Effects of epidermal growth factor on early embryonic development after in vitro fertilization of oocytes collected from ewes treated with follicle stimulating hormone.

Epidermal growth factor (EGF) has been shown to enhance the in vitro rate of blastocyst formation in several species. Follicular development was induced in ewes (n=15) by twice daily administration of FSH-P on Days 13 and 14 of the estrous cycle. Cumulus oocyte complexes (COCs) were collected from all visible follicles (n=25+/-2.4/ewe) on Day 15. COCs from each ewe were cultured separately for 24h in maturation medium (containing 10% serum, LH, FSH and estradiol) with (8.2+/-0.9 per ewe) or without (7.8+/-0.8 per ewe) EGF (10 ng/ml). Oocytes were then denuded by hyaluronidase treatment, and healthy oocytes were cultured in the presence of frozen-thawed semen in synthetic oviductal fluid (SOF) medium containing 2% sheep serum. After 18-20 h, zygotes were transferred to SOF medium without glucose and cultured for about 36 h until they reached the 4-8 cell stage. Embryos were transferred to SOF medium with glucose for further development. Medium was changed every other day until blastocyst formation on Day 8 of culture (Day 1=day of fertilization). The rate of embryonic development was evaluated throughout the culture period. After maturation, cumulus cells were more expanded in the presence than in the absence of EGF. The rates of fertilization (overall 75.7+/-3.9%) and morula formation (overall 40.6+/-7.1%) were similar (P>0.05) for COCs cultured with or without EGF. However, EGF increased (P<0.01) the number of blastocysts (1.4+/-0.1 versus 0.6+/-0.2 per ewe) and tended to increase (P<0.1) the rate of blastocyst formation (21.0+/-6.6% versus 13.4+/-4.3% per ewe). These data demonstrate that EGF increases blastocyst formation in FSH-treated ewes. Therefore, EGF is recommended as a supplement to maturation medium to enhance embryonic development in vitro in FSH-treated sheep.

Nonassociation of interleukin-1 receptor antagonist genotypes with bone mineral density, bone turnover status, and estrogen responsiveness in Korean postmenopausal women.

Interleukin-1 receptor antagonist (IL-1ra), a natural inhibitor of interleukin-1 (IL-1), completely inhibits the stimulatory effects of IL-1 on bone resorption. Bioactivity of IL-1 increases in the estrogen-deficient state with an increased IL-1:IL-1ra ratio and decreases after estrogen replacement therapy with a decreased IL-1:IL-1ra ratio. An association was found between an 86 basepair variable number tandem-repeat (VNTR) polymorphism of the IL-1ra gene and an increased production of IL-1ra in a cultured monocyte system. The IL-1ra VNTR polymorphism, therefore, is an attractive candidate gene for osteoporosis susceptibility as well as hormone responsiveness after estrogen replacement. We examined the association of this VNTR polymorphism with bone mass, bone turnover, and the change of bone mineral density (BMD) after 1 year of hormone replacement therapy (HRT). The frequencies of the five alleles were as follows: A1, 90.8% (410 bp, four repeats); A2, 7.2% (240 bp, two repeats); A3, 1.6% (500 bp, five repeats); A4, 0.4% (326 bp, three repeats); and A5, 0% (595 bp, six repeats), in 714 healthy ethnically Korean postmenopausal women, aged 41-74 years (55.2 +/- 6.3 years mean +/- SD). Spine (L2-4) and femoral neck BMD were not significantly different among IL-1ra genotypes, and no significant genotypic differences were found in bone markers. There were no differences in genotypic proportions when we categorized the subjects into a high-loss group and a normal-loss group with regard to levels of bone marker. No significant genotypic differences were found in changes in lumbar and femoral neck BMD and those in bone markers before and after 1 year of HRT in 312 women. Our data suggest that these IL-1ra polymorphisms are not associated with BMD, bone turnover, or the change of BMD after 1 year of HRT in Korean women.

Lack of an intronic Sp1 binding-site polymorphism at the collagen type I alpha1 gene in healthy Korean women.

Osteoporosis is a disease that is strongly genetically influenced. However, the genes responsible for the disease are poorly defined. Recent data show that a G-T transition polymorphism of the Sp1 binding site at the collagen type I alpha1 gene (Sp1 polymorphism) is associated significantly with bone mineral density (BMD) and osteoporotic fracture in British women. To establish the association between the Sp1 genotypes and BMD in Korean women, we examined 200 healthy postmenopausal women of Korean ethnicity, ranging in age from 44 to 66 years (mean+/-SD: 54.7+/-5.3 years). PCR amplification using the same primers as those used previously, with enzyme digestion, revealed no restriction site in our samples. We also performed a single-strand conformational polymorphism (SSCP) analysis in 100 of the 200 samples and could not find any polymorphic sites in the PCR amplification region. Based on our study, the Sp1 polymorphism at the type I collagen alpha1 gene was not found in Korean women. Therefore, we suggest that the Sp1 polymorphism at the type I collagen alpha1 gene is absent or rare in Korean women. Based on the present findings, this polymorphism does not seem to be responsible for the entire genetic contribution to BMD.

[cDNAs encoding the antigenic proteins in pathogenic strain of Entamoeba histolytica].

The differential display reverse transcription polymerase chain reaction (DDRT-PCR) analysis was performed to identify the pathogenic strain specific amplicons. mRNAs were purified from the trophozoites of the pathogenic strain YS-27 and the non-pathogenic strain S 16, respectively. Three kinds of first stranded cDNAs were reverse transcribed from the mRNAs by one base anchored oligo-dT11M (M: A, C, or G) primers. Each cDNA template was used for DDRT-PCR analysis. A total of 144 pathogenic strain specific amplicons was observed in DDRT-PCR analysis using primer combinations of the 11 arbitrary primers and the 3 one base anchored oligo-dT11M primers. Of these, 31 amplicons were verified as the amplicons amplified only from the mRNAs of the pathogenic strain by DNA slot blot hybridization. Further characterization of the 31 pathogenic strain specific amplicons by DNA slot blot hybridization analysis using biotin labeled probes of the PCR amplified DNA of cysteine proteinase genes revealed that 21 of them were amplified from the mRNAs of the cysteine proteinase genes. Four randomly selected amplicons out of the rest 10 amplicons were used for screening of cDNA library followed by immunoscreening and all of them were turned out to be amplified from the mRNA.

Factors affecting in vivo viability of DNA-injected bovine blastocysts produced in vitro.

In vitro matured and fertilized bovine ova were microinjected with pBL1, which consisted of the bovine beta-casein gene promoter, human lactoferrin cDNA and SV40 polyadenylation signal. Of the 2931 zygotes injected, 2505 (85.5%) survived 1 h after DNA injection and were cultured in 50-microl drops of CR1aa medium containing 3 mg/ml BSA under mineral oil at 39 degrees C, 5% CO2 in air. Cleaved (2- to 8-cell) embryos were selected at approximately 48 h after DNA injection and then cultured further in 50-microl drops of CR1aa medium supplemented with 10% (v/v) FBS. Blastocysts were classified into 4 quality grades and 3 developmental stages by morphological criteria. Then all but poor quality blastocysts were nonsurgically transferred to the uterus of heifers 7 to 8 d after natural estrus. Following transfer, the recipients were observed for signs of estrus, and pregnancy was confirmed by palpation per rectum at approximately 60 d of gestation. Although 72.0% (1804/2505 ) of the DNA-injected zygotes reached 2- to 8-cell stages only 5.2% (131/2505) developed to blastocysts. A total of 75 DNA-injected, in vitro cultured blastocysts were transferred to 59 recipients. When 2 blastocysts were transferred to a single recipient, only the better quality embryo was counted. The overall pregnancy rate was 30.5% (18/59 ) and reflected 1) an apparent correlation between the quality of embryos and the pregnancy rate. However, the difference was not statistically significant. 2) expanded blastocysts had a higher pregnancy rate (50.0%, 11/22 ) than early (13.3%, 2 15 ) or mid (22.7%, 5/22 ) blastocysts with a significant difference between expanded and early blastocysts (P < 0.05). 3) the pregnancy rate of DNA-injected blastocysts was higher when they were transferred at Day 7 (34.5%, 10/29 ) or 8 (36.8%, 7/19 ) than at Day 6 (9.0%, 1/11 ). The results indicate that the developmental stage of DNA-injected bovine embryos may be one of contributing factors in improving the pregnancy rate after transfer, although the effects of the quality and culture period of the embryos may not be inconsequential.