The sex ratio of bovine embryos produced in vitro in serum-free oviduct cell-conditioned medium is not altered

The sex ratio of bovine blastocysts produced in vitro in serum-free oviduct cell-conditioned medium was investigated. Bovine embryos reaching the blastocyst stage were removed from culture medium on Days 6, 7, 8 and 9 and were identified as small, mid-sized or expanded blastocysts. One third (29/91) of the blastocysts appeared on Day 6. Twelve from them were small blastocysts (5 males), 7 were mid-sized blastocysts (4 males) and 10 were expanded blastocysts (5 males). On Day 7, 33 blastocysts were obtained: 8 small (5 males), 9 mid-sized (3 males) and 16 expanded (13 males) blastocysts. Finally, on Days 8 and 9, 29 blastocysts were obtained: 12 small (9 males), 9 mid-sized (6 males) and 8 (3 males) expanded blastocysts. Sexing of the 91 blastocysts was performed by using an original polymerase chain reaction (PCR) protocol generating discreet internal control signals from both female and male samples and Y-specific smears from the male samples. Proportions of male embryos on Days 6, 7 and on Days 8+9 were 48, 64 and 62%, respectively. These values did not differ significantly among days and did not differ from 50%. Fifty-nine percent of small blastocysts, 52% of mid-sized blastocyst and 62% of expanded blastocysts were male. No difference between these values or with respect to 50% could be observed. These results show that bovine blastocysts produced in serum-free oviduct cell-conditioned medium do not have an altered sex ratio.     

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.     

Effect of conventional controlled-rate freezing and vitrification on morphology and metabolism of bovine blastocysts produced in vitro

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.     

Impact of adding 5.5 mM glucose to SOF medium on the development,metabolism and quality of in vitro produced bovine embryos from the morula to the blastocyst stage

Although toxic for early stages of embryo development, glucose is a physiological metabolic substrate at the morula and blastocyst stages. We evaluated the effect of adding 5.5 mM glucose from the morula stage on bovine blastocyst development and quality. In vitro matured and fertilised bovine oocytes were cultured in modified Synthetic Oviduct Fluid medium containing 5% fetal calf serum, but without added glucose, up to day 5 post-insemination (pi). Morulae were selected and further cultured in the presence or absence of 5.5 mM glucose. Blastocyst and hatched blastocyst rates were recorded. Oxygen, glucose and pyruvate uptakes as well as lactate release were evaluated. The quality of the resulting blastocysts was evaluated by the cell allocation to the inner cell mass (ICM) and trophectoderm (TE) and by the apoptotic index. Adding glucose increased the blastocyst rate at day 8 pi (80% vs 65%) but had no impact on hatching rate (25% vs 28%). A 22% decrease in oxygen uptake was observed in the presence of glucose, concomitant with an increase in lactate release, although no change was observed in pyruvate uptake. A slight decrease in blastocyst cell number was observed at day 7 in the presence of glucose while neither the ICM/TE cell ratio nor the apoptotic index were affected. In conclusion, adding 5.5 mM glucose from the morula stage has a limited impact on blastocyst rate and quality although important modifications were observed in embryo metabolism. It remains to be determined whether those modifications could influence embryo viability after transfer.     

Role of G(i/o)-Src kinase-PI3K/Akt pathway and caveolin-1 in β₂-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery

Activation of the β₂-adrenoceptor (β₂-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β₂-AR-mediated eNOS activation, with special focus on G_i/o proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β₂-AR agonist procaterol was reduced by inhibitors of G_i/o proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser¹¹⁷⁷, which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr¹⁴, which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β₂-AR is coupled to a G_i/o-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser¹¹⁷⁷ leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr¹⁴, through a G_i/o-Src kinase pathway. Since pulmonary β₂-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.     

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.     

Physiological concept for a blood based CFTR test.

We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) could be involved in the volume regulation of human red blood cells (RBC). Experiments were based on two gadolinium (Gd(3+)) sensitive mechanisms, i.e. inhibition of ATP release (thetaATP(i)) and membrane destabilization. RBC of either cystic fibrosis (CF) patients or healthy donors (non-CF) were exposed to KCl buffer containing Gd(3+). A significantly larger quantity of non-CF RBC (2.55 %) hemolyzed as compared to CF RBC (0.89 %). It was found that both of the Gd(3+) mechanisms simultaneously are needed to achieve hemolysis, since either overriding thetaATP(i) by exogenous ATP addition prevented Gd(3+) induced hemolysis, or mimicking thetaATP(i) by apyrase in absence of Gd(3+) could not trigger hemolysis. Additionally, ion driven volume uptake was found to be a prerequisite for Gd3+ induced hemolysis as chloride and potassium channel blockers reduced the Gd(3+) response. The results show that in non-CF RBC Gd(3+) exerts its dual effect leading to hemolysis. On the contrary, in CF RBC, lacking CFTR dependent ATP release, the sole Gd(3+) effect of membrane destabilization is not sufficient to induce hemolysis similar to non-CF. This concept could form the basis of a novel method suitable for testing CFTR function in a blood sample.     

Evidence for involvement of the PKC-alpha isoform in myogenic contractions of the coronary microcirculation

The role of protein kinase C (PKC) isoforms in myogenic tone of the ferret coronary microcirculation was investigated by measuring fura 2 Ca(2+) signals, PKC immunoblots, contractile responses, and confocal microscopy of PKC translocation. Phorbol ester-evoked contractions were completely abolished in the absence of extracellular Ca(2+) but involved a Ca(2+) sensitization relative to KCl contractions. Immunoblotting using isoform-specific antibodies showed the presence of PKC-alpha and -iota and traces of PKC-epsilon and -mu in the ferret coronary microcirculation. PKC-beta was not detectable. When intraluminal pressure (40 to 60 and 80 mmHg) was increased, ferret coronary arterioles showed a transient increase in fura 2 Ca(2+) signals, whereas the myogenic tone remained sustained. The increase in Ca(2+) and tone was sustained at 100 mmHg. Isolated ferret coronary arterioles were fixed and immunostained for PKC-alpha at 40 and 100 mmHg intraluminal pressure. PKC translocation was determined by confocal microscopy. Increased PKC translocation was observed when vessels were exposed to 100 mmHg relative to that at resting pressure (40 mmHg). These results suggest a link between the Ca(2+) sensitization that occurs during the myogenic contraction and activation of the alpha-isoform of PKC.