Impact of a mild scrotal heat stress on DNA integrity in murine spermatozoa

in Reproduction
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  • 1 MRC Human Reproductive Sciences Unit, The Chancellor’s Building, 49 Little France Crescent, Edinburgh, EH16 4SB, UK

Correspondence should be addressed to P Saunders, MRC Human Reproductive Sciences Unit, 49 Little France Crescent, Edinburgh EH16 4SB, UK; Email: p.saunders@ed.ac.uk

An increase in scrotal temperature can lead to the production of poor quality spermatozoa and infertility. In the present study we have used mice to examine the impact of mild, scrotal heat stress (42 °C for 30 min) on numbers of spermatozoa as well as on the integrity of their DNA. Spermatozoa recovered from the epididymides hours (1 to 24) or days (7 to 32) after treatment were analysed using COMET and sperm chromatin structure (SCSA) assays. The treatment induced a stress response in both the testis and the epididymis that was associated with reduced expression of the cold inducible RNA binding protein (Cirp) and an increase in germ cell apoptosis (Apotag positive cells). Although spermatozoa present in the epididymis at the time of heating contained correctly packaged DNA, its integrity was compromised by heat stress. In addition, although some germ cells, which were present within the testis at the time of heat stress, were removed by apoptosis, many germ cells completed their development and were recovered as motile spermatozoa with damaged DNA. In conclusion, these data demonstrate that scrotal heat stress can compromise the DNA integrity of spermatozoa and this may have clinical implications for patients undergoing IVF and intra-cytoplasmic sperm injection (ICSI).

Abstract

An increase in scrotal temperature can lead to the production of poor quality spermatozoa and infertility. In the present study we have used mice to examine the impact of mild, scrotal heat stress (42 °C for 30 min) on numbers of spermatozoa as well as on the integrity of their DNA. Spermatozoa recovered from the epididymides hours (1 to 24) or days (7 to 32) after treatment were analysed using COMET and sperm chromatin structure (SCSA) assays. The treatment induced a stress response in both the testis and the epididymis that was associated with reduced expression of the cold inducible RNA binding protein (Cirp) and an increase in germ cell apoptosis (Apotag positive cells). Although spermatozoa present in the epididymis at the time of heating contained correctly packaged DNA, its integrity was compromised by heat stress. In addition, although some germ cells, which were present within the testis at the time of heat stress, were removed by apoptosis, many germ cells completed their development and were recovered as motile spermatozoa with damaged DNA. In conclusion, these data demonstrate that scrotal heat stress can compromise the DNA integrity of spermatozoa and this may have clinical implications for patients undergoing IVF and intra-cytoplasmic sperm injection (ICSI).

Introduction

In most mammals, scrotal temperature is 2–8 °C below core temperature and this lower temperature is required for normal spermatogenesis (Harrison & Weiner 1948, Ulberg 1958). The impact of raised scrotal temperature in the rat (Lue et al. 2000), mouse (Jannes et al. 1998) and human (Mieusset et al. 1987) includes decreased testis weight, reduced viability, morphology and motility of spermatozoa, and reduced testicular blood flow and altered vasomotion (Setchell et al. 1995). In the mouse direct effects of scrotal heating on the developing germ cells are reported to include altered synthesis of DNA, RNA and proteins, as well as protein denaturation and abnormal chromatin packing (Steinberger 1991, Sailer et al. 1997).

Previous studies in the mouse have examined the effects on spermatogenesis of exposure of testes to a range of temperatures (38–42 °C) and exposure times (20 min to 1 h). In an extensive study by De Vita et al.(1990), it was shown that exposure to mild heat stress (38 °C) for a short time (20 min) caused cytotoxic effects in some germ cell types and that these effects were more pronounced in spermatocytes than in differentiated spermatogonia. Following heat stress, the epididymis was reported to lose its ability to store and maintain viable spermatozoa, resulting in the gradual and progressive accumulation of dead, decapitated and immotile spermatozoa (Bedford 1991, 1994); the region most affected is reported to be the cauda.

In man, raised scrotal temperature may occur as a result of occupational exposure, lifestyle or a clinical disorder, such as cryptorchidism (Mieusset et al. 1987, Hjollund et al. 2000, 2002). It has been shown that men with a past history of cryptorchidism are likely to exhibit scrotal temperatures above the normal range and are often subfertile or infertile. It is also notable that the spermatozoa in their ejaculates exhibit an increased incidence of abnormalities such as tapered/elongated forms. In two separate studies on normal men, Hjollund et al.(2000, 2002) recorded the diurnal scrotal temperature using continuous monitoring over a 72 or 24 h period. They found evidence that mean scrotal temperature (median 33.3 °C) was increased during sedentary work periods (average 0.7 °C higher) and at night (median 34.8 °C) (Hjollund et al. 2000). In both studies a negative correlation was found between high scrotal temperature and sperm output with sperm concentration being decreased 40% per 1 °C increment of median day time scrotal temperature in a study of 99 men (Hjollund et al. 2002). Studies of infertile males have shown increased scrotal temperature compared with fertile controls (Mieusset et al. 1987, Mieusset & Bujan 1995), but it has not yet been clearly established whether raised scrotal temperature is the cause of the infertility in these men or simply a concomitant symptom of their disorder. Treatment of couples with male factor infertility by IVF/intra-cytoplasmic sperm injection (ICSI) is now common place in clinical practice (Campbell & Irvine 2000). There is some evidence of a loss of genetic integrity within the spermatozoa used for these procedures and this may contribute to reduced fertilization rates, development of fewer blastocysts and poor embryonic development all of which have been reported (Lopes et al. 1998, Evenson et al. 1999).

The aims of the current study were first to investigate the effects of heat stress on the process of spermatogenesis, secondly to identify the cell types in the testis and epididymis susceptible to heat stress and thirdly to examine the integrity of the DNA in spermatozoa which developed from cells exposed to heat either during spermatogenesis or during epididymal transit. The response of the testicular and epididymal cells to a mild heat was monitored using immunohistochemical evaluation of known stress markers and Apotag. The integrity of the DNA in motile spermatozoa at various time points after heat stress was investigated using COMET and sperm chromatin structure assays (SCSA).

Materials and Methods

Animals and heat treatment

Wild type mice (MF1 strain) were bred and maintained at the Medical Faculty Animal Accommodation, University of Edinburgh. All animals were maintained under standard animal house conditions and treated in accordance with Government guidelines as stated in the Animals and Scientific Procedures Act 1986.

Animals were anaesthetised with an i.p. injection of Hypnorm (fentanyl citrate 0.315 mg/ml, fluanisone 10 mg/ml; Research Diagnostics Inc. Flanders, NJ, USA) and Hypnovel (midazolam hydrochloride; Roche, Lewes, East Sussex, UK). Following anaesthesia, the lower third (hind legs, tail and scrotum) of each male was passed through a hole in a polystyrene ‘raft’ which was then placed in a circulating water bath at 42 °C for 30 min. Control animals received anaesthetic only. All animals were returned to their cages, which were placed on a warm mat (20–25 °C) to maintain body temperature, until fully recovered from the anaesthetic.

Animals, three per treatment group plus three controls at each time point, were killed at 1, 2, 4, 6, and 24 h, and 7, 14, 21, 24, 28 and 32 days after treatment. Each time-point was chosen either to allow study of the spermatozoa present within the epididymis at the time of heating (1 to 24 h) or those which developed from the different germ cell types that were present in the testis at the time of heat stress (7 to 32 days); these are summarized in Table 1. For the latter, calculations were based on the stage durations of mouse spermatogenesis as defined by Oakberg (1956) and Russell et al.(1990) and a 7-day epididymal transit time as defined by Cornwall et al.(1990).

Recovery of tissues

Testes were fixed in Bouins for 6 h then transferred to 70% ethanol and processed into paraffin wax for histological analysis. Epididymides were either fixed in Bouins or cleaned of fatty tissue and placed in 1 ml Biggers, Whitten, Whittingham media (BWW; Biggers et al. 1971) containing 20 mM N-[2-hydroxyethyl]piperazine-N′-[2-ethan sulphonic acid] (HEPES; Gibco, Paisley, UK) and 0.3% human serum albumin (HSA; Gibco). In order to recover the motile spermatozoa the tissues from each of the treated animals (n = 3 per group) as well as the controls (n = 3 at each point) were minced with fine scissors and incubated at 34 °C (95% air, 5% CO2) for 30 min prior to careful removal of all the buffer. Samples were diluted to a final volume of 1 ml in BWW, aliquots were taken for the sperm counts and the remainder of the sample stored at −20 °C.

Concentration of spermatozoa

Aliquots (three per sample) prepared from minced epididymides from each of the treated animals (three per group) as well as from two groups of controls (1 h and 7 days, n = 6) were diluted 1:10 with BWW and a 10 μl aliquot placed into the chamber of an improved Neubauer haemocytometer (CamLab, Cambridge, UK). Sperm were counted according to the techniques described by the World Health Organisation (WHO 1999).

Immunohistochemistry

Slides were washed twice in PBS (5 min each) before incubation in a bath of 3% hydrogen peroxide in methanol at room temperature for 30 min to block endogenous peroxidases. After further washes in PBS, slides were blocked with normal swine serum (NSS; 5% BSA, 10% swine serum; Diagnostics Scotland, Carluke, UK) for 1 h at room temperature. A rabbit polyclonal antibody raised against the cold inducible RNA binding protein (Cirp; Nishiyama et al. 1997) was diluted 1:20 000 in NSS and incubated on the sections for 2 h at room temperature; sections were washed in PBS as before. For fluorescent detection (testis sections) slides were incubated with swine anti-rabbit FITC (1:50 in NSS; DAKO, Ely Cambridgeshire, UK) for 1 h at room temperature and washed in PBS before mounting using Permafluor (Coulter-Immunotech, Hamburg, Germany). For colour detection (epididymis) sections were washed twice in tris-buffered saline (TBS) (5 min each) and incubated with swine anti-rabbit biotinylated secondary antibody (DAKO, 1:500 in BSA/TBS) at room temperature for 30 min. Following washes in TBS, single stained sections were incubated with avidin–biotin–HRP complex (DAKO) according to the manufacturer’s instructions. Bound antibody was visualized using 3,3-diaminobenzidine tetrahydrochloride (DAB, DAKO); sections were counterstained with Mayer’s haematoxylin. Sections from the testes and epididymes from each of the treated animals were stained on three separate occasions; staining intensity was compared with sections from the controls killed at the 1 h and 7 day time points (n = 3 per group). Negative controls, omitting the primary antibody, were included in each experiment and the results were negative (not shown). Sections were photographed using a Provis microscope (Olympus Optical, London, UK) and a Kodak DCS330 digital camera (Eastman Kodak, Rochester, NY, USA); images were assembled using Photoshop 6 (Adobe, Mountain View, CA, USA).

Detection of apoptotic cells using Apotag

Sections (three from each animal) were pretreated by incubation with 100 μg/ml proteinase K (Sigma, Gillingham, Dorset, UK) in buffer (3.5 ml 1 M Tris, pH 8, 0.7 ml 0.5 M EDTA, pH 8, made up to 35 ml with dH20) for 10 min at room temperature, and then washed twice in PBS (5 min each). The reaction mix (30 mM Tris/HCl, pH 7.2, 140 mM Na cacodylate, 1.5 mM CoCl) containing 1 μl/ml terminal d-transferase (TdT, Roche, Lewes, Sussex, UK), 5 μl/ml digoxigenin (DIG, Roche) was added and sections were covered with cover-slips which were sealed with cow gum/hexane before they were incubated at 37 °C for 30 min.

Following the removal of cover-slips and washes in PBS, sections were blocked with 20% normal rabbit serum (NRS) in PBS for 10 min at room temperature. Sheep anti-DIG IgG (Boehringer, 1:100 in NRS/PBS) was added and the slides incubated at room temperature for 90 min before further washes in PBS. Rabbit anti-sheep IgG biotinylated (1:500 in NRS/TBS) was added for 30 min at room temperature before washing twice in TBS (5 min each). Bound antibodies were detected using DAB and sections were counterstained with Mayer’s haematoxylin. In order to assess the numbers of Apotag-positive germ cells at each time-point (control and heated animals), all sections were examined using a × 20 objective on an Olympus BH2 microscope fitted with a Prior automatic stage (Prior Scientific Instruments, Cambridge, UK). The number of immunopositive cells in three fields selected using the software provided with the stage was counted and the average value calculated for each animal.

COMET analysis of spermatozoa

Spermatozoa (5 μl) recovered from the epididymis (as above) were mixed with 25 μl Low Melting Agarose (LMA, 37 °C; Trevigen, Gaithersburg, Maryland, USA). This gel/sample mix was dropped onto a ‘COMET’ slide (Trevigen) which was covered with a clean, warm (37 °C) cover-slip, placed horizontally and chilled at 4 °C. After the gels were set cover-slips were removed and the slides equilibrated in lysis buffer (0.75% SDS, 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% sodium lauryl sarcosinate, 0.01% Triton X-100) for 3 h at 37 °C. Electrophoresis was performed by submerging slides in a horizontal gel electrophoresis tank containing alkaline electrophoresis buffer (3 M NaOH, 1 mM EDTA, pH 12.3). Slides were pre-equilibrated for 20 min at room temperature before current was applied (25 V, 300 mA for 10 min). Slides were transferred to ice-cold methanol (100%, 5 min) then ethanol (100%, 5 min) and allowed to dry overnight at room temperature before staining with 50 μl ethidium bromide (1:1000 in dH2O). For each sample, 100 cells were analysed using the Komet Image Analysis System, version 4.0 (Kinetic Imaging Ltd, Liverpool UK). The percentage of head DNA, tail DNA and the COMET moment was calculated for each cell. COMET moment is a measurement that takes into account both the length of the COMET tail and the amount of DNA present in the tail and is expressed as an arbitrary unit (the greater the value, the higher the level of DNA damage). The Mann–Whitney U test was used to compare the results from each heated group against the controls (n = 3 in each group).

Sperm chromatin structure assay (SCSA)

A modified method of the SCSA described by Evenson et al.(1999) was employed. Briefly, motile spermatozoa recovered from the epididymis in BWW were defrosted at room temperature and adjusted to a concentration of 2 × 106 cells/ml with TNE (0.15 M NaCl, 0.1 M Tris, 1 mM EDTA pH 7.4). 100 μl of this sample was mixed with 200 μl of acid detergent solution (0.1% TritonX-100, 0.15 M NaCl, 0.08 M HCl, pH 1.2). After 30 sec at room temperature 600 μl acridine orange staining solution was added (37 mM citric acid, 126 mM Na2HPO4, 1 mM EDTA, 0.15 M NaCl, pH 7.4 with acridine orange (Sigma) added fresh to a final concentration of 6 μg/ml) and left at room temperature for an additional 150 s after which cells were immediately analysed in a Coulter Epics XL Flow Cytometer (Beckman Coulter (UK) Ltd. High Wycombe, Bucks, UK). Red fluorescence (single-stranded DNA) was measured using a 675 nm detector and green fluorescence (double-stranded DNA) measured using a 525 nm detector. The Epics XL was calibrated for each run by adjusting the wild-type control samples to give a mean fluorescence value (arbitrary units) of 145 ± 10 at 675 nm and 445 ± 10 at 525 nm. 10 000 events were collected for each sample and each was run in triplicate. Raw data were exported as Static Dimension data files (LMD File Format) and analysed using FlowJo (Tree Star Inc., Ashland, Oregon, USA). Background contamination (bacterial contaminants and cells other than spermatozoa) were removed by gating and the results expressed in terms of αt (red/[red plus green] fluorescence, expressed as percentage). COMP αt (cells outside the main population) was calculated as the percentage of cells in the population exhibiting an αt value greater than 26% according to the methods described by Evenson et al.(1999).

Results

Expression of the cold-inducible RNA binding protein (Cirp) following scrotal heating

In testes from control mice, Cirp was immunolocalised to spermatocytes (Fig. 1a). Following in vivo heat treatment, a decrease in immunoexpression of Cirp was detected at 2, 4, 6 and 24 h; this was most marked in the samples recovered at 6 h (Fig. 1c) and expression remained low at 24 h (not shown). Expression appeared identical to that of controls in testes recovered 7 (Fig. 1d) or more (not shown) days after treatment. Cirp was immunolocalised to all regions of the epididymis in controls (Fig. 2 a–d). Six hours after heat stress immunoexpression of Cirp was markedly reduced in the initial segment, corpus and cauda but only slightly reduced in the caput (Fig. 2 e–h).

Apotag labelling of testicular germ cells

Consistent with other data obtained from our laboratory from studies on murine testes (Hsai et al. 2003, De Gendt et al. 2004) the number of Apotag-positive germ cells in control animals was low (Fig. 3, open bars). An increase in Apotag-positive germ cells was detectable 2 h after scrotal heating. The number of positive cells was increased further in samples recovered at 24 h, reached a peak at 14 days and thereafter declined to control levels (Fig. 3, shaded bars).

Changes in the number of motile spermatozoa following heat stress

The concentration of motile spermatozoa recovered from the epididymides of control animals (n = 6) was in the range of 5.7 to 7.4 × 106/ml (mean 6.4 × 106/ml). The average number of motile spermatozoa retrieved from the epididymides was decreased 1 h after scrotal heat stress (4.9 × 106/ml; P < 0.05). The number of motile spermatozoa was further reduced at subsequent time points reaching a nadir at 4 h post-heating (1.1 × 106/ml; P < 0.05); numbers remained low (maximum 2.12 × 106/ml) for the duration of the experiment (32 days).

Levels of DNA damage in motile spermatozoa following heat stress

COMET Assay

DNA damage in motile spermatozoa recovered from the epididymides showed a significant increase 1 h following heat treatment (Tail DNA 37.8%; P < 0.05, COMET Moment 15.8, P < 0.05) compared with the control (25.3% COMET tail DNA, 6.9 COMET moment; Fig. 4). COMET values continued to increase until 4 h post heat treatment (Tail DNA 57.8%, P < 0.05, COMET moment 27.7, P < 0.005) and stayed above control levels until 7 days post heat treatment when they were indistinguishable from controls.

In addition there was a second rise in recovery of spermatozoa with COMET values above controls at 21 days after treatment (COMET Tail DNA 34.9%; P < 0.05, COMET Moment 19.4, P < 0.05). Values also remained above controls in samples recovered at 24, 28 and 32 days (Fig. 4; 28 days COMET Tail DNA 41%; P < 0.05, COMET Moment 19.4, P < 0.05).

Sperm chromatin structure assay (SCSA)

SCSA results were expressed as COMP αt which was a measure of the number of spermatozoa exhibiting abnormal DNA integrity (Fig. 5). Compared with control (COMP αt 5.9%), the spermatozoa from heat-treated animals showed a significant increase in COMP αt 1 h following treatment (COMP αt 31.1%; P < 0.05); highest values were obtained 6 h after heat stress (COMP αt 58.2%; P < 0.05). COMP αt values returned to control levels after 24 h. A second increase in COMP αt was seen at day 7 (COMP αt 16.9%; P < 0.05). Significant levels of DNA abnormality were detected in spermatozoa recovered from 14 days to 32 days after treatment (Fig. 5).

Additional information relating to the status of the DNA within the spermatozoa was obtained by evaluating the numbers of spermatozoa in which the level of green fluorescence was over 800 (‘High Green’) as a measure of whether they contained DNA which was not fully condensed and therefore more accessible to acridine orange. Although most samples included 2 to 5% of spermatozoa with High Green values, a significant peak was observed at 14 days post heat stress when 18.3% (P < 0.05) of spermatozoa had a High Green value.

Discussion

In the present study we have shown that subjecting the testis to a transient heat stress results in an increased level of apoptosis in germ cells, but that those cells that do survive can complete spermatogenesis to form spermatozoa that carry damaged DNA. The DNA integrity of epididymal spermatozoa was also compromised if scrotal temperature was raised. We have shown for the first time that the temperature sensitive RNA binding protein Cirp is expressed in the murine epididymis and that its expression is reduced when scrotal temperature is raised. The heating regime (30 min at 42 °C) used in this study was chosen to mimic that of a hot bath. Jannes et al.(1998) had previously reported that subjecting mice to scrotal heating at a temperature of 42 °C for 20 min resulted in the production of poor quality spermatozoa, reduced embryo weight in vivo, and reduced fertilization rates in vitro, a situation that is reminiscent of that associated with some types of human sub-fertility.

Changes in the level of expression of the temperature-regulated RNA binding protein Cirp were consistently detected in both the testis and the epididymis following treatment, confirming that the transient heat stress had had an impact on cell function in both organs. The maximal decrease in Cirp immunoexpression was detected 6 h after treatment. These findings are in agreement with those of Nishiyama et al.(1998) who reported that expression of Cirp in germ cells was reduced 6 h after increasing testicular temperature in mice using a water bath (42 °C) or by placing the testes in the abdomen. Furthermore, these authors also reported that expression of Cirp was reduced in the germ cells of testes from men with varicocele and they proposed a role for Cirp in the regulation and co-ordination of germ cell function during spermatogenesis (Nishiyama et al. 1998). RNA binding proteins, some of which are expressed on the Y chromosome, have been identified as important regulators of male germ cell function in mammals (Cooke & Saunders 2002) and it appears that Cirp also contributes to normal germ cell function by regulating the availability of mRNAs. Studies in vitro have suggested that Cirp expression may have an impact on cell cycle regulation (Hamid et al. 2003) and it is therefore possible that loss of Cirp expression contributed to the increased levels of germ cell apoptosis observed because regulation of mitosis and/or meiosis is disturbed. Other studies have reported that heat stress (via experimental cryptorchidism and scrotal heating) results in increased apoptosis of testicular germ cells (Yin et al. 1997, Lue et al. 1999, Yamamoto et al. 2000, Rockett et al. 2001). In this study we observed both an increase in the numbers of Apotag-positive germ cells as well as an increase in the expression of the pro-apoptotic marker Bax (unpublished observations by S King and P Saunders in the testes after scrotal heating, a finding consistent with a role for Bax in heat-induced apoptosis of germ cells, reviewed in Sinha Hikim et al. 2003). We also observed that the numbers of apoptotic germ cells remained markedly elevated above controls from 2 h to 24 days after the transient heat stress suggesting that the treatment had induced changes in germ cell and/or somatic cell function that had a long term impact on testicular function and further studies are planned to elucidate what these changes might be.

The epididymis has four main functions; the transportation, maturation, storage and protection of spermatozoa. In most mammals, including humans, the epididymis is located within the scrotum that is generally maintained at several degrees below body temperature. In the present study the number of motile spermatozoa retrieved from the heat-stressed epididymis rapidly decreased and remained low for the duration of the experiment. The initial decrease maybe be attributed to increased removal of spermatozoa by the basal cells in the epididymal epithelium which have the ability to act as tissue-fixed macrophages and may also be involved in the detoxification of the epididymal epithelium and lumen (Yeung et al. 1994, Seiler et al. 1999, 2000).

The effect of scrotal heating on DNA integrity in the motile spermatozoa was assessed using a modified alkaline COMET assay as well as the SCSA. The COMET assay, which was originally developed to study the DNA integrity of somatic cells, has been adapted for use in spermatozoa (Singh et al. 1988, Haines et al. 1998, Irvine et al. 2000) and gives a measure of the single and double stranded DNA breaks present within the cell nucleus. In contrast, the SCSA measures the susceptibility of sperm chromatin to acid denaturation (Evenson et al. 1999). This assay is considered to give a measure both of what proportion of spermatozoa contain incorrect packaged/remodeled DNA as well as the prevalence of DNA strand breaks, as both situations will result in DNA more susceptible to acid denaturation (Larson et al. 2000, Evenson et al. 2002). We found that both the mature spermatozoa in the epididymis and the spermatozoa that had developed from germ cells within the testis at the time of the heat stress contained DNA exhibiting higher SCSA values than those from control animals. Evenson et al.(2000) reported that in a fertile patient who had experienced a one day fever of 39.9 °C, 36% of spermatozoa recovered 18 days after the fever contained denatured DNA but that this had decreased to 23% by 39 days. Results obtained comparing pregnancy outcome from patients in which the spermatozoa of the male partner have high SCSA values have reported that this assay can identify the likelihood of achieving a pregnancy more reliably than measurement of conventional semen parameters (Larson-Cook et al. 2003, Virro et al. 2004).

A number of studies have shown that DNA damage in spermatozoa can occur as a result of oxidative stress (Aitken et al. 1989, Aitken et al. 1998, Twigg et al. 1998, Aitken & Krausz 2001). Oxidative stress, which has been shown to affect spermatozoa in the testis and the epididymis, can be caused by factors such as cancer treatments and cigarette smoke (Hinton et al. 1995, Shen et al. 1997). We suggest that the increase in epididymal temperature could have resulted in an increased level of oxidative stress in the epididymis and this induced an increase in DNA strand breaks within the spermatozoa. This would be consistent with reports that raised temperature causes changes in oxygen levels, water and ion transport mechanisms, protein biosynthesis and secretion, and in the cellular structure of the epididymal epithelium (Djakiew & Cardullo 1986, Seiler et al. 2000). It has been proposed that the availability of oxygen in the epididymis is of significant importance in the maintenance and storage of maturing spermatozoa (Djakiew & Cardullo 1986) with lower scrotal temperatures increasing the solubility of oxygen and decreasing the respiration rate of spermatozoa, thereby ensuring that there is sufficient oxygen to sustain large numbers of spermatozoa. An increase in scrotal temperature could therefore result in an unfavorable epididymal environment.

Motile spermatozoa retrieved from the epididymides 7 to 32 days after scrotal heating originated from cells located within the testis at the time of treatment (see Table 1). In the current study, levels of DNA damage in spermatozoa, as measured by COMET and SCSA, were the same in controls and those recovered 7 days after heating. At the time of heat stress these cells would have been developing within in the testis as stage 11 elongating spermatids or more mature elongating spermatids and this suggests that the DNA within these cells was resistant to the effects of treatment. However in contrast, the SCSA revealed that the populations of motile spermatozoa recovered 14 days post heating contained 18% with a High Green value and that 60% had poor DNA integrity (Comp αt value greater than 26%). At the time of heating these cells would have been developing as step 1 to step 11 spermatids. Marcon & Boissonneault (2004) have reported that during the normal process of spermiogenesis strand breaks are introduced in step 9–11 spermatids and that these are associated with the condensation of the germ cell DNA and replacement of histones by protamines, but that after stage 11 all DNA breaks within the normal spermatid have been repaired and the histones have been completely replaced by protamines. Zhao et al.(2004) have shown that transition proteins (TP) involved in the histone to protamine transition are also required for normal chromatin condensation, for reducing the number of DNA breaks and preventing a loss of genetic integrity. In the mouse transition proteins are expressed in step 10 to step 14 spermatids and the onset of protamine expression has been detected in step 10 spermatids using immunohistochemistry. Our results suggest spermatids undergoing DNA remodelling (up to and including step 11 spermatids) are vulnerable to the effects of heating, resulting in a disruption to the histone to TP to protamine transition and in incorrect packaging of DNA within the head of the spermatozoa.

Previous studies have identified pachytene spermatocytes and early spermatids as being susceptible to heat stress (Collins & Lacy 1969, De Vita et al. 1990) and this is not surprising as during meiotic prophase substantial rearrangements of DNA occurs within the nuclei of the leptotene, zygotene and pachytene spermatocytes. Collins & Lacy (1969) identified two critical periods in spermatogenesis (leptotene-pachytene and maturation division) through which cells were unable to progress following heating. Our results extend these earlier findings and have demonstrated that spermatozoa recovered 24, 28 and 32 days after testicular heating, which would have been developing as type A spermatogonia to stage X pachytene spermatocytes, were susceptible to heat-induced alteration in cell function and that this was associated with reduced numbers of motile spermatozoa being recovered up to 4 weeks later. Moreover, although the DNA damage within many of these cells was not fully repaired, they were able to complete their maturation and be released into the epididymis. Evenson and colleagues (Evenson et al. 1999, Larson et al. 2000) have shown that a COMP αt value in human spermatozoa of greater than 27% would predict that pregnancy would not be achieved in the context of IVF and ICSI. Rockett et al.(2001) reported that male mice that had undergone heat stress (43 °C for 20 min) showed a significant decrease in the viable fetuses that they could produce. In the current study, the fertilizing capacity of the spermatozoa was not directly tested by performing IVF or mating experiments and further experiments are therefore required to determine if there is any loss of fertility following the transient heat stress we applied.

It has been shown that raised temperature can lead to oxidative stress within the rat testis; this in turn can trigger apoptosis (Ikeda et al. 1999). The effects of oxidative stress on the genomic integrity of spermatozoa have been studied by inducing oxidative stress in the testis and in mature spermatozoa (Aitken et al. 1989, Lucesoli & Fraga 1995). Within the testis a number of cell types produce high levels of reactive oxygen species (ROS) that may result in oxidative damage to DNA, proteins and cell membranes and to combat this, the testis has developed a very complex antioxidant system (Bauche et al. 1994, Fisher & Aitken 1997). Therefore decreased expression of oxidative stress response genes, including extracellular superoxide dismutase (SOD) 3, manganese SOD 2 and genes involved in the metabolism of glutathione, which has been reported to occur following heating, may leave cells more susceptible to oxidative DNA damage (Barroso et al. 2000, Rockett et al. 2001)

A number of DNA repair mechanisms have been documented to be active during spermatogenesis (Richardson et al. 2000, Aguilar-Mahecha et al. 2001, Aitken & Krausz 2001, Rockett et al. 2001, Hsai et al. 2003). These are responsible for the repair of the DNA strand breaks known to occur accidentally during spermatogenesis, particularly during meiosis, and also those nicks and breaks that facilitate the transition of DNA from the somatic cell histone complex to the tightly packed protamine complex of the mature spermatozoa by relieving the torsional stress created during this process and aiding in remodelling (Ward & Coffey 1991, McPherson & Longo 1992). Normally these strand breaks and nicks are religated before the completion of spermiogenesis (Sakkas et al. 1995, Marcon & Boissonneault 2004). Following heating, the expression of a number of genes in the testis is down-regulated, these genes include several involved in DNA repair and recombination (Exonuclease III, G/T mismatch binding protein, DNA topoisomerase, 8-oxohuanine DNA glycosylase), cell cycle regulation and stress response (Rockett et al. 2001). The reduced levels of expression of these gene products may mean that the testis is less able to deal with the damage caused to the DNA within the germ cells. Alternatively, the level of DNA damage induced by heat stress may simply overwhelm the repair mechanisms in the testis and while a certain amount of DNA damage is repaired some may persist into the mature spermatozoa; further studies are required to test these possibilities.

In summary, it appears that the loss of DNA integrity in mature spermatozoa resulting from heat stressed germ cells may be explained in two ways, first, DNA damage that occurs in the cells at the time of heating is not repaired during spermatogenesis, or secondly, the physiology of the testis is disrupted by the heat stress resulting in sub-optimal support for germ cell development which is detected as a loss of DNA integrity in mature spermatozoa. Alterations in generation of reactive oxygen species or reduced activity of DNA repair proteins could contribute to the persistence of DNA breaks in maturing spermatozoa.

In conclusion, we have demonstrated that mature spermatozoa in the epididymis are not protected from heat-induced DNA damage and that following a transient heat stress the environment of the epididymis itself may contribute to the deterioration of DNA integrity of spermatozoa. Studies involving in vitro heating and culturing of spermatozoa and epididymis could be employed to explore this hypothesis further. Furthermore, we have identified cell types within the testis, which following heat stress, develop into motile, but DNA-damaged, spermatozoa. These findings suggest the need for additional criteria to be taken into consideration when selecting spermatozoa for IVF/ICSI treatment; in particular, the effect on DNA integrity may play a significant role in determining the viability of spermatozoa for use in an ICSI/IVF procedure.

Table 1

Summary of the germ cell types predicted to be afected by heat stress at the time points chosen post heat stress for analysis (timings based on Oakberg 1956).

Time pointCell type at time of treatment
1, 2, 4, 6, 24 (h)Mature spermatozoa in epididymis
7 daysStep 11 spermatid – mature spermatozoa
14 daysStep 1 spermatid – step 11 spermatid
21 daysStage IV pachytene – step 1 spermatid
24 daysStage XII pachytene – stage X pachytene
28 daysPreleptotene – stage III pachytene
32 daysA spermatogonia – zygotene
Figure 1
Figure 1

Immunolocalisation of Cirp to murine testes removed from controls (a) and at the following times after scrotal heating b) 2 h, c) 6 h and d) 7 days. Note that expression of Cirp is intense in spermatocytes in controls (a) but that expression is markedly suppressed in the testes 6 h after a transient heat stress (c).

Citation: Reproduction 129, 4; 10.1530/rep.1.00531

Figure 2
Figure 2

Immunoexpression of Cirp in epididymides from control (a–d) and heat-treated (e–h) males recovered 6 h after heat treatment. Expression of Cirp was detected in the epithelial cells of the initial segment (a), caput (b), corpus (c) and cauda (d) of control animals. Immunoexpression was reduced to undetectable in all but the caput (f) following scrotal heating.

Citation: Reproduction 129, 4; 10.1530/rep.1.00531

Figure 3
Figure 3

Apotag-positive germ cells in testes from controls (open bars) and those subjected to a transient scrotal heat stress (shaded bars). Bars show average values for each group (n = 3 animals per group) and points show the values for individual mice.

Citation: Reproduction 129, 4; 10.1530/rep.1.00531

Figure 4
Figure 4

Levels of DNA damage in motile spermatozoa following scrotal heating as determined by the COMET assay. Values for tail moment (bars) and COMET moment (points) for each group are shown. d, days.

Citation: Reproduction 129, 4; 10.1530/rep.1.00531

Figure 5
Figure 5

Percentage of motile spermatozoa exhibiting DNA damage expressed in terms of Comp αt as determined by the sperm chromatin structure assay (SCSA) following heat stress. Values are means±S.D. (n = 3 animals per group); d, days.

Citation: Reproduction 129, 4; 10.1530/rep.1.00531

Received 9 October 2004
 First decision 9 November 2004
 Revised manuscript received 21 December 2004
 Accepted 14 January 2005

The authors would like to thank Professor H Nishiyama, Faculty of Medicine, Kyoto University, Japan for the kind gift of the antibody directed against cold induced RNA-binding protein and F Watt, formerly of the MRC Human Reproductive Sciences Unit for assistance with immunohistochemistry.

References

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    • Search Google Scholar
    • Export Citation
  • Aitken RJ & Krausz C2001 Oxidative stress, DNA damage and the Y chromosome. Reproduction 122 497–506.

  • Aitken RJ, Clarkson JS & Fishel S1989 Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biology of Reproduction 41 183–197.

    • Search Google Scholar
    • Export Citation
  • Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z & Irvine DS1998 Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biology of Reproduction 59 1037–1046.

    • Search Google Scholar
    • Export Citation
  • Barroso G, Morshedi M & Oehninger S2000 Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Human Reproduction 15 1338–1344.

    • Search Google Scholar
    • Export Citation
  • Bauche F, Fouchard M & Jegou B1994 Antioxidant system in rat testicular cells. FEBS letters 349 392–396.

  • Bedford JM1991 Effects of Elevated Temperaure on the Epididymis and Testis: Experimental Studies. Advances in Experimental Medicine and Biology 286 19–32.

    • Search Google Scholar
    • Export Citation
  • Bedford JM1994 The status and the state of the human epididymis. Human Reproduction 9 2187–2199.

  • Biggers J, Whitten W & Whittingham D1971 The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, pp 86–94. Ed JC David. San Francisco, Freeman Press.

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  • Collins P & Lacy D1969 Studies on the structure and function of the mammalian testis. II. Cytological and histochemical observations on the testis of the rat after a single exposure to heat applied for different lengths of time. Proceedings of the Royal Society of London. Series B 172 17–38.

    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Export Citation
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    • Export Citation
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    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • Hjollund NH, Storgaard L, Ernst E, Bonde JP & Olsen J2002 Impact of diurnal scrotal temperature on semen quality. Reproductive Toxicology 16 215–221.

    • Search Google Scholar
    • Export Citation
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    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • Larson KL, DeJonge CJ, Barnes AM, Jost LK & Evenson DP2000 Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Human Reproduction 15 1717–1722.

    • Search Google Scholar
    • Export Citation
  • Larson-Cook KL, Brannian JD, Hansen KA, Kasperson KM, Aamold ET & Evenson DP2003 Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertility and Sterility 80 895–902.

    • Search Google Scholar
    • Export Citation
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  • View in gallery

    Immunolocalisation of Cirp to murine testes removed from controls (a) and at the following times after scrotal heating b) 2 h, c) 6 h and d) 7 days. Note that expression of Cirp is intense in spermatocytes in controls (a) but that expression is markedly suppressed in the testes 6 h after a transient heat stress (c).

  • View in gallery

    Immunoexpression of Cirp in epididymides from control (a–d) and heat-treated (e–h) males recovered 6 h after heat treatment. Expression of Cirp was detected in the epithelial cells of the initial segment (a), caput (b), corpus (c) and cauda (d) of control animals. Immunoexpression was reduced to undetectable in all but the caput (f) following scrotal heating.

  • View in gallery

    Apotag-positive germ cells in testes from controls (open bars) and those subjected to a transient scrotal heat stress (shaded bars). Bars show average values for each group (n = 3 animals per group) and points show the values for individual mice.

  • View in gallery

    Levels of DNA damage in motile spermatozoa following scrotal heating as determined by the COMET assay. Values for tail moment (bars) and COMET moment (points) for each group are shown. d, days.

  • View in gallery

    Percentage of motile spermatozoa exhibiting DNA damage expressed in terms of Comp αt as determined by the sperm chromatin structure assay (SCSA) following heat stress. Values are means±S.D. (n = 3 animals per group); d, days.

  • Aguilar-Mahecha A, Hales BF & Robaire B2001 Expression of stress response genes in germ cells during spermatogenesis. Biology of Reproduction 65 119–127.

    • Search Google Scholar
    • Export Citation
  • Aitken RJ & Krausz C2001 Oxidative stress, DNA damage and the Y chromosome. Reproduction 122 497–506.

  • Aitken RJ, Clarkson JS & Fishel S1989 Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biology of Reproduction 41 183–197.

    • Search Google Scholar
    • Export Citation
  • Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z & Irvine DS1998 Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biology of Reproduction 59 1037–1046.

    • Search Google Scholar
    • Export Citation
  • Barroso G, Morshedi M & Oehninger S2000 Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Human Reproduction 15 1338–1344.

    • Search Google Scholar
    • Export Citation
  • Bauche F, Fouchard M & Jegou B1994 Antioxidant system in rat testicular cells. FEBS letters 349 392–396.

  • Bedford JM1991 Effects of Elevated Temperaure on the Epididymis and Testis: Experimental Studies. Advances in Experimental Medicine and Biology 286 19–32.

    • Search Google Scholar
    • Export Citation
  • Bedford JM1994 The status and the state of the human epididymis. Human Reproduction 9 2187–2199.

  • Biggers J, Whitten W & Whittingham D1971 The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, pp 86–94. Ed JC David. San Francisco, Freeman Press.

  • Campbell AJ & Irvine DS2000 Male infertility and intracytoplasmic sperm injection (ICSI). British Medical Bulletin 56 616–629.

  • Collins P & Lacy D1969 Studies on the structure and function of the mammalian testis. II. Cytological and histochemical observations on the testis of the rat after a single exposure to heat applied for different lengths of time. Proceedings of the Royal Society of London. Series B 172 17–38.

    • Search Google Scholar
    • Export Citation
  • Cooke HJ & Saunders PT2002 Mouse models of male infertility. Nature Reviews. Genetics 3 790–801.

  • Cornwall GA, Vreeburg J, Holland MK & Orgebin-Crist MC1990 Interactions of labeled epididymal secretory proteins with spermatozoa after injection of 35S-methionine in the mouse. Biology of Reproduction 43 121–129.

    • Search Google Scholar
    • Export Citation
  • De Gendt K, Swinnen JV, Saunders PT, Schoonjans L, Dewerchin M, Devos A, Tan K, Atanassova N, Claessens F, Lecureuil C, Heyns W, Carmeliet P, Guillou F, Sharpe RM & Verhoeven G2004 A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis. PNAS 101 1327–1332.

    • Search Google Scholar
    • Export Citation
  • De Vita R, Calugi A, Chiarantano C, Forte D, Mauro F & Uccelli R1990 Effects of heat on mouse spermatogenesis monitored by flow cytometry. International Journal of Hyperthermia 6 543–551.

    • Search Google Scholar
    • Export Citation
  • Djakiew D & Cardullo R1986 Lower Temperature of the Cauda Epididymis Facilitates the Storage of Sperm by Enhancing Oxygen Availability. Gamete Research 15 237–245.

    • Search Google Scholar
    • Export Citation
  • Evenson DP, Jost LK, Corzett M & Balhorn R2000 Characteristics of human sperm chromatin stucture following an episode of influenza and high fever: a case study. Journal of Andrology 21 739–746.

    • Search Google Scholar
    • Export Citation
  • Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, de Angelis P & Claussen OP1999 Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Human Reproduction 14 1039–1049.

    • Search Google Scholar
    • Export Citation
  • Evenson DP, Larson KL & Jost LK2002 Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. Journal of Andrology 23 25–43.

    • Search Google Scholar
    • Export Citation
  • Fisher HM & Aitken RJ1997 Comparative analysis of the ability of precursor germ cells and epididymal spermatozoa to generate reactive oxygen metabolites. Journal of Experimental Zoology 277 390–400.

    • Search Google Scholar
    • Export Citation
  • Haines G, Marples B, Daniel P & Morris I1998 DNA damage in human and mouse spermatozoa after in vitro-irradiation assessed by the comet assay. Advances in Experimental Medicine and Biology 444 79–91.

    • Search Google Scholar
    • Export Citation
  • Hamid AA, Mandai M, Fujita J, Nanbu K, Kariya M, Kusakari T, Fukuhara K & Fujii S2003 Expression of cold-inducible RNA-binding protein in the normal endometrium, endometrial hyperplasia, and endometrial carcinoma. International Journal of Gynecological Pathology 22 240–247.

    • Search Google Scholar
    • Export Citation
  • Harrison R & Weiner J1948 Abdomino-testicular temperature gradients. Journal of Physiology 18 256–262.

  • Hinton BT, Palladino MA, Rudolph D & Labus JC1995 The epididymis as protector of maturing spermatozoa. Reproduction, Fertility, and Development 7 731–745.

    • Search Google Scholar
    • Export Citation
  • Hjollund NH, Bonde JP, Jensen TK & Olsen J2000 Diurnal scrotal skin temperature and semen quality. The Danish First Pregnancy Planner Study Team. International Journal of Andrology 23 309–318.

    • Search Google Scholar
    • Export Citation
  • Hjollund NH, Storgaard L, Ernst E, Bonde JP & Olsen J2002 Impact of diurnal scrotal temperature on semen quality. Reproductive Toxicology 16 215–221.

    • Search Google Scholar
    • Export Citation
  • Hsai K-T, Millar MR, King S, Selfridge J, Redhead NJ, Melton D & Saunders PTK2003 DNA repair gene Ercc-1 is essential for normal spermatogenesis and oogenesis and for functional integrity of germ cell DNA in the mouse. Development 130 369–378.

    • Search Google Scholar
    • Export Citation
  • Ikeda M, Kodama H, Fukuda J, Shimizu Y, Murata M, Kumagai J & Tanaka T1999 Role of radical oxygen species in rat testicular germ cell apoptosis induced by heat stress. Biology of Reproduction 61 393–399.

    • Search Google Scholar
    • Export Citation
  • Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA & Aitken RJ2000 DNA integrity in human spermatozoa: relationships with semen quality. Journal of Andrology 21 33–44.

    • Search Google Scholar
    • Export Citation
  • Jannes P, Spiessens C, Van der Auwera I, D’Hooghe T, Verhoeven G & Vanderschueren D1998 Male subfertility induced by acute scrotal heating affects embryo quality in normal female mice. Human Reproduction 13 372–375.

    • Search Google Scholar
    • Export Citation
  • Larson KL, DeJonge CJ, Barnes AM, Jost LK & Evenson DP2000 Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Human Reproduction 15 1717–1722.

    • Search Google Scholar
    • Export Citation
  • Larson-Cook KL, Brannian JD, Hansen KA, Kasperson KM, Aamold ET & Evenson DP2003 Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertility and Sterility 80 895–902.

    • Search Google Scholar
    • Export Citation
  • Lopes S, Sun JG, Jurisicova A, Meriano J & Casper RF1998 Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertility and Sterility 69 528–532.

    • Search Google Scholar
    • Export Citation
  • Lucesoli F & Fraga CG1995 Oxidative damage to lipids and DNA concurrent with decrease of antioxidants in rat testes after acute iron intoxication. Archives of Biochemistry and Biophysics 316 567–571.

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