bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of the luteinising hormone surge on regulation of vascular endothelial growth factor and extracellular matrix-degrading proteinases and their inhibitors in bovine follicles
Authors: Berisha Bajram , Steffl Martin , Welter Harald , Kliem Heike , Meyer Heinrich H. D. , Schams Dieter , Amselgruber Werner
Published: January 2008

The aim of the present study was to evaluate the pattern of regulation of vascular endothelial growth factor (VEGF)-A (isoforms 121, 165, 189), VEGF receptor tyrosine kinases (VEGF-R1 and VEGF-R2), matrix metalloproteinase (MMP)-1, MMP-2, MMP-14, MMP-19, tissue-specific inhibitor of metalloproteinases (TIMP)-1, TIMP-2, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) in time-defined follicle classes before (0 h) and after the application of gonadotrophin-releasing hormone (GnRH). Bovine ovaries containing periovulatory follicles or new corpora lutea (CL; Days 1–2) were collected 0, 4, 10, 20 and 25 h (follicles) or 60 h (CL) after the injection of GnRH. Transcripts of VEGF isoforms (VEGF121, VEGF165, VEGF189) were upregulated 4 h after GnRH injection (during the luteinising hormone (LH) surge) and decreased thereafter to lowest levels around ovulation. All VEGF isoforms and their receptors were upregulated again after ovulation. The VEGF peptide concentration in follicular fluid decreased 20 h after GnRH injection, followed by an increase in follicles 25 h after GnRH. Expression of MMP-1 mRNA increased rapidly 4 h after GnRH injection and remained high during the entire experimental period. In contrast, MMP-19 mRNA increased significantly only after ovulation. Expression of TIMP-1 mRNA increased 4 h after GnRH and again after ovulation. Expression of tPA mRNA increased 4 h after GnRH and remained high during the entire experimental period, whereas expression of uPA transcripts increased significantly only after ovulation. Both uPAR and PAI-1 mRNA levels increased in follicles 4 h after GnRH and again after ovulation. The amount of MMP-1 protein (immunolocalisation) increased in follicles 10 h after GnRH: additional staining was observed in the granulosa cell layer. In conclusion, the temporal and spatial pattern of regulation of VEGF and extracellular matrix-degrading proteinases during periovulation suggests they are important mediators of the LH-dependent rupture of bovine follicles and for early CL formation (angiogenesis).
Soluble adenylyl cyclase is required for activation of sperm but does not have a direct effect on hyperactivation
Authors: Marquez Becky , Suarez Susan S.
Published: January 2008

Soluble adenylyl cyclase (SACY) is an essential component of cAMP-signalling cascades that activate sperm motility and capacitate sperm. SACY activity is stimulated by HCO3– and Ca2+. Sperm from Sacy–/– (null) mice were immotile or weakly motile, but cAMP analogues N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate (dbcAMP) and adenosine 3′,5′-cyclic monophosphate acetoxymethyl ester (cAMP-AM) activated motility. Null sperm activated by dbcAMP quickly developed hairpin bends at the junction of the midpiece and principal piece, which could be prevented by omitting HCO3–. Treating Sacy–/– sperm with thimerosal or NH4Cl to raise flagellar cytoplasmic Ca2+ could not substitute for cAMP analogues in activating motility; however, sperm activated with cAMP-AM hyperactivated after thimerosal treatment. Treating activated wild-type sperm with SACY inhibitor KH7 did not prevent hyperactivation from developing during capacitation in vitro, although high doses impaired motility. These results indicate that, while the SACY/cAMP signalling pathway is required for motility activation, it is not directly involved in triggering hyperactivation.
Endometrial modifications during early pregnancy in bonnet monkeys (Macaca radiata)
Authors: Rosario Gracy X. , D'Souza Serena J. , Manjramkar Dhananjay D. , Parmar Vipul , Puri Chander P. , Sachdeva Geetanjali
Published: January 2008

The present study was undertaken to investigate endometrial modifications that occur before embryo invasion in bonnet monkeys (Macaca radiata). These changes were analysed in luminal epithelium, glandular epithelium and stroma of endometrial functionalis on Day 6 post ovulation from pregnant and non-pregnant animals (n = 4 each) by transmission electron microscopy. Distinct features (i.e. loss of columnar shape by epithelial cells, changes in mitochondrial size and diffused apicolateral gap junctions) were observed in the luminal and glandular epithelium in pregnant animals. Stromal compaction was also observed in pregnant animals. Further, immunogold localisation studies demonstrated significantly higher expression (P < 0.05) of oestrogen receptor α, an oestrogen-regulated gene, in the glandular epithelium and stroma of the endometrium in pregnant animals compared with non-pregnant animals. Expression of two other genes known to be regulated by oestradiol, namely β-actin and cyclo-oxygenase-1, were also significantly higher (P < 0.05) in the endometria of pregnant animals. These studies demonstrate marked changes in the endometrium before embryo invasion in bonnet monkeys. These studies also indicate altered oestrogenic activity in the uterine milieu before embryo invasion.
Cysteamine supplementation of in vitro maturation medium, in vitro culture medium or both media promotes in vitro development of buffalo (Bubalus bubalis) embryos
Authors: Anand T. , Kumar D. , Chauhan M. S. , Manik R. S. , Palta P.
Published: January 2008

The effects of supplementation of in vitro maturation (IVM) or in vitro culture (IVC) or both IVM and IVC media with cysteamine on the yield, hatching rate (HR) and total cell number (TCN) of buffalo blastocysts were examined. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF. The IVM or IVC media were supplemented with 0, 50, 100 or 200 µm cysteamine. Supplementation of IVM medium with 50 µm cysteamine increased (P < 0.01) the cleavage rate and blastocyst yield without affecting the HR and TCN whereas a higher concentration of 200 µm significantly (P < 0.05) reduced the blastocyst yield but not TCN. Similar increases in blastocyst yield, without any effect on HR and TCN were observed after supplementation of the IVC medium with 100 (P < 0.01) or 50 µm (P < 0.05) cysteamine, whereas 200 µm cysteamine was ineffective. Supplementation of both IVM medium with 50 µm cysteamine and of IVC medium with 100 µm cysteamine increased the yield of blastocysts and hatched blastocyst by over 100% (P < 0.01) compared with the controls without any adverse effects on HR or TCN. The results of the present study suggest that supplementation of both IVM and IVC media improves the yield of blastocysts without compromising their health.
121 DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED IN SEQUENTIAL OR CONTINUOUS MEDIA
Authors: Amorim L. S. , Walker D. J. , Seidel Jr G. E.
Published: January 2008

Slaughtered bovine females have different characteristics including age, nutritional status, breed, and management system, all of which may affect the results obtained in in vitro embryo production. Another key consideration is that early embryos move from the oviduct to a slightly different environment in the uterus, which has led to development of sequential embryo culture media (e.g. Lane M et al. 2003 Theriogenology 60, 407–419). However, the benefits and importance of using sequential media are not fully known. Therefore, the aim of the present study was to compare developmental rates of oocytes obtained from slaughterhouse-derived ovaries from cows or heifers after culture in sequential media (CDM-1, CDM-2) or in a continuous medium (C-CDM). The experiment was a 3 × 2 × 2 factorial design [bulls (A, B, or C), source (cows or heifers), and medium (sequential or continuous)]. Cumulus–oocyte complexes were aspirated, within 5 h of slaughter, from 3- to 8-mm ovarian follicles of cows (1482 oocytes) and fattened heifers usually fed melengesterol acetate (2818 oocytes). Embryos were produced in vitro as described by De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596, with slight modifications. Presumptive zygotes were vortexed to remove cumulus cells and cultured for 2.5 d in C-CDM (CDM supplemented with 5.0 mm L-lactate, essential and nonessential amino acids, and 0.5% FAF-BSA, or in CDM-1 (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596) at 39°C in a humidified incubator under 5% CO2, 5% O2, and 90% N2. Cleavage was assessed after 2.5 d; 2- to 6-cell embryos were considered as cleaved, but were not cultured further. Embryos at the 7- to 8-cell stage were cultured for an additional 4.5 d in fresh C-CDM or CDM-2. The percentage blastocysts per oocyte was assessed after 7 and 8 days of culture. Data were arcsin-transformed and evaluated by ANOVA. There was a significant interaction between bull and ovary source for both 8-cell embryos and cleavage rate (P < 0.05); however, this interaction was no longer significant for blastocysts. No other interactions were significant nor a source of ovaries. Culturing embryos in CDM-C refreshed after cleavage evaluation (continuous) or culturing embryos in CDM-1 early and CDM-2 after cleavage evaluation (sequential) resulted in similar cleavage and blastocyst rates (Table 1). We conclude that bovine embryos can be produced using a single chemically defined medium (+BSA) with similar efficacy as a system using 2 sequential media.

Table 1. Effects of media on embryonic development (mean ± SE)
166 EXPRESSION OF PLURIPOTENCY-DETERMINING FACTORS IN IN VITRO-PRODUCED BUFFALO EMBRYOS
Authors: Anand T. , Kumar D. , Singh M. K. , Chauhan M. S. , Manik R. S. , Palta P.
Published: January 2008

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocysts. These are pluripotent cells that retain the ability to differentiate into all cell types. Various cell surface antigens, the expressions of which have been widely used as markers to monitor the pluripotency of ESCs, include Oct-4, stage-specific embryonic antigens (SSEAs) such as SSEA-1, SSEA-3, and SSEA-4, and tumor rejection antigens (TRAs) such as TRA-1-60 and TRA-1-81. In this study, the cell surface expression patterns of these markers were examined in in vitro-produced buffalo embryos at the 2-, 4-, 8- to 16-cell, morula, and blastocyst stages using immunofluorescence microscopy. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF, following which the cleaved embryos were cultured for 9 days for production of embryos at different stages (n = 246). The embryos were fixed in 4% paraformaldehyde in Dulbecco's phosphate-buffered saline (DPBS) for 30 min, permeabilized by treatment with 0.1% Triton X-100 in DPBS for 30 min, and incubated first with the blocking solution (4% normal goat serum) for 30 min and then with the primary antibody (Oct-4: clone 9E3; SSEA-1: MC-480; SSEA-3: MC-631; SSEA-4: MC-813-70; TRA-1-60: clone TRA-1-60; and TRA-1-81: clone TRA-1-81, Chemicon� Inc., Temecula, CA, USA) at a dilution of 1:10 to 1:20 for 1 h. After being washed with DPBS, the embryos were incubated with appropriate FITC-labeled second antibody (anti-rat IgM or anti-mouse IgG or IgM, diluted 1:100 to 1:200) for 1 h and then examined under a fluorescence microscope. Oct-4 expression was detected at all embryonic stages starting from the 2-cell to the blastocyst stage, in which ICM, but not trophectoderm cells, exhibited a strong expression. SSEA-4 signal was found to be strongest at the 2-cell stage, with continued expression at all intermediate stages until the blastocyst stage in which there was a strong expression in ICM cells. In contrast, all of the embryonic stages were found to be negative for SSEA-3 expression. The SSEA-1 signal was present at all of the embryonic stages but was very weak. Expression of TRA-1-60 and TRA-1-81, which was detected only on the inner surface of the zona pellucida and in the perivitelline space in early embryonic stages, was absent in morulae and blastocysts. The results of this study indicate that the pluripotency-determining markers are differentially expressed in buffalo embryos and that the pattern of their expression is distinct from that of murine and human embryos but resembles to some extent that of goat embryos. Comparison of the expression pattern of these markers needs to be done between embryonic cells and ESCs for a better understanding of their developmental regulation.
187 IMPRINTED GENE EXPRESSION IN IN VIVO-AND IN VITRO-PRODUCED BOVINE FETUSES AND PLACENTAS
Authors: Perecin F. , Méo S. C. , Yamazaki W. , Ferreira C. R. , Biase F. H. , Merighe G. K. F. , Meirelles F. V. , Garcia J. M.
Published: January 2008

Some gestational alterations associated with bovine somatic cell nuclear transfer (SCNT) are presumably consequences of abnormal imprinted gene expression. This work aimed to evaluate the expression patterns of imprinted genes IGF2 and IGF2R in bovine fetuses and chorioallantoic membranes derived from in vivo- and in vitro-produced embryos. Fetuses were produced by AI (in vivo group, n = 3), IVF (n = 3), parthenogenesis (n = 3), or SCNT (n = 2). Cows with positive pregnancy diagnosis after ultrasonographic examination were slaughtered between Days 33 and 36 of gestation. The reproductive tract was transported on ice to the laboratory, where fetuses and chorioallantoic fragments were collected and stored in liquid nitrogen. Total RNA extraction was performed using TRIzol, according to manufacturer's instructions, and the reverse transcription reaction was carried out with 1 µg of total RNA, 6.75 µm oligo pd(T)12–18, and 50 U of reverse transcriptase (Improm-II, Promega, Madison, WI, USA). The relative quantification of IGF2 and IGF2R transcripts was done using real-time PCR with SYBR Green dye. The average efficiency of PCR amplifications was estimated for each gene using a linear regression on the logarithm of fluorescence per cycle (Ramakers et al. 2003 Neurosci. Lett. 339, 62–66), and the expression ratios were calculated according to the method described previously by Livak and Schmittgen (2001 Methods 25, 402–408). To verify statistical differences, a pair-wise fixed reallocation randomization test (Pfaffl et al. 2002 Nucl. Acids Res. 30, e36) was used. All expression ratios were normalized by glyceraldehyde 3-phosphate dehydrogenase expression and calibrated by the in vivo group (expression assumed as 1.00 for all genes and tissues). The analysis of relative differences on transcript levels of imprinted genes in fetuses revealed IGF2 down-regulation (P < 0.05) in the SCNT (0.19) and parthenogenetic (0.02) groups when compared to the in vivo group and IVF fetuses (2.02). In chorioallantois, IGF2 was down-regulated (P < 0.001) in parthenotes (0.001) when compared to the in vivo, IVF (3.13), and SCNT (0.98) groups. IGF2R was down-regulated (P < 0.001) in SCNT chorioallantois (0.25) when compared to the in vivo group. Low expression of IGF2 in parthenogenetic fetuses and chorioallantois confirms its imprinted status in bovine. Alterations in the relative frequency of IGF2 and IGF2R transcripts were observed in bovine SCNT-derived fetuses and chorioallantoic membranes, respectively, supporting the hypothesis that abnormalities in the expression of imprinted genes are causes for the low efficiency of SCNT procedures in this species. Such alterations suggest modifications in DNA methylation patterns at IGF2 and IGF2R imprinting centers.
195 CORRELATION BETWEEN SPERM PENETRATION ASSAY AND IN VITRO FERTILIZATION USING HAMSTER AND HUMAN OOCYTES
Authors: Al-Anssari S. , Taiyeb A. , Ridha-Albarzanchi M. , Qurjia Z. , Al-Atraqchi S. , Al-Jabar A. , Kanan Z.
Published: January 2008

The objective of this study was to evaluate the diagnostic and therapeutic significance of a sperm penetration assay (SPA) for predicting IVF outcomes in different age groups of infertile male patients. The patients were divided into 4 age groups, including a first fertile control group (G1: n = 20, aged 20 to 60 yr; G2: n = 25, aged 20 to 30 yr; G3: n = 40, aged 31 to 40 yr; and G4: n = 10, aged >41 yr). Seminal fluid analysis, SPA, and IVF were performed. Sperm concentration, motility, motility grade, and morphology were significantly (P < 0.01) higher in G1 compared with the other groups. Sperm parameters in G4 were also significantly (P < 0.05) lower compared with other groups, except in the sperm morphology percentages. Sperm penetration rate (SPR) was significantly higher in G2 v. G3 and G4 (31.6 v. 26.6 and 24.2, respectively; P < 0.05). The sperm penetration index was similar in G2, G3, and G4 (P > 0.05). The SPR in hamster oocytes was positively correlated with the sperm grade of motility (r = 0.80, P < 0.01), and normal sperm morphology (r = 0.81, P < 0.01) in infertile men (n = 75). The sperm penetration percentage in human oocytes was positively correlated with sperm motility, grade of motility, and normal sperm morphology (r = 60, P < 0.01) in infertile men. No positive correlation with human sperm concentrations was observed in hamster and human oocytes (r = 0.34, P < 0.01). The IVF rates were significantly, positively correlated with SPR of zona-free hamster oocytes (P < 0.001, r = 0.7). The results of the present study demonstrate that the SPA is a more sensitive screening tool than the seminal fluid analysis for predicting IVF outcomes, and it is positively correlated with human IVF scores.
The authors thank Baghdad University and the director of Baghdad Teaching Hospital for their kind cooperation. The authors also thank the Ministry of Health and the Ministry of Higher Education and Scientific Research for their financial support.
203 PRETREATMENT OF BOVINE SPERM OR OOCYTE WITH ANTIBODY TO LIPOCALIN-TYPE PROSTAGLANDIN D SYNTHASE INHIBITS FERTILIZATION
Authors: Gonçalves R. F. , Barnabe V. H. , Killian G. J.
Published: January 2008

Lipocalin-type prostaglandin D synthase (L-PGDS) has been identified in cow uterine tube fluid (UTF), and as fertility-associated protein in the Holstein bull seminal plasma, but its function is unclear. A previous study demonstrated that L-PGDS is associated with the bovine zona pellucida, and that antibody incubated with UTF decreased embryo development in vitro. This study was conducted to determine whether IVF of bovine oocytes would be affected by pretreating either the sperm or oocytes, or both, with L-PGDS antibody. In vitro-matured bovine oocytes were incubated for 1 h in IVF TALP medium supplemented with penicillamine, hypotaurine, epinephrine, and heparin containing (a) no antibody, or (b) a rabbit polyclonal antibody against recombinant bovine L-PGDS (α L-PGDS; 1:2000). Frozen–thawed spermatozoa were washed by a 45:90% layered Percoll gradient centrifugation and incubated for 1 h in IVF TALP with (a) no antibody, or (b) α L-PGDS. For this study we had 4 different treatments: (1) no antibody (control), (2) α L-PGDS at fertilization time, (3) α L-PGDS-treated oocytes, or (4) α L-PGDS-treated sperm. Oocytes were inseminated with 10 � 104 washed spermatozoa in 4-well culture dishes. After 18 h (39�C, 5% CO2 in air), oocytes were vortexed to remove cumulus cells and accessory spermatozoa, and fixed in 3.7% paraformaldehyde and 10% Triton X–100 for 1 h. Oocytes were washed and transferred to a solution with PBS, 0.3% BSA, and 1% Triton for 1 h, stained with Hoechst 33342, and observed in the presence of 2 pronuclei in the cytoplasm. There were 4 replicates of 200 to 250 oocytes for fertilization assays. Data were analyzed by SAS. Addition of α L-PGDS with sperm, oocytes, or both significantly decreased fertilization (P < 0.05) compared with the control: (1) 89.2 � 2.0%; (2) 19.4 � 2.0%; (3) 27.2 � 3.1%; or (4) 14.1 � 3.4%. These studies demonstrated that a rabbit polyclonal antibody against recombinant bovine L-PGDS reacts with both oocytes and spermatozoa, resulting in inhibition of fertilization in vitro, and has a possible role in bovine fertilization.
This study was supported by FAPESP #2007/00363-5 and #2006/06008-0, Brazil.
235 DEVELOPMENT OF CAMEL (CAMELUS DROMEDARIUS) OOCYTES AFTER CHEMICAL ACTIVATION
Authors: Wani N. A.
Published: January 2008

Identification of an optimal protocol for activation of the MII oocytes in a species like camel not only allows us to evaluate the quality of oocytes after their in vitro maturation, but also is required for the success of advanced technologies like cloning. The present study was aimed to determine activation of in vitro-matured dromedary (Camelus dromedarius) oocytes using ionomycin or ethanol followed by sequential culture in phosphorylation inhibitor (6-dimethylaminopurine) or the specific maturation promoting factor inhibitor (roscovitine). Cumulus–oocyte complexes (COCs), collected from slaughterhouse ovaries, were randomly distributed to 4-well culture plates (20–25 COCs/well) containing 500 µL of the maturation medium. The maturation medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 20 ng mL–1 epidermal growth factor, 50 µg mL–1 gentamycin, 10 µg mL–1 bFSH, 10 µg mL–1 bLH, 1 µg mL–1 estradiol, and 10% estrous dromedary serum (EDS). The COCs were cultured at 38.5�C in an atmosphere of 5% CO2 in air for 36–40 h. The COCs were either fertilized in vitro (positive control) using epididymal spermatozoa collected from slaughtered males or activated with 5 µm ionomycin for 5 min or 7% ethanol for 7 min, both followed by exposure to 2 mm 6-DMAP or 50 µm roscovitine for 4 h. After being washed thoroughly in embryo culture medium, they were cultured for a period of 7 days at 38.5�C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. The embryo culture medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 50 µg mL–1 gentamicin, 1% insulin-transferrin-selenium (ITS) media supplement, and 10% EDS. First cleavage was recorded on Day 2 and the number of embryos developing to morulas and blastocysts was recorded on Day 7 of culture. The proportions of oocytes cleaved were 58.6 � 4.4, 55.9 � 4.5, 49.1 � 5.3, 43.2 � 6.05, and 54.1 � 3.3%, while the proportions of cleaved oocytes reaching blastocyst stage were 22.5 � 0.9, 19.1 � 2.8, 9.04 � 3.3, 8.2 � 3.8, and 15.2 � 2.3%, and those at morula stage were 61.1 � 4.9, 54.6 � 6.2, 67.1 � 7.2, 57.8 � 4.6, and 53.6 � 5.6% in the ionomycin/ 6-diethylaminopurine, ionomycin/roscovitine, ethanol/6-diethylaminopurine, ethanol/roscovitine, and IVF groups, respectively. The proportions of blastocysts obtained in the ionomycin/6-diethylaminopurine and ionomycin/roscovitine groups were higher (P < 0.05) when compared with the ethanol/6-diethylaminopurine and ethanol/roscovitine groups. Also, the proportion of blastocysts obtained in the ionomycin/6-diethylaminopurine group was higher than that in the in vitro-fertilized group. In summary, methods for oocyte or cytoplast activation in dromedary camel incorporating ionomycin/6-diethylaminopurine and ionomycin/roscovitine giving better results than those incorporating ethanol/6-diethylaminopurine and ethanol/roscovitine.
283 EXPRESSION OF TRANSCRIPTION FACTORS AND MESENCHYMAL DIFFERENTIATION OF STEM CELLS DERIVED FROM WHARTON'S JELLY OF PORCINE UMBILICAL CORD
Authors: Kang E. J. , Mohana Kumar B. , Song H. J. , Kim M. K. , Ock S. A. , Jeon B. J. , Balasubramanian S. , Rho G. J.
Published: January 2008

Wharton's jelly derived from mesoderm of umbilical cord matrix is an abundant source of stem cells such as mesenchymal and more primitive stem cells that don't raise the ethical issues associated with embryonic stem cells. The Oct-4 POU transcription factor, Nanog, and Sox-2 are expressed in embryonic stem cells and have been implicated in embryonic development and maintenance of pluripotency. The present study examined the expression of early transcription factors, Oct-4, Nanog, and Sox-2, and capability of differentiation to osteocytes, adipocytes, and chondrocytes of the cells of Wharton's jelly isolated from porcine umbilical cord. Wharton's jelly was obtained from the umbilical cord of post-natal piglets (immediately after birth). This tissue was diced into 2-mm-diameter explants and attached to tissue culture dishes. Cells were then cultured in DMEM/F12 supplemented with 10% fetal blood serum (FBS), 10 ng mL–1 basic fibroblast growth factor (bFGF), 1 nm dexamethasone, and 10 nm insulin at 38.5�C, 5% CO2 in air. Expression of Oct-4, Nanog, and Sox-2 by these cells was analyzed by immunocytochemistry and RT-polymerase chain reaction (PCR), and compared with expression by porcine bone marrow MSCs established in our laboratory. Osteogenic, adipogenic, and chondrogenic differentiation were induced following the protocols described earlier (Jin et al. 2007 Int. J. Dev. Biol. 51, 85–90; Mohana Kumar et al. 2007 Mol. Cells, in press). Cells isolated from Wharton's jelly expressed Oct-4, Nanog, and Sox-2 especially in colonies/clusters, as observed by immunocytochemistry and gene expression by RT-PCR, and this expression was higher than by MSCs isolated from bone marrow. Osteocytes were documented by the formation of the mineral nodules with alkaline phosphatase-positive cells and deposition of calcium. Differentiation into adipocytes was exhibited by oil red O staining of lipid vacuoles. Alcian blue staining for proteoglycans demonstrated the differentiation of chondrogenic cells. In conclusion, the results of the present study suggest that cells isolated from Wharton's jelly have properties of primitive pluripotency and multilineage differentiation. These stem cells, therefore, can serve as an easily accessible and expandable source possessing potential for preclinical applications using a large animal model.
This work was supported by Grant No. 20070301034040 from Bio-organ, Republic of Korea.
31 ALTERED HYPOTHALAMIC-PITUITARY-ADRENAL AXIS RESPONSE IN WEANED CLONED CALVES
Authors: Green M. P. , Berg M. C. , Lee R. S. F.
Published: January 2008

Somatic cell nuclear transfer (NT) is associated with an increased incidence of abnormal placental and fetal development. Postnatal survival of NT offspring to weaning and even into adult life is lower when compared to contemporary offspring produced by artificial insemination (AI). Neonatal NT calves are hypoglycemic and lethargic initially but show increased frequency of feeding later on. These and other symptoms suggest that NT animals have impaired homeostatic mechanisms. Despite this, NTs that survive beyond weaning appear normal, but underlying health problems can become apparent when NTs are subjected to physiological stressors. The health and physiology of predominantly (75%) Jersey NT bull calves (n = 9), derived from a fetal myogenic cell line, and control Jersey AI bull calves (n = 5) were investigated. At 6 months of age, animals were subjected to a series of physiological intravenous hormonal challenges to test the responses of individual organs. Pancreatic insulin secretion response was assessed via a glucose tolerance test (GTT) and direct adrenal resistance via ACTH administration. The response of the hypothalamic-pituitary-adrenal (HPA) axis was assessed via a glucagon challenge. Except for glucagon, the doses given were adjusted for body weight at the time of challenge and all animals were fasted overnight. Plasma glucose, insulin, glucagon, and cortisol concentrations were measured using commercial assays. All animals were euthanized at approximately 7 months of age and a full postmortem (PM) was undertaken. Calf and organ weights were recorded. Liver glycogen content was also determined. Parameters and challenge data were analyzed by one-way or as repeated measures ANOVA. NT and AI calves appeared healthy at the time of the physiological challenges, and at PM, no major gross or histological organ abnormalities were recorded. There were no differences in the relative mean liver and adrenal weights between NT and AI. Basal plasma glucose concentrations were similar between NT and AI controls but the post-fasting decrease in glucose concentration was greater (P < 0.05) in controls than in NTs. There was no difference in response between the groups to the GTT. The NTs showed a slower response to ACTH than did AI controls. Glucose and insulin secretion were significantly higher (P < 0.05) whereas initial cortisol release was significantly lower (P < 0.05) after glucagon administration. Plasma glucagon levels and liver glycogen content did not differ between NT and controls. The current study indicates that surviving NT calves appear healthy, but when physiologically challenged, demonstrate reduced adrenal sensitivity and altered HPA axis response. These response deviations are indicative of underlying physiological differences and could explain the increased susceptibility of NT animals to physiological stressors. Ultimately, the cloning procedures may affect the long-term health of cloned offspring.
311 RISK ASSESSMENT OF α-LACTALBUMIN TRANSGENIC PIGS
Authors: Wheeler M. B. , Hurley W. L. , Lane S. J. , Bressner G. E. , VanEtten T. , Kim D. , Lima A. S. , Monaco E. , Wilson S. M.
Published: January 2008

Assessment of the general risk posed by transgenic animals is important to their future contributions to society. Identification of potentially harmful properties of transgenic livestock is the initial step in a risk assessment. Direct and indirect impacts of potential harmful properties of transgenic livestock need to be evaluated at 3 levels: (1) characterization of how the transgene, the transgene product, and the transgenic livestock behave in their immediate environment; that is, in their barn or pen; (2) determination of possible impacts of large-scale release of transgenic livestock; that is, if they were to be integrated into the larger population of food animal livestock; and (3) determination of the more complex environmental and safety consequences of their release into the livestock population. We previously developed and characterized transgenic swine containing a mammary-specific transgene (bovine α-lactalbumin, bALAC) that results in increased milk production in sows (Bleck et al. 1998). We are currently determining whether bALAC is expressed in tissues of transgenic (T) swine other than the lactating mammary gland, and whether the transgene (DNA; Tg) crosses into non-transgenic control (C) swine under various physiological and physical conditions. The specific aims addressed in the present study were to determine (1) whether the Tg can be transferred directly from T animals to C animals by physical association or contact, and (2) whether the Tg can be transferred directly from an adult T animal to an adult C animal via mating. The T animals utilized in these studies were in generation 10 at least and have stable incorporation of the Tg. Comparable age- and weight-matched animals, T and C, were housed together allowing for general contact that is normal within swine production, for 180, 220, or 250 days after weaning. Due to the nature of swine behavior, these animals may ingest saliva, regurgitated food, and stool and urinary products as well as other bodily fluids and cells during normal housing and establishment of dominance hierarchy. In a second study, vaginal, cervical, uterine, oviductal, and ovarian tissues were collected from C females on 2 or 7 days after mating to T males. The presence of Tg in tissues from all C animals was tested via PCR. We have analyzed for the presence of the Tg in various tissues, including mammary gland, salivary gland, skin (sebaceous gland), muscle, lung, liver, kidney, brain, ovary, oviduct, uterus, cervix, vagina, and intestine. Preliminary results indicate no presence of the Tg in tissues of C animals (n = 20) after cohabitation for 180, 220, or 250 days (n = 201 samples analyzed) or at 2 (n = 3) or 7 (n = 5) days post-mating (n = 38 and 59 samples analyzed, respectively). This work provides a critical first step toward providing rigorous scientific data for risk assessment of transgenic livestock.
The USDA BRAG Program, Project No. 2005–03799, supported this work.
34 MEIOTIC RECOMBINATION IN SOMATIC CELL NUCLEAR TRANSFER BULLS AND THEIR OFFSPRING
Authors: Hart E. J. , Pinton A. , Powell A. , Wall R. , King W. A.
Published: January 2008

In mammals, homologous chromosome pairing and recombination are essential events for meiosis. The generation of reciprocal exchanges of genetic material ensure both genetic diversity and the proper segregation of homologous chromosomes. With the advent of reproductive biotechnologies such as somatic cell nuclear transfer (SCNT) in the livestock industry, these vital steps have begun to be bypassed. Despite this, there have been few studies carried out on the reproductive characteristics of cloned animals and none to date regarding the consequences on the meiotic process. As these procedures grow in popularity and use, the importance of evaluating the long-term viability, health, and productivity of cloned animals and any subsequent offspring in future generations to validate potential applications of the technology is increasingly evident. Previously, studies of recombination and synapsis have focused on the physical observation of chiasmata formation in meiotic chromosomes; however, in recent years, the characterization of proteins that localize to the sites of crossing-over and of proteins present in the synaptonemal complex have permitted the study of meiotic recombination using a precise direct immunocytogenetic approach. Cytological analysis of meiotic cells obtained from the testicular tissue of five normal bulls of proven fertility, two SCNT transgenic bulls (Powell et al. 2004 Biol. Reprod. 71, 210–216, 2004), and four reproductively mature offspring of SCNT bulls was performed in order to detect the effects of SCNT on the meiotic process. Over 50 pachytene cells per animal were analyzed by immunofluorescence using antibodies against the synaptonemal complex protein 3 (SCP3) and the mismatch repair protein 1 (MLH1) located on the crossover sites. Data were analyzed using a mixed model analysis of variance with repeated measurements to determine group effects (SAS 9.1; SAS Institute, Inc., Cary, NC, USA). The average number of crossovers per spermatocyte for the non-SCNT bulls (42 � 4 SD, min: 33, max: 56), SCNT bulls (43 � 5 SD, min: 35, max: 56), and SCNT offspring (46 � 4 SD, min: 37, max: 58) were quite similar among the cells of the same individual; however, inter-individual variation was observed. These results are the first documentation of the normal range of variability of recombination distribution within the cattle genome and suggest that the SCNT process does not affect meiotic recombination.
This work was funded by NSERC and the CRC program.
75 EFFECT OF PHENAZINE ETHOSULFATE ON PORCINE BLASTOCYST DEVELOPMENT, APOPTOSIS, AND CRYOTOLERANCE AFTER OPEN PULLED STRAW VITRIFICATION
Authors: Gajda B. , Smorag Z. , Bryla M.
Published: January 2008

It is possible to improve the success of cryopreservation of in vitro-produced bovine embryos by modifying the embryos with the metabolic regulator phenazine ethosulfate (PES) (Seidel 2006 Theriogenology 65, 228–235). The PES treatment increased glucose matabolism, tended to increase the pentose phosphate pathway flux of glucose, and clearly reduced accumulation of lipids in cultured bovine embryos (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607). It is known that porcine embryos have a considerably high content of lipids, and the success rates of their cryopreservation appear to be highly correlated with cytoplasmic lipid content. In our preliminary study, we observed that supplementation of NCSU-23 medium with PES has a positive effect on efficiency of pig blastocysts of good quality (Gajda et al.. 2007 Acta Biochim. Pol. 54(Suppl 1), 52 abst). In the present study, the effects of PES on pig blastocyst development, apoptosis, and survival after vitrification were investigated. In Exp. 1, porcine zygotes obtained from superovulated gilts were cultured in NCSU-23 medium supplemented with 0 (control), 0.025, 0.05, or 0.075 µm PES. The culture was performed at 39�C, with 5% CO2 in air, for 96–120 h. Embryo quality criteria were developmental competence (cleavage, morula stage, and blastocyst stage), cell number per blastocyst, and the degree of apoptosis as assessed by TUNEL staining. In Exp. 2, expanded blastocysts cultured with 0.025 µm PES were vitrified in a ethylene glycol and dimethyl sulfoxide mixture using open pulled straw (OPS) technology (Vajta et al. 1997 Acta Vet. Scand. 38, 349–352). After thawing, the blastocysts were cultured in vitro for re-expansion or transferred to synchronized recipients. Data were analyzed by chi-square test. There was a difference between the 0.025 µm PES-treated and the control group in percentage of cleaved embryos (99.0 and 91.4%, respectively; P < 0.05), between all experimental groups and control in percentage of morula stage (90.7, 87.8, 83.8, and 80.0%, respectively), and between 0.025 and 0.05 µm PES-treated and control in percentage of blastocyst rates (70.0, 75.5, and 65.7%, respectively). The number of cells and percentage of TUNEL-positive nuclei per blastocyst were lower in the PES-treated than in the control group. The survival rate of blastocysts after vitrification and thawing was enhanced in the presence of PES compared to that in the PES-free group (45.2 and 38.9%, respectively; P < 0.05). After transfer of 56 expanded blastocysts cultured with PES and vitrified into 3 recipients, two gilts were confirmed pregnant at 35 days of gestation. In conclusion, a higher blastocyst percentage with a low incidence of apoptosis was obtained in the presence of PES compared to control. These blastocysts also had an increased ability to survive cryopreservation.
92 PROCESSING OF POSTMORTEM BOVINE EPIDIDYMAL SPERM AFTER COOLING THE TESTES FOR 24 HOURS
Authors: Saenz J. R. , Guerrero C. A. , Paccamonti D. , Eilts B. , Bondioli K. R. , Godke R. A.
Published: January 2008

Harvesting and cryopreservation of epididymal sperm from postmortem animals is one way to save gametes from genetically valuable males. Before sperm can be properly harvested from the epididymides, usually the testes need to be cooled and transported to a semen laboratory. Previously, it has been recommended to harvest epididymal sperm from cooled testes in a 4°C cold room. However, a walk-in cold room is not always available. The objective of this study was to hold the testes for 22 h at 4°C, and then remove the epididymal sperm at room temperature (22 to 23°C; Treatment A) or in a cooled environment (4°C; Treatment B). Testicles within the scrotum were collected from sexually mature mixed breed bulls (n = 11) at a local abattoir, placed into plastic bags, and transported (2 h) in a Styrofoam ice chest (pre-cooled with frozen gel packs) to the laboratory. Each pair of testes was then removed from the ice chest and placed into a refrigerator at 4°C for 22 h. After 22 h of cooling, each testicle of the pair was removed from the scrotum and randomly assigned to either Treatment A or Treatment B. The sperm were flushed in a retrograde flow out of a small incision made at the medial section of the cauda epididymides. Total motility (TM) and progressive motility (PM) were determined using an inverted Nikon Diaphot microscope. Membrane integrity (MI) was determined using SYBR 14 and propidium iodide staining under a microscope with epifluorescence detection capability. Sperm in a Triladyl® one-step extender were frozen in 0.5-mL plastic straws in the vapor 2 cm above the LN2. A paired t-test was used for statistical analyses. In summary, there was significant decrease (P ≤ 0.05) from pre-freeze (PF) to post-thaw (PT) for all of the sperm parameters within each treatment (Table 1). However, there were no significant differences between PF and PT sperm parameters between treatments for any of the parameters measured. Post-thaw sperm from all samples had a tendency to swim in a circular pattern after warming, which is a known sign of sperm affected by cold shock. It is likely that during their cold storage, testes were cooled too quickly, inducing cold shock. In summary, cooling bull testes for 24 h and processing them at room temperature produced results similar to those for processing the testes in a cool environment. The PT parameters in this study suggest that these sperm could be used for IVF and/or ICSI procedures.

Table 1. Pre-freeze and post-thaw mean values (±SEM) for bull epididymal sperm parameters
Formation of large coronary arteries by cardiac progenitor cells
Authors: Tillmanns Jochen , Rota Marcello , Hosoda Toru , Misao Yu , Esposito Grazia , Gonzalez Arantxa , Vitale Serena , Parolin Carola , Yasuzawa-Amano Saori , Muraski John , De Angelis Antonella , LeCapitaine Nicole , Siggins Robert W. , Loredo Maria , Bearzi Claudia , Bolli Roberto , Urbanek Konrad , Leri Annarosa , Kajstura Jan , Anversa Piero
Published: January 2008

Coronary artery disease is the most common cause of cardiac failure in the Western world, and to date there is no alternative to bypass surgery for severe coronary atherosclerosis. We report that c-
kit
-positive cardiac progenitor cells (CPCs) activated with insulin-like growth factor 1 and hepatocyte growth factor before their injection in proximity of the site of occlusion of the left coronary artery in rats, engrafted within the host myocardium forming temporary niches. Subsequently, CPCs divided and differentiated into endothelial cells and smooth muscle cells and, to a lesser extent, into cardiomyocytes. The acquisition of vascular lineages appeared to be mediated by the up-regulation of hypoxia-inducible factor 1α, which promoted the synthesis and secretion of stromal-derived factor 1 from hypoxic coronary vessels. Stromal-derived factor 1 was critical in the conversion of CPCs to the vascular fate. CPCs formed conductive and intermediate-sized coronary arteries together with resistance arterioles and capillaries. The new vessels were connected with the primary coronary circulation, and this increase in vascularization more than doubled myocardial blood flow in the infarcted myocardium. This beneficial effect, together with myocardial regeneration attenuated postinfarction dilated myopathy, reduced infarct size and improved function. In conclusion, locally delivered activated CPCs generate
de novo
coronary vasculature and may be implemented clinically for restoration of blood supply to the ischemic myocardium.
Osteoblast expression of an engineered G
s
-coupled receptor dramatically increases bone mass
Authors: Hsiao Edward C. , Boudignon Benjamin M. , Chang Wei C. , Bencsik Margaret , Peng Jeffrey , Nguyen Trieu D. , Manalac Carlota , Halloran Bernard P. , Conklin Bruce R. , Nissenson Robert A.
Published: January 2008

Osteoblasts are essential for maintaining bone mass, avoiding osteoporosis, and repairing injured bone. Activation of osteoblast G protein-coupled receptors (GPCRs), such as the parathyroid hormone receptor, can increase bone mass; however, the anabolic mechanisms are poorly understood. Here we use “Rs1,” an engineered GPCR with constitutive G
s
signaling, to evaluate the temporal and skeletal effects of G
s
signaling in murine osteoblasts.
In vivo
, Rs1 expression induces a dramatic anabolic skeletal response, with midfemur girth increasing 1,200% and femur mass increasing 380% in 9-week-old mice. Bone volume, cellularity, areal bone mineral density, osteoblast gene markers, and serum bone turnover markers were also elevated. No such phenotype developed when Rs1 was expressed after the first 4 weeks of postnatal life, indicating an exquisite temporal sensitivity of osteoblasts to Rs1 expression. This pathway may represent an important determinant of bone mass and may open future avenues for enhancing bone repair and treating metabolic bone diseases.
The evolution of gene collectives: How natural selection drives chemical innovation
Authors: Fischbach Michael A. , Walsh Christopher T. , Clardy Jon
Published: January 2008

DNA sequencing has become central to the study of evolution. Comparing the sequences of individual genes from a variety of organisms has revolutionized our understanding of how single genes evolve, but the challenge of analyzing polygenic phenotypes has complicated efforts to study how genes evolve when they are part of a group that functions collectively. We suggest that biosynthetic gene clusters from microbes are ideal candidates for the evolutionary study of gene collectives; these selfish genetic elements evolve rapidly, they usually comprise a complete pathway, and they have a phenotype—a small molecule—that is easy to identify and assay. Because these elements are transferred horizontally as well as vertically, they also provide an opportunity to study the effects of horizontal transmission on gene evolution. We discuss known examples to begin addressing two fundamental questions about the evolution of biosynthetic gene clusters: How do they propagate by horizontal transfer? How do they change to create new molecules?
Rosiglitazone increases dendritic spine density and rescues spine loss caused by apolipoprotein E4 in primary cortical neurons
Authors: Brodbeck Jens , Balestra Maureen E. , Saunders Ann M. , Roses Allen D. , Mahley Robert W. , Huang Yadong
Published: January 2008

Convergent evidence has revealed an association between insulin resistance and Alzheimer's disease (AD), and the peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, rosiglitazone, an insulin sensitizer and mitochondrial activator, improves cognition in patients with early or mild-to-moderate AD. Apolipoprotein (apo) E4, a major genetic risk factor for AD, exerts neuropathological effects through multiple pathways, including impairment of dendritic spine structure and mitochondrial function. Here we show that rosiglitazone significantly increased dendritic spine density in a dose-dependent manner in cultured primary cortical rat neurons. This effect was abolished by the PPAR-γ-specific antagonist, GW9662, suggesting that rosiglitazone exerts this effect by activating the PPAR-γ pathway. Furthermore, the C-terminal-truncated fragment of apoE4 significantly decreased dendritic spine density. Rosiglitazone rescued this detrimental effect. Thus, rosiglitazone might improve cognition in AD patients by increasing dendritic spine density.
In and out of Iraq: A vote-seeking explanation of Berlusconi's Iraq policy
Authors: Davidson Jason W.
Published: January 2008

This article seeks to explain the Iraq policy of Silvio Berlusconi's second government. Why did Berlusconi's Government declare ‘non-belligerency’ when the American-led war with Iraq began in March 2003? Why did the Government send a mission of 3,000 Italian troops to Iraq in April 2003? Why did Berlusconi announce the progressive withdrawal of that contingent in March 2005? This article recognises the Berlusconi Government's ideologically-rooted pro-American tendencies, but draws on liberal international relations theory to stress the importance of the electoral motive to explain the timing and nature of the Government's decisions. As the Italian public was highly critical of the Iraq war, the Berlusconi Government could not actively participate in the war; it had to frame the deployment as a peace mission and had to begin withdrawing Italian troops before Iraq was stable and secure.