In vitro culture of secondary follicles and prematuration of cumulus–oocyte complexes from antral follicles increase the levels of maturation‐related transcripts in bovine oocytes

2019 ◽  
Vol 86 (12) ◽  
pp. 1874-1886
Author(s):  
Francisco Taiã G. Bezerra ◽  
Francisco Edilcarlos O. Lima ◽  
Laís Rayani F. M. Paulino ◽  
Bianca R. Silva ◽  
Anderson W. B. Silva ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Antral Follicles ◽  
Bovine Oocytes

242 USE OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN IN VITRO PREMATURATION OF BOVINE OOCYTES SUBJECTED TO PARTHENOGENETIC ACTIVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 200
Author(s):  
T. H. C. De Bem ◽  
R. Rochetti ◽  
P. R. L. Pires ◽  
F. F. Bressan ◽  
P. R. Adona ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Polar Body ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Parthenogenetic Activation ◽  
Bovine Oocytes ◽  
Hatching Rates

Prematuration provides an additional time for oocyte capacitation and maturation in an attempt to improve in vitro embryo production (IVP) rates and allows media supplementation during this period for IVP. The aim of this study was to use brain-derived neurotropic factor (BDNF) in prematuration to improve maturation of bovine oocytes subjected to parthenogenetic activation and cultured with different media. Oocytes were subjected to prematuration in TCM-199 medium supplemented with 10 µm butyrolactone I, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin for 24 h in the absence of BDNF (control) or in the presence of 10 ng mL–1 BDNF (BD). Oocytes were then in vitro-matured (IVM) in TCM-199 medium supplemented with 10% FCS, 0.5 µg mL–1 FSH, 5.0 µg mL–1 LH, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin at 38.5�C under 5% CO2 in air. After 19 h oocytes were denuded using hyaluronidase and vortexing for 3 min for the 1st polar body (1PB) selection. Those which extruded the 1PB were maintained in IVM until 26 h, when parthenogenetic activation was performed (5 min in 5 µm ionomycin, followed by 3 h in 2 mm 6-DMAP). Activated oocytes were then transferred to in vitro culture (IVC) for embryo development evaluation. Embryos from both groups were cultured in SOF medium with 2.5% FCS, 0.05 g mL–1 BSA, 0.2 mm pyruvate, and 10 mg mL–1 gentamicin. Cleavage rates on the second day of in vitro culture (D2), embryo production at Days 7 and 8 (D7 and D8), and hatching rate at Day 8 were evaluated. Data regarding 1PB extrusion, cleavage, blastocyst development on D7 and D8, and blastocyst D8 hatching rates of three replicates were analyzed by chi-square test at 5% significance using the BIOESTATS 4.0 software. Control and BD, respectively, did not show differences (P > 0.05) regarding 1PB extrusion (n = 164, 63.81%, and n = 175, 66.79%) or cleavage (n = 117, 71.34%, and n = 138, 78.86%). However, for control and BD, respectively, blastocyst development on D7 (n = 63, 38.41%, and n = 89, 50.86%), D8 (n = 63, 38.41%, and n = 91, 52.00%), and hatching on D8 (n = 22, 34.92%, and n = 39, 43.82%) were all significantly higher for BD when compared with control (P < 0.05). In conclusion, BDNF during prematuration improved in vitro embryo development by increasing blastocyst and hatching rates of parthenogenetic embryos.

174 Effects of Melatonin on In Vitro Maturation of Bovine Oocytes and Gene Expression in Cumulus Cells: Preliminary Results

2018 ◽  
Vol 30 (1) ◽  
pp. 226
Author(s):  
F. C. Castro ◽  
L. Schefer ◽  
K. L. Schwarz ◽  
H. Fernandes ◽  
R. C. Botigelli ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Reverse Transcription ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Bovine Oocytes

Melatonin mediates several processes in animal reproduction and has drawn attention for its potent antioxidant, anti-apoptotic, anti-inflammatory action and, more recently, for its benefits on oocyte maturation and embryo development in vitro. The aim of this study was to assess the effect of melatonin during the in vitro maturation (IVM) on nuclear maturation of bovine oocytes and gene expression in their corresponding cumulus cells (CC). Bovine cumulus–oocyte complexes (COC) were obtained by aspiration of follicles (2-6 mm) from slaughterhouse ovaries, selected (grades I and II) and transferred to 4 well plates (25-30 COC/well) containing IVM medium [TCM-199 supplemented with sodium bicarbonate (26 mM), sodium pyruvate (0.25 mM), FSH (0.5 µg mL−1), LH (5.0 µg mL−1), 0.3% BSA, and gentamicin (50 µg mL−1)] with 0, 10−5, 10−7, 10−9 or 10−11 M melatonin and cultured for 24 h at 38.5°C and 5% CO2. At the end of IVM, oocytes were stained with Hoechst 33342 (10 μg mL−1) and evaluated for nuclear maturation rate. The CC were evaluated for the expression of antioxidant (SOD1, SOD2, GPX4), pro-apoptotic (P53, BAX) and expansion-related genes (PTX3, HAS1, HAS2). For transcript detection in CC, RNA isolation was performed with TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Relative quantification of transcripts was performed by RT-qPCR using 3 endogenous controls (β-actin, GAPDH, PPIA). Nuclear maturation rate and gene expression were tested by ANOVA and means were compared by Tukey’s test (6 replicates). In CC, the different concentrations of melatonin did not significantly alter expression of the investigated genes (P > 0.05), although all concentrations provided a numerical increase in the expression of the antioxidant SOD1 and of the expansion-related genes PTX3 and HAS2. Regarding the pro-apoptotic genes, concentrations of 10−11 and 10−9 M were able to reduce only numerically the expression of BAX and P53, respectively. In oocytes, the rate of nuclear maturation was not different among the tested treatments (P > 0.05), but it was numerically higher in the 10−7 M melatonin treated group compared with the control (69.71 ± 13.76% v. 88.1 ± 12.54%). In conclusion, under the studied conditions, melatonin was unable to improve maturation rate or to affect the expression of antioxidant, pro-apoptotic, and expansion-related genes in CC. Melatonin during IVM has shown variable results in different studies and appears to show different effects depending on culture conditions and parameters studied. In order to take advantage of the possible positive antioxidant effects of melatonin, other culture conditions and parameters should be investigated. In a next step, melatonin will be included during in vitro culture of embryos to evaluate its possible cytoprotective role, because such embryos are more exposed to oxidative stress during in vitro culture, and to investigate its benefits on developmental competence in vitro. This reaesrch was funded by FAPESP (2015/20379-0; 2014/17181-0).

scholarly journals In Vitro Culture of Pig Oocytes Collected from Early Antral Follicles.

1998 ◽  
Vol 15 (3) ◽  
pp. 161-166 ◽  
Author(s):  
Takashi Miyano ◽  
Sadanobu Moritake ◽  
Kazumasa Hirata ◽  
Masashi Miyake ◽  
Seishiro Kato
Keyword(s):  
In Vitro Culture ◽  
Antral Follicles

scholarly journals Effects of Oxygen Tension on Survival and Growth In Vitro of Bovine Oocytes from Early Antral Follicles

2002 ◽  
Vol 19 (1) ◽  
pp. 12-20 ◽  
Author(s):  
Yuji Hirao ◽  
Ken-ichi Aizawa ◽  
Naoki Takenouchi ◽  
Takashi Nagai
Keyword(s):  
Oxygen Tension ◽  
Antral Follicles ◽  
Survival And Growth ◽  
Bovine Oocytes

In vitro culture of isolated preantral and antral follicles of goats using human recombinant FSH: Concentration-dependent and stage-specific effect

2018 ◽  
Vol 196 ◽  
pp. 120-129 ◽  
Author(s):  
Anna Clara A. Ferreira ◽  
Jesús Cadenas ◽  
Naiza A.R. Sá ◽  
Hudson H.V. Correia ◽  
Denise D. Guerreiro ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Specific Effect ◽  
Recombinant Fsh ◽  
Antral Follicles

Effect of medium composition on the in vitro culture of bovine pre-antral follicles: morphology and viability do not guarantee functionality

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 125-128 ◽  
Author(s):  
Rafael Rossetto ◽  
Márcia Viviane Alves Saraiva ◽  
Regiane Rodrigues dos Santos ◽  
Cleidson Manoel Gomes da Silva ◽  
Luciana Rocha Faustino ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Media ◽  
Medium Composition ◽  
Minimum Essential Medium ◽  
Antral Follicles ◽  
Suitable Medium

SummaryThis study investigated the effect of three different culture media (α minimum essential medium (α-MEM), McCoy or TCM199 during the in vitro culture (IVC) of bovine isolated pre-antral follicles. Pre-antral follicles greater than 150 μm in size were isolated and cultured for 0 (control), 8 or 16 days in one of the abovementioned culture media. Follicles were evaluated for survival, growth and antrum formation at days 8 and 16. The results showed that TCM199 was the most suitable medium to preserve follicular viability and ultrastructure, resulting in the highest rates of antrum formation. In conclusion, TCM199 promotes the in vitro development of isolated pre-antral follicles without hampering follicular functionality by sustaining in vitro growth and antrum formation.

scholarly journals Partenogenetic development of Bos taurus embryos from oocytes matured in different culture systems

2020 ◽  
Vol 197 (6) ◽  
pp. 66-72
Author(s):  
T. I. KUZMINA
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Cold Shock ◽  
Bos Taurus ◽  
Embryonic Stem ◽  
Structural Components ◽  
Antral Follicles ◽  
Culture Systems ◽  
Bovine Oocytes

Abstract. Identification of the factors determining of donor’s oocyte competence to parthenogenetic development will allow developing an effective method for obtaining parthenotes to solve fundamental problems of regulating gene activity in ontogenesis, creating homozygous embryonic stem cell lines, improving the stages of cloning technology, and modeling of in vitro oocyte maturation media. The purpose of study is to evaluate the potencies of Bos taurus oocytes matured in different culture systems to cold shock-induced parthenogenesis. Methods. For oocyte maturation, culture systems of the following composition were used: 1 – TC-199 with 10 % fetal bovine serum (FBS), 50 μg/ml estradiol, 10 μg/ml luteinizing hormone, 10 μg/ml follicle-stimulating hormone; 2 – TC-199 with 10 % estrous serum of cows; 3 – TC-199 with 50 % fluid from follicles (Ø 3–8 mm); 4 – TC-199 with 50 % protein of follicular fluid (molecular weight of 65 kDa); 5 – TC-199 with 10 % FBS, 1×106 granulosa cells/ml medium; 6 – TC-199 with 10 % FBS and walls of follicles (Ø 6–8 mm); 7 – TC-199 with 10 % FBS, 1×106 granulosa cells/ml medium and walls of follicles (Ø 6–8 mm). After 24 hours of cultivation, the oocytes were activated by cold shock (exposure time 20 minutes, temperature 0…–4 °C. Results. The proportion of embryos at the stages of late morula and blastocysts from oocytes matured in system 7 was 45 % (58/129), which was significantly higher than in other systems: 1 – 28 % (39/141), P < 0.05; 2 – 31 % (42/137), P < 0.05; 3 – 25 % (33/133), P < 0.01; 4 – 18 % (25/139), P < 0.001; 5 – 31 % (41/132), P < 0.05; 6 – 33 % (43/129). The introduction of estradiol or structural components of antral follicles into the oocytes maturation medium contributed to an increase in the proportion of parthenotes at the preimplantation stages of development, including blastocysts, and a decrease in the level of degenerated embryos. Scientific novelty. A comparative morphological analysis of the potentials for parthenogenesis of bovine oocytes matured in various culture systems and activated by cold shock was carried out for the first time. Optimal systems for in vitro maturation of female gametes were proposed. Based on the analysis of the results, we recommend before induction to parthenogenesis bovine oocytes culture in media supplemented with 50 ng/ml estradiol or structural components of antral follicles producing estradiol.

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