The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⁻¹) from Days 0 to 6 and 500 ng mL⁻¹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⁻¹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⁻¹ bovine serum albumin, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 μg mL⁻¹ ascorbic acid (α-MEM⁺). Comparisons of uncultured (0.2 mm) and α-MEM⁺ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.

This study aims to investigate the effects of jacalin and follicle-stimulating hormone (FSH) on activation and survival of goat primordial follicles, as well as on gene expression in cultured ovarian tissue. Ovarian fragments were cultured for 6 days in minimum essential medium (MEM) supplemented with jacalin (10, 25, 50 or 100 μg/ml - Experiment 1) or in MEM supplemented with jacalin (50 μg/ml), FSH (50 ng/ml) or both (Experiment 2). Non-cultured and cultured tissues were processed for histological and ultrastructural analysis. Cultured tissues from Experiment 2 were also stored to evaluate the expression of BMP-15, KL (Kit ligand), c-kit, GDF-9 and proliferating cell nuclear antigen (PCNA) by real-time polymerase chain reaction (PCR). The results of Experiment 1 showed that, compared with tissue that was cultured in control medium, the presence of 50 μg/ml of jacalin increased both the percentages of developing follicles and viability. In Experiment 2, after 6 days, higher percentages of normal follicles were observed in tissue cultured in presence of FSH, jacalin or both, but no synergistic interaction between FSH and jacalin was observed. These substances had no significant effect on the levels of mRNA for BMP-15 and KL, but FSH increased significantly the levels of mRNA for PCNA and c-kit. On the other hand, jacalin reduced the levels of mRNA for GDF-9. In conclusion, jacalin and FSH are able to improve primordial follicle activation and survival after 6 days of culture. Furthermore, presence of FSH increases the expression of mRNA for PCNA and c-kit, but jacalin resulted in lower GDF-9 mRNA expression.

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