The HMG box containing protein (HBP) has a high mobility group domain and involved in the regulation of proliferation and differentiation of tissues. We screened HBP2 in glioblastoma using Suppression Subtractive Hybridization (SSH) and isolated human spermatogonial stem cell‐like cells (hSSC‐like cells) derived from patients of nonobstructive azoospermia (NOA). Expression of HBP2 was analyzed by RT‐PCR in undifferentiated stem cells (human Embryonic Stem Cells, hSSC‐like cells 2P) and spontaneous differentiated stem cells (hSSC‐like cells 4P). It was overexpressed in hESC and hSSC‐like cells 2P but not in hSSC‐like cells 4P. Also, the expression level of HBP2 was downregulated in colon tumor tissues compared to normal tissues. Specifically in synchronized WI‐38 cells, HBP2 was highly upregulated until the G1 phase of the cell cycle and gradually decreased during the S phase. Our results suggest that HBP2 was downregulated during the spontaneous differentiation of hSSC‐like cells. HBP2 was differently expressed in colon tissues and was related to G1‐progression in WI‐38 cells. It may play a role in the maintenance of an undifferentiated hSSC‐like cell state and transits from G1 to S in WI‐38 cells. This research was important that it identified a biomarker for an undifferentiated state of hSSC‐like cells and characterized its involvement to arrest during cell cycle in colon cancer.
The techniques of IVM, IVF and IVC of canine oocytes may provide useful information for gamete salvage programs and the conservation of endangered canidae. This investigation has been made to determine the efficiency of in vitro maturation (IVM) as a basic experiment to study the development of canine oocytes after in vitro fertilization (IVF). The rate of oocytes developing to the MII stage was higher in the hormone treated group (10 IU/ml hCG+eCG, 14.7%, p<0.05) than in the control group (0 IU/ml hCG+eCG, 10.0%). The monospermy and pronuclear rates of canine oocytes were investigated after caffeine treatment on IVF. Canine oocytes were fertilized in the Fert‐TALP medium supplemented with 0, 10, 20 or 30 mM caffeine (Fert I, Fert II, Fert III or Fert IV, respectively). The highest pronuclear formation rate was obtained in the Fert I for 24 h IVF (6.7%, 6/89). Therefore, it is believed that unlike in other mammals, caffeine in canine IVF does not increase the efficiency of fertilization rate, and is not an important factor.
Human embryonic stem (ES) cells retain the capacity for self‐renewal, are pluripotent and differentiate into the three embryonic germ layer cells. The regulatory transcription factors Oct4, Nanog and Sox2 play an important role in maintaining the pluripotency of human ES cells. The aim of this research was to identify unknown genes upregulated in human ES cells along with Oct4, Nanog, and Sox2. This study characterizes an unknown gene, named chromosome 1 open reading frame 31 (C1orf31) mapping to chromosome 1q42.2. The product of C1orf31 is the hypothetical protein LOC388753 having a cytochrome c oxidase subunit VIb (COX6b) motif. In order to compare expression levels of C1orf‐ 31 in human ES cells, human embryoid body cells, vascular angiogenic progenitor cells (VAPCs), cord‐blood endothelial progenitor cells (CB‐EPCs) and somatic cell lines, we performed RT‐PCR analysis. Interestingly, C1orf31 was highly expressed in human ES cells, cancer cell lines and SV40‐immortalized cells. It has a similar expression pattern to the Oct4 gene in human ES cells and cancer cells. Also, the expression level of C1orf31 was shown to be upregulated in the S phase and early G2 phase of synchronized HeLa cells, leading us to purpose that it may be involved in the S/G2 transition process. For these reasons, we assume that C1orf31 may play a role in on differentiation of human ES cells and carcinogenesis.
Silencing of Dicer1 by siRNA did not inhibit development up to the blastocyst stage, but decreased expression of selected transcription factors, including Oct‐4, Sox2 and Nanog, suggesting that Dicer1 gene expression is associated with differentiation processes at the blastocyst stage (Cui et al., 2007). In order to get insights into genes which may be linked with microRNA system, we compared gene expression profiles in Gapdh and Dicer1 siRNA‐microinjected blastocysts using the Applied Biosystem microarray technology. Our data showed that 397 and 737 out of 16354 genes were up‐ and down‐regulated, respectively, following siRNA microinjection (p<0.05), including 24 up‐ and 28 downregulated transcription factors. Identification of genes that are preferentially expressed at particular Dicer1 knock down embryos provides insights into the complex gene regulatory networks that drive differentiation processes in embryos at blastocyst stage.
The objective of this study was to examine the effect of caffeine and sodium bicarbonate in a fertilization medium on the fertilizability of boar spermatozoa that were frozen in straws. Boar spermatozoa were extended with Beltsville F5 extender and frozen in 0.25‐ml straws. In vitro matured porcine oocytes were fertilized in vitro (IVF) with frozen‐thawed boar spermatozoa for 6 h in a modified tris‐buffered medium (mTBM) or in its modified medium by substituting the tris with 25 mM sodium bicarbonate (modified bicarbonate‐buffered medium; mBBM). Some of inseminated oocytes were fixed and stained for examination of sperm penetration. IVF embryos were cultured in a North Carolina State University‐23 medium for embryo development. The percentage of live sperm was 47±4% and morphological abnormality of acrosome was found in 14±3% of spermatozoa. Optimal sperm concentration for IVF was 0.75~1.0×106 sperms/ml when mTBM containing 5 mM caffeine was used as the fertilization medium. Sperm penetration was significantly (p<0.05) stimulated by increasing caffeine concentration in the IVF medium. In addition, mBBM significantly (p<0.05) increased sperm penetration (92%) compared to mTBM (65%). More (p<0.05) blastocysts (22% vs. 32%) developed from the oocytes that were fertilized in mBBM containing 1 mM caffeine than from those fertilized in mTBM with 5 mM caffeine. Our results indicate that boar spermatozoa can be frozen successfully in straws with holding their normal fertilizability and that caffeine and sodium bicarbonate stimulates sperm penetration in vitro.
In the present studies, we have intended to compare the EDS (20% EG + 20% DMSO + 0.4 M sucrose + 10% FCS) and EDT (20% EG + 20% DMSO + 0.3 M trehalose 10% FCS) methods for vitrification of canine oocytes, in order to improve the vitrification methods. The survival rate of vitrified‐thawed oocytes using the EDS method was 15.1±1.8% (p<0.05), which was lower than that of the control group (66.7±2.5%). About 45~55% of the vitrified‐warmed oocytes showed normal morphology, as assessed by PI staining. However, the ratio of survival rate of oocytes showed lower than that of normal morphology in comparison between EDS method and control group. The survival and developmental rates of vitrified‐warmed oocytes by the EDS and EDT methods were 16.7±1.4% and 11.1±0.8% and 8.3±1.4% and 4.4±1.8%, respectively (p<0.05). The results were significantly lower than the control group (66.7±2.5% and 16.7±3.7%). However, the survival rate of vitrified‐warmed oocytes using EDS method showed higher than that in the ETS group.
Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for studying the interactions between an oocyte and somatic nucleus. The object of this study was to investigate the developmental competence of in vitro‐matured porcine oocytes after transfer of the somatic cell nuclei of 2 different species (goat and rabbit). Porcine cumulus oocytes were obtained from the follicles of ovaries and matured in TCM‐199. The reconstructed embryos were electrically fused with 2 DC pulses of 1.1 kV/cm for 30 μs in 0.3 M mannitol medium. The activated cloned embryos were cultured in porcine zygote medium‐3 (PZM‐3), mSOF or RDH medium for 7 days. The blastocyst formation rate of the embryos reconstructed from goat or rabbit fetal fibroblasts was significantly lower than that of the embryos reconstructed from porcine fetal fibroblast cells. However, a significantly higher number of embryos reconstructed from goat or rabbit fetal fibroblasts cultured in mSOF or RDH, respectively, developed to the morular stage than those cultured in PZM‐3. These results suggest that goat and bovine fetal fibroblasts were less efficacious than porcine‐porcine cloned embryos and that culture condition could be an important factor in iSCNT. The lower developmental potential of goat‐porcine and porcine‐bovine cloned embryos may be due to incompatibility between the porcine oocyte cytoplasm and goat and bovine somatic nuclei.
The early diagnosis of bovine pregnancy is an essential component of successful reproductive planning on farms, because lack of bovine pregnancy over the long term results in reproductive failure and low milk yield‐the latter of which is a special concern on dairy farms. This study was designed to identify early pregnancy‐specific whey proteins in bovine, by comparing milk samples collected from cattle during pregnancy (Days 30 and 50) and from non‐pregnant cattle. In this study, differentially expressed proteins in five pregnant and five non‐pregnant Holstein dairy cattle were investigated and compared, using proteomics analysis. The first dimension was applied to a pH 3.0~10.0 strip, by loading a 2‐mg milk protein sample. After the second‐dimension separation was performed, the gels were stained with colloidal Coomassie brilliant blue. The stained gels were scanned and the images were analyzed, to detect variations in protein spots between non‐pregnant and pregnant cattle milk protein spots, using ImageMaster; this was followed by analysis with MALDI TOF‐MS. Analysis of the 2‐DE gel image resulted in a total of approximately 500~600 protein spots, of which 12 spots were differentially expressed, six spots were up‐regulated, and four spots were downregulated; two spots were identified as pregnancy‐specific proteins. These proteins were identified as lactoferrin, NADH dehydrogenase subunit 2, albumin, serum albumin precursor and transferrin. Our results via 2‐D PAGE analysis revealed composite profiles of several milk proteins related to early bovine pregnancy, implying the possible use of these milk proteins in the early detection of bovine pregnancy.
This study was conducted to compare the expression pattern of the specific factors associated with pregnancy and angiogenesis during early pregnancy in Hanwoo. Synchronized female Hanwoo (4~6 year‐old) were inseminated artificially. After 10 weeks after artificial insemination (AI), the pregnancy was tested by rectal palpation method. Three pregnant and non‐pregnant Hanwoo were used in this experiment, respectively. The plasma progesterone level was measured by ELISA. Western blot analysis was performed to detect the expression of pregnancy associated glycoprotein (PAG) or angiogenic factors (VEGF, B‐FGF, ANP‐1, and TIE‐2). The plasma P4 level was increase gradually in pregnant group and maintained high level. The concentration of PAG was significantly higher from 5th weeks in pregnant group compared to that of non‐pregnant group (p<0.05). The concentrations of the VEGF (p<0.05), B‐FGF (p<0.05), and ANP‐1 (p<0.05) were significantly increased from 6th or 7th week after AI in pregnant group, respectively. And the intensity of TIE‐2, ANP‐1 receptor, was well matched with ANP‐1 (p<0.05). Taken together, it can be postulated that the blood vessels connected with fetus and dam were formed dramatically around 40 days after AI, because the expression levels of the angiogenic factors were increased significantly from this time in pregnant Hanwoo.