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

Bovine in vitro oocyte maturation as a model for manipulation of the γ-glutamyl cycle and intraoocyte glutathione

2008 ◽  
Vol 20 (5) ◽  
pp. 579 ◽  
Author(s):  
E. C. Curnow ◽  
J. Ryan ◽  
D. Saunders ◽  
E. S. Hayes
Keyword(s):  
Oocyte Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Buthionine Sulfoximine ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Maturation Medium

Glutathione (GSH) is the main non-enzymatic defence against oxidative stress and is a critical intracellular component required for oocyte maturation. In the present study, several modulators of intracellular GSH were assessed for their effect on the in vitro maturation (IVM) and intracellular GSH content of bovine metaphase (MII) oocytes. Of the five GSH modulators tested, only the cell-permeable GSH donor glutathione ethyl ester (GSH-OEt) significantly increased the GSH content of IVM MII oocytes in a concentration-dependent manner without adversely affecting oocyte maturation rate. The GSH level in IVM MII oocytes was greatly influenced by the presence or absence of cumulus cells and severely restricted when oocytes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine MII oocytes. Supplementation of the maturation medium with bovine serum albumin (BSA) or fetal calf serum (FCS) affected the GSH content of IVM MII oocytes, with greater levels attained under BSA culture conditions. The addition of GSH-OEt to the maturation medium increased the GSH content of IVM MII oocytes, irrespective of protein source. Spindle morphology, as assessed by immunocytochemistry and confocal microscopy, displayed distinct alterations in response to changes in oocyte GSH levels. GSH depletion caused by BSO treatment tended to widen spindle poles and significantly increased spindle area. Supplementation of the IVM medium with GSH-OEt increased spindle length, but did not significantly alter spindle area or spindle morphology. GSH-OEt represents a novel oocyte-permeable and cumulus cell-independent approach for effective elevation of mammalian oocyte GSH levels.

scholarly journals The Number of LH Receptor could Predict the Success of Oocyte Maturity in the Process of In Vitro Maturation

2016 ◽  
Author(s):  
Adek Amansyah
Keyword(s):  
In Vitro Maturation ◽  
Clinical Laboratory ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Maturity ◽  
Lh Receptor ◽  
First Polar Body ◽  
Significant Difference

Objective: To evaluate the relationship between the number of LH receptor and the success of oocyte maturity in the process of in vitro maturation (IVM). Method: This experimental study was conducted in the Permata Hati Infertility Clinical Laboratory, Dr. Sardjito General Hospital, Yogyakarta, with the samples of 300 oocytes obtained through collecting immature cow’s oocytes from the abattoir and grouped the oocytes into 3 (three) groups based on the pattern of oocyte cumulus cells on the vesicle germinal stage 2 - 8 mm with three layers of cumulus cell. The sample of the cumulus cells from these three groups were taken and the LH receptor examination was done with immunohistochemistry. After that, the IVM process was performed to the three groups and its development for 24 hours was evaluated. Its maturation quality was evaluated with the emergence of the first polar body (1PB) and compared to the other groups and related to the number of LH receptor in the three groups. Result: The result of this study indicated that the oocyte cumulus cells showed a difference of function during IVM process. The maturity rate in this study showed that the number of LH receptor was related to the morphological pattern of oocyte cumulus cells with oocyte maturity. The maturity of the cumulus cells which 100% covered the oocyte was higher than that of the cumulus cells which > 50% and < 30% covered the oocytes, namely, 74% compared to 60% and 12%. The result of this study also showed that the average number of LH receptors in the three groups (A, B, and C) was 183.4, 78.8, and 24.0 respectively. A significant difference was found in the three groups (p < 0.0001). When related to IVM maturity, this difference showed that the bigger number of oocyte cumulus cells influenced the oocyte maturity. Conclusion: The number of LH receptor can be used as a prediction to determine the success of oocyte maturation in the process of in vitro maturation. [Indones J Obstet Gynecol 2013; 1-4:183-7] Keywords: IVM, LH receptor, oocyte cumulus cell

253 OOCYTE PREMATURATION IN THE PRESENCE OF MILRINONE IMPROVES NUCLEAR BUT NOT CYTOPLASMIC MATURATION OF MACAQUE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 224 ◽  
Author(s):  
E. C. Curnow ◽  
J. P. Ryan ◽  
D. M. Saunders ◽  
E. S. Hayes
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Molecular Probes ◽  
Developmental Competence ◽  
Oocyte Growth ◽  
North West ◽  
Significant Difference ◽  
Cytoplasmic Maturation

During oocyte growth chromatin configuration of the germinal vesicle (GV) oocyte undergoes modification in relation to changes in transcriptional activity crucial for conferring meiotic as well as developmental competence on the oocyte. In the macaque oocyte, there are 3 distinct GV states: GV1, noncondensed chromatin; GV2, an intermediate state; and GV3, condensed chromatin. The aim of this study was to test the effects of a prematuration culture (PMC) system, using the phosphodiesterase type 3 inhibitor milrinone (MIL), on the synchronization of GV chromatin to the GV3 stage and assess metaphase II (MII) oocyte reduced glutathione (GSH) content as a measure of cytoplasmic maturation. Reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. To assess the effect of PMC on GV chromatin status, immature oocytes retrieved from unstimulated ovaries were either fixed (2% paraformaldehyde+0.1% Triton-X100) immediately after follicular aspiration (t = 0) or after culture in a humidified atmosphere of 6% CO2 in air at 37°C for 24 h in modified Connaught Medical Research Laboratories medium (mCMRL) supplemented with 10% FCS (Hyclone, Logan, UT, USA) and 12.5 μM MIL in the absence (MILNil) or presence of 1.0 IU of FSH (MILFSH). For chromatin assessment, fixed GV oocytes were stained with 5 μg mL–1 of 4′,6-diamidino-2-phenylindole (Molecular Probes, Leiden, the Netherlands) and imaged using confocal microscopy. Following PMC, MILFSH oocytes were transferred to fresh mCMRL+FCS supplemented with 1.0 IU of recombinant human FSH and 1.0 IU of hLH and cultured for a further 30 h. Control and MILFSH oocytes were denuded of cumulus cells and assessed for maturation. The MII oocytes were prepared for GSH analysis, and total GSH content was determined using a commercial 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay kit (North-West Life Science). The MII rates were compared using chi-square. Differences in oocyte GSH content were compared using t-test. Significant differences were determined at P < 0.05. There was no significant difference in the proportion of oocytes remaining at the GV stage following 24 h of PMC in MILNil or MILFSH (42/44, 96% v. 32/35, 91%, respectively). However, there was a significant reduction in GV1 chromatin (15/49, 31% v. 28/54, 52% and 22/58, 38%) and a significant increase in GV3 chromatin (23/49, 47% v. 14/54, 26% and 16/58, 28%) observed in MILFSH oocytes compared with both MILNil and t = 0 oocytes, respectively. The MII rate of MILFSH oocytes following in vitro maturation was significantly higher compared with the MII rate of control in vitro matured oocytes (91/167, 55% v. 83/243, 34%). There was no significant difference in the GSH content of GV oocytes from the time of oocyte collection (t = 0) or GV oocytes following PMC in MILFSH (3.69 ± 0.16 and 4.14 ± 0.28 pmol/oocyte, n = 39–49 oocytes). The GSH content of control in vitro matured MII oocytes was significantly greater than that of MILFSH-treated MII oocytes (3.13 ± 0.16 v. 2.02 ± 0.04 pmol/oocyte, n =53–54 oocytes). The PMC supported high rates of nuclear maturation, but cytoplasmic maturation, assessed by GSH content, was negatively affected. Further assessment following fertilization and development is required to determine the practical utility of PMC in a primate in vitro maturation setting.

353 EFFECTS OF CULTURE DURATION AND CUMULUS CELL REMOVAL ON CANINE OOCYTE IN VITRO MATURATION AND FERTILIZATION USING VASAL SPERM

2007 ◽  
Vol 19 (1) ◽  
pp. 292
Author(s):  
M. Ridha-Albarzanchi ◽  
J. Liu ◽  
M. Kjelland ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Vas Deferens ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Culture Period ◽  
Cell Contact ◽  
Nuclear Maturation ◽  
Sperm Penetration ◽  
Culture Duration

The objective of this study was to test the hypothesis that in vitro maturation (IVM) and fertilization (IVF) rates of canine oocytes could be improved by increasing culture duration or decreasing/increasing cumulus cell contact with the oocytes when using sperm retrieved from the vas deferens. The canine oocyte is ovulated at the germinal vesicle stage, and maturation of the oocyte occurs in the oviduct and requires up to five days. Therefore, an increase in the culture duration may cause an increase in oocyte nuclear maturation. Canine ovaries and testes were collected from a local clinic, placed in warm saline solution, and transported to the laboratory. Two distinct experiments were carried out, one involving IVM (M-II) after cumulus cell removal at 72 h and 96 h for nuclear maturation evaluation, and the second experiment the same but continued up to IVF. The oocytes were recovered from the ovaries by mincing them in warm Medium-199 with Hanks salts, L-glutamine, and HEPES (GIBCO, Grand Island, NY, USA; Invitrogen Co., Carlsbad, CA, USA). Canine oocytes with a dark cytoplasm and at least 2 layers of cumulus cells were cultured in Medium-199 supplemented with Earle&apos;s salts, 2200 mg mL&minus;1 sodium bicarbonate, 25 mM HEPES, 2 mM sodium pyruvate, 5 &micro;g mL&minus;1 progesterone, 100 ng mL&minus;1 epidermal growth factor, 10 IU mL&minus;1 human chorionic gonadotropin (HCG), 5 &micro;g mL&minus;1 insulin, 0.50 mM epinephrine, 10&percnt; estrus bitch serum, 0.01 mM nonessential amino acids, and 20 &micro;g mL&minus;1 gentamicin. The oocytes were cultured for 72, 96, 120, or 144 h at 38.5&deg;C in 5&percnt; CO2 in humidified air. The cumulus cells were removed after either a 72- or 96-h culture period. For the first 48 h, the cumulus&ndash;oocyte complexes were cultured in the modified Medium-199 containing 10 IU mL&minus;1 HCG and then cultured in the same medium free of HCG. The oocytes were denuded by pipetting, stained with Hoechst 33342, and examined for nuclear maturation. ANOVA was used for statistical analysis of the data. The IVM rate (MII) was significantly higher (P &lt; 0.05) at 72 and 96 h compared to 48, 120, and 144 h (15.1&percnt; and 16.9&percnt; vs. 6&percnt;, 12.4&percnt;, and 9.1&percnt;, respectively). The removal of cumulus cells at 72 h and 96 h resulted in 17.9&percnt; (43/240) and 14.8&percnt; (35/236) IVM rate (MII), respectively (P &gt; 0.05). The sperm motility index (SMI &equals; motility percentage &times; sperm activity grade) was significantly higher in sperm retrieved from the vas deferens (vasal sperm) compared to epididymal and testicular sperm (259 vs. 95 and 19.2, respectively, P &lt; 0.05). The mature oocytes were inseminated by vasal sperm following in vitro hyperactivation with HEPES solution supplemented with 3 mg mL&minus;1 bovine serum albumin. The IVF rates of the oocytes following 72 and 96 h of maturation in vitro were 48.2&percnt; and 40&percnt;, respectively (P &gt; 0.05). Sperm penetration was significantly higher at 96 h compared to 72 h, and the number of sperm heads inside the ooplasm was 3.2 for the 72 h group vs. 4.8 for the 96 h group (P &lt; 0.05). In conclusion, increasing the IVM culture period beyond 72 h did not increase the oocyte maturation rates, and increasing the culture time to 96 h without cumulus cells present increased the rate of sperm penetration.

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

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.

250 CYTOPLASMIC DYNEIN INTERMEDIATE CHAIN AND DYNACTIN p150Glued EXHIBIT DISTINCT SPATIAL AND TEMPORAL MICROTUBULE ASSOCIATIONS DURING BOVINE IN VITRO MATURATION AND ARE AFFECTED BY FOLLICLE SIZE

2008 ◽  
Vol 20 (1) ◽  
pp. 204
Author(s):  
S. E. Racedo ◽  
M. C. Branzini ◽  
D. Salamone ◽  
V. Y. Rawe ◽  
H. Niemann
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Chromatin Condensation ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Meiotic Spindle ◽  
Germinal Vesicle Breakdown ◽  
Intermediate Chain

Microtubule molecular motors are critically involved in transporting vesicles during interphase, in building and maintaining spindles during mitosis and meiosis, and also in the localization of various organelles. DYNC1I1 (cytoplasmic dynein 1 intermediate chain) and its cofactor DCTN1 (dynactin p150Glued) are crucial for oocyte maturation but their role during mammalian female meiosis is not yet known. The goal of this study was to analyze the dynamics of these proteins in oocytes collected from different-size follicles at different stages of in vitro maturation (IVM), i.e., germinal vesicle stage (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII), and their association with microtubules. Ovaries were collected at a local abattoir. Cumulus–oocyte complexes (COCs) were aspirated from follicles either <2 mm or 2–8 mm in size and matured in M199, supplemented with 1% fatty acid-free BSA, 10 UI pregnant mare serum gonadotropin (PMSG)/5 UI HCG, and 100 µm cysteamine, at 39�C and 5% CO2. Follicle sizes and time points for fixation were: GV-0 h; GVBD-8 h for oocytes <2 mm and 9 h for oocytes 2–8 mm; MI-15 h; MII-24 h (Racedo et al. 2007, pub. online: 10.1002/mrd.20770). The distribution of the proteins was assessed by immunocytochemistry and laser confocal microscopy. The attached cumulus cells and zona pellucida of oocytes were removed in TALP-HEPES medium containing 1 mg mL–1 hyaluronidase and 2 mg mL–1 pronase, respectively. The oocytes were then incubated in a fixation–permeabilization solution containing 2% formaldehyde and 0.1%Triton X-100 for 1 h. Samples were then blocked for 1 h in 10 mm PBS + 0.3% BSA + 1% fetal calf serum (ICC blocking solution). The primary antibody was applied over night at 4�C, followed by treatment with fluorochrome-conjugated secondary antibodies for 1 h at 37�C in the dark. After RNase treatment, oocytes were incubated with TOTO-3 (Invitrogen, Carlsbad, CA, USA) to visualize the DNA. The material was mounted in an anti-fade medium (Vectashield�, Vector Laboratories, Burlingame, CA, USA) and imaged with a Zeiss laser scanning microscope. Immediately after chromatin condensation (GVBD), dynactin was in close association with the DNA and interacting with the spindles in MI and MII oocytes recovered from large follicles. No clear association with the DNA was observed in GVBD oocytes obtained from small follicles; little dynactin was found in MI and MII spindles. Dynein localization did not differ from dynactin in GVs and was homogeneously distributed in the cytoplasm of both groups of follicles. Dynein was not associated with the DNA in the GVBD stage while at MI and MII it was associated with the meiotic spindle. The association of dynein with microtubules was weak at the MI stage in oocytes from small follicles. Results provide insight into the regulatory mechanisms of oocyte maturation and a possible relationship with oocyte competence.

scholarly journals 184 PRELIMINARY FINDINGS ON CARBOHYDRATE METABOLISM OF INTACT EQUINE CUMULUS-OOCYTE COMPLEXES DURING IN VITRO MATURATION

2016 ◽  
Vol 28 (2) ◽  
pp. 223 ◽  
Author(s):  
N. Lewis ◽  
K. Hinrichs ◽  
D. Brison ◽  
R. Sturmey ◽  
D. Grove-White ◽  
...  
Keyword(s):  
Carbohydrate Metabolism ◽  
Dna Content ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Glucose Consumption ◽  
Cumulus Cells ◽  
Significant Difference ◽  
Spent Media

Production of equine embryos in vitro is gaining popularity, and many differences exist in composition of in vitro maturation (IVM) media. Metabolism of the cumulus-oocyte complex (COC) is essentially unknown in the horse. Here, we describe preliminary data on carbohydrate metabolism of the equine COC during IVM. COC, collected by scraping of the granulosa layer of all visible follicles of abattoir-derived ovaries, were held overnight (12–18 h) at room temperature (~20°C) and then placed in Maturation Medium (M199 with Earle’s salts, 10% FBS, with 25 μg mL–1 gentamicin and 5 mU mL–1 FSH). They were incubated singly in 10-μL droplets under mineral oil for 30 h at 38.3°C in 5% CO2 in air. Control droplets without COC were incubated in the same dish. After incubation, COC were removed and spent media kept at –80°C for later analysis. Oocytes were denuded of cumulus cells by pipetting in the presence of hyaluronidase and evaluated by light microscopy at 500×. Those with a visible polar body were classified as metaphase II (MII); oocytes with an intact oolemma and no polar body were classified as immature intact (INT) and those with an irregular or unapparent oolemma, or shrunken cytoplasm, were classed as degenerating (DEG). To adjust for variation in cumulus cell number, the stripped cumulus cells were frozen at –20°C and later analysed for DNA content using Picogreene. The spent media was analysed for depletion of glucose and appearance of lactate on a BMG Fluostar spectrophotometer using an enzyme-linked ultrafluorometric method. Data were expressed as pmol/ng DNA/hr and analysed by t-test, x2 and logistic regression. Thirty COC were cultured and analysed; 14 were classified as MII, 2 INT and 14 DEG. Seven COC (23%) depleted all the available glucose, indicating that the rate of glucose consumption in those 7 complexes was ≥1866 pmol/COC per hour. DNA content was positively correlated with glucose depletion (P = 0.02). In the COC that did not deplete available glucose, the ratio of glucose consumption:lactate production was 1.82, indicating that the major fate of exogenous glucose was production of lactate by glycolysis. In the 7 oocytes that depleted all the glucose, the ratio of glucose consumption:lactate production was 1.22. One explanation for this may be that when glucose was no longer available, it was conserved for other pathways. It was noteworthy that these COC had more cumulus cells (P < 0.01) and the maturation rate was 4/7 (57%). In the group of COC that did not deplete all of the glucose, there was no significant difference in glucose consumption (13.17 v. 12.25 pmol/ng DNA per hour; P > 0.4) or lactate production (21.48 v. 20.28 pmol/ng DNA per hour; P > 0.4) between COC in which the oocyte reached MII (10/23; 43%), and those which contained a degenerated oocyte at the end of culture, respectively. To the best of our knowledge, this is the first report documenting the metabolism of equine COC. These data underline the importance of further studies to determine optimal conditions for in vitro maturation of equine COC, especially in terms of glucose availability.

297 EFFECT OF CUMULUS CELL REMOVAL DURING IN VITRO MATURATION OF PORCINE CUMULUS–OOCYTE COMPLEXES ON THE APOPTOTIC STATUS AND MEIOTIC PROGRESSION OF THE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 237
Author(s):  
P. Ferré ◽  
H. Funahashi
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Annexin V ◽  
Cumulus Cells ◽  
Dapi Staining ◽  
Meiotic Progression ◽  
Viability Assay ◽  
Significant Difference ◽  
Porcine Oocyte

This study was undertaken to examine the apoptotic status and meiotic progression of oocytes from small (SF) and medium follicles (MF) when the oocytes were denuded from cumulus cells (CC) before, during and after culture for in vitro maturation (IVM). Cumulus-oocyte complexes (COC) were aspirated from SF (0.5–2 mm in diameter) or MF (3–6 mm in diameter) of slaughtered prepubertal gilt ovaries. Only COC with a good morphology of the surrounding cumulus cells were cultured for IVM in modified porcine oocyte medium supplemented with 50 µM β-mercaptoethanol, 1 mM dibutyryl c-AMP, 10 IU mL–1 of eCG, and 10 IU mL–1 of hCG for 20 h at 39°C and 5% CO2 in air and then continued culture in the absence of dibutyryl c-AMP, eCG, and hCG in the same medium for another 24 h. Before and 20 h after the start of IVM culture, some of the oocytes were denuded of CC and the oocytes continued the IVM culture. After IVM culture, oocyte viability and meiotic progression were examined by the annexin V/PI viability assay and DAPI staining. Statistical analyses of 5 replicate data were performed with a 2-way ANOVA and a Tukey's multiple comparisons test. Before IVM culture, there was no significant difference between the viability of SF and MF oocytes, but the incidence of oocytes at the GV0 stage was higher in specimens from SF than MF (24.8 v. 3.3%), and that of oocytes at the GVI stage was the opposite (57.8 in MF v. 22.7% in SF). After IVM culture, apoptotic status of oocytes was only affected by the decumulation timing. The percentage of normal live oocytes was significantly higher when CC were removed after 20 and 44 h of IVM in both SF (39.7 and 39.3 v. 17.7%) and MF (45.4 and 37 v. 22.2%). The incidence of early and late apoptotic oocytes was significantly higher when the CC were removed before IVM culture in both SF (74.3 and 7.4%) and MF (69.4 and 6.7%). The incidence of mature live oocytes was significantly affected by both the origin of COC and the decumulation timing. Although the percentage of mature oocytes was higher in MF, maturation rates were significantly higher when oocytes were denuded at 20 h of IVM culture (SF 65.4%, MF 83.1%) as compared at 0 (SF 27.9%, MF 32.3%) and 44 h (SF 41%, MF 68.5%). However, the percentage of oocytes with normal spindle morphology was significantly higher when oocytes were denuded at 44 h of IVM culture (SF 70.6%, MF 91.5%) than 20 h (SF 66.8%, MF 73%). In summary, regardless of COC from SF and MF, removal of CC at 20 h of IVM culture seems to promote meiotic progression of the oocytes to the MII stage, but factor(s) from or communication with CC during the latter half of IVM culture may be needed to obtain a normal spindle morphology in mature oocytes.

271 NUCLEAR MATURATION OF WOOD BISON (BISON BISON ATHABASCAE) CUMULUS - OOCYTE COMPLEXES

2013 ◽  
Vol 25 (1) ◽  
pp. 283
Author(s):  
M. P. Cervantes ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
J. M. Palomino ◽  
G. P. Adams
Keyword(s):  
In Vitro Maturation ◽  
Transvaginal Ultrasound ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Bison Bison ◽  
Nuclear Maturation ◽  
Wood Bison

Methods of producing wood bison embryos in vivo and in vitro are being developed in an effort to preserve the genetic diversity of this threatened species. Previous data from our laboratory suggest that oocytes collected 24 h after LH treatment had not yet achieved nuclear maturation. The objectives of this study were (1) to determine the optimal interval of time after hCG treatment required for in vivo maturation of cumulus–oocyte complexes (COC) in wood bison, and (2) to compare the maturational characteristics of COC after in vitro v. in vivo maturation. Follicular wave emergence was synchronized among bison cows (n = 25) by follicular ablation (Day –1) from May to June. Ovarian superstimulation was induced with FSH IM diluted in 5 mg mL–1 of hyaluronan (MAP-5, Bioniche, Belleville, Ontario, Canada) given on Day 0 (300 mg) and Day 2 (100 mg). Superstimulated cows were assigned randomly to 5 groups (n = 5/group): COC collected on Day 4 with no maturation (control), or matured in vitro for 24 or 30 h, or collected 24 or 30 h after treatment with 2000 IU of hCG IM on Day 4. The COC were collected by transvaginal ultrasound-guided follicle aspiration. In vitro maturation was done in TCM-199 with 5% calf serum, 5 µg mL–1 of LH, 0.5 µg mL–1 of FSH, and 0.05 µg mL–1 of gentamicin, at 38.5°C and in 5% CO2. To assess nuclear maturation, oocytes were stained with anti-lamin AC/DAPI (4′,6-diamidino-2-phenylindole). Nuclear stages were classified as germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI), or metaphase II (MII). Comparisons among groups were made by ANOVA and Fisher’s exact test (Table 1). A mean (± SEM) of 7.6 ± 0.6 COC was collected per bison; no differences were observed among groups (P = 0.37). Cumulus cell expansion was more extensive after in vivo than in vitro maturation, and the percentage of fully expanded COC was highest in the in vivo 30-h group (97%; P < 0.05). No COC were expanded in the control (0 h) group, and none reached MI. Maximal nuclear maturation was achieved in vitro by 24 h; that is, there was no difference in the proportion of MII-stage COC at 24 versus 30 h. However, between 24 and 30 h of in vivo maturation, the percentage of nuclear stages GV + GVBD decreased from 54 to 24% (P < 0.05), whereas nuclear stages MI + MII increased from 39 to 74% (P < 0.05). In conclusion, nuclear maturation occurred earlier in vitro versus in vivo, but the consequences of this difference are unknown. Although more than one-third of oocytes matured in vivo for 30 h were mature enough to permit immediate IVF, whether additional in vivo maturation time would be beneficial to fertilization rates remains to be tested. Table 1.Nuclear status of wood bison oocytes after in vitro or in vivo maturation Thanks to Bioniche Canada.

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