1 Centre for Reproductive Biology, Clinica Villa Del Sole, and 2 Dipartimento Clinica di Emergenza Ginecologica e Ostetrica e Medicina della Riproduzione, Azienda Universitaria Policlinico, Università degli Studi `Federico II', Naples, Italy
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Abstract |
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Key words: cell cycle/cellular metabolism/IVF/mitochondria/oocytes
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Introduction |
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The rate of cellular respiration depends on two major factors. The first factor is the efficiency of conversion in the oocyte cytoplasm of metabolic precursors such as glucose to pyruvate (Alberts et al., 1983). The second factor is the efficiency of the mitochondrial matrix in the conversion of pyruvate to ATP, the latter being required for diverse cellular processes in developing embryos, including cell division, DNA replication and genomic activation. The metabolism of mammalian oocytes is specialized in that pyruvate is the major utilizable energy source (Biggers et al., 1967
; Bavister, 1995
; Gardner, 1998
). The addition of pyruvate into in-vitro human embryo culture medium therefore reduces the significance of cytoplasmic metabolic enzymes in the supply of metabolic precursors during preimplantation embryo development. This leaves the efficiency of mitochondrial respiration as the major factor involved in the production of ATP during this stage.
Human preimplantation embryos are characterized by diverse rates of development and potential for implantation. Although genetic and paternal factors may contribute to these differences (Janny and Ménézo, 1994; Ménézo and Dale, 1995
; Warner et al., 1998
), one of the major factors that influences the development and implantation rate of human embryos is maternal age (Cummins et al., 1994
; Janny and Ménézo, 1996
; Keefe, 1997
). These data suggest that embryos from older patients have accumulated negative factors lowering the implantation rate (Cummins et al., 1994
). The factors may include errors in the efficiency of ATP production through mitochondrial respiration (Van Blerkom et al., 1995
). This hypothesis is supported by the fact that the transfer of cytoplasm between mouse oocytes increased ATP production in recipients (Van Blerkom et al., 1998
) and may improve the development and implantation rate in human embryos produced after IVF (Cohen et al., 1998
).
JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) is a fluorescent dye that accumulates in mitochondria and reports the membrane potential across the matrix membrane (Reers et al., 1991, 1995
). Because this value is highly related to mitochondrial respiratory rate, JC-1 can be used as an indicator of mitochondrial activity. In this study, JC-1 and ratiometric confocal microscopy were used to monitor the activity of mitochondria in human oocytes and preimplantation embryos. The results suggest that mitochondrial activity is highly correlated with maternal age, and that this is the cause of the slower development rate of embryos observed in these patients.
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Materials and methods |
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Fluorescence labelling and confocal microscopy
The potential-sensitive fluorescence dye JC-1 (Molecular Probes, Eugene, Oregon, USA) was used to measure the activity of mitochondria (Reers et al., 1991, 1995
). The dye was dissolved to a stock concentration of 0.5 mmol/l in dimethylsulphoxide and diluted into pre-equilibrated IVF medium (Medicult), using a vortex to aid the dissolution of the dye, as required. Under these conditions, the dye was found to remain dissolved for up to 1 h, permitting accurate loading of oocytes. An Olympus Fluoview (Olympus, Segrate, Italy) confocal microscope, based on an Olympus IX-70 inverted microscope, was used for all experiments. A Kr/Ar laser was used to produce the excitation laser line at 488 nm, and emission wavelengths were separated by a 530 nm dichroic mirror followed by analysis in a photomultiplier after further filtering through a 515530 nm bandpass filter (green emission) or a 585 nm longpass filter (red emission). Laser power and photomultiplier settings were kept constant for all experiments. Oocytes were positioned with the polar body in the plane of focus where present, and a single scan through the centre of the oocyte was used for the analysis. For embryos, single scans through the centre of focus of each blastomere were used for the analysis. Areas of embryo fragmentation and overlaps between blastomeres were excluded from the analysis by deselecting then with the confocal software. Aged oocytes were scored as unfertilized on day 1 after cycles of IVF. Controls for this group were fresh oocytes analysed on the day of oocyte retrieval, donated by the same patient as the aged material. Oocytes and embryos were used only once before being discarded. Images were processed by the confocal software and Adobe Photoshop.
Statistical analysis
All data were plotted as mean ± SD unless stated otherwise. All plots and statistical analysis were calculated using the Sigma Plot and Sigma Stat software packages [Statistics Package for Social Sciences (SPSS), Erkrath, Germany] except where stated. The strength of the normal distribution was tested using the KolmogorovSmirnov normality test. Regression lines were calculated by the method of least squares, and the significance of the regression lines was tested with the Pearson product-moment test. Partial regression analysis was calculated using the SPSS statistical package. The MannWhitney rank sum test was used to adjust the t-test for small populations of data.
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Results |
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Human oocyte maturation is characterized by changes in mitochondrial aggregation pattern and not mitochondrial membrane potential
The fluorescence dye JC-1 can be used to determine both the activity and localization of mitochondria in human oocytes and preimplantation embryos. First, the localization of mitochondria in human oocytes was determined at various stages of maturation by analysing data from the potential-insensitive confocal channel. Germinal vesicle-stage oocytes obtained on the day of oocyte retrieval were characterized by mitochondria with a granular, clumped aggregation pattern which was termed type A and which coincided with the appearance of the oocyte under Nomarski optics (Figure 2A; Table I
). Maturation of oocytes to metaphase I or II led to the appearance of a second type of pattern, the appearance of which was smooth under both fluorescence and light microscopy (Figure 2A
). This was termed mitochondrial pattern type B. Both metaphase I and II oocytes had a mixture of granular (type A) and smooth mitochondria and in consequence cytoplasm (type B, Figure 2A
; Table I
). These data demonstrate that the mitochondrial aggregation pattern in the oocyte cytoplasm is directly related to the appearance of the oocyte under the light microscope, and further suggest that mitochondrial aggregation is modified during oocyte maturation. Localized areas of heterogeneity in mitochondrial membrane potential have been reported in other cell types (Smiley et al., 1991
). In the present experiments, no difference was noted in membrane potential between the two distinct mitochondrial populations (Figure 2B
). Furthermore, the membrane potential observed in germinal vesicle, metaphase I and metaphase II oocytes obtained at oocyte retrieval was not significantly different (Figure 2C
), suggesting that no major changes in metabolism occur during oocyte maturation. These data suggest that the pattern of aggregation of mitochondria within the human oocyte is independent of the mitochondrial membrane potential.
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Discussion |
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Maturing human oocytes are characterized by diverse cytoplasmic morphological characteristics visible under the light microscope. The data presented here suggest that the aggregation pattern of mitochondria corresponds closely to the gross oocyte morphology. Two major mitochondrial aggregation patterns were visualized. The first pattern (type A) was more common in immature, germinal vesicle-stage oocytes. Oocyte maturation proceeded with the appearance of a second pattern (type B). Of particular interest was the observation that type B was formed mainly towards the centre of the oocyte, whereas type A was confined to the subplasma membrane region. In fact, the two patterns often showed a polarized distribution. These data suggest that mature human oocytes are polarized, at least with respect to mitochondrial aggregation. In this study, the relationship between the mitochondrial aggregation pattern and the position of the meiotic apparatus was not examined, but one hypothesis would be that the polarization of the mitochondrial morphology is an indicator of the animal-vegetal pole of the oocyte. This is particularly interesting because the polarization of mitochondrial aggregation did not correspond with the position of the polar body, which is already known to be an unreliable indicator of the position of the meiotic apparatus (Garello et al., 1999; Van der Westerlaken et al., 1999
). It is not known which cytoplasmic determinant determines mitochondrial aggregation: however, the data presented here suggest that cytoplasmic maturity may play a role. Despite the morphological polarization of oocytes, the data did not reveal any localized heterogeneity of mitochondrial activity, suggesting that the two effects are unrelated. Human preimplantation embryos were also characterized by two distinct populations of mitochondria. Preimplantation embryos were further characterized by a concentration of mitochondria to one side of the nucleus. In the present data, no heterogeneity in mitochondrial activity was observed, although this has been reported previously (Bavister and Squirrel, 2000
). These data indicate that the type of mitochondrial aggregation observed is independent of activity in human oocytes, and suggest that factors localized within the oocyte and embryo cytoplasm determine these patterns.
The pattern of mitochondrial aggregation in oocytes was not found in the present investigation to be correlated with localized regions of mitochondrial activity. However, mitochondrial activity did show distinct relationships to diverse factors in oocytes and embryos. No changes in mitochondrial activity were noted during oocyte maturation. However, it must be noted that these data were obtained using immature oocytes obtained after oocyte retrieval and therefore 36 h after the administration of HCG. The data cannot therefore exclude increases in oocyte metabolism as a direct effect of HCG administration. Fresh oocytes obtained at oocyte retrieval were characterized by a mitochondrial activity that showed a negative correlation with maternal age. Furthermore, the mitochondrial activity in blastomeres of preimplantation embryos also showed a strong negative correlation with maternal age. Mitochondrial activity was positively correlated with the rate of development of human embryos, but only when these embryos were analysed on day 3 after oocyte retrieval. A reasonable explanation for this seemingly odd observation is that day 2 embryos have not always fully entered the cleavage stage of embryo development, and therefore the number of blastomeres present is not indicative of the rate of development. No correlation was observed with the quality of spermatozoa used to inseminate the partner's oocytes. This is perhaps not surprising when it is considered that the sperm mitochondria play no part in the development of the embryo (Hecht et al., 1984); however, the data further demonstrate that the spermatozoa do not introduce factors into the oocyte cytoplasm that directly affect the level of respiration.
Taken together, these data suggest that an accumulation of factors in the ovaries of older women leads to a reduced efficiency of mitochondrial respiration, with a subsequent negative effect on embryo development. It is not known whether these factors are genetic; for example, the accumulation of mutations in mitochondrial DNA (Keefe et al., 1995; but see Brenner et al., 1998; Barritt et al., 1999; Perez et al., 2000; Steuerwald et al., 2000), or environmental, for example in the accumulation of reactive oxygen species-induced damage to oocytes within the ovary (Wallace, 1992; Shigenaga et al., 1994
). However, the present data underlie the previously reported decrease in oocyte quality and consequent increase in aneuploidy in embryos from older patients (Gaulden, 1992
; Munne et al., 1995
; Dailey et al., 1996
; Janny and Ménézo, 1996
) and hence suggest reasons for the lower embryo implantation rate in these couples. The data suggest that the transfer of cytoplasm from young donors to older recipient oocytes (Cohen et al., 1997
, 1998
) increases embryo quality by introducing mitochondria with a higher activity than that of the recipient (Brenner et al., 2000
). The transfer of donor mitochondria may improve the recipient embryo quality during the early stages of embryo development, thus increasing the possibilities for the embryo to implant.
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Acknowledgements |
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Notes |
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References |
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Submitted on October 2, 2000; accepted on February 6, 2001.