| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the * Department of Basic Veterinary Science,
The United Graduate School of Veterinary Sciences, Gifu University, Gifu,
Japan; Laboratory of Veterinary Physiology and
Department of Tissue Physiology, Tokyo
University of Agriculture and Technology, Tokyo, Japan; and
PM2.5/DEP Research Project, ||
Environmental Dioxin Project Group, National
Institute for Environmental Studies, Ibaraki, Japan.
| Correspondence to: Kazuyoshi Taya, DVM, PhD, Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan (e-mail: taya{at}cc.tuat.ac.jp). |
| Received for publication November 4, 2004; accepted for publication February 4, 2005. |
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Key words: Estrogen immunization, gonadotropin
and ERß were widely expressed in the testis and accessory
sex organs throughout development and in adulthood in the male
(Danzo et al, 1983;
Fisher et al, 1997;
Hess et al, 1997; Saunders et
al, 1997,
1998). Studies in male mice,
rats, and monkeys revealed the presence of ER in the Leydig cell, rete
testis, efferent duct, and epididymis, as well as in the pituitary, with some
species-dependent differences (Kuiper et
al, 1997). Expression of ER was particularly intense in the
efferent duct, which transports spermatozoa from the testis to the epididymis
(Hess et al, 2001). In
contrast, ERß was expressed in the testis, including Sertoli cells,
Leydig cells, and germ cells, as well as in the epididymis, prostate, and
seminal vesicle. These results suggest that testicular estrogens directly
affect testicular cells or male reproductive tracts. Although physiological importance of estrogens in male rodents was suggested by previous studies using the rat and mouse, reproductive features of the male vary between species, even within rodents. For example, the golden hamster is a seasonal breeding species. In addition, regulation of pituitary gonadotropin by testicular feedback system in the golden hamster is likely different from that of rat (Kishi et al, 2000). Therefore, it is profitable to study male fertility in many species for understanding common and species-specific features of male reproduction. However, studies on reproductive features of the male golden hamster are quite limited. Therefore, we used this species in this study to extend our knowledge about its reproductive features.
Several previous studies were designed to examine physiological roles of estrogens in male mice and rats by using gene-targeting techniques (Eddy et al, 1996; Fisher et al, 1998; Krege et al, 1998), aromatase inhibitors (Turner et al, 2000; Gerardin and Pereira, 2002), and estrogen antagonists (Gill-Sharma et al, 1993; Balasinor et al, 2001) and have suggested physiological importance of estrogens in male fertility. However, it is difficult to evaluate physiological roles of estrogens during adulthood by knockout studies because the knockout mice have been affected by deficiency of estrogen action throughout their life. In addition, aromatase inhibitors and estrogen antagonists induce some effects besides inhibition of estrogen action. For example, administration of an aromatase inhibitor may cause accumulation of androgens because the drug inhibits conversion of androgens into estrogens. A widely used estrogen antagonist tamoxifen shows genotoxicity (Phillips, 2001) and site-specific effects (Jordan and Morrow, 1999). Therefore, in the present study, two complementary approaches were used to clarify the physiological roles of estrogen in the regulation of sperm motility; the first method is the treatment of low and high amounts of estradiol-17ß (experiment 1), and the second method is immunoneutralization of endogenous estradiol-17ß (experiment 2). The computer-assisted sperm analysis system was used to examine effects of estradiol-17ß on sperm motility parameters.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Experiment 1: Effect of Exogenous Estradiol-17ß
The aim of the first experiment was to determine whether exogenous
estradiol-17ß affects epididymal sperm motility. Steroid treatment
procedure was according to the previous paper
(Ebling et al, 2000). The male
golden hamster was subcutaneously implanted with a 1.0-cm silastic tube
(1.59-mm id, 3.18-mm od; Osteotec, Christchurch, United Kingdom) containing 2%
crystalline estradiol-17ß (Sigma Chemical Co, St Louis, Mo) and 98%
crystalline cholesterol (Sigma) (low E2 group, n = 8) or a 0.5-cm
silastic tube containing crystalline estradiol-17ß alone (high
E2 group, n = 10) under ether anesthesia. The control animal was
implanted with a 1.0-cm tube containing crystalline cholesterol alone (n = 6).
All silastic tubes were plugged at both ends with silicone medical adhesive
(Osteotec). Before the implantation, the silastic tubes were washed three
times in 70% ethanol, then incubated at 37°C for 24 hours in 0.01 M
phosphate-buffered saline (PBS) (pH 7.4) for stabilizing release rate of
estradiol-17ß. Groups of animals were killed by decapitation at 20 days
after the implantation. Blood samples were centrifuged at 1200 x
g at 4°C for 15 minutes and plasma were separated and stored at
-20°C until assayed for FSH, LH, immunoreactive (ir-) inhibin and
testosterone. Testes, epididymides, and seminal vesicle-coagulating gland
complexes (SV+CG) were collected for weighing wet weights. Epididymides were
further subjected to sperm motility analysis.
Experiment 2: Active Immunization Against Endogenous Estradiol-17ß
Estradiol-17ß conjugated with bovine serum albumin
(estriol-6-[O-carboxymethyl]oxime: BSA 6-ketoestriol 6-CMO: BSA) was
purchased from Steraloid Inc (Newport, RI). The conjugate (1 mg) was dissolved
in 1 mL of saline and the solution was mixed with an equal volume of Freund's
complete adjuvant. Eight hamsters were subcutaneously injected with 200 µL
of the suspension four times during the period studied. First, second, and
third immunizations were performed at 2-week intervals and fourth injection
was given at 4 weeks after the third injection. Control animals (n = 6)
received a mixture of saline and the adjuvant. Blood samples were obtained
from jugular vein just before each immunization for checking titer of
antibodies to estradiol-17ß. Further, plasma samples were obtained at 12
and 16 weeks after the first immunization. They were killed by decapitation at
16 weeks after the first immunization. Testes, epididymides, and SV+CG were
collected for weighing wet weights. Epididymides were further subjected to
sperm motility analysis.
Titer Check of Antibodies Against Estradiol-17ß
Changes in titers of anti-estradiol-17ß antibodies in plasma were
determined by measuring the binding of 125I-labeled
estradiol-17ß as reported previously
(Kaneko et al, 1995a). The
plasma was diluted 1:1000 with 0.05 M PBS (pH 7.4) containing 1% BSA, and the
diluted samples were incubated with 5000 counts per minute of
125I-labeled estradiol-17ß at 4°C for 24 hours in a total
volume of 200 µL. To separate bound radioligands, 100 µL of 1% bovine
gamma globulin in PBS and 500 µL of 25% polyethylene glycol in PBS were
added, and the mixture was agitated for 3 minutes. After centrifugation at
1700 x g at 4°C, radioactivity of the precipitate was
counted in a gamma counter. Estradiol-17ß binding activity was expressed
as a percentage of total count added.
Radioimmunoassay (RIA) of FSH, LH, Testosterone, and ir-Inhibin
Plasma concentrations of FSH were measured as previously described
(Bast and Greenwald, 1974;
Kishi et al, 1995) by National
Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) RIA kit for
rat FSH, using anti-rat FSH (S-11), NIDDK rat FSH-I-7 for iodination, and
NIDDK rat FSH (RP-2) as a reference standard. Plasma concentrations of LH were
measured as previously described (Bast and
Greenwald, 1974; Kishi et al,
1995) by NIDDK RIA kit for rat LH, using anti-rat LH (S-10), NIDDK
rat LH-I-8 for iodination, and NIDDK rat LH (RP-2) as a reference standard.
Serial dilutions of female golden hamster serum pools revealed concentration
curves parallel to standard FSH and LH curves in the RIAs (data not shown).
The intra- and interassay coefficients of variation were 4.4% and 14.6% for
FSH and 6.7% and 8.9% for LH, respectively.
Plasma concentrations of ir-inhibin were measured by a double-antibody RIA
(Hamada et al, 1989), which
was shown to be applicable to the hamster
(Kishi et al, 1995). The
antiserum used was raised in rabbits against bovine inhibin (TNDH-1). Purified
bovine 32-kDa inhibin was used for radioiodination and standard. The assay
system does not distinguish dimeric inhibin from -subunit monomer
(Kaneko et al, 1995b). The
intra- and interassay coefficients of variation were 8.8% and 14.4%,
respectively.
Plasma concentrations of estradiol-17ß and testosterone were determined by a double-antibody RIA system using 125I-labeled radioligand, as described previously (Taya et al, 1985). Antiserum against estradiol-17ß (GDN #244) (Korenman et al, 1974) and testosterone (GDN #250) (Gay and Kerlan, 1978) were kindly supplied by Dr G. D. Niswender (Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colo). The intra- and interassay coefficients of variation were 5.8% and 11.4% for estradiol-17ß, and 6.3% and 7.2% for testosterone, respectively.
It should be noted that because the assays for FSH, LH, and inhibin are a heterologous system, values of these hormones are rational.
Computer-Assisted Sperm Motility Analysis
The sperm motility parameters were obtained using C. IMAGING
computer-assisted sperm motion analysis system (Compix Inc, Cranberry
Township, Penn). Sperm in one drop of caudal epididymal fluid were incubated
at 37°C for 3 minutes in medium 199 (Biocell, Cal) containing 2.5 mM HEPES
(pH 7.2) and 0.5% BSA (Sigma). After the incubation, an aliquot of this
solution was diluted 10- to 20-fold with the medium, and 10 µL was placed
into the microcell-HAC chamber, which has a depth of 50 µm (Conception
Technologies, San Diego, Cal). Analyses of motile characteristics were
performed on at least 200 cells for each sample. Sperm motion, as viewed on an
Olympus microscope (4x, pseudodark field optics) with a stage warmer
(37°C) (MP-10DM, Kitazato, Japan), was used by C. IMAGING system. The C.
IMAGING system settings were as follows: frames analyzed = 15; framing rate =
30; maximum velocity = 1200 µm/s; threshold velocity = 45 µm/s; minimum
linearity for mean amplitude of lateral head displacement (ALH) = 3.5; pixel
scale 3.26 µm/pixel; maximum average number of cells/field = 30; cell size
range = 350–1600 pixel. The following characteristics were analyzed: the
percentage of motile spermatozoa, curvilinear velocity (the total distance
traveled divided by the total time the cell was tracked), straight velocity
(straight line distance), ALH (deviation of the sperm head from the mean
trajectory), beat/cross frequency, linearity (ratio of the straight line
distance to the actual tracked distance), and the percentage of circular
cells.
Statistics
One-way analysis of variance was performed. Significance between two means
was evaluated with Student's t test, and significance among more than
two means was determined by Duncan's multiple range test
(Steel and Torrie, 1960). All
data are presented as means ± SEM. Differences were considered
significant when P < .05.
|
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Plasma Concentrations of FSH, LH, Estradiol-17ß, ir-Inhibin, and Testosterone (Figure 2)— Plasma concentrations of estradiol-17ß were increased in a dose-dependent manner (Figure 2c). Plasma concentrations of LH showed a dose-dependent increase in response to the treatment with estradiol-17ß, and the level is significantly high in the high E2 group as compared with the control group (Figure 2b). Plasma concentrations of testosterone were significantly low in the high E2 group as compared with the control group (Figure 2e). Plasma concentrations of FSH and ir-inhibin were not affected by administration of estradiol-17ß (Figure 2a and d).
|
Weights of Reproductive Organs (Figure 3)— Treatment with either low or high dose of estradiol did not affect weights of bodies, epididymides, testes, and SV+CG.
|
|
|
|
|
Plasma Concentrations of FSH, LH, Testosterone, and ir-Inhibin (Figure 6)— Plasma concentrations of FSH were significantly lower in the immunized group than in the control group from 4 to 12 weeks after the primary immunization (Figure 6a). Plasma concentrations of LH were also significantly lower in the immunized group than in the control group from 4 to 16 weeks (Figure 6b). Plasma concentrations of testosterone and ir-inhibin did not differ significantly between the two groups except at 2 weeks for testosterone and 4 weeks for ir-inhibin (Figure 6c and d).
Weights of Reproductive Organs (Figure 7)— Body weights were significantly high in the immunized group as compared with the control group (Figure 7a). There are no significant differences between the immunized and control groups in weights of testes, epididymides, and SV+CG, respectively (Figure 7b through d).
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
knockout (ERKO) mice
suggested that estrogens are important for sperm production
(Eddy et al, 1996) and fluid
reabsorption within the efferent ducts
(Hess et al, 1997). Targeted
disruption of ER resulted in damage to spermatogenesis and subsequent
infertility (Eddy et al, 1996).
The ERgb knockout mice showed hyperplasia of the bladder and prostate
epithelium in older males. However, the reproductive tract appears grossly
normal in two types of ERKO mice (Krege et
al, 1998). On the other hand, aromatase knockout mice have
generated phenotypes implicating a role of estrogen in determining male
fertility (Fisher et al,
1998). Previous papers reported that exposure of the
neonatal/fetal male rodent to exogenous estrogen can cause a range of
abnormalities of the reproductive system, including testes atrophy and
abnormalities of the rete testis (Arai et
al, 1983; Gaytan et al,
1986). It has generally been concluded that the indirect action is
responsible for the adverse effects of neonatal estrogen exposure on the
testis (Arai et al, 1983;
Bellido et al, 1990). Because
the maximal treatment period was 20 days in the present study, only late
spermatids and sperms that were already within the epididymis would be
examined for sperm motility. Therefore, the effects of the treatments on sperm
motility might be due to effects on the epididymis. The hormone profiles in the first experiment of the present study showed that plasma LH levels significantly increased at 20 days after treatment with the high dose of estradiol-17ß. In addition, results of the second experiment clearly showed that plasma concentration of FSH and LH declined after the active immunization of estradiol-17ß. These results corroborate previous findings, which showed a decrease in circulating LH by a low dose of anti-estrogen tamoxifen in the adult rat (Gill-Sharma et al, 1993). Although treatment with estradiol-17ß decreased serum FSH concentrations in wild-type mice (as expected), it induced a small (but significant) rise in circulating FSH levels in male HPG mice (Ebling et al, 2000) and in the neonatal rat (Atanassova et al, 2000). The reason for the discrepancy is not known yet. With respect to testosterone, plasma levels of testosterone declined significantly at 20 days after treatment with high levels of estradiol-17ß, although plasma levels of LH increased. Similarly, active immunization against estradiol-17ß temporarily increased circulating testosterone. These results suggest that estradiol-17ß may directly suppress testicular secretion of testosterone. At present, it is not known whether estradiol-17ß directly affected sperm motility through ERs in spermatozoa. It is also possible that the changes in plasma concentrations of LH and FSH affected sperm motility, although testosterone secretion did not reflect the changes in plasma gonadotropins.
In conclusion, the present study clearly demonstrated that estradiol-17ß has stimulatory effects on sperm motility and secretion of gonadotropin in the golden hamster, and has a suppressive effect on testosterone secretion. Further studies are required to reveal mechanisms responsible for these responses.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Atanassova N, McKinnell C, Turner KJ, Walker M, Fisher JS, Morley
M, Millar MR, Groome NP, Sharpe RM. Comparative effects of neonatal exposure
of male rats to potent and weak (environmental) estrogens on spermatogenesis
at puberty and the relationship to adult testis size and fertility: evidence
for stimulatory effects of low estrogen levels.
Endocrinology. 2000; 141: 3898
-3907.
Balasinor N, Parte P, Gill-Sharma MK, Juneja HS. Effect of tamoxifen on sperm fertilising ability and preimplantation embryo development. Mol Cell Endocrinol. 2001; 178: 199 -206.[Medline]
Bast JD, Greenwald GS. Serum profiles of follicle-stimulating hormone, lutienizing hormone and prolactin during the estrous cycle of the hamster. Endocrinology. 1974; 94: 1295 -1299.[Medline]
Bellido C, Pinilla L, Aguilar R, Gaytan F, Aguilar E. Possible role of changes in post-natal gonadotrophin concentrations on permanent impairment of the reproductive system in neonatally oestrogenized male rats. J Reprod Fertil. 1990;90: 369 -374.
Carreau S. Germ cells: a new source of estrogens in the male gonad. Mol Cell Endocrinol. 2001; 178: 65 -72.[CrossRef][Medline]
Danzo BJ, Eller BC, Hendry WJ III. Identification of cytoplasmic estrogen receptors in the accessory sex organs of the rabbit and their comparison to the cytoplasmic estrogen receptor in the epididymis. Mol Cell Endocrinol. 1983; 33: 197 -209.[CrossRef][Medline]
Ebling FJ, Brooks AN, Cronin AS, Ford H, Kerr JB. Estrogenic
induction of spermatogenesis in the hypogonadal mouse.
Endocrinology. 2000; 141: 2861
-2869.
Eddy EM, Washburn TF, Bunch DO, Goulding EH, Gladen BC, Lubahn DB, Korach KS. Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility. Endocrinology. 1996; 137: 4796 -4805.[Abstract]
Fisher CR, Graves KH, Parlow AF, Simpson ER. Characterization of
mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19
gene. Proc Natl Acad Sci U S A. 1998; 95: 6965
-6970.
Fisher JS, Millar MR, Majdic G, Saunders PT, Fraser HM, Sharpe RM. Immunolocalisation of oestrogen receptor-alpha within the testis and excurrent ducts of the rat and marmoset monkey from perinatal life to adulthood. J Endocrinol. 1997; 153: 485 -495.[Abstract]
Gay VL, Kerlan JT. Serum LH and FSH following passive immunization against circulating testosterone in the intact male rat and in orchidectomized rats bearing subcutaneous silastic implants of testosterone. Arch Androl. 1978;1: 257 -266.[Medline]
Gaytan F, Pinilla L, Aguilar R, Lucena MC, Paniagua R. Effects of
neonatal estrogen administration on rat testis development with particular
reference to Sertoli cells. J Androl. 1986; 7: 112
-121.
Gerardin DC, Pereira OC. Reproductive changes in male rats treated perinatally with an aromatase inhibitor. Pharmacol Biochem Behav. 2002;71: 301 -305.[CrossRef][Medline]
Gill-Sharma MK, Gopalkrishnan K, Balasinor N, Parte P, Jayaraman S, Juneja HS. Effects of tamoxifen on the fertility of male rats. J Reprod Fertil. 1993;99: 395 -402.
Goyal HO, Braden TD, Mansour M, Williams CS, Kamaleldin A,
Srivastava KK. Diethylstilbestrol-treated adult rats with altered epididymal
sperm numbers and sperm motility parameters, but without alterations in sperm
production and sperm morphology. Biol Reprod. 2001; 64: 927
-934.
Hamada T, Watanabe G, Kokuho T, Taya K, Sasamoto S, Hasegawa Y, Miyamoto K, Igarashi M. Radioimmunoassay of inhibin in various mammals. J Endocrinol. 1989; 122: 697 -704.[Abstract]
Hess RA, Bunick D, Bahr J. Oestrogen, its receptors and function in the male reproductive tract—a review. Mol Cell Endocrinol. 2001;178: 29 -38.[CrossRef][Medline]
Hess RA, Gist DH, Bunick D, Lubahn DB, Farrell A, Bahr J, Cooke PS,
Greene GL. Estrogen receptor (alpha and beta) expression in the excurrent
ducts of the adult male rat reproductive tract. J
Androl. 1997;18: 602
-611.
Jordan VC, Morrow M. Tamoxifen, raloxifene, and the prevention of
breast cancer. Endocr Rev. 1999; 20: 253
-278.
Kaneko H, Nakanishi Y, Akagi S, Arai K, Taya K, Watanabe G, Sasamoto S, Hasegawa Y. Immunoneutralization of inhibin and estradiol during the follicular phase of the estrous cycle in cows. Biol Reprod. 1995a;53: 931 -939.[Abstract]
Kaneko H, Kishi H, Watanabe G, Taya K, Sasamoto S, Hasegawa Y. Changes in plasma concentrations of immunoreactive inhibin, estradiol and FSH associated with follicular waves during the estrous cycle of cows. J Reprod Dev. 1995b; 41: 311 -320.[CrossRef]
Kishi H, Itoh M, Wada S, Yukinari Y, Tanaka Y, Nagamine N, Jin W,
Watanabe G, Taya K. Inhibin is an important factor in the regulation of FSH
secretion in the adult male hamster. Am J Physiol Endocrinol
Metab. 2000;278: E744
-E751.
Kishi H, Taya K, Watanabe G, Sasamoto S. Follicular dynamics and secretion of inhibin and oestradiol-17 beta during the oestrous cycle of the hamster. J Endocrinol. 1995; 146: 169 -176.[Abstract]
Korenman SG, Stevens RH, Carpenter LA, Robb M, Niswender GD, Sherman BM. Estradiol radioimmunoassay without chromatography: procedure, validation and normal values. J Clin Endocrinol Metab. 1974; 38: 718 -720.[Medline]
Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar
M, Korach KS, Gustafsson JA, Smithies O. Generation and reproductive
phenotypes of mice lacking estrogen receptor beta. Proc Natl Acad
Sci U S A. 1998;95: 15677
-15682.
Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S,
Gustafsson JA. Comparison of the ligand binding specificity and transcript
tissue distribution of estrogen receptors alpha and beta.
Endocrinology. 1997; 138: 863
-870.
Kula K. Induction of precocious maturation of spermatogenesis in infant rats by human menopausal gonadotropin and inhibition by simultaneous administration of gonadotropins and testosterone. Endocrinology. 1988; 122: 34 -39.[Abstract]
Lindgren S, Damber JE, Carstensen H. Compensatory testosterone secretion in unilaterally orchidectomized rats. Life Sci. 1976;18: 1203 -1205.[CrossRef][Medline]
Pentikainen V, Erkkila K, Suomalainen L, Parvinen M, Dunkel L.
Estradiol acts as a germ cell survival factor in the human testis in vitro.
J Clin Endocrinol Metab. 2000; 85: 2057
-2067.
Phillips DH. Understanding the genotoxicity of tamoxifen?
Carcinogenesis. 2001; 22: 839
-849.
Robaire B, Duron J, Hales BF. Effect of estradiol-filled polydimethylsiloxane subdermal implants in adult male rats on the reproductive system, fertility, and progeny outcome. Biol Reprod. 1987; 37: 327 -334.[Abstract]
Saunders PT, Fisher JS, Sharpe RM, Millar MR. Expression of oestrogen receptor beta (ER beta) occurs in multiple cell types, including some germ cells, in the rat testis. J Endocrinol. 1998; 156: R13 -R17.[Abstract]
Saunders PT, Maguire SM, Gaughan J, Millar MR. Expression of oestrogen receptor beta (ER beta) in multiple rat tissues visualised by immunohistochemistry. J Endocrinol. 1997; 154: R13 -R16.[Abstract]
Steel RGD, Torrie JH. Principles and procedures of statistics. New York: McGraw-Hill; 1960.
Taya K, Watanabe G, Sasamoto S. Radioimmunoassay for progesterone, testosterone, and estradiol-17ß using 125I-iodohistamine radioligands. Jpn J Anim Reprod. 1985; 31: 186 -197.
Turner KJ, Morley M, Atanassova N, Swanston ID, Sharpe RM. Effect of chronic administration of an aromatase inhibitor to adult male rats on pituitary and testicular function and fertility. J Endocrinol. 2000;164: 225 -238.[Abstract]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
