Early preantral follicles of the domestic cat express gonadotropin and sex steroid signaling potential.

Key biomolecular processes, which regulate primordial ovarian follicle dormancy and early folliculogenesis in mammalian ovaries, are not fully understood. The domestic cat is a useful model to study ovarian folliculogenesis and is the most relevant for developing in vitro growth methods to be implemented in wild felid conservation breeding programs. Previously, RNA-sequencing of primordial (PrF), primary (PF), and secondary follicle (SF) samples from domestic cat implicated ovarian steroidogenesis and steroid reception during follicle development. Here, we aimed to identify which sex steroid biosynthesis and metabolism enzymes, gonadotropin receptors, and sex steroid receptors are present and may be potential regulators. Differential gene expression, functional annotation, and enrichment analyses were employed and protein localization was studied too. Gene transcripts for PGR, PGRMC1, AR (steroid receptors), CYP11A1, CYP17A1, HSD17B1 and HSD17B17 (steroidogenic enzymes), and STS (steroid metabolizing enzyme) were significantly differentially expressed (Q values of ≤0.05). Differential gene expression increased in all transcripts during follicle transitions apart from AR which decreased by the secondary stage. Immunohistochemistry localized FSHR and LHCGR to oocytes at each stage. PGRMC1 immunostaining was strongest in granulosa cells, whereas AR was strongest in oocytes throughout each stage. Protein signals for steroidogenic enzymes were only detectable in SFs. Products of these significantly differentially expressed genes may regulate domestic cat preantral folliculogenesis. In vitro growth could be optimized as all early follicles express gonadotropin and steroid receptors meaning hormone interaction and response may be possible. Protein expression analyses of early SFs supported its potential for producing sex steroids.

Steroidogenesis during prenatal testicular development in Spix's cavy Galea spixii.

Spix's cavy is a potentially good experimental model for research on reproductive biology and sexual development. The aim of the present study was to evaluate the ontogeny of the steroidogenic enzymes involved in testicular androgen synthesis during prenatal development. Testes were investigated on Days 25, 30, 40 and >50 of gestation. Immunohistochemistry and immunoblotting were used to establish the site and relative amount of androgenic enzymes, including 5α-reductase, cytosolic 17ß-hydroxysteroid dehydrogenase (17ß-HSDI) and mitochondrial microsomal 3ß-hydroxysteroid dehydrogenase (3ß-HSDII), throughout prenatal development. The testicular parenchyma began to organise on Day 25 of gestation, with the development of recognisable testicular cords. The mesonephros was established after Day 25 of gestation and the ducts differentiated to form the epididymis, as testicular cords were beginning to proliferate and the interstitium to organise by Day 30 of gestation, continuing thereafter. The androgen-synthesising enzymes 5α-reductase, 17ß-HSDI and 3ß-HSDII were evident in Leydig cells as they differentiated at all subsequent gestational ages studied. In addition, immunoblotting showed an increase in immunoreactivity for the enzymes at Days 30 and 40 of gestation (P<0.05) and a decrease at Day 50 of gestation (P<0.05). It is concluded that the increase in androgenic enzymes in Leydig cells coincides with the functional differentiation of the testes, and with the stabilisation and differentiation of mesonephric ducts forming the epididymis.

Characterization of essential domains in HSD17B13 for cellular localization and enzymatic activity.

Human genetic studies recently identified an association of SNPs in the 17-ß hydroxysteroid dehydrogenase 13 (HSD17B13) gene with alcoholic and nonalcoholic fatty liver disease development. Mutant HSD17B13 variants devoid of enzymatic function have been demonstrated to be protective from cirrhosis and liver cancer, supporting the development of HSD17B13 as a promising therapeutic target. Previous studies have demonstrated that HSD17B13 is a lipid droplet (LD)-associated protein. However, the critical domains that drive LD targeting or determine the enzymatic activity have yet to be defined. Here we used mutagenesis to generate multiple truncated and point-mutated proteins and were able to demonstrate in vitro that the N-terminal hydrophobic domain, PAT-like domain, and a putative α-helix/ß-sheet/α-helix domain in HSD17B13 are all critical for LD targeting. Similarly, we characterized the predicted catalytic, substrate-binding, and homodimer interaction sites and found them to be essential for the enzymatic activity of HSD17B13, in addition to our previous identification of amino acid P260 and cofactor binding site. In conclusion, we identified critical domains and amino acid sites that are essential for the LD localization and protein function of HSD17B13, which may facilitate understanding of its function and targeting of this protein to treat chronic liver diseases.

An assessment of the multifactorial profile of steroid-metabolizing enzymes and steroid receptors in the eutopic endometrium during moderate to severe ovarian endometriosis.

BACKGROUND: Previous studies of expression profiles of major endometrial effectors of steroid physiology in endometriosis have yielded markedly conflicting conclusions, presumably because the relative effects of type of endometriosis, fertility history and menstrual cycle phases on the measured variables were not considered. In the present study, endometrial mRNA and protein levels of several effectors of steroid biosynthesis and action in patients with stage III-IV ovarian endometriosis (OE) with known fertility and menstrual cycle histories were compared with the levels in control endometrium to test this concept. METHODS: Endometrial samples were collected from patients without endometriosis (n = 32) or OE stages III-IV (n = 52) with known fertility and cycle histories. qRT-PCR and immunoblotting experiments were performed to measure levels of NR5A1, STAR, CYP19A1, HSD17Bs, ESRs and PGR transcripts and proteins, respectively. Tissue concentrations of steroids (P4, T, E1 and E2) were measured using ELISAs. RESULTS: The levels of expression of aromatase and ERß were lower (P < 0.0001) and 17ß-HSD1 (P < 0.0001) and PRA (P < 0.01) were higher in OE endometrium. Lower aromatase levels and higher 17ß-HSD1 levels were detected in fertile (aromatase: P < 0.05; 17ß-HSD1: P < 0.0001) and infertile (aromatase: P < 0.0001; 17ß-HSD1: P < 0.0001) OE endometrium than in the matched control tissues. Both proliferative (PP) and secretory (SP) phase OE samples expressed aromatase (P < 0.0001) and ERß (PP: P < 0.001; SP: P < 0.01) at lower levels and 17ß-HSD1 (P < 0.0001) and PRA (PP: P < 0.01; SP: P < 0.0001) at higher levels than matched controls. Higher 17ß-HSD1 (P < 0.01) and E2 (P < 0.05) levels and a lower (P < 0.01) PRB/PRA ratio was observed in infertile secretory phase OE endometrium than in control. CONCLUSIONS: We report that dysregulated expression of 17ß-HSD1 and PGR resulting in hyperestrogenism and progesterone resistance during the secretory phase of the menstrual cycle, rather than an anomaly in aromatase expression, was the hallmark of eutopic endometrium from infertile OE patients. Furthermore, the results provide proof of concept that the fertility and menstrual cycle histories exerted relatively different effects on steroid physiology in the endometrium from OE patients compared with the control subjects.

Paraquat exposure delays stem/progenitor Leydig cell regeneration in the adult rat testis.

Paraquat, a widely used nonselective herbicide, is a serious hazard to human health. However, the effects of paraquat on the male reproductive system remain unclear. In this study, adult male Sprague Dawley rats were intraperitoneally injected ethane dimethane sulfonate (EDS, 75 mg/kg) to initiate a regeneration of Leydig cells. EDS-treated rats were orally exposed to paraquat (0.5, 2, 8 mg/kg/day) from post-EDS day 17 to day 28 and effects of paraquat on Leydig and Sertoli cell functions on post-EDS day 35 and day 56 were investigated. Paraquat significantly decreased serum testosterone levels at 2 and 8 mg/kg. Paraquat lowered Leydig cell Hsd17b3, Srd5a1, and Hsd11b1 mRNA levels but increased Hsd3b1 on post-EDS day 35. Paraquat lowered Cyp11a1, Cyp17a1, and Hsd11b1 but increased Srd5a1 on post-EDS day 56. However, paraquat did not alter Leydig cell number and PCNA labeling index. Epididymal staining showed that few sperms were observed in paraquat-treated rats. Primary culture of adult Leydig cells showed that paraquat diminished testosterone output and induced reactive oxygen species generation at 1 and 10 µM and apoptosis rate at 10 µM. In conclusion, a short-term exposure to paraquat delays Leydig cell regeneration from stem/progenitor Leydig cells, causing low production of testosterone and an arrest of spermatogenesis.

The Mussel Mytilus galloprovincialis in the Bay of Naples: New Insights on Oogenic Cycle and Its Hormonal Control.

The aim of the present article was to investigate the oogenic cycle of Mytilus galloprovincialis sampled in the Bay of Naples, and to immunolocalize 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and P450 aromatase, enzymes involved in the synthesis of two sex hormones: testosterone and 17ß-estradiol. We demonstrate that the oogenic cycle starts in late summer-early fall and continues in early winter when the first event of spawning occurs; other spawning events take place until June, when the ovary is spent and contains a few empty ovarian follicles and numerous somatic cells, that is, adipogranular cells and vesicular connective tissue cells. During the oogenic cycle, apoptotic events occur at the level of oogonia, previtellogenic oocytes, as well as follicle cells; by contrast, necrosis events probably take place in vitellogenic oocytes, which, once degenerated, transfer their content to healthy oocytes. Finally, the present data demonstrate that 3ß-HSD, 17ß-HSD, and P450 aromatase are present in the ovary both during the reproductive and nonreproductive phases. The possible role of these enzymes during the Mytilus galloprovincialis reproductive cycle is discussed. Anat Rec, 302:1039-1049, 2019. © 2019 Wiley Periodicals, Inc.

In vitro and in vivo androgen regulation of Dendropanax morbiferus leaf extract on late-onset hypogonadism.

Late-onset hypogonadism (LOH) is associated with advancing age and is caused by a deficiency in serum testosterone levels. The aim of this study was to examine the effect of a Dendropanax morbiferus H.Lév. leaf extract (DME) on LOH using TM3 cells and aging male rats as in vitro and in vivo models, respectively. The in vitro effects of DME on testosterone levels and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) protein expression in TM3 cells were analyzed. In the in vivo experiments, DME was orally administered to rats at three doses (50, 100, and 200 mg/kg/day) for 4 weeks. DME significantly increased the testosterone levels and 3ß-HSD protein expression in TM3 cells. The DME groups showed significantly increased levels of androgenic hormones such as testosterone and dehydroepiandrosterone sulfate. The sex hormone-binding globulin production was significantly lower in the DME groups than that in the control group, while the neurohormone levels in the hypothalamic-pituitary-gonadal axis markedly increased. No significant differences were observed in the glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and prostate-specific antigen levels among the DME and control groups. The triglyceride and low-density lipoprotein cholesterol levels were significantly lower, while the high-density lipoprotein cholesterol levels were significantly higher in the DME groups than those in the control group. The latency time in the rotarod, treadmill, and swimming tests increased with the DME treatment. Furthermore, the sperm counts in the epididymis markedly increased. These results suggest that DME can be effectively used to alleviate the symptoms of LOH.

Immunohistochemical localization of 3ß-Hydroxysteroid dehydrogenase and progesterone receptors in the ovary and placenta during gestation of the placentotrophic lizard Mabuya sp (Squamata: Scincidae).

In squamates, progesterone (P) plays a key role in the inhibition of uterine mobility during egg retention in oviparous species, and during gestation in viviparous species. The corpus luteum (CL) is the main organ responsible for the production of P; however, in some species, the CL degenerates early and the P needed for gestation maintenance should be produced in other tissues. Mabuya sp (Scincidae) is a viviparous lizard with a prolonged gestation, it produces microlecithal eggs and, consequently, has an obligate placentotrophy related with a highly complex placenta. Its CL degenerates at early stages of gestation and therefore, other sources of P should exist. The aim of this study was to determine and localize by immunohistochemistry the production of P by detection of the enzyme 3ß-Hydroxysteroid dehydrogenase (3ß-HSD) and P receptors (PR) during gestation in the ovary and placenta of Mabuya sp. Positive and negative control sections were used. The ovary of this species localizes 3ß-HSD and PR in the same tissues. The CL of the ovaries of females at early stages of gestation were positive for both molecules, whereas they did not localize from mid gestation to the end of pregnancy. Previtellogenic and vitellogenic follicles labelled for both molecules in the follicular epithelium and thecae. The placenta of Mabuya sp. demonstrated the potential for P production from mid gestation to the end of gestation in the uterine and chorionic tissues. PR were located in the uterine tissues throughout gestation, with a decrease towards its completion. Western blot analysis confirmed the presence of 3ß-HSD mainly in the ovary of early pregnant females and in the placental tissues at mid gestation stages. Therefore, the chorioallantoic placenta of Mabuya sp. has an endocrine function producing the P needed for gestation and replacing the CL from mid gestation to the end of pregnancy.

Effects of T-2 toxin on the regulation of steroidogenesis in mouse Leydig cells.

T-2 toxin is one of the mycotoxins, a group of type A trichothecenes produced by several fungal genera including Fusarium species, which may lead to the decrease of testosterone secretion in primary Leydig cells derived from mouse testis. The previous study demonstrated T-2 toxin decrease the testosterone biosynthesis in the primary Leydig cells derived from the mouse testis directly. In this study, we further examined the direct biological effects of T-2 toxin on the process of steroidogenesis, primarily in Leydig cells of mice. Leydig cells of mature mouse were purified by Percoll gradient centrifugation and the cell purity was determined by 3ß-hydroxysteroid dehydrogenase (3ß-HSD) staining. To examine the decrease in T-2 toxin-induced testosterone secretion, we measured the transcription level of three key steroidogenic enzymes including 3ß-HSD-1, cytochrome P450 side-chain cleavage (P450scc) enzyme, and steroidogenic acute regulatory (StAR) protein in T-2 toxin/human chorionic gonadotropin (hCG) co-treated cells. Our previous study showed that T-2 toxin (10(-7), 10(-8), and 10(-9) M) significantly suppressed hCG (10 ng/ml)-induced testosterone secretion. The studies demonstrated that the suppressive effect is correlated with a decrease in the level of transcription of 3ß-HSD-1, P450scc, and StAR (p < 0.05).

Exposure to leachate from municipal battery recycling site: implication as key inhibitor of steroidogenic enzymes and risk factor of prostate damage in rats.

Few or no studies have measured the effect of short- and long-term exposure to industrial leachate. Mature male Wistar strain albino rats (175-220 g) underwent sub-chronic exposure to leachate from the Elewi Odo municipal battery recycling site (EOMABRL) via oral administration for a period of 60 days at different doses (20%, 40%, 60%, 80%, and 100%) per kilogram of body weight to evaluate the toxic effects of the leachate on male reproductive function using steroidogenic enzymes and biomarkers of prostate damage. Control groups were treated equally but were given distilled water instead of the leachate. After the treatment periods, results showed that the treatment induced systemic toxicity at the doses tested by causing a significant (p<0.05) loss in absolute body weight and decline in growth rate. There was a marked significant decrease (p<0.05) in testicular activities of Δ(5)-3ß-hydroxysteroid dehydrogenase and 17ß-hydroxysteroid dehydrogenase. Conversely, the activity of prostatic acid phosphatase, a key marker enzyme for prostrate damage was significantly (p<0.05) elevated in the treated rats. Similarly, the administration of EOMABRL significantly (p<0.05) exacerbated the activity of total acid phosphatase with concomitant increase in the activity of prostatic alkaline phosphatase. These findings conclude that exposure to leachate from a battery recycling site induces sub-chronic testicular toxicity by inhibiting key steroidogenic enzymes and activating key markers linked with prostate damage/cancer in rats.

Expression patterns of 17ß-hydroxysteroid dehydrogenase 14 in human tissues.

17ßHSD enzymes catalyze the stereospecific oxidation/reduction at carbon 17ß of androgens and estrogens, and are important players in intracrine sex hormone synthesis. The biological relevance of 17ßHSD14, first named retSDR3, is largely unknown. We generated and validated an antibody targeting the 17ßHSD14 antigen and used this for immunohistochemical evaluation of expression patterns in 33 healthy human tissues. Furthermore, sex steroid conversional activity in HSD17B14 overexpressing HEK293 and MCF10A cells was investigated by assessing interconversion products of estrone, estradiol, androstenedione, testosterone, and dehydroepiandrosterone. Immunohistochemical staining patterns of 17ßHSD14 with the enzyme being primarily expressed in glandular epithelial tissue reveal an enzyme with possible implications in the secretion or conversion of externally derived compounds. A role for 17ßHSD14 in sex steroid metabolism is supported by the finding that 17HSD14 oxidizes both estradiol and testosterone into less bioactive steroid metabolites estrone and androstenedione, respectively.

Halowax 1051 affects steroidogenesis, 17ß-hydroxysteroid dehydrogenase (17ß-HSD) and cytochrome P450arom (CYP19) activity, and protein expression in porcine ovarian follicles.

We evaluated the effect of Halowax 1051 on ovarian follicular testosterone (T) and estradiol (E2) secretion determined by EIA and on the expression of 17ß-HSD and CYP19 analyzed by Western blot. 17ß-HSD and CYP19 activity were determined indirectly by measuring the conversion of androstenedione (A4) to T and T to E2, respectively. Additionally, CYP19 activity by dibenzylfluorescein assay. Follicles were exposed to 1-1000pg/ml of Halowax 1051 for 24, 48 or 72h. It was found that Halowax 1051, in all doses used, increased basal T secretion concomitant with a decrease in basal E2 secretion. Inhibitory action on 17ß-HSD and stimulatory action on CYP19 (activity and protein expression) under the influence of 1pg/ml, and opposite effect under the influence of 10-1000pg/ml was noted. In conclusion, we suggest non-linear dose-response effect of Halowax 1051 on steroidogenesis and steroidogenic enzymes activity and protein expression.

Modulation of estrogen synthesis through activation of protein kinase A in H295R cells by extracts of estuary sediments.

Sediments from two estuaries within Liaodong Bay, China, were examined for the effects on steroidogenesis using H295R human adrenocortical carcinoma cells. Total extracts (TE) isolated from sediments by Soxhlet extraction were separated into three fractions (F1, F2, and F3) using Florisil columns. After exposing H295R cells to each TE and fractions, the expressions of six steroidogenic genes (cytochrome P450 cholesterol side-chain cleavage [CYP11A], 3ß-hydroxysteroid dehydrogenase type 1 [3ß-HSD1], 3ß-hydroxysteroid dehydrogenase type 2 [3ß-HSD2], cytochrome P450 17-hydroxylase/17-20 lyase [CYP17], cytochrome P450 aromatase [CYP19], 17ß-hydroxysteroid dehydrogenase [17ß-HSD]), and the production of six steroid hormones (progesterone [PGT], 17-hydroxyprogesterone [17-HPT], testosterone [TTR], androstenedione [ADD], estrone [E1], and 17ß-estradiol [17ß-E2]) were measured. The gene expressions of CYP11A, CYP17, 3ß-HSD2, and CYP19, and hormone productions of PGT, 17-HPT, TTR, ADD, E1, and 17ß-E2 were significantly increased after exposure to F3 extracts from the Daliao River. In particular, greater concentrations of E1 (up to 48-fold) and 17ß-E2 (up to 20-fold), as well as up-regulation of CYP19 gene expression (up to tenfold), were caused by exposure to the F3 fraction from the Daliao River, but not from the Daling River. Insight into the mechanism of action was obtained by use of principal component analysis (PCA), the results of which were consistent with unidentified constituents in F3 from the Daliao River activating the protein kinase A (PKA) pathway. This hypothesis was confirmed by reversal of the effects caused by F3 through a co-exposure of a PKA inhibitor (H89) and F3 extract. The H89 down-regulated CYP19 messenger RNA (mRNA) expression with concomitant lesser production of E1 and 17ß-E2 in the co-exposure group, indicating unidentified constituents that could modulate estrogen synthesis, primarily through a mechanism of PKA activation.

Methoxychlor reduces estradiol levels by altering steroidogenesis and metabolism in mouse antral follicles in vitro.

The organochlorine pesticide methoxychlor (MXC) is a known endocrine disruptor that affects adult rodent females by causing reduced fertility, persistent estrus, and ovarian atrophy. Since MXC is also known to target antral follicles, the major producer of sex steroids in the ovary, the present study was designed to test the hypothesis that MXC decreases estradiol (E2) levels by altering steroidogenic and metabolic enzymes in the antral follicles. To test this hypothesis, antral follicles were isolated from CD-1 mouse ovaries and cultured with either dimethylsulfoxide (DMSO) or MXC. Follicle growth was measured every 24 h for 96 h. In addition, sex steroid hormone levels were measured using enzyme-linked immunosorbent assays (ELISA) and mRNA expression levels of steroidogenic enzymes as well as the E2 metabolic enzyme Cyp1b1 were measured using qPCR. The results indicate that MXC decreased E2, testosterone, androstenedione, and progesterone (P4) levels compared to DMSO. In addition, MXC decreased expression of aromatase (Cyp19a1), 17ß-hydroxysteroid dehydrogenase 1 (Hsd17b1), 17α-hydroxylase/17,20-lyase (Cyp17a1), 3ß hydroxysteroid dehydrogenase 1 (Hsd3b1), cholesterol side-chain cleavage (Cyp11a1), steroid acute regulatory protein (Star), and increased expression of Cyp1b1 enzyme levels. Thus, these data suggest that MXC decreases steroidogenic enzyme levels, increases metabolic enzyme expression and this in turn leads to decreased sex steroid hormone levels.

Reproductive toxic potential of panmasala in male Swiss albino mice.

Some ingredients of panmasala have the ability to penetrate the blood-testis barrier but the reproductive toxic potential of panmasala has not been studied. This study is aimed to assess the possible damage caused by panmasala to male reproductive system in mice. Swiss albino male mice were randomly divided into 7 groups receiving either standard control diet or panmasala-containing diet. Three doses (0.5%, 1.5% and 3%) of panmasala plain (PMP) as well as panmasala with tobacco (PMT)-gutkha were given for a period of 6 months. Assessment of organ weight, sperm count and morphology, spermatid count, sperm production, testicular 17ß-hydroxysteroid dehydrogenase (17ß-HSD) activity and histology were conducted. A nonsignificant decrease in absolute and relative weight of testis and epididymis was observed. Spermatid count, sperm count and production were significantly decreased and 17ß-HSD activity was found considerably declined at 3% of both PMP- and PMT-treated groups as compared to control. The histological observations revealed panmasala induced testicular damage. Abnormal morphology of sperm head shape was significantly elevated in higher doses of both types of panmasala-treated groups than control. The results suggests that panmasala has reproductive toxic potential and more alteration is seen with gutkha as compared to panmasala plain, indicating that similar effects might also be possible in humans.

(-)-CHANA, a fluorogenic probe for detecting amyloid binding alcohol dehydrogenase HSD10 activity in living cells.

The association of 17ß-hydroxysteroid dehydrogenase 10 (HSD10) with ß-amyloid in the brain is known to contribute to the progression of Alzheimer's disease. Further, it has been shown that the interaction between the purified HSD10 and ß-amyloid inhibits its enzymatic activity. However, to date no system has been developed to enable the study of HSD10 activity in intact living cells. To address this significant shortcoming, we have developed a novel fluorogenic probe, (-)-cyclohexenyl amino naphthalene alcohol [(-)-CHANA], to observe and measure the activity of HSD10 in living cells. The oxidation of (-)-CHANA by HSD10 results in the production and accumulation of a fluorescent product, which can be measured using real-time fluorescence microscopy. This compound permits the measurement of mitochondrial HSD10 activity and its inhibition by both a small molecule HSD10 inhibitor and by ß-amyloid, in living cells. Herein, we define the parameters under which this probe can be used. This compound is likely to prove useful in future investigations aimed at developing therapeutic compounds targeting the HSD10-ß-amyloid association.

Ethanol induces mouse spermatogenic cell apoptosis in vivo through over-expression of Fas/Fas-L, p53, and caspase-3 along with cytochrome c translocation and glutathione depletion.

Although it has been well established that spermatogenic cells undergo apoptosis when treated with ethanol, the molecular mechanisms behind it remain to be investigated. Adult male mice were given intra-peritoneal injection (IP) of ethanol at a dose of 3 g (15%, v/v) per kg body weight per day during the period of 14 days. Testicular androgenesis and apoptotic germ cell death, along with different interrelated proteins expression, were evaluated. Ethanol treatment induced apoptotic spermatogenic cell death with a decrease in the plasma and intra-testicular testosterone concentration. Western blot analysis revealed that repeated ethanol treatment decreased the expression of steroidogenic acute regulatory protein (StAR), 3 beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17beta-HSD); increased the expression of active caspase-3, p53, Fas and Fas-L; and led to up-regulation of Bax/Bcl-2 ratio and translocation of cytochrome c from mitochondria to cytosol in testis. It has also been shown in our study that repeated ethanol treatment led to up-regulation of caspase-3, p53, Fas, Fas-L transcripts; increase in caspase-3 and caspase-8 activities; diminution of 3beta-HSD, 17beta-HSD, and GPx activities; decrease in the mitochondrial membrane potential along with ROS generation and depletion of glutathione pool in the testicular tissue. The present study has indicated that the ethanol treatment induced apoptosis in the mouse testis through the increased expression of Fas/Fas-L and p53, up-regulation of Bax/Bcl-2 ratio, cytosolic translocation of cytochrome c along with caspase-3 activation and glutathione depletion.

Immunolocalization of estrogen-producing and metabolizing enzymes in benign breast disease: comparison with normal breast and breast carcinoma.

It is well known that estrogens play important roles in the cell proliferation of breast carcinoma. Benign breast disease (BBD) contains a wide spectrum of diseases, and some are considered an important risk factor for subsequent breast carcinoma development. However, the significance of estrogens in BBD has remained largely unknown. Therefore, in this study, we examined tissue concentrations of estrogens and immunolocalization of estrogen-producing/metabolizing enzymes in BBD, and compared these findings with those in the normal breast and ductal carcinoma in situ (DCIS). Tissue concentration of estradiol in BBD (n = 9) was significantly (3.4-fold) higher than normal breast (n = 9) and nearly the same (0.7-fold) as in DCIS (n = 9). Immunoreactivity of estrogen sulfotransferase in BBD was significantly lower (n = 82) than normal breast (n = 28) but was not significantly different from DCIS (n = 28). Aromatase and steroid sulfatase immunoreactivities tended to be higher (P = 0.07) in BBD than in normal breast, and 17ß-hydroxysteroid dehydrogenase type 1 immunoreactivity was significantly higher in BBD than normal breast in the postmenopausal tissues. Immunoreactivity of estrogen and progesterone receptors was also significantly higher in BBD than normal breast. These results suggest that tissue concentration of estradiol is increased in BBD at a level similar to DCIS, which is considered mainly due to loss of estrogen sulfotransferase expression. Increased local estradiol concentration in BBD due to aberrant expression of estrogen-producing/metabolizing enzymes may play important roles in the accumulation of estradiol-mediated growth and/or subsequent development of breast carcinoma.

Identification of a substrate-binding site in a peroxisomal beta-oxidation enzyme by photoaffinity labeling with a novel palmitoyl derivative.

Peroxisomes play an essential role in a number of important metabolic pathways including beta-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various beta-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed photoaffinity labeling of purified rat liver peroxisomes. As a result, an 80-kDa peroxisomal protein was specifically labeled by the photoaffinity ligand, and the labeling efficiency competitively decreased in the presence of palmitoyl-CoA. Mass spectrometric analysis identified the 80-kDa protein as peroxisomal multifunctional enzyme type 2 (MFE2), one of the peroxisomal beta-oxidation enzymes. Recombinant rat MFE2 was also labeled by the photoaffinity ligand, and mass spectrometric analysis revealed that a fragment of rat MFE2 (residues Trp(249) to Arg(251)) was labeled by the ligand. MFE2 mutants bearing these residues, MFE2(W249A) and MFE2(R251A), exhibited decreased labeling efficiency. Furthermore, MFE2(W249G), which corresponds to one of the disease-causing mutations in human MFE2, also exhibited a decreased efficiency. Based on the crystal structure of rat MFE2, these residues are located on the top of a hydrophobic cavity leading to an active site of MFE2. These data suggest that MFE2 anchors its substrate around the region from Trp(249) to Arg(251) and positions the substrate along the hydrophobic cavity in the proper direction toward the catalytic center.

RNAi and iTRAQ reagents united: targeted quantitation of siRNA-mediated protein silencing in human cells.

Bridging the gap between functional genomics and traditional molecular cell biology is a challenge of the next decade. Here, we are aiming to find routines for targeted quantitation of protein silencing in response to RNAi based on complex cellular lysates. A workflow was established adapting siRNA treatment, processing the sample, generating isobaric iTRAQ-reagent-labeled peptides, and analyzing the sample applying MRM-based peptide quantitation to verify protein silencing on a 4000 QTRAP LC/MS/MS mass spectrometer. Subsequently, eight targets were analyzed, mostly with two siRNA designs. Although transcript and protein silencing correlated, the downregulation on the protein level was less pronounced. A time-course analysis of the chaperon HSPA9/mortalin indicated a delayed kinetic of protein versus transcript silencing. Further, the analysis of the functional response on the example of HSD17B4, a multifunctional enzyme essential to generate precursors for cholesterol biosynthesis, confirmed that strong silencing on the transcript level accompanied by moderate reduction of protein is sufficient to generate a physiological significant response. Fifty percent protein silencing resulted in a 3.5-fold induction of low-density lipoprotein and therefore cholesterol uptake in human liver cells. The established routines pave the way for the development of targeted protein quantitation assays suitable for target and biomarker validation.