC in the presence or absence of early embryos.
Materials and methods
Chemicals
All reagents were purchased from Sigma Chemical Química S.A Company unless otherwise stated.
Bovine oviduct epithelial cell isolation and in vitro culture
Bovine oviducts at the early luteal phase (corresponding to days 3–5 of the estrous cycle) were obtained from slaughtered heifers and selected based on ovarian morphology, according to Ireland et al. (1980). Oviduct epithelial cell isolation was performed as described previously (García et al. 2017). Cells were cultured at a final concentration of 2 × 106 cells/mL with TCM 199 supplemented with 10% fetal calf serum (FCS), 2.5% gentamycin and 1% amphotericin, in four-well plates at 38.5°C in an atmosphere of 5% CO2, 20% O2 and saturated humidity until confluence (6–7 days). Half of the media was renewed every 48 h. Twenty-four hours before starting the co-culture with embryos or their CM, the cell culture media was replaced with Synthetic Oviduct Fluid supplemented with amino acids (SOFaa) + 5% FCS (see ‘Experimental design’ section for more details and Fig. 2).
Immunocytochemistry
The epithelial nature of the cultured cells was confirmed by immunocytochemical analysis using antibodies anti-bovine-pancadherin (C1821), anti-bovine-pancytokeratin (C2931) and anti-bovine-vimentin (V2258), as previously described (Lopera-Vásquez et al. 2016) (Fig. 1A).
In vitro production of embryos and their CM
Immature cumulus oocyte complexes recovered by aspirating follicles (2–8 mm in diameter) from ovaries of heifers slaughtered at a local abattoir were submitted to in vitro maturation and fertilization, as described previously (García et al. 2017). Approximately 18–22 h after insemination, presumptive zygotes were completely denuded of cumulus cells by vortexing and groups of 50 were initially cultured in 500 µL of SOFaa supplemented with 5% of FCS in a four-well dish under an atmosphere of 5% CO2, 5% O2 and 90% N2 at 38.5°C. Embryos at the two- and eight-cell stage were selected at 31 and 52 h post insemination (hpi), respectively, and then randomly cultured either in groups of 50 for 48 h to produce embryo CM or co-culture with BOEC (see Experimental design, Fig. 2).
Mesh preparation and co-culture condition
In order to limit the area of contact between the embryos and the BOEC monolayer, a nontoxic woven polyester mesh (Sefar Petex; Sefar, Bury, Lancashire, UK) was used as described previously (García et al. 2017). Grids with size of 41 × 41 openings covering an area of 121 mm2, fitting perfectly inside the well of a Nunc 4-well dish, were used. First, a template grid containing delimited marked areas of 50 openings ((7 × 7) + 1) was placed outside underneath each well of a four-well dish (Fig. 1B). A second mesh square of the same size was washed once with 70% ethanol and three times with PBS followed by SOFaa media, and then it was introduced into the well, so that the external and internal meshes overlapped (Fig. 1B). During the co-culture, 50 embryos (at the two- or eight- cell stage) were placed inside the BOEC well, over the central marked area. After 48 h of co-culture, BOEC were collected from the marked area directly beneath the embryos and as well as from other areas of the well that were not in direct contact with the embryos (Fig. 1B) by mechanical scraping with a micropipette tip and snap frozen in liquid nitrogen and maintained at −80°C until gene expression analysis.
RNA isolation and reverse transcription
mRNA from each experimental group of BOEC was extracted from four biological replicates using Dynabeads mRNA Direct Extraction Kit (Dynal Biotech, Oslo, Norway), following the manufacturer’s instructions with minor modifications as described previously (Bermejo-Alvarez et al. 2008). After 10 min of incubation in lysis buffer with Dynabeads, poly(A) RNA attached to the Dynabeads was extracted with a magnet and washed twice in washing buffer A and washing buffer B. RNA was eluted with Tris–HCl. Immediately after extraction, the reverse transcription reaction was performed as recommended by the manufacturer (Epicentre Technologies Corp, Madison, WI, USA). Briefly, oligo-dT (0.2 μM) and random primers (0.5 µM) were added to the RNA and were then heated for 5 min at 70°C to denature the secondary RNA structure. Next, the tubes were incubated at 25°C for 10 min to promote the annealing of random primers. Then, the RNA was reverse-transcribed for 60 min at 37°C in a final volume of 40 μL containing 0.375 mM dNTPs (Biotools, Madrid, Spain), 6.25 U RNasin RNAse inhibitor (Promega), 10× MMLV-RT buffer with 8 mM dithiothreitol and 5 U MMLV high-performance reverse transcriptase (Epicentre Technologies Corp), followed by incubation at 85°C for 5 min to inactivate the RT enzyme.
Gene expression analysis by quantitative real-time polymerase chain reaction
The mRNA expression levels of the selected genes were determined by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) using specific primers designed with Primer-BLAST software (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) to span exon–exon boundaries when possible (Table 1). Primers were previously validated for adequate primer efficiency and specificity of their PCR products was confirmed by electrophoresis on a 2% agarose gel. All target genes showed efficiencies between 95 and 100% and correlation coefficients close to 1.0.
Set of primers used for qRT-PCR assays.
Gene | Primer sequence (5′–3′)* | Fragment size (bp) | Gene bank accession | |
---|---|---|---|---|
Forward | Reverse | |||
H2AFZ | AGGACGACTAGCCATGGACGTGTG | CCACCACCAGCAATTGTAGCCTTG | 209 | NM_174809 |
ACTG1 | GAGAAGCTCTGCTACGTCG | CCAGACAGCACCGTGTTGG | 255 | NM_001033618.1 |
GPX4 | TGTGGTGAAGCGGTATGGTC | TATTCCCACAAGGCAGCCAG | 266 | NM_174770.4 |
NFE2L2 | GCTCAGCATGATGGACTTGGAG | GGGAATGTCTCTGCCAAAAGC | 390 | NM_001011678.2 |
ROCK1 | ACGTGACCTAGTGCCTTGTG | CCTCAGTGTGCTTTTGTGCC | 164 | XM_002697789.5 |
ROCK2 | CTTGGCTGCTCAACTGGAGA | TGCTCTTGGGCTTCCTTCAG | 276 | NM_174452.2 |
AGR3 | TGTCACACTCAGTTCTGGTCC | GTCATCTCCCCACCCTCTTGA | 119 | NM_001191502.1 |
SMAD6 | GGAGAAATTCGCTCCAAGTGC | CCCTGCCTTTAAAACCCAAGC | 242 | NM_001206145.1 |
TDGF1 | ATGGTGAGAGACGGGCTGCTAG | GCCCTTGTCTCATACAGCTTCC | 201 | NM_001080358.1 |
SCN9A | GTTGATAACCCTGTGCCTGGA | CTTCAAAAGCCAGAGCACCAC | 250 | XM_005202453.3 |
SOCS3 | GCGAGAAGATCCCTCTGGTG | CTAAAGCGGGGCATCGTACT | 167 | NM_174466.2 |
IGFBP3 | GAGTCCAAGCGTGAGACAGAA | GCGGCACTGCTTTTTCTTGTA | 150 | NM_174556.1 |
PRELP | CAGCATCGAGAAAATCAATGGGA | AGCACATCATGAGGTCCAGC | 158 | NM_174434.3 |
EPSTI1 | AAACGACAGCAACAGGAGGAA | CCTTGGAGTCGGTCCAGAAAA | 89 | XM_002691775.5 |
*All the primers were designed with NCBI Primer-BLAST online tool of the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/tools/primer-blast/).
All mRNA transcripts were quantified in duplicate. Each RT-qPCR reaction was performed in a final volume of 20 μL, containing 0.25 mM of forward and reverse primers, 10 μL of GoTaq RT-qPCR Master Mix (Promega) and 2 μL of each cDNA sample derived from BOEC (≈60 ng/μL) using a Rotorgene 6000 Real-Time Cycler (Corbett Research, Sydney, Australia) and SYBR Green as double-stranded DNA-specific fluorescent dye.
Relative expression levels were quantified by the comparative cycle threshold (ΔΔCT) method (Schmittgen & Livak 2008). Values were normalized using two housekeeping genes (H2AFZ and ACTG1). Fluorescence was acquired in each cycle to determine the threshold cycle during the log-linear phase of the reaction at which fluorescence increased above background for each sample. According to the comparative CT method, the ΔCT value was determined by subtracting the mean CT value of the two housekeeping genes for each sample from each gene CT value of the sample. Calculation of ΔΔCT involved using the highest sample ΔCT value (i.e. the sample with the lowest target expression) as an arbitrary constant to subtract from all other ΔCT sample values. Fold changes in the relative gene expression of the target were determined using the formula 2−ΔΔCT (Livak & Schmittgen 2001).
Metabolic analysis
Glucose lactate and pyruvate consumption
Concentrations of glucose, lactate and pyruvate in spent medium following culture of BOEC in the presence or absence of embryos were measured using fluorometric assays based on those described by Gardner & Leese (1990), but with modifications (Guerif et al. 2013). All values were expressed as mM and were relative to a blank control to calculate consumption rate.
Amino acid profiling
The analysis of 18 amino acids in spent medium was performed using high-performance liquid chromatography (HPLC) as previously described (Houghton et al. 2002). All values were expressed in µM and were relativized to blank control to calculate consumption rate.
Experimental design
Experiment 1: Direct and indirect effects of the embryo on the BOEC gene expression
An in vitro co-culture system was established to allow a localized and temporal contact between BOEC and early bovine embryos. Isthmic epithelial cells obtained from a pool of three post-ovulatory stage oviducts on each day (day = replicate, four replicates) were cultured until confluence/monolayer for 6 days. Based on our previous studies (Barrera et al. 2017, García et al. 2017), where the highest proportions of two- and eight-cell embryos were observed at 31 and 52 hpi respectively, in this experiment embryos, at the two- and eight-cell stages were generated by IVF and removed from culture at 31 or 52 hpi respectively to be cultured during 48 h to either generate fresh CM (four replicates) or co-cultured with BOEC (four replicates) (Fig. 2A). BOECs were cultured for 48 h as follows: (1) directly beneath and in direct contact with 50 embryos at the two- or eight-cell stage (BOEC+); (2) in the same well as 50 embryos at the two- or eight-cell stage but not in direct contact with the embryos (BOEC−); (3) with CM from embryos at the two- or eight-cell stage (BOECCM) and (4) without embryos or CM (control, BOEC) (Fig. 2B). Following 48 h of culture, BOECs were collected from the marked area from all groups by mechanical scraping to perform RT-qPCR. The mRNA abundance of genes that had previously been shown to be affected by the presence of embryos from an in vivo study (Maillo et al. 2015: SMAD6, TDGF1, ROCK1, ROCK2, SOCS3, PRELP, AGR3, NFE2L2) and from in vitro studies (Schmaltz-Panneau et al. 2014, 2015: GPX4, SCN9A, EPSTI1, IGFBP3) were analyzed as described earlier.
Experiment 2: Effect of early embryo on BOEC metabolism
After 48 h of co-culture, the media obtained from the previous experimental groups (BOEC+, BOECCM, BOEC) as well as the spent media obtained from embryos cultured alone (EMB) were collected to assess the metabolic changes that may result from interaction of BOEC with the embryos or with their CM, by analyzing the energy substrate and amino acid composition. As blank control, embryo/BOEC-free media was treated in the same manner as the experimental groups (Fig. 2C).
Statistical analysis
Statistical analysis was performed with the SigmaStat software package (Jandel Scientific, San Rafael, CA, USA). The Kolmogorov–Smirnov test was applied to assess data normality. Differences in embryo development were analyzed using chi-square. Differences in the relative mRNA abundance levels and metabolic results were analyzed using one-way ANOVA followed by Holm–Sidak post hoc test, when applicable, to determine the statistical differences between the experimental groups. When normality test failed Kruskal–Wallis, a non-parametric test was conducted. Values were considered significantly different when the P value was lower than 0.05
Results
Effect of BOEC co-culture on early embryo development in vitro
Cleavage rates at 31 hpi (two-cell = 774/1177) and 52 hpi (eight-cell = 652/1197) were 65.5 ± 2.5% and 54.0 ± 7.4% respectively. After 48 h of co-culture, no differences were found in the percentage of two-cell embryos developed to eight-cell stage between embryos co-cultured with (57.0 ± 1.4%) or without BOEC (65.0 ± 2.6%). Similarly, the percentage of 8-cell embryos developed to 16-cell stage was not different between both groups (51.0 ± 4.2% vs 57.7 ± 1.9% for embryos cultured with or without BOEC respectively).
Experiment 1: Direct and indirect effects of the embryo on the BOEC gene expression
The presence of 50 two-cell embryos, in direct contact, or their CM decreased the relative abundance of GPX4, ROCK2 and SCN9A in BOEC in comparison with the control group (P < 0.05). Abundance of SMAD6 was decreased in the presence of embryos, irrespective of direct contact, but was not altered following exposure to CM. The only difference between cells cultured in direct or indirect contact with embryos was observed for NFE2L2 gene, which decreased in cells directly cultured with embryo or their CM in comparison with the culture in indirect contact or control groups. Culture of BOEC with CM increased expression of IGFBP3 (P < 0.05) compared with the control, but no difference was observed with the rest of groups (Fig. 3A). The remaining genes were unaffected by treatment (P > 0.05) (Fig. 3A).
Co-culture in the presence of eight-cell embryos or their CM revealed a different expression pattern of many of the analyzed genes to that observed with two-cell embryos. PRELP was decreased (P < 0.05) in the direct or indirect presence of eight-cell embryos or their CM in comparison with the control group. Also, BOEC directly exposed to eight-cell embryos or their CM exhibited decreased expression of SOCS3 and increased expression of SCN9A in comparison with BOEC cultured in indirect contact with embryos and with the control (P < 0.05). GPX4 was only decreased in BOEC cultured directly with embryos in comparison with the other groups. Furthermore, the expression level of NFE2L2 was increased (P < 0.05) in cells cultured with CM compared to the control but no differences were observed with the rest of groups. Also, abundance of ROCK1 and ROCK2 was increased (P < 0.05) in cells cultured with CM in comparison to the control or to the direct culture with embryos, but it was similar to the indirect contact group. SMAD6 is the only gene that maintained the pattern of expression between BOEC co-cultured with either two- or eight-cell embryos, where it decreased in BOEC cultured in direct or indirect contact with embryos in comparison with the rest of groups (P < 0.05) (Fig. 3B). The remaining genes did not exhibit an alteration of their expression due to treatment (P > 0.05) (Fig. 3B).
Experiment 2: Effect of early embryo on BOEC metabolism
Glucose, lactate and pyruvate consumption
Glucose, lactate and pyruvate consumption by BOEC was not affected by the presence of two-cell or eight-cell embryos (Fig. 4A and B). Indeed, a similar consumption of glucose and pyruvate by BOEC was observed whether they were cultured independently with or without embryos or CM. However, BOEC consumption of those substrates was higher than embryo consumption (P < 0.001 for two-cell embryos; P < 0.005 for eight-cell embryos). In contrast, lactate production was elevated in BOEC cultured with CM from two-cell embryos and in media from two-cell embryos cultured alone in comparison with the other groups (P < 0.001) (Fig. 4A). Lactate consumption was also higher in BOEC cultured alone or with eight-cell embryos and in media from eight-cell embryos cultured alone in comparison with BOEC cultured with CM (P < 0.005) (Fig. 4B). Regarding embryo metabolism, two-cell embryos cultured without BOEC did not reveal a consumption of energy substrates when they were compared to the blank control (data of blank media were not included in the graphs).
Amino acid profiling
The results of HPLC revealed that among the 18 amino acids analyzed, the concentration of ten was affected in BOEC co-cultured with two-cell embryos and 12 in the BOEC co-cultured with eight-cell embryos. These amino acids were significantly highly depleted in BOEC cultured alone or with embryos or with CM compared with both stage embryos cultured alone, except for alanine having the opposite pattern (Fig. 5A and B) (P < 0.005). Similar to alanine, serine had the same tendency of accumulation, but significant differences were observed only for eight-cell embryos in BOEC cultured alone or with embryos or CM compared with eight-cell embryos cultured alone (P < 0.005) (Fig. 5B). Regarding embryo metabolism, two-cell embryos cultured without BOEC did not reveal a consumption of amino acids substrates when they were compared to the blank control (data of blank media were not included in the graphs).
Discussion
We have previously reported that oviduct transcriptome changes produced by the presence of an embryo may be very local in nature (Maillo et al. 2015). The present study aimed to validate these data using an in vitro model which offered the double benefit of immobilizing the embryo on the surface of the BOEC monolayer during co-culture, enabling the analysis of the local interaction between BOEC and embryos. In this model, we chose to use 50 embryos as we wanted to amplify any putative signal, also this number corresponds to the number of embryos transferred in the study of Maillo et al. (2015).
The response of BOEC to two- and eight-cell embryos or their CM was evaluated at gene expression and metabolic levels. The overall transcriptional profile of a panel of candidate genes between BOEC co-cultured with two-cell embryos or with their CM was different from that observed after co-culture with eight-cell embryos or their CM. In cattle, the major activation of transcription occurs during the 8- to 16-cell stage (reviewed by Graf et al. 2014). These events perhaps are accompanied by specific embryo signaling. Evidence is emerging of a dynamic mutual paracrine communication between both the embryonic and the maternal environments, through extracellular vesicles (EVs), at the early stages of preimplantation embryo development (Saadeldin et al. 2015, Almiñana et al. 2017). For example, the secretome of in vitro-cultured human embryos contains EVs that are taken up by the maternal side (Giacomini et al. 2017). In support of the existence of stage-specific effects of the embryo on the reproductive tract, data from Passaro et al. (2018) have shown that bovine endometrial explants respond to the presence of 8-day-old blastocysts and not to earlier stages (oocytes, two-cell, Day 5 morula).
Serum has been associated with reduced blastocyst quality, although it is still used for embryo culture (e.g., 5–10%) (reviewed by Rizos et al. 2017). We have reported that 10% FCS accelerates blastocyst development, with more blastocysts appearing at Day 6 after IVF than when it is absent and that this accelerated development was associated with poorer survival following cryopreservation (Rizos et al. 2003). However, others working in commercial IVF/ET in Brazil routinely use low concentrations (e.g., 2.5%) of FCS and report no difference in survival following cryopreservation or conception rates after transfer (Sanches et al. 2016). Also, BOEC cultures typically require the presence of serum for normal development, cell attachment, growth and proliferation (Brunner et al. 2010). Thus, FCS was supplemented to all experimental groups.
Results indicate that BOEC mRNA abundance was affected directly by embryos or by their secretions, depending on the transcript analyzed, which indicates the complexity of the mechanisms implicated in embryo–maternal communication. The oviductal mRNA abundance of SMAD6 decreased in the presence of both two- and eight-cell embryos, consistent with our previous data indicating the relevance of SMAD6 as a component of the BMP signaling pathway (García et al. 2017).
SOCS3, PRELP and SCN9A expression was affected in BOEC cultured in direct contact with eight-cell embryos and their CM. The fact that eight-cell embryos decreased the expression of SOCS3 and PRELP, implicated in immune defense, supports the idea that embryo may avoid the maternal immune response by decreasing inflammation (Maillo et al. 2015). This agrees with data obtained from the comparison of ipsilateral oviducts from pregnant and cyclic mares, showing that the embryo modifies the expression of immune response-related genes with marked upregulation of interferon-associated genes (Smits et al. 2016). This is also consistent with recent data indicating that BOEC stimulates embryos to produce IFNT, which then acts on immune cells to promote an anti-inflammatory response in the oviduct (Talukder et al. 2018). Expression of SCN9A was upregulated in BOEC co-cultured with eight-cell embryos or their CM. This is consistent with the study of Schmaltz-Panneau et al. (2014) who hypothesized that there is neurotrophin signaling by the embryo at the time of blastocyst formation (Schmaltz-Panneau et al. 2014). Furthermore, SCN9A mediates the voltage-dependent sodium ion permeability of excitable cell membrane. (Chan et al. 2012). Taking this information into account, our results indicate that during its passage through the oviduct, the early embryo might affect the epithelial ion channels modulating the luminal fluid volume and composition according to its needs.
The IGFBP3 transcript was also increased in BOEC cultured with CM of two-cell embryos, a finding consistent with previous reports of an increase of IGFBP3 in BOEC co-cultured for 8 days with 20 early embryos (Schmaltz-Panneau et al. 2014). IGFBP3 belongs to a family of six binding proteins that bind insulin-like growth factor 1 and 2 (IGF-1 and -2). It was demonstrated that the bovine oviduct expresses IGFBP-3 in a region-specific manner in the isthmus epithelium. This pattern of distribution might help to create an IGF-1 gradient along the oviduct as the embryo travels through it (Pushpakumara et al. 2002).
Recently, changes in the oviduct microenvironment in its metabolite levels related to the stage of the cycle and the proximity of ovulation have been reported (Lamy et al. 2018). However, whether the embryo can elicit similar responses is largely unknown. Consequently, we sought to evaluate the effect of the early embryo on the metabolism of energy substrates and amino acids of BOEC cultured in vitro. Results indicate that BOEC metabolism was not affected by the presence of early embryos (two-cell or eight-cell stage) or by their CM. Furthermore, results indicate that BOEC metabolism is different to that of embryos, suggesting that BOEC and embryos have metabolic requirements that may be independently satisfied in vitro by the culture media. BOEC consumed glucose, lactate and pyruvate as well as amino acids, except for serine and alanine, which were produced by cells. It has previously been proposed that BOEC altered the composition of culture medium in a manner consistent with embryo development; indeed, BOEC-depleted glucose concentrations while there was an accumulation of lactate and pyruvate, two key metabolites required for successful development of the early embryo indicating that BOEC might be attempting to create an environment favorable for embryo development (Edwards et al. 1997). However, it is important to note that in their experiment, Edwards et al. (1997) performed the co-culture in SOFaa media supplemented with high levels of carbohydrates, mimicking those present in TCM-199. In contrast, our study used SOFaa that was free of carbohydrates, following the composition previously described (Holm et al. 1999) which may explain the discrepancies in results.
Interestingly, embryo metabolism before EGA seems to be independent of the environment. The results indicated no consumption of energy substrate or amino acids by two- to eight-cell embryos. However, this was not the case for embryos at the 8- to 16-cell stage which displayed an active metabolism. The lack of metabolism by two- to eight-cell embryos may suggest that the embryo has sufficient endogenous energy sources, most likely fat but possibly protein, to support its early development (Sturmey et al. 2009, Leese 2015).
The amino acid analysis revealed the production of serine and alanine by BOEC independent of the presence of embryos. The release of alanine amino acid in the extracellular medium is witness of ammonium detoxification mediated by pyruvate metabolism (reviewed by de Souza et al. 2015). Furthermore, alanine is involved in the regulation of intracellular pH and may have a role in protecting the embryo from osmotic stress (Van Winkle 2001), and it has shown to promote bovine preimplantation development in vitro (Moore & Bondioli 1993). Thus, BOEC could be predisposed to increase alanine concentration in the media to sustain embryo needs.
In conclusion, using an in vitro approach allowing a local and temporal interaction between the embryo and oviduct epithelial cells, this study confirms that the early embryo affects the gene expression of BOEC. The effect was embryo stage specific and resulted from a direct contact with BOEC or from embryo secretions released into the media. Under our experimental conditions the early embryo did not affect BOEC metabolism indicating that during its short passage through the oviduct, it might elicit changes at signaling level necessary for its development without inducing metabolic changes.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2015-70140-R to D Rizos and AGL2015-66145-R to A Gutierrez-Adan). M Hamdi was supported by a fellowship from the Spanish Ministry of Economy and Competitiveness (BES-2013-066767). The authors are also funded by the European Union H2020 Marie Sklodowska-Curie (MSCA) Innovative Training Network (ITN) project ‘Biology and Technology of Reproductive Health – REP-BIOTECH-675526’. The authors are members of the COST Action 16119. In vitro 3D total cell guidance and fitness (Cellfit).
Acknowledgments
The authors would like to thank Almansa Ordóñez A and Barroso Sáenz A for their help. Special thanks are extended to the abattoirs (Transformación Ganadera De Leganés SA; Matadero Madrid Norte, San Agustín de Guadalix; and Cárnica Colmenar SC, in Madrid, Spain) for providing access to the biological material (ovaries) and to the Spanish Association of Breeders of Selected Cattle of the Asturian Valley Breed (ASEAVA) for providing the semen.
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