Stem cells are defined as cells able to both extensively self-renew and differentiate into progenitors. Research data show that more resistant stem cells than common cancer cells exist in cancer patients.To identify unrecognized differences between cancer stem cells and cancer cells might be able to develope effective classification,diagnose and treat ment for cancer.

Stem Cells,Cancer and Cancer Stem Cells

Oxidative stress is involved in the aetiology of defective embryo development. Reactive oxygen species (ROS) may originate from embryo metabolism and/or embryo surroundings. Embryo metabolism generates ROS via several enzymatic mechanisms. The relative contribution of each source seems different depending on the species, the stage of development, and the culture conditions. Several exogenous factors and culture conditions can enhance the production of ROS by embryos. ROS can alter most types of cellular molecules, and also induce development block and retardation. Multiple mechanisms of embryo protection against ROS exist, and these have complementary actions. External protection, present in follicular and tubal fluids, mainly comprises non-enzymatic antioxidants such as hypotaurine, taurine and ascorbic acid. Internal protection mainly comprises antioxidant enzymes: superoxide dismutase, glutathione peroxidase and gamma-glutamylcysteine synthetase. Transcripts encoding for these enzymes are present in the oocyte, embryo and oviduct. It may be important that these transcripts are stored during oocyte maturation in order to allow the embryo to acquire the aptitude to develop. It is now common to add antioxidant compounds to culture media. Nevertheless, maintaining the pro-oxidant-antioxidant equilibrium in embryos through such supplementation is a complex problem. Further studies are necessary to limit oxidative stress during embryo culture.

Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings

The Practice Committee of the American Society for Reproductive Medicine

The onset of clinical assisted reproduction, a quarter of a century ago, required the isolation of motile spermatozoa. As the indication of assisted reproduction shifted from mere gynaecological indications to andrological indications during the years, this urged andrological research to understand the physiology of male germ cell better and develop more sophisticated techniques to separate functional spermatozoa from those that are immotile, have poor morphology or are not capable to fertilize oocytes. Initially, starting from simple washing of spermatozoa, separation techniques, based on different principles like migration, filtration or density gradient centrifugation evolved. The most simple and cheapest is the conventional swim-up procedure. A more sophisticated and most gentle migration method is migration-sedimentation. However, its yield is relatively small and the technique is therefore normally only limited to ejaculates with a high number of motile spermatozoa. Recently, however, the method was also successfully used to isolate spermatozoa for intracytoplasmic sperm injection (ICSI). Sperm separation methods that yield a higher number of motile spermatozoa are glass wool filtration or density gradient centrifugation with different media. Since Percoll® as a density medium was removed from the market in 1996 for clinical use in the human because of its risk of contamination with endotoxins, other media like IxaPrep®, Nycodenz, SilSelect®, PureSperm® or Isolate® were developed in order to replace Percoll®. Today, an array of different methods is available and the selection depends on the quality of the ejaculates, which also includes production of reactive oxygen species (ROS) by spermatozoa and leukocytes. Ejaculates with ROS production should not be separated by means of conventional swim-up, as this can severely damage the spermatozoa. In order to protect the male germ cells from the influence of ROS and to stimulate their motility to increase the yield, a number of substances can be added to the ejaculate or the separation medium. Caffeine, pentoxifylline and 2-deoxyadenosine are substances that were used to stimulate motility. Recent approaches to stimulate spermatozoa include bicarbonate, metal chelators or platelet-activating factor (PAF). While the use of PAF already resulted in pregnancies in intrauterine insemination, the suitability of the other substances for the clinical use still needs to be tested. Finally, the isolation of functional spermatozoa from highly viscous ejaculates is a special challenge and can be performed enzymatically to liquefy the ejaculate. The older method, by which the ejaculate is forcefully aspirated through a narrow-gauge needle, should be abandoned as it can severely damage spermatozoa, thus resulting in immotile sperm.

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Sperm preparation for ART.

Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans, cows, and mice. Two basic cryopreservation techniques rule the field--controlled-rate freezing, the first to be developed, and vitrification, which, in recent years, has gained a foothold. While much progress has been achieved in human medicine, the cattle industry, and in laboratory animals, this is far from being the case for most other mammals and even less so for other vertebrates. The major strides and obstacles in human and other vertebrate oocyte and embryo cryopreservation will be reviewed here.

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Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification

Bovine oocytes were matured, fertilized, and cultured (TCM 199 with serum and co-culture) in vitro (IVMFC) with addition, during different phases of the procedure, of antioxidants: superoxide dismutase (SOD) and reduced glutathione (GSH). The addition of SOD (1,500 or 3,000 IU/ml) did not improve proportions of oocytes undergoing cleavage or the development of embryos to morula and blastocyst stages. The cleavage rates were significantly lower than in the control group (CTR 57.5%) when SOD was present during the insemination interval (IVF) or throughout the entire procedure (IVMFC). Thus when the lower concentration was present for IVF and IVMFC, 35.1% and 36.4% of inseminated oocytes cleaved (P < 0.01 compared to CTR) and cleavage results with the higher concentration during IVF and IVMFC were 38.5% and 29.2% (P < 0.025 and P < 0.001 compared to CTR, respectively). Significant improvements in proportions of oocytes undergoing cleavage (84.5% vs. 57.0%, P < 0.001) and morula/blastocyst development (33.3% vs. 13.9%, P < 0.005) were achieved when GSH (1 mM) was added to the culture medium. In a defined medium for culture (mSOF and BSA) the presence of SOD (3,000 IU/ml) was ineffective, but in a defined medium supplemented with GSH (1 mM) at day 6 postinsemination (i.e., when 90% of developing embryos were in 8–16 cell stages), development to the morula and blastocyst stages was supported for 35.5% of cultured oocytes (P < 0.005 compared to 19.2% for CTR). These data suggest that bovine embryos are sensitive to oxidative stress and that medium supplementation with the radical scavenger glutathione can improve embryo development in vitro. © 1996 Wiley-Liss, Inc.

/pdf/improvement-in-bovine-embryo-production-in-vitro-by-41lwsxx8i6.pdf

Improvement in bovine embryo production in vitro by glutathione-containing culture media.

Factors involved in vivo and in vitro maturation of canine oocytes.

Gamete cryopreservation in the domestic cat

A study was undertaken to estimate gestational age, in terms of days from parturition, in medium and small size dogs by ultrasonographic examination. Serial ultrasonographic examinations were performed in four medium size pregnant bitches throughout two consecutive pregnancies and three small size pregnant bitches throughout one pregnancy, in order to determine the range of variation in the size of selected fetal structures throughout gestation. Formulae were derived to estimate the expected delivery date for both groups of bitches by measuring anatomical fetal structures, so that this method could be applied to a large number of different breeds. The determination of gestational age could be achieved with reasonable precision by selecting fetal structures. Prediction of parturition date was accurate to within one day by ultrasonographic measurement of the diameter of the gestational sac in early pregnancy and the biparietal diameter in late pregnancy in both small size breeds and medium size breeds.

Determination of gestational age in medium and small size bitches using ultrasonographic fetal measurements.

In bitches, the length of gestation is highly variable when measured from the day of mating, thus prediction of parturition may be greatly inaccurate when determined from this point. The ability to precisely predict the duration of pregnancy is of practical importance for managing parturition or planning caesarean section and this review analyses the methods that can be used to accurately forecast the day of parturition. At the time of breeding, determination of the luteinizing hormone surge and the initial rise in progesterone provide reliable information on gestational length of the bitch, but when pregnancy is ascertained, ultrasonography is the most useful tool to predict the delivery day. In fact, by ultrasonographic measurements of the extra-foetal and foetal structures an accurate prediction can be made both in early and late pregnancy.

Gaia Cecilia Luvoni

Papers

Improvement in bovine embryo production in vitro by glutathione-containing culture media.

Factors involved in vivo and in vitro maturation of canine oocytes.

Gamete cryopreservation in the domestic cat

Determination of gestational age in medium and small size bitches using ultrasonographic fetal measurements.

The Prediction of Parturition Date in Canine Pregnancy