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.

256 ROLE OFTHE BOVINE OOCYTE-SPECIFIC PROTEIN JY-1 IN REGULATION OF MESSENGER RNA ABUNDANCE FOR GENES LINKED TO CUMULUS EXPANSION

2010 ◽  
Vol 22 (1) ◽  
pp. 285 ◽  
Author(s):  
G. Wee ◽  
K. B. Lee ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Extracellular Matrix ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Specific Protein ◽  
Negative Control ◽  
Mrna Abundance ◽  
Cumulus Expansion ◽  
Vitro Fertilization ◽  
Maturation Medium

We have previously demonstrated a requirement of the oocyte-specific protein JY-1 for oocyte and early embryonic development in cattle. Microin- jection of JY-1 siRNA into cumulus-enclosed germinal vesicle-stage oocytes impedes progression to metaphase II and cumulus expansion during in vitro maturation and limits subsequent embryonic development following in vitro fertilization. Negative effects of siRNA-mediated reduction in JY-1 on oocyte maturation, cumulus expansion, and initial cleavage divisions following in vitro fertilization can be rescued bysupplementation with recom- binant JY-1 protein during oocyte culture. However, the mechanisms involved in JY-1 regulation of above developmental endpoints are unknown. The objective of the current study was to determine whether JY-1-induced regulation of cumulus expansion during meiotic maturation is linked to alterations in mRNA abundance for genes that promote formation and stabilization of the mucified extracellular matrix characteristic of an expanded cumulus layer. Cumulus-enclosed germinal vesicle stage-bovine oocytes were microinjected with JY-1 siRNA, subjected to negative control siRNA microinjection or served as uninjected controls (n = 10 oocytes/treatment; n = 5 replicates), and cultured for 48 h in maturation medium containing 50 μM S-roscovitine to block spontaneous germinal vesicle breakdown. Cumulus-oocyte complexes were then washed and in vitro matured for an additional 24 h in maturation medium minus S-roscovitine. Additional JY-1 siRNA injected and uninjected cumulus-enclosed oocytes were cultured as described earlier but in the presence of 1 ng/mL of recombinant JY-1 protein (n = 10 oocytes/treatment; n = 5 replicates). Dose of recombinant JY-1 protein utilized was previously shown to reverse inhibitory effects of JY-1 siRNA injection on cumulus expansion. After in vitro maturation, cumulus cells were harvested and RNA isolated and subjected to reverse transcription. Real-time PCR analysis was then conducted to determine the effect of treatments on cumulus-cell mRNA abundance for HAS2, HAS3, PTX3, and TNFAIP6. Effects of JY-1 supplementation or depletion (siRNA injection) on cumulus-cell mRNA abundance for HAS2 and HAS3 (enzymes involved in hyaluronan synthesis) were not observed. However, abundance of mRNA for TNFAIP6 and PTX3 (molecules implicated in stabilization of the hyaluronan-rich extracellular matrix) was reduced (relative to uninjected and negative control siRNA groups) in response to JY-1 siRNA injection (P < 0.05) and effects of JY-1 siRNA were rescued by JY-1 protein treatment (P < 0.05). Effects of JY-1 protein supplementation on cumulus-cell TNFAIP6 and PTX3 mRNA in uninjected controls were not observed. Results support a requirement of the oocyte-specific protein JY-1 for regulation of expression of genes functionally linked to stabilization of the hyaluronan-rich extracellular matrix and cumulus expansion. This research was supported by USDA 2008-35203-19094 to G. W. Smith.

261 IDENTIFICATION OF OOCYTE- AND CUMULUS-DERIVED CO-REGULATED AND DIFFERENTIALLY REGULATED TRANSCRIPTS ASSOCIATED WITH BOVINE MEIOTIC MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 238
Author(s):  
O. V. Patel ◽  
A. Bettegowda ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Oocyte Maturation ◽  
Cdna Array ◽  
Ring Finger ◽  
Germinal Vesicle ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Leukotriene B4 ◽  
Mrna Abundance ◽  
Meiotic Maturation

Understanding the process of oocyte maturation is critical for efficient application of biotechnologies such as in vitro embryo production and nuclear transfer/cloning. Intercellular communication between the oocyte and the encompassing somatic (cumulus) cells is pivotal for successful growth of ovarian follicles and oocyte maturation. Therefore, we utilized global gene expression profiling to determine changes in the transcriptome of oocytes and their adjacent cumulus cells during meiotic maturation in vitro to identify both co-regulated and differentially regulated transcripts within the two cell compartments of the cumulus oocyte complex (COC). Germinal vesicle (GV) and in vitro matured metaphase II (MII) COC (n = 5 pools of 5 COC per group) were denuded and separated into oocytes and cumulus cells. RNA was extracted from the oocytes and cumulus cells and subjected separately to microarray analysis using a bovine cDNA array containing expressed sequence tags (ESTs) representing 15 500 unique genes. A combined total of 1045 genes displaying greater mRNA abundance in GV oocytes and associated cumulus cells compared to MII samples were detected (P < 0.05; false discovery rate (FDR) = 5%). A combined total of 711 genes displaying greater mRNA abundance in MII oocytes and enclosing cumulus cells compared to GV samples were detected (P < 0.05; FDR = 5%). Fourteen transcripts were identified that were co-regulated and of greater abundance in GV or MII oocytes and in their matching cumulus cells (P < 0.05; FDR = 5%). The co-regulated transcripts identified are implicated in metabolism (e.g. heme oxygenase-2, leukotriene B4 12-hydroxydehydrogenase), signal transduction (e.g. caveolin 1, ring finger protein 31), and cell growth (e.g. BTG family member 2, myosin regulatory light chain 2). In contrast, thirteen transcripts differentially regulated in the GV oocyte versus MII cumulus cells were identified (P < 0.05; FDR = 5%). Similarly, nine transcripts differentially regulated in the MII oocyte versus GV cumulus cells were identified (P < 0.05; FDR = 5%). Some of the identified differentially regulated transcripts encode for genes associated with the cytoskeleton (e.g. tropomyosin 1), apoptotic activity (e.g. death effector domain containing protein 2) and DNA replication (e.g. epsilon polymerase). The results provide novel insights into the identity of transcripts whose abundance is co-regulated or differentially regulated between the oocyte and cumulus cells during the transition of a COC from the GV to the MII stage. Characterization of the signaling pathways driving changes in transcript abundance for co-regulated and differentially regulated genes in oocytes versus associated cumulus cells may lead to a better understanding of regulation of meiotic maturation and potential cross-talk between germ cells and somatic cells during the oocyte maturation cascade. This work was supported by the Rackham Foundation and the MI Agriculture Experiment Station.

scholarly journals ATRX is a novel progesterone-regulated protein and biomarker of low developmental potential in mammalian oocytes

Reproduction ◽  
2017 ◽  
Vol 153 (5) ◽  
pp. 671-682 ◽  
Author(s):  
Lynne C O’Shea ◽  
Edward Daly ◽  
Carmel Hensey ◽  
Trudee Fair
Keyword(s):  
Protein Expression ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cell Number ◽  
Cell Nuclei ◽  
Blastocyst Development ◽  
Synthesis Inhibitor ◽  
Developmental Potential ◽  
Oocyte Competence

A multi-species meta-analysis of published transcriptomic data from models of oocyte competence identified the chromatin remodelling factor ATRX as a putative biomarker of oocyte competence. The objective of the current study was to test the hypothesis that ATRX protein expression by cumulus–oocyte complexes (COCs) reflects their intrinsic quality and developmental potential. In excess of 10,000 bovine COCs were utilised to test our hypothesis. COCs were in vitro matured (IVM) under conditions associated with reduced developmental potential: IVM in the presence or absence of (1) progesterone synthesis inhibitor (Trilostane); (2) nuclear progesterone receptor inhibitor (Aglepristone) or (3) an inducer of DNA damage (Staurosporine). ATRX protein expression and localisation were determined using immunocytochemistry and Western blot analysis. A proportion of COCs matured in the presence or absence of Trilostane was in vitro fertilised and cultured, and subsequent embryo development characteristics were analysed. In addition, ATRX expression was investigated in 40 human germinal vesicle-stage COCs. Our results showed that ATRX is expressed in human and bovine germinal vesicle oocytes and cumulus cells. In bovine, expression decreases after IVM. However, this decline is not observed in COCs matured under sub-optimal conditions. Blastocyst development rate and cell number are decreased, whereas the incidence of abnormal metaphase phase spindle and chromosome alignment are increased, after IVM in the presence of Trilostane (P < 0.05). In conclusion, localisation of ATRX to the cumulus cell nuclei and oocyte chromatin, after IVM, is associated with poor oocyte quality and low developmental potential. Furthermore, ATRX is dynamically regulated in response to progesterone signalling.

scholarly journals Comparing four laboratory three-parent techniques to construct human aged non-surrounded nucleolus germinal vesicle oocytes: A case-control study

2020 ◽  
Author(s):  
Sara Darbandi ◽  
Mahsa Darbandi ◽  
Ashok Agarwal ◽  
Hamid Reza Khorram khorshid ◽  
Mohammad Reza Sadeghi ◽  
...  
Keyword(s):  
Case Control Study ◽  
Germinal Vesicle ◽  
Oocyte Donation ◽  
Case Control ◽  
Assisted Reproductive Technique ◽  
Oocyte Competence ◽  
Reproductive Techniques ◽  
Developmental Capacity ◽  
Control Study

Background: The three-parent assisted reproductive technique may increase oocyte competence. Objective: In this case-control study, the suitability of germinal vesicle transfer (GVT), synchronous ooplasmic transfer (sOT), asynchronous ooplasmic transfer using cryopreserved MII oocyte (caOT), and asynchronous ooplasmic transfer using waste MII oocyte (waOT) for maturation of the human-aged non-surrounded nucleolus germinal vesicle-stage (NSN-GV) oocyte were investigated. Materials and Methods: NSN-GV oocytes were subjected to four methods: group A (GVT), B (sOT), C (caOT) D (waOT), and E (Control). The fusion rates, MI, MII, ICSI observations and cleavage at 2-cell, 4-cell, and 8-cell stages were compared in the groups. Results: In GVT, none of the oocytes fused. In sOT, all oocytes fused, 20 achieved the MI, 14 progressed to MII, 8 fertilized, 6 cleaved and 5, 4, and 3 achieved the 2- cells, 4-cells and 8-cells, respectively. In caOT, all oocytes fused and achieved the MI, 8 progressed to MII and fertilized, 6 cleaved and 6, 5, and 5 achieved the 2-cells, 4- cells, and 8-cells respectively. In waOT, all oocytes fused, 5 and 3 progressed to MI and MII, respectively, but only one fertilized, cleaved and reached a 4-cells stage. In group E, 6 and 2 oocytes progressed to MI and MII, respectively, and only one fertilized but arrested at the zygote stage. caOT had the highest survival rate when compared to sOT (p = 0.04), waOT (p = 0.002), and control (p = 0.001). Conclusion: The caOT method was beneficial over sOT, waOT, and GVT in supplementing the developmental capacity of human-aged NSN-GV oocytes. Key words: Assisted reproductive techniques, In vitro oocyte maturation techniques, Nuclear transfer techniques, Oocytes, Oocyte donation.

scholarly journals Actions of colony-stimulating factor 3 on the maturing oocyte and developing embryo in cattle

2020 ◽  
Vol 98 (4) ◽  
Author(s):  
Elizabeth A Jannaman ◽  
Yao Xiao ◽  
Peter J Hansen
Keyword(s):  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Bovine Oocyte ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Serum Free Medium ◽  
Rt Pcr ◽  
Oocyte Competence ◽  
Stimulating Factor ◽  
Therapeutic Administration

Abstract Colony-stimulating factor 3 (CSF3), also known as granulocyte colony-stimulating factor, is used to reduce the incidence of mastitis in cattle. Here, we tested whether recombinant bovine CSF3 at 1, 10, or 100 ng/mL acts on the bovine oocyte during maturation or on the developing embryo to modify competence for development and characteristics of the resultant blastocyst. For experiment 1, oocytes were matured with or without CSF3. The resultant embryos were cultured in a serum-free medium for 7.5 d. There was no effect of CSF3 on cleavage or on development to the blastocyst stage except that 100 ng/mL reduced the percent of putative zygotes and cleaved embryos becoming blastocysts. Expression of transcripts for 93 genes in blastocysts was evaluated by RT-PCR using the Fluidigm platform. Transcript abundance was affected by one or more concentrations of CSF3 for four genes only (CYP11A1, NOTCH2, RAC1, and YAP1). For experiment 2, cumulus-oocyte complexes (COC) were fertilized with either X- or Y-sorted semen. Putative zygotes were cultured in medium containing CSF3 treatments added at the beginning of culture. There was no effect of CSF3, sex, or the interaction on the percent of putative zygotes that cleaved or on the percent of putative zygotes or cleaved embryos becoming a blastocyst. For experiment 3, CSF3 was added from day 4 to 7.5 of development. There was no effect of CSF3 on development to the blastocyst stage. Transcript abundance of 10 genes was increased by 100 ng/mL CSF3, including markers of epiblast (NANOG, SOX2), hypoblast (ALPL, FN1, KDM2B, and PDGFRA), epiblast and hypoblast (HNF4A) and trophectoderm (TJAP1). Results are indicative that concentrations of CSF3 higher than typical after therapeutic administration can reduce oocyte competence and act on the embryo to affect characteristics of the blastocyst.

scholarly journals Predictive value of bovine follicular components as markers of oocyte developmental potential

2014 ◽  
Vol 26 (2) ◽  
pp. 337 ◽  
Author(s):  
Satoko Matoba ◽  
Katrin Bender ◽  
Alan G. Fahey ◽  
Solomon Mamo ◽  
Lorraine Brennan ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Cumulus Cell ◽  
Principal Component ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Potential ◽  
Thecal Cells ◽  
Total Fatty Acids

The follicle is a unique micro-environment within which the oocyte can develop and mature to a fertilisable gamete. The aim of this study was to investigate the ability of a panel of follicular parameters, including intrafollicular steroid and metabolomic profiles and theca, granulosa and cumulus cell candidate gene mRNA abundance, to predict the potential of bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles were dissected from abattoir ovaries, carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through in vitro maturation, fertilisation and culture. The mean (± s.e.m.) follicular concentrations of testosterone (62.8 ± 4.8 ng mL–1), progesterone (616.8 ± 31.9 ng mL–1) and oestradiol (14.4 ± 2.4 ng mL–1) were not different (P > 0.05) between oocytes that formed (competent) or failed to form (incompetent) blastocysts. Principal-component analysis of the quantified aqueous metabolites in follicular fluid showed differences between oocytes that formed blastocysts and oocytes that degenerated; l-alanine, glycine and l-glutamate were positively correlated and urea was negatively correlated with blastocyst formation. Follicular fluid associated with competent oocytes was significantly lower in palmitic acid (P = 0.023) and total fatty acids (P = 0.031) and significantly higher in linolenic acid (P = 0.036) than follicular fluid from incompetent oocytes. Significantly higher (P < 0.05) transcript abundance of LHCGR in granulosa cells, ESR1 and VCAN in thecal cells and TNFAIP6 in cumulus cells was associated with competent compared with incompetent oocytes.

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

scholarly journals Tricellular tight junction-associated angulins in the gill epithelium of rainbow trout

2018 ◽  
Vol 315 (2) ◽  
pp. R312-R322 ◽  
Author(s):  
Dennis Kolosov ◽  
Scott P. Kelly
Keyword(s):  
Tight Junction ◽  
Transcript Abundance ◽  
Barrier Properties ◽  
Mrna Abundance ◽  
Gill Epithelium ◽  
Molecular Physiology ◽  
Permeability Characteristics ◽  
Gill Cells ◽  
Associated Proteins ◽  
Trout Gill

Molecular physiology of the tricellular tight junction (tTJ)-associated proteins lipolysis-stimulated lipoprotein receptor ( lsr, = angulin-1) and an immunoglobulin-like domain-containing receptor ( ildr2, ≈angulin-3) was examined in model trout gill epithelia. Transcripts encoding lsr and ildr2 are broadly expressed in trout organs. A reduction in lsr and ildr2 mRNA abundance was observed during and after confluence in flask-cultured gill cells. In contrast, as high-resistance and low-permeability characteristics developed in a model gill epithelium cultured on permeable polyethylene terephthalate membrane inserts, lsr and ildr2 transcript abundance increased. However, as epithelia entered the developmental plateau phase, lsr abundance returned to initial values, while ildr2 transcript abundance remained elevated. When mitochondrion-rich cells were introduced to model preparations, lsr mRNA abundance was unaltered and ildr2 mRNA abundance significantly increased. Transcript abundance of ildr2 was not altered in association with corticosteroid-induced tightening of the gill epithelium, while lsr mRNA abundance decreased. Transcriptional knockdown of the tTJ protein tricelluin (Tric) reduced Tric abundance, increased gill epithelium permeability, and increased lsr without significantly altering ildr2 transcript abundance. Data suggest that angulins contribute to fish gill epithelium barrier properties but that Lsr and Ildr2 seem likely to play different roles. This is because ildr2 typically exhibited increased abundance in association with decreased model permeability, while lsr abundance changed in a manner that suggested a role in Tric recruitment to the tTJ.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

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