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BOR - Papers in Press, published online ahead of print July 30, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.017566
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BIOLOGY OF REPRODUCTION 69, 1750–1762 (2003)
DOI: 10.1095/biolreprod.103.017566
© 2003 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Gene and Protein Expression in the Epididymis of Infertile c-ros Receptor Tyrosine Kinase-Deficient Mice1

Trevor G. Cooper2,4, Andrea Wagenfeld3,4, Gail A. Cornwall5, Nelson Hsia5, Sin Tak Chu6, Marie-Claire Orgebin-Crist7, Joel Drevet8, Patrick Vernet8, Cosmina Avram4, Eberhard Nieschlag4, and Ching-Hei Yeung4

Institute of Reproductive Medicine of the University,4 D-48129 Münster, Germany Department of Cell Biology and Biochemistry,5 Texas Tech University Health Sciences Center, Lubbock, Texas 79430 Institute of Biological Chemistry,6 Academia Sinica, Taipei, Taiwan, Republic of China Center for Reproductive Biology Research,7 Vanderbilt Medical Center, Nashville, Tennessee Department of Biology,8 Blaise Pascal University, CNRS UMR 6547 GEEM, Reproduction and Development, 63177 Aubière Cedex, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic male mice bearing inactive mutations of the receptor tyrosine kinase c-ros lack the initial segment of the epididymis and are infertile. Several techniques were applied to determine differences in gene expression in the epididymal caput of heterozygous fertile (HET) and infertile homozygous knockout (KO) males that may explain the infertility. Complementary DNA arrays, gene chips, Northern and Western blots, and immunohistochemistry indicated that some proteins were downregulated, including the initial segment/proximal caput-specific genes c-ros, cystatin-related epididymal-spermatogenic (CRES), and lipocalin mouse epididymal protein 17 (MEP17), whereas other caput-enriched genes (glutathione peroxidase 5, a disintegrin and metalloproteinase [ADAM7], bone morphogenetic proteins 7 and 8a, A-raf, CCAAT/enhancer binding protein ß, PEA3) were unchanged. Genes normally absent from the initial segment ({gamma}-glutamyltranspeptidase, prostaglandin D2 synthetase, alkaline phosphatase) were expressed in the undifferentiated proximal caput of the KO. More distally, lipocalin 2 (24p3), CRISP1 (formerly MEP7), PEBP (MEP9), and mE-RABP (MEP10) were unchanged in expression. Immunohistochemistry and Western blots confirmed the absence of CRES in epididymal tissue and fluid and the continued presence of CRES in spermatozoa of the KO mouse. The glutamate transporters EAAC1 (EAAT3) and EAAT5 were downregulated and upregulated, respectively. The genes of over 70 transporters, channels, and pores were detected in the caput epididymidis, but in the KO, only three were downregulated and six upregulated. The changes in these genes could affect sperm function by modifying the composition of epididymal fluid and explain the infertility of the KO males. These genes may be targets for a posttesticular contraceptive.

epididymis, gamete biology, gene regulation, male reproductive tract, sperm maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The proto-oncogene c-ros is an orphan tyrosine kinase receptor usually classified as a member of the subfamily of insulin receptors [1], yet neither its ligand nor substrate is known. Its role has been examined by use of antisense c-ros oligonucleotides that decrease synthesis of extracellular matrix components in tissues including heparin sulphate proteoglycan and laminin [2, 3] and have suggested a role for the gene in epithelial-mesenchyme interactions. Stimulation of chimeric c-ros receptors with relevant ligand has indicated that activated mitosis, morphological transformation, and epithelial cell migration are associated with phosphorylation of early signaling proteins, including MAPK, insulin receptor substrate 1, phospholipase C{gamma}, and inositol triphosphate kinase [4–6]. Other tyrosine kinase receptors involved in these processes include the oncogenes ret and met. SHP-1 is a c-ros-specific protein tyrosine phosphatase that is considered an important downstream regulator of c-ros action, which colocalizes with c-ros in the proximal epididymis, and mice with a natural mutation in this gene display an abnormal proximal caput epithelium [7]. The fertility of the males is reduced but complicated by deficiencies in both steroidogenic and spermatogenic processes [8].

In most organs, including the kidney, intestine, and lung, c-ros disappears soon after birth, but in the proximal part of the epididymis, it persists in the wild-type adult [9, 10]. Male mice homozygous for a deletion in this gene (knockout, KO) are sterile [11] and the initial segment, the most proximal part of the caput epididymidis, fails to differentiate. In contrast, heterozygous males (HET) are fertile and their epididymis contains an initial segment. The infertility by natural mating [11] is foremost due to a failure of sperm to reach the oviduct [12]. This failure reflects the abnormally angulated sperm tails, which are a physical demonstration of swollen cells [13] caused by impaired acquisition of volume regulation during epididymal transit [14]. This has been suggested to reflect reduced epididymal provision of osmolytes as a consequence of a downregulated glutamate transporter in the KO [15]. The genes that are either underexpressed or overexpressed in the caput of the mutant may illuminate mechanisms whereby the initial segment influences spermatozoa during their normal maturation.

The initial segment expresses a number of specific genes [16], some of which have been implicated in sperm maturation [17, 18]. In the mouse, these include BMP7, BMP8a [19], MEP17 [20], PEA3 [21, 22], A-raf [23], and cystatin TE-1 [24]. Other genes are expressed in the initial segment as well as proximal caput (c-ros [11]), CRES (cystatin-related epididymal-spermatogenic, or cystatin 8 [25], B-myc [26]) or the initial segment and whole caput (GPX5 [27–29], the CCAAT/enhancer binding protein C/EPBß [30], the disintegrin and metalloprotease protein, ADAM 7 [31]), or the initial segment and corpus epididymidis (lipocalin 2 [32]).

In this study, cDNA arrays and gene chips were used to examine general gene expression in the caput epididymidis of fertile wild-type and HET mice and infertile c-ros KO mice, and Northern blotting, immunohistochemistry, and Western blotting were used to investigate the expression of initial segment-specific markers. The expression of epididymal proteins lipocalin 2 (lipocalin 2 [32]), CRISP1 (formerly MEP7), PEBP (formerly MEP9), and mE-RABP (murine epididymal retinoic acid binding protein, formerly MEP10) [33] were also examined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimentation

A colony of transgenic c-ros mice was established from heterozygous (HET) animals that were generated by targeted deletions of c-ros [9, 11]. The breeding pairs, derived from crossbred C57BL6 and Ola129 strains, were generously donated by Dr. E. Riethmacher and Prof. Dr. C. Birchmeier from the Max-Delbrück Center of Molecular Medicine, Berlin, Germany. The animals were kept at 22°C under a cycle of 12L:12D and were provided with water and pelleted food (Altromin GmbH, Lage, Germany) ad libitum. Experimental studies were conducted according to the German Federal Law on the Care and Use of Laboratory Animals (license 41/98). Caudal contents were flushed out from a cannula inserted into the vas deferens and centrifuged to remove sperm as previously described [15].

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction

Epididymides from fertile heterozygous (HET, c-ros+/-) and infertile homozygous knockout mice (KO, c-ros-/-) were dissected into proximal caput (including initial segment) or its gross anatomical equivalent regions and remaining caput, corpus, and cauda epididymidis. Tissue from the different epididymal regions and testes of 4–7 animals were pooled, frozen in liquid nitrogen, and stored at -80°C for cDNA expression and Northern blot analysis. Total cellular RNA was isolated using the Ultraspec kit (Biotecx, Houston, TX). Reverse transcription was performed with MMLV reverse transcriptase (Promega, Mannheim, Germany). The rapid dissection of the tissue required to prevent RNA degradation precludes too careful a dissection of the tubule loops, which in the epididymal head are in such an arrangement [34] that exclusion of noninitial segment tissue from the morphologically recognizable initial segment cannot be discounted. Thus, the entire proximal caput was dissected from both genotypes for comparison.

cDNA Expression Analysis

Total RNA was reverse transcribed into a complex cDNA probe in the presence of {alpha}-[32P]-dATP. The reverse transcription was carried out employing a gene-specific primer mix enclosed in the cDNA Expression Array kit (Clontech, Heidelberg, Germany) allowing only the generation of cDNAs detectable by the Atlas mouse cDNA Expression Array. Gene expression analysis was performed using the Atlas Mouse cDNA Expression broad-coverage 1.2 Arrays I and II, containing 1176 single-spotted cDNAs on a nylon membrane as described by the manufacturer. Two micrograms total RNA per tissue were used for the cDNA arrays. The cDNA arrays were hybridized overnight at 68°C in hybridization bottles with the 32P-labeled complex cDNA probes, washed several times at a final stringency of 0.1x saline-sodium citrate (SSC)/0.5% (w/v) SDS at 68°C and scanned in a Storm 860 PhosphorImager (Molecular Dynamics, Freiburg, Germany). The intensity of the hybridization signal was determined automatically and corrected for background. For normalization, housekeeping control cDNAs (GAPDH, UBA52, ACTB, RPS29) were chosen that generated equally intense hybridization signals for the samples compared.

Quantification of the expression of genes in up to five comparisons on Clontech arrays was made by ImaGene 4.1 software (BioDiscovery Inc., Los Angeles, CA). Total fluorescence signals were digitized and probes with signals >2x the average background (noise) and a signal:noise ratio exceeding 10 were further analyzed. A comparison of the difference between KO proximal caput and HET gene signals was made by calculating KO:HET ratios and accepting only those changes exceeding a twofold increase or decrease. Where replicates using different batches of extracted RNA were made (n = 2–5), the ranges and mean ± SD values were calculated.

The Affymetrix gene chips (MG U74A) were washed and incubated in a Fluidics station 400 (Affymetrix, Highwycombe, UK) following the manufacturer's instructions and were scanned with the GeneArray (Affymetrix) scanner. Quantification of the digitized fluorescence signals generated mean values, and comparison of the differences between four pairs of gene chips (1 KO and 1 HET) was analyzed by GeneChip Suite Analysis Software (Affymetrix). This provides numerical data and a call of whether the software considers a gene to be present (P), absent (A), or marginal (either absent or present) (M). Only data considered to be present in the tissue were compared.

Northern Blot Hybridization

Northern blot hybridization of the extracted total RNA (15 µg) was performed on 1% (w/v) agarose/10x (N-morpholino)propane sulfonic acid buffer/formaldehyde gels, blotted onto nylon transfer membranes (Amersham Pharmacia, Freiburg, Germany) and fixed by cross-linkage by ultraviolet irradiation. Filters were prehybridized at 68°C for 2 h in ExpressHyb solution (Clontech) with 0.1 mg/ml sheared and denatured salmon sperm DNA. Hybridization conditions were identical to those for prehybridization but with the addition of the [32P]dCTP-labeled cDNA probe. For CRES, the cDNA probe was identical to the corresponding cDNA fragment on the Atlas Mouse broad-coverage array 1.2. Information about the individual primer sequences was obtained from Clontech. Labeling of the purified cDNA probes (HighPure, Boehringer, Mannheim, Germany) employed High Prime solution (Boehringer), and hybridization was performed overnight at 68°C, followed by washing twice, 30 min each, with continuous agitation in 2x SSC/0.05% (w/v) SDS at 68°C and twice in 0.1x SSC/0.1% (w/v) SDS. The blots were exposed to PhosphorImager screens (Molecular Dynamics, Amersham Biosciences, Uppsala, Sweden) and signal strengths quantified by ImageQuant 5.0 software (Molecular Dynamics). Ribosomal RNA (18 and 28 S) was used to indicate loading of the lanes.

Western Blots

Western blots of CRES were performed as previously described [35] and lipocalin 2 (24p3) was examined using 10 µg proteins [32] on proteins extracted by a Micro-Dismembrator (Braun Biotech International, Melsungen, Germany) from whole tissue and luminal fluid from the cauda.

Immunohistochemistry

Immunohistochemical staining of HET and KO tissue for CRISP1, PEBP, and mE-RABP was performed as previously described [33]. MEP17 staining was performed on paraformaldehyde-fixed tissue by using a specific polyclonal rabbit antibody and a goat anti-rabbit secondary antibody conjugated to alkaline phosphatase (Sigma, Taufkirchen, Germany) with Fuchsin substrate (DAKO, Hamburg, Germany). Immunohistochemistry of lipocalin 2 [32] and CRES [35] was performed on HET and KO epididymal tissues. Immunohistochemical studies on GPX5 were done as described [28].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of Gene Expression by Mouse Clontech cDNA Arrays and Affymetrix Gene Chips

Gene expression in general In order to obtain information about the differences in gene expression of the proximal caput epididymidis (including initial segment) of fertile HET mice and the equivalent proximal caput region of infertile KO mice, Clontech cDNA arrays were hybridized with complex cDNA probes from both genotypes. The standard housekeeping gene GAPDH was no different between genotypes, indicating equal loading of RNA. Using the objective criteria outlined above, 213 genes were considered as upregulated in the KO animal but only 39 as downregulated. Table 1 indicates that, of the regulated proteins, receptors, enzymes, and transcription factors changed the most, with homeobox genes and binding proteins next, but other oncogenes and growth factor genes changed little. The genes that were upregulated or downregulated are listed in Table 2 together with some of the genes displayed in the figures that were unchanged in expression (Tables 2 and 3).


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  		TABLE 1. Classification of 252 genes on Clontech arrays whose expression differed between the proximal caput epididymidis of heterozygous fertile (HET) and infertile homozygous c-ros knockout (KO) mice


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  		TABLE 2. Genes expressed in the proximal caput epididymidis of c-ros knockout male mice and changes relative to the HET as ascertained from Clontech cDNA arrays and Affymetrix gene chips


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  		TABLE 3. Clontech array transporters whose expression was unchanged between genotypes


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  		TABLE 3. Continued

A representative pair of filters is shown in Figure 1, in which genes are located in six groups of related proteins. On each filter, signals from five genes are circled as examples of those that are changed in intensity of expression between HET and KO genotypes in the proximal caput epididymidal region. Two genes with apparent reduced intensity in the KO were lipocalin 2 (24p3) and CRES, and three appeared to be increased in the KO (alkaline phosphatase 2, phosphodiesterase I/ectonucleotide pyrophosphatase 1, and mE-RABP). The relative changes in gene expression between genotypes are listed in Table 2.



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  		FIG. 1. Photographs of two Clontech Array II cDNA filters hybridized with RNA extracted from the proximal caput (including initial segment) and gross anatomical equivalent regions of HET (upper panel) and KO males (lower panel). Signals from five genes are circled to represent genes that are reduced (lipocalin, 24p3; cystatin 8, CRES) or increased (alkaline phosphatase, phosphodiesterase I, mE-RABP) in intensity in the KO mice

A close-up of the region designated F of Array II is depicted in Figure 2 for genes in the HET (Fig. 2a) and the knockout (Fig. 2d). Genes circled in grey are largely unchanged in expression between genotypes, genes circled in black are downregulated in the KO, and genes depicted in white demonstrate greater expression in the KO (see figure legends for the genes identified). Examples of the expression of other genes from Clontech Array I are also depicted in Figure 2, b and e. Several genes (labeled as 1, 2, 4, 7) remain relatively unchanged, but number 5 (EAAC1) is reduced in the KO, whereas number 6 (prostaglandin D2 synthetase) is more strongly expressed. Nine genes that are apparently upregulated in the KO are shown in Figure 2, c and f (see Table 2).



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  		FIG. 2. Close-up photographs of three regions of two Clontech cDNA arrays hybridized with total RNA obtained from the caput epididymidis of HET mice (a–c) and KO mice (d–f). In region F of array II (a, d), genes circled in grey are largely unchanged in expression (1, RNA activating protein inhibitor; 2, cathepsin D; 3, myosin light chain subunit; 4, PA6 stromal protein); genes circled in black are downregulated in the KO; genes circled in white demonstrate greater expression in the KO (5, vitronectin precursor; 6, collagen 2 type 5; 7, brain-specific lipid binding protein; 8, leptin). In Clontech Array I Region I (b, e), genes 9–12 are unchanged in expression but EAAC1 (13) is lacking from the KO and prostaglandin D synthetase (14) is more strongly expressed. In Clontech Array I, Region II (c, f), nine genes are apparently upregulated in the KO (15, vitronectin precursor; 16, laminin ß2 precursor; 17, brain-specific lipid binding protein; 18, obesity protein; 19, collagen {alpha}l type 4; 20, laminin ß3 precursor; 21, neurofilament triplet 3 protein; 22, keratin type 2 skeletal type 4 cytokeratin {alpha}4)

Replicate hybridizations with RNA pooled from different animals revealed several genes on the Clontech arrays that had repeatably high expression in the HET epididymal caput on more than one occasion. Of the regulated genes (KO:HET < 0.5 or > 2.0) found on Clontech arrays, the most expressed was EAAC1 (rank 1), ~2x > heat shock protein HSP86 (rank 2) ~2x > transcription initiation factor TFIID.

Caput gene signals downregulated in the KO compared with HET males As expected for an animal in which the c-ros gene had been functionally targeted, there was downregulation of the oncogene, and the signal detection was categorized as absent by the Affymetrix software. Other proteins displaying a great reduction in expression in the KO included the known epididymal genes CRES (rank 1) and EAAC1 (EAAT3: rank 3). Other genes, coding for inositol polyphospate-5-phosphatase (rank 2), the ionotropic glutamate receptor AMPA2 (alpha 2: rank 4), and the inwardly rectifying potassium channel (rank 7) were also expressed at far lower levels in the KO males than the HET organ (Table 2).

The Clontech arrays confirmed the downregulation of CRES (ranked 1 of genes displaying a more than twofold decrease) and EAAC1 (rank 11), and between them in ranking were cystatin B (rank 4), major urinary protein 1 (rank 5), and ADAM3 (cyritestin: rank 10). Cystatin 7, the ATP-binding cassette subfamily member D3 and cationic amino acid transporter were also downregulated (ranks 27, 15, 34) (Table 2).

Caput gene signals upregulated in the KO compared with HET males The Affymetrix chips confirmed the increase in phosphodiesterase I/ectonucleotide pyrophosphatase 1 (rank 5 of genes upregulated more than fivefold) observed with the Clontech arrays. The Na+-phosphate cotransporter IIb (Npt2b, rank 6), {gamma}-glutamyl transpeptidase (rank 12), and the calcium-activated, voltage-gated potassium channel (shaker subfamily, member 1: rank 14) were all upregulated in the mutant caput epididymidis.

From the genes spotted on the Clontech arrays, the most upregulated (of those with increases exceeding twofold) were homeobox protein A3 (rank 1) and distal-less homeobox protein 7 (rank 2), with phosphodiesterase 1/ectonucleotide pyrophosphatase 1 at rank 5. Several transporters were upregulated; the glutamate transporter EAAT5 (rank 16), a large conductance, calcium-activated, potassium channel (rank 18), an unspecific facilitated diffusion glucose transporter (GLUT, rank 57), and a voltage-gated potassium channel, Shab-related subfamily (rank 206). Glutamate-related receptors were also upregulated: glutamate receptor (NMDA), subunit zeta precursor (rank 81), and the ionotropic glutamate receptors AMPA-1 (rank 137) and delta-1 (rank 169) (see Table 2).

Caput gene signals unchanged between the KO and HET males Table 2 indicates that the majority of genes available on the arrays and chips was expressed at the same level between HET and KO genotypes. Those presented below are of interest because changes may have been anticipated or because of our interest in the sperm phenotype exhibited by the c-ros KO males. Surprisingly, the initial segment-specific genes BMP7, BMP8a, and A-raf and caput-enriched proteins ADAM7, C/EBP{delta}, GPX5, PEA3, and Pem were unaltered in expression in the KO males. The following related, but non-epididymis-specific proteins, displayed no difference in gene expression: ADAM5, ADAM10, ADAM12, GPX2 (intestinal, GSHPx-GI), GPX3 (extracellular, plasma GSHPx-P), GPX4 (phospholipid hydroperoxide glutathione peroxidase, PHGPx), L-myc, N-myc, c-myc, c-ret, and B-raf, met (Table 2).

Because epididymal luminal fluid is important for affecting sperm function, particular interest was shown in transporters, channels, or pores that modulate the composition of epididymal fluid. Of the large number on the arrays and present in the caput tissue, only a few were downregulated (see Tables 2 and 3).

Northern Blots

The expression of the CRES gene in epididymal tissue confirmed a 700-kb transcript solely in the proximal caput (including initial segment) of the wild-type males (Fig. 3). No signals were present in other regions of the wild type or any region of the KO, confirming the cDNA array and gene chip data (Fig. 1). Northern blots of GGT mRNA in the caput of KO males were fivefold higher than that in the proximal caput of HET males (Fig. 4), confirming the change demonstrated in Affymetrix chips (Table 2).



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  		FIG. 3. Northern blot of CRES in epididymides (lanes 1 and 2: proximal caput; lanes 3 and 4: remaining caput; lanes 5 and 6: corpus; lanes 7 and 8: cauda) of HET mice (lanes 1, 3, 5, 7) and KO males (lanes 2, 4, 6, 8). A product of 700 kbp is only observed in the proximal caput of HET males and not in any epididymal region of the KO mice. The amount of RNA loaded is indicated by the ribosomal 18S RNA



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  		FIG. 4. Northern blot of {gamma}-glutamyl transpeptidase (GGT) in epididymides (lanes 1 and 2: proximal caput; lanes 3 and 4: remaining caput; lanes 5 and 6: corpus; lanes 7 and 8: cauda) of HET mice (lanes 1, 3, 5, 7) and KO males (lanes 2, 4, 6, 8). A product of 2.4 kbp is only observed in the caput epididymidis and an upregulation is apparent in the caput of the KO organs. The amount of RNA loaded is indicated by the ribosomal 18S RNA

Immunocytochemistry

The immunolocalization of MEP17, as anticipated for a specific product of the initial segment, was strongly expressed in the principal cells of wild-type tissue (Fig. 5a), but no staining was evident in the KO tissue (Fig. 5b). Higher power magnification revealed strong staining of principal and apical cells in the initial segment (Fig. 6a) but also staining in the adjacent proximal caput of apical cells, stronger than that of principal cells (Fig. 6b). No cell type was positive for MEP17 in the entire caput of the KO male (Fig. 6c). The proteins CRISP1, PEBP, and mE-RABP were unchanged from their normal locations in the proximal corpus, distal caput, and both corpus and distal caput, respectively (Fig. 5, c–h).



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  		FIG. 5. Immunohistochemical localization of murine retinoic acid binding proteins MEP 17 (a, b), CRISP1 (c, d), mE-RABP (e, f), and PEBP (g, h) in the caput and proximal corpus epididymidis of HET (a, c, e, g) and homozygous KO males (b, d, f, h). MEP17 is strongly expressed in the initial segment (IS) of the HET mice (a) but not in the undifferentiated proximal caput of the KO mouse (IS). b) For CRISP1, PEBP, and mE-RABP, there are no differences in location or intensity of staining between genotypes. Scale bars = 100 µm



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  		FIG. 6. Immunohistochemical localization of MEP17 in the caput epididymidis of HET and KO mice. Strong staining of principal and apical cells in the initial segment ([50], region I) in the HET male is observed (a). In the adjacent proximal caput ([50], region II), apical cell staining is stronger than the principal cells (arrowheads, b). In the proximal caput of the KO, no staining is evident (c). Scale bars = 10 µm

Immunolocalization of lipocalin 2 (24p3) confirmed its presence in the caput epithelium and brush border of the wild-type initial segment (Fig. 7a), but staining was stronger in the adjacent proximal caput region (Fig. 7b). More distally, staining became patchy, with adjacent principal cells being either strongly stained or not at all (Fig. 7c), and in the distal caput/proximal corpus lipocalin 2 was observed solely in the lumen and on microvilli, with no epithelial staining (Fig. 7d). In the KO mouse, lipocalin 2 staining was present but in reduced amounts in the proximal caput, notably in supranuclear Golgi and on the microvillous border of short principal cells typical of the proximal caput of the HET (Fig. 7e); more distally, staining was occasionally found in a weak but patchy fashion (Fig. 7f) but mostly only in the lumen and on microvilli (Fig. 7g). Omission of primary antibody removed staining in both genotypes (Fig. 7h).



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  		FIG. 7. Immunohistochemical localization of lipocalin 2 (24p3) in the caput epididymidis of HET mice. a) Lipocalin 2 in the epithelium (E) of the initial segment ([50], region I) with prominent staining of the Golgi (arrowheads) and stereocilia (sc). b) Stronger staining in the adjacent proximal caput region ([50], region II) with intense staining of the luminal contents (L) as well as in the epithelial cells (E) and microvilli (mv). c) In the distal caput region ([50], region III), patchy staining arises from populations of principal cells (E) either staining or not, but luminal staining (L) is maintained. d) Toward the corpus epididymidis, epithelial staining (E) is absent but microvilli and luminal staining persists. e) In the equivalent region to the initial segment in the KO male, lipocalin 2 is expressed in the Golgi (arrowheads), microvilli, and lumen. f) In the adjacent region, only occasional and weak patchy staining of the epithelium (E) persists but the microvilli (mv) and lumen (L) are stained. g) Close to the corpus epididymidis, microvillous and luminal staining in the absence of epithelial staining is observed. h) Control sections of the caput of HET males incubated with preimmune serum are not stained. Scale bars = 10 µm

CRES was present in the apical principal cell cytoplasm of the initial segment in HET males (Fig. 8a) and showed an abrupt loss of signal in the immediately adjacent caput, separated by a connective-tissue septum (not shown). In the KO males, no signal was present in either epithelium or lumen (Figs. 8c). In control sections incubated without primary antibody, no selective staining was observed in either genotype (Figs. 8, b and d). The localization and intensity of GPX5 in the caput epididymidis of the KO was little different from that of the HET (not shown).



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  		FIG. 8. Immunohistochemical localization of CRES protein in the caput epididymidis of HET (a, b) and homozygous mice KO males (c, d). CRES is strongly expressed in the initial segment (IS) of the HET mice (a) but not in the undifferentiated proximal caput (IS) of the KO mouse (c). Controls lacking primary antibody (b, d) are negative. Scale bars = 100 µm (a–d).

Western Blots

Lipocalin 2 (24p3) was found in caput and corpus tissue and caudal fluid (Fig. 9). Quantification of two Western blots revealed a higher level in the proximal caput (set at 1.00 for the proximal caput of the HET organ) than remaining caput (mean 0.44), approximately the same amount in the corpus tissue (0.92) and more in caudal fluid (1.47). A larger isoform (31 kDa) appeared in addition to the expected 25-kDa form in corpus tissue and caudal fluid. There was similar expression of the lower (0.44) and higher (0.48) molecular weight isoforms in corpus tissue as well as cauda fluid (0.71/0.76) of the HET males. The KO tissue retained the two isoforms in corpus tissue and cauda fluid and there was relatively less lipocalin 2 in the proximal (0.27) and remaining caput (0.31), about equal expression in the corpus (0.84) but more in caudal fluid (1.79).



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  		FIG. 9. Western blots of standard lipocalin 2 (24p3) (lane 1), cauda epididymidal fluid (lanes 2 and 9) and tissue from the proximal caput (lanes 3 and 6), remaining caput (lanes 4 and 7), and corpus epididymidis (lanes 5 and 8) from HET mice (lanes 2, 6, 7, 8) and homozygous KO mice (lanes 3, 4, 5, 9). The KO tissue retained the presence of two isoforms in corpus tissue and cauda fluid

The 19-kDa N-glycosylated and 14-kDa nonglycosylated CRES proteins were present in equal abundance in testicular and caudal sperm extracts of HET and KO mice (Fig. 10). The less obvious appearance of the 19-kDa CRES in caudal sperm from the KO mice compared with HET and KO sperm likely reflects sample extraction differences because the 19-kDa protein was readily detectable in other preparations of KO sperm (data not shown). Not surprisingly, CRES was not detected in the cauda luminal fluid from the KO mice while residual 19-kDa and processed 17-kDa CRES was present in cauda fluid from HET mice. Longer exposure of the Western blots also revealed minor amounts of the 14-kDa CRES as well as processed 12-kDa CRES form (data not shown).



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  		FIG. 10. Western blots of CRES in proteins extracted from the testis (lanes 1, 2, 3), luminal fluid from the cauda epididymidis (lanes 4 and 5) and cauda epididymidal spermatozoa (lanes 6 and 7) from HET males (lanes 2, 4, 6), homozygous KO males (lanes 3, 5, 7) and wild-type mice (c-ros+/+) as control (lanes 1 and 8). Note the equally strong presence of CRES in testicular homogenates of all genotypes and the presence of the mature 14-kDa isoform in caudal sperm from HET and KO c-ros males. In luminal fluid from the cauda, most of the proximally secreted 19-kDa form exhibits a slightly lower mass (approximately 17 kDa) in the HET male that is absent from the KO epididymis. The 17- and 12-kDa forms may represent the proteolytically processed forms of the 19- and 14-kDa CRES proteins. Molecular weight standards and the migration of the 14- and 19-kDa forms are indicated to the left and right, respectively


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The c-ros KO mouse is a unique model to examine the way in which the epididymis influences sperm function because the epithelium lacks the initial segment and the males are infertile predominantly because of extreme flagellar angulation of the sperm in the female tract. The missing epididymal segment may directly affect luminal spermatozoa by, e.g., secreting products that interact directly with sperm solely in this region, e.g., GPX5 [29] or MEP17 [20]. However, indirect effects of the initial segment on distal epididymal secretions are also possible through luminal paracrine regulation of the downstream epithelium and its secretion products [36]. In order to elucidate normal control of sperm function by the epididymis, the techniques of cDNA microarrays, gene chips, and Northern blots were utilized here to provide an overview of gene expression in the caput epididymidis of the two genotypes. This was complemented by Western blots and immunohistochemistry of known caput secretions in the tissue and luminal fluids from these males.

Although the trends in gene expression were the same between Clontech arrays and Affymetrix gene chips, the extent of differences varied. Also widely variable were repeated determinations of gene expression using different RNA batches from different animals, which reflects the innate complexity of the coiled tubule segments of the HET and the anatomical changes wrought in the KO organ.

As the main morphological difference between the homozygous and HET males is the absence of the initial segment, decreases in the genes expressed in the differentiated initial segment would be anticipated. Indeed, the oncogene c-ros was absent from the tissue and several genes also expressed in the proximal caput epididymidis were downregulated in the mutant caput. The most drastic decrease in gene expression was experienced by CRES, a proteinase inhibitor expressed both in the testis and proximal caput of the epididymal epithelium [36]. Western blots demonstrated CRES was present as a 14-kDa isoform in caudal sperm [37], indicating maintained production by germ cells in the testis. Other cystatins (cystatins B and 7) were also markedly reduced in the KO caput. The initial segment-specific location of MEP17 in HET males [38] was confirmed and its absence from the proximal caput of the KO demonstrated immunohistochemically. For other genes solely expressed in the initial segment, a decrease in expression in the KO is mandatory unless there is upregulation of gene expression in the undifferentiated tubule.

An unexpected finding was the drastic reduction in expression of ADAM3 (cyritestin). This is an acrosomal protein that undergoes epididymal processing but was not detected at the protein level in the murine epididymis by Linder et al. [39]. Its regional location is unknown and it is unclear if the epididymal gene found in this study is translated. The enzymes responsible for processing ADAM3 are unknown, but inositol polyphosphate 5-phosphatase (inpp5b) is implicated in the epididymal processing of ADAM2 (fertilin-ß) and inpp5b was markedly reduced in the c-ros KO caput. It is noteworthy that inpp5b-deficient male mice are infertile and that their sperm exhibit reduced fertilizing capacity in vitro [40]. For the above proteins, the absence of gene signals in the KO caput suggests an association with, or a dependence on, the presence of the differentiated epithelium of the initial segment, which makes them prime candidates for having a functional role in the infertility of the males.

There is an inherent problem in what valid interpretations can be made by comparing gene expression in the caput epididymidis of HET and KO males because there is no initial segment in the KO male and the proportion of the tissue comprising post-initial-segment epithelium differs between the genotypes. This implies that the interpretation of changed signal strengths in terms of transcriptional status depends on the location of genes within the epididymal caput, i.e., whether they are also expressed in the adjacent caput regions and with greater expression in the initial segment (c-ros, ADAM7, CRES, B-myc, C/EPB), in the proximal caput (A-raf), equally expressed in the three regions (GPX5), or absent from the initial segment (GGT, PGDS). That BMP7, BMP8a, A-raf, C/EBP{delta}, ADAM7, and GPX5 were unchanged in expression indicates they were expressed in the undifferentiated proximal caput of the KO, a region where their expression is normally absent or weak. Thus, paradoxically, an increase in gene transcription may really underlay the apparently unchanged gene expression. More precise interpretation cannot be made without knowledge of the exact proportion of differentiated and undifferentiated epithelium in the samples analyzed.

GPX5, and possibly ADAM7, are sperm-coating proteins: human ADAM7 encodes glycoprotein 83 [41], a secreted epididymal protein that is conjugated to sperm in both man [42] and mice [43] and GPX5 binds to murine spermatozoa over the acrosome [28, 29, 44]. As their expression is maintained in the infertile males, they may have little influence on the sperm phenotype. As expected for related but non-tissue-specific genes, glutathione peroxidases GPX2 (intestinal), GPX3 (plasma), or GPX4 (phospholipid hydroperoxide) and metalloproteinases ADAM5, ADAM10, and ADAM12 were expressed to the same extent in fertile and infertile transgenics.

In the case of {gamma}-glutamyltranspeptidase [45], prostaglandin D2 synthetase [46, 47], alkaline phosphatase [48, 49], Pem [50], and clusterin [51], all of which are absent or weak in the initial segment of the mouse but expressed in the adjacent caput region, the total RNA extracted from the HET caput would include RNA from the initial segment (containing no transcript of these proteins) as well as that of the adjacent caput (which effectively transcribes them all). In the KO tissue, the total caput RNA is derived from only a non-initial-segment tubule, which would be relatively enriched in the transcripts examined. Thus, the upregulation of GGT and alkaline phosphatase may be more apparent than true (increased nucleic acid synthesis) and a reflection of the lack of dilution with transcript-free mRNA. Any upregulation of a gene absent from the initial segment may be interpreted in this way. Thus, for PGDS, Pem, and clusterin, in which no change in gene expression was observed, gene transcription may in reality have been downregulated, as an increase would be anticipated because of dilution with non-transcript RNA.

Several genes were upregulated in the KO epididymis, e.g., the homeobox proteins 7 and A3. This supports the contention that homeobox genes in the adult play a role in maintenance of tissue segmentation [52] and may be involved in the remodeling of the tissue that occurs during development. The observed changes in cytoskeletal and associated elements (vitronectin, keratins, neurofilaments, laminins, collagens) may be expected in tissue with such sharply contrasting cellular architecture as the initial segment and the undifferentiated caput of the KO. Other oncogenes related to tissue differentiation (A-raf, B-raf, c-ret, c-myc, N-myc, L-myc, met) were unchanged in the mutant organ.

The unchanged gene expression of lipocalin 2 (24p3), which is present in all the proximal caput [32], contrasts with an apparent reduced epithelial synthesis of lipocalin 2 at the protein level, as judged from immunohistochemical staining. The novel observation of a higher molecular weight form of the lipocalin 2 protein in corpus tissue and caudal fluid may merely reflect the different genetic backgrounds of the mice because the higher molecular weight isoform was not previously reported [32]; nevertheless, the isoforms did not differ between c-ros genotypes and should not explain the sperm phenotype.

For genes for which the expression pattern has not been determined, the extent of gene transcription, if any, cannot be stated with certainty. The major urinary protein family is a lipocalin-family member [53] that binds odoriferous compounds employed by nocturnal animals for communication. Its presence in the epididymis is surprising because the proteins are excreted in the urine, but this may attest to the mesonephric origin of this organ. The drastic loss of MUP1 in the KO tissue parallels the decline of MEP17 and could indicate its expression in the initial segment. The loss of these lipocalins contrasts with the unchanged protein expression in more distal locations of CRISP1, PEBP, and mE-RABP, the latter being confirmed by Clontech quantification of gene expression. These observations clearly demonstrate that only the more proximal regions of the duct are altered by the absence from birth of the c-ros gene. The unaltered expression of EAAC1 in the corpus and cauda of the c-ros KO has been reported [15].

In addition to secreted coating proteins having an influence on sperm cell survival, low molecular weight epididymal secretions have been implicated in sperm maturation from the observations of raised tissue glutamate in the c-ros KO lacking the glutamate transporter EAAC1 [15]. The present study confirmed the downregulation of EAAC1 (EAAT3) but also demonstrated an upregulation of another glutamate transporter (EAAT5). By contrast, the expression of other glutamate transporter genes (EAAT1, EAAT2, and EAAT4) was unchanged in the mutant organ. Perhaps EAAT5 rather than EAAC1 is responsible for increased glutamate in the KO tissue. The role of epididymal glutamate is unclear, but several glutamate receptors were found in the tissue. The inorganic composition of epididymal fluid in the KO could be influenced by the downregulation of an inwardly rectifying potassium channel and upregulation of three voltage-gated and/or calcium-activated potassium channels and the Na+-phosphate cotransporter IIb.

Many channels and transporters in the Clontech arrays did not differ in signal expression between genotypes. Although failing to explain the phenotype, it indicates for the first time the huge range of transport activities in the murine epididymis, including water channels (AQP1, AQP2, AQP4, AQP7, AQP8); members of the ATP binding cassette family; facilitated glucose transporters (GLUT1, GLUT3, GLUT4); transporters for GABA (GAT1, GT23), noradrenalin, dopamine, serotonin, acetylcholine, folate; for amino acids (glycine, cysteine, glutamate [EAAT1, EAAT2, EAAT3, EAAT4, EAAT5], cationic amino acids); for cations (Cu, Zn); for organic cations (OCT1, OCT2, OCT3, OCTN); for organic anions (monocarboxylates [MCT1, MCT2, MCT3]); anion exchangers (AE1, AE2, AE3); cation exchangers (Na+/Ca2+, Na+/H+); cation cotransporters (Na+/K+/Cl-, Na+/PO43+); mitochondrial transporters (citrate, nucleotides); transporters for protons (V-ATPase); IgG Fc fragments; and orphan transporters. The presence of the taurine transporter in the wild-type murine epididymis has recently been reported, with no change in gene expression in the KO [54]. The fact that no change was indicated in their expression in the caput presumably implies that they are independent of initial segment control and that what they transport does not contribute to the sperm defect, given the proviso discussed above, that they are neither excluded from nor solely expressed in the initial segment.

The present study has provided a wealth of information on gene expression in the KO organ, which should underlie changes in the composition of epididymal fluid and spermatozoa in the KO males. From the data presented here, it seems likely that epididymal luminal fluid is altered in the infertile KO male because a variety of secreted proteins (MEP17) and epithelial transporters (EAAC1, EAAT5, cationic amino acid transporter, Na+/PO43- cotransporter, facilitated glucose transporter) and K+ channels were either upregulated or downregulated in the KO tissue. The significance of this for sperm entering the KO epididymis is that they would be both deprived of the normal secretions elaborated in the initial segment and come into contact with more distal epididymal secretions earlier than is the case for HET sperm. Surrounded by luminal fluid of subnormal composition, sperm in the KO males may be deprived of essential substances such as organic or inorganic osmolytes and become unable to regulate their volume, leading to volume increase, flagellar angulation in the uterus, and impeded progress to the oviduct [13, 14, 55], thus explaining the infertility of the males. By mimicking the current model and upsetting the mechanisms responsible for sperm function, a deliberate induction of the infertile state could be induced and a new contraceptive for men developed.


   ACKNOWLEDGMENTS
 
We thank Joachim Esselmann (Münster) and Nathaly Cormier (Texas) for technical assistance and Martin Heuermann and Günther Stelke (Münster) for taking care of the animals. We appreciate the input of Dr. H. Funke and Frau. B. Foppe from the Integrated Functional Genomics Centre of the University Münster for assistance in the Affymetrix gene chip experiments.


   FOOTNOTES
 
1 This work was supported by the Rockefeller-Schering Research Foundations' Application of Molecular Pharmacology for Post-Testicular Activity [AMPPA] Network (A.W.), the Deutsche Forschungsgemeinschaft 197/3-1 for "The male gamete: production, maturation, function" (C.H.Y.), NIH HD33903 (G.A.C.), and NICH HD36900 (M.C.O.C.). Back

2 Correspondence: T.G. Cooper, Institute of Reproductive Medicine of the University, Domagkstraße 11, D-48129 Münster, Germany. FAX: +49 251 8356093; cooper{at}uni-muenster.de Back

3 Current address: Schering AG, 13342 Berlin, Germany Back

Received: 3 April 2003.

First decision: 2 May 2003.

Accepted: 4 June 2003.


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