Discovery in Silico and Characterization in Vitro of Novel Genes Exclusively Expressed in the Mouse Epididymis

Jenni Penttinen, Dwi Ari Pujianto, Petra Sipilä, Ilpo Huhtaniemi and Matti Poutanen

Department of Physiology, Institute of Biomedicine, and Turku Graduate School of Biomedical Sciences (J.P., P.S.), University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland

Address all correspondence and requests for reprints to: Dr. Matti Poutanen, Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: matti.poutanen{at}utu.fi.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Epididymal proteins interact with sperm during their passage through the epididymis and thus contribute to the maturation and fertilizing capacity of the spermatozoa. In the present study we have discovered five novel epididymis-specific genes through in silico analysis of expressed sequence tags (ESTs) at the UniGene library collection. The strategy used is a powerful way to discover novel epididymis-specific genes. The full-length cDNA sequences were determined, and computational tools were used to characterize the genomic structures and to predict putative functions for the encoded proteins. In vitro analyses revealed that all five genes characterized were highly expressed in the defined areas of the epididymis, and they were not expressed at significant levels in any other tissue. Three of the genes were named on the basis of their putative functions: Spint4 (serine protease inhibitor, Kunitz type 4), and Rnase9 and Rnase10 (ribonuclease, Rnase A family 9 and 10), while for the ESTs AV381130 and AV381126 no putative functions could be predicted. The expression of Spint4, Rnase9, and AV381130 was found to be under a direct or indirect regulation by androgens, while the expression of Rnase10 is regulated by a testicular factor(s) other than androgen. None of the genes were expressed in the immature epididymis, while mRNAs were detected from d 17 onward, at the time of maturation of epididymal epithelium. However, the expression of AV381130 was not detected until d 30 after birth, indicating a close connection between gene expression and puberty.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SPERMATOZOA produced in the testis are passed to the epididymis, where posttesticular maturation of the spermatozoa occurs. During their transit through the epididymis, spermatozoa become fully motile and capable of fertilizing the oocyte. The epididymis is structurally and functionally organized into four major distinctive segments: initial segment, caput, corpus, and cauda. In all segments the epididymal duct consists of epithelial cells attached to a basal lamina, which is surrounded by contractile cells. The duct is coiled and encapsulated within a sheath of a connective tissue of the tunica vaginalis, and it has three distinct functions. One of these is sperm transport, which is achieved by contractions of the smooth muscle cells surrounding the tubule, and by continuous production and movement of fluid from the testis. The second function of the epididymis is maturation of spermatozoa, as during their epididymal transit the spermatozoa acquire a change in their motility pattern and become able to bind to and penetrate the oocyte. The third major function is the storage of sperm, which occurs in the caudal region. (1, 2)

The maturation of spermatozoa is connected to a myriad of biochemical and molecular changes as they traverse the epididymis. These changes are thought to be caused by ion transport and attachment of proteins secreted in the lumen of the epididymal duct (1, 3, 4). The maturational changes commence in the initial segment, which secretes proteins at a greater rate than other parts of the epididymis (5). The importance of the proteins secreted in the initial segment is confirmed by the fact that when the segment is absent, as, for example, in Ros1 tyrosine kinase receptor knockout mice, the animals are sterile even though other parts of the male reproductive system are unaffected (6). Similarly, in transgenic mice expressing the SV40 virus T Ag in the initial segment, the epithelium in this region is slightly hyperplastic, and its protein production is altered, resulting in infertility (7). Recently, several genes expressed in the epididymis having a putative role in sperm maturation have been characterized. Examples are glutathione peroxidase 5 (Gpx5) (8, 9, 10, 11), Ros1 (6, 12, 13), murine epididymal retinoic acid binding protein (Erabp) (14), human epididymal proteins types 1–6 (15, 16, 17), acidic epididymal glycoprotein (Aeg1) (18), and Bin1b (19), and many remain unknown (20, 21).

As proteins secreted into the epididymal lumen are essential for the maturation of spermatozoa, interference with their function represents a promising area with regard to male contraception. The ideal male contraceptive would allow normal testicular sperm production, but not allow sperm to fertilize an oocyte in vivo. The method should also be fully and rapidly reversible. Theoretically, disturbance of posttesticular maturation events essential for the production of fertile spermatozoa should provide such a situation.

Enlarged databases for expressed sequence tags (ESTs) as well as genome databases represent important sources for discovery of novel genes with tissue-specific expression profiles. The information can be explored via the internet at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/). Gene expression information available in silico combined with the data available in protein domain databases and in other computational bioinformatics databases represent a great deal of information for predicting the tissue specificity of gene expression, for analyzing cDNA and gene structure, as well as for predicting the structure and putative function of a novel protein. Most protein signature databases use sequence-motif methods, some focus on divergent domains (e.g. Pfam), some focus on functional site domains (e.g. PROSITE), and others focus on protein family signatures. Homologous proteins can also be directly sought from the SWISS-PROT database. The computational tools for analyzing protein topology and predicting posttranslational modifications are based on statistical analysis and on combining several different databases, organized as an expert system with a knowledge base that is a collection of "if-then"-type rules. Altogether, in silico biology is becoming one of the most rapidly expanding tools in modern biotechnology.

In this study we intended to identify and characterize novel epididymis-specific genes. The genes were initially discovered through in silico analysis of ESTs at NCBI. The structures of the cDNAs, as well as expression profiles and hormonal regulation of the genes were studied using mouse tissue samples in vitro, while the putative functions and genomic structures of the cDNAs were defined in silico.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Discovering the Clones and Identification of the cDNA and Amino Acid Sequences
The UniGene database at NCBI (http://www.ncbi.nlm.nih.gov) includes a mouse epididymis library provided by the RIKEN Genome Exploration Research Group (Tsukuba, Japan), currently consisting of 4976 EST clones. This library was searched in silico for new putative epididymis-specific cDNA sequences by means of two criteria: a high number of ESTs of a given gene was considered to indicate high expression, and if the EST had not been identified in any other EST libraries of the UniGene database, the clone was considered to be putatively epididymis specific. In the present study we characterized five such ESTs provided by RIKEN. The UniGene cluster ID, UniGene code of the EST clones characterized, given name, cDNA size, and chromosomal location are presented in Table 1Go.


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Table 1. UniGene Cluster ID, EST Codes, Given Names, cDNA Sizes, and Chromosomal Locations of the EST Clones Characterized

 
The EST clones were thoroughly sequenced to confirm the accuracy of the sequence present in the UniGene database and to obtain the full-length sequences of the inserts. All of the cDNA sequences received from RIKEN contained a poly-A tail and a polyadenylation signal, confirming proper termination of the 3' ends of the clones. Furthermore, to ensure that we had the full-length 5' ends of the cDNAs, we performed several rapid amplification of 5' cDNA ends (5'-RACE) analyses for all EST clones. Of the clones received, AV381357, AV380943, and AV378971 were considered to be full length, as no 5'-RACE product was obtained, and the size of the cDNA correlated with the mRNA size obtained by Northern analysis. For AV381126, the 5'-RACE approach gave a product of 15 bp, and for AV381130, a product of 175 bp was obtained. The 5'-sequences of the EST inserts were extended accordingly. The cDNA sequences of all of the genes are shown in Figs. 1–5GoGoGoGoGo.



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Fig. 1. The cDNA (Upper Part) and Defined Peptide (Middle) Sequences, and the Intron-Exon Structure (Lower Part) of Spint4

In the cDNA sequence, the ATG codon for the first methionine (bold, underlined), polyadenylation signal (underlined), and poly-A tail (italics) are shown. The protein contains a putative signal sequence (underlined) with a cleavage site between amino acids 24 and 25. The protein also contains a 51-amino-acid Kunitz pancreatic trypsin inhibitor consensus sequence (bold, italics).

 


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Fig. 2. The cDNA (Upper Part) and Defined Peptide (Middle) Sequences, and the Intron-Exon Structure (Lower Part) of Rnase9

In the cDNA sequence, the ATG codon for the first methionine (bold, underlined), polyadenylation signal (underlined), and poly-A tail (italics) are shown. RNASE9 contains a putative signal sequence (underlined) with a cleavage site between amino acids 26 and 27. The protein contains a 103-amino-acid-long sequence showing 55% similarity to the pancreatic RNase region of a predicted murine protein to olfactory receptor MOR104-3 (bold, italics).

 


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Fig. 3. The cDNA (Upper Part) and Defined Peptide (Middle) Sequences, and the Intron-Exon Structure (Lower Part) of Rnase10

In the cDNA sequence the ATG codon for the first methionine (bold, underlined), polyadenylation signal (underlined), and poly-A tail (italics) are shown. RNASE10 contains a 115-amino-acid-long fragment with 47% similarity to putative human pancreatic RNAse (underlined).

 


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Fig. 4. The cDNA (Upper Part) and Two Defined Putative Peptide (Middle) Sequences, and the Intron-Exon Structure (Lower Part) of AV381130

In the cDNA sequence ATG codons for the first methionines (bold, underlined), polyadenylation signal (underlined), and poly-A tail (italics) are shown. The putative translation product of AV381130, shown uppermost, contains a 28-amino-acid-long fragment, and the other possible product (shown below) contains a 50-amino-acid-long fragment homologous to an amidase region of a hypothetical zinc finger protein (underlined). Two short regions of the cDNA sequence (41 and 40 bp in length) could not be found in the genome databases, and therefore, their exact localization in the genomic sequence could not be confirmed. These fragments are represented in the figure as exon 1' and exon 3'.

 


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Fig. 5. The cDNA (Upper Part) and Defined Peptide (Middle) Sequences, and the Intron-Exon Structure (Lower Part) of AV381126

In the cDNA sequence ATG codon for the first methionine (bold, underlined), polyadenylation signal (underlined), and poly-A tail (italics) are shown. AV381126 contains an 80-amino-acid-long fragment homologous to a hypothetical protein XM149964 (underlined).

 
Translation of the cDNAs into amino acid sequences revealed that the mRNAs coded for peptides of 62–184 amino acids in length (Figs. 1–5GoGoGoGoGo). Both cDNA and peptide sequences were used in BLAST analysis against the Standard nucleotide-nucleotide BLAST (blastn) and Standard protein-protein BLAST (blastp; http://www. ncbi.nlm.nih.gov/BLAST/) databases. PROSITE and PFAM HMM were used to predict the presence of various protein patterns and profiles (http://us.expasy.org). The analysis showed that all of the cDNAs encoded novel mouse proteins that are not yet characterized. However, certain similarities with known proteins and protein domains were found in three of the five peptides, which helped us to suggest putative functions for these proteins.

AV380943 contains a signal sequence that is putatively cleaved between amino acids 24 and 25. The other profound feature of this sequence is that the protein contains a 51-amino-acid Kunitz pancreatic trypsin inhibitor consensus sequence (Fig. 1Go). Hence, the protein is expected to be a novel epididymal protease inhibitor secreted into the lumen of the epididymal duct, and it was therefore named SPINT4 (serine protease inhibitor, Kunitz type 4). Similarly to SPINT4, AV378971 contains a signal sequence that is putatively cleaved between amino acids 26 and 27 (Fig. 2Go). This protein is predicted to be a novel secreted epididymal ribonuclease (RNase). Based on the 103- amino-acid-long region with significant similarity (55%) to the pancreatic RNase region of a mouse olfactory receptor protein, MOR104-3 (GenBank accession no. XM139041), the protein was named RNASE9 (RNase A family 9). Another putative epididymal RNase was identified on the basis of the fact that the AV381357 sequence contains a 115-amino-acid-long fragment showing 47% similarity to a putative human pancreatic RNase (Fig. 3Go), and the protein was therefore named RNASE10 (RNase A family 10). RNASE10 has a possible transmembrane helix from amino acids 39–69, but was not predicted to have a signal sequence for secretory function. The peptide sequences of RNASE9 and RNASE10 also showed significant similarity to those of several other RNases (Fig. 6Go). The names of the discovered genes and the proteins encoded by them were approved by The Jackson Laboratory (Bar Harbor, ME) nomenclature committee.



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Fig. 6. Tissue Distribution of the Characterized Genes According to Quantitative Real-Time RT-PCR

Spint4 is highly expressed in corpus. Rnase9 is expressed especially in caput and corpus. Rnase10 is expressed mainly in initial segment, with residual expression in caput. AV381130 is expressed in caput and corpus, and AV381126 is especially in caput and corpus, with a low level of expression in cauda.

 
For AV381130 two possible peptide sequences could be predicted, both with regions showing similarity to the amidase region of a hypothetical zinc finger protein, FLJ14011 (GenBank accession no. XM145320). The two possible peptide sequences are shown in Fig. 4Go. The AV381126 peptide contains a 50-amino-acid-long region with significant similarity (43%) to a predicted mouse protein, LOC2110321 (GenBank accession no. XM149964) of unknown function. A hydrophobic region was also identified in AV381126 between amino acids 99 and 110 (Fig. 5Go). These proteins did not show any significant homology with known genes, and no putative functional domains could be found.

Gene Structure and Chromosomal Localization
To determine the chromosomal locations and exon-intron structures of the genes, the cDNA sequences were blasted into the NCBI Mouse Genome Resources and into the Wellcome Trust Sanger Institute Mouse Genome Server. All five cDNAs were found to consist of relatively short genomic fragments composed of two to nine exons, expanded mainly by short introns (the exception being AV381130), which is characteristic of highly expressed genes (22) (Figs. 1–5GoGoGoGoGo). The two putative RNases are both located in chromosome region 14C1, only 28 kb apart. Furthermore, nine additional RNases were located at the same genomic region: Rnase4 (GeneBank accession no. AB041044), RnaseK6 (AK020595), eosinophilic cationic-type RNase 4 (Ear4; AF017259), eosinophilic cationic-type RNase 5 (Ear5; AF017260), RNase pancreatic precursor (Rib1; M27814), angiogenin precursor (Ang; U22516), angiogenin-related protein precursor (Angrp; U22519), a predicted protein similar to human epididymal secretory protein E3 (HE3{alpha}), and eosinophil- associated RNase 11 (AY015178). These genes thus form a dense gene cluster for the RNase A family. Spint4 is located in chromosome region 2H3, AV381130 in 7A1, and AV381126 in 7D2, while no clusters of structurally similar genes were identified in these regions.

Characterization of mRNA Expression
To investigate the tissue distribution of gene expression, Northern blot hybridization and quantitative RT-PCR were carried out on total RNA extracted from different tissues. For all of the genes, one major mRNA was identified in Northern blot analysis, of the expected size determined from the cDNAs characterized. Both the Northern and RT-PCR analyses showed that each of the genes was highly expressed in the epididymis. No signal was found in any of the other tissues examined by Northern blot analysis (data not shown), and real-time RT-PCR analyses further confirmed the epididymis specificity of the gene expression (Fig. 6Go). The real-time RT-PCR analyses indicated that the level of expression outside epididymis was below or at the level of detection limit for the different genes, and were always less than 1% of that found in the defined epididymal regions.

We next determined whether the expression of the newly discovered genes was regulated by androgens or other factors released by the testis. For this purpose, we analyzed gene expression in the epididymal caput, corpus, and cauda regions of gonadectomized mice (1, 4, 7, and 14 d after gonadectomy), including mice that were given testosterone replacement therapy immediately after gonadectomy (treated for 7 or 14 d). The data showed that the expression of Spint4, Rnase9, Rnase10, and AV381130 had clearly diminished 1–3 d postgonadectomy and had completely disappeared 7 d after gonadectomy (Fig. 7Go). Interestingly, the expression of Rnase10 disappeared within 4 h postgonadectomy (data not shown) and was not restored by testosterone replacement (Fig. 7Go). This suggests that the expression of Rnase10 is not regulated by testosterone, but is likely to be controlled by some other testicular factors. The expression of the other three genes ceased after a longer period of time and was efficiently restored by testosterone replacement, suggesting androgen dependence (direct or indirect) of their expression. Furthermore, there was only mild alteration in the expression of AV381126 after gonadectomy (Fig. 7Go), which may be a result of shrinkage of the epithelial cell layer. Hence, we concluded that the expression of AV381126 is not dependent on testicular factors, and therefore, it served as a good control for studies on hormonal regulation.



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Fig. 7. Northern Hybridization Analysis in Testis- and Androgen-Dependent Expression of the Characterized Genes

Expression was analyzed in nongonadectomized (d 0) mice, and 1–14 d after gonadectomy (1d, 3d, 7d, 14d). Furthermore, expression was analyzed in gonadectomized mice that had received testosterone replacement therapy for 7 and 14 d immediately after the operation (7d+T, 14d+T). The expression of Spint4, Rnase9, Rnase10, and AV381130 diminished and ceased after gonadectomy. The expression of Spint4, Rnase9, and AV381130 was clearly restored by testosterone replacement, whereas the expression of Rnase10 was not. In addition, there was only mild alteration in the expression of AV381126 after gonadectomy.

 
RT-PCR analyses further indicated that none of the genes characterized was expressed in immature epididymis, whereas detectable expression for Spint4, Rnase9, Rnase10, and AV381126 was found at the age of 17 d. The mRNA for AV381130 appeared somewhat later and was not detected until 30 d of age (Fig. 8Go).



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Fig. 8. RT-PCR Analysis of Epididymal Gene Expression at Various Ages

The expression of Spint4, Rnase9, and Rnase10, and AV381126 was detected in the epididymis at the age of 17 d, whereas the expression of AV381130 was not detected until the age of 30 d.

 
Localization of mRNA Expression in the Epididymis
To determine the exact sites of expression of the genes, we performed in situ hybridization analysis. Interestingly, for some of the genes the signal was seen only in well defined regions of the epididymis, indicating highly segment-specific expression (Figs. 9Go and 10Go). Spint4 is expressed in a fraction of epithelial cells lining the duct at segment IV, while in segment V of the distal caput (23) and in a narrow region of the proximal part of the corpus expression is seen in almost every epithelial cell. Then, toward the corpus epididymis, the expression vanishes with a chess board-type appearance (Fig. 10Go). Rnase10 is expressed in only a part of the initial segment [segment I (23)]. The expression level of Rnase10 is extremely high in the small segment where it is expressed, as indicated by the fact that the slides were slightly overexposed despite the very short exposure time used. Furthermore, the expression of Rnase10 diminished toward the distal caput in a similar fashion as noted for Spint4. AV381130 is highly expressed in segments II–V of the caput epididymis, while AV381126 and Rnase9 are more widely expressed in the different epididymal regions. Rnase9 and AV381126 showed high expression in the distal caput region, and the expression continued throughout the corpus, albeit gradually vanishing. For Rnase9, some signal was detected even in the caudal region.



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Fig. 9. Localization of mRNAs in the Mouse Epididymis Using in Situ Hybridization

The images were counterstained with hematoxylin, whereas the hybridization signal appears black. Spint4 is expressed in the epithelial cells of the distal caput and early corpus. Rnase10 is expressed by only a part of the initial segment. AV381130 is expressed in segments II–V of the caput epididymis. Rnase9 and AV381126 show high expression in the distal caput region; expression continues throughout the corpus, gradually vanishing, and for Rnase9 some signal was detected even in the cauda region.

 


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Fig. 10. Localization of mRNAs in the Mouse Epididymal Epithelia by in Situ Hybridization

The images were counterstained with Hoechst stain (blue; no counterstain in F, I, and J); the specific hybridization signals appear white. Very strong signals were seen in the epididymal epithelial cells. A and B, Spint4; C and D, Rnase9; E and F, Rnase10; G and H, AV381130; I and J, AV381126. Most of the genes show a chess board-type expression at the border areas.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The public EST and genome databanks contain a large amount of unexplored information about gene expression and structure. Currently the largest and most widely used EST database is UniGene (24), which also identifies the cDNA library from which an individual EST originates. The ESTs in UniGene are organized into clusters, each of which is composed of transcripts that share overlap in sequence with at least one other member of the same cluster, but not with members of any other cluster. Thus, each cluster is likely to contain the sequence information of transcription products of a single gene. The RIKEN Genome Exploration Research Group has established a mouse epididymis cDNA library (25, 26, 27) containing a total of 4976 EST clones. This library is included in the UniGene database and is the only epididymal library in UniGene. Of the ESTs in the library, several sequences are currently listed as sequences found mainly in the epididymal library. The strategy used in the present work is an efficient and powerful way to discover novel epididymis-specific genes by analyzing the UniGene database in silico. A similar strategy in the search for gonad-specific genes has recently been described (28, 29, 30, 31). Using this strategy, we have identified the cDNA and peptide sequences, genomic structures, and expression patterns of five novel epididymis- specific genes. Some predictions of the functions of the novel genes identified could be made, but more detailed functional studies are needed. The epididymis specificity and region-specific expression of the mRNAs are consistent with their putative role in creating the microenvironment necessary for sperm maturation in the epididymis. Furthermore, none of the genes was expressed in the immature epididymal epithelium, but, rather, they were expressed at the time of functional maturation of the epididymis, occurring after the postnatal d 16 (32).

Proteases are known to have important roles in multiple physiological processes. It is also known that regulation of the function of proteases by their inhibitors is important for maintaining homeostasis of protein degradation (33). Previous results suggest that some of the modifications in spermatozoa during their maturation result from very specific proteolytic processing of sperm surface proteins (34, 35, 36). Supporting the idea that proteolytic processing occurs in the epididymis, several proteases have been found to be present in epididymal fluid (37). Several proteases have also been found attached to the sperm surface membrane (36, 38). Hence, protease inhibitors might have an important role in the epididymis in inhibiting the proteolytic activities of proteases involved in the acrosome reaction until they are needed. Equally, it has long been suggested that protease inhibitors could actually be involved in the process of capacitation and fertilization (39). In addition, the integrity of the very tight blood-epididymal barrier must be controlled by a very specific process involving equilibrium between proteases and protease inhibitors (37).

In this report we have described the discovery of SPINT4, a new epididymis-specific protein containing a Kunitz pancreatic trypsin inhibitor domain in its peptide sequence, suggesting that the protein is a protease inhibitor. Certain protease inhibitors have been characterized previously in the epididymis. Cystatin 8 (Cst8) (40, 41) is a cysteine protease inhibitor expressed in the epididymis, testis, and anterior pituitary gland. Serine proteinase inhibitor 5 (Serpina5), also known as protein C inhibitor (42), is a nonspecific serine protease inhibitor found as a plasma protein and present also at high concentrations in the male reproductive tract. Cystatin 11 (Cst11) is an epididymis-specific cysteine protease inhibitor (43), HE4 is a human epididymal protein containing two WAP-type protease domains (44), and Eppins are proteins containing both the Kunitz- and WAP-type protease inhibitor domains. Eppin1 is expressed in the testis and epididymis, and Eppin2 is expressed only in the epididymis (45). In addition to SPINT4, Eppins are the only epididymal proteins shown to have a Kunitz protease inhibitor domain. SPINT4 as well as Eppin-1 contain signal sequences, whereas Eppin-2 seems to be an intracellular protein (45). The roles of these inhibitors in the epididymis have not been confirmed. However, it has been found that male mice lacking protein C inhibitor (Serpina5) are infertile, apparently as a result of abnormal spermatogenesis because of destruction of the Sertoli cell barrier. In addition to the testicular changes there were also changes in the epididymal duct; the cells of the lining epithelium were of irregular shape, and the epithelium was missing at some sites (42).

RNASE9 and RNASE10 both have a pancreatic RNase A domain in their sequence. The locations of the genes showed them to be present in an RNase cluster on chromosome 14. Eosinophilic cationic-type RNases 4 and 5 (Ear4 and Ear5) are also located in the cluster. Ear4 and Ear5 show exceptionally high homology with each other and are therefore believed to have emerged via multiple duplications of a single murine ribonuclease gene (46). Rnase9 and Rnase10 do not show such a high homology with each other or with other RNases in the gene cluster and therefore cannot be considered to be duplicates of a known RNase gene. Little is known about the functions of RNases in the epididymis. Gupta et al. (47) showed RNase II activity in monkey epididymides, especially in the caudal region. The activity was markedly reduced after castration and was partially restored by androgen replacement. Androgen dependency is consistent with our findings on Rnase9. However, Rnase10 seems to have a more sensitive testicular regulation system, as its expression disappeared within a very short time after gonadectomy, and it was not restored by testosterone replacement. An RNase has also been identified in bovine semen; it is expressed in bull ampullary glands and seminal vesicles, and it has been demonstrated to have aspermatogenic, embryotoxic, antitumor, and immunosuppressive activities (48). Immunosuppressive substances such as RNases could have a role in the protection of spermatozoa in the female reproductive tract, and it has also been shown that eosinophilic ribonucleases can inhibit retroviral transduction of human target cells (49). Together these findings indicate that there is a possibility that certain RNases have diverged to promote specific host defense-related activities, and RNASE9 and RNASE10 are potential new members of epididymal proteins involved in host defense.

In summary, we have identified five novel genes of the epididymal epithelium with segment-specific expression patterns. Four of the five genes identified were under regulation by testicular factors and were up-regulated in the epididymis, in line with increased postnatal maturation of epididymal epithelium. One of the proteins is a putative protease inhibitor in epididymal fluid, hence putatively involved in sperm maturation processes. Two of the proteins exhibit structural features of RNases, and are putatively novel members of epididymal proteins involved in host defense. To explore fully the roles of these proteins in epididymal physiology, the development of knockout mouse models would be a preferred approach.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Riken EST Clones
We identified 4976 EST sequences in the RIKEN full-length enriched, adult mouse male epididymis library deposited at the NCBI database (http://www.ncbi.nlm.nih.gov/UniGene/library.cgi?ORG=Mm&LID=2606). We used in silico analysis to search for ESTs that have a high expression level and are expressed only in the epididymis. The EST clones were provided by the RIKEN Genomic Sciences Center, RIKEN Yokohama Institute (Yokohama, Japan). The clones were obtained in pBluescript I KS+ vectors (Stratagene, La Jolla, CA), modified at the cloning site. The clones were named according to the UniGene identification code: AV380943, AV378971, AV381357, AV381130, and AV381126.

Sequencing
The EST clones were sequenced with an ABI PRISM 377-XL DNA sequencer using ABI PRISM BigDye Terminators version 3.0 Cycle Sequencing Kits (PE Applied Biosystems, Foster City, CA). The reactions were performed three times in the 5' to 3' direction, twice using the primer Seq1 and once using the T7 primer (Table 2Go). Furthermore, the inserts were sequenced once in the 3' to 5' direction using a T3 primer from the vector.


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Table 2. A, Primers Used in the 5'-RACE and RT-PCR; B, Primers Used for Quantitative and Nonquantitative RT-PCR

 
5'-RACE
All of the selected EST clones contained a poly-A tail; thus, we could conclude that the clones contained the full 3'-ends of the cDNAs. We then used the 5'-RACE approach to obtain the full 5'-end of the cDNA. A GeneRacer Kit (Invitrogen, Groningen, The Netherlands) was used for clone AV381126, and SMART RACE cDNA Amplification Kits (Clontech, Palo Alto, CA) were used for clones AV380943, AV378971, AV381357, and AV381130. Both types of kit were used according to the manufacturers’ protocols. The gene-specific primers used in the RT-PCR were rev1a for AV381126, rev1b for AV380943, rev1c for AV378971, rev1d for AV381130, and rev1e for AV381357 (Table 2Go). The RT-PCR products generated were cloned into pCR 4-TOPO vectors (Invitrogen) and sequenced as describe above (PE Applied Biosystems) with the primers used for RT-PCRs.

Bioinformatics
The cDNA sequences of the EST clones were translated into the corresponding peptide sequences, which were analyzed by the means of several computational bioinformatics tools. TMpred (http://www.ch.embnet.org), DAS (http://www.sbs.su.se), and HMMTOP (http://www.enzim.hu) were used to predict the transmembrane regions and protein orientation; PROSITE and PFAM HMM were used to predict the presence of various protein patterns and profiles (http://us.expasy.org); SignalP (http://www.cbs.dtu.dk) was used to analyze the presence of putative signal peptides and their cleavage sites; PSORT II (http://psort.nibb.ac.jp) was used to predict protein sorting signals and intracellular localization; and GOR IV (http://pbil.ibcp.fr) was used to predict the secondary structure of the proteins. The cDNA sequences obtained were also blasted into the NCBI Mouse Genome Resources (http://www.ncbi.nlm.nih.gov/genome/guide/mouse/) and into the Wellcome Trust Sanger Institute Mouse Genome Server (http://www.ensembl.org/mus_musculus/) to reveal chromosomal locations and intron exon structures of the genes.

Mouse Models and RNA Extraction
Wild-type FVB/N male mice were used throughout the study, and all mice were handled in accordance with the institutional animal care policies of University of Turku (Turku, Finland). The mice were specific pathogen free and were fed complete pelleted chow and tap water ad libitum in a room with controlled light (12 h of light, 12 h of darkness) and temperature (21 ± 1 C). To analyze tissue distribution of the genes, 2- to 3-month-old mice were used for RNA extraction. For these studies the mice were killed by cervical dislocation, and various tissues (hypothalamus, brain, heart, muscle, spleen, pancreas, liver, kidney, adrenal gland, seminal vesicle, testis, vas deferens, and caput, corpus, and cauda epididymis) were dissected out, snap-frozen in liquid nitrogen, and stored at -70 C. To characterize the androgen dependency of gene expression, 24 sexually mature male mice were gonadectomized under anesthesia. Thereafter, the epididymides were collected 1, 3, 7, and 14 d after gonadectomy (four mice/group). Furthermore, eight gonadectomized male mice were treated with a supraphysiological dose of testosterone for 7 and 14 d; both groups contained 4 mice. The testosterone replacement therapy was performed by inserting a SILASTIC brand (Dow Corning, Midland, MI) tube (2 mg of testosterone) sc in the back of the mice. For analyzing gene expression in the epididymis at different ages (0, 4, 7, 13, 17, 20, 30, 40, and 60 d), adult and juvenile animals were killed by decapitation or cervical dislocation. The epididymides were dissected out, immediately frozen in liquid nitrogen, and stored at -70 C, and total RNA from the tissues was isolated using the single step method (50).

Northern Hybridization
For Northern blot analysis, 15 µg denatured total RNA were resolved on a 1% denaturing agarose gel and transferred onto nylon membranes (Hybond-XL, Amersham Pharmacia Biotech, Little Chalfont, UK). The membranes were hybridized with the [{alpha}-32P]CTP-labeled cDNAs of the EST clones using standard techniques. To generate the probes, the cDNA of each EST was cut from the vector by SfiI restriction enzyme (Promega, Madison, WI), and used for labeling. Hybridization signals were detected by autoradiography using x-ray film (Fuji Film Ltd., Tokyo, Japan) or a phosphorimager (Fuji). Hybridizations with a cDNA fragment for 28S rRNA was used to control equal loading of the RNA.

RT-PCR
For analyzing the appearance of gene expression in epididymis at the different ages, RT-PCR analysis was carried out. For the analysis, 100 ng deoxyribonuclease I (amplification grade, Invitrogen)-treated total RNA was used, and the reactions were performed using the QuantiTect SYRB-Green RT-PCR Kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. The primers and annealing temperatures used are described in Table 2Go. After 35 cycles, the amplification products were resolved in agarose gel and visualized with ethidium bromide.

For analyzing the tissue distribution of gene expression, quantitative real-time RT-PCR measurements were performed using the DNA Engine Opticon system (MJ Research, Inc., Waltham, MA), with continuous fluorescence detection. One hundred nanograms of deoxyribonuclease I (Invitrogen)-treated total RNA were used for each tissue, and the reactions were performed using a QuantiTect SYRB-Green RT-PCR Kit (Qiagen), according to the manufacturer’s instructions. The samples and standard curves were run in triplicate, and analyses were repeated with identical results. The relative standard curve method (51) was used to calculate relative gene expression, and RT-PCR results for the ß-actin were used as endogenous normalization controls. The primers and annealing temperatures were the same as those described for the nonquantitative RT-PCR.

In situ hybridization
The epididymides were excised free from adipose tissue, fixed in 4% paraformaldehyde (PFA), and embedded in paraffin. Five-micrometer-thick sections were cut and rehydrated in xylene and a descending ethanol series. This was followed by denaturation [20 min in 0.2 N HCl and 15 min in 2x standard saline citrate (SSC) at 70 C], fixation (10 min in 4% PFA), proteinase K treatment (1 mg/liter in 50 mM Tris-HCl and 5 mM EDTA, pH 8.0) for 15 min at 37 C, another fixation (10 min in 4% PFA), acetylation (5 min in 0.25% acetic anhydride in 0.1 M triethanolamine and 5 min in 0.5% acetic anhydride in 0.1 M triethanolamine), and dehydration in ethanol. The slides were then stored at -72 C until hybridization. The cDNAs in pBluescript I KS+ vectors were cut with appropriate restriction enzymes to obtain DNA fragments containing 100- to 750-bp fragments of the EST cDNA and either the T3 or T7 promoter site from the vector. The DNA fragments were used as templates for sense and antisense [{alpha}-35S]UTP-labeled probes generated by in vitro transcription with T3 and T7 RNA polymerases (Promega).

The probes were denatured by heating at 70 C for 5 min, and they were dissolved in hybridization buffer [50% formamide, 0.3 M NaCl, 20 mM Tris-HCl, 5 mM EDTA, 50 mM dithiothreitol (DTT), 0.5 mg tRNA/ml, 1x Denhardt’s solution, and 10% dextran sulfate] to obtain a specific activity of about 100,000 cpm/µl. The probes were applied to the tissue sections and overlayed with Parafilm, and the slides were incubated in a moisturized chamber at 55 C overnight. After hybridization, the slides were washed with 2x SSC/50% formamide/10 mM DTT for 30 min at 55 C and with 0.2x SSC/50% formamide/10 mM DTT for 30 min at 55 C. Thereafter, the sections were treated with RNase A solution (10 µg/ml in 0.5 M NaCl, 10 mM Tris-HCl, and 5 mM EDTA, pH 8.0) for 30 min at 37 C, washed again with 2x SSC/50% formamide/10 mM DTT for 15 min at 55 C, dehydrated with ethanol, and air-dried. The slides were then dipped in NTB2 emulsion (Eastman Kodak Co., Rochester, NY) and exposed in the dark at 4 C for 1–5 d. The slides were developed with Dektol developer (Eastman Kodak Co.) for 2 min, rinsed in distilled water, fixed for 5 min in Kodak Fixer (Eastman Kodak Co.), and rinsed in distilled water. The slides were counterstained with hematoxylin and Hoechst 33258 (Sigma-Aldrich Corp., St. Louis, MO), after which they were mounted with fluorescent mounting medium (DAKO Corp., Carpinteria, CA). Hybridization with a sense probe was used as a control, and no hybridization signals were detected in any of the slides analyzed (results not shown).


    ACKNOWLEDGMENTS
 
We thank Johanna Vesa for technical assistance, and Dr. Yoshihide Hayashizaki from RIKEN Genomic Sciences Center for providing the EST clones.


    FOOTNOTES
 
This work was supported by grants from the Academy of Finland, Turku University Foundation, and Turku Graduate School of Biomedical Sciences.

Abbreviations: DTT, Dithiothreitol; EST, expressed sequence tag; PFA, paraformaldehyde; RACE, rapid amplification of cDNA ends; RNase, ribonuclease; SSC, standard saline citrate.

Received for publication January 9, 2003. Accepted for publication August 5, 2003.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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