The effect of cryptorchidism on the quantitative histology, histochemistry and hydrolytic enzyme activity of the rat testis.

Cryptorchidism of the mature rat testis led to degeneration of the seminiferous tubules and changes in enzyme patterns and activities. Spermatogenic stages 1-4, containing pachytene primary spermatocytes in late meiotic prophase, and stage 5, containing recently formed round spermatids, were damaged by 48 h. Within 96 h stages showed a loss of germinal cells into the lumen and this was almost complete by 192 h. Acid phosphatase showed increased histochemical activity in the basal area of the seminiferous tubule up to 96 h of cryptorchidism, and at 192 h much of the activity was located in large lipidcontaining bodies within the remaining seminiferous epithelium. Total and free biochemical acid phosphatase decreased during cryptorchidism in parallel with cell loss; there were no significant changes in total cathepsin D activity but free enzyme activity was increased throughout the experimental period indicating increased lability of lysosomes in the Sertoli cell. Lactate dehydrogenase activity was mainly tubular but succinate dehydrogenase also showed interstitial activity. Lipoamide dehydrogenase (NADH) was found mainly in the interstitium. During cryptorchidism both lactate and succinate dehydrogenase activity decreased in the tubules parallel to the loss of germinal cells, whereas lipoamide dehydrogenase (NADH) activity increased in both interstitial and tubular areas. It is suggested that the initial lesion in the seminiferous epithelium, produced by cryptorchidism is in the Sertoli cell and that germ cell damage may result from reduced function of the Sertoli cell.     

Radioprotective properties of ATP and modification of acid phosphatase response after a lethal dose of whole body p(66MeV)/Be neutron radiation to BALB/c mice.

The intraperitoneal administration of exogenous ATP prior to a lethal dose (7 Gy) of whole body neutron irradiation increased the radioresistance of BALB/c mice. This radiation used the beam from a neutron therapy facility produced by the reaction p(66 MeV)/Be. Survival of the mice, determined 7 days post-irradiation as the endpoint, was increased from 26% to 86% by the action of the exogenous ATP. Furthermore, the response of acid phosphatase activity as an indicator of the acute radiation effects showed a marked augmentation in both tissues studied, testes and small intestine. The activity of the enzyme after neutron irradiation with prior administration of ATP showed smaller increases when compared with the increases observed after neutron irradiation alone. This implies that exogenous ATP reduces the effect of the lytic enzyme and, hence, damage. Finally, changes were observed in the activity of acid phosphatase in the testes and intestine with different concentrations of exogenous ATP. In both tissues there was a monotonic decrease in the activity of the enzyme with increase of the concentration of exogenous ATP administrated before radiation. These results reflect the protective ability of exogenous ATP as an adaptive defence mechanism to reduce radiation damage in normal tissues after a lethal dose of neutron radiation.     

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.     

Myoinositol biosynthesis by Sertoli cells, and levels of myoinositol biosynthetic enzymes in testis and epididymis.

Levels of glucose-6-phosphate cyclase (myoinsitol-1-phosphate synthase, EC 5.5.1.4) and myoinositol-1-phosphate phosphatase (myoinositol-1-phosphatase, EC 3.1.3.25) were determined in extracts of testes from10-, 20-, and 30-day-old rats, and in extracts of Sertoli cells, germinal cells, and epididymides. The specific activity of the cyclase was approximately 1/10th that of the phosphatase in all extracts found to contain either enzyme. Among cells in the testis examined, Sertoli cells had highest levels of enzymes required for inositol biosynthesis from glucose, while spermatocytes and round spermatids did not have detectable activity. Spermatozoa from the epididymis also had no detectable cyclase or phosphatase activity. In contrast, extracts of washed epididymides contained exceedingly high specific activities of these enzymes. Primary cultures of Sertoli cells, maintained in a chemically defined medium without added inositol, released inositol into the medium during three successive 24-h periods. The amounts released were greater in cells stimulated by dibutyryl cyclic AMP. Results were interpreted to indicate that inositol in the fluid of seminiferous tubules most probably originates from Sertoli cells, which synthesize inositol from glucose. Additional inositol in the fluid of epididymal tubules could readily be provided by metabolism of glucose by epididymal epithelial cells     

Ultrastructural localisation of alkaline phosphatase in the intertubular tissue of the testis in the domestic fowl

Alkaline phosphatase activity in the intertubular tissue of the testes of the domestic fowl was examined using an ultracytochemical technique based on the lead capture method. In the interstitial tissue, the Leydig cells, transitional cells and the fibroblasts displayed enzyme activity on their cell membranes. Vacuoles located in the transitional cells were lined by reaction products of enzyme activity, whereas the vacuoles representing extracted lipid droplets and present mainly in the Leydig cells were free of enzyme activity. In the peritubular tissue the cell processes of fibroblasts showed enzyme activity on the cell membranes and in pinocytotic vesicles. Cell processes lying adjacent to blood vessels showed pronounced activity. In the blood vessel itself some activity was present in the basement membrane and the endothelium. The surface of the red blood cell showed moderate activity. The possible role of alkaline phosphatase in the transfer of hormone from the Leydig cells to the seminiferous tubules and from the seminiferous tubules to the interstitium is discussed. The myoid cells and their processes were devoid of enzyme activity.     

Histochemical demonstration of certain testicular enzymes of the Indian desert gerbil Meriones hurrianae jerdon and their response to unilateral cryptorchidism

Summary Active male gerbils were rendered unilaterally cryptorchid and the enzymatic changes were studied after 48, 60 and 100 days. The histochemical observations revealed inhibition of alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine-tri-phosphatase and succinic dehydrogenase activities in the germinal elements of the seminiferous tubules. The non-specific esterase and succinic dehydrogenase activities increased in the Leydig cells to certain extent. The sudanophilic lipids increased in the Sertoli cells and the interstitial cells. An attempt has been made to approach the intricate problem of the spermatogenesis in order to understand the sequence of certain enzymatic activities in the gerbil testis in which spermatogenesis is suppressed when one of the testes is lodged inside the abdominal cavity.     

Modification of the effects of hyperthermia and neutron radiation on the activity of acid phosphatase in CaNT tumors.

Acid phosphatase activity was measured in implanted murine CaNT tumors of varying volumes. There is a clear monotonically increasing relation between acid phosphatase activity and tumor volume. Also the tumors were subjected to either induced artificial hypoxia or hyperthermia (41.0 degrees C) alone, or combined with neutron irradiation (3.8 Gy). Changes in the activity of this enzyme following radiation damage could reflect tissue damage associated with metabolic disturbances. The effect on enzyme activity after sequential hyperthermia and neutron irradiation is not synergistic, as is shown in the quantitative experimental data. This implies that the mechanisms of heat damage differ from that of neutron beam damage, as reflected by acid phosphatase activity. The CaNT tumor was also shown to be thermosensitive after administration of mitoxantrone. Finally, the role of exogenous ATP was shown to provide heat protection by modification of those thermal effects resulting in the activity of acid phosphatase. The augmentation of this hydrolytic enzyme probably represents initial metabolic damage in the tumor after different modalities of radiation alone, or combined with mitoxantrone and exogenous ATP.     

Immunolocalization and activity of aromatase in the bank vole testes.

A direct approach to identify the cellular source of P450 aromatase in the bank vole testes (seasonally breeding rodents) is the use of immunohistochemistry with a specific antibody that recognizes this enzyme. To confirm the presence of functional aromatase, its activity was measured in microsomal preparations of whole testes and of seminiferous tubules by means of biochemical assay with tritiated androstenedione. The assay was validated using increasing concentrations of both microsomal preparations. Immunoreactive aromatase was found in Leydig cells, Sertoli cells, and germ cells, especially in spermatocytes and spermatids. The aromatase activity was present in microsomal fractions of whole testis and seminiferous tubules. The immunolocalization of P450 aromatase and aromatase activity have been found as photoperiod-dependent.     

Testicular involution following optic enucleation

Summary The testes of adult male Syrian hamsters underwent involution within six weeks after optic enucleation. The diameter of the seminiferous tubules was 39% less than controls. Sertoli cells, spermatogonia, and primary spermatocytes were still present, but all steps of spermatids were completely absent from the involuted testes. Lipid droplets filled the Sertoli cell cytoplasm and often encroached upon the nucleus. Sertoli cells had sparse mitochondria and smooth endoplasmic reticulum, but Golgi cisternae were abundant. Typical SertoliSertoli junctions attached contiguous Sertoli cells. With lanthanum tracers it was demonstrated that these junctions were impenetrable; therefore, the bloodtestis barrier was deemed intact. Irregularly shaped protrusions often arose from the peritubular tissue and extended inward toward the seminiferous epithelium, often displacing the cytoplasm of the Sertoli cells and spermatogonia. The core of these protrusions consisted of irregular extensions of myoid cell cytoplasm surrounded by the myoid cells' basal lamina. External to the myoid cell basal lamina were bundles of collagen filaments with the basal lamina of the seminiferous epithelium forming the outermost layer of these protrusions. The apices of the Sertoli cells gave rise to numerous leaf-like processes that extended into and obliterated the lumen of the tubules. The Sertoli cell basal cytoplasm often contained phagocytized degenerating germ cells that appeared to give rise to the lipid droplets that filled the Sertoli cell cytoplasm. Acid phosphatase rich lysosome-like organelles were seen fusing with the degenerating germ cells and lipid droplets. The degenerating germ cells also were shown to contain acid phosphatase activity.     

An ultrastructural and histochemical study of autoimmune aspermatogenesis in the rat testis

Summary Immune aspermatogenesis was induced in young rats by the method of Freundet al. (1954) and testes were studied by electron microscopic and histochemical methods. At time of sacrifice the testes of several animals were markedly atrophic as demonstrated by reduction in weight. Sections of seminiferous tubules exhibited primarily profiles of Sertoli cells but germinal elements were sparse or absent. The ultrastructure of Sertoli cells appeared to be normal except for the presence of areas of dilated smooth endoplasmic reticulum and fragments of phagocytized germ cells in the cytoplasm.Light microscopic sections showed an apparent hyperplasia of intertubular tissue. Electron micrographs revealed a moderate to extreme vesiculation of the smooth endoplasmic reticulum in many interstitial cells. Macrophages and lymphocytes were often observed in contact with these cells.There was increased localization of non-specific esterase and acid phosphatase associated with lipid bodies of the tubules and in intertubular areas.This work was supported in part by USPHS Grant FR 5391.     

Studies on the toxic effect of cadmium in the rat. I. Testicular changes induced by a single subcutaneous injection of cadmium chloride.

Adult male rats of Wistar strain were subcutaneously injected with a single dose of 0.025 mM CdCl2/kg body weight. The autopsies were performed 2 and 12 weeks post cadmium injection. Experimental animals showed a normal gain of body weight while a marked lowering of the testes weight was observed. The lowering of testes weight of cadmium treated rats occured mainly due to the necrosis of seminiferous tubules, volume of which is markedly lower if compared to intact control rats. After 2 weeks of experiment a marked enlargement of the interstitial gland of the testis occured while after 12 weeks the gland is markedly lower if compared to control rats and to rats studied 2 weeks post cadmium injection. Histochemical reactions for lactic, succinic, alpha-glycerophosphate and 3beta-hydroxy steroid dehydrogenases, NADH tetrazolium reductase, acid phosphatase and nonspecific esterases showed for irreversible necrotic changes of seminiferous tubules of cadmium treated rats while the damage to interstitial gland is only temporary.     

Response to cryptorchidism of the testis and epididymis of the opossum (Didelphis virginiana).

Adult male opossums, Didelphis virginiana, were rendered hemicryptorchid for 35 days. The cyrptorchid testis exhibited a significant reduction in weight, while the contralateral testis had a compensatory weight gain compared with testes of untreated animals. Histological changes in the cryptorchid testis included fibrosis of the tunica propria, involution of the seminiferous tubules and an apparent increase in the interstitial tissue. Many seminiferous tubules were empty and germinal cells were absent. Some Sertoli cells persisted, but the cytoplasm was vacuolated. Cryptorchid testes were characterized by mononuclear leucocytic invasion around the tubules, and some eosinophils were observed. Cryptorchidism in the opossum may induce a reaction similar to experimental orchitis.     

Electrophysiological changes of Sertoli cells produced by the acute administration of amino acid and FSH.

Electrophysiological studies were carried out using seminiferous tubules of "Sertoli cell enriched testes" of rats irradiated in utero. Sertoli cells were inserted with glass microelectrodes in a superfusion chamber continuously perfused with KRb buffer. The topical application of FSH (4.0 mU/ml) produced a biphasic effect characterized by a rapid hyperpolarization (less than 3 s) followed by depolarization. The depolarizing effect of FSH was prolonged and potentiated in the presence of 5 mmol/l alpha-methylamino-isobutyric acid in the bath medium of the superfusion chamber. Verapamil, at a dose (250 mumol/l) that nullified the stimulatory action of FSH in the amino acid transport, suppressed the depolarizing effect of FSH. It was concluded that in immature rat testes FSH produces electrophysiological changes that mediate the stimulatory action of the amino acid transport.     

Impact of fine needle aspiration (FNA) and of the number of punctures on the feline testis: clinical, gross anatomy and histological assessment

The safety of testicular fine needle aspiration (FNA) has been proven in dogs but has not been fully established in men, while studies in rats have given contradictory results. Furthermore, the extent of damage inflicted by multiple punctures is unknown. The aim of this study was to determine the impact of FNA and of the number of punctures on the feline testis with clinical, gross anatomy and histological examinations. Twenty-seven sexually mature healthy laboratory Domestic Shorthair cats were randomly assigned to two groups: 5 cats in which no FNA was performed (control group), and 22 cats which had their left and right testis punctured with a 26 ga needle towards 3 and 8 directions, respectively (experimental group). Two cats at a time were orchiectomized 5 or 30 min, 1, 2, 4, 7 or 14 days or 1, 2, 3 or 4 mo post-aspiration. The cats of the control group were also orchiectomized. During the first week post-aspiration clinical examination revealed vaginal cavity hematoma (8/44 testes), while the histological findings were focal hemorrhagic areas (20/24 testes), erythrocytes inside the seminiferous tubules' lumen (9/24 testes), and germinal cell degeneration in <1.94% of the seminiferous tubules (15/24 testes). After the first week the histological findings were germinal cell degeneration in <2.14% of the seminiferous tubules (19/20 testes) and enlargement of the lumen of <5.16% of the seminiferous tubules (7/20 testes). The germinal epithelium and interstitium had an overall normal appearance. No significant differences were observed between the left and right testis. The results of the study indicate that testicular FNA should be considered a safe procedure in the cat when up to 8 punctures are performed.     

Localization of gamma-butyrobetaine hydroxylase in the rat testis

The activity of gamma-butyrobetaine hydroxylase in testes is comparable on a protein basis to the amount of activity found in liver and accounts for a significant amount of the total activity found in the rat. The enzyme is localized to the seminiferous tubules of the testes. In attempting to further localize this enzyme, activity remained the same when reported on a protein basis in cryptorchid testes as compared to controls, and activity was found in bovine sperm cells obtained from the caput epididymis. These results are consistent with the localization of this enzyme in both the Sertoli cells and the germ cells.     

Expression of calbindin-D28k in developing and growing chick testes.

Calbindin, a 28-kDa vitamin D-dependent calcium-binding protein was localized immunohistochemically in developing and growing chick testes. The protein first appeared in the germinal epithelium of developing testes of the eight-day-old embryo and remained therein throughout development. Calbindin was not present in the germinal epithelium after hatching. Calbindin was next detected in the spermatogonia and spermatocytes of one-week-old and growing chick testes. Calbindin-positive spermatogonia and spermatocytes gradually increased in number and staining intensity as the seminiferous tubules further developed. A few interstitial Leydig cells were positive for calbindin from five-week-old and older chicks. Comparison of the time-course of appearance and increase in calbindin content in spermatogonia and spermatocytes with spermatogenesis in chickens suggests that calbindin may be involved in the mitotic process in spermatogonia and spermatocytes.     

Effect of aqueous leaf extract of Azadirachta indica on the reproductive organs in male mice

Effect of oral administration (50, 100, and 200 mg/kg body weight/day, for 28 days) of aqucous leaf extract of neem (Azadirachta indica) on the male reproductive organs of the Parkes (P) strain mice was investigated. The treatment had no effect on body weight and the reproductive organs weight. In treated mice, testes showed both normal and affected seminiferous tubules in the same sections; the affected seminiferous tubules showed intraepithelial vacuolation, loosening of germinal epithelium, marginal condensation of chromatin in round spermatids, occurrence of giant cells, mixing of germ cell types in stages of spermatogenesis and degenerated appearance of germ cells. In severe cases, the tubules were lined with Sertoli cells only, Sertoli cells and rare germ cells, or with Sertoli cells and several germ cells but without cellular association patterns. Also, the frequency of affected seminiferous tubules in testes of the extract-treated mice was significantly higher than the controls, though this remained unaffected in mice treated at 50 mg/kg body weight of the extract. Doses at 50 or 100 mg/kg body weight of neem leaf extract did not cause appreciable alterations in histological appearance of the epididymis, while a dose of 200 mg/kg body weight caused marked alterations both in histological appearance and the level of sialic acid in the duct. The treatment also had adverse effects on motility, morphology, and number of spermatozoa in the cauda epididymidis, level of fructose in the seminal vesicle, and on litter size. After 42 days of withdrawal of the treatment, the alterations induced in the reproductive organs recovered to control levels. Our results suggested that treatment with neem leaf extract caused reversible alterations in the male reproductive organs of P mice.     

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.     

Effect of 17 alpha-cyanomethyl-17 beta-hydroxy-estra-4, 9-dien-3-one on reproductive organs of the male laboratory mouse

The effect of subcutaneous administration (10, 15 and 20 mg/kg body weight/day, for 21 days; and 20 mg/kg body weight/day, for 28 days) of 17 alpha-cyanomethyl-17 beta-hydroxy- estra-4, 9-dien-3-one (STS 557) on the male reproductive organs of the Parkes strain mouse was investigated. The effect of the treatment on the testis was not uniform; both regressed and normal seminiferous tubules were observed in the same section of the organ. Furthermore, the histological changes observed in the seminiferous tubules in testes of STS 557--treated mice were not different in different dosage groups. In general, in moderately affected seminiferous tubules, the germinal epithelium was thin and consisted of Sertoli cells, spermatogonia, spermatocytes and spermatids; such tubules showed presence of many vacuoles in the epithelium. In severe cases, the tubules had collapsed and were lined by mainly Sertoli cells, spermatogonia and spermatocytes. The treatment also caused marked depression in motility and concentration of spermatozoa in cauda epididymidis, weight of accessory sex glands and in the levels of sialic acid and fructose in the epididymis and seminal vesicle, respectively. By 56 days of drug withdrawal, the alterations induced in the reproductive organs returned to control levels, suggesting that STS 557 treatment induces reversible alterations in the male reproductive organs of Parkes strain mouse.     

Effect of dietary zinc and copper on peripheral blood plasma cholesterol, testosterone and histomorphology of testes in rats

Total plasma cholesterol (mg/dl) significantly (P less than 0.01) decreased from 70.8 to 54.01 as the dietary Cu levels increased from 2.5 to 5 ppm at 12 pm Zn concentrations in male weanling rats. A similar trend was observed in the blood peripheral testosterone concentration at 12 ppm Zn and 2.5 ppm Cu. Histological examination of testes revealed smaller seminiferous tubules with atrophy of germinal epithelium. Also a marked loss of spermatogenic cells was observed in Zn and Cu deficient rats.