Effects of Genistein Isoflavone (4',5',7-Trihydroxyisoflavone) and Dexamethasone on Functional Characteristics of Spermatozoa

Caudal epididymal spermatozoa were used to study the influence of genistein isoflavone and dexamethasone (dxm) on the functional characteristics of spermatozoa. The effects of genistein alone and in combination with dxm on sperm motility, sperm morphology, spontaneous acrosome reaction (AcR), and ionophore A23187-induced AcR were investigated. The FITC-PSA/Hoechst 33258 staining procedure was used to assess sperm cell viability and AcR status and thus to differentiate between true AcR and acrosome degeneration. The overall results indicated that (1) lower doses of genistein alone, or in combination with dxm, did not significantly influence sperm motility or sperm morphology; (2) ionophore A23187 induced AcR in rat spermatozoa; (3) there appeared to be no direct correlation between sperm motility and AcR, (4) higher doses of genistein, alone or in combination with dxm, significantly interfered with percentage sperm motility and caused significant detachment of sperm heads but did not cause morphological defects; and (5) higher doses of genistein caused significant decrease in sperm acrosome reactivity with long duration of exposure. In view of the fact that sperm capacitation and AcR are physiological prerequisites for successful fertilization of oocytes, the findings suggest that chronic exposure of spermatozoa to high doses of genistein could be associated with infertility problems through suppression/inhibition of AcR and sperm motility. Dexamethasone did not appear to influence the effect of genistein on the functionality of postspermatogenic spermatozoa.     

受精调控机制的研究进展

人类和哺乳动物的受精机制是近年来的研究热点.通过生物化学、转基因和基因敲除等研究得出的受精机制主要分为4个部分：①精子获能;②精子结合至卵子透明带;③精子与透明带结合诱发顶体反应（AR）;④精子与卵子的结合和膜融合.笔者拟就近期研究阐明的精子获能、AR及精、卵融合的新信息、新观点,进行综述如下.     

Baboon spermatozoa-zona pellucida binding assay

This study developed a baboon in vitro system that allows transport of sperm from a treatment facility to an off-site location for subsequent evaluation of sperm functional capacity. We further described a sperm functional assay that evaluates baboon sperm binding to homologous zona pellucida, a baboon hemizona assay (HZA). Semen samples were collected from baboons via electroejaculation directly into refrigeration transport buffer. Postshipment semen characteristics were analyzed and each specimen prepared for assessment of sperm-zona pellucida interaction. Optimization of the baboon HZA included determination of the relationship between motile sperm concentration and zona pellucida binding. The effect of the sperm activators, caffeine and dbcAMP, on computerized sperm motion characteristics and HZ binding was also determined. A significant motile sperm concentration dependent increase was observed in sperm-zona pellucida binding. Maximal binding was observed at approximately 1-2 million motile sperm/mL. Treatment with the sperm activators, caffeine and dbcAMP, resulted in a significant increase in sperm progressive motility, straightline velocity (VSL), and amplitude of lateral head displacement (ALH), p <0.05 and a highly significant increase in curvilinear velocity (VCL), p <0.01. Treatment with caffeine and dbcAMP was not an absolute requirement for sperm-zona pellucida binding, inasmuch as binding did occur in the absence of activators. However, treatment with the two activators, caffeine and dbcAMP, resulted in a highly significant increase in HZ binding, p <0.0001. This system allows for the short-term maintenance of baboon sperm in a semiquiescent state until stimulation with the activators, caffeine and dbcAMP. It further provides a novel approach to delineating a contraceptive regimen's or agent's (ie, sperm vaccine) impact on specific cellular events occurring in the male gamete during fertilization.     

Effects of hypothermic liquid storage and cryopreservation on basal and induced plasma membrane phospholipid disorder and acrosome exocytosis in boar spermatozoa

Flow cytometry was utilised to determine whether short-term (Day 1) or long-term hypothermic liquid storage (Day 5), or cryopreservation of boar spermatozoa (1) caused changes in plasma membrane phospholipid disorder (MPLD) and acrosome exocytosis (AE), indicative of an advanced stage of capacitation or acrosome status, and (2) facilitated or inhibited the induction of capacitation and the acrosome reaction. Merocyanine with Yo-Pro-1 and peanut agglutinin-fluorescein isothiocyanate with propidium iodide were used to identify MPLD and AE, respectively, in viable spermatozoa. The incidence of basal sperm MPLD and AE in fresh semen was very low (1.1 and 2.2%, respectively) and was increased (P < 0.05) only a small amount in Day 5 and cryopreserved semen (3-8%). Compared to no bicarbonate, incubation with bicarbonate increased MPLD, but the response was greatest (P < 0.05) in fresh sperm (52.3%) compared with Day 1 (36.6%), Day 5 (13.9%) and cryopreserved sperm (13.6%). Incubation with calcium ionophore A23187 increased AE in spermatozoa, but the response was less (P < 0.05) for fresh (34%) and cryopreserved (27%) semen than for Day 1 (45%) and Day 5 (57%) semen. In summary, hypothermic liquid storage and cryopreservation of boar spermatozoa did not advance capacitation or acrosome status in viable spermatozoa, but did alter their responses to induction of capacitation and the acrosome reaction.     

Toxic potential of dietary genistein isoflavone and beta-lapachone on capacitation and acrosome reaction of epididymal spermatozoa

We determined if acrosomal reaction was influenced by exposure of sperm cells to two dietary phytochemicals, genistein isoflavone and beta-lapachone, using the rat model. Spermatozoa were capacitated in capacitating medium with or without genistein isoflavone and beta-lapachone, and the percentage of posttreatment acrosome reaction compared with controls was assessed with two fluorescent probes, chlortetracycline (CTC) and fluorescein isothiocyanate- Pisum sativum ag-glutinin conjugate (FITC-PSA). Spermatozoa were permeabilized in ethanol and labeled with the FITC-PSA or CTC to determine the acrosome status. The results revealed that calcium ionophore could induce acrosome reaction in spermatozoa and that acrosome-reacted sperm cells showed obvious darkness in the head region, whereas acrosome-intact sperm displayed bright fluorescence over the entire sperm head. The basic response and pattern of acrosome reaction status were significantly similar in both CTC and FITC assays and in both treatment (genistein and beta-lapachone) groups. It was observed that higher doses of both genistein and beta-lapachone significantly suppressed acrosome reaction and that this inhibitory effect was both dose- and time-dependent. It was stipulated that the observed genistein inhibition of acrosome reaction could be due to suppression of protein kinase C, and that beta-lapachone could inhibit acrosome reaction through direct cytotoxic effects on sperm cell membrane at higher doses. However, light microscopic examination indicated that both phytochemicals had no significant effect on sperm morphology. It is concluded that, in view of the fact that acrosome reaction is a physiological prerequisite for fertilization of most mammalian eggs, both genistein and beta-lapachone could potentially suppress male fertility via suppression of acrosome reaction at higher doses, but could enhance fertility by promoting acrosome reaction at lower doses. This bimodal mode of action of both phytochemicals could offer a potentially new dimension in the search for causes of male infertility and possibly for male contraceptive development.     

Factors Regulating Sperm Capacitation

Capacitation is broadly defined as the functional modifications rendering sperm competent to fertilize, encompassing the ability of the sperm to bind the zona pellucida and subsequently undergo the acrosome reaction, hyperactivated motility, and the capacity to fuse with the oocyte. Although discovered in 1951, research over the past 15 years has considerably clarified the mechanisms leading to capacitation. The purpose of this review is to discuss the challenges of studying capacitation and to summarize recent notions regarding its regulation. Of particular interest is an atypical soluble adenylyl cyclase that is stimulated by bicarbonate to activate protein kinase A and drive sperm protein tyrosine phosphorylation. The identities of the phosphorylated sperm-protein substrates and the kinase(s) responsible for their tyrosine phosphorylation have fostered major questions regarding this pathway. Recent investigations, however, have made exciting advances toward resolving these queries. Advanced proteomic approaches have revealed the tyrosine phosphorylated substrates to be implicated in a diverse range of cellular activities. SRC tyrosine kinase is a particularly interesting candidate as the mediator of the protein kinase A-driven sperm protein tyrosine phosphorylation. Future studies are merited to fully characterize additional signaling mediators such as phosphatases and other kinases that may be involved, to elucidate the functional importance of the tyrosine phosphorylation on those particular substrates and to appreciate the differences that may exist among species.     

醋酸乌利司他体外对人精子参数与功能影响的研究

目的：醋酸乌利司他（ulipristal acetate,UPA）是一种孕激素受体调节剂,目前作为紧急避孕药物广泛应用于临床。本研究探索在体外UPA对人精子参数与功能的影响。方法：选择15例精液参数正常的标本,液化后以G-IVF调节精子浓度为2×106/mL。每例标本再分为6份,每份0.5 mL,共6组（n=15）。密度梯度离心后,其中4组分别在含0.04、0.4、4、40 μmol/L UPA的培养液中孵育1 h;另2组为空白对照组、二甲基亚砜（DMSO）对照组。检测各组精子活力、存活率、形态、DNA完整性、顶体反应、精子超活化、精子内游离钙离子浓度。结果：不同浓度的UPA体外处理精子,精子存活率、活力、形态与对照组（空白+DMSO）比较差异无统计学意义（P＞0.05）。UPA浓度达到0.4 μmol/L时处理精子,精子损伤的比例和彗尾长度显著增加,精子超活化的比例降低（P＜0.01）;顶体反应被抑制,精子内钙离子浓度也显著降低（P＜0.01）。结论：UPA在体外可抑制精子的顶体反应和超活化,降低精子内钙离子浓度,可能与UPA致精子损伤有关。     

Polyspermic penetration in porcine IVM-IVF systems

Although techniques for in vitro production of porcine embryos have proceeded very rapidly during the past decade, polyspermic penetration still remains a persistent obstacle to porcine in vitro fertilization (IVF) systems. Considerable research on in vitro polyspermic penetration in porcine in vitro-matured (IVM) oocytes has been undertaken to try to solve this problem. In the current paper, recent advancements in overcoming the problems of polyspermy in porcine IVF systems are reviewed. Partial induction of the acrosome reaction of boar spermatozoa in IVF media that contain caffeine is likely to be one of the major causes of polyspermy. A reduction in the number of incompletely acrosome-reacted spermatozoa, which can bind tightly to the zona pellucida and mask free sperm receptors of the zona pellucida, could reduce the incidence of polyspermic penetration; however, morphological differences in the reaction of the zona pellucida have been observed between IVM and ovulated oocytes, which suggests that altered zona morphology may be another cause of polyspermic penetration. It has been shown that the developmental ability of polyspermic porcine embryos to the blastocyst stage is similar to that of normal embryos but that developmental competence to term is much lower. To overcome the current problems of polyspermy, it is suggested that future efforts should be focused on controlling boar sperm function and/or sperm-zona binding to achieve the final maturation associated with normal zona modifications of porcine oocytes at fertilization.     

哺乳动物精子顶体反应相关的蛋白及信号传导

哺乳动物精子顶体反应是自然受精起始过程中的重要环节,有多种精子膜蛋白和卵子透明带糖蛋白参与。这些蛋白一方面通过精子膜跨膜信号传导引起精子的顶体反应,另一方面,也为精子穿过透明带及精卵融合等提供必要的条件。精子顶体反应过程中涉及多种细胞信号传导的过程,如G蛋白耦联的信号传导、受体酪氨酸激酶(RTK)途径等。研究精子顶体反应过程中相关分子作用的信号传导通路,对进一步深入了解受精的分子机制具有重要的影响。     

Effect of L-Carnitine on Motility and Acrosome Reaction of Human Spermatozoa

L-carnitine added to the suspension medium decreases the glucose-sustained progressive motility of human spermatozoa. Addition of 20 mM L-carnitine to the capacitation medium causes an inhibition of the occurrence of the acrosome reaction parallel to a viability enhancement and negligible changes of the cellular content of ATP. The cellular efflux of glutamate-oxaloacetate transaminase was also inhibited by L-carnitine. A possible role of L-carnitine on membrane stability and metabolism of spermatozoa is briefly discussed.     

Effects of aqueous crude extract of Echeveria gibbiflora on mouse sperm function

The present study evaluates the possible antifertility effect of aqueous crude extract (OBACE) of Echeveria gibbiflora, a plant that belongs to the crassulaceae family, used in traditional Mexican medicine as a vaginal post coital rinse to prevent pregnancy and shown to have an immobilization/agglutination effect on sperm of different mammal species. We evaluated the effect of OBACE on functional parameters of mouse sperm, such as viability, capacitation, and acrosome reaction. In addition, due to the high concentrations of calcium bis-(hydrogen-1-malate) hexahydrate [Ca (C₄H₅O₅)₂?6H₂O] present in this plant extract, we evaluated its effect on Ca²⁺ influx in mouse sperm under capacitating conditions. Moreover, we determined the acute toxicity of OBACE and its in vivo effect in mouse sperm motility administering a single daily dose of 50 and 100 mg/kg during seven days, intraperitoneally. The sperm viability was not affected by the presence of different concentrations of OBACE, however, the capacitation and acrosome reaction suffered a significant decrease in a concentration-dependent manner, coinciding with the reduction of Ca²⁺ influx. Furthermore, OBACE displayed an LD₅₀ of 3,784.42 mg/kg and can be classified as a low toxic substance. Also, in vivo OBACE showed an inhibition of total and progressive motility on mouse sperm alongside a significant decrease of motility kinematic parameters and IVF rates. The results confirm the antifertility effect of this plant used in Mexican folk medicine. Further study on OBACE as a possible contraceptive treatment is warranted because of its activity and low in vivo toxicity.

Abbreviations: ALH: lateral amplitude; AP: acid phosphatase; BCF: beat frequency; BSA: bovine serum albumine; CTC: chlortetracycline; FDA: fluorescein diacetate; Fura-2 AM: fura-2-acetoxymethyl ester; HIV: human immunodeficiency virus; IVF: in vitro fertilization; OBACE: aqueous crude extract of Echeveria gibbiflora; PI: propidum iodide; SN: supernatant; VAP: average path velocity; VCL: track speed; VSL: straight line velocity     

The effects of levonorgestrel on various sperm functions

Two doses of 750-μg levonorgestrel at 12 h apart is one of the regimens for emergency contraception. The mechanism of action of this regimen is not fully known. We investigated whether levonorgestrel influences sperm functions and thereby, exerts contraceptive activity. The motility, acrosome reaction, zona binding capacity, and oocyte fusion capacity of human spermatozoa treated with 1, 10, and 100 ng/mL levonorgestrel for 3 h were evaluated. Levonorgestrel decreased the curvilinear velocity of the treated spermatozoa in a dose-dependent manner. A significant decrease in straight-line velocity, average path velocity and linearity were also found with 100 ng/mL levonorgestrel treatment. This concentration of levonorgestrel, but not others, also marginally decreased (p = 0.045) the zona binding capacity of the treated spermatozoa. The steroid had no effect on acrosome reaction but had a dose-dependent inhibition on spermatozoa-oocyte fusion. These data show that levonorgestrel affects sperm function only at high concentration and the contribution of these effects to emergency contraception is unlikely to be significant.     

精子顶体发育相关蛋白的研究进展

受精是新生命诞生的第一步,在此过程中,精子将所携带的单倍体遗传物质与卵子的单倍体遗传物质融合成双倍体合子。哺乳动物的受精过程涉及到一系列复杂而精细的活动,如精子的超活化与获能、顶体反应以及精卵结合等。精子顶体是一种特殊的膜细胞器,具有帽状结构并覆盖在精子的细胞核前部,在受精过程中发挥着关键作用。顶体的形成主要包括囊泡形成、囊泡运输、囊泡融合和顶体与细胞核结合等过程,其独特的蛋白运输机制需要内质网、高尔基体等细胞器以及某些特殊结构之间的相互配合。顶体形成是精子细胞分化为精子的关键步骤之一,受多基因精细调控,其发育缺陷与包括圆头精子症在内的多种男性不育症有关。综述近年精子顶体发育过程中主要生物学事件的发生顺序中相关蛋白的研究,并介绍了自噬相关蛋白对顶体发育过程作用的新近研究,以期为男性不育症的诊疗提供新思路。     

POTASSIUM INCREASES INTRACELLULAR CALCIUM SIMULATING PROGESTERONE ACTION IN HUMAN SPERM

Progesterone (P) and zona pellucida are known to induce acrosome reaction in human sperm by increasing cytosolic calcium. High concentrations of potassium ions (K⁺) improve the rate of acrosome reaction in human sperm in vitro. This article determined whether the effect of K⁺ on the acrosome in human sperm is mediated by increasing intracellular calcium ([Ca²⁺]i). The effect of K⁺ on [Ca²⁺]i was examined by using Fura 2 as the fluorescent indicator. The effect of K⁺ and P on [Ca²⁺]i in sperm and the involvement of ion channels was compared. Motile sperm were collected by the swim-up method from semen of healthy volunteers and capacitated overnight in BWW containing 0.5% BSA. Incubation of capacitated sperm with different concentrations of potassium chloride (1.25-20 mM) resulted in dose-dependent increase in [Ca²⁺]i similar to that observed with P. The increase in [Ca²⁺]i by K⁺ and P was blocked by the addition of EGTA, a Ca²⁺ chelator. K+-induced change in [Ca²⁺] was not altered by the addition of dihydropyridine derivatives. The combined treatment of K+ (20 mM) and P (0.75 mug/mL) caused an additive effect on the increase in [Ca²⁺]i. It would appear that human sperm plasma membrane possess different Ca2+ channels responsive to P and K⁺.     

弱精子症发病机制的研究进展

弱精子症是男性不育的主要原因之一,多由染色体异常、微生物感染、内分泌紊乱、精索静脉曲张和自身免疫等因素引起,目前仍有相当比例弱精子症的发病机制未明。精子鞭毛结构蛋白对维持精子正常形态和运动能力十分重要,微管相关蛋白Tektins和细胞骨架重要组成成分Septins等基因发生突变或表达量减少均可导致精子的运动能力下降;精子细胞内的Ca2+、Cl-等离子浓度影响精子内磷酸化级联反应,使精子不断适应外界环境变化并完成获能、顶体反应等重要生理功能;Catspers、富半胱氨酸分泌蛋白（CRISPs）等离子通道及离子通道调节蛋白的表达异常与弱精子症发病密切相关;线粒体内的氧化磷酸化反应为精子运动提供能量,氧化磷酸化酶和呼吸链基因突变以及线粒体相关微小RNA表达异常都可能降低精子的运动能力;另外,JAK-STAT信号转导通路与精子的发生相关,而线粒体介导的精子细胞凋亡通路也与精子活动力低下有关。现就弱精子症发病机制的最新研究进展进行综述。     

Plasma Membrane Cytoskeletal Complex of the Mammalian Spermatozoa

Human and ram sperm were incubated in vitro in Tris buffer with (1) calcium ions, (2) ionophore A23187, (3) calcium plus A23187, (4) without any substance, as described in [13], and were reincubated in situ with the protein S-1 of heavy meromyosin (1 mg/ml). These spermatozoa were examined with the electron microscope to study and characterize the cytoskeletal structures after negative staining, freeze fracturing, and thin sectioning. The formation of the cytoskeletal complex, associated with the plasma membrane in the postacrosome region, appeared to be triggered by S-1 in those sperm that were earlier treated with the ionophore. The dynamic nature of actin present in the postacrosomal dense matrix becomes evident with the formation of a cytoskeletal basket in that region. The cytoskeletal complex may play an important role during the exocytotic acrosome reaction, helping to retain the shape of the sperm head and protect the genetic material until fertilization.     

Use of oocytes that failed to be fertilized in vitro to study human sperm-oocyte interactions: comparison of sperm-oolemma and sperm-zona pellucida binding, and relationship with results of IVF

A test for human sperm binding to the oolemma was developed with oocytes that failed to be fertilized in vitro. The zonae pellucidae of the oocytes were removed under a dissecting microscope by brief exposure to dilute HCl (pH 2.5-3.0) in 0.9% NaCl. The zona-free oocytes (ZFOs) were incubated with a mixture of equal numbers of motile sperm from men to be tested and fertile donors. The sperm was differentially labelled with fluorescein or rhodamine and the results expressed as a ratio of the number of test to control sperm bound to several ZFOs in order to control for variability in the ability of the oolemma to bind sperm. The number of sperm bound to the oolemma increased with time and sperm concentration. The sperm-oolemma binding ratio determined for 32 patients undergoing in vitro fertilization (IVF) was significantly correlated with the sperm-zona pellucida (ZP) binding ratio but was not correlated with other sperm tests. The sperm-oolemma binding ratio was also related to the IVF rate, but this was not significant if the sperm-ZP binding ratio was included in the logistic regression model. Only four of the 32 patients had failure of fertilization in vitro. The human sperm-oolemma binding test may be useful for studying the interaction between gametes, but the test is unlikely to be as useful clinically as the sperm-ZP binding test for predicting fertilization in vitro.     

Contraceptive responses of female hamsters immunized with recombinant sperm protein P26h

BACKGROUND: A number of antigens have been characterized and proposed as potential candidates for immunocontraception. P26h, a 26-kDa hamster sperm protein located on the acrosomal cap, is known to be involved in sperm-zona pellucida interactions. Furthermore, in vivo fertilization can be blocked by active immunization of male hamsters against P26h or maltose-binding protein recombinant P26h (MBP-P26h). OBJECTIVE: The aim of this study was to investigate the immune response and reproductive function of female hamsters immunized against MBP-P26h. RESULTS: Active immunization against MBP-P26h resulted in anti-P26h circulating antibodies, with enzyme-linked immunosorbent assay (ELISA) titers showing interindividual variability. The antibodies produced by the animals immunized against MBP-P26h reacted with the native P26h protein in ELISA, in Western blot analysis and in immunostaining performed on cauda epididymal spermatozoa. Mating of immunized female hamsters resulted in a significant decrease in the number of viable fetuses only in females with high titers of anti-P26h circulating antibodies. DISCUSSION: This result is in agreement with the sperm-zona pellucida binding assay's results. Indeed, sera collected from immunized animals, and not from control animals, significantly blocked sperm-zona pellucida binding in vitro. Histological studies showed that active immunization did not cause any pathology in the reproductive tissues. CONCLUSION: These findings suggest that P26h is a potential candidate for the development of a contraceptive vaccine in both males and females.     

Influence of hyaluronic acid synthesis and cumulus mucification on bovine oocyte in vitro maturation, fertilisation and embryo development

During cumulus-oocyte complex (COC) maturation, cumulus expansion involves the deposition of mucoelastic compounds, especially hyaluronic acid, synthesised from glucose via the hexosamine biosynthesis pathway. The aim of the present study was to determine the effects of uridine monophosphate (UMP) and 6-diazo-5-oxo-L-norleucine (DON), inhibitors of hyaluronic acid synthesis, during bovine oocyte in vitro maturation (IVM) on cumulus expansion, glucose uptake, protein synthesis, cumulus cell number, meiotic maturation, cleavage rate and subsequent embryo development. A further aim of the study was to examine the effect of hyaluronic acid on sperm capacitation and acrosome reaction in relation to the capacity of COCs to be fertilised in vitro. A low correlation between glucose uptake and degree of cumulus expansion was observed. Total and partial inhibition of cumulus expansion was observed with DON and UMP, respectively, and was accompanied by a decrease in glucose uptake with DON. Total protein content and cumulus cell number per COC increased during IVM, but was unaffected by the presence of DON or UMP, as was oocyte meiotic maturation. Rates of cleavage and blastocyst development decreased in oocytes matured with DON and UMP, although this inhibition was reversed when the in vitro fertilisation (IVF) medium contained heparin. Hyaluronic acid induced capacitation and the acrosome reaction, and in IVF medium prevented the inhibition of cleavage and blastocyst development by DON in a similar fashion to heparin. Hyaluronic acid synthesis during cumulus mucification contributes to the penetration and fertilisation of bovine oocytes, most likely by facilitating the processes of capacitation and acrosome reaction. Mucification during IVM is independent of cumulus cell proliferation, COC protein content, oocyte meiotic maturation and subsequent developmental competence once fertilised.