scholarly journals 78 SURVIVAL OF PORCINE OOCYTES AT GERMINAL VESICLE STAGE AFTER VITRIFICATION WITH OPEN PULLED STRAW METHOD

2005 ◽  
Vol 17 (2) ◽  
pp. 189 ◽  
Author(s):  
A. Bali Papp ◽  
T. Somfai ◽  
E. Varga ◽  
M. Marosán
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Penicillin G ◽  
National Committee ◽  
Ministry Of Education ◽  
Germinal Vesicle Breakdown ◽  
Maturation Medium ◽  
Maturation Rate ◽  
Nuclear Stage

The present study was performed to assess the survival of immature denuded or cumulus-covered porcine oocytes (COCs). Immature porcine oocytes were collected from 2–6 mm follicles of slaughterhouse ovaries and subjected to open pulled straw (OPS) vitrification, according to the method of Vajta et al. (1998 Mol Reprod. Dev. 51, 53–58). After vitrification, oocytes were matured in vitro for 48 h at 39°C, 5% CO2 in air. The maturation medium was TCM199 supplemented with 10% pig follicular fluid, 1.25 mM L-glutamine, 0.9 mM Na pyruvate, 150 μM cysteamine, 0.1 mg/mL streptomycin sulfate, 100 IU/mL PG penicillin g potassium, 10 IU/mL PMSG, and 25 IU/mL hCG. After IVM, to assess nuclear stage, all oocytes were fixed with acetic acid–alcohol (1:3) for at least three days and then stained with 0.1% orcein and examined under a phase-contrast microscope at 100× magnification. All data were analyzed by χ2 test (P < 0.05). Immediately after collection, all oocytes were at the germinal vesicle (GV) stage with an intact GV membrane. After vitrification, significantly fewer oocytes had normal morphology (intact plasma membrane) in the denuded and COC groups (4.7% and 8.5%, respectively) than did the denuded and COC control groups (95% and 92%, respectively). By the end of IVM, significantly fewer oocytes were surrounded by expanded cumulus after vitrification of COCs than were the COC controls (28.1% and 63.5%, respectively). After IVM, more of the COC control oocytes underwent germinal vesicle breakdown than did the denuded controls (95% and 78.2%, respectively); the rate of MII oocytes was higher for the COC controls than for the denuded controls (80% and 54.5%, respectively). After vitrification, the number of oocytes that underwent GVBD was significantly less for both the denuded and the COC groups (2.0% and 7.0%, respectively); the percentage of oocytes that reached MII was also lower (0.64% and 2.78%, respectively). Most of the vitrified oocytes had a damaged GV with disrupted membrane and cluster-like or scattered chromatin in both the denuded and the COC groups (96.4% and 90.7%, respectively). These data suggest that vitrification of cumulus-enclosed immature porcine oocytes is preferable compared to vitrification of denuded ones. Loss of cumulus cells compromises competence of oocytes to resume meiosis, which might result in a lower maturation rate after IVM. This research was supported by the grants of the Hungarian Scientific Research Fund (T 031758), the Hungarian National Committee of the Technical Development at the Ministry of Education (00796/2003), and the Ministry of Education (OM-KMUFA; BIO-00086/2002).

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

Time course of the meiotic arrest in sheep cumulus–oocyte complexes treated with roscovitine

Zygote ◽  
2015 ◽  
Vol 24 (2) ◽  
pp. 310-318 ◽  
Author(s):  
Letícia Ferrari Crocomo ◽  
Wolff Camargo Marques Filho ◽  
Camila Louise Ackermann ◽  
Daniela Martins Paschoal ◽  
Midyan Daroz Guastali ◽  
...  
Keyword(s):  
Exposure Time ◽  
Cell Expansion ◽  
Time Course ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Nuclear Maturation ◽  
Maturation Medium

SummaryTemporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus–oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 μM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.

scholarly journals Does the Apoptosis Value of Cumulus Cells Play a Role in Rescue Oocyte in Vitro Maturation?

2020 ◽  
pp. 1
Author(s):  
Tulay Irez ◽  
Sinem Ercan Dogan ◽  
Enver Ciraci ◽  
Saadet Busra Aksoyer ◽  
Muhammet Sait Toprak ◽  
...  
Keyword(s):  
Experimental Study ◽  
Luteinizing Hormone ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Immature Oocytes ◽  
Maturation Rate

<p><strong>OBJECTIVE:</strong> In this study, we aimed to investigate the role of the cumulus cell’s apoptosis parameter in the maturation of immature rescue oocytes. </p><p><strong>STUDY DESIGN:</strong> In this experimental study, donated immature germinal vesicle oocytes were cultured for, in vitro maturation, embryo development in matured germinal vesicle oocytes were compared with apoptotic properties of cumulus cells. </p><p><strong>RESULTS:</strong> In all of the immature oocytes after oocyte in vitro maturation, the maturation rate has been observed as 56.1% and 2PN rate as 63.0%. Afterin vitro maturation of germinal vesicle oocytes, there was no difference in apoptosis rates of the cumulus cells between mature and immature oocytes (p&gt; 0.05). The ratio of 2PN in matured germinal vesicle oocytes showing embryo development was 35.4%. A positive correlation was found between luteinizing hormone values on day 3 and E2 values during HCG days during oocyte maturation and embryo development (p=0.021, p=0.020). In addition, it has been observed that the germinal vesicle oocytes, which have completed their maturation and developed into embryos, have high E2 values during HCG days (p=0.020).</p><p><strong>CONCLUSION:</strong> In our study, it has been demonstrated that in vitro maturation in rescue oocytes from stimulated cycles, embryo development potential could not be explained by the apoptosis parameter.</p>

341 IN VITRO MATURATION AND IN VITRO FERTILIZATION OF ALPACA (VICUGNA PACOS) OOCYTES: EFFECT OF TIME OF INCUBATION ON NUCLEAR MATURATION AND CLEAVAGE

2010 ◽  
Vol 22 (1) ◽  
pp. 327 ◽  
Author(s):  
W. Huanca ◽  
R. Condori ◽  
J. Cainzos ◽  
M. Chileno ◽  
L. Quintela ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Hypodermic Needle ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Maturation Time ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Maturation Medium

Experiments were carried out to evaluate the effect of incubation time on nuclear maturation (Experiment 1) and determine the cleavage rate of alpaca oocytes after of IVF time (Experiment 2) In Experiment 1, CCOs were collected from slaughterhouse ovaries and transported to the laboratory in a thermos flask containing a saline solution 0.9% with antibiotic antimycotic at 35°C. CCOs were aspirated from follicles >2 mm and pooled in a conical tube to sedimentation previous to evaluation under stereomicroscope and CCOs with a cytoplasm homogeneous and 2 or more layers of cumulus cells were transferred to plates with a 40-μL drop of maturation medium TCM-199 supplemented with 10% FCS (v : v) plus 0.5 μg mL-1 FSH, 10 μg mL-1 hCG, 0.2 mM sodium pyruvate, 50 μg mL-1 gentamicine, and 1 μg mL-1 Estradiol under mineral oil with 10-12 oocytes/drop. Oocytes were incubated under the following maturation times: 30, 34, and 38 h at 39°C in an atmosphere of 5% CO2 and high humidity. After each maturation time, CCOs were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1 mgmL-1 of hyaluronidase and fixed in ethanol: acetic acid (3 : 1). Oocytes were placed on the slide with minimum medium and stained with 1% orcein for 5 min The slides were examined under a phase contrast microscope at × 400 to evaluate status of nuclear maturation and classified as germinal vesicle (GV); metaphase I (M-I), anaphase-telophase; metaphase II (M-II) and degenerated. Experiment 2: The same maturation method as Experiment 1 was used. Testes were collected of mature males from slaughterhouse and transported to the laboratory. Caudal epididymide was isolated. A prick was made on the convoluted tubules with a sterile hypodermic needle and the fluid, rich in spermatozoa, was aspirated in syringes containing 2 mL of Tris-fructose egg yolk extender. Motile spermatozoa were obtained by centrifugation: 700 g on a Percoll discontinuous gradient (22.5 :45.0%) for 25 min. The supernatant was removed by aspiration and pellet (containing viable spermatozoa) was resuspended in TL stock. Spermatozoa and oocytes were co-incubated for 18-20 h at 39°C with 5% CO2 and then cultivated in TCM-199 supplemented with 10% FCS (v: v), 0.2 mM sodium pyruvate, and 50 μg mL-1 gentamicine and evaluated at 48 h. Data were subjected to ANOVA. For Experiment 1, the proportions of oocytes reaching M-II stage was 18.9 ± 15.7, 42.9 ± 16.2, and 65.8 ± 8.1% for the 30, 34, and 38 h of culture, respectively, with difference to maturation time (P < 0.05). For Experiment 2, the cleavage rate was 9.5, 7.7, and 15.4% to 30, 34, and 38 h after of fertilization time 48 h culture. These results indicate that 38 or more h is required for the maturation and fertilization of alpaca oocytes. Grant 064 FINCyT-PIBAP 2008.

Phosphatidylinositol 3-kinase in cumulus cells is responsible for both suppression of spontaneous maturation and induction of gonadotropin-stimulated maturation of porcine oocytes

2003 ◽  
Vol 179 (1) ◽  
pp. 25-34 ◽  
Author(s):  
M Shimada ◽  
J Ito ◽  
Y Yamashita ◽  
T Okazaki ◽  
N Isobe
Keyword(s):  
High Activity ◽  
Cell Function ◽  
Kinase Inhibitor ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Basic Medium ◽  
Phosphatidylinositol 3 Kinase ◽  
Germinal Vesicle Breakdown ◽  
Phosphatidylinositol 3

In this study, we investigated the mechanisms of protein kinase B (PKB) activation and its role in cumulus cells during in vitro meiotic resumption of porcine oocytes. PKB activity in cumulus cells was significantly decreased by 12 h cultivation of cumulus-oocyte complexes (COCs) in basic medium. However, the addition of phosphodiesterase inhibitors, hypoxanthine or 3-isobutyl-1-methylxanthine, maintained the level of PKB activity in cumulus cells at comparable with that in cumulus cells just after collection from their follicles. When COCs were cultured with phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, PKB activity was significantly decreased, and both caspase 3 activity and the proportion of apoptotic cells were significantly increased as compared with those in cumulus cells just after collection from their follicles. Moreover, the inhibitory effect of hypoxanthine on spontaneous meiotic resumption was overcome by addition of LY294002. On the other hand, markedly high activity of PKB and high intensity of the phosphorylated PKB band were observed in cumulus cells of COCs which were cultured with FSH. The addition of 20 microM LY294002 to FSH-containing medium induced an apoptosis of cumulus cells, whereas little apoptotic-positive signal was detected in COCs cultured with 5 microM LY294002 and FSH. However, the inhibitory effects of LY294002 on progesterone production by cumulus cells and germinal vesicle breakdown in oocytes reached a maximum at 5 microM. Thus, high activity of the PI 3-kinase-PKB pathway in cumulus cells plays an important role in FSH regulation of cell function. Judging from these results, it is estimated that PI 3-kinase in cumulus cells is required for both the suppression of spontaneous meiotic resumption and the induction of gonadotropin-stimulated meiotic resumption.

Relationship between DNA fragmentation and nuclear status of in vitro-matured porcine oocytes: role of cumulus cells

2004 ◽  
Vol 16 (8) ◽  
pp. 773 ◽  
Author(s):  
Pimprapar Wongsrikeao ◽  
Takeshige Otoi ◽  
Masako Murakami ◽  
Ni Wayan Kurniani Karja ◽  
Agung Budiyanto ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Critical Role ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Fresh Medium ◽  
Tunel Method ◽  
Maturation Rate ◽  
Total Proportion

The present study was conducted to investigate the effects of the attachment of cumulus cells to oocytes and coculture with cumulus cells during maturation culture on the nuclear status and DNA fragmentation of porcine denuded oocytes (DOs). In the first experiment, cumulus cells were removed from cumulus–oocyte complexes (COCs) at 0, 8, 16, 24 or 32 h after the onset of maturation culture and the DOs were then cultured in their original droplets until 42 h of culture was reached. In the second experiment, all COCs were denuded before the onset of culture and the DOs were cocultured with their removed cumulus cells. The DOs were transferred into fresh medium at 0, 8, 16, 24 or 32 h after the onset of coculture with cumulus cells and then cultured until 42 h of culture was reached. After culture, DNA fragmentation and the nuclear status of oocytes were examined using the terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL) method. When the DOs were returned to the same droplets after removal of the cumulus cells, the removal of the cumulus cells after 16 h of culture significantly decreased the proportion of oocytes remaining at the germinal vesicle (GV) stage. However, coculture treatment of DOs in the presence of their removed cumulus cells had no significant effects on the GV breakdown (GVBD) of oocytes. There were no significant differences in the proportion maturing to MII oocytes among the groups following removal of cumulus cells after the onset of maturation culture; however, DOs cocultured with cumulus cells until the end of maturation culture exhibited an increased maturation rate compared with DOs cocultured for 8 and 16 h. The total proportion of TUNEL-positive oocytes of oocytes remaining at the GV stage was higher than that of oocytes reaching other stages, irrespective of the removal of cumulus cells and coculture treatments. However, coculture for more than 16 h decreased the total proportion of TUNEL-positive oocytes. Our results indicate that the attachment of cumulus cells to oocytes may have a critical role for oocytes undergoing GVBD and that coculture with cumulus cells promotes the ability of oocytes to complete maturation. Moreover, coculture with cumulus cells may assist the oocyte to avoid undergoing DNA fragmentation.

140 EXPRESSION OF PERILIPIN IN PORCINE OOCYTES AND CUMULUS CELLS AT DIFFERENT STAGES OF IN VITRO MATURATION

2016 ◽  
Vol 28 (2) ◽  
pp. 200
Author(s):  
A. Oh ◽  
J.-X. Jin ◽  
S. Lee ◽  
G. A. Kim ◽  
B. C. Lee
Keyword(s):  
Messenger Rna ◽  
Lipid Droplets ◽  
In Vitro Maturation ◽  
Statistical Significance ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Intracellular Lipid ◽  
Germinal Vesicle Breakdown ◽  
Interacting Protein

Perilipin, one of the perilipin adipophilin tail-interacting protein of 47 kDa (PAT) family, has been found to coat the surface of intracellular lipid droplets. It limits the interaction of lipases with intracellular lipid droplets and is involved in the formation and regulation of lipids in various kinds of cells. However, little is known about the effect of perilipin on porcine oocytes and cumulus cells. Therefore, this study aimed to detect the expression of perilipin1 (PLIN1), perilipin2 (PLIN2), perilipin3 (PLIN3), and perilipin4 (PLIN4) in porcine oocytes and cumulus cells at 4 stages of in vitro maturation (IVM) by quantitative real-time PCR (RT-qPCR). Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) stage were found to occur predominantly at 18 to 24 h, after 24 h, 30 to 36 h, and 36 to 44 h of IVM, respectively. Cumulus-oocyte complexes (COC) were cultured in IVM medium and oocytes and cumulus cells were isolated after different durations of IVM (20, 26, 32, and 44 h). The data were analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Software, Sn Diego, CA, USA) and the threshold for statistical significance was set at P < 0.05. Messenger RNA expression of PLIN1 was not detected in either oocytes or cumulus cells during all periods of IVM. PLIN2, on the other hands, showed a significant lower expression in GVBD, MI, and MII oocytes compared with the GV oocytes, but showed no difference in cumulus cells. PLIN3 expression was significantly decreased in oocytes of MI stage, whereas PLIN3 from cumulus cells was expressed significantly lower in GVBD and MII stage compared with GV stage. Expression of PLIN4 was significantly decreased in only cumulus cells of GVBD and MI stage. These findings suggest that PLIN2 may have important roles in lipid metabolism during porcine oocyte maturation, whereas PLIN4 may play a major role in cumulus cells. PLIN3 can be hypothesised as a common lipid droplet-associated protein in both oocytes and cumulus cells. Further studies should be conducted to characterise the expression and distribution of PLIN1, PLIN2, PLIN3, and PLIN4 in porcine oocytes and cumulus cells. This study was supported by Ministry of Trade, Industry and Energy (#10048948), Korea IPET (#311011-05-4-SB010), Research Institute for Veterinary Science, and the BK21 plus program.

340 EFFECT OF GLUCOSE ON THE MORPHOLOGY OF GERMINAL VESICLE AND MEIOTIC PROGRESSION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM

2007 ◽  
Vol 19 (1) ◽  
pp. 285
Author(s):  
H. Funahashi ◽  
T. Koike
Keyword(s):  
Critical Role ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Defined Medium ◽  
Pentose Phosphate ◽  
Chemically Defined Medium ◽  
Dibutyryl Camp ◽  
Germinal Vesicle Breakdown ◽  
Maturation Medium ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose metabolism through the pentose phosphate pathway (PPP) seems to play a critical role in meiotic resumption in mouse oocytes (Downs et al. 1998 Biol. Reprod. 58, 1084–1094). However, the role is not clear in porcine oocytes. In the present study, we examined whether glucose affects morphological change of germinal vesicles and the resumption of meiosis in porcine oocytes in a chemically defined medium. In the first experiment, porcine cumulus–oocyte complexes (COCs) were collected from 3–6-mm follicles of slaughterhouse ovaries and cultured in a chemically defined medium, mNCSU37-PVA with/without 5.55 mM glucose in the presence of eCG, hCG, and dibutyryl cAMP for 20–22 h and then in the absence of eCG, hCG, and dibutyryl cAMP for 24 h. In the second experiment, 5.55 mM glucose in the maturation medium was replaced with the same concentration of Na pyruvate. In the third experiment, the PPP inhibitor 6-aminonicotinamide (6-AN) was added to the maturation medium at various concentrations (0, 10, 50, and 100 �M). To determine the activity of glucose-6-phosphate dehydrogenase (G6PD), OCCs were fixed, blocked, and treated with anti-G6PD polyclonal antibody and the secondary antibody labeling a fluorescent material. Results from 3–5 replicates were analyzed by ANOVA and Duncan&apos;s multiple range test. When OCCs were cultured in glucose-free chemically defined maturation media, regardless of the presence of hormones and dibutyryl cAMP, germinal vesicle breakdown (GVBD) of oocytes was inhibited (10.0&ndash;21.3&percnt;), as compared with OCCs cultured in the presence of glucose and hormones (91.4&ndash;92.0&percnt;). In a majority of oocytes in which GVBD was inhibited, the arrest occurred at the GV-I stage. When OCCs were cultured in maturation media in which glucose was replaced with Na pyruvate, GVBD was not inhibited any more than in control samples that were cultured in the presence of glucose (97.4&percnt; vs. 97.1&percnt;). However, the incidence of oocytes developing to the metaphase II stage was significantly lower in this condition than in controls (4.8&percnt; vs. 49.9&percnt;, respectively). A majority of the oocytes were at the metaphase I stage (86.0&percnt; vs. 45.5&percnt; in controls). The presence of 6-AN in maturation media significantly inhibited GVBD of oocytes (77.3, 29.0, 7.4, and 8.4&percnt; at 0, 10, 50, and 100 &micro;M, respectively) and arrested the oocytes at the GV-I stage. Immunocytochemistry with anti-G6PD demonstrated the activity of G6PD in cumulus cells of OCCs. In conclusion, these results demonstrate that glucose plays a critical role in the release of porcine oocytes arrested at the GV-I stage, probably through PPP of cumulus cells. The current results also suggest the possibility of gluconeogenesis in porcine OCCs when glucose in maturation media was replaced with Na pyruvate.

76 EFFECTS OF REDUCED ENVIRONMENTAL LIGHT ON THE IN VITRO MATURATION OF PIG OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 167
Author(s):  
L. Y. Parra-Forero ◽  
A. Góngora ◽  
S. Romo-García ◽  
E. P. López Damian ◽  
G. D. Mendoza ◽  
...  
Keyword(s):  
Light Exposure ◽  
Red Light ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Penicillin G ◽  
Ambient Light ◽  
Embryo Viability ◽  
Group 2 ◽  
Group 1

The effects of light on the steps of embryo manipulation have been described in several species, including humans. There are reports in which exposure of these cells to UV rays from sunlight affects them epigenetically, which could lead to meiotic arrest that would prevent normal maturation from the germinal vesicle (GV) to metaphase II (MII) stage, which would compromise subsequent embryo viability. Development to the blastocyst was not evaluated. The objective of the study was to observe the difference in maturation capacity (GV to MII) of prepubertal gilt oocytes under conditions of reduced ambient light. Thirty Duroc ovaries were recovered at slaughter and immersed in saline (0.9% NaCl) supplemented with penicillin-G (100 IU mL–1) and streptomycin sulfate (100 mg mL–1) at 29 to 34°C for transport to the laboratory where they were punctured with an 18-gauge needle attached to a 10-mL syringe. GV oocytes were selected with at least 3 layers of compact cumulus cells and were incubated with TCM-199 for 42 h in total with replacement with fresh maturation medium at 22 h. Oocytes were subsequently denuded using 0.1% hyaluronidase, fixed with 2% formaldehyde, and stained with Hoechst 33342 (10 min). Thereafter, oocytes were evaluated under a fluorescence microscope for the stage of meiosis: GV, metaphase I (MI), anaphase I/telophase I (ATI), and MII. GV oocytes were divided into two groups of 60 each as follows: Group 1 = handling in ambient light (1 change of culture medium and evaluation at the stereoscope); Group 2 = handling with reduced light (the change of medium was carried out in a dark room with red light on the stereoscope. Chi-square and Student’s t-test with Minitab 16.0 statistical program were used, and differences were considered significant at P < 0.05. There were significant differences in the percentages of maturation stages between Group 1 and Group 2, respectively: GV = 3.33, 3.33% (P = 0.45); MI = 20, 13.3% (P = 0.037); ATI = 33.3, 26.6% (P = 0.03); and MII = 43.3, 56.6% (P = 0.019). In conclusion, rate of maturation to MII was significantly higher with decreased light exposure. There was no difference in the GV groups probably because there was no manipulation at this stage. Further studies are necessary to determine the amount of light needed to optimize oocyte viability and to assess any potential effects on blastocyst production.

scholarly journals Effects of cryodevice type and donors' sexual maturity on vitrification of minke whale (Balaenoptera bonaerensis) oocytes at germinal vesicle stage

Zygote ◽  
2004 ◽  
Vol 12 (4) ◽  
pp. 333-338 ◽  
Author(s):  
Hiroshi Iwayama ◽  
Shinichi Hochi ◽  
Megumi Kato ◽  
Masumi Hirabayashi ◽  
Masashige Kuwayama ◽  
...  
Keyword(s):  
Polar Body ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Minke Whale ◽  
Minke Whales ◽  
Maturation Medium ◽  
First Polar Body ◽  
Maturation Rate ◽  
Cell Layers

Germinal-vesicle-stage oocytes enclosed with compact cumulus cell layers (COCs) were recovered from adult or prepubertal minke whale ovaries, and were vitrified in a solution containing 15% ethylene glycol, 15% DMSO and 0.5 M sucrose using either a Cryotop or an open-pulled straw (OPS) as the cryodevice. The post-warm COCs with normal morphology were cultured for 40 h in a 390 mosmol in vitro maturation medium, and oocytes extruding the first polar body were considered to be matured. The proportion of morphologically normal COCs after vitrification and warming was higher when the COCs were cryopreserved by Cryotop (adult origin, 88.4%; prepubertal origin, 80.8%) compared with the OPS (adult origin, 67.7%; prepubertal origin, 64.2%). The oocyte maturation rate was higher in the adult/Cryotop group (29.1%) compared with those of the prepubertal/Cryotop group (14.4%), the adult/OPS group (14.3%) and the prepubertal/OPS group (10.6%). These results indicate that the Cryotop is a better device than the OPS for vitrification of immature oocytes from adult minke whales.

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