Journal: Molecular Biology of the Cell

Article Title: NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa

doi: 10.1091/mbc.E13-09-0523

Figure Lengend Snippet: TPC1 expression in reproductive tissue and mammalian spermatozoa. (A, B) Expression and membrane localization of TPC1 in testicular and epididymal tissue. Equal amounts of crude tissue starting material (supernatant 1 [S 1 ]), as well as cytoplasmic (supernatant 2 [S 2 ]) and membrane extracts (P 2 ) of mouse wild-type (TPC1 [+/+]) and TPC1-deficient (TPC1 [−/−]) testis (A) and epididymis (B), were subjected to SDS–PAGE and assayed for TPC1 immunoreactivity using the anti-TPC1NK antibody. Equal protein loading of tissue fractions derived from both genotypes was verified using an antibody recognizing the raft marker protein caveolin-1 (Cav-1). Note that in wild-type samples the anti-TPC1NK IgG-labeled immunoreactive bands were centered at a molecular mass of ∼94 kDa in testis (A) and epididymis (B), whereas in reproductive tissue of TPC1-null animals no immunostaining was detectable. Comparing the labeling in the cytosolic (S 2 ) and membrane fractions (P 2 ), it is evident that the immunoreactive bands were always enriched in the corresponding membrane protein fractions, whereas the cytosolic extracts showed no obvious immunoreactivity. (C–E) Identification of TPC1 in rodent and human spermatozoa by immunoblot analysis. Equal amounts of membrane fractions of mouse spermatozoa of TPC1 wild-type ([+/+]) and TPC1-knockout mice ([−/−]; C) and membrane fractions of rat spermatozoa (D), as well as human sperm membranes (E), were separated by SDS–PAGE and probed with the anti-TPC1NK IgG. On monitoring of sperm preparations of the three species for immunoreactivity, bands with the expected size for TPC1 of ∼94 kDa for rodents and ∼100 kDa for humans were visible. The immunoreactive bands with higher molecular masses indicate glycosylated forms of TPC1. Specificity of immunostaining in rat and human sperm was confirmed upon neutralization of the primary antibody with the immunogenic peptide (D and E, Ab + BP). For mouse sperm, germ cells of TPC1-knockout animals were examined as negative control (C, [−/−]). A, B, and D show representatives of at least three independent experiments with tissue preparations of different animals. For separated mouse (C, 19 animals) and human sperm (E, ejaculates of three healthy volunteers), germ cells were pooled and fractionated; membrane proteins were subjected at least three times to Western blot analyses. The caveolin-1 blot (Cav-1) served as loading control for epididymal mouse sperm. Left, positions of the molecular weight standards in kilodaltons for each Western blot.

Article Snippet: To identify TPC1 and TPC2 in mouse and rat spermatozoa, we used the following antibodies: a polyclonal rabbit anti-TPC1 antibody recognizing a synthetic peptide from the C-terminal region of mouse TPC1 (ab80961; Abcam, Cambridge, United Kingdom), as well as a polyclonal goat anti-TPC1 raised against an epitope mapping the C-terminus of TPC1 of human origin, which was also suggested to recognize TPC1 of mouse and rat (sc-67973; Santa Cruz, Biotechnology, Heidelberg, Germany).

Techniques: Expressing, SDS Page, Derivative Assay, Marker, Labeling, Immunostaining, Western Blot, Knock-Out, Neutralization, Negative Control, Molecular Weight

Journal: Molecular Biology of the Cell

Article Title: NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa

doi: 10.1091/mbc.E13-09-0523

Figure Lengend Snippet: Immunoelectron microscopic analysis of TPC1 in adult mouse testis. Ultrathin sections of adult mouse testis of wild-type (left, TPC1 [+/+]) and TPC1-knockout males (right, TPC1, [−/−]) were labeled with anti-TPC1NK antibody, followed by incubation with goat anti-rabbit IgG conjugated to colloidal gold particles. TPC1-null testis tissue and the midpiece (mp) and proximal part of the principal piece (pp) of sperm tails of wild-type males did not show any precipitated immunogold particles. However, in wild-type spermatides, immunogold particles were only detected at a narrow area between the plasma membrane (pm) and the subacrosomal space (ss). This area holds membranes lining the acrosome. Although these membranes were only poorly preserved due to the fixation and embedding procedure, gold particles were found to follow the contour of the outer acrosome membrane. Images in the second line show different tissue areas with similar gold particle distribution. nm, nuclear membrane; nu, nucleus.

Article Snippet: To identify TPC1 and TPC2 in mouse and rat spermatozoa, we used the following antibodies: a polyclonal rabbit anti-TPC1 antibody recognizing a synthetic peptide from the C-terminal region of mouse TPC1 (ab80961; Abcam, Cambridge, United Kingdom), as well as a polyclonal goat anti-TPC1 raised against an epitope mapping the C-terminus of TPC1 of human origin, which was also suggested to recognize TPC1 of mouse and rat (sc-67973; Santa Cruz, Biotechnology, Heidelberg, Germany).

Techniques: Knock-Out, Labeling, Incubation

Journal: Molecular Biology of the Cell

Article Title: NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa

doi: 10.1091/mbc.E13-09-0523

Figure Lengend Snippet: Localization of NAADP-binding sites and TPC1 in mouse spermatozoa. (A) Subcellular localization of trans -Ned-19–binding sites in murine sperm. Epididymal mouse sperm were incubated with 100 μM fluorescent NAADP antagonist trans -Ned-19 (Ned19), and subsequently binding of trans- Ned-19 was examined microscopically. To visualize autofluorescence under the excitation/emission conditions of trans -Ned-19, control sperm were incubated in the corresponding buffer only. Note that trans -Ned-19–loaded sperm not only show autofluorescence-derived tail staining, as observed for untreated sperm (control, arrow), but in addition are characterized by an intense blue fluorescence signal located at the neck region (Ned19, arrowhead], as well as at the acrosomal cap (Ned19, arrowhead). The dotted lines in the phase-contrast picture (right, top micrograph) border the sperm's head and the flagellum. Micrographs show representative images of at least three different sperm preparations with comparable results. (B) Trans -Ned-19 binding is localized to the acrosomal region. Isolated epididymal mouse sperm were probed with trans -Ned-19 (Ned19, blue), the fluorescent acidotropic dye LysoTracker Red (Lyso, red), or the FITC-conjugated acrosomal marker PNA (green), illustrating the position of the hook-shaped acrosome. Note that staining with PNA as well as with LysoTracker Red shows the same subcellular distribution as labeling with trans -Ned-19. The light micrograph of the mouse sperm head (top) marks the sperm nucleus (nu), the apical acrosomal region (a), and part of the midpiece of the sperm tail (mp). Arrowheads mark positive sperm acrosomal labeling. (C) Distribution of TPC1 in epididymal mouse spermatozoa visualized via immunogold electron microscopy. Ultrathin sections of adult mouse epididymis were labeled sequentially with anti-TPC1NK antibody, followed by incubation with gold-conjugated anti-rabbit IgG. Note that immunogold particles are predominantly localized at the acrosomal region of the sperm head between residues of the plasma membrane (pm) and the nuclear membrane (nm). A few colloidal particles were also found associated with the condensed DNA in the nucleus (nu).

Article Snippet: To identify TPC1 and TPC2 in mouse and rat spermatozoa, we used the following antibodies: a polyclonal rabbit anti-TPC1 antibody recognizing a synthetic peptide from the C-terminal region of mouse TPC1 (ab80961; Abcam, Cambridge, United Kingdom), as well as a polyclonal goat anti-TPC1 raised against an epitope mapping the C-terminus of TPC1 of human origin, which was also suggested to recognize TPC1 of mouse and rat (sc-67973; Santa Cruz, Biotechnology, Heidelberg, Germany).

Techniques: Binding Assay, Incubation, Derivative Assay, Staining, Fluorescence, Isolation, Marker, Labeling, Electron Microscopy

Journal: Molecular Biology of the Cell

Article Title: NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa

doi: 10.1091/mbc.E13-09-0523

Figure Lengend Snippet: Fertility and outcome of systematic breeding of TPC1-deficient mice.

Article Snippet: To identify TPC1 and TPC2 in mouse and rat spermatozoa, we used the following antibodies: a polyclonal rabbit anti-TPC1 antibody recognizing a synthetic peptide from the C-terminal region of mouse TPC1 (ab80961; Abcam, Cambridge, United Kingdom), as well as a polyclonal goat anti-TPC1 raised against an epitope mapping the C-terminus of TPC1 of human origin, which was also suggested to recognize TPC1 of mouse and rat (sc-67973; Santa Cruz, Biotechnology, Heidelberg, Germany).

Techniques:

Journal: Molecular Biology of the Cell

Article Title: NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa

doi: 10.1091/mbc.E13-09-0523

Figure Lengend Snippet: Genotype distribution of pups produced by heterozygous TPC1 breeding pairs. Frequency of genotypes of offspring from heterozygous TPC1 mating pairs was determined in a continuous breeding study. Columns represent the percentage of the registered genotype of offspring; dashed red lines indicate expected frequency based on Mendelian inheritance. Note that heterozygous intercrosses produced fewer TPC1-mutant mice ([−/−]) than predicted from the Mendelian distribution. Only 128 of 681 weaned pups from 105 litters were homozygous for TPC1, resulting in a significant deviation from Mendelian distribution of TPC1-null pups. Data present the ratio of offspring of 26 heterozygous TPC1 breeding pairs. Numbers in the columns represent numbers of pups for each genotype. The significance of deviation from expected Mendelian ratio was tested using chi-squared statistics.

Article Snippet: To identify TPC1 and TPC2 in mouse and rat spermatozoa, we used the following antibodies: a polyclonal rabbit anti-TPC1 antibody recognizing a synthetic peptide from the C-terminal region of mouse TPC1 (ab80961; Abcam, Cambridge, United Kingdom), as well as a polyclonal goat anti-TPC1 raised against an epitope mapping the C-terminus of TPC1 of human origin, which was also suggested to recognize TPC1 of mouse and rat (sc-67973; Santa Cruz, Biotechnology, Heidelberg, Germany).

Techniques: Produced, Mutagenesis

Journal: Molecular Biology of the Cell

Article Title: NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa

doi: 10.1091/mbc.E13-09-0523

Figure Lengend Snippet: Effect of TPC1 deficiency on testis weight and number of caudal epididymal sperm.

Article Snippet: To identify TPC1 and TPC2 in mouse and rat spermatozoa, we used the following antibodies: a polyclonal rabbit anti-TPC1 antibody recognizing a synthetic peptide from the C-terminal region of mouse TPC1 (ab80961; Abcam, Cambridge, United Kingdom), as well as a polyclonal goat anti-TPC1 raised against an epitope mapping the C-terminus of TPC1 of human origin, which was also suggested to recognize TPC1 of mouse and rat (sc-67973; Santa Cruz, Biotechnology, Heidelberg, Germany).

Techniques:

Journal: Molecular Biology of the Cell

Article Title: NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa

doi: 10.1091/mbc.E13-09-0523

Figure Lengend Snippet: Comparison of trans -Ned-19–induced Ca 2+ increase and acrosome reaction rates in TPC1-deficient and wild-type sperm. (A) Time course of trans -Ned-19–induced increase in [Ca 2+ ] i in TPC1 wild-type and TPC1-deficient sperm. To compare directly the effect of TPCN1 gene deletion on trans -Ned-19–induced increase in [Ca 2+ ] i , capacitated wild-type [ +/+ ] and TPC1-null sperm [− / −] were loaded with the Ca 2+ fluorescent dye Fluo8-AM (10 μM). Fluorescence intensity was determined at the excitation wavelength of Fluo8-AM (485 nm) using a yellow fluorescent protein filter and a microscope-based imaging system. After a 30-s baseline interval to determine the Fluo8 fluorescence intensity in the head region of adhered sperm, 100 μM trans -Ned-19 was added. Note that sperm of both genotypes responded with a comparable transient increase in [Ca 2+ ] i to stimulation with trans -Ned-19. Fluorescence intensity, recorded in 2-s intervals, was normalized to the initial values of each single cell and is presented as percentage of basal Fluo8 emission (F/F 0 ). Data show mean values ± SEM of sperm preparations of three TPC1 wild-type and knockout animals (total number of measured sperm, 14–35 cells/animal). (B) Comparison of spontaneous acrosome reaction rates of TPC1-deficient and wild-type spermatozoa. Spontaneous loss of the acrosomal vesicle was quantified via incubation of spermatozoa of wild-type and TPC1-deficient animals for either 90 min in capacitation buffer or KRB buffer supplemented with SLO (120 min). In addition, permeabilized sperm were treated with 10 mM CaCl 2 (Ca 2+ ) or 10 mM CaCl 2 together with 10 μM of the Ca 2+ ionophore A23187 (Ca 2+ + A23187). CaCl 2 , as well as CaCl 2 plus A23187, markedly increased acrosomal secretion rates in sperm of both genotypes when compared with the basic level of spontaneously acrosome-reacted spermatozoa ([ +/+ ], 16.58 ± 1.81%; [−/−], 16.58 ± 0.68%). However, in calculating statistical differences between acrosome reaction rates between sperm of both genotypes, no significant differences ( p ≤ 0.05) were detected. (C) NAADP-induced acrosome reaction in sperm of TPC1-null mice compared with wild-type spermatozoa. Capacitated and permeabilized epididymal sperm of TPC1-knockout and wild-type animals were stimulated with NAADP, and acrosome reaction rates were determined. Because dose-dependent relationship analyses show three response peaks for NAADP, NAADP doses inducing maximal acrosomal secretion rates were applied (50 nM, 1 μM, 50 μM). Quantification of the acrosomal status of treated sperm revealed that very low (50 nM) and high NAADP doses (50 μM) elicited strong increase in acrosome reaction rates in TPC1-null sperm, which was not significantly different from the percentage of acrosome reaction in wild-type sperm. However, comparing the elevation in the percentage of acrosome reaction induced upon application of 1 μM NAADP wild-type sperm shows the expected increase in acrosome reaction rate. In contrast, TPC1-deficient spermatozoa did not show responsiveness to this NAADP concentration. Data presented as acrosomal exocytosis index are mean values ± SEM of independent experiments with different mouse sperm preparations ([ +/+ ], n = 10; [− / −], n = 9). Statistical analysis was done using an unpaired Student's t test comparing the acrosomal exocytosis index of sperm of both genotypes. *** p < 0.001; n.s., not significant.

Article Snippet: To identify TPC1 and TPC2 in mouse and rat spermatozoa, we used the following antibodies: a polyclonal rabbit anti-TPC1 antibody recognizing a synthetic peptide from the C-terminal region of mouse TPC1 (ab80961; Abcam, Cambridge, United Kingdom), as well as a polyclonal goat anti-TPC1 raised against an epitope mapping the C-terminus of TPC1 of human origin, which was also suggested to recognize TPC1 of mouse and rat (sc-67973; Santa Cruz, Biotechnology, Heidelberg, Germany).

Techniques: Fluorescence, Microscopy, Imaging, Knock-Out, Incubation, Concentration Assay