114. HUMAN CUMULUS - OOCYTE COMPLEXES SECRETE CUMULUS EXPANSION ENABLING FACTOR(S)

Interactions between the oocyte and its companion somatic cells are crucial to establish and maintain a highly specialized microenvironment required for oocyte viability. Specifically, cumulus cell expansion in the mouse is reliant on oocyte-secreted factors (OSF). Little is know about factors secreted by the human oocyte and how they may interact with cumulus cells. Therefore, the aim of this study was to establish whether human cumulus oocyte complexes (COC) produce OSF that induces cumulus expansion. COC of patients undergoing routine clinical IVF were cultured individually for 6h following oocyte retrieval. The human oocyte conditioned medium (HOCM) was collected. The bioactivity of OSF in the HOCM was assessed using an established assay of cumulus expansion of mouse oocytectomized complexes (OOX). Cumulus expansion was assessed blinded using the scoring system; 1 (no expansion) to 4 (maximally expanded) and gene expression was assessed by real time RT-PCR. Culture of OOX in control media with or without FSH did not induce expansion. Similarly, OOX cultured in HOCM without FSH did not expand. However, culture of OOX in HOCM with FSH significantly induced expansion (2.4±0.1 compared with control 1.1±0.04, P<0.05). Furthermore, this expansion was not different to OOX co-cultured with human (2.9±0.1) or mouse (2.6±0.1) denuded oocytes. Cumulus/OOX gene expression of hyaluronan synthase-2 and cyclooxygenase-2 was significantly up-regulated 4-5 fold when OOX were cultured in HOCM compared to control (P<0.05). Interestingly, different patients produced HOCM which resulted in different levels of expansion (range from 1.5-3.7). This study has established that human COC secrete paracrine factor(s) that enable cumulus expansion. This expansion was dependent on the presence of FSH. The identity of these factor(s) are currently unknown however it appears that COC from different patients produce differing levels of these cumulus expansion enabling factor(s).

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60 EFFECTS OF AY9944 A-7 ON MEIOTIC RESUMPTION OF PORCINE OOCYTES AND CUMULUS CELL EXPANSION

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab60 ◽

2016 ◽

Vol 28 (2) ◽

pp. 160

Author(s):

S. Lee ◽

C. Khoirinaya ◽

J.-X. Jin ◽

G. A. Kim ◽

B.-C. Lee

Keyword(s):

Gene Expression ◽

Oocyte Maturation ◽

Cell Expansion ◽

Cholesterol Biosynthesis ◽

Cumulus Cell ◽

Cumulus Cells ◽

Mitogen Activated Protein Kinase ◽

Control Group ◽

Cumulus Expansion

In vitro studies on mammalian oocytes have shown that follicular fluid-meiosis activating sterol (FF-MAS) can overcome the inhibitory effect of hypoxanthine (Hx) on the resumption of meiosis. FF-MAS, an intermediate in the cholesterol biosynthesis pathway, is converted to testis meiosis–activating sterol by a sterol Δ14-reductase. AY9944 A-7, an inhibitor of Δ14-reductase and Δ7-reductase, induces accumulation of FF-MAS by inhibiting its metabolism. The aim of this study was to evaluate the effects of AY9944 A-7 on meiotic resumption of porcine oocytes, cumulus cell expansion, and gene expression related to M-phase-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and oocyte maturation in oocytes and related to cumulus expansion in cumulus cells. In experiment 1, 1136 cumulus-oocyte complexes (COCs) were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in addition to a meiotic inhibitor (Hx, 4 mM) for 44 h. Oocytes treated with 10 and 20 μM AY9944 A-7 in the presence of Hx had significantly higher GVBD and M2 rates than the control group. However, 40 μM AY9944 A-7 significantly decreased GVBD and M2 rates and increased degeneration of oocytes compared with other groups. In experiment 2, 600 COCs were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in the absence of Hx for 44 h. Cumulus expansion of 40 μM AY9944 A-7 treated group was significantly decreased compared with other groups. In experiment 3, we evaluate the effects of AY9944 A-7 on gene expression, and the experiment was replicated four times. Data on gene expression were analysed using Student’s t-test. Oocytes treated with 10 μM AY9944 A-7 increased expression of genes involved in MPF (Cyclin B and Cdc2), MAPK (C-mos), and oocyte maturation (GDF9 and BMP15). Cumulus cells treated with 10 μM AY9944 A-7 decreased cumulus expansion-related genes (Has2, Tnfaip6, Ptgs2, and Ptx-3). In conclusion, our results suggest that although 10 μM AY9944 A-7 decreased cumulus expansion-related genes, there was no difference in cumulus expansion and it induced meiotic resumption of porcine oocytes with increased MPF, MAPK, and oocyte maturation-related genes. Further studies are needed to evaluate the effect of AY9944 A-7 on porcine embryo development. This study was supported by Ministry Of Trade, Industry & Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

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GDF-9 and BMP-15 mRNA Levels in Canine Cumulus Cells Related to Cumulus Expansion and the Maturation Process

Animals ◽

10.3390/ani10030462 ◽

2020 ◽

Vol 10 (3) ◽

pp. 462 ◽

Cited By ~ 3

Author(s):

George Ramirez ◽

Jaime Palomino ◽

Karla Aspee ◽

Monica De los Reyes

Keyword(s):

Gene Expression ◽

Estrous Cycle ◽

Expression Patterns ◽

Cumulus Cells ◽

Mrna Levels ◽

Cumulus Expansion ◽

Polymerase Chain ◽

Specific Regulation ◽

Reproductive Phases

The competence to undergo expansion is a characteristic of cumulus cells (CCs). The aim was to investigate the expression of GDF-9 and BMP-15 mRNA in canine cumulus cells in relation to cumulus expansion and meiotic development over the estrous cycle. CCs were recovered from nonmatured and in vitro-matured (IVM) dog cumulus oocyte complexes (COCs), which were obtained from antral follicles at different phases of the estrous cycle. Quantitative real-time polymerase chain reaction (q-PCR) was used to evaluate the relative abundance of GDF-9 and BMP-15 transcripts from the CCs with or without signs of expansion. The results were evaluated by ANOVA and logistic regression. The maturity of the oocyte and the expansion process affected the mRNA levels in CCs. There were differences (p < 0.05) in GDF-9 and BMP-15 gene expression in CCs isolated from nonmatured COCs when comparing the reproductive phases. Lower mRNA levels (p < 0.05) were observed in anestrus and proestrus in comparison to those in estrus and diestrus. In contrast, when comparing GDF-9 mRNA levels in IVM COCs, no differences were found among the phases of the estrous cycle in expanded and nonexpanded CCs (p < 0.05). However, the highest (p < 0.05) BMP-15 gene expression in CCs that did not undergo expansion was exhibited in anestrus and the lowest (p < 0.05) expression was observed in estrus in expanded CCs. Although the stage of the estrous cycle did not affect the second metaphase (MII )rates, the expanded CCs obtained at estrus coexisted with higher percentages of MII (p < 0.05). In conclusion, the differential expression patterns of GDF-9 and BMP-15 mRNA transcripts might be related to cumulus expansion and maturation processes, suggesting specific regulation and temporal changes in their expression.

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332 GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) ENHANCES CUMULUS CELLS EXPANSION OF IN VITRO-MATURED BOVINE CUMULUS - OOCYTE COMPLEXES IN A CHEMICALLY DEFINED MEDIUM

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab332 ◽

2010 ◽

Vol 22 (1) ◽

pp. 322

Author(s):

D. D. Bücher ◽

M. A. Castro ◽

M. E. Silva ◽

M. A. Berland ◽

I. I. Concha ◽

...

Keyword(s):

Cell Viability ◽

In Vitro Maturation ◽

Cumulus Cell ◽

Cumulus Cells ◽

Cell Number ◽

Control Group ◽

Cumulus Expansion ◽

Gm Csf ◽

Nuclear Stage

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine that stimulates proliferation, differentiation and function in different cells types. We have previously demonstrated (Bücher DD et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 146 abst.) that both subunits of GM-CSF receptor are expressed in granulosa cells from antral follicles in bovine ovaries. Also, we determined that the cytokine enhances glucose uptake through facilitative hexose transporters in granulosa cells in primary culture. The goals of the present study were to characterize the expression of GM-CSF receptor in cumulus cells and oocytes from bovine antral follicles and to determine its effects on in vitro-matured bovine COCs in a chemically defined medium. To determine the presence of a and |5 subunits of GM-CSF receptor, COCs were aspirated from follicles <8 mm in diameter, fixed, and submitted to immunocytochemistry. To study the effect of GM-CSF on in vitro maturation of oocytes, COCs (n =481) were cultured using serum-free medium (SOF) containing 0, 1, 10, and 100 ng mL-1 of human recombinant GM-CSF (R&D Systems, Inc., Minneapolis, MN, USA) for 22 h at 39°C, 5% CO2 in humidified air. Nuclear stage, cumulus expansion, cumulus cell number, and viability were analyzed after in vitro maturation. Cumulus expansion was assessed using the cumulus expansion index (CEI) (Fagbohun C and Down S 1990 Biol. Reprod. 42, 413-423). Nuclear stage was evaluated using aceto-orcein stain. To determine cumulus cell viability and number, COCs (n = 10-12 per group) were transferred into an Eppendorf tube and cumulus cells were removed by vortexing for 3 min, stained with trypan blue and counted with a hemocytometer. The study was conducted in 6 replicates. Data from cumulus expansion and cell number were analyzed by Kruskal-Wallis analysis. Data for nuclear stage and cell viability were analyzed by chi-square analysis and one way ANOVA, respectively. Both receptor subunits were present in cumulus cells and oocytes from COCs. COCs cultured in 10 and 100 ng mL-1 GM-CSF had CEI scores (0.8 and 1.22, respectively) greater (P < 0.01) than controls (0.2), but the proportion of COCs displaying second metaphase did not differ (P = 0.5) among treatment groups. GM-CSF at a concentration of 100 ng mL-1 increased (P < 0.01) cumulus cell viability by more than 20% compared to the control group. Similarly, GM-CSF at concentrations of 10 and 100 ng mL-1 increased (P < 0.05) cumulus cell number by more than 20% and 45%, respectively, from the control group. The use of a specific inhibitor of PI3 kinase (Ly294002; 10 and 100 μM) blocked the stimulatory effect of GM-CSF on cumulus expansion, cell viability, and cell number. In conclusion, the results of the study suggest a plausible modulator role of GM-CSF in the metabolism and function of cumulus cells and oocytes during in vitro maturation. Funding from Faculty of Veterinary Sciences, Universidad Austral de Chile, MECESUP AUS-0005, AUS-0601, and DID D-2006-24 and from Universidad Católica de Temuco, research grant 2007 DGI-CDA-04.

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192 The effect of invitro maturation on the PI3K-Akt pathway in bovine cumulus cells

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab192 ◽

2020 ◽

Vol 32 (2) ◽

pp. 224

Author(s):

G. Andrade ◽

M. Del Collado ◽

R. Nociti ◽

W. J. Da Silva ◽

F. Meirelles ◽

...

Keyword(s):

Gene Expression ◽

Rna Extraction ◽

Cumulus Cell ◽

Principal Component ◽

Cumulus Cells ◽

Culture Conditions ◽

Signalling Pathway ◽

Differentially Expressed ◽

Akt Pathway ◽

Apoptosis Protein

Oocyte quality is influenced by invitro oocyte maturation (IVM) because the culture conditions can alter the metabolism and gene expression of cumulus cells. Proper oocyte development requires fine regulation of signalling pathways involved with cell proliferation and survival, such as the PI3K-Akt (phosphatidylinositol-3-kinase/protein kinase B) signalling pathway. The aim of this study was to determine the effect of IVM on the expression of PI3K-Akt-related genes in bovine cumulus cells. To this aim, cumulus cells associated with immature oocytes, associated with oocytes invitro-matured for 24h, or associated with oocytes invivo-matured were compared in terms of gene expression. Pools (n=4) of cumulus cells from 20 cumulus-oocyte complexes (COCs) per group were submitted to total RNA extraction using the TRizol protocol, libraries were prepared with TruSeq stranded mRNA sample prep kit (Illumina Inc.), and the sequencing was performed in the HisEqn 2500 V4 (Illumina Inc.). After quality check with FastQC and filtering with Trim Galore, read alignment was performed with STAR and analysis of differential gene expression was done using DESEqn 2 in R considering the Benjamini-Hochberg method for adjusted P-values<0.10, and absolute value of log2-fold change >0.5. Principal component analysis was able to separate, with 94% cumulative variance (81% and 13% for PC1 and PC2, respectively), the cumulus cells groups, especially the immature from the matured counterparts. Gene ontology and enrichment analysis showed that the PI3K-Akt signalling pathway was affected in immature cumulus cells compared with cumulus cells from invitro- or invivo-matured oocytes, with 77 and 88 genes from PI3K-Akt pathway being differentially expressed, respectively. A total of 51 genes were common in invivo- and invitro-matured oocytes cumulus cells groups compared with immature group. Regarding cumulus cells after the maturation process, 48 genes from the PI3K-Akt pathway were differentially expressed; of those, 26 genes were upregulated in cumulus cells from invitro-matured oocytes and 22 genes were upregulated in cumulus cells from invivo-matured oocytes. Comparing the invitro and invivo cumulus cells, the main genes of the pathway (AKT, PI3K, and PTEN) were not differentially expressed. The differences in expression between invitro and invivo cumulus cells were in genes responsible for different cellular functions controlled by the PI3K-Akt pathway, such as apoptosis, protein synthesis, and DNA repair, among others, which, in general, were increased in cumulus cells from invitro-matured oocytes. These results demonstrated the effect of culture conditions on cumulus cell gene expression modulating important pathways involved in oocyte competence acquisition, such as PI3K-Akt signalling. Financial support was provided by FAPESP grants 2014/22887-0, 2018/01431-9, and 2018/13155-6.

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251.Regulation of mouse cumulus expansion by oocyte-secreted growth differentiation factor-9 (GDF-9)

Reproduction Fertility and Development ◽

10.1071/srb04abs251 ◽

2004 ◽

Vol 16 (9) ◽

pp. 251

Author(s):

R. A. Dragovic ◽

L. J. Ritter ◽

F. Amato ◽

S. J. Scott ◽

M. Cranfield ◽

...

Keyword(s):

Cumulus Cell ◽

Cumulus Expansion ◽

Maximum Expansion ◽

Paracrine Signalling ◽

Differentiation Factor ◽

Protein Receptor ◽

Morphogenetic Protein ◽

Growth Differentiation Factor 9 ◽

Hyaluronan Synthase 2 ◽

Candidate Molecule

Oocyte paracrine signalling is necessary for mouse cumulus cell expansion, an important preovulatory process. The oocyte-secreted factor growth differentiation factor-9 (GDF-9) signals through the bone morphogenetic protein receptor-II (BMPR-II) and is currently the primary candidate molecule for the cumulus expansion enabling factor (CEEF). The present study was conducted to determine whether in the mouse GDF-9 is the CEEF. Cumulus oocyte complexes (COC) were collected from eCG-primed mice and the oocyte was microsurgically removed to generate an oocytectomised complex (OOX). An established scoring system was used to measure FSH-induced cumulus expansion; 0 (no expansion) to +4 (maximum expansion). OOX complexes treated with FSH alone failed to expand (score: 0), whereas expansion was significantly (P�<�0.05) induced by either recombinant mouse GDF-9 (score; mean +/– SEM: 2.7 +/– 0.1), recombinant TGF-μ1 (score: 2.6 +/– 0.2) or co-culture with oocytes (score: 2.3 +/– 0.2). A GDF-9 neutralising antibody mAb-53, raised against hGDF-9, was effective in neutralising the response of OOX complexes to GDF-9 (score: 0.1 +/– 0.1), but had no significant effect on the expansion of OOX complexes co-cultured with oocytes (score: 2.3 +/– 0.2). Likewise, a TGF-μ antagonist neutralised (P�<�0.05) TGF-μ-induced, but not oocyte-induced, expansion of OOX complexes. A soluble portion of the BMPR-II ectodomain, a known GDF-9 antagonist, failed to neutralise oocyte-induced cumulus expansion (P�>�0.05) at the highest dose implying that BMPR-II is not a critical receptor involved in regulating cumulus expansion. Using real-time RT-PCR, hyaluronan synthase-2 (HAS2) mRNA expression by OOXs was upregulated 6- to 7-fold by oocytes and GDF-9. The GDF-9 neutralising antibody mAb-53, partially neutralised GDF-9-induced OOX HAS2 expression, but not oocyte-induced HAS2 expression. This study provides evidence that like TGF-μ1, GDF-9 can enable FSH-induced cumulus expansion, however more importantly demonstrates that neither GDF-9 nor TGF-μ1 alone account for the crucial oocyte-secreted factor regulating cumulus expansion in the mouse.

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Impact of oocyte-secreted factors on its developmental competence in buffalo

Zygote ◽

10.1017/s0967199417000156 ◽

2017 ◽

Vol 25 (3) ◽

pp. 313-320 ◽

Cited By ~ 2

Author(s):

Swati Gupta ◽

Sriti Pandey ◽

Mehtab S. Parmar ◽

Anjali Somal ◽

Avishek Paul ◽

...

Keyword(s):

Cumulus Cell ◽

Cumulus Cells ◽

Developmental Competence ◽

Marker Genes ◽

Enabling Factors ◽

Secreted Factors ◽

Morphogenetic Protein ◽

Growth Differentiation Factor 9 ◽

Follicle Size

SummaryOocyte-secreted factors (OSFs) play an important role in the acquisition of oocyte developmental competence through bidirectional cross-talk between oocyte and cumulus cells via gap junctions. Thus, the present study was designed to investigate the effect of two OSFs, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the developmental competence of buffalo oocytes derived from two different follicle sizes. Cumulus–oocyte complexes (COCs) from large follicles (LF, >6 mm) or small follicles (SF, <6 mm) were collected and matured in vitro either in the presence of GDF9 or BMP15, or both, or with the denuded oocytes (DOs) as a source of native OSFs. Cleavage and blastocyst rates were significantly (P < 0.05) higher in LF-derived than SF-derived oocytes. Cleavage and blastocyst rates were significantly higher (P < 0.05) in the DOs and the combination groups compared with the control, GDF9 alone and BMP15 alone groups, both in LF-derived and SF-derived oocytes, although the cleavage and blastocyst rates did not differ significantly (P > 0.05) between DOs and combination groups. Relative mRNA analysis revealed significantly higher (P > 0.05) expression of the cumulus cell marker genes EGFR, HAS2, and CD44 in LF-derived than SF-derived oocyte; the expression of these markers was significantly higher (P > 0.05) in DOs and combination groups, irrespective of the follicle size. These results suggested that LF-derived oocytes have a higher developmental competence than SF-derived oocytes and that supplementation of GDF9 and BMP15 modulates the developmental competence of buffalo oocytes by increasing the relative abundance of cumulus-enabling factors and thereby increasing cleavage and the quality of blastocyst production.

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Effects of nitric oxide synthase inhibitors on porcine oocyte meiotic maturation

Zygote ◽

10.1017/s0967199404002953 ◽

2005 ◽

Vol 13 (1) ◽

pp. 1-9 ◽

Cited By ~ 28

Author(s):

Yong Tao ◽

Huirong Xie ◽

Haiyan Hong ◽

Xiufen Chen ◽

Jie Jang ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Dna Fragmentation ◽

Cumulus Cell ◽

Cumulus Cells ◽

Meiotic Maturation ◽

Cumulus Expansion ◽

Wide Range ◽

Oxide Synthase ◽

Oocyte Meiotic Maturation

As an important biological messenger, nitric oxide (NO) exhibits a wide range of effects during physiological and pathophysiological processes, including mammalian oocyte meiotic maturation. The present study investigated whether NO derived from two nitric oxide synthase (NOS) isoforms, inducible NOS (iNOS) or endothelial NOS (eNOS), is involved in the meiotic maturation of porcine oocytes. Meanwhile, the cumulus cells' function in meiotic maturation and their interaction with oocyte development and degeneration were also investigated using cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs). Different inhibitors for NOS were supplemented to the medium. Cumulus expansion, cumulus cell DNA fragmentation and oocyte meiotic resumption were evaluated 48 h after incubation. Aminoguanidine (AG), a selective inhibitor for iNOS, suppressed cumulus expansion and inhibited CEOs to resume meiosis (p<0.05), but did not inhibit cumulus cell DNA fragmentation. Both Nω-nitro-L-arginine (L-NNA) and Nω-nitro-L-arginine methyl ester (L-NAME), inhibitors for both iNOS and eNOS, delayed cumulus expansion, inhibited cumulus cell DNA fragmentation and inhibited CEOs to resume meiosis. Such effects were not seen in DOs. These results indicate that iNOS-derived NO is necessary for cumulus expansion and meiotic maturation by mediating the function of the surrounding cumulus cells, and eNOS-derived NO is also involved in porcine meiotic maturation.

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132 Influence of oocyte retrieval methods and maturation media on invitro development of polar body extrusion in pig oocytes

Reproduction Fertility and Development ◽

10.1071/rdv33n2ab132 ◽

2021 ◽

Vol 33 (2) ◽

pp. 174

Author(s):

K. M. Honneysett ◽

M. L. Mphaphathi ◽

A. M. Maqhashu ◽

E. C. Webb

Keyword(s):

Follicular Fluid ◽

Cell Layer ◽

Polar Body ◽

Cumulus Cell ◽

Oocyte Retrieval ◽

Cumulus Cells ◽

Reproductive Technology ◽

Maturation Stage ◽

Significant Difference ◽

Polar Body Extrusion

Oocyte recovery is a reproductive technology that can be done by using two techniques, aspiration and slicing. Invitro maturation (IVM) is an additional reproductive technology used to advance an oocyte to a maturation stage; thereafter, it may be used during IVF. The objectives of the present study were (1) to compare two different oocyte retrieval methods (aspiration and slicing) from pig ovaries on oocyte quality and quantity, and (2) to compare three different IVM media [NCSU 37, TCM-199, and modified porcine follicular fluid (mpFF=porcine follicular fluid+FSH+LH] on oocytes’ polar body extrusion. During aspiration, an 18G needle was attached to a 10-mL syringe and all visible follicles were aspirated. During slicing, a surgical blade was used to slice the ovaries held in mDPBS. Follicular fluid collected from both methods was assessed for the presence of oocytes with the aid of a microscope. The collected oocytes were then categorized as Grade A, B, or C: Grade A=oocytes with compacted, multilayered cumulus cells and a homogeneous ooplasm; Grade B=oocytes with a compact cumulus cell layer with homogeneous ooplasm; Grade C=oocytes with a less compact cumulus cell layer with irregular ooplasm containing dark granules. The IVM media were placed in a four-well multidish; thereafter Grades A and B oocytes were allocated per treatment groups and matured for 44h. The treatment means were compared using the Fisher’s protected t-test least significant difference. The results showed significant differences between the grades of oocytes (P<0.05) with Grade A and B oocytes accounting for 50.8% of total oocytes (193.8) for aspiration and 58.7% of total oocytes (488.6) for slicing. The oocytes polar body extrusion was recorded as 25.3, 84.2, and 73.8% for NCSU 37 (P<0.05) and TCM-199 and mpFF respectively (P>0.05). In conclusion, the slicing method proved to be better than aspiration with regards to the retrieval of Grades A and B oocytes as well as the total number of oocytes retrieved. The TCM-199 and mpFF media had a higher percentage of oocytes with polar body extrusion than NCSU 37.

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Effect of FSH and LH hormones on oocyte maturation of buffalo and gene expression analysis of their receptors and Cx43 in maturing oocytes

Zygote ◽

10.1017/s096719940999030x ◽

2010 ◽

Vol 18 (3) ◽

pp. 231-234 ◽

Cited By ~ 9

Author(s):

Alok Pandey ◽

S.C. Gupta ◽

Neelam Gupta

Keyword(s):

Gene Expression ◽

Oocyte Maturation ◽

Connexin 43 ◽

Gene Expression Analysis ◽

Gene Expression Regulation ◽

Polar Body ◽

Cumulus Cells ◽

Mrna Abundance ◽

Cumulus Expansion ◽

Cx43 Mrna

SummaryFollicle stimulating hormone (FSH) and luteinizing hormone (LH) are commonly added to maturation media to improve cumulus expansion known to be a predictor of oocyte maturation. Therefore, effects of various concentrations of FSH (1000 ng/ml), LH (1000 ng/ml) and FSH + LH (1000 ng/ml each) in comparison with control (without FSH + LH) cultured oocytes were investigated. FSH and LH (1000 ng/ml each) induced significantly more cumulus expansion and polar body numbers, as compared with control and treatments of 1000 ng/ml FSH and 1000 ng/ml LH alone. Expression of FSH receptor (r), LHr and Cx43 mRNAs was determined by real-time PCR in cumulus–oocyte complexes (COCs) and denuded oocytes at different maturation times. Expression of all three genes was higher in COCs compared with denuded oocytes, confirming the importance of cumulus cells in oocyte maturation. FSHr and connexin 43 (Cx43) mRNA abundance in both COCs and denuded oocytes was highest at 0–6 h of maturation and decreased subsequently. However, LHr mRNA abundance increased from 6 h up to 24 h of maturation. The study concluded that FSH, LH receptors and Cx43 gene expression regulation is an index related to oocyte maturation.

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MicroRNA-378 regulates oocyte maturation via the suppression of aromatase in porcine cumulus cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00480.2014 ◽

2015 ◽

Vol 308 (6) ◽

pp. E525-E534 ◽

Cited By ~ 50

Author(s):

Bo Pan ◽

Derek Toms ◽

Wei Shen ◽

Julang Li

Keyword(s):

Oocyte Maturation ◽

Cumulus Cell ◽

Specific Effect ◽

Germinal Vesicle ◽

Cumulus Cells ◽

Blastocyst Stage ◽

Cumulus Expansion ◽

Vitro Fertilization ◽

And Function

We sought to investigate whether miR-378 plays a role in cumulus cells and whether the manipulation of miRNA levels in cumulus cells influences oocyte maturation in vitro. Cumulus-oocyte complexes (COCs) from ovarian follicles had significantly lower levels of precursor and mature miR-378 in cumulus cells surrounding metaphase II (MII) oocytes than cumulus cells surrounding germinal vesicle (GV) oocytes, suggesting a possible role of miR-378 during COC maturation. Overexpression of miR-378 in cumulus cells impaired expansion and decreased expression of genes associated with expansion ( HAS2, PTGS2) and oocyte maturation ( CX43, ADAMTS1, PGR). Cumulus cell expression of miR-378 also suppressed oocyte progression from the GV to MII stage (from 54 ± 2.7 to 31 ± 5.1%), accompanied by a decrease of growth differentiation factor 9 ( GDF9), bone morphogenetic protein 15 ( BMP15), zona pellucida 3 ( ZP3), and CX37 in the oocytes. Subsequent in vitro fertilization resulted in fewer oocytes from COCs overexpressing miR-378 reaching the blastocyst stage (7.3 ± 0.7 vs. 16.6 ± 0.5%). miR-378 knockdown led to increased cumulus expansion and oocyte progression to MII, confirming a specific effect of miR-378 in suppressing COC maturation. Aromatase (CYP19A1) expression in cumulus cells was also inhibited by miR-378, leading to a significant decrease in estradiol production. The addition of estradiol to IVM culture medium reversed the effect of miR-378 on cumulus expansion and oocyte meiotic progression, suggesting that decreased estradiol production via suppression of aromatase may be one of the mechanisms by which miR-378 regulates the maturation of COCs. Our data suggest that miR-378 alters gene expression and function in cumulus cells and influences oocyte maturation, possibly via oocyte-cumulus interaction and paracrine regulation.

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