209 A COMPARISON OF THE EFFECTS OF DEFINED, SEMI-DEFINED, AND UNDEFINED MATURATION MEDIA ON CLEAVAGE AND SUBSEQUENT EMBRYO DEVELOPMENT OF SHEEP OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 202
Author(s):  
H. Karami Shabankareh ◽  
M. Zandi
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Statistical Significance ◽  
In Vitro Production ◽  
Efficient System ◽  
Significant Difference ◽  
Igf I ◽  
17Β Estradiol

The objectives were to determine the effects of addition of growth factor(s) and antioxidant to defined (DMM), semi-defined (SDMM), or undefined (UDMM) sheep oocyte maturation media on cleavage and subsequent embryo development. Exogenous epidermal growth factor (EGF, 10 ng mL–1), insulin-like growth factor-I (IGF-I, 50 ng mL–1), or cysteamine (Cys, 100 m) was added to DMM, SDMM, or UDMM. In the experiments, 967 oocytes were collected from 1160 ovaries (Experiment 1), 770 oocytes were collected from 831 ovaries (Experiment 2), and 778 oocytes were collected from 845 ovaries (Experiment 3). After in vitro fertilization with fresh ram semen in SOF medium, groups of 10 oocytes were stripped free of cumulus cells and transferred into 50 μL of a 2-step SOF medium. The incubations were conducted under paraffin oil at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. In all experiments, data were analyzed by ANOVA. Duncan’s multiple range test was used to determine the difference between 2 means after ANOVA. Results were expressed as mean ± SEM, and statistical significance was accepted from P < 0.05. In Experiment 1, the effects of IGF-I and EGF in DMM (TCM-199 supplemented with FSH, LH, 17β-estradiol, Na pyruvate, gentamycin sulfate, and polyvinyl alcohol) were evaluated in 4 treatment groups (DMM as a control, DMM + EGF, DMM + IGF-I, and DMM + EGF + IGF-I). Cleavage, morula, and blastocyst production rates were higher in the DMM + EGF + IGF-I (83.1 ± 0.5, 47.7 ± 0.5, and 30.8 ± 1.2) than in the DMM (72.9 ± 1.3, 37.0 ± 1.1, and 20.2 ± 1.2), DMM + EGF (77.2 ± 1.0, 43.2 ± 2.1, and 26.1 ± 1.8), or DMM + IGF-I (79.9 ± 0.6, 43.3 ± 1.2, and 25.7 ± 1.3) groups (P < 0.05). In Experiment 2, the effects of Cys in DMM were evaluated. The addition of Cys to DMM + EGF + IGF-I resulted in a mean blastocyst rate of 35.0 ± 0.9% compared with 31.5 ± 1.3% in DMM + EGF + IGF-I alone (P < 0.05); however, mean cleavage rates (84.5 ± 1.3 v. 82.8 ± 1.0) did not differ (P = 0.32). In Experiment 3, the effects of DMM + EGF + IGF-I + Cys, SDMM (TCM-199 supplemented with FSH, LH, 17β-estradiol, Na pyruvate, gentamycin sulfate, and BSA) + EGF + IGF-I + Cys, and UDMM (TCM-199 supplemented with FSH, LH, 17β-estradiol Na pyruvate, gentamycin sulfate, and FBS) + EGF + IGF-I + Cys on cleavage and embryo developmental were compared. The UDMM supplemented with EGF, IGF-I, and Cys resulted in higher proportions of cleavage, morula yields, and blastocyst yields (P < 0.05) than the DMM or SDMM supplemented with EGF + IGF-I + Cys. There was no significant difference between DMM and SDMM in the proportions of oocytes reaching the morula and blastocyst stages. In conclusion, an efficient system for in vitro production of sheep blastocysts was developed by using a defined oocyte maturation system combined with growth factors, hormones, and an antioxidant, but the UDMM resulted in higher cleavage, morula yields, and blastocyst yields than the DMM or SDMM.

174 EFFECT OF HUMAN ENDOTHELIAL PROGENITOR CELLS ON IN VITRO MATURATION OF PORCINE OOCYTES AND PARTHENOGENETIC EMBRYO DEVELOPMENT COMPETENCE

2017 ◽  
Vol 29 (1) ◽  
pp. 195
Author(s):  
S. H. Lee ◽  
H. J. Oh ◽  
M. J. Kim ◽  
G. A. Kim ◽  
E. M. N. Setyawan ◽  
...  
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Polar Body ◽  
Blastocyst Formation ◽  
First Polar Body ◽  
Significant Difference

In oocyte maturation, hepatocyte growth factor and vascular endothelial growth factor (VEGF) contribute to promote granulosa cell proliferation and cumulus cell expansion. It is well known that human endothelial progenitor cells (hEPC), which are isolated from monocytes and macrophages, secrete a variety of growth factors, such as hepatocyte growth factor and VEGF, and improve the process of angiogenesis. Therefore, the aim of this study was to investigate the effects of hEPC on in vitro oocyte maturation and subsequent embryo development in pigs. To isolate and culture hEPC, human peripheral blood sample was collected from a healthy donor and peripheral blood mononuclear cells were separated. The peripheral blood mononuclear cells were seeded into flask with defined Keratinocyte-SFM-based medium and incubated at 37°C, 5% CO2. The hEPC were cultured and cryopreserved until use for co-culturing with porcine oocytes obtained from a local slaughterhouse ovaries. Cumulus-oocyte complexes were randomly cultured in 2 groups; 1) co-culturing with hEPC and 2) culturing without hEPC. Cumulus-oocyte complexes were cultured in the in vitro maturation (IVM) medium containing TCM-199 supplemented with 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μL mL−1 of insulin-transferrin-selenium solution 100X (Invitrogen, Seoul, South Korea), 10% porcine follicular fluid, 10 IU mL−1 of eCG, and 10 IU mL−1 of hCG. After IVM, the first polar body extrusion was observed under the microscope. To evaluate embryo development competence, the matured oocytes were activated with electrical stimulus and cultured in porcine zygote medium-5 for 7 days. The cleavage and blastocyst formation rates were observed on Day 2 and 7, respectively. Also, blastocysts were stained with Hoechst 33342 and total blastocyst cell numbers were evaluated under a fluorescence microscope. As a result, the oocyte maturation rate or first polar body extrusion rate of the hEPC co-culture group (90.06 ± 0.75) was significantly higher than the control group (90.06 ± 0.75 v. 85.79 ± 0.59; P < 0.05). There was no significant difference between the hEPC co-cultured and the control groups in cleavage rate. However, a significant difference in blastocyst formation rate was observed between the hEPC co-cultured and the control groups (28.45 ± 4.92 v. 15.87 ± 2.27; P < 0.05), whereas total blastocyst cell numbers did not show significant difference between the 2 groups. The all data were analysed by unpaired t-test using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Values are means ± standard error of mean. In conclusion, the results in the present study demonstrated that co-culturing with hEPC improved the in vitro oocyte maturation and blastocyst formation rate. Also, we are underway in analysing the concentration of VEGF families in the hEPC co-culture medium after IVM. For further study, we will analyse the genes of the VEGF signaling pathway in the cumulus cells and matured oocytes derived from the 2 groups. This research was supported by Nature Cell (#550-20150030), global PH.D Fellowship Program through NRF funded by the Ministry of Education (NRF-20142A1021187), and Research Institute for Veterinary Science, the BK21 plus program.

scholarly journals The Effect of HT-2 Toxin on Ovarian Steroidogenesis and Its Response to IGF-I, Leptin and Ghrelin in Rabbits

2017 ◽  
pp. 705-708 ◽  
Author(s):  
A. KOLESAROVA ◽  
N. MARUNIAKOVA ◽  
A. KADASI ◽  
M. HALENAR ◽  
M. MARAK ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fusarium Species ◽  
Endocrine System ◽  
Control Group ◽  
Trichothecene Mycotoxin ◽  
Ovarian Steroidogenesis ◽  
Estradiol Secretion ◽  
Igf I ◽  
17Β Estradiol

T-2 toxin and its metabolite HT-2 toxin are one of the most toxic mycotoxins of type A-trichothecenes, which are produced mainly by Fusarium species. Therefore, study of Fusarium toxins T-2 toxin and HT-2 toxin is an essential issue because they could also play role in failures of reproductive functions as well as endocrine system of domestic animals. Assessment of the effect of A-trichothecene mycotoxin HT-2 toxin alone or combined with insulin-like growth factor (IGF-I), leptin and ghrelin on estradiol secretion by rabbit ovarian fragments in vitro was done. Rabbit ovarian fragments were incubated without (control group) or with HT-2 toxin, or its combinations with IGF-I, leptin and ghrelin at various concentrations for 24 h. Secretion of 17β-estradiol was determined by ELISA. Firstly, HT-2 toxin at the doses 10 and 100 ng.ml-1, but not at 1 ng.ml-1 decreased 17β-estradiol secretion by ovarian fragments. Secondly, 17β-estradiol secretion was not affected by HT-2 toxin exposure combined with growth factor IGF-I, metabolic hormones leptin and ghrelin. In conclusion, HT-2 toxin has potent direct dose-dependent effects on ovarian steroidogenesis in rabbits. These direct effects of HT-2 mycotoxin on ovarian steroidogenesis could impact negatively on the reproductive performance of rabbits.

Influence of vascular endothelial growth factor and Cysteamine on in vitro bovine oocyte maturation and subsequent embryo development

2015 ◽  
Vol 39 (10) ◽  
pp. 1090-1098 ◽  
Author(s):  
Juan Mateo Anchordoquy ◽  
Juan Patricio Anchordoquy ◽  
Juan Alberto Testa ◽  
Matías Ángel Sirini ◽  
Cecilia C. Furnus
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Vascular Endothelial ◽  
Bovine Oocyte ◽  
Endothelial Growth Factor

260 EVALUATION OF PORCINE IN VITRO PRODUCTION WITH THE ADDITION OF CHITOSAN TO CULTURE MEDIA

2015 ◽  
Vol 27 (1) ◽  
pp. 219 ◽  
Author(s):  
F. García ◽  
Y. Ducolomb ◽  
S. P. Miranda-Castro ◽  
J. F. De la Torre-Sánchez ◽  
S. Romo
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Culture Media ◽  
Biological Properties ◽  
In Vitro Production ◽  
Porcine Oocyte ◽  
Main Component ◽  
Vitro Production ◽  
Positive Effect

Chitosan is a partially deacetylated polymer obtained from the alkaline deacetylation of chitin, which is a glucose-based unbranched polysaccharide widely distributed in nature as the main component of exoskeletons of crustaceans and insects. Chitosan has a variety of physicochemical and biological properties resulting in numerous applications. In addition to its lack of toxicity and allergenicity, its biocompatibility, biodegradability, and bioactivity make it a very attractive substance for diverse applications as a biomaterial in pharmaceutical and medical fields. Chitosan stimulates cell growth and it has been used in fibroblast culture, increasing cell proliferation. For these reasons, it is important to evaluate if this polymer has a positive effect on embryo production. The aim of this study was to evaluate porcine oocyte maturation and embryo development, comparing the effect of supplementing different concentrations of chitosan to the maturation (MM) and development media (DM). Cumulus-oocyte complexes (COC) were aspirated from ovarian follicles of slaughtered sows. The COC were matured in supplemented TCM-199 (MM) and incubated for 44 h. All incubations were performed at 38.5°C, with 5% CO2 in air and humidity at saturation. After maturation IVF was performed, frozen-thawed semen from the same boar was used and gametes were co-incubated in MTBM for 7 h. Then, putative zygotes were cultured in NCSU-23 (DM) for 144 h. The following experiments were performed: 1) addition of 0 (control), 35, 50, 100, and 150 ppm chitosan to the MM (n = 1353), 2) addition of 0, 50, 100, and 150 ppm chitosan to the DM (n = 739), 3) addition of 0, 50, 100, and 150 ppm of chitosan to the MM first and then the same concentrations to the DM (n = 702). When chitosan was added to the MM, the highest percentage of matured oocytes (metaphase II) was obtained in the 50 ppm treatment (87%, P < 0.05) when compared with the control, 100, and 150 ppm groups (78, 78, and 82%, respectively). Regarding the percentage of blastocysts, there were no differences when comparing the treatment and the control groups (ranging from 12 to 13%). After addition of chitosan to the putative zygotes in the DM, the percentage of morulae in the 150 ppm treatment was significantly increased with regard to the other groups (54 v. 46%, respectively, P < 0.05). When adding chitosan to both MM and DM, there was no effect on embryo development. It is concluded that the addition of chitosan to the MM at a concentration of 50 ppm significantly improved oocyte maturation and a concentration of 150 ppm in the DM increased the percentage of morulae. Chitosan had a positive effect on oocyte maturation and embryo development. These results justify further investigations to find out if chitosan can be useful as a supplement for chemically defined media.

scholarly journals Use of Melatonin in the In Vitro Production of Bovine Embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Alan da Silva LIRA ◽  
Ricardo de Macedo CHAVES ◽  
Felipe de Jesus MORAES JUNIOR ◽  
Sergio Henrique COSTA JUNIOR ◽  
Brenda Karine Lima do AMARAL ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Maturation Rate ◽  
Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

276 FIBROBLAST GROWTH FACTOR 2 PROMOTES BOVINE OOCYTE MEIOTIC MATURATION AND DEVELOPMENTAL COMPETENCE

2011 ◽  
Vol 23 (1) ◽  
pp. 236 ◽  
Author(s):  
K. Zhang ◽  
P. J. Hansen ◽  
A. D. Ealy
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Relative Abundance ◽  
Cumulus Cells ◽  
Fibroblast Growth Factor 2 ◽  
Fibroblast Growth ◽  
Cumulus Expansion

Oocyte competency is acquired during the course of folliculogenesis and is controlled by various endocrine and paracrine signals. One of these is fibroblast growth factor 2 (FGF2). Its expression is up-regulated in theca and granulosa cells during final maturation of a bovine follicle, and its cognate receptors are expressed in cumulus cells and oocytes throughout the final stages of oocyte maturation. The overall goal of this work was to describe how supplementing FGF2 during oocyte maturation in vitro affects oocyte maturation and subsequent embryo development. Cumulus–oocyte complexes (COC) were collected from bovine ovaries obtained from a local abattoir and cultured in defined TCM-based oocyte maturation medium. Depending on the study, oocytes were examined either during (6 h) or after (21 h) maturation or were fertilized in vitro and examined throughout in vitro embryo development in modified SOFF. Data were analysed with least-squares ANOVA using GLM of SAS. Adding 0.5 to 50 ng mL–1 of FGF2 did not affect cleavage rate or the percentage of 8 to 16 cell embryos at day 3 post-IVF. However, the blastocyst rate at day 7 was greater when oocytes were exposed to 0.5 ng mL–1 of FGF2 during maturation [30.0 ± 1.9% (17/109) v. 16.0 ± 2.6% (23/77) for nontreatment control; 4 replicates; P < 0.05], whereas higher doses of FGF2 did not affect blastocyst rates when compared with controls. Total cell number per blastocyst was not affected by FGF2 addition. The effects of FGF2 on oocyte maturation and cumulus expansion were examined to better understand how FGF2 improves oocyte competency. Adding 0.5 ng mL–1 of FGF2 did not affect the percentage of oocytes containing condensed chromatin after 6 h IVM or metaphase II (MII) rate after 21 h IVM, but 0.5 ng mL–1 of FGF2 treatment increased the cumulus expansion index score after 21 h IVM (P < 0.05). Interestingly, adding 5 ng mL–1 but not 50 ng mL–1 of FGF2 increased MII rate [61.5 ± 4.3% (53/120) for 5 ng mL–1 of FGF2 v. 46.9 ± 5.9% (64/104) for nontreatment controls; 7 replicates; P < 0.05], but neither FGF2 affected rates of chromatin condensation and cumulus expansion. Changes in the relative abundance for several putative oocyte competency markers and maternal genes (CTSB, Sprouty2, EGFR, FSHR, Has2, BMP15, GDF9, JY-1, Follistatin, H2A) were examined at 6 and 21 h after treatment with 0.5 ng mL–1 of FGF2 by quantitative RT-PCR. Relative amounts of 18S RNA was used as an internal control, and 2-ΔΔCT was used to quantify relative gene expression. The relative abundance of most of the transcripts examined was not affected by FGF2, but EGFR mRNA levels were greater after 6 h but not 21 h IVM in cumulus cells isolated from FGF2-supplemented COC (P = 0.057). In summary, improvements in blastocyst development were achieved by FGF2 treatment during oocyte maturation. The reason for the enhanced oocyte competency remains unclear, but it may occur in part because of improvements in cumulus expansion and production of EGFR. This project was supported by NRICGP number 2008-35203-19106 from the USDA-NIFA.

Influence of vasoactive intestinal peptide (VIP), atrial natriuretic peptide (ANP) and insulin-like growth factor-I (IGF-I) on in vitro maturation of prepubertal and adult sheep oocytes

Zygote ◽  
1996 ◽  
Vol 4 (04) ◽  
pp. 343-348 ◽  
Author(s):  
S. Ledda ◽  
L. Bogliolo ◽  
G. Leoni ◽  
P. Calvia ◽  
S. Naitana
Keyword(s):  
Growth Factor ◽  
Atrial Natriuretic Peptide ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Optimal Conditions ◽  
Factor I ◽  
Igf I

Much effort has been focused on establishing optimal conditions for obtainingin vitromaturation of oocytes from different species with results comparable to those achieved afterin vivodevelopment (reviewed by Brackett, 1992). However, even though extraordinary progress has been made, thein vitrotechnology for oocyte maturation lags far behind thatin vivoand improvements are needed to increase the quantity and quality of the embryos produced from these matured oocytes.

scholarly journals In Vitro Changes in Secretion Activity of Rat Ovarian Fragments Induced by Molybdenum

2014 ◽  
pp. 807-809 ◽  
Author(s):  
S. ROYCHOUDHURY ◽  
L. DETVANOVA ◽  
A. V. SIROTKIN ◽  
R. TOMAN ◽  
A. KOLESAROVA
Keyword(s):  
Growth Factor ◽  
Ammonium Molybdate ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Study Results ◽  
Igf I ◽  
17Β Estradiol ◽  
Growth Factor I ◽  
Dose Dependent

The aim of this in vitro study was to examine the secretion activity (progesterone, 17β-estradiol and insulin-like growth factor-I) of rat ovarian fragments after molybdenum (Mo) addition. Rat ovarian fragments were incubated with ammonium molybdate (NH4)6Mo7O24.4H2O at the doses 90, 170, 330 and 500 µg.ml-1 for 24 h and compared with control group without Mo addition. Release of progesterone (P4), estradiol (17β-estradiol) and insulin-like growth factor I (IGF-I) by ovarian fragments was assessed by radioimmunoassay (RIA). Data show that P4 release by ovarian fragments was not affected by (NH4)6.Mo7O24.4H2O addition at all the doses used (90-500 µg.ml-1). However, addition of ammonium molybdate was found to cause a significant (P<0.05) dose-dependent decrease (at the doses 90, 170 and 500 µg.ml-1) in release of 17β-estradiol by ovarian fragments in comparison to control. Also, addition of ammonium molybdate significantly (P<0.05) inhibited IGF-I release at all the doses (90-500 µg.ml-1) used in the study. Results suggest ammonium molybdate induced inhibition in the release of growth factor IGF-I and its dose-dependent effect on secretion of steroid hormone 17β-estradiol but not progesterone. These data contribute to new insights regarding the mechanism of action of Mo on rat ovarian functions.

336 SHEEP OOCYTES MATURED IN A SIMPLEX MEDIUM (HECM-1), AN ANSWER TO IN VITRO FERTILIZATION

2006 ◽  
Vol 18 (2) ◽  
pp. 275
Author(s):  
C. Navarro-Maldonado ◽  
Y. Ducolomb-Ramirez ◽  
A. Galindo-Rodriguez ◽  
A. Rosado-Garcia
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Sperm Quality ◽  
Complete Control ◽  
Vitro Fertilization ◽  
Significant Difference

In vitro maturation and in vitro fertilization (IVM and IVF) of mammalian oocytes still show unsatisfactory results when applied to the study of embryo development. This is probably due to inadequate information about the use of media components and supplements for oocyte maturation and to a discrepancy between results obtained by focusing strictly on oocyte maturation and those that are focused on IVF. A conventional medium that provides adequate results in studies of oocyte maturation (TCM-199) contains hypoxanthine, phosphate ions, and glucose, all known to inhibit embryo development in vitro in some species. In contrast, it has been shown that a simpler medium (HECM-9) increases embryo development in bovine although its use for oocyte maturation has not been defined. This medium contains taurine (an amino acid that reduces intracellular peroxide content) and is supplemented with polyvinyl alcohol (PVA) instead of using protein components, making it a simple defined medium that reduces variability in embryo development. Adding sodium panthothenate to media also confers cell protection against reactive oxygen species. Finally, supplements such as epidermal growth factor (EGF) increase the number of oocytes that complete maturation (Metaphase II, MII) and facilitate embryo development. An adequate combination of our knowledge about in vitro maturation and fertilization of oocytes, together with the requirements for embryo development, is important for the preparation of culture media to study regulatory mechanisms for embryo development and to increase the number of viable and normal term individuals. In this study we compared the effects of HECM-9 (containing panthothenate) vs. TCM-199 (both media supplemented with PVA, EGF, and FSH/LH) on the integrated processes involving IVM and IVF. No significant differences were found between the results obtained with these media in relation to oocyte maturation (65% MII for HECM-9 vs. 71% for TCM-199); however, those oocytes matured in HECM-9 showed a highly significant difference in in vitro fertilization using a conventional IVF medium (SOFm) (25% in HECM-9 vs. 6% in TCM-199). Maturation results were relatively low but in accordance with those reported by other groups, whereas IVF results are lower than those reported in the literature, perhaps because we have been using frozen and thawed samples and do not have complete control over the sperm quality. At present, we are extending our investigation using fresh semen samples.

191 SUPPLEMENTATION OF MATURATION MEDIUM WITH FOLLICULAR FLUID, EPIDERMAL GROWTH FACTOR AND NEUREGULIN AFFECTS MITOCHONDRIAL DNA REPLICATION, OOCYTE MATURATION AND EMBRYO DEVELOPMENT IN PIGS

2012 ◽  
Vol 24 (1) ◽  
pp. 208
Author(s):  
J. Mao ◽  
K. M. Whitworth ◽  
L. D. Spate ◽  
E. M. Walters ◽  
J. Zhao ◽  
...  
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Copy Number ◽  
In Vitro Maturation ◽  
Meiotic Maturation ◽  
Mtdna Copy Number ◽  
Maturation Medium

Mitochondria supply the majority of ATP in a cell. Mitochondrial DNA (mtDNA) copy number in oocytes might be used as a marker of viability and might be a key determinant of pre-implantation embryo development. However, little is known about mtDNA copy number changes during porcine oocyte maturation and its regulation by extracellular growth factors. The objectives of the current study were to determine the effects of supplementation of in vitro maturation medium with porcine follicular fluid (pFF; 0, 10, 20 and 30%), epidermal growth factor (EGF; 10 ng mL–1), neuregulin 1 (NRG; 20 ng mL–1) and NRG + IGF1 (insulin-like growth factor-1; 100 ng mL–1 + NRG, 20 ng mL–1) during in vitro maturation on mtDNA copy number, oocyte meiotic maturation and subsequent embryo development after parthenogenic activation. Follicular fluid used for the pFF supplementation experiment was prepared from medium-sized (3–6 mm in diameter) healthy follicles. Cumulus–oocyte complexes (COCs) were collected from antral follicles (3–6 mm in diameter), cultured in LH- and FSH-containing maturation medium for 22 h at 38.5°C, transferred into basic maturation medium without FSH and LH and cultured for another 22 h. The basic maturation medium was TCM-199 supplemented with 0.1% polyvinylalcohol (w/v), 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 10 μg mL–1 of gentamicin, 0.57 mM cysteine and without or with different growth factors depending on the experimental design. In total, 177 germinal vesicle (GV) oocytes and 3837 MII oocytes were used for this study. All data were analyzed by the general linear model (GLM) procedure of SAS software (V9.2). The mtDNA copy number in oocytes increased (P < 0.05) from GV to MII stage oocytes (MII oocytes from all treatment groups pooled). Supplementation of IVM media with 10% pFF decreased mtDNA copy number (P < 0.05), whereas 20 and 30% pFF had no major effect on mtDNA copy number, resulting in a quadratic correlation between percentage of pFF and mtDNA copy number. There was a negative linear correlation between percentage of pFF and oocyte meiotic maturation, with a higher percentage of pFF inhibiting meiotic maturation (73.2 ± 5.2, 71.9 ± 4.8, 64.1 ± 8.5 and 65.8 ± 6.4% for 0, 10, 20 and 30% pFF groups, respectively). The mtDNA copy numbers in EGF and NRG-treated MII oocytes were significantly higher than those in GV oocytes, whereas the control was not different (EGF, 237 042.6 ± 22 198.2; NRG, 281 293.4 ± 22 893.5; and control, 231 856.8 ± 21 883.5 in MII oocytes vs 192 288.7 ± 21 675.4 in GV oocytes). The EGF, NRG and NRG+IGF1 treatments enhanced oocyte maturation as well. There was no difference in Day-7 blastocyst formation between EGF, NRG+IGF1 and the control, whereas the NRG treatment enhanced blastocyst formation as compared to the control (23.8 ± 2.4 vs 15.1 ± 2.1%; P < 0.05). This study demonstrated that there was an increase in mtDNA copy number during in vitro maturation. The EGF and NRG treatments stimulated mitochondria biogenesis, which may provide new means to increase oocyte quality and enhance embryonic development.

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