In the female reproductive tract, mammalian sperm undergo a regulated sequence of prefusion changes that "prime" sperm for fertilization. Among the least understood of these complex processes are the molecular mechanisms that underlie sperm guidance by environmental chemical cues. A "hard-wired" Ca 2؉ signaling strategy that orchestrates specific motility patterns according to given functional requirements is an emerging concept for regulation of sperm swimming behavior. The molecular players involved, the spatiotemporal characteristics of such motility-associated Ca 2؉ dynamics, and the relation between a distinct Ca 2؉ signaling pattern and a behavioral sperm phenotype, however, remain largely unclear. Here, we report the functional characterization of two human sperm chemoreceptors. Using complementary molecular, physiological, and behavioral approaches, we comparatively describe sperm Ca 2؉ responses to specific agonists of these novel receptors and bourgeonal, a known sperm chemoattractant. We further show that individual receptor activation induces specific Ca 2؉ signaling patterns with unique spatiotemporal dynamics. These distinct Ca 2؉ dynamics are correlated to a set of stimulus-specific stereotyped behavioral responses that could play vital roles during various stages of prefusion sperm-egg chemical communication.On their journey to locate the oocyte, navigating mammalian sperm depend on both chemical (1) and physical (2, 3) cues to regulate flagellar motion, switch flagellar beat modes, and, thus, direct their movement. How such environmental signals are detected and translated into changes in motility, however, remains largely unknown.Sperm acquire motility upon vaginal deposition (4). This initial swimming behavior is referred to as activated motility and characterized by a relatively low amplitude/high frequency sinusoidal flagellar motion (5). Motile sperm that pass the cervical mucus barrier must locate and enter the oviduct. Only a fraction of sperm present in the uterus (ϳ10% in humans (6)) accomplishes this task. Within the oviductal isthmus, sperm frequently attach to the highly convoluted epithelial surface (3,7,8), forming a sperm reservoir in close proximity to the fertilization site. Upon ovulation, capacitated sperm that adopt a hyperactivated state (i.e. displaying asymmetrical and relatively high amplitude/low frequency flagellar beating) (4), generate sufficient propulsion force to detach from the epithelium, enter the ampulla, and eventually penetrate the cumulus and zona pellucida surrounding the egg (9).Given the small fraction of sperm that reaches the oviduct, a "competitive race" scenario has fallen out of favor in recent years. Instead, effective sperm guidance mechanisms are required to assure a synchronized arrival and encounter of both gametes at the fertilization site (1). Current models propose a complex multistep process of sperm navigation along thermal (2) and chemical (10) gradients. Accordingly, chemical guidance cues are secreted by both the oocyte and the surroundi...

The zona pellucida (ZP) is a glycoprotein matrix surrounding mammalian oocytes. Upon fertilization, ZP hardening prevents sperm from binding to and penetrating the ZP. Here, we report that targeted gene deletion of the liver-derived plasma protein fetuin-B causes premature ZP hardening and, consequently, female infertility. Transplanting fetuin-B-deficient ovaries into wild-type recipients restores fertility, indicating that plasma fetuin-B is necessary and sufficient for fertilization. In vitro fertilization of oocytes from fetuin-B-deficient mice only worked after rendering the ZP penetrable by laser perforation. Mechanistically, fetuin-B sustains fertility by inhibiting ovastacin, a cortical granula protease known to trigger ZP hardening. Thus, plasma fetuin-B is necessary to restrain protease activity and thereby maintain ZP permeability until after gamete fusion. These results also show that premature ZP hardening can cause infertility in mice.

SUMMARYGlutamatergic transmission converging on calcium signaling plays a key role in dendritic differentiation. In early development, AMPA receptor (AMPAR) transcripts are extensively spliced and edited to generate subunits that differ in their biophysical properties. Whether these subunits have specific roles in the context of structural differentiation is unclear. We have investigated the role of nine GluA variants and revealed a correlation between the expression of flip variants and the period of major dendritic growth. In interneurons, only GluA1(Q)-flip increased dendritic length and branching. In pyramidal cells, GluA2(Q)-flop, GluA2(Q)-flip, GluA3(Q)-flip and calcium-impermeable GluA2(R)-flip promoted dendritic growth, suggesting that flip variants with slower desensitization kinetics are more important than receptors with elevated calcium permeability. Imaging revealed significantly higher calcium signals in pyramidal cells transfected with GluA2(R)-flip as compared with GluA2(R)-flop, suggesting a contribution of voltage-activated calcium channels. Indeed, dendritic growth induced by GluA2(R)-flip in pyramidal cells was prevented by blocking NMDA receptors (NMDARs) or voltage-gated calcium channels (VGCCs), suggesting that they act downstream of AMPARs. Intriguingly, the action of GluA1(Q)-flip in interneurons was also dependent on NMDARs and VGCCs. Cell class-specific effects were not observed for spine formation, as GluA2(Q)-flip and GluA2(Q)-flop increased spine density in pyramidal cells as well as in interneurons. The results suggest that AMPAR variants expressed early in development are important determinants for activity-dependent dendritic growth in a cell type-specific and cell compartment-specific manner.

Non-technical summary In mammalian testes, Sertoli cells play a key physiological role in germ cell development. Previous research has implicated local ATP release as a potential mechanism of Sertoli cell stimulation. We show that, in mouse Sertoli cells, two different receptor proteins are activated by ATP. Receptor activation, in turn, causes elevation of calcium ion levels inside the cells. By using a novel method to visualize such calcium signals, we identify mitochondria as essential elements of calcium regulation in the testis.Abstract Intimate bidirectional communication between Sertoli cells and developing germ cells ensures the integrity and efficiency of spermatogenesis. Yet, a conceptual mechanistic understanding of the physiological principles that underlie Sertoli cell autocrine and paracrine signalling is lacking. Here, we characterize a purinergic Ca 2+ signalling network in immature mouse Sertoli cells that consists of both P2X2 and P2Y2 purinoceptor subtypes, the endoplasmic reticulum and, notably, mitochondria. By combining a transgenic mouse model with a dedicated bioluminescence imaging device, we describe a novel method to monitor mitochondrial Ca 2+ mobilization in Sertoli cells at subcellular spatial and millisecond temporal resolution. Our data identify mitochondria as essential components of the Sertoli cell signalling 'toolkit' that control the shape of purinergic Ca 2+ responses, and probably several other paracrine Ca 2+ -dependent signals.

Male germ cell development takes place within the testes of mammals, but little is known about its regulation. Fleck et al. record from spermatogonia and Sertoli cells, both in vitro and in situ, and find evidence for P2X4- and P2X7-mediated ATP-gated currents as well as a Ca2+-activated BK conductance.