Rat age-related benign prostate hyperplasia is concomitant with an increase in the secretion of low ramified α-glycosydic polysaccharides.

This work analysed the expression of prostate polysaccharides in rats with age-related benign prostatic hyperplasia (BPH) for a better understanding of the possible relationship between prostate polysaccharides secretion and BPH onset. For this, prostatic glands from 1 month-old, 3 months-old, 6 months-old and 12 months-old Sprague-Dawley rats were processed in order to identify their overall polysaccharide content. Additionally, serum testosterone was also determined. One-month old rats showed significantly (P < 0.05) lower testosterone levels (0.77 ng/mL±0.12 ng/mL) compared with the other groups, which showed no significant difference among them. PAS staining showed positive polysaccharides markings in both the prostatic lumen and inside of luminal prostatic cells in all groups. Semiquantitative analysis of intraluminal PAS showed that one month-old rats had significantly (P < 0.005) lower PAS intensity when compared with all other groups (100.0 ± 0.5, arbitrary units vs. 107.3 ± 0.6, arbitrary units in 3 months-old ones), whereas 12 months-old ones showed significantly (P < 0.005) higher values when compared with all other groups (133.6 ± 3.5, arbitrary units in 12 months-old rats vs. 108.6 ± 1.4, arbitrary units in 6 months-old ones). The PAS + content practically disappeared when tissues were pre-incubated with either α-amylase or amyloglucosidase, regardless of a previous incubation with proteinase K. Incubation of prostate extracts from 12 months-old rats for 2 h with α-amylase yielded a significantly higher amount of free glucose (1.47 nmol/mg protein±0.23 nmol/mg protein vs. 0.32 nmol/mg protein±0.01 nmol/mg protein in untreated extracts). Similar results were obtained when extracts were pre-incubated with amyloglucosidase. Contrarily, pre-incubation with N-glycosidase induced a significantly (P < 0.05), much lower increase of free glucose. Pre-treatment with proteinase K did not significantly modify these results, which indicate that BPH is related to an increase in the secretion of low ramified ductal α-glycosydic polysaccharides that were not protected against lysis by any type of protein protective core. These changes seem to not be related with concomitant variations in serum testosterone levels.

The onset of age-related benign prostatic hyperplasia is concomitant with increased serum and prostatic expression of VEGF in rats: Potential role of VEGF as a marker for early prostatic alterations.

The onset of age-related benign prostate hyperplasia (BPH) is linked with changes in the expression of specific prostatic chemokines. The aim of this work was to characterize those most relevant changes through the simultaneous analysis of 34 chemokines in both prostatic tissue and serum in rats at different ages with the aim to identify clinically workable parameters for the detection of early prostatic alterations. The study included 28 healthy Sprague-Dawley male rats that were distributed in four groups, 1 month-old (prepuberal; n = 7), 3 months-old (young; n = 7), 6 months-old (mature; n = 7) and 12 months-old (elder; n = 7). Chemokines were analyzed through a commercial mini-array system specially designed for rat tissues. Serum testosterone levels and prostatic histological status were also evaluated. Histological lesions indicative of BPH were detected in three mature rats and in all elder ones. Mini-arrays from prostatic tissue showed that young animals had an overall decreased expression of most of the analyzed chemokines when compared with prepuberal rats, with the exception of agrin, which showed a significant (P < 0.05) increase (100.0 ± 1.3, arbitrary units in prepuberal rats vs.148.2 ± 4.1, arbitrary units in young ones). Older animals showed further specific changes in 4 out 34 analyzed chemokines, namely agrin, platelet-derived growth factor (PDGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) and vascular endothelial growth factor (VEGF). Additionally, elder rats showed the lowest intensity levels of agrin combined with the highest ones for PDGF, TIMP1 and VEGF when compared with all other groups. Finally, a significant increase of serum VEGF was detected in elder, BPH-affected rats when compared with young ones. Results indicated that the onset of both rat puberty and BPH would be related with specific changes in the prostatic expression of chemokines such as VEGF. Otherwise, the observed changes in serum VEGF levels could suggest the future possible utilization of serum VEGF levels to detect early pathological prostatic processes.

The achievement of boar sperm in vitro capacitation is related to an increase of disrupted disulphide bonds and intracellular reactive oxygen species levels.

The aim of this work was to determine the relationship of intracellular reactive oxygen species (ROS) and the disulphide bonds established between sperm proteins with the achievement of capacitation in boar spermatozoa. With this purpose, spermatozoa were incubated in a specifically designed in vitro capacitation medium (CM) in the presence or absence of reduced glutathione (GSH). Incubation of boar spermatozoa in CM for 4 h significantly (p < 0.05) increased free cysteine residues, which is a marker of disrupted disulphide bonds, and also intracellular ROS levels. The addition of GSH to the medium prevented most capacitation-like changes in sperm motility, membrane lipid disorder, mitochondrial membrane potential, intracellular calcium levels and localization of tyrosine-phosphorylated proteins (pTyr), but not in tyrosine phosphorylation of P32. These effects were accompanied by the inhibition of the ability of sperm cells to trigger the acrosome exocytosis in response to progesterone. When GSH was added together with progesterone after 4 h of incubation, acrosome exocytosis was not altered, but the subsequent decrease in intracellular calcium observed in controls cells was inhibited. Furthermore, co-incubation of oocytes with spermatozoa previously incubated in CM in the presence of GSH for 4 h significantly (p < 0.05) increased the number of spermatozoa attached to the oocyte surface but decreased normal fertilization rates. Our results suggest that boar sperm capacitation is related to an increase in disrupted disulphide bonds and intracellular ROS levels and that both events are related to the regulation of hyperactivated motility, intracellular calcium dynamics, sperm binding ability to the oocyte and achievement of proper nuclear decondensation upon oocyte penetration.

Intracellular calcium movements of boar spermatozoa during 'in vitro' capacitation and subsequent acrosome exocytosis follow a multiple-storage place, extracellular calcium-dependent model.

This work analysed intracellular calcium stores of boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced acrosome exocytosis (IVAE). Intracellular calcium was analysed through two calcium markers with different physico-chemical properties, Fluo-3 and Rhod-5N. Indicative parameters of IVC and IVAE were also evaluated. Fluo-3 was located at both the midpiece and the whole head. Rhod-5N was present at the sperm head. This distribution did not change in any of the assayed conditions. Induction of IVC was concomitant with an increase in both head and midpiece Ca(2+) signals. Additionally, while IVC induction was concurrent with a significant (p < 0.05) increase in sperm membrane permeability, no significant changes were observed in O2 consumption and ATP levels. Incubation of boar spermatozoa in the absence of calcium showed a loss of both Ca(2+) labellings concomitantly with the sperm's inability to achieve IVC. The absence of extracellular calcium also induced a severe decrease in the percentage of spermatozoa exhibiting high mitochondrial membrane potential (hMMP). The IVAE was accompanied by a fast increase in both Ca(2+) signalling in control spermatozoa. These peaks were either not detected or much lessened in the absence of calcium. Remarkably, Fluo-3 marking at the midpiece increased after progesterone addition to sperm cells incubated in a medium without Ca(2+) . The simultaneous addition of progesterone with the calcium chelant EGTA inhibited IVAE, and this was accompanied by a significant (p < 0.05) decrease in the intensity of progesterone Ca(2+) -induced peak, O2 consumption and ATP levels. Our results suggest that boar spermatozoa present different calcium deposits with a dynamic equilibrium among them and with the extracellular environment. Additionally, the modulation role of the intracellular calcium in spermatozoa function seems to rely on its precise localization in boar spermatozoa.

'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.

"In vitro" capacitation and subsequent acrosome reaction are related to changes in the expression and location of midpiece actin and mitofusin-2 in boar spermatozoa.

The induction of "in vitro" capacitation (IVC) and subsequent, progesterone-induced "in vitro" acrosome reaction (IVAR) was concomitant with an increase in actin polymerization, also showing an increase in actin presence at the apical area of the midpiece. The presence of mitofusin-2, a protein involved in the regulation of the coordinated mitochondrial function, expanded from midpiece to the principal piece after IVC and IVAR. All of these results indicate that the increase of boar sperm mitochondrial activity during IVC and the first minutes of IVAR is concomitant with changes in the expression and location of both actin and mitofusin-2. Our results suggest that both actin and mitofusin-2 play important roles in the modulation of boar sperm mitochondrial function, both by originating changes in the protein membrane environment and by changes in the mitochondrial structure itself.

Freezing-thawing induces alterations in histone H1-DNA binding and the breaking of protein-DNA disulfide bonds in boar sperm.

The main aim of this work is to gain insight into the mechanisms by which freezing-thawing alters the nucleoprotein structure of boar sperm. For this purpose, the freezing-thawing-related changes of structure and location of histones-DNA domains in the boar sperm head were analyzed through Western blot and immunocytochemistry. Afterwards, it was analyzed whether freezing-thawing induced changes in tyrosine phosphorylation levels of both protamine 1 and histone H1, through Western blot analyses in samples previously subjected to immunoprecipitation. This analysis was completed with the determination of the changes induced by freezing-thawing on the overall levels of sperm-head disulfide bonds through analysis of free-cysteine radicals levels. Freezing-thawing induced significant changes in the histones-DNA structures, which were manifested in the appearance of a freezing-thawing-linked histone H1-DNA aggregate of about a 35-kDa band and in the spreading of histone H1-positive markings from the caudal area of the sperm head to more cranial zones. Freezing-thawing did not have any significant effect on the tyrosine phosphorylation levels of either protamine 1 or histone H1. However, thawed samples showed a significant (P < 0.05) increase in the free cysteine radical content (from 3.1 ± 0.5 nmol/µg protein in fresh samples to 6.7 ± 0.8 nmol/µg protein). In summary, our results suggest that freezing-thawing causes significant alterations in the nucleoprotein structure of boar sperm head by mechanism/s linked with the rupture of disulfide bonds among the DNA. These mechanisms seem to be unspecific, affecting both the protamines-DNA unions and the histones-DNA bonds in a similar way. Furthermore, results suggest that the boar-sperm nuclear structure is heterogeneous suggesting the existence of a zonated pattern, differing in their total DNA density and the compactness of the precise nucleoprotein structures present in each zone.

'In vitro' capacitation and acrosome reaction are concomitant with specific changes in mitochondrial activity in boar sperm: evidence for a nucleated mitochondrial activation and for the existence of a capacitation-sensitive subpopulational structure.

The main scope of this manuscript is to analyse the dynamics of mitochondrial activity in boar sperm subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced 'in vitro' acrosome reaction (IVAR). This was determined after analysis of the rhythm of O(2) consumption and concomitant changes in the mitochondria activity-specific JC-1 staining. Results showed that IVC, and especially IVAR, was concomitant with a peak in O(2) consumption (from 1.61 ± 0.08 nmol O(2)/min/10(7) viable sperm at 0 h of incubation to 2.62 ± 0.12 nmol O(2) /min/10(7) viable sperm after 5 min of IVAR induction). These results were accompanied by parallel changes in the mean intensity of JC-1 staining. Based on JC-1, mitochondrial activation followed a nucleated pattern, with specific, activation starting points at the midpiece from which mitochondrial activation was spread. Moreover, four separate sperm subpopulations were detected following the JC-1 orange-red/green ratio, and the observed changes in the mean JC-1 staining during IVC and IVAR were related to concomitant changes in both the orange-red/green JC-1 ratio and the percentage of sperm included in each subpopulation. All of these results indicate that IVC and the first minutes of IVAR are accompanied by a progressive increase in mitochondrial activity, which reached a peak coincidental with the achievement of IVAR. Moreover, results suggest the presence of separate sperm subpopulations, which show a different mitochondrial sensitivity to IVC and IVAR. Finally, mitochondrial activation, at least under JC-1 staining, seems to originate in concrete nucleation points at the midpiece, thus suggesting thus a well-coordinated pattern in boar-sperm mitochondrial activity modulation.

Differential effects of glucose and fructose on hexose metabolism in dog spermatozoa.

Incubation of dog spermatozoa with 10 mmol l(-1) glucose or fructose rapidly increased the intracellular content of glucose 6-phosphate and fructose 6-phosphate, although the effect of fructose was greater. These effects were correlated with increases in ATP, ribose 5-phosphate and glycogen contents, and in the rates of formation of L-lactate and CO2. In all cases, except for ATP and glycogen, the effect of fructose was greater than that of glucose. The total hexokinase activity of the crude extracts of dog spermatozoa was more sensitive to fructose than to glucose at lower concentrations (0.1-3.0 mmol l(-1)). Both monosaccharides induced a fast and intense increase in the overall tyrosine phosphorylation of dog spermatozoa, although their specific induced-phosphorylation patterns differed slightly. Glut 3 and Glut 5 hexose transporters were the main hexose transporters in dog spermatozoa; however, other possible SGLT family-related hexose transporters were also localized. These data indicate that, at concentrations from 1 mmol l(-1) to 10 mmol l(-1), fructose has a stronger effect than glucose on hexose metabolism of dog spermatozoa. These differences appear to be related to variations in the sensitivity of hexokinase activity. Moreover, the differential hexose metabolism induced by the two sugars had distinct effects on the function of dog spermatozoa, as revealed by the diverse patterns of tyrosine phosphorylation.

Role of glucose 6-phosphate in the translocation of glycogen synthase in rat hepatocytes.

Incubation of rat hepatocytes with glucose induces the translocation of glycogen synthase from soluble fractions to fractions which sediment at 10,000 g. Incubation of the cells with fructose, galactose, 2-deoxyglucose or 5-thioglucose, which activate glycogen synthase, also resulted in the translocation of the enzyme, whereas 3-O-methylglucose, 6-deoxyglucose and 1,5-anhydroglucitol, which do not cause the activation of the enzyme, were ineffective. Adenosine and carbonyl cyanide m-chlorophenylhydrazone, although activating glycogen synthase, did not induce its translocation. Mannoheptulose, which inhibits glucose phosphorylation, impaired the translocation of glycogen synthase induced by glucose. Furthermore, the extent of the translocation of the enzyme triggered by glucose and other sugars showed a high positive correlation with the intracellular concentration of glucose 6-phosphate. Microcystin, which blocks the activation of glycogen synthase by glucose, but not the accumulation of glucose 6-phosphate, did not affect the translocation of the enzyme. These results indicate that glucose 6-phosphate plays a role in the translocation of glycogen synthase in rat hepatocytes.