June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
TESTOSTERONE AND PROGESTERONE IN HUMAN MEIBOMIAN GLAND EPITHELIAL CELLS DETECTED USING LC-MS
Author Affiliations & Notes
  • Blanka Golebiowski
    School of Optometry and Vision Science, University of New South Wales, Sydney, New South Wales, Australia
  • Minh Anh Thu Phan
    School of Optometry and Vision Science, University of New South Wales, Sydney, New South Wales, Australia
  • Martin Bucknall
    School of Optometry and Vision Science, University of New South Wales, Sydney, New South Wales, Australia
  • Michele C Madigan
    School of Optometry and Vision Science, University of New South Wales, Sydney, New South Wales, Australia
  • Mark Willcox
    School of Optometry and Vision Science, University of New South Wales, Sydney, New South Wales, Australia
  • Fiona Stapleton
    School of Optometry and Vision Science, University of New South Wales, Sydney, New South Wales, Australia
  • Footnotes
    Commercial Relationships   Blanka Golebiowski, None; Minh Anh Thu Phan, None; Martin Bucknall, None; Michele Madigan, None; Mark Willcox, None; Fiona Stapleton, None
  • Footnotes
    Support  ARC Discovery Grant
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 714. doi:
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      Blanka Golebiowski, Minh Anh Thu Phan, Martin Bucknall, Michele C Madigan, Mark Willcox, Fiona Stapleton; TESTOSTERONE AND PROGESTERONE IN HUMAN MEIBOMIAN GLAND EPITHELIAL CELLS DETECTED USING LC-MS. Invest. Ophthalmol. Vis. Sci. 2021;62(8):714.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Sex hormones may regulate lipid production in the meibomian glands. Sex hormone receptors and mRNAs of key steroidogenic enzymes are present in human meibomian glands and in meibomian gland cells, suggesting these hormones are locally synthesised and metabolised. However, the presence of sex hormones in meibomian gland cells or tissue has not yet been reported. This study aimed to determine whether androgens or estrogens can be synthesized in immortalised human meibomian gland cells (iHMGEC) from the precursor dehydroepiandrosterone (DHEA).

Methods : Undifferentiated and PPARγ-agonist-differentiated iHMGEC were cultured in serum-free media and treated for 3 days with DHEA (0.1 and 1 μM). Culture supernatant and cell lysates were extracted with tert-butyl methyl ether. Detection of sex hormones in the extracts was carried out by liquid chromatography-mass spectrometry on a Thermo QExactive Plus quadrupole-orbitrap MS. The extracts were examined for the presence of testosterone, dihydrotestosterone, estrone, estradiol, estriol, androstenedione, progesterone and DHEA. All experiments and analyses were run in duplicate.

Results : Testosterone, androstenedione and DHEA were detected in cell lysates of DHEA-treated cells cultured with and without PPARγ-agonist (rosiglitazone) and in the culture supernatants, whereas progesterone was detected in cell lysates only. When cells were cultured without DHEA, testosterone, androstenedione, progesterone and DHEA were detected in the cell lysates only, but not the supernatant. Signal intensity (ion counts) indicated that the detected sex hormones were present in much greater abundance in the supernatant than in cell lysate. Dihydrotestosterone and estrogens were not detectable in any of the samples using our current methods.

Conclusions : iHMGEC synthesize the androgens testosterone and androstenedione from the precursor DHEA, followed by the release of these sex hormones from cells into the culture medium. The synthesis of dihydrotestosterone and the estrogens will be confirmed using a more sensitive MS approach.

This is a 2021 ARVO Annual Meeting abstract.

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