Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print April 20, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.040956
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/2/366    most recent
biolreprod.105.040956v2
biolreprod.105.040956v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Related articles in Biol Reprod
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thouas, G. A.
Right arrow Articles by Jones, G. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thouas, G. A.
Right arrow Articles by Jones, G. M.
Agricola
Right arrow Articles by Thouas, G. A.
Right arrow Articles by Jones, G. M.
BIOLOGY OF REPRODUCTION 73, 366–373 (2005)
DOI: 10.1095/biolreprod.105.040956
© 2005 by the Society for the Study of Reproduction, Inc.

Effect of Female Age on Mouse Oocyte Developmental Competence Following Mitochondrial Injury1

George A. Thouas 2 , Alan O. Trounson , and Gayle M. Jones 

Monash Immunology and Stem Cell Laboratories (MISCL), Monash University, Clayton, Victoria 3800, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oocytes from aging ovaries contain mitochondria with morphological and genetic flaws. How these flaws relate to phenotypes of oocyte developmental compromise associated with clinical infertility is not well understood. This study was conducted to investigate the role of mitochondria in the developmental compromises observed with female aging using a mouse model of mitochondrial dysfunction. Oocytes obtained from aging (30–40 wk) (C57BL/6J x CBACaH)F1 (B6CBAF1) hybrid female mice were photosensitized with mitochondrial fluorophore rhodamine-123 for variable durations and compared to similarly treated oocytes derived from pubertal mice (4–6 wk). Blastocyst development of normally fertilized oocytes from both age-groups correlated negatively in mathematically unique profiles with irradiation time, with a more sudden decline in development for oocytes from aging mice. Complete inhibition of blastocyst development occurred following a shorter duration of photosensitization for oocytes from aging compared to pubertal animals (60 vs. 90 sec). Prolonged photosensitization resulted in mitochondrial uncoupling and promoted localized generation of reactive oxygen species, mitochondrial permeabilization, and apoptotic phenotypes. Thus, aging oocytes are more developmentally sensitive to mitochondrial damage than pubertal oocytes but undergo similar metabolic and apoptotic responses. These and future findings may encourage further optimization of laboratory-based strategies to minimize mitochondrial injury to oocytes, particularly those from older women, and improve clinical outcomes for women with age-related etiologies of infertility.

aging, apoptosis, embryo, in vitro fertilization, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondrial pathophysiology is a characteristic cytoplasmic marker of the complex degenerative processes of aging in many cell types, particularly in metabolically active cells such as those of the heart and central nervous system [1]. Metabolic dysfunction in mitochondria can result in oxidative damage of cytoplasmic and nuclear structures as well as an increased likelihood of apoptotic and necrotic degeneration [2]. Progressive age-related degenerative processes such as telomere attrition have also been linked to mitochondrial dysfunction [3]. At present, the extent to which ooplasmic mitochondrial dysfunction influences age-related declines in oocyte developmental competence, a common and often unexplained observation in women undergoing IVF treatment for clinical infertility, remains a relatively unresolved issue [4–8].

Human oocytes and early embryos from aging, clinically infertile women contain mitochondria with ultrastructural features similar to those of senescing or degenerating somatic cells including vacuolation, swelling, and mineral deposition [9, 10]. Sundstrom and colleagues [11] reported an age-related increase in the appearance of small ooplasmic mitochondrion-vesicle complexes that could be interpreted as an indication of mitochondrial degradation or autophagy. Müller-Hocker and colleagues [12] measured a significant increase in the mitochondrial surface area and volume fraction in oocytes derived from older women compared with a younger age-group, indicating that number or size of ooplasmic mitochondria potentially increases with age. Such an increase may reflect mitochondrial swelling as observed in apoptotic somatic cells [13] or a reduced rate of mitochondrial degradation [14].

A progressive decline in mitochondrial charge represents a functional alteration that has been reported relative to decreasing embryo cell number and increasing female age [15]. Decreased mitochondrial charge may result in metabolic defects, such as mitochondrial proton or electron leakage, that increase progressively with age in somatic cells [1, 16, 17] and can lead to increased peroxidative damage. Furthermore, levels of oocyte ATP have been shown to correlate with pre- and postimplantation developmental outcomes [18], and so developmental delay has been speculated to result from metabolic energy deficits. Decreased mitochondrial charge has also been linked to increased levels of genetic mosaicism in human blastocysts [19]. In addition, human ooplasmic mitochondria have been reported to possess higher levels of mitochondrial DNA (mtDNA) point mutations and rearrangements with advancing female age [20]. Such genetic defects have been thought to have a more delayed pathological significance in the developing fetus as mitochondria undergo maturation and active replication as tissues differentiate.

Previous attempts to understand the influence of somatic age on oocyte developmental competence have involved the use of rodent oocytes that have undergone prolonged retention within the ovary, oviduct, or in vitro environment [21]. These so-called pre- or postovulatory aging oocytes have been attributed with cellular and biochemical traits that parallel those of oocytes from aging animals. Mouse oocytes aging in vitro contain decreased levels of ATP [22] perhaps because of acquired mitochondrial dysfunction or excessive energy expenditure. In vitro aging oocytes also have a reduced ability to initiate development because of deficient calcium signaling [23, 24], an important ooplasmic mechanism recently shown to require the direct involvement of oocyte mitochondria [25].

Other models, such as the senescence-accelerated mouse (SAM), are perhaps more clinically relevant to systemic aging processes. SAM mice exhibit artificially induced reductions in life expectancy and systemic mitochondrial dysfunction. The same strain also exhibits reduced reproductive capacity that may be associated with intrinsic ooplasmic defects, such as chromosomal misalignment in the meiotic spindle [26–28]. To date, naturally aging animals, including rodents, remain important models for the investigation of age-related links between naturally occurring declines in reproductive capacity and impaired viability of the oocyte. Although readily amenable to research, these models remain imperfect for drawing parallels to oocyte-related etiologies of infertility in aging women. In the laboratory mouse, for example, complicating factors include strain-specific differences in ovarian follicle pool size [29] and age of onset of reproductive senescence as well as the potential influence of intrinsic ovarian aging [30]. The first-generation hybrids (F1) of inbred mouse strains have traditionally been used to study early embryo developmental competence in vitro [31–33] and as a bioassay system for clinical IVF [34]. The popularity of hybrids in this context has been attributed not only to their physical hardiness but also to their responsiveness to ovulation induction and the resistance of preimplantation embryos derived from superovulated oocytes to undergo developmental arrest in vitro compared to the founder strains [35]. Although exhibiting increased longevity and a slower decline in breeding performance relative to its inbred founders, variants of the (C57BL/6J x CBA/CaH)F1 (B6CBAF1) strain have recently been demonstrated with an age-related decrease in ovulation numbers as well as increased incidence of morphological and chromosomal abnormalities in oocytes from stimulated females [36]. Interestingly, these abnormalities coincided with a threefold increase in the proportion of normal MII oocytes with abnormal mitochondrial clusters occurring between 12 and 40 wk of age. This mitochondrial abnormality was also pronounced in oocytes that had undergone prolonged in vitro aging [36].

In the present study, the B6CBAF1 mouse strain was therefore used to investigate the role of mitochondria in embryonic developmental compromises observed with increasing female aging. The same strain was used previously to optimize a method of photosensitization of the preloaded mitochondrion specific fluorescent probe rhodamine-123 (R123) to induce mitochondrial injury in living oocytes before insemination [37]. Blastocyst development, zygotic metabolic function, and apoptotic phenotype were subsequently assessed after the same treatment was applied to oocytes derived from pubertal (4–6 wk) and aging (30–40 wk) female mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

In all experiments, B6CBAF1 hybrid mice were used. Mice were housed in an environmentally controlled room at 22–24°C with a 12L:12D photoperiod with food and water available ad libitum. For oocyte harvest, 4–6-wk (pubertal) female and 30–40-wk (aging) mice were superovulated by i.p. injection of 5 IU eCG (Folligon; Intervet, Bendigo East, VIC, Australia) followed approximately 48 h later with an i.p. injection of 5 IU hCG (Chorulon; Intervet). Pubertal animals at the specified age were used to maximize the yield of oocytes in response to hormone stimulation, as previously recommended [38]. For in vitro insemination of oocytes, mature sperm were isolated from whole epididymes of sexually mature (10– 12 wk) male mice. Both female and male mice were killed by cervical dislocation before gamete removal. For simplicity of reference, oocytes from pubertal and aging animals are henceforth referred to as pubertal or aging oocytes.

Ethics of Experimentation

This study was approved by Monash Medical Center Animal Ethics Committee A, Monash Medical Center, Clayton, Victoria, Australia, approval numbers A2003/40. Experiments were conducted in accordance with the 1997 NH & MRC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and the Victorian Prevention of Cruelty to Animals Act and Regulations, 1986.

Oocyte Isolation, In Vitro Fertilization, and Blastocyst Culture

Oocyte collection, sperm preparation, and in vitro fertilization were performed as described by Lacham-Kaplan and colleagues [39]. Normally fertilized zygotes were then cultured for 96 h to the blastocyst stage in a humidified atmosphere of 5% CO2 at 37°C in a thermostat-controlled incubator. Zygotes were cultured in groups of 10 in 20-µl droplets of bicarbonate buffered potassium modified simplex-optimized medium with amino acids (KSOMaa)[40] supplemented with 1 mg/ml bovine serum albumin (BSA) (Invitrogen, Carlsbad, CA) in a sterile plastic Petri dish (Becton-Dickinson, Franklin Lakes, NJ) overlaid with mineral oil (Sigma-Aldrich, St. Louis, MO). Embryos were cultured in three replicate experimental groups of at least 30 zygotes, and blastocyst formation was expressed as a percentage of normally fertilized oocytes. Inseminated oocytes from pubertal females were cultured alongside those from aging females.

Photosensitization of Oocytes Using Fluorescence Microscopy

Isolated and denuded oocytes were photosensitized using the mitochondrion-specific fluorophore rhodamine 123 (R123, 25 µg/ml; Molecular Probes, Eugene, OR) prepared from a stock solution of 100 mg/ml R123 in dimethyl sulfoxide (DMSO; Sigma Aldrich) as described previously [37]. Pubertal and aging test oocytes were irradiated for periods of 0, 20, 40, 60, or 90 sec before insemination. Control oocytes from the same batch of mice were allocated to one of three groups: 1) not loaded with R123 and irradiated for the maximal time period ("Light"), 2) incubated in R123 vehicle DMSO for 15 min before insemination but not loaded or irradiated ("Vehicle"), or 3) not loaded or irradiated ("No treatment"). "Maximal" photosensitization treatment was defined as the duration for which blastocyst development was decreased by at least 80%; hence, an extended time point of 90 sec for loaded pubertal oocytes was adopted as the maximal exposure duration for this age-group.

Laser-Scanning Confocal Microscopic Determination of Mitochondrial Membrane Potential ({Delta}{psi})

Assessment of mitochondrial {Delta}{psi} was performed by ratiometric quantitation of 5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimdazoylcarbocyanine iodide (JC-1) red to green fluorescence levels using laser-scanning confocal microscopy (LSCM), as described previously [37]. Briefly, a test group of at least 20 zygotes formed from R123 loaded oocytes that were irradiated for 60 sec (for aging oocytes) or 90 sec (for pubertal oocytes) was assessed and compared to a similar-sized control group of zygotes from the same age-groups that had fertilized normally from R123 loaded, nonirradiated oocytes. LSCM was also used to capture equatorial sections of zygotes derived from loaded (irradiated or nonirradiated) oocytes for a qualitative visual assessment of oocyte cytoplasmic distribution of R123 (520-nm emission).

Fluorimetric Assessment of Zygote Metabolism and Apoptosis

Quantitative detection of autofluorescence at 340-nm excitation (UV light) was performed using microfluorimetry as described previously for whole zygotes as an indicator of cytoplasmic NADH-NADPH content [37]. Similar test and control groups of zygotes as described for determination of {Delta}{psi} quantitation were selected. Absolute values of autofluorescence intensity were expressed in arbitrary values on a scale of 0–850. All values were corrected for background fluorescence and internal reflectance. Cytoplasmic ATP content was also determined microfluorometrically, using the luciferin-luciferase reaction as described previously [37]. Test and control groups for ATP quantitation were also similar to those described for {Delta}{psi} quantitation, although sample numbers represent pooled batches of 10 zygotes.

Photosensitization has been reported to result in transient, specific production of the highly reactive oxygen intermediate singlet oxygen [41], which is also related to local variations in cytoplasmic oxidation-reduction (REDOX) status by direct reaction with electron carriers such as NADH and NADPH [42, 43]. Singlet oxygen levels in zygotes were therefore quantitated as an indicator of the ability of mitochondria to support the generation of reactive oxygen intermediates in response to photosensitization. Zygotes derived from test and control groups were stained for 15 min in a warmed solution of 20 µg/ml trans-methoxy (1,2-vinyl) pyrene (TMVP) (Molecular Probes) dissolved in Hepes KSOMaa. Zygotes were rinsed twice in the same medium and then analyzed singly using microfluorimetry. Probe emission after 0.5-sec excitations using 340 nm (UV) light resulted in blue fluorescence ranging from 420 to 460 nm that was measured in a similar manner to that described for autofluorescence detection. Chromatin and plasma membrane integrity were assessed using the fluorophore propidium iodide. Detection of activation of the apoptosis effector enzyme caspase-3 was performed using an immunofluorescent antibody protocol as described previously [37].

Statistical Analysis

The mean proportion of zygotes developing to blastocyst by 96 h in vitro was calculated for test and control treatments and exponentially transformed for comparison using ANOVA. Mean values for metabolic endpoint measures between age-groups were compared using Student t-test. Differences between means were considered biologically significant at a P-value of less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Blastocyst Development after Photosensitization of Pubertal and Aging Oocytes

Blastocyst development of normally fertilized zygotes derived from photosensitized oocytes was negatively affected by the duration of exposure in both age-groups. Of zygotes derived from aging oocytes, 52.7%, 46.8% and 7.1% formed blastocysts at each of the photosensitization exposure times of 20, 40, and 60 sec, respectively (Fig. 1A). The blastocyst formation rates for oocytes of all durations (20–60 sec) were significantly lower (P < 0.05) than for loaded and nonirradiated aging oocytes (0 sec; 67.9%) as well as "light" (74.5%), "vehicle" (78.3%), and "no treatment" (76.5%) control values.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 1. Blastocyst development after photosensitization of aging and pubertal oocytes. A) Graph of mean blastocyst formation rates of zygotes derived from aging oocytes photosensitized (hatched bars) for 0 sec (n = 113), 20 sec (n = 112), 40 sec (n = 120), and 60 sec (n = 115). Control groups (hollow bars) represent aging oocytes that were (a) not loaded but maximally irradiated ("light 60 sec," n = 92), (b) incubated briefly in the R123 diluent DMSO ("vehicle," n = 115), or (c) not treated ("no treatment," n = 120). Bars sharing like superscripts (1, 2, 3) are not statistically different. B) Graph of mean blastocyst formation rates of zygotes derived from pubertal oocytes photosensitized (hatched bars) for 0, 20, 40, 60, and 90 sec (n = 90). Control groups (hollow bars) represent pubertal oocytes that were (a) not loaded but maximally irradiated ("light 90 sec," n = 90), (b) incubated briefly in the R123 diluent DMSO ("vehicle," n = 110), or (c) not treated ("no treatment," n = 100). Bars sharing like superscripts (1, 2) are not statistically different. Small bars above larger bars indicate ± SEM for all values

Zygote-to-blastocyst development of photosensitized oocytes from pubertal females was similar to previously reported observations [37]. Of zygotes derived from treated oocytes, 68%, 41%, 22%, and 0% developed to blastocyst for exposure times of 20, 40, 60, and 90 sec, respectively (Fig. 1B). These blastocyst formation rates were significantly lower (P < 0.01) than for pubertal oocytes that were loaded and nonirradiated (0 sec; 82%) as well as "light" (80%), "vehicle" (74%), and "no treatment" (78%) control values.

Blastocyst development of photosensitized pubertal and aging oocytes correlated with photosensitization duration (R2 = 1) according to the third-order polynomial relationships y = –0.0009x3 + 0.065x2 – 1.6917x + 68 and y = 0.0002x3 – 0.0191x2 – 0.3994x + 80.496, respectively, where x represents photosensitization time (min) and y represents the proportion of fertilized oocytes that developed to blastocyst (Fig. 2). The trend for aging oocytes indicated that a minimum of 62 sec of photosensitization was required for complete inhibition of blastocyst development, whereas the minimum equivalent photosensitization period to inhibit blastocyst formation from pubertal oocytes was longer (90 sec).



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 2. Blastocyst development of aging oocytes compared to pubertal oocytes after photosensitization treatment. Point plots of mean formation rates of blastocyst derived from aging oocytes (open squares; n = 90 per group) in comparison to pubertal oocytes (diamonds; n = 90 per group). Corresponding polynomial curves of best fit have been included (solid line, aging oocytes; broken line, young oocytes; small bars above larger bars indicate ± SEM for all values)

Mitochondrial Metabolism in Zygotes after Photosensitization of Pubertal and Aging Oocytes

Microfluorimetric analysis revealed that zygotic pools of NADH-NADPH after loading and maximal irradiation of aging oocytes were lower (P < 0.05) than after loading without irradiation (88.6 ± 1.4 vs. 98.9 ± 2.2 arbitrary units), as were levels for pubertal oocytes (96.7 ± 2.3 vs. 106 ± 3.8 arbitrary units; P < 0.05; Table 1). Zygotes derived from loaded and maximally irradiated aging oocytes contained lower ATP levels (P < 0.05) than after loading without irradiation (0.60 ± 0.13 vs. 1.15 ± 0.13 pmol/zygote). Similarly, ATP levels in zygotes derived from loaded and maximally irradiated pubertal oocytes were lower (P < 0.05) than after loading without irradiation (0.62 ± 0.13 vs. 1.46 ± 0.12 pmol/zygote; Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Age-related effect of oocyte photosensitization on endpoint measures of zygote mitochondrial physiology.a

Mitochondrial Membrane Permeability in Zygotes after Photosensitization of Pubertal and Aging Oocytes

Loading in the absence of photosensitization revealed a punctate staining pattern (Fig. 3A) similar to nontreated controls (results not shown). Maximal irradiation of loaded aging oocytes resulted in leakage of rhodamine-123 from the mitochondria into the ooplasm to produce a diffuse staining pattern (Fig. 3B). This morphological evidence of a mitochondrial permeability transition was associated with the reductions observed in mitochondrial metabolic activity. Quantitative LSCM analysis of zygotes derived from maximally irradiated aging oocytes revealed a significantly lower {Delta}{psi} (Table 1) compared to loaded, nonirradiated control zygotes (1.19 ± 0.02 vs. 1.29 ± 0.02 arbitrary units; P < 0.05). A similar result was observed for zygotes derived from maximally irradiated oocytes from pubertal females compared to loaded, nonirradiated control zygotes (1.27 ± 0.04 1.46 ± 0.02 vs. arbitrary units; P < 0.05). In addition to decreases after treatment, zygotes from loaded, nonirradiated aging oocytes had a significantly lower {Delta}{psi} than pubertal oocytes (1.29 ± 0.02 vs. 1.46 ± 0.02 arbitrary units; P < 0.01).



View larger version (76K):
[in this window]
[in a new window]
  		FIG. 3. Microscopic evidence of mitochondrial damage in zygotes after prolonged photosensitization of aging oocytes. Images of mouse zygotes represent equatorial sections taken approximately 6–9 h after insemination by laser-scanning confocal microscopy, unless otherwise indicated. A) Zygote derived from an aging oocyte loaded with R123 but not irradiated. B) An arrest zygote derived from a loaded and maximally photosensitized aging oocyte. C) Fluorescence and (D) phase contrast microscopic images of a zygote derived from a maximally photosensitized aging oocyte stained with trans-methoxy (1,2-vinyl) pyrene. E) Phase contrast image of arrested zygote from B. F) The same zygote from B after staining with propidium iodide. G and H) Caspase-3 antibody staining of zygotes derived from two maximally photosensitized and (I) a loaded nonirradiated oocyte. J and K) Arrested syngamal zygote and two-cell embryo derived from maximally photosensitized oocytes. L) Degenerated zygote derived from an aging maximally photosensitized oocyte stained with JC-1. BL, Membrane bleb; CC, condensed chromatin; F, cytoplasmic fragment; P, pronucleus; V, vacuolation; ZP, zona pellucida. Bars = 25 µm

Evidence of Oxidative Damage and Apoptosis in Zygotes Derived from Pubertal and Aging Photosensitized Oocytes

Regardless of oocyte age, developmental and metabolic changes after maximal photosensitization were associated with generation of reactive oxygen species, mitochondrial permeabilization, and apoptosis. Qualitatively, singlet oxygen fluorescence in zygotes derived from maximally photosensitized oocytes occupied discrete locations in the zygote cytoplasm (Fig. 3C) even when zygotes showed no overt apoptotic morphology (Fig. 3D). Levels of TMVP fluorescence in zygotes were higher after maximal photosensitization treatment of pubertal (610 ± 24 vs. 495 ± 14) and aging (603 ± 18 vs. 522 ± 21) oocytes compared to loaded nonirradiated controls (Table 1).

Apoptotic progression after maximal photosensitization of aging oocytes was indicated by phenotypes of blebbing (Fig. 3E) and vacuole formation (Fig. 3B). Maximal photosensitization also led to plasma membrane permeabilization, resulting in the cytoplasmic uptake of propidium iodide that revealed densely compacted pronuclear chromatin (Fig. 3F). Apoptosis was further evidenced by immunolocalization of the cleaved form of caspase-3 (Fig. 3, G and H) compared to the loaded nonirradiated control (Fig. 3I) as well as mitochondrial clumping in perinuclear regions in association with zygote (Fig. 3J) and two-cell embryo arrest (Fig. 3K). An additional morphological feature unique to degenerating zygotes derived from maximally photosensitized aging oocytes was retraction of mitochondria away from the plasma membrane (Fig. 3L) with evidence of an intact plasma membrane. Similar apoptotic phenotypes were observed for maximally photoesensitized pubertal oocytes (results not shown).

Maximally photosensitized aging oocytes were pretreated with the mitochondrial permeability pore antagonist and antiapoptotic agent cyclosporin-A in a similar manner to pubertal oocytes as described previously [37]. However, cyclosporin-A pretreatment was not found to be beneficial for developmental competence or mitochondrial function of embryos derived from maximally photosensitized aging oocytes (results not shown) as was reported for pubertal oocytes [34].


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Photosensitization of oocytes from aging female mice led to a dosage-dependent reduction in developmental competence, similar to that observed for oocytes from pubertal female mice. Compared to pubertal oocytes, aging oocytes underwent developmental arrest sooner, with a more plateau-like profile. Zygote mitochondrial membrane potential and ATP levels were depressed after treatment, with control values exhibiting a significant age-related reduction compared to that observed for zygotes derived from pubertal oocytes. Zygote autofluorescence, indicative of REDOX potential, was also depressed after oocyte photosensitization treatment in both age-groups. Photosensitization also resulted in production of the reactive oxygen intermediate singlet oxygen, a likely trigger for the observed apoptotic phenotypes. Cyclosporin-A treatment was, however, ineffective in ameliorating this apoptotic progression in photosensitized oocytes from aging females.

The age-related decline in zygote mitochondrial membrane potential that was observed without photosensitization is in agreement with a previously reported trend for human preimplantation embryos from aging infertile women [15] and aging somatic cells [44]. Similarly, the observed age-related decrease in zygotic ATP levels, while related to the reduced mitochondrial membrane potential, is also relevant to the observation that human oocytes with decreased ATP content are likely to have a compromised developmental outcome [18]. Regardless of the starting basal levels, maximal photosensitization of mouse oocytes from both age-groups after loading with rhodamine-123 led to a significant decrease in zygotic ATP content and mitochondrial membrane potential. Similarly, a decrease in zygote NADH/NADPH levels was seen, suggesting a continued consumption of REDOX substrates separate from ATP generation and hence mitochondrial uncoupling as described previously [37]. Since the ATP content and mitochondrial membrane potential of aging oocytes were lower relative to pubertal oocytes before photosensitization and further lowered by injury, an age-related mitochondrial energy deficiency may explain the faster developmental arrest in response to photosensitization observed for oocytes from this group. A compensatory generation of ATP from alternative sources, such as from exhaustible cytoplasmic reserves, may further explain the rather precipitous decline in development compared to pubertal oocytes following injury. An age-related increase in subpopulations of mitochondria with pathological function may also lead to a developmental profile with a pronounced threshold of demise, as described for several somatic cell types [45].

Prior to the sharp decline in blastocyst development for photosensitized oocytes from aging mice, blastocyst development was apparently at a plateau. In this case, it is possible that age-related decreases in mitochondrial membrane potential are responsible for a decrease in the mitochondrial uptake of rhodamine-123. This may result in a weaker localized toxicity response to photosensitization and a flattening of the curve relative to younger oocytes until an absolute threshold was reached. Second, amelioration of this phototoxic effect may result from a nonuniform spatial and temporal response of individual mitochondria to injury, as observed for cultured neural cells after photosensitization [46].

Third, a cytoplasmic up-regulation of survival factors in response to altered REDOX status [47] after mitochondrial injury may more readily occur with age and contribute to the attenuation of blastocyst development. A fourth, more delayed contributing factor may be the beneficial effect of partial uncoupling on later stages of development, as observed for chemically treated bovine morulae, suggested to be due to compensatory ATP generation from glycolysis [48]. Aging oocytes with marginally increased mitochondrial uncoupling in association with decreased membrane potential, similar to aging somatic cells [16], could therefore be somewhat protective against developmental arrest at sublethal photosensitization doses.

Gradual age-related inefficiencies in mitochondrial metabolism in somatic cells are also associated with gradual increases in generation of reactive oxygen species because of inefficient decomposition of oxygen [49]. The increased production of singlet oxygen in zygotes after maximal photosensitization of oocytes confirms that mitochondria are likely inducers of oxidative damage, although this increase was not age related, nor did aging oocytes have a higher threshold level of singlet oxygen before injury. In any case, the observation does not rule out the possible up-regulation of other reactive oxygen species [7, 50, 51] or down-regulation of antioxidant defense systems [52] with female age. So it remains likely that singlet oxygen is a key factor responsible for morphological phenotypes of mitochondrial permeabilization [43] and apoptotic progression [41] in zygotes in response to photosensitization. This observation confirms that oocyte and early embryo mitochondria maintain an inherent susceptibility for oxidative damage that triggers cellular demise. Intriguingly, the antiapoptotic agent cyclosporin-A, which acts by blocking mitochondrial permeability transition pores, was not effective in protecting maximally photosensitized oocytes from cell death. This was contrary to a previous observation for pubertal oocytes [37] and may represent an age-related insensitivity to the agent, a relative down-regulation of the blocked pore protein cyclophilin-D, a relative up-regulation of anti-apoptotic factors, or a less efficient active uptake of the agent into mitochondria.

Though not addressed in the present study, the role of intracellular calcium signaling and storage is an important regulatory factor that conceptually links mitochondrial metabolism, oocyte activation, cell cycle progression, respiratory control of early embryo development [25], apoptosis, and underlying age-related dysfunction [24]. For example, mitochondria from aging cells have a reduced capacity to accumulate and sequester calcium, resulting in elevated cytoplasmic levels and increased risk of mitochondrial calcium overload [53]. Sudden release or overload of calcium beyond "safe" threshold limits [54] in aging oocytes in response to photosensitization may thus trigger apoptosis rather than signal the onset of development and transient up-regulation of respiration [24, 55]. This may help explain the increased susceptibility and more sudden demise of development in aging oocytes after prolonged photosensitization.

The effects of aging on physical and biochemical interactions between mitochondria and other important ooplasmic organelles also merit further investigation. This concept arises in light of the potentially important observation that maximally photosensitization of an aging oocyte led to mitochondrial clustering in the resultant arrested two-cell embryo, a critical developmental stage when zygotic gene transcriptions begins to increase [56]. Mitochondrial condensation of an atypical nature has been observed in oocytes from aging B6CBAF1 mice and has been interpreted as evidence of apoptosis [36]. Similar atypical mitochondrial condensations have been observed in early mouse embryos that spontaneously arrested in vitro [57] and in arrested mouse embryos with disturbed microfilament distributions [58]. Conversely, disturbance of microfilament organization by chemical inhibition has been reported to interfere with perinuclear mitochondrial patterning in mouse zygotes [59]. Ooplasmic cytoskeletal modification is a dynamic, cell cycle- and ATP-dependent process [60] that is potentially compromised in aging oocytes [61]. Thus, oocyte developmental compromise induced by mitochondrial injury may be enhanced in the presence of other age-related ooplasmic defects. This enhancement may help explain the age-related sensitivity to developmental arrest after maximal photosensitization as well as insensitivity to pore antagonism by cyclosporin A.

In summary, the present study confirms that mitochondria maintain an important role in the maintenance of oocyte developmental competence with age via metabolic regulation of energy production and apoptosis. Subtle age-related declines in mitochondrial energy generation may contribute to a lowering and sensitizing of a developmental "threshold" that may be further perturbed by absence of sufficient antiapoptotic and antioxidant defense mechanisms. Further investigations of mitochondrial function in human oocytes are required, particularly in regard to age-related alterations in mitochondrial gene expression and protein expression that ultimately regulate physiological processes. Particular focus should be paid to the specific biological effects of clinical and laboratory interventions such as ovarian stimulation and in vitro manipulation and culture to reduce the potential for mitochondrial insult. The photosensitized oocyte from aging mice may thus be a suitable model for optimization of in vitro conditions toward prevention of the compromised preimplantation embryo development in aging populations of infertile women. Furthermore, the model may assist in improving the efficacy of clinical laboratory interventions aimed at preventing mitochondrial defects, such as autologous mitochondrial transfer [62].


   ACKNOWLEDGMENTS
 
This study was undertaken as part of a Ph.D. project. The authors wish to thank Dr. Peter Crack for his donation of the caspase-3 antibody and Dr. Juan Tarin for reprints of his previous publications.


   FOOTNOTES
 
1 Supported by departmental funding based on private donation, with scholarship support from the Helen Macpherson-Smith Trust. Back

2 Correspondence: George A. Thouas, Monash Immunology and Stem Cell Laboratories (MISCL), STRIP—Building 75, Monash University, Wellington Road, Clayton, VIC 3800, Australia. FAX: 61 3 9905 0680; george.thouas{at}med.monash.edu.au Back

Received: 13 February 2005.

First decision: 21 March 2005.

Accepted: 18 April 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lee HC, Wei YH. Role of mitochondria in human aging. J Biomed Sci 1997 4:319-326[CrossRef][Medline]
  2. Desagher S, Martinou JC. Mitochondria as the central control point of apoptosis. Trends Cell Biol 2000 10:369-377[CrossRef][Medline]
  3. Liu L, Trimarchi JR, Smith PJ, Keefe DL. Mitochondrial dysfunction leads to telomere attrition and genomic instability. Aging Cell 2002 1:40-46[CrossRef][Medline]
  4. Navot D, Bergh PA, Williams MA, Garrisi GJ, Guzman I, Sandler B, Grunfeld L. Poor oocyte quality rather than implantation failure as a cause of age-related decline in female fertility. Lancet 1991 337:1375-1377[CrossRef][Medline]
  5. Janny L, Menezo YJ. Maternal age effect on early human embryonic development and blastocyst formation. Mol Reprod Dev 1996 45:31-37[CrossRef][Medline]
  6. Jansen RP. Germline passage of mitochondria: quantitative considerations and possible embryological sequelae. Hum Reprod 2000 15:suppl_2112-128
  7. Eichenlaub-Ritter U, Vogt E, Yin H, Gosden R. Spindles, mitochondria and redox potential in ageing oocytes. Reprod Biomed Online 2004 8:45-58[Medline]
  8. Armstrong DT. Effects of maternal age on oocyte developmental competence. Theriogenology 2001 55:1303-1322[CrossRef][Medline]
  9. Motta PM, Nottola SA, Makabe S, Heyn R. Mitochondrial morphology in human fetal and adult female germ cells. Hum Reprod 2000 15:suppl 2129-147
  10. de Bruin JP, Dorland M, Spek ER, Posthuma G, van Haaften M, Looman CW, te Velde ER. Age-related changes in the ultrastructure of the resting follicle pool in human ovaries. Biol Reprod 2004 70:419-424[Abstract/Free Full Text]
  11. Sundstrom P, Nilsson BO, Liedholm P, Larsson E. Ultrastructure of maturing human oocytes. Ann N Y Acad Sci 1985 442:324-331[Medline]
  12. Muller-Hocker J, Schafer S, Weis S, Munscher C, Strowitzki T. Morphological-cytochemical and molecular genetic analyses of mitochondria in isolated human oocytes in the reproductive age. Mol Hum Reprod 1996 2:951-958[Abstract/Free Full Text]
  13. Lee HC, Wei YH. Mitochondrial alterations, cellular response to oxidative stress and defective degradation of proteins in aging. Biogerontology 2001 2:231-244[CrossRef][Medline]
  14. Kowald A, Kirkwood TB. Accumulation of defective mitochondria through delayed degradation of damaged organelles and its possible role in the ageing of post-mitotic and dividing cells. J Theor Biol 2000 202:145-160[CrossRef][Medline]
  15. Wilding M, Dale B, Marino M, di Matteo L, Alviggi C, Pisaturo ML, Lombardi L, De Placido G. Mitochondrial aggregation patterns and activity in human oocytes and preimplantation embryos. Hum Reprod 2001 16:909-917[Abstract/Free Full Text]
  16. Shigenaga MK, Hagen TM, Ames BN. Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci U S A 1994 91:10771-10778[Abstract/Free Full Text]
  17. Cadenas E, Davies KJ. Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med 2000 29:222-230[CrossRef][Medline]
  18. Van Blerkom J, Davis PW, Lee J. ATP content of human oocytes and developmental potential and outcome after in-vitro fertilization and embryo transfer. Hum Reprod 1995 10:415-424[Abstract/Free Full Text]
  19. Wilding M, De Placido G, De Matteo L, Marino M, Alviggi C, Dale B. Chaotic mosaicism in human preimplantation embryos is correlated with a low mitochondrial membrane potential. Fertil Steril 2003 79:340-346[CrossRef][Medline]
  20. Barritt JA, Brenner CA, Cohen J, Matt DW. Mitochondrial DNA rearrangements in human oocytes and embryos. Mol Hum Reprod 1999 5:927-933[Abstract/Free Full Text]
  21. Tarin JJ, Perez-Albala S, Cano A. Consequences on offspring of abnormal function in ageing gametes. Hum Reprod Update 2000 6:532-549[Abstract/Free Full Text]
  22. Chi MM, Manchester JK, Yang VC, Curato AD, Strickler RC, Lowry OH. Contrast in levels of metabolic enzymes in human and mouse ova. Biol Reprod 1988 39:295-307[Abstract]
  23. Jones KT, Whittingham DG. A comparison of sperm- and IP3-induced Ca2+ release in activated and aging mouse oocytes. Dev Biol 1996 178:229-237[CrossRef][Medline]
  24. Fissore RA, Kurokawa M, Knott J, Zhang M, Smyth J. Mechanisms underlying oocyte activation and postovulatory ageing. Reproduction 2002 124:745-754[Abstract]
  25. Dumollard R, Marangos P, Fitzharris G, Swann K, Duchen M, Carroll J. Sperm-triggered [Ca2+] oscillations and Ca2+ homeostasis in the mouse egg have an absolute requirement for mitochondrial ATP production. Development 2004 131:3057-3067 Epub 2004 May 26 [Abstract/Free Full Text]
  26. Liu L, Keefe DL. Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice. Hum Reprod 2002 17:2678-2685[Abstract/Free Full Text]
  27. Mori A, Utsumi K, Liu J, Hosokawa M. Oxidative damage in the senescence-accelerated mouse. Ann N Y Acad Sci 1998 854:239-250[CrossRef][Medline]
  28. Mizutani J, Chiba T, Tanaka M, Higuchi K, Mori M. Unique mutations in mitochondrial DNA of senescence-accelerated mouse (SAM) strains. J Hered 2001 92:352-355[Abstract/Free Full Text]
  29. Dorland M, van Kooij RJ, te Velde ER. General ageing and ovarian ageing. Maturitas 1998 30:113-118[CrossRef][Medline]
  30. Canning J, Takai Y, Tilly JL. Evidence for genetic modifiers of ovarian follicular endowment and development from studies of five inbred mouse strains. Endocrinology 2003 144:9-12[Abstract]
  31. Brown JJ, Whittingham DG. The roles of pyruvate, lactate and glucose during preimplantation development of embryos from F1 hybrid mice in vitro. Development 1991 112:99-105[Abstract]
  32. Harlow GM, Quinn P. Development of preimplantation mouse embryos in vivo and in vitro. Aust J Biol Sci 1982 35:187-193[Medline]
  33. Summers MC, Bhatnagar PR, Lawitts JA, Biggers JD. Fertilization in vitro of mouse ova from inbred and outbred strains: complete preimplantation embryo development in glucose-supplemented KSOM. Biol Reprod 1995 53:431-437[Abstract]
  34. Weiss TJ, Warnes GM, Gardner DK. Mouse embryos and quality control in human IVF. Reprod Fertil Dev 1992 4:105-107[CrossRef][Medline]
  35. Goddard MJ, Pratt HP. Control of events during early cleavage of the mouse embryo: an analysis of the "2-cell block.". J Embryol Exp Morphol 1983 73:111-133[Medline]
  36. Tarin JJ, Perez-Albala S, Cano A. Cellular and morphological traits of oocytes retrieved from aging mice after exogenous ovarian stimulation. Biol Reprod 2001 65:141-150[Abstract/Free Full Text]
  37. Thouas GA, Trounson AO, Wolvetang EJ, Jones GM. Mitochondrial dysfunction in mouse oocytes results in preimplantation embryo arrest in vitro. Biol Reprod 2004 71:1936-1942[Abstract/Free Full Text]
  38. Hogan B, Costantini F, Lacey E. Manipulating the Mouse Embryo: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 1986
  39. Lacham-Kaplan O, Shaw J, Sanchez-Partida LG, Trounson A. Oocyte activation after intracytoplasmic injection with sperm frozen without cryoprotectants results in live offspring from inbred and hybrid mouse strains. Biol Reprod 2003 69:1683-1689[Abstract/Free Full Text]
  40. Biggers JD, McGinnis LK, Raffin M. Amino acids and preimplantation development of the mouse in protein-free potassium simplex optimized medium. Biol Reprod 2000 63:281-293[Abstract/Free Full Text]
  41. Cosso RG, Turim J, Nantes IL, Almeida AM, Di Mascio P, Verces AE. Mitochondrial permeability transition induced by chemically generated singlet oxygen. J Bioenerg Biomembr 2002 34:157-163[CrossRef][Medline]
  42. Petrat F, Pindiur S, Kirsch M, de Groot H. "Mitochondrial" photochemical drugs do not release toxic amounts of 1O(2) within the mitochondrial matrix space. Arch Biochem Biophys 2003 412:207-215[CrossRef][Medline]
  43. Petrat F, Pindiur S, Kirsch M, de Groot H. NAD(P)H, a primary target of 1O2 in mitochondria of intact cells. J Biol Chem 2003 278:3298-3307[Abstract/Free Full Text]
  44. Leprat P, Ratinaud MH, Julien R. A new method for testing cell ageing using two mitochondria specific fluorescent probes. Mech Ageing Dev 1990 52:149-167[CrossRef][Medline]
  45. Rossignol R, Faustin B, Rocher C, Malgat M, Mazat JP, Letellier T. Mitochondrial threshold effects. Biochem J 2003 370:751-762[CrossRef][Medline]
  46. Peng TI, Jou MJ. Mitochondrial swelling and generation of reactive oxygen species induced by photoirradiation are heterogeneously distributed. Ann N Y Acad Sci 2004 1011:112-122[CrossRef][Medline]
  47. Harvey AJ, Kind KL, Thompson JG. REDOX regulation of early embryo development. Reproduction 2002 123:479-486[Abstract]
  48. Thompson JG, McNaughton C, Gasparrini B, McGowan LT, Tervit HR. Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos cultured in vitro. J Reprod Fertil 2000 118:47-55
  49. Genova ML, Pich MM, Bernacchia A, Bianchi C, Biondi A, Bovina C, Falasca AI, Formiggini G, Castelli GP, Lenaz G. The mitochondrial production of reactive oxygen species in relation to aging and pathology. Ann N Y Acad Sci 2004 1011:86-100[CrossRef][Medline]
  50. Agarwal A, Allamaneni SS. Role of free radicals in female reproductive diseases and assisted reproduction. Reprod Biomed Online 2004 9:338-347[Medline]
  51. Nasr-Esfahani MH, Aitken JR, Johnson MH. Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo. Development 1990 109:501-507[Abstract]
  52. Tarin JJ, Gomez-Piquer V, Pertusa JF, Hermenegildo C, Cano A. Association of female aging with decreased parthenogenetic activation, raised MPF, and MAPKs activities and reduced levels of glutathione S-transferases activity and thiols in mouse oocytes. Mol Reprod Dev 2004 69:402-410[CrossRef][Medline]
  53. Nicholls DG. Mitochondrial function and dysfunction in the cell: its relevance to aging and aging-related disease. Int J Biochem Cell Biol 2002 34:1372-1381[CrossRef][Medline]
  54. Jacobson J, Duchen MR. Interplay between mitochondria and cellular calcium signalling. Mol Cell Biochem 2004 256: –257 209-218
  55. Gordo AC, Rodrigues P, Kurokawa M, Jellerette T, Exley GE, Warner C, Fissore R. Intracellular calcium oscillations signal apoptosis rather than activation in in vitro aged mouse eggs. Biol Reprod 2002 66:1828-1837[Abstract/Free Full Text]
  56. Schultz RM. Regulation of zygotic gene activation in the mouse. Bioessays 1993 15:531-538[CrossRef][Medline]
  57. Muggleton-Harris AL, Brown JJ. Cytoplasmic factors influence mitochondrial reorganization and resumption of cleavage during culture of early mouse embryos. Hum Reprod 1988 3:1020-1028[Abstract/Free Full Text]
  58. Neganova IE, Sekirina GG, Eichenlaub-Ritter U. Surface-expressed E-cadherin, and mitochondrial and microtubule distribution in rescue of mouse embryos from 2-cell block by aggregation. Mol Hum Reprod 2000 6:454-464[Abstract/Free Full Text]
  59. Van Blerkom J. Microtubule mediation of cytoplasmic and nuclear maturation during the early stages of resumed meiosis in cultured mouse oocytes. Proc Natl Acad Sci U S A 1991 88:5031-5035[Abstract/Free Full Text]
  60. Brevini TA, Vassena R, Francisci C, Gandolfi F. Role of adenosine triphosphate, active mitochondria, and microtubules in the acquisition of developmental competence of parthenogenetically activated pig oocytes. Biol Reprod 2005 72:1218-1223[Abstract/Free Full Text]
  61. Pickering SJ, Johnson MH, Braude PR, Houliston E. Cytoskeletal organization in fresh, aged and spontaneously activated human oocytes. Hum Reprod 1988 3:978-989[Abstract/Free Full Text]
  62. St. John JC. Ooplasm donation in humans: the need to investigate the transmission of mitochondrial DNA following cytoplasmic transfer. Hum Reprod 2002 17:1954-1958[Abstract/Free Full Text]

Related articles in Biol Reprod:

Highlights
Mary Ann Handel, John Eppig, and Ursula Eichenlaub-Ritter
Biol Reprod 2005 73: 213. [Full Text]  



This article has been cited by other articles:


Home page
 Hum ReprodHome page
R. Fukuhara, S. Fujii, R. Nakamura, E. Yuzawa, H. Kimura, A. Fukui, and H. Mizunuma
Erythrocytes counteract the negative effects of female ageing on mouse preimplantation embryo development and blastocyst formation
Hum. Reprod., September 1, 2008; 23(9): 2080 - 2085.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
C. Tatone, F. Amicarelli, M. C. Carbone, P. Monteleone, D. Caserta, R. Marci, P. G. Artini, P. Piomboni, and R. Focarelli
Cellular and molecular aspects of ovarian follicle ageing
Hum. Reprod. Update, March 1, 2008; 14(2): 131 - 142.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
S. Cukurcam, I. Betzendahl, G. Michel, E. Vogt, C. Hegele-Hartung, B. Lindenthal, and U. Eichenlaub-Ritter
Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes
Hum. Reprod., March 1, 2007; 22(3): 815 - 828.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
G. A. Thouas, A. O. Trounson, and G. M. Jones
Developmental Effects of Sublethal Mitochondrial Injury in Mouse Oocytes
Biol Reprod, May 1, 2006; 74(5): 969 - 977.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
73/2/366    most recent
biolreprod.105.040956v2
biolreprod.105.040956v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Related articles in Biol Reprod
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thouas, G. A.
Right arrow Articles by Jones, G. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thouas, G. A.
Right arrow Articles by Jones, G. M.
Agricola
Right arrow Articles by Thouas, G. A.
Right arrow Articles by Jones, G. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS