Acute changes in hippocampal metabolism after anesthesia and surgery: Implications for perioperative neurocognitive disorder.

BACKGROUND: The risk of developing dementia is higher in individuals who suffer from perioperative neurocognitive disorder (PND), including postoperative cognitive dysfunction (POCD) and delirium. Recent studies have indicated correlations between anesthesia, surgery and PND. Acute metabolic changes induced by anesthesia and surgery may be related to cognitive impairments. Despite a paucity of research on acute metabolic changes in the hippocampus during surgery, there are conflicting about specific metabolites. METHODS: We developed a mouse model of cognitive impairment induced by isoflurane anesthesia and unilateral nephrectomy. Cognition was evaluated by Y maze and fear conditioning test (FCT). The hippocampus was harvested after the surgery. LC-MS (liquid chromatography-mass spectrometry) was performed. The differential metabolites involved in lipid, amino acid, nucleotide, carbohydrate metabolism were analyzed. RESULTS: Anesthesia and surgery exposure induced cognition decline. A total of 49 metabolites were significantly up-regulated and 122 down-regulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of the metabolites identified purine, glutathione, nicotinate and nicotinamide metabolism. Metabolites involved in lipid, amino acid, nucleotide, carbohydrate metabolism were identified including nicotinamide adenine dinucleotide (NAD), 1-Methylnicotinamide, propionic acid, histidine, adenosine, and guanosine cyclic monophosphate. Some metabolites exhibited a consistent change trend in the hippocampus of aging mice. CONCLUSIONS: The study indicates that anesthesia and surgery can induce acute alterations in hippocampal metabolomics, including metabolites involved in lipid, amino acid, nucleotide, and carbohydrate metabolism. These metabolites may play a role in modulating PND through the regulation of neuroinflammation, oxidative stress, blood-brain barrier (BBB) permeability.

Targeting Endothelial Necroptosis Disrupts Profibrotic Endothelial-Hepatic Stellate Cells Crosstalk to Alleviate Liver Fibrosis in Nonalcoholic Steatohepatitis.

Chronic liver diseases affect over a billion people worldwide and often lead to fibrosis. Nonalcoholic steatohepatitis (NASH), a disease paralleling a worldwide surge in metabolic syndromes, is characterized by liver fibrosis, and its pathogenesis remains largely unknown, with no effective treatment available. Necroptosis has been implicated in liver fibrosis pathogenesis. However, there is a lack of research on necroptosis specific to certain cell types, particularly the vascular system, in the context of liver fibrosis and NASH. Here, we employed a mouse model of NASH in combination with inducible gene knockout mice to investigate the role of endothelial necroptosis in NASH progression. We found that endothelial cell (EC)-specific knockout of mixed lineage kinase domain-like protein (MLKL), a critical executioner involved in the disruption of cell membranes during necroptosis, alleviated liver fibrosis in the mouse NASH model. Mechanistically, EC-specific deletion of Mlkl mitigated the activation of TGFß/Smad 2/3 pathway, disrupting the pro-fibrotic crosstalk between endothelial cells and hepatic stellate cells (HSCs). Our findings highlight endothelial MLKL as a promising molecular target for developing therapeutic interventions for NASH.

Endothelins (EDN1, EDN2, EDN3) and their receptors (EDNRA, EDNRB, EDNRB2) in chickens: Functional analysis and tissue distribution.

Endothelins (EDNs) and their receptors (EDNRs) are reported to be involved in the regulation of many physiological/pathological processes, such as cardiovascular development and functions, pulmonary hypertension, neural crest cell proliferation, differentiation and migration, pigmentation, and plumage in chickens. However, the functionality, signaling, and tissue expression of avian EDN-EDNRs have not been fully characterized, thus impeding our comprehensive understanding of their roles in this model vertebrate species. Here, we reported the cDNAs of three EDN genes (EDN1, EDN2, EDN3) and examined the functionality and expression of the three EDNs and their receptors (EDNRA, EDNRB and EDNRB2) in chickens. The results showed that: 1) chicken (c-) EDN1, EDN2, and EDN3 cDNAs were predicted to encode bioactive EDN peptides of 21 amino acids, which show remarkable degree of amino acid sequence identities (91-95%) to their respective mammalian orthologs; 2) chicken (c-) EDNRA expressed in HEK293 cells could be preferentially activated by chicken EDN1 and EDN2, monitored by the three cell-based luciferase reporter assays, indicating that cEDNRA is a functional receptor common for both cEDN1 and cEDN2. In contrast, both cEDNRB and cEDNRB2 could be activated by all three EDN peptides with similar potencies, indicating that both receptors can function as common receptors for the three EDNs and share functional similarity. Moreover, activation of three EDNRs could stimulate intracellular calcium, MAPK/ERK, and cAMP/PKA signaling pathways. 3) qPCR assay revealed that cEDNs and cEDNRs are widely, but differentially, expressed in adult chicken tissues. Taken together, our data establishes a clear molecular basis to uncover the physiological/pathological roles of EDN-EDNR system in birds and helps to reveal the conserved actions of EDN-EDNR signaling across vertebrates.

Comprehensive review of potential drugs with anti-pulmonary fibrosis properties.

Pulmonary fibrosis is a chronic and progressive lung disease characterized by the accumulation of scar tissue in the lungs, which leads to impaired lung function and reduced quality of life. The prognosis for idiopathic pulmonary fibrosis (IPF), which is the most common form of pulmonary fibrosis, is generally poor. The median survival for patients with IPF is estimated to be around 3-5 years from the time of diagnosis. Currently, there are two approved drugs (Pirfenidone and Nintedanib) for the treatment of IPF. However, Pirfenidone and Nintedanib are not able to reverse or cure pulmonary fibrosis. There is a need for new pharmacological interventions that can slow or halt disease progression and cure pulmonary fibrosis. This review aims to provide an updated overview of current and future drug interventions for idiopathic pulmonary fibrosis, and to summarize possible targets of potential anti-pulmonary fibrosis drugs, providing theoretical support for further clinical combination therapy or the development of new drugs.

Effect of IVIG therapy on pregnant women with unexplained recurrent spontaneous abortion: a systematic review and meta-analysis.

Objective: To assess the effect of intravenous immunoglobulin (IVIG) therapy on unexplained recurrent spontaneous abortion (URSA). Methods: We retrieved all randomized controlled trials (RCTs) related to the effect of IVIG therapy on URSA in the following databases: PubMed, Embase, Web of Science, and Cochrane Central Register of Controlled Trials before April 30, 2023, according to the PRISMA statement. The therapeutic effect of IVIG was measured by live birth rates. Quality assessment was conducted independently by two reviewers, based on the Newcastle-Ottawa scale. For the meta-analysis, we used odds ratios (random effects model and fixed effects model). The between-study heterogeneity was assessed by the Q test. Publication bias was assessed by funnel plots. Results: A total of 12 studies with 751 participants were included in this meta-analysis. There was no statistical significance [OR = 1.07, 95%CI (0.65, 1.75), P=0.80] between the IVIG group and the non-IVIG group, including low molecular weight heparin (LMWH) plus low-dose aspirin (LDA), intralipid, multivitamins, albumin, and normal saline. A subgroup analysis was conducted according to the different treatment regimens of the non-IVIG group. Compared to the placebo group, including multivitamins, albumin, and saline, the live birth rate of the IVIG group is superior, but there was no statistical significance [OR =1.43, 95%CI (0.99, 2.07), P=0.05]. Another subgroup analysis was performed according to URSA with positive for antiphospholipid antibodies (aPLs). Results showed the live birth rate of IVIG on URSA with positive for aPLs is inferior to that of LMWH plus LDA [OR = 0.25, 95%CI (0.11, 0.55), P=0.0007]. Conclusions: IVIG didn't increase the live birth rate of URSA compared to placebo. Conversely, compared with the IVIG, the LMWH plus LDA treatment schedule can increase the live birth rate of URSA with positive for aPLs.

Alterations in microbiota of patients with COVID-19: implications for therapeutic interventions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently caused a global pandemic, resulting in more than 702 million people being infected and over 6.9 million deaths. Patients with coronavirus disease (COVID-19) may suffer from diarrhea, sleep disorders, depression, and even cognitive impairment, which is associated with long COVID during recovery. However, there remains no consensus on effective treatment methods. Studies have found that patients with COVID-19 have alterations in microbiota and their metabolites, particularly in the gut, which may be involved in the regulation of immune responses. Consumption of probiotics may alleviate the discomfort caused by inflammation and oxidative stress. However, the pathophysiological process underlying the alleviation of COVID-19-related symptoms and complications by targeting the microbiota remains unclear. In the current study, we summarize the latest research and evidence on the COVID-19 pandemic, together with symptoms of SARS-CoV-2 and vaccine use, with a focus on the relationship between microbiota alterations and COVID-19-related symptoms and vaccine use. This work provides evidence that probiotic-based interventions may improve COVID-19 symptoms by regulating gut microbiota and systemic immunity. Probiotics may also be used as adjuvants to improve vaccine efficacy.

Lung regeneration: diverse cell types and the therapeutic potential.

Lung tissue has a certain regenerative ability and triggers repair procedures after injury. Under controllable conditions, lung tissue can restore normal structure and function. Disruptions in this process can lead to respiratory system failure and even death, causing substantial medical burden. The main types of respiratory diseases are chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and acute respiratory distress syndrome (ARDS). Multiple cells, such as lung epithelial cells, endothelial cells, fibroblasts, and immune cells, are involved in regulating the repair process after lung injury. Although the mechanism that regulates the process of lung repair has not been fully elucidated, clinical trials targeting different cells and signaling pathways have achieved some therapeutic effects in different respiratory diseases. In this review, we provide an overview of the cell type involved in the process of lung regeneration and repair, research models, and summarize molecular mechanisms involved in the regulation of lung regeneration and fibrosis. Moreover, we discuss the current clinical trials of stem cell therapy and pharmacological strategies for COPD, IPF, and ARDS treatment. This review provides a reference for further research on the molecular and cellular mechanisms of lung regeneration, drug development, and clinical trials.

BRD4: an effective target for organ fibrosis.

Fibrosis is an excessive wound-healing response induced by repeated or chronic external stimuli to tissues, significantly impacting quality of life and primarily contributing to organ failure. Organ fibrosis is reported to cause 45% of all-cause mortality worldwide. Despite extensive efforts to develop new antifibrotic drugs, drug discovery has not kept pace with the clinical demand. Currently, only pirfenidone and nintedanib are approved by the FDA to treat pulmonary fibrotic illness, whereas there are currently no available antifibrotic drugs for hepatic, cardiac or renal fibrosis. The development of fibrosis is closely related to epigenetic alterations. The field of epigenetics primarily studies biological processes, including chromatin modifications, epigenetic readers, DNA transcription and RNA translation. The bromodomain and extra-terminal structural domain (BET) family, a class of epigenetic readers, specifically recognizes acetylated histone lysine residues and promotes the formation of transcriptional complexes. Bromodomain-containing protein 4 (BRD4) is one of the most well-researched proteins in the BET family. BRD4 is implicated in the expression of genes related to inflammation and pro-fibrosis during fibrosis. Inhibition of BRD4 has shown promising anti-fibrotic effects in preclinical studies; however, no BRD4 inhibitor has been approved for clinical use. This review introduces the structure and function of BET proteins, the research progress on BRD4 in organ fibrosis, and the inhibitors of BRD4 utilized in fibrosis. We emphasize the feasibility of targeting BRD4 as an anti-fibrotic strategy and discuss the therapeutic potential and challenges associated with BRD4 inhibitors in treating fibrotic diseases.

Dopaminylation of endothelial TPI1 suppresses ferroptotic angiocrine signals to promote lung regeneration over fibrosis.

Lungs can undergo facultative regeneration, but handicapped regeneration often leads to fibrosis. How microenvironmental cues coordinate lung regeneration via modulating cell death remains unknown. Here, we reveal that the neurotransmitter dopamine modifies the endothelial niche to suppress ferroptosis, promoting lung regeneration over fibrosis. A chemoproteomic approach shows that dopamine blocks ferroptosis in endothelial cells (ECs) via dopaminylating triosephosphate isomerase 1 (TPI1). Suppressing TPI1 dopaminylation in ECs triggers ferroptotic angiocrine signaling to aberrantly activate fibroblasts, leading to a transition from lung regeneration to fibrosis. Mechanistically, dopaminylation of glutamine (Q) 65 residue in TPI1 directionally enhances TPI1's activity to convert dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (GAP), directing ether phospholipid synthesis to glucose metabolism in regenerating lung ECs. This metabolic shift attenuates lipid peroxidation and blocks ferroptosis. Restoring TPI1 Q65 dopaminylation in an injured endothelial niche overturns ferroptosis to normalize pro-regenerative angiocrine function and alleviate lung fibrosis. Overall, dopaminylation of TPI1 balances lipid/glucose metabolism and suppresses pro-fibrotic ferroptosis in regenerating lungs.

Role of ferroptosis in pregnancy related diseases and its therapeutic potential.

Ferroptosis is a form of regulated cell death characterized by iron overload, overwhelming lipid peroxidation, and disruption of antioxidant systems. Emerging evidence suggests that ferroptosis is associated with pregnancy related diseases, such as spontaneous abortion, pre-eclampsia, gestational diabetes mellitus, intrahepatic cholestasis of pregnancy, and spontaneous preterm birth. According to these findings, inhibiting ferroptosis might be a potential option to treat pregnancy related diseases. This review summarizes the mechanisms and advances of ferroptosis, the pathogenic role of ferroptosis in pregnancy related diseases and the potential medicines for its treatment.

The role of cGAS-STING signaling in pulmonary fibrosis and its therapeutic potential.

Pulmonary fibrosis is a progressive and ultimately fatal lung disease, exhibiting the excessive production of extracellular matrix and aberrant activation of fibroblast. While Pirfenidone and Nintedanib are FDA-approved drugs that can slow down the progression of pulmonary fibrosis, they are unable to reverse the disease. Therefore, there is an urgent demand to develop more efficient therapeutic approaches for pulmonary fibrosis. The intracellular DNA sensor called cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) plays a crucial role in detecting DNA and generating cGAMP, a second messenger. Subsequently, cGAMP triggers the activation of stimulator of interferon genes (STING), initiating a signaling cascade that leads to the stimulation of type I interferons and other signaling molecules involved in immune responses. Recent studies have highlighted the involvement of aberrant activation of cGAS-STING contributes to fibrotic lung diseases. This review aims to provide a comprehensive summary of the current knowledge regarding the role of cGAS-STING pathway in pulmonary fibrosis. Moreover, we discuss the potential therapeutic implications of targeting the cGAS-STING pathway, including the utilization of inhibitors of cGAS and STING.

Machine learning identified MDK score has prognostic value for idiopathic pulmonary fibrosis based on integrated bulk and single cell expression data.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease that poses a significant challenge to medical professionals due to its increasing incidence and prevalence coupled with the limited understanding of its underlying molecular mechanisms. In this study, we employed a novel approach by integrating five expression datasets from bulk tissue with single-cell datasets; they underwent pseudotime trajectory analysis, switch gene selection, and cell communication analysis. Utilizing the prognostic information derived from the GSE47460 dataset, we identified 22 differentially expressed switch genes that were correlated with clinical indicators as important genes. Among these genes, we found that the midkine (MDK) gene has the potential to serve as a marker of Idiopathic pulmonary fibrosis because its cellular communicating genes are differentially expressed in the epithelial cells. We then utilized midkine and its cellular communication-related genes to calculate the midkine score. Machine learning models were further constructed through midkine and related genes to predict Idiopathic pulmonary fibrosis disease through the bulk gene expression datasets. The midkine score demonstrated a correlation with clinical indexes, and the machine learning model achieved an AUC of 0.94 and 0.86 in the Idiopathic pulmonary fibrosis classification task based on lung tissue samples and peripheral blood mononuclear cell samples, respectively. Our findings offer valuable insights into the pathogenesis of Idiopathic pulmonary fibrosis, providing new therapeutic directions and target genes for further investigation.

Research progress on perioperative blood-brain barrier damage and its potential mechanism.

The blood-brain barrier (BBB) is an important barrier separating the central nervous system from the periphery. The composition includes endothelial cells, pericytes, astrocytes, synapses and tight junction proteins. During the perioperative period, anesthesia and surgical operations are also a kind of stress to the body, which may be accompanied by blood-brain barrier damage and brain metabolism dysfunction. Perioperative blood-brain barrier destruction is closely associated with cognitive impairment and may increase the risk of postoperative mortality, which is not conducive to enhanced recovery after surgery. However, the potential pathophysiological process and specific mechanism of blood-brain barrier damage during the perioperative period have not been fully elucidated. Changes in blood-brain barrier permeability, inflammation and neuroinflammation, oxidative stress, ferroptosis, and intestinal dysbiosis may be involved in blood-brain barrier damage. We aim to review the research progress of perioperative blood-brain barrier damage and its potential adverse effects and potential molecular mechanisms, and provide ideas for the study of homeostasis maintenance of brain function and precision anesthesia.

Deficiency of endothelial FGFR1 signaling via upregulation of ROCK2 activity aggravated ALI/ARDS.

Vascular leakage and inflammation are pathological hallmarks of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Endothelial cells (ECs) serve as a semipermeable barrier and play a key role in disease progression. It is well known that fibroblast growth factor receptor 1 (FGFR1) is required for maintaining vascular integrity. However, how endothelial FGFR1 functions in ALI/ARDS remains obscure. Here, we revealed that conditional deletion of endothelial FGFR1 aggravated LPS-induced lung injury, including inflammation and vascular leakage. Inhibition of its downstream Rho-associated coiled-coil-forming protein kinase 2 (ROCK2) by AAV Vec-tie-shROCK2 or its selective inhibitor TDI01 effectively attenuated inflammation and vascular leakage in a mouse model. In vitro, TNFα-stimulated human umbilical vein endothelial cells (HUVECs) showed decreased FGFR1 expression and increased ROCK2 activity. Furthermore, knockdown of FGFR1 activated ROCK2 and thus promoted higher adhesive properties to inflammatory cells and higher permeability in HUVECs. TDI01 effectively suppressed ROCK2 activity and rescued the endothelial dysfunction. These data demonstrated that the loss of endothelial FGFR1 signaling mediated an increase in ROCK2 activity, which led to an inflammatory response and vascular leakage in vivo and in vitro. Moreover, inhibition of ROCK2 activity by TDI01 provided great value and shed light on clinical translation.

Single-cell RNA sequencing analysis of lung cells in COVID-19 patients with diabetes, hypertension, and comorbid diabetes-hypertension.

Background: There is growing evidence that the lung is a target organ for injury in diabetes and hypertension. There are no studies on the status of the lungs, especially cellular subpopulations, and related functions in patients with diabetes, hypertension, and hypertension-diabetes after combined SARS-CoV-2 infection. Method: Using single-cell meta-analysis in combination with bulk-RNA analysis, we identified three drug targets and potential receptors for SARS-CoV-2 infection in lung tissues from patients with diabetes, hypertension, and hypertension-diabetes, referred to as "co-morbid" patients. Using single-cell meta-analysis analysis in combination with bulk-RNA, we identified drug targets and potential receptors for SARS-CoV-2 infection in the three co-morbidities. Results: The single-cell meta-analysis of lung samples from SARS-CoV-2-infected individuals with diabetes, hypertension, and hypertension-diabetes comorbidity revealed an upregulation of fibroblast subpopulations in these disease conditions associated with a predictive decrease in lung function. To further investigate the response of fibroblasts to therapeutic targets in hypertension and diabetes, we analyzed 35 upregulated targets in both diabetes and hypertension. Interestingly, among these targets, five specific genes were upregulated in fibroblasts, suggesting their potential association with enhanced activation of endothelial cells. Furthermore, our investigation into the underlying mechanisms driving fibroblast upregulation indicated that KREMEN1, rather than ACE2, could be the receptor responsible for fibroblast activation. This finding adds novel insights into the molecular processes involved in fibroblast modulation in the context of SARS-CoV-2 infection within these comorbid conditions. Lastly, we compared the efficacy of Pirfenidone and Nintedanib as therapeutic interventions targeting fibroblasts prone to pulmonary fibrosis. Our findings suggest that Nintedanib may be a more suitable treatment option for COVID-19 patients with diabetes and hypertension who exhibit fibrotic lung lesions. Conclusion: In the context of SARS-CoV-2 infections, diabetes, hypertension, and their coexistence predominantly lead to myofibroblast proliferation. This phenomenon could be attributed to the upregulation of activated endothelial cells. Moreover, it is noteworthy that therapeutic interventions targeting hypertension-diabetes demonstrate superior efficacy. Regarding treating fibrotic lung conditions, Nintedanib is a more compelling therapeutic option.

Trimethylamine-N-oxide (TMAO) mediates the crosstalk between the gut microbiota and hepatic vascular niche to alleviate liver fibrosis in nonalcoholic steatohepatitis.

Liver fibrosis is one main histological characteristic of nonalcoholic steatohepatitis (NASH), a disease paralleling a worldwide surge in metabolic syndromes with no approved therapies. The role of the gut microbiota in NASH pathogenesis has not been thoroughly illustrated, especially how the gut microbiota derives metabolites to influence the distal liver in NASH. Here, we performed 16S rDNA amplicon sequencing analysis of feces from a mouse NASH model induced by a Western diet and CCl4 injury and found genera under Streptococcaceae, Alcaligenaceae, Oscillibacter, and Pseudochrobactrum, which are related metabolites of TMAO. Injection of the gut microbial metabolite TMAO reduced the progression of liver fibrosis in the mouse NASH model. Further analysis revealed that the anti-fibrotic TMAO normalized gut microbiota diversity and preserved liver sinusoidal endothelial cell integrity by inhibiting endothelial beta 1-subunit of Na (+), K (+)-ATPase (ATP1B1) expression. Collectively, our findings suggest TMAO-mediated crosstalk between microbiota metabolites and hepatic vasculature, and perturbation of this crosstalk disrupts sinusoidal vasculature to promote liver fibrosis in NASH.

Evidence for Neuropeptide W Acting as a Physiological Corticotropin-releasing Inhibitory Factor in Male Chickens.

In vertebrates, adrenocorticotropin (ACTH), released by the pituitary gland, is a critical part of the stress axis and stress response. Generally, the biosynthesis and secretion of ACTH are controlled by both hypothalamic stimulatory factors and inhibitory factors [eg, ACTH-releasing inhibitory factor (CRIF)], but the identity of this CRIF remains unrevealed. We characterized the neuropeptide B (NPB)/neuropeptide W (NPW) system in chickens and found that NPW could directly target the pituitary to inhibit growth hormone (GH) and prolactin (PRL) secretion via neuropeptide B/W receptor 2 (NPBWR2), which is completely different from the mechanism in mammals. The present study first carried out a series of assays to investigate the possibility that NPW acts as a physiological CRIF in chickens. The results showed that (1) NPW could inhibit ACTH synthesis and secretion by inhibiting the 3',5'-cyclic adenosine 5'-monophosphate/protein kinase A signaling cascade in vitro and in vivo; (2) NPBWR2 was expressed abundantly in corticotrophs (ACTH-producing cells), which are located mainly in cephalic lobe of chicken pituitary, as demonstrated by single-cell RNA-sequencing, immunofluorescent staining, and fluorescence in situ hybridization; (3) dexamethasone could stimulate pituitary NPBWR2 and hypothalamic NPW expression in chicks, which was accompanied by the decease of POMC messenger RNA levels, as revealed by in vitro and subcutaneous injection assays; and (4) the temporal expression profiles of NPW-NPBWR2 pair in hypothalamus-pituitary axis and POMC in pituitary were almost unanimous in chicken. Collectively, these findings provide comprehensive evidence for the first time that NPW is a potent physiological CRIF in chickens that plays a core role in suppressing the activity of the stress axis.

Transcriptomic analysis of granulosa cell populations proximal and distal to the germinal disc of chicken preovulatory follicles.

Within the oocytes of chicken preovulatory follicles, the engulfed yolk constitutes 99% of the oocyte content, while the small germinal disc (GD) (which contains the nucleus and 99% ooplasm) occupies only less than 1%. Relative to the position of the GD, the single granulosa cell layer surrounding the oocyte can be sub-divided into two sub-populations: granulosa cells proximal (named Gp cells) and distal (Gd cells) to the GD. It was reported that Gp cells and Gd cells differ in their morphology, proliferative rate and steroidogenic capacity, however, the underlying mechanism controlling granulosa cell heterogeneity remains unclear. Here we analyzed the transcriptomes of Gd and Gp cells of preovulatory (F5 and F1) follicles in chicken ovaries. We found that: (1) genes associated with cell cycle and DNA replication (CDK1, CCNB3 etc.) have comparatively higher expression levels in Gp cells than in Gd cells, while genes associated with steroidogenesis (CYP51A1, DHCR24) are highly expressed in Gd cells, indicating that Gp cells are likely more mitotic and less steroidogenic than Gd cells; (2) genes associated with extracellular matrix remodeling, cell adhesion and sperm binding (ZP3, ZP2) are differentially expressed in Gp and Gd cells; (3) Furthermore, signaling molecules (WNT4/IHH) and receptors for NGF (NGFR), epidermal growth factor (EGFR), gonadotropins (FSHR/LHR) and prostaglandin (PTGER3) are abundantly but differentially expressed in Gp and Gd cells. Taken together, our data strongly supports the notion that Gp and Gd cells of preovulatory follicles differ in their proliferation rate, steroidogenic activity, ECM organization and sperm binding capacity, which are likely controlled by gonadotropins and local ovarian factors, such as GD-derived factors.

Single-Cell RNA Sequencing Analysis of Chicken Anterior Pituitary: A Bird's-Eye View on Vertebrate Pituitary.

It is well-established that anterior pituitary contains multiple endocrine cell populations, and each of them can secrete one/two hormone(s) to regulate vital physiological processes of vertebrates. However, the gene expression profiles of each pituitary cell population remains poorly characterized in most vertebrate groups. Here we analyzed the transcriptome of each cell population in adult chicken anterior pituitaries using single-cell RNA sequencing technology. The results showed that: (1) four out of five known endocrine cell clusters have been identified and designated as the lactotrophs, thyrotrophs, corticotrophs, and gonadotrophs, respectively. Somatotrophs were not analyzed in the current study. Each cell cluster can express at least one known endocrine hormone, and novel marker genes (e.g., CD24 and HSPB1 in lactotrophs, NPBWR2 and NDRG1 in corticotrophs; DIO2 and SOUL in thyrotrophs, C5H11ORF96 and HPGDS in gonadotrophs) are identified. Interestingly, gonadotrophs were shown to abundantly express five peptide hormones: FSH, LH, GRP, CART and RLN3; (2) four non-endocrine/secretory cell types, including endothelial cells (expressing IGFBP7 and CFD) and folliculo-stellate cells (FS-cells, expressing S100A6 and S100A10), were identified in chicken anterior pituitaries. Among them, FS-cells can express many growth factors, peptides (e.g., WNT5A, HBEGF, Activins, VEGFC, NPY, and BMP4), and progenitor/stem cell-associated genes (e.g., Notch signaling components, CDH1), implying that the FS-cell cluster may act as a paracrine/autocrine signaling center and enrich pituitary progenitor/stem cells; (3) sexually dimorphic expression of many genes were identified in most cell clusters, including gonadotrophs and lactotrophs. Taken together, our data provides a bird's-eye view on the diverse aspects of anterior pituitaries, including cell composition, heterogeneity, cell-to-cell communication, and gene expression profiles, which facilitates our comprehensive understanding of vertebrate pituitary biology.

Functional differences between TSHR alleles associate with variation in spawning season in Atlantic herring.

The underlying molecular mechanisms that determine long day versus short day breeders remain unknown in any organism. Atlantic herring provides a unique opportunity to examine the molecular mechanisms involved in reproduction timing, because both spring and autumn spawners exist within the same species. Although our previous whole genome comparisons revealed a strong association of TSHR alleles with spawning seasons, the functional consequences of these variants remain unknown. Here we examined the functional significance of six candidate TSHR mutations strongly associated with herring reproductive seasonality. We show that the L471M missense mutation in the spring-allele causes enhanced cAMP signaling. The best candidate non-coding mutation is a 5.2 kb retrotransposon insertion upstream of the TSHR transcription start site, near an open chromatin region, which is likely to affect TSHR expression. The insertion occurred prior to the split between Pacific and Atlantic herring and was lost in the autumn-allele. Our study shows that strongly associated coding and non-coding variants at the TSHR locus may both contribute to the regulation of seasonal reproduction in herring.