scholarly journals 99MACROMOLECULES FOR VITRIFYING BOVINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 171
Author(s):  
G. Horvath ◽  
G.E. Seidel
Keyword(s):  
Fetal Calf Serum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Defined Medium ◽  
Standard Procedures ◽  
Zona Hardening ◽  
Maturation Medium ◽  
Experimental Treatments ◽  
Bovine Oocytes

Vitrification of oocytes would make them available for research and clinical purposes wherever and whenever needed. However, development rates and quality of blastocysts arising from vitrified oocytes have been low. Zona hardening following exposure to vitrification solutions and cooling could contribute to low fertilization rates. Addition of fetal calf serum (FCS) to handling media and vitrification solutions can prevent zona hardening, but FCS may be detrimental to resulting embryos and spread viral diseases, and its composition varies among batches. Fetuin is the component responsible for the protective effect of FCS (Landim-Alvarenga FC et al., 2002 Anim. Reprod. Sci. 71, 181–191). Our objective was to determine whether fetuin is a suitable substitute for FCS during vitrification. Oocytes derived from slaughterhouse ovaries were matured in a chemically defined medium with hormones and with or without 1mgmL−1 fetuin at 39°C in 5% CO2 in air. At 22h after the start of maturation, oocytes were transferred to one of the following handling media: 2% BSA, 2% BSA+1mgmL−1 fetuin, or 20% FCS in TCM-199+HEPES (HTCM-199). In the same media plus 100IUmL−1 hyaluronidase, cumulus cells were partly removed by gentle pipetting. Oocytes with approximately 3 layers of cumulus were chosen for two-step vitrification. First, they were exposed to VS1 (10% ethylene glycol (EG), 10% DMSO, 6% PVP in HTCM-199) for 30s, then to VS2 (20% EG, 20% DMSO, 6% PVP, 0.48M galactose in HTCM-199) for 25s, loaded into cryoloops in groups of five, and plunged into liquid nitrogen. Rapidly warmed oocytes were moved stepwise in 0.5, 0.25, 0.125, and 0M galactose in HTCM-199+20% FCS, 3 min each. All procedures were conducted at 39°C. Warmed oocytes were placed in maturation medium for an additional hour, and then fertilized and cultured according to standard procedures (Olson SE and Seidel GE Jr 2000 J. Anim. Sci. 78, 152–157). About 2000 oocytes were used in 11 replicates with semen of 4 bulls. The experimental treatments and controls were: A: maturation BSA, handling BSA; B: maturation BSA, handling FCS; C: maturation BSA, handling BSA+fetuin; D: maturation BSA+fetuin, handling BSA+fetuin; E: non-vitrified control, maturation BSA; F: non-vitrified control, maturation BSA+fetuin (Table 1). Controls did not differ (P>0.1) from each other, nor were there differences among vitrification treatments. Controls resulted in greater cleavage and more 8-cell embryos and blastocysts than vitrified treatments, and also tended to have higher cell numbers. In summary, fetuin can replace FCS during handling without decreasing the success of vitrification. Table 1 Development of vitrified oocytes and controls

165 FETAL CALF SERUM INFLUENCES CYCLIC GMP PATHWAY, WHICH IN TURN AFFECTS THE LIPID CONTENT IN IN VITRO-MATURED BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 196
Author(s):  
K. R. L. Schwarz ◽  
R. C. Botigelli ◽  
F. C. Castro ◽  
M. R. Chiaratti ◽  
C. L. V. Leal
Keyword(s):  
Lipid Content ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Maturation Medium ◽  
Cgmp Pathway ◽  
Bovine Oocytes ◽  
Synthesis And Degradation

The sensitivity of IVP embryos to cryopreservation is often associated with lipid accumulation in the cytoplasm induced by the presence of fetal calf serum (FCS) during culture. Intracellular levels of cyclic (c)AMP and cGMP are involved in the regulation of lipolysis in adipocytes; high levels stimulate lipolysis whereas low levels lead to lipogenesis. Both nucleotides are present in bovine oocytes, together with the enzymes for their synthesis and degradation. The aim of this study was to analysis the effect of FCS on the cGMP pathway and the influence of cGMP on cytoplasmic lipids in bovine oocytes. In experiments 1 and 2, cumulus–oocyte complexes (COC) were cultured for 24 h in maturation medium with different proportions of FCS (2 and 10%) and a control group was matured with 0.4% BSA. After this period, transcripts for cGMP pathway were assessed by real-time PCR (GUCY1B3 and PDE5, cGMP synthesis and degradation enzymes, respectively; experiment 1) in oocytes and cumulus cells, and cGMP levels were measured in COC using commercial enzyme immunoassay kits (EIA; experiment 2). In experiments 3 and 4, COC were matured for 24 h with 0.4% BSA and different concentrations of the phosphodiesterase (PDE)5 inhibitor (0, 10–7, and 10–5 M sildenafil) to inhibit cGMP degradation and a control group was matured with 0.4% BSA. The nucleotide levels were measured in COC (experiment 3) and the oocytes were stained with Nile Red (1 μg mL–1) for evaluation of lipid content (experiment 4). Statistical analyses were performed by ANOVA followed by Tukey post hoc test using SAS software (SAS Institute Inc., Cary, NC, USA). Data for gene expression from 5 replicates and for cGMP measurements and lipid content from 3 replicates were log10-transformed into before analyses. The level of significance was 5%. The presence of FCS reduced GUCY1B3 expression in both cells and increased PDE5A in cumulus cells (P < 0.05). In experiment 2, the groups treated with 2 (0.64 fmol/COC) and 10% FCS (1.04 fmol/COC) showed decreased cGMP levels compared with control (9.46 fmol/COC; P < 0.05). In experiment 3, inhibition of PDE5A increased cGMP levels in the treated groups (36 and 56 fmol/COC for 10–7 and 10–5 M sildenafil, respectively) compared with control (9.5 fmol/COC; P < 0.05). Therefore, sildenafil showed inverse effects compared with FCS (experiment 2). In experiment 4, oocytes treated with 10–7 and 10–5 M sildenafil showed a reduced lipid content compared with controls (11.6 ± 9.4 v. 13.9 μm2 fluorescence intensity, respectively; P < 0.05). The results suggest that FCS in maturation medium affects the cGMP pathway, interfering with the transcription of genes that control its levels, which in turn results in nucleotide reduction. Inhibition of PDE5 increases cGMP levels and reduces the lipid content of oocytes, indicating that changes in this pathway caused by FCS may affect lipid metabolism of oocytes. More studies are underway to better understand this mechanism. The authors acknowledge FAPESP 2012/00170-0 for financial support.

164 EFFECTS OF CUMULUS CELLS AND PITUITARY HORMONES ON AGE-ASSOCIATED CELLULAR CHANGES DURING THE PROLONGED CULTURE OF BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 196
Author(s):  
I. Lebedeva ◽  
G. Singina ◽  
A. Lopukhov ◽  
N. Zinovieva
Keyword(s):  
Fetal Calf Serum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Pituitary Hormones ◽  
Meiotic Arrest ◽  
Parthenogenetic Activation ◽  
Cellular Changes ◽  
Prolonged Culture ◽  
Bovine Oocytes

In vivo and in vitro aging of mature mammalian oocytes heavily reduces their quality and developmental capacity. Therefore, the knowledge of physiological factors modulating the speed of oocyte aging is of great importance for successful reproduction. The goal of the present research was to study effects of cumulus cells (CC) and two related pituitary hormones, prolactin (PRL) and growth hormone (GH), on the dynamics of age-associated cellular changes during the prolonged culture of bovine oocytes in vitro. Bovine cumulus–oocyte complexes (COC) were cultured for 20 h in the following maturation medium: TCM 199 containing 10% fetal calf serum, 10 μg mL–1 porcine FSH, and 5 μg mL–1 ovine LH. After IVM, COC were transferred to the aging medium consisting of TCM-199 supplemented with 10% fetal calf serum and cultured for 0, 12, 24, or 36 h in the absence (Control) or presence of 50 ng mL–1 bovine PRL (Research Center for Endocrinology, Moscow, Russia) or 10 ng mL–1 recombinant bovine GH (Monsanto, St. Louis, MO, USA). A portion of in vitro-matured oocytes were denuded of their CC and cultured in the control aging medium. At the end of culture, the state of the nuclear material in oocytes and embryos was evaluated by Tarkowski's cytogenetic method. The number of oocytes undergoing spontaneous parthenogenetic activation in the respective groups was determined by summarising the numbers of embryos cleaved and oocytes reaching anaphase-II to telophase-II stages or containing a pronucleus. Destructive changes in CC were assessed using the morphological signs of apoptosis. The data from 3 to 5 replicates were analysed by ANOVA. In the control group of COC, a rise in the rate of metaphase-II (M-II) oocytes with abnormal chromosome configurations occurred by 12 h of aging [31.8 ± 4.6% (12 h) v. 17.5 ± 2.6% (0 h); P < 0.05] and persisted up to 36 h (70.4 ± 2.0%; P < 0.001). At the same time, the frequency of oocyte parthenogenetic activation markedly increased only between 0 and 36 h of aging (from 0% to 20.7 ± 3.4%; P < 0.001). The addition of PRL or GH to the aging medium or removal of CCs resulted in a decline in the rate of M-II oocytes with degenerative changes of chromosomes throughout the culture period (at least P < 0.05). Furthermore, PRL and GH reduced the frequency of the oocyte activation at 36 h of the prolonged culture (up to 5.4 ± 2.5 and 1.7 ± 1.7%, respectively; P < 0.01), although CC did not influence meiotic arrest at M-II. Meanwhile, the rate of degenerated CC steadily increased as the culture time increased from 0 h (10.3 ± 1.1%) to 36 h (22.7 ± 2.2%; P < 0.001) and was unaffected by both hormones. The data suggest that, in bovine COC, CC accelerate abnormal changes in the chromosomal structure of aging M-II oocytes, whereas PRL and GH may decelerate these changes and support meiotic arrest during the prolonged culture of in vitro-matured oocytes. This research was partially supported by RFBR (project no. 13-04-01888).

scholarly journals 87 VITRIFICATION OF BOVINE OOCYTES TREATED WITH CHOLESTEROL-LOADED METHYL-β-CYCLODEXTRIN

2005 ◽  
Vol 17 (2) ◽  
pp. 193
Author(s):  
G. Horvath ◽  
L. Solti ◽  
G. Seidel
Keyword(s):  
Fatty Acid ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Chemically Defined Media ◽  
Defined Media ◽  
Standard Procedures ◽  
Maturation Medium ◽  
Cell Embryo ◽  
Bovine Oocytes ◽  
Better Than

Addition of cholesterol-loaded cyclodextrin (CLC) can increase sperm cryosurvival (Purdy et al. 2000 Cryobiology 48, 36–45). The purpose of this study was to determine if cryosurvival of vitrified oocytes could be improved by incubation with CLC prior to vitrification. Slaughterhouse-derived cumulus oocyte complexes were matured in a chemically defined medium with fatty acid-free BSA and hormones for 21 h followed by partial cumulus removal with 100 U/mL hyaluronidase and gentle pipetting. For an additional hour, oocytes were placed into maturation medium supplemented with 0.5% PVA instead of BSA with or without 2.5 mg/mL CLC. At 22 h after the start of maturation, oocytes were transferred to handling media containing 20% FCS or 0.5% PVA in TCM-199 + HEPES (HTCM-199). Oocytes with approximately 3 layers of cumulus were vitrified in two steps. First, they were exposed to VS1 (10% ethylene glycol (EG), 10% DMSO, 6% PVP, or 20% FCS, in HTCM-199) for 30 s, then exposed to VS2 (20% EG, 20% DMSO, 6% PVP, or 20% FCS, 0.48 M galactose in HTCM-199) for 25 s, loaded into cryoloops in groups of five, and plunged into liquid nitrogen. Rapidly warmed oocytes were moved stepwise through 0.5, 0.25, 0.125, and 0 M galactose in HTCM-199 + 20% FCS, 3 min each. All procedures were conducted at 39°C. Warmed oocytes were placed in maturation medium for an additional hour, fertilized with semen from 3 bulls, 3 replicates each, and cultured according to standard procedures (Zhang et al. 2003 Theriogenology 60, 1657–1663). For each replicate, 30 oocytes were assigned to the following treatments: A: chemically-defined media with PVA for the last hour of maturation, handling and vitrification; B: same as A except CLC treatment, for 1 h before vitrification; C: chemically defined media for maturation, but with 20% FCS for HM, VS1 and VS2. Data were analyzed by ANOVA. CLC treatment resulted in higher cleavage rates and 8- to 16-cell embryo production, but not higher blastocyst (bl) production (Table 1). Non-vitrified oocytes developed better than vitrified ones (means: cleavage, 76%; 8- to 16-cell, 64%; bl D8, 21%; bl D9, 24%). Further studies with vitrification of cholesterol-loaded cyclodextrin-treated oocytes and chemically defined media are warranted. Table 1. Development of vitrified oocytes (LS means ± SE)

Influence of day-0 and day-7 superovulated cow serum during development of bovine oocytes in vitro

1994 ◽  
Vol 6 (2) ◽  
pp. 261 ◽  
Author(s):  
A Boediono ◽  
M Takagi ◽  
S Saha ◽  
T Suzuki
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Culture Media ◽  
Calf Serum ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Low Concentrations ◽  
Maturation Medium ◽  
Bovine Oocytes

Oocytes were matured in medium supplemented with 5% serum collected from superovulated cows at oestrus (Day-0 SCS) or at the time of embryo collection (Day-7 SCS), or in medium supplemented with fetal calf serum (FCS). After insemination using frozen-thawed sperm, oocytes were cultured in vitro with medium supplemented with 5% Day-0 SCS or 5% Day-7 SCS or 5% FCS. The proportions of embryos that cleaved were not significantly different among treatments, whereas development of the embryo to a blastocyst was significantly higher in the presence of SCS than FCS. When the four possible combinations of Day-0 SCS and Day-7 SCS were used in the maturation and culture media, there were no differences among treatments, except that the cleavage rate was significantly higher (P < 0.05) with Day-0 SCS in the maturation medium and Day-7 SCS in the culture medium than with Day-7 SCS in the maturation medium and Day-0 SCS in the culture medium. The proportions of embryos that cleaved and developed to blastocysts were not related with the level of progesterone and luteinizing hormone in the serum added to the maturation and culture media. However, the use of serum with low concentrations of glucose, fatty acids and cholesterol in the maturation medium and the culture medium tended to be associated with a higher rate of cleavage and blastocyst development.

scholarly journals Delaying meiotic resumption during transportation of bovine cumulus–oocyte complexes: effects on development, apoptosis and caspases activity of in vitro-produced embryos

Zygote ◽  
2017 ◽  
Vol 25 (6) ◽  
pp. 740-750 ◽  
Author(s):  
Priscila Chediek Dall'Acqua ◽  
Beatriz Caetano da Silva Leão ◽  
Nathália Alves de Souza Rocha-Frigoni ◽  
Fernanda Patrícia Gottardi ◽  
Gisele Zoccal Mingoti
Keyword(s):  
Bovine Serum Albumin ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
The Other ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Control Groups ◽  
Meiotic Inhibitors

SummaryThis study examined the effects of meiosis inhibition during bovine oocyte transportation on developmental competence and quality of produced embryos. The transportation medium was supplemented with: 100 μM butyrolactone I (BL), 500 μM IBMX + 100 μM forskolin (mSPOM), 100 μM milrinone (MR) or follicular fluid (bFF), and was carried out in a portable incubator for 6 h. Next, oocytes were in vitro matured (IVM) for 18 h, without the meiotic inhibitors, with the exception of mSPOM group, in which was added 20 μM cilostamide. The three control groups were IVM with 10% fetal calf serum (FCS) (Control Lab FCS) or 0.6% bovine serum albumin (BSA) (Control Lab BSA) in a CO2 in air incubator or in the portable incubator with 0.6% BSA (Control Transp BSA). Higher cleavage rates (P < 0.05) were obtained in the Control Lab FCS group (84.5 ± 5.3%) compared with the other groups (59.6 ± 3.4% to 70.9 ± 2.3%). Embryonic development was higher (P < 0.05) in the Control Lab FCS group (39.8 ± 4.7%) than in the Control Transp BSA (22.7 ± 3.4%) and MR (21.6 ± 2.3%) groups. However, they were similar (P > 0.05) to the other groups (23.6 ± 3.3% to 28.8 ± 2.7%). The total number of blastomeres was higher (P < 0.05) in the Control Lab FCS group (85.2 ± 5.6) than in Control Lab BSA (53.6 ± 2.9), Control Transp BSA (55.5 ± 4.4), BL (58.2 ± 3.0), mSPOM (57.9 ± 4.9) and MR (59.2 ± 3.9), but all these treatments did not differ (P > 0.05) from bFF (67.7 ± 4.2). No differences (P > 0.05) were found in apoptosis by the activity of caspases (139.0 ± 3.2 to 152.4 ± 6.5, expressed in fluorescence intensity) as well as the percentage of TUNEL-positive cells (12.3 ± 2.0% to 15.7 ± 1.7%). In conclusion, the transportation of oocytes over 6 h with BL, mSPOM or bFF enabled the acquisition of developmental competence at similar rates to the Control Lab FCS group.

6 TIMING OF DEADENYLATION OF GDF-9 AND CYCLIN 3′ UTR CONSTRUCTS IN BOVINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 121
Author(s):  
D. J. Walker ◽  
C. J. Wilusz ◽  
G. E. Seidel Jr
Keyword(s):  
In Vitro Maturation ◽  
Control Point ◽  
Tail Length ◽  
Cumulus Cells ◽  
Cyclin B1 ◽  
Defined Medium ◽  
Untranslated Regions ◽  
Labeled Rna ◽  
Bovine Oocytes

The maternal pool of mRNA undergoes major changes during oocyte maturation and early embryonic development. Specific genes are activated or degraded in response to changes in poly-(A) tail length. However, little is known about how the oocyte targets specific transcripts for degradation or translation in a timely manner. The objective of this study was to determine how poly-(A) tail length of different transcripts is affected in bovine oocytes by time of in vitro maturation. Cyclin B1 and GDF-9 32 untranslated regions (UTRs) were cloned into modified p-GEM plasmids containing a poly-(A) tract of 60 or 0 adenosines (A60 or A0, respectively). Each 32 UTR was transcribed in vitro with (A60) or without (A0) a poly-(A) tail to generate UTP32-labeled RNA. Transcriptions producing at least 200 000 counts per min (cpm) per �L were used for subsequent injections into denuded bovine oocytes. Cumulus-oocyte complexes (COCs) recovered from slaughterhouse-derived ovaries (n = 216) were vortexed to remove cumulus cells immediately after aspiration, after 3 h of in vitro maturation, or after 19 h of maturation in a chemically defined medium supplemented with FSH, LH, EGF, and cysteamine. After vortexing, denuded oocytes were injected and snap frozen, or matured in vitro for 1 or 3 h. Eight oocytes were injected with ~0.5 nL (~100 cpm/oocyte) labeled RNA at each time point in 3 replicates. Total RNA was isolated from injected oocyte pools and loaded onto a 5% denaturing acrylamide gel for size separation. Radiolabeled A0 was used as a control point of reference for deadenylation. Gels were dried, and RNA was visualized on a phosphoimager after 24 h exposure to a phosphor screen. Changes in polyadenylation status (transcript size) were evaluated by comparing shifts in bands from gene-specific A60

143 EFFECT OF FSH OR EPIDERMAL-GROWTH-FACTOR-LIKE PEPTIDE SUPPLEMENTATION TO MATURATION MEDIUM ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES DERIVED FROM FULLY DEVELOPED FOLLICLES INDUCED BY SUPER-STIMULATION

2017 ◽  
Vol 29 (1) ◽  
pp. 180
Author(s):  
T. Yamanouchi ◽  
S. Sugimura ◽  
H. Matsuda ◽  
M. Ohtake ◽  
Y. Goto ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Formation Rate ◽  
Calf Serum ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Maturation Medium ◽  
Epidermal Growth ◽  
Bovine Oocytes

Bovine oocytes obtained by ovum-pick-up (OPU) following follicle growth treatment (FGT) have improved quality and competence (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). However, the effect of the presence of FSH or epidermal growth factor (EGF) like peptide during in vitro maturation (IVM) on the developmental competence of FGT oocytes has not been well known. This study was undertaken to examine the developmental competence of FGT oocytes following IVM in the presence of FSH (recombinant human FSH) or EGF-like peptide (amphiregulin; Areg) and IVF. Japanese Black cows (n = 17) were used as donors. Five days after arbitrary OPU (opu group), follicles ≥8 mm in diameter were aspirated again, a controlled internal drug release (CIDR) was inserted into the vagina, and then pFSH was injected twice a day from the evening of Day 6 to the morning of Day 10 with decreasing doses (total of 20 AU; 4, 4, 3, 3, 2, 2, 1, 1 AU/day). On the evening of Day 8, PGF2α (0.5 mg of cloprostenol) was administered. On Day 11, oocytes were aspirated from follicles with ≥5 mm in diameter of the treated donors by OPU (fgt group). The cumulus-oocyte complexes (COC) were cultured in the absence (opu-cont and fgt-cont groups) or presence of 0.1 IU mL−1 FSH (opu-fsh and fgt-fsh groups) or 100 ng mL−1 Areg (opu-areg and fgt-areg groups) in IVM medium (mTCM199 containing 5 mg mL−1 BSA) for 20 to 22 h (1 COC/5 µL, total of 162–171 COC per group), and then co-cultured with 3 × 106 sperm/mL for 6 h. The presumptive zygotes were continued to culture in mCR1aa supplemented with 5% newborn calf serum for 216 h (1 zygote/5 µL) using micro-well culture dishes (Dai-Nippon-Print). When repeating this opu-fgt session in the same cow, an interval at least for 50 days was kept, and the session was performed 28 times. Statistical analysis was carried out by Mann-Whitney’s U-test (between opu and fgt groups) or Steel-Dwass test after Kruskal-Wallis test (among all groups). The number of follicles ≥5 mm increased in the fgt than opu group (17.8 v. 2.9; P < 0.01). The number of COC collected was not different between the opu and fgt groups (23.1 v. 19.6; P > 0.05). The blastocyst formation rate was higher in the fgt than opu group (36.9 v. 23.1%; P < 0.01). Within 6 groups, the blastocyst formation rate was higher in the fgt-fsh (43.3%; P < 0.01) and fgt-areg (39.5%; P < 0.05) groups than the opu-cont (16.3%) group. The rate in the fgt-fsh group was also higher than that in the opu-fsh group (43.3 v. 18.7%; P < 0.01). These results suggested that FGT improved the developmental competence of bovine oocytes, probably through improving the ability of the COC to react against FSH/Areg.

Effect of fetal calf serum on the quality of in vitro produced cattle embryos

1999 ◽  
Vol 51 (1) ◽  
pp. 257 ◽  
Author(s):  
H Tricoire ◽  
J-L Touzé ◽  
P Mermillod
Keyword(s):  
Fetal Calf Serum ◽  
Calf Serum

Astaxanthin counteracts the effects of heat shock on the maturation of bovine oocytes

2018 ◽  
Vol 30 (9) ◽  
pp. 1169 ◽  
Author(s):  
J. Ispada ◽  
T. A. Rodrigues ◽  
P. H. B. Risolia ◽  
R. S. Lima ◽  
D. R. Gonçalves ◽  
...  
Keyword(s):  
Heat Shock ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Negative Effects ◽  
Cellular Mechanisms ◽  
Sod Activity ◽  
Oocyte Competence ◽  
Maturation Medium ◽  
Bovine Oocytes

The cellular mechanisms induced by elevated temperature on oocytes are not fully understood. However, there is evidence that some of the deleterious effects of heat shock are mediated by a heat-induced increase in reactive oxygen species (ROS). In this context, carotenoid antioxidants might have a thermoprotective effect. Therefore, the objective of this study was to determine the role of astaxanthin (AST) on oocyte ROS production and on the redox profile and developmental competency of cumulus-oocyte complexes (COCs) after 14 h heat shock (41°C) during in vitro maturation (IVM). Exposure of oocytes to heat shock during IVM increased ROS and reduced the ability of the oocyte to cleave and develop to the blastocyst stage. However, 12.5 and 25 nM astaxanthin rescued these negative effects of heat shock; astaxanthin counteracted the heat shock-induced increase in ROS and restored oocyte developmental competency. There was no effect of astaxanthin on maturation medium lipid peroxidation or on glutathione peroxidase and catalase activity in oocytes and cumulus cells. However, astaxanthin stimulated superoxide dismutase (SOD) activity in heat-shocked cumulus cells. In conclusion, direct heat shock reduced oocyte competence, which was restored by astaxanthin, possibly through regulation of ROS and SOD activity in oocytes and COCs.

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.

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