Immunohistochemical evidence for estrogen receptors in meibomian glands

doi:10.1016/S0161-6420(99)00040-8

Ophthalmology

Volume 107, Issue 1, January 2000, Pages 180-184

Presented in part as a poster at the annual meeting of the American Academy of Ophthalmology, New Orleans, Louisiana, November 1998.

https://doi.org/10.1016/S0161-6420(99)00040-8 Get rights and content

Abstract

Purpose

To look for sex hormone receptor distribution in three structures contributing to the normal human tear film: the conjunctiva, the accessory lacrimal glands, and the meibomian glands.

Design

An immunohistochemical study.

Tissues and controls

Forty-one upper eyelid specimens were collected from 15 male and 26 female patients (age range, 1.5–85 years) during blepharoptosis surgery via posterior tarsoconjunctival mullerectomy (Fasanella-Servat or Gavaris). In addition, control sections of histologically normal breast, prostate, and skin tissue were obtained.

Methods

Immunohistochemical staining using mouse monoclonal antibodies against estrogen, progesterone, and androgen receptors was performed on all tissues and controls. Quantitation of the receptors was performed and expressed as percentage nuclear positivity. Specimens were divided into three groups based on the age of the patient: <12 years (n = 9); 18–55 years (n = 1); >55 years (n = 12).

Results

Forty-one specimens contained conjunctiva. All were negative for estrogen, progesterone, and androgen receptors. Twenty-four specimens contained accessory lacrimal glands of Wolfring. All were negative for the three receptors. Twenty-two specimens contained meibomian glands. All were positive for estrogen receptors; one was positive for progesterone receptors and one for androgen receptors. Using Minitab statistical software (Minitab Inc. State College, PA), analysis of variation revealed no statistical difference between sexes or between age groups studied. The sebaceous glands of skin were uniformly positive for androgen receptors. Sebaceous glands of the face and scalp (3 of the 15 skin samples) were also positive for estrogen receptors.

Conclusions

Estrogen receptors are present in the meibomian glands of the upper eyelid. Unlike sebaceous glands elsewhere on the skin, the meibomian glands lack androgen receptors. Estrogen receptors may play a role in modulation of the lipid layer of the tear film, and their activity may be linked to meibomian gland dysfunction and dry eye syndrome.

Section snippets

Tissues

Forty-one upper eyelid specimens were collected from 15 male and 26 female patients (age range, 1.5–85 years) during routine blepharoptosis surgery via posterior tarsoconjunctival mullerectomy (Fasanella-Servat or Gavaris) as described previously.5 All specimens were fixed in 4% formaldehyde in 0.075 M phosphate buffer, pH 7.4, for 24 hours, transferred to 70% v/v ethanol and stored for up to 7 days before paraffin wax processing. Processing was carried out in an enclosed tissue processor

Results

The demographic data including sex and age for each patient and a summary of the immunohistochemical findings for each structure are given in Table 1. All procedural control sections showed appropriate immunoreactivity and contained nuclei that were positive for the antibodies tested. Figure 1, Figure 2, Figure 3.

Forty-one specimens contained conjunctiva. All were negative for estrogen (with either 6F11 or 1D5 clone in the subset tested with the latter antibody) (Fig 4), progesterone, and

Discussion

There are approximately 30 to 40 meibomian glands in the upper eyelid and 20 to 30 in the lower lid. Meibomian glands are modified sebaceous glands that consist of alveolar units of secretory cells that empty into a duct. Secretion occurs by the degeneration of the lipid droplet-containing cells in the center of the lobule into the ducts.6, 7 Each gland has an orifice that opens on the lid margin. The lipid layer of the tear film is produced by the meibomian glands; it increases the surface

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mRNAs of steroidogenic enzymes are found in human tear-producing tissues including meibomian glands and lacrimal glands (Butovich et al., 2019; Rocha et al., 2000; Schirra et al., 2006). Receptors for androgen, oestrogen, and progesterone are present in the ocular surface tissues (Auw-Haedrich and Feltgen, 2003; Esmaeli et al., 2000; Fuchsjäger-Mayrl et al., 2002; Schröder et al., 2016; Suzuki et al., 2001), suggesting sex steroids influence the structure, functions, and pathology of these tissues (Rocha et al., 2000). In vitro human studies show that 5α-dihydrotestosterone (DHT) enhances genes for lipogenesis in meibomian gland epithelial cells and upregulates genes involved in wound healing and regeneration in corneal epithelial cells (Khandelwal et al., 2012).
Sex steroids play a role in regulation of tear film function and may exert their action locally at the ocular surface. However, measurement of sex steroids in tears is difficult due to small-volume tear samples and very low concentrations of the hormones. This short communication highlights what has been achieved to date in the analysis of tear sex steroids using ultra-performance LC-MS (UPLC-MS) as previously published, and reports further and more recent investigations toward optimising mass spectrometry method sensitivity and accuracy. The published UPLC-MS method successfully measured progesterone, androsterone glucuronide and 5α-androstane-3α,17β-diol in pooled basal tears of postmenopausal women, and fourteen sex steroid standards in methanol. Limitations included sub-optimal limits of detection (LOD) and lower limits of quantification (LLOQ) for some analytes (particularly oestrogens), exclusion of sample matrix effects and no use of internal standards. This update reports on further experiments carried out to improve sensitivity and accuracy. Sample matrix effects, internal standard spiking, and derivatisation with dansyl chloride and oximes were investigated. Dansylation significantly improved the LOD and LLOQ of oestrogens and their metabolites, by a factor of 10 for oestradiol and a factor of 5 for oestrone, but sensitivity of this updated method is not sufficient however for analysis of these oestrogens in human tears. Using gas chromatography-mass spectrometry (GC-MS) as an alternative technique to LC-MS, improved sensitivity for derivatised oestradiol is reported. This work demonstrates the need to develop higher sensitivity methods and points researchers towards specific MS ionisation techniques for future analysis of sex steroids in tears, in order to progress current understanding of the role of sex steroids in tear function and dry eye.
Impact of aging on the pathophysiology of dry eye disease: A systematic review and meta-analysis
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Membrane-associated mucin (MAMs) expression, such as MUC4, MUC5AC, and MUC5B, was altered in elderly females who received treatment for DED [56]. Age-related changes in the meibomian gland were investigated from 10 human and 3 mouse studies [35,46,54,65–74] (Table 5). The structural changes of meibomian gland in humans and mice included meibomian gland density and diameter decreasing while meibomian gland dropout and obstruction rate increasing with age [46,54,69,74].
Dry eye disease (DED) is a common age-related ocular surface disease. However, it is unknown how aging influences the ocular surface microenvironment. This systematic review aims to investigate how the aging process changes the ocular surface microenvironment and impacts the development of DED.
An article search was performed in PubMed, EMBASE, and Web of Science. 44 studies reporting on age-related ocular changes and 14 large epidemiological studies involving the prevalence of DED were identified. 8 out of 14 epidemiological studies were further analyzed with meta-analysis. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines were followed. Study-specific estimates (impact of aging on the prevalence of DED) were combined using one-group meta-analysis in a random-effects model.
Meta-analysis revealed the prevalence of DED in the elderly aged 60 years old or older was 5519 of 60107 (9.2%) and the odds ratio of aging compared to younger age was 1.313 (95% confidence interval [CI]; 1.107, 1.557). With increasing age, the integrity of the ocular surface and tear film stability decreased. Various inflammatory cells, including senescent-associated T-cells, infiltrated the ocular surface epithelium, lacrimal gland, and meibomian gland, accompanied by senescence-related changes, including accumulation of 8-OHdG and lipofuscin-like inclusions, increased expression of p53 and apoptosis-related genes, and decreased Ki67 positive cells.
The aging process greatly impacts the ocular surface microenvironment, consequently leading to DED.
Evaluation estrogen, progesteron and androgen receptor expressions in corneal epithelium in keratoconus
2019, Contact Lens and Anterior Eye
To evaluate the role of sex steroid hormone receptors in corneal epithelium in etiopathogenesis of keratoconus (KC).
Thirty patients with KC who were planned for corneal collagen-crosslinking and 20 patients who were planned for excimer laser for refractive errors included in this study. Corneal epitheliums were curated mechanically during surgeries. Right eyes were evaluated immunohistochemically and left eyes were evaluated by quantitative polymerase chain reaction (qPCR) to investigate estrogenα, estrogenβ, progesterone and androgen receptors.
Immunohistochemically, staining for progesterone and androgen receptors did not significantly differ between KC and control groups (p > 0.05). None of the cases had staining for estrogenα and estrogenβ receptors. qPCR showed that mRNA expressions of estrogenα and androgen receptors were significantly higher in the KC group (p < 0.001).
A significantly higher rate of estrogenα and androgen receptor expressions in corneal epithelium from patients with KC through qPCR supports a possible relation between KC and sex steroid hormones.
TFOS DEWS II Sex, Gender, and Hormones Report
2017, Ocular Surface
One of the most compelling features of dry eye disease (DED) is that it occurs more frequently in women than men. In fact, the female sex is a significant risk factor for the development of DED. This sex-related difference in DED prevalence is attributed in large part to the effects of sex steroids (e.g. androgens, estrogens), hypothalamic-pituitary hormones, glucocorticoids, insulin, insulin-like growth factor 1 and thyroid hormones, as well as to the sex chromosome complement, sex-specific autosomal factors and epigenetics (e.g. microRNAs).
In addition to sex, gender also appears to be a risk factor for DED. “Gender” and “sex” are words that are often used interchangeably, but they have distinct meanings. “Gender” refers to a person’s self-representation as a man or woman, whereas “sex” distinguishes males and females based on their biological characteristics. Both gender and sex affect DED risk, presentation of the disease, immune responses, pain, care-seeking behaviors, service utilization, and myriad other facets of eye health.
Overall, sex, gender and hormones play a major role in the regulation of ocular surface and adnexal tissues, and in the difference in DED prevalence between women and men. The purpose of this Subcommittee report is to review and critique the nature of this role, as well as to recommend areas for future research to advance our understanding of the interrelationships between sex, gender, hormones and DED.
Role of topical dehydroepiandrosterone in ameliorating isotretinoin-induced Meibomian gland dysfunction in adult male albino rat
2017, Annals of Anatomy
Citation Excerpt :
Androgens and estrogens have been considered to regulate the Meibomian glands through hormone binding receptors (Krenzer et al., 2000). Sex steroid receptors for androgens (Rocha et al., 2000) and estrogen (Esmaeli et al., 2000; Auw-Haedrich and Feltgen, 2003) have been identified on the Meibomian glands and are presumed to have an influence on synthesis of meibum (Suzuki et al., 2008). Where androgens stimulate the expression of genes related to lipid production in the Meibomian glands (Schirra et al., 2005), estrogens cause a decrease in their expression (Suzuki et al., 2008).
Isotretinoin broad effectiveness is limited by its detrimental effect on the Meibomian glands. Androgens have been reported to regulate the Meibomian gland function. Dehydroepiandrosterone (DHEA) is an androgen precursor considered as an efficient and physiological anti-ageing skin agent. This study aimed to investigate the role of the topical DHEA in ameliorating Meibomian gland dysfunction caused by isotretinoin employing different histological and immunohistochemical techniques. Twenty-four adult male albino rats were divided into four equal groups; control group, DHEA-treated group (1% twice daily for 3 months), isotretinoin-treated group (0.5 mg/kg/day for 3 months), and both isotretinoin and DHEA-treated group. Meibomian gland specimens were processed for light microscopy. Immunohistochemical study was carried out using antibodies for proliferating cell nuclear antigen (PCNA), androgen receptor (AR) and estrogen receptor-alpha (ER-α). Sections from the isotretinoin-treated group revealed a reduction in the number and size of acini. Thickening and keratinization of the epithelial lining of the ducts were observed. Multiple degenerated acini and casts of acinar cells in the ducts of Meibomian glands were detected. Some dilated congested blood vessels and mononuclear cells were occasionally seen. A significant decrease in PAS reaction and a significant increase in collagen fiber content were detected. Immunohistochemical study revealed a significant increase in immunoexpression of PCNA in basal ductal cells coupled with decreased expression in basal acinar cells. Both AR and ER-α immunoexpression was significantly decreased. Minimal alterations were observed upon concomitant treatment with isotretinoin and DHEA as compared to the control group. Topical DHEA could prove to be beneficial in ameliorating isotretinoin-induced Meibomian gland dysfunction most probably through its androgenic effect.
In vitro effects of sex hormones in human meibomian gland epithelial cells
2016, Experimental Eye Research
Meibomian gland dysfunction (MGD) is considered the most common cause of dry eye disease (DED). Sex hormones seem to play a role in the pathogenesis of MGD although their involvement is not completely understood. Therefore, in the present study we evaluated the effect of dihydrotestosteron (DHT) and estradiol (β-Est) on an immortalized human meibomian gland epithelial cell line (HMGEC). Protein expression of sex hormone receptors in HMGEC was investigated by western blot. Ultrastructural morphology, Sudan III lipid staining, cell proliferation as well as vitality assays were performed. Furthermore, expression of MGD-associated markers for keratinization (hornerin, involucrin and CK6), proliferation (CK5 and CK14) and lipid synthesis (fatty acid synthase and stearoyl-CoA desaturase) were analyzed by real time RT-PCR. Western blot revealed presence of androgen receptor (AR), estrogen receptors α and -β (ERα, ERβ) and progesterone receptor (PR) in HMGEC. PR, ERα and ERβ expression was significantly induced under cultivation with serum, whereas sex hormones stimulation showed no further effect on protein expression of PR, ERα and ERβ. Our results showed no impact of MGD-associated sex hormones to cellular morphology and lipid accumulation in HMGEC. Cell proliferation was slightly induced through application of sex hormones and supplementation of calcium. However, both sex hormones and calcium altered gene expression of MGD-associated markers. Especially keratinization genes hornerin (HRNR) and cornulin (COR) were induced after application of sex hormones and calcium in serum-free cultivated HMGEC. This may promote keratinization processes that are associated with MGD. Further investigations are necessary to analyze the (hyper)keratinization processes that occur during MGD and using HMGEC as an in vitro model.

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^☆: Supported by a grant from the Research Committee at St. Joseph Mercy Hospital, Ann Arbor, Michigan.

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Immunohistochemical evidence for estrogen receptors in meibomian glands☆

Abstract

Purpose

Design

Tissues and controls

Methods

Results

Conclusions

Section snippets

Tissues

Results

Discussion

Ophthalmology

Exp Eye Res

Ophthalmology

Ophthalmology

Obstet Gynecol

Dermatol Clin

The frequency of sicca syndrome in an elderly female population

J Rheumatol