Mfn2 regulates mitochondria and mitochondria-associated endoplasmic reticulum membrane function in neurodegeneration induced by repeated sevoflurane exposure.

Repeated sevoflurane exposure in neonatal mice can leads to neuronal apoptosis and mitochondrial dysfunction. The mitochondria are responsible for energy production to maintain homeostasis in the central nervous system. The mitochondria-associated endoplasmic reticulum membrane (MAM) is located between the mitochondria and endoplasmic reticulum (ER), and it is critical for mitochondrial function and cell survival. MAM malfunction contributes to neurodegeneration, however, whether it is involved in sevoflurane-induced neurotoxicity remains unknown. Our study demonstrated that repeated sevoflurane exposure induced mitochondrial dysfunction and dampened the MAM structure. The upregulated ER-mitochondria tethering enhanced Ca2+ transition from the cytosol to the mitochondria. Overload of mitochondrial Ca2+ contributed to opening of the mitochondrial permeability transition pore (mPTP), which caused neuronal apoptosis. Mitofusin 2(Mfn2), a key regulator of ER-mitochondria contacts, was found to be suppressed after repeated sevoflurane exposure, while restoration of Mfn2 expression alleviated cognitive dysfunction due to repeated sevoflurane exposure in the adult mice. These evidences suggest that sevoflurane-induced MAM malfunction is vulnerable to Mfn2 suppression, and the enhanced ER-mitochondria contacts promotes mitochondrial Ca2+ overload, contributing to mPTP opening and neuronal apoptosis. This paper sheds light on a novel mechanism of sevoflurane-induced neurotoxicity. Furthermore, targeting Mfn2-mediated regulation of the MAM structure and mitochondrial function may provide a therapeutic advantage in sevoflurane-induced neurodegeneration.

Dysregulation of iron homeostasis and ferroptosis in sevoflurane and isoflurane associated perioperative neurocognitive disorders.

In recent years, sevoflurane and isoflurane are the most popular anesthetics in general anesthesia for their safe, rapid onset, and well tolerant. Nevertheless, many studies reported their neurotoxicity among pediatric and aged populations. This effect is usually manifested as cognitive impairment such as perioperative neurocognitive disorders. The wide application of sevoflurane and isoflurane during general anesthesia makes their safety a major health concern. Evidence indicates that iron dyshomeostasis and ferroptosis may establish a role in neurotoxicity of sevoflurane and isoflurane. However, the mechanisms of sevoflurane- and isoflurane-induced neuronal injury were not fully understood, which poses a barrier to the treatment of its neurotoxicity. We, therefore, reviewed the current knowledge on mechanisms of iron dyshomeostasis and ferroptosis and aimed to promote a better understanding of their roles in sevoflurane- and isoflurane-induced neurotoxicity.

Effects of alcohol on the symptoms of gouty arthritis and taxonomic structure of gut microbiota in C57BL/6 mice.

Gout is an acute arthritis caused by the elevated levels of serum uric acid (UA), and its prevalence has been rapidly increasing. Alcohol abuse could lead to a series of health problems. Multiple pieces of evidence suggest that alcohol intake affects the development and progression of gout, while the gut microbiota plays an important role in the development of gout and the long-term alcohol consumption could affect the stability of the gut microbiota. This study aimed to explore the effects of alcohol intake at different concentrations on gouty arthritis based on the gut microbiota. We investigated the effects of different concentrations of alcohol on gouty arthritis in mouse models of acute gouty arthritis established by injection of monosodium urate (MSU) crystals into C57BL/6 mice. The results indicated that the high-alcohol consumption not only exacerbated joint swelling and pain, increased the levels of UA, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), but also showed dramatic effects on the composition and structure of the gut microbiota in gouty mice. Two key microorganisms, Parasutterella and Alistipes, could aggravate gout symptoms through lipopolysaccharide biosynthesis, riboflavin metabolism, phenylalanine metabolism, and arginine and proline metabolisms. In conclusion, our study suggested that high-concentrations of alcohol altered the gut microbiota structure in gouty mice induced by MSU crystals, which could exacerbate gouty symptoms by enhancing pro-inflammatory pathways.

General anesthetic action profile on the human prefrontal cortex cells through comprehensive single-cell RNA-seq analysis.

The cellular and molecular actions of general anesthetics to induce anesthesia state and also cellular signaling changes for subsequent potential "long term" effects remain largely elusive. General anesthetics were reported to act on voltage-gated ion channels and ligand-gated ion channels. Here we used single-cell RNA-sequencing complemented with whole-cell patch clamp and calcium transient techniques to examine the gene transcriptome and ion channels profiling of sevoflurane and propofol, both commonly used clinically, on the human fetal prefrontal cortex (PFC) mixed cell cultures. Both propofol and sevoflurane at clinically relevant dose/concentration promoted "microgliosis" but only sevoflurane decreased microglia transcriptional similarity. Propofol and sevoflurane each extensively but transiently (<2 h) altered transcriptome profiling across microglia, excitatory neurons, interneurons, astrocytes and oligodendrocyte progenitor cells. Utilizing scRNA-seq as a robust and high-through put tool, our work may provide a comprehensive blueprint for future mechanistic studies of general anesthetics in clinically relevant settings.

Bicuculline and Bumetanide Attenuate Sevoflurane-Induced Impairment of Myelination and Cognition in Young Mice.

Sevoflurane (Sevo) is one of the most commonly used general anesthetics for infants and young children. We investigated whether Sevo impairs neurological functions, myelination, and cognition via the γ-aminobutyric acid A receptor (GABAAR) and Na+-K+-2Cl- cotransporter (NKCC1) in neonatal mice. On postnatal days 5-7, mice were exposed to 3% Sevo for 2 h. On postnatal day 14, mouse brains were dissected, and oligodendrocyte precursor cell line level lentivirus knockdown of GABRB3, immunofluorescence, and transwell migration assays were performed. Finally, behavioral tests were conducted. Multiple Sevo exposure groups exhibited increased neuronal apoptosis levels and decreased neurofilament protein levels in the mouse cortex compared with the control group. Sevo exposure inhibited the proliferation, differentiation, and migration of the oligodendrocyte precursor cells, thereby affecting their maturation process. Electron microscopy revealed that Sevo exposure reduced myelin sheath thickness. The behavioral tests showed that multiple Sevo exposures induced cognitive impairment. GABAAR and NKCC1 inhibition provided protection against Sevo-induced neurotoxicity and cognitive dysfunction. Thus, bicuculline and bumetanide can protect against Sevo-induced neuronal injury, myelination impairment, and cognitive dysfunction in neonatal mice. Furthermore, GABAAR and NKCC1 may be mediators of Sevo-induced myelination impairment and cognitive dysfunction.

Association between vitamin D concentration and delirium in hospitalized patients: A meta-analysis.

BACKGROUND: Now the occurrence of delirium is more concerning to clinicians and psychiatrists. It has been reported that vitamin D deficiency may be a relevant factor in the development of delirium in hospitalized patients. STUDY OBJECTIVE: To investigate the association between vitamin D concentration and delirium in hospitalized patients. DESIGN: Meta-analysis. METHODS: A systematic literature search was conducted using PubMed, EMBASE, and the Cochrane Library. The primary outcome was the occurrence of delirium in the inpatient setting. Odds ratios (OR) were calculated with random or fixed effects models. RESULTS: In this article, we define the normal range of vitamin D concentrations as greater than 75 nmol / L, 50-75 nmol / L as vitamin D insufficiency, 25-50 nmol / L as vitamin D deficiency, and less than 25 nmol / L as vitamin D severe deficiency. The Results showed that severe vitamin D deficiency (OR: 1.98 [1.41-2.79], P<0.001) and vitamin D deficiency (OR: 1.50 [1.12-2.00], P = 0.006) were more likely to develop delirium than normal vitamin D levels. Subgroup analysis also revealed that low vitamin D concentrations were associated with a higher incidence of delirium, whether the cutoff point was 25 nmol/L (OR: 1.52 [1.40-1.64], P<0.001), 50 nmol/L (OR: 1.47 [1.19-1.82], P<0.001), or 75 nmol/L (OR: 1.54 [1.21-1.96], P<0.001). The included studies scored medium and high on the Newcastle-Ottawa quality assessment scale. CONCLUSION: Compared with normal vitamin D levels, severe vitamin D deficiency and vitamin D deficiency, but not vitamin D insufficiency, are associated with a higher incidence of delirium in hospitalized patients. TRIAL REGISTRATION: This review was registered in the PROSPERO database under identifier CRD42021271347. https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021271347.

Ropivacaine inhibits proliferation and invasion and promotes apoptosis and autophagy in bladder cancer cells via inhibiting PI3K/AKT pathway.

Application of a certain concentration of local anesthetics during tumor resection inhibits the progression of tumor. The effects of ropivacaine in bladder cancer (BC) have never been explored. We explored the effects of ropivacaine on the progression of BC in vitro and in vivo. CCK8 assay and EDU staining was conducted to examine cell proliferation. Flow cytometry and transwell assay were performed to evaluate apoptosis and invasion, respectively. Expression of light chain 3 (LC3) was observed through immunofluorescence. Furthermore, the xenograft tumor model of BC was built to detect the effects of ropivacaine in vivo. IHC and TUNEL assay were conducted to detect cell proliferation and apoptosis in vivo. Ropivacaine inhibited the proliferation of T24 and 5639 cells with the 50% inhibitory concentration (IC50) of 20.08 and 31.86 µM, respectively. Ropivacaine suppressed the invasion ability and induces the apoptosis of cells. Besides, ropivacaine triggers obvious autophagy in BC cells. Moreover, ropivacaine blocks the PI3K/AKT signal pathway in BC cells. The impact of ropivacaine on cell viability, motility, and autophagy was reversed by 740 Y-P, the activator of PI3K/AKT signal pathway. The in vivo experiments demonstrated that ropivacaine inhibited the proliferation and mobility of BC. Ropivacaine has anti-carcinoma effects in BC via inactivating PI3K/AKT pathway, providing a new theoretical reference for the use of local anesthetics in the treatment of BC.

The research landscape of ferroptosis in the brain: A bibliometric analysis.

Background: Ferroptosis is a newly proposed concept of programmed cell death and has been widely studied in many diseases during the past decade. However, a bibliometric study that concentrates on publication outputs and research trends of ferroptosis related to the brain is lacking. Methods: We retrieved publication data in the field of ferroptosis in the brain from the Web of Science Core Collection on 31 December 2021. A bibliometric analysis was performed using VOSviewer and CiteSpace software. Results: Six hundred fifty-six documents focusing on ferroptosis in the brain were published from 2012 to 2021. The number of publications in this field has shown a steady increase in recent years. Most publications were from China (338) and the United States (166), while the most productive organizations were at the University of Melbourne (34) and University of Pittsburgh (23). Ashley I. Bush was the most productive author, while Scott J Dixon was the most co-cited author. The journal Free Radical Biology and Medicine published the most articles in this field, while Cell was the most cited journal. Among 656 publications, top 10 cited documents were cited at least 300 times. Among the top 20 references with the strongest citation bursts, half of the papers had a burst until 2021. The keywords analysis suggests that the top 20 keywords appeared at least 40 times. Additionally, "amyloid precursor protein" was the keyword with strongest bursts. Conclusion: Research on ferroptosis in the brain will continue to be highly regarded. This study analyzed the research landscape of ferroptosis in the brain and offers a new reference for researchers in this field.

Identification and Validation of Ferroptosis-Related Genes in Sevoflurane-Induced Hippocampal Neurotoxicity.

Background: Sevoflurane is one of the most popular inhalational anesthetics during perioperative period but presenting neurotoxicity among pediatric and aged populations. Recent experiments in vivo and in vitro have indicated that ferroptosis may contribute to the neurotoxicity of sevoflurane anesthesia. However, the exact mechanism is still unclear. Methods: In current study, we explored the differential expressed genes (DEGs) in HT-22 mouse hippocampal neuronal cells after sevoflurane anesthesia using RNA-seq. Differential expressed ferroptosis-related genes (DEFRGs) were screened and analyzed by Gene Ontology (GO) and pathway enrichment analysis. Protein-to-protein interaction (PPI) network was constructed by the Search Tool for the Retrieval of Interacting Genes (STRING). Significant modules and the hub genes were identified by using Cytoscape. The Connectivity Map (cMAP) was used for screening drug candidates targeting the identified DEFRGs. Potential TF-gene network and drug-gene pairs were established towards the hub genes. In final, we validated these results in experiments. Results: A total of 37 ferroptosis-related genes (18 upregulated and 19 downregulated) after sevoflurane exposure in hippocampal neuronal cells were finally identified. These differentially expressed genes were mainly involved into the biological processes of cellular response to oxidative stress. Pathway analysis indicated that these genes were involved in ferroptosis, mTOR signaling pathway, and longevity-regulating pathway. PPI network was constructed. 10 hub genes including Prkaa2, Chac1, Arntl, Tfrc, Slc7a11, Atf4, Mgst1, Lpin1, Atf3, and Sesn2 were found. Top 10 drug candidates, gene-drug networks, and TFs targeting these genes were finally identified. These results were validated in experiments. Conclusion: Our results suggested that ferroptosis-related genes play roles in sevoflurane anesthesia-related hippocampal neuron injury and offered the hub genes and potential therapeutic agents for investigating and treatment of this neurotoxicity after sevoflurane exposure. Finally, therapeutic effect of these drug candidates and function of potential ferroptosis targets should be further investigated for treatment and clarifying mechanisms of sevoflurane anesthesia-induced neuron injury in future research.

Sevoflurane exposure induces neurotoxicity by regulating mitochondrial function of microglia due to NAD insufficiency.

Developmental neurons received with sevoflurane, the commonly used inhalational anesthetic agent in clinical surgery, several times tend to be destroyed. Microglia, the resident immune cells of the central nervous system (CNS), are activated after sevoflurane exposure, accompanied by releasing proinflammatory cytokines that damage developing neurons. The sevoflurane-induced neurotoxicity could be attributed to activated microglia presenting proinflammatory and anti-inflammatory functions. Proinflammatory microglia release cytokines to impair the CNS, while anti-inflammatory microglia engulf damaged neurons to maintain CNS homeostasis. Sevoflurane exposure promotes the secretion of proinflammatory cytokines by microglia, inhibiting the microglial phagocytic function. Microglia with poor phagocytic function cannot engulf damaged neurons, leading to the accumulation of damaged neurons. The mechanism underlying poor phagocytic function may be attributed to mitochondrial dysfunction of microglia induced by sevoflurane exposure, in which affected mitochondria cannot generate adequate ATP and NAD to satisfy the energy demand. We discovered that sevoflurane treatment impaired the mitochondrial metabolism of microglia, which resulted in NAD deficiency and couldn't produce sufficient energy to clear damaged neurons to maintain CNS development. Our findings provide an explanation of a new mechanism underlying sevoflurane-induced neurotoxicity.

Role of GABAA receptor depolarization-mediated VGCC activation in sevoflurane-induced cognitive impairment in neonatal mice.

Background: In neonatal mice, anesthesia with sevoflurane depolarizes the GABA Type A receptor (GABAAR), which leads to cognitive impairment. Calcium accumulation in neurons can lead to neurotoxicity. Voltage-gated calcium channels (VGCCs) can increase intracellular calcium concentration under isoflurane and hypoxic conditions. The underlying mechanisms remain largely unknown. Methods: Six-day-old mice were anesthetized with 3% sevoflurane for 2 h/day for 3 days. The Y-Maze, new object recognition (NOR) test, the Barnes maze test, immunoassay, immunoblotting, the TUNEL test, and Golgi-Cox staining were used to assess cognition, calcium concentration, inflammatory response, GABAAR activation, VGCC expression, apoptosis, and proliferation of hippocampal nerve cells in mice and HT22 cells. Results: Compared with the control group, mice in the sevoflurane group had impaired cognitive function. In the sevoflurane group, the expression of Gabrb3 and Cav1.2 in the hippocampal neurons increased (p < 0.01), the concentration of calcium ions increased (p < 0.01), inflammatory reaction and apoptosis of neurons increased (p < 0.01), the proliferation of neurons in the DG area decreased (p < 0.01), and dendritic spine density decreased (p < 0.05). However, the inhibition of Gabrb3 and Cav1.2 alleviated cognitive impairment and reduced neurotoxicity. Conclusions: Sevoflurane activates VGCCs by inducing GABAAR depolarization, resulting in cognitive impairment. Activated VGCCs cause an increase in intracellular calcium concentration and an inflammatory response, resulting in neurotoxicity and cognitive impairment.

The role of depolarizing activation of Na+-Ca2+ exchanger by oligodendrocyte progenitor cells in the effect of sevoflurane on myelination.

AIMS: The aim of this study was to investigate the role of depolarizing activation of Na+-Ca2+ exchanger (NCX) by oligodendrocyte progenitor cells (OPC) in the effect of sevoflurane on myelination. MAIN METHODS: On postnatal days 7, 8, and 9, mice were exposed to 3 % sevoflurane for 2 h per day. The proliferation, differentiation, and myelin sheath of OPC were observed with immunofluorescence, quantitative real-time polymerase chain reaction (QRT-PCR), and transmission electron microscopy (TEM) at various time points. The open field, Y maze, and new object recognition tests were used to measure spatial learning and memory. siRNA was used for the knockdown NCX1 in human OPC (HOPC) before sevoflurane exposure; the Transwell migration assay was used to measure cell migration ability and Fluo 4-AM was used to measure intracellular Ca2+ concentration. KEY FINDINGS: Pretreatment with an NCX inhibitor attenuated the proliferation and differentiation of OPC induced by sevoflurane and induced a remarkable increase in platelet-derived growth factor receptor-alpha (PDGFRα), 2, 3-cyclic nucleotide 3-phosphodiesterase (CNPase), oligodendrocyte transcription factor 2 (Olig2), and homeodomain protein NK2 homeobox 2 (NKX2.2) levels. Pretreatment with an NCX inhibitor alleviated the sevoflurane-induced myelination disorder and cognitive impairment. The decreased cell migration and increased intracellular Ca2+ concentration observed in the siRNA-negative control group was reversed in the sevoflurane plus siRNA-NCX1 group. SIGNIFICANCE: This study suggests that repeated sevoflurane exposure in newborn mice leads to depolarization of OPC, which leads to Ca2+ influx through NCX and affects OPC proliferation, migration, differentiation, and myelination, ultimately leading to cognitive impairment.

Effect of Anesthesia on Oligodendrocyte Development in the Brain.

Oligodendrocytes (OLs) participate in the formation of myelin, promoting the propagation of action potentials, and disruption of their proliferation and differentiation leads to central nervous system (CNS) damage. As surgical techniques have advanced, there is an increasing number of children who undergo multiple procedures early in life, and recent experiments have demonstrated effects on brain development after a single or multiple anesthetics. An increasing number of clinical studies showing the effects of anesthetic drugs on the development of the nervous system may mainly reside in the connections between neurons, where myelin development will receive more research attention. In this article, we review the relationship between anesthesia exposure and the brain and OLs, provide new insights into the development of the relationship between anesthesia exposure and OLs, and provide a theoretical basis for clinical prevention of neurodevelopmental risks of general anesthesia drugs.

The TLR4/NF-κB/MAGI-2 signaling pathway mediates postoperative delirium.

PURPOSE: To evaluate the TLR4/NF-κB/MAGI-2 signaling pathway in postoperative delirium. METHODS: Elderly patients aged 65-80 years who received unilateral hip arthroplasty under subarachnoid anesthesia were included. Pre-anesthesia cerebrospinal fluid and perioperative blood samples were collected. After follow-up, patients were divided into two groups according to the occurrence of postoperative delirium (POD) after surgery. The potential differentially expressed proteins in the two groups were determined by proteomics assay and subsequent western blot validation. A POD model of aged mice was established, and the TLR4/NF-κB/MAGI-2 signaling pathway was determined. MAIN FINDINGS: The IL-1ß and TNF-α levels in pre-anesthesia cerebrospinal fluid and postoperative blood were higher in patients who developed POD than in those patients who did not. Compared with non-POD patients, MAGI-2 was highly expressed in POD patients, as validated by proteomics assays and western blotting. Higher p-NF-κB-p65, TLR4 and MAGI-2 in POD patients were detected by western blot. The POD model in aged mice was successfully established and verified by three behavioral tests. Postoperative inflammatory cytokines and the TLR4/NF-κB/MAGI-2 signaling pathway were increased in mice with POD. Inhibiting TLR4/NF-κB/MAGI-2 signaling pathway could reduce postoperative delirium. CONCLUSIONS: The TLR4/NF-κB/MAGI-2 signaling pathway mediates POD.

An atlas of dynamic peripheral blood mononuclear cell landscapes in human perioperative anaesthesia/surgery.

BACKGROUND: The number of patients receiving anaesthesia is increasing, but the impact of general anaesthesia on the patient's immune system remains unclear. The aim of the present study is to investigate dynamics of systemic immune cell responses to anaesthesia during perioperative period at a single-cell solution. METHODS: The peripheral blood mononuclear cells (PBMCs) and clinical phenomes were harvested and recorded 1 day before anaesthesia and operation, just after anaesthesia (0 h), and 24 and 48 h after anaesthesia. Single-cell sequencing of PBMCs was performed with 10× genomics. Subsequently, data analysis was performed with R packages: Seurat, clusterProfiler and CellPhoneDB. RESULTS: We found that the cluster of CD56+ NK cells changed at 0 h and the cluster of monocytes increased at 24 and 48 h after anaesthesia. The characteristic genes of CD56+ NK cells were mainly enriched in the Jak-STAT signalling pathway and in cell adhesion molecules (24 h) and carbon metabolism (48 h). The communication between CD14+ monocytes and other cells decreased substantially 0 and 48 h after operation. The number of plasma cells enriched in protein export in men was substantially higher than that in women, although the total number in patients decreased 24 h after operation. CD14+ monocytes dominated that cell-cell communications appeared in females, while CD8+ NKT cells dominated that cell-cell communications appeared in male. The number of plasma cells increased substantially in patients with major surgical trauma, with enrichments of pentose phosphate pathway. The communications between plasma cells with other cells varied between surgical severities and anaesthetic forms. The intravenous anaesthesia caused major alterations of cell types, including CD14+ monocytes, plasmas cells and MAIT cells, as compared with inhalation anaesthesia. CONCLUSION: We initially reported the roles of perioperative anaesthesia/surgery in temporal phenomes of circulating immune cells at a single-cell solution. Thus, the protection against immune cell changes would benefit the recovery from anaesthesia/surgery.

Values of Single-Cell RNA Sequencing in Development of Cerebral Cortex.

The single-cell RNA sequencing (scRNA-seq) is a powerful tool for exploring the complexity, clusters, and specific functions of the brain cells. Using scRNA-seq, the heterogeneity and changes in transcriptomic profiles of a single neuron were defined during dynamic development and differentiation of cells in cerebral cortex regions, and in the pathogenesis of neurological diseases. One of the great challenges is that the brain sample is susceptible to interference and confounding. More advanced methodologies of computational systems biology need to be developed to overcome the inherent interference and technical differences in the detection of single-cell signals. It is expected that scRNA-seq will be extended to metabolic profiles of the single neuron cell on basis of transcriptional profiles and regulatory networks. It is also expected if the transcriptional profiles can be integrated with molecular and functional phenomes in a single neuron and with disease-specific phenomes to understand molecular mechanisms of brain development and disease occurrence. scRNA-seq will provide the new emerging neurological disciple of the artificial intelligent single neuron for deep understanding of brain diseases.

The alteration of RhoA geranylgeranylation and Ras farnesylation breaks the integrity of the blood-testis barrier and results in hypospermatogenesis.

Non-obstructive azoospermia (NOA) severely affects male infertility, however, the deep mechanisms of this disease are rarely interpreted. In this study, we find that undifferentiated spermatogonial stem cells (SSCs) still exist in the basal compartment of the seminiferous tubules and the blood-testis barrier (BTB) formed by the interaction of neighbor Sertoli cells (SCs) is incomplete in NOA patients with spermatogenic maturation arrest. The adhesions between SCs and germ cells (GCs) are also broken in NOA patients. Meanwhile, the expression level of geranylgeranyl diphosphate synthase (Ggpps), a key enzyme in mevalonate metabolic pathway, is lower in NOA patients than that in obstructive azoospermia (OA) patients. After Ggpps deletion specifically in SCs, the mice are infertile and the phenotype of the SC-Ggpps-/- mice is similar to the NOA patients, where the BTB and the SC-GC adhesions are severely destroyed. Although SSCs are still found in the basal compartment of the seminiferous tubules, fewer mature spermatocyte and spermatid are found in SC-Ggpps-/- mice. Further examination suggests that the defect is mediated by the aberrant protein isoprenylation of RhoA and Ras family after Ggpps deletion. The exciting finding is that when the knockout mice are injected with berberine, the abnormal cell adhesions are ameliorated and spermatogenesis is partially restored. Our data suggest that the reconstruction of disrupted BTB is an effective treatment strategy for NOA patients with spermatogenic maturation arrest and hypospermatogenesis.

Spermatogenesis improved by suppressing the high level of endogenous gonadotropins in idiopathic non-obstructive azoospermia: a case control pilot study.

BACKGROUND: Elevated plasma gonadotropins were associated with desensitization of Sertoli and Leydig cells in the male testis. Testis spermatogenesis ability would be improved via inhibiting high endogenous gonadotropin in patients with severe oligozoospermia. Whether it would be beneficial for non-obstructive azoospermia (NOA) patients was still unclear. METHODS: Goserelin, a gonadotropin releasing hormone agonist (GnRHα) was used to suppress endogenous gonadotropin levels (gonadotropin reset) in the NOA patients, improving the sensitization of the Sertoli and Leydig cells. Then human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG) were injected to stimulate them to ameliorate the ability of testicular spermatogenesis. The main outcome measure was the existence of spermatozoa in the semen or by testicular sperm extraction (TESE). Elevation of inhibin B and/or ameliorative expression pattern of ZO-1 was the secondary objective. RESULTS: A total of 35 NOA men who failed to retrieve sperm via TESE were enrolled. Among these, 10 patients without treatment were selected as control group and secondary TESE was performed 6 months later. Of the 25 treated men, inhibin B was elevated in 11 patients in the first 4 weeks (Response group), while only 5 patients had constant increase in the following 20 weeks (Response group 2). Of the 5 men, 2 men acquired sperm (Response group 2B), while 3 failed (Response group 2A). Immunofluorescence of mouse vasa homologue (MVH) and ZO-1 showed that both positive MVH signals and ZO-1 expression were significantly increased in the Response group 2, but only Response group 2B showed ameliorative ZO-1 distribution. CONCLUSIONS: Gonadotropin reset, a new therapeutic protocol with GnRHα, was able to improve the ability of testicular spermatogenesis in the NOA patients through restoring the sensitivity of Sertoli and Leydig cells, which were reflected by elevated inhibin B and ameliorative ZO-1 expression and distribution. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02544191 .

GGPP-Mediated Protein Geranylgeranylation in Oocyte Is Essential for the Establishment of Oocyte-Granulosa Cell Communication and Primary-Secondary Follicle Transition in Mouse Ovary.

Folliculogenesis is a progressive and highly regulated process, which is essential to provide ova for later reproductive life, requires the bidirectional communication between the oocyte and granulosa cells. This physical connection-mediated communication conveys not only the signals from the oocyte to granulosa cells that regulate their proliferation but also metabolites from the granulosa cells to the oocyte for biosynthesis. However, the underlying mechanism of establishing this communication is largely unknown. Here, we report that oocyte geranylgeranyl diphosphate (GGPP), a metabolic intermediate involved in protein geranylgeranylation, is required to establish the oocyte-granulosa cell communication. GGPP and geranylgeranyl diphosphate synthase (Ggpps) levels in oocytes increased during early follicular development. The selective depletion of GGPP in mouse oocytes impaired the proliferation of granulosa cells, primary-secondary follicle transition and female fertility. Mechanistically, GGPP depletion inhibited Rho GTPase geranylgeranylation and its GTPase activity, which was responsible for the accumulation of cell junction proteins in the oocyte cytoplasm and the failure to maintain physical connection between oocyte and granulosa cells. GGPP ablation also blocked Rab27a geranylgeranylation, which might account for the impaired secretion of oocyte materials such as Gdf9. Moreover, GGPP administration restored the defects in oocyte-granulosa cell contact, granulosa cell proliferation and primary-secondary follicle transition in Ggpps depletion mice. Our study provides the evidence that GGPP-mediated protein geranylgeranylation contributes to the establishment of oocyte-granulosa cell communication and then regulates the primary-secondary follicle transition, a key phase of folliculogenesis essential for female reproductive function.

Alteration of protein prenylation promotes spermatogonial differentiation and exhausts spermatogonial stem cells in newborn mice.

Spermatogenesis in adulthood depends on the successful neonatal establishment of the spermatogonial stem cell (SSC) pool and gradual differentiation during puberty. The stage-dependent changes in protein prenylation in the seminiferous epithelium might be important during the first round of spermatogenesis before sexual maturation, but the mechanisms are unclear. We have previous found that altered prenylation in Sertoli cells induced spermatogonial apoptosis in the neonatal testis, resulting in adult infertility. Now we further explored the role of protein prenylation in germ cells, using a conditional deletion of geranylgeranyl diphosphate synthase (Ggpps) in embryonic stage and postmeiotic stage respectively. We observed infertility of Ggpps(-/-) Ddx4-Cre mice that displayed a Sertoli-cell-only syndrome phenotype, which resulted from abnormal spermatogonial differentiation and SSC depletion during the prepubertal stage. Analysis of morphological characteristics and cell-specific markers revealed that spermatogonial differentiation was enhanced from as early as the 7(th) postnatal day in the first round of spermatogenesis. Studies of the molecular mechanisms indicated that Ggpps deletion enhanced Rheb farnesylation, which subsequently activated mTORC1 and facilitated spermatogonial differentiation. In conclusion, the prenylation balance in germ cells is crucial for spermatogonial differentiation fate decision during the prepubertal stage, and the disruption of this process results in primary infertility.