Introduction
The fibroblast growth factor (FGF) family is composed of 22 proteins, which are proteins that span the plasma membrane of a cell and contain an extracellular domain that binds to its ligands, and an intracellular domain important for signalling relay. FGFs have been associated with several biological activities including angiogenesis, embryonic development, endocrine signalling pathway, cell proliferation and differentiation (Böttcher and Niehrs, Reference Böttcher and Niehrs2005; Ocón-Grove et al., Reference Ocón-Grove, Cooke, Alvarez, Johnson, Ott and Ealy2008).
As a member of the FGF family, FGF18 has been detected in theca and granulosa cells (Jiang et al., Reference Jiang, Guerrero-Netro, Juengel and Price2013). FGF18 is secreted by theca cells and acts on granulosa cells during atresia. Intrafollicular injection experiments in cows revealed that exogenous FGF18 supplementation caused the interruption of follicular growth and steroidogenesis (Portela et al., Reference Portela, Machado, Buratini, Zamberlam, Amorim, Goncalves and Price2010; da Silva et al., Reference da Silva, Yang, Caixeta, Castilho, Amorim, Price, Fortune and Buratini2019).
The granulosa cells have a close connection with the oocyte and the cumulus cells. In this regard, whereas mouse oocytes express FGF18 (da Silva et al., Reference da Silva, Yang, Caixeta, Castilho, Amorim, Price, Fortune and Buratini2019), the growth factor has not been detected in bovine oocytes (Portela et al., Reference Portela, Machado, Buratini, Zamberlam, Amorim, Goncalves and Price2010). The oocyte interacts intimately with cumulus cells forming the cumulus–oocyte complex (COC), which is essential for the formation and development of the female gamete. Such an intimate interaction helps in the nuclear and cytoplasmic maturation of the oocyte, leading to oocyte competence in supporting early embryonic development (Sánchez and Smitz, Reference Sánchez and Smitz2012). Preliminary studies by this group showed that the oviduct secretes FGF18 during embryonic development, therefore indicating that this protein may have an important role in cell proliferation during the initial embryonic development in cattle.
The Hippo pathway has a crucial role in the regulation of embryonic development as, in its inactive state, it leads to delayed embryonic development or embryonic death (Lorthongpanich and Issaragrisil, Reference Lorthongpanich and Issaragrisil2015). When the Hippo pathway is inactive, transcription activators YAP and TAZ are maintained unphosphorylated, being allowed to translocate into the nucleus and interact mainly with transcription factor TEA domain (TEAD1/2/3/4) family members. In the nucleus, the complex YAP/TAZ–TEAD acts as transcription factors for target genes such as CTGF and CYR61 (Serrano et al., Reference Serrano, McDonald, Lock, Muller and Dedhar2013), which are known regulators of cell proliferation and tissue growth.
Oocyte maturation can be understood as the set of biological modifications by which the oocyte acquires competence to support fertilization and the beginning of embryonic development. Several molecules and pathways that regulate cell proliferation are closely related to the regulation of oocyte maturation and early embryonic development. This paper aims to determine whether the set of target molecules regulated by FGF18 include the Hippo-related regulators of cell proliferation, CTGF and CYR61, during oocyte maturation and early embryonic development.
Material and methods
Cow ovaries were obtained from a local abattoir and transported to the laboratory in saline solution (0.9% NaCl; 30°C) containing 100 UI/ml of penicillin and 50 µg/ml of streptomycin sulfate. COCs from 3 to 8 mm diameter follicles were aspirated with a vacuum pump (vacuum rate of 20 ml of water/min). COCs were recovered and selected under a stereomicroscope. Grades 1 and 2 COCs were randomly distributed into 500 μl of maturation medium in four-well plates (Nunc, Roskilde, Denmark) and cultured in incubators at 38.5°C (101.3°F) in a 5% CO2 and 95% air saturation humidity atmosphere for 22–24 h. The maturation medium consisted of TCM199, containing Earle’s salts and l-glutamine (Gibco Laboratories, Grand Island, NY, USA), supplemented with 25 mM HEPES, 0.2 mM pyruvic acid, 2.2 mg/ml sodium bicarbonate, 5.0 µg/ml luteinizing hormone (LH) (Lutropin-V®), 0.5 µg/ml follicle-stimulating hormone (FSH) (Follitropin-V®), and 10% fetal bovine serum (FBS; Gibco Laboratories, Grand Island, NY, USA), 100 UI/ml penicillin and 50 µg/ml streptomycin sulfate.
In vitro fertilization (IVF)
After in vitro maturation (IVM), bovine oocytes were fertilized in vitro with tested semen after thawing and separation through a discontinuous Percoll (GE Healthcare, São Paulo, SP, Brazil) gradient. Sperm cells were diluted and added to the COC plate with the final concentration adjusted to 2 × 106 sperm/ml in Fert-TALP medium containing 10 µg/ml heparin, 30 µg/ml penicillamine, 15 mM hypotaurine and 1 mM epinephrine. Fertilization was carried out by co-culture of sperm and oocytes for 18–20 h in four-well plates under the same atmospheric conditions used for maturation. The day of IVF was considered as day 0 of embryo development.
In vitro embryo culture (IVC)
After IVF, presumptive bovine zygotes were denuded by vortexing, and then cultured in a culture chamber at 38.5°C and a 5% CO2, 5% O2 and 90% N2 saturated humidity atmosphere in 500 µl of synthetic oviductal fluid (SOF) medium in four-well plates (Nunc, Roskilde, Denmark). Cleavage rates were evaluated 48 h after fertilization and blastocyst rates were assessed on day 7 of embryo development. Blastocysts assessed on day 7 were rinsed three times in phosphate-buffered saline (PBS) and collected in TRIzol® reagent at 80°C for subsequent RNA extraction.
RNA extraction, reverse transcription and real time PCR
Total RNA was extracted from 25 COCs and five day-7 blastocysts in accordance with TRIzol instructions. Briefly, the extraction used 1000 µl TRIzol reagent (Thermo Fischer, Waltham, MA, USA) and 200 µl chloroform, followed by purification of the aqueous phase with GlycoBlue (Thermo Fisher, Walthan, MA, USA) and 700 µl isopropyl alcohol. Quantification and assessment of RNA purity was performed using a NanoDrop spectrophotometer (Thermo Fisher, Walthan, MA, USA; 260/280 nm absorbance ratio). Complementary DNA was synthesized from 200 ng of RNA, which was treated with 0.1 U DNase Amplification Grade (Thermo Fisher, Walthan, MA, USA) for 15 min at 27°C to neutralize any DNA molecules. DNase was inactivated with 1 µl ethylenediaminetetraacetic acid (EDTA) for 10 min at 65°C. Reverse transcription was performed adding 1 U iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) for 5 min at 25°C followed by 30 min at 42°C and 5 min at 85°C. Quantitative polymerase chain reaction (qPCR) was conducted in a thermocycler (Bio-Rad, Hercules, CA, USA) using 2.5 ng of cDNA in 2 µl and 8 µl of Mix containing forward and reverse bovine specific primers (Table 1), nuclease-free water and GoTaq® Master Mix (Promega Corporation, Madison, USA). Amplification was performed with initial denaturation at 95°C for 5 min followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 30 s. To optimize the qPCR assays, serial dilutions of cDNA templates were used to generate a relative standard curve. Samples were run in duplicate and the results of all genes were analyzed as relative fold difference, using RPS18 and GAPDH as reference genes, according to Pfaffl (Reference Pfaffl2001).
Primers used for expression analysis of candidate genes
Experimental design
The study assessing the effect of FGF18 exposure during IVM on COC expression of Hippo-related molecules, CTGF and CYR61, used an FSH-induced cumulus expansion model, and it was divided into two experiments. Experiment 1 assessed the effects of different concentrations of recombinant human FGF18 (0, 10 and 100 ng/ml) in FSH-induced (100 ng/ml) cumulus expansion, at different time points, while each sampling time had three treatment groups. The COCs were distributed randomly in 400 μl of maturation medium in a Nunc four-well plate, where they were matured and were collected at 6, 12 and 24 h of in vitro maturation (IVM). After sampling, the COCs were stored in TRIzol at −80°C for later RNA extraction. The experiment was replicated five times and, in total, 25 COCs per replicate were used for each treatment condition. This experiment was conducted especially to assess the effect of different FGF18 concentrations on Hippo pathway-related target molecules (i.e. CTGF and CYR61) during IVM.
In the second experiment, the IVM treatment groups were 0 and 100 ng/ml of FGF18 in FSH-induced (100 ng/ml) cumulus expansion. In this experiment, the COCs were also randomly distributed into 400 μl of maturation medium and then collected after 0, 3, 6 or 9 h of IVM.
In the third experiment, early embryonic development was evaluated. In this experiment, three groups were placed: control group (untreated), IVM group (treated with 100 ng/ml of FSH and FGF18 only during IVM), and IVC group (100 ng/ml of FSH and FGF18 added to the presumptive embryo only during the embryo culture period). As there was no difference between the concentrations tested in experiment 1, FGF18 concentration was 100 ng/ml. All groups were collected at embryonic day 7 and stored in TRIzol until RNA extraction. The experiment was replicated five times; 150 COCs were used for each treatment group.
Statistical analysis
The data were tested for normal distribution using the Shapiro–Wilk test and normalized when necessary. All data were analyzed through analysis of variance with the treatment as main effect and replicates as a random variable. Differences between the means were tested using the Tukey multiple comparison test and JMP software [Tukey–Kramer honest significant difference (HSD) test] using the JMP software (SAS Institute Inc., Cary, NC, USA). The results are presented as mean ± standard error of the mean. A P-value < 0.05 was considered significant.
Results
Concentration–effect relationship of FGF18 and mRNA expression for CTGF and CYR61 in COCs during 6-h intervals of oocyte maturation (Experiment 1)
The effect of different concentrations of FGF18 (0, 10 and 100 ng/ml) on Hippo-dependent gene expression, in FSH-induced cumulus expansion, was assessed at 6, 12 and 24 h of IVM. CTGF mRNA abundance revealed that FGF18 affected the Hippo pathway during bovine oocyte maturation. There was increased CTGF mRNA expression in COCs induced by 100 ng/ml of FGF18 at 12 h of in vitro maturation (P < 0.05). However, there was no effect of FGF treatment on CYR61 mRNA levels at all the examined time intervals and on CTGF at 6 and 24 h of IVM (Fig. 1).
Relative abundance of target gene mRNA of the Hippo pathway (CTGF and CYR61), during IVM of bovine COCs produced in vitro. COCs were matured in the presence of different concentrations of recombinant human FGF18 (0, 10 or 100 ng/ml) in FSH-induced (100 ng/ml) cumulus expansion for 6 h (a, d), 12 h (b, e) or 24 h (c, f). Cultures were replicated five times with, in total, 25 COCs. *Indicates statistical difference (P < 0.05).
Effect of FGF18 on the expression of Hippo pathway-related genes (CTGF and CYR61) in bovine COCs during 3-h intervals up to 9 h of oocyte maturation (Experiment 2)
In this experiment, COCs were cultured in the presence of 100 ng/ml of FGF18 and the abundance of CTGF and CYR61 mRNA was assessed at 0, 3, 6 and 9 h of oocyte maturation. CTGF mRNA abundance was reduced (P < 0.05) at 3 h compared with the untreated control group. Hippo pathway target gene expression did not change at 0, 6 and 9 h of IVM. The mRNA level for CYR61 was not affected by FGF18 treatment in any of the evaluated times (Fig. 2).
Relative abundance of target gene mRNAs of the Hippo pathway (CTGF and CYR61), during IVM of bovine COCs produced in vitro. COCs were matured in the presence of recombinant human FGF18 (0 or 100 ng/ml) in FSH-induced (100 ng/ml) cumulus expansion for 0 h (Control; a, e), 3 h (b, f), 6 h (c, g) or 9 h (d, h). Cultures were replicated five times with, in total, 25 COCs. a,bIndicates statistical difference (P < 0.05).
Regulation of Hippo pathway-related gene expression (CTGF and CYR61) at day 7 embryo after FGF18 exposure during IVM or IVC of bovine embryos (Experiment 3)
The presence of 100 ng/ml FGF18 during embryo development impaired blastocyst rate (c. 20%) at day 7 post-insemination (unpublished data). CTGF and CYR61 mRNA abundance was assessed in a study of FGF18 exposure, either during oocyte maturation or during embryo culture, to analyze whether FGF18 would affect the Hippo pathway in different moments of in vitro embryo production. Treatment with FGF18, during oocyte maturation or during embryonic culture, did not affect mRNA levels for CYR61 in the blastocyst. However, embryos cultured in the presence of FGF18 had higher levels of CTGF mRNA (P < 0.05) compared with embryos cultured without FGF18, or those embryos derived from oocytes matured in the presence of FGF18 (Fig. 3).
Relative abundance of mRNA of Hippo pathway genes (CTGF and CYR61) in day 7 blastocyst. COCs were matured in the presence of 100 ng/ml of FSH or without [Control and in vitro embryo culture (IVC)] or with FGF18 (100 ng/ml, IVM). In the IVC group, the presumptive embryos were cultured with FGF18 (100 ng/ml). The experiment was replicated five times and, in total, 150 COCs were used for each treatment group. Different letters indicate statistical significance (P < 0.05) among the groups.
Discussion
This exploratory study on FGF18 sheds light on its effect on Hippo pathway-regulated genes during early embryonic development (preimplantation). This study is the first to assess the effect of FGF18 on CTGF and CYR61 mRNA expression during early embryonic development. It was possible to demonstrate that, whereas FGF18 alters CTGF mRNA expression, no effect on CYR61 mRNA abundance was observed. These findings strongly suggested that FGF18 regulates YAP/TAZ. The increase in CTGF mRNA levels occurs mainly when YAP/TAZ were dephosphorylated and transported from the cytoplasm to the nucleus, binding to TEADs for the transcription of target genes (Boopathy and Hong, Reference Boopathy and Hong2019).
In the first experiment, the FGF18 concentration was determined and, in the same experiment, its action at different critical moments (6, 12 and 24 h) of oocyte maturation was evaluated. It is important to note that differential abundance in mRNA must be caused by FGF18 stimulation in cumulus cells, because oocytes removed from the follicle resume meiosis and cease transcription (Pincus and Enzmann, Reference Pincus and Enzmann1935; Hyttel et al., Reference Hyttel, Viuff, Fair, Laurincik, Thomsen, Callesen, Vos, Hendriksen, Dieleman, Schellander, Besenfelder and Greve2001). Supplementation of FGF18 to culture medium in maturation did not change mRNA levels for CYR61 in any experiment. However, the abundance of CTGF mRNA increased at 12 h when COCs were treated with FGF18. CTGF acts on the activation of MAP kinase (MAPK) and Smad-dependent pathways, which are important for oocyte maturation (Ohashi et al., Reference Ohashi, Naito, Sugiura, Iwamori, Goto, Naruoka and Tojo2003; Jiang et al., Reference Jiang, Guerrero-Netro, Juengel and Price2013; Higaki et al., Reference Higaki, Kishi, Koyama, Nagano, Katagiri, Takada and Takahashi2017). These findings are important because, in cattle, MAPK is activated during GVBD, reaches the maximum activity in MI, and remains elevated until the formation of pronuclei, not decreasing in MII (Tian et al., Reference Tian, Lonergan, Jeong, Evans and Yang2002). Therefore, there is evidence that the Hippo pathway, through MAPK, is important for maintaining the oocyte in MII and entering interphase. In this study, CTGF was regulated by FGF18, which may activate MAPK, however we did not investigate those pathways.
After determining the concentration at 6-h intervals, the effect of FGF18 was evaluated from the beginning of oocyte maturation until the end of the GVBD at 3-h intervals (0, 3, 6 and 9 h). Interestingly, FGF18 caused downregulation of CTGF in COCs at 3 h of maturation. At these time points, bovine oocytes undergo GVBD and cumulus granulosa cells go through expansion and mucification. These results agreed with previous studies by Liu and colleagues that observed an increase in CTGF expression at the end of cumulus expansion (Liu et al., Reference Liu, Zhang, Wen, Feng, Zhang, Xiang, Cao, Tong, Ji and Xue2018).
The level of CTGF mRNA was increased when zygotes were cultured in vitro up to the blastocyst stage in the presence of FGF18. This finding is interesting because CTGF plays an important role in embryonic development (Krupska et al., Reference Krupska, Bruford and Chaqour2015). During embryonic development, the abundance of CTGF (but not CYR61) mRNA was higher in day 7 blastocysts compared with the control when the embryos were treated with FGF18. CTGF and CYR61 genes seem to be expressed by the trophectoderm in the blastocyst (Sanz-Ezquerro et al., Reference Sanz-Ezquerro, Münsterberg and Stricker2017). The Hippo pathway may participate in the regulation of cell proliferation and differentiation during embryo development, as observed in other cell types (Yu and Guan, Reference Yu and Guan2013). Although we have not directly investigated this mechanism, there is evidence that FGF18 regulates, through the Hippo pathway, cell proliferation and, probably, differentiation processes during embryonic development.
The preliminary data presented here show that FGF18 modulates CTGF mRNA expression at critical periods of oocyte nuclear maturation, cumulus expansion and early embryonic development in cattle.
Acknowledgements
The authors would like to thank Frigorífico El’Golli for providing the bovine ovaries.
Ethical approval
Not applicable
Funding
This work was supported by grants and fellowships from the National Council for Scientific and Technological Development (CNPq; Brazil), Coordination for the Improvement of Higher Education Personnel (CAPES; Brazil) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS).
Conflict of interest
The authors declare that there is no conflict of interest regarding the publication of this article.
