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Although studies of both humans and animals suggest detrimental effects of psychological (restraint) stress on reproduction, reports concerning the direct effect of psychological (restraint) stress on the oocyte are few and conflicting. In the present study, a restraint system that allows mice free intake of feed and water while restraining their movement was established, and effects of maternal restraint on oocyte competence were examined by observing embryo development in vitro and in vivo. The results indicated that restraint stress applied to both gonadotropin-stimulated and unstimulated females during oocyte growth and maturation increased their plasma cortisol level but impaired ovulation and oocyte developmental potential. Injection of cortisol also decreased oocyte developmental potential in both stimulated and unstimulated mice. However, whereas restraint stress reduced the plasma follicle-stimulating hormone (FSH) level of unstimulated mice, injection of cortisol did not. Because the stimulated mice had received very high doses of FSH and luteinizing hormone from injection with equine chorionic gonadotropin injection, the results suggested that whereas cortisol acts directly on the ovary to damage the oocyte, restraint stress impairs oocyte competence by actions on both the hypothalamic-pituitary-gonadal and the hypothalamic-pituitary-adrenal axes. However, exposing the cumulus-oocyte complexes (COCs) to physiological levels of cortisol did not affect oocyte nuclear and cytoplasmic maturation in vitro. Thus, cortisol might have impaired ovulation and oocyte potential by an indirect effect on ovarian tissues other than the COCs.

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The objectives of this study were to investigate the effect of heat stress during in vitro maturation on the developmental potential of mouse oocytes and to determine whether the deleterious effect was on the nuclear or cytoplasmic component. While rates of oocyte nuclear maturation (development to the metaphase II stage) did not differ from 37 to 40 8C, rates for blastocyst formation decreased significantly as maturation temperature increased from 38.5 to 39 8C. Chromosome spindle exchange showed that while blastocyst formation did not differ when spindles matured in vivo or in vitro at 37, 40 or 40.7 8C were transplanted into in vivo matured cytoplasts, no blastocyst formation was observed when in vivo spindles were transferred into the 40 8C cytoplasts. While oocytes reconstructed between 37 8C ooplasts and 37 or 40 8C karyoplasts developed into 4-cell embryos at a similar rate, no oocytes reconstituted between 40 8C ooplasts and 37 8C spindles developed to the 4-cell stage. Immunofluorescence microscopy revealed impaired migration of cortical granules and mitochondria in oocytes matured at 40 8C compared with oocytes matured at 37 8C. A decreased glutathione/GSSG ratio was also observed in oocytes matured at 40 8C. While spindle assembling was normal and no MAD2 was activated in oocytes matured at 37 or 40 8C, spindle assembling was affected and MAD2 was activated in some of the oocytes matured at 40.7 8C. It is concluded that 1) oocyte cytoplasmic maturation is more susceptible to heat stress than nuclear maturation, and 2) cytoplasmic rather than nuclear components determine the pre-implantation developmental capacity of an oocyte.

Studies in both humans and animals suggest detrimental effects of psychological stress on reproduction. Although our recent study shows that maternal-restraint stress diminishes oocyte developmental potential, the mechanism behind this effect is unknown. This prompted us to study the potential role of maternal-restraint stress in the genesis of aneuploidy during meiosis I. At 24 h after equine chorionic gonadotropin injection, mice were subjected to restraint stress for 24 h. After the restraint, some mice were killed to recover immature oocytes for in vitro maturation, while others were injected with human chorionic gonadotropin to recover in vivo matured oocytes. Analysis on chromosome complements of both mature oocytes and parthenotes confirmed that maternal restraint increased aneuploidy in both in vivo and in vitro matured oocytes and that the percentage of aneuploid oocytes were three times higher in the earlier matured oocytes than in the later matured ones. Further observations indicated that maternal restraint 1) impaired metaphase I (MI) spindle assembly while inhibiting MAPK activities, 2) accelerated progression of anaphase I while down-regulating the expression of spindle assembly checkpoint (SAC) proteins, and 3) induced intraoocyte oxidative stress. The following possible model was proposed to explain the results. Maternal-restraint stress increased oocyte aneuploidy by impairing MI spindle assembly and decreasing the SAC. Whereas abnormal spindles would affect centromere attachments, a reduction in SAC would accelerate the anaphase I progression. Failure of centromere attachment, together with the hastened anaphase, would result in nondisjunction of the unattached chromosomes. Furthermore, maternal-restraint stress might also impair spindle assembly and SAC function by inducing intraoocyte oxidative stress, which would then reduce MAPK activity, a critical regulator of microtubule assembly and the establishment and maintenance of the SAC during oocyte maturation.

This study tested the hypothesis that oocyte aging could be prevented for a longer time by reducing the culture temperature while supplementing the culture medium with more pyruvate. Newly ovulated mouse oocytes were cultured at various temperatures for various times in HCZB medium (Kimura and Yanagimachi, Biol Reprod 1995; 52:709-720) containing various concentrations of pyruvate before examining for aging parameters and developmental potential. The increase in susceptibility to activating stimuli was efficiently prevented when oocytes were cultured in HCZB with 10.27 mM pyruvate at 37°C for 6 h, 25°C for 24 h, 15°C for 96 h, and 5°C for 48 h. Satisfactory blastocyst development of both parthenotes and fertilized zygotes was achieved after oocyte culture in HCZB containing 10.27 mM pyruvate at 37°C for 6 h, 25°C for 24 h, 15°C for 36 h, and 5°C for 24 h. Transfer of two-cell embryos or blastocysts showed no difference between newly ovulated control oocytes and oocytes cultured at 15°C for 36 h in either term pregnancy, live young per pregnant recipient, live young/transferred embryos, or birth weight of young. Oocytes with impaired developmental potential after culture at 15°C for 96 h and at 5°C for 48 h showed unrecoverable decreases in the content of glutathione, the glutathione/oxidized glutathione ratio, the BCL2 content, and in the numbers of oocytes with normal spindles and cortical granule distribution, suggesting induction of oxidative stress, which caused oocyte apoptosis and cytoskeleton alterations by downregulating BCL2. Because oocytes cultured at 15°C for 36 h were activated or fertilized after a 6-h recovery culture, aging of ovulated mouse oocytes has been successfully prevented for 42 h without impairing their developmental potential.