202 EFFECTS OF ASCORBIC ACID ON IN VITRO CULTURE OF CATTLE PREANTRAL FOLLICLES

2010 ◽  
Vol 22 (1) ◽  
pp. 259
Author(s):  
E. R. Andrade ◽  
R. van den Hurk ◽  
L. A. Lisboa ◽  
M. F. Hertel ◽  
F. A. Melo-Sterza ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
In Vitro Culture ◽  
Ovarian Tissue ◽  
Immunohistochemical Analysis ◽  
Development Stage ◽  
Nuclear Antigen ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Ovarian Cortex

The mechanisms that regulate the gradual exit of ovarian follicles from the nongrowing, primordial pool are poorly understood. The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine the effects of this addition on the growth activation and viability of preantral follicles. The ovarian cortex was divided into small fragments; 1 fragment was immediately fixed in Bouin’s solution (control). The other fragments were cultured for 2, 4, 6, or 8 days on culture plates in minimum essential medium (MEM) supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxantine, BSA, and antibiotics (MEM+) or in MEM+ plus ascorbic acid (5, 25, 50, 100, or 200 μg mL-1). Ovarian tissue was processed for classical histology, TEM, and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Preantral follicles were classified according to their development stage (primordial, intermediate, primary, and secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey’s procedure. Chi-square test was used to compare percentages of follicles with PCNA-positive granulosa cells. All analyses were done with Statistical Analysis System (SAS Institute, Cary, NC, USA); P ≤ 0.05 was considered significant. Compared with control fragments, the percentage of primordial follicles was reduced (P ≤ 0.05) and the percentage of growing follicles was increased (P ≤ 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (P ≤0.05), but not when cultures were supplemented with 25, 50, and 100 μg mL-1 of ascorbic acid. Ultrastructural and immunohistochemical analysis of ovarian cortical fragments cultured for 8 days, however, showed the integrity and viability of follicles only when fragments were cultured in the presence of 50 μg mL-1 of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg mL-1 not only stimulates the activation and subsequent growth of cattle primordial follicles that are cultured in vitro for 8 days but also safeguards the viability of these preantral follicles. E. R. Andrade and A. A. Alfieri are recipients of the PRODOC/CAPES fellowship.

scholarly journals Effects of ascorbic acid on in vitro culture of bovine preantral follicles

Zygote ◽  
2012 ◽  
Vol 20 (4) ◽  
pp. 379-388 ◽  
Author(s):  
Evelyn R. Andrade ◽  
Robert van den Hurk ◽  
Lívia A. Lisboa ◽  
Mariana F. Hertel ◽  
Fabiana A. Melo-Sterza ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Early Stage ◽  
Ovarian Tissue ◽  
Immunohistochemical Analysis ◽  
Nuclear Antigen ◽  
Primordial Follicles ◽  
Ovarian Cortex ◽  
Growth Activation ◽  
The Media

SummaryThe objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine their effects on growth activation and viability of early-stage follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed (control) and the other fragments were cultured in minimum essential medium (MEM) supplemented or not with various doses of ascorbic acid. Ovarian tissue was processed for histology, transmission electron microscopy (TEM) and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Compared with control fragments, the percentage of primordial follicles was reduced (p < 0.05) and the percentage of growing follicles had increased (p < 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (p < 0.05), but not when cultures were supplemented with 25, 50 or 100 μg/ml of ascorbic acid. Ultrastructural and immunohistochemical analysis of 8 day cultured ovarian cortical fragments, however, showed the integrity and viability of follicles only when fragments were cultured in presence of 50 μg/ml of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg/ml not only stimulates the activation of 8 day in vitro cultured cattle primordial follicles and subsequent growth of activated follicles, but also safeguards the viability of these early-stage follicles.

325 VIABILITY AND GROWTH OF CATTLE PREANTRAL FOLLICLES AFTER IN VITRO CULTURE OF OVARIAN FRAGMENTS IN α-TOCOPHEROL

2010 ◽  
Vol 22 (1) ◽  
pp. 318 ◽  
Author(s):  
L. A. Lisboa ◽  
E. R. Andrade ◽  
M. F. Hertel ◽  
F. A. Melo-Sterza ◽  
K. Moreno ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Ovarian Tissue ◽  
Nuclear Antigen ◽  
Cortical Tissue ◽  
Chi Square ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Chi Square Test ◽  
Proliferating Cell

The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. The aims of the present study were to investigate the effects of α-tocopherol on survival, activation, and growth of cattle preantral follicles using histological and immunohistochemistry proliferating cell nuclear antigen (PCNA) studies. The ovarian cortex was divided into small fragments; one fragment was immediately fixed in Bouin (control). The other fragments were cultured for 2, 4, 6, or 8 d in culture plates with Minimum Essential Medium supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxanthine, BSA, and antibiotics (MEM+); and MEM+ plus α-tocopherol (5, 25, 50, 100, or 200 ng mL-1). Preantral follicles were classified according to their developmental stage (primordial, intermediate, primary, or secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey’s procedure. Chi-square test was used to compare the percentage of follicles with PCNA-positive granulosa cells. All analyses were done with the SAS software (SAS Institute, Cary, NC, USA), and P < 0.05 was considered significant. 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 of a-tocopherol. 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 6 days of culture in MEM plus 200 ng mL-1 of a-tocopherol. The PCNA analysis confirmed the viability of follicles cultured with 200 ng mL-1 of a-tocopherol after 6 d. After 8 days of in vitro culture, we observed severe follicular degeneration in all media tested, suggesting that other supplements are recommended for longer periods of culture. In conclusion, the results of the present study indicate that 200 ng mL-1 of a-tocopherol maintains the survival of cattle preantral follicles and promotes activation of primordial follicles after 6 days of in vitro culture. Financial support: L. A. Lisboa is a recipient of CAPES support; E. R. Andrade and A. A. Alfieri are recipients of PRODOC/CAPES fellowships.

scholarly journals Effects of α-tocopherol and ternatin antioxidants on morphology and activation of goat preantral follicles in vitro cultured

2009 ◽  
Vol 61 (1) ◽  
pp. 57-65 ◽  
Author(s):  
I.B. Lima-Verde ◽  
M.H.T. Matos ◽  
J.B. Bruno ◽  
F.S. Martins ◽  
R.R. Santos ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Ovarian Tissue ◽  
Control Culture ◽  
Ultrastructural Analysis ◽  
Minimum Essential Medium ◽  
Preantral Follicles ◽  
Ovarian Cortex

The effects of α-tocopherol and ternatin on the morphology, activation, and growth of goat preantral follicles in vitro cultured, for one or five days, were evaluated. Ovarian fragments were immediately fixed (non-cultured control) or in vitro cultured for one or five days in Minimum Essential Medium (MEM) with or without α-tocopherol or ternatin supplementation, both at concentrations of 5, 10, or 15µM, corresponding to the following treatments: MEM, TOC5, TOC10, TOC 15, TER5, TER10, and TER15. The percentages of morphologically normal preantral follicles in non-cultured ovarian tissue (control) was 73.2% and after five days of culture, there was a decrease on these percentages in all treatments (P<0.05) when compared with non-cultured control. Culture of ovarian cortex for five days increased the percentages of follicular activation in all treatments (P<0.05). Ultrastructural analysis did not confirm the integrity of caprine preantral follicles cultured for five days in medium containing antioxidants. This study demonstrated that α-tocopherol and ternatin can promote follicular activation; however, addition of these antioxidants in the tested concentrations reduced the follicular viability after in vitro culture.

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

2008 ◽  
Vol 20 (8) ◽  
pp. 916 ◽  
Author(s):  
F. S. Martins ◽  
J. J. H. Celestino ◽  
M. V. A. Saraiva ◽  
M. H. T. Matos ◽  
J. B. Bruno ◽  
...  
Keyword(s):  
Ovarian Tissue ◽  
Development Stage ◽  
Cortical Tissue ◽  
Minimum Essential Medium ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Differentiation Factor ◽  
Transmission Electron ◽  
Before And After

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.

scholarly journals Transforming growth factor-β (TGF-β) maintains follicular ultrastructure and stimulates preantral follicle growth in caprine ovarian tissue cultured in vitro

2014 ◽  
Vol 66 (2) ◽  
pp. 411-416 ◽  
Author(s):  
G.Q. Rodrigues ◽  
I.M.T. Lima ◽  
R.N. Chaves ◽  
R. Rossetto ◽  
S.L. Costa ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Ovarian Tissue ◽  
Transforming Growth Factor Β ◽  
Preantral Follicle ◽  
Follicle Growth ◽  
Minimum Essential Medium ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Ultrastructural Studies

The objectives of this study were to investigate whether TGF-β affect the survival, activation and further growth of goat primordial follicles enclosed in ovarian cortex after in vitro culture. Goat ovaries were collected from an abattoir and pieces of ovarian tissues were cultured for one or seven days in a supplemented alpha Minimum Essential Medium, alone or containing TGF-β (1, 5, 10 or 50ng/mL). Ovarian tissues from the fresh control as well as those cultured were processed for histological and ultrastructural studies. The results showed that when compared with fresh control, there was decrease in the percentages of histologically normal follicles in all treatments only after seven days culture. TGF-β did not affect the activation of preantral follicles regardless of its concentration, however, larger follicles diameter (P<0.05) was observed using 10ng/mL TGF-β than in the fresh control and other treatments. Moreover, this concentration maintained the normal ultrastructure after seven days of culture. In conclusion, TGF-β showed additional effect on the follicle growth and the maintenance of ultrastructural integrity of goat preantral follicles enclosed in ovarian tissue when used at 10ng/mL during seven days of culture.

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

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 537-549 ◽  
Author(s):  
Regislane P. Ribeiro ◽  
Antonia M.L.R. Portela ◽  
Anderson W.B. Silva ◽  
José J.N. Costa ◽  
José R.S. Passos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ovarian Tissue ◽  
Follicle Stimulating Hormone ◽  
Primordial Follicle ◽  
Nuclear Antigen ◽  
Primordial Follicles ◽  
Primordial Follicle Activation ◽  
Follicle Activation ◽  
Stimulating Hormone

SummaryThis 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.

scholarly journals Interval in the replacement of in vitro culture medium affects the integrity and development of equine preantral follicles

2018 ◽  
Vol 38 (12) ◽  
pp. 2284-2288
Author(s):  
Camila Bizarro-Silva ◽  
Suellen M. González ◽  
Isabela Búfalo ◽  
Andressa G. Lindquist ◽  
Fabiana D. Sarapião ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture System ◽  
Culture Medium ◽  
Follicular Development ◽  
Significance Level ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Medium Exchange ◽  
Significant Difference

ABSTRACT: The efficiency of a culture system is related to the elaboration and replacement of a medium with conditions suitable for follicular development. Recent investigations suggested that in vitro culture medium should be replaced after specific time periods in various species. However, the suitable interval for the exchange of in vitro culture medium has not yet been established in equine species. The objective of this investigation was to evaluate the effect of medium exchange intervals of 24 hours (T24) or 48 hours (T48) for in vitro culture of preantral follicles at 2 or 6 days. At the end of the culture period, the fragments were processed using classical histology. Equine preantral follicles were classified according to morphological integrity and developmental stage. Data analysis was performed using Fisher’s test with a significance level of p<0.05. Out of a total of 399 follicles evaluated, 174 (43.6%) were primordial follicles, 225 (56.4%) were in development, and 63.76% were morphologically intact. In the in vitro culture performed over two days, there was no significant difference in relation to follicular integrity after medium replacement (p>0.05). Compared to the medium replacement at six days of culture, there was a statistically significant difference for T24 (68.9%, p<0.05). Therefore, we suggest changing the medium for equine species at 48 hours after the start of culture followed by subsequent daily replacements.

Effects of melatonin on morphology and development of primordial follicles during in vitro culture of bovine ovarian tissue

2019 ◽  
Vol 54 (12) ◽  
pp. 1567-1573 ◽  
Author(s):  
Barbara N. Cavalcante ◽  
Bruno G. Matos‐Brito ◽  
Lais R. F. M. Paulino ◽  
Bianca R. Silva ◽  
Antonio Wesley Melo Aguiar ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Ovarian Tissue ◽  
Primordial Follicles

scholarly journals Extract of Amburana cearensis maintains the survival of ovine preantral follicles during long-term ovarian tissue transport and promotes primordial follicle activation after in vitro culture

2018 ◽  
Vol 39 (5) ◽  
pp. 2001
Author(s):  
Vanúzia Gonçalves Menezes ◽  
Ricássio De Sousa Barberino ◽  
Bruna Bortoloni Gouveia ◽  
Rodrigo José de Souza Gonçalves ◽  
Jackson Roberto Guedes da Silva Almeida ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Ovarian Tissue ◽  
Primordial Follicle ◽  
Chi Square ◽  
Preantral Follicles ◽  
Primordial Follicle Activation ◽  
Chi Square Test

This study evaluated the effect of Amburana cearensis extract as a preservation or culture medium for ovine ovarian tissue. Ovarian fragments were fixed in 4% buffered formaldehyde for 18 h (fresh control), stored in Minimal Essential Medium (MEM) or in A. cearensis extract (0.1; 0.2 or 0.4 mg/mL) at a temperature of 4ºC for 6, 12 or 24 h (preservation - experiment 1) or cultured for 7 days in ?-MEM+ or in A. cearensis extract without (0.1; 0.2 or 0.4 mg/mL) or with supplements (0.1+ ; 0.2+ or 0.4+ mg/ mL; experiment 2). The percentages of morphologically normal follicles and follicular activation were submitted to analysis of variance (ANOVA) and Tukey´s test. The values of TUNEL-positive cells were submitted to Chi-square test (P < 0.05). The storage of fragments for 6 h in MEM showed higher percentages of normal follicles (62%) and a lower rate of TUNEL positive cells (36.17%) compared to other treatments (normal follicles: 46%; 43% and 52%; TUNEL positive cells: 58.57%; 55.30% and 55.63% for Amb 0.1; Amb 0.2 and Amb 0.4 mg/mL, respectively). However, after 12 or 24 h, MEM (12 h: 48%; 24 h: 45%) and Amb 0.2 mg/mL (12 h: 37%; 24 h: 39%) showed similar percentages of normal follicles and TUNEL positive cells (MEM - 12 h: 43.26%; 24 h: 58%; Amb 0.2 mg/mL - 12 h: 50%; 24 h: 61%). After culture, ?-MEM+ recorded a higher percentage of normal follicles (58.25%) than A. cearensis treatments (32.8%; 25.4% and 34.2% for Amb 0.1; Amb 0.2 and Amb 0.4 mg/mL, and 22.25%; 20.0% and 36.6% for Amb 0.1+ ; Amb 0.2+ and Amb 0.4+ mg/mL, respectively) (P < 0.05). Follicular activation increased in all treatments (52.5%; 36.73%; 54.05%; 47.5% and 58.19% for ?-MEM+ ; Amb 0.1; Amb 0.1+ ; Amb 0.2+ and Amb 0.4+ mg/mL, respectively) compared to the fresh control (11.65%), except for Amb 0.2 mg/mL (23.69%) and Amb 0.4 mg/mL (28.85%) (P > 0.05). Moreover, after in vitro culture, A. cearensis at a concentration of 0.1 mg/mL maintained the percentage of TUNEL positive cells (30.0%) in a way that is similar to that observed in the fresh control (22%) (P > 0.05). In conclusion, ovine preantral follicles can be preserved at 4°C in MEM for 6 h. For longer periods of preservation (24 h), MEM and 0.2 mg/mL A. cearensis are recommended. Moreover, after in vitro culture, A. cearensis extract (0.1 mg/mL) showed higher activation and lower DNA fragmentation in ovine preantral follicles.

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