Effects of co-culture with multipotent endometrial stromal cells during in vitro production of bovine embryosABSTRACT
In vitro embryo production (IVP) protocols generally use supplementation with fetal bovine serum (FBS) in its culture media. Although it contains essential nutrients and growth factors for the embryo, exposure to serum can affect embryonic morphology and biochemistry, in addition to being possibly related to increased fetal weight at birth. In this sense, multipotent stromal cells (MSCs), given the wide range of secreted factors, could help improve IVP protocols. This study verified whether cell proliferation factors secreted by multipotent stromal cells obtained from endometrial tissue (eMSCs) stimulate the development and production of better quality embryos. For that purpose, bovine embryos produced in vitro were co-cultured with eMSCs and evaluated for cleavage index and blastocyst formation, as well as total cell count, trophoblast cell count and apoptotic cell count. It is concluded that the production of bovine embryos associated with co-culture with eMSCs presented similar results to the use of traditional media, showing an improvement regarding the total number of cells, in which the embryos cultivated in the presence of eMSCs and absence of FBS presented a higher number of total cells than those cultured in the absence of eMSCs and FBS.
Keywords:
embryos; bovines; IVP; co-culture; stem cells
RESUMO
Os protocolos de produção in vitro de embriões (PIVE), em geral, utilizam a suplementação com soro fetal bovino (SFB) em seus meios de cultivo. Embora contenha nutrientes essenciais e fatores de crescimento para o embrião, a exposição ao soro pode afetar a morfologia e a bioquímica embrionárias, além de estar possivelmente relacionada com o aumento de peso do feto ao nascimento. Nesse sentido, células estromais multipotentes (CTMs), diante da grande gama de fatores secretados, poderiam auxiliar no melhoramento dos protocolos de PIVE. Este estudo verificou se os fatores de proliferação celular secretados por células estromais multipotentes obtidas de tecido endometrial (eCTMs) estimulam o desenvolvimento e a produção de embriões de melhor qualidade. Para tanto, embriões bovinos produzidos in vitro foram cocultivados com eCTMs e avaliados quanto ao índice de clivagem e à formação de blastocistos, bem como à contagem de células totais, células do trofoblasto e células apoptóticas. Conclui-se que a produção de embriões bovinos associada ao cocultivo com eCTMs apresentou resultados similares ao uso de meios tradicionais, revelando melhora em relação ao número total de células, uma vez que os embriões cultivados na presença das eCTMs e na ausência de SFB apresentaram um maior número de células totais em comparação àqueles cultivados na ausência de eCTMs e SFB.
Palavras-chave:
embriões; bovinos; PIVE; cocultivo; células-tronco
INTRODUCTION
Among the events experienced by the embryo during the preimplantation there are: the first cleavages, taken as indicators of subsequent potential development (Alberio, 2019); activation of transcription of the embryonic genome, which occurs in two phases, called major and minor, with the smallest occurring in the 2-cell stage and the largest in the 4- to 8-cell stage, in which there is a change in maternal genomic control, given by proteins and mRNAs stored in the oocyte, now regulated by proteins synthesized by the embryo (Graf et al., 2014; Jiang et al., 2014); compaction of the morula, establishing cell-cell contact between the blastomeres and, finally, the formation of the blastocyst, involving the formation of the embryo's two main cell types, the trophectoderm and the inner cell mass (Alberio, 2019). The succession of events experienced by the embryo during this period holds crucial importance, as they can affect the implantation process and consequently gestational development (Alberio, 2019).
MSCs are multipotent stromal cells that have the capacity for self-renewal and are responsible for tissue maintenance (Gargett et al., 2016). This group of cells includes MSCs from endometrial tissue (eMSCs). In humans, there is evidence that eMSCs may be responsible for regeneration of the endometrium during the reproductive phases (reviewed by Gargett et al., 2009) and a population of progenitor/stem cells present in the basal portion of the endometrium in humans can originate a functional endometrial tissue, with the presence of glands, vessels and stroma (Gargett and Masuda, 2010). In cattle, endometrial tissue has also been studied as a source of MSCs since it is highly regenerative and contains undifferentiated cells, with high plasticity, capacity for growth and differentiation during the estrous cycle and pregnancy (Łupicka et al., 2015; Donofrio et al., 2008). The therapeutic application of eMSCs has already been described in humans in models of cardiac (Bockeria et al., 2013) and neurological (Wollf et al., 2011) diseases, and are proposed as an alternative in the endometrial regeneration treatment (Meng et al., 2007).
Assisted reproductive technologies, such as in vitro fertilization (IVF), embryo transfer and cloning, do not yet have an ideal commercial impact due to the high cost and low efficiency in producing high-quality embryos (Kupka et al., 2014; Lonergan and Fair, 2008). The use of conventional systems for in vitro production of bovine embryos results in approximately 40% blastocyst production, which is considered low in comparison to in vivo fertility rates observed for the species (Lonergan and Fair, 2008). Therefore, new approaches based on the use of co-cultivation systems of oocytes and/or embryos with stem cells of adult or embryonic origin have been carried out to increase embryo quality and production (Miranda et al., 2016).
Laboratories generally use supplementation with fetal bovine serum (FBS) in its culture media. However, the role of serum during embryonic development is still undefined. Although it contains essential nutrients and growth factors (Chelladurai et al., 2021) and protects embryos against the deleterious effects reactive oxygen species (ROS), its composition is highly undefined and often varies from one batch to another (Moore et al., 2007). Furthermore, serum is known to have a biphasic effect when added to the culture medium: the presence of serum can initially prevent the first cleavages from appearing prematurely, in addition to, in a second moment, promoting and accelerating the development of the embryo to the morula and blastocyst stage (Rizos et al., 2003). Other studies show that prolonged exposure of embryos to serum can greatly affect embryonic morphology and biochemistry (Reis et al., 2003) and is possibly related to increased fetal weight at birth (Farin et al., 2001). Moreover, Rizos et al. (2003) demonstrates that omitting serum can increase cryotolerance, in addition to altering transcript profiles related to development.
Besides the limitations imposed using FBS, it is recognized among in vitro embryo production biotechniques that bovine embryonic development is arrested between 8 and 16 cell stage (Gonçalves et al., 2008), a fact that is not verified in in vivo development. It is important to highlight that this retention is concomitant with the change from maternal to embryonic genomic control (Barnes and Eyestone, 1990), which allows to infer that the culture conditions and media traditionally used, such as serum, for example, are scarce in molecules related to the transcriptional activation of the embryo. In an attempt to increase the production rate and quality of embryos, in addition to suppressing the retention of embryonic development, an alternative to in vitro embryo production is the co-culture of embryos with somatic cells (Orsi & Reischl, 2007).
The use of the co-cultivation systems is also established through the fact that somatic cells have the ability to secrete several important substances, such as cytokines, growth factors, hormones and even microRNAs (included in exosomal microvesicles), thus providing paracrine factors important for embryonic development; Furthermore, they can contribute to the removal of toxic components and ROS (Tan and Tan, 2003). Therefore, somatic cells allow the possibility of modulating the microenvironment around them (Kapur and Katz, 2013). Studies demonstrate beneficial effects of co-cultivating mesenchymal stem cells with embryos on oocyte maturation and embryonic development (Park et al., 2013). Additionally, the use of conditioned media, produced by these cell types, in which these substances are present, also highlights the beneficial development of embryos (Cintra et al., 2022).
Miranda et al. (2016) demonstrated the beneficial effect of these cells during the cultivation of bovine embryos, observing higher embryo production rates than with the use of pre-conditioned medium from the same stem cells and even than co-culture with granulosa cells, which are traditionally used in co-culture systems. The use of mesenchymal cells derived from adipose tissue in co-culture improved the quality of embryos, as it increased the total number of cells, and the levels of transcripts related to pluripotency and energetic metabolism.
MATHERIALS AND METHODS
Ethical aspects. This study was conducted in accordance with the rules of the National Council of Animal Experimentation Control (CONCEA) through protocol CEUA 0193/2017 of the host institution.
Reagents: All chemicals and reagents were obtained from Sigma-Aldrich® (St. Louis, MO, USA), unless otherwise cited.
Preparation, cultivation and storage of eMSCs. This study used endometrium-derived multipotent stromal cells (eMSCs) in 3rd passage, previously isolated, characterized (Moraes et al., 2017) and maintained in a cryobiological bank in the laboratory, at -196°C. Samples were thawed and reconstituted using parts of the protocol described by Chaytor et al. (2012). Thawed cells were subjected to centrifugation with cell maintenance medium (DMEM) for 10 minutes at FCR 940G, aiming to improve viability. After 2 cycles, the supernatant was discarded and subsequent counting in the Neubauer chamber with Trypan blue dye was performed, using cell viability = number of living cells/number of dead cells to check cell viability.
After counting, the cells were homogenized and then plated in a 168 cm2 KASVI® culture bottle, containing 18 ml of culture medium, composed of DMEM High Glucose + F12 + 20% FBS (Gibco®, New York, NY, USA) + penicillin/streptomycin (10 μl/ ml) + amphotericin (12 μl/mL) and amikacin (0.07 μl/ml) (Gibco®, New York, NY, USA), at 37.5ºC in a humid atmosphere containing 95% air and 5% 6 CO2. The medium was changed every 48 hours until a minimum cell confluence of 90%.
For use in embryo production routines, eMSCs were placed in cryovials composed of 90% filtered FBS + 10% DMSO. The cryotubes containing 10x104 cells were stored in a Mr. Frosty™ cryogenic container and placed at -80°C for 4 days, and then transferred to the laboratory's liquid nitrogen cylinders, at -196°C.
Follicular aspiration and in vitro maturation. The ovaries were obtained from nearby slaughterhouses and transported to the laboratory in a 0.9% NaCl solution at an approximate temperature of 35°C to 37°C. Follicles between 2 and 8 mm in diameter were aspirated with a 10 mL syringe and 40x12 mm needle to obtain oocytes. After follicular aspiration, grade 1 and 2 oocytes were selected in complete HBSS saline solution (Gibco®, New York, NY, USA) plus 0.5% FBS under a stereomicroscope for in vitro maturation (IVM). The criteria for selecting oocytes is the same as that used by Caixeta and Dode (2010). After selection, oocytes were washed in drops of BotuFIV MIV oocyte maturation medium (Botupharma®, Botucatu, SP, Brazil). IVM was performed in Petri dishes, in 90µl drops, containing oocyte maturation medium plus 10% FBS, covered with sterile mineral oil. The plates were placed in an incubator at 38.5°C with a 5% CO2 atmosphere for a period of 22 to 24 hours.
In vitro fertilization. After maturation, the oocytes were subjected to in vitro fertilization (IVF). The matured oocytes were washed in BotuFIV FIV fertilization medium (Botupharma®, Botucatu, SP, Brazil), and were subsequently placed in petri dishes containing 90 µl drops of the same medium (containing up to 20 oocytes per drop). Semen straws were thawed in NaCl 0.9% solution heated to 40ºC for 20 seconds, proceeding with the preparation of the semen by centrifugation using a sperm separation gradient, which was done by adding it to a tube containing 250µl of BotuFIV Sperm (Botupharma®, Botucatu, SP, Brazil) and 250 µl of BotuFIV Cap (Botupharma®, Botucatu, SP, Brazil), homogenizing them and proceeding with the addition of 500µl of BotuFIV Sperm to the bottom of the tube. Centrifugation was performed for 5 minutes at 5000 RPM. The volume of the pellet obtained was measured with a pipette, and the appropriate assessments were made in the Neubauer chamber regarding motility (from a sample of 2.5µl of the pellet in 125µl of heated fertilization medium). and sperm concentration (from a sample of 2.5 µl of the pellet in 125 µl of water).
The following calculation was made to determine the amount of fertilization medium to be added to dilute the semen: Fertilization medium to be added to the sperm pellet = Pellet volume x Sperm motility x Sperm concentration x 0.001. The amount of fertilization medium obtained from the account was added to the pellet, resulting in a concentration of 1 million sptz/mL. The oocytes, already in fertilization medium, were fertilized with a volume of 7 µl of semen, and then returned to the incubator for a period of 12 to 18 hours.
In vitro cultivation. After 12-18 hours of IVF, the possible zygotes were denuded with the aid of a pipette and cultured according to the experimental groups: 1 - Negative control - BotuFIV CIV embryonic culture medium (Botupharma®, Botucatu, SP, Brazil) in the absence of cell co-cultivation. 2 - Positive control - BotuFIV CIV medium plus 2.5% FBS in the absence of cell co-culture. 3 - eMSCs - BotuFIV CIV medium in the presence of cell co-culture and 4 - eMSCs+FBS - BotuFIV CIV medium plus 2.5% FBS in the presence of cell co-culture. The zygotes were plated in drops of 150µl of BotuFIV CIV. In the case of the last two groups, in which eMSCs co-culture was present, an amount of approximately 5 x 103 cells was added subsequently in the drops containing the embryos, resulting in a concentration of approximately 30 x 103 cells/mL, considering 150µl drops. The cells were prepared by thawing the cryovials, followed by centrifugation at 940G for 10 minutes in 1mL of BotuFIV CIV medium and subsequent counting. The cells reached confluence within 24 hours after the start of embryo cultivation. All embryos were cultivated in petri dishes covered with mineral oil, and embryos were fed every 48 hours, in all groups, discarding half of the medium and adding half of new, already stabilized medium.
After 48 hours, in D3, cleavage was evaluated, with structures that were not cleaved being discarded. Blastocyst formation was assessed on D8. Blastocysts were also classified according to their stage of development, evaluating the quality and viability of embryonic production in each experimental group. The embryos were classified into the Morula (MO), Initial Blastocyst (BI), Blastocyst (BL), Expanded Blastocyst (BX), Hatching Blastocyst (BN) and Hatched Blastocyst (BE) stages, as recommended by the International Embryo Technology Society (Stringfellow and Seidel, 1998). Therefore, the production of blastocysts at the expanded stage (BX) or higher in each group was also verified in relation to the total production. At the end of the experiment, a total of 10 IVPE repetitions were performed.
Determination of the total number of total cells (T) and trophoblast cells (TF). The morphological quality of blastocysts was assessed by the proportion of the number of TF trophoblast cells in relation to the total number (T) of cells. To this end, embryos with the zona pellucida intact were initially incubated for 10 seconds in 50 µl of BSA-free buffer solution containing 1% Triton X-100 and 100 µg/ml of Propidium iodide. The blastocysts were then immediately transferred to 400µL of fixation solution with 100% ethanol and 25µg/mL bisbenzimide (Hoechst-33258) and stored at 4ºC for at least 30 minutes (Thouas et al., 2001). The fixed and stained embryos were transferred directly from the fixation solution to a drop of glycerol, avoiding excessive fixative medium. The blastocysts were mounted in glycerol in a slide and coverslip and evaluated under ultraviolet light in an inverted epifluorescence microscope (Olympus IX71) (excitation, 340-380 nm; emission, 440-480 nm), in which total cells appeared in blue and trophoblast cells in red.
TUNEL Assay. The TUNEL technique (terminal deoxynucleotidyl transferase-mediated XdUTP nick end labeling, Roche Diagnostics, Indianapolis, IN, USA) was used to detect DNA fragmentation in the final stages of apoptosis (programmed cell death). To this end, the embryos were washed in 3 drops of washing solution, containing 100 µl of HBSS saline solution plus BSA (1mg/ml) and then incubated for 25 seconds in a permeabilizing solution, containing PBS plus BSA (2mg/mL) and Triton X-100 (2 mg/mL). Afterwards, the embryos were washed again in LAV solution and incubated for 5 minutes in a new permeabilizing solution, this containing PBS + Triton X-100 (1mg/mL) + Sodium Citrate (1 mg/mL). Subsequently, the embryos were washed again in washing solution and incubated for 1 hour in Eppendorf tubes, in 10 µl of Mix TUNEL solution (In Situ Cell Death Detection Kit, Roche ®), containing 9 parts of marker solution (pink) and 1 part of enzyme solution (blue), in a humid atmosphere at 37.5°C. After this incubation period, the embryos were washed in LAV solution and placed on slides, in 10 µl drops of glycerol 11 covered with a coverslip. The visualization of green fluorescent nuclei (TUNEL assay; 450-490nm filter and 515nm emission filter) indicated cells undergoing apoptosis, with DNA fragmentation.
Statistic analysis. The sequence of analysis was followed by performing the data normality test and homogeneity of variances between groups (ANOVA), and the probability test of individual differences using the Statistical Analysis System software (SAS Institute™).
RESULTS AND DISCUSSION
According to Miranda et al. (2016), embryonic production using co-culture with a concentration of 10x103 MSCs/mL derived from bovine adipose tissue increased blastocyst production rates when compared to those using conventional media. In the present study, no significance was observed between the cleavage or blastocyst rates between the treatments (p>0.05), as shown in Table 1. It should be noted that the origin of the MSCs used by Miranda et al. (2016) is from adipose tissue and in the present study, the cells are from endometrial tissue. Although MSCs from different tissues may have similar therapeutic properties, the secretome of MSCs from different tissues are generally different (Schauwer et al., 2014). Another difference concerns the number of cells plated in the culture drops, which was higher in the present protocol (30x103 cells/mL). Despite a higher concentration of eMSCs, the medium was changed every 48 hours, replacing que drop with 50% new medium, in order to avoid competition for nutrients as well as the accumulation of harmful metabolites such as ammonia that could be produced due to the high concentration of cells. This, in addition to blastocyst production rates above 40% on average, suggests that a greater number of mesenchymal cells did not cause deleterious effects on the embryos.
The MSCs used in the study by Miranda et al. were plated in embryonic culture medium 24 hours before the addition of zygotes, with these being added after this period. In the present study, eMSCs were plated at the time of transferring the embryos from the fertilization drop to culture. Tests were carried out in which cell plating occurred between 48 and 72 hours before the addition of zygotes. Cells were plated in 150μl drops of DMEM/F12 cell culture medium plus 10% FBS. At the time of embryonic culture, the drops were washed, removing cell maintanance medium and adding embryonic culture medium.
However, the results in embryo production following this methodology were not favorable. Despite having high cleavage rate (>90%), the production of blastocysts was close to zero. This suggests a blocking situation caused by a possible interaction of MSCs with pre-cavitation embryos, preventing or substantially altering the activation of the embryonic genome. In fact, it has already been verified that an excess of nutrients in the culture medium, such as that produced by a layer of confluent MSCs, is harmful to the embryonic development of bovine embryos (Kim et al., 2011). After the methodology was revised and the cells began to be prepared and added at the same time as the zygotes, favorable results were obtained in the production of blastocysts. In this case, the cells began adhering to the plate and reached confluence within 24 hours of cultivation while the zygotes began the first cleavages.
More studies are needed to understand this mechanism, however it is known that embryos in early stages of cleavage have different metabolism and nutritional needs than embryos after the maternal-fetal transition (Lane et al., 2003; Meireles et al., 2004). These biological differences are apparently responsible for greater embryonic production in a medium containing a lower concentration of factors secreted by MSCs.
Total number of embryonic cells, trophoblast cells and cells undergoing apoptosis. A total of 30 embryos were stained using the Hoechst 33258 and Propidium iodide staining protocol, and another 23 embryos were subjected to the TUNEL protocol, to reveal cells in the final stage of apoptosis. The results can be seen in Fig. 1.
Blastocysts in D8 stained with Hoechst 33342 to determine the total number of cells, Propidium iodide showing trophoblast cells and TUNEL, performed to identify cells in the final stage of apoptosis. Negative Control (a-c), Positive Control (d-f), eMSCs (g-i) and eMSCs + FBS (j-l). 50μm scale.
As shown in Table 2, the embryos with the lowest number of cells were those from the Negative Control group, and those with the highest number of cells were from the eMSCs group, without the addition of FBS. There was significance between these two treatments (p=0.0438), showing a beneficial effect of co-cultivation with mesenchymal stem cells in an experimental group in which FBS was not used, agreeing with the conclusions of Miranda et al. (2016). Furthermore, it is known that embryos with a higher number of cells have a greater capacity for development and to generate full-term fetuses (Van Soom et al., 2007).
Total cell count, with significance (p=0.0438) between the Negative Control and eMSCs groups
In an experiment previously carried out by the same research group (Cintra et al., 2021), it was observed that the addition of 20% eMSCs-conditioned medium in the absence of FBS also led to the production of embryos with a higher total number of cells and number of cells in the inner cell mass compared to embryos produced in the absence of FBS and with the addition of 2.5% FBS. However, no differences were observed. Furthermore, the production of blastocysts in the group in which FBS was used was statistically higher than that observed in the absence of FBS and with the addition of 20% conditioned medium. It is possible to observe that the presence of eMSCs and the constant secretion of the factors produced by it were efficient in equaling the embryonic production to that obtained with the addition of FBS.
The values obtained from staining with propidium iodide did not show a significant difference when compared to each other, as shown in table 3. This effect was also observed in the study by Cintra et al. (2021), but was contrary to Miranda et al. (2016) who observed embryos with a greater number of trophoblast cells in the presence of MSCs derived from adipose tissue.
Evaluating the embryo solely based on the number of cells is not enough. Another way to assess embryonic quality is the apoptosis index. Byrne et al. (1999) demonstrated that a high rate of apoptosis is associated with reduced embryonic viability. The results of the TUNEL assay can be seen in table 4, and there was no significance when comparing the groups to each other, which shows that in this study MSCs did not influence embryonic apoptosis rates.
CONCLUSION
In vitro production of embryos associated with co-culture with eMSCs presented similar results to the use of traditional media, associated or not with FBS, in terms of cleavage and blastocyst rates, number of trophoblast cells and stage end of apoptosis. This finding indicates that the presence of eMSCs added components, such as bioactive molecules and growth factors, to the culture medium, similar and/or equivalent to those added by the presence of FBS. These components seem to have had a positive influence on the growth of embryos, indicating the possibility of removing FBS from cultivation systems, thus reducing its deleterious effects. In this experiment, an evaluation of the gene expression of the embryos was not carried out as a way of estimating embryonic quality. Carrying out these additional experiments is of crucial importance for determining the quality of the embryos produced and their development into full-term fetuses.
ACKNOWLEDGEMENTS
This study was funded by the State of São Paulo Research Foundation (FAPESP), process 2018/10340-7.
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