Functional analysis of rare anti-Müllerian hormone protein-altering variants identified in women with PCOS.

Recently, rare heterozygous AMH protein-altering variants were identified in women with polycystic ovary syndrome (PCOS), causing reduced anti-Müllerian hormone (AMH) signaling. However, the exact functional mechanism remains unknown. Here, we analyzed the processing, secretion, and signaling of these AMH variants. Functional analysis of six PCOS-specific AMH variants (V12G, P151S, P270S, P352S, P362S, H506Q) and one control-specific variant (A519V) was performed in the mouse granulosa cell-line KK-1. Human (h) AMH-151S and hAMH-506Q have ∼90% decreased AMH signaling compared to wild-type (wt) AMH signaling. Coexpression of hAMH-151S or hAMH-506Q with wt-hAMH dose-dependently inhibited wt-hAMH signaling. Western blotting revealed that hAMH-151S and hAMH-506Q proteins were detected in the cell lysate but not in the supernatant. Confocal microscopy showed that HEK293 cells expressing hAMH-151S and hAMH-506Q had higher cellular AMH protein levels with endoplasmic reticulum (ER) retention compared to cells expressing wt-hAMH. Using two AMH ELISA kits, hAMH-151S was detected in the cell lysate, while only very low levels were detected in the supernatant. Both hAMH-362S and hAMH-519V were detectable using the automated AMH ELISA but showed severely reduced immunoactivity in the manual ELISA. Surprisingly, hAMH-506Q was undetectable in both the cell lysate and supernatant using either ELISA. However, in PCOS cases, heterozygous carriers of the P151S and H506Q variants still had detectable AMH in both assays. Thus, P151S and H506Q disrupt normal processing and secretion of AMH, causing ER retention. Additionally, AMH variants can impair the AMH immunoactivity. An AMH variant may be considered when serum AMH levels are relatively low in PCOS cases.

Experimental tests of host-virus coevolution in natural killer yeast strains.

Fungi may carry cytoplasmic viruses that encode anticompetitor toxins. These so-called killer viruses may provide competitive benefits to their host, but also incur metabolic costs associated with viral replication, toxin production and immunity. Mechanisms responsible for the stable maintenance of these endosymbionts are insufficiently understood. Here, we test whether co-adaptation of host and killer virus underlies their stable maintenance in seven natural and one laboratory strain of the genus Saccharomyces. We employ cross-transfection of killer viruses, all encoding the K1-type toxin, to test predictions from host-virus co-adaptation. These tests support local adaptation of hosts and/or their killer viruses. First, new host-virus combinations have strongly reduced killing ability against a standard sensitive strain when compared with re-constructed native combinations. Second, viruses are more likely to be lost from new than from original hosts upon repeated bottlenecking or the application of stressful conditions. Third, host fitness is increased after the re-introduction of native viruses, but decreased after the introduction of new viruses. Finally, rather than a trade-off, original combinations show a positive correlation between killing ability and fitness. Together, these results suggest that natural yeast killer strains and their viruses have co-adapted, allowing the transition from a parasitic to a mutualistic symbiosis.

Back to the basics of ovarian aging: a population-based study on longitudinal anti-Müllerian hormone decline.

BACKGROUND: Anti-Müllerian hormone (AMH) is currently used as an ovarian reserve marker for individualized fertility counseling, but very little is known of individual AMH decline in women. This study assessed whether the decline trajectory of AMH is uniform for all women, and whether baseline age-specific AMH levels remain consistently high or low during this trajectory. METHODS: A total of 3326 female participants from the population-based Doetinchem Cohort Study were followed with five visits over a 20-year period. Baseline age was 40 ± 10 years with a range of 20-59 years. AMH was measured in 12,929 stored plasma samples using the picoAMH assay (AnshLabs). Decline trajectories of AMH were studied with both chronological age and reproductive age, i.e., time to menopause. Multivariable linear mixed effects models characterized the individual AMH decline trajectories. RESULTS: The overall rate of AMH decline accelerated after 40 years of age. Mixed models with varying age-specific AMH levels and decline rates provided the significantly best fit to the data, indicating that the fall in AMH levels over time does not follow a fixed pattern for individual women. AMH levels remained consistent along individual trajectories of age, with an intraclass correlation coefficient (ICC) of 0.87. The ICC of 0.32 for AMH trajectories with time to menopause expressed the large variation in AMH levels at a given time before the menopause. The differences between low and high age-specific AMH levels remained distinguishable, but became increasingly smaller with increasing chronological and reproductive age. CONCLUSIONS: This is the first study to characterize individual AMH decline over a long time period and broad age range. The varying AMH decline rates do not support the premise of a uniform AMH decline trajectory. Although age-specific AMH levels remain consistently high or low with increasing age, the converging trajectories and variance of AMH levels at a given time before menopause shed doubt on the added value of AMH to represent individualized reproductive age.

Presence of inclusions positive for polyglycine containing protein, FMRpolyG, indicates that repeat-associated non-AUG translation plays a role in fragile X-associated primary ovarian insufficiency.

STUDY QUESTION: Does repeat-associated non-AUG (RAN) translation play a role in fragile X-associated primary ovarian insufficiency (FXPOI), leading to the presence of polyglycine containing protein (FMRpolyG)-positive inclusions in ovarian tissue? SUMMARY ANSWER: Ovaries of a woman with FXPOI and of an Fmr1 premutation (PM) mouse model (exCGG-KI) contain intranuclear inclusions that stain positive for both FMRpolyG and ubiquitin. WHAT IS KNOWN ALREADY: Women who carry the FMR1 PM are at 20-fold increased risk to develop primary ovarian insufficiency (FXPOI). A toxic RNA gain-of-function has been suggested as the underlying mechanism since the PM results in increased levels of mRNA containing an expanded repeat, but reduced protein levels of fragile X mental retardation protein (FMRP). Recently, RAN translation has been shown to occur from FMR1 mRNA that contains PM repeat expansions, leading to FMRpolyG inclusions in brain and non-CNS tissues of fragile X-associated tremor/ataxia syndrome (FXTAS) patients. STUDY DESIGN, SIZE, DURATION: Ovaries of a woman with FXPOI and women without PM (controls), and ovaries from wild-type and exCGG-KI mice were analyzed by immunohistochemistry for the presence of inclusions that stained for ubiquitin and FMRpolyG . The ovaries from wild-type and exCGG-KI mice were further characterized for the number of follicles, Fmr1 mRNA levels and FMRP protein expression. The presence of inclusions was also analyzed in pituitaries of a man with FXTAS and the exCGG-KI mice. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human ovaries from a woman with FXPOI and two control subjects and pituitaries from a man with FXTAS and a control subjects were fixed in 4% formalin. Ovaries and pituitaries of wild-type and exCGG mice were fixed in Bouin's fluid or 4% paraformaldehyde. Immunohistochemistry was performed on the human and mouse samples using FMRpolyG, ubiquitin and Fmrp antibodies. Fmr1 mRNA and protein expression were determined in mouse ovaries by quantitative RT-PCR and Western blot analysis. Follicle numbers in mouse ovaries were determined in serial sections by microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: FMRpolyG-positive inclusions were present in ovarian stromal cells of a woman with FXPOI but not in the ovaries of control subjects. The FMRpolyG-positive inclusions colocalized with ubiquitin-positive inclusions. Similar inclusions were also observed in the pituitary of a man with FXTAS but not in control subjects. Similarly, ovaries of 40-week-old exCGG-KI mice, but not wild-type mice, contained numerous inclusions in the stromal cells that stained for both FMRpolyG- and ubiquitin, while the ovaries of 20-week-old exCGG-KI contained fewer inclusions. At 40 weeks ovarian Fmr1 mRNA expression was increased by 5-fold in exCGG-KI mice compared with wild-type mice, while Fmrp expression was reduced by 2-fold. With respect to ovarian function in exCGG-KI mice: (i) although the number of healthy growing follicles did not differ between wild-type and exCGG-KI mice, the number of atretic large antral follicles was increased by nearly 9-fold in 40-week old exCGG-KI mice (P < 0.001); (ii) at 40 weeks of age only 50% of exCGG-KI mice had recent ovulations compared with 89% in wild-type mice (P = 0.07) and (iii) those exCGG-KI mice with recent ovulations tended to have a reduced number of fresh corpora lutea (4.8 ± 1.74 versus 8.50 ± 0.98, exCGG-KI versus wild-type mice, respectively, P = 0.07). LIMITATIONS, REASONS FOR CAUTION: Although FMRpolyG-positive inclusions were detected in ovaries of both a woman with FXPOI and a mouse model of the FMR1 PM, we only analyzed one ovary from a FXPOI subject. Caution is needed to extrapolate these results to all women with the FMR1 PM. Furthermore, the functional consequence of FMRpolyG-positive inclusions in the ovaries for reproduction remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that a dysfunctional hypothalamic-pituitary-gonadal-axis may contribute to FXPOI in FMR1 PM carriers. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from NFXF, ZonMW, the Netherlands Brain Foundation and NIH. The authors have no conflict of interest to declare.

Decreased serum anti-Müllerian hormone levels in girls with newly diagnosed cancer.

STUDY QUESTION: Are anti-Müllerian hormone (AMH) levels reduced in girls with newly diagnosed cancer before the start of treatment? SUMMARY ANSWER: AMH levels are already compromised in girls at the time of cancer diagnosis compared with healthy girls. WHAT IS KNOWN ALREADY: In women diagnosed with cancer, evidence of reduced ovarian function has been described even before treatment has started. In girls with newly diagnosed cancer, no data are available. STUDY DESIGN, SIZE, DURATION: We performed an age-matched case-control study in girls with newly diagnosed cancer. PARTICIPANTS/MATERIALS, SETTING, METHODS: We determined serum AMH levels in a cohort of 208 girls with newly diagnosed cancer, up to 18 years of age at diagnosis, and compared them with AMH levels of 250 age-matched healthy girls. The diagnoses included were acute lymphoblastic leukaemia, acute myeloid leukaemia, Hodgkin lymphoma, non-Hodgkin lymphoma, nephroblastoma, sarcoma and neuroblastoma. MAIN RESULTS AND THE ROLE OF CHANCE: The median age was 6.6 years (range 0.0-17.4), comparable with that in the control group (median 6.3 years, range 0.3-18.0). Girls with childhood cancer presented with significantly lower serum AMH levels compared with healthy age-matched controls (standard deviation scores (SDS) -0.8, P < 0.001). Median AMH level in patients was 1.4 µg/l (0.1-10.2) versus 3.0 µg/l (0.1-18.3) in controls. Specifically, 84% of all patients had AMH levels below the 50th percentile of normal AMH levels, and 19% below the 10th percentile. Surrogate markers of general health status (temperature, C-reactive protein and haemoglobin levels at diagnosis) were significantly correlated with AMH SDS. LIMITATIONS, REASONS FOR CAUTION: Some caution is warranted because AMH levels increase with age in healthy children but the cases and controls were age-matched in our study. Although our sample size was large, additional studies are still required in an independent cohort. WIDER IMPLICATIONS OF THE FINDINGS: Our study shows that AMH levels are reduced in girls with newly diagnosed cancer even before the cancer treatment has started. AMH levels correlate with impairment of general health status in girls. Therefore, besides (pre) antral follicle number, other factors may influence serum AMH levels. Longitudinal studies during and after childhood cancer are currently being performed in order to evaluate possible ovarian recovery after discontinuation of treatment. STUDY FUNDING/COMPETING INTEREST(S): W.v.D. is supported by the Paediatric Oncology Centre Society for Research (KOCR), Rotterdam, The Netherlands. J.S.E.L. has received grants from the following companies (in alphabetical order): Ferring, Genovum, Merck Serono, Merck Sharp and Dome, Organon, Serono, Shering Plough and Shering. All other authors have nothing to disclose.

Adiponectin secretion by perivascular adipose tissue supports impaired vasodilation in a mouse model of accelerated vascular smooth muscle cell and adipose tissue aging.

OBJECTIVE: Perivascular adipose tissue (PVAT) function during aging has not been investigated in detail so far and its effect on vasodilation remains to be fully elucidated. The aim of this study was to investigate endothelium-dependent vasodilation of thoracic aorta in a mouse model of accelerated, selective vascular smooth muscle and PVAT aging, induced by SM22α-Cre-driven genetic deletion of the endonuclease ERCC1 (SMC-KO mice) versus healthy littermates (LM). We hypothesized that PVAT enhances vasodilation in LM, possibly through adiponectin secretion, which might be compromised in SMC-KO animals. METHODS: Thoracic aorta was isolated from SMC-KO animals and LM and segments with and without PVAT were mounted in wire myography setups. The endothelium-dependent vasodilation was assessed via acetylcholine dose-response curves and pathway contribution was studied. Moreover, adiponectin secretion was measured after stimulating the aortic segments with PVAT with acetylcholine. RESULTS: Adiponectin, secreted by PVAT, led to increased NO-contribution to endothelium-dependent vasodilation in healthy LM, although this did not increase maximum relaxation due to loss of EDH. Endothelium-dependent vasodilation was decreased in SMC-KO animals due to reduced NO-contribution and complete EDH loss. Despite strong lipodystrophy the PVAT partially compensated for lost vasodilation in SMC-KO. LM PVAT contained acetylcholinesterase that attenuated acetylcholine responses. This was lost in SMC-KO. CONCLUSIONS: PVAT-derived adiponectin is able to partially compensate for age-related decline in NO-mediated vasodilation, even during strong lipodystrophy, in conditions of absence of compensating EDH. In aorta with healthy PVAT acetylcholinesterase modulates vascular tone, but this is lost during aging, further compensating for decreased acetylcholine responsiveness. Thus, preservation of adiponectin levels, through relatively increased production in lipodystrophic PVAT, and reduction of cholinesterase might be regulatory mechanisms of the PVAT to preserve cholinergic vasodilation during aging.

Genetic variation may modify ovarian reserve in female childhood cancer survivors.

STUDY QUESTION: Are genetic polymorphisms, previously identified as being associated with age at menopause in the healthy population, associated with ovarian reserve and predicted age at menopause in adult long-term survivors of childhood cancer? SUMMARY ANSWER: The CT genotype of rs1172822 in the BRSK1 gene is associated with lower serum anti-Müllerian hormone (AMH) levels and a younger predicted age at menopause in adult survivors of childhood cancer. WHAT IS KNOWN ALREADY: Gonadotoxicity is a well-known late side effect of chemotherapy and radiotherapy in adult survivors of childhood cancer. In the healthy population, several genetic polymorphisms are associated with age at natural menopause. Currently, data on the impact of previously identified variants in gene loci associated with ovarian reserve in adult long-term survivors of childhood cancer are lacking. STUDY DESIGN, SIZE, DURATION: We performed a pilot study in a single-centre cohort of adult female Caucasian childhood cancer survivors (n = 176). PARTICIPANTS/MATERIALS, SETTING, METHODS: We determined serum AMH levels (a marker of ovarian reserve) in adult survivors of childhood cancer (n = 176) and studied single nucleotide polymorphisms (SNPs) previously reported to be associated with age at natural menopause: BRSK1 (rs1172822), ARHGEF7 (rs7333181), MCM8 (rs236114), PCSK1 (rs271924), IGF2R (rs9457827) and TNF (rs909253). Association analysis was performed using the additive genetic model. Linear regression was conducted to assess the effect of significant polymorphisms in two previously published menopause prediction models. MAIN RESULTS AND THE ROLE OF CHANCE: The CT genotype of rs1172822 in the BRSK1 (BR serine/threonine kinase 1) gene was negatively associated with serum AMH levels in our cohort (odds ratio: 3.15, 95% confidence interval: 1.35-7.32, P = 0.008) and significantly associated with the predicted age at menopause (P = 0.04). The other five SNPs were not associated with serum AMH levels. LIMITATIONS, REASONS FOR CAUTION: This is a pilot study showing preliminary data which must be confirmed. To confirm our findings and enlarge the project, a nationwide genome-wide association (GWA) project on the ovarian reserve in female survivors of childhood cancer should be performed, including a replication cohort. WIDER IMPLICATIONS OF THE FINDINGS: Our findings support the hypothesis that previously identified genetic polymorphisms associated with age at menopause in healthy women may have an effect on the onset of menopause in female survivors of childhood cancer. Our study highlights a new aspect of the influences on the ovarian reserve after childhood cancer, which should be investigated further in a nationwide GWA study. Eventually, this information can help us to improve counselling on fertility preservation prior to cancer treatment based on genetic factors in individual patients. STUDY FUNDING AND CONFLICT OF INTEREST: W.D. is supported by the Paediatric Oncology Centre Society for Research (KOCR), Rotterdam, The Netherlands. J.S.E.L. has received fees and grant support from the following companies (in alphabetic order): Ferring, Genovum, Merck-Serono, Organon, Schering Plough and Serono. All other authors have nothing to disclose.

Acute effects of acylated and unacylated ghrelin on total and high molecular weight adiponectin inmorbidly obese subjects.

BACKGROUND: Energy homeostasis and body weight are regulated by a highly complex network involving the brain, the digestive tract, and white adipose tissue (WAT). Knowledge about signaling pathways connecting digestive tract and WAT is limited. Gut hormone ghrelin and adipokine adiponectin are both decreased in obesity and they share a potent effect on insulin sensitivity: both adiponectin and the combination of acylated (AG) and unacylated ghrelin (UAG) improve insulin sensitivity. AIM: In the present study, we evaluated whether acute administration of UAG alone or combined with AG affects adiponectin concentrations. SUBJECTS AND METHODS: Eight morbidly obese non-diabetic subjects were treated with either UAG 200 µg, UAG 100 µg + AG 100 µg (Comb), or placebo in 3 episodes in a double blind randomized cross-over design. Study medication was administered as single iv bolus injections at 09:00 h after an overnight fast. High molecular weight (HMW) and total adiponectin, glucose, insulin, and total ghrelin and AG were measured up to 1 h after administration. RESULTS: HMW and total adiponectin concentrations did not change after administration of either UAG or Comb, nor were they different from placebo. Insulin concentrations decreased significantly after acute administration of Comb, reaching a minimum at 20 min: 58.2 ± 3.9% of baseline. CONCLUSIONS: Acute iv administration of UAG and the combination of UAG and AG in morbidly obese non-diabetic subjects without overt diabetes does not affect total or HMW adiponectin concentrations, neither directly nor indirectly by changing insulin concentrations.

Assessment of ovarian reserve in adult childhood cancer survivors using anti-Müllerian hormone.

BACKGROUND: The aim was to assess possible treatment-induced gonadal damage in a cohort of adult female childhood cancer survivors (CCS) using anti-Müllerian hormone (AMH), the most sensitive marker of ovarian reserve. METHODS: A total cohort of 185 survivors was compared with 42 control subjects. The median follow-up time was 18.1 years (range 4.1-43.2 year). RESULTS: Median AMH concentrations in the analysed cohort were not different from controls (median 1.7 versus 2.1 microg/l; P = 0.57). However, AMH levels were lower than the 10th percentile of normal values in 27% (49/182) of our survivors. In addition, 43% (79/182) had AMH levels lower than 1.4 microg/l, a previously established cut-off value which predicts ongoing pregnancy after assisted reproduction. There were no differences in AMH levels in subgroups classified according to disease. However, survivors treated with three or more procarbazine containing chemotherapy cycles had significantly lower AMH levels than controls (median 0.5 microg/l; P = 0.004). Also survivors treated with abdominal or total body irradiation had significantly lower AMH levels than controls (median < 0.1 microg/l; P < 0.001). CONCLUSIONS: AMH can be used to identify subgroups of CCS at risk for decreased fertility or premature ovarian failure. In these survivors, options for fertility preservation should be considered prior to starting treatment since they may be at risk for poor chances of pregnancy after assisted reproductive treatment.

Anti-Müllerian hormone: a marker for oocyte quantity, oocyte quality and embryo quality?

Serum anti-Müllerian hormone (AMH) concentrations decline with increasing age and constitute a sensitive marker for ovarian ageing. In addition, basal serum AMH concentrations predict ovarian response during IVF cycles. Concomitantly, oocyte quantity and embryo quality decrease with advancing age. Hence, it was postulated that AMH in serum constitutes a marker for embryo quality. Women aged 37 years and younger with regular menstrual cycles, normal body mass index and partners with normal semen parameters were randomly assigned to either a standard or mild stimulation protocol for IVF treatment. Blood samples were drawn at cycle day 3 and at the day of human chorionic gonadotrophin administration. Embryo quality was assessed using embryo morphology score and preimplantation genetic screening. Serum AMH concentrations on cycle day 3 were correlated with the number of oocytes retrieved in both groups. AMH and embryo morphology were correlated after mild stimulation, but not after conventional ovarian stimulation. AMH and the chromosomal competence of embryos were not correlated. Serum AMH is predictive for ovarian response to stimulation. However, the lack of a consistent correlation with embryo morphology and embryo aneuploidy rate is not in favour of a direct relationship between oocyte quantity and embryo quality.

[Polymicrobial infections: ecosystems with special properties]. / Polymicrobiële infecties: ecosystemen met bijzondere eigenschappen.

Bacteria often live together in complex communities. Insight into these microbial ecosystems is essential to make it possible to intervene when these ecosystems lead to disease. Bacteria do not only respond to their host, but they also affect each other, which may have far-reaching consequences for the course of the disease. In this article we describe that clinical isolates in a polymicrobial infection can be seen as ecosystems. These ecosystems often have properties that separate isolates do not have; they may, for example, be more virulent or more resistant to antibiotics. We therefore emphasise that the whole is greater than the sum of its parts, even for infections.

Differential coreceptor expression allows for independent evolution of non-syncytium-inducing and syncytium-inducing HIV-1.

We demonstrated previously that CD45RA(+) CD4(+) T cells are infected primarily by syncytium-inducing (SI) HIV-1 variants, whereas CD45RO(+) CD4(+) T cells harbor both non-SI (NSI) and SI HIV-1 variants. Here, we studied evolution of tropism for CD45RA(+) and CD45RO(+) CD4(+) cells, coreceptor usage, and molecular phylogeny of coexisting NSI and SI HIV-1 clones that were isolated from four patients in the period spanning SI conversion. NSI variants were CCR5-restricted and could be isolated throughout infection from CD45RO(+) CD4(+) cells. SI variants seemed to evolve in CD45RO(+) CD4(+) cells, but, in time, SI HIV-1 infection of CD45RA(+) CD4(+) cells equaled infection of CD45RO(+) CD4(+) cells. In parallel with this shift, SI HIV-1 variants first used both coreceptors CCR5 and CXCR4, but eventually lost the ability to use CCR5. Phylogenetically, NSI and SI HIV-1 populations diverged over time. We observed a differential expression of HIV-1 coreceptors within CD45RA(+) and CD45RO(+) cells, which allowed us to isolate virus from purified CCR5(+) CXCR4(-) and CCR5(-) CXCR4(+) CD4(+) cells. The CCR5(+) subset was exclusively infected by CCR5-dependent HIV-1 clones, whereas SI clones were preferentially isolated from the CXCR4(+) subset. The differential expression of HIV-1 coreceptors provides distinct cellular niches for NSI and SI HIV-1, contributing to their coexistence and independent evolutionary pathways.

Nanowire sensors monitor bacterial growth kinetics and response to antibiotics.

Miniaturized and cost-efficient methods aiming at high throughput analysis of microbes are of great importance for the surveillance and control of infectious diseases and the related issue of antimicrobial resistance. Here we demonstrate a miniature nanosensor based on a honeycomb-patterned silicon nanowire field effect transistor (FET) capable of detection of bacterial growth and antibiotic response in microbiologically relevant nutrient media. We determine the growth kinetics and metabolic state of Escherichia coli cells in undiluted media via the quantification of changes in the source-drain current caused by varying pH values. Furthermore, by measuring the time dependent profile of pH change for bacterial cultures treated with antibiotics, we demonstrate for the first time the possibility of electrically distinguishing between bacteriostatic and bactericidal drug effects. We believe that the use of such nanoscopic FET devices enables addressing parameters that are not easily accessible by conventional optical methods in a label-free format, i.e. monitoring of microbial metabolic activity or stress response.

IGF system targeted therapy: Therapeutic opportunities for ovarian cancer.

The insulin-like growth factor (IGF) system comprises multiple growth factor receptors, including insulin-like growth factor 1 receptor (IGF-1R), insulin receptor (IR) -A and -B. These receptors are activated upon binding to their respective growth factor ligands, IGF-I, IGF-II and insulin, and play an important role in development, maintenance, progression, survival and chemotherapeutic response of ovarian cancer. In many pre-clinical studies anti-IGF-1R/IR targeted strategies proved effective in reducing growth of ovarian cancer models. In addition, anti-IGF-1R targeted strategies potentiated the efficacy of platinum based chemotherapy. Despite the vast amount of encouraging and promising pre-clinical data, anti-IGF-1R/IR targeted strategies lacked efficacy in the clinic. The question is whether targeting the IGF-1R/IR signaling pathway still holds therapeutic potential. In this review we address the complexity of the IGF-1R/IR signaling pathway, including receptor heterodimerization within and outside the IGF system and downstream signaling. Further, we discuss the implications of this complexity on current targeted strategies and indicate therapeutic opportunities for successful targeting of the IGF-1R/IR signaling pathway in ovarian cancer. Multiple-targeted approaches circumventing bidirectional receptor tyrosine kinase (RTK) compensation and prevention of system rewiring are expected to have more therapeutic potential.

An experimental test for synergistic epistasis and its application in Chlamydomonas.

Theoretically, one of the most general benefits of sex is given by its function in facilitating selection against deleterious mutations. This advantage of sex may be deterministic if deleterious mutations affect the fitness of an individual in a synergistic way, i.e., if mutations increase each others' negative fitness effect. We present a new test for synergistic epistasis that considers the skewness of the log fitness distribution of offspring from a cross. We applied this test to data of the unicellular alga Chlamydomonas moewussii. For this purpose, two crosses were made: one between two strains that are presumed to have accumulated slightly deleterious mutations, the other between two strains without a history of mutation accumulation. Fitness was measured by estimating the two parameters of logistic growth in batch culture, the maximum growth rate (r) and the carrying capacity (K). The finding of a negatively skewed distribution for K in the accumulation cross suggests synergism between mutations affecting the carrying capacity, while the absence of skewness for tau in both crosses is consistent with independent effects of mutations affecting this parameter. The results suggest a possible alternative explanation for the general observation that sex is related to constant environments, where selection on K predominates, while asexual reproduction is found in more variable environments, where selection on r is more important.

The serine/threonine transmembrane receptor ALK2 mediates Müllerian inhibiting substance signaling.

Müllerian inhibiting substance (MIS or anti-Müllerian hormone) is a member of the transforming growth factor-beta family and plays a pivotal role in proper male sexual differentiation. Members of this family signal by the assembly of two related serine/threonine kinase receptors, referred to as type I or type II receptors, and downstream cytoplasmic Smad effector proteins. Although the MIS type II receptor (MISRII) has been identified, the identity of the type I receptor is unclear. Here we report that MIS activates a bone morphogenetic protein-like signaling pathway, which is solely dependent on the presence of the MISRII and bioactive MIS ligand. Among the multiple type I candidates tested, only ALK2 resulted in significant enhancement of the MIS signaling response. Furthermore, dominant-negative and antisense strategies showed that ALK2 is essential for MIS-induced signaling in two independent assays, the cellular Tlx-2 reporter gene assay and the Müllerian duct regression organ culture assay. In contrast, ALK6, the other candidate MIS type I receptor, was not required. Expression analyses revealed that ALK2 is present in all MIS target tissues including the mesenchyme surrounding the epithelial Müllerian duct. Collectively, we conclude that MIS employs a bone morphogenetic protein-like signaling pathway and uses ALK2 as its type I receptor. The use of this ubiquitously expressed type I receptor underscores the role of the MIS ligand and the MIS type II receptor in establishing the specificity of the MIS signaling cascade.

Effect of prenatal exposure to diethylstilbestrol on Müllerian duct development in fetal male mice.

The clinical use of diethylstilbestrol (DES) by pregnant women has resulted in an increased incidence of genital carcinoma in the daughters born from these pregnancies. Also, in the so-called DES-sons abnormalities were found, mainly, the presence of Müllerian duct remnants, which indicates that fetal exposure to DES may have an effect on male sex differentiation. Fetal regression of the Müllerian ducts is under testicular control through anti-Müllerian hormone (AMH). In male mice, treated in utero with DES, the Müllerian ducts do not regress completely, although DES-exposed testes do produce AMH. We hypothesized that incomplete regression in DES-exposed males is caused by a diminished sensitivity of the Müllerian ducts to AMH. Therefore, the effect of DES on temporal aspects of Müllerian duct regression and AMH type II receptor (AMHRII) messenger RNA (mRNA) expression in male mouse fetuses was studied. It was observed that Müllerian duct regression was incomplete at E19 (19 days post coitum), upon DES administration during pregnancy from E9 through E16. Furthermore, analysis of earlier time points of fetal development revealed that the DES treatment had clearly delayed the onset of Müllerian duct formation by approximately 2 days; in untreated fetuses, Müllerian duct formation was complete by E13, whereas fully formed Müllerian ducts were not observed in DES-treated male fetuses until E15. Using in situ hybridization, no change in the localization of AMH and AMHRII mRNA expression was observed in DES-exposed male fetuses. The mRNA expression was quantified using ribonuclease protection assay, showing an increased expression level of AMH and AMHRII mRNAs at E 13 in DES-exposed male fetuses. Furthermore, the mRNA expression levels of Hoxa 11 and steroidogenic factor-1 (SF-1) were determined as a marker for fetal development. Prenatal DES exposure had no effect on Hoxa 11 mRNA expression, indicating that DES did not exert an overall effect on the rate of fetal development. In DES-exposed male fetuses, SF-1 showed a similar increase in mRNA expression as AMH, in agreement with the observations that the AMH gene promoter requires an intact SF-1 DNA binding site for time- and cell-specific expression, although an effect of DES on SF-1 expression in other tissues, such as the adrenal and pituitary gland, cannot be excluded. However, the increased expression levels of AMH and AMHRII mRNAs do not directly explain the decreased sensitivity of the Müllerian ducts to AMH. Therefore, it is concluded that prenatal DES exposure of male mice delays the onset of Müllerian duct development, which may result in an asynchrony in the timing of Müllerian duct formation, with respect to the critical period of Müllerian duct regression, leading to persistence of Müllerian duct remnants in male mice.

Anti-müllerian hormone and anti-müllerian hormone type II receptor messenger ribonucleic acid expression during postnatal testis development and in the adult testis of the rat.

Anti-müllerian hormone (AMH) induces degeneration of the müllerian ducts during male sex differentiation and may have additional functions concerning gonadal development. In the immature rat testis, there is a marked developmental increase in AMH type II receptor (AMHRII) messenger RNA (mRNA) expression in Sertoli cells, concomitant with the initiation of spermatogenesis. AMHRII mRNA is also expressed at a high level in Sertoli cells in adult rats. To obtain information about the possible functions of AMH in the testis, we investigated the postnatal expression patterns of the genes encoding AMH and AMHRII in the rat testis in more detail. Using RNase protection assays, AMH and AMHRII mRNA expression was measured in total RNA preparations from testes or testicular tubule segments isolated from control rats and from rats that had received various treatments. The testicular level of AMHRII mRNA was found to be much higher than that of AMH mRNA in adult rats. AMH mRNA was detected at a maximal level at stage VII of the spermatogenic cycle and at a low level at the other stages. AMHRII mRNA increases from stage XIII, is highest at stages VI and VII, and then rapidly declines at stage VIII to almost undetectable levels at stages IX-XII. It was found that the increase in testicular AMHRII mRNA expression during the first 3 weeks of postnatal development also occurs in sterile rats (prenatally irradiated), and hence, is independent of the presence or absence of germ cells. Yet, the total testicular level of AMHRII mRNA was decreased in sterile adult rats (prenatally irradiated or experimental cryptorchidism), as compared with intact control rats. However, treatment of adult rats with methoxyacetic acid or hydroxyurea, which resulted in partial germ cell depletion, had no effect on total testicular AMHRII mRNA expression. We conclude that a combination of multiple spermatogenic cycle events, possibly involving changes of Sertoli cell structure and/or Sertoli cell-basal membrane interactions, regulate autocrine AMH action on Sertoli cells, in particular at stage VII of the spermatogenic cycle.

Mutational meltdown in laboratory yeast populations.

In small or repeatedly bottlenecked populations, mutations are expected to accumulate by genetic drift, causing fitness declines. In mutational meltdown models, such fitness declines further reduce population size, thus accelerating additional mutation accumulation and leading to extinction. Because the rate of mutation accumulation is determined partly by the mutation rate, the risk and rate of meltdown are predicted to increase with increasing mutation rate. We established 12 replicate populations of Saccharomyces cerevisiae from each of two isogenic strains whose genomewide mutation rates differ by approximately two orders of magnitude. Each population was transferred daily by a fixed dilution that resulted in an effective population size near 250. Fitness declines that reduce growth rates were expected to reduce the numbers of cells transferred after dilution, thus reducing population size and leading to mutational meltdown. Through 175 daily transfers and approximately 2900 generations, two extinctions occurred, both in populations with elevated mutation rates. For one of these populations there is direct evidence that extinction resulted from mutational meltdown: Extinction immediately followed a major fitness decline, and it recurred consistently in replicate populations reestablished from a sample frozen after this fitness decline, but not in populations founded from a predecline sample. Wild-type populations showed no trend to decrease in size and, on average, they increased in fitness.

Micro-CO2-incubator for use on a microscope.

A simple micro-CO2-incubator designed for use on the stage of an inverted microscope is described. This micro-incubator is easy to use, offers a handy tool for the culture of cells under the microscope and its performance compares well with that of a conventional CO2-incubator. A standard disposable culture dish can be placed in the micro-incubator. The culture medium is covered by a gas-permeable layer of mineral oil, this protects the culture from the environment without affecting the culture conditions and allows easy cell manipulation under microscopical control.