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Regular article |
Has a Functional Role in the Mouse Rete Testis and Efferent Ductules1
a Department of Veterinary Bioscience, University of Illinois, Urbana, Illinois 61802
b Department of Animal Science, University of Illinois, Urbana, Illinois 61801
c Department of Biochemistry and
d Department of Veterinary Biomedical Sciences, University of Missouri, Columbia, Missouri 65211
ABSTRACT
Previous studies of the estrogen receptor-alpha knockout (
ERKO) in the male mouse demonstrate that the rete testis and efferent ductules are targets of estrogen. Because the ERKO mouse lacks a functional estrogen receptor alpha (ER) throughout development, it was not known whether the morphological and physiological abnormalities observed in the ERKO male were due to developmental defects or to dysfunctions concurrent with the lack of ER in the tissue. This study was designed to determine if treatment of normal wild-type (WT) mice with the pure antiestrogen, ICI 182,780, (ICI) could reproduce the morphological characteristics seen in ERKO mice. Thirty-day-old male mice were treated for 35 days with either castor oil or ICI. Age-equivalent ERKO mice were used for comparison. Light microscopic examinations of the reproductive tracts revealed dramatic changes in the efferent ductules of treated mice: a 1.7-fold increase in luminal diameter, a 56% reduction in epithelial cell height, a 60% reduction in brush boarder height of nonciliated cells, and an apparent reduction of the number of observable lysosomes and endocytotic vesicles. Testes of ICI-treated mice showed swollen rete testes area (6.5 times larger than control) and a 65% reduction in rete testis epithelium height. However, there were no significant changes in body and testis weights. These results indicate that ER blockage with ICI in WT mice results in morphological changes of the efferent ductules resembling those seen in ERKO siblings of the same age. Based on this study, we conclude that ER has a functional role in the mouse reproductive tract and the aberrant morphology observed in the efferent ductules of the ERKO mouse is likely the result of a concurrent response to the lack of functional ER, and not solely due to the lack of ER during early developmental times.
epididymis, estradiol, estradiol receptor, male reproductive tract, testes
INTRODUCTION
The estrogen receptor- knockout mouse (ERKO) provides the first unequivocal evidence that estrogens play a role in male fertility and reproductive tract structure and function [1–4]. In this transgenic animal model, early morphological differences, dilation of seminiferous tubules and thinning of seminiferous epithelium, are seen in the testis at 20 days after birth [1]. At 10–12 weeks of age the ERKO rete testis is grossly dilated with fluid and degeneration of the seminiferous tubules is observed [1]. Our previous study has shown that testis weight is significantly increased beginning around Day 30 and continues until about Day 80, when there is a sharp decrease due to atrophy of the seminiferous epithelium [2]. Other regions of the male reproductive tract showed distinct morphological differences compared with the wild-type (WT) mouse. The efferent ductules of the ERKO are dilated and have a thin epithelium that is characterized by shortened microvilli and reductions in the endocytotic apparatus and lysosomal granules [2, 4]. These histopathological changes in the ERKO efferent ductules are consistent with the apparent inhibition of fluid reabsorption from the lumen of these ductules [2].
Results from these studies imply that the disruption of estrogen receptor-alpha (ER) in the male alters both morphology and physiology of the male reproductive tract, which leads to an accumulation of fluid in the lumen and eventual backup within the testis. However, the ERKO mouse lacks a functional ER from the time of fertilization and throughout development. Therefore, we questioned whether the morphological and physiological abnormalities observed in the reproductive tract of ERKO males were due to the propagation of developmental defects, or if they were dysfunctions that were concurrent with the loss of ER, regardless of the age of the animal. To test the hypothesis that the abnormal morphological appearance seen in the testis and efferent ductules of ERKO male mice are not due to the propagation of developmental defects, we proposed treating normal, young adult, WT mice with the pure antiestrogen, ICI.
In this study, young adult WT (ERKO-sibling) male mice were treated with ICI for 35 days to block estrogen receptor (ER) function. WT control and ERKO mice of the same age were used for morphological comparison. The results from this study indicate that many of the aberrant morphological features observed in the reproductive tract of the ERKO male can be reproduced by antiestrogen treatment of normal WT mice. Moreover, the results suggest that ER is directly responsible for normal structure and function of the efferent ductules in adult male mice.
MATERIALS AND METHODS
Animals and Treatment
Thirteen 24- to 25-day-old (11.3–17.9 g body weight) homozygous ERKO-sibling WT male mice (57BL65/129SVJ) were obtained from a resident breeding colony maintained at the University of Illinois College of Veterinary Medicine. The mice were individually housed under controlled conditions and given ad libitum food (TekLad mouse chow, University of Illinois) and water for the entire experimental period. The mice were randomly divided into two experimental groups, a WT control (n = 5) and a treated group (n = 8). At 30 days of age, each WT control mouse was s.c. injected with 0.1 ml of castor oil as vehicle (as recommended by Dr. Wakeling, Zeneca Pharmaceuticals, Macciesfield Cheshire, UK). For each treated mouse, 0.1 ml of ICI 182,780, (a pure ER/ERß antagonist) as "Faslodex" formulation (corresponding to 5 mg ICI, Zeneca) was injected s.c. All injections were performed once a week for 35 days. Four 65-day-old homozygous ERKO mice were obtained from the same colony, and used for comparison of the ICI-treatment effects.
Tissue Preparation
At the end of the experiment, mice were weighed and anesthetized by i.p. injection with 0.1–0.12 ml of sodium pentobarbital (University of Illinois). The reproductive tracts were fixed by vascular perfusion as previously described [5]. Just before the perfusion, one caudal epididymis from a mouse was surgically removed, weighed, and stored in -20°C, and later used for obtaining a sperm count. After fixation, testes were weighed, stored in the fixative for 24 h, and then transferred to 0.1 M cacodylate buffer. Later, they were embedded in glycol methacylate, and sectioned into 2.5 µm thickness, and stained with periodic acid-Schiffs reagent (PAS) followed by counterstaining with hematoxylin. The proximal and distal efferent ductules were excised and processed for light microscopic studies in either glycol methacylate or epoxy resin. To obtain a glycol methacylate section, the tissues were cut into sections (3 µm thickness), and stained with PAS followed by counterstaining with hematoxylin [5]. For epoxy resin sections, tissues were first postfixed with 1% osmium tetroxide for 90 min at 4°C, and followed by 1% osmium tetroxide plus 1.5% ferrocyanide for 30 min at 4°C. After the postfixation, the tissues were embedded in Eponate 12 resin. The tissue blocks were cut (1 µm thickness) and stained with toluidine blue. Tissues were photographed using a Spot-2 (Diagnostic Instruments, Sterling Heights, MI) digital camera and imaged using PhotoShop software (Adobe Systems, San Jose, CA). All chemicals used were purchased from Sigma (St. Louis, MO).
Sperm Count in Caudal Epididymis
All procedures were conducted as previously described [6]. Briefly, the caudal epididymis was homogenized in a solution containing 0.05% Triton X-100 for 1 min. The homogenate was diluted and mixed with saline and 0.4% trypan blue. The number of sperm heads was determined with the use of a hemacytometer. The final values were expressed as the number of sperm per cauda tissue.
Histological Analysis and Statistical Analysis
The digital images were analyzed using NIH Image software (public domain). Rete testis (RT) epithelial cell heights were determined by measurements from the base to the apex of RT epithelial cells. From a representative tissue section, 30 RT epithelial cell heights were measured per mouse. Each RT area was measured five times and averaged. For the proximal efferent ductules, the degree of dilation was determined by measuring the widest distance from one side to the other at the tip of the brush border in the cross-sectional area. The diameters of two to six ductules per mouse, were measured three times and averaged to give a mean and SEM for each mouse.
Epithelial cell height was determined by measuring the distance from the basal membrane to the base of the brush border on nonciliated cells. Final values were determined by measuring and averaging the heights of 20 nonciliated cells per mouse from a representative tissue section. Brush border heights were determined by measurements from the tip to the base of the microvilli of 20 nonciliated cells per animal. The percentage of ciliated cells was determined by counting a total of 100 cells per animal in proximal efferent ductule cross-sections and identifying them as ciliated or nonciliated. Nuclear height was determined by linear measurement of nonciliated cell nuclei from the base to the apex in cells cut approximately in the center of the nucleus. In order to determine cilia number in ciliated cells of the proximal efferent ductules, the number of basal bodies were counted per 20 ciliated cells per animal and expressed as the average number of basal bodies per 5 µm. Data are reported as means ± SEM. Differences between experimental groups for each end point were analyzed using ANOVA, followed by the unpaired Student's t-test. In all cases, results were considered significant if P < 0.05.
RESULTS
Changes of Body Weight, Testis Weight, and Sperm Numbers in Caudal Epididymis
There were no significant differences in total body weight among the experimental groups on the day they were killed (data not shown). Testis weight was not changed in ICI-treated mice compared with WT controls but did show a 30% increase in ERKO mice (Fig. 1a). The concentration of sperm in the caudal epididymis of the ICI-treated mice was reduced, as was the concentration in ERKO mice, compared with WT controls (Fig. 1b). Total sperm number per cauda was reduced by 11% and 22% in the ICI-treated mice and ERKO mice, respectively, compared with WT controls (Fig. 1b). Although the difference between WT controls and ICI-treated mice was not statistically significant, likely because of a high variation in the ICI-treated mice, no statistical difference was found between ICI-treated and ERKO mice.
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Testis and Rete Testis
In order to study the effects of 35 days of the ICI treatment on the testis, PAS-stained testis sections were examined under light microscopy. No apparent histological changes were observed in the treated testes (Fig. 2b), compared with WT control testes (Fig. 2a). Testes in ERKO mice showed the expected disruption of spermatogenesis with dilated seminiferous tubules and rete testis (Fig. 2c). The testes of mice treated for 35 days with ICI exhibited a swollen RT lumen (Fig. 2b) compared with WT controls but there were no observable changes in the seminiferous tubules (Fig. 2b). The degree of RT dilation, however, was much greater in ERKO mice than in the ICI-treated mice (Figs. 2c and 3a). The 35 days of ICI treatment caused approximately a 5.5-fold increase in the RT area. The ERKO mice at the same age had a 48.2-fold larger RT area than WT controls, which was 6.5-fold greater than the ICI-treated RTs. Observed at high magnification, the RT epithelial cell height was reduced by 36% and 47% in ICI-treated mice and ERKO mice, respectively (Fig. 2, e through f; Fig. 3b) compared with WT controls (Figs. 2d and 3b).
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Changes of Luminal Diameter, Epithelial Cell Heights, and Brush Borders in Proximal Efferent Ductules
Treatment with ICI resulted in a 1.7-fold increase in luminal diameter (Figs. 4b and 5a), than in WT controls (Figs. 4a and 5a). The degree of dilation observed in the efferent ductules of treated mice was nearly 40% greater than that observed in the ERKO mice (Figs. 4c and 5a). At high magnification (Fig. 4, d through f), the epithelial height was determined to be reduced by 56% and 76% in treated and ERKO mice, respectively (Fig. 5b).
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The height of the microvillus brush border was reduced by 40% in ICI-treated mice, compared with WT controls (Figs. 4h and 5c). In ERKO mice, microvilli were further reduced in length, being 60% shorter than in WT controls. The brush border of WT control mice was well-organized and homogenous in height along the entire efferent ductule tubule (Fig. 4g). However, in treated mice the brush border was less organized and had a heterogeneous appearance along the efferent ductule (Fig. 4h), which was similar to that observed in ERKO mice (Fig. 4i).
Changes of Cilia Number, Nuclear Shape, and Percentage of Ciliated Cell Numbers in Proximal Efferent Ductules
The number of cilia per ciliated cell was significantly reduced in ICI-treated ductules (47% reduction) and in ERKO tissue (65% reduction), compared with WT controls (Fig. 4, g through i; Fig. 5d); however, no significant difference was found in the percentage of ciliated cells among all three experimental groups (Fig. 5f). The ICI treatment also resulted in a change in the shape of the nucleus from columnar (Fig. 4, d and g) to more flattened (Fig. 4, e and h), as seen in ERKO mice (Fig. 4, f and i). Nuclear height was reduced by 39% in ICI-treated mice and 50% in ERKO mice compared with WT control mice (Fig. 5e). In addition, ICI treatment resulted in a reduction in the abundance of endocytotic vesicles and lysosomal granules in the efferent ductule epithelium (Fig. 4, e and h). A more severe decline in abundance of these organelles in the efferent ductule epithelium was observed in ERKO mice (Fig. 4, f and i).
Changes in Distal Efferent Ductules
To determine whether the 35-day ICI treatment affected the entire efferent ductules, distal efferent ductules were also examined. Similar to the proximal efferent ductules, ICI treatment led to the dilation of the lumen (Fig. 6, a through c), a reduction in epithelial height (Fig. 6, d through f), and a reduction in the height of the microvillus brush border. Also, the number of ciliary basal bodies per cell (data not shown) and nuclear height (Fig. 6, d through f) were significantly reduced in the distal efferent ductules in both treated and ERKO mice. However, as with the proximal efferent ductules, there was no change in the percentage of ciliated cells (data not shown). The abundance of endocytotic vesicles and lysosomal granules was also reduced in treated mice and even more so in ERKO mice (Fig. 6, d through f).
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Glycogen-Containing Cells in ICI-Treated Mice
Occasionally, as in an ERKO mouse [4], epithelial cells filled with cytoplasmic glycogen particles were found in the RT/efferent ductule junction in treated mice (Fig. 7). These cells were never found in WT control mice efferent ductules. The accumulation of glycogen particles was noted primarily in nonciliated cells, but occasionally ciliated cells also contained the granules (Fig. 7). Endocytotic vesicles were observed in glycogen-containing cells (Fig. 7). Light microscopy showed that the glycogen particles were PAS-positive, and the granules showed typical ultrastructural rosettes in electron microscopy (data not shown).
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DISCUSSION
This is the first study to demonstrate an experimental animal model of ER disruption in the adult male without the histological disturbance of spermatogenesis. It is demonstrated that 35 days of treatment with the pure antiestrogen, ICI, is able to produce an ERKO-like effect in adult male mice. In this study, many of the effects previously seen in an ERKO male [1, 2, 4] were reproduced, including the following: a) decrease in cauda sperm concentration, b) luminal dilation in the RT and efferent ductules, c) a reduction in the epithelial height in RT and efferent ductules, d) a decrease in number and height of microvilli, e) a decrease in the endocytotic apparatus and number of lysosomes, f) a decrease in the number of cilia, and g) the appearance of glycogen granules in epithelial cells at the RT/efferent ductule junction.
A decrease in cauda sperm concentration occurred in both ICI-treated and ERKO mice, and was comparable to prior studies in ERKO males [1, 4, 7]. This decrease in concentration in the presence of histologically normal spermatogenesis further supports our earlier work on isolated efferent ductules that suggest they are not reabsorbing luminal fluids when ER is blocked with ICI [2].
Dilation of the RT and efferent ductules after ICI treatment was conspicuous and resembled that seen in ERKO mice [1, 2, 4]. It is within the efferent ductules that the majority of testicular fluid is reabsorbed [8–13]. The removal of fluid results in the crucial concentration of sperm so that maturation can occur in the epididymis. From our earlier study, it was presumed that the abnormalities seen in the ERKO testis were likely due to the back-pressure accumulation of luminal fluids in the dysfunctional efferent ductules [2]. However, because this information was obtained from adult ERKO mice, it remained unclear if the aberrant morphology found in this transgenic animal was due to developmental defects or if there was a functional deficit in the adult efferent ductules. The current study answers this question by showing that antiestrogen treatment over a 35-day period in the male mouse will induce similar dilation of the efferent ductules. Yet, some differences were observed between the ERKO and ICI-treated mice. The degree of efferent ductule luminal dilation was approximately 29% greater in the ICI-treated mice than it was in the ERKO mouse, while the RT was 6.5-fold larger in the ERKO male.
At least two hypotheses may explain this difference in efferent ductule dilation. First, the RT is enlarged in the ERKO mouse prior to puberty [1] and it remains larger at puberty than it does in ICI-treated animals. This early enlargement in ERKO could provide added luminal space to accommodate some of the fluid backup in the testis. Second, we have shown that the ERKO testis secretes less fluid than the WT control testis does [2] but this may not hold true for ICI-treated mice. The treated mice may have a normal (greater than ERKO) level of secretion and develop a greater internal pressure caused by greater fluid accumulation in the efferent ductules, resulting in greater swelling. Future studies will determine the rate of fluid secretion under the ICI-treated conditions. In addition, the disparity in swelling could be due to the shorter period of time the treated animals are lacking ER function, 30 days at most versus 60 days for the ERKO. The fact that the RT in ICI-treated males showed less dilation, but efferent ductules showed more than in ERKO mice supports the hypothesis that in ERKO mice, the RT displays a developmental overgrowth, which would explain its tremendous size in the adult.
Treatment with ICI caused structural changes in the epithelium lining both the RT and efferent ductules. The reduction in epithelial height was less than that seen in ERKO [2, 4] mice, but was significantly shorter than in WT mice. Because luminal dilation was greater in the ICI-treated ductules than it was in ERKO, it is unlikely that fluid accumulation alone causes the epithelium to become squamous in appearance. Instead, the loss of epithelial cell structures (i.e., microvilli, cytoplasmic organelles) is likely associated with the flattening of the epithelium.
In ERKO mice, the microvillus borders are partially or almost entirely lost on the surface of many epithelial cells lining the efferent ductules [2, 4]. This organizational heterogeneity in the brush border was also found after ICI treatment, but was slightly less severe than in ERKO mice. Several studies have demonstrated that microvilli formation is under estrogen regulation in culture [14]. Szego et al. [15] showed that endometrial epithelial cells in the female reproductive tract quickly responded to estrogen treatment by increasing number and height of microvilli. However, it is not known how estrogen is involved in the regulation of the apical surface of epithelial cells.
The complex endocytotic process in efferent ductule epithelial cells has been well documented by Hermo et al. [16]. The endocytotic apparatus is commonly found in nonciliated cells of the normal efferent ductule and is an indication of active fluid reabsorption [16–19]. Endocytosis begins with the formation of short, tubular coated pits between the microvilli, and ends with the lysosome transferring enzymes to the late endosome [20]. Several studies [21, 22] showed that structural changes in microvilli can alter fluid reabsorption. Because the initial steps of endocytosis appear to require the presence of microvilli [23], it is possible that estrogen is responsible for the maintenance of microvillus structure. Thus, in the absence of ER function microvilli are lost and endocytosis is decreased, with a subsequent decreases in the number of lysosomes.
Ciliary basal bodies were significantly reduced when ER function was disrupted by ICI treatment, which is in agreement with our previous observations in the ERKO mouse [4]. Several studies have shown that ciliogenesis in the female reproductive tract is regulated by estrogen [23–25]. The present study would suggest that estrogen is also important for maintenance of cilia in the male reproductive tract.
Another strong indication of the effectiveness of the ICI treatment in reproducing most of the morphological abnormalities seen in the ERKO male was the appearance of glycogen-containing cells at the junction of the RT and efferent ductules. The same abnormality was found in ERKO efferent ductules [4]. This would suggest that the accumulation of glycogen is not a developmental effect, but rather a direct response of the epithelium to a lack of functional ER.
One caveat to the results of the current study is that ICI inhibits the function of both ER and ERß [26]. However, phenotypic changes seen in efferent ductules from mice treated with ICI182–780 treated mice are very similar to those seen the ERKO male. Although both ER and ERß are coexpressed in the efferent ductules [27], ER is the predominant form. In contrast, it has been found that ERß knockout (ßERKO) mice are fertile and the male reproductive tract appears normal, with no resemblance to the ERKO abnormalities [28]. Recently, ER and ERß double knockout mice were produced. The double knockout animals also show the ERKO-like pathology in the male reproductive tract [7 and personal communication]. Several recent studies suggest that ERß is more important in nonreproductive tissues, such as brain [29, 30], cardiovascular system [31], bone [32, 33], and accessory reproductive organs, such as prostate [31, 34–37] and seminal vesicles [31]. The present study also suggests that ER, not ERß, plays the major role in efferent ductule function in males.
The ERKO mouse is deficient in a functional ER throughout all of development. Our previous study implied that certain morphological features in the ERKO male reproductive tract were due to developmental anomalies [4]. These defects were not observed in the ICI-treated males. First, the ERKO mouse had an increased number of blind-ending efferent tubules [4]. ICI treatment of adult WT mice did not have an observed effect on the formation of blind-ending tubules. This observation implies that the formation of blind-ending tubules is a developmental response to the lack of ER. Second, an unusual growth of initial segment epithelial cells was observed in the ERKO efferent ductules [4]. This abnormal phenotype was found in the most proximal and the common zones of the ductules. [4]. In the current study, such abnormal growth did not occur following ICI treatment, indicating that the misplaced growth of initial segment epithelia in ERKO mice was also of developmental origin.
It remains uncertain to what degree, if any, dysfunction of the efferent ductules, due to the lack of ER, is a direct contributing factor to infertility in the ERKO male. However, it is likely to be less important than the eventual degradation of the seminiferous epithelium as several ICI-treated male mice we tested for fertility at 65 days of age all sired litters, whereas ERKO mice were not able to do so (unpublished data). The recent study by Mahato et al. [3] showed conclusively that ERKO sperm are capable of fertilizing an oocyte, if the sperm are developed and transported in a normal reproductive tract. Thus, infertility in the ERKO male prior to degeneration of the seminiferous epithelium could be due in part or in combination to decreased libido, dysfunction of the efferent ductules, unrecognized abnormalities in somatic cells of the testis, and changes in the initial segment and caput regions of the epididymis [4]. Studies have now shown that most of the epididymis can be surgically bypassed and yet fertility in the rodent can be maintained; however, if the initial segment and proximal caput regions are bypassed, there is a reduction in fertility [38].
Our earlier analysis of ICI effects on efferent ductule function under in vitro conditions [2], and this analysis of ERKO and ICI-treated mice, strongly suggests that ER plays a direct role in efferent ductule function with fluid reabsorption being a major casualty of disrupting ER function. Yet the efferent ductules also contain androgen receptors [39, 40], and the contribution of other steroids to the regulation of these ducts remains unclear. In one study, rats were administered daily doses of testosterone propionate, flutamide (antiandrogen), 17ß-estradiol 3-benzoate, or tamoxifen for 7 days [41]. The results of this 7-day treatment appear to contradict the findings for ICI and the ERKO mouse. Testosterone caused a small increase in fluid reabsorption, but estrogen showed a large increase in the rate of fluid flow, suggesting an inhibition of reabsorption [41]. The antiandrogen, flutamide, caused a greater reduction in fluid reabsorption than testosterone. Tamoxifen, used because it is recognized as an antiestrogen, showed the greatest stimulation of fluid reabsorption. In contrast, estradiol treatment decreased reabsorption. Thus, the only other study to have examined whole-animal effects of steroid hormones on fluid reabsorption in efferent ductules appears to raise conflicting results. Clulow and colleagues [42] explained these data by equating estrogen and tamoxifen effects to an antiandrogen action and suggesting that flutamide would cause an increase in systemic androgen concentrations.
One explanation would be the potential effects of treatments on the physiological feedback on the hypothalamus-pituitary-testis axis [43]. Systemic treatment with hormones and antihormones will either decrease or increase testosterone and, thus, there is the possibility that spermatogenesis is altered [44]. Indeed, flutamide and estradiol treatments showed decreases in daily sperm production [41]. In addition, the flow of RT fluid is affected by gonadotropins or hypophysectomy after 24 h [45]. The only study to address the question of hormonal regulation of ER in the male reproductive tract found that androgens were sufficient to maintain ER presence in the efferent ductules [46]. Therefore, any decrease in testosterone resulting from estrogen treatment could down-regulate ER in the efferent ductal epithelium, which would further decrease fluid reabsorption, based upon the ERKO model [4].
In conclusion, ICI treatment in the adult mouse reproduced a majority of the morphological abnormalities observed in RT and efferent ductules of the ERKO male. This study clearly indicates that the aberrant morphology, associated with the inhibition of fluid reabsorption in the male ERKO reproductive tract, is most likely the result of a direct response to the absence of ER during adulthood and not due to the disruption of normal developmental processes. Thus, ER plays an important role in maintaining an appropriate morphology and physiology in the RT and efferent ductules during pubertal and adult ages in the mouse.
ACKNOWLEDGMENTS
The authors are grateful to Drs. A.E. Wakeling and B.M. Vose of Zeneca Pharmaceuticals for their support of this work through the generous gift of Faslodex (ICI 182, 780).
FOOTNOTES
First decision: 11 April 2000.
1 This work was supported in part by NIH grant HD-35126. Part of this work was presented at the XV North American Testis Workshop, Louisville, Kentucky. 
2 Correspondence: David Bunick, University of Illinois, Department of Veterinary Bioscience, 2001 South Lincoln Ave., Urbana, IL 61801. FAX: 217 244 1652; d-bunick{at}uiuc.edu
Accepted: July 20, 2000.
Received: March 14, 2000.
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S. Aquila, D. Sisci, M. Gentile, E. Middea, L. Siciliano, and S. Ando Human Ejaculated Spermatozoa Contain Active P450 Aromatase J. Clin. Endocrinol. Metab., July 1, 2002; 87(7): 3385 - 3390. [Abstract] [Full Text] [PDF]
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C. A. Oliveira, Q. Zhou, K. Carnes, R. Nie, D. E. Kuehl, G. L. Jackson, L. R. Franca, M. Nakai, and R. A. Hess ER Function in the Adult Male Rat: Short- and Long-Term Effects of the Antiestrogen ICI 182,780 on the Testis and Efferent Ductules, without Changes in Testosterone Endocrinology, June 1, 2002; 143(6): 2399 - 2409. [Abstract] [Full Text] [PDF]
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R. Nie, Q. Zhou, E. Jassim, P. T.K. Saunders, and R. A. Hess Differential Expression of Estrogen Receptors {alpha} and {beta} in the Reproductive Tractsof Adult Male Dogs and Cats Biol Reprod, April 1, 2002; 66(4): 1161 - 1168. [Abstract] [Full Text] [PDF]
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