In vitro development of primordial follicles after long-term culture of goat ovarian tissue

This study aims to investigate the effects of follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on the survival and growth of caprine preantral follicles. Ovarian tissues were cultured for 1, 7, 14, 21 or 28 days in medium supplemented with FSH (FSH-2d or FSH-7d, i.e., with replacement of the culture medium every 2 or 7 days, respectively) or FSH + FGF-2 (replacement of the medium every 2 days). Non-cultured (control) and cultured ovarian fragments were processed for histological and ultrastructural analysis. After 28 days of culture, the media supplemented with FSH-2d was the most effective in maintaining the percentage of normal follicles and in promoting follicular growth. Furthermore, both treatments with FSH increased the percentage of the primary follicles. However, ultrastructural studies did not confirm follicular integrity from 14 days of culture onward. In conclusion, culturing tissue for up to 7 days in medium containing FSH alone or combined with FGF-2 maintains caprine preantral follicle integrity and promotes their growth 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 100 μg/mL), FSH (50 ng/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 50 μg/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 50 μg/mL with or without FSH, and ascorbic acid at 100 μg/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 50 μg/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 50 μg/mL of ascorbic acid and FSH after 14 d. In conclusion, the combination of 50 μg/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

Levels of mRNA for bone morphogenetic proteins, their receptors and SMADs in goat ovarian follicles grown in vivo and in vitro

This study investigated the stability of housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase, β-tubulin, β-actin, phosphoglycerate kinase (PGK), 18S rRNA, ubiquitin and ribosomal protein 19) and the levels of mRNA for bone morphogenetic protein-2 (BMP-2), -4 (BMP-4), -6 (BMP-6), -7 (BMP-7) and -15 (BMP-15), their receptors (BMPR-IA, -IB and -II) and Similar to Mothers Against Decapentaplegic (SMADs) (-1, -5 and -8) in goat follicles of 0.2, 0.5 and 1.0 mm, as well as in secondary follicles before and after culture for 18 days. β-tubulin and PGK were the most stable housekeeping genes and the levels of mRNA for BMP-2 in follicles of 0.2 mm were higher than in follicles of 0.5 and 1.0 mm. For BMP-4, -6 and -7, the highest levels of mRNA were found in follicles of 1.0 mm. The expression of BMPR-IB was higher in follicles of 0.2 mm, whereas the levels of BMPR-II were higher in follicles of 0.5 mm. The levels of mRNA for SMAD-5 were higher in follicles of 0.2 mm, whereas SMAD-8 had higher levels in 0.5-mm follicles. After culture, follicles showed increased levels of mRNA for BMP-2 and reduced mRNA for BMP-4, BMP-7, BMPR-IA and SMAD-5. In conclusion, β-tubulin and PGK are the most stable reference genes, and BMPs, their receptors and SMADs have variable levels of mRNA in the follicular size classes analysed.</jats:p

Expression of growth and differentiation factor 9 (GDF-9) and its effect on the in vitro culture of caprine preantral ovarian follicles

AbstractThis study examined the expression of growth and differentiation factor 9 (GDF-9) in caprine ovarian follicles, and the effect of GDF-9 with or without FSH on the in vitro culture of preantral follicles. To evaluate the expression of GDF-9 in Experiment 1, follicles were recovered from 32 goat ovaries and the total RNA isolated and transcribed for real-time polymerase chain reaction (PCR). Experiments 2 and 3 each used a further 32 goat ovaries to provide preantral follicles of ≥150μm. These follicles were isolated and cultured individually in 100μL drops. In each experiment at least 45 follicles were used per treatment. Every 6 days, follicles were evaluated for viability, antrum formation and growth rate. At the end of the culture period, oocytes were submitted to in vitro maturation (IVM), viability tests and chromatin evaluation. In Experiment 2, follicles were cultured in a basal medium (control) or this medium supplemented with GDF-9 at a concentration of 100ng/mL (GDF-9 100) or 200ng/mL (GDF-9 200). The same media were used in Experiment 3, supplemented with recombinant FSH at a level of 100ng/mL from day 0, 500ng/mL from day 6 to 12 and 1000ng/mL from day 12 to 18 of culture to form the three treatments: control FSH, GDF-9 (100) plus FSH and GDF-9 (200) plus FSH. Relative GDF-9 expression (Experiment 1) was greater in the secondary (18units) than the primordial (1unit) and the primary (1unit) preantral follicles (P<0.05). In the antral follicles, GDF-9 expression was significantly higher in the cumulus–oocyte complexes COC's<3mm (1.6units) than those of >3mm diameter (1unit; P<0.05), and in COC's<3mm and >3mm (319.2 and 200.1units, respectively), compared to their respective granulosa and theca cells (1unit for each category, P<0.05). In Experiment 2, GDF-9 supplementation significantly improved the survival of the follicles (60.8%, 66.0% and 77.4% for the control, GDF-9 100 and GDF-9 200, respectively; P<0.05), follicular growth rate and antrum formation following 18 days of culture. Oocyte survival was approximately 100% in all treatments. More oocytes were submitted to IVM from GDF-9 100 (78.0%; P<0.05), compared to GDF-9 200 (48.1%), but no suitable oocytes could be retrieved from the control (58.8%). The proportion of oocytes showing a resumption of meiosis, was not significantly different between treatments (41.4%, 35.9% and 36.0% for the control, GDF-9 100 and GDF-9 200, respectively). The addition of GDF-9 to the media supplemented with FSH (Experiment 3) did not significantly affect any of the variables studied. The proportion of oocytes submitted to IVM in Experiment 3 was 53.3%, 56.5% and 63.8% for the control FSH, GDF-9 100 plus FSH and GDF-9 200 plus FSH, respectively (no statistical differences). The resumption of meiosis was 75.0%, 60.9% and 60.7% for the control FSH, GDF-9 100 plus FSH and GDF-9 200 plus FSH, respectively (NS). The occurrence of metaphase II was very low in both experiments. It was concluded that the supplementation of a basal medium with GDF-9 had a positive effect on the survival and development of caprine preantral follicles, but had no real effect in the presence of FSH

Interaction between growth differentiation factor 9, insulin-like growth factor I and growth hormone on the in vitro development and survival of goat preantral follicles

The objective of this study was to determine the effects of GDF-9, IGF-I, and GH alone or combined on preantral follicle survival, activation and development after 1 and 7 days of in vitro culture. Either fresh (non-cultured) or cultured ovarian tissue was processed for histological and fluorescence analysis. For all media tested, the percent of normal follicles was greater when compared to minimum essential medium supplemented (MEM+) alone, except when ovarian tissue was cultured with GDF-9/IGF-I or GDF-9/GH (P < 0.05). Fluorescence analysis showed that the percent of viable follicles after 7 days of culture was similar for non-cultured tissue and for all treatments tested. The percent of primordial follicles was reduced (P < 0.05) and there was a significant and concomitant increase in the percent of intermediate and primary follicles in all treatments tested after 7 days of culture when compared to non-cultured tissue. After 7 days of culture, the highest percent of intermediate follicles was observed with IGF-I/GH (61.3%), and the highest percent of primary follicles was achieved with IGF-I (57.7%). After 7 days of culture in MEM+ containing GDF-9, IGF-I and GH alone or in all associations, a significant increase in follicular diameter was observed when compared to MEM+ alone and non-cultured tissue. In conclusion, GDF-9, IGF-I and GH alone or in combination maintain preantral follicle survival and promote primordial follicle activation. Nevertheless, the data showed that IGF-I/GH and IGF-I alone are efficient in promoting the transition from primordial to intermediate follicles and from intermediate to primary follicles, respectively