Sperm morphophysiology in different sections of the rooster reproductive tract / Morfofisiología espermática en diferentes secciones del tracto reproductivo del gallo

It is important to know the morphological changes that occur in the spermatozoa of rooster during their passage through the reproductive tract, which help to understand what they acquire their fertilization capacity. The morphophysiological changes related to the capacitation and acrosomal reaction processes in the different segments of the rooster reproductive system were analyzed. Samples were obtained from various regions of the rooster reproductive tract and dorso-ventral massage to obtain ejaculates, 25 roosters were used Rhode Island Red with proven fertility, assessments were performed with chlortetracycline and Lectin WGA-FITC to determine the morphophysiological parameters. Sperm motility increases (p<0.05) during the passage of spermatozoa from the testis until they are ejaculated. The parameters of viability and morphology also show differences (p<0.05) in the different segments of the tract. Sperm morphometry shows a spermatic contraction (p<0.05) in the cranial and medial segments of the vas deferens. The acrosomal reaction capacity evaluated with chlortetracycline (CTC) or Wheat germ agglutinin (WGA), was evident increasing the parameters (p<0.05) with the use of the perivitelline layer in the spermatozoa of the reproductive tract and of the ejaculate. Spermatozoa of the reproductive tract of the rooster demonstrate acrosomal reaction capacity without requiring a previous sperm capacitation condition. On the other hand, they do not show parameters of incapacity, which implies that they cannot be stored in any segment of the reproductive tract.

Es importante conocer los cambios morfológicos que se producen en los espermatozoides del gallo durante su paso por el tracto reproductivo y que ayudan a comprender como adquieren su capacidad de fertilización. Se analizaron cambios morfofisiológicos relacionados con los procesos de capacitación y reacción acrosomal de los espermatozoides presentes en los diferentes segmentos del tracto reproductor del gallo. Se obtuvieron espermatozoides de diferentes regiones del tracto reproductor del gallo y de espermatozoides de eyaculado. Se usaron 25 gallos Rhode Island Red con fertilidad probada. Se realizaron evaluaciones básicas, con clortetraciclina (CTC) y lectina Wheat germ agglutinin conjugada con isotiosionato de fluoresceína (WGA-FITC) para determinar los parámetros morfofisiológicos. La motilidad del esperma aumenta (P<0,05) durante el paso de los espermatozoides desde el testículo hasta que son eyaculados. Los parámetros de viabilidad y morfología también muestran diferencias (P <0,05) en los diferentes segmentos del tracto. La morfometría mostró una contracción de los espermatozoides (P<0,05) en los segmentos craneal y medial del conducto deferente. La capacidad de reacción acrosomal evaluada con clortetraciclina CTC o WGAFITC, fue evidente al aumentar los parámetros (P<0,05) con el uso de membrana perivitelina en los espermatozoides del tracto reproductivo y del eyaculado. los espermatozoides del tracto reproductivo del gallo demuestran capacidad de reacción acrosomal sin requerir una condición previa de capacitación espermática. Por otro lado, no muestran parámetros de descapacitación espermática lo que implica que no pueden almacenar en ningún segmento del tracto reproductivo.

Resveratrol protects human sperm against cryopreservation-induced injury / 中华男科学杂志

<p><b>Objective</b>To investigate the effects of resveratrol in the cryopreservation medium on the quality and function of post-thaw sperm.</p><p><b>METHODS</b>Semen samples were obtained from 50 normozoospermic and 50 oligoasthenozoospermic men, liquefied and then cryopreserved in the glycerol-egg yolk-citrate (GEYC) medium with or without 30 μmol/L resveratrol. Sperm motility, viability and acrosome reaction (AR) were examined before and after thawing. Sperm lipid peroxidation and the level of reactive oxygen species (ROS) were measured using commercial malondialdehyde (MDA) and the ROS assay kit. Sperm mitochondrial membrane potential (MMP) and DNA damage were determined by Rhodamine 123 staining and TUNEL.</p><p><b>RESULTS</b>The percentage of progressively motile sperm (PMS), total sperm motility, sperm viability, MMP and AR were significantly decreased (P <0.05) while the levels of sperm ROS, MDA and DNA fragmentation index (DFI) remarkably increased in both the normozoospermia and oligoasthenozoospermia groups after cryopreservation as compared with those in the fresh ejaculate (P <0.05). In comparison with the non-resveratrol control, the post-thaw sperm cryopreserved with 30 μmol/L resveratrol showed markedly higher PMS (［32.7 ± 4.8］ vs ［43.1 ± 6.3］ %, P <0.05), total motility (［44.8 ± 6.9］ vs ［56.9 ± 7.4］ %, P <0.05), viability (［52.3 ± 6.1］ vs ［67.5 ± 5.6］ %, P <0.05), MMP (［56.5 ± 7.0］ vs ［63.4 ± 7.5］ %, P <0.05) and AR (［16.6 ± 3.8］ vs ［26.3 ± 4.7］ %, P <0.05) but lower ROS, MDA and DFI (all P <0.05) in the normozoospermia group, and so did the post-thaw sperm in the oligoasthenozoospermia group, with a particularly lower DFI (［28.5 ± 4.8］ vs ［36.3 ± 5.7］%, P <0.01).</p><p><b>CONCLUSIONS</b>Resveratrol in the cryopreservation medium can improve the quality and function of post-thaw human sperm by reducing cryopreservation-induced sperm injury and the level of ROS.</p>

Plasminogen activator inhibitor 1 and Antipain preserve acrosome integrity of bovine spermatozoa during cryopreservation / Inibidor do ativador do plasminogênio 1 e antipaína preservam a integridade do acrossoma de espermatozoides bovinos durante a criopreservação

Seminal plasma contains serine proteases and serine protease inhibitor, which are involved in mammalian fertilization, and the inhibitors can be applied to prevent cold-induced sperm capacitation. The effects of different concentrations of two serine protease inhibitors were analyzed, Plasminogen activator inhibitor 1 - PAI-1 (70ƞg, 140ƞg and 210 ƞg) and Antipain (10µg, 50µg and 100µg) as supplementation to bovine semen cryopreservation extender. The effects of the inhibitors on the sperm parameters (sperm kinetics - CASA, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, sperm defects and acrosome reaction rate) were evaluated in the post-thaw semen. Cryopreservation of sperm with Antipain decreased post-thaw kinetic parameters of MP, VSL, LIN, SRT and the percentage of hyper-activated sperm while PAI-1 (210 ƞg) decreased VSL and LIN. Antipain and PAI-1 had no effect on the integrity parameters of the plasma membrane, mitochondrial membrane potential and sperm defects. Sperm cryopreserved in the presence of Antipain and PAI-1 (70 and 140 ƞg) preserved acrosome integrity, as they were able to complete the in vitro acrosome reaction. In conclusion, the serine protease inhibitors, Antipain and PAI-1 (70 and 140ƞg) are able to preserve the acrosome integrity of cryopreserved bovine sperm.(AU)

A criopreservação é parcialmente prejudicial à fertilidade do sêmen de bovinos e induz mudanças semelhantes à capacitação em espermatozoides. O plasma seminal contém serina-proteases e inibidores de serina-proteases que estão envolvidos na fertilização de mamíferos, e os inibidores podem ser aplicados para evitar uma capacitação espermática induzida pelo frio. Analisaram-se os efeitos de diferentes concentrações de dois inibidores de serina-proteases, inibidor do ativador do plasminogênio 1 - PAI-1 (70ƞg, 140ƞg e 210ƞg) e antipaína (10µg, 50µg e 100µg) na suplementação ao diluidor de criopreservação de sêmen bovino. Trinta e seis ejaculados de quatro bovinos Curraleiro Pé-Duro foram usados para criopreservação. Os efeitos dos inibidores sobre os parâmetros dos espermatozoides (cinética espermática - CASA, integridade acrossomal, integridade da membrana plasmática, potencial de membrana mitocondrial, defeitos espermáticos e taxa de reação acrossomal) foram avaliados no sêmen pós-descongelamento. A criopreservação de espermatozoides com antipaína diminuiu os parâmetros cinéticos pós-descongelamento de MP, VSL, LIN, SRT e a porcentagem de espermatozoides hiperativados, PAI-1 (210ƞg) diminuiu VSL e LIN. Antipaína e PAI-1 não tiveram efeitos nos parâmetros de integridade da membrana plasmática, no potencial de membrana mitocondrial e nos defeitos espermáticos. Espermatozoides criopreservados na presença de antipaína e PAI-1 (70 e 140ƞg) preservaram a integridade acrossomal, assim como foram capazes de completar a reação acrossômica in vitro. Em conclusão, os inibidores de serina-proteases, antipaína e PAI-1 (70 e 140ƞg) são capazes de preservar a integridade acrossomal de espermatozoides criopreservados de bovinos.(AU)

Efecto de la hiperviscosidad seminal sobre la integridad acrosómica y la movilidad espermática antes y después de la criopreservación. / Effect of seminal hyperviscosity on acrosomal integrity and sperm motility before and after cryopreservation

La criopreservación del semen es una herramienta útil en la reproducción asistida, la cual puede tener impacto en las características espermáticas durante el congela miento y el descongelamiento. El objetivo de este estudio fue valorar la integridad del acroso ma y la movilidad de los espermatozoides criopreservados y descongelados provenientes de muestras hiperviscosas y no viscosas. Se realizó el espermograma, la integridad del acrosoma, el espermocultivo y los niveles de los marcadores de glándulas accesorias en 60 muestras de semen. Cada alícuota de semen fue inmersa en un crioprotector comercial para congelar a -196°C. Transcurridos 30 días, éstas fueron descongeladas y en el sedimento celular espermá ticesuspendido se evaluó la movilidad y la integridad acrosómica, disminuyendo significa tivamente la movilidad progresiva (p<0,05), la vitalidad espermática (p<0,005) y la integridad acrosómica (p<0,05); dicho descenso fue más evidente en las muestras hiperviscosas. La viscosidad del semen fresco se relacionó inversamente con la movilidad y la integridad del acrosoma antes y después del congelamiento (p<0,05). En veinte muestras de semen se iden tificó la presencia de microorganismos y de anticuerpos IgA anti C. trachomatis , de las cuales quince muestras en la reproducción hiperviscosas. El aumento de la viscosidad seminal y los niveles de ácido cítrico están asociados con disfunción prostática, baja movilidad espermática y reacción prematura del acrosoma, lo que puede reducir la capacidad fecundante de un esper matozoide. La etiología de la hiperviscosidad sigue siendo compleja; sin embargo, para pre servar la movilidad y la integridad del acrosoma, previamente deben investigarse sus causas en las muestras seminales que van a ser sometidas a la criopreservación.

Semen cryopreservation is a useful tool in assisted reproduction, which may have impact on sperm characteristics during freezing and thawing. The aim of this study was to assess the integrity of the acrosome and motility of cryopreserved and thawed spermatozoos in hyperviscous and no viscous samples. In semen samples spermiogram, glandular markers, acrosome integrity, culture and the levels markers accessory glands were measured. Each ali quot of semen was immersed in cryoprotectant and maintained in a commercial freezer at -196 ° C. After 30 days, these were thawed and in the cell pellet resuspended, spermatic motility and acrosomal integrity were evaluated. In thawed samples, there were significant decreases in progressive motility (p <0.05), vitality (p <0.005) and acrosome integrity (p <0.05) with respect to fresh sperm, this decline was most evident in hyperviscous samples. The viscosity of fresh semen was inversely related to motility and acrosome integrity before and after freezing (p <0.05). Twenty semen samples showed the presence of microorganisms and C. trachomatis IgA antibodies, of which fifteen showed hyperviscosity. Biochemical analysis demonstrated that semen samples with low levels of citric acid had less acrosomal integrity both before and after freezing (p <0.05). The viscoelasticity and citric acid levels are associated with prostate dys function, low sperm motility and premature acrosome reaction, which can reduce the fertilizing capacity of sperm. The etiology of hyperviscosity remains complex; however, to preserve mo tility and acrosome integrity, its causes must be investigated previously in the seminal samples to be subjected to cryopreservation.

Impact of sperm DNA and acrosome integrity and acrosome reaction rate on outcomes of rescue intracytoplasmic sperm injection / 南方医科大学学报

<p><b>UNLABELLED</b>Objective To explore the effects of sperm DNA integrity rate, acrosome integrity rate and acrosome reaction rate on the outcomes of rescue intracytoplasmic sperm injection (ICSI).</p><p><b>METHODS</b>This retrospective analysis was conducted among 97 infertile couples receiving rescue ICSI due to failure of in vitro fertilization procedures in our Reproductive Medicine Center. Of these 97 women, 41 had clinical pregnancy and 56 did not, and the effects of sperm DNA integrity rate (estimated by DNA fragmentation index, DFI), acrosome integrity rate and acrosome reaction rate on rescue ICSI outcomes were analyzed.</p><p><b>RESULTS</b>No significant difference was found in paternal age, testosterone value, testicular volume, FSH, female patient' age or the number of eggs retrieved between the two groups (P>0.05), but the infertility years was significantly shorter in the pregnancy group than in the non-pregnancy group (P<0.05). The fertilization rate and cleavage rate were similar between the two groups (P>0.05), but the good embryo rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity or acrosome reaction rate did not differ significantly between the two groups (P>0.05), but the acrosome integrity rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity rate, acrosome integrity or acrosome reaction rate were not correlated with the fertilization rate, cleavage rate or good embryo rate (P>0.05). The pregnancy rate, twin and single fetus rates were 42.3%, 10.3% and 32.0% in this cohort after recue ICSI, respectively.</p><p><b>CONCLUSION</b>Rescue ICSI is an effective treatment after failed in vitro fertilization procedure, and sperm acrosome integrity rate is associated with the outcome of rescue ICSI.</p>

Quality characteristics of human spermatozoa with hyaluronic acid receptors / 中华男科学杂志

<p><b>OBJECTIVE</b>To analyze the quality characteristics of human spermatozoa with hyaluronic acid (HA) receptors and search for a new indicator for the assessment of sperm quality.</p><p><b>METHODS</b>Using sperm-HA binding assay with HA-coated slides, we determined the binding rate of motile sperm with HA receptors and analyzed its correlation with routine semen parameters, sperm membrane function, sperm fertilizing function and diminished/arrested sperm maturation.</p><p><b>RESULTS</b>The motile sperm with HA binding sites in the acrosomal region showed significantly higher acrosomal integrity ([95.4 +/- 3.9]%) and mitochondrial membrane potential (MMP) ([97.8 +/- 2.1]%) than those in the initial semen ([68.8 +/- 6.2]% and [72.8 +/- 7.4]%) (P < 0.01). The sperm-HA binding scores were correlated mildly with many routine semen parameters (r = 0.195-0.268, P < 0.05), positively with the acrosome reaction level after ionophore challenge (r = 0.666, P < 0.01) and normal sperm morphology (r = 0.417, P < 0.01), and negatively with sperm nucleoprotein immaturation (r = -0.266, P < 0.01), DNA fragmentation (r = -0. 308, P < 0.01) and excessive residual cytoplasm (r = -0.218, P < 0.05).</p><p><b>CONCLUSION</b>Sperm with HA receptors in the acrosomal region exhibit significant advantages in plasma membrane structure, fertilizing potential and maturation. The sperm-HA binding assay, which is based on a relationship between sperm receptors for zona pellucida and HA, is likely to become a new independent indicator for assessing the multiple qualities of spermatozoa.</p>

Effects of nonylphenol and cadmium on sperm acrosome reaction in vitro in mice / 中华男科学杂志

<p><b>OBJECTIVE</b>To study the effects of nonylphenol and cadmium on acrosome reaction in vitro in mouse spermatozoa.</p><p><b>METHODS</b>Sperm were collected from the vas deferens of mice, capacitated in vitro and stimulated with A23187 at 30 micromol/L to induce acrosome reaction. Then the sperm suspension was treated with nonylphenol at 10, 20, 30, 60 and 100 micromol/L or cadmium at 500, 2500 and 5 000 micromol/L, and the control group treated with the carrier solvent. Acrosome reaction of the sperm was analyzed by FITC-PSA staining.</p><p><b>RESULTS</b>Compared with the control group, nonylphenol significantly inhibited acrosome reaction at the concentration of > 60 micromol/L (P < 0.01), but not at < 30 micromol/L (P > 0.05), and the sperm survival rate was reduced with increased concentration of nonylphenol. However, cadmium exhibited no significant influence on either acrosome reaction (P > 0.05) or sperm survival rate at 500 - 5 000 micromol/L.</p><p><b>CONCLUSION</b>Nonylphenol and cadmium affect the spermatogenesis of mice in different ways; the former directly inhibits sperm acrosome reaction, while the latter has no direct effect on it.</p>

NYD-SP27, a novel intrinsic decapacitation factor in sperm / 亚洲男科学杂志(英文版)

Prior to fertilization sperm has to undergo an activation process known as capaciation, leading to the acrosome reaction. Till now, little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved. In this study, we report that NYD-SP27, an isoform of phospholipase C Zeta 1 (PLCZ1), is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody. Western blot and double staining analyses show NYD-SP27 becomes detached from sperm, as they undergo capacitation and acrosome reaction. The absence of HCO3-, a key factor in activating capacitation, from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm. The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm, reduced the number of capacitated sperm, inhibited the acrosome reaction induced by ATP and progesterone, and inhibited agonist-induced PLC-coupled Ca2+ mobilization in sperm, which can be mimicked by the PLC inhibitor, U73122. These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.

Destabilization of acrosome and elastase influence mediate the release of secretory phospholipase A2 from human spermatozoa / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To determine the cellular distribution of secretory phospholipase A(2) (sPLA(2)) in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes.</p><p><b>METHODS</b>Acrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA(2) and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used.</p><p><b>RESULTS</b>Although sPLA(2) was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA(2) was associated with enhanced binding of annexin V-fluoroscein isothiocyanate (FITC) to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA(2), disturbance of acrosome structure, and loss of vitality.</p><p><b>CONCLUSION</b>The ability of spermatozoa to release secretory phospholipase A(2) is related to the acrosomal state. Premature destabilization of the acrosome and loss of sPLA(2) can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA(2) in intact spermatozoa might be an additional parameter for evaluating sperm quality.</p>

Effect of caspase inhibitors on the post-thaw motility, and integrity of acrosome and plasma membrane of cryopreserved equine spermatozoa.

The present study was designed to test the hypothesis that addition of anticaspase cocktails (inhibiting caspases and thus blocking apoptosis) to the extenders increases the post-thaw viability of equine spermatozoa. The addition of caspase inhibitors failed to improve the acrosome and plasma membrane integrity of spermatozoa, suggesting that in equine sperm cryopreservation protocols, the addition of these caspase inhibitors to cryopreservation medium may not be beneficial in protecting the sperm from the stress of cryopreservation.

Protein detection in spermatids and spermatozoa of the butterfly Euptoieta hegesia (Lepidoptera)

This study was undertaken to detect protein components in both sperm types of the butterfly Euptoieta hegesia. These spermatozoa possess complex extracellular structures for which the composition and functional significance are still unclear. In the apyrene sperm head, the proteic cap presented an external ring and an internal dense content; basic proteins were detected only in external portions. In the tail, the paracrystalline core of mitochondrial derivatives and the axoneme are rich in proteins. The extratesticular spermatozoa are covered by a proteic coat, which presented two distinct layers. In eupyrene spermatozoa, acrosome and nucleus were negatively stained, probably because of their high compaction. In the tail, there is no paracrystalline core and the axoneme presented a very specific reaction for basic proteins. The lacinate and reticular appendages are composed of cylindrical sub-units and presented a light reaction to E-PTA and a strong reaction to tannic acid. A complex proteic coat also covers the extratesticular spermatozoa. We found similarities between both extratesticular coats, indicating a possible common origin. Both spermatozoon types are rich in proteins, especially the eupyrene appendages and the extratesticular coats. We believe that both coats are related to the sperm maturation and capacitation processes.

Seminal plasma angiotensin II detection and its clinical implication / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the variation of seminal plasma angiotension II (Ang II) in infertile men and its clinical implication.</p><p><b>METHODS</b>Ang II values in paired blood plasma and seminal plasma from 43 infertile men(13 azoospermia, 8 asthenozoopermia, 17 asthenozoospermia and 5 cases with normal semen parameters) and 10 normal controls were obtained by SPE-HPLC-RIA. All semen samples with spermatozoa were analyzed by CASA for sperm count, motility and other parameters. Acrosome reaction rate (AR) was assessed by triple-stain.</p><p><b>RESULTS</b>The mean concentration of seminal plasma Ang II was 4 times as high as that of blood plasma in all patients and controls (P < 0.01), but there was no correlation between them. The seminal plasma Ang II of azoospermic patients was higher than that of other infertile men and controls(P < 0.05), but no difference was found between the latter two groups. There was no correlation between seminal plasma Ang II values and other traditional parameters of sperm together with AR.</p><p><b>CONCLUSIONS</b>Seminal plasma Ang II may be secreted locally in male reproductive tract. In addition to testis and epididymis, prostate and/or seminal vesicle may also be the source of it. The reason why seminal plasma Ang II of azoospermic patients is higher than that of others remains unknown. Further study is required to clarify the exact role of seminal plasma Ang II in the mechanisms of male fertility regulation.</p>

Comparison of Sperm Morphology Evaluation Using Strict Criteria, Acrosome Reaction Following Ionophore Challenge and Zona-free Hamster Ova Sperm Penetration Assay as Prognostic Factors in Diagnosis of Male Infertility and In Vitro Fertilization / 대한불임학회지

OBJECTIVE: This study was designed to investigate the interrelationship and clinical usefulness of sperm morphology by strict criteria (SM), acrosome reaction following ionophore challenge test (ARIC) and sperm penetration assay (SPA) using zona-free hamster ova as prognostic factors in in vitro fertilization. MATERIALS AND METHODS: Semen samples were provided by 83 patients undergoing IVF. We first evaluated the differences between normal fertilization group and poor fertilization group on three andrologic tests. Secondly, we analyzed the relationship between the three andrologic tests and in vitro fertilization on IVF settings. Finally, we evaluated the effectiveness of the three andrologic tests as the prognostic indicators for fertilizing ability. RESULTS: The fertilization rate of all men in the poor fertilization group was less than 30%; but there was no evidence that this poor fertilization was due to oocyte defects. The results of three andrologic tests were significatly higher in normal fertilization group. Fertilization rate (%) in vitro was highly correlated (p<0.001) with % normal sperm by SM, ARIC value (%), and SPA result. By using Receiver-Operator-Characteristic curve (ROC), we evaluated the effectiveness of these three tests. The sensitivity and specificity of SM, ARIC test and SPA in predicting fertilization potential in IVF setting were 76% and 75%, 84% and 90%, and 76% and 95%, respectively. CONCLUSION: Our data suggest that the three andrologic tests can be reliable tools as prognostic factors of sperm fertilizing ability. Among these test, ARIC test and SPA gave more accurate information on fertilizing capacity. ARIC test was shown to have a predictive value for fertilizing ability comparable to that of SPA that appears to be a simple and cost-effective addition to current andrology laboratory. Combined application of these three tests may give more information on predicting sperm fertilizing capacity.

Detection of human sperm morphology and acrosome reaction with Coomassie brilliant blue staining / 中华男科学杂志

<p><b>OBJECTIVES</b>To evaluate the application of Coomassie brilliant blue (CBB) G250 staining for the detection of human sperm deformity rate, rate of intact acrosome and acrosome reaction.</p><p><b>METHODS</b>The smear of spermatozoa before and after capacitation and induced acrosome reaction with progesterone (P) were stained with 0.05% CBB G250 and Wright-Giemisa solution respectively, and visualized with light microscopy. The deformity rate of spermatozoa, rate of intact acrosome and acrosome reaction were calculated.</p><p><b>RESULTS</b>There was no any difference in detection of deformity rate of spermatozoa and rate of intact acrosome with CBB G250 and Wright-Giemisa staining(P < 0.05). The sperm population of acrosome reaction with induced P was divided by CBB staining into two types: positive staining with dark violet blue on acrosome cap and pale or negative staining on the same area. The rate of the latter was increasing with increasing inductive time, maybe representative of the rate of acrosome reaction. The mean rate was(75.1 +/- 3.8)% after induced for 1 h.</p><p><b>CONCLUSIONS</b>CBB G250 staining is a reliable method for assessment of the human sperm morphology and acrosome reaction.</p>

Acrosome intactness and seminal hyaluronidase activity: relationship with conventional seminal parameters.

Seminal hyaluronidase activity was estimated after liquefaction in semen samples of 100 male partners of infertile couples including 16 azoospermic (no spermatozoon) men and 48 fertility proven men by a method based on measurement of the area of digestion of substrate (hyaluronic acid) in agar plate. Semen samples were also evaluated for Acrosomal Intactness (AI) test except the azoospermics of the studied samples. Seminal hyaluronidase activity was completely absent in azoospermic specimens confirming its cellular origin. Seminal hyaluronidase activity was found to be significantly correlated, statistically, with sperm density (r = 0.708, p < 0.001), % motility (r = 0.6478, p < 0.001) and % normal sperm morphology (r = 0.5724, p < 0.001). Acrosomal Intactness (AI) test scores were also well correlated with sperm density (r = 0.6477, p < 0.001), % motility (r = 0.5965, p < 0.001) and % normal morphology (r = 0.6237, p < 0.001). Both values were higher in semen samples with normal routine parameters (proven fertility and normozoospermic infertile groups) than those compared with abnormal routine parameters (oligozoospermic). We also found very highly significant correlation (r = 0.8442) between seminal hyaluronidase activity and Acrosomal Intactness scores, statistically (p < 0.001). This could be because; normal germinal semineferous epithelium generates abundant number of sperms with normal motility and morphology that are also having intact acrosome. Intact acrosome prevents loss of acrosomal enzymatic activity (e.g. hyaluronidase) until released after liquefaction during seminal analysis and during acrosomal reaction in female genital tract prior to fertilization. Seminal hyaluronidase activity, thus determined, is primarily dependent upon the intact status of acrosome. As each sperm contributes to the seminal hyaluronidase activity, it is directly correlated with sperm density; but at the same time it exhibits goods correlation with % motility and % normal morphology. Therefore AI score and seminal hyaluronidase activity can be considered as good indicators of sperm function.

Analysis of Calcium-Binding Proteins of Mouse Spermatozoa by 2-Dimentional SDS-PAGE / 대한남성과학회지

PURPOSE: To verify the expression of calcium-binding proteins in mouse spermatozoa. MATERIALS AND METHODS: Mouse sperm proteins were subjected to 2-dimentional SDS-PAGE combined with calcium shift in the presence or absence of Ca2+, Zn2+ or EDTA. RESULTS: In the epididymal sperm extracts, 10 kinds of protein spots showed mobility shift in the gel containing Ca2+. Most of the CBPs disappeared after the acrosome reaction (AR) induced by Ca2+-ionophore A23187, suggesting that they originated from acrosome and/or the plasma membrane overlaying the acrosome. CONCLUSIONS: The CBPs might be involved in acrosome reaction of mouse spermatozoa. Protein database of sperm CBPs might be useful for diagnosis of male infertility.

Effect of Fertilization Promoting Peptide on Kinematic Parameters, Capacitaion and Acrosome Reaction in Human Spermatozoa / 대한불임학회지

OBJECTIVE: This study has been carried out to evaluation the effect of fertilization promoting peptide (FPP) on the kinematic parameters, capacitation and acrosome reaction of the frozen-thawed human spermatozoa. METHODS: After FPP treatment, we examined kinematic parameters, capacitation and acrosome reaction, using the methods of computer-aided sperm analysis (CASA) and chlortetracycline (CTC) fluorescence analysis. RESULTS: We have obtained the evidence that FPP can promote the capacitation and inhibit the spontaneous acrosome reaction of frozen-thawed human spermatozoa in vitro. Fpp (25~100 nM) induced a significant increase in the proportion of B-pattern capacitated spermatozoa, and a significant decrease in the proportion of F-pattern uncapacitated ones without significant stimulation of acrosomal exocytosis. In the kinematic parameters treatment, FPP treated groups maintained higher LIN, BCF and STR than those of control. The VAP, VSL, VCL and ALH were not different. Therefore it is suggested that FPP in human seminal plasma may play a positive role in promoting human sperm function.

Observation of the Incidence of Acrosome Reaction in Human Spermatozoa Treated with Mibefradil as a T-type Ca2+i Channels Inhibitor / 대한불임학회지

OBJECTIVE : The sperm acrosome reaction is a Ca2+ -dependent exocytotic event that is triggered by adhesion to the mammalian egg's zona pellucida. Previous studies suggested a role of Ca2+ channels in acrosome reactions. This study was conducted to investigate the T-type calcium channel is operated in acrosome reaction of human spermatozoa. METHOD : Human semen samples were obtained from healthy donors with nomal criteria. The spermatozoa were divided into five groups: Group 1 were non-treated as a control; Group 2 where spermatozoa were exposed to 5 micrometer Ca2+ A23187 (Ca2+i); Group 3 where spermatozoa were exposed 5 micrometer Ca2+i and mibefradil; Group 4 where spermatozoa were exposed 5 micrometer Ca2+i and nifedipine, and Group 5 where spermatozoa were treated with 5 micrometer Ca2+i and both of mibefradil and nifedipine. Spermatozoa in all groups were retrieved after incubation for 15 and 30 minutes at 37degrees C. After staining with PSA-FITC, fluorescence was observed under a fluorescence microscope, and AR was evaluated on a total >100 spermatozoa/side. RESULT AND CONCLUSION : We observed on acrosome reaction inhibition rate in human spermatozoa the various of concentration of mibefradil, nifedipine. Maximum response was noted with 1.0 micrometer mibefradil and the decrease of acrosome reaction inhibition rate 45%. Nifedipine in acrosome reaction inhibition rate was only about 25%. The Ca2+i-induced AR of spermatozoa was significantly suppressed by mibefradil. Incidence of the suppression was depending on concentration of mibefradil. Results from the present study suggest that the human spermatozoa possess T-type channel. The observation that reversible inhibitor of T channels in male germ cells provides a new mechanism of contraceptive action.

Peritoneal fluid modulates the sperm acrosomal exocytosis induced by N-acetylglucosaminyl neoglycoprotein

The effect of peritoneal fluid (PF) on the human sperm acrosome reaction (AR) was tested. Sperm was pre-incubated with PF and the AR was induced by calcium ionophore A23187 and a neoglycoprotein bearing N-acetylglycosamine residues (NGP). The AR induced by calcium ionophore was inhibited 40 percent by PF from controls (PFc) and 50 percent by PF from the endometriosis (PFe) group, but not by PF from infertile patients without endometriosis (PFi). No significant differences were found in the spontaneous AR. When the AR was induced by NGP, pre-incubation with PFc reduced (60 percent) the percentage of AR, while PFe and PFi caused no significant differences. The average rates of acrosome reactions obtained in control, NGP- and ionophore-treated sperm showed that NGP-induced exocytosis differed significantly between the PFc (11 percent) and PFe/PFi groups (17 percent), and the ionophore-induced AR was higher for PFi (33 percent) than PFc/PFe (25 percent). The incidence of the NGP-induced AR was reduced in the first hour of pre-incubation with PFc and remained nearly constant throughout 4 h of incubation. The present data indicate that PF possesses a protective factor which prevents premature AR

The Effects of Sperm Motility Stimulants on the Hyperactivation, Acrosomal Reaction of Sperm and their in vitro Fertilization / 대한산부인과학회잡지

OBJECTIVES: To evaluate the effects of sperm motility stimulants on the hyperactivation (HA), acrosomal reaction (AR) and sperm penetration assay (SPA) in fresh and frozen-thawed spermatozoa from fertile men. METHODS: We treated the semen samples obtained from 20 normospermic men (fresh semens from 10 and cryopreserved ones from 10) with pentoxiphylline (PF) and 2-deoxyadenosine (2-DXA) to evaluate the change of the patterns of motility using the computerized motility analyzer. The semen samples treated with motility stimulants were incubated in the medium with calcium ionophore A23187 for the examination of the proportion of acrosome lost spermatozoa. Finally we performed SPA in both groups for the evaluation of fertilizing capacity after stimulant treatments. RESULTS: In both fresh and cryopreserved semen samples, the addition of PF and 2-DXA significantly altered the patterns of motility (ALH, VCL, HA) known to have association with sperm quality without increasing the number of sperms with progressive motility and velocity. A23187 induced AR was also augmented by the treatment with PF and 2-DZA. Although the treatment with PF did not increase the mean rates of egg penetration significantly, in selected cases in the cryopreserved semen group, the improvement of the motility pattern was impressive. CONCLUSION: PF and 2-DXA can improve the quality of sperm function in both fresh and frozen-thawed semen from normal fertile men and may increase the sperm penetration rate of zona-free hamster eggs in selected samples of the frozen-thawed semen. The results suggest that PF and 2-DXA pretreatment can be used in the clinical practice for intrauterine insemination (IUI) program with frozen-thawed sperms as well as with samples from men with abnormal semen parameters. In addition, it may be a cost- effective therapy to try IUI combined with such a pretreatment for the couples planned to enter into the ART program.