1 Reproductive Biology Associates, 1150 Lake Hearn Dr., Suite 600, Atlanta, GA 30342, 2 Department of Molecular, Cellular and Developmental Biology University of Colorado, Boulder, CO 80309 and 3 Colorado Reproductive Endocrinology, Rose Medical Center, Denver, CO 80220, USA
4 To whom correspondence should be addressed. Email: hesikaij{at}aol.com
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Abstract |
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Key words: ATP/calcium/embryo competence/mitochondrial polarity/oocyte cryopreservation
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Introduction |
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While the underlying cause(s) of poor outcomes with thawed human oocytes are unknown, numerical chomosomal disorders (aneuploidy and mosaicism) resulting from cooling-induced meiotic spindle defects are generally assumed to be the primary factor adversely affecting embryo competence (Pickering et al., 1990; Almeida and Bolton, 1995
; Wang et al., 2001
). However, several studies report no significant increase in levels of chomosomal disruption after thawing of human oocyte (Gook et al., 1993
, 1994
; Van Blerkom and Davis, 1994
; Baka et al., 1995
). Cryopreservation may have adverse downstream consequences if the normality of embryogenesis is influenced by molecular and cellular activities in the oocyte that are labile to damage or disruption during the freezingthawing process (Ménézo and Guerin, 2004
). For example, cellular alterations associated with osmotic forces produced during oocyte dehydrationrehydration cycles may have differential effects on cytoplasmic activities including mitochondrial metabolism and intracellular signalling pathways, such as those mediated by Ca.2+
In previous studies (Van Blerkom et al., 2002, 2003
), we suggested that high-polarized pericortical mitochondria in oocytes and early embryos may be involved in the modulation of focal levels of intracellular free Ca2+ and ATP. Here, mitochondrial polarity, ATP generation, and levels of intracellular free Ca2+ were examined in thawed MII human oocytes that had been cryopreserved with a standard protocol used for oocytes and pronuclear/cleavage stage embryos.
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Materials and methods |
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Cryopreservationthawing
Denuded oocytes were dehydrated for 20 min at room temperature in HEPES-buffered human tubal fluid (HTF) containing 1.5 mol/l 1,2-propanediol (PROH) and 12% human serum albumin (HSA) followed by an additional 10 min in a second solution containing 1.5 mol/l PROH and 0.2 mol/l sucrose (Tucker et al., 1996). At the end of the dehydration phase, oocytes were transferred to plastic cryostraws and cooled at a control rate of 2°C/min from room temperature to 7°C, at which point manual seeding was performed. After seeding, samples were cooled to 36°C at 0.3°C/min and then plunged directly into liquid nitrogen. Oocytes were thawed by exposing straws to air for 30 s followed by immersion in a water-bath for 45 s at 31°C. PROH was removed in two steps by passage of oocytes in 1 ml of HSA-supplemented HEPES-buffered HTF containing 0.5 mol/l (10 min) and 0.2 mol/l sucrose (10 min) respectively. Intact oocytes were transferred to normal embryo culture medium (Sage Cleavage medium, Sage BioPharma) supplemented with 10% HSA and pre-equilibrated in an atmosphere containing 5% CO2 in air.
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Analysis of mitochondrial polarity (![]() ![]() |
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Cortical granule staining
Cortical granule staining followed protocols previously described for the human MII oocyte (Van Blerkom and Davis, 1994). Briefly, oocytes were exposed to an acidic Tyrode's solution to remove the zona pellucida and were immediately fixed in phosphate-buffered saline (PBS) containing 3.7% formaldehyde for 30 min. After fixation, oocytes were subjected to thee sequential 10 min washes in PBS followed by a 30 min incubation in modified PBS containing 3% bovine serum albumin (BSA), 0.1 mol/l glycine and 0.1% Triton X-100. Fixed and washed oocytes were exposed to biotinylated Lens culinaris lectin (LCA; 5 µg/ml) for 30 min, washed as above for 1 h, and stained with Texas Redstreptavidin (2 µg/ml) in modified PBS for 30 min followed by a 1214 h incubation in modified PBS at 4°C. Oocytes were placed on glass coverslips in droplets of Slow-fade anti-quenching solution (Molecular Probes, USA) and examined by scanning laser confocal microscopy (SLCM).
Microscopic analysis of JC-1 fluorescence
JC-1-stained oocytes were maintained in modified HTF under oil at 37°C and examined by epifluorescence microscopy in the fluorescein isothiocynate (FITC) and rhodamine isothiocynate (RITC) channels using narrow band path filter sets (Van Blerkom et al., 2002).
Estimation of free Ca2+
Estimation of free Ca2+ levels by SLCM measured the average maximum relative fluorescence intensity (RFI) in individual control and thawed oocytes using the same analytical protocol described for the MII mouse oocyte (Van Blerkom et al., 2003). Briefly, oocytes were preloaded for 60 min in HEPES-buffered HTF supplemented with 4% BSA and 20 µmol/l Fluo-4 AM (Molecular Probes), followed by a 30 min wash in normal medium. Oocytes were transferred to
T dishes containing 1 ml of serum-free HEPES-buffered medium on an inverted microscope with temperature maintained at precisely at 37°C by means of a
T controller (Bioptics, USA). Five micrometre scans taken though the approximate centre of the oocyte established baseline RFI levels. Quantification of RFI used a long path filter with emission detection >510 nm and processing of the digital images with ImageSpace software (v3.10; Molecular Dynamics, USA). Specimens were examined at the same photomultiplier detector gain and laser intensity at 5 s intervals for up to 150 s after the addition of the Ca2+ ionophore A23187 at 10 µmol/l as previously described (Van Blerkom et al., 2003
).
Measurement of net cytoplasmic ATP content
Measurements of net ATP content followed a previously described protocol (Van Blerkom et al., 1995). Briefly, individual oocytes were rapidly frozen to 80°C in 200 ml of ultrapure water. ATP levels were quantified by measuring the luminescence (Berthold LB 9501 luminometer) generated in an ATP-dependent luciferin-luciferase bioluminescence assay (Bioluminescence Somatic Cell Assay System; Sigma, USA). A standard curve containing 14 ATP concentrations from 5 fmol to 5 pmol was generated for each series of analyses.
Effects of cryoprotectants
Representative oocytes were dehydrated and rehydrated as described above but without cryopreservation. After equilibration in normal embryo culture medium (10 min), oocytes were stained with JC-1 and examined by fluorescence microscopy.
Statistical analysis
The occurrence of J-aggregate fluorescence and differences in levels of intracellular free Ca2+ in ionophore-exposed fresh and thawed oocytes were analysed by Fisher's exact test and unpaired t-test respectively.
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Results |
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In a third series of experiments, fresh GV , MI
and MII
oocytes from fertile ovum donors (day 0) were stained (at 37°C) with JC-1 during or after dehydration. Approximately 90% (25/28) of these oocytes showed normal pericortical J-aggregate fluorescence (similar to images in Figure 1B and C) indicating that exposure to cryoprotectants alone, under the conditions used in this study, does not appear to depolarize mitochondria.
Cortical granule staining
Twenty-six thawed MII oocytes obtained from day 0 and day 1 groups (all ICSI-failed fertilization; n = 12) were stained to resolve cortical granules by SLCM. While morphometric assessments were beyond the scope of this study, all day 0 and 75% (9/12) of day 1 oocytes exhibited intense circumferential cortical granule staining comparable in density and distribution to fresh oocytes (Figure 1L; Table I). Thee day 1 ICSI oocytes displayed cortical granule staining but at reduced intensity (images not shown).
Intracellular free calcium and ATP levels
Owing to the limited availability of oocytes, the following preliminary studies were conducted to determine ATP contents or levels of intracellular free Ca2+ . Changes in the RFI of Fluo-4-stained oocytes exposed to A23187 were examined in fresh day 1 oocytes and in thawed oocytes, randomly selected from day 0
and day 1 groups
. The maximum average increase (untreated, Figure 1M) in RFI above background in fresh oocytes was
400% (80 s after the addition of ionophore, Figure 1N), while for thawed human oocytes (untreated, Figure 1O) the maximum average increase at 90 s after ionophore treatment was
50% (Figure 1P), and these differences were significant
(Table I). RFI was determined from pseudocolour images where the increase in the intensity of Fluo-4 fluorescence was measured on a scale from 0 to 255 (colour bar, Figure 1N). We did not determine whether the oocytes used for Fluo-4 analysis were J-aggregate positive or negative owing to mutual fluorescence of Fluo-4 and the JC-1 monomer in the FITC channel. However, as noted above, all thawed day 0 and most day 1 oocytes were J-aggregate negative.
In a second series of analyses, the net ATP content of individual day 1 oocytes was determined after thawing and culture (6 h). The average ATP contents of J-aggregate positive (n = 17; 1.96 pmol/oocyte±200 fmol) and negative (n = 29; 1.91 pmol/oocyte±200 fmol) oocytes were not significantly different and were comparable to values previously reported for fresh MII oocyes (Van Blerkom et al., 1995).
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Discussion |
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We report that cryopreservation had two negative consequences for the oocyte: (i) loss of the ability of pericortical mitochondria to retain high polarity and form J-aggregates and (ii) a significant diminution in the ability of the thawed oocyte to up-regulate Ca2+ in response to the Ca2+ ionophore A23187. The extent to which loss of high polarity contributed to the diminished ability to up-regulate Ca2+ is unknown, and the present studies do not preclude the possibility that cryopreservation-associated defects or damage to other Ca2+ -regulating elements that act in concert with mitochondria, such as the smooth endoplasmic reticulum (SER), may be involved (Hajnoczky et al., 2000; Liu et al., 2001
). However, it is an intriguing possibility that the relationship between polarization, mitochondrial respiration and the regulation of intracellular free Ca2+ described for somatic cells (Ichas et al., 1997
; Smaili and Russell, 1999
) and mouse oocytes (Liu et al., 2001
; Dumollard et al., 2003
; Van Blerkom et al., 2003
) also pertains to the human female gamete.
Ozil and Huneau (2001) reported that a reduction in the amplitude of the first Ca2+ transient during the first seconds of oocyte activation was associated with growth retardation and developmental anomalies in rabbit embryos that were not evident until day 11.5 of embryogenesis. In the human MII oocyte, the pericortical distribution of J-aggregate fluorescence is associated with clusters of mitochondria that surround or are embedded within discrete, spheroidal SER networks (Van Blerkom et al., 2002
). The possibility that some proportion of embryos derived from thawed oocytes are unable to maintain normal Ca2+ signalling owing to cryopreservation-induced defects in mitochondrial polarity warrants further investigation. In this respect, irreversible mitochondrial depolarization coupled with sustained high levels of intracellular free Ca2+ and diminished ATP production are cellular events leading to activation of the apoptotic pathway (Kroemer and Reed, 1997
; Duchen, 2000
). However, loss of mitochondrial hyperpolarization does not appear to be associated with apoptosis in thawed human oocytes because: (i) they exhibit a diminished capacity to increase intracellular Ca2+ , (ii) they show levels of ATP comparable to fresh oocytes, (iii) they are fertilizable by ICSI and capable of significant in vitro development, and (iv) they remained intact after 4 days of culture in the present study.
The ability of pericortical mitochondria to form J-aggregates during dehydration indicates that high polarity may be lost during the slow cool stages that precede or follow seeding. The detection of an apparently normal level of cortical granules thoughout the circumference of J-aggregate negative thawed oocytes suggests that if Ca2+ is released during this stage, it is either below levels that can induce cortical granule exocytosis or that their exocytosis is inhibited at reduced temperatures. In the short term, loss of high m did not appear to compromise the ability of the thawed human oocyte to generate ATP at levels comparable to their fresh counterparts. Therefore, the relationship between
m and respiratory activity in fresh and thawed human oocytes needs further investigation that includes kinetic analyses of ATP turnover. For the human, one possible explanation for compromised competence currently under investigation is that after thawing, the inability of mitochondria to hyperpolarize may have downstream effects on respiratory capacity during stages of development where higher demands for ATP may exist, especially if persistent hypopolarization makes mitochondria refractory to changes in ambient Ca2+ that could up-regulate respiration.
Support for a mitochondrial polarity association with competence would be indicated if the relatively small proportion of thawed oocytes that retained the ability to form pericortical J-aggregates are shown to be responsible for successful outcomes, or if new protocols of oocyte cryopreservation prevent the apparent irreversible changes in mitochondrial polarity we describe. The present results confirm our earlier report that a variable proportion of fresh MII oocytes obtained from infertile women are J-aggregate negative (Van Blerkom et al., 2002). These preliminary findings suggest the possibility that maintenance of high polarized mitochondria may be related to oocyte competence, and, as such, it may be useful to determine whether loss of polarity is associated with certain aetiologies of infertility or outcomes in IVF cycles. If an association between J-aggregate fluorescence and human oocyte competence exists, as suggested for the early human embryo (Wilding et al., 2003
), the finding that some proportion of fresh MII oocytes (in vivo- and in vitro-matured) are J-aggregate negative may need to be considered when oocyte cryopreservation is contemplated.
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Submitted on November 18, 2003; accepted on April 22, 2004.