Quantitative and qualitative changes of mitochondria in human preimplantation embryos.

PURPOSE: The oxygen consumption rates (OCRs) in mice and cattle have been reported to change during preimplantation embryogenesis. On the other hand, mitochondrial DNA (mtDNA) copy number has been shown to be unchanged in mice and changed in cattle and pigs. The interactions between mitochondrial functions and mtDNA copy numbers in human embryos during preimplantation development remain obscure. METHODS: Sixteen oocytes and 100 embryos were used to assess mtDNA copy numbers and OCR. Three oocytes and 12 embryos were used to determine cytochrome c oxidase activity. All specimens were obtained between July 2004 and November 2014, and donated from couples after they had given informed consent. Mature oocytes and embryos at 2-14-cell, morula, and blastocyst stages were used to assess their OCR in the presence or absence of mitotoxins. The mtDNA copy number was determined using the samples after analysis of OCR. The relationships between developmental stages and OCR, and developmental stages and mtDNA copy number were analyzed. Furthermore, cytochrome c oxidase activity was determined in oocytes and 4-cell to blastocyst stage embryos. RESULTS: The structure of inner mitochondrial membranes and their respiratory function developed with embryonic growth and the mtDNA copy numbers decreased transiently compared with those of oocytes. The undifferentiated state of inner cell mass cells appears to be associated with a low OCR. On the other hand, the mtDNA copy numbers increased and aerobic metabolism of mitochondria increased in trophectoderm cells. CONCLUSIONS: The mitochondrial respiratory function of human embryos developed along with embryonic growth although the copy numbers of mtDNA decreased transiently before blastulation. OCRs increased toward the morula stage ahead of an increase of mtDNA at the time of blastulation. Data regarding changes in mitochondrial function and mtDNA copy number during preimplantation development of human embryos will be useful for the development of ideal culture media.

Dynamic changes in mitochondrial distribution in human oocytes during meiotic maturation.

PURPOSE: The change of mitochondrial distribution in human oocytes during meiotic maturation was assessed using 223 human oocytes donated from patients undergoing fertility treatment between June 2013 and February 2016. METHODS: Live cell images of fluorescence-labelled mitochondria in human oocytes were analysed to investigate dynamic changes in mitochondrial distribution during meiotic maturation using a confocal microscope combined with an incubator in the presence or absence of colchicine and cytochalasin B, inhibitors for tubulin and actin filament, respectively. Subcellular distribution of mitochondria in human oocytes was also assessed at various stages using a transmission electron microscope (TEM). RESULTS: Live cell imaging analysis revealed that the mitochondria-occupied cytoplasmic area decreased from 83 to 77 % of the total cytoplasmic area around 6 h before germinal vesicle breakdown (GVBD) and that mitochondria accumulated preferentially close to the perinuclear region. Then, the mitochondria-distributed area rapidly increased to 85 % of total cytoplasm at the time of GVBD. On the other hand, there was no significant change in mitochondrial distribution before and after polar body extrusion. Such changes in mitochondrial localization were affected differently by colchicine and cytochalasin B. Most of mitochondria in the cytoplasm formed cluster-like aggregates before GVBD while they distributed homogeneously after GVBD. CONCLUSIONS: Most mitochondria localized predominantly in the non-cortical region of the cytoplasm of GV stage-oocytes, while the mitochondria-occupied area decreased transiently before GVBD and increased rapidly to occupy the entire area of the cytoplasm at GVBD by some cytoskeleton-dependent mechanism.

[Trends of refractive correction in the Japanese Ground Self-Defense Forces: examination after the Great East Japan earthquake].

PURPOSE: To investigate the trends in refractive correction in Japanese Ground Self-Defense Forces' (JGSDF) soldiers. METHODS: A questionnaire was distributed to 519 soldiers of the Camp Funaoka in the northeastern region. RESULTS: Five hundred and sixteen subjects (99.4%) responded. In total, 246 soldiers (47.7%) wore spectacles or contact lenses. Among the contact lens wearers, 52 had experienced problems previously and 35 reported problems that occurred during military exercises. With regard to military exercises, 66.9% and 63.5% of the spectacle or disposable soft contact lens-users reported inconvenience. Among contact lens users, 33.1% reported that they changed to new sterilized contact lenses only occasionally and 61.9% did not change their contact lenses at all during exercises. During disaster-relief work, subjects worried 'very' (21.5%) or 'somewhat' (46.9%) about problems associated with their spectacles or contact lenses. Twenty-four had undergone refractive surgery (4.9%). CONCLUSION: About one-half of the JGSDF soldiers surveyed required refractive correction. Attention to convenient and safe refractive correction in the military is warranted.

Effect of maternal age on the ratio of cleavage and mitochondrial DNA copy number in early developmental stage bovine embryos.

Age-associated deterioration in both the quality and quantity of mitochondria occurs in older women. The main aim of this study was to examine the effect of age on mitochondrial DNA copy number (mtDNA number) in early developmental stage bovine embryos as well as the dynamics of mtDNA number during early embryo development. Real-time PCR was used to determine mtDNA number. In vitro-produced embryos 48 h after insemination derived from Japanese black cows, ranging in age from 25 to 209 months were categorized based on their cleavage status. There was an overall negative relationship between the age of the cow and cleavage status, to the extent that the ratio of embryos cleaved over the 4-cell stage was greater in younger cows. The mtDNA number did not differ among the cleaved status of embryos. In the next experiment, oocytes collected from each donor cow were divided into 2 groups containing 10 oocytes each, in order to compare the mtDNA number of mature oocytes and early developmental stage embryos within individuals. Upon comparing the mtDNA number between oocytes at the M2 stage and early developmental stage 48 h post insemination, mtDNA number was found to decrease in most cows, but was found to increase in some cows. In conclusion, age affects the cleaving ability of oocytes, and very old cows (> 180 months) tend to have lower mtDNA numbers in their oocytes. The change in mtDNA number during early development varied among individual cows, although overall, it showed a tendency to decrease.

Effect of bovine age on the proliferative activity, global DNA methylation, relative telomere length and telomerase activity of granulosa cells.

Granulosa cells influence the growth and acquisition of the developmental competence of oocytes. We investigated the effects of ageing on the proliferative activity, global genomic DNA methylation, relative telomere length and telomerase activity of bovine granulosa cells. The proliferative activity of cells was examined by bromodeoxyuridine (BrdU) assay, genomic DNA methylation was examined by enzyme-linked immunosorbent assay (ELISA), and relative telomere length and telomerase activity were examined by real-time polymerase chain reaction. We first compared the proliferative activity of the granulosa cells of the medium follicles between in dominant phase ovaries and growth phase ovaries. We observed that the proliferative activity of the granulosa cells of dominant phase ovaries was significantly lower than those of growth phase ovaries. In addition, the proliferative activity of granulosa cells was inversely associated with follicular size. Based on the results, we used granulosa cells harvested from the medium follicles (3-5 mm in diameter) on the surfaces of the dominant phase ovaries collected from cows at a slaughterhouse. The proliferative activity of the granulosa cells harvested from the ovaries of old cows (N = 8; average age 165.1 months) was lower than that of the cells from young cows (N = 8; average age 30.9 months). Global loss of cytosine methylation was detected in the granulosa cells of old cows (N = 12; average age 141.0 months) compared with young cows (N = 15; average age 27.4 months). Although the relative telomere lengths of cumulus cells were similar in the two age groups, the relative telomere lengths and telomerase activity of the granulosa cells from old cows (N = 17 and 9; average age, 164.6 and 151.3 months, respectively) tended to be shorter than those of the cells from young cows (N = 17 and 10; average age 30.6 and 28.1 months, respectively); however, this difference was not significant p = 0.09 and 0.053, respectively). In conclusion, the proliferative activity and genomic global DNA methylation significantly decreased, and the relative telomere lengths and telomerase activity of granulosa cells tended to be shorter with the age of donor cows.

Effect of maternal age on mitochondrial DNA copy number, ATP content and IVF outcome of bovine oocytes.

The primary aim of the present study was to examine the effect of maternal age (in months) on mitochondrial DNA copy number (Mt number), ATP content and IVF outcome of bovine oocytes. We also compared the Mt number of oocytes with fertilisation outcome and ATP content. Oocytes were collected from cows aged 20-204 months and the Mt number was determined by real-time polymerase chain reaction. The Mt number in immature and mature oocytes was determined to be 368,118 and 807,794, respectively; the ATP content in these oocytes was 1.2 and 2.0 pM, respectively. Both Mt number and ATP content increased during oocyte maturation. However, after 90 months of age, the Mt number of mature oocytes decreased with increasing maternal age, whereas the ATP content of mature oocytes was positively correlated with maternal age (P<0.01); there was no obvious relationship observed between Mt number and ATP content. Furthermore, maternal age was positively correlated with the abnormal fertilisation rate (P<0.01). Mt number and fertilisation outcome were unrelated, but the nature of this relationship differed between young (21-89 months) and old (>89 months) cows. Thus, we conclude that Mt number, the ATP content and fertilisation outcome of bovine oocytes are affected by maternal age.

Light scattering and optical aberrations as objective parameters to predict visual deterioration in eyes with cataracts.

PURPOSE: To predict the visual deterioration of eyes with cortical (CC) or nuclear (NC) cataract from objective data on ocular higher-order aberration (HOA) and forward (FLS) and backward light scattering (BLS). SETTING: Osaka University Medical School, Osaka, Japan. METHODS: Twenty-two eyes with mild NC, 41 eyes with mild CC, and 11 normal eyes were examined. Higher-order aberrations were calculated with the Zernike polynomials up to the fourth order from the values obtained by wavefront analysis using the Hartmann-Shack aberrometer. Forward light scattering was calculated from the size of the aberrometer spot images for the central 4 mm, and backward light scattering (BLS) was calculated from the optical density of the Scheimpflug images. The relationship between the area under the log contrast sensitivity function (AULCSF) curve and HOAs, FLS, and BLS was examined. RESULTS: Area under the log contrast sensitivity function was moderately correlated with the HOAs, FLS, and BLS. Multiple linear regression analysis revealed that the AULCSF was predicted by the linear combination of these variables (R(2)=.484, P<.001). Area under the log contrast sensitivity was predicted by BLS and HOA (R(2)=.555) in the NC group and by FLS and HOAs (R(2)=.540) in the CC group. CONCLUSIONS: Loss of contrast sensitivity was predominantly due to BLS and HOA in eyes with NC and FLS and HOA in eyes with CC. Higher-order aberrations, FLS, and BLS, variables that are obtained objectively, can be used to predict quantitatively the visual deterioration in cataractous eyes.

[The effect of substituting latanoprost 0.005% for unoprostone 0.12%].

PURPOSE: To evaluate the effect of substituting latanoprost(LAT) 0.005% for unoprostone(UNO) 0.12% after a trial of unilateral treatment. METHODS: We treated 30 patients with primary open-angle glaucoma(n = 8), ocular hypertension (n = 1), or normal-tension glaucoma(n = 21) with UNO for 4 weeks in one eye and then substituted LAT for UNO. Four weeks later we measured the intraocular pressure(IOP) in the ipsilateral eye. RESULTS: The mean baseline IOP level was 18.6 +/- 3.8(mean +/- standard deviation) mmHg. The mean IOP levels(reduction rates) after UNO and LAT therapy were 16.7 +/- 3.1 mmHg (16.6%) and 14.1 +/- 3.2 mmHg (28.9%), respectively(p < 0.001). All patients who responded to UNO also responded to LAT; however, 55% of those who did not respond to UNO responded to LAT. CONCLUSIONS: If LAT is substituted for UNO, it can be predicted that 63.3% of the patients will respond.