Time Post-activation Research Articles

Development of an activated microglia cell line model for screening lipid nanoparticle formulations of BDNF siRNA for neuropathic pain therapy

Purpose Brain-derived neurotrophic factor (BDNF) released from activated spinal microglia via the ATP-P2 × 4R-BDNF signaling pathway plays a central role in initiating pain hypersensitivity in neuropathic pain. Therefore, knockdown of BDNF expression in microglia using molecules like siRNA can be a promising strategy for neuropathic pain therapy. Our lab is developing lipidoid nanoparticles (LNP) for the delivery of BDNF siRNA (siBDNF) for treating neuropathic pain. We characterized the Spontaneously Immortalized Microglia-A9 (SIM-A9) cell line and confirmed the expression of Iba1, P2 × 4R, and BDNF under resting (non-activated) and ATP-stimulated conditions. SIM-A9 cell line was cultured with and without external stimuli, adenosine triphosphate (ATP), and/or lipopolysaccharide (LPS) by varying parameters such as the concentration of stimuli, incubation time, and cell regeneration time post-activation. The cytocompatibility of SIM-A9 cells with ATP and LPS was determined using an ATP assay. Intracellular Iba1, P2 × 4R, and BDNF protein expression in the resting and stimulated cells were characterized using western blotting, in-cell western, and immunocytochemistry. In a pilot study, SIM-A9 cells were transfected with RNAiMAX (a commercial transfection agent)/siBDNF complexes and changes in BDNF expression were evaluated using western blotting. We present two novel findings: first, SIM-A9 cells expressed P2 × 4R and BDNF proteins. Second, ATP at a safe dose showed a time-dependent increase in Iba1 and BDNF expression without intracellular toxicity. At 40 nM siRNA concentration, RNAiMAX/siBDNF complexes showed about a 15% reduction in SIM-A9 BDNF expression. In preliminary studies, LNP/siBDNF at 40 nM were well-tolerated by SIM-A9 cells, however, they only showed negligible BDNF knockdown in activated SIM-A9 cells. Currently, we are optimizing LNP formulations and transfection conditions to increase the extent of BDNF gene silencing. We have developed an ATP-activated SIM-A9 cell line model system that can be utilized for screening of neuropathic pain therapeutics.

Sperm competition, but not major histocompatibility divergence, drives differential fertilization success between alternative reproductive tactics in Chinook salmon

Post-copulatory sexual selection processes, including sperm competition and cryptic female choice (CFC), can operate based on major histocompatibility (MH) genes. We investigated sperm competition between male alternative reproductive tactics [jack (sneaker) and hooknose (guard)] of Chinook salmon (Oncorhynchus tshawytscha). Using a full factorial design, we examined invitro competitive fertilization success of paired jack and hooknose males at three time points after sperm activation (0, 15 and 60s) to test for male competition, CFC and time effects on male fertilization success. We also examined egg-mediated CFC at two MH genes by examining both the relationship between competitive fertilization success and MH divergence as well as inheritance patterns of MH alleles in resulting offspring. We found that jacks sired more offspring than hooknose males at 0s post-activation; however, jack fertilization success declined over time post-activation, suggesting a trade-off between sperm speed and longevity. Enhanced fertilization success of jacks (presumably via higher sperm quality) may serve to increase sneaker tactic competitiveness relative to dominant hooknose males. We also found evidence of egg-mediated CFC (i.e. female×male interaction) influencing competitive fertilization success; however, CFC was not acting on the MH genes as we found no relationship between fertilization success and MH II β1 or MH I α1 divergence and we found no deviations from Mendelian inheritance of MH alleles in the offspring. Our study provides insight into evolutionary mechanisms influencing variation in male mating success within alternative reproductive tactics, thus underscoring different strategies that males can adopt to attain success.

Semen quality and sperm ultrastructure of Himalayan snowtrout Schizothorax plagiostomus Heckel, 1838

The aim of this study was to characterise physical and biochemical aspects of semen as well as to investigate the fine structure of spermatozoa of the Himalayan snowtrout Schizothorax plagiostomus Heckel, 1838 using scanning and transmission electron microscopy. The species breed twice in a year and semen was collected during both seasons, i.e. from 24 males in February and March, 2015 and from 30 males in September and October, 2015. Size of the fish ranged from 13.5 to 36 cm in February-March and 12.3 to 38 cm in September-October. The mean milt volume (ml), sperm density (×1010 ml-1) and spermatocrit (%) values were 2.25±1.26, 2.22±0.53 and 78.87± 8.25 in February-March and 2.12±1.25, 2.12±0.52 and 75.54±8.23 in September-October respectively. Biochemical parameters of seminal plasma viz., total protein (g dl-1), carbohydrates (mg dl-1) and total lipids were 0.312±0.05, 1.348±0.07 and 26.4±2.23 in February-March and 0.340±0.05, 1.34±0.1 and 26.4±3.19 in September-October respectively. Scanning and transmission electron microscopy studies of sperm revealed that the sperm was composed of an ovoid shaped head without acrosome, ellipsoidal midpiece with mitochondria and tail or flagellum. Flagellum had a typical 9+2 axoneme arrangement. The mean length (μm) of head, midpiece, flagella and total length of sperm were 1.82±0.24, 0.35±0.07, 20.18±0.79 and 22.3±3 respectively. For both the breeding seasons, sperm motility decreased significantly with time post-activation.

Seasonal variation of sperm quality in Pacific oyster (Crassostrea gigas)

Seasonal changes of sperm quality which can affect sperm biological parameters throughout the breeding period, have been little studied in mollusc species. Controlling gamete quality would aid the management of gametes in hatcheries and the development of selection programs. The aim of the present study was to describe the changes in sperm quality of wild Pacific oysters through the spawning season by comparing sperm parameters at the beginning (May), middle (July) and end (October) of this period using a panel of bio-descriptors. These parameters were studied over the 2014 breeding season based on shed sperm collected after serotonin injection of wild breeders. A significantly higher percentage of motile sperm was observed at the end of the spawning season (+78% relative to the value observed at the beginning) althought a lower total number of spermatozoa was collected (−59%). The mean condition index of oysters, however, was no different between the three sampling dates. For intratesticular sperm, the increase of the percentage of motile sperm and Velocity of the Average Path (VAP) in relation to time post activation was not significantly different among sperm sampling periods, suggesting that the kinetic of the sperm maturation process was similar. Furthermore, the mean VAP observed on shed sperm did not change through the spawning season. The sub-continuous gametogenesis of Pacific oyster can help to explain why only limited consequences of sperm ageing are observed in this species. Furthermore, the effects of sperm ageing may depend on the annual reproductive pattern of Pacific oyster. Statement of relevanceThis study showed the effect of sperm ageing on sperm quality parameters. This knowledge is useful for aquaculture and would support the recent trends of mollusc farming, allowing a better management of the gametes in hatchery.

Fresh, equilibrated and post-thaw sperm quality of Brycon orbignyanus (Valenciennes, 1850) and Prochilodus lineatus (Valenciennes, 1837) treated with either salmon GnRHa and domperidone or pituitary extract

The effects of reduced doses of Ovaprim™ (GnRHa + domperidone) on sperm release of Brycon orbignyanus and Prochilodus lineatus were evaluated. Furthermore, sperm quality was compared among fresh, equilibrated and post-thaw samples. Males received a single and reduced dose of Ovaprim™ (0.125 or 0.25 ml/kg); control males received pituitary extract (cPE; 3 mg/kg). Fresh sperm was evaluated for volume, concentration, seminal plasma osmolality and seminal plasma pH. Then sperm was diluted in a freezing medium, equilibrated for 15-20 min and frozen in nitrogen vapor vessel (dry-shipper). Sperm motility was analyzed during 60 s post-activation in fresh, equilibrated and post-thaw samples. Sperm quality of males treated with Ovaprim™ (both doses) were not different from that of cPE-treated males, thus these data were pooled. In B. orbignyanus, motility was higher in fresh (99%) than in equilibrated sperm (81%); post-thaw motility dropped to 42%. In P. lineatus, motility was similar in fresh (99%) and equilibrated sperm (92%); post-thaw motility was 73%. Motility decreased as a function of time post-activation, and this decrease was significant after 60 s in fresh and equilibrated sperm, and as soon as 30 s in post-thaw sperm, in both species. Ovaprim™ at 1/4 of the recommended dose can successfully replace cPE.

Effects of tetrabrombisphenol A on DNA integrity, oxidative stress, and sterlet (Acipenser ruthenus) spermatozoa quality variables

The sperm of sterlet (Acispenser ruthenus) was used to investigate the effect of the xenobiotic tetrabrombisphenol A (TBBPA) on sperm quality variables (ATP content, spermatozoa motility, and velocity), DNA integrity, and oxidative stress indices. Sperm was diluted to obtain a spermatozoa density of 5 × 10(8) cells/mL and exposed for 2 h to final concentrations of TBBPA (0.5, 1.75, 2.5, 5, and 10 μg/L). The oxidative stress indices, including lipid peroxidation, carbonyl derivatives of proteins, and antioxidant activity were significantly higher with increased concentrations of TBBPA. There was significantly less intracellular ATP in sperm samples at TBBPA concentrations of 2.5 μg/L and above. Spermatozoa velocity and percent motile sperm were significantly lower at each sampling time post-activation compared to controls. DNA damage expressed as percent DNA in Tail and Olive Tail moment was significantly higher with exposures ≥2.5 μg/L TBBPA. The results demonstrated that TBBPA and other xenobiotics can induce reactive oxygen species stress in fish spermatozoa, which could impair the sperm quality, DNA integrity, ATP content, and the antioxidant defense system. This study confirmed that fish spermatozoa can be used in in vitro assays for monitoring residual pollution in aquatic environments.

Effect of sampling location, release technique and time after activation on the movement characteristics of scallop (Pecten maximus) sperm

Sperm characteristics of scallops have not been well described in the scientific literature. The effects of sperm release technique (thermal shock versus serotonin injection), of sperm collection technique (testis sampling ver- sus serotonin injection), of sperm sampling location along the genital tract, of in vitro sperm maturation, and of time post activation on scallop sperm characteristics were assessed in the present work. Whatever sperm release technique used, no significant differences were observed regarding the percentage of motile spermatozoa and the velocity of the average path (VAP). Compared to testicular sperm, a higher percentage of motile spermatozoa, VAP and intracellular adenosine triphosphate (ATP) content were observed for sperm shed after serotonin injection. From the distal part of testes up to the gonopore, an increase of the percentage of motile spermatozoa and VAP was assessed, suggesting a sperm 'maturation process' along the genital ducts. A higher increase in the percentage of motile sperm was recorded during a 5 min incubation of testicular sperm in seawater containing 2 mM serotonin and seawater containing 10 mM caffein compared to seawater (control). In addition, a higher VAP was assessed, incubating testicular sperm in caf- fein, compared to control or serotonin. Then, the percentage of motile spermatozoa, VAP and intracellular ATP content exhibited a progressive reduction during the 10 h swimming period. Mean values of the percentage of motile spermato- zoa, VAP, sperm track linearity (LIN) and intracellular ATP content recorded at the beginning of the movement period for sperm samples collected after intragonadal serotonin injection, were 82 ± 7%, 162 ± 15 µ ms −1 ,0 .33 ± 0.12 and 212 ± 133 nmol ×10 −9 spermatozoa (n = 9 males), respectively. The present study confirms the existence of a sperm maturation process along scallop genital ducts. In addition, the cessation of scallop sperm movement can be explained by the exhaustion of ATP content at the end of the movement phase.

Role of Ooplasm in Nuclear and Nucleolar Remodeling of Intergeneric Somatic Cell Nuclear Transfer Embryos during the First Cell Cycle

Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one-cell stage embryos were processed at different points in time post activation (2 hpa, 4 hpa, 8 hpa, and 12 hpa) for detailed nuclear and nucleolar analysis by TEM, and immunofluorescence for visualization of nucleolar proteins related to transcription (UBF) and processing (fibrillarin). Bovine and porcine intergeneric SCNT embryos were compared to their parthenogenetic counterparts to assess the effects of the introduced somatic cell. Despite the absence of morphological remodeling (premature chromatin condensation, nuclear envelope breakdown), reconstructed embryos showed nuclear and nucleolar precursor body (NPB) morphology similar to the host ooplasm, which, together with detected posttranslational activity of somatic cell introduced into the bovine ooplasm, suggests a universal function of ooplasmic factors. However, the lack of distinct UBF localization in intergeneric embryos indicates failures in sequence-specific interactions between the ooplasm and chromatin of another genus. In conclusion, the results demonstrate a possible reason why the intergeneric SCNT embryos never reached the full term.

Changes in motility, ATP content, morphology and fertilisation capacity during the movement phase of tetraploid Pacific oyster (Crassostrea gigas) sperm

Changes in sperm features during the movement phase are especially interesting to study in external fertilization species whose sperm duration movement is long because this implies a significant adaptation of moving cells to the external medium. This study describes the changes in tetraploid Pacific oyster sperm characteristics in relation to time post activation. Sperm individually collected on three tetraploid males were activated in seawater. Their features were analysed over a 24h period and compared to a sperm pool collected on three diploid males as a reference. The percentage of motile spermatozoa, the intracellular ATP content, and the fine structure of spermatozoa were studied in relation to time post activation. Furthermore, the fertilisation capacity of sperm individually collected on five diploid males was assessed after 1 and 24h post activation. A forward progressive movement was maintained for at least a 20h duration. Compared to diploid males, the percentage of motile spermatozoa was lower in tetraploid males. The intracellular ATP concentration was higher in spermatozoa from tetraploid males than in spermatozoa from diploid males. A decrease in ATP content was observed in the first 6h post activation and severe alterations were observed in sperm morphology after 24h. Then, a lower fertilisation capacity of sperm from diploid males was observed at the end of the movement phase. The cessation of Pacific oyster sperm motility was unlikely caused by ATP consumption as ATP concentration was still high at the end of sperm movement but rather caused by drastic changes in sperm morphology. Compared to sperm collected on diploid males, the lower quality of sperm from tetraploid males was emphasized by a shorter movement duration and deeper morphological alterations at the end of the movement phase.

Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua

The objective was to investigate changes, throughout the spawning season, in body size attributes and quantitative semen characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua L. Sperm velocity increased significantly throughout the spawning season of cod from both origins. Curvilinear velocity (VCL; 30 sec post-activation) increased from 78.9 ± 6.5 to 128.2 ± 6.5 μm/sec (mean ± SEM) between the beginning and end of the spawning season, respectively, for wild-caught cod, whereas for cultivated fish, it increased from 26.6 ± 2.4 to 48.9 ± 3.1 μm/sec between January and March. Spermatocrit did not undergo a significant seasonal change in wild-caught cod but did thicken for cultivated cod (24.6 ± 4.2% in January to 40.5 ± 4.4% in April; P < 0.01). Sperm head area, perimeter, length, and width declined significantly at the end of the spawning season of cod from both origins (all P values < 0.01). Seminal plasma osmolality and Na + ion concentration followed a dome-shaped function through the spawning season for both wild-caught and cultivated cod (P < 0.05). For cultivated cod, seminal plasma pH was significantly lower at the start of the spawning season (P < 0.001), whereas Ca 2+ increased then decreased (P < 0.05). Body size attributes, spermatocrit, and seminal plasma constituents had significant relationships with sperm activity variables. These relationships varied as a function of time post-activation, month, and fish origin. Our findings may be used to (i) assess spermiation stage without killing males; (ii) optimize semen collection for hatchery production; (iii) characterize the potential impact of farming on sperm quality; and (iv) improve success of sperm cryopreservation and short-term storage.

Effects of osmolality on sperm morphology, motility and flagellar wave parameters in Northern pike ( Esox lucius L.)

Northern pike ( Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 μm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 μm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg −1 (average: 283.88 ± 33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media ( P < 0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg −1. Osmolality above 375 mOsmol kg −1 inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile sperm and spermatozoa velocity due to changes in flagellar wave parameters.

Measurements of Activation Products Associated with Havar Foils from a GE PETtrace Medical Cyclotron Using High Resolution Gamma Spectroscopy

Havar foils are specially engineered for beam-line windows used in the General Electric (GE) PETtrace medical cyclotron that can withstand the high pressure differentials and also temperatures developed near the target with proton bombardment. These foils effectively separate components along the beam line of the cyclotron. Various activation products are produced in the foils from the primary proton beam and other secondary radiations. An accurate estimate of the activation products is necessary for the disposal of these foils. The foils were assayed using two High Purity Germanium (HPGe) detectors calibrated over a wide energy range. These were positioned at different distances (0.10 m, 0.24 m, and 1.74 m) away from the detector faces to accommodate their high activities. A summary of the anticipated relative abundance of each activation product and a scatter plot of the average exposure rate per unit charge incident on the foil vs. time post-activation are provided. A detailed spectral analysis of the foils in the energy ranges between 12 keV to 300 keV and 12 keV through 2,500 keV revealed the residual activation products 56Co, 57Co, 58Co, 54Mn, and 183Re at 264 d post-irradiation. Spectral examinations of the different foils removed between 2003 and 2005 show the same activation products regardless of the irradiation time or foil position in the target assembly. The information presented in this paper can be used along with the integrated charge incident on the foils in estimating the activity of Havar foils for the purpose of disposal.

Changes of sperm morphology, volume, density and motility and seminal plasma composition inBarbus barbus(Teleostei: Cyprinidae) during the reproductive season

Eighteen spermiating males were randomly selected from a hatchery-reared stock and electronically tagged to record changes in their sperm quality parameters (spermatozoa morphology, ultrastructure and motility, ionic composition and osmolality of the seminal plasma, and sperm volume and density) during the spawning season. Stripping was performed at the beginning of March, April and May. The Barbus barbus spermatozoon has a head without acrosome, a midpiece with 4–6 mitochondria and proximal and distal centrioles, and a flagellum with the typical 9+2 pairs of microtubules. Apart from posterior width of the midpiece, morphological and ultrastructural parameters changed significantly during the reproductive season; generally by decreasing toward the end of reproductive season. Sperm volume also decreased from 0.42 in March to 0.15 ml in May, and density from 18.81 in March to 12.45 × 109 spz ml−1 in May. Osmolality (mOsmol kg−1) was 268 ± 4, 276 ± 2 and 268 ± 2 in March, April and May respectively. Chloride, sodium, calcium and potassium ion concentrations (mM) did not show significant differences between March and April (Cl−: 125.3 vs. 120.5, Na+: 75.7 vs. 69.7, Ca2+: 0.4 vs. 0.3 and K+: 84.7 vs. 84.0). The percentage of motile spermatozoa at 15 s post activation did not show a significant difference between dates, but the highest spermatozoa velocity at 15 s post activation was observed in April (91.4 ± 3.2 µm s−1) and then decreased significantly towards the end of the reproductive season (80.6 µm s−1 in May). However, lowest spermatozoa velocity was measured in March (70.4 ± 1.9 µm s−1). This study supports the hypothesis that longer spermatozoa swim faster. Within one stripping, velocity and percentage motility decreased significantly with time post activation. In conclusion, changes observed in B. barbus sperm parameters during the reproductive season, suggest there is association between such changes and spermatozoa aging processes.

Development of a novel CASA system based on open source software for characterization of zebrafish sperm motility parameters

Although computer-assisted sperm analysis (CASA) outperforms manual techniques, many investigators rely on non-automated analysis due to the high cost of commercial options. In this study, we have written and validated a free CASA software primarily for analysis of fish sperm. This software is a plugin for the free National Institutes of Health software ImageJ and is available with documentation at http://rsb.info.nih.gov/ij/plugins/casa.html. That it is open source makes possible external validation, should improve quality control and enhance the comparative value of data obtained among laboratories. In addition, we have improved upon the traditional velocity straight line (VSL) algorithm, eliminating inaccurate characterization of highly curved fish sperm paths. Using this system, the motion of zebrafish ( Danio rerio) sperm was characterized relative to time post-activation and the impact of acquisition conditions upon data analysis determined. There were decreases in velocity and path straightness (STR), but not linearity (LIN), relative to time. From 30 to 300 frames/s, frame rate significantly affected curvilinear velocity (VCL) and STR measurements. Sperm density in the field of view did not affect any measured parameter. There was significant inter-male variation for VCL, VSL, velocity average path (VAP), percent motility, path character (STR, LIN), and duration of motility. Furthermore, relative sperm output (a measure reflecting both semen volume and concentration) was positively correlated to percent motility. For all motion parameters measured (except duration), the average CV was ≤10%, comparable to values obtained using commercial systems.