Comparative analysis of granulosa cell gene expression in association with oocyte competence in FSH-stimulated Holstein cows

2017 ◽  
Vol 29 (12) ◽  
pp. 2324 ◽  
Author(s):  
David A. Landry ◽  
Chloé Fortin ◽  
Anne-Marie Bellefleur ◽  
Rémi Labrecque ◽  
François-Xavier Grand ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Granulosa Cells ◽  
Ovarian Stimulation ◽  
Insulin Signalling ◽  
Effective Means ◽  
Developmental Competence ◽  
Holstein Cows ◽  
In Vitro Production ◽  
Oocyte Competence

Ovarian stimulation with exogenous FSH followed by FSH withdrawal or ‘coasting’ is an effective means of increasing the number of oocytes obtainable for the in vitro production of cattle embryos. However, the quality of the oocytes thus obtained varies considerably from one cow to the next. The aim of the present study was to gain a better understanding of the follicular conditions associated with low oocyte developmental competence. Granulosa cells from 94 Holstein cows in a commercial embryo production facility were collected following ovarian stimulation and coasting. Microarray analysis showed 120 genes expressed with a differential of at least 1.5 when comparing donors of mostly competent with donors of mostly incompetent oocytes. Using ingenuity pathway analysis, we revealed the main biological functions and potential upstream regulators that distinguish donors of mostly incompetent oocytes. These are involved in cell proliferation, apoptosis, lipid metabolism, retinol availability and insulin signalling. In summary, we demonstrated that differences in follicle maturity at collection could explain differences in oocyte competence associated with individual animals. We also revealed deficiencies in lipid metabolism and retinol signalling in granulosa cells from donors of mostly incompetent oocytes.

scholarly journals Superovulation following follicular synchronization with GnRH at random stages of the oestrous cycle in heifers: oocyte competence and in vitro embryo production

2010 ◽  
Vol 55 (No. 5) ◽  
pp. 190-194
Author(s):  
H. Kohram ◽  
V. Vahedi ◽  
A. Farahavar
Keyword(s):  
Late Phase ◽  
Oestrous Cycle ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Embryo Production ◽  
Oocyte Competence ◽  
The Mean ◽  
Follicular Puncture ◽  
Vitro Production

The objective of this study was to develop a superovulatory program based on the synchronization of follicular waves with GnRH which could be applied regardless of the stage of the oestrous cycle. In this experiment, GnRH was given to 30 heifers in lactation between Days 0 and 7 (n = 13), 8 and 12 (n = 12), 13 and 16 (n = 5) of the oestrous cycle. Twenty-four heifers were used as controls and did not receive any GnRH. All follicles ≥ 6 mm were punctured 4 days after GnRH treatment in treated animals and between Days 8 and 12 of the oestrous cycle in control heifers. Two days after the follicular puncture, all heifers were superstimulated with 160 mg Folltropin-V given twice daily over 2 days. Oocytes were collected 42 h after the last FSH treatment. The oocytes were subjected to IVM/IVF and the developmental competence of embryos was compared. In vitro production of embryos was affected only by the stages of the oestrous cycle when the GnRH treatment was given and not by the GnRH treatment. No difference (P > 0.1) in the mean number of oocytes, cleavage and embryo production was noted between the control animals and the animals treated with GnRH in the late phase of the oestrous cycle. The mean number of blastocysts was higher (P < 0.05) in heifers treated with GnRH in the mid and the late phase of the oestrous cycle than in the early phase. In conclusion, the in vitro production of embryos was compromised in the present study with heifers following the follicular synchronization with GnRH. This procedure is advantageous for the in vitro production of bovine embryos since the spontaneous oestrus is eliminated. However, more investigations are needed to increase the competence of oocytes obtained following this procedure.

P–178 Mural granulosa cells of the human follicles indirectly show death molecular signals not depending on different ovarian stimulation protocols

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Ruvolo ◽  
F Geraci ◽  
M C Roccheri ◽  
R Alessandro ◽  
L Bosco
Keyword(s):  
In Vitro Culture ◽  
Granulosa Cells ◽  
Pregnancy Outcomes ◽  
Protein Level ◽  
Ovarian Stimulation ◽  
Embryo Quality ◽  
Cumulus Cells ◽  
Oocyte Competence

Abstract Study question Could the expression of the anti-apoptotic molecules AKT, p-Akt and ERK1/2 in Mural Granulosa Cells (MGC) be considered as marker of oocyte quality? Summary answer MGCs activate cell death pathways in analyzed follicles and it is not influenced by different stimulation protocols and it is not correlated to oocyte competence. What is known already It has been previously demonstrated that apoptosis rate of mural granulosa or cumulus cells (CC) were correlated with follicular oocyte number, age, embryo numbers in IVF/ICSI and also clinical pregnancy. Moreover, our previous data demonstrated that in selected patients, who received recombinant LH associated with recombinant FSH (rFSH), the DNA fragmentation in cumulus cells was significantly lower and pregnancy rate was higher, compared to patients treated with rFSH alone. However, to date little is known about the differences between MGC and CC regarding death/survival pathways and whether the two cell types respond in the same way. Study design, size, duration Molecular study on MGCs to investigate the role of the surviving/apoptotic molecules AKT, p-Akt and ERK1/2 and their relationship with the administration of exogenous r-LH combined with r-FSH administration in ovarian stimulation comparing with r-FSH alone. We analyzed also the oocyte competence, for each follicle, according to the embryo development during in vitro culture and the pregnancy outcome. We included fifty-three normo-responder women undergoing ICSI in two years. Participants/materials, setting, methods Patients were divided into two groups: 1) 34 women were stimulated with r-FSH and used as control group, 2) 19 women were stimulated with r-FSH combined with r-LH. Mural granulosa cells isolated singularly from 255 MII oocytes of the 53 patients were used for the study. The study was conducted in public university laboratory. MGCs obtained from each single follicle were suspended in medium, without serum. For immunoreaction anti-AKT, p-AKT, ERK1/2 antibodies were used. Main results and the role of chance Out of 255 MII oocytes collected, 197 were fertilized and the derived embryos had the following evolution: 117 transferred, 57 vitrified and 23 arrested during in vitro culture. 58 oocytes were not analyzed because of failed fertilization or because of their immature condition (GV or MI). In the MGCs isolated from the follicle of each oocyte generating an embryo, the expression AKT, pAKT and ERK1/2 was analyzed and associated with embryo quality and pregnancy outcomes. Immunoblot analysis on granulose cells showed no statistically significant differences in protein level in MGCs isolated from oocytes that have generated transferred embryos (blastocyst at day 5 or 6) comparing with embryos who arrested during in vitro culture. No differences were found also in MGCs collected from the follicles derived from r-FSH ovarian stimulation compared to r-FSH+r-LH. Moreover, no difference was highlighted even between protein level and pregnancy outcomes. The results seem to demonstrate that the MGCs primarily have an endocrine function and support the growth of the follicle, and finally follow a specific death pathway. This condition is not influenced by different ovarian stimulation protocol, in contrast with CC, and is not correlated to oocyte competence, embryo quality and clinical outcomes. Limitations, reasons for caution Only a limited number of patients have been observed. Wider implications of the findings: Our current and past results suggest that the evaluation of cumulus cells and mural granulosa cells in the same follicle show different expression of molecules involved in the apoptotic pathway and therefore they cannot be used, as molecular markers, in the same way, to assess the competence of oocytes. Trial registration number Not applicable

Supplementation of c-type natriuretic peptide during in vitro growth period benefits the development of murine preantral follicles

Zygote ◽  
2020 ◽  
pp. 1-5
Author(s):  
Li Ang ◽  
Cao Haixia ◽  
Li Hongxia ◽  
Li Ruijiao ◽  
Guo Xingping ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Granulosa Cells ◽  
Culture System ◽  
Early Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Follicle Development ◽  
Control Groups ◽  
Preantral Follicles

Summary The present study investigated the effects of c-type natriuretic peptide (CNP) on the development of murine preantral follicles during in vitro growth (IVG). Preantral follicles isolated from ovaries of Kunming mice were cultured in vitro. In the culture system, CNP was supplemented in the experimental groups and omitted in the control groups. In Experiment 1, CNP was only supplemented at the early stage and follicle development was evaluated. In Experiments 2 and 3, CNP was supplemented during the whole period of in vitro culture. In Experiment 2, follicle development and oocyte maturity were evaluated. In Experiment 3, follicle development and embryo cleavage after in vitro fertilization (IVF) were assessed. The results showed that in the control groups in all three experiments, granulosa cells migrated from within the follicle and the follicles could not reach the antral stage. In the experimental groups in all three experiments, no migration of granulosa cells was observed and follicle development was assessed as attaining the antral stage, which was significantly superior to that of the control group (P < 0.0001). Oocyte meiotic arrest was effectively maintained, hence giving good developmental competence. In conclusion, CNP supplementation in the culture system during IVG benefited the development of murine preantral follicles.

In vitro production of progesterone and estradiol by rat granulosa cells regulated by cabergoline and prolactin

1998 ◽  
Vol 24 (2) ◽  
pp. 195-203 ◽  
Author(s):  
H. Negishi ◽  
I. Furuta ◽  
K. Ushigoe ◽  
S. Fujimoto ◽  
S. S. Koide
Keyword(s):  
Granulosa Cells ◽  
In Vitro Production ◽  
Vitro Production

scholarly journals Oocyte Competence Biomarkers Associated With Oocyte Maturation: A Review

2021 ◽  
Vol 9 ◽  
Author(s):  
Batara Sirait ◽  
Budi Wiweko ◽  
Ahmad Aulia Jusuf ◽  
Dein Iftitah ◽  
R. Muharam
Keyword(s):  
Oocyte Maturation ◽  
Blastocyst Stage ◽  
Current Approach ◽  
Developmental Competence ◽  
Matrix Remodeling ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Oocyte Developmental Competence ◽  
Cumulus Oocyte Complex

Oocyte developmental competence is one of the determining factors that influence the outcomes of an IVF cycle regarding the ability of a female gamete to reach maturation, be fertilized, and uphold an embryonic development up until the blastocyst stage. The current approach of assessing the competency of an oocyte is confined to an ambiguous and subjective oocyte morphological evaluation. Over the years, a myriad of biomarkers in the cumulus-oocyte-complex has been identified that could potentially function as molecular predictors for IVF program prognosis. This review aims to describe the predictive significance of several cumulus-oocyte complex (COC) biomarkers in evaluating oocyte developmental competence. A total of eight acclaimed cumulus biomarkers are examined in the study. RT-PCR and microarray analysis were extensively used to assess the significance of these biomarkers in foreseeing oocyte developmental competence. Notably, these biomarkers regulate vital processes associated with oocyte maturation and were found to be differentially expressed in COC encapsulating oocytes of different maturity. The biomarkers were reviewed according to the respective oocyte maturation events namely: nuclear maturation, apoptosis, and extracellular matrix remodeling, and steroid metabolism. Although substantial in vitro evidence was presented to justify the potential use of cumulus biomarkers in predicting oocyte competency and IVF outcomes, the feasibility of assessing these biomarkers as an add-on prognostic procedure in IVF is still restricted due to study challenges.

Modification of ovary stock solution with magnesium and raffinose improves the developmental competence of oocytes after long preservation

Zygote ◽  
2005 ◽  
Vol 13 (4) ◽  
pp. 303-308 ◽  
Author(s):  
H. Iwata ◽  
T. Hayashi ◽  
H. Sato ◽  
K. Kimura ◽  
T. Kuwayama ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Germinal Vesicle ◽  
Storage Period ◽  
Cell Number ◽  
Developmental Competence ◽  
Germinal Vesicle Breakdown ◽  
Storage Solutions ◽  
Stock Solutions ◽  
Phosphate Buffered Saline

During ovary storage oocytes lose some of their developmental competence. In the present study, we maintained storage solutions of phosphate-buffered saline (PBS) at various temperatures (20 or 35 °C) or supplemented them with magnesium (Mg), raffinose and sucrose. Subsequently, we examined the kinetics of electrolytes in the follicular fluid (FF) during the ovary storage period (9h), the survival rate of granulosa cells in the follicles, and the developmental competence of oocytes after the storage. Lowering the temperature from 35 to 20 °C increased the total cell number of blastocysts that developed at 7 days after in vitro maturation and in vitro fertilization of oocytes. In stock solution with supplements of 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose or sucrose, a significantly higher number of oocytes developed into blastocysts with a large number of cells in each blastocyst, and a significantly higher number of living granulosa cells were obtained as compared with stock solutions without any supplements. During ovary storage, the concentrations of potassium and chloride in the FF were increased, and the addition of Mg to the stock solution increased the concentration of Mg in the FF. Germinal vesicle breakdown in oocytes that were collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM of raffinose occurred at a slower rate than that in oocytes collected from ovaries stored in PBS alone. On the other hand, the oocytes collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose reached the metaphase II (MII) stage more rapidly than the oocytes collected from ovaries stored in the PBS alone. In conclusion, the modification of stock solution by the addition of Mg and raffinose improved the developmental competence of oocytes obtained from ovaries preserved for a long period.

221 EFFECT OF ROCKING ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 209
Author(s):  
Y. Serita ◽  
C. Kubota ◽  
T. Kojima
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Humid Atmosphere ◽  
Vitro Fertilization ◽  
Rate Of Development ◽  
Fetal Bovine ◽  
Vitro Production

This study tested whether embryo development yield using in vitro fertilization (IVF) could be improved by rocking cultures. Bovine ovaries were obtained at a slaughterhouse and transported to the laboratory within 6 h. Cumulus–oocyte complexes were collected and 20–25 were transferred in 100-μL drops of TCM-199 containing 10% fetal bovine serum and antibiotics under paraffin oil. Maturation was for 20–24 h at 38.5°C under 5% CO2 and 95% air in a humid atmosphere (IVM). In vitro fertilization was carried out for 6 h using frozen–thawed sperm from a single bull in modified Brackett and Oliphant (BO) medium. Presumptive zygotes were cultured in CR1aa supplemented with 10 mg mL–1 of BSA or 5% FBS for 9 d at 38.5°C under 5% CO2, 5% O2, and 90% N2 in a humid atmosphere (IVC). Rocking was performed to a height of 6 cm every 7 s using a Profile Rocker (New Brunswick Scientific Co., Edison, NJ, USA) in an incubator. Dishes were placed at a 15-cm distance from the fulcrum of the rocker. The conventional method (no rocking) served as a control, and every experiment was replicated 3 times. For Experiment 1, the effect of the period of rocking on developmental competence was examined when COC or zygotes were subjected to rocking for IVM, IVF, or IVC (IVM-move, IVF-move, and IVC-move). There were no significant differences in rates of oocyte maturation, cleavage, and development for IVM-move v. the control, or for rate of development between IVC-move and the control. However, the rate of fertilization for IVF-move was higher than that of the control (88.9 v. 67.5%; P < 0.01), and the rate of development was higher for IVF-move than for the control (39.0 v. 25.7%; P < 0.05). For Experiment 2, the effect of rocking frequency during IVF on development was determined. The IVF cultures were rocked every 7, 3.5, and 1.5 s (IVF-1move, IVF-2move, IVF-3move). The rates of cleavage on IVF-1move, IVF-2move, IVF-3move, and the control were 74.3, 69.8, 68.8, and 60.4%, and the rates of development were 39.0, 48.3, 26.2, and 25.7%, respectively. The rates of development on IVF-1move and IVF-2move were significantly different from the control and IVF-3move (P < 0.01). These results showed that rocking during IVF improved fertilization and embryo yield, and that frequency of rocking affected embryo development.

285 RELATIONSHIP BETWEEN FOLLICLE FLUID STEROID CONCENTRATIONS, GRANULOSA CELL GENE EXPRESSION, AND BOVINE OOCYTE DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 299
Author(s):  
S. Matoba ◽  
S. Mamo ◽  
E. Gallagher ◽  
A. G. Fahey ◽  
T. Fair ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Target Genes ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Oocyte Competence ◽  
Cell Gene Expression ◽  
The Relationship

The ability to culture oocytes and embryos in an individually identifiable manner facilitates the study of the relationship between follicle param- eters and oocyte development, in order to identify markers of competent oocytes. The aim of this study was to examine the predictive value of intrafollicular steroid concentrations and granulosa cell transcript abundance on the ability of immature bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles (n = 214, 11 replicates, 49 animals) were dissected from the ovaries of slaughtered animals. Following measure- ment of diameter, follicles were carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through maturation, fertilization, and culture on the cell adhesive Cell-Tak (20 oocytes/100 μL; Matoba and Lonergan 2009 Reprod. Fertil. Dev. 21, 160). Cleavage and blastocyst rates were assessed on Days 2 and 9, respectively. Follicular fluid was recovered and stored at -80°C until analysis for concentrations of the steroids estradiol, progesterone, and testosterone by RIA. Granulosa cells were collected from each follicle for analysis of gene expression by quantitative RT-PCR. Primers were designed for 7 target genes (AMH, CYP19A, ESR1, ESR2, FSHR, HSD3B1 and LHCGR) and 2 reference genes (PPIA and H2AZ). Transcript abundance of target genes in granulosa cells associated with embryos that cleaved and developed to the blastocyst stage (competent) and those that cleaved but failed to develop (incompetent) was examined. Mean steroid concentrations were compared by ANOVA and Spearman correlations, and logistical regression were used to test the relationship between follicle size and steroid con- centration and the ability of steroid concentration to predict developmental competence. Gene expression data were analyzed using the delta-delta CT (cycle threshold) method. Values were normalized to the average values of the reference genes and means were compared by the Student’s t-test In total, 79.1% of oocytes cleaved after IVF and 28.3% developed to the blastocyst stage. The mean (±SEM) follicular concentrations of testosterone (62.8 ± 4.8 ng mL-1), progesterone (616.8 ± 31.9 ng mL-1), or estradiol (14.4 ± 2.4 ng mL-1 were not different (P ≥ 0.05) between competent and incompetent oocytes. Follicular diameter was negatively correlated with testosterone, progesterone, testosterone:estradiol, and pro- gesterone:estradiol (P ≤ 0.01) and positively correlated with estradiol (P ≤ 0.01). Logistical regression analysis showed that steroid concentrations or the ratio of steroids were not satisfactory predictors of oocyte competence. Transcript abundance of AMH, ESR1, ESR2, FSHR, and HSD3B1 was significantly higher (P ≤ 0.05) in granulosa cells associated with competent compared with incompetent oocytes. In conclusion, follicular steroid concentrations were not associated with oocyte development. In contrast, granulosa cell gene expression may be a useful predictor of oocyte competence. Supported by Science Foundation Ireland (07/SRC/B1156).

204 PERIPHERAL PROGESTERONE CONCENTRATION AFFECTS BOVINE OOCYTE QUALITY AT THE MOLECULAR LEVEL

2013 ◽  
Vol 25 (1) ◽  
pp. 250
Author(s):  
N. Schlüter ◽  
A. Hanstedt ◽  
H. Stinshoff ◽  
K. Knauer ◽  
S. Wilkening ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Relative Abundance ◽  
Clinical Signs ◽  
Follicular Phase ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Glucose Transporter 1 ◽  
Vitro Production

The developmental competence of cumulus–oocyte complexes (COC) used for in vitro production is dependent on several factors including the stage of the oestrus cycle. In a recent study, we were able to show that circulating progesterone (P4) had no effect on follicle number, size, recovery rate, or in vitro production suitability of recovered COC (Schlüter et al. 2012 Reprod. Fertil. Dev. 24, 175–176). The aim of the present study was to determine the influence of circulating P4 concentrations on the molecular quality of bovine COC collected during repeated OPU sessions. The COC were aspirated twice per week for 5 to 6 weeks from 12 Holstein Friesian heifers. The first OPU session took place on Day 7 of the oestrous cycle after spontaneous ovulation (ovulation = Day 0). Blood samples were taken at the time of each OPU session, and P4 concentrations were determined using a radioimmunoassay. All animals showed clinical signs of oestrus and large follicles (≥8.5 mm) during the course of the OPU sessions. Following the aspiration of a large follicle, a CL-like structure (induced CL) could be detected. According to the P4 concentrations, the cycle was divided into 3 phases: CL phase after spontaneous ovulation (oCL; P4: ≥1 ng mL–1), follicle phase 1 (Fp; P4 <1 ng mL–1), and induced CL phase (iCL; P4: ≥1 ng mL–1). The length of the cycle after spontaneous ovulation did not differ significantly from that after induced ovulation (22.4 ± 3.1 days v. 23.8 ± 1.8 days, respectively). During the oCL-phase, blood P4 concentrations were significantly higher than during the iCL-phase (4.9 ± 2.3 ng mL–1 v. 3.0 ± 1.6 ng mL–1). For mRNA analysis, denuded COC were individually frozen at –80°C to analyse the relative transcript abundance using RT-qPCR. The transcripts studied play important roles during oocyte development [growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), glucose transporter 1 (SCL2A1), hypoxia inducible factor 2α (HIF2α), progesterone receptor (PGR), progestin and adipoQ receptor 5 (PAQR5), progesterone receptor membrane component 1 and 2 (PGRMC1, PGRMC2)]. Data were tested using analysis of variance (ANOVA) followed by multiple pairwise comparisons using Tukey’s test. A P-value of ≤0.05 was considered significant. The relative abundance of all transcripts except SCL2A1 was significantly increased in oocytes collected from follicles of the oCL phase compared with that from oocytes that had been aspirated during the iCL phase. A significant increase in the relative amount of PGR, PGRMC1, PGRMC2, and BMP15 transcripts was detected in oocytes stemming from the follicular phase to those from the iCL phase. No differences in the relative abundance of all transcripts were seen comparing oocytes from oCL phase and oocytes from the follicular phase. In summary, circulating P4 concentrations had an effect on the molecular quality of COC recovered during repeated OPU session, which might affect further development. The financial support of the FBF (Förderverein Biotechnologieforschung) e.V. is gratefully acknowledged.

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