As shown in Table 1, ultrasound guided ovum pick up (OPU) of sixty cyclic heifers showed that ovaries of both growth and dominance phases had greater number of small follicles (3–5 mm) than medium and large follicles. Moreover, a higher number of small and medium follicles (n = 333) has been found at growth phase compared to dominance phase (n = 262). Consequently, the average number of oocytes collected per cow was greater during growth phase (7.7 ± 3.5) than in the dominance phase (4.9 ± 2.1).

All differentially regulated transcripts were functionally classified based on the criteria of Gene Ontology Consortium classifications 29. The resulting data were supplemented with additional information from various tools available in National Center for Biotechnology Information 30. Accordingly, the differentially regulated transcripts represent genes with known function (36/51), genes with unknown function (6/51) and novel transcripts (9/51) (Fig. 3). Regarding the molecular function, transcripts with known function showed to be involved in protein biosynthesis (18%), transcription (10%), cytoskeleton (8%), cell cycle (8%), NADH dehydrogenase activity (4%), calcium ion binding (4%), nucleotide binding (4%) and other functions (10%). Oocytes recovered at growth phase (Tab. 2) were enriched with genes regulating protein biosynthesis (RPLP0, RPL8, RPL24, ARL6IP, RpS14, RpS15, RpS4x and RPS3A), translation elongation (EF1A), ATP binding (ATP5A1), NADH dehydrogenase activity (FL396 and FL405), cytoskeleton (Actin, beta-Actin, H2AZ and KRT8), calcium ion binding (S100A10 and ANXA2), signal transduction (G-beta like protein), (.)inorganic diphosphatase (PP) and thiol-disulfide exchange intermediate (TXN). On the other hand, oocytes recovered at dominance phase (Tab. 3) were encoded transcripts controlling transcription (MSX1, PTTG1, FANK1 and PWP1), cell cycle (CCNB1, CKS2, UBE2D3 and CDC31), aldehyde reductase activity (AKR1B1), nucleotide binding (TUBA6 and K-ALPHA-1), growth factor (BMP15) and fertilization (ZP4).

Real-time PCR analysis using independent oocyte samples was conducted to validate the array results of 10 differentially regulated transcripts. No differences in relative abundance of the internal control gene (GAPDH) were observed in all samples. Based on this, the quantitative real-time PCR has confirmed the relative abundance of 8 transcripts (80%) to be in agreement with microarray results (Fig. 4A, B, C). The mRNA relative abundance of mRNAs for ANAXA2, S100A10, RPL24, and PP was higher (P < 0.05) in growth phase compared to dominance phase oocytes. Greater transcript abundance for MSX1 and BMB15 was observed in dominance phase versus growth phase oocytes. However, differences in transcript abundance of FL396 and PTTG1 between the two oocyte groups were not statistically significant. The array results of CKS2 and CCNB1 could not be confirmed by real-time quantitative PCR (Fig. 4A).

To avoid any biasness in the number of cumulus cells used as an input for mRNA isolation and subsequent real-time PCR, cumulus cells were derived from equal number of cumulus oocyte complexes (COCs) with similar morphological appearance. After mRNA isolation and subsequent cDNA synthesis, the relative abundance of GAPDH gene was tested and showed no variation between these samples. Two transcripts (MSX1 and FL396) were highly abundant (P < 0.05) in cumulus cells of dominance phase than that of growth phase (Fig. 5). On the other hand, RPL24 and CKS2 transcripts were found to be more abundant in cumulus cells of growth phase compared to that of dominance phase. The relative abundance of mRNA for S100A10 was nearly the same in cumulus cells of both phases.

Immature oocytes screened for developmental competence based on BCB staining were used for validation of the expression profile of five transcripts (BMP15, RPL24, PP, MSX1 and PTTG1). The cDNA synthesized from BCB⁺ (stained blue) and BCB^- (stained colourless) oocyte samples were quantified using real-time PCR. After confirming that there were no significant differences in the relative abundance of GAPDH between the samples, all transcripts were quantified using independent real-time PCR runs. The relative abundance for MSX1 and PTTG1 were higher (P < 0.05) in BCB^- oocytes than BCB⁺ ones (Fig. 6). The BMP15 was also more abundant in BCB^- than BCB⁺ oocytes but differences were not significant. On the other hand, greater mRNA abundance (P < 0.05) for RPL24 and PP has been observed in BCB⁺ than BCB^- oocytes (Fig. 6).

Immunofluorescent labelling of MSX1 gene in ovarian samples collected at the time of estrus (day 0), ovulation (day 1), growth phase (day 3) and dominance phase (day 7) showed the presence of this protein in all stages of follicular development under investigation. More specifically, this protein was found to be more localized in the oocyte cytoplasm than the enclosed cumulus cells (Fig. 7d, h, p) or other cellular layers of the growing follicle (Fig. 7b, f, n) at all stages of follicular development except at growth phase (Fig. 7j, l). Furthermore, MSX1 protein was found to be dispersed in the cytoplasm of immature and matured oocytes and early zygote (Fig. 8a, b, c) but tends to be localized around the nucleus at advanced zygote, 2-cell, 4-cell and 8-cell embryos (Fig. 8d, e, f, g). Comparative analysis of protein signals between oocytes showed that green fluorescence signals were reduced after maturation (Fig. 8b). Moreover, in situ hybridization experiment showed that MSX1 mRNA was localized in the oocytes, cumulus cells and follicular wall throughout all stages of follicular turnover (Fig. 9A, B, C, D, E, F, G, H).

The experimental protocol was carried out according to the rules and regulations of the German law of animal protection.

Sixty Simmental cyclic heifers 24 to 30 months old, were selected based on general clinical examination and normal ovarian cyclicity as determined by ultrasound scanning. The animals were housed as one group and were fed grass silage ad libitum. Estrus and subsequent ovulation were synchronized by two administrations of prostaglandin PGF2α (Estrumate, 2 ml i.m, Fa. Essex, Germany) at 11 days interval followed by GnRH injection (Receptal, 2500 IU i.m, Intervet, Unterschleissheim, Germany) on the day of estrus onset. Estrus was detected after two days of the last prostaglandin PGF2α treatment. Common signs of estrus were monitored by visual observation followed by careful palpation of ovaries for confirmation.

OPU was performed at two different phases of follicular development during the first follicular wave using a total of 60 heifers at growth phase (day 3 after estrus, n = 30) and dominance phase (day 7 after estrus, n = 30) for two sessions of OPU in each phase. The heifers were restrained in a chute, and given 5 ml of epidural anesthesia (procaine hydrochloride 2%, Selectavet, Munich, Germany). Using transvaginal ultrasound scanner (Pie Medical 400 Vet, Maastricht, Netherlands) with a 7.5 MHz sector probe the follicles were visualized on the monitor and counted. The follicles were classified according to their size into three categories: small (3–5 mm), medium (6–8 mm) and large follicles (≥ 9 mm). A 50-cm-long 18-gauge needle was passed through a needle guide along the polyethylene housing of the transducer, and carried into the fornix vagina. After fixing the ovary against the transducer, the needle was advanced to puncture the vaginal wall to enter the ovarian follicle. The needle was attached via Teflon tubing to a 50-ml Falcon tube and vacuum pressure provided with a regulated vacuum pump (K-MAR-5000B; William Cook Europe) and adjusted to create a flow rate of 16–20 ml/min. The follicular content of each heifer was aspirated individually into modified Parker maturation medium (MPMM) supplemented with 15% oestrus cow serum (OCS), 0.5 mM L-glutamine, 0.2 mM pyruvate, 50 μg/ml gentamycin sulphate, 10 μl/ml FSH (Folltropin, Vetrepharm, Canada) and kept at 39°C in thermos. The follicular fluid contents were poured into a square grid dish to facilitate finding of oocytes under a stereomicroscope. The collected oocytes were kept in the same maturation medium used for collection before being separated from the surrounding cumulus cells. Oocytes from each follicular size category and phase were handled and frozen separately.

In this study, only COCs from the small follicles of the growth and dominance phases were used for the analysis of transcript abundance. Cumulus cells were removed from the oocytes of each phase mechanically in maturation medium supplemented with hyaluronidase 1 mg/ml (Sigma). Separation of cumulus cells was carefully checked under a stereomicroscope. Cumulus free oocytes and the corresponding cumulus cells of each phase were washed two times in PBS (Sigma) and snap frozen separately in cryo-tubes containing 20 μl of lysis buffer [0.8 % IGEPAL (Sigma), 40 U/μl RNasin (Promega Madison WI, USA), 5 mM dithiothreitol (DTT) (Promega Madison WI, USA)]. Finally, samples were stored at -80°C until RNA extraction.

Messenger RNA isolation of oocytes and cumulus cells was performed at four different points during the whole experiment. 1) A total of six pools, each containing 20 oocytes from growth and dominance phases of follicular development, were used for array analysis after amplification, 2) A total of six pools, each containing 20 oocytes from growth and dominance phases, were used for real-time validation of array results, 3) The cumulus cells detached from the oocytes at the growth and dominance phases were also used for mRNA isolation and subsequent expression analysis of selected transcripts, 4) A total of 8 pools of oocytes, each containing 50 oocytes from BCB⁺ and BCB^- categories were used to validate the expression profile of selected transcripts in oocytes with different developmental competences. In all cases, mRNA isolation was performed using Dynabead oligo (dT)25 (Dynal Biotech, Oslo, Norway) according to manufacturer's instructions. Briefly, oocytes or cumulus cells in lysis buffer were mixed with 40 μl binding buffer [20 mM Tris HCl with pH 7.5, 1 M LiCl, 2 mM EDTA with pH 8.0] and incubated at 70°C for 5 min to obtain complete lysis of and to release RNA. Ten microlitres of oligo (dT)25 attached magnetic bead suspension were added to the samples, and incubated at room temperature for 30 min. The hybridized mRNA and magnetic beads were washed three times using washing buffer (10 mM Tris HCL with pH 7.5, 0.15 mM LiCl, 1 mM EDTA with pH 8.0). For each sample, cDNA synthesis has been performed using oligo (dT)23 primer and superscript reverse transcriptase II (Invitrogen, Karlsruhe, Germany) except for samples used in array analysis where the reverse transcription was performed using T7 promotor attached oligo d(T)21 primer.

First and second-strand cDNA synthesis were carried out as described in our previous study 25. Ten amplification cycles were used during second strand synthesis as this showed less bias on the representativeness of the original mRNA population after in vitro transcription 57. The cDNA was purified and used for in vitro transcription using AmpliScribe T7 transcription kit (Epicentre technologies, Oldendorf, Germany) according to manufacturer's instructions. Then the amplified RNA (aRNA) was purified using RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Finally, the aRNA was eluted in 30 μl RNase free water from which 8 μl was taken to estimate the yield, purity of aRNA by gel electrophoresis and UV absorbance reading at A260/280 using Ultrospec™ 2100 pro UV/Visible Spectrophotometer (Amersham Bioscience, Freiburg, Germany).

MIAME (Minimum Information About Microarray Experiments) guidelines were adhered to the experimental design. Two independent labelling reactions were carried out per aRNA sample pertinent to each biological replicate for dye-swap hybridizations. Accordingly, 3 μg of aRNA from each oocyte pool representing each follicular phase of development (growth or dominance) was used as template in reverse transcription reactions incorporating amino-modified dUTPs into the cDNA using the CyScribe Post-Labelling Kit (Amersham Biosciences, Freiburg, Germany) as described previously 2557. The aminoallyl labelled cDNA samples were purified using CyScribe™ GFX™ Purification kit (Amersham Biosciences) after adding 10 μl of 2 M HEPES. The purified Aminoallyl labelled cDNA was then eluted in 60 μl 0.1 M sodium bicarbonate. The cDNA samples from each phase were differentially labelled indirectly using N-hydroxysuccinate-derived Cy3 and Cy5 dyes and incubated for 1.5 hrs at room temperature in dark. At the end of incubation, non reacting dyes were quenched by adding 15 μl of 4 M hydroxylamine solution (Sigma) and incubated for 15 min at room temperature in dark. To avoid variation due to dye coupling, aRNA samples from the same follicular phase were labelled reversibly either with Cy3 or Cy5 for dye swaps hybridizations. The reaction was then purified with CyScribe™ GFX™ Purification kit (Amersham Biosciences, Freiburg, Germany). Samples were finally eluted in 60 μl elution buffer.

Pre-hybridization of the slides was performed by placing the array slides into a corning GAPS II slide container as described in El-Sayed et al. 25. Hybridization and post-hybridization washes were carried out as previously described elsewhere 58 with slight modifications. Samples that were going to be hybridized on specific array were mixed and dried in speedvac centrifuge (GMI, Inc. Minnesota, USA) then the pellet was re-suspended in pre-warmed (42°C) formamid based hybridization buffer [15 μl hybridization buffer (Amersham Bioscience, Freiburg, Germany), 30μl 100% Formamide, and 15μl DEPC water]. Yeast tRNA (4 mg/ml) and 2.5 μl of Cot-human DNA (1 mg/ml) (Invitrogen, Karlsruhe, Germany) were added in a volume of 2.5 μl each to avoid non specific hybridisation. The pellet was denatured at 95°C for 5 min, centrifuged briefly and hybridized to the array. The arrays were covered with glass cover slips (ROTH, Karlsruhe, Germany) and fixed in the hybridisation cassettes (TeleChem International, Inc, CA, USA.) before incubation in a hybridisation chamber (GFL, Dülmen, Germany) at 42°C for 16–20 hrs. After hybridization, slides were washed twice with 2× SSC-0.1% SDS buffer for 5 min at 42°C, then once with 1× SSC, 0.2× SSC and 0.1× SSC for 5 min each at room temperature. Finally, the slides were rinsed in RNA free water then in isopropanol for 1 min each and centrifuged at ≥ 2000 rpm for 2 min.

Ready made bovine cDNA array (BlueChip) 59 provided by Centre de Recherche en Biologie de la Reproduction was used in this study. The glass slide contains 4928 spots divided into two sub-arrays. Each sub-array was composed of 2304 ESTs randomly selected clones obtained from four different subtraction suppressive hybridizations (SSH) made with bovine embryos and tissues (First SSH: GV oocytes subtracted from somatic tissues, second SSH: GV oocytes subtracted from day-8 blastocysts, third SSH: day-8 blastocysts subtracted from GV oocytes and fourth SSH: day-8 blastocysts subtracted from somatic tissues). All the clones were spotted in each sub-array for a total of four replicates per slide. Eleven more samples namely vide (32 spots), alien1 (8 spots), alien2 (8 spots), GFP (4 spots), GFP1 (4 spots), GFP1/2 (4 spots), GFP1/4 (4 spots), GFP1/8 (4 spots), GFP 1/16 (4 spots) and H20/DMSO (50 spots) were spotted to be used as negative controls for determination of hybridisation background during the statistical analysis. Housekeeping genes including tubulin (8 spots), ubiquitin (8 spots), β-actin (6 spots) and actin (8 spots) were also added as positive controls.

The slides were scanned using Axon GenePix 4000B scanner (Axon Instruments, Foster City, CA, USA). The GenePix^® Pro 4.0 software (Axon Instruments, CA, USA) was used to process the images, to find spots, to integrate robot-spotting files and finally to create reports of spot intensity data. The LOWESS normalization of microarray data was performed using GProcessor 2.0a software 60. The normalised data were used to calculate intensity ratios of all replicates and to obtain one value per clone. Ratios were finally log₂ transformed and submitted to SAM analysis. Microarray data analysis was performed using SAM, free software developed at Stanford University 61. To get truly differentially expressed genes, the FDR was set at 10% and P-value of < 0.05. Hierarchical clustering and heatmap of log₂-transformed data for up and down regulated genes were generated using PermutMatrix (version 1.8.2) available at 62. In addition, average linkage clustering algorithm method was employed 63. Genes expressed equally in both samples were not included in the hierarchical clustering.

To validate the results obtained from microarray analysis, 10 candidate transcripts were selected for further analysis using real-time quantitative PCR (Tab. 4). Five of these 10 candidates were further quantified in the corresponding cumulus cells from the oocytes of growth and dominance phases of follicular development. Furthermore, five transcripts were subjected to real-time quantitative PCR using cDNA synthesized form BCB⁺ and BCB^- oocytes. In all cases, quantitative analysis of cDNA samples was performed as described previously 25 in comparison with the bovine GAPDH gene (endogenous control), and was run in separate wells using ABI PRISM^® 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA). Finally quantitative analysis was done using the relative standard curve method and results were reported as the relative expression or fold change as compared to the calibrator after normalization of the transcript level to the endogenous control 64.

Eight Simmental cyclic heifers, two of each at day of estrus (day 0), day of ovulation (day 1), growth phase (day 3) and dominance phase (day 7) were slaughtered to obtain ovaries for MSX1 protein localization using immunohistochemistery. Ovaries were embedded in Tissue-Tek (Sakura Finetek Europe, Zoeterwoude, Netherlands) and snap-frozen in liquid nitrogen and stored at -80°C until sectioning. Serial sections of 5μm were cut at -20°C using rapid sectioning cryostat (Leica microsystem Nussloch GmbH, Heidelberger, Germany). The sections were mounted on poly-L-lysine coated slides (Menzel GmbH & Co. KG, Braunschweig, Germany) then washed twice in PBS for 5 min and fixed in 4% (w/v) paraformaldehyde in PBS for 45 min at room temperature. The fixed specimens were permeabilized for 5 min with 0.2% (v/v) Triton-X100 (Sigma) in PBS and washed three times with PBS. In order to inhibit non-specific binding of the antibodies, samples were subsequently blocked in 3% (w/v) bovine serum albumin (BSA) in PBS for 1 hr at 37°C. After triple washing with 0.3% (w/v) BSA in PBS slides were incubated for 15 hrs at 4°C with anti-MSX1 primary polyclonal antibody (Sigma) with a dilution of 1:100. After three consecutive washes with 0.3% (w/v) BSA in PBS, slides were further incubated for 1 hr with 1:100 dilutions of secondary anti-rabbit IgG FITC conjugated antibody (Sigma). Negative controls were processed in the same manner by omitting the primary antibody.

Iin vitro produced bovine oocytes (immature, matured) and early cleavage stages embryos (zygotes, advanced zygotes, 2-cell, 4-cell, 8-cell) were used for immunohistological localization of MSX1 protein. Ten oocytes or embryos from each stage were processed similar to ovarian sections with some modifications. Specimens were fixed in 4% (w/v) paraformaldehyde in PBS overnight at 4°C and permeabilized by 0.5% (v/v) Triton-X100 (Sigma) in PBS. In order to inhibit non-specific binding of the antibodies, samples were subsequently blocked in 3% (w/v) BSA in PBS for 1 hr. The oocytes and embryos were then incubated for 1 hr at 39°C with 1:100 dilution of anti-MSX1 primary polyclonal antibody (Sigma-Aldrich, St Louis, MO, USA). Oocytes and embryos were finally counterstained with 0.5 μg/ml propidium iodide (Sigma-Aldrich, St Louis, MO, USA) for 15 min, followed by washing with PBS. Slides were mounted with Vectashield mounting medium, covered with coverslip and viewed under confocal laser scanning microscope (CLSM LSM-510, Carl Zeiss, Germany).

Digoxigenin-labeled riboprobes (MSX1 cDNA, sense and antisense) were generated by SP6 and T7 polymerases from linearized pGEM^®-T Vector plasmid containing a 382 bp MSX1 cDNA insert (details of primers used Tab. 4) using Dig-labeling kit (Roche Diagnostics, Mannheim, Germany), according to the manufacturer's protocol. The same tissue samples that have been used for immunohistochemistery were serially sectioned of 5μm at -20°C as mentioned above. All sections were fixed in 4% paraformaldehyde at room temperature for 15 min. The RNA probes (50 ng/μl, sense or antisense) were added to hybridisation buffer [50% Dextran sulphate, 2.5 M NaCL, Formamide, 20× SSC, Yeast tRNA (10 mg/ml), 50× Denhardt's solution, Fish sperm DNA (10 mg/ml)], denatured at 80°C for 5 min and incubated overnight at 52°C in humidified chamber. Posthybridization washes were in 2× SSC (pH = 8), 50% formamide twice at 45°C and three times at room temperature for 10 min each. Then sections were treated with 5 μg of RNase A and 50 units of RNase T1 (Fermentas, Steinheim, Germany) in 2× SSC at room temperature for 30 min, followed by three times washing with 2× SSC at room temperature for 10 min each. After washing with TN buffer (0.1 M Tris, 0.15 M NaCl, pH 7.5) at room temperature for 5 min, the slides were incubated with 5% (w/v) blocking buffer (PerKin Elmer, Rodgau Juegesheim, Germany). Then the slides were incubated in a humidified chamber overnight at room temperature with sheep anti-DIG antibody conjugated with horseradish peroxidase (Roth, Karlsruhe, Germany), diluted to 1:100 in buffer 1 containing 1% blocking reagent. After three times washing with TNT buffer (0.05% Tween 20 in TN buffer) for 5 min each, the immunoreactions were visualized by incubating the sections with 0.02% (w/v) TSA™-Plus Fluorescent System (Perkin Elmer, Rodgau Juegesheim, Germany). Slides were counterstained with propidiumiodide (0.5 μg/ml of TNT buffer) and finally mounted with drop of Vecta shield and covered with a cover slip before viewed under fluorescence microscope.

Oocytes aspirated form slaughter house ovaries were used for BCB staining. The procedure of BCB staining is done as described in our previous studies 5253. Briefly a total of 500 morphologically good quality immature oocytes were subjected to 26 μM BCB (B-5388, Sigma-Alderich, Taufenkirchen, Germany) diluted in mDPBS for 90 min at 38.5°C in a humidified air atmosphere. After washing the stained COCs were examined under stereomicroscope and categorized into two groups according to their cytoplasm colouration: oocytes with any degree of blue colouration in the cytoplasm (BCB⁺) and oocytes without visual blue colouration (BCB^-). From each group, four pools of oocytes each with 50 oocytes (a total of 200) were used for mRNA isolation and subsequent real-time PCR after removal of cumulus cells as described before.

The mRNA expression analysis for the studied genes was performed based on the relative standard curve method. The relative expression data were analysed using General Linear Model (GLM) of the Statistical Analysis System (SAS) software package version 8.0 (SAS Institute Inc., NC, USA). Differences among the mean values were tested using ANOVA followed by a multiple pair wise comparison using t-test. Differences of P ≤ 0.05 were considered to be significant.