Profiling of relaxin and its receptor proteins in boar reproductive tissues and spermatozoa

Abstract

Background: Relaxin levels in seminal plasma have been associated with positive effects on sperm motility and quality, and thus having potential roles in male fertility. However, the origin of seminal relaxin, within the male reproductive tract, and the moment of its release in the vicinity of spermatozoa remain unclear. Here, we assessed the longitudinal distribution of relaxin and its receptors RXFP1 and RXFP2 in the reproductive tract, sex accessory glands, and spermatozoa of adult boars.

Methods: Spermatozoa were harvested from three fertile boars and reproductive tract (testes and epididymis) and sex accessory gland (prostate and seminal vesicles) tissues were collected post-mortem from each boar. Epididymis ducts were sectioned into caput, corpus, and cauda regions, and spermatozoa were mechanically collected. All samples were subjected to immunofluorescence and/or western immunoblotting for relaxin, RXFP1, and RXFP2 detection. Immunolabeled-spermatozoa were submitted to flow cytometry analyses and data were statistically analyzed with ANOVA.

Results: Both receptors were detected in all tissues, with a predominance of mature and immature isoforms of RXFP1 and RXFP2, respectively. Relaxin signals were found in the testes, with Leydig cells displaying the highest intensity compared to other testicular cells. The testicular immunofluorescence intensity of relaxin was greater than that of other tissues. Epithelial basal cells exhibited the highest relaxin immunofluorescence intensity within the epididymis and the vas deferens. The luminal immunoreactivity to relaxin was detected in the seminiferous tubule, epididymis, and vas deferens ducts. Epididymal and ejaculated spermatozoa were immunopositive to relaxin, RXFP1, and RXFP2, and epididymal corpus-derived spermatozoa had the highest immunoreactivities across epididymal sections. Both vas deferens-collected and ejaculated spermatozoa displayed comparable, but lowest immunofluorescence signals among groups. The entire sperm length was immunopositive to both relaxin and receptors, with relaxin signal being robust in the acrosome area and RXFP2, homogeneously distributed than RXFP1 on the head of ejaculated spermatozoa.

Conclusions: Immunolocalization indicates that relaxin-receptor complexes may have important roles in boar reproduction and that spermatozoa are already exposed to relaxin upon their production. The findings suggest autocrine and/or paracrine actions of relaxin on spermatozoa, either before or after ejaculation, which have possible roles on the fertilizing potential of spermatozoa.

Fig. 1

Validation of specificity and immunogenicity of anti-human relaxin antibody on porcine ovarian tissues. Relaxin is detected (Green-FITC) in sections of superficial (a/d) and deep (b/e) areas of the corpus luteum, as well as the medulla sections of the sow ovary. Micrographs a, b and c correspond to the labeling with commercial antibody (Santa Cruz Inc., Biotechnology) and micrographs d, c, and f with the homemade porcine antibody (gift from Dr. Bagnell). GL = Granulosa lutein cells, TL = Theca lutein cells, Magnification = 200×. Nuclei are counterstained in blue with DAPI

Fig. 2

Immuno-fluorescence comparison of two commercial anti-human RXFP1 antibodies on porcine ovarian tissues. Micrograph show control (no primary antibody; black frame), ACRIS (blue frame), and Santa Cruz (red frame) antibodies. Relaxin receptor RXFP1 is generally stained in Green-FITC and mostly seen in the superficial or plasma membrane regions of (mural granulosa) cells lining the internal follicle wall. Upper panel micrographs indicate cells counterstained with DAPI for the nuclei visualization, while the lower panel indicate overlays imaging with visible and fluorescence lights. FC = follicular cavity or antrum

Fig. 3

Immunofluorescence of control samples for relaxin, RXFP1, and RXFP2 detection. Images correspond to negative controls, without the primary antibody in sperm (a), cumulus-oocyte complex (b) and ovary section (e) preparations. Absence or weaker green FITC fluorescence signals were observed following sample incubations with FITC-conjugated antibody only. An illustration is shown in micrograph c. Cumulus-oocyte complexes incubated with anti-relaxin are shown in micrograph d. Micrographs f, g, and h are ovary sections incubated with anti-relaxin, anti-RXFP1, and anti-RXFP2 antibodies, respectively. Cells nuclei are counterstained in blue with DAPI, while protein of interests appeared green (FITC). Micrographs a, b, c, and d show stained nuclei, bright-light, green FITC-fluorescence, and the combination of all. Arrow head and arrow respectively indicate the cumulus cells and the oocyte. GC = Granulosa cells; GL = Granulosa Lutein cells; TC = Theca cells; FA = follicle antrum

Fig. 4

Representative RXFP1 and RXFP2 western immunoblotting gels. Equal amounts (20–60 μg) of total proteins obtained from various pig tissues were resolved on 4–12.5 % SDS-PAGE gels, transferred to PVDF membranes, and immunoblotted with commercial anti-RXFP1 (sc-50328) and anti-RXFP2 (sc-50327) antibodies. Proteins were extracted from prostate (PR), seminal vesicles (SV), vas deferens (VD), and epididymis sections of cauda (CA), corpus (CO), caput (CP), and testicular homogenate (TE). Total protein extracts of sow ovarian corpus lutea (CL) were used as the positive control. Immunodetected proteins were revealed using a colorimetric technique (Novex® HRP Chromogenic). MW1 and MW2 correspond to two different molecular markers, MagicMark™ XP Western Protein Standard (1 = 220 kDa; 2 = 120 kDa; 3 = 100 kDa; 4 = 80 kDa; 5 = 60 kDa; 6 = 50 kDa; 7 = 40 kDa; 8 = 30 kDa; 9 = 20 kDa

Fig. 5

Immunodetection of relaxin in boar reproductive tract and accessory glands. Relaxin signal (green fluorescence) is detected in boar testes (a), prostatic glands (b), and seminal vesicles (c). In the testis, relaxin is detected in interstitial or Leydig cells (LC) and lumen (Lu) of seminiferous tubules (in A-1) and vicinity of spermatids or Sp (in A-2), in spermatocytes (SP) and Sertoli cells or SC (in A-3), and blood vessels (Ve). Scale bars = 10 μm

Fig. 6

Detection of relaxin in boar epididymis and vas deferens. Relaxin (green fluorescence) is detected in the epididymal caput (a), corpus (b), and cauda (c). Detection is also found in the proximal (d), middle (e), and distal (f) sections of the vas deferens. Signals are usually found in the basal cells (white arrows) and lumen of all epididymal and vas deferens sections, in the vicinity of spermatozoa (white arrow heads). Red arrows indicate relaxin signals found in the blood vessels. Nuclei are counter-stained in blue. Ar = Fluorescence artefact area; SLM = Smooth Longitudinal Muscle; SCM = Smooth Circular Muscle; 200× magnification

Fig. 7

Graphical representations of relaxin, RXFP1, and RXFP2. The figure represents FlowJo graphical layouts of flow cytometry analyses, following immunostaining of ejaculated (freshly collected), epididymal (caput, corpus, and cauda), and vas deferens spermatozoa. Controls corresponded to spermatozoa incubated without any (No-Ab) or only FITC-conjugated secondary (FITC) antibodies. Signal detection peaks of both controls, in each targeted protein, were comparable at the lowest levels (Green lines and arrows) in comparison to other groups

Fig. 8

Mean fluorescence detection of relaxin, RXFP1, and RXFP2 in epididymal and freshly ejaculated boar spermatozoa using flow cytometry. ^*,**,*** For each protein target (relaxin, RXFP1, and RXFP2), asterisks indicate significant differences between groups (caput, corpus, cauda, and ejaculated). Letters (a, b, c) show significant differences between the protein targets within the same group (P < 0.05; ANOVA-1). Data are means ± sem of 3 boars

Fig. 9

Detection of relaxin, RXFP1, and RXFP2 in epididymal boar spermatozoa. Spermatozoa were collected from epididymal caput (a, d, and g), corpus (b, e, and h), and cauda (c, f, and i) of boars, for the immunodetection of relaxin (a, b, and c), RXFP1 (d, e, and f), and RXFP2 (g, h, and i). Scale bars = 10 μm

Fig. 10

Detection of relaxin, RXFP1, and RXFP2 in ejaculated boar spermatozoa. The green-FITC fluorescence indicates relaxin (a/d), RXFP1 (b/e), and RXFP2 (c/f) detection. Corresponding micrographs d, e, and f show the blue nuclei counterstained with DAPI. Inserts in micrographs a and d are zooms of the sperm head and mid-piece