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-beta). 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-beta. 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 o...

Using reverse transcriptase-amplified fragment length polymorphism (RT-AFLP) analysis of differential mRNA expression and semiquantitative reverse transcriptase-polymerase chain reaction, we compared mRNA expression in bovine blastocysts from 4 sources, known to differ in quality in terms of their ability to withstand cryopreservation: 1) in vitro culture in synthetic oviduct fluid of in vitro-matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with granulosa cells (coculture) of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, artificial insemination, and nonsurgical embryo recovery. Total mRNA was isolated from pools of blastocysts and reverse transcription was performed. Triplicate reactions from each sample were displayed, and only consistent banding variations were recorded. Using AFLP-differential display assay, we found that cDNA banding patterns are highly conserved between the 4 groups of blastocysts studied; however, there was a difference of 7% in bands either missing or expressed across the groups. Fifty bands were reamplified, and a sequence comparison search revealed similarity of 14 isolated fragments to ribosomal and mitochondrial genes, 16 matched to described cDNA, and 20 corresponded to unknown sequences that may represent novel genes. The study of 7 differentially expressed mRNAs known to be involved in developmental process in the embryo suggests roles for apoptosis, oxidative stress, gap junctions, and differentiation in the determination of embryo quality. The aberrant transcription patterns detected in in vitro-produced bovine embryos compared with those produced in vivo may explain their reduced quality in terms of viability after cryopreservation.

The objective of this study was to examine the time during the postfertilization period that gene expression patterns in in vitro-cultured bovine embryos diverge from those of their in vivo-cultured counterparts. Presumptive bovine zygotes were produced by in vitro maturation and fertilization of immature oocytes collected from the ovaries of slaughtered animals. Approximately 20 h post insemination (hpi), zygotes were denuded and randomly divided into two groups for culture either in vitro, in synthetic oviduct fluid medium, or in vivo, in the ewe oviduct. Embryos were recovered from both systems at approximately 30 hpi (2-cell), 2 (4-cell), 3 (8-cell), 4 (16-cell), 5 (early morula), 6 (compact morula), or 7 (blastocyst) days post insemination. On recovery, they were examined for stage of development and snap frozen in liquid nitrogen for the analysis of transcript abundance using real-time polymerase chain reaction. The transcripts studied were glucose transporter 5, sarcosine oxidase, mitochondrial Mn-superoxide dismutase, connexin 43, interferon tau, insulin-like growth factor II, apoptosis regulator box-alpha and insulin-like growth factor-I receptor, most of which are known from our previous work to differ in terms of transcript abundance in blastocysts derived from culture in vitro or in vivo. The results demonstrate that the relative abundance of the transcripts studied varies throughout the preimplantation period and is strongly influenced by the culture environment. In addition, the data demonstrate that changes in transcript abundance in blastocyst stage embryos are in many cases a consequence of perturbed transcription earlier in development. Depending on the transcript, these differences may be evident by as little as 10 h of initiation of culture. Such information has implications not only for basic biology but also for human assisted reproduction in which there is a move toward culturing embryos to the blastocyst stage, necessitating prolonged culture in vitro under potentially deleterious conditions.

Evidence exists to suggest detrimental effects of heat stress on male fertility. This study was designed to assess the effects of scrotal heat stress on mature and developing sperm in a mouse model. After receiving shock heat treatment (42 degrees C for 30 min), mature spermatozoa were recovered from the epididymis hours (6) or Days (7, 14, 21, 28, 60) later, to determine the variables: number of spermatozoa, sperm viability, motility and progressive motility, sperm DNA integrity as established by the TUNEL method, embryo implantation rate, and sex ratio of the fetuses conceived using the heat-exposed spermatozoa. Our results indicate that transient mild heat treatment does not affect in the same way the different types of male germ cells. Spermatocytes present within the testis at the time of heat stress resulted into a lower concentration of spermatozoa with reduced viability and low motility. Even though, DNA integrity of spermatozoa resulting from spermatocytes was also compromised by heat stress, the higher degree of DNA damage was found among spermatozoa resulting from spermatids present within the testis at the time of heat stress. At last, heat shock effect on spermatozoa present in the epididymis at the time of thermal stress resulted into a sex ratio distortion. These findings point to a higher sensitivity of spermatocytes to heat exposure and also suggest a different response of X and Y chromosome-bearing spermatozoa to heat stress that warrants further investigation.

Recent data have demonstrated that fast-cleaving embryos produced in vitro are more likely to develop to blastocyst stage, and that the postfertilization culture system used impacts considerably on the mRNA expression and quality of blastocysts produced. The present study is the first to investigate the relationship between the developmental speed of embryos produced in vivo or in vitro and the temporal transcription pattern. Genes related to important preimplantation events are monitored during the first 4 days of embryo development in embryos with fast or slow development. The set of genes analyzed in the present study characterizes several important physiological processes including: transport and metabolism of fructose (Glut-5), stress (SOX), mitochondrial activity and detoxification of reactive oxygen species (MnSOD), cell communication (Cx43), maternal recognition of pregnancy (IFN-tau), imprinting (IGF-II), apoptosis (Bax), growth factor binding and metabolism (IGF-IR), and oxidative stress (G6PD). Using real time PCR, we have found that for all the genes analyzed there are differences in mRNA expression between embryos with fast and slow developmental speed produced both in vitro and in vivo. Frequently, genes that may be stress induced such as SOX, MnSOD, BAX, IFtau, and G6PD were highly transcribed in in vitro produced embryos and in embryos with slow developmental speed. On the other side, transcripts from genes related with metabolism, growth, and differentiation (Glut-5, Cx 43, IGF-II, and IGF-IR) were detected in higher amounts in in vivo produced embryos and in embryos with fast developmental speed. Moreover, it is interesting to stand out that for some genetic markers (such as SOX and G6PD) there are in vivo and in vitro differences that can be observed even before materno-zygotic transition, which probably reflects a differential mRNA degradation. These transcription patterns reflects the embryonic response to the adverse in vitro culture conditions, and connect the low quality of embryos which slow developmental speed produced in vivo and in vitro, with the mRNA expression pattern of some embryonic genes.

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Recent studies comparing bovine oocyte maturation, fertilization and embryo culture in vivo and in vitro have demonstrated that the origin of the oocyte is the main factor affecting blastocyst yield, while the post-fertilization culture environment is critical in determining blastocyst quality, measured in terms of cryotolerance and relative transcript abundance, irrespective of the origin of the oocyte. Production of embryos in vitro, particularly when using an extended period of in-vitro culture, may predispose the embryo to phenomena such as the large offspring syndrome, which is likely to alter gene expression, particularly of imprinted genes. It is clear now that the post-fertilization culture environment has a profound effect on the relative abundance of gene transcripts within the embryo, and culture under suboptimal conditions for as little as 1 day can lead to perturbations in the pattern of expression.

During the preimplantation period, in vitro cultured males have a higher metabolic rate, different gene expression, and grow faster than females. It has been suggested that under some stress conditions male embryos are more vulnerable than females; however, the biological fragility of male embryos is little understood. Since many forms of stress result in the overproduction of cellular reactive oxygen species (ROS), we addressed the hypothesis that the connection between female advantage during early developmental stages and heat stress involves ROS and differential gene expression of G6PD, an X-linked gene related to oxidative stress. We have found that after compaction, female heat-stressed embryos have less relative amounts of H2O2 than males, and female embryos survive better than males under in vivo or in vitro heat stress situations. In addition, in vitro produced female embryos grow slower than male embryos, have differential mRNA transcription of G6PD and also of some genes situated on autosomal-chromosomes (Sox, Bax, and Oct-4). Moreover, by inhibiting G6PD, all differences generated by oxidative stress between male and female embryos disappear. For the first time, we provide an experimental demonstration of a mechanism that explains why following exposure to heat stress-induced ROS, female preimplantation embryos are more resistant than males.