In Vitro Growth, Maturation, Fertilization, and Embryonic Development of Oocytes from Porcine Preantral Follicles

Abstract

This study was conducted to identify an in vitro culture system that would support intact porcine follicle growth from preantral follicle to antral stages, oocyte maturation, fertilization, and embryonic development; and to evaluate factors that influence porcine preantral follicle growth in vitro. Preantral follicles isolated from prepubertal porcine ovaries were cultured for 4 days in the presence of different concentrations of porcine serum and FSH, and with different numbers of follicles per well. A series of experiments showed that porcine antral follicles can be grown at a high frequency in vitro from healthy preantral follicles with intact theca when cultured in North Carolina State University 23 medium supplemented with 1.5 ng/ml FSH, 7.5% serum, and when cultured with three follicles per well. After 4 days of culture, 68% healthy cumulus-enclosed oocytes from these follicles were obtained, and 51% of the oocytes completed meiotic maturation to the metaphase II stage. Fifty-three percent of the mature oocytes underwent fertilization, 43% of the fertilized oocytes cleaved, and 13% developed to the blastocyst stage. The results show 1) that porcine preantral follicles can grow efficiently to the antral stage using these culture conditions, and 2) that oocytes from in vitro-matured porcine preantral follicles can acquire meiotic competence and undergo fertilization and embryonic development.

Effects of luteinizing hormone and follicle stimulating hormone on the developmental competence of porcine preantral follicle oocytes grown in vitro

“…In contrast, without FSH, preantral follicles failed to grow to the antral stage and none became mature oocytes. These results support previous studies [9,10,21,[24][25][26][27] suggesting that FSH plays an important role in the development of preantral follicles in vitro. FSH promotes preantral follicle growth by inducing granulosa cell proliferation and differentiation, because granulosa cell growth accounts for the majority of this follicle expansion [28].…”

“…The preantral follicles were collected and cultured as previously described [21,22]. The ovaries were cut into small pieces (1-3 mm) and preantral follicles were isolated mechanically using watchmaker's forceps in DPBS with 3 mg/ml BSA.…”

“…Maturation of OCCs was evaluated as described by Wu et al [21] After the culture was completed, the follicles were opened using two needles and the OCCs were flushed into DPBS supplemented with 3 mg/ml BSA. After washing three times in NCSU23 medium supplemented with 0.23 mM pyruvate and 10% porcine serum, the OCCs were cultured for 48 h in the same medium, which was further supplemented with 0.12 μg/ml OFSH, 2.5 μg/ml OLH, 20 ng/ml epidermal growth factor (EGF), 50 μg/ml L-ascorbic acid and 10-20 antral follicular shell pieces.…”

“…Porcine sperm (SGI, Cambridge, MA, USA) was prepared as described previously [21,23]. The prepared sperm suspension (50 μl) was added to 50 μl oocytecontaining medium (final concentration of 5×10 5 cells/ml).…”

Opportunities and challenges in applying genomics to the study of oogenesis and folliculogenesis in farm animals

“…In vitro growth culture systems where oocyte and somatic cells are cultured together have been developed in both pig and bovine species, albeit with limited success (Wu et al 2001, Hirao et al 2004, Hashimoto et al 2007. In these species, culture has been limited to cumulus-oocyte complexes issued from antral follicles, whereas earlier stages can be grown in mouse.…”