Franz Dessy

Glucose metabolism during bovine preimplantation development: analysis of gene expression in single oocytes and embryos.

Glucose metabolism of the bovine embryo is low during the first cleavages and increases sharply after the major resumption of the genome (8–16 cells). The mRNA level for genes involved in glucose metabolism was tested by RT‐PCR on individual oocytes and embryos at different stages of development. These genes were: glucose transport GLUT‐1, hexokinase (HK), glucose‐6‐phosphate‐dehydrogenase (G6PDH), and glucose‐phosphate‐isomerase (GPI); actin was used as a reference transcript. RT‐PCR results revealed three types of oocytes or embryos: positive with a PCR signal for each transcript considered, nul with no signal for any transcript, and heterogeneous with a PCR signal for some transcripts and none for others. The number of nul and heterogeneous samples was higher for slow than for fast‐cleaving embryos (81% vs. 36%), and the proportion of positive embryos increased significantly at the 16‐cell and morula stages (P < 0.002), suggesting a correlation between mRNA content and developmental capacity. In positive embryos, GLUT‐1 mRNA level was reduced by half during maturation and fertilization. Actin and hexokinase mRNA levels decreased during the first cleavages, but significantly increased at the 16‐cell and morula stages, respectively. GPI transcript remained stable throughout development, whereas there was a significant rise for G6PDH at the 4‐cell stage, perhaps due to a polyadenylation process. Finally, the absence or decrease in intensity of several transcripts at the blastocyst stage suggests suboptimal culture conditions. Mol. Reprod. Dev. 48:216–226, 1997.

Effects of co-culture and embryo number on the in vitro development of bovine embryos.

It is generally accepted that culturing embryos in groups or with somatic cells improves both the yield and quality of the blastocysts obtained. The aims of this study were 1) to compare the yield and quality of the embryos obtained after culture in several number conditions and in several culture systems and 2) to assess the effect of co-culture started at various stages of embryo development. Under cell-free culture conditions (modified synthetic oviduct fluid [mSOF] supplemented with 10% fetal calf serum [FCS] 48 h post insemination, the rate of Day 10 blastocysts was lower when embryos were cultured in small groups (1 to 6 per drop) than in large groups (4 versus 23% ; P < 0.01). There was no group effect when embryos were co-cultured either with Buffalo rat liver (BRL) cells in TCM 199, or in a culture system allowing the progressive development of cumulus cells in mSOF, even if co-culture started at 66 or 114 h post insemination. However, embryos cultured singly had lower cell numbers than embryos cultured in large groups when co-culture started at 114 h post insemination. This suggests that 1) somatic cells improve the development of singly cultured bovine embryos up to the blastocyst stage after the 9-16 cell stage; 2) co-culture affects blastocyst cell number of singly cultured embryos by acting roughly between the 5-8 and the 9-16 cell stage; and 3) cooperation between embryos could replace the effect of co-culture either on the yield of blastocysts or on blastocyst cell number. Blastocysts appeared significantly earlier in co-culture with cumulus cells in mSOF than in co-culture with BRL cells in TCM 199 (detection of the blastocysts: 7.3 +/- 0.1 d post insemination with cumulus cells versus 8.1 +/- 0.1 d with BRL cells; P < 0.001) and had a significant higher number of cells (143 +/- 9 versus 85 +/- 11; P < 0.001). This system thus seems suitable for the culture of small numbers of embryos resulting from in vitro maturation and fertilization of oocytes from individual donor cows.

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.

Bovine embryos cultured in serum-poor oviduct-conditioned medium need cooperation to reach the blastocyst stage

Immature bovine oocytes were matured and fertilized in vitro, and the resulting zygotes were cultured to the blastocyst stage in droplets of tissue culture medium 199 (TCM 199) conditioned by oviduct cells in the absence of serum. In Experiment 1, the effect of the number of zygotes in a constant culture volume was investigated by culturing 1, 4 or 40 zygotes in 40 microl of culture medium. The cleavage rate was low with a single embryo (36%) but increased with the number of embryos, to reach 50% with 4 embryos/40 microl and 59% with 40 embryos/40 microl. Blastocyst formation was nil with 1 embryo per 40 microl, reaching 2.5% with 4 embryos/40 microl and 18% with 40 embryos/40 microl. The effect of the size of the drop was assessed in Experiment 2, the concentration of embryos remaining constant (1 embryo/1 microl). The volumes tested were 10, 20, 30 and 40 microl. Development into blastocysts increased gradually from 12% in the 10 10 group to 20% in the 40 40 group. Experiment 3 was designed to find a minimal droplet volume able to support the development of a single embryo to the blastocyst stage. The minimum tested volume was 5 microl and was not successful. These results show that bovine embryos cultured in oviduct-conditioned TCM 199 need to cooperate to reach the blastocyst stage. The mechanism of this cooperation is not known, but some autocrine/paracrine factors, probably growth factors, could promote embryo development as was demonstrated in mice. From Experiment 2 we can hypothesize that the surface volume ratio of the droplets could play a role in the culture conditions by interfering with the exchanges between the culture medium and the surrounding environment.

Effect of Conventional Controlled-Rate Freezing and Vitrification on Morphology and Metabolism of Bovine Blastocysts Produced In Vitro

Abstract This study compares the effects of conventional controlled-rate freezing and vitrification on the morphology and metabolism of in vitro-produced bovine blastocysts. Day 7 expanded blastocysts cultured in synthetic oviduct fluid with 5% fetal calf serum were frozen in 1.36 M glycerol, 0.25 M sucrose or vitrified in 25% glycerol, 25% ethylene glycol. Cell alterations and in vitro development were evaluated immediately after thawing or after 72 h. The effect of cryopreservation on inner cell mass and trophectoderm (TE) cell number as well as glucose, pyruvate, and oxygen uptakes, and lactate release by blastocysts were evaluated. Immediately after thawing, blastocysts showed equivalent cell membrane permeabilization after both cryopreservation procedures, while alterations in nuclear staining were more frequent in vitrified embryos. After culture, similar survival and hatching rates were observed. Both procedures decreased cell number immediately after thawing and after 72 h. However, the number of TE cells was lower in frozen embryos than in vitrified ones. In relation to this, frozen blastocysts showed a decrease in glucose, pyruvate, and oxygen uptake, although those parameters were not altered in vitrified embryos. An increased glycolytic activity was also observed in frozen embryos, indicating a stress response to this procedure.

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.

Pyruvate prevents peroxide-induced injury of in vitro preimplantation bovine embryos.

The impact of oxidative stress on the in vitro development of bovine embryos in synthetic oviduct fluid medium (mSOF) was assessed by using H2O2 as a stress inducer. In a preliminary experiment, a chemiluminescent method was used to measure the antioxidative capacity of the mSOF culture medium. Pyruvate was the mSOF component displaying the highest H2O2 degrading ability. Essential and nonessential amino acids also significantly reduced the H2O2 concentration, whereas lactate and glutamine were ineffective. The effect on further development of a short exposure of zygotes, 9–16‐cell stage embryos and blastocysts to 0 M; 10−7 M ; 10−6 M, and 10−5 M H2O2 in pyruvate‐free mSOF was evaluated. Developmental rates of the H2O2‐treated zygotes to the 5–8‐cell or blastocyst stages and survival of H2O2‐treated blastocysts were reduced in a dose‐dependent manner whereas the 9–16‐cell embryos were unaffected by those treatments. Blastocysts treated with H2O2 also tended to have lower numbers of bisbenzimide‐stained nuclei and showed increased nuclear fragmentation. Including pyruvate in the mSOF culture medium during a 10−5 M H2O2 pulse highly reduced the H2O2 concentration as measured by chemiluminescence and improved zygote and blastocyst development, but failed to prevent blastocyst nuclei degradation. These experiments suggest that bovine embryos show developmental change in sensitivity to exogenous H2O2, the 9–16‐cell embryos being more resistant than zygotes and blastocysts and that H2O2 and its toxic effects can be attenuated by including pyruvate in the medium. Mol. Reprod. Dev. 52:149–157, 1999.

Embryo production by ovum pick up in unstimulated calves before and after puberty.

The possibility of producing embryos from oocytes repeatedly collected from unstimulated calves was tested, and results obtained before and after puberty were compared in the same animals. Ovum pick-up (OPU) coupled with in vitro embryo production was used on 2 sets of 7 and 9 calves, aged 7 to 10 m.o. at the start of the experiment. The oocytes were collected twice a week during a 2-m.o. period before puberty and a 1-m.o. period after puberty. Oocytes were fertilized and co-cultured with cumulus cells in modified synthetic oviduct fluid (SOF) up to Day 7 post insemination. Some Day 7 blastocysts were vitrified and transferred to recipient heifers. An average of 3.8 to 6.8 follicles was punctured per OPU session; 1.9 to 3.1 oocytes were collected, of which more than 60% were of good quality. The number of punctured follicles and collected oocytes varied between donors. Blastocyst rates of 19 to 27% were obtained for the 2 sets. Three normal calves were born from the transfer of 20 vitrified embryos. While no significant difference was observed for the first set of calves, a significant decrease in the number of punctured follicles was observed after puberty in the second set. A direct correlation was also obtained between the number of follicles punctured before and after puberty in the same animal. In conclusion, oocytes can be collected by repeated OPU in calves 7 to 10 m.o. old without affecting their growth or the onset of puberty. An average of 5 to 11 (range 0 to 16) blastocysts per donor was produced over 2 month. However, important variations were found between donors. The correlation observed for the number of follicles punctured before and after puberty suggests that this parameter is determined before puberty.

Comparative immunohistochemical distribution of connexin 37 and connexin 43 throughout folliculogenesis in the bovine ovary

Among gap junctional proteins previously identified in the mouse ovary, connexins (Cx) Cx37 and Cx43 appeared to be essential for normal follicular growth. The aim of this work was to detect Cx37 expression in the bovine ovary, then to quantify and compare its follicular distribution pattern with that of Cx43 using quantitative analysis of immunofluorescently labeled ovary sections viewed with a confocal laser scanning microscope. Cx37 immunoreactivity was detected in bovine ovarian follicles and was predominantly localized at preantral stages. Unlike follicular Cx43 expression which was restricted to granulosa cells, Cx37 staining was observed in both oocyte and granulosa cell compartments. While no changes were seen during early follicular growth, the level of Cx37 expression decreased significantly at the onset of antral cavity formation (P ≤ 0.01). On the contrary to what was found for Cx37, Cx43 was weakly expressed in preantral follicles. Concomitant with antrum formation, the level of Cx43 expression increased significantly (P ≤ 0.01). A further increase was correlated with antral follicular size (P ≤ 0.01). Cx43 immunoreactivity declined significantly in morphologically atretic follicles (P ≤ 0.01). A comparative analysis showed that Cx37 and Cx43 expression patterns were differentially regulated and could reflect specific physiological roles for each gap junction protein throughout folliculogenesis in cow. Mol. Reprod. Dev. 57:60–66, 2000.

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.