Characterization and multilineage differentiation of embryonic stem cells derived from a buffalo parthenogenetic embryo.

Embryonic stem (ES) cells derived from mammalian embryos have the ability to form any terminally differentiated cell of the body. We herein describe production of parthenogenetic buffalo (Bubalus Bubalis) blastocysts and subsequent isolation of an ES cell line. Established parthenogenetic ES (PGES) cells exhibited diploid karyotype and high telomerase activity. PGES cells showed remarkable long-term proliferative capacity providing the possibility for unlimited expansion in culture. Furthermore, these cells expressed key ES cell-specific markers defined for primate species including stage-specific embryonic antigen-4 (SSEA-4), tumor rejection antigen-1-81 (TRA-1-81), and octamer-binding transcription factor 4 (Oct-4). In vitro, in the absence of a feeder layer, cells readily formed embryoid bodies (EBs). When cultured for an extended period of time, EBs spontaneously differentiated into derivatives of three embryonic germ layers as detected by PCR for ectodermal (nestin, oligodendrocytes, and tubulin), mesodermal (scleraxis, alpha-skeletal actin, collagen II, and osteocalcin) and endodermal markers (insulin and alpha-fetoprotein). Differentiation of PGES cells toward chondrocyte lineage was directed by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and dexamethasone. Moreover, when PGES cells were injected into nude mice, teratomas with derivatives representing all three embryonic germ layers were produced. Our results suggest that the cell line isolated from a parthenogenetic blastocyst holds properties of ES cells, and can be used as an in vitro model to study the effects of imprinting on cell differentiation and as an a invaluable material for extensive molecular studies on imprinted genes.

Complete deficiencies of complement C4A and C4B including 2-bp insertion in codon 1213 are genetic risk factors of systemic lupus erythematosus in Thai populations.

The complement component C4 is encoded by two genes: C4A and C4B on human chromosome 6p in the major histocompatibility complex (MHC). Most studies have linked the deficiencies in C4 with systemic lupus erythematosus (SLE) in Angio-Irish, North American, Black American, Mexican American, Australian and Japanese populations. Null alleles at either locus (C4AQ0 or C4BQ0) are relatively common in Americans occurring at the C4A and C4B loci in approximately 10% and 16% of normal individuals, respectively. In the present study, we extensively examined the possible association between homozygous C4Q0 and SLE in a large cohort of Thai populations diagnosed as SLE and further attempted to identify the genetic basis of C4Q0. One hundred and eighteen cases of SLE patients and 145 matched controls were genotyped by touchdown PCR. The results confirmed the previous studies that 5.93% (7/118) of C4 null genes: 2.54% (3/118) of C4AQ0 and 3.39% (4/118) of C4BQ0 were found in SLE patients. In contrast to other studies, we found no cases of C4 null genes in normal control (0 from 145 samples). To further investigate the genetic basis of C4 deficiency, all genomic DNAs were also analyzed for 2-bp (TC) insertion at codon 1213 in exon 29 which is a common mutation in many C4A null genes and a novel 1-bp deletion (C) at codon 522 in exon 13 that is common in most C4B null genes. Both mutation results in a flame-shift mutation and premature stop codon using sequence specific primers PCR (SSP-PCR) and direct sequencing. The results showed that there was 2-bp insertion in exon 29 of mutant C4B gene in one SLE patient carrying C4AQ0. There was no 2-bp insertion in exon 29 of both C4A and C4B genes in normal individual and the rest of SLE patients. All patients with C4AQ0 exhibited more than 5 ACR criteria including malar rash, oral ulcers, renal disorder, immunological disorder, anti-nuclear antibody, without hematological disorder. In contrast, all of C4BQ0 SLE patients showed 5 or 6 ACR criteria including hematological disorder, malar rash, oral ulcers, renal disorder, immunological disorder and anti-nuclear antibody. A patient who possesses C4AQ0 and 2-bp insertion in exon 29 of mutant C4B showed 9 ACR criteria but no discoid rash and hematological disorder. In conclusion, both C4AQ0 and C4BQ0 are the strong predisposing factors for SLE in Thais. It was supported by the absence of either C4A or C4B deletion in healthy control. We suggested that the different racial and genetic backgrounds could alter the thresholds for requirement of C4A or C4B protein levels in immune tolerance and regulation.

In view of an optimal gut antifungal therapeutic strategy: an in vitro susceptibility and toxicity study testing a novel phyto-compound.

OBJECTIVE: In view of the raising concern for gut fungal infection, the aim of the present research was to carry out a systematic in vitro study testing the antifungal activity and possible toxicity of a polygodyal-anethole compound (Kolorex) in several strains of Candida albicans and in other fungal pathogens. METHODS: The in vitro susceptibility tests were carried out on 4 strains of C. albicans (C. krusei, C. lipolytica, C. tropicalis, C. utilis), Aspergillus flavus and A. fumigatus. Cultures were also analyzed by varying medium, pH and inoculum size, and a time-course killing test was carried out. RESULTS: In the present study the polygodyal-anethole compound showed remarkable in vitro activity against the most common fungi, which was significantly better than polygodyal alone. Moreover, such mixture compound was shown to exert its activity against a wide spectrum of fungi, including C. lipolytica and C. tropicalis, which required significantly higher MIC of polygodyal to be unfeasible in clinical application. The activity of the polygodyal-anethole compound was significantly better than polygodyal alone with high inoculum size and low pH. Moreover, it proved to exert a significantly faster biological activity against low inoculum. CONCLUSIONS: This study suggests that the mixture compound Kolorex has a very good profile of antifungal activity in terms of effectiveness and spectrum of action while being devoid of any significant toxicity.

Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa using flow cytometry: the effects of extender and cryoprotectant.

Although the development of semen cryopreservation in the African elephants (Loxodonta africana) has been accomplished, effective procedures for cryopreservation of Asian elephant (Elephas maximus) spermatozoa have not been established. In the present study, we investigate the freezing methods for conservation of Asian elephant spermatozoa under field conditions and identify the most suitable freezing protocols which provide acceptable post-thaw semen quality. Semen was collected from two Asian elephant bulls (EM1 and EM2, 10 ejaculates from each bull) by manual manipulation and were assessed for volume, pH, sperm cell concentration, and progressive motility. Eight out of 20 ejaculates were of acceptable quality (progressive motility >/= 60%), and were used for cryopreservation studies. Semen were frozen in TEST + glycerol, TEST + DMSO, HEPT + glycerol, or HEPT + DMSO. The post-thaw progressive sperm motilities were assessed, and sperm cells were stained with PI and FITC-PNA for membrane and acrosomal integrity assessment using flow cytometry. Post-thaw progressive motility of spermatozoa (EM1: 42.0 +/- 4.3%; EM2: 26.0 +/- 17.3%) and the percentage of membrane and acrosome intact spermatozoa (EM1: 55.5 +/- 8.1%; EM2: 46.3 +/- 6.4%) cryopreserved in TEST + glycerol were significantly higher than (P < 0.05) those frozen in the other medium investigated choices for cryopreservation of Asian elephant spermatozoa. The data support the use of TEST + glycerol as an acceptable cryopreservation media of Asian elephant semen for the establishment of sperm banks.

Development of swamp buffalo (Bubalus bubalis) embryos after parthenogenetic activation and nuclear transfer using serum fed or starved fetal fibroblasts.

The knowledge of oocyte activation and somatic cell nuclear transfer in the swamp buffalo (Buballus bubalis) is extremely rare. The objectives of this study were the following: (1) to investigate the various activation treatments on the parthenogenetic development of buffalo oocytes, (2) to examine the events of nuclear remodeling and in the in vitro development of cloned buffalo embryos reconstructed with serum fed or starved fetal fibroblasts, and (3) to investigate the in vivo development of cloned embryos derived from serum fed or starved cells after transfer into the recipients. The rates of cleavage and blastocyst development were found to be significantly higher (P < 0.05) when the oocytes were activated by the combination treatment of calcium ionophore (A23187) or ethanol followed by 6-DMAP than those activated by electrical pulses and 6-DMAP or other single treatments. Flow cytometric analysis revealed that the percentage in the G0/G1 phase in serum starved cells was significantly (P < 0.05) higher than that in serum fed cells (88.8 +/- 6.2 vs. 68.2 +/- 2.6). At 1 h post fusion (hpf), most of the transferred nuclei (71%) from serum fed cells did not change in size, and the nuclear envelope remained intact, whereas 29% underwent NEBD and PCC. When serum starved cells were used, 83% of the transferred nuclei underwent NEBD and PCC whereas 17% remained intact. The nuclear swelling and pronucleus (PN) formation were observed at 2-4 and 12 h post activation (hpa), respectively. The remodeled nuclei underwent mitotic division and developed to the 2-cell stage within 18-24 hpa. Fifty-five percent of oocytes reconstructed with serum fed cells were 2PN and 45% were 1PN, whereas 79% of the embryos reconstructed from starved cells were 1PN and 21% were 2PN. The percentage of blastocyst development of the embryos derived from starved cells was higher than that from the serum fed cells (35% vs. 21%, P < 0.05). Pregnancy was detected after the transfer of cloned blastocysts into the recipients but no recipients supported the development to term. The results of this work can be used to establish effective activation protocols for buffalo oocytes which can be used during nuclear transfer experiments.

Somatic cell nuclear transfer in domestic cat oocytes treated with IGF-I for in vitro maturation.

Oocyte maturation and somatic cell nuclear transfer (NT) studies conducted in the domestic cat can provide valuable insights that are relevant to the conservation of endangered species of felids. The present investigation focuses on the in vitro maturation (IVM) of domestic cat oocytes stimulated by insulin-like growth factor-I (IGF-I) and their possible use as recipient cytoplasts for somatic cell NT. In Experiment I, the effects of IGF-I on cat oocyte IVM were monitored. Cumulus-oocyte complexes (COCs) were recovered in TALP-HEPES medium following ovarian follicular aspiration and were classified under a stereomicroscope into four grades using criteria based on cumulus cell investment and the uniformity of ooplasm. The COCs were either cultured in Dulbecco's modified Eagle medium (DMEM) alone as a control group or supplemented with 100 ng/ml IGF-I. After culturing for 32-34 h, oocytes were denuded and maturation rate was evaluated by observing the extrusion of the first polar body and staining with aceto-orcein. The percentages of maturation of Grades 1 and 2 oocytes were significantly increased (P<0.05) in IGF-I supplemented medium compared with medium alone (85.8 versus 65.5 and 70.3 versus 51.8, respectively) whereas the maturation rates of Grades 3 and 4 oocytes were not different. The IVM of Grade 1 oocytes was significantly higher (P<0.05) than for all other grades in both control and experimental groups. In Experiment II, the in vitro development of cat NT embryos using cumulus cells, fetal or adult fibroblasts as donor nuclei was investigated. The IVM oocytes in medium containing IGF-I were enucleated and fused with cumulus cells, fetal or adult fibroblasts between passages 2 and 4 of culture. Reconstructed embryos were cultured and monitored every 24h for progression of development through Day 9. There was no significant difference in the percentage of fusion of NT embryos using different donor nuclei whereas the cleavage rates of NT embryos reconstructed with fetal fibroblasts and cumulus cells were significantly higher (P<0.05) than those reconstructed with adult fibroblasts (72.5 and 70.7% versus 54.8%, respectively). Development of NT embryos reconstructed with adult fibroblast to the morula stage was significantly lower (P<0.05) compared with cumulus cell or fetal fibroblast donor cells (25.8% versus 37.9 or 47.5%, respectively). However, no difference was observed in development to the blastocyst stage. These results demonstrated that IGF-I promoted the IVM of domestic cat oocytes. The enucleated IVM oocytes could be used as recipient cytoplasm for fetal and adult somatic cell nuclei resulting in the production of cloned cat embryos.

Xenonuclear transplantation of buffalo (Bubalus bubalis) fetal and adult somatic cell nuclei into bovine (Bos indicus) oocyte cytoplasm and their subsequent development.

Bovine oocyte cytoplasm has been shown to support the development of nuclei from other species up to the blastocyst stage. Somatic cell nuclei from buffalo fetal fibroblasts have been successfully reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts. The aim of this study was to compare the in vitro development of fetal and adult buffalo cloned embryos after the fusion of a buffalo fetal fibroblast, cumulus or oviductal cell with bovine oocyte cytoplasm. The fusion of oviductal cells with enucleated bovine oocytes was higher than that of fetal fibroblasts or cumulus cells (83% versus 77 or 73%, respectively). There was a significantly higher cleavage rate (P < 0.05) for fused nuclear transferred embryos produced by fetal fibroblasts and oviductal cells than for cumulus cells (84 or 78% versus 68%, respectively). Blastocyst development in the nuclear transferred embryos produced by fetal fibroblasts was higher (P < 0.05) than those produced either by cumulus or oviductal cells. Chromosome analysis of cloned blastocysts confirmed the embryo was derived from buffalo donor nuclei. This study demonstrates that nuclei from buffalo fetal cells could be successfully reprogrammed to develop to the blastocyst stage at a rate higher than nuclei from adult cells.