Maria Helena Tavares de Matos

Essential role of follicle stimulating hormone in the maintenance of caprine preantral follicle viability in vitro

The aims of the present study were to investigate the effects of follicle-stimulating hormone (FSH) on survival, activation and growth of caprine primordial follicles using histological and ultrastructural studies. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM - control medium) supplemented with different concentrations of FSH (0, 10, 50 or 100 ng/ml). Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days in a specific medium were processed for classical histology and transmission electron microscopy (TEM). Additionally, effects of FSH on oocyte and follicle diameter of cultured follicles were evaluated. The results showed that the lowest percentage of normal follicles was observed after 7 days of culture in control medium. After 1 day of culture, a higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FSH. In the presence of 10 and 50 ng/ml of FSH, an increase in diameter of both oocyte and follicle on day 7 of culture was observed. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in MEM plus 50 ng/ml FSH, but did not confirm the integrity of those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that FSH at concentration of 50 ng/ml not only maintains the morphological integrity of 7 days cultured caprine preantral follicles, but also stimulate the activation of primordial follicles and the growth of activated follicles.

Chilling ovarian fragments during transportation improves viability and growth of goat preantral follicles cultured in vitro

The aim of the present study was to evaluate the effects of storage of goat ovarian fragments at different temperatures and for different incubation times on the viability and growth of cultured preantral follicles in vitro. Caprine ovaries were collected and divided into 19 fragments, with one fragment being fixed immediately (fresh control). The remaining fragments were placed in minimal essential medium (MEM) and maintained at 4, 20 or 35 degrees C for 2 or 4 h. After each incubation period, some of the fragments were fixed (non-cultured), whereas others were cultured in vitro for 1 or 7 days. Fragments were processed to enable routine histological and transmission electron microscopic examination. After 7 days of culture, only ovarian fragments stored at 4 degrees C for 4 h maintained a percentage of morphologically normal follicles similar to that in the fresh control. For all other treatments groups, there was a significant increase in follicular activation observed. In addition, there was an increase in oocyte and follicular diameter after culture of ovarian cortex that had been chilled previously at 4 degrees C for 2 or 4 h. In conclusion, the present study demonstrated that chilling ovarian fragments at 4 degrees C during transportation is best for maintaining follicle viability and to increase follicular growth during in vitro culture.

Growth and differentiation factor-9 stimulates activation of goat primordial follicles in vitro and their progression to secondary follicles.

The aim of the present study was to investigate the effects of growth and differentiation factor-9 (GDF-9) on the survival and activation of preantral follicles, as well as their subsequent progression to secondary follicles, using goat ovarian cortical culture in vitro. Pieces of ovarian cortex were cultured for 1 and 7 days in minimum essential medium (MEM) with or without different concentrations of GDF-9 (1-200 ng mL(-1)). On Day 0 and after 1 and 7 days of culture, cortical pieces were fixed for histological and transmission electron microscopy evaluation. Preantral follicles were classified according to their development stage (primordial, intermediate, primary and secondary) and on the basis of morphological features (normal or degenerated). In addition, follicular and oocyte diameters were determined before and after culture. The results showed that, compared with non-cultured cortical tissue (Day 0), the culture of ovarian tissue significantly reduced (P < 0.05) the percentage of normal follicles in all media tested, except for tissue cultured in the presence of 200 ng mL(-1) GDF-9. Furthermore, in all media tested, the percentage of primordial follicles was significantly reduced (P < 0.05), with a concomitant increase in the percentage of developing follicles. The highest percentage of secondary follicles was observed after 7 days of culture in MEM plus 200 ng mL(-1) GDF-9. At all concentrations of GDF-9 tested, follicular diameter increased significantly after 7 days of culture compared with non-cultured cortical tissue. In conclusion, the results of the present study indicate that 200 ng mL(-1) GDF-9 maintains the survival of preantral follicles and promotes activation of primordial follicles. Furthermore, GDF-9 stimulates the transition from primary to secondary follicles, maintaining ultrastructural integrity of the follicles.

Expression of follicle-stimulating hormone receptor (FSHR) in goat ovarian follicles and the impact of sequential culture medium on in vitro development of caprine preantral follicles

This study evaluated the expression of FSH receptors (FSHR) in the different stages of goat follicle development and investigated whether the addition of increasing concentrations of FSH throughout the culture period influences the survival, growth and antral formation of in vitro-cultured caprine preantral follicles. The expression of FSHR was analysed before and after culturing follicles using real-time RT-PCR. For the culture, preantral follicles (≥150 μm) were isolated from ovarian fragments and cultured for 18 days in α-MEM+ alone or associated with recombinant FSH (rFSH: 100 or 1000 ng/ml), or in α-MEM+ supplemented with increasing concentrations of FSH throughout culture periods as follows: (a) sequential medium 1: FSH 100 ng/ml (from day 0 to 6), FSH 500 ng/ml (from day 6 to 12) and FSH 1000 ng/ml (from day 12 to 18); and (b) sequential medium 2: FSH 500 ng/ml (from day 0 to 9) and 1000 ng/ml (from day 9 to 18). Follicle development was evaluated on the basis of antral cavity formation, follicular and oocyte growth, and cumulus-oocyte complex health. The expression of FSHR in isolated caprine follicles increased from the preantral to antral phase. Regarding the culture, after 18 days, sequential medium 1 promoted follicular survival, antrum formation and a reduction in oocyte extrusion. Both sequential media promoted a higher rate of meiotic resumption compared with the other treatments. In conclusion, the addition of increased concentrations of FSH (sequential medium) has a significant impact on the in vitro development of caprine preantral follicles.

In vitro survival and development of goat preantral follicles in two different oxygen tensions

The aim of the present study was to evaluate the effect of two different oxygen (O(2)) concentrations on survival and development of preantral follicles of goats cultured in vitro. Preantral ovarian follicles (> or =150 microm) were isolated from ovarian cortex fragments of goats and individually cultured for 30 days under two different O(2) concentrations (5% and 20% O(2)). Follicle development was evaluated on the basis of antral cavity formation, increase in follicular diameter, presence of healthy cumulus oocyte complexes and fully grown oocytes. Results showed with progression of culture period from 6 to 12 days, a decrease in follicular survival was observed in both O(2) concentrations (P<0.05). When the O(2) tensions were compared to each other in the different days of culture, 20% O(2) was more efficient in promoting an increase in follicular diameter from day 24 of culture onward than 5% O(2) (P<0.05). However, follicles cultured with 5% O(2) had an increased percentage of antrum formation from 12 days to the end of culture, compared with 20% O(2) (P<0.05). Moreover, there was no difference in percentage of fully developed oocytes with the different O(2) tensions. However, only oocytes (16.7%) from follicles cultured in 20% O(2) resumed meiosis. In conclusion, concentration of 20% O(2) was more efficient in promoting follicular growth and oocyte meiosis resumption from preantral follicles of goats when grown in vitro.

Interaction between ascorbic acid and follicle-stimulating hormone maintains follicular viability after long-term in vitro culture of caprine preantral follicles.

This study evaluates the effects of ascorbic acid and its interaction with follicle-stimulating hormone (FSH) on the morphology, activation, and in vitro growth of caprine preantral follicles. Ovarian fragments were cultured for 1, 7, or 14 d in minimum essential medium (MEM) containing ascorbic acid (50 or 100microg/mL), FSH (50ng/mL), or both of these substances. Ovarian tissue that was either fresh (control) or cultured for 1, 7, or 14 d was processed for histological and ultrastructural evaluation. The results showed that after 14 d of culture, medium supplemented with 50microg/mL of ascorbic acid alone or combined with FSH showed higher rates of follicular survival compared with MEM. After 7 d of culture, FSH, ascorbic acid at 50microg/mL with or without FSH, and ascorbic acid at 100microg/mL increased the percentage of follicular activation compared to fresh control. In addition, FSH alone significantly increased the percentage of growing follicles after 14 d. The combination of 50microg/mL of ascorbic acid and FSH promoted a significant increase in oocyte and follicular diameter after 7 d of culture. Ultrastructural and fluorescent analysis confirmed the integrity of follicles cultured with 50microg/mL of ascorbic acid and FSH after 14 d. In conclusion, the combination of 50microg/mL of ascorbic acid and FSH maintained follicular integrity and promoted follicular activation and growth after long-term in vitro culture of caprine preantral follicles.

Follicle stimulating hormone and fibroblast growth factor-2 interact and promote goat primordial follicle development in vitro

The aims of the present study were to investigate the effects of the interaction between follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on survival, follicular growth initiation and further growth of caprine preantral follicles. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM) supplemented with FSH, FGF-2 or FSH + FGF-2. Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days were processed for classical histology and transmission electron microscopy (TEM) to verify follicular morphology and growth. The results showed that, after 7 days culture, the highest percentages of normal follicles were observed in medium supplemented with FSH. After 7 days culture, the interaction between FSH and FGF-2 was most effective to promote the initiation of primordial follicles growth and oocyte growth. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in all treatments, except in those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that the interaction between FSH and FGF-2 stimulates the initiation of primordial follicles growth and the subsequent growth of developing follicles. Furthermore, these data showed that FSH is important to maintain follicular integrity after 7 days culture.

Steady-state level of kit ligand mRNA in goat ovaries and the role of kit ligand in preantral follicle survival and growth in vitro.

The aims of this study were to investigate steady‐state level of Kit Ligand (KL) mRNA and its effects on in vitro survival and growth of caprine preantral follicles. RT‐PCR was used to analyze caprine steady‐state level of KL mRNA in primordial, primary, and secondary follicles, and in small (1–3 mm) and large (3–6 mm) antral follicles. Furthermore, ovarian fragments were cultured for 1 or 7 days in Minimal Essential Medium (MEM+) supplemented with KL (0, 1, 10, 50, 100, or 200 ng/ml). Noncultured (control) and cultured fragments were processed for histology and transmission electron microscopy (TEM). RT‐PCR demonstrated an increase in steady‐state level of KL mRNA during the transition from primary to secondary follicles. Small antral follicles had higher steady‐state levels of KL mRNA in granulosa and theca cells than large follicles. After 7 days, only 50 ng/ml of KL had maintained the percentage of normal follicles similar to control. After 1 day, all KL concentrations reduced the percentage of primordial follicles and increased the percentage of growing follicles. KL at 10, 50, 100, or 200 ng/ml increased primary follicles, compared to MEM+ after 7 days. An increase in oocyte and follicular diameter was observed at 50 ng/ml of KL. TEM confirmed ultrastructural integrity of follicles after 7 days at 50 ng/ml of KL. In conclusion, the KL mRNAs were detected in all follicular categories. Furthermore, 50 ng/ml of KL maintained the integrity of caprine preantral follicle cultured for 7 days and stimulated primordial follicle activation and follicle growth. Mol. Reprod. Dev. 77: 231–240, 2010.

Effects of Fibroblast Growth Factor-2 on the in vitro Culture of Caprine Preantral Follicles

The aims of the present study were to evaluate the effects of fibroblast growth factor-2 (FGF-2) on survival, activation and growth of caprine early-staged (preantral) follicles using histological and ultrastructural studies. Fragments of caprine ovarian cortex were cultured for 1 or 5 days in an enriched minimum essential medium, supplemented or not with different concentrations of FGF-2 (10, 50 or 100 ng/ml). Fragments from non-cultured ovarian tissue (control) and from tissues cultured for 1 or 5 days in a specific medium were processed for transmission electron microscopy (TEM) or classical histology to evaluate the morphological quality of caprine preantral follicles and to calculate the percentages of normal follicles. Additionally, effects of FGF-2 on oocyte and follicle diameter of cultured preantral follicles were investigated. Our results showed that, although the percentages of histologically normal follicles were lower in cultured than in non-cultured ovarian tissue fragments, there were no differences in this regard among treatments, neither on day 1 nor on day 5 of culture. After 1 and 5 days of culture, a significantly higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FGF-2. This FGF-2 treatment furthermore resulted in an increase in diameter of both oocytes and follicles that were cultured for 5 days. TEM showed that the ultrastructural integrity of caprine preantral follicles was maintained during their 5-day culture in the presence of 50 ng/ml FGF-2. In conclusion, this study demonstrated that at a concentration of 50 ng/ml FGF-2 not only maintains the morphological integrity of caprine preantral follicles cultured for 5 days, but also stimulates the activation of primordial follicles and the growth of activated follicles.

Expression of vascular endothelial growth factor (VEGF) receptor in goat ovaries and improvement of in vitro caprine preantral follicle survival and growth with VEGF.

The aim of the present study was to evaluate the effect of vascular endothelial growth factor (VEGF) on the survival and growth of goat preantral follicles after in vitro culture and to verify the expression of VEGF receptor (VEGFR)-2 in goat ovaries. Ovarian fragments were cultured for 1 or 7 days in minimal essential medium (MEM) with different concentrations of VEGF (1, 10, 50, 100 or 200 ng mL(-1)). Non-cultured (fresh control) and cultured tissues were processed for histological and ultrastructural studies. The results showed that 200 ng mL(-1) VEGF resulted in a similar percentage of normal preantral follicles after 1 and 7 days of culture compared with control. Compared with basic culture medium alone, an increase in follicular and oocyte diameters was observed in the presence of 10 ng mL(-1) VEGF after 7 days culture. Ultrastructural analysis confirmed follicular integrity after 7 days culture in the presence of 200 ng mL(-1) VEGF. Immunohistochemical studies demonstrated the expression of VEGFR-2 in oocytes and granulosa cells of all follicular stages, except in granulosa cells of primordial follicles. In conclusion, the present study has shown that VEGF maintains follicular ultrastructural integrity and promotes follicular growth. In addition, VEGFR-2 is expressed in oocytes of caprine ovarian follicles at all developmental stages and in granulosa cells of developing follicles.