Estradiol supports in vitro development of bovine early antral follicles

Abstract

Antrum formation and estradiol (E 2 ) secretion are specific features of oocyte and granulosa cell complexes (OGCs). This study investigates the effect of E 2 on the in vitro development of bovine OGCs derived from early antral follicles as well as on the expression of genes in granulosa cells (GCs). The supplementation of culture medium with either E 2 or androstenedione (A 4 ) improved the in vitro development of OGCs and the nuclear maturation of enclosed oocytes. When OGCs were cultured in medium containing A 4 , developmentally competent OGCs secreted more E 2 than OGCs that were not competent. In addition, fulvestrant inhibited the effect of both E 2 and A 4 on OGCs development. Comprehensive gene expression analysis using next-generation sequence technology was conducted for the following three types of GCs: i) GCs of OGCs cultured for 4 days with E 2 (1 mg/ml; E 2 (C)), ii) GCs of OGCs cultured for 4 days without E 2 (E 2 (K)) or iii) OGCs that formed clear antrum after 8 days of in vitro culture in medium containing E 2 (1 mg/ml; AF group). GCs of the E 2 (C) group had a similar gene expression profile to the profile reported previously for the in vivo development of large follicles. This genetic profile included factors implicated in the up-regulation of E 2 biosynthesis and down-regulation of cytoskeleton and extracellular matrices. In addition, a novel gene expression profile was found in the AF group. In conclusion, E 2 impacts the gene expression profile of GCs to support the in vitro development of OGCs.Reproduction (2013) 145 85-96

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

“…A recent report showed that E 2 improved antrum formation of bovine OGCs cultured in vitro, and gene expression between OGCs cultured with high E 2 and those with low E 2 . The use of NGS showed that most of the marker genes that are associated with the large healthy follicles were expressed at higher levels in granulosa cells of OGCs cultured with E 2 than in those cultured without E 2 [24]. In the present gene expression analysis, the genes that are associated with healthy large follicles were expressed at lower levels in OGCs of aged cows.…”

“…Ovaries of Japanese Black Cows (Bos taurus) were collected from a slaughterhouse, maintained at 30°C in phosphate-buffered saline (PBS) containing antibiotics, 10 mM sucrose, and 10 mM glucose, and transported to the laboratory within 4 h. The medium used for the in vitro culture of OGCs collected from EAFs was based on a previous report [24] with a slight modification; our medium contained 0.1 or 10 μg/mL 17β-estradiol. TCM-199 medium supplemented with 10 % FCS (570H, ICN; Costa Mesa, CA, USA) and antibiotics was used for in vitro maturation (IVM medium).…”

“…These media were IVC medium containing 0.1 μg/mL estradiol and an IVC medium containing 10 μg/mL estradiol. Concentration of estradiol was set on the basis of the previous reports on estradiol concentration of bovine follicles [23], and that high concentration of estradiol 10 μg/mL enhanced in vitro growth of oocytes of EAFs derived from young cows and gilts [24,30]. For replicates, about 30-40 oocytes were collected from the group of young or aged cows (Table 1) and the experiment was repeated 10 times.…”

“…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.…”

Brain-derived neurotrophic factor promotes human granulosa-like tumor cell steroidogenesis and proliferation by activating the FSH receptor-mediated signaling pathway

“…Estradiol and progesterone are major hormones that regulate ovarian growth and physiological function 31–34 . In mammals, circulating estrogens are mainly produced by the granulosa cells, and required for the development of secondary sex characters in females 35 .…”