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Nuclear status of immature and mature stallion spermatozoa
Theriogenology 66 (2006) 354–365 www.journals.elsevierhealth.com/periodicals/the
Nuclear status of immature and mature stallion spermatozoa G.M. Dias, C.A. Retamal, L. Tobella, A.C.V. Arnholdt, M.L. Lo´pez * Centro de Biocieˆncias e Biotecnologia, LBCT, Setor Biologia da Reproduc¸a˜o, Universidade Estadual do Norte Fluminense, Av. Alberto Lameˆgo 2000, Horto, Campos dos Goytacazes CEP: 28013600, RJ, Brazil Received 25 August 2005; accepted 16 October 2005
Abstract ’The highly packed chromatin of mature spermatozoa results from replacement of somatic-like histones by highly basic arginine- and cysteine-rich protamines during spermatogenesis, with additional conformational changes in chromatin structure during epididymal transit. The objective of the present study was to compare the nuclear characteristics of immature and mature epididymal stallion spermatozoa, using a variety of experimental approaches. Resistance to in vitro decondensation of chromatin, following exposure to SDS-DTT and alkaline thioglycolate, increased significantly in mature spermatozoa. Evaluation of the thioldisulfide status (monobromobimane labeling) demonstrated that immature cells obtained from ductulli efferentes contained mostly thiol groups, whereas these groups were oxidized in mature cells collected from the cauda epididymidis. Based on atomic absorption spectrophotometry, maturation of stallion spermatozoa was accompanied by a 60% reduction in the Zn2+ content of sperm cells, concomitant with increased concentrations of this ion in epididymal fluid. Furthermore, the degree of disulfide bonding was inversely correlated with susceptibility of chromatin to acid denaturation (SCSA). Collectively, these data were consistent with the hypothesis that maturation of stallion spermatozoa involves oxidation of sulphydryl groups to form intra- and intermolecular disulfide links between adjacent protamines, with loss of zinc as an integral feature. These changes endow mechanical and chemical resistance to the nucleus, ensuring efficient transmission of the paternal genome at fertilization. # 2005 Elsevier Inc. All rights reserved. Keywords: Sperm maturation; Chromatin decondensation; Zinc2+; Thiol-disulfide status; Stallion
1. Introduction During spermiogenesis, the nucleus of spermatids undergoes complex morphological, biochemical and physiological alterations. Their shape, size and condensation state change dramatically, due to exchange of histones by transition proteins and highly basic arginineand cysteine-rich protamines [1,2]. These changes in the complex DNA-proteins eliminate the nucleosomal organization of chromatin, resulting in a tightly packed toroidal-like structure, containing up to 60 kb of DNA, in which the transcription and repair activities are
* Corresponding author. Tel.: +55 22 27261690. E-mail address: [email protected] (M.L. Lo´pez). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.10.024
inactivated [3,4]. The primary factor that induces compaction is thought to be protamine binding; it neutralizes the negative charge on the phosphodiester backbone of DNA [5]. In vitro studies of the kinetics of DNA condensation and decondensation induced by protamines and synthetic peptides have shown that the number of clustered arginine residues present in the DNA binding domain is the most important factor affecting the condensation and stability of the DNA-protamine complex, prior to the formation of inter-protamine disulfide cross-links [4]. Although the exact structure of the complex has not been determined, spectroscopic studies suggested that protamine binds to DNA in an extended conformation, with a footprint that covers 11 bp [5]. Protamine binding experiments, conducted in the presence of Zn2+, suggested that a zinc finger would
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facilitate the binding of P2 protamine to DNA [6]. Although zinc-induced conformational changes in protamines have not been demonstrated, their presence appears to be important for sperm chromatin function. In the epididymis, the gross morphology of the spermatozoon does not change, but biophysical and biochemical changes affect sperm architecture and composition; these changes include progressive formation of disulfide bonds that covalently link adjacent protamine chains [7]. Other sperm structures, e.g. outer dense fibers of the flagellum, also become stabilized by S–S linkages during epididymal transit. Thiol oxidation is also involved in the functional competence of the outer dense fibers, thereby affecting sperm motility [8]. There is abundant evidence regarding the structural organization of chromatin and its association with gene regulation [9]; chromatin condensation during sperm formation and its subsequent decondensation in the oocyte are essential for a successful transmission of the male genome. A serine/threonine protein kinase in mouse spermatids that seems to be essential for DNA condensation and male fertility was recently reported [10]. In the last decades, particularly since the development of in vitro fertilization techniques, nuclear status has been used for assessment of potential male fertility. However, there is still uncertainty regarding the fine architecture of the nucleus, the mechanism of disulfide bond formation, and the influence of Zn2+ in sperm physiology. Furthermore, species-specific differences in the chromatin-packing pattern, its susceptibility to reducing agents, and its zinc content, have been reported [11]. The objective of this study was to analyze the nuclear status of immature and mature epididymal stallion spermatozoa, utilizing decondensation tests, ultrastructural study of condensed and decondensed cells, sperm chromatin structure assays (SCSA), determination of thiol-disulfide status, and Zn2+ relative content. 2. Materials and methods 2.1. Chemicals Unless otherwise indicated, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Sample collection Twenty healthy, thoroughbred stallions, 3–5 years of age, were surgically castrated. The epididymides were dissected from the testes; the proximal caput, corpus
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and cauda regions were separated, incised and squeezed to liberate their luminal contents. Spermatozoa were initially collected in a 0.9% NaCl solution, centrifuged (700 g, 10 min) and then washed three times in 0.9% NaCl by centrifugation (700 g, 5 min) before processing for each assay. 2.3. Sperm chromatin stability Chromatin stability was tested by in vitro assays of decondensation induced by: (a) an anionic detergent, 0.5% sodium dodecyl sulfate (SDS; Calbiochem-Nova Biochem Co., San Diego, CA, USA) with 2 mM dithiothreitol (DTT; Bio-Rad (Richmon, CA, USA), in 0.05 M borate buffer, pH 9 [12]; (b) 0.4 M sodium thioglycolate, pH 9 [12]; and (c) 0.5% SDS in 6 mM EDTA [13]. Incubation times were 8, 10 and 30 min for SDS-DTTand 8, 10 and 15 min for alkaline thioglycolate (at room temperature), whereas incubation times were 15, 30 and 60 min (at 37 8C) for SDS-EDTA. The reactions were stopped by the addition of a fixative solution. Control (without treatment) and treated sperm cells obtained from caput, corpus and cauda epididymides regions were fixed in 4% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M phosphate buffer, washed twice in PBS, and processed for electron microscopy. For light microscopy, samples were fixed in 3:1 methanol/acetic acid (10 min) and stained with Giemsa, or directly observed by phase contrast microscopy (Zeiss-Axioplan, Oberkochen, Germany). Morphology was assessed by one observer, using pre-established criteria (form, size and light diffraction), as follows: (a) Group I or stable, with ordinary light diffraction or fully retained staining properties, no observable changes in area and configuration of sperm head (head length 6.5–7 mm); (b) Group II, with moderately swollen spermatozoa (head length >7.0 to <8 mm), partially refringent or lightly stained; and (c) Group III, with grossly swollen head (head length 8 mm), decreased light diffraction or further reduction of nuclear stain. Sperm head measurements were made with ‘‘Soft Imaging System AnalysisTM’’, with a Zeiss Axioplan video microscope; the percentages of stable, partially and decondensed spermatozoa were calculated. At least 200 sperm cells were evaluated and classified per each epididymal region and experimental condition. The effects of each decondensation treatment on spermatozoa obtained from proximal caput, corpus and cauda epididymides regions were tested by analysis of variance (one way ANOVA) and comparison of means by Tukey HSD test. Analysis of variance and comparison of means were also carried out between stable spermatozoa after SDS-DTT, SDS-EDTA and alkaline thioglycolate.
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These analyses were performed with procedures available in Statistic software (StatSoft, Inc., Tulsa, OK, USA). Values were considered to be statistically significant when P < 0.05. 2.4. Nuclear morphology Sperm samples were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, processed for routine electron microscopy, and observed at 80 kV in a transmission electron microscope (Zeiss 900; Oberkochen, Germany). 2.5. Cytochemical tests 2.5.1. Labeling of thiol groups Spermatozoa from the proximal caput and cauda epididymidis were washed twice by centrifugation in a saline solution, re-suspended and incubated for 20 min in 2 mM fluorescent monobromobimane (mBBr) solution (prepared from a stock solution of 50 mM mBBr in acetonitrile; Calbiochem-Nova Biochem Co. San Diego, CA, USA) [14,15]. Labeling was carried out in the dark and was followed by two washes. To block reactive thiol, some samples were previously treated with 0.5 M iodoacetamide at 37 8C (control). Fixed and unfixed sperm samples were examined with a Zeiss-Axiophot microscope with epifluorescence optics (Zeiss-Axioplan, Oberkochen, Germany). Sperm samples were also analyzed in an Elite/ESP Coulter flow cytometer (Coulter Inc., Miami, FL, USA). For this purpose, aliquots of washed spermatozoa (with or without 1 mM DTT, for 10 min) obtained from proximal caput and cauda epididymides regions were incubated with mBBr, as already described. Sperm cells were excited with a 488 nm argon ion laser (15 mW) and the emitted fluorescence was optically collected with DL500/BP525 band pass filter (green). Ten-thousand events for each sample were acquired and the results were presented as a frequency histogram of relative cell numbers versus log of green fluorescence intensity generated by WinMDI 2.8 (Window Multiple Document Interface for flow cytometry). 2.5.2. Zn2+ determination Spermatozoa (6 106 spermatozoa/mL), with or without 200 mM N,N,N0 ,N0 tetrakis-2-pyridylmethyl ethylene diamine (TPEN; Calbiochem-Nova Biochem Co. San Diego, CA, USA) pre-treatment (40 min at room temperature), EDTA 6 mM (30 min at 37 8C), or both, were processed for a zinquin test [16].
Zinquin (Poliscience Inc. Warrington, PA, USA) was used at a final concentration of 25–50 mM and the incubation time was 30 min at 37 8C. After washing twice in PBS and mounted in microscopy slides, sperm samples were examined in a fluorescence microscope using an excitation wavelength of 488 nm. Replicate samples were analyzed by flow cytometry. Fixed and unfixed sperm smears were also stained with dithizone [17]. In some experiments, before the zinquin or dithizone labeling, aliquots of mature and immature sperm suspension were previously treated with 500 mM to 1 mM ZnSO4 to determine the uptake of zinc from the incubation medium. Parafin sections of rat dorso-lateral prostate were used as a positive control for Zn2+ cytochemical determination with dithizone. 2.5.3. DNA determination Semen smears, previously fixed in methanol–acetic acid, were stained with Feulgen technique [17] and evaluated in a cytophotometer (Microscope Photometer 01 K—Zeiss, Oberkochen, Germany), with an epifluorescence kit—exitation filter 510–560; emission filter Lp 590 (magnification, 2000). 2.5.4. Sperm chromatin structure assays (SCSA) This procedure, recommended by Evenson et al. [18], involved staining with acridine orange, followed by flow cytometry. Sperm samples were treated with 0.01 M Tris, 0.15 M NaCl and 1 mM EDTA (pH 7.4). Aliquots of these samples (200 mL, concentration of 1 to 2 106 sperm/mL) were added to 400 mL of the denaturing solution (0.1% Triton X-100, 0.15 M NaCl and 0.08 N HCl; pH 1.2). After 30 s, cells were stained by adding 1.2 mL of a solution containing 6 mg acridine orange/mL of buffered saline (pH 6.0). As a control, samples were incubated in 0.1% Triton X-100 and 0.15 M NaCl for 30 s. The susceptibility to acid denaturation was quantified by flow cytometry (Elite/ ESP Coulter flow cytometer) of the green shift (native DNA) to red shift (denatured DNA). The acridine orange-stained cells were excited with a 488 nm argon ion laser (15 mW) and the emitted fluorescence was optically separated and collected with DL500/BP525 band pass filter (green) and 675 nm BP long pass filter (red). Raw data were expressed as dual parameter scattergrams of 10,000 cells, with each dot representing the amount of green and/or red fluorescence per cell. A frequency histogram of red and green fluorescence distribution, including the relative cell number outside the main population (COMP), was generated by WinMDI 2.8.
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Plate 1. Transmission electron microscope images of stallion epididymal spermatozoa after SDS-DTT (A) alkaline thioglycolate (B) and SDS-EDTA treatment (C). There were different degrees of nuclear decondensation in samples obtained from the proximal caput (Panels 1) and corpus (Panels 2) epididymides regions. Spermatozoa with a homogeneously dense nucleus were predominant in cells obtained from the cauda epididymides (Panels 3).
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2.5.5. Atomic absorption spectrophotometry Spermatozoa were separated from the epididymal fluid by centrifugation through a discontinuous density gradient, composed of three layers of Ficoll in normal saline (densities 1.060, 1.0934 and 1.1432, and osmolarities 336, 340 and 343 mosmol/L, respectively). According to Lindholmer and Eliasson [19], 0.5 mL of each sample was placed in the top of the gradient, and the tubes were centrifuged at 550 g for 15 min, followed by 15 min at 375 g. The epididymal fluid was directly assayed, and zinc concentration was expressed as mg/mL. The sperm fraction (trapped at the bottom of the middle layer) was aspirated and diluted with 0.5 mL saponin-acetic acid solution (800 mg saponin, 75 mL acetic acid and distilled water up to 1.5 L). After counting the sperm cells, 0.5 mL 4N NaOH were added and incubated for 30 min. The final dilution was made by adding 1.5 mL of strontium chloride in distilled water (final concentration, 600 mg/ mL). The Zn2+ content was determined in a PerkinElmer 303 atomic absorption spectrophotometer
(Perkin-Elmer CA, USA) and expressed as mg/108 cells. Results are presented as means S.E.M. Data on zinc content of cells collected from caput and cauda epididymal were analyzed by t-tests for independent samples using the StatSoft, Inc. 4.5 statistical software package (Tulsa, OK, USA). Analysis of variance and comparison of means by Tukey HSD test was also used. A probability value of P < 0.05 was considered significant. 3. Results Samples obtained from the caput and corpus epididymidis region had a morphologically heterogeneous sperm population, with a wide spectrum of decondensed sperm heads following treatment with S–S reducing agents (Plate 1, Panels 1 and 2). Sperm cells of normal size with a homogeneously dense nucleus were predominant in samples collected from the cauda epididydimis region (Plate 1, Panels 3). The morphological effects of the three applied treatments were
Plate 2. The figure shows the percentage of stable, moderately and grossly decondensed spermatozoa in samples collected from the proximal caput, corpus and cauda epididymides region of the stallion, after SDS-DTT (A), sodium thioglycolate (B) and SDS-EDTA (C and D) assays. Incubation times—SDS-DTT: 10 min; alkaline thioglycolate: 8 min; SDS-EDTA 15 and 30 min, respectively (C and D). The percentage of decondensed sperm heads was higher (P < 0.005) in spermatozoa from the caput vs. the cauda epididymides.
G.M. Dias et al. / Theriogenology 66 (2006) 354–365 Plate 3. Photomicrograph of stallion spermatozoa labeled with the fluorescent agent monobromobimane. Sperm cells obtained from the caput epididymidis (A) had a stronger fluorescence than those from the cauda epididymides (B) region (1000). Frequency histogram of mBBr labeled sperm cells obtained from caput epididymidis region (left column) and from cauda epididymis regions (right column) obtained by flow cytometry. At basal conditions [1], mBBr bind principally to immature cells. After treatment with DTT [2], both population cells were labeled. The mean fluorescence intensity (MFI) is showed by the numbers at the right corner of the histograms. Mature spermatozoa displayed an increase of the MFI when treated with DTT prior to labeling.
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relatively similar, and include varying degrees of swelling of the sperm head, chromatin decondensation, alteration of perinuclear structures, disruption or loss of the sperm membranes, and flagellar detachment. At the ultrastructural level, some differences were observed (Plate 1, Panels 1–3). Sperm membranes were more drastically affected after SDS-DTT treatment (A) compared with alkaline thioglycolate-treated samples
(B). The decondensation pattern of thioglycolatetreated samples had a fibrillar network that was apparently different from the granular structures produced by SDS-DTT and SDS-EDTA (C). The head posterior region was the first decondensed sperm region following treatment with SDS-DTT and SDS-EDTA, whereas thioglycolate-treated cells were better preserved, with an increased nuclear contrast near the
Plate 4. Representative sperm chromatin structure assay data from stallion spermatozoa. A green vs. red fluorescence flow cytogram (subpopulation of cells containing intact native DNA and denaturated DNA after acid denaturation), collected from proximal caput (Panel 1), distal caput (Panel 2) corpus (Panel 3) and cauda (Panel 4) epididymides region. The flow cytogram shows untreated cells (left column) and acid treated cells (middle column). The COMP (cells outside the main population) frequency histogram is shown in the right column. Note the increased red fluorescence, in immature sperm populations (Panels 1 and 2) compared with mature spermatozoa (Panel 4) indicating an increased DNA denaturation in sperm cells obtained from the proximal caput epididymides region.
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implantational fossa. High electron density granules were regularly distributed at the nuclear periphery, especially after SDS-DTT and SDS-EDTA treatment. Disruption and loss of the dense lamina were also observed (Plate 1). The number of decondensed spermatozoa varied according to the concentration and nature of the reducing agent, incubation time and origin and quality of the sperm sample. However, independent of incubation time, methodology or inter-individual variations, the percentage of decondensed cells was always highest (P < 0.005) in the immature sperm population, obtained
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from the proximal caput epididymides (Plate 2). The resistance to in vitro chromatin decondensation increased during sperm transit through the epididymis, especially between the caput and corpus regions. For stallion spermatozoa, the standard incubation time was set at 10 min for SDS-DTT, 8 min for alkaline thioglycolate and 15 min for SDS-EDTA. However, the results were not different when other incubation times were used. Incubation times over 30 min produced a greater degree of swelling and sperm lysis. Sperm suspensions were also incubated with mBBr, a fluorescent labeling agent that penetrates intact
Plate 5. Stallion spermatozoa obtained from the proximal caput (Panel 1a) and cauda (Panel 1b) epididymidis regions after zinquin labeling (1000). At basal conditions, immature sperm heads exhibited a brighter fluorescence than mature cells, indicating a higher Zn2+ content. Zinc uptake was principally associated with the connecting and midpiece of the flagellum of spermatozoa collected from the proximal caput epididymides, as is shown in (Panels 2a and 3b) (spermatozoa treated with zinquin and dithizone, respectively, 1000). A positive control (prostate section) for dithizone staining is observed in (Panel 3b) (1000). The relative number of cells (events) displaying positive fluorescence is significantly higher in samples obtained from the caput epididymidis, as shown in the flow cytometry histogram (Panel 4a). After incubation in ZnSO4, the mean fluorescence intensity was increased, especially in sperm cells obtained from the caput epididymis region (Panel 4b). Atomic absorption spectrophotometry corroborated the higher Zn2+ content of spermatozoa (53 8.7 mg/108 cells obtained from proximal caput, in relation to mature spermatozoa (20.1 4.6 mg/108 cells) obtained from cauda epididymidis region. Data from epididymal fluid samples collected from the proximal caput and cauda epididymal regions are also shown (4.5 0.9 and 6.1 2.1 mg/mL, respectively) (Panel 5).
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spermatozoa and binds to free thiol groups in a stable covalent bond under physiological conditions. With this approach, the nucleus of immature cells (obtained from proximal caput) had a brighter fluorescence than those of mature sperm cells (collected from the cauda epididymidis) (Plate 3). Differences in the thiol content of sperm within individual samples were also observed. Non-labeling was found when spermatozoa were first treated with iodoacetamide. Flow cytometric analysis of monobromobimane-labeled spermatozoa corroborated the differences in the thiol content between the immature and mature sperm populations. Pre-treatment with DTT increased the mBBr fluorescence labeling, especially of the mature sperm population obtained from the cauda epididymis region (Plate 3). Sperm cells displayed a strong positive reaction for Feulgen and acridine orange staining at basal conditions. Cytophotometric data for Feulgen reaction revealed minor differences in the DNA staining intensity between immature and mature cells (2.77 0.14 versus 3.47 0.2). When the sperm cells were stained with acridine orange and subsequently analyzed by flow cytometry, differences in the susceptibility to acid denaturation were observed between the sperm populations obtained from the different epididymal regions (Plate 4). Analysis of mature spermatozoa showed low cell numbers drifted towards the red fluorescence (COMP region). In contrast, the analysis of immature cells obtained from the caput epididymides region showed a broad area at COMP, corresponding to exposed denatured DNA. Although the fluorescence displayed by sperm cells treated with the fluorochrome-zinquin was faint and showed some heterogeneity in patterns of fluorescence within a given population, immature spermatozoa (collected from ductulli efferentes) consistently had a more intense reaction than mature populations (Plate 5, Panel 1a and b). Cytochemical detection of Zn2+ with dithizone was unsuccessful. However, control sections of rat and stallion dorsolateral prostate, reacted positively in the epithelial luminal border (Plate 5, Panel 3b). The effect of exogenous zinc on the sperm labeled with both techniques was an increased reaction on the post-acrosomal region, middle piece and cytoplasmic drop of immature sperm cells (Plate 5, Panels 2a and 3a). Flow cytometry of zinquin-labeled spermatozoa corroborated a stronger fluorescence on immature cells compared with the labeling of mature cells (Plate 5, Panel 4a). Zinc chelators, as TPEN and EDTA, decreased the positive fluorescence of the sperm cells, whereas ZnSO4 increased the fluorescence intensity of immature cells (Plate 5, Panel 4b).
Atomic absorption spectrophotometry demonstrated differences in the zinc content of spermatozoa collected from caput and cauda epididymides regions. Sperm maturation was accompanied by a 60% reduction in the Zn2+ content (Plate 5, Panel 5). The mean of Zn2+ was 53 8.7 mg/108 cell in immature cells versus 20.1 4.6 mg/108 cells in mature spermatozoa obtained from the cauda epididymides region (P < 0.05). By contrast, in the epididymal fluid, the relative zinc concentration increased from proximal caput to cauda epididymis (4.5 0.93 mg/mL versus 6.1 2.15 mg/ mL; Plate 5, Panel 5). 4. Discussion Most sperm selection methods for in vitro fertilization have focused on sperm motility and morphological quality. However, in addition to these end points, spermatozoa must have a resistant and undamaged chromatin that permits successful sperm–oocyte interaction. In the present study, we documented the establishment of disulfide linkage during the sperm maturation process. The occurrence of disulfide crosslinking was demonstrated by reductive cleavage, through treatment of spermatozoa with SDS-DTT and alkaline thioglycolate. As thioglycolate does not require detergent to penetrate the cells (due to its low molecular weight), membranes were better preserved using this procedure. Both assays clearly demonstrated that the rate of response to reductive agents was significantly faster in spermatozoa collected from the caput epididydimides. The bromobimane assays to determine the extent of sulfhydryl oxidation also showed differences in the thiol-disulfide status of stallion immature and mature spermatozoa. Some heterogeneity in the thiol content of sperm within individual samples was observed. Similar results have been described in other species (rat, mouse, bull and human) [20,21]. Thiol labeling of oligozoospermic and normozoospermic samples from human ejaculates [22] exhibited a relatively similar pattern in the ratio of high/low fluorescent cells of the immature and mature spermatozoa observed in our assays. It has been suggested that the enhanced susceptibility of sperm DNA to denaturation in infertile compared with fertile men might be associated with incomplete oxidation of sperm – SH groups [23]. The progressive oxidation of thiol groups further stabilized the compacted stallion sperm nuclear chromatin. An appropriate degree of oxidation is required, since spermatozoa are very susceptible to oxidative damage and chromatin condensation favors the resistance of these cells to the genotoxic effects of reactive oxygen species. The mechanisms underlying the
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selective oxidation of sperms thiol proteins to disulfide in the epididymis are not completely understood. The participation of thiol-oxidase activity and non-protein thiol glutathione (GSH), in addition to playing a role as antioxidant, have been implicated in the formation or maintenance of sperm disulphide bounds [24,25]. It is not clear whether non-protein thiols and non-protein disulfides are involved in sperm protein thiol oxidation or whether GSH catabolism in the epididymis can serve as a pathway for sperm protein thiol oxidation [24]. Recently, it has been suggested that a selenoprotein thioredoxin-glutathione reductase participates in this process [26]. Further studies are necessary to determine if there is an interaction between thiol present in the epididymal fluid and sperm proteins. Additional approaches to diagnose the status of the sperm chromatin were Feulgen and acridine orange staining (SCSA). We inferred a differential packing of the DNA-protein complex of immature and mature spermatozoa, not only because of the different stain ability of both cell populations, but also because immature sperm cells were more affected by acid denaturation, as indicated by the broader COMP distribution of immature versus mature spermatozoa. Other authors have shown that the SCSA is a sensitive measurement of the denaturability of sperm DNA in situ [18,27]. We inferred that residual DNA strand breaks were more accessible in immature versus mature spermatozoa. The DNA strand breaks probably occur due to altered protamine deposition or improper protamine–protamine interaction during sperm differentiation, that are accessible due to chromatin packing problems. Normal mature sperm chromatin may contain DNA breaks that are concealed by deposition of protamine molecules during sperm differentiation [28,29]. The DNA of normally condensed and stabilized nuclei has a low vulnerability to environmental changes. However, normal spermatozoa with underprotaminated regions can be naturally nicked, partially denatured, or both [27,30]. The low pH of the assay medium apparently caused partial DNA denaturation in spermatozoa with an altered chromatin structure [31]. Incubation with SDS-EDTA produced similar morphological effects as SDS-DTT, probably because EDTA induced zinc removal, favoring the oxidation of protamine –SH groups. It has been postulated that a fraction of zinc in sperm cells is associated with sulfhydril groups, contributing to the stability of the quaternary structure of the chromatin [32]. Experimental DNA condensation, induced by transition proteins and protamines, showed that both proteins condense free DNA and that zinc facilitates condensation by P2
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protamine. Further analyses demonstrated that zinc binds specifically P2 protamine, and that each protamine molecule binds a single zinc ion [33]. Experimental data indicated that Zn2+ were incorporated into spermatozoa during the later stages of rat sperm differentiation [34]. However, contradictory findings are reported in the literature concerning the origins, content and metabolism of sperm Zn2+ [35–39]. We detected a high concentration of zinc in the stallion epididymal spermatozoa, independent of any contribution from prostatic fluid. After zinquin treatment, sperm heads from samples collected from the proximal caput epididymides region display a bright fluorescence, whereas spermatozoa from the cauda epididymides exhibited a faded fluorescence. It was difficult to verify the localization of Zn2+ in the stallion sperm cell using dithizone (diphenyl thiocarbazone, 3 mercapto 1,5 diphenyl formazan), probably due to its solubility and relatively low quantity. Based on the literature, Zn2+ must be present at a concentration of 50 mg/g of tissue to be demonstrated histochemically [17]. The use of atomic absorption spectrophotometry showed the presence of Zn2+ in the stallion sperm cells and epididymal fluid; both displayed regional differences in their content. Our results clearly demonstrated a marked reduction (60%) in the zinc content of mature spermatozoa. Concomitantly, a higher level of this ion was detected in the epididymal fluid collected from the distal epididymal region, suggesting zinc absorption by the epididymal epithelium. It is known that Zn2+ plays regulatory and structural roles in different cell types; a deficiency leads to impaired-testicular development, reduced spermatogenesis and male sterility [40,41]. Zinc appears to be an indispensable element in reproduction also for another reason. The gonads are the most rapidly growing tissues and vital enzymes involved in nucleic acid and protein synthesis are metalloenzymes [40]. It has been also suggested that several proteins essential for sperm differentiation are androgen-dependent and influenced by the availability of zinc [42]. Since zinc interacts with DNA-protamine complex, a regulatory role in chromatin condensation–decondensation of sperm nucleus has been postulated for this ion [40,43]. According to Zirkin et al. [44,45] the timing of sperm nucleus decondensation after fertilization may depend, in part, on the sperm nuclear disulfide content. The importance of zinc elimination for the functional competence of the outer dense fiber of the flagellum has been reported [8]. It has been observed that zinc, present in the flagellum of bull sperm cells, starts to be mobilized in the caput epididymidis and is absorbed by corpus epididymal epithelium [36]. The fluorescence
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observed in the connecting and midpiece of the flagellum of immature cells after incubation in ZnSO4 solution was consistent with findings in other species, which showed higher uptake Zn2+ in these sperm regions [16]. In vitro assays have shown that additional zinc decreased sperm motility and capacity [46]. The degree of chromatin condensation and zinc content seem to be related with the fertilizing ability of spermatozoa [47,48]. It has been reported that the Zn2+ content of fertile men is <10 mg/106 cells, versus higher concentrations in infertile men [47]. It has been also shown that semen from normospermic men had a significantly lower concentration of cellular zinc and – SH reactive sites than a teratospermic group [12,49]. Our results were consistent with the hypothesis that sperm maturation involves oxidation of sulphydryl groups to form inter- and intra-molecular disulphide links in chromatin proteins (stabilizing this structure) and that the loss of zinc, weakly bound to –SH groups, is an integral part of the process. The biological importance of sperm chromatin stability is not well defined. However, a stabilized nucleus makes the cell less susceptible to physical, chemical or mutagenic agents during epididymal storage, and facilitates transport to the site of fertilization. Furthermore, the level of intra- and inter-molecular disulphide bonds in the protamine molecules may be critical during the post-fertilization decondensation process. Sperm chromatin packing assessments as a complement of routine spermiograms, using any of the above mentioned procedures, might be useful as diagnostic tools for predicting the fertilization potential, since the instability of the nucleus is associated with male infertility, post-fertilization reproductive failure, or both. Acknowledgements This work was supported by Faperj, CNPq and TECNORTE Foundation. The authors are grateful to C.M. Mariano and Arthur Rodrigues for technical collaboration. We gratefully acknowledge Professor E. Hansen for reviewing the manuscript. References [1] Hecht NB. Molecular mechanisms of male germ cell differentiation. Bioassays 1998;20:555–5561. [2] Brewer L, Corzet M, Balhorn RJ. Condensation of DNA by spermatid basic nuclear proteins. Biol Chem 2002;227:38895– 900. [3] Kierszenbaum AL. Transition nuclear proteins during spermiogenesis: unrepaired DNA breaks not allowed. Mol Reprod Dev 2001;58:357–8.
[4] Brewer L, Corzet M, Lau EY, Balhorn R. Dynamics of protamine 1 binding to single DNA molecules. J Biol Chem 2003;278: 42403–8. [5] Hud NV, Milanovich FP, Balhorn R. Evidence of novel secondary structure in DNA bound protamine is revealed by Raman spectroscopy. Biochemistry 1994;33:7528–35. [6] Bianchi F, Rousseaux-Prevost R, Bailly C, Rousseaux J. Interaction of human P1 and P2 protamines with DNA. Biochem Biophys Res Commun 1994;201:1197–204. [7] Seligman J, Zipser Y, Kosower NS. Tyrosine phosphorylation, thiol status and protein tyrosine phosphatase in rat epididymal spermatozoa. Biol Reprod 1999;71:1009–15. [8] Henkel R, Bittner J, Weber R, Hu¨ther F, Miska W. Relevance of zinc in human sperm flagella and its relation to motility. Fertil Steril 1999;71:1138–43. [9] Fenselfeld G. Chromatin as an essential part of the transcription mechanism. Nature 1992;355:219–24. [10] Spiridonov NA, Wong L, Zerfas P, Starost MF, Pack SD, Pawelrtz CP, et al. Identification and characterization of SSTK, a serine/treonine protein kinase essential for male fertility. Mol Cell Biol 2005;25:4250–61. [11] Perrault SD, Barbee RR, Elstein KH, Zucker RM, Keefer CL. Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry. Biol Reprod 1988;39:157–67. [12] Bustos-Obregon E, Leiva S. Chromatin packing in normal and teratozoospermic human ejaculated spermatozoa. Andrology 1983;5:468–78. [13] Kvist U, Kjellberg S, Bjo¨rndahl L, Soufir JC, Arver S. Seminal fluid from men with agenesis of the wolfian ducts: zinc binding properties and effects on sperm chromatin stability. Int J Androl 1990;13:24–52. [14] Kosower NS, Kosower EM. Thiol labeling with monobrobimane. Methods Enzymol 1987;143:76–84. [15] Shalgi R, Seligman J, Kosower NS. Dynamics of the thiol status of rat spermatozoa during maturation: analysis with the fluorescence labeling agent monobromobimane. Biol Reprod 1989;40:1037–45. [16] Zalewski PD, Jian X, Soon LL, Breed WG, Seamark RF, Linciln SF, et al. Changes in distribution of labile zinc in mouse during maturation in the epididymis by the fluorescent fluorochrome zinquin. Reprod Fertil Dev 1996;8:1097–2005. [17] Kiernan JA. Histological and histochemical methods. NY, USA: Theory & Practice Pergamon Press; 1981. [18] Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 1980;210:1131–3. [19] Lindhomer C, Eliasson R. Zinc and magnesium in human spermatozoa. Int J Fertil 1972;17:153–60. [20] Dadoune JP. The nuclear status of human sperm cells. Micron 1995;26:323–45. [21] Seligman J, Shalgi R. Protein thiols in spermatozoa and epididymal fluid of rats. J Reprod Fertil 1991;93:399–408. [22] Rufas O, Fish B, Seligman J, Tadir Y, Ovadia J, Shalgi R. Thiol status in human sperm. Mol Reprod Dev 1991;29:282–8. [23] Zini A, Kamal K, Phang D. Free thiols in human spermatozoa: correlation with sperm DNA integrity. Urology 2001;58:80–4. [24] Seligman J, Newton G, Fahey RC, Shalgi R, Kosower N. Nonprotein thiols and disulfides in rat epididymal spermatozoa and epididymal fluid: role of glutamyl transpeptidases in sperm maturation. J Androl 2005;26(5):629–37 (discussion 638–640).
G.M. Dias et al. / Theriogenology 66 (2006) 354–365 [25] Conrad M, Moreno SG, Sinowats F, Ursini F, Kolle S, Roveri A, et al. The nuclear form of phospholipids hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability. Mol Cell Biol 2005;25:7637– 44. [26] Su D, Novoselov SV, Sun Q, Moustafa ME, Zhou Y, Oko R, et al. Mammalian selenoprotein thioredoxin-glutathione reductase roles in disulfide bond formation and sperm maturation. J Biol Chem 2005;280:26491–8. [27] Sailer BL, Jost K, Evenson DP. Mammalian sperm DNA susceptibility to in situ desnaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. J Androl 1995;16:80–7. [28] McPherson S, Longo FJ. Chromatin structure function alterations during mammalian spermatogenesis: DNA nicking and repair in elongating spermatids. Eur J Histochem 1993;37:109– 28. [29] McPherson S, Longo FJ. Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage dependent patterns and unique sensitivity of elongating spermatids. Mol Reprod Dev 1992;31:268–79. [30] Bianchi PG, Manicardi GC, Bizzaro D, Bianchi U, Sakkas D. Effect of deoxyribonucleic acid protamination on fluorochrome staining an in situ nick-translation of murine and human spermatozoa. Biol Reprod 1993;49:1083–8. [31] Evenson DP, Higgins PH, Grueneberg D, Ballachey BE. Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea. Cytometry 1985;6:253–83. [32] Kvist U. Sperm nuclear chromatin decondensation ability. Acta Physiol Scand Suppl 1980;486:1–24. [33] Bench G, Corzett MH, Kramer CE, Grant PG, Balhorn R. Zinc is sufficiently abundant within mammalian sperm nuclei to bind stoichiometrically with protamine 2. Mol Reprod Dev 2000;56:512–9. [34] Kruczynski D, Passia D, Haider SH, Glassmeyer M. Zinc transport through residual bodies in the rat testis: a histochemical study. Andrologia 1985;17:98–103. [35] Henkel R, Baldauf Z, Schill WB. Resorption of the element zinc from spermatozoa by epididymal epithelium. Reprod Dom Anim 2003;38:97–101.
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Nuclear status of immature and mature stallion spermatozoa G.M. Dias, C.A. Retamal, L. Tobella, A.C.V. Arnholdt, M.L. Lo´pez * Centro de Biocieˆncias e Biotecnologia, LBCT, Setor Biologia da Reproduc¸a˜o, Universidade Estadual do Norte Fluminense, Av. Alberto Lameˆgo 2000, Horto, Campos dos Goytacazes CEP: 28013600, RJ, Brazil Received 25 August 2005; accepted 16 October 2005
Abstract ’The highly packed chromatin of mature spermatozoa results from replacement of somatic-like histones by highly basic arginine- and cysteine-rich protamines during spermatogenesis, with additional conformational changes in chromatin structure during epididymal transit. The objective of the present study was to compare the nuclear characteristics of immature and mature epididymal stallion spermatozoa, using a variety of experimental approaches. Resistance to in vitro decondensation of chromatin, following exposure to SDS-DTT and alkaline thioglycolate, increased significantly in mature spermatozoa. Evaluation of the thioldisulfide status (monobromobimane labeling) demonstrated that immature cells obtained from ductulli efferentes contained mostly thiol groups, whereas these groups were oxidized in mature cells collected from the cauda epididymidis. Based on atomic absorption spectrophotometry, maturation of stallion spermatozoa was accompanied by a 60% reduction in the Zn2+ content of sperm cells, concomitant with increased concentrations of this ion in epididymal fluid. Furthermore, the degree of disulfide bonding was inversely correlated with susceptibility of chromatin to acid denaturation (SCSA). Collectively, these data were consistent with the hypothesis that maturation of stallion spermatozoa involves oxidation of sulphydryl groups to form intra- and intermolecular disulfide links between adjacent protamines, with loss of zinc as an integral feature. These changes endow mechanical and chemical resistance to the nucleus, ensuring efficient transmission of the paternal genome at fertilization. # 2005 Elsevier Inc. All rights reserved. Keywords: Sperm maturation; Chromatin decondensation; Zinc2+; Thiol-disulfide status; Stallion
1. Introduction During spermiogenesis, the nucleus of spermatids undergoes complex morphological, biochemical and physiological alterations. Their shape, size and condensation state change dramatically, due to exchange of histones by transition proteins and highly basic arginineand cysteine-rich protamines [1,2]. These changes in the complex DNA-proteins eliminate the nucleosomal organization of chromatin, resulting in a tightly packed toroidal-like structure, containing up to 60 kb of DNA, in which the transcription and repair activities are
* Corresponding author. Tel.: +55 22 27261690. E-mail address: [email protected] (M.L. Lo´pez). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.10.024
inactivated [3,4]. The primary factor that induces compaction is thought to be protamine binding; it neutralizes the negative charge on the phosphodiester backbone of DNA [5]. In vitro studies of the kinetics of DNA condensation and decondensation induced by protamines and synthetic peptides have shown that the number of clustered arginine residues present in the DNA binding domain is the most important factor affecting the condensation and stability of the DNA-protamine complex, prior to the formation of inter-protamine disulfide cross-links [4]. Although the exact structure of the complex has not been determined, spectroscopic studies suggested that protamine binds to DNA in an extended conformation, with a footprint that covers 11 bp [5]. Protamine binding experiments, conducted in the presence of Zn2+, suggested that a zinc finger would
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facilitate the binding of P2 protamine to DNA [6]. Although zinc-induced conformational changes in protamines have not been demonstrated, their presence appears to be important for sperm chromatin function. In the epididymis, the gross morphology of the spermatozoon does not change, but biophysical and biochemical changes affect sperm architecture and composition; these changes include progressive formation of disulfide bonds that covalently link adjacent protamine chains [7]. Other sperm structures, e.g. outer dense fibers of the flagellum, also become stabilized by S–S linkages during epididymal transit. Thiol oxidation is also involved in the functional competence of the outer dense fibers, thereby affecting sperm motility [8]. There is abundant evidence regarding the structural organization of chromatin and its association with gene regulation [9]; chromatin condensation during sperm formation and its subsequent decondensation in the oocyte are essential for a successful transmission of the male genome. A serine/threonine protein kinase in mouse spermatids that seems to be essential for DNA condensation and male fertility was recently reported [10]. In the last decades, particularly since the development of in vitro fertilization techniques, nuclear status has been used for assessment of potential male fertility. However, there is still uncertainty regarding the fine architecture of the nucleus, the mechanism of disulfide bond formation, and the influence of Zn2+ in sperm physiology. Furthermore, species-specific differences in the chromatin-packing pattern, its susceptibility to reducing agents, and its zinc content, have been reported [11]. The objective of this study was to analyze the nuclear status of immature and mature epididymal stallion spermatozoa, utilizing decondensation tests, ultrastructural study of condensed and decondensed cells, sperm chromatin structure assays (SCSA), determination of thiol-disulfide status, and Zn2+ relative content. 2. Materials and methods 2.1. Chemicals Unless otherwise indicated, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Sample collection Twenty healthy, thoroughbred stallions, 3–5 years of age, were surgically castrated. The epididymides were dissected from the testes; the proximal caput, corpus
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and cauda regions were separated, incised and squeezed to liberate their luminal contents. Spermatozoa were initially collected in a 0.9% NaCl solution, centrifuged (700 g, 10 min) and then washed three times in 0.9% NaCl by centrifugation (700 g, 5 min) before processing for each assay. 2.3. Sperm chromatin stability Chromatin stability was tested by in vitro assays of decondensation induced by: (a) an anionic detergent, 0.5% sodium dodecyl sulfate (SDS; Calbiochem-Nova Biochem Co., San Diego, CA, USA) with 2 mM dithiothreitol (DTT; Bio-Rad (Richmon, CA, USA), in 0.05 M borate buffer, pH 9 [12]; (b) 0.4 M sodium thioglycolate, pH 9 [12]; and (c) 0.5% SDS in 6 mM EDTA [13]. Incubation times were 8, 10 and 30 min for SDS-DTTand 8, 10 and 15 min for alkaline thioglycolate (at room temperature), whereas incubation times were 15, 30 and 60 min (at 37 8C) for SDS-EDTA. The reactions were stopped by the addition of a fixative solution. Control (without treatment) and treated sperm cells obtained from caput, corpus and cauda epididymides regions were fixed in 4% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M phosphate buffer, washed twice in PBS, and processed for electron microscopy. For light microscopy, samples were fixed in 3:1 methanol/acetic acid (10 min) and stained with Giemsa, or directly observed by phase contrast microscopy (Zeiss-Axioplan, Oberkochen, Germany). Morphology was assessed by one observer, using pre-established criteria (form, size and light diffraction), as follows: (a) Group I or stable, with ordinary light diffraction or fully retained staining properties, no observable changes in area and configuration of sperm head (head length 6.5–7 mm); (b) Group II, with moderately swollen spermatozoa (head length >7.0 to <8 mm), partially refringent or lightly stained; and (c) Group III, with grossly swollen head (head length 8 mm), decreased light diffraction or further reduction of nuclear stain. Sperm head measurements were made with ‘‘Soft Imaging System AnalysisTM’’, with a Zeiss Axioplan video microscope; the percentages of stable, partially and decondensed spermatozoa were calculated. At least 200 sperm cells were evaluated and classified per each epididymal region and experimental condition. The effects of each decondensation treatment on spermatozoa obtained from proximal caput, corpus and cauda epididymides regions were tested by analysis of variance (one way ANOVA) and comparison of means by Tukey HSD test. Analysis of variance and comparison of means were also carried out between stable spermatozoa after SDS-DTT, SDS-EDTA and alkaline thioglycolate.
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These analyses were performed with procedures available in Statistic software (StatSoft, Inc., Tulsa, OK, USA). Values were considered to be statistically significant when P < 0.05. 2.4. Nuclear morphology Sperm samples were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, processed for routine electron microscopy, and observed at 80 kV in a transmission electron microscope (Zeiss 900; Oberkochen, Germany). 2.5. Cytochemical tests 2.5.1. Labeling of thiol groups Spermatozoa from the proximal caput and cauda epididymidis were washed twice by centrifugation in a saline solution, re-suspended and incubated for 20 min in 2 mM fluorescent monobromobimane (mBBr) solution (prepared from a stock solution of 50 mM mBBr in acetonitrile; Calbiochem-Nova Biochem Co. San Diego, CA, USA) [14,15]. Labeling was carried out in the dark and was followed by two washes. To block reactive thiol, some samples were previously treated with 0.5 M iodoacetamide at 37 8C (control). Fixed and unfixed sperm samples were examined with a Zeiss-Axiophot microscope with epifluorescence optics (Zeiss-Axioplan, Oberkochen, Germany). Sperm samples were also analyzed in an Elite/ESP Coulter flow cytometer (Coulter Inc., Miami, FL, USA). For this purpose, aliquots of washed spermatozoa (with or without 1 mM DTT, for 10 min) obtained from proximal caput and cauda epididymides regions were incubated with mBBr, as already described. Sperm cells were excited with a 488 nm argon ion laser (15 mW) and the emitted fluorescence was optically collected with DL500/BP525 band pass filter (green). Ten-thousand events for each sample were acquired and the results were presented as a frequency histogram of relative cell numbers versus log of green fluorescence intensity generated by WinMDI 2.8 (Window Multiple Document Interface for flow cytometry). 2.5.2. Zn2+ determination Spermatozoa (6 106 spermatozoa/mL), with or without 200 mM N,N,N0 ,N0 tetrakis-2-pyridylmethyl ethylene diamine (TPEN; Calbiochem-Nova Biochem Co. San Diego, CA, USA) pre-treatment (40 min at room temperature), EDTA 6 mM (30 min at 37 8C), or both, were processed for a zinquin test [16].
Zinquin (Poliscience Inc. Warrington, PA, USA) was used at a final concentration of 25–50 mM and the incubation time was 30 min at 37 8C. After washing twice in PBS and mounted in microscopy slides, sperm samples were examined in a fluorescence microscope using an excitation wavelength of 488 nm. Replicate samples were analyzed by flow cytometry. Fixed and unfixed sperm smears were also stained with dithizone [17]. In some experiments, before the zinquin or dithizone labeling, aliquots of mature and immature sperm suspension were previously treated with 500 mM to 1 mM ZnSO4 to determine the uptake of zinc from the incubation medium. Parafin sections of rat dorso-lateral prostate were used as a positive control for Zn2+ cytochemical determination with dithizone. 2.5.3. DNA determination Semen smears, previously fixed in methanol–acetic acid, were stained with Feulgen technique [17] and evaluated in a cytophotometer (Microscope Photometer 01 K—Zeiss, Oberkochen, Germany), with an epifluorescence kit—exitation filter 510–560; emission filter Lp 590 (magnification, 2000). 2.5.4. Sperm chromatin structure assays (SCSA) This procedure, recommended by Evenson et al. [18], involved staining with acridine orange, followed by flow cytometry. Sperm samples were treated with 0.01 M Tris, 0.15 M NaCl and 1 mM EDTA (pH 7.4). Aliquots of these samples (200 mL, concentration of 1 to 2 106 sperm/mL) were added to 400 mL of the denaturing solution (0.1% Triton X-100, 0.15 M NaCl and 0.08 N HCl; pH 1.2). After 30 s, cells were stained by adding 1.2 mL of a solution containing 6 mg acridine orange/mL of buffered saline (pH 6.0). As a control, samples were incubated in 0.1% Triton X-100 and 0.15 M NaCl for 30 s. The susceptibility to acid denaturation was quantified by flow cytometry (Elite/ ESP Coulter flow cytometer) of the green shift (native DNA) to red shift (denatured DNA). The acridine orange-stained cells were excited with a 488 nm argon ion laser (15 mW) and the emitted fluorescence was optically separated and collected with DL500/BP525 band pass filter (green) and 675 nm BP long pass filter (red). Raw data were expressed as dual parameter scattergrams of 10,000 cells, with each dot representing the amount of green and/or red fluorescence per cell. A frequency histogram of red and green fluorescence distribution, including the relative cell number outside the main population (COMP), was generated by WinMDI 2.8.
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Plate 1. Transmission electron microscope images of stallion epididymal spermatozoa after SDS-DTT (A) alkaline thioglycolate (B) and SDS-EDTA treatment (C). There were different degrees of nuclear decondensation in samples obtained from the proximal caput (Panels 1) and corpus (Panels 2) epididymides regions. Spermatozoa with a homogeneously dense nucleus were predominant in cells obtained from the cauda epididymides (Panels 3).
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2.5.5. Atomic absorption spectrophotometry Spermatozoa were separated from the epididymal fluid by centrifugation through a discontinuous density gradient, composed of three layers of Ficoll in normal saline (densities 1.060, 1.0934 and 1.1432, and osmolarities 336, 340 and 343 mosmol/L, respectively). According to Lindholmer and Eliasson [19], 0.5 mL of each sample was placed in the top of the gradient, and the tubes were centrifuged at 550 g for 15 min, followed by 15 min at 375 g. The epididymal fluid was directly assayed, and zinc concentration was expressed as mg/mL. The sperm fraction (trapped at the bottom of the middle layer) was aspirated and diluted with 0.5 mL saponin-acetic acid solution (800 mg saponin, 75 mL acetic acid and distilled water up to 1.5 L). After counting the sperm cells, 0.5 mL 4N NaOH were added and incubated for 30 min. The final dilution was made by adding 1.5 mL of strontium chloride in distilled water (final concentration, 600 mg/ mL). The Zn2+ content was determined in a PerkinElmer 303 atomic absorption spectrophotometer
(Perkin-Elmer CA, USA) and expressed as mg/108 cells. Results are presented as means S.E.M. Data on zinc content of cells collected from caput and cauda epididymal were analyzed by t-tests for independent samples using the StatSoft, Inc. 4.5 statistical software package (Tulsa, OK, USA). Analysis of variance and comparison of means by Tukey HSD test was also used. A probability value of P < 0.05 was considered significant. 3. Results Samples obtained from the caput and corpus epididymidis region had a morphologically heterogeneous sperm population, with a wide spectrum of decondensed sperm heads following treatment with S–S reducing agents (Plate 1, Panels 1 and 2). Sperm cells of normal size with a homogeneously dense nucleus were predominant in samples collected from the cauda epididydimis region (Plate 1, Panels 3). The morphological effects of the three applied treatments were
Plate 2. The figure shows the percentage of stable, moderately and grossly decondensed spermatozoa in samples collected from the proximal caput, corpus and cauda epididymides region of the stallion, after SDS-DTT (A), sodium thioglycolate (B) and SDS-EDTA (C and D) assays. Incubation times—SDS-DTT: 10 min; alkaline thioglycolate: 8 min; SDS-EDTA 15 and 30 min, respectively (C and D). The percentage of decondensed sperm heads was higher (P < 0.005) in spermatozoa from the caput vs. the cauda epididymides.
G.M. Dias et al. / Theriogenology 66 (2006) 354–365 Plate 3. Photomicrograph of stallion spermatozoa labeled with the fluorescent agent monobromobimane. Sperm cells obtained from the caput epididymidis (A) had a stronger fluorescence than those from the cauda epididymides (B) region (1000). Frequency histogram of mBBr labeled sperm cells obtained from caput epididymidis region (left column) and from cauda epididymis regions (right column) obtained by flow cytometry. At basal conditions [1], mBBr bind principally to immature cells. After treatment with DTT [2], both population cells were labeled. The mean fluorescence intensity (MFI) is showed by the numbers at the right corner of the histograms. Mature spermatozoa displayed an increase of the MFI when treated with DTT prior to labeling.
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relatively similar, and include varying degrees of swelling of the sperm head, chromatin decondensation, alteration of perinuclear structures, disruption or loss of the sperm membranes, and flagellar detachment. At the ultrastructural level, some differences were observed (Plate 1, Panels 1–3). Sperm membranes were more drastically affected after SDS-DTT treatment (A) compared with alkaline thioglycolate-treated samples
(B). The decondensation pattern of thioglycolatetreated samples had a fibrillar network that was apparently different from the granular structures produced by SDS-DTT and SDS-EDTA (C). The head posterior region was the first decondensed sperm region following treatment with SDS-DTT and SDS-EDTA, whereas thioglycolate-treated cells were better preserved, with an increased nuclear contrast near the
Plate 4. Representative sperm chromatin structure assay data from stallion spermatozoa. A green vs. red fluorescence flow cytogram (subpopulation of cells containing intact native DNA and denaturated DNA after acid denaturation), collected from proximal caput (Panel 1), distal caput (Panel 2) corpus (Panel 3) and cauda (Panel 4) epididymides region. The flow cytogram shows untreated cells (left column) and acid treated cells (middle column). The COMP (cells outside the main population) frequency histogram is shown in the right column. Note the increased red fluorescence, in immature sperm populations (Panels 1 and 2) compared with mature spermatozoa (Panel 4) indicating an increased DNA denaturation in sperm cells obtained from the proximal caput epididymides region.
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implantational fossa. High electron density granules were regularly distributed at the nuclear periphery, especially after SDS-DTT and SDS-EDTA treatment. Disruption and loss of the dense lamina were also observed (Plate 1). The number of decondensed spermatozoa varied according to the concentration and nature of the reducing agent, incubation time and origin and quality of the sperm sample. However, independent of incubation time, methodology or inter-individual variations, the percentage of decondensed cells was always highest (P < 0.005) in the immature sperm population, obtained
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from the proximal caput epididymides (Plate 2). The resistance to in vitro chromatin decondensation increased during sperm transit through the epididymis, especially between the caput and corpus regions. For stallion spermatozoa, the standard incubation time was set at 10 min for SDS-DTT, 8 min for alkaline thioglycolate and 15 min for SDS-EDTA. However, the results were not different when other incubation times were used. Incubation times over 30 min produced a greater degree of swelling and sperm lysis. Sperm suspensions were also incubated with mBBr, a fluorescent labeling agent that penetrates intact
Plate 5. Stallion spermatozoa obtained from the proximal caput (Panel 1a) and cauda (Panel 1b) epididymidis regions after zinquin labeling (1000). At basal conditions, immature sperm heads exhibited a brighter fluorescence than mature cells, indicating a higher Zn2+ content. Zinc uptake was principally associated with the connecting and midpiece of the flagellum of spermatozoa collected from the proximal caput epididymides, as is shown in (Panels 2a and 3b) (spermatozoa treated with zinquin and dithizone, respectively, 1000). A positive control (prostate section) for dithizone staining is observed in (Panel 3b) (1000). The relative number of cells (events) displaying positive fluorescence is significantly higher in samples obtained from the caput epididymidis, as shown in the flow cytometry histogram (Panel 4a). After incubation in ZnSO4, the mean fluorescence intensity was increased, especially in sperm cells obtained from the caput epididymis region (Panel 4b). Atomic absorption spectrophotometry corroborated the higher Zn2+ content of spermatozoa (53 8.7 mg/108 cells obtained from proximal caput, in relation to mature spermatozoa (20.1 4.6 mg/108 cells) obtained from cauda epididymidis region. Data from epididymal fluid samples collected from the proximal caput and cauda epididymal regions are also shown (4.5 0.9 and 6.1 2.1 mg/mL, respectively) (Panel 5).
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spermatozoa and binds to free thiol groups in a stable covalent bond under physiological conditions. With this approach, the nucleus of immature cells (obtained from proximal caput) had a brighter fluorescence than those of mature sperm cells (collected from the cauda epididymidis) (Plate 3). Differences in the thiol content of sperm within individual samples were also observed. Non-labeling was found when spermatozoa were first treated with iodoacetamide. Flow cytometric analysis of monobromobimane-labeled spermatozoa corroborated the differences in the thiol content between the immature and mature sperm populations. Pre-treatment with DTT increased the mBBr fluorescence labeling, especially of the mature sperm population obtained from the cauda epididymis region (Plate 3). Sperm cells displayed a strong positive reaction for Feulgen and acridine orange staining at basal conditions. Cytophotometric data for Feulgen reaction revealed minor differences in the DNA staining intensity between immature and mature cells (2.77 0.14 versus 3.47 0.2). When the sperm cells were stained with acridine orange and subsequently analyzed by flow cytometry, differences in the susceptibility to acid denaturation were observed between the sperm populations obtained from the different epididymal regions (Plate 4). Analysis of mature spermatozoa showed low cell numbers drifted towards the red fluorescence (COMP region). In contrast, the analysis of immature cells obtained from the caput epididymides region showed a broad area at COMP, corresponding to exposed denatured DNA. Although the fluorescence displayed by sperm cells treated with the fluorochrome-zinquin was faint and showed some heterogeneity in patterns of fluorescence within a given population, immature spermatozoa (collected from ductulli efferentes) consistently had a more intense reaction than mature populations (Plate 5, Panel 1a and b). Cytochemical detection of Zn2+ with dithizone was unsuccessful. However, control sections of rat and stallion dorsolateral prostate, reacted positively in the epithelial luminal border (Plate 5, Panel 3b). The effect of exogenous zinc on the sperm labeled with both techniques was an increased reaction on the post-acrosomal region, middle piece and cytoplasmic drop of immature sperm cells (Plate 5, Panels 2a and 3a). Flow cytometry of zinquin-labeled spermatozoa corroborated a stronger fluorescence on immature cells compared with the labeling of mature cells (Plate 5, Panel 4a). Zinc chelators, as TPEN and EDTA, decreased the positive fluorescence of the sperm cells, whereas ZnSO4 increased the fluorescence intensity of immature cells (Plate 5, Panel 4b).
Atomic absorption spectrophotometry demonstrated differences in the zinc content of spermatozoa collected from caput and cauda epididymides regions. Sperm maturation was accompanied by a 60% reduction in the Zn2+ content (Plate 5, Panel 5). The mean of Zn2+ was 53 8.7 mg/108 cell in immature cells versus 20.1 4.6 mg/108 cells in mature spermatozoa obtained from the cauda epididymides region (P < 0.05). By contrast, in the epididymal fluid, the relative zinc concentration increased from proximal caput to cauda epididymis (4.5 0.93 mg/mL versus 6.1 2.15 mg/ mL; Plate 5, Panel 5). 4. Discussion Most sperm selection methods for in vitro fertilization have focused on sperm motility and morphological quality. However, in addition to these end points, spermatozoa must have a resistant and undamaged chromatin that permits successful sperm–oocyte interaction. In the present study, we documented the establishment of disulfide linkage during the sperm maturation process. The occurrence of disulfide crosslinking was demonstrated by reductive cleavage, through treatment of spermatozoa with SDS-DTT and alkaline thioglycolate. As thioglycolate does not require detergent to penetrate the cells (due to its low molecular weight), membranes were better preserved using this procedure. Both assays clearly demonstrated that the rate of response to reductive agents was significantly faster in spermatozoa collected from the caput epididydimides. The bromobimane assays to determine the extent of sulfhydryl oxidation also showed differences in the thiol-disulfide status of stallion immature and mature spermatozoa. Some heterogeneity in the thiol content of sperm within individual samples was observed. Similar results have been described in other species (rat, mouse, bull and human) [20,21]. Thiol labeling of oligozoospermic and normozoospermic samples from human ejaculates [22] exhibited a relatively similar pattern in the ratio of high/low fluorescent cells of the immature and mature spermatozoa observed in our assays. It has been suggested that the enhanced susceptibility of sperm DNA to denaturation in infertile compared with fertile men might be associated with incomplete oxidation of sperm – SH groups [23]. The progressive oxidation of thiol groups further stabilized the compacted stallion sperm nuclear chromatin. An appropriate degree of oxidation is required, since spermatozoa are very susceptible to oxidative damage and chromatin condensation favors the resistance of these cells to the genotoxic effects of reactive oxygen species. The mechanisms underlying the
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selective oxidation of sperms thiol proteins to disulfide in the epididymis are not completely understood. The participation of thiol-oxidase activity and non-protein thiol glutathione (GSH), in addition to playing a role as antioxidant, have been implicated in the formation or maintenance of sperm disulphide bounds [24,25]. It is not clear whether non-protein thiols and non-protein disulfides are involved in sperm protein thiol oxidation or whether GSH catabolism in the epididymis can serve as a pathway for sperm protein thiol oxidation [24]. Recently, it has been suggested that a selenoprotein thioredoxin-glutathione reductase participates in this process [26]. Further studies are necessary to determine if there is an interaction between thiol present in the epididymal fluid and sperm proteins. Additional approaches to diagnose the status of the sperm chromatin were Feulgen and acridine orange staining (SCSA). We inferred a differential packing of the DNA-protein complex of immature and mature spermatozoa, not only because of the different stain ability of both cell populations, but also because immature sperm cells were more affected by acid denaturation, as indicated by the broader COMP distribution of immature versus mature spermatozoa. Other authors have shown that the SCSA is a sensitive measurement of the denaturability of sperm DNA in situ [18,27]. We inferred that residual DNA strand breaks were more accessible in immature versus mature spermatozoa. The DNA strand breaks probably occur due to altered protamine deposition or improper protamine–protamine interaction during sperm differentiation, that are accessible due to chromatin packing problems. Normal mature sperm chromatin may contain DNA breaks that are concealed by deposition of protamine molecules during sperm differentiation [28,29]. The DNA of normally condensed and stabilized nuclei has a low vulnerability to environmental changes. However, normal spermatozoa with underprotaminated regions can be naturally nicked, partially denatured, or both [27,30]. The low pH of the assay medium apparently caused partial DNA denaturation in spermatozoa with an altered chromatin structure [31]. Incubation with SDS-EDTA produced similar morphological effects as SDS-DTT, probably because EDTA induced zinc removal, favoring the oxidation of protamine –SH groups. It has been postulated that a fraction of zinc in sperm cells is associated with sulfhydril groups, contributing to the stability of the quaternary structure of the chromatin [32]. Experimental DNA condensation, induced by transition proteins and protamines, showed that both proteins condense free DNA and that zinc facilitates condensation by P2
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protamine. Further analyses demonstrated that zinc binds specifically P2 protamine, and that each protamine molecule binds a single zinc ion [33]. Experimental data indicated that Zn2+ were incorporated into spermatozoa during the later stages of rat sperm differentiation [34]. However, contradictory findings are reported in the literature concerning the origins, content and metabolism of sperm Zn2+ [35–39]. We detected a high concentration of zinc in the stallion epididymal spermatozoa, independent of any contribution from prostatic fluid. After zinquin treatment, sperm heads from samples collected from the proximal caput epididymides region display a bright fluorescence, whereas spermatozoa from the cauda epididymides exhibited a faded fluorescence. It was difficult to verify the localization of Zn2+ in the stallion sperm cell using dithizone (diphenyl thiocarbazone, 3 mercapto 1,5 diphenyl formazan), probably due to its solubility and relatively low quantity. Based on the literature, Zn2+ must be present at a concentration of 50 mg/g of tissue to be demonstrated histochemically [17]. The use of atomic absorption spectrophotometry showed the presence of Zn2+ in the stallion sperm cells and epididymal fluid; both displayed regional differences in their content. Our results clearly demonstrated a marked reduction (60%) in the zinc content of mature spermatozoa. Concomitantly, a higher level of this ion was detected in the epididymal fluid collected from the distal epididymal region, suggesting zinc absorption by the epididymal epithelium. It is known that Zn2+ plays regulatory and structural roles in different cell types; a deficiency leads to impaired-testicular development, reduced spermatogenesis and male sterility [40,41]. Zinc appears to be an indispensable element in reproduction also for another reason. The gonads are the most rapidly growing tissues and vital enzymes involved in nucleic acid and protein synthesis are metalloenzymes [40]. It has been also suggested that several proteins essential for sperm differentiation are androgen-dependent and influenced by the availability of zinc [42]. Since zinc interacts with DNA-protamine complex, a regulatory role in chromatin condensation–decondensation of sperm nucleus has been postulated for this ion [40,43]. According to Zirkin et al. [44,45] the timing of sperm nucleus decondensation after fertilization may depend, in part, on the sperm nuclear disulfide content. The importance of zinc elimination for the functional competence of the outer dense fiber of the flagellum has been reported [8]. It has been observed that zinc, present in the flagellum of bull sperm cells, starts to be mobilized in the caput epididymidis and is absorbed by corpus epididymal epithelium [36]. The fluorescence
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observed in the connecting and midpiece of the flagellum of immature cells after incubation in ZnSO4 solution was consistent with findings in other species, which showed higher uptake Zn2+ in these sperm regions [16]. In vitro assays have shown that additional zinc decreased sperm motility and capacity [46]. The degree of chromatin condensation and zinc content seem to be related with the fertilizing ability of spermatozoa [47,48]. It has been reported that the Zn2+ content of fertile men is <10 mg/106 cells, versus higher concentrations in infertile men [47]. It has been also shown that semen from normospermic men had a significantly lower concentration of cellular zinc and – SH reactive sites than a teratospermic group [12,49]. Our results were consistent with the hypothesis that sperm maturation involves oxidation of sulphydryl groups to form inter- and intra-molecular disulphide links in chromatin proteins (stabilizing this structure) and that the loss of zinc, weakly bound to –SH groups, is an integral part of the process. The biological importance of sperm chromatin stability is not well defined. However, a stabilized nucleus makes the cell less susceptible to physical, chemical or mutagenic agents during epididymal storage, and facilitates transport to the site of fertilization. Furthermore, the level of intra- and inter-molecular disulphide bonds in the protamine molecules may be critical during the post-fertilization decondensation process. Sperm chromatin packing assessments as a complement of routine spermiograms, using any of the above mentioned procedures, might be useful as diagnostic tools for predicting the fertilization potential, since the instability of the nucleus is associated with male infertility, post-fertilization reproductive failure, or both. Acknowledgements This work was supported by Faperj, CNPq and TECNORTE Foundation. The authors are grateful to C.M. Mariano and Arthur Rodrigues for technical collaboration. We gratefully acknowledge Professor E. Hansen for reviewing the manuscript. References [1] Hecht NB. Molecular mechanisms of male germ cell differentiation. Bioassays 1998;20:555–5561. [2] Brewer L, Corzet M, Balhorn RJ. Condensation of DNA by spermatid basic nuclear proteins. Biol Chem 2002;227:38895– 900. [3] Kierszenbaum AL. Transition nuclear proteins during spermiogenesis: unrepaired DNA breaks not allowed. Mol Reprod Dev 2001;58:357–8.
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