Non-invasive assessment of culture media from goat cloned embryos associated with subjective morphology by gas chromatography - mass spectroscopy-based metabolomic analysis.

Pre-implantation embryo metabolism demonstrates distinctive characteristics associated with the development potential of embryos. We aim to determine if metabolic differences correlate with embryo morphology. In this study, gas chromatography - mass spectroscopy (GC-MS)-based metabolomics was used to assess the culture media of goat cloned embryos collected from high-quality (HQ) and low-quality (LQ) groups based on morphology. Expression levels of amino acid transport genes were further examined by quantitative real-time PCR. Results showed that the HQ group presented higher percentages of blastocysts compared with the LQ counterparts (P < 0.05). Metabolic differences were also present between HQ and LQ groups. The culture media of the HQ group showed lower levels of valin, lysine, glutamine, mannose and acetol, and higher levels of glucose, phytosphingosine and phosphate than those of the LQ group. Additionally, expression levels of amino acid transport genes SLC1A5 and SLC3A2 were significantly lower in the HQ group than the LQ group (P < 0.05, respectively). To our knowledge, this is the first report which uses GC-MS to detect metabolic differences in goat cloned embryo culture media. The biochemical profiles may help to select the most in vitro viable embryos.

Age-associated and tissue-specific expression of osteopontin in male Hu sheep reproductive tract.

Osteopontin (OPN) is indispensable in mammalian reproduction, but the role of OPN in male reproductive tract and fertility remains unclear. The objective of this study is to elucidate the function of OPN by unveiling the localization and expression of OPN in the reproductive tract (testis, epididymis, and ductus deferens) of male Hu sheep in different ages (10-days, 4-months, and 8-months). To accomplish this, the localization, mRNA and protein expression patterns of OPN in all samples were investigated. Immune staining showed that OPN was present in the testicular interstitium of prepubertal Hu sheep testis (10-days and 4-months group), while it was immunostained in acrosomes of spermatids nearby adluminal compartment of seminiferous tubules in sexual maturity Hu sheep testis (8-months group). The localization of OPN in epididymis gradually changed from the loose connective tissue to the apical region of principal cells (pseudostratified columnar epithelium) with growing (10-days to 8-months). In addition, increase trend was observed in the mRNA expression levels of OPN with growing in the same reproductive tissues (P<0.05). Furthermore, two different OPN isoforms of 30kDa and 34kDa were detected in the reproductive tract of male Hu sheep by western blot. Immunofluorescence detection showed that OPN was localized in the cauda epididymal spermatozoa. These results suggested that the expression of OPN might be closely related to spermatogenesis and spermatozoa function in Hu sheep. This will be helpful for us to understand how OPN regulate the high reproductive capacity in Hu sheep.

Using cysteine/cystine to overcome oxidative stress in goat oocytes and embryos cultured in vitro.

Assisted reproductive techniques expose gametes to excessive concentrations of reactive oxygen species. The present study aimed to evaluate the effects of oxidative stress on apoptosis in goat oocytes and embryonic development. The results demonstrated that the addition of 100 µM hydrogen peroxide (H2O2) into media produces an oxidative environment during oocyte maturation. The number of cumulus cells positive for terminal deoxynucleotidyl transferase UTP nick end labeling, and the activity of caspase 3 in mature oocytes were increased, compared with the control group (P<0.05). In addition, the expression levels of mitochondrial regulators, including peroxisome proliferator­activated receptor Î³ coactivator-1 α (PGC-1α) and nuclear respiratory factor­1 (NRF­1) were increased in the oxidative oocytes, compared with those in the control group (P<0.05). The ratio of the proapoptotic gene, B cell lymphoma (Bcl-2)-associated X protein (BAX), to the anti­apoptotic gene, BCL­2, was higher in the H2O2 group, compared with the control group (P<0.05). To overcome oxidative stress in oocytes and embryos cultured in vitro, 200 µM cysteine and 200 µM cystine were added to the media, thereby increasing the concentration of intracellular glutathione (GSH) and assisting in maintaining the redox state of the cells. In conclusion, cysteine and cystine reduced the oxygen tension caused by H2O2, thereby providing a novel strategy for optimizing in vitro embryonic development systems.

Growth regulation, imprinting, and epigenetic transcription-related gene expression differs in lung of deceased transgenic cloned and normal goats.

Somatic cell nuclear transfer (SCNT) is a promising technique to produce mammalian transgenic clones. Only a small proportion of manipulated embryos, however, can develop into viable offspring. The abnormal growth and development of cloned animals, furthermore, are accompanied by aberrant lung development. Our objective was to investigate molecular background of lung developmental problems in transgenic (random insertion of exogenous DNA) cloned goats. We examined expression of 15 genes involved in growth regulation, imprinting, and epigenetic transcription in lung tissue of deceased transgenic cloned and normal goats of various ages. Compared with normal goats of the same age from conventional reproduction, expression of 13 genes (BMP4, FGF10, GHR, HGFR, PDGFR, RABP, VEGF, H19, CDKNIC, PCAF, MeCP2, HDAC1, and Dnmt3b) decreased in transgenic cloned goats that died at or shortly after birth; Expression of eight genes (FGF10, PDGFR, RABP, VEGF, PCAF, HDAC1, MeCP2, and Dnmt3b) decreased in fetal death of transgenic cloned goats. Expression of two epigenetic transcription genes (PCAF and Dnmt3b) decreased in disease death of transgenic cloned goats (1-4 months old). Disruptions in gene expression might be associated with the high neonatal mortality in transgenic cloned animals. These findings have implications in understanding the low efficiency of transgenic cloning.

Age-associated changes in gene expression of goat oocytes.

Oocyte aging severely decreases the quality of oocytes, which hampers fertilization and subsequent embryo development. In the present study, age-dependent molecular changes in goat oocytes were investigated. First, the quality of goat oocytes with various in vitro culture times (24, 30, 36, 48, and 60 hours) was evaluated on the basis of developmental rates of parthenogenetically activated embryos and apoptosis of cumulus cells (CCs). Second, relative gene expression of six genes (mitochondrial genes: PGC-1α and NRF-1; epigenetic modification genes: SNRPN and HAT1; mitotic spindle checkpoint protein: SMAD2; and hyaluronan synthase gene: HAS3) were analyzed during oocyte aging. Third, we further studied the changes of seven genes (PGC-1α and NRF-1; apoptotic-related genes: BAX and BCL2; hyaluronan synthase gene: HAS2; metabolism-related gene: STAR; and superoxide dismutase gene: SOD1) in CCs during oocyte aging. In these studies, the blastocyst rate gradually decreased and the number of apoptotic cells significantly increased as the culture time increased (P < 0.05). Moreover, relative gene expressions of PGC-1α, NRF-1 and SMAD2 significantly decreased from 24 to 36 hours (P < 0.05), whereas the levels of HAT1 and HAS3 slowly increased as culture was prolonged. Furthermore, the levels of PGC-1α, BCL2, HAS2 and SOD1 quickly reduced, and BAX significantly increased from 24 to 36 hours in aged CCs (P < 0.05). In conclusion, goat oocytes started to age at 30 hours in vitro culture, and gene expression patterns of oocytes and CCs significantly changed as the oocytes aged. Gene expression pattern changes in CCs may provide a convenient and effective way to detect oocyte aging without compromising oocyte integrity.

Production of myostatin-targeted goat by nuclear transfer from cultured adult somatic cells.

Myostatin, a member of the transforming growth factor-ß family, acts as a negative regulator of skeletal muscle mass. In this study, myostatin-targeted caprine fibroblasts were obtained and subjected to SCNT to determine whether myostatin-knockout goats could be created. Fibroblasts from a 2-mo-old goat were transfected with a myostatin-targeted vector to prepare transgenic donor cells for nuclear transfer. After serum-starvation (for synchronization of the cell cycle), the percentage of transgenic fibroblasts in the G(0)/G(1) phase increased (66.2% vs. 82.9%; P < 0.05) compared with that in the control group, whereas the apoptosis rate and mitochondrial membrane potential were unaffected (P > 0.05). There were no significant differences between in vivo- and in vitro-matured oocytes as recipient cytoplasts for rates of fusion (86.5% vs. 78.4%), pregnancy (21.6% vs. 16.7%), or kidding (2.7% vs. 0%). One female kid from an in vivo-matured oocyte was born, but died a few hours later. Microsatellite analysis and polymerase chain reaction identification confirmed that this kid was genetically identical to the donor cells. Based on Western blot analysis, myostatin of the cloned kid was not expressed compared with that of nontransgenic kids. In conclusion, SCNT using myostatin-targeted 2-mo-old goat fibroblasts as donors has potential as a method for producing myostatin-targeted goats.

Efficiency of donor cell preparation and recipient oocyte source for production of transgenic cloned dairy goats harboring human lactoferrin.

The objective was to investigate the effects of the transgenic donor cell synchronization method, oocyte sources, and other factors, on production of hLF-gene nucleus transfer dairy goats. Three transfected cell lines from ear biopsies from three 3-mo-old Saanen dairy goats (designated Number 1, Number 2, and Number 3, respectively) were selected as karyoplast donors for somatic cell nuclear transfer (SCNT) after detailed identification (including PCR and sequencing of PCR products). In donor cell cycle synchronization studies, the apoptosis rate of hLF transgenic fibroblasts was not different (P > 0.05) after 3 days of serum starvation or 2 days of contact inhibition. Additionally, there was no effect (P > 0.05) on developmental capacity of reconstructed embryos; however, the kidding rate of recipients in the serum starvation group was higher than that in the contact inhibition group (18 vs. 0%, respectively). The production efficiency of the transgenic cloned goats using donor cells from the Number 1 dairy goat cell line was higher than those using the Number 2 and the Number 3 cell lines (kidding rates were 18, 2, and 0%, respectively, P < 0.05). The oocyte source did not significantly affect the pregnancy rate of hLF-transgenic cloned dairy goats, but more fetuses were aborted when using in vitro matured oocytes compared to in vivo matured oocytes. In summary, utilizing transfected 3-mo-old dairy goat fibroblasts as donor cells, seven live offspring were produced, and the hLF gene was successfully integrated. This study provided additional insights into preparation of donor cells and recipient oocytes for producing transgenic cloned goats through SCNT.