ORIGINAL ARTICLE
A simple sperm nuclear vacuole assay with propidium iodide W.-J. Zhu1 & J. Li2 1 Department of Developmental and Regenerative Biology, College of Life Science and Technology, Jinan University, Guangzhou, China; 2 Department of Pathophysiology, Medical College, Jinan University, Guangzhou, China
Keywords Chromatin—nuclear vacuole—propidium iodide—sperm—varicocele Correspondence Wei-Jie Zhu, Ph.D, Department of Developmental and Regenerative Biology, College of Life Science and Technology, Jinan University, 601# Huang Pu Da Dao Xi, Guangzhou City 510632, China. Tel.: +86-20-85225718; E-mail: [email protected] Accepted: June 16, 2014 doi: 10.1111/and.12328
Summary Our aim was to develop a new simple sperm nuclear vacuole assay (SNVA) with propidium iodide (PI) to determine the status of nuclear vacuole (NV) of individual spermatozoa. After PI staining, sperm nuclei were classified into the 14 categories according to both nuclear morphology and the status of NV. The incidence was 57.8% (range 28–84%) in fertile controls (n = 40), and 85.1% (range 67– 99%) in men with varicocele (n = 40). In the fertile group, normal nuclear-shaped spermatozoa without NV or with one small NV located in the ante-nuclear region were significantly more in comparison with the varicocele group. In the varicocele group, abnormal nuclear-shaped spermatozoa with one large NV and with multiple NVs located in the ante-nuclear region were most frequent findings. Besides, spermatozoa with NVs in both ante- and post-nuclear regions in the varicocele group were significantly more than those in the fertile group. In both fertile and varicocele groups, normal or abnormal nuclear-shaped spermatozoa with one or more vacuoles only located in the post-nuclear region occurred sparingly. The SNVA provides a useful additional approach to identify the status of NV in human spermatozoa for diagnostic purposes. A good sperm sample would have more spermatozoa without NV or with one small NV located in the ante-nuclear region.
Introduction Sperm nuclear vacuoles (NVs) are common phenomena within chromatin, probably occurring in the process of spermiogenesis (Zamboni, 1987, 1992). During this stage, sperm chromatin undergoes complex and highly coordinated changes involving histone replacement and chromatin rearrangement (Ward & Coffey, 1991), leading to providing a highly organised and compact structure that reflects the level of chromatin integrity. Defective, uneven processes or disturbances in chromatin condensation or organisation may result in the formation of NVs (Zamboni, 1987, 1992; Franco et al., 2012). Thus, the status of NVs can in part reflect the level of chromatin integrity. According to the semen analysis manual of the WHO (2010), large NVs in sperm head observed in Papanicolaoustained semen smears should be considered as a type of head morphological defect. In recent years, clinical and laboratory evidence has demonstrated a negative correlation between spermatozoa with large NVs and DNA damage or low fertility potential (Berkovitz et al., 2005; Franco et al., 2008; Garolla et al., 2008; Oliveira et al., 2010; Boitrelle © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
et al., 2011). Microinjection of spermatozoa with NVs can reduce pregnancy outcomes (Berkovitz et al., 2006; Vanderzwalmen et al., 2008). Therefore, the evaluation of NVs should be an important parameter for the understanding of sperm quality related to chromatin integrity and fertile status. Propidium iodide (PI) is a fluorescent nuclear dye that can permeate damaged sperm membranes and stains to sperm chromatin. Several methods have been established to determine sperm viability, membrane integrity, DNA fragmentation, sperm apoptosis and sperm necrosis by using PI in combination with flow cytometry (Collodel & Moretti, 2008; Martınez-Pastor et al., 2008; Muratori et al., 2008). However, there is no assay in detecting sperm NVs with PI by using microscopy. On the other hand, Papanicolaou staining and the motile sperm organelle morphology (MSOME) method can evaluate NVs (Berkovitz et al., 2006; WHO, 2010); however, the former takes a long staining procedure and the latter could not be used for routine analysis in a common laboratory. The aim of this study was to develop a new simple sperm nuclear vacuole assay (SNVA) based on the use of 1
Sperm nuclear vacuole assay with PI
PI to determine the status of NVs of individual spermatozoa. Using the SNVA, we quantitatively analysed the status of NVs of sperm samples from fertile men and men with varicocele to evaluate the applicability of this assay. Materials and methods Study subjects and semen samples Semen samples were obtained from 40 men with varicocele (aged 25–43 years) who attended an infertility clinic. Their wives had failed to conceive after 2 years of unprotected intercourse and had not significant female-factor infertility (tubal obstruction or ovarian failure). Varicocele was graded according to the criteria described by the WHO manual (WHO, 1992). Based on their grades and the experimental design, only men with grades II or III were included in the present study. The controls consisted of 40 normal fertile donors (aged 27–37 years) who had fathered a child in the past 12 months (fertile group). All semen samples were collected from the First Affiliated Hospital of Jinan University, China. The study protocol was approved by the local research ethics committee. Samples were produced by masturbation after 3–7 days of sexual abstinence. Semen examination was performed according to the WHO manual (WHO, 2010). Sperm morphology was assessed using strict criteria after slide staining with the Papanicolaou staining method.
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filter; E400, Nikon, Japan) at 91000 magnification under oil immersion (cat. No. 16242, Nikon, Japan). At least 200 spermatozoa were observed on each slide. Sperm nuclei were displayed orange fluorescence. The neck and tail of spermatozoa were observable due to their nonspecific fluorescent staining, depending on the type of oil used (fluorescent absorbing or nonabsorbing). Images were acquired with a CCD camera (Qimaging, Surrey, BC, Canada) and were pseudocoloured using IPP 5.1 (Media Cybernetics, Inc., Rockville, MD, USA) colour image. For long time preservation, the stained slides were placed in 50% ethanol (10 dips), 70% ethanol (10 dips), 95% ethanol (10 dips), absolute ethanol 92 (1 min in each), xylene 92 (1 min in each) after the step of removing excess stain. The stained slides then were mounted, and kept in a dark box for preservation. For quality control of analysis of NV, the acceptable differences between two duplicate assessments of percentage of vacuole were determined according to the method by the WHO manual (see table 2.1 from the WHO manual) (WHO, 2010). Sperm nucleus Sperm nuclei were classified into the 14 categories described below according to both normal oval or nonoval nuclear shape and the status of vacuole within nucleus, and the schematic representation is shown in Fig. 1. The percentage of each type of sperm nuclei was calculated.
Sperm nuclear vacuole assay PI solutions were prepared as follows: PI stock solution (0.1%, 1 mg/ml): 1 mg PI (Sigma-Aldrich, USA) was dissolved in 1 ml distilled water in a 1.5-ml Eppendrof tube wrapped in aluminium foil, and stored at 4 °C for 2 years. PI working solution (0.002%, 20 lg ml-1): 0.2 ml of PI stock solution was added into 9.8 ml distilled water in a 15-ml conical centrifuge tube wrapped in aluminium foil. This solution could be stored at 4 °C for 12 months for the assay. Each slide was cleaned thoroughly with 95% ethanol before use. No more than 5 ll of semen was used on the slide to make the smear as thin as possible. Two smears were prepared from each sample for duplicate assessments. The slides were air-dried and fixed in 95% ethanol for 15 min. After fixation, the slides were air-dried. 200 ll of PI working solution was applied on the smear. The smear should be fully covered, and then horizontally placed in a dark box for 5 min. The stained slides were gently rinsed with distilled water for 1 minute to remove excess stain, and allowed to air-dry. After drying, the stained slides were examined using a fluorescence microscope (535/617 nm excitation/barrier 2
Normal oval nuclear shape Type a: normal oval nuclear shape, without vacuole (Fig. 1 a). Type b: normal oval nuclear shape, with one small vacuole. Vacuole was located in the ante-nuclear region (ante-half of the nucleus). The length or width of the vacuole was approximately less than the width of normal sperm neck (about 0.6 lm) (Fig. 1 b). Type c: normal oval nuclear shape, with one small vacuole. Vacuole was located in the post-nuclear region (post-half of the nucleus). The length or width of the vacuole was approximately less than the width of normal sperm neck (Fig. 1c). Type d: normal oval nuclear shape, with one large vacuole. Vacuole was located in the ante or post-nuclear region. The length or width of the vacuole was more than the width of the neck (Fig. 1d). Type e: normal oval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the ante-nuclear region. (Fig. 1e). Type f: normal oval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the post-nuclear region (Fig. 1f). © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
Sperm nuclear vacuole assay with PI
W.-J. Zhu and J. Li
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
Fig 1 Schematic representation of sperm nuclei according to both nuclear morphology and the status of vacuole within nucleus. (a) Normal oval nuclear shape, without vacuole. (b) Normal oval nuclear shape, with one small vacuole. Vacuole was located in the ante-nuclear region (ante-half of the nucleus). The length or width of the vacuole was approximately less than the width of normal sperm neck. (c) Normal oval nuclear shape, with one small vacuole. Vacuole was located in the post-nuclear region (post-half of the nucleus). The length or width of the vacuole was approximately less than the width of normal sperm neck. (d) Normal oval nuclear shape, with one large vacuole. Vacuole was located in the ante or post-nuclear region. The length or width of the vacuole was more than the width of the neck. (e) Normal oval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the ante-nuclear region. (f) Normal oval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the post-nuclear region. (g) Nonoval nuclear shape, without vacuole. (h) Nonoval nuclear shape, with one small vacuole. Vacuole was located in the ante-nuclear region. The length or width of the vacuole was approximately less than the width of normal sperm neck. (i) Nonoval nuclear shape, with one small vacuole. Vacuole was located in the post-nuclear region. The length or width of the vacuole was approximately less than the width of normal sperm neck. (j) Nonoval nuclear shape, with one large vacuole. Vacuole was located in the ante or post-nuclear region. The length or width of the vacuole was more than the width of the neck. (k) Nonoval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the ante-nuclear region. (l) Nonoval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the post-nuclear region. (m) nonoval or oval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the ante- and post-nuclear regions. (n) Degenerating nucleus.
Nonoval nuclear shape Type g: nonoval nuclear shape, without vacuole (Fig. 1g). Type h: nonoval nuclear shape, with one small vacuole. Vacuole was located in the ante-nuclear region. The length or width of the vacuole was approximately less than the width of normal sperm neck (Fig. 1h). Type i: nonoval nuclear shape, with one small vacuole. Vacuole was located in the post-nuclear region. The length or width of the vacuole was approximately less than the width of normal sperm neck (Fig. 1i).
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Type j: nonoval nuclear shape, with one large vacuole. Vacuole was located in the ante or post-nuclear region. The length or width of the vacuole was more than the width of the neck (Fig. 1j). Type k: nonoval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the ante-nuclear region (Fig. 1k). Type l: nonoval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the post-nuclear region (Fig. 1l). Type m: nonoval or oval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the ante- and postnuclear regions (Fig. 1m). Type n: degenerating nucleus (Fig. 1n). 3
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Statistical analysis Data were expressed as mean SD. The difference between values obtained by the two sperm populations was evaluated by independent-samples t-test. A difference with P < 0.05 was considered significant. The correlation between values of vacuoles following PI-stained spermatozoa and values of vacuoles following Papanicolaou-stained spermatozoa was evaluated by the linear regression analysis and correlation coefficient determinations. All statistical analysis was performed using SPSS for Windows Version 16.0.
significantly more than those in varicocele group (P < 0.0001). In the varicocele group, the normal nuclear-shaped spermatozoa without vacuole (type a) were less, and abnormal nuclear-shaped spermatozoa with one large vacuole (type j) and with multiple vacuoles located in the ante-nuclear region (type k) were most frequent findings. In addition, spermatozoa with vacuoles in both ante- and post-nuclear regions (type m) in varicocele group were significantly more than those in fertile group (P < 0.01). In both fertile and varicocele groups, types of c, f, i and l had very low incidences.
Results Comparison of values of sperm NVs obtained by PI-staining and Papanicolaou staining methods
Characteristics of NVs All ejaculates in this study had spermatozoa with NVs. Due to the fact that only PI-stained nuclei were shown under fluorescence, the nuclear shapes were slightly smaller in size than those under visible light. NVs showed a variety in number, size, shape, location and distribution. Figure 2 shows representative examples of NVs following PI-stained sperm nuclei. Degenerative nucleus showed a loose, coarsely granular appearance with vacuoles of various sizes, and shown weak fluorescent intensity. NVs appeared anywhere within nucleus such as anterior, posterior region, nuclear edge, or near the neck. The characteristics of NVs in fertile and varicocele groups analysed are presented in Table 1. There was a large variation of spermatozoa with NVs between individual sperm samples in both fertile and varicocele groups. The incidence of NV was significantly lower in the fertile group than that in the varicocele group (P < 0.0001). In the fertile group, the normal nuclear-shaped spermatozoa without vacuole (type a) and with one small vacuole located in the ante-nuclear region (type b) were significantly more in comparison with varicocele group (P ≤ 0.001). In addition, nonoval nuclear-shaped spermatozoa without vacuole (type g) in fertile group were also (a)
After Papanicolaou staining, NVs were analysed based on criteria described in Figure 1. The means of vacuole in fertile and varicocele groups were 60.3 15.3% (range: 30– 88%), 86.5 8.7% (range: 70–99%), respectively, which did not differ compared with the values of vacuole of PIstaining in both groups (P > 0.05). A significant correlation between values of vacuole of Papanicolaou staining and those of PI-staining was found in both fertile (r = 0.846, P < 0.001) and varicocele (r = 0.678, P < 0.001) groups. Discussion A healthy, fertile spermatozoon has good chromatin integrity. Poor chromatin integrity would be a hidden cause of male infertility. NVs may be one of the factors affecting chromatin integrity. Recently, increasing evidence has shown that the presence of NVs is associated with abnormal embryonic development, low implantation and early abortion (Berkovitz et al., 2005, 2006; Franco et al., 2008; Vanderzwalmen et al., 2008; Oliveira et al., 2010; Knez et al., 2012). Thus, the development of relatively simple assays that can identify the status of sperm
(b)
Fig 2 Staining of sperm nuclei with PI. (a) Phase contrast photograph of spermatozoa. (b) Fluorescent photograph of spermatozoa stained with PI. Nuclear vacuoles (arrows). Bar = 5 lm.
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Sperm nuclear vacuole assay with PI
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Table 1 The characteristics of nuclear vacuoles in fertile and varicocele groups Pvalue
Variable
Fertile group
Varicocele group
No. of individuals Vacuole incidence (%) % type a of sperm % type b of sperm % type c of sperm % type d of sperm % type e of sperm % type f of sperm % type g of sperm % type h of sperm % type i of sperm % type j of sperm % type k of sperm % type l of sperm % type m of sperm % type n of sperm
40
40
57.8 13.4 (28–84)
85.1 8.8 (67–99)
0.000
24.9 11.6 (11–66)
4.5 3.6 (0–12)
0.000
6.8 5.3 (0–20)
3.8 2.5 (0–8)
0.001
0.2 0.4 (0–1)
0.1 0.2 (0–1)
–
1.5 1.8 (0–7)
0.6 0.8 (0–3)
–
1.3 2.4 (0–11)
0.7 1.4 (0–6)
–
0
0
–
17.9 8.7 (2–40)
10.8 7.1 (1–26)
0.000
13.0 7.8 (2–36)
11.7 7.3 (2–31)
0.452
0.4 0.7 (0–3)
0.4 0.7 (0–3)
–
17.6 6.2 (5–33)
29.3 12.6 (13–77)
0.000
15.1 8.9 (2–31)
31.4 13.1 (11–68)
0.000
0.03 0.2 (0–1)
0.03 0.2 (0–1)
–
1.2 2.7 (0–12)
3.3 3.7 (0–18)
–
1.1 2.6 (0–15)
2.5 3.7 (0–14)
–
Note: Results were expressed as mean SD. The definition of types a-n is given in the text.
NVs should provide information relevant to chromatin integrity and aid better understanding certain types of male infertility or reproductive failure. PI is a fluorescent vital dye that binds to nuclear DNA, but without membrane permeability. Viable spermatozoa with intact membrane has its resistance to PI penetration. After ethanol fixation, sperm plasma membrane is ruptured. The damaged head membrane allows PI to permeate into the nuclei and the sperm chromatin can be stained, which displays orange fluorescence. With the application of PI, the established SNVA clearly demonstrates the status of NVs of individual spermatozoon, which is suitable to identify the status of sperm NVs in an ejaculate for diagnostic purposes. As shown in Table 1, spermatozoa from fertile group exhibited a high proportion of types a, b and g, which indicated that sperm chromatin from fertile men under© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
went a better packaging that results in decreased levels of nuclear vacuolation. NVs have been considered to occur during spermiogenesis, however, the actual mechanism(s) of formation of NVs remain unknown. Protamines play an essential role in the normal sperm chromatin packaging (Balhorn, 1982; Braun, 2001). During the process of spermiogenesis, sperm nucleoproteins (especially histones) are substituted by protamines, which are highly basic proteins, providing spermatozoa with a highly condensed and compact nucleus. Due to the tight packaging afforded by the protamines, the statue of these nucleoproteins would influence the level of chromatin compaction or condensation. Basically, human sperm nuclei usually contain approximately 85% protamines (Gatewood et al., 1987; Bench et al., 1996), residual 15% histones, which are less basic than protamines, leads to the formation of a less tightly compact chromatin structure. These relatively high residual histones would probably be the biological basis for the origin of NVs of human spermatozoa. Mouse sperm nuclei contain more than 95% protamines in their nucleoprotein component (Bizzaro et al., 1998), and have few or no nuclear vacuole (authors’ observation). Infertile men possess an increased sperm histone to protamine ratio when compared with fertile controls (Zhang et al., 2006). The spermatozoa with increased histone to protamine ratio can potentially result in defective chromatin compaction and an increased susceptibility to DNA damage (Aoki et al., 2006; Torregrosa et al., 2006; Simon et al., 2011). Furthermore, protamine-deficient spermatozoa also show higher levels of DNA damage and lower head intensity (Mohammad et al., 2005). Consequently, any modification or the absence of protamines leads to an anomaly in the packaging process of sperm nucleus, which may facilitate sperm nuclear vacuolation. In this study, the presence of one or more small vacuoles (types b, e, h and k) in anterior nuclear region were most frequent findings, which might be related to different chromatin density or different residual histones content in ante- and post-nuclear regions. Maybe the anterior region has less chromatin density or more residual histones content than the posterior region, leading to the occurrence of NVs more common. The significance of physiology and pathophysiology of 14 categories of sperm nuclei described in this study needs further investigation. From our observations, there were different characteristics on the status of NVs between fertile and varicocele men. In general, (i) for a good sperm sample, types a and b had high proportions, even type g also had a high incidence. Types j and k did not contribute to high incidences. (ii) For a poor sperm sample, types a and b had low proportions. Obviously, type j or k would be the highest incidence. (iii) As common features for an ejaculate, types c, f, i and l occurred sparingly. In other words, 5
Sperm nuclear vacuole assay with PI
if a spermatozoon has one or more NVs located in the post-nuclear region, it means that this is a rare event and the spermatozoon would be recognised as severe aberration on chromatin integrity. Varicocele has been widely considered as a common cause of male infertility. The role of varicocele in male infertility is complex and multifactorial. One of the responsible factors for this disorder in patients with varicocele is associated with elevated intratesticular temperature (Naughton et al., 2001). Scrotal hyperthermia largely impaired sperm chromatin integrity (Ahmad et al., 2012). Elevation in heat stress during spermiogenesis could increase abnormal sperm chromatin condensation (Sadek et al., 2011). Spermatozoa from infertile men with varicocele have significant high levels of DNA damage (Saleh et al., 2003; Talebi et al., 2008; Wu et al., 2009; Zini & Dohle, 2011). In this study, men with varicocele demonstrated a higher proportion of spermatozoa with one large vacuole (type j) and with multiple NVs located in the ante-nuclear region (type k). It indicated that varicocele could cause impaired chromatin packing during spermiogenesis, leading to an increased level of NVs. Repair of varicocele has been shown to usually improve spermatogenesis and has the beneficial effect on sperm chromatin or DNA damage (Zini et al., 2005; Baazeem et al., 2011; Sadek et al., 2011; Schauer et al., 2012). Thus, analysis of NVs would be useful for an additional quantitative evaluation of efficacy of treatment on chromatin integrity before and after varicocelectomy. Both the Papanicolaou staining method and the SNVA can be used to observe NVs of human spermatozoa and have high positive relationship on vacuoles between them. On the other hand, these two assays have different methodology. The Papanicolaou staining shows the whole spermatozoa, and the SNVA only demonstrates sperm nuclei which are clearer for observing vacuoles especially small vacuoles. In addition, the SNVA also has advantages as follows: (i) The PI-stained nuclei are only displayed single orange fluorescence, no transition or differential colour, which is easily analysed. (ii) Under the microscopy, the length or width of a vacuole exceeding the width of the neck was considered as a large vacuole (Fig. 1, types d, j). This definition makes a small or large vacuole evaluation easier and fast, which has a practical value for laboratories that have busy work on the analysis of sperm samples. (iii) Observation at 91000 magnification, the immersion oil does not require a strict fluorescent absorbing property. Slight nonspecific fluorescence can provide a background that aids to observe the neck and tail of sperm. (iv) The fluorescence fading is relatively slow, which allows to have ample time for detailed examination of both nuclei and NVs or repeated observation. (v) Staining is stable. The PIstained slides can be kept over 6 years long (authors’ obser6
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vation). (vi) This assay has fast staining (only 5 min) and is a less work intensive process. In conclusion, the SNVA described in this paper has demonstrated its applicability, which provides a useful additional approach to identify the status of NVs in human spermatozoa for diagnostic purposes. Spermatozoa from men with mid- or severe varicocele have high levels of NVs that reflects poor chromatin integrity. A good sperm sample would have more spermatozoa without NV or with one small vacuole located in antenuclear region. Sperm NV would become an important, independent parameter of sperm quality. Acknowledgement The authors would like to thank the staff of Department of Urology, the first Affiliated Hospital of Jinan University for their kind clinical collaboration. References Ahmad G, Moinard N, Esquerre-Lamare C, Mieusset R, Bujan L (2012) Mild induced testicular and epididymal hyperthermia alters sperm chromatin integrity in men. Fertil Steril 97:546–553. Aoki VW, Emery BR, Liu L, Carrell DT (2006) Protamine levels vary between individual sperm cells of infertile human males and correlate with viability and DNA integrity. J Androl 27:890–898. Baazeem A, Belzile E, Ciampi A, Dohle G, Jarvi K, Salonia A, Weidner W, Zini A (2011) Varicocele and male factor infertility treatment: a new meta-analysis and review of the role of varicocele repair. Eur Urol 60:796–808. Balhorn R (1982) A model for the structure of chromatin in mammalian sperm. J Cell Biol 93:298–305. Bench G, Friz A, Corzett M, Morse D, Balhorn R (1996) DNA and total protamine masses in individual sperm from fertile mammalian subjects. Cytometry 23:263–271. Berkovitz A, Eltes F, Yaari S, Katz N, Barr I, Fishman A, Bartoov B (2005) The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm. Hum Reprod 20:185–190. Berkovitz A, Eltes F, Ellenbogen A, Peer S, Feldberg D, Bartoov B (2006) Does the presence of nuclear vacuoles in human sperm selected for ICSI affect pregnancy outcome? Hum Reprod 21:1787–1790. Bizzaro D, Manicardi G, Bianchi P, Bianchi U, Mariethoz E, Sakkas D (1998) In-situ competition between protamine and fluorochromes for sperm DNA. Mol Hum Reprod 4:127–132. Boitrelle F, Ferfouri F, Petit J, Segretain D, Tourain C, Bergere M, Bailly M, Vialard F, Albert M, Selva J (2011) Large human sperm vacuoles observed in motile spermatozoa
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under high magnification: nuclear thumbprints linked to failure of chromatin condensation. Hum Reprod 26:1650–1658. Braun RE (2001) Packaging paternal chromosomes with protamine. Nat Genet 28:10–12. Collodel G, Moretti E (2008) Morphology and meiotic segregation in spermatozoa from men of proven fertility. J Androl 29:106–114. Franco J Jr, Baruffi R, Mauri A, Petersen C, Oliveira J, Vagnini L (2008) Significance of large nuclear vacuoles in human spermatozoa: implications for ICSI. Reprod Biomed Online 17:42–45. Franco J Jr, Mauri A, Petersen C, Massaro F, Silva L, Felipe V, Cavagna M, Pontes A, Baruffi R, Oliveira J (2012) Large nuclear vacuoles are indicative of abnormal chromatin packaging in human spermatozoa. Int J Androl 35:46–51. Garolla A, Fortini D, Menegazzo M, De Toni L, Nicoletti V, Moretti A, Selice R, Engl B, Foresta C (2008) High-power microscopy for selecting spermatozoa for ICSI by physiological status. Reprod Biomed Online 17:610–616. Gatewood J, Cook G, Balhorn R, Bradbury E, Schmid C (1987) Sequence-specific packaging of DNA in human sperm chromatin. Science 236:962–964. Knez K, Tomazevic T, Zorn B, Vrtacnik-Bokal E, Virant-Klun I (2012) Intracytoplasmic morphologically selected sperm injection improves development and quality of preimplantation embryos in teratozoospermia patients. Reprod Biomed Online 25:168–179. Martınez-Pastor F, Fernandez-Santos M, Del Olmo E, Domınguez-Rebolledo A, Esteso M, Montoro V, Garde J (2008) Mitochondrial activity and forward scatter vary in necrotic, apoptotic and membrane-intact spermatozoan subpopulations. Reprod Fertil Dev 20:547–556. Mohammad HN, Mohammad S, Shahnaz R, Maryam A, Shahla R, Fariba M, Mohammad M (2005) Effect of sperm DNA damage and sperm protamine deficiency on fertilization and embryo development post-ICSI. Reprod Biomed Online 11:198–205. Muratori M, Marchiani S, Tamburrino L, Tocci V, Failli P, Forti G, Baldi E (2008) Nuclear staining identifies two populations of human sperm with different DNA fragmentation extent and relationship with semen parameters. Hum Reprod 23:1035–1043. Naughton CK, Nangia AK, Agarwal A (2001) Varicocele and male infertility: Part II: pathophysiology of varicoceles in male infertility. Hum Reprod Update 7:473–481. Oliveira JB, Massaro FC, Baruffi RL, Mauri AL, Petersen CG, Silva LF, Vagnini LD, Franco JG Jr (2010) Correlation between semen analysis by motile sperm organelle morphology examination and sperm DNA damage. Fertil Steril 94:1937–1940. Sadek A, Almohamdy AS, Zaki A, Aref M, Ibrahim SM, Mostafa T (2011) Sperm chromatin condensation in infertile men with varicocele before and after surgical repair. Fertil Steril 95:1705–1708.
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Sperm nuclear vacuole assay with PI
Saleh RA, Agarwal A, Sharma RK, Said TM, Sikka SC, Thomas AJ Jr (2003) Evaluation of nuclear DNA damage in spermatozoa from infertile men with varicocele. Fertil Steril 80:1431–1436. Schauer I, Madersbacher S, Jost R, H€ ubner WA, Imhof M (2012) The impact of varicocelectomy on sperm parameters: a meta-analysis. J Urol 187:1540–1547. Simon L, Castillo J, Oliva R, Lewis SE (2011) Relationships between human sperm protamines, DNA damage and assisted reproduction outcomes. Reprod Biomed Online 23:724–734. Talebi A, Moein M, Tabibnejad N, Ghasemzadeh J (2008) Effect of varicocele on chromatin condensation and DNA integrity of ejaculated spermatozoa using cytochemical tests. Andrologia 40:245–251. Torregrosa N, Domınguez-Fandos D, Camejo MI, Shirley CR, Meistrich ML, Ballesca JL, Oliva R (2006) Protamine 2 precursors, protamine 1/protamine 2 ratio, DNA integrity and other sperm parameters in infertile patients. Hum Reprod 21:2084–2089. Vanderzwalmen P, Hiemer A, Rubner P, Bach M, Neyer A, Stecher A, Uher P, Zintz M, Lejeune B, Vanderzwalmen S (2008) Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles. Reprod Biomed Online 17:617–627. Ward WS, Coffey D (1991) DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol Reprod 44:569–574. World Health Organization (1992) WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge University Press, Cambridge. World Health Organization (2010) WHO laboratory manual for the examination and processing of human semen, 5th edn. WHO, Geneva, p. 2010. Wu GJ, Chang FW, Lee SS, Cheng YY, Chen CH, Chen I (2009) Apoptosis-related phenotype of ejaculated spermatozoa in patients with varicocele. Fertil Steril 91:831–837. Zamboni L (1987) The ultrastructural pathology of the spermatozoon as a cause of infertility: the role of electron microscopy in the evaluation of semen quality. Fertil Steril 48:711–734. Zamboni L (1992) Sperm structure and its relevance to infertility. An electron microscopic study. Arch Pathol Lab Med 116:325. Zhang X, Gabriel MS, Zini A (2006) Sperm nuclear histone to protamine ratio in fertile and infertile men: evidence of heterogeneous subpopulations of spermatozoa in the ejaculate. J Androl 27:414–420. Zini A, Dohle G (2011) Are varicoceles associated with increased deoxyribonucleic acid fragmentation? Fertil Steril 96:1283–1287. Zini A, Blumenfeld A, Libman J, Willis J (2005) Beneficial effect of microsurgical varicocelectomy on human sperm DNA integrity. Hum Reprod 20:1018–1021.
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A simple sperm nuclear vacuole assay with propidium iodide W.-J. Zhu1 & J. Li2 1 Department of Developmental and Regenerative Biology, College of Life Science and Technology, Jinan University, Guangzhou, China; 2 Department of Pathophysiology, Medical College, Jinan University, Guangzhou, China
Keywords Chromatin—nuclear vacuole—propidium iodide—sperm—varicocele Correspondence Wei-Jie Zhu, Ph.D, Department of Developmental and Regenerative Biology, College of Life Science and Technology, Jinan University, 601# Huang Pu Da Dao Xi, Guangzhou City 510632, China. Tel.: +86-20-85225718; E-mail: [email protected] Accepted: June 16, 2014 doi: 10.1111/and.12328
Summary Our aim was to develop a new simple sperm nuclear vacuole assay (SNVA) with propidium iodide (PI) to determine the status of nuclear vacuole (NV) of individual spermatozoa. After PI staining, sperm nuclei were classified into the 14 categories according to both nuclear morphology and the status of NV. The incidence was 57.8% (range 28–84%) in fertile controls (n = 40), and 85.1% (range 67– 99%) in men with varicocele (n = 40). In the fertile group, normal nuclear-shaped spermatozoa without NV or with one small NV located in the ante-nuclear region were significantly more in comparison with the varicocele group. In the varicocele group, abnormal nuclear-shaped spermatozoa with one large NV and with multiple NVs located in the ante-nuclear region were most frequent findings. Besides, spermatozoa with NVs in both ante- and post-nuclear regions in the varicocele group were significantly more than those in the fertile group. In both fertile and varicocele groups, normal or abnormal nuclear-shaped spermatozoa with one or more vacuoles only located in the post-nuclear region occurred sparingly. The SNVA provides a useful additional approach to identify the status of NV in human spermatozoa for diagnostic purposes. A good sperm sample would have more spermatozoa without NV or with one small NV located in the ante-nuclear region.
Introduction Sperm nuclear vacuoles (NVs) are common phenomena within chromatin, probably occurring in the process of spermiogenesis (Zamboni, 1987, 1992). During this stage, sperm chromatin undergoes complex and highly coordinated changes involving histone replacement and chromatin rearrangement (Ward & Coffey, 1991), leading to providing a highly organised and compact structure that reflects the level of chromatin integrity. Defective, uneven processes or disturbances in chromatin condensation or organisation may result in the formation of NVs (Zamboni, 1987, 1992; Franco et al., 2012). Thus, the status of NVs can in part reflect the level of chromatin integrity. According to the semen analysis manual of the WHO (2010), large NVs in sperm head observed in Papanicolaoustained semen smears should be considered as a type of head morphological defect. In recent years, clinical and laboratory evidence has demonstrated a negative correlation between spermatozoa with large NVs and DNA damage or low fertility potential (Berkovitz et al., 2005; Franco et al., 2008; Garolla et al., 2008; Oliveira et al., 2010; Boitrelle © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
et al., 2011). Microinjection of spermatozoa with NVs can reduce pregnancy outcomes (Berkovitz et al., 2006; Vanderzwalmen et al., 2008). Therefore, the evaluation of NVs should be an important parameter for the understanding of sperm quality related to chromatin integrity and fertile status. Propidium iodide (PI) is a fluorescent nuclear dye that can permeate damaged sperm membranes and stains to sperm chromatin. Several methods have been established to determine sperm viability, membrane integrity, DNA fragmentation, sperm apoptosis and sperm necrosis by using PI in combination with flow cytometry (Collodel & Moretti, 2008; Martınez-Pastor et al., 2008; Muratori et al., 2008). However, there is no assay in detecting sperm NVs with PI by using microscopy. On the other hand, Papanicolaou staining and the motile sperm organelle morphology (MSOME) method can evaluate NVs (Berkovitz et al., 2006; WHO, 2010); however, the former takes a long staining procedure and the latter could not be used for routine analysis in a common laboratory. The aim of this study was to develop a new simple sperm nuclear vacuole assay (SNVA) based on the use of 1
Sperm nuclear vacuole assay with PI
PI to determine the status of NVs of individual spermatozoa. Using the SNVA, we quantitatively analysed the status of NVs of sperm samples from fertile men and men with varicocele to evaluate the applicability of this assay. Materials and methods Study subjects and semen samples Semen samples were obtained from 40 men with varicocele (aged 25–43 years) who attended an infertility clinic. Their wives had failed to conceive after 2 years of unprotected intercourse and had not significant female-factor infertility (tubal obstruction or ovarian failure). Varicocele was graded according to the criteria described by the WHO manual (WHO, 1992). Based on their grades and the experimental design, only men with grades II or III were included in the present study. The controls consisted of 40 normal fertile donors (aged 27–37 years) who had fathered a child in the past 12 months (fertile group). All semen samples were collected from the First Affiliated Hospital of Jinan University, China. The study protocol was approved by the local research ethics committee. Samples were produced by masturbation after 3–7 days of sexual abstinence. Semen examination was performed according to the WHO manual (WHO, 2010). Sperm morphology was assessed using strict criteria after slide staining with the Papanicolaou staining method.
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filter; E400, Nikon, Japan) at 91000 magnification under oil immersion (cat. No. 16242, Nikon, Japan). At least 200 spermatozoa were observed on each slide. Sperm nuclei were displayed orange fluorescence. The neck and tail of spermatozoa were observable due to their nonspecific fluorescent staining, depending on the type of oil used (fluorescent absorbing or nonabsorbing). Images were acquired with a CCD camera (Qimaging, Surrey, BC, Canada) and were pseudocoloured using IPP 5.1 (Media Cybernetics, Inc., Rockville, MD, USA) colour image. For long time preservation, the stained slides were placed in 50% ethanol (10 dips), 70% ethanol (10 dips), 95% ethanol (10 dips), absolute ethanol 92 (1 min in each), xylene 92 (1 min in each) after the step of removing excess stain. The stained slides then were mounted, and kept in a dark box for preservation. For quality control of analysis of NV, the acceptable differences between two duplicate assessments of percentage of vacuole were determined according to the method by the WHO manual (see table 2.1 from the WHO manual) (WHO, 2010). Sperm nucleus Sperm nuclei were classified into the 14 categories described below according to both normal oval or nonoval nuclear shape and the status of vacuole within nucleus, and the schematic representation is shown in Fig. 1. The percentage of each type of sperm nuclei was calculated.
Sperm nuclear vacuole assay PI solutions were prepared as follows: PI stock solution (0.1%, 1 mg/ml): 1 mg PI (Sigma-Aldrich, USA) was dissolved in 1 ml distilled water in a 1.5-ml Eppendrof tube wrapped in aluminium foil, and stored at 4 °C for 2 years. PI working solution (0.002%, 20 lg ml-1): 0.2 ml of PI stock solution was added into 9.8 ml distilled water in a 15-ml conical centrifuge tube wrapped in aluminium foil. This solution could be stored at 4 °C for 12 months for the assay. Each slide was cleaned thoroughly with 95% ethanol before use. No more than 5 ll of semen was used on the slide to make the smear as thin as possible. Two smears were prepared from each sample for duplicate assessments. The slides were air-dried and fixed in 95% ethanol for 15 min. After fixation, the slides were air-dried. 200 ll of PI working solution was applied on the smear. The smear should be fully covered, and then horizontally placed in a dark box for 5 min. The stained slides were gently rinsed with distilled water for 1 minute to remove excess stain, and allowed to air-dry. After drying, the stained slides were examined using a fluorescence microscope (535/617 nm excitation/barrier 2
Normal oval nuclear shape Type a: normal oval nuclear shape, without vacuole (Fig. 1 a). Type b: normal oval nuclear shape, with one small vacuole. Vacuole was located in the ante-nuclear region (ante-half of the nucleus). The length or width of the vacuole was approximately less than the width of normal sperm neck (about 0.6 lm) (Fig. 1 b). Type c: normal oval nuclear shape, with one small vacuole. Vacuole was located in the post-nuclear region (post-half of the nucleus). The length or width of the vacuole was approximately less than the width of normal sperm neck (Fig. 1c). Type d: normal oval nuclear shape, with one large vacuole. Vacuole was located in the ante or post-nuclear region. The length or width of the vacuole was more than the width of the neck (Fig. 1d). Type e: normal oval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the ante-nuclear region. (Fig. 1e). Type f: normal oval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the post-nuclear region (Fig. 1f). © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
Sperm nuclear vacuole assay with PI
W.-J. Zhu and J. Li
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
Fig 1 Schematic representation of sperm nuclei according to both nuclear morphology and the status of vacuole within nucleus. (a) Normal oval nuclear shape, without vacuole. (b) Normal oval nuclear shape, with one small vacuole. Vacuole was located in the ante-nuclear region (ante-half of the nucleus). The length or width of the vacuole was approximately less than the width of normal sperm neck. (c) Normal oval nuclear shape, with one small vacuole. Vacuole was located in the post-nuclear region (post-half of the nucleus). The length or width of the vacuole was approximately less than the width of normal sperm neck. (d) Normal oval nuclear shape, with one large vacuole. Vacuole was located in the ante or post-nuclear region. The length or width of the vacuole was more than the width of the neck. (e) Normal oval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the ante-nuclear region. (f) Normal oval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the post-nuclear region. (g) Nonoval nuclear shape, without vacuole. (h) Nonoval nuclear shape, with one small vacuole. Vacuole was located in the ante-nuclear region. The length or width of the vacuole was approximately less than the width of normal sperm neck. (i) Nonoval nuclear shape, with one small vacuole. Vacuole was located in the post-nuclear region. The length or width of the vacuole was approximately less than the width of normal sperm neck. (j) Nonoval nuclear shape, with one large vacuole. Vacuole was located in the ante or post-nuclear region. The length or width of the vacuole was more than the width of the neck. (k) Nonoval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the ante-nuclear region. (l) Nonoval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the post-nuclear region. (m) nonoval or oval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the ante- and post-nuclear regions. (n) Degenerating nucleus.
Nonoval nuclear shape Type g: nonoval nuclear shape, without vacuole (Fig. 1g). Type h: nonoval nuclear shape, with one small vacuole. Vacuole was located in the ante-nuclear region. The length or width of the vacuole was approximately less than the width of normal sperm neck (Fig. 1h). Type i: nonoval nuclear shape, with one small vacuole. Vacuole was located in the post-nuclear region. The length or width of the vacuole was approximately less than the width of normal sperm neck (Fig. 1i).
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
Type j: nonoval nuclear shape, with one large vacuole. Vacuole was located in the ante or post-nuclear region. The length or width of the vacuole was more than the width of the neck (Fig. 1j). Type k: nonoval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the ante-nuclear region (Fig. 1k). Type l: nonoval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the post-nuclear region (Fig. 1l). Type m: nonoval or oval nuclear shape, with ≥2 vacuoles. Vacuoles were located in the ante- and postnuclear regions (Fig. 1m). Type n: degenerating nucleus (Fig. 1n). 3
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Statistical analysis Data were expressed as mean SD. The difference between values obtained by the two sperm populations was evaluated by independent-samples t-test. A difference with P < 0.05 was considered significant. The correlation between values of vacuoles following PI-stained spermatozoa and values of vacuoles following Papanicolaou-stained spermatozoa was evaluated by the linear regression analysis and correlation coefficient determinations. All statistical analysis was performed using SPSS for Windows Version 16.0.
significantly more than those in varicocele group (P < 0.0001). In the varicocele group, the normal nuclear-shaped spermatozoa without vacuole (type a) were less, and abnormal nuclear-shaped spermatozoa with one large vacuole (type j) and with multiple vacuoles located in the ante-nuclear region (type k) were most frequent findings. In addition, spermatozoa with vacuoles in both ante- and post-nuclear regions (type m) in varicocele group were significantly more than those in fertile group (P < 0.01). In both fertile and varicocele groups, types of c, f, i and l had very low incidences.
Results Comparison of values of sperm NVs obtained by PI-staining and Papanicolaou staining methods
Characteristics of NVs All ejaculates in this study had spermatozoa with NVs. Due to the fact that only PI-stained nuclei were shown under fluorescence, the nuclear shapes were slightly smaller in size than those under visible light. NVs showed a variety in number, size, shape, location and distribution. Figure 2 shows representative examples of NVs following PI-stained sperm nuclei. Degenerative nucleus showed a loose, coarsely granular appearance with vacuoles of various sizes, and shown weak fluorescent intensity. NVs appeared anywhere within nucleus such as anterior, posterior region, nuclear edge, or near the neck. The characteristics of NVs in fertile and varicocele groups analysed are presented in Table 1. There was a large variation of spermatozoa with NVs between individual sperm samples in both fertile and varicocele groups. The incidence of NV was significantly lower in the fertile group than that in the varicocele group (P < 0.0001). In the fertile group, the normal nuclear-shaped spermatozoa without vacuole (type a) and with one small vacuole located in the ante-nuclear region (type b) were significantly more in comparison with varicocele group (P ≤ 0.001). In addition, nonoval nuclear-shaped spermatozoa without vacuole (type g) in fertile group were also (a)
After Papanicolaou staining, NVs were analysed based on criteria described in Figure 1. The means of vacuole in fertile and varicocele groups were 60.3 15.3% (range: 30– 88%), 86.5 8.7% (range: 70–99%), respectively, which did not differ compared with the values of vacuole of PIstaining in both groups (P > 0.05). A significant correlation between values of vacuole of Papanicolaou staining and those of PI-staining was found in both fertile (r = 0.846, P < 0.001) and varicocele (r = 0.678, P < 0.001) groups. Discussion A healthy, fertile spermatozoon has good chromatin integrity. Poor chromatin integrity would be a hidden cause of male infertility. NVs may be one of the factors affecting chromatin integrity. Recently, increasing evidence has shown that the presence of NVs is associated with abnormal embryonic development, low implantation and early abortion (Berkovitz et al., 2005, 2006; Franco et al., 2008; Vanderzwalmen et al., 2008; Oliveira et al., 2010; Knez et al., 2012). Thus, the development of relatively simple assays that can identify the status of sperm
(b)
Fig 2 Staining of sperm nuclei with PI. (a) Phase contrast photograph of spermatozoa. (b) Fluorescent photograph of spermatozoa stained with PI. Nuclear vacuoles (arrows). Bar = 5 lm.
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Sperm nuclear vacuole assay with PI
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Table 1 The characteristics of nuclear vacuoles in fertile and varicocele groups Pvalue
Variable
Fertile group
Varicocele group
No. of individuals Vacuole incidence (%) % type a of sperm % type b of sperm % type c of sperm % type d of sperm % type e of sperm % type f of sperm % type g of sperm % type h of sperm % type i of sperm % type j of sperm % type k of sperm % type l of sperm % type m of sperm % type n of sperm
40
40
57.8 13.4 (28–84)
85.1 8.8 (67–99)
0.000
24.9 11.6 (11–66)
4.5 3.6 (0–12)
0.000
6.8 5.3 (0–20)
3.8 2.5 (0–8)
0.001
0.2 0.4 (0–1)
0.1 0.2 (0–1)
–
1.5 1.8 (0–7)
0.6 0.8 (0–3)
–
1.3 2.4 (0–11)
0.7 1.4 (0–6)
–
0
0
–
17.9 8.7 (2–40)
10.8 7.1 (1–26)
0.000
13.0 7.8 (2–36)
11.7 7.3 (2–31)
0.452
0.4 0.7 (0–3)
0.4 0.7 (0–3)
–
17.6 6.2 (5–33)
29.3 12.6 (13–77)
0.000
15.1 8.9 (2–31)
31.4 13.1 (11–68)
0.000
0.03 0.2 (0–1)
0.03 0.2 (0–1)
–
1.2 2.7 (0–12)
3.3 3.7 (0–18)
–
1.1 2.6 (0–15)
2.5 3.7 (0–14)
–
Note: Results were expressed as mean SD. The definition of types a-n is given in the text.
NVs should provide information relevant to chromatin integrity and aid better understanding certain types of male infertility or reproductive failure. PI is a fluorescent vital dye that binds to nuclear DNA, but without membrane permeability. Viable spermatozoa with intact membrane has its resistance to PI penetration. After ethanol fixation, sperm plasma membrane is ruptured. The damaged head membrane allows PI to permeate into the nuclei and the sperm chromatin can be stained, which displays orange fluorescence. With the application of PI, the established SNVA clearly demonstrates the status of NVs of individual spermatozoon, which is suitable to identify the status of sperm NVs in an ejaculate for diagnostic purposes. As shown in Table 1, spermatozoa from fertile group exhibited a high proportion of types a, b and g, which indicated that sperm chromatin from fertile men under© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–7
went a better packaging that results in decreased levels of nuclear vacuolation. NVs have been considered to occur during spermiogenesis, however, the actual mechanism(s) of formation of NVs remain unknown. Protamines play an essential role in the normal sperm chromatin packaging (Balhorn, 1982; Braun, 2001). During the process of spermiogenesis, sperm nucleoproteins (especially histones) are substituted by protamines, which are highly basic proteins, providing spermatozoa with a highly condensed and compact nucleus. Due to the tight packaging afforded by the protamines, the statue of these nucleoproteins would influence the level of chromatin compaction or condensation. Basically, human sperm nuclei usually contain approximately 85% protamines (Gatewood et al., 1987; Bench et al., 1996), residual 15% histones, which are less basic than protamines, leads to the formation of a less tightly compact chromatin structure. These relatively high residual histones would probably be the biological basis for the origin of NVs of human spermatozoa. Mouse sperm nuclei contain more than 95% protamines in their nucleoprotein component (Bizzaro et al., 1998), and have few or no nuclear vacuole (authors’ observation). Infertile men possess an increased sperm histone to protamine ratio when compared with fertile controls (Zhang et al., 2006). The spermatozoa with increased histone to protamine ratio can potentially result in defective chromatin compaction and an increased susceptibility to DNA damage (Aoki et al., 2006; Torregrosa et al., 2006; Simon et al., 2011). Furthermore, protamine-deficient spermatozoa also show higher levels of DNA damage and lower head intensity (Mohammad et al., 2005). Consequently, any modification or the absence of protamines leads to an anomaly in the packaging process of sperm nucleus, which may facilitate sperm nuclear vacuolation. In this study, the presence of one or more small vacuoles (types b, e, h and k) in anterior nuclear region were most frequent findings, which might be related to different chromatin density or different residual histones content in ante- and post-nuclear regions. Maybe the anterior region has less chromatin density or more residual histones content than the posterior region, leading to the occurrence of NVs more common. The significance of physiology and pathophysiology of 14 categories of sperm nuclei described in this study needs further investigation. From our observations, there were different characteristics on the status of NVs between fertile and varicocele men. In general, (i) for a good sperm sample, types a and b had high proportions, even type g also had a high incidence. Types j and k did not contribute to high incidences. (ii) For a poor sperm sample, types a and b had low proportions. Obviously, type j or k would be the highest incidence. (iii) As common features for an ejaculate, types c, f, i and l occurred sparingly. In other words, 5
Sperm nuclear vacuole assay with PI
if a spermatozoon has one or more NVs located in the post-nuclear region, it means that this is a rare event and the spermatozoon would be recognised as severe aberration on chromatin integrity. Varicocele has been widely considered as a common cause of male infertility. The role of varicocele in male infertility is complex and multifactorial. One of the responsible factors for this disorder in patients with varicocele is associated with elevated intratesticular temperature (Naughton et al., 2001). Scrotal hyperthermia largely impaired sperm chromatin integrity (Ahmad et al., 2012). Elevation in heat stress during spermiogenesis could increase abnormal sperm chromatin condensation (Sadek et al., 2011). Spermatozoa from infertile men with varicocele have significant high levels of DNA damage (Saleh et al., 2003; Talebi et al., 2008; Wu et al., 2009; Zini & Dohle, 2011). In this study, men with varicocele demonstrated a higher proportion of spermatozoa with one large vacuole (type j) and with multiple NVs located in the ante-nuclear region (type k). It indicated that varicocele could cause impaired chromatin packing during spermiogenesis, leading to an increased level of NVs. Repair of varicocele has been shown to usually improve spermatogenesis and has the beneficial effect on sperm chromatin or DNA damage (Zini et al., 2005; Baazeem et al., 2011; Sadek et al., 2011; Schauer et al., 2012). Thus, analysis of NVs would be useful for an additional quantitative evaluation of efficacy of treatment on chromatin integrity before and after varicocelectomy. Both the Papanicolaou staining method and the SNVA can be used to observe NVs of human spermatozoa and have high positive relationship on vacuoles between them. On the other hand, these two assays have different methodology. The Papanicolaou staining shows the whole spermatozoa, and the SNVA only demonstrates sperm nuclei which are clearer for observing vacuoles especially small vacuoles. In addition, the SNVA also has advantages as follows: (i) The PI-stained nuclei are only displayed single orange fluorescence, no transition or differential colour, which is easily analysed. (ii) Under the microscopy, the length or width of a vacuole exceeding the width of the neck was considered as a large vacuole (Fig. 1, types d, j). This definition makes a small or large vacuole evaluation easier and fast, which has a practical value for laboratories that have busy work on the analysis of sperm samples. (iii) Observation at 91000 magnification, the immersion oil does not require a strict fluorescent absorbing property. Slight nonspecific fluorescence can provide a background that aids to observe the neck and tail of sperm. (iv) The fluorescence fading is relatively slow, which allows to have ample time for detailed examination of both nuclei and NVs or repeated observation. (v) Staining is stable. The PIstained slides can be kept over 6 years long (authors’ obser6
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vation). (vi) This assay has fast staining (only 5 min) and is a less work intensive process. In conclusion, the SNVA described in this paper has demonstrated its applicability, which provides a useful additional approach to identify the status of NVs in human spermatozoa for diagnostic purposes. Spermatozoa from men with mid- or severe varicocele have high levels of NVs that reflects poor chromatin integrity. A good sperm sample would have more spermatozoa without NV or with one small vacuole located in antenuclear region. Sperm NV would become an important, independent parameter of sperm quality. Acknowledgement The authors would like to thank the staff of Department of Urology, the first Affiliated Hospital of Jinan University for their kind clinical collaboration. References Ahmad G, Moinard N, Esquerre-Lamare C, Mieusset R, Bujan L (2012) Mild induced testicular and epididymal hyperthermia alters sperm chromatin integrity in men. Fertil Steril 97:546–553. Aoki VW, Emery BR, Liu L, Carrell DT (2006) Protamine levels vary between individual sperm cells of infertile human males and correlate with viability and DNA integrity. J Androl 27:890–898. Baazeem A, Belzile E, Ciampi A, Dohle G, Jarvi K, Salonia A, Weidner W, Zini A (2011) Varicocele and male factor infertility treatment: a new meta-analysis and review of the role of varicocele repair. Eur Urol 60:796–808. Balhorn R (1982) A model for the structure of chromatin in mammalian sperm. J Cell Biol 93:298–305. Bench G, Friz A, Corzett M, Morse D, Balhorn R (1996) DNA and total protamine masses in individual sperm from fertile mammalian subjects. Cytometry 23:263–271. Berkovitz A, Eltes F, Yaari S, Katz N, Barr I, Fishman A, Bartoov B (2005) The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm. Hum Reprod 20:185–190. Berkovitz A, Eltes F, Ellenbogen A, Peer S, Feldberg D, Bartoov B (2006) Does the presence of nuclear vacuoles in human sperm selected for ICSI affect pregnancy outcome? Hum Reprod 21:1787–1790. Bizzaro D, Manicardi G, Bianchi P, Bianchi U, Mariethoz E, Sakkas D (1998) In-situ competition between protamine and fluorochromes for sperm DNA. Mol Hum Reprod 4:127–132. Boitrelle F, Ferfouri F, Petit J, Segretain D, Tourain C, Bergere M, Bailly M, Vialard F, Albert M, Selva J (2011) Large human sperm vacuoles observed in motile spermatozoa
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