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Testis |
Institute of Biochemistry,3 National Yang-Ming University, Taipei, 112 Taiwan
Institute of Molecular Biology,4 Academia Sinica, Taipei, 115 Taiwan
Department of Obstetrics and Gynecology,5 Tri-Service General Hospital, Taipei, 114 Taiwan
Department of Life Science,6 National Taiwan Normal University, Taipei, 116 Taiwan
Graduate Institute of Medical Science,7 Taipei Medical University, Taipei, 110 Taiwan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ovary, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several recently identified prd-like homeobox genes were classified into the PEPP subfamily, which include Pem, Esx1, Psx1, and Psx2 (also called Gpbox) from mouse [4–7], and OTEX, ESXR1, hPEPP1, and hPEPP2 from human [8–10]. All of the members of the PEPP subfamily are located on the X chromosome and feature two introns between residues 31, 32 and residues 46, 47 of the HD. PEPP subfamily expression is mainly confined to the placenta and reproductive organs, such as the testis and ovary [4–10]. The functions of PEPP subfamily members still remain unclear. Esx1-deficient mice present placenta abnormalities and fetal-growth retardation [11], whereas Pem and Gpbox knockout mice show no obvious phenotypes [12, 13].
Due to the important role that homeobox genes play in the processes of development, this study focused on characterizing unstudied homeobox genes associated with gametogenesis in the reproductive organs of mice. One of the PEPP subfamily members cloned from a mouse testis cDNA library was named Tox (testis and ovary specifically expressed homeobox gene). We found that Tox, like other PEPP family members, maps to the X chromosome and is expressed in reproductive organs, testis, and ovary. In this study, we identified the sequence features and expression pattern of this gene.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Degenerate oligonucleotides were designed based on the highly conserved helix 3 sequence of HD, and PCR screening of cDNA libraries was performed as described previously [14]. DNA sequences of PCR products containing a putative novel HD were used to search EST databases to obtain additional sequence information from which PCR primers could be designed for amplification of a longer cDNA fragment.
Animals
FVB strain mice were used in this study. All animals were maintained in a specific pathogen-free environment under standard laboratory conditions and handled following the guidelines of the institutional animal committee.
Cloning of Tox Full-Length cDNA
The Tox cDNA fragment (bp 380–740) was PCR amplified and used as a probe to screen a mouse testis cDNA library (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. A total of 106 colonies were plated out for screening. Four positive clones with insert sizes longer than 1 kb were sequenced. The longest clone was 1117 bp with poly-A termination. The 5' end of the cDNA was extended by using a 5'-RACE kit (Life Technologies). Ten micrograms of total RNA isolated from FVB strain mouse testis was treated with calf intestinal phosphatase to remove the 5' phosphates, and then the dephosphorylated RNA was treated with tobacco acid pyrophosphate to remove the 5' cap structure from the intact full-length mRNA. A GeneRacer RNA Oligo was ligated to the decapped RNA using T4 RNA ligase. GeneRacer Oligo dT Primer together with Superscript II RT was used to reverse transcribe the ligated mRNA. The RACE-ready cDNA was amplified using the GeneRacer 5' primer and the Tox-specific oligonucleotides, 5'-CTGCAGCCATGGCCCCCAACAAA-3' (nt 279–301 of Tox cDNA). One microliter of the PCR product was used as a template in a nested PCR using GeneRacer 5' nested primer and the Tox specific oligonucleotides, 5'-CCAACAAAGGGCAGTCCTGTATACCT-3' (nt 268–286 of Tox cDNA). The PCR products were gel purified and subcloned into pGEM-T Easy vector (Promega, Madison, WI) for sequence analysis.
RNA Isolation and Northern Blot Analysis
Total RNA was extracted with Trizol reagent (Life Technologies) from FVB strain mouse testes of different postnatal stages. Twenty micrograms each of total RNA was loaded and sized-fractionated on a 1.5% agarose/formaldehyde gel and transferred to nylon membranes (Amersham Pharmacia Biotech, Piscataway, NJ). The blot was prehybridized in buffer (0.2% SDS, 5x SSPE, 5x Denhardt solution, 100 µg/ml sheared salmon sperm DNA, 50% formamide, 10% dextran sulfate) for 2 to 4 h at 42°C. The blot was then hybridized overnight at 42°C with a 32P-labeled Tox DNA fragment (bp 685–1044) in the same buffer system. Following hybridization, the blot was washed with buffer (2x SSC, 1% SDS) at 65°C for 20 min and autoradiographed.
Reverse Transcription (RT)-PCR Analysis
One microgram of total RNA, isolated with Trizol reagent (Life Technologies) from different organs of mature mouse, was used in reverse transcription with the Superscript II system (Life Technologies). Tox and Gapdh were amplified using the following primers: Tox1a, 5'-ACCGTCAACAGCATCCATGCTA-3'; Tox1b, 5'-AAATGAGAGAGGGGGGTGGTAA-3'; Gapdh 1a, 5'-CCCTTCATTGACCTCAACTA-3'; Gapdh 1b, 5'-CCAAAGTTGTCATGGATGAC-3'. The following PCR conditions were used: 95°C for 5 min; 32 cycles of 95°C for 30 sec, 58°C for 30 sec, and 72°C for 1 min and 40 sec; followed by 72°C for 10 min; PCR amplification of the Gapdh gene was the same as for the Tox gene except that the annealing temperature was 57°C.
In Situ Hybridization
The Tox cDNA fragment (bp 685–1044) was subcloned into the pGEM-T Easy vector (Promega). Sense and antisense riboprobes were prepared with digoxigenin-UTP (Boehringer Mannheim, Indianapolis, IN) by in vitro transcription using T7 and SP6 polymerase, respectively. Testes were isolated from Postnatal Day (dpp) 7 and 8-wk-old FVB strain mice. Three mice were used in each group. Ovaries were isolated from 4-wk-old female mice after FSH stimulation for 2, 4, 6, and 12 h. Two mice at each time point were used to identify ovaries, including follicles of different stages. Testes and ovaries were fixed in 4% paraformaldehyde and OCT embedded. Seven-micrometer sections were cut and air-dried at room temperature overnight. The sections were washed with PBS-T (0.1% Tween-20) and then treated with proteinase K (10 µg/ml) for 10 min, followed by glycine treatment immediately after the sections were washed with PBS-T, and acetylation was carried out with 0.1 M TEA (triethanolamine) for 10 min. After washing with PBS-T, the sections were covered with prehybridization buffer and incubated at 65°C for 2 h. The prehybridization buffer was removed; the sections were then covered with hybridization buffer (prehybridization buffer plus 200 ng/ml antisense and sense probes prepared previously). Hybridization was carried out at 65°C overnight. After hybridization, the sections were washed with buffer I (50% formamide, 2x SSC pH 4.5, 0.1% Tween-20) and buffer II (0.2x SSC pH 4.5, 0.1% Tween-20) at 65°C, three times respectively. The sections were treated with maleic acid buffer (100 mM maleic acid, 150 mM NaCl) three times and then blocked with 10% blocking reagent (in maleic acid buffer) at 4°C for 3 h. A 1:1000 dilution of anti-Dig secondary antibody was prepared in AP buffer (100 mM NaCl, 100 mM Tris pH 9.5, 50 mM MgCl2, 0.1% Tween-20). After removing blocking reagent, the sections were incubated with the secondary antibody at 4°C overnight. When secondary antibody hybridization was finished, the sections were washed with TBS-T (0.1% Tween-20) three times and rinsed with AP buffer three times. The sections were then developed by the addition of a 1:20 dilution of NBT/BCIP (Pierce Biochemicals, Rockford, IL) (in AP buffer). Upon signals detection, the sections were washed with PBS to stop the reaction and then counterstained with methyl green for 10 min. These sections were finally mounted with mounting solution for observation.
Southern Blot Analysis
A Southern blot was carried out on NheI digested genomic DNA from male and female mice, respectively. Digested DNA was sized-fractionated on a 0.7% agarose gel and transferred to a Hybond-N membrane (Amersham). The blot was hybridized with a 1.2 kb Tox genomic DNA fragment, which was a PCR product amplified by the primer pair (forward, 5'-AGCAGAAATCATCAGTAGCCCC-3'; backward, 5'-GGAGGGACTTGAACTC ATTACG-3'). The Southern blot was performed in hybridization buffer (1M NaCl, 1% SDS, 0.1M Tris-Cl pH 7.4, 0.1% dextran sulfate) at 65°C for 16 to 20 h. Following hybridization, the blot was washed with buffer (2x SSC, 1% SDS) at 65°C for 40 min and autoradiographed.
Phylogenetic Alignment of HD Sequence
The HD sequences of family members were obtained from GenBank, and the phylogenetic alignment of HD sequences was performed by the MacVector computer program with the Felsenstein method [15]. The penalty parameter of gap was set as 10.0.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In order to isolate novel homeobox genes that may be involved in male germ line development, we performed degenerate oligonucleotide PCR screening of mouse testis cDNA libraries. Partial cDNA fragments from the PCR amplification were used as probes for the cDNA library screening (Life Technologies). We identified several uncharacterized homeobox genes from these screenings, one of which we named Tox because of its testis- and ovary-specific expression. The Tox cDNA obtained from the testis cDNA library is 1117 bp in length and contains a polyadenylation signal at its 3' end. The 293 amino acids protein encoded by the cDNA contains a 60 amino acid HD at its C-terminal. Since there was no stop codon upstream of the ATG start codon, we performed 5' RACE PCR, using testis total RNA as the template, to identify additional 5' sequences. An additional 120-nucleotide 5' extension fragment was obtained, and it provided another start codon and an in-frame stop codon upstream of the ATG start codon (Fig. 1). The 1237-bp cDNA sequence was submitted to GenBank (accession AY329370). Special features of Tox include the HD being located very near the C-terminal, and two long repeats of glutamic acid (E) of 26 and 25 residues in the middle portion of this protein. The residues between the two long poly E stretches are also rich in glutamic acid. The proportion of these glutamic acids to the total number of amino acids in this protein is 35%, which makes Tox a very acidic protein with a predicted pI value of 3.75.
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Genomic DNA Analysis of Tox
To obtain Tox genomic DNA, we searched Genome databases for BAC or PAC clones with Tox cDNA sequences using the NCBI BLAST program (available at http://www.ncbi.nlm.nih.gov/BLAST/). A mouse BAC clone, RPCI-23, encompassing the complete Tox cDNA was purchased from the Children's Hospital Oakland Research Institute, and its nucleotide sequence was determined (accession AL590629). The genomic DNA mapped to the A2 region of the mouse X chromosome. Alignment between Tox cDNA and genomic DNA showed that Tox cDNA was organized into four exons and three introns (Fig. 2A). Furthermore, the nucleotide sequences of the boundaries between exons and introns followed the GT-AG rule (Table 1). We also performed Southern blot analysis to confirm the copy number of Tox in the mouse genome. A 4.2-kb NheI fragment was detected with a genomic ratio of females to males of about 1.87 (Fig. 2B). This corresponds to the X chromosome allele numbers that exist in the female and male genome.
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Comparison of the Tox Deduced HD Sequences
Prd-class HDs are classified into three subclasses on the basis of the encoded residue at HD position 50, which determines DNA binding specificity. Prd-type genes contain a serine residue at position 50 and a second DNA-binding domain, the prd domain, usually localized upstream of the HD. Q50 Prd-like and K50 Prd-like homeobox genes are characterized by not having the prd domain, with a Q or K residue at their HD position 50, respectively [16]. HD alignment analysis indicated that Tox belongs to the Prd-like subclass, for which the Drosophila protein aristaless, represents the prototype (Fig. 3A). Further analysis indicated that Tox is one member of the recently identified PEPP subfamily, because it shares some common features with this family, such as X chromosome localization (Fig. 2A) and a genomic structure of two introns interrupting the HD (Fig. 3B).
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Tox Expression in Mouse Testis and Ovary
To identify the tissue specificity of Tox, we examined its expression through RT-PCR analysis of total RNA isolated from a variety of adult mouse tissues. Tox was present significantly in testis and ovary (Fig. 4A). Time course expression of Tox in testis was characterized by a Northern blot analysis of total RNA derived from different developmental stages of male testes. A message size of 1.3 kb was detected at each developmental stage: the highest expression shown was between Days 7 and 21, with lower but detectable expression at later times (Fig. 4B). Since most of the PEPP genes, so far identified, are expressed in embryos and in the placenta during embryonic development, we investigated Tox expression in these tissues from Embryonic Day 12 to 19. However, no significant signals were detected from RT-PCR analyses (data not shown).
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Since strong expression of Tox in mouse testis was detected during the first 2 wk after birth, we further examined the localization of Tox in Day 7 testis by in situ hybridization. Tox was expressed in most of the cells, mainly the spermatogonia and Sertoli cells, of the seminiferous tubule (Fig. 5, A and B). The expression of Tox in 8-month-old mature testis seminiferous tubules was mainly in late spermatids and mature sperm (steps 8–16; Fig. 5, C–E). The expression of Tox in ovary was predominantly detected in the somatic cells of follicles by in situ hybridization analysis. In the preantral follicle, Tox was only observed in theca cells (Fig. 6A), whereas in the antral follicle, Tox was expressed both in mural granulosa and theca cells (Fig. 6, C and D). No Tox signal was detected in the corpus luteum (Fig. 6E).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Spermatogenesis and folliculogenesis are complex processes leading to gamete formation. In this study, we found that Tox is expressed during these two processes. Spermatogenesis takes about 35 days in mice. The first wave of spermatogenesis is initiated at dpp 6, and there are a few haploid spermatids present in the seminiferous tubules at 20 dpp [17]. Sertoli cells play very important roles in supporting and feeding germ cells inside the seminiferous tubules. Tox was expressed in the testis postnatally, with signals found in Sertoli cells and spermatogonia before puberty, and in spermatids and spermatozoa after puberty. The result from in situ hybridization (Fig. 5) correlated well with the data from the Northern blot analysis (Fig. 4B), which showed that the Tox expression increased gradually to 21 dpp in testis, a stage in which increasingly spermatids are being produced. These expression phenomena indicate that Tox may be involved in both the early- and late-stage differentiation of male germ cells. The Tox expression appeared to be limited in late spermatids spanning steps from 8 to 16. Since transcription termination occurs several steps earlier, the accumulation of Tox mRNA implies that Tox is translationally regulated. On the other hand, folliculogenesis is a process of oocyte growth and maturation, during which granulosa and theca cell proliferation and development take place in each functional follicle unit [18]. In the preantral or early antral follicles, the theca interna commences its epithelial differentiation and has a low responsiveness to gonadotropin. During the antral stage, both the theca and granulosa cells become more responsive to gonadotropin. FSH, activin, and epidermal growth factor act synergistically to strongly enhance the proliferation of granulosa cells [19]. The expressions of Tox in both Sertoli cells within the testis and in somatic cells of the ovary suggest that FSH may be involved in the expression of Tox. FSH is produced in the gonadotroph cells of the anterior pituitary and acts on Sertoli cells of the testis and granulosa cells of the ovary. During normal folliculogenesis, oocyte growth and maturation are coordinated with granulosa and theca cell proliferation and differentiation [18]. FSH stimulates granulosa cell proliferation, aromatization of androgens to estrogens, and LH receptor expression [20], whereas LH stimulates theca cell androgen production. Without enough FSH, most multilayer preantral and early antral follicles undergo atresia, as evidenced by mice lacking the FSH with folliculogenesis blocked at the preantral follicle stages [21]. Recently, evidence has suggested a direct role for prolactin in modifying granulosa cell function at the level of the ovary. In vitro studies have demonstrated that prolactin acts directly on granulosa cells to both potentiate and inhibit steroid biosynthesis and luteinizing hormone receptor (LH-R) acquisition. In this study, however, no Tox expression was detected in the corpus luteum, which suggests that prolactin might not be involved in the ontogeny and function of Tox during ovary development. Alternatively, FSH function in spermatogenesis to regulate Sertoli cell proliferation and, ultimately, the size and spermatogenic capacity of the testis. These arguments were proved from several mouse mutants, such as FSH-deficient male mice being fertile but having small testes [21], whereas young FSH receptor knock-out males had underdeveloped testis with a 50% reduction in Sertoli cells [22]. Whether FSH is involved in Tox expression during germ cell development has yet to be elucidated.
Two members of the PEPP family, Pem and OTEX, have been shown to be regulated by androgens [7, 9]. The actions of androgens are dependent on their binding to the androgen receptor, igniting a cascade of molecular events on Sertoli cells and peritubular cells that culminates in male sexual differentiation in utero and initiation and maintenance of spermatogenesis. During the folliculogenesis process, theca cells produce androgens through LH stimulation, and the estradiol is further converted from androgen in granulosa cells [19, 20]. Homeostasis among these paracrine/endocrine systems in the follicles is tightly regulated to support oocyte maturation [23, 24]. The expressions of Tox in somatic cells of gonads and peaking around the pubertal stage indicate that androgen may be involved in any role Tox expression may play in female germ line development. Whether Tox is involved in the regulation of the paracrine/endocrine system during folliculogenesis and spermatogenesis remains to be explored.
PEPP family members have previously been shown to be expressed in reproductive organs, such as testis, ovaries, and placenta [4–10]. Pem has also been detected in adult testicular Sertoli cells and ovarian follicle cells [7, 25]. In addition, Pem, Gpbox, and Psx1 have similar expression patterns in the extra-embryonic placenta and within the germ cells of the embryonic gonad [6]. Another PEPP gene, Esx1, is also exclusively expressed in extra-embryonic placenta and adult testicular germ cells [5]. Although these PEPP family members were detected as being expressed in the embryo and placenta, we found no convincing proof of Tox expression in these tissues, even upon a sensitive RT-PCR analysis. This result suggests that Tox may not have a significant function during embryonic development. However, the in vivo function of Tox will be further elucidated from the phenotypes of Tox gene knockout mice.
DNA repeat expansion is the genetic basis for a growing number of neurological disorders. The largest subset of these diseases results in an increase in the length of a polyglutamine (CAG repeats) tract in the protein encoded by the affected gene, such as Spinocerebellar ataxia 1, 2, 3, 6, and 7 [26–28]. The only report of GAA repeats existing inside a gene so far is the two long GAA/TCC repeats in intron 1 of the human frataxin gene on chromosome 9. These two expanded GAA/TTC repeats present in the untranslated region of frataxin have been proposed to act as sticky DNA to reduce frataxin transcriptions, which cause the progressive neurodegenerative disease Friedreich ataxia [29]. However, the two long stretches of GAA repeats in Tox encode poly E, which make Tox a very acidic protein (pI 3.75). Many transcriptional activators are rich in acidic amino acids, such as the yeast GAL4 and GCN4, the first described activation proteins [30, 31]. HNF-4 is another acidic activator reported to be able to bind to the TFIIB- and TATA-binding protein [32]. Tox may act as a transcription factor to activate downstream target genes, not only through its homeodomain binding to target DNAs, but also via its acidic domain interacting with other transcription factors present on the promoters of target genes. A repetitive domain rich in glutamic acid residues has also been identified in an X chromosome gene, RPGR. More than 60% of X-linked retinitis pigmentosa (XLRP) patients have mutations in this glutamic acid-rich exon of the RPGR gene [33]. However, the function of the glutamic acid-rich domain is still not clear. Whether these Tox GAA repeats also act as a transcriptional inhibitor, as the repeats in frataxin may do, or if they are involved in transactivation or protein-protein interactions remains to be explored.
In conclusion, this study found that Tox, a PEPP gene, was mainly expressed in the testes and ovaries of adult mice. The extended poly E region present in the Tox gene indicates that Tox might possess regulatory functions during gametogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Hsiu Mei Hsieh-Li, Department of Life Science, National Taiwan Normal University, Taipei, 116 Taiwan. FAX: 886 2 2931 2904; hmhsieh{at}cc.ntnu.edu.tw
Received: 8 July 2003.
First decision: 25 July 2003.
Accepted: 7 November 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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