Search

Abstract Eighty percent of the estimated 600 million domestic cats in the world are free-roaming. These cats typically experience suboptimal welfare and inflict high levels of predation on wildlife. Additionally, euthanasia of healthy animals in overpopulated shelters raises ethical considerations. While surgical sterilization is the mainstay of pet population control, there is a need for efficient, safe, and cost-effective permanent contraception alternatives. Herein, we report evidence that a single intramuscular treatment with an adeno-associated viral vector delivering an anti-Müllerian hormone transgene produces long-term contraception in the domestic cat. Treated females are followed for over two years, during which transgene expression, anti-transgene antibodies, and reproductive hormones are monitored. Mating behavior and reproductive success are measured during two mating studies. Here we show that ectopic expression of anti-Müllerian hormone does not impair sex steroids nor estrous cycling, but prevents breeding-induced ovulation, resulting in safe and durable contraception in the female domestic cat.

Summary: Cancer-associated mesothelial cells (CAMCs) in the tumor microenvironment are thought to promote growth and immune evasion. We find that, in mouse and human ovarian tumors, cancer cells express anti-Müllerian hormone (AMH) while CAMCs express its receptor AMHR2, suggesting a paracrine axis. Factors secreted by cancer cells induce AMHR2 expression during their reprogramming into CAMCs in mouse and human in vitro models. Overexpression of AMHR2 in the Met5a mesothelial cell line is sufficient to induce expression of immunosuppressive cytokines and growth factors that stimulate ovarian cancer cell growth in an AMH-dependent way. Finally, syngeneic cancer cells implanted in transgenic mice with Amhr2−/− CAMCs grow significantly slower than in wild-type hosts. The cytokine profile of Amhr2−/− tumor-bearing mice is altered and their tumors express less immune checkpoint markers programmed-cell-death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Taken together, these data suggest that the AMH/AMHR2 axis plays a critical role in regulating the pro-tumoral function of CAMCs in ovarian cancer.

Exposure to phthalates, endocrine-disrupting chemicals commonly used as plasticizers and in consumer products, has been associated with infertility and premature ovarian failure. Our objective was to investigate whether urinary phthalate metabolite concentrations were associated with pre-ovulatory follicular fluid (FF) anti-müllerian hormone (AMH) concentrations in women undergoing fertility treatment. This cross-sectional analysis included 138 women with urinary phthalate data available in the Environment and Reproductive Health (EARTH) Study (2010–2016) in whom FF AMH concentrations were quantified using a sandwich enzyme-linked immunosorbent assay (ELISA). We also quantified 8 phthalate metabolite concentrations using tandem mass spectrometry in 1–2 urine samples per cycle (total 331 urines) and calculated the cycle-specific geometric mean for each metabolite. We applied cluster-weighted generalized estimating equation models (CWGEE) to evaluate the associations of tertiles of urinary phthalate metabolite concentrations with log-transformed FF AMH concentrations adjusting for potential confounders. Study participants had median age of 34.0 years (IQR 32.0, 37.0), 83% were white, and median BMI of 23.1 kg/m2 (IQR 21.2, 26.1). The following stimulation protocols were used: luteal phase agonist (70%), antagonist (14%), or flare (16%). Urinary concentrations of select phthalate metabolites were negatively associated with FF AMH. For example, women whose urinary mEOHP was in the lowest tertile (range 0.30–4.04 ng/ml) had an adjusted mean FF AMH of 0.72 ng/mL (95% CI = 0.36, 1.44), compared to women in the highest tertile (range 9.90–235), who had an adjusted mean of 0.24 ng/mL (95% CI = 0.12–0.48, p

Anti-Müllerian hormone (AMH) and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-β)/bone morphogenetic protein (BMP) signaling and epithelial-mesenchymal transition (EMT) in lung cancer. AMH is a TGF-β/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3) also used by the type II receptor for BMP (BMPR2). AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB) and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC). AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independent induction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90) inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-β/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT.

The Mullerian ducts are the anlagen of the female reproductive tract, which regress in the male fetus in response to MIS. This process is driven by subluminal mesenchymal cells expressing Misr2, which trigger the regression of the adjacent Mullerian ductal epithelium. In females, these Misr2+ cells are retained, yet their contribution to the development of the uterus remains unknown. Here, we report that subluminal Misr2+ cells persist postnatally in the uterus of rodents, but recede by week 37 of gestation in humans. Using single-cell RNA sequencing, we demonstrate that ectopic postnatal MIS administration inhibits these cells and prevents the formation of endometrial stroma in rodents, suggesting a progenitor function. Exposure to MIS during the first six days of life, by inhibiting specification of the stroma, dysregulates paracrine signals necessary for uterine development, eventually resulting in apoptosis of the Misr2+ cells, uterine hypoplasia, and complete infertility in the adult female.

In our search for genes required for the development and function of mouse gonads, we identified Gng13 (guanine nucleotide binding protein 13, gamma), a gene with an embryonic expression pattern highly restricted to the ovary. Based on reverse transcriptase‐polymerase chain reaction (RT‐PCR) and whole‐mount in situ hybridization, Gng13 is expressed in both XX and XY gonads at embryonic day (E) 11.5, but becomes up‐regulated in the XX gonad by E12.5. Expression is retained after treatment with busulfan, a chemical known to eliminate germ cells, pointing to the soma as a site of Gng13 transcription. In situ hybridization of embryonic ovarian tissue sections further localized the expression to the cortex of the developing XX gonad. Gng13 expression in the adult is also highly restricted. Northern blot analyses and Genomic Institute of the Novartis Research Foundation expression profiling of adult tissues detected very high expression in the cerebrum and cerebellum, in addition to, a weaker signal in the ovary. Gng13 belongs to a well‐known family of signal transduction molecules with functions in many aspects of development and organ physiology. Here, we report that, in the developing mouse embryo, expression of Gng13 mRNA is highly restricted to the cortex of the XX gonad during sexual differentiation, suggesting a role for this gene during ovarian development. Developmental Dynamics 236:1991–1996, 2007. © 2007 Wiley‐Liss, Inc. [ABSTRACT FROM AUTHOR]

The estrous cycle is regulated by rhythmic endocrine interactions of the nervous and reproductive systems, which coordinate the hormonal and ovulatory functions of the ovary. Folliculogenesis and follicle progression require the orchestrated response of a variety of cell types to allow the maturation of the follicle and its sequela, ovulation, corpus luteum formation, and ovulatory wound repair. Little is known about the cell state dynamics of the ovary during the estrous cycle and the paracrine factors that help coordinate this process. Herein, we used single-cell RNA sequencing to evaluate the transcriptome of >34,000 cells of the adult mouse ovary and describe the transcriptional changes that occur across the normal estrous cycle and other reproductive states to build a comprehensive dynamic atlas of murine ovarian cell types and states.

Congenital diaphragmatic hernia (CDH) is a severe birth defect that is often accompanied by other congenital anomalies. Previous exome sequencing studies for CDH have supported a role of de novo damaging variants but did not identify any recurrently mutated genes. To investigate further the genetics of CDH, we analyzed de novo coding variants in 362 proband-parent trios including 271 new trios reported in this study. We identified four unrelated individuals with damaging de novo variants in MYRF (P = 5.3x10(-8)), including one likely gene-disrupting (LGD) and three deleterious missense (D-mis) variants. Eight additional individuals with de novo LGD or missense variants were identified from our other genetic studies or from the literature. Common phenotypes of MYRF de novo variant carriers include CDH, congenital heart disease and genitourinary abnormalities, suggesting that it represents a novel syndrome. MYRF is a membrane associated transcriptional factor highly expressed in developing diaphragm and is depleted of LGD variants in the general population. All de novo missense variants aggregated in two functional protein domains. Analyzing the transcriptome of patient-derived diaphragm fibroblast cells suggest that disease associated variants abolish the transcription factor activity. Furthermore, we showed that the remaining genes with damaging variants in CDH significantly overlap with genes implicated in other developmental disorders. Gene expression patterns and patient phenotypes support pleiotropic effects of damaging variants in these genes on CDH and other developmental disorders. Finally, functional enrichment analysis implicates the disruption of regulation of gene expression, kinase activities, intra-cellular signaling, and cytoskeleton organization as pathogenic mechanisms in CDH.

CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue-specific protein isoforms. The most ubiquitous, CD44 standard, has been used as a cancer stem cell marker in ovarian and other cancers. Expression of the epithelial CD44 variant containing exons v8-10 (CD44v8-10) has been associated with more chemoresistant and metastatic tumors in gastrointestinal and breast cancers, but its role in ovarian cancer is unknown; we therefore investigated its use as a prognostic marker in this disease. The gene expression profiles of 254 tumor samples from The Cancer Genome Atlas RNAseqV2 were analyzed for the presence of CD44 isoforms. A trend for longer survival was observed in patients with high expression of CD44 isoforms that include exons v8-10. Immunohistochemical (IHC) analysis of tumors for presence of CD44v8-10 was performed on an independent cohort of 210 patients with high-grade serous ovarian cancer using a tumor tissue microarray. Patient stratification based on software analysis of staining revealed a statistically significant increase in survival in patients with the highest levels of transmembrane protein expression (top 10 or 20%) compared to those with the lowest expression (bottom 10 and 20%) (p = 0.0181, p = 0.0262 respectively). Expression of CD44v8-10 in primary ovarian cancer cell lines was correlated with a predominantly epithelial phenotype characterized by high expression of epithelial markers and low expression of mesenchymal markers by qPCR, Western blot, and IHC. Conversely, detection of proteolytically cleaved and soluble extracellular domain of CD44v8-10 in patient ascites samples was correlated with significantly worse prognosis (p

Congenital diaphragmatic hernia is associated with pulmonary hypoplasia and respiratory distress, which result in high mortality and morbidity. Although several transgenic mouse models of lung hypoplasia exist, the role of miRNAs in this phenotype is incompletely characterized. In this study, we assessed microRNA expression levels during the pseudoglandular to canalicular phase transition of normal human fetal lung development. At this critical time, when the distal respiratory portion of the airways begins to form, microarray analysis showed that the most significantly differentially expressed miRNA was miR-449a. Prediction algorithms determined that N-myc is a target of miR-449a and identified the likely miR-449a:N-myc binding sites, confirmed by luciferase assays and targeted mutagenesis. Functional ex vivo knock-down in organ cultures of murine embryonic lungs, as well as in ovo overexpression in avian embryonic lungs, suggested a role for miR-449a in distal epithelial proliferation. Finally, miR-449a expression was found to be abnormal in rare pulmonary specimens of human fetuses with Congenital Diaphragmatic Hernia in the pseudoglandular or canalicular phase. This study confirms the conserved role of miR-449a for proper pulmonary organogenesis, supporting the delicate balance between expansion of progenitor cells and their terminal differentiation, and proposes the potential involvement of this miRNA in human pulmonary hypoplasia.

Abstract Aberrant organ development is associated with a wide spectrum of disorders, from schizophrenia to congenital heart disease, but systems‐level insight into the underlying processes is very limited. Using heart morphogenesis as general model for dissecting the functional architecture of organ development, we combined detailed phenotype information from deleterious mutations in 255 genes with high‐confidence experimental interactome data, and coupled the results to thorough experimental validation. Hereby, we made the first systematic analysis of spatio‐temporal protein networks driving many stages of a developing organ identifying several novel signaling modules. Our results show that organ development relies on surprisingly few, extensively recycled, protein modules that integrate into complex higher‐order networks. This design allows the formation of a complicated organ using simple building blocks, and suggests how mutations in the same genes can lead to diverse phenotypes. We observe a striking temporal correlation between organ complexity and the number of discrete functional modules coordinating morphogenesis. Our analysis elucidates the organization and composition of spatio‐temporal protein networks that drive the formation of organs, which in the future may lay the foundation of novel approaches in treatments, diagnostics, and regenerative medicine.