Expression Pattern Of Oct-4 Research Articles

Expression Pattern of OCT-4 and PLZF Transcription Factors during the Early Events of Spermatogenesis in Mice

Spermatogenesis in mice is typically characterized by self-renewal and proliferation of spermatogonial stem cells (SSCs), which further give rise to mature sperm. The process of SSC self-renewal is regulated by number of genes. Oct-4 (POU family transcription factor) and Promyelocytic Leukemia Zinc Finger (Plzf) transcription factors are very well characterized and are known markers of undifferentiated spermatgonia. They are essential for self-renewal but their pattern of expression during the early events of spermatogenesis in mice has not been reported. Using real time PCR and Western blot we demonstrate marked increase in the expression of Oct-4 and Plzf in the testes of 10 days post partum (dpp) mice compared to 5 dpp both at the transcription and translation levels. The expression was found to be maximum in 10 dpp, declined in 20 dpp and was least in the testes of 35 dpp mice. However, our Immunohistochemistry (IHC) data showed the expression of OCT-4 & PLZF in the undifferentiated spermatogonia of 5, 10 & 20 dpp testes while in the testes of 35 dpp mice expression was seen in the undifferentiated as well as in differentiated spermatogonia. In conclusion, the present study reveals the expression pattern of Oct-4 and Plzf in the early stages of spermatogenesis of mice which sets the basis of spermatogenesis process in adults

Expression of OCT4 in human esophageal squamous cell carcinoma is significantly associated with poorer prognosis

To explore the expression pattern of OCT4 in human esophageal squamous cell carcinoma and its significance in diagnosis and prognosis. Using real-time polymerase chain reaction (PCR), Western blotting, immunocytochemistry and immunohistochemistry, the expression of OCT4 in three esophageal squamous cancer cell lines, KYSE70, KYSE140 and KYSE450, was characterized. OCT4 expression was investigated in a series of 153 esophageal squamous cell carcinoma samples using immunohistochemistry and explored its association with clinicopathological features. Immunohistochemically, OCT4 positive immunostaining was observed in cancer cell nuclei. OCT4 was variably expressed in three esophageal squamous cancer cell lines. Among 153 specimens, 105 (68.7%) were negative or weakly positive for OCT4 staining; 21 (13.7%) were moderately positive and 27 (17.6%) were strongly positive. Higher expression level of OCT4 was significantly associated with higher histological grade (P < 0.001) and poor clinic outcome (P < 0.001). The expression of OCT4 enables the tumor to have a higher degree of stemness, which in turn results in a poorer clinical outcome for patients with esophageal squamous cell carcinoma.

Gene expression profiling of pluripotency and differentiation-related markers in cat oocytes and preimplantation embryos

During mammalian preimplantation development, two successive differentiation events lead to the establishment of three committed lineages with separate fates: the trophectoderm, the primitive endoderm and the pluripotent epiblast. In the mouse embryo, the molecular mechanisms underlying these two cell fate decisions have been studied extensively, leading to the identification of lineage-specific transcription factors. Species-specific differences in expression patterns of key regulatory genes have been reported, raising questions regarding their role in different species. The aim of the present study was to characterise the gene expression patterns of pluripotency (OCT4, SOX2, NANOG) and differentiation (CDX2, GATA6)-related markers during feline early development using reverse transcription-quantitative polymerase chain reaction. In addition, we assessed the impact of in vitro development on gene expression by comparing transcript levels of the genes investigated between in vitro and in vivo blastocysts. To normalise quantitative data within different preimplantation embryo stages, we first validated a set of stable reference genes. Transcript levels of all genes investigated were present and changed over the course of preimplantation development; a highly significant embryo-stage effect on gene expression was observed. Transcript levels of OCT4 were significantly reduced in in vitro blastocysts compared with their in vivo counterparts. None of the other genes investigated showed altered expression under in vitro conditions. The different gene expression patterns of OCT4, SOX2, CDX2 and GATA6 in cat embryos resembled those described in mouse embryos, indicative of a preserved role for these genes during early segregation. However, because of the absence of any upregulation of NANOG transcription levels after embryonic genome activation, it is unlikely that NANOG is a key regular of lineage segregation. Such results support the hypothesis that the behaviour of early lineage markers can be species specific. The present study also revealed a pool of maternal NANOG mRNA transcripts, the role of which remains to be elucidated. Comparing transcription levels of these genes between in vivo and in vitro blastocysts revealed low levels of OCT4 mRNA in the latter, which may contribute to the reduced developmental competence of embryos under suboptimal conditions.

IMAGING REDOX STATE HETEROGENEITY WITHIN INDIVIDUAL EMBRYONIC STEM CELL COLONIES.

Redox state mediates embryonic stem cell (ESC) differentiation and thus offers an important complementary approach to understanding the pluripotency of stem cells. NADH redox ratio (NADH/(Fp + NADH)), where NADH is the reduced form of nicotinamide adenine dinucleotide and Fp is the oxidized flavoproteins, has been established as a sensitive indicator of mitochondrial redox state. In this paper, we report our redox imaging data on the mitochondrial redox state of mouse ESC (mESC) colonies and the implications thereof. The low-temperature NADH/Fp redox scanner was employed to image mESC colonies grown on a feeder layer of gamma-irradiated mouse embryonic fibroblasts (MEFs) on glass cover slips. The result showed significant heterogeneity in the mitochondrial redox state within individual mESC colonies (size: ∼200-440 μm), exhibiting a core with a more reduced state than the periphery. This more reduced state positively correlates with the expression pattern of Oct4, a well-established marker of pluripotency. Our observation is the first to show the heterogeneity in the mitochondrial redox state within a mESC colony, suggesting that mitochondrial redox state should be further investigated as a potential new biomarker for the stemness of embryonic stem cells.

Expression Pattern of Oct-4, Sox2, and c-Myc in the Primary Culture of Human Dental Pulp Derived Cells

Dental pulp cells (DPCs) have shown promising potential in dental tissue repair and regeneration. However, during in vitro culture, these cells undergo replicative senescence and result in significant alteration in cell proliferation and differentiation. Recently, the transcription factors of Oct-4, Sox2, c-Myc, and Klf4 have been reported to play a regulatory role in the stem cell self-renewal process, namely cell reprogramming. Therefore, it is interesting to know whether the replicative senescence during the culture of dental pulp cells is related to the diminishing of the expression of these transcription factors. In this study, we investigated the expression of the reprogramming markers Oct-4, Sox2, and c-Myc in the in vitro explant cultured dental pulp tissues and explant cultured dental pulp cells (DPCs) at various passages by immunofluorescence staining and real-time polymerase chain reaction analysis. Our results demonstrated that Oct-4, Sox2, and c-Myc translocated from nucleus in the first 2 passages to cytoplasm after the third passage in explant cultured DPCs. The mRNA expression of Oct-4, Sox2, and c-Myc elevated significantly over the first 2 passages, peaked at second passage ( P < .05), and then decreased along the number of passages afterwards ( P < .05). For the first time we demonstrated that the expression of reprogramming markers Oct-4, Sox2, and c-Myc was detectable in the early passaged DPCs, and the sequential loss of these markers in the nucleus during DPC cultures might be related to the cell fate of dental pulp derived cells during the long-term in vitro cultivation under current culture conditions.

123 AGGREGATION OF CLONED EQUINE EMBRYOS: IMPROVEMENT OF IN VITRO AND IN VIVO DEVELOPMENT

Development of cloned equine embryo is still inefficient. The aim of our study was to assess the aggregation of zona-free genetically identical cloned embryos as a strategy to improve in vitro and in vivo development. Oocyte collection, maturation, cloning, and activation procedures were performed as described by (Lagutina et al. 2007 Theriogenology 67, 90–98). After activation, reconstructed embryos (RE) were cultured in DMEM/F12 with 5% of FBS in the well of well system in 3 different groups: I, only one RE per well; II, two RE per well; and III, three RE per well. Cleavage and blastocyst formation (7 to 8 days) of all experimental groups was assessed. At day 8, some embryos of each group were either fixed to determine Oct-4 expression by immunocytochemistry or transferred transcervically to a synchronized mare. Pregnancies were assessed by ultrasound from 7 days after embryo transfer until day 45 to 50 of pregnancy every 7 to 10 days, and sizes of vesicles and embryos were measured. In advanced pregnant mares, combined thickness of the uterus and the placenta (CTUP) and serum progesterone levels were also determined. The remaining embryos obtained from each group were maintained in culture from day 7 until day 15. Blastocysts growth was determined every 24 h. In vitro development, on a per-well and RE basis, was compared using the chi-square test. Statistical differences were observed in cleavage among groups I and II (P = 0.0088) and groups I and III (P = 0.0004): (I: 91/111, 82%; II: 74/78, 95%; III: 62/62, 100%). Blastocyst rates differed between groups I and III (I: 10/111, 9%; III: 23/62, 37%); no difference was observed with group II (11/78, 14%). There was no difference on blastocyst rates based on the number of aggregated RE (I: 10/111, 9%; II: 11/156, 7%; III: 23/184, 12.5%). The highest pregnancy rate was obtained in group III (I: 1/3, 33%; II: 2/5, 40%; III: 3/4, 75%). Sizes of vesicles and embryos did not differ statistically in such groups. The CTUP and serum progesterone levels were considered normal (&lt;1.2 cm; &gt;8 ng mL–1, respectively) in ongoing pregnancies. We did not observe any differences in Oct-4 expression patterns among groups. Even though statistical differences were found, surprisingly all embryos grew in vitro until day 15 with good rates and the biggest embryo reached 4.25 mm. Embryo aggregation improved in vitro development of equine cloned embryos until day 7, and pregnancies rates were higher. The in vivo sizes of vesicles and embryos were normal for all groups, and in vitro development beyond day 7 showed the high viability of embryos. To conclude, aggregation of cloned equine embryo does not imply extra oocytes because there is no statistical difference in the number of blastocysts obtained per oocytes used to achieve RE. It is also a good strategy to improve in vitro embryo development without alterations on in vivo progress. This is the first report of pregnancies from aggregated equine cloned embryos, and the first healthy cloned foal from South America, confirmed by STR analysis, was born recently derived from group II. Stumpo, Ignacio, Paola Barboza, and Don Antonio staff.

63 OOCYTE GENOME CLONING USED IN TRANSGENIC BOVINE EMBRYO PRODUCTION

Oocyte genome cloning refers to obtaining haploid maternal embryos in such a way that parthenogenetic haploid blastomeres (PHB) from these embryos should be considered as a clone of the original gamete. Our main objective was to generate oocyte genome replicates and use them to reconstruct biparental embryos by fusion with haploid male hemizygotes. Moreover, we generated biparental homogeneous transgene-expressing embryos using PHB that express a transgene (EGFP). In the first experiment, parthenogenetic haploid embryos were generated by incubation in 5 μM ionomycin for 4 min followed by culture in SOF for 3 h to permit second polar body extrusion and subsequently treated with 1.9 mM DMAP for 3 h. To generate transgene-expressing PHB, parthenogenetic embryos were injected 3 h post-activation with pCX-EGFP–liposome complexes. These treatments were analysed by Fisher test (P &lt; 0.05). The cleavage rate of the haploid and diploid parthenogenetic embryos control was 87.3% (103/118) and 88.5% (108/122) respectively; (P &gt; 0.05). When the haploid activated oocytes were injected with pCX-EGFP–liposome complexes, the cleavage rate was 84.4% (54/64). These embryos showed 54.7% (35/54) of EGFP expression. In the second experiment, haploid parthenogenetic embryos (4 to 16 cells) were disaggregated and the PHB obtained were fused with a zona-free haploid male hemizygotes in order to reconstruct biparental embryos. Haploid male hemizygotes were generated by fertilization of enucleated oocytes. The haploid condition of parthenogenetic embryos and male hemizygotes was confirmed by karyotype analyzes. Reconstructed embryos were cultured in SOF medium in well of well system in 5% O2, 5% CO2, and 90% N2 at 39°C. The development of the reconstructed embryos expressing or not expressing EGFP reached 78.4% (29/37) and 61.1% (80/131) of cleavage; and 10.8% (4/37) and 8.4% (11/131) of blastocysts, respectively; (P &gt; 0.05). EGFP expression was observed in 100% of the reconstructed embryos, with 96.6% (28/29) of homogenic expression. The biparental IVF control showed 64.4% of cleavage and 22.5% of blastocysts (n = 98). The mean cell number of blastocysts were significantly different among the reconstructed biparental embryos (mean = 72.8) and the biparental IVF-embryos control (mean = 97.0). Additionally, the Oct-4 pattern expression was examined in the blastocysts, by inmunocytochemistry. Blastocysts displayed a pattern of Oct-4 expression similar to IVF-blastocyst control. Finally, biparental reconstructed blastocysts (n = 2) were transferred in recipient cows obtaining 1 pregnancy detected at 2 months after embryo transfer. In conclusion, our results proved that it is possible to use oocyte genome replicates to reconstruct biparental bovine embryos and that it is an efficient technique to generate homogeneous transgene-expressing embryos. Haploid oocyte genome replication would have high potential in livestock production, as it increases the number of embryos produced from a single oocyte, and allows the selection of favourable genetic characteristics before biparental embryo reconstruction.

Cloning efficiency following ES cell nuclear transfer is influenced by the methylation state of the donor nucleus altered by mutation of DNA methyltransferase 3a and 3b

The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodeling of cloned embryos. In this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed with embryonic stem (ES) cell nuclei. Our results confirmed that deletion of the DNA methyltransferase 3a (Dnmt3a) and DNA methyltransferase 3b (Dnmt3b) distinctly decreases the level of DNA methylation in ES cells. In contrast to wild type ES cells (J1), Dnmt3a − / − 3b − / − (DKO) and Dnmt3b − / − (3bKO) donor cells significantly elevated the percentage of embryonic stem cell nuclear transfer (ECNT) morula, blastocysts and postimplantation embryos (P < 0.05). However, the efficiency of establishment of NT-ES cell lines derived from DKO reconstructed blastocysts was not improved, and the expression pattern of OCT4 and CDX2 in cloned blastocysts and postimplantation embryos was not altered either. Our results suggest that the DNA methylation state of the donor nucleus is an important factor in regulation of the donor nuclear reprogramming.

6 EFFICIENT TRANSGENESIS IN BOVINE EMBRYOS BY FERTILIZATION WITH ANDROGENETIC TRANSGENIC BLASTOMERES

Pronuclear microinjection and intracytoplasmic sperm injection-mediated gene transfer (ICSI-mgt) are useful techniques to obtain transgenic animals. Nevertheless, a high frequency of mosaic expression is observed in embryos and offspring produced by these techniques. A possible explanation is that the transgene integrates in the embryo genome after the first cell division. Our main objective was to develop a new technique to generate transgenic bovine embryos without mosaic expression and with high efficiency. We hypothesize that fertilizing metaphase II (MII) oocytes with transgenic androgenetic haploidblastomeres (AHB) (from mosaic embryos) would result in non-mosaic transgenic embryos. To this aim, in the first experiment we generated AHB by enucleating IVM MII oocytes, before or after injecting with a single spermatozoon incubated with pCX-EGFP (enhanced green fluorescent protein) plasmid. These treatments were analyzed by Fisher test (P &lt; 0.05). The rate of cleavage of the androgenetic transgenic embryos enucleated before and after ICSI-mgt was 35.1% (34/97) and 61.2% (71/116), respectively (P &lt; 0.05). These embryos showed expression of EGFPof 11.8% (4/34) and 42.3% (30/71) (P &lt; 0.05) with 0% (0/34) and 9.9% (7/71) of non-mosaic expression. The haploid condition of the androgenetic embryos was confirmed by karyotype analysis. After this first approach, we chose the procedure of enucleation after ICSI for successive experiments. In the second experiment, the haploid androgenetic embryos (4 to 16 cells) were disaggregated, and the AHB obtained were used to fertilize MII oocytes. Fertilization was carried out by fusing a single AHB to a zona-free MII oocyte, followed by chemical activation. Presumptive zygotes were cultured in SOF medium in the well of the well (WOW) system. To confirm fertilization, single AHB produced with sexed Y spermatozoa and embryos generated with them were checked by PCR using Y- and X-specific sequence primers. PCR analysis confirmed Y-specific sequences in all the AHB and XY-specific sequences in each of the analyzed embryos. FISH analysis on blastocysts was performed with a specific probe for a Y chromosome sequence, confirming the sexed sperm genome in all blastocyst cells. Additionally, the expression pattern of Oct-4 (pluripotent marker gene) was examined in the blastocysts by inmunocytochemistry with a confocal microscope. Blastocysts displayed a pattern of Oct-4 expression similar to that of IVF embryos, indicating efficient nuclear reprogramming. Finally, we fertilized MII oocytes with EGFP-AHB to produce transgenic bovine embryos without mosaic expression. The development reached 85.1% of cleavage and 9.0% of blastocysts (n = 84). One hundred percent of the embryos showed EGFP expression, with 90.1% non-mosaic expression. In conclusion, our results proved that it is possible to use AHB for fertilization of MII oocyte, and that fertilization with transgenic AHB is a highly efficient technique for the generation of transgenic non-mosaic bovine embryos.

Inner Cell Mass Localization of NANOG Precedes OCT3/4 in Rhesus Monkey Blastocysts

The mechanism by which the inner cell mass (ICM) and trophectoderm (TE) become specified is poorly understood. Considerable species variation is evident in the expression of lineage-specific and embryonic stem cell (ESC) regulatory markers. We sought to investigate localization patterns of these markers in rhesus macaque compact morulae and blastocysts. NANOG protein was restricted to the ICM of blastocysts. In contrast to a previous report, the expression of CDX2 was detected in the primate blastocyst, localized specifically to the TE. Unlike the mouse embryo, OCT4 protein was detected using two different antibodies in both the ICM and TE. The ubiquitous pattern of OCT4 expression is consistent with observations in human, cow, and pig embryos. Significantly, lack of restricted OCT4 protein, and ICM localization of NANOG in primate blastocysts, suggests that NANOG may determine inner cell mass fate more specifically during primate development or may be less susceptible to culture artifacts. These results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of NANOG to constitute a complex interactive network. This difference may also underlie observations that regulatory mechanisms in ESC differ between mice and primates.

35 DEVELOPMENT OF INTERSPECIES CLONED MOUSE EMBRYOS RECONSTRUCTED WITH PORCINE ENUCLEATED OOCYTES

Interspecies somatic cell nuclear transfer (iSCNT) offers significant opportunities for the derivation of embryonic stem cells (ESCs) that can model diseases. In this study, we investigated the development of murine-porcine iSCNT embryos and compared these to outcomes associated with porcine-porcine SCNT embryos. Enucleation was performed on in vitro-matured porcine oocytes using the Oosight spindle viewer system (CRI, UK). Either murine embryonic fibroblasts or porcine cumulus cells were injected into porcine ooplasm using a piezo-electric micromanipulator. After being exposed to the porcine ooplasm for 1 h, the reconstructed oocytes were activated by a combination of A23187 and 6-DMAP for 3.5 h. For the murine-porcine embryos (n = 9375), we used a serial culture method, namely porcine zygote medium (PZM) for 48 h followed by incubation with KSOM for another 5 days. We found that the majority of murine-porcine embryos (73.58%) arrested at the 4- to 8-cell stage, whilst a very small minority progressed through to blastocyst (0.064%). Porcine-porcine SCNT reconstructed oocytes (n = 142) cultured in PZM achieved a 15% blastocyst development rate. In the murine-porcine embryos, 10/44 embryos had chromosomal DNA loss associated with apoptosis, as determined by immunocytochemistry using the H2A.X antibody. We also found that at later stages, morula to blastocyst, the mouse-to-pig reconstructed embryos did not exhibit murine-specific Oct-4 expression patterns. Furthermore, murine mitochondrial DNA tended to be eliminated rather than preserved by the murine nuclei, which could render any subsequently derived ESCs dysfunctional due to 2 diverse genomes contributing genes to the electron transfer chain.

In vitro and in vivo derived porcine embryos possess similar, but not identical, patterns of Oct4, Nanog, and Sox2 mRNA expression during cleavage development

In vitro culture conditions stress the cleavage stage mammalian embryo and can contribute to reduced developmental potential of cultured embryos. One process that may be altered during embryo culture is the establishment and maintenance of pluripotency. Pluripotency is largely controlled by three genes: Oct4, Nanog, and Sox2. The objective of this study was to determine the expression pattern of Oct4, Nanog, and Sox2 in cleavage stage porcine embryos obtained in vivo or by in vitro fertilization and parthenogenetic activation. We used quantitative, real time PCR to assess the relative amount of each transcript in cleavage stage embryos. We found that Oct4 was transiently activated at the 2-cell stage (P-value <0.05) while Nanog and Sox2 were activated at the 4-cell stage (P-value <0.05) in in vitro embryos. Embryos derived in vivo showed a similar but not identical pattern of expression of Nanog mRNA been in highest abundance both at the 4 cell and the blastocyst stage. The activation observed at the 4-cell stage for Nanog and Sox2 was shown to be RNA polymerase II dependent (P-value <0.05). This study showed that Oct4, Nanog, and Sox2 possess similar, but not identical, patterns of expression between in vitro and in vivo derived porcine embryos. The difference between the amount of transcripts may reflect the reduced developmental potential observed in in vitro cultured embryos.

Development of Interspecies Cloned Monkey Embryos Reconstructed with Bovine Enucleated Oocytes

This study was carried out to determine whether culture media reconstructed with bovine enucleated oocytes and the expression pattern of Oct-4 could support dedifferentiaton of monkey fibroblasts in interspecies cloned monkey embryos. In this study, monkey and bovine skin fibroblasts were used as donor cells for reconstruction with bovine enucleated oocytes. The reconstructed monkey interspecies somatic cell nuclear transfer (iSCNT) embryos were then cultured under six different culture conditions with modifications of the embryo culture media and normal bovine and monkey specifications. The Oct-4 expression patterns of the embryos were examined at the two-cell to blastocyst stages using immunocytochemistry. The monkey iSCNT embryos showed similar cleavage rates to those of bovine SCNT and bovine parthenogenetic activation (PA). However, the monkey iSCNT embryos were not able to develop beyond the 16-cell stage under any of the culture conditions. In monkey and bovine SCNT embryos, Oct-4 could be detected from the two-cell to blastocyst stage, and in bovine PA embryos, Oct-4 was detectable from the morula to blastocyst stage. These results suggested that bovine ooplasm could support dedifferentiation of monkey somatic cell nuclei but could not support embryo development to either the compact morula or blastocyst stage. In conclusion, we found that the culture conditions that tend to enhance monkey iSCNT embryo development and the expression pattern of Oct-4 in cloned embryos (monkey iSCNT and bovine SCNT) are different than in bovine PA embryos.

OCT4 is superior to CD30 in the diagnosis of metastatic embryonal carcinomas after chemotherapy

Correctly diagnosing a metastatic germ cell tumor after chemotherapy may be challenging because of the diverse morphological manifestations of postchemotherapy tumors. Both OCT4 and CD30 are sensitive markers for the identification of primary embryonal carcinomas; however, loss of expression of CD30 (65%) has been reported in metastatic embryonal carcinomas after chemotherapy. The present study was conducted to evaluate the expression patterns of OCT4 and CD30 in postchemotherapy metastatic embryonal carcinomas and to compare their utility as diagnostic tools. Twenty-five cases of metastatic embryonal carcinoma after chemotherapy were immunohistochemically analyzed for CD30, OCT4, and cytokeratin AE1/AE3 expression. The staining intensities and the percentages of positively staining tumor cells were recorded. Nineteen (76%) of 25 cases revealed diffuse, moderate to strong nuclear OCT4 staining in postchemotherapy embryonal carcinomas. Among these 19 OCT4-positive cases, 8 also revealed diffuse and moderate to strong membranous CD30 staining. Seven of these OCT4-positive cases retained focal and weak CD30 expression. The remaining 4 OCT4-positive cases demonstrated a complete loss of CD30 expression. The 19 OCT4-positive cases showed a positive but variable cytokeratin AE1/AE3 expression pattern. Six (24%) of 25 cases were negative for both CD30 and OCT4 but demonstrated diffuse and strong cytokeratin AE1/AE3 staining. OCT4 is a useful diagnostic marker to identify metastatic embryonal carcinomas after chemotherapy, with a better sensitivity than CD30.

Gonadotropins affect Oct‐4 gene expression during mouse oocyte growth

The transcription factor Oct-4 is required for the maintenance of stem cells pluripotency and is involved in the regulation of the expression of a number of developmental genes. Oct-4 is also expressed in the female gamete during folliculogenesis, but the role it plays is largely unknown. Its upstream and promoter regions have some characteristic features that make this gene a possible target of hormonal regulation. To further our understanding of Oct-4 gene expression during oocyte growth, we tested whether changes to the hormonal milieu of the ovary may affect its transcription. Using a semi-quantitative single-cell-sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay, we investigated the pattern of Oct-4 expression during mouse oocyte growth in females intraperitoneally injected with pregnant mare serum gonadotropin (PMSG) alone or PMSG followed by human chorionic gonadotropin (hCG). The results of this study show that gonadotropins induced two major increases in Oct-4 expression during folliculogenesis: (1) 48 hr after PMSG injection, in oocytes isolated from primordial follicles; and (2) following a surge of hCG, in preovulatory antral oocytes. These results suggest a potential twofold role for this gene in the recruitment of oocytes for initiating growth and in the selection of oocytes for ovulation. Also, they may contribute to our knowledge of the molecular bases of oocyte growth, meiosis resumption, and acquisition of a developmental competence.

144 Oct-4 EXPRESSION PATTERN IN THE EQUINE EMBRYO

Oct-4 is a key transcription factor in the control of early embryonic development and maintenance of a pluripotent cell population. Variation in Oct-4 expression patterns during embryo development have been reported among species, and have been related to the time of placental development in those species. This study was conducted to investigate Oct-4 expression pattern during early embryonic development in the horse, a species with relatively delayed placentation. In vitro-produced embryos were obtained from in vitro-matured oocytes via fertilization by intracytoplasmic sperm injection. Ex vivo blastocysts were recovered from mares that had been artificially inseminated. Oct-4 status was determined by immunocytochemistry; photomicrographs were taken at 4 standardized settings to aid in qualitative comparison of the amount of fluorescence. A total of 106 oocytes and embryos were evaluated. Immature oocytes showed Oct-4 expression in the nucleus and cytoplasm, as did early-cleaved embryos (2 to 5 cells, 1 to 2 days). Oct-4 expression in embryos at 3 to 4 days (6 to 12 cells) decreased and was restricted to the cytoplasm. From 5 to 6 days (15 cells to morulae), Oct-4 intensity increased and was exclusively found in the nuclei. In vitro-produced blastocysts (7 to 8 days) expressed Oct-4 equivalently in the trophectoderm and inner cell mass nuclei; culture for 2 to 3 more days (10 to 11 days) did not alter Oct-4 expression. However, when in vitro-produced blastocysts were transferred to the uteri of mares and recovered after 2 to 3 days (IVP-ET), the embryos showed strong expression of Oct-4 within the inner cell mass and limited expression in the trophectoderm, and a similar pattern was seen for ex vivo-recovered embryos. In bigger embryos (such as a 1779-�m ex vivo embryo and a 1121-�m IVP-ET embryo), the trophectoderm lost staining completely. These results suggest that Oct-4 expression is present in both nucleus and cytoplasm in equine oocytes and early-cleaved embryos as a result of maternal mRNA accumulation. Oct-4 protein decreases over the first few days of embryonic development as these stores are used. The shift to greater expression, in the nucleus only, during further embryo development suggests embryonic genome activation. Oct-4 expression in the trophectoderm of in vitro-produced blastocysts was different from that in blastocysts that had been exposed to the uterus (both ex vivo and IVP-ET); this indicates that differentiation of the trophectoderm is dependent upon factors present in the uterine environment. The Oct-4 expression in the trophectoderm of in vitro-produced equine blastocysts thus appears to be an artifact due to in vitro culture; this finding may be applicable to the reported patterns of Oct-4 expression in embryos of other species. This work was supported by the Link Equine Research Endowment Fund, Texas A&amp;amp;M University.

167 EXPRESSION OF INTERFERON τ IN BOVINE EMBRYOS DERIVED FROM PARTHENOGENETIC ACTIVATION

Parthenogenetic activation (PA) is a useful technique for reproductive technologies such as somatic cell nuclear transfer. Furthermore, there is a possibility of embryonic stem cell establishment deriving from PA embryos (Cibelli et al. 2002 Science 295, 819). Understanding of the ability of development and differentiation in PA embryos is important for the application. This study was designed to assess the gene expression of octamer-binding transcription factor (OCT-4) and interferon τ (IFNτ) in bovine PA embryos at the blastocyst (BC) and elongated (EL) stages, and the protein secretion of IFNτ at the EL stage. PA embryos were produced from oocytes matured in vitro (24 h), activated with Ca-ionophore (5 min) and electric pulse, and then treated with cytochalasin B and cycloheximide (5 h). In vivo-produced (Vivo) embryos were obtained non-surgically at Day 8 (Day 0 = estrus) from superovulated donor cows. PA or Vivo embryos were transferred to recipient cows (PA: 10 embryos/cow; Vivo: 1 embryo/cow) at Day 8, and then recovered non-surgically with uterine flushings at Day 16. Total RNA in single BC and EL embryos were reverse transcribed for PCR. Quantification of mRNA abundance was performed by real-time PCR. The expression of each mRNA was normalized to the abundance of GAPDH. IFNτ secretion of uterine flushings was estimated by RIA (Takahashi et al. 2005 Theriogenology 63, 1050–1060). The cleavage and blastocyst developmental rates of PA oocytes were 65.8 and 29.7%, respectively. Most embryos had positive signals of OCT-4 and IFNτ regardless of the origin and stage of embryos. The relative abundance (mean � SEM) of OCT-4 expression in PA and Vivo embryos dropped to its lowest level (1.78 � 0.32 and 1.14 � 0.45, respectively) at the EL stage, and it was significantly lower than that at the BC stage (1018.87 � 148.69 and 696.29 � 151.80, respectively; P &amp;lt; 0.05). The transcript level of OCT-4 was not significantly different between PA and Vivo embryos at both stages. Although the transcript level of IFNτ in PA and Vivo embryos increased significantly at the EL stage (0.36 � 0.06 and 7.68 � 2.01, respectively) from the BC stage (0.03 � 0.01 and 0.01 � 0.004, respectively; P &amp;lt; 0.05), that in PA embryos was significantly lower than that in Vivo embryos at the EL stage (P &amp;lt; 0.01). The total amount (mean � SEM) of IFNτ in uterine flushings from cows with transferred PA embryos was 3.38 � 0.35 �g (the number of embryos in each uterine flushing was unknown), and it was low compared with that from cows with Vivo embryos (13.40 � 3.03 �g). Our results indicate that bovine PA embryos have the ability to secrete IFNτ in the uteri of recipient cows at the EL stage, and there is a similar expression pattern of OCT-4 for Vivo embryos.

53 EVALUATION OF NUCLEAR REPROGRAMMING IN CLONED MINIATURE PIG EMBRYOS USING GENES OF Oct-4 FAMILY

Xenotransplantation as a source of organs is a rapidly expanding field which can save thousands of human lives each year. Cloned miniature pigs have been considered as a model system for xenotransplantation. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several aberrancies. Possible explanation of the developmental failure of SCNT embryos is related to insufficient reprogramming of the somatic cell nucleus. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and undifferentiated germ cell nuclei with Oct-4 and four Oct-4-related genes (Ndp5211, Dppa2, Dppa3, and Dppa5) as molecular markers using quantitative reverse transcription-polymerase chain reaction (RT-PCR). Oct-4 expression patterns were similar among IVF-derived embryos and cloned embryos derived from fibroblasts or germ cells during pre-implantation embryo development. However, the expression level was significantly lower (P &lt; 0.05) in hatched blastocysts of fibroblast clones compared to other hatched blastocysts. Also, 9 of 13 cloned morulae and 12 of 40 cloned blastocysts failed to reactivate at least one of the five tested genes, whereas all of the germ cell clones and control embryos correctly expressed these genes. Analysis with miniature pig fetuses collected at Day 30 of gestation revealed that normal and cloned fetuses successfully expressed these genes. In conclusion, our results suggest that analysis of expression of Oct-4 and related genes could be a reliable marker for evaluating the reprogramming status of transplanted donor nuclei in cloned embryos. The reprogramming of fibroblast cloned embryos is highly error-prone. This may contribute to their embryonic lethality because cloned embryos that fail to reactivate the marker genes may fail to be successfully implanted. This study was supported by grants from the Ministry of Science and Technology (Top Scientist Fellowship), and the Biogreen 21-1000520030100000.

43 DNA SYNTHESIS, PREIMPLANTATION DEVELOPMENT AND Oct-4 EXPRESSION OF BOVINE CLONES RECONSTRUCTED WITH OOCYTES PREACTIVATED OR ENUCLEATED AFTER SPINDLE DISASSEMBLY

Enucleation procedures applied in mammalian cloning remove not only the oocyte's chromosomes but presumably also the spindle-associated factors. If these factors are beneficial for reprogramming, alternative protocols that limit enucleation of factors in addition to the chromosomes may improve cloning efficiency. In this study, we evaluated the enucleation in combination with various activation protocols on clone development and gene expression. Clones produced by nuclear transfer into pre-activated bovine oocytes rather than non-activated oocytes can develop in vitro (Kurosaka et al. 2002 Biol. Reprod. 67, 643–647; Tani et al. 2003 Biol. Reprod. 69, 1890–1894). We produced bovine clones using four different nuclear transfer protocols and, in clones of all groups, examined timing of DNA synthesis in the first cell cycle, pre-implantation development, and gene expression at the blastocyst stage. Protocols applied were: (A) donor cells were fused with non-activated oocytes; (B) donor cells were fused with oocytes at 2 hours after activation with ethanol (7%, 7 min); (C) oocytes were enucleated after spindle disassembly with nocodazole treatment (0.3 μg/mL, 30 min) and donor cells were fused with non-activated oocytes; and (D) oocytes were enucleated after spindle disassembly and donor cells were fused with oocytes at 2 h after activation. Fused couplets in all treatment groups were treated with 10 μg/mL cycloheximide for 6 h, and cultured in vitro in SOF supplemented with fetal bovine serum at 39°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. The onset of DNA synthesis was determined by an immunofluorescence assay of 5-bromo-deoxyuridine incorporation at 6 and 9 h post-fusion (hpf). Oct-4 mRNA distribution in clone blastocysts was examined by whole mount in situ hybridization using a bovine Oct-4-specific antisense riboprobe. Data were statistically analyzed with Student's t-test. In the majority of clones DNA synthesis had not commenced 6 hpf but had initiated 9 hpf. Although the cell cycle of activated oocytes (protocols B and D) was 2 hours advanced compared to non-activated oocytes (protocols A and C), clones produced by all protocols had a similar onset of DNA synthesis at 6 to 9 h post-fusion. Developmental rates to the blastocyst stage of clones were not significantly different between the four protocols (48.5%–57.7%, P &lt; 0.05). Oct-4 distribution in clones produced by all four protocols was not different from that of IVF embryos used as a control in that Oct-4 mRNA signal was typically restricted to the ICM (87.0%–100.0%, P &lt; 0.05). We conclude that in bovine clones produced in this study, nocodazole-treated enucleation and activation status of recipient oocyte did not influence the pre-implantation development and spatial pattern of Oct-4 expression. This work was supported by the Lalor Foundation.

Oct-4 mRNA and protein expression during human preimplantation development

The transcription factor OCT-4 is regarded as a critical factor in controlling mammalian early embryonic development because of its role in toti-/pluripotency. In human preimplantation embryos, OCT-4 studies are limited to RNA analysis of abnormally developing embryos. This study thoroughly investigated the expression pattern of OCT-4 throughout the human preimplantation development. Expression was examined by single-cell RT-PCR or indirect immunocytochemistry in 36 single oocytes of various maturity and 112 normally developing preimplantation embryos at the level of single blastomeres, morulas, blastocysts, or inner cell mass (ICM) and trophectoderm (TE) samples. Oocytes and cleavage stage embryos revealed a variable OCT-4 expression pattern, concomitant with a pure cytoplasmic localization of the protein. During compaction, the variability in expression faded away indicating embryonic OCT-4 expression and the protein appeared in the nucleus implying biological activity. In blastocysts, OCT-4 transcripts and proteins were present in the ICM and the TE. At protein level, blastocysts displayed different spatial expression patterns within a cell for the splice variants of OCT-4, which may endow them with different functional properties. As OCT-4 transcripts were also found in various differentiated cells, the presence of OCT-4 transcripts or proteins may not be sufficient for identifying undifferentiated cell lines in humans. Further, we suggest to examine the localization of OCT-4 proteins within a cell rather than to look for the presence and/or amount of transcripts.