Identification of Two Soxl7 Messenger RNA Isoforms, with and without the High Mobility Group Box Region, and Their Differential Expression in Mouse Spermatogenesis

Two different mRNA isoforms of the mouse Soxl 7 gene were isolated from adult mouse testis cDNAs. One form (referred to as form Sox17) encodes an Styrelated protein of 419 amino acids containing a single high mobility group box near the NH2 terminus, while the other form (referred to as form t-Sox17) shows a unique mRNA isoform of the Soxl 7 gene with a partial deletion of the HMG box region. Analysis of genomic DNA revealed that these two isoforms were produced at least by alternative splicing of the exon corresponding to the 5' untranslated region and NH2-terminal 102 amino acids. RNA analyses in the testis revealed that form Soxl7 is expressed in spermatogonia, and the expression clearly declines from the early pachytene spermatocyte stage onward. In contrast, expression of form t-Soxl7 began at the pachytene spermatocyte stage and was highly accumulated in round spermatids. Protein analyses revealed that t-Soxl7 isoforms, as well as Sox17 isoforms, were translated into the protein products in the testis, although the amount of t-Soxl7 products is lower in comparison to the high accumulation of t-Soxl7 mRNA. By the electrophoretic mobility-shift assay and the random selection assay using recombinant Soxl7 and t-Sox17 proteins, Soxl7 protein is a DNA-binding protein with a similar sequence specificity to Sry and the other members of Sox family proteins, while t-Soxl7 shows no apparent DNA-binding activity. Moreover, by a cotransfection experiment using a luciferase reporter gene, Soxl7 could stimulate transcription through its binding site, but t-Soxl7 had little effect on reporter gene expression. Thus, these findings suggest that Soxl7 may function as a transcriptional activator in the premeiotic germ cells, and that a splicing switch into t-Soxl7 may lead to the loss of its function in the postmeiotic germ cells. T HE Sry gene on human and mouse Y chromosomes is the testis-determining gene that induces differentiation of the genital ridge into the testicular pathway (Sinclair et al., 1990; Gubbay et al., 1990; Koopman et al., 1991). It potentially encodes a DNA-binding protein containing the high mobility group (HMG) 1 box that is present in several transcription factors such as T cell-specific factor TCF-1 (van de Wetering et al., 1991) and RNA Please address all correspondence to Yoshiakira Kanai, D.V.M., Ph.D., Department of Ultrastructural Research, The Tokyo Metropolitan Institute of Medical Science, 18-22 Honkomagome 3-chome, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-03-3823-2101 (ext.5163); Fax: 81-03-3823-2965. 1. Abbreviations used in this paper: DIG, digoxigenin; EMSA, electrophoretic mobility-shift assay; GST, glutathione S-transferase; HMG, high mobility group; ORF, open reading frame; RT, reverse transcription; UTR, untranslated region. polymerase I transcription factor UBF (Jantzen et al., 1990). Because the Sry protein synthesized in vitro binds to double-stranded DNA in a sequence-dependent manner (Harley et al., 1992, 1994), it is believed to function in testis determination by regulating the expression of other specific genes. Until now, several genes encoding Sryrelated HMG bo.~x (termed as Sox gene) have been identified in mouse and human cDNA, and a member of this family has also been isolated in a number of other vertebrate and invertebrate species (Denny et al., 1992a,b; Chardard et al., 1993; Wright et al., 1993; Goz6 et al., 1993). The amino acid sequences of Sox family genes are known to be highly conserved from humans to Drosophila (Denny et al., 1992a), and such strong evolutionary conservation suggests that Sox family genes may play important roles in the differentiation and development of the testis and the other cell types and tissues in many species. For example, Soxl, Sox2, Sox3, and Sox11 are known to © The Rockefeller University Press, 0021-9525/96/05/667/15 $2.00 The Journal of Cell Biology, Volume 133, Number 3, May 1996 667~81 667 on Jauary 8, 2018 jcb.rress.org D ow nladed fom be expressed in the developmental nervous system (Stevanovi6 et al., 1993; Uwanogho et al., 1995; Jay et al., 1995; Kamachi et al., 1995). Sox4 is expressed in T lymphocytes and in some pre-B lymphocytes, and Sox4 protein mediates the enhancer effects of the AACAAAG motif in lymphocytes (van de Wetering et al., 1993). Recently, the human SOX9 gene was isolated from a translocation chromosome breakpoint of a sex-reversed patient with campomelic dysplasia, and mutation analysis and expression patterns indicated that Sox9 may play a critical role both in testicular differentiation and skeletal development (Foster et al., 1994; Wagner et al., 1994; Wright et al., 1995). In the mature adult testis, several Sox family genes are expressed. Sox5 is expressed stage specifically during spermatogenesis (Denny et al., 1992b). Sry is also expressed at a high level in the adult testis (Koopman et al., 1991; Rossi et al., 1993), and the most abundant Sry transcripts in adult testes are present as circular RNA molecules (Capel et al., 1993), although their functions in spermatogenesis are uncertain. Sox4 (van de Wetering et al., 1993), Sox6 (SoxLZ) (Denny et al., 1992b; Takamatsu et al., 1995; Connor et al., 1995), and Sox9 (Foster et al., 1994; Wagner et al., 1994) are also reported to be expressed in the adult testis. Although functions of these Sox family genes in the adult testis remain elusive, such testicular expression leads us to the postulation that several members of this gene family may exert an important effect on mammalian spermatogenesis. In the present study, we isolated two different mRNA isoforms of the mouse Sox17 gene (form Soxl7 and t-Soxl7) from adult mouse testis cDNAs. Form Soxl7 encodes an Sry-related protein of 419 amino acids containing a single HMG box near the NH2 terminus, while form t-Soxl7 shows a unique mRNA isoform of the Sox17 gene with a partial deletion of the HMG box region. Moreover, we examined the expression patterns of these two Soxl7 isoforms in the testis and revealed that the isoform with an intact HMG box region was expressed in spermatogonia, and that such expression decreased at the pachytene spermatocyte stage. In contrast, expression of the unique isoform without the HMG box region began at the pachytene spermatocyte stage and was accumulated in round spermatids. Protein analyses revealed that t-Soxl7 isoforms, as well as Sox17 isoforms, were translated into the protein products in the testis, although the amount of t-Soxl7 products is lower in comparison to the high accumulation of t-Soxl7 mRNA. To test the functional difference between Soxl7 and t-Soxl7, we examined their DNA-binding activity by the electrophoretic mobility-shift assay (EMSA) and the transactivation activity by a cotransfection experiment using a luciferase reporter gene. By EMSA, t-Soxl7 showed no apparent DNA-binding activity, but Soxl7 can specifically bind the AACAAT motif. Moreover, by a cotransfection experiment, Sox17 could stimulate transcription through its binding site, but t-Soxl7 had little effect on reporter gene expression. Thus, the Sox17 gene is the first Sox family gene that is found to produce multiisoforms with different expression profiles and DNA-binding activities. The biological significance of the differences between these two isoforms in spermatogenesis will be discussed. Materials and Methods Isolation of Soxl 7 cDNAs and Genomic DNA and Sequencing To isolate Sty-related eDNA expressed in the testis, a mouse testis cDNA library constructed with h g t l l (Clontech Laboratories, Palo Alto, CA) was screened at high stringency with a Sox9 HMG box probe. The Sox9 HMG box fragments were isolated from testis cDNAs by the degenerate PCR approach, as previously described (Denny et al., 1992a; Wright et al., 1993). As a result, two independent positive clones that coded a novel isoform of Sox17 eDNA (referred to as form t-Soxl7) were isolated. Because these two clones encoded a unique isoform of the Soxl7 gene with a partially deleted HMG box region, we further isolated the Soxl7 cDNA (referred to as form Soxl7) encoding an intact HMG box from testis cDNAs using an LA-PCR kit (Takara Biomedicals, Kyoto, Japan) in combination with two sets of primers (forward primer: 5 ' -ATGGCCCACTCACACTGCTGGCGGGT-3 ' [+375 to +400] x reverse primer: 5 ' -CCAACCGCTTGCGTTCGTCTqTGGC-3 ' [+884 to +908]; forward primer: 5 'AGGCTAGC/TCCGATCCCTGCCTC-3 ' [+564 to +587] × reverse primer: 5 ' -GCTTCTGGCCCTCAGGTCGGGTCGGCAA-3 ' [+1913 to + 1940]). Moreover, the mouse genomic library constructed with h FIX II (Stratagene, La Jolla, CA) was screened, and three independent clones were isolated. Inserted or amplified DNA fragments were subcloned into pBluescript KS + (Stratagene). Nucleotide sequences were determined by the dideoxy chain termination method (Sanger et al., 1977) using a BcaBEST sequencing kit (Takara Biomedicals). Generation of Probes For Northern and Southern blot hybridization, Apal-digested (+1249 to +1482; probe A in Fig. 2 a) and EcoRI-digested (+1750 to +2375; probe B) fragments of t-Soxl7 were used as probes that recognize both isoforms. DNA fragments corresponding to the spliced exon (+564 to +863; probe C), which were isolated by PCR, were used as the probe specific for the Soxl7 form. For the reverse transcription (RT) PCR analysis, the fragment digested with SacI and ApaI (+336 to +1248 of t-Soxl7) was used for detection of both Soxl7 and t-Soxl7 cDNAs. The isolated DNA fragments were labeled with [a-32p]dCTP (3,000 Ci/mmol) by a Megaprime DNA labeling system (Amersham, Buckinghamshire, UK). For in situ hybridization analysis, the DNA fragments corresponding to probe B or C were subcloned into pBluescript. Each clone was linearized with the appropriate restriction enzyme, and the sense and antisense RNA probes were generated by in vitro transcription using digoxigenin (DIG)-UTP with T3 and T7 RNA polymerase (Boehringer Mannheim Biochemica, Mannheim, Germany). The resulting sense and antisense DIG-labeled RNA probes were reduced to ~150-bp fragments by limited alkaline hydrolysis before hybridization to tissue sections. Southern Blot Hybridization Genomic DNAs were prepared from the liver of adult male mice and digested with BamHI, EcoRI, HindIII, or PstI. 10 p~g of each DNA preparation were electrophoresed in 0.6% agarose-TBE gels and transferred to a nylon membrane (Hybond N; Amersham). Blots were hybridized with appropriate 32p-labeled DNA probes in a solution containing 50% formamide, 5× SSC, 5× Denhardt's, 1% SDS, 0.2 M sodium phosphate (pH 6.8), and 200 ~g/ml denatured salmon sperm DNA (ssDNA) at 42°C for 12-14 h. Filters were finally washed with 0.1x SSC-0.1% SDS at 65°C for 1 h and autoradiographed with an x-ray film (X-OMAT-AR; Eastman Kodak, Rochester, NY). Chromosomal Mapping of Soxl 7 Gene Interspecific F1 hybrids of C3H/HeJ and a Mus spretus-derived strain, SEG, were back-crossed to C3H/HeJ. 50 interspecific back-cross progeny were scored for the segregation of RFLP of the Soxl7 gene and microsatellite markers, DIMitl , DIMit4, D1MitMit11, D1Mitl2, DIMit14, and DIMit17. Map manager v2.5 was used to analyze the data generated in