Donor-host mitochondrial compatibility improves efficiency of bovine somatic cell nuclear transfer

Background  

The interaction between the karyoplast and cytoplast plays an important role in the efficiency of somatic cell nuclear transfer (SCNT), but the underlying mechanism remains unclear. It is generally accepted that in nuclear transfer embryos, the reprogramming of gene expression is induced by epigenetic mechanisms and does not involve modifications of DNA sequences. In cattle, oocytes with various mitochondrial DNA (mtDNA) haplotypes usually have different ATP content and can further affect the efficiency of in vitro production of embryos. As mtDNA comes from the recipient oocyte during SCNT and is regulated by genes in the donor nucleus, it is a perfect model to investigate the interaction between donor nuclei and host oocytes in SCNT.     

Coexistence of Bos taurus and B. indicus mitochondrial DNAs in nuclear transfer-derived somatic cattle clones

We investigated the mitochondrial DNA (mtDNA) composition in one of the largest adult somatic mammalian clones (n = 20) reported so far. The healthy cloned cattle were derived from nuclear transfer of an identical nuclear genetic background (mural granulosa donor cells including surrounding cytoplasm) into enucleated oocytes with either Bos indicus or B. taurus mtDNA. Here we report the first cases of coexisting mtDNAs of two closely related subspecies following nuclear transfer. Heteroplasmy (0.6-2.8%) was found in 4 out of 11 cross-subspecies cloned cattle. Quantitation was performed using "amplification refractory mutation system (ARMS) allele-specific real-time PCR." We determined that the ratio of donor cell to recipient cytoplast mtDNA copy number was 0.9% before nuclear transfer. Therefore, we concluded that the percentage of donor cell mtDNA in the heteroplasmic intersubspecific cloned animals is in accordance with neutral transmission of donor mtDNA. We determined an amino acid sequence divergence of up to 1.3% for the two subspecies-specific mtDNA haplotypes. In addition, intrasubspecific B. indicus heteroplasmy of approximately 1% (but up to 7.3 and 12.7% in muscle and follicular cells of one animal) was detected in 7 out of the 9 B. indicus intrasubspecific cloned cattle.     

Coordination between donor cell type and cell cycle stage improves nuclear cloning efficiency in cattle

Several studies have shown that both quiescent and proliferating somatic donor cells can be fully reprogrammed after nuclear transfer (NT) and result in viable offspring. So far, however, no comparative study has conclusively demonstrated the relative importance of donor cell cycle stage on nuclear cloning efficiency. Here, we compare two different types of bovine fetal fibroblasts (BFFs) that were synchronized in G(0), G(1), and different phases within G(1). We show that for non-transgenic (non-TG) fibroblasts, serum starvation into G(0) results in a significantly higher percentage of viable calves at term than synchronization in early G(1) or late G(1). For transgenic fibroblasts, however, cells selected in G(1) show significantly higher development to calves at term and higher post-natal survival to weaning than cells in G(0). This suggests that it may be necessary to coordinate donor cell type and cell cycle stage to maximize overall cloning efficiency.     

Serial bull cloning by somatic cell nuclear transfer

Although the list of species successfully cloned continues to grow, serial cloning has not been reported in species other than the mouse. Here we describe two live births of second-generation clones of a bull. Clones of the first and second generations appear healthy and have normal telomere lengths. Our attempts to produce the third generation of clones were unsuccessful.     

Recent advancements in cloning by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.     

Identical marker alleles in Podolic cattle (Bos taurus) and Indian zebu (Bos indicus)

In the context of biochemical marker research and in order to add new information on native breeds, the present work focuses on a local Southern Italy cattle, namely Italian Podolic. We provide the complete structural characterisation of alpha-lactalbumins and beta-globin chains isolated from Podolic cattle (Bos taurus). Given the unavailability of the complete sequence for alpha-lactalbumin A of taurine cattle in the literature, we intended to check its structure in order to ascertain the absence of any possible silent mutation. Screening the Podolic cattle, we found a new beta-globin variant not detectable by conventional methods. The presence of such a new variant might be helpful in the study of the Podolic population genetic structure and for a better knowledge of the gene pool per se, and in comparison with the other breeds. Structural analyses showed that the new beta-globin Podolic variant exhibited the same sequence as beta-globin Azebu. The alpha-lactalbumin A was the same as that isolated from zebu cattle (Bos indicus). The results are discussed in relation to the possible involvement of the two markers in the debate on the origin of the Podolic breed.     

Inbreeding and population dynamics of the Chillingham cattle (Bos taurus)

The Chillingham herd of white cattle has been isolated and confined in a park in northern England for several centuries. Blood grouping confirmed the cattle to be remarkably homozygous. Random fixation of harmful alleles and consequent extinction have presumably been prevented by selection, and this paper discusses possible selective processes and the ways in which these have changed over the last hundred years. Herd records from 1862 to 1899 and 1953 to 1985 show that, in the former period, but not the latter, culling and castration took place. In both periods, breeding was not seasonal. Herd fertility (calves born per female) was higher in the latter period. Between 1953 and 1985, calves which survived for at least 12 months had a median date of birth (25 June) a month later than that of calves which did not survive. Conception intervals were rather longer and fecundity lower than those observed in commercial cattle. K-factor analysis showed mortality to differ in its causes between the sexes. A multiple regression model showed January-May rainfall, and population size on 1 January to influence mortality rate of the January-May period. The Chillingham cattle have evidently been, and continue to be, subjected to rigorous selection and this presumably underlies the survival of this herd.     

Estimate of the population of preantral follicles in the ovaries of Bos taurus indicus and Bos taurus taurus cattle

The number of oocytes recovered from Bos taurus indicus females subjected to ovum pick-up averaged two to four times greater compared to Bos taurus taurus females. The objective of the present study was to test the hypothesis that this difference in oocyte yield was due to more preantral follicles in the ovaries of Bos indicus females. Ovaries (n = 64) from Nelore (Bos indicus) fetuses (n = 10), heifers (n = 12), and cows (n = 10), and Aberdeen Angus (Bos taurus) fetuses (n = 10), heifers (n = 12), and cows (n = 10) were cut longitudinally into halves, fixed, and processed for histological evaluation. The number of preantral follicles was estimated by counting them in each histological section, using the oocyte nucleus as a marker and employing a correction factor. The average number of preantral follicles in the ovaries of Bos indicus vs Bos taurus was (mean ± SD) 143,929 ± 64,028 vs 285,155 ± 325,195 for fetuses, 76,851 ± 78,605 vs 109,673 ± 86,078 for heifers, and 39,438 ± 31,017 vs 89,577 ± 86,315 for cows (P > 0.05). The number of preantral follicles varied greatly among individual animals within the same category, as well as between breeds. In conclusion, we inferred that the higher oocyte yield from Bos indicus females was not due to a greater ovarian reserve of preantral follicles. Therefore, mechanisms controlling follicle development after the preantral stage likely accounted for differences between Bos indicus and Bos taurus females in number of oocytes retrieved at ovum pick-up.     

Isozyme characterization of cattle (Bos taurus) and American buffalo (Bison bison) cell cultures

Four Bovidae cell lines (BEK-1, MDBK, Bu and EBTr) were characterized by means of enzymatic biochemical markers. Out of 15 enzymatic systems, 3 — adenosine dea-minase [Ada), phosphoglucomutase (Pgm) and nucleoside phosphorylase (Np) —were found to be polymorphic and quite suitable for biochemical identification of each cell line. The Bu cell line has shown a Np phenotypic pattern which could be distinctive of the Bison bison species.     

Influence of the breed of bull (Bos taurus indicus vs. Bos taurus taurus) and the breed of cow (Bos taurus indicus, Bos taurus taurus and crossbred) on the resistance of bovine embryos to heat

In vitro studies have shown that Bos taurus indicus (B. t. indicus) embryos submitted to heat shock at early stages of development are better able to survive as compared to Bos taurus taurus embryos. Embryo genotype influences resistance to heat shock thus leading to the question as to whether embryos sired by thermo-tolerant breeds exhibit the same resistance to heat shock. In the present study the influence of both oocyte and semen, on the resistance to heat shock (HS) at early stages of in vitro development, was assessed in B. t. indicus [Nelore (N) breed], B. t. taurus [Holstein (H) and Angus (A) breeds] and crossbreds. In Experiment 1, Nelore and crossbred oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from Nelore and Angus bulls. Presumptive embryos were collected and randomly assigned to control (39 degrees C) or HS at 12, 48 or 96 h post insemination (hpi; 41 degrees C for 12h) treatments. The cleavage rates and proportion of embryos developing to the blastocyst and hatched blastocyst stages were recorded on Days 2, 8 and 10, respectively. Heat shock treatment decreased development of both Nelore and crossbred embryos. There was a significant interaction between time (12, 48 or 96 hpi) and temperature for blastocyst rates, i.e., the embryos became more thermotolerant as development proceeded. In Experiment 2, oocytes from Nelore and Holstein cows were fertilized with semen from bulls of either Nelore or Angus breeds, and subjected to 12 h HS at 96 hpi. Heat shock at 96 hpi, decreased embryo development. Additionally, cowxtreatment and bullxtreatment interactions were significant for blastocyst rates, i.e., both breed of cow and breed of bull affected the decline in blastocyst rate caused by heat shock treatment. In conclusion, the present results indicate that Nelore embryos (indicus) are more resistant to heat shock than Holstein (taurus) at early stages of in vitro development, and that embryos become more thermo-tolerant as development proceeds. Additionally, the resistance to heat shock was a result of the genetic contribution from both oocyte and spermatozoa.     

Proliferation of donor mitochondrial DNA in nuclear transfer calves (Bos taurus) derived from cumulus cells

In embryos derived by nuclear-transfer (NT), fusion of donor cell and recipient oocyte caused mitochondrial heteroplasmy. Previous studies from other laboratories have reported either elimination or maintenance of donor-derived mitochondrial DNA (mtDNA) from somatic cells in cloned animals. Here we examined the distribution of donor mtDNA in NT embryos and calves derived from somatic cells. Donor mitochondria were clearly observed by fluorescence labeling in the cytoplasm of NT embryos immediately after fusion; however, fluorescence diminished to undetectable levels at 24 hr after nuclear transfer. By PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, donor mtDNAs were not detected in the NT embryos immediately after fusion (less than 3-4%). In contrast, three of nine NT calves exhibited heteroplasmy with donor cell mtDNA populations ranging from 6 to 40%. These results provide the first evidence of a significant replicative advantage of donor mtDNAs to recipient mtDNAs during the course of embryogenesis in NT calves from somatic cells.     

Isozyme characterization of cattle (Bos taurus) and American buffalo (Bison bison) cell cultures.

Four Bovidae cell lines (BEK-1, MDBK, Bu and EBTr) were characterized by means of enzymatic biochemical markers. Out of 15 enzymatic systems, 3--adenosine deaminase (Ada), phosphoglucomutase (Pgm) and nucleoside phosphorylase (Np)--were found to be polymorphic and quite suitable for biochemical identification of each cell line. The Bu cell line has shown a Np phenotypic pattern which could be distinctive of the Bison bison species.     

Superovulatory response of dairy cattle (Bos taurus ) in a tropical environment

Dairy (Bos taurus) heifers and cows (n = 40) in a tropical environment were treated during mid-luteal phase using either SUPER-OV(R) or OVAGEN to induce superovulatory response after synchronization of the superovulatory estrus with a synthetic progestagen and cloprostenol (PG). Estrous cattle were inseminated twice using frozen-thawed semen, and embryos were recovered nonsurgically, on-farm, 7 d later. Between initiation of gonadotrophin treatment and recovery of embryos, 4 blood samples per animal were collected from 26 animals for determination of plasma progesterone (P4) concentration. Two (5%), 28 (70%) and 10 (22%) of the animals were observed in estrus 1.5, 2 and 2.5 to 3 d after PG, respectively. There was no difference (P = 0.7) in the number of palpable CL between animals treated with SUPER-OV (7.6 +/- 1.0; n = 18) and those treated with OVAGEN (7.9 +/- 1.1; n = 22). There was also no significant difference (P > 0.05) between Jersey vs Ayrshire breeds or heifers vs cows in the ovarian response as estimated by the number of palpable CL. However, a higher proportion of Ayrshire cattle and donors treated with OVAGEN yielded a higher total number and viable/transferable embryos than Jersey and SUPER-OV-treated cattle. There was a significant (P < 0.05) correlation between the number of CL and total number of embryos (r = 0.65); the number of transferable embryos was also significantly related to the total number of embryos per recovery (r = 0.85; P < 0.05). For 15 animals with normal P4 profiles, the mean (+/-SEM) plasma P4 concentration was 14.4 +/- 0.8, 0.5 +/- 0.2, 5.4 +/- 1.1 and 39.4 +/- 3.0 nmol L at initiation of gonadotrophin treatment, superovulatory estrus and Days 3 and 7, respectively. The mean (+/-SEM) interval between a PG injection given after embryo recovery and the induced estrus was 7.1 +/- 0.7 d (range 3 to 14 d) and the length of the superovulatory cycle was 24.1 +/- 3.2 d (range 12 to 35 d).     

Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality.