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Biology of Reproduction 67, 1189-1196 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Abdominal Temperature Induces Region-Specific p53-Independent Apoptosis in the Cauda Epididymidis of the Mouse1

Marco Jaraa, Pedro Espondaa, and Rosa Carballada2,a

a Centro de Investigaciones Biológicas, CSIC, 28006 Madrid, Spain


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is widely accepted that temperature regulates gene expression and function in the epididymis. However, the significance of reduced temperature of the scrotum in cell survival had not often been examined. Our hypothesis was that the experimental increase of the temperature could induce apoptosis. Using a surgical method that consists of surgically reflecting the cauda epididymidis in the abdomen, we have been able to show that this is the case. Apoptosis was examined by histologic procedures and by visualization of DNA fragmentation in agarose gels. We determined that the apoptosis is region-specific and affects only the principal cells of the proximal region of the cauda. It starts 12 h after surgery and ends by the third day. The apoptotic cells are eliminated by extrusion into the lumen and phagocytosis by adjacent cells. The complete molecular mechanism of apoptosis in this case remains unknown, but we have used the techniques of immunocytochemistry, Western blot, and reverse transcription-polymerase chain reaction to determine the role of some molecules. We have seen no significant role of androgens, the tumor suppressor p53, nor two heat shock proteins, hsp-25 and hsp-70. Nevertheless, we have detected a strong induction of bax and bcl-2 gene products. While the former should be responsible for the apoptosis observed, the latter would promote the survival of most of the cells of the cauda epididymis.

androgen receptor, apoptosis, epididymis, male reproductive tract, male sexual function


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The epididymis has a major role in mammalian reproduction, ensuring sperm concentration, maturation, transport, and storage [1–3]. The epididymis is regionalized from an anatomic and functional point of view. The proximal regions are mainly devoted to sperm maturation [4], while in the distal region, termed the cauda, sperm storage takes place [5]. The epididymis is controlled by several factors [6]. The best known are androgens, i.e., testosterone and its metabolite, dihydrotestosterone. Androgens regulate specific gene expression in the epididymal epithelium [7–9], and androgen withdrawal produces dramatic changes in the pattern of secreted proteins in every region [9]. Most of these changes are reversed by androgen administration [10, 11]. The role of androgens is not restricted to regulating gene expression, but it also ensures cell survival. In the rat, after castration, an apoptotic wave can be detected, spreading cell death through the different regions [12]. This apoptotic wave starts at the proximal regions at 18 h after surgery and declines in the distal regions by Day 7 [12]. Androgen treatment at the time of orchidectomy can prevent apoptotic death in all regions of the epididymis except the initial segment [12]. The second group of molecules that influences epididymal function is testicular factors. The nature of these factors is largely unknown, but in rats, efferent duct ligation has a dramatic effect on both gene expression [13–16] and cell survival [17]. Although the intraluminal androgens may be partially responsible for these effects, it is clear that there are other factors involved [15]. Finally, temperature is another major factor that influences epididymal physiology [5]. Temperature regulates sperm storage in the cauda epididymidis of the rat and the hamster [5, 18, 19]. In addition, specific gene expression in the epididymis is controlled by temperature in rats and rabbits [20–24]. The effects of temperature on epididymal epithelial cell secretion are not mediated by androgens [21–24], although both factors can act synergistically, and the mechanism of action in the rat might be related to mRNA stability [25]. Nevertheless, there are no reports to date on temperature effects on apoptosis in the epididymis.

In this study, we used a surgical model developed by Bedford [26], termed cryptepididymis, to study the effects of temperature on cell survival in the cauda epididymidis of the mouse. We have also tried to elucidate the role of several molecules in the process.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Surgery

Adult male mice of the CD-1 strain bred in our colony were used for all the experiments. Animals were housed at constant temperature in a 12L:12D cycle with food and water ad libitum. The mice (a total of 40) were anesthetized with a mixture of ketamine (50 mg Ketolar; Parke-Davis, Barcelona, Spain) and Rompún (Bayer, Leverkusen, Germany). Typically, 0.8 ml of Ketolar were mixed with 0.15 ml of Rompún and 9 ml of PBS, and 0.8 ml of the mixture was injected intraperitoneally. Then the animals were subjected to unilateral cryptepididymis following the procedure described by Bedford [26]. Briefly, the testis and epididymis from one side were drawn into the abdomen through the inguinal canal. Then the gubernaculum was severed and the mesorchium between testis and epididymis incised to the level of the distal caput. The epididymis was reflected into the abdominal cavity and the cauda was retained by a linen suture between its connective tissue and the abdominal wall. The testis was also fixed to the scrotum by a linen suture. Animals were allowed to recover and then killed at 12, 24, or 48 h or at 20 days after surgery.

The animal protocol used was in accordance with law 223/88 on Animal Protection of Spain and the European Union Agreement about Vertebrate Animal Protection (3/18/1986) and has been approved by the CSIC (Spanish Research Council) ethical committee.

Histologic Detection of Apoptosis

Mice were injected with heparin (100 IU; Byk Leo, Arganda del Rey, Spain) and subjected to vascular perfusion through the left ventricle for 10–15 min with 120 ml of PBS and then another 10–15 min with 120 ml of buffered neutral formalin [27]. After fixation, they were dehydrated through an alcohol series (ethanol 70%, 96%, and 100%) followed by xylol and then embedded in paraffin. Serial 7-µm sections were cut with a microtome and mounted on slides. Sections were deparaffinized with xylol, hydrated, and stained with Weigert iron hematoxylin solution [27]. The stained sections were subsequently washed in distilled water and destained with a saturated solution of picric acid to increase contrast. Then they were dehydrated, mounted with Entellan (Merck, Whitehouse Station, NJ), and scored under bright field optics in a Labophot-2 microscope (Nikon, Tokyo, Japan) for the presence of apoptotic cells. The criterion to define a cell as apoptotic was the presence of a fragmented nucleus with condensed chromatin granules. The scrotal, contralateral caudae were used as controls. In addition, caudae from non- and sham-operated animals were evaluated. At least three different samples were considered for any of the time points examined.

Several series of sections were used for detection of DNA fragmentation in situ using the TUNEL detection kit supplied by Roche (Basel, Switzerland), following the protocols provided. Nuclei were counterstained with a solution of 10-6 M Hoechst 33258 (Sigma, St. Louis, MO). Results were assessed under epifluorescence using the appropriate filters (excitation 450–490 nm for fluorescein isothiocyanate [FITC] and excitation 365/10 nm for Hoechst).

Electron Microscopy

Three animals killed 24 h after surgery were used as the source of tissue. Small pieces of tissue were fixed by immersion in 2 ml of a solution of 2% glutaraldehyde, 1% p-formaldehyde, and 0.1 M sucrose in 0.5 M cacodylate buffer (pH 7.3). Fixation proceeded for 1 h at room temperature and samples were then thoroughly washed with the buffer and postfixed for 1 h at room temperature in 2% osmium tetroxide in cacodylate buffer. Then the tissues were dehydrated in an alcohol series and embedded in an epoxy resin (Epon, Fluka, Alcobendas, Spain). Ultrathin sections were obtained using a LKB Ultratome (type III) (LKB Instruments, Rockville, MD) and were stained with uranyl acetate and lead citrate. Finally, they were observed and photographed in a Philips 400 electron microscope.

Detection of DNA Fragmentation on Agarose Gels

Samples from six different animals obtained 24 h after surgery were used for DNA isolation according to the following procedure. The caudae epididymidis from two operated males were isolated and freed from fat tissue. Then they were cut in small pieces and digested with collagenase (10 mg/ml in RPMI 1640 medium) for 30 min at 37°C. The tissue fragments were pelleted by centrifugation at 1500 x g and fresh collagenase solution added. After a new 30-min digestion, the cells were washed two to three times and the pellet was subjected to extraction of extranuclear DNA as described by Fabregat et al. [28]. Total DNA isolated from scrotal and cryptepididymal caudae were labeled with 32P-dCTP using terminal transferase (Roche) and then cleaned up using the QIAquick Nucleotide Removal Kit (QIAGEN, Valencia, CA). The labeled DNA was loaded on a 1.5% agarose gel and run at 40–45 V for 5 h. Then it was dried and exposed to an X-OMAT-AR film (Kodak, Rochester, NY) to visualize the DNA ladder.

Androgen Receptor and p53 Immunocytochemistry

Sections of the control and experimental caudae were obtained using a cryostat and were used for androgen receptor and p53 immunocytochemistry. Three animals killed 24 h after surgery were used and at least three sections per animal scored. The sections were air dried and fixed for 10 min in cold acetone. The sections were then blocked by incubation in 3% BSA in PBS for 1 h. Then they were incubated in the primary antibody solution overnight at 4°C. For androgen receptor detection, the primary antibody used was PG21 (Upstate Biotechnology, Lake Placid, NY). For p53 detection, the antibody was the mouse monoclonal Pab 122, a generous gift from Dr. Augusto Silva (CSIC, Spain). Both antibodies were used at a dilution of 2 µg/ml in PBS. The secondary antibodies used were an anti-rabbit IgG and an anti-mouse IgG, both FITC-labeled, purchased from Sigma and used at a dilution of 1:200 in PBS. After incubation, the slides were mounted with Vectashield (Vector Labs, Burlingame, CA) and the positive reaction scored by epifluorescence.

Western Blot Analyses

Tissue extracts from six scrotal and six cryptepididymal caudae were obtained. For every extraction, two caudae were homogenized using a glass homogenizer in 100 µl of RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 1% SDS, 2 mM EDTA, pH 7.5) containing protease inhibitors (10 mM PMSF, leupeptin, antipain, chymostatin, and pepstatin at 1 µg/ml and benzamidine at 10 µg/ml). Previously, the organs had been isolated and cut in small pieces in PBS to release the spermatozoa. The protein concentration was measured using the BioRad Protein Assay (BioRad, Hercules, CA). Four hundred micrograms of protein were loaded per lane and separated in a 10% acrylamide gel following standard procedures. The proteins were subsequently transferred to nitrocellulose sheets (0.2-µm pore) using a Semidry Electroblotting Apparatus (Millipore, Molsheim, France) and reversibly stained by Ponceau Red to assess the extent of transfer. The filters were blocked overnight at 4°C in blocking solution (Roche) and then incubated with the primary antibody. The antibodies used were PG-21 (Upstate Biotechnology) for androgen receptor detection, PAb 122 for p53 detection, and SPA-801 and SPA-810 (StressGen, Victoria, Canada) for detection of hsp-25 and hsp-70. Primary antibodies were used at concentrations of 1 µg/ml. The secondary antibodies were all conjugates to alkaline phosphatase and the positive reaction was revealed by the use of the chemoluminescent substrate CDP-Star (Roche) following manufacturer instructions.

RT-PCR Detection of Bax, bcl-2, and Caspase3

Total RNA was extracted from the experimental and control caudae of three operated mice (24 h after surgery) using the Rneasy Mini Kit (QIAGEN). One microgram of total RNA was used for reverse transcription using the First Strand cDNA Synthesis Kit for reverse transcription-polymerase chain reaction (RT-PCR), provided by Roche, according to manufacturer's instructions. One microliter of the RT mixture was then used as template for the PCR reaction. The primers used were 5'-TACCGTCGTGACTTCGCAGAG-3' and 5'-GGCAGGCTGAGCAGGGTCTT-3' (bcl-2), 5'-CGGCGAATTGGAGATGAACTG-3' and 5'-GCAAAGTAGAAGAGGGCAACC-3' (bax), 5'-AGGGGTCATTTATGGGACA-3' and 5'-TACACGGGATCTGTTTCTTTG-3' (caspase3), and 5'-TACCACAGGCATTGTGATGG-3' and 5'-AATAGTGATGACCTGGCCGT-3' (ß-actin, the loading control). Conditions for the PCR were a 5-min hot start at 94°C, 35 cycles at 94°C (1 min), 55°C (1 min), and 72°C (1 min), and a last elongation cycle at 72°C for 5 min. The PCR products were resolved in a 2% agarose gel and viewed and photographed under ultraviolet light.

Cell Counts and Statistical Analyses

Apoptotic cells were counted using the hematoxylin-stained sections as stated above. A minimum of 16 sections including at least 250 tubules per animal were scored. The distance between sections was at least 70 µm, so there was no risk of counting the same cell twice. Cell counts were also carried out in the contralateral scrotal caudae and caudae from normal and sham-operated males. Differences among groups were estimated by analysis of variance (ANOVA) followed by the Student-Newman-Keul test (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of Apoptosis in the Cryptepidymides

Histologic examination of the cryptepididymal caudae showed the presence of apoptotic bodies in the epithelium (Fig. 1, A–D), but they were restricted to the proximal regions of the cauda. None or few apoptotic cells were observed in the distal tubules of the cauda (Fig. 1B). Some cells undergoing apoptosis were observed in the proximity of the basal regions (Fig. 1A), but others were seen in the apical regions (Fig. 1C) or even in the lumen of the tubules (Fig. 1D). That those cells were indeed undergoing programmed cell death was assessed by the TUNEL assay (Fig. 1, E–H). Again, positive nuclei in both apical (Fig. 1G) and basal (Fig. 1H) positions were found. It is also noteworthy that some cells appeared as being extruded into the lumen (Fig. 1G).



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  		FIG. 1. Detection of apoptotic cells in the epithelium of the cryptepididymal cauda 24 h after surgery. A–D) Photomicrographs from the cryptepididymis viewed under bright-field optics after hematoxylin staining. A) Section of the proximal region of a cryptepididymal cauda. Two apoptotic cells (arrow) are shown close to the basal region. The shrunken cytoplasm as well as the condensed chromatin granules are fully visible. Bar = 20 µm. B) Section of the distal region of the same cryptepididymal cauda. No apoptotic cells are detected here. Bar = 20 µm. C) High magnification of the epithelium of a cryptepididymal proximal cauda. An apoptotic cell located close to the lumen of the tubule is indicated by an arrow. Bar = 10 µm. D) Section of the lumen of one tubule of a cryptepididymal proximal cauda. Several apoptotic bodies (arrows) are shown interspersed with the spermatozoa. Bar = 20 µm. E–H) Detection of apoptotic cells in the cryptepididymal caudae by TUNEL. E) Phase contrast/fluorescence photomicrograph of a cryptepididymal proximal cauda showing two positive cells (arrow) in one of the tubules. The inset shows a higher (x5 more) magnification of these cells. Bar = 50 µm. F) Phase contrast/fluorescence photomicrograph of the contralateral scrotal proximal cauda. No positive cells are seen. Bar = 50 µm. G) Phase contrast/fluorescence photomicrograph of a cryptepididymal cauda showing a TUNEL-positive cell (indicated by the arrow) in the apical part of the epithelium being extruded into the lumen. The nuclei were counterstained using Hoechst 33258 and are seen in pale gray. Bar = 10 µm. H) Fluorescence photomicrograph of a cryptepididymal proximal cauda showing two positive cells (arrows) close to the basement membrane. The nuclei were counterstained using Hoechst 33258 and are seen in pale grey. Sperm heads are seen inside the lumen in the right side of the photomicrograph. Bar = 10 µm

Electron microscopic analyses showed cells at different stages of apoptosis (Fig. 2). Early stages of apoptosis could be distinguished by the lobulation of the nucleus and chromatin condensation (Fig. 2A). We found also phagocyted apoptotic bodies in the cytoplasms of principal cells (Fig. 2, B–D). The apoptotic bodies showed partially degraded nucleus and cytoplasm and condensed chromatin. In some of them, most of the chromatin had been already degraded (Fig. 2B).



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  		FIG. 2. Electron micrographs of apoptotic cells in the cryptepididymis 24 h after surgery. A) Epithelial cell at an early stage of apoptosis. The nucleus has started chromatin condensation (arrows) and is now irregular in its shape. The changes are more obvious when it is compared with the nuclei (N) of adjacent cells. The cytoplasm is less electron dense, but the organelles are still intact. Bar = 2 µm. B) Principal cells containing two degraded apoptotic bodies (ab). Most chromatin has already degenerated and there are only three small chromatin granules (cg) visible in one of them. The nucleus of a principal cell (N) is shown below. mv, Microvilli. Bar = 2 µm. C) Principal cell containing a phagocytosed apoptotic body in its apical region. The apoptotic body shows a nuclear fragment with condensed chromatin (asterisks) on the right and some organelles representing the rests of the cytoplasm on the left (arrows). mv, Microvilli. Bar = 1 µm. D) Two partially degraded apoptotic bodies (ab) in the cytoplasm of principal cells. Granules of compacted chromatin (asterisks) are seen in both. Two normal nuclei (N) belonging to the surrounding cells are seen. Bar = 2 µm

A typical DNA ladder was seen in the DNA obtained from the proximal cauda cells in the cryptepididymis (Fig. 3). This is indicative of the DNA fragmentation that takes place during apoptosis. Although controls also showed some fragmentation, this was clearly much reduced and it is consistent with the observations in the tissue. In control caudae, although the apoptotic index was certainly very low, it was not zero (Table 1).



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  		FIG. 3. Analysis of DNA fragmentation in the epididymal epithelial cells on agarose gels. Total extranuclear DNA extracted from two scrotal (S) and two cryptepididymal (C) caudae was obtained 24 h after surgery and processed as described in the text. Molecular weights in kb of the markers (Roche no. XIV) are indicated on the left. The DNA ladder, indicative of apoptosis, is clearly visible in both samples and the intensity of the signal is clearly higher on the right lane


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  		TABLE 1. Percentage of tubules with apoptotic cells and number of apoptotic cells per 100 tubules in the control and experimental caudae. Values are expressed as mean ± SEM.*

Quantitative Analysis of Histologic Sections

The hematoxylin-stained sections were used for estimation of the apoptotic indexes (number of apoptotic cells per tubule and percentage of positive tubules). Results are shown in Table 1. The apoptotic wave was initiated 12 h after surgery, reached its peak at 24 h, and declined by 48 h. Examination at longer times (20 days) showed values similar to controls. The apoptosis seemed to be region dependent because it was restricted to the proximal tubules of the cauda and was almost nonexistent in the large tubules from the distal cauda (Table 1).

Immunological Detection of Androgen Receptor, p53, and Heat Shock Proteins in the Cryptepididymis

The immunohistochemical procedures showed that neither the androgen receptor expression nor its nuclear localization were affected by temperature (Fig. 4). Regarding p53, we were unable to detect the protein in the tissue by either immunocytochemistry (data not shown) or Western blot (Fig. 4C). Finally, the heat shock proteins analyzed, hsp-25 and hsp-70, did not show any significant alteration produced by the temperature increase (Fig. 4C).



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  		FIG. 4. Immunological detection of androgen receptor p53 and heat shock proteins in the cryptepididymis 24 h after surgery. Detection of androgen receptor in tissue sections of the cryptepididymal (A) and scrotal (B) cauda. The androgen receptor is located in the nuclei in both and does not show significant differences. Bar = 20 µm. C) Western blot detection of androgen receptor p53, hsp-70, and hsp-25 in tissue extracts from cryptepididymal and scrotal caudae. A tumor cell line, colo 205, which is known to express p53, was used as control. No differences among the cryptepididymal and scrotal cauda are observed. Numbers on the right represent the molecular weights (kDa) of the protein bands. D) Detection of bcl-2, bax, and caspase3 by RT-PCR. The PCR-amplified products from ß-actin, bcl-2, bax, and caspase3 in cryptoepididymis (C) and scrotal cauda (S) are shown. Numbers on the left show the base pairs corresponding to each fragment

Detection of bcl-2, Bax, and Caspase3 Gene Expression by RT-PCR

The results from the RT-PCR are summarized in Figure 4D. From the three gene products analyzed, caspase3 did not show major changes. Nevertheless, the bcl-2 family members analyzed (bcl-2 and bax) showed a strong overexpression in the cryptepididymis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There are a variety of stimuli that can trigger programmed cell death [29]. In the male genital tract, androgen withdrawal is one of the major inducers of apoptosis in testes [30–32], epididymis [12, 33], and accessory sex glands [34–36]. The role of others factors, such as temperature, has received considerably less attention. Our results show that temperature is indeed one factor that can trigger apoptosis in the cauda epididymidis. Temperature-induced apoptosis has already been described in the testes in cases of cryptorchidism, both spontaneous and experimental [37–40]. It is noteworthy that, in our case, this temperature-induced apoptosis was region dependent, being restricted to the proximal tubules of the cauda. This region-specific responsiveness was also observed in castrated rats, in which apoptosis is higher in proximal regions of the epididymis [12]. In this case, the specificity has been related to the different concentration of androgens in every segment [41]. In our model, this does not seem to be the case because both proximal and distal cauda are subjected to the same temperature in the scrotum. Moreover, when temperature is experimentally increased in the cauda epididymidis, the more profound changes observed anatomically are those of the more distal regions [5, 6]. These changes, of course, are observed 15–30 days after abdominal implantation of the cauda, while apoptosis takes place mainly in the first 2 days. Our results indicate that both regions are functionally distinct and that the proximal tubule cells are the most sensitive in terms of survival, but we have no clue as to why this is so. Regarding the cell type affected, it is likely to be the principal cell. Principal cells are the most abundant cell type in the epididymal epithelium [42]. In addition, the ultrastructural characteristics that we found in the cytoplasms of the apoptotic cells are consistent with this hypothesis. The apoptotic cells seem to be eliminated from the epithelium in two complementary ways, extrusion to the lumen and phagocytosis. The role of leukocytes and macrophages in disposing of the apoptotic cells has already been described in several epithelia [43–45] as well as phagocytosis by adjacent epithelial cells [44, 45]. The role of extrusion to the lumen in the removal of apoptotic cells has been more controversial. While some authors do not consider the shed cells as apoptotic [46], others clearly do [47]. We have seen apoptotic cells that seem to be undergoing extrusion, so we think that this mechanism is also taking place. On the other hand, it is noteworthy that apoptosis affects only a small fraction of the total cell population. This is not surprising because similar results have been shown in cases of orchidectomy or efferent duct ligation [12, 17, 33]. This could be explained by the existence of different subpopulations of principal cells, but it may also reflect the capacity of the cells to react to the changes in their microenvironment. A fraction of them cannot adapt and die in a "clean," apoptotic fashion and the rest survive, although it may change in terms of gene expression [20–22] or morphologic and physiologic characteristics [19, 20]. In fact, in many systems, it is not the phenotype, but the period in the life cycle of those cells, that makes them susceptible to apoptosis [48]. Along the same line of thought, an increase of the temperature from 37 to 39°C induces apoptosis in only a small population of peritoneal macrophages [49]. More drastic conditions (41°C) are needed to induce apoptosis in most of the cells. In this case, again, the population is considered homogeneous, and the differences that might explain why only a certain percentage of cells undergo apoptosis must be subtle. Perhaps they are cells that are already damaged or have deficiencies in their DNA repair mechanisms. In this regard, it has been shown that deficiencies in DNA repair mechanism greatly increases the risk of apoptosis [50].

Regarding the mechanism of action of the temperature to induce apoptosis, this is not mediated by the androgen receptor because no changes in its content or localization in the tissue were detected. This is in agreement with previous reports on temperature effects on epididymis [21–24]. We also examined the role of a protein, p53, that has been classically involved in regulating apoptotic processes [51]. The role of p53 in mediating apoptosis in the male genital tract has been examined in several cases. Apoptosis induced by androgen withdrawal in the prostate [52, 53] and in the seminal vesicles [52] or by deprivation of luminal factors in the epididymis [17] is p53 independent. However, p53 plays a role in the initiation of apoptosis in mouse testicular germ cells after experimental cryptorchidism [39]. In the epididymis, however, it seems that p53 does not play a significant role since we were unable to detect any significant induction of the protein in the epididymal cells. Besides, the putative roles of two heat shock proteins, hsp-25 and hsp-70, in the process were also examined. Heat shock proteins have been involved in the response to temperature stress in several systems [54], and they are recognized regulators of apoptosis [55, 56]. Moreover, hsp-70 levels increase in the prostate after castration [57] and in the cryptorchid testes [58] in parallel with the increase in apoptosis. Small shock proteins (i.e., hsp-25) have been shown to inhibit apoptosis in a cultured prostatic cell line [59]. Nevertheless, in our model, we have not seen any change in the levels of these proteins, and although we cannot discount any role of other members of the family, at least for these two, no significant role should be played.

There is a role, however, for members of the bcl-2 family. These proteins have been involved in induction or prevention of apoptosis [60]. Bax is a proapoptotic gene and is likely to be overexpressed in the cells undergoing apoptosis. On the contrary, bcl-2 has antiapoptotic properties and is probably responsible for the adaptation of the tissue and survival of most cells at the abdominal temperature. Interestingly, both bax and bcl-2 have been involved in the apoptosis induced by hyperthermia in the testes [61]. We do not know, though, whether bax and bcl-2 induction is directly caused by temperature or mediated by other gene product(s). Finally, no effects on caspase3 levels were found.

In conclusion, we have shown that temperature in the cauda regulates not only gene expression but also cell survival. The molecular mechanisms through which temperature exerts its regulation involve the bcl-2 family members.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Augusto Silva and Dr. Jorge Gamonal from the Center of Biological Investigations (CIB/CSIC) in Madrid for the anti-p53 antibody as well as technical assistance and helpful suggestions for performing the p53 immunohistochemistry. Special thanks to Ascension González for her invaluable help in obtaining the cryosections. We are also indebted to Dr. T.G. Cooper (Münster, Germany) and C. Kirchhhoff (Hamburg, Germany) for helpful suggestions.


   FOOTNOTES
 
1 This work was supported by grant BCM2000-0899. M.J. is grateful for support from the AECI (Spanish Agency for International Cooperation). Back

2 Correspondence: Rosa Carballada, Centro de Investigaciones Biológicas, Velázquez 144, 28006 Madrid, Spain. FAX: 34 915627518; rosac{at}mncn.csic.es Back

Received: 17 October 2001.

First decision: 14 November 2001.

Accepted: 8 May 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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