Porcine endometrial 3D co-culture: Morphological changes in 3D endometrium tissues according to hormonal changes.

Cells cultured as monolayers proliferate well, but do not sustain their differentiation characteristics. Previous studies have investigated the interactions between cells and growth factors or cytokines by establishing either in vivo or in vitro three-dimensional (3D) cultures. Using porcine uterine epithelial cells and endometrial cells, the current study was designed to develop a 3D uterine culture system and investigate the response to hormone treatment. Formation of the 3D uterine model was similar to that of uterus from the group supplemented with calcium and magnesium, and the addition of these ions altered the spectrum of basement membrane degrading enzyme expression and activity. In particular, the epithelial cell junctions in the 3D model most closely resembled those of an actual uterus when the medium was supplemented with calcium and magnesium; the intercellular basement membrane structure was also tall under these conditions. The study confirmed that Casp-3 expression was lowest in the P4 (progesterone) treatment group, and this hormone was the most potent stimulus for formation of the endometrial cell layer. Therefore, the addition of calcium and magnesium plays an important role in the formation of a 3D uterine model, and the addition of P4 hormone mimics uterine thickening by stimulating growth of the epithelial cell layer.

Morphological changes in mouse ovary due to hormonal hypersecretion and matrix metalloproteinase -2 activity.

We analyzed whether aberrant gonadotropin secretion affects the morphological remodeling of murine ovarian tissues facilitated by activated matrix metalloproteinase (MMP) enzymes. Six mice were intraperitoneally injected with 5 IU of pregnant mare serum gonadotropin (PMSG) or human chorionic gonadotropin (HCG) every two days after estrus synchronization. Morphology and expression of various MMPs were assessed following the successful induction of hormonal secretion in these tissues. HCG treatment, but not PMSG treatment, resulted in the expanded production of granulose second follicular cells. In addition, the number of developing follicular cells in the HCG group increased compared with that in the PMSG group. Ovarian diameters were also very small in the PMSG group. Immunohistochemistry revealed decreased MMP-2 protein activity in the HCG group and increased MMP-2 activity in the PMSG group. Activity was particularly high in theca and granulose cells of the PMSG group, but only partial activity was observed in the theca cells of the HCG group. Vascular endothelial growth factor activity was increased in both the external and internal theca cell walls in the PMSG group while the HCG group showed high overall expression of this protein in the internal theca cells. These data indicate that follicular cell activity and remodeling of the ovaries differ based on the type of secretory hormone signals they receive. Inappropriate gonadotropin secretion may induce functional changes in the ovaries, and follicular remodeling may be facilitated by the activity of various MMPs.

Matrix metallopeptidases regulate granulosa cell remodeling through the hormone signaling pathway.

OBJECTIVE: Granulosa cells (GCs) play a very important role in reproductive physiology due to their effect on developmental and functional changes. However, there are differing views regarding the mechanism by which hormones stimulate GCs. Therefore, our study aims to determine whether GCs, in the absence of initial stimulation (17ß-estradiol), select specific types of MMPs that reconstitute cells by stimulation of major hormones [follicle-stimulating hormone (FSH) or/and luteinizing hormone (LH)]. MATERIALS AND METHODS: Early GCs were extracted from immature follicles of the porcine ovary to analyze the MMPs levels. Using early GCs in pigs, the cell development rate was evaluated by adding 17ß-estradiol, FSH, LH, or FSH + LH, respectively, to the DMEM containing 10% FBS. Real-time PCR, zymography, enzyme-linked immunosorbent assay, western blot, and immunofluorescence analysis were also performed to determine the MMPs activation in the GCs. RESULTS: Our results confirm that FSH or LH stimulation regulates cell development and intracellular MMPs. In particular, FSH activity kept the MMP-2 and MMP-9 expressions constant in GCs. Conversely, LH activity initially led to rapid increases in the MMP-9 expression, which 96 h later was similar to the MMP-2 expression. Simultaneous utilization of FSH + LH maintained a steady MMP-9 expression and the development of GCs increased. Additionally, when FSH and LH were processed simultaneously, the number of cells increased without changes in cell size, while the cell size changed when LH alone was used. CONCLUSION: Therefore, the results of this study confirm that even without the initial stimulation of GCs, physiological changes occur according to hormonal changes in the environment, and there is variability in the expression of MMPs.

Regulatory effect of apoptosis on morphological changes in cell mass of porcine blastocyst through supplementation of rapamycin during in vitro culture.

OBJECTIVE: The study of an in vitro embryosis is crucial in genetics for breed improvement and reproduction in livestock, identifying the causes of infertility, and stem cell application. Meanwhile, the problem of nucleic acid denaturation observed during embryo development is yet to be resolved. This study was set out to analyze the nucleic acid denaturation during the development of in vitro embryos. MATERIALS AND METHODS: Using an in-vitro fertilization-embryo in porcine, the cell development and apoptosis were evaluated by adding rapamycin by concentration to the TCM-199 containing 10% FBS or 10% porcine follicle fluid (pFF). Real-time PCR, zymography, DNA fragment, Western blot, and immunofluorescence analysis were also carried out to determine the development rate of inner cell mass in the in-vitro fertilization-embryo. RESULTS: The findings indicated that the addition of rapamycin to the 10% pFF group during in vitro maturation led to an increase in the rates of cleavage and blastocyst development and the expression of active matrix metallopeptidase (MMP-9), while nucleic acid denaturation was suppressed. In other words, the addition of rapamycin was found to increase the expression of MMP-2 in the inner cell mass and trophoblast, while it inhibited apoptosis. CONCLUSION: The addition of rapamycin influences the regulation of apoptosis and MMPs, and based on this, it is presumed to have a positive effect on blastocyst development.

Expression of matrix metalloproteinases to induce the expression of genes associated with apoptosis during corpus luteum development in bovine.

Here we investigated the expressions of apoptosis-associated genes known to induce programed cell death through mRNA expressions of two matrix metalloproteinases (MMPs) that are involved in the degradation of collagen and basal membrane in luteal cells cultured in the treatment media. Our results show that the activity of MMP-2 gelatinase was higher in the CL2 and CL1 of luteal phase, was gradually decreased in the CH2 and CH3 of luteal phase. In particular, the expressions of P4-r and survival-associated genes (IGFr, PI3K, AKT, and mTOR) were strongly induced during CL3 stage, whereas the levels of these genes in corpus luteum (CL) were lower during CL2 and CL1 stages. In the cultured lutein cells analyzed, we found that as MMPs increase, genes related to apoptosis (20α-hydroxy steroid dehydrogenase and caspase-3) also increase. In other words, the results for P4-r and survival-related gene expression patterns in the luteal cells were contrary to the MMPs activation results. These results indicate that active MMPs are differentially expressed to induce the expression of genes associated with programed cell death from the degrading luteal cells. Therefore, our results suggest that the MMPs activation may lead to luteal cell development or death.

Characterization of EST Gene in the Bovine Corpus Luteum during the Estrous Cycle.

The objective of this study was to investigate the expression of bovine luteum expressed sequence tags (ESTs), vascular endothelial growth factor (VEGF), and tumor necrosis factor receptor 1 (TNFR1) and the presence of functional ESTs in the bovine corpus luteum (CL) during different stages of the estrus cycle. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a difference in the expression of ESTs during the CL stage. Concentration of ESTs in the CL tissue increased significantly from the mid-luteal stage and decreased thereafter. RT-PCR analysis showed higher levels of the EST genes in the CL of the mid-luteal stage than in other stages, and the same level of expression of VEGF. Immunohistochemistry analysis of the tissue from CL formation to regression showed low cytosol and aggregation of the nucleus. And activity caspase 3 (apoptosis detector) was most strongly detected in the CL1 stage of bovine. During the estrous cycle, the cytosol was magnified and differentiation of the nucleus was clearly manifested. The ESTs affected the CL, and the relationship between VEGF and TNFR1 played a pivotal role for CL development and activation, dependent on the stage of CL. These results suggest local production of ESTs, the presence of functional ESTs in the bovine CL, and that ESTs play a role in regulating the function of cell death in bovine CL.

Microarray-based analysis of the differential expression of melanin synthesis genes in dark and light-muzzle Korean cattle.

The coat color of mammals is determined by the melanogenesis pathway, which is responsible for maintaining the balance between black-brown eumelanin and yellow-reddish pheomelanin. It is also believed that the color of the bovine muzzle is regulated in a similar manner; however, the molecular mechanism underlying pigment deposition in the dark-muzzle has yet to be elucidated. The aim of the present study was to identify melanogenesis-associated genes that are differentially expressed in the dark vs. light muzzle of native Korean cows. Using microarray clustering and real-time polymerase chain reaction techniques, we observed that the expression of genes involved in the mitogen-activated protein kinase (MAPK) and Wnt signaling pathways is distinctively regulated in the dark and light muzzle tissues. Differential expression of tyrosinase was also noticed, although the difference was not as distinct as those of MAPK and Wnt. We hypothesize that emphasis on the MAPK pathway in the dark-muzzle induces eumelanin synthesis through the activation of cAMP response element-binding protein and tyrosinase, while activation of Wnt signaling counteracts this process and raises the amount of pheomelanin in the light-muzzle. We also found 2 novel genes (GenBank No. NM-001076026 and XM-588439) with increase expression in the black nose, which may provide additional information about the mechanism of nose pigmentation. Regarding the increasing interest in the genetic diversity of cattle stocks, genes we identified for differential expression in the dark vs. light muzzle may serve as novel markers for genetic diversity among cows based on the muzzle color phenotype.

Matrix metalloproteinases are important for follicular development in normal and miniature pigs.

To determine the differences in the follicular development process of normal and miniature pigs, we compared the expression of matrix metalloproteinases (MMP) in these two breeds throughout folliculogenesis. In normal pigs, MMP-9 was highly expressed in all stages of the follicular development as well as during ovulation. The follicular development exhibited strong gelatinase activity. The expression of MMP-2 remained insignificant throughout folliculogenesis in both breeds. However, for the follicles of miniature pigs, MMP-2 level was higher than that in normal pigs. Thus MMP types may regulate the remodelling of follicular tissue in the ovaries of normal and miniature pigs. The differential expression of MMPs observed in this study reflected the mechanisms underlying ovarian follicular development in these two breeds.

Effects of hormones on the expression of matrix metalloproteinases and their inhibitors in bovine spermatozoa.

Proteases and protease inhibitors play key roles in most physiological processes, including cell migration, cell signaling, and cell surface and tissue remodeling. Among these, the matrix metalloproteinase (MMPs) pathway is one of the most efficient biosynthetic pathways for controlling the activation of enzymes responsible for protein degradation. This also indicates the association of MMPs with the maturation of spermatozoa. In an attempt to investigate the effect of MMP activation and inhibitors in cultures with various hormones during sperm capacitation, we examined and monitored the localization and expression of MMPs (MMP-2 and MMP-9), tissue inhibitors of metalloproteinases (TIMP-2 and TIMP-3), as well as their expression profiles. Matured spermatozoa were collected from cultures with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and Lutalyse at 1 h, 6 h, 18 h, and 24 h. ELISA detected the expression of MMP-2, MMP-9, TIMP-2, and TIMP-3 in all culture media, regardless of medium type (FSH-supplemented fertilization Brackett-Oliphant medium (FFBO), LH-supplemented FBO (LFBO), or Lutalyse-supplemented FBO (LuFBO)). TIMP-2 and TIMP-3 expression patterns decreased in LFBO and LuFBO. MMP-2 and MMP-9 activity in FBO and FFBO progressively increased from 1 h to 24 h but was not detected in LFBO and LuFBO. The localization and expression of TIMP-2 and TIMP-3 in sperm heads was also measured by immunofluorescence analysis. However, MMPs were not detected in the sperm heads. MMP and TIMP expression patterns differed according to the effect of various hormones. These findings suggest that MMPs have a role in sperm viability during capacitation. In conjunction with hormones, MMPs play a role in maintaining capacitation and fertilization by controlling extracellular matrix inhibitors of sperm.

Metformin enhances the action of insulin on porcine granulosa-lutein cells in vitro.

Metformin is an oral antidiabetic drug extensively used to treat the polycystic ovary syndrome in women. Metformin increases insulin-stimulated glucose uptake and has direct effects on ovarian steroidogenesis in humans. However, the molecular mechanisms of metformin' action on the ovary are not clear. To investigate the effects of this drug on the insulin-signaling pathway in porcine granulosa cells as an alternative model for human research, we examined the mRNA expressions of porcine insulin receptor (INSR), insulin-like growth factor-1 receptor (IGF-1R), insulin receptor substrate-1 (IRS-1), and the protein activity (activation and phosphorylation) of downstream targets including Raf, mitogen-activated protein kinase (MEK)1/2, extracellular signal regulated kinase (ERK), phosphoinositide-dependent 1 kinase (PDK1), mammalian target of rapamycin (TOR), p70, and nuclear factor-κB (NF-κB) in a primary culture system consisting of porcine granulosa-lutein cells (pGLs) incubated with 10(-5)M metformin and/or 100ng/ml insulin for 24h in a serum-free medium. We also investigated the luciferase activity of transcription factors activator protein-1 (AP-1) and NF-κB. Metformin with insulin significantly increased mRNA expressions of INSR, IGF-1R, and IRS-1, while metformin alone had no significant effect. And metformin with insulin had the significant effect on the protein activity (activation and phosphorylation) of downstream targets of INSR signaling pathway. Metformin with insulin significantly elicited an induction of luciferase activity in the transfection of AP-1 and NF-κBreporter, while metformin alone did not. In conclusion, we examined the activity of metformin and insulin on pGLS in vitro and metformin enhanced the action of insulin on the intracellular signaling pathways. These results suggest that metformin could change the function of ovarian granulosa cells.

Differential expression of programmed cell death on the follicular development in normal and miniature pig ovary.

Follicles are important in oocyte maturation. Successful estrous cycle requires remodeling of follicular cells, and proper execution of programmed cell death is crucial for normal follicular development. The objectives of the present study were to understand programmed cell death during follicle development, to analyze the differential follicle development patterns, and to assess the patterns of apoptosis and autophagy expression during follicle development in normal and miniature pigs. Through the analysis of differential patterns of programmed cell death during follicular development in porcine, MAP1LC3A, B and other autophagy-associated genes (ATG5, mTOR, Beclin-1) were found to increase in normal pigs, while it decreased in miniature pigs. However, for the apoptosis-associated genes, progression of genes during follicular development increased in miniature pigs, while it decreased in normal pigs. Thus, results show that normal and miniature pigs showed distinct patterns of follicular remodeling manifesting that programmed cell death largely depends on the types of pathway during follicular development (Type II or autophagy for normal pigs and Type I or apoptosis for miniature pigs).

Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin.

BACKGROUND: Dimeric human erythropoietin (dHuEPO) peptides are reported to exhibit significantly higher biological activity than the monomeric form of recombinant EPO. The objective of this study was to produce transgenic (tg) mice expressing dHuEPO and to investigate the characteristics of these mice. METHODS: A dHuEPO-expressing vector under the control of the goat beta-casein promoter, which produced a dimer of human EPO molecules linked by a 2-amino acid peptide linker (Asp-Ile), was constructed and injected into 1-cell fertilized embryos by microinjection. Mice were screened using genomic DNA samples obtained from tail biopsies. Blood samples were obtained by heart puncture using heparinized tubes, and hematologic parameters were assessed. Using the microarray analysis tool, we analyzed differences in gene expression in the spleens of tg and control mice. RESULTS: A high rate of spontaneous abortion or death of the offspring was observed in the recipients of dHuEPO embryos. We obtained 3 founder lines (#4, #11, and #47) of tg mice expressing the dHuEPO gene. However, only one founder line showed stable germline integration and transmission, subsequently establishing the only transgenic line (#11). We obtained 2 F1 mice and 3 F2 mice from line #11. The dHuEPO protein could not be obtained because of repeated spontaneous abortions in the tg mice. Tg mice exhibited symptoms such as short lifespan and abnormal blood composition. The red blood cell count, white blood cell count, and hematocrit levels in the tg mice were remarkably higher than those in the control mice. The spleens of the tg mice (F1 and F2 females) were 11- and -21-fold larger than those of the control mice. Microarray analysis revealed 2,672 spleen-derived candidate genes; more genes were downregulated than upregulated (849/764). Reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) were used for validating the results of the microarray analysis of mRNA expression. CONCLUSIONS: In conclusion, dHuEPO tg mice caused excessive erythrocytosis that led to abnormal blood composition, short lifespan, and abnormal splenomegaly. Further, we identified 2,672 genes associated with splenomegaly by microarray analysis. These results could be useful in the development of dHuEPO-producing tg animals.

Expression of aldo-keto reductase family 1 member C1 (AKR1C1) gene in porcine ovary and uterine endometrium during the estrous cycle and pregnancy.

BACKGROUND: The aldo-keto reductase family 1 member C1 (AKR1C1) belongs to a superfamily of NADPH-dependent reductases that convert a wide range of substrates, including carbohydrates, steroid hormones, and endogenous prostaglandins. The 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) is a member of AKR family. The aims of this study were to determine its expression in the ovary and uterus endometrium during the estrous cycle and pregnancy. METHODS: Rapid amplification of cDNA ends (RACE) experiments were performed to obtain the 5' and 3' ends of the porcine 20 alpha-HSD cDNA. Reverse-transcriptase-PCR (RT-PCR), real-time PCR, northern blot analysis, and western blot analysis were performed to examine the expression of porcine 20 alpha-HSD. Immunohistochemical analysis was also performed to determine the localization in the ovary. RESULTS: The porcine 20 alpha-HSD cDNA is 957 bp in length and encodes a protein of 319 amino acids. The cloned cDNA was virtually the same as the porcine AKR1C1 gene (337 amino acids) reported recently, and only differed in the C-terminal region (the AKR1C1 gene has a longer C-terminal region than our sequence). The 20 alpha-HSD gene (from now on referred to as AKR1C1) cloned in this paper encodes a deletion of 4 amino acids, compared with the C-terminal region of AKR1C1 genes from other animals. Porcine AKR1C1 mRNA was expressed on day 5, 10, 12, 15 of the cycle and 0-60 of pregnancy in the ovary. The mRNA was also specifically detected in the uterine endometrium on day 30 of pregnancy. Western blot analysis indicated that the pattern of AKR1C1 protein in the ovary during the estrous cycle and uterus during early pregnancy was similar to that of AKR1C1 mRNA expression. The recombinant protein produced in CHO cells was detected at approximately 37 kDa. Immunohistochemical analysis also revealed that pig AKR1C1 protein was localized in the large luteal cells in the early stages of the estrous cycle and before parturition. CONCLUSIONS: Our study demonstrated that AKR1C1 mRNA and protein are coordinately expressed in the luteal cell of ovary throughout the estrous cycle and in the uterus on day 30 of pregnancy. Thus, the porcine AKR1C1 gene might control important mechanisms during the estrous cycle.

Molecular characterization of bovine placental and ovarian 20α-hydroxysteroid dehydrogenase.

The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to its inactive form, 20α-hydroxyprogesterone. This enzyme plays a critical role in the regulation of luteal function in female mammals. In this study, we conducted the characterization and functional analyses of bovine 20α-HSD from placental and ovarian tissues. The nucleotide sequence of bovine 20α-HSD showed significant homology to that of goats (96%), humans (84%), rabbits (83%), and mice (81%). The mRNA levels increased gradually throughout the estrous cycle, the highest being in the corpus luteum (CL) 1 stage. Northern blot analysis revealed a 1.2  kb mRNA in the bovine placental and ovarian tissues. An antibody specific to bovine 20α-HSD was generated in a rabbit immunized with the purified, recombinant protein. Recombinant 20α-HSD protein produced in mammalian cells had a molecular weight of ∼37  kDa. Bacterially expressed bovine 20α-HSD protein showed enzymatic activity. The expression pattern of the 20α-HSD protein in the pre-parturition placenta and the CL1 stage of the estrous cycle was similar to the level of 20α-HSD mRNA expression. Immunohistochemical analysis also revealed that bovine 20α-HSD protein was intensively localized in the large luteal cells during the late estrous cycle.

Bovine adipose triglyceride lipase is not altered and adipocyte fatty acid-binding protein is increased by dietary flaxseed.

In this paper, we report the full-length coding sequence of bovine ATGL cDNA and analyze its expression in bovine tissues. Similar to human, mouse, and pig ATGL sequences, bovine ATGL has a highly conserved patatin domain that is necessary for lipolytic function in mice and humans. This suggests that ATGL is functionally intact as a triglyceride lipase in cattle. Tissue distribution of ATGL gene expression was highest in fat and muscle (skeletal and cardiac) tissue, while protein expression was solely detectible in the adipose tissue. The effect of 109 days of flaxseed supplementation on ATGL and adipocyte fatty acid-binding protein (FABP4 or A-FABP, E-FABP or FABP5) expression was examined in Angus steers. Supplemented steers had greater triacylglycerol (TAG) content in the muscle compared with unsupplemented ones. Additionally, supplementation increased A-FABP expression and decreased stearoyl-CoA desaturase 1 (SCD-1) expression in muscle, while total ATGL expression was unaffected. In summary, supplementation of cattle rations with flaxseed increased muscle TAG concentrations attributed in part to increased expression of key enzymes involved in lipid trafficking (A-FABP) and metabolism (SCD-1).

A two-dimensional electrophoresis reference map for the bovine placenta during late pregnancy.

An understanding of bovine placental gene expression is essential for the study of animal reproductive physiology. Recent reports have found that placental abnormalities occur frequently in cloned bovines and mice. However, the molecular mechanisms underlying bovine placenta function remain unclear. Here, we present a preliminary description of the bovine placenta proteome. Proteins within the isoelectric point ranging from 4.0 to 7.0 and 6.0 to 9.0 were analyzed separately using 2-DE, using three replicates of bovine placenta. Approximately 2000 spots were detected in a placental 2-D gel stained with Coomassie blue. Subsequent excision of 380 spots from gels and MALDI-TOF MS analysis allowed the identification of 273 proteins. Our results revealed the composite profiles of key proteins in the bovine placenta during late pregnancy. These protein profiles will shed light on placental function during pregnancy and assist with functional analysis of the proteins.

Rams genetically superior for IGF-I do not exhibit improved male reproductive traits.

Insulin-like growth factors (IGFs) play an important role in regulating normal physiology, and may be involved in the control of reproduction. The aim of this study was to define the relationship between IGF-I concentrations and reproductive performance over the breeding and non-breeding seasons in lines of New Zealand Romney rams that had been selected for low and high blood serum IGF-I concentration. Yearling rams from two selection lines (13 from the high line and 19 from the low line) were examined in July (winter), September (autumn) and November (summer) 2006 and March (spring) 2007. Scrotal circumference including the inguinal skin was recorded. Semen was collected by electroejaculation on 4 occasions over a 12-month period. Semen was evaluated according to standard procedures (volume, motility, density and morphology). Samples were collected from four animals from each group for measurements of mRNA for IGF-I and the IGF type 1 receptor (IGF 1R) in the testis, and IGF-I, IGF 1R and the insulin receptor (IR) in the liver. Blood samples were collected via jugular venipuncture for the measurement of IGF-I, insulin and testosterone. The incidences of morphologically abnormal sperm cells, the scrotal circumference and sperm motility were higher in the breeding than in non-breeding season. Seasonal changes were found in the percentage of abnormal sperm, scrotal circumference, sperm motility and sperm density, but there were no differences between lines in any reproductive parameters. IGF-I mRNA levels were higher in the high than the low line in the liver but not in the testis, whereas the opposite was found for levels of IGF 1R mRNA. mRNA levels for the insulin receptor in the liver were higher in the high line. Plasma testosterone concentrations did not differ between lines, whereas the concentrations of IGF-I and insulin were higher in the high line. The results suggest that IGF-I may be locally produced in the liver and the testis, and that selection for high IGF-I may not be associated with improved reproductive performance in rams.

Delayed parturition in cloned calves associated with persistently elevated placentomal TGF-beta1 expression.

The objective of this study was to investigate hormonal and TGF-beta(1) characterizations of delayed parturition in the SCNT recipients (Korean native beef cattle: Hanwoo). The SCNT blastocysts produced by Hanwoo fetal fibroblast cells were transferred into the synchronized Hanwoo recipients. The artificially inseminated Hanwoo recipients (AI-R) were used as control. All AI-R were labored by natural delivery. The SCNT recipients (SCNT-R) with no signs of delivery were operated by Caesarean section. The blood and placentomes were collected during parturition. The weight of placentomes in SCNT-R (n=12, 301+/-41.22 g) was significantly higher than that of AI-R (n=10, 204.8+/-24.89 g) (p<0.05). There were significantly lower E2 (p<0.05) or higher P4 (p<0.01) and TGF-beta(1) (p<0.01) levels in the SCNT-R compared to that of AI-R, respectively. The SCNT-R showed a higher placentomal TGF-beta(1) protein level compared to that of AI-R (p<0.01). Interestingly, the TGF-beta(1) protein level in SCNT-R with normal delivery was dramatically decreased as same as AI-R, but it was highly maintained in C-sec at days 250 of pregnancy in AI-R. These results suggest that delayed parturition in clone calving may be associated with persistence of elevated TGF-beta-1 expression in late pregnancy.

Establishment and characterization of three immortal bovine muscular epithelial cell lines.

We have established three immortal bovine muscular epithelial (BME) cell lines, one spontaneously immortalized (BMES), the second SV40LT-mediated (BMEV) and the third hTERT-mediated (BMET). The morphology of the three immortal cell lines was similar to that of early passage primary BME cells. Each of the immortal cell lines made cytokeratin, a typical epithelial marker. BMET grew faster than the other immortal lines and the BME cells, in 10% FBS-DMEM medium, whereas neither the primary cells nor the three immortal cell lines grew in 0.5% FBS-DMEM. The primary BME cells and the immortal cell lines, with the exception of BMES, made increasing amounts of p53 protein when treated with doxorubicin, a DNA damaging agent. On the other hand, almost half of the cells in populations of the three immortal cell lines may lack p16(INK4a) regulatory function, compared to primary BME cells that were growth arrested by enforced expression of p16(INK4a). In soft-agar assays, the primary cells and immortal cell lines proved to be less transformed in phenotype than HeLa cells. The three immortal epithelial-type cell lines reported here are the first cell lines established from muscle tissue of bovine or other species.

Protein profiles of bovine placenta derived from somatic cell nuclear transfer.

Practical application of animal cloning by somatic cell nuclear transfer (SCNT) has been hampered by an extremely low success rate. To address whether placental dysfunction in SCNT causes fetal loss during pregnancy, we have used a global proteomics approach using 2-DE and MS to analyze the differential protein patterns of three placentae from the afterbirth of cases of postnatal death, derived from SCNT of Korean Native cattle, and three normal placentae obtained from the afterbirth of fetuses derived from artificial insemination. Proteins within a pI range of 4.0-7.0 and 6.0-9.0 were analyzed separately by 2-DE in triplicate. A total of approximately 2000 spots were detected in placental 2-DE gels stained with CBB. In the comparison of normal and SCNT samples, 60 spots were identified as differentially expressed proteins, of which 33 spots were up-regulated proteins in SCNT placentae, while 27 spots were down-regulated proteins. Most of the proteins identified in this analysis appeared to be related with protein repair or protection, cytoskeleton, signal transduction, immune system, metabolism, extracellular matrix and remodeling, transcription regulation, cell structure or differentiation and ion transport. One of up-regulated proteins in SCNT was TIMP-2 protein known to be related to extracellular matrix and remodeling during pregnancy. Western blot analysis showed an increased level of TIMP-2 in SCNT placenta compared to normal. Our results revealed composite profiles of key proteins involved in abnormal placenta derived from SCNT, and suggested expression abnormality of these genes in SCNT placenta, resulting in fetal losses following SCNT.