RNASET2 in human spermatozoa and seminal plasma: a novel relevant indicator for asthenozoospermia

Authors


Correspondence:

Zhide Ding, Department of Histology and Embryology, School of Medicine, Shanghai Jiao Tong University, No.280, Chongqing Road (South), Shanghai 200025, China. E-mail: zding@shsmu.edu.cn

Summary

Adequate sperm motility is requisite for human fertilization, whereas the underlying causes or mechanisms of impaired sperm motility, asthenozoospermia, still remain largely unknown. RNASET2 (Ribonuclease T2) may be one of the effectors modulating human sperm motility. We determined if there is a correlation between RNASET2 expression levels in human semen from asthenozoospermia and fertile individuals. Thus, RNASET2 expression levels in spermatozoa and seminal plasma of healthy and asthenozoospermia individuals were evaluated using Western blot, laser scanning confocal microscope analysis, ELISA and flow cytometry. The results revealed that RNASET2 expression was identified in both human spermatozoa and seminal plasma. In spermatozoa from fertile individuals, it was localized to the acrosome, neck and the middle piece of tail regions. However, in spermatozoa from asthenozoospermia individuals (n = 67), RNASET2 staining was especially more frequent and evident in the neck and middle piece than that in fertile individuals (n = 59, < 0.01). Similarly, higher RNASET2 expression was also apparent in seminal plasma from asthenozoospermia than in fertile individuals (< 0.01). Moreover, purified RNASET2 had an inhibitory effect on sperm motility, especially on progressive motility (n = 23, < 0.05). In conclusion, higher expression of RNASET2 in the semen of asthenozoospermia individuals may contribute to sperm motility impairment.

Introduction

Asthenozoospermia, or poor sperm motility, is a common cause of human male infertility. One clinical study showed that, more than 80% of the spermatozoa from infertile patients revealed defects in motility and 19% of them had asthenozoospermia without any other defects in sperm number or morphology (Curi et al., 2003). Undoubtedly, sperm motility plays a critical role in fertilization and is dependent on flagellar function in the sperm tail.

Actin is a major cytoskeletal protein and has well-established roles in regulating cell shape, migration and interaction with extracellular matrices through reversible transformations between monomeric G-actin and filamentous F-actin (McNiven et al., 2000; Insall & Machesky, 2009). Human spermatozoa are motile and rich in actin, which is present in the acrosome, post-acrosomal area, neck (connecting piece) and the sperm tail (Flaherty et al., 1988; Dvoráková et al., 2005).

Interestingly, recent cancer studies suggested that one of the RNase T2 family members, ACTIBIND, produced by Aspergillus niger, could compete with angiogenin binding to cytoplasmic actin of various cancerous cells. Its interference with the actin network suppressed cell growth and migration and thereby enhanced antiangiogenic and anticarcinogenic characteristics (Roiz et al., 2006; Schwartz et al., 2007). In humans, the only member of RNases T2 family, called ribonuclease T2 (RNASET2), is found to be encoded by a tumour suppressor gene located on chromosome 6q27 (Acquati et al., 2001; Lin & Morin, 2001; Acquati et al., 2005).

Thus, these relevant facts prompted us to determine whether RNASET2 is present in human semen. Secondly, if it is there, we asked if: (i) this RNase could be involved in the control of sperm motility; (ii) decreased sperm motility in human asthenozoospermia (sperm motility impairment only) is associated with higher RNASET2 levels than in fertile semen.

We report here on a correlation between increases in RNASET2 content in human asthenozoospermia semen and declines in sperm motility. This was carried out by first identifying RNASET2 expression levels in the human spermatozoa and seminal plasma of healthy and asthenozoospermia individuals. Then, immunocytochemistry was performed to determine if RNASET2 co-localizes with the actin containing cytoskeletal network in fertile human spermatozoa. Furthermore, we compared RNASET2 localization with that of asthenozoospermic spermatozoa. Finally, purified RNase T2 protein was employed to determine if it has an inhibitory effect on human sperm motility.

Materials and methods

Specimens

Human semen specimens were obtained from Shanghai Jiai Genetics & IVF Institute-China USA Center. Use of the semen samples was approved by the Ethics Committee of this unit and all donors (20–35 years old) gave written informed consent for the use of their leftover semen samples when all IVF treatments finished. Semen analysis was performed using a computer assisted semen analyser (CASA) according to World Health Organization guidelines (WHO, 1999). The percentage of motile spermatozoa (total motility) was 16.9 ± 6.6% from asthenozoospermia patients (n = 70) and 59.5 ± 7.9% from the fertile donors (n = 98). The sperm density was higher than 2 × 107 sperm/mL in all semen samples and no samples were selected from asthenozoospermia patients for this study if the number of spermatozoa with abnormal morphology was higher than the prescribed WHO guidelines.

Prokaryotic expression and purification of recombinant RNASET2

Human epididymal cDNA was a kind gift from Dr. Qiang Liu at the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. A segment of RNASET2 was amplified with the forward primer 5′-GCGGATCCCCCGATAAAAGTGAAGGA- 3′ (containing BamHI site, underlined) and the reverse primer 5′-GCAAGCTTAGGTGGGGGATAGAAGAC- 3′ (containing HindIII site, underlined). Prokaryotic expression and purification of recombinant RNASET2 were performed as previously described (Qu et al., 2007). The new recombinant prokaryotic expression vector was named pET-28a(+)/RNASET2 and propagated in Escherichia coli BL21 (DE3) host cells cultured with 0.8 mm isopropyl-β-d-thiogalactopyranoside (IPTG) for 5 h at 37 °C with gentle shaking. Recombinant protein was purified based on its His6-tag by affinity chromatography using a Ni-NTA His-bind resin (Novagen, San Diego, CA, USA) and the purified recombinant RNASET2 was identified by liquid chromatography-mass spectrometry (LC-MS/MS), using a LTQ-Orbitrap hybrid mass spectrometer (Thermo Finnigan, Bremen, Germany) equipped with a Finnigan Dynamic nano-spray source.

Production of polyclonal rabbit anti-rRNASET2 antibodies

Polyclonal anti-recombinant RNASET2 antibodies were produced by immunizing white New Zealand rabbits (~6 months old, body weight ~2.5 kg; purchased from Shanghai SLAC Laboratory Animal Co., Shanghai, China), as previously described (Qu et al., 2007). The titre of rabbit anti-rRNASET2 sera was detected by ELISA at 450 nm in conjunction with an Anthos Zenyth 1100 multimode detector (Anthos Labtec Instruments GmbH, Wals, Austria) with a 5-s pre-read shaker. Finally, the immunized rabbit IgG (including anti-RNASET2 IgG) was purified through immunoaffinity chromatography from crude rabbit sera, using the ImmunoPure (G) IgG Purification kit (Pierce, Rockford, IL, USA). In the meantime, normal rabbit IgG was purified from pre-immunized rabbit sera.

Identification of RNASET2 in human spermatozoa and seminal plasma

Fresh human semen specimens were centrifuged (800 g, 15 min, 4 °C) to separate spermatozoa from seminal plasma. Spermatozoa were washed thrice with PBS and lysed with 8 m urea containing protease inhibitor cocktails (Ameresco, Solon, OH, USA). After centrifugation at 12 000 g, 10 min, 4 °C, the supernatant containing sperm protein was stored at −80 °C immediately for further analyses. Seminal plasma was diluted with 20 volumes of PBS and centrifuged (12 000 g, 10 min, 4 °C) to remove the dissolvable components and seminal plasma protein was also stored at −80 °C immediately until following analyses.

Western blot was performed as previously described (Qu et al., 2007). Protein samples (30 μg) were separated by SDS-PAGE, then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) using a semi-dry transfer apparatus (Bio-Rad, Hercules, Calif, USA); membranes were blocked with 5% bovine serum albumin (BSA) in TBS containing 0.2% Tween-20. Immunoblotting was performed with purified rabbit anti-RNASET2 polyclonal antibodies at a 1 : 2000 dilution. Normal rabbit IgG (Upstate, Temecula, CA, USA) was used as negative control at the same dilution, followed by incubation with goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (Abgent, San Diego, CA, USA) at a 1 : 10 000 dilution. Signals were detected by enhanced chemiluminescence (Millipore) according to the manufacturers' protocol. Meanwhile, β-actin (1 : 5000; Abcam, Cambridge, MA, USA) was treated as the internal reference. Finally, the average grey scale levels of the corresponding protein blot were calculated.

RNASET2 and actin co-localization on human spermatozoa analysed with indirect immunofluorescence

To identify the localization of RNASET2 on human spermatozoa, high quality spermatozoa was obtained by a discontinuous 47/90% Percoll density gradient centrifugation at 800 g for 30 min at 4 °C as described (Qu et al., 2007) and subsequently washed thrice with PBS. Acrosome reaction (AR) was induced using a calcium ionophore, 10 μm A23187 (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C. For immunofluorescence studies, the sperm suspension was added onto slides, fixed with pre-cooled acetone for 15 min. All subsequent incubations were performed in a humid chamber. Then, the slides were blocked with 5% BSA in PBS for 30 min at room temperature, and later incubated with the mixture of purified rabbit anti-RNASET2 polyclonal antibodies (1 : 100 diluted in PBS) and mouse anti-human actin monoclonal antibody (1 : 50 dilution; AbD Serotec, Kidlington, Oxford, UK) overnight at 4 °C. Meanwhile, the mixture consisted of normal rabbit IgG (1 : 100 dilution; Millipore) and normal mouse IgG (1 : 50 dilution; Millipore) was used as negative control. After washing, the sperm samples were incubated with a mixture of fluorescein isothiocyanate (FITC) labelled goat anti-mouse IgG (1 : 100 dilution; Invitrogen, Eugene, Oregon, USA) and Rhodamine-conjugated goat anti-rabbit IgG (1 : 100 dilution; Pierce) for 1 h at 37 °C. For acrosome staining, Alexa Fluor® 647-conjugated lectin PNA from Arachis hypogaea (peanut) (Invitrogen) was applied to trace the sperm acrosome. Finally, the fluorescent stained sperm samples were viewed under laser scanning confocal microscope (LSCM; Carl Zeiss LSM-510, Jena, Germany). The immunofluorescence test was repeated three times and semen from four fertile donors was combined for each co-localization experiment.

Identification of RNASET2 binding to actin in human seminal plasma

ELISA probed in human seminal plasma for RNASET2 binding to actin. Actin (5 μg/mL) protein isolated from human platelet (Cytoskeleton, Denver, CO, USA), diluted with bicarbonate buffer (6.17 mg/mL sodium bicarbonate, 1 mg/mL sodium carbonate, pH 9.5), was coated directly onto COSTAR EIA/RIA plate (Corning, NY, USA) over night at 4 °C. Then, the plate was blocked with 5% (w/v) BSA in PBS containing 0.25% Tween-20 (PBST) at room temperature for 1 h and subsequently incubated with human seminal plasma (1 : 100 diluted in PBST) at 4 °C over night. The plate was washed three times with wash buffer (2 mm Tris-HCl, 0.25% Tween-20, pH 7.4) for 10 min each at room temperature and then was incubated with either rabbit anti-RNASET2 polyclonal antibodies (1 : 1000 diluted in PBST) or normal rabbit IgG (Millipore) at the same dilution at 37 °C for 1 h. After three times washing, 1 μg/mL HRP-conjugated goat anti-rabbit IgG (Upstate) was added to the plate. Signals were generated by TMB solution (Ameresco) and then the relevant optical absorbance was detected at 450 nm with Anthos Zenyth 1100 multimode detector. Meanwhile, to ensure the specificity of this indirect ELISA test, human seminal plasma and RNase A protein (5 μg/mL, a negative control) were individually coated onto a COSTAR EIA/RIA plate, and then exposed to human actin protein (1 μg/mL), followed by mouse anti-actin monoclonal antibody (AbD Serotec) and corresponding secondary antibodies. Finally, signals were generated by TMB solution and the relevant optical absorbance was monitored as described as above.

Identification of RNASET2 binding to actin in human spermatozoa

Immunoprecipitation combined with Western blot was applied to detect RNASET2 binding to actin in human spermatozoa. Human spermatozoa separated from semen by Percoll density gradient centrifugation were lysed with RIPA buffer (Pierce) supplied with protease inhibitors (Ameresco) and kept in a rotating ice bath for at least 30 min to allow proteins to dissolve completely. Centrifugation (12 000 g, 10 min) was then applied to get rid of the sediment and 500 μL of sperm protein sample was incubated with 10 μg of either rabbit anti-RNASET2 antibodies or mouse anti-actin monoclonal antibody overnight at 4 °C. Meanwhile, normal rabbit IgG (Millipore) and normal mouse IgG (Millipore) were used as negative controls. Then, 50 μL of Protein G agarose slurry was added to the sperm-antibody complex and the reaction was incubated with gentle mixing for 4 h at 4 °C. The agarose beads were washed with TBS by centrifugation three times. Twenty microlitre of reducing protein sample buffer (Thermo Scientific, Rockford, IL, USA) was then added and incubated for 5 min at 95 °C. Finally, the supernatant was collected and identified by Western blot as previously described. For Western blot analyses, the samples precipitated with mouse anti-actin monoclonal antibody or normal mouse IgG were detected with rabbit anti-RNASET2 antibodies. Accordingly, the samples precipitated with rabbit anti-RNASET2 antibodies or normal rabbit IgG were identified by mouse anti-actin monoclonal antibody as well.

Comparison of RNASET2 between normozoospermic and asthenozoospermic semen

Detection of RNASET2 on spermatozoa was carried out using indirect immunofluorescence in conjunction with flow cytometry and LSCM. Indirect immunofluorescent labelling of spermatozoa from both healthy donors and asthenozoospermia was performed as following. Spermatozoa and seminal plasma were separated by centrifugation (800 g, 15 min, 4 °C). The spermatozoa containing suspensions were washed thrice with PBS and fixed with 4% paraformaldehyde for 20 min. Then, the sperm samples were incubated with rabbit anti-RNASET2 polyclonal antibodies (1 : 100 diluted in PBS) overnight at 4 °C. After washing, samples were incubated with FITC-conjugated goat anti-rabbit IgG (1 : 100 dilution; Invitrogen, Chicago, IL, USA) for 1 h at 37 °C. Finally, sperm samples were analysed by flow cytometry on a Becton Dickinson FACS Calibur Flow Cytometry System (Becton Dickinson, Beckman Coulter, Brea, CA, USA). For each suspension, 3 × 104 spermatozoa were analysed for RNASET2-positive signal percentage and means of fluorescence intensity. Emission data were collected and analysed using CellQuest software (Beckman Coulter). The alteration of RNASET2 of spermatozoa between that from healthy donors and asthenozoospermia was further confirmed by LSCM analysis.

Detection of RNASET2 in the seminal plasma from both healthy donors and asthenozoospermia was performed using the ELISA as described above. Seminal plasma (1 : 200 diluted with bicarbonate buffer) was coated onto COSTAR EIA/RIA plate and reacted against the rabbit anti-RNASET2 polyclonal antibodies (1: 1000 diluted in PBST), followed by exposure to HRP-conjugated goat anti-rabbit IgG (1 : 2000 diluted in PBST, Upstate) antibody. Signals were generated by TMB solution and then the relevant optical absorbance was detected at 450 nm with Anthos Zenyth 1100 multimode detector. RNASET2 levels in the both spermatozoa and seminal plasma were further confirmed by Western blot analysis as described above.

RNase T2 purification, identification and inhibitory effect assessments on human sperm motility

Extracellular RNase T2 secreted by Aspergillus niger, also named ACTIBIND, was purified as described (Roiz et al., 2000). Briefly, purification of RNase T2 from growth medium of A. niger consisted of two steps. The crude fractions around the RNase T2 peak were eluted at 0.62 m NaCl from an EMD-TMAE column and then, the RNase T2 was eluted from a Mono Q column at 0.5 m NaCl. The eluant containing relative pure RNase T2 protein was ultrafiltrated and dialysed with PBS at 4 °C overnight. Subsequently, the concentration of purified RNase T2 was determined by BCA assay. Finally, it was identified by both SDS-PAGE and Western blot using anti-RNASET2 antibodies and stored in −80 °C for further functional assay.

For determination of the RNase T2 inhibitory effect on human sperm motility, motile, intact spermatozoa were obtained using the swim-up method as described (Liu et al., 2012). Sperm aliquots were incubated in Tyrode's buffer (Sigma-Aldrich) at 37 °C and 5% CO2. All the sperm suspension used in the assay contained 15–30 million sperm/mL and at least 85% of the spermatozoa were motile (total motility). Each sperm suspension was divided into three groups and incubated with either purified RNase T2 protein, or RNase A (a negative control), or without any treatment (blank) for 1–2 h. Sperm motility analysis was performed using a CASA.

Statistical analyses

Data were analysed using the SPSS software (Chicago, IL, USA), and all values are reported as mean ± SEM except for results of sperm motility inhibiting tests presented as mean ± SD. Group comparisons were made using Student's t-test, as appropriate. One-way analysis of variance (anova) test was used assuming a two-tail hypothesis with < 0.05. Correlations were performed using Pearson's rank correlation test (Spearman's rho) and simple linear regression.

Results

Production of recombinant RNASET2 and anti-RNASET2 polyclonal antibodies

A 549 bp fragment was successfully amplified from human epididymal cDNA with designed RNASET2 primers by PCR and separated on a 1% agarose gel (Fig. 1a). The target fragment of RNASET2 was then cloned into the prokaryotic expression vector, which was named pET-28a(+)/RNASET2. This construct was predicted to encode a 230 amino acid-containing recombinant protein with molecular weight of ˜32 kDa. Use of a 15% SDS-PAGE gel revealed that it was successfully overexpressed by inducing it with 0.8 mm IPTG at 37 °C, but was absent in non-induced cells (Fig. 1b). The recombinant protein was purified with Ni+ Sepharose and then confirmed by SDS-PAGE analysis (Fig. 1b). Finally, the purified recombinant protein was identified by LC-MS/MS to be RNASET2 precursor (Homo sapiens) (data not presented).

Figure 1.

Prokaryotic expression and purification of recombinant RNASET2 fragment, and production of polyclonal anti-human RNASET2 antibodies and identification of RNASET2 in human semen. (a) PCR product of RNASET2 fragment (549 bp). (b) Expression and purification of recombinant RNASET2 fragment in Escherichia coli BL21 (DE3) host cells. Different fractions of E. coli were separated on a Coomassie blue-stained 15% SDS-PAGE gel. Arrow shows the purified recombinant RNASET2 protein band. Lane M, protein molecular weight markers; Lane 1, total cell lysates containing pET-28a(+)/RNASET2 fragment before IPTG induction; Lane 2, total cellular protein after IPTG induction; Lane 3, RNASET2 protein eluted in PBS by purification. (c) Values of ELISA absorbance at 450 nm using anti-RNASET2 antibodies in rabbit sera were higher than that of pre-immunized sera (control). (d) Identification of the anti-RNASET2 antibodies (Lane 1) paralleling with anti-His tag antibody (Lane 3) reacted with recombinant RNASET2. Lane 2 and Lane 4 are negative controls corresponding to Lane 1 and Lane 3. (e) Identification of RNASET2 in human spermatozoa (Lane 1) and seminal plasma (Lane 3) by Western blot with raised antibodies. Lane 2 and Land 4 are negative controls corresponding to Lane 1 and Lane 3.

Polyclonal antibodies against the recombinant RNASET2 protein were generated by immunization of white New Zealand rabbits. On day 35, a rabbit was killed and its serum was collected. The results indicated that immunization of rabbit with recombinant RNASET2 induced a very strong IgG response against the antigen, as determined by indirect ELISA at 450 nm. The titres of rabbit anti-RNASET2 sera were fairly high compared with that of pre-immune serum (control), reaching 1 : 1 024 000 (Fig. 1c). Eventually, anti-RNASET2 antibodies (without sodium azide) were then purified from crude rabbit sera, confirmed by Western blotting (Fig. 1d) and stored at −80 °C for further in vitro functional analysis.

Identification of RNASET2 in human spermatozoa and seminal plasma

Western blot analysis characterized RNASET2 expression in human semen. Our generated anti-RNASET2 polyclonal antibodies were employed to react with extracts of human spermatozoa or seminal plasma. A ~36 kDa band indicative of RNASET2 was detected in both human spermatozoa and seminal plasma (Fig. 1e).

Co-localization of RNASET2 with actin in human spermatozoa

Immunofluorescence co-localization assay was applied to identify the relationship between RNASET2 and actin protein in human spermatozoa. A mixture of anti-RNASET2 polyclonal antibodies and anti-actin monoclonal antibody as the primary antibodies was applied, and two secondary antibodies conjugated with different fluorochromes were visualized using LSCM analysis. Even though their staining patterns were quite distinct from one another, some co-localization was obvious in some regions of the spermatozoa (Fig. 2a and b). Anti-actin antibody stained the sperm acrosome, equatorial segment, neck and tail regions (middle piece). Similarly, some anti-RNASET2 antibodies frequently stained these regions predominantly in the neck and middle piece. Thus, much of the staining with both antibodies was mainly co-localized in the acrosome region, sperm neck and tail, especially in the neck and middle piece. After AR, fluorescence of actin on sperm head was weakened, especially reduced in the equatorial segment. However, the localization of RNASET2 was changed only a little. It remained co-localized based on staining of the two proteins in the sperm neck and tail (Fig. 2b).

Figure 2.

Localization of RNASET2 and actin on human spermatozoa by indirect immunofluorescence analysis. (a) The results showed that the both RNaseT2 and actin have similar localization on the acrosome, neck and tail regions of the human spermatozoa, especially on the neck and middle piece. a1, Immunofluorescence analysis of actin. a2, Immunofluorescence analysis of RNASET2. a3, Differential interference contrast image. a4, Merged image of a1–3. a5–8, negative control. (b) Magnified figures showed the localization of RNASET2 and actin on normal ejaculated spermatozoa and acrosome-reacted spermatozoa. b1–5, showed the localization of RNASET2 and actin on normal ejaculated spermatozoa similar to (A). b6–10, showed the localization of RNASET2 and actin on acrosome-reacted spermatozoa. It remained co-localized based on staining of the two proteins in the sperm neck and tail.

Identification of RNASET2 in human seminal plasma interacted with actin

Binding of RNASET2 in human seminal plasma to actin was analysed by indirect ELISA. A positive signal was detected when a plate was treated with anti-RNASET2 polyclonal antibodies (1.03 ± 0.05, n = 6), however, its intensity dramatically decreased when exposed to rabbit normal IgG (0.03 ± 0.005, n = 6, < 0.01, Fig. 3a). This decline suggested that RNASET2 in seminal plasma was bound to immobilized actin. Similarly, the signal was also significant when human seminal plasma was coated onto a plate and then incubated with human actin protein overnight followed by actin detection (mouse anti-actin monoclonal antibody: 0.58 ± 0.06, n = 6; mouse normal IgG: 0.05 ± 0.023, n = 6, < 0.01, Fig. 3a). As a negative control, RNase A was coated onto plate and it faintly reacted with actin, which generated a relatively weak signal (0.15 ± 0.03, n = 6, < 0.01, Fig. 3a).

Figure 3.

Identification of RNASET2 binding to actin in human semen. (a) Identification of RNASET2 in human seminal plasma binding to actin analysed with indirect ELISA. Intensive positive signals were observed when seminal plasma was reacted with immobilized actin detected by anti-RNASET2 antibodies ( 0.01). On the other hand, intense positive signals are also obvious when exogenous actin was reacted with immobilized seminal plasma and detected with anti-actin monoclonal antibody ( 0.01). Meanwhile, RNase A served as a negative control. (b) Identification of RNASET2 in human spermatozoa binding to actin analysed with immunoprecipitation. A distinct protein band at ~36 kDa indicative of RNASET2 was identified when sperm sample precipitated with mouse anti-actin antibody and probed with rabbit anti-RNASET2 antibodies (Lane 1). Similarly, A distinct protein band at ~43 kDa indicative of actin was identified when sperm sample precipitated with rabbit anti-RNASET2 antibodies and probed with mouse anti-actin antibody (Lane 3). Lane 2 and lane 4 were negative controls in which sperm samples were precipitated with either normal mouse IgG or normal rabbit IgG.

Identification of RNASET2 in human spermatozoa interacted with actin

Binding of RNASET2 in human spermatozoa to actin was analysed using immunoprecipitation followed by Western blot. A distinct protein band at ~36 kDa indicative of RNASET2 was identified when a sperm sample was precipitated with mouse anti-actin antibody and probed with rabbit anti-RNASET2 antibodies (Fig. 3b, Lane 1). Similarly, a distinct protein band at ~43 kDa indicative of actin was identified when a sperm sample was precipitated with rabbit anti-RNASET2 antibodies and probed with mouse anti-actin antibody (Fig. 3b, Lane 3). However, no signal was detected when sperm samples were precipitated with either normal mouse IgG or normal rabbit IgG. (Fig. 3b, Lane 2 and Lane 4).

RNASET2 expression levels in semen from healthy and asthenozoospermic males

Indirect immunofluorescence followed by flow cytometry analysis identified RNASET2 content in human spermatozoa. Each sample contained 3 × 104 spermatozoa. The donor populations for each group were 59 healthy and 67 asthenozoospermic individuals. Fluorescence analysis revealed that spermatozoa obtained from asthenozoospermia individuals were labelled with a higher frequency (54.57 ± 2.60%, n = 67) and higher fluorescence intensity (52.50 ± 3.76) compared with those from healthy donors (33.51 ± 2.35%, 36.54 ± 2.84, respectively, n = 59, < 0.01, Fig. 4a–c). Moreover, LSCM analysis found that the aggregation of RNASET2 was mainly in the neck and middle piece of tail regions (Fig. 5).

Figure 4.

Flow cytometry and ELISA analyses of RNASET2 expression in human semen samples from healthy donors and asthenozoospermia. (a–c) Flow cytometry analysis of two different sperm samples with RNASET2 immunofluorescent staining. Both the percentage of RNASET2-positive spermatozoa (b) and the mean of fluorescence intensity of RNASET2 (c) in asthenozoospermic spermatozoa expression are significantly higher than those from healthy donors ( 0.01). (d) Expression of RNASET2 based on ELISA analysis of seminal plasma is similar to that in spermatozoa ( 0.01). (e) RNASET2 content in seminal plasma closely correlates with those in corresponding spermatozoa for each individual semen samples (r = 0.78).

Figure 5.

Comparison of RNASET2 expression in human spermatozoa from healthy donors and asthenozoospermia by indirect immunofluorescence analysis. (N1) Immunofluorescence analysis of RNASET2 in healthy donors' spermatozoa. (a1) Immunofluorescence analysis of RNASET2 in asthenozoospermic spermatozoa. (a2) and (N2) Single spermatozoa in (a1) and (N1) were magnified to show the precise localization of immunofluorescent staining. White arrows indicated the aggregation of green fluorescence in the neck and middle piece of flagella from asthenozoospermic spermatozoa.

ELISA of RNASET2 in seminal plasma from healthy donors' and asthenozoospermic semen was performed accompanied by immunofluorescent analysis of the sperm sample. RNASET2 expression was up-regulated in the seminal plasma from asthenozoospermia (0.57 ± 0.04, n = 67) compared with that from healthy semen specimens (0.40 ± 0.03, n = 59, < 0.01, Fig. 4d). These results were also confirmed by Western blot analysis of RNASET2 expression in both asthenozoospermic and nomozoospermic spermatozoa and seminal plasma with raised antibodies (Fig. 6). Furthermore, we observed that the content of RNASET2 in human spermatozoa detected by immunofluorescence and conjugated with flow cytometry analysis was positively correlated with that in seminal plasma detected by ELISA (r = 0.78, Fig. 4e).

Figure 6.

Western blot analyses of RNASET2 expression in human semen samples from healthy donors and asthenozoospermia. (a) Identification with Western blot analysis of RNASET2 expression in asthenozoospermic spermatozoa and seminal plasma (Lane 1–4) and in nomozoospermic spermatozoa and seminal plasma (Lane 5–8) with raised antibodies. Meanwhile, β-actin was treated as the internal reference. (b) The average grey scale levels of the corresponding protein blot were calculated (* 0.05, ** 0.01).

Identification of purified RNase T2 from Aspergillus niger

Purified RNase T2 from A. niger was identified by both 12.5% SDS-PAGE and Western blot analyses. It exhibited a distinct protein band at ~32 kDa by SDS-PAGE (Fig. 7a) and a ~32 kDa band indicative of RNASET2 was also detected by Western blot (Fig. 7b). These data therefore provided direct evidences that RNase T2 was successfully separated and purified from the growth medium of A. niger.

Figure 7.

Purification and identification of RNase T2 from Aspergillus niger. (a) Proteins purified from growth medium of A. niger were separated by 12.5% SDS-PAGE and assessed using Coomassie blue stain. Arrow showed a protein band ~32 kDa indicative of RNase T2. Lane M, protein molecular weight markers; Lane 1, purified protein. (b) Identification of purified protein by Western blot. Lane 1, purified protein detected with anti-RNASET2 antibodies, revealed a positive band at ~32 kDa. Lane 2, a negative control using normal rabbit IgG as primary antibody.

Inhibitory effect of RNase T2 on human sperm motility

To determine the optimal RNase T2 concentration that efficiently inhibits human sperm motility, final concentrations of either 0.05, 0.10, 0.20, 0.50 or 1.00 mg/mL proteins were individually mixed with a sperm suspension. The inhibition efficiency on both sperm motility and progressive motility was measured with CASA. We found that 0.20 mg/mL protein inhibited both sperm motility (Fig. 8a, n = 6) as well as sperm progressive motility (Fig. 8b, n = 6). At this concentration level, spermatozoa treated with RNase T2 for 60 min had a lower motility (65.00 ± 18.94, n = 23) compared with non-treated spermatozoa (76.70 ± 13.98, n = 23, < 0.05) and on the other hand, there was no difference between spermatozoa treated with either RNase T2 or RNase A (a negative control, 73.09 ± 13.02, n = 23)(Fig. 8c). However, the progressive motility of RNase T2-treated group (34.83 ± 16.08, n = 23) was lower than that of both non-treated and control groups (46.52 ± 15.90 and 44.00 ± 13.85, respectively, n = 23, < 0.05) (Fig. 8d). Moreover, in spermatozoa treated for 120 min, both motility and progressive motility of RNase T2-treated group (58.91 ± 13.66 and 26.57 ± 13.84, respectively, n = 23) were lower than that of both non-treated and control groups (Blank: 71.52 ± 16.22 and 35.65 ± 13.66; Control: 70.13 ± 15.25 and 38.26 ± 13.54, respectively, n = 23, < 0.05) (Fig. 8c and d).

Figure 8.

Inhibitory effect of RNase T2 on human sperm motility. (a) Inhibitory effects of RNase T2 at 0.05, 0.10, 0.20, 0.50 or 1.00 mg/mL levels showed different efficiency to sperm motility, whereas RNase A acted as a negative control at same levels had no or tiny effects. (b) Different levels of RNase T2 had a variety of inhibitory effects on sperm progressive motility. (c) At the 0.20 mg/mL level, spermatozoa treated with RNase T2 for 60 min had a lower motility compared with non-treated spermatozoa (n = 23, * 0.05). In spermatozoa treated for 120 min, sperm motility of RNase T2-treated group was lower than that of both non-treated and control groups (n = 23, * 0.05). (d) At the 0.20 mg/mL level, spermatozoa treated with RNase T2 for 60 min or 120 min had a lower progressive motility compared with both non-treated and control groups (n = 23, * 0.05).

Discussion

Sperm fertility is dependent on its motility and its cytoskeletal architecture (Gibbons, 1996; Clark et al., 2004; Azamar et al., 2007; Correa et al., 2007). Sperm tail has an axoneme structure, surrounded by unique cytoskeletal structures, such as a fibrous sheath and outer dense fibres (Eddy et al., 2003; Inaba, 2003, 2011). The outer dense fibres have been suggested to be involved in modulating the flagellar curvature that also contributes to sperm fertility (Gastmann et al., 1993; Mariappa et al., 2010). Numerous studies demonstrated that in mammalian spermatozoa, a major cytoskeletal protein, actin is mainly found in the tail regions of as well as in the head and neck (Flaherty et al., 1988; Dvoráková et al., 2005). The ultrastructural level analysis indicates that actin is in the tail regions of human spermatozoa, and localized around the connecting piece of the neck and on the surface of the fibrous sheath (Flaherty et al., 1988). In addition, actin polymerization in spermatozoa plays an important role during capacitation and AR in several mammalian species including humans (Brener et al., 2003; Cabello-Agüeros et al., 2003; Etkovitz et al., 2007; Azamar et al., 2007). Actin's functional role was also demonstrated by showing that an anti-actin monoclonal antibody could dramatically inhibit human spermatozoa hyperactivation and that actin-binding protein significantly suppressed sperm motility and zona pellucida-induced acrosomal reaction in vitro (Liu et al., 2002; Capková et al., 2007).

Ribonuclease T2 family is widely distributed in a variety of species in the biosphere ranging from microbes, plants to animals. It is a non selective base acid RNase, whose family members are generally glycosylated secretory protein found in the extracellular milieu. Its expression is associated with many functions, such as scavenging of nucleic acids, degradation of self-RNA, modulating host immune response and serving as extra- or intracellular cytotoxins respectively (Schein, 1997; Strydom, 1998; Maeda et al., 2002). In cancer biology research, ACTIBIND, an RNase T2 family member, attracted much interest because of its newly discovered anti-cancer activities. We became interested in ACTIBIND because it could bind to actin with a molar ratio of 1 : 2 in vitro and in cancer cells it also disrupted the intracellular actin network by competing with angiogenin. On the other hand, several reports indicated that RNASET2, the only member of the T2 family of RNases in humans, is down-regulated in some human malignancies (Acquati et al., 2001; Lin & Morin, 2001; Acquati et al., 2005; Monti et al., 2008). It was established that RNASET2 binds to actin in vitro assays and controlled metastasis, tumorigenesis and angiogenesis (Smirnoff et al., 2006; Monti et al., 2008), but its catalytic RNase activity was unrelated to antitumorigenic activity. This difference strongly indicated that the antitumorigenic activity of the RNase T2 family members may be associated with actin binding rather than RNase activity (Smirnoff et al., 2006).

Although the biological function of RNASET2 in the male genital system remains still unclear, it is obvious that RNASET2 has RNase activity and actin-binding capability (Smirnoff et al., 2006; Luhtala & Parker, 2010). Thus, in this study, we focused on a newly reported actin-binding protein RNASET2. Our objective was to determine if its expression levels are different in human fertile semen and asthenozoospermia (sperm motility impairment only). Using the specific anti-RNASET2 antibodies, we firstly detected RNASET2 expression in human spermatozoa and seminal plasma with Western blot analysis. Immunofluorescence assay results showed that RNASET2 is localized in the acrosome, neck and tail regions of the spermatozoon, especially in the neck and middle piece. When probing for its localization with of actin, we found that their staining was not completely identical. Anti-actin monoclonal antibody stained the sperm acrosome, equatorial segment, neck and tail regions (middle piece). Thus, this enabled us to realize in human spermatozoa that they mainly co-localize with one another in the tail regions (connecting and middle pieces). This region provides sperm motility as this is where flagellum wagging occurs. Their close association clearly suggested a potential correlation between RNASET2 and sperm motility. Furthermore, the actin-binding capability of RNASET2 in both human spermatozoa and seminal plasma was confirmed by immunoprecipitation and indirect ELISA, which agrees with previous reports in which their identity was established based on membrane blotting and a cross-linking assay (Roiz et al., 2006; Smirnoff et al., 2006).

It was determined whether RNASET2 expression levels were different in the semen of fertile from those in asthenozoospermia individuals (sperm motility impairment only) based on ELISA detection, immunofluorescence and Western blot analyses of RNASET2. These assays evaluated its expression and frequency of occurrence in spermatozoa as well as its content in human seminal plasma from semen samples with different quality, including semen from healthy fertile men and asthenozoospermia. We found more frequent and high expression levels of RNASET2 on the spermatozoa from asthenozoospermic patients than in normozoospermic samples. A more detailed examination with LSCM analysis revealed that RNASET2 aggregation was mainly in the neck and middle pieces of tail regions of spermatozoa from asthenozoospermic semen, which was associated with sperm motility suppression. However, it should be pointed out that, in our experiments, the RNASET2 stain on individual spermatozoa was not completely identical within a single sample or among different samples. The reason for this discrepancy may be attributable to the fact that the spermatozoa in semen can be easily divided into different groups based on their motility statuses and there is considerable disparity in the constituents of semen from different men even though they were either fertile or asthenozoospermia. On the other hand, the content of RNASET2 in seminal plasma from asthenozoospermic semen was generally higher than that from healthy donors' semen. Nevertheless, there were still seven samples in the normozoospermic group (n = 59) with relatively high RNASET2 expression or content (the absorbance > 0.65 at 450 nm). Such outliers might account for their high sperm concentration in semen (>108/mL).

Finally, to confirm the inhibitory effect of RNASET2 on human sperm motility, we purified ACTIBIND and found that this RNase T2 efficiently inhibited human sperm motility in a concentration-dependant manner. At the 0.2 mg/mL level, it reduced sperm motility, especially for progressive motility during 60 min to 120 min treatment. These data therefore might account for why RNASET2 expression is high level of in asthenozoospermic semen. As it has already been described, excess expression of actin-binding protein on spermatozoa was associated with poor semen quality and more frequent fertilization failure (Capková et al., 2007).

Thus, herein we concluded that under physiological conditions, in human semen, RNASET2 might act as an actin-binding protein. Such an interaction makes it a natural suppressor or regulator of sperm motility. In this way, it prevents spermatozoa hyperactivation, but in semen RNASET2 up-regulation might instead lead to lower sperm motility and result in asthenozoospermia.

Acknowledgements

The authors thank Ms. Meige Lu, Ms. Ruyao Wang, Ms. Yanqin Hu (Shanghai Key Laboratory for Reproductive Medicine) for their technical assistance and Ms. Guiying Shi (Department of Biochemistry and Molecular Cell Biology, School of Medicine, Shanghai Jiao Tong University) for her work in flow cytometry. We are very grateful to Ms. Xiaofeng Tang, Ms. Ying Chen, Mr. Yulin Liu (Shanghai Jiai Genetics & IVF Institute-China USA Center), Dr. Qiang Liu (Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) and Prof. Jiaqi Xiao (Department of Pathogen Biology, School of Medicine, Shanghai Jiao Tong University) for providing human semen samples, human epididymal cDNA and growth medium of A. niger. The authors also thank Prof. Peter Reinach for his editorial assistance. This research project was supported by grants from Shanghai Municipal Education Commission (no.12ZZ108), the Science and Technology Commission of Shanghai Municipality (no.09ZR1416300, no.10DZ2270600 and no.11JC1404800), the Shanghai Municipal Health Bureau (no.2010058), the Shanghai Leading Academic Discipline Project (no.S30201). The authors declare that there is no conflict of interest that would prejudice the impartiality of this work.

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