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BOR - Papers in Press, published online ahead of print October 17, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.008177
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BIOLOGY OF REPRODUCTION 68, 781–788 (2003)
DOI: 10.1095/biolreprod.102.008177
© 2003 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Effect of Vasectomy on Gene Expression in the Epididymis of Cynomolgus Monkey1

Karine Doirona, Christine Légaréa, Fabrice Saeza, and Robert Sullivan2,a

a Centre de Recherche en Biologie de la Reproduction and Département d'Obstétrique-Gynécologie, Faculté de Médecine, Université Laval, Ste-Foy, Quebec, Canada G1V 4G2


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vasectomy has been shown to affect the pattern of mRNA expression of P34H, a human sperm protein added to the acrosomal cap during epididymal transit. It has been reported that vasectomy alters the histology of the reproductive tract in various species as a result of the increased pressure in the epididymis. The aim of this study was to evaluate if other epididymis-specific mRNAs, which are expressed in different patterns along the duct, are altered by vasectomy as well. We analyzed the expression of P31m (a monkey homologue of human P34H) and three different HE-like (HE-l) mRNAs along the epididymis in the cynomolgus monkey (Macaca fascicularis). Sexually mature cynomolgus monkeys were vasectomized unilaterally; then the epididymides were surgically removed at different time points. The ipsilateral normal epididymis was used as a control. Histomorphometric measurements showed that the height of the epididymal epithelial cells started to be affected only at 14 wk postsurgery. However, Northern blot and in situ hybridization analysis showed that the expression pattern of P31m, HE1, and HE5-like mRNA along the epididymis was not affected by vasectomy. Only the HE2-like mRNA predominantly expressed in the normal corpus epididymidis was significantly lowered 14 wk after vasectomy. Thus, ductal obstruction differentially alters mRNA expression along the epididymis of the cynomolgus monkey.

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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
After their production in the testis, mammalian spermatozoa undergo fundamental maturation steps in the epididymis [1–5]. The major changes, i.e., the acquisition of the motility and of the zona pellucida binding capacity, occur in the caput and the corpus epididymidis [6, 7]. Among the modifications, spermatozoa acquire several secreted proteins present in the epididymal fluid [8]. The more distal segment of the epididymis, the cauda, is specialized in sperm storage [9, 10]. These three regions of the epididymis are distinguished by their epithelial cell morphology [11, 12] and by their specific patterns of gene expression. Many studies have reported complex and highly regulated gene expression in the epithelium of the epididymis [13–16] with subregions of distinct expression patterns suggesting specialized functions.

P34H, previously described by our laboratory, is a protein secreted by the epididymis and transferred to the acrosomal cap of the human spermatozoa. This sperm surface protein is involved in the interaction of the sperm cells with the zona pellucida [17, 18]. A correlation has been shown between low amounts of P34H and a decreased ability of spermatozoa to interact with the zona pellucida [19]. P34H can thus be used as an epididymal marker of human sperm maturation [20]. Studies have shown that the obstruction resulting from vasectomy alters the pattern of P34H mRNA expression when compared with human unobstructed excurrent duct [21]. Moreover, the epididymal lumen was shown to be distended along its entire length, with the height of the epithelium being maximal in the caput [21]. This could have functional consequences on the epididymis, as it has been demonstrated that, in the adult epididymis, protein synthesis is proportional to the height of the epithelium [22].

Vasectomy is a widely used contraceptive method in North America. However, this procedure could have deleterious consequences on the epididymal function in cases of vasectomy reversal. It thus seems that vasectomy affects the expression of at least one gene involved in the maturation steps of the sperm cells and that this deleterious effect can be maintained after vasovasostomy [21].

The cynomolgus monkey (Macaca fascicularis) homologue of human P34H, P31m, was previously identified by our group, and its cDNA shares 97% nucleotide sequence identity with P34H [23]. The expression of human epididymal (HE) proteins HE-1, HE-2, and HE-5 [15] is regulated in a region-specific manner throughout the epididymis. These proteins are thus good candidates to evaluate the influence of vasectomy on gene expression along the epididymis. Homologous gene products showing highly similar expression patterns have been found in other mammals [15, 24–28] and are also present in the cynomolgus epididymis. These proteins will be called HE1-like, HE2-like, and HE5-like (HE1-l, HE2-l, and HE5-l, respectively).

This study was undertaken to further elucidate the effects of ductal obstruction on physiological functions of the epididymis and on sperm maturation with regard to specific gene expression patterns. Here we report the morphometric analysis of the epididymal epithelium as well as the expression patterns of P31m, HE1-l, HE2-l, and HE5-l along normal and vasectomized cynomolgus monkey epididymides as detected by Northern blot analysis and in situ hybridization.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Preparation

Sexually mature cynomolgus monkeys (Macaca fascicularis) were vasectomized unilaterally by cutting the vas deferens in the scrotal portion followed by ligation of both ends with surgical silk. The monkeys used in this study were then castrated 3, 7, and 14 wk postvasectomy. The controls and vasectomized epididymides for each time after vasectomy were immediately excised, defatted, and dissected into six segments corresponding to the proximal and distal parts of the caput, corpus, and cauda epididymidis. Half of the tissues were directly frozen in liquid nitrogen and kept at -80°C for later RNA extraction; the other half was fixed in freshly prepared 4% (w/v) paraformaldehyde in PBS, embedded in cutting medium, and stored at -80°C until use for histologic examination and in situ hybridization.

Histologic Examination

Cryosections (8 µm) of the epididymal segments prepared to perform in situ hybridization were stained with hematoxylin-phloxin-safran [29] for light microscopic observation. Histomorphometric measurements were performed using the Bioquant NOVA (R&M Biometrics, Nashville, TN), a semiautomated image analysis system, and a Summa Sketch III professional (Summagraphics, Anaheim, CA) digitalizing tablet coupled to a Leitz Aristoplan microscope (Leica, Montreal, QC, Canada) and Dage MTI CCD-300-RC1/2; Black and White cooled camera. System calibration and validation were done periodically with a calibration scale bar, 20-µm spacing (Leica), and area and perimeter reference preparations (coefficient of variation [%] = 1.35).

The results are presented as the mean ± SEM of 20 and 10 different measurements for the epithelial thickness and the lumen area, respectively. At least 10 different histologic sections from all epididymal segments of each animal were evaluated. Statistical analysis was performed by ANOVA using super ANOVA software (Abacus Concepts, Berkeley, CA). Results were compared by Scheffe test. Differences were deemed significant at P < 0.01.

RNA Extraction and Northern Blot Analysis

Messenger RNAs were extracted from epididymides of each animal and analyzed separately. Northern blots were performed at least three times on each mRNA sample. Tissues from different epididymal segments kept at -80°C were homogenized with a Poltroon (InterSciences, Markham, ON, Canada) in a homogenization buffer (4 M guanidium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M 2-ß-mercaptoethanol). Extracts then underwent CsCl fractionation according to the method described by Chirgwin et al. [30]. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. The RNA pellets were resuspended in solution (10 mM Tris-HCl, 5 mM EDTA, 1% SDS, pH 7.4), extracted once with phenol/chloroform 1:1 and twice with chloroform/isoamyl alcohol 24:1. RNAs were precipitated in 0.1 volume of 3 M sodium acetate, pH 5.2, and 2.5 volumes 95% ethanol. The RNA pellets were resuspended in diethylpyrocarbonate (DEPC)-treated water and quantified by spectrophotometry at 260 nm. RNAs were stored at -80°C until used. All solutions were treated with DEPC.

Ten micrograms of total RNA samples isolated from proximal and distal caput, corpus, and cauda epididymidis were denatured in 50% formamide at 65°C for 15 min and electrophoresed on 1% agarose containing 2.2 M formaldehyde [30]. The RNA was then transferred to a nylon membrane (Qiagen, Santa Clarita, CA) with 20x SSC (3 M NaCl, 0.3 M sodium citrate) and UV cross-linked. The P31m probe contained the complete cDNA [23]. As for HE1-l, HE2-l, and HE5-l, the probes used were partial sequences amplified by reverse transcription-polymerase chain reaction using primers deduced from human HE1, HE2, or HE5 sequences [31–33] (Table 1). The cDNA probes were random-prime labeled using the T7 Quick Prime kit (Pharmacia Biotech, Baie D'Urfé, QC, Canada) and purified with the QIAquick PCR Purification kit (Qiagen, Mississauga, ON, Canada).


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  		TABLE 1. Oligonucleotide primers used in PCR assays and length of the obtained fragments. The primers were deduced from human HE1, HE2, and HE5 mRNA sequences [26, 31, 33]. Fw, Forward; Rev, reverse

The RNA was prehydridized at 42°C for 4 h in a prehybridization solution (50% formamide [v/v], 0.75 M NaCl, 0.05 M NaH2PO4, 0.005 M EDTA, 2x Denhardt reagent; 5x SSPE [0.9 M NaCl, 50 mM NaH2PO4, 5 mM EDTA; pH 7.7], 10% dextran sulfate, 1% SDS, 0.02% salmon sperm DNA [ICN Biomedicals, Aurora, OH], 5x Denhardt). The hybridization was performed overnight at 42°C in the same buffer to which 106 cpm/ml probe encoding the P31m, HE1-l, HE2-l, HE5-l, or actin cDNA [{alpha}32P]dCTP was added. The membrane was then washed two or three times for 30 min at 65°C with 0.1x SSC and 0.1% SDS, followed by an exposure on Kodak Bio-Max Ms films (Eastman Kodak, Rochester, NY) with intensifying screens at -80°C. An RNA ladder (1.6–7.4 kilobase; Gibco, Burlington, ON, Canada) was used to determine the length of the mRNA, and an actin probe was used as a constitutive internal control. The mRNAs were quantified by densitometric scanning and expressed as a ratio of actin transcript.

In Situ Hybridization

In situ hybridization was performed on epididymal tissues of each animal using digoxigenin (DIG)-labeled cRNA probes as previously described [34]. Epididymis cryosections were fixed with freshly prepared 4% (w/v) paraformaldehyde in PBS for 5 min at room temperature, incubated for 10 min in 95% ethanol/5% acetic acid at -20°C, and rehydrated by successive baths of decreasing concentrations of ethanol diluted with DEPC-treated water. Target RNA was unmasked by enzymatic digestion with 10 µg/ml proteinase K (Roche Diagnostics, Laval, QC, Canada) in PBS for 15 min at 37°C, followed by a 5-min incubation in 0.2% glycine. Sections were postfixed for 5 min with 4% paraformaldehyde in PBS, acetylated with 0.25% acetic anhydride, 0.1 M triethanolamine, pH 8.0, for 10 min, and finally washed with PBS.

Tissues were prehybridized for 1 h at 42°C with 250 µg/ml salmon sperm DNA (ICN Biomedicals) in a hybridization solution (0.3 M NaCl, 0.01 M Tris-HCl, pH 7.5, 1 mM EDTA, 1x Denhardt solution, 5% dextran sulfate, 0.02% SDS, and 50% formamide). Sections were then incubated overnight at 42°C under coverslips with 25 µl of 5 µg/ml (HE1-l and HE5-l) or 10 µg/ml (P31m and HE2-l) heat-denatured antisense or sense cRNA, probed with DIG according to the supplier's instructions. Sections were washed twice for 5 min in 2x SSC, followed by a RNAase treatment (10 mg/ml), 10 min in 2x SSC, and another 10 min in 2x SSC at room temperature. Sections were then washed twice for 10 min at 42°C in 1x SSC and 0.2x SSC.

Hybridization reactions were detected by immunostaining with alkaline phosphatase-conjugated DIG antibodies (Roche Diagnostics). Nonspecific staining was blocked by 1 h preincubation with 5% (v/v) heat-inactivated sheep serum in Tris-HCl/NaCl buffer (0.2 M Tris-HCl, 0.2 M NaCl, 3% Triton X-100). Sections were then incubated for 2 h at room temperature with the alkaline phosphatase-conjugated anti-DIG antibodies diluted 1:1000 in blocking solution, washed with Tris-HCl/NaCl buffer, and incubated with 0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, and 0.01 M MgCl2. The hybridization signal was revealed with the phosphatase substrate, nitroblue tetrazolium chloride, and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (Gibco BRL, Gaithersburg, MD). Levamisole (2 mM; Sigma) was added to the reaction mixture to inhibit endogenous alkaline phosphatase. Microscope slides were immersed in 1 mM EDTA, 0.01 M Tris-HCl, pH 7.5, washed for 5 min in water, counterstained with neutral red, dehydrated through ethanol baths, cleared in xylene, and mounted with Permount (Fisher Scientific, Nepean, ON, Canada). Epididymis sections were processed in parallel to allow comparison between vasectomized and control epididymal sections of the same monkey.

Images were acquired in color directly from the stained tissue with a Zeiss Axioskop 2 plus microscope linked to a digital camera (Spot Insight) using Image Pro software analysis (Carsen Medical Scientific, Markham, ON, Canada) and digitization on a Pentium III computer. Blue staining intensity in sections of epididymis was quantified as follows using Image Pro-Plus. For each section, measurements were taken with the count/size command. The ‘select color’ button was used to specify the range of intensities/colors that defines the tissue sections in the active image. The ratio of the mean density values of P31m, HE1-l, HE2-l, or HE5-l mRNA was calculated for each section of the monkey epididymis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The histology of monkey epididymis was not affected at 3 and 7 wk postvasectomy (data not shown). As for the tissues from 14 wk postsurgery, only the epithelial height was affected.

The diameter of the lumen increased throughout the epididymis in all vasectomized and normal tissues, to reach a maximum in the distal cauda epididymis (Fig. 1A). The epithelium height varied between epididymal segments, with a maximum reached in the distal caput epididymis (Fig. 1B). The epithelium heights were comparable between ligated and control epididymides except at 14 wk postsurgery. Only the distal caput of the vasectomized epididymis had a significant decrease in epithelium height (Fig. 1B).



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  		FIG. 1. Lumen area (A) and epithelial height (B) of monkey epididymal proximal (A) and distal (B) caput, proximal (C) and distal (D) corpus, and proximal (E) and distal (F) cauda, after 14 wk of vasectomy. Histomorphometric measurements from normal and vasectomized monkey epididymides were performed using semiautomated image analysis (lumen and height measurements: n = 6 and n = 10, respectively). Designations (*) are significantly different at P < 0.01.

Considering that vasectomy affects the epididymal histology only 14 wk postsurgery, P31m, HE1, HE2, and HE5-l mRNA expression was affected only at that time postvasectomy. The results of Northern blot analysis, performed on total RNA from 14-wk vasectomized monkeys, revealed a single transcript for each probe: P31m, HE1-l, HE2-l, and HE5-l cDNAs detected transcripts of approximately 1000, 900, 700, and 600 base pairs, respectively (Fig. 2). The actin cDNA serving as an internal control confirmed the quantity and the quality of the mRNA (Fig. 2). The length of HE2 mRNA detected in Northern blots of caput segments was slightly longer than the one detected in more distal segments of the epididymidis of the same animal (Fig. 2, lanes 1 and 2 compared with lanes 3 and 5). This corresponds to different HE2 isoforms derived from alternative splicing of a single gene encoding for multiple peptides [35, 36]. These peptides have antibacterial activity, acting in the defense system of the excurrent duct [36].



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  		FIG. 2. Northern blot analysis of total RNA from normal (lanes 1–6) and vasectomized (lanes 7–12) monkey epididymides. Sections of monkey proximal (lanes 1, 7) and distal (lanes 2, 8) caput, proximal (lanes 3, 9) and distal (lanes 4, 10) corpus, and proximal (lanes 5, 11) and distal (lanes 6, 12) cauda were probed with a P31m, HE1-l, HE2-l, or HE5-l cDNA. An actin probe was also used as an internal control. The probes used for each essay are indicated on the left.

In general, when the signals obtained for each mRNA were expressed as a ratio of actin mRNA, they appeared at slightly lower intensities in vasectomized compared with normal tissues (Fig. 3). However, in the proximal corpus epididymidis, excurrent duct obstruction particularly decreased the expression of HE2-l mRNA (Fig. 3C).



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  		FIG. 3. Ratio intensity of P31m (A), HE1-l (B), HE2-l (C), and HE5-l (D)/actin mRNA was evaluated by densitometric scanning of the Northern blots shown in Figure 2

In situ hybridization was performed to quantify P31m, HE1-l, HE2-l, and HE5-l mRNA expression along vasectomized and ipsilateral normal epididymides of Cynomolgus monkeys at 14 wk postsurgery. As obtained in the Northern blot experiments, the P31m, HE1-l, and HE5-l mRNAs staining were not different between normal and vasectomized tissues (data not shown). An HE2-l transcript was detected in the proximal corpus epididymidis of normal tissues at a much higher intensity than that observed in the same region of vasectomized tissues (Fig. 4). As illustrated in Figure 4E*, the mRNA was located in the perinuclear region of the epididymal principal cells. No signal was detected when sense strand HE2-l cRNA was used as negative control (Fig. 4F*).



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  		FIG. 4. Typical results of in situ localization of HE2-l mRNA along normal (A, C, E, G, I, and K) and vasectomized (B, D, F, H, J, and L) monkey epididymidis 14 wk after unilateral vasectomy. Cryosections of monkey proximal (A, B) and distal (C, D) caput, proximal (E, F) and distal (G, H) corpus, and proximal (I, J) and distal (K, L) cauda segments of the epididymis were probed with DIG-labeled antisense or sense (negative control; F*) HE2-l cRNA. The mRNA was detected by immunostaining using an anti-DIG antibody coupled to an alkaline phosphatase, which appears as blue staining following incubation with NBT/BCIP (4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate) substrate. Tissues were counterstained with neutral red. Magnification x20 (A–L). E*) Higher magnification showing detail of E

The quantification of the HE2-l staining, using Image-Pro plus, was also in accordance with the Northern blot results, confirming the tremendous drop in the expression of this mRNA in the proximal corpus of the vasectomized epididymides (Fig. 5).



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  		FIG. 5. Relative intensity of HE2-l mRNA from monkey epididymal proximal (A) and distal (B) caput, proximal (C) and distal (D) corpus, and proximal (E) and distal (F) cauda. Quantification of epididymides from normal and vasectomized tissues was performed using a Zeiss Axioskop 2 plus microscope linked to a digital camera (Spot Insight; Carsen Medical Scientific, Markham, ON, Canada) using Image Pro software analysis (Carsen Medical Scientific) and digitization on a Pentium III computer. The represented values correspond to the pictures shown in Figure 4


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Following vasectomy, the testis continues to produce spermatozoa. The obstruction imposed by vasectomy leads to an increase in the intraluminal pressure, especially in the epididymis [37]. Studies on rhesus monkeys have demonstrated that vasectomy affects the reproductive tract; 3 wk after vasectomy, a distinct and firm enlargement of the cauda epididymidis appeared in some rhesus monkeys as a result of the pressure built up within the duct [38]. In this study, the histology of the epididymides of cynomolgus monkeys was not affected at 3, 7, or even 14 wk postvasectomy. The fact that we did not see the firm enlargement of the cauda epididymidis at 3, 7, or 14 wk of vasectomy as reported by Bedford [38] illustrates that not all animals react the same way to obstruction resulting from vasectomy. In fact, under the conditions imposed by vasectomy, the fate of the epididymis depends on the elasticity of the epididymal tubule, the amount of spermatozoa produced, and the resorption capacity of the epididymis. In this study, morphologic evaluation as well as histologic parameters indicates that, 14 wk after vasectomy, no granulomata sufficient to build up intraluminal pressure has yet developed. Postvasectomy histologic alterations of the epididymis have been reported but in studies performed on monkeys vasectomized for a much longer period of time. In fact, a marked increase in the diameter of the intraluminal compartment in all segments of the epididymis has been reported at 6 mo postvasectomy in cynomolgus monkeys [39]. Moreover, in another study, a 2- to 4-fold increase in the diameter of the efferent ducts in rhesus monkeys that remained vasectomized for 1 yr has been reported [40]. Our study was designed to detect as soon as possible the effect of vasectomy on the epididymal histology so that alteration of gene expression could be assessed at this early stage. The rationale was to evaluate epididymal gene expression pattern when the epididymal morphology is minimally affected by granulomata or other physical stress. Factors other than intraluminal pressure thus affect the epididymal gene expression. HE2 mRNA expression being almost completely abolished after 14 wk postvasectomy, the proximal corpus appears to be more sensitive to these factors, which remain to be determined.

P34H is among the few proteins secreted in the lumen of human epididymis that has been shown to interact with the sperm surface and to be involved in sperm maturation [18, 41]. In a study using vasectomized tissues, P34H mRNA expression shifted to the more proximal region of the epididymis [21] compared with the normal pattern of expression in unobstructed excurrent duct [34]. Furthermore, the effect of vasectomy on the histology of the human epididymis resulted in a distended lumen throughout the duct of vasectomized tissues compared with normal. The height of the epithelium was also different, with a maximum being reached in the proximal caput epididymidis of vasectomized tissues compared with normal tissues, where the maximum was reached in the distal caput-corpus epididymidis [21]. Interestingly, in that study, the mRNA expression of P34H was greatest in the segment with the highest epithelium height, and that was true for both normal and vasectomized tissues [21]. P31m shares common antigenic properties with P34H [23]. We also reported that P31m mRNA was mostly expressed in the corpus and cauda epididymidis, which was also verified in this study. However, contrary to human P34H, the expression of P31m was not affected by vasectomy at the time we investigated it (14 wk postvasectomy). We cannot exclude the possibility that a longer time of vasectomy would also change the expression pattern of P31m.

Among the human epididymal secretory proteins that have been cloned by an epididymal cDNA library screening [42], homologues in macaque monkeys showing high degrees of identity have been found for HE1 [32, 43, 44] and HE5 [31, 45]. As for HE1-l and HE5-l mRNA expression, no differences between normal and vasectomized tissues were observed. The expression of these mRNAs does not seem to be affected before 14 wk postvasectomy in cynomolgus monkeys. HE1-l mRNA was expressed in almost every segment of the epididymis, as was human HE1 [46]. This pattern seems to be conserved in other mammals [15, 16]. The pattern of expression of the HE5-l mRNA was also in agreement with reports [15] that, in all species studied, epididymal CD52 is highly expressed in a regionalized pattern, with maximum levels presenting in the distal parts of the epididymis. For both HE1-l and HE5-l messengers, the Northern blot results, expressed as a ratio of actin mRNA, seem to show a slight difference between normal and vasectomized tissues. However, the in situ hybridization results clearly show there is no significant difference between them (data not shown). The latter is probably more reliable for evaluating mRNA expression because the technique is more sensitive [47].

In normal tissues, the maximum expression of HE2-l mRNA occurs in proximal corpus epididymidis, with almost no expression appearing in other parts of the organ. In human, HE2 has been shown to be specific to distal caput epididymidis with no expression in other segments [46]. Although they seem to be only poorly conserved among mammals [15], the expression pattern in cynomolgus monkeys is very similar to that of the human. In situ expression of HE2-l mRNA appeared to be strongly affected by the obstruction. In fact, after 14 wk of vasectomy, HE2-l mRNA expression drastically decreased, showing weak expression along the duct. These results were in accordance with the Northern blot analysis. HE2 is secreted by the epididymis and localized on sperm acrosomal and equatorial segments, suggesting involvement in sperm maturation [33]. Vasectomy could thus affect expression of epididymal gene necessary for the acquisition of sperm fertilizing ability.

These overall results thus suggest that vasectomy provokes a disregulation of gene expression in the epididymis, as supported by the results obtained with HE2-l. This effect could to be time dependent, considering that changes in epididymal histology are detectable after 14 wk of vasectomy but not after 3 or 7 wk. This could have important clinical consequences in cases of vasectomy reversal. Indeed, the level of P34H on human spermatozoa can be abnormally low in certain men after vasovasostomy, thus lowering their fertility [48]. It was also shown that a negative correlation exists between the time period after vasectomy and the reproductive capacity of sperm (as assessed by pregnancy and implantation rates after intracytoplasmic sperm injection) obtained by epididymal aspiration [49].

The varying periods of vasectomy used with these cynomolgus monkeys resulted in alterations in the histology of the reproductive tract only at 14 wk postvasectomy. This period of obstruction is probably not sufficient to cause an increase in intraluminal pressure. The mRNA expression was affected in a differential way. HE2-l mRNA expression dramatically decreased in the proximal corpus of the vasectomized epididymis, contrary to P31m, HE1-l, and HE5-l mRNA, which showed no difference. Understanding the factors affecting mRNA expression along the epididymis of vasectomized monkeys could help in understanding the effects of obstruction on excurrent duct physiological functions and the mechanisms regulating differential gene expression along the epididymis.


   ACKNOWLEDGMENTS
 
We thank Dr. J. Gourdon for providing us with primate reproductive tissues and Nancy Verville for technical assistance.


   FOOTNOTES
 
1 This research was supported by a Canadian Institutes for Health Research grant to R.S. Back

2 Correspondence: Robert Sullivan, Unité d'Ontogénie-Reproduction, Centre de Recherche, Centre Hospitalier de l'Université Laval, 2705 Blvd. Laurier, Ste-Foy, PQ, Canada G1V 4G2. FAX: 418 654 2765; e-mail: robert.sullivan{at}crchul.ulaval.ca Back

Received: 10 June 2002.

First decision: 8 July 2002.

Accepted: 4 September 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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