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Relatively, little is known about the corpus luteum (CL) function in early pregnancy after the successful treatment of luteal phase deficiency in repeat-breeder dairy cows when exposed to extreme environments under tropical climate. To investigate the influence of increased tissues of corpora lutea (CLs) by inducing secondary CL based on progesterone (P4) concentration and fertility in repeat-breeder dairy cows undergoing the fixed-time artificial insemination (FTAI) protocol, 32 cows were treated with gonadotropin releasing hormone (GnRH) on day 5 post-induction (experiment 1). In experiment 2, 213 cows were bred using the short-term FTAI protocol. On day 5 post-FTAI, cows were divided into two groups: treatment with (GnRH5-treated group) or without (GnRH5-untreated group) GnRH. The temperature-humidity index ranged from 77.3 to 82.8. Cows bearing two CLs had greater P4 concentrations than cows bearing only one CL on their ovaries (P 0.05). Pregnancy rates were greater in GnRH5-treated group than the GnRH5-untreated group (P 0.01). Moreover, repeat-breeder cows bearing two CLs had a greater likelihood of pregnancy (odds ratio = 20.86) than cows bearing only one CL on their ovaries (P 0.01). Under heat stress condition, the results highlighted that increasing luteal tissues by creating secondary CL leads to enhanced peripheral P4 concentrations and improved pregnancy outcomes in repeat-breeder dairy cows.

The hypothalamus is the coordinating center for maintaining temperature homeostasis. In this study, global protein expression in the hypothalami of layer-type Taiwan country chickens in response to acute heat stress was investigated. Twelve 30-week-old female TCCs were divided into three acute heat-stressed groups, namely acute heat stress at 36 °C for 4 h with 0 h (without recovery, H4R0), 2 h (H4R2), or 6 h (H4R6) of recovery. A control group was maintained at 25 °C. Hypothalamus samples were collected at the end of each time point for proteomic analysis. The analysis results revealed that 134 protein spots representing 118 distinct proteins exhibited differential expressions after acute heat stress treatment. Results of gene ontology analysis showed that most of the differentially expressed proteins are involved in carbohydrate metabolism, cellular processes, actin cytoskeleton organization, and responses to stimuli. Functional pathway analysis results suggested that the proteins are associated with networks of carbon metabolism, glycolysis, and gluconeogenesis. Upregulation of the expression of triosephosphate isomerase, phosphoglycerate kinase, pyruvate kinase, alpha-enolase, glycogen phosphorylase (brain form), phosphoglucomutase, L-lactate dehydrogenase A chain and downregulation of 6-phosphogluconolactonase expression indicated an increase in the glycolytic activity and glucose supply for ATP production in the hypothalami in response to heat stress. By contrast, upregulated expressions of heat shock protein 90 alpha, glutathione S-transferase 2s, peroxiredoxin-1, and dihydropyrimidinase-like 2 suggested that acute heat stress adversely affects the hypothalamus; thus, it induces mechanisms that prevent oxidative damage and endoplasmic reticulum stress. In conclusion, acute heat stress induces differential protein expression in the hypothalami of the L2 strain Taiwan country chickens, which may manifest detrimental effects. Furthermore, differential expression is a critical response in the hypothalamus for the regulation of thermotolerance.

SummaryMuch effort has been devoted to improving the efficiency of animal cloning. The aim of this study was to investigate the effect of BRG1 contained in Xenopus egg extracts on the development of cloned mouse embryos. The results showed that mouse NIH/3T3 cells were able to express pluripotent genes after treatment with egg extracts, indicating that the egg extracts contained reprogramming factors. After co-injection of Xenopus egg extracts and single mouse cumulus cells into enucleated mouse oocytes, statistically higher pronucleus formation and development rates were observed in the egg Extract- co-injected group compared with those in the no egg extract-injected (NT) group (38-66% vs 18-34%, Plt;0.001). Removal of BRG1 protein from Xenopus egg extracts was conducted, and the BRG1-depleted extracts were co-injected with single donor cells into recipient oocytes. The results showed that the percentages of pronucleus formation were significantly higher in both BRG1-depleted and BRG1-intact groups than that in the nuclear transfer (NT) group (94, 64% vs 50%, Plt;0.05). Furthermore, percentages in the BRG1-depleted group were even higher than in the BRG1-intact group (94% vs 64%). More confined expression of Oct4 in the inner cell mass (ICM) was observed in the blastocyst derived from the egg extract-injected groups. However, Nanog expression was more contracted in the ICM of cloned blastocysts in the BRG1-depleted group than in the BGR1-intact group. Based on the present study, BRG1 might not play an essential role in reprogramming, but the factors enhancing pronucleus formation and development of cloned mouse embryos are contained in Xenopus egg extracts.

Many animals can change the size, shape, texture and color of their regenerated coats in response to different ages, sexes, or seasonal environmental changes. Here, we propose that the feather core branching morphogenesis module can be regulated by sex hormones or other environmental factors to change feather forms, textures or colors, thus generating a large spectrum of complexity for adaptation. We use sexual dimorphisms of the chicken to explore the role of hormones. A long-standing question is whether the sex-dependent feather morphologies are autonomously controlled by the male or female cell types, or extrinsically controlled and reversible. We have recently identified core feather branching molecular modules which control the anterior-posterior (bone morphogenetic orotein [BMP], Wnt gradient), medio-lateral (Retinoic signaling, Gremlin), and proximo-distal (Sprouty, BMP) patterning of feathers. We hypothesize that morpho-regulation, through quantitative modulation of existing parameters, can act on core branching modules to topologically tune the dimension of each parameter during morphogenesis and regeneration. Here, we explore the involvement of hormones in generating sexual dimorphisms using exogenously delivered hormones. Our strategy is to mimic male androgen levels by applying exogenous dihydrotestosterone and aromatase inhibitors to adult females and to mimic female estradiol levels by injecting exogenous estradiol to adult males. We also examine differentially expressed genes in the feathers of wildtype male and female chickens to identify potential downstream modifiers of feather morphogenesis. The data show male and female feather morphology and their color patterns can be modified extrinsically through molting and resetting the stem cell niche during regeneration.

This study investigated global gene and protein expression in the small yellow follicle (SYF; 6–8 mm in diameter) tissues of chickens in response to acute heat stress. Twelve 30-week-old layer-type hens were divided into four groups: control hens were maintained at 25 °C while treatment hens were subjected to acute heat stress at 36 °C for 4 h without recovery, with 2-h recovery, and with 6-h recovery. SYFs were collected at each time point for mRNA and protein analyses. A total of 176 genes and 93 distinct proteins with differential expressions were identified, mainly associated with the molecular functions of catalytic activity and binding. The upregulated expression of heat shock proteins and peroxiredoxin family after acute heat stress is suggestive of responsive machineries to protect cells from apoptosis and oxidative insults. In conclusion, both the transcripts and proteins associated with apoptosis, stress response, and antioxidative defense were upregulated in the SYFs of layer-type hens to alleviate the detrimental effects by acute heat stress. However, the genomic regulations of specific cell type in response to acute heat stress of SYFs require further investigation.

The application of appropriate animal models and techniques for the study of osteoporosis is important. Lanyu pigs, a local miniature breed, have been widely used in various biomedical studies in Taiwan. This study aimed to induce bone loss in Lanyu pigs and to examine whether porcine induced pluripotent stem cell (piPSC)-derived osteoblast-like cells could recover bone mass of tibiae via local cell transplantation. piPSCs were directed to differentiate into osteoblast-like cells using osteogenic medium, and differentiated cells expressed osteogenic markers and phenotypes. Twenty mature female Lanyu pigs were divided into four groups, including control (C, 1% calcium diet), treatment 1 (T1, ovariectomy + 1% calcium diet), treatment 2 (T2, ovariectomy + 0.5% calcium diet), and treatment 3 (T3, ovariectomy + 0.5% calcium diet + 1 mg/kg of prednisolone) and were subjected to bone loss induction for twelve months. Micro-CT images revealed that the lowest trabecular bone parameters, such as trabecular bone volume, thickness, separation, number, and total porosity, were detected in the T3 group. The lowest proportions of cortical bone in the proximal metaphysis, proximal diaphysis, and distal diaphysis were also found in the T3 group. These results indicate that ovariectomy, calcium restriction, and prednisolone administration can be applied to induce proper bone loss in Lanyu pigs. After bone loss induction, pigs were subjected to cell transplantation in the left tibiae and were maintained for another six months. Results showed that transplanted piPSC-derived osteoblast-like cells significantly improved trabecular bone structures at transplanted sites and maintained cortical bone structures in the proximal metaphysis. In conclusion, the therapeutic potential of piPSC-derived osteoblast-like cells was confirmed via cell transplantation in the left tibiae of Lanyu pigs. These findings reveal the therapeutic potential of piPSCs for glucocorticoid-induced bone loss in pig models.

Mucin 1 (Muc1) is an integral transmembrane mucin glycoprotein expressed on the apical surface of most epithelia. It is considered to be a barrier to the regulation of embryo implantation by inhibiting attachment of the embryo to the endometrium. Therefore, loss of Muc1 on the surface of uterine epithelial cells is necessary for embryo implantation. Studies have demonstrated that microRNAs (miRNAs) play a key role in enhancing embryo implantation in mammals. In this study, we investigated the regulatory role of two miRNAs (let-7a and let-7b) on the expression of Muc1 in mouse uteri during implantation. Western blotting indicated that Muc1 expression was highest on day1 of pregnancy and constantly decreased thereafter until day 4. In contrast to Muc1 expression, increased expression of let-7a and let-7b was evident on day 4 of pregnancy as measured by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). We demonstrated direct binding of let-7a and let-7b to the 3'untranslated region of muc1. Furthermore, Muc1 expression was suppressed after transfection of mouse uterine epithelial cells isolated from day 1 of pregnancy with let-7a and let-7b. In summary, the present study provides evidence that Muc1 is a direct target of let-7a and let-7b. Additionally, the current study suggests that miRNAs are novel targets which can be used to facilitate a successful pregnancy and repair implantation failure.

The hypothalamus is a critical center for regulating heat retention or dissipation. This study investigated global protein changes in the hypothalamus of broiler-type Taiwan country chickens (TCCs) after acute heat stress. Twelve TCC hens aged 30 weeks were allocated to groups subjected to acute heat stress at 38°C for 2 hr without recovery, with 2 hr of recovery, and with 6 hr of recovery; a control group was maintained at 25°C. Hypothalami were collected for protein expression analysis at the end of each time point. The results showed 114 protein spots differentially expressed after acute heat stress. Most of the differentially expressed proteins were involved in cellular processes, metabolism, transport, and cellular component organization. Functional annotation analysis suggested that these proteins were related to cellular defensive responses against heat and oxidative stress, detoxification and toxin export/delivery, cytoskeleton integrity, oxygen transport, and neural development. The results of this study suggest that acute heat stress damages the hypothalamus of broiler-type TCCs through oxidative stress and provokes a series of responses to stabilize protein structures, degrade misfolded proteins, and remodel cytoskeletons for attenuating the detrimental effects by acute heat stress.

Inconsistent reproductive performance has been reported in superovulated mice. Hence, the aim of this study was to analyze the effect and possible mechanism of superovulation timing on mouse reproductive performance. The results showed that mice superovulated at the metestrous (23.08±6.08%) and diestrous stages (33.33±11.45%) presented significantly lower pregnancy rates compared with those superovulated at the estrous stage (66.67±9.20%). After superovulation at the proestrous and estrous stages, mucin 1 (MUC1) and let-7a/let-7b microRNA (miRNA) expression levels were significantly attenuated and enhanced on embryonic day 3.5 (E3.5), respectively, whereas no significant differences in the expression level were found in mice superovulated at the other two stages. A higher number of developing and Graafian follicles was observed in the ovarian sections 48h after the administration of pregnant mare serum gonadotropin (PMSG) at the proestrous and estrous stages. The sections from mice treated at the metestrous and diestrous stages, however, presented more corpora lutea. Therefore, mice superovulated at the proestrous and estrous stages exhibited the best pregnancy rates. Furthermore, the disordered expression of MUC1 and let-7a/let-7b miRNA in mice superovulated at the metestrous and diestrous stages may impair reproduction performance.

In vivo and in vitro approaches were used to elucidate mechanisms of palmitate-induced cytotoxicity of follicle granulosa cells in fuel-overloaded broiler hens. In contrast to their energy-restricted counterparts, broiler breeder hens fed ad libitum for 2 wk had dyslipidemia, atresia within hierarchical ovarian follicles, and a 34% reduction in egg production (P < 0.05). Based on vital staining of freshly isolated granulosa cells with annexin V/propidium iodide, there were increases in apoptosis consistent with suppressed Akt activation (P < 0.05). Supplementing primary granulosa cell cultures with 0.5 mM palmitate for 48 or 96 h increased apoptosis (P < 0.05). Palmitate-induced cell death was accompanied by increased acyl-CoA oxidase, carnitine palmitoyl transferase-1, serine palmitoyl transferase, and sphingomyelinase transcripts and increased concentrations of proinflammatory interleukin-1β (P < 0.05). Triacsin-C inhibition of fatty acyl-CoA synthesis blunted interleukin-1β production and rescued granulosa cultures from palmitate-induced cell death. That there was partial to complete prevention of cell death with addition of the free radical scavenger pyrrolidine dithiocarbamate, the sphingomyelinase inhibitor imipramine, or the de novo ceramide synthesis inhibitor fumonisin B1, supported the notion that palmitate-induced granulosa cell cytotoxicity operated through a palmitate-derived metabolite. Palmitoyl-CoA may be channeled into β-oxidation and/or into bioactive metabolites that increase free radical generation, an inflammatory response, and ceramide production. In conclusion, palmitate-derived metabolites activated apoptotic machinery in avian granulosa cells, which caused ovarian follicular atresia and reduced egg production in fuel-overloaded broiler breeder hens.

Let-7b, one of the let-7 family members, was studied for its regulative role in endometrial cells during early pregnancy in mice. According to real-time RT-PCR analysis, the expression of let-7b in epithelial cells increased gradually from day 1 to day 4 of preimplantation stages and reached the highest level on day 4. On the other hand, the highest level of let-7b in stromal cells was observed on day 1, although the expression was decreased on day 2 and increased significantly on day 4. By in situ hybridization, let-7b was also found to express in uteri during days 6-8 of pregnancy. Endometrial cells isolated from prepubertal mice were treated with steroid hormones, progesterone (P4), estradiol (E2) and P4 plus E2. After 96 h of culture in the presence of steroid hormones, the expression levels of let-7b were increased in the endometrial cells, although significant differences were only observed after P4 treatment in stromal cells and after individual E2 and P4 treatments in the epithelial cells. In association with the increased let-7b expression, the cell proliferation slope, measured by a MTT assay, significantly decreased in the presence of P4 and P4 plus E2 compared with the nonhormone and E2 treatment groups during 72-108 h of culture. Furthermore, results from transfection of let-7b into stromal cells isolated from day 4 pregnant mice or prepubertal mice demonstrated that let-7b attenuated the proliferation during the periods of time examined. After transfection of let-7b into mouse stromal cells isolated from day 7 of pregnancy, the expression of Basigin (Bsg), a matrix metalloproteinase (MMP) inducer, was suppressed, as well as that of MMP-9. In conclusion, this study clarifies the expression pattern of let-7b in uterine epithelial and stromal cells during preimplantation stages in mice, as well as the inhibitory effect of let-7b associated with steroid hormones on stromal cell proliferation and on the expression of MMP-9.

The objectives of this study were to characterize the nuclear and cytoskeletal changes of pig oocytes during in vitro maturation (IVM) and the development of the reconstructed embryos after injection with membrane intact or disintegrated donor cells. Cumulus-oocyte complexes (COCs) were collected from abattoir ovaries by follicle (2-8mm) aspiration. In Experiment 1, COCs were cultured in NCSU-23 medium for 0, 11, 22, 33, and 44 h. Oocytes were fixed at different time points for nuclear and cytoskeletal labeling. Forty-three percent and 75% oocytes progressed to MII stage at 33 and 44 h after IVM culture, respectively. Dynamic shift of spindle and cytoplasmic microtubules was evident. In Experiment 2, matured oocytes were injected with either the whole cumulus cell with or without intact cell membranes after enucleation. The reconstructed oocytes were fixed at 0, 2, or 4 h after cell injection for nuclear and cytoskeletal evaluation. When an intact cumulus cell was injected, the injected cell remained intact within 4h after injection. When a cell with disintegrated membrane was injected, 59-63% (n=146) of the injected cell underwent premature chromosome condensation (PCC). In Experiment 3, the reconstructed pig oocytes received membrane-disintegrated cumulus cells or fetal fibroblasts were cultured in PZM medium. The blastocyst rate of the fibroblast-injected embryos was 10%, which was lower than the non-cloned parthenotes (33%, P0.05) but higher than the cumulus cell-injected embryos (2.7%). These results suggest that pig oocytes are subjected to nuclear and cytoskeletal reorganization during maturation. Pig oocytes injected with membrane-disintegrated fibroblast cells support better blastocyst development of the cloned embryos.

The precise physiological causes that result in reduced development of oocytes after heat shock (HS) are not clear. In this study, apoptosis, heat shock protein70 (hsp70), and in vitro development of porcine oocytes were evaluated after HS. Porcine cumulus-oocyte complexes (COCs) were subjected to in vitro maturation for 42 h. The matured oocytes were then heated at 41.5 degrees C for 0 h (control, C0h), 1 h (HS1h), 2 h (HS2h), or 4 h (HS4h). An additional group of oocytes was cultured for 4 h without HS (control, C4h). In Experiment 1, expression of hsp70 was detected by Western-blotting and no difference between controls and HS groups was observed. In Experiment 2, apoptosis of matured oocytes after HS was examined by Annexin V-FITC and TUNEL. No significant TUNEL-positive signals were detected in the heated oocytes compared to the controls, but the intensity of Annexin V-FITC labeling among different groups increased with length of HS and in vitro culture (P

Oocytes undergo spontaneous germinal vesicle breakdown (GVBD) after being released from the follicular environment; this potentially prevents manipulation of the oocyte at the germinal vesicle (GV) stage. The objectives of this study were to investigate the effects of indirubin, a potent cdc2 kinase inhibitor, on GVBD and microtubular structure of porcine oocytes. Cumulus-oocyte-complexes (COCs) were collected from abattoir-derived ovaries and were randomly allocated to different concentrations of indirubin treatments (0, 10, 25, 50, and 100 microM in Experiment 1 and 0, 50, 75, and 100 microM in Experiment 2) during 44 h of IVM. The influences on the GVBD, microtubules, and maturation rates were evaluated using epifluorescence microscopy. The percentages of oocytes remaining at the GV stage were 0, 16, 26, 69, and 85% for oocytes treated with 0, 10, 25, 50, and 100 microM of indirubin, respectively, which differed among treatment groups (P0.05). However, there were no significant differences between the oocytes treated with 75 and 100 microM (79 and 81%). The cytoplasmic microtubules were fragmented in oocytes maintained at the GV stage and the chromatin became condensed or aggregated. When COCs were incubated with indirubin (50-75 microM) for 22 h and then transferred to maturation medium for 44 h (Experiments 3-5), the percentages of oocytes reaching the metaphase II stage were generally higher than when the COCs were cultured in the presence of the drug for 44 h (62-65% versus 44-46%). However, the parthenogenetic development of the oocytes in Experiment 6 was reduced significantly in drug-treated oocytes. In summary, treatment with 50-75 microM of indirubin effectively prevented GVBD in porcine oocytes, but the developmental competence of the oocytes was compromised.

This study investigated global gene expression in the small yellow follicles (6-8 mm diameter) of broiler-type B strain Taiwan country chickens (TCCs) in response to acute heat stress. Twelve 30-wk-old TCC hens were divided into four groups: control hens maintained at 25°C and hens subjected to 38°C acute heat stress for 2 h without recovery (H2R0), with 2-h recovery (H2R2), and with 6-h recovery (H2R6). Small yellow follicles were collected for RNA isolation and microarray analysis at the end of each time point. Results showed that 69, 51, and 76 genes were upregulated and 58, 15, 56 genes were downregulated after heat treatment of H2R0, H2R2, and H2R6, respectively, using a cutoff value of two-fold or higher. Gene ontology analysis revealed that these differentially expressed genes are associated with the biological processes of cell communication, developmental process, protein metabolic process, immune system process, and response to stimuli. Upregulation of heat shock protein 25, interleukin 6, metallopeptidase 1, and metalloproteinase 13, and downregulation of type II alpha 1 collagen, discoidin domain receptor tyrosine kinase 2, and Kruppel-like factor 2 suggested that acute heat stress induces proteolytic disintegration of the structural matrix and inflamed damage and adaptive responses of gene expression in the follicle cells. These suggestions were validated through gene expression, using quantitative real-time polymerase chain reaction. Functional annotation clarified that interleukin 6-related pathways play a critical role in regulating acute heat stress responses in the small yellow follicles of TCC hens.

Nanosilicate platelets (NSP), the form of natural silicate clay that was exfoliated from montmorillonite (MMT), is widely used as a feed additive for its high non-specific binding capacity with mycotoxins such as fumonisin B1 (FB1), and has been evaluated its safety for biomedical use including cytotoxicity, genotoxicity, and lethal dosage (LD). In the study, we further examined its toxicity on the development of CD1 mouse embryos and its capacity to prevent teratogenesis-induced by FB1. In vitro cultures, NSP did not disturb the development and the quality of intact pre-implantation mouse embryos. Further, newborn mice from females consumed with NSP showed no abnormalities. NSP had an unexpected high adsorption capacity in vitro. In contrast to female mice consumed with FB1 only, a very low residual level of FB1 in the circulation, reduced incidence of neutral tube defects and significantly increased fetal weight were observed in the females consumed with FB1 and NSP, suggesting a high alleviation effect of NSP on FB1 in vivo. Furthermore, FB1 treatment disturbed the gene expression of sphingolipid metabolism enzymes (longevity assurance homolog 5, LASS 5; sphingosine kinase 1, Sphk1; sphingosine kinase 2, Sphk2; sphingosine 1- phosphate lyase, Sgpl1; sphingosine 1-phosphate phosphatase, Sgpp1) in the maternal liver, uterus, fetus, and placenta, but NSP administration reversed the perturbations. Based on these findings, we conclude that NSP is a feasible and effective agent for supplementary use in reducing the toxicity of FB1 to animals.

Studies with intact preimplantation mouse embryos and some types of chimaeric aggregates have shown that the most advanced cells are preferentially allocated to the inner cell mass (ICM) rather than the trophectoderm. Thus, differences between 4-cell and 8-cell stage embryos could contribute to the tendency for tetraploid cells to colonise the trophectoderm more readily than the ICM in 4-cell tetraploid[harr ]8 cell diploid chimaeras. The aim of the present study was to test whether 4-cell stage embryos in 4-cell diploid[harr ]8-cell diploid aggregates contributed equally to all lineages present in the E12.5 conceptus. These chimaeras were compared with those produced from standard aggregates of two whole 8-cell embryos and aggregates of half an 8-cell embryo with a whole 8-cell embryo. As expected, the overall contribution of 4-cell embryos was lower than that of 8-cell embryos and similar to that of half 8-cell stage embryos. In the 4-cell[harr ]8-cell chimaeras the 4-cell stage embryos did not contribute more to the trophectoderm than the ICM derivatives. Thus, differences between 4-cell and 8-cell embryos cannot explain the restricted tissue distribution of tetraploid cells previously reported for 4-cell tetraploid[harr ]8-cell diploid chimaeras. It is suggested that cells from the more advanced embryo are more likely to contribute to the ICM but, for technical reasons, are prevented from doing so in simple aggregates of equal numbers of whole 4-cell and whole 8-cell stage embryos.

Acute heat stress severely impacts poultry production. The hypothalamus acts as a crucial center to regulate body temperature, detect temperature changes, and modulate the autonomic nervous system and endocrine loop for heat retention and dissipation. The purpose of this study was to investigate global gene expression in the hypothalamus of broiler-type B strain Taiwan country chickens after acute heat stress. Twelve 30-week-old hens were allocated to four groups. Three heat-stressed groups were subjected to acute heat stress at 38 °C for 2 hours without recovery (H2R0), with 2 hours of recovery (H2R2), and with 6 hours of recovery (H2R6). The control hens were maintained at 25 °C. At the end, hypothalamus samples were collected for gene expression analysis. The results showed that 24, 11, and 25 genes were upregulated and 41, 15, and 42 genes were downregulated in H2R0, H2R2, and H2R6 treatments, respectively. The expressions of gonadotropin-releasing hormone 1 (GNRH1), heat shock 27-kDa protein 1 (HSPB1), neuropeptide Y (NPY), and heat shock protein 25 (HSP25) were upregulated at all recovery times after heat exposure. Conversely, the expression of TPH2 was downregulated at all recovery times. A gene ontology analysis showed that most of the differentially expressed genes were involved in biological processes including cellular processes, metabolic processes, localization, multicellular organismal processes, developmental processes, and biological regulation. A functional annotation analysis showed that the differentially expressed genes were related to the gene networks of responses to stress and reproductive functions. These differentially expressed genes might be essential and unique key factors in the heat stress response of the hypothalamus in chickens.

Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP's cytotoxic effect is selective for malignant cells. UP's cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspase 9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-X(L), Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP's cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status.

The objective was to characterize the potential laying traits and underlying transcripts expressed in the hypothalamus and pituitary gland that were associated with egg production variability in five genetic stocks of chickens: two commercial lines, Red- (n = 12) and Black-feather (n = 14) Taiwan country chickens (TCCs); two selected lines of TCCs, B (high body weight/comb size; n = 17) and L2 (high-egg production; n = 14); and a commercial single comb White Leghorn (WL; n = 17). Six laying traits, age at first egg, clutch length, pause length, oviposition lag within clutch, follicle rapid growth period, and rate of yolk accumulation were measured. The significance of differential values among five chicken stocks and correlation coefficients between laying traits and number of eggs to 50 weeks of age or laying rate after first egg, and the expression level of 33 transcripts were determined. Longer clutch length and shorter oviposition lag within clutch contributed to a higher number of eggs to 50 weeks of age or laying rate after first egg in L2 (P < 0.05) and WL strains (P < 0.05). However, their rate of yolk accumulation (P < 0.05) and follicle rapid growth period (P < 0.05) were different, indicating the accumulation of different alleles after long-term, independent selection. Across all five strains, numbers of eggs to 50 weeks of age were positive correlated with average clutch length (P < 0.05) as well as the rate of yolk accumulation (P < 0.05). Expressions of PLAG1, STMN2, PGDS, PARK7, ANP32A, PCDHA@, SCG2, BDH and SAR1A transcripts contributed to number of eggs to 50 weeks of age (P < 0.05) or laying rate after first egg (P < 0.05). Analysis of correlation coefficients indicated that PLAG1 additionally played roles in decreasing average pause length. Two transcripts, PRL and GARNL1, specifically contributed to number of eggs to 50 weeks of age or laying rate after first egg by reducing oviposition lag within clutch (P < 0.05) and/or increasing average clutch length (P < 0.05), respectively. Expression level of NCAM1, contributed to laying rate after first egg by association with a shorter oviposition lag within clutch (P < 0.05). The current study attributed egg production phenotype in five strains into several laying traits; correlations between these traits and expression levels of underlying transcripts expressed in the hypothalamus and pituitary gland were also established.

To identify transcripts related to high egg production expressed specifically in the hypothalamus and pituitary gland of the chicken, two subtracted cDNA libraries were constructed. Two divergently selected strains of Taiwan Country Chickens (TCCs), B (sire line) and L2 (dam line) were used; they had originated from a single population and were further subjected (since 1982) to selection for egg production to 40 wk of age and body weight/comb size, respectively. A total of 324 and 370 clones were identified from the L2-B (L2-subtract-B) and the B-L2 subtracted cDNA libraries, respectively. After sequencing and annotation, 175 and 136 transcripts that represented 53 known and 65 unknown non-redundant sequences were characterized in the L2-B subtracted cDNA library. Quantitative reverse-transcription (RT)-PCR was used to screen the mRNA expression levels of 32 randomly selected transcripts in another 78 laying hens from five different strains. These strains included the two original strains (B and L2) used to construct the subtracted cDNA libraries and an additional three commercial strains, i.e., Black- and Red-feather TCCs and Single-Comb White Leghorn (WL) layer. The mRNA expression levels of 16 transcripts were significantly higher in the L2 than in the B strain, whereas the mRNA expression levels of nine transcripts, BDH, NCAM1, PCDHA@, PGDS, PLAG1, PRL, SAR1A, SCG2 and STMN2, were significantly higher in two high egg production strains, L2 and Single-Comb WL; this indicated their usefulness as molecular markers of high egg production.

Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, approximately 50 and approximately 25%, respectively.

In a previous study of mouse tetraploid[harr ]diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in postimplantation stage tetraploid[harr ]diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid[harr ]diploid chimaeric blastocysts.

Embryo implantation is a complicated process involving interactions between the blastocyst and the luminal epithelium of the receptive uterus. Mucin 1 (MUC1) is an integral membrane glycoprotein expressed apically by secretory epithelial cells and the glandular epithelium in different organs, including the uterus. It is believed that loss of MUC1 on the surface of uterine epithelial cells is necessary for embryo implantation. The endogenous non-protein coding microRNAs (miRNAs) of 21–24 nucleotides are found in diverse organisms. It has been shown that miRNAs participate in a range of cellular processes by regulating gene expression at the post-transcriptional level. In the present study, the regulatory role of miRNA-199a on the expression of MUC1 in mouse uterus during implantation was investigated for its effect on embryo implantation. Western blotting and immunohistochemistry results showed high MUC1 expression on Day 0.5 and low expression by Day 4.5 of pregnancy. In contrast with MUC1 expression, increased miRNA-199a expression was evident at Day 4.5 of pregnancy, as measured by real-time reverse transcription–polymerase chain reaction. In addition, we demonstrated direct binding of miRNA-199a to the 3′-untranslated region of MUC1. Transfection of miRNA-199a into mouse uterine epithelial cells isolated from Day 0.5 of pregnancy also downregulated expression of MUC1. Therefore, the present study provides evidence that MUC1 is a direct target of miRNA-199a and suggests that development of novel strategies to facilitate a successful pregnancy and repair implantation failure humans may include miRNA.