Secretory cell types and cell proliferation of human bronchial epithelial cells in an organ-culture system

We examined the localization of steroidogenic cells in rainbow trout (Oncorhynchus mykiss) testis during spermatogenesis by using polyclonal antibodies generated against rainbow trout cholesterol side-chain cleavage enzyme cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17α-hydroxylase/C17,21 lyase (P450c17), and aromatase cytochrome P450 (P450arom) as markers of steroid production. Since we had previously produced specific antibodies against 3β-HSD and P450arom, antibodies against oligopeptides corresponding to C-terminal sequences of P450scc and P450c17, predicted from rainbow trout P450scc and P450c17 cDNAs, were produced in this study. These two antibodies recognized 54-kDa (P450scc) and 59-kDa (P450c17) bands specifically in several steroidogenic organs, i.e., testis, ovary, and interrenal tissue (head kidney) in Western blots. Immunohistochemically, immunoreactive P450scc, P450c17, and 3β-HSD, but not P450arom, were found only in interstitial Leydig cells of immature and mature testes. Immunoreactive P450arom was not detected in either testis. This study suggests that Sertoli cells and germ cells of rainbow trout testis do not contain P450scc, P450c17, P450arom, or 3β-HSD.     

EST analysis of gene expression in testis of the ascidian Ciona intestinalis

To explore the gene expression underlying spermatogenesis, a large-scale analysis has been done on the cDNAs from testis of the ascidian, Ciona intestinalis. A set of 5,461 expressed sequence tags was analyzed and grouped into 2,806 independent clusters. Approximately 30% of the clusters showed significant sequence matches to the proteins reported in DDBJ/GenBank/EMBL database including a set of proteins closely related to the gene regulation during spermatogenesis, functional and morphological changes of spermatogenic cells during spermiogenesis, and physiological functions of sperm, as well as those with housekeeping functions commonly expressed in other cells. Some clones show similarities to the proteins present in vertebrate lymphocytes, suggesting a primitive immune system in ascidians. We have also found some genes that are known to participate in hormonal regulation of spermatogenesis in vertebrates. The large majority of the genes expressed in Ciona testis show no significant matches to known proteins and the further analysis of these genes may shed new light on the molecular mechanism of spermatogenesis and sperm functions.     

Effect of lithium chloride on spermatogenesis and testicular delta 5-3 beta, 17 beta-hydroxysteroid dehydrogenase activities in the 'antiserum to luteinizing hormone' treated toad (Bufo melanostictus)

Activities of delta 5-3 beta- and 17 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD and 17 beta-HSD), Leydig cell nuclear area (LCNA) and spermatogenesis in the testis were observed after injection of lithium chloride in the 'antiserum to luteinizing hormone (LH)' treated toad. A significant decrease in the activities of steroidogenic enzymes, LCNA and spermatogenesis were noticed after the injections of 'antiserum to LH' to toads. Further decrease in the activities of the above parameters was observed in the lithium chloride--'antiserum to LH' treated toad. It is suggested that lithium chloride may inhibits testicular function without modulating the pituitary activity.     

Feminization of the quail by early diethylstilbestrol treatment: histoenzymological investigations on steroid dehydrogenases in the gonads.

Early treatment of Quail eggs by DES promotes a transient feminization of the gonads in genetic males and a strong stimulation of the Müllerian ducts. The left ovotestis results from the juxtaposition of a testicular medulla and an induced female-type cortex, which develops follicles and a characteristic 17 beta-HSD activity. The right testis is reduced but keeps a consistent structure. The medulla of the treated gonads shows, in both sexes, an inhibition of delta 5-3 beta HSD activity during embryonic development. After hatching, this specific enzyme then develops in the steroidogenic cells. These results are compared with others obtained with estradiol and also in chick. The discussion deals also with the effects of these estrogens on the endogenous abilities and specific responses of the gonads in relation to sex differentiation factors.     

Effects of gold on testicular steroidogenic and gametogenic functions in immature male albino rats

The present study was undertaken to evaluate the effects of gold chloride, a metallic earth salt, on steroidogenic and gametogenic functions of testis in immature rats. Immature rats of Wistar strain, were injected (s.c.) with gold chloride at the dose of 0.3 mg and 0.5 mg/kg body weight/day for 26 days. All the treated animals along with the vehicle-treated controls were sacrificed 24 hours after last injections. Testicular steroidogenic activity was evaluated by measuring the activities of two steroidogenic key enzymes, Delta5-3beta-hydroxysteroid dehydrogenase (Delta5-3beta-HSD) and 17-beta hydroxysteroid dehydrogenase (17-beta HSD). Gametogenic capacity was determined by counting the number of germ cells at stage VII of seminiferous cycle. Plasma levels of testosterone (T) was measured by radioimmunoassay (RIA). Administration of gold chloride at a dose of 0.3 mg/ kg body weight for 26 days led to insignificant changes of testicular Delta5-3beta-HSD,17beta-HSD activities and gametogenesis along with plasma T. In contrast 0.5 mg gold chloride treatment for 26 days caused a significant increase in plasma T (p < 0.001) along with stimulation of testicular Delta5-3beta-HSD activity (p < 0.001) and 17beta-HSD activity (p < 0.001). Gametogenic activity exhibited a significant increase in the number of step 7 spermatids (7Sd) (p < 0.001) at stage VII of seminiferous cycle when compared to control. The results of our experiment suggest that gold chloride treatment might be associated with significant stimulatory effects on testicular activities. Furthermore, since hormonal changes, altered steroidogenic enzymes and gametogenic activities were evident to a specific dose of gold chloride treatment, our data may have some clinical implication on the stimulation of fertility.     

Allorecognition mechanisms during ascidian fertilization

Ascidians (primitive chordates) are hermaphroditic animals, releasing sperm and eggs nearly simultaneously. But, many ascidians, including Ciona intestinalis and Halocynthia roretzi, show self-sterility or preference for cross-fertilization rather than self-fertilization. The molecular mechanisms underlying this allorecognition process are only poorly understood. We recently identified the genes responsible for self-incompatibility in C. intestinalis by a positional cloning: sperm-borne polycystin 1-like receptor, referred to as s-Themis, and its fibrinogen-like ligand called v-Themis on the vitelline coat (VC) are highly polymorphic and appear to be responsible for allorecognition in the fertilization of C. intestinalis. In H. roretzi, on the other hand, we revealed that HrVC70, a 70-kDa main component of the VC consisting of 12 epidermal-growth-factor (EGF)-like repeats, is a candidate allorecognition protein, since the attachment of this protein to the VC during oocyte maturation and its detachment by weak acid are closely linked to the gain and the loss of self-sterility, respectively, and also since nonself-sperm rather than self-sperm efficiently bound to HrVC70-agarose. As a binding partner of HrVC70, a 35-kDa GPI-anchored glycoprotein in sperm lipid rafts, referred to as HrUrabin, was identified: HrUrabin appears to play a key role in allorecognizable sperm binding to HrVC70 during fertilization. In the present review, we describe the current progress on the molecular bases of allorecognition, or self-incompatibility, during ascidian fertilization, by considering the SI systems in another organisms including fungies and flowering plants.     

Expression of steroidogenic enzymes and synthesis of sex steroid hormones from DHEA in skeletal muscle of rats

The functional importance of sex steroid hormones (testosterone and estrogens), derived from extragonadal tissues, has recently gained significant appreciation. Circulating dehydroepiandrosterone (DHEA) is peripherally taken up and converted to testosterone by 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD, and testosterone in turn is irreversibly converted to estrogens by aromatase cytochrome P-450 (P450arom). Although sex steroid hormones have been implicated in skeletal muscle regulation and adaptation, it is unclear whether skeletal muscles have a local steroidogenic enzymatic machinery capable of metabolizing circulating DHEA. Thus, here, we investigate whether the three key steroidogenic enzymes (3beta-HSD, 17beta-HSD, and P450arom) are present in the skeletal muscle and are capable of generating sex steroid hormones. Consistent with our hypothesis, the present study demonstrates mRNA and protein expression of these enzymes in the skeletal muscle cells of rats both in vivo and in culture (in vitro). Importantly, we also show an intracellular formation of testosterone and estradiol from DHEA or testosterone in cultured muscle cells in a dose-dependent manner. These findings are novel and important in that they provide the first evidence showing that skeletal muscles are capable of locally synthesizing sex steroid hormones from circulating DHEA or testosterone.     

Dose-response effects of phytoestrogens on the activity and expression of 3beta-hydroxysteroid dehydrogenase and aromatase in human granulosa-luteal cells

There is evidence that certain phytoestrogens can inhibit key steroidogenic enzymes although most studies have been carried out on microsomal or purified enzyme preparations, some using cell lines. This study was designed to test the hypothesis that low doses of phytoestrogens, at concentrations that would be attained through the diet, could inhibit 3beta-hydroxysteroid dehydrogenase (HSD) and/or aromatase in primary cultures of human granulosa-luteal (GL) cells and that this effect was due to a decrease in the expression of these proteins. Based on published evidence, eight compounds were selected for investigation and these included the flavones apigenin and quercetin, the isoflavones genistein, biochanin A and daidzein, the lignans, enterodiol and enterolactone, and the mycotoxin zearalenone. Human GL cells were cultured for 48 h in the presence of these phytoestrogens at concentrations ranging from 0.01 to 100 microM and after addition of fresh media the conversion of pregnenolone to progesterone or androstenedione to oestradiol over a 4h period was measured. Biochanin A was the only phytoestrogen that displayed any dose-dependent inhibition of 3beta-HSD, others showing inhibition at doses >/=10 microM. Apigenin and quercetin only inhibited aromatase/17beta-HSD at high doses as did genistein, biochanin A and daidzein. The lignans had weak inhibitory effects on aromatase/17beta-HSD, whilst zearalenone showed potent inhibition at 0.1 microM. Phytoestrogens did not exert any significant effects on protein expression of 3beta-HSD or aromatase as determined by Western blots. It is concluded that steroidogenic enzymes are inhibited by phytoestrogens in primary cultures of human GL cells but these cells are less sensitive to the effects of phytoestrogens than cell-free systems. This may be due to poor lipid solubility or cellular metabolism. We have also shown for the first time that phytoestrogens do not act by inhibiting the cellular concentration of 3beta-HSD and aromatase even though exposure time would have allowed for changes in gene expression.     

The effects of estradiol and progesterone on rat ovarian 17-hydroxylase and 3 beta-hydroxysteroid dehydrogenase activities

Testosterone biosynthesis by Leydig cells can be modulated by estradiol. This modulation appears to occur at the 17-hydroxylase and 17,20-desmolase stage. In this study we have examined the effects of estradiol and progesterone on the activities of the 17-hydroxylase (17-OH) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in rat ovarian tissue, to examine the hypothesis that estradiol may regulate these enzymes in the ovary as well as in the testis. Estradiol capsule implants produced a decrease in 17-OH activity (0.5 +/- 0.05 vs. 2.1 +/- 0.1 nmol/mg protein/min, mean +/- SEM, p less than 0.001), and an increase in 3 beta-HSD activity (15.5 +/- 0.9 vs 9.7 +/- 0.7 nmol/mg protein/min p less than 0.001). Progesterone injections produced a decrease in both 17-OH (0.9 +/- 0.1 vs. 2.3 +/- 0.2 p less than 0.005) and 3 beta-HSD (2.5 +/- .4 vs. 8.6 +/- 0.5; p less than 0.005) activities. We conclude that estradiol decreases 17-OH activity in the ovary as it does in the testis. This, coupled with an increase in 3 beta-HSD may explain the pre-ovulatory increase in progesterone seen in many species. Progesterone seems to decrease the steroidogenic activity of the ovarian tissue, perhaps offering an explanation for the gonadotropin resistance seen in corpus luteus bearing ovaries.     

Oxidative 3alpha-hydroxysteroid dehydrogenase activity of human type 10 17beta-hydroxysteroid dehydrogenase

In vitro enzyme assays have demonstrated that human type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSD10) catalyzes the oxidation of 5alpha-androstane-3alpha,17beta-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17beta-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17beta-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17beta-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17beta-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3alpha-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17beta-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.     

Occurrence and possible biological role of the endocannabinoid system in the sea squirt Ciona intestinalis

A cannabinoid receptor orthologue (CiCBR) has been described in the sea squirt Ciona intestinalis. Here we report that CiCBR mRNA expression is highest in cerebral ganglion, branchial pharynx, heart and testis of C. intestinalis, and that this organism also contains cannabinoid receptor ligands and some of the enzymes for ligand biosynthesis and inactivation. Using liquid chromatography-mass spectrometry, the endocannabinoid anandamide was found in all tissues analysed (0.063-5.423 pmol/mg of lipid extract), with the highest concentrations being found in brain and heart. The endocannabinoid 2-arachidonoylglycerol (2-AG) was fivefold more abundant than anandamide, and was most abundant in stomach and intestine and least abundant in heart and ovaries (2.677-50.607 pmol/mg of lipid extract). Using phylogenomic analysis, we identified orthologues of several endocannabinoid synthesizing and degrading enzymes. In particular, we identified and partly sequenced a fatty acid amide hydrolase (FAAH) orthologue, showing 44% identity with human FAAH and containing nearly all the amino acids necessary for a functional FAAH enzyme. Ciona intestinalis also contained specific binding sites for cannabinoid receptor ligands, and an amidase enzyme with pH-dependency and subcellular/tissue distribution similar to mammalian FAAHs. Finally, a typical C. intestinalis behavioural response, siphon reopening after closure induced by mechanical stimulation, was inhibited by the cannabinoid receptor agonist HU-210, and this effect was significantly attenuated by mammalian cannabinoid receptor antagonists.     

Isolation of estrophilic fractions of hydroxysteroid dehydrogenase from rabbit liver

Estrophilic forms of rabbit liver cytosolic hydroxysteroid-dehydrogenase (HSD) were obtained as a highly purified preparations by means of fractionation with ammonium sulfate, gel-filtration, ion-exchange chromatography on DEAE-Sephadex A-50, affinity chromatography on estradiol-Sepharose and ion-exchange chromatography on DEAE-Toyopearl 650M. The protein express 4 different kinds of NADP-dependent activities: 3 alpha, 3 beta- and 17 beta-HSD activities with androgens and 20 alpha-HSD with progesterone as substrates. Revealed multiplicity of HSD enzymatic activity is demonstrated here for the first time. 17 beta-HSD activity of the protein preparations with estradiol is extremely low. Absence of a real metabolic activity of the protein with a ligand interacting with it rather intensively suggests that the isolated HSD forms can act not only as an enzyme, but also as a buffer-reserving mechanism for some steroids.     

Morphofunctional changes in Leydig cells throughout the continuous spermatogenesis of the freshwater teleost fish, Serrasalmus spilopleura (Characiformes, Characidae): an ultrastructural and enzyme study

The freshwater fish Serrasalmus spilopleura (piranha) has a continuous type of reproduction; gametes are constantly produced and released during the reproductive cycle. The testes do not undergo seasonal morphological changes but exhibit two constant regions throughout the year: the medullar region (involved with spermatogenesis) and the cortical region (involved with spermiation and sperm storage). We have evaluated the ultrastructure of the Leydig cells and the activity of 3beta-HSD (an essential enzyme related to steroid hormone biosynthesis) and acid phosphatase (AcPase; lysosomal marker enzyme) in these two regions. The activity of 3beta-HSD is stronger in the medullar region, and the Leydig cells in this region have a variety of cytological features that reflect differences in hormone synthesis and/or that could be linked to steroidogenic cells under various degrees of hormonal activity. In the cortical region, 3beta-HSD activity is weak and the Leydig cells exhibit signs of degeneration, as confirmed by their ultrastructure and intense AcPase activity. These degenerative signs are indicative of cytoplasmic remodelling to degrade steroidogenic enzymes, such as 3beta-HSD, that could lead to senescence or even to autophagic cell degeneration. S. spilopleura thus constitutes an interesting model for increasing our understanding of steroidogenesis control in freshwater teleost fish.     

Testicular organogenesis in the fetal calf: interstitial endocrine (Leydig) cell development

The development of the interstitial endocrine (Leydig) cells of the fetal testis in the calf is described and correlated with a swelling reaction of the gubernaculum and normal, prenatal descent of the testis. An hydroxysteroid dehydrogenase (HSD) procedure is used to determine the onset of functional activity for the interstitial endocrine cells (IEC). The NADH control procedure was strongly positive for the IECs at all ages investigated, indicating that these cells utilize the pyridine nucleotide as a coenzyme for oxireduction conversions. The 3 alpha- and 3 beta-HSD reactions were strongly positive and lightly positive, respectively, demonstrating that these cells contain the HSDs commonly utilized in the early steroidogenesis. TEM revealed structural evidence of this differentiating steroidogenic capability within IECs. During the period of the swelling reaction there is a functional IEC population, but there is no evidence presented by this study for a causal relationship of the gubernacular swelling reaction and subsequent normal descent of the testis into the scrotum.     

Functional genome analysis indicates loss of 17beta-hydroxysteroid dehydrogenase type 2 enzyme in the zebrafish

Among the family of 17beta-hydroxysteroid dehydrogenases, the type 2 (17beta-HSD 2) is the main enzyme responsible for inactivation of estrogens and androgens, catalyzing the oxidation of the C17 hydroxyl group. 17beta-HSD 2 has been studied only in mammals, its occurrence and function in other vertebrates hardly known. We investigated the presence of homologs in non-mammalian species and found sequences of 17beta-HSD 2 and its closest homolog 11beta-HSD 2 in zebrafish (Danio rerio), Takifugu rubripes, Tetraodon nigroviridis, Xenopus tropicalis and chicken databases. Furthermore, we cloned zebrafish 17beta-HSD 2 from ovarian tissue and found high expression also in the testis of adult fish and throughout embryogenesis. The enzyme, though, is inactive likely due to a non-sense N-terminal region including a dysfunctional cofactor binding motif. Replacement of the affected part by the corresponding human 17beta-HSD 2 sequence fully restored enzymatic activity. Comparison of all retrieved 17beta-HSD 2 sequences indicates that this functional loss may have occurred only in zebrafish, where steroid inactivation at position C17 seems to pursue without the protein studied. The closely related 11beta-HSD 2 is unlikely to substitute for 17beta-HSD 2 since in our hands it did not catalyze the respective oxidation of testosterone or estradiol.     

Studies on the onset of Leydig precursor cell differentiation in the prepubertal rat testis

Leydig cells of the adult rat testis differentiate postnatally from spindle-shaped cells in the testis interstitium during the neonatal-prepubertal period. Which spindle-shaped cell types are the precursor for Leydig cells and the stimulus for initiation of their differentiation are, however, two unresolved issues. In the present study, our objectives were to identify unequivocally which spindle-shaped cells are the precursors to Leydig cells and to test whether the initiation of their differentiation into Leydig cells depends on LH. Testes from fifteen groups of Sprague-Dawley rats (n = 4 per group) from 7-21 days of age were fixed in Bouin solution and embedded in paraffin. Immunoexpression of 3beta-hydroxysteroid dehydrogenase (3betaHSD), cytochrome P450 side-chain cleavage (P450(scc)), 17alpha-hydroxylase cytochrome P450 (P450(c17)), and LH receptors (LHR) in interstitial cells (other than fetal Leydig cells) was observed using the avidin biotin method. Of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for all three steroidogenic enzymes, beginning from the 11th postnatal day. All three enzymes were expressed simultaneously in these cells, and their numbers increased significantly thereafter. Immunoexpression of LHR in a few of these cells was just evident for the first time on postnatal Day 12 (i.e., after acquiring the steroidogenic enzyme activity). Their numbers gradually increased with time. The number of immunolabeled cells per 1000 interstitial cells (excluding fetal Leydig cells and capillary endothelial cells) was not significantly different for the three steroidogenic enzymes tested at all ages; however, a lower value was observed for LHR at each time-point. Based on these observations, we suggest that 1) the precursor cell type for the adult generation of Leydig cells in the postnatal rat testis is the peritubular mesenchymal cells, 2) precursor cells acquire 3beta-HSD, P450(scc), and P450(c17) enzyme activity simultaneously during Leydig cell differentiation, and 3) onset of precursor cell differentiation during Leydig cell development does not depend on LH.     

Estrogen-producing steroidogenic pathways in parietal cells of the rat gastric mucosa

Recently we demonstrated the presence of aromatase (P450(arom)), estrogen synthetase, and the active production of estrogen in parietal cells of the rat stomach. We therefore investigated the steroidogenic pathways of estrogen and also other steroid metabolites in the gastric mucosa of male rats, by showing the mRNA expression of steroidogenic enzymes using RT-PCR and in situ hybridization histochemistry, and by measuring the blood concentration of steroids in the artery and the portal vein. RT-PCR analysis showed the strong mRNA expression of 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), 17beta-hydroxysteroid dehydrogenase (HSD) type III and P450(arom), and the weak mRNA expression of 17beta-HSD type II, 5alpha-reductase type I and 3alpha-HSD. The other mRNAs of steroidogenic enzymes examined were not detected. In situ hybridization histochemistry demonstrated the localization of mRNAs for P450(17alpha), 17beta-HSD type III and P450(arom) in the parietal cells. Higher levels of progesterone and testosterone were found in the artery compared with the portal vein. Higher amounts of estrone and 17beta-estradiol, by contrast, were present in the portal vein compared with the artery. These results indicate that parietal cells of rat stomach convert circulating progesterone and/through androstenedione and testosterone to synthesize both estrone and 17beta-estradiol, which then enter the liver via the portal vein.     

环腺苷一磷酸对人Ⅰ型17β羟类固醇脱氢酶在绒癌细胞系中表达的调节作用

由HSD17B1基因编码的人Ⅰ型17β-羟类固醇脱氢酶(17β-hydroxysteroid dehydrogenasetype 1,简称Ⅰ型17HSD)催化雌酮与雌二醇之间的转化。本文研究环腺苷一磷酸简称(cAM-P)对该酶在培养的绒癌细胞系(JAR和JEG-3)中表达的调节作用。用8-bromo-cAMP处理两种绒癌细胞后,观察到在伴随1.3 kbⅠ型17 HSDmRNA表达的同时,Ⅰ型17 HSD蛋白浓度也显著上升。标记基因分析表明,cAMP可诱导HSD 17 B1基因启动子在JAR和JEG-3细胞系中的转录活性,参与调节这一诱导作用的区域位于HSD 17 B1基因编码区上游-659至-550处。凝胶阻滞实验显示这一区域可同JAR、JEG-3、T-47 D和HeLa细胞核抽提物形成特异的DNA-蛋白复合物。本结果首次证实cAMP激活HSD 17 B1基因启动子在绒癌细胞中的转录。