Identification of tyrosine-phosphorylated proteins in seminal vesicle fluid of adult male ratsAbstract
Tyrosine phosphorylated (TyrPho) proteins have a significant function in sperm capacitation and acrosome reaction. Previously, valproic acid (VPA), used for anti-epileptic and cancer treatments, was shown to alter TyrPho protein abundance in seminal vesicle tissue (SVT) and fluid (SVF), containing a major protein called seminal vesicle secretory protein 4 (SVS4) to prevent precocious acrosome exocytosis. Although the proteomics of SVF has been documented in many species, the characterization of its TyrPho proteins with changes after VPA treatment has been undocumented. This study attempted to identify the seminal TyrPho proteins and investigate a potential protein expression in VPA-treated rats. Adult male rats were divided into 3 experiments. In experiment 1 (to characterize TyrPho proteins), the SVF collected from ten animals was immunoprecipitated with TyrPho antibody and further identified using mass spectrometry (MS). In experiment 2 (to examine the binding between sperm and SVS4 in SVF, epididymal sperm were incubated with diluted SVF during capacitation. Moreover, the changes of SVS4 expression in rat seminal vesicles treated with VPA were observed in experiment 3. The immunofluorescence (IF) and immunoblotting (IB) were applied to the samples obtained from experiments 2 and 3. The MS could identify the immunoprecipitated 10 TyrPho proteins in SVF, including SVS4. It was found that the bindings of SVS4 to sperm in SVF-capacitation medium were obviously increased on the sperm head and tail via IF as same as the increase of intensity shown in IB result. Additionally, VPA could significantly increase SVS4 expression in both SVF and SVT. This study has characterized the TyrPho proteins in seminal vesicles. SVS4 is a TyrPho protein that highly binds on sperm membrane during capacitation. However, it is unclear how the increased SVS4 expression in VPA seminal vesicles is mediated, and further investigations are needed. In conclusion, this study demonstrates that VPA alters key seminal fluid components by upregulating the capacitation-associated TyrPho protein SVS4, suggesting that alterations in SVS4 expression may serve as a crucial molecular indicator of VPA-induced male subfertility.
Keywords:
tyrosine phosphorylated protein; valproic acid; seminal vesicle; capacitation; seminal vesicle secretory protein 4 (SVS4)
Resumo
As proteínas tirosina-fosforiladas (TyrPho) desempenham um papel significativo na capacitação espermática e na reação acrossômica. Anteriormente, demonstrou-se que o ácido valproico (VPA), utilizado em tratamentos antiepilépticos e oncológicos, altera a abundância de proteínas TyrPho no tecido da vesícula seminal (TVS) e no fluido da vesícula seminal (FVS), que contém uma proteína principal denominada proteína secretora da vesícula seminal 4 (SVS4), responsável por prevenir a exocitose acrossômica precoce. Embora a proteômica do FVS tenha sido documentada em diversas espécies, a caracterização de suas proteínas TyrPho e as alterações decorrentes do tratamento com VPA ainda não foram documentadas. Este estudo teve como objetivo identificar as proteínas TyrPho seminais e investigar uma potencial expressão proteica em ratos tratados com VPA. Ratos machos adultos foram divididos em três grupos experimentais. No experimento 1 (para caracterizar as proteínas TyrPho), o SVF coletado de dez animais foi imunoprecipitado com anticorpo TyrPho e posteriormente identificado por espectrometria de massa (MS). No experimento 2 (para examinar a ligação entre espermatozoides e SVS4 no SVF), espermatozoides epididimários foram incubados com SVF diluído durante a capacitação. Além disso, as alterações na expressão de SVS4 em vesículas seminais de ratos tratados com VPA foram observadas no experimento 3. Imunofluorescência (IF) e immunoblotting (IB) foram aplicados às amostras obtidas nos experimentos 2 e 3. A espectrometria de massas (MS) identificou as 10 proteínas TyrPho imunoprecipitadas no SVF, incluindo SVS4. Observou-se que a ligação de SVS4 aos espermatozoides no meio de capacitação com SVF aumentou significativamente na cabeça e na cauda dos espermatozoides por meio de IF, assim como o aumento de intensidade mostrado no resultado do IB. Adicionalmente, o VPA aumentou de maneira significativa a expressão de SVS4 tanto no SVF quanto nas vesículas seminais. Este estudo caracterizou as proteínas TyrPho em vesículas seminais. A SVS4 é uma proteína TyrPho que se liga fortemente à membrana espermática durante a capacitação. No entanto, ainda não está claro como esse aumento ocorre. A expressão de SVS4 nas vesículas seminais de VPA é mediada, e mais investigações são necessárias. Em conclusão, este estudo demonstra que o VPA altera os principais componentes do fluido seminal, regulando positivamente a proteína SVS4 TyrPho associada à capacitação, sugerindo que alterações na expressão do SVS4 podem servir como um indicador molecular crucial da subfertilidade masculina induzida pelo VPA.
Palavras-chave:
proteína tirosina-fosforilada; ácido valproico; vesícula seminal; capacitação; proteína secretora da vesícula seminal 4 (SVS4)
1. Introduction
Tyrosine phosphorylation (TyrPho) is a post-translational modification process important in cell regulatory mechanisms to control many processes including signaling transduction, enzyme activity, gene expression, cell cycle growth, and other biological functions (Li et al., 2021; Song et al., 2022). These include the pathological and physiological processes in the reproductive biology (Ruiz-Díaz et al., 2020; Song et al., 2022). In males, TyrPho proteins have been implicated in sperm production, maturation, capacitation, and acrosome reaction (Kwon et al., 2014). Such proteins have been localized at the sperm tail and head region during capacitation related to hyperactivated motility (Sati et al., 2014). Moreover, many TyrPho proteins have been identified in testicular and epididymal tissue with its fluid (Iamsaard et al., 2020; Arun et al., 2021; Chaimontri et al., 2021). Recently, some TyrPho proteins were observed in the seminal vesicle (Tongpan et al, 2019; Iamsaard et al., 2021; Sawatpanich et al., 2022; Iamsaard et al., 2023). Therefore, it is suggested that TyrPho proteins are functionally involved in the male reproductive tracts and seminal plasma.
Valproic acid (VPA), a potent histone deacetylase (HDAC) inhibitor, is primarily used to treat many neurological and psychiatric disorders including mood disorders, migraine, bipolar disorders, and other psychiatric conditions (Sixto-López et al., 2020; Yokoyama et al., 2020). Previously, several studies have demonstrated that VPA could affect the TyrPho protein expressions in the testes (Iamsaard et al., 2017; Sukhorum and Iamsaard, 2017), epididymis (Sawatpanich et al., 2018), and seminal vesicle and its seminal vesicle fluid (SVF), respectively (Tongpan et al, 2019). The SVF is known to contain several energy substrates, proteins, peptides, and soluble ions, essential for sperm physiological changes within the female reproductive tract (Patlar, 2022). Major proteins found in the SVF are called seminal vesicle secretory protein family (SVS1-SVS7) (Noda and Ikawa, 2019). Among those proteins, the SVS4 was shown to be the most crucial protein in early fertilization process and localized on the sperm head (Araki et al., 2016).
Although the proteomic profiles of SVF were reported in some pathological disorders such as varicocele, smoking, spinal cord injury, and hypogonadism (Camargo et al., 2018), the unknown TyrPho proteins have never been characterized in VPA treatments associated with male subfertility. This study hypothesizes that 1) SVF contains functional TyrPho proteins identified by IP-MS, 2) SVS4 binds capacitated sperm, and 3) VPA increases SVS4 via HDAC inhibition, respectively. Therefore, the objectives of this research are characterization of seminal TyrPho proteins present in the SVF and investigation of its changes with SVS4 expression after VPA treating in the rat model.
2. Materials and Methods
2.1. Animal and design of experiments
Thirty-five male Sprague-Dawley rats (200-220 g, age 7-8 weeks) were obtained from the Nomura Siam International Company, Pathum Wan District, Bangkok, Thailand, and housed within plastic cages at Northeast Laboratory Animal Center, Khon Kaen University, Thailand. All experimental protocols were ethically authorized by Institutional Animal Care and Use Committee of Khon Kaen University in accordance with the National Research Council of Thailand's Animal Experimentation Ethics guidelines (Approval reference: IACUC‐KKU‐33/64). After acclimatization, animals were divided for 3 experiments as follows.
In experiment 1, to characterize TyrPho proteins in rat SVF, the SVF collected from 10 animals was immunoprecipitated (IP) with TyrPho antibody and characterized by mass spectrometry (MS).
For experiment 2, to examine the binding of SVS4 (a TyrPho protein) in SVF on plasma membrane of capacitated sperm, a paired contralateral design was used in 5 rats. The cauda epididymal sperm from one side of each rat was incubated with SVF, while the sperm from the contralateral side served as the control group (without SVF), prior to detecting sperm binding complex by immunofluorescence and immunoblotting.
In experiment 3, to investigate the expression of SVS4 in seminal vesicle induced with valproic acid (VPA), SVT and SVF of 10 rats treated with VPA at 500 mg/kg BW via intraperitoneal injection for 10 continuous days were examined for SVS4 expression by immunofluorescence and immunoblotting compared to control.
2.2. Seminal vesicle fluid and tissue collections
After each experimental completion, rats received thiopental sodium (60 mg/kg BW) intraperitoneally for anesthesia before being euthanized via cervical dislocation. After cleaning the thoracoabdominal wall with 70% ethanol, the abdominopelvic wall was opened for harvesting seminal vesicle plus prostate gland. Before dissecting the seminal vesicle, the surrounding fat pads and all parts of prostate glands were separated. To collect the SVF and SVT, the seminal vesicle was punctured and gently squeezed to allow its fluid flowing into a sterilized Eppendorf tube. The remaining SVT wall and its epithelium were stored at -20 °C before using. The right seminal vesicle glands from experiment 3 were fixed in 10% formalin fixative for 48 hours before paraffin tissue processing.
2.3. Immunoprecipitation (IP) of TyrPho proteins
To extract proteins in SVF, the SVF was mixed with 1X RIPA buffer supplemented with protease inhibitor cocktails (dilution 2:1). The mixed sample was homogenized and sonicated (VCX 130 ultrasonic liquid processor, USA) for 10 seconds, 3 times on ice. Following centrifugation (15,000 rpm, 4°C, 15 minutes), the supernatant containing seminal proteins was taken to determine the total protein concentrations by using spectrophotometers at an absorbance of 280 nm (NanoDrop 2000 Spectrophotometers, Thermo Fisher Scientific, USA).
To link TyrPho antibodies on protein G magnetic beads, 60 μg of the beads (1 μg/ 1 μl, Merck Millipore, USA) were incubated with 4 μl of phosphotyrosine antibody (1 μg/ 1 μl; 4G10, Merck Millipore, USA) diluted in phosphate buffered saline (PBS) at 4 °C for 1 hour with gentle shaking. Then, the antibody-bead complexes were magnetically separated to remove the unbound antibodies before washing with PBS with 0.1% Tween 20 (0.1% PBST). Then, anti-TyrPho-beads were mixed with total SVF proteins at 500 μg (1 μg/ 1 μl) at 4ºC for 1 hour with shaking. Subsequently, to immunoprecipitate the seminal TyrPho proteins, the antigen-antibody complex beads were pooled down using a magnetic and washing with 0.1% PBST. The immunoprecipitated TyrPho proteins were eluted with 0.1% sodium dodecyl sulfate (SDS) and its protein concentration was further determined. To evaluate non-specific binding and ensure protocol specificity, a beads-only control was concurrently prepared by incubating total SVF proteins directly with Protein G magnetic beads in the absence of the anti-phosphotyrosine primary antibody, followed by the identical washing and elution steps. To confirm and determine seminal TyrPho proteins, the total precipitated proteins were separated by using 12% SDS-polyacrylamide gels before observing under western blot analysis.
2.4. Mass spectrometry (MS)
Each separated TyrPho protein band on SDS gel was cropped and cut into small pieces (1x1 mm). The gels were then equilibrated with 200 μl of 20 mM ammonium bicarbonate (AmB, Sigma‐Aldrich, USA) for 10 minutes. After destaining, gels were dehydrated with acetonitrile (Fluka, USA) for 10 minutes. To break the disulfide bond, the proteins in gels were incubated with 10 mM DL-Dithiothreitol (Fluka, USA) in AmB at 56 °C for 45 min. For alkylation process, the gels were incubated with 55 mM iodoacetamide (Sigma‐Aldrich, Germany) for 30 minutes at RT. For in-gel digestion, the shrunken gels were incubated with fresh trypsin solution (V5111, Promega, USA) at 4 °C for 30 minutes. Then, the peptides in gel were extracted with 1% formic acid (Fluka, USA) and centrifuged to collect the supernatant containing peptides. Such supernatant solution was evaporated to desalt for 2 hours.
To characterize seminal TyrPho proteins, the peptide solution was subjected into nano-liquid chromatography system (nano-LC, EASY-nLC II, Bruker Daltonics, Germany) coupled with the quadrupole time-of-flight tandem mass spectrometer (Q-TOF, micrOTOF-Q II, Bruker Daltonics, Germany) equipped with an electrospray ionization (ESI) nano-sprayer (ESI-Q-TOF). To identify the mass and charge of ions, the particle ions were determined by quadrupole and time-of-flight (TOF) mass spectrometer analyzers. LC-MS/MS spectra were processed through Compass Data Analysis version 4.0. All mass spectrometry instruments were under the service of Khon Kaen University Research Instrument Center, Thailand. The identified peptide fragments were systemically compared to NCBI’s protein database using the protein database MASCOT search software.
2.5. Sperm collection and SVS4-sperm binding assay
In experiment II, sperm mass from the epididymis (cauda) plus ductus deferens was squeezed and immersed in 1 ml of human tubal fluid (HTF) medium for washing its epididymal fluid by centrifugation (250 g, 37 °C, 10 minutes). Then, sperm pellet was added with HTF medium (1 ml) and incubated in 5% CO2 incubator (37 °C for 30 minutes) to allow sperm swimming up (Pérez-cerezales et al., 2018). Subsequently, the motile sperm (1/3 upper supernatant) were transferred into HTF medium supplemented with 1% bovine serum albumin (BSA) to induce sperm capacitation. For SVS4-sperm binding assay, the motile sperm were divided into incubated with SVF (+SVF) and incubated without SVF (-SVF) groups. Each group contained 10 million sperm/ ml. In the +SVF group, the sperm were incubated with 5 ml of SVF (final concentration: 10 million sperm/ ml) to allow SVS4 binding during sperm capacitation period (37°C, 5% CO2 incubator, 1 hour), whereas sperm in the -SVF group were incubated in capacitated medium without SVF under the same conditions. Then, the unbound proteins including SVS4 in SVF and capacitated medium were washed out and the sperm pellet was resuspended with 1 ml of PBS. The binding of SVS4 to sperm membrane was evaluated by using immunofluorescence localization.
2.6. Immunofluorescence (IF) assay
To localize the SVS4 protein on sperm and SVT, immunofluorescence (IF) was performed. The epididymal sperm were preserved in 2% paraformaldehyde for 48 hours. After washing with PBS, 20 μl of preserved sperm suspension was smeared and dried on the gelatin-coated glass slide overnight. For seminal vesicles, the paraffin-embedded tissues were sectioned (5 μm thickness; ERM‐3100; Heston histology equipment). Subsequently, paraffinized tissue sections were processed through deparaffinization and rehydration before retrieval of antigens with citrate buffer (10 mM citric acid, 0.05% Tween-20, pH 6.0).
For IF method, the dried sperm or seminal section (n=5/group) was circled with a peroxidase-antiperoxidase pen (Merck Millipore, USA) to create hydrophobic barrier area for holding reagents within a targeted area. For endogenous peroxidase activity blocking, the samples were incubated with 3% H2O2 for 30 minutes followed by PBS washing. To permeabilize the plasma membranes, the samples were incubated with 0.2% Triton X-100 for 10 minutes at room temperature (RT), followed by PBS rinsing and then incubated with 0.3% BSA for 1 hour to block nonspecific protein binding. Subsequently, samples were probed overnight with the anti-SVS4 (X-P02783-N (SVS4-(C) [unknown]), Abmart, USA) in PBS (1:200, v/v), while the negative control was omitted for primary antibody in a moisture chamber. After washing unbound antibodies, the Ag-Ab complexes on samples were exposed to goat anti‐rabbit IgG (H+L) secondary antibody, Alexa Fluor 488 (dilution 1:200 [v/v], A11001, Invitrogen, USA) for 90 minutes in a dark chamber and rinsed with PBS. For nucleus visualization, the specimens were subsequently dyed with Hoechst 33342 diluted in PBS (1: 10,000 [v/v], ab228551, Abcam, UK) before mounting with glycerol. The positive immunoreactivity (green fluorescence) on samples was observed using a Nikon ECLIPSE 80i fluorescence microscope equipped with fluorescein isothiocyanate (FITC) filter and images were captured using a DXM1200 digital camera (Nikon).
2.7. Immunoblotting analysis
The SVS4 protein expressions on sperm, SVT, and SVF were evaluated using immunoblotting. In brief, such samples were extracted by adding RIPA buffer and protease inhibitor cocktails to homogenize and sonicate with an ultrasonic sonicator (VCX 130 ultrasonic liquid processor, USA) for 10 seconds on ice. Then each homogenized sample was centrifuged (15,000 rpm, 4 °C, 15 minutes) to obtain total supernatant proteins before measuring protein concentrations using spectrophotometer at an absorbance of 280 nm (NanoDrop 2000 Spectrophotometer, Thermo Fisher Scientific, USA).
The total proteins of sperm (200 µg), SVT (100 µg), SVF (40 µg), or IP SVF (10 µg) were loaded on an acrylamide gel (12% gel) and electroblotted to the nitrocellulose membrane. After blocking nonspecific binding proteins with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (0.1% TBST), each protein membrane was exposed overnight with individual primary antibodies: anti-SVS4 (1:2,000 [v/v], X-P02783-N (SVS4-(C)), Abmart, USA), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:20,000 [v/v], Santa Cruz, USA), or anti-phosphotyrosine (1:1,500 [v/v], 4G10, Merck Millipore, USA), and washed with 0.1% TBST. Subsequently, the membranes were probed with mouse IgG linked with HRP secondary antibody (1:2,000 [v/v], Santa Cruz, USA) for 1 hour and washed with 0.1% TBST. The positive protein immunoreactions were detected using an enhanced chemiluminescence substrate kit (ECL Prime, GE Healthcare, USA) and visualized with a chemiluminescence imaging system (Amersham Imager 600, GE Healthcare, USA). For tyrosine phosphorylation protein expression, epidermal growth factor (EGF) and BSA served as positive and negative controls, respectively. The GAPDH served as a loading control and intensity values of the protein band between groups were quantified in triplicate using ImageJ software (version 1.51). In SVF sample, SDS‐PAGE stained with Coomassie blue was used to confirm equal loading of protein.
2.8. Statistical analysis
All data are recorded and reported as mean ± standard deviation. To compare the differences between groups, an independent t-test was used. Statistically significant differences were considered as P < 0.05. The IBM SPSS Statistics 27 (IBM SPSS Statistics for Mac OS X, version 27, IBM Corporation, New York, USA), downloaded from KKU Microsoft campus license was utilized to analyze statistical data.
3. Results
3.1. Tyrosine phosphorylated (TyrPho) proteins in seminal vesicle fluid
The gel electrophoresis showed the SVF protein profiles of non-immunoprecipitated (non-IP) and immunoprecipitated-TyrPho (IP-TyrPho) samples (Figure 1). The non-IP sample revealed 9 protein bands at 95, 75, 72, 62, 50, 43, 26, 17, and 10 kDas, respectively. It was noted that the 72, 17, and 10 kDas were more intensified as compared among them. In total IP-TyrPho protein sample, the 10 bands are identified and further grouped as 5 ranges (R1 [78, 72, 68, 65, 62 kDas]; R2 [50 kDa]; R3 [44 kDa]; R4 [26 kDa]; R5 [17, 10 kDas]) to be cut for mass spectrometry analysis.
Profiles of immune-precipitated TyrPho proteins in SVF. Comparison of seminal proteins determined by SDS-PAGE between non-IP and IPSVF-TyrPho fractions. kDa; kilodalton, non-IPSVF; non-immunoprecipitated proteins in seminal vesicle fluid, IPSVF-TyrPho; immunoprecipitated proteins with tyrosine phosphorylated antibody in seminal vesicle fluid, R; range of cut-protein bands.
3.2. Identification of specific TyrPho proteins in seminal vesicle fluid
The proteins of IPSVF-anti-TyrPho fraction in 5 range gels were detected and identified based on the time-of-flight mass spectrometry (TOF) as shown in Table 1. The results revealed the total identified 22 protein fragments including 4 (R1), 5 (R2), 4 (R3), 5 (R4), and 4 (R5) proteins, respectively (Table 1). It was noted that the whole sequence of each TyrPho protein identified contains amino acid of tyrosine (Y). Interestingly, it was found that the seminal vesicle secreted protein 4 (SVS4), responsible for sperm binding, anti-apoptosis, anti-inflammatory, and immunomodulatory activities, was detected in the range 5 (Table 1). Importantly, no corresponding proteins or specific bands were detected in the beads-only negative control, confirming the high specificity of the identified TyrPho proteins and ruling out non-specific matrix contamination.
Using LC-MS/MS, Figure 2 revealed peptide spectrums of immunoprecipitated TyrPho proteins that have three proteins associated with sperm functions (Araki et al., 2016; Wang et al., 2021; Álvarez-Rodríguez et al., 2024; Mohanty et al., 2024). The results showed the peptide spectrum and amino acid sequences of quiescin sulfhydryl oxidase 1 (QSOX1), containing IYMADLESALHYILR (ion score: 109) corresponding to its residues of 302-316 and peptide fragment covered 42% of the protein sequence (Figure 2A). In Figure 2B, spectrum and peptide sequences of albumin (ALB) were identified as LGEYGFQNAILVR (ion score: 15) having residues at 422-434 (underlined in 2B) with 2% covered peptide sequences. 30. For vesicle secretory protein 4 (SVS4), its spectrum and peptide sequences are SSGGSNMEGESSYAK (ion score: 61, underlined in 2C) corresponding to residues of 86-100 and 28% peptide fragments coverage as shown in Figure 2C.
The spectra of IpSVF∝TyrPho proteins in rats by using ESI-Q-TOF-MS analysis. QSOX1; quiescin sulfhydryl oxidase 1, ALB, albumin; SVS4; seminal vesicle secretory protein 4. Red letters; fragment identifications and underlines; spectrum fragments.
The western blotting has confirmed the high yield of SVS4 protein obtained from Ip-TyrPho immunoprecipitation (Figure 3).
Immunoblotting of SVS4 in seminal vesicle fluid fractions detected in both non-IP and IP-TyrPho samples.
3.3. Expression of SVS4 on epididymal sperm incubated with SVF
The positive immunofluorescent signals of the SVS4 on caudal epididymal spermatozoa were demonstrated in Figure 4. It was observed that SVS4 was localized on the head and tail region of sperm in both incubated without (-SVF, Figures 4BC) and incubated with seminal vesicle fluid (+SVF, Figures 4EF) groups as compared to negative control (Figures 4H, I). Obviously, the spermatozoa +SVF group (Figure 4EF) revealed that the fluorescence signal intensity of SVS4 was greater than that of the -SVF group (Figure 4BC).
Immunofluorescence microphotographs of SVS4-sperm binding. Green fluorescence, positive immunoreactivity of SVS4. The sperm nuclei stained with Hoechst 33342 (blue fluorescence). Omitted primary antibody was used for negative control (figures 4GI). -SVF; sperm incubated without seminal vesicle fluid (figures 4AC), +SVF; sperm incubated with seminal vesicle fluid (figures 4DF). Scale bar as 20 μm.
The expression of SVS4 protein on capacitated sperm compared between incubated with SVF (+SVF) and without SVF (-SVF) groups was shown in Figure 5. In Figure 5A, the capacitated sperm with +SVF showed a higher SVS4 expression as compared to sperm incubated without SVF (-SVF) group. Indeed, its intensity of SVS4 expression was significantly increased in the +SVF group (SVS4/GAPDH: -SVF 1.00 ± 0.06 vs. +SVF 2.43 ± 0.14, p = 0.0023) as shown in Figure 5B.
Comparison of SVS4 expression (A, western blot and B, relative intensity) between sperm-seminal vesicle fluid binding and sperm without seminal vesicle fluid groups. Data are expressed as mean ± SD (n = 5 rats per group; image quantification was performed in triplicate). *p < 0.01, statistically significant difference, GAPDH; Glyceraldehyde‐3‐phosphate dehydrogenase used as internal control, -SVF; incubated without seminal vesicle fluid, +SVF; incubated with seminal vesicle fluid.
3.4. Localization and expression of SVS4 on seminal vesicle
The results showed that SVS4 was localized on SVT epithelium of both control and VPA-treated groups (Figures 6BF). In contrast, the positive immunofluorescence signals for SVS4 were obviously noted in the VPA group as compared to those of control (Figure 6).
Representative immunofluorescence microphotograph of SVS4 on seminal vesicle tissue. The positive immunoreactivity as shown in green fluorescence. The nuclei staining with Hoechst 33342 (blue fluorescence). The green fluorescence signals (SVS4) expressed on apical epithelium cells of seminal vesicle tissue in both control (figures 6AC) and VPA‐treated (figures 6DF) groups. Omitted primary antibody was used as a negative control (figures 6GI). Scale bars represent as 20 μm.
Furthermore, the expressions of SVS4 in SVF and SVT shown in Figure 7. In VPA-treated rats, the SVS4 level in seminal vesicle tissue was significantly higher than that of control (SVS4/GAPDH in tissue: Control 1.00 ± 0.07 vs. VPA 1.38 ± 0.15, p = 0.021; Figure 7B). Interestingly, the SVS4 expression in the seminal vesicle fluid (SVF) in VPA group was also significantly increased as compared to control (Figure 7C and D; SVS4 relative intensity in fluid: Control 1.00 ± 0.00 vs. VPA 11.14 ± 0.12, p = 0.00003). Figure 7E showed the protein profiles of seminal vesicle fluid, which was used to confirm equal loading.
Expression and relative intensity of SVS4 in seminal vesicle tissue (A, B) and SVF (seminal vesicle fluid, C, D) compared between control and VPA groups. Data are expressed as mean ± SD (n = 10 rats per group; image quantification was performed in triplicate). *p < 0.05 or **p < 0.01 statistically significant difference as compared between groups; GAPDH and SDS-PAGE of seminal vesicle fluid (figure 7E); used as internal protein control.
4. Discussion
Currently, there are many techniques for protein precipitation, including ammonium sulfate, polyethyleneimine, isoelectric, thermal, polyethylene glycol, and immunoprecipitation. In this study, we precipitated seminal TyrPho proteins using magnetic beads linked with TyrPho antibodies for the first time. Previous studies reported that magnetic protein beads conjugated with antibodies increased the efficiency of capturing interested proteins (Brechmann et al., 2021).
Previous studies have investigated the protein profile of seminal plasma (SP) by using SDS-PAGE gels in men (Bianchi et al., 2018), rams (Silva et al., 2021), buffalos (Almadaly et al., 2023), and bulls (Magalhães Junior et al., 2016; Westfalewicz et al., 2017). Moreover, with the same protein separation, the various proteins in SVF were also identified in bulls (Westfalewicz et al., 2017), guinea pigs (Lundwall et al., 2020), rats (Tongpan et al., 2019; Iamsaard et al., 2020; Sawatpanich et al., 2022), and mice (Morohoshi et al., 2021; Ou et al., 2021). More detectable TyrPho proteins were shown in IP group because total protein amounts used in IP were greater (5 folds) than that of non-IP sample.
The SVF proteins have been demonstrated to be essential for coagulum formation, sperm movement regulation, preservation of sperm DNA stability, and immune modulation in the female genital tract (Noda and Ikawa, 2019). Previous studies characterized SP and SVF proteins using mass spectrometry approaches in men, bull, buffalo, ram, rabbit, and mice (Batruch et al., 2011; Souza et al., 2012; Westfalewicz et al., 2017; Brito et al., 2018; Bezerra et al., 2019; Bayram et al., 2020; Skerrett-Byrne et al., 2021). In this study, the 10 TyrPho proteins were identified in rat SVF for the first time. Most of those proteins have been also previously reported in SP and SVF (Batruch et al., 2011; Souza et al., 2012; Westfalewicz et al., 2017; Brito et al., 2018; Bezerra et al., 2019; Bayram et al., 2020; Skerrett-Byrne et al., 2021). However, the C-C motif chemokine 8 (CCL8) and immunoglobulin kappa constant (IGKC) were detected in rat SVF for the first time. Among the characterized proteins, the five TyrPho proteins including quiescin sulfhydryl oxidase 1, albumin, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, and seminal vesicle secretory protein 4 (SVS4), have been demonstrated to involve in sperm maturation, motility, and sperm capacitation (Araki et al., 2016; Wang et al., 2021; Álvarez-Rodríguez et al., 2024; Mohanty et al., 2024). Although the rest of the TyrPho proteins identified in this study (HBA, HBB1, DNS2B, IGKC, ALMS, IGG2B, CCL8) are not documented for actual functions in the reproduction, their functions have been investigated in others such as cell death and survival, cellular growth and proliferation, glycolysis, and immune response (Tarayrah-Ibraheim et al., 2021; Naderi et al., 2022; Chelladurai et al., 2024).
The SVS proteins, secreted from seminal vesicle epithelium, have been reported and classified to be SVS1 to SVS7, involved in copulatory plug formation and sperm viability and capacitation (Araki et al., 2016; Noda et al., 2019). Among them, SVS4 not only plays the role of protecting early sperm capacitation, but also has been shown to have anticoagulant, immunosuppressive, and anti-inflammatory properties (Romano-Carratelli et al., 1995; Tufano et al., 1996; Araki et al., 2016). In this study, the SVS4 protein was localized on head and tail of capacitated sperm incubated with SVF, but a previous study demonstrated its localization only at the post-acrosomal region (Araki et al., 2016). As such result, it is possible that the increased expression of SVS4 on sperm membrane in this study may be associated with SVF substances that facilitate binding process (Rodríguez-Martínez et al., 2011). Such interaction may be involved in sperm capacitation via combining with SVS2 (Araki et al., 2016). In addition, the different concentrations of albumin commonly used to induce sperm capacitation may also affect membrane protein binding sites (Ickowicz et al., 2012). Mechanistically, SVS4 functions as a membrane stabilizer during early capacitation, a process driven by cholesterol efflux, membrane potential shifts, and elevated intracellular calcium via adenylyl cyclase and cAMP dependent protein kinase A cascades (Visconti et al., 1995; Ickowicz et al., 2012). During capacitation, premature or excessive membrane fluidization can trigger an untimely acrosome reaction, which renders sperm non functional before reaching the oocyte. The binding of SVS4 to the sperm head matrix likely acts as a transient decapacitation factor. This protective role may be carried out specifically by the tyrosine phosphorylated form of SVS4, which alters its conformation and strengthens its membrane binding affinity to control cholesterol efflux and reduce premature calcium influx. Consequently, this controlled suppression preserves sperm viability and ensures that full capacitation is achieved in a coordinated manner. Furthermore, we uniquely observed SVS4 localization at the sperm tail, which was not previously reported by Araki et al. (2016). This novel finding suggests that SVS4 may also regulate flagellar signaling during capacitation through interaction with fibrous sheath anchored GAPDH, which supplies glycolytic ATP to dynein ATPases driving axonemal motion.
In this study, SVS4 protein was demonstrated in the seminal vesicle tissue (SVT) epithelium and overexpressed in the VPA-treated rats. It could be explained that the VPA, a histone deacetylase (HDAC) inhibitor, also was involved in upregulating many gene expressions and protein translations (Wulhfard et al., 2010; Larsson et al., 2012). Such stimulation of histone acetylation pathway by VPA may modulate SVS4 overexpression shown in our study and other TyrPho proteins as demonstrated previously (Tongpan et al., 2019). Regarding the VPA HDAC mechanism, VPA acts as a class I and II HDAC inhibitor that prevents the deacetylation of lysine residues on histone tails H3 and H4 in seminal vesicle epithelial cells (Wulhfard et al., 2010). Under normal conditions, HDACs keep chromatin compacted at the promoter regions of secretory protein genes to maintain protein production at normal physiological levels. When VPA inhibits HDACs, histone hyperacetylation occurs, which weakens histone DNA interactions and opens the chromatin structure. Consequently, transcription factors and RNA polymerase II can easily access the promoter regions (Larsson et al., 2012). This open state leads to the overexpression of secretory genes, including SVS4, resulting in excessive protein accumulation in the SVT lumen and SVF. The excess SVS4 in SVF may impair sperm function by oversaturating membrane binding sites. This overload prolongs decapacitation like suppression beyond the normal timeframe, which delays or reduces the capacitation response required for fertilization. Therefore, this VPA HDAC SVS4 pathway provides a possible molecular explanation for the male subfertility observed in VPA exposed rat models (Tongpan et al., 2019). However, SVS4 specific knockdown or rescue experiments are still needed to confirm this mechanism directly.
5. Conclusion
In conclusion, the SVF contains many potential TyrPho proteins including SVS4. Indeed, such TyrPho proteins are involved in the sperm capacitation process, and VPA treatment significantly increases SVS4 expression, likely via histone deacetylase (HDAC) inhibition. These findings suggest that alterations in SVS4 expression within the seminal plasma may serve as a crucial molecular indicator of VPA-induced reproductive changes and male subfertility.
Acknowledgements
Saranya Tongpan was supported by Invitation Research (Grant Number: IN65120), Faculty of Medicine, Khon Kaen University, Thailand.
Data Availability Statement
Research data is only available upon request.
References
-
ALMADALY, E.A., ABDEL-SALAM, A.S., SAHWAN, F.M., KAHILO, K.A., ABOUZED, T.K. and EL-DOMANY, W.B., 2023. Fertility-associated biochemical components in seminal plasma and serum of buffalo (Bubalus bubalis) bulls. Frontiers in Veterinary Science, vol. 9, pp. 1043379. https://doi.org/10.3389/fvets.2022.1043379 PMid:36733430.
» https://doi.org/10.3389/fvets.2022.1043379 -
ÁLVAREZ-RODRÍGUEZ, M., NIETO-CRISTOBAL, H. and DE MERCADO, E., 2024. Bovine serum albumin inclusion in the thawing extender improves boar sperm membrane and acrosomal integrity. Reproduction in Domestic Animals = Zuchthygiene, vol. 59, suppl. 3, pp. e14592. https://doi.org/10.1111/rda.14592
» https://doi.org/10.1111/rda.14592 -
ARAKI, N., KAWANO, N., KANG, W., MIYADO, K., YOSHIDA, K. and YOSHIDA, M., 2016. Seminal vesicle proteins SVS3 and SVS4 facilitate SVS2 effect on sperm capacitation. Reproduction : The Official Journal of the Society for the Study of Fertility, vol. 152, no. 4, pp. 313-321. https://doi.org/10.1530/REP-15-0551 PMid:27486266.
» https://doi.org/10.1530/REP-15-0551 -
ARUN, S., CHAIYAMOON, A., LAPYUNEYONG, N., BUNSUEB, S., WU, A.T. and IAMSAARD, S., 2021. Chronic stress affects tyrosine phosphorylated protein expression and secretion of male rat epididymis. Andrologia, vol. 53, no. 3, pp. e13981. https://doi.org/10.1111/and.13981 PMid:33469986.
» https://doi.org/10.1111/and.13981 -
BATRUCH, I., LECKER, I., KAGEDAN, D., SMITH, C.R., MULLEN, B.J., GROBER, E., LO, K.C., DIAMANDIS, E.P. and JARVI, K.A., 2011. Proteomic analysis of seminal plasma from normal volunteers and post-vasectomy patients identifies over 2000 proteins and candidate biomarkers of the urogenital system. Journal of Proteome Research, vol. 10, no. 3, pp. 941-953. https://doi.org/10.1021/pr100745u PMid:21142078.
» https://doi.org/10.1021/pr100745u -
BAYRAM, H.L., FRANCO, C., BROWNRIDGE, P., CLAYDON, A.J., KOCH, N., HURST, J.L., BEYNON, R.J. and STOCKLEY, P., 2020. Social status and ejaculate composition in the house mouse. Philosophical Transactions of the Royal Society B: Biological Sciences, vol. 375, no. 1813, pp. 20200083. https://doi.org/10.1098/rstb.2020.0083
» https://doi.org/10.1098/rstb.2020.0083 -
BEZERRA, M.J.B., ARRUDA-ALENCAR, J.M., MARTINS, J.A.M., VIANA, A.G.A., VIANA NETO, A.M., RÊGO, J.P.A., MOURA, A.A., MORENO, F.B., MONTEIRO-MOREIRA, A.C. and MOURA, A.A., 2019. Major seminal plasma proteome of rabbits and associations with sperm quality. Theriogenology, vol. 128, pp. 156-166. https://doi.org/10.1016/j.theriogenology.2019.01.013 PMid:30772659.
» https://doi.org/10.1016/j.theriogenology.2019.01.013 -
BIANCHI, L., CARNEMOLLA, C., VIVIANI, V., LANDI, C., PAVONE, V., LUDDI, A., DE LEO, V., PIOMBONI, P. and BINI, L., 2018. Soluble protein fraction of human seminal plasma. Journal of Proteomics, vol. 174, pp. 85-100. https://doi.org/10.1016/j.jprot.2017.12.015 PMid:29288815.
» https://doi.org/10.1016/j.jprot.2017.12.015 -
BRECHMANN, N.A., SCHWARZ, H., ERIKSSON, P.O., ERIKSSON, K., SHOKRI, A. and CHOTTEAU, V., 2021. Antibody capture process based on magnetic beads from very high cell density suspension. Biotechnology and Bioengineering, vol. 118, no. 9, pp. 3499-3510. https://doi.org/10.1002/bit.27776 PMid:33811659.
» https://doi.org/10.1002/bit.27776 -
BRITO, M.F., AULER, P.A., TAVARES, G.C., REZENDE, C.P., ALMEIDA, G.M.F., PEREIRA, F.L., CHAPEAU, E.I., CARVALHO, A.F., SOUZA, G.H.M.F., POZZA, M.S.S., TEIXEIRA, M.C. and SANTOS, L.L., 2018. Label-free proteome of water buffalo (Bubalus bubalis) seminal plasma. Reproduction in Domestic Animals = Zuchthygiene, vol. 53, no. 5, pp. 1243-1246. https://doi.org/10.1111/rda.13206
» https://doi.org/10.1111/rda.13206 -
CAMARGO, M., INTASQUI, P. and BERTOLLA, R.P., 2018. Understanding the seminal plasma proteome and its role in male fertility. Basic and Clinical Andrology, vol. 28, pp. 6. https://doi.org/10.1186/s12610-018-0071-5 PMid:29881623.
» https://doi.org/10.1186/s12610-018-0071-5 -
CHAIMONTRI, C., ARUN, S., SAWATPANICH, T., YANNASITHINON, S., TANGSRISAKDA, N., BUNSUEB, S. and IAMSAARD, S., 2021. The effect of Dolichandrone serrulata (wall. ex DC.) Seem. flower extract containing antioxidant capacity and terpenoids on the male reproductive system. Andrologia, vol. 53, no. 3, pp. e13966. https://doi.org/10.1111/and.13966 PMid:33427326.
» https://doi.org/10.1111/and.13966 -
CHELLADURAI, M., XU, D., IZRAELY, S., BEN-MENACHEM, S., BENGAIEV, R., SAGI-ASSIF, O., WITZ, I.P., ALMOG, O. and SHANI, J., 2024. A heterodimer of α and β hemoglobin chains functions as an innate anticancer agent. International Journal of Cancer, vol. 154, no. 3, pp. 561-572. https://doi.org/10.1002/ijc.34702 PMid:37675956.
» https://doi.org/10.1002/ijc.34702 -
IAMSAARD, S., ARUN, S., BURAWAT, J., YANNASITHINON, S., TONGPAN, S., BUNSUEB, S., SUKHORUM, W., CHAIYAMOON, A. and TANGSRISAKDA, N., 2020. Evaluation of antioxidant capacity and reproductive toxicity of aqueous extract of Thai Mucuna pruriens seeds. Journal of Integrative Medicine, vol. 18, no. 3, pp. 265-273. https://doi.org/10.1016/j.joim.2020.03.003 PMid:32249078.
» https://doi.org/10.1016/j.joim.2020.03.003 -
IAMSAARD, S., KIETINUN, S., SATTAYASAI, J., BUNLUEPUECH, K., WU, A.T. and CHOOWONG-IN, P., 2023. Prevention of seminal vesicle damage by Mucuna pruriens var. pruriens seed extract in chronic unpredictable mild stress mice. Pharmaceutical Biology, vol. 61, no. 1, pp. 89-99. https://doi.org/10.1080/13880209.2022.2157018 PMid:36565036.
» https://doi.org/10.1080/13880209.2022.2157018 -
IAMSAARD, S., SUKHORUM, W., SAMPANNANG, A. and SRIPANIDKULCHAI, B., 2017. Protective effect of Momordica cochinchinensis (L.) spreng aril extract on essential testicular markers in rats induced with valproic acid. International Journal of Morphology, vol. 35, no. 3, pp. 992-999. https://doi.org/10.4067/S0717-95022017000300030
» https://doi.org/10.4067/S0717-95022017000300030 -
IAMSAARD, S., TONGPAN, S., YANNASITHINON, S., ARUN, S., WU, A.T.H. and SUKHORUM, W., 2021. Effect of chronic stress on expression and secretion of seminal vesicle proteins in adult rats. Andrologia, vol. 53, no. 1, pp. e13800. https://doi.org/10.1111/and.13800 PMid:32816406.
» https://doi.org/10.1111/and.13800 -
ICKOWICZ, D., FINKELSTEIN, M. and BREITBART, H., 2012. Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases. Asian Journal of Andrology, vol. 14, no. 6, pp. 816-821. https://doi.org/10.1038/aja.2012.81 PMid:23001443.
» https://doi.org/10.1038/aja.2012.81 -
KWON, W.S., RAHMAN, M.S. and PANG, M.G., 2014. Diagnosis and prognosis of male infertility in mammal: the focusing of tyrosine phosphorylation and phosphotyrosine proteins. Journal of Proteome Research, vol. 13, no. 11, pp. 4505-4517. https://doi.org/10.1021/pr500524p PMid:25223855.
» https://doi.org/10.1021/pr500524p -
LARSSON, P., ULFHAMMER, E., MAGNUSSON, M., BERGH, N., LUNKE, S., EL-OSTA, A., MEDCALF, R.L., SVENSSON, S., KARLSSON, L. and JERN, S., 2012. Role of histone acetylation in the stimulatory effect of valproic acid on vascular endothelial tissue-type plasminogen activator expression. PLoS One, vol. 7, no. 2, pp. e31573. https://doi.org/10.1371/journal.pone.0031573 PMid:22363677.
» https://doi.org/10.1371/journal.pone.0031573 -
LI, W.J., WANG, C.W., TAO, L., YAN, Y.H., ZHANG, M.J., LIU, Z.X., ZHAO, Y.Y. and LIU, J., 2021. Insulin signaling regulates longevity through protein phosphorylation in Caenorhabditis elegans. Nature Communications, vol. 12, no. 1, pp. 4568. https://doi.org/10.1038/s41467-021-24816-z PMid:34315882.
» https://doi.org/10.1038/s41467-021-24816-z -
LUNDWALL, Å., PERSSON, M., HANSSON, K. and JONSSON, M., 2020. Identification of the major rabbit and guinea pig semen coagulum proteins and description of the diversity of the REST gene locus in the mammalian clade Glires. PLoS One, vol. 15, no. 10, pp. e0240607. https://doi.org/10.1371/journal.pone.0240607 PMid:33052982.
» https://doi.org/10.1371/journal.pone.0240607 -
MAGALHÃES JUNIOR, M.J., MARTINS, L.F., SENRA, R.L., SANTOS, T.F., OKANO, D.S., PEREIRA, P.R., FARIA-CAMPOS, A., CAMPOS, S.V., GUIMARÃES, J.D. and BARACAT-PEREIRA, M.C., 2016. Differential abundances of four forms of Binder of SPerm 1 in the seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability. Theriogenology, vol. 86, no. 3, pp. 766-777.e2. https://doi.org/10.1016/j.theriogenology.2016.02.030 PMid:27118515.
» https://doi.org/10.1016/j.theriogenology.2016.02.030 -
MOHANTY, G., SANCHEZ-CARDENAS, C., PAUDEL, B., TOURZANI, D.A., SALICIONI, A.M., SANTI, C.M. and VISCONTI, P.E., 2024. Differential role of bovine serum albumin and HCO3- in the regulation of GSK3 alpha during mouse sperm capacitation. Molecular Human Reproduction, vol. 30, no. 3, pp. gaae007. https://doi.org/10.1093/molehr/gaae007 PMid:38341666.
» https://doi.org/10.1093/molehr/gaae007 -
MOROHOSHI, K., YAMAZAKI, T., KITO, K., SATO, B., KANG, W., HIBINO, T., NIEMI, S., LIU, Z., JOKI-ERKKILÄ, M. and MIYADO, K., 2021. Identification of an antibacterial polypeptide in mouse seminal vesicle secretions. Journal of Reproductive Immunology, vol. 148, pp. 103436. https://doi.org/10.1016/j.jri.2021.103436 PMid:34700103.
» https://doi.org/10.1016/j.jri.2021.103436 -
NADERI, A., FARMAKI, E., CHAVEZ, B., CAI, C., KAZA, V., ZHANG, Y., ARYANNA, S. and MEERAN, M., 2022. Beneficial effects of CCL8 inhibition at lipopolysaccharide-induced lung injury. iScience, vol. 25, no. 12, pp. 105520. https://doi.org/10.1016/j.isci.2022.105520 PMid:36404927.
» https://doi.org/10.1016/j.isci.2022.105520 -
NODA, T. and IKAWA, M., 2019. Physiological function of seminal vesicle secretions on male fecundity. Reproductive Medicine and Biology, vol. 18, no. 3, pp. 241-246. https://doi.org/10.1002/rmb2.12282 PMid:31312102.
» https://doi.org/10.1002/rmb2.12282 -
NODA, T., FUJIHARA, Y., MATSUMURA, T., OURA, S., KOBAYASHI, S. and IKAWA, M., 2019. Seminal vesicle secretory protein 7, PATE4, is not required for sperm function but for copulatory plug formation to ensure fecundity. Biology of Reproduction, vol. 100, no. 4, pp. 1035-1045. https://doi.org/10.1093/biolre/ioy247 PMid:30452524.
» https://doi.org/10.1093/biolre/ioy247 -
OU, C.M., LEE, R.K., LIN, M.H., LU, C.H., YANG, T.H., YEH, L.Y., KUO, Y.W. and CHEN, Y.J., 2021. A mouse seminal vesicle-secreted lysozyme c-like protein modulates sperm capacitation. Journal of Cellular Biochemistry, vol. 122, no. 6, pp. 653-666. https://doi.org/10.1002/jcb.29894 PMid:33469950.
» https://doi.org/10.1002/jcb.29894 -
PATLAR, B., 2022. On the Role of Seminal Fluid Protein and Nucleic Acid Content in Paternal Epigenetic Inheritance. International Journal of Molecular Sciences, vol. 23, no. 23, pp. 14533. https://doi.org/10.3390/ijms232314533 PMid:36498858.
» https://doi.org/10.3390/ijms232314533 -
PÉREZ-CEREZALES, S., LAGUNA-BARRAZA, R., DE CASTRO, A.C., SÁNCHEZ-CALABUIG, M.J., CANO-OLIVA, E., DE CASTRO-PITA, F.J., GUTIÉRREZ-ADÁN, A. and PINTADO, B., 2018. Sperm selection by thermotaxis improves ICSI outcome in mice. Scientific Reports, vol. 8, no. 1, pp. 2902. https://doi.org/10.1038/s41598-018-21335-8 PMid:29440764.
» https://doi.org/10.1038/s41598-018-21335-8 -
RODRÍGUEZ-MARTÍNEZ, H., KVIST, U., ERNERUDH, J., SANZ, L. and CALVETE, J.J., 2011. Seminal plasma proteins: what role do they play? American Journal of Reproductive Immunology : AJRI, vol. 66, no. suppl. 1, pp. 11-22. https://doi.org/10.1111/j.1600-0897.2011.01033.x PMid:21726334.
» https://doi.org/10.1111/j.1600-0897.2011.01033.x -
ROMANO-CARRATELLI, C., GALDIERO, M., NUZZO, I., BENTIVOGLIO, C., PORTA, R., PELUSO, G., RAVAGNAN, G. and METAFORA, S., 1995. In vivo inhibition of cell-mediated and humoral immune responses to cellular antigens by SV-IV, a major protein secreted from the rat seminal vesicle epithelium. Journal of Reproductive Immunology, vol. 28, no. 1, pp. 15-30. https://doi.org/10.1016/0165-0378(94)00900-R PMid:7738913.
» https://doi.org/10.1016/0165-0378(94)00900-R -
RUIZ-DÍAZ, S., GRANDE-PÉREZ, S., ARCE-LÓPEZ, S., TAMARGO, C., OLEGARIO HIDALGO, C. and PÉREZ-CEREZALES, S., 2020. Changes in the cellular distribution of tyrosine phosphorylation and its relationship with the acrosomal exocytosis and plasma membrane integrity during in vitro capacitation of frozen/thawed bull spermatozoa. International Journal of Molecular Sciences, vol. 21, no. 8, pp. 2725. https://doi.org/10.3390/ijms21082725 PMid:32326382.
» https://doi.org/10.3390/ijms21082725 -
SATI, L., CAYLI, S., DELPIANO, E., SAKKAS, D. and HUSZAR, G., 2014. The pattern of tyrosine phosphorylation in human sperm in response to binding to zona pellucida or hyaluronic acid. Reproductive Sciences, vol. 21, no. 5, pp. 573-581. https://doi.org/10.1177/1933719113504467 PMid:24077441.
» https://doi.org/10.1177/1933719113504467 -
SAWATPANICH, T., ARUN, S., TONGPAN, S., CHAICHUN, A., SAMPANNANG, A., SUKHORUM, W. and IAMSAARD, S., 2018. Localization and changes of tyrosine phosphorylated proteins and β actin in epididymis of rats treated with valproic acid. International Journal of Morphology, vol. 36, no. 3, pp. 835-840. https://doi.org/10.4067/S0717-95022018000300835
» https://doi.org/10.4067/S0717-95022018000300835 -
SAWATPANICH, T., CHAIMONTRI, C., WU, A.T., IAMSAARD, S. and YANNASITHINON, S., 2022. Dolichandrone serrulata flower improves seminal biochemical parameters and proteins in T2DM rats induced by a high-fat diet and streptozotocin. Pharmaceutical Biology, vol. 60, no. 1, pp. 1935-1943. https://doi.org/10.1080/13880209.2022.2124279 PMid:36205598.
» https://doi.org/10.1080/13880209.2022.2124279 -
SILVA, L.S., RUIVO, M.A., DA CRUZ, T.M., MENEGUELLO, J.E., PILAU, E.J., CARUSO, Í.P., MARANGONI, S., DE OLIVEIRA, C.I. and DE SOUZA, F.P., 2021. Structural and biophysical characterization of the major proteins from the seminal plasma of Dorper rams. Theriogenology, vol. 166, pp. 64-70. https://doi.org/10.1016/j.theriogenology.2021.02.019 PMid:33689929.
» https://doi.org/10.1016/j.theriogenology.2021.02.019 -
SIXTO-LÓPEZ, Y., BELLO, M. and CORREA-BASURTO, J., 2020. Exploring the inhibitory activity of valproic acid against the HDAC family using an MMGBSA approach. Journal of Computer-Aided Molecular Design, vol. 34, no. 8, pp. 857-878. https://doi.org/10.1007/s10822-020-00304-2 PMid:32180123.
» https://doi.org/10.1007/s10822-020-00304-2 -
SKERRETT-BYRNE, D.A., TRIGG, N.A., BROMFIELD, E.G., DUN, M.D., BERNSTEIN, I.R., ANDERSON, A.L., STANGER, C.J., JONDI, J.A., BANYA, J., XAVIER, M.J., DE IULIIS, G.N., SCOTT, R.J., SMITH, N.D. and NIXON, B., 2021. Proteomic dissection of the impact of environmental exposures on mouse seminal vesicle function. Molecular & Cellular Proteomics, vol. 20, pp. 100107. https://doi.org/10.1016/j.mcpro.2021.100107 PMid:34089863.
» https://doi.org/10.1016/j.mcpro.2021.100107 -
SONG, W., HU, L., MA, Z., YANG, L. and LI, J., 2022. Importance of tyrosine phosphorylation in hormone-regulated plant growth and development. International Journal of Molecular Sciences, vol. 23, no. 12, pp. 6603. https://doi.org/10.3390/ijms23126603 PMid:35743047.
» https://doi.org/10.3390/ijms23126603 -
SOUZA, C.E., REGO, J.P., LOBO, C.H., OLIVEIRA, J.T., NOGUEIRA, F.C., DOMONT, G.B., FIORAMONTE, M., MOURA, A.A. and MOURA, A.A., 2012. Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams. Journal of Proteomics, vol. 75, no. 14, pp. 4436-4456. https://doi.org/10.1016/j.jprot.2012.05.039 PMid:22659383.
» https://doi.org/10.1016/j.jprot.2012.05.039 -
SUKHORUM, W. and IAMSAARD, S., 2017. Changes in testicular function proteins and sperm acrosome status in rats treated with valproic acid. Reproduction, Fertility, and Development, vol. 29, no. 8, pp. 1585-1592. https://doi.org/10.1071/RD16205 PMid:27511211.
» https://doi.org/10.1071/RD16205 -
TARAYRAH-IBRAHEIM, L., MAURICE, E.C., HADARY, G., BEN-HUR, S., KOLPAKOVA, A., BRAUN, T., LI, J., GROSS-THENIK, N. and REIFER, J., 2021. DNase II mediates a parthanatos-like developmental cell death pathway in Drosophila primordial germ cells. Nature Communications, vol. 12, no. 1, pp. 2285. https://doi.org/10.1038/s41467-021-22622-1 PMid:33863891.
» https://doi.org/10.1038/s41467-021-22622-1 -
TONGPAN, S., SUKHORUM, W., ARUN, S., SAWATPHANICH, T. and IAMSAARD, S., 2019. Valproic acid changes the expression of tyrosine-phosphorylated proteins in rat seminal vesicle. Andrologia, vol. 51, no. 7, pp. e13303. https://doi.org/10.1111/and.13303 PMid:31033021.
» https://doi.org/10.1111/and.13303 -
TUFANO, M.A., PORTA, R., FARZATI, B., DI PIERRO, P., ROSSANO, F., CATALANOTTI, P., SOMMESE, L. and METAFORA, S., 1996. Rat seminal vesicle protein SV-IV and its transglutaminase-synthesized polyaminated derivative SPD2-SV-IV induce cytokine release from human resting lymphocytes and monocytes in vitro. Cellular Immunology, vol. 168, no. 2, pp. 148-157. https://doi.org/10.1006/cimm.1996.0061 PMid:8640860.
» https://doi.org/10.1006/cimm.1996.0061 -
VISCONTI, P.E., BAILEY, J.L., MOORE, G.D., PAN, D., OLDS-CLARKE, P. and KOPF, G.S., 1995. Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development (Cambridge, England), vol. 121, no. 4, pp. 1129-1137. https://doi.org/10.1242/dev.121.4.1129 PMid:7743926.
» https://doi.org/10.1242/dev.121.4.1129 -
WANG, T.E., YEH, L.Y., KUO-KUANG LEE, R., LU, C.H., YANG, T.H., KUO, Y.W., CHEN, Y.J. and OU, C.M., 2021. Secretory mouse quiescin sulfhydryl oxidase 1 aggregates defected human and mouse spermatozoa in vitro and in vivo. iScience, vol. 24, no. 10, pp. 103167. https://doi.org/10.1016/j.isci.2021.103167 PMid:34667943.
» https://doi.org/10.1016/j.isci.2021.103167 -
WESTFALEWICZ, B., DIETRICH, M.A., MOSTEK, A., PARTYKA, A., BIELAS, W., NIŻAŃSKI, W. and CIERESZKO, A., 2017. Analysis of bull (Bos taurus) seminal vesicle fluid proteome in relation to seminal plasma proteome. Journal of Dairy Science, vol. 100, no. 3, pp. 2282-2298. https://doi.org/10.3168/jds.2016-11866 PMid:28041731.
» https://doi.org/10.3168/jds.2016-11866 -
WULHFARD, S., BALDI, L., HACKER, D.L. and WURM, F., 2010. Valproic acid enhances recombinant mRNA and protein levels in transiently transfected Chinese hamster ovary cells. Journal of Biotechnology, vol. 148, no. 2-3, pp. 128-132. https://doi.org/10.1016/j.jbiotec.2010.05.003 PMid:20510314.
» https://doi.org/10.1016/j.jbiotec.2010.05.003 -
YOKOYAMA, S., YASUI-FURUKORI, N., NAKAGAMI, T., MIYAZAKI, K., ISHIOKA, M., TARAKITA, N., KUROGAWAGUCHI, K. and NAKAMURA, K., 2020. Association between the serum carnitine level and ammonia and valproic acid levels in patients with bipolar disorder. Therapeutic Drug Monitoring, vol. 42, no. 5, pp. 766-770. https://doi.org/10.1097/FTD.0000000000000778 PMid:32482992.
» https://doi.org/10.1097/FTD.0000000000000778
Edited by
-
Editor:
Marcelo A.M. Esquisatto
