Introduction
Progesterone is released from cumulus cells surrounding the oocyte and has been demonstrated to act as a chemoattractant for human and rabbit sperm [
Studies examining the effects of progesterone on boar sperm cells have largely focussed on its effects on the acrosome reaction [
Material and Methods
Semen supply
Semen was supplied commercially by The Pig Improvement Company (PIC) from their stock of boars (PIC337 Champion species). Semen was diluted 1:1 upon collection into TriXCell (IMV International, Minneapolis, MN, USA) semen extender according to the manufacturer instructions. Semen was delivered overnight in flatpacks at ambient temperature. Upon receipt, the diluted semen was then aliquoted into 15ml polypropylene centrifuge tubes and stored at 16°C in accordance with the manufacturer instructions until use. Assays were completed within 2 days of delivery. No information is available regarding their fertility.
Vitality test
Vitality testing using propridium iodide (PI) staining based on the method by Pintado and colleagues [
Acrosome staining
The acrosome status of sperm was investigated using the method of He et al [
Mobility assay solutions
The mobility assay was based on the validated method of Vizcarra & Ford [
Mobility assay
Cuvettes containing the working Accudenz solution were pre-warmed to 37°C. Data presented in each figure were derived using a split-ejaculate method such that cells under control and treatment conditions were taken from the same insemination dose. 1×108 live sperm cells (in maximally 150µl) were carefully pipetted onto the surface of the Accudenz solution and the change in absorbance (550nm) over time was monitored using a spectrophotometer. This treatment is hereafter called the mobility control. A negative control was prepared by first heating 1×108 sperm cells at 60°C for 60 minutes. A positive control was prepared by mixing 1×108 cells with 1.5ml working Accudenz solution prior to loading into a cuvette. Progesterone was added to the working Accudenz solution to a final concentration of 1, 10 or 100nM and used to monitor sperm penetration rate in response to progesterone concentration. Absorbance readings were recorded at time intervals over 50 minutes (1, 5, 10, 15, 20, 30, 40 and 50 minutes) and cuvettes were incubated at 37°C for the duration of this time period.
To construct data sets within a figure, multiple runs were taken from an individual boar sample and multiple boars were used as indicated in the figure legends. A total of 17 different boars were used by the end of the study.
Capacitating conditions
Capacitating media (CM) was composed of 5mM KCl, 1mM KH2PO4, 95mM NaCl, 5.55mM glucose, 25mM NaHCO3, 2mM CaCl2, 0.4% BSA and 2.5mM pyruvate (pH 7.4). Non-capacitating media (NCM) lacked calcium, bicarbonate and BSA and consisted of 2.7mM KCl, 1.5mM KH2PO4, 8.1mM Na2HPO4, 137mM NaCl, 5.55mM glucose and 2.5mM pyruvate (pH 7.4). 1×108 cells were pelleted and resuspended in 1ml of either CM or NCM and incubated at 37°C for 3hours.
Following capacitation the samples were pelleted, resuspended in mobility buffer and the mobility assay ran as described for non-capacitated cells.
SDS-PAGE and Western Blotting
1×108 cells were incubated in NCM or CM for 3 hours, pelleted at 17000 × g then heated at 95°C in Laemmli sample buffer and frozen for subsequent use in SDS-PAGE. Sperm proteins were separated on 10% polyacrylamide gels and transferred to PVDF membranes by electrophoresis. The membranes were incubated overnight at 4°C with anti phospho-tyrosine primary antibodies (Cell Signaling Technology, UK) at 1:20 000 dilution in Tris-buffered saline (25mM Tris-HCl pH7.4, 150mM NaCl) containing 0.1%(v/v) Tween 20 and 5%(w/v) bovine serum albumin. The detection method involved the use of a WesternDot™625 western blot kit in accordance with the manufacturer instructions (Life Technologies, UK) and imaging with a Biorad Gel Doc XR system.
Data analysis
Data represented mean ± SEM for four ejaculates measured in quadruplet (Graphpad Prism). Curves are fitted using non-linear regression model and Km values (minutes) represent the time at which absorbance was half maximal (Graphpad Prism). P < 0.05 is taken as statistically significant.
To test for differences in the rate of mobility between control and progesterone treated sperm cells data were first transformed to linearize then an ANCOVA was carried out on regression plots of the linearized data using R (open source statistical software http://www.r-project.org/. R version 2.15.1 (2012-06-22) “Roasted Marshmallows”. Copyright 2012 The R Foundation for Statistical Computing). Data were linearized using ln(y) = m*ln(x)+ln(c), where y = mean absorbance and x = 1/time (min). The effect of mibefradil on sperm mobility was analysed using ANOVA followed by Tukeys post-hoc test for three time points (10, 20 and 50 minutes).
Chemicals and reagents
Unless stated all chemicals and reagents were obtained from Sigma-Aldrich, UK.
Mibefradil was obtained from Tocris Bioscience, Bristol, UK.
Results
Non-capacitated boar sperm penetrated the working Accudenz solution with a Km = 18.0±2.2 min. Accudenz solutions containing increasing concentrations of progesterone (1, 10, 100nM) were prepared in order to monitor a change in mobility induced by this putative chemoattractant. Only 100nM progesterone treatment produced a significant increase in sperm mobility as measured by an increase in absorption over time in the sperm mobility assay (Figure 1). Analysis of the progesterone effect using ANCOVA required linearised plots so data from Figure 1 was first transformed (ln(y) = m*ln(x)+ln(c) where y = mean absorbance and x = 1/time (min)) and the gradient and intercept of the regression lines were then compared. The independent variable in the regression analysis was ln(1/time), the dependent variable was ln(mean absorbance) and the factor was +/-progesterone treatment. Initial ANCOVA analysis revealed that there was no significant interaction between progesterone treatment and time (p = 0.76) and therefore progesterone treatment could be analysed for its effect alone on mobility. For non-capacitated sperm, the intercepts of the regression lines were significantly higher in the presence of 100nM progesterone. The intercept of the regression line for hormone treatment was -3.32 and for controls, the intercept was -3.49, (F= 8.997, p= 0.01). This result indicates progesterone significantly increases the rate of penetration of non-capacitated cells. Indeed 100nM progesterone caused over a two-fold increase in the rate of penetration rate (Km = 8.0±0.8min). There were no significant differences between the gradients for 100nM progesterone treated and control sperm for either capacitated (see below) or non-capacitated sperm indicating that there was no interaction between hormone treatment and time and that the relationship between absorbance and time could be modelled by a simple additive regression model without variable interaction.
CatSper is sensitive to the Cav blocker mibefradil [
Capacitation is associated with alterations of the membrane of sperm [
Progesterone (100nM) induced the acrosome reaction in significantly greater number of cells in capacitated cells only, although approximately half of the population remained unreacted (Figure 7). Finally, the capacitation status of the cells was confirmed by assessing the total protein tyrosine phosphorylation status (Figure 8).
Discussion
Progesterone has been demonstrated to induce a number of calcium dependent mechanisms in sperm including hyperactivation [
Previous studies examining the effect of progesterone on human sperm cell motility have typically involved the use of CASA systems and report significant effects on hyperactivation [
As prematurely acrosome reacted sperm cannot undertake fertilisation [
The pattern of response to progesterone of boar sperm cells is interesting due to the molecular mechanism by which progesterone has been proposed to act in stimulating sperm motility. Progesterone potentiates CatSper-mediated currents in human but not mouse sperm, with the constitutive activity of CatSper being much higher in the latter species. CatSper is conserved between species [
In summary, for the first time we have demonstrated that progesterone can sustainably increase the mobility of boar sperm cells in an extracellular calcium dependent manner and that the increased distance travelled with time presents a selective advantage for cells that remain acrosome intact.
Acknowledgements
The authors would like to thank The Carnegie Trust for the Universities of Scotland for the studentship award for JMC which permitted her to complete her work.
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