scholarly journals 280 EFFECTS OF β-ENDORPHIN AND NALOXONE ON INTRACELLULAR CALCIUM LEVELS IN CUMULUS CELLS OF EQUINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 290
Author(s):  
T. De Santis ◽  
M.E. Dell'Aquila ◽  
F. Maritato ◽  
V. Casavola ◽  
P. Minoia
Keyword(s):  
Intracellular Calcium ◽  
Opioid Receptor ◽  
Oocyte Maturation ◽  
Single Cells ◽  
Cumulus Cells ◽  
High Concentration ◽  
Before And After ◽  
Different Seasons ◽  
Μ Opioid Receptor ◽  
Μ Receptor

Changes in intracellular calcium levels in the cumulus oocyte complex (COC) have a crucial role in oocyte maturation. In previous studies we demonstrated that the μ-opioid receptor is expressed in the bovine COC and participates in the signaling associated with oocyte maturation, by inducing an intracellular calcium increase (Dell'Aquila ME et al. 2002 Mol. Reprod. Dev. 63, 210–222). In this work we evaluated modifications of intracellular calcium induced by β-endorphin (β-end) or Naloxone (Nx) in cumulus cells of equine oocytes in relation to the time of the year and cumulus morphology at retrieval. Cumulus cells, isolated by mechanical treatment from compact (Cp, n = 120) or expanded (Exp, n = 120) COCs, recovered from the ovaries of slaughtered mares (follicles <20 mm in diameter) during anestrus, breeding season, spring transition, and autumnal transition, were cultured for 24 h and loaded with 5 μM Fura2-AM for microspectrofluorometric measurements of cytoplasmic ionized calcium (Dell'Aquila et al., 2002). The changes in β-end (30 μM)- or Nx (1mM and 10 μM)-induced calcium concentration were calculated in single cells (n = 194) and are expressed as Δ fluorescence (Fmaximal effect – Fbaseline) before and after 1-min perfusion with the drugs. The use of 1 mM Nx induced a significant increase of intracellular calcium levels in cumulus cells of oocytes recovered in all periods of the year in both Cp and Exp (P < 0.01). The addition of 10 μM Nx or 30 μM β-end significantly increased intracellular calcium only in cumulus cells from oocytes recovered in anestrus (P < 0.05). These results confirm previous observations, carried out on bovine oocytes, in which Nx behaved as a μ-receptor agonist when used at high concentration (Dell'Aquila et al. 2002). The effects of β-end and Nx may be explained in terms of a binding of the two subtances at the μ-receptor with consequent intracellular calcium increases due to extracellular calcium entry or depletion of intracellular stores. These findings could be related to differential espression and/or activation status of the μ-opioid receptor in COCs retrieved in different seasons. These substances can be used to modulate intracellular calcium in the equine COCs, and consequent effects on the stimulation/inhibition of oocyte maturation in this species need to be further investigated. This work was supported by Grant MIUR COFIN PRIN 2003.

Cyclopiazonic acid induces accelerated progress of meiosis in pig oocytes

Zygote ◽  
1997 ◽  
Vol 5 (3) ◽  
pp. 193-205 ◽  
Author(s):  
Jaroslav Petr ◽  
Jirří Rozinek ◽  
František Jílek
Keyword(s):  
Intracellular Calcium ◽  
Oocyte Maturation ◽  
Ryanodine Receptors ◽  
Cyclopiazonic Acid ◽  
Cumulus Cells ◽  
Ruthenium Red ◽  
Sensitive Period ◽  
Calcium Deposits ◽  
Transient Elevation

SummaryIn mammalian oocytes, calcium plays an important role in the regulation of meiotic maturation. In our study, we used the mycotoxin cyclopiazonic acid (CPA), an inhibitor of calcium-dependent ATPases, to mobilise intracellular calcium deposits during in vitro maturation of pig oocytes. The CPA treatment of maturing oocytes significantly accelerated the progress of their maturation. Oocytes entered the CPA-sensitive period after 21 h of in vitro culture. A very short (5 min) exposure to CPA (100 mM) is sufficient to accelerate maturation and it seems that accelerated maturation can be triggered by a transient elevation of intracellular calcium levels. The effect of CPA is not mediated through the cumulus cells, because maturation is accelerated by CPA treatment even in oocytes devoid of cumulus cells. Culture of oocytes with the calcium channel blocker verapamil (concentrations ranging from 0.01 to 0.04 mM) blocked the progress of oocyte maturation beyond the stage of metaphase I. This block can be overcome by the mobilisation of intracellular calcium deposits after CPA treatment (100 nM). The microinjection of heparin (20 pl, 50.1 mg/;ml), the inhibitor of inositol triphosphate receptors, before CPA treatment prevented the acceleration of oocyte maturation. This indicates that CPA mobilises the release of calcium deposits through inositol trisphosphate receptors. On the other hand, the microinjection of procaine (20 pl, 200 nM) or the microinjection of ruthenium red (20 pl, 50 mM), both inhibitors of ryanodine receptors, did not prevent accelerated maturation in CPA-treated oocytes. If present in pig oocytes, ryanodine receptors evidently play no part in the liberation of calcium from intracellular stores after CPA treatment.

μ-Opioid receptor mRNA expression in the rat CNS: comparison to μ-receptor binding

1994 ◽  
Vol 643 (1-2) ◽  
pp. 245-265 ◽  
Author(s):  
Alfred Mansour ◽  
Charles A. Fox ◽  
Robert C. Thompson ◽  
Huda Akil ◽  
Stanley J. Watson
Keyword(s):  
Mrna Expression ◽  
Opioid Receptor ◽  
Receptor Binding ◽  
Receptor Mrna ◽  
Μ Opioid Receptor ◽  
Μ Receptor ◽  
Rat Cns

scholarly journals Fatty Acid Synthesis and Oxidation in Cumulus Cells Support Oocyte Maturation in Bovine

2014 ◽  
Vol 28 (9) ◽  
pp. 1502-1521 ◽  
Author(s):  
Laura Sanchez-Lazo ◽  
Daphné Brisard ◽  
Sébastien Elis ◽  
Virginie Maillard ◽  
Rustem Uzbekov ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Oocyte Maturation ◽  
Binding Protein ◽  
Cumulus Cells ◽  
Meiotic Maturation ◽  
Germinal Vesicle Breakdown ◽  
Mitochondrial Fatty Acid ◽  
Before And After

Oocyte meiotic maturation requires energy from various substrates including glucose, amino acids, and lipids. Mitochondrial fatty acid (FA) β-oxidation (FAO) in the oocyte is required for meiotic maturation, which is accompanied by differential expression of numerous genes involved in FAs metabolism in surrounding cumulus cells (CCs) in vivo. The objective was to elucidate components involved in FAs metabolism in CCs during oocyte maturation. Twenty-seven genes related to lipogenesis, lipolysis, FA transport, and FAO were chosen from comparative transcriptome analysis of bovine CCs before and after maturation in vivo. Using real-time PCR, 22 were significantly upregulated at different times of in vitro maturation (IVM) in relation to oocyte meiosis progression from germinal vesicle breakdown to metaphase-II. Proteins FA synthase, acetyl-coenzyme-A carboxylase, carnitine palmitoyltransferase, perilipin 2, and FA binding protein 3 were detected by Western blot and immunolocalized to CCs and oocyte cytoplasm, with FA binding protein 3 concentrated around oocyte chromatin. By mass spectrometry, CCs lipid profiling was shown to be different before and after IVM. FAO inhibitors etomoxir and mildronate dose-dependently decreased the oocyte maturation rate in vitro. In terms of viability, cumulus enclosed oocytes were more sensitive to etomoxir than denuded oocytes. In CCs, etomoxir (150μM) led to downregulation of lipogenesis genes and upregulated lipolysis and FAO genes. Moreover, the number of lipid droplets decreased, whereas several lipid species were more abundant compared with nontreated CCs after IVM. In conclusion, FAs metabolism in CCs is important to maintain metabolic homeostasis and may influence meiosis progression and survival of enclosed oocytes.

LACTATE CONCENTRATIONS IN PRE-OVULATORY FOLLICLES OF PRO-OESTROUS RATS BEFORE AND AFTER ONSET OF OOCYTE MATURATION

1977 ◽  
Vol 86 (2) ◽  
pp. 380-383 ◽  
Author(s):  
G. H. Zeilmaker ◽  
C. M. P. M. Verhamme
Keyword(s):  
Energy Source ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Limiting Factor ◽  
Lh Surge ◽  
Before And After ◽  
High Concentrations ◽  
Ovulatory Follicles

ABSTRACT Lactate concentrations were determined in pre-ovulatory follicles of rats at pro-oestrus before and after onset of oocyte maturation. It appeared that high concentrations prevail before and after the time of the expected LH surge (27 mM). The level in serum was about 5 mm. Explanted oocytes obtained from pre-puberal rats and surrounded by cumulus cells, matured in the presence of 20 mm. lactate as sole exogenous energy source. It is argued that oxygen may be the limiting factor suppressing oocyte maturation in vivo.

scholarly journals Immunohistochemical localization of inhibin/activin alpha, betaA and betaB subunits and follistatin in bovine oocytes during in vitro maturation and fertilization

Reproduction ◽  
2003 ◽  
pp. 33-42 ◽  
Author(s):  
CC Silva ◽  
NP Groome ◽  
PG Knight
Keyword(s):  
Oocyte Maturation ◽  
Zona Pellucida ◽  
Cumulus Cells ◽  
Immunohistochemical Localization ◽  
Intense Staining ◽  
Immature Oocytes ◽  
Before And After ◽  
Alpha Beta ◽  
Quantitative Changes

The aim of this study was to evaluate the distribution of inhibin/activin alpha, beta(A) and beta(B) subunits and follistatin in immature oocytes and in matured oocytes before and after IVF. Denuded oocytes were submitted to a whole-mount immunofluorescence procedure. Specimens were imaged and fluorescent intensities quantified by scanning laser confocal microscopy. Immunoreactivity for inhibin alpha subunit (both alpha(C) and pro-alpha regions), abundant in the ooplasm of immature oocytes, decreased after maturation (a 68% and 88% decrease, respectively; P < 0.001), but increased after IVF by 2- and 5.7-fold, respectively (P < 0.01). Intense staining for beta(A) was detected in immature oocytes (predominantly in the outer ooplasm and zona pellucida) but after maturation and fertilization it was localized mainly in the zona pellucida, perivitelline space and oolemma. Immunoreactivity for beta(A) in the ooplasm decreased by 58% after maturation (P < 0.001) but increased again by 75% after fertilization (P < 0.01). Immunoreactivity for beta(B) was localized mainly in the zona pellucida and did not change after maturation. However, immunoreactivity for beta(B) was not detected in the zona pellucida after fertilization, but remained unchanged in unfertilized oocytes. Immunoreactivity for follistatin was detected in the ooplasm and zona pellucida of immature oocytes but decreased progressively in the ooplasm after maturation (a 63% decrease; P < 0.001) and did not change after IVF. Examination of partially denuded cumulus-oocyte complexes confirmed abundant expression of alpha(C), pro-alpha, beta(A) and follistatin immunoreactivity in cumulus cells, whereas beta(B) subunit staining was weak or absent in cumulus cells, but intense in the zona pellucida. In conclusion, the present study shows that qualitative and quantitative changes in the distribution of inhibin/activin subunits and follistatin accompany oocyte maturation and fertilization. The possibility, indicated by these observations, that activin A and activin B may play distinct roles in bovine oocyte maturation and fertilization warrants further study.

scholarly journals Genes of cellular components of morphogenesis in porcine oocytes before and after IVM

Reproduction ◽  
2017 ◽  
Vol 154 (4) ◽  
pp. 535-545 ◽  
Author(s):  
Joanna Budna ◽  
Artur Bryja ◽  
Piotr Celichowski ◽  
Rotem Kahan ◽  
Wiesława Kranc ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Cellular Migration ◽  
Pcr Analysis ◽  
Cresyl Blue ◽  
Before And After ◽  
Developmental Capacity ◽  
Cellular Modifications ◽  
Cellular Components

Proper oocyte maturation in mammals produces an oocyte capable of monospermic fertilization and embryo preimplantation. The cumulus-oocyte complexes (COCs), surrounding an oocyte, play a significant role in oocyte maturation. During this process, when the COCs undergo cumulus expansion wherein tightly compact cumulus cells (CCs) form a dispersed structure, permanent biochemical and molecular modifications occur in the maturing oocytes, indicating that the gene expression between immature and mature oocytes differs significantly. This study focuses on the genes responsible for the cellular components of morphogenesis within the developing oocyte. Brilliant cresyl blue (BCB) was used to determine the developmental capability of porcine oocytes. The immature oocytes (GV stage) were compared with matured oocytes (MII stage), using microarray and qRT-PCR analysis to track changes in the genetic expression profile of transcriptome genes. The data showed substantial upregulation of genes influencing oocyte’s morphology, cellular migration and adhesion, intracellular communication, as well as plasticity of nervous system. Conversely, downregulation involved genes related to microtubule reorganization, regulation of adhesion, proliferation, migration and cell differentiation processes in oocytes. This suggests that most genes recruited in morphogenesis in porcine oocytein vitro,may have cellular maturational capability, since they have a higher level of expression before the oocyte’s matured form. It shows the process of oocyte maturation and developmental capacity is orchestrated by significant cellular modifications during morphogenesis.

The regulatory role of miR-20a in bovine cumulus cells and its contribution to oocyte maturation

Zygote ◽  
2021 ◽  
pp. 1-10
Author(s):  
Eryk Andreas ◽  
Hari Om Pandey ◽  
Michael Hoelker ◽  
Dessie Salilew-Wondim ◽  
Samuel Gebremedhn ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Maturation Process ◽  
Progesterone Synthesis ◽  
Before And After ◽  
Maturation Rate ◽  
Transcriptional Gene Regulation

Summary Dynamic changes in microRNAs in oocyte and cumulus cells before and after maturation may explain the spatiotemporal post-transcriptional gene regulation within bovine follicular cells during the oocyte maturation process. miR-20a has been previously shown to regulate proliferation and differentiation as well as progesterone levels in cultured bovine granulosa cells. In the present study, we aimed to demonstrate the function of miR-20a during the bovine oocyte maturation process. Maturation of cumulus–oocyte complexes (COCs) was performed at 39°C in an humidified atmosphere with 5% CO2 in air. The expression of miR-20a was investigated in the cumulus cells and oocytes at 22 h post culture. The functional role of miR-20a was examined by modulating the expression of miR-20a in COCs during in vitro maturation (IVM). We found that the miR-20a expression was increased in cumulus cells but decreased in oocytes after IVM. Overexpression of miR-20a increased the oocyte maturation rate. Even though not statistically significant, miR-20a overexpression during IVM increased progesterone levels in the spent medium. This was further supported by the expression of STAR and CYP11A1 genes in cumulus cells. The phenotypes observed due to overexpression of miR-20a were validated by BMP15 supplementation during IVM and subsequent transfection of BMP15-treated COCs using miR-20a mimic or BMPR2 siRNA. We found that miR-20a mimic or BMPR2 siRNA transfection rescued BMP15-reduced oocyte maturation and progesterone levels. We concluded that miR-20a regulates oocyte maturation by increasing cumulus cell progesterone synthesis by simultaneous suppression of BMPR2 expression.

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