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First published online March 30, 2006
Journal of Experimental Biology 209, 1560-1572 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02136

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Sperm selection and competition in pigs may be mediated by the differential motility activation and suppression of sperm subpopulations within the oviduct

Nana Satake^1,2, Roslyn M. A. Elliott¹, Paul F. Watson² and William V. Holt^1,*

¹ Institute of Zoology, Zoological Society of London, Regent's Park, London NW1 4RY, UK
² Royal Veterinary College, Royal College Street, London NW1 0TU, UK

View larger version (19K): [in a new window]   Fig. 1. (A–D) Bicarbonate induction of increase in linear velocity of boar spermatozoa. Samples of washed spermatozoa were pre-incubated in Tyrode's medium before the addition of 15 mmol l–1 bicarbonate. Subsamples were taken for video recording of motility shortly before (`zero time') and at intervals after bicarbonate addition. Motility parameter values were obtained by analysis of individual sperm tracks using the Hobson Sperm Tracker. These are presented as scatterplots (VAP vs LIN) (average path velocity vs linearity) of individual spermatozoa from a combination of two representative boars; each point represents a single sperm trajectory. In the absence of bicarbonate/CO2 (A,C) most spermatozoa exhibit low VAP and LIN, although a small number of solubilised apical plasma membrane protein fraction (sAPM)-treated spermatozoa show high velocity (>60 µm s–1) and straight tracks (LIN >60%); (highlighted in the box in the upper right corner). 7 min after the addition of bicarbonate/CO2, a sizeable proportion of spermatozoa show activation (B,D); the boxes in the upper right of these panels also highlight spermatozoa showing high velocity (>60 µm s–1) and straight tracks (LIN >60%). The density of spermatozoa within the upper right hand box is higher in the absence than in the presence of sAPM (compare B and D). (E) Representative trajectories of spermatozoa activated in the absence of sAPM. Track 3 represents the most activated trajectory (fast and linear), while tracks 1 and 2 represent subpopulations that would be classified as slow and/or non-linear. (F) Two representative fast-linear tracks activated in the presence of sAPM. These tracks show high linearity because there is relatively little deviation from the average path. Black dots represent x–y coordinates measured at 20-ms intervals; red lines are fitted spline curves.

View larger version (19K): [in a new window]   Fig. 2. (A) Straight line velocity (µm s–1 VSL) and (B) beat cross frequency (Hz; BCF) responses of boar spermatozoa to the addition of 15 mmol l–1 bicarbonate/CO2. Data are means (± s.e.m.) calculated from treatment means for eight individual boars. The points at 22 min represent the NaCl-control samples, which had not been exposed to bicarbonate/CO2. ***Bicarbonate/CO2 vs no bicarbonate/CO2 planned contrasts; VSL effect (F1,87=61.9), P<0.001 and BCF effect (F1,87=49.9), P<0.001. Changes in mean frequencies (± s.e.m. from eight individual boars) of group 1 (fast and linear) spermatozoa plotted against the time elapsed after bicarbonate/CO2 addition. Points at 22 min represent the NaCl-control samples. (C,D) The difference between the absence and presence of sAPM, respectively.

View larger version (14K): [in a new window]   Fig. 3. Scatterplots showing relationships between the proportions of fast-linear spermatozoa present before (time 0 min) and after (time 7 min) bicarbonate/CO2 activation in the absence (A) and presence (B) of solubilised apical plasma membrane protein fraction (sAPM). Each data point represents a separate boar ejaculate; N=7 in this graph because matched data was not available for one boar. (C) Ranking of boars in descending order of the proportion of fast-linear spermatozoa after 7 min in the absence of sAPM; control); the equivalent ranking is changed in the presence of sAPM.

View larger version (19K): [in a new window]   Fig. 4. (A) Solubilised apical plasma membrane protein fraction (sAPM) dose response of the fast-linear sperm subpopulation in the presence (filled circles) and absence (open squares) of bicarbonate/CO2. The data summarise group sperm frequencies (%), calculated for each individual boar (N=6) and treatment (mean % ± s.e.m.). Data representing bicarbonate/CO2-treated spermatozoa are from treatments 7 min after bicarbonate/CO2 addition. Data from the `bicarbonate-absent' groups are from the NaCl control samples, measured at 0 and 17 min. (B) Within the fast-linear sperm subpopulation, the mean (± s.e.m.) LIN (linearity) values increased in relation to the log(base 2) sAPM concentration.

View larger version (18K): [in a new window]   Fig. 5. (A) Bar chart showing the mean (±s.e.m.) frequency (%) of the fast-linear sperm population present in control, solubilised apical plasma membrane protein fraction (sAPM) and 100 µmol l–1 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) treatments. Both treatments caused reduction of bicarbonate-induced activation (P<0.05). (B,C) In comparison to the control treatment, both sAPM and 100 µM DIDS significantly reduced beat cross frequency (Hz; BCF) in the fast-linear sperm group if bicarbonate/CO2 was absent. In the presence of bicarbonate/CO2, there was no inhibitory effect of sAPM on BCF, but DIDS still induced significant reduction of flagellar beat frequency (F1,24536=18.9; P<0.001). Data are mean ± s.e.m; N=5 replicates). (D) Three representative trajectories of spermatozoa exposed to DIDS show high linearity because there is relatively little deviation from the average path. Black dots represent x-y coordinates measured at 20 ms intervals; grey lines are fitted spline curves.

View larger version (15K): [in a new window]   Fig. 6. Effects of 100 µmol l–1 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (A), control treatment with neither DIDS nor solubilised apical plasma membrane protein fraction (sAPM) (B) and 50 µg ml–1 sAPM (C) on the intracellular pH of boar spermatozoa incubated for 1 h in the presence (filled circles) and absence (open squares) of bicarbonate/CO2. Whereas bicarbonate/CO2 induced increased pHi in all treatments, DIDS significantly inhibited (F1,252=93.3; P<0.001) and sAPM significantly enhanced (F1,356=52.45; P<0.001) the pH increase with respect to the control. (Data are means ± s.e.m.; N=6

View larger version (139K): [in a new window]   Fig. 7. Sperm binding to solubilised apical plasma membrane protein fraction (sAPM)-coated beads. (A) Low magnification view of the sAPM-coated beads showing that although some spermatozoa are bound to the bead surfaces many unbound spermatozoa are also present. Bar, 100 µm. (B,C) Spermatozoa interact with the bead surface via the anterior acrosomal region (arrows) while the flagellae are unbound. Bars, 5 µm.