Motility Activity Research Articles

Characterization of a sperm motility signalling pathway in a gonochoric coral suggests conservation across cnidarian sexual systems.

Most stony corals liberate their gametes into the water column via broadcast spawning, where fertilization hinges upon the activation of directional sperm motility. Sperm from gonochoric and hermaphroditic corals display distinct morphological and molecular phenotypes, yet it is unknown whether the signalling pathways controlling sperm motility are also distinct between these sexual systems. Here, we addressed this knowledge gap using the gonochoric, broadcast spawning coral Astrangia poculata. We found that cytosolic alkalinization of sperm activates the pH-sensing enzyme soluble adenylyl cyclase (sAC), which is required for motility. Additionally, we demonstrate for the first time in any cnidarian that sAC activity leads to protein kinase A (PKA) activation, and that PKA activity contributes to sperm motility activation. Ultrastructures of A. poculata sperm displayed morphological homology with other gonochoric cnidarians, and sAC exhibited broad structural and functional conservation across this phylum. These results indicate a conserved role for pH-dependent sAC-cAMP-PKA signalling in sperm motility across coral sexual systems, and suggest that the role of this pathway in sperm motility may be ancestral in metazoans. Finally, the dynamics of this pH-sensitive pathway may play a critical role in determining the sensitivity of marine invertebrate reproduction to anthropogenic ocean acidification.

Serum lipid profile levels and semen quality: new insights and clinical perspectives for male infertility and men's health.

Several clinical scenarios regulate the final ejaculated semen, which is pivotal to reproductive success. Sperm motility and plasma membrane fusogenic activity primarily rely on the peculiar sperm lipid composition, influenced by the patient's metabolism, genetics, nutritional, environmental status, and concomitant clinical entities such as varicocele. This study aimed to determine the relationship between serum lipid profile and testicular function (semen quality and testosterone levels). This retrospective study uses medical charts of 278 infertile men who attended andrological care between 2000 and 2019. Seminal analysis data, lipid profile, and total serum testosterone were collected. A multiple linear regression analysis was performed to evaluate the influence of the lipid parameters on the seminal variables. Statistical analyses were carried out with p ≤ 0.05 considered statistically significant. Seminal creatine kinase activity (p = 0.024) is negatively related to HDL (p = 0.032) and triglycerides (p = 0.037), while total testosterone (p < 0.0001) and seminal volume (p = 0.046) appeared both to be negatively related to triglycerides (p = 0.030 and p = 0.033, respectively). Medical advice commonly advocated to prevent endothelial dysfunction and cardiovascular disease and improve HDL-cholesterol and triglyceride levels in dyslipidemic patients should also be given to infertile men. Physicians should give patients a thorough assessment, including the blood lipid profile, hormonal status, and routine seminal examinations. We propose a more comprehensive men´s health check-up for the infertile male population, not limited to a simple evaluation of basic sperm parameters.

P-074 In-vitro vitamin D treatment on human sperm improves sperm motility, capacitation, and oocyte activation

Abstract Study question Does in-vitro vitamin D treatment on fertile and infertile human sperm improves sperm motility and alter protein markers involved in capacitation and oocyte-activation. Summary answer In-vitro vitamin D treatment enhance functionality of sperm by increase intracellular calcium ion, thus increasing sperm motility and capacitation status. What is known already Many studies have been done to investigate the relationship between vitamin D and male infertility. It is known that low serum vitamin D in human contribute to male infertility. However, the effect of in-vitro vitamin D treatment on sperm remains unclear, particularly on the effect of vitamin D following ejaculation into female reproductive tract. This includes processes involved in fertilisation such as capacitation, hyperpolarization, oocyte activation and sperm-oocyte fusion. Therefore, we examined whether in-vitro vitamin D treatment on sperm can improve the sperm parameters, hence will then improve fertilisation and pregnancy. Study design, size, duration Randomised controlled trial with prospective interventions (in-vitro vitamin D treatment). Based on power analysis, 40 subjects both fertile and infertile men who came for semen analysis were recruited at University Malaya Medical Centre from January 2022 to January 2023. All men were consented to participate in this study. Each washed semen sample were aliquoted to serve as control and receive in-vitro vitamin D treatment. Participants/materials, setting, methods Total of 40 samples were included for final analysis. All semen samples were washed and treated with 1nM vitamin D in-vitro for 45 minutes at 37ºC. Motility, intracellular calcium released, capacitation status, mitochondria membrane potential and other basic semen parameters were measured after in-vitro vitamin D treatment for fertile and infertile samples. Furthermore, immunofluorescence of sperm origin oocyte activation factor, Phospholipase C Zeta (PLCζ), was done to investigate fluorescence intensity per cell using Image J. Main results and the role of chance In both fertile and infertile sample group, total motility is increased after in-vitro vitamin D treatment (MD = 8.727%, P &amp;lt; 0.001). Fluo-4 AM stain shows there are increase of intracellular calcium release after in-vitro vitamin D, with mean difference of Corrected Total Cell Fluorescent (CTCF) for Fluo-4 AM (MD = 25043, P = 0.0228. Chlortetracyclin (CTC) test for capacitation status and immunofluorescence of phosphotyrosine shows there are increase CTCF (MD = 0.02595, P = 0.0142) after in-vitro vitamin D treatment. Staining of JC-1 shows there are more hyperpolarised mitochondria membrane in in-vitro vitamin D treatment group compare to the control group. Ratio of red to green fluorescent is 0.639 in control group and 2.22 in in-vitro vitamin D treatment group. Moreover, immunofluorescence of sperm origin oocyte activation factor, Phospholipase C Zeta (PLCζ) show in-vitro vitamin D improve the expression of these protein. Mean difference of CTCF between control and vitamin D treatment group for PLCζ is 8.013x 10-3, P = 0.0226. Thus, in-vitro vitamin D enhance functionality of sperm by increase intracellular calcium ion, thus increasing sperm capacitation status and sperm membrane hyperpolarization. Limitations, reasons for caution Although power analysis is used to calculate sample size, however bigger sample size always needed to increase the accuracy. Moreover, patients’ sample are examined and treated after 3 hours due to limited collection room in the facility. Wider implications of the findings Vitamin D can be used as a tool in in-vitro fertilisation (IVF) procedure and can be added to sperm preparation medium due to its implication in increasing capacitation and hyperpolarised sperm membrane to increase chance of successful IVF. Trial registration number not applicable

O-020 What is the recovery time for sperm parameters in men who have suffered a mild Covid-19 infection?

Abstract Study question What is the time required in men who have suffered a mild Covid-19 infection, for the seminal parameters to be restored to the pre-Covid values? Summary answer The effect of Covid-19 infection on the concentration and sperm motility is clearly confirmed and is maintained over time, at least beyond 100 days. What is known already Although several studies have been performed on the effect of SARS-CoV-2 on reproductive health in the short term vs. healthy controls, and the negative effect on sperm parameters has been demonstrated, there is a scarcity of data showing the degree to which the virus affects the semen parameters in one group of patients after coronavirus infection compared to before infection. Moreover, there are no conclusive data regarding the long-term effects of SARS-CoV-2 on semen parameters. Study design, size, duration This prospective study was carried out in 6 reproductive centers between February 2020 and October 2022 including 45 male patients with a confirmed diagnosis of mild COVID-19. For each of the semen parameters (volume, sperm count, active and total motility, normal morphology, and vitality) a standardized index was calculated by dividing the value of the parameter from the subsequent semen analysis by that from the semen analysis of the same individual prior to infection. Participants/materials, setting, methods The median age of the participants was 31 years, proving the absence of a relationship between age and any of the parameters analyzed. The post-Covid test was performed between 17 and 516 days after infection, with a median of 100 days. The time elapsed between both samples was a median of 238 days. The effect of time on the affectation of semen parameters was studied, setting 100 days as the cut-off point. Main results and the role of chance There were significant differences in semen parameters before and after COVID-19 in terms of semen volume, sperm concentration, progressive motility, total motility, and vitality which decreased significantly (p &amp;lt; 0,05) after SARS-CoV-2 infection compared to the pre-infection values. Morphology was not significantly affected. The total sperm count is the most clearly affected seminal parameter. Half of the individuals (median) obtained a total count in the second sample less than 57% of the initial one. Considering the time of spermatogenesis, the subjects were separated into two groups: those who performed the semen analysis in the first 100 days after infection and those who performed it later. No differences in any parameter were found between the two groups with U Mann-Withney test. Using the Wilcoxon test for paired samples, it was demonstrated that the deleterious effect of infection on sperm concentration and motility not only does not improve with time, but it becomes more significant in late sperm analysis, performed more than one hundred days after the infection. Limitations, reasons for caution It should be mentioned that impairment of semen parameters may not be due to a direct effect of the SARS-CoV-2 virus. There are likely to be additional factors that contribute to long-term sperm parameters decrease, but whose identity is currently unknown. Wider implications of the findings The present study revealed the long-term impacts of SARS-CoV-2 on various parameters of sperm quality even with a mild infection. Further efforts should be made to study the long-time effects of COVID-19 infection on male reproduction. Trial registration number Not Applicable

Uncharacterized and lineage-specific accessory genes within the Proteus mirabilis pan-genome landscape.

Proteus mirabilis is a Gram-negative bacterium recognized for its unique swarming motility and urease activity. A previous proteomic report on four strains hypothesized that, unlike other Gram-negative bacteria, P. mirabilis may not exhibit significant intraspecies variation in gene content. However, there has not been a comprehensive analysis of large numbers of P. mirabilis genomes from various sources to support or refute this hypothesis. We performed comparative genomic analysis on 2,060 Proteus genomes. We sequenced the genomes of 893 isolates recovered from clinical specimens from three large US academic medical centers, combined with 1,006 genomes from NCBI Assembly and 161 genomes assembled from Illumina reads in the public domain. We used average nucleotide identity (ANI) to delineate species and subspecies, core genome phylogenetic analysis to identify clusters of highly related P. mirabilis genomes, and pan-genome annotation to identify genes of interest not present in the model P. mirabilis strain HI4320. Within our cohort, Proteus is composed of 10 named species and 5 uncharacterized genomospecies. P. mirabilis can be subdivided into three subspecies; subspecies 1 represented 96.7% (1,822/1,883) of all genomes. The P. mirabilis pan-genome includes 15,399 genes outside of HI4320, and 34.3% (5,282/15,399) of these genes have no putative assigned function. Subspecies 1 is composed of several highly related clonal groups. Prophages and gene clusters encoding putatively extracellular-facing proteins are associated with clonal groups. Uncharacterized genes not present in the model strain P. mirabilis HI4320 but with homology to known virulence-associated operons can be identified within the pan-genome. IMPORTANCE Gram-negative bacteria use a variety of extracellular facing factors to interact with eukaryotic hosts. Due to intraspecies genetic variability, these factors may not be present in the model strain for a given organism, potentially providing incomplete understanding of host-microbial interactions. In contrast to previous reports on P. mirabilis, but similar to other Gram-negative bacteria, P. mirabilis has a mosaic genome with a linkage between phylogenetic position and accessory genome content. P. mirabilis encodes a variety of genes that may impact host-microbe dynamics beyond what is represented in the model strain HI4320. The diverse, whole-genome characterized strain bank from this work can be used in conjunction with reverse genetic and infection models to better understand the impact of accessory genome content on bacterial physiology and pathogenesis of infection.

Enhanced BCAT1 activity and BCAA metabolism promotes RhoC activity in cancer progression.

Increased expression of branched-chain amino acid transaminase 1 or 2 (BCAT1 and BCAT2) has been associated with aggressive phenotypes of different cancers. Here we identify a gain of function of BCAT1 glutamic acid to alanine mutation at codon 61 (BCAT1E61A) enriched around 2.8% in clinical gastric cancer samples. We found that BCAT1E61A confers higher enzymatic activity to boost branched-chain amino acid (BCAA) catabolism, accelerate cell growth and motility and contribute to tumor development. BCAT1 directly interacts with RhoC, leading to elevation of RhoC activity. Notably, the BCAA-derived metabolite, branched-chain α-keto acid directly binds to the small GTPase protein RhoC and promotes its activity. BCAT1 knockout-suppressed cell motility could be rescued by expressing BCAT1E61A or adding branched-chain α-keto acid. We also identified that candesartan acts as an inhibitor of BCAT1E61A, thus repressing RhoC activity and cancer cell motility in vitro and preventing peritoneal metastasis in vivo. Our study reveals a link between BCAA metabolism and cell motility and proliferation through regulating RhoC activation, with potential therapeutic implications for cancers.

Establishment of a cascaded microfluidic single cell analysis system for molecular and functional heterogeneity analysis of circulating tumor cells

Single cell heterogeneity analysis of circulating tumor cells (CTCs) is expected to reveal important tumor biological information for the evaluation and prediction of tumor stage and metastatic progression. Although various methods have been proposed to isolate and detect CTCs, consecutive CTCs sorting and in-situ multi-dimensional heterogeneity analysis in a cascaded system remain challenging. Here, a cascaded microfluidic chip was constructed, successively integrating a spiral structure for size-based removal of blood cells and a single cell array structure for the subsequent capture of CTCs, complementing the advantages of high throughput and high separation purity for both structures. Importantly, the molecular and functional heterogeneity analysis of CTCs can be in-situ realized and real-time observed at the single-cell level. Using this system, we captured CTCs and situ-analyzed the heterogeneity of single CTCs from different types of cancer patients, finding that mesenchymal phenotype is present in more CTCs at the advanced stage, and CTCs show active motility and division ability in metastatic patients. Therefore, the cascaded microfluidic chip offers an effective strategy for studying the molecular and functional heterogeneity of single CTCs, providing critical information for the evaluation of tumor progression and metastasis.

Triptonodiol, a Diterpenoid Extracted from Tripterygium wilfordii, Inhibits the Migration and Invasion of Non-Small-Cell Lung Cancer.

Lung cancer is the most prevalent oncological disease worldwide, with non-small-cell lung cancer accounting for approximately 85% of lung cancer cases. Tripterygium wilfordii is a traditional Chinese herb that is widely used to treat rheumatism, pain, inflammation, tumors, and other diseases. In this study, we found that Triptonodiol extracted from Tripterygium wilfordii inhibited the migration and invasion of non-small-cell lung cancer and inhibited cytoskeletal remodeling, which has not been previously reported. Triptonodiol significantly inhibited the motility activity of NSCLC at low toxic concentrations and suppressed the migration and invasion of NSCLC. These results can be confirmed by wound healing, cell trajectory tracking, and Transwell assays. We found that cytoskeletal remodeling was inhibited in Triptonodiol-treated NSCLC, as evidenced by the reduced aggregation of actin and altered pseudopod morphology. Additionally, this study found that Triptonodiol induced an increase in complete autophagic flux in NSCLC. This study suggests that Triptonodiol reduces the aggressive phenotype of NSCLC by inhibiting cytoskeletal remodeling and is a promising anti-tumor compound.

Role of Panchakarma in Male Infertility w.s.r to High Altitude Induced Asthenozoospermia: A Case Report

Background of the Study: Infertility is a diseased condition of the reproductive system which can be defined as failure to achieve a clinical pregnancy after 12 months or more of regular unprotected intercourse. It is a major health issue affecting 8%-12% of couples worldwide. Male factor contributes to 20-30%, in India the contribution rate is 23%. Asthenozoospermia is a common cause of male infertility defined as reduced sperm motility, lower than 40%. Present study is looking at the impact of chronic exposure of high altitude (environmental hypoxia) on male infertility. Hypoxia acts directly on testicular seminiferous tubules resulting in impaired quantity and quality of sperm cells. Ayurveda explains male oriented infertility under the wide classification of Ashtshukradosha and asthenozoospermia which can be correlated with Ksheena Shukra. In case of Ksheenshukrta there is predominance of vitiated Vata and Pitta. Ayurvedic literature clearly mentioned Virechan Karma in Ksheena Shukra. Aim &amp; Objectives: The main aim of this study was to evaluate the effect of high-altitude exposure on seminal parameters along with its successful management with Virechana Karma. Result: The current case study of a 29 years old male visiting the OPD section with 20% active sperm motility treated with Virechana Karma after Snehana and Swedana which resulted in the positive effect and the female partner conceived the very next month after Virechana Karma. Conclusion: The case study established the positive impact of Virechan karma in male infertility due to asthenozoospermia.

Influence of different types of tobacco smoking on seminal fluid parameters and sex hormones among males from the Gaza Strip

Background: Smoking is a global problem that negatively affects human health and fertility and the endocrine functions of the male reproductive system. Objectives: The present study aims to investigate the influence of the different types of tobacco smoking (cigarette, hookah, and electronic) on semen parameters and sex hormones in males from the Gaza Strip. Methods and Materials: A total of 206 samples were classified into 156 cases and 50 controls. The cases have been classified into three groups depending on the type of tobacco smoking (cigarette smoking "n=52", hookah "n=55", and electronic "n=49"). Semen samples were collected to investigate the semen parameters in smokers compared to non-smokers. Additionally, blood samples were also collected to assess the level of sex hormones (testosterone, follicular stimulating hormone (FSH), and luteinizing hormone (LH)). Results: A significant increase in FSH and LH levels has been found in smokers compared to non-smokers (P &lt; 0.001), while a significant decrease in testosterone level has been observed in smokers’ men compared to non-smokers (P &lt; 0.001). Significant variations have been shown in the sperm concentration (P &lt; 0.001) and the percentages of sperm total motility (P &lt; 0.001), active progressive motility (P &lt; 0.001), slow progressive motility (P = 0.004), and immotile spermatozoa (P &lt; 0.001) among the different tobacco smoking groups. Furthermore, significant variations have been found in the levels of testosterone, FSH, and LH among the different tobacco smoking groups (P &lt; 0.001). The results showed significant positive correlations between sperm concentration, total motility, active progressive motility, slow progressive motility, and testosterone level (P &lt; 0.001) in smokers. Conclusions: The different types of tobacco smoking have negative influences on semen parameters and the levels of testosterone, FSH, and LH of smokers in the Gaza Strip.

Emerging microfluidic technologies for sperm sorting

Sorting high-quality sperm with intact DNA, normal morphology, and active motility is crucial for clinically assisted reproductive technology, which influences the success of treatment and the health of offspring. Currently, microfluidic technology has been developed as a powerful platform for sperm sorting owing to its ability to manipulate fluid at the microscale and handle small samples. Specifically, microfluidic technology provides the necessary stimuli including fluid stimulus, chemical induction, and shape sift, which supports researchers in developing various sperm-sorting devices. According to the sorting principle, these devices can be divided into three categories: active sorting devices based on sperm rheological properties, passive sorting devices based on sperm physical properties, and external stimuli-induced sorting devices. Hence, we review a broad range of researches about sperm sorting with microfluidics and briefly present the properties of sperm and female reproductive tract to assist the design of microfluidic sperm sorting devices.

Bacterial development in semen stored at ambient temperature in INRA96®

Sperm motility and mitochondrial activity can be maintained at room temperature for 24h in INRA96®. However bacterial development remains possible. Furthermore, very few studies evaluated the sensitivity of bacteria to the antibiotics included in INRA96. In experiment 1, semen from 8 stallions (28 ejaculates) was diluted to 20 × 106 sperm/mL in INRA96. The total amount of bacteria, dead or alive, was evaluated using Easykit6® (IMV-Technologies) immediately after dilution, then after 24h and 48h. Bacterial concentration remained stable during 48h cooled storage in Neopor® box (Minitube): mean±sem 9.8±2 × 105, 8.5±2 × 105, 8.4±12 × 105 bacteria/mL at 0h, 24h, 48h, respectively. Bacterial concentration significantly increased after 48h at ambient temperature between 13 and 26°C: 9.7±2 × 105a, 10±2 × 105ab, 28±6 × 105b bacteria/mL at 0h, 24h, 48h, and was significantly higher at ambient temperature than in cooled samples both after 24h and 48h. However, when data were analysed individually, the significant increase occured only for 3 of the 8 stallions. In experiment 2, semen from 7 stallions with 3 ejaculates each was evaluated in duplicate after 0h, 24h and 48h at ambient temperature. Easykit6® showed a significant increase in bacterial concentration for 2 stallions. Conventional bacteriology demonstrated an increase in CFU concentration for 3 stallions including the 2 mentioned above, and a decrease for another stallion. For example, stallion S showed 8±8a, 592±298ab, 8567±4493bCFU/mL at 0h, 24h, 48h, respectively, whereas stallion L showed 1259±504b, 92±53a,117±72aCFU/mL. Sixteen bacterialspecies (38 strains) and one yeast were identified. All the strains of Enterococcus fecalis, Listeria grayi, Streptococcus zooepidemicus, and some strains of Aerococcus viridans, Aeromonas salmonicida, Pasteurella multocida, Pseudomonas sepacia, Staphylococcus hominis, Staphylococcus xylosus, were resistant to both antibiotics of INRA96: 0.028g/L penicillinG and 0.077g/L gentamicin. Ten of the 20 strains identified at 0h and resistant to those antibiotics were not observed anymore at 24h. Eighteen of the 20 strains identified at 48h were resistant to those antibiotics. Eight of them have not been detected at 0h. Candida famata and the 17 bacterial strains identified at 0h and sensitive to those antibiotics were not observed anymore at 24h.The antibiotics and amphotericin B present in INRA96 seem enough to prevent microbial development at ambient temperature, provided that micro-organisms are sensitive to those molecules. Competition occurs between strains: those sensitive to the antibiotics of the extender cannot develop during storage, whereas others, even under the level of detection in raw semen, grow during storage because they are resistant. Bacterial concentration was not significantly correlated with sperm quality (motility, morphological abnormalities). Such bacterial level may not affect spermatozoa. Whether it could induce endometritis in inseminated mares needs investigation. Financial support: IFCE scientific council (Spermicrobes).

Assessment of methods to activate epididymal sperm motility

Horse epididymal sperm has satisfactory motility activation (Mot-Act) after harvesting and cryopreservation; however, donkeys typically display poor Mot-Act despite high-plasma-membrane integrity (PMI) with high-mitochondrial-membrane-potential (ΔΨM). Mot-Act can be achieved in murine epididymal sperm with centrifugation by removal of sperm-bound seminal plasma (SP) proteins; also, in other mammals, the dilution of epididymal sperm with SP promotes Mot-Act. Dilution of cryopreserved sperm with an extender without cryoprotectants aids Mot-Act in other species. It is possible that all these methods can enhance donkey and horse epididymal Mot-Act and that there might be positive interactions between methods; however, these hypotheses have yet to be tested. Single-layer colloid-centrifugation (SLC) is a powerful tool to remove sperm-bound particles. Thus, this study sought to assess methods and potential interactions to promote Mot-Act in epididymal sperm by comparing an equid with satisfactory Mot-Act (i.e., the horse) with another poor Mot-Act (i.e., the donkey). Mot-Act, PMI, and ΔΨM were assessed by submitting horse and donkey cryopreserved sperm to re-extension (1:3) on the same egg-yolk extender used for cryopreservation (BC), also the same extender lacking CPA, and donkey- and horse-SP. The Mot-Act, PMI, and ΔΨM were carried out 10-, 30-, and 60-min after incubation. Results showed that donkey and horse epididymal sperm had lower Mot-Act, PMI, and ΔΨM when dilutedwith donkey- and horse-SP (P<0.05). Conversely, Mot-Act was similar between horse-SP, BC, and CPA (P>0.05), and it all increased after 10 min of incubation (P<0.05). PMI and ΔΨM were not different across groups in horse epididymal sperm (P>0.05). Then the same processing methods were applied before and after SLC for cryopreserved horse and donkey epididymal sperm, and then Mot-Act, PMI, and ΔΨM were assessed. The results showed that donkey epididymal sperm had higher Mot-Act when diluted in the same egg-yolk extender compared to donkey-SP or CPA (P<0.05); Horse epididymal sperm had lower PMI and ΔΨM when diluted in donkey-SP compared to BC and horse-SP (P<0.05). The sperm recovery after SLC was similar between donkey and horse epididymal sperm (P>0.05). Next, zona-pellucida binding was carried out as an in vitro functional assessment of Mot-Act, as it is necessary for fertilization. Dilution with SP from either species' sperm did not enhance zona-pellucida binding (P>0.05) as it does for other mammals. Previously, it was suggested that donkey sperm does not bind to heterologous zona-pellucida (i.e., bovine); however, herein, it did bind similarly to horse sperm (P<0.05). This study is a foundation to expand the knowledge in equine sperm biology, showing that donkey epididymal sperm can have Mot-Act enhanced using re-extension with BC, and other standard methods cannot. Also, this is the first report to demonstrate that donkey sperm binds to heterologous zona-pellucida.

The multifaceted roles of embryonic microglia in the developing brain.

Microglia are the resident immune cells of the central nervous system (CNS). Microglia originate from erythromyeloid progenitors in the yolk sac at the early embryonic stage, and these progenitors then colonize the CNS through extensive migration and proliferation during development. Microglia account for 10% of all cells in the adult brain, whereas the proportion of these cells in the embryonic brain is only 0.5-1.0%. Nevertheless, microglia in the developing brain widely move their cell body within the structure by extending filopodia; thus, they can interact with surrounding cells, such as neural lineage cells and vascular-structure-composing cells. This active microglial motility suggests that embryonic microglia play a pivotal role in brain development. Indeed, recent increasing evidence has revealed diverse microglial functions at the embryonic stage. For example, microglia control differentiation of neural stem cells, regulate the population size of neural progenitors and modulate the positioning and function of neurons. Moreover, microglia exert functions not only on neural lineage cells but also on blood vessels, such as supporting vascular formation and integrity. This review summarizes recent advances in the understanding of microglial cellular dynamics and multifaceted functions in the developing brain, with particular focus on the embryonic stage, and discusses the fundamental molecular mechanisms underlying their behavior.

Understanding the impact of varicocele on sperm capacitation

To study the relationship between the seminal sample quality of men with varicocele and sperm capacitation. Cross-sectional observational study. Academic hospital. Seventy-six men (19 control and 57 with varicocele) were analyzed. Semen samples were submitted to a discontinuous density gradient for sperm selection. Sperm capacitation was induced using a human tubal fluid medium supplemented with bovine serum albumin. After capacitation induction, the sperm were assessed by capacitation state, computer-assisted sperm motility, mitochondrial activity, membrane integrity, acrosome reaction, and intracellular oxidative stress. The capacitation period increased sperm motility, showing an increase in the average path velocity and a decrease in the straightness compared with sperm before capacitation (paired analysis). After capacitation, the rate of capacitated sperm, motility, and mitochondrial activity showed differences between groups (control and varicocele). The varicocele group showed lower mitochondrial activity and capacitation than the control group. On the other hand, no significant differences were observed in the other variables evaluated. Varicocele men showed less viable sperm and mitochondrial activity than control men after capacitation sperm. The induction of capacitation altered motility by increasing path velocity and decreasing straightness in all of the studied groups, evidencing the occurrence of hyperactivation.

Astrocytes in the spinal cord contributed to acute stress-induced gastric damage via the gap junction protein CX43

Rat restraint water-immersion stress (RWIS) is a compound stress of high intensity and is widely used to study the pathological mechanisms of stress gastric ulcers. The spinal cord, as a part of the central nervous system, plays a dominant role in the gastrointestinal tract, but whether the spinal cord is involved in rat restraint water-immersion stress (RWIS)-induced gastric mucosal damage has not been reported. In this study, we examined the expression of spinal astrocytic glial fibrillary acidic protein (GFAP), neuronal c-Fos, connexin 43 (Cx43), and p-ERK1/2 during RWIS by immunohistochemistry and Western blotting. In addition, we intrathecally injected the astrocytic toxin L-a-aminoadipate (L-AA), gap junction blocker carbenoxolone (CBX), and ERK1/2 signaling pathway inhibitor PD98059 to explore the role of astrocytes in the spinal cord in RWIS-induced gastric mucosal damage and its possible mechanism in rats. The results showed that the expression of GFAP, c-Fos, Cx43, and p-ERK1/2 was significantly elevated in the spinal cord after RWIS. Intrathecal injection of both the astrocyte toxin L-AA and the gap junction blocker CBX significantly attenuated RWIS-induced gastric mucosal damage and decreased the activation of astrocytes and neurons induced in the spinal cord. Meanwhile, the ERK1/2 signaling pathway inhibitor PD98059 significantly inhibited gastric mucosal damage, gastric motility and RWIS-induced activation of spinal cord neurons and astrocytes. These results suggest that spinal astrocytes may regulate the RWIS-induced activation of neurons via CX43 gap junctions and play a critical role in RWIS-induced gastric mucosa damage through the ERK1/2 signaling pathway.

Tumor suppressive functions of hsa‑miR‑34a on cell cycle, migration and protective autophagy in bladder cancer.

Bladder cancer (BC) cells exhibit a high basal level of autophagy activity, which contributes to the development of a protective mechanism for cellular survival against current treatments. Hsa‑microRNA‑34a (miR‑34a) presents anti‑tumor function in several types of cancer. However, the functional mechanism of miR‑34a in regulating tumor aggressiveness and protective autophagy of BC remains largely unknown. First, transfected BC cells with miR‑34a mimic exhibited LC3‑II and p62 accumulation through immunofluorescence staining. It was demonstrated that syntaxin 17 (STX17), which is required for autophagosome‑lysosome fusion, was downregulated upon miR‑34a mimic treatment. Mechanistically, miR‑34a reduced the expression of STX17 proteins that directly bind on STX17 3'‑untranslated regions and thus suppressed STX17 mRNA translation to eventually inhibit protective autophagy in BC. Cell viability and colony formation assays revealed that overexpression of miR‑34a in BC cells enhances the chemosensitivity of cisplatin, doxorubicin, epirubicin and mitomycin C. Furthermore, miR‑34a inhibited cell proliferation and triggered G0/G1 cell cycle arrest by inhibiting cyclin D1 and cyclin E2 protein expression. Moreover, miR‑34a suppressed cell motility through the downregulation of epithelial‑mesenchymal transition. In summary, miR‑34a inhibits cell proliferation, motility and autophagy activity in BC, which can benefit BC treatment.

Peperomia pellucida (L.) Kunth Piperaceae has the Expected Reproductive Toxicity of Antimalarial Drugs?

In folklore medicine, Peperomia pellucida (L.) Kunth (Piperaceae), has demonstrated a good activity in malaria treatment and this has also been proven pharmacologically. Most antimalarial agents have demonstrated negative effects on spermatogenesis. For effective development and formulation of this plant resource into a dosage form that will be well tolerated by humans, the leaf extract of the plant was therefore evaluated for reproductive toxicity associated with antimalarial drugs in male Wistar rats at 250, 500 and 1000mgkg-1 doses for it effect on the sperm characteristics as well as the histology of the testes. Rats, divided into three groups (n=6) administered with 250, 500 and 1000mgkg-1 doses of the extract and a fourth one which was the control (administered with distilled water), were used for a period of twenty days following standard procedure. The result indicated, although the normal sperm morphology was not significantly affected, a significant reduction of sperm count and active motility by 93 and 71 (p&lt;0.05) observed at 1000 mg/kg and interstitial necrosis of the testes at 500 which became severe at 1000 mg/kg. The extract was however safe at 250 mg/kg. The usual spermatotoxicty of antimalarials was therefore indicated at 500 and 1000mg/kg. The study has proven that P. pellucida leaf educed the usual antimalarial spermatotoxicity.

Alpha4 beta7 integrin controls Th17 cell trafficking in the spinal cord leptomeninges during experimental autoimmune encephalomyelitis.

Th1 and Th17 cell migration into the central nervous system (CNS) is a fundamental process in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Particularly, leptomeningeal vessels of the subarachnoid space (SAS) constitute a central route for T cell entry into the CNS during EAE. Once migrated into the SAS, T cells show an active motility behavior, which is a prerequisite for cell-cell communication, in situ reactivation and neuroinflammation. However, the molecular mechanisms selectively controlling Th1 and Th17 cell trafficking in the inflamed leptomeninges are not well understood. By using epifluorescence intravital microscopy, we obtained results showing that myelin-specific Th1 and Th17 cells have different intravascular adhesion capacity depending on the disease phase, with Th17 cells being more adhesive at disease peak. Inhibition of αLβ2 integrin selectively blocked Th1 cell adhesion, but had no effect on Th17 rolling and arrest capacity during all disease phases, suggesting that distinct adhesion mechanisms control the migration of key T cell populations involved in EAE induction. Blockade of α4 integrins affected myelin-specific Th1 cell rolling and arrest, but only selectively altered intravascular arrest of Th17 cells. Notably, selective α4β7 integrin blockade inhibited Th17 cell arrest without interfering with intravascular Th1 cell adhesion, suggesting that α4β7 integrin is predominantly involved in Th17 cell migration into the inflamed leptomeninges in EAE mice. Two-photon microscopy experiments showed that blockade of α4 integrin chain or α4β7 integrin selectively inhibited the locomotion of extravasated antigen-specific Th17 cells in the SAS, but had no effect on Th1 cell intratissue dynamics, further pointing to α4β7 integrin as key molecule in Th17 cell trafficking during EAE development. Finally, therapeutic inhibition of α4β7 integrin at disease onset by intrathecal injection of a blocking antibody attenuated clinical severity and reduced neuroinflammation, further demonstrating a crucial role for α4β7 integrin in driving Th17 cell-mediated disease pathogenesis. Altogether, our data suggest that a better knowledge of the molecular mechanisms controlling myelin-specific Th1 and Th17 cell trafficking during EAE delevopment may help to identify new therapeutic strategies for CNS inflammatory and demyelinating diseases.

PIM1 phosphorylates ABI2 to enhance actin dynamics and promote tumor invasion.

Distinguishing key factors that drive the switch from indolent to invasive disease will make a significant impact on guiding the treatment of prostate cancer (PCa) patients. Here, we identify a novel signaling pathway linking hypoxia and PIM1 kinase to the actin cytoskeleton and cell motility. An unbiased proteomic screen identified Abl-interactor 2 (ABI2), an integral member of the wave regulatory complex (WRC), as a PIM1 substrate. Phosphorylation of ABI2 at Ser183 by PIM1 increased ABI2 protein levels and enhanced WRC formation, resulting in increased protrusive activity and cell motility. Cell protrusion induced by hypoxia and/or PIM1 was dependent on ABI2. In vivo smooth muscle invasion assays showed that overexpression of PIM1 significantly increased the depth of tumor cell invasion, and treatment with PIM inhibitors significantly reduced intramuscular PCa invasion. This research uncovers a HIF-1-independent signaling axis that is critical for hypoxia-induced invasion and establishes a novel role for PIM1 as a key regulator of the actin cytoskeleton.