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Estrogen receptors α and β in the inner ear of the ‘Turner mouse’ and an estrogen receptor β knockout mouse
Hearing Research 166 (2002) 1^8 www.elsevier.com/locate/heares
Estrogen receptors K and L in the inner ear of the ‘Turner mouse’ and an estrogen receptor L knockout mouse A.E. Stenberg a , H. Wang b , L. Sahlin b , P. Stierna c , E. Enmark c , M. Hultcrantz a
a;
Department of Otorhinolaryngology, Karolinska Hospital, 171 76 Stockholm, Sweden Department of Woman and Child Health, Karolinska Hospital, Stockholm, Sweden Department of Medical Nutrition, Karolinska Institutet, NOVUM, Huddinge, Sweden
b c
Received 22 March 2001; accepted 21 November 2001
Abstract Estrogen receptors have earlier been shown in the normal mouse, rat and human inner ear. If estrogens are important in normal hearing and development of presbyacusis in the normal population is not known. However it is known that patients with Turner syndrome, where a lack of estrogens is one of the main characteristics, commonly develop an early presbyacusis. A ‘Turner mouse’ has been developed, as a model for the ear problems in Turner syndrome, and it shows otitis media and a premature aging of the hearing. Estrogen receptors exist in an K and a L form. In this study inner ear tissue, from the Turner mouse and an estrogen receptor L knockout mouse (LERKO), was investigated regarding estrogen receptor K and L using immunohistochemistry. Results show that the Turner mouse has the same pattern of inner ear labeling, both concerning the estrogen receptor K and L, as that of a normal CBA/Ca mouse, with positive staining in the organ of Corti and spiral ganglion. The LERKO mice show close to normal inner ear morphology and positive estrogen receptor K immunostaining at the same locations as the CBA/Ca mouse. 4 2002 Elsevier Science B.V. All rights reserved. Key words: Turner syndrome; Estrogen receptor; Mouse; Estrogen receptor L knockout mouse; Inner ear; Immunohistochemistry
1. Introduction Estrogen - the female sex hormone - is classically known to in£uence growth, di¡erentiation and function of the female reproductive tract. However, the target ¢eld of estrogen action has been shown to be much wider (for review see Pettersson and Gustafsson, 2001). It includes male regulation of sexual behavior and normal function of the testis and prostate. Moreover, protective e¡ects have been observed on the maintenance of the skeleton, on cardiovascular system and
* Corresponding author. Tel.: +46 (8) 51775653; Fax: +46 (8) 51776267. E-mail address: [email protected] (M. Hultcrantz). Abbreviations: ABR, auditory brainstem response; LERKO, estrogen receptor L knockout mouse; ER, estrogen receptor
in the brain (Losordo et al., 1994; Turner et al., 1994, Garcia-Segura et al., 2001). There are suggestions that estrogens may have an e¡ect on the ear and hearing, but the association is still poorly understood. The well-known gender di¡erence in hearing, both pure tone threshold levels with age and auditory brainstem response (ABR), cannot solely be explained by environmental and anatomical factors (Jerger and Hall, 1980; Jerger and Johnson, 1988; Rosenhall et al., 1990; Wharton and Church, 1990; Jo«nsson et al., 1998). In Turner syndrome (45,X), where a lack of estrogens is one of the predominant characteristics, ear and hearing problems are common with both a mid-frequency loss and an early developing presbyacusis (Andersson et al., 1969; Watkin, 1989; Hultcrantz et al., 1994). Also longer ABR latencies are seen among these patients (Gu«ngo«r et al., 2000). In females, the ovaries are the major source of estrogens, but also non-endocrine tissues, such as adipose
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tissue, adrenals and brain, produce estrogens by the aromatization of androgens to estrogens. The latter is also true for males. In Turner syndrome the lack of estrogens is due to underdeveloped ovaries. The physiological actions of estrogens are primarily mediated through the estrogen receptor alpha (ERK) and the newly discovered estrogen receptor beta (ERL) (Kuiper et al., 1996). Both are members of a nuclear receptor super family that regulates gene expression. In addition non-genomic hormone actions exists (Wheling, 1997). Currently it is unclear what biological consequence the two ER subtypes have. It may be an explanation for the selective actions of estrogens in various target tissues and it is known that di¡erent estrogen compounds have di¡erent relative binding a⁄nities for ERK versus ERL. The expression of ERK and ERL varies in di¡erent tissues and also in-between species. Estrogen receptors have been shown in uterus, ovary, prostate, breast, testis, epididymis, kidney, adrenal gland, bladder, brain, pituitary, heart, blood vessels, bone, liver, intestine, lung and thyroid (Kumagami, 1994; Saunders et al., 1997; Kuiper et al., 1998, Taylor and Al-Azzawi, 2000). Presence of ERs in the inner ear has earlier been shown in the inner ear of normal mouse and rat (Stenberg et al., 1999) using immunohistochemistry. The pattern is speci¢c and staining is found especially in tissues that generates hearing (hair cells and ganglion cells) and where the inner ear homeostasis is regulated (stria vascularis). However, in-situ hybridization on rat cochlea has not been able to verify these ¢ndings (Nathan et al., 1999), which could be due to a slow rate of protein turnover, leading to low mRNA levels. Also a lower sensitivity of the probe compared to the antibody is a possible explanation. In humans, ERK has been shown
in the spiral ganglion cells and ERL in the stria vascularis (Stenberg et al., 2001). ERs are also present in the epithelial cells of the endolymphatic sac (Kumagami, H., 1994). The aim of this study was to map ERK and ERL in the inner ear in an animal model a¡ected with Turner syndrome, the ‘Turner mouse’, known to have similar hearing problems as the Turner patients (Hultcrantz et al., 2000) and in a mouse lacking the ERL, the estrogen receptor knockout (LERKO) mouse, in order to further investigate the possible in£uence of estrogen on inner ear and hearing. The Turner mice are smaller than normal and at the time for usually reaching sexual maturity, the Turner mice only have half of the weight of their controls. The inner ears show a swollen stria vascularis and dilated a¡erent nerve endings on the inner hair cells (Hultcrantz et al., 2000). The LERKO mouse develops normally, but a degeneration of cortical neurons increasing with age has been found in the brain (Wang et al., 2001).
2. Materials and methods 2.1. Animals Five Turner mice (X,0) and ¢ve homozygous mutant LERKO mice (ERL 3/3) were used in the present study. The animals were all adult females, aged 2^ 6 months. As controls to the Turner mice served ¢ve normal mice (three NMRI mice and two CBA mice in earlier runs) and to the LERKO mice served one homozygous wild-type (ERL+/+) and two heterozygous (ERL3/+) mice. The mice lacking estrogen receptor L (ERL 3/3)
Table 1 Overview of nuclear staining of inner ear tissues of the di¡erent strains of mice ERK
Organ of Corti
Spiral ganglion Reissner’s membrane Stria vascularis
Spiral ligament Utricle Ampulla Blood vessels
Inner hair cells Outer hair cells Phalangeal cells Pillar cells Type I cells Type II cells Marginal cells Intermediate cells Basal cells Sensory cells Sensory cells Endothelium Leukocytes
ERL
CBA
X,0
+/+
3/3
CBA
X,0
+/+
3/3
++ + ++ ++ ++ + ++ ++ 3 ++ + ++ ++ + ++
++ + ++ ++ ++ + ++ ++ 3 ++ + ++ ++ + 3
++ + ++ + + + ++ ++ 3 ++ + ++ ++ ++ ++
++ + ++ + + + + ++ 3 ++
++ 3 + 3 + + ++ ++ 3 + 3 3 ++ 3 ++
(+) + + (+) + + ++ (+) 3 + 3 3 (+) 3 ++
(+) 3 + 3 + + ++ 3 3 + + 3 (+) 3 (+)
3/(+) 3 3/(+) 3 3/(+) 3/(+) 3 3 3 3/(+) 3/(+) 3 3/(+) 3 3/(+)
++ ++ ++ ++
3 = no staining, (+) = faint staining, + = moderate staining, ++ = strong staining. CBA = CBA/Ca (control mouse), X,0 = Turner mouse, +/+ = wild-type, 3/3 = homozygous LERKO.
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Fig. 1. Turner mouse. (a) Organ of Corti in a Turner mouse stained with antibodies against ERK. The inner hair cell (arrow), the outer hair cell, the phalangeal cells and the pillar cells are all intensely stained. Bar = 20 Wm. (b) Spiral ganglion in a Turner mouse, labeled with antibodies against ERK. The nuclear staining of the type I (arrow), but also, though fainter, of the type II ganglion cells is clearly visualized. Bar = 20 Wm. (c) Control Turner mouse, where rabbit serum replaced the primary ERK antibody. There is no visible staining in the spiral ganglion cells. Bar = 20 Wm. (d) Staining of a Turner mouse organ of Corti using antibodies against ERL. There is very faint nuclear staining of the phalangeal cells (arrow), while the inner hair cells in this specimen are unstained. Bar = 20 Wm. (e) Utricle from a Turner mouse showing ERL staining in the supporting cells, while the sensory cells are devoid of the brownish positive staining, only showing blue nuclei (white arrow). Bar = 20 Wm. (f) Control Turner mouse utricle where the primary antibody against ERL was replaced with chicken serum. There is no nuclear staining, but some background staining can be seen in the cytoplasm of some cells. Bar = 20 Wm.
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were produced by insertion of a neomycin resistance gene into exon 3 of the coding gene by using homologous recombination in embryonic stem cells (Krege et al., 1998). Screening for correct targeted stem cells was accomplished by using PCR and Southern blot was used to con¢rm targeted stem cells. Male chimeras were mated to the female strain C57BL/6J and the F1 heterozygotes were intercrossed and gave homozygous mutant mice (3/3), which were compared to the homozygous wild-type (+/+), and the heterozygous mice (3/+).
Following primary antibody binding, the sections were incubated with the secondary antibody : a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA, USA) diluted in normal goat serum. The tissue sections were treated as described in Stenberg et al. (1999). Negative controls were obtained by replacing the primary antibody with non-immune serum of the equivalent concentration on inner ear sections. Rat uterus served as positive and negative controls.
2.2. Immunohistochemistry
Tissue sections were incubated with 1:500 dilution of ERL (503) antibody overnight at 4‡C in PBS with 3% bovine serum albumin. After washing, sections were incubated with peroxidase-conjugated rabbit anti-chickº lvsjo«, Sweden) for 1 h en IgG (Dako, Dakopatts AB, A at room temperature. The peroxidase substrate diaminobenzidine was used to visualize the reaction (SK 4100; Vector Laboratories). Thereafter the procedure was as described before (Wang et al., 1999). In addition, a polyclonal rabbit anti-rat antibody (PA1-310, A⁄nity Bioreagents, UK) was also used to detect ERL immunoreactivity in the mouse ear. The results were similar although with a higher background staining, therefore the ERL 503 antibody was used for this study. To obtain negative controls, incubations were done with the absorbed antibody on inner ear tissue. Pig ovaries served as positive and negative controls. A microscope (Leica), connected via a video camera (Sony) to a computer, was used to assess immunostaining photographs. A semi-quanti¢cation of nuclear staining was performed. Classi¢cation into a three-point scale was done by three di¡erent researcher, 3 = no staining, + = moderate staining and ++ = strong staining.
The temporal bones were removed after decapitation. Before immersing in 4% paraformaldehyde for one h, the stapes was removed, the oval and round windows opened, a small fenestra was made in the bony shell of the cochlear apex and the cochlea was perfused. Specimens were decalci¢ed for approximately two weeks in 0.1 M EDTA. When soft enough for sectioning the inner ears were embedded in para⁄n. Sections of 5 Wm were put on glass slides (Superfrost) for further staining. A standard immunohistochemical technique (avidin^ biotin^peroxidase) was used to visualize distribution of immunolocalization of ERK and ERL as previously described (Stenberg et al., 1999). A polyclonal rabbit antihuman antibody was used for detection of ERK (ZS080174, Zymed Laboratories, San Francisco, CA, USA) in the inner ear of the mouse. A polyclonal chicken anti-human ERL (503) antibody was used for ERL immunostaining. The preparation of this antibody is described by Saji et al. (2000). The ERL antibody, preabsorbed with an ERL protein (Panvera, Madison, WI, USA) (1:50 (v/v) overnight at 4‡C), was used to demonstrate antigen speci¢city. The speci¢city of the ERK antibody has earlier been con¢rmed.
2.4. ERL
2.3. ERK
3. Results
The polyclonal rabbit anti-human ERK antibody was further diluted 1:5 in phosphate-bu¡ered saline (PBS) and incubated on the murine sections at 4‡C overnight.
The mutant LERKO mouse develops normally and shows no gross phenotypical di¡erences from the wildtype mouse. The inner ears of the LERKO mice were
C Fig. 2. LERKO mouse. (a) ERK immunostaining of a LERKO mouse, showing strong staining of the spiral ganglion cell nuclei of both the type I and II cells. A cytoplasmic background staining is visible. Bar = 20 Wm. (b) Strong ERK nuclear staining of the supporting cells (black arrow) as well as the sensory cells (white arrow) of the ampulla in a 3/3 LERKO mouse. Bar = 20 Wm. (c) As positive control of ERK immunostaining a rat uterus is shown. The brown positive staining is located in the nuclei of the luminal epithelium (LE) and some of the cells in the stroma (str). Bar = 30 Wm. (d) Negative control of the same specimen as in c, where the primary antibody has been replaced by non-immune rabbit serum. Bar = 30 Wm. (e) ERL immunostaining in the LERKO mouse. In the spiral ganglion in some of the samples, a very faint staining of grayish nuclei in the ganglion cells could be noted. However, most specimens, like this one, appeared negative. Bar = 20 Wm. (f) No clear staining of the organ of Corti in the LERKO mouse is visible. The cell nuclei of hair cells, pillar cells and phalangeal cells are negative (blue). Bar = 20 Wm. (g) Positive staining of ERL in the granulosa cells (GC) of a pig ovary. Bar = 30 Wm. (h) Negative control. Same specimen as in g, except that the primary antibody was replaced with non-immune chicken serum. Bar = 30 Wm.
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normally developed and showed a normal anatomy, however, some swollen a¡erent nerve endings could be detected. The Turner mouse showed, as earlier seen, a swollen stria vascularis and dilated a¡erent nerve endings on the inner hair cells (Hultcrantz et al., 2000). No hearing tests were performed. ERK and ERL nuclear staining was seen in cells in the inner ear, showing a unique distribution pattern (Table 1). The ERs are present in both the cytoplasm and nucleus, but have their action in the nucleus. Therefore, only nuclear staining is considered true.
negative. The cells of the inner ears of LERKO mice were generally very faintly stained, if at all. There was no major di¡erence in the localization of the staining pattern between the wild-type and the heterozygous mice. Labeling was di⁄cult to recognize in the homozygous ERL knockout mouse. The positive control showed strong immunostaining of the granulosa cells in the pig ovary (Fig. 2g). The negative control is shown in Fig. 2h.
4. Discussion 3.1. ERK ERK appeared to be expressed in the inner and outer hair cells (Fig. 1a) as well as in the nuclei of the spiral ganglion with strong staining of the type I and faint staining of the type II cells in both mouse strains (Figs. 1b and 2a). The pillar cells as well as the phalangeal cells in the organ of Corti were positive (Fig. 1a). In the lateral wall, the cell nuclei of mainly the marginal cells of the stria vascularis of all species were stained along with some cell nuclei of the spiral ligament. Some of the cells of Reissner’s membrane were also immunopositive. The utricle and the ampulla (Fig. 2b) in both strains of mice showed a heavy staining of the sensory as well as the supporting cells. The smooth muscle cells of the blood vessels were positively stained. The negative controls showed no staining (Figs. 1c and 2d). The positive control showed strong immunostaining in the luminal epithelium and stroma of the rat uterus (Fig. 2c), the negative control is shown in Fig. 2d. 3.2. ERL The immunolocalization of ERL was not entirely similar to that of ERK. In the Turner mouse ERL was faintly present in the nuclei of the inner hair cells, and faint staining could be detected in the outer hair cells of the second and mainly the third rows (Fig. 1d). The pillar and phalangeal cells of the organ of Corti were stained. In the Turner mouse also the spiral ganglion was faintly immunopositive in the type I and II cells, stronger in type II. Furthermore the marginal and basal cells of the stria vascularis and some cells of Reissner’s membrane were positively stained. The utricle showed staining of the supporting cells while the sensory cells were negative (Fig. 1e; and negative control Fig. 1f). In the ampullas also the supporting cells were stained, however in some sections a few scattered stained sensory cells were noted. In all mice smooth muscle cells of the vessels were
In the present study antibodies against ERK have the same staining pattern in the inner ear of Turner mice as in the normal control mice. However, the Turner mouse generally showed a fainter ERL staining of the inner hair cell of the organ of Corti and not of the sensory cells of the utricle and ampulla, which is a di¡erence compared to the normal mice. This could be due to a downregulation of the receptors by the lack of estrogen. This could also be a reason why Nathan et al. (1999) failed to ¢nd mRNA encoding ER in inner ear tissue, as they used ovariectomized rats. The Turner mouse was generated with a X,0 genetic aberration. The chromosomal pattern is quite like the aberration found in Turner syndrome (Lindsten, 1963), where no or a very low estrogen production is one of the main characteristics. Other typical characteristics are short stature and infertility. Also ear/hearing problems are common. The ear problems are due to middle ear (acute, serous and chronic otitis media) and inner ear (progressive sensorineural dip and premature aging) derangements (Lindsten, 1963; Andersson et al., 1969; Watkin, 1989; Hultcrantz et al., 1994; Stenberg et al., 1998). These problems often require hearing aids. Young girls today receive growth hormone at an early age to augment height and estrogen to induce puberty. The syndrome does not include mental retardation. In a previous morphologic and physiologic study of the Turner mouse results indicated that hearing problems in the Turner mouse seems to be of cochlear origin, with an eighth nerve component (Hultcrantz et al., 2000). Otitis media was found in some of these X,0 animals, a symptom seldom found in control animals. The ABR of the Turner mouse showed a progressive hearing loss in the high frequency region that exceeded the normal age-related hearing loss of control mice. Morphologically a swollen net of synapses was found below the inner hair cells and outer hair cell loss was apparent in the basal turn of Turner mice. The results of the present study, with a normal appearance of ERs, indicate that in the Turner mouse it is not a lack of receptors in the inner ear that causes the inner ear disorders.
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Studying the inner ears of the homozygous knockout (3/3) LERKO mice, swollen synapses were found more frequently. No swollen stria vascularis was seen. In the wild-type (+/+) and heterozygous mice (3/+) mice the inner and middle ears looked normal. The developmental production of estrogen and/or stimulation of the ERK in the inner ear seem to be enough or ERL does not have any impact during maturation of the ear in the LERKO mice. This can be interpreted as if ERL is not essential during ontogeny of the inner ear. An impact on the brain with a survival of neurons has, however, been shown connected to ERL. A recent study of the brain of the LERKO mouse showed that there is a severe neuronal de¢cit in the somatosensory cortex that progresses with age. This is interpreted to indicated that ERL is necessary for neuronal survival (Wang et al., 2001). Whether this has any impact on neurons in the audiological cortex or in the inner ear is not known. How the inner ears look morphologically and functionally with increasing age in the LERKO mouse has not yet been investigated. Why a very faint ERL staining of the cells was found in the ganglion cells and in some supporting cells of the inner ear in the LERKO mouse can be explained by some exons in the receptor not being totally blocked, but still not making the receptor functionally active. In all animals investigated in the present study staining of ERK shows the same distribution pattern in the wild-type, the heterozygous and homozygous LERKO and the Turner mice as compared to the controls. The Turner mouse also showed generally a faint ERL staining of the inner hair cells of the organ of Corti and not of the sensory cells of the utricle and ampulla, which is a di¡erence compared to the normal control strains. The wild-type and heterozygous ERL knockout mice had the same labeling pattern with very faint staining. The inner ear development was normal in spite of the lack of ERL. One can only speculate whether this ER subtype has any function in the inner ear or not. The hearing and inner ear function in the ERL knockout mouse with increasing age has not been tested ; we aim to do this in future studies.
Acknowledgements Grants NR 00720 and S-03972 from the Medical Research Council; Magnus Bergvalls Foundation and Karolinska Institute supported the study.
References Andersson, H., Filipsson, R., Fluur, E., Koch, B., Lindsten, J., We-
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denberg, E., 1969. Hearing impairment in Turner’s syndrome. Acta Otolaryngol. (Stockh.) Suppl. 247. Garcia-Segura, L.M., Azcoitia, I., DonCarlos, L.L., 2001. Neuroprotection by estradiol. Prog. Neurobiol. 63, 29^60. Gu«ngo«r, N., Bo«ke, B., Belgin, E., TuncWbilek, E., 2000. High frequency hearing loss in Ullrich-Turner syndrome. Eur. J. Pediatr. 159, 740^ 744. Hultcrantz, M., Sylve¤n, L., Borg, E., 1994. Ear and hearing problems in 44 middle-aged women with Turner’s syndrome. Hear. Res. 76, 127^132. Hultcrantz, M., Stenberg, A.E., Fransson, A., Canlon, B., 2000. Characterization of hearing in a X/0 ‘Turner mouse’. Hear. Res. 143, 182^188. Jerger, J., Hall, J., 1980. E¡ects of age and sex on auditory brainstem response. Arch. Otolaryngol. 106, 387^391. Jerger, J., Johnson, K., 1988. Interactions of age, gender, and sensorineural hearing loss on ABR latency. Ear Hear. 9, 168^176. Jo«nsson, R., Rosenhall, U., Gause-Nilsson, I., Steen, B., 1998. Auditory function in 70-and 75-year-olds of four age cohorts. Scand. Audiol. 27, 81^93. Krege, J.H., Hodgin, J.B., Couse, J.F., Enmark, E., Warner, M. et al., 1998. Generation and reproductive phenotypes of mice lacking estrogen receptor L. Proc. Natl. Acad. Sci. USA 95, 15677^15682. Kuiper, G.G.J.M., Enmark, E., Pelto-Huikko, M., Nilsson, S., GusY ., 1996. Cloning of a novel estrogen receptor extafsson, J.-A pressed in rat prostate and ovary. Proc. Natl. Acad. Sci. USA 93, 5925^5930. Kuiper, G.G.J.M., Shughrue, P.J., Merchenthaler, I., Gustavsson, Y ., 1998. the estrogen receptor L subtype: A novel mediator J.-A of estrogen action in neuroendocrine systems. Front. Neuroendocrinol. 19, 253^285. Kumagami, H., 1994. Sex hormones in the kidney and in the endolymphatic sac. An immunohistological study. Acta Otorhinolaryngol. (Stockh.) 114, 48^51. Lindsten, J., 1963. The Nature and Origin of X Chromosome Aberrations in Turner’s Syndrome. Thesis, Almqvist and Wiksell, Stockholm. Losordo, D.W., Kearney, M., Kim, E.A., Jekanowski, J., Isner, J.M., 1994. Variable expression of the estrogen receptor in normal and atherosclerotic coronary arteries of premenstrual women. Circulation 89, 1501^1510. Nathan, C.-H.O., Kim, T.S., Harris, J.P., Koutnouyan, H.A., Ryan, A.F., 1999. Absence of mRNA encoding estrogen receptor in the rat cochlea. Acta Otolaryngol. (Stockh.) 119, 853^857. Y ., 2001. Role of estrogen receptor beta Pettersson, K., Gustafsson, J.-A in estrogen action. Annu. Rev. Physiol. 63, 165^192. Rosenhall, U., Pedersen, K., Svanborg, A., 1990. Presbycusis and noise-induced hearing loss. Ear Hear. 11, 257^263. Saji, S., Jensen, E.V., Nilsson, S., Rylander, T., Warner, M., Gustafsson, J.A., 2000. Estrogen receptors alpha and beta in the rodent mammary gland. Proc. Natl. Acad. Sci. USA 97, 337^342. Saunders, P.T.K., Maguire, S.M., Gaughan, J., Millar, M.R., 1997. Expression of oestrogen receptor L (ER L) in multiple rat tissue visualized by immunohistochemistry. J. Endocrinol. 154, R13^ R16. Stenberg, A.E., Nyle¤n, O., Windh, M., Hultcrantz, M., 1998. Otological problems in children with Turner’s syndrome. Hear. Res. 124, 85^90. Stenberg, A.E., Wang, H., Fish, J., Schrott-Fisher, A.-L., Sahlin, L., Hultcrantz, M., 2001. Estrogen receptors in the normal adult and developing human inner ear and in Turner’s syndrome. Hear. Res. 157, 87^92. Stenberg, A.E.., Wang, H., Sahlin, L., Hultcrantz, M., 1999. Mapping of estrogen receptors K and L in the inner ear of the mouse and rat. Hear. Res. 136, 29^34.
HEARES 3850 29-5-02 Cyaan Magenta Geel Zwart
8
A.E. Stenberg et al. / Hearing Research 166 (2002) 1^8
Taylor, A.H., Al-Azzawi, F., 2000. Immunolocalisation of oestrogen receptor beta in human tissues. J. Mol. Endocrinol. 24, 145^155. Turner, R.T., Riggs, B.L., Spelsberg, T.C., 1994. Skeletal e¡ects of estrogen. Endocr. Rev. 15, 275^296. Wang, H., Masironi, B., Eriksson, H., Sahlin, L., 1999. A comparative study of estrogen receptors K and L in the rat uterus. Biol. Reprod. 61, 955^964. Y ., 2001. MorWang, L., Andersson, S., Warner, M., Gustafsson, J.-A
phological abnormalities in the brains of estrogen receptor beta knockout mice. Proc. Natl. Acad. Sci. USA 98, 2792^2796. Watkin, P., 1989. Otological disease in Turner’s syndrome. J. Laryngol. Otol. 103, 731^738. Wharton, J.A., Church, G.T., 1990. In£uence of menopause on the auditory brainstem response. Audiology 29, 196^201. Wheling, M., 1997. Speci¢c, nongenomic actions of steroid hormones. Annu. Rev. Physiol. 59, 365^393.
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Estrogen receptors K and L in the inner ear of the ‘Turner mouse’ and an estrogen receptor L knockout mouse A.E. Stenberg a , H. Wang b , L. Sahlin b , P. Stierna c , E. Enmark c , M. Hultcrantz a
a;
Department of Otorhinolaryngology, Karolinska Hospital, 171 76 Stockholm, Sweden Department of Woman and Child Health, Karolinska Hospital, Stockholm, Sweden Department of Medical Nutrition, Karolinska Institutet, NOVUM, Huddinge, Sweden
b c
Received 22 March 2001; accepted 21 November 2001
Abstract Estrogen receptors have earlier been shown in the normal mouse, rat and human inner ear. If estrogens are important in normal hearing and development of presbyacusis in the normal population is not known. However it is known that patients with Turner syndrome, where a lack of estrogens is one of the main characteristics, commonly develop an early presbyacusis. A ‘Turner mouse’ has been developed, as a model for the ear problems in Turner syndrome, and it shows otitis media and a premature aging of the hearing. Estrogen receptors exist in an K and a L form. In this study inner ear tissue, from the Turner mouse and an estrogen receptor L knockout mouse (LERKO), was investigated regarding estrogen receptor K and L using immunohistochemistry. Results show that the Turner mouse has the same pattern of inner ear labeling, both concerning the estrogen receptor K and L, as that of a normal CBA/Ca mouse, with positive staining in the organ of Corti and spiral ganglion. The LERKO mice show close to normal inner ear morphology and positive estrogen receptor K immunostaining at the same locations as the CBA/Ca mouse. 4 2002 Elsevier Science B.V. All rights reserved. Key words: Turner syndrome; Estrogen receptor; Mouse; Estrogen receptor L knockout mouse; Inner ear; Immunohistochemistry
1. Introduction Estrogen - the female sex hormone - is classically known to in£uence growth, di¡erentiation and function of the female reproductive tract. However, the target ¢eld of estrogen action has been shown to be much wider (for review see Pettersson and Gustafsson, 2001). It includes male regulation of sexual behavior and normal function of the testis and prostate. Moreover, protective e¡ects have been observed on the maintenance of the skeleton, on cardiovascular system and
* Corresponding author. Tel.: +46 (8) 51775653; Fax: +46 (8) 51776267. E-mail address: [email protected] (M. Hultcrantz). Abbreviations: ABR, auditory brainstem response; LERKO, estrogen receptor L knockout mouse; ER, estrogen receptor
in the brain (Losordo et al., 1994; Turner et al., 1994, Garcia-Segura et al., 2001). There are suggestions that estrogens may have an e¡ect on the ear and hearing, but the association is still poorly understood. The well-known gender di¡erence in hearing, both pure tone threshold levels with age and auditory brainstem response (ABR), cannot solely be explained by environmental and anatomical factors (Jerger and Hall, 1980; Jerger and Johnson, 1988; Rosenhall et al., 1990; Wharton and Church, 1990; Jo«nsson et al., 1998). In Turner syndrome (45,X), where a lack of estrogens is one of the predominant characteristics, ear and hearing problems are common with both a mid-frequency loss and an early developing presbyacusis (Andersson et al., 1969; Watkin, 1989; Hultcrantz et al., 1994). Also longer ABR latencies are seen among these patients (Gu«ngo«r et al., 2000). In females, the ovaries are the major source of estrogens, but also non-endocrine tissues, such as adipose
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tissue, adrenals and brain, produce estrogens by the aromatization of androgens to estrogens. The latter is also true for males. In Turner syndrome the lack of estrogens is due to underdeveloped ovaries. The physiological actions of estrogens are primarily mediated through the estrogen receptor alpha (ERK) and the newly discovered estrogen receptor beta (ERL) (Kuiper et al., 1996). Both are members of a nuclear receptor super family that regulates gene expression. In addition non-genomic hormone actions exists (Wheling, 1997). Currently it is unclear what biological consequence the two ER subtypes have. It may be an explanation for the selective actions of estrogens in various target tissues and it is known that di¡erent estrogen compounds have di¡erent relative binding a⁄nities for ERK versus ERL. The expression of ERK and ERL varies in di¡erent tissues and also in-between species. Estrogen receptors have been shown in uterus, ovary, prostate, breast, testis, epididymis, kidney, adrenal gland, bladder, brain, pituitary, heart, blood vessels, bone, liver, intestine, lung and thyroid (Kumagami, 1994; Saunders et al., 1997; Kuiper et al., 1998, Taylor and Al-Azzawi, 2000). Presence of ERs in the inner ear has earlier been shown in the inner ear of normal mouse and rat (Stenberg et al., 1999) using immunohistochemistry. The pattern is speci¢c and staining is found especially in tissues that generates hearing (hair cells and ganglion cells) and where the inner ear homeostasis is regulated (stria vascularis). However, in-situ hybridization on rat cochlea has not been able to verify these ¢ndings (Nathan et al., 1999), which could be due to a slow rate of protein turnover, leading to low mRNA levels. Also a lower sensitivity of the probe compared to the antibody is a possible explanation. In humans, ERK has been shown
in the spiral ganglion cells and ERL in the stria vascularis (Stenberg et al., 2001). ERs are also present in the epithelial cells of the endolymphatic sac (Kumagami, H., 1994). The aim of this study was to map ERK and ERL in the inner ear in an animal model a¡ected with Turner syndrome, the ‘Turner mouse’, known to have similar hearing problems as the Turner patients (Hultcrantz et al., 2000) and in a mouse lacking the ERL, the estrogen receptor knockout (LERKO) mouse, in order to further investigate the possible in£uence of estrogen on inner ear and hearing. The Turner mice are smaller than normal and at the time for usually reaching sexual maturity, the Turner mice only have half of the weight of their controls. The inner ears show a swollen stria vascularis and dilated a¡erent nerve endings on the inner hair cells (Hultcrantz et al., 2000). The LERKO mouse develops normally, but a degeneration of cortical neurons increasing with age has been found in the brain (Wang et al., 2001).
2. Materials and methods 2.1. Animals Five Turner mice (X,0) and ¢ve homozygous mutant LERKO mice (ERL 3/3) were used in the present study. The animals were all adult females, aged 2^ 6 months. As controls to the Turner mice served ¢ve normal mice (three NMRI mice and two CBA mice in earlier runs) and to the LERKO mice served one homozygous wild-type (ERL+/+) and two heterozygous (ERL3/+) mice. The mice lacking estrogen receptor L (ERL 3/3)
Table 1 Overview of nuclear staining of inner ear tissues of the di¡erent strains of mice ERK
Organ of Corti
Spiral ganglion Reissner’s membrane Stria vascularis
Spiral ligament Utricle Ampulla Blood vessels
Inner hair cells Outer hair cells Phalangeal cells Pillar cells Type I cells Type II cells Marginal cells Intermediate cells Basal cells Sensory cells Sensory cells Endothelium Leukocytes
ERL
CBA
X,0
+/+
3/3
CBA
X,0
+/+
3/3
++ + ++ ++ ++ + ++ ++ 3 ++ + ++ ++ + ++
++ + ++ ++ ++ + ++ ++ 3 ++ + ++ ++ + 3
++ + ++ + + + ++ ++ 3 ++ + ++ ++ ++ ++
++ + ++ + + + + ++ 3 ++
++ 3 + 3 + + ++ ++ 3 + 3 3 ++ 3 ++
(+) + + (+) + + ++ (+) 3 + 3 3 (+) 3 ++
(+) 3 + 3 + + ++ 3 3 + + 3 (+) 3 (+)
3/(+) 3 3/(+) 3 3/(+) 3/(+) 3 3 3 3/(+) 3/(+) 3 3/(+) 3 3/(+)
++ ++ ++ ++
3 = no staining, (+) = faint staining, + = moderate staining, ++ = strong staining. CBA = CBA/Ca (control mouse), X,0 = Turner mouse, +/+ = wild-type, 3/3 = homozygous LERKO.
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Fig. 1. Turner mouse. (a) Organ of Corti in a Turner mouse stained with antibodies against ERK. The inner hair cell (arrow), the outer hair cell, the phalangeal cells and the pillar cells are all intensely stained. Bar = 20 Wm. (b) Spiral ganglion in a Turner mouse, labeled with antibodies against ERK. The nuclear staining of the type I (arrow), but also, though fainter, of the type II ganglion cells is clearly visualized. Bar = 20 Wm. (c) Control Turner mouse, where rabbit serum replaced the primary ERK antibody. There is no visible staining in the spiral ganglion cells. Bar = 20 Wm. (d) Staining of a Turner mouse organ of Corti using antibodies against ERL. There is very faint nuclear staining of the phalangeal cells (arrow), while the inner hair cells in this specimen are unstained. Bar = 20 Wm. (e) Utricle from a Turner mouse showing ERL staining in the supporting cells, while the sensory cells are devoid of the brownish positive staining, only showing blue nuclei (white arrow). Bar = 20 Wm. (f) Control Turner mouse utricle where the primary antibody against ERL was replaced with chicken serum. There is no nuclear staining, but some background staining can be seen in the cytoplasm of some cells. Bar = 20 Wm.
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were produced by insertion of a neomycin resistance gene into exon 3 of the coding gene by using homologous recombination in embryonic stem cells (Krege et al., 1998). Screening for correct targeted stem cells was accomplished by using PCR and Southern blot was used to con¢rm targeted stem cells. Male chimeras were mated to the female strain C57BL/6J and the F1 heterozygotes were intercrossed and gave homozygous mutant mice (3/3), which were compared to the homozygous wild-type (+/+), and the heterozygous mice (3/+).
Following primary antibody binding, the sections were incubated with the secondary antibody : a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA, USA) diluted in normal goat serum. The tissue sections were treated as described in Stenberg et al. (1999). Negative controls were obtained by replacing the primary antibody with non-immune serum of the equivalent concentration on inner ear sections. Rat uterus served as positive and negative controls.
2.2. Immunohistochemistry
Tissue sections were incubated with 1:500 dilution of ERL (503) antibody overnight at 4‡C in PBS with 3% bovine serum albumin. After washing, sections were incubated with peroxidase-conjugated rabbit anti-chickº lvsjo«, Sweden) for 1 h en IgG (Dako, Dakopatts AB, A at room temperature. The peroxidase substrate diaminobenzidine was used to visualize the reaction (SK 4100; Vector Laboratories). Thereafter the procedure was as described before (Wang et al., 1999). In addition, a polyclonal rabbit anti-rat antibody (PA1-310, A⁄nity Bioreagents, UK) was also used to detect ERL immunoreactivity in the mouse ear. The results were similar although with a higher background staining, therefore the ERL 503 antibody was used for this study. To obtain negative controls, incubations were done with the absorbed antibody on inner ear tissue. Pig ovaries served as positive and negative controls. A microscope (Leica), connected via a video camera (Sony) to a computer, was used to assess immunostaining photographs. A semi-quanti¢cation of nuclear staining was performed. Classi¢cation into a three-point scale was done by three di¡erent researcher, 3 = no staining, + = moderate staining and ++ = strong staining.
The temporal bones were removed after decapitation. Before immersing in 4% paraformaldehyde for one h, the stapes was removed, the oval and round windows opened, a small fenestra was made in the bony shell of the cochlear apex and the cochlea was perfused. Specimens were decalci¢ed for approximately two weeks in 0.1 M EDTA. When soft enough for sectioning the inner ears were embedded in para⁄n. Sections of 5 Wm were put on glass slides (Superfrost) for further staining. A standard immunohistochemical technique (avidin^ biotin^peroxidase) was used to visualize distribution of immunolocalization of ERK and ERL as previously described (Stenberg et al., 1999). A polyclonal rabbit antihuman antibody was used for detection of ERK (ZS080174, Zymed Laboratories, San Francisco, CA, USA) in the inner ear of the mouse. A polyclonal chicken anti-human ERL (503) antibody was used for ERL immunostaining. The preparation of this antibody is described by Saji et al. (2000). The ERL antibody, preabsorbed with an ERL protein (Panvera, Madison, WI, USA) (1:50 (v/v) overnight at 4‡C), was used to demonstrate antigen speci¢city. The speci¢city of the ERK antibody has earlier been con¢rmed.
2.4. ERL
2.3. ERK
3. Results
The polyclonal rabbit anti-human ERK antibody was further diluted 1:5 in phosphate-bu¡ered saline (PBS) and incubated on the murine sections at 4‡C overnight.
The mutant LERKO mouse develops normally and shows no gross phenotypical di¡erences from the wildtype mouse. The inner ears of the LERKO mice were
C Fig. 2. LERKO mouse. (a) ERK immunostaining of a LERKO mouse, showing strong staining of the spiral ganglion cell nuclei of both the type I and II cells. A cytoplasmic background staining is visible. Bar = 20 Wm. (b) Strong ERK nuclear staining of the supporting cells (black arrow) as well as the sensory cells (white arrow) of the ampulla in a 3/3 LERKO mouse. Bar = 20 Wm. (c) As positive control of ERK immunostaining a rat uterus is shown. The brown positive staining is located in the nuclei of the luminal epithelium (LE) and some of the cells in the stroma (str). Bar = 30 Wm. (d) Negative control of the same specimen as in c, where the primary antibody has been replaced by non-immune rabbit serum. Bar = 30 Wm. (e) ERL immunostaining in the LERKO mouse. In the spiral ganglion in some of the samples, a very faint staining of grayish nuclei in the ganglion cells could be noted. However, most specimens, like this one, appeared negative. Bar = 20 Wm. (f) No clear staining of the organ of Corti in the LERKO mouse is visible. The cell nuclei of hair cells, pillar cells and phalangeal cells are negative (blue). Bar = 20 Wm. (g) Positive staining of ERL in the granulosa cells (GC) of a pig ovary. Bar = 30 Wm. (h) Negative control. Same specimen as in g, except that the primary antibody was replaced with non-immune chicken serum. Bar = 30 Wm.
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normally developed and showed a normal anatomy, however, some swollen a¡erent nerve endings could be detected. The Turner mouse showed, as earlier seen, a swollen stria vascularis and dilated a¡erent nerve endings on the inner hair cells (Hultcrantz et al., 2000). No hearing tests were performed. ERK and ERL nuclear staining was seen in cells in the inner ear, showing a unique distribution pattern (Table 1). The ERs are present in both the cytoplasm and nucleus, but have their action in the nucleus. Therefore, only nuclear staining is considered true.
negative. The cells of the inner ears of LERKO mice were generally very faintly stained, if at all. There was no major di¡erence in the localization of the staining pattern between the wild-type and the heterozygous mice. Labeling was di⁄cult to recognize in the homozygous ERL knockout mouse. The positive control showed strong immunostaining of the granulosa cells in the pig ovary (Fig. 2g). The negative control is shown in Fig. 2h.
4. Discussion 3.1. ERK ERK appeared to be expressed in the inner and outer hair cells (Fig. 1a) as well as in the nuclei of the spiral ganglion with strong staining of the type I and faint staining of the type II cells in both mouse strains (Figs. 1b and 2a). The pillar cells as well as the phalangeal cells in the organ of Corti were positive (Fig. 1a). In the lateral wall, the cell nuclei of mainly the marginal cells of the stria vascularis of all species were stained along with some cell nuclei of the spiral ligament. Some of the cells of Reissner’s membrane were also immunopositive. The utricle and the ampulla (Fig. 2b) in both strains of mice showed a heavy staining of the sensory as well as the supporting cells. The smooth muscle cells of the blood vessels were positively stained. The negative controls showed no staining (Figs. 1c and 2d). The positive control showed strong immunostaining in the luminal epithelium and stroma of the rat uterus (Fig. 2c), the negative control is shown in Fig. 2d. 3.2. ERL The immunolocalization of ERL was not entirely similar to that of ERK. In the Turner mouse ERL was faintly present in the nuclei of the inner hair cells, and faint staining could be detected in the outer hair cells of the second and mainly the third rows (Fig. 1d). The pillar and phalangeal cells of the organ of Corti were stained. In the Turner mouse also the spiral ganglion was faintly immunopositive in the type I and II cells, stronger in type II. Furthermore the marginal and basal cells of the stria vascularis and some cells of Reissner’s membrane were positively stained. The utricle showed staining of the supporting cells while the sensory cells were negative (Fig. 1e; and negative control Fig. 1f). In the ampullas also the supporting cells were stained, however in some sections a few scattered stained sensory cells were noted. In all mice smooth muscle cells of the vessels were
In the present study antibodies against ERK have the same staining pattern in the inner ear of Turner mice as in the normal control mice. However, the Turner mouse generally showed a fainter ERL staining of the inner hair cell of the organ of Corti and not of the sensory cells of the utricle and ampulla, which is a di¡erence compared to the normal mice. This could be due to a downregulation of the receptors by the lack of estrogen. This could also be a reason why Nathan et al. (1999) failed to ¢nd mRNA encoding ER in inner ear tissue, as they used ovariectomized rats. The Turner mouse was generated with a X,0 genetic aberration. The chromosomal pattern is quite like the aberration found in Turner syndrome (Lindsten, 1963), where no or a very low estrogen production is one of the main characteristics. Other typical characteristics are short stature and infertility. Also ear/hearing problems are common. The ear problems are due to middle ear (acute, serous and chronic otitis media) and inner ear (progressive sensorineural dip and premature aging) derangements (Lindsten, 1963; Andersson et al., 1969; Watkin, 1989; Hultcrantz et al., 1994; Stenberg et al., 1998). These problems often require hearing aids. Young girls today receive growth hormone at an early age to augment height and estrogen to induce puberty. The syndrome does not include mental retardation. In a previous morphologic and physiologic study of the Turner mouse results indicated that hearing problems in the Turner mouse seems to be of cochlear origin, with an eighth nerve component (Hultcrantz et al., 2000). Otitis media was found in some of these X,0 animals, a symptom seldom found in control animals. The ABR of the Turner mouse showed a progressive hearing loss in the high frequency region that exceeded the normal age-related hearing loss of control mice. Morphologically a swollen net of synapses was found below the inner hair cells and outer hair cell loss was apparent in the basal turn of Turner mice. The results of the present study, with a normal appearance of ERs, indicate that in the Turner mouse it is not a lack of receptors in the inner ear that causes the inner ear disorders.
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Studying the inner ears of the homozygous knockout (3/3) LERKO mice, swollen synapses were found more frequently. No swollen stria vascularis was seen. In the wild-type (+/+) and heterozygous mice (3/+) mice the inner and middle ears looked normal. The developmental production of estrogen and/or stimulation of the ERK in the inner ear seem to be enough or ERL does not have any impact during maturation of the ear in the LERKO mice. This can be interpreted as if ERL is not essential during ontogeny of the inner ear. An impact on the brain with a survival of neurons has, however, been shown connected to ERL. A recent study of the brain of the LERKO mouse showed that there is a severe neuronal de¢cit in the somatosensory cortex that progresses with age. This is interpreted to indicated that ERL is necessary for neuronal survival (Wang et al., 2001). Whether this has any impact on neurons in the audiological cortex or in the inner ear is not known. How the inner ears look morphologically and functionally with increasing age in the LERKO mouse has not yet been investigated. Why a very faint ERL staining of the cells was found in the ganglion cells and in some supporting cells of the inner ear in the LERKO mouse can be explained by some exons in the receptor not being totally blocked, but still not making the receptor functionally active. In all animals investigated in the present study staining of ERK shows the same distribution pattern in the wild-type, the heterozygous and homozygous LERKO and the Turner mice as compared to the controls. The Turner mouse also showed generally a faint ERL staining of the inner hair cells of the organ of Corti and not of the sensory cells of the utricle and ampulla, which is a di¡erence compared to the normal control strains. The wild-type and heterozygous ERL knockout mice had the same labeling pattern with very faint staining. The inner ear development was normal in spite of the lack of ERL. One can only speculate whether this ER subtype has any function in the inner ear or not. The hearing and inner ear function in the ERL knockout mouse with increasing age has not been tested ; we aim to do this in future studies.
Acknowledgements Grants NR 00720 and S-03972 from the Medical Research Council; Magnus Bergvalls Foundation and Karolinska Institute supported the study.
References Andersson, H., Filipsson, R., Fluur, E., Koch, B., Lindsten, J., We-
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denberg, E., 1969. Hearing impairment in Turner’s syndrome. Acta Otolaryngol. (Stockh.) Suppl. 247. Garcia-Segura, L.M., Azcoitia, I., DonCarlos, L.L., 2001. Neuroprotection by estradiol. Prog. Neurobiol. 63, 29^60. Gu«ngo«r, N., Bo«ke, B., Belgin, E., TuncWbilek, E., 2000. High frequency hearing loss in Ullrich-Turner syndrome. Eur. J. Pediatr. 159, 740^ 744. Hultcrantz, M., Sylve¤n, L., Borg, E., 1994. Ear and hearing problems in 44 middle-aged women with Turner’s syndrome. Hear. Res. 76, 127^132. Hultcrantz, M., Stenberg, A.E., Fransson, A., Canlon, B., 2000. Characterization of hearing in a X/0 ‘Turner mouse’. Hear. Res. 143, 182^188. Jerger, J., Hall, J., 1980. E¡ects of age and sex on auditory brainstem response. Arch. Otolaryngol. 106, 387^391. Jerger, J., Johnson, K., 1988. Interactions of age, gender, and sensorineural hearing loss on ABR latency. Ear Hear. 9, 168^176. Jo«nsson, R., Rosenhall, U., Gause-Nilsson, I., Steen, B., 1998. Auditory function in 70-and 75-year-olds of four age cohorts. Scand. Audiol. 27, 81^93. Krege, J.H., Hodgin, J.B., Couse, J.F., Enmark, E., Warner, M. et al., 1998. Generation and reproductive phenotypes of mice lacking estrogen receptor L. Proc. Natl. Acad. Sci. USA 95, 15677^15682. Kuiper, G.G.J.M., Enmark, E., Pelto-Huikko, M., Nilsson, S., GusY ., 1996. Cloning of a novel estrogen receptor extafsson, J.-A pressed in rat prostate and ovary. Proc. Natl. Acad. Sci. USA 93, 5925^5930. Kuiper, G.G.J.M., Shughrue, P.J., Merchenthaler, I., Gustavsson, Y ., 1998. the estrogen receptor L subtype: A novel mediator J.-A of estrogen action in neuroendocrine systems. Front. Neuroendocrinol. 19, 253^285. Kumagami, H., 1994. Sex hormones in the kidney and in the endolymphatic sac. An immunohistological study. Acta Otorhinolaryngol. (Stockh.) 114, 48^51. Lindsten, J., 1963. The Nature and Origin of X Chromosome Aberrations in Turner’s Syndrome. Thesis, Almqvist and Wiksell, Stockholm. Losordo, D.W., Kearney, M., Kim, E.A., Jekanowski, J., Isner, J.M., 1994. Variable expression of the estrogen receptor in normal and atherosclerotic coronary arteries of premenstrual women. Circulation 89, 1501^1510. Nathan, C.-H.O., Kim, T.S., Harris, J.P., Koutnouyan, H.A., Ryan, A.F., 1999. Absence of mRNA encoding estrogen receptor in the rat cochlea. Acta Otolaryngol. (Stockh.) 119, 853^857. Y ., 2001. Role of estrogen receptor beta Pettersson, K., Gustafsson, J.-A in estrogen action. Annu. Rev. Physiol. 63, 165^192. Rosenhall, U., Pedersen, K., Svanborg, A., 1990. Presbycusis and noise-induced hearing loss. Ear Hear. 11, 257^263. Saji, S., Jensen, E.V., Nilsson, S., Rylander, T., Warner, M., Gustafsson, J.A., 2000. Estrogen receptors alpha and beta in the rodent mammary gland. Proc. Natl. Acad. Sci. USA 97, 337^342. Saunders, P.T.K., Maguire, S.M., Gaughan, J., Millar, M.R., 1997. Expression of oestrogen receptor L (ER L) in multiple rat tissue visualized by immunohistochemistry. J. Endocrinol. 154, R13^ R16. Stenberg, A.E., Nyle¤n, O., Windh, M., Hultcrantz, M., 1998. Otological problems in children with Turner’s syndrome. Hear. Res. 124, 85^90. Stenberg, A.E., Wang, H., Fish, J., Schrott-Fisher, A.-L., Sahlin, L., Hultcrantz, M., 2001. Estrogen receptors in the normal adult and developing human inner ear and in Turner’s syndrome. Hear. Res. 157, 87^92. Stenberg, A.E.., Wang, H., Sahlin, L., Hultcrantz, M., 1999. Mapping of estrogen receptors K and L in the inner ear of the mouse and rat. Hear. Res. 136, 29^34.
HEARES 3850 29-5-02 Cyaan Magenta Geel Zwart
8
A.E. Stenberg et al. / Hearing Research 166 (2002) 1^8
Taylor, A.H., Al-Azzawi, F., 2000. Immunolocalisation of oestrogen receptor beta in human tissues. J. Mol. Endocrinol. 24, 145^155. Turner, R.T., Riggs, B.L., Spelsberg, T.C., 1994. Skeletal e¡ects of estrogen. Endocr. Rev. 15, 275^296. Wang, H., Masironi, B., Eriksson, H., Sahlin, L., 1999. A comparative study of estrogen receptors K and L in the rat uterus. Biol. Reprod. 61, 955^964. Y ., 2001. MorWang, L., Andersson, S., Warner, M., Gustafsson, J.-A
phological abnormalities in the brains of estrogen receptor beta knockout mice. Proc. Natl. Acad. Sci. USA 98, 2792^2796. Watkin, P., 1989. Otological disease in Turner’s syndrome. J. Laryngol. Otol. 103, 731^738. Wharton, J.A., Church, G.T., 1990. In£uence of menopause on the auditory brainstem response. Audiology 29, 196^201. Wheling, M., 1997. Speci¢c, nongenomic actions of steroid hormones. Annu. Rev. Physiol. 59, 365^393.
HEARES 3850 29-5-02 Cyaan Magenta Geel Zwart