Cathepsin C activity as related to some histochemical substrates.
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Animals↗
Biomedical subjects
T Vanha-Perttula
Publications and source records attributed to T Vanha-Perttula.
A dipeptide naphthylamidase from hog kidney.
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Amidohydrolases↗
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.
Acrosome↗
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.
Animals↗
Biochemical studies on dipeptidyl peptidases I to IV of the human placenta.
Homogenates of human term placentae were utilized to demonstrate the presence of four dipeptidyl peptidases (DPP I to IV) after sequential fractionations with gel filtration and anion-exchange chromatography. DPP I was assayed with SerTyrNA as substrate and showed the characteristics of a thiol-dependent serine enzyme with optimum at pH 4.5 and a molecular weight of 210,000. DPP II, analysed with LysAlaNA as substrate, had an optimum at pH 5.5 and a molecular weight of 130,000 with no dependence on thiol groups or divalent ions. DPP III was studied with ArgArgNA as substrate. It was optimally hydrolysed at pH 8.8 and had a molecular weight of 84,000. The enzyme was highly suppressed by chelating agents but could be reactivated by Co2+ and some other divalent ions. DPP IV, with GlyProNA as substrate, displayed an optimum at pH 8.2. The fractionations displayed multiple forms of the enzyme, possibly indicating the presence of proto- and polymeric DPP IV activities. The protomer had a molecular weight of 260,000 and showed no dependence on thiol groups but was sensitive to Zn2+.
Chromatography, Gel↗
Purification and partial characterization of human placental particle-bound aminopeptidase.
Particle-bound aminopeptidase was purified from the human term placenta by separation of the particles from the soluble fraction by centrifugation and solubilization of the particle fraction with 0.5 per cent (v/v) Triton X-100 followed by CM-cellulose chromatography, Sepharose 6B gel filtration, DE-cellulose and Con A-Sepharose 4B affinity chromatography. Polyacrylamide gel electrophoresis indicated enzymatic and protein homogeneity of the purified enzyme. The enzyme seemed to be a glycoprotein with a molecular weight of 320 000. The purified enzyme did not endure freezing but was fairly stable at 4 degrees C. At 60 degrees C more than half and at 65 degrees C all enzyme activity disappeared in 15 min. The pH dependence of the purified enzyme varied between 6.75 and 7.5, and 6.0 and 6.5, depending on the substrate used. The enzyme hydrolysed most rapidly LeuNA (Km 0.095 +/- 0.008 mmol) followed by beta-naphthylamide derivatives of alanine (Km 0.222 +/- 0.02 mmol), arginine (Km 0.041 +/- 0.002 mmol), lysine (Km 0.084 +/- 0.005 mmol), methionine (Km 0.085 +/- 0.004 mmol) and cystine (Km 0.048 +/- 0.004 mmol). Thus LeuNA was the most specific among the substrates for the enzyme. The purification process revealed, however, that CysNA was the most selective substrate for the particulate enzyme, which readily hydrolysed also Leu-p-na (Km 0.865 +/- 0.023 mmol) and Bz-Cys-p-na (Km 0.248 +/- 0.034 mmol).
Amino Acids↗
Acid phosphatase of the rat epididymis. III. Histochemical and biochemical responses in experimental conditions.
The specific activities of three acid phosphatases were followed in the epididymis of growing rats. All activities were constantly rising but revealed two steeper parts probably corresponding to the increase of androgen secretion and the arrival of spermatozoa in the epididymis. Simultaneously an increase in the histochemical staining was obtained in all epididymal segments. Both biochemical and histochemical studies showed that castration reduced the activity of all acid phosphatases and this could be restored with testosterone-estradiol treatment. Estradiol had no effect on the activities in the castrated animal, but in the normal animal it caused an elevation of Enzyme I activity. In the histochemical study estradiol seemed to restore the acid phosphatase activity. Enzyme I reacted slower than the others to both castration and hormone treatment. Increased elimination caused by ligation of the epididymis caused a reduction in the activity of all enzymes within four days. It was greatest in the area of the corpus. This study, however, rendered no further elucidation for the assumption that acid phosphatases participate in the elimination of excessive and defective spermatozoa.
Acid Phosphatase↗
Acid phosphatases in the human testis after prolonged estrogen treatment.
The testes of 2 autopsied adult men and 6 subjects, suffering from prostatic carcinoma, were analysed for acid phosphatase activities, Two of the prostatic patients had been receiving estrogen treatment at least for a year and had completely regressed testes. Testes of other subjects contained well-defined tubules with different spermatogenic cells in abundance. The total acid phosphatase activity, assayed in the homogenate, showed a marked reduction in the testes of estrogen-treated subjects. Enzymes were separated by cellulose chromatography or by gel filtration combined with cellulose chromatography. Three activity peaks were resolved by the former and four by the latter technique, when homogenates of the control testes were used. In contrast, two to three strongly reduced activities could be discerned from the testes of estrogen-treated subjects. The specific activity of each enzyme after fractionation was compared between control and regressed testes. No difference was observed in the activities of enzyme I. Enzyme II was markedly reduced in the regressed testes, but was clearly present. Enzymes III and IV were either totally absent or showed a marked reduction in the regressed testes. It is suggested that a correlation exists between the destruction of germ cells, as a consequence of estrogen treatment, and the marked reduction in the activity of enzymes III and IV.
Acid Phosphatase↗
Further studies on human testicular acid phosphatases.
Four acid phosphatases were separated by gel filtration on Sepharose 6B and subsequent chromatography on DE-52 cellulose from human homogenate. The enzymes differed from each other in substrate preference, Km-values, modifier characteristics and molecular weights. The evidence obtained confirms that also in the human testis acid phosphatases of multiple molecular forms are present. Some of these may be specific for the testicular tissue.
Acid Phosphatase↗
Ultrastructure of a tail stump sperm defect in an Ayrshire bull.
The sperm cells in the ejaculate of a sterile Ayrshire bull were studied by light microscopy as well as scanning and transmission electron microscopy. The spermatozoa showed an almost total lack of tails, whereas the heads appeared morphologically normal. The cytoplasmic residue at the caudal end of the head contained numerous membrane structures as well as mitochondria and incomplete elements of the proximal centriole and striated column. The basic disturbance appears to be a hereditary defect in the spermiogenesis.
Animals↗
Distribution of lectin binding in rat testis and epididymis.
Seven lectins (PNA, RCA I, SBA, Con A, WGA, UEA I, DBA) conjugated with rhodamine were employed to analyse the staining pattern of glycoproteins with varying sugar residues in the testis and epididymis of adult Wistar rats. Some lectins (UEA I, SBA, DBA) gave rather specific staining of the mature acrosome, while others (PNA, RCA I) showed affinity for the early stages of acrosome formation or had a wide affinity for germinal and non-germinal cells and structures (Con A, WGA). In the epididymis the sperm mass had a homogeneous staining reaction with some lectins (PNA, RCA I, Con A, WGA, DBA) which also showed a rather strong reaction on the epithelial surface. It was concluded that this reaction is at least partially due to the secretory products synthetized by principal, apical, narrow and light cells of the epididymal epithelium. Some differences in the staining pattern of these cells were recorded indicating specialization of the cells for the production of distinct glycoproteins. The staining pattern of the interstitial and intertubular compartment of the testis and epididymis was also recorded.
Animals↗
Lectin staining of rat testis and epididymis: effect of cyproterone acetate and testosterone.
Control and castrated (C) adult rats were treated with cyproterone acetate (CA), testosterone (T) or their combination (CA + T) for 15 days and the affinity of the testicular and epididymal tissue for seven rhodamine-conjugated lectins was analysed in fluorescence microscopy. In the testis CA caused the appearance of degenerating cells in the basal part of the seminiferous epithelium. These cells (spermatogonia, early spermatocytes) appear to be the most vulnerable germinal cells to anti-androgen treatment and this effect could be prevented by simultaneous administration of T. Castration caused a marked reduction in lectin staining of principal and light cells of distal caput (Dcp) and distal cauda (Dcd) epididymidis accompanied by the disappearance of the intratubular sperm mass. In castrated animals CA caused a moderate and T as well as CA + T a marked increase in the stainability of these cells and the appearance of homogeneous secretory material in the tubules. This material gave moderate or strong affinity for most of the lectins. Some minor differences were found in the staining pattern of the cells and the secretory material in Dcp and Dcd as well as after different treatments. This is consistent with local and qualitative differences in the epididymal secretory and absorptive activity, which can be further modified by the hormonal milieu.
Animals↗
Effect of castration on lectin staining in rat epididymis.
Seven rhodamine-conjugated lectins (PNA, RCA I, Con A, WGA, UEA I, SBA, DBA) were used to follow the staining pattern of the rat epididymis at different time points after castration. The affinity of the intratubular sperm mass for the lectins increased rapidly with concurrent augmentation of the staining in the principal cells but a decline of the reaction in the light cells. The light cells showed some differences in their response to castration, which was compatible with secretory/absorptive activity in caput and absorptive activity in cauda. The active phase of sperm mass destruction and epithelial involution was accompanied by local accumulation of macrophages and round cells, which also acquired an increased affinity for most of the lectins. It is concluded that the androgen-deprived epididymis is rapidly programmed for autolytic and phagocytic processes, which include the destruction of macromolecules including glycoproteins of the spermatozoa.
Animals↗
Incorporation of selenium-75 into seminal plasma and spermatozoa of the bull.
75Se was given intravenously into 5 bulls. Multiple blood and semen samples were taken and during slaughter 5, 10, 15, 20 and 80 days later samples of various reproductive and other organs were collected. After injection, 75Se in blood reached a peak at 6 h followed by a rapid decline. The label was mainly found in serum with very low levels in erythrocytes. Initially the serum 75Se was bound to a macromolecule with a mw of 80 kDa, but later a larger molecule (100 kDa) was observed. In semen 75Se was first mainly found in seminal plasma, where a plateau level was reached at 5 d followed by a gradual decline after 12 d. The total semen level, however, increased after 14 d and this increase was due to a rapid appearance of the label in spermatozoa. The sperm 75Se level reached a plateau at 20 d and remained high until 40 days, after which a gradual decline ensued. The seminal plasma 75Se eluted in gel filtration coincident with glutathione peroxidase. The highest levels of 75Se were found in the kidney followed by seminal vesicles and testicles. The seminal vesicle secretion was particularly rich in 75Se and its fractionation resembled that of the seminal plasma. 75Se appeared in the epididymal caput within 5 days and passed through the epididymis in 20 days. It is concluded that 75Se is actively incorporated in the bull seminal vesicles into GSH-Px, while in the testis it is incorporated into a structural sperm protein during spermatogenesis.
Animals↗
Human seminal plasma cadmium: comparison with fertility and smoking habits.
Cadmium, selenium and zinc were determined in seminal plasma and serum of 64 men by atomic absorption spectrometry (AAS). The mean (+/- SD) cadmium concentrations in seminal plasma and serum were 0.22 +/- 0.22 micrograms and 0.28 +/- 0.10 micrograms, respectively, but they did not correlate with each other. Smokers (n = 31) had significantly (p less than 0.01) higher serum cadmium concentrations than non-smokers (n = 31). Also seminal plasma cadmium in smokers was elevated, but a significant difference to non-smokers was only found if more than 20 cigarettes were consumed daily. No differences were found in semen quality and fertility between smokers and non-smokers. The seminal plasma cadmium had no correlation to selenium or zinc which, however, displayed a positive correlation (r = 0.852, p less than 0.001) to each other. It is concluded that smoking increases the exposure to cadmium. Although no obvious reproductive suppression was observed, heavy smoking may possibly enhance toxic effects in men under other detrimental exposures.
Adult↗
Acid phosphatases of the rat epididymis. II. Biochemical characteristics, subcellular distribution and histochemical localization.
After separation of three epididymal acid phosphatases their biochemical properties were differently studied. With appropriate substrate and inhibitor selection the distribution of the enzymes in different segments as well as the subcellular fractions of the rat epididymis was also demonstrated. The same biochemical differences were also utilized in the histochemical localization of the enzymes. It was found that Enzyme I had a pH-optimum at 5.0, a molecular weight of 97 000 and Km-constant of 0.901 mM. It was highly sensitive to tartrate and fluoride and it was localized in lysosomes as well as in the epididymal spermatozoa. Enzyme II had an optimum at pH 5.7, a molecular weight of 67 000 and Km-constant of 0.806 mM. It was also inhibited by fluoride but more resistant to tartrate. Its subcellular site was also particulate, but it was also found in the epididymal fluid. Enzyme III had an optimum at pH 5.2, a molecular weight of 135 000 and Km-constant of 0.685 m. It was resistant to low concentrations of fluoride and tartrate but sensitive to heavy metal ions. The enzyme was soluble and it behaved incoherently in thermal inactivation. All enzymes revealed the highest activity in the thin middle segments of the epididymis. Histochemical naphthol substrates gave a diffuse reaction in the epididymal epithelial cells. With the lead salt methods glycerophosphates and p-nitrophenylphosphate gave somewhat different results depending on their specificity as substrates for the epididymal enzymes. Both substrates gave a strong reaction supranuclearly in the Golgi area of the chief cells. This activity was inhibited by tartrate and was most probably due to Enzyme I. The epididymal corpus and cauda showed additionally a very strong apical activity in the chief cells with p-nitrophenylphosphate. This activity was resitant to tartrate but sensitive to fluoride. It was concluded that this enzyme represents Enzyme II activity. Similar activity was also found in the dissolving "holocrine" cells of the corpus and the cauda. The activity of the soluble Enzyme III could not be revealed with the present methods and the spermatozoa in the tubular lumina remained unstained.
Acid Phosphatase↗