W. Allan King

Telomerase activity and telomere detection during early bovine development.

The ends of mammalian chromosomes are composed of repeated DNA sequences of (TTAGGG)(n) known as telomeres. Telomerase is a ribonucleoprotein that synthesizes telomeric DNA to replenish the 50-200 bp lost during cell replication. Cellular aging and senescence are associated with a lack of telomerase activity and a critical shortening of the telomere. The objectives of this study were to confirm the presence of TTAGGG repeats on the chromosomes of bovine embryos using in situ hybridization and assess the relative amounts of telomerase activity using a telomeric repeat amplification protocol (TRAP) during oocyte maturation and early embryo development. Applying a telomere DNA probe to the chromosomes of blastocysts and adult fibroblasts, telomeres were identified on the terminal ends of the p and q arms of chromosomes in all cells examined. Immature oocytes, matured oocytes, zygotes, 2- to 5-cell embryos, 6- to 8-cell embryos, morulae, and blastocysts were lysed in NP-40 lysis buffer and telomerase activity was assayed using the TRAP assay. Telomerase activity was detected in all developmental stages examined. Relative telomerase activity (based on telomerase internal standards and positive controls) appeared to decrease during oocyte maturation and subsequent development to the 8-cell stage but significantly increased (P < 0.05) by approximately 40-fold at the morula and blastocyst stages. It was concluded that the telomeres of bovine chromosomes contain TTAGGG repeats and that telomerase activity is up-regulated in morulae and blastocysts.

The Influence of Nuclear Content on Developmental Competence of Gaur × Cattle Hybrid In Vitro Fertilized and Somatic Cell Nuclear Transfer Embryos

Abstract In nondomestic and endangered species, the use of domestic animal oocytes as recipients for exotic donor nuclei causes the normal pattern of cytoplasmic inheritance to be disrupted, resulting in the production of nuclear-cytoplasmic hybrids. Evidence suggests that conflict between nuclear and cytoplasmic control elements leads to a disruption of normal cellular processes, including metabolic function and cell division. This study investigated the effects of nuclear-cytoplasmic interactions on the developmental potential of interspecies embryos produced by in vitro fertilization and somatic cell nuclear transfer: cattle × cattle, gaur × cattle, hybrid × cattle. Cattle control and hybrid embryos were examined for development to the blastocyst stage and blastocyst quality, as determined by cell number and allocation, apoptosis incidence, and expression patterns of mitochondria-related genes. These analyses demonstrated that a 100% gaur nucleus within a domestic cattle cytoplasmic environment was not properly capable of directing embryo development in the later preimplantation stages. Poor blastocyst development accompanied by developmental delay, decreased cell numbers, and aberrant apoptotic and related gene expression profiles, all signs of disrupted cellular processes associated with mitochondrial function, were observed. Developmental potential was improved when at least a portion of the nuclear genome corresponded to the inherited cytoplasm, indicating that recognition of cytoplasmic components by the nucleus is crucial for proper cellular function and embryo development. A better understanding of the influence of the cytoplasmic environment on embryonic processes is necessary before interspecies somatic cell nuclear transfer can be considered a viable alternative for endangered species conservation.

The oxidative stress adaptor p66Shc is required for permanent embryo arrest in vitro

BackgroundExcessive developmental failure occurs during the first week of in vitro embryo development due to elevated levels of cell death and arrest. We hypothesize that permanently arrested embryos enter a stress-induced senescence-like state that is dependent on the oxidative stress-adaptor and lifespan determinant protein p66Shc. The aim of this study was to selectively diminish p66Shc gene expression in bovine oocytes and embryos using post-transcriptional gene silencing by RNA-mediated interference to study the effects of p66Shc knockdown on in vitro fertilized bovine embryos.ResultsApproximately 12,000–24,000 short hairpin (sh)RNAi molecules specific for p66Shc were microinjected into bovine germinal vesicle stage oocytes or zygotes. Experiments were comprised of a control group undergoing IVF alone and two groups microinjected with and without p66Shc shRNAi molecules prior to IVF. The amount of p66Shc mRNA quantified by Real Time PCR was significantly (P < 0.001) lowered upon p66Shc shRNAi microinjection. This reduction was selective for p66Shc mRNA, as both histone H2a and p53 mRNA levels were not altered. The relative signal strength of p66Shc immuno-fluorescence revealed a significant reduction in the number of pixels for p66Shc shRNAi microinjected groups compared to controls (P < 0.05). A significant decrease (P < 0.001) in the incidence of arrested embryos upon p66Shc shRNAi microinjection was detected compared to IVF and microinjected controls along with significant reductions (P < 0.001) in both cleavage divisions and blastocyst development. No significant differences in p66Shc mRNA levels (P = 0.314) were observed among the three groups at the blastocyst stage.ConclusionThese results show that p66Shc is involved in the regulation of embryo development specifically in mediating early cleavage arrest and facilitating development to the blastocyst stage for in vitro produced bovine embryos.

Role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer

BackgroundSomatic cell nuclear transfer (SCNT) provides an appealing alternative for the preservation of genetic material in non-domestic and endangered species. An important prerequisite for successful SCNT is the availability of good quality donor cells, as normal embryo development is dependent upon proper reprogramming of the donor genome so that embryonic genes can be appropriately expressed. The characteristics of donor cell lines and their ability to produce embryos by SCNT were evaluated by testing the effects of tissue sample collection (DART biopsy, PUNCH biopsy, post-mortem EAR sample) and culture initiation (explant, collagenase digestion) techniques.ResultsDifferences in initial sample size based on sample collection technique had an effect on the amount of time necessary for achieving primary confluence and the number of population doublings (PDL) produced. Thus, DART and PUNCH biopsies resulted in cultures with decreased lifespans (<30 PDL) accompanied by senescence-like morphology and decreased normal chromosome content (<40% normal cells at 20 PDL) compared to the long-lived (>50 PDL) and chromosomally stable (>70% normal cells at 20 PDL) cultures produced by post-mortem EAR samples. Chromosome stability was influenced by sample collection technique and was dependent upon the cultures initial telomere length and its rate of shortening over cell passages. Following SCNT, short-lived cultures resulted in significantly lower blastocyst development (≤ 0.9%) compared to highly proliferative cultures (11.8%). Chromosome stability and sample collection technique were significant factors in determining blastocyst development outcome.ConclusionThese data demonstrate the influence of culture establishment techniques on cell culture characteristics, including the viability, longevity and normality of cells. The identification of a quantifiable marker associated with SCNT embryo developmental potential, chromosome stability, provides a means by which cell culture conditions can be monitored and improved.

Cloning in companion animal, non-domestic and endangered species: can the technology become a practical reality?

Somatic cell nuclear transfer (SCNT) can provide a unique alternative for the preservation of valuable individuals, breeds and species. However, with the exception of a handful of domestic animal species, successful production of healthy cloned offspring has been challenging. Progress in species that have little commercial or research interest, including many companion animal, non-domestic and endangered species (CANDES), has lagged behind. In this review, we discuss the current and future status of SCNT in CANDES and the problems that must be overcome to improve pre- and post-implantation embryo survival in order for this technology to be considered a viable tool for assisted reproduction in these species.

Identification of the homologue of the bovine Rob(1;29) in a captive gaur (Bos gaurus).

Robertsonian translocations have been well documented in domestic cattle, with the most commonly occurring fusion involving chromosomes 1 and 29. The widespread nature of this translocation is indicative of its ancient origin. The gaur (Bos gaurus) is one of many wild cattle species currently listed as vulnerable or endangered. Due to the small founder stock and 50 years of restricted breeding, the captive herd is showing signs of inbreeding and reduced fertility. Recent cytogenetic analysis of a female gaur at Toronto Zoo found that the individual contained 2n=57 chromosomes instead of the normal 2n=58, with an extra submetacentric and the loss of two acrocentric chromosomes being observed. This study was undertaken to identify the translocation in this individual and to examine the karyotype of immediate family members. Chromosome analysis of fibroblast cell cultures was carried out using GTG-banding, C-banding and FISH (bovine 1 and 29 paints) techniques to characterize the translocation. Results from the GTG-banding and FISH analyses confirm that the two autosomes involved in the translocation are the bovine homologues 1 and 29. A monocentric centromere was observed by C-banding. Chromosome abnormalities have not been detected in other gaur tested to date. This study demonstrates the importance of cytogenetic analysis for the establishment of screening protocols for the assessment of reproductive potential in this and other exotic bovinae.

Systemic and local anti-Mullerian hormone reflects differences in the reproduction potential of Zebu and European type cattle

This study was conducted to evaluate plasma anti-Mullerian hormone (Pl AMH), follicular fluid AMH (FF AMH) and granulosa cell AMH transcript (GC AMH) levels and their relationships with reproductive parameters in two cattle subspecies, Bos taurus indicus (Zebu), and Bos taurus taurus (European type cattle). Two-dimensional ultrasound examination and serum collection were performed on Zebu, European type and crossbreed cows to determine antral follicle count (AFC), ovary diameter (OD) and Pl AMH concentration. Slaughterhouse ovaries for Zebu and European type cattle were collected to determine FF AMH concentrations, GC AMH RNA levels, AFC, oocyte number, cleavage and blastocyst rate. Additionally GC AMH receptor 2 (AMHR2) RNA level was measured for European type cattle. Relationship between AMH and reproductive parameters was found to be significantly greater in Zebu compared to European cattle. Average Pl AMH mean ± SE for Zebu and European cattle was 0.77 ± 0.09 and 0.33 ± 0.24 ng/ml respectively (p = 0.01), whereas average antral FF AMH mean ± SE for Zebu and European cattle was 4934.3 ± 568.5 and 2977.9 ± 214.1 ng/ml respectively (p < 0.05). This is the first published report of FF and GC AMH in Zebu cattle. Levels of GC AMHR2 RNA in European cattle were correlated to oocyte number (p = 0.01). Crossbred animals were found more similar to their maternal Zebu counterparts with respect to their Pl AMH to AFC and OD relationships. These results demonstrate that AMH reflects differences between reproduction potential of the two cattle subspecies therefore can potentially be used as a reproductive marker. Furthermore these results reinforce the importance of separately considering the genetic backgrounds of animals when collecting or interpreting bovine AMH data for reproductive performance.

Fetal sex alters maternal anti-Mullerian hormone during pregnancy in cattle

Anti-Mullerian hormone (AMH) is expressed by both male and female fetuses during mammalian development, with males expressing AMH earlier and at significantly higher concentration. The aim of the current study was to explore the potential impact of pregnancy and fetal sex on maternal AMH and to determine if plasma (Pl) AMH or placenta intercotyledonary membrane and cotyledonary AMH receptor 2 (AMHR2) mRNA expression differ in pregnant cows carrying male vs. female fetuses. AMH levels in blood were measured using a bovine optimized ELISA kit. Cows pregnant with a male fetus were observed to have a significantly greater difference in Pl AMH between day 35 and 135 of gestation. Average fetal AMH level between 54 and 220days of gestation was also observed to be significantly higher in male vs. female fetuses. Intercotyledonary membranes and cotyledons were found to express AMHR2 between days 38 and 80 of gestation at similar levels in both fetal sexes. These findings support the hypothesis that fetal sex alters maternal Pl AMH during pregnancy in cattle.