In mammals, the study of gene expression in the preimplantation embryo has been difficult because the standard procedures used to quantify mRNA generally require large amounts of starting material. The development of protocols using different quantitative strategies generally involving the polymerase chain reaction (PCR) has provided new tools for exploration of gene expression in preimplantation embryos. However, the use of an internal standard, often referred as a housekeeping gene, is essential to normalize the mRNA levels. RNA levels of eight housekeeping genes were quantified using real time PCR throughout the preimplantation period of the bovine embryo to find the most suitable gene to be used as standard. Histone H2a was the best internal standard because the transcript levels were constant across the preimplantation period. Linear amplification of antisense RNA using the T7 promotor for in vitro transcription of the entire RNA pool was evaluated as a suitable way to preamplify the starting material prior to quantification and was effective in providing accurate RNA abundance profiles throughout the preimplantation period. However, the amplification appears to be template dependent because the amplification factors were higher for some genes.

Maternal-zygotic transition (MZT) is a complex phenomenon characterized by the initiation of transcription in the embryo and the replacement of maternal mRNA with embryonic mRNA. In order for this to occur, transcriptional activation requires various factors and conditions. Our hypothesis is that the lack of transcription in the bovine pre-MZT embryo is due, in part, to an incomplete or dormant transcriptional apparatus. Therefore, in accordance with this hypothesis, functioning transcriptional mechanisms should appear in the eight-cell bovine embryo to facilitate embryonic transcription during the MZT. With this in mind, we examined the presence of selected transcription factors during preimplantation embryo development to establish how their transcript levels change in bovine pre-MZT embryos. To achieve this goal, real-time reverse transcription-polymerase chain reaction was used to quantify the mRNA level of several different transcription factors (YY1, HMGA1, RY-1, P300, CREB, YAP65, HMGN1, HMGB1, NFAR, OCT-4, TEAD2, ATF-1, HMGN2, MSY2, and TBP) in germinal vesicle (GV) and metaphase II (MII) bovine oocytes and in two-, four-, eight-cell, and blastocyst stage embryos produced in vitro. Our results demonstrate that all genes examined can be grouped into five different categories according to their mRNA expression patterns at the developmental stages observed. To summarize, all transcription factors studied were present in pre-MZT embryos and the expression pattern of many of them suggest a potential role in MZT.

Upon their transit through the female genital tract, bovine spermatozoa bind to oviduct epithelial cells, where they are maintained alive for long periods of time until fertilization. Although carbohydrate components of the oviduct epithelial cell membrane are involved in these sperm/oviduct interactions, no protein candidate has been identified to play this role. To identify the oviduct factors involved in their survival, sperm cells were preincubated for 30 min with apical membranes isolated from oviduct epithelial cells, washed extensively, and further incubated for up to 12 h in the absence of apical membranes. During this incubation, sperm viability, motility, and acrosomal integrity were improved compared with cells preincubated in the absence of apical membranes. This suggests that, during the 30-min preincubation with apical membrane extracts, either an oviductal factor triggered intracellular events resulting in positive effects on spermatozoa or that such a factor strongly attached to sperm cells to promote a positive action. Similarly, spermatozoa were incubated with apical membranes isolated from oviduct epithelial cells labeled with [35S]-methionine and, upon extensive washes, proteins were separated by two-dimensional (2-D) gel electrophoresis to identify the factors suspected to have beneficial effects on spermatozoa. The six major proteins, according to their signal intensity on the autoradiographic film, were extracted from a 2-D gel of oviduct epithelial cell proteins run in parallel and processed for N-terminal sequencing of the first 15 amino acids. Of these, one was identical to heat shock protein 60 (HSP60) and one to the glucose-regulated protein 78 (GRP78). Their identities and association with spermatozoa were confirmed using an antibody directed against these proteins. This paper reports the localization of both GRP78 and HSP60 on the luminal/apical surface of oviduct epithelial cells, their binding to spermatozoa, and the presence of endogenous HSP60 in the sperm midpiece.

Mammalian oocytes are very unique cells with an unlimited developmental potential. These totipotent cells are able to remove existing gene-expression patterns and to impose new ones. However, genome reprogramming is still a mystery. Posttranslational modifications by acetylation of the N-termini portion of histones composing the nucleosome are involved in genome reprogramming. These modifications alter the higher-order chromatin structure to render the DNA accessible to the regulatory and transcriptional machinery. In the present study, we have investigated, to our knowledge for the first time, precise expression patterns of seven genes involved in chromatin structure throughout bovine embryo development. Oocytes harvested from bovine ovaries were used for in vitro production of germinal vesicle oocytes, metaphase II oocytes, 2- and 8-cell embryos, and blastocysts. Total RNA was extracted from pools (triplicates) of 20 oocytes or from embryos of each developmental stage. By means of quantitative reverse transcription-polymerase chain reaction using SYBR Green to detect double-stranded DNA, mRNA expression profiles for histone deacetylases (HDAC1, HDAC2, HDAC3, and HDAC7), histone acetyltransferases (GCN5 and HAT1), and histone H2A were established. Transcripts for all genes were detected at all stages from the oocyte to the blastocyst. The HDAC1, HDAC2 (class I HDAC), and HAT1 (type B HAT) revealed similar expression profiles. The HDAC3 (class I HDAC) tends to have an expression profile similar to those of HDAC1, HDAC2, and HAT1, whereas the HDAC7 (class II HDAC) and GCN5 (type A HAT) profiles were different from those three. These results indicate variable levels of histone deacetylases and histone acetyltransferases throughout embryonic development and may indicate the ones that are involved in somatic remodeling.

Only competent oocytes are able to undergo complete maturation and normal embryonic development. Therefore, the identification of genes that are differentially expressed in competent oocytes would contribute to our understanding of the factors controlling competency. It is well known that time of cleavage after insemination in vitro is highly correlated with embryonic developmental potential and this can be used to distinguish between oocytes of different quality. The main objective of this study was to identify genes associated with competency and rapid cleavage. We examined the expression of 16 candidate genes (IDH, YEAF Cathepsin B, RAD50, TCP1 NCOR1, HUEL, STK6, ZNF403, AOP2, EEF1A1, Hsp90, Hsp40, AKR1B1, PGRMC1, and DMRT2) in early and late cleaving embryos, by real time PCR. These transcripts were derived from previous study in our laboratory using cDNA coming from a suppressive subtraction hybridization (SSH) between early cleaving versus late cleaving embryos spotted on a microarray slide. Of the 16 genes evaluated, 3 (IDH, YEAF, and H2A) showed statistical difference (P < 0.05) between early and late cleaving embryos. However, some genes such as Cathepsin B (P = 0.0677), RAD50 (P = 0.0899), and TCP1 (P = 0.0824) tended to show higher expression in the early cleaving than in the late cleaving embryo. In conclusion, we have identified three genes (YEAF, IDH, H2A) that were differentially expressed in the early cleaving embryos, and their expression can be associated with greater developmental competence.

New insights into the early development of large mammals are becoming available through the measurement of differential mRNA levels in oocytes and preimplantation embryos. These advances in knowledge are rapidly picking up in pace, mainly owing to the advantages brought by new molecular biology approaches being developed. The possibility of amplifying the starting material and therefore making measurements in single embryo units is now feasible. With these tools, the evaluation of variations in gene expression patterns during the preimplantation period or the impact of culture on mRNA levels is now possible. However, it is important to keep in mind that these methods still have limitations associated with sample preparation or the use of the appropriate controls. Even proper methods of analysis are very important to achieve the full benefit of the application of these tools. The present paper describes some of the potential, as well as limitations, of mRNA level analysis in early embryos, especially for microarray analysis. We have generated a bovine cDNA array (>2000 clones) that contains expressed sequence tags (ESTs) collected from various preimplantation development stages. Using this chip, we have initiated the characterisation of global mRNA level patterns of several key developmental stages from the immature oocyte to the blastocyst stage. As expected, the hybridisation results indicate very different expression profiles involving hundreds of genes when comparing oocyte and blastocyst samples to a reference mRNA sample made from a pool of ESTs from pooled somatic tissues. Although this array is still in its preliminary stage and the EST bank has not been processed to contain only unigenes, it is already a very useful tool for discovering candidate genes that may play important roles during early embryonic life.

Protein synthesis from maternal mRNA is needed to sustain oocyte maturation and embryo development prior to the maternal-embryonic transition (MET). Therefore, proteins that are expressed throughout this time are important and may be considered as maternal housekeeping proteins (MHKP). Our objectives were first, identify the translated protein patterns of bovine embryo development and secondly, determine the MHKP. Proteins synthesized during oocyte maturation and embryo development (2, 4 and 8-cell stages) were labeled using [S(35)]-Met and [S(35)]-Cys, and visualized by 2-DE. Embryos were cultured with alpha-amanitine to inhibit new transcription. Only 46 proteins were present throughout all stages. Ten spots were identified by MALDI-TOF and MS/MS: HSC71; HSP70; CypA; UCH-L1; GSTM5; Cct5; E-FABP; 2,3-BPGM, ubiquitin-conjugating enzyme E2D3; and beta-actin/gamma-actin. A new method called in silico protein identification confirmation was developed using EST databases. This method is a promising approach for use in rare tissue or from species with an incomplete protein database. This study has revealed that the translated protein patterns show a transition that brings the embryo to the MET. The needs in translated proteins between oocyte maturation and embryo development are different. In summary, this study represents the bases for future proteomics studies on bovine oocytes and embryos.

To investigate protein synthesis during bovine oocyte maturation in vitro, oocytes were put in culture with 35S-methionine for 4 hr periods from time zero to 28 hr. Pools of 10 oocytes were then prepared for two-dimensional gel electrophoresis (2-DE). For each time interval, three gels were obtained, digitalized, and analyzed to detect proteins. Then, the gel containing the most proteins was chosen as the reference gel and compared with the others. An averaged gel was created with proteins present in at least two gels of the three. Our results indicate that the rate of protein synthesis is higher at the beginning of maturation until the appearance of metaphase I (MI, 8-12 hr) and then it decreases and stays relatively constant. Percentages of initial proteins (0-4 hr) and remaining present during the progression decrease progressively from 100% to 53%. In contrast, when we compare proteins synthesized from the 4 to 8 hr period with proteins from the 8 to 12 or the 12 to 16 hr intervals, percentages of overall protein matching are stable with values of 81 and 79%, respectively. Comparison of proteins from 20 to 24 hr with proteins from 16 to 20 or 24 to 28 hr intervals also gives stable percentages of overall protein matching with values of 83 and 84%, respectively. Furthermore, a higher number of new proteins is observed at 4-8 hr (n=130) and 16-20 hr (n=136) of maturation. Thus, three major patterns of protein synthesis were observed during bovine oocyte maturation in vitro: one at the beginning of maturation (0-4 hr), another one in the middle (4-16 hr), and the last one after the completion of MI stage (16-28 hr).