Association between HSPA5 Promoter Polymorphisms and a Reduced Risk of Normal Tension Glaucoma.

INTRODUCTION: In normal tension glaucoma (NTG), factors other than elevated intraocular pressure (IOP) are likely to play a role in the pathogenesis of optic neuropathy. The potential similarities between Alzheimer's disease (AD) and NTG in cellular apoptosis leading to neurodegeneration have been shown in recent studies. Heat Shock Protein family A member 5 (HSPA5) promoter polymorphisms have been reported to be associated with a risk of AD. The purpose of our study was to investigate the role of HSPA5 promoter polymorphisms in NTG patients. METHODS: A total of 222 patients with NTG, along with 236 normal controls were enrolled in this study. Genomic DNA was amplified through a polymerase chain reaction (PCR) and identified for the polymorphic HSPA5 (-415 and -370) by Xmn1 and BstY1 restriction digestion, respectively. PCR fragments with potential polymorphic HSPA5 (-180) were subjected to sequence-analyses by a Hex-labeled primer. Genotypes for both NTG patients and control groups were compared for statistically significant differences. RESULTS: Polymorphisms (-415) G/A and (-180) del/G were completely linked in our population. The genotype and allele frequency distribution at the -415 G/A and -180 del/G sites showed a significant difference between the NTG cases and controls. The genotype frequency of HSPA5 (-415) AA/(-180) GG and the allele frequency of HSPA5 (-415) A/(-180) G were significantly lower (p = 0.04 and p = 0.01, respectively) in the NTG patients when compared with those in the control group. There was no significant difference in genotype or allele frequency distribution of the HSPA5 (-370) C/T between the NTG and control groups. There was a reduced risk of NTG associated with the carriers for the HSPA5 (-415) A/(-180) G allele compared with that in the control population (p = 0.01). CONCLUSION: HSPA5 (-415) A and (-180) G allele polymorphisms may be protective factors in the development of NTG.

Effects of electromagnetic waves on oocyte maturation and embryonic development in pigs.

Our living environment has been full of electromagnetic radiation (EMR) due to the prevailing electronic devices and equipment. Intermediate frequency electromagnetic field (IF-EMF) or waves constitute a significant part of EMR; therefore, an increasing number of household electrical appliances have become a source of IF-EMF, and concerns about IF-EMF on health are gaining more attention. However, little information is available about its impact on female reproductive traits, such as germ cell viability and early embryonic development, particularly at the cellular and molecular levels. In this study, we used porcine oocytes as a model system to explore the effect of IF-EMF at various intensities on the in vitro maturation (IVM) of oocytes and their subsequent embryonic development. Our results showed that no difference in oocyte maturation rates was detected among groups, but the cleavage and blastocyst rates of parthenotes derived from EMF-treated oocytes decreased with the weaker IF-EMF intensity (25 and 50 Gauss) groups compared to the control group (P < 0.05). For cytoplasmic maturation, the weaker IF-EMF intensity groups also showed a peripheral pattern of mitochondrial distribution resembling that of immature oocytes and increased autophagy activity. No obvious differences in cytoskeletal distribution and total cell numbers of blastocysts were investigated in the four IF-EMF treatments compared to those in the control group. Although the underlying mechanism associated with EMF effects on oocytes and embryos is still elusive, we have demonstrated that low intensity IF-EMF exerts harmful effects on porcine oocytes during the maturation stage, carrying over such effects to their subsequent embryonic development.

Production of viable cloned miniature pigs by aggregation of handmade cloned embryos at the 4-cell stage.

The aim of the present study was to improve the quality of handmade cloned porcine embryos by multiple embryo aggregations. Embryos derived from aggregation of three cloned embryos (3×) had a better blastocyst rate than cloned control (1×) embryos (73.6% vs 35.1%, respectively; P<0.05), but did not differ from those produced by aggregation of two cloned embryos (2×; 63.0%). Total cell numbers differed among treatments (P<0.05), with the greatest cell numbers (126) in the 3× group and the lowest (55) in the control group. The ratio of inner cell mass:total cell number was comparable in the 2× and 3× groups (25.1% vs 26.1%, respectively) and was significantly better than that in the control group (15.3%). The proportion of apoptotic cells in 2× and 3× groups was lower than that in the control group (2.7% and 2.2% vs 4.7%, respectively; P<0.05). Expression of Oct4 and Cdx2 was higher, whereas that of Bax was lower (P<0.05), in the 3× compared with non-aggregate group. Seven piglets were born to two surrogate mothers after embryo transfer of 3× aggregated blastocysts. In conclusion, aggregated embryos had greater total cell numbers and better pluripotency gene expression, with reduced expression of the pro-apoptosis gene Bax. Collectively, these improvement may be associated with the development of cloned embryos to term.

Ascorbic acid improves the developmental competence of porcine oocytes after parthenogenetic activation and somatic cell nuclear transplantation.

In this study, a dose-response assessment was performed to understand the relation between supplementation of media with L-ascorbic acid or vitamin C and porcine oocyte maturation and the in vitro development of parthenotes (PA) and handmade cloned (HMC) embryos. Various concentrations (0, 25, 50 and 100 µg/ml) of vitamin C supplemented in in vitro maturation (IVM) and culture (IVC) media were tested. None of these vitamin C additions affected nuclear maturation of oocytes, yet supplementation at 50 µg/ml led to significantly increased intracellular glutathione (GSH) levels and reduced reactive oxygen species (ROS). When cultured in IVM- and/or IVC-supplemented media, the group supplemented with 50 µg/ml of vitamin C showed improved cleavage rates, blastocyst rates and total cell numbers per blastocyst (P<0.05) compared with other groups (control, 25 µg/ml and 100 µg/ml). In contrast, supplementation with 50 µg/ml vitamin C decreased (P<0.05) the apoptosis index as compared with the groups supplemented with 100 µg/ml. In addition, even with a lower blastocyst rate to start with (37.6 vs. 50.3%, P<0.05), supplementation of HMC embryos with vitamin C ameliorated their blastocyst quality to the extent of PA embryos as indicated by their total cell numbers (61.2 vs. 59.1). Taken together, an optimized concentration of vitamin C supplementation in the medium not only improves blastocyst rates and total cell numbers but also reduces apoptotic indices, whereas overdosages compromise various aspects of the development of parthenotes and cloned porcine embryos.

Sonic hedgehog supplementation of oocyte and embryo culture media enhances development of IVF porcine embryos.

We investigated the expression of sonic hedgehog (SHH) receptor PTCH1 and its co-receptor smoothened (SMO) in fertilized porcine embryos. Effects of exogenous SHH on embryonic development and expressions of survival- and pluripotency-related genes were also determined. We found that PTCH1 and SMO are expressed from two-cell to blastocyst embryos. When oocytes or fertilized embryos were respectively cultured in the maturation or embryo culture medium supplemented with SHH (0.5 µg/ml), their blastocyst rates and total cell numbers increased (P<0.05) compared with the untreated control. When cultured simultaneously in the in vitro maturation (IVM) and in vitro culture (IVC) media supplemented with SHH, the oocytes gained increased blastocyst rates and total cell numbers in an additive manner, with reduced apoptotic indices (P<0.05). Interestingly, SHH treatment did not affect the expression of the BCL2L1 (BCL-XL) gene, yet reduced BAX expression. Blastocysts cultured with various SHH regimes had similar pluripotency-related gene (POU5F1 (OCT-4) and CDX2) expression levels, but blastocysts derived from SHH treatment during IVM had higher ZPF42 (REX01) expression (P<0.05). The highest ZPF42 expression was observed in the blastocysts derived from SHH-supplemented IVC and from dual IVM and IVC treatments. The levels of acetylated histone 3 (AcH3K9/K14) increased in the two-cell and the four-cell embryos when IVM and/or IVC media were supplemented with SHH (P<0.05). Our findings indicate that SHH conferred a beneficial effect on preimplantation development of porcine embryos, particularly when both IVM and IVC media were supplemented with SHH, and the effects may be further carried over from IVM to the subsequent embryonic development.

Ultrastructural Characterization of Porcine Growing and In Vitro Matured Oocytes.

This study aimed to investigate ultrastructural changes of growing porcine oocytes and in vitro maturated oocytes. Light microscopy was used to characterize and localize the primordial, primary, secondary, and tertiary follicles. During oocyte growth and maturation, the morphology of mitochondria was roundish or ovoid in shape depending on the differentiation state, whereas their mean diameters oscillated between 0.5 and 0.7 µm, respectively, from primary and secondary follicles. Hooded mitochondria were found in the growing oocytes of the tertiary follicles. In addition to the pleomorphism of mitochondria, changes in the appearance of lipid droplets were also observed, along with the alignment of a single layer of cortical granules beneath the oolemma. In conclusion, our study is apparently the first report to portray morphological alterations of mitochondria that possess the hooded structure during the growth phase of porcine oocytes. The spatiotemporal and intrinsic changes during oogenesis/folliculogenesis are phenomena at the ultrastructural or subcellular level of porcine oocytes, highlighting an in-depth understanding of oocyte biology and impetus for future studies on practical mitochondrion replacement therapies for oocytes.

Nucleus, Cytoskeleton, and Mitogen-Activated Protein Kinase p38 Dynamics during In Vitro Maturation of Porcine Oocytes.

The mitogen-activated kinase (MAPK) p38, a member of the MAPK subfamily, is conserved in all mammalian cells and plays important roles in response to various physiologic cues, including mitogens and heat shock. In the present study, MAPK p38 protein expression in porcine oocytes was analyzed during in vitro maturation (IVM) by Western blotting and immunocytochemistry. The levels of p-p38 or activated p38 and p38 expression were at the lowest in the germinal vesicle (GV) stage oocyte, gradually rising at the germinal vesicle breakdown (GVBD) and then reaching a plateau throughout the IVM culture (p < 0.05). Similarly, the expression level of total p38 was also lower in the GV oocyte than in the oocyte of other meiotic stages and uprising after GVBD and remained high until the metaphase III (MII) stage (p < 0.05). In the GV stage, phosphorylated p38 (p-p38) was initially detectable in the ooplasm and subsequently became clear around the nucleus and localized in the ooplasm at GVBD (18 h post-culture). During the metaphase I (MI) and metaphase II (MII) stages, p-p38 was evenly distributed throughout the ooplasm after IVM for 30 or 42 h. We found that the subcellular localization increased in p-p38 expression throughout oocyte maturation (p < 0.05) and that dynamic reorganization of the cytoskeleton, including microfilaments and microtubules, was progressively changed during the course of meiotic maturation which was likely to be associated with the activation or networking of p38 with other proteins in supporting oocyte development. In conclusion, the alteration of p38 activation is essential for the regulation of porcine oocyte maturation, accompanied by the progressive reorganization and redistribution of the cytoskeleton and MAPK p38, respectively, in the ooplasm.

Developmental Toxicity of Mycotoxin Fumonisin B1 in Animal Embryogenesis: An Overview.

A teratogenic agent or teratogen can disturb the development of an embryo or a fetus. Fumonisin B1 (FB1), produced by Fusarium verticillioides and F. proliferatum, is among the most commonly seen mycotoxins and contaminants from stale maize and other farm products. It may cause physical or functional defects in embryos or fetuses, if the pregnant animal is exposed to mycotoxin FB1. Due to its high similarity in chemical structure with lipid sphinganine (Sa) and sphingosine (So), the primary component of sphingolipids, FB1 plays a role in competitively inhibiting Sa and So, which are key enzymes in de novo ceramide synthase in the sphingolipid biosynthetic pathway. Therefore, it causes growth retardation and developmental abnormalities to the embryos of hamsters, rats, mice, and chickens. Moreover, maternal FB1 toxicity can be passed onto the embryo or fetus, leading to mortality. FB1 also disrupts folate metabolism via the high-affinity folate transporter that can then result in folate insufficiency. The deficiencies are closely linked to incidences of neural tube defects (NTDs) in mice or humans. The purpose of this review is to understand the toxicity and mechanisms of mycotoxin FB1 on the development of embryos or fetuses.

Sonic Hedgehog promotes in vitro oocyte maturation and term development of embryos in Taiwan native goats.

The aim of this study was to investigate the effects of Shh (Sonic Hedgehog) protein on caprine oocyte maturation, early embryo development, and developmental competence after embryo transfer of vitrified-thawed in vitro-produced embryos. Cumulus-oocyte complexes (COCs) derived from abattoir were randomly allocated to the in vitro maturation (IVM) medium supplemented with 0 (Control), 0.125, 0.25, 0.5, or 1.0 µg mL-1 recombinant mouse Shh protein. After IVM, COCs were fertilized with frozen-thawed semen and the presumptive zygotes were cultured on goat oviduct epithelial monolayers in M199 medium for 9 days. Our results showed that supplementation of Shh (0.25 or 0.5 µg mL-1) enhanced oocyte maturation as compared with the control group (92.4% and 95.0% vs. 86.2%, P < 0.05), yet the effect could be reversed by the simultaneous addition of cyclopamine (an inhibitor of Shh signaling by direct binding to the essential signal transducer Smo). Subsequently, an improved blastocyst rate (66.3 ± 10.9, P < 0.05) was observed for the embryos derived from the oocytes matured in the presence of 0.5 µg mL-1 Shh compared with the control group (41.4 ± 12.9). Expressions of Shh, SMO and Gli1 were observed in the ovaries, granulosa cells, COCs, cumulus cells, oocytes and oviduct epithelia. Notably, Ptch1 was expressed in nearly all of the aforementioned tissues and cells except cumulus cells. The embryos exhibited a higher survival rates in the Shh-supplemented group (37.5%) compared to those without Shh supplementation (14.8%; P < 0.05) after embryo transfer. This study demonstrated the beneficial effects of Shh supplementation on oocyte maturation and subsequent embryo development both in vitro and in vivo, suggesting a functional existence of Shh signaling during the final stage of folliculogenesis and early embryogenesis in caprine.

Derivation and characterization of putative embryonic stem cells from cloned rabbit embryos.

The present study aimed to establish embryonic stem (ES) cell lines, i.e., ntES cells, using rabbit blastocyst stage embryos cloned by somatic cell nuclear transfer. First, we investigated the development of cloned rabbit embryos reconstructed with normal fibroblasts and fibroblasts transfected with enhanced green fluorescence protein (eGFP). Blastocyst rates were 27.4% and 23.9%, respectively, for the embryos reconstructed with normal fibroblasts and fibroblasts transfected with eGFP compared with that from the parthenogenetic group (43.1%). One ntES cell line was established from embryos reconstructed with eGFP-transfected fibroblasts (1 of 17, 5.9%), and three ntES cell lines were derived from those with normal fibroblasts (3 of 17, 17.6%). All the ntES cell lines retained alkaline phosphatase activity and expressed ES cell-specific markers SSEA-4, Oct-4, TRA-1-60, and TRA-1-81. The pluripotency was further confirmed by reverse transcription-polymerase chain reaction analyses of Oct-4, Nanog, and Sox-2 expressions in ntES cell lines. The differentiation capacity of ntES cells was also examined in vitro and in vivo, by which these ntES cell lines were able to differentiate into all three germ layers through embryoid bodies and teratomas. In conclusion, it is apparent that the efficiency of ntES cells derived using eGFP-transfected donor cells is lower than that with nontransfected, normal fibroblasts donor cells. Similar to those from parthenogenetic embryos, all ntES cell lines derived from cloned rabbit embryos are able to express pluripotency markers and retain their capability to differentiate into various cell lineages both in vitro and in vivo.

Stage-dependent expression of extra-embryonic tissue-spermatogenesis-homeobox gene 1 (ESX1) protein, a candidate marker for X chromosome-bearing sperm.

Extra-embryonic tissue-spermatogenesis-homeobox gene 1 (Esx1) encodes an X-linked homeobox protein. Despite the fact that the temporal and spatial mRNA expression pattern of the protein has been studied extensively in the testis, specific localisation of ESX1 in the testis remains to be determined. In the present study, we generated ESX1 antiserum to investigate the stage- and tissue-specific expression of ESX1 in the mouse. Western blotting and immunofluorescent analyses revealed that general localisations of ESX1 were consistent with its RNA expression patterns; that is, it was restricted mainly to the placenta and testis. Immunofluorescent studies demonstrated that ESX1 existed in the testes after 3 weeks of age, coincident with the appearance of round spermatids in the seminiferous tubules. Moreover, ESX1 expression became more abundant in the luminal regions of the seminiferous tubules as the development of round spermatids progressed into spermatozoa. In contrast, reduced expression of ESX1 was observed in experimentally induced cryptorchid testes. The later expression of ESX1 suggests a role in post-meiotic germ cell development. To further understand ESX1 expression in sperm with respect to X chromosome-bearing sperm, we used ESX1 antiserum to immunostain sperm by confocal laser microscopy. Approximately half the sperm population was recognised by the ESX1 antiserum. On the basis of results of the present study, we suggest that ESX1 could be used as a protein marker for X chromosome-bearing sperm.

Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

Leukemia inhibitory factor and fibroblast growth factor 2 critically and mutually sustain pluripotency of rabbit embryonic stem cells.

Effects of leukemia inhibitory factor (LIF) and fibroblast growth factor 2 (FGF2) on establishment and maintenance of rabbit embryonic stem cell (rESC) lines were assessed. When grown on MEF feeders, rESC lines derived from fertilized embryos were established and maintained in medium containing paracrine factors LIF (via STAT3) and/or FGF2 (via MEK-ERK1/2 and PI3K-AKT). However, high levels of ERK1/2 and AKT activities in rESCs were crucial for maintaining their undifferentiated proliferation. Although rESCs under the influence of either LIF (500, 1,000, and 2,000 U/ml) or FGF2 (5, 10, and 20 ng/ml) alone had enhanced expression of pluripotency markers, peak expression occurred when both LIF (1,000 U/ml) and FGF2 (10 ng/ml) were applied. Induced dephosphorylation of STAT3, ERK1/2, and AKT by specific inhibitors limited growth of rESCs and caused remarkable losses of self-renewal capacity; therefore, we inferred that STAT3, ERK, and AKT had essential roles in maintaining rESC proliferation and self-renewal. We concluded that LIF and FGF2 jointly maintained the undifferentiated state and self-renewal of rESCs through an integrative signaling module.

Proteomic profiling of rabbit embryonic stem cells derived from parthenotes and fertilized embryos.

Rabbit embryonic stem (rES) cells can be derived from various sources of embryos. However, understanding of the gene expression profile, which distincts embryonic stem (ES) cells from other cell types, is still extremely limited. In this study, we compared the protein profiles of three independent lines of rabbit cells, i.e., fibroblasts, fertilized embryo-derived stem (f-rES) cells, and parthenote-derived ES (p-rES) cells. Proteomic analyses were performed using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Collectively, the expression levels of 100 out of 284 protein spots differed significantly among these three cell types (p<0.05). Of those differentially expressed spots, 91% were identified in the protein database and represented 63 distinct proteins. Proteins with known identities are mainly localized in the cytoplasmic compartments (48%), nucleus (14%), and cytoskeletal machineries (13%). These proteins were majorly involved in biological functions of energy and metabolic pathways (25%), cell growth and maintenance (25%), signal transduction (14%), and protein metabolisms (10%). When protein expression levels among cell types were compared, six proteins associated with a variety of cellular activities, including structural constituents of the cytoskeleton (tubulins), structural molecule (KRT8), catalytic molecules (α-enolase), receptor complex scaffold (14-3-3 protein sigma), microfilament motor proteins (Myosin-9), and heat shock protein (HSP60), were found highly expressed in p-rES cells. Two proteins related to HSP activity and structural constituent of cytoskeleton in f-rES cells, and one structural molecule activity protein in fibroblasts showed significantly higher expression levels (p<0.05). Marker protein expressions in f-rES and p-rES cells were further confirmed by Western blotting and immunocytochemical staining. This study demonstrated unique proteomic profiles of the three rabbit cell types and revealed some novel proteins differentially expressed between f-rES and p-rES cells. These analyses provide insights into rES cell biology and would invite more in-depth studies toward rES cell applications.

Trichostatin A and ascorbic acid assist in the development of porcine handmade cloned embryos via different physiologic pathways.

The objective was to determine the effects of ascorbic acid (AA), trichostatin A (TSA), and their combined treatment (TA) on reprogramming and development of cloned porcine embryos. Embryos treated with AA (50 and 100 µg/mL) had a higher blastocyst rate than controls (49.6% and 44.0% vs 30.7%, P < .05). Blastocyst rates of handmade cloned (HMC) embryos were nearly 60% in both the 30 and 40 nmol/L TSA treatment groups, which were higher (P < .05) than the control (29.4%). The TA treatment groups had a higher blastocyst rate compared with the AA treatment alone (58.9% vs 43.5%, P < .05). Histone acetylation was much higher in the TSA and TA treatments (primarily in 2- and 4-celled embryos) but was not significantly different between AA-treated and untreated embryos. Both AA and TA treatments reduced apoptotic rates of blastocysts. In conclusion, AA supplementation improved blastocyst development in porcine HMC embryos mainly by a traditional antioxidant pathway rather than by cellular reprogramming.

LIF and FGF cooperatively support stemness of rabbit embryonic stem cells derived from parthenogenetically activated embryos.

We investigated the individual and combined effects of leukemia inhibitory factor (LIF) and basic fibroblast growth factor 2 (bFGF2) on the derivation and maintenance of rabbit embryonic stem cell lines isolated from parthenogenetic activated embryos (p-rES). First, we demonstrated that p-rES cell lines can be prevented from differentiation via LIF (STAT3) and bFGF2 (MEK-ERK1/2 and PI3K-AKT) signaling on MEF feeders. High levels of ERK1/2 and AKT activities were crucial for maintaining p-rES cells in an undifferentiated state. Although the p-rES cells under the influence of LIF (500, 1000, and 2000 U/mL) or bFGF2 (5, 10, and 20 ng/mL) alone showed enhanced expression in the pluripotency markers, the highest levels of marker expressions coincided with the simultaneous presence of LIF (1000 U/mL) and bFGF2 (10 ng/mL). The phosphorylation status of LIF and bFGF2 downstream signaling molecules including STAT3, ERK, and AKT was also intensively involved in the maintenance of p-rES cell proliferation and self-renewal. Induced dephosphorylation of STAT3, ERK1/2, and AKT by specific inhibitors caused remarkable losses of self-renewal capacity of p-rES cells. We conclude that bFGF2 and LIF by itself are self-sufficient in maintaining the state of undifferentiation and self-renewal of rabbit p-ES cells, yet are most effective when acting concomitantly.

Sonic Hedgehog improves in vitro development of porcine parthenotes and handmade cloned embryos.

This study investigated the expression of Sonic Hedgehog (Shh) signaling pathway and its effect on porcine parthenogenetic (PA) embryo development. The Shh receptor Patched (Ptc1) and co-receptor Smoothened (Smo) were expressed at various stages of PA porcine embryos, at both mRNA and protein levels. Furthermore, the transcriptional activator Gli1 mRNA was first present in the 2-cell stage embryos, and was readily detected at the 4-cell stage and beyond. Culture medium supplemented with 0.5 µg/mL Shh optimized blastocyst rates (58.6 vs. 41.1%; P < 0.05) and the total number of cells per blastocyst (56.4 vs. 45.6 cells; P < 0.05); however, this response was prevented by simultaneous addition of 1 mM cyclopamine (an Shh inhibitor). Moreover, blastocysts that developed in medium containing 0.5 µg/mL Shh had lower apoptotic indices and reduced DNA damage (evaluated by TUNEL and comet assays, respectively). Based on Western-blot analysis, expression of phosphorylated Akt protein in Shh-treated blastocysts was higher than that of the control group (1.22- vs. 0.66-fold, P < 0.05), and less total PARP-1/2 protein was accumulated (0.7-fold, P < 0.05) in treated blastocysts compared to untreated controls. Furthermore, supplementation of Shh (1 µg/mL) also supported development of handmade cloned embryos (50.3 vs. 26.8%; P < 0.05) with reduced apoptotic rates (2.8 vs. 6.3%; P < 0.05). We inferred that the Shh signaling pathway existed in porcine PA embryos and we concluded that Shh supplementation improved the quality and developmental competence of early PA embryos, at least in part, by increasing cell proliferation and reducing apoptosis of the developing embryos.

Characterization of embryonic stem cell lines derived from New Zealand white rabbit embryos.

The purposes of this study were to examine technical details in deriving and maintaining rabbit embryonic stem (rES) cell lines and to analyze their characteristics. When STO cells were used as feeder cells, no rES cell lines were established using either intact blastocysts or inner cell masses (ICMs). On the mouse embryonic fibroblasts (MEF) feeder, rES cell lines were efficiently (24%) derived. Addition of leukemia inhibitory factor (LIF) to the cells cultured on the MEF feeders further increased the derivation efficiency (57%) of rES cells. The fact that LIF induced serine-phosphorylation of STAT3 suggested LIF-dependent maintenance of rES cells. Most of the rES cell lines expressed AP, SSEA-4, Oct4, TRA-1-60, and TRA-1-81. Western blot or RT-PCR analysis also confirmed the expression of Oct4, Nanog, and Sox2. When induced to form EBs in vitro or injected to the severe combined immunodeficiency (SCID) mice, the rES cells generated embryoid bodies (EBs) and teratomas with three germ layers expressing the marker genes including MAP2, Desmin, and GATA4, respectively. In conclusion, rabbit ES cell lines can be efficiently established using our current protocols with LIF supplement. These ES cells express pluripotent stem cell markers and retain their capability to differentiate into different tissue cells. Furthermore, rES cells depend on LIF for self-renewal, likely via the JAK-STAT pathway.