Localization of delta 5-3 beta- and 17 beta-hydroxysteroid dehydrogenase activity in the efferent ducts, epididymis and vas deferens of the rabbit, hamster and marmoset monkey

The presence and distribution of delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD: EC 1.1.1.51) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD: EC 1.1.1.51) were studied histochemically in the excurrent ducts of the rabbit, hamster and marmoset monkey. Dehydroepiandrosterone (DHEA) and testosterone were used as substrates for delta 5-3 beta-HSD and 17 beta-HSD respectively, while phenanthroline monohydrate was used to eliminate non-specific staining due to other tissue dehydrogenases. The rabbit possessed least enzyme activity, which was confined to tubules in the middle segment of the epididymis. Enzyme activity was demonstrable throughout the excurrent ducts of the hamster and marmoset, with maximal staining occurring in the middle segment of the epididymis in both species. The region of maximum activity of hydroxysteroid dehydrogenase is where spermatozoa first develop their fertilizing capacity.     

ULTRASTRUCTURAL LOCALIZATION OF DIALYZED IRON-REACTIVE MUCOSUBSTANCE IN RABBIT HETEROPHILS, BASOPHILS, AND EOSINOPHILS

For ultrastructural localization of acid mucosubstances in rabbit granulocytes, bone marrow and buffy coat specimens were fixed with formalin, glutaraldehyde, or osmium tetroxide, sectioned at 40 µ, and stained with the Rinehart and Abul-Haj solution of dialyzed iron (DI). Heterophils revealed DI staining on the outer surface of the plasma membrane, in the Golgi complex involved in primary granulogenesis, and in primary granules. The intragranular distribution of DI-stained material varied at different stages in the maturation of primary granules. Immature granules of heterophils fixed by any of the three methods contained a peripheral concentric band of DI-positive material; however, fully mature primary granules possessed a core of DI-reactive material in heterophils fixed with osmium tetroxide, but they contained little or no staining in heterophils fixed with formalin or glutaraldehyde. Secondary granules of rabbit heterophils failed to stain with DI. Tertiary granules, observed only in late heterophils, contained distinct DI-positive particles. Basophil granules exhibited intensely DI-stained material distributed in an orderly pattern throughout the granule. In eosinophils, DI staining was localized in the Golgi complex and in the rims of a few immature cytoplasmic granules.     

Role of rabbit prostate granules on sperm viability and acrosome reaction evaluated with different methods

In the present study, the role of rabbit seminal granules was observed. Their influence on motility, capacitation and acrosome reaction, as well as the presence of apoptosis and the morphology of rabbit sperm, were compared in different conditions. Ejaculated sperm from five mature New Zealand White rabbit bucks during three series of collections were studied, comparing raw semen, Percoll-selected sperm and Percoll-selected sperm plus prostate granules. We observed sperm motility kinetic traits by computer-assisted sperm analyzer (CASA) analysis in each sample. Acrosome status was evaluated by FITC-labeled Pisum sativum Agglutinin staining and chlortetracycline fluorescence assay, phosphatidylserine translocation was determined by AnnexinV/Propidium iodide assay and sperm morphology was studied using transmission electron microscopy (TEM). All traits were observed after 30 min incubation at 37 °C in 5% CO₂. Data showed that sperm motility and viability markedly improved in the presence of prostate granules, whereas capacitation, acrosome reaction and phosphatidylserine translocation were lowered. TEM confirmed these results. In conclusion, the role of granules was confirmed in synchronizing sperm capacitation and acrosome reaction with egg availability; indeed, rabbit ovulation occurs only 6 to 10 h after mating.     

Species differences in ciprofibrate induction of hepatic cytochrome p450 4A1 and peroxisome proliferation

Six species (CD-1 mouse, Fischer 344 rat, Syrian golden hamster, Duncan-Hartley guinea pig, half-lop rabbit and marmoset monkey) were treated orally with ciprofibrate, a potent oxyisobutyrate hypolipidaemic drug for 14 days. A dosedependent liver enlargement was observed in the mouse and rat and at the high dose level in the hamster. A marked dose-dependent increase in the 12-hydroxylation of lauric acid was observed in the treated mouse, hamster, rat, and rabbit, associated with a concomitant elevation in the specific content of cytochrome P-450 4A1 apoprotein, determined by an ELISA technique. Similarly, in these responsive species, an increase in mRNA levels coding for cytochrome P450 4A1 was observed. Lauric acid 12-hydroxylation was unchanged in the guinea pig and marmoset after ciprofibrate pre-treatment, and cytochrome P-450 4A1 was not detected immunochemically in liver microsomes from these latter species. In the untreated mouse, hamster, rat, and rabbit, the 12-hydroxylation of lauric acid was more extensive than the 11-hydroxylation, whereas in the guinea pig and marmoset the activity ratios were reversed, with 11-hydroxylation predominating. Peroxisomal fatty acid β-oxidation was markedly induced in the mouse, hamster, rat, and rabbit on treatment at the higher dose level (39-, 3-, 13- and 5-fold, respectively) and was slightly increased in the marmoset (2-fold), yet was unchanged in the guinea pig following treatment. In the marmoset the increase in peroxisomal β-oxidation was 3- to 4-fold at the high dose level; however, the dose levels used in the marmoset were 20 and 100 mg/kg as opposed to 2 and 20 mg/kg in the other species. The differences in the foregoing hepatic enzyme responses to ciprofibrate between the species examined in our studies indicate a specific pattern of enzyme changes in responsive species. In the responsive species (rat, mouse, hamster, and rabbit), cytochrome P-450 4A1 specific content and related enzyme activity were increased concomitant with elevated peroxisomal β-oxidation. By contrast, the marmoset and guinea pig lack the coordinate hepatic induction of peroxisomal and microsomal parameters and may be categorized as less responsive species. Accordingly, the rat hepatic responses to peroxisome proliferators cannot confidently be used to predict biological responses in primates, with obvious implications for the extrapolation of animal data to man.     

Ultrastructural identification of round-granule EC cells in rabbit fundic mucosa

In endocrine (EC) cells of rabbit fundic mucosa, it is practically impossible to obtain unequivocal ultrastructural identification of all cells found in order to perform morphometric analysis. In addition to classic EC cells with pleomorphic granules, a cell type with entirely round granules is encountered which can be confused with non-EC cells. To solve this problem, all EC cells in our study were first identified by their 5-HT (immunocytochemistry) and argentaffinity. Examination of the fine structures of reactive cells then revealed that the round granules of EC cells were differentiated from those of non-EC cells by the existence of a dense core surrounded by a less dense halo, a feature providing unequivocal ultrastructural identification. EC cells with round granules showed less argentaffinity and less immunoreactivity to 5-HT as compared with classic EC cells. After labelling with [3H]L-dopa, EC cells with round-granules displayed an overall staining index higher than that of classic EC cells and comparable with that of D cells; however, the nuclear staining index was higher than that of D cells.     

Unmasking and redistribution of lysosomal sulfated glycoconjugates in phagocytic polymorphonuclear leukocytes

Rabbit heterophil and human neutrophil primary granules contain sulfated glycosaminoglycans (GAGs) and acid phosphatase, which can be readily stained in immature but not mature lysosomes. To determine whether this loss of staining represents masking of reactive components or removal of these components, we examined rabbit heterophils to see if high-iron diamine (HID)-reactive sulfate and acid phosphatase staining reappears in phagocytic vacuoles. Rabbit heterophils, obtained by peritoneal lavage, were incubated in vitro with latex beads or Pseudomonas aeruginosa for 15-60 min. Pre-embedment HID staining was enhanced in thin sections of unosmicated specimens with thiocarbohydrazide and silver proteinate (TCH-SP). Phagocytosis of latex beads or bacteria was progressively more prominent with time. Primary granules that were degranulated or in the process of degranulating into phagocytic vacuoles demonstrated intense sulfate staining with large (13 +/- 7 nm) HID-TCH-SP stain deposits. Smaller (6 +/- 1 nm) HID-TCH-SP stain deposits were present in tertiary granules, which were less frequently observed degranulating into phagosomes. Acid phosphatase staining was most intense during early phagolysosome formation. HID-TCH-SP staining was also observed in extracellular degranulated lysosomal matrices and on the surface of many peritoneal heterophils. These results indicate that loss of sulfate staining in mature heterophil granules is the result of masking by intragranular substances rather than of removal, and that these components may be unmasked during phagocytosis and/or redistributed to the cell surface after exocytosis.     

Species variability in the expression of met- and leu-enkephalin-like immunoreactivity in mammalian Merkel cell dense-core granules

Summary Immunogold staining failed to show met-enkephalin immunoreactivity in the Merkel cell dense-core granules of rats when examined by electron microscopy, but showed gold particle staining in the Merkel cell dense-core granules of mice and nude mice. Merkel cells of hamster, guinea pig, rabbit, cat and dog were also examined using a similar method, and different antisera dilutions. Immunogold particles were consistently found in the dense-core granules of mice and nude mice at all antisera dilutions, but not in the other species, except in the dog, where a very low labelling response was encountered. Merkel cells from skin touch domes or sinus hair follicles, did not exhibit any difference in peptide expression as far as met-enkephalin immunoreactivity was concerned. In addition, all species studied, including mice and nude mice, did not show leu-enkephalin immunoreactivity in their Merkel cell dense-core granules. It is concluded that species variability in peptide expression occurs in the Merkel cell dense-core granules, and may be closely related to the different methodologies used.     

Proacrosin and the differentiation of the spermatozoa

Using the indirect immunofluorescence staining technique, the occurrence and localization of proacrosin, the zymogen form of acrosin, was studied during spermatogenesis in the bull, ram, boar and rabbit. Proacrosin staining was demonstrable for the first time in the early haploid spermatid and increased with the differentiation of the spermatid to spermatozoon. The spermatozoon is covered by a cap-like structure of uniform fluorescence corresponding to the acrosomal compartment of the male gamete. No fluorescence could be found in diploid spermatogenic cells, i.e., in spermatogonia and spermatocytes. An identical developmental pattern of proacrosin was observed with the indirect immunoperoxidase staining technique. However, with this staining technique a distinct distribution of proacrosin staining was observed in the acrosome of epididymal and ejaculated spermatozoa of the bull, ram, boar, rabbit and man. Proacrosin seems to be distributed in the acrosome in granules rather than in the homogeneous form, as was indicated by the results of indirect immunofluorescence staining.     

Localization of the guinea pig eosinophil major basic protein to the core of the granule

The localization of the guinea pig eosinophil major basic protein (MBP) within the cell was investigated by the use of immunoelectron microscopy and by isolation of the granule crystalloids. First, by immunoperoxidase electron microscopy, we found that the MBP of eosinophil granules is contained within the crystalloid core of the granule. Specific staining of cores was present when rabbit antiserum to MBP was used as the first stage antibody in a double antibody staining procedure, whereas staining was not seen when normal rabbit serum was used as the first stage antibody. Second, crystalloids were isolated from eosinophil granules by disruption in 0.1% Triton X-100 and centrifugation through a cushion of 50% sucrose. Highly purified core preparations yielded essentially a single band when analyzed by electrophoresis on polyacrylamide gels containing 1% sodium dodecyl sulfate (SDS). The E1%1cm of the core protein was 26.8 +/- 1.0 (X +/- SEM); the E1%1cm for the MBP was 26.3. The core protein could not be distinguished from the MBP by radioimmunoassay (RIA) and essentially all of the protein in the core preparations could be accounted for as MBP. The results indicate that the MBP is contained in the core of the guinea pig eosinophil granule and that it is probably the only protein present in the core.     

Quantitative immunocytochemistry of pituitary receptors for luteinizing hormone-releasing hormone

In Araldite sections of male rat pituitaries, stained after embedding by the unlabeled antibody enzyme method with antisera to native luteinizing hormone-releasing hormone (LH-RH) or LH-RH azo-conjugated to bovine serum albumin, localization is confined mainly to the interior of the large, and to a lesser extent to that of the small, secretion granules of the gonadotrophic cells. Plasma membranes are not demonstrated. Except for weak staining in the granules of corticotrophs, no other pituitary cell is stained. Pretreatment of sections with LH-RH (to dilutions of 4 pg/mul) increases staining intensity in the gonadotrophic granules. Other cells are unaffected. The lesser the gonadotroph staining intensity without pretreatment, the greater the increase (more than 23-fold reactivity). Augmented staining is measurable (P less than 0.001) to antiserum dilutions of 1:240000. Pretreatment with des-Glu-1-LH-RH, porcine corticotropin or rat prolactin has no effect. LH-RH-Gly-10(des-amide) inhibits. Rat glycoprotein hormones enhance staining with anti-azo-conjugated LH-RH. With antinative LH-RH these hormones enhance weak staining, but inhibit strong staining. Thick vibrotome sections of male rat or rabbit pituitaries stained before embedding reveal specific localization on plasma membrane and gonadotrophic secretion granules provided the sections have been pretreated with LH-RH (250 pg/mul). The data show that LH-RH after reaction with receptor is not sterically hindered from binding specific antibodies. Receptor may be found in secretion granules, both in the free state or combined with LH-RH. Plasma membrane receptor, on the other hand, was free under the conditions of the experiments. Immunization with LH-RH elicits not only heteroimmune antibodies specific for LH-RH, but also a group of still ill defined autoimmune antibodies, some of which may conceivably be reactive with glycoprotein hormone alpha-chains.     

Staining of rabbit eosinophil and pseudoeosinophil leukocytes.

Air-dried rabbit blood was stained by HE, PAS and a modification of the Undritz II method. Eosin stained granules red in the eosinophil leukocytes. PAS was negative and the modified Undritz method failed to give consistent results. Cells with eosinophilic granules appeared in the corneal stroma 1 h after removing the corneal epithelium. They were stained red consistently by both eosin and the modified Undritz II method. Electron micrographs failed to demonstrate crystalloids in the granules. Because of the staining characteristics and the lack of crystalloids in their granules these cells were classified as pseudoeosinophil leukocytes. The electron micrographs showed some glycogen 12 h after denuding the cornea, however, glycogen was not well stained by PAS until 18 h after denuding.     

Ultrastructural visualization of sulphated complex carbohydrates in blood and epithelial cells with the high iron diamine procedure

Synopsis The high iron diamine (HID) method has been found to impart density at the ultrastructural level selectively to sites known to contain sulphated complex carbohydrates. Thus, immature primary granules in rabbit heterophils, immature précrystalloid granules in rabbit eosinophils, all granules of rabbit basophils, mouse and rat mast cells and the nucleoids of -granules of rabbit platelets were stained by HID. Granules of mast cells in rat cervical lymph node varied in the distribution pattern of the HID-reactive component. Mucous droplets within goblets of mouse colonic epithelial cells varied in HID reactivity. Sites known to contain sialomucin but no sulphates, such as mucous cells and apical plasmalemmae in mouse rectosigmoid colon, failed to stain with HID in contrast to their reactivity for dialysed iron at the ultrastructural level. The surface of mast cells and blood cells lacked affinity for HID, indicating that the dialysed iron binding at the surfaces can be attributed to neuraminic acid. HID proved more effective than dialysed iron in visualizing acid mucosubstance in precursor forms of the crystalloid granules in the eosinophil and in mast cell granules. Inclusion of 0.5% glycerol in the HID solution enhanced staining in mouse colon.     

Assessment of sperm viability in boar,rabbit and rooster: a modification of the fluorometric ethidium bromide exclusion procedure

The ethidium bromide (EtBr) exclusion procedure, a fluorometric method for measuring sperm cell viability, was studied to optimize the use of this technique on boar, rabbit and rooster semen. Diluted semen was used for boars and roosters. Diluted rabbit semen did not allow for reliable fluorescence readings; the interference of granules characteristic of rabbit seminal plasma was suggested as its cause. Therefore, rabbit semen was washed on several Percoll and Optiprep density gradients, with the aim of removing the granules from the sperm suspension. The complete absence of granules was not obtained, however, the best result was provided by the 35/70% Percoll density gradient. Most spermatozoa formed a loose pellet with low contamination. Although the washing procedure resulted in a selective action, Percoll washed semen was used to assess the EtBr procedure. The fluorescence intensities of stained fresh and stained digitonin-permeabilized samples were corrected, respectively, for the nonspecific fluorescence measures of fresh and digitonin-permeabilized samples both unstained. The contribution of the dye was subtracted from the corrected values, then the ratio between the corrected values of fresh and permeabilized cells provided the proportion of damaged cells in the sample. The working cell concentration range giving a constant proportion of damaged cells was set using diluted semen for boars and roosters (8-32 x 10(6) cell/ml) and Percoll washed semen for rabbits (4-16 x 10(6) cell/ml). The reliability of the fluorometric method was compared with the traditional nigrosin-eosin (NE) staining technique. The intactness of sperm samples containing known proportions of fresh and killed cells was measured in defined working cell ranges. For boars and roosters the values determined by fluorometry agreed closely with those determined using the NE method.     

Fine structural localization of arylsulfatase B activity in the rabbit blood platelets.

Fine structural localization of arylsulfatase in the rabbit blood platelets has been investigated in this study. Among many cell organellae, reaction products were exclusively observed in the alpha granules of the platelets. Within the alpha granules, arylsulfatase activity appeared to localize in variable patterns, i.e. reaction products confined mainly at the peripheral region in many granules, while they deposited heavily throughout the granule matrices in some others. In a blood platelets, each alpha granule showed the different staining pattern which indicated more variable functional heterogeneity in the granules.     

Ultrastructural localization of glycogen in the granulocytes of normal rabbit bone marrow.

The glycogen of rabbit granulocytes has been studied in glutaraldehyde and osmium tetroxide fixed bone marrow by the periodic acid-thiocarbohydrazide-silver proteinate procedure (PA-TCH-SP). The PA-TCH-SP procedure involved the staining of intracytoplasmic glycogen more densely than the routine lead citrate staining. The PA-TCH-SP procedure demonstrated the intracytoplasmic glycogen in all three kinds of granulocytes. Though a sequence of intensity was observed in each stage of cell maturation, intracytoplasmic glycogen increased generally in accordance with cell maturation in the granulocytes. Functional significance of the glycogen in the granulocytes was discussed in relation to its staining. A very weak reaction in the granules of the granulocytes was described in relation to their contents.     

Cytochemical detection of carbohydrate and immunocytochemical detection of relaxin in the same secretory granule

We administered estradiol and progesterone to spayed guinea pigs, with resultant accumulation of secretory granules in endometrial gland cells. By initially employing protein A-colloidal gold immunolocalization of relaxin, followed by cytochemical staining of carbohydrate with the thiocarbohydrazide-silver proteinate method on the same section, we showed clearly that the secretory granules were composed of a central core containing relaxin and a cortex of carbohydrate-rich material. Use of normal rabbit serum rather than relaxin antiserum, and omission of periodic acid, demonstrated the specificity of the technique.     

Ultrastructural localization of glycogen in the granulocytes of normal rabbit bone marrow

Summary The glycogen of rabbit granulocytes has been studied in glutaraldehyde and osmium tetroxide fixed bone marrow by the periodic acid-thiocarbohydrazide-silver proteinate procedure (PA-TCH-SP). The PA-TCH-SP procedure involved the staining of intracytoplasmic glycogen more densely than the routine lead citrate staining. The PA-TCH-SP procedure demonstrated the intracytoplasmic glycogen in all three kinds of granulocytes. Though a sequence of intensity was observed in each stage of cell maturation, intracytoplasmic glycogen increased generally in accordance with cell maturation in the granulocytes. Functional significance of the glycogen in the granulocytes was discussed in relation to its staining. A very weak reaction in the granules of the granulocytes was described in relation to their contents.     

Immunocytochemical identification of growth hormone cells in the adenohypophysis of the golden hamster

Somatotrophs or growth hormone (GH) cells in the adenohypophysis of golden hamsters were identified by immunocytochemical staining with polyclonal rabbit anti-human GH. They were oval or columnar in shape, and had secretory granules of two size ranges, 90-150 nm and 280-320 nm, which were present in the same cells; no subtypes of GH cells were observed. Secretory granules were located in the peripheral portion of the cytoplasm or concentrated at the vascular pole of the cell. Flattened cisternae of the rough endoplasmic reticulum in parallel array and a moderately developed Golgi apparatus were often found in the cytoplasm. No sex difference was noticed in the population ratio of GH cells. Immunocytochemical staining with anti-GH or anti-prolactin (PRL) antibodies on separate adjacent sections revealed that the GH and PRL were stored in two different cell types.     

Immunoreactivity of normal rabbit serum with epinephrine (E) cells of the rat adrenal medulla after microwave antigen retrieval

Normal rabbit serum (NRS) produces intense staining of epinephrine (E) cells in microwave-heated sections of rat and mouse adrenal gland. This staining is not eliminated by liver adsorption, complement inactivation, high salt buffer, Triton X-100 or dilution in normal goat serum and bovine serum albumin (BSA), suggesting that it may result from specific antigen-antibody interactions. Western blots of adrenal medullary protein probed with NRS reveal several bands. The major band does not correspond to rat chromogranin A, which is a major constituent of E-cell secretory granules. The findings suggest that NRS may contain autoantibodies against a secreted rabbit E-cell protein with a homologous counterpart in rats and mice, and that this protein may be immunologically unmasked in situ by microwave heating. This phenomenon is a potential source of error in immunohistochemical studies of the adrenal medulla, and has potential biological significance in neuroimmunology.     

DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES : II. Cytochemistry and Electron Microscopy of Bone Marrow Cells

In the previous paper we presented findings which indicated that enzyme heterogeneity exists among PMN leukocyte granules. From histochemical staining of bone marrow smears, we obtained evidence that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appeared to be restricted to one of the two types. Clear results were obtained with alkaline phosphatase, but those with a number of other enzymes were suggestive rather than conclusive. Since the approach used previously was indirect, it was of interest to localize the enzymes directly in the granules. Toward this end, we carried out cytochemical procedures for five enzymes on normal rabbit bone marrow cells which had been fixed and incubated in suspension. The localization of reaction product in the granules was determined by electron microscopy. In accordance with the results obtained on smears, azurophil granules were found to contain peroxidase and three lysosomal enzymes: acid phosphatase, arylsulfatase, and 5'-nucleotidase; specific granules were found to contain alkaline phosphate. Specific granules also contained small amounts of phosphatasic activity at acid pH. Another finding was that enzyme activity could not be demonstrated in mature granules with metal salt methods (all except peroxidase); reaction product was seen only in immature granules. The findings confirm and extend those obtained previously, indicating that azurophil granules correspond to lysosomes whereas specific granules represent a different secretory product.