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Ras-related proteins (Rab) are key proteins related to male fertility following a unique activation mechanism
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Original article
Ras-related proteins (Rab) are key proteins related to male fertility following a unique activation mechanism Jeong-Won Baea, So-Hye Kima, Dae-Hyun Kimb, Jae Jung Hab, Jun Koo Yib, Seongsoo Hwangc, ⁎ Buom-Yong Ryud, Myung-Geol Pangd, Woo-Sung Kwona,e, a
Department of Animal Science and Biotechnology, Kyungpook National University, Sangju, Gyeongsangbuk-do 37224, Republic of Korea Gyeongbuk Livestock Research Institute, Yeongju Gyeongsangbuk-do 36052, Republic of Korea c Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju-gun, Jeollabuk-do 55365, Republic of Korea d Department of Animal Science & Technology, Chung-Ang University, Anseong, Gyeonggi-do 456-756, Republic of Korea e Department of Animal Biotechnology, Kyungpook National University, Sangju, Gyeongsangbuk-do 37224, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Rab proteins Spermatozoa Capacitation Acrosome reaction Male fertility
Ras-related protein Rab (Rab) proteins, member of Ras superfamily of monomeric G proteins, are well known key regulators of intracellular vesicular transport. Recently, it has been reported that Rab 2A and 3A are related to acrosomal exocytosis in spermatozoa and Rab 2A can be used to fertility-related biomarker in male. However, the role and mechanism of Rab proteins in spermatozoa has not been fully understood yet. Therefore, the study to analyze the expression and location of various Rab proteins in spermatozoa is required to understand the role and mechanism of Rab proteins in spermatozoa. In present study, to analyze the expression level and location of various Rab proteins (Rab 2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, and Rab34) and Rab protein regulators (RabGAP, RabGDI, RabGEF) in spermatozoa following capacitation, immunofluorescence and western blot analysis were performed. All of 11 Rab proteins were expressed in acrosomal region and tail of spermatozoa. Furthermore, all Rab proteins and Rab protein regulators, except RabGAP, have decreased expression patterns after capacitation. Taken together, Rab proteins were located in sperm head and tail. In addition, expression patterns of Rab proteins in spermatozoa were altered following capacitation. Therefore, our results suggested that Rab proteins may be key proteins related with capacitation as well as playing important role with uniquely activation pathway for male fertility.
1. Introduction Ras-related proteins (Rab), which are members of Ras superfamily of monomeric G proteins, are well-known key regulators of intracellular vesicular transport [1,2]. To date, approximately 70 different types of Rab proteins that perform various roles related to membrane trafficking in eukaryotic cells, such as vesicle formation, vesicle movement, and membrane fusion, have been identified [3–7]. In addition, they regulate various vesicular traffic steps related to the biogenesis of transport carriers for moving the cytoskeleton and facilitate binding with target membranes by interacting with effectors such as molecular motors, connecting complexes, scaffolding proteins, and lipid kinases [5,4–7]. It has been reported that certain Rab proteins play a key role in establishing and preserving the Golgi structure by regulating Golgi trafficking [8]. Especially, the Golgi apparatus is an important organelle
in the primordial cells of spermatozoa because the Golgi apparatus derive to acrosome in the sperm head during spermatogenesis [9–11]. To penetrate into the oocyte, the sperm cells have to undergo a special event called “capacitation” during migration through the oviduct tract [12,13]. Subsequently, only acrosome-reacted spermatozoa can fuse with oocytes after capacitation. It has been reported that various proteins contribute to acrosome formation during spermatogenesis [14]. Various studies have reported that certain Rab proteins are key factors for critical mechanisms including acrosomal biogenesis and acrosomal reaction [15–18]. Previous researchers have reported that Rab2A and Rab3A are related to acrosomal exocytosis following capacitation [15,19,20]; some of those researchers also suggested that Rab2A can be used as a fertility-related biomarker because Rab2A expression was altered following male fertility [21,22]. Although various studies related to Rab proteins have been
⁎ Corresponding author at: Department of Animal Science and Biotechnology and Department of Animal Biotechnology, Kyungpook National University, Sangju, Gyeongsangbuk-do 37224, Republic of Korea. E-mail address: [email protected] (W.-S. Kwon).
https://doi.org/10.1016/j.repbio.2019.10.001 Received 19 August 2019; Received in revised form 30 September 2019; Accepted 2 October 2019 1642-431X/ © 2019 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Jeong-Won Bae, et al., Reproductive Biology, https://doi.org/10.1016/j.repbio.2019.10.001
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divided into two groups: before capacitation (BC) and after capacitation (AC). The AC group was incubated for 90 more minutes at 37 °C under 0.5% CO2, in accordance with several previous studies [23–25]. The BC group was used for further experimentation without any additional incubation.
conducted, the roles and mechanism of Rab proteins in spermatozoa have not been fully understood. Therefore, this study was conducted to analyze the expression and location of various Rab proteins and activation process of Rab proteins in spermatozoa. In this study, the location of Rab proteins present in spermatozoa were identified by immunocytochemistry, and their expression levels before and after capacitation were evaluated by western blotting. Finally, the expression levels of various Rab protein regulators such as Rab GTPase-activating protein (GAP), Rab GDP-dissociation inhibitor (GDI), and Rab GTP exchange factor (GEF) were analyzed by western blotting.
2.4. Computer-assisted sperm analysis Ten microliters of each sample group were placed in a Makler counting chamber (Sefi-Medical Instruments, Haifa, Israel) which was heated to 37 °C. Total sperm motility (%) and motion kinematics [VCL, curvilinear velocity (μm/s); VSL, straight-line velocity (μm/s); VAP, average path velocity (μm/s); ALH, mean amplitude of head lateral displacement (μm)] were measured using 10 × objective phase-contrast mode. To measure the motility and motion kinematics, computerassisted sperm analysis program (CASA) (FSA2016, Medical supply, Seoul, Korea), OLYMPUS BX43 phase-contrast microscope (Olympus, Tokyo, Japan), CMOS, CAMERA, 2048 × 1536 (300 M pixel), and 60 Frame (Medical supply, Seoul, Korea) were used.
2. Materials and methods 2.1. Ethical statement All procedures were performed in accordance with the guidelines for the ethical treatment of animals and approved by Institutional Animal Care and Use Committee of Kyungpook National University. 2.2. Chemicals and media
2.5. Combined H33258/chlortetracycline fluorescence (H33258/CTC) assessment of spermatozoa
All reagents were purchased from Sigma-Aldrich (St Louis, MO, USA). We used Modified Tyrode’s medium as the basic medium (BM) (97.84 mM NaCl, 1.42 mM KCl, 0.47 mM MgCl2·6H2O, 0.36 mM NaH2PO4·H2O, 5.56 mM D-glucose, 25 mM NaHCO3, 1.78 mM CaCl2.2H2O, 24.9 mM Na-lactate, 0.47 mM Na-pyruvate, 50 μg/ml gentamycin, and 0.005 mM phenol red). For capacitation, BM was prepared with 0.4% bovine serum albumin 1 day prior to the experiment [23,24]. The basic media was maintained at a pH of 7.2 ± 0.2 [25].
To determine whether or not spermatozoa has become capacitated, dual staining method (Combined Hoechst 33258/chlortetracycline fluorescence assessment) was employed [23–25]. Spermatozoa in both BC and AC groups were centrifuged at 100× g for 2.5 min. Except in 135 μL samples, in all other samples, supernatant was removed. In the 135 μL sample, 15 μL of H33258 solution (10 μg H33258/mL DPBS) was added and incubated for 2 min at RT. After 2 min, 250 μL of 2% (w/v) polyvinylpyrrolidone in DPBS was added and the sample was centrifuged at 100×g for 2.5 min. The supernatant liquid was fully removed from centrifuged samples and the samples were resuspended in 100 μL chlortetracycline fluorescence (CTC) solution (750 mM CTC in 5 μL buffer: 20 mM Tris, 130 mM NaCl, and 5 mM cysteine, pH 7.4) and 100 μL DPBS. After the samples were refrigerated for 20 min under dark conditions, 10 μL of each sample was smeared on the slides. Finally, at least 400 spermatozoa per slide for each sample were evaluated using OLYMPUS BX43 under epifluorescence illumination using ultraviolet BP 340–380/LP 425 and BP 450–490/LP 515 excitation/emission filters for H33258 and CTC, respectively (Olympus, Tokyo, Japan). According to the capacitation status, spermatozoa were divided into four groups: dead (D pattern, blue fluorescence), live non-capacitated (F pattern, bright green fluorescence distributed uniformly over entire sperm head), live capacitated (B pattern, green fluorescence over the acrosomal region and a dark postacrosome), and live acrosome-reacted (AR pattern, sperm showing a mottled green fluorescence over the head or
2.3. Preparation and treatment of spermatozoa Eight to twelve-week-old mature ICR male mice were used (Nara Biotech, Seoul, Korea). The mice were housed individually and maintained at a temperature of 25 ± 2 °C under optimal humidity and light (12-h light/dark) conditions. All mice were provided ad-libitum feeding (Cargil Agripurina, Seongnam, Korea) and water. Mice were sacrificed by cervical dislocation, and their epididymis was collected and dissected to isolate the cauda epididymis. The cauda epididymis, which was trimmed to eliminate fat content, was further used to extract spermatozoa. Using a 1 mL syringe attached to a 26 G needle, spermatozoa was extracted in 35 mm-diameter sterile cell culture dishes containing BM with 0.4% BSA. To facilitate release of the extracted spermatozoa, they were incubated at 37 °C under 0.5% CO2 for 12 min. After releasing, to rule out individual variation, each extracted spermatozoa from 3 mice was pooled. Then the spermatozoa were
Fig. 1. Capacitation status in before capacitation (BC) and after capacitation (AC) groups of spermatozoa. (A) Patterns of live non-capacitated (F pattern). (B) Patterns of live capacitated (B pattern). (C) Patterns of live acrosome reacted (AR pattern). (D) Patterns of dead (D pattern). (E) Proportion of capacitation status in live spermatozoa in the two groups. Data represent mean ± SEM, n = 6. Superscript (*) indicates significant difference between before and after capacitation groups as tested by one-way ANOVA (P < 0.05). 2
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2.97 ± 0.42 94.53 ± 1.01 93.28 ± 1.91
Sperm motility and motion kinematics are presented as mean ± SEM, n = 4. MOT = sperm motility (%); HYP = hyperactivated sperm (%), VCL = curvilinear velocity (μm/s); VSL = straight-line velocity (μm/s); VAP = average path velocity (μm/s); ALH = mean amplitude of head lateral displacement (μm). *P < 0.05.
2.94 ± 0.28 82.56 ± 2.45 82.02 ± 2.71 58.19 ± 2.55* 66.69 ± 1.85 124.24 ± 4.81*
Before capacitation Before capacitation Before capacitation After capacitation
After capacitation
VCL
Before capacitation
Student’s two-tailed t-test using the SPSS software (Version 25.0, IBM, Armonk, NY, USA) was conducted to compare between BC and AC groups. Each experiment was carried out at least 3 times. Significant differences were defined as the confidence level if P values were < 0.05. Numerical data are represented as means ± SEM.
HYP
2.8. Statistical analysis
MOT
Table 1 Sperm motility and motion kinematics BC (Before capacitation) and AC (After capacitation).
After capacitation
VSL
Expression levels of Rab proteins and Rab protein regulators were evaluated by Western blot analysis. The samples were washed two times with DPBS and centrifuged at 10,000× g for 5 min. Modified Laemmli sample buffer (315 mM Tris, 10% glycerol, 10% SDS, 5% 2mercaptoethanol, 5% bromophenol blue, HPLC) was used to re-suspend the samples; the samples were then incubated at RT for 10 min and then heated at 95 °C for 3 min. Subsequently, the samples were segregated according to molecular weight using 12% SDS-PAGE gel (MiniPROTEIN Tetra Cell, Bio-Rad, USA). The separated proteins were transferred onto PVDF membranes (Bio-Rad, USA). Samples were blocked by incubating at RT for 2 h after addition of 5% skim milk (Becton Dickinson and Company, Franklin Lakes, NJ, USA) as the blocking solution. The following Rab proteins were detected in the study samples: Rab 2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, Rab34, RabGEF, RabGDI, and RabGAP. Moreover, antibodies (Supplementary Table 1) were diluted with 5% skim milk at a ratio of 1:1000 and incubated overnight at 4 °C. Goat anti-mouse IgG H&L (HRP) was used as a secondary antibody for Rab27A and diluted at a ratio of 1:2000 with 5% skim milk. Goat anti-rabbit IgG H&L (HRP) was used as a secondary antibody for Rab2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, and Rab34. It was diluted with 5% skim milk at a ratio of 1:1000 and incubated at RT for 2 h. After 2 h, PVDF membranes were washed three times with DPBS. The membranes were stripped and re-probed with anti-α-tubulin antibody (Abcam) (1:5000) and goat anti-mouse IgG H&L (HRP) that was used as an internal control. Proteins were detected by ImageQuant LAS 500 (GE Healthcare, Chicago, IL, USA) by employing the enhanced chemiluminescence (ECL) technique using ECL reagents. Then Image Studio Lite (Version 5.0, LI-COR Corporate, Lincoln, NE, USA) was used for analysis. The bands were evaluated according to the ratios of α-tubulin for each treatment.
98.40 ± 9.39
2.7. Western blot analysis
Before capacitation
After capacitation
VAP
After capacitation
ALH
To evaluate the existence and expression of Rab proteins in spermatozoa, immunocytochemistry was performed. The BC and AC samples were smeared on slides and dried. The sample was fixed with 3.7% paraformaldehyde (PFA) (7.4% PFA 0.5 mL/mL; 50%, 10 × PBS 0.1 mL/mL; 10%, HPLC 0.4 mL/mL; 40%) at 4 °C for 30 min and washed two times with PBS-T. Subsequently, blocking solution (5% BSA in PBS-T) was treated at 37 °C for 1 h. After blocking, anti-Rab antibodies (Abcam, Cambridge, UK) (1:200), diluted using blocking solution (5% BSA in PBS-T), were treated with lectin peanut agglutinin (PNA) conjugates Alexa Fluor 647 (Molecular Probes, Eugene, OR, USA), diluted using blocking solution (1:200), at 4 °C for 1 day. Anti-rabbit IgG FITC conjugated antibody (Abcam) (1:200) was used as a secondary antibody. The samples were stored with secondary antibody at RT for 2 h and then mounted with VectaShield mounting media including 4’,6’diamidino-2-phenylindole (DAPI). Marked cells were observed using Nikon TS-1000 microscope that employed NIS Elements image software (Nikon, Tokyo, Japan) under 600 × magnification [23,24].
36.23 ± 3.51*
2.6. Immunocytochemistry
Before capacitation
After capacitation
no fluorescence over the head) (Fig. 1A–D). Finally, ratio of capacitation status were evaluated following patterns (F, B, and AR) in total live spermatozoa without dead spermatozoa.
4.13 ± 0.21*
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Fig. 2. Localization and expression of Rab proteins before and after capacitation in mouse spermatozoa. (A1, B1, C1 D1, E1, F1,G1, H1, I1, J1, and K1) are images of Rab2A, 3A, 4, 5, 8A, 9, 11, 14, 25, 27A, and 34A before capacitation, respectively (green). (A2, B2, C2 D2, E2, F2, G2, H2, I2, J2, and K2) are merged images of the nucleus (DAPI, blue) and acrosome (lectin PNA, red) for Rab2A, 3A, 4, 5, 8A, 9, 11, 14, 25, 27A, and 34A proteins before capacitation, respectively (green). (A3, B3, C3 D3, E3, F3, G3, H3, I3, J3, and K3) are images of Rab2A, 3A, 4, 5, 8A, 9, 11, 14, 25, 27A, and 34A after capacitation, respectively (green). (A4, B4, C4, D4, E4, F4, G4, H4, I4, J4, and K4) are merged image of nucleus (DAPI, blue) and acrosome (lectin PNA, red) for the abovementioned Rab proteins after capacitation, respectively (green). Images were obtained using a Nikon TS-1000 microscope and NIS Elements imaging software (Nikon, Japan). Bar =15 μm.
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3. Results
4. Discussion
3.1. Differences in sperm motility, and motion kinematics before and after capacitation
Rab proteins are monomeric GTPase belonging to the Ras superfamily. They are well-known key regulators of intracellular vesicular transport and membrane trafficking in eukaryotic cells [1,2]. To date, approximately 70 Rab proteins have been found in humans [3–7]. In eukaryotic cells, Rab proteins are present in two states which regulate cell activity, namely guanine triphosphate (GTP)-bound state (active state) and guanine diphosphate (GDP)-bound state (inactive state); these states are alternatively induced. In the active stage, guanine nucleotide exchange factor (GEF) and GDP dissociation inhibitor (GDI) influence the levels of G proteins. In contrast, GTPase-activating protein (GAP) convert the “active” state to “inactive” state [26,27]. In spermatozoa, Rab proteins are involved in Golgi trafficking and play a role in the formation and maintenance of Golgi apparatus. [2]. Golgi apparatus is a crucial organelle that derive acrosomal regions in the sperm head via spermatogenesis [9–11]. In previous studies, Rab 2A and 3A were reported to be related to acrosomal formation and exocytosis in spermatozoa; furthermore, Rab 2A can be used as a fertility-related biomarker in males [20–22]. Even though the previous study reported that Rab proteins are key proteins in spermatozoa, their mechanisms and roles have not yet been fully understood. Therefore, in the present study, we tracked the expression patterns of Rab proteins and Rab protein regulators in spermatozoa following capacitation. The ejaculated spermatozoa have to reach their full capacity for fertilization [12,13]. Sperm motion kinematics changes during capacitation. Finally, acrosomal exocytosis occurs in capacitated spermatozoa. This is called the acrosomal reaction, in which several subfamily proteins of Rab protein family are involved [16–18]. In the present study, to evaluate the expression patterns of Rab proteins and Rab protein regulators following capacitation, capacitation was induced in vitro according to previously described methods [23–25]. Subsequently, CASA and H33258/CTC were performed to evaluate sperm motility, motion kinematics, and capacitation status to validate capacitation. In present study, we selected the 11 Rab proteins among 70
Sperm motility and VAP did not present significant capacitationbased difference. However, HYP, VCL and ALH increased but VSL decreased after capacitation (P < 0.05) (Table 1). In addition, in the AC group, AR (acrosome reacted) and B (capacitated) patterns were significantly increased (P < 0.05) (Fig. 1E), whereas F pattern (non-capacitated) was significantly decreased (P < 0.05) (Fig. 1E). 3.2. Localization of Rab proteins in spermatozoa As a result of immunocytochemistry, Rab proteins signals in spermatozoa were indicated by FITC (Green) and nuclear spermatozoa were stained by DAPI (Blue). Lectin PNA (Red) stained the acrosomal region of spermatozoa following capacitation (Fig. 2). Rab proteins were localized in the head and tail of spermatozoa (Fig. 2). Especially, the signal of Rab2A, 4, 8A, 11, and 27A in midpiece were stronger than principal piece. The signal of Rab25 has strong signal in principal piece. However, the other Rab proteins were localized in entire sperm tail. All Rab proteins signal were reduced in capacitated spermatozoa (Fig. 2). 3.3. Correlations between the expression level of Rab proteins in spermatozoa and capacitation status To evaluate the expression of Rab proteins in spermatozoa, western blot analysis was performed. Rab2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, and Rab34 were detected as bands of 25 kDa, 25 kDa, 26 kDa, 26 kDa, 24 kDa, 23 kDa, 22 kDa, 24 kDa, 32 kDa, 25 kDa, and 29 kDa, respectively. RabGAP, RabGDI, and RabGEF (Rab protein regulators) were detected as bands of 130 kDa, 55 kDa and 57 kDa, respectively. After capacitation, the expression of all Rab proteins and Rab protein regulators, except Rab GAP, was significantly decreased (P < 0.05) (Fig. 3, Supplementary Fig. 1).
Fig. 3. The expression of Rab proteins before and after capacitation in mouse spermatozoa. (A) Ratios of Rab2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, Rab34, RabGAP, RabGDI, and RabGEF (optical density [OD × mm]/α-tubulin [OD × mm]) in spermatozoa belonging to both BC and AC groups. (Navy bar: Before capacitation, diagonal line bar: After capacitation). (B) The Rab2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, Rab34, RabGAP, RabGDI, and RabGEF were probed with anti-Rab 2A, anti-Rab3A, anti-Rab4, anti-Rab5, anti-Rab8A, anti-Rab9, anti-Rab11, anti-Rab14, anti-Rab25, anti-Rab27A, anti-Rab34, anti-RabGAP, anti-RabGDI, and anti-RabGEF antibodies. Data represent the mean ± SEM, at least 3 replicates were analyzed (*P < 0.05). 5
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Fig. 4. Hypothetical illustration demonstrates the activation mechanism of Ras-related proteins in spermatozoa. Activation model of Ras-related protein in sperm cell: Ras-related protein maintains inactive condition under inhibition of GDP dissociation by RabGDI. When GDP bound with Ras-related protein convert to GTP by RabGEF, the Ras-related protein can be activated. However, activated Ras-related protein cannot convert to inactive condition because RabGAP did not properly work in sperm cell after capacitation. GTP: guanine triphosphate, GDP: guanine diphosphate, RabGEF: guanine nucleotide exchange factor, RabGDI: GDP dissociation inhibitor, RabGAP GTPase-activating protein.
GEF, that exchange GDP to GTP, were used drastically, but in GAP, which induce an inactivate Rab proteins to activate status, there was no significant difference (Fig. 3). This result suggested that in contrast to the somatic cells, Rab proteins in spermatozoa may carry out an irreversible pathway and not return to the inactivated state after capacitation (Figs. 3 and 4). This is the first study to elucidate existence, location, and expression of Rab proteins in spermatozoa following capacitation as well as the mechanism for Rab protein regulation in spermatozoa. Taken together, our data revealed that various Rab proteins existed in the head and tail of spermatozoa. Furthermore, reduction in Rab protein expression was observed after capacitation. Finally, the expression of Rab protein regulators were reduced after capacitation, regardless of the levels of GAP. Therefore, we suggest that Rab proteins play a key role related to male fertility by their unique activation mechanism in spermatozoa. Moreover, we anticipate that the Rab proteins can be used as fertility related biomarkers for prognosis and diagnosis of male fertility. To this, in-depth studies are required to understand the functional mechanism of Rab proteins.
different types Rab proteins. Rab2A, Rab3A, and Rab 27A are proved to exist in spermatozoa based on references [15–22] and another 8 Rab proteins (Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, and Rab34) were randomly selected. The study related to 8 Rab proteins (Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, and Rab34) in spermatozoa are not performed yet. Existence, location, and expression of Rab proteins in spermatozoa were evaluated via immunocytochemistry and western blot analysis in the present study. It was confirmed that a total 11 Rab proteins (Rab2A, 3A, 4, 5, 8A, 9, 11, 14, 25, 27A, and 34A) were present in the acrosomal region and tail of spermatozoa (Fig. 2). Interestingly, expression levels of all Rab proteins significantly decreased after capacitation (Figs. 2 and 3, Supplementary Fig. 1). Previously, it has been reported that Rab 3 and Rab 27 were localized in the membranes rather than the cytosol of human spermatozoa [18]. According to that report, reduction in Rab protein levels after capacitation may be owing to the occurrence of acrosomal exocytosis which causes dislocation of the proteins from sperm membranes. However, in the present study, the expression levels of Rab proteins were reduced under an increase in the percent of acrosome-reacted spermatozoa as well as capacitated spermatozoa. (Figs. 1A and 3). Moreover, the signal of Rab proteins were reduced in sperm tails after capacitation (Fig. 2). It has been suggested that alteration in the expression levels of Rab proteins is not only caused by acrosomal reaction. However, more studies are needed to validate this issue. Furthermore, the expression levels of Rab protein regulators such as RabGAP, RabGDI, and RabGEF were also evaluated. In most eukaryotic cells, Rab proteins are well-known to be the key regulators of intracellular vesicular transport owing to their ability to switch between the inactive (GDP-bound) and active (GTP-bound) forms [1–3]. It has been known that Rab proteins regulate membrane fusion in most eukaryotic cells. To perform this event, various Rab proteins regulators (RabGAP, RabGDI, and RabGEF) perform their roles organically. GDP binds to inactivated Rab proteins by converting RabGDI to GTP; the Rab proteins are subsequently activated by RabGEF. Simultaneously, membrane fusion occurs by effector proteins. Eventually, RabGAP stimulates hydrolysis of bound GTP and converts it to GDP, owing to which Rab proteins return to the inactive state [1,2,5,6,26–30]. In the present study, the levels of RabGDI and RabGEF were significantly reduced after capacitation. However, there was no significant difference in the level of RabGAP. In our findings, GDI, which suppress GDP, and
Authors’ roles J.W.B., S.H.K. D.H.K, J.J.H, J.K.Y., S.H., B.Y.R., M.G.P., and W.S.K. performed the experiments, analyzed the data, and drafted the manuscript. W.S.K. supervised the critical study design and data analysis as well as revised the manuscript. All authors contributed toward revisions that were critical to the intellectual content and approved the final version for publication.
Declaration of Competing Interest The authors have declared that no competing interests exist.
Acknowledgement This research was supported by Kyungpook National University Research Fund, 2017. 6
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Appendix A. Supplementary data
[15] Mountjoy JR, Xu W, McLeod D, Hyndman D, Oko R. RAB2A: a major subacrosomal protein of bovine spermatozoa implicated in acrosomal biogenesis. Biol Reprod 2008;79(2):223–32. [16] Lopez CI, Belmonte SA, De Blas GA, Mayorga LS. Membrane-permeant Rab3A triggers acrosomal exocytosis in living human sperm. FASEB J 2007;21(14):4121–30. [17] Ruete MC, Lucchesi O, Bustos MA, Tomes CN. Epac, Rap and Rab3 act in concert to mobilize calcium from sperm’s acrosome during exocytosis. Cell Commun Signal 2014;12:43. [18] Bustos MA, Lucchesi O, Ruete MC, Mayorga LS, Tomes CN. Rab27 and Rab3 sequentially regulate human sperm dense-core granule exocytosis. Proc Natl Acad Sci U S A 2012;109(30):E2057–66. [19] Yunes R, Michaut M, Tomes C, Mayorga LS. Rab3A triggers the acrosome reaction in permeabilized human spermatozoa. Biol Reprod 2000;62(4):1084–9. [20] Kwon WS, Rahman MS, Lee JS, Kim J, Yoon SJ, Park YJ, et al. A comprehensive proteomic approach to identifying capacitation related proteins in boar spermatozoa. BMC Genomics 2014;15:897. [21] Kwon WS, Rahman MS, Lee JS, Yoon SJ, Park YJ, Pang MG. Discovery of predictive biomarkers for litter size in boar spermatozoa. Mol Cell Proteomics 2015;14(5):1230–40. [22] Kwon WS, Rahman MS, Ryu DY, Park YJ, Pang MG. Increased male fertility using fertility-related biomarkers. Sci Rep 2015;5:15654. [23] Kwon WS, Park YJ, Kim YH, You YA, Kim IC, Pang MG. Vasopressin effectively suppresses male fertility. PLoS One 2013;8:e54192. [24] Kwon WS, Park YJ, Mohamed el-SA, Pang MG. Voltage-dependent anion channels are a key factor of male fertility. Fertil Steril 2013;99(2):354–61. [25] Kwon WS, Kim YJ, Ryu DY, Kwon KJ, Song WH, Rahman MS, et al. Fms-like tyrosine kinase 3 is a key factor of male fertility. Theriogenology 2019;126:145–52. [26] Pfeffer SR, Dirac-Svejstrup AB, Soldati T. Rab GDP dissociation inhibitor: putting rab GTPases in the right place. J Biol Chem 1995;270(29):17057–9. [27] Goody PR. Intrinsic protein fluorescence assays for GEF, GAP and post-translational modifications of small GTPases. Anal Biochem 2016;51:22–5. [28] Schalk I, Zeng K, Wu SK, Stura EA, Matteson J, Huang M, et al. Structure and mutational analysis of Rab GDP-dissociation inhibitor. Nature 1996;381(6577):4–28. [29] Oesterlin LK, Goody RS, Itzen A. Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor. Proc Natl Acad Sci U S A 2012;109(15):5621–6. [30] Gavriljuk K, Itzen A, Goody RS, Gerwert K, Kötting C. Membrane extraction of Rab proteins by GDP dissociation inhibitor characterized using attenuated total reflection infrared spectroscopy. Proc Natl Acad Sci U S A 2013;110(33):13380–5.
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.repbio.2019.10.001. References [1] Wu YW, Oesterlin LK, Tan KT, Waldmann H, Alexandrov K, Goody RS. Membrane targeting mechanism of Rab GTPases elucidated by semisynthetic protein probes. Nat Chem Biol 2010;6(7):534–40. [2] Goody RS, Müller MP, Wu YW. Mechanisms of action of Rab proteins, key regulators of intracellular vesicular transport. Biol Chem 2017;398(5–6):565–75. [3] Pereira-Leal JB, Seabra MC. The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily. J Mol Biol 2000;301(4):1077–87. [4] Alory C, Balch WE. Organization of the Rab-GDI/CHM superfamily: the functional basis for choroideremia disease. Traffic 2001;2(8):532–43. [5] Stenmark H. Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 2009;10(8):513–25. [6] Hutagalung AH, Novick PJ. Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 2011;91(1):119–49. [7] Goud B, Liu S, Storrie B. Rab proteins as major determinants of the Golgi complex structure. Small GTPases 2018;9(1-2):66–75. [8] Liu S, Storrie B. Are Rab proteins the link between Golgi organization and membrane trafficking? Cell Mol Life Sci 2012;69(24):4093–106. [9] Smith CE, Hermo L, Fazel A, Lalli MF, Bergeron JJ. Ultrastructural distribution of NADPase within the Golgi apparatus and lysosomes of mammalian cells. Prog Histochem Cytochem 1990;21(3):1–120. [10] Clermont Y, Oko R, Hermo L. Cell and molecular biology of the testis. In: Desjardins C, Ewing L, editors. Cell biology of mammalian spermatogenesis. New York: Oxford University Press; 1993. p. 332–76. [11] Oko R, Clermont Y. Spermiogenesis. Knobil E, Neill JD, editors. Encyclopedia of reproduction, 4. San Diego: Academic Press; 1998. p. 602–9. [12] Austin CR. Observations on the penetration of the sperm in the mammalian egg. Aust J Sci Res B 1951;4(4):581–96. [13] Chang MC. Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 1951;168(4277):697–8. [14] Peterson RN, Bozzola J, Polakoski K. Protein transport and organization of the developing mammalian sperm acrosome. Tissue Cell 1992;24(1):1–15.
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Reproductive Biology journal homepage: www.elsevier.com/locate/repbio
Original article
Ras-related proteins (Rab) are key proteins related to male fertility following a unique activation mechanism Jeong-Won Baea, So-Hye Kima, Dae-Hyun Kimb, Jae Jung Hab, Jun Koo Yib, Seongsoo Hwangc, ⁎ Buom-Yong Ryud, Myung-Geol Pangd, Woo-Sung Kwona,e, a
Department of Animal Science and Biotechnology, Kyungpook National University, Sangju, Gyeongsangbuk-do 37224, Republic of Korea Gyeongbuk Livestock Research Institute, Yeongju Gyeongsangbuk-do 36052, Republic of Korea c Animal Biotechnology Division, National Institute of Animal Science, RDA, Wanju-gun, Jeollabuk-do 55365, Republic of Korea d Department of Animal Science & Technology, Chung-Ang University, Anseong, Gyeonggi-do 456-756, Republic of Korea e Department of Animal Biotechnology, Kyungpook National University, Sangju, Gyeongsangbuk-do 37224, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Rab proteins Spermatozoa Capacitation Acrosome reaction Male fertility
Ras-related protein Rab (Rab) proteins, member of Ras superfamily of monomeric G proteins, are well known key regulators of intracellular vesicular transport. Recently, it has been reported that Rab 2A and 3A are related to acrosomal exocytosis in spermatozoa and Rab 2A can be used to fertility-related biomarker in male. However, the role and mechanism of Rab proteins in spermatozoa has not been fully understood yet. Therefore, the study to analyze the expression and location of various Rab proteins in spermatozoa is required to understand the role and mechanism of Rab proteins in spermatozoa. In present study, to analyze the expression level and location of various Rab proteins (Rab 2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, and Rab34) and Rab protein regulators (RabGAP, RabGDI, RabGEF) in spermatozoa following capacitation, immunofluorescence and western blot analysis were performed. All of 11 Rab proteins were expressed in acrosomal region and tail of spermatozoa. Furthermore, all Rab proteins and Rab protein regulators, except RabGAP, have decreased expression patterns after capacitation. Taken together, Rab proteins were located in sperm head and tail. In addition, expression patterns of Rab proteins in spermatozoa were altered following capacitation. Therefore, our results suggested that Rab proteins may be key proteins related with capacitation as well as playing important role with uniquely activation pathway for male fertility.
1. Introduction Ras-related proteins (Rab), which are members of Ras superfamily of monomeric G proteins, are well-known key regulators of intracellular vesicular transport [1,2]. To date, approximately 70 different types of Rab proteins that perform various roles related to membrane trafficking in eukaryotic cells, such as vesicle formation, vesicle movement, and membrane fusion, have been identified [3–7]. In addition, they regulate various vesicular traffic steps related to the biogenesis of transport carriers for moving the cytoskeleton and facilitate binding with target membranes by interacting with effectors such as molecular motors, connecting complexes, scaffolding proteins, and lipid kinases [5,4–7]. It has been reported that certain Rab proteins play a key role in establishing and preserving the Golgi structure by regulating Golgi trafficking [8]. Especially, the Golgi apparatus is an important organelle
in the primordial cells of spermatozoa because the Golgi apparatus derive to acrosome in the sperm head during spermatogenesis [9–11]. To penetrate into the oocyte, the sperm cells have to undergo a special event called “capacitation” during migration through the oviduct tract [12,13]. Subsequently, only acrosome-reacted spermatozoa can fuse with oocytes after capacitation. It has been reported that various proteins contribute to acrosome formation during spermatogenesis [14]. Various studies have reported that certain Rab proteins are key factors for critical mechanisms including acrosomal biogenesis and acrosomal reaction [15–18]. Previous researchers have reported that Rab2A and Rab3A are related to acrosomal exocytosis following capacitation [15,19,20]; some of those researchers also suggested that Rab2A can be used as a fertility-related biomarker because Rab2A expression was altered following male fertility [21,22]. Although various studies related to Rab proteins have been
⁎ Corresponding author at: Department of Animal Science and Biotechnology and Department of Animal Biotechnology, Kyungpook National University, Sangju, Gyeongsangbuk-do 37224, Republic of Korea. E-mail address: [email protected] (W.-S. Kwon).
https://doi.org/10.1016/j.repbio.2019.10.001 Received 19 August 2019; Received in revised form 30 September 2019; Accepted 2 October 2019 1642-431X/ © 2019 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Jeong-Won Bae, et al., Reproductive Biology, https://doi.org/10.1016/j.repbio.2019.10.001
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divided into two groups: before capacitation (BC) and after capacitation (AC). The AC group was incubated for 90 more minutes at 37 °C under 0.5% CO2, in accordance with several previous studies [23–25]. The BC group was used for further experimentation without any additional incubation.
conducted, the roles and mechanism of Rab proteins in spermatozoa have not been fully understood. Therefore, this study was conducted to analyze the expression and location of various Rab proteins and activation process of Rab proteins in spermatozoa. In this study, the location of Rab proteins present in spermatozoa were identified by immunocytochemistry, and their expression levels before and after capacitation were evaluated by western blotting. Finally, the expression levels of various Rab protein regulators such as Rab GTPase-activating protein (GAP), Rab GDP-dissociation inhibitor (GDI), and Rab GTP exchange factor (GEF) were analyzed by western blotting.
2.4. Computer-assisted sperm analysis Ten microliters of each sample group were placed in a Makler counting chamber (Sefi-Medical Instruments, Haifa, Israel) which was heated to 37 °C. Total sperm motility (%) and motion kinematics [VCL, curvilinear velocity (μm/s); VSL, straight-line velocity (μm/s); VAP, average path velocity (μm/s); ALH, mean amplitude of head lateral displacement (μm)] were measured using 10 × objective phase-contrast mode. To measure the motility and motion kinematics, computerassisted sperm analysis program (CASA) (FSA2016, Medical supply, Seoul, Korea), OLYMPUS BX43 phase-contrast microscope (Olympus, Tokyo, Japan), CMOS, CAMERA, 2048 × 1536 (300 M pixel), and 60 Frame (Medical supply, Seoul, Korea) were used.
2. Materials and methods 2.1. Ethical statement All procedures were performed in accordance with the guidelines for the ethical treatment of animals and approved by Institutional Animal Care and Use Committee of Kyungpook National University. 2.2. Chemicals and media
2.5. Combined H33258/chlortetracycline fluorescence (H33258/CTC) assessment of spermatozoa
All reagents were purchased from Sigma-Aldrich (St Louis, MO, USA). We used Modified Tyrode’s medium as the basic medium (BM) (97.84 mM NaCl, 1.42 mM KCl, 0.47 mM MgCl2·6H2O, 0.36 mM NaH2PO4·H2O, 5.56 mM D-glucose, 25 mM NaHCO3, 1.78 mM CaCl2.2H2O, 24.9 mM Na-lactate, 0.47 mM Na-pyruvate, 50 μg/ml gentamycin, and 0.005 mM phenol red). For capacitation, BM was prepared with 0.4% bovine serum albumin 1 day prior to the experiment [23,24]. The basic media was maintained at a pH of 7.2 ± 0.2 [25].
To determine whether or not spermatozoa has become capacitated, dual staining method (Combined Hoechst 33258/chlortetracycline fluorescence assessment) was employed [23–25]. Spermatozoa in both BC and AC groups were centrifuged at 100× g for 2.5 min. Except in 135 μL samples, in all other samples, supernatant was removed. In the 135 μL sample, 15 μL of H33258 solution (10 μg H33258/mL DPBS) was added and incubated for 2 min at RT. After 2 min, 250 μL of 2% (w/v) polyvinylpyrrolidone in DPBS was added and the sample was centrifuged at 100×g for 2.5 min. The supernatant liquid was fully removed from centrifuged samples and the samples were resuspended in 100 μL chlortetracycline fluorescence (CTC) solution (750 mM CTC in 5 μL buffer: 20 mM Tris, 130 mM NaCl, and 5 mM cysteine, pH 7.4) and 100 μL DPBS. After the samples were refrigerated for 20 min under dark conditions, 10 μL of each sample was smeared on the slides. Finally, at least 400 spermatozoa per slide for each sample were evaluated using OLYMPUS BX43 under epifluorescence illumination using ultraviolet BP 340–380/LP 425 and BP 450–490/LP 515 excitation/emission filters for H33258 and CTC, respectively (Olympus, Tokyo, Japan). According to the capacitation status, spermatozoa were divided into four groups: dead (D pattern, blue fluorescence), live non-capacitated (F pattern, bright green fluorescence distributed uniformly over entire sperm head), live capacitated (B pattern, green fluorescence over the acrosomal region and a dark postacrosome), and live acrosome-reacted (AR pattern, sperm showing a mottled green fluorescence over the head or
2.3. Preparation and treatment of spermatozoa Eight to twelve-week-old mature ICR male mice were used (Nara Biotech, Seoul, Korea). The mice were housed individually and maintained at a temperature of 25 ± 2 °C under optimal humidity and light (12-h light/dark) conditions. All mice were provided ad-libitum feeding (Cargil Agripurina, Seongnam, Korea) and water. Mice were sacrificed by cervical dislocation, and their epididymis was collected and dissected to isolate the cauda epididymis. The cauda epididymis, which was trimmed to eliminate fat content, was further used to extract spermatozoa. Using a 1 mL syringe attached to a 26 G needle, spermatozoa was extracted in 35 mm-diameter sterile cell culture dishes containing BM with 0.4% BSA. To facilitate release of the extracted spermatozoa, they were incubated at 37 °C under 0.5% CO2 for 12 min. After releasing, to rule out individual variation, each extracted spermatozoa from 3 mice was pooled. Then the spermatozoa were
Fig. 1. Capacitation status in before capacitation (BC) and after capacitation (AC) groups of spermatozoa. (A) Patterns of live non-capacitated (F pattern). (B) Patterns of live capacitated (B pattern). (C) Patterns of live acrosome reacted (AR pattern). (D) Patterns of dead (D pattern). (E) Proportion of capacitation status in live spermatozoa in the two groups. Data represent mean ± SEM, n = 6. Superscript (*) indicates significant difference between before and after capacitation groups as tested by one-way ANOVA (P < 0.05). 2
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2.97 ± 0.42 94.53 ± 1.01 93.28 ± 1.91
Sperm motility and motion kinematics are presented as mean ± SEM, n = 4. MOT = sperm motility (%); HYP = hyperactivated sperm (%), VCL = curvilinear velocity (μm/s); VSL = straight-line velocity (μm/s); VAP = average path velocity (μm/s); ALH = mean amplitude of head lateral displacement (μm). *P < 0.05.
2.94 ± 0.28 82.56 ± 2.45 82.02 ± 2.71 58.19 ± 2.55* 66.69 ± 1.85 124.24 ± 4.81*
Before capacitation Before capacitation Before capacitation After capacitation
After capacitation
VCL
Before capacitation
Student’s two-tailed t-test using the SPSS software (Version 25.0, IBM, Armonk, NY, USA) was conducted to compare between BC and AC groups. Each experiment was carried out at least 3 times. Significant differences were defined as the confidence level if P values were < 0.05. Numerical data are represented as means ± SEM.
HYP
2.8. Statistical analysis
MOT
Table 1 Sperm motility and motion kinematics BC (Before capacitation) and AC (After capacitation).
After capacitation
VSL
Expression levels of Rab proteins and Rab protein regulators were evaluated by Western blot analysis. The samples were washed two times with DPBS and centrifuged at 10,000× g for 5 min. Modified Laemmli sample buffer (315 mM Tris, 10% glycerol, 10% SDS, 5% 2mercaptoethanol, 5% bromophenol blue, HPLC) was used to re-suspend the samples; the samples were then incubated at RT for 10 min and then heated at 95 °C for 3 min. Subsequently, the samples were segregated according to molecular weight using 12% SDS-PAGE gel (MiniPROTEIN Tetra Cell, Bio-Rad, USA). The separated proteins were transferred onto PVDF membranes (Bio-Rad, USA). Samples were blocked by incubating at RT for 2 h after addition of 5% skim milk (Becton Dickinson and Company, Franklin Lakes, NJ, USA) as the blocking solution. The following Rab proteins were detected in the study samples: Rab 2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, Rab34, RabGEF, RabGDI, and RabGAP. Moreover, antibodies (Supplementary Table 1) were diluted with 5% skim milk at a ratio of 1:1000 and incubated overnight at 4 °C. Goat anti-mouse IgG H&L (HRP) was used as a secondary antibody for Rab27A and diluted at a ratio of 1:2000 with 5% skim milk. Goat anti-rabbit IgG H&L (HRP) was used as a secondary antibody for Rab2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, and Rab34. It was diluted with 5% skim milk at a ratio of 1:1000 and incubated at RT for 2 h. After 2 h, PVDF membranes were washed three times with DPBS. The membranes were stripped and re-probed with anti-α-tubulin antibody (Abcam) (1:5000) and goat anti-mouse IgG H&L (HRP) that was used as an internal control. Proteins were detected by ImageQuant LAS 500 (GE Healthcare, Chicago, IL, USA) by employing the enhanced chemiluminescence (ECL) technique using ECL reagents. Then Image Studio Lite (Version 5.0, LI-COR Corporate, Lincoln, NE, USA) was used for analysis. The bands were evaluated according to the ratios of α-tubulin for each treatment.
98.40 ± 9.39
2.7. Western blot analysis
Before capacitation
After capacitation
VAP
After capacitation
ALH
To evaluate the existence and expression of Rab proteins in spermatozoa, immunocytochemistry was performed. The BC and AC samples were smeared on slides and dried. The sample was fixed with 3.7% paraformaldehyde (PFA) (7.4% PFA 0.5 mL/mL; 50%, 10 × PBS 0.1 mL/mL; 10%, HPLC 0.4 mL/mL; 40%) at 4 °C for 30 min and washed two times with PBS-T. Subsequently, blocking solution (5% BSA in PBS-T) was treated at 37 °C for 1 h. After blocking, anti-Rab antibodies (Abcam, Cambridge, UK) (1:200), diluted using blocking solution (5% BSA in PBS-T), were treated with lectin peanut agglutinin (PNA) conjugates Alexa Fluor 647 (Molecular Probes, Eugene, OR, USA), diluted using blocking solution (1:200), at 4 °C for 1 day. Anti-rabbit IgG FITC conjugated antibody (Abcam) (1:200) was used as a secondary antibody. The samples were stored with secondary antibody at RT for 2 h and then mounted with VectaShield mounting media including 4’,6’diamidino-2-phenylindole (DAPI). Marked cells were observed using Nikon TS-1000 microscope that employed NIS Elements image software (Nikon, Tokyo, Japan) under 600 × magnification [23,24].
36.23 ± 3.51*
2.6. Immunocytochemistry
Before capacitation
After capacitation
no fluorescence over the head) (Fig. 1A–D). Finally, ratio of capacitation status were evaluated following patterns (F, B, and AR) in total live spermatozoa without dead spermatozoa.
4.13 ± 0.21*
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Fig. 2. Localization and expression of Rab proteins before and after capacitation in mouse spermatozoa. (A1, B1, C1 D1, E1, F1,G1, H1, I1, J1, and K1) are images of Rab2A, 3A, 4, 5, 8A, 9, 11, 14, 25, 27A, and 34A before capacitation, respectively (green). (A2, B2, C2 D2, E2, F2, G2, H2, I2, J2, and K2) are merged images of the nucleus (DAPI, blue) and acrosome (lectin PNA, red) for Rab2A, 3A, 4, 5, 8A, 9, 11, 14, 25, 27A, and 34A proteins before capacitation, respectively (green). (A3, B3, C3 D3, E3, F3, G3, H3, I3, J3, and K3) are images of Rab2A, 3A, 4, 5, 8A, 9, 11, 14, 25, 27A, and 34A after capacitation, respectively (green). (A4, B4, C4, D4, E4, F4, G4, H4, I4, J4, and K4) are merged image of nucleus (DAPI, blue) and acrosome (lectin PNA, red) for the abovementioned Rab proteins after capacitation, respectively (green). Images were obtained using a Nikon TS-1000 microscope and NIS Elements imaging software (Nikon, Japan). Bar =15 μm.
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3. Results
4. Discussion
3.1. Differences in sperm motility, and motion kinematics before and after capacitation
Rab proteins are monomeric GTPase belonging to the Ras superfamily. They are well-known key regulators of intracellular vesicular transport and membrane trafficking in eukaryotic cells [1,2]. To date, approximately 70 Rab proteins have been found in humans [3–7]. In eukaryotic cells, Rab proteins are present in two states which regulate cell activity, namely guanine triphosphate (GTP)-bound state (active state) and guanine diphosphate (GDP)-bound state (inactive state); these states are alternatively induced. In the active stage, guanine nucleotide exchange factor (GEF) and GDP dissociation inhibitor (GDI) influence the levels of G proteins. In contrast, GTPase-activating protein (GAP) convert the “active” state to “inactive” state [26,27]. In spermatozoa, Rab proteins are involved in Golgi trafficking and play a role in the formation and maintenance of Golgi apparatus. [2]. Golgi apparatus is a crucial organelle that derive acrosomal regions in the sperm head via spermatogenesis [9–11]. In previous studies, Rab 2A and 3A were reported to be related to acrosomal formation and exocytosis in spermatozoa; furthermore, Rab 2A can be used as a fertility-related biomarker in males [20–22]. Even though the previous study reported that Rab proteins are key proteins in spermatozoa, their mechanisms and roles have not yet been fully understood. Therefore, in the present study, we tracked the expression patterns of Rab proteins and Rab protein regulators in spermatozoa following capacitation. The ejaculated spermatozoa have to reach their full capacity for fertilization [12,13]. Sperm motion kinematics changes during capacitation. Finally, acrosomal exocytosis occurs in capacitated spermatozoa. This is called the acrosomal reaction, in which several subfamily proteins of Rab protein family are involved [16–18]. In the present study, to evaluate the expression patterns of Rab proteins and Rab protein regulators following capacitation, capacitation was induced in vitro according to previously described methods [23–25]. Subsequently, CASA and H33258/CTC were performed to evaluate sperm motility, motion kinematics, and capacitation status to validate capacitation. In present study, we selected the 11 Rab proteins among 70
Sperm motility and VAP did not present significant capacitationbased difference. However, HYP, VCL and ALH increased but VSL decreased after capacitation (P < 0.05) (Table 1). In addition, in the AC group, AR (acrosome reacted) and B (capacitated) patterns were significantly increased (P < 0.05) (Fig. 1E), whereas F pattern (non-capacitated) was significantly decreased (P < 0.05) (Fig. 1E). 3.2. Localization of Rab proteins in spermatozoa As a result of immunocytochemistry, Rab proteins signals in spermatozoa were indicated by FITC (Green) and nuclear spermatozoa were stained by DAPI (Blue). Lectin PNA (Red) stained the acrosomal region of spermatozoa following capacitation (Fig. 2). Rab proteins were localized in the head and tail of spermatozoa (Fig. 2). Especially, the signal of Rab2A, 4, 8A, 11, and 27A in midpiece were stronger than principal piece. The signal of Rab25 has strong signal in principal piece. However, the other Rab proteins were localized in entire sperm tail. All Rab proteins signal were reduced in capacitated spermatozoa (Fig. 2). 3.3. Correlations between the expression level of Rab proteins in spermatozoa and capacitation status To evaluate the expression of Rab proteins in spermatozoa, western blot analysis was performed. Rab2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, and Rab34 were detected as bands of 25 kDa, 25 kDa, 26 kDa, 26 kDa, 24 kDa, 23 kDa, 22 kDa, 24 kDa, 32 kDa, 25 kDa, and 29 kDa, respectively. RabGAP, RabGDI, and RabGEF (Rab protein regulators) were detected as bands of 130 kDa, 55 kDa and 57 kDa, respectively. After capacitation, the expression of all Rab proteins and Rab protein regulators, except Rab GAP, was significantly decreased (P < 0.05) (Fig. 3, Supplementary Fig. 1).
Fig. 3. The expression of Rab proteins before and after capacitation in mouse spermatozoa. (A) Ratios of Rab2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, Rab34, RabGAP, RabGDI, and RabGEF (optical density [OD × mm]/α-tubulin [OD × mm]) in spermatozoa belonging to both BC and AC groups. (Navy bar: Before capacitation, diagonal line bar: After capacitation). (B) The Rab2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, Rab34, RabGAP, RabGDI, and RabGEF were probed with anti-Rab 2A, anti-Rab3A, anti-Rab4, anti-Rab5, anti-Rab8A, anti-Rab9, anti-Rab11, anti-Rab14, anti-Rab25, anti-Rab27A, anti-Rab34, anti-RabGAP, anti-RabGDI, and anti-RabGEF antibodies. Data represent the mean ± SEM, at least 3 replicates were analyzed (*P < 0.05). 5
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Fig. 4. Hypothetical illustration demonstrates the activation mechanism of Ras-related proteins in spermatozoa. Activation model of Ras-related protein in sperm cell: Ras-related protein maintains inactive condition under inhibition of GDP dissociation by RabGDI. When GDP bound with Ras-related protein convert to GTP by RabGEF, the Ras-related protein can be activated. However, activated Ras-related protein cannot convert to inactive condition because RabGAP did not properly work in sperm cell after capacitation. GTP: guanine triphosphate, GDP: guanine diphosphate, RabGEF: guanine nucleotide exchange factor, RabGDI: GDP dissociation inhibitor, RabGAP GTPase-activating protein.
GEF, that exchange GDP to GTP, were used drastically, but in GAP, which induce an inactivate Rab proteins to activate status, there was no significant difference (Fig. 3). This result suggested that in contrast to the somatic cells, Rab proteins in spermatozoa may carry out an irreversible pathway and not return to the inactivated state after capacitation (Figs. 3 and 4). This is the first study to elucidate existence, location, and expression of Rab proteins in spermatozoa following capacitation as well as the mechanism for Rab protein regulation in spermatozoa. Taken together, our data revealed that various Rab proteins existed in the head and tail of spermatozoa. Furthermore, reduction in Rab protein expression was observed after capacitation. Finally, the expression of Rab protein regulators were reduced after capacitation, regardless of the levels of GAP. Therefore, we suggest that Rab proteins play a key role related to male fertility by their unique activation mechanism in spermatozoa. Moreover, we anticipate that the Rab proteins can be used as fertility related biomarkers for prognosis and diagnosis of male fertility. To this, in-depth studies are required to understand the functional mechanism of Rab proteins.
different types Rab proteins. Rab2A, Rab3A, and Rab 27A are proved to exist in spermatozoa based on references [15–22] and another 8 Rab proteins (Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, and Rab34) were randomly selected. The study related to 8 Rab proteins (Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, and Rab34) in spermatozoa are not performed yet. Existence, location, and expression of Rab proteins in spermatozoa were evaluated via immunocytochemistry and western blot analysis in the present study. It was confirmed that a total 11 Rab proteins (Rab2A, 3A, 4, 5, 8A, 9, 11, 14, 25, 27A, and 34A) were present in the acrosomal region and tail of spermatozoa (Fig. 2). Interestingly, expression levels of all Rab proteins significantly decreased after capacitation (Figs. 2 and 3, Supplementary Fig. 1). Previously, it has been reported that Rab 3 and Rab 27 were localized in the membranes rather than the cytosol of human spermatozoa [18]. According to that report, reduction in Rab protein levels after capacitation may be owing to the occurrence of acrosomal exocytosis which causes dislocation of the proteins from sperm membranes. However, in the present study, the expression levels of Rab proteins were reduced under an increase in the percent of acrosome-reacted spermatozoa as well as capacitated spermatozoa. (Figs. 1A and 3). Moreover, the signal of Rab proteins were reduced in sperm tails after capacitation (Fig. 2). It has been suggested that alteration in the expression levels of Rab proteins is not only caused by acrosomal reaction. However, more studies are needed to validate this issue. Furthermore, the expression levels of Rab protein regulators such as RabGAP, RabGDI, and RabGEF were also evaluated. In most eukaryotic cells, Rab proteins are well-known to be the key regulators of intracellular vesicular transport owing to their ability to switch between the inactive (GDP-bound) and active (GTP-bound) forms [1–3]. It has been known that Rab proteins regulate membrane fusion in most eukaryotic cells. To perform this event, various Rab proteins regulators (RabGAP, RabGDI, and RabGEF) perform their roles organically. GDP binds to inactivated Rab proteins by converting RabGDI to GTP; the Rab proteins are subsequently activated by RabGEF. Simultaneously, membrane fusion occurs by effector proteins. Eventually, RabGAP stimulates hydrolysis of bound GTP and converts it to GDP, owing to which Rab proteins return to the inactive state [1,2,5,6,26–30]. In the present study, the levels of RabGDI and RabGEF were significantly reduced after capacitation. However, there was no significant difference in the level of RabGAP. In our findings, GDI, which suppress GDP, and
Authors’ roles J.W.B., S.H.K. D.H.K, J.J.H, J.K.Y., S.H., B.Y.R., M.G.P., and W.S.K. performed the experiments, analyzed the data, and drafted the manuscript. W.S.K. supervised the critical study design and data analysis as well as revised the manuscript. All authors contributed toward revisions that were critical to the intellectual content and approved the final version for publication.
Declaration of Competing Interest The authors have declared that no competing interests exist.
Acknowledgement This research was supported by Kyungpook National University Research Fund, 2017. 6
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Appendix A. Supplementary data
[15] Mountjoy JR, Xu W, McLeod D, Hyndman D, Oko R. RAB2A: a major subacrosomal protein of bovine spermatozoa implicated in acrosomal biogenesis. Biol Reprod 2008;79(2):223–32. [16] Lopez CI, Belmonte SA, De Blas GA, Mayorga LS. Membrane-permeant Rab3A triggers acrosomal exocytosis in living human sperm. FASEB J 2007;21(14):4121–30. [17] Ruete MC, Lucchesi O, Bustos MA, Tomes CN. Epac, Rap and Rab3 act in concert to mobilize calcium from sperm’s acrosome during exocytosis. Cell Commun Signal 2014;12:43. [18] Bustos MA, Lucchesi O, Ruete MC, Mayorga LS, Tomes CN. Rab27 and Rab3 sequentially regulate human sperm dense-core granule exocytosis. Proc Natl Acad Sci U S A 2012;109(30):E2057–66. [19] Yunes R, Michaut M, Tomes C, Mayorga LS. Rab3A triggers the acrosome reaction in permeabilized human spermatozoa. Biol Reprod 2000;62(4):1084–9. [20] Kwon WS, Rahman MS, Lee JS, Kim J, Yoon SJ, Park YJ, et al. A comprehensive proteomic approach to identifying capacitation related proteins in boar spermatozoa. BMC Genomics 2014;15:897. [21] Kwon WS, Rahman MS, Lee JS, Yoon SJ, Park YJ, Pang MG. Discovery of predictive biomarkers for litter size in boar spermatozoa. Mol Cell Proteomics 2015;14(5):1230–40. [22] Kwon WS, Rahman MS, Ryu DY, Park YJ, Pang MG. Increased male fertility using fertility-related biomarkers. Sci Rep 2015;5:15654. [23] Kwon WS, Park YJ, Kim YH, You YA, Kim IC, Pang MG. Vasopressin effectively suppresses male fertility. PLoS One 2013;8:e54192. [24] Kwon WS, Park YJ, Mohamed el-SA, Pang MG. Voltage-dependent anion channels are a key factor of male fertility. Fertil Steril 2013;99(2):354–61. [25] Kwon WS, Kim YJ, Ryu DY, Kwon KJ, Song WH, Rahman MS, et al. Fms-like tyrosine kinase 3 is a key factor of male fertility. Theriogenology 2019;126:145–52. [26] Pfeffer SR, Dirac-Svejstrup AB, Soldati T. Rab GDP dissociation inhibitor: putting rab GTPases in the right place. J Biol Chem 1995;270(29):17057–9. [27] Goody PR. Intrinsic protein fluorescence assays for GEF, GAP and post-translational modifications of small GTPases. Anal Biochem 2016;51:22–5. [28] Schalk I, Zeng K, Wu SK, Stura EA, Matteson J, Huang M, et al. Structure and mutational analysis of Rab GDP-dissociation inhibitor. Nature 1996;381(6577):4–28. [29] Oesterlin LK, Goody RS, Itzen A. Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor. Proc Natl Acad Sci U S A 2012;109(15):5621–6. [30] Gavriljuk K, Itzen A, Goody RS, Gerwert K, Kötting C. Membrane extraction of Rab proteins by GDP dissociation inhibitor characterized using attenuated total reflection infrared spectroscopy. Proc Natl Acad Sci U S A 2013;110(33):13380–5.
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.repbio.2019.10.001. References [1] Wu YW, Oesterlin LK, Tan KT, Waldmann H, Alexandrov K, Goody RS. Membrane targeting mechanism of Rab GTPases elucidated by semisynthetic protein probes. Nat Chem Biol 2010;6(7):534–40. [2] Goody RS, Müller MP, Wu YW. Mechanisms of action of Rab proteins, key regulators of intracellular vesicular transport. Biol Chem 2017;398(5–6):565–75. [3] Pereira-Leal JB, Seabra MC. The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily. J Mol Biol 2000;301(4):1077–87. [4] Alory C, Balch WE. Organization of the Rab-GDI/CHM superfamily: the functional basis for choroideremia disease. Traffic 2001;2(8):532–43. [5] Stenmark H. Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 2009;10(8):513–25. [6] Hutagalung AH, Novick PJ. Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 2011;91(1):119–49. [7] Goud B, Liu S, Storrie B. Rab proteins as major determinants of the Golgi complex structure. Small GTPases 2018;9(1-2):66–75. [8] Liu S, Storrie B. Are Rab proteins the link between Golgi organization and membrane trafficking? Cell Mol Life Sci 2012;69(24):4093–106. [9] Smith CE, Hermo L, Fazel A, Lalli MF, Bergeron JJ. Ultrastructural distribution of NADPase within the Golgi apparatus and lysosomes of mammalian cells. Prog Histochem Cytochem 1990;21(3):1–120. [10] Clermont Y, Oko R, Hermo L. Cell and molecular biology of the testis. In: Desjardins C, Ewing L, editors. Cell biology of mammalian spermatogenesis. New York: Oxford University Press; 1993. p. 332–76. [11] Oko R, Clermont Y. Spermiogenesis. Knobil E, Neill JD, editors. Encyclopedia of reproduction, 4. San Diego: Academic Press; 1998. p. 602–9. [12] Austin CR. Observations on the penetration of the sperm in the mammalian egg. Aust J Sci Res B 1951;4(4):581–96. [13] Chang MC. Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 1951;168(4277):697–8. [14] Peterson RN, Bozzola J, Polakoski K. Protein transport and organization of the developing mammalian sperm acrosome. Tissue Cell 1992;24(1):1–15.
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