Epididymal Bull Research Articles

Electron microscopic study of the secretion process in bovine reproductive organs.

The origin and mechanism of the secretion of membrane-bound particles in bovine seminal plasma were studied with transmission (TEM) and scanning (SEM) electron microscopy of the epididymis, vas deferens, ampulla, and seminal vesicle of adult bulls. In the SEM study, all these organs were found to contain apical protrusions in the lining of the epithelial cells. Eventually the protrusions became detached and formed secretory bodies within the lumina of these organs. In the epididymis, the TEM study disclosed a granular and rather homogeneous content in the protrusions and bodies, whereas in the vas deferens they contained dilated cisternae of smooth endoplasmic reticulum. In the ampulla and seminal vesicle, the formation of the apical protrusions was associated with an accumulation of membrane-bound vesicles. These vesicles were found to be released from the storage bodies into the secretory fluid of the lumen. Both could be harvested from isolated seminal vesicle secretions by Percoll gradient centrifugation. It was concluded that various parts of the bovine reproductive organs discharge their secretory products at least partly by an apocrine mechanism. The membrane-bound particles in the seminal plasma, however, appear to be mainly derived from the ampulla and seminal vesicle.

Regulation of oxidative phosphorylation in mitochondria of epididymal bull spermatozoa

The regulation of oxidative phosphorylation was studied with digitonin-treated epididymal bull spermatozoa in which mitochondria are directly accessible to low molecular compounds in the extracellular medium. Due to the high extramitochondrial ATPase activity in this cell preparation, it was possible to stimulate respiration to a small extent only by added hexokinase in the presence of glucose and adenine nucleotides. Added pyruvate kinase plus phospho enolpyruvate, however, strongly suppressed the respiration. Under these conditions, the respiration was found to depend on the extramitochondrial [ ATP] [ ADP] ratio in the range of 1–100. The contribution of the adenine nucleotide translocator to this dependence was determined by titration with the irreversible inhibitor carboxyatractyloside in the presence of ADP. Using lactate plus malate as substrate, the active state respiration was controlled to about 30% by the translocator, whereas 12 and 4% were determined in the presence of l-glycerol-3-phosphate and malate alone, respectively. In order to compare the results with those for intact cells, the adenine nucleotide patterns were determined in intact and digitonin-treated spermatozoa under conditions of controlled respiration in the presence of vanadate and carboxyatractyloside, respectively. About 21% of total cellular adenine nucleotides were found in digitonintreated cells representing the mitochondrial compartment. While allowing for the intramitochondrial amount of adenine nucleotides, the cytosolic [ ATP] [ ADP] ratio was estimated to be 6-times higher than the mitochondrial ratio in intact cells. It is concluded from the data presented that the principal mechanism by which oxidative phosphorylation in sperm mitochondria is regulated via the extramitochondrial [ ATP] [ ADP] ratio is the same as that demonstrated for other isolated mitochondria.

Molecular cloning of the cDNA for androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis.

cDNA clones coding for two closely related androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis were selected by screening an epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the glycoproteins. The largest clone of 956 nucleotides provided coding information for a protein of 246 amino acids of which the first 19 residues comprise a putative signal peptide sequence which when cleaved would produce a mature protein of 227 residues and a molecular mass of 26 kDa. Confirmation of the identity of the clone was provided by a match between the amino acid sequence predicted from the cDNA sequence and the actual amino acid sequence determined for a tryptic peptide fragment of one of the pure glycoproteins. It is probable that the primary amino acid sequence of the two glycoproteins is identical. Northern blot and slot-blot analysis revealed that the mRNA for the glycoproteins is approximately 1250 nucleotides long and that the concentration of the mRNA in the epididymis is androgen-dependent. The glycoproteins and their mRNAs were unique to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from skin, brain, liver, kidney, heart, skeletal muscle and testis. Cross-reacting proteins of slightly smaller apparent molecular mass were detected in extracts of mouse and guinea-pig epididymis, but not rabbit or bull epididymis. Comparison with existing protein data bases revealed that the epididymal glycoproteins display significant sequence homology with yeast carboxypeptidase Y.

Molecular cloning of the cDNA for two major androgen-dependent secretory proteins of 18.5 kilodaltons synthesized by the rat epididymis.

cDNA clones representing two closely related androgen-dependent secretory proteins of 18.5 kDa were selected by screening a rat epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the 18.5-kDa proteins. The entire amino acid sequence of the 18.5-kDa secretory proteins and a putative signal sequence of 18 amino acids was derived from 682 base pairs of the nucleotide sequence of overlapping cDNA clones. Confirmation of the identity of the cDNA clones was obtained by matching a partial amino acid sequence obtained for the N terminus of the pure protein with that of the sequence derived from the nucleotide code of the cDNA. Evidence is presented that the difference between the two closely related proteins may be associated with differential post-translational modification of the N terminus of the protein following cleavage of the signal sequence. Northern blot analysis revealed that the mRNA for the proteins is approximately 850 nucleotides long and that the concentration of the mRNA in the tissue is androgen-dependent. The proteins and their mRNAs were restricted to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from the skin, brain, liver, kidney, heart, skeletal muscle, and testis. With the exception of a weak cross-reaction with mouse epididymis, the proteins were not detected by Western blots of extracts of guinea pig, rabbit, or bull epididymis. The two proteins account for a substantial proportion of the total protein in epididymal luminal fluid and become incorporated as components of the sperm plasma membrane where they may play a specific role in the post-testicular phase of sperm development.

Motility of undiluted bull epididymal spermatozoa collected by micropuncture.

Motility of whole undiluted semen collected from different regions of the bull epididymis by micropuncture was determined by examining a droplet under paraffin oil. Bull caudal spermatozoa showed vigorous motility in undiluted semen. This motility was less in samples collected from nearer the testis: samples from the distal caput showed weak but detectable motility while those from the proximal and mid-caput were completely quiescent. Motility of spermatozoa from the distal caput and the proximal corpus was markedly increased after incubation at 34 or 37 degrees C for 1 h, but was depressed by incubation at 25 degrees C. Similar but smaller effects were observed with spermatozoa collected from the mid-corpus and the mid-cauda, except that motility of spermatozoa from the mid-corpus was reduced after incubation for 1 h at 37 degrees C. The inhibitory effect of low temperature was completely reversible. Incubation of caudal spermatozoa under anaerobic conditions produced partial and reversible inhibition of sperm motility. The results suggested that bull epididymal spermatozoa may not be completely quiescent in their native environment as previously assumed.

Lectin‐binding Pattern of Bull Testis and Epididymis

Seven rhodamine-conjugated lectins (PNA, RCA I, SBA, Con A, WGA, UEA I, DBA) were used to study the distribution of glycoproteins in the testis and epididymis of immature, juvenile, and adult bulls. A marked change was found in the staining pattern of the lectins in the seminiferous tubules during acrosomal development, and the Sertoli cells seemed to have a cyclic affinity for some of the lectins. The distribution of lectin staining in six regions of the bull epididymis showed some typical differences that were associated with the secretory and absorptive functions of the organ. Region 1 was characterized by strong surface and villous staining and a patchy reaction in the principal cells. Regions 2 and 3 showed a strongly reactive apical Golgi zone and secretory material. In regions 4 and 5, the Golgi zone was subapical but strongly reactive with most lectins, while in region 6 a weakly reactive apical Golgi zone was found. During sexual maturation, an increasing number of basal cells with a strong affinity for some lectins was found at the periphery of the epithelium in regions 2 to 6. These regions also had lectin-stained material along the basal border of the principal cells. These findings suggest that the basal cells may be active in the digestion of absorbed material and that they derive from the principal cells, which may be active in transporting absorbed material to them. The staining pattern of the spermatozoa changed during their transit through the epididymis. The degenerating cells in the testis and epididymal tubules also showed an altered affinity for the lectins.

The effect of substrate cycling on the ATP yield of sperm glycolysis.

The conversion of glucose to lactate via the Embden-Meyerhof pathway yields a maximum of 2 mol of ATP per mol of glucose degraded with lesser amounts of ATP obtained if metabolic intermediates are removed for biosynthesis or if substrate cycling occurs during carbohydrate degradation. Bull sperm are an ideal test system for a quantitative estimate of ATP yield because they contain enzymes necessary for potential substrate cycling and do not carry out significant biosynthesis. Experiments utilized specifically labeled glucose and fructose to test for substrate cycling between glucose in equilibrium glucose-6-PO4, fructose in equilibrium fructose-6-PO4, and fructose-6-PO4 in equilibrium bisphosphate. Flux through the pathway (low = less than or equal to 0.05; moderate = 0.1; high = greater than 0.5 mol of carbohydrate consumed per h/10(8) cells) was altered by changing incubation temperature and/or by adding metabolic effectors. The data (in situ rates of kinases and phosphatases and metabolite crossover plots) obtained under these conditions were used to establish if the generally accepted regulatory enzymes (hexokinase and phosphofructokinase) approach the overall flux through the glycolytic pathway, i.e. that of a classical kinetically limiting rate-determining step. In summary: (a) At low flux, the rates of phosphorylation greatly exceeded overall flux. This resulted in extensive substrate cycling at all points and a net ATP yield of less than or equal to 0 mol/mol of glucose initially phosphorylated. (b) At moderate flux, rate of hexokinase approached that of overall flux through the glycolytic pathway but "excessive" phosphofructokinase activity led to substrate cycling between fructose-6-PO4 and fructose 1,6-bisphosphate and resulted in a low net ATP yield (0-0.6 mol/mol of glucose). (c) At high flux, rates of phosphofructokinase and hexokinase approached that of the overall flux. Minimal substrate cycling occurred between fructose-6-PO4 and fructose 1,6-bisphosphate, and the net ATP yield approached 1.8 mol/mol of glucose. These experiments established that the theoretical stoichiometric ATP yield of the pathway was rarely achieved during these incubations because of extensive substrate cycling. Indeed, for cauda epididymal bull sperm isolated and incubated in vitro, the glycolytic pathway serves to generate lactate, an intermediate product that is further metabolized in the mitochondria to yield the ATP that is associated with degradation of exogenous carbohydrate.

Microphotometric analysis of protein-bound thiols and disulfides with an azogenic maleimide.

The chromogenic thiol reagent N-(4-aminophenyl)-maleimide (APM) was used to provide a sensitive procedure for the determination of protein-bound thiol group, both before and after reduction of disulfide bonds by tri-n-butylphosphine. The bound reagent is diazotized and coupled at low pH to N-(1-naphthyl)ethylenediamine to form an intense magenta label (lambda max 547 nm) with a high molar absorbancy (epsilon 547 = 49,800 cm2/mmol). The validity of absolute quantitation afforded by the method was shown by comparing the results with standard chemical analyses of thiol contents in a model system. Furthermore, the applicability of the procedure to biological objects was shown on isolated epididymal bull sperm heads.

The inhibitory activities of 2-acetamido-2,3-dideoxy- D-hex-2-enonolactones on 2-acetamido-2-deoxy-β- D-glucosidase

Treatment of 2-acetamido-2-deoxy- D-mannono-1,4-lactone with dicyclohexylamine in ethanolic solution afforded an unsaturated 1,4-lactone,2-acetamido-2,3-dideoxy- D-erythro-hex-2-enono-1,4-lactone (1), in good yield. 2-Acetamido-2,3-dideoxy- D- threo-hex-2-enono-1,4-lactone(2) was similarly prepared from 2-acetamido 2-deoxy- D-galactono-1,4-lactone. An unsaturated 1,5-Iactone, 2-acetamido-2,3-dideoxy- D- threo-hex-2-enono-I,5-Iactone (4),was obtained through the oxidation of 2-acetamido-2-deoxy-4,6- O-isopropylidene- D-gaIactopyranose with silver carbonate on Celite, followed by mild hydrolysis. The inhibitory activity of four isomeric 2-acetamido-2,3-dideoxy- D-hex-2-enonolactones [1,2,4, and 2-acetamido-2,3-dideoxy- D- erythro-hex-2-eneno-I,5-lactone (3)] was assayed against 2-acetamido-2-deoxy-,β- D-glucosidase from bull epididymis. Only the erythro lactones 1 and 3 are weak competitive inhibitors whereas the threo lactones 2 and 4 are practically inactive. The 1,4-lactone 1 inhibited 2-acetamido-2-deoxy-,β- D-glucosidase more strongly than the 1,5-Iactone 3. The lactones 1-4 were found to be quite stable in aqueous solution or under inhibitory assay conditions. In addition, two 2-acetamido-2-deoxy- D-glycals, 2-acetamido-1,5-anhydro 2-deoxy- D-arabino-hex-l-enitol (6) and 2-acetamido-I,5-anhydro-2-deoxy- D- lyxo hex-l-enitol (7) were tested; both are 10 times as active as 1.

Extraction of ATP-phosphohydrolase activity from epididymal bull sperm and ultrastructure of flagellar degradation

ATP-phosphohydrolase activity was solubilized from isolated epididymal bull spermatozoa flagella by a variety of extractants. The effects of isolation and chemical degradation on specific structural components within the flagellum were visualized electron microscopically. Low ionic strength solutions solubilized 30% of the ATPase activity and 10% of the protein content. High ionic strength KCl solubilized 50% of the ATPase activity and 10% of the protein content. Distinctions between mechanical effects of isolation and mechanical and chemical effects of extraction were determined. As demonstrated by transmission electron microscopy while 10 mM Tris/250 mM sucrose, pH 7.4, caused slight and generalized extraction of all flagellar components, alkaline 0.5 M KCl caused increased discontinuity and disorganization of flagellar structure as well as the solubilization of specific flagellar structures, including B and central pair microtubules, dynein, nexin, radial arms, secondary fibers and an extractable matrix from the outer dense fiber/satellite fiber complex. The A microtubules of the axoneme and the substructure of the outer dense fiber/satellite fiber complex exhibited similar structural stability. Simple criteria for reducing subjectivity in ultrastructural data are proposed.

Purification of N-acetyl β-D-hexosaminidase from bull epididymis by affinity chromatography

Purification of N-acetyl β-D-hexosaminidase from bull epididymis by affinity chromatography

The inhibitory activity of 2-acetamido-2-deoxy- D-gluconolactones and their isopropylidene derivatives on 2-acetamido-2-deoxy-β- D-glucosidase

2-Acetamido-2-deoxy- D-glucono-1,4-lactone ( 1) and 2-acetamido-2-deoxy- D-gluconic acid ( 3) have been examined for inhibitory activity against 2-acetamido-2-deoxy-β- D-glucosidase from bull epididymis. Crystalline 1 and 3 were compared with the known, crystalline 2-acetamido-2-deoxy- D-glucono-1,5-lactone ( 2), and a correlation of the activities of these compounds with various factors is presented. The inhibition constant of the 1,5-lactone 2 is lower (0.45μ M) than that (4.43μ M) of the 1,4-lactone 1. The effect of time is the opposite; whereas the activity of solutions of 2 decreases with time, solutions of 1 show an increase in inhibitory power, but both reach an equilibrium after 5 h. The free acid 3 exhibits no inhibitory activity. 2-Acetamido-2-deoxy-5,6- O-isopropylidene- D-glucono- 1,4-lactone ( 4) and 2-acetamido-2-deoxy-4,6- O-isopropylidene- D-glucono-1,5-lactone ( 5), which are appropriately protected to prevent conversion into the other lactone isomer, were also tested; 4 has 1/1000th the activity of 5.

Proteins in bull rete testis and cauda epididymidis plasma

1.1. The protein composition of bull (Bos taurus frontosus—male) epididymal plasma changes from the rete testis to the cauda epididymidis was examined.2.2. Bull rete testis and cauda epididymidis plasma contents from the aspect of specific protein composition and serum proteins were also considered.3.3. Chromatographic, electrophoretic and immunological evidence is presented for the presence of serum albumin and transferrin in bull rete testis and cauda epididymidis plasma.4.4. Changes in the protein composition in the bull epididymis support the hypothesis concerning both resorption and secretion of proteins in the epididymis.

Microheterogeneity of N-acetyl- -D-hexosaminidase of bull epididymis.

SummaryMost (90%) of the total activity of N-acetyl-β-D-hexosaminidase (EC 3.2.1.30) of bull epididymis homogenate was found in the soluble fraction. Electrophoresis of the soluble fraction on polyacrylamide gel (5.3%) at pH 9.5 separated the enzyme into as many as 12 enzymatically active bands. The bands were successfully stained on the gel using naphthol AS-BI N-acetyl-β-D-glucosaminide as substrate; freed naphthol being coupled with fast garnet GBC. Under similar conditions, human aortic N-acetyl-β-D-hexosaminidase was separated into two bands, namely the A and B forms.In both preparations, 1 mMN-acetyl-glucosaminolactone completely inhibited staining.Incubation of bull epididymal preparation with neuraminidase resulted in the disappearance of some of the fast-moving bands and the concomitant deeper staining of the slowmoving bands. Some changes in relative mobility were also observed.

Purification and separation of A and B forms of N-acetyl- -D-hexosaminidase from human aorta.

Abstract Human aorta has been shown to possess multiple forms of N-Acetyl-6-D-hexosaminidase (β-2-acetamido-2-deoxy-D-glucoside-acetamido-deoxyglucohydro-lase, EC 3.2, 1.30). The enzyme was separable, by gel electrophoresis, into 2 enzymatically active bands representing A and B forms. By gel electro-focussing, A and B forms were further subdivided into at least 5 and 8 bands, respectively. The B form consisted of 4 bands (B1) and 4 bands (B2), which were not inactivated at 50° for 3 hr. (at pH 4.4) in the presence of serum; whereas, the 5 bands found in A form were completely inactivated. All forms of the enzyme were active towards naphthol-AS-BI-N-acetyl-β-D-glucosaminide and the corresponding galactosaminide (about one-eighth of the hydrolysis rate of the former), suggesting each single enzyme acts on both substrates. The N-acetyl-hexosaminidases of bull epididymis, by comparison, were also found to be active towards both substrates and to possess 13 bands having pis more alkaline than those of the B f...

Bull spermatozoa as osmometers.

Summary. The response of epididymal bull spermatozoa to hypotonic media was investigated. Experiments in which spermatocrits were plotted versus the reciprocal of medium osmolality indicate that the swelling of the spermatozoa takes place linearly with a decrease in medium osmolality. The mean volume and area of swollen spermatozoa at critical medium osmolality (44 mosmol) were calculated as 126 μm3 and 145 μ2, respectively. Data available on the isotonic volume, area and water content of the spermatozoa indicate that, at critical medium tonicity, the cells have increased their volume 3·78 to 3·94 times without an apparent increase in area and that, according to conventional osmotic theory, they may be regarded as osmometers with a value of the Ponder's `R' correction factor in the range 0·81 to 0·91, i.e. they seem to deviate from the state of a `perfect osmometer' by 9 to 19%.

Water content, specific gravity and concentrations of electrolytes in bull spermatozoa.

Summary. The water content of epididymal bull spermatozoa in media of osmolalities corresponding to those of seminal plasma and to the fluids from the cauda epididymidis was determined by a gravimetricvolumetric method as 0·586 ± 0·0052 and 0·512 ±0·0086 ml per ml of cells, respectively. It was related to the ionic concentrations in mmol/ litre of spermatozoa determined by the conversion of the mean values of the major cations expressed as mequiv./litre, mg/100 g, or mg/100 ml, established by previous authors. The osmolalities of the intracellular fluids were also calculated. They were compared with the osmolalities of the extracellular fluids, the comparison making it possible to perform an introductory analysis of the osmotic-equilibrium states of bull spermatozoa. The calculated osmolality of the cytosol of epididymal spermatozoa was found to be about 40 mosmol less than that of the epididymal fluids. This discrepancy from the state of osmotic equilibrium is not serious when considering the errors, which are probably inherent in the values of both the ion and water-content determinations. The osmolality of the cytosol of ejaculated spermatozoa was calculated as 1·7 times greater than that established for seminal plasma. If the calculated value is reasonably correct, this should imply that, for the existence of identical concentrations of osmotically active particles on both sides of the cell membrane of spermatozoa suspended in seminal plasma, a fraction of the intracellular ions, amounting to about 38%, must be assumed to be 'bound'. Determinations of the water-content of the spermatozoa furnished data from which it was possible to calculate the specific gravity of the cells. This was compared with those actually established by gradient centrifugation in various media by different authors.

Immunoelectrophoretic study of esterase in blood serum, spermatozoa and genital tract fluids of bulls

SummaryImmunoelectrophoretic methods were used to observe the formation of precipitates with esterase activity in the blood serum, spermatozoa and the genital tract fluid of bulls, and also in the follicular fluid of cows. Fractions with esterase activity were found in the seminal vesicle fluid and the ampullar fluid of all investigated animals. Considerable individual differences in the formation of precipitates with esterase activity were found in the mentioned fluids. Ejaculated and ampullar spermatozoa, spermatozoa from the tail of the epididymis and the testicle fluid formed fractions with esterase activity only in some individuals. Esterase activity was not detected in the fluid of the tail of the epididymis of bulls nor in the follicular fluid of cows.

Cardiac glycoside binding and alkali cation transport in mature and epididymal bull spermatozoa.

Cardiac glycoside binding and alkali cation transport in mature and epididymal bull spermatozoa.

On the mechanism of sperm immobilization induced by normal sera and anti-semen sera

Experimental evidence indicates that the primary cause of the immobilization of bull spermatozoa induced by normal rabbit sera (NS) and rabbit antisera against bull semen (ABS) is a membrane change effected by the action on the cells of antibodies and complement. Owing to this change, which initiates a transient swelling of the sperms, presumably of a colloid osmotic nature, the sperms become permeable to ATP, albumin and generally also to Ficoll. The functional integrity of the sperm motor apparatus is, however, not destroyed: the flagellation of serumimmobilized sperms is restored by the addition of ATP to the medium. This ATP-reactivated flagellation is not confined to a physiological milieu: for short periods of time it may continue, in spite of a greatly reduced ionic strength. The agglutination patterns of the sperms which developed in the sera were of the same type as those observed by earlier investigators. Thus, in NS there was a predominant head-to-head agglutination, whereas in ABS both head-to-head and tail-to-tail agglutination were observed with no real preference for either type. NS and ABS induced no haemolysis of bovine erythrocytes. This indicates that no overlapping serological reaction occurs between bull spermatozoa and bovine erythrocytes. On the other hand, both kinds of sera were haemolytic against sheep erythrocytes. This haemolytic activity was abolished by a previous absorption of the sera at 0 °C with a preparation of horse kidney, which specifically binds Forssman antibodies. However, the absorbed sera were spermicidal. This indicates that the haemolysins were of the Forssman-antibody type, but that they were not identical with the sperm lysins. The haemolytic activity was greatlv reduced after absorption of the sera with epididymal bull spermatozoa pretreated with bull seminal plasma. On the other hand, absorptions with epididymal spermatozoa per se had no significant influence on the haemolytic activity. The meaning of these findings is discussed. Rabbit erythrocytes, like bovine erythrocytes, were unaffected by NS and ABS. However, they were rendered susceptible to haemolysis in both NS and ABS by previous treatment with bull seminal plasma, but not by treatment with extract from epididymal bull spermatozoa or with hyaluronidase. This suggests that the seminal plasma factor, which, by coating, sensitized the cells to subsequent complement haemolysis in NS and ABS, was a true constituent of the seminal plasma, and not present in this liquid due to leakage from the sperms. The spermicidal and haemolytic activities were extended into higher serum dilutions in the presence of agglutinin- and lysin-free guinea-pig serum. This indicates that the limiting factor in the lytic strength of NS and ABS per se was the complement concentration rather than the lysin concentration. Dextran sulphate ( M ̄ w ~ 2 × 10 6 ) in concentrations of 10 −5 to 10 −7 M blocked the spermiolytic and haemolytic activities of the sera. This fact seems to reflect the binding of the actual antibodies by the negatively charged polysaccharide.