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- Original Article -

Mutations of t-complex testis expressed gene 5 transcripts in the testis of sterile t-haplotype mutant mouse

Yibing Han¹, Xue-Xiong Song², Huai-Liang Feng³, Che-Kwok Cheung¹, Po-Mui Lam¹, Chi-Chiu Wang^1,4, Christophe John Haines¹

¹Department of Obstetrics and Gynaecology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China
²Department of Animal Sciences, Laiyang Agricultural College, Qingdao, Shandong 266109, China
³Center for Human Reproduction, North Shore University Hospital, NYU School of Medicine, Manhasset, NY 11030, USA
⁴Li Ka Shing Institute of Health Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China

Abstract

Aim: To determine the possible roles of the t-complex testis expressed gene 5 (Tctex5) on sperm functions, the full-length sequence of mRNA was studied and compared in the testis between the normal wild-type and the sterile t-haplotype mutant mice. Methods: We applied rapid amplification of cDNA ends, Northern blot and reverse transcription polymerase chain reaction to analyze the full length of Tctex5 mRNAs isolated from testes of the wild-type and the t-haplotype mice. Reverse transcription polymerase chain reaction was used to semi-quantitatively compare expression of Tctex5 transcripts in the 16 tissues and 9.5 day stage embryos in the wild-type mice. E-translation was applied to estimate the amino acid sequences.  Results: One long and one short transcript of Tctex5 mRNA were discovered in mouse testis of wild-type (Tctex5^long-+ and Tctex5^short-+) and t-haplotype (Tctex5^long-t and Tctex5^short-t) mice, respectively. Being enhanced only in the testis, Tctex5^long-t had 17 point mutations and one 15-bp-deletion in the exon 1 region, comparing with the Tctex5^long-+, whereas the Tctex5^short-t was similar to the Tctex5^short-+. The short isoforms of Tctex5 mRNAs in the two models encoded exactly the same peptides, but the long isoforms did not. The estimated peptide encoded by Tctex5^long-t had significant mutations on putative sites of phosphorylation and PP1 binding.  Conclusion: We established that mutations that occur in the Tctex5 long transcript of the t-haplotype mice are important for normal sperm function, whereas the short transcript of Tctex5 might have a conserved function among different tissues. (Asian J Androl 2008 Mar; 10: 219_226)

Keywords: t-complex testis expressed gene 5; transcripts; testis; mice

Correspondence to: Dr Yibing Han, Department of Obstetrics and Gynaecology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China.
Tel: +852-2632-1538 Fax: +852-2636-0008
Email: ybhan@cuhk.edu.hk
Received 2006-12-27 Accepted 2007-05-20

DOI: 10.1111/j.1745-7262.2008.00323.x

1 Introduction

Protein phosphorylation and dephosphorylation take part in spermatogenesis, especially for the G2/M transition evidenced by the fact that pachytene spermatocytes entering metaphase within 4_6 h under stimulation in vitro with the protein phosphatase 1 inhibitor okadaic acid [1]. In contrast to protein kinases that add a phosphate group to the hydroxyl oxygen of serine, threonine or tyrosine residues, protein phosphatases (PP) remove the phosphate group. There are mainly four groups of PP according to their catalytic subunits: PP1, PP2A, PP2B and PP2C. Until now, PP1 (including subtypes PP1α, PP1β, PP1γ1 and PP1γ2), PP2A and PP2B have been found to be expressed in the testis [2]. The regulators of PP, such as inhibitor 2 (Inh2), inhibitor 3 (Inh3), sds22, 14-3-3 and hsp90, are also associated with spermatozoa both in the testis and in the epididymis [3_6].

Tctex5 is defined as t-complex testis expressed 5 [7]. The human homologue of Tctex5 was discovered in 1996, and has been confirmed to be a protein phosphatase 1 regulator 11 (ppp1r11), and termed inhibitor-3 (Inh3) [8, 9]. Inh3 in humans is co-localized to the nucleoli and centrosomes with PP1γ1 and PP1α, respectively [10]. In a sterile male mice model (t-haplotype mutant mice), Tctex5 and its function on sperm have drawn our attention as a candidate for the "Curlicue" and "Stop" (sperm oolemma penetration of t-haplotype mice) phenotype [11]. T-haplotypes are mutations in the proximal one-third of mouse chromosome 17. The t true homozygous embryos will die at very early developmental stages in the uterus, whereas the secondary "t-homozygotes", which are homozygous for two non-complimentary t-haplotypes (like t^w32 and t^w5 in the present study) originated from different genetic background (B6 and 129 mice, respectively), can survive. However, the male "t-homozygotes" (t^w32/t^w5) are absolutely sterile because of the expression of mutation-bound factors that perturb numerous and distinct sperm functions, such as motility, zona-pellucida binding and oolemma penetration [12, 13]. "Curlicue" and "Stop" are phenotypes shown by individual spermatozoa isolated from t-homozygotes male mice epididymis in vitro. The t sperm show extra curvicues ("Curlicue") when exposed to increased levels of Ca²⁺ in the medium, and they can not penetrate the oolemma ("Stop") when exposed to the zona-free oocytes. The genetic factors leading to these two phenotypes have been mapped to "Ccu" (include "Ccua" and "Ccub") and "Stop" (include "Stop1p" and "Stop1d") regions in chromosome 17 [14]. Being located to both the "Ccub" and "Stop1d" locus, Tctex5 might be involved in the regulation pathways for sperm function, including motility control, sperm flagella structure development and sperm-oolemma penetration. In our previous studies, Tctex5 has shown a specific expression pattern in the testis undergoing spermatogenesis and a similar distribution of PP1γ2 in the spermatozoa [4]. In addition to the binding of Tctex5 to PP1γ2 in vitro [15], our previous research indicates the important role of Tctex5 in normal sperm function through PP1γ2 or other isoforms of PP1. To reveal the role(s) of Tctex5 during spermatogenesis, in the present study, we have investigated the Tctex5 expression pattern in the testis and compared the sequences of each transcript between the wild-type and the t-haplotype mice.

2 Materials and methods

2.1 Production and genotyping of mice

Wild-type male mice were produced by crossing 129-+/+ and C57BL/6- +/+ mice. All t^w32/t^w5 mice (hereafter we called t-haplotype mice) were obtained by breeding of t^w5/+ and t^w32/+ mice and were congenic to the +/+ mice. The proximal portion of mouse chromosome 17 was genotyped by restriction fragment length polymorphism analysis of genomic DNA extracted from tail-tip biopsies. Briefly, digested genomic DNAs were separated by electrophoresis in agarose gels and blotted to nylon membrane. DNAs were bound to the membrane by ultraviolet light and hybridized with radio-labeled probes that were informative for the chromosome 17 genotype. The presence of complete t haplotypes was detected using the marker DNA clones Tu48, Tu89. Pim1A was used to distinguish between t^w32 and t^w5 haplotypes.

2.2 mRNA preparation and rapid amplification of cDNA ends

mRNA from the testes of the wild-type and the t-haplotype (t^w32/t^w5) mice was isolated using the Oligotex Direct mRNA Mini Kit (Qiagen, Valencia, CA, USA). Full-length 5' and 3' ends of Tctex5 were obtained using the GeneRacer kit (Invitrogen, Carlsbad, CA, USA) based on RNA ligase-mediated and oligo-capping rapid amplification of cDNA ends (RACE). Referring to genomic Tctex5 sequences in the GeneBank (No. AK005379), Tctex5 specific 5' and 3' primers and nested primers were designed (Table 1). Sequencing of full length cDNA was carried out by Macrogen (Seoul, Korea). Experiments were carried out twice.

2.3 Reverse transcription polymerase chain reaction (RT-PCR), semi-quantitative analysis of Tctex5 transcripts in tissues and northern blot

After the long and short transcripts of Tctex5 mRNA were found by RACE in the wild-type mice and the short transcript in the t-haplotype mice, we designed two primer sets that discriminate the long and short Tctex5 transcript: the Tctex5 complete and Tctex5 lower, respectively (Table 1 and Figure 1A). RT-PCR was performed using mRNA isolated from the wild-type and t-haplotype testis as template to confirm the existence of the transcripts. A new long transcript in the t-haplotype mice testis was discovered. Expression of each transcript of Tctex5 mRNA was screened using the mouse rapid scan gene expression panels (Origene MSCB-101, Rockville, MD, USA) in three dilutions (100 ×, 10 × and 1 ×) with the lowest dilution concentration (1 ×) with approximately 1 pg cDNA. Using the panels as templates, we carried out RT-PCR using the following cycle conditions: 95ºC for 3 min; 95ºC for 30 s, 55ºC for 30 s and 72ºC for 90 s for 35 cycles; 72ºC for 10 min, and 4ºC thereafter. Beta-actin was amplified as an internal control. Experiments were carried out twice.

Five µg of total RNA isolated from wild-type and t-haplotype testis and RNA markers were blotted onto membrane (Hybond-XL; Amersham, Piscataway, NJ, USA). Northern blot was then used for hybridizations. The probe was synthesized by RT-PCR using Tctex5-complete primer sets. Probe recovered from 1.5% low melting gel was labeled using α-³²P dCTP (Perkin Elmer, Waltham, MA, USA) and purified with ProbeQuant G-50 micro columns (Amersham, Piscataway, NJ, USA). The blot membrane was hybridized overnight at 65ºC. After washing, the blot was exposed to Kodak BioMax MS film (Kodak, Rochester, NY, USA).

2.4 Computer-assisted sequence analysis

Sequence analysis and Tctex5 intron/exon analysis used in the present study were obtained from http://www.ensemble.org. E-translation of known mRNA and protein structure prediction were carried out using the following websites: http://ca.expasy.org/tools/dna.html and http://www.predictprotein.org, respectively.

3 Results

3.1 Isoforms transcribed in the testis

There were two isoforms of Tctex5 mRNA transcribed in the testis. Significant mutations occurred in the long transcript of Tctex5 between the t-haplotype and the wild-type mice. The long isoform mRNA was enhanced in the testis whereas the short isoform was more ubiquitous.

Using RACE and RT-PCR, we identified a long and a short isoform of Tctex5 (Tctex5^long-+ and Tctex5^short-+ in wild-type, Tctex5^long-t and Tctex5^short-t in t-haplotype mutant) transcribed in the testis. The cDNA sequences of the Tctex5^long-+, Tctex5^short (Tctex5^short-t had exactly the same reading frames as the Tctex5^short-+; therefore, we called them Tctex5^short) and Tctex5^long-t had been sent to GenBank and been given the IDs EF528576, EF528579 and EF528577, respectively. The full lengths of Tctex5^long-+, Tctex5^short-+ and Tctex5^short-t were 650 bp, 435 bp and 495 bp, respectively. As Tctex5^long-t was discovered by RT-PCR, its full length was not known. The full length transcript (Tctex5^long-+) of Tctex5 contained three exons. In the Tctex5^long-+ mRNA, exon 1 spanned from 169 bp upstream of the first initiating ATG to 91 bp downstream of ATG; exon 2 spanned 92_310 bp downstream of the first initiating ATG; exon 3 spanned 311_481 downstream of the first initiating ATG (Figure 1A). Seventeen point mutations and one 15-bp-deletion in exon 1 (from 44 to 58 bp downstream of the first initiating ATG) occurred in the Tctex5^long-t mRNA. The most intensive point mutation (hereafter called the highly mutational region) occurred in an area 7_23 bp downstream of the first initiating ATG, which was just 11 bp upstream of the deletion region. Tctex5^short-t and Tctex5^short-+ shared a second initiating codon ATG in exon 2 (103 bp downstream of the first initiating ATG in Tctex5^long-+ and Tctex5^long-t) without the amino acid reading frame shift comparing with their long isoform counterparts. They were similar in sequences except in the 5' UTR region.

Northern blot analysis of the Tctex5 transcripts transcribed in the testis of the wild-type and the t-haplotype mice showed that the sizes of Tctex5^long-+, Tctex5^short-+, Tctex5^long-t and Tctex5^short-t were approximately 1.2 Kb, 0.6 Kb, 1.0 Kb and 0.6 Kb, respectively. The short isoforms were much more abundant than the long isoforms in both models (Figure 1B). Rapid-scan gene expression panel showed that, in wild-type mice, only the long transcript of Tctex5 was enhanced in the testis. In contrast, the short transcript of Tctex5 was not just enhanced in the testis, but also in other tissues, such as heart, kidney, liver, stomach, small intestine, muscle and lung (Figure 2).

3.2 Estimated mutations of Tctex5 peptide

The short Tctex5 mRNAs encoded for a shorter truncated peptide compared with the long mRNAs with no reading shift. Estimated peptide sequences from the two short Tctex5 mRNA transcripts were exactly the same in the wild-type and the t-haplotype mice. In contrast, estimated peptide sequences from the two long Tctex5 mRNA transcripts were different. Two out of three sites for casein kinase II phosphorylation, one N-myristoylation site, one ASN-glycosylation site, one protein kinase C phosphorylation site, one Amidation site and the estimated PP1 binding site were mutated in the Tctex5^long-t compared with its Tctex5^long-+ counterpart (Figure 1D).

4 Discussion

Among the two verified Tctex5 mRNA transcripts in GenBank, only 29 bps of the AK077455 (1170 bp in length) isolated from day 8 mouse embryo whole body shared 100% identity with the genomic sequences of Tctex5 in GenBank; whereas the BC027737 (652 bp in length) isolated from mouse mammary tumor was very similar to the long isoform mRNA sequence discovered in the present study (with only 21 bp discrepancies in the most 5' end). The long and short transcripts of Tctex5 in the wild-type mice discovered here might be either consequences of alternative splicing in the exon1/exon2, or products of different promoters. The long transcript covered all three exons, whereas the short transcript covered only partial exon 2 and complete exon 3 in the wild-type mice (the Tctex5^short-t covers partial exon 1 and complete exon 2 and 3 in the t-haplotype). In the t-haplotype mice, there were also two transcripts of Tctex5. There were several important mutations in the Tctex5^long-t compared with Tctex5^long-+. Unlike its wild-type counterpart, Tctex5^short-t did not have mutations, but had a different 5' UTR. The two transcripts in both mouse models were not equal. Therefore, we assumed that: (i) the two transcripts in the wild-type and t-haplotype mice might not originate from alternative splicing of mRNAs but from different promoters and (ii) there were two different Tctex5 alleles (t^w32 and t^w5, respectively) in the t-haplotype mouse testis that might be under the control of different promoters (Figure 1C).

We have searched the transcription factor binding sites using bioinformatics tools available at http://www.cbrc.jp/research/db/TFSEARCH.html. There were putative trans-acting binding sites covering both the transcription start site of the long and the short transcripts in the wild-type mice. We estimated that promoter 1 and promoter 2 located just upstream of exon 1 and exon 2, respectively, produced the long and short isoform of Tctex5 in the wild-type mouse. Using northern blot analysis, the Tctex5^long-t mRNA was found to be significantly shorter than the Tctex5^long-+ mRNA. Although the RNA ladder on the northern blot shifted the difference in size (approximately 0.2 Kb) between the Tctex5^long-+ and Tctex5^long-t was even longer than the whole upstream sequences of the initiating ATG in the Tctex5^long-+ (169 bp). Therefore, we assumed that the Tctex5^long-t, which was obtained from RT-PCR, had both shorter 5' and 3' ends than those of Tctex5^long-+. We anticipated that in the t-haplotype allele 1, promoter 2 might be inactivated by mutations; therefore, only promoter 1 worked and Tctex5^long-t was produced. The Tctex5^long-t might be either much less abundant than the Tctex5^long-+, or be a splice variant of the longer transcript or hold a modified 5'-Cap structure because we could not detect it using RACE. Instead, the Tctex5^long-t was obtained by RT-PCR using mRNAs isolated from the t-haplotype testis. In the t-haplotype allele 2, promoter 1 might be inactivated by some unknown mutations; therefore, only promoter 2 worked and the Tctex5^short was produced (Figure 1C).

On average, there were 3.1 human putative alternative promoters (PAP) per gene and the richest tissue sources of the tissue-specific PAP were testis and brain [16]. Tctex5 was expressed on both the germ cells and somatic cells isolated from testicular seminiferous epithelial cells (Han et al., unpublished data). Functions of the two transcripts of Tctex5 in the testis might be different and we anticipated that the long transcript might be a relatively testis-specific isoform. Enhanced expression of the long isoform in primary breast tumor cell lines and metastasic colon tumor cell lines cultured in vitro [17] might indicate a relation of Tctex5 expression and cell division. The long transcript of Tctex5 might have an important role in sperm function. As a candidate gene for "Curlicue" and "Stop", Tctex5^long-t showed a variety of mutations when compared with Tctex5^long-+. The normal Tctex5^short could not compensate the function loss caused by the Tctex5^long-t. Therefore, we predicted that the secondary structure of the Tctex5 long peptide might be important for this function. The phosphorylation state of Tctex5^long might be important and had an on/off function for the signaling pathway(s). Although which pathways Tctex5 might be involved in during spermatogenesis in the testis it is so for unknown, PP1 or other PP pathways were expected to be candidates, implied by the importance of PP1-biding site mutation in the Tctex5^long-t. Tctex5 was very much like a signaling factor itself connecting PP1 or other PP to the signaling pathway(s) important for pachytene-to-metaphase transition in spermatogenesis.

Acknowledgment

This research is supported by Direct grant C001-2041219 and the Li Ka Shing Institute of Health Sciences Grant, the Chinese University of Hong Kong, Hong Kong, China.

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