784
Vasectomy-Dependent Dysregulation of a Local
Renin-Angiotensin System in the Epididymis of the
Cynomolgus Monkey (Macaca fascicularis)
FABRICE SAEZ,* CHRISTINE LE´ GARE´, JULIE LAFLAMME, AND ROBERT SULLIVAN
From the Centre de Recherche en Biologie de la Reproduction et De´partement d’Obste´trique-Gyne´cologie, Faculte´ de Me´decine, Universite´ Laval, Que´bec, Canada.
ABSTRACT: The mammalian epididymis is a fundamental organ for sperm cell maturation; it allows mammals to acquire their fer-tilizing ability. We have previously shown that during obstruction in cases of vasectomy, gene expression profiles were modified in hu-man and cynomolgus monkey epididymides. Paracrine factors thus appear to be key elements in local gene expression along the ep-ididymis. Local renin-angiotensin systems (RAS) have been de-scribed in many other organs as paracrine regulators of gene ex-pression. This work demonstrates the presence of a local RAS in the epididymis of the cynomolgus monkey and investigates the va-sectomy-dependent changes occurring in this system. After unilat-eral vasectomy in 4 monkeys (two for 3 days and two others for 7 days), the presence of two major components of the RAS (ie, an-giotensinogen [ANG] and the type 1 receptor to angiotensin II [AT-I]) was evaluated in the vasectomized and the normal controlateral epididymides of each monkey. We also show by in situ hybridiza-tion that the principal cells of the epididymis express ANG and
AT-I mRNAs and immunohistochemistry permitted to verify the co-lo-calization of the AT-I protein and mRNA. Quantitative comparisons of individual variations in the mRNA and protein profiles for ANG and AT-I revealed that vasectomy altered the RAS expression pro-files in an individual manner, thus confirming its role as a local system. This study provides a good basis for further investigation of the possible implications of the RAS in the physiology of the epididymis. Furthermore, the individual dependent modifications are in accordance with the very fluctuating results obtained in the fertility status of human patients undergoing a vasectomy reversal. The variations observed in the RAS expression profiles may be a good model to study the causes of the overall epididymal gene expression dysregulation that follows vasectomy and potentially af-fects fertility.
Key words: Excurrent duct, sperm maturation, angiotensinogen, type 1 receptor to angiotensin II.
J Androl 2004;25:784-796
V
asectomy is very widely used as a male contracep-tion technique because of its high rate of efficiency. In 2001, almost 100 million men worldwide relied on this method for family planning purposes (Weiske, 2001). However, mainly as a result of changes in their personal lives (divorce or secondary partners), some men have a vasectomy reversal. Data are scarce concerning the pro-portion of these men, but it was estimated that in 1983, around 250 000 to 300 000 men in the United States aloneSupported by a Canadian Institutes for Health Research grant to R.S. and by a Lalor Postdoctoral Fellowship to F.S.
Correspondence to: Robert Sullivan, Unite´ d’Ontoge´nie-Reproduction, Centre de Recherche, Centre Hospitalier de l’Universite´ Laval, 2705 Blvd Laurier, Ste-Foy, PQ, Canada, G1V 4G2 (e-mail: robert.sullivan@ crchul.ulaval.ca).
*Present address: Laboratoire ‘‘Epididyme et maturation des game`tes,’’ Universite´ Blaise Pascal, CNRS UMR 6547 GEEM, 24 avenue des Lan-dais, 63177 Aubie`re Cedex, France.
Received for publication January 6, 2004; accepted for publication March 18, 2004.
underwent vasectomy reversal (Cos et al, 1983). Despite surgery, the fertility status of these men is not always reestablished and is subject to individual variations.
The epididymis is very important in the posttesticular maturation process of the sperm cells, mainly as it con-cerns the acquisition of male fertilizing ability. In this context, we have previously shown that the epididymal expression profiles of fertility-related genes are dramati-cally modified following vasectomy: examples include the P34H in humans (Le´gare´ 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 ep-ididymal 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 ex-pressed 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 ep-ididymis. Among the possible local factors involved, a local renin-angiotensin system (RAS) in the rat epididy-mis 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, immuno-histochemistry, and Western blot analysis have demon-strated 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, equa-torial, 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 im-paired neonatal survival. These effects were dependent on male mice, as breeding heterozygous males with homo-zygous wild-type females also leads to lower litter num-ber (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 eval-uating the quantitative modifications of the ANG and AT-I expression in vasectomized cynomolgus monkey epi-didymides. Studies concerning vasectomy most often con-sider long-term effects of this situation on different as-pects 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 consequenc-es, a point rarely investigated. In this regard, early acti-vation 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 sys-tem 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 Mul-vany, 2003).
In this article, the mRNA and protein expression profiles of ANG and AT-I are described in normal cynomolgus mon-key epididymides. Furthermore, changes in the RAS ex-pression 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.
Materials and Methods
Animals and Tissue Processing
Crab-eating macaques, also known as cynomolgus monkeys
(Ma-caca fascicularis), were used in this study. This Old World
mon-key has a relatively small adult body weight and breeds at any time of the year. This species has previously been used for many studies on reproduction. Sexually mature male M fascicularis (5 to 10 years of age) were individually housed at the animal facility on the campus of Laval University (Que´bec, Canada). They were fed with standard diet and had free access to water. These diurnal males were kept on a 12 hour light/12 hour dark schedule. Four animals were unilaterally vasectomized (two for a period of 3 days and two for a period of 7 days), bilaterally orchidectomized after the respective time periods, and then offered to other researchers in our academic community. The epididymidis of each monkey were excised and processed as follows: they were dissected into 6 different sections (ie, the proximal and distal parts of the caput, corpus, and cauda epididymides) (Figure 1). For each section, tissue pieces were frozen in liquid nitrogen and maintained at
2808C for RNA and protein extraction. Other pieces were fixed
overnight in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 48C and then included in paraffin for immunohistochem-istry, or in OCT (Optimal Cutting Temperature; Canemco Sup-plies, Saint-Laurent, Canada) in order to make cryosections for in situ hybridization purposes.
The ethical committee of Laval University has approved this protocol.
Figure 1. Micrograph of a normal cynomolgus monkey epididymis and representation of the different segments analyzed for renin-angiotensin system expression.
Table 1. Properties of the 3 polymerase chain reaction (PCR) fragments used in this study
Gene Amplified
GenBank
Access Number Primers
Nucleotide Position PCR Fragment Length (bp) GAPDH BC023632 Forward: 59-GAA-GAC-TGT-GGA-TGG-CCC-CTC-39 Reverse: 59-GTT-GAG-GGC-AAT-GCC-AGC-CCC-39 588–608 925–945 358 ANG K02215 Forward: 59-GGA-AGC-CCA-AAG-ACC-CCA-39 Reverse: 59-ACA-GCC-GTT-GGG-GAG-AGG-39 218–235 428–445 228 AT-I NM-009585 Forward: 59-GAT-GAT-TGT-CCC-AAA-GCT-GG-39 Reverse: 59-TAG-GTA-ATT-GCC-AAA-GGG-CC-39 343–362 578–597 255
* GAPDH indicates glyceraldehyde phosphate dehydrogenase; ANG, angiotensinogen; and AT-I, angiotensin II type I receptor.
RNA Extraction
Tissues from different epididymal segments kept at2808C 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- b-mercap-toethanol). Extracts were then processed on columns from Stra-tagene (‘‘Absolutely RNA’’ Kit, Vancouver, Canada) in order to isolate the nucleic acids, following the manufacturer’s instruc-tions. Nucleic acids were then subjected to a DNAse treatment for 30 minutes at 378C 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 at2208C. The RNA pellets were resuspended in diethylpyrocarbonate–treated water and quantified by spectro-photometry 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 at2808C 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 epidid-ymal 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 428C, under coverslips, with 25 mL of 1 mg/mL or 10 mg/mL, respectively; heat-denaturated anti-sense or anti-sense ANG, or AT-I cRNA probed with DIG. The rev-elation method is based on the use of alkaline phosphatase–con-jugated anti-DIG antibodies. The hybridization signals were vi-sualized after a 10- to 15-minute incubation period with the phosphatase substrate, nitroblue tetrazolium chloride and 5-bro-mo-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 mM
dNTPs, and 10mM random primer p(dN)6; Roche Diagnostics, Que´bec, Canada) in a final volume of 20mL. 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-AT-IAT-I. 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
Table 2. Lightcycler parameters for the 3 amplified fragments*
Operation Global Parameters GAPDH ANG AT-I
Activation of the polymerase Denaturation Annealing Elongation Number of PCR cycles 10 min, 958C 0 s, 958C 588C 728C, 20 s 40 628C 728C, 20 s 50 588C 728C, 20 s 40 Melting Cooling
Melting peak temperature of amplified fragments
758C to 958C 958C to 408C
91.58C 908C 848C
* GAPDH indicates glyceraldehyde phosphate dehydrogenase; ANG, angiotensinogen; AT-I, angiotensin II type I receptor; and PCR, polymerase chain reaction.
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 pGEMt-T vector sys-tem (Promega, Madison, Wis) and sequenced in order to confirm their identities.
Quantitative PCR—Each mRNA was quantified as a ratio to
the mRNA encoding the housekeeping gene GAPDH (glyceral-dehyde phosphate dehydrogenase), as this gene appeared to be expressed at similar levels among the different epididymal sec-tions (data not shown).
The standard curves were prepared as follows: known con-centrations 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 ap-paratus (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 temper-atures of the primer dimers and the specific product. The effi-ciency 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 fluo-rescence 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-mL aliquot of the RT dilutions was used. The standard curve was performed using the DNA prepared as described above, and 5 serial dilu-tions 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 728C and taking measurements every 0.18C until 958C 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 melt-ing 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 ver-ified by sequencing and BLAST analysis prior the experiments. 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 (3mm). Sections were deparaffinized in xylene and re-hydrated 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 temper-ature, washed 3 times in PBS for 5 minutes, and incubated over-night at 48C with antiserum raised against human AT-II type 1 receptor (AT-I; 0.2mg/mL, rabbit polyclonal immunoglobulin G [IgG], sc-1173; Santa Cruz Biotechnology Inc, Santa Cruz, Ca-lif). One section was incubated with a rabbit control serum at a concentration corresponding to 0.2mg/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 mount-ed in a water-basmount-ed mmount-edium (Sigma).
Image Acquisition
For both in situ hybridization and immunohistochemistry, slides were observed with a Zeiss Axioskop 2 plus microscope (To-ronto, Canada) linked to a digital camera from Diagnostics In-struments (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 2808C, 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 3 g for 15 minutes at 48C, and the supernatants were kept. Proteins of the supernatants were quan-tified 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, Ca-lif) (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 48C 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 Bir-mingham, 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 visu-alized using a chemiluminescent substrate of peroxidase, accord-ing 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-Cab-inet DE-400; Alpha Innotech Corporation, San Leandro, Calif). Unless mentioned otherwise in the figure legends, the total den-sitometry 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.
Results
In Situ Hybridization
Specific PCR fragments were cloned for ANG and AT-I from cynomolgus monkey epididymal reverse-transcribed RNA. The primers were selected based on human se-quences (Table 1), and the cloned fragments were verified by sequencing before in situ hybridization experiments were carried out.
Considering that in situ hybridization was performed to determine the cellular types expressing the mRNAs, these experiments were only done on 3 different epidid-ymal sections (ie, proximal caput, proximal corpus, and distal cauda of a normal epididymis).
The in situ hybridization for ANG revealed an expres-sion of the mRNA in the perinuclear region of the prin-cipal 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).
Concerning AT-I, the mRNA appeared at the same cel-lular location as ANG, thus being in the perinuclear re-gion of the principal cells (Figure 2E, arrows). The
inten-sity 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 pres-ence 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 individ-ual 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 epidid-ymides, with, however, a comparable level in all individ-uals (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 vasec-tomy 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 va-sectomy 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 mon-key.
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 het-erogeneous 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-Figure 2. Hybridization of the mRNAs for angiotensinogen (ANG) (A to D) and type 1 receptor to angiotensin II (AT-I) (E to H), as well as immuno-histochemistry for AT-I (I to L). The mRNAs appear stained in blue, as pointed by the arrows in A and E. The results obtained with sense oligonu-cleotides are shown in D for ANG and in H for AT-I. Immunohistochemistry was performed using an anti-AT-I antibody (I to L) (see ‘‘Materials and Methods’’ for details), and the signal appears as a brown staining shown by the arrows in I. The negative control serum is shown in L. These experiments were carried out on 3 different epididymal sections: proximal caput (A, E, and I), proximal corpus (B, F, and J), and distal cauda epididymidis (C, G, and K). Both antisense probes and control serum were assessed on the proximal corpus epididymidis. The figures presented are representative of 3 independent experiments, for each mRNA or for the AT-I protein. Scale bar5100mm.
Figure 3. Real-time polymerase chain reaction (PCR) quantification (Lightcycler) of the angiotensinogen mRNA in the 4 normal epididymides (solid lines) as well as in the 4 vasectomized epididymides (dashed lines). A and C represent the 3-day vasectomized monkeys and B and D represent the 7-day vasectomized monkeys. Results are expressed as a ratio to the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH). Each value represents the mean6SEM of 3 independent measurements. *P,.05 compared to the same section of the normal epididymis. Epididymal segments are listed as follows: 15proximal caput, 25distal caput, 35proximal corpus, 45distal corpus, 55proximal cauda, and 65distal cauda.
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 obser-vation could be made in any of the 3 other monkeys.
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
mon-keys. 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 pro-tein expression profiles in any of the 4 monkeys studied. AT-I Protein—As for ANG, the individual AT-I ex-pression 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 anti-body with a blocking peptide (data not shown). The upper
Figure 4. Real-time polymerase chain reaction (PCR) quantification (Lightcycler) of the angiotensin II type I receptor mRNA in the 4 normal epididymides (solid lines) as well as in the 4 vasectomized epididymides (dashed lines). A and C represent the 3-day vasectomized monkeys and B and D represent the 7-day vasectomized monkeys. Results are expressed as a ratio to the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH). Each value represents the mean6SEM of 3 independent measurements. *P,.05 compared to the same section of the normal epididymis. Epididymal segments are listed as follows: 15proximal caput, 25distal caput, 35proximal corpus, 45distal corpus, 55proximal cauda, and 65distal cauda.
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 dis-cussed 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.
Discussion
This article characterizes the short-term vasectomy-de-pendent changes in the expression of two major compo-nents of the RAS, ANG and AT-I, along the epididymis of the cynomolgus monkey, in a quantitative manner. The first step taken in this study was to demonstrate the pres-ence of the local RAS in the normal epididymis of the cynomolgus monkey. With regard to this step, in situ hy-bridization showed that the principal cells actually ex-pressed the mRNAs of ANG and AT-I all along the epi-didymal duct. Furthermore, the immunohistochemistry experiments performed in this study showed that AT-I
Figure 5. Protein quantification by Western blotting of angiotensinogen in the 4 normal epididymides (solid lines) as well as in the 4 vasectomized epididymides (dashed lines). A and C represent the 3-day vasectomized monkeys and B and D represent the 7-day vasectomized monkeys. The corresponding blots are represented above each graph (each figure being representative of 3 different experiments). The densitometric results are expressed as percentages of the total densitometric values from the 6 bands and are presented as mean6SEM for 3 independent experiments. *P
,.05 compared to the same section of the normal epididymis. Epididymal segments are listed as follows: 15proximal caput, 25distal caput, 35 proximal corpus, 45distal corpus, 55 proximal cauda, 65 distal cauda, N5normal, 3-day V5 3-day vasectomized, and 7-day V57-day vasectomized.
was present at the basal side of the epithelium but also at the apical pole. This parameter is different than what was shown in the rat epididymis, in which AT-I was pres-ent at the apical side only in the developing rats (Leung
et al, 1997). Here, the presence of AT-I at the apical side strongly indicates an interaction with the epididymal fluid content, and possibly some kind of feedback loop regu-lation between the secretions (proteolytic degradation of
Figure 6. Protein quantification by Western blotting of angiotensin II type I receptor in the 4 normal epididymides (solid lines) as well as in the 4 vasectomized epididymides (dashed lines). A and C represent the 3-day vasectomized monkeys and B and D represent the 7-day vasectomized monkeys. The corresponding blots are represented above each graph (each figure being representative of 3 different experiments). The densitometric results are expressed as percentages of the total densitometric values from the 6 bands and are presented as mean6SEM for 3 independent experiments. *P,.05 compared to the same section of the normal epididymis. Epididymal segments are listed as follows: 15proximal caput, 25 distal caput, 35proximal corpus, 45distal corpus, 55proximal cauda, 65distal cauda, N5normal, 3-day V53-day vasectomized, and 7-day V57-day vasectomized.
ANG into A-II) and the expression level of the RAS el-ements. It is well known in other, different local RAS, particularly in vascular smooth muscle cells, that the stim-ulation of I by A-II leads to the modstim-ulation of the
AT-I expression in either a positive or a negative feedback loop (Xu and Murphy, 2000; Sodhi et al, 2003; Wang et al, 2003), and this may also be the case in the epididymis. We could not perform immunohistochemistry
experi-ments with the anti-ANG antibody because of the high background levels appearing on the slides, which proba-bly occurred because the antibody was not specific to monkey ANG. Although we do not demonstrate that the ANG protein is produced by the epididymal epithelium, the in situ and Western blot results strongly suggest its epithelial production. These elements seem to confirm the local ANG production, and the ACE, which converts A-I into the active A-II, has already been shown to be present in the epididymal lumen of the ram (Metayer et al, 2001), dog (Yamaguchi et al, 1989), sheep (Udupa and Rao, 1998), rat (Wong and Uchendu, 1990), and human (Krass-nigg et al, 1989). Altogether, these elements are in ac-cordance with the very likely presence of a complete RAS in the epithelium and the lumen of the cynomolgus mon-key epididymis. The presence of an epididymal RAS, and more generally the influence of different RAS systems along the male reproductive tract, have been discussed in a recent review article (Leung and Sernia, 2003). The structure of the human epididymis is closer to that of the monkey than to that of the rodent in terms of structure, and the expression pattern of the RAS in the monkey epididymis may thus reflect the human situation, a fact that will, however, require confirmation.
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 ex-pression was generally higher in the proximal caput and distal cauda epididymidis. It was previously demonstrated in the rat epididymis that overall gene expression is high-er in cauda and caput epididymidis, whhigh-ereas it is the low-est 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 ele-ments are present in the segele-ments of the epididymis where protein synthesis is the most active. As the mor-phological segmentation of the epididymis is fundamen-tally 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 pat-tern 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 ep-ididymides 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 regula-tion of the mRNA processing in the segments of the ep-ididymis. 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 pro-duction 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 in-teresting: 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 posttranscrip-tional 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 com-position 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 fun-damental 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 system-atically restored, despite a normal spermogram. It is also known that after vasectomy, each individual does not re-spond 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 in-fluencing 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.
Acknowledgment
We thank Dr Mathias Leblanc for performing surgery on monkeys used in this study.
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