Effects of chelating calcium in cryopreservation extender on frozen-thawed dog semen / [Efeitos da quelação de cálcio no meio de criopreservação no sêmen descongelado de cão]

We evaluated the effect of reducing free calcium in the cryopreservation medium, using the calcium chelator ethylene diamine tetracetic acid (EDTA) at 0.3% and 0.5% concentrations. Three male mixed breed dogs were subjected to semen collection by digital manipulation (n=16). Each ejaculate was divided in three aliquots, and each one was diluted in TRIS-glucose-egg yolk extender with 6% glycerol and 0.5% Equex STM Paste® (TGE, control); and added with 0.3% EDTA (EDTA 0.3) or 0.5% EDTA (EDTA 0.5). Calcium concentration reduced in EDTA 0.3 and all the calcium ions were chelated in EDTA 0.5. The EDTA addition did not affect sperm morphology or plasma membrane integrity; however, by removing all free calcium (EDTA 0.5), the sperm motility reduced (64.7% in TGE and 45% in EDTA 0.5; p<0.05). Acrosome integrity and sperm binding ability were not improved by calcium chelation. The failure to prevent the premature AR may explain why sperm longevity was not affected by calcium removal. Thus, the partial or complete calcium removal, through EDTA addition, is not able to prevent acrosomal damage or premature acrosomal reaction, and therefore does not improve the dog sperm binding ability.(AU)

Avaliou-se o efeito da redução do cálcio livre no meio de congelamento, usando-se o quelante de cálcio etilenodiaminotetracético (EDTA) a 0,3% e 0,5%. Três cães machos sem raça definida foram submetidos à coleta de sêmen por manipulação digital (n=16). Cada ejaculado foi diluído em diluidor controle com TRIS-glicose - gema de ovo (TGE, controle), ou em diluidor TGE enriquecido com 0,3% (EDTA 0,3) ou 0,5% de EDTA (EDTA 0,5). A concentração de cálcio reduziu no meio EDTA 0,3, e todos os íons de cálcio foram quelados no meio EDTA 0,5. A adição do EDTA e a consequente quelação do cálcio não afetaram a morfologia espermática ou a integridade da membrana plasmática, no entanto, ao remover todo o cálcio do meio (EDTA 0,5), a motilidade espermática se reduziu (64,7% no TGE e 45% no EDTA 0,5; P<0,05). A integridade do acrossoma e a capacidade de ligação do espermatozoide não melhoraram com a quelação do cálcio. Apesar da influência da concentração de cálcio sobre a motilidade espermática após o descongelamento, a falha em prever a reação acrossomal prematura pode explicar por que a longevidade espermática não foi afetada pela remoção do cálcio no meio. Dessa forma, a remoção parcial ou total do cálcio, por meio da adição de EDTA, não é capaz de prevenir o dano no acrossoma ou a reação acrossomal prematura e, portanto, não aumenta a capacidade do espermatozoide de se ligar ao oócito.(AU)

Participation of the inositol 1,4,5-trisphosphate-gated calcium channel in the zona pellucida- and progesterone-induced acrosome reaction and calcium influx in human spermatozoa / 亚洲男科学杂志(英文版)

The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.

Protein kinase A inhibition induces EPAC-dependent acrosomal exocytosis in human sperm / 亚洲男科学杂志(英文版)

To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 μmol l-1), whereas higher concentrations (>5 μmol l-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca2+-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.

Onion peel extract and its constituent, quercetin inhibits human Slo3 in a pH and calcium dependent manner

Sperm function and male fertility are closely related to pH dependent K⁺ current (KSper) in human sperm, which is most likely composed of Slo3 and its auxiliary subunit leucine-rich repeat-containing protein 52 (LRRC52). Onion peel extract (OPE) and its major active ingredient quercetin are widely used as fertility enhancers; however, the effect of OPE and quercetin on Slo3 has not been elucidated. The purpose of this study is to investigate the effect of quercetin on human Slo3 channels. Human Slo3 and LRRC52 were co-transfected into HEK293 cells and pharmacological properties were studied with the whole cell patch clamp technique. We successfully expressed and measured pH sensitive and calcium insensitive Slo3 currents in HEK293 cells. We found that OPE and its key ingredient quercetin inhibit Slo3 currents. Inhibition by quercetin is dose dependent and this degree of inhibition decreases with elevating internal alkalization and internal free calcium concentrations. Functional moieties in the quercetin polyphenolic ring govern the degree of inhibition of Slo3 by quercetin, and the composition of such functional moieties are sensitive to the pH of the medium. These results suggest that quercetin inhibits Slo3 in a pH and calcium dependent manner. Therefore, we surmise that quercetin induced depolarization in spermatozoa may enhance the voltage gated proton channel (Hv1), and activate non-selective cation channels of sperm (CatSper) dependent calcium influx to trigger sperm capacitation and acrosome reaction.

Phyllanthus emblica leaf extract ameliorates testicular damage in rats with chronic stress / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Stress affects the male reproductive system and can cause sub-fertility or infertility. Although Phyllanthus emblica L. (PE) extract has been shown to have high antioxidant capacity and protective properties in damaged tissue, the preventive effects of PE extract on testicular function from stress-related impairment have never been demonstrated. This study aimed to investigate the effects of PE aqueous leaf extract on testicular impairment and protein marker changes in rats suffering from chronic stress. Adult male rats were divided into four groups: a control group, a chronic stress (CS) group, and two groups with CS that received different doses of PE extract (50 or 100 mg/kg body weight (BW)). In the treatment groups, the animals were given PE extract daily before stress induction for 42 consecutive days. Stress was induced through immobilization (4 h/d) followed by forced cold swimming (15 min/d). Sperm quality and the histology of the testes and caudal epididymis were examined, as were levels of serum corticosterone, testosterone, and malondialdehyde (MDA). The expressions of testicular steroidogenic acute regulatory (StAR) and tyrosine-phosphorylated proteins were investigated using immuno-Western blot analysis, as these proteins are assumed to play important roles in spermatogenesis and androgen synthesis. The results showed that PE (50 mg/kg BW) significantly increased sperm concentration and testosterone levels, while decreasing corticosterone levels, MDA levels, sperm head abnormalities, and acrosome-reacted sperm in CS rats. In addition, PE at both doses was found to diminish testicular histopathology in the CS rats. We also found that 50 mg/kg BW of PE significantly improved StAR protein expression and altered the intensities of some tyrosine-phosphorylated proteins in testis. We conclude that PE leaf extract at 50 mg/kg BW can prevent testicular damage in rats with CS.

Clinical assessment of the male fertility

The evaluation of infertility in males consists of physical examination and semen analyses. Standardized semen analyses depend on the descriptive analysis of sperm motility, morphology, and concentration, with a threshold level that must be surpassed to be considered a fertile spermatozoon. Nonetheless, these conventional parameters are not satisfactory for clinicians since 25% of infertility cases worldwide remain unexplained. Therefore, newer tests methods have been established to investigate sperm physiology and functions by monitoring characteristics such as motility, capacitation, the acrosome reaction, reactive oxygen species, sperm DNA damage, chromatin structure, zona pellucida binding, and sperm-oocyte fusion. After the introduction of intracytoplasmic sperm injection technique, sperm maturity, morphology, and aneuploidy conditions have gotten more attention for investigating unexplained male infertility. In the present article, recent advancements in research regarding the utilization of male fertility prediction tests and their role and accuracy are reviewed.

Teofilina como agente capacitante do sêmen bovino / Theophylline as a capacitating agent of bovine semen

Objetivou-se avaliar a teofilina como agente capacitante substituto ou associado à heparina sobre a reação acrossômica dos espermatozoides e o desenvolvimento de embriões produzidos in vitro. O experimento foi realizado com quatro touros e três tratamentos, totalizando 12 grupos experimentais. O sêmen dos touros foi avaliado nos tratamentos descritos a seguir: tratamento 1 (HEP): heparina - 10µg/mL; tratamento 2 (TEO): teofilina - 5mM; tratamento 3 (HEP + TEO): heparina (10µg/mL) + teofilina (5mM), por zero, seis, 12 e 18 horas, corados com trypan blue/Giemsa para avaliação da reação acrossômica. Para a produção dos embriões, os agentes capacitantes foram adicionados aos meios de fertilização. Na análise espermática, a taxa de reação acrossômica verdadeira foi maior (P<0,05) no tempo zero hora, enquanto para espermatozoides mortos, as maiores taxas (P<0,05) foram nos tempos de 12h (84,46±5,82) e 18h (86,75±4,19). A taxa de embriões produzidos (37,97±13) e a taxa de eclosão (33,50±14) foram maiores (P<0,05) para o tratamento HEP. Não foi observada diferença (P>0,05) entre touros na análise de reação acrossômica nem na PIVE. A utilização da teofilina foi tão eficiente quanto a da heparina na indução da reação acrossômica, no entanto resultou em menores taxas de produção embrionária.(AU)

The sperm capacitating process should take special attention during in vitro embryo production (IVEP) once that affects the success of embryo production. The study aimed to evaluate theophylline as substitute capacitating agent or in combination with heparin on the sperm acrosome reaction and development of embryos produced in vitro. The experiment was carried out using 4 bulls and 3 treatments, establishing 12 experimental groups. Each bull was evaluated in the following treatments: Treatment 1 (HEP): Heparin - 10mg/mL; Treatment 2 (THEO): Theophylline - 5mM; Treatment 3 (HEP + THEO), Heparin (10mg/mL) + Theophylline (5mM). The semen of bulls was incubated in each treatment for 0, 6, 12 and 18h, stained with Trypan blue / Giemsa and analyzed by electron microscopy for assessment of acrosome reaction. Using sperm of same bulls, capacitating agents were added to the fertilization media, for IVEP. In sperm analysis, the true acrosome reaction rate was higher (P<0.05) in time 0h, while sperm dead rates were highest (P<0.05) at 12h (84.46±5, 82), and 18h (86.75±4.19). The produced embryos rate (37.97±13) and hatching rate (33.50±14) were larger (P<0.05) for HEP treatment. There was no difference (P>0.05) between bulls in acrosome reaction analysis neither for IVEP. The use of theophylline was as effective as heparin in the induction of the acrosome reaction, although it resulted in lower embryo production rates.(AU)

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>

Coatis (Nasua nasua) semen cryopreservation / Criopreservação do sêmen de quatis (Nasua nasua)

Carnivore semen cryopreservation procedures started with semen washing and centrifuging in culture media for seminal plasma removal and microorganisms elimination. The objective of this study was to perform coatis semen cryopreservation comparing the effects between two extenders Hams F-10 and M199 for washing and centrifugation before cryopreservation using Dilutris medium. Semen samples (n = 36) were collected by electroejaculation from six adult male coatis (Nasua nasua) between May and October of 2008 at the Universidade Federal de Mato Grosso Zoo. Sperm total motility (%), progressive sperm motility (0-5), plasma membrane integrity spermatozoa rates (%), and acrosome integrity (%) were analyzed. These fresh semen samples were divided in two fractions, diluted in 1 ml of Hams F-10 (Hams F-10, Nutricel S.A., Brazil) or M199 (M199, Nutricel S.A., Brazil) and centrifuged at 300 g for 10 min. The supernatant was discarded and pellets resuspended in 1 ml of Dilutris (Dilutris, Minitube®, Brazil), stored at 5ºC for 3 hours, transferred to 0.25 ml straws, placed in liquid nitrogen vapor for 20 min, and immersed in liquid nitrogen. The means/SD for fresh semen and cryopreserved semen using Hams F-10/Dilutris and M199/Dilutris were, respectively: 84.28 ± 11.57, 45.38 ± 27.26, and 44.61 ± 25.03 for total motility; 3.64 ± 1.44, 2.15 ± 1.14, and 2.07 ± 1.03 for progressive sperm motility; 92.76 ± 3.46, 84.69 ± 15.77, and 89.76 ± 13.97 for live spermatozoa rate; and 94.76 ± 2.89, 92.35 ± 4.73, and 90.58 ± 7.17 for acrosome integrity. No significant difference (P 0.05) were observed between the values obtained from the Hams F-10/Dilutris or M199/Dilutris treatments. Both treatments demonstrated to be suitable for freezing semen from this species.

Os procedimentos de criopreservação do sêmen em carnívoros devem ser iniciados após a lavagem e centrifugação em meios de cultura para retirada do plasma seminal e eliminação de microrganismos. O objetivo deste estudo foi realizar a criopreservação do sêmen de quatis comparando os efeitos entre dois extensores Hams F-10 e M199 para lavagem e centrifugação do sêmen antes do congelamento com o meio Dilutris. Amostras de sêmen (n = 36) foram coletadas por eletroejaculação em seis quatis (Nasua nasua) machos adultos entre maio e outubro de 2008, no Zoológico da Universidade Federal de Mato Grosso. Motilidade total (%), motilidade espermática progressiva (0-5), integridade da membrana plasmática (%) e integridade do acrossoma (%) foram analisados. Amostras de sêmen a fresco foram divididas em duas frações, diluídas em 1 mL de Hams F-10 (Hams F-10, Nutricel S.A., Brasil) ou M199 (M199, Nutricel S.A., Brazil) e centrifugado a 300 g por 10 min. O sobrenadante foi descartado e pellets ressuspendidos em 1 mL de Dilutris (Dilutris, Minitube®, Brasil), armazenados a 5ºC durante 3 horas, transferidos para palhetas de 0,25 mL, colocadas em vapor de nitrogênio líquido por 20 min e imersos em nitrogênio líquido. Os resultados para sêmen a fresco e sêmen congelado utilizando Hams F-10/Dilutris e M199/Dilutris foram, respectivamente: 84,28 ± 11,57, 45,38 ± 27,26 e 44,61 ± 25,03 para motilidade total; 3,64 ± 1,44, 2,15 ± 1,14 e 2,07 ± 1,03 para a motilidade progressiva; 92,76 ± 3,46, 84,69 ± 15,77 e 89,76 ± 13,97 para integridade da membrana plasmática; e 94,76 ± 2,89, 92,35 ± 4,73 e 90,58 ± 7,17 para integridade do acrossoma. Não houve diferença significativa (P 0,05) entre os valores obtidos nos tratamentos Hams F-10/Dilutris ou M199/Dilutris. Ambos os tratamentos demonstraram ser adequados para a congelação de sêmen desta espécie.

Evaluating the role of silver nanoparticles on acrosomal reaction and spermatogenic cells in rat

Nanoparticles have wide range of application while there are some reports regarding their probable effects on male reproductive system and spermatozoa. The aim of this study was to evaluate the effect of different doses of silver nanoparticles [AgNPs] [70nm] on acrosome of rat spermatozoa and number of spermatogenic cells. In this experimental study, in experimental group, 32 male wistar rats [8 rats/group] received oral feeding AgNPs every 12 hr in one spermatogenesis period [48 days] by means of gavages in 25, 50, 100 and 200 mg/kg concentration [experimental groups 1-4, respectively]. The control group [8 rats] was treated on schedule with distilled water. Spermatozoa were stained by triple staining protocol for acrosome reaction. Histological evaluation on testis sections was performed using tissue processing and hematoxylin-eosin [H and E] staining. There was significant difference between the control group and the experimental group 1 for acrosome reaction [11.00 +/- 0.00 and 24.25 +/- 3.68, respectively, p=0.01]. There was only significant reduction in spermatogonia cells in experimental group 4. Experimental groups 2, 3 and 4 showed a significant reduction in the number of primary spermatocytes and spermatids as well as spermatozoa. But there were no significant differences between different groups for Sertoli cell number and seminiferous tubule diameter. It seems that Ag NPs have acute and significant effects on spermatogenesis and number of spermatogenic cells and also on acrosome reaction in sperm cells. More experimental investigations are necessary to elucidate better conclusion regarding the safety of nanoparticles on male reproduction system.

Sperm-oocyte interaction and in vitro fertilization clinical outcomes in patients with unexplained infertility / 中南大学学报(医学版)

OBJECTIVE@#To determine the sperm-zona pellucida (ZP) binding and ZP-induced acrosome reaction in patients with unexplained infertility, and to discuss the relationship between ZP-induced acrosome reaction and fertilization rate.@*METHODS@#We compared the fertilization rate and good embryo rate in patients with unexplained infertility after fertilization in 2 ways. Based on the causes of infertility, patients were divided into an unexplained infertility group (Group A) and a pure female tubal factor group (Group B). Oocytes which were obtained by super ovulation from 25 patients with unexplained infertility were randomly divided into 2 groups with conventional in vitro fertilization (IVF) (Group A1) and intracytoplasmic sperm injection (ICSI) fertilization (Group A2). The pure female tubal factor group (Group B) had conventional IVF. We conducted sperm-ZP binding and ZP-induced acrosome reaction experiments with 2 groups of men's sperms separately. We compared the number of sperm-egg binding and ZP-induced acrosome reaction rate and discussed the relationship between the ZP-induced acrosome reaction and fertilization rate, and also the fertilization rate, good embryo rate and pregnancy rate in patients with unexplained infertility after fertilization in 2 ways.@*RESULTS@#The average number of sperm-egg binding (78.29 ± 16.31) and the ZP-induced acrosome reaction rate (55.87 ± 27.69) % in Group A were lower than those of Group B [94.63 ± 6.72, (82.53 ± 17.99)%]. The difference between the average number of sperm-egg binding and the ZP-induced acrosome reaction was significant (P <0.01). The fertilization rate of Group A1 was significantly lower than that of Group B and Group A2 (P <0.01). But there was no significant difference in the good embryo rate among the 3 groups. There was no significant difference between Group A2 and B in fertilization rate and good embryo rate (P <0.05). There was no significant difference in pregnancy rate between Group A and B (P <0.05). Fertilization rate and the rate of acrosome reaction had marked positive correlation with statistical significance (r =0.932, P <0.01).@*CONCLUSION@#ZP binding and ZP-induced acrosome reaction are very important experiments in sperm function test for patients with unexplained infertility. It can not only effectively avoid no embryo transferring due to complete failure of fertilization but also get a desirable outcome of pregnancy using half-ICSI fertilization in patients with unexplained infertility.

Sperm acrosome reaction detection in intrauterine insemination / 中南大学学报(医学版)

OBJECTIVE@#To investigate the relationship between sperm acrosome reaction (AR) and the clinical pregnancy rate of intrauterine insemination (IUI).@*METHODS@#We detected the sperm spontaneous AR rate and Ca2+ ionophore A23187-induced AR rate in 128 patients who accepted IUI treatment, collected their clinical data and analysed the relationship between sperm AR rate and clinical pregnancy rate of IUI.@*RESULTS@#There was no statistical difference between the spontaneous AR rates in the pregnant group and the non-pregnant group (7.7% vs. 7.0%, P>0.05), but there was statistical difference between the induced AR rates(51.9 % vs. 43.5%, P<0.05). There was statistical difference in the clinical pregnancy rate among the 3 IUI groups divided by induced AR rate (≤20.0%, 20.1%-49.9%, and ≥50.0%; 4.8% vs. 12.5% vs. 18.6%, P<0.05).@*CONCLUSION@#The spontaneous AR rate has nothing to do with the clinical pregnancy rate of IUI, but the induced AR rate is associated with the clinical pregnancy rate of IUI.

Protease activity involvement in the passage of mammalian sperm through the zona pellucida

The interaction between acrosome-reacted sperm and zona pellucida proteins is not yet fully understood. Serine protease acrosin and its zymogen proacrosin have been proposed to fulfill this function due to their capacity to bind zona pellucida glycoproteins. However, the molecular mechanism underlying this interaction has been merely speculative. Here we show that fucoidan (a sulfated polysaccharide) and solubilized zona pellucida glycoproteins, but not soybean trypsin inhibitor, are able to detach bound spermatozoa, which suggests that live sperm binds to the zona pellucida in a non-enzymatical way. Interestingly, mild proteolytic digestion with acrosin or trypsin does not modify the structure of the zona pellucida, but rather results in fewer spermatozoa binding to the zona. These results agree with a model where the active site of acrosin digests the zona pellucida and binds through the polysulfate-binding domain through a three-dimensional zona structure rather than a single ligand.

Modulation of spermatozoon acrosome reaction

Spermatozoon acrosome reaction is an exocytotic event of the utmost importance for the development of mammalian fertilisation. Current evidence shows that the triggering of the acrosome reaction (AR) could be regulated by the action of diverse compounds, namely, metabolites, neurotransmitters and hormones. The aim of the present review is to describe the modulating effects of several compounds that have been classified as inductors or inhibitors of acrosome reaction. Among AR inductors, it is necessary to mention progesterone, angiotensin II, atrial natriuretic peptide, cathecolamines, insulin, leptin, relaxin and other hormones. Regarding the inhibitors, oestradiol and epidermal growth factor are among the substances that retard AR. It is worth mentioning that gamma-aminobutyric acid, a neurotransmitter known to be an inhibitor in the central nervous system, has been shown to induce AR. The multiple hormones located in the fluids of the female reproductive tract are also likely to act as subtle regulators of AR, constituting a fundamental aspect for the development of successful fertilisation. Finally, it is necessary to emphasise that the study of regulation exerted by hormones and other compounds on AR is essential for further understanding of mammalian reproductive biology, especially spermatozoon physiology.

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>

Acrosome reaction in the cumulus oophorus revisited: involvement of a novel sperm-released factor NYD-SP8

Fertilization is a process involving multiple steps that lead to the final fusion of one sperm and the oocyte to form the zygote. One of the steps, acrosome reaction (AR), is an exocytosis process, during which the outer acrosome membrane fuses with the inner sperm membrane, leading to the release of acrosome enzymes that facilitate sperm penetration of the egg investments. Though AR has been investigated for decades, the initial steps of AR in vivo, however, remain largely unknown. A well elucidated model holds the view that AR occurs on the surface of the zona pellucida (ZP), which is triggered by binding of sperm with one of the ZP glycosylated protein, ZP3. However, this model fails to explain the large number of 'falsely' acrosome-reacted sperms found within the cumulus layer in many species examined. With the emerging evidence of cross-talk between sperm and cumulus cells, the potential significance of AR in the cumulus oophorus, the outer layer of the egg, has been gradually revealed. Here we review the acrosome status within the cumulus layer, the cross-talk between sperm and cumulus cells with the involvement of a novel sperm-released factor, NYD-SP8, and re-evaluate the importance and physiological significance of the AR in the cumulus in fertilization.

Induction of the acrosome reaction test to in vitro estimate embryo production in Nelore cattle / Teste de indução da reação acrossomal na estimativa de produção in vitro de embriões Nelore

The effectiveness of induction of the acrosome reaction (AR) test as a parameter to in vitro estimate embryo production (IVP) in Nelore breed and the AR pattern by the Trypan Blue/Giemsa (TB) stain were evaluated. Frozen semen samples from ten Nelore bulls were submitted to AR induction and were also evaluated for cleavage and blastocyst rates. The treatments utilized for AR induction were: control (TALP medium), TH (TALP medium + 10μg heparin), TL (TALP medium + 100μg lysophosphatidylcholine) and THL (TALP medium + 10μg heparin + 100μg lysophosphatidylcholine). Sperm acrosomal status and viability were evaluated by TB staining at 0 and after 4h incubation at 38ºC. The results obtained for AR presented a significant difference (P<0.05) in the percentage of acrosome reacted live sperm after 4h of incubation in the treatments that received heparin. The cleavage and blastocyst rates were 60 percent and 38 percent respectively and a significant difference was observed among bulls (P<0.05). It was founded a satisfactory model to estimate the cleavage and blastocyst rates by AR induction test. Therefore, it can be concluded that the induction of the AR test is a valuable tool to predict the IVP in Nelore breed.

Avaliou-se a eficiência da técnica de indução da reação acrossomal (RA) como parâmetro para estimar a produção in vitro (PIV) de embriões Nelore e analisou-se o padrão de RA pela técnica de coloração Azul de Tripan/Giemsa (TB). Amostras de sêmen congelado de dez touros foram submetidas à indução da RA e avaliadas quanto a taxa de clivagem e blastocisto. Os tratamentos utilizados para indução da RA foram: controle (meio TALP), TH (meio TALP + 10μg heparina), TL (meio TALP + 100μg lisofosfatidilcolina) e THL (meio TALP + 10μg heparina + 100μg lisofosfatidilcolina). Avaliou-se viabilidade espermática e acrossomal pela coloração TB a zero e após 4h de incubação a 38ºC. Os resultados obtidos para RA mostram uma diferença significativa (P<0,05) na porcentagem de espermatozoides vivos com acrossoma reagindo após 4h de incubação nos tratamentos que receberam heparina. As taxas de clivagem e blastocisto obtidas foram 60 por cento e 38 por cento respectivamente e observou-se uma diferença significativa entre touros (P<0,05). Delineou-se um modelo satisfatório para estimar as taxas de clivagem e blastocisto. Desta forma, conclui-se que o teste de indução da RA é uma ferramenta valiosa para predizer a PIV na raça Nelore.

Characterization of arylsulphatase A in a 70 kDa protein isolated from goat spermatozoa having Na+, K+-ATPase inhibitory activity.

A protein having inhibitory effect on Na+, K+-ATPase as well as showing arylsulphatase A activity (ASA) was isolated from the cytosolic fraction of goat spermatozoa and characterized biochemically. The molecular mass of the protein was found to be 70 kDa (P70) on 10% SDS-PAGE after 35% ammonium sulphate precipitation, followed by hydroxyapatite column chromatographic separation. The isoelectric point (pI) of the protein was found to be 4.9. The sequencing results of first ten N-terminal amino acid residues of protein showed 100%, 90%, and 80% homology with N-terminal 18-27 amino acid residues of mice, pig and human testicular ASA, respectively. The optimum pH, temperature and incubation time for maximum ASA activity of the protein was 5.5, 37°C and 30 min respectively. The ASA activity of protein and AS from a commercial source was studied with respect to the sensitivity to different metal ions, vanadate, carbonyl compounds and ascorbate. Inhibition of AS activity of P70 by silver nitrate suggested that it was related to ASA. Comparable effects of different polyunsaturated fatty acids (eicosapentaenoic and docosahexaenoic acids) and purified anti P70-antibody on P70 and AS from commercial source were observed. The findings suggested that protein was novel in nature, having both regulatory and catalytic functions and showed similarities with the ASA reported from different sources.

Efeito da adição de trolox e pentoxifilina na motilidade, integridade do acrossoma e do DNA de espermatozoides equinos após descongelação / Effect of trolox and pentoxifylline on motility and integrity of, acrossome and DNA of equine spermatozoa after thawing

Três garanhões foram utilizados para estudar o efeito da adição de trolox e pentoxifilina na motilidade, integridade do acrossoma e DNA de espermatozoides pós-descongelação. Para congelação, utilizou-se Tris-gema com glicerol (5 por cento) em máquina de congelação de sêmen. As amostras foram descongeladas a 37ºC durante 30 segundos e tratadas com: T1= 150µL de sêmen + 150µL de Tris; T2= 150µL de sêmen + 150µL de Tris + 120µM/mL de trolox; T3= 150µL de sêmen + 150µL de Tris + 3,5mM de pentoxifilina e T4= 150µL de sêmen + 150µL de Tris + 3,5mM de pentoxifilina + 120µM/mL de trolox. Após 0, 60 e 120 minutos de incubação (37ºC), as amostras foram analisadas quanto à motilidade, vigor, integridade de acrossoma e DNA. Não houve diferença (P>0,05) entre tratamentos após 0 e 60 minutos de incubação em todos os parâmetros estudados. Após 120 minutos de incubação, verificou-se maior porcentual (P<0,05) de células com motilidade total e progressiva nas amostras do T2. Conclui-se que a adição de trolox após descongelação do sêmen equino preserva a motilidade total e progressiva dos espermatozoides submetidos à incubação a 37ºC durante 120 minutos.

Three stallions were used to study the effect of trolox and pentoxifylline addition on the motility and integrity of acrossome and DNA equine spermatozoa after thawing. Tris-egg-yolg diluent with glycerol (5 percent) were used to freeze the semen samples in a freezing machine. The samples were thawed at 37ºC during 30 seconds and treated with: T1=150µL of semen + 150µL of Tris; T2= 150µL of semen + 150µL of Tris +150mM/mL of trolox; T3= 150µL of semen + 150µL Tris +3.5mM of pentoxifylline; and T4= 150µL of semen + 150µL of Tris + 3.5mM of pentoxifylline + 150mM of trolox. After 0, 60, and 120 minutes of incubation (37ºC), the samples were analyzed to motility, vigor, and integrity of acrossome and DNA. There was no difference (P>0.05) among treatments considering 0 and 60 minutes of incubation in all studied parameters. After 120 minutes of incubation, it was observed higher percentage (P<0.05) of cells with total and progressive motility in the samples of T2. It can be concluded that the trolox addition after thawing of equine semen preserved total and progressive motility of the sperm incubated at 37ºC during 120 minutes.

Tyrosine phosphorylation pattern in sperm proteins isolated from normospermic and teratospermic men

In mammalian system, spermatozoa are not able to fertilize the oocyte immediately upon ejaculation, thus they undergo a series of biochemical and molecular changes which is termed capacitation. During sperm capacitation, signal transduction pathways are activated which lead to protein tyrosine phosphorylation. Tyrosine phosphorylated proteins have an important role in sperm capacitation such as hyperactive motility, interaction with zona pellucida and acrosome reaction. Evaluation of tyrosine phosphorylation pattern is important for further understanding of molecular mechanisms of fertilization and the etiology of sperm dysfunctions and abnormalities such as teratospermia. The goal of this study is to characterize tyrosine phosphorylation pattern in sperm proteins isolated from normospermic and teratospermic infertile men attending Avicenna Infertility Clinic in Tehran. Semen samples were collected and the spermatozoa were isolated using Percoll gradient centrifugation. Then the C with 5% CO[2] in 3% Bovine Serum spermatozoa were incubated up to 6h at 37 Albumin-supplemented Ham's F-10 for capacitation to take place. The total proteins from spermatozoa were extracted and were subjected to SDS-PAGE before and after capacitation. To evaluate protein tyrosine phosphorylation pattern, western blotting with specific antibody against phosphorylated tyrosines was performed. The results upon western blotting showed: 1] at least six protein bands were detected before capacitation in the spermatozoa from normospermic samples. However, comparable levels of tyrosine phosphorylation was not observed in the spermatozoa from teratospermic samples. 2] The intensity of protein tyrosine phosphorylation appears to have been increased during capacitation in the normospermic relative to the teratospermic group. For the first time, these findings demonstrate and suggest that the differences in the types of proteins and diminished tyrosine phosphorylation efficiency in sperm from teratospermic men may be responsible for their compromised capacitation and low fertilization success rates