In vitro growth of bovine oocytes in oocyte-cumulus cell complexes and the effect of follicle stimulating hormone on the growth of oocytes

Abstract

Several successful in vitro culture experiments have used oocyte-cumulus cell-mural granulosa cell complexes (OCGCs) from early antral follicles (0.5-0.7 mm) for the growth of bovine oocytes. However, in studies related to in vitro oocyte maturation and in vitro embryo production, oocyte-cumulus cell complexes (OCCs) that have no mural granulosa cells have been widely used instead of OCGCs. The purpose of this study was to determine whether cumulus cells alone support oocyte growth. First, OCCs and OCGCs were cultured in vitro for 14 days to compare the integrity of the complexes as well as antrum formation. After 14 days, the diameter and meiotic competence of oocytes in OCCs and OCGCs were examined. Oocytes in OCCs grew fully and acquired meiotic competence similar to OCGCs, whereas antrum formation occurred later in OCCs as compared to OCGCs. Subsequently, the effects of follicle stimulating hormone (FSH) on in vitro growth of OCCs were examined for 14 days. When FSH was added to the culture medium, OCCs formed antrum-like structures one day earlier than those cultured without FSH. Oocytes cultured with 1 mIU/ml FSH grew fully and acquired meiotic competence. In contrast, when oocytes were cultured in media containing high concentrations of FSH, some of the OCCs collapsed and the number of degenerated oocytes increased. In conclusion, bovine oocytes in OCCs grow and acquire meiotic competence similar to OCGCs and, 1 mIU/ml FSH supports the development of OCCs and oocyte growth as observed in our culture system.

Keywords: Bovine oocyte; Cumulus cell; Follicle stimulating hormone (FSH); In vitro growth.

Fig. 1.

Typical morphologies of bovine oocyte-cumulus cell-mural granulosa cell complexes (OCGCs; A) and oocyte-cumulus cell complexes (OCCs; B) during in vitro growth culture. OCGCs and OCCs were cultured for 14 days. A0–A14 and B0–B14 represent Days 0–14, respectively. After OCGCs and OCCs attached onto a membrane sheet, they increased in size, and some of them formed antrum-like structures. The scale bars represent 200 µm.

Fig. 2.

Integrity of bovine oocyte-cumulus cell-mural granulosa cell complexes (OCGCs) and oocyte-cumulus cell complexes (OCCs) (A) and formation of antrum-like structures by OCGCs and OCCs (B) during in vitro growth culture. Complexes with cytoplasmic degenerative oocytes, detachment of granulosa cells or cumulus cells from the zona pellucida, and collapsed complexes were classified as disintegrated complexes; all others were regarded as complexes maintaining their integrity. Antrum formation by OCGCs and OCCs was observed every day by identifying visible spaces surrounded by granulosa cells or cumulus cells. The number of complexes (n) used in each group is shown in each graph (A and B). * Values are significantly different from those of OCGCs (P < 0.05).

Fig. 3.

Diameters of bovine oocytes in oocyte-cumulus cell-mural granulosa cell complexes (OCGCs) and oocyte-cumulus cell complexes (OCCs) after in vitro growth culture. The number of oocytes (n) used in each group is shown at the bottom of each box. The mean (± SEM) diameters of oocytes are shown at the top of each box. ¹⁾ The diameters of oocytes collected from early antral follicles (0.5–0.7 mm) before culture. ²⁾ The diameters of fully grown oocytes collected from antral follicles (4–6 mm). * Values are significantly different from those of oocytes before culture (Day 0) (P < 0.05). ^{a, b} Different alphabets denote significantly different values (P < 0.05).

Fig. 4.

Typical morphologies of bovine oocyte-cumulus cell complexes (OCCs) during in vitro growth culture with follicle stimulating hormone (FSH) (A) and representative images of histological sections of OCCs after 14 days of culture (B). OCCs were cultured for 14 days with varying concentrations of FSH ranging between 1–50 mIU/ml. The pictures represent OCCs on Days 1, 7 and 14 (A). After OCCs attached onto a membrane sheet, they increased in size, and some of the OCCs formed antrum-like structures (A). After 14 days of culture, antrum-like structures were observed inside the complexes (B). When OCCs were cultured with 10 or 50 mIU/ml FSH, some of the antrum-like structures collapsed before the end of the culture period. OCCs cultured with higher concentrations of FSH included degenerated oocytes. The scale bars represent 200 µm.

Fig. 5.

Integrity of bovine oocyte-cumulus cell complexes (OCCs) (A) and formation of antrum-like structures by OCCs (B) during in vitro growth culture with follicle stimulating hormone (FSH). Complexes with cytoplasmic degenerative oocytes, detachment of cumulus cells from the zona pellucida, and collapsed complexes were classified as disintegrated complexes; all others were regarded as complexes maintaining their integrity. Antrum formation by OCCs was observed every day by identifying visible spaces surrounded by cumulus cells. The number of complexes (n) used in each group is shown in each graph (A and B). * Values are significantly different from those of OCCs cultured without FSH (0 mIU/ml) (P < 0.05).

Fig. 6.

Diameters of bovine oocytes in oocyte-cumulus cell complexes (OCCs) after in vitro growth culture with follicle stimulating hormone (FSH). The number of oocytes (n) used in each group is shown at the bottom of each box. The mean (± SEM) diameters of oocytes are shown at the top of each box. ¹⁾ The diameter of oocytes collected from early antral follicles (0.5–0.7 mm) before culture. ²⁾ The diameter of fully grown oocytes collected from antral follicles (4–6 mm). * Values are significantly different from those of oocytes before culture (Day 0) (P < 0.05). ^a–c Different alphabets denote significantly different values (P < 0.05).