Sperm motility initiation factor is a minor component of the Pacific herring egg chorion

Polyclonal antibodies were generated to the 105 kDa herring sperm motility initiation factor (SMIF) and used to explore the role of SMIF in sperm-egg interaction. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with SMIF antibodies, it was demonstrated that SMIF is present as a minor (4–7% of total chorion protein) component of the chorion. The major polypeptides in the chorion migrated at 117 kDa and in a grouping between 48–54 kDa, with other minor bands above and below. The only detectable glycosylated component was the 105 kDa band, which was resolved at two isoelectric points (8.22 and 8.31) after isoelectric focusing gel electrophoresis. Using antibodies to SMIF, fertilization was blocked, sperm motility was inhibited in vitro in the presence of solubilized SMIF and SMIF binding sites on sperm were localized. Lastly, SMIF was localized to the region of the herring egg that encircles the micropyle.     

The micropyle: a sperm guidance system in teleost fertilization

The micropylar region of the Rosy barb, Barbus conchonius, egg consists of 7-10 grooves and ridges, which drain directly into a funnel-shaped vestibule, the only point on the chorion through which sperm-egg contact is achieved during fertilization. Results of time-lapse video microscope study and computer-aided analysis of sperm motility pattern in the micropylar region showed that the fertilizing sperm, usually the first to enter the micropylar region, always travelled preferentially along the grooves into the micropylar pit. Subsequently, 86% of sperm arriving the micropylar region within 30 s travelled preferentially along the grooves into the immediate vicinity of the micropylar pit. The sperm guidance role of the micropylar region was calculated to enhance chances of egg penetration/fertilization by as much as 99.7% once sperm were within the micropylar region, possibly in response to some form of chemo-attractant(s) from the egg. Sperm agglutination post-fertilization was also found to occur preferentially along the grooves. Results of our in vitro fertilization experiments showed association between point of sperm entry and blastodisc formation: the blastodisc formed directly beneath the micropyle in all undisturbed eggs.     

Sperm motility-initiating substance in newt egg-jelly induces differential initiation of sperm motility based on sperm intracellular calcium levels

Sperm motility-initiating substance (SMIS), a novel motility inducer from newt egg-jelly, is activated by the release from associated jelly substances at the beginning of internal fertilization and affects female-stored sperm. We examined motility initiation kinetics of newt sperm in response to SMIS by monitoring the changes of sperm intracellular calcium ([Ca2(+)](i)). In quiescent non-motile sperm loaded with the Ca2(+) indicator Fluo-4, intracellular free Ca2(+) was observed around mitochondria using confocal scanning laser microscopy. A slight increase in [Ca2(+)](i) occurred simultaneously and transiently at motility initiation in sperm treated with either heated jelly extract (hJE) containing activated SMIS, or a low osmotic solution, which naturally initiates motility in externally-fertilizing amphibians and can initiate motility in urodele sperm. When the increase of [Ca2(+)](i) at motility-initiation was monitored using spectrofluorometry, large increases in [Ca2(+)](i) occurred immediately in the low osmotic solution and within 1.5 min in the hJE. In the intact jelly extract (no heating), small increases of [Ca2(+)](i) irregularly occurred from around 1 min and for about 4 min, during which motility was differentially initiated among sperm. These results indicate that the SMIS induces differential initiation of sperm motility depending on the activational states of the SMIS and its overall activity. The motility initiation in the jelly extract was delayed in sperm whose intracellular Ca2(+) had been chelated with BAPTA-AM. The relative levels of [Ca2(+)](i) were variable with a mean of 414 ± 256 nmol/L among resting sperm, suggesting that the level of [Ca2(+)](i) in the resting sperm modulates the responsiveness to the SMIS.     

Factors Controlling Sperm Entry into the Micropyles of Salmonid and Herring Eggs

When the micropyle area of salmonid (trout and salmon) eggs was observed continuously from the moment of insemination, spermatozoa were seen moving along the surface of the chorion and entering the micropyle one by one in a directed fashion. The ability of spermatozoa to enter the micropyle was reduced after the treatment of chorions with pronase; this reduction in sperm entry was observed even before the outer opening of the micropyle channel was narrowed due to gradual swelling of the chorion by pronase treatment. Herring spermatozoa, unlike spermatozoa of most other marine fishes, were motionless in seawater. However, they became vigorously motile on contact with the micropyle area of the herring egg chorion and entered the micropyle rapidly and efficiently. Motility initiation of herring spermatozoa in the micropyle area was dependent on extracellular calcium and potassium. Sodium also appears to be intricately involved in this process as demonstrated by the initiation of sperm movement in sodium-free seawater. When herring eggs were treated with acidic seawater, organic solvents, or glutaraldehyde, spermatozoa did not initiate movement in the micropyle area, and sperm entry was not observed. Herring spermatozoa did not initiate movement in the micropyle area of salmonid eggs. These and other observations suggest that the micropyle areas of salmonid and herring eggs possess some sperm guidance factors which facilitate entry of homologous spermatozoa into the micropyle.     

Adaptation and strategy for fertilization in the sperm of teleost fish

Discovery of the environmental triggers for the initiation of sperm motility ( Movisawa and Suzuki, 1980 ) was one of the starting points for understanding the mechanisms regulating sperm behaviour, activation and chemotaxis, which occur as a prologue to fertilization. Further discoveries on egg-derived factors triggering the activation of sperm motility in Pacific herring ( Movisawa et al., 1992 ; Yanagimachi et al., 1992 ) also demonstrated the cooperation of two different proteins playing an important role for the successful encounter of sperm and egg at fertilization. The purpose of this paper was to review the details of the cell-signaling factors for the initiation and activation of sperm motility in teleosts. An additional aim was to introduce the conceptual idea of regulatory mechanisms for sperm motility reflecting evolutionary processes as well as strategies for establishing successful fertilization mechanisms in teleosts.     

Characteristics of sperm motility induced on the egg-jelly in the internal fertilization of the newt,Cynops pyrrhogaster

Most urodeles undergo internal fertilization and sperm are directly inseminated onto the surface of egg-jelly. Feature of sperm motility induced on the egg-jelly was examined in the newt, Cynops pyrrhogaster. When sperm were directly inseminated onto an egg-jelly, sperm motility was immediately induced on its surface. The egg-jelly of C. pyrrhogaster was composed of six sublayers that were added by turns in oviduct. When the eggs with various sets of the sublayers were obtained and sperm were inseminated onto the egg-jelly, the immediate activity for the initiation of sperm motility was observed only on the outermost sublayer. Similarly, the immediate initiation of sperm motility was induced in the sperm suspended in the extract of the egg-jelly (JE). The initiation of sperm motility was affected by the external pH, and the motility was activated in the moving sperm. A K(+)-channel antagonist, charybdotoxin (CTX), or a Ca(2+)-channel antagonist, gallopamil inhibited the initiation of sperm motility in a dose dependent manner. These results demonstrated the feature of the mechanism regulating sperm motility under stable surroundings in the internal fertilization of amphibians.     

Regulation of activation state and flagellar wave form in epididymal rat sperm: evidence for the involvement of both Ca2+ and cAMP

Rat sperm from the cauda epididymis exhibit increased motility, longevity, and a distinct circular pattern of flagellar curvature in response to 5 mM procaine-HCl or 0.1 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), reagents that are thought to play a role in the immobilization of free cellular calcium. Triton X-100-extracted sperm models will exhibit the same pattern of motility and curvature as procaine- or TMB-8-activated cells, but only when calcium is removed by a strong chelating agent, and in the presence of cAMP (3 microM). Demembranated sperm models produced from epididymal rat sperm are quiescent unless cAMP is added. In these sperm models, the presence or absence of free calcium mediates a transition in flagellar curvature. The increased activity of the procaine-treated intact cells was not accompanied by a change in cellular ATP content, nor was ATP availability the limiting factor in the quiescent sperm. Therefore, the increased motility produced by procaine is probably mediated by a fall in free intracellular Ca2+ accompanied by a rise in cAMP. Our finding that calcium controls the curvature of sperm flagella may explain altered patterns of flagellar beating, such as the hyperactivated motility that sperm exhibit in the female reproductive tract.     

Changes in Intracellular Concentrations of Cyclic Nucleotides during the Initiation Process of Starfish Sperm Motility

Changes in the intracellular concentrations of cyclic nucleotides during the initiation of starfish sperm motility were examined. The intracellular concentration of cGMP decreased just after dilution of sperm with sea water, whereas that of cAMP increased concomitant with initiation of sperm motility. In acidic sea water, the intracellular concentration of cGMP decreased but no increase in that of cAMP was observed and sperm remained immotile. The presence of a phosphodiesterase inhibitor enhanced the rate of increase in the intracellular concentration of cAMP and the sperm motility.
These results indicate that cAMP is involved in the initiation of sperm motility in starfish.     

家养鱼类受精生物学的研究 Ⅱ.几种淡水鱼类成熟卵球的精孔器与精子入卵通路的光镜与扫描电镜观察

作者对我国四种淡水养殖鱼类——团头鲂、草鱼、白鲢和花鲢卵球的精孔器作了光学显微镜和扫描电子显微镜的比较描述,在扫描电子显微镜下观察到这几种鱼类的精子均直接经精孔器前庭穿过精孔管进入卵内,并对精孔细胞、受精孔与精子入卵的关系以及精孔的位置进行了讨论。       

Extracellular adenosine 5'-triphosphate alters motility and improves the fertilizing capability of mouse sperm

Extracellular adenosine 5'-triphosphate (ATPe) treatment of human sperm has been implicated in improving in vitro fertilization (IVF) results. We used the mouse model to investigate mechanisms of action of ATPe on sperm. ATPe treatment significantly enhanced IVF success as indicated by both rate of pronuclear formation and percentage cleavage to the 2-cell stage. However, ATPe did not increase the percentage of sperm undergoing spontaneous acrosomal exocytosis nor change the pattern of protein tyrosine phosphorylation normally observed in capacitated sperm. ATPe altered sperm motility parameters; in particular, both noncapacitated and capacitated sperm swam faster and straighter. The percentage of hyperactivated sperm did not increase in capacitated ATPe-treated sperm compared to control sperm. ATPe induced a rapid increase in the level of intracellular calcium that was inhibited by two distinct P2 purinergic receptor inhibitors, confirming that these receptors have an ionotropic role in sperm function. The observed motility changes likely explain, in part, the improved fertilizing capability when ATPe-treated sperm were used in IVF procedures and suggest a mechanism by which ATPe treatment may be beneficial for artificial reproductive techniques.     

Changes in the chorion and sperm entry into the micropyle during fertilization in the teleostean fish, Oryzias latipes

Specific antibodies against the major chorionic glycoproteins (ZI1 -2 and ZI3) of unfertilized eggs were used to analyze the differences in the chorion and its surrounding constituents before and after fertilization. The glycoproteins in the inner layers of the chorion and its surrounding material were specifically stained by both of the antibodies. Thirty and 60 min after activation, the thickness of the chorion's inner layers was already reduced and the micropylar canal was closed. At the same time, the broadly diluted mucous area (DMA) of glycoproteins on the outermost layer of the chorion in unfertilized eggs was modified to a thin, compact layer. When unfertilized eggs were treated with trypsin, the inner third portion of the micropylar canal closed and the glycoproteins in the DMA were digested. The incidence of sperm entry into the micropyle of these eggs was extremely reduced. These results suggest that in medaka eggs, the chorionic glycoproteins in the DMA on the chorion surface, which have an affinity for spermatozo, play an important role in sperm guidance into the micropyle.     

Molecular Reproduction & Development Volume 78, Issue 12 cover image

Signal transduction across plasma membrane is prerequisite for activation of sperm motility and sperm chemotaxis at fertilization. An oscillatory increase in intracellular calcium occurs upon activation of tunicate sperm (shown in false color in the foreground, clockwise displaying later time points; starting at 11:00). Zhu and Inaba (this issue) report that lipid rafts are essential for these calcium dynamics and for subsequent activation of adenylyl cyclase. Background image is of a single tunicate, Ciona intestinalis.     

A comparison of intracellular changes in porcine eggs after fertilization and electroactivation.

The experiments compare intracellular changes in porcine eggs induced by electrical activation with those induced by sperm penetration. Adequate electrostimulation induces changes in both cortical granule exocytosis and protein synthesis similar to those induced by sperm during fertilization. However, ionic changes induced by electrostimulation differ markedly from those initiated at fertilization. Thus, dynamic video imaging using Fura-2 as a Ca2+ probe provides evidence that parthenogenetic activation induced by electrostimulation is initiated by a single sharp rise in the concentration of intracellular free calcium ([Ca2+]i) in the egg. The intracellular Ca2+ transient increase is triggered by an influx of extracellular Ca2+ immediately after electrostimulation. The amplitude of the intracellular Ca2+ transient increase is a function both of the extracellular Ca2+ concentration and of electric field parameters (field strength and pulse duration). Imaging demonstrates further that a single electrical pulse can only induce a single Ca2+ transient which usually lasts three to five minutes; no further Ca2+ transients are observed unless additional electrical stimuli are applied. By contrast, sperm-induced activation is characterised by a series of Ca2+ spikes which continue for at least 3 hours after sperm-egg fusion. The pattern of Ca2+ spiking after fertilization is not consistent during this period but changes both in frequency and amplitude. Overall, the results demonstrate that, although electrostimulation induces both cortical granule exocytosis and protein reprogramming in porcine eggs, it does not reproduce the pattern of [Ca2+]i changes induced by sperm entry at fertilization.     

Deletion of type IIalpha regulatory subunit delocalizes protein kinase A in mouse sperm without affecting motility or fertilization.

Cyclic AMP stimulates sperm motility in a variety of mammalian species, but the molecular details of the intracellular signaling pathway responsible for this effect are unclear. The type IIalpha isoform of protein kinase A (PKA) is induced late in spermatogenesis and is thought to localize PKA to the flagellar apparatus where it binds cAMP and stimulates motility. A targeted disruption of the type IIalpha regulatory subunit (RIIalpha) gene allowed us to examine the role of PKA localization in sperm motility and fertility. In wild type sperm, PKA is found primarily in the detergent-resistant particulate fraction and localizes to the mitochondrial-containing midpiece and the principal piece. In mutant sperm, there is a compensatory increase in RIalpha protein and a dramatic relocalization of PKA such that the majority of the holoenzyme now appears in the soluble fraction and colocalizes with the cytoplasmic droplet. Unexpectedly the RIIalpha mutant mice are fertile and have no significant changes in sperm motility. Our results demonstrate that the highly localized pattern of PKA seen in mature sperm is not essential for motility or fertilization.     

Roles of Calmodulin and Calcium/Calmodulin-Dependent Protein Kinase in Flagellar Motility Regulation in the Coral Acropora Digitifera

In the corals Acropora spp., eggs secrete substances that induce sperm motility regulation. An elevation of intracellular pH ([pH]i) and a regulation of intracellular Ca²⁺ concentration ([Ca²⁺]) are involved in the sperm motility regulation cascade. However, the detailed molecular aspects of flagellar motility regulation have not been fully demonstrated in Acropora. In this study, we determined the presence and roles of both calmodulin (CaM) and calcium/calmodulin dependent-protein kinase (CaMK) in the sperm flagellar motility regulation of Acropora. A ⁴⁵Ca²⁺-overlay assay and an immunoblot analysis showed that sperm contain an acidic 16-kDa protein that was CaM, and an immunoblot analysis revealed the presence of CaMK in coral sperm. In addition, a specific inhibitor of CaMK, KN-93, and a CaM antagonist, W-7, inhibited sperm motility activation induced by NH₄Cl treatment. NH₄Cl treatment causes an increase in intracellular [pH]i of sperm, suggesting that CaM and CaMK are involved in sperm motility initiation caused by an increase in [pH]i. The involvement of CaM and CaMK in motility regulation in coral highlights the importance of these molecules throughout the animal kingdom.     

泥鳅精子入卵的动力作用

在扫描电镜下,泥鳅成熟卵卵膜孔外围呈现完整的左涡旋状结构,受精时精子是顺着涡旋的流线进入卵膜孔。涡旋纹理接近对数螺线。本文分析了真骨鱼类的受精因素,除已知的化学因素外,还存在物理因素。也讨论了泥鳅成熟卵卵膜孔形态形成的必然性。     

金鱼精子入卵过程的扫描电镜观察

本文采用扫描电镜观察了金鱼(Carassius auratus)卵壳膜(chorion)表面结构和精子入卵过程。在壳膜的卵膜孔(micropyle)区有5—10条沟和嵴。位于精孔管下面,卵的质膜为一束较长的微绒毛组成的精子穿入部(sperm entry site)。授精5s,精子头的顶部已附着于精子穿入部,随即两者的质膜发生融合,而围于精子头部四周的微绒毛迅速伸长形成一受精锥,它不断将精子头部包裹。授精110s,精子的头部和颈部已完全进入卵内,受精锥本身也渐趋消失,但精子尾部仍平躺于卵的表面。皮层小泡是在授精30s后才开始破裂并释放其内含物,导致卵子表面呈蜂窝状,并在无膜内表面附着了大量球状物。     

Possible role of calcium on oocyte development after intracytoplasmic sperm injection in quail (Coturnix japonica)

Although a rise in intracellular calcium concentration of vertebrate oocytes plays a pivotal role for the initiation of fertilization or oocyte activation, no study on this subject has been reported in birds. This study was conducted to study the role of intracellular calcium in relation to fertilization in avian oocytes. First, immediately after a quail oocyte was injected with a sperm, it was treated with strontium chloride as an inducer for intracellular calcium rise at doses of 0, 2.5, 5, 7.5, 10 mM for 4 hr in the culture medium and was followed by 20-hr culture. Treatment with 5 mM of strontium chloride induced blastodermal development in 24.2% of injected eggs, although no oocytes developed without strontium treatment. Second, quail oocytes were injected with a sperm and 0.1 M calcium chloride or a sperm and saline solution, cultured without calcium for 4 hr and was followed by 20-hr culture without strontium. The calcium solution induced blastodermal development in 20.5% of the oocytes, although no oocytes developed without calcium treatment. Third, quail oocytes were injected with 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) as a calcium chelator, cultured with strontium (5 mM) for 4 hr followed by 20-hr culture without strontium. Only one oocyte developed after BAPTA and strontium treatment of 36 oocytes examined. Developmental stages of all the oocytes ranged from IV to VII. These results suggest that intracellular calcium rise may participate in quail oocyte activation and allow fertilization and blastodermal development.     

Paradoxical stimulation of human sperm motility by 2-deoxyadenosine

Exposure of cryostored human spermatozoa to 2-deoxyadenosine resulted in significant increases in percentage motility, the linear velocity of progression and the frequency of sperm head rotation, which were maximal at a dose of 2.5 mM. At the same dose both adenosine and caffeine significantly increased percentage motility, although neither compound influenced the quality of sperm movement as assessed by time-exposure photomicrography. 2-Deoxyadenosine was also significantly more effective than caffeine in sustaining the motility of cryostored spermatozoa as well as in enhancing the motility of fresh and washed preparations of human spermatozoa. The ability of caffeine and 2-deoxyadenosine to influence sperm motility was counteracted by the presence of calcium in the external medium although the latter was less susceptible to such inhibition and still enhanced motility in the presence of calcium levels (1.7 mM) typical of media used for in-vitro fertilization. The mechanism of action of 2-deoxyadenosine was associated with an increase of intracellular cAMP levels, which were sustained over a time course lasting from 5 to 180 min and exhibited significant dose dependency over the range 1-10 mM. The response to 2-deoxyadenosine did not involve any changes in the steady state levels of ATP and was augmented by the presence of the phosphodiesterase inhibitors, IBMX and caffeine. We conclude that 2-deoxyadenosine is a powerful stimulator of human sperm motility and that this effect involves an increase of intracellular cAMP levels via mechanisms which do not involve the classical 'R'-site receptor mediated pathway.     

革胡子鲶受精过程的扫描电镜观察

应用扫描电镜观察和描述了革胡子鲶成熟卵和精子的形态、卵壳膜的表面结构和形态、受精孔的位置和结构、精子入卵过程的程序和变化。讨论了精子入卵过程及精孔细胞在解体之后可能转变为一种能够吸引精子在精孔区聚集的“受精素”物质等问题。