Pre-implantation development of cattle embryos produced from fresh bull semen enriched for X- chromosome-bearing spermatozoa using a monoclonal antibody.

Immunological approaches are gaining attention as a convenient and economical method for sex-sorting mammalian spermatozoa. A monoclonal antibody (WholeMom™) has previously been reported to cause agglutination of Y-chromosome-bearing spermatozoa in frozen-thawed semen for gender preselection. However, its usefulness for gender preselection in fresh semen and subsequent in vitro fertilization (IVF) after freeze-thawing has not been reported. This study investigated the in vitro development of cattle embryos produced from fresh bull semen pre-treated with WholeMom™ monoclonal antibody. Results showed that antibody-treated, non-agglutinated spermatozoa (presumably X-chromosome-bearing spermatozoa) could fertilize cattle oocytes in vitro. However, embryos generated from non-agglutinated (enriched in X-chromosome-bearing spermatozoa) had a lower (p < 0.05) ability to cleave (66.4 ± 2.5% vs. 75.1 ± 3.3%) than those of non-treated control sperm. Nevertheless, the percentage of blastocysts developed from cleaved embryos did not differ (p > 0.05) between the groups (34.8 ± 3.7% vs. 35.8 ± 3.4%). Duplex PCR of blastocysts, using a bovine-specific universal primer pair and a Y-chromosome-specific primer pair, showed a sex ratio of 95.8% females from sex-sorted spermatozoa, which was higher than those of non-treated control spermatozoa (46.4%). In conclusion, the results of the present study suggest that monoclonal antibody-based enrichment of X- chromosome-bearing spermatozoa can be applied to fresh bull semen without compromising their post-fertilization early embryonic development to the blastocyst stage. Future studies should investigate the term development and sex ratio of calves from antibody-treated spermatozoa.

5-Azacytidine improves the meiotic maturation and subsequent in vitro development of pig oocytes.

Treatment of donor cells and/or cloned embryos with cytidine analogues, having an Aza group at its 5th carbon (5-Aza), such as 5-Azacytidine (5-Aza-C) or 5-Aza-2'-deoxycytidine (5-Aza-dC) improves the in vitro development of cloned embryos produced by somatic cell nuclear transfer (SCNT). In vitro maturation (IVM) of immature pig oocytes treated with 5-Aza-C not only results in greater (P < 0.05) meiotic maturation to the MII stage but also enhances the capacity of 5-Aza-C treated oocytes for early embryonic development after parthenogenetic activation (PA), in vitro fertilization (IVF) or SCNT in a dose-dependent manner (0-10 µM). Cloned embryos generated from 5-Aza-C (0.01 µM) treated oocytes had an increased capacity to develop to the blastocyst stage (14.1 ±â€¯1.5% compared with 9.6 ±â€¯1.8%), greater probability of hatching (61.8 ±â€¯1.5% compared with 45.0 ±â€¯3.9%) and contained a greater number of cells per blastocyst (38.5 ±â€¯4.4 compared with 30.5 ±â€¯3.4) than those produced from non-treated control oocytes (P < 0.05). Data from the present study indicate that treatment of oocytes with 5-Aza-C may be an important approach to enhance the meiotic maturation and subsequent in vitro development of pig embryos. Future studies should be conducted to determine the underlying mechanism of improved early embryonic development of 5-Aza-C treated oocytes.

TIP60 contributes to porcine embryonic development by regulating DNA damage response.

The acetyltransferase TIP60 (also known as Kat5) is a member of the MYST family of histone acetyltransferases and was initially identified as a cellular protein. TIP60 acetylates histone and non-histone proteins and is involved in diverse biological processes, including apoptosis, cell cycle, and DNA damage responses. In this study, a specific inhibitor of TIP60 was used to detect the function of TIP60 in porcine parthenogenetic embryos. The results showed that TIP60 inhibition impaired porcine parthenogenetic embryonic development. The mechanism of TIP60 was also determined. We found that the TIP60 inhibition impaired embryonic development by ROS induced DNA damage, as demonstrated by the number of γH2A in the nuclei. TIP60 inhibition triggered DNA damage through the regulation of p53-p21 pathway and TIP60 played a role in DNA repair. TIP60 inhibition decreased the efficiency of DNA repair by regulating 53BP1-dependent repair after DNA damage. Inhibition of TIP60 also increased the adaptive response, autophagy, by modulating LC3. Therefore, TIP60 plays a role in early porcine parthenogenetic embryonic development by regulating DNA damage and repair.

Mammary alveolar cell as in vitro evaluation system for casein gene expression involved in glucose level.

OBJECTIVE: Glucose is an essential fuel in the energy metabolism and synthesis pathways of all mammalian cells. In lactating animals, glucose is the major precursor for lactose and is a substrate for the synthesis of milk proteins and fat in mammary secretory (alveolar) epithelial cells. However, clear utilization of glucose in mammary cells during lactogenesis is still unknown, due to the lack of in vitro analyzing models. Therefore, the objective of this study was to test the reliability of the mammary alveolar (MAC-T) cell as an in vitro study model for glucose metabolism and lactating system. METHODS: Undifferentiated MAC-T cells were cultured in three types of Dulbecco's modified Eagle's medium with varying levels of glucose (no-glucose: 0 g/L, low-glucose: 1 g/L, and high-glucose: 4.5 g/L) for 8 d, after which differentiation to casein secretion was induced. Cell proliferation and expression levels of apoptotic genes, Insulin like growth factor-1 (IGF1) receptor, oxytocin receptor, αS1, αS2, and ß casein genes were analyzed at 1, 2, 4, and 8 d after differentiation. RESULTS: The proliferation of MAC-T cells with high-glucose treatment was seen to be significantly higher. Expression of apoptotic genes was not affected in any group. However, expression levels of the mammary development related gene (IGF1 receptor) and lactation related gene (oxytocin receptor) were significantly higher in the low-glucose group. Expressions of αS1-casein, αS2-casein, and ß-casein were also higher in the low-glucose treated group as compared to that in the no-glucose and high-glucose groups. CONCLUSION: The results demonstrated that although a high-glucose environment increases cell proliferation in MAC-T cells, a low-glucose treatment to MAC-T cells induces higher expression of casein genes. Our results suggest that the MAC-T cells may be used as an in vitro model to analyze mammary cell development and lactation connected with precise biological effects.

Biochanin A Ameliorates Arsenic-Induced Hepato- and Hematotoxicity in Rats.

Biochanin A (BCA) is a natural organic compound of the phytoestrogenic isoflavone class that has antioxidant and metal chelator properties in the presence of transition metal ions, however, its efficacy in animal models is still obscure. Therefore, the objective of this study was to investigate the protective effects of BCA against arsenic-induced hepatic injury and hematotoxicity in rats. The results suggest that arsenic intoxicated rats showed significantly higher levels of plasma hepatic markers than normal control rats. Furthermore, an increase in lipid peroxidation with depletion of reduced glutathione (GSH) and activities of superoxide dismutase (SOD) and catalase (CAT) occurred in the livers of rats exposed to arsenic. Administration of BCA (20 mg/kg·bw/day) and selenium (3 mg/kg·bw/day) resulted in a significant reversal of hepatic and oxidative stress markers in arsenic-intoxicated rats. A low dose of BCA (10 mg/kg·bw/day) did not show any preventive effect, while a high dose of BCA (40 mg/kg·bw/day) partially prevented all hepatotoxicity events. These biochemical perturbations were supported by histopathological observations of the liver. Our results suggest that administration of BCA (20 mg/kg·bw/day) attenuated the arsenic hepatotoxicity, a property that could contribute to the therapeutic approaches for chronic liver diseases.

CRISPR/Cas9 nuclease-mediated gene knock-in in bovine-induced pluripotent cells.

Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3ß and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.

Transcriptional coactivator undifferentiated embryonic cell transcription factor 1 expressed in spermatogonial stem cells: a putative marker of boar spermatogonia.

Spermatogenesis is initiated from spermatogonial stem cells (SSCs), which are derived from gonocytes. Although some rodent SSC markers have been investigated, other species- and developmental stage-specific markers of spermatogonia have not been identified. The objective of this study was to characterize the expression of undifferentiated embryonic cell transcription factor 1 (UTF1) gene as a potential marker for spermatogonia and SSCs in the boar testis. In boar testis tissue at pre-pubertal stages (tissues collected at 5, 30, and 60 days of age), UTF1 gene expression was detected in almost all spermatogonia cells that expressed a protein gene product 9.5 (PGP9.5), and immunocytochemical analysis of isolated total testicular cells showed that 91.14% of cells staining for PGP9.5 also stained for UTF1. However, in boar testis tissue at pubertal and post-pubertal stages (tissues collected at 90, 120, 150, and 180 days of age), UTF1 was not detected in all PGP9.5-positive cells in the basement membrane. While some PGP9.5-positive cells stained for UTF1, other cells stained only for PGP9.5 or UTF1. PGP9.5, UTF1, and NANOG was assessed in in vitro cultures of pig SSCs (pSSCs) from testes collected at 5 days of age. The relative amounts of PGP9.5, NANOG, and UTF1 mRNA were greater in pSSC colonies than in testis and muscle tissue. Thus, the UTF1 gene is expressed in PGP9.5-positive spermatogonia cells of pigs at 5 days of age, and its expression is maintained in cultured pSSC colonies, suggesting that UTF1 is a putative marker for early-stage spermatogonia in the pre-pubertal pig testis. These findings will facilitate the study of spermatogenesis and applications in germ cell research.

Development of a method of vitrification, thawing, and transfer of mammalian blastocysts using a single closed cryo-straw.

BACKGROUND: There are different methods for cryopreservation of mammalian embryos with variable degrees of success. These methods require specific vessels for embryo vitrification, thawing, and transfer. OBJECTIVE: Here, we report a simple and inexpensive way to vitrify, thaw and transfer mammalian blastocysts in one straw. METHODS: This in-straw vitrification solution with microdrop (ISVDM) was compared with EM grid and normal 0.25 mL straw methods. RESULTS: There were no differences in the rates of re-expanded and hatching-to-hatched murine and bovine blastocysts exposed to 1, 0.5, and 0.3 M of sucrose in the diluent that was loaded into the straw. Low re-expanded and hatching-to-hatched rates of murine and bovine blastocysts were observed with PBS only. The pregnancy rates of control murine blastocysts (57.1%) and blastocysts exposed to 0.3 M sucrose in diluent and ISVDM (71.4%) were significantly higher (p < 0.05) than those exposed to 1, 0.5, and 0 M sucrose and those loaded into 0.25 mL straws. The rate of offspring delivery was highest in the control group. There was no significant difference (p < 0.05) in the rate of offspring delivery among ISVDM, 0.25 mL straw, and EM grid groups. CONCLUSION: Our results indicate that vitrified embryos can be warmed and diluted in a single straw and that this one-step method enables farm animal embryo transfer without a microscope or other laboratory equipment.

Vitrification of immature mouse oocytes by the modified-cut standard straw method.

The feasibility of using the modified-cut standard straw (M-CSS) method for the vitrification of immature mouse oocytes has been tested. The effects of different vitrification methods on oocyte survival, cytoskeletal organization, the distribution of cortical granules (CGs), and apoptosis have also been compared. Immature mouse oocytes were vitrified-thawed using electron microscope grid or M-CSS method, and cultured to meiosis II (MII) stage. Oocyte development, cytoskeletal organization, CG distribution, and the expression of apoptosis-related genes were evaluated. Rates of recovery (91.7 vs. 74.9%) and survival (89.0 vs. 62.6%) were significantly higher in M-CSS group than in EM grid group. The number of oocytes with normal chromosome alignment at the spindle and spindle morphology were similar in both groups. However, the actin cap was significantly degraded in EM grid groups (52.6 vs. 35.1%, respectively). Abnormal release of CGs also frequently occurred in EM grid groups (42.6 vs. 32.7%, respectively). Pro-apoptosis-related gene expression levels of Bax, caspase 3 were expressed lower than control in MII stage oocytes derived from M-CSS group; anti apoptosis-related genes, survivin and heat shock factor-1 (Hsf-1) were slightly increased. However, all genes expression was significantly increased in MII stage oocytes derived from EM grid groups. Vitrification reduces the survival rate of immature mouse oocytes, alters cytoskeletal organization and CG distribution, and promotes apoptosis. However, these effects are less pronounced in vitrified oocytes generated by M-CSS than in those generated by EM grid method. Therefore, the novel M-CSS is a feasible approach for the cryopreservation of immature mouse oocytes.

Clinical outcomes of vitrified-thawed embryo transfer using a pull and cut straw method.

OBJECTIVE: To compare the clinical outcomes of patients with vitrified-thawed embryos transferred using either the 0.25 mL straw method and the pull and cut straw (PNC) method. To evaluate the clinical outcomes of patients with transferred embryos that underwent assisted hatching at the cleaved embryo (day 3) or the blastocyst (day 5) stage. METHODS: The study population consisted of women who underwent vitrified-warmed embryo transfer between May 2000 and December 2011 and assisted hatching was performed after warming of embryos. Cycles of thawing between assisted hatching treated and non treated groups were compared for survival and pregnancy rates. RESULTS: The PNC vitrification method improved survival and pregnancy rates in partial lysed embryos. While assisted hatching did not affect the developmental and clinical pregnancy rates of the vitrified-warmed blastocyst group, it did increase the pregnancy rate of poor quality vitrified-warmed cleaved embryos. CONCLUSION: These results suggest that PNC may increase the number of clinical pregnancies via the vitrification of both cleaved embryos and blastocysts. In addition, selective assisted hatching treatment of embryos that show a poor prognosis after warming may increase the rate of clinical pregnancy.

Knockdown of maternal homeobox transcription factor SEBOX gene impaired early embryonic development in porcine parthenotes.

A number of germ cell-specific transcription factors essential for ovarian formation and folliculogenesis have been identified and studied. However, the role of these factors during early embryonic development has been poorly explored. In the present study, we investigated the role of SEBOX, a maternal homeobox transcription factor, during early embryonic development in porcine parthenotes. mRNA for SEBOX is preferentially expressed in oocytes, and expression persists until embryonic genome activation (EGA). Knockdown of SEBOX by siRNA disrupted early embryonic development, but not oocyte maturation. Many maternal genes essential for early embryonic development were upregulated in SEBOX-depleted embryos. Moreover, some pluripotency-associated genes, including SOX2 and NANOG, were upregulated when SEBOX was knocked down. Therefore, our data demonstrate that SEBOX is required for early embryonic development in pigs and appears to regulate the degradation of maternal transcripts and the expression of pluripotency genes.

Transgenic chicken, mice, cattle, and pig embryos by somatic cell nuclear transfer into pig oocytes.

This study explored the possibility of producing transgenic cloned embryos by interspecies somatic cell nuclear transfer (iSCNT) of cattle, mice, and chicken donor cells into enucleated pig oocytes. Enhanced green florescent protein (EGFP)-expressing donor cells were used for the nuclear transfer. Results showed that the occurrence of first cleavage did not differ significantly when pig, cattle, mice, or chicken cells were used as donor nuclei (p>0.05). However, the rate of blastocyst formation was significantly higher in pig (14.9±2.1%; p<0.05) SCNT embryos than in cattle (6.3±2.5%), mice (4.2±1.4%), or chicken (5.1±2.4%) iSCNT embryos. The iSCNT embryos also contained a significantly less number of cells per blastocyst than those of SCNT pig embryos (p<0.05). All (100%) iSCNT embryos expressed the EGFP gene, as evidenced by the green florescence under ultraviolet (UV) illumination. Microinjection of purified mitochondria from cattle somatic cells into pig oocytes did not have any adverse effect on their postfertilization in vitro development and embryo quality (p>0.05). Moreover, NCSU23 medium, which was designed for in vitro culture of pig embryos, was able to support the in vitro development of cattle, mice, and chicken iSCNT embryos up to the blastocyst stage. Taken together, these data suggest that enucleated pig oocytes may be used as a universal cytoplast for production of transgenic cattle, mice, and chicken embryos by iSCNT. Furthermore, xenogenic transfer of mitochondria to the recipient cytoplast may not be the cause for poor embryonic development of cattle-pig iSCNT embryos.

Superovulation induces defective methylation in line-1 retrotransposon elements in blastocyst.

BACKGROUND: Series of epigenetic events happen during preimplantation development. Therefore assistant reproduction techniques (ART) have the potential to disrupt epigenetic regulation during embryo development. The purpose of this study was to investigate whether defects in methylation patterns in blastocyst due to superovulation originate from abnormal expression of Dnmts. METHODS: Low- (6 IU) and high- (10 IU) dosage of PMSG was used to stimulate the female mice. The metaphase II(MII) oocytes, zygotes and blastocyst stage embryos were collected. Global methylation and methylation at H3K9 in zygote, and methylation at repeated sequence Line 1 and IAP in blastocysts were assayed. In addition, expression of Dnmts was examined in oocytes and zygotes. RESULTS: Global DNA methylation and methylation at H3K9 in zygotes derived from females after low- or high-dosage hormone treatment were unaltered compared to that in controls. Moreover, DNA methylation at IAP in blastocysts was also unaffected, regardless of hormone dosage. In contrast, methylation at Line1 decreased when high-dose hormone was administered. Unexpectedly, expression of Dnmt3a, Dnmt3b, Dnmt3L as well as maintenance Dnmt1o in oocytes and zygotes was not disrupted. CONCLUSIONS: The results suggest that defects in embryonic methylation patterns do not originate from the disruption of Dnmt expression.

Greatwall kinase is required for meiotic maturation in porcine oocytes.

Meiotic maturation in many species is initiated by the activation of maturation-promoting factor (MPF) with concomitant inactivation of counteracting phosphatases, most notably protein phosphatase 2A (PP2A). Recently, Greatwall (GWL) has been identified as a cell cycle regulator that inhibits PP2A activity. In this study, we demonstrate that GWL is required for meiotic maturation in porcine oocytes. GWL expression increases from germinal vesicle (GV) to metaphase II (MII) stages of porcine oocytes and dramatically decreases with progression of the meiotic cell cycle. GWL is initially localized in the nucleus of GV oocytes and is associated with spindle fibers following GV breakdown. Depletion of GWL inhibited or delayed meiotic maturation secondary to defects in chromosome congression and spindle formation. Conversely, overexpression of GWL overcame meiotic arrest and initiated progression to MII stage. However, these oocytes had severe spindle defects. Furthermore, MII oocytes depleted of GWL progressed to pronuclear formation. Taken together, our data demonstrate that GWL is required not only for meiotic maturation but also for maintenance of MII arrest in porcine oocytes.

Identifying MicroRNA and mRNA expression profiles in embryonic stem cells derived from parthenogenetic, androgenetic and fertilized blastocysts.

MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions. In this study, miRNA and messenger RNA (mRNA) profiles were examined by Illumina microarray in mouse embryonic stem cells (ESCs) derived from parthenogenetic, androgenetic, and fertilized blastocysts. The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression. Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs (aESCs) and fertilized ESCs (fESCs), a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs (pESCs) and fESCs, and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs. In addition, a total of 575, 5 and 376 miRNA-mRNA target pairs were observed in aESCs vs. fESCs, pESCs vs. fESCs, and aESCs vs. pESCs, respectively. Furthermore, 15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR. Finally, transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3, Dyrk1b, and Prrg2 mRNA, inhibition of miR-363 increased the expression of Nfat5 and Soat1 mRNA, and inhibition of miR-883b-5p increased Nfat5, Tacstd2, and Ppapdc1 mRNA. These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.

Production of transgenic Korean native cattle expressing enhanced green fluorescent protein using a FIV-based lentiviral vector injected into MII oocytes.

The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins. Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer, but these approaches have been largely ineffective; however, a third approach, lentivirus-mediated transgenesis, has successfully produced transgenic livestock. In this study, we generated transgenic (TG) Korean native cattle using perivitelline space injection of viral vectors, which expressed enhanced green fluorescent protein (EGFP) systemically. Two different types of lentiviral vectors derived from feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) carrying EGFP were injected into the perivitelline space of MII oocytes. EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group (47.5%±2.2% v.s. 22.9%±2.9%). Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers. Ten heifers were successfully impregnated and delivered 10 healthy calves. All of these calves expressed EGFP as detected by in vivo imaging, PCR and Southern blotting. In addition, we established an EGFP-expressing cell line from TG calves, which was followed by nuclear transfer (NT). Recloned 8-cell embryos also expressed EGFP, and there were no differences in the rates of fusion, cleavage and development between cells derived from TG and non-TG calves, which were subsequently used for NT. These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle. Moreover, our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells.

Proteins associated with reproductive disorders in testes of human erythropoietin gene-harboring transgenic boars.

To investigate reproductive disorder in human erythropoietin (EPO)-expressing pig, we performed comparative proteomic analyses of testicular tissues from human erythropoietin (hEPO) gene-harboring transgenic pigs and wild type pigs born from natural conception. In hEPO TG pigs, we found relatively low sperm motility and higher death rate indicating impaired sperm development. Consistently, plasma concentration of testosterone was significantly lower in the transgenic post-pubertal boars compared with wild type boars. Normalized protein spots showing higher than 2-fold differential expression intensity in two-dimensional polyacrylamide gel electrophoresis were selected for matrix associated laser desorption/ionization time-to-flight mass spectrometry analysis. Specific proteins were identified by searching the NCBI protein sequence databases. Among 55 proteins selected, 12 proteins were identified as those differentially expressed between transgenic and wild type pigs. Three downregulated proteins (ß-globin, carbonyl reductase 1, and peroxiredoxin 6) and nine upregulated proteins (cytoskeletal ß-actin, α 2,3-sialyltransferase, apolipoprotein A-I, tubulin α-1A chain, tropomodulin 3, thioredoxin, heat shock Protein 70.2, ch4/domains of swine IgM, and albumin), all of which are closely related to apoptosis and cytoskeletal development, were found in the transgenic boar testes. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay confirmed the increased occurrence of apoptosis in the transgenic boar testes compared with the wild type boar testes. Reproductive defects of the hEPO-expressing transgenic pigs may be caused by the abnormal expression of the genes identified in this study.

Autophagy influences maternal mRNA degradation and apoptosis in porcine parthenotes developing in vitro.

Autophagy, an essential process for cellular maintenance, cell viability, and development, is the bulk degradation of proteins and organelles. This study investigated the expression levels of autophagy-related genes and the effect of 3-methyladenine (3-MA, an autophagy inhibitor) or rapamycin (an autophagy inducer) on maternal gene degradation and apoptosis in porcine parthenotes developing in vitro. LC3, which is essential for the formation of autophagosomes, was widely expressed in porcine parthenotes. High levels of autophagy-related genes, Atg5, Beclin1 and Lc3 transcripts were expressed in the 1-cell (1C) stage and gradually decreased through the 2-cell (2C) to blastocyst stages. The mRNA expression of Gdf9, c-mos and cyclin B maintained high levels in 2C and 4-cell (4C) embryos treated with 3-MA compared with the control. The Bmp15 and cyclin B mRNA levels were significantly reduced in embryos treated with rapamycin compared with the control. These results suggest that autophagy influences the degradation of these maternal genes. Furthermore, 3-MA-treated embryos exhibited significantly reduced developmental rates, decreased total cell numbers and increased rates of apoptosis. Expression of Atg5, Beclin1 and Lc3 and synthesis of LC3 protein were significantly reduced at the blastocyst stage. Although rapamycin treatment did not affect the developmental rate, it decreased the cell number and increased the rate of apoptosis, and the expression of Atg5, Beclin1 and Lc3 and LC3 protein synthesis were increased. Finally, blastocysts derived following treatment with 3-MA or rapamycin exhibited significantly decreased expression of selected transcription factors, including Pou5f1, Sox2 and Nanog. In conclusion, our results demonstrate that autophagy influences maternal mRNA degradation and apoptosis at the blastocyst stage and suggest that autophagy plays an important role in early embryo development in the pig.

Production of somatic chimera chicks by injection of bone marrow cells into recipient blastoderms.

Several types of cells, including blastoderm cells, primordial germ cells, and embryonic germ cells were injected into early-stage recipient embryos to produce chimera avians and to gain insights into cell development. However, a limited number of studies of avian adult stem cells have also been conducted. This study is, to the best of our knowledge, the first to evaluate chicken bone marrow cells' (chBMC) ability to differentiate into multiple cell lineages and capability to generate chimera chicks. We induced random differentiation of chBMCs in vitro and injected immunologically selected pluripotent cells in chBMCs into the blastoderms of recipient eggs. The multipotency of BMCs from the barred Plymouth rock (BPR) was confirmed via AP staining, RT-PCR, immunocytochemistry, and FACS using specific markers, such as Oct-4 and SSEA-1, 3 and 4. Isolated chBMCs were found to be able to induce in vitro differentiation to multiple cell lineages. Approximately 5,000 chBMCs were injected into the blastoderms of white leghorn (WL) recipients and proved able to contribute to the generation of somatic chimera chicks with a frequency of 2.7% (2 of 73). Confirmation of chimerism in hatched chicks was achieved via PCR analysis using D-loop-specific primers of BPR and WL. Our study demonstrated the successful production of chimera chicks using chBMC. Therefore, we propose that the use of adult chBMCs may constitute a new possible approach to the production of chimera poultry, and may provide helpful studies in avian developmental biology.

Bone marrow cell-mediated production of transgenic chickens.

The transplantation of adult stem cells into recipients is a method used widely in mammals to determine the fate of transferred cells, and for the production of progenies. This study is the first report, to our knowledge, to demonstrate the successful production of chickens using cells transdifferentiated from adult chicken bone marrow cells (BMCs) transplanted into the testes. BMCs from the enhanced green fluorescent protein (eGFP) transgenic (Tg) chickens were induced via in vitro transdifferentiation to male germ cells and injected into the testes of normal recipients. The multipotency of BMC was found with RT-PCR, immunocytochemistry, and FACS using specific markers, such as OCT4 and SSEA-1, -3, and -4. Localization and in vivo transdifferentiation of injected cells in the seminiferous tubules of recipients were traced for up to 40 days' post-injection by GFP expression and immunocytochemical analyses. The integration of the eGFP and the neo(R) genes in sperm gDNAs of recipient was confirmed via PCR analysis. A subsequent testcross of the recipient roosters with non-Tg hens resulted in the production of eGFP Tg progenies, demonstrating the successful transdifferentiation of the adult BMC to the germ cells in the testis. Therefore, we suggest that the use of adult BMCs is a new and promising approach to the production of Tg poultry, and may prove helpful in the study of avian developmental biology.