Flow Cytometric Analysis of Ca2+-Induced Membrane Permeability Transition of Isolated Rat Liver Mitochondria

The membrane permeability transition (MPT) of mitochondria plays an important role in the mechanism of apoptotic cell death in various cells. Classic type MPT is induced by Ca2+ in the presence of inorganic phosphate and respiratory substrate, and is characterized by various events including generation of reactive oxygen species (ROS), membrane depolarization, swelling, release of Ca2+ and high sensitivity to cyclosporine A. However, the sequence of these events and the effect of antioxidants on their events remain obscure. Flow cytometry is a convenient method to investigate the order of events among various functions occurring in MPT using a limited amount of mitochondria (200 µl of 0.02 mg protein/ml) without contamination by other organelles. Flow cytometric analysis revealed that Ca2+ sequentially induced ROS generation, depolarization, swelling and Ca2+ release in mitochondria by a cyclosporine A-inhibitable mechanism. These results were supported by the finding that Ca2+-induced MPT was inhibited by antioxidants, such as glutathione and N-acetylcysteine. It was also revealed that various inhibitors of Ca2+-induced phospholipase A2 suppressed all of the events associated with Ca2+-induced MPT. These results suggested that ROS generation and phospholipase A2 activation by Ca2+ underlie the mechanism of the initiation of MPT

生殖補助医療を行っている患者において卵胞液中のキスペプチン濃度は卵成熟および性腺ホルモン値と関連している

Purpose: To assess the kisspeptin concentrations in follicular fluid and their relationship with clinical outcomes during assisted reproductive technology. Methods: Thirty-nine patients who were aged 24-40 years and underwent oocyte retrieval for in vitro fertilization/intracytoplasmic sperm injection participated in this study. In 65 follicular fluid samples that had been obtained from 30 patients and their blood samples, the kisspeptin levels were measured in order to investigate the correlations with their gonadal hormone levels. Venous blood samples were collected from 14 patients to investigate their plasma kisspeptin levels across different phases of assisted reproductive technology. Results: The follicular fluid kisspeptin level was significantly higher than that of the plasma level and was positively associated with the follicular fluid estradiol concentration and with the serum estradiol and number of mature oocytes. In the plasma, the maximum concentration of kisspeptin was observed on the day of ovum pick-up and on the day of embryo transfer during ovarian stimulation for assisted reproductive technology. Conclusion: Kisspeptin was present in the follicular fluid and the plasma kisspeptin concentration was affected by ovarian stimulation. Kisspeptin appears to affect oocyte maturation and ovulation

サイトカイン誘発好中球化学誘引物質の発現はTNFα発現を抑制することにより卵胞の閉鎖およびアポトーシスから回避させる

Aim: Cytokine-induced neutrophil chemoattractant (CINC/gro) is a CXC family chemokine, similar to interleukin-8 in rats, and is one of the factors that regulates ovulation. However, the mechanism that regulates atresia of the ovaries postovulation is not clearly defined. Methods: Whether antibody-blocking of CINC/gro can alter the number of ovulated oocytes and modulate neutrophil infiltration was investigated. The effect of the antibody on the level of inflammatory cytokine production and follicular atresia was examined. Apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and via analysis of the messenger RNA expression of Bcl-2 and Bcl2-associated X (Bax). Results: The anti-CINC/gro antibody treatment decreased the number of ovulated oocytes. The messenger RNA levels of cyclooxygenase-2 and interleukin-1 beta were decreased by the antibody treatment, whereas that of tumor necrosis factor (TNF) alpha was increased. The TUNEL analysis revealed a larger number of apoptotic cells in the antibody group, compared with those in the control group, as well as a significant increase in the Bax/Bcl-2 ratio 24 hours after human chorionic gonadotropin administration. Conclusion: These findings suggest that ovulation is accelerated by neutrophil infiltration into the theca layer. The CINC/gro appears to synergize with interleukin-1 beta for ovulation. By contrast, the data suggest that CINC/gro expression suppresses TNF alpha expression and that CINC/gro expression therefore prevents the follicles from undergoing atresia and apoptosis

OVARIAN Kiss1 mRNA IN THE PREPUBERTAL PERIOD

Kisspeptin, which is encoded by the Kiss1 gene, and its receptor, the G protein-coupled receptor 54 (Kiss1r), play important roles in the regulation of reproductive functions in mammals. Several studies have shown that the Kiss1 and Kiss1r genes are expressed in the rat, primate, and human ovaries, and that the ovarian kisspeptin system plays a pivotal role in ovulation at the proestrous stage in adulthood. The purpose of this study was to evaluate development-related changes in the expression of ovarian Kiss1 and Kiss1r genes and in kisspeptin levels, and to identify the regulatory factors for these genes during the prepubertal period. The serum kisspeptin level was also measured to examine whether ovarian kisspeptin affects serum kisspeptin levels. Variations in the ovarian Kiss1 and Kiss1r mRNA levels were observed during the prepubertal period in female rats, with levels peaking around postnatal days 20 and 15, respectively. Nevertheless, the ovarian kisspeptin content per total protein level was stably maintained. Serum kisspeptin levels at postnatal days 30 and 35 were higher than those at earlier postnatal days. The pattern of the ovarian Kiss1 mRNA levels was similar to that of the serum luteinizing hormone (LH) levels, and the ovarian Kiss1 mRNA level increased after injection with human chorionic gonadotropin (HCG) on postnatal day 20, but not on postnatal days 10 and 30. These data indicate that ovarian Kiss1 and Kiss1r mRNA levels are increased on postnatal days 20 and 15, respectively, and that changes in the serum LH level and the ovarian sensitivity to LH may be involved in the alteration of ovarian Kiss1 mRNA levels

A novel PCOS rat model and an evaluation of its reproductive, metabolic, and behavioral phenotypes

Background: Although animal models of PCOS have been used in many studies, none of them can reproduce both the reproductive and metabolic phenotypes of PCOS. In addition, behavioral parameters have not been evaluated in PCOS animal models. Purpose: We tried to produce an improved rat model of PCOS, and the reproductive, metabolic, and behavioral phenotypes of the model rats were evaluated. Methods: Female rats were implanted with silicon tubes containing oil-dissolved dihydrotestosterone (Oil-DHT) as a new PCOS model. Their phenotypes were compared with those of conventional PCOS model rats (DHT), into which tubes containing crystalline DHT were implanted, and non-DHT-treated rats (control). Results: Both the Oil-DHT and DHT rats showed greater body weight gain, food intake, and fat depot weight than the control rats. Furthermore, these groups showed fewer estrous stages and increased numbers of cystic follicles. The DHT rats exhibited lower ovarian and uterine weights than the control rats, whereas no such changes were observed in the Oil-DHT rats. The Oil-DHT and DHT rats showed less locomotor activity in the light phase than the control rats. Conclusions: Our proposed PCOS model reproduced both the reproductive and metabolic phenotypes of PCOS and may have potential for PCOS research

Effect of semen characteristics on pregnancy rate following intrauterine insemination

Objective : To assess the effects of semen characteristics on the success of intrauterine insemination (IUI). Design : A retrospective study. Settings : The Department of Obstetrics and Gynecology, Tokushima University Hospital, Japan. Patients : Between 2004 and 2008, 1,177 IUI cycles in 283 couples were studied. Intervention : IUI cycles were preceded with ovarian stimulation. Main Outcome Measure : Clinical pregnancy. Result : A total of 82 clinical pregnancies were obtained (7.0% pregnancy rate per cycle, 28.9% per case). Their subsequent outcomes of pregnancies were 18 miscarriages (21.9%), 2 ectopic pregnancies (2.4%) and 60 live births (73.2%). Of the 82 clinical pregnancies, 2 were twin pregnancies (2.4%). There was no triple or higher order multiple pregnancies. At the end of the sixth cycle, 73 clinical pregnancies had been achieved (89.0%). After diagnostic laparoscopy, the pregnancy rate per cycle for patients 35 years age was 18%, which is significantly higher than that of patients 35 years of age. Pregnancies occurred up to the fifth cycle after laparoscopy. The pregnancy rate (PR) per cycle was significantly higher in cases of sperm movement rates more than 30% (PR9.3%) and total motile sperm counts more than 10 106/ml (PR 8.2%). A study comparing the washed and unwashed cases did not reveal any differences. Conclusion : In male sub-fertility cases of sperm parameters as motility rates 30% and motile sperm concentration 10 106/ml, IUI could be a useful option for infertility treatment J. Med. Invest. 5

Biotin levels in blood and follicular fluid

It has been shown that biotin, a water-soluble vitamin (B7), plays roles in reproductive functions, such as oocyte maturation and embryo development, in experimental animals. On the other hand, little is known about the clinical effects of biotin on human reproduction. In this study, serum and follicular fluid biotin levels were measured in patients who underwent in vitro fertilization / intracytoplasmic sperm injection (IVF / ICSI), and their associations with reproductive outcomes were evaluated. As a result, biotin was detected in follicular fluid, as well as serum, and the biotin levels of follicular fluid were found to be positively correlated with those of serum. The biotin levels of serum were higher than those of follicular fluid, suggesting that biotin may be taken up into the follicular fluid from the blood. Although serum and follicular fluid biotin levels tended to be higher in pregnant patients than in non-pregnant patients, these data did not show the significant statistical difference. These findings indicate that biotin does not contribute to the maintenance of oocyte quality, and hence, it does not increase fertilization and pregnancy rates

On the origin and evolution of the asteroid Ryugu: A comprehensive geochemical perspective

Presented here are the observations and interpretations from a comprehensive analysis of 16 representative particles returned from the C-type asteroid Ryugu by the Hayabusa2 mission. On average Ryugu particles consist of 50% phyllosilicate matrix, 41% porosity and 9% minor phases, including organic matter. The abundances of 70 elements from the particles are in close agreement with those of CI chondrites. Bulk Ryugu particles show higher δ18O, Δ17O, and ε54Cr values than CI chondrites. As such, Ryugu sampled the most primitive and least-thermally processed protosolar nebula reservoirs. Such a finding is consistent with multi-scale H-C-N isotopic compositions that are compatible with an origin for Ryugu organic matter within both the protosolar nebula and the interstellar medium. The analytical data obtained here, suggests that complex soluble organic matter formed during aqueous alteration on the Ryugu progenitor planetesimal (several 10’s of km), <2.6 Myr after CAI formation. Subsequently, the Ryugu progenitor planetesimal was fragmented and evolved into the current asteroid Ryugu through sublimation

A pristine record of outer Solar System materials from asteroid Ryugu’s returned sample

Volatile and organic-rich C-type asteroids may have been one of the main sources of Earth’s water. Our best insight into their chemistry is currently provided by carbonaceous chondritic meteorites, but the meteorite record is biased: only the strongest types survive atmospheric entry and are then modified by interaction with the terrestrial environment. Here we present the results of a detailed bulk and microanalytical study of pristine Ryugu particles, brought to Earth by the Hayabusa2 spacecraft. Ryugu particles display a close compositional match with the chemically unfractionated, but aqueously altered, CI (Ivuna-type) chondrites, which are widely used as a proxy for the bulk Solar System composition. The sample shows an intricate spatial relationship between aliphatic-rich organics and phyllosilicates and indicates maximum temperatures of ~30 °C during aqueous alteration. We find that heavy hydrogen and nitrogen abundances are consistent with an outer Solar System origin. Ryugu particles are the most uncontaminated and unfractionated extraterrestrial materials studied so far, and provide the best available match to the bulk Solar System composition