Amino Acid Turnover by Bovine Oocytes Provides an Index of Oocyte Developmental Competence In Vitro1

Retinoic acid (RA) can induce cell differentiation and plays a role in controlling events within the cell cycle, but little is known of RA post-transcriptional modifications in the oocyte. Bovine oocyte and cumulus cells express most of RA receptors, and the presence of 9-cis-RA during in vitro prematuration and maturation (IVM) improves oocyte developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). This work analyzes the mRNA stability in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were cultured in defined medium with polyvinyl alcohol (DM). Those COCs undergoing prematuration were cultured for 24h in DM with 25μM roscovitine. For IVM, COCs were cultured in DM containing pFSH, LH and E2 for 24h, and some prematured COCs were then allowed to mature. Incubations were made at 39°C in 5% CO2 in air and high humidity. Within experiments, COCs were cultured with 5nM 9-cis-RA, in 1% ethanol (both as a vehicle and as an inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Groups of 10 COCs per treatment were cultured, and oocytes detached from cumulus cells were analyzed. Poly(A) mRNA quantification was based on the pyrophosphorylation property of the DNA polymerase (Klenow). ATP production was measured by luminometric assay as a function of numbers of poly(A) tails. Data (4 replicates) were analyzed by ANOVA and Duncan’s test (v,x,y,zP<0.01; a,bP<0.05), and poly(A) mRNA (pg oocyte−1) was expressed as LSM±SE. After prematuration, poly(A) mRNA contents differed between 9-cis-RA (125.7±4.8x) and untreated (95.5±4.8y) oocytes, as compared to 1% ethanol (72.2±4.8z) and immature (71.5±4.8z) oocytes. After IVM, untreated oocytes (23.0±2.2v) showed the lowest poly(A) mRNA amount, and poly(A) mRNA in 9-cis-RA (36.2±2.2y) basically equalled that in 1% ethanol (35.2±2.2y), while 3% (44.5±2.2yz) and 5% ethanol (52.0±2.2z) increased poly(A) mRNA levels. All groups of matured oocytes showed poly(A) mRNA contents lower than in immature (71.5±4.8x). After prematuration+maturation, poly(A) mRNA values were 34.2±2.2v (untreated+untreated), 36.5±2.2v (9-cis-RA+untreated), 49.5±2.2xa (untreated+9-cis-RA), 41.0±2.2vxb (9-cis-RA+9-cis-RA) and 59.0±2.2y (untreated+1% ethanol). Levels of poly(A) mRNA from prematured+matured oocytes were again lower than in immature (71.5±4.8x). Our study shows that beneficial effects of RA on the oocyte developmental competence can be represented in part as a gain in the quality of mRNAs stored. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).

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Maturation, fertilisation and culture of bovine oocytes and embryos in an individually identifiable manner: a tool for studying oocyte developmental competence

Reproduction Fertility and Development ◽

10.1071/rd09277 ◽

2010 ◽

Vol 22 (5) ◽

pp. 839 ◽

Cited By ~ 27

Author(s):

Satoko Matoba ◽

Trudee Fair ◽

Patrick Lonergan

Keyword(s):

Blastocyst Stage ◽

Developmental Competence ◽

Standard Group ◽

Success Rates ◽

Group Culture ◽

Polyester Mesh ◽

Oocyte Developmental Competence ◽

Development In Vitro ◽

Bovine Oocytes

The ability to successfully culture oocytes and embryos individually would facilitate the study of the relationship between follicle parameters and oocyte developmental competence, in order to identify markers of competent oocytes, as well as the ability to use small numbers of oocytes from an individual donor such as when ovum pick-up is carried out. Using a total of 3118 oocytes, the aim of the present study was to develop a system capable of supporting the development of immature bovine oocytes to the blastocyst stage in an individually identifiable manner. Initially, post-fertilisation embryo culture in the Well-of-the-Well (WOW) system, on the cell adhesive Cell-Tak or in polyester mesh was tested and shown to result in similar development to embryos cultured in standard group culture. The results demonstrate that it is possible to culture bovine oocytes to the blastocyst stage in an individually identifiable manner in all three culture systems with comparable success rates. This permits the localisation and identification of individual embryos throughout preimplantation development in vitro while retaining the developmental benefits of group culture. In terms of ease of preparation and use, culture in isolation within the strands of a polyester mesh is preferable.

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The Effect of Granulosa Cells Apoptosis on the Cumulus Expansion and the Developmental Competence of Bovine Oocytes

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.997.251 ◽

2014 ◽

Vol 997 ◽

pp. 251-254 ◽

Cited By ~ 1

Author(s):

Wei Guo Feng ◽

Zhi Fang Pan

Keyword(s):

Granulosa Cells ◽

Culture System ◽

Developmental Competence ◽

Bovine Oocyte ◽

Cumulus Expansion ◽

Oocyte Developmental Competence ◽

Bovine Oocytes

In this study, we used the well in the well (WIW) culture system to study the effect of granulosa cells apoptosis on the cumulus expansion and the developmental competence of bovine oocytes. Results: The apoptosis of granulosa cells affect the cumulus expansion of bovine oocytes significantly. Especially when the percentage of granulosa cells apoptosis exceed 40%, the cumulus expansion was worse. The cumulus expansion affect the oocyte developmental competence of bovine oocytes significantly. The developmental competence of bovine oocyte increases with the increasing of cumulus expansion.

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The Effects of Floating Granulosa Cells Density on the Developmental Competence of Bovine Oocytes: A Study Using the Well-in-Well Culture System

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.884-885.544 ◽

2014 ◽

Vol 884-885 ◽

pp. 544-547

Author(s):

Wei Guo Feng ◽

Chun E Ren ◽

Zhi Fang Pan

Keyword(s):

Granulosa Cells ◽

Culture System ◽

Developmental Competence ◽

Group System ◽

Apoptosis Rate ◽

Oocyte Developmental Competence ◽

Bovine Oocytes ◽

Hatching Rates

In this study, we succeed to establish the well in the well (WIW) culture system. Using this system we studied the effect of floating granulosa cells density on oocyte developmental competence. Results: Apoptosis rate of the floating cells were high (73.576.3%) and did not differ (P > 0.05) among follicles of different sizes. Hatching rates of blastocysts did not differ (P > 0.05) between oocytes cultured in the WIW system (13.3%) and those cultured in the group system (16.3%). In 4-8mm follicles, oocyte developmental competence is higher (P < 0.05) in follicles with 1030×105/mL floating cells than in follicles with < 10 or >30%×105/mL floating cells.

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292 INHIBITION OF HSP90 AGGRAVATES THE EFFECTS OF HEAT SHOCK ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab292 ◽

2015 ◽

Vol 27 (1) ◽

pp. 235

Author(s):

E. D. Souza ◽

N. C. Rabelo ◽

T. D. Araujo ◽

C. M. Assunção ◽

C. C. R. Quintão ◽

...

Keyword(s):

Heat Shock ◽

In Vitro Maturation ◽

Calf Serum ◽

Developmental Competence ◽

Cleavage Rate ◽

Oocyte Developmental Competence ◽

Significant Difference ◽

Blastocyst Rate ◽

Bovine Oocytes

The heat shock protein 90kDa (HSP90) is a chaperone involved in protein homeostasis under normal and stress conditions. Its inhibition by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma, St. Louis, MO, USA) for 12 or 24 h during in vitro maturation reduces the oocyte's ability to develop after in vitro fertilization (Souza et al. 2014 Reprod. Fert. Dev. 26, 197). This study aimed to evaluate the effect of treatment with 17AAG during the heat shock on oocyte developmental competence. Immature bovine COC were randomly allocated in 4 treatments during IVM: control = no heat shock or 17AAG; HS = heat shock (41.5°C) for the first 12 h of IVM; 17AAG = 2 µM 17AAG for the first 12 h of IVM; and 17AAG + HS = 2 µM 17AAG plus heat shock for the first 12 h of IVM. In vitro maturation was performed in Nunc plate containing 400 µL of TCM199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2 in air, 95% humidity, and 38.5°C for 24 h. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) and oocytes were in vitro fertilized for 20 h with 2 × 106 spermatozoa mL–1 under the same IVM atmospheric conditions. Presumptive zygotes were completely denuded in a PBS solution with 0.1% 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 postfertilization and blastocyst rate was evaluated at Day 7 (D7) and 8 (D8). Data from 7 replicates were submitted to analysis of variance and means were compared by Student Newman Keul's test. There was no difference (P > 0.05) on cleavage rate among treatments. Heat shock or treatment with 17AAG, both for 12 h of IVM, decreased (P < 0.05) the blastocyst rate at D7 and D8 when compared to control but no significant difference between HS and 17AAG treatments was found (Table 1). However, the lowest (P < 0.05) blastocyst rate at D7 and D8 was achieved when oocytes were submitted simultaneously to 17AAG and heat shock for 12 h of IVM (17AAG + HS treatment, Table 1). In conclusion, the treatment with 17AAG during IVM worsens the deleterious effect of heat shock on oocyte developmental competence and suggests that HSP90 may also play role on cellular protection during heat shock in bovine oocytes. Table 1.Cleavage and blastocyst (Bl) rates at D7 and D8 for control, 17AAG, Heat Shock (HS), and 17AAG plus HS treatments Financial support comes from CNPq, FAPEMIG, and FAPES.

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