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. 2000 Jan;84(1):76–84. doi: 10.1136/bjo.84.1.76

Identification of androgen receptor protein and 5α-reductase mRNA in human ocular tissues

E Rocha 1, L Wickham 1, L A da Silveira 1, K Krenzer 1, F Yu 1, I Toda 1, B Sullivan 1, D Sullivan 1
PMCID: PMC1723240  PMID: 10611104

Abstract

BACKGROUND/AIMS—Androgens have been reported to influence the structural organisation, functional activity, and/or pathological features of many ocular tissues. In addition, these hormones have been proposed as a topical therapy for such conditions as dry eye syndromes, corneal wound healing, and high intraocular pressure. To advance our understanding of androgen action in the eye, the purpose of the present study was twofold: firstly, to determine whether tissues of the anterior and posterior segments contain androgen receptor protein, which might make them susceptible to hormone effects following topical application; and, secondly, to examine whether these tissues contain the mRNA for types 1 and/or 2 5α-reductase, an enzyme that converts testosterone to the very potent metabolite, dihydrotestosterone.
METHODS—Human ocular tissues and cells were obtained and processed for histochemical and molecular biological procedures. Androgen receptor protein was identified by utilising specific immunoperoxidase techniques. The analysis of type 1 and type 2 5α-reductase mRNAs was performed by the use of RT-PCR, agarose gel electrophoresis, and DNA sequence analysis. All immunohistochemical evaluations and PCR amplifications included positive and negative controls.
RESULTS—These findings show that androgen receptor protein exists in the human lacrimal gland, meibomian gland, cornea, bulbar and forniceal conjunctivae, lens epithelial cells, and retinal pigment epithelial cells. In addition, our results demonstrate that the mRNAs for types 1 and 2 5α-reductase occur in the human lacrimal gland, meibomian gland, bulbar conjunctiva, cornea, and RPE cells.
CONCLUSION—These combined results indicate that multiple ocular tissues may be target sites for androgen action.



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Figure 1  .

Figure 1  

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Identification of androgen receptor protein in epithelial cell nuclei of the lacrimal gland. The tissue was obtained from an elderly man during ocular surgery and processed for immunoperoxidase procedures. To confirm the specificity of first antibody staining (A), an aliquot of the rabbit anti-human androgen receptor was preincubated with an excess amount of peptide "AR1-21" (B).

Figure 2  .

Figure 2  

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Presence of androgen receptor protein in the nuclei of acinar epithelial cells of the meibomian gland. The tissue was obtained from a 70 year old woman during plastic surgery for lid reconstruction. Immunoperoxidase staining of the tissue sample was performed with a first antibody that had been preincubated with either peptide "AR462-478" (A) or peptide "AR1-21" (B).

Figure 3  .

Figure 3  

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Location of androgen receptor protein in epithelial cell nuclei of the conjunctiva. The tissue was obtained from a 79 year old woman during an extracapsular crystalline extraction and then processed for immunoperoxidase procedures. To verify the specificity of first antibody staining (A), an aliquot of the rabbit anti-human androgen receptor was preincubated with an excess amount of peptide "AR1-21" (B).      

Figure 4  .

Figure 4  

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Identification of androgen receptor protein in the nuclei of apical epithelial cells of the cornea. The tissue was obtained from a 65 year old woman during keratoplastic surgery. Immunoperoxidase analysis of the tissue sample was conducted with a first antibody that had been preincubated with either peptide "AR462-478" (A) or peptide "AR1-21" (B).

Figure 5  .

Figure 5  

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Presence of androgen receptor protein in the nuclei of lens epithelial cells. The lens cells were obtained from a 13 year old male undergoing cataract surgery and were then cultured, cytocentrifuged onto poly-L-lysine coated glass slides (105 cells/slide), fixed in acetone for 5 minutes at 4°C, and processed for immunoperoxidase procedures. Staining was performed with a first antibody that had been preincubated with either peptide "AR462-478" (A) or peptide "AR1-21" (B). Competition with this latter peptide resulted in background staining.    

Figure 6  .

Figure 6  

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Identification of androgen receptor protein in the nuclei of retinal pigment epithelial cells. The cells were obtained from a 71 year old man following a cardiac arrest and were cultured until utilisation in this immunoperoxidase study. For analysis, cultured cells were cytocentrifuged onto poly-L-lysine coated glass slides (105 cells/slide), fixed in acetone for 5 minutes at 4°C, and then processed for receptor staining. To confirm the specificity of the first antibody staining (A), an aliquot of the rabbit anti-human androgen receptor was preincubated with an excess amount of peptide "AR1-21" (B).

Figure 7  .

Figure 7  

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Presence of type 1 5α-reductase mRNA (Type 1) and type 2 5α-reductase mRNA (Type 2) in human ocular tissues and cells. Samples were processed for RT-PCR, agarose gel electrophoresis and ethidium bromide staining, as explained in Materials and methods. Photographs of agarose gels were obtained with a Polaroid camera (Polaroid Corporation, Cambridge, MA, USA) and images on the internegatives were then captured with a CCD-72S video camera (Hamamatsu Photonics, Japan), imported into Adobe Photoshop 4.01 and printed with a Kodak XLS 8600 printer. Samples 1-10 and 11-13 were run on parallel gels and bands have been aligned according to the molecular size of the bands. The numbers refer to the following samples: (1) male meibomian gland; (2) female meibomian gland; (3) female lacrimal gland; (4) male lacrimal gland; (5) prostate (surgical specimen); (6) Hs68 cells; (7) female conjunctiva (n = 3 combined tissues); (8) male conjunctiva (n = 3 combined tissues); (9) female cornea (n = 3 combined tissues); (10) male cornea (n = 3 combined tissues); (11) retinal pigment epithelial cells; (12) LNCaP cells; (13) prostate (RNA from Clontech).

Selected References

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