Figure 1.
Detection of Tas1r-transcripts from cDNA of murine vallate papillae and testicular tissue using RT-PCR.
Primer sets specific for the murine Tas1r1 and Tas1r3 yielded amplification products with the expected size ([Tas1r1]; 468 bp; ([Tas1r3]; 510 bp) from cDNA derived from taste [VP] as well as from testicular tissue ([Te]), whereas the primer pair for the Tas1r2 only resulted in the generation of an amplification product in taste cDNA ([Tas1r2]; 403 bp [VP]), but not in testicular cDNA ([Te]). cDNA quality was assured determining amplification products with a primer pair against the housekeeping gene beta-actin (right panel, [actin]; 425 bp]). Negative controls present samples in which water was used instead of cDNA ([H2O]). The identities of amplified taste receptor subtypes are indicated on the top of each panel. The corresponding 500 bp DNA size marker is shown on the left of both panels.
Figure 2.
Expression of Tas1r3 in murine taste buds and epididymal spermatozoa.
[A] Immunohistochemical analysis of Tas1r3 localization in taste cells of murine vallate papillae. The two applied Tas1r3-specific antisera ([Tas1r3M]; [Tas1r3A]) labeled a subset of spindle-shaped cells within taste buds (arrowheads); neutralization of the Tas1r3M primary antiserum with an excess of the corresponding antigenic peptide ([Tas1r3M+BP]) resulted in elimination of the fluorescence signals. Sections incubated with the secondary antiserum alone showed no immunoreactivity (left panel; [control]). The dotted lines in the control panel highlight the border of individual taste buds. [B] Subcellular localization of Tas1r3 in murine spermatozoa determined by indirect immunofluorescence. Isolated murine sperm were fixed with ice-cold methanol and subsequently incubated with one of the two above mentioned Tas1r3 antisera ([Tas1r3M]; [Tas1r3A]). Bound primary antiserum was visualized by a FITC-conjugated anti-rabbit IgG. Nuclear staining was performed with propidium iodide (shown in blue). An application of both Tas1r3 antisera resulted in a strong immunostaining (green fluorescence) which was restricted to the convex side of the sperm head (arrowheads) and the principle piece of the sperm flagellum([Tas1r3M] and [Tas1r3A], arrows). Pre-incubation of the Tas1r3M antiserum with the immunogenic peptide completely prevented the immunoreactivity ([Tas1r3M+BP]). Negative controls represent samples incubated with the secondary antiserum alone (right panel; [control]). The inserts in the upper panels show regions presented at higher magnifications in the micrographs below. [C] Acrosomal localization of Tas1r3 in murine sperm. To determine the precise subcellular localization of Tas1r3 in mouse spermatozoa, freshly isolated epididymal mouse sperm were probed with one of the two rabbit anti-Tas1r3 antisera ([Tas1r3M], [Tas1r3A]) (green) and the acrosomal marker peanut agglutinin ([PNA]) conjugated to TRITC (red). Note that overlay of each of the two antiserum-derived fluorescence staining patterns with the labeling signals of the fluorochrome-conjugated PNA resulted in an orange-yellow fluorescence color in the acrosomal cap ([Tas1r3M+PNA]; [Tas1r3A+PNA] ¸ arrowhead), indicating a localization of the Tas1r3 within the acrosomal region. Presented experiments show representative results of experiments which were repeated at least three times with different tissue and cell preparation.
Figure 3.
Tas1r1 mCherry reporter expression and co-localization with Tas1r3.
[A] Localization of the Tas1r1 reporter protein mCherry and the Tas1r3 receptor in a fungiform papilla of the tongue. Coronal sections of a fungiform papilla of a Tas1r1/mCherry reporter mouse were incubated with a Tas1r3 specific antiserum ([Tas1r3A]) which was visualized using a FITC-coupled secondary antiserum (green). Subsequently, fluorescence labeling patterns were imaged using confocal microscopy. Note that mCherry fluorescence (red), reflecting activity of the Tas1r1 promoter in the taste bud, and staining with the Tas1r3 specific antiserum are visible in the same cells of the papilla (right panels; ([mCherry+Tas1r3A]). However, while the mCherry fluorescence signal is located in the cytoplasm of the immune-positive cells (lower left panel; [mCherry], arrowhead), the Tas1r3 immunostaining is mainly observed at the plasma membrane (lower middle panel; [Tas1r3A], arrowhead). The superimposed boxes in upper panels represent higher magnifications shown in lower panels. [B] Tas1r1mCherry reporter expression and co-localization with Tas1r3 in testicular tissue. In the upper left panel, a schematic drawing of a single seminiferous tubule with different stages of developing germ cells during spermatogenesis is shown. Note that germ cells of a distinct developmental stage are organized in concentric layers within the tubule: In the most basal cell layer, the spermatogonial stem cells (middle blue) are located, followed by spermatocytes (light blue), round spermatids and finally the most mature elongating spermatids concentrated in the luminal region of the tubule (dark blue). Monitoring localization of the taste dimerization partner by applying a Tas1r3 specific IgG ([Tas1r3M]; green). mCherry expressing tubules also showed immunoreactivity for the Tas1r3 antiserum ([mCherry+Tas1r3M]). The dotted lines in the overview in the top panel mark higher magnifications of three representative tubules with distinct combination of germ cell generations depicted below ([i], [ii], [iii]). Pictures of the fluorescence channels (green, [Tas1r3M]; red, [mCherry]) are merged with the corresponding transmitted-light channels, in the lower panels, only the FITC-derived fluorescence is shown. Micrographs show representative pictures of different Tas1r1/mCherry male mice with comparable results.
Figure 4.
Tas1r1 expression in mammalian spermatozoa.
[A] Extrusion of the mCherry protein during sperm maturation in the epididymis. Cryosections of the caput of the epididymis of a Tas1r1/mCherry reporter mouse were incubated with an anti-mCherry antiserum (red; [mCherry]) and counterstained with the nuclear dye TO-PRO-3 (blue; [TOPRO]). ([mCherry+TOPRO], inset, arrowhead). [B] mCherry fluorescence is not detectable in mature epididymal sperm. Isolated sperm of the mutant mouse line were fixed with PFA and counterstained with the FITC-coupled acrosomal marker PNA (middle panel; arrow; [PNA]). Imaging sperm for mCherry fluorescence revealed that the fluorescent protein was completely lost during epididymal maturation (left panel [mCherry]). Insets in the right panels show higher magnification of the tubule's lumen [A] or a sperm's acrosome [B], respectively. [C] Expression of Tas1r1 in human spermatozoa. Ejaculated human sperm were incubated with a human specific Tas1r1 antiserum; bound primary antiserum was visualized applying a FITC-conjugated anti-rabbit IgG. The two representative confocal micrographs document that the anti-Tas1r1 IgG ([Tas1r1]) showed a staining in the flagellum (arrow) and in the post-acrosomal region as well as at the equatorial segment (arrowheads). Immunostaining in both subcellular compartments was extinguished upon neutralizing the primary antiserum with an excess of the corresponding immunogenic peptide (lower panels; [Tas1r1+BP]), thus confirming specificity of the detected immunolabeling. Negative controls, in which the primary antiserum was omitted, did not show any labeling (data not shown). Confocal images were produced by an overlay of corresponding fluorescence channels (propidium iodide, [red]; FITC-conjugated secondary antiserum, [green]) and the transmission channel. Boxes indicate regions that are magnified in insets in the right panels. Experiments were repeated with at least three independent sperm preparations from different donors, showing comparable results.
Table 1.
Reproductive success of homozygote and heterozygote Tas1r1-deficient mice compared to wild-type mice.
Table 2.
Genotype distribution of offspring from heterozygous Tas1r1 mating pairs.
Table 3.
Effect of Tas1r1 deficiency on total body weight and weight of testes.
Figure 5.
Morphological defects during spermatogenesis upon Tas1r1 gene deletion in Tas1r1/mCherry knock-in mice.
Hematoxylin-Eosin stained sections of seminiferous tubules of wild-type and Tas1r1 knock out littermates were examined for abnormalities during spermatogenesis. Comparing testis of wild-type animals ([+/+]) and Tas1r1-deficient mice ([−/−]),Tas1r1 loss resulted in an increase in the number of spermatocytes which were abnormally found to be localized to the tubule's lumen instead of being concentrated to the basal cell layer (inserts with higher magnifications). In addition, some multinucleated giant cells were visible in single knock-out animals (lower right panel; arrowhead). The images are representatives of histological analyses of 4 adult Tas1r1 knock-out and wild-type littermate animals.
Figure 6.
Determination of apoptotic cells in testicular sections of wild-type and Tas1r1/mCherry knock-in mice.
[A] Paraffin sections of Bouin-fixed wild-type and Tas1r1-deficient testes were used in a fluorescent TUNEL assay and counterstained with DAPI to visualize nuclei and thus cellular compartmentalization. The two photomicrographs for each genotype document representative staining patterns of TUNEL positive cells of 5 male littermates per genotype. Note that in wild-type animals [+/+] as well as in Tas1r1-deficient mice [−/−], spatial localization of TUNEL-reactive cells (red) showed the usual accumulation within the basal cell layer of the testicular tubules. Moreover, apoptotic cells for each genotype did not show obvious differences in their morphology (higher magnifications presented in the inserts in the two upper panels). Micrographs are composed by an overlay of the two fluorescent channels (TUNEL, [red]; DAPI, [blue]); apoptotic TUNEL-positive cells are highlighted by insets. [B] Quantitative analysis of apoptotic cells in testes of wild-type, heterozygous and Tas1r1 null animals. Numbers of TUNEL-positive cells of the three genotypes are presented as apoptotic cells per visual field. Note that Tas1r1-deficient mice ([−/−]) show a significantly increased rate of apoptosis compared to wild-type ([+/+]) and heterozygous ([+/−]) animals. Data presented are mean values ± SEM; statistical analysis was done using a paired Student's t-test comparing apoptotic rates of corresponding littermates (*: p≤0.05; **: p<0.01). Testes of littermate animals (n = 5) of each genotype were analyzed, and sections were taken from two different regions. 3–4 tissue sections of each testicular domain were quantified for TUNEL positive germ cells counting 3–4 randomly chosen microscopic fields containing 25–30 seminiferous tubules each.
Figure 7.
Sperm count and testosterone level of Tas1r1/mCherry knock-in mice.
[A] Total number of caudal epididymal sperm in Tas1r1 null-mutant mice. Number of sperm in the caudal part of the epididymis were counted in male wild-type [+/+], heterozygous [+/−] and homozygous [−/−] mutantTas1r1 animals with identical strain background. Data are mean values ±SEM of 17–46 animals of the three genotypes. [B] Serum testosterone levels in Tas1r1-deficient male mice. Testosterone concentrations were measured in 4–6 month old male littermates of wild-type [+/+], heterozygous [+/−] and homozygous [−/−] Tas1r1 mice by a commercial enzyme-linked immunoassay. Data, expressed as means ± SEM, are obtained from 3 animals of each genotype with triplicate determinations; statistical analysis was done by a paired T-test; a p-value of ≤0.05 was considered to be statistically significant.
Figure 8.
Morphology of Tas1r1-null sperm from the Tas1r1/mCherry mouse line.
[A] Analysis of sperm morphology of wild-type and Tas1r1-deficient sperm. Isolated epidydymal sperm from C57BL/6 wild-type animals [+/+] and Tas1r1-deficient mice [−/−] were fixed, stained with Coomassie blue and subsequently subjected to bright field light microscopy. [B and C] Quantitative morphometric analysis of the sperm head of Tas1r1-deficient mice. To quantify dimensions of the sperm head, the length from the tip of the acrosome to the sperm neck ([I]) and to the post-acrosomal region ([II]) was scaled; in addition, circumference of sperm head ([III]) and the area of the whole sperm head ([IV]) were determined (for overview s. [B]). Data represent mean values ± SEM of the determined parameter which were obtained from 5 Tas1r1-deficient (black bars) and wild-type animals (grey bars); 8–15 sperm from each preparation were analyzed.
Figure 9.
Effect of glutamate on intracellular calcium concentrations in wild-type and Tas1r1/mCherry knock-in mice.
To evaluate the effect of MSG on intracellular Ca2+ concentration ([Ca2+]i) in sperm lacking the Tas1r1 receptor, capacitated cells were loaded with Fura-2/AM and subsequently fluorescence intensity of sperm populations was determined in a plate reader. Therefore, 90 µl of a capacitated sperm suspension (450,000–900,000 cells) were stimulated with different concentrations of MSG (1 mM MSG, 10 mM MSG, 50 mM MSG) by injecting 10 µl of a concentrated tastant stock solution. The concentration of the cation ionophore ionomycin used as a positive control was 5 µM; HS/NaHCO3 buffer alone served as negative control. Fura-2 fluorescence was recorded with excitation wavelengths of 340 and 380 nm; subsequently data were calculated as ratio (F340/F380) and plotted against the time in seconds. Presented data are mean values ± SD of sperm of wild-type [+/+] and Tas1r1-deficient [−/−] mice measured in triplicates, which were representative for 3 experiments per genotype.
Table 4.
Motility analysis of wild-type and Tas1r1-deficent sperm.
Figure 10.
Capacitation and acrosome reaction in Tas1r1 null sperm from the Tas1r1/mCherry mouse line.
[A] Capacitation dependent efflux of cholesterol in Tas1r1-deficient mice. To quantify capacitation dependent cholesterol release in isolated epididymal sperm of wild-type and Tas1r1 null mutant animals, equal amounts of a homogeneous sperm suspension were incubated for different time periods (0 min, 30 min, 60 min, 90 min, 120 min) in HS/BSA/NaHCO3 as described in Materials and Methods. At the indicated time points, aliquots of the supernatant were collected and used to measure cholesterol release using a fluorometric-based quantification kit. Obtained data were calculated as cholesterol efflux per cell after subtracting basal cholesterol content at the beginning of the incubation (0 min: [+/+]: 42±3 ng cholesterol/106 sperm; [−/−]: 37±2 ng cholesterol/106 sperm). Time-dependent sterol release in sperm of both genotypes increased over time and showed no significant difference (p≤0.05). Data, presented as mean values ± SEM, are the average of nine independent sperm preparations of C57BL/6wild-types and Tas1r1-deficient animals from the same colony. [B] A23187 and zona pellucida induced acrosomal secretion in Tas1r1 null sperm. To assess whether Tas1r1-deficient sperm show a defect in the acrosomal exocytotic machinery or in recognizing the egg's coat, respectively, in vitro capacitated spermatozoa of wild-type and Tas1r1 null mutant littermates were either treated with 10 µM A23187 [A23187] or alternatively with solubilised zona pellucida [ZP] at 37°C for 30 min. Subsequently, aliquots of sperm were stained with Commassie blue G.250 and acrosomal status was quantified by counting at least 200 cells for each condition. Data, calculated as absolute percentages of acrosome reacted sperm represent mean values ± SEM of independent experiments with different mouse sperm preparations ([A23187], n = 15; [ZP], n = 7). Spontaneously occurring secretion rates were determined incubating sperm in corresponding buffer used to dilute the stimulating compounds [buffer with DMSO: wild-type [+/+]: 28.1±2.2%; Tas1r1 [−/−]: 35.2±2.5%; ZP buffer alone: wild-type [+/+]: 33.1±3.5%; Tas1r1 [−/−]: 37.7±3.0%). Statistical analysis was done using a Student's t-test comparing acrosome reacted sperm of both genotypes.
Figure 11.
Tas1r1 deletion results in increased spontaneous acrosome reaction and elevated cytosolic Ca2+ and cAMP levels.
[A] Incidence of spontaneous loss of the acrosomal vesicle in sperm from Tas1r1 knock-out mice compared to control wild-type sperm. To quantify spontaneous acrosome reaction of uncapacitated and fully capacitated sperm, epididymal spermatozoa of wild-type and Tas1r1 null mutant mice with identical genetic background were either directly assessed for acrosomal secretion rates or incubated for 90 min in capacitation medium (HS/BSA/NaHCO3). Data shown are mean values ± SEM of 15 independent experiments of different mouse sperm preparations. Obtained data were subjected to a Student's t-test for determination of significant differences (*: p≤0.05) between pairs of both genotypes. [B] Comparison of [Ca2+]i, of wild-type and Tas1r1-deficient spermatozoa. To determine basal [Ca2+]i in the head region of wild-type ([+/+], grey rhombs and squares) and Tas1r1-deficient ([−/−], black rhombs and squares) spermatozoa, epididymal sperm cells were either directly loaded with Fura-2AM ([uncapacitated], rhombs on the left side), or capacitated for 60 min prior Fura-2 loading ([capacitated], squares on the right side). Subsequently, Fura-2 fluorescence at 510 nm was measured at excitation wavelengths of 340 and 380 nm using a microscope based imaging system (TillPhotonics, Graefelfing, Germany). Fura-2 ratios (F340/F380) were determined for at least 14 cells per sperm preparation (total number of measured sperm cells: uncapacitated: 151 [+/+], 136 [−/−]); capacitated sperm: 168 [+/+], 181 [−/−]). [Ca2+]i was calculated using the mean Fura-2 ratio of each animal (F340/F380) according to [84]. Only spermatozoa that showed [Ca2+]i, increases upon stimulation with the calcium ionophore ionomycin were considered. Shown are vertical scatter plots of Fura-2 ratios of isolated spermatozoa of 5 animals for each genotype (littermates and animals with matched genetic background); the mean Fura-2 ratio is indicated by a bar. Mean values ± SEM of calculated [Ca2+]i, for each genotype are given in numbers in the lower part of the graph.Statistical analyses were done using a paired Student's t-test (**: p<0.01). [C] Vertical scatter plot of basal cAMP concentration in uncapacitated spermatozoa. Shown are basal cAMP concentrations of epididymal sperm isolated in HS buffer. Littermate animals and animals with identical genetic background were prepared and assayed in parallel. cAMP values of corresponding animal pairs are connected by a line. Note that in 13 of 15 analyzed animal pairs, cAMP concentrations were higher in Tas1r1 -deficient [−/−] mice than in wild-type [+/+] animals. [D–E] cAMP concentrations in Tas1r1-deficient sperm compared to sperm of wild-type animals. Epididymal sperm of wild-type [+/+] and Tas1r1-deficient [−/−] mice were either isolated in HS (for 15 min) [uncapacitated] or in capacitation buffer (HS/BSA/NaHCO3 for 60 min; [capacitated]), and subsequently treated for 5 min at 37°C with buffer alone [D] (uncapacitated: n = 15; capacitated: n = 11) or with 0.5 mM IMBX [E] (uncapacitated: n = 13; capacitated: n = 9). After shock-freezing the cells in liquid nitrogen, cAMP was extracted with PCA (7%), and quantified using a commercially available EIA kit. Data are mean values ± SEM. Sperm of littermate animals and animals with identical genetic background and age were assayed in parallel and compared using a paired student's T-Test (*: p≤0.05; **: p<0.01).
Figure 12.
Working model illustrating a possible functional role of taste receptor signaling in taste cells and spermatozoa.
[A] Model for the transduction cascade of the umami receptor in taste cells. On the left, a schematic drawing of the onion-like structure of a single taste bud formed by elongated taste cells is shown. The peripheral ends of the 50–100 taste cells in one taste bud terminate at the gustatory pore; taste information is coded by afferent nerve fibers which innervate the taste buds and come close to type II receptor cells but only form conventional chemical synapses with the basolateral membrane of type III taste cells. In taste cells, the Tas1r1 and Tas1r3 receptors form a functional dimer which is able to recognize amino acids such as MSG. Upon ligand binding, the umami receptor activates a trimeric G Protein consisting of α-gustducin [αGus] and β3 and γ13 [βγ]. The βγ subunit activates phopholipase Cβ2 [PLC] which cleaves phosphatidylinositol 4, 5-bisphosphate [PIP2] to inositol trisphoshate [IP3] and diacylglycerol [DAG]. IP3 mediates an increase in intracellular calcium by activation of calcium channels in the endoplasmic reticulum [ER] and subsequently an influx of calcium through ion channels in the plasma membrane [TRPM5]. Simultaneously, released α-gustducin can activate phosphodiesterase, resulting in a decrease of intracellular levels of cyclic adenosine monophosphate [cAMP]. A crosstalk between the two pathways exists through a cAMP regulated activation of protein kinas A [PKA] which inhibits PLC and the IP3-receptor in the ER. This mechanism may ensure adequate Ca2+ signaling to taste stimuli by keeping the taste cell in a tonically suppressed state. The drawing was modified from Ref. [45] and [109]. [B] Putative model of Tas1 taste receptor signaling in spermatozoa. The schematic drawing in the left signifies the sperm's journey in the different sections of the female genital tract [uterus, oviduct, ampulla] which sperm have to transit to reach the egg in the ampullar region of the oviduct (dotted red line). In sperm cells, the Tas1r1 protein [Tas1r1] may dimerize with its taste partner Tas1r3 or with a yet not identified receptor [R?]. G protein activation results in the release of a G protein α-subunit [Gα] which activates phosphodiesterase [PDE], thus leading to the hydrolysis of cAMP. In this model, an activation of the receptor dimer [Tas1r1/R?] by chemosensory ligands within the different regions of the female genital tract (red rhoms) or a constitutively active receptor may ensure low cAMP levels, thereby preventing cAMP-triggered maturation processes of the sperm, like capacitation, motility or acrosome reaction, before the sperm reaches the egg in the ampullary part of the oviduct. If the simultaneously released Gβγ complex [βγ] indeed stimulates PLC in analogy to taste cells or alternatively activates potassium [K+] channels in sperm, is currently not clear. Constant cAMP hydrolysis can be overcome during sperm maturation either by an decrease in taste receptor activation controlled by changes in the composition of chemical components in the different fluids of the female genital tract or by an increase in [Ca2+]i, or high bicarbonate concentration which would lead to an activation of the soluble adenylatecyclase [sAC] in spermatozoa. For seek of simplicity, regulatory effects of PKA activation or EPAC stimulation on calcium channels or the IP3 receptor are omitted in the model.