pH Regulation in Mouse Sperm: Identification of Na+-, Cl−-, and[formula]Dependent and Arylaminobenzoate-Dependent Regulatory Mechanisms and Characterization of Their Roles in Sperm Capacitation

AbstractIntracellular pH (pHi) regulates several aspects of mammalian sperm function, although the transport mechanisms that control pHiin these cells are not understood. The pHiof mouse cauda epididymal sperm was determined from the fluorescence excitation ratio of 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein and calibrated with nigericin and elevated external [K+]. Two acid efflux mechanisms were identified following imposition of acid loads. One pathway has many anticipated characteristics of the somatic Na+-dependent Cl−–[formula]exchanger, although sperm and somatic mechanisms can be distinguished by their ion selectivity and inhibitor sensitivity. Sperm may have an isoform of this exchange pathway with novel functional characteristics. The second acid-export pathway does not require extracellular anions or cations and is inhibited by arylaminobenzoates (flufenamic acid, diphenylamine-2-carboxylate). Mouse sperm also recover spontaneously from intracellular alkalinization. Recovery rates inN-methyl-D-glucamine+Cl−or in 0.25Msucrose are not significantly different from that in a complex culture medium. Thus, recovery from alkalinization does not utilize specific, ion-dependent transport mechanisms. Other widely distributed acid-efflux mechanisms, such as the Na+–H+antiport pathway and the Na+-independent Cl−–[formula]exchanger are not major regulators of mouse sperm pHi. Sperm capacitation results in pHiincreases (from 6.54 ± 0.08 to 6.73 ± 0.09) that require a functional Na+-, Cl−-, and[formula]-dependent acid-efflux pathway. Inhibition of this regulatory mechanism attenuates alkaline shifts in pHiduring capacitation as well as the ability of sperm to produce a secretory response to zona pellucida agonists. These data suggest that one aspect of mouse sperm capacitation is the selective activation of one major pHiregulator

A Sustained Increase in Intracellular Ca2+ Is Required for the Acrosome Reaction in Sea Urchin Sperm

AbstractThe acrosome reaction (AR), necessary for fertilization in many species, requires an increase in intracellular Ca2+ ([Ca2+]i). In sea urchin sperm, the AR is triggered by an egg-jelly factor: the associated [Ca2+]i elevation lasts minutes and involves two Ca2+ permeable channels. Both the opening of the second channel and the onset of the AR occur ∼5 s after treatment with egg factor, suggesting that these events are linked. In agreement, removal of Ca2+ from sea water or addition of Ca2+ channel blockers at the time when opening of the second channel is first detected inhibits AR and causes a “rapid” (t1/2 = 3–15 s) decrease in [Ca2+]i and partial inhibition of the intracellular pH change associated with the AR. Simultaneous addition of NH4Cl and either EGTA, Co2+, or Ni2+ 5 s after egg factor prevents the partial inhibition of the evoked pHi change observed but does not reverse AR inhibition. Therefore, the sustained increase in [Ca2+]i caused by the second Ca2+ channel is needed for the sperm AR. Experiments with agents that induce capacitative Ca2+ uptake (thapsigargin and cyclopiazonic acid) suggest that the second channel opened during the AR could be a store-operated Ca2+ channel

Sperm Competition, Sperm Numbers and Sperm Quality in Muroid Rodents

Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An “overall sperm quality” parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and developmental pathways underlying processes of sperm formation, maturation, transport in the female reproductive tract, and preparation for fertilization must all evolve in concert

Mechanisms of sperm-egg interactions: between sugars and broken bonds

A model of the early events of mammalian fertilization has emerged during the past 30 years. However, studies during the past decade have used newly available mouse models to readdress these processes. Here, we will consider these new data in light of the existing model and point to areas of reconciliation and of controversy

Mechanisms of sperm-egg interactions: between sugars and broken bonds

A model of the early events of mammalian fertilization has emerged during the past 30 years. However, studies during the past decade have used newly available mouse models to readdress these processes. Here, we will consider these new data in light of the existing model and point to areas of reconciliation and of controversy

Sperm Membrane Potential: Hyperpolarization during Capacitation Regulates Zona Pellucida-Dependent Acrosomal Secretion

AbstractMembrane potential (VM) was investigated in mouse and bovine sperm populations. VM was determined from the fluorescence emission of the lipophilic anion, bis(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC2(3)), and from the lipophilic cation, 3,3′-dipropylthiodicarbocyanine iodide (DiSC3(5)). Fluorescent signals were corrected for contributions of mitochondrial potentials and apparent VM values were obtained by calibrations in sperm selectively permeabilized with valinomycin or with gramicidin D. The calculated VM values of uncapacitated mouse and bovine sperm were approximately -35 and -30 mV, respectively. In contrast, capacitated populations of mouse and bovine sperm have VM values of -50 to -60 mV. Membrane hyperpolarization is due in part to an enhanced K+ permeability. The development of zona pellucida-activated signal transducing mechanisms during capacitation is dependent upon hyperpolarization. It is suggested that VM alterations regulate the activation state of sperm, thereby suppressing premature acrosome reactions in uncapacitated sperm and permitting capacitated sperm to respond to zona pellucida stimuli

A polycystin-1 controls postcopulatory reproductive selection in mice

Pkdrej, a member of the polycystin-1 gene family, is expressed only in the male germ line. Male mice that are homozygous for a targeted mutation in the Pkdrej allele (Pkdrejtm/tm) are fertile in unrestricted mating trials, but exhibit lower reproductive success when competing with wild-type males in sequential mating trials and in artificial insemination of mixed-sperm populations. Following mating, sperm from Pkdrejtm/tm mice require >2 h longer than those of wild-type males to be detected within the egg/cumulus complex in the oviduct. Sperm from mice of both genotypes are able to capacitate in vitro. However, one of the component processes of capacitation, the ability to undergo a zona pellucida–evoked acrosome reaction, develops more slowly in sperm from Pkdrejtm/tm animals than in sperm from wild-type males. In contrast, a second component process of capacitation, the transition to hyperactivated flagellar motility, develops with a similar time course in both genotypes. These two behavioral consequences of capacitation, exocytotic competence and altered motility, are therefore differentially regulated. These data suggest that Pkdrej controls the timing of fertilization in vivo through effects on sperm transport and exocytotic competence and is a factor in postcopulatory sexual selection