CaMKIIalpha interacts with multi-PDZ domain protein MUPP1 in spermatozoa and prevents spontaneous acrosomal exocytosis

The success of acrosomal exocytosis, a complex process with a variety of inter-related steps, relies on the coordinated interaction of participating signaling molecules. Since the acrosome reaction resembles Ca(2+)-regulated exocytosis in neurons, we investigated whether cognate neuronal binding partners of the multi-PDZ domain protein MUPP1, which recruits molecules that control the initial tethering and/or docking between the acrosomal vesicle and the plasma membrane, are also expressed in spermatozoa, and whether they contribute to the regulation of acrosomal secretion. We observed that CaMKIIalpha colocalizes with MUPP1 in the acrosomal region of epididymal spermatozoa where the kinase selectively binds to a region encompassing PDZ domains 10-11 of MUPP1. Furthermore, we found that pre-treating mouse spermatozoa with a CaMKII inhibitor that directly blocks the catalytic region of the kinase, as well as a competitive displacement of CaMKIIalpha from PDZ domains 10-11, led to a significant increase in spontaneous acrosomal exocytosis. Since Ca(2+)-calmodulin releases CaMKIIalpha from the PDZ scaffolding protein, MUPP1 represents a central signaling platform to dynamically regulate the assembly and disassembly of binding partners pertinent to acrosomal secretion, thereby precisely adjusting an increase in Ca(2+) to synchronized fusion pore formation

Expression of Tas1 Taste Receptors in Mammalian Spermatozoa: Functional Role of Tas1r1 in Regulating Basal Ca2+ and cAMP Concentrations in Spermatozoa

Background: During their transit through the female genital tract, sperm have to recognize and discriminate numerous chemical compounds. However, our current knowledge of the molecular identity of appropriate chemosensory receptor proteins in sperm is still rudimentary. Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined. Methodology/Principal Findings: The present manuscript documents that Tas1r1 and Tas1r3, which form the functional receptor for monosodium glutamate (umami) in taste buds on the tongue, are expressed in murine and human spermatozoa, where their localization is restricted to distinct segments of the flagellum and the acrosomal cap of the sperm head. Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities. In addition, a significant increase in spontaneous acrosomal reaction was observed in Tas1r1 null mutant sperm whereas acrosomal secretion triggered by isolated zona pellucida or the Ca2+ ionophore A23187 was not different from wild-type spermatozoa. Remarkably, cytosolic Ca2+ levels in freshly isolated Tas1r1-deficient sperm were significantly higher compared to wild-type cells. Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes. Conclusions/Significance: Since Ca2+ and cAMP control fundamental processes during the sequential process of fertilization, we propose that the identified taste receptors and coupled signaling cascades keep sperm in a chronically quiescent state until they arrive in the vicinity of the egg - either by constitutive receptor activity and/or by tonic receptor activation by gradients of diverse chemical compounds in different compartments of the female reproductive tract

Clinical Experience with Noninvasive Prenatal Testing in Twin Pregnancy Samples at a Single Center in Germany

In this study we wanted to determine the performance of a paired-end sequencing-based noninvasive prenatal testing (NIPT) assay in the detection of common fetal trisomies in twin pregnancy samples. Samples from patients with a twin pregnancy were collected from at least 10 weeks of gestation and analyzed at a single prenatal center in Germany. Results of Anomaly Detected (i.e., high risk) or No Anomaly Detected (i.e., low risk) for trisomy 21, trisomy 18, or trisomy 13 were reported. Follow-up confirmatory outcomes were requested for all cases. A total of 1,658 patients with twin pregnancies submitted samples during the study period; only two of these samples failed resulting in a low failure rate of 0.12%. Of the remaining 1,656 cases, there were 1,625 (98.1%) low-risk and 31 (1.9%) high-risk NIPT samples in our cohort. Of these, follow-up information was available for 301 (18.5%) of the low-risk samples and 19 (61.3%) of the high-risk samples. All of the low-risk cases with follow-up were determined to be true negatives giving an estimated negative predictive value of 100%. Seventeen of the 19 high-risk samples with follow-up were true positives, resulting in an overall positive predictive value of 89.5%. Sensitivities of > 99.9% were noted for both trisomy 21 and trisomy 18, with high specificities of ≥ 99.7% observed for all three trisomies. In conclusion, our study showed strong performance of the NIPT assay in the detection of common fetal trisomies in twin pregnancy samples, with high sensitivities, specificities, and positive predictive values observed based on known clinical outcomes along with a low failure rate

Detection of Tas1r-transcripts from cDNA of murine vallate papillae and testicular tissue using RT-PCR.

<p>Primer sets specific for the murine Tas1r1 and Tas1r3 yielded amplification products with the expected size ([<i>Tas1r1</i>]; 468 bp; ([<i>Tas1r3</i>]; 510 bp) from cDNA derived from taste [VP] as well as from testicular tissue ([<i>Te</i>]), whereas the primer pair for the Tas1r2 only resulted in the generation of an amplification product in taste cDNA ([<i>Tas1r</i>2]; 403 bp [<i>VP</i>]), but not in testicular cDNA ([<i>Te</i>]). cDNA quality was assured determining amplification products with a primer pair against the housekeeping gene beta-actin (right panel, [<i>actin</i>]; 425 bp]). Negative controls present samples in which water was used instead of cDNA ([<i>H₂O</i>]). 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.</p

Effect of Tas1r1 deficiency on total body weight and weight of testes.

<p>Adult male homozygous ([<i>−/−</i>]), heterozygous ([<i>+/−</i>]) and wild-type animals ([<i>+/+</i>]) were analyzed for their total body and testis weight. Data represent mean values ± SEM of 17–46 animals of each genotype with no significant differences between Tas1r1-deficient mice and wild-type animals. Statistical analyses were performed using the Student's t-test. A p-value≤0.05 was considered to be statistically significant.</p

Working model illustrating a possible functional role of taste receptor signaling in taste cells and spermatozoa.

<p>[<b>A</b>] 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 [<i>αGus</i>] and β₃ and γ₁₃ [<i>βγ</i>]. The βγ subunit activates phopholipase Cβ₂ [<i>PLC</i>] which cleaves phosphatidylinositol 4, 5-bisphosphate [<i>PIP₂</i>] to inositol trisphoshate [<i>IP₃</i>] and diacylglycerol [<i>DAG</i>]. IP₃ mediates an increase in intracellular calcium by activation of calcium channels in the endoplasmic reticulum [<i>ER</i>] and subsequently an influx of calcium through ion channels in the plasma membrane [<i>TRPM5</i>]. Simultaneously, released α-gustducin can activate phosphodiesterase, resulting in a decrease of intracellular levels of cyclic adenosine monophosphate [<i>cAMP</i>]. A crosstalk between the two pathways exists through a cAMP regulated activation of protein kinas A [<i>PKA</i>] 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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032354#pone.0032354-Kinnamon2" target="_blank">[45]</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032354#pone.0032354-Clapp1" target="_blank">[109]</a>. [<b>B</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 [<i>uterus</i>, <i>oviduct</i>, <i>ampulla</i>] 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 [<i>Tas1r1</i>] may dimerize with its taste partner Tas1r3 or with a yet not identified receptor [<i>R?</i>]. G protein activation results in the release of a G protein α-subunit [<i>Gα</i>] which activates phosphodiesterase [<i>PDE</i>], thus leading to the hydrolysis of cAMP. In this model, an activation of the receptor dimer [<i>Tas1r1/R?</i>] 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 [<i>βγ</i>] 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 [<i>sAC</i>] 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.</p

Reproductive success of homozygote and heterozygote Tas1r1-deficient mice compared to wild-type mice.

<p>In a continuous mating study, intervals between mating and delivery of pubs [<i>time to litter</i>], time to first delivery [<i>time to first litter</i>] and number of weaned pubs per litter [litter size] were determined for wild-type C57BL/6 animals [<i>(+/+)×(+/+)</i>] and for Tas1r1 mCherry heterozygous [<i>(+/−)×(+/−)</i>] and homozygous [<i>(−/−)×(−/−)</i>] breeding pairs. Given data are mean values ± SEM; 7–14 breeding pairs with 31–50 litters were analyzed per genotype; p-values were determined using an unpaired Student's t test (two-tailed).</p

Motility analysis of wild-type and Tas1r1-deficent sperm.

<p>Computer-assisted sperm analysis (CASA) was performed using an IVOS sperm analyzer (Hamilton Thorne, Berverly, USA). Parameters analyzed are given on the left. Motility values of wild-type [<i>+/+</i>] and Tas1r1 heterozygous [<i>+/−</i>] and homozygous [<i>−/−</i>] sperm are shown as mean values ± SEM of 3 littermate animals for each genotype. Additionally, p-values of a paired Student's T-Test [<i>p values</i>] are given. The following parameters are shown: Percentage of motile sperm [<i>Mot</i>], percentage of sperm with active motility [<i>Prog</i>], averaged path velocity [<i>VAP</i>], straight line velocity [<i>VSL</i>], curvilinear velocity [<i>VCL</i>], amplitude of lateral head displacement [<i>ALH</i>], beat cross frequency [<i>BCF</i>], straightness [<i>STR</i>], linearity [<i>LIN</i>]. A minimum of 2000 spermatozoa was analyzed per animal. Note that wild-type sperm and Tas1r1-deficient spermatozoa did not show any significant differences (p-value≤0.05) in the analyzed motility parameters.</p

Morphology of Tas1r1-null sperm from the Tas1r1/mCherry mouse line.

<p>[<b>A</b>] Analysis of sperm morphology of wild-type and Tas1r1-deficient sperm. Isolated epidydymal sperm from C57BL/6 wild-type animals [<i>+/+</i>] and Tas1r1-deficient mice [−/−] were fixed, stained with Coomassie blue and subsequently subjected to bright field light microscopy. [<b>B and C</b>] 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>I</i>]) and to the post-acrosomal region ([<i>II</i>]) was scaled; in addition, circumference of sperm head ([<i>III</i>]) and the area of the whole sperm head ([<i>IV</i>]) were determined (for overview s. [<b>B</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.</p

Effect of glutamate on intracellular calcium concentrations in wild-type and Tas1r1/mCherry knock-in mice.

<p>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-<i>2</i> fluorescence was recorded with excitation wavelengths of 340 and 380 nm; subsequently data were calculated as ratio (<i>F340/F380</i>) and plotted against the time in seconds. Presented data are mean values ± SD of sperm of wild-type [<i>+/+</i>] and Tas1r1-deficient [<i>−/−</i>] mice measured in triplicates, which were representative for 3 experiments per genotype.</p