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

2013 ◽  
Vol 25 (6) ◽  
pp. 955 ◽  
Author(s):  
A. M. C. V. Alves ◽  
R. N. Chaves ◽  
R. M. P. Rocha ◽  
L. F. Lima ◽  
P. M. Andrade ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Period ◽  
Oocyte Diameter ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Differentiation Factor ◽  
Follicle Diameter ◽  
Growth Differentiation Factor 9

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.

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 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.

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.

224. Platelet derived growth factors and receptors in the rat ovary contribute towards preantral follicle growth

2005 ◽  
Vol 17 (9) ◽  
pp. 87
Author(s):  
L. S. Sleer ◽  
C. C. Taylor
Keyword(s):  
Growth Factors ◽  
Preantral Follicle ◽  
Follicle Growth ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Follicle Diameter ◽  
The Family ◽  
Rat Ovary ◽  
In Cells

In this study, the family of platelet derived growth factors (PDGF) and receptors were identified and characterized in the rat ovary and a role in contributing towards growth of preantral follicles was revealed. Real-time polymerase chain reaction revealed the presence of mRNA for all platelet derived growth factors (PDGF-A, PDGF-B, PDGF-C and PDGF-D) and receptors (PDGF-Rα and PDGF-Rβ). In situ hybridization identified oocytes of primordial/primary follicles and cells of the theca layer as a source of PDGF-B, PDGF-C and PDGF-D mRNA. Protein expression was explored through immunohistochemistry. In rats aged days 0 and 4, PDGF-Rα, PDGF-A and PDGF-C immunoreactivity was observed within oocyte clusters, and PDGF-Rβ and PDGF-B immunoreactivity in cells surrounding oocyte clusters. In primordial follicles, PDGF-Rβ and PDGF-C was observed in the oocyte, and PDGF-Rα and A in the either the oocyte or pregranulosa cells. In primary follicles, PDGF-A, PDGF-C, PDGF-Rα and PDGF-Rβ are expressed in the oocyte. PDGF-Rβ is also expressed in cells surrounding primordial and primary follicles, possibly the precursors to theca cells. In secondary and antral follicles, all four PDGF isoforms and both receptors are expressed in either theca or vascular cells of the theca layer, and PDGF-Rα and A are also expressed in some granulosa cells in rats aged day 20 and older. A role in preantral follicle growth was identified by in vitro culture of preantral follicles. Preantral follicles cultured in serum free medium increased in diameter by 11.0 ± 1.57% over 5 days. Addition of PDGF-AA, PDGF-AB or PDGF-BB to the medium resulted in increases in follicle diameter after 5 days of 18.32 ± 2.18%, 17.72 ± 2.3% and 17.6 ± 1.81%, respectively, representing a significant increase over control diameters. In summary, this study has identified and characterized the presence and localization of all members of the family of platelet derived growth factors and receptors in the rat ovary and revealed a role for these growth factors in positively influencing early follicle growth.

Long-Term in vitro Culture of Bovine Preantral Follicles by Using Different Culture Media

2008 ◽  
Vol 8 (2) ◽  
pp. 386-391
Author(s):  
Khairy M.A. Zoheir ◽  
Nermin El-Halawan ◽  
Xiang Li ◽  
Moaeen-ud- Din ◽  
Li-Guo Yang
Keyword(s):  
In Vitro Culture ◽  
Culture Media ◽  
Preantral Follicles

Effect of growth differentiation factor 9 (GDF-9) on in vitro development of collared peccary preantral follicles in ovarian tissues

2021 ◽  
Vol 226 ◽  
pp. 106717
Author(s):  
Lívia B. Campos ◽  
Andreia M. Silva ◽  
Érica C.G. Praxedes ◽  
Luana G.P. Bezerra ◽  
Jeferson L.S. Freitas ◽  
...  
Keyword(s):  
In Vitro Development ◽  
Preantral Follicles ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9 ◽  
Collared Peccary ◽  
Vitro Development

scholarly journals Equol: A Microbiota Metabolite Able to Alleviate the Negative Effects of Zearalenone during In Vitro Culture of Ovine Preantral Follicles

Toxins ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 652
Author(s):  
Talyne Emilia Santos Silva ◽  
Danielle Cristina Calado de Brito ◽  
Naiza Arcângelo Ribeiro de Sá ◽  
Renato Felix da Silva ◽  
Anna Clara Accioly Ferreira ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Cell Types ◽  
Culture Conditions ◽  
Dna Double Strand Breaks ◽  
Female Reproduction ◽  
Negative Effects ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
The Impact

The impact of zearalenone (ZEN) on female reproduction remains an issue, since its effects may differ among exposed cell types. Besides the use of decontaminants in animal diet, other approaches should be considered to minimise ZEN effects after exposure. Since the first organ in contact with ZEN is the gastrointestinal tract, we hypothesise that products of microbiota metabolism may play a role in ZEN detoxification. We aimed to evaluate the effect of 1 µmol/L ZEN and 1 µmol/L equol (a microbial metabolite), alone or in combination, on the survival and morphology of in vitro cultured ovarian preantral follicles. Ovaries from 12 sheep were collected at a local abattoir and fragmented, and the ovarian pieces were submitted to in vitro culture for three days in the presence or absence of the test compounds. The follicular morphology was impaired by ZEN, but equol could alleviate the observed degeneration rates. While ZEN decreased cell proliferation in primary and secondary follicles, as well as induced DNA double-strand breaks in primordial follicles, all these observations disappeared when equol was added to a culture medium containing ZEN. In the present culture conditions, equol was able to counteract the negative effects of ZEN on ovarian preantral follicles.

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