Immunohistochemical Localization of Fibroblast Growth Factor‐2 in the Sheep Ovary and its Effects on Pre‐antral Follicle Apoptosis and Development In Vitro

Abstract

Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF-2 protein expression in ovine ovaries and to verify the effect of FGF-2 on the morphology, apoptosis and growth of ovine pre-antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM(+) ) alone or supplemented with FGF-2 at different concentrations (1, 10, 50, 100 or 200 ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF-2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10 ng/ml FGF-2, and significantly higher than those observed in 50, 100 or 200 ng/ml FGF-2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200 ng/ml FGF-2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200 ng/ml FGF-2 (p < 0.05). However, the percentage of apoptotic cells only decreased (p < 0.05) in ovarian tissues cultured in 1 or 10 ng/ml FGF-2 compared with the control medium and other FGF-2 treatments. In conclusion, this study demonstrated the presence of FGF-2 in ovine ovaries. Furthermore, 10 ng/ml FGF-2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis.

Follicular survival, activation of primordial follicles and DNA fragmentation after storage of goat ovaries at 35ºC in supplemented Minimal Essential Medium

“…For a more in-depth evaluation of follicular quality in the treatments, the ovarian tissue was subjected to the terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) assay (SANTOS et al, 2014). Briefly, sections (5 µm) were mounted on glass slides and submitted to antigen retrieval with sodium citrate buffer (pH 6.0; Dinâmica) in a microwave for 6 min.…”

“…The DNA fragmentation in preantral follicles was evaluated as described previously (SANTOS et al, 2014). Only follicles containing an oocyte nucleus were analyzed.…”

“…The importance of small ruminants as providers of essential food, meat and dairy products, has been well reported all over the world (SOUZA-FABJAN et al, 2014). In addition, besides acting as an important source of fertilizable oocytes for further in vitro embryo production (GUPTA et al, 2007), the use of culture systems for preantral follicles could help in improving our knowledge about the mechanisms involved in early folliculogenesis in mammals (SANTOS et al, 2014).…”

Felis catus ovary as a model to study follicle biology in vitro

“…Follicle culture at different stages of development has been used to study diverse biological processes (folliculogenesis, steroidogenesis, and oocyte maturation) in various species, such as goat [9,10], mouse [11][12][13], rat [14], sheep [15,16], dog [17][18][19], and monkey [20][21][22][23]. Regarding the domestic cat, efforts to date have focused on oocytes [7,[24][25][26][27][28][29][30] or preantral follicle cultures [19,31].…”

Effect of ovarian tissue storage in Morus nigra extract on the morphology and DNA fragmentation of ovine preantral follicles

“…Preantral follicles apoptosis was evaluated as described previously (SANTOS et al, 2014). Only follicles that contained an oocyte nucleus were analyzed for apoptotic assay.…”