Isabelle Donnay

Apoptosis at the time of embryo implantation in mouse and rat.

The aim of this review is to summarize the information currently available regarding the occurrence of apoptosis in the developing embryo and in the receptive uterus during the peri-implantation period of gestation. Cell death is detected in the inner cell mass of late pre-implantation embryos as the result of an eliminative process that helps trim the embryonic cell lineages of surplus or dysfunctional stem cells. Cell death is also detected in the epiblastic core of early post-implantation embryos, where the process is implicated in the formation of the pro-amniotic cavity. On the maternal side, uterine epithelial cells situated around the attachment site undergo cell death during the initial phase of implantation in order to facilitate embryo anchorage and access to maternal blood supply. Uterine stromal cells closest to the implantation chamber first transform into decidual cells and then commit suicide to make room for the rapidly growing embryo. Although apoptosis is well recognized as a crucial determinant of successful peri-implantation development, our understanding of the cellular and molecular mechanisms regulating this process clearly lags behind the comprehension of cell death control in other systems.

Cloning of bovine peroxiredoxins-gene expression in bovine tissues and amino acid sequence comparison with rat, mouse and primate peroxiredoxins.

The peroxiredoxin (PRDX) family is a recently identified family of peroxidases found in organisms ranging from bacteria to mammals. In mammals, six PRDX isoforms have been characterized in human (Homo sapiens), rat (Rattus norvegicus) and mouse (Mus musculus). PRDXs are cytosolic, secreted or targeted to organelles such as peroxisomes, mitochondria and the nucleus. Some PRDXs are synthesized as larger precursor proteins with a presequence that is cleaved to produce the mature form. To study the expression of the six PRDXs in bovine (Bos taurus), we first cloned cDNAs coding for PRDX1, PRDX2, PRDX4 and PRDX5. PRDX3 and PRDX6 had previously been cloned and characterized in bovine. The comparison of bovine PRDXs with their rat, mouse and primate orthologues reveals a minimum of 95% similarity of mature proteins. Even though mitochondrial or export signal presequences are normally less conserved, the unprocessed proteins still present a minimum of 84% similarity. Nevertheless, a major divergence lies at the N-terminus of bovine PRDX2, where a Cys-Val-Cys motif was identified. The expression of the six PRDXs in 22 bovine tissues has been studied by RT-PCR. Our results point out the ubiquity of the different PRDX transcripts in bovine tissues. The important conservation of the different PRDXs, the multiple processes they have been associated with, as well as the ubiquity of all the members of the family analyzed in this study for the first time altogether, suggest that they play a major role in the basal metabolism of mammalian cells.

Cell Cycle Duration at the Time of Maternal Zygotic Transition for In Vitro Produced Bovine Embryos: Effect of Oxygen Tension and Transcription Inhibition

Abstract Early embryonic cleavages are mostly regulated by maternal components then control of development progressively depends on newly synthesized zygotic products. The timing of the first cleavages is a way to assess embryo quality. The goal of this study was to evaluate the duration of the fourth cell cycle, at the time of maternal-to-zygotic transition (MZT) in in vitro-produced bovine embryos by means of cinematographic analysis. We found that 75% of the embryos displayed a long fourth cycle (43.5 ± 5.4 h) whereas the remaining embryos had a very short fourth cell cycle (8.9 ± 2.9 h). Both groups did not differ in cleavage rhythm up to the eight-cell stage and timing of cavitation and blastocyst expansion was identical. However, embryos with a short fourth cell cycle had a better blastocyst rate than embryos with a long cycle (59% versus 38%, P < 0.01). Total cell number, inner cell mass (ICM):total cell ratio, and hatching rate were identical for blastocysts produced from embryos with either a long or a short fourth cell cycle. In a second experiment, we showed that increasing the oxygen tension, from 5% to 20%, decreased the percentage of embryos with a short fourth cell cycle, from 25% to 11% (P < 0.01), indicating that suboptimal culture conditions can influence the length of this cycle. Finally, we investigated whether fourth cell cycle duration could be influenced by transcription inhibition. With alpha-amanitin added at 18 h postinsemination (HPI), cleavage was reduced (66% versus 79%) and, at 70 HPI, the 9- to 16-cell rate increased (50% versus 25%) concomitantly with a 5- to 8-cell rate decrease (16% versus 47%). A similar pattern was observed when the drug was added at 6 HPI or 42 HPI but not at 0 HPI. Cinematographic analysis revealed that alpha-amanitin increased the first cell cycle duration whereas the second and third cell cycles were not affected. With the drug, one third of the embryos could develop up to the 9- to 16-cell stage and they all had a short fourth cell cycle (11.2 ± 3.7 h) with a good synchrony of cleavage between blastomeres. These results suggest that duration of the fourth cell cycle of bovine embryo, during the MZT, is under a zygotic transcriptional control that can be affected by oxidative conditions.

Addition of β-mercaptoethanol or Trolox® at the morula/blastocyst stage improves the quality of bovine blastocysts and prevents induction of apoptosis and degeneration by prooxidant agents

This study was conducted to evaluate the effect of beta-mercaptoethanol (a stimulator of glutathione synthesis) and Trolox (an hydrosoluble analogue of Vitamin E) on bovine embryos cultured from the morula stage (Day 5 post-insemination; pi) under oxidative stress conditions. Culture of embryos with increased doses of Trolox showed a dose-dependent embryotoxicity on Day 8 pi. The use of 400 microM Trolox as well as beta-mercaptoethanol at 100 microM prevented at least partly (P < 0.05) the prooxidant-induced blastocyst degeneration on Day 8. Hatching rates of surviving blastocysts were significantly increased by both antioxidants and beta-mercaptoethanol alone improved their mean cell numbers, which was significant in the ICM (P < 0.05). Analysis of their effect on Day 7 pi showed that both the antioxidants significantly reduced the prooxidant-induced apoptosis and beta-mercaptoethanol diminished the physiological level of apoptosis as well as it stimulated the glutathione synthesis (P < 0.05). In addition, a comparison between in vitro- and in vivo-produced embryos showed that the levels of apoptosis were similar at the same age post-insemination (morulae and blastocysts) but increased steadily with the embryonic age in in vitro ones. In conclusion, beta-mercaptoethanol and Trolox added separately from the morula stage protected embryos against oxidative stress and improved the quality of the resulting blastocysts.

EXPRESSION OF CU/ZN AND MN SUPEROXIDE DISMUTASES DURING BOVINE EMBRYO DEVELOPMENT: INFLUENCE OF IN VITRO CULTURE

Temporal pattern of expression of Cu/Zn and Mn superoxide dismutases (SODs) was investigated in bovine oocytes and embryos produced in vitro in two different culture conditions and in vivo after superovulation. SODs were examined at a transcriptional level in single oocytes and embryos by reverse transcriptase–polymerase chain reaction (RT‐PCR) and, at a protein level, by Western blotting on pools of embryos. mRNA encoding Cu/Zn SOD were detected in in vitro bovine embryos throughout preattachment development as well as in in vivo derived morulae and blastocysts. Transcripts for Mn SOD gene were detected in most immature and in vitro matured oocytes as well as in some zygotes and 5‐ to 8‐cell embryos while no transcript was found at the 9‐ to 16‐cell stage in both culture conditions. In vitro embryonic expression of Mn SOD was detected earlier in the presence of serum. Half of the morulae showed the transcript if cultured with 5% serum while none without serum. At the blastocyst stage Mn SOD could be detected independently of culture conditions. For in vivo–derived embryos Mn SOD transcripts were detected both in morulae and blastocysts. Immunoblotting analyses revealed that Cu/Zn SOD and Mn SOD were also present at a protein level in in vitro‐derived zygotes and blastocysts. Together these data demonstrate, for the first time, that Mn SOD is transcribed and that Cu/Zn and Mn SOD proteins are expressed in preimplantation bovine embryos. Finally, they suggest that Mn SOD transcription is altered by in vitro culture conditions. Mol. Reprod. Dev. 58:45–53, 2001.

Embryo metabolism during the expansion of the bovine blastocyst

Embryo metabolism was evaluated during re‐expansion of in vitro produced bovine blastocysts collapsed with cytochalasin D (CCD) and incubated in the presence and absence of ouabain, a specific inhibitor of the Na+, K+ pump. Day 8 expanded blastocysts were treated for 2 to 4 hr with 20 μg/ml CCD. Four conditions were tested: untreated embryos and embryos collapsed with CCD and allowed to re‐expand for 4 hr in the presence of 0 M, 1 nM, or 1 μM ouabain. Incubation of collapsed embryos for 4 hr in the presence of 1 nM or 1 μM ouabain significantly inhibited blastocyst re‐expansion. Glucose, pyruvate, and amino lactate uptake/release were not significantly affected by ouabain treatment and did not correlate with the degree of blastocyst re‐expansion. Few variations in the uptake/release of amino acids by the embryos were observed. Ouabain treatment significantly decreased oxygen uptake which directly correlated with the degree of blastocyst re‐expansion. For embryos allowed to re‐expand in the presence or absence of ouabain, a direct correlation was observed between the uptake of oxygen and of glucose. One mM cyanide or 2,4 dinitrophenol inhibited blastocyst re‐expansion although 0.01 and 0.1 mM were ineffective. This study indicates a role for oxidative metabolism in providing the energy necessary for blastocoel expansion in the bovine. Nevertheless, blastocyst expansion is relatively insensitive to inhibition of oxidative phosphorylation indicating the ability of the bovine blastocyst to adapt to hypoxic conditions. Mol. Reprod. Dev. 53:171–178, 1999.

Vitrification of in vitro produced bovine blastocysts: methodological studies and developmental capacity.

Methodological studies were undertaken to test the validity of a three-step vitrification procedure for bovine in vitro produced embryos using glycerol and ethylene glycol as cryoprotectants. Embryos were produced in a low-phosphate culture system (medium VT1 + 10% foetal calf serum) and vitrified at day 7 post-insemination either in a mixture of 25% glycerol--25% ethylene glycol or a mixture of 10% glycerol--40% ethylene glycol. In the first mixture 67% (n = 283) of blastocysts were re-expanded after 72 h of culture and 53% were hatched while in the second one (n = 65) only 5% survived. The mean number of cells of the surviving blastocysts was correlated with the rate of survival (R2 = 0.47; P = 0.0024). Embryo size (diameter < or > to 180 microm) did not influence blastocyst survival or cell number, but hatching rate was higher for embryos > 180 microm. Embryo survival, hatching rate and cell number 72 h post-warming were not affected by the mode of vitrification (direct plunging into nitrogen liquid or vitrification into nitrogen liquid vapour), the mode of preparation of the vitrification solutions (molar or molal basis) or by the concentration of galactose used as a diluent (0 to 0.85 M). Only one calf was born after transfer of 22 vitrified blastocysts. These results confirm the apparent lack of correlation for cryopreserved embryos between in vitro survival or hatching and viability after transfer.

Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification

The ability of bovine blastocysts to recover after cryopreservation and thawing procedures is often assessed by evaluating their re-expansion during in vitro co-culture. However, the influence of factors such as feeder cell type and gas atmosphere on blastocyst survival and evolution have never been considered. This study therefore compared two cell co-culture systems and two different gas atmospheres to assess survival of in vitro produced bovine blastocysts after vitrification. Day-7 blastocysts (n = 181) were vitrified in a mixture of 25% glycerol/25% ethylene glycol. After warming and dilution, they were co-cultured either on Buffalo rat liver cells (BRL CC cell line) or on granulosa cells (GR CC primary culture) in TCM 199 supplemented with 10% FCS and under an atmosphere of 5% or 20% O2. Surviving and hatching rates were recorded at 24 h intervals for 3 days. After 72 h of culture, surviving blastocysts were treated for differential counting of inner cell mass (ICM) and trophectoderm cells. Blastocyst survival rates were higher when BRL and granulosa co-culture were performed under 20% oxygen as compared to 5% oxygen (20% O2: 62% vs. 5% O2: 25%, P < 0.0001). However, the quality of blastocysts surviving in the granulosa co-culture condition was lower under 20% O2 than under 5% O2 as indicated by lower total and trophectoderm cell numbers (respectively 79 +/- 6 and 56 +/- 6 at 20% O2 vs. 100 +/- 10 and 74 +/- 10 at 5% O2, P < 0.05), by an altered ICM/trophectoderm ratio (20% O2: 28% vs. 5% O2: 23%, P < 0.05), by a higher total nuclear fragmentation (20% O2: 3.7% vs. 5% O2: 1.5%, P < 0.05) and a trend to decreased hatching (20% O2: 32% vs. 5% O2: 81%, P = 0.07). Whereas, for BRL co-culture, 20% O2 yielded higher quality blastocysts than 5% O2 as evaluated by higher ICM and trophectoderm cell numbers (19 +/- 1 and 71 +/- 5 at 20% O2 vs. 15 +/- 2 and 48 +/- 9 at 5% O2, respectively, P < 0.05), by lower nuclear fragmentation in the ICM (20% O2: 2.2% vs. 5% O2: 6.7%, P < 0.05). In conclusion, co-culture conditions may influence blastocysts survival and quality after cryopreservation. In our conditions, co-culture with BRL cells under 20% O2 seems to be the best combination to evaluate blastocyst survival and quality after vitrification.

Peroxiredoxin 6 Is Upregulated in Bovine Oocytes and Cumulus Cells During In Vitro Maturation: Role of Intercellular Communication

Abstract Peroxiredoxins are peroxidases involved in antioxidant defense and intracellular signaling. Expression of transcripts coding for peroxiredoxin 6 (PRDX6) has been previously described to be upregulated in oocytes after in vitro maturation, a period during which general transcription decreases dramatically in oocytes. The aim of the present work was to evaluate PRDX6 regulation in bovine cumulus-oocyte complexes in relation to maturation and intercellular communication. PRDX6 expression was analyzed by reverse transcription-PCR and Western blotting in oocytes and cumulus cells before and after in vitro maturation. PRDX6 was found to be upregulated at the mRNA and protein levels in both cell types after maturation. The effect of paracrine and gap junctional communication on PRDX6 expression was then assessed by culturing cumulus clusters in the presence or absence of denuded oocytes. While PRDX6 upregulation in oocytes required intact cumulus-oocyte junctions, the presence of denuded oocytes was necessary but sufficient for the upregulation to occur in cumulus cells. Finally, the influence of recombinant mouse growth differentiation factor-9 (GDF-9) on PRDX6 expression in cumulus cells was studied. GDF-9 induced cumulus expansion and PRDX6 upregulation in bovine cumulus clusters. Altogether, our data suggest that PRDX6 upregulation in cumulus-oocyte complexes during in vitro maturation is mutually regulated by both cell types: PRDX6 upregulation in oocytes would require gap junctions with cumulus cells, while upregulation in cumulus would depend on secretion of oocyte paracrine factor(s) with GDF-9 being a likely candidate.

Short-term exposure to hydrogen peroxide during oocyte maturation improves bovine embryo development

Recent studies have shown that short-term exposure of oocytes to a stressor such as hydrostatic pressure or osmotic stress might induce stress tolerance in embryos. The aim of the present study was to investigate the consequences of short-term hydrogen peroxide (H(2)O(2)) exposure to bovine in vitro matured cumulus-oocyte complexes (COCs) on subsequent preimplantation embryo development and apoptosis. In the first experiment, mature COCs were incubated in H(2)O(2) at concentrations ranging between 0.01 and 100 micromol/l, and subsequently fertilized and cultured. Oocyte incubation with 50-100 micromol/l of H(2)O(2) resulted in a significantly higher blastocyst yield (47.3%) in comparison with control medium (31.8%), while apoptotic cell ratio was inversely related with H(2)O(2) concentration. In the second experiment, we showed that the stress tolerance after H(2)O(2) exposure was not mediated by increased glutathione content in treated oocytes nor by enhanced fertilization or penetration. Further research should concentrate on the potential role of players that have been associated with stress tolerance in somatic cell lines.