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Evidence for estrogen receptor expression during medullary bone formation and resorption in estrogen-treated male Japanese quails (Coturnix coturnix japonica).
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PMID:  23000578     Owner:  NLM     Status:  MEDLINE    
The temporal expression of estrogen receptor (ER)-α and ER-β mRNA was examined in male Japanese quails. Femurs of quails receiving 17β-estradiol underwent RT-PCR and histochemical analysis 1 to 15 days after treatment. Untreated quails were used as controls (day 0). Between days 0 and 5, cells lining the bone endosteal surface differentiated into osteoblasts, which in turn formed medullary bone. Expression of ER-α was already observed on day 0 and increased slightly during bone formation whereas ER-β was hardly detected throughout this process. After osteoclasts appeared on the medullary bone surface, this type of bone disappeared from the bone marrow cavity (days 7˜15). ER-α expression simultaneously decreased slightly and ER-β levels remained very low. These results suggest that estrogen activity mediated by ER-α not only affects medullary bone formation but also bone resorption.
Shinji Hiyama; Toshie Sugiyama; Seiji Kusuhara; Takashi Uchida
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of veterinary science     Volume:  13     ISSN:  1976-555X     ISO Abbreviation:  J. Vet. Sci.     Publication Date:  2012 Sep 
Date Detail:
Created Date:  2012-09-24     Completed Date:  2013-02-19     Revised Date:  2013-07-11    
Medline Journal Info:
Nlm Unique ID:  100964185     Medline TA:  J Vet Sci     Country:  Korea (South)    
Other Details:
Languages:  eng     Pagination:  223-7     Citation Subset:  IM    
Department of Oral Biology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8553, Japan.
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MeSH Terms
Bone Resorption / genetics
Bone and Bones / chemistry,  cytology,  metabolism*
Cells, Cultured
Coturnix / metabolism*
Estradiol / pharmacology*
Estrogen Receptor alpha / genetics,  metabolism*
Estrogen Receptor beta / genetics,  metabolism*
Gene Expression Regulation
Osteoblasts / chemistry,  cytology,  metabolism*
Osteogenesis / genetics
RNA, Messenger / metabolism
Reverse Transcriptase Polymerase Chain Reaction
Reg. No./Substance:
0/Estrogen Receptor alpha; 0/Estrogen Receptor beta; 0/RNA, Messenger; 50-28-2/Estradiol

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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Journal Information
Journal ID (nlm-ta): J Vet Sci
Journal ID (iso-abbrev): J. Vet. Sci
Journal ID (publisher-id): JVS
ISSN: 1229-845X
ISSN: 1976-555X
Publisher: The Korean Society of Veterinary Science
Article Information
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Copyright © 2012 The Korean Society of Veterinary Science
Received Day: 23 Month: 8 Year: 2011
Accepted Day: 17 Month: 3 Year: 2012
Print publication date: Month: 9 Year: 2012
Electronic publication date: Day: 20 Month: 9 Year: 2012
Volume: 13 Issue: 3
First Page: 223 Last Page: 227
PubMed Id: 23000578
ID: 3467396
DOI: 10.4142/jvs.2012.13.3.223

Evidence for estrogen receptor expression during medullary bone formation and resorption in estrogen-treated male Japanese quails (Coturnix coturnix japonica)
Shinji Hiyama1
Toshie Sugiyama2
Seiji Kusuhara2
Takashi Uchida1
1Department of Oral Biology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8553, Japan.
2Department of Animal Science, Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan.
Correspondence: Corresponding author: Tel: +81-82-257-5624; Fax: +81-82-257-5689;


The lack of estrogen is known to be a causative factor of osteoporosis [14]. It has therefore been suggested that estrogen is related to the differentiation and/or activities of bone cells, and many studies over the last few decades have investigated the relationships between bone metabolism and estrogen. Investigations have been performed in vivo using estrogen-injected [5,25] and ovariectomized animals [1,29], and the effects of estrogen on the differentiation and/or proliferation of osteoblasts and/or osteoclasts have been examined in vitro [11,22]. In addition, the expression of estrogen receptors (ERs) in bone cells has been evaluated both in vivo [7,30] and in vitro [17,26]. In the 1980s, a novel ER subtype, ER-β, was cloned in addition to the previously identified ER-α [9,31]. This discovery further complicated the relationship between bone metabolism and estrogen.

Formation and resorption of medullary bone in female birds is under the control of circulating estrogen [3,21]. Medullary bone can also be formed in the bone marrow cavity of male birds by estrogen administration [10,12]. ERs have been found in osteoblasts, bone-lining cells, and bone marrow cells in these experimental animal models [15,16]. As found in a previous study, ER-α mRNA, but not ER-β mRNA, is expressed at constant levels throughout the differentiation of osteogenic cells isolated from medullary bone [6]. Although these experiments were performed during the bone formation period, expression of ER-α and/or ER-β during bone resorption has not been assessed. In the present study, we analyzed the temporal expression of ER-α and ER-β mRNA during medullary bone formation and resorption in estrogen-treated male Japanese quails using reverse transcription-polymerase chain reaction (RT-PCR).

Materials and Methods

Male Japanese quails (Coturnix coturnix japonica; Quail Cosmos, Japan) 8~9 weeks old were used in our study. 17β-estradiol (E2) (Progynon-Depot, 20 mg/kg; Fuji Pharma, Japan) was injected into the breast muscle at 1, 2, 3, 5, 7, 10, and 15 days before the femurs were removed (n = 3~4). Control birds (day 0) were not treated with E2. All animal experiments were carried out in strict accordance with the Institutional Guidelines of the Committee of Research Facilities for Laboratory Animal Science, Hiroshima University, Japan.

Histological analysis

E2-treated and untreated (control) quails were sacrificed by decapitation and exsanguination. The right femurs were cut at the center of the diaphyses and fixed in 3.7% paraformaldehyde in phosphate buffered saline (PBS) (pH 7.4) at 4℃ for 5~7 days. The bone samples were then decalcified by immersion in PBS with 10% EDTA (pH 7.4) at 4℃ for 14~21 days. This solution was changed every 3 days. After decalcification, the proximal diaphyses were dehydrated in ascending grades of ethanol and embedded in plastic (Technovit 7100 kit; Heraeus Kulzer, Germany). Transverse sections of the diaphyses (2-µm thick) mounted on glass slides (Matsunami Glass Ind., Japan) were stained with hematoxylin and eosin.

RT-PCR analysis

The left femurs were quickly frozen in liquid nitrogen and stored at -80℃ until RNA was extracted. The diaphyses were crushed in liquid nitrogen and total RNA was obtained using a Sepasol RNA I Super commercial extraction kit (Nacalai Tesque, Japan). cDNA was synthesized from 3 µg of total RNA using a ReverTra Ace-α first-strand cDNA synthesis kit (Toyobo, Japan) and amplified with Taq DNA polymerase (Qiagen, Germany). Primers specific for ER-α (GenBank accession No. X03805), ER-β (GenBank accession No. AF045149), and β-actin (GenBank accession No. L08165) were designed using Primer 3 as a software for primer design (NCBI, USA). Table 1 summarizes the PCR amplification conditions with the specific primer sets. PCR was performed for 25 to 36 cycles. The amplified products were subjected to electrophoresis in 1.75% agarose gels and visualized with ethidium bromide staining. Band density was determined by densitometric analysis (ATTO Densitograph; ATTO, Japan). β-actin was used as an internal control.

Statistical analysis

Quantitative data were analyzed using a non-parametric Scheffe test by StatView (SAS, USA) for the statistical software. Data are presented as the mean ± SD.

Histological analysis

To examine histochemical changes in the bone marrow cavity following E2 treatment, transverse sections of femurs were stained with hematoxylin and eosin. In untreated male quails (day 0), the bone-lining cells appeared flat and were arranged on the endosteal bone surface. The bone marrow cavity was filled with bone marrow cells and adipose tissue (Fig. 1A). After E2 treatment, the bone-lining cells differentiated into cuboidal osteoblasts (day 1; Fig. 1B). These cells formed a part of the matrix between the endosteal surface and cells lining the bone (day 2; Fig. 1C). On day 3, matrices formed by the osteoblasts extended towards the bone marrow cavity and contained embedded osteocytes (Fig. 1D). These matrices were reticularly developed and many mature osteoblasts were seen on their surfaces on day 5 (Fig. 1E). Seven days after E2 treatment, volume of the matrices in the bone marrow cavity was reduced. This decrease was accompanied by an increase in osteoclasts and a decrease in osteoblasts on the surface of the matrices (Fig. 1F). After 10 days, the bone matrices were only observed in the vicinity of cortical bone while the marrow cavity began to refill with bone marrow cells and adipose tissue (Fig. 1G). After 15 days, the matrices had disappeared from the bone marrow cavity and the endosteal surface was again covered with flat bone-lining cells (Fig. 1H).

RT-PCR analysis of ER-α and ER-β expression

To assess the expression patterns of ER-α and ER-β mRNA during medullary bone formation and resorption, a semi-quantitative RT-PCR analysis was performed (Figs. 2 and 3). On day 0 (untreated quails), ER-α mRNA was already expressed but ER-β mRNA was barely detectable. The level of ER-α mRNA expression increased during bone formation although no significant difference was observed between days 0 and 5. Following this, ER-α mRNA expression decreased insignificantly from day 7 to day 15. ER-β mRNA was very weakly expressed throughout this period.


In the present study, we measured the temporal expression of ER-α and ER-β mRNA during medullary bone formation and resorption in male Japanese quails treated with E2. Histochemical analysis demonstrated that medullary bone was formed in the bone marrow cavity by osteoblasts derived from bone-lining cells on the endosteal surface following E2 treatment. Many osteoclasts appeared on the surface of this bone 5 days after E2 administration. Following this, medullary bone disappeared from the bone marrow cavity and bone-lining cells reappeared on the endosteal surface after 15 days. This medullary bone model is therefore useful for examining the process of bone remodeling along with the relationship between estrogen and bone metabolism. This process of bone formation and resorption has not previously been reported in an experimental mammalian model. Several studies have previously investigated the relationship between mammalian osteoblasts and estrogen. Estrogen treatment stimulates cancellous bone formation in female rats [5]. Samuels et al. [24,25] demonstrated that the bone marrow cavity is filled with bone arising from cancellous bone in the proximal metaphysis after administering high doses of estrogen to mice. In addition, many studies using ovariectomized rats have reported the prevention of bone loss by estrogen [1,13,29].

In humans, ER-α and ER-β are expressed in cortical and cancellous bone, respectively, in cells such as osteoblasts, osteocytes, and osteoclasts [2,4,7,17,23]. Thus, the two ER isoforms may have different functions in different types of bone [4]. Batra et al. [2] demonstrated that the expression of ER-α and ER-β in human bone varied according to age, gender, and cell type. Oreffo et al. [17] found that ER-α mRNA is expressed in preosteoclasts but not mature osteoclasts. On the other hand, both ER-α and ER-β are expressed in osteoblasts, osteocytes, bone-lining cells, and osteoclasts on metaphyseal trabecular bone in rodents [4,7,32]. The maturation of osteoclasts found among human peripheral blood mononuclear cells [23] as well as murine bone marrow monocytes and RAW264.7 cells [26] is directly inhibited by estrogen. Additionally, the inhibition of bone resorption by estrogen via ER-α is mediated by a reduction of human osteoclastogenesis rather than by suppressing resorptive activity [27]. These results suggest that estrogen may affect bone formation and resorption through both ER-β and ER-α. Thus, it is not clear whether one or both types of ER is involved in the effect of estrogen on bone metabolism in humans and rodents.

The presence of ERs in avian medullary bone was previously examined during the early and active periods of medullary bone formation [15,16] using an ER-α-specific antibody [28]. Consequently, ERs were found in osteogenic cells such as bone-lining cells, osteoblasts, and alkaline phosphatase-positive bone marrow cells [16]. Using in situ hybridization, Imamura et al. [8] demonstrated that osteoblasts express ER-α mRNA but not ER-β mRNA. Furthermore, a previous study we performed indicated that osteogenic cells derived from medullary bone express ER-α mRNA, but not ER-β mRNA, during bone formation [6]. These results suggest that estrogen, acting through ER-α but not ER-β, might influence medullary bone formation by osteogenic cells.

Results of the RT-PCR analysis in the present study demonstrated that ER-β mRNA levels were very low while ER-α mRNA was stably expressed at higher levels during medullary bone formation and resorption. Although these results showed that expression of ER-α mRNA increased slightly, there was no significant change throughout the bone formation period. The variation in ER-α mRNA expression patterns might be due to differences between in vivo and in vitro studies, or could be related to the isolation of osteogenic cells during active medullary bone formation. Avian osteoclasts have been shown to express ERs [18,19], but Imamura et al. [8] recently demonstrated that osteoclasts do not express either ER-α or ER-β mRNA during the active medullary bone formation period. Although the presence of ERs in osteoclasts from medullary bone is debatable, the resorption activity of osteoclasts does seem to be inhibited by estrogen, suggesting that estrogen may regulate the expression of lysosomal genes [19] and the expression level of ERs in osteoclasts [20]. Reduced numbers of osteoclasts, as well as reduced osteoblast numbers and activity, might therefore be responsible for the slight decrease in ER-α expression we observed during bone resorption. As suggested by our RT-PCR analysis of total RNA extracted from the diaphyses of femurs containing cortical bone, medullary bone, bone cells, and other bone marrow cells, estrogen may act on osteoclasts via ER-α during medullary bone resorption.

In conclusion, our results showed that estrogen might affect both the formation and resorption of medullary bone through ER-α but not ER-β. Further studies are required to clarify which cell types, osteoblasts and/or osteoclasts, express ER-α during these processes. Moreover, we found that estrogen-induced medullary bone formation in male Japanese quails is a useful model for examining the relationship between bone metabolism and estrogen.


This study was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (18791351 in 2006-2007 to S.H.).

1. Andersson N,Islander U,Egecioglu E,Löf E,Swanson C,Movérare-Skrtic S,Sjögren K,Lindberg MK,Carlsten H,Ohlsson C. Investigation of central versus peripheral effects of estradiol in ovariectomized miceJ EndocrinolYear: 200518730330916293778
2. Batra GS,Hainey L,Freemont AJ,Andrew G,Saunders PT,Hoyland JA,Braidman IP. Evidence for cell-specific changes with age in expression of oestrogen receptor (ER) α and β in bone fractures from men and womenJ PatholYear: 2003200657312692843
3. Bloom W,Bloom MA,McLean FC. Calcification and ossification. Medullary bone changes in the reproductive cycle of female pigeonsAnat RecYear: 194181443475
4. Bord S,Horner A,Beavan S,Compston J. Estrogen receptors α and β are differentially expressed in developing human boneJ Clin Endocrinol MetabYear: 2001862309231411344243
5. Chow JW,Lean JM,Chambers TJ. 17 beta-estradiol stimulates cancellous bone formation in female ratsEndocrinologyYear: 1992130302530321572310
6. Hiyama S,Sugiyama T,Kusuhara S,Uchida T. Evidence for the expression of estrogen receptors in osteogenic cells isolated from hen medullary boneActa HistochemYear: 200911150150718835015
7. Hoyland JA,Mee AP,Baird P,Braidman IP,Mawer EB,Freemont AJ. Demonstration of estrogen receptor mRNA in bone using in situ reverse-transcriptase polymerase chain reactionBoneYear: 19972087929028531
8. Imamura T,Sugiyama T,Kusuhara S. Expression and localization of estrogen receptors α and β mRNA in medullary bone of laying hensAnim Sci JYear: 200677223229
9. Krust A,Green S,Argos P,Kumar V,Walter P,Bornert JM,Chambon P. The chicken oestrogen receptor sequence: homology with v-erbA and the human oestrogen and glucocorticoid receptorsEMBO JYear: 198658918973755102
10. Kusuhara S,Schraer H. Cytology and autoradiography of estrogen-induced differentiation of avian endosteal cellsCalcif Tissue IntYear: 1982343523586814725
11. Masuyama A,Ouchi Y,Sato F,Hosoi T,Nakamura T,Orimo H. Characteristics of steroid hormone receptors in cultured MC3T3-E1 osteoblastic cells and effect of steroid hormones on cell proliferationCalcif Tissue IntYear: 1992513763811458342
12. Miller SC,Bowman BM. Medullary bone osteogenesis following estrogen administration to mature male Japanese quailDev BiolYear: 19818752637286421
13. Mödder UIL,Riggs BL,Spelsberg TC,Fraser DG,Atkinson EJ,Arnold R,Khosla S. Dose-response of estrogen on bone versus the uterus in ovariectomized miceEur J EndocrinolYear: 200415150351015476452
14. Notelovitz M. Estrogen therapy and osteoporosis: principles & practiceAm J Med SciYear: 19973132129001160
15. Ohashi T,Kusuhara S. Immunoelectron microscopic detection of estrogen target cells in the bone marrow of estrogen-treated male Japanese quailBone MinerYear: 19932031398453320
16. Ohashi T,Kusuhara S,Ishida K. Estrogen target cells during the early stage of medullary bone osteogenesis: immunohistochemical detection of estrogen receptors in osteogenic cells of estrogen-treated male Japanese quailCalcif Tissue IntYear: 1991491241271913292
17. Oreffo ROC,Kusec V,Virdi AS,Flanagan AM,Grano M,Zambonin-Zallone A,Triffitt JT. Expression of estrogen receptor-alpha in cells of the osteoclastic lineageHistochem Cell BiolYear: 199911112513310090573
18. Oursler MJ,Osdoby P,Pyfferoen J,Riggs BL,Spelsberg TC. Avian osteoclasts as estrogen target cellsProc Natl Acad Sci USAYear: 199188661366171907373
19. Oursler MJ,Pederson L,Pyfferoen J,Osdoby P,Fitzpatrick L,Spelsberg TC. Estrogen modulation of avian osteoclast lysosomal gene expressionEndocrinologyYear: 1993132137313808440193
20. Pederson L,Kremer M,Foged NT,Winding B,Ritchie C,Fitzpatrick LA,Oursler MJ. Evidence of a correlation of estrogen receptor level and avian osteoclast estrogen responsivenessJ Bone Miner ResYear: 1997127427529144340
21. Riddle O,Rauch VM,Smith GC. Action of estrogen on plasma calcium and endosteal bone formation in parathyroidectomized pigeonsEndocrinologyYear: 1945364147
22. Robinson JA,Harris SA,Riggs BL,Spelsberg TC. Estrogen regulation of human osteoblastic cell proliferation and differentiationEndocrinologyYear: 1997138291929279202236
23. Saintier D,Burde MA,Rey JM,Maudelonde T,de Vernejoul MC,Cohen-Solal ME. 17β-estradiol downregulates β3-integrin expression in differentiating and mature human osteoclastsJ Cell PhysiolYear: 200419826927614603529
24. Samuels A,Perry MJ,Goodship AE,Fraser WD,Tobias JH. Is high-dose estrogen-induced osteogenesis in the mouse mediated by an estrogen receptor?BoneYear: 200027414610865207
25. Samuels A,Perry MJ,Tobias JH. High-dose estrogen induces de novo medullary bone formation in female miceJ Bone Miner ResYear: 1999141781869933470
26. Shevde NK,Bendixen AC,Dienger KM,Pike JW. Estrogens suppress RANK ligand-induced osteoclast differentiation via a stromal cell independent mechanism involving c-Jun repressionProc Natl Acad Sci USAYear: 2000977829783410869427
27. Sørensen MG,Henriksen K,Dziegiel MH,Tankó LB,Karsdal MA. Estrogen directly attenuates human osteoclastogenesis, but has no effect on resorption by mature osteoclastsDNA Cell BiolYear: 20062547548316907645
28. Stossi F,Barnett DH,Frasor J,Komm B,Lyttle CR,Katzenellenbogen BS. Transcriptional profiling of estrogen-regulated gene expression via estrogen receptor (ER) α or ERβ in human osteosarcoma cells: distinct and common target genes for these receptorsEndocrinologyYear: 20041453473348615033914
29. Turner RT,Colvard DS,Spelsberg TC. Estrogen inhibition of periosteal bone formation in rat long bones: down-regulation of gene expression for bone matrix proteinsEndocrinologyYear: 1990127134613512387257
30. Westerlind KC,Sarkar G,Bolander ME,Turner RT. Estrogen receptor mRNA is expressed in vivo in rat calvarial periosteumSteroidsYear: 1995604844877482634
31. White R,Lees JA,Needham M,Ham J,Parker M. Structural organization and expression of the mouse estrogen receptorMol EndocrinolYear: 198717357442484714
32. Windahl SH,Norgård M,Kuiper GGJM,Gustafsson JÅ,Andersson G. Cellular distribution of estrogen receptor β in neonatal rat boneBoneYear: 20002611712110678405

Article Categories:
  • Original Article

Keywords: estrogen receptor α, estrogen receptor β, medullary bone, osteoblasts, osteoclasts.

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