Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte–cumulus cell complexes in vitro

Abstract

During oocyte growth and follicle development, oocytes closely communicate with cumulus cells. We examined the effects of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the growth and acquisition of meiotic competence of porcine oocytes collected from early antral follicles (1.2–1.5 mm). First, we confirmed that GDF9 and BMP15 mRNAs were expressed almost exclusively in the oocytes. Oocyte–cumulus cell complexes (OCCs) collected from early antral follicles were cultured in growth medium supplemented with 0–100 ng/ml of GDF9 or BMP15 for 5 days. GDF9 dose-dependently increased the OCC diameter, while BMP15 did not. GDF9 and BMP15 had no significant effects on oocyte growth (P > 0.05). When OCCs that had been cultured with 50 and 100 ng/ml BMP15 were subjected to a subsequent maturation culture, they expanded fully by gonadotropic stimulation and 49% and 61% of oocytes matured to metaphase II (MII), respectively. In contrast, GDF9 did not promote cumulus expansion, and < 10% of oocytes matured to MII. Based on the difference in cumulus expansion, we compared the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and follicle stimulating hormone receptor (FSHR) mRNAs in cumulus cells. The level of LHCGR mRNA was increased in cumulus cells of the BMP15 group, although there were no significant differences in FSHR mRNA levels among the groups. These results suggest that GDF9 promotes the growth of OCCs and that BMP15 promotes LHCGR mRNA expression in cumulus cells during oocyte growth culture, which may contribute to cumulus expansion and oocyte maturation.

The basic functional unit of the ovary is the ovarian follicle. This follicle consists of an oocyte surrounded by cumulus cells, granulosa cells, basal lamina, and theca cells. Oocytes grow in follicles and acquire nuclear and cytoplasmic competence for meiotic maturation and fertilization. After gonadotropic surge, the cumulus expands and fully grown oocytes resume meiosis and mature to the second metaphase in antral follicles. It has been suggested that oocytes are the central regulators of follicle function and play critical roles in follicle development, ovulation, and thus female fertility [1]. Conversely, oocyte growth is strongly dependent on the surrounding somatic cells, which supply essential metabolic substrates and regulatory molecules [2].

Oocyte-secreted factors may form the basis of a complex bidirectional communication network between the oocyte and its surrounding somatic cells [1, 3, 4]. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor-beta (TGF-β) family and have been identified as oocyte-secreted factors [1]. In GDF9-null mice, proliferation of granulosa cells is retarded in the follicles, and folliculogenesis does not progress beyond the primary follicle stage [5]. BMP15-null female mice exhibit subfertility [6], ewes carrying BMP15 mutations are infertile, and their follicular development is arrested at the primordial stage [7]. BMP15-knockdown gilts show abnormal ovarian follicular development and ovulation [8].

In culture experiments of oocytes with cumulus/granulosa cells, oocyte-secreted factors have been suggested to target these cells to regulate a broad range of functions, such as proliferation [9], glycolysis [10], luteinization [11], and cumulus expansion in mice [2]. The role of oocytes in cumulus expansion has been examined by physically removing oocytes from oocyte–cumulus/granulosa cell complexes [2, 12, 13], and the oocyte-derived cumulus expansion has been attributed to GDF9 [14].

The direct effects of exogenous GDF9 and BMP15 have been examined using cultured granulosa cells. GDF9 reportedly promoted the proliferation of mouse granulosa cells [15] and suppressed progesterone and estradiol production in rat granulosa cells [16] and apoptosis of rat cumulus cells [17]. Similarly, BMP15 promoted the proliferation of rat granulosa cells [18] and inhibited progesterone production in rat granulosa cells [18], in addition to inhibiting apoptosis of bovine cumulus cells [19]. GDF9 was reported to promote follicle development in organ-cultured rat ovaries [20] and cultured human ovarian cortical tissues [21]. Recently, we reported that GDF9 and BMP15 promoted the formation of antrum-like structures in bovine oocyte–granulosa cell complexes [22]. Studies in pigs have demonstrated that GDF9 promotes proliferation of mural granulosa cells [23] and GDF9 and BMP15 prevent cumulus cell apoptosis [24, 25].

In the final stage of oocyte growth in porcine antral follicles, oocytes become able to mature [26] and cumuli become able to expand [27]. Although GDF9 and BMP15 are thought to participate in the bidirectional communication network between oocytes and surrounding cumulus cells at this stage, the effects of exogenous GDF9 and BMP15 on the growth and acquisition of meiotic competence of oocytes have not been studied in pigs. In this study, we dissected early antral follicles (1.2–1.5 mm in diameter) containing growing oocytes from porcine ovaries. We confirmed the expression of GDF9 and BMP15 mRNAs in oocytes. Next, we cultured oocyte–cumulus cell complexes (OCCs) collected from the early antral follicles over 5 days to examine the effects of GDF9 and BMP15 on OCC and oocyte growth. The cultured OCCs were stimulated by gonadotropin in GDF9- and BMP15-free media to induce oocyte maturation and the maturational competence of the in vitro grown oocytes was compared. The results suggested that the presence of BMP15 during the oocyte growth culture period promoted cumulus expansion and oocyte maturation. Thus, we considered that the cumulus cells in OCCs cultured with BMP15 became more sensitive to gonadotropin. To investigate this hypothesis, we further compared the expression of the mRNAs of luteinizing hormone/choriogonadotropin receptor (LHCGR) and follicle stimulating hormone receptor (FSHR) in cumulus cells from cultured OCCs.

Materials and Methods

Chemicals

All chemicals were purchased from MilliporeSigma (St. Louis, MO, USA) unless otherwise noted.

Collection of OCCs

Porcine ovaries were collected at a local slaughterhouse and transported to the laboratory at room temperature (20–25°C). The ovaries were washed once with 0.2% (w/v) cetyltrimethylammonium bromide (FUJIFILM Wako Pure Chemical, Osaka, Japan) and three times with Dulbecco’s phosphate buffered saline (PBS) containing 0.1% (w/v) polyvinyl alcohol (PBS-PVA). The ovaries were sliced with a surgical blade (No. 21; Keisei Medical Industrial, Niigata, Japan), and the slices were pooled in 25 mM HEPES-buffered medium 199 (HEPES-199; Nissui Pharmaceutical, Tokyo, Japan) containing 0.1% (w/v) PVA, 0.85 mg/ml sodium bicarbonate, and 0.08 mg/ml kanamycin sulfate. Early antral follicles (1.2–1.5 mm in diameter) were collected from the ovarian slices and pooled in HEPES-199. The follicles were opened using a surgical blade (No. 21) and OCCs containing growing oocytes were collected. OCCs containing oocytes that were partly or completely denuded and whose ooplasms were not homogenous were excluded from the experiment.

As an in vivo control, OCCs containing fully grown oocytes were collected from antral follicles (4.0–6.0 mm in diameter). The follicles were dissected using surgical blades (No. 11; Feather Safety Razor, Osaka, Japan) and opened to collect OCCs. OCCs with oocytes that were partly or completely denuded and whose ooplasms were not homogenous were excluded.

RNA extraction and cDNA synthesis

After washing in PBS-PVA, cumulus cells were removed from a group of 30 OCCs using a fine pipette to collect denuded oocytes. The 30 denuded oocytes, along with a group of 30 OCCs, were each transferred into a 1.5 ml microtube with a minimum volume of PBS-PVA and stored at –80°C until use. The cumulus cells collected from the 30 OCCs and mural granulosa cells derived from 30 follicles were each transferred into a 1.5 ml microtube with PBS-PVA. The microtubes were centrifuged at 10,000 rpm (9,100 × g) for 5 min. After discarding the supernatant, the cumulus cells and mural granulosa cells were washed three times in 300 µl PBS-PVA by centrifuging for 5 min each time. After washing, they were stored at –80°C with a minimum volume of PBS-PVA until use.

Total RNA was extracted from the collected OCCs, oocytes, cumulus cells, and mural granulosa cells using an RNeasy Plus Micro Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcription was performed using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions.

Reverse transcription PCR (RT-PCR) and real-time quantitative PCR (qPCR)

RT-PCR was performed using PCR Master Mix (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Primers for GDF9, BMP15, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) for internal control were designed according to known sequences in GenBank (accession numbers: porcine GDF9, NM_001001909; BMP15, NM_001005155; and GAPDH, NM_001206359) and were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Primer sequences are shown in Supplementary Table 1. The cycling conditions for the amplification were 95°C for 2 min, followed by 30 cycles of 95°C for 30 sec, 60°C for 1 min, and 72°C for 1 min, followed by 72°C for 5 min with a final hold at 4°C. RT-PCR products were electrophoresed in 2% (w/v) agarose gels containing ethidium bromide at 120 V and 180 mA for 30 min and visualized by exposure to ultraviolet (UV) light.

A QIAquick gel extraction kit (QIAGEN) was used to purify RT-PCR products from the agarose gels according to the manufacturer’s instructions. Sequencing of gel-purified products was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and ABI 3130 Genetic Analyzer (Applied Biosystems). The nucleotide sequences of the RT-PCR products were checked against the porcine cDNA sequences of GDF9, BMP15, and GAPDH.

The qPCR reactions were performed using the THUNDERBIRD SYBR qPCR Mix (TOYOBO) according to the manufacturer’s instructions. Primers for GDF9, BMP15, LHCGR, FSHR, GAPDH, and actin beta (ACTB) were designed according to known sequences in GenBank (accession numbers: porcine GDF9, NM_001001909; BMP15, NM_001005155; FSHR, XM_021085881.1; GAPDH, NM_001206359; and ACTB, XM_003124280) or a previous report (LHCGR, [28]). They were purchased from Thermo Fisher Scientific (Supplementary Table 1). GAPDH and ACTB were used as internal controls. Results are indicated only with GAPDH because those of ACTB were almost identical. The cycling conditions for amplification were 95°C for 1 min, followed by 40 cycles of 95°C for 15 sec and 63°C (for GDF9 and BMP15) or 61.4°C (for LHCGR and FSHR) for 30 sec. Melt curve analyses were carried out for all genes. The specificity of the PCR products was checked by a single peak.

In vitro growth culture of OCCs

Porcine OCCs collected from early antral follicles were cultured as previously reported [22] with some modifications. Briefly, OCCs were cultured individually for 5 days in 96-well culture plates (BioCoat Collagen I Cellware; Becton Dickinson, San Diego, CA, USA) containing 200 µl of growth medium in each well under a humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂ at 38.5°C. The basic growth medium was Minimum Essential Medium α (GIBCO Invitrogen, Inchinnin, Scotland, UK) supplemented with 2.2 mg/ml sodium bicarbonate, 0.08 mg/ml kanamycin sulfate, 2% (w/v) polyvinylpyrrolidone (molecular weight 360,000), 55 µg/ml L-cysteine, 0.05 µM dexamethasone, 4.0 mM hypoxanthine, 50 µg/ml ascorbic acid 2-glucoside (Hayashibara, Okayama, Japan), and 5% (v/v) fetal bovine serum (ICN Biomedicals, Solon, OH, USA). The medium was also supplemented with 10 µM 17β-estradiol and 0.01 IU/ml recombinant human follicle stimulating hormone (rhFSH; MSD, Tokyo, Japan). In the experimental groups, 10, 50, or 100 ng/ml of GDF9 (recombinant mouse GDF-9 protein: 739-G9; R&D Systems, Minneapolis, MN, USA) or BMP15 (recombinant human BMP-15 protein: 5096-BM; R&D Systems) were further added to the culture medium. Mouse GDF9 (Gly307-Arg441) and human BMP15 (Gln268-Arg392) are bioactive mature forms that share 88% and 80% amino acid sequence identity with porcine GDF9 and BMP15, respectively. In addition, their effects on cumulus/granulosa cells or OCCs have been reported in several mammalian species, including pigs [22, 29,30,31,32,33].

Half (100 µl) of the culture medium was replaced with fresh medium on Day 3. At the start of culture (Day 0), Day 3 and Day 5, OCC morphologies were assessed and photographed. The OCC diameters were measured using the ImageJ software (NIH, Bethesda, MD, USA). Oocytes completely wrapped with cumulus cells were considered to be surviving. The formation of antrum-like structures was examined on Day 3 and Day 5 by identifying the visible spaces surrounded by cumulus cells. OCCs showing cytoplasmic degeneration of oocytes, detachment of cumulus cells from the zona pellucida, or loss of the aggregated structure of cumulus cells were classified as disintegrated complexes; all other complexes were considered to have maintained their integrity. In vitro grown OCCs were used for the measurement of oocyte diameters, nuclear stage analysis, in vitro maturation culture, and qPCR. For the measurement of oocyte diameters, the oocytes were denuded and the diameter excluding the zona pellucida was measured using an ocular micrometer (Olympus, Tokyo, Japan).

In vitro maturation culture of OCCs

After 5 days of growth culture, oocytes were assessed for their meiotic competence. In vitro grown OCCs were recovered and further cultured in maturation medium (0.5 ml) in 4-well multi dishes (Thermo Fisher Scientific) in an atmosphere of 5% CO₂ in air at 38.5°C for 48 h. The maturation medium was bicarbonate-buffered medium 199 supplemented with 2.2 mg/ml sodium bicarbonate, 0.08 mg/ml kanamycin sulfate, 0.1 mg/ml sodium pyruvate (Nacalai Tesque, Kyoto, Japan), 10% (v/v) fetal bovine serum, and 0.1 IU/ml human menopausal gonadotropin (hMG; ASKA Pharmaceutical, Tokyo, Japan). After 48 h of maturation culture, oocytes were denuded by gently pipetting with 0.1% (w/v) hyaluronidase.

In some experiments, oocytes after growth culture were denuded and used to induce spontaneous maturation to eliminate the effect of cumulus cells on oocyte maturation. In addition, some OCCs after growth culture were subjected to maturation culture with 10 ng/ml epidermal growth factor (recombinant human EGF: 236-EG; R&D systems) [34] instead of hMG.

OCCs containing growing oocytes before growth culture and OCCs containing in vivo grown oocytes were used as controls.

Analysis of nuclear stages of oocytes

Oocytes before and after growth culture and maturation culture were denuded mechanically, fixed in aceto-ethanol, and stained with 1% (w/v) aceto-orcein. The stained oocytes were then classified based on the morphologies of the chromatin and nuclear envelope [35, 36]. Oocytes containing the nuclear envelope were classified into filamentous chromatin (FC), stringy chromatin (SC), or germinal vesicle (GVI–IV) stages. Oocytes after germinal vesicle breakdown (GVBD) were classified as diakinesis (D), metaphase I (MI), anaphase I (AI), telophase I (TI), and metaphase II (MII). Oocytes showing cytoplasmic or nuclear abnormalities were considered to be degenerated oocytes.

Statistical analysis

The frequencies of antrum formation and oocyte nuclear stage were compared using a chi-squared test. The mean diameters of the OCCs were subjected to a two-way analysis of variance (ANOVA) followed by a Tukey–Kramer test. The mean diameters of oocytes and the quantity of mRNAs were subjected to one-way ANOVA followed by the Tukey–Kramer test. Statistical significance was set at P < 0.05.

Results

Expression of GDF9 and BMP15 mRNAs in porcine follicles

Expression of GDF9 and BMP15 mRNAs in OCCs (Fig. 1A), oocytes (Oos; Fig. 1B), cumulus cells (CCs; Fig. 1C), and mural granulosa cells (MGCs; Fig. 1D) from early antral follicles (1.2–1.5 mm) was examined by RT-PCR (Fig. 1E). OCCs and Oos from both early antral follicles and antral follicles (4–6 mm) showed GDF9 and BMP15 PCR product bands of the expected sizes. In CCs and MGCs from both types of follicles, GDF9 bands were faintly detected and BMP15 bands were undetectable.

Fig. 1.

Expression of GDF9 and BMP15 mRNAs in porcine oocyte–cumulus cell complexes (OCCs; A), oocytes (Oos; B), cumulus cells (CCs; C), and mural granulosa cells (MGCs; D) from early antral follicles (1.2–1.5 mm) and antral follicles (4.0–6.0 mm) was examined by RT-PCR (E) and qPCR (F and G). The RT-PCR product bands specific to porcine GDF9, BMP15, and GAPDH (internal control) mRNAs are shown in the top, middle, and bottom panels, respectively (E). Relative expression levels of GDF9 (F) and BMP15 (G) mRNAs are shown as the mean ± S.E. from more than three replicates. Porcine GAPDH was used as an internal control.

The qPCR results supported the expression of GDF9 and BMP15 in oocytes from both types of follicles (Figs. 1F and G). GDF9 and BMP15 mRNAs were mainly expressed in oocytes, whereas they were scarcely detected in CCs and MGCs. Oocytes from the early antral follicles expressed higher levels of GDF9 and BMP15 mRNAs. These levels decreased with follicle development, although the decrease was not statistically significant.

Effects of GDF9 and BMP15 on OCC growth

The typical morphologies of OCCs from early antral follicles during growth culture with GDF9 or BMP15 are shown in Fig. 2. Before culture (Day 0), the oocytes were covered with two to three layers of cumulus cells. During growth culture, the diameters of OCCs gradually increased and some of the OCCs formed antrum-like structures. The percentages of antrum formation were 10–30% in all groups. There were no obvious effects of GDF9 and BMP15. More than 85% of OCCs were completely covered with cumulus cells in all groups after culture.

Fig. 2.

Typical morphologies of porcine oocyte–cumulus cell complexes (OCCs) cultured with GDF9 (A) and BMP15 (B) during growth culture. The top, middle, and bottom panels in each set of pictures show results from Day 0, 3, and 5 of incubation, respectively. The different concentrations (ng/ml) of growth factors are shown in increasing order from left to right. The scale bar represents 200 µm.

Figures 3A and 3B show the changes in the diameter of OCCs during culture. Before culture (Day 0), the diameters of the OCCs were 170–180 µm. The diameters gradually increased in all groups, reaching more than 300 µm in the GDF9 group and 250 µm in the BMP15 group on Day 5. GDF9 induced a concentration-dependent increase in OCC diameter, whereas higher concentrations of BMP15 did not promote a further increase in OCC diameter. The mean diameter of OCCs cultured with 100 ng/ml GDF9 at Day 5 was 349.9 ± 9.0 µm (mean ± S.E.), which was significantly larger than the diameter in the control OCCs (302.0 ± 7.5 µm).

Fig. 3.

Effects of GDF9 and BMP15 on the diameters of porcine oocyte–cumulus cell complexes (OCCs) during growth culture (A and B), and the diameters of oocytes after growth culture (C and D). OCCs were cultured in the growth medium supplemented with GDF9 (A and C) and BMP15 (B and D). The diameters of OCCs excluding disintegrated complexes were measured at Day 0, 3, and 5. The diameters of OCCs including fully grown oocytes collected from antral follicles (4.0–6.0 mm) were measured as in vivo controls (FO) in (A) and (B). The diameters of growing oocytes (GO) from early antral follicles (1.2–1.5 mm) and FO were measured as in vivo controls in (C) and (D). GDF9 and BMP15 concentrations are shown in ascending order under each set of boxes. The numbers of OCCs or oocytes (n) used for each experiment are shown at the bottom of each box. The mean diameter ± S.E. is shown at the top of each box. Boxes with different letters (a–g) in each figure are significantly different (two-way ANOVA for OCC diameters, and one-way ANOVA for oocyte diameters; P < 0.05). The experiments were replicated more than three times.

Effects of GDF9 and BMP15 on oocyte growth

After growth culture, more than 80% of oocytes showed normal morphology with no significant differences among the experimental groups. Figs. 3C and 3D show the diameters of the oocytes after growth culture. The diameters of cultured oocytes increased significantly compared to those before culture (approximately 110 µm). In all groups, the mean diameter of oocytes after culture was approximately 125 µm, which was similar to the mean diameters of in vivo fully grown oocytes (FO in Figs. 3C and D). There were no significant effects of GDF9 or BMP15 on oocyte growth.

The effects of GDF9 and BMP15 on the meiotic stage of oocytes were examined after growth culture (Supplementary Table 2). Most of the growing oocytes were at the FC or SC stage immediately after collection from early antral follicles. However, in vivo fully grown oocytes from antral follicles were at the GV (I–IV) stage. More than 70% of in vitro grown oocytes were at the GV stage and there were no significant effects of GDF9 and BMP15 on the meiotic stage of oocytes.

Effects of GDF9 and BMP15 on oocyte meiotic competence

To examine the meiotic competence of oocytes grown with GDF9 or BMP15, OCCs after growth culture were subjected to in vitro maturation culture. Figs. 4A–C show cumulus expansion after maturation culture. OCCs from antral follicles fully expanded (Fig. 4A, FO), while those from early antral follicles barely expanded (Fig. 4A, growing oocytes [GO]). OCCs cultured without growth factors swelled after maturation culture with cumulus cells loosely connected in the mass (Figs. 4B and C). OCCs that had been cultured with GDF9 showed swelling similar to that of the OCCs without growth factors (Fig. 4B). In contrast, OCCs cultured with 50 and 100 ng/ml BMP15 expanded fully after maturation culture in a manner similar to that of OCCs collected from antral follicles (Fig. 4C).

Fig. 4.

Typical morphologies of the cumulus expansion of oocyte–cumulus cell complexes (OCCs, A–C). After growth culture for 5 days with 0, 10, 50, or 100 ng/ml GDF9 (B) or BMP15 (C), OCCs were futher cultured for maturation without GDF9 and BMP15. OCCs collected from early antral follicles (1.2–1.5 mm; A, GO) and antral follicles (4.0–6.0 mm; A, FO) were cultured in the maturation medium as in vivo controls. The scale bar represents 500 µm. Relative expression levels of LHCGR (D) and FSHR (E) mRNAs in porcine cumulus cells after 5 days of growth culture with GDF9 (100 ng/ml) or BMP15 (100 ng/ml). Cumulus cells from early antral follicles (GO: 1.2–1.5 mm; open bar) and antral follicles (FO: 4.0–6.0 mm; grey bar) were used as in vivo controls. Porcine GAPDH was used as an internal control. Values with different letters (a–c) are significantly different (one-way ANOVA; P < 0.05). As LHCGR mRNA of GO was not detected in several analyses (shown as N.D.), it was excluded from the statistical analysis. Data are shown as the mean ± S.E. from three replicates.

We further examined the meiotic stage of the oocytes after maturation culture. The GVBD rate of the growing oocytes from early antral follicles was low; none matured to MII, while almost all in vivo fully grown oocytes collected from antral follicles underwent GVBD and matured to MII (Table 1). Approximately 20% of in vitro grown oocytes in the control groups cultured without GDF9 and BMP15 underwent GVBD, and 15–16% matured to MII after maturation culture. Similarly, less than 10% of oocytes cultured with GDF9 (50 and 100 ng/ml) underwent GVBD and matured to MII. On the other hand, the percentage of oocytes that underwent GVBD and reached MII after culturing with BMP15 increased dose-dependently, and the MII rates in the 50 and 100 ng/ml BMP15 groups (49% and 61%, respectively) were significantly higher than those of the control oocytes (16%).

Table 1. Nuclear stages of porcine oocytes after in vitro maturation culture with hMG.

These data led us to speculate that the presence of high concentrations of BMP15 during the growth culture period promoted cumulus expansion after gonadotropic stimulation, ultimately resulting in oocyte maturation. Next, to eliminate the effect of cumulus cells on oocyte maturation, OCCs were cultured with GDF9 or BMP15 for 5 days, after which the oocytes were denuded from cumulus cells and subjected to maturation culture (Table 2). We used 100 ng/ml of GDF9 and BMP15 for this experiment, because 100 ng/ml GDF9 yielded the greatest increase in OCC diameter and 100 ng/ml BMP15 showed the greatest promotion of oocyte maturation. After maturation culture of denuded oocytes, approximately half of the oocytes underwent GVBD and more than 40% of oocytes matured to MII in the control (41%) and GDF9 groups (45%). These percentages were not significantly different from those in the BMP15 group (70%). By removing the cumulus cells, the oocyte maturation rates of the control and GDF9 groups were improved compared to the rates in Table 1.

Table 2. Nuclear stages of porcine oocytes after in vitro maturation culture by the denudation of oocytes, and with EGF.

It has been reported that the EGF network transmits the LH signal from the somatic cells of the follicle to the oocyte to promote oocyte meiotic resumption [37, 38]. Thus, we also examined the effects of GDF9 and BMP15 on EGF-stimulated maturation of in vitro grown oocytes (Table 2). There were no significant differences in both GVBD and MII rates among the control, GDF9, and BMP15 groups. These results indicated that the oocytes in any group acquired meiotic competence after growth culture. However, the cumulus cells of the control and GDF9 groups did not acquire sufficient ability to respond to gonadotropic stimulation for oocyte maturation.

Effects of GDF9 and BMP15 on the expression of LHCGR and FSH mRNAs

We considered that the cumulus cells of the BMP15 group responded more sensitively to gonadotropin than the cumulus cells of the other groups. To investigate this, we collected cumulus cells from OCCs cultured with GDF9 and BMP15 for 5 days and subjected them to qPCR to compare their expression of LHCGR and FSHR mRNAs (Figs. 4D and E). The expression of LHCGR mRNA increased with follicle development (from GO to FO in Fig. 4D), while the expression of FSHR mRNA decreased (Fig. 4E). For the LHCGR mRNA, cumulus cells from the control, GDF9, and BMP15 groups expressed higher levels of LHCGR mRNA than cumulus cells from the early antral and antral follicles, while the BMP15 group showed the highest level of expression. The expression level of FSHR mRNA was higher in cumulus cells from the GDF9 group than in those from the control and BMP15 groups; however, these differences were not significant.

Discussion

Porcine GDF9 mRNA was expressed mainly in oocytes in early antral follicles and faintly in cumulus cells and mural granulosa cells, whereas BMP15 mRNA was expressed specifically in oocytes. Oocyte-specific expression of GDF9 mRNA has been reported in mouse [39,40,41,42], rat [43], cow [22, 44], sheep [44], and human [45] follicles. In pigs, GDF9 mRNA is produced by oocytes and also by cumulus cells and mural granulosa cells [46]. BMP15 mRNA expression was also shown to be oocyte-specific in mouse [41, 47], rat [44], human [45], and bovine [22] follicles. One study reported that the expression of GDF9 mRNA in porcine granulosa cells was less than 1/30 that of BMP15 mRNA [48]. The results of the present study are in good agreement with these reports. The present results suggest that, in pigs, GDF9 mRNA is produced mainly in oocytes, whereas BMP15 mRNA is produced exclusively in oocytes.

In the present study, the expression levels of GDF9 and BMP15 mRNAs did not change significantly during antral follicle development. In previous reports, the mRNA levels of GDF9 were higher in bovine oocytes in antral follicles than in oocytes in secondary follicles [49]. Furthermore, GDF9 and BMP15 mRNAs were expressed constantly in bovine oocytes throughout the antral follicle stage [50]. The present results indicate that GDF9 and BMP15 mRNAs are also expressed constantly in oocytes throughout the development of antral follicles in pigs.

Under our culture conditions, the diameter of OCCs increased in all groups after the growth culture. GDF9 increased the OCC diameter in a dose-dependent manner, whereas the effects of BMP15 on the diameter were not dose-dependent. Studies in different species have shown that GDF9 promotes the proliferation of cultured granulosa cells in mice [15], rats [51], cows [52], and pigs [23]. The diameters of secondary follicles cultured with GDF9 were increased in mice [53] and rats [51]. Our findings suggest that GDF9 probably promotes cumulus cell proliferation and increases the diameter of porcine OCCs.

Studies on the effects of GDF9 and BMP15 on oocyte growth are limited. In the present study, GDF9 and BMP15 had no significant effect on either oocyte growth or meiotic arrest after growth culture. However, when OCCs cultured with GDF9 for 5 days were subjected to a subsequent maturation culture, gonadotropin did not induce oocyte maturation, similar to the findings in control OCCs. In contrast, oocytes cultured with higher concentrations of BMP15 matured to MII. Moreover, OCCs cultured with GDF9 expanded loosely, whereas those with high concentrations of BMP15 expanded fully in the manner of in vivo grown OCCs. When the oocytes were denuded after growth culture and subjected to maturation culture, there were no significant differences in maturation rates among the experimental groups.

BMP15 treatment during the growth culture period promoted cumulus expansion during the subsequent maturation culture, whereas GDF9 did not. This difference may be related to the different oocyte maturation rates. We added hMG, which has both FSH and LH activities [54], to the maturation culture medium. It has been suggested that LH induces the production of EGF-like peptides in the granulosa cells and that the peptides bind to the EGF receptor in the cumulus cells and activate the signaling pathways leading to cumulus expansion and oocyte meiotic resumption [37, 38]. Therefore, we conducted two experiments. In one, OCCs after growth culture were matured with EGF instead of hMG. In the second, expression of LHCGR and FSHR mRNAs in cumulus cells of OCCs that had been cultured with GDF9 or BMP15 for 5 days was compared. When OCCs after growth culture were stimulated by EGF, there were no significant differences in oocyte GVBD and MII rates among the experimental groups. These results suggest that the cumulus cells gained the ability to respond to EGF after growth culture regardless of the supplementation of GDF9 or BMP15. However, the LHCGR mRNA level was significantly increased by BMP15, although there were no significant differences in FSHR mRNA levels among the culture groups.

The expression of LHCGR mRNA in cumulus cells from early antral follicles before growth culture was quite low, whereas the expression of LHCGR mRNA in cumulus cells from antral follicles was substantial. In several species, including pigs, LHCGR mRNA has been reported to be absent in cumulus cells, or to be present at much lower levels than in mural granulosa cells [55,56,57,58]. In contrast, upregulation of LHCGR mRNA in cumulus cells was observed in all culture groups in the present study. This upregulation was probably because we supplemented the growth medium with FSH, which is essential for the growth of porcine oocytes [59]. It has been reported that FSH promotes differentiation of granulosa cells and upregulates LHCGR mRNA in mouse [60, 61] and porcine [62, 63] OCCs. The addition of BMP15 during growth culture further enhanced LHCGR mRNA expression in cumulus cells in the present study. As LHCGR is expressed in mural granulosa cells in the follicle [58], it is possible that the outer layer of cumulus cells of OCCs after growth culture with BMP15 could differentiate into mural granulosa cell-like cells. Considering that OCCs from the GDF9 and control groups did not show a full expansion and the oocytes did not mature to MII, the in vitro cultured OCCs may require a higher level of LHCGR mRNA to achieve cumulus expansion in response to LH.

These results suggest that GDF9 is produced in oocytes and promotes the growth of OCCs and that BMP15 produced by oocytes promotes the expression of LHCGR mRNA in cumulus cells during oocyte growth culture, which may contribute to cumulus expansion and oocyte maturation.

Conflict of interests

The authors declare that there are no conflicts of interest related to this manuscript.