K. R. Bondioli

Effect of Epigenetic Modifications of Donor Somatic Cells on the Subsequent Chromatin Remodeling of Cloned Bovine Embryos

Evidence indicates that failure of nuclear transfer (NT) embryos to develop normally can be attributed, at least partially, to the use of differentiated cells as the donor karyoplast. Blastocyst production and development to term of cloned embryos has been hypothesized to differ between population doublings of the same cell line as a consequence of changes in the levels of DNA methyltransferase 1 (DNMT1) and methylated DNA during in vitro culture. The objective of this study was to determine embryo production, developmental potential, and gene expression patterns of prehatched and posthatched embryos generated using donor cells with different levels of DNMT1 transcript. Day 7 embryos generated using donor cells with high and low levels of DNMT1 mRNA were transferred to recipient cows. Embryos recovered on Day 13 were morphologically characterized or used for gene expression analysis of DNMT, INFT, and MHC1. A higher proportion of 8- to 16-cell embryos developed to the blastocyst stage when cells with low levels of DNMT1 mRNA were used as donor nuclei. Day 13 NT embryos generated using donor cells with decreased levels of DNMT1 mRNA and capable of developing beyond the 8- to 16-cell stage produced a larger number of apparently developing embryos, larger conceptuses, and a higher expression of DNMT3A transcript than NT embryos reconstructed using cells with high levels of DNMT1 mRNA. However, abnormal gene expression of DNMT, INFT, and MHC1 was noted in the majority of cloned embryos, indicating inefficient nuclear reprogramming and retarded embryo development. Furthermore, aberrant DNMT1 expression may partially contribute to the inefficient nuclear reprogramming observed in cloned embryos.

Isolation and Characterization of Porcine Adipose Tissue-Derived Adult Stem Cells

Background: Stem cell characteristics such as self-renewal, differentiation and expression of CD34 and CD44 stem cell markers have not been identified in porcine adipose tissue-derived adult stem (ADAS) cells. The objective of this study was to develop a protocol for the isolation and culture of porcine adipose tissue-derived cells and to determine stem cell-like characteristics. Methods: Primary cultures were established and cell cultures were maintained. Cloning capacity was determined using a ring cloning procedure. Primary cultures and clones were differentiated and stained for multiple differentiated phenotypes. CD34 and CD44 messenger ribonucleic acid (mRNA) was isolated and reverse transcriptase polymerase chain reaction was used to compare expression profiles. Results: An average of 2,700,000 nucleated cells/ml was isolated; 26% were adherent, and cells completed a cell cycle approximately every 3.3 days. Ring cloning identified 19 colonies. Primary cultures and clones were determined to differentiate along osteogenic, adipogenic and chondrogenic tissue lineages. The mRNA expression profiles showed CD34 expression was higher for undifferentiated ADAS cells versus differentiated cell types and the CD34 expression level was lower than that of CD44 among differentiated cells. Conclusion: Improved culture conditions and defined cellular characteristics of these porcine ADAS cells have been identified. Porcine ADAS can self-renew, can differentiate into multiple tissue lineages and they express CD34.

Ooplasm transfer and interspecies somatic cell nuclear transfer: heteroplasmy, pattern of mitochondrial migration and effect on embryo development.

Although interspecies somatic cell nuclear transfer (iSCNT) has potential applications in the conservation of exotic species, an in vitro developmental block has been observed in embryos produced by this approach. It has been suggested that mitochondrial mismatch between donor cell and recipient oocyte could cause embryonic developmental arrest. A series of experiments was conducted to investigate the effect of mixed mitochondrial populations (heteroplasmy) on early development of iSCNT-derived cloned embryos. The effect of combining the techniques of ooplasm transfer (OT) and somatic cell nuclear transfer (SCNT) was examined by monitoring in vitro embryonic development; the presence and pattern of migration of foreign mitochondria after OT was analysed by MitoTracker staining. In addition, the effect of transferring caprine ooplasm (iOT) into the bovine enucleated oocytes used in iSCNT was analysed. There was no significant effect of the sequence of events (OT-SCNT or SCNT-OT) on the number of fused, cleaved, blastocyst or hatched blastocyst stage embryos. MitoTracker Green staining of donor oocytes used for OT confirmed the introduction of foreign mitochondria. The distribution pattern of transferred mitochondria most commonly remained in a distinct cluster after 12, 74 and 144 h of in vitro culture. When goat ooplasm was injected into bovine enucleated oocytes (iSCNT), there was a reduction (p < 0.05) in fusion (52 vs. 82%) and subsequent cleavage rates (55 vs. 78%). The procedure of iOT prior to iSCNT had no effect in overcoming the 8- to 16-cell in vitro developmental block, and only parthenogenetic cow and goat controls reached the blastocyst (36 and 32%) and hatched blastocyst (25 and 12%) stages, respectively. This study indicates that when foreign mitochondria are introduced at the time of OT, these organelles tend to remain as distinct clusters without relocation after a few mitotic divisions. Although the bovine cytoplast appears capable of supporting mitotic divisions after iOT-iSCNT, heteroplasmy or mitochondrial incompatibilities may affect nuclear-ooplasmic events occurring at the time of genomic activation.

Sex Ratio of Bovine Embryos and Calves Originating from the Left and Right Ovaries

An asymmetric distribution of the sexes within the left and right uterine horns has been described in multiple species. A series of experiments were conducted to evaluate the sex ratio (% male) of calves gestated in the left and right uterine horns, as well as the sex ratio of embryos originating from the left and right ovaries of cattle. The sex ratio of calves gestated in the right uterine horn of naturally mated cows was significantly higher compared with the sex ratio of calves gestated in the left uterine horn. In addition, the sex ratio of the left and right uterine horns differed significantly from parity. The sex ratio of embryo transfer calves born following transfer to the left and right uterine horns was not significantly different. Additionally, the proportion of male embryos collected from the right uterine horns was significantly greater than from the left uterine horns of superovulated cows. The sex ratio of embryos collected from the left and right uterine horns of unilaterally ovariectomized cows was not significantly different. However, more female than male embryos were produced when left ovary oocytes fertilized in vitro. In conclusion, the results of these experiments demonstrate that a significantly greater proportion of males are gestated in the right uterine horn of cattle and a greater proportion of females in the left. Additionally, the data indicate that sex-specific selection pressure may be applied to embryos by ovarian factors rather than by the uterine environment.

Isolation, culture and characterisation of somatic cells derived from semen and milk of endangered sheep and eland antelope

Semen and milk are potential sources of somatic cells for genome banks. In the present study, we cultured and characterised cells from: (1) cooled sheep milk; (2) fresh, cooled and frozen-thawed semen from Gulf Coast native (GCN) sheep (Ovis aries); and (3) fresh eland (Taurotragus oryx) semen. Cells attached to the culture surface from fresh (29%), cooled (43%) and slow-frozen (1 degrees C/min; 14%) ram semen, whereas no attachment occurred in the fast-frozen (10 degrees C/min) group. Proliferation occurred in fresh (50%) and cooled (100%) groups, but no cells proliferated after passage 1 (P1). Eland semen yielded cell lines (100%) that were cryopreserved at P1. In samples from GCN and cross-bred milk, cell attachment (83% and 95%, respectively) and proliferation (60% and 37%, respectively) were observed. Immunocytochemical detection of cytokeratin indicated an epithelial origin of semen-derived cells, whereas milk yielded either fibroblasts, epithelial or a mixture of cell types. Deoxyribonucleic acid microsatellite analysis using cattle-derived markers confirmed that eland cells were from the semen donor. Eland epithelial cells were transferred into eland oocytes and 12 (71%), six (35%) and two (12%) embryos cleaved and developed to morulae or blastocyst stages, respectively. In conclusion, we have developed a technique for obtaining somatic cells from semen. We have also demonstrated that semen-derived cells can serve as karyoplast donors for nuclear transfer.

Inhibition of DNA methyltransferase 1 expression in bovine fibroblast cells used for nuclear transfer

The aberrant expression of DNA methyltransferase 1 (DNMT1) in cloned embryos has been implicated as a possible factor in the improper donor genome reprogramming during nuclear transfer. DNMT1 is responsible for maintaining DNA methylation and the subsequent differentiation status of somatic cells. The presence of DNMT1 transcript in the donor cell may contribute to perpetuation of the highly methylated status of the somatic nuclei in cloned embryos. The objective of the present study was to determine the methylation pattern of cloned embryos reconstructed with cells treated with DNMT1-specific small interfering RNA (siRNA). Bovine fibroblasts were transfected with a DNMT1-specific siRNA under optimised conditions. The expression patterns of DNMT1 were characterised by Q-PCR using the DeltaDeltaC(T) method. The level of DNMT1 was successfully decreased in bovine fibroblast cells using a DNMT1-specific siRNA. Additionally, reduction in the expression of DNMT1 mRNA and DNMT1 protein led to a moderate hypomethylation pattern in the siRNA-treated cells. The use of siRNA-treated cells as donor nuclei during nuclear transplantation induced a reduction in methylation levels compared with controls but did not reduce methylation levels to that of IVF embryos. Further studies are required to determine if this level of reduced methylation is sufficient to improve subsequent development.

Distribution of sexes within the left and right uterine horns of cattle.

In cattle, limited data are available regarding the sex ratio of the offspring in relation to the horn of gestation. Therefore, the objective of this study was to evaluate the sex ratio of fetuses gestated in the left and right uterine horns of cattle (Bos taurus, Bos indicus and crosses). The distribution of male and female fetuses in the left and right uterine horn was analyzed on gravid, abattoir-derived reproductive tracts and artificially inseminated crossbred cows. The total number of fetuses/calves and the sex of the fetuses/calves gestated in each uterine horn were used as the end point for side comparisons using the Glimmix Procedure. Of 64 gravid reproductive tracts evaluated, 29 (45.3%) pregnancies occurred in the left uterine horn, whereas 35 (54.7%) occurred in the right. The sex ratio (% males) of fetuses in the left uterine horn (37.9%) was significantly lower than the sex ratio detected in the right uterine horn (65.7%). Of 113 pregnancies evaluated in artificially inseminated heifers, 53 (46.9%) occurred in the left uterine horn, whereas 60 (53.1%) occurred in the right uterine horn. The sex ratio of calves gestated in the left uterine horn (35.8%) was significantly lower than the sex ratio of calves gestated in the right uterine horn (63.3%). In conclusion, in these experiments, a significantly greater proportion of males were gestated in the right uterine horn of cattle and a greater proportion of females in the left uterine horn. Further investigation is needed to determine the mechanisms underlying the observed disparity of the expected sex ratio within the uterine horns of cattle.

Production of Transgenic and Knockout Pigs by Somatic Cell Nuclear Transfer

Xenotransplantation is one alternative to transplantation of human organs which has been investigated. It is generally accepted that the pig represents the most logical choice of animals to serve as organ donors for xenotransplantation. Moreover, the implementation of cloning by somatic cell nuclear transfer (SCNT) and transgenic techniques have resulted in the production of numerous transgenic pigs than can be used for xenotransplantation purposes as well as models for human diseases.

The effect of equine chorionic gonadotropin (eCG) on pregnancy rates of white-tailed deer following fixed-timed artificial insemination.

Control of the white-tailed does reproductive cycle is not well documented. The objective was to determine the effects of giving equine chorionic gonadotropin (eCG) at progesterone device removal on fixed time artificial insemination (FTAI) pregnancy rates in white-tailed does. All does (n = 74) were synchronized with a vaginal progesterone implant (CIDR; 0.3 g progesterone), inserted on Day 0 (without regard to stage of estrous cycle), removed 14 days later, and subjected to FTAI, on average, 60 h post-CIDR removal. Of these, 34 were given 200 IU (im) of eCG at CIDR removal. Overall, FTAI pregnancy rate was 50% across 2 yrs (effect of year, P = 0.35). Administration of eCG at CIDR removal did not affect (P = 0.16) pregnancy rate (eCG = 59%; no eCG = 43%). Pregnancy rates were not affected by vulva score or doe disposition. Does that were ≤ 4 yrs old were more likely (P = 0.01) to become pregnant than does > 4 yrs of age. Does inseminated ≥ 60.5 h after CIDR removal were 22 times more likely (P = 0.002) to become pregnant to FTAI than does inseminated < 60.5 h. When frozen-thawed semen was deposited in the cervix or uterus, does were 17 times more likely (P = 0.005) to become pregnant compared with those receiving intravaginal insemination. Fecundity was not different (P = 0.73) across treatment groups (1.6 ± 0.11; no eCG vs. 1.7 ± 0.10; eCG). Furthermore, fecundity of does pregnant to FTAI was not different (P = 0.72) compared with does pregnant to clean-up bucks (1.7 ± 0.08; AI does vs. 1.7 ± 0.09; clean-up bucks). In summary, white-tailed does were successfully inseminated using a 14 days FTAI protocol, eCG may not be essential for acceptable pregnancy rates, and increased pregnancy rates may result when FTAI is done ≥ 60.5 h after progesterone device removal.

Inhibition of DNA Methylation in Somatic Cells

DNA methylation plays a significant role in the expression of the genetic code and affects early growth and development through its influence on gene expression. DNA methyltransferase 1 (Dnmt1) is the enzyme responsible for maintaining the methylation marks through cell division. However, the de novo methyltransferases, Dnmt3a and Dnmt3b, can also contribute to the maintenance of the methylation pattern. Manipulation of these enzymes, especially Dnmt1, provides a means to alter DNA methylation levels. Manipulation of the DNA methylation pattern of somatic cells will allow a better understanding of the different molecular process associated with chromatin structure and gene expression. Different approaches to artificially manipulate the expression of Dnmt1 in somatic cells include the addition of 5-azacytidine, culture of cells for an extended period of time, and the use of small interfering RNA technologies.