Confocal Raman micro-spectroscopy for rapid and label-free detection of maleic acid-induced variations in human sperm Ning Li,1, 2 Diling Chen,1 Yan Xu,4 Songhao Liu,2, 3 and Heming Zhang1,* 2
1 Southern Institute of Pharmaceutical Research, South China Normal University, Guangzhou, 510631, China School of Information and Optoelectronic Science and Engineering, South China Normal University, Guangzhou, 510006, China 3 Ministry of Education Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China 4 College of Pharmaceutical Sciences, Southwest University, Chongqing, 400716, China * [email protected]
Abstract: Confocal Raman microspectroscopy is a valuable analytical tool in biological and medical research, allowing the detection of sample variations without external labels or extensive preparation. To determine whether this method can assess the effect of maleic acid on sperm, we prepared human sperm samples incubated in different concentrations of maleic acid, after which Raman spectra from the various regions of sperm cells were recorded. Following the maleic acid treatment, Raman spectra indicated significant changes. Combined with other means, we found that the structures and chemical compositions of sperm membranes were damaged, and even the sperm DNA was damaged by the incorporation of maleic acid. Thus, this technique can be used for detection and identification of maleic acid-induced changes in human sperm at a molecular level. Although this particular application of Raman microspectroscopy still requires further validation, it has potentially promise as a diagnostic tool for reproductive medicine. © 2014 Optical Society of America OCIS codes: (300.6450) Spectroscopy, Raman; (170.0170) Medical optics and biotechnology; (170.5660) Raman spectroscopy; (170.1530) Cell analysis.
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1. Introduction Approximately 15 to 20% of couples face the fertility problem worldwide [1]. Even when fertilization is successful, poor-quality embryos, low implantation rates, and high miscarriage rates are still serious problems for some couples [2–5]. In general, male factor infertility is responsible for 40–50% of cases [6]. Various reasons may result in male infertility including genetic defects, illnesses, injuries, environmental factors, or lifestyle choices [7]. And semen quality is closely linked to the probability of infertility. At present, numerous methods are available for the measurement of sperm status and assessment of sperm quality. Computer-aided sperm analysis (CASA) and computer-aided sperm morphometric assessment (CASMA) are employed to evaluate sperm physiological parameters and morphological parameters, respectively, including sperm concentration, motility, ellipticity, and regularity. However, relevant studies suggest that methods only using the above parameters lack sufficient evidence to evaluate sperm’s potential for fertilization [8–10]. Other analytical techniques with high spatial resolution, such as optical or electron microscopy, X-ray imaging, or secondary ion-mass spectroscopy, either damage the sample during analysis, or do not provide detailed information about structure and chemical composition simultaneously [11]. In any case, several methods are currently available for the analysis of sperm nuclear DNA (nDNA) quality, such as sperm chromatin dispersion, the comet assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), and sperm chromatin structure assay [12–15]. However, these methods also face a similar problem, in that the processes required may lead to the destruction of the sperm sample. What is required clinically, then, is a reliable, non-invasive, and non-destructive analytical technique that provides precise information on the quality of a sperm while not affecting the integrity of the cell, thereby qualifying it for use in assisted reproductive technology (ART). Raman spectroscopy has been recognized as a valuable analytical technique by providing chemical fingerprint information of substances without external labels or extensive
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1692
preparation. This can be used to identify and characterize biomolecules, cells, or tissues. Using a confocal microscope affords three-dimensional spatial resolution, permitting the identification of molecules in organelles [11,16,17]. In biomedicine, Raman microspectroscopy has been used as a powerful tool in the discrimination, classification and diagnosis of pathological conditions, such as various malignancies and tumors [18–20]. Being non-invasive, it has also been employed in the evaluation of various living cells without any adverse reaction experienced by the cells themselves [21–23]. Recent studies have successfully demonstrated the utility of Raman microspectroscopy for investigating the molecular compositions and sub-cellular organelles of human sperm [11, 24–28]. Previous research showed that adding maleic acid to human sperm and golden hamster sperm can reduce their motility and penetration, and was even almost instantaneously spermicidal [29,30]. In addition, several synthetic polymers based upon maleic acid also have exhibited strong inhibition of sperm motility [31]. Thus, maleic acid or its chemically modified compounds exhibit promise as cervical or vaginal contraceptives. Meanwhile, it is very necessary to obtain more information about the changes of structures and chemical compositions of sperm maleic acid-induced at a molecular level. Consequently, the aim of our study is to evaluate the possibility of label-free, rapid identification of human sperm damage caused by maleic acid, using Raman microspectroscopy. In this study, we prepared normal human sperm cells for Raman scanning, incubating them in different concentrations of maleic acid reagent, in order to study the effects on the regions of acrosome, nucleus, and middle piece. 2. Materials and methods 2.1 Sample preparation This study was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. Semen samples were obtained from five healthy donors after securing informed consent to use their gametes for the purposes of this study. All ejaculates were provided by masturbation after 2-6 days of sexual abstinence and were examined before use. They all had normal analytic parameters according to current World Health Organization (WHO) standards. To achieve viable sperm, both density-gradient centrifugation method [27] and the commonly known swim-up protocol [32] were then performed. The resulting sperm suspension was divided into five aliquots. One aliquot was left untreated, while the remaining four were subjected to the increasing amounts of maleic acid reagent (0.01 M, 0.02 M, 0.04 M, 0.08 M). After 45 minutes of incubation at 37 °C in ambient air with 5% CO2, the sperm were washed with PBS (800 × g, 10 min, 4 °C). Subsequently, the supernatants were discarded and the pellets resuspended in PBS. In addition, each sample was further divided into three aliquots, which were assessed for sperm quality by Raman micro-spectroscopy, flow cytometry, and hypo-osmotic swelling (HOS) testing [33]. 2.2 Confocal Raman microspectroscopy A drop of 15 μL sperm suspensions were smeared onto aluminium slices and left to air-dry. This kind of pure metal has no Raman spectral features and very low background interference. For Raman spectral measurement, a confocal micro-Raman spectroscopy system (Renishaw Invia, UK) with an excitation wavelength of 514.5 nm generated by an Ar+ laser (~10mw) was employed, in which a × 50 objective lens is used to focus the laser beam and to collect the Raman signal. The lateral resolution of the instrument was about 1 μm, and the power of the laser beam measured at the sample was 1.5 mw approximately. Raman spectra were recorded by a Peltier-cooled charge-coupled device (CCD) camera with an integration time of 30 s, and three accumulations. At least five sperm cells were scanned for each sample.
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1693
Three scanning points were selected for each individual sperm according to anatomical structure, including the acrosome, nucleus, and middle piece. 2.3 Flow cytometry The flow-cytometric method based on acridine orange was used to assess the degree of sperm DNA damage, as described elsewhere [4]. Briefly, samples were diluted with TNE buffer (0.01 M Tris·Cl, 0.15 M NaCl, 1 mM EDTA, pH 7.4) and transferred into tubes for flow cytometry (Lab test tubes; Beckmann Coulter); 0.4 mL of acid detergent solution (0.08 N HCl, 0.15 M NaCl, 0.1% Triton X-100, pH 1.2) was added, and, after 30 seconds of gentle agitation, 1.2 mL of acridine orange staining solution (6 μg/mL) was added. The samples were kept for 3 min at 4 °C in the dark and then analyzed by flow cytometry (Cytomics FC 500, Beckman Coulter, USA). Fluorescence data were acquired from a total of 5,000 sperm per sample. Excitation by a 488-nm-wavelength light source resulted in the emission of red fluorescence (single-stranded DNA) and green fluorescence (double-stranded DNA). The results were expressed as a DNA fragmentation index (DFI, Flow cytometry software: FCS Express, version 3), which represents the percentage of cells exhibiting increased red fluorescence. 2.4 Data analysis Before further analysis, the raw spectra were processed using the software packages R2.8.1 and Origin 8.0. In order to compare the related spectra changes, we chose the intensity of band at 1665cm−1 to normalize the spectra. First, using R2.8.1, a baseline correction was applied, in which the spectral background was subtracted. Then the spectra were smoothed, averaged, and normalized using Origin 8.0. Standard principal component analysis (PCA) was performed on the data of the Raman spectra from both the control sperm cells and the sperm cells incubated with maleic acid. Computations were performed using the MATLAB (R2012b) software system. In this paper, we chose the first two principal components accounted for more than 75% of the accumulative total contribution for analysis. 3. Results and discussion 3.1 The Raman spectra of sperm Figure 1(a) shows a bright-field image of a normal sperm cell; it exhibits a regular outline and a clearly distinguished acrosomal cap. Note that the sperm acrosome outline is clearly visible without any staining treatment. Single point Raman spectra of the nucleus, middle piece and acrosome regions in normal sperm cells are presented in Fig. 1(b).
Fig. 1. (A) Bright-field image of sperm cell and (B) averaged Raman spectra correspond to the regions of nucleus (a), middle piece (b), and acrosome (c) in normal human sperm, respectively.
The averaged Raman spectra of control sperm cells and the treated sperm cells with different concentrations of maleic acid are shown in Fig. 2. In order to quantitatively identify
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1694
how maleic acid caused the variation in cellular components, we selected specific Raman peaks corresponding to DNA, proteins and lipids and compared the changes in their spectral intensities (Fig. 3). The band at 787 cm−1 is assigned to the thymine and cytosine vibrations as well as the DNA backbone. The peak at 988cm−1 corresponds to the vibration of desoxyribose. The 1094 cm−1 peak represents the vibration of phosphodioxy groups PO2- in the DNA backbone. The bands observed at around 1337, 1344, and 1421 cm−1 are related to adenine and guanine, respectively. The main changes related to the proteins can be observed at 1004 (the ring stretching of phenylalanine), 1238 (β-sheet of amide III) and 1303 cm−1 (αhelix of amide III). The peak at 1446 cm−1 can be attributed to proteins and lipids [34–40].
Fig. 2. Averaged Raman spectra obtained from the nucleus region (A), the middle piece (B), and the acrosomal region (C) of control sperm and treated sperm with different concentrations of maleic acid, respectively.
Fig. 3. Spectral intensities of Raman peaks that occur in the nucleus, A: 787, 1094, 1337, 1421, 1446cm−1; in the middle piece, B: 988, 1004, 1344 cm−1; and in the acrosome, C: 1238, 1303 cm−1, after 0.01 M, 0.02 M, 0.04 M, 0.08 M and for control sperm cells without maleic acid.
3.2 Effect on the sperm nucleus region In order to quantitatively identify how maleic acid treatment influenced the variation in DNA, we selected specific Raman peaks and compared the changes in their spectral intensities. The results are shown in Fig. 3(a). For sperm cells exposed to maleic acid, the Raman intensities corresponding to DNA, such as 787, 1094, 1337 and 1421 cm−1, were reduced in comparison with the control. The peak at 988 cm−1 noticeably shifted to 973 cm−1 (Fig. 4). It is possible #208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1695
that broken covalent bonds between desoxyribose and phosphodiester or desoxyribose and base, caused by the maleic acid damage, can change the groups and their force-bearing environments, changing their Raman activities [41]. All of these changes indicate the destruction of the DNA structures.
Fig. 4. Comparison of the spectra of control and treated sperm cells with the different concentrations of maleic acid in the region of 900 cm−1 to 1050 cm−1 from the nucleus region of human sperm..
3.3 Effect on the sperm middle-piece region According to previous research [42], the ratio of 855/836 cm−1 relevant to the environment of tyrosine residue was used to estimate the conformational change of tyrosine. For the “exposure” conformation, the interval of the 855/836 cm−1 ratio is from 0.3 to 1.5. This ratio ranges from 1.7 to 2.5 for the “buried” conformation [43]. From Fig. 5(a), we see that the intensities of peaks at 836 and 855 cm−1 changed in comparison with the control. As shown in Fig. 5(b), the 855/836 cm−1 ratio of the control group is 1.76 ± 0.04 and those of the treatment groups range from 1.44 to 1.01. This demonstrates that in the control cells, most of the tyrosine residues in membranous proteins from the middle piece region of sperm are “buried,” which is good for maintaining the stability of the protein spatial structure. However, this conformation turns into “exposure” after treatment, which indicates that the environment of the tyrosine residues has been changed [42]. In addition, as shown in Fig. 3(b), the band intensities at 1004 cm−1 also decreased. This decrease corresponds to the ring stretching of phenylalanine, which is a very important component of cell proteins. These changes suggest damage to the protein spatial structure.
Fig. 5. (A) Comparison of the spectra of control and treated sperm cells with the different concentrations of maleic acid in the region of 800 cm−1 to 900 cm−1 from the middle piece in human sperm and (B) correlation of the maleic acid concentration with the peak intensity ratios 855/836cm−1 of the spectra.
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1696
The Raman peaks at 1128 cm−1 and 1071 cm−1, belonging to the trans stretching vibrations of C-C skeleton, and 1086 cm−1, belonging to the gauche vibrations of C-C skeleton were chosen for data analysis. These peaks are very sensitive to the change of the conformation of membrane lipids. The longitudinal order-parameters in chains (Strans) [44] was used to assess the damage to the middle piece membranous lipids. The more content of the trans conformation, the higher order of the vertical chain. Increased content of the trans conformation also results in less liquidity of the middle piece membrane [45]. The value of Strans is calculated following Eq. (1). For the solid dipalmitoylphosphatidylcholine (DPPC), the intensity ratio of 1128cm−1/1086 cm−1 was 1.77 [44]. Our calculated results (Table 1) mean that the percentage of trans conformation increased and gauche conformation decreased in the membrane, as compared to the control sample, and so the liquidity and ionic permeability of the membrane in the middle piece region was reduced.
strans =
( I 1128 / I 1086) sample ( I 1128 / I 1086) sample = ( I 1128 / I 1086) DPPC of solid 1.77
(1)
Table 1. Longitudinal order-parameters in chains Strans and the rate of variation in the middle piece membrane before and after the effects of maleic acid. Sample Control 0.01M 0.02M 0.04M 0.08M
Strans 0.54 ± 0.08 0.69 ± 0.06 0.80 ± 0.05 0.88 ± 0.10 0.92 ± 0.08
ΔStrans — 27.8 48.1 63.0 70.4
Mitochondria, which are concentrated in the middle piece of human sperm cells, were also affected by the incorporation of maleic acid. As shown in Fig. 3(b), the intensities of the peaks at 988cm−1 assigned to the vibration of desoxyribose and 1344 cm−1 related to adenine decreased. In addition, the peaks at 1068cm−1 corresponds to the vibration of desoxyribose. The bands observed at around 1250, 1374, 1421, and 1583 cm−1 are related to adenine, guanine, cytosine, and thymine. These five peaks decreased or even disappeared in comparison with the control. All of these changes suggest damage to the DNA structure of mitochondria. Mitochondria produce adenosine triphosphate (ATP) for the cell through aerobic respiration. A high concentration of ATP is expected to be in the middle piece region, as it contributes to providing energy for the flagellum and overall sperm mobility [11]. If the normal structures and components of mitochondria are damaged, the functional integrity of mitochondria will be affected in different degrees. 3.4 Effect on the sperm acrosome region Next, we discuss the peaks at 1238 and 1303 cm−1, which are assigned to the β-sheet and αhelix of amide III, respectively. The α-helix and the β-sheet are both common secondary structures in proteins and are maintained by hydrogen bonds. Amide III is very sensitive to changes of the main chain’s conformation in membrane proteins [45]. Its quantity decreased with increasing maleic acid concentrations as indicated in Fig. 3(c). This suggests the occurrence of denaturation and conformational changes to the acrosome membrane proteins. This change can be identified as the rupture of the peptide and hydrogen bonds in membrane proteins. Thirty-four Raman spectra of control sperm cells (CSC) and 36 Raman spectra of treated sperm cells (TSC) were used for PCA. All the Raman spectra were from the acrosome region of the sperm cells. The result in Fig. 6 shows that although there are similarities, spectra from the control sample are sufficiently distinguishable and significantly different from those
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1697
obtained after incubation with different concentrations of maleic acid. All of these changes indicate the destruction of the acrosome membrane.
Fig. 6. PC1 vs PC2 plot for the spectra from the acrosome region in the control sperm cells (CSC) and treated sperm cells (TSC) with maleic acid incubation.
3.5 Evaluation of human sperm damage To verify the results obtained with Raman microspectroscopy, we performed the HOS test and flow-cytometry. A functional and intact membrane is required for sperm to be fertile. The HOS test can evaluate the integrity of the sperm’s plasma membrane and also serves as a useful indicator of fertility potential [46–48]. It predicts membrane integrity by determining the ability of the sperm membrane to maintain equilibrium between the sperm cell and its environment. Influx of the fluid due to hypo-osmotic stress causes the sperm tail to coil and balloon or “swell.” A higher percentage of swollen sperm indicates the presence of sperm having functional and intact plasma membranes [33]. Changes to membranous structure can affect the functional integrity of the sperm’s plasma membrane. The results of HOS test indicate that the sperm membrane has been damaged after the incorporation of maleic acid (Fig. 7). This result is in agreement with the Raman results. Since the membrane is subjected to damage, the intracelluar organelles and molecules will lose its protection.
Fig. 7. Evaluation of sperm membrane integrity with HOS test.
The flow-cytometric method based on acridine orange was used to assess the degree of sperm DNA damage. The results were expressed as DNA fragmentation index (DFI). Analysis of the DNA fragmentation by flow cytomtry confirmed that increasing levels of
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1698
sperm DNA damage occurred after the treatments with maleic acid (Fig. 8). This result is also in agreement with the Raman results.
Fig. 8. Flow-cytometric assessment of sperm DNA damage showing the different levels of fragmention induced by maleic acid.
4. Conclusion In summary, our study has shown that Raman micro-spectroscopy is capable of assessing the variations and damage at a molecular level in human sperm cells induced by maleic acid treatment. Following the maleic acid treatment, Raman spectra indicated significant changes in different regions of the sperm cells. All of these results suggest destruction and conformational changes in proteins and lipids, and damage to DNA structure, including nDNA and mitochondrial DNA. We can see that maleic acid can affect the quality of sperm. In contrast, maleic acid or its chemically modified compounds exhibit promise as cervical or vaginal contraceptives because it disrupts the structural and functional integrity of sperm via either electrostatic or pH-lowering effects. In the future, non-invasive and label-free Raman micro-spectroscopy could prove to be a promising diagnostic tool with further potential to identify normal sperm, allowing for their routine selection in ART. This method can also be applied to the exploration of the biochemical and molecular mechanisms of human sperm function with the chemical fingerprints we obtained. Acknowledgments We gratefully acknowledge the financial support for this work via grants from china national ministry of science and technology plan projects (2011BAI01B02), and Guangdong science and technology plan projects (2012A030100005), and Guangdong Natural Science Foundation Grant (S2013040016159).
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1699
1 Southern Institute of Pharmaceutical Research, South China Normal University, Guangzhou, 510631, China School of Information and Optoelectronic Science and Engineering, South China Normal University, Guangzhou, 510006, China 3 Ministry of Education Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China 4 College of Pharmaceutical Sciences, Southwest University, Chongqing, 400716, China * [email protected]
Abstract: Confocal Raman microspectroscopy is a valuable analytical tool in biological and medical research, allowing the detection of sample variations without external labels or extensive preparation. To determine whether this method can assess the effect of maleic acid on sperm, we prepared human sperm samples incubated in different concentrations of maleic acid, after which Raman spectra from the various regions of sperm cells were recorded. Following the maleic acid treatment, Raman spectra indicated significant changes. Combined with other means, we found that the structures and chemical compositions of sperm membranes were damaged, and even the sperm DNA was damaged by the incorporation of maleic acid. Thus, this technique can be used for detection and identification of maleic acid-induced changes in human sperm at a molecular level. Although this particular application of Raman microspectroscopy still requires further validation, it has potentially promise as a diagnostic tool for reproductive medicine. © 2014 Optical Society of America OCIS codes: (300.6450) Spectroscopy, Raman; (170.0170) Medical optics and biotechnology; (170.5660) Raman spectroscopy; (170.1530) Cell analysis.
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1. Introduction Approximately 15 to 20% of couples face the fertility problem worldwide [1]. Even when fertilization is successful, poor-quality embryos, low implantation rates, and high miscarriage rates are still serious problems for some couples [2–5]. In general, male factor infertility is responsible for 40–50% of cases [6]. Various reasons may result in male infertility including genetic defects, illnesses, injuries, environmental factors, or lifestyle choices [7]. And semen quality is closely linked to the probability of infertility. At present, numerous methods are available for the measurement of sperm status and assessment of sperm quality. Computer-aided sperm analysis (CASA) and computer-aided sperm morphometric assessment (CASMA) are employed to evaluate sperm physiological parameters and morphological parameters, respectively, including sperm concentration, motility, ellipticity, and regularity. However, relevant studies suggest that methods only using the above parameters lack sufficient evidence to evaluate sperm’s potential for fertilization [8–10]. Other analytical techniques with high spatial resolution, such as optical or electron microscopy, X-ray imaging, or secondary ion-mass spectroscopy, either damage the sample during analysis, or do not provide detailed information about structure and chemical composition simultaneously [11]. In any case, several methods are currently available for the analysis of sperm nuclear DNA (nDNA) quality, such as sperm chromatin dispersion, the comet assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), and sperm chromatin structure assay [12–15]. However, these methods also face a similar problem, in that the processes required may lead to the destruction of the sperm sample. What is required clinically, then, is a reliable, non-invasive, and non-destructive analytical technique that provides precise information on the quality of a sperm while not affecting the integrity of the cell, thereby qualifying it for use in assisted reproductive technology (ART). Raman spectroscopy has been recognized as a valuable analytical technique by providing chemical fingerprint information of substances without external labels or extensive
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1692
preparation. This can be used to identify and characterize biomolecules, cells, or tissues. Using a confocal microscope affords three-dimensional spatial resolution, permitting the identification of molecules in organelles [11,16,17]. In biomedicine, Raman microspectroscopy has been used as a powerful tool in the discrimination, classification and diagnosis of pathological conditions, such as various malignancies and tumors [18–20]. Being non-invasive, it has also been employed in the evaluation of various living cells without any adverse reaction experienced by the cells themselves [21–23]. Recent studies have successfully demonstrated the utility of Raman microspectroscopy for investigating the molecular compositions and sub-cellular organelles of human sperm [11, 24–28]. Previous research showed that adding maleic acid to human sperm and golden hamster sperm can reduce their motility and penetration, and was even almost instantaneously spermicidal [29,30]. In addition, several synthetic polymers based upon maleic acid also have exhibited strong inhibition of sperm motility [31]. Thus, maleic acid or its chemically modified compounds exhibit promise as cervical or vaginal contraceptives. Meanwhile, it is very necessary to obtain more information about the changes of structures and chemical compositions of sperm maleic acid-induced at a molecular level. Consequently, the aim of our study is to evaluate the possibility of label-free, rapid identification of human sperm damage caused by maleic acid, using Raman microspectroscopy. In this study, we prepared normal human sperm cells for Raman scanning, incubating them in different concentrations of maleic acid reagent, in order to study the effects on the regions of acrosome, nucleus, and middle piece. 2. Materials and methods 2.1 Sample preparation This study was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. Semen samples were obtained from five healthy donors after securing informed consent to use their gametes for the purposes of this study. All ejaculates were provided by masturbation after 2-6 days of sexual abstinence and were examined before use. They all had normal analytic parameters according to current World Health Organization (WHO) standards. To achieve viable sperm, both density-gradient centrifugation method [27] and the commonly known swim-up protocol [32] were then performed. The resulting sperm suspension was divided into five aliquots. One aliquot was left untreated, while the remaining four were subjected to the increasing amounts of maleic acid reagent (0.01 M, 0.02 M, 0.04 M, 0.08 M). After 45 minutes of incubation at 37 °C in ambient air with 5% CO2, the sperm were washed with PBS (800 × g, 10 min, 4 °C). Subsequently, the supernatants were discarded and the pellets resuspended in PBS. In addition, each sample was further divided into three aliquots, which were assessed for sperm quality by Raman micro-spectroscopy, flow cytometry, and hypo-osmotic swelling (HOS) testing [33]. 2.2 Confocal Raman microspectroscopy A drop of 15 μL sperm suspensions were smeared onto aluminium slices and left to air-dry. This kind of pure metal has no Raman spectral features and very low background interference. For Raman spectral measurement, a confocal micro-Raman spectroscopy system (Renishaw Invia, UK) with an excitation wavelength of 514.5 nm generated by an Ar+ laser (~10mw) was employed, in which a × 50 objective lens is used to focus the laser beam and to collect the Raman signal. The lateral resolution of the instrument was about 1 μm, and the power of the laser beam measured at the sample was 1.5 mw approximately. Raman spectra were recorded by a Peltier-cooled charge-coupled device (CCD) camera with an integration time of 30 s, and three accumulations. At least five sperm cells were scanned for each sample.
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1693
Three scanning points were selected for each individual sperm according to anatomical structure, including the acrosome, nucleus, and middle piece. 2.3 Flow cytometry The flow-cytometric method based on acridine orange was used to assess the degree of sperm DNA damage, as described elsewhere [4]. Briefly, samples were diluted with TNE buffer (0.01 M Tris·Cl, 0.15 M NaCl, 1 mM EDTA, pH 7.4) and transferred into tubes for flow cytometry (Lab test tubes; Beckmann Coulter); 0.4 mL of acid detergent solution (0.08 N HCl, 0.15 M NaCl, 0.1% Triton X-100, pH 1.2) was added, and, after 30 seconds of gentle agitation, 1.2 mL of acridine orange staining solution (6 μg/mL) was added. The samples were kept for 3 min at 4 °C in the dark and then analyzed by flow cytometry (Cytomics FC 500, Beckman Coulter, USA). Fluorescence data were acquired from a total of 5,000 sperm per sample. Excitation by a 488-nm-wavelength light source resulted in the emission of red fluorescence (single-stranded DNA) and green fluorescence (double-stranded DNA). The results were expressed as a DNA fragmentation index (DFI, Flow cytometry software: FCS Express, version 3), which represents the percentage of cells exhibiting increased red fluorescence. 2.4 Data analysis Before further analysis, the raw spectra were processed using the software packages R2.8.1 and Origin 8.0. In order to compare the related spectra changes, we chose the intensity of band at 1665cm−1 to normalize the spectra. First, using R2.8.1, a baseline correction was applied, in which the spectral background was subtracted. Then the spectra were smoothed, averaged, and normalized using Origin 8.0. Standard principal component analysis (PCA) was performed on the data of the Raman spectra from both the control sperm cells and the sperm cells incubated with maleic acid. Computations were performed using the MATLAB (R2012b) software system. In this paper, we chose the first two principal components accounted for more than 75% of the accumulative total contribution for analysis. 3. Results and discussion 3.1 The Raman spectra of sperm Figure 1(a) shows a bright-field image of a normal sperm cell; it exhibits a regular outline and a clearly distinguished acrosomal cap. Note that the sperm acrosome outline is clearly visible without any staining treatment. Single point Raman spectra of the nucleus, middle piece and acrosome regions in normal sperm cells are presented in Fig. 1(b).
Fig. 1. (A) Bright-field image of sperm cell and (B) averaged Raman spectra correspond to the regions of nucleus (a), middle piece (b), and acrosome (c) in normal human sperm, respectively.
The averaged Raman spectra of control sperm cells and the treated sperm cells with different concentrations of maleic acid are shown in Fig. 2. In order to quantitatively identify
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1694
how maleic acid caused the variation in cellular components, we selected specific Raman peaks corresponding to DNA, proteins and lipids and compared the changes in their spectral intensities (Fig. 3). The band at 787 cm−1 is assigned to the thymine and cytosine vibrations as well as the DNA backbone. The peak at 988cm−1 corresponds to the vibration of desoxyribose. The 1094 cm−1 peak represents the vibration of phosphodioxy groups PO2- in the DNA backbone. The bands observed at around 1337, 1344, and 1421 cm−1 are related to adenine and guanine, respectively. The main changes related to the proteins can be observed at 1004 (the ring stretching of phenylalanine), 1238 (β-sheet of amide III) and 1303 cm−1 (αhelix of amide III). The peak at 1446 cm−1 can be attributed to proteins and lipids [34–40].
Fig. 2. Averaged Raman spectra obtained from the nucleus region (A), the middle piece (B), and the acrosomal region (C) of control sperm and treated sperm with different concentrations of maleic acid, respectively.
Fig. 3. Spectral intensities of Raman peaks that occur in the nucleus, A: 787, 1094, 1337, 1421, 1446cm−1; in the middle piece, B: 988, 1004, 1344 cm−1; and in the acrosome, C: 1238, 1303 cm−1, after 0.01 M, 0.02 M, 0.04 M, 0.08 M and for control sperm cells without maleic acid.
3.2 Effect on the sperm nucleus region In order to quantitatively identify how maleic acid treatment influenced the variation in DNA, we selected specific Raman peaks and compared the changes in their spectral intensities. The results are shown in Fig. 3(a). For sperm cells exposed to maleic acid, the Raman intensities corresponding to DNA, such as 787, 1094, 1337 and 1421 cm−1, were reduced in comparison with the control. The peak at 988 cm−1 noticeably shifted to 973 cm−1 (Fig. 4). It is possible #208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1695
that broken covalent bonds between desoxyribose and phosphodiester or desoxyribose and base, caused by the maleic acid damage, can change the groups and their force-bearing environments, changing their Raman activities [41]. All of these changes indicate the destruction of the DNA structures.
Fig. 4. Comparison of the spectra of control and treated sperm cells with the different concentrations of maleic acid in the region of 900 cm−1 to 1050 cm−1 from the nucleus region of human sperm..
3.3 Effect on the sperm middle-piece region According to previous research [42], the ratio of 855/836 cm−1 relevant to the environment of tyrosine residue was used to estimate the conformational change of tyrosine. For the “exposure” conformation, the interval of the 855/836 cm−1 ratio is from 0.3 to 1.5. This ratio ranges from 1.7 to 2.5 for the “buried” conformation [43]. From Fig. 5(a), we see that the intensities of peaks at 836 and 855 cm−1 changed in comparison with the control. As shown in Fig. 5(b), the 855/836 cm−1 ratio of the control group is 1.76 ± 0.04 and those of the treatment groups range from 1.44 to 1.01. This demonstrates that in the control cells, most of the tyrosine residues in membranous proteins from the middle piece region of sperm are “buried,” which is good for maintaining the stability of the protein spatial structure. However, this conformation turns into “exposure” after treatment, which indicates that the environment of the tyrosine residues has been changed [42]. In addition, as shown in Fig. 3(b), the band intensities at 1004 cm−1 also decreased. This decrease corresponds to the ring stretching of phenylalanine, which is a very important component of cell proteins. These changes suggest damage to the protein spatial structure.
Fig. 5. (A) Comparison of the spectra of control and treated sperm cells with the different concentrations of maleic acid in the region of 800 cm−1 to 900 cm−1 from the middle piece in human sperm and (B) correlation of the maleic acid concentration with the peak intensity ratios 855/836cm−1 of the spectra.
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1696
The Raman peaks at 1128 cm−1 and 1071 cm−1, belonging to the trans stretching vibrations of C-C skeleton, and 1086 cm−1, belonging to the gauche vibrations of C-C skeleton were chosen for data analysis. These peaks are very sensitive to the change of the conformation of membrane lipids. The longitudinal order-parameters in chains (Strans) [44] was used to assess the damage to the middle piece membranous lipids. The more content of the trans conformation, the higher order of the vertical chain. Increased content of the trans conformation also results in less liquidity of the middle piece membrane [45]. The value of Strans is calculated following Eq. (1). For the solid dipalmitoylphosphatidylcholine (DPPC), the intensity ratio of 1128cm−1/1086 cm−1 was 1.77 [44]. Our calculated results (Table 1) mean that the percentage of trans conformation increased and gauche conformation decreased in the membrane, as compared to the control sample, and so the liquidity and ionic permeability of the membrane in the middle piece region was reduced.
strans =
( I 1128 / I 1086) sample ( I 1128 / I 1086) sample = ( I 1128 / I 1086) DPPC of solid 1.77
(1)
Table 1. Longitudinal order-parameters in chains Strans and the rate of variation in the middle piece membrane before and after the effects of maleic acid. Sample Control 0.01M 0.02M 0.04M 0.08M
Strans 0.54 ± 0.08 0.69 ± 0.06 0.80 ± 0.05 0.88 ± 0.10 0.92 ± 0.08
ΔStrans — 27.8 48.1 63.0 70.4
Mitochondria, which are concentrated in the middle piece of human sperm cells, were also affected by the incorporation of maleic acid. As shown in Fig. 3(b), the intensities of the peaks at 988cm−1 assigned to the vibration of desoxyribose and 1344 cm−1 related to adenine decreased. In addition, the peaks at 1068cm−1 corresponds to the vibration of desoxyribose. The bands observed at around 1250, 1374, 1421, and 1583 cm−1 are related to adenine, guanine, cytosine, and thymine. These five peaks decreased or even disappeared in comparison with the control. All of these changes suggest damage to the DNA structure of mitochondria. Mitochondria produce adenosine triphosphate (ATP) for the cell through aerobic respiration. A high concentration of ATP is expected to be in the middle piece region, as it contributes to providing energy for the flagellum and overall sperm mobility [11]. If the normal structures and components of mitochondria are damaged, the functional integrity of mitochondria will be affected in different degrees. 3.4 Effect on the sperm acrosome region Next, we discuss the peaks at 1238 and 1303 cm−1, which are assigned to the β-sheet and αhelix of amide III, respectively. The α-helix and the β-sheet are both common secondary structures in proteins and are maintained by hydrogen bonds. Amide III is very sensitive to changes of the main chain’s conformation in membrane proteins [45]. Its quantity decreased with increasing maleic acid concentrations as indicated in Fig. 3(c). This suggests the occurrence of denaturation and conformational changes to the acrosome membrane proteins. This change can be identified as the rupture of the peptide and hydrogen bonds in membrane proteins. Thirty-four Raman spectra of control sperm cells (CSC) and 36 Raman spectra of treated sperm cells (TSC) were used for PCA. All the Raman spectra were from the acrosome region of the sperm cells. The result in Fig. 6 shows that although there are similarities, spectra from the control sample are sufficiently distinguishable and significantly different from those
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1697
obtained after incubation with different concentrations of maleic acid. All of these changes indicate the destruction of the acrosome membrane.
Fig. 6. PC1 vs PC2 plot for the spectra from the acrosome region in the control sperm cells (CSC) and treated sperm cells (TSC) with maleic acid incubation.
3.5 Evaluation of human sperm damage To verify the results obtained with Raman microspectroscopy, we performed the HOS test and flow-cytometry. A functional and intact membrane is required for sperm to be fertile. The HOS test can evaluate the integrity of the sperm’s plasma membrane and also serves as a useful indicator of fertility potential [46–48]. It predicts membrane integrity by determining the ability of the sperm membrane to maintain equilibrium between the sperm cell and its environment. Influx of the fluid due to hypo-osmotic stress causes the sperm tail to coil and balloon or “swell.” A higher percentage of swollen sperm indicates the presence of sperm having functional and intact plasma membranes [33]. Changes to membranous structure can affect the functional integrity of the sperm’s plasma membrane. The results of HOS test indicate that the sperm membrane has been damaged after the incorporation of maleic acid (Fig. 7). This result is in agreement with the Raman results. Since the membrane is subjected to damage, the intracelluar organelles and molecules will lose its protection.
Fig. 7. Evaluation of sperm membrane integrity with HOS test.
The flow-cytometric method based on acridine orange was used to assess the degree of sperm DNA damage. The results were expressed as DNA fragmentation index (DFI). Analysis of the DNA fragmentation by flow cytomtry confirmed that increasing levels of
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1698
sperm DNA damage occurred after the treatments with maleic acid (Fig. 8). This result is also in agreement with the Raman results.
Fig. 8. Flow-cytometric assessment of sperm DNA damage showing the different levels of fragmention induced by maleic acid.
4. Conclusion In summary, our study has shown that Raman micro-spectroscopy is capable of assessing the variations and damage at a molecular level in human sperm cells induced by maleic acid treatment. Following the maleic acid treatment, Raman spectra indicated significant changes in different regions of the sperm cells. All of these results suggest destruction and conformational changes in proteins and lipids, and damage to DNA structure, including nDNA and mitochondrial DNA. We can see that maleic acid can affect the quality of sperm. In contrast, maleic acid or its chemically modified compounds exhibit promise as cervical or vaginal contraceptives because it disrupts the structural and functional integrity of sperm via either electrostatic or pH-lowering effects. In the future, non-invasive and label-free Raman micro-spectroscopy could prove to be a promising diagnostic tool with further potential to identify normal sperm, allowing for their routine selection in ART. This method can also be applied to the exploration of the biochemical and molecular mechanisms of human sperm function with the chemical fingerprints we obtained. Acknowledgments We gratefully acknowledge the financial support for this work via grants from china national ministry of science and technology plan projects (2011BAI01B02), and Guangdong science and technology plan projects (2012A030100005), and Guangdong Natural Science Foundation Grant (S2013040016159).
#208156 - $15.00 USD Received 14 Mar 2014; revised 20 Apr 2014; accepted 23 Apr 2014; published 29 Apr 2014 (C) 2014 OSA 1 May 2014 | Vol. 5, No. 5 | DOI:10.1364/BOE.5.001690 | BIOMEDICAL OPTICS EXPRESS 1699
