Shu Huei Kao

Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility

Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.

Simultaneous Increase of Mitochondrial DNA Deletions and Lipid Peroxidation in Human Aginga

Human mtDNA is a naked circular double-stranded DNA, which is continually exposed to the matrix that contains high levels of ROS and free radicals. High oxidative stress and a lack of proofreading during mtDNA replication and efficient DNA repair mechanisms in the mitochondria have rendered mtDNA extremely vulnerable to oxidative damage. More than one dozen large-scale deletions in mtDNA have been identified in various tissues of old humans. The 4,977-bp and 7,436-bp deletions are the most prevalent and abundant ones. The onset age of various mtDNA deletions varies greatly with tissues of each individual and type of deletion. In this and previous studies, we have demonstrated with PCR techniques that the frequency of occurrence and the proportion of the 4,977-bp and 7,436-bp deleted mtDNAs are significantly increased with the age of the human. The mtDNA deletions are not detectable in any tissues from young healthy subjects or blood cells from normal individuals of any age, which indicates that the deletions are generated and accumulated only in postmitotic cells upon aging. Moreover, we found that these mtDNA deletions occur more frequently and abundantly in tissues with high energy demand (e.g., muscle) as compared to those with low energy demand. On the other hand, we found that the amount of lipid peroxides measured as malondialdehyde and the activity of manganese-superoxide dismutase in the mitochondria exhibit an age-dependent increase in various human tissues. The lipid peroxide level in muscle was significantly higher than that in the other tissues. Moreover, we found a positive correlation between the proportion of the 4,977-bp deleted mtDNA and lipid peroxide content in the mitochondria of human tissues during aging. Muscle the tissue of high energy demand, was found to be more vulnerable to oxidative damage that lead to most abundant mtDNA deletions and lipid peroxidation among all the tissues examined. Taking these results together, we suggest that the enhanced generation of reactive oxygen species and lipid peroxides in the mitochondria during the aging process occur simultaneously with large-scale deletions and the other types of mutations in mtDNA, which are early molecular events and major contributory factors of human aging.

Abnormal mitochondrial structure in human unfertilized oocytes and arrested embryos

Abstract: To clarify the relationship between mitochondria and embryo development, we collected human unfertilized oocytes, early embryos, and arrested embryos. Unfertilized oocytes and poor‐quality embryos were collected, and the ultrastructure of mitochondria was determined by transmission electron micrography. Four criteria for determining the mitochondrial state were mitochondrial morphology, cristae shape, location, and number of mitochondria. In mature oocytes, mitochondria were rounded with arched cristae and a dense matrix and were distributed evenly in the ooplasm. In pronuclear zygotes, the size and shape of mitochondria were similar to those in mature oocytes; however, mitochondria appeared to migrate and concentrate around pronuclei. In this study, 67% of examined unfertilized oocytes had fewer mitochondria in the cytoplasm. A decreased number of mitochondria located near the nucleus was also demonstrated in 60% of arrested embryos. Fewer differentiated cristae were determined in all three arrested blastocyst stages of embryos. The relative expressions of oxidative phosphorylation genes in oocytes and embryos were also determined. These data imply that inadequate redistribution of mitochondria, unsuccessful mitochondrial differentiation, or decreased mitochondrial transcription may result in poor oocyte fertilization and compromised embryo development.

Repeated ovarian stimulations induce oxidative damage and mitochondrial DNA mutations in mouse ovaries

Abstract: Superovulation by injection of exogenous gonadotropin is the elementary method to produce in vivo‐derived embryos for embryo transfer in women. Increased oocyte aneuploidy, embryo mortality, fetal growth retardation, and congenital abnormalities have been studied at higher‐dose stimulations. Ovarian and oocyte biological aging possibly may have adverse implications for human oocyte competence with repeated hyperstimulation. In this study, we found that reduced competence for the human oocyte has been associated with degenerative embryo upsurge during embryo culture and failure to develop into the blastocyst stage in the three, four, five, and six stimulation cycles. On the other hand, the numbers of ovulated oocytes were decreased in the groups with more ovarian stimulation. More aggregated mitochondria were found in the cytoplasm of the repetitively stimulated embryos. Higher amounts of oxidative damage including 8‐OH‐dG, lipoperoxides, and carbonyl proteins were also revealed in the ovaries with more cycle numbers of ovarian stimulation. Higher proportions of mtDNA mutations were also found. The detected molecular size of the mutated band was approximately 675 bp. Increased amounts of carbonyl proteins were also revealed after repeated stimulation. An understanding of the relationship between oocyte competence and ovarian responses to stimulation in the mouse may provide insights into the origin of oocyte defects and the biology of ooplasmic aging that could be of clinical relevance in the diagnosis and treatment of human infertility.

Phenytoin-mediated oxidative stress in serum of female epileptics: A possible pathogenesis in the fetal hydantoin syndrome:

1 The concentration of serum malondialdehyde (MDA) was measured as the index of lipid peroxidation in female epileptics with phenytoin (PHT) monotherapy. Sera from 20 female epileptics with PHT monotherapy, 12 female epileptics without anticonvulsant therapy and 20 female healthy controls were sampled. The levels of serum copper (S-Cu), serum zinc (S-Zn), copper/zinc superoxide dismutase (CuZn-SOD), and reduced glutathione (GSH) were analyzed as inter active factors of the oxidative stress. 2 For the female epileptics with PHT monotherapy, serum MDA concentration (2.6 ± 0.7 μM vs control 1.8 ± 0.6 μM, P < 0.05), CuZn-SOD activity (178.2 ± 63.5 U/dL vs control 97.1 ± 36.4 U/dL, P < 0.01), and S-Cu content (126.2 ± 36.1 μg/dL vs control 98.4 ± 16.7 μg/dL, P < 0.05) were significantly in creased, but GSH level (27.5±6.8 μM vs control 32.2 ± 5.7 μM, P < 0.05) was significantly decreased. The level of serum MDA was associated with the elevation of CuZn-SOD activity (r=0.54, P < 0.05) and S-Cu content ( r=0.44, P < 0.05) in all the samples collected from epileptics and controls. However, there were no significant differences in all the above parameters between the female epileptics without anticonvulsant therapy and healthy controls. 3 These results indicated that oxidative stress was enhanced in the female epileptics with PHT-mono therapy. Apart from the reactive PHT intermediate, the abnormal metabolism of S-Cu, CuZn-SOD, and GSH was highly involved in the PHT-mediated toxicity. Supplement of GSH, modification of CuZn-SOD enzyme activity and reduction of the absorption of copper may prevent the incidence of fetal hydantoin syndrome during pregnancy.

Oxidative Damage and Mitochondrial DNA Mutations with Endometriosis

Abstract: Endometriosis, a frequently encountered disease in gynecology, is a considerable threat to the physical, psychological, and social integrity of women. Moreover, up to 50% of infertile patients have this disease. The etiology and pathogenesis of this important disease are poorly understood; it is defined as an ectopic location for endometrium‐like glandular epithelium and stroma outside of the uterine cavity. It still remains an open question as to what extent the peritoneal environment influences the establishment and/or progression of endometriosis. As a result of such stress, a sterile, inflammatory reaction with the secretion of growth factors, cytokines, and chemokines is generated, which is especially deleterious to successful reproduction. Significantly higher amounts of oxidative damage were detected in endometriotic lesions than in controlled normal endometrium, including mitochondrial DNA (mtDNA) rearrangement, 8‐OH‐deoxyguanosine (8‐OH‐dG), and lipoperoxide contents. There were approximately sixfold increases in 8‐OH‐dG and lipoperoxides in chocolate cysts compared with normal endometrial tissues. A novel 5,335‐bp deletion of mtDNA was identified in endometriotic tissue. According to these results, we propose that oxidative stress and mtDNA mutations might be anticipated in the initiation or progression of endometriosis. Only by understanding the mechanisms involved in the pathogenesis of endometriosis can we develop a basis for new diagnostic and therapeutic approaches.

Expression of the pluripotent transcription factor OCT4 promotes cell migration in endometriosis.

OBJECTIVE To identify the impact of the pluripotent transcription factor OCT4 in endometrial cell migration and endometriosis. DESIGN The OCT4 expression and cell migration study. SETTING Research institution and reproductive medical clinic. PATIENT(S) Nine subjects with normal endometrium, 3 subjects with normal myometrium, 36 patients with hyperplastic endometrium, and 58 patients with endometriosis. INTERVENTION(S) The expression of OCT4 messenger RNA in normal endometrium, normal myometrium, hyperplastic endometrium, and ectopic endometriotic tissues was analyzed using reverse transcription and quantitative real-time polymerase chain reaction (PCR). The effect of OCT4 expression on the migration activity of the endometrial cells was examined. MAIN OUTCOME MEASURE(S) Reverse transcription and quantitative real-time PCR, Western blotting, and wound closure and transwell assays. RESULT(S) The expression of OCT4 and NANOG messenger RNA was significantly higher in ectopic endometriotic tissues, compared with that of the normal endometrium, the normal myometrium, and the hyperplastic endometrium. The level of OCT4 messenger RNA in endometriotic tissues was positively correlated with the expression of genes associated with cell migration. Overexpression of the OCT4 protein in primary human endometriotic stromal cells and human RL95-2 and HEC1A endometrial carcinoma cell lines resulted in decreased levels of E-CADHERIN, the increased expression of the VIMENTIN, TWIST, and SLUG proteins, and an increase in the migration activity of endometrial cells in transwell and wound closure assays. CONCLUSION(S) The transcription of the OCT4 gene is significantly up-regulated in human ectopic endometriotic tissues. The expression of OCT4 may contribute to the pathology of ectopic endometrial growth by stimulating the migration activity of endometrial cells.

A prevalent POLG CAG microsatellite length allele in humans and African great apes.

The human nuclear gene for the catalytic subunit of mitochondrial DNA polymerase γ (POLG) contains within its coding region a CAG microsatellite encoding a polyglutamine repeat. Previous studies demonstrated an association between length variation at this repeat and male infertility, suggesting a mechanism whereby the prevalent (CAG)10 allele, which occurs at a frequency of >80% in different populations, could be maintained by selection. Sequence analysis of the POLG CAG microsatellite region of more than 1000 human chromosomes reveals that virtually all allelic variation at the locus is accounted for by length variation of the CAG repeat. Analysis of POLG from African great apes shows that a prevalent length allele is present in each species, although its exact length is species-specific. In common chimpanzee (Pan troglodytes) a number of different sequence variants contribute to the prevalent length allele, strongly supporting the idea that the length of the POLG microsatellite region, rather than its exact nucleotide or amino acid sequence, is what is maintained. Analysis of POLG in other primates indicates that the repeat has expanded from a shorter, glutamine-rich sequence, present in the common ancestor of Old and New World monkeys.

Smoking-associated mitochondrial DNA mutations in human hair follicles

The mitochondrial DNA (mtDNA) of hair follicles was used for studying the genotoxicity of smoking‐mediated carcinogens. We determined the incidences of the 4,977 bp and 7,436 bp mtDNA deletions, tandem duplication in the D‐loop region and the proportion of the 4,977 bp deleted mtDNA (dmtDNA) in the total DNA of hair follicles from 213 male non‐smokers and 74 male smokers, respectively. Twenty‐three patients with lung cancer were also investigated. We found that the current cigarette smokers had a 3.1 times higher average incidence of the 4,977 bp dmtDNA (RR: 3.1, P < 0.001) as compared with non‐smokers, and this mtDNA deletion was especially prevalent in the old heavy smokers. For the smokers of the age above 70, the average incidence of the 4,977 bp dmtDNA was 3.7 times higher in the group with a smoking index of 401–800 (RR: 3.7, P < 0.005) and 3.2 times higher in the group with a smoking index greater than 800 (RR: 3.2, P < 0.005). However, there was no statistically significant relationship between the incidence of the 7,436 bp dmtDNA and the smoking index, although there was a mild increase in the percentage of the 7,436 bp dmtDNA with the increase of the consumption of cigarettes. No tandem duplication of mtDNA in the D‐loop region was disclosed in either smokers or non‐smokers group. The proportions of the 4,977 bp dmtDNA in hair follicles were found to correlate with age, but did not keep increasing with cigarette consumption except in the group of subjects with a smoking index of less than 400. On the other hand, we found that the average proportion of the 4,977 bp dmtDNA in the hair follicles was 1.201 ± 0.371% for the patients with lung cancer who had a smoking index greater than 400, while that was only 0.146% for the age‐matched healthy smokers with the same smoking index. In conclusion, the high incidence of the 4,977 bp dmtDNA of hair follicles is not only associated with aging but also correlated with the amount of cigarette smoking. A high proportion of the 4,977 bp dmtDNA in the hair follicles may be considered one of the molecular events that are associated with the occurrence of smoking‐associated cancers. Environ. Mol. Mutagen. 30:47–55, 1997

Celecoxib induces heme-oxygenase expression in glomerular mesangial cells.

Abstract: Nonsteroidal anti‐inflammatory drugs (NSAIDs) are frequently used as analgesics. They inhibit cyclooxygenases (COX), preventing the formation of prostaglandins, including prostacyclin and thromboxane. A serious side effect of COX‐1 and COX‐2 inhibitors is renal damage. To investigate the molecular basis of the renal injury, we evaluated the expression of the stress marker, heme oxygenase‐1 (HO‐1), in celecoxib‐stimulated mesangial cells. We report here that a COX‐2 selective NSAID, celecoxib, induced a concentration‐ and time‐dependent increase of HO‐1 expression in glomerular mesangial cells. Celecoxib‐induced HO‐1 protein expression was inhibited by actinomycin D and cycloheximide, suggesting that de novo transcription and translation are required in this process. N‐acetylcysteine, a free radical scavenger, strongly decreased HO‐1 expression, suggesting the involvement of reactive oxygen species (ROS). Celecoxib‐induced HO‐1 expression was attenuated by pretreatment of the cells with SP 600125 (a specific JNK inhibitor), but not SB 203580 (a specific p38 MAPK inhibitor), or PD 98059 (a specific MEK inhibitor). Consistently, celecoxib activated c‐Jun N‐terminal kinase (JNK) as demonstrated by kinase assays and by increasing phosphorylation of this kinase. N‐acetylcysteine reduced the stimulatory effect of celecoxib on stress kinase activities, suggesting an involvement of JNK in HO‐1 expression. On the other hand, LY 294002, a phosphatidylinositol 3‐kinase (PI‐3K)‐specific inhibitor, prevented the enhancement of HO‐1 expression. This effect was correlated with inhibition of the phosphorylation of the PDK‐1 downstream substrate Akt/protein kinase B (PKB). In conclusion, our data suggest that celecoxib‐induced HO‐1 expression in glomerular mesangial cells may be mediated by ROS via the JNK‐PI‐3K cascade.