Expression of a novel bHLH-Zip gene in human testis

Jia-Hao SHA^1,2, Zuo-Min ZHOU^1,2,3, Jian-Min LI², Ming LIN², Hui ZHU², Hu ZHU², Ya-Dong ZHOU², Li-Long WANG², Yi-Quan WANG¹, Kai-Ya ZHOU¹

Asian J Androl 2003 Jun; 5: 83-88             

Keywords: basic helix-loop-helix; leucine zipper; testis; spermatogenesis; DNA microarrays

Abstract

Aim: To identify specifically expressed genes in the adult and fetal testes. Methods: A human testis cDNA microarray was established. Then the mRNA of adult and fetal testis was purified and probes were prepared by a reverse transcription reaction with the testis mRNA as template. The microarray was hybridized with probes of adult and fetal testes. The nucleic sequences of differentially expressed genes were determined and homologies were searched in the databases of the GenBank. Results: When hybridized with adult or fetal testis probes, the positive clones were 96.8 % and 95.4 %, respectively. Among these genes, one was a new testis-specific gene, which was named TSP1. TSP1 was highly expressed in human adult testis. The cDNA of TSP1 was 1,484 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AF333098). TSP1 was also determined as Interim Gen Symbol (Unigene, No. Hs.98266). Protein analysis showed that TSP1 contained two functional domains: an N-terminal basic helix-loop-helix (bHLH) and a C-terminal leucine zipper (Zip). Homologous analysis showed that the 430 amino acid sequences deduced from the 1293 bp open reading frame (ORF) had a homology with the human gene FLJ2509 (AK098575). TSP1 had also a sequence homology with Spz 1 protein of mouse. Expression profiles showed that TSP1 was specifically and strongly expressed in the testis. Conclusion: TSP1 is a gene highly expressed in adult testis. It may play an important role in spermatogenesis in the humans.

1 Introduction

The testis has two fundamental functions, steroidogenesis and spermatogenesis. Spermatogenesis is a complex process that begins with the division and differentiation of the spermatogenic stem cell within the seminiferous tubule. The expression of genes is different between testis and other organs as in the former both meiosis and mitosis occur. Moreover, spermatogenesis begins in puberty, so the expression of genes is different in different developmental stages [1-3]. Study on genes specifically expressed in the testis at different stages of development may reveal new genes related to the function of testis, especially spermatogenesis [4].

One approach toward the understanding of the processes involved in germ cell development and maturation is to identify genes that are expressed in testis. Most genes identified in this manner are likely to be structure determining or metabolic activity-controlling genes. The identification 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-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 basic helix-loop-helix (bHLH) motif-containing proteins. The promoter sequences from a computerized data search of a number of Sertoli cell expressed genes indicated the presence of bHLH 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 [5]. Evidence of the involvement of bHLH protein in sex determination and gonadal development in Drosophila [6, 7] has also provided support for the significance of bHLH factors in spermatogenesis.

In this study, we identified a novel human testis specific gene, TSP1, which encodes a putative bHLH-Zip transcription factor. This gene is highly and specifically expressed in adult human testis.

2 Materials and methods

The Human Testis 5'-STRETCH PLUS cDNA library was ordered from Clontech, Palo Alto, USA (Catalog number: HL5503U, Source of insert cDNA came from 25 Caucasians, aged 20 years~65 years). The length of insert cDNA is 3.4 kb on average. According to Clontechs Manual PT3003-1, each l TriplEx2 clone was converted to a p l TriplEx2 clone which required excising with E.coli BM25.8 and the complete plasmid was circularized from the recombinant phage.

The plasmids were extracted by a modified Sam-brooks method [8] of alkaline lysis in our laboratory and the samples were stored in 96-well trays at -70 .

To produce DNA for spotting the microarray, the gene sequence was amplified from the plasmid clone by the polymerase chain reaction (PCR). Primers were designed according to the 5 and 3 sequences of l TriplEx2 vector flanking the insert. 5 primer is CCATTGTGT-TGGTACCCGGGAATTCG, interval 6 bp to insert cDNA site and 3 primer is ATAAGCTTGCTCGAGTCTAGA-GTCGAC, interval 7 bp to insert cDNA site. Altogether 9216 clones randomly selected from cDNA library were amplified. The PCR products were 2.0 kb to 7.0 kb in length and were stored at -20 .

A cDNA array was assembled with 9,216 samples of PCR product. Each sample was dotted on a 8 cm12 cm nylon membrane (ordered from Amersham Pharmacia Biotech, UK, Lot No: YA1103) by using an automatic arrayer (BioRobotics, Cambridge, UK). Two dots for each sample were spotted, so a total of 18,432 dots represented 9,216 samples. DNA was cross-linked to nylon membrane by UV light. Eight house keeping genes were used as positive controls: ribosomal protein S9 (RPS9), actin gamma, glyceraldehyde-3-phosphate dehydrogenase (GAPD), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), H.sapiens mRNA for 23 kD highly basic protein, ubiquitin C(UBC), phospholipase A2 and ubiquitin carboxyl-terminal esterase L1 (UCHL1). TriplEx2 phage DNA and PUC18 plasmid DNA were used as negative controls. For each control cDNA 12 spots were evenly distributed in each membrane.

Testes from deceased human adults (n=2) and six-month fetuses accidentally aborted (n=3) were collected. The testis was homogenized and the total mRNA was extracted according to the Trizol RNA isolation protocol (Gibco BRL, Grand Island, USA) and quantified with a UV spectrometer after electrophoresis. The Poly(A)+ mRNA was purified by using an affinity column filled with poly(dT) resins (Qiagen, Hilden, Germany). The probes were prepared by incorporation of ³³P-labeled dATP in a reverse transcription reaction using 2 mg of purified mRNA as the template and an oligo(dT) as the primer with Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase. Each labeling reaction was carried out with 200 mCi of ³³P-dATP following the manu-facturers instruction (NEN Life Science, Boston, USA)

Nylon membranes spotted with cDNA fragments were prehybridized with 20 mL of prehybridization solution [6sodium chloride/sodium citrate buffer (SSC), 0.5 % SDS, 5Denhardt, 100 mg/L of denatured salmon sperm DNA] at 68 for 3 h. Overnight hybridization with the ³³P-labeled cDNA from testis samples was carried out in 6 mL of hybridization solution (6SSC, 0.5 % SDS, 100 mg/L of denatured salmon sperm DNA) and followed by stringent washing with 20 mL of wash solution (10 % SSC, 0.5 % SDS ) at 65 oC for 1 h. Membranes were exposed to phosphor screen overnight and scanned using a FLA-3000A fluorescent image analyzer (Fuji Photo Film, Tokyo, Japan). The radioactive intensity of each spot was linearly scanned with a 65,536 gray-grade in a pixel of 50 m and read out using the array gauge software (Fuji Photo Film, Tokyo, Japan). After subtraction of the background from an area where no PCR product was spotted, clones with an intensity density over 10 were considered as positive signals. Hybridization data would be considered invalid if any of the 12 control spots for the same control cDNA had a 1.5-fold difference in its intensity between arrays. The hybridization intensity of correspondent dots in adult and fetus were compared. If the difference in values of spot intensity in adult and fetus was more than three fold higher or lower, the correspondent genes were considered as differentially expressed.

For differentially expressed genes, cDNA clones were proliferated. The amplified cDNA plasmids were isolated and purified in mini-preps (QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany) and the inserts were sequenced using the ABI 377 automatic sequencing machine. For each clone, sequence homologies were searched in databases of GenBank. The nucleic and deduced amino acid sequences were also analyzed by using Gene Runner software.

After sequence identification and analysis, a novel testis specific gene, named TSP1, was found. Expression profile of TSP1 was determined by using PCR screening. Multiple tissue cDNA panels were purchased from Clontech (# 1420-1), including testis, thymus, small intestine, colon, spleen, leukocyte, prostate gland, ovary, pancreas, heart, kidney, lung, placenta, liver, brain and skeletal muscle. The TSP1 specific primers were as follows: upstream: 5 GAGTAGAGATTCTCAAGG-AACTCC 3 (nt 1,217- nt1,240) and downstream: 5 ATA-GCTCTTTCTTGGTTGAACC 3 (nt 1,612-nt 1,633). The PCR product was 417 bp in size. b-actin was used as the positive control. The reagents in 30 mL PCR reaction tubes were as follows: buffer 3 mL, 25 mmol/L Mg²⁺ 2.4 mL, 2 mmol/L dNTP 2.4 mL, Tag DNA polymerase (5 u/mL) 0.2 mL, upstream primer 5 pmol, downstream primer 5 pmol and cDNA sample 3 mL (0.6 ng). PCR conditions were as follows: denaturation at 94 for 1 min, annealing at 58 for 1 min and extension at 72 for 1 min. The first cycle had a denaturation period of 5 min. The last cycle had an extension period of 7 min. 35 cycles of PCR were performed. The PCR products were analyzed after electrophoresis.

3 Results

The microarrays of the human testis cDNA were hybridized with cDNA probes prepared from the mRNA of human adult and fetal testes. Signals were obtained from 8,925 spots (about 96.8 % of the cDNA clones) in case of adult testis, and 8,795 spots (about 95.4 % of the cDNA clones) in fetal testis. It appears that the majority of genes were expressed in both adult and fetal testes. The others were expressed only in adult or fetal testis. Among the cDNA clones that gave signals, 731 were differentially expressed in adult or fetal testis. 592 clones were highly expressed in adult testis, whereas 139, expressed in fetal testis. The intensity difference was at least three fold. These genes are candidates for differentially expressed genes in the testis. The reciprocal expression characteristics of these genes indicated that different sets of genes were involved in different developmental stages.

A novel gene, named TSP1 (testis specific protein) was found with special expression in the testis. The level of hybridization intensity of TSP1 in adult and fetus testis were 134.27 and 22.53, respectively (Figure 1, Six-month fetal testis/ adult testis). The expression level in adult testis was 5.96 fold that in fetal testis. The full length of TSP1 cDNA was 1,848 bp and contained an open reading frame from nt 217 to nt 1,509, which encodes a 49.4 kD protein of 430 amino acids (Figure 2). The methionine at nt 217- nt 219 was almost certainly the site of initiation because there is an up-frame upstream stop code at nt 127- nt 129. The cDNA sequence of this clone was deposited with Genbank. The accession number was AF333098. TSP1 was also determined as an Interim Gen Symbol (Unigene). The UniGene number is Hs.98266. Functional and homologous analysis of TSP1 predicted protein showed that TSP1 contained two functional dormains: an N-terminal basic helix-loop-helix (bHLH) and a C-terminal Zip. Homologous analysis by using Blast of Genbank showed that the 430 amino acid sequences deduced from the 1,293 bp open reading frame (ORF) had a homology with human gene FLJ2509 (AK098575). The identity was 98 %. TSP1 had also sequence homology with Spz 1 protein of mouse (Figure 3). The positive rate of homology for full sequences was 58 %. In bHLH and Zip motif the positive rate of homology was 59.5 % and 97.3 %, respectively.

Figure 1(Six-month fetal testis/ adult testis). Partial cDNA hybridization images showing differential expression of TSP1 in fetal and adult testes (red circle). The hybridization intensity in fetal and adult testes was 22.53 and 134.27, respectively.

Figure 2. Nucleic acid and deduced amino acid sequences of the cDNA for TSP1. Underlinings show PCR primers for the determination of expression profile. Boxes show bHLH and Zip mortifs.

Figure 3. Sequence comparison of bHLH and Zip motifs between TSP1 and Spz1. Identical and similar amino acids are shaded in dark and light colors individually.

PCR and electrophoresis showed that this TSP1 gene was particularly and strongly expressed in human testis (Figure 4a, b). There was no expression in thymus, small intestine, colon, spleen, leukocyte, prostate gland, ovary, pancreas, heart, kidney, lung, placenta, liver, brain and skeletal muscle.

Figure 4a, b. Electrophoresis showing expression profiles of TSP1; b-actin was used as control. (A) Amplification of TSP1. PCR product was 417 bp. TSP1 was specifically and strongly expressed in testis. There was no expression in any other organ. (B) Amplification of b-actin as control. All organs had b-actin expression.

4 Discussion

Sprmatogenesis is a complex process that leads to the formation of male gametes. Spermatogenesis takes 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 [9].

A variety of approaches have been developed to obtain information on gene expression levels based on some intensity measurement. Examples of this are whole-mount in situ hybridization [10], differential display technology [11] and cDNA microarray [12]. In this study cDNA microarray was used and a testis specific protein gene, TSP1, was found. Sequence analysis showed that the TSP1 protein contains two functional motifs. One is bHLH (basic helix-loop-helix). The highly charged basic region in each of the dimerization partners immediately upstream of the HLH region forms a specific DNA-binding dormain that can recognize a DNA sequence known as an E-box (CACNAG), an N-box (CACNAG) [13, 14] or a G-box [15]. Proteins contain bHLH motif are often transcription factors of comprising important components in the regulatory network of many developmental pathways [16]. Recent studies have suggested that bHLH proteins regulated Sertoli cell differentiation. For example, transferrin, a marker of differentiation, has an E-box element (bHLH binding dormain) in its promoter that can be activated by E12, a bHLH factor present in the Sertoli cells [5]. Another example relevant to the current study was the requirement of bHLH proteins in sex determination and gonadal development in Drosophilia [6]. TCFL5 (transcription factor-like 5) is another bHLH protein expressed specifically in the primary spermatocytes [17]. The cell-type and stage-specific expression of TCFL5 indicates that this protein may play a crucial role in spermatogenesis as a transcription factor by regulating cell proliferation or differentiation through binding to a specific DNA sequence like other bHLH molecules. All these observations provide additional support for that bHLH factors play an important role in spermatogenesis. Another functional motif is a leucine zipper (Zip). Zips are involved in protein dimerization enhancement. Some dimers effect activation of gene expression while others, repression. Certain members of bHLH family also contain zip motif that is closely contiguous to the C-terminal of the bHLH motif. They are bHLH-Zip proteins [18, 19]. These bHLH-Zip proteins have been implicated in various developmental and cellular processes and some are known to be oncogenes [18, 20-22]. In mouse, a bHLH-zip protein (Spz1) was found [23]. Spz1 was exclusively expressed in spermatogenesis-specific adult tissues, mainly in germ cells, Sertoli cells and Leydig cells. The results showed that Spz1 was related to spermatogenesis in mouse. TSP1 was also a bHLH-zip protein and had homology with Spz1 protein. Compared with TCFL5 protein, TSP1 contained Zip domain. TSP1 is so far the only bHLH-Zip protein expressed in adult human testis. As TSP1 is expressed highly in human adult testis and weakly in embryo testis, it may also play an important role in spermatogenic cells in the humans.

The work was supported by grants from China National 973 (No. G1999055901) and Chinese Natural Science Funds (No. 30170433).

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Correspondence to: Dr. Zuo-Min ZHOU, Key Laboratory of Reproductive Medicine, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, China.
Tel: +86-25-666 2908, Fax: +86-25-650 8960
E-mail: zhouzm@njmu.edu.cn
Received 2003-02-20  Accepted 2003-04-29

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