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From the * Institute of Reproductive Medicine of
the University, D-48129 Münster, Germany; and
Children's Hospital, Department of Metabolic
Disorders, University of Freiburg, D-79106 Freiburg, Germany.
Current address: Schering AG, 13342 Berlin, Germany.
| Correspondence to: Dr TG Cooper, Institute of Reproductive Medicine of the University, Domagkstraße 11, D-48129 Münster, Germany (e-mail: cooper{at}uni-muenster.de). |
| Received for publication April 22, 2002; accepted for publication June 6, 2002. |
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Abstract |
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Key words: Male reproductive tract, gene chips, sperm, gamete biology, cDNA arrays
In c-ros mice, the initial segment of the epididymis, which comprises the most proximal part of the caput epididymidis, also fails to differentiate. This region is typified by a tall epithelium bearing long stereocilia, which reduce the size of the lumen (Abe et al, 1983) and by a rich vasculature (Suzuki, 1982; Le Barr et al, 1987) of fenestrated blood capillaries (Suzuki, 1982; Abe et al, 1984). In addition, the region is more metabolically active than other regions (Eliott, 1965), and it expresses a number of specific genes (Dacheux et al, 1998; Kirchhoff, 1999) that support sperm maturation (Robaire and Hermo, 1994; Cooper, 1998). Many of these genes and epithelial height are under the control of testicular exocrine secretions (Abe and Takano, 1989a,b; Kirchhoff, 1999), but the role of these genes in the process of sperm maturation has not been unequivocally established.
The majority of motile spermatozoa from c-ros knockout mice exhibit a marked angular morphology of the flagellum (Yeung et al, 1998, 1999). A consequence of this is that sperm fail to enter the oviduct and fertilize eggs (Yeung et al, 2000). Because abnormal sperm physiology must in some way reflect the lack of the initial segment, identification of the genes that are absent in the knockout epididymis should provide information about the link between epithelial and sperm function. The epididymal genes of interest are likely to be those important for modifying luminal fluid because flagellar angulation reflects a defect in volume regulation (Yeung et al, 1999, 2002a), and the capacity for sperm volume regulation is developed during maturation in the epididymis (Yeung et al, 2002b). In order to select candidate genes that might be involved in the marked phenotype that is characteristic of infertile male c-ros knockout mice, complementary DNA (cDNA) arrays were used in this study to investigate epididymal gene expression in fertile wild-type and heterozygous mice, and in infertile c-ros knockout mice.
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Material and Methods |
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RNA Extraction and Reverse Transcriptase-Polymerase Chain
Reaction
Total cellular RNA was isolated from different epididymal regions and the
testis using Ultraspec (Biotecx, Houston, Tex). In order to generate specific
probes against excitatory amino acid carrier 1 (EAAC1) and glutamate
transporter interacting protein (GTRAP) 3-18 in Northern blot hybridization,
primers were used that generated cDNA probe sizes of 190 bp and 144 bp,
respectively. Reverse transcription was performed with Moloney murine leukemia
virus reverse transcriptase (Promega, Mannheim, Germany).
Complementary DNA Expression Analysis
Total RNA extracted from the initial segment of wild-type mice, and the
equivalent gross anatomical region of knockout mice, were reverse transcribed
into a complex cDNA probe in the presence of (
32P)dATP.
Reverse transcription was carried out employing a gene-specific primer mix
enclosed in a cDNA Expression Array kit (Clontech, Heidelberg, Germany) which
allowed generation of only cDNAs that could be detected by the Atlas mouse
cDNA Expression Array. Gene expression analysis was performed using the Atlas
Mouse cDNA Expression broad-coverage 1.2 Array containing 1176 single-spotted
cDNAs on a nylon membrane as described by the manufacturer. 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) sodium dodecyl
sulfate (SDS) at 68°C, and scanned with 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, and RPS29) were
chosen that generated equally intense hybridization signals for the compared
samples. Two micrograms of total RNA per tissue were used for cDNA arrays.
Quantification of gene expression was performed with ImaGene 4.1 software
(BioDiscovery Inc, Los Angeles, Calif). Gene chips were washed and incubated
in a Fluidics station 400 (Affymetrix, Santa Clara, Calif) following the
manufacturer's instructions and scanned with the HP GeneArray scanner
(Affyme-trix).
Northern Blot Hybridization
Northern blot hybridization of the extracted total RNA (15 µg) was done
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 for cross-linkage with UV
irradiation. Filters were prehybridized at 68°C for 2 hours in ExpressHyb
solution (Clontech, Palo Alto, Calif) with 0.1 mg/mL of sheared and denatured
salmon sperm DNA. Hybridization conditions were identical to those used for
prehybridization but with the addition of (32P)dCTP-labeled cDNA
probe. For EAAC1, the cDNA probe was identical to the corresponding cDNA
fragment on the Atlas Mouse broad-coverage array 1.2. Information on
individual primer sequences was obtained from Clontech. Labeling of purified
cDNA probes (HighPure, Boehringer-Mannheim, Mannheim, Germany) was done with
High Prime solution (Boehringer-Mannheim). Hybridization was performed
overnight at 68°C, followed by washing twice for 30 minutes 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, Sunnyvale, Calif) and signal strengths were
quantified with ImageQuant 5.0 software (Molecular Dynamics and Amersham
Biosciences, Uppsala, Sweden).
Immunohistochemistry
Paraformaldehyde-fixed (4% w/v) tissue was dehydrated, embedded in
paraffin, and cut into 3- to 4-µm sections for localization of EAAC1. After
deparaffinization, the tissue sections were rehydrated and they underwent
antigen retrieval for 20 minutes at 80°C in 0.05 M glycine buffer in a
microwave oven. This was followed by 3 washes in Tris-buffered saline (TBS;
0.15 M NaCl, 0.05 M Tris/HCl pH 7.6). Blocking was performed with 5% (v/v)
normal rabbit serum for 20 minutes at room temperature. Sections were
incubated for 1 hour at room temperature with a polyclonal goat antibody
against a synthetic peptide corresponding to amino acids 504-523 from the
carboxy terminus of the cloned rat EAAC1 (Chemicon, Hofheim/Ts, Germany) at a
dilution of 1:400. Each section was incubated with biotinylated rabbit
anti-goat immunoglobulin (Ig) G (1:1000, DAKO, Hamburg, Germany) for 1 hour at
room temperature. This was followed by incubation with alkaline phosphatase
conjugated to extravidin (Sigma, Taufkirchen, Germany) for 1 hour at room
temperature. Sections were rinsed 3 times for at least 5 minutes in TBS
between each antibody incubation step, and the antibody buffers contained 1%
(w/v) bovine serum albumin (BSA, Sigma). For visualization, Neufuchsin (DAKO)
was used. Sections were counterstained for 5-10 seconds with Mayers
hematoxylin and mounted in Faramount (DAKO). Control staining included
replacing the first antibody with 5% (w/v) BSA.
Analysis of Tissue Glutamate
Epididymides from c-ros+/- and
c-ros-/- mice (n = 8) were dissected into initial segments
or its gross anatomical equivalent region and the remaining caput, corpus, and
cauda regions and placed into 100 µL of phosphate-buffered saline (PBS)
with Protease Inhibitor Cocktail (P-8340; Sigma). Each region was cut into
fine pieces in a small Petri dish and the contents were dispersed for 20
minutes. Luminal contents and tissues were both centrifuged for 11 seconds at
500 x g to separate the sperm and fluid (supernatant) from the
tissue. The Petri dish was rinsed with 100 µL of PBS and after a second
centrifugation (11 seconds at 500 x g), the 2 tissue fractions
from each region were pooled. The tissue fraction was homogenized 3 times at
15 000 rpm for 5 seconds on ice (Omni 2000 hand-held homogenizer,
Südlaborbedarf, Gauting, Germany) and sonicated 3 times for 10 seconds on
ice (1.5 mm tip, 50 Hz, 18 W; Vibra-Cell-Sonicator, Sonics and Materials Inc,
Danby, Colo). The processed probes were centrifuged for 11 seconds at 500
x g and stored at -20°C. Measurements were made separately
on the tissue and supernatant but because the supernatant does not constitute
luminal fluid (particularly in the initial segment where damaged long
stereocilia would release their intracellular contents), the results were
combined to provide whole tissue content.
In other experiments, cauda epididymidal luminal contents from both genotypes were flushed with PBS (with osmolality raised to 430 mmol/kg with NaCl to preserve the cytoplasmic droplets; NaCl430) through a cannula in the vas deferens into the same medium. After centrifugation for 5 minutes at 800 x g, the sperm-free supernatant was removed and the sperm pellet was washed again in NaCl430 and frozen at -20°C after sperm concentration measurement. After thawing, spermatozoa were homogenized and sonicated as above and protein measurements from aliquots of all probes were performed with the BCA Protein Assay kit (Pierce, Rockford, Ill) using albumin as the standard. Glutamate was measured according to the method described by Spackman et al (1958) with an amino acid autoanalyzer LC3000 (Biotronik, Berlin, Germany). For quantification, external standards were used and sensitivity was 3 µM. Values were expressed per milligram of protein, or per 106 spermatozoa.
Statistics
Data on glutamate content of tissue fractions from the 4 epididymal regions
from both genotypes were compared using the Mann-Whitney U test, and
significant differences were determined using the Student-Neuman-Keuls test.
Differences were accepted as significant when P < .05.
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Results |
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In order to obtain information on differences in gene expression of the initial segment from fertile c-ros+/- mice and the anatomically equivalent proximal caput region of infertile c-ros-/- mice, cDNA arrays were hybridized with complex cDNA probes from both genotypes. To avoid high variability in the signal intensities of some genes, experiments were repeated three times with freshly extracted total RNA using the same conditions. Three arrays (with the same charge number) were employed. Figure 1 shows the same section of a cDNA array from 3 experiments with labeled cDNA from heterozygous and homozygous c-ros knockout mice. An obvious and reproducible spot reflecting a strong hybridization signal of EAAC1 cDNA was clearly detectable in all experiments performed with cDNA from the initial segment of c-ros+/- mice, but it was completely missing from all the arrays tested with cDNA from c-ros-/- mice. Visual assessment of the signal strength on a scale of 0 (absent) to 4 (dark spot) rated gene expression as 3, 3, 3, and 3-4 in tissues from wild-type mice, and 0, 0, 0, and 0 in tissues from knockout mice. A total of 8 Affymetrix arrays used on total RNA extracted from 4 wild-type and 4 knockout tissues confirmed an 11.0-fold decrease in EAAC1 expression.
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Northern Blots
To confirm the results on EAAC1 expression level in cDNA array experiments,
Northern blots were performed with a specific cDNA probe for EAAC1, which was
identical to the one spotted onto the commercial cDNA arrays. In addition to
the result from the proximal caput region, including the initial segment, we
investigated EAAC1 expression in other epididymal regions and in the testis.
Northern hybridization revealed 2 bands of high densitometric intensity in the
total RNA lane from the proximal caput of fertile heterozygous mice, whereas
the lane from c-ros-/- mice lacked any signal
(Figure 2). These 2 bands
represent transcripts of EAAC1 with sizes 4.3 and 2.7 kilobases (kb). The
distal caput region showed reduced EAAC1 expression in knockout mice in
comparison with wild-type mice. Low expression was also found in the corpus
epididymidis. In the cauda epididymidis, both genotypes showed 2 strong bands
of comparable signal intensity to that of the proximal caput epididymidis in
c-ros+/- animals.
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Northern blots from the testis revealed no signal for EAAC1 expression in either heterozygous or c-ros knockout mice (Figure 2). Reverse transcriptase-polymerase chain reaction with testicular RNA exhibited a very faint band of the expected size in both genotypes, reflecting a very low level of expression that was not detectable in Northern blots (Figure 2).
Two major gtrap3-18 transcripts of 4.2 and 1.2 kb were detected in the proximal caput epididymidis of c-ros+/- mice, whereas in all other epididymal regions, only 1 major transcript (1.2 kb) was found. In knockout mice, only the 1.2 kb transcript was present in all regions (Figure 3). Densitometric measurement of the 4.2 kb transcript in the proximal caput region of c-ros+/- revealed a signal intensity that was 4 times higher than that of the 1.2 kb transcript.
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Northern blots of 
-glutamyl transpeptidase (GGT) mRNA in the caput
of knockout males was fivefold higher than that in the proximal caput of
wild-type males (not shown). This was also confirmed in Affymetrix arrays
(6.8-fold increase).
Expression of EAAC1 at the Protein Level
The immunohistochemical staining intensity of the adluminal region of the
epithelium of the initial segment from fertile c-ros+/-
mice was strong on the stereocilia and supranuclear Golgi region of the
principal cells (Figures 4a and
5a). In addition to the initial
segment, the adjacent proximal caput region, termed segment II by Abou-Haila
and Fain-Maurel (1984), also
exhibited a strong expression of EAAC1 located on the microvilli
(Figure 4c). Contrary to this
finding, there was no EAAC1 expression in the equivalent regions of infertile
c-ros-/- mice (Figure
4, b and d). Sporadic single-cell staining reaction was found in
the cytoplasm of apical cells in the caput epididymidis of
c-ros-/- mice (Figures
4b and
5b). There was no expression of
EAAC1 in the efferent ducts, and low adluminal expression in the rest of the
caput and proximal to mid corpus region in both genotypes
(Figure 4, e and
f). 
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The distal epididymal region, including the distal corpus and cauda epididymidis, by contrast, exhibited a strong expression of the apical aspect of principal cells comparable to that found in the initial segment or proximal caput epididymidis of wild-type mice, both in fertile c-ros+/- and infertile c-ros-/- mice (Figure 4, g and h). No staining was detected in the clear cells of the distal epididymal regions (Figures 4, g and h; and 5c).
The immunohistochemical data reflect an identical profile at the protein level as that shown at the mRNA level, with a high level of expression in the initial segment of c-ros+/- and no expression in the proximal caput of c-ros-/- animals and strong expression in the cauda of both phenotypes. Immunostaining of testicular sections showed very faint signals from cells of late spermatogenic stages, round and elongated spermatids, but sperm smears from the caput and cauda regions did not reveal specific staining for EAAC1 (not shown).
Glutamate Analysis
The glutamate content of the tissue of the heterozygous c-ros
initial segment and proximal caput epididymidis was significantly lower than
that found in the equivalent proximal caput region of c-ros knockout
animals (Table). The glutamate concentration decreased in the distal regions
of the epididymis, and the corpus and cauda regions exhibited only minor
differences in glutamate content between genotypes. Only 4 of 5 sperm-free
cauda epididymidal fluid samples had measurable glutamate concentrations for
each genotype, which did not differ (-/-, 3.8 ± 0.5 nmol/mg protein;
+/-, 8.0 ± 4.1 nmol/mg protein; ANOVA on ranks, P = .234), but
the glutamate content of caudal sperm was undetectable with high-performance
liquid chromatography in 11 samples from both geno-types.
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Discussion |
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In order to determine how the epididymal epithelium normally controls this aspect of sperm function, the technique of DNA microarrays was used to provide an over-view of gene expression in epididymal tissue of wild-type and knockout genotypes. Of a large number of genes that displayed up- or down-regulation, this study concentrated on a high intensity EAAC1 hybridization signal, reflecting a high gene expression of glutamate transporter in the initial segment from fertile c-ros+/- mice, standing in stark contrast to the lack of signal from the c-ros knockout mice. This was reproducible with different batches of total RNA on the microarrays and confirmed in Affymetrix gene chips, making EAAC1 a useful and prominent candidate gene for further analysis of the role of the epididymal initial segment in fertility.
EAAC1 (human EAAT3) belongs to the solute carrier 1 (SLC1) family, which consists of 5 mammalian isoforms (EAAT 1-5) that all share the structural characteristic of 8 transmembrane domains (Hediger, 1999) and transport glutamate. The transporters are Na+-dependent, and movement of glutamate is coupled to cotransport of Na+ and H+ and the countertransport of K+ (Barbour et al, 1988). Expression of the glutamate transporter EAAC1 has been described in rats (Bjoras et al, 1996), mice (Mukainaka et al, 1995), and humans (Shashidharan et al, 1994), with highest expression in brain and kidney tissues (Shayakul et al, 1997). In the brain it is confined to the neurons in the hippocampus, neocortex, and cerebellum (Velaz-Faircloth et al, 1996) where its fundamental role lies in the clearance from the synapse of glutamate, the major excitatory transmitter released during neurotransmission. In the kidney, high-affinity EAAC1 is responsible for the reabsorption of glutamate, predominantly in the proximal tubules, where it is located on the brush border of the proximal convoluted tubule (Shayakul et al, 1997).
Although it is known that epididymal fluid contains numerous organic and inorganic osmolytes, knowledge about the concentration and absorption of amino acids and amino acid transporting systems in the epididymis is limited (Hinton and Hernandez, 1987; Hinton, 1990; Cooper, 1998). In the rat epididymis, regional differences in neutral amino acid transport have been reported (Hinton, 1990) and an epididymal sodium-dependent dicarboxylate transporter (SDCT1) can transport glutamate and Krebs cycle intermediates (Chen et al, 1998), although this is not its main function. In this study, the EAAC1 gene was detected for the first time at a high level in the proximal and distal parts of the murine epididymis. Northern blots confirmed the results from the array experiments; namely, the lack of EAAC1 expression in the proximal caput epididymidis from c-ros knockout mice. Expression of EAAC1 in all other epididymal regions was similar between both genotypes with lower expression in the corpus and high expression in the cauda epididymidis (c-ros+/-; -/-), comparable to that in the initial segment (c-ros+/-). Two gene transcripts (2.7 kb and 4.3 kb) were found in the epididymal tissue, as reported for the brain and nonnervous tissues in the mouse (Mukainaka et al, 1995).
Strong immunohistochemical localization of EAAC1 protein in the caput, with weaker expression in the corpus and high activity in the cauda, mirrored the regional profile of mRNA transcripts. The Golgi apparatus in principal cells in all epididymal regions was positively stained, which is indicative of its synthesis in these secretory cells. The staining was predominantly confined to the apical cell cytoplasm and particularly to the stereocilia of principal cells in the initial segment and microvilli of the principal cells in corpus and cauda epididymidis, as found in the renal tubules in the rat. The positive cytoplasm of absorptive apical cells in the distal caput region may reflect uptake of surplus EAAC1-positive membranes shed from the principal cells, although the similarly absorptive but more distally situated clear cells of corpus and cauda were negative. Alternatively, the stained apical cells in knockout males, in which there is no expression of EAAC1 by the more proximal region, could indicate synthesis by this cell type.
Identification of a glutamate transporter in the epididymis raises the question of its function in this particular part of the reproductive tract and whether, like the brain and kidney, glutamate is transported by EAAC1 into epithelial cells. To answer this, glutamate concentrations were measured in epididymides from heterozygous and knockout males. Because the very tall epithelium and small lumen in the initial segment preclude perfusion techniques, it was not possible to take measurements of luminal fluid, and glutamate was measured in tissue homogenates. Analysis of the proximal caput epididymidis from c-ros+/- and c-ros-/- revealed a significantly higher glutamate content in the tissue from c-ros knockout mice, but toward the distal epididymal regions (corpus and cauda), a decrease in glutamate content was noted for wild-type mice (as found by Agrawal et al. 1989) and knockout mice.
A greater glutamate content in tissue from males lacking EAAC1 suggests a retention of the amino acid in the tissue. The adluminal location, rather than basolateral location of EAAC1 in the epithelium, might suggest that the normal direction of glutamate transport in the initial segment is out of the cell into the lumen, the opposite mode of transport described in the kidney. Indeed, glutamate introduced into the lumen of the caput is not taken up from the lumen as it is in the cauda (Hinton et al, 1991), indicating regional differences in the direction of glutamate transport in the rat epididymis. Proluminal glutamate transport in the proximal epididymis is supported by micropuncture and microperfusion measurements on the rat epididymal lumen, where an extraordinarily high concentration of glutamate is demonstrated in the caput epididymidis of 50 mM (compare brain, 10 mM [Shashidharan et al, 1997]; and plasma, 25 µM [Peghini et al, 1997]), which should be dissipated by a transporter removing glutamate from the lumen. Furthermore, unlike the kidney, where EAAC1 and GGT are colocalized and act as a unit to synthesize and transport glutamate in the proximal tubule from the renal lumen (Welbourne and Matthews, 1999), the murine initial segment exhibits only low GGT activity, although considerable activity resides in the adjacent caput region (Agrawal et al, 1989).
The luminal location of electrogenic glutamate transporters can be involved in a variety of other cellular processes such as acidification (Shashidharan et al, 1997; Fairman and Amara, 1999). If proluminal transport occurs, additional actions of epididymal EAAC1 might involve generation of low epididymal fluid pH, which would be disturbed in the proximal lumen of knockout males. The osmotic pressure in the distal epididymis of the -/- mouse is not significantly different from that of wild-type mice (Yeung et al, 1999), but more proximal regions have not been studied.
The activity of EAAC1 in transporting glutamate can be regulated by proteins that interact with it, the GTRAP. GTRAP 3-18 is present in several tissues and reduces the substrate affinity of EAAC1, and thereby, glutamate transport (Lin et al, 2001). In this study, expression of the known GTRAP 3-18 transcript was demonstrated in all epididymal segments, but was independent of genotype. A novel, larger mRNA species was restricted to the initial segment of c-ros+/- mice and was absent in knockout animals. The role of this transcript and its relationship to the high EAAC1 expression in the initial segment warrants further study. The absence of this novel transcript in knockout males may be responsible for abnormal glutamate tissue distribution.
Nevertheless, the sodium dependence of glutamate transport by EAAC1, and the high Na+ in testicular fluid entering the caput epididymidis, raise questions about the mechanism and thermodynamics of proluminal transport of glutamate in the epididymis. Glutamate transport through transporters can be reversed, but usually under conditions of hypoxia (Watzke et al, 2000; Rossi et al, 2001). In addition, glutamate can be transported by a range of transporters (Hediger, 1999; Meldrum et al, 1999; Slotboom et al, 1999) and anion channels (Song et al, 1998) that may also be altered in infertile mice.
Another explanation of raised glutamate tissue content in knockout males could be increased glutamate synthesis. The high luminal concentrations of glutamate in wild-type males are unlikely to be generated by the GGT enzyme, a membrane-bound enzyme strongly expressed on microvillous membranes (Kozak and Tate, 1982) because the concentrations of the substrate glutathione entering the epididymal lumen are in the micromolar range (Hinton et al, 1995) and the murine initial segment exhibits only low GGT activity (Agrawal et al, 1989). Thus, the source of glutamate transported by EAAC1 in the initial segment of wild-type males is unclear. However, because the knockout caput lacks the enzymatically inactive initial segment, and considerable GGT activity resides in the adjacent caput region, relatively higher GGT activity should predominate in infertile males, as indeed was confirmed by the gene chips.
In summary, the results demonstrate a reduced expression of the glutamate transporter EAAC1, and an increase in tissue glutamate, solely in the proximal epididymis of c-ros knockout mice. Because glutamate is an osmolyte employed in volume regulation in somatic cells (Song et al, 1998), it could have repercussions for the development of volume regulation properties of maturing spermatozoa (Yeung et al, 2002b). The apparent normal fertility of mice with functional knockout of the EAAC1 gene (Peghini et al, 1997) suggests that the mere absence of EAAC1 from the proximal part of the epididymis is not wholly responsible for the observed infertility of c-ros-/- males. In EAAC1 knockout mice, other osmolytes may compensate for the lack of EAAC1 and redistributed glutamate. In c-ros knockout mice, EAAC1 deficiency is only one of the consequences of an undifferentiated initial segment and could act in concert with other deficiencies. Alternatively, other secretions of the initial segment, acting directly on sperm or indirectly on downstream regions and their secretions, may also be disturbed in knockout males and have an effect on male fertility. Regardless of the ultimate cause, upsetting the mechanism responsible for sperm volume regulation could be taken advantage of for the purposes of male contraception.
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Acknowledgments |
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Footnotes |
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