Acquisition of functional receptivity by the endometrium is assumed to be effected by progesterone-dependent expression and repression of several genes during the implantation window in a menstrual cycle. In the present study, we employed differential display (DD) reverse transcription–polymerase chain reaction (RT–PCR) to identify progesterone-dependent gene/gene fragments that are differentially expressed during the peri-implantation phase in receptive and nonreceptive endometria, obtained from fertile and infertile bonnet monkeys respectively. Receptive endometria were obtained from regularly cycling (n=5) fertile female bonnet monkeys. Endometrial nonreceptivity was induced by treating bonnet monkeys with either 2.5 mg (n=5) or 5.0 mg (n=5) onapristone (ZK 98.299), an antiprogestin, on every third day for one cycle. Ovulation, levels of circulatory hormones (estradiol and progesterone) and menstrual cycle length did not change in treated animals; however, endometrial growth was retarded. DD2, one of the differentially expressed cDNA fragments, showed higher representation in nonreceptive endometria than in receptive endometria. The DD2 sequence was found to be homologous to the sequence of the carboxyl terminal region of Rab coupling protein (RCP), a recently discovered protein involved in intracellular vesicular trafficking. To confirm the identity of DD2 as RCP, RT–PCR studies were carried out with a forward primer deduced from the RCP sequence and a reverse primer from the DD2 sequence. The product (DDRCP) obtained, when sequenced, revealed 95% homology with the nucleotide number 1196–1757 of human RCP cDNA. Furthermore, the pattern of DDRCP expression at transcript level was found to be similar to that shown by DD2; that is, it was higher in nonreceptive endometrium. Northern analysis using labeled DD2 or DDRCP cDNA fragments identified two transcripts of 6.0 and 4.0 kb in human endometrium. In situ hybridization studies using digoxigenin-labeled DD2 revealed significantly higher (P < 0.05) localization of endometrial RCP transcripts in the proliferative phase than in the peri-implantation phase in control animals. The localization was also significantly (P < 0.01) higher in peri-implantation-phase endometria from antiprogestin-treated animals than in control animals. These antiprogestin-treated animals, however, did not demonstrate any concomitant increase in the levels of immunoreactive endometrial Rab4 and Rab11 during the peri-implantation phase. A similar pattern of cycle-dependent RCP expression was observed in human endometrial biopsies. Furthermore, significantly higher (P < 0.05) levels of RCP transcripts were detected during the peri-implantation phase in women with unexplained infertility (n=3) than in fertile women (n=3). This is the first report indicating the endometrial expression of RCP and its hormonal regulation.
V S Patil, G Sachdeva, D N Modi, R R Katkam, D D Manjramkar, I Hinduja and C P Puri
Sucharitha Iyer and Sunita K Agarwal
Epigenetic regulation is emerging as a key feature in the molecular characteristics of various human diseases. Epigenetic aberrations can occur from mutations in genes associated with epigenetic regulation, improper deposition, removal or reading of histone modifications, DNA methylation/demethylation and impaired non-coding RNA interactions in chromatin. Menin, the protein product of the gene causative for the multiple endocrine neoplasia type 1 (MEN1) syndrome, interacts with chromatin-associated protein complexes and also regulates some non-coding RNAs, thus participating in epigenetic control mechanisms. Germline inactivating mutations in the MEN1 gene that encodes menin predispose patients to develop endocrine tumors of the parathyroids, anterior pituitary and the duodenopancreatic neuroendocrine tissues. Therefore, functional loss of menin in the various MEN1-associated endocrine cell types can result in epigenetic changes that promote tumorigenesis. Because epigenetic changes are reversible, they can be targeted to develop therapeutics for restoring the tumor epigenome to the normal state. Irrespective of whether epigenetic alterations are the cause or consequence of the tumorigenesis process, targeting the endocrine tumor-associated epigenome offers opportunities for exploring therapeutic options. This review presents epigenetic control mechanisms relevant to the interactions and targets of menin, and the contribution of epigenetics in the tumorigenesis of endocrine cell types from menin loss.
C Roger, S Lambard, A Bouskine, B Mograbi, D Chevallier, M Nebout, G Pointis, S Carreau and P Fenichel
It is now well established that estrogens participate in the control of normal spermatogenesis and endogenous or environmental estrogens are involved in pathological germ cell proliferation including testicular germ cell tumors. Studying a human testicular seminoma cell line, JKT-1, we show here that 17β-estradiol (10−12 to 10−6 M) induced in vitro a significant dose-dependent decrease of cell growth. This antiproliferative effect was maximum after 4 days of exposure at a physiologically intratesticular concentration of 10−9 M, close to the K d of ER, and reversed by ICI 182780, an ER antagonist, suggesting an ER-mediated pathway. By RT-PCR and Western blot we were able to confirm that JKT-1, like tumoral seminoma cells and normal human testicular basal germ cells, expresses estrogen receptor β (ERβ), including ERβ1 and ERβ2, a dominant negative variant, but not ERα. Using immunofluorescence and confocal microscopy, ERβ was observed as perinuclear intracytoplasmic spots in JKT-1 and tumoral seminoma cells without significant translocation of ERβ into the nucleus, under 17β-estradiol exposure. Double staining observed by confocal microscopy revealed that ERβ colocalized in JKT-1 cells with cytochrome C, a mitochondrial marker. We report for the first time the expression of a functional aromatase complex in seminoma cells as assessed by RT-PCR, Western blot and enzymatic assay. Seminoma cells are able to respond to estrogens through a possible autocrine or paracrine loop. These preliminary results support estrogen-dependency of human testicular seminoma, the most frequent tumor of young men, and suggest potential pharmacological use. Whether this estrogen control, however, involves an ERβ-mediated stimulation of cell apoptosis and/or an ERβ-mediated inhibition of cell proliferation, remains to be further determined.
Magda A Meester-Smoor, Anco C Molijn, Yixian Zhao, Nicole A Groen, Cora A H Groffen, Merel Boogaard, Diny van Dalsum-Verbiest, Gerard C Grosveld and Ellen C Zwarthoff
The IGF-binding protein (IGFBP) family consists of six proteins that are expressed and secreted in different tissues. The proteins are regulators of physiological processes throughout the body by modulating the activity of IGF-I and IGF-II. In this article, we describe the coordinated expression of IGFBP5 and MN1 in meningiomas. MN1 is a transcriptional co-activator and we show that MN1 stimulates the IGFBP5 promoter in Hep3B cells. A CACCC-containing sequence, located 140 bp upstream of the transcription start site of the promoter, is required for MN1 action. This sequence matches with the CACCCAC consensus sequence that was selected in an oligonucleotide selection assay performed for MN1. The CACCC element has also been shown to be important for induction of the IGFBP5 promoter by retinoic acid (RA) and progesterone (Pg). We were unable to confirm the effect of Pg on the promoter in Hep3B and U2-osteosarcoma cells regardless of the presence of MN1. On the other hand, we show that induction of the promoter by RA depends on co-expressed MN1 in Hep3B cells. MN1TEL, a leukemia-related fusion protein containing parts of the MN1 and TEL (ETV6) genes, is capable of stimulating the IGFBP5 promoter but is unable to cooperate with RA in Hep3B cells. This suggests that the effects of RA can be negatively affected in leukemias caused by MN1TEL.
Maya Elena Lee, Aisha Aderayo Tepede, Adel Mandl, Lee Scott Weinstein, Jaydira del Rivero, Sunita K Agarwal and Jenny E. Blau
Gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) comprise a heterogenous and diverse group of neoplasms arising from a common neuroendocrine cell origin. The majority of these tumors occur sporadically while ~20% manifest within the context of hereditary syndromes. Germline MEN1 mutations cause a syndrome with an increased susceptibility to multifocal primary GEP NETs. In addition, MEN1 mutations also occur in sporadic GEP NETs. MEN1 alternations are the most frequent sporadic mutation found in pancreatic neuroendocrine tumors (PNETs). We explore the implication of the loss of the MEN1 encoded protein menin as a key pathogenic driver in subsets of GEP NETs with downstream consequences including upregulation of the oncogenic receptor c-MET (hepatocyte growth factor receptor). This review will summarize the data related to the clinical presentation, therapeutic standards, and outcomes of sporadic and MEN1 associated GEP NETs. We present the data on c-MET expression in GEP NETs, clinical trials using c-MET inhibtors, and provide an overview of the molecular mechanisms by which c-MET inhibition in GEP NETs represents a potential precision-medicine targeted approach.
Steroid hormones can exist in functionally dissociable sulfated and non-sulfated (free) forms and can exert profound effects on numerous aspects of mammalian physiology; the ratio of free-to-sulfated steroids is governed by the antagonistic actions of steroid sulfatase (STS) and sulfotransferase (SULT) enzymes. Here, I examine evidence from human and animal model studies, which suggests that STS and its major substrate (dehydroepiandrosterone sulfate, DHEAS) and product (DHEA) can influence brain function, behaviour and mental health, before summarising how the activity of this axis varies throughout mammalian pregnancy and the postpartum period. I then consider how the steroid sulfate axis might impact upon normal maternal behaviour and how its dysfunction might contribute towards risk of postpartum psychiatric illness. Understanding the biological substrates underlying normal and abnormal maternal behaviour will be important for maximising the wellbeing of new mothers and their offspring.
Joram D Mul
Acute or chronic exposure to stress can increase the risk to develop major depressive disorder, a severe, recurrent and common psychiatric condition. Depression places an enormous social and financial burden on modern society. Although many depressed patients are treated with antidepressants, their efficacy is only modest, underscoring the necessity to develop clinically effective pharmaceutical or behavioral treatments. Exercise training produces beneficial effects on stress-related mental disorders, indicative of clinical potential. The pro-resilient and antidepressant effects of exercise training have been documented for several decades. Nonetheless, the underlying molecular mechanisms and the brain circuitries involved remain poorly understood. Preclinical investigations using voluntary wheel running, a frequently used rodent model that mimics aspects of human exercise training, have started to shed light on the molecular adaptations, signaling pathways and brain nuclei underlying the beneficial effects of exercise training on stress-related behavior. In this review, I highlight several neurotransmitter systems that are putative mediators of the beneficial effects of exercise training on mental health, and review recent rodent studies that utilized voluntary wheel running to promote our understanding of exercise training-induced central adaptations. Advancements in our mechanistic understanding of how exercise training induces beneficial neuronal adaptations will provide a framework for the development of new strategies to treat stress-associated mental illnesses.
R A Gadkari, S Roy, N Rekha, N Srinivasan and R R Dighe
Human chorionic gonadotrophin (hCG) is secreted during early pregnancy and is required for implantation and maintenance of the pregnancy. Active or passive immunoneutralization of hCG results in termination of pregnancy and this forms the basis of the hCG-based female contraceptive vaccine. However, the β subunit of hCG possesses 85% sequence homology with the first 114 amino acids of the β subunit of pituitary human LH (hLH), which is required for ovulation and maintenance of the corpus luteum function during the menstrual cycle. Immunization against hCG or its β subunit leads to generation of antibodies that can neutralize hLH due to many shared epitopes and hence may cause abnormal menstrual cycles. Therefore, it is essential to identify epitopes that are different in the two hormones. In the present study, we report a monoclonal antibody (MAb) specific for hCG that shows no binding to the isolated subunits. Interestingly, the MAb also does not bind hLH at all. The epitope mapping analysis revealed that this antibody recognizes a unique discontinuous epitope present only in the heterodimeric hCG and is distinct from the unique C-terminal extension of hCGβ that is absent in hLHβ. The MAb, either as IgG or its recombinant single-chain variable region fragment, inhibited the response to hCG, but not to hLH. Thus, the epitope recognized by this MAb is an ideal candidate antigen for immunocontraception.
Michel Crépin, Pascal Pigny, Fabienne Escande, Catherine Cardot Bauters, Alain Calender, Sylvain Lefevre, Marie-Pierre Buisine, Nicole Porchet and Marie-Françoise Odou
The identification of mutations in the MEN1 gene causing MEN1 has represented a challenge since the cloning of the gene in 1997 because of the lack of mutation hot-spots in the gene and the lack of phenotype–genotype correlations. The use of denaturing high performance liquid chromatography (DHPLC), a high throughput, reliable and automated heteroduplex-based technique, is the ideal for mutation detection in MEN1. In this work, DHPLC was optimised for the screening of the nine coding exons and splice junctions of MEN1. Thanks to collaboration between two French laboratories recognised as reference centres for genotypic MEN1 diagnosis (Lyon and Lille), a blind retrospective study conducted in a cohort of 160 unrelated MEN1 probands with (or without) known germline mutations was undertaken to evaluate the sensitivity of DHPLC. We were able to detect 101 different sequence variations by DHPLC, distributed in the 10 analysed DNA fragments and corresponding to 100% of mutation detection compared with direct sequencing. 1·2% of samples were considered as false positive, exhibiting a heterogenous profile. DHPLC did not detect five cases of deletion or duplication of complete exons, neither did direct sequencing, showing the limits of the technique. Nevertheless, the method appeared to allow automated, rapid and low-cost mutation detection with high accuracy. Direct sequencing can be then applied to identify the sequence variations on the targeted DNA fragments showing heterozygous profile by DHPLC. In conclusion, genotypic diagnosis of MEN1 can benefit from DHPLC in terms of efficacy, rapidity and cost.
Luca Persani, Raffaella Rossetti and Chiara Cacciatore
Premature ovarian failure (POF) is an ovarian defect characterized by the premature depletion of ovarian follicles before the age of 40 years, representing one major cause of female infertility. POF relevance is continuously growing because women tend to conceive ever more frequently in their thirties and forties. POF can present very early with a pubertal defect. More frequently, it is the end stage of an occult process (primary ovarian insufficiency, POI) affecting ∼1–2% of under-40 women. POI is a heterogeneous disease caused by a variety of mechanisms. Though the underlying cause remains unexplained in the majority of cases, various data indicate that POI has a strong genetic component. These data include the existence of several causal genetic defects in humans, experimental and natural models, as well as the frequent familiarity. The variable expressivity of POI defect in women of the same family may indicate that, in addition to some monogenic forms, POI may frequently be considered as a multifactorial defect resulting from the contribution of several predisposing alleles. The X chromosome-linked defects play a major role among the presently known causal defects. Here, we review the principal X-linked and autosomal genes involved in syndromic and nonsyndromic forms of POI with the wish that this list will soon become upgraded because of the discovery of novel contributing mechanisms. A better understanding of POI pathogenesis will indeed allow the construction of tests able to predict the age of menopause in women at higher risk of POI.