The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 μg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles.

Summary This study investigated the effect of three different culture media (α minimum essential medium (α-MEM), McCoy or TCM199 during the in vitro culture (IVC) of bovine isolated pre-antral follicles. Pre-antral follicles greater than 150 μm in size were isolated and cultured for 0 (control), 8 or 16 days in one of the abovementioned culture media. Follicles were evaluated for survival, growth and antrum formation at days 8 and 16. The results showed that TCM199 was the most suitable medium to preserve follicular viability and ultrastructure, resulting in the highest rates of antrum formation. In conclusion, TCM199 promotes the in vitro development of isolated pre-antral follicles without hampering follicular functionality by sustaining in vitro growth and antrum formation.

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The aim of the present study was to evaluate the effects of different medium replacement intervals on the viability, antral cavity formation, growth and in vitro maturation (IVM) of oocytes from caprine and ovine pre-antral follicles. Pre-antral ovarian follicles (≥ 150 μm) were isolated from the ovarian cortex of goats and sheep and were individually cultured for 24 days using two different medium replacement intervals [2 days (T(1) ) or 6 days (T(2) )]. Follicle development was evaluated on the basis of antral cavity formation, increases in follicular diameter and the presence of healthy cumulus oocyte complexes and fully grown oocytes. For caprine species, results showed a higher percentage (p<0.05) of viable follicles in T(1) than T(2) from day 6 until the end of the culture. In addition, when comparing both treatments after the same culture duration, the rate of antrum formation was significantly higher in T(1) than in T(2) from day 12 onwards. Yet, in ovines, when both treatments were compared on day 24 of the culture, there were more viable follicles in T(2) than in T(1) (p<0.05). In the caprine species, percentages of fully grown oocytes (≥ 110 μm) acceptable for IVM after 24 days of culture were significantly higher in normal follicles cultured in T(1) (30.0%) than in T(2) (6.7%; p<0.05). On the other hand, in ovines, at the end of the culture, the percentage of oocytes destined for IVM was higher in T(2) than in T(1) (23.5% vs 2.9%; p<0.05). In conclusion, under the same conditions, the frequency of medium replacement significantly affected the in vitro development of caprine and ovine pre-antral follicles. To improve the efficiency of the culture system, the medium must be replaced every 2 and 6 days for goat and sheep pre-antral follicles, respectively.

This study evaluated the levels of bone morphogenetic protein receptors BMPRIB and BMPRII mRNA in goat follicles and the effects of bone morphogenetic protein-15 (BMP-15) on the in vitro development of cultured preantral follicles. Real-time polymerase chain reaction (PCR) was used to analyze the levels of BMPRIB and BMPRII mRNA in caprine preantral follicles and in small and large antral follicles. Preantral follicles (≥150 μm) were also isolated from goat ovaries and cultured for 18 days in α-MEM(+) supplemented with or without BMP-15 (10, 50, or 100 ng/ml). At the end of culture, some follicles were fixed for ultrastructural evaluation. Real-time PCR showed a reduction in BMPRII mRNA levels from the primary to secondary follicles. Higher levels of BMPRIB mRNA were observed in granulosa/theca cells from large antral follicles compared with small antral follicles. Moreover, BMPRII mRNA was expressed to a greater extent in cumulus-oocyte complexes from large antral follicles than in their respective granulosa/theca cells. In culture, 50 ng/ml BMP-15 positively influenced antral cavity formation and follicle growth after 18 days and also maintained follicular integrity. Thus, BMPRIB and BMPRII mRNAs are present in all follicular categories. BMP-15 (50 ng/ml) stimulates growth, antrum formation and the ultrastructural integrity of isolated caprine preantral follicles after 18 days of culture.