Sperm-oocyte membrane fusion in the human during monospermic fertilization

Sperm-oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro. Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH-surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7–14 hours in vitro and inseminated with preincubated sperm, fixed between 1–3 hours after insemination, and examined by transmission electron microscopy. Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome-reacted and had been partly incorporated into the ooplasm, while the anterior two-thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized. The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg.     

Effect of Ca2+ channel antagonists on the acrosome reaction of guinea pig and golden hamster spermatozoa

The effects of Ca²⁺ channel antagonists on the motility and acrosome reaction of guinea pig spermatozoa were examined by incubating the spermatozoa continuously in Ca²⁺-containing capacitating media with 10^?6 M to 10^?4 M antagonist. Antagonists tested were four voltage-gated Ca²⁺ channel antagonists (verapamil, nifedipine, nimodipine, and FR–34235) and two ligand-gated channel antagonists (NaNO₂ and Na-nitroprusside). None of these antagonists could block the acrosome reaction. Instead, three antagonists (verapamil, nimodipine, and FR-34235, each at 10^?4 M) accelerated the onset of the acrosome reaction with a subsequent decrease in sperm motility. Nifedipine and Na-nitroprusside at the same concentration caused a complete loss of sperm motility by 4 hr of incubation with no substantial effect on the rate of acrosome reaction. The detrimental effect of antagonists on the motility of spermatozoa appears to be due to a direct, Ca²⁺-independent, membrane-perturbing action of the reagents. The acrosome reaction was not inhibited when guinea pig spermatozoa were precapacitated in Ca²⁺-free medium (with a low concentration of lysolecithin) in the continuous presence of antagonists. An acceleration of the onset of the acrosome reaction by verapamil (10^?4 M) was also demonstrated in the golden hamster. These results may be interpreted as indicating that the entry of extracellular Ca²⁺ into spermatozoa, which triggers the acrosome reaction of guinea pig and hamster spermatozoa, is not mediated by Ca²⁺ channels. This is in marked contrast with the case reported in invertebrate spermatozoa. Possible mechanisms by which some of the antagonists stimulate the acrosome reaction and affect the motility of mammalian spermatozoa are discussed.     

A minimum mechanism for Na+−Ca++ exchange: Net and unidirectional Ca++ fluxes as functions of ion composition and membrane potential

Summary Both simultaneous and consecutive mechanisms for Na⁺–Ca⁺⁺ exchange are formulated and the associated systems of steady-state equations are solved numerically, and the net and unidirectional Ca⁺⁺ fluxes computed for a variety of ionic and electrical boundary conditions. A simultaneous mechanism is shown to be consistent with a broad range of experimental data from the squid giant axon, cardiac muscle and isolated sarcolemmal vesicles. In this mechanism, random binding of three Na⁺ ions and one Ca⁺⁺ on apposing sides of a membrane are required before a conformational change can occur, translocating the binding sites to the opposite sides of the membranes. A similar (return) translocation step is also permitted if all the sites are empty. None of the other states of binding can undergo such translocating conformational changes. The resulting reaction scheme has 22 reaction steps involving 16 ion-binding intermediates. The voltage dependence of the equilibrium constant for the overall reaction, required by the 31 Na⁺Ca⁺⁺ stoichiometry was obtained by multiplying and dividing, respectively, the forward and reverse rate constants of one of the translocational steps by exp(–FV/2RT). With reasonable values for the membrane density of the enzyme (120 sites m²) and an upper limit for the rate constants of both translocational steps of 10⁵·sec^–1, satisfactory behavior was obtainable with identical binding constants for Ca⁺⁺ on the two sides of the membrane (10⁶ m ^–1), similar symmetry also being assumed for the Na⁺ binding constant (12 to 60m ^–1). Introduction of order into the ion-binding process eliminates behavior that is consistent with experimental findings.     

Effects of irradiation level on leaf growth of sunflower

Sunflower, Helianthus annuus L. cv. INRA 6501, plants were grown in a gravel culture subirrigated with Hoagland nutrient solution, at photosynthetically active radiation levels of 15, 30 and 60 W m^-2 at a daylength of 16 h, a temperature of 20°C and a relative humidity of 60% throughout. Development of the plant and growth of the leaves were measured. High irradiance accelerated development proportionally in all phases from germination, through leaf initiation, primordial flower formation and the maturation of all plant organs until anthesis. High irradiance levels stimulated the expansion of the growing shoot, which produced more and larger primordia. Under constant conditions the ratio between leaf initiation rate and mature length of a leaf remained constant, although the growth patterns [relationship between relative growth rate (RGR) and organ age] of successive leaves were not similar. Consequently, it may be assumed that, as in poplar, the increasing size of the growing shoot reflects the increase of the vascular system of sunflower. The growth patterns of the leaves depend on the developmental stage of the plant and, in the young primordial stage, also on irradiance level. In the linear phase of growth the growth pattern is independent of irradiance level.     

Gleichgewichtsbewegungen von Blättern als Reaktion auf asymmetrische Belastung — Isoklinotropismus nach Härdtl (1927) — und ihre Beziehung zum Gravitropismus

1. Die anscheinend in Vergessenheit geratenen älteren Angaben von HÄRDTL (1927 und spärer) und PRINGSHEIM (1931), nach denen einseitig belastete Blätter Gleichgewichtsbewegungen ausführen können (Isoklinotropismus nach HÄRDTL), wurden an mehreren Arten (Chelidonium majus, Aegopodium podagra ria, Ranunculus repens, Sambucus nigra, Coleus blumei und Hibiscus rosa sinensis) bestätigt,— Die einseitige Belastung erfolgte durch Einstecken von Nadeln in die eine Hälfte der Blattspreite bzw. in eine Blattfieder. 2. Bei Blättern, die in Spreite oder Stiel epinastisch gekrtimmt sind (die überwiegende Mehrzahl) oder auch durch die Belastung abwärts gebogen sind, kommt es dabei zu zwei verschiedenen Reaktionen: a) zu einer sofort eintretenden rein physikalischen reversiblen Reaktion, da die einseitige Last eine Torsion der Spreite bewirkt, die die Spreitenspitze nach der der Last gegenüberliegenden Seite [führt, und b) zu einer spärer eintretenden physiologischen Reaktion durch eine Wachstumskrümmung nach derselben Seite, - Beide Komponenten der Bewegung ftihren zu einer Verringerung oder einem Ausgleich des Ungleichgewichtes. 3. Die physiologische Reaktion war meist nach einem oder einigen Tagen erkennbar, bei Chelidonium mitunter schon nach einigen Stunden, bei Hibiscus gelegentlich erst nach etwa einer Woche. Mitunter blieb die Reaktion auch aus. Bei den gefiederten Blattern erfolgte sie in der Rhachis, ober- und unterhalb der belasteten Fieder, mitunter auch im Blattstiel, bei Coleus im Spreitengrund und im Blattstiel, bei Hibiscus im oberen Gelenkpolster (“Sekundargelenk”) des Blattstiels. Auch ältere Blätter reagierten oft noch überraschend gut. Vielfach führte die physiologische Reaktion zu einer vollständigen Ausbalancierung der einseitig belasteten Blattspreite. Auf nachtragliche Entfernung der eingesteckten Nadeln gingen auch die Krtimmungen in 2–3 Tagen wieder weitgehend zurück. 4. Als Ursache ftir die Gleichgewichtsbewegungen der Blätter kommen zwei verschiedene Mechanismen in Betracht: a) Infolge der durch die einseitige Belastung hervorgerufenen Schräglage der Blattspreite sammelt sich das Auxin auf der tiefer liegenden Flanke von Mittelnerv bzw. Rhachis und Blattstiel an, was zu einem stärkeren Wachstum dieser Seite und einer Gleichgewichtskrümmung führen muß. Nach dieser Auffassung ordnet sich der Isoklinotropismus dem Gravitropismus ein. b) Die nach der Belastung sofort eintretende auf der Torsionsspannung beruhende rein physikalische und zunächst reversible Gleichgewichtsreaktion des Blattes wird nach einigen Stunden oder Tagen teilweise irreversibel. (Für die spannungsfreien Abschnitte der Rhachis eines gefiederten Blattes oberhalb der Belastung kann diese Erklärung natürlich nicht gelten). Vermutlich sind beide Mechanismen, vor allern wohl der erstgenannte, bei den einzelnen Arten in verschiedenem Maße, als Ursache der Gleichgewichtsbewegungen wirksam. 5. Der biologische Sinn der Ausbalancierung eines (größeren) Blattes (PRINGSHELM 1931, HÄRDTL 1927, 1937 a) liegt darin, daß a) ein ausbalanciertes Blatt den geringsten Aufwand an mechanischen Elementen erfordert, und b) Photo- und Gravitropismus nur ein ausbalanciertes Blatt ohne Komplikationen in die angestrebte Lage fuhren konnen.     

一种快速、灵敏的血小板释放功能检测方法及其在抗血小板药物研究中的应用

本文报道了一种快速、灵敏的血小板释放功能检测方法:利用荧光素-荧光素酶在有ATP、Mg~(2+)、O_2存在时产生的生物发光素测定血小板ATP的释放量,以反映血小板的释放功能;研究了ADP、AA、胶原、凝血酶等四种诱导剂对血小板释放功能的作用,发现ADP的诱导释放能力较其他三者为弱;观察在不同剂量ADP和AA的诱导下,血小板聚集强度和释放能力之间的关系,研究了血小板数等因素对ATP释放功能测定的影响。应用该方法研究了Aspirin及活血化淤药物川芎嗪,毛冬青甲素对血小板释放功能的影响,发现Aspirin对AA诱导的释放反应有强烈的抑制作用。在以ADP诱导的释放反应中,川芎嗪的抑制作用较毛冬青甲素更为强烈。     

Acrosomal reaction and early events at fertilization in Marthasterias glacialis (Echinodermata: Asteroidea)

When the spermatozoon of M glacialis contacts the mature oocyte jelly it adheres to it. Following this, there is a slight tumefaction of the acrosome, which is followed by the disruption of the apical acrosomal vesicle and cytoplasmic membranes. Acrosomal vesicle contents are liberated and spread along the outer surface of the oocyte jelly. Meanwhile, the acrosomal process begins to extend, penetrates all the jelly extension, then the vitelline layer, and finally contacts the cytoplasmic egg membrane. Nevertheless, the sperm cell continues lying at the outer border of the jelly. From the beginning of the acrosome reaction the dense and finely fibrillar subacrosomal material is connected, by some expansions, to the basal acrosomal vesicle membrane. Both nuclear and mitochondrial diameters have diminished.     

General method for the derivation and numerical solution of epithelial transport models

Summary A general method is presented for the formulation and numerical evaluation of mathematical models describing epithelial transport. The method is based on the principles of conservation of mass, and maintenance of electroneutrality within the cells and bathing solutions. It is therefore independent of the specific membrane transport mechanisms, and can be used to evaluate different models describing arbitrary transport processes (including passive, active and cotransport processes). Detailed numerical methods are presented that allow computation of steady-state and transient responses under open-circuit, current-clamp and voltage-clamp conditions, using a general-purpose laboratory minicomputer. To evaluate the utility of this approach, a specific model is presented that is consistent with the Koefoed-Johnson and Ussing hypothesis of sodium transport in tight epithelia (Acta Physiol. Scand. 42:298–308, 1958). This model considers passive transport of an arbitrary number of permeant solutes, active transport of sodium and potassium, and osmotically induced water transport across the apical and basolateral membranes. Results of the model are compared to published experimental measurements in rabbit urinary bladder epithelium.     

Transient breakdown in the selective permeability of the plasma membrane ofChlorella emersonii in response to hyperosmotic shock: Implications for cell water relations and osmotic adjustment

Summary In osmotic experiments involving cells of the euryhaline unicellular green algaChlorella emersonii exposed to hyperosmotic stress by immersion in a range of low molecular weight organic and inorganic solutes, a temporary breakdown in the selective permeability of the plasma membrane was observed during the initial phase of transfer to media of high osmotic strength (up to 2000 mosmol kg^–1). Thus, although the cells appeared to obey the Boyle-van't Hoff relationship in all cases, showing approximately linear changes in volume (at high salinity) as a function of the reciprocal of the external osmotic pressure, the extent of change was least for the triitols, propylene glycol and glycerol, intermediate for glucose, sorbitol, NaCl and KCl, with greatest changes in media containing the disaccharides sucrose and maltose. In NaCl-treated cells, uptake of external solute and loss of internal ions was observed in response to hyperosmotic treatment while sucrose-treated cells showed no significant uptake of external solute, although loss of intracellular K⁺ was observed. These observations suggest that the widely used technique of estimating cellular turgor, and osmotic/nonosmotic volume by means of the changes in volume that occur upon transfer to media containing increasing amounts of either a low molecular weight organic solute or an inorganic salt may be subject to error. The assumption that all algal cells behave as ideal osmometers, with outer membranes that are permeable to water but not to solutes, during the course of such experiments is therefore incorrect, and the data need to be adjusted to take account of hyperosmotically induced external solute penetration and/or loss of intracellular osmotica before meaningful estimates of cell turgor and osmotic volume can be obtained.