An unusual cAMP signaling system mediates many of the events that prepare spermatozoa to meet the egg. Its components include the atypical, bicarbonate-stimulated, sperm adenylyl cyclase and a cAMP-dependent protein kinase (PKA) with the unique catalytic subunit termed C␣ 2 or Cs. We generated mice that lack C␣2 to determine its importance in the events downstream of cAMP production. Male C␣ 2 null mice produce normal numbers of sperm that swim spontaneously in vitro. Thus, C␣ 2 has no required role in formation of a functional flagellum or the initiation of motility. In contrast, we find that C␣ 2 is required for bicarbonate to speed the flagellar beat and facilitate Ca 2؉ entry channels. In addition, C␣2 is needed for the protein tyrosine phosphorylation that occurs late in the sequence of sperm maturation and for a negative feedback control of cAMP production, revealed here. Consistent with these specific defects in several important sperm functions, C␣ 2 null males are infertile despite normal mating behavior. These results define several crucial roles of PKA in sperm cell biology, bringing together both known and unique PKA-mediated events that are necessary for male fertility. T he terminally differentiated, transcriptionally dormant, and translationally inactive posttesticular spermatozoan has a limited ability to respond to environmental cues encountered as it progresses through the male and female reproductive tracts. During this passage, the bicarbonate anion present in the reproductive fluids (1, 2) has an unexpectedly prominent role in promoting several of the events (3-5), collectively called capacitation, which transform sperm to readiness for fertilization.Much evidence indicates that bicarbonate directly increases production of cAMP by atypical sperm adenylyl cyclase (sAC) (6, 7), and recent work finds that sAC is required for male fertility and normal sperm motility (8). Although cAMP might open ion channels or activate guanine nucleotide exchange factors, most evidence places cAMP-dependent protein kinase (PKA) as the major downstream effector of cAMP signals in sperm. However, only a small number of sperm proteins have been identified as phospho-substrates of PKA (9, 10). We now apply phenotypic analysis of loss-of-function mutants to examine downstream effects of the PKA subunit C␣ 2 . Materials and MethodsTargeted Disruption of C␣2. C␣ 2 null mice were generated from the targeted disruption of exon 1b of the C␣ gene by homologous recombination in embryonic stem (ES) cells. The targeting vector was constructed from a 13.2-kb genomic fragment containing exons 1-3 of C␣. A loxP-flanked neomycin phosphotransferase (NEO) cassette and a mutation of the translational initiation codon were inserted into exon 1b. The linearized targeting vector was electroporated into ES cells derived from 129SV͞J mice as described (11). Germ-line chimeras were bred to C57BL͞6 mice. Pups carrying the NEO were crossed to heterozygous ROSA26-Cre recombinase transgenic mice (from P. Sorriano, Fred Hutchinson Cancer Rese...

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Summary Olfactory signals influence social interactions in a variety of species [1, 2]. In mammals, pheromones and other social cues can promote mating or aggression behaviors, can communicate information about social hierarchies, genetic identity and health status, and can contribute to associative learning [1–5]. However, the molecular, cellular and neural mechanisms underlying many olfactory-mediated social interactions remain poorly understood. Here, we report that a specialized olfactory subsystem that includes olfactory sensory neurons (OSNs) expressing the receptor guanylyl cyclase GC-D, the cyclic nucleotide-gated channel subunit CNGA3 and the carbonic anhydrase isoform CAII (GC-D+ OSNs) [6–11] is required for the acquisition of socially transmitted food preferences (STFPs) in mice. Using electrophysiological recordings from gene-targeted mice, we show that GC-D+ OSNs are highly sensitive to the volatile semiochemical carbon disulfide (CS2), a component of rodent breath and a known social signal mediating the acquisition of STFPs [12–14]. Responses to sub-micromolar concentrations of CS2 in the main olfactory epithelium or in identified GC-D+ OSNs are absent in mice lacking CNGA3 or CAII and drastically reduced in mice lacking GC-D. Mice in which GC-D+ OSN transduction mechanisms have been disrupted fail to acquire STFPs from either live or surrogate demonstrator mice and do not exhibit neuronal activation of the ventral subiculum of the hippocampus, a brain region implicated in STFP retrieval [15]. Our findings indicate that GC-D+ OSNs detect chemosignals that facilitate food-related social interactions.

2؉ channels in epididymal sperm examined prior to capacitation. Involvement of a previously undetected Ca V 2.2 (N-type) channel, suggested by the action of GVIA, is substantiated by immunodetection of Ca 2؉ channel ␣ 1B subunits in sperm and sperm extracts. Resistance to dihydropyridines, calciseptine, MVIIC, and kurtoxin indicates that Ca V 1, Ca V 2.1, and Ca V 3 (L-, P/Q-, and T-type) channels contribute little to this evoked response. Partial sensitivity to 1 M mibefradil and an enhanced sensitivity of the GVIAresistant component of response to Ni 2؉ suggest participation of a Ca V 2.3 (R-type) channel specified by previously found ␣ 1E subunits. Our examination of depolarizationevoked Ca 2؉ entry indicates that mature sperm possess a larger palette of voltage-gated Ca 2؉ channels than previously thought. Such diversity may permit specific responses to multiple cues encountered on the path to fertilization.Identification and characterization of sperm Ca 2ϩ entry channels and the mechanisms that modulate their function as sperm prepare to fertilize an egg remain major unsolved problems in reproductive biology (1-3). These challenges stand unmet largely because of the difficulty of applying patch-clamp methods and the tools of molecular biology to sperm. The poorly understood nature of the modifications to sperm that occur between mating and fertilization ("capacitation") presents an additional barrier to investigation.Several indirect approaches have been informative. Probing of a germ line cell library for mRNA of Ca 2ϩ channel ␣ 1 subunits found predominant message for ␣ 1E (4). Whole cell recording from spermatocytes and spermatids found only transient, low voltage-activated (LVA) 1 Ca 2ϩ currents (5-8). Finally, pharmacological sensitivities for spermatid Ca 2ϩ currents showed some parallels with those for Ca 2ϩ -mediated responses of sperm (8, 9). On the basis of these results, it was proposed that a T-type Ca 2ϩ channel is specified by ␣ 1E and is retained after spermiogenesis to provide the major route for depolarization-evoked entry of Ca 2ϩ into sperm. Newer work has shown that expression of ␣ 1E produces Rtype rather than T-type channels (10) and has increased the number of Ca 2ϩ channels found in the germ line. Message for the ␣ 1G and ␣ 1H subunits, now known to specify T-type channels (11-17), has been found in spermatogenic cells (18). Study by immunological methods showed that sperm contain ␣ 1E , ␣ 1A , and ␣ 1C channel proteins (19). If these proteins are functionally active, then P/Q-and L-type channels also might provide routes for entry of Ca 2ϩ (see Table I). Here, we use the kinetics of depolarization-evoked increases in the intracellular free [Ca 2ϩ ] (Ca i ) of epididymal sperm to monitor the activity of voltage-gated Ca 2ϩ channels and examine their pharmacological sensitivity. The results indicate that a portion of the observed activity requires opening of N-type Ca 2ϩ channels, specified by ␣ 1B Ca 2ϩ channel proteins that are detected here for the first time in sperm and sperm e...