In Vitro Growth and Development of Bovine Oocyte-Granulosa Cell Complexes on the Flat Substratum: Effects of High Polyvinylpyrrolidone Concentration in Culture Medium1

Abstract

The aim of this study was to establish a culture system to support the growth of bovine oocytes as enclosed in granulosa cell complexes that extend on a flat substratum. Such systems have been established for mouse oocytes but are not applicable to larger animals because it is difficult to maintain an appropriate association between the oocyte and companion somatic cells. Growing bovine oocytes with a mean diameter of 95 microm were isolated from early antral follicles: the growing stage corresponds to that of oocytes in preantral follicles of 12-day-old mice. Oocyte-granulosa cell complexes were cultured for 14 days in modified TCM199 medium supplemented with 5% fetal bovine serum, 4 mM hypoxanthine, and 0.1 microg/ml estradiol. The novel modification made for this medium was a high concentration, 4% (w/v), of polyvinylpyrrolidone (PVP; molecular weight of 360000). The flat substratum used was either an insert membrane fit in the culture plate or the bottom surface of the wells of 96-well culture plates. PVP influenced the organization of complexes, resulting in a firm association between the oocyte and the innermost layer of surrounding cells. More oocytes enclosed by a complete cell layer were recovered from the medium supplemented with 4% PVP than from the control medium. Similarly, of the oocytes initially introduced into the growth culture, a significantly larger proportion developed to the blastocyst stage from medium containing 4% PVP than from medium without PVP. When PVP medium was used, the overall yield of blastocysts was similar between the system with the insert membranes (12%) and that with the 96-well culture plates (9%). A calf was produced from one of four embryos derived from oocytes grown in 96-well culture plates, matured, and fertilized in vitro and then transferred to a recipient cow.

Age-associated changes in bovine oocytes and granulosa cell complexes collected from early antral follicles

“…Furthermore, we cultured OGCs derived from EAFs of young and aged cows using two types of media containing low and high concentration of estradiol; low estradiol concentration was based on the corresponding levels in follicular fluid [23]. High concentration of estradiol was based on those reported in previous reports, which yielded high antrum OGCs formation in vitro [24], because antrum formation is a primary marker of follicular development in vitro [25,26]. We also compared the developmental ability of the oocytes grown under in vitro conditions.…”

A feasibility study of prepubertal and over mature aged local goat in relation to results of In Vitro growth culture to obtain additional M-II oocyte resources

“…Follicles with the diameter of 2.0-3.0 mm were isolated and selected using a micro-dissecting method 3 . Preantral follicles of different groups of aged (prepubertal and old mature aged) goat were cultured individually for 14 days in 20 ul drops of sticky medium containing 4% polyvinylpyrrolidone 4 in TCM 199, supplemented with 10% heat activated Fetal Bovine Serum (FBS), 10% follicular fluid, 0.1 IU/ml Follicle Stimulating Hormone (FSH), pen-strep, under paraffin oil in a humidified atmosphere of 5% CO 2 in air at 37 o C.…”

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.…”