Stimulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability

Glutathione (GSH) has been shown to play an important role in embryo development. In a previous study, we demonstrated that cysteamine supplementation of in vitro maturation (IVM) medium increased the intracellular GSH content in bovine oocytes and improved subsequent embryo development to the blastocyst stage. The present study was carried out to evaluate the effect of inhibition by buthionine sulfoximide (BSO) of GSH synthesis during IVM in the presence of cysteamine, on subsequent embryo development, and the effect of cysteamine during IVM on the survival of blastocysts following freezing. The effect of β-mercaptoethanol and cysteine added to the maturation medium on GSH levels in bovine oocytes, as well as the effect of these compounds on de novo GSH synthesis by oocytes during in vitro maturation, was also studied. The inhibitory effect of BSO during in vitro maturation on GSH synthesis was also evaluated. Evidence was found confirming that GSH synthesis occurs intracellularly during IVM of oocytes and is stimulated by cysteamine, β-mercaptoethanol and cysteine. Moreover, the present results suggest that the increase in the rate of embryo development exerted by cysteamine, when present during IVM, was due to its stimulatory effect on GSH synthesis. This increase in GSH levels during IVM improves embryo development and quality, producing more embryos reaching the blastocyst stage on day 6, those most suitable for freezing. © 1996 Wiley-Liss, Inc.     

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.     

Improved development of microspore-derived embryo cultures of Brassica napus cv Topaz following changes in glutathione metabolism

Glutathione has been shown to play an important role during embryo development in both plant and animal systems. The effects of altered glutathione metabolism during microspore-derived embryos (MDEs) of Brassica napus were investigated following exogenous application of reduced glutathione (GSH), its oxidized form (GSSG) and buthionine sulfoximine (BSO), an inhibitor of glutathione de novo synthesis. Applications of BSO which lowered the cellular glutathione redox status, i.e. GSH/(GSH + GSSG), enhanced significantly the quality of the embryos and their ability to convert into viable plants. Histological analyses revealed that inclusions of BSO in the culture medium altered the pattern of storage product accumulation in the embryos and improved the architecture of the shoot apical meristems (SAMs). Compared with their control counterparts which showed severe signs of SAM deterioration, such as the formation of intercellular spaces and differentiation of the meristematic cells, BSO-treated embryos had well-organized SAMs. The improved SAM organization observed in the presence of BSO also correlated with the proper localization pattern of WUSCHEL , a SAM molecular marker gene which was miss-expressed in control embryos. The beneficial effects of BSO on embryo development and conversion were ascribed to the increasing levels of ABA. The concentration of this growth regulator in BSO-treated embryos was always higher than that of control embryos during the second half of the maturation period. Furthermore, many structural alterations induced by BSO could be reproduced in embryos cultured in the presence of ABA. Taken together, these results suggest that a lowering of the glutathione redox status during embryo development may represent a metabolic switch needed for increasing the endogenous levels of ABA, which is required for successful completion of the developmental program.     

Glutamine uptake and utilization by preimplantation mouse embryos in CZB medium

At least 71% of CF1 x B6SJLF1/J embryos developed from the 1-cell stage to the blastocyst stage in an optimum glutamine concentration of 1 mM, as long as glucose was present after the first 48 h of culture. Blastocysts raised under these conditions had significantly more cells than did blastocysts raised in CZB medium alone (glutamine present, glucose absent). Embryos raised in vivo accumulated 170-200 fmol glutamine/embryo/h at the unfertilized egg and 1-cell stages with a decline to 145 fmol/embryo/h at the 2-cell stage, followed by sharp increases to 400 and 850 fmol/embryo/h at the 8-cell and blastocyst stages. The presence or absence of glucose in the labelling medium had no effect on glutamine uptake by these embryos. Embryos raised in vitro accumulated 2-3 times more glutamine at stages comparable to those of embryos raised in vivo. In all cases in which 1-cell to blastocyst development in vitro was successful, glucose was present in the culture medium and the incremental uptake of glutamine between the 8-cell stage and the blastocyst stage was approximately 2-fold. This was also the increment for in-vivo raised embryos. When glucose was not present after the first 48 h, the 8-cell to blastocyst glutamine increment was not significant, and development into blastocysts was reduced. The results also show that glutamine can be used as an energy source for the generation of CO2 through the TCA cycle by all stages of preimplantation mouse development, whether raised in vivo or in vitro from the 1-cell stage. Two-cell embryos raised in vivo converted as much as 70% of the glutamine uptake into CO2, consistent with an important role for glutamine in the very earliest stages of preimplantation development. Cultured blastocysts appeared to convert less glutamine and the presence of glucose in the culture medium seemed to inhibit this conversion.     

Potent and stage-specific action of glutathione on the development of goat early embryos in vitro

The effect of glutathione (GSH) addition on the development of 1- or 2-cell goat early embryos in vitro was examined. Embryos were collected from superovulated Korean black goat (Capra hircus aegagrus) and cultured for 6 days in synthetic oviduct fluid medium supplemented with either bovine serum albumin (BSA) or serum. Without GSH addition, almost all embryos could not develop beyond 8- to 16-cell block. However, GSH addition greatly improved in vitro development of early embryos to blastocyst stage, and its action was highly dependent on the presence and source of proteins supplemented into the culture medium. Among the protein-supplemented cultures, GSH effect was most prominent in 10% FBS-supplemented culture, in which the proportion (91%) of blastocysts developed from early embryos was much higher than that of BSA- (42-64% depending on its content) or goat serum (GS)-supplemented cultures (21%), or even than that of somatic cell-supported co-culture (60%). As well as in terms of the morphological development, mean cell number of blastocysts (185 +/- 12) developed from FBS condition was significantly higher than that of blastocysts developed from any other culture conditions and moreover comparable to that of blastocysts developed in vivo (190 +/- 9). The viability of these blastocysts was finally confirmed by their term development (6/12) from embryo transfer. To delineate action time of GSH during embryo development, GSH was treated at 1-day intervals through 6-days culture periods excepting the last day. In the GSH-treated embryos at day 3 of culture, which corresponds to the time of in vitro 8- to 16-cell block stage, the proportion of blastocyst was markedly increased up to 77% of cultured embryos and conversely that of the arrested embryos was decreased to 7%. In the embryos treated later, however, their developmental potency decreased abruptly. Therefore, these results clearly demonstrated that GSH could greatly improve the in vitro development of goat early embryos by specifically acting on the 8- to 16-cell block stage during in vitro development, suggesting that GSH may be one of the important regulators on the development of goat embryos in vivo.     

Glutathione synthesis during in vitro maturation of buffalo (Bubalus bubalis) oocytes: effects of cysteamine on embryo development

It was demonstrated that cysteamine supplementation during in vitro maturation (IVM) improves embryo development by increasing glutathione (GSH) synthesis in several species. An improved developmental competence of oocytes matured in the presence of cysteamine was also recorded in buffalo species. The purpose of this work was to investigate (1) if glutathione is de novo synthesized during in vitro maturation of buffalo oocytes, (2) if cysteamine improves buffalo embryo development via an increase in GSH synthesis, and (3) if the inhibition of glutathione synthesis by buthionine sulfoximide (BSO), in the presence or absence of cysteamine, affects subsequent embryo development and GSH synthesis.Cumulus-oocytes complexes (COCs), recovered from slaughtered animals, were matured in vitro in TCM199+10% fetal calf serum (FCS), 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17-beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 5mM BSO. Glutathione content was measured by high-performance liquid chromatography (HPLC) and fluorimetric analysis in immature oocytes and in oocytes matured in the different experimental conditions.In a second experiment, the mature oocytes were in vitro fertilized and cultured for 7 days in order to assess development to blastocysts (BLs). It was demonstrated that buffalo oocytes synthesize glutathione during in vitro maturation and that cysteamine increases glutathione synthesis. Furthermore, the promoting effects of cysteamine on embryo development and GSH synthesis were neutralized by buthionine sulfoximide. These results indicate that glutathione plays a critical role on buffalo embryo development.     

Development and DNA polymerase activities in cultured preimplantation mouse embryos: comparison with embryos developed in vivo

Embryos from superovulated female mice that developed in vitro from the two-cell stage were compared with in vivo embryos with respect to yield of blastocytes, number and types of cells, morphology in histologic section, and DNA polymerase activities. Significantly more embryos developed into blastocytes in vitro (93%) than in vivo (18%). Inner cell mass (ICM) cells comprised approximately 30% of total cells in late morula/early blastocyst stage embryos developed either in vitro or in vivo. However, the in vitro embryos developed approximately half the number of total cells as in vivo embryos, did not develop endoderm, and did not develop abembryonic trophoblast cells with morphologic characteristics of late preimplantation in vivo embryos. DNA-dependent DNA polymerase activities in in vitro embryos decreased in correspondence with the decrease in cell number resulting in per cell levels comparable to in vivo embryos. In contrast, the poly (A).oligo(dT)-dependent DNA polymerase activity was the same in embryos developing either in vitro or in vivo, indicating different regulatory mechanisms for the two enzyme activities. A variety of nutrients and growth factors in the culture medium did not increase cell numbers or DNA polymerase activities in embryos cultured for 3 days; extending the culture an additional 24 hours resulted in a loss of ICM cells and decreases in both DNA polymerase activities. These results show that the retarded growth of embryos in vitro is equally distributed between ICM and trophoblast, is not reversed by culture conditions that include serum growth factors, and is not due to decreased cellular levels of DNA polymerase activities.     

Expression and downregulation of WNT signaling pathway genes in rhesus monkey oocytes and embryos

Mammalian WNT genes encode secreted glycoproteins that are conserved homologues of the Drosophila Wingless gene, which plays a crucial role in Drosophila development. Recently, WNT pathway signaling has been implicated in ovarian development, oogenesis, and early development. We sought to evaluate whether these genes may contribute to the formation of healthy human oocytes or embryos, and whether the expression of these genes could provide informative markers of human oocyte and embryo quality. To do this, we employed the primate embryo gene expression resource (PREGER; www.preger.org) to examine expression of mRNAs encoding 38 components of the WNT signaling pathway in rhesus monkey oocytes and embryos as a nonhuman primate model. We observed considerable conservation between rhesus monkey and mouse of expression of WNT, FZD, and effector gene mRNAs, and a generalized downregulation of genes encoding key components of the WNT signaling pathway during preimplantation development. Our results support a role for WNT signaling during oocyte growth or maturation, but not during preimplantation development. Additionally, we observed differences between in vitro cultured and in vivo developing blastocysts, indicating possible effects of culture on WNT signaling during the peri-implantation period.     

Buffalo rat liver cells produce factors that support preimplantation development of mouse embryos cultured in vitro

To examine the effects of buffalo rat liver (BRL) cells on the preimplantation development of mouse embryos in vitro, we first cultured two-cell mouse embryos alone in serum-free Dulbecco modified Eagle medium. As expected, the embryos did not develop to subsequent stages. However, when cocultured with BRL cells, the embryos developed to the blastocyst stage efficiently. Direct contact of embryos with BRL cells was not necessary for development: the medium conditioned by BRL cells contained soluble factors that supported the preimplantation development of mouse embryos. Embryos cultured with BRL-conditioned medium that was replaced at various intervals had a further increased rate of development to the blastocyst stage. This finding indicated that the activities of the factors were maintained only briefly. Seven proteins between 35 and 44 kDa that were detected in the medium were highly beneficial to the development of the embryos. Follistatin-related protein and pigment epithelium-derived factor are believed to be the factors supporting embryo development. The other five proteins also may improve the environment for the development of mouse embryos cultured in vitro.     

Effect of glutathione synthesis stimulation during in vitro maturation of ovine oocytes on embryo development and intracellular peroxide content

Cysteamine and beta-mercaptoethanol supplementation of in vitro maturation (IVM) medium has been found to increase intracellular glutathione (GSH) content in oocytes and to improve embryo development and quality in several species. The objective of this experiment was to study the effect of cysteamine and beta-mercaptoethanol added during IVM of sheep oocytes on GSH synthesis and embryo development. Furthermore, we examined if cysteamine addition (hence GSH production) had an effect on the reduction of the intracellular peroxide content. We matured oocytes obtained from ovaries collected at a slaughterhouse in vitro in the presence of 0, 50, 100, and 200 microM cysteamine (Experiment 1) or with 0, 50, 100, and 200 microM beta-mercaptoethanol (Experiment 2). Following fertilization and embryo development, there was a increasing level of morula and blastocyst development in the presence of cysteamine, reaching significance in the presence of 200 microM (P < 0.05). However, beta-mercaptoethanol did not influence on the rate of embryo development. GSH levels were measured in oocytes matured in the presence or absence of 200 microM cysteamine (Experiment 3) or 50 microM beta-mercaptoethanol (Experiment 4), with or without buthionine sulfoximide (BSO), an inhibitor of GSH synthesis. Results demonstrated that for both cysteamine and beta-mercaptoethanol, intracellular GSH levels increased against control values (P < 0.01), which was abolished in the presence of BSO. Finally, we reduced intracellular peroxide levels, as measured by the relative fluorescence of the intracellular peroxide probe, carboxy-H2DCFDA, in the presence of either 200 microM cysteamine or 50 microM beta-mercaptoethanol (Experiment 5). These results demonstrate that cysteamine, but not beta-mercaptoethanol, when present during IVM, stimulates sheep embryo development; both cysteamine and beta-mercaptoethanol stimulate GSH synthesis; the increase in intracellular GSH is associated with a decrease in peroxide levels within oocytes.     

Maturation of arabidopsis seeds is dependent on glutathione biosynthesis within the embryo

Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, gamma-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoterbeta-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.     

Influence of short-term maternal zinc deficiency on the in vitro development of preimplantation mouse embryos

In this study, we evaluated the use of mouse preimplantation embryos as a model to study zinc deficiency-induced abnormal development. In Experiment 1, the effect of culture medium Zn concentrations on blastocyst development was studied. Preimplantation embryos (2 and 4 cells) obtained from superovulated females developed normally in media containing 0.7-30 microM Zn for up to 72 hr; higher levels of medium Zn resulted in abnormal development. In Experiment 2A, females were fed diets containing 50 (+Zn) or 0.4 (-Zn) micrograms Zn/g (760 vs 6 nmol/g, respectively) from 1 day before to 1 day after mating (3 days total). Preimplantation embryos were removed from the dams and cultured for 72 hr in 0.7 microM Zn medium. Embryos from the -Zn dams were morphologically normal at time zero; however, over the 72-hr period, these embryos tended to develop at a slower rate than controls, although compaction and cavitation frequency were similar. By the end of the 72-hr culture period, embryos from -Zn dams had significantly fewer cells than did embryos from control dams. In Experiment 2B, an extended period of maternal Zn deprivation (6 days) was used to investigate the potential for further impairment of in vitro preimplantation embryo development observed in Experiment 2A. Results from this experiment were consistent with those from Experiment 2A, in addition to providing evidence that the developmental progress of embryos obtained from mice fed Zn-deficient diets for 6 days was significantly impaired. In Experiment 3, the potential for supplemental Zn in culture medium to overcome the impairment in development due to maternal Zn deficiency was investigated. Embryos from female mice subjected to the same dietary regimen described in Experiment 2A were cultured to the blastocyst stage in medium containing Zn at a concentration of either 0.7 or 7.7 microM. Medium Zn supplementation did not improve development of embryos from dams fed Zn-deficient diets. In summary, embryos from mice fed -Zn diets for a 3- or 6-day period encompassing oocyte maturation and fertilization exhibited impaired development in vitro. This impairment was not overcome by medium Zn supplementation.     

Triglyceride content of bovine oocytes and early embryos

A microfluorescence technique was used to measure the triglyceride content of a minimum of two bovine oocytes or preimplantation embryos up to the hatched blastocyst stage. Embryos were produced in vitro from abattoir-derived ovaries and grown in medium containing synthetic oviductal fluid, amino acids and BSA (SOFaaBSA medium); 10% fetal calf serum was added to some of the embryos at the four-cell stage. Before maturation, the triglyceride content of oocytes was 59 +/- 1.37 ng and it decreased (P < 0.05) after maturation to 46 +/- 0.85 ng. A decrease in triglyceride content (P < 0.05) was also observed after fertilization with the formation of the two-cell embryo (34 +/- 1.80 ng). In the absence of serum, the triglyceride content remained relatively constant from the two-cell to the hatched blastocyst stage. The triglyceride content of blastocysts produced in vivo was similar (33 +/- 0.70 ng) to that of blastocysts produced in vitro in the absence of serum. In contrast, the triglyceride content of embryos grown with 10% fetal calf serum increased steadily from the 9-16-cell stage to a value in hatched blastocysts (62 +/- 1.14 ng) almost double that in serum-free conditions. These results indicate that triglyceride may act as energy source during bovine oocyte maturation and fertilization and that the presence of serum causes excessive synthesis or accumulation of triglyceride in early embryos.     

In vitro-produced equine embryos: production of foals after transfer, assessment by differential staining and effect of medium calcium concentrations during culture

Viability of equine embryos produced by oocyte maturation, intracytoplasmic sperm injection and embryo culture to the blastocyst stage in vitro was evaluated after transfer of embryos to recipient mares. No pregnancies were produced after transfer of five blastocysts that had been cultured in G media. Transfer of 10 blastocysts cultured in modified DMEM/F-12 medium produced five pregnancies and three live foals; the two lost pregnancies developed only trophoblast (based on transrectal ultrasonography). To evaluate the status of the inner cell mass, equine blastocysts produced in vivo and in vitro were assessed after differential staining. A discrete inner cell mass could not be appreciated in blastocysts of either source after staining; this was attributed to the presence of a network of cells within the trophoblastic vesicle. Because increased medium calcium concentrations have been reported to decrease the incidence of trophoblast-only pregnancy after transfer of equine nuclear transfer embryos, we investigated the effect of increased calcium concentrations during oocyte maturation or during embryo culture. Increasing calcium concentration of culture medium from 2 to 5.6mM during in vitro oocyte maturation did not affect maturation rate (75 and 68%, respectively) or blastocyst development after fertilization (23 and 27%). However, increasing calcium concentration (from 1.3 to 4.9 mM) of medium used for embryo culture significantly decreased blastocyst development (27% versus 13%, respectively) and adversely affected embryo morphology. More work is needed to optimize culture systems for in vitro production of equine embryos.     

Analysis of HLA-G in maternal plasma, follicular fluid, and preimplantation embryos reveal an asymmetric pattern of expression

Soluble HLA-G (sHLA-G) secretion by human preimplantation embryos in culture has been associated with successful embryo development, and therefore has potential to serve as a noninvasive marker of embryo viability. We have examined the spatial and temporal expression of HLA-G in embryos of varying developmental competence and the role of maternal factors in human embryonic HLA-G expression. Embryos that reached blastocyst stage on day 5 showed a higher frequency of sHLA-G secretion than those at morula or arrested stages (p < 0.05). There was no significant difference in sHLA-G secretion between normal embryos and those diagnosed as chromosomally abnormal by preimplantation genetic diagnosis. HLA-G detected in maternal plasma and follicular fluid did not appear to correlate with HLA-G expressed in the embryo or embryo supernatants. Confocal microscopy analysis indicated that HLA-G protein expression in embryos was not homogeneous; mostly, it was confined to blastocysts localized on trophectoderm and trophectoderm projections. Single-particle fluorescent imaging analysis of HLA-G on the cell surface of JEG-3 cells showed that HLA-G particles were mostly monomeric, but dimeric and higher order oligomers were also observed. These results suggest that HLA-G play an important role in preimplantation embryo development. However, the observed expression of HLA-G in arrested and chromosomally abnormal embryos indicates that HLA-G testing should be used with caution and in conjunction with conventional methods of embryo screening and selection.     

Metabolite availability as a window to view the early embryo microenvironment in vivo

A preimplantation embryo exists independent of blood supply, and relies on energy sources from its in vivo environment (e.g., oviduct and uterine fluid) to sustain its development. The embryos can survive in this aqueous environment because it contains amino acids, proteins, lactate, pyruvate, oxygen, glucose, antioxidants, ions, growth factors, hormones, and phospholipids—albeit the concentration of each component varies by species, stage of the estrous cycle, and anatomical location. The dynamic nature of this environment sustains early development from the one‐cell zygote to blastocyst, and is reciprocally influenced by the embryo at each embryonic stage. Focusing on embryo metabolism allowed us to identify how the local environment was deliberately selected to meet the dynamic needs of the preimplantation embryo, and helped reveal approaches to improve the in vitro culture of human embryos for improved implantation rates and pregnancy outcome.     

A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development

The potential role of endogenous triglyceride in bovine oocyte maturation and preimplantation development has been investigated. Bovine immature oocytes were recovered from abattoir-derived ovaries, matured and fertilised in vitro and the zygotes grown to the blastocyst stage in SOFaaBSA. Methyl palmoxirate (MP) blocks the oxidation of fatty acids by inhibiting mitochondrial carnitine palmitoyltransferase A. The development of zygotes exposed to MP during oocyte maturation, and of zygotes exposed to MP during embryo culture has been assessed in terms of oxygen consumption by oocytes and embryos during a 4-6 hr incubation period in the presence of MP and as blastocyst formation and cell number. Immature oocytes exposed to MP during maturation had reduced capacity to form blastocysts after fertilisation; the same effect was apparent, but to a lesser extent, in zygotes exposed to MP during embryo development. Oxygen consumption values of oocytes and blastocysts in the absence of exogenous substrates were similar to those in control medium containing nutrients. MP-inhibited oxygen consumption of immature oocytes, mature oocytes, cleavage stages embryos and blastocysts by 64, 45, 12 and 13%, respectively. The data are consistent with a role for triglyceride as a key energy source during bovine oocyte maturation and potentially, during preimplantation embryo development.     

Cumulus expansion during in vitro maturation of bovine oocytes: Relationship with intracellular glutathione level and its role on subsequent embryo development

Glutamine (GLN) is a metabolic precursor for hexosamine synthesis and its inclusion in culture medium has been reported to improve cumulus expansion. Glutamine and cysteine share the same transport system. Excess external GLN may act as a competitive inhibitor for the uptake of cysteine and stimulate loss of cellular cysteine, interfering this with GSH synthesis. Experiments were designed to evaluate the effect of 1–3 mM GLN during in vitro maturation (IVM) on bovine-cumulus expansion, intracellular GSH levels in both oocytes and cumulus cells, and subsequent embryo development up to blastocyst stage. Also, GSH content was measured in 6- to 8-cell embryos and a possible relationship between cumulus expansion and GSH synthesis was studied. Intact cumulus cell-oocyte complexes were incubated for 24 hr and cumulus expansion was measured by a computerized image-digitizing system either before or after IVM. IVM/IVF bovine oocytes were cultured up to 6- to 8-cell stage embryos for assessment of GSH content or for 8 days up to blastocyst stage for embryo development. The measurement of total GSH content was performed by an enzymatic method in oocytes, cumulus cells and 6- to 8-cell embryos. The maximal expansion was achieved by addition of 2 mM GLN without affecting GSH levels, in both oocytes and cumulus cells. At 3 mM, the degree of cumulus expansion was lower and the GSH levels decreased. The addition of 2 mM GLN improves cleavage and blastocyst rates, whereas no differences were found between 0, 1, and 3 mM GLN. Moreover, the GSH content in 6- to 8-cell embryos was similar at any GLN concentrations. In order to study the relationship between GSH and cumulus expansion: 6-diazo-5-oxo-1-norleucine (DON), an inhibitor of hexosamine synthesis, or buthionine sulfoximide (BSO), an inhibitor of GSH synthesis, either alone or with GLN was added to IVM medium. GSH level was not affected by the presence of DON. However, the degree of cumulus expansion was reduced in the presence of BSO. In conclusion, bovine oocytes matured in the presence of 2 mM GLN improve their capacity for subsequent embryo development. Nevertheless, GSH level was altered when GLN was added to IVM medium at a high concentration with a reduction in the degree of cumulus expansion. This study provides evidence that optimal cumulus expansion in vitro is partially dependent on hexosamine production and intracellular GSH content. Mol. Reprod. Dev. 51:76–83, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of apoptosis in the bovine blastocyst by insulin and the insulin-like growth factor (IGF) superfamily

Insulin and the insulin-like growth factors, IGF-I and IGF-II, have been reported to exert a mitogenic effect on the preimplantation mammalian embryo. Furthermore, it has been proposed that loss of imprinting of the insulin-like growth factor II receptor gene and the consequent over-production of IGF-II may be involved in the aetiology of the Enlarged Offspring Syndrome, which occurs as an artefact of in vitro embryo production. We have previously shown that apoptosis occurs in the preimplantation bovine embryo and is influenced by in vitro culture conditions. We have therefore sought to establish the effects of insulin, IGF-I and IGF-II on apoptosis and cell proliferation in bovine blastocysts in vitro. Zygotes, obtained by in vitro maturation and fertilization of follicular oocytes, were cultured to blastocysts, with or without exogenous growth factors. Embryos were stained with propidium iodide to label all nuclei and by TUNEL to label apoptotic nuclei and analyzed by epifluorescent and confocal microscopy. IGF-I and IGF-II, but not insulin, were found to increase the proportion of embryos which formed blastocysts. Insulin decreased the incidence of apoptosis without affecting blastocyst cell number. IGF-I acted to decrease apoptosis and increase total cell number and IGF-II increased cell number alone. These data suggest roles for insulin and the IGFs as mitogens and/or apoptotic survival factors during early bovine development. Perturbation of IGF-II regulated growth may be involved in fetal oversize.