Treatment with the MAPK kinase inhibitor U0126 during the first two hours of in vitro maturation improves bovine oocyte developmental competence

2017 ◽  
Vol 53 (1) ◽  
pp. 270-273 ◽  
Author(s):  
T Shinohara ◽  
Y Ohta ◽  
N Kawate ◽  
M Takahashi ◽  
N Sakagami ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Kinase Inhibitor ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Mapk Kinase ◽  
Oocyte Developmental Competence

327 OOCYTE-SECRETED FACTORS DIRECTLY AFFECT OOCYTE DEVELOPMENTAL COMPETENCE DURING IN VITRO MATURATION OF THE BOVINE CUMULUS - OOCYTE COMPLEX

2006 ◽  
Vol 18 (2) ◽  
pp. 271 ◽  
Author(s):  
T. S. Hussein ◽  
R. B. Gilchrist ◽  
J. G. Thompson
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Paracrine Factors ◽  
Cell Functions ◽  
Oocyte Developmental Competence ◽  
Secreted Factors

Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions including proliferation, differentiation, and apoptosis. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this study was to determine if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were obtained by aspiration of >3-mm follicles from abattoir-derived ovaries. IVM was conducted in Bovine VitroMat (Cook Australia, Eight Mile Plains, Brisbane, Australia) supplemented with 0.1 IU/mL rhFSH for 24 h under 6% CO2 in air at 38.5�C. In the first experiment, COCs were co-cultured with denuded oocytes (DOs, 5/COC in 10 �L) beginning at either 0 or 9-h of IVM. To generate the 9-h DO group, COCs were first cultured intact for 9-h and then denuded. In the second experiment, specific OSFs, recombinant bone morphogenetic protein-15 (BMP-15) and growth differentiation factor 9 (GDF-9), were prepared as partially purified supernatants of transfected 293H cells, and used as 10% v/v supplements in Bovine VitroMat. Treatments were: (1) control (no supplement), (2) BMP-15, (3) GDF-9, (4) BMP-15 and GDF-9, and (5) untransfected 293H control. Following maturation, in vitro production of embryos was performed using the Bovine Vitro system (Cook Australia) and blastocysts were examined on Day 8 for development. Developmental data were arcsine-transformed and analyzed by ANOVA, followed by Tukey's test. Cell numbers were analyzed by ANOVA. Co-culturing intact COCs with DOs from 0 or 9 h did not affect cleavage rate, but increased (P < 0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination (50.6 � 1.9 and 61.3 � 1.9%, respectively), compared to COCs cultured alone (40.7 � 1.4%). Therefore, paracrine factors secreted by DOs increased the developmental competence of oocytes matured as COCs. OSFs also improved embryo quality, as co-culture of COCs with DOs (0 or 9 h) significantly increased total cell (156.1 � 1.3 and 159.1 � 1.3, respectively) and trophectoderm (105.7 � 1.3 and 109.8 � 0.4, respectively) numbers, compared to control COCs (total = 148 � 1.2, trophectoderm = 98.2 � 0.8, P < 0.001). BMP-15 alone or with GDF-9 also significantly (P < 0.001) increased the proportion of oocytes that reached the blastocyst stage post insemination (57.5 � 2.4% and 55.1 � 4.5%, respectively), compared to control (41.0 � 0.9%) and 293H-treated (27.1 � 3.1%) COCs. GDF-9 also increased blastocyst yield (49.5 � 3.9%) but not significantly. These results are the first to demonstrate that OSFs, and particularly BMP-15 and GDF-9, directly affect bovine oocyte developmental competence. These results have far-reaching implications for improving the efficiency of IVM in domestic species and human infertility treatment, and support the role of OSF production by oocytes as a diagnostic marker for developmental competence.

scholarly journals Impact of L-carnitine supplementation on the in vitro developmental competence and cryotolerance of buffalo embryos

2021 ◽  
pp. 3164-3169
Author(s):  
Mohamed M. M. El-Sokary ◽  
Al-Shimaa Al-H. H. El-Naby ◽  
Amal R. Abd El Hameed ◽  
Karima Gh. M. Mahmoud ◽  
T. H. Scholkamy
Keyword(s):  
Embryo Culture ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Effective Concentration ◽  
Developmental Competence ◽  
Carnitine Supplementation ◽  
Oocyte Developmental Competence ◽  
High Lipid ◽  
Significant Difference

Background and Aim: Despite many trials, buffalo embryos have poor cryosurvivability because of their high lipid content. L-carnitine was found to be a lipid-reducing agent when added to oocyte and embryo culture media. The study aimed to determine the most effective concentration of L-carnitine to improve the oocyte developmental competence and cryotolerance of buffalo embryos. Materials and Methods: In vitro maturation and embryo culture media were supplemented with four concentrations of L-carnitine: 0 (control), 0.25, 0.5, and 1 mM. Good-quality embryos on 7 days were vitrified using mixtures of dimethyl sulfoxide and ethylene glycol at two concentrations (3.5 and 7 M). Results: The result showed that the cleavage and morula rates were significantly (p<0.05) higher in the 0.5 mM group. Blastocyst rates were significantly (p<0.05) higher at both 0.5 and 1 mM. The rates of viable embryos directly after thawing were significantly (p<0.05) increased in the 0.5 mM group. No significant difference was found in embryos cultured for 24 h after warming among all the groups. Conclusion: The addition of L-carnitine at a concentration of 0.5 mM to the culture media improves the oocyte developmental competence and cryotolerance of buffalo embryos directly after warming but not after 24 h of culture. Nevertheless, further studies must identify how L-carnitine exerts its beneficial micromechanisms.

205 CUMULUS CELL MORPHOLOGY BEFORE AND AFTER IN VITRO MATURATION AS AN INDICATOR OF PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 260
Author(s):  
M. Bertoldo ◽  
P. K. Holyoake ◽  
G. Evans ◽  
C. G. Grupen
Keyword(s):  
Cell Morphology ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Oocyte Developmental Competence ◽  
Before And After ◽  
Follicle Size

Effective in vitro maturation (IVM) is essential for successful in vitro embryo production. The morphology of the cumulus investment before and after IVM may be a useful noninvasive indicator of oocyte quality. In pigs, oocyte developmental competence is reduced during the summer months. The aim of this study was to determine whether the morphology of cumulus-oocyte complexes (COC) before and after IVM are associated with oocyte quality, using COC collected from small and large follicles in summer and winter as models of poor and good oocyte quality. Ovaries were collected from sows slaughtered 4 days after weaning. The COC recovered from small (3-4 mm) and large (5-8 mm) antral follicles were morphologically graded and parthenogenetically activated following IVM during winter (n = 1419; 10 replicates) and summer (n = 2803; 10 replicates). Grade 1 and 2 COC had >2 layers of compact cumulus cells and a homogenous cytoplasm. Grade 3 COC were either partially or fully denuded, had a heterogeneous cytoplasm, or were vacuolated or dark in color. Grade 4 COC had expanded cumulus cells. Cumulus expansion was also assessed subsequent to IVM. The COC recorded as having a cumulus expansion index (CEI) of 1 had the poorest expansion with no detectable response to IVM, whereas those with a CEI of 4 had the greatest amount of expansion, including that of the corona radiata. Data were analyzed using a generalized linear mixed model in GenStat® (release 10, VSN International, Hemel Hempstead, UK). There was an effect of follicle size for Grade 1 COC, with COC from large follicles in both seasons yielding better quality COC (P < 0.05). The proportion of COC in Grade 2 was higher in small follicles during winter compared with large follicles, but there were no differences between follicle sizes during summer (P < 0.05). The proportion of COC with CEI 1 was highest in COC from small follicles during summer (P < 0.05). The proportion of COC from large follicles with CEI 2 was higher during summer compared with winter (P < 0.05). There were no seasonal or follicle size effects on COC with CEI 3 or 4 (P > 0.05). The proportion of oocytes that developed to blastocysts was greater in winter than in summer (39.06% ± 5.67 v. 22.27% ± 4.01; P < 0.05). Oocytes derived from large follicles had a greater ability to form blastocysts compared with those from small follicles (37.13% ± 5.65 v. 23.32% ± 4.56; P < 0.06). Morphological assessment of cumulus cells before and after IVM may be a useful tool to evaluate the effects of follicle size on oocyte developmental competence. However, the results of the present study indicate that cumulus cell morphology is not a good indicator of the effect of season on oocyte developmental competence.

167 INHIBITION OF HEAT SHOCK PROTEIN 90 REDUCES DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 197
Author(s):  
E. D. Souza ◽  
F. B. E. Paula ◽  
C. C. R. Quintao ◽  
J. H. M. Viana ◽  
L. T. Iguma ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
In Vitro Maturation ◽  
Hsp90 Inhibitor ◽  
Stress Conditions ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Cleavage Rate ◽  
Bovine Oocytes

The 90-kDa heat shock protein (HSP90) is a chaperone that is important for maintaing protein homeostasis under stress conditions. HSP90 seems also to be required for maturation of Xenopus oocytes (Fisher et al. 2000 EMBO J. 19, 1516) and first cleavage of mouse zygotes (Audouard et al. 2011 PloS One 6, e17109). This study aimed to evaluate the effect of inhibition of HSP90 by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma St. Louis, MO, USA) during in vitro maturation (IVM) on bovine oocyte developmental competence. Immature cumulus–oocyte complexes (COC) were randomly allocated in 3 treatments during IVM: T0 (control; n = 240), no HSP90 inhibitor; T1: 2 μM HSP90 inhibitor (17AAG; n = 250) for the first 12 h of IVM; and T2: 2 μM HSP90 inhibitor (n = 188) for 24 h of IVM. In vitro maturation was performed in Nunc plates containing 400 μL of TCM-199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2, 95% humidity, and 38.5°C for 24 h. Oocytes were in vitro fertilized for 20 h and incubated under the same IVM conditions. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) an IVF performed with 2 × 106 spermatozoa mL–1. Presumptive zygotes were completely denuded in a PBS solution with hyaluronidase and then cultured in wells with 500 μL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h post-fertilization and blastocyst rates were evaluated at Day 7 and Day 8. Data from 6 repetitions were analysed by generalized linear model procedure of SAS software (version 9.1; SAS Institute Inc., Cary, NC, USA), and means were compared by Student-Newman-Keuls test. Values are shown as mean ± s.e.m. There was a tendency (P = 0.08) for a lower cleavage rate in T2 (52.6 ± 5.8%) than in T0 (control; 74.2 ± 4.1%). Inhibition of HSP90 by 17AAG for 12 h and 24 h of IVM (T1 and T2, respectively) decreased blastocyst rates at Day 7 (20.4 ± 3.0% and 14.3 ± 2.6%, respectively; P < 0.01) and Day 8 (22.6 ± 4.1% and 16.9 ± 2.7%, respectively; P < 0.05) when compared with control (T0 = 31.8 ± 2.5% and 34.1 ± 2.9% for Day 7 and Day 8, respectively). In addition, the inhibition of HSP90 for 24 h decreased (P < 0.05) the proportion of hatched blastocysts at Day 8 (9.5 ± 5.0% for T2, respectively) when compared with control (T0 = 35.8 ± 3.9%), indicating a reduction on embryo quality. In conclusion, inhibition of HSP90 by 17AAG during IVM results in lower developmental competence, suggesting that this protein is also important for bovine oocytes. Further studies are required to investigate if the role of HSP90 on developmental competence of bovine oocyte is affected when under stress conditions. The authors acknowledge CNPq 473484/2011-0, FAPEMIG and FAPES for financial support.

313 PRE-IN VITRO MATURATION WITH CYCLIC AMP AND CYCLIC GMP MODULATORS IMPROVES DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 245 ◽  
Author(s):  
N. W. Santiquet ◽  
A. F. Greene ◽  
W. B. Schoolcraft ◽  
R. L. Krisher
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Nuclear Maturation ◽  
Oocyte Developmental Competence ◽  
Oocyte Collection ◽  
Short Period

In vitro maturation (IVM) of cumulus-oocyte complexes (COC) results in oocytes with reduced quality and is still not as efficient as in vivo maturation in most species. One hypothesis that could explain the low developmental competence of oocytes following IVM is that the oocytes resume meiosis too quickly after being retrieved from the follicles. Studies in mice and bovine have shown that a short period of prematuration in the presence of cAMP modulators, before IVM, enhances oocyte developmental competence. Moreover, other studies have recently demonstrated that cGMP is also a crucial molecule involved in meiotic resumption. Here, our objective was to examine the effect of a cGMP modulator in combination with a cAMP modulator during a short period of prematuration on mouse oocyte nuclear maturation and subsequent embryo development following IVF. The COC were collected (6 replicates) from 2-month-old outbred CF1 mice 48 h after PMSG (5 IU) injection in the presence (pre-IVM) or absence (control) of cGMP and cAMP modulators. Pre-IVM COC (n = 184) were then placed in prematuration medium that also contained these cGMP and cAMP modulators. After 2 h, pre-IVM COC were washed and transferred to our in-house prepared, completely defined IVM medium (Paczkowski et al. 2014 Reprod.) for the remaining 16 h of culture; 10 oocytes per 50 µL drop under oil, at 37°C in 7.5% CO2 and 6.5% O2 due to the increased altitude at our location. Control COC (n = 161) were matured in the same IVM medium under identical conditions for 18 h, without prematuration. After IVM, oocytes were fixed for assessment of nuclear maturation, or fertilized and cultured in vitro and subsequent development (96 and 112 h) was recorded (Paczkowski et al. 2014 Reprod.). Results were analysed by ANOVA. A short 2-h prematuration period in the presence of cGMP and cAMP modulators had no impact on oocyte nuclear maturation to metaphase II after IVM or on embryo cleavage after IVF. However, pre-IVM treatment improved the developmental competence of the oocyte, as demonstrated by increased embryo development. More (P < 0.02) blastocysts (96 h of culture) and hatched blastocysts (112 h of culture) developed in the pre-IVM treatment compared to control (31.0 ± 3.4 v. 19.9 ± 3.2%; 31.5 ± 3.4 v. 19.9 ± 3.2%, respectively). In conclusion, a combination of cGMP and cAMP modulators during oocyte collection and a subsequent short pre-IVM improves oocyte developmental competence and could therefore be a potential tool to improve embryo yield following IVM.

scholarly journals Temporal expression of cumulus cell marker genes during in vitro maturation and oocyte developmental competence

2017 ◽  
Vol 34 (11) ◽  
pp. 1493-1500 ◽  
Author(s):  
Arindam Dhali ◽  
Pradeep Krishna Javvaji ◽  
Atul P. Kolte ◽  
Joseph Rabinson Francis ◽  
Sudhir C. Roy ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Marker Genes ◽  
Cell Marker ◽  
Temporal Expression ◽  
Oocyte Developmental Competence

scholarly journals Follicle-stimulating hormone mediates the consumption of serum-derived glycogen by bovine cumulus-oocyte complexes during in vitro maturation

2021 ◽  
pp. 2512-2517
Author(s):  
Ludymila F. Cantanhêde ◽  
Cristiane T. Santos-Silva ◽  
Marcelo T. Moura ◽  
José C. Ferreira-Silva ◽  
Júnior M. B. Oliveira ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Follicle Stimulating Hormone ◽  
Cumulus Cell ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Independent Manner ◽  
Oocyte Developmental Competence ◽  
Stimulating Hormone

Background and Aim: Oocyte in vitro maturation (IVM) is an appealing approach for several assisted reproductive technologies and dissecting oocyte maturation. Nonetheless, IVM leads to lower developmental competence and usually relies on undefined, serum-containing media. Therefore, biochemical profiling aimed to explore fluctuations in IVM media content during the acquisition of oocyte developmental competence. Materials and Methods: Bovine cumulus-oocyte complexes (COCs) underwent IVM in TCM199 medium with Earle's salts, supplemented with 2.0 mM L-glutamine, 10% fetal bovine serum, antibiotics, and 0.05 IU/mL porcine follicle-stimulating hormone (FSH+) or vehicle control (CTL) medium for 22 h. Results: FSH withdrawal (CTL) diminished several processes associated with the acquisition of oocyte developmental competence, such as reduced cumulus cell expansion, diminished estradiol synthesis (FSH+: 116.0±0.0 pg/mL vs. CTL: 97.6±18.0 pg/mL), and lower oocyte nuclear maturation rate (FSH+: 96.47% vs. CTL: 88.76%). Fresh media formulations (i.e., TCM199 with FSH or vehicle) were indistinguishable under biochemical profiling threshold conditions. Biochemical profiling showed similar total protein and lipid concentrations between groups. Further, total sugar concentrations diminished from fresh media to their post-IVM counterparts, albeit in an FSH-independent manner. Glycogen concentrations remained unaltered after IVM within CTL media, albeit were substantially lower after IVM under FSH+ conditions. Conclusion: FSH mediates the consumption of serum-derived glycogen by bovine COCs during IVM and implies that serum-free media should contain increased glucose concentrations to facilitate the acquisition of oocyte developmental competence.

scholarly journals 94MEIOSIS INHIBITION OF BOVINE OOCYTES: EFFECTS ON SURVIVAL AFTER VITRIFICATION BY OPEN PULLED-STRAW METHOD

2004 ◽  
Vol 16 (2) ◽  
pp. 168
Author(s):  
C. Diez ◽  
M. Carbajo ◽  
L. Fernandez ◽  
C.O. Hidalgo ◽  
S. de la Varga ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
17Β Estradiol ◽  
Bovine Oocytes ◽  
Range Test

Mammalian oocytes remain one of the most difficult cell types to successfully cryopreserve. The in vitro-maturation protocols (IVM) have a large impact on the oocyte maturation. Consequently, inhibition of meiosis has been used to improve developmental competence of oocytes without reducing blastocyst rates. Moreover, the meiotic stage influences the ability of oocytes to survive cryopreservation. This work analyzes the effect of the inhibition of meiosis (prematuration) on the freezability of the bovine oocyte. Cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries. Inmature oocytes (I) with compact cumulus and evenly granulated cytoplasm were selected. Prematuration (PM) was performed by incubating COCs for 22h in TCM199 NaHCO3 and roscovitine 25μM. IVM was accomplished in TCM199 NaHCO3, 10% FCS, FSH-LH and 17β-estradiol. Oocytes were subjected to 5 treatments prior the vitrification (see table). COCs were partially denuded from cumulus cells and vitrified/warmed using the OPS system (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58). Warmed oocytes were fertilized (Day 0) and presumptive zygotes having a normal morphological appearance were cultured in SOFaa+5% of FCS (Day 3); elements with degenerated appearance were discarded and recorded. Fresh oocytes submitted to IVM (c-M) or prematured and matured (c-PM+M) were fertilized and cultured as controls. Data were analyzed by ANOVA and Duncan’s multiple range test and expressed as LSM±SE. Developmental data are referred to the zygotes cultured. Only oocytes vitrified after IVM reached the blastocyst stage, but at lower rates than fresh controls. However, no differences were found between treatments at any developmental stage. Oocytes vitrified both as prematured+matured and immature oocytes showed increased proportions (P&lt;0.01) of degenerated oocytes (37.3±5.9 and 49.9±5.9, respectively), as compared with oocytes matured before vitrification (17.6±5.9). These results show that effects induced by incubation with roscovitine (Lonergan et al. 2003 Mol. Reprod. Dev. 64, 369–378) in combination with cryodamage compromise the oocyte developmental ability. Supported by CICYT, AGL2001-379.

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