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(Investigative Ophthalmology and Visual Science. 2006;47:158-168.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-1003
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Estrogen’s and Progesterone’s Impact on Gene Expression in the Mouse Lacrimal Gland

Tomo Suzuki,1,2 Frank Schirra,1,2 Stephen M. Richards,1,2 Nathaniel S. Treister,1,3 Michael J. Lombardi,4 Patricia Rowley,4 Roderick V. Jensen,4 and David A. Sullivan1,2

1From the Schepens Eye Research Institute and 2Department of Ophthalmology, Harvard Medical School; 3Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine; and 4Department of Physics, University of Massachusetts Boston, Boston, Massachusetts.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. The hypothesis tested in the study was that the effect of estrogen and progesterone on the lacrimal gland is mediated through specific receptors and that hormonal effects involve the regulation of gene expression and protein synthesis.

METHODS. Lacrimal glands were collected from young adult, ovariectomized mice, that were treated with 17ß-estradiol, progesterone, 17ß-estradiol plus progesterone or vehicle for 2 weeks. Glands were pooled according to treatment, processed for the isolation of RNA, and evaluated for differentially expressed mRNAs by using gene microarrays. Bioarray data were analyzed with sophisticated bioinformatics and statistical programs. The expression of selected genes was verified by using gene chips and quantitative real-time PCR methods.

RESULTS. The results demonstrate that 17ß-estradiol, progesterone, or both hormones together significantly influences the expression of hundreds of genes in the mouse lacrimal gland. Sex steroid treatment led to numerous alterations in gene activities related to transcriptional control, cell growth and/or maintenance, cell communication, signal transduction, enzyme catalysis, immune expression, and the binding and metabolism of nucleic acids and proteins. A number of the 17ß-estradiol, progesterone or 17ß-estradiol plus progesterone effects on gene expression were similar, but most were unique to each treatment. Of particular interest was the finding that these hormones seem to contribute little to the known sex-related differences in gene expression of the lacrimal gland.

CONCLUSIONS. These results support the hypothesis that estrogen’s and progesterone’s action on the lacrimal gland involves the regulation of numerous genes. However, these hormone effects do not appear to represent a major factor underlying the sexual dimorphism of gene expression in lacrimal tissue.

Investigators have proposed that estrogens may play an important role in the anatomy, physiology, and sexual dimorphism of the lacrimal gland.1 2 3 4 . In support of this proposition are reports that ovariectomy or antiestrogen treatment lead to acinar cell disruption and necrosis, cellular vacuolization, DNA degradation, inflammation, glandular tissue loss, and dry eye (Jacobs M, et al. IOVS 1986;27:ARVO Abstract page 25; Azzarolo AM, et al. IOVS 1994;35:ARVO Abstract 2500; Huang ZM, et al. IOVS 1995;36:ARVO Abstract 2979; Coles N, et al. IOVS 1988;29:ARVO Abstract page 48).1 2 4 5 6 7 8 Conversely, estrogen administration reportedly corrects these changes in lacrimal gland structure and function, and promotes lacrimal secretion (Jacobs M, et al. IOVS 1986;27:ARVO Abstract page 25; Azzarolo AM, et al. IOVS 1993;34:ARVO Abstract 3773).1 2 7 9 10 11 12 13

However, other researchers have found that neither estrogen insufficiency nor estrogen treatment has any effect on the weight, morphology, total protein content, specific enzyme activity, lymphocyte accumulation, or secretion of the lacrimal gland (Cripps MM, et al. IOVS 1986;27:ARVO Abstract page 25).9 14 15 16 17 18 19 20 21 Yet, other investigators have reported that estrogens have a negative influence on lacrimal tissue and cause glandular regression, suppression of protein production, androgen antagonism, and reduced tear secretion (Azzarolo AM, et al. IOVS 1993;34:ARVO Abstract 3773).5 9 15 22 23 24

Some of these conflicting findings regarding estrogens may be explained by differences in experimental design, hormone dosage, or animal model. It is also possible that these disparate results may be due to variations in the relative levels of progesterone, a hormone that may significantly modify estrogen effects. However, an overriding difficulty with clarification is that the nature and extent of estrogen or progestin action on the lacrimal gland is not known. In fact, no consensus exists concerning the cellular targets for, or the cellular processes that may be controlled by, estrogens or progestins in lacrimal tissue. Indeed, it has not yet even been established whether these hormones have functional receptors in the lacrimal gland.

We hypothesize that estrogen and progesterone action on the lacrimal gland is mediated through specific receptors and that hormonal effects involve the regulation of gene expression and protein synthesis. To begin to test this hypothesis, we examined in the present study whether 17ß-estradiol, progesterone, and both hormones in combination influence gene expression in the female mouse lacrimal gland.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals and Hormone Treatment
Young adult and age-matched BALB/c mice, that were ovariectomized at 8 weeks of age, were obtained from Taconic Laboratories (Germantown, NY). Animals were housed in constant temperature rooms (70–72°F) with fixed light–dark intervals of 12 hours. Ten days after surgery, ovariectomized mice were treated with subcutaneous pellet implants containing placebo (cholesterol, methylcellulose, lactose), 17ß-estradiol (0.5 mg), progesterone (10 mg), or combined 17ß-estradiol plus progesterone. These pellets were purchased from Innovative Research of America (Sarasota, FL) and were designed for the continuous release of vehicle or physiological amounts of hormone throughout the 14-day experimental period. When indicated, mice were killed by CO2 inhalation and exorbital lacrimal glands were removed (n = 7 to 20 mice per condition per experiment), pooled according to group (n = 14 to 40 glands per sample) and processed for molecular biological procedures. All experiments with mice were approved by the Institutional Animal Care and Use Committee of The Schepens Eye Research Institute and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Molecular Biological Procedures
To analyze the effects of 17ß-estradiol and progesterone on lacrimal gland gene expression, total RNA was extracted from tissues by using TRIzol reagent (Invitrogen Corp., Carlsbad, CA). When indicated, samples were also exposed to RNase-free DNase (Invitrogen), examined spectrophotometrically at 260 nm to determine concentration and evaluated on 6.7% formaldehyde/1.3% agarose (Invitrogen-Gibco, Grand Island, NY) gels to verify RNA integrity. The RNA samples were then processed by using several different methods.

The principle method to examine differential gene expression involved the use of CodeLink Uniset Mouse I Bioarrays (~10,000 genes; GE Healthcare, Piscataway, NJ). Toward this end, glandular RNA samples were further purified with RNAqueous spin columns (Ambion, Austin, TX), and the integrity of these preparations was assessed with a RNA 6000 Nano LabChip with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The RNA samples were then processed for CodeLink Bioarray hybridization, as previously described.25 In brief, cDNA was synthesized from RNA (2 µg) with a CodeLink Expression Assay Reagent Kit (Amersham, Piscataway, NJ) and purified with a QIAquick purification kit (Qiagen, Valencia, CA). After sample drying, cRNA was made with a CodeLink Expression Assay Reagent Kit (Amersham), recovered with an RNeasy kit (Qiagen) and quantified with an UV spectrophotometer. Fragmented, biotin-labeled cRNA was then incubated and agitated (300 rpm shaker) on a CodeLink Bioarray at 37°C for 18 hours. The Bioarray was then washed, exposed to streptavidin-Alexa 647, and scanned by using ScanArray Express software and a ScanArray Express HT scanner (Packard BioScience, Meriden, CT) with the laser set at 635 nm, laser power at 100%, and photomultiplier tube voltage at 60%. Scanned image files were evaluated by utilizing CodeLink image and data analysis software (Amersham), which produced both raw and normalized hybridization signal intensities for each array spot. The spot intensities (~10,000) on the microarray image were standardized to a median of 1. Normalized data, with signal intensities exceeding 0.75, were analyzed with GeneSifter.Net software (VizX Labs LLC, Seattle, WA, vizxlabs.com). This comprehensive program also generated gene ontology and z-score reports. The ontologies were organized according to the guidelines of the Gene Ontology Consortium (http://www.geneontology.org/GO.doc.html),26 and included biological processes, molecular functions, and cellular components. Data were evaluated with and without log transformation. Statistical analysis of individual gene expression data was performed with Student’s t-test (two-tailed, unpaired).

The data from the individual Bioarrays (n = 6) are available for download through the National Center for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) via series accession number GSE1582. The data will also be accessible for analyses through GeneSifter (http://genesifter.net/datacenter/).

Differentially expressed mRNAs were also analyzed by utilizing GEM 1 (>8,000 genes) and GEM 2 (>9,500 genes) gene chips (Incyte Genomics, Inc., St. Louis, MO). The GEM chips and CodeLink arrays have over 4,700 sequences in common. Following additional purification with RNAqueous spin columns, poly(A) mRNA was extracted from lacrimal gland RNA samples (400 µg) by using the MicroPoly(A)Pure mRNA Isolation Kit (Ambion, Inc., Austin, TX). The mRNA concentration was determined with a RiboGreen RNA Quantitation Kit (Molecular Probes, Eugene, OR), according to Incyte’s procedures. After designating mRNA samples (750 ng) for use with either cy3 or cy5 probes, preparations were suspended in TE buffer, placed in siliconized RNase-Free Microfuge Tubes (Ambion) and shipped on dry ice to Incyte for hybridization. Microarray data were sent electronically to the Harvard Center for Genomic Research (Cambridge, MA). Results were downloaded into the Resolver Gene Expression Data Analysis System, version 2.0 (Rosetta Inpharmatics, Kirkland, WA), then normalized and evaluated as previously reported.25

Real Time PCR Procedures
Quantitative real-time PCR (qPCR) was used to verify the differential expression of selected genes. Sense and antisense primers were designed by using Primer Express Software, version 1.5a (Applied Biosystems, Inc., Foster City, CA; Table 1 ). The qPCR reactions were carried out according to the manufacturer’s protocol, by using aliquots of lacrimal gland cDNA (0.01–0.3 µL cDNA), optimal primer concentrations, and Applied Biosystems’ SYBR Green PCR Master Mix, MicroAmp Optical 96-Well Reaction Plates, ABI PRISM Optical Adhesive Covers and the GeneAmp 7900 HT Sequence Detection System. Gene expression was determined by employing the Relative Standard Curve Method, as described in User Bulletin #2 ABI Prism 7700 Sequence Detection System (Applied Biosystems; updated version 10/01), and standardizing levels to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Dissociation curves were monitored to confirm the absence of secondary PCR products.


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  		TABLE 1. Oligonucleotide Primers for Real-Time PCR Confirmation of Selected Genes

 

   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Estrogen and Progesterone Regulation of Gene Expression in the Lacrimal Gland
To examine the influence of "female" sex steroids on gene expression in the lacrimal gland, tissues were obtained from ovariectomized mice (n = 7 per treatment group) that had been treated for 2 weeks with 17ß-estradiol, progesterone, or both hormones in combination. Glands were processed for analysis by using CodeLink Uniset Mouse I Bioarrays and GeneSifter software.

Examination of non- and log-transformed data from three different experiments demonstrated that 17ß-estradiol and progesterone exert a very significant impact on gene activity in the lacrimal gland. As shown in Table 2 , these hormones, whether administered alone or together, significantly altered the expression of hundreds of genes. Those genes that were upregulated (e.g., asialoglycoprotein receptor 1 and NF-{kappa}-B-repressing factor) and downregulated (e.g., lymphocyte antigen 6 complex, locus F, and dopamine receptor 2) to the greatest extent (i.e., in terms of ratios) after hormone treatment are listed in Tables 3 4 and 5 .


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  		TABLE 2. Influence of 17ß-Estradiol and/or Progesterone on Gene Expression in the Lacrimal Gland

 

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  		TABLE 3. 17ß-Estradiol Effect on Gene Expression Ratios in the Mouse Lacrimal Gland

 

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  		TABLE 4. Progesterone Influence on Gene Expression Ratios in the Mouse Lacrimal Gland

 

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  		TABLE 5. Impact of 17ß-Estradiol and Progesterone on Gene Expression Ratios in the Mouse Lacrimal Gland

 
Administration of these various hormones had a considerable influence on biological processes, molecular functions, and cellular components in the lacrimal gland. For example, 17ß-estradiol, progesterone, and the combined sex steroid treatment all significantly affected the expression of numerous genes (i.e., ≥34 genes per category) related to nucleic acid and protein metabolism, cell growth and/or maintenance, cell communication, signal transduction, transcriptional regulation, and development. In addition, these treatments all significantly influenced the expression of many genes (i.e., ≥26 genes per category) associated with nucleic acid, protein, adenosine triphosphate (ATP) and metal ion binding, as well as assorted catalytic, hydrolase, receptor, and transferase activities (data not shown). These hormones also affected a diverse array of immune-related genes (Table 6) .


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  		TABLE 6. Sex Steroid Regulation of Various Immune-Related Genes in the Lacrimal Gland

 
Of particular interest was the impact of sex steroids on specific gene ontologies. As shown by z-score analyses, the most significant 17ß-estradiol actions were directed toward the stimulation of signaling pathways, ion transport, enzyme activities, and membrane aspects, whereas genes suppressed by estrogen were related to cell organization and biogenesis, cytokine activity, receptor binding, mitochondria, and intracellular components (Table 7) . Progesterone administration led to a significant increase in the expression of genes linked to signal transduction and cell communication and a decrease in those associated with cell growth and/or maintenance, metabolism, and ion binding (Table 8) . In contrast to these findings, combined 17ß-estradiol and progesterone exposure enhanced the expression of cell death genes and attenuated those related to receptor binding, signal transduction, protein transport, cytokine activity, and development (Table 9) .


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  		TABLE 7. Highest and Lowest Expression of Gene Ontologies in the Lacrimal Glands of Placebo- or 17ß-Estradiol-Treated Mice

 

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  		TABLE 8. Highest and Lowest Expression of Gene Ontologies in the Lacrimal Glands of Placebo- or Progesterone-Treated Mice

 

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  		TABLE 9. Highest and Lowest Expression of Gene Ontologies in the Lacrimal Glands of Placebo- or 17ß-Estradiol- and Progesterone-Treated Mice

 
In these studies the different hormone treatments had analogous or opposite effects on the expression of a number of genes (Table 10) . There were two genes that were significantly (P < 0.05) upregulated by 17ß-estradiol or progesterone, as well as 17ß-estradiol plus progesterone. These genes encode serine/threonine kinase 22B and discoidin domain receptor family, member 2, both of which are involved in phosphate metabolism. Conversely, there were 14 genes that were significantly (P < 0.05) downregulated by all three forms of hormone treatment. These genes (and their ontologies) included cell division cycle 45 homologue (S phase of mitotic cycle), chordin (neurogenesis), lysosomal acid lipase 1 (lipid metabolism), nidogen 1 (cell-matrix adhesion), parathyroid hormone receptor 1 (signal transduction), forkhead box P3 (Foxp3; regulation of transcription), high-mobility group box 2-like 1 (nucleic acid binding), and calpain 9 (protein metabolism).


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  		TABLE 10. Analogous and Opposite Effects of 17ß-Estradiol and Progesterone on Gene Expression in the Lacrimal Gland

 
To confirm in part the CodeLink Bioarray data, additional lacrimal gland mRNA samples (n = 20 mice per group per experiment) were processed for the GEM 1 and 2 gene chip analyses. This approach identified 38 genes on GEM chips that were either up- or downregulated by hormone treatment, and that met our analytical criteria (i.e., GEM signal intensity ≥500; expression ratio ≥1.8 or ≤ –1.8). However, only two of these genes were common to both the GEM and CodeLink platforms (i.e., pancreatic lipase–related protein 1 and asialoglycoprotein receptor 1). Therefore, to verify further the CodeLink results, selected genes were analyzed by qPCR. As shown in Table 11 , this method confirmed the effect of hormones on a number of different genes.


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  		TABLE 11. Verification of Selected CodeLink Bioarray and GEM Chip Results

 
Role of 17ß-Estradiol and Progesterone in the Sex-Related Differences in Gene Expression of the Lacrimal Gland
We have recently found that significant, sex-related differences exist in the expression of more than 500 genes in the mouse lacrimal gland.25 To evaluate whether female sex steroids may contribute to these differences, we determined whether genes expressed to a greater extent in female lacrimal tissues, compared with those of males, are also upregulated by treatment with 17ß-estradiol, progesterone or both hormones together. Moreover, we assessed whether genes expressed to a lower degree in female lacrimal glands, relative to those of males, are downregulated by female sex steroid administration. The ages of the BALB/c mice (n = 5 to 7 mice/sex/experiment) used in the analysis of sex-related differences (n = 3 experiments) in the lacrimal gland were similar to those in the present studies.25 Furthermore, all gene expression data were generated from CodeLink Bioarrays and analyzed with GeneSifter software.

As demonstrated in Table 12 , 17ß-estradiol, progesterone, and combined hormone exposure seemed to account for a minority of the sex-related differences in gene expression of the lacrimal gland. Evaluation of nontransformed data indicated that estrogen, with or without progesterone, appeared to contribute to between 6.6% and 26.8% of the gene activity differences between male and female lacrimal tissues (Table 12) . In contrast, progesterone alone had relatively little influence (i.e., <4%) on these sex differences. Examples of genes affected similarly by sex and sex steroid administration are shown in Table 13 .


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  		TABLE 12. Comparative Influence of Sex and Sex Steroid Treatment on Gene Expression in the Mouse Lacrimal Gland

 

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  		TABLE 13. Similarities between the Effects of Sex and Sex Steroid Administration on the Expression of Specific Genes in the Mouse Lacrimal Gland

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This investigation demonstrates that the administration of 17ß-estradiol, progesterone, or both hormones significantly influenced the expression of hundreds of genes in the mouse lacrimal gland. Sex steroid treatment led to numerous alterations in gene activities related to transcriptional control, cell growth and/or maintenance, cell communication, signal transduction, enzyme catalysis, and the binding and metabolism of nucleic acids and proteins. A number of the 17ß-estradiol, progesterone, or 17ß-estradiol plus progesterone effects on gene expression were similar, but most were unique to each treatment. These results support our hypothesis that the effect of estrogen and progesterone on the lacrimal gland involves the regulation of gene expression.

Some of the most significant effects of 17ß-estradiol were directed toward the control of genes linked to signaling pathways, transport, enzymatic activities, and cellular organization. For example, 17ß-estradiol enhanced the mRNA levels of (1) mitogen-activated protein kinase kinase kinase 4, which activates the CSBP2, P38, and JNK MAPK pathways28 ; (2) serine (or cysteine) proteinase inhibitor, clade A, member 6, which is another name for corticosteroid-binding globulin or transcortin and is the major transport protein for glucocorticoids and progestins28 ; (3) aldo-keto reductase family 1, member C18, which is also called 20-{alpha}- hydroxysteroid dehydrogenase, and catalyzes the conversion of progesterone into the inactive 20-{alpha}-dihydroprogesterone29 ; and (4) pancreatic lipase–related protein 1, which belongs to an enzymatic superfamily of lipases, esterases, and thioesterases.30 In contrast, 17ß-estradiol decreased the content of numerous mRNAs, such as that of dynamin binding protein, which plays a role in membrane trafficking,28 and of metaxin 1, which is involved in the transport of proteins into mitochondria.28

Progesterone administration induced a significant increase in the expression of genes involved in signal transduction and cell communication and a significant decrease in those associated with metabolism and cell growth and/or maintenance. As examples, progesterone enhanced the activity of genes encoding mitogen-activated protein kinase kinase kinase 4, as well as for (1) tachykinin receptor 2, which binds substance K (neurokinin A) and activates a phosphatidylinositol-calcium second messenger system28 ; (2) solute carrier family 2 (facilitated glucose transporter), member 9, which helps to maintain glucose homeostasis28 ; and (3) tumor necrosis factor receptor superfamily, member 19, a protein capable of inducing apoptosis by a caspase-independent mechanism (online). Conversely, progesterone downregulated the expression of genes encoding such proteins as (1) 3-hydroxy-3-methylglutaryl-coenzyme A reductase, which is the rate-limiting enzyme for cholesterol synthesis28 ; (2) dopamine receptor 2, a G protein-coupled receptor that inhibits adenylyl cyclase28 ; (3) solute carrier family 16 (monocarboxylic acid transporters), member 2, which catalyzes the rapid transport of many monocarboxylates across the plasma membrane28 ; (4) cyclin K, which may regulate transcription through the phosphorylation of RNA polymerase II28 ; and (5) RAS p21 protein activator 3, a GTPase-activating protein involved in the control of cell proliferation and differentiation.28

Combined 17ß-estradiol and progesterone exposure elicited many changes in gene expression that were analogous to those of each hormone individually, as well as novel to the combination alone. These sex steroids together altered the activity of numerous cell death, signal transduction, receptor, endocrine, enzymatic, and growth factor genes. Those increased included genes for vascular endothelial growth factor A, discoidin domain receptor 2 (a tyrosine kinase receptor for fibrillar collagen that mediates fibroblast migration and proliferation),28 and the adenosine A2b receptor. Those decreased included genes for insulin-like growth factor I, fibroblast growth factor-10 (may be active in wound healing),28 parathyroid hormone receptor, calpain 9 (a calcium-regulated non-lysosomal thiol-protease),28 and nidogen 1 (participates in the assembly of basement membranes).31

Of particular interest was the influence of 17ß-estradiol, with or without progesterone, on the expression of many immune-related genes. Thus, for example, hormone treatment downregulated the expression of genes linked to histocompatibility 2, O region alpha locus (i.e., MHC class II, a chain),28 CDw131 antigen (a high-affinity receptor for IL-3, IL-5 and granulocyte-macrophage colony-stimulating factor),28 IL-3 (stimulates development of stem cells, granulocytes, macrophages, mast cells, and eosinophils),28 IL-2 receptor ß chain (receptor for IL-2),28 IL-12 {alpha} and ß chains (growth factors for activated T and NK cells),28 small inducible cytokine A28 (chemotactic for resting CD4, CD8 T-cells and eosinophils),28 cell surface glycoprotein CD200 (OX2) receptor (involved in regulation of macrophage function),32 and toll-like receptor 9 (participates in the innate immune response to microbial agents).28 Estrogen also enhanced the mRNA levels of suppressor of cytokine signaling 3, which inhibits the signaling of specific proinflammatory cytokines.33 34 35 36 It is possible that some of these hormonal effects could contribute to an anti-inflammatory action of 17ß-estradiol in the lacrimal gland. Indeed, such an immunologic role for estrogen in lacrimal tissue has been proposed by several investigators.37

However, other effects of 17ß-estradiol, again with or without progesterone, do not appear to be consistent with such a proposition. For instance, hormone administration upregulated the expression of lacrimal gland genes related to transcription factor 7 (involved in T cell differentiation),28 T-cell surface antigen CD2 (promotes T-cell adhesion to other cell types)28 and small inducible cytokine B15 (chemotactic for neutrophils).28 Sex steroid treatment also reduced the gene expression for (1) leukocyte immunoglobulin-like receptor (subfamily B member 4), a receptor for class I MHC antigens, which is involved in the downregulation of the immune response and the development of tolerance,28 and (2) Foxp3, which acts as a rheostat of the immune response.38 A reduction in Foxp3 function, in turn, may attenuate the activity of regulatory CD4+ CD25+ T cells and promote both lymphoproliferation38 and autoimmune disease.39 Of interest, estrogen has opposite effects on Foxp3 expression elsewhere in the body.40 41

Another observation is that 17ß-estradiol exposure significantly increased the mRNA levels of asialoglycoprotein receptor 1. This receptor has been linked to the development of exocrine gland inflammation and keratoconjunctivitis sicca.42 43 44 Collectively, these latter findings suggest that estrogen may promote inflammation and autoimmune disease in the lacrimal gland. If so, such an action may explain why 17ß-estradiol administration to a female mouse model of Sjögren’s syndrome caused a significant increase in the area of lymphoid infiltrates in lacrimal tissue.22 Moreover, a proinflammatory effect would be consistent with estrogen’s known ability to enhance the polyclonal B cell activation, autoantibody formation, and tissue abnormalities encountered in this autoimmune disorder.22 45 46

The mechanism by which 17ß-estradiol and progesterone act on the lacrimal gland may involve classic receptors. Estrogen and progesterone receptor mRNA have been identified in lacrimal glands of rats, rabbits, and humans,47 48 and putative estrogen-binding sites have been detected in a pooled cytosol preparation from rabbit lacrimal glands.24 However, there is no evidence indicating that estrogen or progesterone receptor mRNAs are translated into saturable, high-affinity, specific, and functional proteins.20 49 It should be noted that low-affinity receptors for estrogens appear to exist in rat lacrimal tissue, but these sites may actually represent a low-affinity association of estrogens to androgen receptors.49 A further possibility is that estrogen and progesterone may act on the lacrimal gland indirectly through the control of other hormones (e.g., from the pituitary), or through nonclassic pathways (e.g., signaling through membrane receptors).50

Our study indicated that 17ß-estradiol and progesterone, whether alone or together, seem to contribute little to the known sex-related differences in gene expression of the lacrimal gland.25 Estrogen (15%) and progesterone (2%) influenced only a small percentage of those lacrimal tissue genes that have been reported to vary significantly between males and females.25 In contrast, androgens are involved in more than 70% of these variations51 and may therefore represent the major factor underlying the sexual dimorphism of the lacrimal gland.

In summary, our findings demonstrate that 17ß-estradiol and progesterone exert a considerable impact on gene expression of the lacrimal gland. Our ongoing research is designed to determine the functional significance of these sex steroid actions.


   Acknowledgements
 
The authors thank Christian B. Wade (Seattle, WA) for assistance with the GeneSifter software and the Harvard Center for Genomic Research for help with processing GEM 1 and 2.


   Footnotes
 
Supported by National Eye Institute Grants EY05612, K16, and EY12523, Allergan, Advanced Medical Optics, Bausch & Lomb, the German Research Society (Deutsche Forschungsgemeinschaft) Grants SCHI 562/1-1 and 1-2, and the Japanese Eye Bank Association.

Submitted for publication July 29, 2005; revised September 13 and October 1, 2005; accepted November 28, 2005.

Disclosure: T. Suzuki, None; F. Schirra, None; S.M. Richards, None; N.S. Treister, None; M.J. Lombardi, None; P. Rowley, None; R.V. Jensen, None; D.A. Sullivan, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: David A. Sullivan, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114; sullivan{at}vision.eri.harvard.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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