295 EFFECT OF TREHALOSE DURING IN VITRO MATURATION OF PIG OOCYTES ON OOCYTE MATURATION AND EMBRYONIC DEVELOPMENT AFTER PARTHENOGENESIS

2015 ◽  
Vol 27 (1) ◽  
pp. 236
Author(s):  
Y. Jeon ◽  
B. Baasanjav ◽  
Y. I. Jeong ◽  
Y. W. Jeong ◽  
Y. W. Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Large Scale ◽  
Treated Group ◽  
Developmental Competence ◽  
Control Group ◽  
Scale Production ◽  
Developmental Potential

Autophagy is a critical process for the maintenance of cellular homeostasis and mammalian early embryogenesis. Autophagy can be regulated by various chemical inducers. However, there are few reports about effect of autophagy inducer in vitro maturation (IVM) of porcine oocyte. The present study investigated the effects of supplementary trehalose, a novel mTOR-independent autophagy enhancer, on oocyte maturation and embryonic development after parthenogenetic activation (PA). Immature oocytes were treated with various concentrations (0, 25, 50, and 100 mM) of trehalose in M-199 (Invitrogen, Carlsbad, CA) supplemented with 10 ng mL–1 of epidermal growth factor (EGF; Sigma-Aldrich Corp.), 1 ug mL–1 of insulin (Sigma-Aldrich Corp.), 4 IU mL–1 of pregnant mare serum gonadotropin (PMSG; Intervet, Boxmeer, Holland), 4 IU mL–1 of human chorionic gonadotropin (hCG; Intervet), and 10% (vol/vol) porcine follicular fluid (pFF) for 10 h, and transferred to another IVM medium without trehalose. Osmolality of each groups (0, 25, 50, and 100 mM trehalose) was in the 290 to 295, 310 to 315, 330 to 335, and 375 to 380 osmol range, respectively. After 44 h of IVM, trehalose treatment during IVM did not improve nuclear maturation rates of oocytes in any group (90.7, 92.1, 92.7, and 90.1%, respectively). The developmental competence of oocytes matured with different trehalose concentrations was evaluated after PA. There were no significant differences in cleavage rates. However, blastocyst (BL) formation was different. Oocytes treated with 25 mM of trehalose during IVM had a significantly higher (P < 0.05) BL formation rate (64.2%) after PA compared with the control (52.0%). The BL quality was also improved in the 25 mM trehalose-treated group. Early BL rate significantly reduced in the 25 mM trehalose-treated group as compared to control (19.6 v. 29.9%, P < 0.05). By contrast, expanded BL rate significantly increased in the 25 mM trehalose-treated group than of control (27.7 v. 11.0%, P < 0.05). Total cell numbers of BL were significantly higher (P < 0.05) in the 25 mM trehalose-treated group compared to those in the control group (52.2 v. 36.8). However, BL rate and quality of oocytes treated with 50 and 100 mM trehalose were similar with control group. In conclusion, these results indicate that 25 mM trehalose during IVM improved the developmental potential of porcine embryos. Trehalose will be useful for large-scale production of BL with good quality in porcine in vitro production.This work was supported by a grant from the Next-Generation Bio Green 21 Program (No. PJ009563032014), Rural Development Administration, Republic of Korea.

scholarly journals Effect of Interleukin-7 on In Vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Developmental Potential after Parthenogenetic Activation

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 741
Author(s):  
Dongjin Oh ◽  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Junchul-David Yoon ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Control Group ◽  
Developmental Potential ◽  
Parthenogenetic Activation ◽  
Nuclear Maturation ◽  
Interleukin 7

Interleukin-7 (IL-7) is a cytokine essential for cell development, proliferation and survival. However, its role in oocyte maturation is largely unknown. To investigate the effects of IL-7 on the in vitro maturation (IVM) of porcine oocytes, we analyzed nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, and subsequent embryonic developmental competence after parthenogenetic activation (PA) under several concentrations of IL-7. After IVM, IL-7 treated groups showed significantly higher nuclear maturation and significantly decreased intracellular ROS levels compared with the control group. All IL-7 treatment groups exhibited significantly increased intracellular GSH levels compared with the control group. All oocytes matured with IL-7 treatment during IVM exhibited significantly higher cleavage and blastocyst formation rates after PA than the non-treatment group. Furthermore, significantly higher mRNA expression levels of developmental-related genes (PCNA, Filia, and NPM2) and antioxidant-related genes (GSR and PRDX1) were observed in the IL-7-supplemented oocytes than in the control group. IL-7-supplemented cumulus cells showed significantly higher mRNA expression of the anti-apoptotic gene BCL2L1 and mitochondria-related genes (TFAM and NOX4), and lower transcript levels of the apoptosis related-gene, Caspase3, than the control group. Collectively, the present study suggests that IL-7 supplementation during porcine IVM improves oocyte maturation and the developmental potential of porcine embryos after PA.

scholarly journals Porcine follicular fluid derived from > 8 mm sized follicles improves oocyte maturation and embryo development during in vitro maturation of pigs

Zygote ◽  
2020 ◽  
pp. 1-6
Author(s):  
Ji-Eun Park ◽  
Sang-Hee Lee ◽  
Yong Hwangbo ◽  
Choon-Keun Park
Keyword(s):  
Embryonic Development ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Control Group ◽  
Cumulus Expansion ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Summary The aim of the present study was to investigate the effects of porcine follicular fluid (pFF) from large-sized (LFF; >8 mm in diameter) and medium-sized (MFF; 3–6 mm in diameter) follicles on the maturation and developmental competence of porcine oocytes. Cumulus–oocyte complexes (COCs) were collected from follicles 3–6 mm in diameter. The collected COCs were incubated for 22 h with LFF or MFF (in vitro maturation (IVM)-I stage) and were incubated subsequently for 22 h with LFF or MFF (IVM-II stage). Cumulus expansion was confirmed after the IVM-I stage and nuclear maturation was evaluated after the IVM-II stage. Intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured and embryonic development was evaluated. Relative cumulus expansion and GSH levels were higher in the LFF group compared with in the MFF group after the IVM-I stage (P < 0.05). After the IVM-II stage, the numbers of oocytes in metaphase-II were increased in the LFF group and GSH content was higher in all of the LFF treatment groups compared with in the MFF treatment groups during both IVM stages (P < 0.05). ROS levels were reduced by LFF treatment regardless of IVM stage (P < 0.05). Blastocyst formation and the total numbers of cells in blastocysts were increased in all LFF treatment groups compared with the control group (P < 0.05). These results suggested that pFF from large follicles at the IVM stage could improve nucleic and cytoplasmic maturation status and further embryonic development through reducing ROS levels and enhancing responsiveness to gonadotropins.

Paradoxical effects of kisspeptin: it enhances oocyte in vitro maturation but has an adverse impact on hatched blastocysts during in vitro culture

2012 ◽  
Vol 24 (5) ◽  
pp. 656 ◽  
Author(s):  
Islam M. Saadeldin ◽  
Ok Jae Koo ◽  
Jung Taek Kang ◽  
Dae Kee Kwon ◽  
Sol Ji Park ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Early Period ◽  
Threshold Effect ◽  
Developmental Competence ◽  
Control Group ◽  
Maturation Medium ◽  
Paradoxical Effects

Kisspeptin (Kp) is best known as a multifunctional peptide with roles in reproduction, the cardiovascular system and cancer. In the present study the expression of kisspeptin hierarchy elements (KISS1, GNRH1 and LHB) and their receptors (KISS1R, GNRHR and LHCGR, respectively) in porcine ovary and in cumulus–oocyte complexes (COCs) were investigated, as were its effects on the in vitro maturation (IVM) of oocytes and their subsequent ability to sustain preimplantation embryo competence after parthenogenetic electrical activation. Kp system elements were expressed and affected IVM of oocytes when maturation medium was supplemented with 10–6 M Kp. Oocyte maturation, maternal gene expression (MOS, GDF9 and BMP15), blastocyst formation rate, blastocyst hatching and blastocyst total cell count were all significantly increased when oocytes were matured in medium containing Kp compared with the control group (without Kp). A Kp antagonist (p234) at 4 × 10–6 M interfered with this hierarchy but did not influence the threshold effect of gonadotrophins on oocyte maturation. FSH was critical and permissive to Kp action on COCs by increasing the relative expression of KISS1R. In contrast, Kp significantly increased apoptosis, the expression of pro-apoptotic gene, BAK1, and suppressed trophoblast outgrowths from hatched blastocysts cultured on feeder cells. The present study provides the first functional evidence of the Kp hierarchy in porcine COCs and its role in enhancing oocyte maturation and subsequent developmental competence in an autocrine–paracrine manner. However, Kp supplementation may have a harmful impact on cultured hatched blastocysts reflecting systemic or local regulation during the critical early period of embryonic development.

155 EFFECT OF CO-CULTURE WITH CUMULUS-DERIVED SOMATIC CELLS DURING IN VITRO MATURATION ON PORCINE CUMULUS–OOCYTE COMPLEXES AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION

2014 ◽  
Vol 26 (1) ◽  
pp. 191 ◽  
Author(s):  
J. D. Yoon ◽  
L. Cai ◽  
S. U. Hwang ◽  
Y. Jeon ◽  
E. Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Mrna Expression ◽  
In Vitro Maturation ◽  
Somatic Cells ◽  
The Other ◽  
Control Group ◽  
Cortical Granules ◽  
Developmental Potential ◽  
Significant Difference

The purpose of this study was to investigate the effects of co-culture with cumulus-derived somatic cells (CSC) during porcine in vitro maturation (IVM) and subsequent embryonic development after IVF. The CSC were cultured in Dulbecco's modified Eagle medium for 48 h with various numbers of cumulus-derived somatic cells (0.0, 2.5, 5.0, and 10.0 × 104), and then cultured in TCM-199 for 4 h before the oocytes were added. Cumulus-oocytes complexes from 3- to 6-mm follicles were matured in 500 μL of TCM-199, with eCG and hCG, for 22 h, and then cultured in M199 without hormones for 22 h. Each experiment consisted of at least 4 replicates. Statistical analyses were carried out using SPSS 17.0 software (SPSS Inc., Chicago, IL). Percentage data were compared by one-way ANOVA, followed by Duncan's multiple range test. Data were presented as means ± s.e.m. Differences were considered to be significant if the P-value was 0.05. After IVM, no significant difference (P < 0.05) was observed in nuclear maturation rate among the 0.0, 2.5, 5.0, and 10.0 × 104 groups (88.0 ± 2.37, 81.5 ± 2.17, 87.0 ± 1.98 and 86.0 ± 1.93%, respectively). The 2.5 × 104 group showed a significant (P < 0.05) increase in intracellular glutathione (GSH) levels compared with that of the other groups. Intracellular reactive oxygen species (ROS) levels of mature oocyte in all groups showed no significant differences. The developmental competence of matured oocytes in all groups was evaluated after IVF. The 2.5 and 5.0 × 104 groups showed significantly (P < 0.05) high cleavage rates (60.0 ± 4.7 and 64.52 ± 5.9%, respectively) compared with the 0 and 10.0 × 104 groups (43.15 ± 5.0 and 53.8 ± 5.0%, respectively). The 2.5 × 104 group showed a significantly (P < 0.05) higher BL formation rate (35.7 ± 2.9) than control group (21.0 ± 3.8%, respectively), and higher total cell number (127.25 ± 7.7) compared with the 0 and 10 × 104 groups (89.3 ± 4.0 and 92.6 ± 3.7, respectively). In the analysis of gene expression, IVF-BL derived from the 2.5 and 5.0 × 104 groups showed higher (P < 0.05) mRNA expression of PCNA, which is an essential component of the DNA replication and repair machinery and POU5F1 has been used to evaluate developmental potential in embryos. The 10.0 × 104 group showed higher (P < 0.05) mRNA expression of caspase-3 and Bak as known pro-apoptotic factors, compared with the control group IVF-BL. The results of cortical granules distribution which leads digesting sperm receptor proteins ZP2 and ZP3 to block polyspermy, showed that the 2.5 × 104 group was increased significantly (P < 0.05) compared with the other co-culture groups (13.7 ± 6.1, 29.2 ± 9.5, 18.3 ± 0.8 and 19.52 ± 5.3, respectively). In conclusion, co-culture with 2.5 × 104 cumulus-derived somatic cells during IVM improved the developmental potential of porcine IVF embryos by increasing the intracellular GSH level and distribution of cortical granules during oocyte maturation. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

197 EFFECTS OF PRE-IN VITRO MATURATION WITH CAFFEINE ON BOVINE OOCYTE DEVELOPMENTAL CAPACITY

2016 ◽  
Vol 28 (2) ◽  
pp. 229
Author(s):  
S. M. B. Ulloa ◽  
J. Heinzmann ◽  
D. Herrmann ◽  
U. Baulain ◽  
K.-G. Hadeler ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Developmental Competence ◽  
Differential Staining ◽  
Control Group ◽  
Dependent Manner ◽  
Meiotic Arrest ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Camp Levels

High cyclic adenosine monophosphate (cAMP) concentrations are critical for maintaining oocyte meiotic arrest in vivo. For in vitro maturation (IVM), the oocyte is released mechanically from the follicle, which induces a significant drop in intra-oocyte cAMP levels, triggering non-physiological meiotic resumption. It has been proposed that modulation of cAMP before IVM can increase bovine blastocyst rates in vitro. Caffeine is a nonspecific competitive phosphodiesterases (PDE) inhibitor and can inhibit meiotic resumption of oocytes due to maintenance of cAMP levels. It has been reported that gamete treatment with caffeine can increase developmental potential. The current study evaluated the effects of pre-in vitro maturation culture with different concentrations of caffeine on meiotic progress, developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 6648 cumulus-oocyte complexes were obtained by slicing. Caffeine was used in 5 different concentrations (1, 5, 10, 20, and 30 mM) during slicing, searching, and 2 h pre-IVM culture. A control group, with 2 h pre-IVM without caffeine (0 mM) and a standard control were also included. Oocytes were washed either after standard or pre-IVM treatments and cultured for 24 h in vitro without caffeine. After IVM, oocytes were fertilised in vitro for 19 h, and zygotes were cultured in vitro for 8 days until the blastocyst stage. Subsets of oocytes were fixed in 2% glutaraldehyde at 9, 20, and 24 h after IVM. Hoechst staining was performed to evaluate nuclear status of matured oocytes. Cleavage and blastocyst formation rates were evaluated at Days 3 and 8 after IVF. Expanded blastocysts from all treatments were submitted to differential staining. One-way ANOVA from R software was applied to evaluate differences in cleavage and blastocysts rates and blastocyst cell numbers. Fisher’s exact test complemented by Bonferroni correction was used to determine meiotic progress. Caffeine maintained oocytes in meiotic arrest after 9 h of IVM in a concentration-dependent manner (germinal vesicle: 79.0%, 92.2%, 66.7%, 55.1%, 56.9%, 43.9%, 30.2%, respectively, for 30, 20, 10, 5, 1, 0 mM and standard; P < 0.016). Cleavage rates were similar in all treatments; however 30 mM caffeine decreased blastocyst rates (Table 1; P < 0.05). The number of cells did not differ significantly among in vitro treatments (Table 1; P > 0.05). Developmental competence was not affected by 2 h pre-IVM culture. Caffeine supplementation before IVM delayed resumption of meiosis and affected embryo development. Table 1.In vitro developmental competence of oocytes treated with different caffeine concentrations before IVM

154 THE EFFECT OF ZINC ON PORCINE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION

2014 ◽  
Vol 26 (1) ◽  
pp. 191
Author(s):  
Y. Jeon ◽  
J. D. Yoon ◽  
L. Cai ◽  
S. U. Hwang ◽  
E. Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Inductively Coupled Plasma ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Significant Difference

Zinc (Zn) is one of the abundant transition metals in biology and is an essential component of most cells. However, there are few reports about the effect of Zn in porcine oocytes. The objective was to investigate the effects of supplementary Zn during in vitro maturation (IVM) of porcine oocytes. We investigated nuclear maturation, intracellular glutathione (GSH) levels, reactive oxygen species (ROS) levels, and subsequent embryonic development after IVF. Before the experiment, Zn concentrations in IVM medium and body fluids were measured using inductively coupled plasma spectrophotometer (sensitivity: 1 μM) and treatment concentrations were determined. Zinc concentration was 12.6 μM in porcine plasma and 12.9 μM in porcine follicular fluid. We confirmed that Zn was not detected in IVM medium. A total of 541 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of Zn (0, 6, 12, 18, and 24 μM). After 44 h of IVM, no significant difference was observed in all groups (metaphase II rate: 85.7, 88.7, 90.4, 90.3, and 87.2%, respectively). A total of 100 matured oocytes were examined for the effects of different Zn concentrations (0, 6, 12, 18, and 24 μM) on porcine oocyte intracellular GSH and ROS levels, which were measured through fluorescent staining and image analysis program. The groups of 12, 18, and 24 μM showed a significant (P < 0.05) increase in intracellular GSH levels (1.45, 1.67, and 1.78, respectively) compared with the control and 6 μM group (1.00 and 1.08, respectively). The intracellular ROS level of oocytes matured with 12, 18, and 24 μM (0.82, 0.68, and 0.55) were significantly (P < 0.05) decreased compared with the control and 6 μM groups (1.00 and 1.03, respectively). Finally, the developmental competence of oocytes matured with different concentrations of Zn (0, 6, 12, 18, and 24 μM) was evaluated after IVF. There were no significantly different in cleavage rates. However, cleavage patterns and blastocyst (BL) formation were different. Fragmented embryo ratio of the 12 μM group (14.9%) was significantly lower than that of the other groups (control, 6, 18, and 24 μM: 26.4, 17.8, 18.4, and 18.0%, respectively). Oocytes treated with 12 μM Zn during IVM had a significantly higher BL formation rate (28.2%) after IVF compared with the control (19.8%). In conclusion, these results indicate that Zn treatment as body fluid concentration during IVM improved the developmental potential of IVF in porcine embryos by increasing the intracellular GSH concentration and decreasing the ROS level. This work was supported, in part, by a grant from the Next-Generation Bio Green 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

Gene transcription and regulation of oocyte maturation

2004 ◽  
Vol 16 (2) ◽  
pp. 55 ◽  
Author(s):  
Karina F. Rodriguez ◽  
Charlotte E. Farin
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Transcriptional Control ◽  
Molecular Mechanisms ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Oocyte Developmental Competence ◽  
Specific Factors

The developmental potential of an embryo is dependent on the developmental potential of the oocyte from which it originates. The process of oocyte maturation is critical for the efficient application of biotechnologies such as in vitro embryo production and mammalian cloning. However, the overall efficiency of in vitro maturation remains low because oocytes matured in vitro have a lower developmental competence than oocytes matured in vivo. Furthermore, oocytes that have been exposed to gonadotropins have greater developmental competence than oocytes matured in the absence of gonadotropins. By understanding the molecular mechanisms underlying gonadotropin-induced maturation, improvement in oocyte maturation technologies may be expected as procedures to manipulate specific factors involved in signalling for resumption of meiosis are identified. The present review will focus on transcriptional mechanisms underlying the maturation of mammalian oocytes in vitro, as well as on the acquisition of oocyte developmental competence. In addition, a working model for the transcriptional control of mammalian oocyte maturation is proposed.

scholarly journals Inhibition of miR-152 During in Vitro Maturation Enhances the Developmental Potential of Porcine Embryos

2020 ◽  
Author(s):  
Ahmed Gad ◽  
Matej Murín ◽  
Lucie Nemcova ◽  
Alexandra Bartkova ◽  
Jozef Laurincik ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Potential Role ◽  
Target Genes ◽  
Developmental Competence ◽  
Expression Level ◽  
Developmental Potential ◽  
Blastocyst Rate ◽  
And Control

Abstract Background Oocyte developmental competence is regulated by various mechanisms and molecules including microRNAs (miRNAs). However, the functions of many of these miRNAs in oocyte and embryo development are still unclear. In this study, we managed to manipulate the expression level of miR-152 during oocyte maturation to figure out its potential role in determining the developmental competence of porcine oocytes.Results The inhibition of miR-152 during oocyte maturation does not affect the MII and cleavage rates, however it significantly enhances the blastocyst rate compared to the mimic and control groups. Pathway analysis identified several signaling pathways (including PI3K/AKT, TGFβ, Hippo, FoxO, and Wnt signaling) that are enriched in the predicted target genes of miR-152. Gene expression analysis revealed that IGF1 was significantly up-regulated in the inhibitor group and downregulated in the mimic group of oocytes. Moreover, IGF1R was significantly upregulated in the inhibitor group compared to the control one and IGFBP6 was downregulated in the inhibitor group compared to the other groups. Blastocysts developed from the mimic-treated oocytes exhibited an increase in miR-152 expression, dysregulation in some quality-related genes, and the lowest rate of blastocyst formation. Conclusions Our results demonstrate a negative correlation between miR-152 expression level and blastocyst rate in pigs. This correlation could be through targeting IGF system components during oocyte development.

153 ZINC INSUFFICIENCY DURING PORCINE OOCYTES IN VITRO MATURATION CAUSED MEIOTIC BLOCK AND DEVELOPMENTAL FAILURE

2014 ◽  
Vol 26 (1) ◽  
pp. 190
Author(s):  
E. Kim ◽  
Y. Jeon ◽  
J. D. Yoon ◽  
L. Cai ◽  
S. U. Hwang ◽  
...  
Keyword(s):  
Embryonic Development ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Treated Group ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Total Cell Number ◽  
Nuclear Maturation

The objective was to investigate the effects of zinc (Zn) insufficiency during in vitro maturation (IVM) of porcine oocytes. Zinc insufficiency was induced by treatment of Zn chelator, N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN). In experiment 1, we investigated the effect of duration of Zn insufficiency in IVM on oocytes maturation and subsequent embryonic development after parthenogenetic activation (PA). First, 10 μM TPEN was added to the IVM medium for 0, 7, 15, or 22 h. After TPEN treatment, 10 μM Zn were supplemented on IVM medium except in the 0 h group. Reductions in the nuclear maturation rates were dependent on TPEN duration. The 0-h-treated oocytes showed 83.9 ± 3.9% metaphase II (MII) rate; the 7-h-treated oocytes had significantly lower MII rate (44.8 ± 3.0%) than 0-h-treated oocytes. The majority of 15- and 22-h-treated oocytes were arrested at metaphase I (MI rate: 98.0 ± 1.0 and 97.2 ± 1.7%, MII rate: 0 and 0%, respectively). Embryonic developmental competence was similar to maturation results. Reduction in cleavage and blastocyst (BL) rates were also dependent on duration of TPEN treatment (cleavage rate: 65.3 ± 1.4, 42.6 ± 4.8, 2.6 ± 0.1, and 3.0 ± 1.6%; BL formation rate: 29.3 ± 2.8, 9.2 ± 1.5, 0, and 0% for 0, 7, 15, and 22 h). Total cell number of BL was also significantly different. Total cell number of BL in the 0-h-treated group (51.4 ± 4.5) was significantly higher than that in the 7-h-treated group (23.2 ± 1.6). In experiment 2, to confirm that the Zn insufficiency caused oocyte immaturities and loss of developmental competence in TPEN-treated oocytes, we investigated nuclear maturation and subsequent embryonic development following 3 groups: (1) non treatment (control); (2) 10 μM TPEN treatment during 22 h of IVM; (3) 10 μM TPEN + 10 μM Zn treatment during 22 h of IVM. Only TPEN-treated oocytes and TPEN+Zn-treated oocytes showed contrasting results. Oocyte maturation rates and subsequent embryonic development competence in TPEN with Zn-treated oocytes were similar to control (MII rate: 93.0 ± 1.2 and 92.7 ± 1.8%, BL formation rate: 42.0 ± 6.7 and 40.0 ± 7.5% for TPEN+Zn-treated oocytes and control). These results were significantly different compared with only TPEN-treated oocytes’ results (MII rate: 0.61 ± 0.61%, BL formation rate: 0%). In conclusion, Zn is an essential element for successful oocyte maturation and embryo development in porcine. Zinc insufficiency caused meiotic block and had lasting effects on early embryo development. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

scholarly journals Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes

Reproduction ◽  
2003 ◽  
pp. 369-376 ◽  
Author(s):  
S Ikeda ◽  
H Imai ◽  
M Yamada
Keyword(s):  
Dna Fragmentation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Treated Group ◽  
Developmental Competence ◽  
Pcr Analysis ◽  
Control Group ◽  
Dependent Manner

The aim of this study was to investigate whether apoptosis occurs in cumulus cells during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes (CEOs). The bovine CEOs obtained from ovaries from an abattoir were cultured for 24 h in IVM medium in the presence or absence of 10% (v/v) fetal bovine serum. The developmental competence of enclosed oocytes, as assessed by the development of the blastocyst after IVF, was significantly higher in the serum-treated group than in the control group. The morphological features of apoptosis that were analysed by orcein staining were hardly detectable in the cumulus cells at the start (0 h) of IVM, but were evident at the end (24 h) of IVM both in the control and serum-treated groups. Genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect apoptotic internucleosomal DNA fragmentation. DNA fragmentation was hardly detectable at the start of IVM, but increased in a time-dependent manner as the IVM culture proceeded. DNA fragmentation was not observed in the oocytes, indicating that fragmentation occurs in cumulus cells. The degree of fragmentation was lower in the serum-treated group compared with the control group. The LM-PCR analysis of DNA extracted from CEOs at 24 h of IVM, in which the DNA had been pretreated with Klenow enzyme or T4 DNA polymerase, revealed that the characteristic forms of the DNA ends generated during cumulus cell apoptosis were mainly 3'-overhangs and blunt ends. In conclusion, the results of the present study demonstrate that cumulus cells in bovine CEOs spontaneously undergo apoptosis during IVM. The degree of apoptosis may be correlated with the developmental competence of the enclosed oocytes.

Sign in / Sign up

Export Citation Format

Share Document