Confocal fluorescence assessment of bioenergy/redox status of dromedary camel (Camelus dromedarius) oocytes before and after in vitro maturation

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Abstract

Background

Reproductive biotechnologies in dromedary camel ( Camelus dromedarius) are less developed than in other livestock species. The in vitro maturation (IVM) technology is a fundamental step for in vitro embryo production (IVP), and its optimization could represent a way to increase the success rate of IVP. The aim of the present study was to investigate the bioenergy/oxidative status of dromedary camel oocytes before and after IVM by confocal microscopy 3D imaging.

Methods

Oocytes were retrieved by slicing ovaries collected at local slaughterhouses. Recovered oocytes were examined before and after IVM culture for nuclear chromatin configuration and bioenergy/oxidative status, expressed as mitochondria (mt) distribution and activity, intracellular Reactive Oxygen Species (ROS) levels and distribution and mt/ROS colocalization.

Results

The mean recovery rate was 6 oocytes/ovary. After IVM, 61% of oocytes resumed meiosis and 36% reached the Metaphase II stage (MII). Oocyte bioenergy/redox confocal characterization revealed changes upon meiosis progression. Immature oocytes at the germinal vesicle (GV) stage were characterised by prevailing homogeneous mt distribution in small aggregates while MI and MII oocytes showed significantly higher rates of pericortical mt distribution organized in tubular networks (P < 0.05). Increased mt activity in MI (P < 0.001) and MII (P < 0.01) oocytes compared to GV stage oocytes was also observed. At any meiotic stage, homogeneous distribution of intracellular ROS was observed. Intracellular ROS levels also increased in MI (P < 0.01) and MII (P < 0.05) oocytes compared to GV stage oocytes. The mt/ROS colocalization signal increased in MI oocytes (P < 0.05).

Conclusions

This study provides indications that qualitative and quantitative indicators of bioenergy and oxidative status in dromedary camel oocytes are modified in relation with oocyte meiotic stage. These data may increase the knowledge of camel oocyte physiology, in order to enhance the efficiency of IVP procedures.

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Most cited references 30

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Mitochondrial function in the human oocyte and embryo and their role in developmental competence.

Jonathan van Eyll, JONATHAN VAN BLERKOM (2011)

The role of mitochondria as a nexus of developmental regulation in mammalian oogenesis and early embryogenesis is emerging from basic research in model species and from clinical studies in infertility treatments that require in vitro fertilization and embryo culture. Here, mitochondrial bioenergetic activities and roles in calcium homeostasis, regulation of cytoplasmic redox state, and signal transduction are discussed with respect to outcome in general, and as possible etiologies of chromosomal defects, maturation and fertilization failure in human oocytes, and as causative factors in early human embryo demise. At present, the ability of mitochondria to balance ATP supply and demand is considered the most critical factor with respect to fertilization competence for the oocyte and developmental competence for the embryo. mtDNA copy number, the timing of mtDNA replication during oocyte maturation, and the numerical size of the mitochondrial complement in the oocyte are evaluated with respect to their relative contribution to the establishment of developmental competence. Rather than net cytoplasmic bioenergetic capacity, the notion of functional compartmentalization of mitochondria is presented as a means by which ATP may be differentially supplied and localized within the cytoplasm by virtue of stage-specific changes in mitochondrial density and potential (ΔΨm). Abnormal patterns of calcium release and sequestration detected at fertilization in the human appear to have coincident effects on levels of mitochondrial ATP generation. These aberrations are not uncommon in oocytes obtained after ovarian hyperstimulation for in vitro fertilization. The possibility that defects in mitochondrial calcium regulation or bioenergetic homeostasis could have negative downstream development consequences, including imprinting disorders, is discussed in the context of signaling pathways and cytoplasmic redox state. Copyright © 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

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Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos.

William K. Oh, Peter K. Hwang, Ki Kim … (1998)

In human in-vitro fertilization (IVF)-embryo transfer, the in-vitro culture environment differs from in-vivo conditions in that the oxygen concentration is higher, and in such conditions the mouse embryos show a higher concentration of reactive oxygen species (ROS) in simple culture media. ROS are believed to cause damage to cell membranes and DNA fragmentation in somatic cells. This study was conducted to ascertain the level of H2O2 concentration within embryos and the morphological features of cell damage induced by H2O2. A total of 62 human oocytes and embryos (31 fragmented, 15 non-fragmented embryos, 16 unfertilized oocytes) was obtained from the IVF-embryo transfer programme. The relative intensity of H2O2 concentrations within embryos was measured using 2',7'-dichlorodihydrofluorescein diacetate by Quanti cell 500 fluorescence imaging and DNA fragmentation was observed with transmission electron microscopy and an in-situ apoptosis detection kit. The H2O2 concentrations were significantly higher in fragmented embryos (72.21 +/- 9.62, mean +/- SEM) compared to non-fragmented embryos (31.30 +/- 3.50, P < 0.05) and unfertilized oocytes (30.75 +/- 2.67, P < 0.05). Apoptosis was observed only in fragmented embryos, and was absent in non-fragmented embryos. Electron microscopic findings confirmed apoptotic bodies and cytoplasmic condensation in the fragmented blastomeres. We conclude that there is a direct relationship between increased H2O2 concentration and apoptosis, and that further studies should be undertaken to confirm these findings.

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Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.

Amit Lugade, R P Haugland, Z. Zhang … (1996)

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.

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Author and article information

Journal

Journal ID (nlm-ta): Reprod Biol Endocrinol

Journal ID (iso-abbrev): Reprod. Biol. Endocrinol

Title: Reproductive Biology and Endocrinology : RB&E

Publisher: BioMed Central

ISSN (Electronic): 1477-7827

Publication date Collection: 2014

Publication date (Electronic): 18 February 2014

Volume: 12

Page: 16

Affiliations

[1 ]Section of Clinics and Animal Productions, Department of Emergency and Organ Transplantation (DETO), University of Bari Aldo Moro, Str Prov Casamassima, km 3, Bari Valenzano 70010, Italy

[2 ]National Research Council (CNR), ISPAAM, c/o Istituto tecnico agrario statale “E. De Cellis”, Via Argine 1085, Naples Ponticelli80147, Italy

[3 ]A.I. Lab., Maryout Research Station (MRS), Desert Research Center (DRC), Al-Nasryia St., Al Amryia, Alexandria, Egypt

[4 ]Cairo University Research Park (CURP), Faculty of Agriculture, Cairo University, Giza 12613, Egypt

[5 ]SupAgro, Systèmes d’Elevage Méditerranéens et Tropicaux, UMR_SELMET, Montpellier 34598, France

[6 ]Department of Animal Production, Faculty of Agriculture, Cairo University, Giza, Egypt

Article

Publisher ID: 1477-7827-12-16

DOI: 10.1186/1477-7827-12-16

PMC ID: 3931272

PubMed ID: 24548378

SO-VID: 80b80009-0362-4395-9387-8ff9175e27f4

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Confocal fluorescence assessment of bioenergy/redox status of dromedary camel ( Camelus dromedarius) oocytes before and after in vitro maturation

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Most cited references 30

Mitochondrial function in the human oocyte and embryo and their role in developmental competence.

Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos.

Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.

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