Differential expression of porcine testis proteins during postnatal development.

The development of the testes includes changes in cell morphology and endocrine levels that are essential for the maturation of males. A large number of novel proteins are expressed throughout testis development and play important roles in spermatogenesis. Differences in protein expressions during the development of porcine testes have not been systematically studied. The purpose of this study was to investigate differential protein expression in porcine testes during postnatal development. Testes from four pigs each at 1wk, 3mo, and 1yr of age were used for a proteomic analysis. Expression levels of 264 protein spots were quantified using the Melanie 3 software. In total, 108 protein spots showed more than 2-fold differences (P<0.05) among developmental stages, and 90 of them were successfully identified by mass spectrometry. The proteins were sorted based on whether the expression levels increased with age (36.1%), decreased with age (38.0%), or fluctuated among different developmental stages (25.9%). In total, 69 unique gene products were further classified according to their gene ontology annotations. A majority of the proteins are organelle proteins (41%) with the nucleus and mitochondria being the main organelles. About 45% of the proteins have a protein binding domain and are likely involved in protein-protein interactions. Finally, a large proportion of these differentially expressed proteins are involved in cellular (25%) and metabolic (22%) processes. Identifying these differentially expressed proteins should be valuable for exploring developmental biology and the pathology of male reproduction.

Analysis of chicken serum proteome and differential protein expression during development in single-comb White Leghorn hens.

Serum is believed to harbor thousands of distinct proteins that are either actively secreted or leak from various blood cells or tissues. Exploring protein composition in serum may accelerate the discovery of novel protein biomarkers for specific economic traits in livestock species. This study analyzed serum protein composition to establish a 2-DE reference map, and monitored protein dynamics of single-comb White Leghorn hens at 8, 19 and 23 weeks after hatching. A total of 119 CBB-stained and 315 silver-stained serum protein spots were analyzed by MALDI-TOF MS. Of these, 98 CBB-stained and 94 silver-stained protein spots were significantly matched to existing chicken proteins. The identified spots represented 30 distinctive proteins in the serum of laying hens. To compare protein expression during development, expression levels of 47 protein spots were quantified by relative spot volume with Melanie 3 software. Ten protein spots increased and 3 protein spots decreased as hen age increased. Previous research has suggested that some of these proteins play critical roles in egg production. The differentially expressed proteins with unknown identities will be valuable candidates for further explorations of their roles in egg production of laying hens.

A reference map and identification of porcine testis proteins using 2-DE and MS.

The development of the testis is essential for maturation of male mammals. A complete understanding of proteins expressed in the testis will provide biological information on many reproductive dysfunctions in males. The purposes of this study were to apply a proteomic approach to investigating protein composition and to establish a 2-D PAGE reference map for porcine testis proteins. MALDI-TOF MS was performed for protein identification. When 1 mg of total proteins was assayed by 2-D PAGE and stained with colloidal CBB, more than 400 proteins with a pI of pH 3-10 and M(r) of 10-200 kDa could be detected. Protein expression varied among individuals, with CV between 4.7 and 131.5%. A total of 447 protein spots were excised for identification, among which 337 spots were identified by searching the mass spectra against the NCBInr database. Identification of the remaining 110 spots was unsuccessful. A 2-D PAGE-based porcine testis protein database has been constructed on the basis of the results and will be published on the WWW. This database should be valuable for investigating the developmental biology and pathology of porcine testis.

Developmental changes of heat-shock proteins in porcine testis by a proteomic analysis.

Heat-shock proteins (HSPs) are important in spermatogenesis. This study investigated developmental changes in the expression of major HSPs in porcine testis. The testis from five immature (mean age 2.9+/-0.1 months) and five mature boars (35.7+/-14.0 months) were examined. Two-dimensional polyacrylamide gel electrophoresis was conducted and proteins were identified by Western blotting and/or matrix-assisted laser desorption/ionization mass spectrometry. Moreover, the 90, 70, and 60 kDa HSPs, 70 kDa heat-shock cognate protein (HSC 70), tubulin, and actin were quantified on two-dimensional gels. Protein spots were quantified by densitometry, combined with a computer-assisted image analysis system. Immunohistochemistry was performed to analyze the expression pattern of major HSPs and beta-tubulin in testis. One isoform of HSP 90 (HSP 90 alpha), two isoforms of HSC 70 (HSC 70a and HSC 70c), one isoform of HSP70 (HSP 70e), and tubulin increased after sexual maturation (P<0.05). A testis-specific HSP70 (P70t) was markedly increased in the testes of sexually mature boars. Meanwhile, levels of actin and some isoforms of HSPs including 60 kDa HSP remained similar in both groups. These observations were further confirmed by immunohistochemistry; therefore, the upregulation of protein expression in the adult testis could be attributed to a higher level of protein expression and the number of cells that were HSPs-positive already resided in the immature testis. The differential expression of major HSPs suggested that they may be important in porcine spermatogenesis.

Serum acute phase proteins and swine health status.

The purpose of this study was to investigate the relationship between swine health status and the concentration of the serum acute phase proteins, haptoglobin (HP), and C-reactive protein (CRP). A total of 378 clinically healthy pigs from farms A and B, plus 20 pigs culled from farm A due to poor growth, were used in this experiment. Each pig was examined and blood samples were collected during slaughter. The HP concentration was measured by using an HP-hemoglobin binding assay. The CRP concentration was measured by using a CRP enzyme immunoassay. Gross and histopathological lesions were examined and recorded at slaughter. Representative samples were then collected in order to isolate pathogens. Swine enzootic pneumonia, found in 47.7% of the pigs, was the most common lesion. Other lesions included pleuropneumonia (32.7%), suppurative pneumonia (10.3%), fibrinous pericardititis (4.3%), Ascaris migration in the liver (33.9%), and intestinal serositis (3.0%). On farm A, the percentage of pigs with 1 or more lesions was 88.2%. For culled pigs from farm A, the mean serum concentrations of HP and CRP were 2.23 +/- 0.14 mg/mL and 252.93 +/- 11.62 microg/mL, which were significantly higher than concentrations in clinically normal pigs (1.42 +/- 0.02 mg/mL and 84.88 +/- 2.61 microg/mL, respectively, P < 0.01). Moreover, among clinically normal farm A pigs, the mean HP concentration in pigs with lesions (1.43 +/- 0.02 mg/mL) was significantly higher than in pigs without lesions (1.32 +/- 0.07 mg/mL) (P < 0.05). However, the mean serum CRP concentrations in these animals were not significantly different. On farm B, the percentage of pigs with one or more lesions was 50.0%. Interestingly, the mean serum HP concentration in clinically normal pigs with lesions was significantly lower in farm B pigs (1.23 +/- 0.07 mg/mL) than in the farm A pigs (1.43 +/- 0.02 mg/mL; P < 0.01). However, serum CRP concentrations in farm A and B pigs were not significantly different. Serum HP concentration, which is a better indicator of inflammatory reactions in pig herds than serum CRP concentration, provides an important marker for swine health status.

Serum C-reactive protein in dairy herds.

The purpose of this study was to determine the relationship between the serum level of C-reactive protein (CRP) and lactation and health status. Blood samples were collected every 2 wk for 12 mo from 29 randomly selected dairy cattle on 3 farms. At the time the blood samples were collected, the stage of pregnancy, lactation status, breeding records, general health condition, reproductive status, and body condition score were recorded for each cow. Serum CRP was detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis and western immunoblotting. C-reactive protein levels were measured with a densitometer and expressed as an optimal dose value. C-reactive protein levels were correlated with the body condition score, lactation status, and animal health (P < 0.05), but not with ambient temperature, animal age, or parity. C-reactive protein levels increased with milk production, peaking during high lactation (2 to 4 mo of pregnancy), and decreased when lactation ceased. In addition, the CRP level was highest during naturally occurring infections, such as mastitis and other tissue inflammation. Thus, the CRP level can confirm the presence of inflammation. The stress effect of taking blood samples as measured by the CRP level, was also examined. The CRP level became rapidly elevated 12 h after the blood samples were taken but returned to normal 36 h later. In conclusion, the stresses resulting from overall poor health, heavy lactation, and blood sampling caused the elevation of serum CRP. C-reactive protein is a marker or tool for evaluating the health status of a herd. C-reactive protein should also be considered as a useful criteria to assess the stress levels and may be useful in early surveillance of disease conditions in a dairy herd.

Echocardiographic features of pigs with spontaneous hypertrophic cardiomyopathy.

Ultrasonography is one of the most common, noninvasive techniques used for cardiovascular diagnosis because it provides reliable information and enhances patient safety. Two-dimensional (2-D) and M-mode echocardiography is conducted to assess the severity and distribution of myocardial hypertrophy. Hypertrophic cardiomyopathy (HCM) is a primary myocardial disease that has variable manifestations because interactions between the many facets of systolic and diastolic dysfunction of the heart are complex. The objective of the study reported here was to characterized clinical HCM in pigs. A commercial Vingmed (CFM-800) 3.25 MHz transducer was used to perform 2-D and M-mode echocardiography. Experimental pigs (about 100 kg in body weight) were anesthetized and positioned in left lateral recumbency. Echocardiographic images (2-D) were acquired in parasternal short-axis and long-axis views. The 2-D images provided M-mode under direct anatomic visualization. The pigs were sacrificed for pathologic study after echocardiographic examination. In typical HCM cases (n = 8), the interventricular septum thickness increased, the left ventricular (LV) end-systolic and end-diastolic dimensions decreased, and the left atrial dimensions and the indexes of systolic function, such as ejection fraction and velocity of fiber shortening, increased. The LV outflow tract narrowed, particularly when gross upper septal hypertrophy was evident. Moreover, systolic cranial motion (SCM) of the septal leaflet of the mitral valve was observed. Doppler evidence of mitral regurgitation often was associated with SCM. The echocardiographic findings from pigs with HCM resembled those from humans. Thus, porcine HCM may serve as a spontaneous animal model for the study of HCM in humans.

Ultrastructure of intramural coronary arteries in pigs with hypertrophic cardiomyopathy.

Pigs with hypertrophic cardiomyopathy diagnosed by echocardiographic examination were selected for study from a genetic breeding herd. Under dissecting microscopic examination, intramural coronary arteries in the septum and left ventricular free wall of euthanized pigs were collected for ultrastructural study. The major lesions of wall thickening included degeneration or denudation of endothelium, subendothelial edema, proliferation of collagen fiber, and hyperplasia of smooth muscle cells. Smooth muscle cells proliferated and migrated through the internal elastic lamella into the intima, which caused the early lesion of wall thickening of the intramural coronary arteries. The extent of smooth muscle cell proliferation was related to the severity of endothelial damage. The smooth muscle cells in the intima were identified by immunohistochemical staining (i.e., smooth muscle actin [SMA] stain). Three major types of severe wall thickening with narrow lumen were observed in the intramural coronary arteries. Edema in the intima caused the major lesion of Type I wall thickening. The internal elastic lamella was broken into small interrupted fragments, and fine fragments of elastic fibers surrounded by the cellular processes of smooth muscle were observed in Type I lesions. Many smooth muscle cells proliferated in the intima and media, which constituted the major lesion of Type II wall thickening of the intramural coronary arteries. Many vacuolized, degenerated smooth muscle cells with fewer sarcoplasmic myofilaments could be clearly observed in the Type II lesions. In advanced cases, severe vacuolization and degeneration of smooth muscle cells with the presence of many bizarrely shaped smooth muscle cells in the walls of the intramural coronary arteries could be observed, which caused the major lesion of Type III wall thickening. Pigs with hypertrophic cardiomyopathy, characterized by spontaneously occurring lesions in intramural coronary arteries, may prove a valuable animal model for human disease.

Purification and characterization of porcine testis 90-kDa heat shock protein (HSP90) as a substrate for various protein kinases.

We purified a large quantity of HSP90 from porcine testis by hydroxylapatite (HA-HSP90) and SDS-PAGE/electroelution (eluted-HSP90) to explore the molecular mechanism of HSP90 phosphorylation affecting its metabolism. The purified HSP90 was used as an antigen to raise polyclonal antibodies in rabbits. Immunoblot analysis revealed that most purified HSP90 was HSP90alpha. Compared with the commercial anti-HSP90 antibody, the polyclonal antibody raised in this study could specifically detect the testis HSP90 and immunoprecipitate HSP90 from tissue homogenates or cell extracts. Incubation of the purified HSP90 or HSP90 immunoprecipitated from extracts of human A431 cells, Balb/c 3T3 fibroblasts, and porcine testis with [gamma-32P]ATP/Mg2+ resulted in phosphorylation of HSP90. However, the eluted-HSP90 lost its phosphorylation ability when incubated with [gamma-32P]ATP x Mg2+ alone but could be phosphorylated by various protein kinases, including PKA, CKII, kinase FA/GSK-3 alpha, and AK. The order of phosphorylation of HSP90 by these kinases is PKA = CKII > AK >> kinase FA/GSK-3 alpha.

Whole-body hyperthermia-induced thermotolerance is associated with the induction of heat shock protein 70 in mice.

Molecular mechanisms of whole-body thermotolerance (WBT) in mammals have not been investigated thoroughly. The purpose of this study was to assess the induction of the 70 kDa heat shock protein (HSP70) and antioxidant enzyme activity in animal WBT, which was induced by whole-body hyperthermia (WBH) in mice. As a preconditioning treatment, WBH was applied to mice to induce WBT. Synthesis of inducible HSP70 (HSP70i) and quantification of its increased level in liver were investigated by one- and two-dimensional polyacrylamide gel electrophoresis and immunoblotting. HSP70i synthesis in mice liver was induced by non-lethal WBH (41 degrees C, 30 min). When compared to control animals, the level of liver HSP70i increased substantially (by 3.6-fold; P<0.0001). When exposed to 30 min of hyperthermia preconditioning, and after recovery for 48 h, the survival rate was 88.2 %, which was significantly higher than that of the control group (37.5 %; P<0.01). Moreover, the survival rate of animals subjected to preconditioning for 15 min was 72.2 %, which was also significantly higher than that of the control group (P<0.05). In contrast, the survival rate of animals subjected to preconditioning for 45 min was 63.5 %, which was not different from the control group. Nonetheless, the protection index of the group subjected to 15 min and 30 min of preconditioning was 1.93 and 2.37, respectively. Furthermore, to assess their contributions to WBT, the activities of antioxidant enzymes were also measured. After 48 h of recovery in preconditioned animals, hepatic antioxidant enzyme activities, including superoxide dismutase, catalase and glutathione peroxidase, had not changed significantly. To study the molecular mechanism of WBT, we successfully developed a mouse model and suggest that, rather than the activities of antioxidant enzymes, it is HSP70i that has a role to help animals survive during severe heat stress.

Influence of Age on the Electrocardiographic Waves in Taiwanese Lan-Yu Miniature Pigs.

The purpose of the study reported here was to determine baseline information of the electrocardiogram (ECG) and the influence of age on cardiac function in Taiwanese Lan-Yu (TLY) miniature pigs. Non-anesthetized TLY minipigs (from birth to 6 months of age) were placed on a webbed stanchion, simulating a standing position, for acquisition of an ECG, using six limb leads (I, II, III, aVR, aVL, and aVF), with each as a single-channel ECG recorder. The P and T waves obtained from leads I, III, and aVL were useful in determining the subtle cardiac changes during maturation of TLY minipigs. Interestingly, changes in the QT interval analyzed from all 6 leads were almost indistinguishable. Shortening of the QT interval was induced (p < 0.05) between days 1 and 7 of postnatal life. The QT interval lengthened to a steady state at day 60, and paralleled pigs' physical maturation. The longer QT interval was conversely correlated to heart rate as pigs matured. In the QRS complex interval, only lead aVR was significantly decreased at day 7 (p < 0.05). Further changes in the QRS interval from day 21 were not observed in any lead. Because the duration of the ventricular complex represents the period required for the excitation front to reach the terminals of the Purkinje fibers in the ventricular myocardium, the increase in QRS interval observed within 21 days of birth could be attributed to an increase in the thickness of ventricular myocardium. The data suggest that cardiac maturation was achieved at 60 days of age, although the body weight of minipigs continued to increase beyond 60 days of age. Because the body weight of these newly developed TLY minipigs can be maintained within 25 to 30 kg at one year of age and their major ECG findings did not significantly differ from those of domestic pigs and humans, they may be useful as a model for cardiovascular and pharmacologic research. The similarity of ECG profiles between pigs and humans also was evaluated.