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

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab202 ◽

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.

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Epigallocatechin-3-gallate (EGCG) reduces apoptosis of preantral follicles through the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway after in vitro culture of sheep ovarian tissue

Theriogenology ◽

10.1016/j.theriogenology.2020.05.037 ◽

2020 ◽

Vol 155 ◽

pp. 25-32

Author(s):

R.S. Barberino ◽

J.M.S. Santos ◽

T.L.B.G. Lins ◽

V.G. Menezes ◽

A.P.O. Monte ◽

...

Keyword(s):

Protein Kinase ◽

In Vitro Culture ◽

Signaling Pathway ◽

Protein Kinase B ◽

Ovarian Tissue ◽

Akt Signaling ◽

Akt Signaling Pathway ◽

Phosphatidylinositol 3 Kinase ◽

Preantral Follicles

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

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/1678-41626023 ◽

2014 ◽

Vol 66 (2) ◽

pp. 411-416 ◽

Cited By ~ 2

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.

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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

Semina Ciências Agrárias ◽

10.5433/1679-0359.2018v39n5p2001 ◽

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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Three dimensional in vitro culture of preantral follicles following slow-freezing and vitrification of mouse ovarian tissue

Cryobiology ◽

10.1016/j.cryobiol.2015.11.001 ◽

2015 ◽

Vol 71 (3) ◽

pp. 529-536 ◽

Cited By ~ 11

Author(s):

Fatemeh Asgari ◽

Mojtaba Rezazadeh Valojerdi ◽

Bita Ebrahimi ◽

Roya Fatehi

Keyword(s):

In Vitro Culture ◽

Ovarian Tissue ◽

Three Dimensional ◽

Slow Freezing ◽

Preantral Follicles

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Powdered coconut water (ACP 406®) as an alternative base culture medium for in vitro culture of goat preantral follicles enclosed in ovarian tissue

Animal Reproduction ◽

10.21451/1984-3143-ar2019-0011 ◽

2019 ◽

Vol 16 (4) ◽

pp. 838-845

Author(s):

Olga Juliana Roldan Castañeda ◽

Francisco Léo Nascimento de Aguiar ◽

Naiza Arcângela Ribeiro de Sá ◽

Maria Luana Galdencio dos Santos Morais ◽

Francielli Weber Santos Cibin ◽

...

Keyword(s):

In Vitro Culture ◽

Culture Medium ◽

Ovarian Tissue ◽

Coconut Water ◽

Preantral Follicles

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Platelet-derived growth factor-BB (PDGF-BB) improves follicular survival, oocyte and follicular diameters, in a dose-dependent manner, after the in vitro culture of goat preantral follicles enclosed in ovarian tissue fragments

Animal Reproduction ◽

10.21451/1984-3143-ar935 ◽

2017 ◽

Vol 14 (4) ◽

pp. 1095-1102

Author(s):

Renato Félix da Silva ◽

Ivina Rocha Brito ◽

Laritza Ferreira de Lima ◽

Francisco Léo Nascimento de Aguiar ◽

Giovanna Quintino Rodrigues ◽

...

Keyword(s):

Growth Factor ◽

In Vitro Culture ◽

Ovarian Tissue ◽

Platelet Derived Growth Factor ◽

Dependent Manner ◽

Preantral Follicles ◽

Dose Dependent

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47 THE AVIAN CHORIO-ALLANTOIC MEMBRANE: A SUITABLE SHORT-TERM CULTURE SYSTEM FOR BOVINE OVARIAN TISSUE

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab47 ◽

2015 ◽

Vol 27 (1) ◽

pp. 116

Author(s):

K. L. Beck ◽

J. Singh ◽

M. Anzar

Keyword(s):

In Vitro Culture ◽

Blood Vessels ◽

Culture System ◽

Ovarian Tissue ◽

Short Term ◽

In Ovo ◽

Ovarian Cortex ◽

In Vitro Culture System ◽

Short Term Culture

Successful cryopreservation of bovine ovarian tissue holds enormous potential for long-term maintenance of female gametes to preserve genetic diversity by tissue banking. Traditionally, in vitro culture followed by histopathological examination has been used to assess the post-thaw viability of cryopreserved tissues. Recently, in ovo transplantation of mammalian tissues on the chorio-allantoic membrane (CAM) of a growing chicken embryo has emerged as an alternative method for short-term culture. The purpose of this experiment was to compare CAM culture of bovine ovarian tissue over a 5-day period with the in vitro culture system. Fertilized White Leghorn eggs were incubated at 37°C and 62% relative humidity. A window (1 × 2 cm) was cut into the eggshell on Day 3 of incubation. Ovaries were retrieved from a local abattoir and brought to the laboratory within 6 h. Ovarian cortex pieces (1–2 mm3) were randomly assigned to control, CAM-culture, or in vitro-culture groups. Control-group tissues were fixed immediately in 4% paraformaldehyde. The CAM was traumatized on Day 10 of incubation to expose the underlying blood vessels, and tissue pieces were grafted at the site (one graft per egg). For in vitro culture, the ovarian cortex pieces were placed on tissue culture inserts within 6-well plates containing TCM199 with 1% insulin-transferrin-selenium, 100 mIU mL–1 of FSH, 100 IU mL–1 of penicillin, and 50 μg mL–1 of streptomycin and incubated at 38°C in 5% CO2. Ovarian tissues from the CAM and in vitro culture group were removed on Day 1, 3, and 5 of grafting/culture, fixed, embedded in paraffin, sectioned at 5 μm, stained with hematoxylin-eosin, and analysed under a light microscope. The numbers of normal and degenerated follicles (indicating follicle survival) and number of blood vessels containing bovine and avian red blood cells (indicating angiogenesis) were counted using standard stereological procedures. All ovarian cortex grafts from surviving chick embryos showed adhesion with the CAM and a marked neo-vascularization in the graft areas. Gross and histological examination revealed the circulation of avian blood cells in ovarian stromal vessels with a concomitant decrease in the number of bovine blood vessels over the incubation period. Total follicle densities (mean ± s.e.m.) on Day 1, 3, and 5 were 13.3 ± 5.9, 27.9 ± 6.7, and 36.9 ± 7.3 in the in vitro-cultured group and 36.7 ± 13.0, 73.6 ± 24.0, and 44.02 ± 12.67 per millimeter cubed in the CAM-cultured group, respectively. Overall, total follicle density was higher in the CAM-cultured group (P < 0.05). Likewise, the normal follicle densities on Day 1, 3, and 5 were 10.4 ± 4.9, 15.5 ± 3.6, and 20.7 ± 6.3 in the in vitro-cultured group and 30.5 ± 8.5, 45.7 ± 18.4, and 22.7 ± 7.3 per millimeter cubed in the CAM-cultured group (P > 0.05). In conclusion, in ovo CAM grafting system was as successful as the in vitro-culture system and may be considered an acceptable alternative to the traditional in vitro-culture system for bovine ovarian tissue.

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