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Regular Article |
a Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322
ABSTRACT
In primates, expression of the EP2 gene is androgen-dependent and epididymis-specific. EP2 mRNA expression was investigated in caput, corpus, and cauda regions of rat epididymis and in 15 other rat tissues. Polymerase chain reaction and Northern analyses showed that rat EP2 is expressed predominantly in the proximal caput epididymidis. EP2 mRNA expression was determined in proximal epididymides from castrated, sham-operated, and efferent duct-ligated rats. In castrated rats, EP2 mRNA decreased to <10% of that in sham-operated rats between Days 3 and 4 postcastration, demonstrating the androgen dependence of EP2 expression. In epididymides ligated unilaterally at the efferent ducts, EP2 mRNA levels were approximately equal to those in the unligated contralateral epididymides or in sham-operated rats, indicating that EP2 expression does not depend on testicular factors. In bilaterally castrated rats, immediate and delayed testosterone replacement showed the dependence of EP2 expression on circulating androgens. Injection of testosterone propionate (TP) on Days 0, 1, 2, and 3 postcastration maintained EP2 mRNA levels approximately equal to those in sham-operated rats. Starting at Day 4 postcastration, daily injection of TP for 7 days restored EP2 mRNA to approximately normal levels. These data indicate for the rat that EP2 is expressed specifically in the proximal caput epididymidis and that its expression depends on circulating androgens but not on testicular factors.
epididymis, male reproductive tract, sperm, sperm maturation, testosterone
INTRODUCTION
Through its secretory and endocytotic activities, the mammalian epididymis provides the environment necessary for sperm maturation, protection, and storage [1–3]. Proteins secreted by epididymal epithelial cells interact with sperm [4–6] and may contribute to their acquisition of fertilizing capacity [7–10].
Studies of transcription, translation, and protein secretion demonstrate that normal epididymal function depends on both circulating androgens and testicular factors [11–14]. These factors include a variety of molecules found in the seminiferous tubular fluid, such as luminal androgens [15], androgen-binding protein or sex hormone-binding globulins [12, 16], estrogen [17], retinoids [18], growth factors [19], and other organic molecules. Nonsteroidal testicular factors have been shown to stimulate protein synthesis and secretion by caput epididymal cells [3, 20, 21]. Testicular factors have been shown to participate in the maintenance of epididymal 5
-reductase enzyme activity [22, 23] and to affect the expression of proenkephalin [24], cystatin-related epididymal specific protein (CRES) [25], 
-glutamyl transpeptidase mRNA IV [26], A-raf [27], glutathione peroxidase (GPX5) [28, 29], polyoma virus enhancer 3 (PEA3) [30], a disintegrin and metalloprotease 7 (ADAM 7) [31], and low-density lipoprotein receptor-related protein-2 and clusterin [32].
In the primate, HE2/EP2 has been characterized as a family of epididymis-specific androgen-dependent secretory gene products [33–36]. The EP2 gene codes for at least five different message variants in the chimpanzee [36] and for at least six different message variants in the human [35]. To date, nine different message variants (EP2A–EP2I) that code for eight different secretory proteins have been reported in the primate [35, 36]. However, thus far, only one message variant, corresponding to variant EP2E, has been found in rodents [37].
Thus far, EP2/HE2 has been studied only in the primate. However, because primates are not readily available for experimental use, we explored the potential use of the rodent as a model in which to study the physiological functions of EP2/HE2. To do this, it is necessary first to determine whether the primate EP2 gene and the rodent EP2 gene are homologous. Therefore, we investigated whether rat EP2 expression has the same epididymal tissue specificity and the same androgen dependence as primate EP2. In addition, we used the rat to investigate the potential effect of testicular factors on the expression of EP2.
MATERIALS AND METHODS
Animals
Adult Sprague-Dawley rats, 300–500 g in weight, were obtained from Charles River Laboratories (Wilmington, MA). Animals were housed in a 20–25°C controlled environment at a 10L:14D cycle. They were fed normal rat chow, supplied with water ad libitum and allowed to acclimatize for at least 1 wk. All experimental procedures were conducted according to the guidelines stated in the United States Public Health Service's Guide for the Care and Use of Laboratory Animals and as approved by Emory University's Institutional Animal Care and Use Committee.
Tissue Samples
Immediately following killing, epididymides and samples of thymus, heart, liver, lung, kidney, pancreas, large and small intestine, skeletal muscle, spleen, testis, prostate, and seminal vesicle were obtained from male rats and samples of uterus and ovaries were obtained from female rats. Epididymides were also subdivided into six segments: 1) initial segment, 2) proximal caput, 3) distal caput, 4) corpus, 5) proximal cauda, and 6) distal cauda epididymidis [38]. All tissues were frozen immediately in liquid nitrogen and stored at -80°C prior to use.
Surgical Procedures
On Day 0, male rats were anesthetized by i.m. injection of ketamine-xylazine mixture at a dose of 90 mg/kg (ketamine) and 10 mg/kg (xylazine). The scrotal surface was shaved and prepared for aseptic surgery. Scrotal contents were exposed by scrotal incision [39]. All ligations and sutures were done using silicone-treated nonabsorbable braided #4-0 silk (Sherwood Medical, St. Louis, MO). All surgical procedures, injections, and killing were performed between 1200 and 1500 h.
Castration
After exposure of scrotal contents, the testicular vascular supply was ligated without compromising epididymal blood supply. The testis was dissected from the epididymis and associated fat pad and excised. Epididymis and fat pad were returned, caput first, into the tunica vaginalis, and the incision was sutured. For sham operation (control), testis and epididymis were manipulated and returned into the tunica vaginalis and the incision was sutured. Experimental rats were killed on Days 1, 2, 3, 4, 5, 10, and 15 postcastration, and control rats were killed on Day 15 post-sham operation. The epididymis was removed and trimmed from associated fat and connective tissue. The initial segment plus caput epididymidis were frozen in liquid nitrogen and stored at -80°C prior to use for total RNA extraction.
Unilateral Efferent Duct Ligation
Efferent ductules of the left testis were ligated at their junction with extratesticular rete testes without compromising testicular or epididymal blood supply [40]. As a control, the right testis was manipulated similar to the left one, but its efferent ductules were not ligated. Each testis was returned into its tunica vaginalis and the incisions were sutured. The rats were killed on Days 1, 2, 5, 10, and 15 postligation. Ligated and control epididymides were removed and the initial segment plus caput epididymidis was stored as above.
Testosterone Replacement
All testosterone (T) replacement was done by s.c. injection of 1 mg of testosterone propionate (TP) (Sigma Chemical Co., St. Louis, MO) dissolved at 10 mg/ml in sterile sesame-seed oil as vehicle [39, 40].
Four rats were castrated on Day 0 and used in an immediate T replacement (T maintenance) regimen. Two experimental rats received 100 µl TP in vehicle on Days 0, 1, 2, and 3 postcastration. Two control rats received 100 µl vehicle on Days 0, 1, 2, and 3 postcastration. The rats were killed on Day 4 postcastration. The initial segment plus caput epididymidis were collected and stored as above.
Twelve rats were castrated on Day 0 and used in a delayed T replacement (T repletion) regimen that began on Day 4 postcastration. Two rats received TP on Day 4 and were killed on Day 5. Two rats received TP on Days 4 and 5 and were killed on Day 6. Two rats received TP on Days 4, 5, and 6 and were killed on Day 7. Two rats received TP on Days 4, 5, 6, 7, and 8 and were killed on Day 9. Two rats received TP on Days 4, 5, 6, 7, 8, 9, and 10 and were killed on Day 11 postcastration. As control, two rats received vehicle on Days 4, 5, 6, 7, 8, 9, and 10 and were killed on Day 11 postcastration. Initial segment plus caput epididymidis was collected and stored as above.
Plasma T Determination
Blood was collected from each rat via the tail vein before castration or sham operation and before each TP or vehicle injection and by heart puncture after killing. Plasma T levels were determined by RIA at the Yerkes Regional Primate Research Center Assay Services Laboratory (Atlanta, GA) using kits obtained from Diagnostic Systems Laboratories (Webster, TX). The kit has a sensitivity of 0.05–25 ng/ml, an intra- and interassay coefficient of variation of <3%, and a cross-reactivity of <6%. Testosterone levels in blood collected before castration or sham operation are reported as Day 0 T levels.
Polymerase Chain Reaction Analysis
Total RNA was isolated by acid phenol extraction [41] from frozen segments of rat epididymis, testis, prostate, seminal vesicle, heart, liver, spleen, lung, kidney, pancreas, small intestine, large intestine, skeletal muscle, ovaries, and uterus. Aliquots of 1 µg total RNA were reverse-transcribed using an oligo-dT primer and the SuperScript Preamplification System (Life Technologies, Gaithersburg, MD). Polymerase chain reaction (PCR) analysis was performed using the forward primer RT2PCR1F (5'-GAG AGC GCC ATA AAA CAT GAA GGT-3') and the reverse primer RT2PCR2R (5'-CAT CTG TTC CAG GGG TCA GAG CAA AT-3'). The sequence of the forward and reverse primers are based on the DNA sequence of the Bin1b clone [37]. The PCR protocol used Taq polymerase (Sigma, St. Louis, MO; Qiagen, Valencia, CA) and comprised 30 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C. The PCR amplification products were resolved on 1.5% agarose gels. They were subcloned into pGEM-T Easy (Promega, Madison, WI) and were sequenced at Emory University's DNA sequencing core facility (Atlanta, GA).
Northern Analysis
Total RNA was analyzed using the NorthernMax system kit (Ambion, Austin, TX). RNA (10 µg/lane) was separated electrophoretically on 1.5% glyoxal-containing agarose gels and capillary blotted onto BrightStar Plus charged nylon membranes (Ambion). The blots were hybridized using 32P-labeled antisense cRNA probes.
Hybridization probes for rat EP2, glutaraldehyde phosphate dehydrogenase (GAPDH), and epididymal retinoic acid-binding protein (EP-RABP) were synthesized using the Strip-EZ kit (Ambion) and 32P-CTP. The EP2 probe was transcribed using a plasmid containing the 192-base pair (bp) PCR product obtained with the RT2PCR1F and RT2PCR2R primers. The GAPDH probe was transcribed using the p-Tri-GAPDH plasmid provided by the Strip-EZ kit (Ambion). The EP-RABP probe was transcribed using a plasmid containing a 316-bp PCR fragment insert we constructed using the following PCR primers based on the rat EP-RABP/proteinB/ESP I cDNA sequence of Genbank entries X59832 and M12790: forward primer, 5'-CAA ACT CCG GCT GTA CAG TTT CAT GG-3' and reverse primer, 5'-TGA GAT TGC CTT TGC CTC CAA GAT G-3'.
Membranes were preincubated with prehybridization-hybridization solution for 
1.5 h. Hybridization probes were added to the solution and incubated overnight at 65°C. Unbound probe was removed by washing, and the membrane was exposed to a Storage Phosphor Screen (Molecular Dynamics Inc., Hayward, CA). For reprobing, membranes were stripped using the solutions and protocol provided in the Strip-EZ kit (Ambion). Each membrane was probed for EP2, stripped, and reprobed for GAPDH.
The phosphor screens were scanned using a phosphoimager SI (Molecular Dynamics Inc.). Relative expression of EP2 mRNA was determined by densitometric analysis of the bands using ImageQuaNT version 1.1 (1994) software (Molecular Dynamics Inc.). To normalize the EP2 signals, the intensity of each EP2 band was divided by the intensity of the corresponding GAPDH band. The GAPDH-normalized EP2 band intensities from experimental (operated) rats are expressed as a percentage of GAPDH-normalized EP2 band intensities from control (sham-operated) rats. Representative Northern analysis blots from one of the duplicate experimental series are shown in Figures 3–6.
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RESULTS
Epididymal Specificity of Rat EP2
To determine the tissue expression of rat EP2, we analyzed 16 different tissues by reverse transcription (RT)-PCR. Results are shown in Figure 1. A single PCR amplification product of the expected size, 192 bp, was observed with rat epididymis (Fig. 1, lane 13). No PCR products were observed with uterus, ovaries, thymus, heart, liver, lung, kidney, pancreas, large intestine, small intestine, skeletal muscle, spleen, testis, prostate, or seminal vesicle (Fig. 1, lanes 1–12 and 14–16, respectively). The identity of the 192-bp PCR product was confirmed by sequencing.
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To determine the pattern of EP2 expression within the rat epididymis, we analyzed six different epididymal segments by Northern hybridization. The results are shown in Figure 2. A strong message signal corresponding to approximately 0.4 kb was detected predominantly in the proximal caput (Fig. 2A, lane 2). Weak message signals were detected in the initial segment (Fig. 2A, lane 1) and the distal caput (Fig. 2A, lane 3). However, no message signals were observed in the corpus, proximal cauda, or distal cauda (Fig. 2A, lanes 4–6, respectively).
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To compare the distribution of EP2 to that of EP-RABP, an epididymis-specific and androgen-dependent protein [39, 40, 42], we reprobed the hybridization membranes with an EP-RABP probe. Figure 2B shows that EP-RABP is more broadly distributed than is EP2. EP-RABP is expressed almost equally in the initial segment, proximal caput and distal caput epididymidis (Fig. 2B, lanes 1–3, respectively). Like EP2, EP-RABP was not detected in corpus, proximal, or distal cauda (Fig. 2B, lanes 4–6, respectively).
Effect of Castration on EP2 Expression
Castration resulted in a 90% decrease in the level of EP2 message in rat proximal epididymis by Day 4 (Fig. 3A). The EP2 signal decreased to 85% on Day 1, 74% on Day 2, 33% on Day 3, and 5–10% on Days 4–15 postcastration (Fig. 3B). Testosterone levels were 3.5 ng/ml on Day 0 and were undetectable (<0.05 ng/ml) on Days 0, 1, 2, 3, 4, 5, 10, and 15 postcastration.
Effect of Efferent Duct Ligation on EP2 Expression
To test for possible contribution of testicular factors to the regulation of EP2 expression, unilateral efferent duct ligation was used to prevent the flow of testicular fluid into one epididymis. At 1–15 days after ligation, EP2 levels in ligated and unligated epididymides were similar to each other and to those in the sham-operated rats (Fig. 4, A and B). The normalized hybridization signals varied from 105% to 166% for the ligated epididymides and from 79% to 113% for the unligated epididymides after 15 days of ligation (Fig. 4C). Testosterone levels in efferent duct-ligated rats were similar to T levels in Day 0 rats.
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Testosterone Replacement and EP2 Expression
Testosterone-maintained rats were injected with TP on Days 0, 1, 2, and 3 after castration. Northern analysis showed that in these animals EP2 message was maintained at 93% of sham-operated rats (Fig. 5). In epididymides collected from vehicle-injected rats, EP2 message was 8% of sham-operated rats (Fig. 5). In TP-injected rats, plasma T levels were 9, 11, 12, and 6 ng/ml on Days 1–4, respectively. In vehicle-injected rats, T levels were 
0.07 ng/ml on Day 4.
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In delayed testosterone replacement rats, TP injections were begun 4 days after castration when EP2 levels were <10% of control. On Day 3 of TP injection, EP2 levels increased to 53%, on Day 5 to 66%, and on Day 7 to 73% compared to sham-operated rats (Fig. 6). In vehicle-injected rats, EP2 levels were 9% of sham-operated rats (Fig. 6). Plasma T levels in TP-injected rats were 0.08, 14, 19, 19, 18, 20, 18, and 11 ng/ml on Days 4–11 after castration. T levels in vehicle-injected rats were 0.9 ng/ml on Days 4–11 after castration.
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DISCUSSION
We report that, in the rat, EP2 gene expression is epididymis specific and androgen dependent. Among the 16 rat tissues tested by RT-PCR, only the epididymis provided a detectable amplified product.
In the primate, the EP2 gene expresses at least nine different message variants, EP2A–EP2I [35, 36]. These message variants result from alternative use of two promoters and eight exons [36]. In the rat, the EP2 gene appears, thus far, to code for only one message variant, Bin-1b, which is homologous to EP2E [37]. In the mouse, three epididymal cDNA clones are listed in the EST database (Genbank entries AV379374, AV381254, AV381644), each of which is homologous to EP2E. As EP2E appears to be the major message variant in rodent epididymis, it is likely that our Northern hybridization experiments detected mainly the EP2E variant. Moreover, as the PCR primers used were based on rat Bin-1b cDNA sequence, the RT-PCR experiments amplified only EP2E. However, although EP2E message appears to be the major message detected, we cannot exclude the existence of less abundant EP2 variants.
In the rat, EP2 message was detected predominantly in the proximal portion of the caput region of the epididymis. In the human [35, 43] and in the chimpanzee [34], EP2 message was detected predominantly in the caput epididymidis. However, in the rhesus monkey (Macaca mulatta), EP2 message was detected predominantly in the corpus epididymidis [35]. This difference could result from species-specificity in regional expression of the predominant EP2 variant.
In the rat, the segmental distribution of EP2 is narrower than that of another epididymis-specific androgen-dependent protein, EP-RABP [39, 40, 42]. EP-RABP is expressed in the initial segment and throughout the caput epididymidis [39] (Fig. 2). A narrow region of expression is consistent with the hypothesis that the sequence in which sperm come into contact with epididymal proteins might be important for post-testicular maturation [35].
We also report that, in the rat, EP2 gene expression depends critically on the presence of androgens. Following castration, which eliminates both circulating androgens and testicular factors, T levels decreased to <10% of baseline values within 24 h, whereas EP2 message levels did not decrease to <10% of control levels until Day 4 postcastration. Unesterified testosterone is eliminated from the circulation within 2 h of administration [44]. Therefore, the disappearance of EP2 message lags behind that of T with an estimated half-life of 2–3 days.
However, with castration the effect of circulating androgens could not be distinguished from the effect of androgen-dependent and androgen-independent factors that enter the epididymis from the testis. Testicular factors are reported to affect both positively and negatively protein expression by epididymal epithelial cells [11, 14, 32]. Therefore, to determine whether circulating androgens, testicular factors, or both are necessary for EP2 expression in the rat, we ligated the efferent ducts of one testis and determined EP2 expression. Efferent duct ligation, which prevents testicular factors from entering the epididymis, had no effect on EP2 expression in rat epididymis (Fig. 4). Therefore, we conclude that EP2 expression in rat epididymis depends on circulating androgens and not on testicular factors.
This is confirmed by results of T maintenance experiments in which castrated rats were injected daily with TP beginning immediately after castration. In these experiments, EP2 message levels were approximately equal to those of sham-operated rats (Fig. 5). In T maintenance experiments with castrated rhesus monkeys injected immediately after castration with a single dose of 400 mg testosterone enanthate, EP2 message levels were higher than in castrated monkeys not injected with T but lower than in sham-operated monkeys [35]. Although T maintenance in both studies showed similar effects, the differences reported in EP2 message levels may result from differences in injection protocols or from differences in species.
The direct dependence of EP2 message levels on circulating testosterone also is confirmed by T repletion experiments, in which castrated rats were injected daily with TP beginning on Day 4 postcastration. These experiments showed that over 7 days, EP2 message levels gradually returned to about 75% of those measured in sham-operated rats (Fig. 6). That EP2 levels of T-repleted rats were 25% lower than those of sham-operated rats may reflect experimental errors intrinsic to quantitating Northern hybridization signals rather than to an actual difference in EP2 levels. However, it is possible that complete EP2 restoration requires more than 7 days of TP injections.
These studies demonstrate that EP2 mRNA is expressed in the rat epididymis. However, as antibodies to rat EP2E protein are not currently available, we could not demonstrate that rat EP2 message is translated into a protein. However, as EP2 proteins have been demonstrated in the human epididymis using antibodies to human EP2/HE2 [33, 35], it is reasonable to assume that rat EP2 message also is translated.
The physiological role of EP2 is not known. As the EP2 gene in the primate produces multiple mRNA variants that result in multiple protein variants [36], EP2 may have multiple functions in the epididymis. As most EP2 proteins have no recognizable homology with known proteins, their function cannot be inferred from those of any known protein. However, the cysteine distribution pattern of primate EP2C, EP2D, and EP2E and of rat EP2E is homologous to that of ß-defensins [36, 45], a family of secretory antimicrobial peptides. Therefore, these EP2 protein variants could be new members of the ß-defensin family.
In addition, EP2 protein variants may have roles independent of antimicrobial activity. Antibodies to the EP2A protein bind to the equatorial region of human sperm [33], and antibodies to the C-terminal sequence of the EP2D and EP2E proteins bind to the postacrosomal, head, and neck regions of human sperm [35]. Therefore, it was suggested that these proteins may be involved in sperm maturation in the epididymis [35]. Whether EP2 proteins defend the epididymis against microbial infection, participate in post-testicular sperm maturation, or protect sperm during epididymal transit remains to be demonstrated.
In summary, in the rat, EP2 is expressed predominantly in the proximal region of the caput epididymidis and depends on circulating androgens, not on testicular factors, for its expression. These data show that EP2 expression in the rat has the same epididymal tissue specificity and the same androgen dependence as EP2 expression in the primate and that the rat is a suitable model in which to study the physiological functions of EP2.
ACKNOWLEDGMENTS
We thank Dr. Yong-Lian Zhang, State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai, 200031, China for providing the sequence of rat EP2. We also thank Cecilia Po and Altaf Tadkod for technical assistance.
FOOTNOTES
First decision: 1 February 2001.
1 This work was supported by the National Institutes of Health (RR05994). 
2 Correspondence: Otto Froehlich, Department of Physiology, Emory
University School of Medicine, 1648 Pierce Drive, Atlanta, GA 30322.
FAX: 404 727 2648; froehlich{at}physio.emory.edu
Accepted: April 3, 2001.
Received: December 19, 2000.
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