Follicle-stimulating hormone promotes growth of human prostate cancer cell line-derived tumor xenografts.

Chemical castration in prostate cancer can be achieved with gonadotropin-releasing hormone (GnRH) agonists or antagonists. Their effects differ by the initial flare of gonadotropin and testosterone secretion with agonists and the immediate pituitary-testicular suppression by antagonists. While both suppress luteinizing hormone (LH) and follicle-stimulating hormone (FSH) initially, a rebound in FSH levels occurs during agonist treatment. This rebound is potentially harmful, taken the expression of FSH receptors (R) in prostate cancer tissue. We herein assessed the role of FSH in promoting the growth of androgen-independent (PC-3, DU145) and androgen-dependent (VCaP) human prostate cancer cell line xenografts in nude mice. Gonadotropins were suppressed with the GnRH antagonist degarelix, and effects of add-back human recombinant FSH were assessed on tumor growth. All tumors expressed GnRHR and FSHR, and degarelix treatment suppressed their growth. FSH supplementation reversed the degarelix-evoked suppression of PC-3 tumors, both in preventive (degarelix and FSH treatment started upon cell inoculation) and therapeutic (treatments initiated 3 weeks after cell inoculation) setting. A less marked, though significant FSH effect occurred in DU145, but not in VCaP xenografts. FSHR expression in the xenografts supports direct FSH stimulation of tumor growth. Testosterone supplementation, to maintain the VCaP xenografts, apparently masked the FSH effect on their growth. Treatment with the LH analogue hCG did not affect PC-3 tumor growth despite their expression of luteinizing hormone/choriongonadotropin receptor. In conclusion, FSH, but not LH, may directly stimulate the growth of androgen-independent prostate cancer, suggesting that persistent FSH suppression upon GnRH antagonist treatment offers a therapeutic advantage over agonist.

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model.

BACKGROUND/AIMS: The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. METHODS: We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. RESULTS: PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. CONCLUSION: PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways.

Luteinizing Hormone and GATA4 Action in the Adrenocortical Tumorigenesis of Gonadectomized Female Mice.

BACKGROUND/AIMS: Physiological role of luteinizing hormone (LH) and its receptor (LHCGR) in adrenal remains unknown. In inhibin-α/Simian Virus 40 T antigen (SV40Tag) (inhα/Tag) mice, gonadectomy-induced (OVX) elevated LH triggers the growth of transcription factor GATA4 (GATA4)-positive adrenocortical tumors in a hyperplasia-adenoma-adenocarcinoma sequence. METHODS: We investigated the role of LHCGR in tumor induction, by crossbreeding inhα/Tag with Lhcgr knockout (LuRKO) mice. By knocking out Lhcgr and Gata4 in Cα1 adrenocortical cells (Lhcgr-ko, Gata4-ko) we tested their role in tumor progression. RESULTS: Adrenal tumors of OVX inhα/Tag mice develop from the hyperplastic cells localized in the topmost layer of zona fasciculata. OVX inhα/Tag/LuRKO only developed SV40Tag positive hyperplastic cells that were GATA4 negative, cleaved caspase-3 positive and did not progress into adenoma. In contrast to Lhcgr-ko, Gata4-ko Cα1 cells presented decreased proliferation, increased apoptosis, decreased expression of Inha, SV40Tag and Lhcgr tumor markers, as well as up-regulated adrenal- and down-regulated sex steroid gene expression. Both Gata4-ko and Lhcgr-ko Cα1 cells had decreased expression of steroidogenic genes resulting in decreased basal progesterone production. CONCLUSION: Our data indicate that LH/LHCGR signaling is critical for the adrenal cell reprogramming by GATA4 induction prompting adenoma formation and gonadal-like phenotype of the adrenocortical tumors in inhα/Tag mice.

Transgenic GATA-4 expression induces adrenocortical tumorigenesis in C57Bl/6 mice.

A link between elevated luteinizing hormone (LH) levels, GATA-4 and LH receptor (LHCGR) expression and gonadotropin-dependent adrenocortical tumorigenesis in humans and mice has been shown. To assess the mechanistic tumorigenic interrelationships between these factors, we transgenically expressed Gata4 under the 21-hydroxylase promoter (Cyp21a1, 21-OH) in C57Bl/6N mice. There was a gradual age-dependent increase of GATA-4 expression only in 21-OH-GATA-4 (TG) female adrenals, in association with slowly progressing neoplasia of non-steroidogenic spindle-shaped A cells in the subcapsular cortex. Gonadectomy (GDX), apparently through direct action of elevated serum LH, markedly enhanced the adrenocortical neoplasia, which now also appeared in GDX TG males. The neoplastic areas of the post-GDX TG adrenals contained, besides A cells, larger lipid-laden, steroidogenically active and LHCGR-positive B cells. Prolonged (>10 months) exposure to elevated post-GDX LH levels resulted in formation of adrenocortical adenomas in the TG mice. Intact and GDX TG mouse adrenals displayed elevated FOG-2 and decreased GATA-6 expression. Additionally, increased expression/activation of components of the Inhbb-Acvr2a-Acvr1c-Smad2/3 signaling system was observed in 12-month-old GDX TG adrenals. Our findings show that two distinct GATA-4-dependent populations of neoplastic adrenocortical cells form: non-steroidogenic LH-independent A cells and steroidogenic LH-dependent B cells.

Distinct testicular steroidogenic response mechanisms between neonatal and adult heat-acclimated male rats.

BACKGROUND: In comparison to short-term gonad heat exposure, little is known about the molecular mechanisms that regulate testicular steroidogenesis during long-term whole body heat acclimation. MATERIAL AND METHODS: Testicular slices from neonatal (NHA) and adult (AHA) heat-acclimated Wistar rats were analysed in vitro to assess the mRNA expression and enzymatic activity of steroidogenic enzymes under basal and luteinising hormone (LH) or prolactin (PRL) stimulated conditions compared with control rats (CR). Furthermore, a de-acclimated group (DA) was created by transferring adult NHA rats to control conditions. RESULTS: Heat acclimation significantly increased plasma LH levels in the AHA group and LH and PRL in the NHA group compared with the CR group; however, after heat acclimation, the T and E2 levels did not differ from the control levels. All heat-acclimated groups showed high basal intra-testicular steroid production in vitro. Moreover, basal Cyp11a1 and Hsd3b1 levels were upregulated in vitro in the NHA and DA groups versus the CR group. LH in vitro stimulation upregulated Cyp11a1 expression in the NHA and AHA groups and PRL stimulation upregulated Cyp17a1 levels in the NHA and DA groups compared with the basal expression levels. In the AHA group, decreased basal Star and CYP11A activities but increased HSD3B1 and CYP17A1 activities were found. CONCLUSION: Our data revealed that despite the similar steroid levels in plasma and secreted in vitro by neonatal and adult heat-acclimated rat testicular slices, the molecular mechanisms underlying the steroidogenic response to heat acclimation during these different developmental stages were distinct.

A novel treatment strategy for ovarian cancer based on immunization against zona pellucida protein (ZP) 3.

We tested the principle of treating malignant ovarian tumors by vaccination against their ectopically expressed protein, zona pellucida glycoprotein (ZP) 3, using as the experimental model the granulosa cell tumors that develop in transgenic mice expressing the simian virus 40 T-antigen under the inhibin-α promoter (inhα/Tag). We found high ZP3 expression in granulosa cell tumors of the transgenic mice, in human surface ovarian cancer and granulosa cell lines, and in human granulosa cell tumors and their metastases. Early preventive immunization (between 2 and 5.5 mo of age) of transgenic mice with recombinant human (rh) ZP3 prevented ovarian tumorigenesis, and delayed therapeutic immunization (between 4.5 and 7 mo) reduced weights of existing tumors by 86 and 75%, respectively (P<0.001), compared to vehicle-treated control mice. No objective side effects of the immunizations were observed. Liver metastases were found in nontreated/vehicle-treated controls (n=7/39), but none following active rhZP3 immunizations (n=0/36; P<0.05). Immunization with rhZP3 was highly effective, as demonstrated by the induction of anti-ZP3 antibodies, as well as proliferative responses to the ZP3 antigen. These results signal rhZP3 immunization as a novel strategy to be developed for the immunotherapy of ovarian granulosa cell tumors, as well as for that of other malignancies that may express ZP3.

A tumor cell specific Zona Pellucida glycoprotein 3 RNA transcript encodes an intracellular cancer antigen.

Background: Expression of Zona Pellucida glycoprotein 3 (ZP3) in healthy tissue is restricted to the extracellular Zona Pellucida layer surrounding oocytes of ovarian follicles and to specific cells of the spermatogenic lineage. Ectopic expression of ZP3 has been observed in various types of cancer, rendering it a possible therapeutic target. Methods: To support its validity as therapeutic target, we extended the cancer related data by investigating ZP3 expression using immunohistochemistry (IHC) of tumor biopsies. We performed a ZP3 transcript specific analysis of publicly available RNA-sequencing (RNA-seq) data of cancer cell lines (CCLs) and tumor and normal tissues, and validated expression data by independent computational analysis and real-time quantitative PCR (qPCR). A correlation between the ZP3 expression level and pathological and clinical parameters was also investigated. Results: IHC data for several cancer types showed abundant ZP3 protein staining, which was confined to the cytoplasm, contradicting the extracellular protein localization in oocytes. We noticed that an alternative ZP3 RNA transcript, which we term 'ZP3-Cancer', was annotated in gene databases that lacks the genetic information encoding the N-terminal signal peptide that governs entry into the secretory pathway. This explains the intracellular localization of ZP3 in tumor cells. Analysis of publicly available RNA-seq data of 1339 cancer cell lines (CCLs), 10386 tumor tissues (The Cancer Genome Atlas) and 7481 healthy tissues (Genotype-Tissue Expression) indicated that ZP3-Cancer is the dominant ZP3 RNA transcript in tumor cells and is highly enriched in many cancer types, particularly in rectal, ovarian, colorectal, prostate, lung and breast cancer. Expression of ZP3-Cancer in tumor cells was confirmed by qPCR. Higher levels of the ZP3-Cancer transcript were associated with more aggressive tumors and worse survival of patients with various types of cancer. Conclusion: The cancer-restricted expression of ZP3-Cancer renders it an attractive tumor antigen for the development of a therapeutic cancer vaccine, particularly using mRNA expression technologies.

First-in-Class Small Molecule to Inhibit CYP11A1 and Steroid Hormone Biosynthesis.

Binding of steroid hormones to their cognate receptors regulates the growth of most prostate and breast cancers. We hypothesized that CYP11A inhibition might halt the synthesis of all steroid hormones, because CYP11A is the only enzyme that catalyses the first step of steroid hormone biosynthesis. We speculated that a CYP11A inhibitor could be administered safely provided that the steroids essential for life are replaced. Virtual screening and systematic structure-activity relationship optimization were used to develop ODM-208, the first-in-class, selective, nonsteroidal, oral CYP11A1 inhibitor. Safety of ODM-208 was assessed in rats and Beagle dogs, and efficacy in a VCaP castration-resistant prostate cancer (CRPC) xenograft mouse model, in mice and dogs, and in six patients with metastatic CRPC. Blood steroid hormone concentrations were measured using liquid chromatography-mass spectrometry. ODM-208 binds to CYP11A1 and inhibited its enzymatic activity. ODM-208 administration led to rapid, complete, durable, and reversible inhibition of the steroid hormone biosynthesis in an adrenocortical carcinoma cell model in vitro, in adult noncastrated male mice and dogs, and in patients with CRPC. All measured serum steroid hormone concentrations reached undetectable levels within a few weeks from the start of ODM-208 administration. ODM-208 was well tolerated with steroid hormone replacement. The toxicity findings were considered related to CYP11A1 inhibition and were reversed after stopping of the compound administration. Steroid hormone biosynthesis can be effectively inhibited with a small-molecule inhibitor of CYP11A1. The findings suggest that administration of ODM-208 is feasible with concomitant corticosteroid replacement therapy.

Novel expression of zona pellucida 3 protein in normal testis; potential functional implications.

The expression of the zona pellucida glycoprotein 3 (ZP3), originally thought to be specific for oocytes, was recently extended to ovarian, prostate, colorectal and lung cancers. Earlier successful ZP3 immunization of a transgenic mouse model carrying a ZP3 positive ovarian tumor emphasized the suitability of ZP3 for cancer immunotherapy. This study was carried out to determine whether any other normal tissues besides the ovary in healthy human and mouse tissues may express ZP3, considered important to exclude off-target effects of ZP3 cancer immunotherapy. Strong ZP3 expression was found in normal human and mouse testis. ZP3 protein and mRNA transcripts were localized in spermatogonia, spermatocytes and round and elongated spermatids of both human and mouse testis, as well as in a mouse spermatogonial cell line, but absent in testicular Sertoli, Leydig, spermatogonial stem and progenitor cells. All other normal human and mouse tissues were ZP3 negative. This surprising testicular ZP3 expression has implications for the development of ZP3 cancer immunotherapies, and it also alludes to the potential of using ZP3 as a target for the development of a male immunocontraceptive.

Congenital Hypothyroidism and Hyperthyroidism Alters Adrenal Gene Expression, Development, and Function.

Background: The human adrenal cortex undergoes several rapid remodeling steps during its lifetime. In rodents, similar remodeling occurs postnatally in the "X-zone" layer through unknown mechanisms. Furthermore, little is known regarding the impact of thyroid hormone (TH) on adrenal glands in humans. Methods: To investigate the impact of TH on adrenal pathophysiology, we created two genetic murine models mimicking human nonautoimmune hypothyroidism and hyperthyroidism. Moreover, we analyzed serum thyrotropin (TSH) and steroid hormone concentrations in patients diagnosed with congenital hypothyroidism and premature adrenarche (PA). Results: We found that TH receptor beta-mediated hypertrophy of the X-zone significantly elevated the adrenal weights of hyperthyroid women. In the hypothyroid model, the X-zone was poorly developed in both sexes. Moreover, large reciprocal changes in the expression levels of genes that regulate adrenal cortical function were observed with both models. Unexpectedly, up- and downregulation of several genes involved in catecholamine synthesis were detected in the adrenal glands of the hypothyroid and hyperthyroid models, respectively. Furthermore, TSH and adrenal steroid concentrations correlated positively in pediatric patients with congenital hypothyroidism and PA. Conclusions: Our results revealed that congenital hypothyroidism and hyperthyroidism functionally affect adrenal gland development and related steroidogenic activity, as well as the adrenal medulla.

Immortalization of swine umbilical vein endothelial cells (SUVECs) with the simian virus 40 large-T antigen.

Implementation of the swine umbilical vein endothelial cells (SUVECs) model in vitro can be instrumental in determining the biology of endothelial cells. We have generated an immortalized endothelial cell line, G-1410, using Simian virus 40 T-antigen (SV40 T-ag) primarily to overcome the short life span before the onset of senescence and high variability among enzymatically isolated cells of primary cultures. Fast proliferating cells were selected from cultures and, after a fifth passage, examined for the presence of the SV40 T-ag by PCR and immunocytochemistry. Phase contrast and transmission electron microscopy revealed that G-1410 cells did not differ morphologically from SUVECs. The G-1410 cells exhibited positive staining for vascular endothelial (VE)-cadherin and von Willebrand factor (vWF), and formed capillary-like tube structures on Matrigel. Despite the strong oncogenic signal provided by SV40 T-ag, these transformed G-1410 cells have remained karyotypically normal and non-tumorigenic. G-1410 cells also responded to stimulation with VEGF, FGF-2, and newborn calf serum. Moreover, G-1410 cells showed elevated expression of VEGF120, VEGF164 (VEGF-A), and FGF-2 at both mRNA and protein levels. In conclusion, based on the cytological and functional evaluation of the newly obtained immortalized cell line, it can be concluded that G-1410 cells provide a useful tool for studying the effects of VEGF and FGF systems, and other signal transduction pathways related to angiogenesis.

Mifepristone Treatment Promotes Testicular Leydig Cell Tumor Progression in Transgenic Mice.

The selective progesterone receptor modulator mifepristone (MF) may act as a potent antiproliferative agent in different steroid-dependent cancers due to its strong antagonistic effect on the nuclear progesterone receptor (PGR). Hereby, we analyzed the effects of MF treatment on Leydig cell tumor (LCT) progression in a transgenic mouse model (inhibin-α promoter-driven SV40 T-antigen), as well as on LCT (BLTK-1 and mLTC-1) cell proliferation. MF significantly stimulated the proliferation of LCT in vitro. Similarly, a 1-mo MF or P4 treatment stimulated LCT tumor growth in vivo. Traceable/absent classical Pgr or nonclassical membrane PRs α, ß, γ and Pgrmc2, but abundant membrane Pgrmc1 expression, was found in LCTs. MF did not activate glucocorticoid or androgen receptors in LCTs. Functional analysis showed that PGRMC1 is required for MF and P4 to stimulate the proliferation and invasiveness of LCTs. Accordingly, MF and P4 induced PGRMC1 translocation into the nucleus and thereby stimulated the release of TGFß1 in LCT cells. MF and P4 treatments upregulated Tgfbr1, Tgfbr2, and Alk1 expression and stimulated TGFß1 release in LCT cells. Our findings provide novel mechanistic insights into the action of MF as a membrane PR agonist that promotes LCT growth through PGRMC1 and the alternative TGFß1 signaling pathway.

Extragonadal FSHR Expression and Function-Is It Real?

Expression of the follicle-stimulating hormone receptor (FSHR), besides gonadal tissues, has recently been detected in several extragonadal normal and tumorous tissues, including different types of primary and metastatic cancer and tumor vessel endothelial cells (TVEC). The suggested FSH actions in extragonadal tissues include promotion of angiogenesis, myometrial contractility, skeletal integrity, and adipose tissue accumulation. Non-malignant cells within cancer tissue have been shown to be devoid of FSHR expression, which implies a potential role of FSHR as a diagnostic, prognostic, or even a therapeutic tool. There are shared issues between several of the published reports questioning the validity of some of the conclusion. Firstly, protein expression of FSHR was performed solely with immunohistochemistry (IHC) using either an unavailable "in house" FSHR323 monoclonal antibody or poorly validated polyclonal antibodies, usually without additional methodological quality control and confirmations. Secondly, there is discrepancy between the hardly traceable or absent FSHR gene amplification/transcript data and non-reciprocal strong FSHR protein immunoreactivity. Thirdly, the pharmacological high doses of recombinant FSH used in in vitro studies also jeopardizes the physiological or pathophysiological meaning of the findings. We performed in this review a critical analysis of the results presenting extragonadal expression of FSHR and FSH action, and provide a rationale for the validation of the reported results using additional more accurate and sensitive supplemental methods, including in vivo models and proper positive and negative controls.

Features of the fetal gonad in androgen synthesis in the postpubertal testis are preserved in complete androgen insensitivity syndrome due to a novel genetic splice site donor variant in androgen receptor gene intron 1.

Mutations in the X-linked androgen receptor (AR) gene cause complete androgen insensitivity syndrome (CAIS). CAIS may cause congenital sexual development disorder, which frequently develops into testicular tumors. Here, we describe a novel splice-site intron 1 mutation in AR leading to improper splicing and AR protein absence in CAIS gonads. We characterized a patient's postpubertal gonadal steroidogenic enzyme expression profile. Localization of both CYP11A1 and CYP17A1 enzymes was restricted to both Leydig tumor cells and adjacent to tumor gonadal tissues. Sertoli cells of the CAIS gonad showed abundant HSD17B3 protein, which is an adult Leydig cell marker that enables the conversion of androstenedione to testosterone. Such HSD17B3 expression is typical for fetal-type Sertoli cells in rodents. The postpubertal CAIS gonad of our patient was completely devoid of androgen signaling pathway activity. Plausibly, the postpubertal Leydig cells consisted of two distinct cell populations: postpubertal fetal-type Leydig cells that persisted as androgen-independent cells and immature adult Leydig cells that failed to differentiate. Taken together, in this CAIS postpubertal testis, both Leydig and fetal-type Sertoli cells participated in testosterone production. Our results indicate the importance of molecular analysis as well as the characterization of steroidogenic enzyme profiling in the CAIS patient's gonad.

Molecular mechanisms underlying mifepristone's agonistic action on ovarian cancer progression.

BACKGROUND: Recent clinical trials on ovarian cancer with mifepristone (MF) have failed, despite in vitro findings on its strong progesterone (P4) antagonist function. METHODS: Ovarian cancer human and murine cell lines, cultured high-grade human primary epithelial ovarian cancer (HG-hOEC) cells and their explants; as well as in vivo transgenic mice possessing ovarian cancer were used to assess the molecular mechanism underlying mifepristone (MF) agonistic actions in ovarian cancer progression. FINDINGS: Herein, we show that ovarian cancer cells express traceable/no nuclear P4 receptor (PGR), but abundantly P4 receptor membrane component 1 (PGRMC1). MF significantly stimulated ovarian cancer cell migration, proliferation and growth in vivo, and the translocation of PGRMC1 into the nucleus of cancer cells; the effects inhibited by PGRMC1 inhibitor. The beneficial antitumor effect of high-doses MF could not be achieved in human cancer tissue, and the low tissue concentrations achieved with the therapeutic doses only promoted the growth of ovarian cancers. INTERPRETATION: Our results indicate that treatment of ovarian cancer with MF and P4 may induce similar adverse agonistic effects in the absence of classical nuclear PGRs in ovarian cancer. The blockage of PGRMC1 activity may provide a novel treatment strategy for ovarian cancer. FUND: This work was supported by grants from the National Science Centre, Poland (2013/09/N/NZ5/01831 to DP-T; 2012/05/B/NZ5/01867 to MC), Academy of Finland (254366 to NAR), Moikoinen Cancer Research Foundation (to NAR) and EU PARP Cluster grant (UDA-POIG.05.01.00-005/12-00/NCREMFP to SW).

GnRH antagonist treatment of malignant adrenocortical tumors.

Aberrantly expressed G protein-coupled receptors in tumors are considered as potential therapeutic targets. We analyzed the expressions of receptors of gonadotropin-releasing hormone (GNRHR), luteinizing hormone/chorionic gonadotropin (LHCGR) and follicle-stimulating hormone (FSHR) in human adrenocortical carcinomas and assessed their response to GnRH antagonist therapy. We further studied the effects of the GnRH antagonist cetrorelix acetate (CTX) on cultured adrenocortical tumor (ACT) cells (mouse Cα1 and Y-1, and human H295R), and in vivo in transgenic mice (SV40 T-antigen expression under inhibin α promoter) bearing Lhcgr and Gnrhr in ACT. Both models were treated with control (CT), CTX, human chorionic gonadotropin (hCG) or CTX+hCG, and their growth and transcriptional changes were analyzed. In situ hybridization and qPCR analysis of human adrenocortical carcinomas (n = 11-13) showed expression of GNRHR in 54/73%, LHCGR in 77/100% and FSHR in 0%, respectively. CTX treatment in vitro decreased cell viability and proliferation, and increased caspase 3/7 activity in all treated cells. In vivo, CTX and CTX+hCG (but not hCG alone) decreased ACT weights and serum LH and progesterone concentrations. CTX treatment downregulated the tumor markers Lhcgr and Gata4. Upregulated genes included Grb10, Rerg, Nfatc and Gnas, all recently found to be abundantly expressed in healthy adrenal vs ACT. Our data suggest that CTX treatment may improve the therapy of human adrenocortical carcinomas by direct action on GNRHR-positive cancer cells inducing apoptosis and/or reducing gonadotropin release, directing tumor cells towards a healthy adrenal gene expression profile.

Influence of estradiol-17beta and progesterone on nitric oxide (NO) production in the porcine endometrium during first half of pregnancy.

The purpose of the study was to examine: 1/ endometrial concentrations of nitrate/nitrite (NOx) in pregnant pigs, and 2/ the influence of estradiol-17beta (E(2)) and/or progesterone (P(4)) on NOx production by porcine endometrium during the first half of pregnancy. Total NOx concentrations were determined using a microplate assay method based on the Griess reaction. Evident fluctuations of endometrial NOx content were found during the examined time of pregnancy (days 5, 10, 15, 20, 25, 30, 35, 40 and 60 of pregnancy). The NOx concentration was highest on days 10 and 15, and then lowered until day 60 of pregnancy. In addition, we demonstrated the stimulatory effect of E(2) and/or P(4) on NO in vitro production by porcine endometrial slices. The medium content of NOx depended on the steroid type, treatment dose and day of pregnancy. It is possible that the observed differences in the strength of the stimulatory action of E(2) and/or P(4) on endometrial NOx production are associated with activation of different isoforms of NOS.

The influence of steroid hormones on in vitro NOx production by porcine fetal membranes.

The aim of the study was to examine: 1/ allantochorial concentrations of nitrate/nitrite (NOx) and 2/ plasma concentration of NOx in pigs on days 25, 35, 40 and 60 of pregnancy as well as 3/ the influence of estradiol-17beta (E(2)) and/or progesterone (P(4)) on NOx production by porcine fetal membranes on the studied days of pregnancy. Total NOx concentration was determined using a microplate assay method based on the Griess reaction. Fetal membrane NOx content gradually increased from day 25 to day 60 of gestation. Blood plasma NOx concentration decreased from day 25 to 40, and then plasma NOx concentration significantly increased on day 60. In addition, the stimulatory effect of E(2), P(4) and E(2)+P(4) on NO in vitro production by porcine fetal membranes was demonstrated. The stimulatory effect of steroid hormones on NOx release depended on steroid dose and day of pregnancy. It is possible that the observed differences in the strength of the stimulatory action of E(2), P(4) and E(2)+P(4) on fetal membrane NOx production are associated with an activation of different isoforms of nitric oxide synthase.

The localization and expression of NADPH-diaphorase and isoforms of nitric oxide synthase in the porcine gravid uterus.

The aim of the present study was to investigate the localization and expression of NADPH-diaphorase (NADPH-d) as well as endothelial (eNOS) and inducible (iNOS) nitric oxide synthase isoforms in the porcine uterus on Days 20, 30, 40, 60, 75 and 90 of gestation. The significant increase (p<0.001) in NADPH-d reaction was found in the trophoblast. The positive NADPH-d staining was also observed in the luminal and glandular epithelium and vascular endothelium. eNOS immunoreactivity was found in the endothelium of placental and maternal blood vessels, in the luminal epithelium and endometrial glands. The trophoblast displayed a slight increase in eNOS immunoreactivity starting from Day 20 of gestation. In comparison to eNOS, a stronger iNOS staining was found in the placental and endometrial blood vessels and in the trophoblast cells. Western blot analysis revealed a gradual increase in eNOS protein expression from Day 30 (p<0.001) and iNOS from Day 60 of gestation (p<0.001) in the placental zone. The significant increase in the NADPHd/iNOS expression after day 60 of gestation suggests a contribution of iNOS in uterine NO generation during the second half of pregnancy. A gradual increase in eNOS expression starting on Day 30 of pregnancy may indicate a synergistic, supportive effect on uterine vascular and myometrial contractility in pigs affecting the maintenance of the uterine quiescence.

Constitutively active follicle-stimulating hormone receptor enables androgen-independent spermatogenesis.

Spermatogenesis is regulated by the 2 pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This process is considered impossible without the absolute requirement of LH-stimulated testicular testosterone (T) production. The role of FSH remains unclear because men and mice with inactivating FSH receptor (FSHR) mutations are fertile. We revisited the role of FSH in spermatogenesis using transgenic mice expressing a constitutively strongly active FSHR mutant in a LH receptor-null (LHR-null) background. The mutant FSHR reversed the azoospermia and partially restored fertility of Lhr-/- mice. The finding was initially ascribed to the residual Leydig cell T production. However, when T action was completely blocked with the potent antiandrogen flutamide, spermatogenesis persisted. Hence, completely T-independent spermatogenesis is possible through strong FSHR activation, and the dogma of T being a sine qua non for spermatogenesis may need modification. The mechanism for the finding appeared to be that FSHR activation maintained the expression of Sertoli cell genes considered androgen dependent. The translational message of our findings is the possibility of developing a new strategy of high-dose FSH treatment for spermatogenic failure. Our findings also provide an explanation of molecular pathogenesis for Pasqualini syndrome (fertile eunuchs; LH/T deficiency with persistent spermatogenesis) and explain how the hormonal regulation of spermatogenesis has shifted from FSH to T dominance during evolution.