First results about ProAKAP4 concentration in stallion semen after cryopreservation in two different freezing media.

The quality of fresh or thawed sperm in stallions has been generally determined by the viability and total and progressive motility of the sperm. Today, the expression of ProAKAP4, a protein present in the flagellum of spermatozoa, appears to be an innovative and relevant functional marker to assess semen quality and male fertility. This study aims to compare the concentration of ProAKAP4 in the semen from 5 stallions frozen with two different extenders immediately after thawing (T0) and 4 h post-thawing (T4). Viability, total and progressive motility were measured in parallel. Significant differences for sperm viability and total motility were observed between the two extenders, as was the concentration of ProAKAP4 both at T0 and T4. At T4, all quality parameters and ProAKAP4 content significantly decreased compared to T0, but with a considerably slower decrease in one extender than the other. These preliminary results suggest that measuring the concentration of ProAKAP4 is a promising tool for the comparison of different extenders and the selection of the optimal freezing medium for each stallion ejaculate.

Expression, localization, and concentration of A-kinase anchor protein 4 (AKAP4) and its precursor (proAKAP4) in equine semen: Promising marker correlated to the total and progressive motility in thawed spermatozoa.

A-kinase anchor protein 4 (AKAP4) is playing a central role in flagellar structure, chemotaxis, capacitation and sperm motility. In mammals, AKAP4 is expressed during spermatogenesis. AKAP4 is synthesized as a precursor, proAKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. The proAKAP4 is a good indicator of sperm quality in humans and boars. The aims of this work were to study the expression, the localization and the concentration of proAKAP4 and AKAP4 in equine semen, and to evaluate the possible correlation between the total and progressive motility and the concentration of proAKAP4 measured by ELISA in post-thawed semen. Frozen sperm from 13 different stallions were used. Semen samples (n = 17) were prepared using the INRA Freeze medium to reach a concentration of 150 million spermatozoa/mL, packaged and frozen in 0.5 mL straws. The precursor proAKAP4 and the mature protein AKAP4 both localize to the fibrous sheath of the principle piece of equine sperm flagellum. The concentrations of proAKAP4 were determined in the post-thawed semen using ELISA method (Horse 4MID® kits, 4BioDx, France). The mean concentration of proAKAP4 was then of 7.372 ±â€¯0.79 ng/µL and was significantly correlated with the post-thawed total motility (Pearson coefficient r = 0.66, p = 0.002) and progressive motility (Pearson coefficient r = 0.76, p = 0.0002) and the amount of proAKAP4 represent the amount of spermatozoa that expressed proAKAP4. Taken together, these preliminary results confirm the interest to use proAKAP4 concentrations as a promising marker of stallion sperm quality as close correlation was observed between the proAKAP4 concentration and sperm motility parameters.

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.

Plant protein hydrolysates (plant peptones) as substitutes for animal proteins in embryo culture medium.

The aim of the present study was to improve the sanitary quality of in vitro-produced bovine embryos by using plant protein hydrolysates (plant peptones) as substitutes for animal proteins. Peptones were compared with bovine serum albumin (BSA) as the protein source in synthetic oviduct fluid medium and the quality of the resulting embryos was determined. Two batches of peptones (wheat and cotton) were selected on the basis of their anti-oxidant properties. When added to the culture medium, both peptones (at 0.56 mg mL(-1) for cotton peptone and at 0.18 mg mL(-1) for wheat peptone) led to similar developmental and hatching rates compared with 4 mg mL(-1) BSA and embryos were equally resistant to freezing and able to elongate after transfer. Surprisingly, a significant decrease in reduced glutathione (GSH) content was observed when embryos were produced with plant peptone instead of BSA. Supplementation of the culture medium with precursors of GSH (cysteine and beta-mercaptoethanol) significantly increased the GSH content. A shift of the sex ratio towards male embryos was seen for Day 8 embryos cultured with wheat peptone, whereas no shift was observed for embryos cultured in the presence of BSA or polyvinylpyrrolidone. In conclusion, culture with plant peptones enables embryos to be obtained at a similar rate and of similar quality to that seen following the use of BSA. The use of the plant peptones increased the sanitary quality of the embryos and decreased the cost of embryo production.

Analysing motility parameters on fresh bull semen could help to predict resistance to freezing: a preliminary study.

Batches of straws often need to be thrown away after freezing because of a too few number of motile or progressive sperm cells after thawing. Our objective was to evaluate the possibility to predict before freezing the quality of the semen after freezing/thawing. Computer-Aided Sperm Analysis was performed on motility parameters both before and after freezing of 20 ejaculates from different bulls. Significant variation between bulls was observed both before and after freezing for all the analysed traits (anova2; p < 0.001): proportion of motile (%mot) and progressive (%prog) sperm, velocity on the curved line (VCL), velocity on the straight line (VSL), velocity on the average path (VAP), linearity (VSL/VCL), beat cross frequency and average orientation change of the head. Freezing significantly altered the motility parameters and correlations were found between samples analysed before and after freezing (Pearson coefficient: R = 0.43-0.72; p < 0.05). The mean VAP, VSL and the %prog obtained before freezing were highly correlated to the %mot and %prog observed after freezing (R = 0.75-0.82; p < 0.001). Applying thresholds on mean VAP and VSL values allowed us to predict respectively 6 and 7 of nine batches that would be rejected after freezing due to a too low %prog (<15%). Combining different traits did not add to the precision. In conclusion, analysis of velocity traits on fresh sperm seems more efficient than analysis of %mot or %prog to discard batches that will be of poor quality after freezing.

Set up of a serum-free culture system for bovine embryos: embryo development and quality before and after transient transfer.

It is well known that serum in culture medium negatively affects blastocyst quality. The objective of this work was to develop and test a serum-free culture medium which could improve embryo quality, measured by the resistance to freezing, lipid and glutathione content of the resulting blastocysts, as well as the ability of the blastocysts to elongate after transient transfer to recipient cows. In a first experiment we showed that adding a mixture of insulin, transferrin and selenium to serum-free Synthetic Oviduct Fluid medium (SOF-ITS) improved embryo development and quality. In the second experiment, the addition of BSA to SOF-ITS further improved blastocyst development. Moreover, a reduction in lipid content of morulae was observed in SOF-ITS-BSA by comparison with morulae cultured with serum (SOF-FCS). The resistance to freezing measured by hatching rates 24h post-thawing was also improved for blastocysts with a diameter between 160 and 180 microm cultured in SOF-ITS-BSA by comparison to those produced with serum. In order to evaluate the redox potential of the embryos, reduced glutathione content (GSH) was evaluated both before and after cryopreservation. A significant decrease in glutathione was observed after freezing, whatever the culture medium, but no difference was observed between culture conditions. Transient transfers were performed and elongated D-13 embryos were recovered. Elongation was more pronounced and the embryonic disk more often visible in embryos cultured in SOF-ITS-BSA than in embryos cultured with FCS. In conclusion, the serum-free system we developed to produce in vitro bovine embryos meets the developmental and qualitative requirements for a large-scale use.

Kinetics of fertilization and development, and sex ratio of bovine embryos produced using the semen of different bulls.

The between bulls variation in in vitro fertility and the shift of sex ratio towards male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the kinetics of fertilization, embryo development and the sex ratio of the resulting embryos using the frozen/thawed semen of four different bulls. In a first experiment, the kinetics of pronucleus (PN) formation was evaluated at 8, 12 and 18 h post-insemination (hpi). Based upon the pronuclei sizes and the distance between the two pronuclei, inseminated oocytes were classified in three PN stages. Differences between bulls were observed at each time point, but were more important at 12 hpi. At 8 and 12 hpi bull III showed a significantly faster PN evolution by comparison with the three other bulls (P<0.05), while at 18 hpi, the proportion of the three PN stages was similar to those of bulls I and IV, bull II being delayed. In a second experiment, the kinetics of in vitro embryo development was compared using time-lapse cinematography. The analysis of embryos reaching the blastocyst stage revealed significant differences in the mean time of first cleavage (range of 22.7-25.6h, P<0.05), while the lengths of the subsequent three cell cycles did not differ between bulls. The early mean time of first cleavage with bull III was associated with an early blastulation and a high blastocyst rate at Day 7, in opposition to what was observed with bull II showing a later timing of first cleavage (first cleavage 22.1 hpi versus 25.5 hpi; blastulation 140.4 hpi versus 152.5 hpi; D7 blastocyst rates: 31.3% versus 21.9%; P<0.05). In a third experiment, 65-76 Day 8 blastocysts per bull were sexed by PCR. Only blastocysts obtained with bull III showed a shift in sex ratio towards male embryos (76% male embryos; P<0.05). Such shift was already observed at the 2-cell and morula stages. In conclusion, the bull influences the kinetics of PN formation, of embryo development and the sex ratio of the embryos. Moreover, those parameters might be related.

Freezing of in vitro produced bovine embryos in animal protein-free medium containing vegetal peptones.

Successful cryopreservation is essential for a large-scale dispersal of bovine in vitro produced (IVP) embryos that have been shown to be more sensitive to cryopreservation than their in vivo counterparts. On the other hand, the use of animal proteins in freezing media increases sanitary risks. We first replaced animal proteins, such as bovine serum albumin (BSA) in the freezing medium by plant-derived peptides (vegetal peptones). A batch of wheat peptones was selected after a preliminary experiment showing the absence of toxicity of concentrations<18 mg/mL on in vitro bovine blastocysts. Increasing concentrations of peptones were then added in the freezing medium. The surviving and hatching rates were not affected by comparison with those observed with BSA. No significant difference was observed between groups either for the total number of cells or for the ratio ICM/Total cell, nor for the rate of apoptosis in surviving embryos. When embryos were cryopreserved in 1.8 mg/mL peptone, the hatching rate and embryo quality as assessed at 48 h post-thawing were not significantly different from those of unfrozen embryos. In a second experiment two additives were added in this animal protein-free freezing medium containing 1.8 mg/mL peptones. No beneficial effect of adding 1 mg/mL sodium hyaluronate or 100 microM beta-mercaptoethanol was observed on embryo survival or quality. In conclusion, we have demonstrated that vegetal peptones can replace BSA in freezing media without affecting blastocyst survival and quality.

Assessment of sperm quality parameters of six bulls showing different abilities to promote embryo development in vitro.

Experiments were conducted to investigate the possible origins of variation between six bulls showing various blastocyst rates after in vitro fertilisation. No significant difference was observed for the rates of cleavage and 5-8 cell stages, whereas blastocyst yields at Day 6, 7 and 8 post insemination were significantly different between bulls (P < 0.05). Fertilisation rates ranged from 59.5 to 79.3% (P < 0.05), with no difference in the incidence of polyspermy. The proportions of motile and progressive spermatozoa before and after Percoll separation were analysed. A positive effect of Percoll was noted on both parameters (P < 0.05), leading to the absence of difference between bulls after the separation process. Sperm viability and spontaneous acrosome reaction were assessed during 18 h incubation in fertilisation medium. A sharp decrease in sperm viability was observed for all bulls after 2 h incubation, with only 12.6-21.7% of spermatozoa still viable at 18 h. In contrast, the proportion of reacted acrosomes was low in five out of six bulls (<15% at 18 h). In conclusion, the fertilisation rate was the only parameter to show some correlation with blastocyst rate for all bulls.

The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes.

This study aimed to investigate the use of Nile red, a fluorescent dye specific for intracellular lipid droplets, to quantify the lipid content of single mammalian oocytes. It was hypothesized that a higher amount of lipid present in lipid droplets in an oocyte would result in a higher amount of emitted fluorescent light. Following fixation and subsequent staining of denuded oocytes, the fluorescence of the whole oocyte was visualized by fluorescence microscopy and quantified with a photometer and photomultiplier connected to the microscope. The peak of fluorescence was observed in the yellow spectrum (590 nm) and the fluorescence was restricted to the lipid droplets corresponding to apolar lipids. Nile red concentrations ranging from 0.1 to 10 microg/ml yielded similar results. After fixation, a minimum of 2 h staining was necessary to reach maximal fluorescence which remained stable for several hours. The position of the microscopic focus within the oocyte had no influence on the amount of measured fluorescence. Successive measurements of the same oocyte yielded very similar results indicating the repeatability of the method. Finally, the technique was validated by comparing the lipid content of bovine, porcine and murine immature oocytes, which are known to contain different amounts of lipids. After staining, the fluorescence of murine oocytes was 2.8-fold lower than the fluorescence of bovine oocytes which in turn were 2.4 times less fluorescent than porcine oocytes. Based on this study, it can be said that this rather fast and easy technique allows for the relative quantification of the lipid content (present in the lipid droplets) of one single oocyte. The different amounts of emitted fluorescent light in bovine, porcine and murine oocytes correlated with the known lipid contents in these three species. This technique could be used to compare the lipid content of oocytes originating from different donors, from different sized follicles or cultured in various conditions.

Evaluation of the lipid content in bovine oocytes and embryos with nile red: a practical approach.

In this study, the fluorescent lipid dye Nile Red, was used to demonstrate that the lipid content of immature bovine oocytes is correlated with the morphological appearance of the ooplasm. Oocytes with a uniform dark cytoplasm contained significantly more intracellular lipids in lipid droplets compared with oocytes with a granulated or pale cytoplasm (p < 0.05). Furthermore, this lipid-analysing technique was applied for the first time on single bovine in vitro embryos, showing a significant increase of the lipid content in lipid droplets after culture in the presence of serum (p < 0.05).

Cell cycle duration at the time of maternal zygotic transition for in vitro produced bovine embryos: effect of oxygen tension and transcription inhibition.

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.

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.

Increased cell death in mouse blastocysts exposed to high D-glucose in vitro: implications of an oxidative stress and alterations in glucose metabolism.

AIMS/HYPOTHESIS: Signs of apoptosis have been observed in rodent blastocysts exposed to high d-glucose concentrations in vitro. The mechanism underlying the detrimental influence of glucose remains to be identified. It has been postulated that high d-glucose concentrations induced oxidative stress in rat post-implantation embryos in vitro. A decreased glucose uptake has also been implicated in the embryotoxicity of glucose in pre-implantation mouse embryos. We examined whether the high incidence of cell death in high d-glucose-treated embryos was associated with a disrupted redox status and with alterations in glucose transport and metabolism. METHODS: After blastocysts were incubated in different concentrations of d-glucose for 24 h, they were examined for the proportion of nuclei showing signs of chromatin degradation using the TUNEL technique, for the generation of reactive oxygen species and for the mitochondrial membrane potential using specific fluoroprobes and the confocal microscopy. Glucose transport and metabolism were assessed using radiolabelled 3-O-methylglucose and glucose, respectively. RESULTS: Compared to the control blastocysts, high d-glucose-treated embryos showed a higher incidence of TUNEL-positive nuclei and reactive oxygen species generation principally in the inner cell mass cells. Decreased glucose transport and glycolytic activity but unmodified pentose phosphate pathway activity were detected in these embryos. CONCLUSION/INTERPRETATION: Incubation in high d-glucose concentrations in vitro increased cell death, induced oxidative stress and decreased glucose transport and metabolism in mouse blastocysts. As only glycolysis was affected, however, we suggest that metabolic inhibition occurred downstream glucose transport and glucose-6-phosphate formation.

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.

Identification of caspase-3 and caspase-activated deoxyribonuclease in rat blastocysts and their implication in the induction of chromatin degradation (but not nuclear fragmentation) by high glucose.

Previous investigations have shown that maternal diabetes impairs rodent embryo development during the earliest phase of gestation. Exposure to high concentrations of glucose before implantation results in a decrease in the number of cells per embryo and in a concomitant increase in two nuclear markers of apoptosis, chromatin degradation and nuclear fragmentation. In the present study, we show that two intracellular effectors of apoptosis, caspase-3 and caspase-activated deoxyribonuclease (CAD), are involved in the embryotoxicity of high glucose. Using reverse transcription-polymerase chain reaction and immunocytochemistry, we first demonstrated that these two effectors were expressed in rat blastocysts. The two effectors were detected in all the cells of the blastocysts and the immuno-signals were excluded from the nuclei. Rat blastocysts were incubated for 24 h in either 6 mM or 28 mM glucose in the presence or absence of specific inhibitors (DEVD-CHO [10 microM] against caspase-3 and aurin [1 microM] against CAD). After incubation, blastocysts were examined for the proportion of nuclei showing signs of chromatin degradation or nuclear fragmentation. Addition of DEVD-CHO or aurin was found to inhibit the increase in chromatin degradation induced by high glucose. None of these two inhibitors prevented the increase in nuclear fragmentation triggered by excess glucose. Our data indicate that chromatin degradation and nuclear fragmentation are two nuclear damages that are induced separately by high glucose in rat blastocysts. Chromatin degradation is apparently mediated by the activation of caspase-3 and CAD.

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.

Expression and role of Bcl-2 in rat blastocysts exposed to high D-glucose.

Bcl-2 mRNA expression was detected in rat blastocysts by in situ hybridization. The distribution of mRNA expression was rather heterogenous, with approximately 2% of high-expressing cells. In vitro exposure to 28 mmol/l D-glucose for 24 h resulted in a significant increase in the proportion of these cells compared with control embryos in either 6 mmol/l D-glucose or 28 mmol/l D+L-glucose. Heterogeneity in the expression of Bcl-2 was also observed at the protein level by immunocytochemistry. Exposure to 28 mmol/l D-glucose significantly increased the incidence of chromatin degradation (karyolysis) and nuclear fragmentation (karyorhexis), two nuclear markers of apoptosis in rat blastocysts. When two different antisense oligodeoxynucleotides designed to block Bcl-2 expression were added to 28 mmol/1 D-glucose, the incidence of karyolysis (but not karyorhexis) was increased compared with embryos in 28 mmol/l D-glucose alone. These data suggest that Bcl-2 is involved in the protective response against the induction of karyolysis in blastocysts on their exposure to high concentrations of D-glucose in vitro, whereas karyorhexis appears to result from the activation of an intracellular pathway that is independent of Bcl-2.

Pulsatile activity and hatching of in vitro produced cow blastocysts: effects of serum supplementation.

The effect of serum added to a modified SOF medium on pulsatile activity and hatching of in vitro produced cow blastocysts was investigated by time-lapse cinematography. Embryos were generated from abattoir material and cultured in mSOF without serum or with 10% FCS added at 42h pi. Addition of serum significantly increases pulsatile activity before zona rupture and reduces the time of hatching. Pulsatile activity does not seem to be involved in the hatching process.

Characterization of embryos derived from calf oocytes: kinetics of cleavage, cell allocation to inner cell mass, and trophectoderm and lipid metabolism.

Embryos derived from calf oocytes were compared with adult cow oocyte-derived embryos (1) by studying the kinetics of embryo development using time-lapse cinematography (2) by evaluating the ratio between inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts (3) by measuring the triglyceride content of the blastocysts. The rate of calf oocyte-derived embryos reaching the blastocyst stage was reduced (26 vs. 46% for adult derived embryos). Calf oocyte-derived embryos preferably arrested their development before the 9-cell stage. Those that developed into blastocysts had cleaved earlier to reach the 2-cell or 3-cell stages than embryos that arrested before the 9-cell stage. The 9-cell stage tended to appear later in calf oocyte-derived embryo that reached the blastocyst stage than in adult-derived embryos. This difference became significant at the morula stage. Accordingly, the fourth cell cycle duration was longer for calf oocyte-derived embryos. Day 8 blastocysts from both sources had similar total cell numbers (calf: 89 +/- 20; cow: 100 +/- 30) and cell distribution between TE and ICM. The triglyceride content of day 7 blastocysts was similar for both sources (64 +/- 15 vs. 65 +/- 6 ng/embryo, respectively). In conclusion, calf oocyte-derived embryos are characterized by a higher rate of developmental arrest before the 9-cell stage and by a longer lag phase preceding the major onset of embryonic genome expression. These changes might be related to insufficient "capacitation" of the calf oocyte during follicular growth. Despite these differences, modifications in the quality of the resulting blastocysts were not detected.