RNF4 during rat testis development suggests a role in spermatid maturation

Mechanisms of Development 118 (2002) 247–253 www.elsevier.com/locate/modo

Gene expression pattern

Expression of the nuclear RING finger protein SNURF/RNF4 during rat testis development suggests a role in spermatid maturation Wei Yan a,b, Sirpa J. Hirvonen-Santti c, Jorma J. Palvimo c,d, Jorma Toppari a,b, Olli A. Ja¨nne c,e,* a

Department of Physiology, University of Turku, FIN-20520 Turku, Finland Department of Pediatrics, University of Turku, FIN-20520 Turku, Finland c Biomedicum Helsinki, Institute of Biomedicine, University of Helsinki, FIN-00014 Helsinki, Finland d Institute of Biotechnology, University of Helsinki, FIN-00014 Helsinki, Finland e Department of Clinical Chemistry, University of Helsinki and Helsinki University Central Hospital, FIN-00014 Helsinki, Finland b

Received 8 February 2002; received in revised form 19 July 2002; accepted 19 July 2002

Abstract A small nuclear RING finger protein, termed SNURF (or RNF4), is a coregulator of androgen receptor-dependent transcription. To elucidate the physiological role of SNURF in vivo, cell type-specific localization and changes in SNURF mRNA and protein accumulation were followed during testicular development and spermatogenesis of the rat. Two SNURF transcripts, ,3.0 and 1.6 kb in size, were detected in adult rat testis. Both mRNA species are capable of encoding full-length SNURF protein. The 3.0 kb SNURF mRNA is persistently expressed in Sertoli cells of both immature and mature testes, whereas the expression of the 1.6 kb transcript appears after day 30 of postnatal life and is restricted to step 4–11 spermatids. Increased accumulation of SNURF in step 4–11 spermatids, which do not express the androgen receptor, indicates that SNURF action is not restricted to the regulation of androgen signaling. Germ cell expression of SNURF coincides with the last transcriptional activity of the haploid genome and alterations in chromatin structure, suggesting that SNURF is involved in the regulation of processes required for late steps of spermatid maturation. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Androgen receptor; Coactivator; Coregulatory protein; RNF4; Spermatogenesis; SNURF; Spermatid; Testosterone

1. Results and discussion A small nuclear RING finger protein, SNURF (or RNF4), is a nuclear coregulatory protein (Moilanen et al., 1998; Chiariotti et al., 1998; Poukka et al., 2000; Galili et al., 2000; Lyngsø et al., 2000; Fedele et al., 2000; Ha¨kli et al., 2001; Pero et al., 2001). In addition to a 3.0 kb SNURF mRNA species cloned from rat testis cDNA library, we isolated another SNURF cDNA of ,1.6 kb in size (GenBank accession No. AY050655) from the same library. The 5 0 -untranslated regions of the two mRNAs are identical up to nt 2149 upstream of the translation initiation site, after which they diverge (Fig. 1). The 3 0 -untranslated sequences of the two mRNAs are also different, in that the 1.6 kb mRNA contains a premature poly(A) 1 sequence at 109 nt downstream of the TGA stop codon, thus yielding a truncated 3 0 -untranslated region (Fig. 1). A conserved polyadenylation signal, AATAAA, is not present, but an A-rich region is located within 20 nt preceding the poly(A) 1

* Corresponding author. Tel.: 1358-9-19125040; fax: 1358-9-19125047. E-mail address: [email protected] (O.A. Ja¨nne).

stretch. It is likely that the A-rich sequence acts as an alternative polyadenylation signal (Sheets et al., 1990) to generate the 1.6 kb SNURF mRNA species in rat. To explore the role of SNURF in testicular function, its expression sites during testicular development and spermatogenesis as well as regulation of accumulation in rat testis were examined. Two SNURF mRNA species were detected in adult rat testis (Fig. 2A) when the SNURF-NT probe was used, whereas only the 3.0 kb SNURF mRNA was hybridized to the SNURF-CT probe (Figs. 1 and 2B). The 3.0 kb mRNA species was persistently expressed throughout testicular development, with the levels being somewhat higher in newborn and 20-day-old animals than in other age groups (Fig. 2A). The 1.6 kb SNURF mRNA was not detectable prior to day 30, after which it accumulated to very high levels, suggesting that its expression is confined to late round spermatids and elongating spermatids. Only the 3.0 kb SNURF mRNA species was expressed to moderate levels in Leydig cells of adult rat testis (Fig. 2C). Most intense SNURF mRNA signals were observed in step 4–11 spermatids by in situ hybridization with the SNURF-NT probe (Fig. 3). Signal intensity was higher in

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days 4, 18, or 30 after MAA (Fig. 7). By contrast, the 1.6 kb mRNA content was very low on day 18 after MAA, apparently due to depletion of most of the SNURFexpressing spermatids at stages IV–VI (Bartlett et al., 1988; McKinnell and Sharpe, 1997). It was elevated on days 4 and 30 after MAA treatment possibly due to the relative enrichment of SNURF-expressing spermatids in testes devoid of spermatocytes and elongating spermatids. Together with the constant level of the 3.0 kb mRNA, these data provide further evidence that the 3.0 and 1.6 kb SNURF mRNA

Fig. 1. Schematic presentation of the rat 1.6 and 3.0 kb SNURF mRNA species. The solid black box represents the protein-coding region of the two mRNAs. The alternative polyadenylation site of the 1.6 kb mRNA is located at 190 nt downstream of the TGA stop codon. Positions of the two RNA probes, SNURF-NT and SNURF-CT, used for Northern blotting and in situ hybridization are indicated by arrows. Nucleotide sequences of the 5 0 - untranslated regions of the two SNURF mRNA species are shown below. The translation initiation codon ATG is underlined.

step 6–8 spermatids than in step 4–5 and 9–11 spermatids, and it was more pronounced in step 4–11 spermatids than in Sertoli cells at all stages of the epithelial cycle (see Fig. 6). Much weaker signals were also observed in the basal compartment of the seminiferous epithelium. With the SNURF-CT probe, specific for the 3.0 kb mRNA, the most intense signals were confined to Sertoli cell cytoplasm and probably to spermatogonia as well (Fig. 4). The intensity of signals appeared higher at stages XII–XIV and I–III than at stages IV–XI. The 35 kDa molecular mass SNURF protein was detectable in rat testis at all ages examined. Only Sertoli cell nuclei were stained with anti-SNURF antibody before day 20 (Fig. 5). From day 30 onwards, nuclei of both Sertoli cells and spermatids at certain stages were decorated by anti-SNURF antibody. In the adult rat seminiferous epithelium, SNURF protein levels were high at stages VII–VIII, IX–XII, and II–VI, and low at stages XIII–I. From day 20 onwards, a few Leydig cells were stained with anti-SNURF antibody, and the number of stained Leydig cell nuclei increased with the testicular development. Accumulation and localization of SNURF mRNA and protein during testicular development and spermatogenesis are schematically illustrated in Fig. 6. Spermatocytes at stages I–VI are selectively depleted within 24 h after methoxyacetic acid administration (MAA) (Bartlett et al., 1988; McKinnell and Sharpe, 1997). Consequently, the seminiferous tubules at certain stages are devoid of spermatocytes on day 4 after MAA treatment. On day 18, certain stages lack round spermatids, and on day 30, there are no elongating spermatids at defined stages. MAA treatment was used to validate further the cellular localization data on SNURF mRNA and protein. The levels of the 3.0 kb mRNA remained unchanged on

Fig. 2. Differential accumulation of SNURF mRNA species during testicular development. (A) Northern blot hybridization analysis of testis RNA from rats of different ages using SNURF-NT as a probe. NB, newborn; the postnatal developmental phase is indicated as days after birth (d). (B) The SNURF-CT probe is specific for the 3.0 kb mRNA species. (C) Detection of 3.0 kb SNURF mRNA in Leydig cells (LC) purified from the adult rat testis. The mRNA from the seminiferous tubules (ST) was used as a positive control. The entire experiment was repeated three times with essentially identical results.

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were significantly elevated (Fig. 8B). Four- to five-fold increased accumulation of the 3.0 kb transcript was observed in the EDS 1 T treated rats, whereas the content of the 1.6 kb transcript was three-fold higher than that in controls. Androgen regulation of SNURF mRNA accumulation in Sertoli cells may not represent a direct action of the androgen receptor (AR) on the SNURF promoter, since at least the murine SNURF promoter is not activated by ectopic expression of AR in transfected cells (Hirvonen et al., 2002). Androgen action in germ cells is thought to be mediated primarily through the surrounding cells that express AR, including Sertoli cells, peritubular cells, periarterial cells and Leydig cells (Eddy, 1998; Sar et al., 1990; Takeda et al., 1990, Griswold, 1995). Thus, SNURF accumulation in spermatids is likely to be mediated indirectly by products from the neighboring cells. In conclusion, SNURF is encoded by two mRNA species, one of which is spermatid-specific. SNURF accumulation is

Fig. 3. SNURF mRNA localization in rat testis by in situ hybridization using SNURF-NT RNA probe. Panels A, A 0 , B, and B 0 correspond to in situ hybridization with the antisense SNURF-NT probe, while panels C, C 0 , D, and D 0 depict results with the sense probe. The bright field is shown on the left and the dark field on the right. Roman numerals represent stages of the epithelial cycle. Bar ¼ 0.5 mm. Sc, Sertoli cell; Sg, spermatogonium, Sp, spermatocyte; Sd, spermatid, and LC, Leydig cell. Note that the most intense signals are observed in the adluminal compartment at stages IV–XI. Specific signals can also be seen in the basal compartment of almost all stages.

species accumulate in Sertoli cells and spermatids, respectively. Ethylene dimethane sulfonate (EDS) depletes Leydig cells in rat testis within 24 h. As a consequence, circulating testosterone (T) concentration declines, remains undetectable between days 2 and 15 and starts to increase after day 20 when the repopulated Leydig cells start functioning (Tena-Sempere et al., 1997; Teerds, 1996). The levels of both SNURF mRNA species were significantly reduced on days 2–15 after EDS treatment (Fig. 8A). Accumulation of the two transcripts was elevated on days 20–40, in concert with the increasing T levels. In rats that received EDS and T implants simultaneously, circulating T concentrations are much higher than in untreated animals (Tena-Sempere et al., 1997), and the levels of both SNURF mRNA species

Fig. 4. Localization of the 3.0 kb SNURF mRNA species in adult rat testis detected by using SNURF-CT RNA probe. Panels A, A 0 , B, and B 0 correspond to in situ hybridization with the antisense SNURF-CT probe, while panels C, C 0 , D, and D 0 depict results with the sense probe. The bright field is shown on the left and the dark field on the right. Roman numerals represent the stages of the epithelial cycle. Bar ¼ 0.5 mm. Sc, Sertoli cell; Sg, spermatogonium, Sp, spermatocyte; and Sd, spermatid.

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Fig. 5. Localization of SNURF protein in immature and mature rat testis as revealed by staining with anti-SNURF antibody. NB, newborn; 5–60 days, days 5– 60 after birth; NC, negative control. Roman numerals represent the stages of the epithelial cycle. Pc, peritubular cell; Sc, Sertoli cell; Sg, spermatogonium; Sd, spermatid; and Lc, Leydig cell. Note that in adult rat testis (60 days), spermatids at stages II and XII are negative, while those at stages VII and VIII are positive for immunostaining. Bar ¼ 0.5 mm.

not restricted to Sertoli cells, but this protein is also highly expressed in round and elongating spermatids, suggesting that SNURF plays a role in spermiogenesis. 2. Materials and methods 2.1. Experimental animals and treatments All animal experiments were approved by the Ethical Committee on Animal Experimentation at the University of Turku. Sprague–Dawley male rats were injected intraperitoneally (i.p.) with a single dose (75 mg/kg body weight) of EDS (Jackson and Jackson, 1984; Tena-Sempere, 1997). Control animals received vehicle only. MAA (Aldrich Chemie, Steinheim, Germany) was diluted in physiological saline and a single dose (650 mg/kg body weight) was administered orally; control animals received physiological saline. The animals were sacrificed under CO2 anesthesia, the testes were snap frozen in liquid nitrogen and stored at 2708C.

2.2. Transillumination-assisted microdissection of the seminiferous tubules Seminiferous tubule segments at stages II–VI, VII–VIII, IX–XII, and XIII–I of the epithelial cycle were isolated under a stereomicroscope by transillumination-assisted microdissection technique (Toppari and Parvinen, 1985; Yan et al., 1999). 2.3. Purification of Leydig cells from the adult rat testis Leydig cells were isolated from rat testis as described previously (El-Gehani et al., 1998), and the purity of the enriched cell population was $85%. 2.4. RNA probe preparation The EcoRI/BamHI fragments, corresponding to nt 234– 967 and nt 968–1644 of SNURF cDNA (Moilanen et al., 1998), were subcloned into pGEM-3Z (Promega Corp., Madison, WI). The resulting SNURF-NT and SNURF-CT

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Fig. 7. SNURF mRNA accumulation in rat testes on days 4, 18 and 30 after MAA treatment. As a consequence of selective depletion of spermatocytes within 24 h after MAA, there are no spermatocytes at stages I–VI on day 4, no round spermatids on day 18, and no elongating spermatids on day 30 at certain stages. A representative blot for three experiments is shown.

Fig. 6. SNURF mRNA and protein accumulation during testicular development and spermatogenesis. (A) Diagram illustrating the progression of germ cell development in rat testis during the first 60 days after birth. The main cell types are shown in the top panel. The length of open boxes represents the stages of germ cell development in the postnatal life. NB stands for newborn and 5–60 days for days 5–60 after birth. The solid boxes depict the cell types of SNURF accumulation with the thickness of the boxes reflecting the relative abundance of SNURF mRNA or protein. (B) Organization of the seminiferous epithelial cycle in the adult rat. The SNURF-expressing cells are boxed with the width of the boxes representing the relative abundance of SNURF mRNA or protein. The specific cell associations in the vertical column represent the specific stages (Roman numerals) of the epithelial cycle. Sc, Sertoli cells; A1–4, type A spermatogonia; In, intermediate spermatogonia; B, type B spermatogonia; Pl, preleptotene spermatocytes; L, leptotene spermatocytes; Z, zygotene spermatocytes; P, pachytene spermatocytes; Di, diplotene spermatocytes; m, meiosis.

plasmids were linearized, and the RNA probes were synthesized using SP6 RNA polymerase (Promega) for the antisense and T7 RNA polymerase (Promega) for the sense probe. The probes were labeled with [a- 32P]dUTP and [a- 35S]dUTP (both from Amersham Pharmacia Biotech, Aylesbury, UK) for Northern and in situ hybridizations, respectively. 2.5. RNA preparation and Northern blot hybridization Total testicular RNA was isolated using a single step method (Chomczynski and Sacchi, 1987). Ten micrograms of total RNA were size-fractionated in 1% denaturing agar-

Fig. 8. Regulation of SNURF mRNA accumulation in vivo. (A) Steadystate levels of testicular SNURF mRNA in response to testosterone (T) withdrawal and recovery after ethylene EDS treatment. (B) Steady-state levels of testicular SNURF mRNA in EDS-treated rats that received T implants. Testicular RNA was fractionated and hybridized with SNURFNT cRNA and 28S rRNA cDNA probes, and the data analyzed as described in Section 2. The results are presented in both panels as means ^ SEM of three independent experiments using different rats. **, P , 0:01; *, P , 0:05 as compared to controls.

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ose gel and transferred onto Hybond-N 1 nylon membrane (Amersham Pharmacia Biotech). Prehybridization and hybridization conditions were as described (Yan et al., 1999). After hybridization with SNURF probes, the blots were stripped and then hybridized with 28S rRNA cDNA probe labeled with [a 32P]dCTP. 2.6. In situ hybridization Five microgram thick sections were cut from paraffinembedded samples and mounted onto SuperFrost Plus glass slides (Menzel-Gla¨ ser, Steinheim, Germany). The slides were incubated at 378C overnight and stored at 48C before use. In situ hybridization was performed as described (Kaipia et al., 1992). 2.7. Immunohistochemistry One 5 mm thick section was used for immunohistochemical staining with polyclonal SNURF antibody (1:2000 dilution in TBS containing 1% BSA) (Moilanen et al., 1998), and the other for periodic acid–Schiff–hematoxylin staining (Yan et al., 2000). Visualization of positive cells was performed using Vectastain Elite kit (Vector laboratories, Burlingame CA, USA) according to the manufacturer’s instructions. Preimmune serum or rabbit IgG were used to monitor the specificity of staining. 2.8. Quantitative analysis of Northern hybridization and immunoblotting results Northern hybridization and immunoblotting films were scanned by a UMAX scanner (UMAX Inc., Fremont, CA) and saved as TIFF-type files ( p .tif, Microsoft Co. and Aldus Co., New York, NY). TINA 2.0 densitometric analytical system (Raytest Isotopenmesgerate GmbH, Straubenhardt, Germany) was used for quantification. Northern blot and immunoblot results were normalized to 28S rRNA and actin, respectively. Control values were designated as 100%, and other results were expressed as percentages of controls. Three independent experiments were pooled for calculation of the means ^ standard errors (SEM) and for one-way analysis of variance and Duncan’s new multiple range test to study the significance of differences between groups. The P value ,0.05 was considered statistically significant. Acknowledgements This work was supported by grants from the Medical Research Council (Academy of Finland), Research Program on Environmental Health, Finnish Foundation for Cancer Research, Sigrid Juse´ lius Foundation, Biocentrum Helsinki, Helsinki University Central Hospital, and Turku University Central Hospital. We thank Johanna Vesa for technical assistance and Jukka Kero for help in animal experiments.

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