Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma.

Recently, major advances have been made in the identification of antigens from human melanoma which are recognized by T cells. In spite of this, little is known about the optimal ways to use these antigens to treat patients with cancer. Progress in this area is likely to require accurate preclinical animal models, but the availability of such models has lagged behind developments in human tumor immunology. Whereas many of the identified human melanoma antigens are normal tissue differentiation proteins, analogous murine tumor antigens have not yet been identified. In this paper we identify a normal tissue differentiation antigen, tyrosinase-related protein 2 (TRP-2), expressed by the murine B16 melanoma which was found by screening a cDNA library from B16 with tumor-reactive cytotoxic T lymphocytes (CTL). A peptide conforming to the predicted MHC class I H2-Kb binding motif, TRP-2181-188, was identified as the major reactive epitope within TRP-2 recognized by these anti-B16 CTLs. By site-directed mutagenesis, it was shown that alteration of this epitope eliminated recognition of TRP-2. It was further demonstrated that a CTL line raised from splenocytes by repeated stimulation in vitro with this peptide could recognize B16 tumor and was therapeutic against 3-d-old established pulmonary metastases. The use of TRP-2 in a preclinical model of tumor immunotherapy may be helpful in suggesting optimal vaccination strategies for cancer therapy in patients.

The isolation and partial characterization of the pyrenoid protein of Eremosphaera viridis.

The pyrenoids of Eremosphaera viridis, a green alga, were isolated by density gradient centrifugation and their physical and enzymatic properties were studied. The ultraviolet absorption spectrum of sodium dodecyl sulfate (SDS) extracts of pyrenoids showed a single peak at a wavelength of 277 nm, indicating the presence of protein and the probable absence of nucleic acid. Upon electrophoresis on polyacrylamide gels containing SDS, 16 bands were resolved of which two, together, accounted for 90% of the total protein on the gels. The molecular weights of these two proteins were estimated to be 59,000 and 12,300 and the ratio by weight of the larger to the smaller protein was found to be 2:1. The physical and enzymatic properties of these two proteins were found to closely resemble the properties reported in the literature for the subunits of fraction I protein. Both pyrenoids and fraction I protein are localized in the chloroplast, and both have two principal protein components. The molecular weights and relative ratio of the two pyrenoid components are very similar to those of the two components of fraction I protein. The pyrenoid was found to contain a high specific activity of ribulose-1,5-diphosphate carboxylase which is the same enzymatic activity exhibited by fraction I protein. The presence of ribose-5-phosphate isomerase and ribulose-5-phosphate kinase activities was also noted in pyrenoid preparations. It is suggested that the pyrenoid contains fraction I protein and possibly other enzymes of the Calvin-Bassham carbon dioxide fixing pathway.

Uroporphyrinogen 3 cosynthetase activity in the fox squirrel (Sciurus niger).

The activity of uroporphyrinogen III cosynthetase in hemolyzates and tissue extracts from fox squirrels is much less than in similar preparations from gray squirrels. Low activity of this enzyme explains the production of large amounts of uroporphyrin I by the fox squirrel. Members of this species thus provide a small-animal model for studies of congenital erythropoietic porphyria, a hereditary disease of man and cattle which is associated with a similar partial deficiency of uroporphyrinogen III cosynthetase.

Ruminal biohydrogenation as affected by tannins in vitro.

The aim of the present work was to study the effects of tannins from carob (CT; Ceratonia siliqua), acacia leaves (AT; Acacia cyanophylla) and quebracho (QT; Schinopsis lorentzii) on ruminal biohydrogenation in vitro. The tannins extracted from CT, AT and QT were incubated for 12 h in glass syringes in cow buffered ruminal fluid (BRF) with hay or hay plus concentrate as a substrate. Within each feed, three concentrations of tannins were used (0.0, 0.6 and 1.0 mg/ml BRF). The branched-chain volatile fatty acids, the branched-chain fatty acids and the microbial protein concentration were reduced (P < 0.05) by tannins. In the tannin-containing fermenters, vaccenic acid was accumulated (+23 %, P < 0.01) while stearic acid was reduced ( - 16 %, P < 0.0005). The concentration of total conjugated linoleic acid (CLA) isomers in the BRF was not affected by tannins. The assay on linoleic acid isomerase (LA-I) showed that the enzyme activity (nmol CLA produced/min per mg protein) was unaffected by the inclusion of tannins in the fermenters. However, the CLA produced by LA-I (nmol/ml per min) was lower in the presence of tannins. These results suggest that tannins reduce ruminal biohydrogenation through the inhibition of the activity of ruminal micro-organisms.

Cultured lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis have a diminished capacity to synthesize prostaglandin E2 and to express cyclooxygenase-2.

Prostaglandin E2 (PGE2) inhibits fibroblast proliferation and collagen synthesis. In this study, we compared lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (F-IPF) and from patients undergoing resectional surgery for lung cancer (F-nl) with respect to their capacity for PGE2 synthesis and their expression and regulation of cyclooxygenase (COX) proteins. Basal COX activity, assessed by quantitating immunoreactive PGE2 synthesized from arachidonic acid, was twofold less (P < 0.05) in F-IPF than F-nl. In F-nl, incubation with the agonists PMA, LPS, or IL-1 increased COX activity and protein expression of the inducible form of COX, COX-2, and these responses were inhibited by coincubation with dexamethasone. By contrast, F-IPF failed to demonstrate increases in COX-2 protein expression or COX activity in response to these agonists. Under conditions of maximal induction, COX activity in F-IPF was sixfold less than that in F-nl (P < 0.05). Our data indicate that F-IPF have a striking defect in their capacity to synthesize the antiinflammatory and antifibrogenic molecule PGE2, apparently because of a diminished induction of COX-2 protein. This reduction in the endogenous capacity of F-IPF to down-regulate their function via PGE2 may contribute to the inflammatory and fibrogenic response in IPF. Moreover, we believe that this represents the first description of a defect in COX-2 expression in association with a human disease.

Compositions and biological activities of essential oils of Kadsura longepedunculata and Schisandra sphenanthera.

The chemical compositions, antimicrobial activities, antioxidant activities and cytotoxicities of the essential oils isolated from the root of Kadsura longepedunculata Finet et Gagnep (KLREO) and the fruit of Schisandra sphenanthera Rehd. et Wills. (SSFEO) were investigated.The analyses of gas chromatography-mass spectrometry (GC-MS) showed that cadinane type compounds and their derivatives were rich in both oils (54.2% and 39.7%, respectively) and delta-cadinene was the major component of both oils (13.8% and 25.6%, respectively). The antimicrobial activities of both oils were evaluated against five microorganisms with the disc diffusion and the broth micro-dilution method. Results showed that Gram-positive bacteria were more sensitive to both oils than Gram-negative bacteria and the yeast. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the oil of KLREO were lower than those of SSFEO, indicating that the former possessed slightly stronger antibacterial capability than the latter. The reducing power and lipid peroxidation assays were employed to study the potential antioxidant activities of both oils. Both oils remarkably decreased the content of malondialdehyde (MDA) in rat liver homogenate in a dose dependent manner. The antioxidant activities of KLREO appeared to be more potent than that of SSFEO. The oils of KLREO and SSFEO exhibited concentration-dependent cytotoxicities and were proved to be toxic to HepG2 cells with IC(50) of 147 and 189 mug/ml, respectively.

Comprehensive identification and structural characterization of target components from Gelsemium elegans by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry based on accurate mass databases combined with MS/MS spectra.

This study reports an applicable analytical strategy of comprehensive identification and structure characterization of target components from Gelsemium elegans by using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QqTOF MS) based on the use of accurate mass databases combined with MS/MS spectra. The databases created included accurate masses and elemental compositions of 204 components from Gelsemium and their structural data. The accurate MS and MS/MS spectra were acquired through data-dependent auto MS/MS mode followed by an extraction of the potential compounds from the LC-QqTOF MS raw data of the sample. The same was matched using the databases to search for targeted components in the sample. The structures for detected components were tentatively characterized by manually interpreting the accurate MS/MS spectra for the first time. A total of 57 components have been successfully detected and structurally characterized from the crude extracts of G. elegans, but has failed to differentiate some isomers. This analytical strategy is generic and efficient, avoids isolation and purification procedures, enables a comprehensive structure characterization of target components of Gelsemium and would be widely applicable for complicated mixtures that are derived from Gelsemium preparations. Copyright © 2017 John Wiley & Sons, Ltd.

High performance mass spectrometry based proteomics reveals enzyme and signaling pathway regulation in neutrophils during the early stage of surgical trauma.

PURPOSE: In clinical conditions trauma is associated with high mortality and morbidity. Neutrophils play a key role in the development of multiple organ failure after trauma EXPERIMENTAL DESIGN: To have a detailed understanding of the neutrophil activation at primary stages after trauma, neutrophils are isolated from control and surgical trauma rats in this study. Extracted proteins are analyzed using nano liquid chromatography coupled with tandem mass spectrometry. RESULTS: A total of 2924 rat neutrophil proteins are identified in our analysis, of which 393 are found differentially regulated between control and trauma groups. By using functional pathways analysis of the 190 proteins up-regulated in surgical trauma, we found proteins related to transcription initiation and protein biosynthesis. On the other hand, among the 203 proteins down-regulated in surgical trauma we found enrichment for proteins of the immune response, proteasome degradation and actin cytoskeleton. Overall, enzyme prediction analysis revealed that regulated enzymes are directly involved in neutrophil apoptosis, directional migration and chemotaxis. Our observations are then confirmed by in silico protein-protein interaction analysis. CONCLUSIONS AND CLINICAL RELEVANCE: Collectively, our results reveal that neutrophils drastically regulate their biochemical pathways after the early stages of surgical trauma, showing lower activity. This implies higher susceptibility of the trauma patients to infection and bystander tissues damage.

Purification and partial amino acid sequence of the cyanogen bromide fragments of muconolactone isomerase from Pseudomonas putida.

Muconolactone isomerase is shown to be resistant to proteolytic cleavage by trypsin. Cyanogen bromide cleavage at the methionine residues of the polypeptide is at least 95% complete. Six cyanogen bromide fragments are separated on DEAE-cellulose. One fragment is shown by amino acid analysis and carboxyl-terminal analysis to be an incomplete cleavage product. The five remaining fragments represent the entire polypeptide and have been ordered with respect to the entire muconolactone isomerase sequence. Approximately 50% of the polypeptide sequence could be determined from these fragments by the dansyl-Edman technique. The possible evolutionarily homologous origins of muconolactone isomerase and two analogous isomerases, carboxymuconolactone decarboxylase and sigma5-3-ketosteroid isomerase, are discussed.

A membrane-bound form of protein disulfide isomerase (PDI) and the hepatic uptake of organic anions.

Protein disulfide isomerase (PDI) was considered to be involved in the hepatic uptake of certain organic anions because the protein is photoaffinity labeled by photolabile derivatives of the bile acid taurocholate. Several lines of evidences including photoaffinity labeling experiments indicated a close relationship between the uptake of bile acids and the organic anion bumetanide. The possible involvement of PDI in hepatic transport processes of these organic anions was tested with polyclonal antibodies raised against a PDI-beta-galactosidase fusion protein. Western blot analysis and immunofluorescence of intact hepatocytes showed that protein disulfide isomerase is located in sinusoidal rat liver plasma membranes. This protein is immunologically identical with microsomal PDI prepared from bovine liver. The plasma membrane form of PDI is, however, not labeled by photoactivated bumetanide as revealed by two-dimensional gel electrophoresis. These results indicate that, although a membrane-bound form of the PDI is present in the sinusoidal plasma membrane of rat hepatocytes, this protein is not involved in the hepatocellular uptake of the organic anion bumetanide.

Astrocytes synthesize and secrete prostaglandin D synthetase in vitro.

Prostaglandin D synthetase [PGD-S, prostaglandin-H2 D-isomerase, (5Z, 13E)-(15S)-9alpha, 11 alpha-epidioxy-15-hyrdroxyprosta-5,13-dienoate D-isomerase, EC 5,3,99,2], an enzyme that catalyzes the formation of prostaglandin D2, was originally isolated from homogenates of rat brain and spleen and is known to be a membrane-bound enzyme. Subsequent immunohistochemical studies have shown that PGD-S is associated with neurons in the brain of immature rats, whereas in adult rats it is associated with oligodendrocytes. Several recent studies have shown that the beta-trace protein isolated from human cerebrospinal fluid (CSF), the second most abundant protein in human CSF after albumin, is equivalent to PGD-S. In this paper, we report the preparation of a monospecific polyclonal antibody against purified PGD-S isolated from human CSF and the establishment of a specific radioimmunoassay for this protein. Using this radioimmunoassay in conjunction with immunoblot analysis, PGD-S was detected in various biological fluids including serum, aqueous humor, and rete testis fluid. In addition, an antibody prepared against human PGD-S partially cross-reacted with the PGD-S in the rat and ram. Using a monospecific polyclonal antibody prepared against purified rat PGD-S isolated from rat CSF in conjunction with [35S]methionine incorporation and immunoprecipitation techniques, it was shown for the first time that PGD-S is actively synthesized and secreted by astrocytes cultured in vitro, suggesting the astrocyte is the cellular origin of PGD-S in the CSF. The identification of the astrocyte as the cellular origin of this unique enzyme will allow the use of an in vitro system to study its regulation.

Altered arachidonic acid metabolism during differentiation of the human monoblastoid cell line U937.

The human cell line U937 was used as a model for differentiation along the mononuclear phagocyte lineage. Following treatment with the phorbol ester TPA, PGE2 and TxB2 secretion was induced 50-100-fold, and both PGF2 alpha and PGI2 levels became detectable in the supernatant of TPA-differentiated U937 cells. The content of the prostaglandin precursor, arachidonic acid, remained unchanged in the cellular phospholipids of undifferentiated and TPA-differentiated U937 cells. Of the enzymes involved in the availability and metabolism of arachidonic acid, phospholipase A2 activity was increased 2-fold in the membranes of TPA-differentiated U937 cells, whereas lysophosphatide acyltransferase activity remained unaltered. Cyclooxygenase activity, however, was enhanced 5-10-fold, which was due to enhanced expression of the enzyme as demonstrated by dot-blot analysis. The data suggest that the capacity to secrete prostaglandins is acquired during differentiation with TPA and results mainly from an increased cyclooxygenase activity. Despite the capacity of TPA-differentiated U937 cells to synthesize prostaglandins, none of the known monocytic stimuli further stimulated prostaglandin secretion in TPA-differentiated U937 cells. Generation of leukotrienes appears to represent a later state in the differentiation along the monocyte-macrophage lineage, since neither LTB4 nor cysteinyl-leukotrienes were detectable in the supernatants of either undifferentiated or TPA-differentiated U937 cells.

Disruption of endothelial-cell caveolin-1alpha/raft scaffolding during development of monocrotaline-induced pulmonary hypertension.

BACKGROUND: In the monocrotaline (MCT)-treated rat, there is marked stimulation of DNA synthesis and megalocytosis of pulmonary arterial endothelial cells (PAECs) within 3 to 4 days, followed by pulmonary hypertension (PH) 10 to 14 days later. Growing evidence implicates caveolin-1 (cav-1) in plasma membrane rafts as a negative regulator of promitogenic signaling. We have investigated the integrity and function of endothelial cell-selective cav-1alpha/raft signaling in MCT-induced PH. METHODS AND RESULTS: Although PH and right ventricular hypertrophy developed by 2 weeks after MCT, a reduction in cav-1alpha levels in the lung was apparent within 48 hours, declining to approximately 30% by 2 weeks, accompanied by an increase in activation of the promitogenic transcription factor STAT3 (PY-STAT3). Immunofluorescence studies showed a selective loss of cav-1alpha and platelet endothelial cell adhesion molecule-1 in the PAEC layer within 48 hours after MCT but an increase in PY-STAT3. PAECs with cav-1alpha loss displayed high PY-STAT3 and nuclear immunostaining for proliferating cell nuclear antigen (PCNA). Biochemical studies showed a loss of cav-1alpha from the detergent-resistant lipid raft fraction concomitant with hyperactivation of STAT3. Moreover, cultured PAECs treated with MCT-pyrrole for 48 hours developed megalocytosis associated with hypo-oligomerization and reduction of cav-1alpha, hyperactivation of STAT3 and ERK1/2 signaling, and stimulation of DNA synthesis. CONCLUSIONS: MCT-induced disruption of cav-1alpha chaperone and scaffolding function in PAECs likely accounts for diverse alterations in endothelial cell signaling in this model of PH.

Isozyme frequency patterns in Drosophila pavani associated with geographical and seasonal variables.

Fourteen population samples of Drosophila pavani were obtained from a number of localities in Chile. The populations sampled were dispersed over 7 degrees of latitude and 1800 meters of elevation, and were drawn at three different times. Sixteen electrophoretic loci were assayed for each population; eight of the loci were analyzed statistically for geographic variation; the other eight were essentially monomorphic. For all eight variable loci, variation in allelic frequencies among populations was highly significant. In all cases, a significant portion of the variation among populations was associated with variation in gross environmental variables (latitude, elevation, month of collection). The implications of the evidence were discussed, and the authors concluded that there was suggestive evidence for selection.

Brefeldin A and mannose 6-phosphate regulation of acrosomic related vesicular trafficking.

Acrosomal biogenesis represents a unique system for the molecular analysis of the various processes involved in vesicular membrane transport. To assess the basic membrane trafficking mechanisms used in spermatids, we have used two fluorescent lipid compounds that label: a) the Golgi and Golgi-derived vesicles (C5-DMB-Cer), and b) endocytic vesicles (FM4-64). Incubation of mouse testicular germ cells at 33 degrees C for 1.5 h with C5-DMB-Cer revealed that C5-DMB-Cer labeling is localized in the Golgi and acrosome of early-maid round spermatid stages, with no labeling of the acrosome in late round spermatid stages. Culturing germ cells with FM4-64 for 1.5 h at 33 degrees C, showed that FM4-64 labeling in spermatids was localized in endocytic vesicles and Golgi of early-mid round spermatid stages, whereas in a small population of late round spermatid stages, FM4-64 was also localized in the apex region of the acrosome. Incubation with brefeldin A (BFA) (5 micrograms/ml) inhibited the distribution of C5-DMB-Cer and FM4-64 to the acrosome, however, it did not affect the localization of acrosin-an acrosome-specific protein-indicating that there was no apparent acrosome disassembly in the presence of BFA. Localization of FM4-64 in late round spermatids in the presence of 2.5 mM mannose 6-phosphate was found in endocytic vesicles and the Golgi, but not the acrosome. These results show that there are at least two sources of vesicular transport to the acrosome derived from the Golgi and plasma membrane.

Melanogenesis during the anagen-catagen-telogen transformation of the murine hair cycle.

Melanin synthesis of follicular melanocytes is strictly coupled to the growth stage of the hair cycle (anagen), ceases during follicle regression (catagen), and is absent throughout the resting stage (telogen). Having previously characterized the expression and activity of melanogenesis-related proteins during the telogen-anagen transition of the murine hair cycle (JID 96:172, 1991), we here report a biophysical and biochemical analysis of follicular melanogenesis during the anagen-catagen-telogen transformation of the C57 BL-6 mouse hair cycle. Tyrosinase activity and concentration as well as dopachrome tautomerase activity were compared with melanin synthesis, as measured by electron paramagnetic resonance spectroscopy (EPR). The visible changes in skin color and the histologically appreciable switch-off of melanin formation during the anagen-catagen transformation were accompanied by a steep decline in 1) the melanin-associated EPR signal of full-thickness mouse skin, 2) tyrosinase and dopachrome tautomerase activities, and 3) the skin concentration of 80-85-kD melanogenesis related protein and 66-68-kD tyrosinase protein. Telogen skin displayed a minimum of the EPR amplitude as well as of tyrosinase and dopachrome tautomerase activity detected. By EPR, only eumelanin was identified during all hair cycle stages. The gradual switch-off of melanogenesis during anagen VI started with an unexpectedly early decline of the EPR melanin signal, followed by dopachrome tautomerase activity and the concentration of 80-85-kD melanogenesis related protein. The initiation of catagen was characterized by a significant and rapid decrease in activity and concentration of tyrosinase, and was accompanied by a second drop in dopachrome tautomerase activity. Together, these biochemical and biophysical parameters of follicular melanogenesis serve as novel and differential markers for the imminent termination of anagen and the development of catagen. They also show that the switch-off of melanogenesis during the anagen-catagen-telogen transition is a stochastic process commencing already in mid anagen VI.

Pathogenesis of the platinum (cp) mutation, a model for oculocutaneous albinism.

The platinum mutation at the C (albino) locus in the mouse is a potential model for oculocutaneous albinism in humans other than type IA (tyrosinase-negative) albinism. Although tissues from mice homozygous for the mutation display substantial tyrosinase activity, cutaneous and ocular pigmentation is severely restricted in affected animals. Using specific antipeptide antisera, we demonstrate that ocular extracts from wild-type mice contain two isoforms of tyrosinase bearing either the amino-terminal PEP5 epitope or the carboxy-terminal PEP7 epitope. The latter isoform participates in a high-molecular-weight complex detectable on sucrose density gradients. In platinum mice, antiserum to the PEP7 epitope fails to recognize any protein species, and the high-molecular-weight form of tyrosinase is not detectable. Our results support a key role for this high-molecular-weight complex in melanogenesis, and suggest that mutations that interfere with the ability of tyrosinase to participate in a multimeric complex may be a cause of oculocutaneous albinism in people.

High-molecular-weight forms of tyrosinase and the tyrosinase-related proteins: evidence for a melanogenic complex.

Tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2, (TRP-2, dopachrome tautomerase) were shown by immunoblotting and enzyme assays to copurify from extracts of Cloudman S91 melanoma cells. Antibodies to TRP-1 and TRP-2 immunoprecipitated tyrosinase activity, suggesting a stable interaction (complex) among these proteins. The tyrosine hydroxylase activity of tyrosinase was reduced in the complexed form; treatment with Triton X-100 dissociated the complex and activated the tyrosinase present within it. To further study this complex, we employed sucrose gradient density centrifugation of extracts from cultured murine melanocytes. Tyrosinase, TRP-1, and TRP-2 all existed in high molecular weight "multimers" of approximately 200 to > 700 kilodaltons. Extraction of cells with buffers containing the detergent CHAPS preserved the high molecular weight multimers; Triton X-100 caused their dissociation into monomers. Low pH, low ionic strength, and millimolar concentrations of calcium ions favored the maintenance of multimers. The results of this study demonstrate that the participation of tyrosinase, TRP-1, and TRP-2 in a multimeric complex could have important physiologic consequences, and raise the possibility that some of the well-known interactions between coat color genes may be explained by intermolecular interactions between the gene products.

Opposite effects of prolactin and corticosterone on the expression and activity of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase in rat skin.

In rat skin, type IV is the major 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) isoenzyme expressed. Although types I and II 3 beta-HSD mRNAs are also present in the skin, their level of expression is about two orders of magnitude lower than that of type IV. In this study, we have investigated the control of type IV 3 beta-HSD mRNA levels as well as 3 beta-HSD enzymatic activity in hypophysectomized adult rats of both sexes. Skin 3 beta-HSD activity was measured by the conversion of [14C]-dehydroepiandrosterone into [14C]-androstenedione, whereas ribonuclease protection assay using a specific type IV cRNA probe was used to assess mRNA levels. Intact male and female rats show a similar level of skin 3 beta-HSD activity, although hypophysectomy caused opposite effects, a decrease being observed in males while an increase was observed in hypophysectomized female animals. We next studied the effects of hyperprolactinemia, corticosterone and 1-thyroxine in hypophysectomized animals. L-thyroxine was found to stimulate 3 beta-HSD expression and activity in male rats whereas no significant effect was observed on the already elevated levels in hypophysectomized female rats. Corticosterone caused an inhibition of type IV 3 beta-HSD mRNA levels and activity in both male and female animals. Hyperprolactinemia achieved by pituitary implants inserted under the kidney capsule stimulated the expression of type IV mRNA as well as 3 beta-HSD enzymatic activity in hypophysectomized male and female animals. The present data demonstrate the multihormonal regulation of 3 beta-HSD/isomerase expression and activity in the rat skin.

Light microscopic immunocytochemical localization of 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase in the gonads and adrenal glands of the guinea pig.

The enzyme complex 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) is involved in the biosynthesis of all classes of active steroids, namely glucocorticoids, mineralocorticoids, progesterone, and sex hormone steroids. To obtain precise information about the cellular and subcellular localization of 3 beta HSD in the gonads and adrenals of the guinea pig, we have proceeded to immunocytochemical localization of the enzyme using antibodies developed against purified human placental 3 beta HSD. In the testis, specific immunostaining was restricted to the cytoplasm of the interstitial cells. In the ovary, on the other hand, immunostaining was found in the cytoplasm of the cells of the corpus luteum and theca interna. The granulosa cells of the follicles were not immunolabeled, with the exception of one layer of cells in contact with the zona pellucida. This restricted labeling was observed in growing and mature follicles, but not in primordial and small primary follicles. In the adrenals, the three zones of the cortex were labeled, whereas no staining could be detected in the medulla. Contrary to findings in the testis and ovary, staining was found in both the cytoplasm and nuclei of adrenocortical cells. The present data suggest a functional interaction between the ovocyte and the specialized layer of 3 beta HSD-containing granulosa cells which might play a role in ovocyte maturation.