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Spz1, a novel bHLH-Zip protein, is specifically expressed in testis
Mechanisms of Development 100 (2001) 177±187
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Spz1, a novel bHLH-Zip protein, is speci®cally expressed in testis Shih-Hsien Hsu a,b, Huey-Wen Shyu a,b, Hsiu-Mei Hsieh-Li c, Hung Li a,b,* a
b
Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan Institute of Life Science, National Defense Medical Center, Taipei, Taiwan c Institute of Medical Science, Taipei Medical College, Taipei, Taiwan
Received 7 August 2000; received in revised form 16 October 2000; accepted 16 October 2000
Abstract We isolated a novel bHLH-Zip gene designated Spz1 from a mouse testis cDNA library. Spz1 is expressed speci®cally in the testis and epididymis. Immuno¯uorescence staining detected Spz1 protein in the nuclei of LFG6 Leydig cells. The ability of Spz1 protein to bind to the bHLH consensus-binding site, the E-box, was con®rmed by EMSA, and a 9-bp asymmetric target site was identi®ed by random selection and PCR ampli®cation. Hormonal regulation of Spz1 was investigated and downregulation of Spz1 expression by testosterone and retinoic acid was found. This nuclear transcription factor may play a crucial role in spermatogenesis by regulating cell proliferation or differentiation through binding to speci®c DNA sequences like other bHLH-Zip molecules. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Basic helix±loop±helix; Leucine zipper; Mouse; Spermatogenesis; Testis
1. Introduction The basic helix±loop±helix (bHLH) family of transcription factors comprises important components in the regulatory networks of many developmental pathways (Srivastava et al., 1997; Rawls and Olson, 1997; Murre et al., 1994; Feder et al., 1993). This family is characterized by a conserved structural motif consisting of two amphipathic helixes separated by a loop, the HLH domain, which mediates protein interactions to form homo- and heterodimers (Murre et al., 1994). There are two major categories of bHLH proteins. Class A consists of ubiquitously expressed proteins such as those encoded by differentially spliced transcripts of the E2A (E12, E47, and E2-5/ITF1), E2-2/ITF2, and HEB/HTF4 genes (Goldfarb et al., 1996; Watada et al., 1995; Whyte et al., 1988; Glick et al., 2000). Class B consists of tissue-speci®c bHLH proteins such as MyoD, NeuroD, MASH, and TAL (Arnold and Winter, 1998; Guillemot, 1999; Lo et al., 1994; Xia and Hwang, 1994). These proteins form heterodimers consisting of ubiquitous class A and tissue-speci®c class B bHLH proteins. The highlycharged basic region in each of the dimerization partners immediately upstream of the HLH region forms a speci®c DNA-binding domain that can recognize a DNA sequence known as an E-box (CANNTG), N-box (CACNAG) * Corresponding author. Tel.: 1886-2-2788-0460; fax: 1886-2-27826085. E-mail address: [email protected] (H. Li).
(Oellers et al., 1994; Tietze et al., 1992) or G-box (de Pater et al., 1997). Some members of the bHLH family also contain a leucine zipper (Zip) motif that occurs immediately C-terminal to the bHLH motif; these are the bHLHZip proteins (Blackwood and Eisenman, 1991; FerreD'Amare et al., 1993). These Zip motifs are involved in protein dimerization enhancement. Some dimers result in activation of gene expression while others result in repression. These bHLH-Zip proteins have been implicated in various developmental and cellular processes, and some are known to be oncogenes (Murre et al., 1989; Blackwood and Eisenman, 1991; Atchley and Fitch, 1997). Spermatogenesis is a complex process that leads to the formation of male gametes. Spermatogenesis takes about 40 days in rats and about 63 days in humans. During this period, type-A spermatogonia, originally derived from primordial germ cells (PGC), either cycle as pluripotent stem cells or further differentiate into type-B spermatogonia that give rise to primary spermatocytes. Two meiotic divisions of spermatocytes yield haploid spermatids that undergo morphological and functional differentiation to produce highly specialized spermatozoa (Dym, 1994). Spermatozoa then complete the differentiation process in the epididymis. The epididymis is not only a sojourn for spermatozoa which might last several weeks, but it also promotes the development of the spermatozoa's fertilizing capacity, culminating in their acquisition of full mobility
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and the ability to bind to and penetrate the ovum (Cooper and Baker, 1986; Zhang et al., 1990). One approach toward understanding the processes involved in germ cell development and maturation is to identify genes that are expressed in testis and epididymis and to determine their functions. Most genes identi®ed in this manner are likely to be structure determining or metabolic activity-controlling genes. The identi®cation of regulatory proteins is of particular interest, as they underlie the execution of these diverse, yet linked developmental programs. As in other developmental pathways, germ cell differentiation by testis and epididymis-expressed transcription factors is likely to be promoted at the transcription level. One of the major classes of growth and development regulatory proteins consists of the bHLH motif-containing proteins. The promoter sequences from a computerized data search of a number of Sertoli expressed genes indicated the presence of putative E-box elements. These genes included both embryonic genes, such as SRY and MIS, and adult functional genes, such as androgen-binding protein, inhibin/activin, and transferrin (Chaudhary et al., 1997). Evidence of the involvement of bHLH proteins in sex determination and gonadal development in Drosophila (Granadino et al., 1993; Erickson and Cline, 1993) has also provided support for the signi®cance of bHLH factors in spermatogenesis. In the present study we identi®ed a novel mouse gene,
Spz1, which encodes a putative bHLH-Zip transcription factor. This gene is highly expressed in testis and epididymis in male pubertal mice. The encoded protein is located in the nucleus, and can bind to both the E-box and the Gbox. We also found that Spz1 expression is downregulated by testosterone and retinoic acid. These results suggest that this protein plays an important role during spermatogenesis.
2. Results 2.1. Isolation of Spz1 cDNA The initial goal of this study was to isolate the full-length cDNA of a novel mouse testis-speci®c homeobox gene, Tsec. More than 40 candidate clones were isolated from the screening of a mouse testis library (Life Technologies). Eight clones with insert sizes larger than 1.5 kb were further characterized and three identical clones were found encoding a basic helix±loop±helix leucine zipper (bHLH-Zip) motif instead of a homeodomain. The 1549 bp cDNA sequence of this clone was deposited with Genbank (accession number AF271697) (Fig. 1A). The 378 amino acid sequence deduced from the 1137 bp open reading frame (ORF) showed no identity with any known gene products in Genbank, but some stretches of amino acids showed
Fig. 1. (A) The Spz1 full-length cDNA and the encoded amino acid sequence. The bHLH domain is shaded. The Zip domain and polyadenylation signal are underlined individually. (B) Alignment of bHLH domain and (C) leucine zipper domain of Spz1. Identical and similar amino acids are shaded in dark and light colors individually.
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sequence homology with several leucine zipper proteins. Based upon its pattern of expression, this gene was designated Spz1 (spermatogenic Zip). The protein encoded by the Spz1 cDNA has a deduced size of 44 kDa and consists of two major domains: an N-terminal bHLH (amino acids 115193) region and a C-terminal Zip (amino acids 252±287) motif. The bHLH of Spz1 consists of a basic amino acid region and two a -helix secondary structures interrupted by a 30-amino-acid loop (Fig. 1B). The Spz1 bHLH domain represents a new subtype of the bHLH gene family since it has low homology with the bHLH domains of other bHLH gene families (shown in the alignment-guiding tree in Fig. 1B). The Zip motif of Spz1, however, is a typical Zip motif (Fig. 1C). These data indicated that Spz1 is a novel bHLHZip gene.
Fig. 2. Expression of Spz1 in different adult tissues and embryonic stages determined by Northern blot analysis. (A) Spz1 is speci®cally expressed in adult testis. Lanes: 1, brain; 2, heart; 3, kidney; 4, spleen; 5, thymus; 6, liver; 7, stomach; 8, small intestine; 9, muscle; 10, lung; 11, testes; 12, skin. (B) Spz1 is expressed in both adult testis and epididymis. T, testis; E, epididymis. (C) Expression of Spz1 at different stages of testicular development. (D) Expression of Spz1 at different embryonic stages. NB, newborn mice.
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2.2. Expression pattern of Spz1 To characterize the expression pattern of Spz1, adult multiple tissue Northern blot analysis was performed with a full-length Spz1 cDNA probe. A 1.7 kb transcript was detected exclusively in the testis (Fig. 2A). Spz1 expression in mouse testes of different ages was also examined by Northern blot analysis. As shown in Fig. 2C, although hardly any Spz1 mRNA was detected in day-10 testis, the transcription of the Spz1 gene became abruptly apparent at day-20 testis, increased further (day 40), and then decreased in the adult stage (day 60). To understand the expression pattern of Spz1 during embryonic development, total RNA extracted from different stage embryos (embryonic day 9.5± newborn) was subjected to Northern analysis. As shown in Fig. 2D, Spz1 mRNA is uniformly expressed at all time points examined. Both in situ hybridization and immunohistochemistry
Fig. 3. In situ hybridization and immunohistochemical analyses of Spz1 in testis. (A,C,E) are control testes hybridized with sense probe. (B,D,F) are testes hybridized with antisense probe. Signals are indicated by arrows (Sertoli cells), arrowheads (Leydig cells) and asterisks (germ cells). (A± D,G,H) are 40-day testes; (E,F) are 80-day testes. (G) Control testis treated with control serum. (H) Testis treated with anti-Spz1 polyclonal antibody. (C) £100; (E) £200; (D,F,G,H), £400.
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2.3. Subcellular localization of Spz1 protein We performed an immuno¯uorescence assay to study the Spz1 expression pattern in cellular compartments of LFG6 Leydig cells. Nuclear staining by propidium iodine (PI) was evident in cells (Fig. 6A). FITC-conjugated anti-Spz1 antibody staining revealed that the Spz1 protein is expressed in the nuclei of LFG6 cells (Fig. 6B,C). 2.4. DNA binding target sequence of Spz1 To understand Spz1's DNA binding ability and further identify Spz1's downstream target genes, the Spz1 binding sequences were ®rst determined by an electrophoresis mobility shift assay (EMSA) analysis using random oligonucleotides (Wilson et al., 1993). We gel-isolated the shifted bands and eluted the selected oligonucleotides. After PCR ampli®cation, these oligonucleotides were subcloned and sequenced. A 9 bp consensus-binding site, GG(G/ A)GGG(G/A)(A/T)T, was identi®ed from 30 selected sequences (Fig. 7A). The interaction of Spz1 to this target sequence was further con®rmed by interaction between Spz1 and two oligonucleotides, S2 and S3, which contain this 9 bp consensus sequence. The speci®c binding could be competed by a speci®c competitor (Fig. 7B). To determine whether Spz1 can also bind to conventional bHLH domain recognition sites, both E-box and G-box containing oligo-
Fig. 4. Expression of Spz1 in Leydig cell line (LFG6) and Sertoli cell line (SF7). (A) The RT±PCR analysis of Spz1 expression. RLF (relaxin-like factor), Leydig cell marker; FSHR (FSH receptor), Sertoli cell marker. (B) SDS gel and Western blot analyses. Top panel is total protein extracts from LFG6 and SF7 cells, respectively. Bottom panel is the result of Western blot with anti-Spz1 polyclonal antibody. L, LFG6 Leydig cell line; S, SF7 Sertoli cell line.
were used to further characterize the expression of Spz1 in testes. The expression pattern in day-40 and day-80 testes indicated that Spz1 is expressed in both germ cells and somatic cells (Sertoli and Leydig cells) (Fig. 3). The Spz1 expression in Sertoli and Leydig cells was con®rmed by reverse transcription±polymerase chain reaction (RT± PCR) and Western blot on both Sertoli cell line (SF7) and Leydig cell line (LFG6) (Fig. 4). Using whole-mount in situ hybridization, we found that in addition to expression in testis, Spz1 is expressed in the epididymis (Fig. 5A±D). This expression was also conformed with Northern blot analysis (Fig. 2B) and immunohistochemistry performed on the epididymis indicated that Spz1 expression was in the epithelium (Fig. 5F).
Fig. 5. Spz1 expression in epididymis analyzed by whole-mount in situ hybridization and immunohistochemistry. (A) Forty-day epididymis hybridized with sense probe. (B±D) Twenty-, 40-, 60-day epididymis, respectively, hybridized with antisense probe. Signals are indicated by arrows. (E,F) are treated individually with normal serum and anti-Spzl polyclonal antibody (signals are indicated by arrows). (E,F) £100.
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Fig. 6. Sublocalization of Spz1 in the LFG6 cell line. (A) Cell nuclei were stained with propidium iodine (PI) (red). (B) Spz1 was recognized by FITCconjugated anti-Spz1 polyclonal antibody (green). (C) Combination of Spz1 localization and nuclear staining. (A±C) £200.
nucleotides were also used in EMSA analysis with Spz1. Results are shown in Fig. 7C and suggest that Spz1 can bind to both the E-box and G-box and has a much higher af®nity for the E-box. 2.5. Spz1 expression by hormonal induction The fact that Spz1 is highly expressed in testis at day 20 suggests that Spz1 expression might be under hormonal regulation. Thus, the expression of Spz1 in the testis was analyzed after a 4-day i.p. injection with gonadotropin (GT), progesterone (PG), retinoic acid (RA), or testosterone (TT). As shown in Fig. 8, GT and PG had no effect on Spz1 expression. On the other hand, RA and TT both signi®cantly reduced Spz1 expression. 2.6. Mapping of the Spz1 gene The speci®c labeling of Spz1 BAC clone DNA was observed in the distal region of a chromosome, believed to be chromosome 13 on the basis of DAPI staining. After cohybridization with D13MIT158, a marker of the centromeric region of chromosome 13, we found that Spz1 is located at a position 79% of the distance from the heterochromatic±euchromatic boundary to the telomere of chromosome 13, which corresponds to band 13D1 (Fig. 9). A total of 80 metaphase cells were analyzed, with 71 exhibiting speci®c labeling. 3. Discussion In the present study, we isolated and characterized a novel bHLH-Zip gene, Spz1. Members of the bHLH-Zip family are
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important in controlling both cell proliferation and differentiation (Srivastava et al., 1997; Rawls and Olson, 1997; Blackwood and Eisenman, 1991; Atchley and Fitch, 1997). The bHLH and bHLH-Zip motifs in these proteins mediate dimerization, which results in the formation of both heterodimers and homodimers. Recent studies have suggested that bHLH proteins regulate Sertoli cell differentiation functions. For example, transferrin, a marker of differentiation, has an E-box element in its promoter that can be activated by E12, a bHLH factor present in Sertoli cells (Chaudhary et al., 1997). Another example relevant to the current study is the requirement of bHLH proteins in sex determination and gonadal development in Drosophila (Granadino et al., 1993; Erickson and Cline, 1993). These observations provide additional support for our proposed hypothesis that bHLH factors play an important role in spermatogenesis. The expression patterns of Spz1 mRNA and protein suggest that this gene is under both spatial and temporal controls. Spz1 mRNA is expressed in the adult mouse testis and epididymis but not in any other organs examined. The cell types responsible for the strong signal in testis on Northern blots were determined by in situ hybridization and immunohistochemistry on mouse testicular sections. Signals were found in the germ cells, Sertoli cells, and Leydig cells of day 20±60 testes. The expression of Spz1 in Sertoli and Leydig cells was also con®rmed by Western blot analysis of cell extracts isolated from a Sertoli cell line (SF7) and a Leydig cell line (LFG6). These expression patterns suggest that this bHLH-Zip gene may play a role in regulating genes that in¯uence the early spermatogenesis steps. Development of the spermatogenic cell lineage occurs throughout most of the pre- and postnatal lifetime of male mammals, and is regulated by a well-characterized program of gene expression. In general, 15 000±20 000 different transcripts are present in a speci®c cell population (Zhang et al., 1997), and only a few percent of the genes expressed in testis have been identi®ed (Eddy and O'Brien, 1998). Some of the identi®ed genes encode proteins that have essential roles in structures or functions speci®c to spermatogenic cells, e.g. protamine 1 and 2; some are expressed in developmentally regulated patterns, e.g. RXR; and others are transcribed only in spermatogenic cells or produce mRNAs unique to spermatogenic cells. However, the unique gene expression pattern of genes required for spermatogenesis must be tightly regulated by testis-speci®c transcription factors. Several such transcription factors have been found, e.g. A-myb (Toscani et al., 1997), Nhlh2 (Good et al., 1997) and Zfx (Luoh et al., 1997), but the expression patterns of these genes are not restricted to organs responsible only for spermatogenesis. TCFL5 was the ®rst reported bHLH protein expressed speci®cally in human testis (Maruyama et al., 1998). SOX-LZ and HSS (human sperm surface protein) are Zip proteins whose expression is also restricted to testis (Takamatsu et al., 1995; Shankar et al., 1998). KTT4 contains two Zip motifs and is expressed speci®cally in rat testis and epididymis
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Fig. 7. Spz1 consensus binding site. Puri®ed RGS±His±Spz1 fusion protein was used to select binding sites from a mixture of random sequence oligonucleotides. (A) Thirty binding sites are aligned above and the conserved binding sequence is presented below. (B) Gel mobility shift assay of the in vitro binding consensus sequence. The sequences of control oligonucleotide S1 and oligonucleotides containing the binding site (S2 and S3) are shown at the top. The competitor used in lanes 3 and 7 are £250; in lanes 4 and 8 are £500 and in lanes 5 and 9 are £750: (C) Gel mobility shift assay shows the binding activity of Spz1 to traditional bHLH protein binding sites, G-box and E-box. The consensus binding sites in G-box and E-box are underlined. E-box binding activity was competed by £250; £500 and £1000 cold E-box probe.
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Fig. 8. Northern blot analysis of Spz1 expression after hormonal treatment. Fourteen-day-old male mice were treated with different hormones and killed 4 days later. Twenty micrograms of total RNA was loaded in each lane. C, control (saline solution); GT, gonadotropin (12 IU/dose, twice per day); PG, progesterone (0.15 mg/dose, twice per day); RA, retinoic acid (0.2 mg/dose per day); TT, testosterone (0.15 mg/dose, twice per day).
(Turner et al., 1997). However, Spz1 is so far the only bHLH-Zip protein known that is expressed exclusively in spermatogenesis-speci®c adult tissues. The nuclear localization of Spz1 was determined in the LFG6 Leydig cell line, indicating that Spz1 is a nuclear transcription factor that may bind to speci®c sequences through its basic domain to perform the trans-regulation of target genes. However, a nuclear localization signal (NLS) was not identi®ed in the Spz1 sequence using a computer search. The NLS is usually a short stretch of amino acids exposed at the protein surface, and contains several characteristic basic amino acids and prolines (Boulikas, 1993). Other transcription factors without NLS signals have also been found. For example, a deletion analysis of the USF2 transcription factor demonstrated that the basic region of the bHLH domain and a region upstream of this basic region (USR) are involved in nuclear targeting (Luo and Sawadogo, 1996). No basic residues were present within this USR region. Another example of an NLS present in the basic region of the bHLH domain was reported for sterol regulatory element-binding protein 2 (SREBP2) (Nagoshi et al., 1999). These observations indicate that the NLS of Spz1 might be located in the basic region of the bHLH domain or in regions upstream of the basic region where proline and some basic amino acids are present. To identify the target-binding site of Spz1 protein, a random selection and ampli®cation approach was conducted. The 9 bp consensus binding site, 5 0 GG(G/ A)GGG(G/A)(A/T)-3 0 , determined from 30 selected oligonucleotides indicated that Spz1 prefers to bind to asymmetric G-rich sequences. The consensus DNA sequence targeted by several bHLH proteins has been reported to be
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5 0 -CANNTG-3 0 (Blackwell and Weintraub, 1990). This consensus-binding site, known as the E-box, contains two central bases that control the af®nity of binding of the different bHLH factors. The binding ability of Spz1 to the E-box and G-box was also con®rmed by EMSA analysis (Fig. 8C). Among the bHLH gene family, SREBPs recognize an asymmetric target sequence, the sterol regulatory element (SRE; 5 0 -GTGGGGTGAT-3 0 ), as well as the E-box (Parraga et al., 1998). This SRE site is also rich in G residues. In addition, the putative binding site of Drosophila E (sp1), a bHLH protein upstream of the achaete gene, is 5 0 -CGGCACGCGACG-3 0 (Henry site) (Ohsako et al., 1994; Van Doren et al., 1994), another asymmetric GC-rich sequence. Results from our EMSA analysis showed that Spz1 has a higher af®nity to the E-box than to the G-box and the 9 bp Grich sequences. A previous computerized data search indicated that a number of genes expressed in Sertoli cells have E-box elements present in their promoters, such as SRY, MIS, androgen binding protein, transferrin and inhibin/activin (Chaudhary et al., 1997). This observation suggests that
Fig. 9. Mouse Spz1 mapped to the distal end of chromosome 13 by FISH analysis. The arrows represent the speci®c signal of Spz1 on band 13D1. D13MIT158 was used as a control probe, which was speci®c to chromosome 13 centromere (arrowhead).
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bHLH proteins might be involved in Sertoli cell differentiation, which in turn promotes testicular development and male sex determination. In our study, the initial expression of Spz1 in the testis was detected at postnatal day 10, the time when spermatogenesis is induced by follicle stimulating hormone (FSH) and testosterone. The testicular expression level reached the highest point at day 40, and then gradually decreased. These observations indicate that Spz1 might be regulated by hormones during spermatogenesis. The initiation of spermatogenesis is subject to secondary regulation by endocrine cues transmitted indirectly through surrounding somatic cells (Sertoli cells and Leydig cells) and by short-loop paracrine and autocrine signals (Jegou, 1993). Testosterone and FSH are considered to be the primary hormonal initiators and regulators of spermatogenesis. Testosterone is a steroid hormone produced by LH-induced Leydig cells and affects spermatogenesis indirectly through androgen receptors in Sertoli cells and peritubular cells. The nature of the signals from these cells to germ cells is unknown. Treatment of male rats with ethane dimethane sulfonate (EDS) destroys Leydig cells and disrupts spermatogenesis (Orth et al., 1988). FSH is a peptide hormone produced in the anterior lobe of the pituitary gland. It binds to receptors on the cell surfaces of Sertoli cells, but not germ cells, to activate the signal transduction process. FSH has a role in the progression of Sertoli differentiation and can regulate genes through cAMP response elements (Hamil et al., 1994; Hall et al., 1988; Smith and Baenziger, 1988; Foulkes et al., 1992). Suppression of FSH action with antibodies or receptor antagonists causes reduction in testis weight, germ cell numbers, and sperm output in rats (Zirkin, 1998; Sharpe et al., 1994), but these FSH-de®cient rats were still fertile (Kumar et al., 1997). These studies suggest that FSH supports spermatogenesis, but does not have a signi®cant role in regulating gene expression in germ cells. Progesterone, another steroid hormone, regulates LH secretion (Kishi et al., 1999). In addition to hormones, vitamin A is critical for normal spermatogenesis (Huang and Hembree, 1979). In the testes of vitamin A-de®cient mice and rats, most germ cells in later stages of differentiation disappear, and mainly undifferentiated A spermatogonia remain. Spermatogenesis can be restored in these vitamin A-de®cient rats and mice by providing retinol or retinoic acid (Huang et al., 1983; van Pelt and de Rooij, 1991). Retinoid-binding proteins localized in both Sertoli cells and germ cells have been suggested to facilitate retinoid transportation into the nucleus to bind to retinoid receptors and in turn, activate speci®c RNA transcription (Morales and Griswold, 1987). In the present study, using Northern blot analysis, we found that Spz1 expression in pubertal testis was not changed after gonadtropin treatment. On the other hand, retinoid acid and testosterone reduced the expression level of Spz1. Since both retinoic acid and testosterone have been suggested to be involved in the regulation of cellular differentiation during embryonic and organ
development, the downregulation of Spz1 expression by these two hormones implies that Spz1 may play a repressor role in the differentiation process during spermatogenesis. Our results show that Spz1, a novel bHLH-Zip transcription factor, is expressed during spermatogenesis in postnatal mouse testis, and can be downregulated by retinoic acid and testosterone in pubertal testis. Consequently, we suggest that Spz1 might play an important role in regulating mouse spermatogenesis. 4. Experimental procedures 4.1. Cloning of Spz1 full-length cDNA The DNA insert of EST clone AA606869 was radiolabeled and used as a probe to screen a mouse testis cDNA library (Life Technologies) according to the manufacturer's instructions. A total of 10 6 colonies were plated out and lifted onto duplicate membranes for the primary screening. Secondary and tertiary screenings were performed to con®rm the positive clones from the primary screening. The insert sizes of the positive clones were checked by PCR with T7 and T3 primers. Eight clones with insert sizes longer than 1.5 kb were sequenced. 4.2. Sequence analysis The MacVector program (Kodak) was used for both the DNA and amino acid sequence analyses, which included predictions of the ORF and functional domains, sequence alignments, and a GenBank search. 4.3. RNA isolation and Northern blot analysis The membrane for multiple tissue Northern blot analysis with 2 mg poly(A) 1 RNA from 12 adult mouse tissues in each lane was purchased from OriGene. Total RNA was extracted with Trizol reagent (Life Technologies) from different stages of testes and mouse embryos. Twenty micrograms each of total RNA were loaded and size-fractionated on a 1% agarose/formaldehyde gel and transferred to nylon membranes (Amersham Pharmacia Biotech). Blots were hybridized to the radiolabeled Spz1 cDNA probe (bp 1±1100) and subsequently hybridized with a GADPH control probe to ensure integrity and equal loading of the RNA samples. 4.4. RT±PCR analysis One microgram of total RNA isolated from LFG6 and SF7 cell lines was used in reverse transcription with Superscript II system (Life Technologies). The Spz1, Relaxin-like factor (RLF) and Follicle-stimulating hormone receptor (FSHR) were ampli®ed by using the following primers: Spz1a: 5 0 -GGGGAGCCTTTTGATGATGC-3 0 ; Spz1b: 5 0 CGCTTCGTGGAGATGGAAAT-3 0 ; RLF1: 5 0 -TGCTACTGATGCTCCTGGCT-3 0 ; RLF2: 5 0 -TCAGTGGGGA-
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CACAGACCCA-3 0 : FSHR1: 5 0 -ATGGCCTTGCTCCTGGTCTC-3 0 ; FSHR2: 5 0 -GAGGCTCCCTGGAAAACATC-3 0 . 4.5. In situ hybridization The Spz1 cDNA fragment (bps 478-785) was PCR-ampli®ed and subcloned into pGEM-T vector (Promega). Sense and antisense riboprobes were prepared by in vitro transcription using T7 and SP6 polymerase, respectively, with digoxigenin-UTP (Boehringer Mannheim). Whole-mount in situ hybridization of testis and epididymis was performed as previously described (Nieto et al., 1992). The in situ hybridization of testis and epididymis cryosections were performed using standard protocols. 4.6. Immunohistochemistry Immunohistochemistry was performed according to the method of Gupta and Aikat (1970). In brief, mouse testes and epididymis were removed and ®xed in 4% paraformaldehyde for 1 h, then embedded in OTC. Tissues were sectioned 6 mm thick onto slides and re®xed with 4% paraformaldehyde for 20 min. Slides were rinsed with phosphatebuffered saline (PBS) and then incubated in 3.5% H2O2 in PBS for 20 min to block endogenous peroxidase. Slides were then incubated with 5% skimmed milk in PBS for 2 h to reduce non-speci®c background. The slides were incubated with anti-Spz1 polyclonal antibody (1:10 000) in PBST (PBS 1 0.2% Tween-20) for 1 h. After washing with PBST for 30 min, slides were incubated with alkaline phosphatase-conjugated anti-rabbit polyclonal antibody (1:8000). Normal rabbit serum was used in the negative control group experiment. Slides were ®nally counterstained with 0.5% methyl green. 4.7. Antibody and Western blot Spz1 cDNA (bp 51±1120) was subcloned into the pQE30 expression vector (Qiagen) to produce His6±Spz1 fusion protein. Spz1 fusion protein expression was induced for 6 h by 1 mM IPTG in E. coli strain SR13009. Cells were lysed with denatured buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris±HCl, pH 8.0). After centrifugation, the fusion protein was puri®ed from a Ni 1-NTA±agarose column (Qiagen) according to the manufacturer's instructions. Anti-Spz1 antibody was raised in New Zealand rabbits according to the protocol described by el-Roeiy et al. (1988). Sodium dodecyl sulfate±polyacrylamide gel electrophoresis (SDS± PAGE) was performed using the buffer system described by Laemmli (1970). Samples were boiled for 5 min in an equal volume of sample buffer containing 2% SDS, 5% 2-mercaptoethanol and electrophoresed on an 8% acrylamide gel. After proteins were transferred onto PVDF membranes (Hoefer, Pharmacia Biotech), the membranes were blocked at room temperature for 1 h in 5% skimmed milk (in PBST) and washed for 40 min with PBST. The membranes were
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then incubated with alkaline phosphatase-conjugated antirabbit antibody for 1 h, and washed with PBST. The membranes were developed using an ECL system (Amersham Pharmacia Biotech) on X-ray ®lm (Kodak). 4.8. Immuno¯uorescence analysis The expression sites of Spz1 were determined by immuno¯uorescence as described by Andersson et al. (1986). LFG6 cells were cultured on coverslides precoated with poly-l-lysine for 16 h. After washing with cold PBS, cells were ®xed with 2% paraformaldehyde in PBS for 30 min on ice, and then the slides were washed with PBS for 30 min. Cells were permeabilized with buffer containing 0.2% Triton X-100 and 1% NGS in PBS for 20 min on ice, and then the slides were washed with PBS to remove excessive detergent. Cells were then incubated in anti-Spz1 polyclonal antibody (1:10 000) for 1 h at room temperature. After washing cells with PBS for 30 min, FITC-conjugated human anti-rabbit polyclonal antibody (1:8000) was then added and incubated for another 1 h at room temperature in a humid chamber. Cells were ®nally counterstained with propidium iodine (PI) to localize the nucleus. 4.9. Immunoprecipitation To isolate native Spz1 protein, LFG6 cells were lysed and Spz1 protein was puri®ed by immunoprecipitation. The method was modi®ed according to Erikson et al. (1977). Cells were cultured in DMEM containing 15% FBS and lysed with detergent solution (1% NP-40, 0.4% DOC, 10 mM EDTA and 10 mM Tris±HCl, pH7.4) on ice for 30 min. After centrifugation, the supernatant was diluted with 4 volumes of PBS, precleared with preimmune rabbit serum for 1 h at 48C, and then mixed with protein A Sepharoseconjugated beads. After centrifugation, the Spz1 protein was precipitated by anti-Spz1 polyclonal antibody and protein A. The Spz1 protein was then eluted by resuspending the protein A-Sepharose beads in lysis buffer. 4.10. In vitro target binding sequence An in vitro binding reaction and PCR ampli®cation were used to identify the Spz1 DNA binding sequences. In vitro binding reaction was performed using a 74-mer doublestrand oligonucleotide (5 0 -ACTAGTCTCGAGGGATCCGATATCNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAGAAAGCTTGTCGACCTGCGG-3 0 ) as probe in which 26 random nucleotides were ¯anked by two known 24-mer sequences. One microgram of native Spz1 protein puri®ed from immunoprecipitation was incubated with 5000 cpm of 32P-labeled probe for 30 min at room temperature. The DNA±protein mixtures were separated on 4% Tris± glycine gels (Buratowski et al., 1989) and then autoradiographed using X-ray ®lm (Kodak). The DNA±Spz1 associated complex was cut from the gel and diluted by resuspending in 100 ml of H2O. Using the two ¯anking
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24-mer sequences as primers, the oligonucleotides that Spz1 protein bound were ampli®ed by PCR and subcloned into pGEM T-Easy vector (Promega) and sequenced. The consensus DNA sequence was determined by alignment of 30 sequences. 4.11. Electrophoresis mobility shift assay (EMSA) The DNA binding reaction was performed in buffer containing 10 mM Tris±HCl, 1 mM EDTA, 100 mM NaCl, 2 mM dithiothreitol (DTT) and 10% glycerol. The 24-mer double-stranded oligonucleotides containing an Ebox (CANNTG) or G-box (CCACGTGG) were used as probes. One microgram of Spz1 protein was incubated with 5000 cpm 32P-labeled oligonucleotides, 2 mg poly(dIdC), and BSA (1 mg/ml) for 30 min at room temperature. The DNA±protein mixtures were separated on a 6% polyacrylamide gel (30:1 bis acrylamide in 0.5£ TBE). 4.12. Hormonal treatment Total RNA was isolated from 18-day-old mouse testis after i.p. injection with gonadotropin (12 IU/10 g body wt., twice per day), progesterone (0.15 mg/10 g body wt., twice per day), retinoid acid (0.2 mg/10 g body wt.), or testosterone (0.15 mg/10 g body wt., twice per day) for 4 days. Twenty micrograms of total RNA was loaded in each lane, and the membrane was hybridized with a full-length Spz1 cDNA probe. 4.13. BAC clone isolation and Spz1 chromosomal localization A Spz1 cDNA fragment (bp 204±1338) was used as a probe to screen a mouse BAC library (Genome System). Three positive clones were isolated, one of which was used as probe for ¯uorescence in situ hybridization (FISH) to identify the chromosomal localization of Spz1. FISH was performed using a previously published procedure (Shi et al., 1997). Acknowledgements We would like to thank Drs W.W. Branford, S.S. Potter and K.B. Choo for their critical reading of the manuscript. This work was supported in part by a research grant NSC89-2311-B-001-214 from the National Science Council, Taiwan. References Andersson, I., Billig, H., Fryklund, L., Hansson, H.A., Isaksson, O., Isgaard, J., Nilsson, A., Rozell, B., Skottner, A., Stemme, S., 1986. Localization of IGF-I in adult rats. Immunohistochemical studies. Acta Physiol. Scand. 126, 311±312. Arnold, H.H., Winter, B., 1998. Muscle differentiation: more complexity to
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Spz1, a novel bHLH-Zip protein, is speci®cally expressed in testis Shih-Hsien Hsu a,b, Huey-Wen Shyu a,b, Hsiu-Mei Hsieh-Li c, Hung Li a,b,* a
b
Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan Institute of Life Science, National Defense Medical Center, Taipei, Taiwan c Institute of Medical Science, Taipei Medical College, Taipei, Taiwan
Received 7 August 2000; received in revised form 16 October 2000; accepted 16 October 2000
Abstract We isolated a novel bHLH-Zip gene designated Spz1 from a mouse testis cDNA library. Spz1 is expressed speci®cally in the testis and epididymis. Immuno¯uorescence staining detected Spz1 protein in the nuclei of LFG6 Leydig cells. The ability of Spz1 protein to bind to the bHLH consensus-binding site, the E-box, was con®rmed by EMSA, and a 9-bp asymmetric target site was identi®ed by random selection and PCR ampli®cation. Hormonal regulation of Spz1 was investigated and downregulation of Spz1 expression by testosterone and retinoic acid was found. This nuclear transcription factor may play a crucial role in spermatogenesis by regulating cell proliferation or differentiation through binding to speci®c DNA sequences like other bHLH-Zip molecules. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Basic helix±loop±helix; Leucine zipper; Mouse; Spermatogenesis; Testis
1. Introduction The basic helix±loop±helix (bHLH) family of transcription factors comprises important components in the regulatory networks of many developmental pathways (Srivastava et al., 1997; Rawls and Olson, 1997; Murre et al., 1994; Feder et al., 1993). This family is characterized by a conserved structural motif consisting of two amphipathic helixes separated by a loop, the HLH domain, which mediates protein interactions to form homo- and heterodimers (Murre et al., 1994). There are two major categories of bHLH proteins. Class A consists of ubiquitously expressed proteins such as those encoded by differentially spliced transcripts of the E2A (E12, E47, and E2-5/ITF1), E2-2/ITF2, and HEB/HTF4 genes (Goldfarb et al., 1996; Watada et al., 1995; Whyte et al., 1988; Glick et al., 2000). Class B consists of tissue-speci®c bHLH proteins such as MyoD, NeuroD, MASH, and TAL (Arnold and Winter, 1998; Guillemot, 1999; Lo et al., 1994; Xia and Hwang, 1994). These proteins form heterodimers consisting of ubiquitous class A and tissue-speci®c class B bHLH proteins. The highlycharged basic region in each of the dimerization partners immediately upstream of the HLH region forms a speci®c DNA-binding domain that can recognize a DNA sequence known as an E-box (CANNTG), N-box (CACNAG) * Corresponding author. Tel.: 1886-2-2788-0460; fax: 1886-2-27826085. E-mail address: [email protected] (H. Li).
(Oellers et al., 1994; Tietze et al., 1992) or G-box (de Pater et al., 1997). Some members of the bHLH family also contain a leucine zipper (Zip) motif that occurs immediately C-terminal to the bHLH motif; these are the bHLHZip proteins (Blackwood and Eisenman, 1991; FerreD'Amare et al., 1993). These Zip motifs are involved in protein dimerization enhancement. Some dimers result in activation of gene expression while others result in repression. These bHLH-Zip proteins have been implicated in various developmental and cellular processes, and some are known to be oncogenes (Murre et al., 1989; Blackwood and Eisenman, 1991; Atchley and Fitch, 1997). Spermatogenesis is a complex process that leads to the formation of male gametes. Spermatogenesis takes about 40 days in rats and about 63 days in humans. During this period, type-A spermatogonia, originally derived from primordial germ cells (PGC), either cycle as pluripotent stem cells or further differentiate into type-B spermatogonia that give rise to primary spermatocytes. Two meiotic divisions of spermatocytes yield haploid spermatids that undergo morphological and functional differentiation to produce highly specialized spermatozoa (Dym, 1994). Spermatozoa then complete the differentiation process in the epididymis. The epididymis is not only a sojourn for spermatozoa which might last several weeks, but it also promotes the development of the spermatozoa's fertilizing capacity, culminating in their acquisition of full mobility
0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00513-X
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and the ability to bind to and penetrate the ovum (Cooper and Baker, 1986; Zhang et al., 1990). One approach toward understanding the processes involved in germ cell development and maturation is to identify genes that are expressed in testis and epididymis and to determine their functions. Most genes identi®ed in this manner are likely to be structure determining or metabolic activity-controlling genes. The identi®cation of regulatory proteins is of particular interest, as they underlie the execution of these diverse, yet linked developmental programs. As in other developmental pathways, germ cell differentiation by testis and epididymis-expressed transcription factors is likely to be promoted at the transcription level. One of the major classes of growth and development regulatory proteins consists of the bHLH motif-containing proteins. The promoter sequences from a computerized data search of a number of Sertoli expressed genes indicated the presence of putative E-box elements. These genes included both embryonic genes, such as SRY and MIS, and adult functional genes, such as androgen-binding protein, inhibin/activin, and transferrin (Chaudhary et al., 1997). Evidence of the involvement of bHLH proteins in sex determination and gonadal development in Drosophila (Granadino et al., 1993; Erickson and Cline, 1993) has also provided support for the signi®cance of bHLH factors in spermatogenesis. In the present study we identi®ed a novel mouse gene,
Spz1, which encodes a putative bHLH-Zip transcription factor. This gene is highly expressed in testis and epididymis in male pubertal mice. The encoded protein is located in the nucleus, and can bind to both the E-box and the Gbox. We also found that Spz1 expression is downregulated by testosterone and retinoic acid. These results suggest that this protein plays an important role during spermatogenesis.
2. Results 2.1. Isolation of Spz1 cDNA The initial goal of this study was to isolate the full-length cDNA of a novel mouse testis-speci®c homeobox gene, Tsec. More than 40 candidate clones were isolated from the screening of a mouse testis library (Life Technologies). Eight clones with insert sizes larger than 1.5 kb were further characterized and three identical clones were found encoding a basic helix±loop±helix leucine zipper (bHLH-Zip) motif instead of a homeodomain. The 1549 bp cDNA sequence of this clone was deposited with Genbank (accession number AF271697) (Fig. 1A). The 378 amino acid sequence deduced from the 1137 bp open reading frame (ORF) showed no identity with any known gene products in Genbank, but some stretches of amino acids showed
Fig. 1. (A) The Spz1 full-length cDNA and the encoded amino acid sequence. The bHLH domain is shaded. The Zip domain and polyadenylation signal are underlined individually. (B) Alignment of bHLH domain and (C) leucine zipper domain of Spz1. Identical and similar amino acids are shaded in dark and light colors individually.
S.-H. Hsu et al. / Mechanisms of Development 100 (2001) 177±187
sequence homology with several leucine zipper proteins. Based upon its pattern of expression, this gene was designated Spz1 (spermatogenic Zip). The protein encoded by the Spz1 cDNA has a deduced size of 44 kDa and consists of two major domains: an N-terminal bHLH (amino acids 115193) region and a C-terminal Zip (amino acids 252±287) motif. The bHLH of Spz1 consists of a basic amino acid region and two a -helix secondary structures interrupted by a 30-amino-acid loop (Fig. 1B). The Spz1 bHLH domain represents a new subtype of the bHLH gene family since it has low homology with the bHLH domains of other bHLH gene families (shown in the alignment-guiding tree in Fig. 1B). The Zip motif of Spz1, however, is a typical Zip motif (Fig. 1C). These data indicated that Spz1 is a novel bHLHZip gene.
Fig. 2. Expression of Spz1 in different adult tissues and embryonic stages determined by Northern blot analysis. (A) Spz1 is speci®cally expressed in adult testis. Lanes: 1, brain; 2, heart; 3, kidney; 4, spleen; 5, thymus; 6, liver; 7, stomach; 8, small intestine; 9, muscle; 10, lung; 11, testes; 12, skin. (B) Spz1 is expressed in both adult testis and epididymis. T, testis; E, epididymis. (C) Expression of Spz1 at different stages of testicular development. (D) Expression of Spz1 at different embryonic stages. NB, newborn mice.
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2.2. Expression pattern of Spz1 To characterize the expression pattern of Spz1, adult multiple tissue Northern blot analysis was performed with a full-length Spz1 cDNA probe. A 1.7 kb transcript was detected exclusively in the testis (Fig. 2A). Spz1 expression in mouse testes of different ages was also examined by Northern blot analysis. As shown in Fig. 2C, although hardly any Spz1 mRNA was detected in day-10 testis, the transcription of the Spz1 gene became abruptly apparent at day-20 testis, increased further (day 40), and then decreased in the adult stage (day 60). To understand the expression pattern of Spz1 during embryonic development, total RNA extracted from different stage embryos (embryonic day 9.5± newborn) was subjected to Northern analysis. As shown in Fig. 2D, Spz1 mRNA is uniformly expressed at all time points examined. Both in situ hybridization and immunohistochemistry
Fig. 3. In situ hybridization and immunohistochemical analyses of Spz1 in testis. (A,C,E) are control testes hybridized with sense probe. (B,D,F) are testes hybridized with antisense probe. Signals are indicated by arrows (Sertoli cells), arrowheads (Leydig cells) and asterisks (germ cells). (A± D,G,H) are 40-day testes; (E,F) are 80-day testes. (G) Control testis treated with control serum. (H) Testis treated with anti-Spz1 polyclonal antibody. (C) £100; (E) £200; (D,F,G,H), £400.
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2.3. Subcellular localization of Spz1 protein We performed an immuno¯uorescence assay to study the Spz1 expression pattern in cellular compartments of LFG6 Leydig cells. Nuclear staining by propidium iodine (PI) was evident in cells (Fig. 6A). FITC-conjugated anti-Spz1 antibody staining revealed that the Spz1 protein is expressed in the nuclei of LFG6 cells (Fig. 6B,C). 2.4. DNA binding target sequence of Spz1 To understand Spz1's DNA binding ability and further identify Spz1's downstream target genes, the Spz1 binding sequences were ®rst determined by an electrophoresis mobility shift assay (EMSA) analysis using random oligonucleotides (Wilson et al., 1993). We gel-isolated the shifted bands and eluted the selected oligonucleotides. After PCR ampli®cation, these oligonucleotides were subcloned and sequenced. A 9 bp consensus-binding site, GG(G/ A)GGG(G/A)(A/T)T, was identi®ed from 30 selected sequences (Fig. 7A). The interaction of Spz1 to this target sequence was further con®rmed by interaction between Spz1 and two oligonucleotides, S2 and S3, which contain this 9 bp consensus sequence. The speci®c binding could be competed by a speci®c competitor (Fig. 7B). To determine whether Spz1 can also bind to conventional bHLH domain recognition sites, both E-box and G-box containing oligo-
Fig. 4. Expression of Spz1 in Leydig cell line (LFG6) and Sertoli cell line (SF7). (A) The RT±PCR analysis of Spz1 expression. RLF (relaxin-like factor), Leydig cell marker; FSHR (FSH receptor), Sertoli cell marker. (B) SDS gel and Western blot analyses. Top panel is total protein extracts from LFG6 and SF7 cells, respectively. Bottom panel is the result of Western blot with anti-Spz1 polyclonal antibody. L, LFG6 Leydig cell line; S, SF7 Sertoli cell line.
were used to further characterize the expression of Spz1 in testes. The expression pattern in day-40 and day-80 testes indicated that Spz1 is expressed in both germ cells and somatic cells (Sertoli and Leydig cells) (Fig. 3). The Spz1 expression in Sertoli and Leydig cells was con®rmed by reverse transcription±polymerase chain reaction (RT± PCR) and Western blot on both Sertoli cell line (SF7) and Leydig cell line (LFG6) (Fig. 4). Using whole-mount in situ hybridization, we found that in addition to expression in testis, Spz1 is expressed in the epididymis (Fig. 5A±D). This expression was also conformed with Northern blot analysis (Fig. 2B) and immunohistochemistry performed on the epididymis indicated that Spz1 expression was in the epithelium (Fig. 5F).
Fig. 5. Spz1 expression in epididymis analyzed by whole-mount in situ hybridization and immunohistochemistry. (A) Forty-day epididymis hybridized with sense probe. (B±D) Twenty-, 40-, 60-day epididymis, respectively, hybridized with antisense probe. Signals are indicated by arrows. (E,F) are treated individually with normal serum and anti-Spzl polyclonal antibody (signals are indicated by arrows). (E,F) £100.
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Fig. 6. Sublocalization of Spz1 in the LFG6 cell line. (A) Cell nuclei were stained with propidium iodine (PI) (red). (B) Spz1 was recognized by FITCconjugated anti-Spz1 polyclonal antibody (green). (C) Combination of Spz1 localization and nuclear staining. (A±C) £200.
nucleotides were also used in EMSA analysis with Spz1. Results are shown in Fig. 7C and suggest that Spz1 can bind to both the E-box and G-box and has a much higher af®nity for the E-box. 2.5. Spz1 expression by hormonal induction The fact that Spz1 is highly expressed in testis at day 20 suggests that Spz1 expression might be under hormonal regulation. Thus, the expression of Spz1 in the testis was analyzed after a 4-day i.p. injection with gonadotropin (GT), progesterone (PG), retinoic acid (RA), or testosterone (TT). As shown in Fig. 8, GT and PG had no effect on Spz1 expression. On the other hand, RA and TT both signi®cantly reduced Spz1 expression. 2.6. Mapping of the Spz1 gene The speci®c labeling of Spz1 BAC clone DNA was observed in the distal region of a chromosome, believed to be chromosome 13 on the basis of DAPI staining. After cohybridization with D13MIT158, a marker of the centromeric region of chromosome 13, we found that Spz1 is located at a position 79% of the distance from the heterochromatic±euchromatic boundary to the telomere of chromosome 13, which corresponds to band 13D1 (Fig. 9). A total of 80 metaphase cells were analyzed, with 71 exhibiting speci®c labeling. 3. Discussion In the present study, we isolated and characterized a novel bHLH-Zip gene, Spz1. Members of the bHLH-Zip family are
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important in controlling both cell proliferation and differentiation (Srivastava et al., 1997; Rawls and Olson, 1997; Blackwood and Eisenman, 1991; Atchley and Fitch, 1997). The bHLH and bHLH-Zip motifs in these proteins mediate dimerization, which results in the formation of both heterodimers and homodimers. Recent studies have suggested that bHLH proteins regulate Sertoli cell differentiation functions. For example, transferrin, a marker of differentiation, has an E-box element in its promoter that can be activated by E12, a bHLH factor present in Sertoli cells (Chaudhary et al., 1997). Another example relevant to the current study is the requirement of bHLH proteins in sex determination and gonadal development in Drosophila (Granadino et al., 1993; Erickson and Cline, 1993). These observations provide additional support for our proposed hypothesis that bHLH factors play an important role in spermatogenesis. The expression patterns of Spz1 mRNA and protein suggest that this gene is under both spatial and temporal controls. Spz1 mRNA is expressed in the adult mouse testis and epididymis but not in any other organs examined. The cell types responsible for the strong signal in testis on Northern blots were determined by in situ hybridization and immunohistochemistry on mouse testicular sections. Signals were found in the germ cells, Sertoli cells, and Leydig cells of day 20±60 testes. The expression of Spz1 in Sertoli and Leydig cells was also con®rmed by Western blot analysis of cell extracts isolated from a Sertoli cell line (SF7) and a Leydig cell line (LFG6). These expression patterns suggest that this bHLH-Zip gene may play a role in regulating genes that in¯uence the early spermatogenesis steps. Development of the spermatogenic cell lineage occurs throughout most of the pre- and postnatal lifetime of male mammals, and is regulated by a well-characterized program of gene expression. In general, 15 000±20 000 different transcripts are present in a speci®c cell population (Zhang et al., 1997), and only a few percent of the genes expressed in testis have been identi®ed (Eddy and O'Brien, 1998). Some of the identi®ed genes encode proteins that have essential roles in structures or functions speci®c to spermatogenic cells, e.g. protamine 1 and 2; some are expressed in developmentally regulated patterns, e.g. RXR; and others are transcribed only in spermatogenic cells or produce mRNAs unique to spermatogenic cells. However, the unique gene expression pattern of genes required for spermatogenesis must be tightly regulated by testis-speci®c transcription factors. Several such transcription factors have been found, e.g. A-myb (Toscani et al., 1997), Nhlh2 (Good et al., 1997) and Zfx (Luoh et al., 1997), but the expression patterns of these genes are not restricted to organs responsible only for spermatogenesis. TCFL5 was the ®rst reported bHLH protein expressed speci®cally in human testis (Maruyama et al., 1998). SOX-LZ and HSS (human sperm surface protein) are Zip proteins whose expression is also restricted to testis (Takamatsu et al., 1995; Shankar et al., 1998). KTT4 contains two Zip motifs and is expressed speci®cally in rat testis and epididymis
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Fig. 7. Spz1 consensus binding site. Puri®ed RGS±His±Spz1 fusion protein was used to select binding sites from a mixture of random sequence oligonucleotides. (A) Thirty binding sites are aligned above and the conserved binding sequence is presented below. (B) Gel mobility shift assay of the in vitro binding consensus sequence. The sequences of control oligonucleotide S1 and oligonucleotides containing the binding site (S2 and S3) are shown at the top. The competitor used in lanes 3 and 7 are £250; in lanes 4 and 8 are £500 and in lanes 5 and 9 are £750: (C) Gel mobility shift assay shows the binding activity of Spz1 to traditional bHLH protein binding sites, G-box and E-box. The consensus binding sites in G-box and E-box are underlined. E-box binding activity was competed by £250; £500 and £1000 cold E-box probe.
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Fig. 8. Northern blot analysis of Spz1 expression after hormonal treatment. Fourteen-day-old male mice were treated with different hormones and killed 4 days later. Twenty micrograms of total RNA was loaded in each lane. C, control (saline solution); GT, gonadotropin (12 IU/dose, twice per day); PG, progesterone (0.15 mg/dose, twice per day); RA, retinoic acid (0.2 mg/dose per day); TT, testosterone (0.15 mg/dose, twice per day).
(Turner et al., 1997). However, Spz1 is so far the only bHLH-Zip protein known that is expressed exclusively in spermatogenesis-speci®c adult tissues. The nuclear localization of Spz1 was determined in the LFG6 Leydig cell line, indicating that Spz1 is a nuclear transcription factor that may bind to speci®c sequences through its basic domain to perform the trans-regulation of target genes. However, a nuclear localization signal (NLS) was not identi®ed in the Spz1 sequence using a computer search. The NLS is usually a short stretch of amino acids exposed at the protein surface, and contains several characteristic basic amino acids and prolines (Boulikas, 1993). Other transcription factors without NLS signals have also been found. For example, a deletion analysis of the USF2 transcription factor demonstrated that the basic region of the bHLH domain and a region upstream of this basic region (USR) are involved in nuclear targeting (Luo and Sawadogo, 1996). No basic residues were present within this USR region. Another example of an NLS present in the basic region of the bHLH domain was reported for sterol regulatory element-binding protein 2 (SREBP2) (Nagoshi et al., 1999). These observations indicate that the NLS of Spz1 might be located in the basic region of the bHLH domain or in regions upstream of the basic region where proline and some basic amino acids are present. To identify the target-binding site of Spz1 protein, a random selection and ampli®cation approach was conducted. The 9 bp consensus binding site, 5 0 GG(G/ A)GGG(G/A)(A/T)-3 0 , determined from 30 selected oligonucleotides indicated that Spz1 prefers to bind to asymmetric G-rich sequences. The consensus DNA sequence targeted by several bHLH proteins has been reported to be
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5 0 -CANNTG-3 0 (Blackwell and Weintraub, 1990). This consensus-binding site, known as the E-box, contains two central bases that control the af®nity of binding of the different bHLH factors. The binding ability of Spz1 to the E-box and G-box was also con®rmed by EMSA analysis (Fig. 8C). Among the bHLH gene family, SREBPs recognize an asymmetric target sequence, the sterol regulatory element (SRE; 5 0 -GTGGGGTGAT-3 0 ), as well as the E-box (Parraga et al., 1998). This SRE site is also rich in G residues. In addition, the putative binding site of Drosophila E (sp1), a bHLH protein upstream of the achaete gene, is 5 0 -CGGCACGCGACG-3 0 (Henry site) (Ohsako et al., 1994; Van Doren et al., 1994), another asymmetric GC-rich sequence. Results from our EMSA analysis showed that Spz1 has a higher af®nity to the E-box than to the G-box and the 9 bp Grich sequences. A previous computerized data search indicated that a number of genes expressed in Sertoli cells have E-box elements present in their promoters, such as SRY, MIS, androgen binding protein, transferrin and inhibin/activin (Chaudhary et al., 1997). This observation suggests that
Fig. 9. Mouse Spz1 mapped to the distal end of chromosome 13 by FISH analysis. The arrows represent the speci®c signal of Spz1 on band 13D1. D13MIT158 was used as a control probe, which was speci®c to chromosome 13 centromere (arrowhead).
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bHLH proteins might be involved in Sertoli cell differentiation, which in turn promotes testicular development and male sex determination. In our study, the initial expression of Spz1 in the testis was detected at postnatal day 10, the time when spermatogenesis is induced by follicle stimulating hormone (FSH) and testosterone. The testicular expression level reached the highest point at day 40, and then gradually decreased. These observations indicate that Spz1 might be regulated by hormones during spermatogenesis. The initiation of spermatogenesis is subject to secondary regulation by endocrine cues transmitted indirectly through surrounding somatic cells (Sertoli cells and Leydig cells) and by short-loop paracrine and autocrine signals (Jegou, 1993). Testosterone and FSH are considered to be the primary hormonal initiators and regulators of spermatogenesis. Testosterone is a steroid hormone produced by LH-induced Leydig cells and affects spermatogenesis indirectly through androgen receptors in Sertoli cells and peritubular cells. The nature of the signals from these cells to germ cells is unknown. Treatment of male rats with ethane dimethane sulfonate (EDS) destroys Leydig cells and disrupts spermatogenesis (Orth et al., 1988). FSH is a peptide hormone produced in the anterior lobe of the pituitary gland. It binds to receptors on the cell surfaces of Sertoli cells, but not germ cells, to activate the signal transduction process. FSH has a role in the progression of Sertoli differentiation and can regulate genes through cAMP response elements (Hamil et al., 1994; Hall et al., 1988; Smith and Baenziger, 1988; Foulkes et al., 1992). Suppression of FSH action with antibodies or receptor antagonists causes reduction in testis weight, germ cell numbers, and sperm output in rats (Zirkin, 1998; Sharpe et al., 1994), but these FSH-de®cient rats were still fertile (Kumar et al., 1997). These studies suggest that FSH supports spermatogenesis, but does not have a signi®cant role in regulating gene expression in germ cells. Progesterone, another steroid hormone, regulates LH secretion (Kishi et al., 1999). In addition to hormones, vitamin A is critical for normal spermatogenesis (Huang and Hembree, 1979). In the testes of vitamin A-de®cient mice and rats, most germ cells in later stages of differentiation disappear, and mainly undifferentiated A spermatogonia remain. Spermatogenesis can be restored in these vitamin A-de®cient rats and mice by providing retinol or retinoic acid (Huang et al., 1983; van Pelt and de Rooij, 1991). Retinoid-binding proteins localized in both Sertoli cells and germ cells have been suggested to facilitate retinoid transportation into the nucleus to bind to retinoid receptors and in turn, activate speci®c RNA transcription (Morales and Griswold, 1987). In the present study, using Northern blot analysis, we found that Spz1 expression in pubertal testis was not changed after gonadtropin treatment. On the other hand, retinoid acid and testosterone reduced the expression level of Spz1. Since both retinoic acid and testosterone have been suggested to be involved in the regulation of cellular differentiation during embryonic and organ
development, the downregulation of Spz1 expression by these two hormones implies that Spz1 may play a repressor role in the differentiation process during spermatogenesis. Our results show that Spz1, a novel bHLH-Zip transcription factor, is expressed during spermatogenesis in postnatal mouse testis, and can be downregulated by retinoic acid and testosterone in pubertal testis. Consequently, we suggest that Spz1 might play an important role in regulating mouse spermatogenesis. 4. Experimental procedures 4.1. Cloning of Spz1 full-length cDNA The DNA insert of EST clone AA606869 was radiolabeled and used as a probe to screen a mouse testis cDNA library (Life Technologies) according to the manufacturer's instructions. A total of 10 6 colonies were plated out and lifted onto duplicate membranes for the primary screening. Secondary and tertiary screenings were performed to con®rm the positive clones from the primary screening. The insert sizes of the positive clones were checked by PCR with T7 and T3 primers. Eight clones with insert sizes longer than 1.5 kb were sequenced. 4.2. Sequence analysis The MacVector program (Kodak) was used for both the DNA and amino acid sequence analyses, which included predictions of the ORF and functional domains, sequence alignments, and a GenBank search. 4.3. RNA isolation and Northern blot analysis The membrane for multiple tissue Northern blot analysis with 2 mg poly(A) 1 RNA from 12 adult mouse tissues in each lane was purchased from OriGene. Total RNA was extracted with Trizol reagent (Life Technologies) from different stages of testes and mouse embryos. Twenty micrograms each of total RNA were loaded and size-fractionated on a 1% agarose/formaldehyde gel and transferred to nylon membranes (Amersham Pharmacia Biotech). Blots were hybridized to the radiolabeled Spz1 cDNA probe (bp 1±1100) and subsequently hybridized with a GADPH control probe to ensure integrity and equal loading of the RNA samples. 4.4. RT±PCR analysis One microgram of total RNA isolated from LFG6 and SF7 cell lines was used in reverse transcription with Superscript II system (Life Technologies). The Spz1, Relaxin-like factor (RLF) and Follicle-stimulating hormone receptor (FSHR) were ampli®ed by using the following primers: Spz1a: 5 0 -GGGGAGCCTTTTGATGATGC-3 0 ; Spz1b: 5 0 CGCTTCGTGGAGATGGAAAT-3 0 ; RLF1: 5 0 -TGCTACTGATGCTCCTGGCT-3 0 ; RLF2: 5 0 -TCAGTGGGGA-
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CACAGACCCA-3 0 : FSHR1: 5 0 -ATGGCCTTGCTCCTGGTCTC-3 0 ; FSHR2: 5 0 -GAGGCTCCCTGGAAAACATC-3 0 . 4.5. In situ hybridization The Spz1 cDNA fragment (bps 478-785) was PCR-ampli®ed and subcloned into pGEM-T vector (Promega). Sense and antisense riboprobes were prepared by in vitro transcription using T7 and SP6 polymerase, respectively, with digoxigenin-UTP (Boehringer Mannheim). Whole-mount in situ hybridization of testis and epididymis was performed as previously described (Nieto et al., 1992). The in situ hybridization of testis and epididymis cryosections were performed using standard protocols. 4.6. Immunohistochemistry Immunohistochemistry was performed according to the method of Gupta and Aikat (1970). In brief, mouse testes and epididymis were removed and ®xed in 4% paraformaldehyde for 1 h, then embedded in OTC. Tissues were sectioned 6 mm thick onto slides and re®xed with 4% paraformaldehyde for 20 min. Slides were rinsed with phosphatebuffered saline (PBS) and then incubated in 3.5% H2O2 in PBS for 20 min to block endogenous peroxidase. Slides were then incubated with 5% skimmed milk in PBS for 2 h to reduce non-speci®c background. The slides were incubated with anti-Spz1 polyclonal antibody (1:10 000) in PBST (PBS 1 0.2% Tween-20) for 1 h. After washing with PBST for 30 min, slides were incubated with alkaline phosphatase-conjugated anti-rabbit polyclonal antibody (1:8000). Normal rabbit serum was used in the negative control group experiment. Slides were ®nally counterstained with 0.5% methyl green. 4.7. Antibody and Western blot Spz1 cDNA (bp 51±1120) was subcloned into the pQE30 expression vector (Qiagen) to produce His6±Spz1 fusion protein. Spz1 fusion protein expression was induced for 6 h by 1 mM IPTG in E. coli strain SR13009. Cells were lysed with denatured buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris±HCl, pH 8.0). After centrifugation, the fusion protein was puri®ed from a Ni 1-NTA±agarose column (Qiagen) according to the manufacturer's instructions. Anti-Spz1 antibody was raised in New Zealand rabbits according to the protocol described by el-Roeiy et al. (1988). Sodium dodecyl sulfate±polyacrylamide gel electrophoresis (SDS± PAGE) was performed using the buffer system described by Laemmli (1970). Samples were boiled for 5 min in an equal volume of sample buffer containing 2% SDS, 5% 2-mercaptoethanol and electrophoresed on an 8% acrylamide gel. After proteins were transferred onto PVDF membranes (Hoefer, Pharmacia Biotech), the membranes were blocked at room temperature for 1 h in 5% skimmed milk (in PBST) and washed for 40 min with PBST. The membranes were
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then incubated with alkaline phosphatase-conjugated antirabbit antibody for 1 h, and washed with PBST. The membranes were developed using an ECL system (Amersham Pharmacia Biotech) on X-ray ®lm (Kodak). 4.8. Immuno¯uorescence analysis The expression sites of Spz1 were determined by immuno¯uorescence as described by Andersson et al. (1986). LFG6 cells were cultured on coverslides precoated with poly-l-lysine for 16 h. After washing with cold PBS, cells were ®xed with 2% paraformaldehyde in PBS for 30 min on ice, and then the slides were washed with PBS for 30 min. Cells were permeabilized with buffer containing 0.2% Triton X-100 and 1% NGS in PBS for 20 min on ice, and then the slides were washed with PBS to remove excessive detergent. Cells were then incubated in anti-Spz1 polyclonal antibody (1:10 000) for 1 h at room temperature. After washing cells with PBS for 30 min, FITC-conjugated human anti-rabbit polyclonal antibody (1:8000) was then added and incubated for another 1 h at room temperature in a humid chamber. Cells were ®nally counterstained with propidium iodine (PI) to localize the nucleus. 4.9. Immunoprecipitation To isolate native Spz1 protein, LFG6 cells were lysed and Spz1 protein was puri®ed by immunoprecipitation. The method was modi®ed according to Erikson et al. (1977). Cells were cultured in DMEM containing 15% FBS and lysed with detergent solution (1% NP-40, 0.4% DOC, 10 mM EDTA and 10 mM Tris±HCl, pH7.4) on ice for 30 min. After centrifugation, the supernatant was diluted with 4 volumes of PBS, precleared with preimmune rabbit serum for 1 h at 48C, and then mixed with protein A Sepharoseconjugated beads. After centrifugation, the Spz1 protein was precipitated by anti-Spz1 polyclonal antibody and protein A. The Spz1 protein was then eluted by resuspending the protein A-Sepharose beads in lysis buffer. 4.10. In vitro target binding sequence An in vitro binding reaction and PCR ampli®cation were used to identify the Spz1 DNA binding sequences. In vitro binding reaction was performed using a 74-mer doublestrand oligonucleotide (5 0 -ACTAGTCTCGAGGGATCCGATATCNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAGAAAGCTTGTCGACCTGCGG-3 0 ) as probe in which 26 random nucleotides were ¯anked by two known 24-mer sequences. One microgram of native Spz1 protein puri®ed from immunoprecipitation was incubated with 5000 cpm of 32P-labeled probe for 30 min at room temperature. The DNA±protein mixtures were separated on 4% Tris± glycine gels (Buratowski et al., 1989) and then autoradiographed using X-ray ®lm (Kodak). The DNA±Spz1 associated complex was cut from the gel and diluted by resuspending in 100 ml of H2O. Using the two ¯anking
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24-mer sequences as primers, the oligonucleotides that Spz1 protein bound were ampli®ed by PCR and subcloned into pGEM T-Easy vector (Promega) and sequenced. The consensus DNA sequence was determined by alignment of 30 sequences. 4.11. Electrophoresis mobility shift assay (EMSA) The DNA binding reaction was performed in buffer containing 10 mM Tris±HCl, 1 mM EDTA, 100 mM NaCl, 2 mM dithiothreitol (DTT) and 10% glycerol. The 24-mer double-stranded oligonucleotides containing an Ebox (CANNTG) or G-box (CCACGTGG) were used as probes. One microgram of Spz1 protein was incubated with 5000 cpm 32P-labeled oligonucleotides, 2 mg poly(dIdC), and BSA (1 mg/ml) for 30 min at room temperature. The DNA±protein mixtures were separated on a 6% polyacrylamide gel (30:1 bis acrylamide in 0.5£ TBE). 4.12. Hormonal treatment Total RNA was isolated from 18-day-old mouse testis after i.p. injection with gonadotropin (12 IU/10 g body wt., twice per day), progesterone (0.15 mg/10 g body wt., twice per day), retinoid acid (0.2 mg/10 g body wt.), or testosterone (0.15 mg/10 g body wt., twice per day) for 4 days. Twenty micrograms of total RNA was loaded in each lane, and the membrane was hybridized with a full-length Spz1 cDNA probe. 4.13. BAC clone isolation and Spz1 chromosomal localization A Spz1 cDNA fragment (bp 204±1338) was used as a probe to screen a mouse BAC library (Genome System). Three positive clones were isolated, one of which was used as probe for ¯uorescence in situ hybridization (FISH) to identify the chromosomal localization of Spz1. FISH was performed using a previously published procedure (Shi et al., 1997). Acknowledgements We would like to thank Drs W.W. Branford, S.S. Potter and K.B. Choo for their critical reading of the manuscript. This work was supported in part by a research grant NSC89-2311-B-001-214 from the National Science Council, Taiwan. References Andersson, I., Billig, H., Fryklund, L., Hansson, H.A., Isaksson, O., Isgaard, J., Nilsson, A., Rozell, B., Skottner, A., Stemme, S., 1986. Localization of IGF-I in adult rats. Immunohistochemical studies. Acta Physiol. Scand. 126, 311±312. Arnold, H.H., Winter, B., 1998. Muscle differentiation: more complexity to
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