Cloning and differential expression of steroid 5 alpha-reductase type 1 (Srd5a1) and type 2 (Srd5a2) from the Harderian glands of hamsters.

In hamsters, the Harderian glands (HGs) exhibit a marked sexual dimorphism which is thought to depend on dihydrotestosterone (DHT); however, it is unclear whether hamster HGs contain one or more 5 alpha-reductases and whether these enzymes are differentially expressed in males and females. In this study, we isolated specific cDNAs for 5 alpha-reductase 1 (Srd5a1) and 5 alpha-reductase 2 (Srd5a2), determined their sequences and investigated their expression in the HG of both sexes. Isozyme 1, cloned from liver mRNA, encodes a protein of 255 amino acids (aa); isozyme 2 cDNA, isolated from the epididymis encodes a 254-aa protein. When assayed in transfected HEK-293 cells, the type 1 isozyme displayed activity over a broad pH range (6.5-8), while isozyme 2 had a pH optimum of 5.5. Both isoenzymes efficiently catalyzed the in vitro transformation of T into DHT, with apparent K(m) values of 7.1 and 1.9 micromol/L for Srd5a1 and Srd5a2, respectively. Real-time PCR analysis revealed higher mRNA levels for Srd5a1 than for Srd5a2. Expression of both isoenzymes increased slightly in HGs of castrated males and showed variations during the estrous cycle in females. Hormonal replacement with 17beta-estradiol administered to spayed females induced the up-regulation of Srd5a2 mRNA levels. Altogether, our results demonstrated that both Srd5a1 and Srd5a2 are expressed in HGs without clear differences between males and females. The biochemical characteristics and relative expression of these 5 alpha-reductases support the view that both isozymes may play a relevant role in modulating androgen signaling in HG.

Inhibition of urethane-induced carcinogenicity in cyp2e1-/- in comparison to cyp2e1+/+ mice.

Urethane is an established animal carcinogen and has been classified as "reasonably anticipated to be a human carcinogen." Until recently, urethane metabolism via esterase was considered the main metabolic pathway of this chemical. However, recent studies in this laboratory showed that CYP2E1, and not esterase, is the primary enzyme responsible for urethane oxidation. Subsequent studies demonstrated significant inhibition of urethane-induced genotoxicity and cell proliferation in Cyp2e1-/- compared to Cyp2e1+/+ mice. Using Cyp2e1-/- mice, current studies were undertaken to assess the relationships between urethane metabolism and carcinogenicity. Urethane was administered via gavage at 1, 10, or 100 mg/kg/day, 5 days/week, for 6 weeks. Animals were kept without chemical administration for 7 months after which they were euthanized, and urethane carcinogenicity was assessed. Microscopic examination showed a significant reduction in the incidences of liver hemangiomas and hemangiosarcomas in Cyp2e1-/- compared to Cyp2e+/+ mice. Lung nodules increased in a dose-dependent manner and were less prevalent in Cyp2e1-/- compared to Cyp2e+/+ mice. Microscopic alterations included bronchoalveolar adenomas, and in one Cyp2e1+/+ mouse treated with 100 mg/kg urethane, a bronchoalveolar carcinoma was diagnosed. Significant reduction in the incidence of adenomas and the number of adenomas/lung were observed in Cyp2e1-/- compared to Cyp2e1+/+ mice. In the Harderian gland, the incidences of hyperplasia and adenomas were significantly lower in Cyp2e1-/- compared to Cyp2e+/+ mice at the 10 mg/kg dose, with no significant differences observed at the high or low doses. In conclusion, this work demonstrated a significant reduction of urethane-induced carcinogenicity in Cyp2e1-/- compared to Cyp2e1+/+ mice and proved that CYP2E1-mediated oxidation plays an essential role in urethane-induced carcinogenicity.

The influence of sex steroid hormones on ferrochelatase gene expression in Harderian gland of hamster (Mesocricetus auratus).

Ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the insertion of ferrous iron into protoporphyrin IX to form protohaem. The Syrian hamster Harderian gland (HG) is known for its ability to produce and accumulate large amounts of protoporphyrins. In this species, the female gland contains up to 120 times more porphyrin than the male gland. Data from biochemical studies suggest that this gland possesses the enzymatic complex for haem biosynthesis but lacks ferrochelatase activity. The abundance of intraglandular haem proteins does not support this idea. To gain more insight into this process, we isolated cDNA for ferrochelatase from hamster liver, using the 5'- and 3'- rapid amplification of complementary DNA ends (RACE), and investigated its expression in HG from males and females. The full-length cDNA comprises an open reading frame of 1269 bp encoding a polypeptide of 422 amino-acid residues. Hamster DNA sequence exhibits 92% identity to mouse and 87% identity to human sequences. The predicted hamster enzyme was shown to have structural features of mammalian ferrochelatase, including a putative NH2- terminal presequence, a central core of about 330 amino-acid residues and an extra 30-50-amino-acid stretch at the carboxyl-terminus. RNA blotting experiments indicated that this cDNA hybridized to a liver mRNA of about 2.1 kb, while a weak hybridization signal was observed with mRNA from HG preparations. RT-PCR assays confirmed the expression of specific transcripts in both tissues. Male glands contained approximately twofold more enzyme mRNA than female glands. Likewise, the intraglandular content of mRNA varied during the oestrous cycle, with the highest levels found in the oestrous phase. These cyclic variations were less evident in liver. Ovariectomy plus treatment with progesterone or 17beta-oestradiol plus progesterone increased ferrochelatase mRNA of the gland. In HG of short- or long-term castrated males, the administration of testosterone did not affect the ferrochelatase mRNA concentration. Based on mRNA expression levels, we conclude that Harderian ferrochelatase may play an active role in maintaining the physiological pool of haem required for processing cytochromes and other glandular haem proteins. Likewise, the sex-steroid hormones appear to have only a modest influence upon Harderian ferrochelatase.

The quaternary structure and activity of newly purified fatty acid synthetase from the Harderian gland of guinea-pig.

Fatty acid synthetase was isolated from the Harderian gland of guinea-pig. The fatty acids synthesized by the purified enzyme were analyzed by mass fragmentography. The purified enzyme had an inherent capacity to utilize methylmalonyl-CoA and synthesize methyl-branched fatty acids. Physicochemical studies indicated that an active enzyme was a dimer, consisted of two subunits of Mr = 2.5 X 10(5). The negatively stained enzyme had an electron micrographic image of an ellipsoidal contour with a continuous middle cleft along the major axis. The major and minor axes were approximately equal to 220 and 150 A, respectively. In a dimer, the subunit had a rod-like structure about 220 A long and 50 A wide. The enzyme was inactivated and dissociated into subunits by incubation at 0 degree C. The inactivated enzyme was fully reactivated by raising the temperature of the solution. The relationship between the quaternary structure of the enzyme and the occurrence of enzymatic activity was studied by high-performance liquid chromatography. Neither active monomers nor inactive dimers were found in inactivation and reactivation processes. The initial velocity of reactivation was proportional to the enzyme concentration over a concentration range of 160-800 micrograms/ml, indicating that the rate-determining step in the reactivation reaction was unimolecular.

A short-chain acyl-CoA: 1-alkyl-2-acyl-sn-glycerol acyltrasnferase from a microsomal fraction of the rabbit Harderian gland.

The 3-position of the alkyldiacylglycerols from the pink portion of the rabbit harderian gland is occupied exclusively by isovaleric acid. We describe a microsomal 1-alkyl-2-acyl-sn-glycerol acyltransferase from this gland which specifically incorporates short-chain acyl-CoA's into the 3-position of alkylacyl-glycerols. The enzyme is most active in the presence of CoA esters with chain lengths similar to isovaleric acid and is inactive in the presence of acetyl CoA and long-chain acyl-CoA's. No evidence was found for an enzyme that would transfer long-chain acyl-CoA's to the same substrate. The specificity of this acyltransferase can account for the exclusion of long-chain acyl moieties from the 3-position of the alkyldiacylglycerols in the harderian gland of rabbits.

N-Acetyltransferase activity of the rat Harderian gland.

Harderian gland extracts from male rats catalyze the conversion of serotonin to N-acetylserotonin and of tryptamine to N-acetyltryptamine. The reaction is linear up to 14 mg tissue and departs from linearity after 10 min. The pH otpimum with tryptamine as substrate is between 8 and 9. Enzymic activity of the gland in vivo does not show diurnal variations. Enzymic activity of tissue in organ culture is not stimulated by 10 micrometer isoproterenol or 100 micrometer dibutyryl cyclic AMP. Harderian gland tissue in culture can acetylate tryptamine and serotonin and can O-methylate the N-acetylserotonin to form melatonin.

Hematopoietic prostaglandin D2 synthase in the chicken Harderian gland.

The Harderian gland (HG), a sero-mucous secreting organ in the eye orbit, has long been recognized as immunologically important in chickens. During experimentation to characterize immune components of the gland, proteomics analysis revealed the presence of hematopoietic prostaglandin D synthase (H-PGDS). Extraction of total RNA followed by RT-PCR produced cDNA of 597 base pairs. DNA sequencing revealed nucleic acid and predicted amino acid sequences that were 99% aligned with the one published sequence for chicken H-PGDS of the spleen. Alignment with murine, rat, and human H-PGDS were 69, 69, and 66%, respectively. Ocular vaccination of chickens with a Newcastle Disease/Infectious Bronchitis vaccine (Mass.-Ark. Strain) induced an increase in H-PGDS expression determined by real-time PCR. Furthermore, immunohistochemistry of frozen HG sections showed positive stained cells for both H-PGDS and mast cell tryptase in the sub-epithelial cell layers of the HG ducts. Based on the potent vasoactive role of PGD(2), it appears that the chicken HG is a site of active mucosal immunity partially mediated by PGD(2) synthesized by H-PGDS in the gland.

Hormonal regulation and characterization of MHG30 gene, a desaturase-like gene of hamster harderian gland.

The harderian gland (HG) is an orbital gland of the vast majority of land vertebrates. In the Syrian hamster these glands display a marked sexual dimorphism. Here we present data on a male specific clone named MHG30. The MHG30 cDNA (1470 bp) has significant sequence homologies with human #15µ10#Δ6-desaturase enzymes. The expression of MHG30 has been found in male HG and in the liver of both sexes, no other tissue showing the presence of MHG30 mRNA. Castration brings the MHG30 levels below detectable level in about 7 days. In in vitro cultures of male hamster HG cells, androgens (A) determine an enhancement of MHG30 expression in a time-dependent manner. Conversely, a continuous decrement has been observed in control cells and in cells treated with A plus flutamide (F) or with A and cycloheximide (Cy). Incubation of cells in cultures supplemented with desamethason (Dex) or thyroid hormone (T3) also increases MHG30 expression while 17ß-estradiol prevents the stimulatory effect exerted by A, Dex and T3. Findings strongly suggest that the MHG30 gene could be involved in supporting the sexual dimorphism and its expression is likely triggered by a series of hormonal interactions.

Melatonin protects against delta-aminolevulinic acid-induced oxidative damage in male Syrian hamster Harderian glands.

Effects of the prooxidant delta-aminolevulinic acid (ALA) and the antioxidant melatonin (MEL) were investigated in the male Syrian hamster Harderian gland (HG). Rodent Harderian glands are highly porphyrogenic organs, which may be used as model systems for studying damage by delta-aminolevulinic acid and its metabolites, as occurring in porphyrias. Chronic administration of delta-aminolevulinic acid (2 weeks) markedly decreased activities of the porphyrogenic enzymes delta-aminolevulinate synthase (ALA-S) and delta-aminolevulinate dehydratase (ALA-D) and of the antioxidant enzymes superoxide dismutase (SOD), glutathione reductase (GR) and catalase (CAT), whereas porphobilinogen deaminase (PBG-D) remained unaffected. This treatment led to increased lipid peroxidation (LPO) and oxidatively modified protein (protein carbonyl) as well as to morphologically apparent tissue damage. Melatonin also caused decreases in delta-aminolevulinate synthase, delta-aminolevulinate dehydratase, superoxide dismutase, glutathione reductase and catalase. Despite lower activities of antioxidant enzymes, lipid peroxidation and protein carbonyl were markedly diminished. The combination of delta-aminolevulinic acid and melatonin led to approximately normal levels of delta-aminolevulinate dehydratase, glutathione reductase, catalase and protein carbonyl, and to rises in superoxide dismutase and porphobilinogen deaminase activities; lipid peroxidation remained even lower than in controls and the appearance of the tissue revealed a protective influence of melatonin. These results suggest that melatonin may have profound effects on the oxidant status of the Harderian gland.

Physiological oxidative stress model: Syrian hamster Harderian gland-sex differences in antioxidant enzymes.

The Syrian hamster Harderian gland, a juxtaorbital organ exhibiting marked gender-associated differences in contents of porphyrins and melatonin, was used as a model system for comparing strong (in females) and moderate (in males) physiological oxidative stress. Histological differences showing much higher cell damage in females were studied in conjunction with lipid peroxidation and activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Lipid peroxidation and enzyme activities were measured throughout the circadian cycle, revealing the importance of dynamical processes in oxidative stress. Especially in lipid peroxidation and in catalase, short-lasting rises exhibited strongest gender differences. Peaks of lipid peroxidation were about three times higher in females, compared to males. Catalase peaks of females exceeded those in males by several hundred-fold. Average levels of superoxide dismutase and glutathione peroxidase were about three or two times higher in females, respectively. A clear-cut diurnally peaking rhythm was found in glutathione peroxidase of females, which was not apparent in males. Glutathione reductase showed differences in time patterns, but less in average activities. The time courses of lipid peroxidation and of protective enzymes are not explained by circulating melatonin, whereas melatonin formed in the Harderian gland should contribute to differences in average levels. Neither damage nor antioxidative defense simply reflect the illumination cycle and are, therefore, not only a consequence of photoreactions.

Expression of type II thyroxine 5'-deiodinase from rat harderian gland in Xenopus laevis oocytes.

The presence of isoenzymes mediating the conversion of thyroxine to 3,5,3'-triiodothyronine has been studied according to characteristic kinetics and physiological regulation. In this paper, we report the expression of type II 5'-deiodinase (5'D) activity in oocytes of Xenopus laevis. Oocytes injected with total RNA extracted from rat Harderian gland, and then incubated up to five days demonstrated a progressive increase in 5'D activity, reaching a maximal value at 24 h; then, 5'D activity remained almost stable for an additional period of four days. Characteristics of the enzyme activity expressed by oocytes included its inhibition by iopanoic acid, but not by propylthiouracil, and its increase during beta-adrenergic agonist treatment and hypothyroidism. The expressed activity manifests characteristics typical of the type II isoenzyme. Deiodinating activity in oocytes also exhibited diurnal variations. In this study, 5'D activity expressed in oocytes exhibited low values when animals were killed during the day, and high values when animals were killed at night. Maximal values were reached 3-4 h before the nocturnal peak of 5'D activity in Harderian gland crude homogenates. Results suggest that the in vivo activation of 5'D by isoproterenol, hypothyroidism, or dark exposure may be caused by an increase in the synthesis and/or maturation of the RNA expressing the enzyme.

Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase.

In this paper we describe isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase (DAP-AT). The enzyme was extracted from rabbit Harderian gland peroxisomes and isolated as a trimeric complex by sucrose density gradient centrifugation. From peptide sequences matching EST-clones were obtained which allowed cloning and sequencing of the cDNA from a human cDNA library. The nucleotide-derived amino acid sequence revealed a protein consisting of 680 amino acid residues of molecular mass 77187 containing a C-terminal type 1 peroxisomal targeting signal. Monospecific antibodies raised against this polypeptide efficiently immunoprecipitated DAP-AT activity from solubilized peroxisomal preparations, thus demonstrating that the cloned cDNA codes for DAP-AT.

Effects of delta-aminolevulinic acid and melatonin in the harderian gland of female Syrian hamsters.

Effects of delta-aminolevulinic acid (ALA) and melatonin were investigated in the female Syrian hamster Harderian gland. This is an organ physiologically exposed to strong oxidative stress due to the highest porphyrinogenic rates known in nature. Enzyme activities of porphyrin biosynthesis and of antioxidative protection, oxidative protein modification, and histological integrity were studied. In the porphyrin biosynthetic pathway, ALA and melatonin acted synergistically by downregulating ALA synthase (ALA-S) and stimulating product formation from ALA; the combination of ALA and melatonin suppressed ALA-S activity, down to about 1% of that in controls. While ALA effects on porphyrinogenesis can be interpreted in terms of homeostasis, melatonin's actions may be seen in relation to seasonality and/or reduction of oxidative stress. Among antioxidant enzymes, superoxide dismutase (SOD) and glutathione reductase (GR) activities were diminished by ALA, presumably due to the vulnerability of their active centers to free radicals, whereas melatonin moderately increased SOD. Both ALA and melatonin strongly stimulated catalase (CAT), thereby counteracting the oxidative stress induced by ALA and its metabolites. Nevertheless, exogenous ALA caused a strong net rise in protein carbonyl and considerable damage of tissue. When given together with ALA, melatonin antagonized these effects and largely protected the integrity of glandular structures.

Melatonin specifically stimulates type-II thyroxine 5'-deiodination in brown adipose tissue of Syrian hamsters.

The response of type-II thyroxine 5'-deiodinase (5'-DII) to melatonin treatment was studied in the Syrian hamster. Male hamsters were treated for 15 days with a s.c. pellet containing melatonin, and 5'-DII activity in brown adipose tissue, anterior pituitary gland, Harderian gland and pineal gland was measured using a radioenzymatic technique. Melatonin-treated animals exhibited enhanced 5'-DII activity restricted to brown adipose tissue; the increase was threefold above the values measured in the control group. Serum concentrations of thyroid hormones were unaffected by melatonin treatment. We conclude that the stimulatory effect of melatonin on type-II thyroxine 5'-deiodination is specifically directed to the isoenzyme located in brown adipose tissue and is not accompanied by changes in serum thyroid hormones.

N-acetyltransferase activity in the Harderian glands of the Syrian hamster, Mesocricetus auratus, is regulated by androgens and by hormones of the pituitary-thyroid axis.

This study tested the hypothesis that activity of the enzyme N-acetyltransferase (NAT) in the Harderian gland of the Syrian hamster is regulated both by androgens and by hormones of the pituitary-thyroid axis. To test the effects of castration and hypothyroidism, intact or castrated male hamsters were given either tap water or methimazole in their drinking water for 3 weeks. Methimazole suppresses iodination of thyroglobulin, thereby decreasing circulating levels of thyroid hormones and increasing TSH levels. Hypothyroidism or castration caused elevated or depressed Harderian gland NAT activities respectively, compared with euthyroid controls. When castration and hypothyroidism were combined, the animals exhibited high NAT activity compared with castrated euthyroid males. To test the effects of castration and hyperthyroidism, male hamsters were given daily injections of thyroxine (T4) or diluent and were either castrated or left intact for 4 weeks. Intact animals given T4 had depressed Harderian NAT activity; serum thyroid hormone levels were elevated and TSH levels were depressed compared with those of intact controls. Castrated animals had depressed NAT activity below that of intact controls; serum thyroid hormone levels were normal but TSH levels were depressed. Castrated animals given T4 injections had NAT activity similar to that of euthyroid castrated hamsters; thyroid hormone levels were elevated but TSH levels were similar to those seen in euthyroid castrated hamsters. In another experiment, both T4 and tri-iodothyronine (T3) were equally effective in decreasing NAT activity in intact males. To determine the effects of the removal of pituitary influences, male hamsters were hypophysectomized. NAT activity in the Harderian glands of these animals was reduced compared with intact controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel regulation of delta-aminolevulinate synthase in the rat harderian gland.

The mode of expression of delta-aminolevulinate synthase (ALAS), as well as that of mRNAs for other heme pathway enzymes, was examined in the rat Harderian gland. Northern blot and in situ hybridization analyses demonstrated that the non-specific ALAS (ALAS-N) mRNA is highly expressed in this tissue, whereas the erythroid-specific ALAS (ALAS-E) mRNA is not. Immunoblot analysis of ALAS also confirmed this finding at the protein level. ALAS-N mRNA was maximally induced in the Harderian gland and was not increased further by treatment of animals with 2-allyl-2-isopropylacetamide (AIA). The levels of mRNAs for other heme pathway enzymes, i.e., delta-aminolevulinate dehydratase, porphobilinogen deaminase, uroporphyrinogen decarboxylase, and coproporphyrinogen oxidase, also were increased markedly in the Harderian gland and not influenced by AIA treatment. The level of ferrochelatase (FeC) mRNA in the gland was, however, lower than that in the liver. The gland contained an extremely high level of protoporphyrin, while heme was undetectable. Microsomal heme oxygenase-1 (HO-1) mRNA levels were significantly higher in the Harderian gland than in the liver. When isolated glands were incubated with hemin in vitro in organ cultures, the level of HO-1 mRNA was increased, whereas the ALAS-N mRNA level was not. These findings indicate that markedly elevated levels of protoporphyrin and extremely low levels of heme in the Harderian gland are the results of both decreased expression of FeC and markedly increased expression of ALAS-N and HO-1. The constitutive expression of the ALAS-N gene in the Harderian gland suggests a novel transcriptional control mechanism of this gene.

Effect of estrogenic perturbations on delta-aminolevulinic acid-induced porphobilinogen deaminase and protoporphyrin IX levels in rat Harderian glands, liver, and R3230AC tumors.

The Harderian gland in rodents highly expresses enzymes of the heme biosynthetic pathway that are responsible for porphyrin production. Interestingly, many of the steps in Harderian gland heme biosynthesis, including protoporphyrin production, are controlled hormonally. We hypothesized that estrogenic alterations, ovariectomy or tamoxifen administration, might also alter the response of porphobilinogen deaminase activity and/or protoporphyrin IX production to delta-aminolevulinic acid administration in the hormonally responsive R3230AC rat mammary adenocarcinoma. We also determined whether the response of the R3230AC tumor, borne on ovariectomized hosts, to delta-aminolevulinic acid-based photodynamic therapy was altered compared with tumors treated on intact hosts. Ovariectomy of female Fischer rats bearing the hormonally responsive R3230AC mammary adenocarcinoma caused a significant reduction in delta-aminolevulinic acid-induced protoporphyrin IX levels and porphobilinogen deaminase activity in tumors compared with levels in tumors from intact animals treated with delta-aminolevulinic acid. In contrast, although porphobilinogen deaminase activity in the Harderian gland from ovariectomized animals was reduced significantly compared with that in glands from intact animals, protoporphyrin IX levels were unaltered. Administration of the anti-estrogen tamoxifen to tumor-bearing rats resulted in a significant increase in porphobilinogen deaminase in both tumor and Harderian gland. Although administration of delta-aminolevulinic acid increased protoporphyrin IX levels in Harderian glands in tamoxifen-treated animals, tumor levels of protoporphyrin IX remained unaltered in the tamoxifen-treated rats. Treatment of R3230AC tumors with delta-aminolevulinic acid-based photodynamic therapy in ovariectomized rats resulted in a significantly reduced response compared with the same treatment regimen in intact animals, 4.9+/-0.39 versus 10.6+/-0.6 days to reach twice the initial tumor volume, respectively. These results indicate that the hormonal status of the host should be considered when treating hormonally sensitive tumors with delta-aminolevulinic acid-based photodynamic therapy.

5-aminolevulinate synthase mRNA levels in the Harderian gland of Syrian hamsters: correlation with porphyrin concentrations and regulation by androgens and melatonin.

The levels of 5-aminolevulinate synthase mRNA were investigated in the Harderian glands of male and female Syrian hamsters by using a cDNA clone from rat liver. Female hamsters showed higher levels of mRNA than those in males, while the administration of testosterone to female hamsters led to a reduction in mRNA levels. Castration of male hamsters caused a marked elevation of mRNA levels, whereas both the exposure to constant darkness or melatonin injections to castrated males partially prevented the effects of castration. Porphyrin concentration of Harderian glands showed a strong correlation with the mRNA levels of 5-aminolevulinate synthase in all the animals studied. These results lead to the conclusion that in this system, porphyrin metabolism is controlled through hormonal regulation of 5-aminolevulinate synthase gene expression.

Gender-associated differences in the development of 5-aminolevulinate synthase gene expression in the harderian gland of Syrian hamsters.

The mRNA levels for aminolevulinate synthase (ALV-S), the rate-limiting enzyme in porphyrin synthesis, were studied in male and female Syrian hamsters during postnatal development. Sex-associated differences in the expression of ALV-S gene were evident at the end of the third week of postnatal development. Serum levels of luteinizing hormone (LH), testosterone, cortisol, thyroid hormones and insulin-like growth factor were also studied in order to correlate their concentrations with the mRNA levels for ALV-S. Among these hormones, serum LH levels showed a positive correlation with the ALV-S mRNA levels. However, the expected negative correlation with testosterone levels was not clearly observed. Thus, in order to test the effects of testosterone on ALV-S gene expression, 11-day-old male and female Syrian hamsters and adult female hamsters were injected with 50 micrograms of testosterone for 4 days. Testosterone administration decreased the levels of ALV-S mRNA in the adult females but did not influence those of young females. The possible explanation for the insensitivity to testosterone during these postnatal stages might involve the maturational state of androgen receptors in the Harderian glands.

Characterization of sn-glycerol 3-phosphate acyltransferase from guinea pig harderian gland microsomes.

Microsomal sn-glycerol 3-phosphate acyltransferase from the guinea pig Harderian gland was studied. Its specific activity (1.0 nmol/min X mg, with palmitoyl-CoA as a substrate) was almost the same as that of the rat liver microsomal enzyme. The enzyme acted on various types of acyl-CoA, the relative reaction rates being as follows: palmitoyl-CoA, 100(%); stearoyl-CoA, 30; oleoyl-CoA, 50; linoleoyl-CoA, 40; and arachidonoyl-CoA, 20. When assayed in the presence of 1 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), the activity on palmitoyl-CoA was inhibited by only 20-30%, whereas those for other acyl-CoAs were completely abolished. The DTNB-resistant activity was inhibited by 0.1 mM dihydroxyacetonephosphate and 0.5 mM dithiothreitol, whereas the DTNB-sensitive activity was not affected. Furthermore, heat treatment at 50 degrees C for 15 min abolished most of the DTNB-sensitive activity, but not the DTNB-resistant activity. These results, taken together, suggested that the microsomal fraction of the guinea pig Harderian gland contained at least two types of sn-glycerol 3-phosphate acyltransferase, and that, in contrast to in the case of rat liver microsomes, a DTNB-resistant enzyme that utilized exclusively palmitoyl-CoA was predominant.