Early pregnancy results in th production of various signal molecules such as steroids, prostaglandins, and many protein factors. The proteins especially produced by the placenta have been used to detect pregnancy for many years in other species. More recently, pregnancy-specific protein B, which is a placental glycoprotein can be measured by RIA or proteomic methods in serum of pregnant cow. And 2D Fluorescence difference gel electrophoresis (DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. For this reason, we are analyzed serum of bovine. The purpose of this study was to apply DIGE technique for identification of bovine pregnancy-specific proteins using bovine pregnant and non-pregnant serum samples. Serums of 2 pregnant Holstein dairy cattle at day 21 after AI and those of 2 non-pregnant were used in this study. The molecular weight and charge matched cyanine dyes enable pre-electrophoretic labeling of non-pregnancy and pregnant serum proteins which are then mixed and labeled with Cy2 were used as an internal standard. Two pools of proteins are labeled with Cy3 and Cy5 fluorescent dyes, respectively. Labeled proteins with Cy2, Cy3 and Cy5 mixed together and separated in same gel and then were detected by fluorescence image analyzer. The 2D DIGE analysis using fluorescence CyDye flour showed higher sensitivity and better reproducible results than conventional 2D gel electrophoresis. Approximately 1,500 protein spots were detected by 2D DIGE. The differentially expressed proteins were identified by MALDI-TOF Mass spectrometer. Total 16 protein spots differentially expressed in the pregnant serum were detected, among which 7 spots were up-regulated proteins identified as conglutinin precursor, modified bovine fibrinogen, IgG1 etc, and 6 spots were down-regulated proteins identified as hemoglobin, complement component 3, bovine fibrinogen, IgG2a etc. These results indicated that DIGE system could be advantageous for the analysis of serum proteomics diversified by physiological conditions.

An understanding of oocyte gene expression is a necessary for the study of early female gamete development. Recently, oocyte has been used in many techniques such as somatic cell nuclear transfer, intracytoplasmic sperm injection and embryonic stem cell derivation. The purpose of this study was to investigate in the proteomes of pig oocytes and identification of differential proteins between using DIGE technique. In this experiment to overcome of limitation of 2D gel method like a low reproducibility and low sensitivity for proteome analysis of very small quantities, 2D fluorescence difference gel electrophoresis (DIGE), which enables co-detection of up to three samples on the same 2DE gels with CyDyes was used for analysis of oocyte proteins. Proteins within an isoelectric point (pI) range of 3 to 10 and a molecular weight (Mw) range of 20～100 kDa were primarily analyzed in DIGE with 2 replications of each sample. Approximately 1000 spots were detected in 2-D gel. Then, image analysis of DeCyder was performed to detect variations in protein spots between mature oocyte and parthenogenesis embryo. In the comparison of mature oocyte and parthenogenesis embryo, 11 spots were identified to be up-regulated proteins and 2 spots to be down-regulated proteins in parthenogenesis embryo, among which proteins were zona pellucida glycoprotein 4, transferrin receptor, apolipoprotein B, L-3-Hydroxyacyl Coa Dehydrogenase Revisited, cytochrome P450 2C33, similar to Monocarboxylate transporter 2, 2'-5' oligoadenylate synthetase 3, interferon alpha/ beta receptor-1, Chloride channel protein 6, pyruvate carboxylase as well as2'-5' oligoadenylate synthetase 3 using MALDI-TOF-MS. These results suggested that differential proteins were present between mature oocyte and parthenogenesis embryo.

An understanding of oocyte gene expression is a necessary for the study of biological development. Recently, Oocyte has been used in many techniques such as somatic cell nuclear transfer (SCNT), intracytoplasmic sperm injection (ICSI) and embryonic stem cell derivation. However, the molecular mechanism underlying porcine oocyte is still unclear. In this study, we present the description of the porcine oocyte proteome. Proteins within the isoelectric point ranges of 3.0 to 10.0 were analyzed separately using 2‐dimensional electrophoresis (2‐DE). About 450 spots were detected in 2‐ D gel of oocytes, stained with Coomassie blue. Subsequent excision of 227 spots from gels and MALDI‐TOF MS analysis allowed the identification of 85 proteins. Our results indicated the composite profiles of proteins in the porcine oocyte. Tubulin beta chain and meiosis‐specific nuclear structural protein 1 antibody was used to confirm those antibody expression levels in immature, mature and parthenogenetic embryo. Western blot analysis showed that expressions of those proteins increased during mature and parthenogenetic embryo. These protein profiles will make available important guides for the study of oocyte function and assist in functional analysis of the proteins.

Pregnancy is a unique event in which a fetus develops in the uterus despite being genetically and immunologically different from the mother, and the underlying mechanisms remain poorly understood. To analyze the differential gene expression profiles in nonpregnant and 7 days post coitus (dpc) pregnant uterus of mice, we performed a global proteomic study by 2‐D gel electrophoresis (2‐DE) and MALDI‐TOF‐MS. The uterine proteins were separated using 2‐DE. Approximately 1,000 spots were detected on staining with Coomassie brilliant blue. An image analysis using Melanie III (Swiss Institute for Bioinformatics) was performed to detect variations in protein spots between pregnant and nonpregnant uterus. Twenty‐one spots were identified as differentially expressed proteins, of which 10 were up‐regulated proteins such as alpha‐fetoprotein, chloride intracellular channel 1, transgelin, heat‐shock protein beta‐1, and carbonic anhydrase II, while 11 were down‐regulated proteins such as X‐box binding protein, glutathione S‐transferase omega 1, olfactory receptor Olfr204, and metalloproteinase‐disintegrin domain containing protein TECADAM. Most of the identified proteins appeared to be related with catabolism, cell growth, metabolism, regulation, cell protection, protein repair, or protection. Our results uncovered key proteins of mouse uterus involved in pregnancy.

Insulin, transferrin and selenium (ITS) complex is reported to improve in vitro development of oocytes and embryos. This study was carried out to investigate the effects of ITS during in vitro culture (IVC) of porcine parthenogenetic and nuclear transfer (NT) embryos on subsequent developmental capacity in vitro. The electrically activated oocytes were cultured in Porcine Zygote Medium (PZM-3) with various concentrations (0, 0.1, 0.5, and 1.0%) of ITS for 7 days. Also, the electrically activated reconstructed embryos were cultured in PZM-3 with various concentrations (0, 0.1, 0.5, and 1.0%) of ITS for 6 days. Addition of ITS to culture medium did not affect development of porcine parthenogenetic embryos in vitro. To test the effect of ITS on the in vitro development of porcine NT embryos, factorial experiments were also performed for in vitro maturation (IVM) medium (TCM-199) with or without 1% ITS and culture medium (PZM-3) with or without 0.5% ITS. Addition of 0.5% ITS to culture medium increased (p<0.05) the proportion of NT blastocysts compared with non-treated group. In contrast, addition of 1% ITS to culture medium was ineffective or had a detrimental effect. Also, addition of ITS only to maturation medium increased (p<0.05) the percentage of NT blastocysts formation compared with the control group. In conclusion, addition of ITS to IVM or IVC medium could improve subsequent blastocyst development of porcine NT embryos.

To examine the differential protein expression pattern in the 11.5 day post-coitus (dpc) and 18.5 dpc placenta of mouse, we have used the global proteomics approach by 2-D gel electrophoresis (2-DE) and MALDI-TOF-MS. The differential protein patterns of 3 placentae at the 11.5 dpc and 18.5 dpc from nature mating mice were analyzed. Proteins within isoelectric point range of 3.0~10.0, separately were analyzed in 2DE with 3 replications of each sample. A total of approximately 1,600 spots were detected in placental 2-D gel stained with Coomassie-blue. In the comparison of 11.5 dpc and 18.5 dpc placentae, a total of 108 spots were identified as differentially expressed proteins, of which 51 spots were up-regulated proteins such as alpha-fetoprotein, mKIAA0635 protein and transferrin, annexin A5, while 48 spots were down-regulated proteins such as Pre-B-cell colony-enhancing factor 1(PBEF), aldolase 1, A isoform, while 4 spots were 11.5 dpc specific proteins such as chaperonin and Acidic ribosomal phosphoprotein P0, while 3 spots were 18.5 dpc specific proteins such as aldo-keto reductase family 1, member B7 and CAST1/ERC2 splicing variant-1. Most identified proteins in this analysis appeared to be related with catabolism, cell growth, metabolism and regulation. Our results revealed composite profiles of key proteins involved in mouse placenta during pregnancy.