Stage-Specific Expression and Cellular Localization of the Heat Shock Factor 2 Isoforms in the Rat Seminiferous Epithelium

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

240, 16–27 (1998)

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Stage-Specific Expression and Cellular Localization of the Heat Shock Factor 2 Isoforms in the Rat Seminiferous Epithelium Tero-Pekka Alastalo,*,† Minna Lo¨nnstro¨m,† Sirpa Leppa¨,†,1 Kai Kaarniranta,†,‡ Markku Pelto-Huikko,§ Lea Sistonen,† and Martti Parvinen*,2 *Department of Anatomy, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland; †Turku Centre for Biotechnology, ˚ bo Akademi University, P.O. Box 123, FIN-20521 Turku, Finland; ‡Department of Anatomy, University of Turku, A University of Kuopio, P.O. Box 6, FIN-70211 Kuopio, Finland; and §Department of Biomedical Sciences, University of Tampere, P.O. Box 607, FIN-33101 Tampere, Finland

members of this family, HSF1, HSF2, and HSF4, have been identified in mammals [2–5]. HSF1 is the best studied member of this transcription factor family and represents the classical HSF that responds to elevated temperatures and other forms of stress, whereas HSF2 is refractory to typical stress stimuli [1]. In addition to stress, heat shock genes are transiently expressed under certain developmental conditions. For example, expression of hsp70 is strongly induced in the mouse preimplantation embryo at the time of zygotic genome activation [6, 7]. As HSF1 is abundantly expressed and localized in the nuclei of one- and two-cell state embryos when the typical stress inducibility is absent, HSF1 has been suggested to be involved in the spontaneous expression of hsp70 during this early stage of embryonic development [8]. However, at later stages (morula-blastocyst), HSF1 and HSF2 are coexpressed, and at the blastocyst stage, when the inducible stress response is restored, HSF2 is abundantly expressed and constitutively active [8, 9]. These results indicate that at these later stages of mouse embryogenesis, HSF1 and HSF2 are involved in the regulation of the inducible and spontaneous expression of hsp70, respectively. The hypothesis that HSF2 plays an important role in the transcriptional regulation of gene expression during development is further supported by the findings that HSF2 is constitutively active in the male germline and in embryonal carcinoma cells representing early embryonic cells [10, 11]. In addition, HSF2 is activated during hemin-mediated erythroid differentiation of human K562 erythroleukemia cells [12]. HSF2 consists of two polypeptides which are generated by alternative splicing [13, 14]. The smaller HSF2b isoform results from exclusion of an 18-amino-acid sequence present in the coding region of the larger HSF2-a isoform [14]. Analysis of a wide range of murine tissues has revealed that although HSF2 is ubiquitously expressed, it is especially abundant in testis and brain [13]. Furthermore, the ratio of HSF2-a and HSF2-b isoforms is regulated in a tissue-dependent manner as HSF2-a is abundantly expressed in mouse

Heat shock transcription factors (HSFs) are generally known as regulators of cellular stress response. The mammalian HSF1 functions as a classical stress factor, whereas HSF2 is active during certain developmental processes, including embryogenesis and spermatogenesis. In the present study, we examined HSF2 expression at specific stages of the rat seminiferous epithelial cycle. We found that expression of the alternatively spliced HSF2-a and HSF2-b isoforms is developmentally regulated in a stage-specific manner. Studies on cellular localization demonstrated that HSF2 is present in the nuclei of early pachytene spermatocytes at stages I–IV and in the nuclei of round spermatids at stages V–VIIab. In contrast a strong HSF2 immunoreactivity was detected in small distinct cytoplasmic regions from zygotene spermatocytes to maturation phase spermatids. Immunoelectron microscopic analysis revealed that these structures are mainly cytoplasmic bridges between germ cells. Our results on cellular localization of HSF2 and stage-specific expression of the HSF2 isoforms indicate that HSF2, in addition to its function as a nuclear transcription factor, may be involved in other cellular processes during spermatogenesis, possibly in the sharing process of gene products between the germ cells. q 1998 Academic Press

INTRODUCTION

Expression of heat shock genes coding for heat shock proteins (Hsps) is primarily regulated on the transcriptional level by specific DNA-binding proteins, called heat shock factors (HSFs). HSFs have been shown to act through a highly conserved upstream response element (heat shock element, HSE) that is found in the promoter region of heat shock genes [1]. To date three 1 Current address: European Molecular Biology Laboratory, P.O. Box 10.2209, D-69012 Heidelberg, Germany. 2 To whom correspondence and reprint requests should be addressed. Fax: /358-2-333 7352. E-mail: [email protected].

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0014-4827/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.

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testis and HSF2-b in heart and brain [14]. Transient transfection experiments have shown that HSF2-a is a more potent transcriptional activator than HSF2-b [14], and stable overexpression of HSF2-b inhibits hemin-induced heat shock gene expression and erythroid differentiation in K562 cells [15]. These results have raised the possibility that the HSF2 isoforms could play distinct roles in regulation of the target genes. Spermatogenesis is a process by which immature male germ cells go through a complex series of differentiation involving mitotic and meiotic cell divisions and finally a series of morphological transformations leading to the formation of mature spermatozoa. One of the striking features of spermatogenesis is its spatial and temporal organization. In the seminiferous epithelium, the differentiating spermatogenic cells are not randomly distributed, but form defined cell associations, also called stages, which follow one another in a wavelike fashion [16]. Each stage contains a different collection of developing cell types and is classified primarily by the morphology of the developing spermatids. The differentiation of haploid cells is called spermiogenesis, which in turn is divided into several steps on the basis of morphological criteria. The synchronous production cycle of spermatogenic cells is an excellent model for studying the regulation of gene expression in the context of differentiation. Several genes are expressed as specific isoforms in somatic cells and in spermatogenic cells due to alternative splicing or alternative start sites of polyadenylation [17, 18]. Although the function of the alternatively processed transcripts in testis is largely unknown, in some cases the alternative processing reverses the function of the protein. For example, a cAMP-responsive element modulator protein, CREM, is expressed as a testis-specific isoform, CREMt, which functions as a transcriptional repressor in spermatocytes but is converted to an activator by alternative splicing in postmeiotic spermatids [19]. Recently, CREMt has been shown to be essential for the differentiation program of spermiogenesis [20]. During spermatogenesis, HSF2 has been shown to be most abundantly expressed in spermatocytes and round spermatids [11], indicating that HSF2 could be regulated in a stage-dependent manner. The specific objectives of this study were to determine the detailed stage-specific and cell type-specific expression of HSF2 during spermatogenesis. Our results revealed that the distinct HSF2 isoforms were differentially regulated and HSF2 was localized not only in the nuclei of spermatocytes and of round spermatids, but also in the cytoplasmic interconnecting bridges. MATERIALS AND METHODS Microdissection of seminiferous tubule. Adult rats were killed by CO2 asphyxiation and the testes were excised and decapsulated. The seminiferous tubules were teased free under a transilluminating ste-

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reomicroscope in a petri dish. The stages of the cycle of the seminiferous epithelium were identified according to light absorption criteria based on the condensation of spermatid nuclei and their relative localization in the tubule [21]. The tubules were microdissected to 10 pools containing segments from stages I, II–III, IV–V, VI, VIIab, VIIcd, VIII, IX–XI, XII, and XIII–XIV [22] and used for Western and Northern blot analyses. An improved modification of the transillumination method was used to prepare squash preparations for immunohistochemistry. After the stages were determined using the transilluminating stereomicroscope, the isolated 0.5- to 1-mm segments were squashed between a microscope slide and a coverslip. By phase-contrast microscope, using the morphological criteria of Leblond and Clermont [23], the exact stage of the cycle was identified from the monolayer of living cells [24]. To remove the coverslips, the slides were frozen in liquid nitrogen and immediately fixed in methanol at 0207C. Antibodies and purification of recombinant HSF2-b. Polyclonal rabbit antisera against HSF1 and HSF2 were obtained from R. I. Morimoto (Northwestern University, Evanston, IL) and were used as previously described [25]. In addition, polyclonal rabbit HSF2 antiserum was prepared by using purified mouse recombinant HSF2b protein as described by Sarge and co-workers [25] (Fig. 1A). This antiserum was affinity-purified with bacterially expressed mouse HSF2-b [25] covalently bound to diazophenylthioether paper according to the manufacturer’s instructions (Schleicher & Schuell, Dasser, Germany). The specificity of the antibody was verified by Western blotting using bacterially expressed HSF2-b as well as human, mouse, and rat cell lysates. The characteristic HSF2 reactivity of the affinity-purified antibody was identical to that obtained with the previously described antiserum (Fig. 1B). Mouse monoclonal antibody SPA-810 (StressGen, Victoria, BC, Canada) was used to detect the inducible form of Hsp70. Rat monoclonal antibody SPA-835 (StressGen) was used to detect Hsp90. Rabbit polyclonal antibody SPA-801 (StressGen) was used to detect Hsp25. Mouse monoclonal antiserum against actin was obtained from Amersham (Buckinghamshire, UK). Horseradish peroxidaseconjugated goat anti-rabbit IgG (Promega, Madison, WI), goat antimouse IgG (Amersham), and goat anti-rat IgG (Promega) were used as secondary antibodies in Western blot analyses. Immunohistochemistry was completed using the Vectastain ABC Kit reagents (Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol. Purified mouse recombinant HSF1 protein was a gift from R. I. Morimoto, and purified human recombinant Hsp70 protein (SPP755) was obtained from StressGen. Whole-cell extracts and immunoblotting. Whole-cell extracts from staged seminiferous tubules and from control, heat-shocked, and nerve growth factor (NGF)-treated rat pheocromocytoma (PC-12) cells were prepared as described previously [26]. Briefly, the samples were pelleted, quick-frozen, and resuspended in a double volume of high-salt buffer containing 20 mM Hepes, pH 7.9, 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl2 , 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol. Extracts were centrifuged at 50,000g at 47C for 5 min. Amounts of total soluble proteins were quantified from the supernatants using the Bradford method [27]. Then 10- to 15-mg protein samples were separated on an 8% SDS–polyacrylamide gel and transferred onto a nitrocellulose filter using a semidry transfer apparatus (Bio-Rad). Western blotting was performed as described [25] with the following modifications. Filters were blocked in PBS containing 5% nonfat dry milk and 0.3% Tween 20 overnight at 47C. Antibody reactions against HSF1, HSF2, Hsp70, Hsp90, Hsp25, and actin were performed in PBS–BSA (5 mg/ml) for 1 h at room temperature (RT). Subsequently, the blots were washed three times for 10 min in PBS and 0.3% Tween 20 followed by incubation with horseradish peroxidase-conjugated secondary antibodies in PBS–BSA (5 mg/ml) and 0.3% Tween 20 for 1 h at RT. After washing the blots were subjected to immunodetection using the ECL method (Amersham). HSF2-a and HSF2-b isoforms were quantitated with

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computerized image analysis (Microcomputer Image Device Version M4, Imaging Research Inc.). Gel mobility shift assay. Whole-cell extracts from staged seminiferous epithelium and heat-shocked (427C, 1 h) whole testis were used to assay HSE-binding activity. Reactions containing 15 mg wholecell extract, 0.1 ng oligonucleotide probe, 1 mg poly(dIrdC)–poly (dIrdC), 10 mg BSA, in 10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 5% (v/v) glycerol, 0.5 mM DTT, 0.5 mM PMSF were incubated for 20 min at room temperature. A consensus HSE oligonucleotide (5*-GTCGACGGATCCGAGCGCGCCTCGAATGTTCTAGAAAAGG-3*) was used to detect DNA binding [13]. Protein–DNA complexes were resolved on a 4% native polyacrylamide gel containing 0.51 TBE and visualized by autoradiography. RNA preparation and Northern blot analysis. Total cellular RNA was isolated from microdissected seminiferous tubule segments and PC-12 cells using the single-step method described by Chomczynski and Sacchi [28]. Ten micrograms of RNA was separated on a 1% agarose gel containing formaldehyde and transferred onto a nylon membrane (Hybond-N, Amersham). Filters were hybridized to [a-32P]dCTP-labeled cDNAs coding for mouse HSF2 [3], human hsp70 [29], rat hst70 [30], human hsp90a [31], and rat glyceraldehyde phosphate dehydrogenase (GAPDH) [32]. Hybridizations were performed according to the instructions of the manufacturer. Quantification of the high-molecular-weight isoforms was performed with computerized image analysis. Immunohistochemistry. For immunohistochemistry, adult anesthesized rats were killed by transcardial perfusion with a fixation solution containing 4% formaldehyde and 0.2% picric acid in PBS. The testes were excised and placed in the same fixative for 1 h at 47C. The tissue blocks were then washed, dehydrated, embedded in paraffin, and sectioned at 6 mm. Before incubations the sections were treated with 0.1 M sodium citrate (pH 5) in a microwave oven for 10 min and washed in PBS. Immunohistochemistry was then completed using the manufacturer’s protocol for the immunostaining of paraffin sections. Immunoreaction was visualized by 0.25 mg/ml 3,3-diaminobenzidine tetrahydrochloride in PBS containing 10 mg/ml ammonium nickel sulfate and 0.005% hydrogen peroxide. In preabsorption controls the HSF1, HSF2, and Hsp70 antisera were preabsorbed with 5 mg of respective recombinant proteins for 12 h in 47C. For accurate stage identification, squash preparations were made from the rat seminiferous tubule segments [24]. The slides were frozen in liquid nitrogen, the coverslips were removed, and the cells were fixed in methanol at 0207C for 15 min, air dried for 1 h, washed in PBS, and microwaved in 0.1 M sodium citrate (pH 5) for 10 min. The slides were then washed in 0.2% Tween 20 in PBS for 10 min and the immunohistochemistry was completed as above. Electron microscopic immunohistochemistry. The rats were anesthesized and perfused with 4% paraformaldehyde, 0.2% glutaraldehyde, and 0.2% picric acid in PBS. The testes were excised, postfixed for 6 h, cryoprotected with 60% sucrose in PBS for several days, and then frozen in liquid nitrogen. Thick sections (É500 mm) were cut, thawed in PBS, and incubated with polyclonal rabbit antiserum against HSF2 (1:500) for 5 days. After several washes, the sections were incubated in biotinylated goat anti-mouse IgG and ABC complex for 12 h each. Diaminobenzidine chromogen was used to reveal ABC complexes. The tissues were then postfixed with 2.5% glutaraldehyde, 1% osmium tetroxide, and 1% uranyl acetate (30 min each), dehydrated, and embedded in Epon. The ultrathin sections were examined with a Jeol 1200EX electron microscope (Jeol USA, Peabody, MA).

RESULTS

HSF2 Isoforms Are Expressed in a Stage-Specific Manner in the Rat Seminiferous Epithelium To determine the stage-specific expression pattern of HSF2 in the rat seminiferous epithelium, whole-cell

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extracts from stages I, II–III, IV–V, VI, VIIab, VIIcd, VIII, IX–XI, XII, and XIII–XIV were analyzed by Western blotting. Both the previously described HSF2 antiserum and affinity-purified antibody (see Materials and Methods; Fig. 1) were used and identical results were obtained. Rat PC-12 cells which express both the HSF2-a and the HSF2-b isoforms were used as controls. As shown and quantified in Fig. 2, the amounts of HSF2 varied markedly between distinct stages of spermatogenesis. The larger HSF2-a isoform was detected at stages I–VIIcd, exhibiting the highest level at stages II–III. At stages VIIcd–XI, HSF2-a could not be detected. In contrast, the smaller HSF2-b isoform was expressed at all stages with a maximum at stages II–III. In all stages, however, the HSF2-b was more abundant than HSF2-a. In addition, we observed several (at least three) additional bands of lower mobility at stages II–VIIcd. PC-12 cells showed no alteration in the amount or ratio of HSF2-a and HSF2-b isoforms upon treatment with NGF or exposure to elevated temperature. In comparison to HSF2, the amounts of HSF1 were analyzed in the same samples (Fig. 2A). Unlike HSF2, the levels of HSF1 did not markedly vary between different stages but a slight change in migration on SDS – PAGE was observed at stages VIIcd – XIV, indicating that HSF1 may be modified during these phases of spermatogenesis. However, the altered migration of HSF1 in the testis samples was not as dramatic as in the heat-shocked sample of PC12 cells, where the slower migration band indicates the hyperphosphorylated and transcriptionally active forms of HSF1 [25, 33]. Since HSFs function as regulators of heat shock gene expression, we also examined the accumulation of Hsp70, Hsp90, and Hsp25 proteins (Fig. 2A), but no correlation was seen between the amounts of HSF2 and Hsps. The highest levels of Hsp70 were detected at stages I–VI, and at stages VIII–XIV Hsp70 was hardly detectable. While only one band was detected in PC-12 cells, an additional band of slower mobility was found using the antibody against the inducible form of Hsp70 in the testis samples, but the origin of this band is not known. Hsp90 was constitutively present throughout all stages, whereas Hsp25 could not be detected during spermatogenesis. Actin was used as a control protein as it was detected at all stages with the most abundant accumulation at stages VIII–XIV, where the lowest levels of HSF2 isoforms were observed. To determine whether HSF DNA-binding activity is present at stages expressing high levels of HSF2 protein, we performed a gel mobility shift assay with a labeled oligonucleotide containing a consensus HSE. Unlike whole testis exposed to elevated temperatures, no stages of the seminiferous epithelial wave contained HSE-binding activity (Fig. 3).

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FIG. 1. Characterization of the recombinant mouse HSF2-b protein used for immunization and specificity of the affinity-purified HSF2 antibody. (A, left) Coomassie blue stained SDS–PAGE showing the purified recombinant mHSF2-b protein (HSF2). 1 mg BSA was used as control, and sizes (kDa) of the molecular weight marker (MW) are indicated on the left. (A, right) Detection of the mHSF2-b protein (HSF2; approx. 10 ng) by Western blotting using the previously described HSF2 antibody [25]. (B) Western blot analysis of untreated (C) and hemin-treated (he; 16 h) human K562 cells and K562 cells overexpressing mouse HSF2-a (a) or HSF2-b (b) isoforms [15]. The results were identical using the previously described HSF2 antiserum (left) and the affinity-purified antibody (right). The endogenous human and exogenous mouse HSF2-a and -b isoforms are indicated by hHSF2-a/b and mHSF2-a/b, respectively.

Expression of HSF2 mRNAs in Seminiferous Tubules To determine the relative amounts of HSF2 mRNAs at different stages of the seminiferous epithelium, total RNA was extracted from equivalent amounts of tissue and analyzed by Northern hybridization (Fig. 4). HSF2 mRNA levels showed a wide variation at different stages of spermatogenesis. The major HSF2 mRNA, which presumably corresponds to the HSF2-a and HSF2-b isoforms, was detected at stages XII–VIIab, being most abundant at stages II–VI. The increase in the HSF2 mRNA was maximally (stages IV–V) over 40-fold, compared to the HSF2 mRNA levels at stages VIII–XIV (Fig. 4B). In comparison, we also analyzed the total RNA from untreated and heat-shocked PC-12 cells, which contained relatively small amounts of HSF2 mRNA. Consistent with our finding of the smaller HSF2 forms detected by Western blotting (Fig. 2A), a novel lower molecular weight transcript, or several transcripts detected in a single band representing a size range of approximately 1.9 kb, was detected at stages II–VIIab. This HSF2 transcript could possibly result from alternative splicing or different lengths of poly(A) tails. We also analyzed steady-state mRNA levels of the putative HSF2 target genes, including the inducible form of hsp70, testis-specific hst70, and hsp90. Transcripts of all these genes were ubiquitously expressed at all stages, and the highest levels of hsp70, hst70, and hsp90 mRNAs were detected at stages XII–XIV (Fig. 4A). Hence, no temporal correlation between the expression of HSF2 and the heat shock genes could be observed on the steady-state mRNA level. Furthermore, the expression patterns of hsp70 or hsp90 mRNAs did not correlate with the corresponding protein levels. In fact, accumulation of hsp70 mRNA preceded the accumulation of Hsp70 protein in the epithelial cycle, raising the possibility that the mRNAs could be stored for subsequent translation. In the heatshocked PC-12 cells, a remarkable increase in the

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hsp70 and hsp90 mRNA levels could be observed, whereas the testis-specific hst70 mRNA could not be detected in either untreated or heat-shocked PC-12 cells. Stage- and Cell Type-Specific Localization of HSF2, HSF1, and Hsp70 during Rat Spermatogenesis Biochemical analyses did not reveal the cell typespecific expression and intracellular distribution of HSF2. Therefore, we examined the localization of HSF2 in rat testis sections by immunohistochemical techniques using the two specific HSF2 antisera (see Materials and Methods; Fig. 1), and identical results were obtained. Due to the convoluted nature of the seminiferous tubules, a cross section exhibits tubules from each of the 14 stages. The expression pattern of HSF2 showed marked variation from almost entirely absent to intense tubules in a stage-specific manner (Figs. 5A and 5C). Closer examination of the seminiferous tubules at each of the 14 stages of the cycle revealed that the most intense HSF2 signal was in the tubules of stages I–IX in primary spermatocytes and round spermatids, whereas tubules from stages X–XIV lacked the intense signal (Figs. 5C–5E, 5G). No immunoreactivity was detected in spermatogonia and preleptotene spermatocytes (data not shown). The HSF2 reactivity also showed stage specificity with respect to the intracellular compartmentalization pattern. At stages I–IV immunoreactivity was localized to a region in the interior of the epithelia which was identified as nuclei of early pachytene spermatocytes (Figs. 5C and 5E). However, the more apically located round spermatids showed only distinct cytoplasmic immunoreactions at these stages (Fig. 5E). An intense signal at stages V–VII appeared to localize to both the nuclei and the cytoplasm of cells closer to the lumen rather than the basal area of the epithelium (Figs. 5C and 5D). These HSF2-positive cells were identified as round spermatids. The basal region of these

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FIG. 2. (A) Accumulation of HSF2 isoforms in rat seminiferous tubules at different stages (I–XIV) of the cycle. Untreated (C), NGFtreated (NGF), and heat-shocked (HS) PC-12 cells were used as controls. Whole-cell extracts (10 mg) were analyzed by SDS–PAGE and Western blotting using antibodies against HSF2 and HSF1. Subsequently, the same blots were analyzed with antibodies against Hsp70, Hsp90, Hsp25, and actin. a, b, and smw (small molecular weight) indicate the distinct isoforms of HSF2. (B) Computerized image analysis was used to quantitate the amounts of HSF2-a and HSF2-b at different stages of rat seminiferous epithelial cycle. The values for the fold induction of HSF2 isoforms are shown relative to the lowest detectable level of HSF2-a at stage XII, which was arbitrarily assigned a fold induction value of 1.

tubules showed only cytoplasmic immunoreactions (Fig. 5D). After stage VII, the HSF2 reactivity decreased gradually so that at stages VIII and IX, a transient reactivity of acrosomic head caps was observed (Fig. 5G). Furthermore, immunohistochemistry of accurately staged squash preparations confirmed the results obtained with paraffin sections (data not shown). Preabsorption of HSF2 antiserum and affinity-purified antibody with the purified HSF2-b protein was

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markedly reduced at most stages (Fig. 5B). However, closer examination revealed that a slight immunoreactivity remained specifically in the nuclei of early pachytene spermatocytes at stages I–IV (Fig. 5F), but the specificity of the nuclear immunoreactions remains to be elucidated. Unlike HSF2, HSF1 was more uniformly distributed during the cycle of the seminiferous epithelium (Figs. 6A and 6C). Using higher magnification, the HSF1 im-

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immunoreactivity and was detected in the cytoplasm of pachytene spermatocytes at stages I–IX (Figs. 7A and 7C). In control preparations, the primary antibodies were preabsorbed with the purified HSF1 and Hsp70 proteins, respectively. No HSF1 immunoreactions could be seen after preabsorption with the purified HSF1 protein (Fig. 6B). Apart from the reactive residual bodies at stage VIII, no Hsp70 immunoreactions could be detected after preabsorption with the purified Hsp70 protein (Fig. 7B). The control preparations incubated with preimmune serum and secondary antibody were also negative (data not shown). Ultrastructural Localization of HSF2

FIG. 3. Analysis of HSE-binding activity in rat seminiferous tubules at different stages of the cycle (I–XIV) compared to heatshocked (HS) whole testis. Whole-cell extracts (15 mg) were analyzed by gel mobility shift assay using a 32P-labeled consensus HSE oligonucleotide probe [13]. HSE–HSF complex (HSE), nonspecific complex (NS), and free probe (FREE) are indicated.

munoreactivity was observed in the nuclei of different types of spermatocytes as well as round and elongating spermatids (Fig. 6C). Hsp70 showed a stage-specific

To study more carefully the specific cytoplasmic immunoreactions, we performed immunoelectron microscopy. HSF2 immunoreactivity was distinctly localized in the membranes of the cytoplasmic bridges or ring canals interconnecting spermatocytes and spermatids (Fig. 8A). In addition, intensively labeled small structures were found inside or in the immediate vicinity of the ring canals (Fig. 8B). Some ring canals also showed immunoreactive projections toward the lumen (Fig. 8C). Furthermore, less intense HSF2 immunoreactivity was observed in other cytoplasmic regions and in the nucleus (data not shown). These cytoplasmic immunoreactions were possibly localized in the chromatoid body or associated structures, and the nuclear HSF2 immunoreactivity appeared in small (80–100 nm) dotlike structures.

FIG. 4. Expression of HSF2 mRNA in rat seminiferous tubules at different stages of the cycle (I–XIV) compared to untreated (C) and heatshocked (HS) PC-12 cells. (A) Equal amounts of total RNA (15 mg) were analyzed by Northern blotting using 32P-labeled cDNA probes for HSF2, hsp70, hst70, hsp90, and GAPDH. The sizes of mRNAs (kb) are indicated on the right. (B) Computerized quantification was used to analyze the abundance of the high-molecular-weight HSF2 mRNA at different stages of the cycle. The value for the fold induction of HSF2 mRNA level is shown relative to the lowest level of HSF2 mRNA at stage VIII, which was arbitrarily assigned a fold induction value of 1.

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FIG. 5. Localization of HSF2 in perfusion-fixed testis sections. Samples were treated with HSF2 antiserum (A, C–E, G) or HSF2 antiserum preabsorbed with purified HSF2-b protein (B, F). Using different magnifications, A, C–E, and G show HSF2 at different stages as indicated by Roman numerals. Black and white asterisks indicate cell layers of pachytene spermatocytes and round spermatids, respectively. Arrows and arrowheads mark specific nuclear and cytoplasmic immunoreactions, respectively. Bars are 50 mm. 22

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DISCUSSION

FIG. 6. Distribution of HSF1 in perfusion-fixed testis sections. Samples were treated with HSF1 antiserum (A, C) or HSF1 antiserum preabsorbed with purified recombinant HSF1 protein (B). Higher magnification (C) shows HSF1 in the nuclei of spermatocytes and round spermatids. Bars are 50 mm.

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Spermatogenesis consists of a complex schedule of stage-specific developmental events. The proper coordination and succession of events during spermatogenesis require accurate control of the temporal and spatial processes, suggesting that regulatory factors are expressed and act in a stage-specific manner. The rapid and dramatic changes in the morphology and biochemistry of the germ cells are the result of a remarkable modulation of gene expression by the concerted action of transcription factors. In the present study, we examined the complex expression and distribution pattern of HSF2, a transcriptional regulator of heat shock genes, during different stages of spermatogenesis. The highest expression levels of the HSF2-a and HSF2-b isoforms were detected at early stages of the epithelial cycle (Fig. 9A). The assumption that the expression of distinct HSF2 isoforms is differentially regulated was supported by our Western analyses, revealing that HSF2-a is maximally expressed at stages II–III and is barely detectable at stages VIIcd–XI, whereas HSF2-b is also expressed after stage III. Furthermore, the quantitative comparison between the HSF2-a and the HSF2-b isoforms indicates that HSF2-b is more abundantly expressed throughout spermatogenesis than HSF2-a, which is in disagreement with a previous study showing a switch in expression from HSF2-b to HSF2-a during postnatal development of testis [14]. In addition to the major HSF2 mRNA, consisting of transcripts for HSF2-a and -b isoforms, we detected a low-molecularweight HSF2 mRNA at stages II–VIIab, which may be the transcript(s) coding for the low-molecular-weight HSF2 protein isoforms detected at stages II–VIIcd. An additional low-molecular-weight HSF2 was also detected in the whole testis preparation by Sarge and co-workers [11], but the number, identity, and stage-specificity of these low-molecular-weight HSF2 isoforms were not determined. Our immunohistochemical studies with testis sections and accurately staged squash preparations confirmed and extended the results obtained by Sarge and co-workers [11]. Strong nuclear HSF2 immunoreactivity was present in primary spermatocytes at stages I–IV and in round spermatids at stages V–VII, suggesting a role for HSF2 as a transcriptional regulator during these developmental phases (Fig. 9B). Interestingly, the strong HSF2 immunoreactivity of round spermatids was identified at the same stages as the small molecular isoforms, raising the possibility that they could be haploid-specific isoforms of HSF2. In addition, the expression of these low-molecular-weight isoforms correlates well with the activation of most haploid-specific genes, particularly with the expression of pituitary adenylate cyclase activating polypeptide [34]. The cytoplasmic localization of HSF2 detected in this study has not been described earlier. Immunoelectron microscopic analysis revealed that the strong HSF2 im-

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FIG. 7. Localization of Hsp70 in perfusion-fixed testis sections. Samples were treated with Hsp70 antiserum (A, C) or preabsorption serum (B). Higher magnification (C) shows Hsp70 in the cytoplasm of pachytene spermatocytes. Arrowheads indicate immunoreactive cytoplasmic regions. Bars are 50 mm.

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munopositive cytoplasmic foci are cytoplasmic bridges or ring canals. Such structures have been demonstrated to interconnect spermatocytes and spermatids [35, 36]. In these cells the formation of cytoplasmic bridges has been suggested to synchronize mitotic and meiotic divisions [37, 38] and to function as channels to transport gene products which make genetically haploid spermatids turn into phenotypically diploid cells [39]. Previous studies on ring canals of Drosophila follicles have shown that actin filaments are key elements of these structures [40, 41]. Actin, together with several other recently characterized proteins, is likely to be essential for normal germ cell differentiation [42]. The unexpected cytoplasmic localization of HSF2 may be due to the acquisition of novel functions of this transcription factor. There is evidence that several proteins, such as cytoskeletal components, secreted growth factors, glycolytic enzymes, kinases, transcription factors, molecular chaperones, transmembrane proteins, and extracellular matrix proteins, exist in cellular compartments other than their conventional sites of action [43]. However, there are no reports of extranuclear locations of active transcription factors. Furthermore, in some electron micrographs, we found small HSF2-immunopositive granules inside the ring canals. This indicates that HSF2 could be involved in mRNA or protein transport, as certain phases of gametogenesis are known to be dependent on stored mRNA species and probably also on presynthesized stored proteins [44]. In the present study, we found that HSF1 and HSF2 have clearly distinct expression patterns in the rat seminiferous epithelium. HSF1 is evenly expressed at all stages and is localized to the nuclei of spermatocytes and spermatids. Furthermore, an altered change in migration on SDS–PAGE was observed at stages VIIcd–XIV, suggesting a stage-specific change in the phosphorylation state of HSF1. Interestingly, a slower migrating putative intermediate form of HSF1 has also been detected after treatment with salicylate or the protein kinase C activator TPA [45, 46]. The partial phosphorylation of HSF1 upon salicylate treatment has been suggested to account for a transcriptionally inert HSF1 which binds to DNA and enhances synergistically the stress response induced with different stress stimuli [45]. Nevertheless, no hyperphosphorylation characteristic of a fully active HSF1 [25, 33] could be seen during spermatogenesis. However, as HSF1 is localized in the nuclei throughout the epithelial cycle, the role of this molecule during spermatogenesis remains to be elucidated. Hsps have been shown to be abundantly expressed during spermatogenesis [47–55]. Previous studies have demonstrated a possible role for HSF2 as a regulator of the transcription of hsp70.2, a member of the mammalian hsp70 gene family [11]. Hsp70.2 has an important role during spermatogenesis as the disruption of this gene results in failed meiosis, germ cell apoptosis, and male infertility [56]. Among the possible target genes of HSF2, we examined the expression of Hsp70, Hsp90, and Hsp25.

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spermatogenesis. Furthermore, the analysis of corresponding mRNAs together with the testis-specific hst70 mRNA revealed no temporal correlation with the HSF2 isoforms. Our finding suggests that these mRNAs are stored for subsequent translation or that heat shock gene expression is not regulated by HSF2 during spermatogenesis. Recently, similar results have been obtained for the central nervous system during mouse embryogenesis, showing that the HSF2 and Hsp expression domains do not coincide during postimplantation development [57]. Thus, there is increasing evidence that HSF2 regulates

FIG. 8. Immunoelectron microscopic localization of HSF2. (A) Electron micrograph of HSF2 immunoreactive ring canals connecting several pachytene spermatocytes. Bar is 1 mm. (B) Small immunopositive granules in the ring canals and marked with arrowheads. Bar is 200 nm. (C) HSF2 immunoreactive wall of a ring canal. Bar is 200 nm.

The inducible form of Hsp70 is abundantly expressed at stages I–VI and Hsp90 is expressed evenly throughout the cycle, whereas Hsp25 could not be detected during

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FIG. 9. Summary of the expression pattern (A) and cell typespecific distribution (B) of HSF2 in the rat seminiferous epithelium illustrated in a schematic map of the 14 stages of the epithelial cycle. Stages are correlated to the light absorption pattern of freshly isolated seminiferous tubule under the transilluminating stereomicroscope [22]. A, type A; IN, intermediate; B, type B; PL, preleptotene; L, leptotene; Z, zygotene; EP, early pachytene; LP, late pachytene; D, diakinetic; DIV, division; RS, round spermatid; ES, elongating spermatid; MS, maturation phase spermatid.

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other target genes apart from the heat shock genes. Consistent with this hypothesis, Drosophila HSF has been reported to be required for oogenesis and early larval development by regulating novel, non-heat-shock genes [58]. Alternatively, HSF2 may be involved in cellular processes other than transcriptional regulation. Unlike previous reports on constitutive HSF DNA-binding activity in testis [11, 13], we did not detect HSE-binding activity at any stage of the seminiferous epithelial cycle. The different results could be due to experimental or technical procedures, such as differences in HSE-containing oligonucleotides or possible in vitro activation/inactivation of HSFs during sample preparation. Interestingly, the HSF2-b isoform is more abundantly expressed than HSF2-a during spermatogenesis. Together with our earlier study showing that overexpression of HSF2-b inhibits the induction of HSF2 DNA-binding activity [15], the present study provides further evidence that the HSF2 isoforms are functionally distinct and that HSF2-b lacks DNA-binding and transactivating properties. Taken together, our results on the cell type-dependent and stage-specific expression of HSF2 during rat spermatogenesis suggest that HSF2 might be involved in several specific functions during differentiation and developmental processes. Future studies are required to determine whether the up- and down-regulation of HSF2 expression is critical for normal developmental timing of spermatogenesis. To this end, the mechanisms responsible for controlling expression of distinct HSF2 isoforms need to be elucidated. Due to the complex nature of this molecule, these studies are likely to reveal interesting insights into the regulation of gene expression, how the same molecules participate in the transcriptional regulation, and other cellular functions.

6.

7.

8.

9.

10.

11.

12.

13.

14.

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16. We are indebted to Zdzislaw Krawczyk for providing the cDNA probe for rat hst70 (p121/3.2d). We thank John E. Eriksson, Diana M. Toivola, Urban Lendahl, and Harri Hakovirta for fruitful discussions and critically reading the manuscript. We are also grateful to Helena Saarento and Leena Simola for skillful technical assistance. This work was supported by The Academy of Finland, the Sigrid Juse´lius Foundation, and the Finnish Cancer Foundation.

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Received September 2, 1997 Revised version received December 5, 1997

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