Glial cell line-derived neurotrophic factor supplementation promotes bovine in vitro oocyte maturation and early embryo development

Theriogenology 113 (2018) 92e101

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Glial cell line-derived neurotrophic factor supplementation promotes bovine in vitro oocyte maturation and early embryo development Dong-Hui Wang 1, Hong-Xia Zhou 1, Shu-Jun Liu, Cheng-Jie Zhou, Xiang-Wei Kong, Zhe Han, Cheng-Guang Liang* State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestock, The Research Center for Laboratory Animal Science, College of Life Science, Inner Mongolia University, Hohhot, Inner Mongolia, People's Republic of China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 September 2017 Received in revised form 9 February 2018 Accepted 15 February 2018 Available online 19 February 2018

Paracrine factors such as glial cell line-derived neurotrophic factor (GDNF), which was originally derived from the supernatants of a rat glioma cell line, play pivotal roles in oocyte maturation and early embryo development in mammals, such as mice, rats, pigs, sheep, and even humans. However, whether GDNF facilitates in vitro oocyte maturation or early embryo development in bovines is not yet known. We show for the first time that GDNF and its receptor, GDNF family receptor alpha-1 (GFRA1), are presented in ovarian follicles at different stages as well as during oocyte maturation and early embryo development. Immunostaining results revealed the subcellular localizations of GDNF and GFRA1 in oocytes throughout follicle development, first in germinal vesicles and during blastocyst embryo stages. The ability of exogenously applied GDNF to promote oocyte maturation and early embryo development was evaluated in culture, where we found that an optimal concentration of 50 ng/mL promotes the maturation of cumulus-oocyte complexes and the nuclei of denuded oocytes as well as the development of embryos after IVF. To further investigate the potential mechanism by which GDNF promotes oocyte maturation, bovine oocytes were treated with morpholinos targeting Gfra1. The suppression of GFRA1 presence blocked endogenous and exogenous GDNF functions, indicating that the effects of GDNF that are essential and beneficial for bovine oocyte maturation and early embryo development occur through this receptor. Furthermore, we show that supplementation with GDNF improves the efficiency of bovine IVF embryo production. © 2018 Elsevier Inc. All rights reserved.

Keywords: GDNF GFRA1 Bovine Oocyte maturation Early embryo development

1. Introduction Recent advances in biotechnology have enabled the creation of cloned or genetically modified bovines via the manipulation of in vitro-produced embryos. Notably, the overall efficiency of this process is still extremely low, and the quality of the embryos produced in vitro is inferior to that of in vivo embryos [1e4]. In vivo, ovarian follicle development requires somatic cell proliferation and differentiation to coordinate with oocyte growth and maturation. This coordination is ensured by paracrine interactions between the

* Corresponding author. State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestock, The Research Center for Laboratory Animal Science, College of Life Science, Inner Mongolia University, No. 24 Zhao Jun Road Hohhot, Inner Mongolia, People's Republic of China. E-mail address: [email protected] (C.-G. Liang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.theriogenology.2018.02.015 0093-691X/© 2018 Elsevier Inc. All rights reserved.

oocytes and the surrounding granulosa cells to promote integrated cellular functions. Many recent findings suggest that neurotrophins can promote oocyte maturation and early embryo development. For example, treatment with brain-derived neurotrophic factor is important for nuclear and cytoplasmic oocyte maturation in vitro and is essential for the development of preimplantation embryos by increasing cellular proliferation and decreasing apoptosis [5,6]. The neurotrophin nerve growth factor was also found to be presented in porcine oocytes, granulosa cells, and theca cells throughout the estrous cycle and may play important roles during oocyte maturation [7]. Glial cell line-derived neurotrophic factor (GDNF) is another neurotrophin that was shown to promote the survival, growth, and differentiation of neurons, such as dopaminergic neurons in the embryonic midbrain [8,9]. The GDNF family includes three structurally related members (neurturin, artemin, and persephin) and is

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distantly related to the transforming growth factor-b superfamily of proteins [8,10]. Many reports indicate that GDNF is presented not only in glial cells but also in other cells of various organs and tissues. For instance, strong cytoplasmic staining for GDNF was found in intraprostatic nerves and in prostate cancer cells and may be involved in their progression and invasion [11]. Recent studies showed that GDNF produced by peritubular myoid cells and sertoli cells promotes spermatogonial stem cell self-renewal and localization [12,13], effects that are synergized by insulin-like growth factor 1 [14]. The supplementation of GDNF could promote porcine oocyte nuclear and cytoplasmic maturation in vitro and early embryo development [15]. A study in rats showed that GDNF promotes primordial follicle development and mediates the autocrine and paracrine interactions required during folliculogenesis. In sheep, GDNF and kit ligand show profound effects on follicle health, development, and differentiation [16]. Human oocytes incubated with GDNF and other ovarian factors (e.g., brain-derived neurotrophic factor, insulin-like growth factor 1, estradiol, basic fibroblast growth factor, and leptin) in the medium show improved nuclear and cytoplasmic maturation, parthenogenesis, and blastocyst development [17]. An earlier study by our group indicated that treatment with the GDNF family member artemin promotes the development of two-cell embryos into expanded and hatched blastocysts [18]. The GDNF family ligand-receptor complex includes two subunits, namely, the receptor tyrosine kinase RET and GDNF family receptor alpha-1 (GFRA1), which is a ligand-specific subunit [19]. As an extracellular membrane-bound protein, GFRA1 mediates most of the biological effects of GDNF [20]. As a ligand, GDNF first associates with GFRA1 to form the complex, which then binds and activates RET [21,22] to regulate the activation of intracellular signaling pathways involved in cell proliferation and differentiation [23]. However, little is known about the effects and functions of GDNF and GFRA1 in bovine oocyte maturation and embryo development. To address this, we conducted the present study with the aim to improve the efficiency and quality of in vitro-produced bovine embryos. 2. Material and methods 2.1. Ethics statement The cattle were slaughtered in accordance with the animal welfare laws of China. All studies adhered to procedures consistent with the National Research Council's guide for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at Inner Mongolia University. 2.2. Chemicals and reagents The materials used in this study included: GDNF recombinant human protein (Invitrogen, Carlsbad, CA, USA), tissue culture medium-199 ([TCM-199] Invitrogen), BSA (Amresco, Solon, OH, USA), Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) containing 1:2000 (v/v) protease inhibitor cocktail and 1:20 (v/v) bmercaptoethanol (Amresco), rabbit anti-GDNF and anti-GFRA1 antibodies (Bioss, Beijing, China), mouse anti-beta actin antibody (Santa Cruz Biotechnologies, Dallas, TX, USA), rabbit IgG (Abcam, Cambridge, UK), precast gels (Bio-Rad), Tris-buffered saline and PBS (CWBIO, Beijing, China), paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA,USA), peroxidase AffiniPure donkey antirabbit and goat anti-mouse IgGs (H þ L) and fluoresceinconjugated and DyLight 549-conjugated donkey anti-rabbit IgGs (H þ L) (Jackson ImmunoResearch Laboratories, West Grove, PA,

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USA), 40 ,6-diamidino-2-phenylindole ([DAPI] Roche, Mannheim, Germany), Vectashield mounting medium (Vector Labs, Burlingame, CA, USA), and Histostain-SP kits (Invitrogen). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.3. Cumulus-oocyte complex (COC) collection and IVM Bovine ovaries were collected from a local abattoir, transported to laboratory within 2 h in a 0.9% sodium chloride solution, and maintained at 35e37  C. Immature COCs were aspirated with an 18-ga needle from obvious follicles (2e8 mm in diameter) and dispersed in 90-mm cell culture dishes after three washes using prewarmed TCM-199. All oocytes with a uniform ooplasm and cumulus cell mass were collected for IVM. COCs derived from follicles were divided randomly into four-well plates (50 COCs per well) and cultured in oocyte maturation medium (TCM-199 supplemented with 10% fetal calf serum, 0.02 IU/mL FSH, 1 IU/mL LH, and 0.1 mg/mL estradiol-17b) in the absence or presence of GDNF. COCs were incubated at 38.5  C in 5% CO2 for 22e24 h. 2.4. Assessment of oocyte nuclear maturation The nuclear maturation of IVM oocytes to the metaphase II (MII) stage was evaluated on the basis of the presence of the first polar body (PB1) extrusion. After 22e24 h of culture, cumulus cells were removed from oocytes by hyaluronidase digestion (0.3 mg/mL). PB1s were identified under an inverted microscope (IX71; Olympus, Japan). The oocytes at the MII stage were counted, and the percent maturation was calculated on the basis of the number of total oocytes used for maturation in each group. 2.5. IVF and IVC of embryos After maturation, COCs were rinsed three times in fertilization medium [24] (supplemented with 5 mg/mL BSA). Approximately 30 COCs were transferred to a 100-mL fertilization droplet covered with mineral oil. Frozen semen (Sai Ke Xing Company; Inner Mongolia, China) was thawed in 37  C water for 10 s. Spermatozoa were rinsed twice in fertilization medium and centrifuged for 5 min at  452 g. Spermatozoa were collected via the swim-up method to yield a final concentration of 106/mL. Matured COCs were incubated with the spermatozoa for 6 h at 38.5  C in 5% CO2. The day of IVF was considered day 0. The procedure for zygote IVC was performed as described previously [25]. Briefly, presumptive zygotes were transferred to culture plates and placed in drops containing 100 mL culture medium (25 presumptive zygotes per drop) covered with mineral oil. The culture medium was synthetic oviduct fluid medium supplemented with 6 mg/mL BSA, 0.5 mg/mL inositol, 30 mL/mL essential amino acids, 10 mL/mL nonessential amino acids, and 29.2 mg/mL glutamine. Embryos were cultured at 38.5  C in an atmosphere composed of 7% O2, 5% CO2, and 88% N2 with saturated humidity, as previously reported [26]. Embryos of two-cell, four-cell, eight-cell, morula, and blastocyst stages were evaluated on day 1, day 2, day 3, day 5, and day 7, respectively. The cleavage rate of two-cell embryos was calculated as the total number of two-cell embryos/the number of all COCs; from the four-cell stage to the blastocyst-stage, the total number of two-cell embryos was counted as the denominator for calculating the embryo development rate. 2.6. Assessment of stages of oocyte maturation and embryo development The stages of oocyte maturation and early embryo development were determined by their phenotypes as described in previous

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studies [27e29]. Oocytes were fixed in PBS with 4% paraformaldehyde for 1 h at room temperature and then transferred to PBS containing 1% Triton X-100 for 2 h at 37  C and rinsed three times in PBS (containing 1:1000 [v/v] Tween 20 and 1:10,000 [v/v] Triton X-100) for 5 min on a shaker. DNA was stained with DAPI (5 mg/mL). Finally, samples were mounted on glass slides with mounting medium and examined under a fluorescence microscope (Observer A1; Zeiss, Germany). Oocytes with intact nuclear envelopes were considered to be at the germinal vesicle (GV) stage, whereas those without were considered as GV breakdown. Oocyte chromosomes with a metaphase plate or punctate status and a polar body were classified as MII. After fertilization, embryos with two blastomeres, four blastomeres, and eight blastomeres were considered two-cell, four-cell, and eight-cell embryos, respectively. Embryos with numerous blastomeres compacted in solid balls were considered morulae. Embryos with fluid-filled cavities as well as two layers, inner cell masses, and trophoblastic cells were considered blastocysts. 2.7. Immunohistochemistry Bovine ovary immunohistochemistry was performed using the streptavidin-biotin immunoperoxidase method [30]. Briefly, ovaries were fixed in 4% paraformaldehyde for 24 h, followed by serial dehydration processing for paraffin embedding. Embedded tissues were cut and mounted on poly-L-lysine-coated slides. After deparaffinization and rehydration, the sections were subjected to antigen retrieval with microwave heating in 10 mM citrate buffer (pH 6.0) for 15 min. Slides were treated with 3% (v/v) hydrogen peroxidase-methanol for 20 min to quench endogenous peroxidase activities, and then nonspecific binding was blocked with 10% (v/v) normal goat serum in humidified chambers for 10 min at room temperature. Sections were incubated with a polyclonal anti-GDNF (dilution, 1:100) or anti-GFRA1 (dilution, 1:100) antibody overnight at 4  C. After washing three times in PBS for 5 min, visualization was performed with the LAB-SA detection system (Invitrogen). Parallel negative control sections were incubated with nonimmune rabbit IgG instead of the primary antibody. Sections were imaged under a microscope (Eclipse Ci; Nikon, Japan).

with b-mercaptoethanol and a protease inhibitor cocktail. All other Western blotting procedures were conducted as previously reported [32]. Briefly, proteins were separated on 4e15% precast gels and electrotransferred to nitrocellulose membranes. The membranes were blocked in 5% skim milk diluted in Tris-buffered saline containing 0.1% Tween 20 for 1 h to eliminate nonspecific binding. We used rabbit anti-GFRA1 (dilution, 1:1000) and mouse anti-b actin (dilution, 1:200) as primary antibodies. Accordingly, peroxidase AffiniPure donkey anti-rabbit (dilution, 1:15,000) and goat anti-mouse (dilution, 1:2000) IgGs (H þ L) were used as the secondary antibodies. Band intensities were calculated using densitometry in Quantity One software (Bio-Rad). 2.10. Morpholino (MO) injections For GFRA1 knockdown in bovine oocytes, we utilized a Gfra1 MO (50 -CGAAGTACAGGGTCGCCAGGAACAT-30 ) or control MO (50 CGTACTACAGGTCCCCAGAACAT-30 ) (Gene Tools, Philomath, OR, USA). Oocytes at the GV stage received microinjections (10 pL) of 2 mM Gfra1 or control MOs. The oocytes were then moved to fresh operating solution (TCM-199 containing 2.5 mM milrinone) for 5 min and then incubated for 20 h in maturation medium also containing 2.5 mM milrinone to allow for the elimination of the endogenous target mRNA (i.e., Gfra1) [33]. The oocytes were then washed three times in maturation medium without milrinone and cultured for another 22e24 h to facilitate the resumption of meiosis and oocyte maturation. 2.11. Statistical analysis Each experiment was repeated three times. The differences between groups were evaluated with Duncan's multiple comparison tests in SPSS 17.0 (IBM Corp., Armonk, NY, USA). Averages, standard deviations, and statistical significances (two-tailed Student's t tests) were calculated using Microsoft Excel software. Results were considered significant at P values of <0.05. Quantified data show the means ± standard deviations from all repeats. 3. Results

2.8. Immunofluorescence and confocal microscopy

3.1. Presence of GDNF and GFRA1 in bovine ovarian follicles

All stages of oocytes or embryos were fixed in PBS with 4% paraformaldehyde for 1 h at room temperature. The samples were then transferred to PBS containing 1% Triton X-100 for 2 h at 37  C, rinsed three times in PBS (containing 1:1000 [v/v] Tween 20 and 1:10,000 [v/v] Triton X-100) for 10 min, and blocked with 2% BSA in PBS for 1 h at room temperature. The samples were then incubated with primary (1:100 dilution of rabbit anti-GDNF or anti-GFRA1) antibodies at 4  C overnight, followed by incubations with secondary antibodies (fluorescein- or DyLight 549-conjugated donkey anti-rabbit IgG [H þ L], respectively). DNA was stained with DAPI (5 mg/mL). For the negative control, nonimmune rabbit IgG was substituted for the primary antibody. Finally, samples were mounted on glass slides with mounting medium and examined with a confocal laser scanning microscope (Nikon A1R). Images of optical sections were captured at 2-mm intervals and analyzed with the NIS-Element AR 3.0 software (Nikon). The fluorescence intensities for GDNF and GFRA1 were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA) as previously reported [31].

The localization of GDNF and GFRA1 in the bovine ovarian follicles was determined by immunohistochemistry. During folliculogenesis, GDNF was always cytoplasmic in oocytes in the primordial follicle, primary follicle, secondary follicle, and Graafian follicle (Fig. 1A, yellow solid arrowheads). In addition, staining was also detected in the cumulus cells of secondary and Graafian follicles (yellow open arrowheads) and in granulosa cells (green solid arrowhead) and theca cells (black solid arrowhead) of Graafian follicles. Similarly, GFRA1 staining was observed in primordial, primary, secondary, and Graafian follicles and was detected in the cytoplasm and nuclei of oocytes (yellow solid arrowheads) (Fig. 1B). GFRA1 was also found in cumulus cells of secondary and Graafian follicles (yellow open arrowheads) and granulosa cells (green solid arrowhead) and theca cells (black solid arrowhead) of Graafian follicles. No GDNF or GFRA1 staining was observed in the negative controls (Fig. 1C). 3.2. Localization of GDNF in bovine oocytes and during early embryo development

2.9. Western blotting A total of 50 oocytes were collected in Laemmli sample buffer

Immunofluorescence staining revealed the localization of GDNF during oocyte maturation and early embryo development

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Fig. 1. Presence of GDNF and GFRA1 in ovarian follicles. (A) GDNF (brown) was presented in oocyte cytoplasm (yellow solid arrowheads), cumulus cells (yellow open arrowheads), granulosa cells of follicles (green solid arrowhead), and theca cells (black solid arrowhead). (B) GFRA1 (brown) was presented in oocyte cytoplasm and nuclei (yellow solid arrowheads), cumulus cells (yellow open arrowheads), granulose cells of follicles (green solid arrowhead), and theca cells (black solid arrowhead). (C) Sections incubated with nonimmune IgG served as the negative control. Nuclei were stained with hematoxylin (blue). Black frames indicate areas shown in detail (800  ). Scale bar ¼ 100 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

(Fig. 2A). At GV and MII stages, GDNF was distributed throughout the cytoplasm. After fertilization, GDNF was detected in the cytoplasm of each blastomere. No positive staining was detected in oocytes or embryos in the negative control (Fig. 2B). A relative quantitation of immunofluorescence intensity showed that GDNF was highly presented in GV, MII, two-cell, and four-cell stages. However, the presence dramatically decreased at the eight-cell stage compared with that at all other stages (P < 0.01) (Fig. 2C). When developed to the morula and blastocyst stages, GDNF presence was restored. In blastocysts, the intensity of immunofluorescence was 3efold higher in the cytoplasm than in the nucleus (P < 0.01) (Fig. 2D).

3.3. Localization of GFRA1 in bovine oocytes and during early embryo development The localization of GFRA1 was also assessed during oocyte maturation and early embryo development, where it was detected in both the cytoplasm and cytomembranes from GV to blastocyst stages (Fig. 3A). No positive staining was observed in the negative controls (Fig. 3B). A relative quantitation of immunofluorescence intensity showed that GFRA1 was also highly presented in GV, MII, two-cell, and four-cell stages, with a corresponding decrease at the eight-cell stage (Fig. 3C). Higher presence levels were found in morula- and blastocyst-stage embryos (P < 0.01). In blastocysts, the

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Fig. 2. Localization of GDNF during bovine oocyte maturation and early embryo development. (A) GDNF (green) was distributed throughout the cytoplasm of GV oocytes and blastocyst-stage embryos. White open arrowhead, nuclei of the trophectoderm cells; white solid arrowhead, nuclei of the inner cell mass cells. DNA was stained with DAPI (blue). Scale bar ¼ 30 mm. (B) Sections incubated with nonimmune IgG served as the negative control. (C) Relative densitometry of GDNF from GV to blastocyst stages. The immunofluorescence staining level in GV oocytes was set as 1.00 for standardization. (D) Relative GDNF densitometry in the cytoplasm of blastomeres relative to that in nuclei of blastocysts (set as 1.00 for standardization). Quantified data are the means ± standard deviations. Different letters on the bars indicate statistical difference (P < 0.01) by Student's t tests. a.u., arbitrary units. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

intensity of immunofluorescence was 2efold higher in the cytomembrane than in the cytoplasm (P < 0.01) (Fig. 3D). 3.4. Facilitation by GDNF of oocyte maturation and early embryo development The potential for GDNF to act as a paracrine factor during in vitro oocyte maturation and embryo development was investigated. First, we determined the optimal concentration of GDNF for facilitating COCs. Groups of COCs derived from bovine ovarian follicles were cultured in oocyte maturation medium in the presence or

absence of 1, 5, 15, 50, or 150 ng/mL recombinant human GDNF. Supplementation of GDNF at the dose of 15 or 50 ng/mL improved the rate of PB1 extrusion, and 50 ng/mL GDNF produced the best nuclear maturation result (50 ng/mL vs 15 ng/mL, P < 0.05; 50 ng/ mL vs control, 1 ng/mL, and 5 ng/mL, P < 0.01) (Fig. 4A). Interestingly, when a higher concentration of GDNF (150 ng/mL) was added to the culture medium, the PB1 extrusion rate was reduced to the level of the control, indicating a possible negative feedback loop at high concentrations (Fig. 4A). Treatment with 50 ng/mL GDNF increased PB1 extrusion in denuded oocytes (DOs) (P < 0.05) (Fig. 4B). However, the rate of PB1 extrusion in the presence of

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Fig. 3. Localization of GFRA1 during bovine oocyte maturation and early embryo development. (A) GFRA1 (red) was mainly distributed in the cytomembranes but also in the cytoplasm of GV oocytes and blastocyst-stage embryos. DNA was stained with DAPI (blue). Scale bar ¼ 30 mm. (B) Sections incubated with nonimmune IgG served as the negative control. (C) Relative densitometry of GFRA1 from GV to blastocyst stages. The immunofluorescence staining level of GV oocytes was set as 1.00 for standardization. (D) Relative GFAR1 densitometry in the cytoplasm of blastomeres (set as 1.00 for standardization) relative to that in cytomembranes of blastocysts. Quantified data are the means ± standard deviations. Different letters on the bars indicate statistical difference (P < 0.01) by Student's t tests. a.u., arbitrary units. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

GDNF was higher in COCs than in DOs (P < 0.05) (Fig. 4C). We next investigated whether GDNF could facilitate early embryo development. The results showed that supplementation with 50 ng/mL GDNF facilitated the development of embryos at two-cell, four-cell, eight-cell, morula, and blastocyst stages compared with that of the untreated control group (all Ps < 0.05) (Fig. 4D).

3.5. Depletion of GFRA1 at GV stage causes bovine oocyte maturation failure To explore the possible mechanism by which GDNF increases the maturation rate of bovine oocytes, we inhibited Gfra1 expression with a specific antisense MO. Western blotting (Fig. 5A) and densitometry (Fig. 5B) showed that GFRA1 protein presence was

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Fig. 4. GDNF supplementation promotes bovine oocyte maturation and early embryo development. (A) Percentages of cumulus-oocyte complexes (COCs) with first polar body (PB1) extrusions. The COCs were cultured with or without different doses of recombinant human GDNF. (B) Percentages of denuded oocytes (DOs) with PB1 extrusions when cultured with or without 50 ng/mL GDNF. (C) Percentages of COCs and DOs with PB1 extrusions when cultured with 50 ng/mL GDNF. (D) Percentages of embryo development when cultured without (Ctrl) or with 50 ng/mL GDNF. Total numbers of oocytes counted (n) are indicated in the graphs. Quantified data are the means ± standard deviations. Different letters on the bars indicate statistical difference (P < 0.05) by Student's t tests.

markedly reduced in oocytes treated with Gfra1 MOs compared with that in cells receiving the control MO (P < 0.05), illustrating the efficiency of GFRA1 depletion via MO injection. Immunofluorescence staining shown that less GFRA1 (red) was detected in the group treated with Gfra1 MOs (Fig. 5C), which further confirmed the efficiency of the Gfra1 MO. Interestingly, oocytes with GFRA1 depletion could not mature, and most were arrested at anaphase/ telophase I, even when treated with supplemental GDNF. Indeed, the stimulatory effect of GDNF on oocyte maturation was blocked by GFRA1 depletion compared with that in cells receiving the control MO (P < 0.05) (Fig. 5D). 4. Discussion The results from previous studies in various mammals indicate that GDNF has potential paracrine functions, such as promoting primordial follicle development [16,34,35], the completion of meiosis I [15], and the development of the early embryo [36]. Although GDNF supplementation, along with other ovarian factors, was shown to promote the development of blastocysts from human cumulus-free oocytes [17], this effect had not been investigated in bovine ovaries. Here, we report for the first time that GDNF promotes bovine oocyte maturation at the end of the folliculogenesis process and also enhances the developmental competence of early embryos via GFRA1. The oocyte maturation is obtained at the end of the folliculogenesis process. For this competence, there is a series of events, beginning at the primordial follicle stage upon reactivation of prophase I and continuing to the antral follicle stage, where it is arrested again in MII. These represent two major stages of

oogenesis, one stage that is independent of gonadotropins and the other in which the gonadotropins FSH and LH stimulate oocyte maturation. The immunohistochemical analyses in this study indicate that the localization of GDNF is independent of gonadotropins, which contrasts with results from our previous study in mice showing that GDNF was not present in oocytes of preovulatory follicles and small antral follicles [34]. However, the observation in the present study is similar to that from a previous report in which GDNF was presented in rat follicles from primordial to antral stages [37]. Thus, the discrepancy may be explained by the difference of species. The localization of GFRA1 during oogenesis varies among species. For example, we and others have shown that Gfra1 transcript levels remain stable in oocytes isolated at different stages of follicular development [15,34]. Conversely, a study in rats found that GFRA1 localized to primordial and primary follicles but weakly presented in oocytes of the antral follicle [37]. In the present study, we observed constitutive presence of GFRA1 in the cytoplasm and nuclei of oocytes during all stages of follicular development, independent of gonadotropins. This is the first report of GFRA1 presenting in the oocyte nuclei of follicles and the similar localization was also observed in GV and in vitro matured MII stage oocytes (Fig. 3A). However, this special nucleic localization disappeared after two-cell stage. We proposed that this shift in GFRA1 localization is related to its different functions in oocyte and embryo. Genes that are directly activated by the maternal contribution can be seen as a “first wave” of transcription, compared to “subsequent wave” genes that depend on factors encoded by the zygotic genome for their expression [38]. During oocyte growth, the genome is

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Fig. 5. Depletion of GFRA1 via treatment with MOs impaired bovine oocyte maturation. Western blotting (A) and densitometry (B) of GFRA1 presence after GV stage oocytes were treated with control or Gfra1 MOs. b-Actin was used as a loading control. Experiments were performed in triplicates. One representative experiment is shown. (C) Confocal images of oocytes treated with MOs at the GV stage and then cultured with 0 or 50 ng/mL GDNF for an additional 22 h without milrinone. Red, GFRA1; blue, DNA. Scale bar ¼ 30 mm. (D) Percentages of oocytes with PB1 extrusion after injections with MOs at GV stage and then cultured with 0 or 50 ng/mL GDNF for an additional 22 h without milrinone. Total numbers of oocytes counted (n) are indicated in the graphs. Quantified data are the means ± standard deviations. Different letters on the bars indicate statistical difference (P < 0.05) by Student's t tests. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

actively transcribed. Some transcripts are readily translated into proteins [39]. Our study showed that GFRA1 was abundant in oocyte nucleus during oogenesis, in which maternal-effect genes are transcribed, suggesting that GFRA1 may act not only as a GDNF receptor but also as a transcription factor for the oocyte genome transcription in bovine oocyte. However, maternal transcripts involved in meiotic processes, but not embryonic development, are rapidly degraded upon fertilization [40]. For the zygote to acquire totipotency, the parental genomes must be undergo extensive epigenetic reprogramming, which involves global DNA demethylation and the cell cycle machinery must switch from meiotic to mitotic chromosome segregation [41,42]. Upon these modifications, GFRA1 may not act as a transcription factor any more in embryo. Another possibility is that GFRA1 plays roles as an oocytespecific transcription factor like others such as Obox1, Obox5 and Obox6 which only expressed in the ovary and are abundantly present in unfertilized oocytes [43,44]. In mouse spermatogonial stem cells, GFRA1 localizes to type A spermatogonia and it is coexpressed with transcription factor Pou5f1 (Oct4) [45]. However, further studies are needed to confirm the potential functions of GFRA1. Another interesting phenomena in our study is that during folliculogenesis, GDNF and GFRA1 were presence only in oocytes of primordial and primary follicles, after secondary follicles both of them were presence in follicular cells. This means that oocyte begins to synthesize GDNF and GFRA1 before the activation of meiosis, and the process of secretion become when the oocyte is going to mature. In female mammals, the primordial follicles remain quiescent until they are recruited into the primary stage. There are various autocrine/paracrine factors that control the ovarian follicle activation [46e48]. In humans, GDNF was reported to be involved in regulating primordial follicular activation [49]. In

sheep, both GDNF and kit ligand significantly improved the activation of primordial follicles and had profound effects on follicle health, development, and differentiation [16]. Supplementation of rat ovaries cultured in vitro with exogenous GDNF stimulated the primordial-to-primary follicle transition, increased the percentage of developing follicles, and mediated autocrine/paracrine cellular interactions during folliculogenesis [37]. In addition, GDNF enhanced the developmental competence of oocytes from antral follicles in pigs [15]. Thus, we believe that large amount of GDNF and GFRA1 is synthesized in oocytes for primordial to primary follicles transition through autocrine pathway, then secreted to the surrounding follicular cells through both autocrine and paracrine pathways in developing antral follicles. And this result is consist with the previous study in rat [37]. Another possibility is that after the stage of secondary follicle, follicular cells proliferation and maturation initiates the synthesis of GDNF and GFRA1. Presence of GDNF and GFRA1 in follicular cells in turn promotes the communication between the oocytes and the surrounding somatic cells. And this communication is involved in the control of ovarian follicle activation and growth, which is similar to the findings in human that GDNF and BDNF were synthesized and secreted by the granulosa cells for promoting oocyte maturation [50]. The localization and relative presence of GDNF and GFRA1 during bovine oocyte maturation and early embryo development were determined in this study by immunohistochemistry. Whereas GDNF was found in the cytoplasm of oocytes and blastomeres, GFRA1 was found mainly in cytomembranes, suggesting that it acts as a membrane receptor in an autocrine/paracrine manner. Our results support those from previous studies showing that GFRA1 is a cell surface receptor for GDNF in a motoneuron cell line [51] and that the GDNF receptor is localized in the plasma membranes of early human embryos [52]. Moreover, GFRA1 was also detected in

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the cytoplasm of oocytes and blastomeres, and this finding further suggests that GFRA1 may have functions in bovine oocytes and early embryos other than acting as a membrane receptor. Oocyte growth is accompanied by the accumulation of maturation-promoting proteins, factors, and stored maternal mRNAs necessary for successful fertilization and early embryo development [53e55]. It is noteworthy that GDNF is presented before the eight-cell embryo stage, during which zygote genome activation occurs in bovines [56], suggesting it is essential for early embryo development. In addition, the abundance of GDNF in follicles during oocyte growth and maturation indicates that maternal GDNF accumulates to sustain these processes and the maternal-tozygotic transition. Similarly, maternal GFRA1 accumulates in follicles, oocytes, and early embryos before zygote genome activation, suggesting there is cooperation between GDNF and GFRA1. After the eight-cell stage, additional GDNF and GFRA1 are produced during zygote genome activation for subsequent embryo development. The developmental potential of oocytes matured in vitro is much lower than those in vivo due in part to insufficient nuclear and cytoplasmic maturation. The supplementation of mammalian oocyte culture medium with autocrine/paracrine factors is an efficient approach for improving in vitro oocyte maturation and early embryo development [57e60]. Our earlier study in mice found that GDNF supplementation in COC IVM medium promoted PB1 extrusion but had no effect on cytoplasmic maturation nor on the development of two-cell stage embryos to the morula stage [34]. However, other studies have shown that supplemental GDNF increases the levels of the protein cyclin B1, an indicator of oocyte cytoplasmic maturation, and improves oocyte competence for supporting blastocyst formation and hatching in pigs [15,35,36]. Our research on bovine oocytes clearly shows that treatment with GDNF significantly promotes PB1 extrusion, suggesting that GDNF supplementation is beneficial for nuclear maturation. As maternal mRNA and proteins important for early embryo development accumulate during oocyte maturation until the activation of the zygotic genome, we evaluated GDNF supplementation during these stages. We found that exogenous GDNF improved early embryo development beginning from the two-cell stage and continuing throughout the blastocyst stages, which is consistent with results from a study in pigs [15]. Thus, the application of GDNF to culture medium is different between rodents and domestic animals and further confirms that GDNF not only provides benefits for oocyte nuclear maturation but also for cytoplasmic maturation in bovines. The interactions between oocytes and the surrounding cumulus cells play key roles for producing and transmitting paracrine factors. To determine if cumulus cells participate in the utilization of GDNF during oocyte maturation, we employed a culture model with COCs and DOs. The culture of bovine DOs in the presence of GDNF significantly facilitated PB1 extrusion, demonstrating that oocytes themselves have the ability to utilize GDNF in vitro. This result was consistent with the results from a previous study showing that human DOs are capable of increasing nuclear maturation and blastocyst formation when cultured with ovarian factors [17]. The PB1 extrusion rate of COCs was higher than that in DOs when cultured with GDNF. Immunohistochemical staining of follicles showed a large amount of GDNF in surrounding cumulus cells, indicating that GDNF also plays a role in these cells. Thus, the stimulatory effect of GDNF on bovine oocyte maturation is exerted not only directly on oocytes but also indirectly on surrounding cumulus cells. It is well established that the binding of GDNF to GFRA1 results in the activation of the RET tyrosine kinase coreceptor [20,61e63]. Our previous report found an autocrine/paracrine role of the artemin-GFRA3 signaling system in regulating early embryonic

development. Here, by inhibiting GFRA1 translation with a specific Gfra1 MO, we show that blocking GFRA1 significantly impairs PB1 extrusion, an effect that is not reversed by the addition of exogenous GDNF. This finding confirms that the GDNF effects observed depend on GFRA1. Similar results were obtained when GFRA1 was blocked via antibody treatments to reduce porcine oocyte nuclear maturation and embryo development [15]. Thus, GFRA1 depletion leads to an impairment of GDNF function, which in turn impairs the signal transduction for oocyte nuclear maturation. In conclusion, we propose that GDNF combines with GFRA1 to form a paracrine ligand-receptor complex that functions in oocyte maturation and early embryo development in bovines. Thus, GDNF supplementation may be valuable for improving the efficiency of in vitro bovine oocyte maturation and IVF. Funding This work was supported by the NSF of China (31671560 and 31371454), the Natural Science Foundation of Inner Mongolia (2015JQ02), and an open project of the Collaborative Innovation Center of Grassland Ecological Animal Husbandry and the State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestock. Declarations of interest None. Author contributions Conceived of and designed the experiments: C.G.L., D.H.W. Performed the experiments: D.H.W., H.X.Z, S.J.L., C.J.Z, X.W.K., Z.H. Analyzed the data: D.H.W., C.G.L. Contributed to writing the manuscript: D.H.W., C.G.L. References [1] Bauersachs S, Ulbrich SE, Zakhartchenko V, Minten M, Reichenbach M, Reichenbach HD, et al. The endometrium responds differently to cloned versus fertilized embryos. Proc Natl Acad Sci USA 2009;106:5681e6. [2] Farin PW, Piedrahita JA, Farin CE. Errors in development of fetuses and placentas from in vitro-produced bovine embryos. Theriogenology 2006;65: 178e91. [3] Su JM, Yang B, Wang YS, Li YY, Xiong XR, Wang LJ, et al. Expression and methylation status of imprinted genes in placentas of deceased and live cloned transgenic calves. Theriogenology 2011;75:1346e59. [4] Yang X, Smith SL, Tian XC, Lewin HA, Renard JP, Wakayama T. Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning. Nat Genet 2007;39:295e302. [5] Kawamura K, Kawamura N, Mulders SM, Sollewijn Gelpke MD, Hsueh AJ. Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos. Proc Natl Acad Sci USA 2005;102: 9206e11. [6] Kawamura K, Kawamura N, Fukuda J, Kumagai J, Hsueh AJ, Tanaka T. Regulation of preimplantation embryo development by brain-derived neurotrophic factor. Dev Biol 2007;311:147e58. [7] Jana B, Koszykowska M, Czarzasta J. Expression of nerve growth factor and its receptors, TrkA and p75, in porcine ovaries. J Reprod Dev 2011;57:468e74. [8] Lin LF, Doherty DH, Lile JD, Bektesh S, Collins F. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 1993;260: 1130e2. [9] Tomac A, Lindqvist E, Lin LF, Ogren SO, Young D, Hoffer BJ, et al. Protection and repair of the nigrostriatal dopaminergic system by GDNF in vivo. Nature 1995;373:335e9. [10] Airaksinen MS, Saarma M. The GDNF family: signalling, biological functions and therapeutic value. Nat Rev Neurosci 2002;3:383e94. [11] Baspinar S, Bircan S, Ciris M, Karahan N, Bozkurt KK. Expression of NGF, GDNF and MMP-9 in prostate carcinoma. Pathol Res Pract 2017;213:483e9. [12] Caires KC, de Avila J, McLean DJ. Endocrine regulation of spermatogonial stem cells in the seminiferous epithelium of adult mice. BioResearch Open Access 2012;1:222e30. [13] Oatley MJ, Racicot KE, Oatley JM. Sertoli cells dictate spermatogonial stem cell niches in the mouse testis. Biol Reprod 2011;84:639e45.

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