The relationship among psychological factors, neglect-like symptoms and postoperative pain after total knee arthroplasty.

BACKGROUND: Persistent postoperative pain has a significant relationship with patient health and satisfaction. OBJECTIVES: To investigate the prevalence and association of neglect-like symptoms (NLS) and other psychological factors on postoperative pain in patients following total knee arthroplasty (TKA). NLS are defined as the loss of perception of the limb with pain and excessive effort required to move the limb. The authors hypothesized that NLS were an important contributor to postoperative pain. METHODS: The factors influencing pain were investigated using a longitudinal study with assessments at three and six weeks postsurgery. The relationships among demographic factors (age, body weight, body mass index), psychological factors (State-Trait Anxiety Inventory and Pain Catastrophizing Scale [PCS]) and NLS with postoperative pain were investigated in 90 patients after TKA. The associations among motor functions (muscle strength of knee extension, range of motion), sensory functions (joint position sense and two-point discrimination in the thigh) and NLS were also investigated. RESULTS: At three and six weeks after surgery, 36% and 19% of patients, respectively, experienced NLS. In hierarchical multiple regression analysis, NLS and PCS scores were significantly associated with postoperative pain, while joint position sense and range of motion were significantly associated with NLS. CONCLUSIONS: These results suggest that facilitation of sensory integration is important in rehabilitation after TKA because NLS appears to result from impaired sensory integration. The association of PCS scores with postoperative pain and NLS suggests the need to provide appropriate postoperative education to reduce persistent negative thoughts regarding future pain.

Growth, reproductive performance, carcass characteristics and meat quality in F1 and F2 progenies of somatic cell-cloned pigs.

The objective of this study was to examine the health and meat production of cloned sows and their progenies in order to demonstrate the application of somatic cell cloning to the pig industry. This study compared the growth, reproductive performance, carcass characteristics and meat quality of Landrace cloned sows, F1 progenies and F2 progenies. We measured their body weight, growth rate and feed conversion and performed a pathological analysis of their anatomy to detect abnormalities. Three of the five cloned pigs were used for a growth test. Cloned pigs grew normally and had characteristics similar to those of the control purebred Landrace pigs. Two cloned gilts were bred with a Landrace boar and used for a progeny test. F1 progenies had characteristics similar to those of the controls. Two of the F1 progeny gilts were bred with a Duroc or Large White boar and used for the progeny test. F2 progenies grew normally. There were no biological differences in growth, carcass characteristics and amino acid composition among cloned sows, F1 progenies, F2 progenies and conventional pigs. The cloned sows and F1 progenies showed normal reproductive performance. No specific abnormalities were observed by pathological analysis, with the exception of periarteritis in the F1 progenies. All pigs had a normal karyotype. These results demonstrate that cloned female pigs and their progenies have similar growth, reproductive performance and carcass quality characteristics and that somatic cell cloning could be a useful technique for conserving superior pig breeds in conventional meat production.

Arthroscopic bone marrow stimulation techniques for osteochondral lesions of the talus: prognostic factors for small lesions.

BACKGROUND: The defect size of an osteochondral lesion of the talus is one of the most important prognostic factors for deciding clinical outcomes. However, the prognostic factors for small osteochondral lesions of the talus are unknown. PURPOSE: To investigate the significant prognostic factors for small osteochondral lesions of the talus using arthroscopic bone marrow stimulation techniques. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Fifty ankles in 50 patients treated with arthroscopic bone marrow stimulation techniques for an osteochondral lesion of the talus (<150 mm(2)) were evaluated for prognostic factors. The patients were 22 men and 28 women (mean age, 35.0 years). Outcomes were measured using the American Orthopaedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot Scale, Berndt and Harty scale, and Saxena criteria. RESULTS: The mean lesion size was 62 mm(2) (range, 7-119 mm(2)). The mean AOFAS score improved from 74 (range, 18-90) preoperatively to 90 (range, 67-100) postoperatively. The Saxena criteria results were excellent, good, fair, and poor in 36 (72%), 8 (16%), 5 (10%), and 1 (2%) patients, respectively. The Berndt and Harty scale results were good in 34 (68%), fair in 6 (12%), and poor in 10 (20%) patients. Linear regression analyses showed prognostic significance for lesion depth and outcome. Medial lesions had a significantly higher incidence of poor outcomes than lateral lesions (P < .05). Among the medial lesions, lesions uncovered with the medial malleolus had inferior outcomes compared with covered lesions (P < .0001). There was no association between clinical outcome and lesion size or body mass index. In older patients (≥40 years), there was a significant trend toward inferior clinical outcomes (P < .05). CONCLUSION: Arthroscopic bone marrow stimulation techniques provided satisfactory clinical outcomes. However, older patients, deep lesions, and medial lesions uncovered with the medial malleolus were associated with inferior clinical outcomes.

Efficient establishment of pig embryonic fibroblast cell lines with conditional expression of the simian vacuolating virus 40 large T fragment.

The pig is an important animal for both agricultural and medical purposes. However, the number of pig-derived cell lines is relatively limited when compared with mouse- and human-derived lines. We established in this study a retroviral conditional expression system for the Simian vacuolating virus 40 large T fragment (SV40T) which allowed us to efficiently establish pig embryonic fibroblast cell lines. The established cell lines showed high levels of cell proliferation and resistance to cellular senescence. A chromosome analysis showed that 84% of the cells had the normal karyotype. Transient expression of the Cre recombinase allowed us to excise the SV40T fragment from the genome. The development of this research tool will enable us to quickly establish new cell lines derived from various animals.

Comparative proteomic analysis of liver mitochondrial proteins derived from cloned adult pigs reconstructed with Meishan pig fibroblast cells and European pig enucleated oocytes.

Somatic cell nuclear transfer (SCNT) has been exploited in efforts to clone and propagate valuable animal lineages. However, in many instances, recipient oocytes are obtained from sources independent of donor cell populations. As such, influences of potential nuclear-cytoplasmic incompatibility, post SCNT, are largely unknown. In the present study, alterations in mitochondrial protein levels were investigated in adult SCNT pigs produced by microinjection of Meishan pig fetus fibroblast cells into enucleated matured oocytes (maternal Landrace genetic background). Mitochondrial fractions were prepared from liver samples by mechanical homogenization and differential centrifugation. Liver mitochondria were then subjected to two-dimensional difference gel electrophoresis (2-D DIGE). Protein expression changes were confirmed with a volume ratio greater than 2 fold (P<0.05). 2-D DIGE analysis further revealed differential expression of three proteins between the Meishan (n=3) and Landrace (n=3) breeds. Differential expression patterns of 16 proteins were detected in SCNT pig liver tissue (n=3) when compared with Meishan control samples. However, none of the 16 proteins correlated with the three differentially expressed Meishan and Landrace liver mitochondrial proteins. In summary, alteration of mitochondrial protein expression levels was observed in adult SCNT pigs that did not reflect the breed difference of the recipient oocytes. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.

Comparison of liver mitochondrial proteins derived from newborn cloned calves and from cloned adult cattle by two-dimensional differential gel electrophoresis.

Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. The inability to establish functional interactions between donor nucleus and recipient mitochondria is also likely responsible for such a developmental deficiency. However, detailed knowledge of protein expression during somatic cell nuclear transfer (SCNT) in cattle is lacking. In the present study, variations in mitochondrial protein levels between SCNT-derived and control cattle, and from calves derived by artificial insemination were investigated. Mitochondrial fractions were prepared from frozen liver samples and subjected to two-dimensional (2-D) fluorescence differential gel electrophoresis (DIGE) using CyDye™ dyes. Protein expression changes were confirmed with a volume ratio greater than 2.0 (P < 0.05). 2D-DIGE analysis revealed differential expression of three proteins for SCNT cattle (n = 4) and seven proteins for SCNT calves (n = 6) compared to controls (P < 0.05). Different protein patterning was observed among SCNT animals even if animals were generated from the same donor cell source. No differences were detected in two of the SCNT cattle. Moreover, there was no novel protein identified in any of the SCNT cattle or calves. In conclusion, variation in mitochondrial protein expression concentrations was observed in non-viable, neonatal SCNT calves and among SCNT individuals. This result implicates mitochondrial-related gene expression in early developmental loss of SCNT embryos. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.

Microinjection of serum-starved mitochondria derived from somatic cells affects parthenogenetic development of bovine and murine oocytes.

Microinjection of isolated mitochondria into oocytes is an effective method to introduce exogenous mitochondrial DNA. In nuclear transfer procedures in which donor cell mitochondria are transferred with nuclei into recipient oocytes; development and survival rates of reconstructed embryos may be also directly influenced by mitochondrial viability. Mitochondrial viability is dramatically affected by cell culture conditions, such as serum starvation prior to nuclear transfer. This study was conducted to examine the influence of exogenous mitochondria using bovine and mouse parthenogenetic models. Mitochondria were isolated from primary cells at confluency and after serum starvation. The bovine oocytes injected with serum-starved mitochondria showed lower rates of morula and blastocyst formation when compared to uninjected controls (P<0.05). However, the developmental rates between non-starved mitochondria injection and controls were not different (P>0.05). The murine oocytes injected with serum-starved mitochondria showed lower rates of development when compared with non-starved mitochondria and controls (P<0.01). In contrast to mitochondria transfer, ooplasm transfer did not affect murine or bovine parthenogenetic development (P>0.05). The overall results showed that injection of serum-starved mitochondria influenced parthenogenetic development of both bovine and murine oocytes. Our results illustrate that the somatic mitochondria introduction accompanying nuclei has the capacity to affect reconstructed embryo development; particularly when using serum-starved cells as donor cells.

Characterization of a donor mitochondrial DNA transmission bottleneck in nuclear transfer derived cow lineages.

In embryos derived by nuclear-transfer (NT), fusion of donor cells with recipient oocytes resulted in varying patterns of mitochondrial DNA (mtDNA) transmission in NT animals. Distribution of donor cell mtDNA (D-mtDNA) found in offspring of NT-derived founders may also vary from donor cell and host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to G(1) offspring. Eleven NT founder cows were produced by fusion of enucleated oocytes (Holstein/Japanese Black) with Jersey/ Holstein oviduct epithelial cells, or Holstein/Japanese Black cumulus cells. Transmission of mtDNA was analyzed by PCR mediated single-strand conformation polymorphism of the D-loop region. In six of seven animals sampled postmortem, heteroplasmy were detected in various tissues, while D-mtDNA could not be detected in blood or hair samples from four live animals. The average proportion of D-mtDNA detected in one NT cow was 7.6%, and those in other cows were <5%. Heteroplasmic NT cows (n = 6) generated a total 12 G(1) offspring. Four of 12 G(1) offspring exhibited high percentages of D-mtDNA populations (range 17-51%). The remaining eight G(1) offspring had slightly or undetectable D-mtDNA (<5%). Generally, a genetic bottleneck in the female germ-line should favor a homoplasmic state. However, proportions of some G(1) offspring maintained heteroplasmy with a much higher percentage of D-mtDNA than their NT dams, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrate that D-mtDNA in NT cows is transmitted to G(1) offspring with varying efficiencies.

Bone landmarks are more reliable than tensioned gaps in TKA component alignment.

Two distinct techniques have been used to achieve alignment and ligament balance in TKA. We hypothesized a bone landmark technique would result in normal alignment, stability, and load-bearing characteristics and that the tensioned gap technique results in malalignment. We studied 12 normal cadaveric knees (six with each technique) for stability, alignment, load-bearing stress transfer characteristics, and patellar groove position after TKA. The tensioned gap technique used tensioners to establish equal loads in the medial and lateral ligaments at 90 degrees flexion and to resect the posterior femoral surfaces parallel to the cut tibial surfaces. The bone landmarks technique aligns the anterior and posterior femoral cuts perpendicular to the median sagittal plane as defined by the anteroposterior axis and then resects bone to match implant thickness. The tensioned gap technique maintained nearly equal laxity medially and laterally but the knee shifted into varus malalignment in flexion. Compressive stress in the knee shifted medially in flexion, and the patellar groove shifted laterally. The bone landmarks technique produced near normal varus and valgus and rotational stability; alignment, patellar groove position, and load transfer characteristics remained near normal throughout flexion.

Differentiation of female primordial germ cells in the male testes of chicken (Gallus gallus domesticus).

In our previous studies, we demonstrated that female primordial germ cells (PGCs) have the ability to differentiate into W chromosome-bearing (W-bearing) spermatozoa in male gonads of germline chimeric chickens. In this study, to investigate the differentiation pattern of female PGCs in male gonads in chickens, three germline chimeric chickens were generated by injecting female PGCs into the male recipient embryos. After these male chimeras reached sexual maturity, the semen samples were analyzed for detecting W-bearing cells by PCR and in situ hybridization analyses. The results indicated that the female PGCs had settled and differentiated in their testes. A histological analysis of the seminiferous tubule in those chimeras demonstrated that the W-bearing spermatogonia, spermatocytes, and round spermatids accounted for 30.8%, 32.7%, and 28.4%, respectively. However, the W-bearing elongating spermatid was markedly lower (7.7%) as compared to the W-bearing round spermatid. The W-bearing spermatozoa were hardly ever observed (0.2%). We concluded that although female PGCs in male gonads are capable of passing through the first and second meiotic division in adapting themselves to a male environment, they are hardly complete spermiogenesis.

Tibial tubercle transfer on a medial periosteal pedicle--a report of a new technique.

Tibial tubercle transfer is still probably the most widely used procedure of the numerous operative procedures described to realign the patella and extensor mechanism and to prevent a recurrent dislocation. Although this procedure most likely disturbs the blood supply to the tibial tubercle and thus may lead to a delayed union. Tibial tubercle transfer is also considered to play a role in the incidence of a tibial tubercle delayed union. Furthermore, a fracture of the tibial metaphysis has been reported to occur in some cases. We therefore devised a new procedure in which the periosteum of the medial side of the proximal portion of the tibia was left intact when tibial tubercle transfer is performed. The current paper describes the results of new technique in 25 knees with patellar maltracking. Eighty-four percent of the patients had good or excellent results at a mean follow-up time of 49 months. All of the patients achieved complete healing radiographically within 2 months after the operation. Serious complications such as compartment syndrome, infection and skin slough were also completely avoided in all cases. This new procedure that the use of a protective maneuver for the periosteum of the medial side of the tibia may thus reduce the incidence of a delayed union and thereby promote early postoperative rehabilitation after tibial tubercle transfer.

Transmission of mitochondrial DNA in pigs and progeny derived from nuclear transfer of Meishan pig fibroblast cells.

In embryos derived by nuclear transfer (NT), fusion, or injection of donor cells with recipient oocytes caused mitochondrial heteroplasmy. Previous studies have reported varying patterns of mitochondrial DNA (mtDNA) transmission in cloned calves. Here, we examined the transmission of mtDNA from NT pigs to their progeny. NT pigs were created by microinjection of Meishan pig fetal fibroblast nuclei into enucleated oocytes (maternal Landrace background). Transmission of donor cell (Meishan) mtDNA was analyzed using 4 NT pigs and 25 of their progeny by PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, PCR-RFLP, and a specific PCR to detect Meishan mtDNA single nucleotide polymorphisms (SNP-PCR). In the blood and hair root of NT pigs, donor mtDNAs were not detected by PCR-SSCP and PCR-RFLP, but detected by SNP-PCR. These results indicated that donor mtDNAs comprised between 0.1% and 1% of total mtDNA. Only one of the progeny exhibited heteroplasmy with donor cell mtDNA populations, ranging from 0% to 44% in selected tissues. Additionally, other progeny of the same heteroplasmic founder pig were analyzed, and 89% (16/18) harbored donor cell mtDNA populations. The proportion of donor mtDNA was significantly higher in liver (12.9 +/- 8.3%) than in spleen (5.0 +/- 3.9%), ear (6.7 +/- 5.3%), and blood (5.8 +/- 3.7%) (P < 0.01). These results demonstrated that donor mtDNAs in NT pigs could be transmitted to progeny. Moreover, once heteroplasmy was transmitted to progeny of NT-derived pigs, it appears that the introduced mitochondrial populations become fixed and maternally-derived heteroplasmy was more readily maintained in subsequent generations.

Chromosomal instability in the cattle clones derived by somatic cell nuclear-transfer.

Cytogenetic analysis was performed on peripheral lymphocytes collected from 20 cattle clones (19 showed no overt phenotypic abnormalities except for high birth weight while 1 exhibited left forelimb contracture), the donor cell cultures from which they were derived and lymphocytes from six insemination produced control cattle. All animals and cell cultures had a modal chromosome number of 60. The frequency of abnormal cells for donor cell cultures, clones, and controls was 6.68+/-0.30%, 5.30+/-5.49%, and 5.08+/-1.04%, respectively, and did not differ significantly among the groups. There were, however, two clones derived from different donor cell cultures with high incidences of 21.29% and 20.13%, of abnormal cells consisting of pseudodiploid (near-diploid), near-triploid and near-tetraploid, and tetraploid cells. Among these two clones, one had only a few endoreduplicated nuclei although further studies are necessary to precisely define the cytological origin and nature of the abnormal cells. The clones were evaluated at multiple time points for up to 20 months of age and the incidence of abnormal lymphocytes remained stable indicating that the chromosomally abnormal nuclei found in cloned animals was not a transient event. These results show that the majority of phenotypically normal clones have normal chromosomal make up but that instability of chromosome number can occur in clones that are phenotypically normal. Therefore, cytogenetical evaluation of peripheral lymphocytes and other tissues with follow up of the phenotypical consequences of these abnormalities is warranted even in phenotypically normal clones.

Low oxygen tension during in vitro maturation of porcine follicular oocytes improves parthenogenetic activation and subsequent development to the blastocyst stage.

To establish a reliable in vitro maturation system for activation and subsequent development as nuclear recipients for the effective production of pig clones, we assessed maturation, activation and parthenogenetic development in response to the following: (1) type of immature oocytes (cumulus-oocyte complexes (COCs) or parietal granulosa plus cumulus-oocyte complexes (GCOCs)); (2) oxygen (O(2)) tension (5 or 20%); and (3) maturation period (36-60 h). The rate of nuclear maturation to metaphase-II (M-II) in the GCOC group (73.0 +/- 3.1%) was higher than that in the COC group (P < 0.05, 60.6 +/- 3.5%), but the rates did not differ between the 5 and 20% O(2) tension groups. M-II rate increased (P < 0.05) to about 70% after 42 h and then remained constant until 60 h of culture. When oocytes were matured under 5% O(2) tension and stimulated, the rate of normal oocyte activation (a female pronucleus formation and emission of the second polar body) was higher (P < 0.05, 38.5 +/- 3.9%) than when oocytes were matured under 20% O(2) tension (24.5 +/- 3.9%). On the other hand, the rate of normal activation was not significantly different between the COC and GCOC groups, and the highest (P < 0.05) normal activation rate was obtained in oocytes cultured for 48 and 54 h (48.4 +/- 5.5% and 47.9 +/- 8.2%, respectively). When COC and GCOC matured for 48 h under 5 and 20% O(2) tension were stimulated and subsequently cultured in vitro for 6 days, the rate of blastocyst formation did not differ between the oocyte types nor between the O(2) tension groups. However, blastocyst quality, as measured by mean total cell number, was significantly higher in the 5% O(2) group (P < 0.05, 34.6 +/- 2.0 for COC; 33.8 +/- 1.8 for GCOC) compared with the 20% O(2) group (25.9 +/- 1.8 for COC; 27.0 +/- 2.0 for GCOC). In conclusion, low O(2) tension (5%) during in vitro maturation of porcine oocytes promoted their ability to be activated normally and improved the quality of parthenogenetic blastocysts developed in vitro in modified NCSU-37 solutions. This knowledge may be applicable for preparation of in vitro matured oocytes with good quality as recipient oocytes for generating pig clones.

Microinjection of cytoplasm or mitochondria derived from somatic cells affects parthenogenetic development of murine oocytes.

Cloned mammals are readily obtained by nuclear transfer using cultured somatic cells; however, the rate of generating live offspring from the reconstructed embryos remains low. In nuclear transfer procedures, varying quantities of donor cell mitochondria are transferred with nuclei into recipient oocytes, and mitochondrial heteroplasmy has been observed. A mouse model was used to examine whether transferred mitochondria affect the development of the reconstructed oocytes. Cytoplasm or purified mitochondria from somatic cells derived from the external ear, skeletal muscle, and testis of Mus spretus mice or cumulus cells of Mus musculus domesticus mice were transferred into M. m. domesticus (B6SJLF1 and B6D2F1) oocytes to observe parthenogenetic development through the morula stage. All B6D2F1 oocytes injected with somatic cytoplasm or mitochondria showed delayed development when compared to oocytes injected with buffer. The developmental rates were not different among injected cell sources, with the exception of testis-derived donor cells injected into B6SJLF1 oocytes (P < 0.01). The developmental rate of B6D2F1 oocytes injected with buffer alone (98.8% survival) was different from those injected with somatic cytoplasm (60.8% survival) or somatic mitochondria (56.5% survival) (P < 0.01). Conversely, injection of ooplasm into B6D2F1 oocytes did not affect parthenogenetic development (100% survival). Our results indicate that injection of somatic cytoplasm or mitochondria affected parthenogenetic development of murine oocytes. These results have further implications for in vitro fertilization protocols employing ooplasmic transfer where primary oocyte failure is not confirmed.

Effects of caffeine treatment on aged porcine oocytes: parthenogenetic activation ability, chromosome condensation and development to the blastocyst stage after somatic cell nuclear transfer.

The possibility of using aged porcine oocytes treated with caffeine, which inhibits the decrease in M-phase promoting factor activity, for pig cloning was evaluated. Cumulus-oocyte complexes (COCs) were cultured initially for 36 h and subsequently with or without 5 mM caffeine for 24 h (in total for 60 h: 60CA+ or 60CA- group, respectively). As a control group, COCs were cultured for 48 h without caffeine (48CA-). The pronuclear formation rates at 10 h after electrical stimulation in the 60CA+ and 60CA- groups decreased significantly (p < 0.05) compared with the 48CA- group. However, the fragmentation rate was significantly higher (p < 0.05) in the 60CA- group than in the 60CA+ and 48CA- groups. When the stimulated oocytes were cultured for 6 days, the 60CA+ group showed significantly lower blastocyst formation and higher fragmentation or degeneration rates (p < 0.05) than the 48CA- group. However, the number of total cells in blastocysts was not affected by maturation period or caffeine treatment. When somatic cell nuclei were injected into the non-enucleated oocytes and exposed to cytoplasm for a certain duration (1-11 h) before the completion of maturation (48 or 60 h), the rate of nuclear membrane breakdown after exposure to cytoplasm for 1-2 h in the 60CA- oocytes was significantly lower (p < 0.05) than in the other experimental groups. The rate of scattered chromosome formation in the same 60CA- group tended to be lower (p = 0.08) than in the other groups. After the enucleation and transfer of nuclei, blastocyst formation rates in the 60CA+ and 60CA- groups were significantly lower (p < 0.05) than in the 48CA- group. Blastocyst quality did not differ among all the groups. These results suggest that chromosome decondensation of the transplanted somatic nucleus is affected by both the duration of exposure to cytoplasm and the age of the recipient porcine oocytes, and that caffeine treatment promotes nuclear remodelling but does not prevent the decrease in the developmental ability of cloned embryos caused by oocyte aging.

Infection of human fibroblast-like synovial cells with Chlamydia trachomatis results in persistent infection and interleukin-6 production.

Recent studies have shown that the urogenital pathogen Chlamydia trachomatis to be a major bacterium triggering reactive arthritis (ReA), and is able to induce interleukin-6 (IL-6) production in human fibroblast-like synovial cells (FSC) in vitro. In the present study, we examined the correlation between IL-6 production and multiplication of chlamydia in FSC. All FSC from five patients secreted highly increased quantities of IL-6 in a dose-dependent and time-dependent fashion. Heat and UV inactivated chlamydia failed to enhance production of IL-6. When azithromycin was added to infected cultures of FSC at 0 or 48 h after infection, the level of IL-6 production was very low. Transmission electron microscopy of such infected cultures revealed many abnormal forms of chlamydia within the inclusions in FSC. From one step-growth curve experiments, it was suggested that C. trachomatis hardly multiplied in FSC. In contrast, in C. trachomatis infected HeLa 229 cells, chlamydia multiplied as usual, but little IL-6 production were found. These observations indicated that live chlamydia and the persistence of chlamydia may be essential for stimulating the synthesis of IL-6 in FSC.

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.

Mitochondrial activity in response to serum starvation in bovine (Bos taurus) cell culture.

In nuclear transfer procedures, in addition to nuclei, donor cell mitochondria are routinely transferred into recipient oocytes, and mitochondrial heteroplasmy has been reported. However, various protocols have resulted in either homoplasmy for recipient oocyte mitochondria or varying heteroplasmic levels in cloned animals. In nuclear transfer protocols, donor cells are subjected to serum-starvation prior to electroporation. Therefore, the relationship between culture conditions and mitochondrial activity was explored. Fibroblast cell lines were propagated from bovine ear epithelium, skin, skeletal muscle, or cumulus cells. In vitro mitochondrial viability was assessed in proliferative and confluent cells, cultured under serum-starvation or supplemented conditions. Cells were stained with MitoTracker Red CMXRos and comparative fluorescence intensities were assessed. The mitochondrial activity per cell was highest under proliferation, significantly lower at confluency (p < 0.001), and remained depressed after serum starvation for within a week (p < 0.001). Serum starvation induced an increase in mitochondrial viability in confluent cells. These results demonstrate that mitochondrial viability is dramatically affected by cell culture conditions. Consequently, specific cell culture parameters provide one explanation for the varying incidence of heteroplasmy identified in cloned animals. Future research should reveal whether specific cell culture parameters represent one of the factors for the varying incidence of heteroplasmy identified in cloned animals.