The interactions between cysteamine, cystine and cumulus cells increase the intracellular glutathione level and developmental capacity of goat cumulus-denuded oocytes.

To improve in vitro maturation (IVM) of denuded oocytes (DOs), we observed the interactive effects of cysteamine, cystine and cumulus cells on the glutathione (L-gamma-glutamyl-L-cysteinyl-glycine; GSH) level and developmental capacity of goat IVM oocytes. Cysteamine supplementation increased the GSH level and blastocyst rates of both cumulus-oocyte complexes (COCs) and DOs, while the addition of cystine increased the GSH level and blastulation only in the presence of cumulus cells (COCs or DOs co-cultured on a cumulus cell monolayer). Simultaneous supplementation of cysteamine and cystine increased the GSH content and blastulation of co-cultured DOs to a level similar to that of COCs matured without thiol supplementation. Co-culture without thiol supplementation improved DOs' GSH synthesis but not blastulation. The results suggest that DOs cannot utilize cystine for GSH synthesis unless exogenous cysteamine is supplied by either cumulus cells or supplementation. Thus, while the addition of cystine alone is enough to improve IVM of COCs, improvement of DOs requires supplementation of both cystine and cysteamine. Synergic actions between cysteamine, cystine and cumulus cells restore the GSH level and developmental capacity of goat DOs.

Zhou P ，Wu YG ，Li Q ，Lan GC ，Wang G ，Gao D ，Tan JH ... -  《-》

Mouse cumulus-denuded oocytes restore developmental capacity completely when matured with optimal supplementation of cysteamine, cystine, and cumulus cells.

Our objectives were to study how cysteamine, cystine, and cumulus cells (CCs), as well as oocytes interact to increase oocyte intracellular glutathione (GSH) and thereby to establish an efficient in vitro maturation system for cumulus-denuded oocytes (DOs). Using M16 that contained no thiol as maturation medium, we showed that when supplemented alone, neither cystine nor cysteamine promoted GSH synthesis of mouse DOs, but they did when used together. Although goat CCs required either cysteamine or cystine to promote GSH synthesis, mouse CCs required both. In the presence of cystine, goat CCs produced cysteine but mouse CCs did not. Cysteamine reduced cystine to cysteine in cell-free M16. When TCM-199 that contained 83 microM cystine was used as maturation medium, supplementation with cysteamine alone had no effect, but supplementation with 100 microM cysteamine and 200 microM cystine increased blastulation of DOs matured with CC coculture to a level as high as achieved in cumulus-surrounded oocytes (COCs). Similar numbers of young were produced after two-cell embryos from mouse COCs or CC-cocultured DOs matured with optimal thiol supplementation were transferred to pseudopregnant recipients. It is concluded that 1) mouse CCs can use neither cysteamine nor cystine to promote GSH synthesis, but goat CCs can use either one; 2) goat CCs promote mouse oocyte GSH synthesis by reducing cystine to cysteine, but how they use cysteamine requires further investigation; and 3) mouse DOs can use neither cystine nor cysteamine for GSH synthesis, but they restore developmental capacity completely when matured in the presence of optimum supplementation of cysteamine, cystine, and CCs.

Zhou P ，Wu YG ，Wei DL ，Li Q ，Wang G ，Zhang J ，Luo MJ ，Tan JH ... -  《-》

Glutathione content and glutathione peroxidase expression in in vivo and in vitro matured equine oocytes.

The in vitro developmental competence of equine oocytes is still low in comparison with other domestic mammals. A major factor affecting the viability of cells during in vitro culture is the increased oxidative stress. Oxidative modifications could be responsible for oocyte incompetence for in vitro maturation (IVM). Cysteamine, a glutathione (GSH) synthesis enhancer, has been shown to increase intracellular GSH content and to improve embryo development when added during IVM of bovine, porcine, and ovine oocytes. The aim of the present study was (1) to determine whether equine cumulus-oocyte complexes (COCs) benefit from the addition of cysteamine during IVM, (2) to compare the GSH content of oocytes after in vivo maturation and IVM, (3) to assess whether cysteamine administration during IVM of equine oocyte enhances early embryonic developmental capability following ICSI, (4) to study the glutathione peroxidase (GPX) mRNA level in COCs. In vivo matured COCs were collected by aspiration from preovulatory follicles, and analyzed at collection. Immature COCs were collected in vivo or from slaughterhouse ovaries and matured in culture media supplemented or not with 100 microM cysteamine. After nuclear stage assessment, oocytes were analyzed for GSH concentration and both oocytes and cumulus cells were analyzed for GPX and GAPDH mRNA. Our data showed that the maturation capability was similar in both in vivo aspirated oocytes and in those isolated from slaughterhouse ovaries. Moreover, the addition of cysteamine during IVM affected neither GSH content nor maturation rate. At the time of collection, intra-oocyte GSH content was not influenced by the chromatin status. GSH concentration was similar in in vivo and in vitro matured metaphase II (MII) stage oocytes, and was significantly higher in MII than immature germinal vesicle stage oocytes. Moreover, the presence of serum inhibited whereas its absence stimulated the accumulation of GSH within oocytes during IVM. After ICSI, a similar proportion of zygotes in each group developed beyond the two-cell stage after 72 hr of culture. Cumulus cells expressed GPX mRNA, while GPX transcript was absent in both immature and mature oocytes. Cumulus expression of GPX mRNA was significantly higher when analyzed at collection than after IVM. Taken together, our results demonstrate that in equine oocytes, GSH increases during IVM but the relative intra-oocyte content of this thiol does not affect maturation and early development efficiency after fertilization. We hypothesize that factor(s) other than GSH/GPX are responsible for the limited in vitro early developmental capability of equine oocytes.

Luciano AM ，Goudet G ，Perazzoli F ，Lahuec C ，Gérard N ... -  《MOLECULAR REPRODUCTION AND DEVELOPMENT》

Effects on in vitro embryo development and intracellular glutathione content of the presence of thiol compounds during maturation of prepubertal goat oocytes.

The low number of embryos produced from in vitro matured, fertilized, and cultured (IVM-IVF-IVC) oocytes of prepubertal goat is mainly due to a low incidence of sperm head decondensation at fertilization (Martino et al., 1995: Theriogenology 43:473-485; Mogas et al., 1997: Theriogenology 48:815-829). Thiol compounds stimulate glutathione (GSH) synthesis and improve the rates of male pronucleus (MPN) formation and embryo development. The present study was carried out to determine whether supplementation of the IVM medium with 100 microM of cysteamine, 100 microM of beta-mercaptoethanol, 0.57 mM of cysteine, and 0.57 mM cystine might improve the embryo development and intracellular GSH level of prepubertal goat oocytes. After 27 hr post IVM, a sample of oocytes was frozen and the intracytoplasmic GSH content was evaluated by spectrophotometry. IVM-oocytes were inseminated with fresh semen and cultured in SOF medium. Only the addition of cysteamine to IVM media significantly improved the percentage of the morula plus blastocyst yield compared to the control group (oocytes matured in absence of thiol compounds) (22.2 vs. 6.4%, respectively; P < 0.05). The percentage of expanded blastocysts in cysteamine and control groups was 13.0 and 2.6%, respectively, and the mean cell number per blastocyst was 86.8 and 60.5, respectively. None of the other thiol compounds studied significantly improved the percentage of embryos obtained. It has been demonstrated that prepubertal goat oocytes synthesize GSH during IVM and that thiol compounds increase this GSH synthesis. In conclusion, only the addition of 100 microM of cysteamine to the maturation medium improves embryo development from prepubertal goat oocytes although all the thiol compounds used in this study increased intracellular GSH content.

Rodríguez-González E ，López-Bejar M ，Mertens MJ ，Paramio MT ... -  《MOLECULAR REPRODUCTION AND DEVELOPMENT》

Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development.

The purpose of this study was to evaluate whether enriching the oocyte in vitro maturation medium with cystine, in the presence of cysteamine, would improve the in vitro embryo production efficiency in buffalo by further increasing the GSH reservoir created by the oocyte during maturation. Cumulus-oocytes complexes were matured in vitro in TCM 199 + 10% FCS, 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 0.3mM cystine. In Experiment 1, glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in representative samples of oocytes matured in the four different experimental conditions. In Experiment 2, oocytes were fixed and stained to assess nuclear maturation and normal pronuclear development following IVM and IVF respectively. In Experiment 3, mature oocytes were in vitro fertilized and cultured to assess development to blastocysts. In all supplemented groups the intracytoplasmic GSH concentration was significantly higher than the control, with the highest GSH levels in oocytes matured in the presence of both thiol compounds (3.6, 4.7, 5.4 and 6.9 picomol/oocyte in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Cystine supplementation of IVM medium, both in the presence or absence of cysteamine, significantly increased the proportion of oocytes showing two normal synchronous pronuclei following fertilization. In all supplemented groups, cleavage rate was significantly improved compared to the control (55, 66.1, 73.5 and 78.4% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Similarly, blastocyst yield was also increased in the three enriched groups compared to the control (17.1, 23.8, 29.3, 30.9% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Overall, the addition of cystine to a cysteamine-enriched medium resulted in a significant increase of cleavage rate and transferable embryo yield compared to the medium supplemented with only cysteamine.

Gasparrini B ，Boccia L ，Marchandise J ，Di Palo R ，George F ，Donnay I ，Zicarelli L ... -  《THERIOGENOLOGY》