Background
Sertoli cells build tight junctions among themselves in the testes to form a blood-testis barrier that maintains an enclosed microenvironment favoring normal sperm production. Upon their release within the seminiferous tubule lumen, newly produced spermatozoa are exposed to various molecules that contribute to the acquisition of their final maturation during the epididymal transit [
1,
2]. The epididymis is a long duct consisting of various sections with specific functions, known to secrete and reabsorb a variety of molecules that locally interact with spermatozoa to influence their fertilizing potential [
3‐
6]. Relaxin might be one of these molecules, whose presence in the seminal plasma contributes to the crucial roles of this fluid in mammalian fertilization through beneficial effects on sperm motility [
7‐
10].
Relaxin peptide was discovered in the early part of the twentieth century and is characterized as hormone of pregnancy because of its roles during parturition [
11]. The first report on relaxin used pregnant guinea pig serum relaxin to demonstrate its beneficial effect on widening the birth canal of non-pregnant animals [
12]. Further works later confirmed its positive effects during pregnancy and parturition in various species such as monkeys, pigs, and rodents [
13‐
15]. At present, numerous studies have detected the presence of relaxin in both female (i.e., uterus, ovary) and male (i.e., testes, seminal vesicles, prostate) reproductive tissues, and its functions have been reported in various reproductive and non-reproductive (i.e., brain, pancreas, and kidney) tissues [
11].
The tissular distribution of relaxin varies across species and its pleiotropic biological effects are exerted through membrane receptors, known as relaxin family peptide receptor 1 (RXFP1) and 2 (RXFP2) and corresponding to the former leucine-rich repeat-containing G-protein-coupled receptor 7 (LGR7) and 8 (LGR8), respectively [
16]. The cellular and physiological effects of relaxin-receptor interactions are better characterized in females, mainly during early and late pregnancy [
13,
17,
14,
18], and the co-localization of both relaxin and its receptors in various tissues (i.e., oocytes, cervix, uterus, mammary gland) indicate the existence of possible autocrine and paracrine actions of relaxin [
19,
20,
11,
21]. Yet, the presence of both relaxin receptors mRNA and proteins in male reproductive organs is still controversial among studies and species, and consequently, little remains known about relaxin’s presence and roles in male reproductive organs; especially its contribution to the fertilizing potential of spermatozoa [
21].
Relaxin is found in male reproductive tissues and accessory glands of various species, and its main production sites appear species-specific [
21,
11], with testes, seminal vesicles, and prostate being reported as the major sources of production in various species [
21,
22]. Although there are still controversies about the major site of relaxin production in boars [
23,
24,
22,
25,
26], a recent study tends to support the testes as the major site of relaxin production, as both RNA transcript and protein were detected in Leydig cells during porcine post-natal development [
24]. Nonetheless, the potential roles of testicular relaxin in male reproduction are still controversial, despite various knock-out studies conducted in rodents [
21,
27].
Available data in the literature body imply that spermatozoa may be exposed to relaxin within the reproductive tract. The confirmation of this assumption in pigs will contribute to understanding the physiological effects of testicular relaxin on male fertility, as its levels in semen ejaculates appear correlated to sperm motility [
8]. Additionally,
in vitro studies revealed positive effects of relaxin usually extracted from female tissues (e.g., ovary) on post-mating events such as, cervical mucus penetration [
28], acrosome reaction, mitochondrial potential, hyperactivation of spermatozoa [
29,
30], and oocyte maturation [
31]. Hence, it becomes reasonable to question whether it is the male- and/or the female-produced relaxin that provide such effects in the physiological conditions.
From the available literature, it appears that the dynamic expression of relaxin and its receptors throughout the boar reproductive tract, which will provide additional insights into the reproductive impacts of relaxin on male fertility, have not been the focus of previous studies. The current study was undertaken to investigate the main source (s) of relaxin accumulation and the presence of its receptors RXFP1 and RXFP2 in boar reproductive tract (testis, epididymis, and vas deferens) and sex accessory glands (prostate and seminal vesicles). In addition, we profiled the presence of both relaxin and receptor proteins in porcine epididymal and ejaculated spermatozoa. The major findings are that: 1) relaxin and its receptors are present in both reproductive tract and accessory glands, 2) relaxin mainly accumulates within the Leydig cells of the testis, and lower levels were detected in the prostate and seminal vesicles, 3) relaxin is found within the lumen and epithelia of seminiferous tubules and epididymis, in the vicinity of produced and maturing spermatozoa, 4) spermatozoa possess both relaxin and receptor proteins with the amount varying significantly during the epididymal transit, and 5) ejaculated spermatozoa contain both relaxin and receptors RXFP1 and RXFP2 that likely support the long-term roles of male-born relaxin within the female genital tract, through possible autocrine and/or paracrine action.
Methods
Unless otherwise indicated, all chemicals and reagents were purchased from Sigma-Aldrich (Saint Louis, MI) for general purpose and from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA) for antibodies.
Sample preparation
Animals and fresh semen collection
Three fertile cross-breed boars of approximately 2.7 ± 0.06 (mean ± sem) years old were used in this study. Fresh semen from each boar was harvested using standard protocol by technicians of a commercial boar stud (Prestage Farms, West Point, MS) and diluted in the Beltsville Thawing Solution (BTS; Minitube of America, Verona, WI). Semen doses were prepared and shipped to our laboratory for experiments. Approximately four hours post-semen collection, all three boars were killed at a local abattoir and reproductive tracts and sex accessory glands (prostate and seminal vesicles) were collected and immediately transported on ice to our laboratory for tissue collections. Ovarian corpus lutea were collected from post-mortem sows at the abattoir for validation studies.
Tissue and spermatozoa collections
Reproductive tracts of all boars (n = 3) were dissected into testes, epididymis (caput or head, corpus or body, and cauda or tail), vas deferens, and accessory glands (prostate and seminal vesicles). All tissue samples were recovered and kept on ice. Spermatozoa within each section of the epididymis and vas deferens were mechanically collected (by aspiration with syringes, flushing with a pre-warmed PBS-PVP, and squeezing) and transferred into petri-dishes containing pre-warmed PBS-PVP. Ejaculated and non-ejaculated spermatozoa collected above were subjected to a purification procedure using a single layer percoll gradient (PorciPure, Nidacon: Mölndal, Sweden), as previously described [
32]. After centrifugation (600 g - 30 minutes), pelleted spermatozoa devoid of any contaminations (cell debris, extender components, and somatic cells), were washed twice with a cold PBS-PVP through centrifugation (250 g – 5 minutes each). Purified spermatozoa were aliquoted and stored at −20 °C until use for protein analyses. In parallel, subsets of dissected tissues (testis, epididymal caput, corpus and cauda, vas deferens, prostate, and seminal vesicles) were stored at room temperature in containers filled with 10 % formalin for immunofluorescence, while the other subsets were wrapped in aluminum foils, snap-frozen, and stored at −20 °C for protein analyses.
Western immunoblotting
All samples were thawed at room temperature and total proteins were extracted using complete RIPA buffer containing a protease inhibitor cocktail (Santa Cruz Biotechnologies; Santa Cruz, CA). Total extracted protein were quantified using the Pierce BCA kit (Thermo Fisher Scientific; Rockford, IL) and equivalents of 20 μg were resolved onto 4–12.5 % SDS-PAGE NuPage gels and transferred to PVDF membranes (Millipore Corp, Belford, USA). Similar amounts of ovarian corpus luteum protein extracts were also loaded into the gels for analyses. All membranes were incubated with 500× diluted anti-human relaxin (sc-20652), RXFP1 (sc-50328), or RXFP2 (sc-50327) antibodies, which immunogenicities with pig tissues were previously tested [
19]. The immunodetection of proteins was revealed by using the Novex® HRP Chromogenic Western Blot Immunodetection kit (Life Technologies; Grand Island, NY). In parallel, gel electrophoreses loaded with more protein samples (40–60 μg/well) were ran together with the MagicMark™ XP Western Protein Standard (Life Technologies; 10 μl/well) for a longer resolution to better determine the protein sizes after immunoblotting.
Protein immunofluorescence detection
In spermatozoa
Purified sperm samples were fixed in 4 % methanol-free paraformaldehyde (30–60 minutes), permeabilized (30 min) in 1 % Triton-×100, and non-specific binding sites were blocked (60 min) in PBS-PVP solution containing 5 % BSA (v/v). Sperm suspensions were incubated overnight (4 °C) with 100× diluted rabbit anti-human relaxin 1 (sc-20652), RXFP1 (sc-50328), and RXFP2 (sc-50327) in the blocking buffer. Then after, spermatozoa were incubated one hour with 200× diluted FITC labeled goat anti-rabbit secondary antibody in the blocking buffer. Subsets of labeled-spermatozoa were kept in suspensions for a flow cytometry evaluation, while the other subsets were smeared on microscope slides and air-dried under dark. Slides were immediately covered with a DAPI-contained mounting medium to counterstain sperm nuclei for a fluorescence evaluation using a confocal microscope (LSM-510). Samples were washed three times by centrifugation (250 g× 5 min) with PBS-PVP-0.1 % Tween 20 between steps and all procedures were performed at room temperatures otherwise indicated. Samples without any or either primary or secondary antibodies were used as negative controls.
In tissues
Tissues were fixed in 10 % formalin at room temperature (30–60 minutes) and blocks were sliced in sections (4–6 μm) and placed on histological slides. Tissue sections were deparafinized, submitted to antigen retrieval (microwave), washed with PBS-PVP and submitted to immediate standard in situ immunofluorescence detection of relaxin, RXFP1, and RXFP2 proteins. Additionally, pig ovary sections were processed with the anti-synthetic human RXFP1 antibody (AP23448SU-S; ACRIS Antibodies Inc., San Diego, CA) used at 1/4000 dilution for immunofluorescence signal comparisons with the Santa Cruz anti-human RXFP1.
Reagents used and all procedures were performed as described above for spermatozoa, and samples on the microscope slides were covered with a DAPI-contained mounting medium to counterstain nuclei. The immunofluorescence detection was assessed under a confocal microscope (LSM 510). Samples incubated without primary or secondary antibodies were used as negative controls.
Flow cytometry evaluation
Suspensions of spermatozoa labeled with relaxin, RXFP1, and RXFP2 antibodies were diluted to approximately 106 cells in 0.8 ml PVP-PVP (1 mg/ml). Samples were analyzed on a Becton Dickinson FACS Aria II flow cytometer (BD Biosciences; San Jose, CA) by excitation with a blue laser (488 nm). The emission signal was measured in FL2 channel centered at 585/42 nm and sorting was performed with a total of 10,000 events analyzed per sample. Data were acquired with the BD FACSDiva™ Version 6.1.3 software and images were treated with the FlowJo software (FlowJo, LLC; Ashland, OR). Three independent experiments were performed and data were expressed as means ± sem. The mean fluorescence of stained cells with both primary and secondary antibodies minus the mean fluorescence of stained cells with only the secondary antibody was used to calculate the percent of labeled cells.
Confocal microscope imaging
Immediately after immunolabeling, slides containing specimens (spermatozoa and tissue sections) were submitted to fluorescence signal visualization using a Zeiss Laser Scanning Microscope System (LSM 510; Carl Zeiss MicroImaging GmbH, Jena, Germany) with a 488 nm excitation. A (DAPI/Fluorescein/Transmission) filter set was used in single channel mode imaging. Excitation wavelengths of 405 nm/488 nm, Band Pass Emission wavelengths of 420–480 nm (Blue), and Long Pass wavelengths of 505 nm (Green) were acquired at 1024× 1024 pixel formats for imaging purposes.
Statistical analyses
Only flow cytometry data were statistically analyzed by ANOVA using the General Linear Model procedures of SPSS, version 22 (IBM statistic package package, version 22, Armonk, NY). The homogeneity of variances was performed using the Levene’s test and boars were considered as the random event to evaluate the effect of epididymal section on the immunofluorescence intensity of spermatozoa. The Fisher’s Least Square Difference (LSD) test was performed for pairwise comparisons between protein targets (relaxin, RFXP1, or RXFP2), within and between groups (caput, corpus, cauda, and ejaculated sperm). The threshold of statistical significance was fixed at p ≤ 0.05. Data are mean ± sem.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JMF designed the study, performed all experiments, and drafted the manuscript. JMG participated for semen collection and analyses and contributed to the manuscript. HLS-R assisted for tissue collection and preparation for immunofluorescence. JVS performed the flow cytometry analyses. MAC, STW, and PLR participated to the research design and data interpretation. All authors read and approved the final manuscript.