The protective effect of Stevia rebaudiana Bertoni on serum hormone levels, key steroidogenesis enzymes, and testicular damage in testes of diabetic rats.

Diabetes Mellitus (DM) is a kind of metabolic endocrine diseases, which has various effects on the gonadal system. The current study aimed to examine the effect of Stevia rebaudiana Bertoni extract on the mRNA expression involved in testosterone synthesis, and stereological parameters in rat testes, for improving diabetes complications. In this study, 48 rats were randomly classified into control, diabetic (streptozocin 60 mg/kg + nicotinamide 120 mg/kg), diabetic + Stevia (400 mg/kg), and diabetic + metformin (500 mg/kg) groups. Finally, Fasting Blood Sugar (FBS) level, the serum level of LH and testosterone, the Star, Cyp11a1, and Hsd17b3 gene expressions, and changes in the testis histology were evaluated. The results indicated a decrease in body weight, serum LH and testosterone level, the star gene expression, stereological changes of testes, and an increase in the FBS level in diabetic group, compared with the control group (P<0.05). Nonetheless, Stevia significantly reduced the FBS and increased the serum LH level, in comparison with diabetic rats (P<0.05), but no significant differences in the serum testosterone level and the Star gene expression has been found. Stevia also resulted in an increase in weight, testis volume, the number of sexual lineage cells, and sperm count and motility, compared to diabetic rats (P<0.05). Due to its antioxidant properties, Stevia enhanced the alteration in spermatogenesis and stereological characteristics in diabetic rat testes. Hence, Stevia could diminish the reproductive system problems and improve infertility in diabetic male rats.

Hydroxysteroid (17ß) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility.

The pituitary gonadotrophins and testosterone are the main hormonal regulators of spermatogenesis, but estradiol is also known to play a role in the process. The hormonal responses in the testis are partially mediated by somatic Sertoli cells that provide nutritional and physical support for differentiating male germ cells. Hydroxysteroid (17ß) dehydrogenase 1 (HSD17B1) is a steroidogenic enzyme that especially catalyzes the conversion of low potent 17keto-steroids to highly potent 17ß-hydroxysteroids. In this study, we show that Hsd17b1 is highly expressed in Sertoli cells of fetal and newborn mice, and HSD17B1 knockout males present with disrupted spermatogenesis with major defects, particularly in the head shape of elongating spermatids. The cell-cell junctions between Sertoli cells and germ cells were disrupted in the HSD17B1 knockout mice. This resulted in complications in the orientation of elongating spermatids in the seminiferous epithelium, reduced sperm production, and morphologically abnormal spermatozoa. We also showed that the Sertoli cell-expressed HSD17B1 participates in testicular steroid synthesis, evidenced by a compensatory up-regulation of HSD17B3 in Leydig cells. These results revealed a novel role for HSD17B1 in the control of spermatogenesis and male fertility, and that Sertoli cells significantly contribute to steroid synthesis in the testis.-Hakkarainen, J., Zhang, F.-P., Jokela, H., Mayerhofer, A., Behr, R., Cisneros-Montalvo, S., Nurmio, M., Toppari, J., Ohlsson, C., Kotaja, N., Sipilä, P., Poutanen, M. Hydroxysteroid (17ß) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility.

Expression and regulation of CYP17A1 and 3ß-hydroxysteroid dehydrogenase in cells of the nervous system: Potential effects of vitamin D on brain steroidogenesis.

Steroids are reported to have diverse functions in the nervous system. Enzymatic production of steroid hormones has been reported in different cell types, including astrocytes and neurons. However, the information on some of the steroidogenic enzymes involved is insufficient in many respects. Contradictory results have been reported concerning the relative importance of different cell types in the nervous system for expression of CYP17A1 and 3ß-hydroxysteroid dehydrogenase (3ß-HSD). 3ß-HSD is important in all basic steroidogenic pathways and CYP17A1 is required to form sex hormones. In the current investigation we studied the expression of these enzymes in cultured primary rat astrocytes, in neuron-enriched cells from rat cerebral cortex and in human neuroblastoma SH-SY5Y cells, a cell line often used as an in vitro model of neuronal function and differentiation. As part of this study we also examined potential effects on CYP17A1 and 3ß-HSD by vitamin D, a compound previously shown to have regulatory effects in steroid hormone-producing cells outside the brain. The results of our study indicate that astrocytes are a major site for expression of 3ß-HSD whereas expression of CYP17A1 is found in both astrocytes and neurons. The current data suggest that neurons, contrary to some previous reports, are not involved in 3ß-HSD reactions. Previous studies have shown that vitamin D can influence gene expression and hormone production by steroidogenic enzymes in some cells. We found that vitamin D suppressed CYP17A1-mediated activity by 20% in SH-SY5Ycells and astrocytes. Suppression of CYP17A1 mRNA levels was considerably stronger, about 50% in SH-SY5Y cells and 75% in astrocytes. In astrocytes 3ß-HSD was also suppressed by vitamin D, about 20% at the enzyme activity level and 60% at the mRNA level. These data suggest that vitamin D-mediated regulation of CYP17A1 and 3ß-HSD, particularly on the transcriptional level, may play a role in the nervous system.

Estrogen Metabolism in Abdominal Subcutaneous and Visceral Adipose Tissue in Postmenopausal Women.

Context: In postmenopausal women, adipose tissue (AT) levels of estrogens exceed circulating concentrations. Although increased visceral AT after menopause is related to metabolic diseases, little is known about differences in estrogen metabolism between different AT depots. Objective: We compared concentrations of and metabolic pathways producing estrone and estradiol in abdominal subcutaneous and visceral AT in postmenopausal women. Design, Setting, Patients, and Interventions: AT and serum samples were obtained from 37 postmenopausal women undergoing surgery for nonmalignant gynecological reasons. Serum and AT estrone, estradiol, and serum estrone sulfate (E1S) concentrations were quantitated using liquid chromatography-tandem mass spectrometry. Activity of steroid sulfatase and reductive 17ß-hydroxysteroid dehydrogenase enzymes was measured using radiolabeled precursors. Messenger RNA (mRNA) expression of estrogen-converting enzymes was analyzed by real-time reverse transcription quantitative polymerase chain reaction. Results: Estrone concentration was higher in visceral than subcutaneous AT (median, 928 vs 706 pmol/kg; P = 0.002) and correlated positively with body mass index (r = 0.46; P = 0.011). Both AT depots hydrolyzed E1S to estrone, and visceral AT estrone and estradiol concentrations correlated positively with serum E1S. Compared with visceral AT, subcutaneous AT produced more estradiol from estrone (median rate of estradiol production, 1.02 vs 0.57 nmol/kg AT/h; P = 0.004). In visceral AT, the conversion of estrone to estradiol increased with waist circumference (r = 0.65; P = 0.022), and estradiol concentration correlated positively with mRNA expression of HSD17B7 (r = 0.76; P = 0.005). Conclusions: Both estrone and estradiol production in visceral AT increased with adiposity, but estradiol was produced more effectively in subcutaneous fat. Both AT depots produced estrone from E1S. Increasing visceral adiposity could increase overall estrogen exposure in postmenopausal women.

Secretory expression, purification and functional characterization of 17ß-hydroxysteroid dehydrogenase type 1 from mammalian HEK293T cells.

17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) mainly catalyzes the reduction of estrone into estradiol. The enzymatic conversion is a critical step in estradiol accumulation in breast tissue, which is a valuable prognosis index of breast cancer disease. However, the source of 17ß-HSD1 for inhibitor design is limited. In this study, the fragment encoding human 17ß-HSD1 was successfully cloned and expressed in human embryonic kidney (HEK) 293T mammalian cells. The recombinant protein was purified by immobilized metal ion affinity chromatography yielding above 17 mg of purified 17ß-HSD1 protein per liter of cell culture, with a specific activity of 8.54 µmoL/min/mg of protein for conversion of estradiol into estrone, with NAD+ as cofactor at pH 9.2. Enzyme characterization studies revealed that the protein has estrogenic activity and the Km value for estrone is about 20 nM. The recombinant protein purified from transfected HEK293T cells had higher specific activity compared to that of the enzyme purified directly from placenta. The present data show that the mammalian cell expression system can provide active 17ß-HSD1 which is functionally identical to its natural counterpart and easy to purify in qualities suitable for its structure-function study.

Loss of PI3K p110α in the Adipose Tissue Results in Infertility and Delayed Puberty Onset in Male Mice.

Deletion of PI3K catalytic subunit p110α in adipose tissue (aP2-Cre/p110αflx/flx, α-/- hereafter) results in increased adiposity, glucose intolerance, and liver steatosis. Because this endocrine organ releases hormones like leptin, which are important in reproductive physiology, we investigated the reproductive phenotype of α-/- males. Compared to controls, α-/- males displayed delayed onset of puberty accompanied by a reduction in plasma LH levels and testicular weight. At postnatal day 30, α-/- mice exhibited normal body weight but elevated fasted plasma leptin levels. Testicular leptin gene expression was increased, whereas expression of the cholesterol transporter StAR and of P450 cholesterol side chain cleavage enzyme was decreased. Adult α-/- males were infertile and exhibited hyperandrogenemia with normal basal LH, FSH, and estradiol levels. However, neither sperm counts nor sperm motility was different between genotypes. The mRNA levels of leptin and of 17-beta-dehydrogenase 3, and enzyme important for testosterone production, were significantly higher in the testis of adult α-/- males. The mRNA levels of ERα, an important regulator of intratesticular steroidogenesis, were lower in the testis of adult and peripubertal α-/- males. We propose that chronic hyperleptinemia contributes to the negative impact that disrupting PI3K signaling in adipocytes has on puberty onset, steroidogenesis, and fertility in males.

Effects of Etomidate on the Steroidogenesis of Rat Immature Leydig Cells.

BACKGROUND: Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3ß-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established. METHODOLOGY: Immature Leydig cells isolated from 35 day-old rats were cultured with 30 µM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3-30 µM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity. RESULTS AND CONCLUSIONS: In intact Leydig cells, 30 µM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 µM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 µM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 µM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.

Hydroxysteroid (17ß)-dehydrogenase 1-deficient female mice present with normal puberty onset but are severely subfertile due to a defect in luteinization and progesterone production.

Hydroxysteroid (17ß)-dehydrogenase type 1 (HSD17B1) catalyzes the conversion of low active 17-ketosteroids, androstenedione (A-dione) and estrone (E1) to highly active 17-hydroxysteroids, testosterone (T) and E2, respectively. In this study, the importance of HSD17B1 in ovarian estrogen production was determined using Hsd17b1 knockout (HSD17B1KO) mice. In these mice, the ovarian HSD17B enzyme activity was markedly reduced, indicating a central role of HSD17B1 in ovarian physiology. The lack of Hsd17b activity resulted in increased ovarian E1:E2 and A-dione:T ratios, but we also observed reduced progesterone concentration in HSD17B1KO ovaries. Accordingly with the altered steroid production, altered expression of Star, Cyp11a1, Lhcgr, Hsd17b7, and especially Cyp17a1 was observed. The ovaries of HSD17B1KO mice presented with all stages of folliculogenesis, while the corpus luteum structure was less defined and number reduced. Surprisingly, bundles of large granular cells of unknown origin appeared in the stroma of the KO ovaries. The HSD17B1KO mice presented with severe subfertility and failed to initiate pseudopregnancy. However, the HSD17B1KO females presented with normal estrous cycle defined by vaginal smears and normal puberty appearance. This study indicates that HSD17B1 is a key enzyme in ovarian steroidogenesis and has a novel function in initiation and stabilization of pregnancy.

Effects of individual polychlorinated naphthalene (PCN) components of Halowax 1051 and two defined, artificial PCN mixtures on AHR and CYP1A1 protein expression, steroid secretion and expression of enzymes involved in steroidogenesis (CYP17, 17ß-HSD and CYP19) in porcine ovarian follicles.

In this study we tried to answer a question which component of Halowax 1051 is responsible for, observed in previously published study, androgenic effects of the mixture, and whether it is possible to draw conclusions about the action of mixtures by examining the effect of an indicator congener. Ovarian follicles were incubated with individual congeners of an artificial mixture for 6-24h. At the end of the incubation period, media were collected for determination of progesterone (P4), androstenedione (A4), testosterone (T) and estradiol (E2) levels by enzyme immunoassay, and follicles were retained for an examination of aryl hydrocarbon receptor (AHR), cytochrome p450 enzymes (CYP1A1, CYP17, CYP19), and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) protein expression by Western blotting. CN73 in dose 50pg/ml after 6h had no effect and decreased AHR expression after 24h, while at dose 400pg/ml increased AHR protein expression after 6h of exposure which remained elevated after 24h. CN74 and CN75 at both concentrations tested (25 and 50pg/ml) stimulated AHR protein expression after 6h and decreased it after 24h of exposure. Individual congeners induced a rapid increase in CYP1A1 protein expression, with a rank order of efficacy of CN73>CN74=CN75. All congeners increased P4/A4 and T/E2 secretion ratios in association with a decrease in the A4/T ratio, pointing to androgenic and anti-estrogenic properties of PCNs in ovarian follicles. The most potent congener in this context was CN73. The effects of mixtures were comparable to those of CN74 and CN75, and were not as strong as those observed for CN73. Collectively, these data suggest antagonistic actions of single congeners in a mixture, indicating that the actions of a mixture cannot be predicted based on the actions of individual congeners.

Expression of membrane transporters and metabolic enzymes involved in estrone-3-sulphate disposition in human breast tumour tissues.

Two-thirds of newly diagnosed hormone-dependent (HR?) breast cancers are detected in post-menopausal patients where estrone-3-sulphate (E3S) is the predominant source for tumour estradiol. Understanding intra-tumoral fate of E3S would facilitate in the identification of novel molecular targets for HR? post-menopausal breast cancer patients. Hence this study investigates the clinical expression of (i) organic anion-transporting polypeptides (OATPs), (ii) multidrug resistance protein (MRP-1), breast cancer resistance proteins (BCRP), and (iii) sulphatase (STS), 17ß-hydroxysteroid dehydrogenase (17ß-HSD-1), involved in E3S uptake, efflux and metabolism, respectively. Fluorescent and brightfield images of stained tumour sections (n = 40) were acquired at 4× and 20× magnification, respectively. Marker densities were measured as the total area of positive signal divided by the surface area of the tumour section analysed and was reported as % area (ImageJ software). Tumour, stroma and non-tumour tissue areas were also quantified (Inform software), and the ratio of optical intensity per histologic area was reported as % area/tumour, % area/stroma and % area/non-tumour. Functional role of OATPs and STS was further investigated in HR? (MCF-7, T47-D, ZR-75) and HR-(MDA-MB-231) cells by transport studies conducted in the presence or absence of specific inhibitors. Amongst all the transporters and enzymes, OATPs and STS have significantly (p < 0.0001) higher expression in HR? tumour sections with highest target signals obtained from the tumour regions of the tissues. Specific OATP-mediated E3S uptake and STS-mediated metabolism were also observed in all HR? breast cancer cells. These observations suggest the potential of OATPs as novel molecular targets for HR? breast cancers.

Bacterial lipopolysaccharide differently modulates steroidogenic enzymes gene expressions in the brain and testis in rats.

Bacterial lipopolysaccharide (LPS) is a major component of the cell wall of gram negative bacteria contributing to the pathogenesis of bacterial infection, in particular in those diseases affecting central nervous system and reproductive tissues. The present work is an attempt to study the regulation of steroidogenic enzymes gene expression in the brain and testis in LPS induced rats. Adult male albino rats were administered LPS (5mg/kg BW) to induce acute inflammation. LPS administration induced severe oxidative damage in the brain and testicular tissue which was evident from decreased activities of enzymic antioxidants and increased lipid peroxidation levels. The mRNA expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD) and androgen receptor corepressor-19kDa (ARR19) in the brain and testis were determined. The mRNA expression of 3ß-HSD and 17ß-HSD was increased in the brain with significant decrease in the testis at 24h and 48h in LPS treated animals. The results also demonstrated an interesting finding that LPS treatment completely represses ARR19 in the brain, while not in the testis. These findings show ARR19 might play a crucial role in regulation of neuronal and testicular steroidogenesis in inflammatory diseases.

Characteristics of 17ß-hydroxysteroid dehydrogenase 8 and its potential role in gonad of Zhikong scallop Chlamys farreri.

17ß-Hydroxysteroid dehydrogenases (17ß-HSDs) are important enzymes catalyzing steroids biosynthesis and metabolism in vertebrates. Although studies indicate steroids play a potential role in reproduction of molluscs, little is known about the presence and function of 17ß-HSDs in molluscs. In the present study, a full-length cDNA encoding 17ß-HSD type 8 (17ß-HSD8) was identified in the Zhikong scallop Chlamys farreri, which is 1104bp in length with an open reading frame of 759bp encoding a protein of 252 amino acids. Phylogenetic analysis revealed that the C. farreri 17ß-HSD8 (Cf-17ß-HSD8) belongs to the short chain dehydrogenase/reductase family (SDR) and shares high homology with other 17ß-HSD8 homologues. Catalytic activity assay in vitro demonstrated that the refolded Cf-17ß-HSD8 expressed in Escherichia coli could effectively convert estradiol-17ß (E2) to estrone (E1), and weakly catalyze the conversion of testosterone (T) to androstenedione (A) in the presence of NAD(+). The Cf-17ß-HSD8 mRNA was ubiquitously expressed in all tissues analyzed, including gonads. The expression levels of Cf-17ß-HSD8 mRNA and protein increased with gametogenesis in both ovary and testis, and were significantly higher in testis than in ovary at growing stage and mature stage. Moreover, results of in situ hybridization and immunohistochemistry revealed that the mRNA and protein of Cf-17ß-HSD8 were expressed in follicle cells and gametes at all stages except spermatozoa. Our findings suggest that Cf-17ß-HSD8 may play an important role in regulating gametogenesis through modulating E2 levels in gonad of C. farreri.

17ß-Estradiol and estradiol fatty acyl esters and estrogen-converting enzyme expression in adipose tissue in obese men and women.

CONTEXT: Obesity is associated with increased circulating 17ß-estradiol (E2), but less is known about E2 concentrations in adipose tissue. In addition to E2, adipose tissue synthesizes E2 fatty acyl esters (E2-FAE). OBJECTIVE: The aim was to compare estrogen concentrations and expression of estrogen-converting enzymes in adipose tissue between severely obese men and women. DESIGN AND SETTING: Tissue samples were obtained during elective surgery in University Central Hospital in the years 2008 through 2011. PATIENTS: We studied 14 men and 22 premenopausal women undergoing bariatric surgery and 10 control women operated for nonmalignant reasons. INTERVENTIONS: Paired samples were taken from abdominal sc and visceral adipose tissue and serum and analyzed for E2 and E2-FAE by fluoroimmunoassay and liquid chromatography-tandem mass spectrometry. mRNA expression of genes was analyzed by quantitative PCR. RESULTS: Compared with men, E2 levels in sc adipose tissue in obese women were higher, along with higher relative mRNA expression of steroid sulfatase and 17ß-hydroxysteroid dehydrogenases 1, 7, and 12. In men, E2-FAE concentrations in adipose tissue were similar to E2 but in women significantly lower compared with E2. Adipose tissue E2-FAE and serum E2-FAE levels correlated positively in obese subjects. Serum E2 did not significantly correlate with E2 concentration or mRNA expression of genes in adipose tissue in obese men or women. CONCLUSIONS: The production of E2 by the large adipose mass was not reflected by increased circulating E2 concentrations in severely obese men or women. However, adipose tissue may contribute to concentrations of serum E2-FAE.

Postnatal development of gastric aromatase and portal venous estradiol-17ß levels in male rats.

Gastric parietal cells synthesize and secrete estradiol-17ß (E2) into gastric veins joining the portal vein, and a large amount of gastric E2 first binds to its receptors in the liver. However, the role of the gastric E2 is not entirely clear during postnatal development. The objective of this study was to reveal the onset of aromatase and other steroid-synthesizing enzymes in the gastric mucosa; to determine the period of rising E2 levels in the portal vein; and to further understand the relationship between gastric E2 and liver estrogen receptor α (ERα). The immunoblot bands and the immunohistochemistry of gastric mucosa revealed that aromatase protein began to express itself at 20 days and then increased in the levels of aromatase protein from 20 days onward. Expression of mRNAs for gastric aromatase (Cyp19a1) and other steroid-synthesizing enzymes, 17α-Hydroxylase (Cyp17a1) and 17ß-hydroxysteroid dehydrogenase (HSD17b3), also increased similar to the increment of aromatase protein. Portal venous E2 levels were elevated after 20 days and increased remarkably between 23 and 30 days, similar to gastric aromatase mRNA levels. The E2 level was approximately three times higher at 40 days than that at 20 days. The liver weight and Esr1 levels began to increase after 20 days and the increment was positively correlated with the change of portal venous E2 levels. These findings suggest that some changes may occur around 20 days to regulate the gastric E2 synthesis and secretion.

Synthesis of 5α-androstane-17-spiro-δ-lactones with a 3-keto, 3-hydroxy, 3-spirocarbamate or 3-spiromorpholinone as inhibitors of 17ß-hydroxysteroid dehydrogenases.

We synthesized two series of androstane derivatives as inhibitors of type 3 and type 5 17ß-hydroxysteroid dehydrogenases (17ß-HSDs). In the first series, four monospiro derivatives at position C17 were prepared from androsterone (ADT) or epi-ADT. After the protection of the alcohol at C3, the C17-ketone was alkylated with the lithium acetylide of tetrahydro-2-(but-3-ynyl)-2-H-pyran, the triple bond was hydrogenated, the protecting groups hydrolysed and the alcohols oxidized to give the corresponding 3-keto-17-spiro-lactone derivative. The other three compounds were generated from this keto-lactone by reducing the ketone at C3, or by introducing one or two methyl groups. In the second series, two dispiro derivatives at C3 and C17 were prepared from epi-ADT. After introducing a spiro-δ-lactone at C17 and an oxirane at C3, an aminolysis of the oxirane with L-isoleucine methyl ester provided an amino alcohol, which was treated with triphosgene or sodium methylate to afford a carbamate- or a morpholinone-androstane derivative, respectively. These steroid derivatives inhibited 17ß-HSD3 (14-88% at 1 µM; 46-94% at 10 µM) and 17ß-HSD5 (54-73% at 0.3 µM; 91-92% at 3 µM). They did not produce any androgenic activity and did not bind steroid (androgen, estrogen, glucocorticoid and progestin) receptors, suggesting a good profile for prostate cancer therapy.

Steroidogenic capacity of the placenta as a supplemental source of progesterone during pregnancy in domestic cats.

BACKGROUND: Until recently, the corpus luteum (CL) was considered to be the main source of progesterone (P4) during pregnancy in the domestic cat (Felis catus). However, other possible sources of P4 have not been ruled out. Although feline placental homogenates were found to be capable of synthesizing P4, expression of the respective steroidogenic enzymes has not been investigated at the molecular level. Therefore, in the present study, expression of the two major factors involved in the synthesis of P4 - 3beta-hydroxysteroid dehydrogenase (3betaHSD) and steroidogenic acute regulatory protein (StAR) - was investigated in the feline CL and placenta during the course of pseudopregnancy and pregnancy. METHODS: The mRNA levels of StAR and 3betaHSD were determined using Real Time PCR and their localizations were determined by immunohistochemistry. Placental P4 concentrations, after ethyl extraction, were measured by EIA. RESULTS: Luteal 3betaHSD and StAR mRNA levels were strongly time-dependent, peaking during mid-pregnancy. The placental 3betaHSD mRNA level was significantly upregulated towards the end of pregnancy. In the CL, 3betaHSD and StAR protein were localized in the luteal cells whereas in the placenta they were localized to the maternal decidual cells. Placental P4 concentrations were low in early pregnant queens, but increased along with gestational age. CONCLUSIONS: These results confirm that the placenta is an additional source of P4 in pregnant queens and can thereby be considered as an important endocrine organ supporting feline pregnancy.

Progesterone and levonorgestrel regulate expression of 17ßHSD-enzymes in progesterone receptor positive breast cancer cell line T47D.

The use of combined hormone replacement therapy (HRT) with oestrogens and progestins in postmenopausal women has been associated with an increased risk for developing breast cancer. The reasons are not fully understood, but influence of HRT on endogenous conversion of female sex hormones may be involved. The expression of 17ß hydroxysteroid dehydrogenases (17ßHSD), which are enzymes catalysing the conversion between more or less potent oestrogens, may partly be regulated by progestins. The breast cancer cell lines T47D, MCF7 and ZR75-1 were treated with progesterone, medroxyprogesterone acetate (MPA) or levonorgestrel for 48 and 72 h at 10(-7) and 10(-9)M to investigate influence on 17ßHSD1, 17ßHSD2 and 17ßHSD5 mRNA expression measured by real time PCR. The expression of 17ßHSD1 increased in progesterone and levonorgestrel treated T47D cells (48 h 10(-7)M P=0.002; P<0.001) and 17ßHSD5 increased after progesterone treatment (48 h 10(-7)M P=0.003), whereas the expression of 17ßHSD2 decreased after the (48 h 10(-7)M P=0.003; P<0.001). Similar, but less prominent effects were seen in MCF7 and ZR75-1. The progestin effects on 17ßHSD-expression were lost when T47D cells were co-treated with progestins and the progesterone receptor (PgR) inhibitor mifprestone. We show that both reductive (17ßHSD1 and 17ßHSD5) and oxidative (17ßHSD2) members of the 17ßHSD-family are under control of progesterone and progestins in breast cancer cell lines. This is most clear in T47D cells which have high PgR expression. 17ßHSD-enzymes are important players in the regulation of sex steroids locally in breast tumours and tumoural expression of various 17ßHSD-enzymes have prognostic and treatment predictive relevance. We propose a mechanism for increased breast cancer risk after HRT in which hormone replacement affects the expression of 17ßHSD-enzymes, favouring the expression of reductive enzymes, which in turn could increase levels of bioactive and mitogenic estrogens in local tissue, e.g. breast tissue.

The industrial chemical bisphenol A (BPA) interferes with proliferative activity and development of steroidogenic capacity in rat Leydig cells.

The presence of bisphenol A (BPA) in consumer products has raised concerns about potential adverse effects on reproductive health. Testicular Leydig cells are the predominant source of the male sex steroid hormone testosterone, which supports the male phenotype. The present report describes the effects of developmental exposure of male rats to BPA by gavage of pregnant and lactating Long-Evans dams at 2.5 and 25 µg/kg body weight from Gestational Day 12 to Day 21 postpartum. This exposure paradigm stimulated Leydig cell division in the prepubertal period and increased Leydig cell numbers in the testes of adult male rats at 90 days. Observations from in vitro experiments confirmed that BPA acts directly as a mitogen in Leydig cells. However, BPA-induced proliferative activity in vivo is possibly mediated by several factors, such as 1) protein kinases (e.g., mitogen-activated protein kinases or MAPK), 2) growth factor receptors (e.g., insulin-like growth factor 1 receptor-beta and epidermal growth factor receptors), and 3) the Sertoli cell-secreted anti-Mullerian hormone (also called Mullerian inhibiting substance). On the other hand, BPA suppressed protein expression of the luteinizing hormone receptor (LHCGR) and the 17beta-hydroxysteroid dehydrogenase enzyme (HSD17B3), thereby decreasing androgen secretion by Leydig cells. We interpret these findings to mean that the likely impact of deficits in androgen secretion on serum androgen levels following developmental exposure to BPA is alleviated by increased Leydig cell numbers. Nevertheless, the present results reinforce the view that BPA causes biological effects at environmentally relevant exposure levels and its presence in consumer products potentially has implication for public health.

Increased 5α-reductase type 2 expression in human breast carcinoma following aromatase inhibitor therapy: the correlation with decreased tumor cell proliferation.

Tumor cell proliferation and progression of breast cancer are influenced by female sex steroids. However, not all breast cancer patients respond to aromatase inhibitors (AI), and many patients become unresponsive or relapse. Recent studies demonstrate that not only estrogens but also androgens may serve as regulators of estrogen-responsive as well as estrogen-unresponsive human breast cancers. However, the mechanism underlying these androgenic actions has remained relatively unknown. Therefore, in this study, we evaluated the effects of AI upon the expression of enzymes involved in intratumoral androgen production including 17ß-hydroxysteroid dehydrogenase type 5 (17ßHSD5), 5α-reductase types 1 and 2 (5αRed1 and 5αRed2) as well as androgen receptor (AR) levels and correlated the findings with therapeutic responses including Ki67 labeling index (Ki67). Eighty-two postmenopausal invasive ductal carcinoma patients were enrolled in CAAN study from November 2001 to April 2004. Pre- and post-treatment specimens of 29 cases were available for this study. The status of 17ßHSD5, 5αRed1, 5αRed2, and Ki67 in pre- and post-treatment specimens were evaluated. The significant increments of 5αRed2 as well as AR were detected in biological response group whose Ki67 LI decreased by more than 40% of the pre-treatment level. This is the first study demonstrating an increment of 5αRed2 and AR in the group of the patients associated with Ki67 decrement following AI treatment. These results suggest that increased 5αRed2 and AR following AI treatment may partly contribute to reduce the tumor cell proliferation through increasing intratumoral androgen concentrations and its receptor.