Effect of cryopreservation on the ability of granulosa cells to support in vitro development of oocytes derived from porcine early antral follicles.

Granulosa cells (GCs) contribute to oocyte development. The present study addressed the effect of cryopreservation on the ability of GCs to support oocyte development. GCs were collected from antral follicles. Oocyte granulosa cell complexes (OGCs) derived from early antral follicles were cultured with additional fresh-GCs or frozen-thawed-GCs for 14 days, and the developmental ability and characteristics of the oocytes grown in vitro were examined. Furthermore, fresh- or frozen-thawed-GCs were cultured for two days, and the effects of cryopreservation on the characteristics of GCs were examined. The developmental ability of blastocysts and the acetylation levels of H4K12 in oocytes grown in vitro did not significantly differ among the three culture conditions: OGCs cultured with additional fresh-GCs, frozen-thawed-GCs, or without additional GCs. Although both fresh- and frozen-thawed-GCs exhibited increased ATP content compared with that in oocytes developed without additional GCs, only fresh-GCs showed significantly increased lipid content in oocytes grown in vitro. ATP content, reactive oxygen content, mitochondrial membrane potential, and mitochondrial DNA copy number were greater in cultured frozen-thawed-GCs compared with fresh-GCs. In contrast, lipid content of cultured frozen-thawed-GCs was lower than that of fresh-GCs. Both fresh- and frozen-GCs support oocyte growth, but cryopreservation changes the properties of GCs in a manner that affects the energy status of oocytes grown in vitro.

Follicular factors determining the developmental competence of porcine oocyte.

PURPOSE: This study aimed to examine the relationship between granulosa cells (GCs), number of follicles, and the ability of follicular fluid to support in vitro growth of oocytes. METHODS: The culture medium was supplemented with follicular fluid (FF) collected from GC-rich ovaries and GC-poor ovaries, and its effect on in vitro growth and quality of oocytes derived from early antral follicles (EAFs) was assessed. RESULTS: GC-rich FF treatment enhanced oocyte growth and augmented changes in the chromatin configuration and lipid content of oocytes when compared to oocytes treated with GC-poor FF. Moreover, oocytes treated with GC-rich FF had a higher ability to progress to the blastocyst stage, than oocytes derived from large antral follicles (3-5 mm in diameter). In addition, supplementation of the culture medium with either GC-rich or GC-poor FF enhanced histone acetylation in oocytes grown in vitro. CONCLUSION: GC-rich FF contains key factors that support in vitro oocyte growth; hence, oocytes grown in GC-rich FF medium had high developmental competence, which was comparative to the oocytes grown in vivo.

Modification of the medium volume and gel substrate under in vitro culture conditions improves growth of porcine oocytes derived from early antral follicles.

This study compared the effects of different volumes of culture medium for the in vitro growth of oocytes derived from porcine early antral follicles (EAFs). Oocyte granulosa cell complexes (OGCs) were collected from EAFs (0.5-0.7 mm in diameter) and individually cultured for 14 days. When OGCs were cultured in 1 ml of medium with or without polyacrylamide gel (PAG), the presence of PAG supported granulosa cell (GC) proliferation and oocyte growth. When OGCs were cultured in 0.2 or 1 ml of medium on PAG, the number of GC in the OGC culture and the developmental ability of the oocytes cultured in vitro were significantly higher for the 1 ml of culture medium group than for the 0.2 ml group. In conclusion, a combination of a large volume of culture medium with PAG improved the growth and developmental ability of the oocytes cultured in vitro, which were comparable to the oocytes collected from large antral follicles.

Addition of granulosa cells collected from differential follicle stages supports development of oocytes derived from porcine early antral follicles.

PURPOSE: Improvement of in vitro oocyte growth by addition of granulosa cells derived from differential developmental stages of follicles. METHODS: Granulosa cells (GCs) collected from either early antral follicles (EAFs) or antral follicles (AFs) were added to oocyte-granulosa cell complexes (OGCs) derived from EAFs, and the in vitro growth of the oocytes was evaluated. RESULTS: Granulosa cells were incorporated into OGCs to form new OGCs within 2 days of culture. After 14 days of culture, the number of GCs surrounding oocytes was similar among the three OGCs conditions (unmanipulated "natural OGCs," "EAF-GCs add OGCs," and "AF-GCs add OGCs"), whereas the survival rate of the GCs and diameter of oocytes grown in vitro were the greatest for "AF-GCs added OGCs." After parthenogenetic activation, developmental rate till the blastocyst stage tended to be higher for "AF-GCs add OGCs" compared with other groups. Addition of AF-GCs significantly increased a hypoxic marker (pimonidazole staining) and increased the lipid content in oocytes grown in vitro compared with unmanipulated OGCs. CONCLUSION: Addition of GCs derived from more advanced stages of follicles to the OGCs changes the metabolism of oocytes and is beneficial for in vitro growth of oocytes derived from EAFs.