Influence of epidermal growth factor supplementation during in vitro maturation on nuclear status and gene expression of canine oocytes
ABSTRACT This study evaluated the effect of epidermal growth factor (EGF) supplementation during in vitro maturation on the meiotic status and the expression of EGF receptor (EGFr), luteinizing hormone receptor (LHr) and gap junction protein α 5 (GJA5) in canine cumulus-oocyte-complexes (COCs). COCs of ≥110 μm diameter, exhibiting dark pigmentation and completely surrounded by three or more layers of cumulus cells collected from anestrus stage ovaries in natural cycle were matured in TCM-199 supplemented with 10% fetal bovine serum, 0.57 mM cysteine, 10 μg/ml LH and FSH, and different concentrations of EGF (0, 10 and 30 ng/ml). Oocytes cultured for 72 h were fixed to assess the nuclear maturation. Expression of EGFr, LHr and GAJ5 was assessed by immunocytochemistry and real-time PCR. Proportion of metaphase II status of oocytes cultured in in vitro maturation (IVM) medium supplemented with 10 ng/ml EGF for 72 h was significantly (P<0.05) higher than 0 and 30 ng/ml EGF supplemented IVM medium (9.8% vs. 6.5% and 5.2%). In both cumulus cells and oocytes, EGFr protein was undetectable, LHr protein level of expression was low and a strong expression of GJA5 protein was observed. The relative abundance (RA) of EGFr transcript revealed low levels and the LHr expression decreased steadily with addition of EGF. However it did not vary among different concentrations of EGF supplementation. The RA of GJA5 transcript exhibited lower level at 10 ng/ml EGF supplementation. In conclusion, the supplementation of 10 ng/ml EGF in IVM media exerted a positive influence on the progression of maturation to MII phase and the expression level of GJA5 at 72 h, but did not demonstrate any stimulatory role on the expression of EGFr and LHr during the maturation of the canine IVM oocytes.
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- "SDF-1a is believed to promote T-cell recruitment and granulosa cell survival, thereby creating an environment favourable to oocyte maturation; thus, in this respect, SDF-1a may have potential as a candidate biomarker of oocyte maturation and fertilisation (Kryczek et al., 2005). As outlined earlier, EGF and EGF-like peptides have been implicated in oocyte maturation in vivo, as reflected by the fact that these are frequently used as culture medium supplements to promote in vitro maturation of germinal vesicle-stage oocytes in various mammalian species (Park et al., 1997; Goud et al., 1998; Merriman et al., 1998; Oyamada et al., 2004; Merlo et al., 2005; Bolamba et al., 2006; Lee et al., 2011; Song et al., 2010; "
ABSTRACT: Cytokines are key regulators of ovarian physiology, particularly in relation to folliculogenesis and ovulation, where they contribute to creating an environment supporting follicle selection and growth. Their manifold functions include regulating cellular proliferation/differentiation, follicular survival/atresia, and oocyte maturation. Several cytokines, such as TGFβ-superfamily members, are involved at all stages of folliculogenesis while the production of others is stage-dependent. This review draws upon evidence from both human and animal models to highlight the species-specific roles at each milestone of follicular developmental. Given these pivotal roles and their ease of detection in follicular fluid, cytokines have been considered as attractive biomarkers of oocyte maturational status and of successful assisted reproductive outcome. Despite this, our understanding of cytokines and their interactions remains incomplete, and is still frequently limited to overly simplistic descriptions of their interrelationships. Given our increased appreciation of cytokine activity in complex and highly regulated networks, we put forward the case for using Bayesian modelling approaches to describe their hierarchical relationships in order to predict causal physiological interactions in vivo. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.Molecular Reproduction and Development 04/2014; 81(4). DOI:10.1002/mrd.22285 · 2.68 Impact Factor
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- "Indeed, the metabolism of lipids represents the main energy source for protein synthesis during oocyte nuclear maturation and early embryo development [34, 35]. Simultaneously, adequate communication between oocyte and CCs and appropriate assembly and organization of the CC matrix are required for both oocyte maturation and competence [36–38]. Most of the genes, identified in the present investigation as differentially expressed in CCs treated with HP-hMG and rFSH, were reported for the first time, except for TNFAIP6 and GJA5 (connexin 40) which have been previously identified as potential markers of oocyte competence in CCs from bovine preovulatory follicles  and biomarker of oocyte maturation in canine cumulus-oocyte complexes matured in vitro, respectively . "
ABSTRACT: In in vitro fertilization cycles, both HP-hMG and rFSH gonadotropin treatments are widely used to control human follicle development. The objectives of this study are (i) to characterize and compare gene expression profiles in cumulus cells (CCs) of periovulatory follicles obtained from patients stimulated with HP-hMG or rFSH in a GnRH antagonist cycle and (ii) to examine their relationship with in vitro embryo development, using Human Genome U133 Plus 2.0 microarrays. Genes that were upregulated in HP-hMG-treated CCs are involved in lipid metabolism (GM2A) and cell-to-cell interactions (GJA5). Conversely, genes upregulated in rFSH-treated CCs are implicated in cell assembly and organization (COL1A1 and COL3A1). Interestingly, some genes specific to each gonadotropin treatment (NPY1R and GM2A for HP-hMG; GREM1 and OSBPL6 for rFSH) were associated with day 3 embryo quality and blastocyst grade at day 5, while others (STC2 and PTX3) were related to in vitro embryo quality in both gonadotropin treatments. These genes may prove valuable as biomarkers of in vitro embryo quality.09/2013; 2013:354582. DOI:10.1155/2013/354582
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- "These impressive results first described the strong effect of growth factors on the meiotic competence of canine oocytes (49% of total oocytes resume meiosis) (Alhaider and Watson 2009). The effect of EGF on meiotic competence of canine oocytes as well as the expression of maturation related genes, such as: EGF receptor (EGFr), luteinizing hormone receptor (LHr) and gap junction protein h 5 (GJA5) was investigated by Song et al. (2010) "
ABSTRACT: The maturation of oocytes is one of the most important steps determining their developmental competence. Due to the low percentage of oocytes of bitches that reach the MII stage, searching for reagents that may stimulate the growth and maturation of oocytes is still present in this species of mammals. The most important media supplements include gonadotropins (LH, FSH, hCG), growth factors (IGF, TGF, EGF, FGF), progesterone and follicular fluid. It is suggested that the supplement of EGF, and/or follicular cells may have an important influence on the percentage of cells that reach the MII stage. Despite plenty of research based on the improvement of bitch oocytes in vitro culture, the results obtained are still unsatisfactory. Moreover, in the long stages of canine oocytes maturation many molecular and morphological modifications (including changes in mitochondria structure and configuration in the cytoplasm) are involved. In this article, the influence of selected media supplements on the efficiency of bitch oocytes in vitro maturation was described. The molecular and morphological modifications during canine oocytes maturation were also considered in the text.Polish journal of veterinary sciences 01/2012; 15(1):199-205. DOI:10.2478/v10181-011-0134-4 · 0.71 Impact Factor