Capacitation Medium Research Articles

Avaliação da capacidade de penetração de sêmen congelado de onça pintada (Panthera onca) em oócitos heterólogos

Assisted reproductive technologies can be viewed as one potential approach for safeguarding wild species. In this study were evaluated the fertility of captivity male jaguar (Panthera onca) and different capacitation media using the golden hamster zona free oocyte penetration assay. We used frozen/thawed semen from 3 animals housed at Bosque dos Jequitibas, Campinas/SP, to test the Percoll gradient, Swim-up and Swim-up + 1 h incubation (5% CO2 / 38°C), considering as penetration the spermatozoa head descondensation visualized within the oocyte. The results rate was greater for Percoll (26.5%) as compared to Swim-up (8.1%) (X2 = 19.93; p < 0.05). It was not observed penetration with Swim-up + 1 h incubation (5% CO2 / 38°C). It is concluded that Percoll and Swim-up are efficient methods to perform the golden hamster zona free oocyte penetration assay using frozen/thawed jaguar (Panthera onca) spermatozoa. The low rate of penetration could be related to the high rate of morphological abnormal spermatozoa observed in the samples examined.

Chlortetracycline staining patterns of frozen-thawed bull spermatozoa treated with beta-cyclodextrins, dibutyryl cAMP and progesterone.

Efforts to achieve complete chemical definition of media used for in vitro capacitation of bovine spermatozoa including removal of heparin purified from porcine intestinal mucosa are presented. Fluorescent staining with chlortetracycline (CTC), known to reflect changes coincident with sperm capacitation in certain species, was studied following treatments of frozen-thawed bull spermatozoa with beta-cyclodextrins, dibutyryl cAMP (dbcAMP) and progesterone in comparison with heparin. The CTC staining patterns (F, B and AR) were confirmed to correlate with known conditions that effectively prepare cryopreserved bull spermatozoa for fertilisation in vitro. In the absence of glucose, the routinely employed heparin-containing capacitating medium caused an increase in spermatozoa displaying the AR pattern. Both progesterone (100 microM) and dbcAMP (0.01-0.1 mM) were able to increase the proportion of B pattern stained sperm cells more than after exposure to control (mDM) conditions without a significant reduction in motility. Exposure to either dbcAMP or beta-cyclodextrins was accompanied by an increase in proportions of spermatozoa displaying the AR pattern over those seen in controls. Exposure to beta-cyclodextrins did not increase the proportion of B pattern stained spermatozoa. Comparison of spermatozoa from two bulls revealed differential responses of spermatozoa from different males to treatments with heparin and progesterone. In vitro fertilisation results demonstrated that previously cryopreserved bull spermatozoa could be capacitated in chemically defined conditions devoid of heparin or other biological components.

Reduction of intramembranous particles in the periacrosomal plasma membrane of boar spermatozoa during in vitro capacitation: a statistical study.

Membrane remodeling in the periacrosomal plasma membrane (PAPM) of boar spermatozoa during incubation in capacitation medium was examined by the freeze-fracture technique. In the preservation medium (PM) group, the major small (about 8 nm) intramembranous particles (IMP) and the minor large (> 10 nm) IMP were distributed evenly in the PAPM. The IMP-free area increased during capacitation. To correct the IMP-free area, arithmetically redistributed (ARD)-IMP density was used for statistical analysis. In the PM group, the mean density +/- SD of large IMP was 379 +/- 64 and 266 +/- 58/microm2, and that of small IMP was 1450 +/- 155 and 672 +/- 252/microm2 in protoplasmic (P) and external (E) faces, respectively. During capacitation, the significant (P < 0.01) reduction of large IMP density was encountered only in the E face of a few incubation groups, while that of the small IMP density occurred in the P face by 2 h. Consequently, reduction of the total IMP density of both faces was not significant in the large IMP, but it was significant (P < 0.01) in the small IMP. One-fifth of the total small IMP density reduced by 2 h. Filipin-sterol complexes (FSC) were numerous in the PAPM, and FSC-free areas also increased during capacitation. The mechanism of IMP-free area formation and the behavior of the small IMP in the PAPM during capacitation were discussed in relation to membrane stability.

Immunolocalization of proacrosin/acrosin in bovine sperm and sperm penetration through the zona pellucida.

The aim of the present work was to immunolocalize acrosin in bull spermatozoa incubated for up to 6 h in capacitating culture medium (TALP-heparin), in order to study the kinetics of its release during the acrosome reaction and in vitro sperm penetration. Six replicates from semen of one bull were used. Acrosin was localized by the silver-enhanced immunogold technique using anti-bovine acrosin monoclonal antibody ACRO-C2E5. Spermatozoa thus showed the presence of acrosin only at the acrosomal region. Four different patterns were seen: (1) no labeling: (2) intense labeling on the rim of the portion of the acrosome; (3) diffuse label over the entire acrosomal region; and (4) intense label over the entire acrosomal region. Spermatozoa incubated in capacitating medium for 4 h showed that unlabeled (pattern 1) spermatozoa decreased from 72% to 28% difference that was found to be significant (p<0.05). Patterns 3 and 4 increased from about 10% to 20-29%, (p<0.05). With further incubation (4-6 h), pattern 1 increased while patterns 3 and 4 decreased differences were not significant (p0.05). The incidence of pattern 2 did not change through the whole incubation period. Sperm penetration through the zona pellucida of in vitro matured bovine oocytes (57%) or empty zonae pellucida (70.5%) increased (p<0.05) as a function of sperm incubation time in capacitating medium. The presence of acrosin, as determined by the silver-enhanced immunogold technique, was highly correlated with sperm penetration of in vitro mature bovine oocyte (r=0.98) and cryopreserved zonae pellucidae (r=0.93) (p<0.01).

Effect of sperm diluents on the acrosome reaction in canine sperm

In this study we investigated the influence of sperm diluting media and temperature on the incidence of the acrosome reaction in dog sperm. Ejaculates were collected from 5 dogs, diluted with six different media and then incubated at 37 °C and 20 °C. Fluorescein isothiocynate conjugated peanut agglutinin (FITC-PNA) and ethidium homodimer as a vital stain were used in combination to determine the acrosomal status of viable spermatozoa, the technique was validated using electron microscopy. The outer acrosomal membrane of dog spermatozoa was shown to be the specific binding site for FITC-PNA. After 6 h of incubation, ejaculates diluted in media with a high Ca 2+ concentration showed a significantly higher percentage (means ± SD) of acrosome reacted spermatozoa [64 ± 7 and 58 ± 9 in sperm capacitation medium with (SP-TALP-1) and without BSA (SP-TALP-2), respectively] than those diluted in media with a low Ca 2+ concentration [36 ± 5, 39 ± 4, 18 ± 2 and 20 ± 4 in Canine Capacitation Medium (CCM), Egg Yolk Tris dog semen extender (EXT-1), Modified Egg Yolk Tris extender (EXT-2) and Modified CCM (MCCM), respectively]. The increase in the percentage of acrosome reaction (AR) was slower at 20 °C than at 37 °C. In addition, the percentage of viable acrosome reacted spermatozoa increased significantly from 19 ± 5 and 22 ± 3 in non-bound sperm to 27 ± 4 and 30 ± 6 in zona pellucida bound sperm (diluted in EXT-2 and MCCM, respectively). We conclude that the composition of the spermatozoa diluent has a marked effect on the incidence of the acrosome reaction. Therefore, both the media used to dilute dog sperm and the temperature at which the spermatozoa are handled are important factors to consider when processing spermatozoa for artificial insemination, IVF procedures or preservation.

Changes in the surface structure of the hamster sperm head associated with maturation, in vitro capacitation and acrosome reaction: an atomic force microscopic study.

Structural changes of the hamster sperm head surface associated with maturation, capacitation and acrosome reaction were examined by atomic force microscopy. Spermatozoa were taken from the initial segment and distal cauda of the epididymis, washed in a modified Tyrode solution and fixed by glutaraldehyde. Some sperms taken from distal cauda epididymides were incubated with the capacitation medium before fixation. All samples were attached on the glass slide, dried in a critical point drier and observed by atomic force microscopy. The sperm head surface was characterized by the presence of numerous round particles, approximately 40 and 60 nm in diameter. The distribution and density of these particles on the sperm surface were significantly different between the equatorial segment and post-acrosomal region in each sperm, and also between sperms under different conditions. The surface of the equatorial segment was rather smooth in sperms from the initial segment of the epididymis, but had many large (60 nm) particles in sperms from the distal cauda epididymides, suggesting that the large particles were glycoproteins which were secreted from the epididymis and attached to the sperm surface during maturation. The number of these particles dramatically decreased in both capacitated acrosome-unreacted and acrosome-reacted sperms. This finding supports the idea that glycoproteins are removed from the sperm surface during capacitation. Atomic force microscopic studies of the sperm head surface are expected to be used for future molecular studies on the cell surface components involved in the mechanism of maturation, capacitation and acrosome reaction.

Induction of dog sperm capacitation by glycosaminoglycans and glycosaminoglycan amounts of oviductal and uterine fluids in bitches.

Ejaculated sperm collected from 12 beagle dogs were incubated in canine capacitation medium (CCM), supplemented with 5 microg/ml chondroitin sulfate A (CS), 5 microg/ml hyaluronic acid (HA), or 5 microg/ml heparin (HP) for 7 hr at 38 degrees C in a 5% CO2 in air atmosphere to investigate the effects of glycosaminoglycans (GAGs) on dog sperm capacitation. The percentages of motile sperm, hyperactivated sperm (%HY), and acrosome-reacted sperm (%AR) in all media were examined after 4 hr and 7 hr of incubation. The oviducts and uteri of 9 anestrous and 18 estrous beagle bitches were removed under halothane inhalation anesthesia to measure the total GAG amounts in oviductal and uterine fluids. The lumens of the ampulla of the oviducts, isthmus of the oviducts, and the uterine horns were each flushed with 1 ml HEPES-EDTA fluid. Total GAG amounts in the flush fluids obtained were measured with a spectrophotometer. Sperm motility (51-59%), %HY (79-86%), and %AR (31-36%) in CCM supplemented with CS, HA, or HP were significantly higher after 7 hr of incubation than when incubated in CCM without GAGs (P<0.01 or 0.05). The mean total GAG amounts in the fluids from the ampulla and isthmus of the oviducts and the uterine horns in the estrous bitches were higher than in the anestrous bitches. These results indicate that GAGs in the oviductal and uterine fluids in estrous bitches are associated with in vivo sperm capacitation.

In vitro capacitation of fresh, chilled and frozen-thawed dog spermatozoa assessed by the chloretetracycline assay and changes in motility patterns.

The effect of preservation on capacitation status of dog spermatozoa was investigated. Split ejaculates from six dogs were assessed as fresh, chilled for 24 h and rewarmed, and frozen-thawed samples. Capacitation-like status was assessed using the chlortetracycline (CTC)-assay and the measurement of sperm motility patterns using a computer-assisted sperm analyzer. Evaluations were performed on washed spermatozoa immediately after dilution in a Tris-fructose-citrate buffer (TFC) or in canine capacitation medium (CCM), and at 2-h intervals during 8 h of incubation in 5% CO2 in air, at 37 degrees C. Preservation decreased significantly the proportion of uncapacitated spermatozoa. In TFC, at hour 0, chilled-rewarmed and frozen-thawed samples had a significantly lower proportion of uncapacitated, viable spermatozoa than the fresh samples (P<0.05) according to the CTC-assay. The time course of capacitation was accelerated in the preserved samples, compared to the fresh ones. During incubation in CCM, the mean time from hour 0 to when, according to the CTC-assay, the highest proportion of capacitated spermatozoawas present in the samples (time-to-peak), was 4 h for fresh and 2 h for chilled-rewarmed and frozen-thawed samples (P<0.1). The highest values for curvilinear line velocity (VCL) and lateral head displacement (LHD), thought to be descriptive of sperm hyperactivation, were also observed 4 and 2 h after incubation began, in the fresh and the preserved samples, respectively. The difference in time-to-peak for VCL and LHD between fresh, chilled-rewarmed and frozen-thawed semen samples was statistically significant (P<0.02). It can be concluded that based on the CTC-assay and the analysis of motility patterns, capacitation-like changes in dog semen seem to be both initiated and accelerated by the preservation procedures.

In vitro capacitation of dog spermatozoa as assessed by chlortetracycline staining

We developed an assay for detecting capacitation and acrosome status in dog spermatozoa using chlortetracycline (CTC) as a fluorescent probe. Sperm cells were stained after incubation in modified canine capacitation medium (mCCM). Calcium ionophore A23187 permitted the induction of acrosomal exocytosis of capacitated sperm cells. Spermac ® staining and transmission electron microscopy were used as control tests to detect acrosome-reacted spermatozoa. Three different patterns of CTC distribution in the spermatozoa were found. These patterns were similar to those observed in other mammalian species. The CTC test was used to monitor the time course of the capacitation process in dogs. It was found that the kinetics of capacitation in canine sperm cells are similar to those observed in other mammals.

Comparison of the longevity of motility of stallion spermatozoa incubated at 38°C in different capacitating media and containers

This study was designed to compare the effects of different media and containers on longevity of motility of spermatozoa during in vitro incubation at 38°C in either air or 5% CO 2 atmosphere. Three ejaculates were collected from each of 4 stallions. The media tested were skim milk-glucose, modified Krebs/Ringer and Hank's salts solution for incubation in an air atmosphere, and modified Krebs/Ringer and Brackett and Oliphant (BO) defined medium for incubation in a 5% CO 2 atmosphere. All samples were incubated in 5-mL borosilicate glass tubes filled with 3 mL of extended spermatozoa, 5-mL borosilicate tubes filled with 6 mL (topped) of extended spermatozoa, 35-mm Petri dishes filled with 3 mL of extended spermatozoa, and 35-mm Petri dishes with 200-μL microdroplets of extended spermatozoa under sterile mineral oil. For all treatments, individual samples were removed at 2, 4, 6 and 12 h of incubation to determine the percentage of motile cells. Overall, spermatozoa incubated in Petri dishes in both 3-mL and microdroplet treatments had significantly higher motility than those incubated in glass tubes (P<0.01). At 6 and 12 h of incubation in Petri dishes, progressive motility was significantly higher for spermatozoa extended in the Hank's salts solution than in the other media. Both the medium and container used significantly affected the longevity of motility of spermatozoa incubated at 38°C.

Penetration by stallion sperm of zona-free bovine oocytes matured in equine preovulatory follicular fluid

The purpose of this study was to determine how long canine spermatozoa remain motile and with intact membranes when maintained within epididymides stored at 4 °C, and to determine whether such stored spermatozoa are able to bind to canine zonae pellucidae. Testes with attached epididymides, obtained from 32 dogs (26 purebred; six mixed breeds) at orchiectomy, were refrigerated at 4 °C, and spermatozoa were collected from caudae epididymides at nine time intervals ranging from 5 to 192 h. The effects on spermatozoa that had been refrigerated within epididymides for various times were determined by assaying sperm motility, integrity of plasma membranes and of acrosomes, and measuring binding of membrane-intact spermatozoa to canine zonae pellucidae. Membrane integrity was assessed using a double fluorescent dye, and acrosome integrity by staining with Pisum sativum agglutinin. For the zona-binding assay at various refrigeration time points, duplicate sets of six oocytes each, isolated from ovaries retrieved at elective ovariohysterectomy, were placed into 100 μl droplets of sperm capacitation medium containing 5×106 spermatozoa/ml. One minute later, oocytes were rinsed vigorously by pipetting, and then incubated for 1 h at 38.5 °C in a humidified atmosphere of 5% CO2 in air; the number of membrane-intact spermatozoa bound to zonae were counted. There was no significant decrease in membrane integrity and acrosome integrity of spermatozoa recovered from epididymides stored at 4 °C within the first 48 h of refrigeration. In contrast, sperm motility decreased significantly within the first 5 h of refrigeration (P<0.05), but then declined more gradually thereafter. Some spermatozoa recovered from epididymides that had been refrigerated for 192 h retained their capability to bind to zonae pellucidae, although the mean number of refrigerated spermatozoa (0.4) bound to zonae was less than that of fresh samples (9.0). Membrane integrity of spermatozoa recovered from epididymides refrigerated for various times was highly correlated (r=0.88) with sperm motility. Even after storage for 192 h (8 days) at 4 °C, motile spermatozoa could be recovered from the epididymides, and such refrigerated spermatozoa were capable of binding to zonae. We interpreted these data to indicate that it might be possible to recover functional spermatozoa from postmortem specimens of domestic and nondomestic canids.

Effects of medium containing heparin and theophylline on capacitation and metabolic enzyme activities of ejaculated spermatozoa from dogs with asthenozoospermia.

The percentages of motile sperm (%MO), hyperactivated sperm (%HA), and acrosome-reacted sperm (%AR) of four beagle dogs with asthenozoospermia (AS) and five normal beagle dogs were determined during 7 h of incubation. The metabolic enzyme activities of the sperm was examined after 0 and 4 h of incubation. The sperm were incubated in canine capacitation medium (CCM) and CCM containing either 20 microg ml(-1) heparin (HE), 10 microg ml(-1) theophylline (TH) or 20 microg ml(-1) HE + 10 microg ml(-1) TH in glass tubes at 38 degrees C under 5% CO2 in air. The %HA and %AR were determined by counting the sperm exhibiting star-spin like movement and by the triple stain technique. The spermatozoa in HE + TH CCM were homogenized and centrifuged, and the metabolic activities of hexokinase, fructokinase, glucose-6-phosphodehydrogenase (G6PD), and pyruvate kinase in the sperm cytosol in the supernatant was measured with a spectrophotometer. The mean %MO and %HA values of both AS and normal dogs in the four types of CCM were highest in HE + TH CCM, with a mean %HA in HE + TH CCM of 78 +/- 5% (S.E.) after 7 h of incubation. However, there was little difference in %AR among the four types of CCM. The mean activities of the four enzymes in the sperm of AS dogs before incubation was significantly lower than in the sperm of normal dogs (P < 0.05, 0.01). However, after 4 h of incubation the activities of all enzymes in the sperm of both AS and normal dogs was clearly higher in HE + TH CCM than in the control CCM. These findings indicate that HE and TH in the medium are effective inactivating metabolic enzymes, maintaining longer sperm motility, and efficiently inducing HA even of the sperm of AS dogs.

Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa.

While hyperactivated motility is known to be a concomitant of capacitation, and a prerequisite for fertilization, the specific interdependence of capacitation and hyperactivation in human spermatozoa has not been investigated. This study was designed to determine the effect of seminal plasma contamination on the expression of hyperactivated motility and the relationship between hyperactivation and capacitation, since seminal plasma contains decapacitation factor(s). Seminal plasma was obtained by centrifugation of aliquots of liquefied semen layered over 1.5 ml 40.5% Percoll and mixed with human tubal fluid (HTF) medium containing 30 mg/ml human serum albumin (HSA) (HTF) to a final concentration of 5% (v/v) seminal plasma (SP). Motile spermatozoa were isolated from the remainder of the semen by swim-up into either HTF or SP medium. Samples were taken from each treatment immediately post-harvest (0 h) and after 60 min at 37 degrees C (1 h) for hyperactivation and capacitation assessment. The treatments were then divided into two portions, centrifuged and resuspended in either HTF or SP, giving HTF control and SP control treatments and two crossover treatments, 1 h HTF then 1 h SP (H/SP) and 1 h SP then 1 h HTF (SP/H). All tubes were incubated for a further 60 min at 37 degrees C before aliquots were taken for hyperactivation and capacitation assessments. Hyperactivation was estimated using an IVOS v10.6t (Hamilton Thorne Research, Beverly, MA, USA) 60 Hz CASA instrument, and capacitation was estimated using the chlortetracycline (CTC) method. The presence of seminal plasma in the capacitation medium for 60-120 min post-swim-up inhibited the development of hyperactivated motility. This inhibition was reversible, and was not prevented by preincubation for 1 h in HTF medium. There was no difference in the CTC binding patterns between treatments at 2 h, indicating that the capacitation-associated membrane changes were not affected by the presence of a low concentration of seminal plasma. There was no correlation between percentage capacitated and percentage hyperactivated spermatozoa for any treatment. Since the proportions of hyperactivated spermatozoa and capacitated spermatozoa were not related, we conclude that the processes leading to hyperactivation and to the membrane changes associated with capacitation are not tightly interlinked and consider this finding to be due to hyperactivated motility being associated with flagellar movement, while the CTC assay assesses changes in the Ca2+ levels of the sperm head plasma membrane.

Effect of sperm capacitation and fertilization media on IVF and early embryo development of prepubertal goat oocytes

Experiments were carried out to develop an improved IVF system for prepubertal goat oocytes matured in vitro. Cumulus oocyte complexes (COC) were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM199 supplemented with 20% estrous goat serum (EGS) + 10 μ/mL FSH + 10 μg/mL LH + 1 μg/mL estradiol 17β for 27 h at 38.5°C in 5% CO 2 in air. In Experiments 1 and 2, freshly ejaculated spermatozoa were capacitated in 1 of 3 media: TALP/H, modified Defined Medium (mDM) and mH-M199 with 50 μg/mL heparin for 45 min. Matured oocytes were fertilized in TALP, mDM or mH-M199 in Experiment 1 and in TALP in Experiment 2. In Experiment 3, three media were used for sperm capacitacion and fertilization: Treatment A (control group): spermatozoa were capacitated in mDM with 50 μg/mL heparin for 45 min and fertilized in TALP medium with 1 μg/mL hypotaurine; Treatment B: spermatozoa were capacitated in mDM with 50 μg/mL heparin + 388 μg/mL caffeine for 30 min and fertilized in TALP medium without hypotaurine; Treatment C: spermatozoa were capacitated in mDM with 50 μg/mL heparin for 45 min and fertilized in TALP medium with PHE (20 μM penicillamine, 10 μM hypotaurine and 2μM epinephrine). At 24 h post insemination, the ova were transferred to a granulosa cell monolayer, and early embryo development was evaluated until Day 8. In experiment 2, the results show, that mDM plus heparin for sperm capacitation and TALP medium with hypotaurine for oocyte fertilization provided the highest proportion of penetrated oocytes, both total number (79.6%) and normal fertilization (55.1%), whereas the use of caffeine (44.6 and 31.2%, total and normal fertilization rate, respectively) and PHE (31.8 and 20.6%, total and normal fertilization rate, respectively) as motility enhancers did not improve the results obtained in the control group (48.7% and 37.2%, total and normal fertilization rate, respectively). These were no differences for the results of morulae and blastocysts.

Sperm capacitation is, after all, a prerequisite for both partial and complete acrosome reaction

The acrosome reaction (AR) – an essential step in mammalian fertilization – can occur, according to the consensus, only in capacitated spermatozoa. In apparent contrast, recent reports have demonstrated that human spermatozoa incubated in vitro in an albumin-free medium and therefore believed to be non-capacitated, do undergo the AR. With the aim of determining unequivocally whether or not capacitation is required for the AR and whether albumin is essential for capacitation, we compared the potential to undergo partial and complete AR (induced by phorbol myristate ester or by the Ca 2+ ionophore A23187) between human spermatozoa incubated in a capacitating medium, albumin-free medium, and non-capacitating medium. The results clearly demonstrate that capacitation is, after all, a prerequisite for both partial and complete AR. Albumin, on the other hand, is essential only for acquiring the capacity to undergo complete, not partial AR.

Increase of Intracellular Calcium Is Not a Cause of Pentoxifylline-Induced Hyperactivated Motility or Acrosome Reaction in Human Sperm

Objective: To investigate the effects of the phosphodiesterase inhibitor pentoxifylline on hyperactivated motility and acrosome reaction in human sperm and to determine whether its stimulatory effects occur via increased intracellular calcium levels. Design: Prospective study. Setting: Academic tertiary care facility. Participant(s): Healthy male donors. Intervention(s): The effects of pentoxifylline on hyperactivated motility, acrosome reaction, and intracellular calcium were studied and compared with the effects of progesterone. Thapsigargin, a known mobilizer of intracellular calcium, also was used as positive control. Main Outcome Measure(s): Hyperactivated motility was assessed by computer-assisted sperm motion analysis using the HTM-IVOS, acrosome reaction was evaluated with the fluorescent probe fluorescein isothiocyanate-labeled Pisum sativum agglutinin, and intracellular calcium was determined by fura-2 using spectrofluorometry. Result(s): Pentoxifylline significantly increased both hyperactivated motility and acrosome reaction. Enhancement of hyperactivated motility by pentoxifylline in the capacitation medium persisted for up to 5 hours after pentoxifylline was washed from the medium. It also enhanced the percentage of acrosome-reacted spermatozoa after 4 hours of incubation. These effects occurred in the presence of a marginally significant decrease in intracellular calcium. Conclusion(s): Pentoxifylline stimulates hyperactivated motility and acrosome reaction in spermatozoa from fertile men. Its stimulatory effects occur through mechanism(s) other than increase in intracellular calcium.

Factors influencing human sperm kinematic measurements by the Celltrak computer-assisted sperm analysis system

This study examines the effect of varying several factors, both extrinsic and technology-dependent, on the reconstruction of human sperm trajectories and the derived kinematic measurements using videotapes and the Motion Analysis Celltrak/S instrument. In semen samples from normal healthy men, curvilinear (VCL) and straight line velocities (VSL) were found to increase 1.5-fold, and linearity (LIN) of trajectories and amplitude of lateral head displacement (ALH) increased 1.25-fold when the temperature of analysis was raised from 24 to 37 degrees C. Only VCL and VSL were found to increase significantly between 24 and 37 degrees C for sperm samples selected by Percoll gradient and incubated in a capacitating medium. An analysis chamber of 20 microm depth was found to be adequate for seminal sperm samples while for Percoll-selected sperm samples the analysis in a 50 microm depth provided the highest proportions of spermatozoa with the highest VCL and the largest ALH. The grey level detection threshold required careful adjustment: using a threshold lower than the optimal threshold produced spurious sperm trajectories for seminal sperm samples and rejected some trajectories for Percoll-selected sperm samples. Definition of the appropriate frame rate and maximum burst speed was critical for valid trajectory reconstruction and therefore adequate derived kinematic measurements. Optimal values of these parameters were found to be 30 Hz and 400 microm/s for seminal spermatozoa and 60 Hz and 700 microm/s for selected spermatozoa. The optimal values of 'ALH path-smoothing factor' used to calculate average path and ALH were 5-10 points for seminal spermatozoa analysed at 30 Hz and 15-20 points for selected spermatozoa analysed at 60 Hz. We propose a set of standard conditions for reliable kinematic analysis of human spermatozoa using the Celltrak/S system.

Treatment of human spermatozoa with seminal plasma inhibits protein tyrosine phosphorylation.

It has long been known that seminal plasma contains factors that influence the fertilizing capacity of spermatozoa in many different ways. However, little is understood of the biochemical cascades triggered when spermatozoa and seminal plasma interact. In this study, we examined how incubation with seminal plasma affected protein tyrosine phosphorylation in human spermatozoa. Increased protein tyrosine phosphorylation is a hallmark of sperm capacitation in several mammalian species, including human. Seminal plasma blocks protein tyrosine phosphorylation when added to washed, non-capacitated spermatozoa. Removal of seminal plasma and incubation in capacitating medium led to partial recovery of the tyrosine phosphorylation cascade. Addition of seminal plasma to a suspension of spermatozoa previously incubated for 5 h under capacitating conditions decreased the level of tyrosine phosphorylation on all proteins in a dose-dependent manner. In this case, the phosphotyrosine signal did not increase upon removal of seminal plasma followed by overnight incubation in fresh capacitating media, indicating that removal of seminal plasma was necessary but not sufficient for protein tyrosine phosphorylation to occur. These results indicate that human seminal plasma contains factors that influence the tyrosine phosphorylation status of human spermatozoa.

Changes in semen quality and in vitro sperm capacitation during various frequencies of semen collection in dogs with both asthenozoospermia and teratozoospermia.

Eight male dogs with both asthenozoospermia and teratozoospermia were used in this study. In experiment 1, semen was collected 10 times at intervals of 48 hr, 24 hr and 12 hr in 4 of the 8 dogs, and the semen quality was evaluated. In experiment 2, semen was collected 5 times at 24-hr intervals in the other 4 dogs. The spermatozoa collected on day 1 and day 5 were incubated for 4-6 hr in Canine Capacitation Medium, and the percentages of hyperactivated sperm (%HA), acrosome-reacted sperm (%AR), and the zona pellucida-binding sperm count (ZP sperm count) were assessed. The results of experiment 1 showed that the percentage of motile sperm increased and the percentage of abnormal sperm decreased markedly as the intervals between semen collections became shorter. When semen was collected at 12-hr intervals, the percentage of motile sperm increased from about 65%, the value before frequent collection was started, to about 80%, and the percentage of sperm with abnormal tails decreased from 30% to 15%. In experiment 2, the percentages of HA and AR, and the ZP sperm count in specimens collected on day 5 were higher than those in specimens collected on day 1, and the differences in % HA and in ZP sperm count were significant (P<0.05). The results demonstrated that sperm motility, abnormality, and potential fertility in dogs with asthenozoospermia and teratozoospermia can be temporarily improved by frequent semen collection.

Induction of dog sperm capacitation by oviductal fluid.

Four estrous beagles were inseminated with 1 x 10(8) sperm into both the right and left uterine horns, and the uterine horn and oviduct on one side were removed under anesthesia after 7 hr and 24 hr, respectively. The lumen of the uterine horns and oviducts was flushed with canine capacitation medium (CCM), and movement of the sperm in CCM was assessed by phase-contrast microscopy. In a second experiment, ejaculated sperm obtained from 5 normal beagles was incubated in CCM supplemented with oviductal flush fluid (OF-CCM) at 38 degrees C with 5% CO2 in air. Motility of sperm, and percentages of hyperactivated sperm (%HA) and acrosome-reacted sperm (%AR) among freely swimming (FS) sperm were investigated until 24 hr after the start of incubation. After 7 hr of incubation the sperm was coincubated with canine oocytes in OF-CCM for 2 hr, and the number of zona pellucida-binding (zona-binding) sperm was then counted. The %HA among the sperm in the oviductal flush fluid both 7 hr (mean +/- S.E.; 15.0 +/- 2.4%) and 24 hr (77.5 +/- 5.2%) after intrauterine insemination were significantly higher than in the uterine flush fluid (P < 0.05, 0.01, respectively). The motility and %HA among FS-sperm in OF-CCM were higher than in the control medium without oviductal fluid. However, there was no difference in the %AR between OF-CCM and control medium. The number of zona-binding sperm in OF-CCM (8 +/- 1) was significantly greater than in control medium (5 +/- 1) (P < 0.05). These results suggest that oviductal fluid in the estrous bitch maintains sperm motility and induces sperm capacitation.