Molecular cloning and identification of mouse epididymis-specific gene mHong1, the homologue of rat HongrES1

Abstract

Previous studies have shown that rat epididymis-specific gene HongrES1 plays important roles in sperm capacitation and fertility. In this study, we cloned the mouse homologue gene by sequence alignment and RT-PCR methods and designated it as mHong1. The mHong1 gene is located on chromosome 12p14, spanning five exons. The cDNA sequence consists of 1257 nucleotides and encodes a 419 amino-acid protein with a predicted N-terminal signal peptide of 20 amino acids. The mHong1 mRNA shows similarity with HongrES1 in the expression patterns: (i) specific expression in epididymal tissue, especially in the cauda region; and (ii) androgen-dependence but testicular fluid factor independence. Its protein product shows 71% similarity with HongrES1 and contains a classical serpin domain as does HongrES1. A polyclonal antibody against mHong1 with high specificity and sensitivity was raised. Like HongrES1, the mHong1 protein shows a checker-board expression pattern in the epididymal epithelium and is secreted into the epididymal lumen. The mHong1 protein shows higher glycosylation than HongrES1. Although both of them are deposited onto the sperm head surface, mHong1 is localized to the equatorial segment, which is different from that of HongrES1. The mHong1 protein can be removed from the sperm membrane by high ionic strength and therefore can be classed as an extrinsic membrane protein. Collectively, we conclude that mHong1 is the homologue of HongrES1 and the present work paves the way for establishing animal models to elucidate the precise functions of HongrES1 and mHong1.

Figure 1

Cloning of the full-length mHong1. (a) The rat HongrES1 cDNA sequence was used to interrogate the mouse genome. Two fragments (224 bp and 653 bp) with significant similarity were obtained. On the basis of the sequences of these two fragments, two pairs of primers (FP1/RP1, FP2/RP2) were synthesized. (b) Amplification of cDNA fragments of mHong1 by RT-PCR. The cDNA fragments of 224 bp, 653 bp and 1524 bp were amplified with primers FP1/RP1, FP2/RP2 and FP1/RP2, respectively. (c) Northern blot analysis of mHong1 mRNA in the epididymis and testis probed with different cDNA regions (1–224 bp, 225–871 bp and 872–1524 bp). 18s rRNA was used as loading control. Ep, epididymis; Te, testis.

Figure 2

The cDNA and deduced amino acid sequence analysis and the intron–exon structure of mHong1. (a) The mHong1 open reading frame contains 1257 bp coding for a 418-amino acid protein, and the initial and terminal codons are boxed. The protein contains a putative signal peptide (in italics) with a cleavage site between amino acids 20 and 21. The N-glycosylation (N_{31, 151, 223, 228, 263, 322 and 383}) sites are shadowed grey. The serpin motif from 380th to 390th amino acid is in bold and boxed. (b) The mHong1 gene diagram shows the relative lengths of exons 1–5 and intervening introns. The shadowed grey box represents the open reading frame of mHong1 mRNA. (c) Amino-acid sequence alignment of mouse mHong1 and rat HongrES1. Dashes show alignment gaps. The conserved serpin motif residues are indicated by grey box.

Figure 3

Tissue distribution, androgen-dependent and testicular factor-independent expression of mHong1 mRNA by Northern blot analysis. (a) Total RNAs (15 µg per lane) from various tissues were used. The mHong1 mRNA is highly expressed in the mouse epididymis, especially in the corpus and cauda, and weakly expressed in vas deferens but not in the other tissues. Cap, caput; Cor, Corpus; Cau, Cauda; Te, testis; Ep, epididymis; Va, vas deferens; Sv, seminal vesicle; Li, liver; He, heart; Ki, kidney; St, stomach; Br, brain; In, intestine; Th, thymus; Sp, spleen; Lu, lung; Ad, adrenal. (b) Northern blot analysis of adult mouse epididymal RNAs from pre-castration (d0) and bilateral castration for 1,3, 5 and 7 days (d1, d3, d5 and d7) as well as for 1, 3, 5 and 7 days after the initial injection of testosterone propionate to the 7-day castrated rats (d7+1, d7+3, d7+5 and d7+7). The total RNAs were pooled from six animals at each time point. (c) The relative expression levels of mHong1 mRNA (hybridisation density of mHong1 mRNA/18s ribosomal RNA) in the mouse epididymis and the serum testosterone level (expressed in nmol l⁻¹) during androgen manipulation. (d) Northern blot analysis of mHong1 mRNA and 18s rRNA in the epididymis of efferent duct-ligated mice and control mice. The left side of each rat epididymis (1L, 2L, 3L, 4L) was ligated, and the other side (1R, 2R, 3R, 4R) served as the control. The RNAs were pooled with four animals per group. (e) The relative expression levels of transcripts (hybridisation density of mHong1 mRNA/18s ribosomal RNA) in the mouse epididymis.

Figure 4

Western blot analysis of mHong1 native protein in tissues. (a) Rabbit polyclonal antisera were raised against mHong1 C-terminal peptide (antigen) and the sensitivity of the antibody towards antigen was verified by Western blotting: 0.1, 0.5, 2 and 5 ng of antigen peptide were loaded. (b) Protein extracts (12 µg per lane) from various tissues were analysed. Te, testis; Va, vas deferens; Sv, seminal vesicle; Li, liver; He, heart; Ki, kidney; St, stomach; Ep, epididymis; Ad, adrenal; Lu, lung; Sp, spleen; Th, thymus; In, intestine; Br, brain. (c–e) Western blot analysis of mHong1 in epithelium (c), lumen (d) and sperm (e) protein extracts from caput (Cap), corpus (Cor) and cauda (Cau) epididymidis. GAPDH was used as an internal control in (c) and (e). The same amount of protein was separated by electrophoresis and stained by Coomassie blue to demonstrate equal loading in (d). (f) The change of molecular masses of mHong1 protein in total tissue protein of cauda epididymidis before (−) and after (+) deglycosylation by peptide N-glycosidase F (PNGase-F).

Figure 5

The region-specific localisation of mHong1 in the mouse epididymis. (a) The expression pattern of mHong1 in the whole mouse epididymis. Bars=2 mm. (b) The magnified photographs for individual fields of (a): initial segment (b1), caput (b2), distal caput (b3), proximal corpus (b4), corpus (b5, b6), distal corpus (b7), proximal cauda (b8) and distal cauda (b9). Bars=50 µm.

Figure 6

The localisation of mHong1 protein on spermatozoa by indirect immunofluorescence assays. (a) Immunolocalisation of mHong1 (FITC-labelled) on spermatozoa isolated from different epididymal regions. Cauda spermatozoa detected by pre-immune serum as negative control. Bars=10 µm. (b) mHong1 localisation on spermatozoa from the cauda epididymidis. (b1) Phase contrast view of spermatozoa in (b2). (b2) The immunofluorescence of mHong1 (FITC-labelled). (b3) Sperm nucleus detected by propidium iodide (PI). (b4) Merged micrograph of (b1) and (b2). (b5) Merged micrograph of (b1), (b2) and (b3). (b6) Phase contrast view of spermatozoa in (b7). (b7) Cauda spermatozoa detected by pre-immune serum as negative control. (b8) Sperm nucleus detected by propidium iodide (PI). (b9) Merged micrograph of (b6) and (b7). (b10) Merged micrograph of (b6), (b7) and (b8). Bars=50 µm. (c) mHong1 can be removed from cauda spermatozoa by washing with high-salt solution (HSS) but not phosphate-buffered saline (PBS). Bars=10 µm.