Molecular characteristics of granulosa and cumulus cells and oocyte competence in Nelore cows with low and high numbers of antral follicles

2018 ◽  
Vol 53 (4) ◽  
pp. 921-929 ◽  
Author(s):  
CO Rosa ◽  
LSR Marinho ◽  
PRA da Rosa ◽  
MP De Cesaro ◽  
PA Lunardelli ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Molecular Characteristics ◽  
Oocyte Competence ◽  
Antral Follicles

334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 275
Author(s):  
H. S. Lee ◽  
Y. I. Seo ◽  
X. J. Yin ◽  
S. G. Cho ◽  
I. H. Bae ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Uv Light ◽  
Cumulus Cells ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Oviduct Fluid

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

193 DIFFERENTIAL GENE EXPRESSION IN CUMULUS CELLS OF DEVELOPMENTALLY COMPETENT V. CHALLENGED BOVINE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 195 ◽  
Author(s):  
R. R. Payton ◽  
L. A. Rispoli ◽  
J. L. Edwards
Keyword(s):  
Gene Expression ◽  
Gene Ontology ◽  
Heat Stress ◽  
Empirical Bayes ◽  
Rna Extraction ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Oocyte Competence ◽  
Extracellular Region

It is well established that exposure of cumulus–oocyte complexes (COC) to heat stress during the first 12 h of maturation reduces blastocyst development by 42 to 65%. Previous research supports the notion that some of the effects of heat stress on oocyte competence may be cumulus-mediated. To determine the extent to which this may occur, COC were matured at 38.5°C for 24 h (control) or 41°C for the first 12 h of maturation followed by 38.5°C for remaining 12 h (heat stress). A subset of COC underwent IVF with Percoll-prepared sperm and then was cultured in KSOM containing 0.5% BSA to assess developmental competence. Remaining oocytes were denuded. Cumulus cells, kept separate by treatment, were stored in lysis buffer at –80°C until RNA extraction. Total RNA from cumulus was amplified prior to hybridization to bovine Affymetrix GeneChips (Affymetrix Inc., Santa Clara, CA, USA; n = 8 pools per treatment collected on 8 different occasions; n = 16 chips). Following pre-processing using the MAS5.0 algorithm, microarray data were subjected to linear modeling and empirical Bayes analyses (Bioconductor, Limma package). False discovery rate was controlled using the Benjamini and Hochberg method, and differentially expressed genes were selected by an adjusted P-value (P < 0.05). Functional annotation of selected genes was performed using NetAffx (Affymetrix Inc.) and Database for Annotation, Visualization and Integrated Discovery (DAVID; NIAID, NIH, Bethesda, MD, USA). Heat stress of COC reduced blastocyst development (27.2 v. 16.1% for control v. heat stress, respectively; SEM = 1.6; P < 0.002). Approximately 66 and 65% of 24 000 possible genes were called present (i.e. expressed) in RNA from cumulus of competent (control) v. challenged (heat-stressed) oocytes, respectively. In cumulus from developmentally challenged COC, increased abundance of 42 genes (36 currently annotated) was noted. Use of DAVID demonstrated enrichment of genes important for electron transport and energy generation (NOS2A, MAOB, CYP11A1, HSD11B1L, LTB4DH). Further examination of gene ontology identified genes associated with mitochondrial function (SLC25A10, MAOB, CYP11A1), cell signaling (similar to JAK-3, FSHR, CYP11A1, WNT2B), cytoskeleton (ACTA1), antioxidant activity (GSTA1), and extracellular region (FMOD). In contrast, cumulus from developmentally competent COC had increased expression of 22 genes (20 currently annotated), of which 15% were related to protein binding (CAV1, MMP9, TGFB2) according to DAVID. Further analysis using gene ontology revealed genes associated with extracellular matrix formation (MMP9, MMP19, PCOLCE2) and neural tissue (METRNL). In summary, alterations in cumulus gene expression were associated with differences in developmental competence of oocytes. Additional research is necessary to examine the extent to which identified genes account for functional differences in oocyte competence. This research was supported in part by National Research Initiative Competitive Grant no. 2004-35203-14772 from the USDA Cooperative State Research, Education, and Extension Service.

214 EFFECT OF OVARIAN SUPERSTIMULATION ON EXPRESSION OF GENES ASSOCIATED WITH THE OOCYTE DEVELOPMENTAL COMPETENCE OF NELORE COWS

2013 ◽  
Vol 25 (1) ◽  
pp. 255
Author(s):  
R. A. Satrapa ◽  
E. M. Razza ◽  
A. G. Pupulim ◽  
A. C. S. Castilho ◽  
B. Loureiro ◽  
...  
Keyword(s):  
Animal Health ◽  
Rna Extraction ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Oocyte Competence ◽  
Embryo Yield ◽  
Expression Of Genes

The P36 protocol has contributed to the genetic improvement of Brazilian herd through its successful use in embryo transfer programs. We aimed to investigate the effect of P36 protocol on embryo yield and mRNA expression of genes correlated with the competence of cumulus–oocyte complex (COC): receptors of FSH (FSHR), EGF (EGFR), and pentraxin 3 (PTX3) in cumulus cells; receptors of LH (LHR) and angiotensin 2 (AT2) in granulosa cells; and GDF9, BMP15, and histone H2A (H2A) in oocytes. Multiparous Nelore cows were allocated in control and P36 groups. Control group (non-superovulated, n = 15) received a progesterone intravaginal device (P4, 1.0 g, Primer®, Tecnopec, Sao Paulo, Brazil) and 2.5 mg of oestradiol benzoate (EB, IM, BER-BE®, Syntex, Buenos Aires, Argentina) at a random day of the oestrous cycle (Day 0). A PGF2α analogue (150 mg d-cloprostenol, IM, Prolise®, RARS SRL) was administered (Day 8) and Primer® was removed. The P36 group (n = 10) received a Primer® and 2.0 mg of EB (Day 0). The FSH treatment (160 mg Folltropin®, Bioniche Animal Health, Ontario, Canada) was initiated at decreasing doses: 40, 30, 20, and 10% of the total dose twice daily for 4 days (Day 5). The PGF2α analogue was administered (Day 8) and after 36 h primer was removed. Animal slaughter to ovary collection was performed 12 h after Primer® removal (Day 9). Some of the oocytes were matured (TCM199), fertilized with Nelore semen (n = 6), and cultured (SOF-synthetic oviduct fluid) to the blastocyst stage. Embryos were removed from culture (Day 6), allocated in 5 pools with 5 embryos in each group, and subjected to RNA extraction. Remaining oocytes were denuded from cumulus and zona pellucida (vortex and Protease®, Sigma-Aldrich, St. Louis, MO, USA). Pools of 20 oocytes and of their respective cumulus cells (n = 6 pools; control group and n = 4 pools, P36 group) were subjected to RNA extraction (RNeasy kit, Qiagen, Valencia, CA, USA). Gene expression was performed by real-time RT-PCR using oligo-dT in reverse transcription and bovine-specific primers. Expression of cyclophilin A was used as endogenous control. Change to developmental rates to the blastocyst stage and transcript abundance were compared by t-test and significance was considered when P < 0.05. Blastocyst rates were also similar (P > 0.05) in groups P36 (40/99; 40%) and control (16/43; 37%). Expression of H2A, EGFR, FSHR, and PTX3 in cumulus cells did not differ (P > 0.05) among treatment groups. The expression of GDF9 and BMP15 in cumulus cells was higher (P < 0.05) in the P36 group, but in oocytes these transcripts were more expressed in the control group (P < 0.05). Although important genes (GDF9 and BMP15) were less expressed in oocytes from superstimulated cows, the maintenance of H2A in oocytes, as well as PTX3, EGFR, and FSHR, and the increases in GDF9 and BMP15 expression in cumulus cells do not seem to affect oocyte competence due to the similar embryo yield of both groups. Supported by FAPESP.

scholarly journals Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro

Reproduction ◽  
2008 ◽  
Vol 136 (1) ◽  
pp. 9-21 ◽  
Author(s):  
Ikkou Kawashima ◽  
Tetsuji Okazaki ◽  
Noritaka Noma ◽  
Masahide Nishibori ◽  
Yasuhisa Yamashita ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Chorionic Gonadotropin ◽  
Follicular Fluid ◽  
Culture System ◽  
Expression Patterns ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Antral Follicles

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression ofLHCGRandPGRin cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-inducedLHCGRexpression in cumulus cells in culture. The expression ofLHCGRmRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation ofPGRandLHCGRin cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observedin vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.

scholarly journals Metabolic markers of developmental competence for in vitro-matured mouse oocytes

Reproduction ◽  
2005 ◽  
Vol 130 (4) ◽  
pp. 475-483 ◽  
Author(s):  
Kimberly A Preis ◽  
George Seidel ◽  
David K Gardner
Keyword(s):  
Lactate Production ◽  
Glucose Consumption ◽  
Cumulus Cells ◽  
Defined Medium ◽  
Developmental Competence ◽  
Metabolic Markers ◽  
Oocyte Competence ◽  
Carbohydrate Consumption ◽  
Mouse Oocytes

In vitro maturation of oocytes has enormous potential in assisted reproductive technology, but its use has been limited due to insufficient knowledge of oocyte physiology during this dynamic period and lack of an adequate maturation system. The aim of this study was to characterize the metabolic profiles of three groups of oocytes throughout maturation: cumulus–oocyte complexes (COCs), denuded oocytes, and denuded oocytes co-cultured with cumulus cells. Mouse oocytes were collected from 28-day-old unstimulated females and matured in a defined medium. Oocytes were matured individually and transferred into fresh 0.5 μl drops of medium at 4 h intervals until 16 h. Ultramicrofluorimetry was used to quantitate carbohydrate consumption from and metabolite release into the medium. Glucose consumption and lactate production of COCs increased (P < 0.001) over the maturation interval (0–16 h). Glucose consumption by COCs that subsequently fertilized was higher between 8–12 h of maturation than by COCs that did not fertilize (38 versus 29 pmol/COC per h, respectively; P < 0.01). Lactate production by COCs that subsequently fertilized was higher between 8–16 h of maturation, than by oocytes that did not fertilize (8–12 h, 66 versus 46 pmol/COC per h, P < 0.01; 12–16 h, 56 versus 40 pmol/COC per h, respectively; P < 0.05). These data indicate that the final hours of maturation may hold a unique marker of oocyte competence, as during this time fertilizable COCs take up more glucose and produce more lactate than those not subsequently fertilized.

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.

Effect of superstimulatory treatments on the expression of genes related to ovulatory capacity, oocyte competence and embryo development in cattle

2013 ◽  
Vol 25 (1) ◽  
pp. 17 ◽  
Author(s):  
Ciro M. Barros ◽  
Rafael A. Satrapa ◽  
Anthony C. S. Castilho ◽  
Patrícia K. Fontes ◽  
Eduardo M. Razza ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Granulosa Cells ◽  
Embryo Quality ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Treatment Protocol ◽  
Cumulus Cells ◽  
Fixed Time ◽  
Treatment Protocols ◽  
Oocyte Competence

Multiple ovulation (superovulation) and embryo transfer has been used extensively in cattle. In the past decade, superstimulatory treatment protocols that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-time AI (FTAI), have been developed for zebu (Bos indicus) and European (Bos taurus) breeds of cattle. There is evidence that additional stimulus with LH (through the administration of exogenous LH or equine chorionic gonadotrophin (eCG)) on the last day of the superstimulatory treatment protocol, called the ‘P-36 protocol’ for FTAI, can increase embryo yield compared with conventional protocols that are based on the detection of oestrus. However, inconsistent results with the use of hormones that stimulate LH receptors (LHR) have prompted further studies on the roles of LH and its receptors in ovulatory capacity (acquisition of LHR in granulosa cells), oocyte competence and embryo quality in superstimulated cattle. Recent experiments have shown that superstimulation with FSH increases mRNA expression of LHR and angiotensin AT2 receptors in granulosa cells of follicles >8 mm in diameter. In addition, FSH decreases mRNA expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes, but increases the expression of both in cumulus cells, without diminishing the capacity of cumulus–oocyte complexes to generate blastocysts. Although these results indicate that superstimulation with FSH is not detrimental to oocyte competence, supplementary studies are warranted to investigate the effects of superstimulation on embryo quality and viability. In addition, experiments comparing the cellular and/or molecular effects of adding eCG to the P-36 treatment protocol are being conducted to elucidate the effects of superstimulatory protocols on the yield of viable embryos.

scholarly journals Profiling of superoxide dismutase isoenzymes in compartments of the developing bovine antral follicles

Reproduction ◽  
2010 ◽  
Vol 139 (5) ◽  
pp. 871-881 ◽  
Author(s):  
Catherine M H Combelles ◽  
Emily A Holick ◽  
Louis J Paolella ◽  
David C Walker ◽  
Qiaqia Wu
Keyword(s):  
Superoxide Dismutase ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Antral Follicle ◽  
Supporting Evidence ◽  
Sod Activity ◽  
Antral Follicles ◽  
Sod Isoforms

The antral follicle constitutes a complex and regulated ovarian microenvironment that influences oocyte quality. Oxidative stress is a cellular state that may play a role during folliculogenesis and oogenesis, although direct supporting evidence is currently lacking. We thus evaluated the expression of the three isoforms (SOD1, SOD2, and SOD3) of the enzymatic antioxidant superoxide dismutase in all the cellular (granulosa cells, cumulus cells, and oocytes) and extracellular (follicular fluid) compartments of the follicle. Comparisons were made in bovine ovaries across progressive stages of antral follicular development. Follicular fluid possessed increased amounts of SOD1, SOD2, and SOD3 in small antral follicles when compared with large antral follicles; concomitantly, total SOD activity was highest in follicular fluids from smaller diameter follicles. SOD1, SOD2, and SOD3 proteins were expressed in granulosa cells without any fluctuations in follicle sizes. All three SOD isoforms were present, but were distributed differently in oocytes from small, medium, or large antral follicles. Cumulus cells expressed high levels of SOD3, some SOD2, but no detectable SOD1. Our studies provide a temporal and spatial expression profile of the three SOD isoforms in the different compartments of the developing bovine antral follicles. These results lay the ground for future investigations into the potential regulation and roles of antioxidants during folliculogenesis and oogenesis.

scholarly journals Functional genomics studies of oocyte competence: evidence that reduced transcript abundance for follistatin is associated with poor developmental competence of bovine oocytes

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 95-106 ◽  
Author(s):  
Osman V Patel ◽  
Anilkumar Bettegowda ◽  
James J Ireland ◽  
Paul M Coussens ◽  
Patrick Lonergan ◽  
...  
Keyword(s):  
Hormone Secretion ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Positive Association ◽  
Transcript Abundance ◽  
Mrna Abundance ◽  
Molecular Characteristics ◽  
Α Subunit ◽  
Oocyte Competence ◽  
Early Embryos

Poor oocyte competence contributes to infertility in humans and livestock species. The molecular characteristics of such oocytes are generally unknown. Objectives of the present studies were to identify differences in RNA transcript abundance in oocytes and early embryos associated with reduced oocyte competence and development to the blastocyst stage. Microarray experiments were conducted using RNA isolated from germinal vesicle stage oocytes collected from adult versus prepubertal animals (model of poor oocyte competence). A total of 193 genes displaying greater mRNA abundance in adult oocytes and 223 genes displaying greater mRNA abundance in prepubertal oocytes were detected. Subsequent gene ontology analysis of microarray data revealed significant overrepresentation of transcripts encoding for genes in hormone secretion classification within adult oocytes and such genes were selected for further analysis. Real-time PCR experiments revealed greater abundance of mRNA for βA and βB subunits of inhibin/activin and follistatin, but not the α subunit in germinal vesicle stage oocytes collected from adult versus prepubertal animals. Cumulus cell follistatin and βB subunit mRNA abundance were similar in samples collected from prepubertal versus adult animals. A positive association between time of first cleavage (oocyte competence) and follistatin mRNA abundance was noted. Follistatin, βB, and α subunit mRNAs were temporally regulated during early bovine embryogenesis and peaked at the 16-cell stage. Collectively, results demonstrate a positive association of follistatin mRNA abundance with oocyte competence in two distinct models and dynamic regulation of follistatin, βB, and α subunit mRNAs in early embryos after initiation of transcription from the embryonic genome.

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