'In vitro' capacitation and further 'in vitro' progesterone-induced acrosome exocytosis are linked to specific changes in the expression and acrosome location of protein phosphorylation in serine residues of boar spermatozoa

The aim of this study is to determine changes in the expression and location of protein serine phosphorylation (pSer) during 'in vitro' capacitation (IVC) and 'in vitro' acrosome exocytosis (IVAE) in boar spermatozoa. This was performed in both mono- and bi-dimensional analyses of protein expression through Western blot, as well as through immunocytochemistry. Furthermore, IVC was induced through incubation in an IVC medium, and afterwards, progesterone-induced IVAE was performed. The mono-dimensional Western blot analysis showed the presence of a predominant pSer band of approximately 70-75 kDa, which was accompanied by fainter bands, especially three with molecular weights of approximately 50, 35 and 32 kDa. Neither IVC nor IVAE significantly modified this pattern. Bi-dimensional analyses showed a more complex pattern, with at least five protein clusters. The attainment of IVC caused the disappearance of the proteins with the highest molecular weight concomitantly with the appearance of pSer proteins of 75-kDa/pI 9.5 and 80-kDa/pI 10. The induction of IVAE caused the appearance of new pSer proteins of a 75-kDa/pI 6.5-7.5 and 75-kDa/pI 10. Immunocytochemistry showed that the main pSer expression in boar expression before the attainment of IVC was located at the midpiece. The IVC induced the appearance of acrosomal pSer, which was greatly increased during IVAE. Our results indicate that the changes in serine protein phosphorylation associated with IVC and IVAE comprise not only the appearance of specific phosphorylated proteins, such as the pSer-75 kDa, but also changes in pI and displacements in the sperm location of phosphorylated proteins, like the specific acrosomal pSer signal induced during IVC.     

Influence of different cellular concentrations of boar sperm suspensions on the induction of capacitation and acrosome reaction

We aimed to analyze the influence of different cellular concentrations of boar sperm suspensions on the induction of capacitation and acrosome reaction. When spermatozoa were incubated at 100 or 200 mill/ml, significant increases in protein tyrosine phosphorylation in the p32 protein were observed, compared to those at 50 mill/ml. In addition, sperm concentration-dependent increases were observed in plasma membrane lipid disorganization (50 mill/ml vs. 200 mill/ml), induction of the acrosome reaction (50 mill/ml vs. 100 mill/ml and 200 mill/ml), and sperm viability (50 mill/ml vs. 100 mill/ml and 200 mill/ml). Our data indicate that an increase in sperm concentration stimulates the induction of capacitation and acrosome reaction in boars.     

Cytochrome c upregulation during capacitation and spontaneous acrosome reaction determines the fate of pig sperm cells: linking proteome analysis

To identify the mechanisms underlying capacitation, we undertook a high-resolution differential proteomic analysis of pig sperm cells. Two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry analyses led to identification of 56 differentially expressed proteins. After induction of capacitation in vitro, the well-established markers of the capacitation (lactadherin P47, acrosomal protein SP-10 precursor, prohibitin, proteasomes, DJ-1 protein and arylsulfatase-A) and TCA cycle proteins (isocitrate dehydrogenase, malate dehydrogenase and pyruvate dehydrogenase) were identified. During induction, cytochrome c expression via the p53 pathway increased, however apoptotic executors, such as caspase-3, decreased significantly. Therefore, we tested the hypothesis that cytochrome c upregulation in spermatozoa is capable of activating tyrosine phosphorylation for capacitation, rather than apoptosis. Exposure of sperm cells to soluble Na2CrO4 [Cr (VI)], which induces cytochrome c upregulation, caused a dose- and time-dependent increase in tyrosine phosphorylation of sperm proteins in non-capacitating medium. In contrast, supplementation of cyclosporin A, which blocks cytochrome c upregulation, inhibited tyrosine phosphorylation of sperm proteins. Furthermore, spermatozoa in capacitation medium or non-capacitation media supplemented with soluble Cr (VI) showed similar levels of capacitation. These findings indicate that differential expression of many of these proteins has previously been unrecognized in sperm cells incubated in capacitation medium also suggest that a gradual increase of cytochrome c during incubation to induce capacitation determines sperm cell fate, i.e., apoptosis or further development for fertilization.     

“In vitro” capacitation and further progesterone‐induced acrosome exocytosis are linked to specific changes in the expression and location of threonine phosphorylation of boar spermatozoa

The aim of this study was to determine if the achievement of the “in vitro” capacitation (IVC) status and subsequent progesterone‐induced “in vitro” acrosome exocytosis (IVAE) was accompanied with overall changes in threonine phosphorylation (pThre) of boar spermatozoa. For this purpose, mono‐ and bi‐dimensional Western blot analyses as well as immunocytochemistry studies against pThre were performed in boar sperm subjected to IVC and subsequent IVAE. Mono‐dimensional Western blot in non‐capacitated samples showed that launching of IVC did induce an overall increase in signal intensity in all observed bands that was followed by a subsequent decrease afterwards. Bi‐dimensional Western blot analysis showed the presence of four main signal protein clusters. The attainment of IVC induced an overall decrease in the number and intensity of spots of Clusters A, B and C and a concomitant increase in the intensity of spots of Cluster D. The IVAE launching caused a rapid increase in the intensity of spots of Clusters B, C and D, which was followed by a subsequent decrease of the intensity together with a concomitant pI displacement of Cluster C. Finally, immunocytochemistry showed that the pThre signal of non‐capacitated cells was located at the whole sperm. The IVC did not induce prominent changes in this location. In contrast, the induction of IVAE caused the appearance of an additional an intense acrosome and tail pThre signal that subsequently decreased. In conclusion, our results indicate that IVC and further IVAE induced specific changes in the intensity and appearance of pThre protein phosphorylation which were linked to changes of specific protein characteristics as pI. These results support, thus, the existence of a specific role of pThre in IVC/IVAE of boar sperm.     

Administration of flutamide alters sperm ultrastructure, sperm plasma membrane integrity and its stability, and sperm mitochondrial oxidative capability in the boar: in vivo and in vitro approach

Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 μg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p < 0.05) in flutamide-treated boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p < 0.05). No further decrease in the membrane integrity was found when the effect of anti-androgen lasted for 24 h. On the other hand, a decrease in sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p < 0.05). Characterization of sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal.     

Effects of protein phosphatase inhibitor calyculin a on the postacrosomal protein serine/threonine phosphorylation state and acrosome reaction in boar spermatozoa incubated with a cAMP analog

In order to reveal the involvement of the sperm postacrosomal region in the acrosome reaction, we examined the effects of the protein phosphatase inhibitor calyculin A on the postacrosomal protein serine/threonine phosphorylation state and acrosome morphology in boar spermatozoa incubated with a cAMP analog. Proteins were highly phosphorylated on the serine/threonine residues only in the postacrosomal region before incubation. After 90-min incubation without calyculin A, the protein phosphorylation state declined in the postacrosomal region irrespective of the capacitation state while it remained under the detectable level in the other regions of the sperm head. However, addition of calyculin A effectively suppressed the decline in protein phosphorylation state and increased an inactive form of protein phosphatase 1 in the postacrosomal region. On the other hand, this inhibitor had no influence on the protein phosphorylation state in the acrosome and equatorial segment. After incubation without calyculin A for 180 or 360 min, many spermatozoa exhibited acrosomal changes and loss that indicated occurrence of the acrosome reaction. However, addition of calyculin A significantly blocked these events. These results are consistent with our suggestion that postacrosomal serine/threonine-phosphorylated proteins are involved in suppression of the acrosome reaction in boar spermatozoa in vitro.     

New strategies of boar sperm cryopreservation: Development of novel freezing and thawing methods with a focus on the roles of seminal plasma

Cryopreservation of boar spermatozoa offers an effective means of long‐term storage of important genetic material. Many researchers have investigated how to improve reproductive performance by artificial insemination (AI) using cryopreserved boar spermatozoa. Recently, we and other groups reported that high conception rates (70–80%) can be achieved by AI with frozen‐thawed boar spermatozoa using a modified temperature program during freezing, or a novel cryopreservation extender to improve sperm quality (including sperm survivability, motility, membrane status and fertilization ability) after thawing, or a novel sperm infusion method, deep intra uterine insemination. However, these techniques have not yet been used for commercial pig production. The variation in sperm freezability among boars or among ejaculations in an identical boar is one of the main reasons for this problem. In our previous study, it was revealed that some components of seminal plasma have a negative effect on the freezability of boar sperm. One of these factors is bacteria‐released endotoxin (lipopolysaccharide: LPS). LPS binds to Toll‐like receptor‐4 (TLR‐4) expressed on the sperm surface, resulting in induction of apoptosis. On the other hand, seminal plasma suppresses cryo‐capacitation induced by thawing stress. On the basis of these findings, we designed a novel protocol of AI using frozen‐thawed boar sperm.     

Genetic structure in Atlantic brown trout (Salmo trutta L.) populations in the Iberian peninsula: evidence from mitochondrial and nuclear DNA analysis

Brown trout (Salmo trutta L.) was sampled in rivers belonging to three different Spanish basins in order to analyse the distribution of genetic variability. The genetic analysis was performed by using two systems and techniques: nuclear DNA was screened through random amplified polymorphic DNAs (screening 2 × 10⁵ bp of the whole genome), and mitochondrial DNA (mtDNA) through sequencing of the hypervariable control region. Genetic distances between the populations were similar using either analysis although some differences arise. For example, some populations of the Tajo basin were very close through nuclear analysis but more distant using mtDNA. Differences between the two DNA sources could be the result of a different evolutionary rate, and the fact that mtDNA is maternally transmitted and differences in sex migration rates will influence the patterns of genetic variation between the transmitted DNAs. Total variation was partitioned using amova showing a clear subdivision among basins although intrapopulation variation remained as high as 62%. A correspondence analysis defined the differences in a three‐dimensional way, clustering the populations according to their common basin. When mtDNA was sequenced, higher variability was noted in the segment between 400 and 600bp of the whole D‐loop sequence, suggesting that these 200bp improved the analysis of the variability more than sequencing the t‐RNA ends of the control region. A comparison was made between the t‐RNA^Pro ends of the 10 populations screened here and the rest of the published sequences found in the literature, leading to a concentration of these populations in group IV which includes all trouts which originate in the Atlantic. The analyses performed suggest that a high genetic variability is present in all populations and that although there has been a probable interference from stocked strains introduced to increase population density, this was only detectable through the variance between rivers which reflect different policies according to the region where the basin is located. However, the genetic analysis using the two approaches allows the control of the natural populations avoiding a loss of their genetic potential.     

Genetic structure in Atlantic brown trout (Salmo trutta L.) populations in the Iberian peninsula: evidence from mitochondrial and nuclear DNA analysis

Brown trout (Salmo trutta L.) was sampled in rivers belonging to three different Spanish basins in order to analyse the distribution of genetic variability. The genetic analysis was performed by using two systems and techniques: nuclear DNA was screened through random amplified polymorphic DNAs (screening 2 × 10⁵ bp of the whole genome), and mitochondrial DNA (mtDNA) through sequencing of the hypervariable control region. Genetic distances between the populations were similar using either analysis although some differences arise. For example, some populations of the Tajo basin were very close through nuclear analysis but more distant using mtDNA. Differences between the two DNA sources could be the result of a different evolutionary rate, and the fact that mtDNA is maternally transmitted and differences in sex migration rates will influence the patterns of genetic variation between the transmitted DNAs. Total variation was partitioned using amova showing a clear subdivision among basins although intrapopulation variation remained as high as 62%. A correspondence analysis defined the differences in a three‐dimensional way, clustering the populations according to their common basin. When mtDNA was sequenced, higher variability was noted in the segment between 400 and 600 bp of the whole D‐loop sequence, suggesting that these 200 bp improved the analysis of the variability more than sequencing the t‐RNA ends of the control region. A comparison was made between the t‐RNA^Pro ends of the 10 populations screened here and the rest of the published sequences found in the literature, leading to a concentration of these populations in group IV which includes all trouts which originate in the Atlantic. The analyses performed suggest that a high genetic variability is present in all populations and that although there has been a probable interference from stocked strains introduced to increase population density, this was only detectable through the variance between rivers which reflect different policies according to the region where the basin is located. However, the genetic analysis using the two approaches allows the control of the natural populations avoiding a loss of their genetic potential.     

BF, HP, DQB and DRB are associated with haemolytic complement activity, acute phase protein reaction and antibody response in the pig

In order to examine the loci factor B (BF), C3, corticotropin releasing hormone (CRH), DQB, DRB, haptoglobin (HP) and transforming growth factor beta 1 (TGFB1) for association with traits of humoral, specific and unspecific defence F2-animals of a porcine resource family were genotyped at single nucleotide polymorphisms within these loci. Haemolytic complement activity in the alternative and classical pathway, C3c and haptoglobin serum concentration and antibody titres were determined immediately prior and at days 4 and 10 after vaccinations against Mycoplasma hyopneumoniae (Mh), Aujeszky's disease virus, and porcine reproductive and respiratory syndrome virus at 6, 14 and 16 weeks of age, respectively. Analysis of variance revealed association of BF, HP and DRB with C3c serum concentration. The trend of haemolytic complement activity and C3c serum concentration during the experiment was affected by the interaction of DQB genotype and time of measurement. Association with antibody titres were found for BF, DQB and DRB. Results of the mixed model analyses were confirmed by quantitative transmission disequilibrium test that showed linkage and association with antibody titres, complement activity and acute phase reaction at certain times of measurement. The findings promote the importance of the candidate genes for humoral mechanisms of unspecific and specific defence that provide natural resistance against many pathogens.     

Dogs and pigs are transport hosts of Necator americanus: Molecular evidence for a zoonotic mechanism of human hookworm transmission in Ghana

Hookworm infection (Necator americanus and Ancylostoma spp) causes significant morbidity in resource‐limited countries. Dog and pig ownership is associated with human infection, although the mechanism through which animals increase risk remains unknown. We first confirmed this association in Kintampo North, Ghana, using a retrospective analysis and serology, followed by a prospective molecular study of animal faeces. As a proxy of exposure to dog faeces, we analysed immunoreactivity of human serum to the zoonotic nematode Toxocara canis. Anti‐Toxocara antibodies were present in 62% of samples (n = 89), and reactivity was associated with dog ownership. A subsequent prospective study revealed that 43% of dog and 56% of pig faecal samples contained hookworm eggs by microscopy. PCR analysis confirmed the presence of N. americanus DNA in 47% of samples from dogs and 56% pig samples. Nematode larvae were successfully cultured from samples collected from 36 dogs and seven pigs. These results demonstrate that dogs and pigs have a likely role in the transmission of N. americanus in endemic communities.     

Reproduction in Javanese sheep: evidence for a gene with large effect on ovulation rate and litter size

Three breeds of Javanese sheep are described briefly and data suggesting the segregation of a gene with large effect on ovulation rate and litter size are presented. The three breeds are Javanese Thin Tail (JTT), Javanese Fat Tail (JFT) and Semarang (SEM), the last possibly a substrain of JTT. All three breeds have mean mature ewe weights under 30 kg. Ovulation rate and litter size did not differ significantly among the three; all had litter sizes of up to 4 or 5 with a mean for mature ewes of approximately 2. Ovulation rate ranged from 1 to 5 and had an average within-breed repeatability of .8 within season and .65 between seasons. Within-breed repeatability of litter size was .35 +/- .06. Prenatal survival in pregnant ewes with two, three and four or more ovulations averaged 93, 88 and 86% over two seasons. Dams that had at least one ovulation rate or litter size record greater than or equal to 3 produced two groups of daughters in approximately equal numbers: one group with many records greater than or equal to 3 and mean ovulation rate and litter size of 2.73 and 2.31, respectively, and one group with ovulation rates and litter sizes of 1 or 2 and corresponding means of 1.39 and 1.38. Dams with ovulation rate or litter size records of only 1 or 2 produced daughters in which over 90% had records of only 1 or 2. Estimated heritabilities for the mean of approximately three ovulation rate or litter size records from these daughter-dam comparisons exceeded .7. These results suggest segregation of a Booroola-type gene, one copy of which increases ovulation rate by about 1.3 and litter size by .9 to 1.0. Relationships between duration of estrus and ovulation rate, and between timing of release of luteinizing hormone and number of eggs shed, resemble the pattern in Booroola Merino more closely than that in Finnish Landrace or Romanov, supporting the hypothesis of a major gene.     

Serum thymidine kinase activity in dogs with malignant lymphoma: a potent marker for prognosis and monitoring the disease

Serum thymidine kinase (sTK) activity was evaluated as a tumor marker for canine malignant lymphoma (ML). The objective was to investigate if sTK, as in humans, could be used as a prognostic marker for survival time in dogs with ML and if sTK could identify early signs of progression of disease in treated dogs. Serum samples from 52 dogs with ML were tested for initial TK activity. Samples from 21 normal dogs and 25 dogs with nonhematologic neoplasms were used for comparison. Forty-four dogs with ML were treated. Serum TK activity was measured in treated dogs before each treatment and every 4 weeks thereafter until relapse. Dogs with ML had 2-180 times higher TK activity (TK 5-900 U/L) than normal dogs (TK <7 U/L) based on the mean + 2 standard deviations. In the group of other neoplasms, only 2 dogs had a moderate increase (6.4 and 7.5 U/L) compared with the controls. Mean sTK activities in the dogs with ML that had gone into complete remission (CR) were not significantly different from activities in healthy controls (P = .68). Mean sTK at least 3 weeks before and at the time of relapse was significantly higher than activity measured at CR (P < .0001). Dogs with ML that initially had sTK >30 U/L had significantly shorter survival times (P < .0001). Furthermore, sTK activity reflected the clinical staging of ML. Measuring sTK can be used as a powerful objective tumor marker for prognosis and for predicting relapse before recurrence of clinically detectable disease in dogs with ML undergoing chemotherapy.     

Associations between the c-myc proto-oncogene and carcass quality traits in the pig: evidence for epistasis with the Ryr1-gene

c-myc is an ubiquitous nuclear phosphoprotein involved in the regulation of cell-growth, differentiation and apoptosis. Higher nuclear levels of c-myc block adipogenesis and trigger the onset of myogenesis and folliculogenesis. The c-myc proto-oncogene has the potential of a mediator between second messengers such as calcium and cAMP and gene expression. As both are involved in stress-susceptibility and carcass quality in the pig, this study investigates associations between c-myc genotypes and carcass quality traits as well as interactions with the Ryr1 genotype in this species. An association between c-myc and carcass fat traits was evident, but did not reach genome-wide significance levels. Significance niveaus, explained variance and mean differences between the homozygotes of the Ryr1-gene which clearly decreased from the AA-genotype to the BB-genotype of c-myc are indicating a gene interaction between both genes. Mean differences in lean percentage (expressed in SD) decreased by 89.2% in ME × PI and by 86.3% in WS × PI. The existing but weakly developed significances of the association between c-myc and carcass fat traits are discussed under the aspect of epistatic nullification.     

Biomarkers for the activation of calcium metabolism in dairy cows: elevation of tartrate-resistant acid phosphatase activity by lowering dietary cation-anion difference is associated with the prevention of milk fever

In our previous study, it was demonstrated that the administration of anion salts, which slightly lower the dietary cation-anion difference (DCAD), in the prepartum period is safe and effective for preventing milk fever in multiparous cows. In the present study, several biomarkers, which might show activation of Ca metabolism, were analyzed using stored samples in the previous study to investigate the mechanism of the preventive effect on milk fever by lowering DCAD. Changes in bone-specific alkaline phosphatase activity, osteocalcin and insulin-like growth factor I concentrations in serum were almost the same among the three groups of multiparous cows with or without the oral administration of anion salts, while the levels of these serum biomarkers in the group of primiparous cows (heifer group) were much higher compared with those in the three multiparous groups throughout the experimental period. Urinary deoxypyridinoline excretion was not a useful biomarker for dairy cows because it hardly changed during the peripartum period in all groups. However, serum tartrate-resistant acid phosphatase (TRAP) activity, which is known as a biomarker of osteoclast activity, was well associated with the administration of anion salts lowering DCAD because among the three multiparous groups, only the group of multiparous cows fed the anion salts (anion group) showed an increased level, which rose to the level in the heifer group, and was markedly higher than those in the other control groups of multiparous cows. The increased activity of serum TRAP in the anion group suggested that Ca in the plasma pool was mobilized smoothly from bone-bound Ca via mature osteoclasts at parturition, which might be due to prior activation under mild acidosis induced by slightly lowering DCAD. Therefore, TRAP was the best biomarker to monitor the activation of Ca metabolism in dairy cows fed anion salts.     

Intraruminal administration of milk in the calf as a model for ruminal drinking: Morphological and enzymatical changes in the jejunal mucosa

In order to develop a calf model for studying the syndrome of ruminal drinking (RD) in veal calves, three dual-fistulated calves were used to test the effect of intraruminal administration of milk replacer on the jejunal mucosa. Biopsies of the proximal jejunal mucosa were taken through a jejunal fistula and the mucosal morphology and the activities of two brush border enzymes, lactase and alkaline phosphatase, were determined.Means of villus length and brush border enzyme activities decreased during the period of intraruminal administration of milk. The hyperplastic villus atrophy in this model was similar to that found in chronic RD patients in previous studies. This could not be associated with isolation of pathogenic micro-organisms from the faeces and is probably the consequence of the intraruminal milk feeding procedure itself.Clinical recovery from the signs of RD occurred rapidly after intraruminal administration of milk ceased and was followed by restoration of villus length and brush border enzyme activities 3–4 weeks later.     

Interaction of bovine immunoglobulin gamma 2 (IgG2) with staphylococcal protein A: evidence for IgG2a and IgG2b sub-subclasses in the bovine

The reactivity of bovine IgG with protein A is confusing with respect to which of the bovine IgG class and subclasses are reactive. We have, therefore, re-examined the interaction of bovine immunoglobulins with protein A. The results presented in this paper indicated that at pH 8.0 protein A binds only immunoglobulin of the IgG2 subclass. The bound IgG2 can be readily recovered from an immobilized protein A column at pH 5.0. Furthermore, the antigenic IgG2 eluted demonstrated two charged species which could readily be separated by ion-exchange chromatography. These results indicate that IgG2 in the bovine exists in two sub-subclasses, IgG2a and IgG2b. The two sub-subclasses of IgG2 could be rapidly isolated with a good yield in two-steps namely protein A affinity chromatography followed by ion exchange chromatography.