Human benign Leydig cell tumor - Biochemical evaluation.

Recently we reported expressional alterations in 219 genes and their transcripts in Leydig cell tumors but nowadays there is still a lack of full basic biochemical characteristics of these tumors. The discovery of potential biochemical markers for tumor management from early detection, treatments, and control of therapy results may markedly supplement genetic data. Leydig cell micronodules were obtained from patients with azoospermia who were qualified for testicular biopsy. The biochemistry of Leydig cell tumors was analyzed using histological staining and spectrophotometric measurements of total proteins, carbohydrates, lipids, and nucleic acids. In addition, the levels of calcium (Ca2 +), copper (Cu2 +), zinc (Zn2 +), and selenium (Se2 +) ions were measured. When compared to healthy testis we revealed, for the first time, that in the interstitial tissue with Leydig cell tumors, great amounts of proteins, carbohydrates, lipids, and acids were dislocated from the seminiferous tubules. Measurements of organic compounds showed a decrease (P < 0.05) only in the Cu2 + content in Leydig cell tumors which may be related to their altered biochemical structure. This specific result may be promising for designing further approaches to manage this tumor based on combining morphological and molecular data.

ß-Catenin alterations in testicular Leydig cell tumour: a immunohistochemical and molecular analysis.

BACKGROUND: Testicular Leydig cell tumours (LCTs) are the most common type of sex cord-stromal tumour in men, representing 1%-3% of all testicular neoplasms. Among testicular sex cord-stromal tumours, CTNNB1 mutations and nuclear expression of ß-catenin have been typically associated with Sertoli cell tumour. Recent genomic analyses have shown that CTNNB1 variants are also identified in a subset of LCTs; however, the frequency and clinicopathologic associations of ß-catenin alterations remain incompletely understood in this tumour type. METHODS: In this study, we evaluated 32 LCTs (five malignant/metastasizing, 27 nonmetastasizing) using ß-catenin immunohistochemistry and DNA sequencing. RESULTS: Immunohistochemistry revealed focal or multifocal nuclear ß-catenin expression in 47% of the tumours. Diffuse nuclear ß-catenin expression (in >50% of the tumour cells) was not detected in any of the cases analysed herein. Comparison of ß-catenin-positive and ß-catenin-negative cases did not show significant differences in the frequency of adverse histopathologic findings or malignant clinical behaviour. DNA sequencing performed de novo on a subset of seven cases revealed the presence of exon 3 CTNNB1 variants in four of them (4/7, 57%), with variant allele frequencies (VAF) ranging from 7 to 33%. Two additional ß-catenin-positive cases that had been sequenced as part of a previous study harboured exon 3 CTNNB1 variants at VAF of 28% and 7%, respectively. CONCLUSION: These results demonstrate that ß-catenin alterations are relatively common in LCT, most likely occurring as subclonal events that are not enriched in cases with aggressive features. Further studies are needed to clarify the oncogenic role of ß-catenin in this tumour type.

Histidine decarboxylase inhibitors: a novel therapeutic option for the treatment of leydigioma.

Recent reports indicate an increase in Leydig cell tumor (LCT) incidence. Radical orchiectomy is the standard therapy in children and adults, although it entails physical and psychosocial side effects. Testis-sparing surgery can be a consideration for benign LCT of 2.5 cm or less in size. Malignant LCTs respond poorly to conventional chemotherapy, so new treatment modalities are needed. In this study, we observed increased histidine decarboxylase expression and pro-angiogenic potential in LCT surgically resected from pediatric patients (fetal to pubertal) vs control samples from patients without endocrine or metabolic disorders which were collected at necropsy. We, therefore, evaluated for the first time the antitumor efficacy of two histidine decarboxylase inhibitors (α-methyl-dl-histidine dihydrochloride (α-MHD) and epigallocatechin gallate (EGCG)), alone and combined with carboplatin, in two preclinical models of LCT. MA-10 and R2C Leydig tumor cells, representing two different LCT subtypes, were used to generate syngeneic and xenograft mouse LCT models, respectively. In the syngeneic model, monotherapy with α-MHD effectively reduced tumor growth and angiogenesis. In the xenografts, which showed co-expression of histidine decarboxylase and CYP19, the combination of EGCG plus carboplatin was the most effective therapy, leading to LCT growth arrest and undetectable levels of plasmatic estradiol. Testicular and body weights remained unaltered. On the basis of this study, histidine decarboxylase may emerge as a novel pharmacological target for LCT treatment.

New-onset hirsutism.

This postmenopausal patient developed hirsutism following a surgical procedure. Thorough lab work directed us to the unusual cause.

Comparative molecular analysis of testicular Leydig cell tumors demonstrates distinct subsets of neoplasms with aggressive histopathologic features.

Testicular Leydig cell tumor (LCT), the most common sex-cord stromal tumor in men, represents a small fraction of all testicular tumors (~1 to 3%). Although most testicular LCTs are indolent and cured by radical orchiectomy, 5-10% have aggressive biology and metastatic potential. In primary LCTs, large size, cytologic atypia, necrosis, increased mitotic activity, and vascular invasion have been associated with clinically aggressive tumors. From a molecular perspective, the characteristics of aggressive LCTs and the differences between aggressive and nonaggressive LCTs remain largely unexplored. This study compares the genomic landscape of aggressive and nonaggressive testicular LCTs. Twenty-six cases were analyzed using next-generation DNA sequencing (NGS) and immunohistochemistry. Cases were classified as aggressive LCT if they met published criteria for malignancy in primary (i.e., testicular) tumors or if they had pathology-proven metastatic disease; otherwise, cases were considered nonaggressive. This multi-institutional series included 18 aggressive LCTs (14 primary/testicular, 4 metastatic) and 8 nonaggressive LCTs. Two cases (2/26, 8%; both aggressive LCTs) failed sequencing and had negative (i.e., uninformative) FH immunohistochemistry results. One additional primary aggressive LCT failed sequencing but had informative FH immunohistochemistry results. Combined NGS and immunohistochemical analysis demonstrated FH inactivation in 5/26 cases (19%). In addition, NGS demonstrated CTNNB1 mutations or biallelic APC inactivation in 9/23 cases (39%), copy number changes without recurrent mutations in 6/23 (26%) cases, and no alterations in 4/23 cases (17%). CTNNB1 mutations were present in both aggressive and nonaggressive LCTs. In contrast, FH inactivation and multiple copy number changes were only identified in aggressive LCTs. In conclusion, three distinct subgroups of aggressive LCTs were characterized by FH inactivation, Wnt pathway activation, and copy number changes without recurrent mutations, respectively. Nuclear translocation of ß-catenin and Wnt pathway activation appear to be early driver events that provide an environment conducive for progression to aggressive biology in a subset of LCTs.

Dynamic Remodeling of Membranes and Their Lipids during Acute Hormone-Induced Steroidogenesis in MA-10 Mouse Leydig Tumor Cells.

Lipids play essential roles in numerous cellular processes, including membrane remodeling, signal transduction, the modulation of hormone activity, and steroidogenesis. We chose steroidogenic MA-10 mouse tumor Leydig cells to investigate subcellular lipid localization during steroidogenesis. Electron microscopy showed that cAMP stimulation increased associations between the plasma membrane (PM) and the endoplasmic reticulum (ER) and between the ER and mitochondria. cAMP stimulation also increased the movement of cholesterol from the PM compared to untreated cells, which was partially inhibited when ATPase family AAA-domain containing protein 3 A (ATAD3A), which functions in ER and mitochondria interactions, was knocked down. Mitochondria, ER, cytoplasm, PM, PM-associated membranes (PAMs), and mitochondria-associated membranes (MAMs) were isolated from control and hormone-stimulated cells. Lipidomic analyses revealed that each isolated compartment had a unique lipid composition, and the induction of steroidogenesis caused the significant remodeling of its lipidome. cAMP-induced changes in lipid composition included an increase in phosphatidylserine and cardiolipin levels in PAM and PM compartments, respectively; an increase in phosphatidylinositol in the ER, mitochondria, and MAMs; and a reorganization of phosphatidic acid, cholesterol ester, ceramide, and phosphatidylethanolamine. Abundant lipids, such as phosphatidylcholine, were not affected by hormone treatment. Our data suggested that PM-ER-mitochondria tethering may be involved in lipid trafficking between organelles and indicated that hormone-induced acute steroid production involves extensive organelle remodeling.

Hyperandrogenism caused by ovarian Leydig cell tumour: finding the needle in a haystack.

Leydig cell tumours (LCTs) of the ovary are rare ovarian tumours that usually present with hyperandrogenism. Conventional radiological imagings are helpful in localising these tumours. However, some tumours may be too small to be localised before curative surgical removal. It is important to identify these androgen-secreting neoplasms which originate mostly from adrenals or ovaries because they are potentially malignant and require specific treatment. When conventional imagings are unrevealing, selective ovarian and adrenal venous sampling (SOAVS) is the next option. We report a case of LCT that was localised by SOAVS after results from other imaging modalities remained inconclusive.

Interstitial Leydig Cell Tumorigenesis-Leptin and Adiponectin Signaling in Relation to Aromatase Expression in the Human Testis.

Although epidemiological studies from the last years report an increase in the incidences of Leydig cell tumors (previously thought to be a rare disease), the biochemical characteristics of that tumor important for understanding its etiology, diagnosis, and therapy still remains not completely characterized. Our prior studies reported G-protein coupled estrogen receptor signaling and estrogen level disturbances in Leydig cell tumors. In addition, we found that expressions of multi-level-acting lipid balance- and steroidogenesis-controlling proteins including peroxisome proliferator-activated receptor are altered in this tumor. In order to get deeper into the other molecular mechanisms that regulate lipid homeostasis in the Leydig cell tumor, here we investigate the presence and expression of newly-described hormones responsible for lipid homeostasis balancing (leptin and adiponectin), together with expression of estrogen synthase (aromatase). Samples of Leydig cell tumors (n = 20) were obtained from patients (31-45 years old) and used for light and transmission electron microscopic, western blotting, and immunohistochemical analyses. In addition, body mass index (BMI) was calculated. In tumor mass, abundant lipid accumulation in Leydig cells and various alterations of Leydig cell shape, as well as the presence of adipocyte-like cells, were observed. Marked lipid content and various lipid droplet size, especially in obese patients, may indicate alterations in lipid homeostasis, lipid processing, and steroidogenic organelle function in response to interstitial tissue pathological changes. We revealed significantly increased expression of leptin, adiponectin and their receptors, as well as aromatase in Leydig cell tumors in comparison to control. The majority of patients (n = 13) were overweight as indicated by their BMI. Moreover, a significant increase in expression of phospholipase C (PLC), and kinases Raf, ERK which are part of adipokine transductional pathways, was demonstrated. These data expand our previous findings suggesting that in human Leydig cell tumors, estrogen level and signaling, together with lipid status, are related to each other. Increased BMI may contribute to certain biochemical characteristics and function of the Leydig cell in infertile patients with a tumor. In addition, altered adipokine-estrogen microenvironment can have an effect on proliferation, growth, and metastasis of tumor cells. We report here various targets (receptors, enzymes, hormones) controlling lipid balance and estrogen action in Leydig cell tumors indicating their possible usefulness for diagnostics and therapy.

Towards understanding leydigioma: do G protein-coupled estrogen receptor and peroxisome proliferator-activated receptor regulate lipid metabolism and steroidogenesis in Leydig cell tumors?

Leydig cell tumors (LCT) are the most common type of testicular stromal tumor. Herein, we investigate the G protein-coupled estrogen receptor (GPER) and peroxisome proliferator-activated receptor (PPAR) implication in regulation of lipid homeostasis including the expression of steroidogenesis-controlling molecules in clinical specimens of LCTs and tumor Leydig cells (MA-10). We showed the general structure and morphology of LCTs by scanning electron and light microscopy. In LCTs, mRNA and protein analyses revealed increased expression of GPER and decreased expression of PPARα, ß, and γ. Concomitantly, changes in expression pattern of the lutropin receptor (LHR), protein kinase A (PKA), perilipin (PLIN), hormone sensitive lipase (HSL), steroidogenic acute regulatory protein (StAR), translocator protein (TSPO), HMG-CoA synthase, and reductase (HMGCS, HMGCR) were observed. Using MA-10 cells treated with GPER and PPAR antagonists (alone and in combination), we demonstrated GPER-PPAR-mediated control of estradiol secretion via GPER-PPARα and cyclic guanosine monophosphate (cGMP) concentration via GPER-PPARγ. It is assumed that GPER and PPAR can crosstalk, and this can be altered in LCT, resulting in a perturbed lipid balance and steroidogenesis. In LCTs, the phosphatidylinositol-3-kinase (PI3K)-Akt-mTOR pathway was disturbed. Thus, PI3K-Akt-mTOR with cGMP can play a role in LCT outcome and biology including lipid metabolism.

Leydig cell tumorigenesis - implication of G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and xenoestrogen exposure. In vivo and in vitro appraisal.

The etiology and molecular characteristics of Leydig cell tumor (LCT) are scarcely known. From the research data stems that estrogen can be implicated in LCT induction and development, however it is not investigated in detail. Considering the above, herein we analyzed the relation between G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and insulin-like family peptides (insulin-like 3 peptide; INSL3 and relaxin; RLN) expressions as well as estrogen level with impact of xenoestrogen (bisphenol A; BPA, tetrabromobisphenol A; TBBPA, and tetrachlorobisphenol A; TCBPA). While in our previous studies altered GPER-PPAR partnership was found in human LCT being a possible cause and/or additionally effecting on LCT development, here mouse testes with experimentally induced LCT and mouse tumor Leydig cell (MA-10) treated with BPA chemicals were examined. We revealed either diverse changes in expression or co-expression of GPER and PPAR in mouse LCT as well as in MA-10 cells after BPA analogues when compared to human LCT. Relationships between expression of INSL3, RLN, including co-expression, and estrogen level in human LCT, mouse LCT and MA-10 cells xenoestrogen-treated were found. Moreover, involvement of PI3K-Akt-mTOR pathway or only mTOR in the interactions of examined receptors and hormones was showed. Taken together, species, cell of origin, experimental system used and type of used chemical differences may result in diverse molecular characteristics of LCT. Estrogen/xenoestrogen may play a role in tumor Leydig cell proliferation and biochemical nature but this issue requires further studies. Experimentally-induced LCT in mouse testis and MA-10 cells after BPA exposure seem to be additional models for understanding some aspects of human LCT biology.

Inhibition of progesterone biosynthesis induced by deca-brominated diphenyl ether (BDE-209) in mouse Leydig tumor cell (MLTC-1).

Polybrominated Diphenyl Ethers (PBDEs) have been extensively applied as flame retardants in different polymeric materials since the 1970s, which have become a group of long-lasting environmental pollutants. They have been reported from previous studies to accumulate and then disrupt the endocrine system in humans. However, the mechanisms are still little known. In the present study, mouse Leydig tumor cells were utilized to investigate steroidogenic activity influenced by deca-brominated diphenyl ether (BDE-209). Our data showed that BDE-209 did not change intracellular cAMP level in the presence of human Chorionic Gonadotropin (hCG), cholera toxin (CT), and forskolin, which indicated that reduction of progesterone may not be related to the hCG-cAMP signal pathway in MLTC-1 cells. Furthermore, the reduction of progesterone generation was not shifted by 8-Br-cAMP, an analog of cAMP, indicating that BDE-209 may inhibit post-cAMP sites. In addition, mRNA expression levels of P450 side-chain cleavage enzyme (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) presented a concentration-dependent decrease. In conclusion, this study suggested that BDE-209 may attenuate the progesterone secretion mainly through lowering the expression of these two enzymes.

The Orphan Nuclear Receptors Steroidogenic Factor-1 and Liver Receptor Homolog-1: Structure, Regulation, and Essential Roles in Mammalian Reproduction.

Nuclear receptors are intracellular proteins that act as transcription factors. Proteins with classic nuclear receptor domain structure lacking identified signaling ligands are designated orphan nuclear receptors. Two of these, steroidogenic factor-1 (NR5A1, also known as SF-1) and liver receptor homolog-1 (NR5A2, also known as LRH-1), bind to the same DNA sequences, with different and nonoverlapping effects on targets. Endogenous regulation of both is achieved predominantly by cofactor interactions. SF-1 is expressed primarily in steroidogenic tissues, LRH-1 in tissues of endodermal origin and the gonads. Both receptors modulate cholesterol homeostasis, steroidogenesis, tissue-specific cell proliferation, and stem cell pluripotency. LRH-1 is essential for development beyond gastrulation and SF-1 for genesis of the adrenal, sexual differentiation, and Leydig cell function. Ovary-specific depletion of SF-1 disrupts follicle development, while LRH-1 depletion prevents ovulation, cumulus expansion, and luteinization. Uterine depletion of LRH-1 compromises decidualization and pregnancy. In humans, SF-1 is present in endometriotic tissue, where it regulates estrogen synthesis. SF-1 is underexpressed in ovarian cancer cells and overexpressed in Leydig cell tumors. In breast cancer cells, proliferation, migration and invasion, and chemotherapy resistance are regulated by LRH-1. In conclusion, the NR5A orphan nuclear receptors are nonredundant factors that are crucial regulators of a panoply of biological processes, across multiple reproductive tissues.

Postmenopausal androgen-secreting ovarian tumors: challenging differential diagnosis in two cases.

Postmenopausal hyperandrogenism constitutes a very rare condition of tumoral or non-tumoral origin primarily residing either in the ovary or in the adrenal glands. We present herein two cases with this condition; one with abnormal postmenopausal genital bleeding and mild increase in facial hair, and the second with slow-developing hirsutism and virilization. Both cases shared a notorious increase in libido. The laboratory tests showed high levels of testosterone (>100 ng/ml). A normal value of dehydroepiandrosterone sulfate and a normal cortisol level at 9 am after 1 mg of dexamethasone administered at midnight (Nugent test) made an adrenal etiology very unlikely. On the other hand, a high level of inhibine B oriented to an ovarian source. Transvaginal sonography failed to demonstrate an ovarian tumor, but an abdominal and pelvic computed tomography scan or magnetic resonance imaging detected an ovarian tumor and normal adrenal glands. A laparoscopic oophorectomy was performed, and the histological study demonstrated a steroidal cell tumor in the first case and a Leydig cell tumor in the second.

The methanolic extract of Guibourtia tessmannii (caesalpiniaceae) and selenium modulate cytosolic calcium accumulation, apoptosis and oxidative stress in R2C tumour Leydig cells: Involvement of TRPV1 channels.

This study evaluated the effects of the methanolic extract of Guibourtia tessmannii (GT) and selenium (Se) on cell viability, intracellular calcium concentration ([Ca2+ ]i ), apoptosis and oxidative stress through transient receptor potential vanilloid 1 (TRPV1) channel activity in CCL-97 (R2C) tumour Leydig cells. The cells were divided into nine groups and treated as follows: (a)-Control, (b)-Capsazepine (CPZ, 0.1 mM, a TRPV1 channel blocker), (c)-Capsaicin (CAP, 0.01 mM, a TRPV1 channel activator), (d)-GT (500 µg/ml), (e)-GT+CPZ, (f)-GT+CAP, (g)-Se (200 nM), (h)-Se+CPZ and (i)-Se+CAP. After treatments, cell viability, [Ca2+ ]i , apoptosis, caspase 3/9, reactive oxygen species (ROS) and mitochondrial membrane depolarisation (MMD) were evaluated. The [Ca2+ ]i , apoptosis, caspase 3/9, MMD and ROS levels were significantly (p < 0.001) increased in CAP group, but lowered in CPZ group. Interestingly, these parameters were significantly (p < 0.001) improved by GT and Se, compared to the CAP group. Moreover, the co-administration of GT+CAP or Se+CAP inhibited the cytotoxicity of CAP. Thus, the modulatory properties of GT and Se on Ca2+ influx, apoptosis and oxidative stress require the integrity of TRPV1 channel in CCL-97 Leydig cells. These results suggest that GT and Se might be used in the management of cytotoxicity in the testes, involving TRPV1 channel activity.

Biphasic effects of perfluorooctanoic acid on steroidogenesis in mouse Leydig tumour cells.

Perfluorooctanoic acid (PFOA) is a persistent organic pollutant, which may possess endocrine disrupting properties. Herein, we investigated the possible mechanism(s) of toxicity and steroidogenesis in mouse Leydig cells. MLTC-1 (mouse Leydig tumour cells) cells were exposed to 0, 50, 100 or 200 µM PFOA for 48 h to ascertain their effects on the nuclear (membrane) receptor responses, steroidogenesis pathway and related regulated gene expression and steroid hormone secretion profiles. Our results reveal that nuclear receptors PXR, SR-B1 and LHR are sensitive to PFOA exposure. PFOA can accumulate in mitochondria and alter cholesterol precursor (fatty acid) mitochondrial transport process-related gene expression and thus inhibit steroid hormone precursor (cholesterol) production. In particular, PFOA exhibits biphasic effects on testosterone and progesterone production at differing levels of exposure. These findings indicate the potential endocrine-related effects of PFOA on steroid hormone secretion in Leydig cells and point to a novel disruption model.

INSL3 Expression in Leydig Cell Hyperplasia and Leydig Cell Tumors.

Insulin-like 3 (INSL3) is a hormone produced by Leydig cells (LCs) and leads to physiological testicular descent during embryonic development. We investigated the expression of INSL3 by immunohistochemistry in normal LCs, in Leydig cell tumor (LCT) (n=17 including 15 testes and 2 ovaries) and in Leydig cell hyperplasia (LCH) (n=10). Normally distributed LCs showed strong immunostaining in the cytoplasm in all cases. All 10 cases (100%) of LCH were strongly and diffusely positive in the intertubular areas. Six cases of LCH had nodules raging in size from 0.2 to 0.9 cm with variable INSL3 staining. Fifteen of 17 (88.2%) LCTs showed marked decrease INSL3 staining, 10/17 (58.8%) were completely negative, and 5/17 (29.4%) were only focally positive. Two cases with multifocal LCTs showed strong and diffuse cytoplasmic staining of LCs around seminiferous tubules while the LCTs were negative. Two cases diagnosed as LCT were strongly positive for INSL3. Other sex cord stromal tumors tested were consistently negative including Sertoli-cell tumor (n=4), granulosa cell tumor (n=2), and fibrothecoma (n=1). In conclusion, our results contrast with those of previously published studies, and show that the great majority of LCTs are negative or have decreased expression of INSL3 while its expression is retained in LCH. INSL3 negative nodules within LCH may represent early LCTs. INSL3 immunostaining could be helpful to highlight LCs in cases where it is difficult to identify them (ie, small testicular biopsies performed for infertility workup) and in the differential diagnosis between florid LCH and LCT.

Modulation of the transcriptomic profile of the R2C tumor Leydig cell line by the adipose tissue derived hormone leptin.

Leptin is an important adipose derived hormone being secreted by adipocytes and involved in appetite regulation. Disruption of normal plasma levels of leptin has been associated with the pathogenesis of obesity. It is now well established that obesity is associated to an increased risk of cancer development, including testicular cancers. Recently, we have shown that high levels of leptin have inhibitory effects on cAMP-dependent steroidogenic genes expression in MA-10 Leydig cells. Hence, we examined if leptin could alter the transcriptome of the constitutively steroidogenic rat tumor Leydig cell line R2C. These cells were treated with high levels of leptin (1000 ng/ml) for 4 h, followed by mRNA extraction and RNA-Seq analysis. Interestingly, leptin had no effect on steroidogenic gene expression or pathways promoting tumorigenesis of this cell line. Genes being upregulated or downregulated by leptin were enriched in biological processes that were non-relevant to Leydig cell function. Surprisingly, lepr isoforms were weakly expressed in R2C cells and may explain in part the low levels of leptin's response for tumor Leydig cells. Hence, the current findings suggest that acute treatments of tumor Leydig cells with high levels of leptin have negligible effects on transcriptomics and rather supports that leptin would have a central effect on the hypothalamus to influence testicular function.

FGF9/FGFR2 increase cell proliferation by activating ERK1/2, Rb/E2F1, and cell cycle pathways in mouse Leydig tumor cells.

Fibroblast growth factor 9 (FGF9) promotes cancer progression; however, its role in cell proliferation related to tumorigenesis remains elusive. We investigated how FGF9 affected MA-10 mouse Leydig tumor cell proliferation and found that FGF9 significantly induced cell proliferation by activating ERK1/2 and retinoblastoma (Rb) phosphorylations within 15 minutes. Subsequently, the expressions of E2F1 and the cell cycle regulators: cyclin D1, cyclin E1 and cyclin-dependent kinase 4 (CDK4) in G1 phase and cyclin A1, CDK2 and CDK1 in S-G2 /M phases were increased at 12 hours after FGF9 treatment; and cyclin B1 in G2 /M phases were induced at 24 hours after FGF9 stimulation, whereas the phosphorylations of p53, p21 and p27 were not affected by FGF9. Moreover, FGF9-induced effects were inhibited by MEK inhibitor PD98059, indicating FGF9 activated the Rb/E2F pathway to accelerate MA-10 cell proliferation by activating ERK1/2. Immunoprecipitation assay and ChIP-quantitative PCR results showed that FGF9-induced Rb phosphorylation led to the dissociation of Rb-E2F1 complexes and thereby enhanced the transactivations of E2F1 target genes, Cyclin D1, Cyclin E1 and Cyclin A1. Silencing of FGF receptor 2 (FGFR2) using lentiviral shRNA inhibited FGF9-induced ERK1/2 phosphorylation and cell proliferation, indicating that FGFR2 is the obligate receptor for FGF9 to bind and activate the signaling pathway in MA-10 cells. Furthermore, in a severe combined immunodeficiency mouse xenograft model, FGF9 significantly promoted MA-10 tumor growth, a consequence of increased cell proliferation and decreased apoptosis. Conclusively, FGF9 interacts with FGFR2 to activate ERK1/2, Rb/E2F1 and cell cycle pathways to induce MA-10 cell proliferation in vitro and tumor growth in vivo.

Interstitial Cells in the Pineal Gland of Pregnant and Nonpregnant Viscachas (Lagostomus maximus maximus): A Morphometric and Biochemical Study.

The pineal gland of mammals undergoes morphological and biochemical changes throughout the gestation period. In viscachas, a seasonal breeding rodent, pregnancy lasts approximately 154 days and 3 stages can be defined, i.e., early, mid, and late pregnancy. The purpose of this study is to analyze morphometric variations in the expression of S-100 protein, glial fibrillary acidic protein (GFAP), and vimentin in the interstitial cells (IC) in pregnant and nonpregnant viscachas by immunohistochemistry (IHC). We also aim to evaluate a probable relation between glandular activity and pregnancy. The immunopositive percentage area (%IA) for the studied proteins and the number of immunoreactive cells against the S-100 protein with a visible nucleus (nº IC-S-100) were analyzed. Estradiol and progesterone serum levels were also determined by RIA. Variations in the expression of the S-100 protein and GFAP, as well as changes in the nº IC-S-100 related to serum hormone levels, were found between pregnant and nonpregnant viscachas. Viscachas in mid pregnancy exhibited the highest values of %IA for the analyzed proteins, followed by females in late and early pregnancy, while the nonpregnant ones showed the lowest values for all of the groups studied. Likewise, the nº IC-S-100 also varied following the same pattern. Thus, these variations seem to indicate a direct relationship between glandular activity and gonadal hormone levels. On these grounds, we may conclude that IC undergo changes in relation to ovarian hormone levels and participate in the regulation of glandular activity during pregnancy. However, further research is necessary to elucidate this relationship.