Aggregated chromosomes/chromatin transfer: a novel approach for mitochondrial replacement with minimal mitochondrial carryover: the implications of mouse experiments for human aggregated chromosome transfer.

Nuclear transfer techniques, including spindle chromosome complex (SC) transfer and pronuclear transfer, have been employed to mitigate mitochondrial diseases. Nevertheless, the challenge of mitochondrial DNA (mtDNA) carryover remains unresolved. Previously, we introduced a method for aggregated chromosome (AC) transfer in human subjects, offering a potential solution. However, the subsequent rates of embryonic development have remained unexplored owing to legal limitations in Japan, and animal studies have been hindered by a lack of AC formation in other species. Building upon our success in generating ACs within mouse oocytes via utilization of the phosphodiesterase inhibitor 3-isobutyl 1-methylxanthine (IBMX), this study has established a mouse model for AC transfer. Subsequently, a comparative analysis of embryo development rates and mtDNA carryover between AC transfer and SC transfer was conducted. Additionally, the mitochondrial distribution around SC and AC structures was investigated, revealing that in oocytes at the metaphase II stage, the mitochondria exhibited a relatively concentrated arrangement around the spindle apparatus, while the distribution of mitochondria in AC-formed oocytes appeared to be independent of the AC position. The AC transfer approach produced a marked augmentation in rates of fertilization, embryo cleavage, and blastocyst formation, especially as compared to scenarios without AC transfer in IBMX-treated AC-formed oocytes. No significant disparities in fertilization and embryo development rates were observed between AC and SC transfers. However, relative real-time PCR analyses revealed that the mtDNA carryover for AC transfers was one-tenth and therefore significantly lower than that of SC transfers. This study successfully accomplished nuclear transfers with ACs in mouse oocytes, offering an insight into the potential of AC transfers as a solution to heteroplasmy-related challenges. These findings are promising in terms of future investigation with human oocytes, thus advancing AC transfer as an innovative approach in the field of human nuclear transfer methodology.

Ageing and chronic disease-related changes in the morphometric characteristics of ovarian follicles in cynomolgus monkeys (Macaca fascicularis).

STUDY QUESTION: How is the localisation of ovarian follicles affected by ageing and chronic diseases? SUMMARY ANSWER: Ovarian follicles shift deeper towards the medulla, due to thickening of the tunica albuginea (TA), with ageing and some major common chronic diseases. WHAT IS KNOWN ALREADY: The ovary undergoes morphological and functional changes with ageing. The follicular pool follows these changes with alterations in the amount and distribution of residual follicles. Diseases causing a chronic inflammatory process are associated with morphological changes and impaired ovarian function. STUDY DESIGN, SIZE, DURATION: We conducted a cross-sectional study, examining 90 ovaries from 90 female monkeys. The samples were collected from April 2018 to March 2019 at Tsukuba Primate Research Center in National Institutes of Biomedical Innovation, Health and Nutrition, Japan. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian samples were obtained from cynomolgus monkeys that died from natural causes or were euthanised. Ovarian sections were stained with haematoxylin and eosin (H&E) for histological analyses. In ovarian sections from 64 female macaques aged 0-25 years, a total of 13 743 follicles at different developmental stages (primordial, intermediary, primary, early secondary and late secondary) were assessed to determine the depth of each follicle from the outer surface of the ovarian cortex to the far end of the follicle, by using a digital imaging software. TA thickness was measured as sum of basal membrane and tunica collagen layer for each ovary under H&E staining. To explore the possibility of age-related trends in ovarian morphometric characteristics, samples were divided into four different age groups (0-3 years (pre-menarche), 4-9 years, 10-14 years and 15-20 years). To evaluate the effect of common chronic diseases on ovarian morphometric characteristics, macaques with diabetes mellitus (DM) (n = 10), endometriosis (n = 8) or inflammatory bowel disease (IBD) (n = 8) were compared to age-matched controls without chronic diseases. MAIN RESULTS AND THE ROLE OF CHANCE: Ovarian morphometric analysis revealed that the relative location of follicles became deeper in all age groups according to development of follicles (P < 0.05). Total follicle distance from the ovarian surface was increased with ageing (P < 0.05). In a sub-analysis according to developmental stage, only primordial and intermediary follicles were localised deeper with increasing age (P < 0.05). TA thickness was also increased with ageing (P < 0.05). The localisation of the total number of follicles became deeper in ovaries from monkeys with DM, endometriosis or IBD as compared to the control group (P < 0.05). With DM, analysis of follicles distance at almost each developmental stage was significantly deeper compared to controls (P < 0.05) with the exception of early secondary follicles. With endometriosis, follicles at primary and early and late secondary stages were significantly deeper compared to controls (P < 0.05). Also with IBD, follicles at primary and early and late secondary follicles were significantly deeper compared to controls (P < 0.001). The TA was thicker with DM and endometriosis compared to controls (P < 0.05), but not with IBD (P = 0.16). LARGE SCALE DATA: NA. LIMITATIONS, REASONS FOR CAUTION: Two-dimensional histology was used to assess follicle localisation. The possibility of minimal variations between the measured distance to the actual distance in a spherical structure cannot be excluded. Additionally, the severity of disease was not assessed. WIDER IMPLICATIONS OF THE FINDINGS: This study is the first step towards enhancing our understanding of how ageing and chronic diseases affect the relative localisation of dormant and developing follicles. These observations, combined with possible future human studies, may have managerial implications in the field of fertility preservation and other conditions involving ovarian tissue cryopreservation. STUDY FUNDING/COMPETING INTEREST(S): The present work was supported by the Grant-in-Aid for Scientific Research B (19H03801) (to K.K.), Challenging Exploratory Research (18K19624), Japan Agency for Medical Research and Development, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Takeda Science Foundation and Naito Foundation (to K.K.). All authors have no conflicts of interest directly relevant to the content of this article.

Spontaneous endometriosis in cynomolgus monkeys as a clinically relevant experimental model.

STUDY QUESTION: Does spontaneous endometriosis in cynomolgus monkeys have the characteristics required of a good experimental model? SUMMARY ANSWER: Spontaneous endometriosis in cynomolgus monkeys exhibited similar clinicopathological characteristics to the human disease and was useful as an experimental model. WHAT IS KNOWN ALREADY: The prevalence of endometriosis in autopsied cynomolgus monkeys (Macaca fascicularis) in a breeding colony was reported to be 28.7% in 1993. The histopathological findings we reported recently showed that components of spontaneous endometriosis were not only endometriotic epithelium and stromal cells (CD10-positive) with hemorrhage and inflammation, but also smooth muscle metaplasia and nerve fibers. STUDY DESIGN, SIZE, DURATION: During routine medical examinations at a research facility from 2008 to 2012, 614 female cynomolgus monkeys of reproductive age (6-25 years) were screened for endometriosis by the presence of regular menstrual bleeding, serum CA125 levels and palpation of the abdomen. In total, 29 monkeys were selected as subjects for the following study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 29 monkeys selected, 15 were diagnosed with endometriosis by laparoscopy and/or open surgery. The monkeys were monitored by observing their general condition, and eight of these were monitored using laparoscopy and MRI. In addition, to investigate appropriate screening parameters and endometriosis-associated biological parameters in monkeys, we retrospectively examined general laboratory parameters that correlate to the menstrual cycle and disease status. MAIN RESULTS AND THE ROLE OF CHANCE: The combination of CA125 serum levels (this was a useful marker for chocolate cysts), palpation of the abdomen, and fecal abnormalities was the most efficient screening method for diagnosing monkeys with endometriosis. Each animal could be diagnosed and assigned a disease stage by laparoscopy. While monitoring the disease stage by laparoscopy and/or MRI, disease status in individual monkeys was mainly stable or was progressive for 2-7 months. The detection rate by screening was low (15/614) but age-specific analysis suggests that screening would be more efficient if a colony for an endometriosis model is maintained with 11-20-year olds. As an endometriosis-associated biological parameter, the decrease in food consumption that coincided with menstruation was selected and correlated well (R2 value = 0.8239) with disease status (according to a modified adhesion revised American Fertility Society score). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Peritoneal fluid was not analyzed because a smaller amount is produced in cynomolgus monkeys than in baboons. Although clinical endometriosis-associated pain is evaluated in women using a visual analog scale, pain could not be directly evaluated in this non-human primate model. WIDER IMPLICATIONS OF THE FINDINGS: Although cynomolgus monkeys are relatively small (2-5 kg) primates, laparoscopy and MRI make it possible to evaluate spontaneous endometriosis in these monkeys and to monitor its development over time. Spontaneous endometriosis in cynomolgus monkeys is a useful model for evaluating disease progress and drug efficacy because they have similar lesions to those in humans, and conventional laboratory methods and parameters for assessment are well established. STUDY FUNDING/COMPETING INTEREST(S): No external funds were used for this study. A.N.-K., K.T., H.T., A.K. and M.S. are full-time employees of Chugai Pharmaceutical Co., Ltd. R.K. received a consultancy fee from Chugai Pharmaceutical Co., Ltd. and lecture fees from Chugai Pharmaceuticals, Japan Vaccine Co. Ltd., Merck & Co., Mochida Co. Ltd., Roche Diagnostics, and BD, unrelated to the submitted work. S.N., S.O. and T.S. have nothing to declare.

Characteristics of histologically confirmed endometriosis in cynomolgus monkeys.

STUDY QUESTION: What are the characteristics of spontaneous endometriosis in cynomolgus monkeys? SUMMARY ANSWER: Spontaneous endometriosis in cynomolgus monkeys exhibited similar characteristics to the human disease. WHAT IS KNOWN ALREADY: One previous report described the prevalence and the basic histopathology of spontaneous endometriosis in cynomolgus monkeys. STUDY DESIGN, SIZE, DURATION: Endometriotic lesions that had been histologically confirmed in 8 female cynomolgus monkeys between 5 and 21 years old were subjected to study. PARTICIPANTS/MATERIALS, SETTING, METHODS: The monkeys died of, or were sacrificed because of, sickness consequent on endometriosis. Specimens were evaluated histopathologically with haematoxylin and eosin staining, iron staining and immunohistochemistry (CD10, CD31, α-SMA and PGP9.5), and by observing them under a microscope. MAIN RESULTS AND THE ROLE OF CHANCE: Endometriotic and stromal cells (CD10-positive) with haemorrhage and inflammation were observed. Smooth muscle metaplasia and nerve fibres were also noted in the endometriotic lesions. Endometriotic lesions in lymph nodes were incidentally found. LIMITATIONS AND REASONS FOR CAUTION: Since laparoscopic analysis for monitoring the disease state was not set as a parameter of the current study, time course changes (progression) of the disease were not assessed. WIDER IMPLICATIONS OF THE FINDINGS: Further investigation of spontaneous endometriosis in cynomolgus monkeys may contribute to better understanding of the disease pathobiology. STUDY FUNDING/COMPETING INTERESTS: No external funds were used for this study. A.N.K., S.M., S.H., T.I., O.K., A.K. and M.S. are full-time employees of Chugai Pharmaceutical Co., Ltd. R.K. received lecture fees from Chugai Pharmaceutical Co., Ltd., unrelated to the submitted work. S.N., S. O., L.Y., K.Y. and T.S. have nothing to declare. TRIAL REGISTRATION NUMBER: N/A.

Vitrification and transfer of cynomolgus monkey (Macaca fascicularis) embryos fertilized by intracytoplasmic sperm injection.

Protocols for cryopreservation of monkey embryos are not well established. The objective of the current study was to determine the efficacy of the polypropylene strip method for cryopreserving cynomolgus monkey embryos. Cynomolgus monkey embryos, 63 and 56 at the 4- to 8-cell and 56 blastocyst stages, respectively, were produced by intracytoplasmic sperm injection and in vitro culture, and vitrified using a polypropylene strip. For these two stages, 95 and 86% survived after thawing and pregnancy rates were 29.2% (7 pregnant females/24 recipients, with three live births) and 0% (n = 16 recipients). These were apparently the first live births obtained from embryos fertilized by ICSI. In conclusion, 4- to 8-cell preimplantation cynomolgus monkey embryos were successfully cryopreserved using a polypropylene strip.

Transthyretin amyloidosis and two other aging-related amyloidoses in an aged vervet monkey.

An aged male vervet monkey showed severe cardiac arrhythmia for more than 3 years. A multifocal amyloid consisting of transthyretin was deposited in all areas of the heart wall, especially in the extracellular stroma among muscle fibers and external tunica of arterioles. Moreover, the amyloid was deposited in the stroma and arterioles of other systemic organs except the liver and spleen. These characteristics are consistent with senile systemic amyloidosis in humans. A second amyloid consisting of amyloid beta protein was in senile plaques and cerebral amyloid angiopathy in the cerebral cortex. A third amyloid consisting of islet amyloid polypeptide was deposited in islets of the pancreas. Apolipoprotein E and amyloid P component colocalized with the 3 amyloids. Thus, 3 different aging-related amyloids were found in an aged vervet monkey. In particular, to our knowledge, this is the first report on spontaneous transthyretin amyloidosis in animals.

Comparison of methods to stimulate ovarian follicular growth in cynomolgus and African green monkeys for collection of mature oocytes.

The objective was to compare various gonadotropin-based methods to stimulate ovarian follicular growth in female cynomolgus (n=16) and African green monkeys (n=8) for collection of mature oocytes. On the 1st day of menstruation, the monkeys were treated with 3.75 mg leuprorelin acetate (a GnRH agonist). Starting 2-3 weeks later, ovarian follicular growth was stimulated as follows: (a) 25 IU/kg of human FSH (hFSH) in a glycerol solution given once daily for 9 d; (b) 200 IU of eCG given six times during a 9-d interval; (c) 75 IU/kg hFSH in a glycerol solution given three times (72 h intervals) during a 6-d interval. In addition, the monkeys were given 1200 or 4000 IU of hCG 36 h (Methods A and B) or 60 h (Method C) after the last gonadotropin treatment, and oocyte collection was attempted 36-38 h after hCG. Although there were no significant differences among methods in the number of oocytes collected, in cynomolgus monkeys, hFSH (Methods A and C) was better than eCG (Method B; 12 and 10 versus 7 mature oocytes, respectively), whereas in African green monkeys, eCG (Method B) was more effective than hFSH (Method A; 12 versus 7 mature oocytes). Furthermore, in cynomolgus monkeys, Method C was nearly as effective as Method A; using a glycerol solution as a solvent decreased the frequency of hFSH administration from nine to three times. In conclusion, in cynomolgus and African green monkeys, ovarian response depended on the species and on the individual, and in cynomolgus monkeys, hFSH in a glycerol solvent was effective.

Characterization of an immortalized oviduct cell line from the cynomolgus monkey (Macaca fascicularis).

To establish reproductive biological techniques in mammals, it is important to understand the growth environment of the embryo. Oviduct epithelial cells are in close proximity to the embryo during pre-implantation development. We, therefore, established an immortalized oviduct epithelial cell line from the cynomolgus monkey, evaluated the usefulness of these cells as feeder cells for embryo culture, and investigated the gene expression of several growth factors and cytokines in the cells. The immortalized cells were positive for the anti-cytokeratin antibody, as determined by immunocytochemistry, indicating that they are epithelial. They also expressed oviductin, which is specific to oviduct epithelial cells, glyceraldehyde-3-phosphate dehydrogenase (control), leukemia inhibitory factor, vascular endothelial growth factor, epidermal growth factor, insulin-like growth factor 1, transforming growth factor beta-2, and interleukin 4. Mouse embryo development was improved when the immortalized cells were used as feeder cells. This cell line is also useful for studying the factors secreted by oviduct epithelial cells.

Behavioral compensations in a positional learning and memory task by aged monkeys.

The present experiment assessed learning and memory of a positional task by evaluating behavioral strategies as well as accuracy of a task in four young and four aged monkeys. They were tested in a delayed response (DR) task that has been widely used to study animal models of aging. The task consisted of two phases; an acquisition of the task and a positional memory test with five delay times (1-30 s). There was no clear difference between age groups in the number of trials needed for acquisition of the task. However, an analysis of behavior revealed differences in behavioral characteristics displayed during testing. The young monkeys showed various irrelevant behaviors during the execution of the task. In contrast, the aged monkeys consistently concentrated on the task exhibiting no behaviors irrelevant to the task. These results showed than the aged monkeys' performance was supported by a different behavioral strategy from the young monkeys. The results of the memory test were similar to those of the acquisition on the accuracy and the behavior. The aged monkeys depended on behavioral cues to preserve their positional memory, especially during the task. The present study suggests that cognitive impairments in aged monkeys can be compensated for by employing behavioral strategies.

Cryopreservation-induced acrosomal vesiculation in live spermatozoa from cynomolgus monkeys (Macaca fascicularis).

BACKGROUND: Cryopreserved spermatozoa are known to undergo accelerated capacitation and require a shorter incubation time for fertilization. However, details of their acrosomal membranes following cryopreservation remain unclear. METHODS: Percoll density gradient centrifugation was used to remove dead spermatozoa; thus >90% live spermatozoa were recovered after cryopreservation, and acrosomal status was compared among non-incubated and incubated fresh and cryopreserved spermatozoa. RESULTS: Transmission election microscopy (TEM) using microwave methods and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) staining revealed that 21.1 and 61.6% respectively of non-incubated, cryopreserved spermatozoa were intact, whereas 97.6% (TEM) or 91.9% (FITC-PSA) of non-incubated fresh spermatozoa were intact. TEM revealed that 28.8% of the cryopreserved spermatozoa were swollen, and probably included among those counted as intact by FITC-PSA staining. The non-incubated cryopreserved spermatozoa had fused plasma and outer acrosomal membranes, and 36.4% of them had vesiculation when observed by TEM. FITC-PSA staining indicated that 22% of the live spermatozoa were acrosome reacted. CONCLUSIONS: Acceleration of the acrosome reaction was evident by both TEM and FITC-PSA. Incubation of cryopreserved spermatozoa for 2 h accelerated vesiculation to a state similar to that of fresh spermatozoa that had been incubated for 8 h. These results reveal that in cryopreserved spermatozoa, the process of acrosome reaction begins before incubation.

Immunolocalization of proliferating cell nuclear antigen (PCNA) in cynomolgus monkey (Macaca fascicularis) testes during postnatal development.

The age-related distribution of proliferating cell nuclear antigen (PCNA) in the testes of cynomolgus monkeys (Macaca fascicularis) during postnatal development was detected using light-microscopic immunohistochemistry. In neonatal testes, some PCNA-positive spermatogonia, Sertoli cells, peritubular cells, and Leydig cells were detected. In early infantile testes, only a few of these cell types were positive. In late infantile testes, the numbers of positive cells were greater than in the earlier developmental stages. In pubertal testes, the numbers of positive spermatogonia, spermatocytes, Sertoli cells, peritubular cells, and Leydig cells were considerably higher. In adult testes, a larger percentage of spermatogonia and spermatocytes was positive, and peritubular cells and Leydig cells were occasionally positive; secondary spermatocytes, spermatids, and Sertoli cells were not positive. We concluded that immunolocalization of PCNA can serve as a tool for studying proliferation status in developing testes of cynomolgus monkeys. A relatively low proliferative activity in early infantile testes and a remarkable increase of proliferative activity in pubertal testes correlate with the fluctuations of steroidogenic functions during postnatal development in cynomolgus monkeys.

[Trends in the incidence of cardiovascular diseases and risk factors among urban and rural Japanese males].

OBJECTIVES: To examine long-term trends in the incidence of coronary heart disease, stroke, and their risk factors among Japanese populations, we explored 32 years of surveillance data for male residents in urban and rural areas in Japan. METHODS: The surveyed populations were 40-79 year-old male residents in M community (population over 40 years old in 1995 was 11,121) of Y City in Osaka (urban area; Osaka) and I town (n = 3,571) in Akita prefecture (rural area; Akita). Incidence rates of coronary heart disease (myocardial infarction, angina pectoris), sudden cardiac death, percutaneous transluminal coronary angioplasty (PTCA) and stroke per 1,000 person-years were calculated for 1964-71, 1972-79, 1980-87 and 1988-95. Risk factors were evaluated by cross-sectional surveys conducted in the median years for each period. Dietary intake was examined by the 24-hour recall method in the latter three periods. RESULTS: Age-adjusted incidence of coronary heart disease per 1,000 men increased progressively from 0.27 in 1964-71 to 0.90 in 1988-95 (P = 0.222 for trend) among 40-59 year old residents in Osaka. Among their 60-79 year old counterparts, though the incidence was 2.62-3.11 and did not change over the periods studied, the combined rates for coronary heart disease and men who had a PTCA reached 3.79 in 1988-95. In contrast, the incidence of coronary heart disease among Akita residents did not change over time and stroke declined 70 percent between 1964-71 and 1988-95 (P < 0.001) in both 40-59 and 60-79 year age group: The decrease in cerebral infarction was less marked between 1980-87 and 1988-95 among 60-79 year old individuals. Significant increases in diastolic blood pressure, total serum cholesterol, body mass index, salt intake and total fat percent of total energy in Osaka, were associated with the elevation in the incidence of coronary heart disease. In Akita, blood pressure leveled off over the study period, but decreased less during the last decade whereas the prevalence of obesity increased. CONCLUSION: Trends in cardiovascular diseases and their risk factors differ among different geographical areas. The present long-term study, in particular, demonstrated an increase in the incidence of coronary heart disease among urban Japanese male residents in Osaka between the 1960s and the 1990s.

Activity of a sperm-borne oocyte-activating factor in spermatozoa and spermatogenic cells from cynomolgus monkeys and its localization after oocyte activation.

It is widely accepted that mature mammalian oocytes are induced to resume meiosis by a sperm-borne oocyte-activating factor(s) (sperm factor, SF) immediately after normal fertilization or intracytoplasmic sperm injection. The SF is most likely a soluble factor that is localized within the cytoplasm of mature spermatozoa, but the exact stage at which it appears during spermatogenesis and its localization after oocyte activation is not fully understood, except in the mouse. First, we injected mature spermatozoa and spermatogenic cells from cynomolgus monkeys into mouse oocytes to assess their oocyte-activating capacity. More than 90% of mouse oocytes were activated after injection of monkey spermatozoa. Round spermatids and primary spermatocytes (late pachytene to diplotene) also activated oocytes (93% and 79%, respectively). Injection of monkey spermatozoa and spermatids induces intracellular Ca(2+) oscillations in a pattern similar to that seen following normal fertilization. Most spermatocytes did not produce typical intracellular Ca(2+) oscillations. Second, we transferred pronuclei or cytoplasts from mouse oocytes that had been activated by monkey spermatozoa or spermatids into intact mature mouse oocytes by electrofusion in order to examine the localization of the SF after pronuclear formation. Some of the SF was localized within the pronuclei, but some stayed in the ooplasm. This study demonstrated that spermatogenic cells of cynomolgus monkeys acquire oocyte-activating capacity at much earlier stages than those of mice, and that the monkey SF has a pronucleus-directing nature, although to a lesser extent than the mouse SF.

Short-term nonfrozen storage of mouse epididymal spermatozoa.

Six simple methods for short-term (up to 8 d), nonfrozen (5 to 20 degrees C) storage of mouse epididymides were compared with respect to the motility and fertility of spermatozoa. A high percentage of progressively motile spermatozoa was obtained from epididymis stored for 8 d at 5 degrees C in mineral oil (78.3%), covered with body fat (80.0%), or stored in the intact body of the euthanized donor animal (77.5%). Fertilized eggs (6.4% fertilization rate) were obtained by IVF using spermatozoa that had been stored in mineral oil at 5 degrees C for at least 8 d, and offspring were obtained from 77.5% of transferred eggs that were fertilized by spermatozoa stored for 2 d. These methods inhibited moisture loss from the preserved epididymal spermatozoa, thereby allowing spermatozoa to be stored for a few days without loss of either motility or fertility. These methods make possible such wide-ranging applications as the long-distance transport of epididymis spermatozoa. While in storage at 5 degrees C, the tail of each recovered spermatozoon was bent midway along the tail, possibly owing to damage to the plasma membranes and due to the spermatozoa's hardening in the phospholipid by exposure to the low temperature.

Prospective study of depressive symptoms and risk of stroke among japanese.

BACKGROUND AND PURPOSE: We sought to examine the relationship between depressive symptoms and the incidence of stroke among Japanese men and women. METHODS: A 10.3-year prospective study on the relationship between depressive symptoms and the incidence of stroke was conducted with 901 men and women aged 40 to 78 years in a rural Japanese community. Depressive symptoms were measured at baseline with the use of the Zung Self-Rating Depression Scale (SDS). The incidence of stroke was ascertained under systematic surveillance. RESULTS: During the 10-year follow-up, 69 strokes (39 ischemic strokes, 10 intracerebral hemorrhages, 10 subarachnoid hemorrhages, and 10 unclassified strokes) occurred. Age- and sex-adjusted prevalence of mild depression (SDS scores >/=40) at baseline was 25% among subjects with incident stroke and 12% among subjects without stroke (P<0.01). Persons with SDS scores in the high tertile had twice the age- and sex-adjusted relative risk of total stroke as those with scores in the low tertile. The excess risk was confined to ischemic stroke. After we adjusted for body mass index, systolic blood pressure level, serum total cholesterol level, cigarette smoking, current treatment with antihypertensive medication, and history of diabetes mellitus, these relative risks remained statistically significant for total stroke (1.9; 95% CI, 1.1 to 3.5) and ischemic stroke (2.7; 95% CI, 1.2 to 6.0). CONCLUSIONS: Depressive symptoms predict the risk of stroke, specifically ischemic stroke among Japanese.

Serum triglycerides and risk of coronary heart disease among Japanese men and women.

To examine the relation of triglycerides with coronary heart disease among populations with low mean total cholesterol, the authors conducted a 15.5-year prospective study ending in 1997 of 11,068 Japanese aged 40-69 years (4,452 men and 6,616 women with mean total cholesterol = 4.73 mmol/liter and 5.03 mmol/liter, respectively), initially free of coronary heart disease or stroke. There were 236 coronary heart disease events comprising 133 myocardial infarctions, 68 angina pectoris events, and 44 sudden cardiac deaths. The coronary heart disease incidence was greater in a dose-response manner across increasing quartiles of nonfasting triglycerides for both sexes. The multivariate relative risk of coronary heart disease adjusting for coronary risk factors and time since last meal associated with a 1-mmol/liter increase in triglycerides was 1.29 (95% confidence interval (CI): 1.09, 1.53; p = 0.004) for men and 1.42 (95% CI: 1.15, 1.75; p = 0.001) for women. The trend was similar for myocardial infarction, angina pectoris, and sudden cardiac death. The relation of triglycerides with coronary heart disease was not influenced materially by total cholesterol levels or, in a subsample analysis (51% of total sample), by high density lipoprotein cholesterol levels. Nonfasting serum triglycerides predict the incidence of coronary heart disease among Japanese men and women who possess low mean values of total cholesterol. Further adjustment for high density lipoprotein cholesterol suggests an independent role of triglycerides on the coronary heart disease risk.

Short-term preservation of mouse oocytes at 5 degrees C.

The temporary preservation of oocytes without freezing would be useful for some experiments. ICR mouse oocytes were kept in a preservation medium under mineral oil for 1, 2, 3, 4 or 7 days at 5 degrees C, and 1 or 2 days at 37 degrees C. In vitro fertilization was attempted on oocytes rinsed with TYH medium after preservation. More than 70% of morphologically normal oocytes were recovered from each preservation group. Fertilization rates of oocytes preserved for 1, 2, 3, 4 or 7 days at 5 degrees C were 69.9, 66.5, 45.3, 26.7 and 8.8% respectively. Fertilization rates of oocytes preserved for 1 or 2 days at 37 degrees C were 9.6 and 1.6%, respectively. Preservation of oocytes at 5 degrees C has some capability as a method of short-term storage without freezing.

Comparison of the effects of two fixatives for immunolocalization of testosterone in the testes of the cynomolgus monkey, mouse and rat.

We compared the effect of two fixatives, Bouin's fixative and neutralized buffered 4% formaldehyde (10% formalin), for immunolocalization of testosterone in the testes of cynomolgus monkeys, mice and rats. In the samples fixed with Bouin's fixative, immunoreactive testosterone was detected as intense deposits in the cytoplasm of Leydig cells of monkeys and mice. Immunoreactive testosterone was detected not only in Leydig cells of rats but also moderately shown within tubules. Immunoreactive testosterone could not be detected in the testes of monkeys, mice or rats fixed with neutralized buffered formalin because of the poor morphology caused by the fixative. It is concluded that Bouin's fixative is a suitable fixative for immunolocalization of testosterone in the testes of cynomolgus monkeys, mice and rats.

Angiotensinogen T174M and M235T variants, sodium intake and hypertension among non-drinking, lean Japanese men and women.

OBJECTIVE: To examine the interaction of sodium intake with genetic variations of the angiotensinogen gene and hypertension. DESIGN: A community-based case-reference study. SETTING: Two rural Japanese communities. PARTICIPANTS: Non-overweight and non-drinking Japanese men and women: 229 hypertensives and 229 age-, sex- and community-matched normotensives aged 32 to 83 years. METHODS: Polymorphisms of the angiotensinogen gene detected by an allele-specific polymerase chain reaction. A priori hypothesis is individuals with 174M (threonine-to-methionine substitution) or 235T (methionine-to-threonine substitution) allelic variations may have an elevated risk of hypertension when they have a high sodium intake, estimated by 24-h urine collection and a dietary questionnaire. RESULTS: The genotypic frequency of the haplotype including both the 174M and 235T alleles was higher among hypertensives than among normotensives (23 versus 14%, P= 0.02). The frequency of the 174M allele was specifically higher among hypertensives than normotensives (12 versus 7%, P=0.01), and the odds ratio of hypertension associated with the 174M (versus 174T) allele was 1.8 [95% confidence interval (CI) 1.1-3.0, P=0.01]. The frequency of the 235T allele did not vary between the two groups (80 versus 82%, P= 0.40). The relationship between the 174M allele and hypertension was more evident among persons who had higher urinary sodium excretion (> = 166 mmol/day) than those with lower excretion (< 166 mmol/day): odds ratio 2.5 (95% CI, 1.2-5.2), P=0.01 versus 1.5 (95% CI, 0.7-3.1), P= 0.31; P for interaction = 0.04, and this trend was primarily observed for early-onset hypertension (< 55 years at onset). A similar but nonsignificant association was observed when stratified using present and past sodium intake scores derived from questionnaires. CONCLUSION: Angiotensinogen genotype may affect the development of early-onset hypertension among Japanese, particularly in those who have a high sodium intake.