Estrogens and epididymal function - CiteSeerX

Review

Reprod. Fertil. Dev., 2001, 13, 273–283

Estrogens and epididymal function Rex A. HessAD, Qing ZhouA, Rong NieA, Cleida OliveiraB, Hyun ChoC, Masaaki NakaiA and Kay CarnesA AReproductive

Biology and Toxicology, Veterinary Biosciences, University of Illinois, 2001 S. Lincoln, Urbana, IL 61802-6199, USA. BDepartments of Morphology and Physiology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. CDepartment of Biology, Sunchon National University, Sunchon, 540-742, South Korea. DTo whom correspondence should be addressed. email: [email protected] Abstract. Estrogen is synthesized in the male reproductive system and is found in high concentrations in rete testis and seminal fluids. This luminal estrogen targets estrogen receptors (ER) along the male reproductive tract, and in particular the efferent ductules, where ERα is abundant. However, both ERα and ERβ are found in various regions of the male reproductive tract. The transgenic ER knockout mice (αERKO and βERKO) have been used to help define the role of ER in the male. In the αERKO animal model, the efferent ductules are dramatically altered, forming an epithelium in which fluid reabsorption is inhibited and epithelial cells have greatly reduced numbers of lysosomes and organelles associated with endocytosis. The βERKO male reproductive tract appears normal. Because these animals are transgenic and lack ER throughout development, we developed animal models using pure antiestrogen ICI 182,780 treatments in adult males. The data show that ERα participates in the regulation of the apical cytoplasm of non-ciliated cells of the efferent ductules, narrow cells of initial segment epididymis and clear cells in the remaining segments of the epididymis. There appears to be no effect on vas deferens. The inhibition of ERα function in the male leads to decreases in sperm concentrations and eventually to infertility. The current literature leaves the mechanisms of estrogen action in the male reproductive tract unsettled and raises the question of androgen’s contribution to the regulation of fluid transport, especially in the efferent ductules. Extra keywords: efferent ductules, estrogen receptor, male, testis.

Estrogen concentrations Estrogen is present in very high concentrations in rete testis and seminal fluids of several species (Table 1). Current evidence indicates that germ cells, in addition to Leydig cells, synthesize estrogen, and that sperm in the upper epididymis serve as the major source of estrogen in the male reproductive tract (Nitta et al. 1993; Carreau et al. 1999; Hess 2000). The presence of P450 aromatase in male germ cells has now been demonstrated in several species, including mouse, rat, brown bear and rooster (Nitta et al. 1993; Tsubota et al. 1993; Hess et al. 1995; Kwon et al. 1995; Janulis et al. 1998). Its presence in germ cells and spermatozoa was recently confirmed and shown to represent approximately 62% of the total testicular aromatase activity (Levallet et al. 1998; Carreau et al. 1999). Others have shown the absence of aromatase in the epididymis (Schleicher et al. 1989). Thus, the conversion of androgens to estrogens by sperm remains the primary source of estrogen in the lumen of the tract. In the reproductive tract, estrogen can reach relatively high concentrations. In rete testis fluid of the rat, estradiol is approximately 250 pg mL–1 (Free and Jaffe 1979), which is higher than the average serum concentrations in the female (Overpeck et al. 1978; Robaire © CSIRO 2001

and Hermo 1988). Estrogens are also abundant in semen (Table 1). Estrogen receptors It has been known for many years that an estrogen receptor-like protein exists in epididymal tissues (Danzo et al. 1975). This early work in the rabbit showed that an estrogen receptor (ER) had the highest concentration in cauda epididymis (Danzo et al. 1977, 1978, 1981, 1983; Danzo and Eller 1979). However, further studies showed that a protease was present, which was responsible for removing the DNA-binding portion of the ER, thus making estrogen less active in the adult epididymis (Hendry and Danzo 1986). Because of this discovery, Danzo concluded that estrogen was more likely to be important during development of the epididymis than in adult function. However, in the 1980s, estrogen-binding activity was again shown in epididymal tissues in other species, including the dog (Younes et al. 1979; Younes and Pierrepoint 1981), human (Murphy et al. 1980), guinea-pig (Danzo et al. 1981), turtle (Dufaure et al. 1983), monkey (Kamal et al. 1985; West and Brenner 1990), ram (Tekpetey and Amann 1988), and rat (Kuiper et al. 1997). Autoradiography has 10.1071/RD00100

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Table 1.

Estrogen concentrations in rete testis and seminal fluids

Source

Estrogen concentration

Species

Reference

Rete testis fluid

14–195 pg mL–1 249 pg mL–1 11.5 pg mL–1 0.38 nM (total estrogens) 8.60 nM (estrone sulfate)

Monkey Rat Bull Boar

Waites and Einer-Jensen (1974) Free and Jaffe (1979) Ganjam and Amann (1976) Setchell et al. (1983)

Semen

14–162 pg mL–1

Man

73 pg mL–1 (estradiol) 4116 pg mL–1 (estrone sulfate) 50–890 pg mL–1

Horse

Adamopoulos et al. (1984) Bujan et al. (1993) Claus et al. (1992)

430 pg mL–1 (estradiol) 860 pg mL–1 (estrone)

Boar

Bull

also shown that 3H-estradiol binds to epithelial and stromal tissues throughout the male reproductive system (Schleicher et al. 1984; Hess et al. 1997b). The second form of ER (ERβ) has now been found in testis, efferent ductules, epididymis and prostate (Kuiper et al. 1996, 1997; Hess et al. 1997b; Saunders et al. 1997, 1998; Krege et al. 1998; Prins et al. 1998; Rosenfeld et al. 1998; van Pelt et al. 1999). However, a function for ERβ in the male reproductive tract awaits further investigation, as the transgenic ERβ knockout mouse (βERKO) has been shown to be fertile and appears to have a normal testis and epididymis (Krege et al. 1998). Binding assays do not differentiate between ERα and ERβ. Therefore, immunocytochemistry (ICC), in situ hybridization and Northern blot analysis have been used to further differentiate ER presence in the male (Tables 2, 3). However, these techniques have not provided consistent results among laboratories and between species. The only Table 2.

Eiler and Graves (1977) Ganjam and Amann (1976) Claus et al. (1985)

data that have been consistent show the presence of high concentrations of ER in the epithelium of efferent ductules (Tables 2, 3) across all species (West and Brenner 1990; Iguchi et al. 1991; Sato et al. 1994; Ergun et al. 1997; Fisher et al. 1997; Goyal et al. 1997a, 1998; Hess et al. 1997b; Kwon et al. 1997). In the goat and monkey, only non-ciliated cells of the efferent ductal epithelium stained ER-positive (West and Brenner 1990; Goyal et al. 1997a). After the discovery of ER subtypes and the production of specific antibodies, ERα localization in the epididymis has improved, but the results are equivocal (Fisher et al. 1997; Goyal et al. 1997a; Hess et al. 1997b; Kwon et al. 1997). In the mouse at 90 days of age, the efferent ductule epithelium was strongly positive for ERα immunostaining, using the H222 antibody (Iguchi et al. 1991). Other epithelia along the epididymis were only slightly positive. In the vas deferens, H222 gave no reactivity in the epithelium, but stromal nuclei were positive. This immunostaining is some-

Localization of estrogen receptor α in the adult male reproductive system by immunohistochemistry, in situ hybridization or RNA analysis

Organ Testis Rete testis Efferent ductule Epididymis Vas deferens Seminal vesicles Prostate Reference

Rat + + + –

1

+

+

+

+ + +

+ 2

+ 3

Mouse Goat +

+

+

4

+ – 5

+ 6

+ + – + +

+ + + +

7

8

9

Bird

Monkey

Human

+ – + –

+

+ –

+ –

+ – + –

10

11

12

13

14

+ +

+

15

16

1. LC (Fisher et al. 1997); 2. Echeverria et al. (1994); 3. S in Vas (Hess et al. 1997b); 4. Zhai et al. (1996); 5. LC, GC spermatids; E and S in Sv (Pelletier et al. 2000); 6. Kuiper et al. (1996, 1997); 7. S in Vas, E and S in Sv (Williams et al. 2000); 8. Yuasa et al. (1997, 1999); 9. LC; S in Vas (Iguchi et al. 1991); 10. ED-N (Goyal et al. 1997b); 11. Kwon et al. (1997); 12. ED-N (Fisher et al. 1997); 13. ED-N (West and Brenner 1990); 14. Heikinheimo et al. (1995); 15. Ergun et al. (1997) #3664; 16. Palacios (1993) #5919. Cp, caput-corpus; Cd, cauda; E, epithelium; ED-N, non-ciliated cells only in efferent ductules; ED-NC, non-ciliated and ciliated cells in efferent ductules; GC, germ cells; GP, guinea-pig; IS, initial segment epididymis; LC, Leydig cells; P, prostate; RT, rete; S, stroma; Sv, seminal vesicles; Vas, vas deferens.

Estrogens and epididymal function

275

Table 3. Localization of estrogen receptor β in the adult male reproductive system by immunohistochemistry, in situ hybridization or RNA analysis Organ Testis Rete testis Efferent ductule Epididymis Vas deferens Seminal vesicles Prostate Reference

Rat +

+ –

+ + + +

Mouse Monkey Human +

+ +

+ +

+ +

+ + + + + 1 2 3 4 5 6 7

+

+

+ 9

10

+ + +

8

1. Kuiper et al. (1997, 1998); 2. Hess et al. (1997b); 3. GC and SC (Saunders et al. 1997, 1998); 4. Couse et al. (1997); 5. E (Prins et al. 1998); 6. SC, E in P, E in Sv (Pelletier et al. 2000; Taylor and Al-Azzawi 2000); 7. E and S in Vas and Sv (Williams et al. 2000); 8. LC, GC, IS (Rosenfeld et al. 1998); 9. SC, GC (Pelletier et al. 1999). 10. GC spermatids only (Enmark et al. 1997). E, epithelium; GC, germ cells; IS, initial segment epididymis; LC, Leydig cells; P, prostate; S, stroma; SC, Sertoli cells; Sv, seminal vesicles; Vas, vas deferens.

what similar to the autoradiographic data previously shown by Schleicher et al. (1984). 3H-Estradiol showed binding in clear cells of the cauda and vas deferens and in fibroblasts along the entire tract. In contrast, ERα localization in the rat has been more controversial (Table 2). In one study, using a mouse monoclonal antibody (6F11) against the A/B region of the human ERα, positive staining was found only in epithelial cells of the efferent ductules (Fisher et al. 1997). The epididymal tissues were negative. Our laboratory repeated this study using the 6F11 antibody (Novocastra, UK) and the data were in complete agreement with the Fisher study, showing staining only in epithelia of the efferent ductules. In another study using frozen sections and the ER21 antibody, which is made against a peptide containing the first 21 amino acids of the rat and human ER (and which does not cross-react with ERβ), we also found predominant staining in efferent ductules (Hess et al. 1997b), as shown for all species examined to date. However, the initial segment of epididymis was also strongly positive and the remaining regions of the epididymis were moderately positive. This study was also repeated, but antigen retrieval methods were used instead of frozen sections. The results differ only slightly. This difference in staining in the rat between the two antibodies, 6F11 and ER21, raises serious questions regarding the description in the literature of ER localization in the male reproductive tract using ICC alone. Autoradiography and estradiol-binding assays indicate that ER is present in the rat epididymis. Reverse transcription-polymerase chain reaction data also show that ERα is present in epididymal tissues (Heikinheimo et al. 1995; Hess et al. 1997b). Therefore, future studies should focus on methods that clarify ER presence in cell types of the epididymis.

Estrogen function The efferent ductules and initial segment of the epididymis are dependent on hormones and/or testicular factors derived from the lumen for normal maintenance of epithelial function. Both regions contain androgen receptors and bind labeled dihydrotestosterone (DHT) (Schleicher et al. 1984), and show epithelial regression after ductal ligation or castration followed by androgen re-supplementation (Fawcett and Hoffer 1979; Hermo and Morales 1984; Ilio and Hess 1994). Thus, these regions do not show androgen responsiveness compared with the remaining epididymal segments. Instead, estrogen appears to play a more important role in regulating efferent ductule function (Hess et al. 1997a), similar to the role that DHT has in the initial segment of the epididymis (Robaire and Viger 1995). The primary function of efferent ductules is to reabsorb luminal fluids (Ilio and Hess 1994; Clulow et al. 1998). These ductules transport sperm and reabsorb water, ions and proteins. Many physiological and micropuncture studies on the proximal segments of the excurrent ducts in different species have confirmed the original findings of Crabo (1965) that more than 90% of the fluid secreted by the seminiferous epithelium is reabsorbed in the efferent ductules (Jones and Jurd 1987; Clulow et al. 1994; Man et al. 1997). The importance of the remaining epididymal regions in the reabsorption process is less clear, as epididymal by-pass surgery appears to show that the initial segment and proximal caput epididymal regions are essential, but that more distal regions are not essential for normal sperm maturation (Temple-Smith et al. 1998). Although the efferent ductules are now recognized as the major site for rete testis fluid reabsorption, the underlying mechanisms for absorption remain unsettled. However, the work of several laboratories suggests that the primary mechanism of fluid movement in the ductules involves the coupling of water and active ion transport (Jones and Clulow 1987; Jones and Jurd 1987; Ilio and Hess 1992; Clulow et al. 1994, 1996, 1998; Chan et al. 1995; Man et al. 1997; Hansen et al. 1999). Non-ciliated cells in the efferent ductules have a well-developed endocytotic system that is specialized for fluid reabsorption. Apical cytoplasm is characterized by the presence of a microvillus brush border (Fig. 1), a profusion of apical canaliculi and vesicles, and a variety of large vacuoles and membrane-bound bodies of different shapes, sizes and staining intensities (Hermo et al. 1988b, 1991; Robaire and Hermo 1988; Ilio and Hess 1994). Markers for electron microscopy have demonstrated that coated pits, apical tubules, endosomes, multivesicular bodies and lysosomes are components of an elaborate system for fluid phase, adsorptive and receptor-mediated endocytosis (Morales and Hermo 1983; Hermo and Morales 1984; Byers et al. 1985; Hermo et al. 1985; Veeramachaneni and Amann

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1990, 1991; Veeramachaneni et al. 1990; Ilio and Hess 1994). Rete testis fluid is taken up by the endocytotic apparatus from coated pits to multivesicular bodies and then to lysosomes for digestion by hydrolytic enzymes (Hermo and Morales 1984). The lateral plasma membranes in the basal and supranuclear regions of the efferent ductules form a well-localized ‘tubular network’ (Ramos and Dym 1977; Jones and Jurd 1987; Robaire and Hermo 1988; Ilio and Hess 1994). The intercellular spaces become dilated when absorption is active (Pudney and Fawcett 1984). The occurrence of these dilated intercellular channels strongly suggests that fluid movement in this part of the tract may be coupled to active solute transport (Suzuki and Nagano 1978). However, these tubular networks show considerably less amplification than is normally found in a reabsorptive epithelium such as the proximal convoluted tubules of the kidney (Ilio and Hess 1992). Although Jones and Jurd (1987) showed by morphometry that the efferent ductules are capable of moving substantial amounts of fluid by transcytosis in vesicles and vacuoles, they found that such a route could not account for the tremendous quantity of fluid reabsorbed by this epithelium. In 1993, a transgenic mouse was developed lacking a functional ERα gene (αERKO) (Lubahn et al. 1993). This animal, provided for the first time a direct observation of the importance of ER in the regulation of male fertility and male reproductive tract function. Although androgens are traditionally considered the more important male hormone, the αERKO male was found to be infertile. The cauda sperm were abnormal with significantly reduced sperm concen-

trations, and testicular atrophy was seen in the older males (Eddy et al. 1996). A key observation was the excessive dilation of the rete testis and efferent ductules in this animal (Hess et al. 1997a). Two hypotheses were formulated to explain these observations. (1) There could be excessive fluid reabsorption in the efferent ductules, which would increase the concentration of sperm and cause the luminal contents to become compacted. This rapid response would induce occlusions in the ductules and produce fluid build-up and subsequent back-pressure atrophy of the testis, as seen after toxicant exposures (Cooper and Jackson 1973; Carter et al. 1987; Hess et al. 1991; Nakai et al. 1992). (2) An opposite mechanism could occur, in which fluid reabsorption in the efferent ductules is inhibited. This would cause the retention of water, producing fluid accumulation in the ductal lumen. The excess fluid would overload the funnel-like ductal system and cause testicular accumulation of fluid, an increase in testis weight and subsequent back-pressure atrophy. Thus, either hypothesis would lead to the same conclusion that the accumulation of fluid would cause subsequent atrophy of the testis and infertility. Histological observations of the αERKO male support the second hypothesis that luminal fluid is not reabsorbed by the efferent ductules (Hess et al. 1997a). The efferent ductal epithelium in αERKO tissue was reduced in height by nearly half (Fig. 1), with a loss of cellular organelles, a flattening of the nucleus and the loss or shortening of the microvillus border (Hess et al. 2000). All of these changes are consistent with a decrease in fluid reabsorption (Hess et al. 1997a). The endocytotic apparatus, including apical vesicles and PAS-positive lysosomal granules (Fig. 1), which are

Fig. 1. (a) Wild-type (WT) efferent ductule epithelium is columnar, with non-ciliated and ciliated (Ci) cells. The non-ciliated cells contain a prominent brush border (arrow) on the apical surface and an extensive endocytotic apparatus in the apical cytoplasm (double arrow). Nu, nucleus. (b) Estrogen receptor α knockout (αERKO) efferent ductule epithelium, which is cuboidal in shape. Some non-ciliated cells show the loss of microvilli (arrow), whereas others have microvilli that are greatly reduced in height (circle). The apical cytoplasm is reduced in area and contains fewer organelles of the endocytotic apparatus. Nu, nucleus.


277

Fig. 2. (a) Electron microscopic view of the apical cytoplasm in a wild-type (WT) efferent ductule non-ciliated cell. The microvillus border is denoted between the two hatched lines. Evidence of endocytosis is noted by the apical endosomes (En) and coated pits and tubules (arrows). Mi, mitochondria. (b) Electron microscopic view of the apical cytoplasm in an estrogen receptor α knockout (αERKO) efferent ductule non-ciliated cell showing extensive loss of apical microvilli (between the two hatched lines). The apical cytoplasm contains small vesicles, but the typical endosomes and coated pits and tubules are lacking. Mi, mitochondria; Nu, nucleus.

prominent in non-ciliated cells of normal efferent ductules (Morales and Hermo 1983; Hermo and Morales 1984; Ilio and Hess 1994), was greatly reduced in the αERKO efferent ductule epithelium. The apical surface (Fig. 2) took on the appearance of a non-absorbing epithelium, with decreases in the number and size of microvilli and endosomes (Hess et al. 2000; Lee et al. 2000; Nakai et al. 2001). The ability of this epithelium to reabsorb fluid was tested in vitro using small segments of adult efferent ductules in organ culture. The tubular ends were ligated, preventing the inward flow of culture medium, and the lumen was observed over a 24-h period. Efferent ductules from wild-type males were capable of rapidly reabsorbing the luminal fluid, which resulted in a collapse of the ductule walls. However, the luminal area in αERKO ductules did not collapse, but instead showed a dramatic increase in area (Hess et al. 1997a). Thus, these data support the conclusion that the αERKO efferent ductule epithelium not only lacks the ability to reabsorb fluids, but also permits the movement of fluid into the lumen. As predicted in the second hypothesis, a transient increase in testis weight in αERKO males was noted between 32 and 81 days of age and then a continual decrease in weight up to 185 days of age. This suggested that long-term atrophy of testes in the knockout mouse was caused by back pressure of the accumulating luminal fluids, as a result of the inhibition of fluid reabsorption in the efferent ductules (Hess et al. 1997a, 2000). This type of pathological process is noted in other experimental animals,

in which the efferent ductules are dysfunctional (Hess and Nakai 2000). Further evidence that the pathological changes responsible for αERKO infertility in the male are associated with the reproductive tract and not with the testis, came from a study in which αERKO germ cells were transplanted into a normal, ERα-positive testis that had been treated to remove native germ cells. Sperm from the αERKO testis were found to be capable of fertilization when transplanted, thus suggesting that a normal reproductive tract is lacking in the knockout animals (Mahato et al. 2000). Therefore, the αERKO mouse provides strong evidence that estrogen, or more specifically, a functional ERα, is involved in the regulation of fluid transport in the male reproductive tract and is responsible for increasing the concentration of sperm as they enter the head of the epididymis. The αERKO mouse also exhibits an abnormal epididymis (Hess et al. 2000). The effects are not as evident as observed in efferent ductules and are relegated to specific cell types along the tract. The narrow cells of the initial segment develop a pyramid-shaped protrusion into the lumen, with a thin cell forming a tight cap over the other cells. The apical cells in a specific region of the caput accumulate PAS-positive granules that distort the cell size and nuclear shape. Finally, the clear cells in the distal epididymal regions are also slightly abnormal, containing larger endocytotic vesicles and PAS-positive granules. Interestingly, these are the specific cell types that were originally identified as binding 3H-estradiol in an autoradiographic

278

study (Schleicher et al. 1984). These same cell types show a similar response to treatments with an antiestrogen chemical (see below), and thus their normal functions are likely regulated by ERα, rather than ERβ. The functions of these cells are purported to be ion transport and endocytosis (Hermo et al. 1988a, 1991; Adamali and Hermo 1996), functions similar to the epithelial cells of the efferent ductules, where ERα is found in abundance (Hess et al. 1997a). At the present time, it is not possible to know the importance of these epididymal changes because the pathophysiology seen in the αERKO efferent ductules is so severe that it overwhelms any epididymal effects. However, it is reasonable to speculate that the sperm abnormalities observed in cauda epididymis (Eddy et al. 1996) could be owing to abnormal function of the narrow, apical and clear cells. Dysfunction of these cells could lead to abnormal luminal concentrations of ions and the accumulation of cytoplasmic droplet materials that are normally reabsorbed by the clear cells (Cooper 1998). There is some controversy over the types and amounts of ER present in the epididymis and vas deferens (Tables 2, 3). However, binding studies suggest that estrogen does have activity in this region of the male tract during development and possibly in the adult. In one of the first experiments to suggest that estrogen could influence epididymal function, Meistrich et al. (1975) simply injected intact adult male mice with estradiol benzoate, testosterone proprionate, or both hormones, and looked for effects on sperm concentrations in the epididymis and the kinetics of sperm transport. They found a decrease in sperm transit times with estradiol and estradiol plus testosterone. However, estradiol alone was more effective. More importantly, this rapid transport of sperm through the epididymis resulted in the passage of immature sperm and in total sterility. Other studies have shown that estrogen treatments, even in the presence of maintenance levels of testosterone, produces harmful effects on the epididymis and reduce the fertilizing ability of epididymal sperm (Lubicz-Nawrocki 1974). There are also numerous physiological experiments involving hormonal treatment, or castration or hypophysectomy followed by hormonal supplementation of estrogen. However, it is unclear in these studies whether the effects of estrogen on the epididymis are direct or indirect. In general, the effects of castration on the epididymis are reversible by testosterone administration, and estrogen is antagonistic of the androgen response (Jones et al. 1980; Grandmont et al. 1983; Robaire and Hermo 1988). Therefore, it remains an open question as to whether estrogen plays an important or minor role in the regulation of the epididymis and vas deferens. The αERKO mouse lacks a functional ERα throughout development. This raises many questions regarding developmental defects and adult dysfunction in the absence of ER. Further evidence of this possibility was found when we

R. A. Hess et al.

examined the perinatal efferent ductules in the developing αERKO mouse. During the period just after birth, the efferent ductules appeared dilated and the epithelial height was shorter than in wild-type, indicating that the epithelium never differentiated into the adult phenotype (K. Carnes, unpublished observations). However, to experimentally test this hypothesis, adult mice were treated with the pure antiestrogen ICI 182,780 (AstraZeneca, Macclesfield, Cheshire, UK), for 35–59 days. Table 4 compares the results from αERKO and ICI treatments, and clearly demonstrates that many of the effects seen in the αERKO mouse are representative of adult dysfunction owing to the absence of ERα (Lee et al. 2000). In αERKO and ICI-treated mice, the efferent ductule epithelium shows a loss of endocytotic organelles, a flattening of the nucleus and the loss or shortening of the microvillus border (Hess et al. 1997a, 2000; Lee et al. 2000). All of these changes are consistent with a decrease in fluid reabsorption, which was observed in the αERKO male (Hess et al. 1997a). In mice, the endocytotic apparatus, including apical vesicles and PAS-positive lysosomal granules, which are prominent in non-ciliated cells of normal efferent ductules (Morales and Hermo 1983; Hermo and Morales 1984; Ilio and Hess 1994), was greatly reduced in the αERKO and ICI-treated animals. Thus, the apical surface of this reabsorbing epithelium appears to be transformed into a non-absorbing structure, when ERα is inhibited, either by transgenic deletion or by chemical competition with a potent antiestrogen. In the ICI-treated rats there was an increase in lysosomes in efferent ductule epithelial cells during the early periods of treatment, but in most areas these decreased with further treatment (Oliveira et al. 2001). Although these data from the αERKO mouse clearly show that the loss of ERα function decreases fluid reabsorption in the efferent ductules, placing a greater emphasis on the role of estrogen in male reproduction than previously recognized, we must remember that the male tract also contains androgen receptors (Cooke et al. 1991; Goyal et al. 1997b). However, androgen contribution to the regulation of efferent ductules remains unknown. To date, few studies have attempted to compare estrogens and androgens from a physiological perspective (Ilio and Hess 1994; Hansen et al. 1997). In the Hansen study, male rats were administered daily doses of testosterone propionate, flutamide (antiandrogen), 17β-estradiol 3-benzoate, or tamoxifen (antiestrogen) for 7 days. The results of that study seem to contradict findings in the αERKO mouse (Hess et al. 1997a); estrogen appeared to inhibit the reabsorption of fluid in the efferent ductules (Hansen et al. 1997). The data are also confusing because an antiandrogen, flutamide, caused a greater reduction in fluid flow (a higher rate of fluid reabsorption) than did testosterone. A further complication was seen with tamoxifen, which showed the greatest


Table 4.

279

Characteristics of the male reproductive systems in estrogen receptor α knockout αERKO) and ICI antiestrogen-treated mice mice (α

Characteristic

αERKO1

ICI-182,7802,3

yes yes yes yes yes yes yes yes yes yes

yes no no/yes4 no/yes4 no/yes5 not determined yes yes not determined no/yes5

yes yes yes yes yes yes yes yes yes yes yes yes yes

yes yes yes/no6 yes yes yes yes no no yes yes yes not determined

Rete testis dilation Rete testis over growth Seminiferous tubule dilation Period of testis weight increase Testicular atrophy Increased serum testosterone concentration Efferent ductule dilation Cauda sperm number decreased Decreased sperm motility Decreased fertility Efferent duct epithelium: decreased height decreased endocytosis decreased lysosomes decreased microvillus border decreased nuclear height decreased numbers of cilia Presence of glycogen granules in efferent ductules Increase in blind ending efferent ductules Abnormal growth of initial segment epididymis Abnormal narrow cells of the epididymis Abnormal epididymal cells with PAS-positive granules Abnormal clear cells Normal expression of ERβ in efferent ductules 1Eddy

et al. (1996); Hess et al. (2000); Lee et al. (2000); Lubahn et al. (1993). formulation (AstraZeneca, Macclesfield, Cheshire, UK); 5 mg/mouse/week for 5 weeks. 3Lee et al. (2000). 4In one experiment there was no increase in testis weight (Lee et al. 2000), however, subsequent studies have shown an increase over time (H. Cho, personal communication). 5Fertile after 59 days of treatment (H. Cho, personal communication), but decreased fertility and testicular atrophy in rats (C. Oliveira, personal communication). 6In the rat, at first there was an increase, followed by a decrease in most epithelium with further treatment. 2Faslodex

stimulation of fluid reabsorption. Thus, the only treatment that appeared to decrease reabsorption was estradiol. Clulow et al. (1998) explained these conflicting data by equating estrogen and tamoxifen effects with antiandrogen actions, and suggesting that flutamide would cause an increase in systemic androgen concentrations. These studies make it difficult to reach a satisfactory conclusion regarding the involvement of androgens and estrogens in the reabsorption of efferent ductal fluid. However, in the αERKO male, testosterone is elevated (Eddy et al. 1996), not reduced, and fluid reabsorption is suppressed (Hess et al. 1997a). Therefore, other explanations of these results must be considered. For example, the potential effects of hormonal treatments on the physiological feedback in the hypothalamus–pituitary–testis axis (Barton and Andersen 1998) could complicate the interpretation. Systemic treatments with hormones and antihormones will either decrease or increase testosterone. It is possible that a decrease in testosterone would decrease ER

and increase androgen receptor expression in the efferent ductule epithelium, as there is now evidence that androgens can maintain these receptors in the efferent ductules after castration (Goyal et al. 1998). Conclusions The current literature leaves the mechanisms of estrogen action in the male reproductive tract unsettled and raises the question of the role of androgen in regulating fluid transport. It is also unclear how best to test the hypothesis that estrogen stimulates fluid reabsorption under normal physiological conditions. Treatments with steroid hormones, with or without castration or rete testis ligation, will create complications owing to interference with the feedback regulation of gonadotrophin release. Castration and rete testis ligation experiments remove luminal contents and thus eliminate a major pathway of entry by regulatory factors and steroids along the apical surface of the ductal epithelium. Future studies must address these problems and attempt to

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reconcile the apparent contradictions seen in the effects of androgens and estrogens. Regardless, it is now well established that the loss of ER activity in the male, interferes with fluid reabsorption in the efferent ductules. Antiestrogen treatment of adult mice and rats has proven that the observed effects in the transgenic αERKO males are not simply developmental in origin, but are representative of adult dysfunction. Thus, it appears that estrogen is required for normal fertility in the male. However, the mechanisms of estrogen action in this reabsorptive epithelium, as well as in the epididymis and vas deferens, remain to be determined. Acknowledgment We would like to acknowledge several former and current students including Hiro Nitta, Soga Kwon, Yu-Chyu Chen, Lynn Janulis, Sarah Janssen, Ki-Ho Lee and Eman Jassim. We also acknowledge the fruitful collaboration of colleagues and scientists at the University of Illinois, David Bunick and Janice Bahr, and from other institutions, Dennis Lubahn, Ken Korach, Lane Clark, Dan Gist, Howard Lien, and Alan Verkman, in the study of estrogens in the male. This study was supported by NIH Grant no. HD35126. References Adamali, H. I., and Hermo, L. (1996). Apical and narrow cells are distinct cell types differing in their structure, distribution, and functions in the adult rat epididymis. J. Androl. 17, 208–22. Adamopoulos, D., Lawrence, D. M., Vassilopoulos, P., Kapolla, N., Kontogeorgos, L., and McGarrigle, H. H. (1984). Hormone levels in the reproductive system of normospermic men and patients with oligospermia and varicocele. J. Clin. Endocrinol. Metab. 59, 447–52. Barton, H. A., and Andersen, M. E. (1998). A model for pharmacokinetics and physiological feedback among hormones of the testicular–pituitary axis in adult male rats: a framework for evaluating effects of endocrine active compounds. Toxicol. Sci. 45, 174–87. Bujan, L., Mieusset, R., Audran, F., Lumbroso, S., and Sultan, C. (1993). Increased oestradiol level in seminal plasma in infertile men. Hum. Reprod. 8, 74–7. Byers, S. W., Musto, N. A., and Dym, M. (1985). Culture of ciliated and nonciliated cells from rat ductuli efferentes. J. Androl. 6, 271–8. Carreau, S., Genissel, C., Bilinska, B., and Levallet, J. (1999). Sources of oestrogen in the testis and reproductive tract of the male. Int. J. Androl. 22, 211–23. Carter, S. D., Hess, R. A., and Laskey, J. W. (1987). The fungicide methyl 2-benzimidazole carbamate causes infertility in male Sprague-Dawley rats. Biol. Reprod. 37, 709–17. Chan, H. C., Lai, K. B., Fu, W. O., Chung, Y. W., Chan, P. S., and Wong, P. Y. (1995). Regional differences in bioelectrical properties and anion secretion in cultured epithelia from rat and human male excurrent ducts. Biol. Reprod. 52, 192–8. Claus, R., Schopper, D., and Hoang-Vu, C. (1985). Contribution of individual compartments of the genital tract to oestrogen and testosterone concentrations in ejaculates of the boar. Acta Endocrinol. 109, 281–8. Claus, R., Dimmick, M. A., Gimenez, T., and Hudson, L. W. (1992). Estrogens and prostaglandin F2α in the semen and blood plasma of stallions. Theriogenology 38, 687–93.

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Manuscript received 28 September 2000; revised and accepted 2 February 2001.

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