337 EFFECTS OF CANINE SYNTHETIC OVIDUCT FLUID MEDIUM SUPPLEMENTED WITH THE VARIOUS ENERGY SUBSTRATES ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 276
Author(s):  
H. J. Oh ◽  
M. K. Kim ◽  
Y. H . Fibrianto ◽  
G. Jang ◽  
H. J. Kim ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
The Other ◽  
Germinal Vesicle Breakdown ◽  
Energy Substrates ◽  
Fluid Medium ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Oviduct Fluid

In most mammals, maturation occurs within the ovarian follicle, and preovulatory oocytes are ovulated and ready for fertilization within the oviduct. In contrast, bitch ovulate primary oocytes, over a three day period, undergo both maturation and fertilization within the oviduct. The present study was conducted to evaluate the effects of canine synthetic oviduct fluid (cSOF) supplemented with the various energy substrates on in vitro maturation of canine oocytes. Oocytes were recovered by mincing ovaries collected after ovariohysterectomy in bitches at the follicular stage. Only oocytes with more than two layers of cumulus cells and with homogeneous cytoplasm >100 mm in diameter were selected. Then, oocytes cultured in tissue culture medium (TCM)-199 (control) or cSOF supplemented with various concentrations of glucose (0, 1.11, 3.89, or 5.56 mM, Exp. 1) or fructose (0, 1.11, 3.89, or 5.56 mM, Exp. 1), pyruvate (0, 0.1, 0.25, or 0.5 mM, Exp. 2) or lactate (0, 0.5, 1.0, or 5.0 mM, Exp. 3). In Exp. 4, the combined effects of glucose (1.11 mM), pyruvate (0.5 mM) and lactate (5.0 mM) on nuclear maturation of canine oocytes were investigated. A total of 2990 canine oocytes from 205 ovaries were used for experiments with replication at least three times. The oocytes were cultured for 72 h at 38.5�C in a humidified atmosphere of 5% CO2 in air. After 72 h, the oocytes were stained with 1.9 �g/mL Hoechst 33342 in glycerol and then evaluated under UV light to determine the stage of meiosis as follows: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII) with first polar body. The results of Exp. 1 showed that maturation of canine oocytes to MII was significantly higher (P < 0.05) in medium supplemented with 1.11 mM glucose (4.8%) than for the control (1.8%) and the other glucose-supplemented groups (0 to 1.8%). In Exp. 2, oocytes cultured in cSOF supplemented with 0.5 mM pyruvate showed a significantly higher (P < 0.05) maturation rate to MII (6.3%) than did the other pyruvate-supplemented (0, 0.8, or 2.5%) groups or the control (2.4%). In Exp. 3, more oocytes were matured to the MII stage in cSOF supplemented with 5.0 mM lactate (7.3%) than were the other lactate-supplemented groups (0 to 2.4%) or the control (2.5%). Results of Exp. 4 showed more oocytes progressed to MII in cSOF supplemented with 0.5 mM pyruvate (8.2%), 1.11 mM glucose + 0.5 mM pyruvate (7.4%), or 1.11 mM glucose + 0.5 mM pyruvate 0.5 + 5.0 mM lactate (7.3%) than did the other combination groups (2.2 to 5.2%). In conclusion, the present study demonstrated that supplementing cSOF with 1.11 mM glucose, 0.5 mM pyruvate, or 5.0 mM lactate significantly increased the maturation of canine oocytes to MII, and the combined supplementation of 1.11 mM glucose, 0.5 mM pyruvate, and 5.0 mM lactate further promoted oocyte nuclear maturation compared to 1.11 mM glucose alone and the control. This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00).

171 IN VIVO AND IN VITRO MATURATION OF WOOD BISON (BISON BISON ATHABASCAE) CUMULUS–OOCYTE COMPLEXES DURING THE OVULATORY SEASON

2014 ◽  
Vol 26 (1) ◽  
pp. 199
Author(s):  
M. P. Cervantes ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
J. M. Palomino ◽  
G. P. Adams
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Bison Bison ◽  
Germinal Vesicle Breakdown ◽  
Cumulus Expansion ◽  
Nuclear Maturation ◽  
Wood Bison

Technologies are being developed to conserve the genetic diversity of wood bison. Knowledge of the characteristics of in vivo and in vitro maturation of the cumulus–oocyte complex (COC) are needed in wood bison to design efficient in vitro embryo production protocols. The objectives were to (1) determine the optimal interval after hCG treatment for in vivo maturation of COC in superstimulated wood bison, and (2) compare the characteristics of COC after in vitro and in vivo maturation. Ovarian synchronization was induced in 25 bison during October and November by giving a luteolytic dose of prostaglandin followed 8 days later by follicular ablation (Day –1). Ovarian superstimulation was induced with FSH (Folltropin-V) given i.m. on Day 0 (300 mg) and Day 2 (100 mg). A second luteolytic dose of prostaglandin was given on Day 3. Bison were assigned randomly to 5 groups (n = 5/group). The COC were collected by transvaginal follicle aspiration on Day 4 and were either assessed immediately (0 h, control), or matured in vitro for 24 or 30 h (in vitro maturation), or collected on Day 5 (in vivo maturation), 24 or 30 h after bison were given 2000 IU of hCG i.m. on Day 4. In vitro maturation was done in TCM-199 with 5% calf serum, 5 μg mL–1 LH, 0.5 μg mL–1 FSH, and 0.05 μg mL–1 gentamicin, at 38.5°C and in a 5% CO2 humidified atmosphere. Nuclear maturation was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), or metaphase II (MII) with anti-lamin AC/DAPI staining. Groups were compared by analysis of variance and Fisher's exact test (Table 1). A mean (±s.e.m.) of 7.3 ± 1.7 COC were collected per bison, with no difference among groups. The COC in the control (0 h) group were at the nonexpanded GV stage. Cumulus cells were more expanded after in vivo than in vitro maturation, and the percentage of fully expanded COC was the highest in the 30-h in vivo maturation group (87%; P < 0.05). The greatest number of oocytes reached MII stage after 24 h of in vitro maturation, and 30 h of in vivo maturation. In conclusion, nuclear maturation occurred more quickly in vitro compared with in vivo, but the degree and incidence of cumulus expansion was greater after in vivo maturation. The competence of oocytes to undergo fertilization and develop into embryos remains to be investigated. Table 1.Cumulus expansion and nuclear maturation of wood bison oocytes

Effect of GnRH treatment on the maturation and in vitro development of oocytes collected from 4- to 6-week-old Merino lambs

2007 ◽  
Vol 19 (8) ◽  
pp. 947 ◽  
Author(s):  
Jennifer M. Kelly ◽  
David O. Kleemann ◽  
W. M. Chis Maxwell ◽  
Simon K. Walker
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation ◽  
Oocyte Collection ◽  
Vitro Development

Two experiments were conducted in Merino lambs to examine the effects of gonadotrophin-releasing hormone (GnRH) treatment on the developmental competence of oocytes collected after pretreatment with follicle stimulating hormone (FSH). The first experiment examined the effects of six GnRH treatment times (control and GnRH administered 2, 4, 6, 8 and 10 h before oocyte collection) and four in vitro maturation (IVM) periods (18, 20, 22, 24 h) on the rate of oocyte nuclear maturation. The second experiment examined the effect of five GnRH treatment times (control and GnRH administered 2, 4, 6 and 8 h before oocyte collection) and three IVM periods (20, 22, 24 h) on the development of oocytes and embryos after in vitro maturation, fertilisation and culture. In Experiment 1, GnRH treatment did not influence the mean number of cumulus-oocyte-complexes (COCs) collected or COC morphology at the time of collection. However, treatment changed (P < 0.01) the distribution of follicle size and this was primarily due to a marked reduction in the number of follicles with diameters <2 mm. In addition, GnRH treatment at 6 and 8 h increased (P < 0.01) the proportion of oocytes that developed to Metaphase II (MII) (63.2 and 72.6%, respectively) compared with other treatment times (range 52.9–59.9%). Nuclear maturation was influenced by a significant (P < 0.05) interaction between GnRH treatment and IVM period due to a disproportionately greater number of oocytes at the germinal vesicle breakdown (GVBD) stage for the 2 and 4 h GnRH treatments compared with other treatments. In Experiment 2, cleavage rate (range 63.5–85.9%) was highest when GnRH was administered 8 h before collection but the percentage of cleaved oocytes that developed into blastocysts (range 10.0–35.0%) was significantly (P < 0.05) lower for the 6 and 8 h GnRH treatments compared with the control and the 2 h GnRH treatment. These results demonstrate that GnRH treatment before oocyte collection can improve nuclear maturation and cleavage rates in lamb oocytes but that these improvements are not reflected in improved rates of blastocyst development. It is speculated that this discrepancy may result from GnRH treatment either adversely affecting cytoplasmic maturation or inducing asynchrony between the maturation of the nuclear and cytoplasmic components of the oocyte.

scholarly journals 289 EFFECTS OF THE STORAGE OF BOVINE OVARIES ON THE NUCLEAR MATURATION AND DEVELOPMENT OF IN VITRO PRODUCED EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 295
Author(s):  
M. Narita ◽  
S. Goda ◽  
Y. Inaba ◽  
K. Imai ◽  
S. Matoba ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Saline Group ◽  
The Other ◽  
Control Group ◽  
Maturation Time ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Stage

The objectives of this study were to investigate effects of storage of bovine ovaries on the maturation of oocytes and to determine the optimal maturation time for oocytes obtained from the stored ovaries. Ovaries were obtained at a local abattoir and transported in physiological saline to the laboratory (18°C, 3 h; storage group). As a control, oocytes were collected from ovaries without storage. Other ovaries were kept in a plastic bag without solution (Bag-group) or with saline (Saline-group). These ovaries were preserved at 20°C for 18 h. Then cumulus-oocyte complexes were collected and maturated in TCM-199 + 5% CS. In Experiment 1, to investigate effects of the storage methods of bovine ovaries on the timing of germinal vesicle breakdown (GVBD) and the progression to MII in oocytes obtained from ovaries, oocytes were fixed every 2 h (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 h) from the start of in vitro maturation, and then stained for examination of their nuclear stage. In Experiment 2, to investigate effects of length of in vitro maturation (18, 20, 22, 24 h) of oocytes (18-h, 20-h, 22-h and 24-h group, respectively) obtained from the ovaries stored in a saline for 18 h at 20°C on the subsequent in vitro development after IVF and IVC. Following insemination, the presumptive zygotes were cultured in CR1aa + 5% CS for 6 days to assess the development of embryos on Day 2 (Day 0 = the day of IVF) for rates of cleavage and on Day 6 for rates of embryo development to morulae (M), compacted morulae (CM), and blastocyst (BL) stages. The data of nuclear stage were analyzed by ANOVA after transformation to arcsine, and the rates of embryo development were analyzed by chi-square. There were two peaks of GVBD in the storage group, one occurred at 2 h of maturation culture, the other at 4–8 h of culture as control. There were between-treatment differences in the timing of increase in the rates of oocytes to reach MII. After 12 h of culture 21.2 ± 1.1% of oocytes in the Saline-group and 11.6 ± 4.6% of oocytes in the Bag-group reached MII, but no oocytes in the control group reached MII (P < 0.05). Furthermore, the rate of oocytes in the Saline-group matured to MII at 20 h of culture was lower than that of the control group (Bag-group: 67.9 ± 7.3%; Saline-group: 61.2 ± 14.5%; control: 82.9 ± 5.3%) (P < 0.05). The rates of embryos that cleaved after IVF of IVM oocytes in the 18-h group (90.2 ± 7.0%) was higher than those of the other groups (20-h group: 81.3 ± 8.2%, 22-h group: 80.5 ± 13.2%, 24-h group: 75.8 ± 6.0%) (P < 0.05). The rate of embryos developed to M, CM, and BL stages in the 18-h group (48.4 ± 6.7%) was the highest among the treatments, and significantly higher than that of the 24-h group (36.2 ± 6.7%) (P < 0.05). These results indicated that the timing of undergoing GVBD and reaching MII of oocytes obtained from the stored ovaries was earlier than that of oocytes obtained from the non-preserved ovaries, and the optimal maturation time for oocytes obtained from stored ovaries was 18 h. This work was supported by The Ito Foundation, Tokyo, Japan.

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

336 MEIOTIC DEVELOPMENT AND CORTICAL GRANULES DISTRIBUTION IN CANINE OOCYTES DURING IN VITRO MATURATION

2010 ◽  
Vol 22 (1) ◽  
pp. 324 ◽  
Author(s):  
M. De los Reyes ◽  
D. Luna ◽  
J. Palomino
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Fluorescein Isothiocyanate ◽  
Cumulus Cells ◽  
Homogeneous Distribution ◽  
Cortical Granules ◽  
Dapi Staining ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Low development of IVM canine oocytes could be in part attributed to an impaired cytoplasmic maturation. In mammalian oocytes, migration and the redistribution of cortical granules (CGs) around the periphery of the oocyte contribute to the inhibition of polyspermy and it is an important criterion to evaluate cytoplasmic maturation. The state of nuclear maturation and the distribution of CGs were evaluated in canine oocytes cultured for different periods in order to compare the synchrony of nuclear and cytoplasmic maturation during in vitro maturation. Bitch ovaries at different stages of the estrous cycle were obtained following ovariectomy. COCs with compact cumulus cells showing a homogeneous cytoplasm were selected for experiments. Thirty-six COCs were processed at immature stage, placed in PBS medium until evaluation. A total of 275 COCs were matured in vitro for 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM Hepes, 10% FCS, 0.25 mM pyruvate, 10 IU mL-1 of hCG, 300 IU mL-1 penicillin, and 20 mg mL-1 streptomycin, at 38.5°C and 5% CO2. At each culture period, the oocytes were stained with Lens culinaris agglutinin (LCA), labeled with fluorescein isothiocyanate, and the CGs distributions were examined under a fluorescent microscope. The nuclear status of the denuded oocytes was determined by DAPI staining under a fluorescence microscope. For each treatment, at least four replicates were performed and the data was analyzed by ANOVA using Tukey’s test to determine the differences P < 0.05. Three types of CGs distribution were distinguished during canine oocyte maturation: (1) homogeneous distribution throughout the cytoplasm including the cortex; (2) heterogeneous (clusters) within the cytoplasm and (3) densely distributed beneath the oolemma. Nuclear stages were classified as immature or germinal vesicle (GV) stage; resumption of meiosis or germinal vesicle break down (GVBD); metaphase I to telophase I (MI toTel I); and mature or second metaphase (MII). The distribution patterns of GCs were different (P < 0.05) among oocytes cultured for different periods and the nuclear maturation status also differed between oocytes cultured for different intervals (P < 0.05). Most (>84%) of the immature oocytes at GV showed a uniform distribution of CGs throughout the cytoplasm. At 48 h of culture, CGs distribution was mainly Type 2 (25%) and 3 (61%) and the oocytes were at GVBD (33%) and MI-Tel I (33%) stages. Most nuclei of the type 3 oocytes were in the MI (40%) and MII (11%) stages, corresponding to those oocytes matured for 72 (88%) or 96 h (71%). These results indicate that canine oocytes migrate to the cortex during IVM and this process is not finished before 72 h of culture. In addition, although the re-distribution of the CGs occurred in parallel with nuclear maturation, the oocytes cannot always proceed to the MII stage; however, in such oocytes the CGs are distributed beneath the oolemma. Supported by Grant FONDECYT 1080618.

scholarly journals Delay of nuclear maturation and reduction in developmental competence of pig oocytes after mineral oil overlay of in vitro maturation media

Reproduction ◽  
2002 ◽  
pp. 557-564 ◽  
Author(s):  
M Shimada ◽  
N Kawano ◽  
T Terada
Keyword(s):  
Steroid Hormones ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Mineral Oil ◽  
Developmental Competence ◽  
Cdc2 Kinase ◽  
Germinal Vesicle Breakdown ◽  
High Concentrations ◽  
P34 Cdc2

Steroid hormones, such as progesterone, oestrogen, androgen and meiosis activating sterols, are secreted from cumulus cells that are stimulated by gonadotrophins during maturation of oocytes in vitro. These steroid hormones may be absorbed by mineral oil or paraffin oil; however, in vitro maturation of pig oocytes is commonly performed using medium covered by oil. In this study, high concentrations of progesterone, oestradiol and testosterone were detected in the culture medium after pig cumulus-oocyte complexes (COCs) were cultured with FSH and LH for 44 h in medium without an oil overlay. However, high concentrations of these steroid hormones were not detected in medium when COCs were cultured with the mineral oil overlay. When high concentrations of these steroid hormones were secreted by COCs, germinal vesicle breakdown (GVBD) and the activation of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase in oocytes occurred earlier in comparison with oocytes cultured in medium covered with mineral oil. Moreover, a decrease in p34(cdc2) kinase activity during meiotic progression beyond metaphase I was observed in oocytes cultured in conditions under which high concentrations of steroid hormones were secreted by COCs. In addition, the rate of development to the blastocyst stage after IVF was higher in oocytes matured in medium without an oil overlay. These adverse effects of oil may be explained by absorption by the oil of cumulus-secreted steroids or by the release of toxic compounds into the medium.

Molecular Mechanisms of Prophase I Meiotic Arrest Maintenance and Meiotic Resumption in Mammalian Oocytes

2018 ◽  
Vol 26 (11) ◽  
pp. 1519-1537
Author(s):  
Maxim Filatov ◽  
Yulia Khramova ◽  
Maria Semenova
Keyword(s):  
In Vitro Maturation ◽  
Molecular Mechanisms ◽  
Germinal Vesicle ◽  
Follicular Cells ◽  
Meiotic Arrest ◽  
Germinal Vesicle Breakdown ◽  
Prophase I ◽  
The Stability ◽  
Multiple Molecules

Mechanisms of meiotic prophase I arrest maintenance (germinal vesicle [GV] stage) and meiotic resumption (germinal vesicle breakdown [GVBD] stage) in mammalian oocytes seem to be very complicated. These processes are regulated via multiple molecular cascades at transcriptional, translational, and posttranslational levels, and many of them are interrelated. There are many molecular cascades of meiosis maintaining and meiotic resumption in oocyte which are orchestrated by multiple molecules produced by pituitary gland and follicular cells. Furthermore, many of these molecular cascades are duplicated, thus ensuring the stability of the entire system. Understanding mechanisms of oocyte maturation is essential to assess the oocyte status, develop effective protocols of oocyte in vitro maturation, and design novel contraceptive drugs. Mechanisms of meiotic arrest maintenance at prophase I and meiotic resumption in mammalian oocytes are covered in the present article.

344 PENTOSE PHOSPHATE PATHWAY ACTIVITY CONTROLS NUCLEAR MATURATION OF PORCINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 279 ◽  
Author(s):  
L. Tubman ◽  
A. Peter ◽  
R. Krisher
Keyword(s):  
Pentose Phosphate Pathway ◽  
Control Level ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Germinal Vesicle ◽  
Pentose Phosphate ◽  
Meiotic Stage ◽  
Germinal Vesicle Breakdown ◽  
Pathway Activity ◽  
Nuclear Maturation

Diphenyleneiodonium (DPI), an inhibitor of the pentose phosphate pathway (PPP), arrests nuclear maturation of porcine oocytes. This inhibition is reversed using products or cofactors of PPP such as nicotinamide adenine dinucleotide phosphate (NADP), phosphoribose diphosphate (PRPP), and ribose-5-phosphate (R5P). The objective of this study was to determine the relationship between DPI-mediated meiotic inhibition, reversal of this inhibition, and metabolism of in vitro-matured (IVM) porcine oocytes. Oocytes were aspirated, searched, and selected in the presence of DPI, with the exception of control oocytes. Oocytes were then matured in one of five treatments for 40 h in 7% CO2 in air at 39°C in defined Purdue Porcine Medium for maturation (PPMmat). Treatments included control, 50 nM DPI (DPI), DPI + 5 mM NADP (NADP), DPI + 12.5 mM PRPP (PRPP), and DPI + 10 mM R5P (R5P). Following IVM, oocytes were denuded by vortexing. Glycolysis and PPP activities were measured in 4 μL hanging drops containing labeled glucose (0.0125 mM 5-3H glucose and 0.482 mM 1-14C glucose, respectively) for 3 h in 6% CO2. Oocytes were then individually fixed in a 3:2:1 solution of ethanol:acetic acid:chloroform and stained with aceto-orcein for determination of meiotic stage (germinal vesicle = 1 through metaphase II = 7). Data were analyzed using one-way ANOVA. The use of DPI inhibited PPP and nuclear maturation; additionally glycolysis was decreased by DPI compared to control. Addition of NADP and PRPP increased both metabolic pathways and nuclear maturation compared to DPI. R5P restored glycolysis and nuclear maturation to control levels, and PPP to above the control level. There were no significant differences among meiotic stages relative to glycolytic activity. PPP activity was significantly different (values with different superscripts; P < 0.05) among oocytes of different meiotic stages (germinal vesicle = 0.24 ± 0.03ad, germinal vesicle breakdown = 0.40 ± 0.05bcde, condensed chromatin = 0.44 ± 0.05bcd, metaphase I = 0.45 ± 0.12abcd, anaphase = 0.76 ± 0.50abcde, telophase = 0.92 ± 0.17be, metaphase II = 0.74 ± 0.08be). Percentages of oocytes reaching MII were 43.48 (control), 2.08 (DPI), 28.30 (NADP), 18.18 (PRPP), and 46.94 (R5P). These results demonstrate that the PPP is a critical control mechanism for nuclear maturation of porcine oocytes, as inhibition of this metabolic pathway resulted in arrest of nuclear maturation. Addition of PPP cofactors or end products to the arresting medium led to reversal of inhibition as demonstrated by restoration of PPP activity resulting in nuclear maturation. Table 1. Meiotic stage, glycolysis, and pentose phosphate pathway activity after in vitro maturation of porcine oocytes

61 THE EFFECTS OF COLLECTION SEASON AND STORAGE DURATION IN LIQUID NITROGEN ON POST-WARMING SURVIVAL AND NUCLEAR MATURATION OF IMMATURE PORCINE OOCYTES PRESERVED BY SOLID SURFACE VITRIFICATION

2015 ◽  
Vol 27 (1) ◽  
pp. 123
Author(s):  
T. Nagai ◽  
T. Somfai ◽  
N. T. Men ◽  
H. Kabeko ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Solid Surface ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Storage Duration ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Duration Of Storage ◽  
And Storage

We investigated the effects of collection season and storage duration of vitrified porcine oocytes in liquid nitrogen (LN2) on their survival and maturation ability after warming. A total of 3338 cumulus-enclosed oocytes were vitrified using solid surface vitrification, preserved, and warmed according to previous report (Somfai et al. 2014 PLoS One 9, e97731) in 26 occasions between October 2012 and March 2014. Vitrified oocytes were stored in LN2 for various durations from 0 (vitrified but without storage) to 243 days. The date of preservation and length of storage (days) of vitrified oocytes in LN2 were recorded. Warming of vitrified oocytes was conducted on a hotplate set at 42°C. After warming, oocytes were subjected to in vitro maturation according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041). Then oocytes were denuded and their live/dead status and nuclear maturation were assessed under stereo microscope based on their morphology and the presence of the first polar body. After linear regression analysis, it was found that there was no correlation between the duration of storage of vitrified oocytes in LN2 for up to 243 days and their survival rate after warming (R = 0.254; P = 0.210) or the maturation rate of surviving oocytes (R = 0.147; P = 0.471). Vitrification during spring (March 1–May 31) resulted in significantly higher rates of survived oocytes compared with vitrification during winter (December 1–February 28; 86.9 and 73.1%, respectively; P < 0.05), whereas the mean survival rates of oocytes vitrified during summer (June 1–August 31; 79.0%) and autumn (September 1–November 31; 81.9%) did not differ significantly from those of other seasons (ANOVA). After in vitro maturation, nuclear maturation of surviving oocytes did not differ significantly among oocytes vitrified at different seasons (ranging between 59.1 and 67.8%). The results indicate that the oocyte collection season affects survival of vitrified oocytes, whereas storage duration in LN2 does not affect this parameter. Furthermore, nuclear maturation of oocytes that survive after vitrification and warming is not affected by their collection season and storage length.This work was supported by JSPS KAKENHI Grant Number 26870839.

scholarly journals Glycogen synthase kinase 3B in bovine oocytes and granulosa cells: possible involvement in meiosis during in vitro maturation

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 235-246 ◽  
Author(s):  
Svetlana Uzbekova ◽  
Mohamad Salhab ◽  
Christine Perreau ◽  
Pascal Mermillod ◽  
Joëlle Dupont
Keyword(s):  
Granulosa Cells ◽  
Map Kinases ◽  
Glycogen Synthase ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Glycogen Synthase Kinase ◽  
Aurora A Kinase ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body

Glycogen synthase kinase 3 (GSK3) regulates cellular metabolism and cell cycle via different signalling pathways. In response to insulin and growth factors GSK3 is serine-phosphorylated and inactivated. We analysed GSK3B expression and activation in bovine cumulus cells (CC) and oocytes at different meiotic stagesin vitroin parallel with MAP kinases ERK (MAPK3/MAPK1) and p38 (MAPK14). GSK3B localised to cytoplasm in granulosa cells and in oocytes throughout folliculogenesis. In mature metaphase-II (MII) oocytes, GSK3B was concentrated to the region of midzone between the oocyte and the first polar body, as well as active phospho-Thr Aurora A kinase (AURKA). Duringin vitromaturation (IVM), in oocytes, phospho-Ser9-GSK3B level increased as well as phospho-MAPK3/MAPK1, while phospho-MAPK14 decreased. In CC, phospho-MAPK14 increased upon germinal vesicle breakdown (GVBD)/metaphase-I (MI) and then decreased during transition to MII. Administration of inhibitors of GSK3 activity (lithium chloride or 2′Z,3′E -6-bromoindirubin-3′-oxime) rapidly increased phospho-Ser9-GSK3B, and led to transient decrease of phospho-MAPK3/MAPK1 and to durable enhancing of phospho-MAPK14 in granulosa primary cell culture. GSK3 inhibitors during IVM diminished cumulus expansion and delayed meiotic progression. In cumulus, phospho-MAPK14 level was significantly higher in the presence of inhibitors, comparing with control, through the time of MI/MII transition. In oocytes, phospho-GSK3B was increased and phospho-MAPK3/MAPK1 was decreased before GVBD and oocytes were mainly arrested at MI. Therefore, GSK3B might regulate oocyte meiosis, notably MI/MII transition being the part of MAPK3/1 and MAPK14 pathways in oocytes and CC. GSK3B might be also involved in the local activation of AURKA that controls this transition.

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