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Piyathip Setthawong Praopilas Phakdeedindan Mongkol Techakumphu Theerawat Tharasanit 《Reproduction in domestic animals》2021,56(8):1104-1116
Overall efficiency of cell reprogramming for porcine fibroblasts into induced pluripotent stem cells (iPSCs) is currently poor, and few cell lines have been established. This study examined gene expression during early phase of cellular reprogramming in the relationship to the iPSC colony morphology and in vitro pluripotent characteristics. Fibroblasts were reprogrammed with OCT4, SOX2, KLF4 and c-MYC. Two different colony morphologies referred to either compact (n = 10) or loose (n = 10) colonies were further examined for proliferative activity, gene expression and in vitro pluripotency. A total of 1,697 iPSC-like colonies (2.34%) were observed after gene transduction. The compact colonies contained with tightly packed cells with a distinct-clear border between the colony and feeder cells, while loose colonies demonstrated irregular colony boundary. For quantitative expression of genes responsible for early phase cell reprogramming, the Dppa2 and EpCAM were significantly upregulated while NR0B1 was downregulated in compact colonies compared with loose phenotype (p < .05). Higher proportion of compact iPSC phenotype (5 of 10, 50%) could be maintained in undifferentiated state for more than 50 passages compared unfavourably with loose morphology (3 of 10, 30%). All iPS cell lines obtained from these two types of colony morphologies expressed pluripotent genes and proteins (OCT4, NANOG and E-cadherin). In addition, they could aggregate and form three-dimensional structure of embryoid bodies. However, only compact iPSC colonies differentiated into three germ layers. Molecular signature of early phase of cell reprogramming coupled with primary colony morphology reflected the in vitro pluripotency of porcine iPSCs. These findings can be simply applied for pre-screening selection of the porcine iPSC cell line. 相似文献
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Chommanart THONGKITTIDILOK Theerawat THARASANIT Nucharin SONGSASEN Thanida SANANMUANG Sirirak BUARPUNG Mongkol TECHAKUMPHU 《The Journal of reproduction and development》2015,61(4):269-276
This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group
or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages. 相似文献
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Narong TIPTANAVATTANA Araya RADTANAKATIKANON Poul HYTTEL Hanne HOLM Supranee BURANAPRADITKUN Piyathip SETTHAWONG Mongkol TECHAKUMPHU Theerawat THARASANIT 《The Journal of reproduction and development》2015,61(6):581-588
The development of germ cells has not been entirely documented in the cat especially the transition phase of
the gonocyte to the spermatogonial stem cell (G/SSC). The aims of study were to examine testicular development
and to identify the G/SSC transition in order to isolate and culture SSCs in vitro. Testes
were divided into 3 groups according to donor age (I, < 4 months; II, 4–6 months; and III, > 6 months).
In Exp. 1, we studied testicular development by histology, transmission electron microscopy and
immunohistochemistry. In Exp. 2, we determined the expression of GFRα-1, DDX-4 and c-kit and performed flow
cytometry. The SSCs isolated from groups II and III were characterized by RT-PCR and TEM (Exp. 3).
Chronological changes in the G/SSC transition were demonstrated. The size, morphology and ultrastructure of
SSCs were distinguishable from those of gonocytes. The results demonstrated that group II contained the
highest numbers of SSCs per seminiferous cord/tubule (17.66 ± 2.20%) and GFRα-1+ cells (14.89 ±
5.66%) compared with the other groups. The findings coincided with an increased efficiency of SSC derivation
in group II compared with group III (74.33 ± 2.64% vs. 23.33 ± 2.23%). The colonies expressed
mRNA for GFRA1, ZBTB16, RET and POU5F1.
Our study found that the G/SSC transition occurs at 4–6 months of age. This period is useful for isolation and
improves the establishment efficiency of cat SSCs in vitro. 相似文献
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Vibuntita CHANKITISAKUL Nutthee AM-IN Theerawat THARASANIT Tamas SOMFAI Takashi NAGAI Mongkol TECHAKUMPHU 《The Journal of reproduction and development》2013,59(1):66-71
Failure of male pronucleus formation has hampered the success of intracytoplasmic sperm
injection (ICSI) in swamp buffalo. The aim of the present study was to improve male
pronucleus formation by pretreating sperm with various chemicals before ICSI. In
Experiments1 and 2, sperm were treated according to one of the following protocols: (1)
0.1% Triton-X 100 (TX) for 1 min, (2) 10 µM calcium ionophore (CaI) for 20 min, (3)
freezing and thawing (FT) without any cryoprotectant, or (4) no treatment (control). These
sperm treatment groups then either did or did not receive additional sperm treatment with
5 mM dithiothreitol (DTT) for 20 min. Acrosomal integrity (Experiment 1) and DNA
fragmentation (Experiment 2) were evaluated in the sperm before ICSI. In Experiment 3,
oocytes matured in vitro were subjected to ICSI using pretreated sperm as
described above and then were cultured either with or without activation. The TX- and
CaI-treated sperm caused an increase in the number of acrosome-loss sperm, whereas the FT
treatment and control increased the proportion of acrosome-reacted sperm (P<0.05). The
DNA fragmentation did not differ among treatments (P>0.05). At 18 h post-ICSI,
pronucleus (PN) formation was found only in activated oocytes. The majority of the
activated ICSI oocytes contained intact sperm heads. Normal fertilization was observed in
the CaI and FT treatment groups and control group when sperm were treated with DTT before
ICSI. In conclusion, DTT treatment of sperm with reacted acrosomes before ICSI together
with activation of the ICSI oocytes is important for successful male pronucleus
formation. 相似文献
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Epigenetic modulation on cat‐cow interspecies somatic cell nuclear transfer embryos by treatment with trichostatin A 
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下载免费PDF全文 Manita Wittayarat Yoko Sato Lanh Thi Kim Do Kaywalee Chatdarong Theerawat Tharasanit Mongkol Techakumphu Masayasu Taniguchi Takeshige Otoi 《Animal Science Journal》2017,88(4):593-601
This study aimed to determine the acetylation at lysine 9/18/23 of histone H3 (H3K9ac/H3K18ac/H3K23ac; H3K9/18/23 ac) and the di‐methylation at lysine 9 of histone H3 (H3K9me2) during early embryogenesis among trichostatin A (TSA)‐treated interspecies somatic cell nuclear transfer (iSCNT) cat‐cow (TSA‐iSCNT) embryos, TSA‐untreated iSCNT cat‐cow control (control) embryos and bovine in vitro fertilization (IVF) embryos, because TSA‐iSCNT embryos can develop to blastocysts. Compared to the control embryos, higher expressions of H3K9/18/23 ac were observed in TSA‐iSCNT embryos and IVF embryos at most following stages (2 h post‐fusion / post‐insemination (PF/PI) to eight‐cell stage). At 6 h PF/PI the expression of H3K9/23 ac in TSA‐iSCNT embryos and IVF embryos were lower than those in control embryos, and the expression of H3K18ac was no difference among the three groups. The expression of H3K9/23 ac increased in TSA‐iSCNT embryos and IVF embryos at pronuclear (PN) stages. The expression of H3K9me2 in TSA‐iSCNT embryos resembled that of IVF embryos at 2 h PF/PI to PN stages, and these expression levels were greater than those of control embryos. These results suggest that treatment of iSCNT embryos with TSA modifies the patterns of histone modification at certain lysine residues in a manner that is comparable with that seen in IVF during early embryogenesis. 相似文献
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Narong TIPTANAVATTANA Mongkol TECHAKUMPHU Theerawat THARASANIT 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2015,77(11):1347-1353
The efficiency of spermatogonial stem cell (SSC) isolation and culture from pubertal
donors is currently poor primarily, because of contamination with other testicular cells.
This study aimed to purify SSC-like cells using different extracellular matrixes and a
discontinuous gradient density. In experiment 1, testes (n=6) were analyzed for histology
and SSC-related protein expressions (laminin, SSEA-4, DDX-4 and GFRα-1). After enzymatic
digestion, the cell suspension was plated onto either a laminin- or gelatin-coated dish.
The number of SSC-like cells was determined at 15, 30 and 60 min of culture (experiment
2). Experiment 3 was performed to test whether or not the additional step of Percoll
gradient density centrifugation could really improve purification of SSC-like cells.
Testicular histology revealed complete spermatogenesis with laminin expression essentially
at the basal lamina of the seminiferous tubules. SSEA-4 and GFRα-1 co-localized with DDX-4
in the spermatogonia. The relative percentage of SSC-like cells, as determined by cells
expressing SSEA-4 (59.42 ± 2.18%) and GFRα-1 (42.70 ± 1.28%), revealed that the highest
SSC-like cell purity was obtained with the 15-min laminin-coated dish compared with other
incubation times and gelatin treatment (P<0.05). Percoll treatment
prior to laminin selection (15 min) significantly improved SSC-like cell recovery (91.33 ±
0.14%, P<0.001) and purity (83.82 ± 2.05% for SSEA-4 and 64.39 ± 1.51%
for GFRα-1, P<0.05). These attached cells demonstrated a typical
SSC-like cell morphology and also expressed POU5F1, RET
and ZBTB16 mRNA. In conclusion, double enrichment with Percoll gradient
density centrifugation and laminin plating highly enriched the SSC-like cells
population. 相似文献
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de Graaf-Roelfsema E Tharasanit T van Dam KG Keizer HA van Breda E Wijnberg ID Stout TA van der Kolk JH 《American journal of veterinary research》2005,66(11):1907-1913
OBJECTIVE: To determine the effects of short-term IV administration of hydrocortisone or equine growth hormone (eGH) or long-term IM administration of eGH to horses on tissue sensitivity to exogenous insulin. ANIMALS: 5 Standardbreds and 4 Dutch Warmblood horses. PROCEDURE: The euglycemic-hyperinsulinemic clamp technique was used to examine sensitivity of peripheral tissues to exogenous insulin 24 hours after administration of a single dose of hydrocortisone (0.06 mg/kg), eGH (20 microg/kg), or saline (0.9% NaCl) solution and after long-term administration (11 to 15 days) of eGH to horses. The amounts of metabolized glucose (M) and plasma insulin concentration (I) were determined. RESULTS: Values for M and the M-to-I ratio were significantly higher 24 hours after administration of a single dose of hydrocortisone than after single-dose administration of eGH or saline solution. After long-term administration of eGH, basal I concentration was increased and the mean M-to-I ratio was 22% lower, compared with values for horses treated with saline solution. CONCLUSIONS AND CLINICAL RELEVANCE: Increases in M and the M-to-I ratio after a single dose of hydrocortisone imply that short-term hydrocortisone treatment increases glucose use by, and insulin sensitivity of, peripheral tissues. Assuming a single dose of hydrocortisone improves sensitivity of peripheral tissues to insulin, it may be an interesting candidate for use in reducing insulin resistance in peripheral tissues of horses with several disease states. In contrast, long-term administration of eGH decreased tissue sensitivity to exogenous insulin associated with hyperinsulinemia. Therefore, increased concentrations of growth hormone may contribute to insulin resistance in horses with various disease states. 相似文献
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