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Biology of Reproduction 67, 767-775 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Cellular and Molecular Deviations in Bovine In Vitro-Produced Embryos Are Related to the Large Offspring Syndrome1

Giovanna Lazzaria, Christine Wrenzyckib, Doris Herrmannb, Roberto Duchia, Theo Kruipc, Heiner Niemann2,b, and Cesare Gallic

a Laboratorio di Tecnologie della Riproduzione, Cremona, Italy b Department of Biotechnology, Institute for Animal Science, Mariensee, 31535 Neustadt, Germany c Department of Reproduction, Institute for Animal Science & Health, Lelystad, The Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The large offspring syndrome (LOS) is observed in bovine and ovine offspring following transfer of in vitro-produced (IVP) or cloned embryos and is characterized by a multitude of pathologic changes, of which extended gestation length and increased birthweight are predominant features. In the present study, we used bovine blastocysts to analyze cellular parameters, i.e., the number of cells in Day 7 blastocysts and the size of Day 12 elongating blastocysts, and molecular parameters, i.e., the relative abundance of developmentally important genes: glucose transporter (Glut) 1, Glut-2, Glut-3, Glut-4, heat shock protein (Hsp) 70.1, Cu/Zn-superoxide dismutase (SOD), histone H4.1, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) I receptor (R), and IGFII-R. Some blastocysts were produced by in vitro maturation and fertilization followed by in vitro culture in synthetic oviduct fluid medium supplemented with BSA or human serum or by in vivo culture in the sheep oviduct. Other blastocysts were derived in vivo from the uterine horns of superovulated donors. The findings made in the early embryos were related to a representative number of calves obtained from each production system and from artificial insemination (AI). In vitro culture of bovine embryos in the presence of high concentrations of serum or BSA significantly increased the number of cells in Day 7 blastocysts, the size of blastocysts on Day 12, and the relative abundance of the transcripts for Hsp70.1, Cu/Zn-SOD, Glut-3, Glut-4, bFGF, and IGFI-R when compared with embryos from the in vivo production groups. Birthweights of calves derived from IVP embryos were significantly higher than those of calves derived from sheep oviduct culture, superovulation, or AI. The results support the hypothesis that persistence of early deviations in development is causally involved in the incidence of LOS, in particular in increased birthweights. The cellular and molecular parameters analyzed in this study can be considered early markers of LOS in cattle.

embryo, gene expression, in vitro production, large offspring syndrome


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bovine in vitro-produced (IVP) embryos display numerous marked differences from their in vivo-derived counterparts with regard to morphology [1], timing of development [2], resistance to low temperature [3, 4], embryonic metabolism [5], and gene expression [6]. The large offspring syndrome (LOS) has been observed in bovine and ovine offspring following transfer of IVP or cloned embryos [7–9]. LOS is characterized by a multitude of pathologies, including high birthweights, increased gestation length, frequent dystocia, elevated abortion and perinatal mortality rates, and various morphological deviations. Frequency and severity of the symptoms of LOS vary from one study to another even under similar experimental conditions [10, 11]. Early deviations from the normal developmental pattern, particularly with regard to embryonic gene expression, may be involved in this phenomenon [6, 12].

Whether these deviations from the normal pattern of gene expression and development observed in the preimplantation embryo persist throughout fetal development and even up to birth has not yet been determined. Following culture of embryos in the presence of serum and/or somatic cells, aberrant ovine fetal growth was detected soon after implantation at Day 21 [13] and later at Day 61 of gestation [14]. Furthermore, levels of insulin-like growth factor (IGF) II mRNA were significantly elevated at Day 63 of gestation in bovine fetuses derived from IVP compared with their in vivo counterparts [15]. Methylation and expression levels of the IGFII receptor (IGFII-R) gene were reduced by 30–60% in plasma, liver, and muscle of oversized fetuses in midgestation compared with the control group, thereby altering the subtle regulation of expression of the IGF family [16].

In the present study, we analyzed cellular and molecular parameters in bovine embryos and the incidence of LOS in calves derived from transfer of embryos produced with different in vitro and in vivo systems. Cellular parameters were the number of cells in Day 7 blastocysts and the size of the elongating embryo on Day 12. Alterations in the relative abundance of several developmentally important genes, as determined in single embryos by a highly sensitive semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay [17, 18], served as indicators for early deviations at the molecular level. Embryos employed in this study were obtained via in vitro maturation and in vitro fertilization (IVF) and cultured using two different systems based on synthetic oviduct fluid (SOF) medium [19] supplemented with either serum or BSA and a third system that included a temporary in vivo culture period from zygote to blastocyst in the sheep oviduct. Serum versus BSA supplementation was compared because in previous studies addition of serum to the culture medium exaggerated the syndrome whereas replacement of serum with BSA significantly reduced its occurrence [20, 21]. The results show for the first time a relationship between the incidence of early embryonic deviations and LOS in IVP calves.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All chemicals were purchased from Sigma-Aldrich (Milan, Italy) unless otherwise indicated.

Experiment 1

Production of Day 7 embryos Bovine cumulus-oocyte complexes (COCs) were collected by dissection of ovaries collected from cattle (Holstein-Friesian) from a local abattoir, and COCs with at least three layers of cumulus cells and a homogenous cytoplasm were matured in tissue culture medium (TCM) 199 supplemented with 10% fetal calf serum (FCS), ITS (insulin, transferrin, sodium selenite, 1 µl/ml), Long-IGFI (100 ng/ml), Long-EGF (epidermal growth factor, 50 ng/ml), and FSH and LH (0.1 IU each; Pergovet, Serono, Italy) for 22 h with 5% CO2 in air [22]. Frozen/thawed semen from a single Holstein-Friesian bull of proven fertility was used for IVF. After thawing, the semen was separated on a Percoll gradient and resuspended at a concentration of 0.5 x 106 sperm/ml in SOF medium [19], Hepes buffered and supplemented with 6 mg/ml fatty acid-free (FAF) BSA, modified Eagle medium (MEM) amino acids, 1 µg/ml heparin, 20 µM penicillamine, 1 µM epinephrine, and 10 µM hypotaurine. Matured oocytes were coincubated with the sperm suspension for 18–20 h at 38.5°C in a gas mixture consisting of 5% CO2, 5% O2, and 90% N2. At termination of gamete coculture, the cumulus cells were completely removed and cumulus-free presumptive zygotes were randomly transferred into one of three culture systems in which they were cultured for up to Day 7 (Day 0 = IVF): 1) in vitro, in 20-µl drops of SOF medium supplemented with MEM amino acids and 16 mg/ml FAF BSA (SOF-BSA group), 2) in vitro, in 20-µl drops of SOF medium without amino acids and BSA [23] but supplemented with a 20% solution of human serum previously shown to be associated with a high incidence of LOS in sheep (L. Young, personal communication; SOF-serum group), and 3) in vivo, in ligated oviducts of sheep that received an intravaginal progesterone-releasing device (EAZI-BREED CIDR; InterAg, Hamilton, New Zealand) irrespective of the stage of estrous cycle at the time of transfer of zygotes (sheep oviduct group). On Day 7, embryos from all groups were scored for the rate of blastocyst development. A representative number of blastocysts was fixed and stained with lacmoid to determine total cell numbers. For mRNA analysis (see experiment 3), embryos were washed three times in PBS containing 0.1 % polyvinyl alcohol (PVA) and stored individually at -80°C in a minimum volume (<=5 µl) of medium until experimental use.

Production of Day 12 embryos Groups of 20–40 morphologically intact Day-7 blastocysts derived from the three different production systems (SOF-BSA, SOF-serum, sheep oviduct) were transferred nonsurgically into the uterine horn ipsilateral to the corpus luteum of 12 synchronized recipient heifers for a total of 5 days. On Day 12, the embryos were collected with the aid of a Dissi catheter (19003/0016; Minitüb, Tiefenbach, Germany) by flushing the uterine horns of the temporary recipient heifers using Dulbecco PBS (DPBS) supplemented with 1% FCS as the medium. The fluid was recovered in sterile plastic containers, and the embryos were allowed to settle. The supernatant was aspirated, and the embryos were collected from the bottom of the containers and placed into Hepes-buffered SOF medium supplemented with MEM amino acids and 6 mg/ml BSA fraction V. All embryos were measured individually using a micrometer scale on a stereomicroscope or a calibrated ruler for the larger embryos; maximum length and width were determined.

Transfer on Day 12 into final recipient heifers Embryos (n = 27) within each group and within each production system that were considered to be at an advanced stage of development (>0.60 ± 0.50 mm length and width) were selected for transfer into final recipients. Seven embryos from the SOF-serum group that were considered to be at the least advanced stage of development (<0.35 ± 0.35 mm length and width) were also transferred into final recipients. In total, 34 embryos were transferred singly into synchronized Day 12 recipient heifers using routine nonsurgical embryo transfer procedures. Pregnancies were monitored regularly at 40, 60, 90, 120, and 180 days and were allowed to go to term. Pregnancy rates, incidence of abortions, type of delivery, and birthweight and sex of calves were recorded. Control calves that were derived from artificial insemination (AI) employing the same bull as used in IVF and were born in the same herd during the same period of time were included in this experiment.

Experiment 2: Production of Calves from Day 7 Embryos Derived from In Vitro and Temporary In Vivo Culture

Blastocysts were produced employing the same protocol described in experiment 1 with the exception that different bulls were used for IVF. On Day 7, morphologically intact embryos (grade I of the International Embryo Transfer Society grading system) were frozen in 0.25-ml straws with 1.5 M ethylene glycol in Hepes-buffered SOF medium supplemented with 6 mg/ml BSA fraction V. After seeding at -6°C, the embryos were cooled at 0.5°C/min to -32°C, plunged into liquid nitrogen, and after storage for several days thawed in air and then directly transferred into heifers and cows serving as final recipients [24]. Pregnancy rates, incidence of abortions, and birthweight and sex ratio of calves were recorded. Control calves that were derived from frozen-thawed embryos produced by superovulation and were born in the same herds during the same period of time were included in this experiment.

Experiment 3: Determination of Relative mRNA Abundances in Individual Embryosby Semiquantitative RT-PCR

Poly(A)+ RNA was isolated from single blastocysts generated in the different culture systems as previously described [17, 18] and was used immediately for RT, which was carried out in a total volume of 20 µl using 2.5 µM random hexamers (Perkin-Elmer, Vaterstetten, Germany). Prior to RNA isolation, 1 pg of rabbit globin RNA (Gibco BRL, Gaithersburg, MD) was added as an internal standard. The reaction mixture consisted of 1x RT buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3; Perkin-Elmer), 5 mM MgCl2, 1 mM of each dNTP (Amersham, Braunschweig, Germany), 20 IU RNase inhibitor (Perkin-Elmer), and 50 IU murine leukemia virus reverse transcriptase (Perkin-Elmer). The mixture was overlaid with mineral oil to prevent evaporation. The RT reaction was carried out at 25°C for 10 min and at 42°C for 1 h, followed by a denaturation step at 99°C for 5 min and flash cooling on ice. The PCR was performed with embryo equivalents as described in Table 1 from different embryos generated in different IVP runs and 50 fg of globin RNA (corresponding to 0.05 embryo equivalents) in a final volume of 50 µl of 1x PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl; Gibco BRL, Eggenstein, Germany), 1.5 mM MgCl2, 200 µM of each dNTP, and 1 µM of each sequence-specific primer (globin: 0.5 µM) using a PTC-200 thermocycler (MJ Research, Watertown, MA). To ensure specific amplification, a "hot start" PCR was employed by adding 1 IU Taq DNA polymerase (Gibco) at 72°C. PCR primers were designed from the coding regions of each gene sequence using the OLIGO-program. The sequences of the primers used, the annealing temperatures, the fragment sizes, and the sequence references are summarized in Table 1.


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  		TABLE 1. Primers and conditions used for PCR

The PCR program employed an initial step of 97°C for 2 min and 72°C for 2 min (hot start) followed by different numbers of cycles (see Table 1) of 15 sec each at 95°C for DNA denaturation, 15 sec at different temperatures for annealing of primers, and 15 sec at 72°C for primer extension. The final cycle was followed by 5 min of extension at 72°C and then cooling to 4°C. As negative controls, tubes were prepared in which RNA or reverse transcriptase was omitted during the RT reaction (data not shown).

The RT-PCR products were subjected to electrophoresis on a 2% agarose gel in 1x TBE buffer (90 mM Tris, 90 mM borate, 2 mM EDTA, pH 8.3) containing 0.2 µg/ml ethidium bromide. Ethidium bromide was also added to the running buffer to the same concentration as the gel. The image of each gel was recorded using a CCD camera (Quantix, Photometrics, München, Germany) and the IP Lab Spectrum program (Signal Analytics Corp., Vienna, VA). The intensity of each band was assessed by densitometry using an image analysis program (IP Lab Gel; Signal Analytics). The relative amount of the mRNA of interest was calculated by dividing the intensity of the band for each developmental stage by the intensity of the globin band for the corresponding stage. Experiments were repeated 10 times with different embryos for each gene transcript. In vivo-derived embryos recovered after superovulation were included as controls. The relative abundance was calculated on a per cell basis per embryo because the average number of cells per embryo differed significantly between production systems. This approach is accurate enough to determine significant differences among single embryos derived from different systems [17, 18] and thus provides valid data.

For each pair of gene-specific primers, semilog plots of the fragment intensity as a function of cycle number were used to determine the range of cycle number over which linear amplification occurred, and the number of PCR cycles was kept within this range [25]. Because the overall efficiency of amplification for each set of primers during each cycle is not known, this assay can only be used to compare relative abundances of one mRNA among different samples [26].

Statistical Analysis

Relative abundances were analyzed using the SigmaStat 2.0 (Jandel Scientific, San Rafael, CA) software package. After testing for normality (Kolmogorov-Smirnov test with Lilliefor correction) and testing for equal variance (Levene Median test), an ANOVA followed by multiple pairwise comparisons using a Tukey test was employed. Differences between treatment groups of embryos or calves were calculated by chi-square or Student t-test if appropriate. Differences at P <= 0.05 were considered significant. Data are presented as mean ± SD for cell counts, birthweights, and gestation length and as mean ± SEM for the relative abundances.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1

Effects of different culture systems on embryo development to Day 7 Overall, 2783 bovine oocytes were matured and fertilized. The development of these oocytes to blastocysts is shown in Table 2. The proportion of blastocysts relative to the number of oocytes and cleavage stages was similar in all groups (SOF-BSA: 19.5% and 28.9%; SOF-serum: 18.1% and 27.8%; sheep oviduct: 21.2% and 31.5%, respectively). The cell number was not different between embryos produced in SOF-BSA (183 ± 37) and SOF-serum (179 ± 34), whereas it was significantly decreased (P < 0.05) in the sheep oviduct group (142 ± 34). In vivo-produced embryos had an average number of 158 ± 46 cells.


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  		TABLE 2. Bovine blastocyst development and cell number after culture in SOF-BSA, SOF-serum, or sheep oviduct. Values within columns with different superscripts are significantly different (P < 0.05)

Effects of different culture systems on embryo development from Day 7 to Day 12 A total of 338 Day 7 embryos from the three production systems were transferred into 12 synchronized recipient heifers from which 173 embryos (51.2%) were collected 5 days later, corresponding to a recovery rate of 57.5% (65/113) for the SOF-BSA group, 58.3% (60/103) for the SOF-serum group, and 39.3% (48/122) for the sheep oviduct group, respectively. Average length and width were similar in the SOF-BSA (0.84 ± 0.41 and 0.62 ± 0.24 mm) and SOF-serum (0.83 ± 0.23 and 0.65 ± 0.19 mm) groups, but the embryos derived from the sheep oviduct group were significantly smaller (0.43 ± 0.15 and 0.40 ± 0.12 mm, respectively; P < 0.05). No differences in recovery rates and embryo size and development were found on Day 12 with regard to the different amounts of Day-7 blastocysts transferred per uterine horn.

Viability of Day 12 embryos after transfer into final recipients From the 27 advanced-stage (i.e., large) embryos, 17 pregnancies (63.0%) were obtained, but only 1 pregnancy resulted from transfer of 7 small embryos (14.3%; P < 0.05). With regard to the origin of the embryos, pregnancy rates were 54.5% (6/11) for the SOF-BSA group, 80.0% (8/10) for the SOF-serum group, and 50% (3/6) for the sheep oviduct group. Four of 18 (22.2%) recipients aborted spontaneously between Days 60 and 180 of pregnancy. Three pregnancies were lost in the SOF-BSA group, and one was lost in the SOF-serum group. The aborted fetuses were grossly intact, and no signs of infective agents were observed.

Type of delivery and birthweights of calves All calves were naturally delivered with the exception of one that was born by Caesarean section; no perinatal pathology was observed. Gestation length was similar among the groups: SOF-BSA: 283.3 ± 9.6 days (n = 3); SOF-serum: 279.9 ± 6.9 days (n = 8); sheep oviduct: 281.0 ± 0.4 days (n = 3); AI: 281.7 ± 4.3 days (n = 24). A significant difference (P < 0.05) was observed between average birthweight of the in vitro-derived calves (SOF-BSA: 52.0 ± 11.3 kg; SOF-serum: 51.8 ± 9.0 kg) and that of calves derived from the sheep oviduct (44.2 ± 2.8 kg) or AI (43.4 ± 4.3 kg) groups.

Experiment 2: Viability of Day 7 Frozen-Thawed Embryos after Transfer into Final Recipients and Characteristicsof Calves

Pregnancy rates were 36.9% (24/65) for SOF-BSA embryos, 16.7% (4/24) for SOF-serum embryos, 53.8% (35/65) for sheep oviduct embryos, and 55.2% (69/125) for in vivo embryos derived from superovulated donors. A significant difference (P < 0.05) with regard to pregnancy rate existed between SOF-BSA or SOF-serum embryos and those derived from sheep oviduct culture or in vivo.

The overall proportion of pregnancies lost was 12.1% (16/132): 16.7% (4/24) in the SOF-BSA group, 50% (2/4) in the SOF-serum group, 11.4% (4/35) in the sheep oviduct group, and 8.7% (6/69) in the in vivo group. Perinatal mortality was 12.9% (15/116): 15.0% (3/20) in the SOF-BSA group, 0 (0/2) in the SOF-serum group, 12.9% (4/31) in the sheep oviduct group, and 12.7% (8/63) in the in vivo group. Sex ratio (female:male) was not different among groups: SOF-BSA, 35% (7:13); SOF-serum, 50% (1:1); sheep oviduct, 41.9% (13:18); and in vivo, 42.8% (27:36). The average birthweights were 52.9 ± 9.1 kg, 76.5 ± 8.5 kg, 44.1 ± 5.7 kg, and 41.1 ± 3.0 kg for the SOF-BSA, SOF-serum, sheep oviduct, and in vivo groups, respectively. Gestation length was 280.7 ± 6.7 days for the SOF-BSA group, 281.5 ± 0.5 days for the SOF-serum group, 278.9 ± 5.6 days for the sheep oviduct group, and 279.4 ± 5.1 days for the in vivo group.

In experiments 1 and 2, the embryos were produced by identical culture protocols. The combined data are shown in Table 3. Because of the small number of observations in the SOF-serum group in experiment 2 (n = 2), no statistical analysis was conducted for the SOF-serum group in experiment 1. There were no significant differences between experiments 1 and 2 with regard to gestation length and birthweight in the other three groups. Embryos derived from the SOF-BSA and SOF-serum systems gave rise to calves that were significantly heavier (P < 0.05) than those derived from embryos grown in the sheep oviduct or in vivo. The incidence of abnormally large calves (>50 kg body weight) was significantly higher (P < 0.05) in the SOF-BSA and SOF-serum groups than in the sheep oviduct and in vivo groups.


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  		TABLE 3. Gestation length and birthweight of calves derived from in vitro and in vivo production (combined data from experiments 1 and 2). Values within columns with different superscripts are significantly different (P < 0.05)

Experiment 3: Effect of Different Embryo Production Systems on the Relative Abundance of Developmentally Important Gene Transcripts

Representative gel photographs of a semiquantitative RT-PCR assay of the analyzed gene transcripts in single bovine embryos of different origin are shown in Figure 1. Alterations in mRNA abundances of the specific gene transcripts in relation to the different production systems are depicted in Figure 2. The relative abundance for Hsp70.1 was significantly increased (P < 0.05) in blastocysts derived from the SOF-serum system compared with their counterparts produced in sheep oviducts or by superovulation. The relative abundance of Cu/Zn-SOD was significantly increased (P < 0.05) in both SOF-BSA- and SOF-serum-generated embryos compared with their counterparts produced in vivo or in the sheep oviduct. Transcript levels of Glut-3 and Glut-4 were significantly (P < 0.05) upregulated in IVP embryos (SOF-BSA, SOF-serum) compared with embryos generated in the sheep oviduct or in vivo. For Glut-3, a significant increase (P < 0.05) in relative abundance was detected between in vivo-derived embryos and those cultured in the sheep oviduct. The relative abundance of Glut-1 transcripts was similar in embryos from all production systems. Glut-2 transcripts were never detected in bovine blastocysts regardless of their origin. With regard to genes involved in growth factor signalling, IGFI-R transcripts were significantly increased (P < 0.05) in BSA- and serum-derived embryos compared with embryos generated in vivo or in the sheep oviduct. The relative abundance of bFGF mRNA was significantly increased (P < 0.05) in embryos grown in SOF supplemented with BSA compared with embryos grown in serum-enriched medium, in vivo, or in the sheep oviduct. No differences in transcript levels in embryos of different origins were detected for the IGFII-R and the H4 gene.



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  		FIG. 1. Representative gels of a semiquantitative RT-PCR analysis of various developmentally important gene transcripts in bovine blastocysts of different origin. Lane 1: SOF-serum; lane 2: SOF-BSA; lane 3: ligated sheep oviduct; lane 4: in vivo; lane 5: negative control



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  		FIG. 2. Expression patterns of various developmentally important gene transcripts in bovine blastocysts of different origin. Vertical striped bars: in vivo; open bars: ligated sheep oviduct; shaded bars: SOF-BSA; solid bars: SOF-serum. Bars with different superscripts differ significantly (a:b, P <= 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study is the first to link deviations in early embryonic development at the level of mRNA expression in Day 7 bovine embryos and cell proliferation in Day 7 and Day 12 embryos with gestation length and birthweight of the derived calves. Expression levels in embryos derived from the sheep oviduct in vivo culture system were analyzed and compared with those in embryos derived from two IVP systems or produced in vivo. Both IVP systems were associated with a significantly elevated incidence of deviations in embryonic development and a higher proportion of calves with increased birthweight and other symptoms of LOS. The findings support our hypothesis that persistent deviations in embryonic expression patterns of developmentally important genes are critically involved in the incidence of LOS [6, 12]. Deviations in the expression of several nonimprinted genes are thought to lead to perturbation of placental and fetal growth attributed to cumulative action [27, 28].

Embryos derived from the SOF-BSA or SOF-serum system possessed significantly more cells than those derived from the sheep oviduct or in vivo production, indicating enhanced cell proliferation and accelerated development under our in vitro conditions. This increased rate of development was maintained at Day 12 of gestation, when the average length and width of the embryos from the SOF-BSA and SOF-serum groups were significantly greater than those of embryos from the sheep oviduct group. Upon transfer to recipients, this difference may lead to asynchronous development with potentially deteriorating effects on embryonic and fetal development [29]. In a previous study, the majority of embryos derived in vitro had lower numbers of cells than did in vivo embryos at a comparable stage of development [30]. These diverging observations reflect the great range of effects exerted on early embryos by current in vitro production protocols.

The success of embryo recollection on Day 12 from the temporary recipient heifers was similar (51.2%) to that in previous reports [31, 32] after surgical flushing of superovulated donors on Day 12 after insemination, suggesting that the loss of embryos at Day 12 in this study cannot be attributed to poor quality of the embryos transferred on Day 7. Therefore, the low pregnancy rate after transfer of the small, less developed Day 12 embryos can be explained by compromised developmental potential. The high degree of variability in embryo morphology between Days 7 and 12 is thought to be involved in embryonic losses during this period of bovine gestation [31, 33]. Pregnancy rate and the proportion of embryos lost after transfer of well-developed Day 12 embryos in the present study were similar to those of previous studies after transfer of Day 10 to Day 16 embryos [32].

The present results demonstrate a distinct effect of in vitro culture conditions, irrespective of the presence of serum or BSA, on the incidence of early cellular and molecular embryonic deviations and LOS. Changing culture supplements from serum to BSA had previously been shown to reduce the frequency of LOS [21, 34]. The contrary findings of this study can be explained by the sporadic nature of LOS [10] and/or batch-to-batch variability of BSA [35]. BSA and serum have been replaced by macromolecules such as PVA to generate chemically defined media without detrimental effects on early embryonic development [36–38]. The expression pattern of several developmentally important genes in bovine embryos grown in TCM or SOF medium supplemented with PVA was closer to that of in vivo embryos, whereas embryos produced in the presence of BSA or serum showed more deviations in their expression patterns [25, 38]. In the present study, embryos cultured in the ovine oviduct were characterized by an expression pattern that was nearly identical to that of their in vivo-produced counterparts. This finding is remarkable because bovine embryos travel from the oviducts to the uterus at Day 4 after insemination and are thus subject to a dramatic change in environment. However, other genes may show deviations from the normal expression pattern in embryos cultured in the sheep oviduct system. Recently, similar results were found for a panel of seven gene transcripts analyzed in bovine embryos derived from IVP systems and the sheep oviduct [39].

The different pregnancy rates observed in experiments 1 and 2 may have been affected by the fact that in experiment 2 only a few transfers were done in some subgroups. Previously, an increased incidence of stillbirth, perinatal death, and problems in adapting to extrauterine life was reported in offspring afflicted by LOS [40–42], whereas in another study no such results were found [43], in agreement with the study reported here. Cryopreservation did not seem to affect the developmental capacity of IVP embryos, those derived from the sheep oviduct, or in vivo, and the pregnancy rates were even lower in experiment 1, in which fresh embryos had been transferred. Similar to previous studies, pregnancy rates in experiment 2 were reduced after transfer of embryos from both IVP systems [4, 7, 22]. Cryopreservation of bovine embryos is routinely used in commercial embryo transfer and has not been associated with LOS [44]. Thus, symptoms of LOS should have originated from the IVP process, and the different relative abundances probably reflect the status of embryonic origin (IVP, oviduct, in vivo) and are not related to freezing.

Significant differences in the relative abundances of developmentally important gene transcripts were detected in embryos obtained from the different production systems. Gene transcripts known to be sensitive indicators of stress, i.e., Hsp70.1 and Cu/Zn-SOD, were increased in embryos from both IVP systems. Hsp70.1 transcripts were upregulated in embryos generated in serum-enriched medium [25, 38]. Heat shock proteins are thought to have two major physiological functions: 1) acting as chaperones in facilitating protein folding and assembly and 2) stabilizing damaged proteins to prevent aggregation of fragments, thereby allowing repair or degradation [43, 45]. Cellular stress is likely caused by an increased production of peroxides during in vitro culture, regardless of the protein supplement. Therefore, we also analyzed the expression of the antioxidative enzyme Cu/Zn-SOD [46, 47], which is critically involved in protection against oxidative stress. The significant increase of Cu/Zn-SOD transcripts in IVP embryos further supports the hypothesis that current in vitro culture systems are associated with a considerable amount of oxidative stress. The early embryo attempts to protect itself against these deteriorating effects by upregulating a battery of protective genes.

With the blastocyst stage, embryos become dependent on aerobic metabolism and acquire the capacity to utilize glucose [48]. The expression pattern of the facilitative glucose transporter family has recently been studied in IVP bovine embryos [49]. In the present study, the Glut-1 expression pattern was nearly identical in embryos from the different origins, suggesting that this isoform may not play a dominant role in embryonic glucose uptake from the environment. The observation that Glut-2 was not transcribed in preimplantation bovine embryos up to the blastocyst stage irrespective of the production system supports recent findings [49]. Glut-3 serves as a high-affinity transporter to ensure the glucose supply from the external environment. In mouse embryos, Glut-3 plays a crucial role in the uptake of maternal glucose by the blastocyst [50]. Glut-3 transcription was upregulated in all in vitro-derived embryos and those cultured in the ligated sheep oviduct compared with embros from in vivo production, indicating that this isoform could serve as a sensitive marker for a suboptimal embryo environment. Upregulation of the insulin-dependent Glut-4 in IVP embryos might be attributed to insulin contaminations of the serum and BSA supplements. In the mouse blastocyst, glucose transport is responsive to IGFI and insulin, and the effects are mediated via the IGFI-R [51].

Upregulation was also determined for bFGF and IGFI-R transcripts in IVP embryos. The potent mitogen bFGF promotes early bovine embryonic development in vitro during the fourth cell cycle synergistically with transforming growth factor ß [52], and FGF-4 mRNA expression has been considered a suitable candidate for evaluating the quality of nuclear transfer-derived bovine embryos [53]. In human tumor cells, bFGF expression was induced by metabolic stress [54]. Whether bFGF expression in bovine embryos is regulated by a similar mechanism is unknown.

Phenotypes similar to those described for LOS in cattle and sheep have been found in mice and humans and were related to altered expression of imprinted genes [55]. Genomic imprinting has been demonstrated in >40 genes in the mouse, in a few human genes, but in only 1 gene from farm animals [56]. Alterations in the expression of imprinted genes such as IGFI-R may be critically involved in ovine fetal overgrowth after embryo culture [16]. Consistent with recent findings for nuclear transfer-derived embryos [17], in the present study the relative abundance of IGFII-R transcripts was not affected by the different production systems. However, timing and magnitude of expression of genes from the IGF family in bovine IVP blastocysts were altered by the employed culture system [57]. Aberrant expression of several imprinted genes was obviously not sufficient to induce a pathological phenotype in cloned mice derived from embryonic stem cells [58].

Another factor involved in the regulation of gene expression is chromatin structure, which undergoes maturation prior to the first step of differentiation at the blastocyst stage [59, 60]. Histones are key structural components of chromatin. In mice, at the time of genomic activation the nuclear periphery becomes enriched with H4 [59, 61]. In the present study, H4 expression was stable in embryos derived from different origins. In a similar finding, the expression of the transcription factor Oct-4 was not affected by the mode of embryo production [62]. The compensatory mechanism employed by the early embryo to cope with suboptimal culture conditions preferentially affects genes for which expression can vary within a certain range without being detrimental to preimplantation development [12]. A microarray panel of selected cDNAs of genes sensitive to changes in early embryonic environment would significantly extend the possibilities of unraveling differences in gene expression patterns. We have recently developed the first microarray for single embryos [63] and are currently exploring this exciting possibility.

Results of this study demonstrate that in vitro culture of bovine embryos in the presence of high levels of proteins alters the kinetics of embryo development, the expression of developmentally important genes, and ultimately the weight of the calves at birth compared with embryos produced in vivo or by temporary in vivo culture. The cellular and molecular parameters analyzed in this study could be used as indicators of accelerated development and may be considered early features of LOS. Results of this study provide a solid basis for further exploration of the causative mechanism of LOS.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Lorraine Young (Roslin Institute, Edinburgh, U.K.) for providing the batch of serum used in the present study.


   FOOTNOTES
 
First decision: 28 February 2002.

1 This study was supported by a grant from the European Union (FAIR-CT98-4339). Back

2 Correspondence: Heiner Niemann, Institut für Tierzucht und Tierverhalten, 31535 Neustadt, Germany. FAX: 49 0 5034 871 101; niemann{at}tzv.fal.de. Reprint requests: Giovanna Lazzari, Laboratorio di Tecnologie d. Riproduzione, Cremona, Italy. FAX: 39 0372 436 133;lazzaltr{at}tin.it Back

Accepted: April 2, 2002.

Received: February 12, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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