Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA)

Abstract

Background: Opioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology.

Methods: We analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltrindole and DPDPE by means of a computer assisted sperm analyzer and a fluorescent confocal microscope. The evaluation of viability, capacitation and acrosome reaction was carried out by the double CTC/Hoechst staining, whereas mitochondrial activity was assessed by means of MitoTracker Orange dye.

Results: We showed that in equine sperm cells, delta opioid receptor is expressed as a doublet of 65 and 50 kDa molecular mass and is localized in the mid piece of tail; we also demonstrated that naltrindole, a delta opioid receptor antagonist, could be utilized in modulating several physiological parameters of the equine spermatozoon in a dose-dependent way. We also found that low concentrations of the antagonist increase sperm motility whereas high concentrations show the opposite effect. Moreover low concentrations hamper capacitation, acrosome reaction and viability even if the percentage of cells with active mitochondria seems to be increased; the opposite effect is exerted at high concentrations. We have also observed that the delta opioid receptor agonist DPDPE is scarcely involved in affecting the same parameters at the employed concentrations.

Conclusions: The results described in this paper add new important details in the comprehension of the mammalian sperm physiology and suggest new insights for improving reproduction and for optimizing equine breeding.

Figure 1

Western blot for DOR in equine spermatozoa (A) proteins from ejaculated sperm cells were subjected to gradient 4-12% SDS-polyacrylamide gel and immunoblotted with rabbit anti DOR antiserum. Two bands of 50 and 65 kDa were observed (arrows). (B) negative control: blot containing proteins from ejaculated sperm cells was incubated with a solution of the primary antibody adsorbed with a molar excess of the immunizing peptide. (C and D) same filters re-blotted with rabbit anti β-actin as loading control. Results shown are representative of three separate experiments.

Figure 2

Localization of DOR and evaluation of mitochondrial activity in equine sperm cells by fluorescence and confocal laser scanning microscopy. (A) Reactivity staining (green fluorescence) detected by the rabbit anti-DOR antibody is observed exclusively on the mid piece of the sperm tail; sperm cells were counterstained by Evans blue to visualize sperm morphology. Picture was captured by Nikon coolpix 990 digital camera. (B) The highly selective fluorescent tripeptide analogue of the DOR antagonist DMT-TIC detected DOR on the same sperm region; image captured by confocal laser scanning microscope. (C), (D) negative controls for the antibody and the fluorescent tripeptide: in (C), the primary antibody was omitted, only the secondary antibody-FITC conjugated was employed; in (D) spermatozoa were incubated in the presence of 0.2 μmol l^-1 naltrindole to block binding sites for the tripeptide. (E) The mid piece of an equine sperm cell containing active mitochondria was intensively labelled by MitoTracker orange (arrow), the cell with inactive mitochondria was only faint coloured in the same region (double arrow). Scale bars, 9.0 μm.

Figure 3

Dose dependent effects of naltrindole and DPDPE on total and progressive equine sperm cell motility. Panel A: total equine sperm cell motility affected by different concentrations of DPDPE and naltrindole at T 0, T 30 min, T 90 min and T 180 min. Panel B: progressive equine sperm cell motility affected by different concentrations of DPDPE and naltrindole at T 0, T 30 min, T 90 min and T 180 min. Data are reported as mean ± SD, n = 6. The statistical analysis conducted by Chi square test, after comparison with the control condition at the same time point, was considered significant for p < 0.05. b = p < 0.05 and c = p < 0.001. Nal-4 = 10^-4 M naltrindole; Nal-5 = 10^-5 M naltrindole; Nal-6 = 10^-6 M naltrindole; Nal-7 = 10^-7 M naltrindole; DPDPE-5 = 10^-5 M DPDPE; DPDPE-6 = 10^-6 M DPDPE; DPDPE-7 = 10^-7 M DPDPE.

Figure 4

Dose dependent effects of naltrindole and DPDPE on capacitation, AR, vitality and mitochondrial activity: Different concentrations of DPDPE and naltrindole affect the number of capacitated (Panel a), AR (Panel B), dead (Panel C) equine sperm cells and those with active mitochondria (Panel D) after 4.5 h of incubation. Data are reported as mean ± SD, n = 6. The statistical analysis conducted by Chi square test, after comparison with the control condition at the same time point, was considered significant for p < 0.01. a = p < 0.01; b = p < 0.05 and c = p < 0.001. Nal-4 = 10^-4 M naltrindole; Nal-5 = 10^-5 M naltrindole; Nal-6 = 10^-6 M naltrindole; Nal-7 = 10^-7 M naltrindole; DPDPE-5 = 10^-5 M DPDPE; DPDPE-6 = 10^-6 M DPDPE; DPDPE-7 = 10^-7 M DPDPE.