A study on the technique of hemocytometric determination of sperm motility and sperm concentration in bull semen.
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Animals↗
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Phosphatidylinositol 3-kinase inhibition enhances human sperm motility and sperm-zona pellucida binding.
Various signalling pathways are involved in the regulation of sperm motility, capacitation, acrosome reaction and sperm-zona binding. Recent data pointed out an important role for phosphatidylinositol 3-kinase (PI3K) in human sperm motility. However, no study as of yet has been carried out to determine the effect of sperm treatment with the PI3K inhibitor LY294002 on other sperm parameters. In the present study, we investigated the role of PI3K on human sperm motility, acrosome reaction and sperm-oocyte binding by using this inhibitor. We demonstrate that in vitro incubation of washed unselected spermatozoa with LY294002 increased the percentage motility and progressive motility in asthenozoospermia patients as evaluated by computer-aided sperm analysis. The compound furthermore did not influence the acrosome reaction, whilst it (further) slightly enhanced sperm-oocyte binding. Our results therefore imply that PI3K negatively affects sperm motility and oocyte binding and might suggest a possible therapeutic role for PI3K inhibitors in the treatment regime for asthenozoospermia.
Acrosome Reaction↗
Effect of sperm morphology and motile sperm count on outcome of intrauterine insemination in oligozoospermia and/or asthenozoospermia.
Eighty-six couples with long-standing infertility and poor postcoital test, due to oligozoospermia and/or asthenozoospermia (68 cases) or mucus hostility (18 cases), were treated by 411 intrauterine inseminations (IUI) with motile sperm suspensions from the husband's semen. The pregnancy rate per couple in the group with abnormal semen was lower than in the group with mucus hostility (22% versus 38.9%). Influence of seminal and other parameters on outcome of IUI was assessed by discriminant analysis, and a significant correlation with pregnancy rate was found for motile sperm count and sperm morphology. Teratozoospermia (normal morphology less than 50%) affected the outcome of IUI both when associated with moderate oligozoospermia and/or asthenozoospermia (motile sperm count greater than or equal to 5 X 10(6)/mL) (success rate per couple: 11.1%), and, even more, when associated with severe oligozoospermia and/or asthenozoospermia (motile sperm count less than 5 X 10(6)/mL), where no pregnancy was achieved. In the absence of teratozoospermia, the success rate per couple both in severe and in moderate oligozoospermia and/or asthenozoospermia had similar results (33.3% versus 35.7%). In conclusion, the absence of teratozoospermia appears to be an effective criterion for selecting couples with infertility due to oligozoospermia and/or asthenozoospermia who may benefit from IUI.
Adult↗
Structural and immunocytochemical characterization of the Ginkgo biloba L. sperm motility apparatus.
Ginkgo biloba and the cycads are the only extant seed plants with motile sperm cells. However, there has been no immunocytochemical characterization of these gametes to determine if they share characteristics with the flagellated sperm found in bryophytes and pteridophytes or might give clues as to the relationships to nonflagellated sperm in all other seed plants. To determine characteristics of proteins associated with the motility apparatus in these motile sperm, we probed thin sections of developing spermatogenous cells of Ginkgo biloba with antibodies to acetylated and tyrosinated tubulin and monoclonal antibodies that recognize mammalian centrosomes and centrin. The blepharoplast that occurs as a precursor to the motility apparatus consists of an amorphous core, pitted with cavities containing microtubules and a surface studded with probasal bodies. The probasal bodies and microtubules within the blepharoplast cavities are labeled with antibodies specific to acetylated tubulin. Positive but weak reactions of the blepharoplast core occur with the centrosomereactive antibodies MPM-2 and C-9. Reactions to centrin antibodies are negative at this developmental stage. From this pre-motility apparatus structure, an assemblage of about 1,000 flagella and associated structures arises as the precursor to the motility apparatus for the sperm. The flagellar apparatus consists of a three-layered multilayered structure that subtends a layer of spline microtubules, a zone of amorphous material similar to that in the blepharoplast, and the flagellar band. Centrin antibodies react strongly with the multilayered structure, the transition zone of the flagella, and fibrillar material near the flagellar base at the surface of the amorphous material. Both the spline microtubules and all of the tubules in the flagella react strongly with the antibodies to acetylated tubulin. These localizations are consistent with the localizations of these components in pteridophyte and bryophyte spermatogenous cells, although the blepharoplast material surrounding and connecting flagellar bases does not occur in the seedless (nonseed) land plants. These data indicate that despite the large size of ginkgo gametes and the taxonomic separation between pteridophytes and Ginkgo biloba, similar proteins in gametes of both groups perform similar functions and are therefore homologous among these plants. Moreover, the presence of acetylated tubulin in bands of microtubules may be a characteristic shared with more derived non-flagellated sperm of other conifers and angiosperms.
Cell Movement↗
Sperm motility improvement: comparison of two methodologies of reproductive techniques.
The objective of this study was to compare two methods for the extraction of high motility sperm; washed semen and swim-up techniques, on 172 ejaculates from 53 patients with suspected infertility. High quality motile sperm had been essential for the success of the assisted reproductive procedures, in which these patients were enrolled. Semen analyses were performed according to the criteria recommended by the WHO. In both methods Ham's F-10 medium plus 7.5% human cord serum were used. The incubations were carried out at 37 degrees C +/- 1. The percentage of motile spermatozoa with forward progression was measured in the fresh sample and after incubation with both methods. The results were analyzed by the two-way analysis of variance. Motility rates were significantly higher in swim-up than fresh samples (mean 41.158%, SD 20.1 vs. 27.572%, SD 16.6; p < 0.0001) and washed semen compared with fresh sample (mean 36.966%, SD 16.3 vs. 27.572% SD 16.6; p < 0.0001).
Adult↗
Influence of system parameter settings on human sperm motility analysis using CellSoft.
The effect of changing CellSoft system parameter settings on measurements of motile sperm concentration and sperm movement characteristics (curvilinear and linear velocities, amplitude of lateral head displacement and beat/cross frequency) was investigated in semen. Specifically, the use of either four or 15 frames to distinguish motile from immotile cells was used in conjunction with a threshold curvilinear velocity of either 10 or 20 micron/s (recommended values: 4 and 10). In addition, a threshold for progressive motility based on linear velocity was also used by manual re-analysis of CellSoft Individual Cell Data printouts. Known reliable semen analysis methods or the analysis of manually reconstructed trajectories from videotape were used as standard methods for comparison. A curvilinear velocity threshold of 20 micron/s gave improved correspondence between CellSoft and standard methods for all characteristics evaluated. Whether a four or 15 frame setting was better varied with the measurement being made. However, using a linear velocity threshold to define the progressively motile sperm population provided the most acceptable values for sperm movement characteristics. Consequently, while substantial improvements in the measurements made by CellSoft can be obtained using different system parameters to those usually recommended, more reliable and certainly more useful information could probably be obtained if all analyses were performed on a defined population of progressively motile, and therefore potentially functional, spermatozoa.
Humans↗
Role of seminal osmolarity in the reduction of human sperm motility.
Mammalian sperm at ejaculation are suspended in the seminal plasma, a heterogeneous mixture deriving from the testicular/epididymal fluid and from secretions of seminal vesicles, prostate and bulbourethral glands. Biochemical characteristics of seminal fluid change along the male reproductive tract when considering its inorganic and organic composition and pH but it is known that in each region of the male genital tract seminal osmolarity is higher than that of serum. It has been previously demonstrated that in invertebrate and vertebrate sperm, seminal plasma osmolarity influences sperm motility and activity, and human sperm have been shown to possess osmosensitive calcium entry pathway that controls important functions such as acrosome reaction and oocyte penetration. In the present study, we have determined seminal plasma osmolarity in a large number of normozoospermic fertile and asthenozoospermic infertile subjects correlating it with sperm motility percentages and kinematic characteristics determined utilizing a computerized motion analysis system. Our results confirm that seminal plasma osmolarity is higher than that of serum (336.1 +/- 20.2 vs. 291.1 +/- 6.9 mOsm/L, respectively). Normozoospermic subjects show seminal osmolarity values that are significantly lower with respect to asthenozoospermic patients (317.8 +/- 12.2 vs. 345.2 +/- 22.6 mOsm/L, p<0.001), irrespective of the cause of asthenozoospermia. Seminal plasma osmolarity negatively correlates with sperm progressive motility percentages and kinetic characteristics (curvilinear velocity, linear velocity, linear coefficient and lateral displacements of sperm head). Furthermore, when sperm from fertile subjects were suspended in medium with an osmolarity increasing from 300 to 600 mOsm, sperm motility percentages and kinetics characteristics were progressively reduced and nearly abolished when medium osmolarity was 600 mOsm. On the contrary, when sperm from asthenozoospermic subjects with high semen osmolarity were resuspended in medium with lower osmolarity, sperm motility parameters improved significantly. Sperm motility parameters did not correlate with seminal plasma concentrations of sodium, potassium, chloride with a weak correlation only with seminal calcium concentration. No correlations are present between seminal plasma osmolarity and ionic composition. In conclusion, the present study confirms and extends the knowledge that, in human, seminal plasma osmolarity is higher than that of serum and demonstrates that seminal osmolarity influences sperm motility characteristics and then it may contribute to the pathogenesis of some forms of asthenozoospermia and male infertility.
Humans↗
Effects of nitric oxide on human spermatozoa: evidence that nitric oxide decreases sperm motility and induces sperm toxicity.
Endogenous nitric oxide (NO) is an important functional mediator in several physiological systems, including the reproductive system. However, when generated in excessive amounts for long periods, mainly during immunological reactions, NO is cytotoxic and cytostatic for invading microbes, as well as for the cells generating it and the tissues present around it. Since infertility associated with urogenital tract infection in males and females is also accompanied by reduced sperm motility and viability, it is possible that reduced fertility in these patients is due to NO-induced sperm toxicity. We therefore evaluated the direct effects of NO, chemically derived from S-nitroso-N-acetylpenicillamine (SNAP, 0.012-0.6 mM) and sodium nitroprusside (SNP, 0.25-2.5 mM), on the motility and viability of human spermatozoa. Furthermore, we tested whether inhibition of NO synthesis prevents sperm motility and viability by incubating washed total cells present in the semen (spermatozoa, round cells) with N-nitro-L-arginine-methyl-ester (L-NAME), a NO synthesis inhibitor. Treatment of purified spermatozoa with SNAP or SNP decreased forward progressive sperm motility and straight line velocity, and also increased the percentage of immotile spermatozoa in a concentration-dependent manner. Furthermore, the percentage of immotile spermatozoa positively correlated with the percentage of dead spermatozoa. In contrast to freshly prepared SNAP, SNAP preincubated for 48 h had no effect on the motility and viability of the spermatozoa. Furthermore, as compared to untreated controls, a significantly higher percentage of forward progressive sperm motility as well as viability (P < 0.05) was maintained in washed semen incubated with L-NAME (0.15 mM).(ABSTRACT TRUNCATED AT 250 WORDS)
Arginine↗
Multiple exposure photographic (MEP) technique: an objective assessment of sperm motility in infertility management.
Multiple exposure photography (MEP), an objective technique, was used in determining the percentage of motile sperms in the semen samples from 41 males being investigated for infertility. This technique was compared with the conventional subjective ordinary microscopy method of spermatozoal motility assessment. A satisfactory correlation was observed in percentage sperm motility assessment using the two methods but the MEP estimation was more consistent and reliable. The value of this technique of sperm motility study in the developing world is discussed.
Humans↗
The concentration of nitrite in seminal plasma does not correlate with sperm concentration, sperm motility, leukocytospermia, or sperm culture.
OBJECTIVE: To correlate the concentration of nitrite (the stable metabolite of nitric oxide) in seminal plasma with sperm number and motility, leukocytospermia, and sperm culture. DESIGN: Prospective study. SETTING: Academic research institution. PATIENT(S): Seventy normozoospermic or dyspermic men enrolled in an artificial insemination/in vitro fertilization program. INTERVENTION(S): Semen samples (n = 70) were checked for sperm concentration, total sperm count, sperm motility, seminal leukocyte concentration, and sperm culture; similarly, the concentration of nitrite in seminal plasma was measured by Griess reaction. MAIN OUTCOME MEASURE(S): Measurement of nitrite concentration in seminal plasma and its correlation with sperm concentration, total sperm count, sperm motility, leukocytospermia, and sperm culture. RESULT(S): The concentration of nitrite in seminal plasma does not correlate with sperm concentration, total sperm count, or with the proportion of immotile or rapid-forward motile spermatozoa. Moreover, the concentration of nitrite in seminal plasma is not significantly increased when sperm culture is positive, nor does it correlate with leukocyte concentration in semen. CONCLUSION(S): Our results do not support the hypothesis that in vivo nitric oxide synthesis affects sperm function; alternatively, our results could suggest that nitrite in the seminal plasma is not a sensitive marker of in vivo nitric oxide synthesis.
Fertilization in Vitro↗
Influence of complement on sperm motility.
Isolated sperm from normo-, oligo- and astheno-spermic men were incubated for 20 h in medium supplemented with 8% heat-inactivated or untreated human serum, and in medium with heated or untreated serum deficient in complement factor C3. Before and after incubation, sperm motility was assessed by means of a computer-assisted semen analyser. The results did not show significant differences between the motility of sperm incubated in heated or untreated serum. It is concluded that heating of homologous serum is not necessary for preserving sperm motility and in some cases may even be disadvantageous.
Blood↗
Enhancement of mouse sperm motility by the PI3-kinase inhibitor LY294002 does not result in toxic effects on preimplantation embryo development.
BACKGROUND: A reduced number of progressively motile sperm (as may occur in cases of asthenozoospermia or when cryopreserved spermatozoa are used for fertilization) limits the possibility of applying various assisted reproductive techniques (ARTs). We previously showed that incubation of sperm with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 increases sperm progressive motility and enhances the number of sperm recovered by capacitation protocols used in ART. METHODS AND RESULTS: In the present study, we investigate the motility-enhancing effects of this compound in epididymal mouse sperm, and examine the use of the mouse system to investigate the effect of LY294002 on oocyte fertilization and preimplantation embryo development. Our results show that neither pre-incubation of mouse spermatozoa with the inhibitor during in vitro capacitation nor the direct addition of LY294002 to the sperm-oocyte mixture significantly affects the process of fertilization and preimplantation development of embryos produced even when they developed in the presence of LY294002. CONCLUSIONS: The present data encourage the design of new drugs based on the molecular structure of LY294002, which may open up new options for the in vitro treatment of human/animal asthenozoospermia.
Animals↗
[A new method for determining sperm motility: the clinical application of sperm motile efficiency (author's transl)].
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Female↗
Comparison of four fluorochromes for the detection of the inner mitochondrial membrane potential in human spermatozoa and their correlation with sperm motility.
BACKGROUND: Sperm motility evaluation is associated with fertility in IVF programmes. The visual estimation of sperm motility is extremely subjective. Hence, alternative methods are required. Among them, determination of mitochondrial membrane potential (Deltapsi(m)) changes of spermatozoa using potentiometric dyes may be a reliable test to determine sperm quality. However, the use of the potentiometric dyes in sperm samples has not been compared. METHODS: We have studied sperm samples from 28 infertile patients enrolled in an IVF programme in flow cytometry after staining of spermatozoa with four commonly used potentiometric dyes. Sperm motility was evaluated visually. RESULTS: As expected, JC-1 seems to detect specifically Deltapsi(m) changes, CMX-Ros, DiOC(6)(3) and TMRE fluorescence is easily analysed and the latter three fluorochromes are particularly suitable for multiparametric staining. Irrespective of the Deltapsi(m)-dependent fluorochromes used to stain spermatozoa, a positive correlation was found between the percentage of Deltapsi(m)(high) cells and forward motility and also with high fertilization rates after IVF. CONCLUSION: The four fluorochromes may be useful for evaluation of sperm samples from infertile patients. The choice of the potentiometric dyes will depend on their fluorescence characteristics in order to use them in combination with other fluorescent markers.
Adult↗
Differential sperm motility scoring and sperm ATP concentrations.
Sperm motility is one of the most important parameters in semen analysis. In this study the motility scoring method of Hellinga was used. Adenosine triphosphate (ATP) is the main energy source used by the sperm flagellum to initiate and propagate forward motility. This study was conducted to evaluate the interrelationships between sperm ATP and differential motility. The differential motility method supplemented the conventional by which the quantitative and qualitative motility were estimated. There was a good correlation between the differential motility scoring method and sperm [ATP], although the latter did not necessarily improve the diagnostic capacity.
Adenosine Triphosphate↗
Bicarbonate stimulation of boar sperm motility via a protein kinase A-dependent pathway: between-cell and between-ejaculate differences are not due to deficiencies in protein kinase A activation.
Because poorly motile sperm samples can often be stimulated by treatments that increase intracellular levels of cyclic adenosine monophosphate (cAMP), it has been supposed that such samples are unable to maintain an adequate supply of the cyclic nucleotide with which to activate protein kinase A (PKA). To investigate this hypothesis, we incubated boar sperm samples with bicarbonate (a stimulator of adenylyl cyclase) and compared its effect with that of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate (cBIMPS, a highly permeable and stable cAMP analog). Videomicroscopy assessment of motility was followed by computer analysis of the sperm tracks to produce motility descriptor values for many individual cells in each sample, whence "cluster" analysis of these data identified groups of spermatozoa that differed in motility characteristics. Bicarbonate stimulation of motility was characterized by an increase in the linearity (LIN) and progressive velocity of part of the sperm population only. The size of this "fast linear" subpopulation varied considerably between ejaculates. However, treatment with cBIMPS did not induce significantly more "fast linear" sperm than treatment with bicarbonate. In further experiments investigating the role of protein kinases in motility control, bicarbonate stimulation was greatly inhibited by H89 (a specific inhibitor of PKA), whereas GF109203X and lavendustin A (inhibitors of protein kinase C [PKC] and protein tyrosine kinase [PTK], respectively) had essentially no effect. While inclusion of the protein phosphatase inhibitor calyculin stimulated motility, it failed to increase the overall percentage of "fast linear sperm" induced by bicarbonate. We conclude that intersperm and interejaculate differences in boar sperm motility are not due to inadequacy in cAMP supply or to ineffective PKA activity.
Animals↗
Nature's motility blockers: controlling human sperm motility machinery from the outside. Chemical characterization of a peritoneal fluid lipid that induces sperm immobilization.
A molecule isolated from the peritoneal fluids of women undergoing laparoscopy for in-vitro fertilization techniques has been chemically characterized and identified as 1-palmitic-3-phosphorylcholine (lysophosphatidylcholine, LPC). This lipid is able, at physiological concentrations, to completely inhibit sperm motility in vitro in a dose-dependent way. Synthetic LPC induced rapid and complete arrest of sperm motility when added to sperm suspensions at physiological concentrations without any damage to cell membranes. Taken together, these results suggest that LPC may represent a previously unrecognized in-vivo modulator of human sperm motility.
Ascitic Fluid↗
Comparison of sperm motility test methods (except computer-assisted sperm analysis) in rats under the condition of alfa-chlorohydrin treatment--collaborative investigation.
A comparison among rat sperm motility test methods including percent of motile sperm (% Motile), scoring method (Scoring), Ishii's method, Progressive Motility Test (PMT) and Sperm Quality Analyzer (SQA), was conducted using data gathered from eleven laboratories. As a unified study design, mature male rats were orally treated daily for approximately 1 week with alpha-chlorohydrin (ACH), which is known to affect the sperm motility at the epididymis, at dose levels of 2.5, 5 and 10 mg/kg, and then subjected to more than two test methods for sperm motility in each laboratory. Scoring (4 or 5 grades), Ishii's method, PMT and SQA showed high sensitivity for the detection of the effects of ACH, which were not considered to be inferior to a computer-assisted sperm analyzer (CASA). Longer incubation time before testing was considered to contribute to detecting the effects of ACH. In particular, we realized that Scoring was a favorable method even if the demerit of poor objectivity was allowed for. Percent Motile showed lower sensitivity than other test methods. The differences in sensitivity between % Motile and other methods were considered to be based on whether the defects of progressive motion could be detected. Although % Motile cannot clearly judge whether immotile sperm are dead or alive, the value is a great help for the interpretation of the result from other methods. Based on the characters for detectability, objectivity and efficiency, the most suitable method of sperm motility should be selected according to the purpose of the toxicity study.
Animals↗