A transgenic pig carrying MMTV/v-Ha-ras was produced by microinjection of DNA into pronuclear stage embryos. Reproductive technologies, such as superovulation, insemination methods and synchronization of recipients were also investigated to establish the efficient systems to obtain pronuclear stage embryos and transgenic pigs aiming at improving efficiency of the protocol for producing transgenic pigs. Integration of the transgene was detected by Southern hybridization analysis in one female among 29 piglets which had been born after transfer of 195 DNA-injected embryos to 8 recipients. Expression of the transgene was detected by RT-PCR in 8 of 17 organs of the founder transgenic pig examined. The transgenic pig transmitted the transgene to 42.3% of 26 offspring. However, development of tumour was not found either in the founder or its progeny during the observation period for 30 and 1-8 months, respectively. The average number of ova collected from donors superovulated with 1500 IU eCG was significantly higher than the number collected from gilts which were superovulated with 1000 IU (26.3 vs 6.8, P<0.05). Donors served by artificial insemination yielded nearly the same number of embryos per head as those mated naturally (14.8 vs 16.1 ). Recipients of which the estrus was synchronized through administration of either 1500 or 750 IU eCG gave rise to pregnancies (5/5 vs 3/4). These data indicate that transgenic pigs carrying activated oncogene can be produced, though induction of the developmental disorders is yet to be investigated.
The fine morphology of the testes and epididymes collected from breeding Zebu (Bos indicus) bulls with normal or decreased testicular consistency (TC) at clinical palpation was studied. The ultrastructure of testicles with a slight to moderate reduction in consistency showed more obvious, albeit slight, degenerative changes in the seminiferous epithelium, with cellular debris present in Sertoli cells, compared with control animals. Abnormalities in the condensation of the chromatin and acrosomes during spermiogenesis were also common findings in cases of decreased TC. The cauda epididymides of these bulls did not show clear morphological deviations compared with controls, although the macrophagic activity of the epithelium was more pronounced, and cell debris as well as foreign cells were apparent in the lumen. The morphological findings confirm the relationship between a decreased TC at clinical palpation and the lowered normality of the testicular parenchyma, thus supporting the value of palpating scrotal contents as part of the field andrological clinical evaluation of breeding Zebu bulls in the tropics.
Newly developed serum-free media (IVD101 in the absence of bovine cumulus/granulosa cell (BCGC) coculture and IVMD101 in the presence of BCGC coculture) have recently been found to improve the yield and quality of bovine blastocysts from in vitro matured and fertilized oocytes . The present study was undertaken with two objectives: first, to examine whether bovine nuclear transfer embryos could develop to the blastocyst stage when cultured in the serum-free media (IVD101 and IVMD101): second, to confirm the potency of the nuclear transfer blastocysts to develop to viable offspring after transfer to recipient cows. In vivo-derived embryos were used as donor nuclei and in vitro-derived oocytes were used as recipient oocytes for nuclear transfer. Recipient oocytes at the second metaphase were enucleated and the fusion of donor blastomere with the recipient oocytes was carried out by electrostimulation. The nuclear transfer embryos cultured in IVMD101 with BCGC coculture and IVD101 without BCGC coculture resulted in 20.4% and 27.5% blastocyst formation rate, respectively, which were similar to that (23.4%) of nuclear transfer embryos cultured in a conventional serum-supplemented medium (CR1aa+5% calf serum; CS) with BCGC coculture. Transfer of the nuclear transfer blastocysts developed in IVMD101, IVD101 and CR1aa+5%CS resulted in one pregnancy (1/7), two pregnancies (2/6), and two pregnancies (2/5), respectively, when examined on Day 40. The corresponding numbers of normal live calves born were 1, 1, and 1. Our results indicate that the two different serum-free media may be useful for the in vitro development of nuclear transfer embryos and the subsequent production of normal calves after transfer to recipient cows.
We cloned cDNA of βig-h3, a TGF-β responsive gene, from the caprine uterus by RT-PCR method combined with the 5'- and 3'- RACE. The caprine βig-h3 cDNA contains a single open reading frame (ORF) encoding a 683-amino-acid protein which is highly conserved among the three species so far examined, i. e., goats, mice and humans. The hydropathy plots of caprine p68βig-h3 (formerly βIG-H3) protein reveals that hydrophilic and hydrophobic domains are regularly repeated. The hydrophobic domain spanning from Met1-Ala23 reprents the signal peptide sequence indicating that caprine p68βig-h3, like in other species, is a secretory protein. On Northern blot analysis, the pregnant uterus exhibited a strong signal at 2.7 kb, while in the immature uterus and the placenta, signal levels were very low.
Studies of the cervix are important for the understanding of structural and physiological changes occurring during the estrous cycle and the second half of pregnancy. The aim of the present study was to illustrate the structural differences seen in the porcine cervix, in relation to the estrogen/progesterone status of the animal. Tissue samples from the uterine portion of the cervix were collected from 23 gilts and assayed with solution hybridization for their contents of mRNA for the estrogen receptor (ER), progesterone receptor (PR), insulin-like growth factor-I (IGF-I) and thioredoxin, all known to vary with reproductive status. The mRNA levels of ER and PR were highest prior to standing estrus. The PR mRNA level was higher at term compared to mid pregnancy, whereas the serum progesterone concentration was the opposite. The thioredoxin mRNA level was highest around standing estrus and at term pregnancy. The IGF-I mRNA level had a peak just after standing estrus, while no significant changes were seen during pregnancy. Thus, steroid hormone involvement in the changes of the porcine cervix during estrus and term pregnancy are likely to be mediated via variations in the expression of gonadal steroid hormone receptors and substances with growth factor or cytokine-like activities.
This study regionalises the epididymal duct of the domestic cat according to the characteristics of the epithelium. The epididymes of nine postpubertal domestic cats were immersion-fixed in glutaraldehyde and plastic-embedded. Semi-thin sections were conventionally stained with H&E, buffered toluidine blue or with PAS. Morphometry was performed on clearly perpendicular sections. Principal cells were most abundant with intermingled basal cells and scattered apical cells. In addition, lymphocyte-like cells could be found among the epithelial cells. The duct could be clearly divided in six different regions according to the histological characteristics. The width of the lumen decreased from region 1 to region 3 where it had the smallest diameter, to widen again, reaching maximum diameter in region 6. The epithelium was high in regions 1 and 2 (58.9 and 60.7 μm), lower in region 4 (47.7 μm) and low in regions 3, 5 and 6 (41.1, 40.2 and 31.2 μm). Stereocilia were long in region 1 and 2, decreasing thereafter to be very short in regions 5 and 6 where they resembled a brush border. Cytoplasmic granules were most abundant in region 2 where they almost completely filled the cytoplasm of the principal cells. Intraepithelial cysts were found in regions 5 and 6. In conclusion this study clearly shows that six different regions can be distinguished in the epididymis of the domestic cat.
To promote the utilization of chicken primordial germ cells (PGCs) in transgenic manipulation, the in vitro culture of these cells was conducted. The PGCs were isolated from the blood vessels of chicken embryos, labeled with PKH26 red fluorescent cell linker, refined by picking up with micromanipulator attached with a fine glass pipette and cultured on feeder cells derived from chicken germinal ridges in medium DMEM plus F10 supplemented with 10% fetal calf serum (FCS) in 96 well plate at 39 C, 5% CO2 in air. The result of cell counting showed that in this condition chicken PGC number increased at least 29 times when the cells were cultured for up to 17 days and the proliferation of chicken PGCs was directly observed through cell tracing experiment, suggesting that feeder cells from germinal ridges might provide some kinds of essential factor(s) for PGC division.
The present study examined whether delipated porcine oocytes and embryos at various stages of development can be cryopreserved by conventional slow cooling or vitrification. Most (93%) of the 27 delipated morulae developed to blastocysts after freezing with 1.5 M propanediol + 0.1 M sucrose. Late morulae and early blastocysts delipated at 2-4 cell stage and cultured in vitro survived freezing either with 1.5 M glycerol + 0.25 M sucrose (10/18, 56%) or 1.8 M ethylene glycol + 0.25 M sucrose (14/19, 74%). Delipated 2-4 cell stage embryos and oocytes could be cryopreserved by vitrification with 40% ethylene glycol, 1 M sucrose and 20% fetal calf serum. Half (7/14) of the vitrified, delipated embryos developed to blastocysts after thawing. Of 48 delipated oocytes, 27 (56%) maintained an intact outline of the ooplasm after vitrification and underwent subzonal sperm injection. Fertilization was confirmed in 12 (25%) of these oocytes and 3 (6%) developed to morula stage. This study also aimed at developing a non-invasive method for cryopreserving porcine embryos after reducing their cytoplasmic lipid content without micromanipulation. Morulae and early blastocysts were centrifuged in the presence of cytochalasin B and cryoprotectants and then frozen immediately. More than half (14/24, 58%) of the centrifuged morulae developed to blastocycts after freezing with 1.5 M propanediol + 0.1 M sucrose. Greater than 70% of centrifuged early blastocysts survived freezing either with 1.5 M propanediol (30/31, 97%), 1.5 M glycerol (22/29, 76%) or 1.8 M ethylene glycol (21/29, 72%). These results demonstrated that delipation (lipid removal) from porcine oocytes and embryos at various stages enables their cryopreservation. A new insight into the development of a non-invasive method for cryopreserving porcine embryos was also provided.
In recent years, much attention has been paid to the manipulation of primordial germ cells (PGCs) to produce transgenic chicken. For this purpose, the in vitro proliferation of PGCs would make gene targeting possible, promoting the efficiency of transgenes. However, few researches have been carried out on this topic. In the present study, we tried to culture chicken PGCs outside the body. The PGCs were isolated from circulating blood of chicken embryos by the method of Ficoll density centrifugation, and labeled with PKH26 red fluorescent cell linker. The cells were refined by selection with a fine glass pipette and a micromanipulator in order to avoid the contamination of PGCs with red cells. The refined cells were then cultured for a given period. The PGCs proliferated when they were cultured on feeder cells derived from the germinal ridges of chicken embryos at stage 27. During a 5 day culture period, the highest rate of increased PGCs was about 51% for one of the two test groups. Although some kinds of growth factors were supplemented in the culture media, no synergistic effect of chicken stem cell factor (chSCF) with murine leukemia inhibitory factor (LIF) and human basic fibroblast growth factor (bFGF) on the proliferation of PGCs was found. This result implies that the LIF and bFGF influencing the proliferation of PGCs may not be conserved between mammals and birds.
A solid-phase extraction procedure for estradiol-17β from bovine blood plasma with a reversed phase cartridge was developed for the measurement of its concentration by radioimmunoassay. Estradiol-17β retained in the cartridge was eluted quantitatively with 70%(v/v) methanol, although estrone was recovered in the same fraction. The apparent values measured by dextran-coated charcoal radioimmunoassay with an eluent of 75%(v/v) methanol in the solid-phase extraction declined approximately by 10% as applied plasma volume was increased by 1 ml (r=-0.98, p<0.001). However, the values by double antibody radioimmunoassay were elevated approximately 8% per 1 ml plasma increase (r=0.82, p<0.001). The recovery of estradiol-17β added to bovine plasma in the extraction and radioimmunoassay with dextran-coated charcoal separation was approximately 75% under conditions of 2 ml plasma application, and concentrations calculated using buffer standards. Matrix effects on the assay recovery were negligible in the addition of solid-phase extract of castrated bull plasma to the standards. On the other hand, the average recoveries calculated with buffer and process standards in double antibody radioimmunoassay were 170% and 110%, respectively. The assay value in the solid-phase extraction showed good agreement with the value in the liquid-liquid extraction and liquid chromatography after correction with the recovery rate (r=0.99). In conclusion, solid-phase extraction with a reversed phase cartridge is a practical extraction method for the measurement of estradiol-17β during the estrous cycle in cattle.