Changes in granulosa cells' gene expression associated with increased oocyte competence in bovine

One of the challenges in mammalian reproduction is to understand the basic physiology of oocyte quality. It is believed that the follicle status is linked to developmental competence of the enclosed oocyte. To explore the link between follicles and competence in cows, previous research at our laboratory has developed an ovarian stimulation protocol that increases and then decreases oocyte quality according to the timing of oocyte recovery post-FSH withdrawal (coasting). Using this protocol, we have obtained the granulosa cells associated with oocytes of different qualities at selected times of coasting. Transcriptome analysis was done with Embryogene microarray slides and validation was performed by real-time PCR. Results show that the major changes in gene expression occurred from 20 to 44 h of coasting, when oocyte quality increases. Secondly, among upregulated genes (20–44 h), 25% were extracellular molecules, highlighting potential granulosa signaling cascades. Principal component analysis identified two patterns: one resembling the competence profile and another associated with follicle growth and atresia. Additionally, three major functional changes were identified: i) the end of follicle growth (BMPR1B,IGF2, andRELN), involving interactions with the extracellular matrix (TFPI2); angiogenesis (NRP1), including early hypoxia, and potentially oxidative stress (GFPT2,TF, andVNN1) and ii) apoptosis (KCNJ8) followed by iii) inflammation (ANKRD1). This unique window of analysis indicates a progressive hypoxia during coasting mixed with an increase in apoptosis and inflammation. Potential signaling pathways leading to competence have been identified and will require downstream testing. This preliminary analysis supports the potential role of the follicular differentiation in oocyte quality both during competence increase and decrease phases.

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

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab285 ◽

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).

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Evaluation of oocyte quality: morphological, cellular and molecular predictors

Reproduction Fertility and Development ◽

10.1071/rd06103 ◽

2007 ◽

Vol 19 (1) ◽

pp. 1 ◽

Cited By ~ 159

Author(s):

Qiang Wang ◽

Qing-Yuan Sun

Keyword(s):

Transforming Growth Factor ◽

Polar Body ◽

Morphological Characteristics ◽

Transforming Growth Factor Β ◽

Oocyte Quality ◽

Developmental Competence ◽

Meiotic Spindle ◽

Oocyte Competence ◽

Cumulus Oocyte Complex ◽

Glucose 6 Phosphate Dehydrogenase

Mounting evidence that oocyte quality profoundly affects fertilisation and subsequent embryo development drives the continued search for reliable predictors of oocyte developmental competence. In the present review, we provide an overall summary and analysis of potential criteria that can be used to evaluate oocyte quality. These criteria are specifically classified as morphological and cellular/molecular predictors. Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus–oocyte complex, polar body and/or meiotic spindle. Although the use of morphological characteristics as predictors of oocyte quality is controversial, such a grading system can provide valuable information for the preselection of oocytes with higher developmental competence and, therefore, may maximise embryo developmental outcome. Several intrinsic markers (such as mitochondrial status and glucose-6-phosphate dehydrogenase l activity) and extrinsic markers (such as apoptosis of follicular cells and levels of the transforming growth factor-β superfamily in follicular fluid or serum) have also been reported as useful indicators of oocyte competence and embryo quality. Compared with the morphological parameters, these cellular and molecular predictors of oocyte quality may prove to be more precise and objective, although further studies and refinement of techniques are needed.

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

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab193 ◽

2009 ◽

Vol 21 (1) ◽

pp. 195 ◽

Cited By ~ 1

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.

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What can we learn from gene expression profiling of mouse oocytes?

Reproduction ◽

10.1530/rep-07-0430 ◽

2008 ◽

Vol 135 (5) ◽

pp. 581-592 ◽

Cited By ~ 22

Author(s):

Toshio Hamatani ◽

Mitsutoshi Yamada ◽

Hidenori Akutsu ◽

Naoaki Kuji ◽

Yoshiyuki Mochimaru ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Molecular Mechanisms ◽

Expression Patterns ◽

Oocyte Quality ◽

Preimplantation Development ◽

Preimplantation Embryos ◽

Gene Expressions ◽

Oocyte Competence

Mammalian ooplasm supports the preimplantation development and reprograms the introduced nucleus transferred from a somatic cell to confer pluripotency in a cloning experiment. However, the underlying molecular mechanisms of oocyte competence remain unknown. Recent advances in microarray technologies have allowed gene expression profiling of such tiny specimens as oocytes and preimplantation embryos, generating a flood of information about gene expressions. So, what can we learn from it? Here, we review the initiative global gene expression studies of mouse and/or human oocytes, focusing on the lists of maternal transcripts and their expression patterns during oogenesis and preimplantation development. Especially, the genes expressed exclusively in oocytes should contribute to the uniqueness of oocyte competence, driving mammalian development systems of oocytes and preimplantation embryos. Furthermore, we discuss future directions for oocyte gene expression profiling, including discovering biomarkers of oocyte quality and exploiting the microarray data for ‘making oocytes’.

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Effect of follicle size on mRNA expression in cumulus cells and oocytes of Bos indicus: an approach to identify marker genes for developmental competence

Reproduction Fertility and Development ◽

10.1071/rd08201 ◽

2009 ◽

Vol 21 (5) ◽

pp. 655 ◽

Cited By ~ 60

Author(s):

Ester Siqueira Caixeta ◽

Paula Ripamonte ◽

Maurício Machaim Franco ◽

José Buratini Junior ◽

Margot Alves Nunes Dode

Keyword(s):

Gene Expression ◽

Bos Indicus ◽

Germinal Vesicle ◽

Cumulus Cells ◽

Developmental Competence ◽

Marker Genes ◽

Oocyte Competence ◽

Follicular Size ◽

Follicle Size

To identify the genes related to oocyte competence, we quantified transcripts for candidate genes in oocytes (H1Foo, H2A, H3A, GHR, GDF9, BMP15, OOSP1) and cumulus cells (FSHR, EGFR, GHR, PTX3, IGFII) using the follicle size model to select oocytes of better developmental quality. Follicles were dissected and distributed into four groups according to diameter as follows: 1.0–3.0, 3.1–6.0, 6.1–8.0 and ≥8.1 mm. Cumulus–oocyte complexes (COCs) were released, classified morphologically, matured, fertilised and cultured in vitro or denuded for measurement of diameter and determination of gene expression. Denuded germinal vesicle oocytes and their cumulus cells were used for gene expression analysis by reverse transcription–polymerase chain reaction. The blastocyst rate was highest for oocytes recovered from follicles >6 mm in diameter. In the oocyte, expression of the H2A transcript only increased gradually according to follicle size, being greater (P < 0.05) in oocytes from follicles ≥8.1 mm in diameter than in oocytes from follicles <6.0 mm in diameter. In cumulus cells, expression of FSHR, EGFR and GHR mRNA increased with follicular size. In conclusion, we confirmed the importance of H2A for developmental competence and identified important genes in cumulus cells that may be associated with oocyte competence.

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307 MATURATION RATE AND GENE EXPRESSION ANALYSIS OF GOAT OOCYTES SELECTED BY FOLLICLE SIZE AND BRILLIANT CRESYL BLUE STAINING

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab307 ◽

2015 ◽

Vol 27 (1) ◽

pp. 242

Author(s):

M. Yang ◽

S. Hu ◽

L. Cox ◽

M. Regouski ◽

H. Rutigliano ◽

...

Keyword(s):

Gene Expression ◽

Oocyte Maturation ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Oocyte Quality ◽

Developmental Competence ◽

Brilliant Cresyl Blue ◽

Relative Expression ◽

Cresyl Blue ◽

Maturation Rate

Oocyte quality plays a critical role in determining the success of embryo development. Studies on cattle and goats indicate that oocytes derived from large follicles (LFO) have greater developmental competence than those derived from small follicles (SFO). Brilliant cresyl blue (BCB) staining determines the activity of glucose-6-phosphate dehydrogenase and is a commonly used noninvasive marker of oocyte competence. Studies in pigs, goats, cows, mice, and dogs showed that the maturation and blastocyst developmental rate of BCB+ oocytes is significantly higher than BCB– oocytes. The aim of this study was to evaluate the maturation rate of goat oocytes selected based on follicular size and BCB staining and compare their relative patterns of gene expression. Maturation rate and gene expression profile were expected to be different in these oocyte groups. Cumulus-oocyte complexes were recovered from abattoir-derived ovaries using a slicing technique. Eleven rounds of oocyte maturation and 4 rounds of BCB staining were carried out. During each replicate, oocytes from large (≥3 mm) and small (<3 mm) follicles were collected separately from the same group of ovaries. Oocyte maturation rates were 54.3 ± 5.4% for LFO (n = 378) and only 33.5 ± 3.7% for SFO (n = 981; P < 0.01). The BCB+ (n = 223) oocytes yielded a significantly higher maturation rate than the BCB– (n = 194) oocytes (56.1 ± 1.8 v. 20.6 ± 3.8%, respectively; P < 0.001). Gene expression analysis was conducted on individual MII oocytes (21 oocytes per group). Specific target amplification was performed on a single oocyte directly by using the CellsDirect One-Step qRT–PCR Kit (Invitrogen). Quantitative real-time PCR was then performed using the 48.48 BioMark platform from Fluidigm. Forty two genes were selected from the following categories: growth factors, transcription factors, metabolism, pluripotency, cell cycle, apoptosis, and oocyte-specific genes. Relative expression values were calculated using the ΔΔCT (fold change) method and analysed by ANOVA. The significance was assigned at P < 0.05. The relative expression of CCNA2, CDK2, CCNB1, POU5F1, SOX2, EGF, FGF2, GDF9, ZP3, BCL2, GJA1, DDR1, PFKFB3, IGF2R, and GRB10 was significantly greater (P < 0.05) in both LFO and BCB+ oocytes compared to SFO and BCB– oocytes, respectively. The proapoptotic gene BAX, the ACSL3 gene involved in fatty acid oxidation, and the growth factor IGF1 were expressed significantly higher (P < 0.05) in SFO compared to LFO. By investigating these differentially expressed transcripts, we will better understand pathways involved in oocyte developmental competence and potentially use them as markers of oocyte quality. We expect that the ability to select oocytes of better quality based on BCB staining will improve outcomes of IVF and SCNT.

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304 EFFECT OF THE SEASONAL INFERTILITY PERIOD ON OOCYTE COMPETENCE IN PIGS

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab304 ◽

2015 ◽

Vol 27 (1) ◽

pp. 241

Author(s):

A. M. Giraldo ◽

D. Hylan ◽

R. R. Payton ◽

J. L. Edwards

Keyword(s):

Reproductive Performance ◽

Late Summer ◽

Oocyte Quality ◽

Developmental Competence ◽

Blastocyst Development ◽

Oocyte Competence ◽

Cell Numbers ◽

Oocyte Developmental Competence ◽

Development Rates ◽

Early Fall

Photoperiod is the principal regulator of seasonal breeding; however, effects of photoperiod on the fertility of the domestic sow are inconclusive. Some evidence indicates that the modern sow exhibits a period of impaired reproductive performance during the late summer and early fall. Seasonal variation in oocyte developmental competence has been described as a contributing factor. Alterations in oocyte quality, along with reductions in blastocyst rates and cell numbers in embryos from summer-sourced oocytes, may be attributed to an alteration in follicular fluid (FF) composition. The objectives of this study were to determine whether seasonal variations in blastocyst development rates are associated with changes in cumulus-oocyte complex (COC) morphology and oocyte developmental competence in sows. This study also compared the effect of FF collected in spring v. summer during in vitro maturation (IVM) on oocyte competence. In experiment 1, oocytes from 3- to 8-mm follicles were aspirated from sow ovaries during 1 calendar year for a total of 77 replicates. Only oocytes with homogeneous dark cytoplasm and at least 2 layers of cumulus cells underwent IVM. Mature oocytes were electrically activated and the resulting embryos were cultured for 6 days. In experiment 2, a total of 1256 good quality COC were divided into 2 groups and cultured in IVM medium containing 10% FF collected in either spring or late summer. Metaphase II oocytes were electrically activated and cultured to generate diploid embryos. Differences between experimental groups were assessed using Student's t-test or X2. The percentage of ovaries exhibiting good-quality follicles and the number of COC per ovary remained constant during the entire calendar year (60% and 6.2 COC/ovary, respectively). However, oocyte quality decreased significantly from 3.6 to 3.2 during late August throughout early October in a 1 to 4 scale. The percentage of good-quality COC decreased significantly during late summer and early fall compared with the rest of the year (54.5 v. 65.5%). However, maturation, cleavage, and blastocyst rates did not show significant differences between the summer and the other seasons (85.5 v. 87.6, 87.8 v. 87.7, and 27.8 v. 27.0%, respectively). The presence of FF collected in either spring or summer in the IVM medium did not affect maturation, cleavage, or blastocyst rates (88.9 v. 87.7, 90.7 v. 90.5, and 42.1 v. 43.7%, respectively). Blastocyst cell numbers (Day 6) did not differ when FF from spring and summer antral follicles was used for supplementing IVM medium (43.6 v. 46.1 cells, respectively). In summary, impaired reproductive performance of domestic sows during late summer and early fall is coincident with a decreased in the number and quality of COC. However, efforts to use strict selection criteria for COC during this time period may result in maturation and development rates comparable to the rest of the seasons. Additionally, the presence of FF collected in either spring or summer in the IVM medium does not seem to affect oocyte maturation and subsequent embryo development.

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54 Effect of clinical endometritis on the follicle growth dynamics, oocyte recovery, oocyte quality, and invitro developmental competence of oocytes using ovum pickup in Sahiwal cattle

Reproduction Fertility and Development ◽

10.1071/rdv33n2ab54 ◽

2021 ◽

Vol 33 (2) ◽

pp. 134

Author(s):

M. Saleem ◽

M. Nawaz ◽

M. Yaseen ◽

M. R. Yousuf ◽

A. G. Bajwa ◽

...

Keyword(s):

Growth Dynamics ◽

Oocyte Quality ◽

Developmental Competence ◽

Follicle Growth ◽

Cleavage Rate ◽

Healthy Group ◽

Nuclear Maturation ◽

Oocyte Recovery ◽

Sahiwal Cattle ◽

Blastocyst Rate

Sahiwal cattle is the premium quality milk breed of cattle in Pakistan. Uterine infections often lead to culling of valuable animals from a herd, resulting in genetic drain. The genetic potential of problematic females could be reaped by invitro embryo production. The objective of the present study was to evaluate the effect of clinical endometritis on follicle growth dynamics, recovery, quality, and invitro developmental competence of oocytes using ovum pickup (OPU) in Sahiwal cattle. The animals, 5–7 years of age, third or fourth parity, and 160 to 170 days in milk (DIM), were inspected for any discharge at the vulva or inside the vagina. Then, B-mode ultrasonography was performed to measure the diameter of cervix and to examine the uterus for the presence of pus. The animals (n=12) were divided into 2 groups: (1) healthy (n=6), and (2) clinical endometritis (n=6), based on the presence or absence of pus at the vulva or in the vagina. The first OPU was performed after 7 days of dominant follicle puncture and subsequently repeated OPUs (54 and 50), after every 7 days over 9 OPU sessions, were performed in the healthy group and clinical endometritis group, respectively. Follicles were aspirated using transvaginal ultrasound–guided needle. Viable COCs were considered for further processing only and were placed in the 100-µL droplets of BO-IVM medium and incubated at 37°C, 5% CO2, and 95% humidity for 24h. Nuclear maturation was estimated by staining the oocytes with Hoechst 33342. Frozen semen from the same Sahiwal bull was thawed and processed for IVF throughout the study. Sperm were prepared using swim-up protocol. Sperm and COCs were co-incubated in 100-µL droplets of BO-IVF for 18h. Finally, presumptive zygotes were cultured in 100-µL drops of BO-IVC medium at 37°C, 5% CO2, 5% O2, and 95% humidity for a period of 7 days. Cleavage rate and blastocyst rate were recorded on Day 2 and 7 following IVF, respectively. The data were analysed using the GLIMMIX procedure of SAS (SAS Institute Inc.). The results revealed that the number of medium-sized follicle (1.32±0.11 vs. 0.56±0.11) and total follicles (9.14±0.70 vs. 6.58±0.72) were higher (P<0.05) in the healthy group than in the clinical endometritis group, respectively. Similarly, the number of oocytes recovered (5.05±0.39 vs. 2.78±0.41), viable oocytes (2.87±0.25 vs. 1.46±0.26), COCs with grade AB, having minimum of 2 cumulus cell layers and homogeneous cytoplasm, (33 vs. 20%) and nuclear maturation (68 vs. 55%) were also higher (P<0.05) in the healthy group than in the clinical endometritis group, respectively. However, cleavage rate (55 vs. 46%) and blastocyst rate (29 vs. 26%) did not differ (P>0.05) between the groups. In conclusion, clinical endometritis has a negative effect on follicle growth dynamics, oocyte recovery, oocyte quality, and nuclear maturation; however, the developmental competence of COCs is not compromised by it.

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151. LIPOTOXICITY MEDIATED ENDOPLASMIC RETICULUM STRESS, MITOCHONDRIAL DYSFUNCTION AND APOPTOSIS CONTRIBUTE IMPAIRED OOCYTE QUALITY IN RESPONSE TO OBESITY

Reproduction Fertility and Development ◽

10.1071/srb09abs151 ◽

2009 ◽

Vol 21 (9) ◽

pp. 69

Author(s):

L. L. Y. Wu ◽

X. Yang ◽

K. R. Dunning ◽

R. J. Norman ◽

R. L. Robker

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Potential ◽

Er Stress ◽

Mitochondrial Dysfunction ◽

Granulosa Cells ◽

Oocyte Quality ◽

Developmental Competence ◽

Tunel Staining ◽

Marker Genes ◽

Er Homeostasis

In obesity, accumulation of lipid in non-adipose tissues, a process termed lipotoxicity, is associated with endoplasmic reticulum (ER) stress, mitochondrial dysfunction and ultimately apoptosis . We have previously shown that diet-induced obesity in mice causes impaired oocyte developmental competence, but whether this is due to activation of lipotoxicity pathways in the ovary is not known. The present study examined the hypothesis that diet-induced lipid accumulation in the cumulus oocyte complex (COC) disrupts ER homeostasis and mitochondrial membrane potential which leads to apoptosis. COCs and mural granulosa cells were collected from ovaries of adult mice fed a high fat (HFD) or control diet for 4 weeks. ER homeostasis was assessed by measuring expression of known ER stress marker genes, GRP78, ATF4 and CHOP. COCs from mice fed HFD showed significantly increased expression of GRP78 and ATF4. There was a similar trend towards increased expression in granulosa cells. Mitochondrial function was assessed by measuring membrane potential using the dual emission probe JC-1. In COCs from mice fed HFD there were reduced numbers of active mitochondria but instead large aggregated clusters of inactive mitochondria. Apoptosis in granulosa cells was determined by DNA laddering assay which showed significantly increased DNA fragmentation in cells from mice fed HFD. Apoptosis was also assessed by TUNEL staining of paraffin embedded ovaries from identical treatment groups. Ovaries from HFD mice appeared to have increased TUNEL positivity in both granulosa and cumulus cells. Our results demonstrate that the ER stress, mitochondrial dysfunction and apoptosis are markedly increased in granulosa cells and COCs from mice fed HFD, suggesting that lipotoxicity contributes to the impaired oocyte quality and reduced fertility observed in response to obesity.

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Granulosa cell biomarkers to predict pregnancy in ART: pieces to solve the puzzle

Reproduction ◽

10.1530/rep-16-0500 ◽

2017 ◽

Vol 153 (2) ◽

pp. R69-R83 ◽

Cited By ~ 15

Author(s):

Richard J Kordus ◽

Holly A LaVoie

Keyword(s):

Gene Expression ◽

Granulosa Cell ◽

Live Birth ◽

Granulosa Cells ◽

Expression Profiles ◽

Ovarian Follicle ◽

Critical Discussion ◽

Developmental Competence ◽

Published Data

Cumulus and mural granulosa cells of the ovarian follicle surround and interact with the developing oocyte. These follicular cells reflect the oocyte’s overall health and may indicate subsequent developmental competence of embryos. Biomarkers of granulosa cells associated with individual oocytes could potentially be used in assisted reproduction to indicate which embryos have the best chance of implanting in the uterus and completing gestation. In this review, we have performed a comprehensive assessment of the recent literature for human cumulus and mural granulosa cell mRNA biomarkers as they relate to pregnancy and live birth. A critical discussion of variables affecting granulosa gene expression profiles for in vitro fertilization patients, including patient demographics and ovarian stimulation regimens, is presented. Although studies with microarray data were evaluated, this synopsis focuses on expressed genes that have been validated by quantitative RT-PCR. Furthermore, we summarize the current published data that support or refute identified granulosa expressed genes as potential biomarkers of embryos that give rise to ongoing pregnancy and live birth. Finally, we review studies that offer predictive models for embryo selection for uterine transfer based on biomarkers that show differential gene expression.

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