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First published online 2 November 2004
doi: 10.1242/jcs.01507

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Functional characterization of a mouse testicular olfactory receptor and its role in chemosensing and in regulation of sperm motility

¹ Department of Integrated Biosciences, The University of Tokyo, Chiba 277-8562, Japan
² Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
³ Genome Information Research Center, Osaka University, Osaka 565-0871, Japan

View larger version (106K): [in a new window]   Fig. 1. Expression and localization of MOR23-encoding transcripts in mouse testis. (A) Tissue distribution of MOR23-encoding transcripts. Expression of MOR23 was assessed by RT-PCR. F, female; G3P-DH, glyceraldehyde-3-phosphate dehydrogenase; M, male. (B-E) The distribution pattern of the MOR23-encoding transcript was analysed by in situ hybridization. (B,D) Detection of MOR23-encoding mRNA with an antisense probe. MOR23-encoding mRNA was detected in a few seminiferous tubes using a TSA biotin system with AP- or HRP-conjugated streptavidin. (C) Control for nonspecific staining using a MOR23 sense probe; no signal was observed. (E) A high-magnification image of the MOR23 antisense probe staining indicates that the MOR23-encoding transcript is localized in spermatogenic cells. Hybridization signals detected with AP-streptavidin followed by NBT/BCIP are stained purple (B,C), whereas those detected with HRP-streptavidin followed by DAB are stained brown and nuclei are counterstained with methyl green (D,E). Scale bars, 100 µm (B-D), 20 µm (E).

View larger version (74K): [in a new window]   Fig. 2. Developmental expression pattern of MOR23 in mouse testis. (A) In situ hybridization using protamine 1 or H1t antisense probe was performed in serial sections to determine the stages of the seminiferous tubes expressing MOR23. (B) Individual tubules are numbered in the image, and the presence (+) or absence (–) of MOR23, protamine 1 and H1t in the corresponding tubules are shown in the table. MOR23-encoding transcripts were observed in seminiferous tubules from which protamine-1-encoding transcripts were absent but H1t transcripts were present. (C) Expression patterns of MOR23 and H1t in the corresponding areas of serial sections. MOR23-encoding transcripts are localized in the round spermatid layer, whereas H1t-encoding mRNA was found in spermatocytes. (D) Diagram of the 12-stage growth cycle of mouse spermatogenesis (Russell, 1990) showing the stages expressing MOR23 (magenta), protamine 1 (blue) and H1t (green) transcripts. MOR23 was expressed during stages VI-VIII. Scale bar, 100 µm.

View larger version (31K): [in a new window]   Fig. 3. Effect of lyral on Ca2+ levels in HEK293 cells expressing MOR23. (A) Chemical structures of odorants used in this study. (B) Effect of various treatments on Ca2+ levels. (top) Pseudocolored images of fura-2-loaded HEK293 cells stimulated with 3 mM odorant or isoproterenol (Iso). Iso, which causes Ca2+ increase in HEK293 cells via intrinsic ß-adrenergic receptors and transfected G15, was used as a control for G15 co-transfection. Scale bar, 20 µm. (bottom) The Ca2+ response profile of MOR23-expressing HEK293 cells. Odorants were applied for 15 seconds during the time indicated by the bars, and cells were continuously washed with buffer between stimulant applications. B, bourgenonal; H, heptanal; LY, lyral.

View larger version (17K): [in a new window]   Fig. 4. Effect of lyral on Ca2+ levels in spermatogenic cells and spermatozoa. (A) Pseudocolored image and representative trace of Ca2+ levels in spermatogenic cells. Different concentrations of lyral were applied for 15 seconds during the times indicated by the bars and cells were continuously washed with buffer between stimulant applications. K8.6, a high potassium solution, was used as a positive control for cell viability. (top) Pseudocolored images of the Ca2+ levels. The cells indicated with white arrows display dose-dependent effects of lyral (scale bar, 20 µm). (bottom) Recordings of the change in fluorescence from single spermatogenic cell. (B) Pseudocolored image and representative trace of Ca2+ levels in cauda epididymal sperm. The response profile shows the change of F ratio calculated in the entire area of the responding sperm head. Arrows indicate the application of an odorant or K8.6. Lyral (LY) but not bourgeonal (Bour) increased the level of Ca2+ in epididymal sperm. Scale bar, 4 µm.

View larger version (54K): [in a new window]   Fig. 5. Construction of transgenic (Tg) mice expressing MOR23 in the testis under the control of the calmegin promoter. (A) The transgene used to generate the Tg mouse line. The arrows show the primers used for genome PCR analysis. (B) Genome PCR analysis with primer pairs I and III demonstrate the insertion of transgene. PCR products with primer pairs II and III were derived from endogenous MOR23. (C) In situ hybridization using DIG-labeled antisense MOR23 probe (MOR23 AS) revealed high-level expression of the MOR23 transgene in Tg mouse testis. Hybridization signals were detected without the TSA system. Calmegin antisense probe (Clgn AS) was used as a positive control. WT, wild-type mouse. Scale bar, 100 µm.

View larger version (20K): [in a new window]   Fig. 6. Effect of lyral on Ca2+ levels in spermatogenic cells and cauda epididymal sperm from MOR23 Tg and wild-type mice. (A) (left) A representative response of Tg mouse testis spermatogenic cells to increasing concentrations of lyral. (middle) A dose-response curve for lyral in spermatogenic cells from Tg (filled squares) and wild-type (filled triangles) mouse testis (± s.e., n=3). (right) The proportion of spermatogenic cells responding to 3 mM lyral in Tg and wild-type mice. **, P<0.01 (Student's t test, ± s.e., n=18). (B) (left) A representative response of an epididymal spermatozoon isolated from a Tg mouse to lyral and K8.6. (middle) Proportion of spermatozoa from Tg and wild-type mice that respond to 2.5 mM lyral. **, P<0.01 (Student's t test, ± s.e., n=6). (right) The average amplitude of the response to 2.5 mM lyral in Tg and wild-type mouse sperm. LY, lyral; WT, wild type.

View larger version (68K): [in a new window]   Fig. 7. Effect of gradients of various stimulants on sperm accumulation. (A) Representative images of mouse spermatozoa from Tg mice showing sperm accumulation around the tip of a glass microcapillary containing 50 mM lyral or buffer. Scale bar, 100 µm. (B) Time course of spermatozoa accumulation in a 200-µm radius circular area around the tip of the microcapillary. Images are representative of six independent experiments for lyral and three for buffer. The graph shows the average numbers of sperm within the accumulation area as a function of time after the ejection of lyral (filled squares) or buffer (empty squares) (± s.e.; buffer, n=4; LY, n=6). A significant difference was observed between lyral and buffer after 4-7 (P<0.01) and 8-10 (P<0.05) minutes. (C) The numbers of Tg and wild-type spermatozoa attracted toward gradients of buffer, 50 mM lyral (LY), 50 mM dihydromyrcenol (DM), 50 mM bourgeonal (B), 10x concentrated K8.6 (K8) or 10-mM 8-Br-cAMP (cAMP). The graph shows the number of spermatozoa that accumulated in the 200-µm radius circle around the tip of each microcapillary. The numbers in parentheses represent the number of experiments performed for each reagent. *, P<0.05; **, P<0.01 (Student's t test, ± s.e.).

View larger version (51K): [in a new window]   Fig. 8. Tracking analysis of the movement of mouse spermatozoa towards a gradient of lyral and analysis of flagellar configuration. (A) A representative trace of wild-type spermatozoa towards a capillary containing 50 mM lyral or buffer. Traces were made from the sperm accumulation assay at 1 second intervals 3-6 minutes after the ejection of the stimulant. Traces exhibiting directional changes are colored. Insets show an enlargement of the red and green traces. Scale bar, 100 µm. (B) Effect of lyral on flagellar configuration. Representative images of the flagellar shape of wild-type mouse spermatozoa exposed to 2.5 mM lyral, 2.5 mM bourgeonal (Bour) or buffer. Arrested spermatozoa with fishhook-shaped flagella (arrowheads) were found in lyral, whereas this configuration was not observed in other odorants or in Ca2+-free buffer containing 2.5 mM lyral. Scale bar, 50 µm. (C) The numbers of spermatozoa with fishhook-like flagellar configuration at various concentrations of lyral (*, P<0.05, **, P<0.01, Student's t test, ± s.e.; 25 µM, n=3; 0.25-5.0 mM, n=4). The effect of lyral on flagellar configuration was dose dependent. (D) Effects of extracellular Ca2+ on a lyral-mediated flagellar configurational change. Wild-type spermatozoa were dispersed in Ca2+-free or Ca2+-containing buffer (disperse – or +, respectively). The dispersed sperm were diluted in Ca2+-free or Ca2+-containing buffer (assay – or +, respectively) and then stimulated with 2.5 mM lyral (± s.e., n=3).