Linear Motility Research Articles

Zinc supplementation in male infertility

Background: Inferile males have been shown to have lower levels of seminal plasma zinc which have been associated with reduced levels of zinc in their blood. Supplementation improve semen parameters by improving zinc level in blood. Objective: To fnd out whether zinc supplementation is effective in improving semen parameters in oligo­asthenozoospermic patients.Method: The study was carried out in the Infertility unit of the Dept of Obs & Gynae, BSMMU during the period of March 2011 to February 2012. Seventy five oligo-asthenozoospermic patients having no history of medical treatment were recruited for the study. The patients were divided into two groups by odd and even numbers. Odd numbers received tablet zinc 20 mg twice daily (Group A) and even numbers received placebo (Group B).Serum zinc level and seminal zinc level estimation were done by Graphite Furnace Atomic Absorption Spectropho­tometry and semen analysis was done according to WHO guidelines (1999). Data analysis was done using software SPSS (version 16) by applying ANOVA (PostHock) and Paired Student's 't' test.Results: Serum zinc level was low in oligo­zoospermic patients which showed significant improvement with zinc supplementation (A+ 197.83 mmol/1, P<0.01). Mean (±SD) seminal plasma zinc level showed significant improvement in group A following zinc supplementation (+942.39 mmol/L, P<0.001). The mean increase in sperm count, sperm motility, sperm rapid linear motility, sperm morphology in group A following zinc supplementation for 12 weeks was 14.83 million/ml (P<0.01), 16.30% (P<0.01), 11.96% (P<0.01), 4.26% (P<0.001) respectively, which was statistically significant.Conclusion: The study shows zinc deficiency affects sperm count, sperm motility, rapid linear motility and sperm morphology and with zinc supplementa­tion there can be significant improvement in semen parameters.

In vitro characterisation of fresh and frozen sex-sorted bull spermatozoa.

This study sought to compare the in vitro characteristics of fresh and frozen non-sorted (NS) and sex-sorted (SS) bull spermatozoa. Experiment 1: Holstein-Friesian ejaculates (n=10 bulls) were split across four treatments and processed: (1) NS fresh at 3×106 spermatozoa, (2) X-SS frozen at 2×106 spermatozoa, (3) X-SS fresh at 2×106 spermatozoa and (4) X-SS fresh at 1×106 spermatozoa. NS frozen controls of 20×106 spermatozoa per straw were sourced from previously frozen ejaculates (n=3 bulls). Experiment 2: Aberdeen Angus ejaculates (n=4 bulls) were split across four treatments and processed as: (1) NS fresh 3×106 spermatozoa, (2) Y-SS fresh at 1×106 spermatozoa, (3) Y-SS fresh at 2×106 spermatozoa and (4) X-SS fresh at 2×106 spermatozoa. Controls were sourced as per Experiment 1. In vitro assessments for progressive linear motility, acrosomal status and oxidative stress were carried out on Days 1, 2 and 3 after sorting (Day 0=day of sorting. In both experiments SS fresh treatments had higher levels of agglutination in comparison to the NS fresh (P<0.001), NS frozen treatments had the greatest PLM (P<0.05) and NS spermatozoa exhibited higher levels of superoxide anion production compared with SS spermatozoa (P<0.05). Experiment 1 found both fresh and frozen SS treatments had higher levels of viable acrosome-intact spermatozoa compared with the NS frozen treatments (P<0.01).

EFFECT OF α–CHYMOTRYPSIN AND GELATIN SUPPLEMENTATION IN RABBIT SEMEN EXTENDER ON ANTI-SPERM ANTIBODYS AND FERTILITY

Three experiments were carried out to study the effects of α-chymotrypsin with gelatin on anti-sperm antibodies (ASAs) in rabbit's semen and fertilization rate. In the first experiment, all rabbits males (20 bucks) were tested for the presence of ASAs in seminal plasma. The secondexperiment, aimed to define the effect of different levels of α-chymotrypsin supplementation (0, 100, 200 and 400 mg/100ml extender) with gelatin in tris-based extender in presence ASAs on progressive sperm motility%, live and abnormal spermatozoa%. In the third experiment, does were inseminated with semen diluted in tris-based extender (1:3) as described in experiment II. Insemination was carried out using 80 does divided into five equal experimental groups (16 does/group) and inseminated artificially by diluted semen containing 50x106 spermatozoa. Kindling rate and litter size were calculated. Results showed significant (P 1/10) as compared with ASAs-negative ejaculates (titer<1/10). Semen ejaculates positive-ASAs supplemented with gelatin and α-chymotrypsin were exhibited ASAs titer 1/10 in seminal plasma that represents a significant change (P<0.05) compared with control group (C). Most amino acids were significantly (P<0.05) increased by the inclusion of gelatin alone or gelatin plus α-chymotrypsin. Supplementation of gelatin and α-chymotrypsin significantly (P<0.05) decreased ammonia in semen, ALT and AST activities in seminal plasma, TBARS concentration, sperm abnormalities, dead spermatozoa, glutathione and superoxide dismutase, while, increased percentages of progressive linear motility as compared to the control. Kindling rate was improved as compared with the control group. In addition, the total and live litter size and bunny weights at birth were considerably higher for supplemented groups α-chymotrypsin and gelatin than those of the control group. The levels of α-chymotrypsin 200 and 400 mg/100 ml extender with gelatin represented the same significant differences with other experimental groups. In conclusion, supplementation of rabbit semen with gelatin and α- chymotrypsin not only enhanced the semen quality but it also improved anti-sperm antibodies status and reproductive performance.

Nanometric features of myosin filaments extracted from a single muscle fiber to uncover the mechanisms underlying organized motility

The single muscle fiber in vitro motility assay (SF-IVMA) is characterized by organized linear motility of actin filaments, i.e., actin filaments motility showing a parallel or anti-parallel direction with similar speed independent of direction in the central part of the flow-cell where density of myosin is high. In contrast, the low myosin density region in the flow-cell exhibits random filament movements, but the mechanisms underlying the organized motility remain unknown. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) imaging techniques have been combined to investigate the morphological features of myosin extracted from single muscle fiber segments in the flow cell. Nanometric scale imaging of myosin filaments in the SF-IVMA showed intact spatial distances between myosin heads being essential for myosin filament function. However, angular spectrum analyses of myosin filaments in the high myosin density region showed organized myosin filament orientation only in small areas, while unorganized filament orientation were dominantly presented when larger areas were analyzed. Thus, parallel myosin filament organization is a less likely mechanism underlying the organized motility of actin filaments and the high myosin density per se is therefore forwarded as the primary “driver” that promotes organized linear motility.

MP74-16 DEVELOPMENT OF A PRACTICAL ANIMAL MODEL FOR HUMAN SPERMATOGONIAL STEM CELL TRANSPLANTATION

probenecid but did not reach statistical significance (18 2.6% vs. 22 3.7%, p1⁄40.19, Figure 1A). The most pronounced improvement was in the grade of sperm motility. Rapid linear motility increased significantly after 4 weeks of treatment (5.0 1.3% vs. 16 3.2%, p < 0.005, Figure 1B), and continued after the cessation of treatment (5.0 1.3% vs. 15 8.3%, p < 0.005, Figure 1B). Sperm concentrations remained normal throughout the course of the study. The medication was well-tolerated by all patients. CONCLUSIONS: This is the first study to report improved sperm motility from an oral medication in men with spinal cord injury. Planned placebo-controlled trials will determine optimal dosage regimens. Probenecid holds promise as a simple, effective therapy for low sperm motility in this severely affected group of patients.

MP74-15 ORAL PROBENECID IMPROVES SPERM MOTILITY IN MEN WITH SPINAL CORD INJURY (SCI)

You have accessJournal of UrologyInfertility: Therapy1 Apr 2015MP74-15 ORAL PROBENECID IMPROVES SPERM MOTILITY IN MEN WITH SPINAL CORD INJURY (SCI) Emad Ibrahim, Teodoro Aballa, Juan Pablo de Rivero Vaccari, Robert Keane, W. Dalton Dietrich, Charles Lynne, and Nancy Brackett Emad IbrahimEmad Ibrahim More articles by this author , Teodoro AballaTeodoro Aballa More articles by this author , Juan Pablo de Rivero VaccariJuan Pablo de Rivero Vaccari More articles by this author , Robert KeaneRobert Keane More articles by this author , W. Dalton DietrichW. Dalton Dietrich More articles by this author , Charles LynneCharles Lynne More articles by this author , and Nancy BrackettNancy Brackett More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2670AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Most men with SCI have abnormally low sperm motility which contributes to infertility. Our previous research has shown that the inflammasome signaling mechanism contributes to this condition [Fertil Steril 2013;99:118-24, Hum Reprod 2014;29:2368-73]. Probenecid is a uricosuric drug that inhibits the inflammasome signaling mechanism via interference with pannexin-1 channels. The objective of this study was to determine if oral administration of probenecid resulted in improved sperm motility in men with SCI. METHODS Subjects were 18 men with SCI who ejaculated regularly by penile vibratory stimulation or electroejaculation. Probenecid tablets (Watson Pharma Inc., Corona, CA) were administered in two phases. Phase 1: Subjects received 250 mg orally twice a day for 1 week. Phase 2: Subjects who completed Phase 1 with no complications were administered 500 mg orally twice a day for 3 weeks. Semen analysis was performed at three time points: Pre-treatment (Pre-Rx), 1-2 days before Phase 1; Post-treatment (Post-Rx), 1-2 days after completion of Phase 2; Follow-up (F/U), 4 weeks after completion of Phase 2. Outcomes were compared by repeated measures analysis of variance. RESULTS Oral probenecid treatment resulted in improved sperm motility in 17 of 18 men. After 4 weeks of treatment, mean sperm motility increased significantly from 18 ± 2.6% to 25 ± 3.7%, p < 0.005, Figure 1A). The improvement continued 4 weeks after cessation of oral probenecid but did not reach statistical significance (18 ± 2.6% vs. 22 ± 3.7%, p=0.19, Figure 1A). The most pronounced improvement was in the grade of sperm motility. Rapid linear motility increased significantly after 4 weeks of treatment (5.0 ± 1.3% vs. 16 ± 3.2%, p < 0.005, Figure 1B), and continued after the cessation of treatment (5.0 ± 1.3% vs. 15 ± 8.3%, p < 0.005, Figure 1B). Sperm concentrations remained normal throughout the course of the study. The medication was well-tolerated by all patients. CONCLUSIONS This is the first study to report improved sperm motility from an oral medication in men with spinal cord injury. Planned placebo-controlled trials will determine optimal dosage regimens. Probenecid holds promise as a simple, effective therapy for low sperm motility in this severely affected group of patients. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e944-e945 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Emad Ibrahim More articles by this author Teodoro Aballa More articles by this author Juan Pablo de Rivero Vaccari More articles by this author Robert Keane More articles by this author W. Dalton Dietrich More articles by this author Charles Lynne More articles by this author Nancy Brackett More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Cholesterol-loaded-cyclodextrins improve the post-thaw quality of stallion sperm

An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-β-cyclodextrin–cholesterol (MβCD)/mL (MβCD0.75); TALP+1.5mg MβCD-cholesterol/mL (MβCD1.5); and Equipro. Following 15min incubation, samples were centrifuged and diluted to 100×106sperm/mL, frozen in 0.5mL straws and stored in liquid nitrogen. Sperm from each treatment was assessed for progressive linear motility (PLM) and acceptable membrane integrity under hypotonic conditions on a phase contrast microscope at 1000× while viability, membrane fluidity and superoxide generation were assessed by flow cytometry. The MβCD1.5 and MβCD0.75 treatments had a greater proportion of viable sperm than the TALP treatment (P<0.01). There was no effect of treatment on PLM or membrane integrity. The MβCD1.5 treatment had a greater proportion of viable sperm positive for membrane fluidity than the TALP treatment (P<0.05). The MβCD1.5 and MβCD0.75 treatments had a lesser proportion of viable sperm positive for superoxide generation than the TALP treatment (P<0.001). This study has demonstrated that adding cholesterol to stallion sperm prior to cryopreservation increases post-thaw viability, with these viable sperm being of better quality in terms of increased membrane fluidity and reduced superoxide generation.

Reducing sperm concentration is critical to limiting the oxidative stress challenge in liquid bull semen

Because of the short breeding season, the use of liquid bull semen is a viable option in seasonal grass-based dairy systems such as Ireland. Currently in Ireland, liquid bull semen contains approximately 5 million sperm per insemination dose and is used within 2.5d of collection. The hypothesis of this study was that reducing the sperm number per insemination dose would enable bull sperm to be stored for longer. Semen was collected at a commercial AI center and diluted to 1 (T1), 2 (T2), 3 (T3), 4 (T4), and 5 (T5) million sperm per 0.25-mL dose in caprogen diluent. On d 0.25 (6 h postcollection), 1, 2, 3, 4, and 5 postcollection, viability, oxidative stress, and mitochondrial activity were assessed using flow cytometry and the fluorescent probes propidium iodide, CM-H2DCFDA, and rhodamine 123, respectively. On the same days, glucose consumption, total antioxidant capacity, and progressive linear motility were assessed. We observed an effect of day and treatment on sperm cell viability, with the highest percentage live found in T 0005 and the lowest in T 0025 on all days. Oxidative stress in live sperm increased with duration of storage and was affected by treatment, being highest in T 0025 and lowest in T 0005 on all days (d 5: 56.4±2.76% and 28.8±1.22%, respectively; mean ± SEM). Both the total antioxidant capacity and percentage of live sperm positive for rhodamine 123 were unaffected by treatment. The concentration of glucose in caprogen declined with time and was lowest in T 0025 and highest in T 0005 on d 5. In conclusion, higher concentrations of sperm have detrimental effects on sperm cell viability and increase oxidative stress but have no effect on the mitochondrial activity of sperm.

Directional Freezing of Reproductive Cells and Organs

Directional freezing is based on a simple thermodynamic principle where ice crystals are precisely controlled through the sample by regulating the velocity of the sample movement through the predetermined temperature gradient. Directional freezing permits a precise and uniform cooling rate in both small and large volume samples. Directional freezing was used for slow and rapid freezing, as well as for vitrification of oocytes and embryos using the minimum drop size technique. Sperm samples from a wide range of domestic and wild animals were successfully cryopreserved using the directional freezing method. The method enabled, for the first time, successful freezing of a whole ovary and freeze-drying of mammalian cells followed by thawing and transplantation and rehydration, respectively.

A New Human Spermatozoon Selection Method Based on Penetration of Cervical Mucus for Intracytoplasmic Sperm Injection

Abstract: Spermatozoa used for intracytoplasmic sperm injection (ICSI) are selected based on their motility and morphology. To explore other methods for selecting better spermatozoa, we developed a spermatozoa-sorting method using a physiologically natural selection system involving penetration into cervical mucus (CM). In addition, we analyzed the spermatozoa that penetrated the CM (CM-penetrating spermatozoa). The results were as follows. The CM-penetrating spermatozoa traveled a longer distance with better linear motility than spermatozoa in semen. Also, in comparison with spermatozoa obtained by density gradient centrifugation (DGC) and the swim-up method, the proportion of spermatozoa with normal morphology was higher, although, the proportion of spermatozoa with the head vacuole did not change. No DNA fragmentation was detected in the CM-penetrating spermatozoa. This method has several advantages. The technical procedure is simple and easy. Physical damage to spermatozoa is reduced because it does n...

Zinc-binding proteins from boar seminal plasma -- isolation, biochemical characteristics and influence on spermatozoa stored at 4°C.

Affinity chromatography on Chelating Sepharose Fast Flow Gel-Zn(2+) was used for fractionation of boar seminal plasma proteins. Approximately 30% of total boar seminal plasma proteins showed affinity for zinc ions (ZnBP fraction). Native electrophoresis (PAGE) of ZnBP revealed six protein fractions which separated into 27 bands under denaturing conditions (SDS/PAGE). Two-dimensional electrophoresis (2D PAGE) showed 148 polypeptides with isoelectric points mostly in the basic and neutral pH range. The zinc-binding proteins comprise mainly 10-20 kDa polypeptides which are probably members of the spermadhesin family. ZnBP present in the incubation mixture of spermatozoa stored for 1 or 24 h at 4 °C allowed preservation of a higher percentage of cells exhibiting linear motility in comparison to a control sample stored in PBS. Presented results indicate that proteins binding Zn(2+) ions have a shielding effect on the sperm plasma membrane and acrosome of spermatozoa, protecting these structures against consequences of cold shock.

Mitochondrial membrane potential profile and its correlation with increasing sperm motility

To investigate sperm mitochondrial integrity through analysis of mitochondrial membrane potential (Δψ) and to correlate the energy status with variations in sperm motility. Experimental study. Seminology Laboratory, University of Rome, Italy. Two hundred thirteen semen samples from the same number of patients, divided into two groups on the basis of their motility: group A, 185 samples with linear motility and group B, 28 samples with nonlinear motility. Evaluation of sperm motility. Sperm mitochondrial integrity evaluated with a fluorimetric method using the cationic lipophilic stain JC-1. The mean FL2 (percentage of sperm with high and low ∆ψ) for group A was 46.19±23.25 and for group B, it was 48.32±24.43. There was no significant difference between the groups. There was a positive correlation between both FL2 and linear motility and FL2 and sperm vitality in group A; both correlations were statistically significant. In group B, there was a positive correlation between FL2 and nonlinear motility and FL2 and sperm vitality; again, both correlations were statistically significant. Our data reveal a positive correlation between total motility and Δψ, suggesting that sperm motility may be dependent on the functional integrity of the mitochondria.

Single layer centrifugation with androcoll-p can be scaled-up to process larger volumes of boar semen.

The objective of this study was to scale-up the procedure for Single Layer Centrifugation (SLC) through AndrocollTM-P, as a preliminary step towords processing the whole ejaculate. The first experiment compared Single Layer Centrifugation using 4.5 mL and 15 mL extended ejaculate (SLC-4.5 and SLC-15, resp.), assessing sperm quality by objective motility analysis, morphology, viability, and the production of reactive oxygen species (ROS). In the second experiment, SLC-4.5 was compared to Single Layer Centrifugation with 25 mL extended ejaculate (SLC-25) using motility analysis and morphology. In both experiments, normal morphology and linear motility were significantly higher in the SLC-selected samples than in the uncentrifuged controls (P < .001), whereas total motility and membrane integrity were unchanged. Although ROS production was higher in the SLC-selected samples than in the controls (P < .01), this might have been due to the presence of antioxidants in seminal plasma in the latter. In conclusion, there was no difference in sperm quality between SLC-4.5 and SLC-15 samples, or between SLC-4.5 and SLC-25 samples, indicating that the SLC method can be scaled-up successfully.

Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction

Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe 2+) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n = 13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe 2+, EE-CAT plus Fe 2+, EE-GPx plus Fe 2+ and EE-SOD plus Fe 2+). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6 h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6 h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2 h after thawing (P < 0.05). Catalase supplementation, however, improved DNA integrity at 4 h (P < 0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6 h, linear motility at 6 h, mitochondrial activity at 6 h, membrane integrity at 2 and 6 h, and DNA integrity at 4 h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2 h after thawing (P < 0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2 h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe 2+ negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P < 0.05). After thawing, there were, however, no significant differences between the control plus Fe 2+ and the antioxidative enzymes supplementation plus Fe 2+ groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.

Effects of clinical stage and immunological status on semen analysis results in human immunodeficiency virus type 1-seropositive men.

Complete semen analyses including computer-assisted sperm motility and morphology assessments were performed to determine if semen and sperm differed between HIV-seropositive men and fertile controls, or differed with symptoms, or CD4+ peripheral cell count categories. Previous studies included small numbers of men and presented conflicting conclusions. Two hundred and fifty non-vasectomized HIV-seropositive men and 38 fertile controls each provided one semen sample. Non-parameteric statistics were used to analyse both continuous and nominal data. Fertile men had significantly greater semen volume, sperm concentration, percent motility, percent rapid and linear motility and total strictly normal spermatozoa than HIV seropositive men. Neither total number nor subtypes of leukocytes in semen differed between the two groups. Among the HIV seropositive men, significant differences in semen analyses were found between CD4+ cell count, clinical, and AIDS categories. Lower CD4+ cell counts (< 200 mm-3) were associated with significantly lower percent motility, percent normal sperm morphology by strict criteria, significantly more spermatids in semen, and higher percentages of teratozoospermia, oligoasthenoteratozoospermia and leukocytospermia. Healthier men, based on clinical categories, had significantly more normal shaped spermatozoa and fewer had azoospermia, oligoasthenoteratozoospermia or leukocytospermia. Many HIV-seropositive men have normal semen analyses, but as the disease progresses more defects are found, particularly in strict criteria sperm morphology.

231 SPERM SUBPOPULATIONS IN THE KOALA (PHASCOLARCTOS CINEREUS) EJACULATE

Koala semen contains a heterogeneous mixture of sperm morphotypes, mainly attributable to extreme degree of shape variability displayed by the hooked sperm head. By analogy with other species, we anticipate that the morphotypes may exhibit correspondingly different sperm-motility behaviors, largely caused by the differences in hydrodynamic interactions with the suspending media. This trend has been shown in human spermatozoa where motility behavior was demonstrably correlated with the sperm head morphology (Overstreet et al. 1981). In this study, we have investigated the heterogeneity of koala sperm motility profiles in semen in an effort to determine whether distinct sperm subpopulations within ejaculates are recognizable by the use of computer-assisted sperm motility analysis. Ejaculates from 5 males were collected by electroejaculation, then diluted and transported in Tris-citrate-glucose (TCG) diluent. Spermatozoa were washed through a 35–60% Percoll gradient to separate seminal plasma and the majority of the prostatic bodies from spermatozoa. Spermatozoa from the washed pellet were then diluted in TCG at 35°C, incubated for 10 min, and video recorded using a negative phase ×10 objective. Sperm motion parameters were then analyzed using the Hobson sperm tracker (Hobson Vision Systems, UK: Holt et al. 1996 J. Androl. 17, 587–596). Multivariate pattern analysis (PATN; CSIRO Australia; Abaigar 1999 Biol. Reprod. 60, 32–41) was used to distinguish 3 sperm subgroups, consistently shown in each ejaculate, within the data (1936 tracks × 6 kinetic parameters; VCL, VAP, MAD, BCF, ALH, LIN). After group allocation by PATN, all parameters showed significant differences between each of the groups (P &lt; 0.0001). Group 1, approximately 25% of the sperm tracks, showed profiles of spermatozoa with fast, non-linear motility (VCL 106.88 ± 28.15; BCF 3.23 ± 3.81; LIN 14.08 ± 10.20). Group 2, approximately 27% of sperm tracks, showed profiles of fast, linear motility (VCL 63.92 ± 13.50; BCF 7.90 ± 3.42; LIN 28.10 ± 12.15). Group 3, 48% of sperm tracks, showed profiles of slow, non-linear or circular patterns of motility (VCL 39.05 ± 11.92; BCF 0.02 ± 0.35; LIN 5.15 ± 4.88). The recognition of 3 clearly identifiable subgroups supports our hypothesis that heterogeneity of sperm motility patterns exists within koala ejaculates. These may be a reflection of the heterogeneity in sperm-head morphotypes in koala semen, but that remains to be investigated in more detail. The clear distinctions between these groups, and the observation that all 3 subpopulations exist in each of the ejaculates, also suggest that the spermatozoa exhibit functional differences, possibly related to biochemical or maturational status. Many thanks to Dr. Michael Pyne and Dr. Vere Nicholson and their teams and animals at Currumbin Wildlife Sanctutary and Dreamwolrd QLD for all their help and support for the collection of samples.

Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapedesis

The Rho GTPases comprise one of the eight subfamilies of the Ras superfamily of monomeric GTP-binding proteins and are involved in cytoskeletal organization. Previously, using a dominant negative construct, we demonstrated a role for RhoC GTPase in conferring invasive capabilities to PC-3 human prostate cancer cells. Further, we demonstrated that inactivation of RhoC led to morphological changes commensurate with epithelial to mesenchymal transition (EMT) and was accompanied by increased random, linear motility and decreased directed migration and invasion. EMT was related positively to sustained expression and activity of Rac GTPase. In the current study we analyze the individual roles of RhoA, RhoC and Rac1 GTPases in PC-3 cell directed migration, invasion and tumor cell diapedesis across a human bone marrow endothelial cell layer in vitro. Use of specific shRNA directed against RhoA, RhoC or Rac1 GTPases demonstrated a role for each protein in maintaining cell morphology. Furthermore, we demonstrate that RhoC expression and activation is required for directed migration and invasion, while Rac1 expression and activation is required for tumor cell diapedesis. Inhibition of RhoA expression produced a slight increase in invasion and tumor cell diapedesis. Individual Rho GTPases are required for critical aspects of migration, invasion and tumor cell diapedesis. These data suggest that coordinated activation of individual Rho proteins is required for cells to successfully complete the extravasation process; a key step in distant metastasis.

Two egg-derived molecules in sperm motility initiation and fertilization in the Pacific herring (Clupea pallasi)

Sperm of the Pacific herring are immotile at spawning. Two egg-derived molecules are capable of initiating sperm motility. One is herring sperm activating protein(s) (HSAPs) and the other is sperm motility initiation factor (SMIF). These two motility initiators differ in their location and association with the chorion, and in their isoelectric points and molecular weights. In this study we have investigated the roles of these two inducers with respect to motility and fertilization. Using computer analysis of sperm motility, we found that HSAPs, as well as the C-terminal HSAPs peptide, elicit a linear motility pattern, while SMIF induced a highly circular and asymmetric pattern. HSAPs induced a two-fold increase in intracellular calcium, whereas SMIF induced a four-fold increase of motility initiation. SMIF-exposed sperm, preloaded with BAPTA-AM, showed a more linear motility and this motility trajectory decreased with their fertilizing capability. The difference in intracellular calcium levels between HSAPs and SMIF is consistent with the observed linear and circular motility. In the absence of SMIF, HSAPs do not support fertilization. Fertilization is rescued in these experiments if SMIF is reintroduced. We propose that diffusible HSAPs are not essential for fertilization, but enhance sperm-egg collisions via linear motility. SMIF, which is bound to the micropylar region of the chorion, is required for fertilization and induces circular motility that is a prerequisite for sperm to enter the micropylar canal and fertilize the egg.

Directional Freezing of Equine Semen in Large Volumes

Despite its potential impact on the horse industry, sperm cryopreservation is not an established technology throughout the industry, for a number of reasons that include a reduction in pregnancy rate and increased cost per pregnancy. We have evaluated a novel directional freezing technique, based on a multi-thermal gradient (MTG), by comparing it with the conventional, controlled-rate cryopreservation method (CRCM). Ninety-seven ejaculates with > or =50% motility, collected from 31 stallions were each divided into two parts and subsequently frozen by either MTG or CRCM. Frozen samples were then stored in liquid nitrogen until thawing. The two treatments were evaluated by three methods: progressive linear motility (PLM), viability stain and hypoosmotic swelling (HOS) test. High correlation was found between the three evaluation methods for all post-thaw samples. Eighty-eight per cent of the ejaculates frozen by MTG had post-thaw PLM > or =35%, whereas only 59% of the ejaculates frozen by CRCM had such motility. Post-thaw evaluations of samples frozen by MTG and CRCM were: PLM - 50.2 +/- 1.5% and 37.4 +/- 1.5%, respectively; viability - 53.6 +/- 1.5% and 39.5 +/- 1.4%, respectively; membrane integrity, as evaluated by HOS - 36.2 +/- 1.3% and 26.5 +/- 1.1%, respectively. The differences according to all the evaluation methods were highly significant (p < 0.001), and the results indicate that freezing stallion semen by MTG is superior to CRCM.

Controlled cooling during semen cryopreservation does not induce capacitation of spermatozoa from two portions of the boar ejaculate

Cryopreservation imposes dramatic changes in boar sperm survivability but it is as yet unclear which part of the process affects the spermatozoa the most. The present study monitored, along the entire process of cryopreservation, the stability (PMS) of the architecture of the lipid plasma membrane and its integrity (PMI), as well as the kinetics of the processed spermatozoa using two portions from the boar ejaculate (P1 = the first 10 mL of the sperm-rich fraction, SRF; P2 = the rest of the ejaculate), frozen in a recently developed package, the MiniFlatPack (MFPs, 0.5 x 10(9) sperm/dose). Evaluation was made at four specific stages, viz. S1 = after collection (suspended in Beltsville thawing solution, BTS); S2 = at 15 degrees C (suspended in lactose-egg yolk, LEY); S3 = at 5 degrees C (suspended in LEY plus glycerol); and S4 = post-thaw. Both sperm kinetics (using computer-assisted sperm analysis, CASA) and PMS [i.e. the degree of lipid disorder and of the exteriorization of phosphatidylserine (PS) in the plasma membrane, measured by flow cytometry using Merocyanine-540 (M-540), and Annexin-V (AV) respectively], as well as plasma membrane integrity [PMI, i.e. the degree of membrane damage, measured using Yo-Pro-1 or propidium iodide (PI)] were assessed after incubation in BTS at 38 degrees C. Moreover, spermatozoa were challenged by incubation in modified Brackett-Oliphant medium (mBO+) with 37 mm of bicarbonate at 38 degrees C for 30 min, and their PMS and PMI further explored. Total sperm motility was significantly higher in P1 than in P2 along the entire process (S1-S4; p < 0.01), decreasing significantly at S4 for both fractions (p < 0.0001). The proportion of spermatozoa showing linear motility (LinM) was similar between ejaculate portions (P1 and P2), with a significant increase post-thaw (S4; p < 0.0001). During cooling (S1-S3) but not post-thaw (S4), lateral head displacement (LHD) differed between portions and changed along the stages (p < 0.01). Sperm velocity differed between portions in S1 (p < 0.01), but remained similar, independently of the portion, thereafter (S2-S4). Both PMS and the total number of live spermatozoa remained similar between S1 and S3 while incubated in BTS for both ejaculate portions. Sperm mortality increased post-thaw (S4) in both portions but the degree of lipid disorder remained low in the live cells (1.28% for P1; 1.55% for P2). Exposure to mBO+, on the other hand, significantly increased membrane lipid disorder along cooling (S1-S3; p < 0.0001), increasing the percentages of dead spermatozoa, especially post-thaw (around 70%, both portions). PS-exteriorization (AV) was not evident along the cryopreservation process in control (BTS) samples and exposure to mBO+ only induced minor variations. The data showed that kinetics, PMS and PMI of boar spermatozoa suspended in BTS (S1), LEY (S2) or LEY plus glycerol (S3) were maintained during controlled cooling but were altered by thawing, showing more characteristics of cell injury than of sperm capacitation. The spermatozoa were able to capacitate but the bicarbonate challenge destabilized the plasma membrane during initial cooling and accelerated membrane changes post-thaw. We conclude that capacitation of boar spermatozoa does not occur during controlled cooling.