NTR-1's essential contribution to asymmetric mating between two sibling nematode Species: Bursaphelenchus xylophilus and B. Mucronatus.

The pine-wood invasive species nematode Bursaphelenchus xylophilus causes great forestry damage globally, particularly in Eurasia. B. xylophilus can hybridize with its native sibling, Bursaphelenchus mucronatus, with whom it shares an interestingly asymmetric mating behavior. However, the molecular mechanism underlying interspecific asymmetric mating has yet to be clarified. ntr-1, a nematocin receptor gene, is involved in an oxytocin/vasopressin-like signaling system that can regulate reproduction. Structural analysis using bioinformatics revealed that both Bxy- and Bmu-ntr-1 encode 7TM-GPCR, a conserved sequence. In situ hybridization and qPCR showed that both Bxy- and Bmu-ntr-1 were highly expressed in adult nematodes. Specifically, Bxy-ntr-1 was expressed in the vulva of females and caudal gonad of males, whereas Bmu-ntr-1 was expressed in the postal vulva and uterus of females and the whole gonads of males. Furthermore, RNAi of ntr-1 further demonstrated the biological function of interspecific mating: ntr-1 can regulate mating behavior, lead to male-female specificity, and ultimately result in interspecific differences. In B. mucronatus, ntr-1 influenced male mating more than female mating success, while downregulation of ntr-1 in B. xylophilus resulted in a significant decline in the female mating rate. Competitive tests revealed that the mating rate of the cross significantly declined after downregulation of Bxy♀- and Bmu♂-ntr-1, but no obvious change occurred in the reciprocal cross. Thus, we speculate that ntr-1 may be the key factor behind interspecific asymmetric mating. The current study (1) demonstrated the regulatory function of ntr-1 on mating behavior and (2) theoretically revealed the molecular basis of interspecific asymmetric mating.

Characteristic analysis of epileptic brain network based on attention mechanism.

Constructing an efficient and accurate epilepsy detection system is an urgent research task. In this paper, we developed an EEG-based multi-frequency multilayer brain network (MMBN) and an attentional mechanism based convolutional neural network (AM-CNN) model to study epilepsy detection. Specifically, based on the multi-frequency characteristics of the brain, we first use wavelet packet decomposition and reconstruction methods to divide the original EEG signals into eight frequency bands, and then construct MMBN through correlation analysis between brain regions, where each layer corresponds to a specific frequency band. The time, frequency and channel related information of EEG signals are mapped into the multilayer network topology. On this basis, a multi-branch AM-CNN model is designed, which completely matches the multilayer structure of the proposed brain network. The experimental results on public CHB-MIT datasets show that eight frequency bands divided in this work are all helpful for epilepsy detection, and the fusion of multi-frequency information can effectively decode the epileptic brain state, achieving accurate detection of epilepsy with an average accuracy of 99.75%, sensitivity of 99.43%, and specificity of 99.83%. All of these provide reliable technical solutions for EEG-based neurological disease detection, especially for epilepsy detection.

Discovery and Optimization of Potent, Selective, and Brain-Penetrant 1-Heteroaryl-1H-Indazole LRRK2 Kinase Inhibitors for the Treatment of Parkinson's Disease.

Inhibition of leucine-rich repeat kinase 2 (LRRK2) kinase activity represents a genetically supported, chemically tractable, and potentially disease-modifying mechanism to treat Parkinson's disease. Herein, we describe the optimization of a novel series of potent, selective, central nervous system (CNS)-penetrant 1-heteroaryl-1H-indazole type I (ATP competitive) LRRK2 inhibitors. Type I ATP-competitive kinase physicochemical properties were integrated with CNS drug-like properties through a combination of structure-based drug design and parallel medicinal chemistry enabled by sp3-sp2 cross-coupling technologies. This resulted in the discovery of a unique sp3-rich spirocarbonitrile motif that imparted extraordinary potency, pharmacokinetics, and favorable CNS drug-like properties. The lead compound, 25, demonstrated exceptional on-target potency in human peripheral blood mononuclear cells, excellent off-target kinase selectivity, and good brain exposure in rat, culminating in a low projected human dose and a pre-clinical safety profile that warranted advancement toward pre-clinical candidate enabling studies.

Regulatory roles of Bxy-laf-1 in reproductive behaviour of Bursaphelenchus xylophilus.

Bursaphelenchus xylophilu is a worldwide quarantine nematode, causing huge economic losses and ecological disasters in many countries. The sex ratio of B. xylophilus plays an important role in the nematode infestation. The laf-1-related genes are highly conserved in animals, playing crucial roles in sex determination. Therefore, we investigated the expression pattern and biological function of its orthologue, Bxy-laf-1 in B. xylophilus. Bxy-laf-1 has two typical conserved DNA-binding domains, DEAD and Helicase C. The real-time quantitative PCR data revealed that Bxy-laf-1 expression was required throughout the entire life of B. xylophilus, with the maximum expression in the J2 stage and the lowest expression in the adult stage. mRNA in situ hybridization showed that Bxy-laf-1 is mainly located in the cephalopharynx and reproductive organs of B. xylophilus. RNA interference (RNAi) indicated that the head swing frequency was dramatically decreased. The RNA interference results displayed that a significant reduction in motility was observed in the hatched larvae. The female to male sex ratio was also decreased in the F0 and F1 generations, but recovered in the F2 generation. The tail of female adults with eggs in the belly appeared deformities. This phenomenon appeared in the F0 and F1 generations, but recovered in the F2 generation. Bxy-laf-1 is a typical sex-determination gene with distinct expression patterns in males and females. As demonstrated in other species, the sex ratio was altered after knocking down Bxy-laf-1 expression. The results of this study contribute to our understanding of the molecular processes of Bxy-laf-1 in B. xylophilus, which may provide clues about how to control pine wilt disease by inhibiting ontogenic growth and reducing nematode fertility.

Ileocolonic-Targeted JAK Inhibitor: A Safer and More Effective Treatment for Inflammatory Bowel Disease.

Janus kinase (JAK) inhibitors, such as tofacitinib (Xeljanz) and filgotinib (Jyseleca), have been approved for treatment of ulcerative colitis with several other JAK inhibitors in late-stage clinical trials for inflammatory bowel disease (IBD). Despite their impressive efficacy, the risk of adverse effects accompanying the use of JAK inhibitors has brought the entire class under scrutiny, leading to them receiving an FDA black box warning. In this study we investigated whether ileocolonic-targeted delivery of a pan-JAK inhibitor, tofacitinib, can lead to increased tissue exposure and reduced systemic exposure compared to untargeted formulations. The stability of tofacitinib in the presence of rat colonic microbiota was first confirmed. Next, in vivo computed tomography imaging was performed in rats to determine the transit time and disintegration site of ileocolonic-targeted capsules compared to gastric release capsules. Pharmacokinetic studies demonstrated that systemic drug exposure was significantly decreased, and colonic tissue exposure increased at 10 mg/kg tofacitinib dosed in ileocolonic-targeted capsules compared to gastric release capsules and an oral solution. Finally, in a rat model of LPS-induced colonic inflammation, targeted tofacitinib capsules significantly reduced concentrations of proinflammatory interleukin 6 in colonic tissue compared to a vehicle-treated control (p = 0.0408), unlike gastric release tofacitinib capsules and orally administered dexamethasone. Overall, these results support further development of ileocolonic-targeted tofacitinib, and potentially other specific JAK inhibitors in pre-clinical and clinical development, for the treatment of IBD.

madd-4 plays a critical role in light against Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is a notorious invasive species, causing extensive losses to pine ecosystems globally. Previous studies had shown that the development of B. xylophilus was seriously suppressed by light. However, the mechanism involved in the inhibition is unknown. Here, it is the first report that Bxy-madd-4 is a light-regulated gene, plays a potential role in B. xylophilus in responding to the blue light. Transcriptome sequencing revealed that the expression level of Bxy-madd-4 declined by 86.39% under blue light. The reverse transcription quantitative real-time PCR results were in accord with the transcriptome sequencing, confirming the expression level of Bxy-madd-4 was suppressed by blue light. Bxy-madd-4 promoter::mCherry reporter constructed in Caenorhabditis elegans were utilized to mimic the spatiotemporal expression patterns of Bxy-madd-4. Bxy-madd-4A promoter activity had a strong continuity throughout all development stages in C. elegans. Further RNA interference indicated that only 36.8% of the Bxy-madd-4 dsRNA treated embryos were hatched. Moreover, 71.6% of the hatched nematodes were abnormal, such as particles on the body surface and concave tissues. Our findings contribute towards a better understanding of the mechanism of light against the destructive invasive nematode, providing a promising hint for control of the destructive invasive nematode.

Molecular characterization and functional analysis of Bxy-octr-1 in Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is an invasive plant-parasitic nematode causing the notorious pine wilt disease (PWD) worldwide, which results in huge economic losses. G protein-coupled receptors (GPCRs) play an essential role in mating and reproduction behavior of animals. As a unique biogenic amine in invertebrates, octopamine (OA) can regulate a variety of physiological and behavioral responses by binding specific GPCRs. These specific GPCRs are also called octopamine receptors (OARs), and octr-1 is one of them. However, Bxy-octr-1 is unknown in B. xylophilus. Therefore, we investigated the expression pattern and biological function of Bxy-octr-1. Bioinformatics analysis indicated that Bxy-octr-1 was evolutionarily conserved. The real-time quantitative PCR data revealed that Bxy-octr-1 expression was required throughout the entire life of B. xylophilus. mRNA in situ hybridization showed that Bxy-octr-1 was mainly located in the cephalopharynx, body wall muscle, intestine, and gonadal organs of B. xylophilus. RNA interference (RNAi) showed that embryo hatching rates and locomotion speeds were both dramatically decreased. Obvious abnormal phenotypes were observed in the second-stage of juveniles after RNAi treated. Furthermore, its ontogenesis was stunting. Lack of Bxy-octr-1 reduced fecundity of females, of which 31.25% of them could not successfully ovulate. In addition, the error positioning ratio of the nematode was significantly increased. Our study suggests that Bxy-octr-1 is indispensable for locomotion, early ontogenesis and mating behavior in B. xylophilus.

A Dissipation Function-Based Method for Calculating the Energy Loss of Intracranial Aneurysms.

At present, the energy loss (EL) mechanism of intracranial aneurysm (IA) rupture is explored based on the global EL calculated by Bernoulli equation, but the details of EL are still unclear. This study aimed to explore the temporal and spatial characteristics of EL of IAs and reveal its mechanism. A novel method for calculating the EL of IAs based on dissipation function (DF) was proposed. DF was derived from the differential form of the energy equation and reflected the irreversible conversion from mechanical energy to internal energy caused by the friction between the fluid micelles. Eight sidewall IAs located at the posterior communicating segment of the internal carotid artery were collected; the three-dimensional (3D) geometric models of IAs were established employing image segmentation and 3D reconstruction. Computational fluid dynamics was applied to obtain hemodynamic parameters of IAs. The temporal and spatial characteristics of EL of IAs were achieved utilizing our proposed method. The simulation results indicated that EL occurred mainly in the boundary layer and the region adjacent to high-velocity inflow jet, EL increased rapidly during cardiac systole and reached its maximum at end-systolic phase and then decreased gradually during diastole until the end of cardiac cycle. The proposed method achieved some improvements over the traditional Bernoulli equation-based method by acquiring the temporal and spatial characteristics of EL, and it could provide insights into the EL of IAs and contribute to further rupture mechanism investigation.

Combination of EP4 antagonist MF-766 and anti-PD-1 promotes anti-tumor efficacy by modulating both lymphocytes and myeloid cells.

Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP1-4). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE2-mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP4 receptor. EP4 inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8+ T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE2-mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE2-mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8+ T-cells by PGE2 and impaired suppression of CD8+ T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE2 high TME. Our studies demonstrate that the combination of EP4 blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles.

Projected Dose Optimization of Amino- and Hydroxypyrrolidine Purine PI3Kδ Immunomodulators.

The approvals of idelalisib and duvelisib have validated PI3Kδ inhibitors for the treatment for hematological malignancies driven by the PI3K/AKT pathway. Our program led to the identification of structurally distinct heterocycloalkyl purine inhibitors with excellent isoform and kinome selectivity; however, they had high projected human doses. Improved ligand contacts gave potency enhancements, while replacement of metabolic liabilities led to extended half-lives in preclinical species, affording PI3Kδ inhibitors with low once-daily predicted human doses. Treatment of C57BL/6-Foxp3-GDL reporter mice with 30 and 100 mg/kg/day of 3c (MSD-496486311) led to a 70% reduction in Foxp3-expressing regulatory T cells as observed through bioluminescence imaging with luciferin, consistent with the role of PI3K/AKT signaling in Treg cell proliferation. As a model for allergic rhinitis and asthma, treatment of ovalbumin-challenged Brown Norway rats with 0.3 to 30 mg/kg/day of 3c gave a dose-dependent reduction in pulmonary bronchoalveolar lavage inflammation eosinophil cell count.

The role of mab-3 in spermatogenesis and ontogenesis of pinewood nematode, Bursaphelenchus xylophilus.

BACKGROUND: Bursaphelenchus xylophilus is one of the most destructive invasive species, causing extensive economic losses worldwide. The sex ratio of female to male of B. xylophilus plays an important role in the nematode infestation. However, little is known about the processes of its sex determination. The double sex/mab-3-related family of transcription factors are highly conserved in animals, playing crucial roles in sex determination, spermatogenesis and ontogenesis. We therefore investigated its orthologue, Bxy-mab-3, in B. xylophilus. RESULTS: Bxy-mab-3 has two typical conserved DNA-binding domains. It was observed in J2 (the second-stage of juveniles), J3, J4 and male adults (specifically on the spicules), but not in eggs or female adults via mRNA in situ hybridization. RNA-Seq indicated significantly higher expression in males. RNAi showed that the body size and sperm size of male adults were markedly smaller than those of the controls. Meanwhile, almost all the RNAi-treated males failed to mate with the normal females, even 26.34% of interfered males did not produce sperm. However, RNAi of Bxy-mab-3 had no effect on the sex ratio of B. xylophilus. CONCLUSION: Bxy-mab-3 is indispensable for spermatogenesis, ontogenesis and mating behavior. It is a typical sex-determination gene with differential expression in males and females. However, knocking down Bxy-mab-3 expression could not alter the sex ratio as seen in other species. Our findings contribute towards a better understanding of the molecular events of Bxy-mab-3 in B. xylophilus, which provides promising hints for control of pine wilt disease by blocking ontogenesis and decreasing nematode fecundity.

LRRK2 inhibitors induce reversible changes in nonhuman primate lungs without measurable pulmonary deficits.

The kinase-activating mutation G2019S in leucine-rich repeat kinase 2 (LRRK2) is one of the most common genetic causes of Parkinson's disease (PD) and has spurred development of LRRK2 inhibitors. Preclinical studies have raised concerns about the safety of LRRK2 inhibitors due to histopathological changes in the lungs of nonhuman primates treated with two of these compounds. Here, we investigated whether these lung effects represented on-target pharmacology and whether they were reversible after drug withdrawal in macaques. We also examined whether treatment was associated with pulmonary function deficits. We conducted a 2-week repeat-dose toxicology study in macaques comparing three different LRRK2 inhibitors: GNE-7915 (30 mg/kg, twice daily as a positive control), MLi-2 (15 and 50 mg/kg, once daily), and PFE-360 (3 and 6 mg/kg, once daily). Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated 2 weeks after drug withdrawal for lung function. All compounds induced mild cytoplasmic vacuolation of type II lung pneumocytes without signs of lung degeneration, implicating on-target pharmacology. At low doses of PFE-360 or MLi-2, there was ~50 or 100% LRRK2 inhibition in brain tissue, respectively, but histopathological lung changes were either absent or minimal. The lung effect was reversible after dosing ceased. Lung function tests demonstrated that the histological changes in lung tissue induced by MLi-2 and GNE-7915 did not result in pulmonary deficits. Our results suggest that the observed lung effects in nonhuman primates in response to LRRK2 inhibitors should not preclude clinical testing of these compounds for PD.

Bxy-fuca encoding α-L-fucosidase plays crucial roles in development and reproduction of the pathogenic pinewood nematode, Bursaphelenchus xylophilus.

BACKGROUND: The pine wood nematode (PWN) Bursaphelenchus xylophilus is the causal agent of pine wilt disease (PWD). This disease is a serious threat to pine forests globally. The fuca gene encodes α-L-fucosidase, which plays crucial roles in numerous biological and pathological processes in bacteria, fungi, plants and animals. To find promising control strategies against PWD, we investigated the expression and functions of Bxy-fuca in B. xylophilus. RESULTS: Bxy-fuca encoding α-L-fucosidase is highly conserved within the deduced functional domains and key residues. It is expressed continuously across all developmental stages of B. xylophilus. mRNA in situ hybridization demonstrated that Bxy-fuca was mainly localized in the body wall muscles and intestine. RNA interference indicated that the zygotic expression of Bxy-fuca was indispensable for embryogenesis. The rate of B. xylophilus egg hatch was significantly decreased after Bxy-fuca was interfered. Postembryonic silence of Bxy-fuca resulted in a dramatic decrease in the longevity of and the total number of eggs produced by B. xylophilus. In addition, the motility of the nematode was greatly hampered with a significant drop in head thrashing frequency. CONCLUSION: Bxy-fuca plays crucial roles in development, lifespan and reproduction of B. xylophilus. Our results provide promising hints for control of PWD by blocking embryogenesis and ontogenesis, decreasing nematode fecundity, and disrupting the connection between B. xylophilus and its vector beetle by preventing nematode movement into the tracheal system. © 2019 Society of Chemical Industry.

Estimation of Fraction Dissolved After Intratracheal Delivery of a Potent Janus Kinase Inhibitor, iJAK-001, with Low Solubility in Rat and Sheep: Impact of Preclinical PKPD on Inhaled Human Dose Projection.

Background: A highly potent pan-Janus kinase (JAK) inhibitor with excellent kinome selectivity was developed for topical delivery to treat severe asthma. This poorly soluble drug discovery candidate, iJAK-001, is expected to exhibit long duration of JAK/STAT pathway inhibition at low doses in asthmatics because of depot effect after dry powder inhalation. Human dose projection for inhaled molecules with low aqueous solubility remains to be a daunting challenge because of several limitations: (1) bioanalytical measurement of dissolved fraction after inhalation of solid particles is uncertain; (2) distribution of these particles is not homogenous in the lung; (3) in vitro solubility measurements to estimate fraction dissolved may not be a reflection of local surface lung concentration; (4) lack of a surrogate biomarker of lung target engagement, and (5) invasive procedure needed to sample human lung tissue in the clinic. Methods: We leveraged in silico, in vitro, and in vivo tools preclinically and found significant differences in lung to plasma partition ratio when iJAK-001 was given intravenously (IV) or intratracheally in a solution-based formulation versus that in suspension, as well as pharmacodynamic response in preclinical asthma models when delivered systemically via IV infusion versus inhaled. Results and Conclusion: The combined results from above suggest that caution must be exercised using either lung or plasma exposure for human dose projection. Instead, using the local inhibitor concentration estimate based on delivery efficiency, dose, fraction absorbed, and rate of absorption normalized by lung (cardiac) blood flow may be more appropriate for dose projection.

Structure Overhaul Affords a Potent Purine PI3Kδ Inhibitor with Improved Tolerability.

PI3Kδ catalytic activity is required for immune cell activation, and has been implicated in inflammatory diseases as well as hematological malignancies in which the AKT pathway is overactive. A purine PI3Kδ inhibitor bearing a benzimidazolone-piperidine motif was found to be poorly tolerated in dog, which was attributed to diffuse vascular injury. Several strategies were implemented to mitigate this finding, including reconstruction of the benzimidazolone-piperidine selectivity motif. Structure-based design led to the identification of O- and N-linked heterocycloalkyls, with pyrrolidines being particularly ligand efficient and kinome selective, and having an improved safety pharmacology profile. A representative was advanced into a dog tolerability study where it was found to be well tolerated, with no histopathological evidence of vascular injury.

Characterizing Pharmacokinetic-Pharmacodynamic Relationships and Efficacy of PI3Kδ Inhibitors in Respiratory Models of TH2 and TH1 Inflammation.

We leveraged a clinical pharmacokinetic (PK)/pharmacodynamics (PD)/efficacy relationship established with an oral phosphatidylinositol 3-kinase (PI3K)δ inhibitor (Idelalisib) in a nasal allergen challenge study to determine whether a comparable PK/PD/efficacy relationship with PI3Kδ inhibitors was observed in preclinical respiratory models of type 2 T helper cell (TH2) and type 1 T helper cell (TH1) inflammation. Results from an in vitro rat blood basophil (CD63) activation assay were used as a PD biomarker. IC50 values for PI3Kδ inhibitors, MSD-496486311, MSD-126796721, Idelalisib, and Duvelisib, were 1.2, 4.8, 0.8, and 0.5 µM. In the ovalbumin Brown Norway TH2 pulmonary inflammation model, all PI3Kδ inhibitors produced a dose-dependent inhibition of bronchoalveolar lavage eosinophils (maximum effect between 80% and 99%). In a follow-up experiment designed to investigate PK attributes [maximum (or peak) plasma concentration (Cmax), area under the curve (AUC), time on target (ToT)] that govern PI3Kδ efficacy, MSD-496486311 [3 mg/kg every day (QD) and 100 mg/kg QD] produced 16% and 93% inhibition of eosinophils, whereas doses (20 mg/kg QD, 10 mg/kg twice per day, and 3 mg/kg three times per day) produced 54% to 66% inhibition. Our profiling suggests that impact of PI3Kδ inhibitors on eosinophils is supported by a PK target with a ToT over the course of treatment close to the PD IC50 rather than strictly driven by AUC, Cmax, or Cmin (minimum blood plasma concentration) coverage. Additional studies in an Altenaria alternata rat model, a sheep Ascaris-sensitive sheep model, and a TH1-driven rat ozone exposure model did not challenge our hypothesis, suggesting that an IC50 level of TE (target engagement) sustained for 24 hours is required to produce efficacy in these traditional models. We conclude that the PK/PD observations in our animal models appear to align with clinical results associated with a TH2 airway disease.

Effects of androgen and oestrogen on IGF pathways controlling phallus growth.

The development of the mammalian phallus involves hormone-dependent mesenchymal-epithelial signalling mechanisms that contribute to urethral closure and regulation of phallus elongation and growth. In marsupials, most differentiation and growth of the phallus occurs post-natally, making them amenable to direct hormone treatment. Expression of IGFs, FGFs, EFNB2, MAFB, DLX5 and AP-1 mRNAs in the phallus at day 50 post-partum (pp) were altered after treatment of tammar wallaby young from day 20 to 40 pp with androgen, oestrogen or after castration at day 25 pp. However, the most interesting changes occurred in the IGF pathway genes. Androgen treatment upregulated IGF1 in female phalluses and oestrogen treatment upregulated IGF1 in male phalluses, but it was downregulated by castration. IGFBP3 was higher in female phalluses and downregulated by androgen. IGF1 expression was higher in all untreated male than in female phalluses from day 50 to 150 pp, but IGFBP3 had the reverse pattern. At day 90 pp, when urethral closure in males is progressing and male phallus growth is accelerating. IGF1 and PCNA protein were only detected in the male urorectal septum, suggesting for the first time that closure and elongation may involve IGF1 activation of cell proliferation specifically in male phalluses. These effects of sex steroids on gene expression and on the IGF1 signalling pathway in particular, suggest that the developing phallus may be especially susceptible to perturbation by exogenous hormones.

Transcriptomic Analysis of MAP3K1 and MAP3K4 in the Developing Marsupial Gonad.

MAPKs affect gonadal differentiation in mice and humans, but whether this applies to all mammals is as yet unknown. Thus, we investigated MAPK expression during gonadal differentiation and after treatment with oestrogen in a distantly related mammal, the marsupial tammar wallaby, using our model of oestrogen-induced gonadal sex reversal. High-throughput RNA-sequencing was carried out on gonads collected from developing tammar 2 days before birth to 8 days after birth to characterise MAPK and key sexual differentiation markers. Day 25 foetal testes were cultured for 120 h in control medium or medium supplemented with exogenous oestrogen and processed for RNA-seq to identify changes in gene expression in response to oestrogen. MAPK pathway genes in the tammar were highly conserved at the sequence and amino acid level with those of mice and humans. Marsupial MAP3K1 and MAP3K4 clustered together in a separate branch from eutherian mammals. There was a marked decrease in the expression of male-determining genes SOX9 and AMH and increase in the female marker FOXL2 in oestrogen-treated male gonads. Only MAP3K1 expression increased in male gonads in response to oestrogen while other MAPK genes remained unaffected. This study suggests that MAP3K1 can be influenced by exogenous oestrogens during gonadal differentiation in this marsupial.

Hormone-responsive genes in the SHH and WNT/ß-catenin signaling pathways influence urethral closure and phallus growth.

Environmental endocrine disruptors (EEDs) that affect androgen or estrogen activity may disrupt gene regulation during phallus development to cause hypospadias or a masculinized clitoris. We treated developing male tammar wallabies with estrogen and females with androgen from day 20-40 postpartum (pp) during the androgen imprinting window of sensitivity. Estrogen inhibited phallus elongation but had no effect on urethral closure and did not significantly depress testicular androgen synthesis. Androgen treatment in females did not promote phallus elongation but initiated urethral closure. Phalluses were collected for transcriptome sequencing at day 50 pp when they first become sexually dimorphic to examine changes in two signaling pathways, sonic hedgehog (SHH) and wingless-type MMTV integration site family (WNT)/ß-catenin. SHH mRNA and ß-catenin were predominantly expressed in the urethral epithelium in the tammar phallus, as in eutherian mammals. Estrogen treatment and castration of males induced an upregulation of SHH, while androgen treatment downregulated SHH. These effects appear to be direct since we detected putative estrogen receptor α (ERα) and androgen receptor (AR) binding sites near SHH. WNT5A, like SHH, was downregulated by androgen, while WNT4 was upregulated in female phalluses after androgen treatment. After estrogen treatment, WIF1 and WNT7A were both downregulated in male phalluses. After castration, WNT9A was upregulated. These results suggest that SHH and WNT pathways are regulated by both estrogen and androgen to direct the proliferation and elongation of the phallus during differentiation. Their response to exogenous hormones makes these genes potential targets of EEDs in the etiology of abnormal phallus development including hypospadias.

Androgen and Oestrogen Affect the Expression of Long Non-Coding RNAs During Phallus Development in a Marsupial.

There is increasing evidence that long non-coding RNAs (lncRNAs) are important for normal reproductive development, yet very few lncRNAs have been identified in phalluses so far. Unlike eutherians, phallus development in the marsupial tammar wallaby occurs post-natally, enabling manipulation not possible in eutherians in which differentiation occurs in utero. We treated with sex steroids to determine the effects of androgen and oestrogen on lncRNA expression during phallus development. Hormonal manipulations altered the coding and non-coding gene expression profile of phalluses. We identified several predicted co-regulatory lncRNAs that appear to be co-expressed with the hormone-responsive candidate genes regulating urethral closure and phallus growth, namely IGF1, AR and ESR1. Interestingly, more than 50% of AR-associated coding genes and lncRNAs were also associated with ESR1. In addition, we identified and validated three novel co-regulatory and hormone-responsive lncRNAs: lnc-BMP5, lnc-ZBTB16 and lncRSPO4. Lnc-BMP5 was detected in the urethral epithelium of male phalluses and was downregulated by oestrogen in males. Lnc-ZBTB16 was downregulated by oestrogen treatment in male phalluses at day 50 post-partum (pp). LncRSPO4 was downregulated by adiol treatment in female phalluses but increased in male phalluses after castration. Thus, the expression pattern and hormone responsiveness of these lncRNAs suggests a physiological role in the development of the phallus.