M. V. A. Saraiva

The bone morphogenetic protein system and the regulation of ovarian follicle development in mammals.

The bone morphogenetic protein (BMP) family consists of several growth factor proteins that belong to the transforming growth factor-β (TGF-β) superfamily. BMPs bind to type I and type II serine-threonine kinase receptors, and transduce signals through the Smad signalling pathway. BMPs have been identified in mammalian ovaries, and functional studies have shown that they are involved in the regulation of oogenesis and folliculogenesis. This review summarizes the role of the BMP system during formation, growth and maturation of ovarian follicles in mammals.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles.

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.

Protein and messenger RNA expression of interleukin 1 system members in bovine ovarian follicles and effects of interleukin 1β on primordial follicle activation and survival in vitro

This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows and to evaluate the effects of IL-1β on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM(+) supplemented with IL-1β (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1β, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1β (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1β promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis.

Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles.

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.

N-acetylcysteine improves morphologic and functional aspects of ovarian grafts in rats

PURPOSE To evaluate morphological and functional aspects of the ovarian graft in transplanted rats treated with NAC. METHODS Female Wistar rats, virgin, 3 to 4 months old, weighing 200-250 grams were used in experiments. The rats have been kept in proper sanitary conditions, receiving food and water ad libitum. Five groups (n=10, each) were constituted: 4 groups treated subcutaneously with NAC, at doses of 150, 300, 600 and 1200 mg/kg (NAC150, NAC300, NAC600 and NAC1200, respectively), one hour of before the ovarian transplantation and control group (GTx) - treated with physiological solution and submitted to ovarian transplantation. The rats were anesthetized and submitted to autologous left ovarian transplantation, without anastomosis in retroperitoneum, and contralateral oophorectomy. During follow-up of 4 or 15 days, the estrous cycle was evaluated by vaginal smears to determine cycle regularity. At the end of 4th or 15th days, rats were re-anesthetized and blood and graft were obtained to estradiol analysis and morphological assessment. Data were analysed by One Way Analysis of Variance (ANOVA) or ANOVA on ranks complemented by Student-Newman-Keuls test. RESULTS At 4th day, viable follicles in the graft did not altered by NAC treatments. The NAC300 and NAC600 groups showed increasing in follicle atresia (p=0.012) compared to GTx and NAC1200 group. At 15th day, 50% of GTx, NAC150, and NAC300 rats showed regular oestrous cycle; 83% of NAC600 and 100% of NAC1200 rats returned to regular cycle. NAC1200 group showed increasing in primordial follicle compared to GTx, NAC150 or NAC300 (p=0.011). NAC did not interfere in estradiol levels after 4 or 15 days of transplantation. CONCLUSION In autologous ovarian transplantation, high dose of NAC promotes graft viability with recovery of estrous cycle.

Effects of bone morphogenetic protein 4 (BMP4) on in vitro development and survival of bovine preantral follicles enclosed in fragments ovarian tissue

The aim of this study was to evaluate the effects of different concentrations of BMP4 on activation, development and mRNA expression of GDF9, BMP15, PCNA, Bax and Bcl2 in cultured bovine follicles enclosed in ovarian tissues. Ovarian tissue fragments were cultured for 6 days in α-MEM+ alone or supplemented with different concentrations of BMP4 (10, 50 or 100 ng/ml). Classical histology was performed to analyze follicle growth and morphology, while real-time PCR was used to analyze mRNA levels in fresh and cultured tissues. After 6 days, the culture of ovarian tissue in α-MEM+ alone or supplemented with 10, 50 or 100 ng/ml BMP4 promoted follicular activation. The different concentrations of BMP4 maintained the percentage of normal follicles similar to results of the control. The presence of 100 ng/ml BMP-4 in culture medium increased oocyte and follicular diameters of primary and secondary follicles when compared with those follicles from uncultured control or cultured in α-MEM+ alone (P < 0.05). The tissues cultured in the presence of increasing concentrations of BMP4 had an increase in mRNA expression of the tested genes, but despite this the differences were not statistically significant. In conclusion, 100 ng/ml BMP4 promotes an increase in diameters of follicles and oocytes of primary and secondary follicles after 6 days of in vitro culture.

Effects of jacalin and follicle-stimulating hormone on in vitro goat primordial follicle activation, survival and gene expression.

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.