GTPases Arf5 and Arl2 function partially distinctly during oocyte meiosis.

Mammalian female meiosis must be tightly regulated to produce high-quality mature oocytes for subsequent regular fertilization and healthy live birth of the next generation. GTPases control many important signal pathways involved in diverse cellular activities. ADP-ribosylation factor family members (Arfs) in mice possess GTPase activities, and some members have been found to function in meiosis. However, whether other Arfs play a role in meiosis is unknown. In this study, we found that Arl2 and Arf5 are the richest among Arfs in mouse oocytes, and they are more abundant in oocytes than in granular cells. Furthermore, Arl2 and Arf5 depletion both impeded meiotic progression, but by affecting spindles and microfilaments, respectively. Moreover, Arl2 and Arf5 depletion both significantly increased regular reactive oxygen species levels and decreased mitochondrial membrane potential and autophagy, indicating that oocyte quality was damaged by Arl2 and Arf5 depletion. These results suggest that Arl2 and Arf5 are two novel essential GTPases required for oocyte meiosis and quality control.

GPR30 receptor promotes preoperative anxiety-induced postoperative hyperalgesia by up-regulating GABAA-α4ß1δ subunits in periaqueductal gray in female rats.

BACKGROUND: G-protein coupled estrogen receptor 30 (GPR30) was proved the specific estrogen receptor relating to mechanical hyperalgesia. Studies have shown that the GABAA receptor subunits α4, ß1, and δ in the periaqueductal gray (PAG) neurons promote the descending facilitation system. This study inquired into whether and how GPR30 and GABAA-α4ß1δ in the PAG promote preoperative anxiety-induced postoperative hyperalgesia in female rats. METHODS: All the female rats were subjected to the single prolonged stress (SPS) to stimulate preoperative anxiety. Subsequently, mechanical allodynia was evaluated before and after the incision, based on the paw withdrawal mechanical threshold (PWMT). The selective GPR30 agonist G1 and antagonist G15 were locally microinjected into the PAG. The expression of GPR30, protein kinase A (PKA), and GABAA receptor subunits α4, ß1, and δ in the PAG neurons were detected using western blotting and immunofluorescence. RESULTS: Behavioral testing revealed that Group S and Group I decreased the nociceptive threshold levels of PWMT in female rats. PWMT in Group S + I decreased more than that of Group S and Group I. Further, results of western blotting showed the expression of GPR30, PKA, and GABAA α4, ß1, and δ subunits significantly up-regulated in Group S + I, and immunofluorescence indicated that the neurons of PAG in Group S + I appeared simultaneously immunopositive for GPR30 and GABAA α4, ß1, and δ receptors. After microinjection of G1 into the PAG, female rats with plantar incision continued to exhibit significant hyperalgesia until postoperative 48 h. On the other hand, microinjection of G15 with SPS and plantar incision procedure relieved postoperative hyperalgesia in female rats. Western blotting demonstrated that intra-PAG injection of G15 markedly decreased the GPR30, PKA, and GABAA α4, ß1, and δ levels in Group G15 + I. CONCLUSIONS: Our results indicate that the GPR30-PKA-GABAAα4ß1δ pathway in the PAG promotes preoperative anxiety-induced postoperative hyperalgesia in female rats. This mechanism might be a potential novel therapeutic target for hyperalgesia in females.

Pediatric Langerhans cell histiocytosis of the temporal bone: clinical and imaging studies of 27 cases.

BACKGROUND: We aimed to evaluate the clinical and imaging presentations of Langerhans cell histiocytosis (LCH) in the pediatric temporal bone. METHODS: This retrospective study included 27 pediatric cases with pathological confirmed LCH of the temporal bone. The clinical and imaging features of the cases were analyzed. The involvement of ossicular chain and otic capsule was also evaluated. RESULTS: A total of 38 lesions (27 cases) with 11 bilateral involvement were identified. For the 27 cases, the most common complaint was periauricular swelling (12/27, 44.4%), followed by otorrhea (9/27, 33.3%) and otalgia (5/27, 18.2%). The mastoid process was the most common involved subsite (31/38, 81.6%) among the 38 lesions. Ten (26.3%, 10/38) lesions belonged to the group of the diffuse involvement, 22 (57.9%, 22/38) were divided into the group of partial involvement and six (15.8%,6/38) localized lesions with punched-out appearance. Erosion of ossicular chains and otic capsule were found in three and seven lesions respectively. CONCLUSION: The results indicate that the most common subsite for LCH of the pediatric temporal bone was the mastoid process. The location and extent of pediatric LCH of the temporal bone varied a lot between each other. The ossicular chains usually remain intact and the erosion of otic capsule can occur in some lesions.

Lamin A/C proteins in the spermatid acroplaxome are essential in mouse spermiogenesis.

Spermiogenesis is a complex process of terminal differentiation that is necessary to produce mature sperm. Using protein expression profiles of mouse and human testes generated from our previous studies, we chose to examine the actions of lamin A/C in the current investigation. Lamin A and lamin C are isoforms of the A-type lamins that are encoded by the LMNA gene. Our results showed that lamin A/C was expressed in the mouse testis throughout the different stages of spermatogenesis and in mature sperm. Lamin A/C was also expressed in mouse haploid germ cells and was found to be localized to the acroplaxome in spermiogenesis, from round spermatids until mature spermatozoa. The decreased expression of lamin A/C following injections of siRNA against Lmna caused a significant increase in caudal sperm head abnormalities when compared with negative controls. These abnormalities were characterized by increased fragmentation of the acrosome and abnormal vesicles, which failed to fuse to the developing acrosome. This fragmentation also caused significant alterations in nuclear elongation and acrosome formation. Furthermore, we found that lamin A/C interacted with the microtubule plus-end-tracking protein CLIP170. These results suggest that lamin A/C is critical for proper structural and functional development of the sperm acrosome and head shape.

Tuning of Ti-doped mesoporous silica for highly efficient enrichment of phosphopeptides in human placenta mitochondria.

Extraction of phosphopeptides from rather complex biological samples has been a tough issue for deep and comprehensive investigation into phosphoproteomes. In this paper, we present a series of Ti-doped mesoporous silica (Ti-MPS) materials with tunable composition and controllable morphology for highly efficient enrichment of phosphopeptides. By altering the molar ratio of silicon to titanium (Si/Ti) in the precursor, the external morphology, Ti content, internal long-rang order, and surface area of Ti-MPS were all modulated accordingly with certain regularity. Tryptic digests of standard phosphoprotein α- and ß-casein were employed to assess the phosphopeptide enrichment capability of Ti-MPS series. At the Si/Ti molar ratio of 8:1, the optimum enrichment performance with admirable sensitivity and capacity was achieved. The detection limit for ß-casein could reach 10 fmol, and 15 phosphopeptides from the digest of α-casein were resolved in the spectrum after enrichment, both superior to the behavior of commercial TiO(2) materials. More significantly, for the digest of human placenta mitochondria, 396 phosphopeptides and 298 phosphoproteins were definitely detected and identified after enrichment with optimized Ti-MPS material, demonstrating its remarkable applicability for untouched phosphoproteomes. In addition, this research also opened up a universal pathway to construct a composition-tunable functional material in pursuit of the maximum performance in applications.

Gm364 coordinates MIB2/DLL3/Notch2 to regulate female fertility through AKT activation.

Many integral membrane proteins might act as indispensable coordinators in specific functional microdomains to maintain the normal operation of known receptors, such as Notch. Gm364 is a multi-pass transmembrane protein that has been screened as a potential female fertility factor. However, there have been no reports to date about its function in female fertility. Here, we found that global knockout of Gm364 decreased the numbers of primordial follicles and growing follicles, impaired oocyte quality as indicated by increased ROS and γ-H2AX, decreased mitochondrial membrane potential, decreased oocyte maturation, and increased aneuploidy. Mechanistically, Gm364 directly binds and anchors MIB2, a ubiquitin ligase, on the membrane. Subsequently, membrane MIB2 ubiquitinates and activates DLL3. Next, the activated DLL3 binds and activates Notch2, which is subsequently cleaved within the cytoplasm to produce NICD2, the intracellular active domain of Notch2. Finally, NICD2 can directly activate AKT within the cytoplasm to regulate oocyte meiosis and quality.

Clinical and magnetic resonance imaging features of 14 patients with trilateral retinoblastoma.

BACKGROUND: Our study aimed to comprehensively investigate the age of onset, magnetic resonance imaging (MRI) features, and prognosis of children with trilateral retinoblastoma (TRB). METHODS: We included 14 patients with TRB diagnosed or followed up in our hospital. The age of onset and MRI features of the intraocular tumor and intracranial lesions were evaluated. A follow-up study was also conducted. RESULTS: A total of 11 participants were diagnosed with concurrent TRB at the age of 11.1±7.4 months, and 3 participants had late-onset TRB at age 37±19.1 months. The incidence of TRB with unilateral eye involvement was 7.1% (1/14). The intraocular tumors showed intense enhancement in contrast-enhanced T1-weighted images (WI) and significant diffusion restriction in diffusion WI (DWI) with an apparent diffusion coefficient (ADC) of (0.619±0.22)×10-3 mm2/s. The intracranial lesions showed similar DWI aspects with an ADC value of (0.680±0.206)×10-3 mm2/s. Therapeutically, 8 participants had a period of intraocular tumor stabilization and significant intracranial lesion volume reduction after chemotherapy, and 6 participants had given up treatment. Only 2 participants who simultaneously received high-dose chemotherapy and autologous hematopoietic stem cell rescue were still alive with no recurrence at 24 and 54 months of follow-up. The 1-, 2-, and 3-year overall survival (OS) rates were 80%, 18.75%, and 12.5%, respectively. CONCLUSIONS: Patients with unilateral or bilateral RB can develop TRB. The intraocular and intracranial tumors showed slightly different ADC values. High-dose chemotherapy, combined with stem cell rescue can significantly improve survival. A long term and scheduled follow-up before 60 months of age is necessary for screening later-onset TRB patients.

Rat BAT xenotransplantation recovers the fertility and metabolic health of PCOS mice.

Polycystic ovarian syndrome (PCOS) is a major severe ovary disorder affecting 5-10% of reproductive women around the world. PCOS can be considered a metabolic disease because it is often accompanied by obesity and diabetes. Brown adipose tissue (BAT) contains abundant mitochondria and adipokines and has been proven to be effective for treating various metabolic diseases. Recently, allotransplanted BAT successfully recovered the ovarian function of PCOS rat. However, BAT allotransplantation could not be applied to human PCOS; the most potent BAT is from infants, so voluntary donors are almost inaccessible. We recently reported that single BAT xenotransplantation significantly prolonged the fertility of aging mice and did not cause obvious immunorejection. However, PCOS individuals have distinct physiologies from aging mice; thus, it remains essential to study whether xenotransplanted rat BAT can be used for treating PCOS mice. In this study, rat-to-mouse BAT xenotransplantation, fortunately, did not cause severe rejection reaction, and significantly recovered ovarian functions, indicated by the recovery of fertility, oocyte quality, and the levels of multiple essential genes and kinases. Besides, the blood biochemical index, glucose resistance, and insulin resistance were improved. Moreover, transcriptome analysis showed that the recovered PCOS F0 mother following BAT xenotransplantation could also benefit the F1 generation. Finally, BAT xenotransplantation corrected characteristic gene expression abnormalities found in the ovaries of human PCOS patients. These findings suggest that BAT xenotransplantation could be a novel therapeutic strategy for treating PCOS patients.

Proteomic analysis of proteins involved in spermiogenesis in mouse.

Spermiogenesis is a unique process in mammals during which haploid round spermatids mature into spermatozoa in the testis. Its successful completion is necessary for fertilization and its malfunction is an important cause of male infertility. Here, we report the high-confidence identification of 2116 proteins in mouse haploid germ cells undergoing spermiogenesis: 299 of these were testis-specific and 155 were novel. Analysis of these proteins showed many proteins possibly functioning in unique processes of spermiogenesis. Of the 84 proteins annotated to be involved in vesicle-related events, VAMP4 was shown to be important for acrosome biogenesis by in vivo knockdown experiments. Knockdown of VAMP4 caused defects of acrosomal vesicle fusion and significantly increased head abnormalities in spermatids from testis and sperm from the cauda epididymis. Analysis of chromosomal distribution of the haploid genes showed underrepresentation on the X chromosome and overrepresentation on chromosome 11, which were due to meiotic sex chromosome inactivation and expansion of testis-expressed gene families, respectively. Comparison with transcriptional data showed translational regulation during spermiogenesis. This characterization of proteins involved in spermiogenesis provides an inventory of proteins useful for understanding the mechanisms of male infertility and may provide candidates for drug targets for male contraception and male infertility.

The 5.8S pre-rRNA maturation factor, M-phase phosphoprotein 6, is a female fertility factor required for oocyte quality and meiosis.

OBJECTIVES: M-phase phosphoprotein 6 (MPP6) is important for 5.8S pre-rRNA maturation in somatic cells and was screened as a female fertility factor. However, whether MPP6 functions in oocyte meiosis and fertility is not yet known. We aimed to address this. MATERIALS AND METHODS: Mouse oocytes with surrounded nucleus (SN) or non-surrounded nucleus (NSN) were used for all experiments. Peptide nanoparticle-mediated antibody transfection was used to deplete MPP6. Immunofluorescence staining, immunohistochemistry and live tracker staining were used to examine MPP6 localization and characterize phenotypes after control or MPP6 depletion. High-fidelity PCR and fluorescence in situ hybridization (FISH) were used to examine the localization and level of 5.8S rRNAs. Western blot was used to examine the protein level. MPP6-EGFP mRNA microinjection was used to do the rescue. RESULTS: MPP6 was enriched within ovaries and oocytes. MPP6 depletion significantly impeded oocyte meiosis. MPP6 depletion increased 5.8S pre-rRNA. The mRNA levels of MPP6 and 5.8S rRNA decreased within ageing oocytes, and MPP6 mRNA injection partially increased 5.8S rRNA maturation and improved oocyte quality. CONCLUSIONS: MPP6 is required for 5.8S rRNA maturation, meiosis and quality control in mouse oocytes, and MPP6 level might be a marker for oocyte quality.

Anxiety-induced hyperalgesia in female rats is mediated by cholecystokinin 2 receptor in rostral ventromedial medulla and spinal 5-hydroxytryptamine 2B receptor.

BACKGROUND: Preoperative anxiety is associated with postoperative hyperalgesia; however, few studies have investigated the mechanism underlying this association in female surgical patients. Research has suggested that ON cells in the rostral ventromedial medulla (RVM) receive nerve impulses via cholecystokinin 2 (CCK2) receptors, facilitating hyperalgesia. Additionally, the downstream serotonergic projection system from the RVM to the spinal cord has a dual regulating effect on pain responses, and the 5-hydoxytryptophan 2B (5-HT2B) receptor in spinal dorsal horn neurons is critically involved in mechanical allodynia. METHODS: Ovariectomized rats were treated with estrogen replacement, single prolonged stress (SPS), and plantar incision. Various receptor agonists and antagonists were then administered into the RVM and spinal cord to study the mechanism underlying postoperative hyperalgesia caused by preoperative anxiety in female rats. RESULTS: Behavioral testing revealed that preoperative SPS induced postoperative hyperalgesia, as well as the expression of the CCK2 receptor in the RVM and the expression of the 5-HT2B receptor, protein kinase Cγ (PKCγ), and phosphorylation of the N-methyl-d-aspartate receptor1 (p-NR1) in the spinal cord increased confirmed by Western blot. RVM microinjection of the CCK2 receptor agonist CCK-8 and intrathecal injection of the 5-HT2B receptor agonist BW723C86 both produced hyperalgesia in female rats after plantar incision, whereas the CCK2 receptor antagonist YM022, the 5-HT2B receptor antagonist RS127445, and the PKCγ inhibitor C37H65N9O13 decreased the rats' sensitivity to the same stimulus. Additionally, electrophysiological analysis suggested that activation of the 5-HT2B receptor increased the whole-cell current (IBa) in superficial dorsal horn neurons through the PKCγ pathway. CONCLUSION: Our study demonstrated that preoperative anxiety-induced postoperative hyperalgesia in female rats is associated with descending pain pathways. The CCK2 receptor in the RVM and spinal 5-HT2B receptor may play a role in this hyperalgesic effect.

Repeated restraint stress increases seizure susceptibility by activation of hippocampal endoplasmic reticulum stress.

A growing body of evidence suggests that stress triggers a variety of pathophysiological responses. Recent studies show that stress produces enduring effects on structure and function of hippocampus, which is one of the most important structures involved in epilepsy. In the present study, we determined the effect of repeated restraint stress exposure on the susceptibility of pentylenetetrazole (PTZ)-induced seizures and the possible mechanisms involved using a rodent model. Our results show that mice subjected to repeated restraint stress exhibited shorter latency to PTZ-induced tonic-clonic seizures and higher seizure severity, suggesting chronic restraint stress increases seizure susceptibility. Following repeated restraint stress, we observed an increased level of endoplasmic reticulum (ER) stress as well as oxidative stress in the hippocampus. Moreover, our results show that chronic restraint stress exposure causes neuron loss in the hippocampus. Inhibition of ER stress with chemical chaperone, tauroursodeoxycholic acid (TUDCA), however, protects against chronic restraint stress-induced neuron loss, suggesting repeated restraint stress-induced neuronal degeneration is dependent on ER stress activation. On the other hand, inhibition of ER stress with TUDCA suppresses restraint stress-induced seizure susceptibility. Taken together, these results indicate that repeated restraint stress increases seizure susceptibility by activation of hippocampal ER stress and ER stress mediated oxidative stress and neurodegeneration. Thus, attenuating ER stress may serve as a potential therapeutic strategy targeted to block stress-induced seizure activities.

Neuronal Nitric Oxide Synthase Contributes to PTZ Kindling-Induced Cognitive Impairment and Depressive-Like Behavior.

Epilepsy is a chronic neurological disease which is usually associated with psychiatric comorbidities. Depsression and cognition impairment are considered to be the most common psychiatric comorbidities in epilepsy patients. However, the specific contribution of epilepsy made to these psychiatric comorbidities remains largely unknown. Here we use pentylenetetrazole (PTZ) kindling, a chronic epilepsy model, to identify neuronal nitric oxide synthase (nNOS) as a signaling molecule triggering PTZ kindling-induced cognitive impairment and depressive-like behavior. Furthermore, we identified that both hippocampal MAPK and PI3K/AKT signaling pathways were activated in response to PTZ kindling, and the increased MAPK and PI3K/AKT signaling activation was paralleled by increased level of reactive oxygen species (ROS) in the hippocampus. However, the PTZ kindling-induced MAPK, PI3K/AKT signaling activities and the ROS level were attenuated by nNOS gene deficiency, suggesting that nNOS may act through ROS-mediated MAPK and PI3K/AKT signaling pathways to trigger cognition deficit and depressive-like behavior in PTZ-kindled mice. Our findings thus define a specific mechanism for chronic epilepsy-induced cognitive impairment and depressive-like behavior, and identify a potential therapeutic target for psychiatric comorbidities in chronic epilepsy patients.

The value of MRI in evaluating the efficacy and complications with the treatment of intra-arterial chemotherapy for retinoblastoma.

Retinoblastoma is the most common intraocular malignant tumor of childhood. Intra-arterial chemotherapy (IAC) is a recently popularized technique used for the treatment of retinoblastoma, to decrease mortality, increase preservation of the eye, and prevent blindness. Along with the extensive use of IAC, it is important to apply noninvasive examination methods to assess the activity of the tumor and the risk factors for disease dissemination without histopathological confirmation. There are few studies that have assessed the value of magnetic resonance imaging (MRI) in evaluating the efficacy and complications of IAC for retinoblastoma. We retrospectively analyzed the MRI features of 60 patients with unilateral retinoblastoma given the primary treatment of IAC from January 2014 to February 2016 in our hospital. Our study showed that MRI could well assess the decreased activity of the tumor after IAC, presenting with diminished tumor size, increased apparent diffusion coefficient (ADC) values (from 0.94 ± 0.24 × 10-3 mm2/s to 2.24 ± 0.40 × 10-3 mm2/s), and a reduced degree of enhancement of the tumor. Our study also showed that MRI can monitor the risk factors of abnormal enhancement of the postlaminar optic nerve, to avoid unnecessary enucleation. Meanwhile, the results showed that the main late complications after IAC included affected eyeball volume reduction, subretinal hemorrhage, vitreous hemorrhage, vitreous opacity, cataractous len, and choroidal vascular ischemia.

Neuronal Nitric Oxide Synthase Contributes to PTZ Kindling Epilepsy-Induced Hippocampal Endoplasmic Reticulum Stress and Oxidative Damage.

Epilepsy is one of the most common chronic neurological disorders which provoke progressive neuronal degeneration. Endoplasmic reticulum (ER) stress has recently been recognized as pivotal etiological factors contributing to epilepsy-induced neuronal damage. However, the specific contribution of epilepsy made to ER stress remains largely elusive. Here we use pentylenetetrazole (PTZ) kindling, a chronic epilepsy model, to identify neuronal nitric oxide synthase (nNOS) as a signaling molecule triggering PTZ kindling epilepsy-induced ER stress and oxidative damage. By genetic deletion of nNOS gene, we further demonstrated that nNOS acts through peroxynitrite, an important member of reactive nitrogen species, to trigger hippocampal ER stress and oxidative damage in the PTZ-kindled mice. Our findings thus define a specific mechanism for chronic epilepsy-induced ER stress and oxidative damage, and identify a potential therapeutic target for neuroprotection in chronic epilepsy patients.

NADPH oxidase activation is required for pentylenetetrazole kindling-induced hippocampal autophagy.

Growing evidence indicates that alterations in autophagy are present in a variety of neurological disorders, ranging from neurodegenerative diseases to acute neurological insults. Only recently has the role of autophagy in epilepsy started to be recognized. In this study, we used pentylenetetrazole (PTZ) kindling, which provides a model of chronic epilepsy, to investigate the involvement of autophagy in the hippocampus and the possible mechanisms involved. Our western blot results showed that autophagy-related proteins were significantly increased after the mice were fully kindled. In addition, immunofluorescence studies revealed a significant increase in the punctate accumulation of LC3 in the hippocampal CA1 region of fully PTZ-kindled mice. Consistent with the upregulation of ATG proteins and punctate accumulation of LC3 in the hippocampal CA1 region, autophagosomal vacuole formation was observed by an ultrastructural analysis, verifying the presence of a hippocampal autophagic response in PTZ-kindled mice. Increased oxidative stress has been postulated to play an important role in the pathogenesis of a number of neurological diseases, including epilepsy. In this study, we demonstrate that PTZ kindling induced reactive oxygen species (ROS) production and lipid peroxidation, which were accompanied by mitochondrial ultrastructural damage due to the activation of NADPH oxidase. Pharmacological inhibition of NADPH oxidase by apocynin significantly suppressed the oxidative stress and ameliorated the hippocampal autophagy in PTZ-kindled mice. Interestingly, pharmacological induction of autophagy suppressed PTZ-kindling progress and reduced PTZ-kindling-induced oxidative stress while inhibition of autophagy accelerated PTZ kindling progress and increased PTZ-kindling-induced oxidative stress. These results suggest that the oxidative stress induced by NADPH oxidase activation may play a pivotal role in PTZ-kindling process as well as in PTZ kindling-induced hippocampal CA1 autophagy.

Expression of Flotilin-2 and Acrosome Biogenesis Are Regulated by MiR-124 during Spermatogenesis.

MicroRNAs (miRNAs) are a class of short non-coding RNA molecules, which diversely regulate gene expression in organisms. Although the regulatory role of these small RNA molecules has been recently explored in animal spermatogenesis, the role of miR-124 in male germ cells is poorly defined. In our previous study, flotillin-2 was investigated as a novel Golgi-related protein involved in sperm acrosome biogenesis. The current study was designed to analyze the contribution of miR-124 in the regulation of flotillin-2 expression during mouse acrosome biogenesis. Luciferase assays revealed the target effects of miR-124 on flotillin-2 expression. Following intratesticular injection of miR-124 in 3-week-old male mice, quantitative real-time RT-PCR and western blot analysis were employed to confirm the function of miR-124 in regulating flotillin-2 after 48 hours. Sperm abnormalities were assessed 3 weeks later by ordinary optical microscopy, the acrosome abnormalities were also assessed by PNA staining and transmission electron microscopy. The results showed the proportion of sperm acrosome abnormalities was significantly higher than that of the control group. The expression of flotillin-2 and caveolin-1 was significantly downregulated during acrosome biogenesis. These results indicated that miR-124 could potentially play a role in caveolin-independent vesicle trafficking and modulation of flotillin-2 expression in mouse acrosome biogenesis.