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BOR - Papers in Press, published online ahead of print October 14, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.007799
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BIOLOGY OF REPRODUCTION 68, 236–243 (2003)
DOI: 10.1095/biolreprod.102.007799
© 2003 by the Society for the Study of Reproduction, Inc.

Embryo

Bovine Embryo Culture in the Presence or Absence of Serum: Implications for Blastocyst Development, Cryotolerance, and Messenger RNA Expression1

D. Rizosa, A. Gutiérrez-Adánb, S. Pérez-Garnelob, J. de la Fuenteb, M.P. Bolanda, and P. Lonergan2,a,b

a Department of Animal Science and Production, University College Dublin, Lyons Research Farm, Newcastle, County Dublin, Ireland b Departamento de Reproducción Animal y Conservación de Recursos Zoogenéticos, INIA, Ctra de la Coruña Km 5.9, Madrid 28040, Spain


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously shown that, while the intrinsic quality of the oocyte is the main factor affecting blastocyst yield during bovine embryo development in vitro, the main factor affecting the quality of the blastocyst is the postfertilization culture conditions. Therefore, any improvement in the quality of blastocysts produced in vitro is likely to derive from the modification of the postfertilization culture conditions. The objective of this study was to examine the effect of the presence or absence of serum and the concentration of BSA during the period of embryo culture in vitro on 1) cleavage rate, 2) the kinetics of embryo development, 3) blastocyst yield, and 4) blastocyst quality, as assessed by cryotolerance and gene expression patterns. The quantification of all gene transcripts was carried out by real-time quantitative reverse transcription-polymerase chain reaction. Bovine blastocysts from four sources were used: 1) in vitro culture in synthetic oviduct fluid (SOF) supplemented with 3 mg/ml BSA and 10% fetal calf serum (FCS), 2) in vitro culture in SOF + 3 mg/ml BSA in the absence of serum, 3) in vitro culture in SOF + 16 mg/ml BSA in the absence of serum, and 4) in vivo blastocysts. There was no difference in overall blastocyst yield at Day 9 between the groups. However, significantly more blastocysts were present by Day 6 in the presence of 10% serum (20.0%) compared with 3 mg/ml BSA (4.6%, P < 0.001) or 16 mg/ml BSA (11.6%, P < 0.01). By Day 7, however, this difference had disappeared. Following vitrification, there was no difference in survival between blastocysts produced in the presence of 16 mg/ml BSA or those produced in the presence of 10% FCS; the survival of both groups was significantly lower than the in vivo controls at all time points and in terms of hatching rate. In contrast, survival of blastocysts produced in SOF + 3 mg/ml BSA in the absence of serum was intermediate, with no difference remaining at 72 h when compared with in vivo embryos. Differences in relative mRNA abundance among the two groups of blastocysts analyzed were found for genes related to apoptosis (Bax), oxidative stress (MnSOD, CuZnSOD, and SOX), communication through gap junctions (Cx31 and Cx43), maternal recognition of pregnancy (IFN-{tau}), and differentiation and implantation (LIF and LR-ß). The presence of serum during the culture period resulted in a significant increase in the level of expression of MnSOD, SOX, Bax, LIF, and LR-ß. The level of expression of Cx31 and Cu/ZnSOD also tended to be increased, although the difference was not significant. In contrast, the level of expression of Cx43 and IFN-{tau} was decreased in the presence of serum. In conclusion, using a combination of measures of developmental competence (cleavage and blastocyst rates) and qualitative measures such as cryotolerance and relative mRNA abundance to give a more complete picture of the consequences of modifying medium composition on the embryo, we have shown that conditions of postfertilization culture, in particular, the presence of serum in the medium, can affect the speed of embryo development and the quality of the resulting blastocysts. The reduced cryotolerance of blastocysts generated in the presence of serum is accompanied by deviations in the relative abundance of developmentally important gene transcripts. Omission of serum during the postfertilization culture period can significantly improve the cryotolerance of the blastocysts to a level intermediate between serum-generated blastocysts and those derived in vivo. The challenge now is to try and bridge this gap.

early development, embryo, female reproductive tract, gene regulation, in vitro fertilization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The quality of in vitro-produced blastocysts continually lags behind that of blastocysts produced in vivo [1–3]. Previous work from our group has clearly demonstrated that, while the intrinsic quality of the oocyte determines the proportion of oocytes developing to blastocysts (i.e., oocyte developmental competence), it is the postfertilization culture environment that has the biggest influence on blastocyst quality, irrespective of the origin of the zygote [4]. For example, culture of in vitro-produced bovine zygotes in vivo in the ewe oviduct can dramatically increase their cryotolerance, to a level similar to that of totally in vivo-produced embryos [4, 5].

Despite the undefined and variable nature of serum composition, the supplementation of bovine embryo culture media with serum is practiced widely. Several studies have shown that serum has a biphasic effect; the presence of serum can inhibit the early cleavage divisions, while it can have an accelerating effect later in development, resulting, e.g., in the appearance of blastocysts earlier in culture [6–10]. There is also evidence to demonstrate that prolonged exposure to serum can greatly alter embryo morphology and biochemistry [11–16]. In addition, long-term effects on fetal development such as increased birth weight have been attributed to the presence of serum in the medium [17–20].

It is not surprising that this period of postfertilization culture is the period having the greatest impact on blastocyst quality [4] when one considers that, during that 6-day window in the bovine embryo, several major developmental events take place. These include the first cleavage division, the timing of which is known to be an important indicator of the subsequent developmental potential of the embryo [21]; the switching on of the embryonic genome [22]; compaction of the morula, which involves the establishment of the first intimate cell-to-cell contacts in the embryo and blastocyst formation, involving the differentiation of two cell types, the trophectoderm and the inner cell mass. Clearly, any modifications of the culture environment, which could affect any or all of these processes, could have a major effect on the quality of the embryo.

Differences in the relative abundance of some developmentally important gene transcripts have been reported between in vivo- and in vitro-produced bovine embryos [23–26]. In addition, it is known that the conditions of culture in vitro can alter gene expression in the embryo [27–34]. The analysis of such differences in mRNA expression may explain the observed differences in cryotolerance between in vivo- and in vitro-produced embryos [4, 5]. In addition, such an approach could allow the opportunity to alter gene expression through modification of culture media and in that way improve the postthaw viability of in vitro-produced embryos.

From the above, it is clear that any improvement in the quality of blastocysts produced in vitro is likely to derive from the modification of the postfertilization culture conditions. The objective of this study was to examine the effect of the presence or absence of serum and the concentration of BSA during the postfertilization embryo culture period in vitro on 1) cleavage rate, 2) the kinetics of embryo development, 3) blastocyst yield, and 4) blastocyst quality, as assessed by cryotolerance and relative transcript abundance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Blastocyst Production

Bovine blastocysts from four sources were used in this study: 1) in vitro culture in synthetic oviduct fluid (SOF) supplemented with 3 mg/ml BSA and 10% fetal calf serum (FCS), 2) in vitro culture in SOF + 3 mg/ml BSA in the absence of serum, 3) in vitro culture in SOF + 16 mg/ml BSA in the absence of serum, and 4) in vivo blastocysts produced by superovulation, artificial insemination, and nonsurgical embryo recovery. Increasing the concentration of BSA from 3 mg/ml to 16 mg/ml has been reported to improve the quality of the blastocysts (C. Galli and G. Lazzari, personal communication).

In vitro maturation Cumulus oocyte complexes (COCs) were obtained by aspirating follicles from the ovaries at slaughter. After four washes in PBS supplemented with 36 µg/ml pyruvate, 50 µg/ml gentamycin. and 0.5 mg/ml BSA (Sigma, St. Louis, MO), groups of up to 50 COCs were placed in 500 µl maturation medium in four-well dishes (Nunc, Roskilde, Denmark) and cultured for 24 h at 39°C under an atmosphere of 5% CO2 in air with maximum humidity. The maturation medium was TCM-199 supplemented with 10% (v/v) FCS and 10 ng/ml epidermal growth factor.

In vitro fertilization For in vitro fertilization (IVF), COCs were washed four times in PBS and then in fertilization medium before being transferred in groups of up to 50 into four-well dishes containing 250 µl of fertilization medium per well (Tyrode medium with 25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg/ml fatty acid-free BSA, and 10 µg/ml heparin-sodium salt (184 units/mg heparin; Calbiochem, San Diego, CA). Motile spermatozoa were obtained by centrifugation of frozen-thawed semen (Dairygold A.I. Station, Mallow, Ireland) on a discontinuous Percoll (Pharmacia, Uppsala, Sweden) density gradient (2.5 ml 45% Percoll over 2.5 ml 90% Percoll) for 8 min at 700 x g at room temperature. Viable spermatozoa, collected at the bottom of the 90% fraction, were washed in Hepes-buffered Tyrode and pelleted by centrifugation at 100 x g for 5 min. Spermatozoa were counted in a hemocytometer and diluted in the appropriate volume of fertilization medium to give a concentration of 2 x 106 spermatozoa/ml. A 250-µl aliquot of this suspension was added to each fertilization well to obtain a final concentration of 1 x 106 spermatozoa/ml. Plates were incubated for 24 h at 39°C under an atmosphere of 5% CO2 in air with maximum humidity. Semen from the same bull was used for all experiments.

In vitro development At approximately 20 h postinsemination (hpi), presumptive zygotes were denuded by gentle vortexing and washed four times in PBS and twice in culture medium before being transferred to 25-µl culture droplets (1 embryo/µl) under mineral oil. Culture took place either in 1) SOF + 3 mg/ml BSA, to which FCS (10%, v/v) was added 24 h after placement in culture (n = 330), 2) SOF + 3 mg/ml BSA, in the absence of serum (n = 391), or 3) SOF + 16 mg/ml BSA (n = 372). Cleavage rate was recorded at 48 hpi and blastocyst development recorded at Days 6, 7, 8, and 9 postinsemination. Seven replicates were carried out.

In vivo embryo production Beef cross heifers were synchronized using a CIDR device (InterAg, Hamilton, New Zealand) for 8 days. Three days before CIDR removal, heifers received 2 ml (15 mg) of prostaglandin F2{alpha} analogue (PG, Prosolvin; Intervet, Dublin, Ireland). Heifers were checked for standing estrus (=Day 0). The dominant follicle was ablated by transvaginal aspiration on Day 8 of the estrous cycle. Beginning on Day 10, animals were superovulated with a total of 180 mg FSH (Folltropin; Vetrepharm Canada Inc., London, ON, Canada) given as twice-daily injections over 4 days on a decreasing dose schedule. Luteolysis was induced with 15 mg PG given on Day 12. Heifers were inseminated with frozen-thawed semen at 48 and 60 h after PG injection. The same semen batch used in IVF was used for artificial insemination. Day 7 embryos were recovered by nonsurgical flushing 9 days after PG.

Blastocyst Vitrification

The ability of the blastocyst to withstand cryopreservation was used as an indicator of quality. Day 7 blastocysts were used (SOF + FCS, n = 85; SOF + 3 mg/ml BSA, n = 85; SOF + 16 mg/ml BSA, n = 80; in vivo blastocysts, n = 48). Approximately half were vitrified and warmed, while the remainder served as a nonvitrified control. Blastocysts were vitrified using the open pulled straw method described by Vajta et al. [35] in a final solution containing 20% ethylene glycol and 20% dimethyl sulfoxide. Warmed blastocysts were cultured in 25-µl droplets of M199 + 10% FCS in the presence of a granulosa cell monolayer [36] in parallel with nonvitrified controls and examined at 24, 48, and 72 h postwarming. Survival was defined as reexpansion of the blastocoel and its maintenance for 24, 48, and 72 h, respectively. The hatching rate was also recorded.

RNA Extraction and Reverse Transcription

Poly(A) RNA was prepared from four groups of pools of 10 Day 7 blastocysts cultured with or without FCS, following the manufacturer's instructions using the QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia, Barcelona, Spain). In addition, during the precipitation step, 10 µl ribonucleic acid transfer (11.3 mg/ml) (R-8508; Sigma) was also added. The precipitated mRNA was dissolved in 50 µl diethyl pyrocarbonate-treated water and stored at -80°C until use.

Four microliters of the sample were used in the reverse transcription-polymerase chain reaction (RT-PCR) for the detection of each transcript. The RT reaction was carried out following the manufacturer's instructions (Promega) using a mix of random and poly(T) primers and AMV reverse transcriptase enzyme in a volume of 20 µl to prime the RT reaction and to produce cDNA. Tubes were heated to 70°C for 5 min to denature the secondary RNA structure, and then the RT mix was completed with the addition of 5 units of Superscript RT enzyme. They were then incubated at room temperature for 10 min and then at 42°C for 30 min to allow the reverse transcription of RNA, followed by 92°C for 1 min to denature the RT enzyme.

Quantitative RT-PCR

The quantification of all gene transcripts was carried out by real-time quantitative RT-PCR. Three replicate PCR experiments were conducted for all genes of interest using blastocysts collected from the four experimental pools. Experiments were conducted to contrast relative levels of each transcript and ß-actin in every sample. PCR was performed adding 4-µl aliquot of each sample to the PCR mix containing the specific primers to amplify ß-actin, mitochondrial Mn-superoxide dismutase (MnSOD), cytosolic Cu/Zn superoxide dismutase (Cu/ZnSOD), sarcosine oxidase (SOX), connexin 43 (Cx43), connexin 31 (Cx31), Bos taurus apoptosis regulator box-{alpha} (Bax), interferon tau (IFN-{tau}), bovine leukemia inhibitory factor (LIF), and bovine leukemia inhibitory factor-receptor-ß (LR-ß). Primer sequences, annealing temperature, and the approximate sizes of the amplified fragments of ß-actin, MnSOD, SOX, Cx43, Cx31, Bax, LIF, and LR-ß are listed in Rizos et al. [34]. Primers for Cu/ZnSOD amplified 220-base pair (bp) fragments [29]. Bovine IFN-{tau} cDNA was amplified with the primers IF1F: 5'-GCCCTGGTGCTGGTCAGCTA-3' and IF2R: 5'-CATCTTAGTCAGCGAGAGTC-3' producing an amplicon of 564 bp.

ß-Actin amplification was used as a standard control of the RT-PCR. For quantification, PCR was performed using a Rotorgene 2000 Real Time Cycler (Corbett Research, Sydney, Australia) and SYBR Green (Molecular Probes, Eurogene, OR) as a double-stranded DNA-specific fluorescent dye. The PCR reaction mixture (25 µl) contained 2.5 µl 10x buffer, 3 mM MgCl2, 2 U Taq Express (MWGAG Biotech, Ebersberg, Germany), 100 µM of each dNTPs, and 0.2 µM of each primer. In addition, the double-stranded DNA dye, SYBR Green (1:3000 of 10 000x stock solution), was included in each reaction. The PCR protocol included an initial step of 94°C (2 min), followed by 40 cycles of 94°C (15 sec), 56–59°C (30 sec), and 72°C (30 sec). Fluorescent data were acquired during the elongation step. For all the genes analyzed, the melt temperature was always higher than 80°C. The melting protocol consisted of a hold temperature at 40°C for 60 sec and then heating from 50 to 94°C, holding at each temperature for 5 sec while monitoring fluorescence. Product identity was confirmed by ethidium-bromide-stained 2% agarose gel electrophoresis. As negative controls, tubes were always prepared in which RNA or reverse transcriptase was omitted during the RT reaction. In addition, amplicon identities were confirmed by appropriate restriction digests of PCR products (data not shown).

The method used for quantification of expression was the relative standard curve method. The quantification was normalized to an endogenous control (the housekeeping gene ß-actin), and standard curves were prepared for each target and the endogenous reference (ß-actin). Fluorescence was acquired in each cycle in order to determine the threshold cycle or the cycle during the log-linear phase of the reaction at which fluorescence rose above background for each sample. The Rotor Gene software generated a best-fit line and extrapolated the unknown concentration from the threshold cycle of titered known quantities (see Fig. 1). For each experimental sample, the amount of mRNA of each transcript and ß-actin were determined from the appropriate standard curve. Subsequently, the quantity of each transcript was divided by ß-actin to obtain a normalized value for each transcript. The sample with the higher value was assigned a value of one. The normalized target values were divided by the calibrator normalized target values to generate the relative expression levels.



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  		FIG. 1. Illustration of real-time quantitative RT-PCR amplification curves for one replicate of MnSOD and ß-actin for the two sets of embryos (eight curves for each gene corresponding to four groups of blastocysts cultured with or without FCS). The sample amplification curves begin rising between the 24th cycle and the 29th cycle of PCR, while the negative control remains horizontal. The negative derivative of fluorescence is plotted to generate a melting peak to indicate the specificity of the reaction

Statistical Analysis

Data on development and cryotolerance were analyzed using chi-square analysis. Data on mRNA expression were analyzed using the SigmaStat (Jandel Scientific, San Rafael, CA) software package. One-way repeated-measures ANOVA (followed by multiple pairwise comparisons using Student-Newman-Keuls method) was used for the analysis of differences in mRNA expression assayed by quantitative RT-PCR. Differences of P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Development

There was no difference in cleavage rate between the three groups (range 83%–85%; Table 1). Similarly, there was no difference in the overall blastocyst yield at Day 9 (range 38%–42%). However, the kinetics of blastocyst appearance were significantly affected by the level of BSA or the presence of serum; significantly more blastocysts were present by Day 6 in the presence of 10% serum (20.0%) compared with 3 mg/ml BSA (4.0%, P < 0.001) or 16 mg/ml BSA (11%, P < 0.01). By Day 7, however, this difference had disappeared.


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  		TABLE 1. Effect of concentration of bovine serum albumin (BSA) and presence or absence of fetal calf serum (FCS) on development of in vitro maturation/fertilization bovine zygotes in vitro

Cryotolerance

Survival of control embryos was similarly high for all groups (range 84.4%–100% at 72 h; Fig. 2). The only difference in hatching rate was between in vivo-derived blastocysts and those generated in the presence of 16 mg/ml BSA (96.0% vs. 67.7%, respectively, P < 0.01). Following vitrification, there was no difference between blastocysts produced in the presence of 16 mg/ml BSA or those produced in the presence of 10% FCS; the survival of both groups was significantly lower than the in vivo controls at all time points and in terms of hatching rate (Fig. 2). In contrast, survival of blastocysts produced in SOF + 3 mg/ml BSA in the absence of serum was intermediate, with no difference remaining at 72 h when compared with in vivo embryos; similarly, there was no difference in hatching rate between these two groups (Fig. 2).



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  		FIG. 2. Survival of bovine blastocysts following vitrification and warming. Blastocysts were produced in synthetic oviduct fluid supplemented with 1) 3 mg BSA (control, n = 37; vitrified, n = 48), 2) 3 mg BSA + 10% fetal calf serum (control, n = 32; vitrified, n = 53), 3) 16 mg/ml BSA (control, n = 31; vitrified, n = 49), or 4) totally in vivo (control, n = 25; vitrified, n = 23). Different superscripts indicate significant differences (P < 0.05) between treatments at a given time point

Relative Transcript Abundance

In order to analyze quantitative differential expression of nine selected genes, gene-specific primers were designed and used for RT-PCR. As illustrated in Figure 1, the level of expression of ß-actin was similar for the two groups of blastocysts analyzed.

Differences in relative transcript abundance between the two groups (Fig. 3) were found for genes related to apoptosis and oxidative stress, such as Bax, MnSOD, CuZnSOD, and SOX; in genes related to communication through gap junctions, such as Cx31 and Cx43; in a gene related to maternal recognition of pregnancy (IFN-{tau}); and in genes related to differentiation and implantation, such as LIF and LR-ß.



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  		FIG. 3. Relative abundance of various developmentally important gene transcripts (four replicates) in bovine blastocysts produced in synthetic oviduct fluid in the presence (black bars) or absence (white bars) of fetal calf serum (FCS). The mean of the four replicates was calculated for both groups and the group with the higher value was assigned a value of 1. Significant differences (P < 0.05) are indicated by an asterisk (*)

The presence of serum during the culture period resulted in a significant increase in the level of expression of MnSOD, SOX, Bax, LIF, and LR-ß. The level of expression of Cx31 and Cu/ZnSOD was also increased, although the difference was not significant. In contrast, the level of expression of Cx43 and IFN-{tau} was decreased in the presence of serum.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Comparative studies on the effects of various medium constituents have generally focused on the percentage of embryos that proceed to the blastocyst stage. While it is reasonable to use such developmental endpoints as markers of the efficacy of culture systems, it is likely that, in some cases, the effects of a given culture method may not manifest themselves during the relatively short period of in vitro culture or indeed that the effect may be on less tangible parameters than blastocyst yield, such as blastocyst quality. For this reason, we have combined measurement of developmental competence (cleavage and blastocyst rates) with qualitative measures such as cryotolerance and relative mRNA abundance to give a more complete picture of the consequences of modifying medium composition on the embryo.

Consistent with previous reports, we observed that the presence of serum in the medium had a stimulatory effect on the speed of development, with more blastocysts appearing by Day 6 of culture than in its absence [7, 8, 10, 37]. Increasing the concentration of BSA from 3 mg/ml to 16 mg/ml also had a stimulatory effect, although this was not as pronounced as in the presence of serum. It was interesting to note that the difference in development rate was no longer apparent by Day 7. This is consistent with our previous observations of a lack of difference in blastocyst cell number on Day 9 between blastocysts produced in SOF supplemented with 3 mg/ml BSA with or without serum [7].

The ability of an embryo to withstand freezing and thawing has been used in the past as a useful indicator of quality [4, 5, 38, 39] and the ranking of treatments based on this parameter has been reflective of differences at the ultrastructural level [11] as well as at the level of mRNA expression [34]. Here we observed that the simple step of omitting serum from the medium can have a dramatic effect on blastocyst cryotolerance, increasing it to a level intermediate between serum-generated embryos and those derived in vivo. Compared with bovine embryos produced in vivo, embryos produced in vitro exhibit ultrastructural differences [11, 14, 15] that may result from the specific culture system used [16, 40]. It has been suggested that the increased lipid content of in vitro embryos may result from the uptake of lipid from serum included in the culture medium [41, 42] or may result from insufficient metabolism by mitochondria, which would be consistent with the observed reduction in volume density of total mitochondria in in vitro-produced blastocysts [15].

The data presented here on relative mRNA abundance confirm previous observations indicating that mRNA levels of genes indicative of various physiological processes during bovine preimplantation embryo development are affected by postfertilization culture conditions. While it should be borne in mind that the presence of the transcript does not necessarily indicate the presence of the protein, we observed that the transcription of the majority of genes studied (with the exception of Cx43 and IFN-{tau}) was increased in the presence of serum. In contrast, in the study of Wrenzycki et al. [31], the transcription of the majority of the genes they studied was increased in polyvinyl alcohol (PVA)-supplemented/serum-free medium-derived embryos compared with their serum-generated counterparts. However, for the only two genes studied by both groups (Cx43 and IFN-{tau}), the pattern of expression was the same.

IFN-{tau}, a Type I interferon that is exclusively secreted by the cells of the trophectoderm, is the primary agent responsible for maternal recognition of pregnancy in cattle [43], exerting its antiluteolytic effect on the maternal corpus luteum by attenuating the pulsatile, luteolytic secretion of PGF2{alpha} by the uterine endometrium. In cattle, embryos begin to express IFN-{tau} as the blastocyst forms, although there is considerable variability between individual embryos in the amount they produce [44], which may be related to the age at which blastocyst formation occurs [45, 46], the group size in which culture takes place [47], the medium composition [46, 48], or the sex of the embryo [49].

IFN-{tau} mRNA has been detected in blastocysts but not in morulae [31, 44, 50], suggesting that the onset of IFN-{tau} production is tightly linked to blastocoel formation. IFN-{tau} can be readily detected in the medium of individually cultured blastocysts in vitro [44, 50] and IFN-{tau} production is apparently independent of blastocyst cell number [45, 47]. Attempts have been made to correlate the amount of IFN-{tau} that is secreted by individual blastocysts with their morphological quality, but results have been inconclusive [45, 50, 51]. Kubisch et al. [46] cultured embryos in SOF containing PVA, BSA, or FCS at comparable concentrations to those used in this study. While there was no difference in the percentage of embryos reaching the blastocyst stage, which is in agreement with our observations, blastocysts produced in PVA had significantly fewer cells, were older at blastocyst formation, and produced significantly more IFN-{tau}. The same authors found that overall secretion of IFN-{tau} by in vivo-derived blastocysts did not differ from that of age-matched blastocysts produced in vitro.

In the present study, we observed a significantly higher level of expression of IFN-{tau} in blastocysts produced in the absence of serum, which would be consistent with the notion that mRNA levels for this transcript are higher in good-quality embryos. In agreement, Wrenzycki et al. [31] reported increased levels of IFN-{tau} mRNA in hatched blastocysts produced in the presence of PVA compared with when serum was present.

The expression of Bax was higher in blastocysts produced in the presence of serum than those produced in its absence, indicating that this gene may represent a useful marker of blastocyst quality. In agreement, Gjorret et al. [52] reported that apoptosis is more frequent in in vitro- than in in vivo-produced blastocysts. Consistent with these observations, a higher incidence of apoptosis has been reported in in vitro-produced blastocysts derived from late-cleaving zygotes than those that cleave earlier [53]. The same authors reported that the incidence of apoptosis increases in bovine embryos produced in the presence of serum. Distortions of apoptosis in the blastocyst may lead to either early embryonic death or the formation of anomalies in the fetus that produce early abortions [54].

Oxidative stress has been implicated as one of the causes of defective embryo development. The overgeneration of intracellular reactive oxygen species (ROS) during culture of mammalian embryos is generally thought to be detrimental to embryo development (reviewed in [55–57]). An increased production of ROS has been measured in mouse embryos produced in vitro compared with those derived in vivo [57]. Transcripts for Cu/ZnSOD have been detected throughout bovine embryonic development [29, 58]. While Harvey et al. [58] failed to observe transcripts for MnSOD in bovine embryos, using a different set of primers, Lequarre et al. [29] demonstrated a culture environment-dependent expression of this transcript. In that study, no expression of MnSOD was detected at the 5- to 8-cell, 9- to 16-cell, and morula stages when culture took place in the absence of serum, while it was detected in almost 80% of blastocysts. In contrast, in the presence of 5% serum or in in vivo-produced embryos, mRNA expression was detectable in 58% of morulae and 74% of blastocysts. Some undefined factors in serum may interact with the expression system; e.g., TNF{alpha} is known as an inductor of MnSOD transcription [59].

We have previously reported a higher level of expression of MnSOD mRNA in in vivo-produced blastocysts and those cultured in the ewe oviduct (i.e., high-quality embryos) compared with those produced by culture in vitro in serum-supplemented SOF [34]. In the present study, the expression of MnSOD was higher in blastocysts produced in the presence of serum. While this may, at first glance, seem paradoxical because those blastocysts produced with serum are of lower quality, it is probably not correct to compare the results directly because, in the present study, both groups of blastocysts were derived in vitro. In addition, in agreement with Lequarre et al. [29], the expression of Cu/ZnSOD was not related to the presence of FCS in culture.

Consistent with our previous observations [34], where expression of the SOX enzyme was higher in blastocysts produced in vitro or in coculture than in those produced in vivo, in the present study, SOX expression was highest in serum-generated blastocysts. SOX is a member of a recently recognized family of enzymes that contain covalently bound flavin and catalyze oxidation reactions [60]. SOX is a peroxisomal enzyme that may be associated with the peroxisomal membrane. Peroxisomas act in detoxification and also in the first two steps of the synthesis of some lipids and in the oxidation of some lipids of more than 18C. Alteration of this transcription could be related to the high level of H2O2 measured in bovine embryos produced in vitro, with high production of glycine and glucose, with lipid peroxidations, and in general with lipid metabolism [57].

At least four connexins contribute to gap junctions in preimplantation development [61]. These junctions are essential for the transport of cryoprotectant and fluids during freezing and thawing. We have analyzed two of these, Cx31 and Cx43. In mice, Cx31 and 43 transcripts are abundant in the zygote and are degraded at the four-cell stage to low levels of Cx31 and undetectable levels of Cx43. Reexpression of Cx43 and 31 mRNA occurs from the compacted morula stage onward. At the blastocyst stage, both connexins are coexpressed in the trophectoderm as well as in the inner cell mass. After implantation, compartmentalization of both connexins is observed. This compartmentalization in connexin expression between the derivatives of the inner cell mass and the trophectoderm may maintain the different developmental programs. Apparently, Cx31 is not related to the first step in trophoblast lineage development and could serve as a compensatory channel during preimplantation development [62].

Wrenzycki et al. [25] reported that Cx43 mRNA was detectable in in vitro-produced bovine embryos from the oocyte to the morula stage but was not detectable in blastocysts or hatched blastocysts, in contrast with its detection in in vivo-derived blastocysts. The same authors subsequently observed that the expression pattern for Cx43 in vitro was altered in the presence of serum, disappearing at the 8- to 16-cell stage and reappearing at the hatched blastocyst stage [31]. In the present study, the level of expression of Cx43 was significantly higher in blastocysts derived from serum-free medium. In contrast, Cx31 mRNA was expressed more strongly in blastocysts produced in the presence of serum. This pattern of expression is consistent with the quality of these blastocysts measured in terms of cryotolerance.

LIF plays an essential role during early differentiation and implantation in embryos. Oviductal cells synthesize LIF to promote and condition the embryo for implantation [63]. LIF has been reported to bind to blastocyst-stage embryos and affect their development in culture [64]. The receptor consists of the two dimerizing subunits, glycoprotein 130 and LR-ß. Human and murine embryos produced in vitro express LIF and LR-ß mRNAs transcripts throughout the preimplantaion period [65]. In cattle, Eckert and Niemann [26] reported differences in the expression of LIF and LR-ß between in vitro- and in vivo-derived embryos. We have detected LIF and LR-ß mRNAs at higher levels in in vitro-produced bovine blastocysts, irrespective of culture system, than in in vivo-derived blastocysts; blastocysts derived from culture in the ewe oviduct were somewhat intermediate [34]. In agreement with these observations, in the present study, expression was highest in blastocysts produced in the presence of serum. As suggested by Eckert and Niemann [26], some perturbation of the mRNA expression patterns of the specific LIF/LIF-receptor system occur during the development of bovine embryos in vitro. This may lead to abnormal development of the inner cell mass and trophectoderm in the blastocyst.

In conclusion, our data indicate that conditions of postfertilization culture, in particular, the presence of serum in the medium, can affect the speed of embryo development and the quality of the resulting blastocysts. The reduced cryotolerance of blastocysts generated in the presence of serum is accompanied by deviations in the relative abundance of developmentally important gene transcripts. As demonstrated here, omission of serum during the postfertilization culture period can significantly improve the cryotolerance of the blastocysts to a level intermediate between serum-generated blastocysts and those derived in vivo. The challenge now is to try and bridge this gap.


   ACKNOWLEDGMENTS
 
The authors thank M. Wade and P. Duffy for excellent technical assistance.


   FOOTNOTES
 
1 D.R. was supported by a grant from the Greek State Scholarships Foundation. P.L. was partially funded by a University College Dublin President's Research Award. Back

2 Correspondence. FAX: 353 1 6288421; pat.lonergan{at}ucd.ie Back

Received: 28 May 2002.

First decision: 24 June 2002.

Accepted: 5 August 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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