Oocytes are matured through mutual interaction of oocytes and their surrounding follicular cells which include granulosa and cumulus cells. When ovarian follicles grow and mature, paracrine growth factors secreted from oocytes play a crucial role. These oocyte-secreted factors (OSF) regulate differentiation of granulosa cells, separating them into two distinct sub-types, cumulus and mural granulosa cells, which become phenotypically and functionally distinct from each other (Eppig et al., 1997;, Li et al., 2000). Such regulation eventually affects maturation of oocytes and later development of preimplantation embryos. Among these factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) regulate many critical processes involved in early folliculogenesis and oocyte maturation. Both have important roles in maturation of oocytes by the functions that GDF9 reduces apoptosis and BMP15 promotes proliferation of granulosa cells (Eppig, 2001;, Hussein et al., 2005).

Culture medium to support in vitro maturation (IVM) of porcine oocytes often includes supplements such as epidermal growth factor, L-cysteine, and porcine follicular fluid that are known to be beneficial for meiotic maturation of oocytes (Abeydeera et al., 1998). However, the developmental ability of oocytes matured in such media is still lower than that of oocytes matured in vivo and needs to be improved (Moor et al., 2001). Therefore, it would be important to study on underlying mechanism of IVM as well as improvement of techniques to produce developmentally competent oocytes. In particular, the beneficial effects of GDF9 and BMP15 on increasing the rates of cleavage and blastocyst development were observed when they were treated in combination during in vitro maturation of porcine oocytes (Lin et al., 2014). However, whether the treatment improves the quality of blastocyst has not been assessed.

In the present study, the effects of recombinant GDF9 and BMP15 treatment during IVM of porcine oocytes upon development both in quantity and quality were investigated.

1. Animal Ethics

All procedures in this study were carried out in accordance with the Code of Practice for the Care and Use of Animals for Scientific Purposes and approved by the Institutional Animal Care and Use Committee, Dankook University.

2. Chemicals and Reagents

All chemicals and reagents were purchased from Sigma- Aldrich (St. Louis, MO, USA), unless stated otherwise.

3. in vitro Maturation of Oocytes

Porcine ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory in a warm box (25∼30℃) within 2 h. Follicular fluid and cumulusoocyte complexes (COC) from follicles (5∼6 mm in diameter) were aspirated using an 18-gauge needle attached to a 5-ml disposable syringe. Compact COC were selected and washed five times in HEPES-buffered tissue culture medium (TCM)- 199 (Invitrogen, Carlsbad, CA, USA). in vitro maturation medium was TCM-199 supplemented with 10 ng/ml of epidermal growth factor, 10 IU/ml equine chorionic gonadotropin (eCG; Intervet, Boxmeer, The Netherlands), 10 IU/ml human chorionic gonadotropin (hCG; Intervet), and 10% (v/v) porcine follicular fluid. A group of 50 COC was cultured in 500 μL of IVM medium at 39℃ in a humidified atmosphere of 5% CO₂ in air. After culturing for 20 h, the COC were transferred to eCGand hCG-free IVM medium and cultured for another 20 h. During 40 h of IVM, 200 ng/ml recombinant human GDF9 or 200 ng/ml recombinant human BMP15 (or both) were added to the IVM medium.

4. Viability Test of Cumulus cells

After 40 h of IVM, cumulus cells were removed from oocytes by repeated pipetting in the IVM medium containing 0.5 mg/ml hyaluronidase for 1 min. Cumulus cells collected from individual oocytes were counted and stained with 0.4% trypan blue to test the viability.

5. Parthenogenetic Activation of Oocytes

The denuded oocytes with clearly extruded polar bodies were considered to be reached to the matured second metaphase (MII) stage and used for subsequent activation and culture. Matured oocytes were transferred to 0.3 M mannitol solution containing 0.5 mM HEPES, 0.05 mM CaCl₂, and 0.1 mM MgCl₂ in a chamber containing two electrodes. Using BTX Electro-Cell Manipulator 2001 (Harvard Apparatus, Holliston, MA, USA), oocytes were activated with a double DC pulse of 1.5 kV/cm for 45 μsec. Activated oocytes were cultured for 4 days in porcine zygote medium-3 (PZM-3) containing 4 mg/ml fatty acid-free BSA and transferred to PZM-3 containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and cultured for another 4 days. All embryos were cultured at 39℃ in humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂. Cleavage and blastocyst formation were evaluated on days 2 and 8 after the activation, respectively.

6. Differential Staining

In blastocysts, the numbers of inner cell mass (ICM) and trophectoderm (TE) cells were determined using differential nuclei staining as described previously (Papaioannou et al., 1988). Briefly, blastocysts on day 8 after activation were transferred to 100 μg/ml propidium iodide (PI) with 0.5% Triton X-100 in phosphate-buffered saline (PBS). Blastocysts were then stained overnight with Hoechst 33342 in ethanol at 4℃. Embryos were then washed in 100% ethanol and mounted in a glycerol drop on a glass slide. Observation was performed under Nikon TE-300 inverted microscope (Nikon Corporation, Tokyo, Japan) equipped with epifluorescence based on ICM and TE cells appeared to be blue and pink, respectively.

7. Statistical Analysis

Statistical analyses were carried out using SPSS version 11.0 for Windows. At least three replicates were conducted for each experiment. Percentage data were subjected to arcsine transformation prior to statistical analysis. All data were analyzed by one-way ANOVA. A value of P<0.05 was considered significant.

None of the treatments including GDF9 or BMP15 (or both) affects the number and viability of cumulus cells (Table 1). Similarly, none of the treatments promote the rates of oocyte maturation, cleavage and blastocyst formation from activated oocytes (Table 2). As shown in Table 3, however, GDF9 treatment significantly increased (P<0.05) the number of total cells in blastocysts due to an increased number of TE cells. The numbers of both ICM and TE cells were significantly increased (P<0.05) by the treatment of BMP15. Combined treatment of GDF9 and BMP15 further increased (P<0.05) the number of cells in blastocysts by increasing both ICM and TE cells.

Table 1. Effect of GDF9 and BMP15 on the number and viability of cumulus cells.

Table 2. Effect of GDF9 and BMP15 on maturation and development of oocytes.

Table 3. Effect of GDF9 and BMP15 on the number of cells in blastocysts.

Oocyte-secreted factors GDF9 and BMP15 regulate many critical processes involved in early folliculogenesis (Hussein et al., 2006). They promote follicle growth beyond the primary stage to reach MII stage by regulating proliferation and apoptosis of granulosa cells (Huang and Dagan, 2010). In the present study, effects of recombinant human GDF9 and BMP15 treatment during IVM of porcine oocytes upon development after parthenogenetic activation were investigated.

As shown in Table 1, neither GDF9 nor BMP15 affected the number and viability of cumulus cells. Combined treatment of GDF9 and BMP15 did not promote survival and proliferation of cumulus cells. Both GDF9 and BMP15 have been known to enhance the function of cumulus cells by simultaneously increasing cell proliferation and reducing apoptosis (Eppig, 2001;,Hussein et al, 2005). However, as shown in the present study, addition of exogenous GDF9 and BMP15 either individually or in combination did not increase proliferation and survival of cumulus cells. Rather, these factors may promote expansion of cumulus cells by weakening extracellular matrix (ECM)-mediated cell attachment as a previous report demonstrating scant ECM in co-culture with denuded oocytes that may secrete OSF (Gomez et al., 2012).

Addition of GDF9 and BMP15 either individually or in combination did not enhance meiotic maturation and subsequent development of parthenogenetically activated oocytes into the cleavage and blastocyst stage (Table 2). Hussein et al. (2006) reported that these two factors could enhance developmental competence of bovine oocytes and substantially increase blastocyst formation. However, a wide range of species variability in GDF9 and BMP15 mRNA levels in oocytes was reported (Crawford and McNatty, 2012). Hence, the species-specific ratio of GDF9:BMP15 mRNA between pigs and cattle might cause variation in developmental competence of oocytes. Recently, Lin et al. (2014) reported slight increases in cleavage and blastocyst rate of parthenogeneticcally activated oocytes after combined treatment of GDF9 and BMP14 during IVM. However, such increases have not been observed in the present study.

As shown in Table 3, GDF9 treatment significantly increased (P<0.05) the number of total cells in blastocysts due to the increased number of TE cells, while the numbers of both ICM and TE cells were significantly increased (P<0.05) by the treatment of BMP15. Combined treatment of GDF9 and BMP15 further increased the number of cells in blastocysts by increasing both ICM and TE cells. Previous studies using denuded oocytes as a source of OSF reported increases in the numbers of ICM cells in mice (Sudiman et al., 2014) and TE cells in cattle (Hussein et al., 2006). In the present study, we demonstrated that exogenous GDF9 more specifically targets TE cells, whereas BMP15 does both ICM and TE cells. Moreover, we showed a synergistic action of two factors to further increase proliferation of both ICM and TE cells. Peng et al. (2013) reported that GDF9 and BMP15 synergistically activate Smad2/3 with 30- to 1,000-fold when compared with either GDF9 or BMP15 alone. This synergistic activation is mediated by binding of GDF9-BMP15 complex with receptors that include ALK4/5/7 and BMPR2.

Overall, the treatment of GDF9 or BMP15 (or both) during IVM enhanced the quality of blastocysts via increased number of ICM and/or TE cells in pigs. Results obtained from the present study may be beneficial not only to study the mechanism involved in maturation of porcine oocytes but also to practically improve efficiency of in vitro fertilization and somatic cell nuclear transfer in pigs which the quality of in vitro matured oocytes are critical.