The effects of plasma-derived extracellular vesicles on cumulus expansion and oocyte maturation in mice

In vitro maturation (IVM) of oocytes in domestic animals is a widespread practice of research and commercial relevance. Gonadotropic hormones are typically supplemented to the IVM medium to stimulate resumption of meiosis, progression to metaphase II (MII), and oocyte developmental competence. The common use of pituitary-derived products presents 2 problems: contamination from other pituitary hormones and inconsistences from batch-to-batch variation. Recombinant hormones can help circumvent these issues and identify specific gonadotropin requirements for in vitro maturation. The aim of the present study was to determine the effect of supplementing recombinant bovine LH and/or FSH (AspenBio) to the maturation of ovine oocytes in terms of cumulus expansion and progression to the MII stage. Abattoir-derived sheep cumulus–oocyte complexes (COC) were obtained from 1- to 5-mm-diameter antral follicles by ovary slicing. Oocytes with a homogeneous cytoplasm surrounded by at least 3 layers of cumulus cells were selected and cultured in serum-free IVM medium (Cotterill et al. 2012 Reproduction 144, 195–207) at 38.5°C and 5% CO2. The COC obtained from 8 replicates were allocated into 4 experimental groups: (1) no hormones; (2) 1.5 μg mL–1 recombinant bovine LH (rbLH); (3) 1.5 μg mL–1 recombinant bovine FSH (rbFSH); and (4) rbLH and rbFSH. The expansion of cumulus cells was recorded in each group after 24 h of IVM and COC classified as (1) very poor or no cumulus expansion (grade 1); (2) limited cumulus expansion (grade 2); and (3) full cumulus expansion (grade 3). Nuclear maturation in the 4 treatments was evaluated by assessing progression to the MII stage via DNA staining with Hoechst 33342 and fluorescence imaging. The effect of treatment on the observed proportion of MII oocytes was evaluated using a mixed logit model including treatment and replicate as fixed and random effects, respectively. Culture in IVM medium in the absence of gonadotropins or in the presence of rbLH resulted in poor cumulus expansion (grade 1). The supplementation of IVM medium with rbFSH (with or without rbLH) yielded a high degree of cumulus expansion (grades 2–3). Likewise, addition of rbFSH enhanced progression of oocytes to the MII stage, whereas use of rbLH, although it had an effect on progression to MII, did not augment the effect of rbFSH (Table 1). These results indicate that rbFSH is necessary and sufficient to induce sheep oocyte maturation in a high proportion of oocytes. Table 1.Cumulus expansion and oocyte nuclear stage after IVM

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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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Interleukin-6 (IL-6) Induces Cumulus Expansion and Improves Oocyte Competence When Present During Mouse In Vitro Oocyte Maturation (IVM).

Biology of Reproduction ◽

10.1093/biolreprod/78.s1.311b ◽

2008 ◽

Vol 78 (Suppl_1) ◽

pp. 311-311 ◽

Cited By ~ 2

Author(s):

Daniel de Matos ◽

Cam Anh Tran ◽

David Kagan ◽

Selvaraj Nataraja ◽

Stephen Palmer

Keyword(s):

Oocyte Maturation ◽

Interleukin 6 ◽

Cumulus Expansion ◽

Oocyte Competence ◽

In Vitro Oocyte Maturation

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296 IMPACT OF CO-CULTURING CUMULUS-ENCLOSED PORCINE OOCYTES WITH DENUDED OOCYTES DURING IN VITRO MATURATION IN A DEFINED MEDIUM ON CUMULUS EXPANSION AND OOCYTE MATURATION

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab296 ◽

2015 ◽

Vol 27 (1) ◽

pp. 237

Author(s):

R. Appeltant ◽

T. Somfai ◽

M. Nakai ◽

S. Bodo ◽

D. Maes ◽

...

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Binary Logistic Regression ◽

Defined Medium ◽

Positive Influence ◽

Developmental Competence ◽

Cumulus Expansion ◽

Nuclear Maturation ◽

Morphological Criteria

Recent research has revealed that oocyte-secreted factors (OSF) affect cumulus expansion and play important roles during maturation and embryo development of mammalian oocytes. The use of denuded oocytes (DO) as supplements during in vitro maturation (IVM) in a nondefined medium improved developmental competence of cumulus-enclosed porcine oocytes (COC; Gomez et al. 2012 Zygote 20, 135–145). We investigated the effect of DO on cumulus expansion and nuclear maturation of COC in pigs during IVM using a defined medium. If the DO exert a positive influence on IVM, the defined medium can then be analysed for the presence of OSF. Immature COC were collected in the slaughterhouse from prepubertal gilts. To obtain DO, some COC were completely denuded by pipetting through a narrow-bore glass pipette. The COC used as a source for DO fulfilled the same morphological criteria as the COC used for IVM. The IVM medium was porcine oocyte medium (POM; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213) with hormone supplementations applied only during the first 20 h of the IVM period. The COC were fixed to the bottom of 35-mm plastic Petri dishes in 3 × 3 grids by Cell-Tak (BD Bioscience, Bedford, MA, USA) in 100-µL droplets POM covered by paraffin oil. Culture droplets (each including 1 COC grid) were supplemented with (DO+ group, n = 179) or without 16 DO (DO– group, n = 143). After 20 h of IVM, the medium was replaced with a preincubated hormone-free POM and oocytes were cultured for an additional 28 h. At 0, 20, and 48 h of IVM, images of each grid were taken at the same magnification. The size of each COC was measured as a 2-dimensional area in pixels by analysing images with ImageJ software. Relative cumulus expansion was calculated at 20 and 48 h of IVM on the basis of the initial COC size at 0 h, which was assigned as 1. At 48 h of IVM, the COC were denuded and examined for oocyte maturation by orcein staining. The experiment was replicated 5 times. Cumulus expansion ratios at 20 and 48 h of IVM were compared between the DO+ and DO– groups by ANOVA. Maturation rates were compared between the DO+ and DO– groups by binary logistic regression. No difference in cumulus expansion between DO– and DO+ could be observed at 20 h (1.83 ± 0.04 and 1.75 ± 0.03, respectively) and 48 h (1.41 ± 0.03 and 1.47 ± 0.02, respectively) of IVM. Nuclear maturation rates of COC in DO– and DO+ groups did not differ significantly (39.0 ± 5.4 and 32.9 ± 8.8%, respectively). In conclusion, addition of DO to the defined IVM medium did not affect the cumulus expansion and oocyte maturation of follicular porcine COC. Further research is needed to assess the effects of DO during IVM on subsequent fertilization. If DO prove to be beneficial for fertilization, the nature of the OSF will be investigated.This study was supported by FCWO of UGent and by FWO-Flanders (grant number FWO11/ASP/276).

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276 FIBROBLAST GROWTH FACTOR 2 PROMOTES BOVINE OOCYTE MEIOTIC MATURATION AND DEVELOPMENTAL COMPETENCE

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab276 ◽

2011 ◽

Vol 23 (1) ◽

pp. 236 ◽

Cited By ~ 2

Author(s):

K. Zhang ◽

P. J. Hansen ◽

A. D. Ealy

Keyword(s):

Growth Factor ◽

Fibroblast Growth Factor ◽

Embryo Development ◽

Oocyte Maturation ◽

Relative Abundance ◽

Cumulus Cells ◽

Fibroblast Growth Factor 2 ◽

Fibroblast Growth ◽

Cumulus Expansion

Oocyte competency is acquired during the course of folliculogenesis and is controlled by various endocrine and paracrine signals. One of these is fibroblast growth factor 2 (FGF2). Its expression is up-regulated in theca and granulosa cells during final maturation of a bovine follicle, and its cognate receptors are expressed in cumulus cells and oocytes throughout the final stages of oocyte maturation. The overall goal of this work was to describe how supplementing FGF2 during oocyte maturation in vitro affects oocyte maturation and subsequent embryo development. Cumulus–oocyte complexes (COC) were collected from bovine ovaries obtained from a local abattoir and cultured in defined TCM-based oocyte maturation medium. Depending on the study, oocytes were examined either during (6 h) or after (21 h) maturation or were fertilized in vitro and examined throughout in vitro embryo development in modified SOFF. Data were analysed with least-squares ANOVA using GLM of SAS. Adding 0.5 to 50 ng mL–1 of FGF2 did not affect cleavage rate or the percentage of 8 to 16 cell embryos at day 3 post-IVF. However, the blastocyst rate at day 7 was greater when oocytes were exposed to 0.5 ng mL–1 of FGF2 during maturation [30.0 ± 1.9% (17/109) v. 16.0 ± 2.6% (23/77) for nontreatment control; 4 replicates; P < 0.05], whereas higher doses of FGF2 did not affect blastocyst rates when compared with controls. Total cell number per blastocyst was not affected by FGF2 addition. The effects of FGF2 on oocyte maturation and cumulus expansion were examined to better understand how FGF2 improves oocyte competency. Adding 0.5 ng mL–1 of FGF2 did not affect the percentage of oocytes containing condensed chromatin after 6 h IVM or metaphase II (MII) rate after 21 h IVM, but 0.5 ng mL–1 of FGF2 treatment increased the cumulus expansion index score after 21 h IVM (P < 0.05). Interestingly, adding 5 ng mL–1 but not 50 ng mL–1 of FGF2 increased MII rate [61.5 ± 4.3% (53/120) for 5 ng mL–1 of FGF2 v. 46.9 ± 5.9% (64/104) for nontreatment controls; 7 replicates; P < 0.05], but neither FGF2 affected rates of chromatin condensation and cumulus expansion. Changes in the relative abundance for several putative oocyte competency markers and maternal genes (CTSB, Sprouty2, EGFR, FSHR, Has2, BMP15, GDF9, JY-1, Follistatin, H2A) were examined at 6 and 21 h after treatment with 0.5 ng mL–1 of FGF2 by quantitative RT-PCR. Relative amounts of 18S RNA was used as an internal control, and 2-ΔΔCT was used to quantify relative gene expression. The relative abundance of most of the transcripts examined was not affected by FGF2, but EGFR mRNA levels were greater after 6 h but not 21 h IVM in cumulus cells isolated from FGF2-supplemented COC (P = 0.057). In summary, improvements in blastocyst development were achieved by FGF2 treatment during oocyte maturation. The reason for the enhanced oocyte competency remains unclear, but it may occur in part because of improvements in cumulus expansion and production of EGFR. This project was supported by NRICGP number 2008-35203-19106 from the USDA-NIFA.

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Fibroblast growth factor 10 enhances bovine oocyte maturation and developmental competence in vitro

Reproduction ◽

10.1530/rep-10-0190 ◽

2010 ◽

Vol 140 (6) ◽

pp. 815-826 ◽

Cited By ~ 53

Author(s):

Kun Zhang ◽

Peter J Hansen ◽

Alan D Ealy

Keyword(s):

Growth Factor ◽

Fibroblast Growth Factor ◽

Oocyte Maturation ◽

Expression Profiles ◽

Cumulus Cells ◽

Fibroblast Growth ◽

Paracrine Factors ◽

Cumulus Expansion ◽

Fibroblast Growth Factor 10

The ability of oocytes to resume meiosis, become fertilized, and generate viable pregnancies is controlled during folliculogenesis by several endocrine and paracrine factors. The aim of this work is to determine whether fibroblast growth factor 10 (FGF10) is an oocyte competent factor. Transcripts for each of the four FGF receptor types (FGFR) were present in cumulus and oocytes after their extraction from the follicles. FGFR1 transcripts predominated in cumulus cells whereas FGFR2 was most abundant in oocytes. Exposing the cumulus–oocyte complexes to FGF10 duringin vitromaturation did not affect cleavage rates, but increases (P<0.05) in the percentage of embryos at the 8–16-cell stage on day 3 and at the blastocyst stage on day 7, which were evident in FGF10-supplemented oocytes. The progression of oocytes through meiosis and cumulus expansion was increased (P<0.05) by FGF10. The importance of the endogenous sources of FGFs was examined by adding anti-FGF10 IgG during oocyte maturation. Blocking endogenous FGF10 activity decreased (P<0.05) the percentage of oocytes developing into blastocysts and limited (P<0.05) cumulus expansion. Expression profiles of putative cumulus and oocyte competency markers were examined for their involvement in FGF10-mediated responses. FGF10 influenced the expression ofCTSBandSPRY2in cumulus cells andBMP15in oocytes. In summary, this work provides new insight into the importance of FGFRs and locally derived FGF10 during oocyte maturation in cattle. Its subsequent impact onin vitroembryo development implicates it as a noteworthy oocyte competent factor.

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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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