Abstract Context Although glial cell line-derived neurotrophic factor (GDNF) and microRNAs (miRNAs) have been shown to regulate mammalian oocyte maturation, little is known about their effects on human oocyte maturation and the underlying molecular mechanisms. Objectives To examine the effects of GDNF on both nuclear and cytoplasmic maturation in cultured immature human oocytes and to investigate the involvement of miRNAs in GDNF-induced oocyte maturation. Design A total of 200 human immature oocytes were used to evaluate the effects of GDNF on oocyte maturation. The involvement of miRNAs in GDNF-induced oocyte maturation was identified by comparing the miRNA expression profiles of cumulus cells (CCs) either with or without GDNF stimulation. Setting An in vitro fertilization center at the Women’s Hospital, Zhejiang University School of Medicine. Methods Agilent human miRNA (8*60K) arrays were used to examine the miRNA expression patterns of human CCs either with or without GDNF stimulation. miR-145-5p inhibitor and mimic transfections were performed to study downstream gene expression in human CCs. Results During the in vitro maturation process, GDNF significantly increased the percentage of metaphase II-stage oocytes and downregulated the expression of miR-145-5p in cultured human CCs. Expression of miR-145-5p in CCs is negatively correlated with oocyte maturation. miR-145-5p mimic significantly decreased the expression of GDNF family receptor-α1, ret proto-oncogene, and epidermal growth factor receptor, whereas miR-145-5p inhibitor reversed these effects. GDNF treatment inhibited cell apoptosis in cultured CCs, and this suppressive effect was reversed by transfection with the miR-145-5p mimic. Conclusion Downregulation of miR-145-5p may contribute to GDNF-induced enhancement of oocyte maturation and of cell viability against cell apoptosis. In vitro maturation (IVM) of human oocytes is a valuable technique that involves the retrieval of immature oocytes with no or minimal gonadotropin administration followed by maturation, cryopreservation, or fertilization in the laboratory. For selected infertile couples, IVM provides a convenient, safe, and less costly alternative to conventional in vitro fertilization (IVF) by avoiding gonadotropin stimulation, which may lead to ovarian hyperstimulation syndrome (1). Although still emerging and undergoing refinement, IVM remains the most challenging assisted reproductive technology because of the poor developmental potential of immature oocytes and a relatively lower pregnancy rate compared with conventional IVF procedures (2). Oocyte maturation is a complex process that involves a series of events, including meiotic resumption, cytoplasmic reorganization, cytoskeletal dynamics, and gamete-somatic cell interactions (3). Oocyte nuclear maturation is characterized by germinal vesicle breakdown (GVBD) and extrusion of the first polar body (3). In addition, reprogramming of the oocyte cytoplasm is an essential event for monospermic fertilization and the development of preimplantation embryos (4). Oocyte maturation is mainly controlled by the endocrine and system driven by the hypothalamic-pituitary–ovarian axis. However, increasing evidence indicates that successful oocyte maturation also relies on locally produced intraovarian factors (5). Previous studies have demonstrated that LH can stimulate the follicular cells [theca, granulosa, and cumulus cells (CCs)] to produce several factors that in turn regulate oocyte maturation in a paracrine/autocrine manner. In mouse oocytes, the preovulatory LH surge promotes GVBD by stimulating the production of epidermal growth factor (EGF)-like factors and endothelin-1 (6, 7). In addition, the LH surge enhances the extrusion of the first polar body by stimulating the secretion of brain-derived neurotrophic factor from granulosa and CCs as well as glial cell line-derived neurotrophic factor (GDNF) from theca, granulosa, and CCs (8, 9). GDNF is a known neurotrophin that plays an important role in promoting cell survival, growth, and differentiation in several classes of neurons. GDNF acts through a two-component receptor system, which consists of the ligand-specific binding subunit, GDNF family receptor-α1 (GFRA1), and the common signal transduction subunit ret proto-oncogene (RET) (10). Outside the nervous system, GDNF and its receptors are expressed in other tissues, including the ovaries (11). GDNF has been shown to play a critical role in regulating mammalian oocyte maturation in both murine and porcine oocytes (9, 12). In cultured human immature oocytes, treatment with GDNF increases the total yield of metaphase II (MII) oocytes (13). At present, the molecular mechanisms by which GDNF regulates oocyte maturation are still not fully understood. MicroRNAs (miRNAs) are a large group of small, noncoding RNAs that regulate gene expression at the posttranscriptional level by directing mRNA degradation, translational repression, or both (14). miRNAs are important regulators of diverse cellular functions, including cell division, differentiation, meiosis, and apoptosis (15, 16). In ovarian tissues, miRNAs have been demonstrated to regulate several ovarian functions, including gonadal development, steroidogenesis, ovulation, and corpus luteum formation (17). Dicer, a type III ribonuclease required for the biogenesis of miRNAs, is essential for the completion of meiotic maturation and early embryo development (18). Studies in animals indicate that miRNAs can regulate oocyte maturation through interactions with CCs. In porcine CCs, miR-378 regulates oocyte maturation via the suppression of aromatase expression (19). Similarly, miR-224 influences the maturation of porcine oocytes by targeting pentraxin 3 expression in CCs (20). Intriguingly, oocyte-specific deletion of DGCR8, a key enzyme in miRNA biogenesis, had no substantial adverse effect on oocyte maturation and preimplantation development (21). Collectively, these studies suggest that CC-derived miRNAs may influence oocyte maturation in a paracrine manner (19, 20). Although the fundamental process of oocyte maturation is highly similar across species, most information on the effect of GDNF is obtained from studies in rodents and pigs. In the current study, we sought to examine the effects of GDNF on the nuclear and cytoplasmic maturation of cultured human immature oocytes. We further investigated the role of miRNAs in GDNF-induced oocyte maturation and the potential underlying molecular mechanisms. Materials and Methods Collection of immature COCs The current study was approved by the Medical Ethics Committee of the Women’s Hospital, Zhejiang University School of Medicine, and informed consent was obtained from all participants. Human immature cumulus-oocyte complexes (COCs) were collected from infertile patients (n = 82) who underwent IVF treatment in our assisted reproductive technology center between January 2015 and November 2015. The demographics of patients included in this study and laboratory characteristics of IVF cycles are listed in Supplemental Table 1. Immature oocytes were collected from IVF patients who underwent ovarian stimulation with normal ovarian reserve. The main types of infertility in these patients are tubal and male factors. The exclusion criteria included (1) any type of ovarian dysfunction, (2) IVF of natural cycle or mild stimulation, (3) preimplantation genetic diagnosis, and (4) fertilization failure in previous IVF treatment. Controlled ovarian stimulation was performed using either the GnRH agonist or antagonist protocol, as previously described (22). Ultrasound-guided transvaginal oocyte retrieval was performed 36 hours after administration of human chorionic gonadotropin (Pregnyl, Merck, Kenilworth, NJ). The flushing fluid containing the COCs was poured into 100-mm culture dishes (Falcon; BD Biosciences, Franklin Lakes, NJ), which were tilted carefully until they had spontaneously expanded. The germinal vesicles (GVs) of the oocytes were inspected using a stereomicroscope. COCs without an intact GV were excluded from this study. To avoid intersubject variability, COCs from the same subject were randomly and equally allocated to each group according to the size and morphology of the COCs. IVM, fertilization, and embryonic development Based on a previous study (13) and the results of our preliminary experiments (Supplemental Table 2), we chose the concentration of 50 nM to evaluate the effects of GDNF on human oocyte maturation in the in vitro study. A total of 200 GV-stage COCs were randomly transferred into the G-IVF (Vitrolife, Sweden AB, Sweden) medium supplemented with 10% synthesis serum substituent with (n = 100) or without (n = 100) 50 nM of recombinant human GDNF (R&D Systems, Minneapolis, MN) and were incubated at 37°C in a controlled atmosphere of 5% O2, 6% CO2, and 89% N2 for 24 hours. For measurement of oocyte maturity, COCs were denuded of CCs by repeated pipetting. Oocytes without the first polar body are regarded as immature and were classified as GV stage or metaphase I (MI) stage according to the presence of a GV. Oocytes that exhibit extrusion of the first polar body (MII stage) were fertilized by intracytoplasmic sperm injection (ICSI) using sperm with normal semen parameters. Semen analysis was performed according to the 2010 World Health Organization laboratory manual (23). Fertilization was assessed by the appearance of two pronuclei (2PN) in the zygote 16 to 18 hours after microinjection. Embryos were cultured and observed every day until reaching the blastocyst stage (approximately 5 to 6 days). Isolation and culture of human CCs Before ICSI, CCs were denuded from oocytes using hyaluronidase (Vitrolife). After three washes with PBS, CCs were seeded into six-well tissue culture dishes (BD Biosciences) and were cultured in serum-free medium composed of Earle’s Balanced Salts Solution (Sigma-Aldrich, St. Louis, MO) at 37°C in a controlled atmosphere of 5% O2, 6% CO2, and 89% N2 for 12 hours before GDNF treatment. miRNA microarray analysis Human CCs (approximately 1 × 106 per well) were serum starved in α-MEM (Life Technologies, Gaithersburg, MD) for 12 hours and then treated with or without 50 nM GDNF for 18 hours. miRNA expression profiles of CCs with or without GDNF treatment were analyzed using an Agilent human miRNA (8*60K) array. Briefly, 5 µg total RNA was extracted and purified using a mirVana miRNA Isolation Kit without phenol (Ambion, Thermo Fisher Scientific, Carlsbad, CA). miRNAs were labeled using the miRNA Complete Labeling and Hyb Kit (Agilent Technologies). For array hybridization, each slide was hybridized with 100 ng Cy3-labeled RNA using the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Palo Alto, CA) in a hybridization oven at 55°C. After being washed in staining dishes, slides were scanned with Agilent Microarray Scanner and Feature Extraction Software 10.7 (Agilent Technologies) using the default settings. Raw data were normalized by the Quantile algorithm (GeneSpring Software 12.6, Agilent Technologies). All procedures were carried out according to the manufacturer’s protocols. Only miRNAs with threefold changes in genes expression in the GDNF-treated group were considered differentially expressed genes. RNA extraction and RT-quantitative polymerase chain reaction To determine the expression levels of miR-145-5p at different maturation stages, CCs were mechanically isolated from COCs before the ICSI procedure. CCs from each COC were transferred individually into a tube containing 200 µL RNAlater reagent (Ambion) and were stored at –80°C until analysis. Cultured CCs that were transfected with the miR-145-5p inhibitor were collected to evaluate the expression levels of GDNF, GFRA1, and RET. RNA extraction of CCs was performed using TRIzol Reagent (Thermo Fisher Scientific). Briefly, approximate 105 to 106 CCs were homogenized in 1000 μL TRIzol Reagent (Thermo Fisher Scientific) and incubated at room temperature for 5 minutes. Then trichloromethane and isopropyl alcohol were added to extract the total RNA. Reverse transcription (RT) was performed using a miScript RT kit (Qiagen, Chatsworth, CA). RT-quantitative polymerase chain reaction (qPCR) was performed on an ABI 7900HT Real-Time PCR System (Applied Biosystems, Framingham, MA) using a miScript SYBR Green PCR Kit (Qiagen). The primers used for has-miR-145-5p were obtained from the 10× miScript Primer Assays (Qiagen). The RNU6B small nuclear ribonucleic acid primer assay (Qiagen) was used as the endogenous reference for miRNA normalization, and glyceraldehyde 3-phosphate dehydrogenase was chosen as the endogenous reference for mRNA normalization. All procedures were carried out according to the manufacturer’s instructions. A quantification cycle (Cq) value <37 was used. The fold change of the miRNAs was calculated using the 2–ΔΔCq method, and the comparison of mRNA or miRNA between different groups was performed using the 2–ΔCq method. All experiments were conducted in triplicate and repeated at least three times. Plasmid vectors and luciferase reporter assays To construct a reporter vector for the GFRA1 gene, we used the method reported by Shimono et al. (24) and followed the steps outlined later: the multiple cloning site of the pGEM-T-Easy vector (Promega, Madison, WI) was amplified by PCR and inserted into the pGL3 control vector (Promega). The primers used in this study were designed by Sangon Biotech Co., Ltd. (Shanghai, China). The 3′-untranslated region (UTR) of the GFRA1 gene, which includes the binding site for miR-145-5p, was amplified by PCR, and the GFRA1 3′-UTR product was cloned onto the 3′ end of the luciferase gene of the pGL3-MC vector (control). To construct the mutant miR-145-5p binding site in the GFRA1 3′-UTR, the 3′UTR GFRA1 amplified fragment was cloned in the antisense orientation. All products were sequenced by Sangon Biotech Co., Ltd. HEK-293T cells that were used for the luciferase reporter assays were cultured in DMEM (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) at 37°C in a 5% CO2 atmosphere. HEK-293T cells were seeded into 96-well plates (1 × 105 cells per well) one day before transfection. The next day, the cells were transfected with 200 ng of the pGL3 luciferase vector containing the 3′-UTR of human GFRA1, 35 ng of the pRL-TK Renilla luciferase vector (Promega), and 20 nM of either hsa-miR-145-5p or the negative control (Thermo Fisher Scientific). The transfection procedure was carried out using the Lipofectamine 3000 reagent (Invitrogen), according to the manufacturer’s instructions. Thirty-six hours after transfection, luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) and normalized using the Renilla luciferase activity (Promega). Experiments were conducted in triplicate and repeated at least three times. Transient transfection of either miR-145-5p inhibitors or miR-145-5p mimics in cultured human CCs Cultured human CCs were transfected with either miR-145-5p mimics or miR-145-5p inhibitors for functional analysis. CCs were seeded in α-MEM (Thermo Fisher Scientific) until they were 70% to 80% confluent at the time of transfection. The CCs were transfected with either 20 nM of the GMR-miR miRNA mimics/inhibitors (GenePharma, Shanghai, China) or 20 nM of the nonrelative RNA duplex, GMR-miR miRNA mimics control/inhibitor control (GenePharma), for 48 hours according to the manufacturer’s protocols using the Lipofectamine 3000 reagent (Invitrogen). Western blot analysis Approximate 2 × 106 cultured human CCs were collected for the following experiments. Total protein was extracted using the Radioimmunoprecipitation lysis buffer (Beyotime, Haimen, China) containing 1% 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride. Proteins were separated by SDS-PAGE using a 4% stacking gel and a 10% separating gel before they were transferred onto the polyvinylidene fluoride membranes. After being blocked with Tris-buffered saline with 0.1% Tween 20 buffer containing 5% BSA, the membranes were incubated with specific primary antibodies (anti-GDNF, anti-GFRA1, anti-RET, and anti-glyceraldehyde 3-phosphate dehydrogenase obtained from Abcam, diluted at 1:400) at 4°C overnight. The membranes were then washed with Tris-buffered saline with 0.1% Tween 20 and were incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (1:2000; Abcam, Cambridge, MA) prior to visualizing the immunoreactive bands using the enhanced chemiluminescence detection system (Thermo Fisher Scientific). Cell apoptosis analysis CCs (1 × 106) were collected after being treated with hyaluronidase without EDTA and were washed twice in PBS. To quantify cell death arising from apoptosis, an Annexin V-FITC/PI apoptosis kit (Multisciences, China) was applied to examine the number of cells at different stages. Briefly, cells were resuspended in 0.5 mL binding buffer, and 5 μL of Annexin V-FITC and PI (Multisciences) were added to each tube. Cells were incubated for 10 minutes at 25°C in the dark before being analyzed by flow cytometry (FACS Calibur; BD Biosciences). Statistical analysis Differences between two groups were evaluated using the two-tailed t test or the nonparametric Mann-Whitney test. Comparison of the proportions of embryo development outcomes were made using the χ2 test or Fisher exact test. Differences among more than two groups were evaluated using an analysis of variance followed by a Holm-Sidak multiple comparison test for post hoc analysis. Data were considered significantly different from each other if P < 0.05. All data were analyzed using the Sigmaplot software version 12.5 (Systat Software Inc., Gmbh, Erkrath, Germany). Results GDNF enhances oocyte IVM To investigate the effect of GDNF on human oocyte IVM, GV-stage COCs were cultured with or without 50 nM GDNF for 24 hours. As shown in Table 1, treatment with GDNF (50 nM) significantly increased the percentage of MII-stage oocyte from 34% to 49% (P = 0.045). However, treatment with GDNF did not affect the GVBD rate (Table 1). To further investigate whether the addition of GDNF can enhance the embryonic developmental potential of oocytes, we fertilized all the MII-stage oocytes by ICSI. The results showed no significant difference in the normal fertilization (2PN) rate between oocytes matured in the standard medium and those matured in the medium supplemented with GDNF. Similarly, treatment with 50 nM GDNF did not increase the proportions of MII oocytes that underwent cleavage or developed into embryos with six to nine blastomeres on day 3. In the current study, the blastocyst formation rate was higher in the GDNF-treated group (45.45% vs 30.00%), but the difference is not statistically significant (P = 0.386). Studies in mice have suggested that the organization of cytoplasmic structures and ribosomal biogenesis are related to developmental competence of oocyte (25). Furthermore, the blastocyst generation rate has been regarded as an indicator of cytoplasmic maturation (4, 9). In this study, we assessed cytoplasmic maturation by calculating blastocyst generation rate, but the result did not show any statistical significant difference. Thus, we speculate that GDNF cannot promote human oocyte cytoplasmic maturation. Table 1. Effect of GDNF on the Outcomes of Embryo Development From Immature Oocytes During IVM Process Parameter GDNF Treatment GDNF Nontreatment P Value No. of oocytes 100 100 GVBD oocytes (%) 55 (55/100) 48 (48/100) 0.396 MII oocytes (%) 49 (49/100) 34 (34/100) 0.045 Normal fertilization rate (% per MII) 67.34 (33/49) 58.82 (20/34) 0.490 Embryo cleavage rate (% per 2PN) 90.90 (30/33) 90.00 (18/20) 1.000 Six to nine cell rate (% per 2PN) 63.63 (21/33) 65.00 (13/20) 1.000 Blastocyst generation rate (% per 2PN) 45.45 (16/33) 30.00 (6/20) 0.386 Parameter GDNF Treatment GDNF Nontreatment P Value No. of oocytes 100 100 GVBD oocytes (%) 55 (55/100) 48 (48/100) 0.396 MII oocytes (%) 49 (49/100) 34 (34/100) 0.045 Normal fertilization rate (% per MII) 67.34 (33/49) 58.82 (20/34) 0.490 Embryo cleavage rate (% per 2PN) 90.90 (30/33) 90.00 (18/20) 1.000 Six to nine cell rate (% per 2PN) 63.63 (21/33) 65.00 (13/20) 1.000 Blastocyst generation rate (% per 2PN) 45.45 (16/33) 30.00 (6/20) 0.386 View Large Table 1. Effect of GDNF on the Outcomes of Embryo Development From Immature Oocytes During IVM Process Parameter GDNF Treatment GDNF Nontreatment P Value No. of oocytes 100 100 GVBD oocytes (%) 55 (55/100) 48 (48/100) 0.396 MII oocytes (%) 49 (49/100) 34 (34/100) 0.045 Normal fertilization rate (% per MII) 67.34 (33/49) 58.82 (20/34) 0.490 Embryo cleavage rate (% per 2PN) 90.90 (30/33) 90.00 (18/20) 1.000 Six to nine cell rate (% per 2PN) 63.63 (21/33) 65.00 (13/20) 1.000 Blastocyst generation rate (% per 2PN) 45.45 (16/33) 30.00 (6/20) 0.386 Parameter GDNF Treatment GDNF Nontreatment P Value No. of oocytes 100 100 GVBD oocytes (%) 55 (55/100) 48 (48/100) 0.396 MII oocytes (%) 49 (49/100) 34 (34/100) 0.045 Normal fertilization rate (% per MII) 67.34 (33/49) 58.82 (20/34) 0.490 Embryo cleavage rate (% per 2PN) 90.90 (30/33) 90.00 (18/20) 1.000 Six to nine cell rate (% per 2PN) 63.63 (21/33) 65.00 (13/20) 1.000 Blastocyst generation rate (% per 2PN) 45.45 (16/33) 30.00 (6/20) 0.386 View Large GDNF treatment induces downregulation of miR-145-5p in cultured human CCs To investigate whether GDNF induces differential miRNA expression, we examined the miRNA expression patterns of human CCs treated with or without GDNF (50 nM) using a miRNA microarray assay. The results revealed 55 significantly differentially regulated miRNAs in CCs (expression fold change >3), of which 28 were upregulated and 27 were downregulated after GDNF treatment (for details, see Fig. 1). Unsupervised cluster analysis (based on the 55 differentially expressed miRNAs) showed two major clusters (Fig. 1). After GDNF treatment, miR-7152-3p, miR-8060, and miR-4417 were the three highest upregulated miRNAs, whereas miR-8071, miR-3059, and miR-145-5p were the three most significantly downregulated miRNAs in cultured CCs. Figure 1. View largeDownload slide Differential miRNA expression patterns in human CCs with or without GDNF treatment. Cluster analysis and heat map of 55 differentially expressed miRNAs (expression fold change greater than 3) in cultured human CCs treated with either a vehicle control or 50 nM GDNF for 18 hours. All 28 upregulated and 27 downregulated miRNAs are listed. The degree of differential miRNA expression compared with control is listed in the table. Figure 1. View largeDownload slide Differential miRNA expression patterns in human CCs with or without GDNF treatment. Cluster analysis and heat map of 55 differentially expressed miRNAs (expression fold change greater than 3) in cultured human CCs treated with either a vehicle control or 50 nM GDNF for 18 hours. All 28 upregulated and 27 downregulated miRNAs are listed. The degree of differential miRNA expression compared with control is listed in the table. Bioinformatics analysis of these most significantly downregulated miRNAs showed that miR-8071 and miR-3059 are not related to the GDNF signaling pathway or to oocyte maturation. Interestingly, miR-145-5p (fold change = 34) is a potential target that may be involved in GDNF-induced oocyte maturation. The bioinformatics analysis suggested that miR-145-5p may target the GFRA1 and the epidermal growth factor receptor (EGFR), and the expression of both receptors in CCs may be correlated with the maturity of the corresponding oocytes. Thus, we next used RT-qPCR to validate the microarray findings, and the results showed that treatment of CCs with GDNF (50 nM) significantly decreased the levels of miR-145-5p (Fig. 2A). Figure 2. View largeDownload slide GDNF induces downregulation of miR-145-5p in cultured human CCs. (A) Human CCs were treated with either a vehicle control or 50 nM GDNF for 24 hours, and miR-145-5p levels were then examined using RT-qPCR. The miR-145-5p levels were normalized to the endogenous U6 small nuclear ribonucleic acid. (B) miR-145-5p expression levels in human CCs from different maturation stages (GV, MI, and MII) of the cumulus-oocyte-complex (n = 10 of each group) were examined using RT-qPCR. The results are expressed as the mean ± SEM of at least three independent experiments. *P < 0.05; ****P < 0.0001. Figure 2. View largeDownload slide GDNF induces downregulation of miR-145-5p in cultured human CCs. (A) Human CCs were treated with either a vehicle control or 50 nM GDNF for 24 hours, and miR-145-5p levels were then examined using RT-qPCR. The miR-145-5p levels were normalized to the endogenous U6 small nuclear ribonucleic acid. (B) miR-145-5p expression levels in human CCs from different maturation stages (GV, MI, and MII) of the cumulus-oocyte-complex (n = 10 of each group) were examined using RT-qPCR. The results are expressed as the mean ± SEM of at least three independent experiments. *P < 0.05; ****P < 0.0001. Expression of miR-145-5p in CCs is negatively correlated with oocyte maturation Because GDNF decreases the level of miR-145-5p in CCs and enhances the oocyte maturation rate, we next investigated whether the miR-145-5p level in CCs is correlated with oocyte maturity (GV stage, MI stage, and MII stage). As shown in Fig. 2B, among three groups, the miR-145-5p expression levels were the highest in GV-stage CCs and were the lowest in MII-stage CCs. Compared with the GV-stage CCs, the miR-145-5p levels in MI- and MII-stage CCs were significantly lower (P < 0.0001). Similarly, the miR-145-5p level in MI-stage CCs was significantly higher than that in MII-stage CCs (P < 0.05). GFRA1 is a target of miR-145-5p in human CCs Given that GDNF significantly downregulated the expression of miR-145-5p in CCs, which negatively correlated with oocyte maturity, we next investigated the potential underlying molecular mechanisms. In silico analysis of potential targets of miR-145-5p was performed using the miRDB (www.mirdb.org) and the TargetScan (www.targetscan.org) miRNA target prediction software; GFRA1, RET, and EGFR were three candidates of interest. GFRA1 is a cell surface receptor that preferentially binds to GDNF and activates downstream intracellular signaling. The TargetScan software predicted that the 3′-UTR of the GFRA1 mRNA contains a highly conserved region that has 8-mer seed matches with positions 2 to 8 of the mature miR-145-5p (the seed + position 8) followed by an “A” (26) (see Supplemental Fig. 1). To determine whether GFRA1 is a direct target for negative regulation by miR-145-5p, we transfected the cells with either the wild-type or mutant GFRA13′-UTR luciferase reporter construct. HEK-293T cells, which do not express miR-145-5p, were cotransfected with the 3′-UTR luciferase vector, the Renilla luciferase vector (for normalization; Promega), and the miR-145-5p mimic. The results showed that the cotransfection with the miR-145-5p mimic significantly reduced the luciferase activity of the vector containing the wild-type GFRA1 3′-UTR by nearly 20% compared with cotransfection with the miRNA mimic control (P < 0.05) (Fig. 3A). However, luciferase reporter activity was not affected by the miR-145-5p mimic when the seeding site was mutated (Fig. 3A), indicating that miR-145-5p directly targets GFRA1. Figure 3. View largeDownload slide miR-145-5p downregulates the mRNA levels of GFRA1 and RET, and GFRA1 is a target of miR-145-5p in human CCs. (A) HEK-293 cells were transfected with either the wild-type or mutant GFRA13′-UTR luciferase reporter constructs before transfection with the 3′-UTR luciferase vector, Renilla luciferase vector (for normalization; Promega), and miR-145-5p mimic for 36 hours. Luciferase activities were measured using a luciferase reporter assay. (B and C) Human CCs were transfected with either the miR-145-5p mimic/miR-145-5p mimic control or the miR-145-5p inhibitor/miR-145-5p inhibitor control for 48 hours, and the mRNA levels of (B) GFRA1 and (C) RET were examined using RT-qPCR. The results are expressed as the mean ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01. Ctrl, control; MUT, mutation. Figure 3. View largeDownload slide miR-145-5p downregulates the mRNA levels of GFRA1 and RET, and GFRA1 is a target of miR-145-5p in human CCs. (A) HEK-293 cells were transfected with either the wild-type or mutant GFRA13′-UTR luciferase reporter constructs before transfection with the 3′-UTR luciferase vector, Renilla luciferase vector (for normalization; Promega), and miR-145-5p mimic for 36 hours. Luciferase activities were measured using a luciferase reporter assay. (B and C) Human CCs were transfected with either the miR-145-5p mimic/miR-145-5p mimic control or the miR-145-5p inhibitor/miR-145-5p inhibitor control for 48 hours, and the mRNA levels of (B) GFRA1 and (C) RET were examined using RT-qPCR. The results are expressed as the mean ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01. Ctrl, control; MUT, mutation. It has been shown that GDNF-induced first polar body extrusion is mediated by its receptor GFRA1 and the common signal transduction subunit RET in mice (9). We next examined the effects of cell transfection with the miR-145-5p inhibitors/mimics on the expression levels of GFRA1 and RET. As shown in Fig. 3B and 3C, transfection with the miR-145-5p mimic for 48 hours significantly decreased the mRNA levels of both GFRA1 (P < 0.05) and RET (P < 0.05) in human CCs. In contrast, transfection of CCs with the miR-145-5p inhibitor for 48 hours significantly increased the mRNA levels of both GFRA1 (P < 0.001) and RET (P < 0.001). These results indicate that miR-145-5p might negatively regulate the expression of GFRA1 and its coreceptor RET. Moreover, time-course studies showed that transfection with the miR-145-5p inhibitor for 12, 24, or 48 hours did not alter the mRNA or protein levels of GDNF in human CCs (Fig. 4A, 4D, and 4G). However, transfection with the miR-145-5p inhibitor significantly increased the mRNA and protein levels of GFRA1 in a time-course–dependent manner, starting at 24 hours and persisting until 48 hours (Fig. 4B, 4E, and 4G). Similarly, transfection with the miR-145-5p inhibitor significantly increased the mRNA and protein levels of RET starting at 48 hours (Fig. 4C, 4F, and 4G). Figure 4. View largeDownload slide miR-145-5p downregulates the mRNA and protein levels of both GFRA1 and RET, but not of GDNF. Human CCs were transfected with either the miR-145-5p inhibitor or the miR-145-5p inhibitor control for 12, 24, or 48 hours, and the mRNA and protein levels of (A and D) GDNF, (B and E) GFRA1, (C and F) and RET were examined using RT-qPCR and western blot analysis, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01; ****P < 0.0001. C/Ctrl, control; Inh, miR-145-5p inhibitor; NC, negative control. Figure 4. View largeDownload slide miR-145-5p downregulates the mRNA and protein levels of both GFRA1 and RET, but not of GDNF. Human CCs were transfected with either the miR-145-5p inhibitor or the miR-145-5p inhibitor control for 12, 24, or 48 hours, and the mRNA and protein levels of (A and D) GDNF, (B and E) GFRA1, (C and F) and RET were examined using RT-qPCR and western blot analysis, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01; ****P < 0.0001. C/Ctrl, control; Inh, miR-145-5p inhibitor; NC, negative control. miR-145-5p downregulates EGFR mRNA levels in human CCs Porcine oocyte IVM studies suggest that GDNF and EGF have synergistic effects on the promotion of oocyte competency and blastocyst quality (27). Moreover, miR-145-5p has been shown to inhibit cell proliferation by downregulating EGFR in human lung adenocarcinoma (28). To examine the effect of miR-145-5p on the expression of EGFR in human CCs, we transfected the cells with the miR-145-5p mimic/mimic control or the miR-145-5p inhibitor/inhibitor control for 48 hours. The RT-qPCR results showed that transfection with the miR-145-5p inhibitor significantly increased the EGFR mRNA levels in CCs (Supplemental Fig. 2), indicating that miR-145-5p may influence EGF-induced oocyte maturation by downregulating the expression of EGFR. Downregulation of miR-145-5p mediates GDNF-induced decreases in CC apoptosis It has been reported that CC apoptosis is inversely correlated with embryonic development and that FSH-induced rescue of CC apoptosis during IVM can enhance the developmental competence of oocytes (29, 30). Apoptosis experiments using flow cytometry showed that GDNF treatment reduced the apoptotic rate of cultured CCs (17.82% ± 1.34% vs 24.60% ± 1.45%, P < 0.05) (Fig. 5A). To examine whether miR-145-5p affects CC apoptosis, we transfected CCs with the miR-145-5p mimic, and the results showed that the miR-145-5p mimic induced an increase in the apoptotic rate of CCs (27.96 ± 1.41% vs 21.46% ± 1.73%, P < 0.05) (Fig. 5B). To further examine whether the downregulation of miR-145-5p induced by GDNF treatment contributes to the increased apoptosis of CCs, we transfected the cultured CCs with either the miR-145-5p mimic or the miR-145-5p mimic control for 48 hours before treatment with GDNF. The results showed that the apoptotic rate of CCs in the group transfected with the miR-145-5p mimic is higher than that of the group transfected with the miR-145-5p mimic control (30.58% ± 2.14% vs 18.89% ± 1.61%, P < 0.05) (Fig. 5C). Together, these findings indicate that miR-145-5p contributes to the increase in CC apoptosis and that GDNF may improve CC apoptosis by downregulating the expression of miR-145-5p. Figure 5. View largeDownload slide Downregulation of miR-145-5p mediates the GDNF-induced inhibition of cell apoptosis in human CCs. (A) Human CCs were treated with either a vehicle control or 50 nM GDNF for 24 hours, and cell apoptosis was examined using an Annexin V-FITC/PI apoptosis kit (Multisciences). (B) Human CCs were transfected with either the miR-145-5p mimic or the miR-145-5p mimic control for 48 hours, and cell apoptosis was examined using an Annexin V-FITC/PI apoptosis kit (Multisciences). (C) Human CCs were transfected with either the miR-145-5p mimic or the miR-145-5p mimic control for 48 hours prior to treatment with 50 nM GDNF for an additional 24 hours, and cell apoptosis was examined using an Annexin V-FITC/PI apoptosis kit (Multisciences). The results are expressed as the mean ± SEM of at least three independent experiments. *P < 0.05. LL, lower left; LR, lower right; UR, upper right. Figure 5. View largeDownload slide Downregulation of miR-145-5p mediates the GDNF-induced inhibition of cell apoptosis in human CCs. (A) Human CCs were treated with either a vehicle control or 50 nM GDNF for 24 hours, and cell apoptosis was examined using an Annexin V-FITC/PI apoptosis kit (Multisciences). (B) Human CCs were transfected with either the miR-145-5p mimic or the miR-145-5p mimic control for 48 hours, and cell apoptosis was examined using an Annexin V-FITC/PI apoptosis kit (Multisciences). (C) Human CCs were transfected with either the miR-145-5p mimic or the miR-145-5p mimic control for 48 hours prior to treatment with 50 nM GDNF for an additional 24 hours, and cell apoptosis was examined using an Annexin V-FITC/PI apoptosis kit (Multisciences). The results are expressed as the mean ± SEM of at least three independent experiments. *P < 0.05. LL, lower left; LR, lower right; UR, upper right. Discussion In the current study, we demonstrate that exogenous GDNF promotes oocyte maturation in cultured human immature oocytes. GDNF and both of its coreceptors, GFRA1 and RET, are highly expressed in oocytes and the surrounding CCs of the antral follicles (31). Additionally, the mature form of the GDNF protein is present at high levels in the follicular fluid of porcine antral follicles (12). Functional studies have demonstrated that in vitro culture with GDNF can enhance the developmental competence of oocytes in several mammals, including pigs, mice, and humans (9, 12, 13). Consistent with our study, an in vitro study showed that GDNF treatment enhanced the expansion rate of porcine small COCs and increased the percentage of MII-stage oocytes without affecting the nuclear maturation of large follicle-derived oocytes (12). In contrast, our study showed that GDNF had a negligible effect on the GVBD of oocytes, and this finding is consistent with a previous study showing that treatment with GDNF and brain-derived neurotrophic factor increased total yields of MII-stage oocytes without affecting their GVBD in cultured human immature oocytes (13). Similarly, our study did not show any effect of GDNF pretreatment during IVM stage on early embryonic development, as treatment with GDNF did not significantly change the fertilization rate (2PN), cleavage rate, or the development of six to nine blastomeres, indicating that GDNF does not influence the induction of cytoplasmic maturation of oocytes. Results from both previous studies and the current study clearly demonstrate a beneficial effect of GDNF on the IVM of mammalian oocytes. However, the underlying molecular mechanism of action remains largely unknown. Using miRNA expression profiles, we identified 55 significantly differentially regulated miRNAs in GDNF-stimulated CCs. Among these differentially regulated miRNAs, miR-145-5p is one of the most significantly downregulated miRNAs, suggesting that it may play an important role in mediating GDNF-induced oocyte maturation. miR-145-5p mainly functions as a tumor suppressor (inhibiting tumor cell growth) and is downregulated in several types of tumors (32). In human embryonic stem cells, miR-145-5p regulates several pluripotency factors (33). In addition, miR-145-5p has been shown to promote cell differentiation and to repress cell proliferation in smooth muscle cells (34). Intriguingly, miR-145 has been identified as one of the most altered expression profiles during oocyte maturation with preferential abundance in the mature stage of bovine oocytes, indicating a potential role for miR145 in regulating oocyte maturation (35). Indeed, the current study shows that the expression of miR-145-5p in CCs is negatively correlated with oocyte maturation. Notably, our findings suggest that beyond the tumor biology, miR-145-5p may also participate in the regulation of ovarian functions. Based on bioinformatics analysis and previous studies, we hypothesized that GFRA1 and EGFR were potential targets of miR-145-5p (28). Using both an miR-145-5p mimic and miR-145-5p inhibition transfection approaches, we further showed that miR-145-5p downregulates the expression of GFRA1, RET, and EGFR in human CCs. We further confirmed that GFRA1 is a direct target for negative regulation by miR-145-5p, as miR-145-5p significantly reduced the luciferase activity of the wild-type GFRA1 3′-UTR but not that of a mutant GFRA1 3′-UTR. In many cell types, GDNF acts through binding to GFRA1 and further activates the RET receptor tyrosine kinase (10). In mouse preovulatory oocytes, GDNF-induced first polar body extrusion is mediated by the receptors GFRA1 and RET (9). Our study demonstrated that GFRA1 is a target of miR-145-5p in human CCs, and the expressions of both GFRA1 and RET in human CCs increase after transfection with the miR-145-5p inhibitor. These results indicate that miR-145-5p can be downregulated by GDNF treatment, leading to the increased expression of the GFRA1-RET receptor complex. Collectively, previous studies and our study suggest that GNDF may exert its action on oocyte maturation more effectively. Although the IVM technique has been widely used in assisted reproductive treatment, the developmental potential of IVM embryos is significantly lower than that of in vivo matured oocytes. Therefore, the average pregnancy rate of IVM cycles is much lower than that of the conventional IVF/ICSI cycles. Even though some IVM oocytes may be competent to complete nuclear maturation, they are unable to develop into the blastocyst stage due to the deficient or defective cytoplasmic maturation of these oocytes (4). GDNF treatment showed no effect on the cytoplasmic maturation of mouse oocytes cultured in serum-free medium (9). However, the addition of GDNF and EGF enhanced the developmental potential of porcine in vitro oocyte maturation (12, 27). These results suggest that GDNF in combination with other factors may induce the cytoplasmic maturation of oocytes. Our results showed that miR-145-5p downregulated the expression of EGFR in human CCs, suggesting that the synergistic effects of GDNF and EGF may be mediated through the action of miRNAs. Previous studies have shown that EGF and EGF-like factors are involved in the regulation of oocyte maturation and cumulus expansion in different species (36–38). In addition to the effect on nuclear maturation, EGF can improve the developmental competence or cytoplasmic maturation of immature oocytes (37). Increasing evidence suggests that EGF exerts its stimulatory effects on the meiotic maturation of oocytes through the activation of EGF receptor signaling, leading to the activation of M-phase promoting factor and MAPK, as well as decreasing the activity of natriuretic peptide receptor 2. The addition of EGF-like factors, cAMP, bone morphogenetic protein 15, and growth differentiation factor 9 into culture medium enhances oocyte meiotic maturation and blastocyst formation, and these beneficial effects are attenuated by cotreatment with an EGF receptor phosphorylation inhibitor (39). Thus, our results indicate that GDNF treatment may improve oocyte developmental competence through the upregulation of EGFR expression by suppressing miR-145-5p. The results from this study have provided a potential molecular mechanism underlying the combination effects of EGF and GDNF. Our findings also suggest that intraovarian paracrine factors may modulate oocyte maturation by interacting with other growth factors to form a complex network. The results generated in this study are based on the cultured CCs in the absence of oocytes. We appreciate that the oocyte and oocyte secreted factors may influence the phenotype and gene expression profiles of the CCs. Previous studies have shown that oocyte secreted factors are essential regulators of the CC functions, including regulation of CCs gene expression, stimulation of CCs proliferation, and inhibition of CCs luteinization (40). Therefore, the gene expression patterns and signal mediators could be different between the in vitro and in vitro systems. Future study aimed at addressing this issue by using a three-dimensional cell culture system that includes both CCs and oocyte will be of great interest. During the IVM process, we found that the morphological characteristics of CCs are better in COCs treated with GDNF. Because CC apoptosis is highly related to blastocyst development of the corresponding gametes (41), we examined the apoptosis rate of CCs cultured with or without GDNF. The results showed that GDNF treatment reduces the apoptosis of cultured CCs and that miR-145-5p is involved in the regulation of apoptosis in human CCs. CCs are essential for the development of oocytes, as they produce specific paracrine factors that influence oocyte developmental competence via functional gap junctions. Furthermore, our results suggest that GDNF may improve oocyte developmental competence by reducing CC apoptosis, which may be mediated through the downregulation of miR-145-5p. In conclusion, our study showed that GDNF treatment promoted the IVM of cultured oocytes to MII stage. Additionally, miR-145-5p was significantly downregulated after GDNF treatment in human CCs, which most likely mediates GDNF-induced enhancement of oocyte maturation. We also demonstrated that miR-145-5p negatively regulated the expression of three major components of GDNF-induced cellular action in human CCs, GFRA1, RET, and EFGR. Furthermore, GDNF may protect human CCs against cell apoptosis, possibly mediated by the downregulation of miR-145-5p. Our findings provide insight into the roles of GDNF and miR-145-5p in the regulation of oocyte maturation and may be clinically applied to improve the developmental potential of immature oocytes from infertile couples undergoing IVM. Abbreviations: Abbreviations: 2PN two pronuclei CC cumulus cell COC cumulus-oocyte complex CQ quantification cycle EGF epidermal growth factor EGFR epidermal growth factor receptor GDNF glial cell line–derived neurotrophic factor GFRA1 GDNF family receptor-α1 GV germinal vesicle GVBD germinal vesicle breakdown ICSI intracytoplasmic sperm injection IVF in vitro fertilization IVM in vitro maturation MI metaphase I MII metaphase II miRNA microRNA qPCR quantitative polymerase chain reaction RET ret proto-oncogene RT reverse transcription UTR untranslated region Acknowledgments Financial Support: This work was supported by Natural Science Foundation of China Grants 81170567 and 81370761 (to Y.Y.) and Canadian Institutes of Health Research Foundation Scheme Grant 143317 (to P.C.K.L.). Disclosure Summary: The authors have nothing to disclose. References 1. Grynberg M , El Hachem H , de Bantel A , Benard J , le Parco S , Fanchin R . In vitro maturation of oocytes: uncommon indications . 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Journal of Clinical Endocrinology and Metabolism – Oxford University Press
Published: Apr 20, 2018
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