Subcellular Localization of Prolyl Endopeptidase During the First Wave of Rat Spermatogenesis and in Rat and Human Sperm

Abstract

Prolyl endopeptidase (PREP) is an enzyme which cleaves several peptide hormones and neuropeptides on the carboxyl side of proline residues and is involved in many biological processes, including cell proliferation and differentiation, glucose metabolism, learning, memory, and cognitive disorders. PREP has also been identified as a binding partner of tubulin, suggesting the involvement of endopeptidase in microtubule-associate processes, independent of its peptidase activity. Furthermore, several reports have implied PREP participation in both male and female reproduction-associated mechanism. We herein assess a potential association of PREP to the morphogenesis of rat testis, profiling its localization versus tubulin, during the first wave of spermatogenesis and in the adult gonad (from 7 to 60 dpp). We show that, in mitotic phases, PREP shares its localization with tubulin in Sertoli cells, gonocytes, and spermatogonia. Later, during meiosis, both proteins are found in spermatocytes, and in the cytoplasm of Sertoli cells protrusions, surrounding the germ cells, while, during spermiogenesis, they both localize in the cytoplasm of round and elongating spermatids. We also found that this enzyme has a peculiar nuclear localization, in the proliferating cells in all phases of analysis. Finally, they are expressed in the flagellum of mature gametes, as corroborated by additional immunolocalization analysis on both rat and human sperm. Our data support the hypothesis of the fundamental role of PREP in reproduction and in cytoskeletal organization during mammalian testis morphogenesis and gamete progression.

Introduction

Prolyl endopeptidase (PREP; EC 3.4.21.26) is a protein belonging to the serine protease family and is conserved throughout evolution.¹ It was identified for the first time in the human uterus,² and subsequently detected in all mammalian tissues, as well as the liver, kidney, heart, spleen, and brain, where the highest enzymatic activity was observed.^3,4 PREP has a typical endopeptidase structure, including the catalytic triad formed by Ser554, Asp641, and His680.⁵ PREP is able to hydrolyze the peptide bond on the carboxyl side of proline residues in oligopeptides comprising no more than about 30 amino acid residues,⁶ as well as peptide hormones and neuropeptides.^7,8 Despite its common intracellular localization and the lack of secretion signal or a lipid anchor sequence,¹ it is believed that PREP may be released from the cells and may act externally by inactivating extracellular neuropeptides.^9–11 PREP has been implicated in several biological processes, including development, cell proliferation^12,13 and differentiation,^14,15 cell death,^16,17 glucose metabolism,¹⁸ celiac disease,^19,20 learning and memory,^21,22 and cognitive disorders.^23,24 Further reports regarding the intracellular activity of PREP suggested an additional physiological role of this enzyme.²⁵ Indeed, PREP was identified as a binding partner of tubulin, indicating novel functions for PREP in vesicle transport and protein secretion.²⁶

Microtubules are highly dynamic cytoskeletal components that play a fundamental role in many cellular processes, such as motility, intracellular transport, division, and cell shape.^27–29 Cytoskeletal remodeling allows the cell to regulate its shape and architecture, and during gametogenesis and reproduction, the germinal compartment and germ cells (GC) undergo a complex series of transformations driven by a major cytoskeletal organization.³⁰ A small number of reports have already suggested PREP participation in both male and female reproduction-associated processes.^31–33 We hereby assess the association of PREP with the morphogenesis of rat testis, by studying and comparing its expression and localization with tubulin, during the first wave of spermatogenesis and in the adult tissue. We also extend our analysis to rat and human spermatozoa (SPZ), to further enhance the profile and to delineate PREP distribution in mature gametes.

Materials and Methods

Animal Care, Tissue Extraction, and Collection of Rat Spermatozoa

Male Sprague–Dawley rats (Rattus norvegicus) were housed under definite conditions (12D:12L) and they were fed with standard food and provided with water ad libitum. Animals at different development stages (7, 14, 21, 28, 35, 42, 60 days postpartum [dpp], and adult) were sacrificed by decapitation under Ketamine anesthesia (100 mg/kg i.p.) in accordance with national and local guidelines covering experimental animals. For each animal testes dissected, one testis was fixed in Bouin’s fluid and embedded in paraffin for histological analysis, and one was quickly frozen by immersion in liquid nitrogen and stored at −80C until protein extraction. In addition, epididymides were removed from adult rats and minced in phosphate-buffer saline (PBS; 13.6 mM NaCl; 2.68 mM KCl; 8.08 mM Na₂HPO₄; 18.4 mM KH₂PO₄; 0.9 mM CaCl₂; 0.5 mM MgCl₂; pH 7.4) to let the SPZ flow out from the ducts. Then, the fluid samples were filtered and examined under a light microscope to exclude contamination by other cell types. Next, aliquots were spotted and air-dried on slides, then stored at −20C, while the remaining samples were centrifuged at 1000 g for 15 min at 4C and stored at −80C until protein extraction.

Collection of Human Spermatozoa

Human sperms were collected from donors at the “Centre for Assisted Fertilization” in Naples (Via Tasso, 480, 80123, Naples, Italy) and the main chemical, physical, and spermatic parameters were evaluated in accordance with the World Health Organization (WHO) guidelines to verify the good quality of the samples (WHO: Standard procedures, 2010). The samples were centrifuged at 800 g for 10 min; the supernatant was removed and the pellet was washed and resuspended in PBS. The samples were examined under a light microscope and aliquots were spotted and air-dried on slides, then stored at −20C, while the remaining samples were centrifuged at 1000 g for 15 min at 4C and stored at −80C until protein extraction. A written informed consent was obtained from all participants to the study, that was approved by the local ethical committee.

Preparation of Total Protein Extracts and Western Blot Analysis

The testes and SPZ (from rat or human, n=4 for each group) were lysed in a specific buffer (1% NP-40, 0.1% SDS, 100 mM sodium ortovanadate, 0.5% sodium deoxycholate in PBS) in the presence of protease inhibitors (4 µg/µl of leupeptin, aprotinin, pepstatin A, chymostatin, PMSF, and 5 µg/µl of tosyl phenylalanyl chloromethyl ketone [TPCK]). The homogenates were sonicated twice by three strokes (20 Hz for 20 sec each); after centrifugation for 30 min at 10,000 g, the supernatants were stored at −80C. Proteins from testis and SPZ (50 µg) were separated by 9% SDS-PAGE and transferred to Hybond-P polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK) at 280 mA for 2.5 hr at 4C. The filters were treated for 3 hr with blocking solution—5% skim milk in TBS (10 mM Tris–HCl pH 7.6, 150 mM NaCl)—containing 0.25% Tween-20 (Sigma–Aldrich Corp., Milan, Italy) before the addition of anti-PREP (Abcam ab58988, Cambridge, UK), or anti-TUBULIN (Sigma–Aldrich Corp.) antibody diluted 1:5000 and 1:10,000, respectively, and incubated overnight at 4C. After three washes in TBST (TBS including 0.1% Tween-20), the filters were incubated with horseradish peroxidase-conjugated antirabbit IgG (Sigma–Aldrich Corp.) for the rabbit anti-PREP antibody, or anti-mouse IgG (Sigma–Aldrich Corp.) for the mouse anti-TUBULIN antibody, both diluted 1:10,000 in the blocking solution. Then, the filters were washed again three times in TBST and the immunocomplexes were revealed using the enhanced chemiluminescence (ECL)-Western blotting detection system (Amersham Pharmacia Biotech).

Preparation of Nuclear Protein Extracts and Western Blot Analysis

For the analysis on nuclear and cytosolic proteins, adult testes (n=4) were gently homogenized using a type B pestle, in seven volumes (w/v) of hypotonic Hepes buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 12% glycerol, 0.1 mM EGTA, 0.5 mM dithiothreitol, 0.5 mM spermidine) with protease inhibitors (4 μg/ml of leupeptin, aprotinin, pepstatin A, chymostatin, PMSF, and 5 μg/ml of TPCK). After centrifugation at 800 g, the supernatant was removed and nuclear pellet, washed three times, was resuspended in 1.2 volumes (1.2 ml/mg pellet) of hypertonic Hepes buffer (10 mM H, pH 7.9, 1.5 mM MgCl₂, 420 mM NaCl, 15% glycerol, 0.1 mM EGTA, 0.5 mM dithiothreitol, 2 mM spermidine) with protease inhibitors (see above), and stirred at 4C for 30 min. Nuclei were pelleted by centrifugation at 10,000 g for 30 min at 4C. The supernatants, containing nuclear proteins, were collected.³⁴ Proteins (50 µg) were separated by 9% SDS-PAGE and transferred to Hybond-P polyvinylidene difluoride membranes (Amersham Pharmacia Biotech) at 280 mA for 2.5 hr at 4C. The filters were treated for 3 hr with blocking solution—5% skim milk in TBS (10 mM Tris–HCl pH 7.6, 150 mM NaCl)—containing 0.25% Tween-20 (Sigma–Aldrich Corp.) before the addition of anti-PREP (Abcam ab58988; Cambridge, UK), anti-PCNA (Santa Cruz, Biotechnology, Inc., Milan, Italy), or anti-GAPDH (Abcam ab9485) antibodies diluted 1:3000 and 1:5000, respectively, and incubated overnight at 4C. After three washes in TBST (TBS including 0.1% Tween-20), the filters were incubated with horseradish peroxidase-conjugated antirabbit IgG (Sigma–Aldrich Corp.) for the rabbit anti-PREP and anti-GAPDH antibodies, or anti-mouse IgG (Sigma–Aldrich Corp.) for the mouse anti-PCNA antibody, both diluted 1:10,000 in the blocking solution. Then, the filters were washed again three times in TBST and the immunocomplexes were revealed using the ECL-Western blotting detection system (Amersham Pharmacia Biotech).

Tissue Quality Control and Classification of Testicular Cell Types

To assess the quality of the tissue samples and their staging, 7 µm-thick rat testis sections of all samples (7, 14, 21, 28, 35, 42, 60 dpp) were prepared and a haematoxylin-eosin staining was performed (see Fig. 1). The cell types for each time point were characterized and confirmed following previously reported classifications.^35,36

Figure 1.

Histology and staging of the developing rat testis. Haematoxylin-eosin staining of tissue sections at 7 (A), 14 (B), 21 (C), 28 (D), 35 (E), 42 (F), 60 (G and H) dpp, in which the most representative cell types are highlighted (for review see Picut et al.³⁵). Pointer legend is provided in the bottom-right table. Scale bars represent 20 μm. Abbreviations: dpp, days postpartum; PT cells, peritubular cells; LC, Leydig cells; SPG, spermatogonia; SC, Sertoli cells; PL SPC, preleptotene primary spermatocytes; L/Z, Leptotene/Zygotene; P, pachytene; RSPT, round spermatids; ESPT, elongating spermatids; SPZ, spermatozoa.

Immunofluorescence Analysis on Testis

For PREP colocalization with both microtubules and the acrosome system, 7 mm-testis sections were dewaxed, rehydrated, and processed as described by Venditti et al.³⁷ Antigen retrieval was performed by pressure cooking slides for 3 min in 0.01 M citrate buffer (pH 6.0). Then, the slides were incubated with 0.1% (v/v) Triton X-100 in PBS for 30 min. Later, nonspecific binding sites were blocked with an appropriate normal serum diluted 1:5 in PBS containing 5% (w/v) BSA before the addition of anti-PREP, or anti-TUBULIN antibody diluted 1:100, for overnight incubation at 4C. After washing in PBS, slides were incubated for 1 hr with the appropriate secondary antibody (Alexa Fluor 488, Invitrogen; FITC-Jackson, ImmunoResearch, Pero MI, Italy; Anti-Mouse IgG 568, Sigma–Aldrich, Milan, Italy) diluted 1:500 in the blocking mixture and with PNA lectin (Alexa Fluor 568, Invitrogen, Monza MB, Italy) diluted 1:50. The slides were mounted with Vectashield + DAPI (Vector Laboratories, Peterborought, UK) for nuclear staining, and then observed with a microscope then observed under the optical microscope (Leica DM 5000 B + CTR 5000) and images where viewed and saved with IM 1000.

Immunofluorescence Analysis on SPZ

To determine PREP and TUBULIN colocalization in rat and human SPZ, the samples were firstly fixed in 4% paraformaldehyde in PBS, and then washed in phosphate buffer (0.01 M PBS, pH 7.4). The slides were incubated with 0.1% (v/v) Triton X-100 in PBS for 30 min. Later, nonspecific binding sites were blocked with an appropriate normal serum diluted 1:5 in PBS containing 5% (w/v) BSA before the addition of the primary antibody (PREP and TUBULIN), as described above, and overnight incubation at 4C. After washing in PBS, slides were incubated for 1 hr with the appropriate secondary antibody (Alexa Fluor 488, Invitrogen; FITC-Jackson, ImmunoResearch; Anti-Mouse IgG 568, Sigma–Aldrich) diluted 1:500 in the blocking mixture and with PNA lectin (Alexa Fluor 568, Invitrogen) diluted 1:50. The slides were mounted with Vectashield + DAPI (Vector Laboratories) for nuclear staining, then observed under the optical microscope (Leica DM 5000 B + CTR 5000) with UV lamp, and images where viewed and saved with IM 1000.

Statistical Analysis

Student’s t-test or ANOVA followed by Tukey’s test for multigroup comparison were performed to evaluate the significance of differences using Prism 5 software (GraphPad, San Diego, CA). All data are reported as mean ± SEM (standard error of the mean).

Results

Expression of PREP and Tubulin During the Postnatal Development of Rat Testis

The expression of PREP during the postnatal development of the gonad was assessed by Western Blot analysis on protein extracts from some of the most representative time points during the first wave of spermatogenesis: 7 dpp (transition of gonocytes from tubule lumen toward the base), 4 dpp (proliferation of Sertoli cells (SC) and A and B spermatogonia, before meiosis), 21 dpp (presence of spermatocytes [SPC], which undertake meiosis; first phases of blood-testis barrier formation), 28 dpp (conclusion of “first wave” meiosis, completion of the blood-testis barrier), 35 dpp (presence of newly formed round spermatids [SPT] in spermiohistogenesis), 42 dpp (final steps of spermiohistogenesis), and 60 dpp (mature testis; presence of SPZ and of all the characteristic germ cell associations). A band of the expected size (80 kDa) was detected for PREP in all samples (Fig. 2A). The relative expression levels of PREP were analyzed and reported as PREP/TUB (TUBULIN) OD ratio (Fig. 2B): PREP expression was persistent in all the stages, with a higher peak at 21 dpp and a smaller one at 35 dpp. We also evaluated the nuclear expression of PREP, finding that it is present both in the cytoplasm and in the nucleus (Fig. 2C), with a relative lower expression in the latter (Fig. 2D).

Figure 2.

Expression of PREP and TUBULIN during the postnatal development of rat testis. (A) Western blot analysis which shows the expression of PREP (80 kDa, top section) and TUBULIN (50 kDa, bottom section) during rat postnatal development, at 7, 14, 21, 28, 35, 42, and 60 days postpartum (dpp). The two proteins are always expressed. (B) Histogram (made with Prism 5 software GraphPad, San Diego, CA) which shows the relative expression levels of PREP in all the analyzed samples. Data were normalized with TUBULIN and reported as PREP/TUB OD ratio. All data represent the mean ± SEM. samples. a: significative difference versus 7 dpp group, p<0.05; a* significative difference versus 7 dpp group, p<0.01; b: significative difference versus 14 dpp group, p<0.05; b*: significative difference versus 14 dpp group, p<0.01; c: significative difference versus 21 dpp group, p<0.05; c*: significative difference versus 21 dpp group, p<0.01; d*: significative difference versus 28 dpp group, p<0.01; e*: significative difference versus 21 dpp group, p<0.01. (C) Expression of PREP in cytoplasm and nucleus of adult rat testis. Western blot analysis on protein extract from cytoplasm (lane C) and nucleus (lane N). PREP (80 kDa, top section) is present in both the samples. To evaluate the purity of the samples, we detected the nuclear expression of PCNA (central section) and cytoplasmic expression of GAPDH (bottom section). (D) Histogram (made with Prism 5 software GraphPad, San Diego, CA) which shows the relative expression levels of PREP in cytoplasm and nucleus. Data were normalized with PCNA and GAPDH, respectively, and reported as PREP/PCNA and PREP/GAPDH ratio. All data represent the mean ± SEM. a*: statistically significant differences versus cytoplasm group (p<0.01). Abbreviation: PREP, Prolyl endopeptidase; TUB, TUBULIN; OD, optical density; PCNA, proliferating cell nuclear antigen; GAPDH, glyceraldeyde 3-phosphate dehydrogenase; SEM, standard error of the mean.

Localization of PREP During the Postnatal Development of Rat Testis

First, tissue quality and staging were checked by performing a haematoxylin-eosin staining on sections of rat testis at the same time points as described in the previous paragraph (Fig. 1).

PREP localization was studied by immunofluorescence analysis on developing testis sections (7, 14, 21, and 28 dpp, Fig. 3; 35, 42, and 60 dpp, Fig. 4). During the initial stages, the protein was detected both in the cytoplasm and the nuclei of the cells: At 7 dpp ( Figs. 3A–C), the protein signal was quite diffuse, and localized in SC, in luminal gonocytes (Fig. 3B, C) and peritubular cells; at 14 dpp ( Fig. 3D–F), the signal was still localized in SC, and it was evident in spermatogonia (SPG) cytoplasm and nucleus (Fig. 3E–F). At 21 to 28 dpp ( Fig. 3G–L) it was detectable inside the nucleus of meiotic I spermatocytes, while a less strong cytoplasmic signal was retained (SPC; Fig. 3J–L, better highlighted by the insets). As for the SC, PREP has a clearly detectable nuclear localization, while in the cytoplasm is evident at the extending region, which could contain more protein than the remaining part. During spermiogenesis, as shown from 28 dpp onward, it was possible to highlight the occurring acrosome formation, thanks to PNA lectin staining ( Fig. 3J–L, P; Fig. 3). In these late stages of the first wave of spermatogenesis, PREP retained its cytoplasmic and nuclear localization in mitotic and meiotic cells, moreover in 35 dpp tubules (Fig. 4A–C), the signal was clear in the cytoplasm of SC, which extends from the base to the lumen, surrounding the developing GC (Fig. 4A–C and insets). Then, the protein localized in elongating SPT at 42 dpp and was also detectable in SC cytoplasm (Fig. 4D–F). Finally, at 60 dpp (Fig. 4G–I), after the conclusion of the first spermatogenetic wave, the signal was comparable to the one seen at 42 dpp, with the protein present in elongating SPT and SC cytoplasm, as well as in both the cytoplasm and nucleus of SPG, SPC, and SC. We also detected the signal outside the tubular compartment, in the Leydig cells (Figs. 4C, 5C–L, and 6F).

Figure 3.

Localization of PREP during the postnatal development of rat testis, part I (7–28 dpp). A, D, G, J. DAPI-fluorescent nuclear staining (blue) and PNA lectin acrosome staining (red). B, E, H, K. PREP fluorescence (green). C, F, I, L, M, N, O, P. Merged fluorescent channels (blue/red/green). A, B, C. 7 dpp testis; PREP-positive fluorescence is diffuse, in the nucleus and cytoplasm of gonocytes, as well as in the Sertoli cells cytoplasm. D, E, F. 14 dpp; fluorescent signal is present in spermatogonia and Sertoli cells. G, H, I. 21 dpp; J, K, L. 28 dpp; positive cells now include meiotic spermatocytes; Sertoli cells cytoplasm is also positive; insets show Sertoli cells protrusions. At 28 dpp, the acrosome formation starts to be visible through PNA lectin staining. M, N, O, P. Negative controls for the same time points obtained by omitting the primary antibody. Scale bars represent 20 μm, except for the insets, where they represent 10 μm. For cell-type pointer legend, see table in Fig. 1. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole, dihydrochloride; dpp, days postpartum; PREP, Prolyl endopeptidase; bg, background/autofluorescence; nc, Negative controls.

Figure 4.

Localization of PREP during the postnatal development of rat testis, part II (35–60 dpp). A, D, G. DAPI-fluorescent nuclear staining (blue) and PNA lectin acrosome staining (red). B, E, H. PREP fluorescence (green). C, F, I, J, K, L. Merged fluorescent channels (blue/red/green). A, B, C. 35 dpp testis PREP is detectable in the nucleus and cytoplasm of spermatocytes, as well as nucleus and cytoplasm of Sertoli cells and in Leydig cells; D, E, F. 42 dpp; PREP is evident in Sertoli cells, nucleus, and cytoplasmic protrusions, which surrounds the germ cells. G, H, I. 60 dpp; PREP localizes in the nucleus and cytoplasm of proliferating cells, and in the Sertoli cells, nucleus (dotted asterisk), and cytoplasm; insets show the latter highlighted. J, K, L. Negative controls for the same time points, obtained by omitting the primary antibody. Scale bars represent 20 μm, except for the insets, where they represent 10 μm. For cell-type pointer legend, see table in Fig. 1. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole, dihydrochloride; dpp, days postpartum; PREP, Prolyl endopeptidase; bg, background/autofluorescence; nc, negative controls.

Figure 5.

Colocalization of PREP and TUBULIN during the postnatal development of rat testis, part I (7–28 dpp). A, D, G, J. DAPI-fluorescent nuclear staining (blue) and TUBULIN staining (red). B, E, H, K. PREP fluorescence (green). C, F, I, L, M, N, O, P. Merged fluorescent channels (blue/red/green). A, B, C. 7 dpp testis; D, E, F. 14 dpp; G, H, I. 21 dpp; J, K, L. 28 dpp. TUBULIN signal is strong in all samples, especially in the cytoplasm of Sertoli cells, where it clearly colocalizes with PREP, which is also detectable in the Leydig cells. M, N, O, P. Negative controls for the same time points, obtained by omitting the primary antibody. Scale bars represent 20 μm. For cell-type pointer legend, see table in Fig. 1. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole, dihydrochloride; dpp, days postpartum; TUB, TUBULIN; PREP, Prolyl endopeptidase; bg, background/autofluorescence; nc, negative controls.

Figure 6.

Colocalization of PREP and TUBULIN during the postnatal development of rat testis, part II (35–60 dpp). A, D, G. DAPI-fluorescent nuclear staining (blue) and TUBULIN staining (red). B, E, H. PREP fluorescence (green). C, F, I, J, K, L. Merged fluorescent channels (blue/red/green). A, B, C. 35 dpp testis; D, E, F. 42 dpp; G, H, I. 60 dpp; Immunopositivity for both proteins is observed in Sertoli cells cytoplasm protrusions from the base to the lumen of the tubules, and in cell–cell junctions. In mature testes, the signal also appears in SPZ. J, K, L. Negative controls for the same time points, obtained by omitting the primary antibody. Scale bars represent 20 μm. For cell-type pointer legend, see table in Fig. 1. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole, dihydrochloride; dpp, days postpartum; TUB, TUBULIN; PREP, Prolyl endopeptidase; bg, background/autofluorescence; nc, negative controls; SPZ, spermatozoa.

Colocalization of PREP and Tubulin During the Postnatal Development of Rat Testis

Given PREP association with Tubulin, the colocalization profile of the two proteins was performed on the same time point described above. The immunofluorescence analysis showed that Tubulin signals resulted in a pattern comparable with the cytoplasmic localization of PREP: Tubulin (Figs. 5 and 6) was expressed in all stages and, to various extent, by all cell types, but it was especially represented inside the somatic SC which nurse the mitotic and meiotic cells during the first phases of spermatogenesis (Fig. 5), as well as the SPT during their differentiation into SPZ (Fig. 6). PREP and Tubulin initially colocalize within GC junctions (Fig. 5C, F, and I). From 28 dpp on, they both are present inside SC cytoplasm, which surrounds the developing GC (Fig. 5L, and Fig. 8C, F), as well as, during spermiohistogenesis, in SPT, and in the epithelial cells which rearrange their architecture to support the path of the evolving GC toward the lumen (Fig. 6F and I).

Figure 8.

Colocalization of PREP and TUBULIN in rat spermatozoa. (A) DAPI-fluorescent nuclear staining (blue) and PNA lectin acrosome staining (red). (B) Fluorescent signal of TUBULIN (red). (C and D) Fluorescent signal of PREP (green). (E and F) Merged fluorescent channels (blue/red/green) including either PREP or TUBULIN, respectively. (C–F) PREP is clearly detectable in the flagellum. (B) TUBULIN marks the region of the tail. (F) PREP and TUBULIN colocalize inside the flagellum. (G and H) Negative controls for PREP or TUBULIN, obtained by omitting the primary antibodies. Scale bars represent 10 μm. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole, dihydrochloride; TUB, TUBULIN; PREP, Prolyl endopeptidase; bg, background/autofluorescence; NC, negative controls.

Expression of PREP and Tubulin in Rat and Human Spermatozoa

The expression of PREP and Tubulin in rat and human SPZ was assessed by Western Blot on protein extracts from epididymal and ejaculated SPZ, respectively (Fig. 7A). The data confirmed the presence of the two proteins in male gametes of both species. The relative expression levels of PREP were analyzed and reported as PREP/TUB OD ratio (Fig. 7B): PREP shows a comparable expression level in both rat and human samples.

Figure 7.

Expression of PREP and TUBULIN in rat and human spermatozoa. (A) Western blot analysis on protein extract from rat (lane 1) and human (lane 2) SPZ. PREP (80 kDa, top section) and TUBULIN (50 kDa, bottom section) are present in both the samples. (B) Histogram (made with Prism 5 software GraphPad, San Diego, CA) which shows the relative expression levels of PREP in rat and human SPZ. Data were normalized with TUBULIN and reported as PREP/TUB OD ratio. All data represent the mean ± SEM. Abbreviations: PREP, Prolyl endopeptidase; TUB, TUBULIN; SPZ, spermatozoa; OD, optical density; SEM, standard error of the mean.

Colocalization of PREP and Tubulin in Rat and Human Spermatozoa

To further expand the profile of PREP localization in male gametes, an immunofluorescence analysis was carried out on rat epididymal SPZ (Fig. 8): There the protein was mainly detectable inside the flagellum (Fig. 8C, E), where it clearly colocalizes with Tubulin (Fig. 8D, F). To obtain more detailed data about PREP expression profile in gametes, we extended the analysis on human ejaculated SPZ (Fig. 9). The signal in human gametes confirmed PREP presence inside the flagellum (Fig. 9C, E), with a weaker signal in the midpiece. Also in this case, PREP and Tubulin colocalize within the flagellum (Fig. 9D, F), showing a comparable expression pattern described in rat SPZ.

Figure 9.

Colocalization of PREP and TUBULIN in human spermatozoa. (A) DAPI-fluorescent nuclear staining (blue) and PNA lectin acrosome staining (red). (B) Fluorescent signal of TUBULIN (red). (C and D) Fluorescent signal of PREP (green). (E and F) Merged fluorescent channels (blue/red/green) including either PREP or TUBULIN, respectively. (C–E) PREP is clearly detectable in the flagellum, with a weaker signal in the midpiece. (B) TUBULIN marks the region of the tail. (F) PREP and TUBULIN colocalize inside the flagellum. (G and H) Negative controls for PREP or TUBULIN, obtained by omitting the primary antibodies. Scale bars represent 10 μm. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole, dihydrochloride; TUB, TUBULIN; PREP, Prolyl endopeptidase; bg, background/autofluorescence; NC, negative controls.

Discussion

In mammals, the postnatal development of the male gonad is a complex process, during which the seminiferous tubules progressively change their size, structural organization, and composition. The first wave of spermatogenesis involves GC migration toward the base of the tubule to initiate proliferation and differentiation, and subsequently produces mature SPZ, which are nurtured and predominated by their association with somatic SC. This process is also marked by a significant cytoskeletal remodeling that endorses the formation of complex structures leading to cell separation (GC), protection, and maintenance.

Tubulin is a fundamental part of these processes, through the regulation of its polymerization and stabilization. Among its many roles, the protein is important in SC in the formation of their wide cytoplasmic protrusions,³⁰ following GC differentiation toward the lumen. In particular, the microtubules are orientated in linear arrays parallel to the long axis of the cell in SC cytoplasm.³⁸ Microtubules are evident in the lateral processes surrounding round and elongating SPT^39,40 illustrating complex changes as GC progress through the various stages of seminiferous cycle.⁴¹

PREP is a serine protease enzyme able to digest small peptides. It is involved in several physiological and pathological processes, such as glucose metabolism,¹⁸ celiac disease,^19,20 learning and memory,^21,22 cognitive disorders,^23,24 development, cell proliferation^12,13 and differentiation,^14,15 and cell death.^16,17 Although its structural and enzymatic features have been well charachterized,⁶ its biological role still remains unclear. PREP was observed in regulating the phosphoinositide pathway,^42,43 acting in cell death and apoptosis,^16,44 and is involved in DNA synthesis^15,45,46 by means of its nuclear localization. Moreover, PREP has been associated to microtubules, in particular with the C-terminus of α-tubulin, suggesting engagement of the endopeptidase in microtubule-associate processes, independent of its peptidase activity.²⁵ Many studies show that the protein may have a very important role in the central nervous system,^7,8 as well as in the physiology of other systems, including the reproductive organs. PREP was originally found as an oxytocin-cleaving enzyme in the human uterus,² and later implicated in male gametogenesis: It was purified from ascidian sperm⁴⁷; then the protein was isolated from herring testis.⁴⁸ PREP was later localized in mouse SPT and SPZ and was indicated in sperm motility.³² Successive analyses on human showed that PREP localizes in the seminiferous tubules and Leydig cells and may regulate the levels of seminal thyrotropin releasing hormone (TRH) analogues, mediating death, associated with necrozoospermia.^49,50 Finally, Dotolo et al.³³ studied the effects of PREP knockdown on testis and sperm in adult mice, demonstrating that the enzyme is essential to provide an efficient reproductive function and a lack of the enzyme may results in marked alterations of the gonads and eventually in the gametes. These reports imply relevant PREP activity in male reproduction. In the present study, we investigate the possible association of PREP with the morphogenetic changes, which occur during the postnatal development of rat testis, selecting a time frame range from 7 to 60 dpp, indicative of the first wave of spermatogenesis.

Western Blot analysis reveals expression of PREP in the developing and adult testis, in different quantities during the various phases, with a maximum peak at 21 dpp, and the lowest at 35 dpp, revealing the regulated nature of PREP expression during testis morphogenesis, most likely due to the hormonal fluctuation throughout the different phases.³⁵ This oscillatory expression undoubtedly requires further investigation, so we hypothesize that PREP is not a housekeeping enzyme, but is tightly regulated.⁵¹ The successive localization analysis highlighted that the protein localizes both in the cytoplasm and the nucleus of proliferating GC as well as in SC cytoplasm during all development phases. Congruence is evident between PREP profile and tubulin distribution in the SC cytoplasmic protrusions, which surround the GC. It is well known that GC translocation, and in particular that of SPT, through the seminiferous epithelium will occur via microtubules-based transport of the apical ectoplasmic specialization (ES), a structural connection between SC and differentiating GC.⁵² It has been proposed that the microtubules in this process act as a “rail” for the relocalization of cellular contents, as well as of translocation of SPT, acquired by the gliding of the entire ES structure with attached SPT along microtubules within SC.³⁰ As already mentioned, PREP has been associated with the C-terminus of α-tubulin, which, in line with our investigation, suggests PREP involvement in the cytoskeletal remodeling.

Conversely, we were able to detect PREP presence within the proliferating and differentiating GC during the first wave of spermatogenesis. Spermatogonia are immature GC which undergo a series of mitotic division, which give rise to a pool of cells that enter in meiosis I and then in meiosis II. Previous studies reported that PREP inhibition suppressed the growth of human neuroblastoma cell¹⁷ and that may be a positive regulator of cell cycle progression in human gastric cancer cell.¹³ Here we can hypothesize that PREP may be involved in the meiotic and postmeiotic phases of GC differentiation. Our supposition is corroborated by the colocalization of this peptidase with tubulin: as well known, microtubules are the main elements of mitotic and meiotic splindles, which will facilitate the division of chromosomes/chromatids into the two daughter cells. Our results showed that, near to the cytoplasmic localization, we were able to find PREP in the nucleus of SPG and SPC. This is not so unexpected: Its nuclear sublocalization in vertebrates and its possible involvement in DNA synthesis have been already reported as aforementioned. Moreover, PREP was found in various cell types in both the cytoplasm and nuclei in mouse whole-body sections, in colocalization with Ki-67, a proliferation marker protein, implying a role in cell proliferation.⁵⁰ Herein, we hypothesize the engagement of PREP could also participate in proliferation of GC, due to its nuclear presence, and its lacking in the one which do not undergo in mitotic and/or meiotic phases, such as SPT and SPZ. Rat PREP is potential single gene product, so the same enzyme is relevant in different cellular compartments.

During spermiogenesis and in adult testis, PREP colocalize with tubulin in the cytoplasm of haploid round and elongating SPT. This may correlate either with the above-mentioned physiological activity of SCs, which maintain and hold the SPTs until their release during spermiation, or with spermiohistogenesis itself, during which PREP may be needed in the appropriate organization and differentiation of SPT. In fact, dynamic microtubules are essential for the assembly of microtubule-based structures that participate in SPT remodeling and physiology, such as the manchette and the sperm flagella. Thus, such wide distribution suggests an involvement of PREP, in view of its enzymatic activity, which may induce the degradation and maturation of small active molecules involved in the process, as well as its association with microtubules and its participation in all microtubule-associated processes occurring during spermatogenesis.

Furthermore, the presence of the enzyme outside the germinal compartment, in Leydig cells, suggests the hypothesis that it may act in the control of germ cell development, by modulating both paracrine and endocrine gonadotropin levels. Indeed, PREP is likely involved in the regulation of GnRH pulsatility⁵³ and, given the crucial role for GnRH in the hypothalamic–pituitary–gonadal axis, the enzyme might consequently control the physiological secretion of hormones which are normally released upon stimulation of the axis, such as luteinizing hormone (LH), follicle-stimulating hormone (FSH) and, ultimately, testosterone. Thus, a relationship may exist between PREP normal function and a proper development and maintenance of the testis. As for the peritubular cells, whose myoid pool is essential for tubule contraction and sperm release, the presence of PREP could be associated with the maturation of small molecules, which contribute to the process, like the known target oxytocin.

Interestingly, the endopeptidase is clearly detectable in the tail of isolated epididymal and human SPZ, as well as tubulin, which suggests PREP involvement in mature sperm function. Our data are partially in line with investigations reported by Kimura et al.³² and Dotolo et al.³³ in mouse sperm, which were able to detect PREP in the head of the flagellum, but with a stronger signal in the midpiece, and a weaker one in the remaining part of the tail. These results support the hypothesis of the role of the enzyme in mammalian sperm motility: considering, the process is driven by the release and uptake of calcium by intracellular stores.^54,55 PREP is a possible regulator of the pathway of inositol 1,4,5 which results in the modulation of cytosolic calcium level,⁶ thus, we consider the contribution of PREP in calcium signaling and propose it as a key participant in the regulation of sperm movement and progression.

In conclusion, our work shows the expression and the localization of PREP during rat spermatogenesis and in rat and human SPZ. The precise functions of this enzyme remain to be elucidated and further investigations are required to better understand the implications of such varied pattern. However, our current data provide a comparative profile of PREP distribution versus the tubulin, a key architectural factor of the germinal compartment, suggesting its possible involvement in morpho-functional remodeling and organization of the male gonad as well as in gametes structure.