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Regular Article |
a School of Molecular Biosciences, Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-4660
b Children's Hospital Research Foundation, Cincinnati, Ohio 45229
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gametogenesis, male reproductive tract, Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A common theme found in several tissues is the association of clusterin with tissue damage or injury. Clusterin is induced at the mRNA and protein levels in regressing, involuting, or injured tissues such as testosterone-deprived regressing prostate [9], involuting mammary gland [10], and tissues affected by neurodegenerative diseases such as Alzheimer disease [11]. A role for clusterin in apoptosis has been proposed because a correlation between clusterin expression and apoptosis has been demonstrated in many organs and cell types including the prostate [9], thymus [12], pancreas [13], and the A431 human epidermoid cancer cell line [14]. The role of clusterin in apoptosis is not clear, but it appears to be expressed by surviving cells in vitro [15] and in vivo [16–18], and it accumulates in apoptotic cells. These studies suggest that clusterin expression is not essential for apoptotic death but instead may play a bioprotective role in response to injury or stress [14, 15, 19].
To understand the role of clusterin in the testis, we analyzed adult clusterin knock-out mice (cluKO) to determine if the absence of clusterin has any consequences for spermatogenesis. In the testis, germ cells are susceptible to apoptosis initiated by heat shock and methoxyacetic acid (MAA) toxicity [20–22]. To test the hypothesis that clusterin plays a bioprotective role, we administered heat to the testis in the form of a single mild exposure, and we injected mice with MAA to determine the toxic effects in the cluKO mouse testis and the wild-type (cluWT) mouse testis. The experiments test the effects of two completely different methods of causing damage to spermatogenesis with the commonality of inducing germ cell apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cluKO mice have been previously described [23]. The cluKO mice which were on a B6 background were screened by the polymerase chain reaction (PCR). Genomic DNA was extracted from tail samples, and 150 ng of DNA was used in the reaction. Parameters for the PCR were denaturation at 94°C, annealing at 60°C, and extension at 72°C for 40 cycles. The PCR products were then visualized on a 1.8% agarose gel stained with ethidium bromide.
Animals and Experimental Protocol
Adult male cluKO and cluWT mice were used in this study. The animals were housed in a standard animal facility under controlled temperature (21–22°C) and photoperiod (12L:12D) with food and water provided ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee.
Heat Treatment
To examine the effects of heat stress on the testis of cluKO mice, the animals were subjected to a short exposure of elevated temperature. Heating the scrota of mice was performed using a modified procedure described previously [24]. Mice were anesthetized with ketamine (110 mg/kg body weight) and xylazine (5.5 mg/kg body weight) by i.p. injection. The testes were manipulated into the scrotum and secured in place by the use of string so as not to interfere with circulation and to prevent withdrawal of the testis into the body cavity. Mice were then lowered into the water bath so that the scrotum and testis were completely immersed for 15 min in a thermostatically controlled water bath at 43°C. Control mice were treated in the same way except that the testes were not immersed. No evidence of thermal damage was observed to the scrotal skin after the short exposure to the elevated temperature of the water bath. Twenty-four adult mice from both cluKO and cluWT strains were assigned to six groups of four mice each. One group of mice was assigned to each of the following time points: 4, 6, 12, 24, 48, and 72 h posttreatment. Three mice per group were exposed to thermal treatment, while the remaining mouse in each group served as an untreated control.
MAA Treatment
To examine the effects of MAA toxicity on spermatogenesis in cluKO mice, 24 adult mice were assigned to six groups of four mice each. One group of mice was assigned to each of the following time points: 4, 6, 12, 24, 48, and 72 h posttreatment. Three mice from each group were treated with MAA (1300 mg/kg body weight; Aldrich, Milwaukee, WI) by i.p. injection, while one control mouse in each group was injected with saline.
Tissue Preparation
After each respective experimental time point, the mice were injected with heparin (130 IU/100 µg body weight, i.p.) 15 min before being given anesthetic as described previously, and body weight was recorded. One testis was removed, weighed, and placed in either Trizol reagent (Invitrogen, Carlsbad, CA) for RNA extraction or fixed by immersion in Bouin solution (acetic acid:37% formaldehyde:saturated picric acid, 1:5:20). Mice were then perfused through the heart with PBS until internal organs cleared, followed by perfusion with Bouin solution for 10 min to fix the tissues. The fixed testis and epididymis were removed and placed in Bouin solution for 6 h, then in 70% ethanol at 4°C until embedded in paraffin.
Sperm Head Counts
The total number of sperm per gram of testis was determined using a procedure described previously [25] with the following modifications. All squares of the hemocytometer were counted to determine the number of homogenization-resistant sperm heads, and four counts were averaged and adjusted for testicular weight per animal. Sperm counts from cluKO and cluWT mice, three mice each, were counted and averaged.
Histologic Techniques
For all of the following procedures, paraffin-embedded testes and epididymides from the preceding experiments were cut into 6-µm sections and mounted onto poly-L-lysine-coated glass slides. Sections were deparaffinized in xylenes, hydrated in a graded series of ethanol solutions, and rinsed in PBS. Sections from the procedures described subsequently were digitally photographed with a Nikon Microphot-FX (Nikon, Tokyo, Japan) microscope using a MagnaFire camera model S99806 and software version 1.0 (both from Olympus, Melville, NY).
Immunohistochemistry and TUNEL Analysis
Immunohistochemical localization of clusterin protein in the testis from treated and control animals was performed as described previously [18]. To assess apoptosis and the stage-related modulation of apoptosis involving specific germ cells, in situ end-labeling of DNA strand breaks was performed on testis sections by the TUNEL technique using the ApopTag-peroxidase kit (Intergen, Purchase, NY) according to the manufacturer's instructions.
Quantitation of apoptosis was carried out by counting apoptotic germ cells that were clearly stained by the TUNEL assay. Apoptotic germ cells were counted at stages I–V, VI–VIII, IX–X, and XI–XII. For each mouse, at least 10 tubules per stage group were examined. The stages were identified according to the criteria proposed by Russell et al. [26]. The rate of germ cell apoptosis was expressed as the number of apoptotic germ cells per tubule.
Data and Statistics
Data are shown as the mean ± SEM of data from three different mice for each time point. Data were examined by the Student t-test using a significance level of P < 0.05.
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RESULTS |
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Adult male and female cluKO mice were viable and fertile, producing normal offspring. The cluKO mice had no overt morphologic differences when compared with cluWT mice. Male cluKO mice had no significant difference in body weight compared with male cluWT mice (Table 1). Adult cluKO males had normal-appearing testicular morphology with testis weights comparable to those of cluWT males (Table 1). The number of homogenization-resistant sperm heads per gram of testis for cluKO and cluWT mice was not significantly different (Table 1).
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Although the cluKO male mice were fertile and their testis contained the same number of sperm as cluWT mice, histologic examination of cluKO testis sections showed that spermatogenesis did not proceed normally at all stages. Spermiation, the process by which sperm disengage from the Sertoli cell and are released into the lumen, was incomplete after stage VIII of spermatogenesis in cluKO mice. Step 16 spermatids are normally released into the lumen at the end of stage VIII in wild-type mice but were still present at stage XI in the cluKO mouse (Fig. 1). All other stages appeared to be normal in the cluKO mouse.
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Response to Treatment with MAA
It has been reported that clusterin is closely associated with dying germ cells in the testes of MAA-treated rats [18]. CluKO mice were treated with MAA to examine the effect of clusterin deletion on the testis with respect to apoptosis and removal of damaged cells. With the exception of the absence of clusterin protein, there were no morphologic differences between the cluKO and cluWT mice at different time points after MAA injection. No morphologic changes were apparent until 6 h after treatment with MAA, at which time some diplotene and pachytene spermatocytes become pyknotic in both animals (Fig. 2, C and I). Pyknosis was not uniform throughout all stages at 6 h. Stages VI–VIII contained the most pyknotic cells, primarily pachytene spermatocytes. By 12 h post-MAA treatment, degenerating spermatocytes could be seen at stages VI–X. As the cells degenerated, they appeared to remain in their respective positions in the tubule (Fig. 2, D and J). By 24 h, degenerating spermatocytes could still be seen in most stages, but most had been removed presumably by the Sertoli cells, and an area devoid of cells could be observed around the seminiferous epithelium where pachytene spermatocytes had been previously located (Fig. 2, E and K). At 48 h posttreatment, degeneration appeared to have slowed, but damage was still evident by the presence of open spaces in the epithelium where spermatocytes and round spermatids had been located (Fig. 2, F and L). Lastly, at 72 h, the tubules looked normal, with the exception of fewer spermatocytes (data not shown).
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CluWT mice showed characteristic immunostaining for clusterin localized to the cytoplasm of Sertoli cells, starting at the basal aspect of the seminiferous tubule and extending towards the lumen and to the heads and tails of late spermatids. CluKO mice showed no immunostaining for clusterin. Germ cells are normally unstained for clusterin, as can be seen in control mice (Fig. 2G); however, by 6 h post-MAA treatment, pachytene spermatocytes at stages VII–X had clusterin localized to their cytoplasm (Fig. 2I). At 12 h post-MAA treatment, clusterin staining was observed in the cytoplasm of spermatocytes and some round spermatids at stages VII–VIII (Fig. 2J). By 48–72 h post-MAA treatment, distribution of clusterin immunostaining was similar to that seen in the controls. Clusterin localization to germ cells in cluWT mice preceded or coincided with degeneration of spermatocytes and round spermatids. The cluKO mouse did not show any clusterin staining, yet the morphologic characteristics of the testis remained the same as in the cluWT mouse.
MAA-induced loss of testicular germ cells occurs through the process of apoptosis [27]. Control testes from cluKO and cluWT showed few apoptotic cells based on TUNEL analysis (Fig. 3, A and G). By 6 h post-MAA treatment, numerous apoptotic pachytene spermatocytes could be observed at stages VI–X in both the cluKO and cluWT mice (Fig. 3, C and I). At 12 and 24 h post-MAA treatment, the most abundant labeling of fragmented DNA was observed, primarily in spermatocytes but also in some round spermatids of stages VI–XII (Fig. 3). At 48 h post-MAA treatment, end-labeling of fragmented DNA had decreased. At all time points, the immunostaining of clusterin was evident before the onset of apoptosis in the cluWT mouse.
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In MAA-treated mice, no significant change in the incidence of germ cell apoptosis was observed between the cluKO and the cluWT mice at any time point (Fig. 4). Overall, the incidence of apoptosis was greatest at 12 and 24 h and rapidly decreased by 48 h post-MAA treatment (Fig. 4). Apoptosis was first observed at 6 h post-MAA treatment, primarily at stages I–VIII (Fig. 5A), and dramatically increased in spermatocytes at 12 h at stages VI–X (Fig. 5B). By 24 h post-MAA treatment, the highest incidence of apoptosis was observed at stages XI–XII, whereas apoptosis at stages VI–VIII had decreased (Fig. 5C), and at 48 h post-MAA treatment, apoptosis had decreased to the level in controls (Fig. 5D). There was no significant difference between the incidence of apoptotic germ cells in the cluKO and cluWT mice at any of the stages. However, a greater number of apoptotic cells were seen at 48 h in cluKO mice at stages IX–X compared with the cluWT mice, but the variability was also high.
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Response to Single Heat Exposure
The clusterin gene is known to have a heat shock element within its promoter, and clusterin mRNA expression and protein biosynthesis have been shown to increase in response to elevated temperatures [28]. In the rat testis, the response of spermatogenesis to heat shock has been well characterized and shows a stage-specific loss of germ cells through the process of apoptosis [24]. CluKO and cluWT mice were treated with a single exposure of mild heat. Both cluKO and cluWT testis sections from 4 h post-heat treatment showed pyknotic pachytene spermatocytes and open spaces in the epithelium when compared with those of untreated controls (Fig. 6). Normal clusterin immunostaining of testis sections was seen in the control testis, but by 4 h post-heat treatment, clusterin staining could be observed in the cytoplasm of some pachytene and diplotene spermatocytes in cluWT mice. At 6 h post-heat treatment, pachytene and diplotene spermatocytes continued to degenerate and became pyknotic, particularly at stages I–IV and stages X–XII in both groups (Fig. 6, C and I). Also at 6 h post-heat treatment, the clusterin immunostaining in the cluWT mouse was most dramatic and apparent at stages I–IV and stages X–XII. By 12 h, degeneration of germ cells could be observed at most stages, and loss of germ cells was indicated by the presence of open spaces in the seminiferous epithelium (Fig. 6, D and J). At 24 h post-heat treatment, pyknosis and loss of germ cells was still evident in both groups of mice (Fig. 6, E and K). Clusterin immunostaining in the cluWT mouse was not as intense at 12 and 24 h post-heat treatment as at 6 h, but staining could still be seen around degenerating spermatocytes and some spermatids at stages XI–XII of spermatogenesis. The distribution of damaged germ cells at each time point was not uniform throughout all seminiferous tubules.
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At 48 h post-heat treatment, a dramatic difference was seen between the cluKO and cluWT mice. The seminiferous tubules of the cluWT mouse became completely disorganized, a severe loss of germ cells was observed, and stages became indistinguishable (Fig. 6, F and L). This pattern was observed throughout the cluWT testis and affected 69% (Table 2) of the tubules. In contrast, at 48 h post-heat treatment, the cluKO testis still had an organized and intact epithelium, with most of the germ cells remaining. This observation was not uniform, and there were a small number of tubules (5%) in the cluKO testis that were beginning to show signs of severe germ cell loss. By 72 h, the cluKO seminiferous tubules became disorganized, and most of the germ cells had been lost (data not shown). The cluWT tubules continued their degeneration with greater loss of germ cells by 72 h post-heat treatment. At 48 and 72 h post-heat treatment, clusterin immunostaining was seen throughout the tubules of the cluWT testis.
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At 4 and 6 h post-heat treatment, both cluKO and cluWT testes showed apoptosis by fragmented DNA end-labeling (Fig. 7). The apoptotic cells were primarily pachytene and diplotene spermatocytes. The most abundant end-labeling of fragmented DNA was noted in both cluKO and cluWT mice at 12 h post-heat treatment (Fig. 7, D and J), primarily in spermatocytes but also in some spermatids. At 24 h post-heat treatment, the incidence of germ cell apoptosis decreased in both animals, and at 48 h, end-labeling appeared confined to degenerating cells in the cluKO testis and to most cells that remained in the cluWT mouse (Fig. 7, F and L). At 72 h post-heat treatment, numerous cells of both the cluKO and cluWT mouse displayed end-labeling, but the background was also high, and determination of cell types was difficult (data not shown).
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There was no difference in the number of apoptotic cells in controls, but by 4 h post-heat treatment, apoptosis in the cluKO mouse was twice that of the cluWT mouse (Fig. 8). Most of the apoptotic cells at the 4-h time point were spermatocytes at stages I–V and XI–XII (Fig. 9A). At 6 h, an increase was seen in the number of apoptotic cells for both groups of animals, with the cluKO mouse having a significantly higher incidence of germ cell apoptosis overall and stages I–V and XI–XII showing the greatest sensitivity (Figs. 8 and 9B). By 12 h, no significant difference in the number of apoptotic cells was seen between the cluKO and cluWT animals (Fig. 8). At 24 h post-heat treatment, cluWT mice had a significantly higher incidence of germ cell apoptosis overall (Fig. 8), and the incidence of apoptosis was highest at stages VI–X. This pattern at 24 h contrasted with the patterns noted at other posttreatment time points, at which significant differences between groups were observed at stages I–V, VI–VIII, and XI–XII (Fig. 9D).
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Testes weights after heat treatment reflected the degeneration and loss of germ cells observed in the seminiferous tubules of both groups of mice. The was no significant difference between testis weights of cluKO and cluWT mice until 48 h post-heat treatment, at which time the weight of the cluWT testes had decreased by approximately half, and the difference between groups was significant (P 
0.05). At 72 h, the weight of the cluKO mouse testes had also decreased to approximately half that at earlier time points and was again similar to that in the cluWT mouse (Fig. 10B). The rapid decrease in testis weight coincided with the dramatic loss of germ cells within the tubules of both animals. In contrast to the weight of the heat-treated testes, the weight of the MAA-treated testes did not differ significantly between the cluKO and cluWT mice (Fig. 10A). Also, there was no dramatic decrease in weight at any of the time points after MAA treatment as seen at 48 h in the heat-treated animals. Rather, there was a steady decrease in the weights after 12 h that reached the lowest point at 48 h and remained steady at 72 h, with no further loss of weight.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One morphologic abnormality observed in the testis of cluKO mice was failed spermiation between stages VIII and IX of spermatogenesis. Incomplete spermiation can be caused by gonadotropin suppression due to hypophysectomy, reproductive toxicants, hormone withdrawal, and spinal cord injury [29, 30]. It has been suggested that incomplete spermiation after hormone withdrawal or toxicant exposure may be a nonspecific reaction to Sertoli cell injury [30].
In most normal tissues, clusterin protein and mRNA are present in relatively low amounts, whereas in the testis, clusterin is secreted constitutively at relatively high amounts [2]. Levels of clusterin mRNA and/or protein secretion have been shown to increase in response to tissue regression, involution, damage, and disease [3]. In the last decade, multiple lines of evidence have suggested that clusterin is associated with apoptosis, and this glycoprotein has been identified in several regressing tissues undergoing apoptotic cell death [9, 19, 31–33]. An increase in germ cell apoptosis is often observed after exposure of laboratory animals to various toxicants, testicular injury, and certain disease states [34, 35]. The results of this study show that pachytene spermatocytes from both cluKO and cluWT mice treated with MAA become pyknotic and have high incidences of apoptosis, as determined using the TUNEL method. A previous study performed in rats demonstrated that MAA treatment causes a localization of clusterin to the cytoplasm of pachytene and diplotene spermatocytes, as shown by immunohistochemistry, and that clusterin localization precluded the apoptotic death of those same cells, as determined by the TUNEL method [18]. Our results were essentially consistent with those observed previously in the rat testis; however, apoptosis was seen earlier in the mouse than in the rat. Clusterin localization in the cluWT testis before apoptosis suggests an association with apoptosis in response to injury. However, clusterin does not appear to protect against apoptosis induced by MAA toxicity. The theory that clusterin protects against apoptosis comes from studies that have shown that overexpression of clusterin protects cells from apoptosis [19, 36], and antisense oligonucleotides that block clusterin biosynthesis cause an increase in cellular apoptosis in vitro [14, 33].
Apoptosis is a complex process that occurs through many pathways [37]. In the testis, at least two pathways are evident. One pathway induced by heat stress affects germ cells at stages I–IV and XII–XIV in the rat, whereas another pathway induced by gonadotropin withdrawal causes apoptosis at stages VII–VIII [38]. In our studies, the apoptotic response to MAA more closely resembled the latter pathway, with apoptosis initially occurring at stages VII–VIII. Clusterin clearly affects the initial kinetics of the heat stress pathway but not that of the MAA-induced pathway. After appearing to delay the initial heat-induced damage, the clusterin in the cluWT mice seemed to play a role in more quickly clearing damaged cells from the tubules after 48 h. In other studies in cluKO mice, it has been shown that clusterin limits the severity of induced autoimmune autocarditis [23] and exacerbates caspase-3-independent brain injury after neonatal hypoxia-ischemia [39]. Thus, there appears to be a duality in the actions of clusterin in that it appears to inhibit some damage processes and enhance others.
This duality of action could be explained by the known properties of clusterin. We have previously proposed that clusterin acts as a biological detergent to aid in the removal or metabolic degradation of hydrophobic molecules during tissue remodeling or damage [3]. This idea is supported by the observation that clusterin binds with high affinity to numerous macromolecules [40] and is capable of mediating the uptake of hydrophobic peptides through the action of LRP-2 (lipoprotein receptor-related protein-2/megalin) receptor [41]. Several investigators have reported that clusterin binds to hydrophobic macromolecules such as lipids [42, 43], proteins [6, 44], and peptides [45, 46]. In addition, clusterin is known to have a chaperone-like function, and it is capable of preventing the precipitation of denatured proteins in vitro [47]. We have proposed that clusterin binds to hydrophobic molecules through specific structures that provide the protein with a dynamic binding site. These structures include multiple amphipathic 
helices and charged residues located within flexible disordered regions (unpublished results). Clusterin could maintain the solubility of potentially toxic cellular debris and prevent the toxic effects of their accumulation in the lumen and could aid in the solubilization and removal of cellular debris. In our study, clusterin could be delaying the initial apoptosis in hyperthermic testes by binding to the apoptosis signaling components while having no effect on the MAA-induced pathway. In addition, clusterin could play a positive role in accelerating the removal of dead cells or cell debris, and this action could explain the observation that after heat treatment, the seminiferous tubules of the cluWT mouse exhibited a rapid loss of germ cells between 1 and 2 days posttreatment, whereas the loss in the cluKO mouse occurred between 2 and 3 days posttreatment. In addition, clusterin is induced in regressing tissues such as the prostate after hormone deprivation induced by castration in rats [9] and the involuting uterus [48]. Clusterin may simply protect tissues from further damage caused by cellular debris from degenerating cells.
An alternative mechanism by which clusterin could delay apoptosis in heat-stressed testes involves the protection of surviving cells from oxidative stress caused by reactive oxygen species. Recently, heat shock has been shown to increase lipid peroxidation in mouse spermatozoa and to elevate intracellular peroxide levels in cultured testicular germ cells [21, 49]. Oxidative stress has been associated with several intracellular apoptotic signals including activation of nuclear factor-
B, activation of caspases, and activation of the Fas system [50–52]. Clusterin has been proposed to be an antioxidant agent and is capable of protecting cells from apoptosis induced by reactive oxygen species [14]. Clusterin has also been shown to associate with lipids and with paraoxonase, a lipoprotein capable of preventing lipid peroxidation [43, 53]. It is possible that clusterin could protect germ cells from apoptosis by indirectly preventing lipid peroxidation caused by heat.
All of the physiologic events associated with the presence of clusterin, including apoptosis, lipid transport, and cell degeneration, require the remodeling of cellular membranes and extracellular matrices. The high constitutive expression and protein levels of clusterin in the testis are consistent with the remodeling that occurs during the division, development, and release of germ cells. Although clusterin is not essential in the cluKO mouse for spermatogenesis, its presence may still be required for efficient spermatogenesis as supported by the abnormal spermiation observed at stage VIII.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH grant 5RO1 HD 30692. 
2 Correspondence. FAX: 509 335 9688; griswold{at}mail.wsu.edu
Accepted: November 6, 2001.
Received: July 9, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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