Min Postactivation Research Articles

Evaluate effects of the dilution medium and holding time on various motility parameters of delta smelt semen

Sperm preservation is an efficient technique used for the recovery, conservation, and management of some endangered fish species. The present study was conducted to explore how preservation time would affect sperm and spawning performance in the endangered delta smelt (Hypomesus transpacificus). Sperm were preserved with the modified Hanks balanced salt solution at 14.7–16.9 °C. The Kruskal-Wallis test of sperm parameters using OpenCASA plugin in ImageJ software showed that sperm (n = 33♂) had significantly higher velocity and motility within the first 5 s after activation than that of other time points, while sperm had the lowest velocity and motility after 3 min post activation (P < 0.001). The findings (n = 30♂) also showed fresh sperm had higher velocity and motility than preserved sperm, while the sperm preserved for over 24 h showed a significantly low performance (P < 0.001). The nonlinear mixed effects models of fertilization results (n = 14♂ × 70♀) indicated the fresh sperm and sperm preserved for 1 h had higher fertilization rates than other preservation times (P < 0.001). The hatching rate (n = 14♂ × 70♀) also showed the fresh sperm and sperm preserved for 3 min and 1 h had higher hatching rates than other preservation times (P < 0.001). Overall, the study showed the best sperm performance in delta smelt was found within the first 5 s post activation, and the best fertilization and hatching rates were found when the sperm were fresh and preserved for 1 h. The findings of this study provide information for the first time about how long the delta smelt's sperm are motile for quality analysis, and how the preservation time can affect sperm quality, fertility, and hatching of this species for future applications.

Effects of post-collection storage conditions on sperm motility longevity in the blunt sea urchin Sphaerechinus granularis

The standardization of procedures for the post-collection management of semen samples is a prerequisite for the development of husbandry techniques, as well as for the use of semen in laboratory research. Despite the relevance of the sea urchin Sphaerechinus granularis as a laboratory model, the basics related to sperm collection and storage have received little attention. The aim of this research was to optimize procedures for the management of S. granularis semen, focusing on the post-collection storage of samples. Specifically, the effects of post-collection storage conditions on sperm motility parameters were evaluated; motility was assessed by computer-assisted analysis (CASA-mot) upon activation (t0), and 15 min, 30 min and 60 min after dilution in freshly collected semen, and in chilled and cryopreserved samples. Motility parameters remained unchanged for up to 60 min after activation in both fresh and chilled semen. In cryopreserved samples, sperm motility upon activation was significantly lower than in fresh semen; however, no differences with respect to t0 values were recorded when thawed semen was cold- incubated for up to 60 min post activation, thus meeting standards required by germplasm cryobanks. Post-activation longevity showed by both fresh and stored S. granularis semen, as well as its resistance to cryopreservation, are positive spermatological features that will allow the running of easy management procedures, encouraging a wider use of S. granularis in hatchery practices, as well as in laboratory activities.

To Vent or Not to Vent: A Loaded Question During Venoarterial Extracorporeal Membrane Oxygenation Support for Cardiogenic Shock

To Vent or Not to Vent: A Loaded Question During Venoarterial Extracorporeal Membrane Oxygenation Support for Cardiogenic Shock

Regulation of sperm motility in Eastern oyster (Crassostrea virginica) spawning naturally in seawater with low salinity.

Oyster aquaculture is expanding worldwide, where many farms rely on seed produced by artificial spawning. As sperm motility and velocity are key determinants for fertilization success, understanding the regulation of sperm motility and identifying optimal environmental conditions can increase fertility and seed production. In the present study, we investigated the physiological mechanisms regulating sperm motility in Eastern oyster, Crassostrea virginica. Sperm motility was activated in ambient seawater with salinity 4–32 PSU with highest motility and velocity observed at 12–24 PSU. In artificial seawater (ASW) with salinity of 20 PSU, sperm motility was activated at pH 6.5–10.5 with the highest motility and velocity recorded at pH 7.5–10.0. Sperm motility was inhibited or totally suppressed in Na+, K+, Ca2+, and Mg2+-free ASW at 20 PSU. Applications of K+ (500 μM glybenclamide and 10–50 mM 4-aminopyridine), Ca2+ (1–50 μM mibefradil and 10–200 μM verapamil), or Na+ (0.2–2.0 mM amiloride) channel blockers into ASW at 20 PSU inhibited or suppressed sperm motility and velocity. Chelating extracellular Ca2+ ions by 3.0 and 3.5 mM EGTA resulted in a significant reduction and full suppression of sperm motility by 4 to 6 min post-activation. These results suggest that extracellular K+, Ca2+, and Na+ ions are involved in regulation of ionic-dependent sperm motility in Eastern oyster. A comparison with other bivalve species typically spawning at higher salinities or in full-strength seawater shows that ionic regulation of sperm motility is physiologically conserved in bivalves. Elucidating sperm regulation in C. virginica has implications to develop artificial reproduction, sperm short-term storage, or cryopreservation protocols, and to better predict how changes in the ocean will impact oyster spawning dynamics.

Effects of gametes post-activation, egg storage and sperm-egg ratio on in vitro fertilization outcomes in the brown-marbled grouper (Epinephelus fuscoguttatus)

Due to some biological characteristics of the brown-marbled grouper, there are challenges in aquaculture enterprises focused on production of this fish. With the aim of facilitating captive breeding in brown-marbled grouper, effects on gamete post-activation, post-stripping egg storage and sperm to egg ratio on fertilization success were investigated. Results indicated eggs were fertile for 4 min after placement in seawater, thereafter, there was a reduction in fertilization. Initial curvilinear velocity (VCL) and total motility of fresh sperm was 129.3 ± 3.7 μm/s and 92.9 ± 2.4 %, both of which decreased as time elapsed post-activation, however, there was no significant difference in fertilization until 6 min post-activation of sperm. Compared with fertilization and hatching rates in eggs used immediately after collection (95.0 ± 2.8 % and 90.1 ± 4.2 %), in vitro storage of eggs had no detectable effect on fertilized egg and hatching rates until 60 min post-stripping (92.0 ± 2.8 % and 89.9 ± 2.7 %), indicating that immediate use of brown-marbled grouper eggs for fertilization is not necessary in aquaculture hatcheries. With regard to optimizing the sperm to egg ratio, there was acceptable fertilization with ratios ranging from 103 to 107, whereas there was a marked decrease in fertilization rate when there were lesser sperm concentrations. When the combined results from this study are considered, there could be improvements of artificial fertilization efficiency in the brown-marbled grouper with utilization of these findings in aquaculture enterprises focused on production of this fish.

Application of hydrostatic pressure shock for retention of the second polar body in sterlet (Acipenser ruthenus)

Induced retention of the second polar body (SPBR) is used for production of triploid fish and/or for restoration of diploidy during induced meiotic gynogenesis (MeiG). Using sterlet, Acipenser ruthenus as model species of sturgeon, a protocol was developed for SPBR with hydrostatic pressure shock, commonly found more considerate for treated eggs of teleosts, instead of heat shock conventionally used hitherto. In the first trial, hydrostatic pressure shock of 55 MPa for 3 min duration applied to eggs 18 min post gamete activation gave the highest fertilization rate (62.14 ± 8.90%) and hatching rate (24.59 ± 8.35%) both on Petri dishes and after pilot testing in Zuger jars (78.55%; 45.75%, respectively). Flow cytometric analysis of relative DNA content confirmed 100% triploidy in all sampled prelarvae from shocked eggs. In the second trial comparing the effectiveness of 55 MPa hydrostatic pressure shock for SPBR with conventional 35 °C heat shock for 3 min duration in order to induce triploidy and/or MeiG, pressure – induced triploids exhibited fertilization rate 62.14 ± 8.90% significantly lower than the control group (79.17 ± 7.64%) but indifferent from that in heat -shocked triploids (60.08 ± 4.65%). In MeiG groups, rediploidization with pressure shock or with heat shock did not reveal significant differences in fertilization rate, triploidization with pressure shock provided significantly higher value (46.54 ± 7.47%) than that obtained with heat shock (13.93 ± 3.46%), while similar to the control group (52.71 ± 1.9%). Hatching rates in both MeiG groups did not differ significantly each other but from values in the control group and in the pressure-induced triploids. Flow cytometric analysis of relative DNA content confirmed 100% and 80% triploidy in prelarvae sampled from the pressure- and heat shocked triploids, respectively. Among the MeiG fish, 26 and 25 analyzed prelarvae out of 30 from each the pressure- and heat shock-rediploidized groups were diploid and the remaining prelarvae were haploid. This experiment confirmed the suitability of 55 MPa hydrostatic pressure shock for SPBR in sturgeons.

Prior muscle activation affects the compound muscle action potential.

This study was performed to evaluate the effect of prior voluntary activation of a muscle on the subsequently-recorded compound muscle action potential (CMAP). The CMAPs from the hypothenar, thenar, and extensor digitorum brevis muscles were recorded in 6 healthy volunteers at rest and for up to 30 min following 5 separate epochs of up to 20 s of voluntary muscle activation. There was consistent, significant (P < 0.02) enhancement of the negative area, amplitude, and duration of the CMAP after activation. The enhancement was maximal, up to 144% of baseline, within about 1 min post-activation; thereafter, the CMAP gradually returned to baseline over about 15 min. Activation of a muscle within several minutes prior to testing enhances the subsequently-recorded CMAP. This observation highlights prior muscle activation as a physiological variable that influences the size of the CMAP during motor nerve conduction studies. Muscle Nerve, 2019.

Analysis of the potential of cancer cell lines to release tissue factor-containing microvesicles: correlation with tissue factor and PAR2 expression.

BackgroundDespite the association of cancer-derived circulating tissue factor (TF)-containing microvesicles and hypercoagulable state, correlations with the incidence of thrombosis remain unclear.MethodsIn this study the upregulation of TF release upon activation of various cancer cell lines, and the correlation with TF and PAR2 expression and/or activity was examined. Microvesicle release was induced by PAR2 activation in seventeen cell lines and released microvesicle density, microvesicle-associated TF activity, and phoshpatidylserine-mediated activity were measured. The time-course for TF release was monitored over 90 min in each cell line. In addition, TF mRNA expression, cellular TF protein and cell-surface TF activities were quantified. Moreover, the relative expression of PAR2 mRNA and cellular protein were analysed. Any correlations between the above parameters were examined by determining the Pearson’s correlation coefficients.ResultsTF release as microvesicles peaked between 30–60 min post-activation in the majority of cell lines tested. The magnitude of the maximal TF release positively correlated with TF mRNA (c = 0.717; p < 0.001) and PAR2 mRNA (c = 0.770; p < 0.001) expressions while the percentage increase correlated with PAR2 mRNA (c = 0.601; p = 0.011) and protein (c = 0.714; p < 0.001). There was only a weak correlation between resting TF release, and microvesicle release. However, TF release in resting cells did not significantly correlate with any of the parameters examined. Furthermore, TF mRNA expression correlated with PAR2 mRNA expression (c = 0.745; p < 0.001).Discussion and ConclusionsIn conclusion, our data suggest that TF and PAR2 mRNA, and PAR2 protein are better indicators of the ability of cancer cells to release TF and may constitute more accurate predictors of risk of thrombosis.

Optimizing Electrical Activation of Porcine Oocytes by Adjusting Pre- and Post-Activation Mannitol Exposure Times

Modifying electrical activation conditions have been used to improve in vitro embryo production and development in pigs. However, there is insufficient information about correlations of porcine embryo development with oocyte pre- and post-activation conditions. The purpose of this study was to compare the developmental rates of porcine oocytes subjected to different mannitol exposure times, either pre- or post-electrical activation, and to elucidate the reason for the optimal mannitol exposure time. Mannitol exposure times around activation were adjusted as 0, 1, 2 or 3min. Blastocyst development were checked on day 7. Exposure of oocytes to mannitol for 1 or 2min before electrical activation produced significantly higher blastocyst rates than exposure for 0 or 3min. There was no significant difference in blastocyst rates when activated oocytes were exposed to mannitol for 0, 1, 2 or 3 min after electrical activation. While exposure of oocytes to mannitol for 1min pre- and 3min post-activation showed significantly higher blastocyst development than 0min pre- and 0min post-activation. It also showed higher maintenance of normal oocyte morphology than exposure for 0min pre- and 0min post-activation. In conclusion, exposure of oocytes to mannitol for 1min pre- and 3min post-activation seems to be optimal for producing higher in vitro blastocyst development of porcine parthenogenetic embryos. The higher blastocyst development is correlated with higher maintenance of normal morphology in oocytes exposed to mannitol for 1min pre- and 3min post-activation.

G Protein–Coupled Receptor Kinase-6 Interacts with Activator of G Protein Signaling-3 To Regulate CXCR2-Mediated Cellular Functions

The IL-8 (CXCL8) receptors CXCR1 and CXCR2 couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. We recently showed that CXCR1 couples predominantly to the G protein-coupled receptor kinase (GRK)2, whereas CXCR2 interacts with GRK6 to regulate cellular responses. In addition to G protein-coupled receptors, GRKs displayed a more diverse protein/protein interaction in cells. In this study, we sought to identify GRK6 binding partner(s) that may influence CXCL8 activities, using RBL-2H3 cells stably expressing CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2), as well as human and murine neutrophils. Our data demonstrated that, upon CXCR2 activation, GRK6 interacts with activator of G protein signaling (AGS)3 and Gαi2 to form a GRK6/AGS3/Gαi2 complex. This complex is time dependent and peaked at 2-3 min postactivation. GTPγS pretreatment blocked GRK6/AGS3/Gαi2 formation, suggesting that this assembly depends on G protein activation. Surprisingly, CXCR2 activation induced AGS3 phosphorylation in a PKC-dependent, but GRK6-independent, fashion. Overexpression of AGS3 in RBL-CXCR2 significantly inhibited CXCL8-induced Ca(2+) mobilization, phosphoinositide hydrolysis, and chemotaxis. In contrast, short hairpin RNA inhibition of AGS3 enhanced CXCL8-induced Ca(2+) mobilization, receptor resistance to desensitization, and recycling to the cell surface, with no effect on receptor internalization. Interestingly, RBL-CXCR2-AGS3(-/-) cells displayed a significant increase in CXCR2 expression on the cell surface but decreased ERK1/2 and P38 MAPK activation. Taken together, these results indicate that GRK6 complexes with AGS3-Gαi2 to regulate CXCR2-mediated leukocyte functions at different levels, including downstream effector activation, receptor trafficking, and expression at the cell membrane.

P38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles

We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles.

Malaria proteases mediate inside-out egress of gametocytes from red blood cells following parasite transmission to the mosquito

Malaria parasites reside in human erythrocytes within a parasitophorous vacuole. The parasites are transmitted from the human to the mosquito by the uptake of intraerythrocytic gametocytes during a blood meal, which in the midgut become activated by external stimuli and subsequently egress from the enveloping erythrocyte. Gametocyte egress is a crucial step for the parasite to prepare for fertilization, but the molecular mechanisms of egress are not well understood. Via electron microscopy, we show that Plasmodium falciparum gametocytes exit the erythrocyte by an inside-out type of egress. The parasitophorous vacuole membrane (PVM) ruptures at multiple sites within less than a minute following activation, a process that requires a temperature drop and parasite contact with xanthurenic acid. PVM rupture can also be triggered by the ionophore nigericin and is sensitive to the cysteine protease inhibitor E-64d. Following PVM rupture the subpellicular membrane begins to disintegrate. This membrane is specific to malaria gametocytes, and disintegration is impaired by the aspartic protease inhibitor EPNP and the cysteine/serine protease inhibitor TLCK. Approximately 15 min post activation, the erythrocyte membrane ruptures at a single breaking point, which can be inhibited by inhibitors TLCK and TPCK. In all cases inhibitor treatment results in interrupted gametogenesis.

Sperm features of captive Atlantic bluefin tuna (Thunnus thynnus)

P The present study aimed to establish some basic characteristics of Atlantic bluefin tuna sperm from captive mature males, treated or untreated by gonadotropin releasing hormones agonist (GnRHa). Intratesticular milt was collected from treated and untreated fish (mean weight +/- SD: 122.9 +/- 29.2 kg, n = 21). There was no significant effect of GnRHa treatment on GSI (1.33 +/- 0.70%, n = 21) or on sperm concentration (3.8 +/- 1.3 x 1010 spermatozoa ml-1, n = 21) estimated by optical density at 260 nm. Similarly, the percentage of motile spermatozoa measured at 30 s post activation: activating medium (AM seawater containing 10 mg ml-1 BSA) was not significantly different between control and GnRHa implanted males. On the other hand, a significantly higher Average Path Velocity (VAP) was assessed for sperm from GnRHa-treated fish, compared to controls. Regardless of hormonal treatment, the percentage of motile sperm decreased after a plateau of 5-6 min post activation, and any forwardly progressing movement ceased after 10-11 min. Linear trajectories of spermatozoa were observed in seawater while tighter circles were assessed when increasing the Ca2+concentration in the AM. The storage capacity at 4 degrees C was significantly lowered when NAM (Non Activating Medium: 50% seawater containing 10 mg ml-1 BSA) was added to sperm at a 1 : 1 dilution. These results demonstrated that treatment with GnRHa had little effects (except on VAP) on sperm characteristics in captive reared Atlantic bluefin tuna. Further investigations are required to improve the knowledge of tuna sperm biology and, in this respect, the comparison between intratesticular and released sperm features should be highly informative.

Physico-biochemical parameters and protein profiles of sperm from beluga Huso huso

Summary Basic physico-biochemical parameters and protein profiles of sperm from beluga (Huso huso), have been evaluated. The results show a stable spermatozoan velocity (ranging from 136.23 ± 5.63 to 105.78 ± 5.27 μms−1) and motility during 2 min post activation, as well as a long duration of the overall spermatozoa motility period (up to 5 min). Mean values were determined for seminal plasma protein concentration (0.29 ± 0.16 mg ml−1), spermatozoa concentration (0.28 ± 0.27 × 109 spz ml−1), seminal plasma osmolality (51.33 ± 4.91 mOsmol kg−1), the pH (8.49 ± 0.01) and Na+ (18.97 ± 3.65 mm), K+ (2.83 ± 1.36 mm), Ca2+ (0.19 ± 0.06 mm), Mg2+ (0.49 ± 0.23 mm) and Cl− (6.33 ± 0.58 mm) concentrations. Moreover, in seminal plasma, five protein bands with molecular weights (MW) of 71, 49, 46, 34, 29 kDa were identified. In spermatozoa, about 100 spots with molecular weights varying from 26.5 to 107 kDa and iso-electric points ranging from 5 to 9.5 were found. The observed physiological and biochemical properties, together with protein patterns, should be considered for the development of methods for controlled reproduction and sperm cryopreservation for the highly endangered beluga sturgeon.

Quantitative semen parameters of Atlantic cod (Gadus morhua) and their physiological relationships with sperm activity and morphology

Summary Aspects of sperm motility and metabolism are important for understanding gamete quality. The objectives of this study were to investigate changes in adenosine 5¢-triphosphate (ATP), and lactate dehydrogenase (LDH) of Atlantic cod sperm over time following activation, and to determine if initial semen parameters (ATP, LDH, Na + ,K + ,C a 2+ , total protein, osmolality, pH, and spermatocrit) are indicative of sperm quality. Analyses of ATP and LDH were also performed on sperm cells at 0.5, 2, and 90 min post-activation, while sperm activity was recorded at 0.5 and 2 min. Initial semen parameters did not appear to be strong indicators of sperm quality but some correlations were observed between pH, LDH, K + , and protein and motility parameters in the first two min post-activation. The lack of significant correlation may be attributed to the low variability of almost all parameters observed between males in this study. There was a significant effect of post-activation time on sperm ATP and LDH with both ATP and LDH decreasing with time after activation. The decrease in ATP levels during the course of sperm motility suggests that the metabolic activity is too low to substitute the energy consumed. Continued research on sperm motility and metabolism will lead to a better understanding of fertilization and gamete quality in this species.

Sperm motility and seminal plasma characteristics in Barbus sharpeyi (Günther, 1874)

Spermatozoa concentration, ionic composition, osmolality, glucose and total protein contents of seminal plasma and sperm motility were determined in Barbus sharpeyi (Cyprinidae, Teleosotei). Spermatozoa concentration ranged from 9.77 to 20.20 × 109 spermatozoa mL−1. Osmolality (mOsmol kg−1) and ionic contents (mM L−1) of the seminal plasma were 274.5±9.0, 70.0±3.4 Na+, 28.8±0.9 K+, 101.7±3.1 Cl−, 0.9±0.1 Mg2+ and 2.1±0.1 Ca2+ respectively. Total protein and glucose were 5.3±0.2 g L−1 and 76.7±4.3 mM L−1 respectively. Sperm motility was initiated in a hypo-osmotic condition, composed of either an ionic (KCl or NaCl) or a non-ionic (sucrose) activation medium. Duration of sperm motility was very short: <2 min after activation in distilled water. Percentage of motile spermatozoa was significantly higher in an activation medium containing NaCl compared with that of distilled water. An activating medium containing NaCl or KCl higher than 150 mM or sucrose higher than 275 mM totally inhibited the activation of sperm motility. Immediately after sperm activation, wave(s) propagated along the flagellum, but waves were restricted to the proximal part of the flagellum (close to the head) at 1 min post activation. Studied characteristics in the present study were compared with those of other cyprinids for understanding inter-species differences.

25 COMPARISON BETWEEN CHEMICALLY ASSISTED, CHEMICALLY INDUCED AND MECHANICAL ENUCLEATION OF MOUSE OOCYTES

Chemically-assisted (AE) and chemically induced (IE) enucleation using demecolcine (DEM) or nocodazole (NOC) have proven to be technically simple procedures to prepare developmentally competent cytoplasts for nuclear transfer (NT) in different species. In this study, we analyzed AE and IE in mouse oocytes in terms of enucleation efficiency, amount of cytoplasmic volume removed and distribution of spindle-associated γ-tubulin after enucleation, and spindle morphology after cytoplast reconstruction by NT. Results were compared to the standard mechanical enucleation (ME) method. Outbred CD-1 and hybrid B6CBAF1 oocytes were collected at 13 to 16 h post-hCG. In AE experiments, oocytes were treated with either 0.4 μg mL–1 DEM or 0.3 μg mL–1 NOC in KSOM for 30 min. Protrusions induced in CD-1 (92.2%, n = 695) and B6CBAF1 (83.3%, n = 370) oocytes were aspirated by piezo-actuated micromanipulation, in H-KSOM with 2.5 μg mL–1 cytochalasin B and 0.05 m sucrose. In IE experiments, oocytes were preactivated with 7% ethanol for 5 min and treated with DEM or NOC in calcium-free KSOM containing 10 mm strontium. At 90 min postactivation (p.a.), completely- and partially-extruded second polar bodies (PBs) were mechanically aspirated. Enucleation efficiencies were higher than 90% both for AE (90.8%, n = 509 CD-1; 90.4%, n = 260 B6CBAF1) and IE methods (90.3%, n = 167 CD-1; 92.9%, n = 197 B6CBAF1), though they were significantly lower than those obtained for ME in nontreated CD-1 (98.4%; n = 126) or B6CBAF1 (100%, n = 498) oocytes. The amount of cytoplasmic volume removed in CD-1 oocytes was smaller in AE than in ME (2.1%, n = 35 and 3.9%, n = 30, respectively). In B6CBAF1 oocytes, used to compare IE (5.4%, n = 60) and ME (4.9%, n = 41), no differences were found. Volumes were calculated using the CellA software on images of cytoplasts and karyoplasts taken after enucleation. Even though both AE and IE methods avoided the removal of the oocyte spindle microtubules, spindle-associated γ-tubulin was eliminated from the cytoplasts generated by all 3 enucleation procedures, as confirmed by immunofluorescence analysis of the cytoplasts and the complementary karyoplasts produced. Finally, spindle morphology was examined in enucleated oocytes reconstructed by NT with a cumulus cell nucleus. Cytoplasts prepared by NOC-AE or NOC-IE displayed morphologically normal bipolar spindles by 2 h post-NT or 18 to 20 h post-activation (hpa), respectively, similar to cytoplasts prepared by ME. However, when DEM was used, microtubule repolymerization was slower and bipolar spindles could not be observed until 4 h post-NT (AE) or 22 to 24 hpa (IE). In conclusion, although enucleation rates are slightly higher for ME, AE and IE protocols allow oocyte enucleation without removal of the meiotic spindle, and a very small cytoplasm volume is eliminated during AE. Treatments with NOC and DEM are reversible, and cytoplasts produced by AE and IE can form morphologically normal spindles after NT, similar to those of cytoplasts produced by ME. MEC BIO 2006-11792; 2005-SGR00437; Portuguese FCT.

Induced meiotic gynogenesis in tench, Tinca tinca (L.) using irradiated heterogenic sperm

Effects of the starting time and duration of cold shock, as well as the source of heterogenic sperm on the percentage of viable gynogenic larvae (PVGL) in tench were studied. The DNA in sperm of red common carp (Cyprinus carpio var singuonensis) was inactivated by ultraviolet radiation prior to use to induce meiotic gynogenetic development in tench. In experiment 1, tench eggs were cold-shocked for 30 min starting at 1, 3, 5, 7, 10 and 15 min post activation. In experiment 2, cold shock began 5 min after activation and lasted for 10, 20, 30, 40, 50 min, respectively. Each experiment was run in triplicate using 3 tench females, and one group not treated with cold shock was included in each experiment to serve as a control group. In experiment 3, sperm of bigmouth buffalo (Ictiobus cyprinellus), red common carp or grass carp (Ctenopharyngodon idella) was used to induce gynogenesis in tench with a 20-min cold shock starting 5 min post activation. The results showed that PVGL from the control group was very low (0.11-0.47%). In experiment 1, the highest average PVGL (9.60%) was observed when cold shock treatment was applied 5 min post activation. When cold shock treatment was started 5 min post activation, duration of cold shock affected PVGL. Cold shock lasting 20 min resulted in the highest average PVGL (12. 57%) among the selected duration of cold shock studied in experiment 2. The average PVGL was 2.3, 8.6, and 9.3%, respectively, for eggs induced by sperm of bigmouth buffalo, red common carp and grass carp. Average PVGL was significantly lower for eggs induced by sperm of bigmouth buffalo, compared with that for eggs induced by sperm of the other two species. However, average PVGL were similar for eggs induced by sperm of red common carp and grass carp. In summary, the optimal conditions for gynogenesis in tench include the use of irradiated sperm of grass carp to activate the eggs and cold shock of 20 min starting 5 min post activation. Since female tench grow much faster to a larger size than male tench, gynogenesis of tench holds great potential for production enhancement.

300MEIOTIC CELL CYCLE PROGRESSION AFTER PARTHENOGENETIC OOCYTE ACTIVATION: EFFECTS OF STRAIN AND ACTIVATION STIMULUS

Parthenogenetic activation of mammalian oocytes can be induced by several stimuli in vitro. In the mouse, ethanol and Sr2+ are commonly used artificial activating agents. The aim of this study was to investigate both the effects of these two activating agents and the genetic background of the oocyte on meiotic cell cycle progression following activation. Metaphase-II (MII) oocytes were collected from superovulated (5 IU eCG/5 IU hCG) 8–10-week-old female mice of hybrid B6D2F1, inbred C57BL/6, outbred CF-1 and nude (NU/+) strains at 16h post-hCG. Oocytes were activated either by a 5-min exposure to 7% ethanol (E), followed by culture in KSOM, or by continuous culture in Ca2+ -free KSOM containing 10mM SrCl2 (S). Starting 5min after the activation stimulus, oocytes were fixed at 5-min intervals, until 20min post-activation (pa), and then at 30-min intervals, until 6hpa. Fixed oocytes were processed for immunofluorescence analysis of chromatin (Hoechst), spindle (α/β-tubulin) and microfilaments (phalloidin) organization. The initial time course of activation (5–20min pa), assessed by exit from MII arrest, was faster in E- than in S-activated oocytes, except in the C57BL/6 strain in which anaphase (AII) onset after ethanol exposure was significantly delayed in comparison to the other three strains. Progression from AII into telophase (TII), at 50 min pa, was faster in CF-1 and C57BL/6 E-activated oocytes (78.3–87.1%) and in B6D2F1 and CF-1 S-activated oocytes (93.1–94.2%) when compared to the other strains (60–68.8% and 41–62.5%, respectively). NU/+ S-activated oocytes showed the lowest activation rates (88.5% v. 100% at 6h pa), and TII entry was also delayed both in comparison to the other strains (41% vs. 62.5–94.2% at 50min pa) and in comparison to E-activated NU/+ oocytes (68.8%). Rotation of the meiotic spindle began at 15min pa in both E- and S-activated C57BL/6 oocytes, at 20min pa in B6D2F1 and CF-1, and at 50min pa in NU/+ oocytes and, in general, spindle rotation was completed more rapidly following E activation. Second polar body (PB2) extrusion was also delayed in NU/+ oocytes (starting at 1h 20min pa) when compared to all other strains (starting at 50min pa) irrespective of the activating agent used. In E-activated oocytes, TII exit commenced earlier in C57BL/6 oocytes (1h 20min pa) than in the other three strains (1h 50min–2h 20min pa), but a significant proportion of CF-1 and NU/+ oocytes (37–49% at 5h 50min pa) remained arrested at metaphase III. S-activation not only accelerated TII exit but also increased the rate of pronuclear formation in both CF-1 and NU/+ oocytes to 100%. In conclusion, the results demonstrate that both the activating stimulus and the genetic background of the oocyte have significant effects on key events of meiotic cell cycle progression following activation. Supported by USDA NRI 2001-35205-09966 and Charles River Labs.

Cannabinoids inhibit the activation of ERK MAPK in PMA/Io-stimulated mouse splenocytes

The mechanism of action of immune suppression by cannabinoids involves suppression of interleukin-2 (IL-2) production in phorbol ester plus calcium ionophore (PMA/Io)-stimulated lymphocytes. This decrease in IL-2 was due to inhibition of activator protein-1 (AP-1) and nuclear factor of activated T cells (NF-AT) transcription factors, both of which depend on proteins that are regulated by the extracellular signal-regulated kinase subgroup of the mitogen-activated protein kinases (ERK MAPK). Thus, the objective of the present study was to characterize the effects of cannabinoid compounds on ERK MAPK under conditions where IL-2 expression was suppressed. Using the MEK inhibitor PD098059 in order to assess the role of ERK MAPK in PMA/Io-stimulated splenocytes (SPLC), it was determined that IL-2 production and expression of c-fos and c-jun nuclear protein expression depended on activation of ERK MAPK. In response to PMA/Io, expression of nuclear phosphorylated ERK MAPK was rapidly induced, peaked at approximately 15 min, and was sustained for up to 240 min. Pretreatment with cannabinol (CBN) inhibited expression of phosphorylated ERK MAPK at several time points up to 240 min post cellular activation. Furthermore, WIN-55212-2, a synthetic cannabinoid, inhibited expression of phosphorylated ERK MAPK at 240 min post cellular activation. CBN did not induce activation of ERK MAPK in the absence of PMA/Io. Collectively, these studies suggest that cannabinoid-induced inhibition of IL-2 in PMA/Io-stimulated splenocytes might be due, in part, to inhibition of ERK MAPK activation.