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Center for Experimental Therapeutics and Department of Pharmacology, and Center for Research on Reproduction and Women’s Health, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: W.-C. Song, Ph.D., University of Pennsylvania School of Medicine, Room 1351, Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail: . song{at}spirit.gcrc.upenn.edu
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Abstract |
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Introduction |
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- and P450 aromatase gene-disrupted mice revealed that estrogen activity is required for maintaining the normal absorptive function of the epithelium of the efferent ducts and for sustaining normal spermatogenesis in the testis (1, 2, 3, 4, 5). On the other hand, estrogens, when given at pharmacologic concentrations to adult male animals, inhibited testicular spermatogenesis and steroidogenesis, and negatively affected epididymal structure and function, resulting in abnormal sperm maturation in the epididymis (6, 7, 8, 9, 10). Thus, although estrogen is biosynthesized in the testis and epididymal sperm, and its action required in both the testis and the epididymis, uncontrolled estrogen activity in the testis and the male reproductive tract can be harmful. Previous work in our laboratory has characterized an estrogen-specific metabolic enzyme, estrogen sulfotransferase (EST), expressed abundantly in Leydig cells (11, 12, 13, 14). Targeted disruption of the EST gene in the mouse led to age-related structural abnormalities in the testis including Leydig cell hyperplasia/hypertrophy and seminiferous tubule damage (14). Development of these structural lesions in the EST gene-disrupted mice could be accelerated by administration of exogenous estradiol, suggesting that they arose in older mutant mice as a result of increased local estrogen activity in the absence of in situ estrogen metabolism by EST (14). Interestingly, in the same study, we also found that epididymal sperm motility in older EST mutant mice was significantly reduced, pointing out the possibility that normal epididymal function may also be impaired in these animals (14). Additionally, the seminal vesicle/coagulating glands in the knockout mice were hypertrophic, displaying markedly increased wet weights (14). These findings indicated that, besides the testicular lesions, structural and functional defects could also develop in other parts of the male reproductive tracts of EST-deficient mice.
Although the observed abnormalities in epididymal sperm motility and seminal vesicle/ coagulating gland structure likely resulted from increased estrogen stimulation, it was not known if this reflected increased estrogen activity in situ or was indicative of a higher concentration of unmetabolized estrogen originating from the testicular fluid. It is also possible that reduced epididymal sperm motility was secondary to the testicular lesions rather than a sign of impaired epididymal function. The purpose of this study was therefore to investigate if EST is expressed in the epididymis and other parts of the male reproductive tract and if so, how it is regulated in these tissues and whether it plays a role in maintaining the functional integrity of the epididymis by regulating luminal estrogen activity locally.
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Materials and Methods |
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and 4). In sperm motility assays, 12-month-old wild-type and EST knockout mice with a mixed 129J/C57BL/6 background were used. All other experiments used mice with a C57BL/6 background. Immediately after the mice were killed, epididymis and the attached efferent duct, vas deferens, prostate, and seminal vesicle/coagulating gland were removed from associated fat and other connective tissue. In some experiments, the epididymis was subdivided into caput (including efferent duct), corpus, and cauda, and each tissue segment was processed separately. Tissues were either processed for histology as described below or frozen in dry ice and stored at -80 C for total RNA or protein extraction. Use of mice in this study was approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.
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Steroid measurements
Blood was obtained from the aortic artery. Serum testosterone and estradiol levels were measured with RIA kits by following the manufacture’s instructions (Diagnostics Inc., Webster, TX). In one experiment, levels of estradiol in the epididymal segments were also determined after organic solvent extraction. For this experiment, the epididymis was separated into a caput segment and a corpus and cauda segment. After dissection, the tissues were quickly homogenized on ice in 1 ml PBS and extracted with 2 ml dichloromethane. After phase separation, the dichloromethane phase was collected and evaporated to a minimum volume to which 1 ml buffer A of the estradiol RIA kit (optimized diluent for serum samples, Diagnostics Inc.) was added. The sample was further dried under a stream of nitrogen to completely remove the residual dichloromethane solvent before RIA assay.
Treatment of mice with human chorionic gonadotropin (hCG), testosterone, and hydroxyflutamide
To examine the effect of LH on EST expression in the male reproductive tract, hypophysectomized male mice were injected with recombinant hCG in PBS or PBS alone (5 IU hCG/d/animal in 200 µl PBS, sc, hCG from Sigma, St. Louis, MO) for 4 d. In another experiment, hypophysectomized mice were treated for 7 d with hCG but starting from d 5 these mice also received concurrent treatment with either an androgen receptor antagonist, hydroxyflutamide, in seed oil or seed oil alone (3 mg hydroxyflutamide/d/animal, ip; hydroxyflutamide was kindly provided by Dr. Ralph Nery, Schering-Plough, Kenilworth, NJ). Starting from d 7 post operation, some castrated mice were treated for 4 d with daily injections of 100 µl testosterone propionate (Sigma) dissolved in seed oil (10 mg/ml). In all experiments described above, caput, corpus, and cauda epididymides and vas deferens were collected at the end of the treatment regimen for total RNA and protein extraction.
Northern and Western blot analysis
Total RNA samples (20 µg per lane) were separated on a 1% formaldehyde-agarose gel and capillary transferred onto a nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Arlington Heights, IL). The membrane was cross-linked under UV and hybridized with a P32-labeled cDNA probe synthesized with random primers from the mouse EST cDNA (11). Hybridization was carried out in QuickHyb solution (Stratagene, La Jolla, CA) at 68 C for 1 h. The membrane was washed first in 2x SSC/0.1% sodium dodecyl sulfate (SDS) at 65 C for 15 min, then in 0.1x SSC/0.1% SDS at 60 C for 10 min twice and exposed to x-ray film. For Western blot analysis, tissues were homogenized at 4 C in 50 mM Tris buffer containing 150 mM NaCl; 1% Nonidet P-40; 0.1% SDS; 0.5% deoxycholic acid; 1 mM EDTA, pH 8.0 in the presence of a protease inhibitor cocktail (Sigma, St. Louis, MO). The homogenate was centrifuged at 15,000 x g for 2 min and the resulting supernatant was used for Western blot analysis (50 µg/lane). Concentrations of the protein samples were determined by the Bradford method using a protein assay kit from Pierce Chemical Co. (Rockford, IL). Samples were electrophoresed on 10% SDS polyacrylamide gels, blotted onto nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA; 0.45 µm) and probed with a rabbit polyclonal antimouse EST antibody, followed by reaction with HRP-conjugated secondary antibody as described (12). Immunodetections were performed with the ECL Western blotting detection system from Amersham. In some experiments, the membranes were stripped and reprobed with an anti-ß-actin antibody (Sigma) to confirm proper sample loading and transfer.
EST activity assays
Sulfotransferase activity was measured with 3H-labeled estradiol ([2,4,6,7-3H(N)]-estradiol, 87.6 Ci/mmol, NEN Life Science Products (Boston, MA), final concentration 1.2 nM) in 200 µl of PBS, pH 7.50, containing 1.25% Triton X-100, 100 mM 3'-phosphoadenosine 5'-phosphosulfate and 135- to 300-µg proteins prepared from tissue homogenate (15,000 x g) in the same buffer. The reaction was initiated by the addition of substrate and continued for 30 min at 37 C. The reaction mixture was extracted with 2 vol of dichloromethane and aliquot of the aqueous phase was counted (11). Enzyme activity was expressed as cpm estrogen sulfate formed per mg of total protein.
Immunohistochemical studies
Paraffin-embedded tissue sections were stained for EST expression using a rabbit antimouse EST polyclonal antibody and the Vectastain ABC Elite kit from Vector Laboratories (Burlingame, CA). Briefly, tissues were fixed in Bouin’s solution overnight at 4 C, dehydrated and paraffin embedded, and cut at 5 µm. Paraffin-sectioned slides were deparaffinized in xylene, passed through graded ethanol solutions, washed in distilled water and 0.05% Tween in PBS, and then treated with 3% H2O2 in methanol for 15 min. After rinsing three times with PBS, 10% normal goat serum was added to the slides to block nonspecific binding and the slides were incubated for 30 min at 37 C. Total IgG partially purified from a rabbit antimouse EST serum or PBS buffer were added, incubated for 30 min at 25 C and rinsed with PBS. EST antigen was localized using reagents provided in the ABC kit by following the manufacturer’s instruction.
Sperm motility assays
Forward progressive sperm motility was assessed in unmanipulated 12-month-old EST knockout and age-matched wild-type mice using a Hamilton Thorn Sperm Analyzer (Hamilton Thorn Research, Beverly, MA). Total sperm motility was also determined by a manual assay (described below) in 3-month-old wild-type and knockout mice. These mice had one side of their testes ligated and were either untreated or received subcutaneous implantation of a 17ß-estradiol pellet (10 µg, 21-d release pellet, Innovative Research America, Sarasota, FL) immediately after the epididymal ligation surgery. They were killed on d 4 post ligation. Sperm were collected from caudal epididymis in Hank’s medium with 2 mg/ml BSA (pH 7.4). After 15 min diffusion, sperm motility was analyzed either automatically with a sperm analyzer or by the manual assay. For the manual assay, sperm motility was assessed visually on a computer screen connected with a phase contrast microscope. For each sample, at least 10 viewing fields, each containing 20–40 sperm, were counted. Total sperm motility was defined as clear head and flagella movement (including forward progressive motility and circular motion). The total number of sperm and the number that showed any motility in a given viewing field were determined to derive % motility. All procedures were performed at 37 C and all equipment that was in contact with sperm were prewarmed to and maintained at 37 C.
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Results |
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shows that EST is expressed in the corpus and cauda but not the caput regions of the mouse epididymis. It is also expressed in the vas deferens, but no signals were easily detected in the prostate or the seminal vesicle/coagulating gland of the mouse. No strain difference (normal CD-1 or C57Bl/6) was observed in this segmental expression of EST along the male reproductive tract (data not shown). There were also no detectable signals in the epididymis and vas deferens of EST gene-disrupted mice (data not shown), indicating that the same EST gene is expressed in the mouse Leydig cells (12, 13, 14) and the epididymis and vas deferens. Enzyme activity assays were largely consistent with the above results and showed that the specific activity was higher in the epididymis (corpus and cauda) and the vas deferens homogenates than in the testis homogenate (Fig. 1C). Interestingly, some EST enzyme activity were detectable in the prostate and the seminal vesicle/coagulating gland (Fig. 1C), suggesting that trace amounts of the enzyme may be expressed in these tissues even though it could not be easily detected by the methods of Northern and Western blot analysis. Immunohistochemical studies revealed that within the cauda epididymis EST is primarily localized to the epithelium, whereas in the vas deferens positive staining was detected both in the epithelial layer and in smooth muscle cells (Fig. 2).
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shows that EST was not expressed in the epididymis and vas deferens of hypophysectomized mice, but its expression in these tissues could be restored by hCG supplement. In a separate experiment, hypophysectomized mice were treated for 7 d with hCG but, starting from d 5 of hCG treatment, they also received an androgen receptor antagonist, hydroxyflutamide, to block the activity of androgen in vivo. Figure 3B shows that hCG-induced expression of EST in the epididymis and vas deferens of hypophysectomized mice could be effectively blocked by hydroxyflutamide. These results suggested that the effect of hCG on EST expression in these tissues was indirect and involved the action of androgen and its receptor.
To provide more evidence that EST expression in the epididymis and vas deferens is directly regulated by testicular androgen(s), we examined EST expression in tissues collected from mice that had been castrated to eliminate the source of testicular androgen. Figure 4A shows that EST expression in the corpus, cauda and vas deferens of sham-operated mice was unaffected when examined at d 7 after the surgery. In contrast, EST expression in the epididymis and vas deferens of castrated mice was significantly reduced at d 4 and completely absent at d 7 post castration (Fig. 4A). However, when the castrated mice received testosterone replacement for 4 d (starting from d 7 post castration), EST expression could be restored in the epididymis and the vas deferens (Fig. 4A). Thus, testicular androgen(s) play an essential and direct role in maintaining EST expression in the male reproductive tract.
Lack of a significant role of other factors in the testicular fluid in controlling EST expression in the epididymis and the vas deferens
It is known that normal epididymal function is maintained by androgen as well as by other factors present in the testicular fluid (16, 17, 18, 19). The castration and testosterone replacement experiments indicated a critical role for androgen in maintaining EST expression in the epididymis and the vas deferens, and implied that other factors in the testicular fluid are not absolutely required. To further evaluate the role of factors other than androgen in the testicular fluid, we surgically ligated the epididymis of the right side while leaving the left epididymis intact. This should have prevented the flow of testicular fluid to the ligated epididymis while still allowing androgens produced in the intact testes to reach the ligated epididymis through systemic circulation. Figure 4C shows that EST expression in the ligated epididymis and the corresponding vas deferens was not significantly reduced when compared with that of the unligated epididymis and the associated vas deferens, either at d 4 or d 7 post ligation. Thus, factors other than androgen in the testicular fluid play a minimal role, if any, in maintaining EST expression in the male reproductive tract.
EST-deficient mouse epididymis is more susceptible to estrogen-induced inhibition in function
In our previous studies, we showed that caudal sperm motility in 18- to 22-month-old EST knockout mice was significantly reduced compared with that in age-matched wild-type controls (14). However, this defect in sperm motility was not apparent in younger (3 month old) knockout mice (14). In the current study, we examined a group of mice that were 12 months old to shed more light on the time course concerning the development of this defect. We found that the forward progressive motility of caudal sperm in 12-month-old EST knockout mice was also significantly reduced as compared with age-matched wild-type controls (26.67 ± 3.99% for wild-type, 6.17 ± 1.57% for knockout, n = 6 for both groups). The reduction in sperm motility in older EST knockout mice was likely a result of increased local estrogen activity in the epididymis (see below) because, as in younger mice (14), there was no significant difference between 18- to 22-month-old wild-type and EST knockout male mice in their serum testosterone or estradiol levels [T: 0.58 ± 0.25 ng/ml (range, 0.33–1.0) for wild-type, 0.48 ± 0.51 ng/ml (range, 0.18–1.51) for knockout, mean ± SD, n = 6; E2: 12.22 ± 9.29 pg/ml (range, 3.82–28.74) for wild-type, 8.35 ± 3.49 pg/ml (range, 2.83–12.32) for knockout, mean ± SD, n = 6].
Because there were Leydig cell and seminiferous tubule abnormalities in aged EST knockout mice (12 months and older), it was not clear if the reduced caudal sperm motility in these mice reflected impaired epididymal function or was secondary to the testicular lesions. The findings described above that EST is discretely expressed and regulated in the middle and distal regions of the epididymis would support the hypothesis that EST also plays a physiological role in regulating local estrogen activity in the epididymis. Thus, lack of epididymal EST expression in the knockout mice would increase local estrogen activity, which over time may impair epididymal function. To test this hypothesis, we employed an epididymal ligation procedure to compare the relative sensitivities of epididymides of wild-type and knockout mice to exogenous estradiol stimulation. We used caudal sperm motility as a functional readout of epididymal function in this experiment. By examining a population of trapped sperm in the ligated epididymis, it would be possible to isolate the effect of estrogen on epididymal function from its well-established inhibitory effect on spermatogenesis, thereby eliminating the potential complication of estrogen-induced sperm damage that may occur in the testis.
As shown in Fig. 5A and consistent with our previous findings (14), there was no significant difference in caudal sperm motility between untreated 3-month-old wild-type and knockout mice, regardless of whether the epididymis had been ligated or not (P = 0.42 and P = 0.23 for unligated and ligated epididymides, respectively, between wild-type and knockout groups). However, as might be expected, epididymal ligation caused a general reduction in sperm motility although the difference between ligated and unligated epididymides reached statistical significance only in the EST knockout group (Fig. 5A, P < 0.05 for knockout mice, P = 0.13 for wild-type mice). Treatment of mice for 4 d with exogenous estradiol (10-µg pellet, 21-d release), significantly reduced caudal sperm motility in unligated EST knockout mouse epididymis but not in that of wild-type mice (P = 0.78 and P < 0.05 for wild-type and knockout mice, respectively, comparing the unligated groups in panels A and B in Fig. 5). In both genotypes, the ligated epididymis was more sensitive to estrogen treatment. Thus, caudal sperm motility in the ligated epididymis was significantly reduced after estrogen treatment in both groups of animals (P = 0.05 and P < 0.0001 for wild-type and knockout mice, respectively, comparing the ligated groups in panels A and B in Fig. 5). However, it is quite evident that the ligated epididymides of EST knockout mice were much more sensitive to the estrogen challenge than that of similarly treated wild-type epididymides. Thus, on average 5.9% of sperm isolated from the ligated and estrogen treated epididymis of knockout mice were motile, whereas 34.1% of sperm from similarly treated wild-type epididymis were motile (Fig. 5B).
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shows that, as expected, estradiol treatment significantly increased the serum and epididymal levels of estradiol of wild-type and knockout mice. There was no significant difference in caput estradiol levels between estrogen-treated wild-type and knockout mice, either with or without ligation (P = 0.48 and P = 0.69, respectively). However, there was a significant difference between wild-type and knockout mice (P = 0.01) in the estradiol levels of ligated corpus and caudal segments. The difference in estradiol levels of the unligated corpus and cauda between the two groups of mice also approached, although did not reach, statistical significance (P = 0.069).
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Discussion |
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We found in this study that EST is discretely expressed and regulated in the male reproductive tract of the mouse. It is present in the epithelium of the corpus and cauda but not the caput epididymis. It is also expressed abundantly in the vas deferens but only in trace amounts in the prostate or the seminal vesicle/coagulating gland (detectable only by activity assays). We also determined that expression of EST in the epididymis and vas deferens is LH and androgen dependent, similar to the regulatory mechanism previously established for Leydig cell EST (12, 13). It is of interest to note that, unlike many other genes studied in the epididymis (16, 17, 18, 19), EST expression in the corpus and cauda did not appear to be significantly influenced by other factors in the testicular fluid besides androgen.
The segmental localization of EST in the epididymis, i.e. being present in the corpus and cauda but not in the caput regions suggests a specific physiological role of this enzyme in regulating local estrogen homeostasis, and thereby in maintaining the proper luminal environment for sperm maturation. Testicular sperm, which are immature, acquire their motility and ability to fertilize the egg through their journey of the epididymis (22, 23, 24, 25). It is well known that the normal function of the epididymis is critically dependent on testicular androgens as well as other factors including estrogen. Several recent studies have addressed the role of estrogen in the efferent ductules and the proximal segment of the epididymis. For example, studies with estrogen receptor knockout mice revealed that estrogen activity is necessary for maintaining the normal absorptive function of the efferent ductules and for the proper morphological differentiation of the caput epididymis (4, 26, 27, 28). However, whether a similar degree of estrogen activity is also required in the corpus and cauda regions of the epididymis is not known. In fact, there is circumstantial evidence to suggest that decreased estrogen activity may be desirable in the distal regions of the epididymis. First, RT-PCR, immunohistochemistry and in situ hybridization experiments demonstrated that estrogen receptor(s) was more easily detected in the efferent duct and the caput region of the epididymis (1, 29, 30, 31, 32). Second, P450 aromatase, which synthesizes estrogen from testosterone, has been localized to the cytoplasm deposit of the sperm tail, and its staining was most prominent in sperm localized in the proximal caput epididymis, decreasing as sperm traveled to the corpus and completely lost in caudal sperm (2, 33, 34, 35). It has been suggested that germ cell-derived estrogen, not circulating estrogen, may serve as the major source of estrogen in the male reproductive tract (36). Thus, the prominent expression of EST as an estrogen inactivation enzyme in the corpus and cauda but not the caput regions of the epididymis may represent part of an elaborate mechanism to keep estrogen activity suppressed in the mid- and distal regions of the epididymis.
Indeed, we have found in our previous studies that caudal sperm motility in 18- to 22-month-old EST knockout mice was greatly reduced compared with that of age-matched wild-type controls (14). We showed here that this deterioration in sperm quality actually occurred much earlier, i.e. on or before the mutant mice had reached the age of 12 months. The deterioration in caudal sperm quality in older knockout mice likely resulted from increased local estrogen activity in the epididymis because serum estradiol and testosterone levels were not significantly elevated in these mice compared with age-matched wild-type controls. To directly address the role of EST in the epididymis, we used an epididymal ligation model to evaluate the effect of exogenous estradiol on epididymal function in wild-type and EST knockout mice. Our result clearly demonstrated a role for epididymal EST in reducing local estrogen level and activity, and thereby in preventing estrogen-induced impairment of epididymal function. Thus, although this acute estrogen challenge regiment had minimal impact on sperm motility in unligated epididymis, it reduced sperm motility in ligated epididymis but a much more profound effect was observed in the EST knockout mice than in the wild-type mice (Fig. 5). The general enhanced sensitivity to estrogen of sperm in the ligated epididymis was likely a refection of the need for factors in the testicular fluid in maintaining optimal epididymal function. It may be expected that chronically increased estrogen activity in the knockout mouse epididymis will eventually lead to progressive impairment in its function, accounting for the observed reduction in caudal sperm motility in older mutant mice (14).
The finding that EST is only minimally expressed in the seminal vesicle/coagulating gland implied that the abnormal morphological changes we previously observed in this organ of older EST knockout mice (14) may have primarily reflected increased estrogen levels in the reproductive tract caused by EST inactivation in Leydig cells and epididymis. It is worth noting that a similar phenotype, i.e. increased seminal vesicle/coagulating gland weight in older mice, was previously described for estrogen receptor knockout mice (1). However, the mechanisms that lead to this phenotype in the two settings are likely to be different. The increased seminal vesicle/coagulating gland weight in the estrogen receptor knockout mice is thought to be caused by elevated testosterone levels that occurred as a consequence of the lack of estrogen receptor-mediated negative feedback in steroidogenesis (1, 37). In the EST knockout mice, the phenotype was likely caused by an increased level of estradiol that produced hyperplasia and metaplasia of the seminal vesicle/coagulating gland. The effect of prenatal or neonatal estrogen exposure on seminal vesicle growth and gene expression is well documented in the literature (38, 39, 40). Estrogen has also been shown to increase (41, 42) or decrease (43) the weights of seminal vesicles in treated adult animals. However, in the latter case, the suppressive effect of estrogen was likely due to its inhibition of systemic testosterone levels (43). As described in this study and in our previous report (14), the systemic testosterone levels in young or old EST knockout mice were not significantly decreased.
In summary, we have extended our previous findings by showing that EST is discretely expressed and regulated in the male reproductive tract of the mouse. We also demonstrated an in vivo function for this enzyme in maintaining the functional integrity of the epididymis through inhibition of local estrogen activity. These findings help to explain our previously observed structural and functional lesions in the male reproductive tract of old EST knockout mice, and lend further support to the conclusion that EST plays a physiologic role in regulating local estrogen homeostasis in the testis and in the male reproductive tract.
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Acknowledgments |
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Footnotes |
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Received February 19, 2002.
Accepted for publication April 12, 2002.
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References |
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has a functional role in the mouse rete testis and efferent ductules. Biol Reprod 63:1873–1880 knockout mice. J Androl 21:107–121[Abstract]
and ß) expression in the excurrent ducts of the adult male rat reproductive tract. J Androl 18:602–611 and -ß mRNAs in the male reproductive system of the rat as revealed by in situ hybridization. J Mol Endocrinol 26:165–174[Abstract]
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