Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level; highlighting the non-random nature of oocyte-mediated reprogramming errors.

BACKGROUND: The limited duration and compromised efficiency of oocyte-mediated reprogramming, which occurs during the early hours following somatic cell nuclear transfer (SCNT), may significantly interfere with epigenetic reprogramming, contributing to the high incidence of ill/fatal transcriptional phenotypes and physiological anomalies occurring later during pre- and post-implantation events. A potent histone deacetylase inhibitor, trichostatin A (TSA), was used to understand the effects of assisted epigenetic modifications on transcriptional profiles of SCNT blastocysts and to identify specific or categories of genes affected. RESULTS: TSA improved the yield and quality of in vitro embryo development compared to control (CTR-NT). Significance analysis of microarray results revealed that of 37,238 targeted gene transcripts represented on the microarray slide, a relatively small number of genes were differentially expressed in CTR-NT (1592 = 4.3 %) and TSA-NT (1907 = 5.1 %) compared to IVF embryos. For both SCNT groups, the majority of downregulated and more than half of upregulated genes were common and as much as 15 % of all deregulated transcripts were located on chromosome X. Correspondence analysis clustered CTR-NT and IVF transcriptomes close together regardless of the embryo production method, whereas TSA changed SCNT transcriptome to a very clearly separated cluster. Ontological classification of deregulated genes using IPA uncovered a variety of functional categories similarly affected in both SCNT groups with a preponderance of genes required for biological processes. Examination of genes involved in different canonical pathways revealed that the WNT and FGF pathways were similarly affected in both SCNT groups. Although TSA markedly changed epigenetic reprogramming of donor cells (DNA-methylation, H3K9 acetylation), reconstituted oocytes (5mC, 5hmC), and blastocysts (DNA-methylation, H3K9 acetylation), these changes did not recapitulate parallel marked changes in chromatin remodeling, and nascent mRNA and OCT4-EGFP expression of TSA-NT vs. CRT-NT embryos. CONCLUSIONS: The results obtained suggest that despite the extensive reprogramming of donor cells that occurred by the blastocyst stage, SCNT-specific errors are of a non-random nature in bovine and are not responsive to epigenetic modifications by TSA.

Improved in vitro development of cloned bovine embryos using S-adenosylhomocysteine, a non-toxic epigenetic modifying reagent.

In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation.

Lentiviral vector-mediated transduction of goat undifferentiated spermatogonia.

Recent studies show that spermatogonial stem cells (SSCs) are able to colonize and form mature spermatozoa following transplantation into germ cell depleted testes of recipient males. Therefore, efficient ways for enrichment and gene transfer into SSCs provides a powerful tool for production of transgenic animals. In order to adapt the technique to goats, three issues were addressed: (i) enrichment of the undifferentiated spermatogonia including SSCs using magnetic activated cell sorting (MACS), (ii) lentiviral vector-mediated transduction of an enhanced green fluorescent protein (EGFP) transgene into enriched cells, and (iii) transplantation of transduced undifferentiated spermatogonia into the germ cell depleted testes of immune-suppressed mice to assess for migration and colony formation ability. Enriched cells were transduced by lentiviral vectors and subsequently analyzed for expression of THY1, PLZF, VASA, UCHL1 and BCL6B genes. Cells were also analyzed for GFP and PLZF by flow cytometry. Enriched transduced cells were transplanted into germ cell depleted mice testis. Quantitative analysis of transcripts revealed that MACS-enrichment significantly increased the expression of SSC-characteristic genes THY1, PLZF, VASA, UCHL1 and BCL6B compared to non-enriched population (P≤0.05). EGFP transduction did not affect the expression levels of SSC-characteristic genes. Flow cytometry revealed that 72% of transduced-enriched cells were positive for EGFP. Finally, transduced-enriched goat SSCs could colonize within the cells into the seminiferous tubules of germ cell depleted recipient mice at higher frequency than non-enriched cells. The results indicated that enrichment of goat undifferentiated spermatogonia by magnetic-activated cell sorting for THY1 antibody combined with lentiviral vector-mediated transduction has the potential to be used for production of transgenic goats.

THY1 as a reliable marker for enrichment of undifferentiated spermatogonia in the goat.

Spermatogonial stem cells are unique cells of testes that can restore fertility upon transplantation into recipient testes. However, use of suitable markers for enrichment of these cells have important potential application. THY1, is an established conserved marker of spermatogonial stem cells in bovine, rodents, and primates, but there is no information available in goats. After three rounds of enzymatic digestion of prepubertal goat testicular tissues, undifferentiated spermatogonia positive for THY1 were isolated by magnetic-activated cell sorting and were used for immunocytochemistry, real-time polymerase chain reaction analysis for gene expression, protein expression, and transplantation into recipient mice. Immunocytochemical analyses showed that significantly higher percentage of THY1(+) cells were positive for PLZF and VASA when compared with unselected population. This result for PLZF was further confirmed at the protein level. Real-time polymerase chain reaction analysis revealed that expression of THY1, PLZF, VASA, BCL6B, and UCHL1 as SCCs characteristic genes in THY1(+) cells was significantly higher than in the initial population. Finally, transplantation of PKH26-labeled cells revealed that THY1(+) cells had higher capacity for colony formation when compared with unselected cells. In conclusion, the results provide indications that THY1 surface marker can be reliably used for enrichment of undifferentiated spermatogonial in the goats.

Improved bovine ICSI outcomes by sperm selected after combined heparin-glutathione treatment.

Despite widespread application of intracytoplasmic sperm injection (ICSI) in human-assisted reproductive techniques (ART), the efficiency of this method is still far from satisfactory in livestock, particularly in the bovine species with its unique sperm condensation. On the basis of the natural chemical structure of chromatin in condensed sperm, we developed a novel combined heparin-reduced glutathione (GSH) sperm pretreatment that improves the efficiency of bovine ICSI via selection of the most appropriate sperm at the time of ICSI. Assessment of sperm DNA integrity revealed that this pretreatment can be considered as a safe and efficient approach for in vitro sperm decondensation when compared to conventional sperm pretreatments with dithiothreitol (DTT). Injection of completely decondensed bull sperm derived from this pretreatment significantly improved fertilization and blastocyst formation rates compared to untreated or intact sperm injection (34.8 ± 2.7 and 29.1 ± 1.5 vs. 12.0 ± 3.2 and 15.9 ± 1.2%, respectively; p<0.05). Real-time PCR analysis revealed that expression of pluripotent and anti-apoptosis markers in blastocysts derived by injection of completely decondensed sperm from heparin-GSH pretreatment were comparable to IVF when compared to the DTT pretreatment and control ICSI groups (p<0.05). The results of this study suggested that the degree of sperm decondensation derived from heparin-GSH pretreatment may affect ICSI efficiency in bovine.

Developmental Competence and Pluripotency Gene Expression of Cattle Cloned Embryos Derived from Donor Cells Treated with 5-aza-2'-deoxycytidine.

BACKGROUND: Reconstructed embryos from terminally differentiated somatic cells have revealed high levels of genomic methylation which results in inappropriate expression patterns of imprinted and non-imprinted genes. These aberrant expressions are probably responsible for different abnormalities during the development of clones. Improvement in cloning competency may be achieved through modification of epigenetic markers in donor cells. MATERIALS AND METHODS: Our objective was to determine if treatment of donor cells for 72 hours with 5-aza-2'-deoxycytidine (5-aza-dc; 0-0.3 µM), a DNA methyl transferase inhibitor, improved development and expression of Oct-4. RESULTS: In comparison with untreated cells, 0.01 and 0.08 µM 5-aza-dc treated cells insignificantly decreased the blastocyst rate (32.1% vs. 28.6% and 27.2%, respectively) while it was significant for 0.3 µM treated cells (6.5%). Embryo quality as measured by the total cell number (TCN) decreased in a dose-related fashion, which was significant at 0.08 and 0.3 µM 5-aza-dc treated cells when compared with 0 and 0.01 µM 5-aza-dc treated cells. Although reconstructed embryos from 0.08 and 0.3 µM 5-aza-dc treated cells showed lower levels of DNA methylation and histone H3 acetylation, development to blastocyst stage was decreased. The epigenetic markers of embryos cloned from 0.01 µM 5-aza-dc remained unchanged. CONCLUSION: These results show that 5-aza-dc is not a suitable choice for modifying nuclear reprogramming. Finally, it was concluded that the wide genomic hypomethylation induced by 5-aza-dc deleteriously impacts the developmental competency of cloned embryos.

Effect of Culture System on Developmental Competence, Cryosurvival and DNA-Fragmentation of In Vitro Bovine Blastocysts.

BACKGROUND: This study investigated the effect of two in vitro embryo culture systems (co-culture system versus cell-free sequential-media) on developmental competence, cryosurvival and DNA- fragmentation of in vitro developed bovine blastocysts. MATERIALS AND METHODS: Bovine presumptive zygotes were cultured in Ménézo's B2 (B2) plus vero-cells or sequential synthetic oviductal fluid (SOF) for eight days. Subsequently, half of the expanded blastocysts developed in both groups were vitrified, warmed within 30 minutes and post- warming embryos along with their corresponding non-vitrified embryos were cultured for two additional days in the same medium used before vitrification. Embryo development, cryosurvival and apoptosis were compared between the groups. RESULTS: For non-vitrified embryos, culture in SOF significantly promoted the potency of embryos to develop into blastocysts compared with the co-culture system. The difference in post vitrification survival rate of SOF blastocysts (83.3%) was insignificant compared with co-culture (84.3%). However, while total cell number of warmed blastocysts in the co-culture system was significantly higher in the co-culture versus the sequential system (215.4 vs. 170.4), the quality of survived embryos in terms of hatching ability and apoptosis was adversely affected by co-culture compared with SOF (65.0% vs. 74.3%, and 13.5% vs. 10.0%, respectively; p<0.05). CONCLUSION: Although co-culture system may increase the viability of embryos following cryopreservation, the potency and dynamics of blastocyst formation significantly increased with sequential media compared to the co-culture system which can compensate for the lower efficiency of sequential media for vitrification/warming purposes.

Effect of ovarian cyclic status on in vitro embryo production in cattle.

BACKGROUND: The relationship between cyclic status of cattle ovaries on in vitro embryo development up to the blastocyst stage was investigated. MATERIALS AND METHODS: Cattle ovaries were collected immediately after slaughter and divided into three categories based on their cyclic status, which included: 1. the presence of a large follicle (LF), 2. the presence of a corpus luteum (CL) and 3. ovaries without LF or CL (WLCF). Oocytes of these ovaries were obtained and used for in vitro maturation and fertilization. Presumptive zygotes were then cultured up to the blastocyst stage in synthetic oviductal fluid culture medium. RESULTS: There were no significant differences between cleavage rates of the three groups. The rate of embryos in the compact morula stage for the CL group was 48.2% which was significantly higher than the related rate of the LF group (36.6%), but non-significantly higher than that of the ST group (45.7%). The highest blastocyst rate belonged to the CL group (54.6%) which was significantly greater than the WLCF group (32.9%) and non-significantly higher than the LF group (52.4%). There was no significant difference in blastocyst rates in the CL and LF groups. CONCLUSION: Preselection of oocyte donor ovaries containing a CL or LF can be used as a feasible and non- invasive criterion to obtain the most competent oocytes capable of development to the blastocyst stage.