Mammalian oviduct and protection against free oxygen radicals: expression of genes encoding antioxidant enzymes in human and mouse.

Genetic expression of five antioxidant enzymes involved in mechanisms protecting embryos against reactive oxygen species (ROS) was studied in human and mouse oviducts. The presence of transcripts encoding for gamma-glutamylcysteine synthetase (GCS), glutathione peroxidase (GPX), Cu-Zn-superoxide dismutase (Cu-Zn-SOD), Mn-superoxide dismutase (Mn-SOD) and catalase was analysed by use of the reverse transcription-polymerase chain reaction (RT-PCR). Different expression profiles of transcripts encoding for these enzymes were observed between human and mouse oviducts. In the mouse, all transcripts encoding for the enzymes tested were present in oviduct. In human, only transcripts encoding for GPX, Cu-Zn-SOD and catalase were also detected in oviduct. However, GCS and Mn-SOD transcripts were never observed in human oviduct. Cu-Zn-SOD transcripts are relatively highly expressed whatever species. These results suggest that different gene expression patterns of these antioxidant enzymes between human and mouse may reflect the variations in the ability of embryos to develop in vivo and in vitro. However, hormone related-expression of the missing transcripts in human cannot be ruled out.

Expression of genes encoding antioxidant enzymes in human and mouse oocytes during the final stages of maturation.

The mRNA expression of five enzymes: catalase, Cu-Zn-superoxide dismutase (Cu-Zn-SOD), Mn-superoxide dismutase (Mn-SOD), glutathione peroxidase (GPX), and gamma-glutamylcysteine synthetase (GCS) each involved in protection against free radicals was studied in human and mouse oocytes. In the mouse, oocytes were collected at different stages of maturation in order to determine the storage of these transcripts. For the human, germinal vesicle (GV) oocytes harvested during intracytoplasmic sperm injection (ICSI) procedures and failed fertilized metaphase II (MII) oocytes were analysed. Human and mouse were compared in order to determine whether the differential developmental capacity of mouse and human preimplantation embryos in culture could be explained by the variations in the patterns of expression for these enzymes. mRNA expression for these enzymes was examined using reverse transcription-polymerase chain reaction (RT-PCR). In the mouse, all transcripts (except for catalase) were detected, whatever the maturation stage. No qualitative differences were detected between GV and MII oocytes. In human, all the enzymes (except for catalase) were expressed in MII oocytes and Cu-Zn-SOD was particularly highly expressed. Transcripts corresponding to GPX and Mn-SOD were not detected at GV stage but only at MII stage, suggesting that storage could occur between GV and MII stages. However, using 3' end-specific primers for GPX and Mn-SOD, instead of the oligo(dT)(12-18) primer, for the reverse transcription reaction, the transcripts for these antioxidants enzymes have been detected in human oocytes at the GV stage. This suggests the presence of maturation-specific polyadenylation of these transcripts. These enzymes can be considered as markers of cytoplasmic maturation.

Glucose metabolism during the final stage of human oocyte maturation: genetic expression of hexokinase, glucose phosphate isomerase and phosphofructokinase.

The low involvement of glucose metabolism in early preimplantation embryos has suggested the presence of metabolic blocks in the glycolytic pathway. Genetic expression of hexokinase (HK), glucose-6-phosphate isomerase (GPI) or phosphofructokinase (PFK) were qualitatively analysed using the reverse transcription nested polymerase chain reaction (RT-nested PCR) in individual human immature oocytes at the germinal vesicle (GV) stage and in single non-fertilised human metaphase II (MII) oocytes, after IVF or ICSI failures. Transcripts encoding for HK, GPI and PFK were detectable in the majority of the oocytes analysed, and with different expression patterns. GPI transcripts were consistently present and appear to be constitutively expressed during GV and MII stages. In contrast, low quantities of transcripts encoding for HK and PFK were observed in all oocytes analysed. Our data revealed that the metabolic block previously reported at GPI or PFK levels underwent post-transcriptional regulation. The expression profiles of HK and PFK transcripts in GV and MII reflect a low level of transcription or active translation of maternal transcripts during oocyte maturation. Nevertheless, enzymatic activities of HK and PFK have previously been determined in human oocytes. This suggests that HK and PFK may be accumulated in protein (enzyme) form instead of maternal mRNA during human oocyte maturation. The expression pathway of glycolytic metabolism reflects the presence of different mechanisms involved in gene expression/regulation at the transcriptional and translational level and their accumulation during human oocyte maturation.

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

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.

Human oocytes and preimplantation embryos express mRNA for growth hormone receptor.

Human genetic expression of growth hormone receptor (GHR) gene was qualitatively analysed using reverse transcription polymerase chain reaction (RT-PCR) in cumulus cells, immature germinal vesicle (GV) and mature metaphase II (MII) stage oocytes and preimplantation human embryos. The transcripts encoding GHR were detected in cumulus cells and also in naked oocytes, either mature or not. In this case, a nested PCR is needed, as for early embryo preimplantation stages, before genomic activation. The GHR gene is highly expressed from the 4-day morula onwards. This suggests that GHR transcription follows a classical scheme associated with genomic activation. It is probable that, in human, growth hormone plays a role in the final stages of oocyte maturation and early embryogenesis as it does for several other mammalian species.

Prenatal diagnosis of common aneuploidies using multiplex quantitative fluorescent polymerase chain reaction.

OBJECTIVE: Prenatal diagnosis of foetal trisomies is usually performed by cytogenetic analysis. This requires lengthy laboratory procedures and it is expensive. Here, we report a retrospective study of quantitative fluorescent polymerase chain reaction (QF-PCR) for prenatal detection of trisomies 13, 18 and 21. METHODS: QF-PCR was performed on a total of 447 amniotic fluids blindly analysed without any knowledge of the cytogenetic results and 43 samples with known karyotype. All samples were tested with at least 4 small tandem repeat markers specific for each chromosome 13, 18 or 21. RESULTS: QF-PCR results on amniotic fluid were consistent with conventional cytogenetic data. QF-PCR detected 5 cases of trisomy 21, 2 cases of trisomy 18, 1 case of trisomy 13 and 1 case with Klinefelter's syndrome. CONCLUSIONS: QF-PCR has proved to be very useful in clinical settings, since it allows the detection of major numerical disorders in a few hours after sampling and thus reduces parental anxiety.