Effect of DNase treatment on RNA extraction from preimplantation murine embryos.

The quality of RNA is crucial when performing microarray experiments. This is particularly important when dealing with preimplantation embryos, from which a minimum yield of RNA of good quality can be produced. We report the optimization of several RNA extraction methods applied to preimplantation embryos at different stages of development. The quality of the samples was confirmed using a microarray and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. A total of 30 cultured two-cell stage embryos of ICR mice were pooled at the 8-cell, morula, and blastocyst stages. The embryos were divided into two groups comprising DNase-treated and non-DNase-treated RNA samples. Total RNA was extracted using a Pico Pure RNA Isolation Kit following the manufacturer protocol, with some modifications. Lysed samples were bound to a silica-based filter, treated with deoxyribonuclease I (DNase I), and washed several times before elution. RNA concentration and integrity were evaluated using an Agilent 2100 Bioanalyzer and an RNA 6000 Pico Assay kit. Although concentrations of non-DNase-treated RNAs were higher than DNase-treated RNA, DNase-treated RNA gave a higher RNA integrity number compared with non-DNase-treated RNA. Inclusion of DNase treatment in the RNA extraction procedure gave the best quality RNA samples from preimplantation embryos, as validated by microarray and RT-qPCR quality control.

Nicotine and endometrial decidual growth in pseudopregnant rats.

PURPOSE: The present study aims to investigate the effects of nicotine on the endometrial decidual growth and levels of estrogen and progesterone in pseudopregnant rats. METHODS: Pseudopregnancy (pc) was induced in cyclic Sprague-Dawley rats by sterile mating. Subcutaneous injection of nicotine tartrate (7.5 mg/kg/day) was scheduled from day 1 through day 5, day 5 through day 9 or day 1 through day 9 of pc. In another group of pseudopregnant rats, concomitant treatment of nicotine tartrate concurrently with progesterone (2 mg/day) was scheduled from day 1 through day 9 pc. Control groups received subcutaneous injections of vehicle only. Endometrial decidualization was induced on day 5 pc. On day 10 pc, animals were sacrificed.The degree of decidual growth and circulating levels of estrogen and progesterone were measured. RESULTS: The decidual growth in all the first three nicotine-treated groups of animals was significantly reduced, particularly in the animals treated with nicotine from day 1 through day 9 pc. Plasma estrogen levels were significantly elevated in animals treated with nicotine from day 1 through day 9 pc. Conversely, levels of plasma progesterone were found to be significantly attenuated in the same group of nicotine-treated animals compared to controls. Exogenous replacement of progesterone, however, caused a higher degree of endometrial decidualization compared to the nicotine-treated group but it was slightly less than when compared to control. CONCLUSIONS: In conclusion, nicotine-induced progesterone deficiency with a corresponding elevation of estrogen may possibly attenuate the degree of endometrial decidualization in pseudopregnant rats.

Ultrastructural studies of developing goat oocytes in vitro.

The structure and distribution of organelles within developing goat oocytes at various stages of incubation were studied. In oocytes with 5 or more layers of cumulus cells, at 0 h of incubation, the zona pellucida had developed although zonation was not evident. Lipid bodies were present but no mitochondria were observed. At 20 h, the zona pellucida had differentiated into thicker and thinner regions. Clusters of membrane-bound electron-transparent bodies were present in the perivitelline space. The mitochondria were fully developed, distributed evenly and usually in close proximity with dilated endoplasmic reticula. Cortical granules were distributed at the periphery. At 40 h of incubation, a number of mitochondria was hooded. In oocytes of 2 to 4 layers of cumulus cells at 0 h, the zona pellucida was penetrated by cumulus cell processes, and the mitochondria were not well developed. However, in 20-h incubated oocytes, fully developed mitochondria, many of which were hooded, could be observed. Clusters of membrane-bound electron-transparent bodies were also observed, while cortical granules were at the periphery. In cumulus-free oocytes, zonation within the zona pellucida was indistinct. Very few vesicles and lipid bodies were observed. At 20 h, mitochondria were sparsely distributed and were not well developed and lacked cristae. At 40 h, the zona pellucida was less compact, and the membrane-bound electron-transparent bodies were less numerous compared with those of the other groups. Endoplasmic reticula were not dilated, and cortical granules were few and had no definite pattern of distribution.

The effects of thyroxine treatment on slow- and fast-contracting skeletal muscle contractions of the cat and their cyclic AMP level.

1. The effects of thyroxine treatment on soleus and extensor digitorum longus (EDL) muscle contractions and their cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels were examined in anaesthetized cats. 2. Thyroxine treatment decreased the tension of incomplete tetanic contractions of the soleus as well as the EDL muscles. The effect on tension of these muscles was not associated with an increase in the cyclic AMP level of the muscle as is the case with a beta 2-adrenoceptor agonist effect. 3. The results do not support the involvement of cyclic AMP in the tension depressant effect of thyroxine on contractions of skeletal muscle. 4. It is suggested that the muscle weakness and tremor observed in thyrotoxicosis and during administration of beta 2-adrenoceptor agonists are mediated by different mechanisms.

Transfer factor in smokers and non-smokers after smoking two cigarettes.

Impairments of lung functionality as long-term effects of cigarette smoking have been well established. To our knowledge, no study on acute recovery patterns in any important lung function index after smoking a very limited number of cigarettes has ever been reported. The present study reports recovery patterns of lung transfer factor (TF) and related parameters in smokers and non-smokers who smoked two Camel cigarettes. Lung transfer factor and other indices were determined by the single breath-holding technique. From our results, the TF and related indices of healthy Malaysians are similar to previously published normals of comparable age. On smoking two cigarettes, male smokers began to recover from the 30th minute; male non-smokers had not begun recovery even by the 50th minute. Extrapolation of the recovery curves suggests that a "safer" interval between cigarettes for male smokers is about 114 minutes.

The effects of 4-methyl-2-thiouracil on fibre type and cross-sectional area in the soleus muscle of the rat.

The effects of 4-methyl-2-thiouracil (MTU, 0.1% in drinking water) on the composition and cross-sectional area of muscle fibres of the rat soleus muscle were studied. The percentage of fast twitch-oxidative-glycolytic (FOG) fibres fell after 2 weeks of treatment with MTU to zero at 8 weeks. In contrast the percentage of FOG fibres in untreated animals fell to 19.2 +/- 2.1% during this period. The mean cross-sectional area of FOG and slow twitch-oxidative (SO) fibres were respectively 39.9% and 23.8% smaller than those of their respective controls 6 weeks after treatment. At 8 weeks the percentage reduction of SO fibre area was 26.8% of the control value. This study indicates that MTU treatment causes atrophy and redistribution of fibre type in the soleus muscle.