Oxidation-reduction potentials and spectral properties of some cytochromes from Thiobacillus versutus (A2)

Antibodies raised against rat hepatic epoxide hydrolase (EC 3.3.2.3) and glutathione S-transferases (EC 2.5.1.18) B, C and E were used to determine the presence and localizations of these epoxide-metabolizing enzymes in testes of sexually immature and mature Wistar and Holtzman rats. Unlabeled antibody peroxidase-antiperoxidase staining for each enzyme was readily detected in rat testes at the light microscopic level. Although significant strain-related differences were not apparent, staining intensity for certain enzymes differed markedly between Leydig cells and seminiferous tubules. Leydig cells of immature and mature rats were stained much more intensely for epoxide hydrolase and glutathione S-transferases B and E than were seminiferous tubules, whereas Sertoli cells, spermatogonia, spermatocytes and spermatids, as well as Leydig cells, were stained intensely by the anti-glutathione S-transferase C. Age-related differences in staining for glutathione S-transferase B were not obvious, while the anti-glutathione S-transferase C stained seminiferous tubules more intensely in immature rats, and antibodies to epoxide hydrolase and glutathione S-transferases C and E stained Leydig cells much more intensely in mature rats. These observations thus demonstrate that testes of both sexually immature and mature rats contain epoxide hydrolase and glutathione S-transferases. Except for glutathione S-transferase C in immature rats, Leydig cells appear to contain much higher levels of enzymes than do seminiferous tubules. During sexual maturation, the testicular level of glutathione S-transferase B appears to remain constant, while levels of epoxide hydrolase and glutathione S-transferases C and E increase within Leydig cells and the level of glutathione S-transferase C decreases within seminiferous tubules.     

Immunohistochemical localization of two angiotensin I-converting isoenzymes in the reproductive tract of the male rabbit

The male reproductive tract contains two different isoenzymes of angiotensin I-converting enzyme (ACE), i.e., pulmonary and testicular ACE. The present study shows selectively the cellular distribution of the ACE isoenzymes in the reproductive tract of male rabbit, using indirect immunofluorescence or immunoperoxidase methods. Testicular ACE was found in the seminiferous tubules of the testes in spermatocytes containing mature spermatids, and in spermatids within the epididymal tubular lumen in sexually mature, but not in immature, rabbits. Epididymal tubular cells contained pulmonary ACE. In the young rabbit, epididymal tissue contained more ACE than that in adult rabbit, since ACE was observed in principal cells in addition to basal cells. In mature rabbit, ACE was observed in basal cells only. Strong staining for pulmonary ACE was observed in cells of the vas deferens in both young and adult rabbit. Therefore, synthesis of epididymal ACE, unlike the testicular isoenzyme, was not stimulated by sexual maturation. Enzymatically active ACE in seminal fluid corresponds to the pulmonary isoenzyme. The present study indicates that this seminal fluid ACE may originate from cells of the epididymal tubules, particularly those of the vas deferens. Endothelial cells of blood vessels lying in the interstitium of both testicular and epididymal tissue contained the pulmonary isoenzyme.     

Changes in the activity of acid glycosidases during posthatch development and regression after light reduction of Japanese quail testes and epididymides

In this study specific activities of four acid glycosidases: beta-N-acethylhexosaminidase (beta-HEX), beta-galactosidase (beta-GAL), alpha- and beta-mannosidase (alpha- and beta-MAN) were investigated in Japanese quail testes and epididymides during posthatch development and regression after light reduction. The specific activity of testicular beta-HEX and beta-GAL increased steadily during posthatch development and assumed maximum values for testes weighing 200-400 mg, when numerous spermatocytes appear in the testes of quail, and then decreased slowly. These enzymes showed much higher specific activity after 15 days of light reduction, and decreased to the control level after 30 days. Activity of alpha- and beta-MAN remained rather constant during testicular development and involution. The epididymal activity of the acid glycosidases was very low in immature individuals, whereas in sexually mature birds it was found to increase several-fold. Short photoperiod resulted in a decreased activity of these enzymes after 30 days to the values found in immature birds. A marked increase in the activity of acid glycosidases in the epididymides of sexually mature animals and a decrease in this activity during epididymidal regression indicate that these enzymes take part in reproductive processes. It is concluded that the activities of beta-HEX, beta-GAL, alpha- and beta-MAN in the development and regression of Japanese quail testes and epididymides change similarly as in mammals.     

Changes in the testicular acid and alkaline phosphatase activities at different durations following prostatectomy: a correlative study with spermatogenesis.

The purpose of this investigation was to make a correlative study between spermatogenesis and testicular acid and alkaline phosphatase activities in mature prostatectomized animals at different post-operative periods. The results demonstrate that there was a significant augmentation in the activity of testicular acid and alkaline phosphatases subsequent to 14 and 21 days of prostatectomy. A parallel quantitative study of spermatogenesis at stage VII of the seminiferous cycle, namely, type A spermatogonia (ASg), preleptotene spermatocytes (pLSc), mid-pachytene spermatocytes (mPSc) and step 7 spermatids (7sd), revealed that there was a significant reduction in the number of step 7 spermatids after 14 and 21 days. No change was observed in the above testicular enzymes and spermatogenesis after 7 days of prostatectomy. Therefore, it is concluded that prostatectomy can alter the above testicular enzyme activities and spermatogenesis in chronic prostatectomized state.     

Localization of epoxide-metablozing enzymes in rat testis

Antibodies raised against rat hepatic epoxide hydrolase (EC 3.3.2.3) and glutathione S-transferases (EC 2.5.1.18) B, C and E were used to determine the presence and localizations of these epoxide-metabolizing enzymes in testes of sexually immature and mature Wistar and Holtzman rats. Unlabeled antibody peroxidase-antiperoxidase staining for each enzyme was readily detected in rat testes at the light microscopic level. Although significant strain-related differences were not apparent, staining intensity for certain enzymes differed markedly between Leydig cells and seminiferous tubules. Leydig cells of immature and mature rats were stained much intensely for epoxide hydrolase and glutathione S-transferase B and E than were seminiferous tubules, whereas Sertoli cells, spermatogonia, spermatocytes and spermatids, as well as Leydig cells, were stained intensely by the anti-glutathione S-transferase C. Age-related differences in staining for glutathione S-transferase B were not obvious, while the anti-glutathione S-transferase C stained seminiferous tubules more intensely in immature rats, and antibodies to expoxide hydrolase and glutathione S-transferases C and E stained Leydig cells much more intensely in mature rats. These observations thus demonstrate that testes of both sexually immature and mature rats contain epoxide hydrolase and glutathione S-transferases. Except for glutathione S-transferase C in immature rats, Leydig cells appear to contain much higher levels of enzymes than do seminiferous tubules. During sexual maturation, the testicular level of glutathione S-transferase B appears to remain constant, while levels of epoxide hydrolase and glutathione S-transferases C and E increase within Leydig cells and the level of glutathione S-transferase C decreases within seminiferous tubules.     

Size changes of protamine 1 mRNA provide a molecular marker to monitor spermatogenesis in wild-type and mutant mice

We utilized a cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine, mouse protamine 1 (MP1), to detect the presence of several classes of differentiating germ cells in testicular extracts from wild-type and male sterile mutant mice. This assay is based on the changes in the poly (A) length of MP1-mRNA during spermatogenesis. Testicular extracts of sexually mature CD-1 mice contain a heterogeneous population of protamine-1 mRNA ranging in length from 450 to 580 nucleotides. When the protamine-1 probe was hybridized to testicular RNA preparations from 16- to 20-day-old animals, no MP1-mRNA was detected. Twenty-four-day-old mice contain only the 580-nucleotide form of MP1-mRNA. This size class of protamine mRNA is also present in purified populations of round spermatids, whereas elongating spermatids and residual bodies contain mRNAs ranging from 450 to 580 nucleotides in length, which are identical in size to those present in the testes of sexually mature animals. When the protamine cDNA probe was used to examine the progression of spermiogenesis in three male sterile mouse mutants, blind sterile (bs), quaking (qk) and testicular feminization (Tfm), the results demonstrated that each mutant is pathologically distinct. Analysis of the bs mutant revealed a diminution in the amount of both size classes of MP1-mRNA, in agreement with the cytological reports of reduced numbers of haploid spermatogenic cells in these animals. The presence of both size classes of protamine mRNA in the qk mutant indicates that germ-cell differentiation has proceeded at least to the step-12 spermatid in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical evidence for the specific localization of aldose reductase in rat Sertoli cells

Evidence that the enzyme aldose reductase (AR) is specifically located in Sertoli cells is presented by means of an established immunocytochemical technique and with a variety of approaches. By staining tissue sections, the enzyme was shown to be present in Sertoli cells at birth and the intensity of the immunocytochemical stain increased by 5 days of age to that found in the testes of older rats. By means of enzyme dispersion of mature testes; the culture of enriched Sertoli cell preparations from the testes of immature rats; and the collection of newly released testicular spermatozoa in rete testis fluid, it was shown that immunoreactive AR was not present in any testicular cell type except the Sertoli cell. The significance of the specific localization in Sertoli cells of a principal enzyme concerned in the sorbitol or polyol pathway for the conversion of aldose sugars to their corresponding ketoses is discussed.     

Correlation of the level of β-citryl-l-glutamic acid with spermatogenesis in rat testes

β-Citryl-l-glutamic acid, which is known to be highly concentrated in the brains of immature animals, is preferentially localized in the testes of various adult animals, including mammals, amphibians and fish, mainly in the germinal cells. In young rats, the citrylglutamate concentration increases with age and coincides with the development of late spermatocytes into early spermatids. Rats with seminiferous tubule failure induced by ductuli efferentes ligation and experimental cryptorchidism are infertile as a result of germ cell depletion, especially spermatocytes and early spermatids. In these animals, the testicular citrylglutamate content was much lower than in normal testes.     

Immunohistochemical study of calmodulin in developing mouse testis

The purpose of this study was to determine the localization of calmodulin in the developing mouse testis by the indirect immunoperoxidase method. In addition, the amount of calmodulin in pachytene spermatocytes, spermatids, and residual bodies isolated from the mouse testis and epididymal spermatozoa was quantitated by the adenylate cyclase activation assay and by enzyme immunoassay. The relative levels of calmodulin in the developing mouse testis and in the isolated testicular germ cells were confirmed by western transfer staining. The level of immunoreactive calmodulin was very low in the testes from immature animals. In testes from the mature mouse, calmodulin was found to be localized in spermatocytes and spermatids, but was not found in spermatogonia, Sertoli cells, and interstitial cells. By contrast, immunochemical staining of tubulin was extremely intense in Sertoli cells. Biochemical determinations also showed that pachytene spermatocytes, round spermatids, spermatozoa, and residual bodies contained 14.9 micrograms, 15.8 micrograms, 2.3 micrograms and 5.2 micrograms of calmodulin per mg of protein, respectively. Both the immunochemical and the biochemical studies revealed that levels of calmodulin were high in the spermatocytes and in the round spermatids, as compared to the level in spermatozoa. This fact strongly suggests that the large amount of calmodulin in mammalian testes may be associated primarily with meiotic divisions and/or spermatogenesis.     

Correlation of the level of β-citryl-l-glutamic acid with spermatogenesis in rat testes

β-Citryl-l-glutamic acid, which is known to be highly concentrated in the brains of immature animals, is preferentially localized in the testes of various adult animals, including mammals, amphibians and fish, mainly in the germinal cells. In young rats, the citrylglutamate concentration increases with age and coincides with the development of late spermatocytes into early spermatids. Rats with seminiferous tubule failure induced by ductuli efferentes ligation and experimental cryptorchidism are infertile as a result of germ cell depletion, especially spermatocytes and early spermatids. In these animals, the testicular citrylglutamate content was much lower than in normal testes.     

DNA polymerases in mouse spermatogenic cells separated by sedimentation velocity.

Activity levels of DNA polymerase alpha and DNA polymerase beta have been measured in mouse spermatogenic cells separated by sedimentation velocity. Testes from prepuberal (17 day old) and sexually mature mice were dissociated and separated by unit gravity sedimentation into 6 populations of cells. Phase contrast microscopy and [3H]thymidine labeling kinetics revealed that at least 85% of the cells in fraction A were pachytene-stage primary spermatocytes, fraction B was enriched for primary spermatocytes and round spermatids, fraction C contained spermatogonia and/or pre-leptotene primary spermatocytes and later stages of spermatids (no spermatids were present in fraction C from the testes of 17 day old mice) and fractions D to F contained mixed populations of cells, many in later stages of spermiogenesis. When expressed as activity in 10(6) cells or as a specific activity, fractions A, B, and C from mature animals population initially loaded onto the gradient while fractions D, E and F had activity levels similar to or below the population of dissociated cells. The ratio of activity between the DNA polymerases was constant in fractions A, B, and C, but in fractions D, E, and F, the ratio decreased due to a more rapid decline of activity of polymerase alpha. A comparison of activity levels in fraction C from prepuberal and sexually mature mice revealed an increase in DNA polymerase alpha activity and a decrease in the activity of DNA polymerase beta in the cells from the 17 day old animals.     

The development of phosphomonesterases in the testes of prepuberal chicks.

1. The biochemical development and histochemical localisation of phosphomonoesterases in the testes of prepuberal chicks have been studied. 2. Maximum acid phosphatase activity was observed at 12 weeks with a decrease in enzyme activity after this age, whereas alkaline phosphatase activity fluctuated with age. 3. Acid phosphatase activity in chicks was similar to that of the cockerel in being tartarate-insensitive. 4. There was a low level of significant correlation between acid phosphatase activity and testes weight. 5. Both alkaline and acid phosphatase activities were observed in the basement membrane of the seminiferous tubules, and acid phosphatase activity also in the various spermatogenic elements. 6. The results suggest that acid phosphatase is more involved in spermatogenesis, and more widely distributed than alkaline phosphatase in testicular tissue during testicular development.     

Effect of depot medroxyprogesterone acetate and testosterone ananthate on the testis of albino rats: ultrastructural and biochemical studies.

Male albino rats were treated with depot medroxyprogesterone acetate (1 mg/animal/day) + testosterone ananthate (100 micrograms/100 g body weight/day) for 30 and 60 days. After 30 days of treatment, all the testicular enzymes like beta-glucuronidase, hyaluronidase, sorbitol dehydrogenase, lactate dehydrogenase, acid and alkaline phosphatase, registered non-significant decrease in their values. Fifty percent of the treated animals achieved sterility after 30 days of treatment. After 60 days of treatment the testis showed degenerative changes in Golgi phase and late spermatids. Changes in the Golgi phase spermatids were related with degeneration of the nuclear membrane. Changes in the late phase spermatids included mitochondrial hypertrophy of the midpieces, membrane lysis, absence of cristae and degeneration of annulus leading to detachment of tail. Cytoplasm of luminal area displayed hypertrophied mitochondria devoid of cristae, prominent appearance of Golgi bodies, intense lysosomal activity and ample vacuolation. Tail fragments of degenerated spermatids prevailed in luminal cytoplasm. Except for beta-glucuronidase which registered a significant decrease, levels of all the other testicular enzymes, viz. hyaluronidase, lactate dehydrogenase, sorbitol dehydrogenase, acid phosphatase and alkaline phosphatase were within their control limits. The ultrastructural and biochemical changes are correlated.     

Zinc metabolism in testicular feminization and surgical cryptorchid testes in rats

Rats with testicular feminization (Tfm) had been reported to have a testis specific zinc deficiency. In this report it is demonstrated that this organ specific zinc deficiency was not corrected by intraperitoneal zinc administration. Normal littermates on the other hand showed a positive testicular response to zinc administration. The increased testicular zinc level in control animals returned to normal 1 week after the zinc treatment probably due to the fast turnover of this element in the testis. Not only surgically induced cryptorchidism but also surgical cryptorchidism and epididymodeferentectomy (to simulate Tfm conditions in normal adult rats) caused a drastic reduction in testicular zinc level. Unlike in Tfm rats, however, the decrease in zinc content in operated animals was not accompanied by a corresponding decrease in alkaline phosphatase activity. Zinc concentration and alkaline phosphatase activity in plasma or other organs were not affected by the surgical procedure. The testicular copper content in the operated animals was higher than that of the unoperated controls.     

Characterization of the testicular cell types present in the rat by in vivo 31P magnetic resonance spectroscopy.

Testes of vitamin A-deficient Wistar rats before and after vitamin A replacement, of rats irradiated in utero, and of control rats were investigated by in vivo 31P magnetic resonance (MR) spectroscopy. The testicular phosphomonoester/ATP (PM/ATP) ratio ranged from 0.79 +/- 0.05 for testes that contained only interstitial tissue and Sertoli cells to 1.64 +/- 0.04 for testes in which spermatocytes were the most advanced cell types present. When new generations of spermatids entered the seminiferous epithelium, this ratio decreased. The testicular phosphodiester/ATP (PD/ATP) ratio amounted to 0.16 +/- 0.06 for testes in which Sertoli cells, spermatogonia, or spermatocytes were the most advanced cell type present. When new generations of spermatids entered the seminiferous epithelium, the PD/ATP ratio rapidly increased and finally reached a value of 0.71 +/- 0.06 for fully developed testes. Taken together, specific patterns of the PM/ATP ratio, the PD/ATP ratio, and pH were obtained that were correlated to the presence of spermatogonia, spermatocytes, round spermatids, and elongated spermatids or to the absence of spermatogenic cells. Hence, a good impression of the status of the seminiferous epithelium in the rat can be obtained by in vivo 31P MR spectroscopy.     

Location of enzymes of androgen and estrogen biosynthesis in the testis of the ground squirrel (Citellus lateralis)

The intratesticular localization of enzymes of androgen and estrogen biosynthesis was studied in the ground squirrel (Citellus lateralis). In mature animals, interstitium and tubules were isolated by manual dissection. Microsomes were prepared and enzymes assayed by analysis of product formation after incubation with appropriate 3H-labeled substrates. In the immature testis, tubules and interstitium are not readily separable; thus, distribution was inferred after analysis of whole testicular microsomes from control, follicle-stimulating hormone (FSH)-treated, and luteinizing hormone (LH)-treated animals. To verify the cellular composition of tissues and the status of steroidogenic organelles in Leydig and Sertoli cells, samples were also analyzed by light and electron microscopy. In mature squirrels, enzymes of androgen biosynthesis were concentrated in the interstitium; however, levels present in the tubules were sufficient to account for a substantial fraction of whole testicular activity (1/3 to 1/5). By contrast, virtually all of the testicular aromatase was accounted for by that in the seminiferous tubules. The purity of these fractions was checked by light microscopy; they showed little cross-contamination. In whole testicular microsomes of immature squirrels, androgen biosynthetic enzymes had a much lower specific activity than in mature animals; however, the opposite was true for aromatase, its activity being approximately 5-fold higher in prepubertal animals. Luteinizing hormone treatment markedly stimulated hydroxylase and lyase but not aromatase. Luteinizing hormone also induced an increase in Leydig cell size and a dramatic proliferation of smooth endoplasmic reticulum. These changes were correlated with increased serum testosterone. As shown previously in rats, 3 beta-hydroxysteroid dehydrogenase was independent of LH control. Follicle-stimulating hormone had no effect on any of the enzymes studied, but induced some increase of agranular reticulum in Sertoli cells. Results from immature squirrels thus corroborate data from mature animals, showing a predominant interstitial location of androgen biosynthetic enzymes. While we cannot explain the absence of FSH stimulation of aromatase activity, the data do not refute the findings in mature animals showing a predominant tubular location of this enzyme. We conclude that the distribution of steroidogenic enzymes in the testis of squirrels differs in several important respects from rats, although both are members of the order Rodentia.     

Changes in mitochondrial protein composition during testicular differentiation in mouse and bull

The morphology of testicular mitochondria changes markedly during spermatogenesis from a form normally seen in somatic cells to a “germ cell” form in which the matrix is diffuse and vacuolated and finally to a form with a condensed matrix seen in spermatozoa. Colloidal silica gel gradients and high-resolution, two-dimensional gel electrophoresis were used to define the changes in density and polypeptide composition that occur in testicular mitochondria during spermatogenesis. Similar densities were observed for mitochondria isolated from the same bovine or murine tissue, but mitochondria from different tissues usually had different densities. Mitochondria from testis of calf, bull, or sexually mature mouse had densities of 1.06 gm/cm³ while liver mitochondria were more dense, having a density of 1.09 gm/cm³. “Somatic-type” testicular mitochondria from calf and “germ cell-type” mitochondria from sexually mature mouse or bull had similar densities, 1.06 gm/cm³, while the density of mitochondria from ejaculated spermatozoa differed, ρ = 1.08 gm/cm³. Analysis of polypeptide composition of somatic and germ cell mitochondria from testes of prepuberal and sexually mature animals and from highly enriched populations of pachytene primary spermatocytes and round spermatids revealed a staining pattern of mitochondrial proteins that was markedly constant throughout development with most polypeptides being conserved and a few specific spots changing in abundance. Marked differences were detected, however, when mitochondria from ejaculated spermatozoa were compared with those from testis with many minor and major polypeptides missing and several new polypeptides present at high concentration.     

Immature versus mature dura mater: II. Differential expression of genes important to calvarial reossification

The ability of immature animals and newborns to orchestrate successful calvarial reossification is well described. This capacity is markedly attenuated in mature animals and in humans greater than 2 years of age. Previous studies have implicated the dura mater as critical to successful calvarial reossification. The authors have previously reported that immature, but not mature, dural tissues are capable of elaborating a high expression of osteogenic growth factors and extracellular matrix molecules. These findings led to the hypothesis that a differential expression of osteogenic growth factors and extracellular matrix molecules by immature and mature dural tissues may be responsible for the clinically observed phenotypes (i.e., immature animals reossify calvarial defects; mature animals do not). This study continues to explore the hypothesis through an analysis of transforming growth factor (TGF)-beta3, collagen type III, and alkaline phosphatase mRNA expression. Northern blot analysis of total RNA isolated from freshly harvested immature (n = 60) and mature (n = 10) dural tissues demonstrated a greater than three-fold, 18-fold, and nine-fold increase in TGF-beta3, collagen type III, and alkaline phosphatase mRNA expression, respectively, in immature dural tissues as compared with mature dural tissues. Additionally, dural cell cultures derived from immature (n = 60) and mature dura mater (n = 10) were stained for alkaline phosphatase activity to identify the presence of osteoblast-like cells. Alkaline phosphatase staining of immature dural cells revealed a significant increase in the number of alkaline phosphatase-positive cells as compared with mature dural tissues (p < 0.001). In addition to providing osteogenic humoral factors (i.e., growth factors and extracellular matrix molecules), this finding suggests that immature, but not mature, dura mater may provide cellular elements (i.e., osteoblasts) that augment successful calvarial reossification. These studies support the hypothesis that elaboration of osteogenic growth factors (i.e., TGF-beta33) and extracellular matrix molecules (i.e., collagen type III and alkaline phosphatase) by immature, but not mature, dural tissues may be critical for successful calvarial reossification. In addition, these studies suggest for the first time that immature dural tissues may provide cellular elements (i.e., osteoblasts) to augment this process.     

Dye permeability and alkaline phosphatase activity of testicular capillaries in the postnatal rat

Summary Staining of testicular and epididymal tissues after intravenous, intraperitoneal or subcutaneous administration of a number of dyes was investigated in rats at different stages of postnatal development. After light green injections heavy staining of both testis and epididymis was visible to the naked eye in neonatal animals up to the age of 10 days, while in rats over 15 days old no appreciable staining of the testis could be seen, although the caput epididymis was strongly coloured. From 3–8 hours after subcutaneous acriflavine administration, the nuclei in the blood vessel walls of the testis, as well as the nuclei in the rete testis, tubuli efferentes and caput epididymis, fluoresced in all age groups. The nuclei of the interstitial and tubular cells were stained intensely until the age of 5 days. Thereafter the intensity gradually diminished until the age of 20 days, when no nuclear fluorescence was visible in the seminiferous tubules and even the interstitial nuclei fluoresced weakly or not at all.The histochemical alkaline phosphatase activity of the testicular capillaries was studied by Gomori's method, using fresh and postfixed cryostat sections from postnatal rat testes. The testicular capillaries exhibited appreciable activity at the age of 10 days.On the basis of the present and previous observations on the permeability of the testicular capillaries, the existence of a blood-testis barrier in the puberal and adult rat testis is suggested.Development of the blood-testis barrier and the alkaline phosphatase activity of the testicular capillaries are suggested to reflect general vascular maturation at the beginning of puberty in the rat.Supported by grants from Yrjö Jahnsson's Foundation and P. O. Klingendahl Foundation.