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From the Centre de Recherche en Biologie de la Reproduction et Département d'Obstétrique-Gynécologie, Faculté de Médecine, Université Laval, Québec, Canada. * Present address: Laboratoire "Epididyme et maturation des gamètes," Université Blaise Pascal, CNRS UMR 6547 GEEM, 24 avenue des Landais, 63177 Aubière Cedex, France.
| Correspondence to: Robert Sullivan, Unité d'Ontogénie-Reproduction, Centre de Recherche, Centre Hospitalier de l'Université Laval, 2705 Blvd Laurier, Ste-Foy, PQ, Canada, G1V 4G2 (e-mail: robert.sullivan{at}crchul.ulaval.ca). |
| Received for publication January 6, 2004; accepted for publication March 18, 2004. |
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Abstract |
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Key words: Excurrent duct, sperm maturation, angiotensinogen, type 1 receptor to angiotensin II
The epididymis is very important in the posttesticular maturation process of the sperm cells, mainly as it concerns the acquisition of male fertilizing ability. In this context, we have previously shown that the epididymal expression profiles of fertility-related genes are dramatically modified following vasectomy: examples include the P34H in humans (Légaré et al, 2001), HE2-like gene in the cynomolgus monkey (Doiron et al, 2003), and CRISP-1 in the rat (Turner et al, 2000). Furthermore, P34H levels remained low on the spermatozoa of certain men after a vasectomy reversal, probably because of epididymal damage caused by the vasectomy (Guillemette et al, 1999). Protein synthesis is also selectively impaired in the rat caput epididymides following vasectomy, and only certain proteins are recovered after vasovasostomy (Turner et al, 2000). These elements strongly suggest that local factors are involved in such specific changes.
Gene expression in the epididymis is characterized by a particular spatial organization, with specific genes expressed in specific segments of the organ (Cornwall and Hann, 1995; Kirchhoff, 1999). The factors regulating this phenomenon are mainly androgens (Lareyre et al, 2000), testicular factors (Cornwall et al, 2001), or other factors such as cis-DNA regulatory elements (PEA-3 and GATA, for example) and transcription factors (for review, see Rodriguez et al, 2001). However, little is known about the overall gene expression regulation in the mammalian epididymis. Among the possible local factors involved, a local renin-angiotensin system (RAS) in the rat epididymis has been shown in the work of Leung et al (reviewed in 1998 and 2003) (Leung et al, 1998; Leung and Sernia, 2003). The principal effector of this system, angiotensin II (A-II), is a powerful systemic vasoconstrictor involved in blood pressure regulation. Its production depends on the processing of a precursor molecule, angiotensinogen (ANG), which is transformed into angiotensin I by the enzyme renin, and which in turn is transformed into A-II via the action of the angiotensin-converting enzyme (ACE). A-II is involved in different local phenomena, and evidence suggests its implication in the physiology of the epididymis. First, angiotensin I, angiotensin II, renin, and ACE are found in the rat epididymis (Wong and Uchendu, 1990, 1991). Second, A-II has been localized in the basal cells of the rat epididymal epithelium (Zhao et al, 1996). Third, studies involving in situ hybridization, immunohistochemistry, and Western blot analysis have demonstrated the expression of mRNA encoding ANG, as well as the protein, in the epithelial cells of the rat epididymis (Leung et al, 1999). In the rat epididymis, the RAS has already been shown to play a role in the control of anion secretion by a paracrine action (Leung et al, 1998). In humans, the RAS also seems to play an important role in the reproductive tract: testis-specific ACE is expressed only in testis and developing spermatozoa (Brentjens et al, 1986; Mukhopadhyay et al, 1995), and it is specifically localized in the plasma membrane of the acrosome, equatorial, and postacrosomal regions (Kohn et al, 1998). Also, AT-I has been shown to be present on the tail of ejaculated spermatozoa and is involved in the regulation of motility (Vinson et al, 1995).
The importance of the RAS in the male reproductive functions is illustrated by the observation made on ANG knock-out mice, in which ANG deficiency was associated with decreased fertility, in utero lethal effect, and impaired neonatal survival. These effects were dependent on male mice, as breeding heterozygous males with homozygous wild-type females also leads to lower litter number (Tempfer et al, 2000). Furthermore, ACE knock-out mice also showed reduced male fertility (Krege et al, 1995; Esther et al, 1996; Ramaraj et al, 1998).
Thus, we investigated whether the epididymal local RAS system could be modified after vasectomy by evaluating the quantitative modifications of the ANG and AT-I expression in vasectomized cynomolgus monkey epididymides. Studies concerning vasectomy most often consider long-term effects of this situation on different aspects of male fertility. However, in cases of vasectomy reversal in men, modifications that could have occurred in the early stages may well have long-term consequences, a point rarely investigated. In this regard, early activation of the RAS system has been described in a model of ureteral obstruction in the fetal sheep kidney (Ayan et al, 2001), and in vitro studies have shown that RAS system is induced by mechanical stress in cardiac myocytes after only 8 to 24 hours (Malhotra et al, 1999), or that A-II can activate pressurized blood vessels after 1 hour in an organ-culture model (Eskildsen-Helmond and Mulvany, 2003).
In this article, the mRNA and protein expression profiles of ANG and AT-I are described in normal cynomolgus monkey epididymides. Furthermore, changes in the RAS expression following short-term vas deferens obstruction (3 and 7 days) strongly support the hypothesis that this system could play a role in the regulation of gene expression along the mammalian epididymis, and thus in the fertility status of the patients after a vasectomy reversal.
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Materials and Methods |
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The ethical committee of Laval University has approved this protocol.
RNA Extraction
Tissues from different epididymal segments kept at -80°C were
homogenized with a Polytron (InterSciences, Markham, Canada) in a
homogenization buffer (4 M guanidium thiocyanate, 25 mM sodium citrate, pH
7.0, 0.5% sarcosyl, and 0.1 M 2-ß-mercaptoethanol). Extracts were then
processed on columns from Stratagene ("Absolutely RNA" Kit,
Vancouver, Canada) in order to isolate the nucleic acids, following the
manufacturer's instructions. Nucleic acids were then subjected to a DNAse
treatment for 30 minutes at 37°C in order to keep only the RNAs; this
process was followed by an extraction in phenol/chloroform 1:1, and once with
chloroform. RNAs were precipitated in 0.1 volume of 3 M sodium acetate, pH
5.2, and 2.5 volume 95% ethanol overnight at -20°C. The RNA pellets were
resuspended in diethylpyrocarbonate–treated water and quantified by
spectrophotometry at 260 nm. The quality of the RNAs was verified on a 1%
agarose gel to ensure that there was no degradation. RNAs were stored at
-80°C until used.
In Situ Hybridization
The protocol used in this study is based on the hybridization of
digoxigenin-labeled cRNA probes on cryosections of the epididymal tissue, and
it is described in detail in Doiron et al
(2003). The specific points
related to this paper are as follows: sections were incubated overnight at
42°C, under coverslips, with 25 µL of 1 µg/mL or 10 µg/mL,
respectively; heat-denaturated antisense or sense ANG, or AT-I cRNA probed
with DIG. The revelation method is based on the use of alkaline
phosphatase–conjugated anti-DIG antibodies. The hybridization signals
were visualized after a 10- to 15-minute incubation period with the
phosphatase substrate, nitroblue tetrazolium chloride and
5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (GIBCO-BRL,
Gaithersburg, Md).
Classical and Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction
Classical Polymerase Chain Reaction—
Five micrograms of total RNA of each epididymal section from each monkey
were used for first-strand cDNA synthesis, using 100 U of SuperScript II
(Gibco-BRL) and reverse transcription (RT) buffer (50 mM Tris, pH 8.3, 75 mM
KCl, 3 mM MgCl2), 10 mM DTT, 200 µM dNTPs, and 10 µM random
primer p(dN)6; Roche Diagnostics, Québec, Canada) in a final volume of
20 µL. The cDNA was diluted 6.25 times in sterile water and used as the
template in the classical and quantitative polymerase chain reaction (PCR)
mixture.
Primers for ANG and AT-I were designed according to the human sequences found in GenBank (Table 1). Primers for AT-I do not amplify AT-II. Submitting them to a BLAST analysis tested the specificity of the primers. For each gene tested, a PCR was performed on cDNA made from monkey total epididymal cDNA. The products were migrated on an agarose gel, and the specific band for each gene studied was extracted from the gel using Qiaquick gel extraction columns (Qiagen, Mississauga, Canada). Fragments were then cloned in a pGEM®-T vector system (Promega, Madison, Wis) and sequenced in order to confirm their identities.
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Quantitative PCR— Each mRNA was quantified as a ratio to the mRNA encoding the housekeeping gene GAPDH (glyceraldehyde phosphate dehydrogenase), as this gene appeared to be expressed at similar levels among the different epididymal sections (data not shown).
The standard curves were prepared as follows: known concentrations of plasmids containing the cloned fragments for ANG, AT-I, or GAPDH were diluted and used to establish the standard curves. The PCRs were conducted in a Lightcycler apparatus (Roche Diagnostics), and products were detected with SYBR Green, which is included in the FastStart Master SYBR Green I mix (Roche Diagnostics). Prior to the quantification, optimization procedures were performed by running PCRs with or without the purified template to identify the melting temperatures of the primer dimers and the specific product. The efficiency of the PCR reaction was evaluated for each amplified fragment in order to perform the amplifications in the optimal conditions. To measure mRNA levels in the samples, the fluorescence values were taken at a temperature corresponding to the end of the elongation step, as our primers did not form any primer-dimers. The reaction parameters for each mRNA are summarized in Table 2. For each quantification, a 5-µL aliquot of the RT dilutions was used. The standard curve was performed using the DNA prepared as described above, and 5 serial dilutions were used, ranging from 500 pg to 5 fg. At the end of the last PCR cycle, the melting curve was generated by starting the fluorescence acquisition at 72°C and taking measurements every 0.1°C until 95°C was reached. The melting curves allowed us to verify that only one fragment was amplified in each reaction and that the melting point of each of those was identical to the melting point of the positive control. Fragment of which sequence had thus been confirmed. The specificity of our reactions was thus verified as the identities of the positive controls were verified by sequencing and BLAST analysis prior the experiments.
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After the completion of the quantitative PCR analysis, the PCR products were verified by migrating them on a standard 1% agarose gel stained with ethidium bromide and were visualized by exposure to ultraviolet light (data not shown).
Immunohistochemistry
Paraffin-embedded tissues from proximal caput and corpus and from distal
cauda epididymidis of one monkey were cut into sections (3 µm). Sections
were deparaffinized in xylene and rehydrated in graded alcohol solutions
before being incubated in 3% hydrogen peroxide in 60% methanol to quench
endogenous peroxidase activity. Sections were blocked in decomplemented goat
serum diluted 1:10 in PBS for 30 minutes at room temperature, washed 3 times
in PBS for 5 minutes, and incubated overnight at 4°C with antiserum raised
against human AT-II type 1 receptor (AT-I; 0.2 µg/mL, rabbit polyclonal
immunoglobulin G [IgG], sc-1173; Santa Cruz Biotechnology Inc, Santa Cruz,
Calif). One section was incubated with a rabbit control serum at a
concentration corresponding to 0.2 µg/mL of IgG in place of the primary
antibody as a negative control. Staining was detected using a biotinylated
secondary antibody in combination with the avidin-biotin-peroxidase method
(Vectastain ABC Kit; Vector Laboratories Inc, Burlingame, Calif) using AEC
(3-amino-9-ethylcarbazole) as chromagen (Sigma Chemical Co, St Louis, Mo).
Sections were counterstained with hematoxylin and mounted in a water-based
medium (Sigma).
Image Acquisition
For both in situ hybridization and immunohistochemistry, slides were
observed with a Zeiss Axioskop 2 plus microscope (Toronto, Canada) linked to a
digital camera from Diagnostics Instruments (Sterling Heights, Mich). Images
were acquired using the Spot software (Diagnostics Instruments) and analyzed
with Image Pro Plus from Media cybernetics (Silver Springs, Md).
Protein Extraction
Tissues from each epididymal segment stored at -80°C, were homogenized
with a Polytron (InterSciences, Markham, Canada) in a homogenization buffer
(10 mM EDTA, 1 mM PMSF, 1% sodium dodecyl sulfate [SDS] in sterile water).
Extracts were then centrifuged at 3000 x g for 15 minutes at
4°C, and the supernatants were kept. Proteins of the supernatants were
quantified using the Bradford method, with a standard curve made of bovine
serum albumin (fraction V, Sigma).
Western Blotting
Proteins extracted from the epididymal sections were subjected to
SDS-polyacrylamide gel electrophoresis
(Laemmli, 1970) and
transferred on nitrocellulose membrane (Bio-Rad, Hercules, Calif) (Towbin et
al, 1979). Briefly, the nitrocellulose membranes were blocked with PBS, pH
7.4, containing 5% skim milk for 1 hour at room temperature. After blocking,
the membranes were washed for 10 minutes with PBS-0.1% Tween 20 (PBS-T), and
incubated overnight at 4°C with anti AT-I polyclonal antibody diluted
1:1000 in PBS-T supplemented with 5% skim milk or with anti-ANG antibody
diluted 1:1000 in 10% PBS-T-10% skim milk. The anti-ANG antibody was a goat
anti-human ANG and was a gift from Dr Suresh KUMAR (University of Birmingham,
United Kingdom). The membranes were further washed 3 times for 15 minutes in
PBS-T and incubated with peroxidase-conjugated goat anti-rabbit IgG for AT-I
revelation, or with peroxidase-conjugated rabbit anti-goat antibody for ANG
revelation (Bio-Rad), both diluted 1:10 000 in PBS supplemented with 5% or 10%
skim milk. Finally, the membranes were visualized using a chemiluminescent
substrate of peroxidase, according to the supplier's instructions (ECL;
Amersham, Life Science, Oakville, Canada). The different intensities of the
revealed bands were assessed by densitometric analysis (MultiImage
Light-Cabinet DE-400; Alpha Innotech Corporation, San Leandro, Calif). Unless
mentioned otherwise in the figure legends, the total densitometry of the
appearing bands was considered.
Statistical Analysis
Statistical analysis was performed by analysis of variance (AN-OVA) using
super ANOVA software (ABACUS Concepts, Berkeley, Calif). Results were compared
with the Fisher test, and differences were considered significant when
P was less than 0.05.
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Results |
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Considering that in situ hybridization was performed to determine the cellular types expressing the mRNAs, these experiments were only done on 3 different epididymal sections (ie, proximal caput, proximal corpus, and distal cauda of a normal epididymis).
The in situ hybridization for ANG revealed an expression of the mRNA in the perinuclear region of the principal cells in the epididymal epithelium, as shown by the blue coloration pointed out by the arrows (Figure 2A). The staining intensity was higher in the proximal caput, and decreased thereafter along the duct (Figure 2A through C).
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Concerning AT-I, the mRNA appeared at the same cellular location as ANG, thus being in the perinuclear region of the principal cells (Figure 2E, arrows). The intensity of the staining seemed slightly higher in the caput epididymidis, and was also decreasing thereafter (Figure 2E through G). No staining was observed when ANG or AT-I sense probes were used as negative controls (Figure 2D and H, respectively).
Immunohistochemistry
The localization of AT-I was investigated, and as for the mRNA
distribution, the presence of the protein was also heterogeneous along the
epididymis (Figure 2I through
L). What is common throughout the duct is the presence of AT-I in
the smooth muscle cells surrounding the tubule (apparent in
Figure 2K), as well as in the
vascular wall. A new element revealed by this experiment is the presence of
AT-I at the apical pole of the epithelial cells, which appeared clearly in all
epididymal segments shown (Figure 2I
through K). As described in the rat epididymis, AT-I was also
present at the basal side of the epithelium, although at very low levels
(Figure 2I and J). No staining
was revealed when a normal rabbit serum was used as a negative control
(Figure 2L).
Attempts were made to characterize the distribution and cellular location of the ANG protein, but the antibody appeared to only be efficient when used in Western blots experiments.
Vasectomy-Dependent Modifications of the Epididymal RAS
Angiotensinogen mRNA—
Figure 3 illustrates the
individual modifications observed concerning the ANG mRNA levels after a 3-day
vasectomy (Figure 3A and C) or
a 7-day vasectomy (Figure 3B and
D). First, the expression profiles of the ANG mRNA in the normal
epididymides (solid lines) appeared heterogeneous among the 4 epididymides,
with, however, a comparable level in all individuals (except for section 4 in
Figure 3C). The variations
observed after vasectomy were very different depending on the individual
concerned. It appeared that the ANG mRNA level can be significantly higher
following vasectomy in certain sections (sections 2 and 5 in
Figure 3D), but the opposite
was also true (sections 1 and 6 in Figure
3A; section 4 in Figure
3C; and section 1 in Figure
3B). Furthermore, there is no general feature related to the
vasectomy duration appearing on this figure. We can thus say that ANG mRNA
varied after vasectomy in a manner that was completely dependent on each
individual monkey.
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AT-I mRNA— As for ANG, the individual AT-I mRNA expression modifications are reported on Figure 4. The AT-I mRNA in the normal epididymides also showed heterogeneous individual profiles (solid lines), with a similar level of expression between individuals as well. Changes were totally related to the individual cases, but there was a tendency toward an increase in the mRNA levels of AT-I following vasectomy, except for in one of the 3-day vasectomized monkeys (Figure 4C). It is worthwhile to notice that in one of the 7-day vasectomized monkeys, both ANG and AT-I mRNAs were significantly higher in the distal caput and in the proximal cauda epididymides (sections 2 and 5, Figures 3D and 4D). No such observation could be made in any of the 3 other monkeys.
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Angiotensinogen Protein— The protein profiles for ANG, along the 4 individual epididymides, are presented in Figure 5. ANG was revealed at the expected molecular weight of around 61 kd as thick bands, because it is a highly glycosylated protein. It appeared on this figure that ANG was expressed at similar levels among the different sections in the normal epididymides (solid lines), with a higher level in the proximal caput (section 1) in all monkeys. This result also showed that despite the differences in the mRNA levels observed, the protein seemed to be expressed at a constant level along the excurrent duct. Vasectomy provoked different significant modifications of the ANG level, in a manner dependent on the individuals, as seen for the ANG mRNA. However, we can make no parallel between the changes in the ANG mRNA and protein expression profiles in any of the 4 monkeys studied.
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AT-I Protein— As for ANG, the individual AT-I expression profiles were determined in the 4 normal (Figure 6, solid lines) and in the 4 vasectomized epididymides (Figure 6, dashed lines). The AT-I protein appeared as two bands at the expected molecular weight of 45 kd, and their specificity was verified by preincubating the antibody with a blocking peptide (data not shown). The upper band probably corresponds to a glycosylated form of the receptor, a fact already observed in other articles. The protein profiles obtained were quite reproducible in the normal epididymides, with a higher AT-I expression in either the proximal caput or the distal cauda (sections 1 and 6) and even in both sections for two monkeys (A and B). This particular profile could be related to specific functions of the AT-I receptor, a point that will be discussed later. It is interesting to note that AT-I and ANG proteins both seem to be expressed at a higher level in the proximal caput of the normal epididymis. Concerning the vasectomy-dependent variations, only a few sections were affected by the obstruction, and once again, each individual reacted differently to this situation.
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Discussion |
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Although the presence of the RAS in the rat epididymis demonstrated the relation to anion secretion, this article brings new insights to the topic of the presence of this system in the cynomolgus monkey epididymis. Indeed, the quantification allowed us to establish the epididymal expression profile of the RAS and revealed that the expression was generally higher in the proximal caput and distal cauda epididymidis. It was previously demonstrated in the rat epididymis that overall gene expression is higher in cauda and caput epididymidis, whereas it is the lowest in the corpus (Jervis and Robaire, 2001). In the same view, protein synthesis has been shown to be the highest in the rat cauda epididymidis (Jones et al, 1980; Brooks, 1983). These data are in accordance with a putative role of the RAS in the gene expression regulation, as its elements are present in the segments of the epididymis where protein synthesis is the most active. As the morphological segmentation of the epididymis is fundamentally related to its functions, we can hypothesize that the most important presence of the RAS in two segments of the monkey epididymis is of functional relevance. It is also tempting to speculate that the RAS expression pattern in the human epididymis is similar to that in the monkey and would thus have close functional properties as well, since these organs have structural similarities, as mentioned above.
The mRNA expression profiles are different in the epididymides of the 4 monkeys, thus showing some inter-individual variation. The segmentation of the epididymis as well as the sensitivity of the quantitative RT-PCR may in part explain the discrepancies observed in the mRNA expression profiles, or there might be differential regulation of the mRNA processing in the segments of the epididymis. The difference in the variations between mRNA and protein for ANG after vasectomy may be due to the fact that the circulating ANG present in the tissue could mask potential variations occurring in the epithelial production of ANG. However, concerning the AT-I protein profiles, the monkeys were quite homogeneous, with a maximum expression either in the proximal caput or in distal cauda, or in both sections for two monkeys. The modifications observed following vasectomy confirmed that the epididymal RAS is really subjected to individual parameters. Even though no general scheme could be drawn from the 4 studied monkeys, some points are interesting: first, the differences appearing at the mRNA level were not true for the protein, thus suggesting some regulatory mechanism, probably at the posttranscriptional level. It is worthwhile to note that the regulation of the AT-I expression has been described in different organs or cell types as being mainly controlled at the posttranscriptional level, by influencing the stability of the mRNA (Krishnamurthi et al, 1999; Xu and Murphy, 2000). This point would concur with the fact that the epididymal RAS is an important local system and thus needs to be tightly regulated. Second, the fact that the AT-I protein is mostly present in the proximal caput and the distal cauda may reflect some important functional properties, as these two sites are very important in the fluid movement and composition in the epididymis: proximal caput is very active in terms of reabsorption of the testicular fluid (Le Lannou et al, 1979), and distal cauda is the location in which sperm cells are stored, and the osmotic regulation is fundamental for their survival (Crichton et al, 1993; Jones and Murdoch, 1996). Modification of the AT-I level could have dramatic effects on these two phenomena because the RAS system is involved in fluid regulation, and we observed changes in the AT-I protein level in the proximal caput of 3 vasectomized epididymides and in 2 cases for the distal cauda. This could also have consequences in the case of vasectomy reversal in humans. In this regard, the outcome of a vasectomy reversal in man is subject to high interindividual variations, and fertility is not systematically restored, despite a normal spermogram. It is also known that after vasectomy, each individual does not respond in the same way in terms of pain, inflammation, anti-sperm antibodies, or granuloma formation (Kessler, 1982; Flickinger et al, 1995; McDonald, 2000). Our data may be taken into account for the explanation of such observations, in the sense that gene expression seems to be altered in an individual-dependent manner following vasectomy. We chose to study short-term vasectomy in this work in order to evaluate the modifications of the RAS as an early event that could have consequences for other genes, a fact that we still need to confirm. It would also be of great interest to test the RAS expression profile in vasovasostomized monkey epididymides in relation with their fertility status.
The role of local RAS in the male reproductive tract needs to be further investigated. The evidence of such a system in the primate epididymis, with marked individual reactions following vasectomy, makes it a good model to use to study the epididymal modifications potentially influencing fertility recovery after a vasectomy reversal. Furthermore, the study of this system may lead to novel improvements in the understanding of the physiology of the epididymis and to new insights in the post-testicular sperm cell maturation process.
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Acknowledgments |
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Footnotes |
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References |
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|---|
Brentjens JR, Matsuo S, Andres GA, Caldwell PR, Zamboni L. Gametes contain angiotensin converting enzyme (kininase II). Experientia.1986;42: 399 -402.[Medline]
Brooks DE. Effect of androgens on protein synthesis and secretion in various regions of the rat epididymis, as analysed by two-dimensional gel electrophoresis. Mol Cell Endocrinol. 1983; 29: 255 -270.[Medline]
Cornwall GA, Collis R, Xiao Q, Hsia N, Hann SR. B-Myc, a proximal
caput epididymal protein, is dependent on androgens and testicular factors for
expression. Biol Reprod. 2001; 64: 1600
-1607.
Cornwall GA, Hann SR. Specialized gene expression in the
epididymis. J Androl. 1995; 16: 379
-383.
Cos LR, Valvo JR, Davis RS, Cockett AT. Vasovasostomy: current state of the art. Urology. 1983; 22: 567 -575.[Medline]
Crichton EG, Suzuki F, Krutzsch PH, Hammerstedt RH. Unique features of the cauda epididymidal epithelium of hibernating bats may promote sperm longevity. Anat Rec. 1983; 237: 475 -481.
Doiron K, Légaré C, Saez F, Sullivan R. Effect of
vasectomy on gene expression in the epididymis of cynomolgus monkey.
Biol Reprod. 2003; 68: 781
-788.
Eskildsen-Helmond YE, Mulvany MJ. Pressure-induced activation of
extracellular signal-regulated kinase 1/2 in small arteries.
Hypertension. 2003; 41: 891
-897.
Esther CR Jr, Howard TE, Marino EM, Goddard JM, Capecchi MR, Bernstein KE. Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility. Lab Invest. 1996;74: 953 -965.[Medline]
Flickinger CJ, Howards SS, Herr JC. Effects of vasectomy on the epididymis. Microsc Res Tech. 1995; 30: 82 -100.[Medline]
Guillemette C, Thabet M, Dompierre L, Sullivan R. Some
vasovasostomized men are characterized by low levels of P34H, an epididymal
sperm protein. J Androl. 1999; 20: 214
-219.
Jervis KM, Robaire B. Dynamic changes in gene expression along the
rat epididymis. Biol Reprod. 2001; 65: 696
-703.
Jones R, Brown CR, Von Glos KI, Parker MG. Hormonal regulation of protein synthesis in the rat epididymis. Characterization of androgen-dependent and testicular fluid–dependent proteins. Biochem J. 1980; 188: 667 -676.[Medline]
Jones RC, Murdoch RN. Regulation of the motility and metabolism of spermatozoa for storage in the epididymis of eutherian and marsupial mammals. Reprod Fertil Dev. 1996; 8: 553 -568.[Medline]
Kessler R. Vasectomy and vasovasostomy. Surg Clin N Am. 1982;62: 971 -980.
Kirchhoff C. Gene expression in the epididymis. Int Rev Cytol. 1999;188: 133 -202.[Medline]
Kohn FM, Dammshauser I, Neukamm C, Renneberg H, Siems WE, Schill
WB, Aumuller G. Ultrastructural localization of angiotensin-converting enzyme
in ejaculated human spermatozoa. Hum Reprod. 1998; 13: 604
-610.
Krassnigg F, Niederhauser H, Fink E, Frick J, Schill WB. Angiotensin converting enzyme in human seminal plasma is synthesized by the testis, epididymis and prostate. Int J Androl. 1989; 12: 22 -28.[Medline]
Krege JH, John SW, Langenbach LL, et al. Male-female differences in fertility and blood pressure in ACE-deficient mice. Nature. 1995;375: 146 -148.[Medline]
Krishnamurthi K, Verbalis JG, Zheng W, Wu Z, Clerch LB, Sandberg K.
Estrogen regulates angiotensin AT1 receptor expression via cytosolic proteins
that bind to the 5' leader sequence of the receptor mRNA.
Endocrinology. 1999; 140: 5435
-5438.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227: 680 -685.[Medline]
Lareyre JJ, Reid K, Nelson C, Kasper S, Rennie PS, Orgebin-Crist
MC, Matusik RJ. Characterization of an androgen-specific response region
within the 5' flanking region of the murine epididymal retinoic acid
binding protein gene. Biol Reprod. 2000; 63: 1881
-1892.
Le Lannou D, Chambon Y, Le Calve M. Role of the epididymis in reabsorption of inhibin in the rat. J Reprod Fertil Suppl. 1979;26: 117 -121.
Légaré C, Thabet M, Picard S, Sullivan R. Effect of
vasectomy on P34H messenger ribonucleic acid expression along the human
excurrent duct: a reflection on the function of the human epididymis.
Biol Reprod. 2001; 64: 720
-727.
Leung PS, Chan HC, Chung YW, Wong TP, Wong PY. The role of local angiotensins and prostaglandins in the control of anion secretion by the rat epididymis. J Reprod Fertil Suppl. 1998; 53: 15 -22.[Medline]
Leung PS, Chan HC, Fu LX, Leung PY, Chew SB, Wong PY. Angiotensin II receptors: localization of type I and type II in rat epididymides of different developmental stages. J Membr Biol. 1997; 157: 97 -103.[Medline]
Leung PS, Sernia C. The renin-angiotensin system and male reproduction: new functions for old hormones. J Mol Endocrinol. 2003;30: 263 -270.[Abstract]
Leung PS, Wong TP, Sernia C. Angiotensinogen expression by rat epididymis: evidence for an intrinsic, angiotensin-generating system. Mol Cell Endocrinol. 1999; 155: 115 -122.[Medline]
Malhotra R, Sadoshima J, Brosius FC III, Izumo S. Mechanical
stretch and angiotensin II differentially upregulate the renin-angiotensin
system in cardiac myocytes. In Vitro Circ Res. 1999; 85: 137
-146.
McDonald SW. Cellular responses to vasectomy. Int Rev Cytol. 2000;199: 295 -339.[Medline]
Metayer S, Dacheux F, Guerin Y, Dacheux JL, Gatti JL. Physiological
and enzymatic properties of the ram epididymal soluble form of germinal
angiotensin I-converting enzyme. Biol Reprod. 2001; 65: 1332
-1339.
Mukhopadhyay AK, Cobilanschi J, Schulze W, Brunswig-Spickenheier B, Leidenberger FA. Human seminal fluid contains significant quantities of prorenin: its correlation with the sperm density. Mol Cell Endocrinol. 1995;109: 219 -224.[Medline]
Ramaraj P, Kessler SP, Colmenares C, Sen GC. Selective restoration of male fertility in mice lacking angiotensin-converting enzymes by sperm-specific expression of the testicular isozyme. J Clin Invest. 1998;102: 371 -378.[Medline]
Rodriguez CM, Kirby JL, Hinton BT. Regulation of gene transcription in the epididymis. Reproduction. 2001; 122: 41 -48.[Abstract]
Sodhi CP, Kanwar YS, Sahai A. Hypoxia and high glucose upregulate
ATI receptor expression and potentiate ANG II-induced proliferation in VSM
cells. Am J Physiol Heart Circ Physiol. 2003; 284: H846
-H852.
Tempfer CB, Moreno RM, Gregg AR. Genetic control of fertility and
embryonic waste in the mouse: a role for angiotensinogen. Biol
Reprod. 2000;62: 457
-462.
Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1079; 76: 4350 -4354.
Turner TT, Riley TA, Vagnetti M, Flickinger CJ, Caldwell JA, Hunt DF. Postvasectomy alterations in protein synthesis and secretion in the rat caput epididymidis are not repaired after vasovasostomy. J Androl. 2000;21: 276 -290.[Abstract]
Udupa EG, Rao NM. Effect of chloride and diamide on angiotensin converting enzyme from sheep testis and epididymis. Indian J Exp Biol. 1998;36: 43 -45.[Medline]
Vinson GP, Puddefoot JR, Ho MM, Barker S, Mehta J, Saridogan E, Djahanbakhch O. Type 1 angiotensin II receptors in rat and human sperm. J Endocrinol. 1995; 144: 369 -378.[Abstract]
Wang CH, Li SH, Weisel RD, et al. C-reactive protein upregulates
angiotensin type 1 receptors in vascular smooth muscle.
Circulation. 2003; 107: 1783
-1790.
Weiske WH. Vasectomy. Andrologia. 2001; 33: 125 -134.[Medline]
Wong PY, Uchendu CN. The role of angiotensin-converting enzyme in the rat epididymis. J Endocrinol. 1990; 125: 457 -465.[Abstract]
Wong PY, Uchendu CN. Studies on the renin-angiotensin system in primary monolayer cell cultures of the rat epididymis. J Endocrinol. 1991;131: 287 -293.[Abstract]
Xu K, Murphy TJ. Reconstitution of angiotensin receptor mRNA
down-regulation in vascular smooth muscle. Post-transcriptional control by
protein kinase a but not mitogenic signaling directed by the
5'-untranslated region. J Biol Chem. 2000; 275: 7604
-7611.
Yamaguchi T, Ikekita M, Kizuki K, Moriya H. Distribution of angiotensin I converting enzyme in male reproductive systems of various vertebrates and properties of the genital enzymes. Adv Exp Med Biol. 1989;247B: 377 -382.[Medline]
Zhao W, Leung PY, Chew SB, Chan HC, Wong PY. Localization and distribution of angiotensin II in the rat epididymis. J Endocrinol. 1996;149: 217 -222.[Abstract]
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