Effects of cadmium on lactate dehydrogenase activities in mouse pre-embryos at various stages

A decline in LDH activity from four-cell to late blastocyst was demonstrated in pre-embryos from F1 (C57 female X A2G male) female mice. An exposure to 0.5 or 1 microgram/ml cadmium did not affect the in vitro development of the four-cell pre-embryos and morulae or their LDH activities. At 5 or 10 micrograms/ml cadmium, the in vitro development of the treated pre-embryos was affected. Although most of the treated four-cell pre-embryos had proceeded to compaction, they became degenerated 24 h after treatment. The LDH activity of these degenerated pre-embryos was higher than that in the control blastocysts. We propose that cadmium may interfere with the general energy metabolism of the cells. This causes a reduced rate of LDH degradation, leading to a slower decline in LDH activity in cadmium-treated pre-embryos. Failure of some critical biochemical processes after cadmium treatment may ultimately lead to the subsequent degeneration of the treated pre-embryos.     

Effect of human uterine flushings collected at various stages of the menstrual cycle on mouse blastocysts in vitro

Mouse blastocysts were cultured in vitro in a defined medium supplemented with uterine flushings (containing 500 microgram protein/ml) obtained from normal women at various stages of the menstrual cycle. With one exception (uterine flushing collected on the last day of a menstrual period) blastocyst hatching and attachment were not impaired by flushings collected before or after ovulation.     

Equilibrium vitrification of mouse embryos at various developmental stages

Previously, we developed a new method by which 2‐cell mouse embryos can be vitrified in liquid nitrogen in a near‐equilibrium state, and then kept at ?80°C for several days. In the present study, we examined whether or not the method was effective for mouse embryos at other developmental stages. Eight‐cell embryos, morulae, and expanded blastocysts of ICR mice were vitrified with ethylene glycol‐based solutions, named EFSc because of their composition of ethylene glycol (30–40%, v/v) and FSc solution. The FSc solution was PB1 medium containing 30% (w/v) Ficoll PM‐70 plus 1.5 M sucrose. The extent of equilibrium was assessed by examining how well vitrified embryos survived after being kept at ?80°C. When 8‐cell embryos and morulae were vitrified with EFS35c or EFS40c and then kept at ?80°C, the survival rate was high even after 4 days in storage and remained high after re‐cooling in liquid nitrogen. On the other hand, the survival of vitrified‐expanded blastocysts kept at ?80°C was low. Therefore, 8‐cell embryos and morulae can be vitrified in a near‐equilibrium state using the same method as for 2‐cell embryos. A high proportion of C57BL/6J embryos at the 2‐cell, 8‐cell, and morula stages vitrified with EFS35c developed to term after transportation on dry ice, re‐cooling in liquid nitrogen, and transfer to recipients. In conclusion, the near‐equilibrium vitrification method, which is effective for 2‐cell mouse embryos, is also effective for embryos at the 8‐cell and morula stages. The method would enable handy transportation of vitrified embryos using dry ice. Mol. Reprod. Dev. 79: 785–794, 2012. © 2012 Wiley Periodicals, Inc.     

Ultrastructural localization of adenosine triphosphatase activity in HeLa cells at various stages of the cell cycle

Summary HeLa cells in a monolayer culture were synchronized to S, G₂ and mitotic phases by use of excess (2.5 mM) deoxythymidine double-block technique. The localizations of Ca⁺⁺-activated adenosine triphosphatase (ATPase) at different phases of the cell cycle were studied using light and electron-microscopic histochemical techniques, and microphotometric comparisons of the densities of reaction products. Enzyme reaction product was always localized in the endoplasmic reticulum, nuclear membrane, mitochondria and Golgi apparatus, but there were qualitative and quantitative differences related to the phases of the cell cycle. In S phase the activity was mainly concentrated in a perinuclear area of the cytoplasm whereas in G₂ and mitosis the activity was scattered throughout the cell. The total activity per cell was maximal in G₂, was less in S phase and least in mitosis. Activity in the mitochondria and endoplasmic reticulum was distinctly less in mitosis than in other phases of the cell cycle. The mitochondrial ATPase differed from the ATPase at other sites in ion dependence and sensitivity to oligomycin. The results suggest that there may be several distinct ATPases in proliferating cells.     

GTP gamma S effects on phosphatidylinositol phospholipase C alpha isoenzyme activity isolated from guinea pig uterine smooth muscle at different stages of pregnancy.

The ability of GTP gamma S to activate release of inositol polyphosphates from isolated permeabilised guinea pig uterine smooth muscle cells and from partially purified PI-PLC alpha has been studied. Streptolysin O permeabilised and [3H]inositol prelabelled cells show a time dependent release of inositol polyphosphates, predominantly inositol 4-phosphate. Ca2+ stimulated IP release with a Ka of 161 +/- 1.1 nM and this was further enhanced in an additive manner by GTP gamma S between 1-100 microM; the Ka for Ca2+ in the presence of 0.1 mM GTP gamma S was 117 +/- 0.7 nM. GTP gamma S activation of IP production did not require Ca2+ in the medium. Permeabilisation of the uterine smooth muscle cells with Streptolysin O readily released PI-PLC activity into the medium. However, unlike studies with isolated membranes 63.4 +/- 6.4% of the enzyme activity remained associated with membranes and/or particulate fractions of the cell. Studies were undertaken with PI-PLC alpha, the predominant isoenzyme form, partially purified from uterine smooth muscle at different stages of pregnancy by Q-Sepharose and Heparin-Agarose chromatography. The enzyme co-purifies with firmly associated GTP-binding activity. Enzyme prepared from near-term uterus is activated by 0.1 mM GTP gamma S, up to 100% when Ca2+ is between 0.1-1 microM, while 10 microM AlF4- under those conditions caused complete inhibition of the enzymes. Responses for enzymes prepared from non-pregnant uteri were broadly similar. In contrast enzyme preparations from guinea pig uteri at 20-60 days of pregnancy show an inhibition of activity in response to 0.1 mM GTP gamma S addition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitrification of mouse embryos at various stages by open-pulled straw (OPS) method

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure-that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method-that is, embryos were first pretreated in 10%E + 10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E + 10%D for 0.5 min, exposed to EDFS30for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.     

Effect of the ionic environment and of various neurotransmitters on spontaneous motility in snail intestine (author's transl)]

A spontaneous rhythmic motility of the isolated intestine in snail, Chryptomphalus hortensis, has been registered at 30 degrees C with the aid of an electronic transductor and an appropriate amplification. These slow regular movements are affected by ionic composition changes in the suspension medium. Na+, K+ and Ca++ ions prove to be important in this motility, and the addition of Ba++ markedly stimulates it. ACh produces hypermotility from 1.8 X 10(-11) g/ml. Its effect decreases in the presence of atropine and increases in that of pyridostigmine. The intestine is sensitive to 5-HT from 10(-10) g/ml, which stimulates its activity. The effect of histamine is weak. Low concentrations of adrenaline tend to increase the amplitude, whereas concentrations from 10(-5) g/ml onward produce a cease of motility in relaxation.     

Effects of ultrasound on the mouse exposed at different stages of gestation: Acute studies

Summary At five stages of gestation, mice were exposed dorsally to ultrasonic irradiation at 1 W/cm² (spatial and temporal average), 2 MHz, and 34 and 37° C (temperature at beginning of exposure). The results indicated that ultrasonic exposure up to at least 100 s is not hazardous to a pregnant mouse or its offspring in utero under the conditions of our experiment. Thresholds for both undesirable and lethal effects on the dam and its embryos or fetuses appeared between 100 and 200 s. Temperature rises in the uterus to more than 40° C and similarity of results obtained with continuous-wave and burst modes of irradiation suggest a thermal mechanism as one cause of these effects. Preliminary conclusions are that thresholds for both tissue damage to the dam and deleterious effects on pregnancy were similar; however, they occurred at spatial average intensity and exposure levels far in excess of those used in clinical diagnostic medicine.     

Presence of uterine peroxidase activity in the rat early pregnancy

Peroxidase has been associated with estrogen action in the uterus. This enzyme plays an important role in the control of hydrogen peroxide levels and in catechol estrogen production. Since the uterus, during early pregnancy, is subjected to estrogen and progesterone regulation, we analyzed the changes of peroxidase activity in relation to receptivity and uterine early response to the embryo. Soluble and microsomal peroxidase activity were determined in the rat uterus during the estrus phase and early pregnancy (days 3 through 6). Soluble peroxidase activity increased significantly (p < 0.01) from day 3 (1.50 +/- 0.24) to day 4 (3.5 +/- 0.3) and 5 (5 +/- 0.5 U/mg protein, mean +/- S.D., n = 6) of pregnancy. During day 6, a significant decrease was noted in both the implantation site and the nonimplantation uterine tissue. Microsomal calcium-extractable peroxidase showed a similar pattern, with lower specific activity than, the soluble peroxidase. During estrus, the uterine tissue showed the highest activity of calcium-extracted peroxidase (8.7 +/- 1.35 U/mg protein), statistically greater when compared with days 3, 4, 5 and 6 of pregnancy. In conclusion, high peroxidase activity was associated with uterine receptivity. The decrease of activity on day 6 might be due to a progesterone-estrogen interaction, and consequently, hydrogen peroxide can be utilized for hydroxile production by means of the Fenton reaction. Lipoperoxidation may be necessary for changes in membrane fluidity for embryo attachment to endometrial epithelium.