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

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.

Oocyte-derived growth factors promote development of antrum-like structures by porcine cumulus granulosa cells <i>in vitro</i>

“…In contrast, BMP15 did not significantly increase the OCC diameter or the formation of antrum-like structures. These results were mostly similar to the results obtained in our previous report [ 24 ], which suggested that GDF9 promoted the proliferation of cumulus cells to increase complex diameter, while BMP15 had little or no effect on OCC development. GDF9 promotes the proliferation of cultured granulosa cells in various species [ 7 , 21 ].…”

“…To collect and handle ovarian follicles and OCCs, 25 mM HEPES-buffered medium 199 (HEPES-199; Nissui Pharmaceutical, Tokyo, Japan) containing 0.1% (w/v) polyvinyl alcohol (PVA), 0.85 mg/ml sodium bicarbonate, and 0.08 mg/ml kanamycin sulfate was used. The basic growth medium was Minimum Essential Medium α (GIBCO Invitrogen, Paisley, 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), 10 µM 17β-estradiol, 0.01 IU/ml Gonalef 75 (recombinant human follicle-stimulating hormone; Merck Biopharma Japan, Tokyo, Japan), and 5% (v/v) fetal bovine serum (ICN Biomedicals, Costa Mesa, CA, USA) as reported previously [ 24 ].…”

“…Porcine OCCs and OXCs were cultured as previously reported [ 24 ], with some modifications. Briefly, OCCs and OXCs were cultured individually for 7 days in 96-well culture plates (Nunclon Sphera low-attachment surface: 174927 for growth culture; 174925 for quantitative PCR (qPCR); Thermo Fisher Scientific, Waltham, MA, USA) containing 200 µl of growth medium in each well under a humidified atmosphere of 5% CO 2 , 5% O 2 , and 90% N 2 at 38.5°C.…”

“…qPCR was performed as previously reported [ 24 ] with some modifications. Briefly, after culture, 15 OXCs from each experimental group were washed with PBS-PVA, transferred into a 1.5 ml microtube with a minimum volume of PBS-PVA, and stored at –80°C until use.…”

“…Studies in pigs have demonstrated that GDF9 promotes the proliferation of mural granulosa cells [ 21 ], and that GDF9 and BMP15 prevent cumulus cell apoptosis [ 22 , 23 ]. In our previous study, we showed that GDF9 promoted the growth of porcine oocyte–cumulus cell complexes collected from early antral follicles, and some of the developing complexes formed antrum-like structures [ 24 ]. However, it has not been determined whether oocyte-derived growth factors are involved in antrum formation in pigs.…”