A. M. C. V. Alves

Role of nerve growth factor (NGF) and its receptors in folliculogenesis

Summary Nerve growth factor (NGF) is a prototype member of the neurotrophins family and has important functions in the maintenance of viability and proliferation of neuronal and non-neuronal cells, such as certain ovarian cells. The present review highlights the role of NGF and its receptors on ovarian follicle development. NGF initiates its multiple actions through binding to two classes of receptors: the high affinity receptor tyrosine kinase A (TrkA) and the low-affinity receptor p75. Different intracytoplasmic signalling pathways may be activated through binding to NGF due to variation in the receptors. The TrkA receptor activates predominantly phosphatidylinositol-3-kinase (PI3K) and mitogenic activated protein kinase (MAPK) to promote cell survival and proliferation. The activation of the phospholipase type Cγ (PLCγ) pathway, which results in the production of diacylglycerol (DAG) and inositol triphosphate (IP3), culminates in the release of calcium from the intracytoplasmic cellular stocks. However, the details of activation through p75 receptor are less well known. Expression of NGF and its receptors is localized in ovarian cells (oocyte, granulosa, theca and interstitial cells) from several species, which suggests that NGF and its receptors may regulate some ovarian functions such as follicular survival or development. Thus, the use of NGF in culture medium for ovarian follicles may be of critical importance for researchers who want to promote follicular development in vitro in the future.

Interaction between melatonin and follicle-stimulating hormone promotes in vitro development of caprine preantral follicles

The aim of this study was to investigate the effects of melatonin and follicle-stimulating hormone (FSH) on the in vitro culture of goat preantral follicles. Ovarian fragments were cultured for 7 d in α-minimum essential medium (α-MEM(+)) containing melatonin (100, 250, 500, or 1,000 pM), FSH (50 ng/mL), or a combination of the 2 hormones and further analyzed by histology and transmission electron and fluorescent microscopy. The results showed that after 7 d of culture, tissues cultured in α-MEM(+) alone or supplemented with FSH alone, melatonin (500 and 1,000 pM), or the combination of FSH and melatonin (1,000 pM) maintained percentages of normal preantral follicles similar to the fresh control. In contrast to the noncultured tissues, the percentage of developing follicles was increased under all culture conditions after 7 d (P < 0.05). The addition of 1,000 pM melatonin associated with FSH to the culture medium increased follicular and oocyte diameters compared with α-MEM(+) alone after 7 d of culture (P < 0.05). Ultrastructural and fluorescent analyses confirmed the integrity of follicles cultured with 1,000 pM of melatonin plus FSH for 7 d. In conclusion, this study demonstrated that the interaction between melatonin and FSH maintains ultrastructural integrity and stimulates further growth of cultured caprine preantral follicles.

Nerve growth factor promotes the survival of goat preantral follicles cultured in vitro.

The aim of this study was to investigate the effects of nerve growth factor (NGF) on the in vitro culture of goat preantral follicles. Ovarian cortex fragments were cultured in α-MEM+ supplemented with 0, 1, 10, 50, 100 or 200 ng/ml NGF for 1 or 7 days. Small fragments of noncultured ovarian tissue as well as those cultured for 1 or 7 days were processed for histology and transmission electron microscopy. The results showed that after 1 or 7 days of culture at all concentrations of NGF, except at 1 ng/ml after 1 day of culture, there was a significant reduction in the percentage of normal follicles compared to noncultured tissues. At higher NGF concentrations (100 and 200 ng/ml) after 7 days of culture, there was a significant reduction in the percentage of normal follicles compared to tissues cultured in α-MEM+ alone or at the other concentrations of NGF. It is important to note that ultrastructural and fluorescent analyses confirmed only the integrity of follicles cultured with 1 ng/ml of NGF after 7 days. In contrast to noncultured control tissues, the percentage of developing follicles was significantly increased at all concentrations of NGF after 1 or 7 days of culture. We observed that follicular diameter was greater at 1 and 10 ng/ml NGF after culture for 7 days than at the other concentrations but was similar to follicles cultured in α-MEM+ alone. In conclusion, NGF improved the survival of goat preantral follicles cultured in vitro in a dose-dependent manner.

Fibroblast growth factor-10 maintains the survival and promotes the growth of cultured goat preantral follicles

The aim of the present study was to investigate the effects of fibroblast growth factor-10 (FGF-10) on the survival, activation (transition from primordial to primary follicles), and growth of goat preantral follicles cultured in vitro. Pieces of ovarian cortex were cultured for 1 and 7 d in the absence or presence of FGF-10 (0, 1, 10, 50, 100, and 200 ng/mL). Noncultured and cultured tissues were processed and analyzed by histology, transmission electron microscopy, and viability testing. Results showed that after 7 d, a greater percentage (79.9%) of morphologically normal follicles (containing an oocyte with regular shape and uniform cytoplasm, and organized layers of granulosa cells without a pyknotic nucleus) was observed when cultured with 50 ng/mL of FGF-10 when compared with other concentrations of FGF-10 (0 ng/mL, 67.3%; 1 ng/mL, 68.2%; 10 ng/mL, 63.3%; 100 ng/mL, 64.4%; 200 ng/mL, 52.7%). Ultrastructural analyses and viability testing using fluorescent markers confirmed the follicular integrity of FGF-10 (50 ng/mL)-treated fragments after 7 d of culture. After 7 d, all FGF-10 concentrations reduced the percentage of primordial follicles and increased the percentage of developing follicles. In the presence of 50 ng/mL of FGF-10, follicles increased in diameter after 7 d of culture when compared with other concentrations tested. In conclusion, this study demonstrates that FGF-10 maintains the morphological integrity of goat preantral follicles and stimulates the growth of activated follicles in culture. The culture conditions identified here contribute to the understanding of the factors involved in goat early follicular development.

Dynamic medium containing growth differentiation factor-9 and FSH maintains survival and promotes in vitro growth of caprine preantral follicles after long-term in vitro culture

The aim of the present study was to evaluate the effects of growth differentiation factor 9 (GDF-9) and FSH on the in vitro development of caprine preantral follicles cultured for 16 days. Ovarian fragments were cultured in αMEM⁺ (α-minimum essential medium, pH 7.2-7.4, 10 μg mL⁻¹ insulin, 5.5 μg mL⁻¹ transferrin, 5.0 ng mL⁻¹ selenium, 2 mM glutamine, 2 mM hypoxanthine and 1.25 mg mL⁻¹ bovine serum albumin) in the absence or presence of 200 ng mL⁻¹ GDF-9 and/or 50 ng mL⁻¹ FSH added during the first (Days 0-8) and/or second (Days 8-16) half of the culture period. Non-cultured and cultured fragments were processed for histological and ultrastructural analyses. After 16 days, all treatments using GDF-9 or FSH showed higher rates of follicular survival compared with αMEM⁺ alone. Compared with non-cultured control, sequential culture media containing GDF-9 and/or FSH significantly increased the percentage of developing follicles and follicle diameter. Moreover, a progressive increase in oocyte diameter was observed only with sequential culture medium containing GDF-9 until Day 8 followed by FSH (GDF-9/FSH) in the second half of the culture period. After 16 days of culture, ultrastructural analysis confirmed the integrity of follicles cultured in the presence of GDF-9/FSH. In conclusion, a dynamic medium containing GDF-9 and FSH (GDF-9/FSH) maintained follicular integrity and promoted activation of primordial follicles and growth during long-term in vitro culture of goat preantral follicles.

Comparison between the additive effects of diluted (rFSH) and diluted/dynamized (FSH 6 cH) recombinant follicle-stimulating hormone on the in vitro culture of ovine preantral follicles enclosed in ovarian tissue

OBJECTIVE This study compared 2 types of recombinant follicle stimulating hormone (rFSH): diluted and diluted/dynamized, on in vitro development of ovine follicles. METHODS In experiment 1, ovarian fragments were cultured for 1 or 7 days in α-MEM(+) in the absence or presence of different concentrations of diluted rFSH to determine the best concentration. In experiment 2, the effect of diluted and diluted/dynamized rFSH (rFSH 6 cH--ultradiluted and succussioned), alone or in combination, was studied. RESULTS In experiment 1, compared to control, 50ng/mL of diluted rFSH induced higher rates of follicular survival after 7 days of culture and higher percentages of growing follicles at day 1 of culture (P<0.05). In experiment 2, compared to control, diluted/dynamized rFSH induced higher follicular diameter and survival rate after 7 days and early follicle activation at day 1 of culture (P<0.05). Compared to diluted rFSH, diluted/dynamized rFSH induced higher rates of follicle activation at day 1 of culture (P<0.05). CONCLUSION In conclusion, compared to the control medium, diluted/dynamized rFSH promoted survival and early activation of follicles, while diluted rFSH promoted higher activation later in the culture. Thus, diluted/dynamized rFSH may be used as an alternative to diluted rFSH for the in vitro culture of ovine preantral follicles.