Beneficial effects of relaxin on motility characteristics of stored boar spermatozoa

Abstract

Background: Relaxin is detected in seminal plasma of many species and its association with sperm motility may be beneficial in some aspects of assisted reproduction. Here, we immunolocalized relaxin receptors and investigated the effects of exogenous relaxin on motility characteristics, viability, and cAMP content of boar spermatozoa after storage.

Methods: Commercial doses of boar semen were obtained on the collection day (Day 0) and kept in shipping containers at room temperature for up to 4 days (Day 4). On Day 0, spermatozoa were fixed for immunofluorescence detection of relaxin receptors RXFP1 and RXFP2 (Experiment 1). Semen aliquots were taken from the same dose at Day 0, Day 1, and Day 2 (Experiment 2a), and Day 2 and Day 4 (Experiment 2b) for analyses. Alive spermatozoa were purified and incubated (1 h-37°C) with 0, 50, or 100 ng relaxin/ml (Experiment 2a) and 0, 100, or 500 ng relaxin/ml (Experiment 2b). Afterward, aliquots of each treatment group were subjected to motility (Experiments 2), viability (Experiment 3) analyses, and cAMP quantification (Experiment 4). Data (3-4 independent replicates) were statistically analyzed (ANOVA followed by pairwise comparisons) and p values less or equal to 0.05 was set for significant difference.

Results: Both RXFP1 and RXFP2 receptors were immunolocalized on the entire spermatozoon. Relaxin concentration of 100 ng/ml significantly improved the proportions of motile, progressive, and rapid spermatozoa up to Day 2. Only 500 ng relaxin/ml provided beneficial effects on Day 4. The viability of spermatozoa was not affected by relaxin (100 ng/ml) during storage, but the extent of mitochondria membrane damages was significantly decreased. Furthermore, relaxin did not affect the cAMP contents of spermatozoa during storage, in our conditions.

Conclusions: Relaxin could be a valuable motility booster of stored- or aged-spermatozoa for assisted reproduction techniques. However, the related-intracellular signaling cascades of relaxin in boar spermatozoa remain undetermined.

Figure 1

Representative micrographs of immunofluorescence detection of RXFP1 and RXFP2 relaxin receptors in boar spermatozoa. The immunofluorescence detection of RXFP1 and RXFP2 (green FITC-staining) receptors of boar spermatozoa are shown in micrographs C and E, respectively. The micrograph A corresponds to the negative control, without a distinctive green FITC staining on spermatozoa. The bottom panel shows overlay images of visible light, blue staining of sperm nuclei (DAPI), and green FITC-staining of the Control (B), RXFP1 (D) and RXFP 2 (F) groups. Scale bars correspond to 10 μm.

Figure 2

Effect of pRLX on sperm straightness (VSL/VAP × 100). Data are mean percentages (+/− SD) of 4 independent replicates, with 48 individual observations or micro-chamber fields during the CASA analysis. Columns with different letters (a, b) differ significantly within the same day (p ≤ 0.05). The presence of pRLX slowed the pace of the straightness decreased of spermatozoa during storage (p < 0.05).

Figure 3

Effect of pRLX on sperm linearity (VSL/VCL × 100). Data are mean percentages (+/− SD) of 4 independent experiments, with 48 individual observations or micro-chamber fields during the CASA analysis. Columns with different letters (a, b) differ significantly within the same day (p ≤ 0.05). The presence of pRLX significantly decelerated the pace of sperm linearity decline during storage (p ≤ 0.05).

Figure 4

Fluorescence microscopy evaluation of sperm viability after storage. Representative images show spermatozoa stained for mitochondria membrane potential (JC-1; Upper panel) or plasma membrane integrity (PI; Lower panel) evaluation. Upon excitation at 488 nm, spermatozoa with intact (high potential) or damaged (low potential) mitochondrial membranes fluoresce red-orange or green, respectively, while those with damaged plasma membrane fluoresce red. Magnification 200X.

Figure 5

Effect of pRLX on sperm cAMP content after storage. Data are means (+/− SD) of three independent replicates, corresponding to nine individual analyses. The cAMP levels of spermatozoa in the control groups (0 ng pRLX/ml) significantly increased during storage (a,b; p < 0.05) and no significant effect of pRLX was found within the same storage day (*; p > 0.05). The insert indicates groups of spermatozoa ran independently to evaluate the effect of forskolin (250 μg/ml), used as positive control for cAMP assessment on Day 0 and Day 2. Data are mean+/− SEM and columns with different letters (a, b) indicating significant differences within the same Day.