In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (E m ) hyperpolarizes. However, the mechanisms that regulate sperm E m are not well understood. Here we show that sperm hyperpolarize when external Mammalian sperm are not able to fertilize after ejaculation. They acquire this ability only after residing in the female uterine tract for a finite period of time that varies depending on the species. The molecular, biochemical, and physiological changes that occur in sperm while in the female tract are collectively referred to as capacitation (1). Capacitation is associated with changes in membrane properties, enzyme activities, and motility that prepare the sperm for the acrosome reaction and for penetration of the egg vestments prior to fertilization. The molecular basis of capacitation has been partially defined and includes: the removal of cholesterol from the sperm plasma membrane by cholesterol acceptors such as bovine serum albumin (2, 3), modifications in plasma membrane phospholipids, fluxes of HCO 3 Ϫ (4) and other intracellular ions, and increased tyrosine phosphorylation of proteins (5-7). These events are likely to play a role in the induction of hyperactivated motility and the ability of the sperm to undergo a regulated acrosome reaction (for review see Ref. 8).Bovine and mouse sperm capacitation is also accompanied by a plasma membrane hyperpolarization. E m decreases in mouse sperm from Ϫ38 to Ϫ55 mV (4, 9, 10) and in bovine sperm from Ϫ33 to Ϫ66 mV (9). Because capacitation prepares sperm for the acrosome reaction, the capacitation-associated hyperpolarization may regulate the ability of sperm to generate transient Ca 2ϩ elevations during the acrosome reaction induced by physiological agonists (e.g. zona pellucida) (11). In this respect, low voltage-activated T-type Ca 2ϩ channels have been detected in mouse spermatogenic cells (12, 13), and these channels are also present in mature mouse sperm (14, 15). One unique property of low voltage-activated Ca 2ϩ channels is that they inactivate at the resting E m of sperm prior to capacitation (around Ϫ35 mV) (12,14). Thus, if low voltage-activated Ca 2ϩ channels are involved in the regulation of the acrosome reaction, the capacitation-associated sperm hyperpolarization may be required to remove this inactivation (11,16,17).Although the molecular mechanisms by which the sperm E m hyperpolarizes during capacitation are not clear, there exist several potential candidates. demonstrated with patch clamp techniques that inward rectifying K ϩ channels are expressed in mouse spermatogenic cells and proposed that these channels may contribute to the capacitation-associated sperm membrane hyperpolarization. An increase in sperm K ϩ permeability should lead to an E m hyperpolarization, according to the K ϩ equilibrium potential (18). Alternatively, the sperm plasma membrane may become less permeable to Na ϩ . The relatively depolarized mamma...

Fertility is a highly complex and regulated phenomenon essential for the survival of any species. To identify Drosophila fertility-specific neural networks, we used a GAL4/UAS enhancer trap genetic screen that selectively inactivates groups of neurons. We identified a GAL4 line (bwktqs) that has a female sterile phenotype only when it expresses the tetanus toxin light chain (TeTxLC). These flies lack oviduct contraction, lay almost no eggs, sperm accumulate in the oviducts, and fewer than normal are seen in the storage organs. In insects, two neuroactive substances are important for oviduct contraction: octopamine (OA), a monoamine that inhibits oviduct contraction, and glutamate (Glu), a neurotransmitter that induces contraction. It is known that octopaminergic neurons of the thoracic abdominal ganglion (TAG) modulate oviduct contraction, however, the glutamatergic neurons that innervate the oviduct have not been identified yet and the interaction between these two neuroactive substances is not well understood. Immunostaining experiments revealed that the bwktqs line trapped an octopaminergic neural network that innervates the genital tract. We show that wt like oviduct contraction in TeTxLC-inactivated flies can only be rescued by simultaneous application of Glu and OA suggesting that the abdominal bwktqs neurons are both octopaminergic and glutamatergic, the use of an agonist and an antagonist for Glu receptors as well as their direct visualization confirmed its participation in this phenomenon. Our work provides the first evidence that adult abdominal type II visceral innervations co-express Glu and OA and allows us to re-evaluate the previous model of neuronal network controlling insect oviduct contraction.

Sperm capacitation is a complex and indispensable physiological process that spermatozoa must undergo in order to acquire fertilization capability. Spermatozoa from several mammalian species, including mice, exhibit a capacitation-associated plasma membrane hyperpolarization, which is necessary for the acrosome reaction to occur. Despite its importance, this hyperpolarization event has not been adequately examined in human sperm. In this report we used flow cytometry to show that a subpopulation of human sperm indeed undergo a plasma membrane hyperpolarization upon in vitro capacitation. This hyperpolarization correlated with two other well-characterized capacitation parameters, namely an increase in intracellular pH and Ca(2+) concentration, measured also by flow cytometry. We found that sperm membrane hyperpolarization was completely abolished in the presence of a high external K(+) concentration (60 mM), indicating the participation of K(+) channels. In order to identify, which of the potential K(+) channels were involved in this hyperpolarization, we used different K(+) channel inhibitors including charybdotoxin, slotoxin and iberiotoxin (which target Slo1) and clofilium (a more specific blocker for Slo3). All these K(+) channel antagonists inhibited membrane hyperpolarization to a similar extent, suggesting that both members of the Slo family may potentially participate. Two very recent papers recorded K(+) currents in human sperm electrophysiologically, with some contradictory results. In the present work, we show through immunoblotting that Slo3 channels are present in the human sperm membrane. In addition, we found that human Slo3 channels expressed in CHO cells were sensitive to clofilium (50 μM). Considered altogether, our data indicate that Slo1 and Slo3 could share the preponderant role in the capacitation-associated hyperpolarization of human sperm in contrast to what has been previously reported for mouse sperm, where Slo3 channels are the main contributors to the hyperpolarization event.

Numerous sperm functions including the acrosome reaction (AR) are associated with Ca 2+ in£ux through voltage-gated Ca 2+ (Ca V ) channels. Although the electrophysiological characterization of Ca 2+ currents in mature sperm has proven di⁄cult, functional studies have revealed the presence of lowthreshold (Ca V 3) channels in spermatogenic cells. However, the molecular identity of these proteins remains unde¢ned. Here, we identi¢ed by reverse transcription polymerase chain reaction the expression of Ca V 3.3 mRNA in mouse male germ cells, an isoform not previously described in these cells. Immunoconfocal microscopy revealed the presence of the three Ca V 3 channel isoforms in mouse spermatogenic cells. In mature mouse sperm only Ca V 3.1 and Ca V 3.2 were detected in the head, suggesting its participation in the AR. Ca V 3.1 and Ca V 3.3 were found in the principal and the midpiece of the £agella. All Ca V 3 channels are also present in human sperm, but only to a minor extent in the head. These ¢ndings were corroborated by immunogold transmission electron microscopy. Tail localization of Ca V 3 channels suggested they may participate in motility, however, mibefradil and gossypol concentrations that inhibit Ca V 3 channels did not signi¢cantly a¡ect human sperm motility. Only higher mibefradil doses that can block high-threshold (HVA) Ca V channels caused small but signi¢cant motility alterations. Antibodies to HVA channels detected Ca V 1.3 and Ca V 2.3 in human sperm £agella. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

This study provides evidence for a novel mechanism of voltage-gated Ca 2+ channel regulation in mammalian spermatogenic cells by two agents that affect sperm capacitation and the acrosome reaction (AR). Patch-clamp experiments demonstrated that serum albumin induced an increase in Ca 2+ T current density in a concentration-dependent manner, and significant shifts in the voltage dependence of both steady-state activation and inactivation of the channels. These actions were not related to the ability of albumin to remove cholesterol from the membrane. In contrast, L L-estradiol significantly inhibited Ca 2+ channel activity in a concentration-dependent and essentially voltage-independent fashion. In mature sperm this dual regulation may influence capacitation and/or the AR. ß