To the Editor:
We read with great interest the article of Tan et al. (1) which reported that thrombospodin-1 (TSP-1), a multifunctional extracellular naturally occurring angiostatic protein is differentially regulated by ovarian steroids. Although it is of great clinical value to understand the mechanism(s) involved in TSP-1 regulation by ovarian steroidal hormones especially in relation to how it is involved in endometriosis. However, there are some points we would like to raise regarding the study of Tan et al. (1).
Tan et al. (1) did not give the age range of the patients and their endometriosis classification in this study.
It has been found that phenol red, which bears a structural resemblance to some non-steroidal estrogens and which is used ubiquitously as a pH indicator in tissue culture media, has significant estrogenic activity. It stimulates the proliferation of estrogen receptors and at the concentrations present in tissue culture media, phenol red causes partial estrogenic stimulation, increasing cell number to 200% and progesterone receptor content to 300% of that found for cells grown in phenol red-free media. The estrogenic activity of phenol red should be considered in any studies that utilize estrogen-responsive cells in culture (2). However, Tan et al. (1) did not indicate whether their serum-free medium has phenol red which may influence estrogenic activities.
Generally, mRNA expression on cultured cells peaks at 6-8 hours and returns to normal in 24 hours, however, Tang et al (1) reported significant maximal estrogenic effect in TSP-1 mRNA expression in 4 hours. Similarly, protein synthesis starts at about 24 hours but in this study, at 2 hours there is already a noticeable effect. Data also obtained by Tang et al (1) on the effect of progesterone on endometrial cells for TSP-1 protein expression was maximum at 4-8 hours and returned to control levels after 24 hours. This seems unusual and no explanation was given for the phenomenon.
Ovarian hormones are known to induce both genomic and non-genomic effects. Tang et al (1) did not include this in their discussion. Interestingly, Mirkin et al (3) did not observe any stimulatory or inhibitory effect on TSP-1 in in-vitro cultured Ishikawa cells also endometrial cells. TSP-1 mRNA and protein expression induced by estrogen in endothelial cells was found to be transient and meditated via estrogen receptor-alpha (4). However, Tang et al (1) did not show whether the increased TSP-1 expression observed in their study was mediated via estrogen receptors despite that estrogen regulate the expression of many genes via interactions with its receptors.
Louis C Ajonuma M.D, Ph.D.a
Mary U Ajuonuma B.Sc., B.Pharm.b
Blessing C Ajuonuma B.Sc., R.N.a,c
Chika L Chukwu M.D, F.R.S.P.H.a,d
Joshua U Ajuonuma B.Sc., B.Pharm.a,e
aSt Mary’s Maternity Hospital
Amakohia, Ihitte, Etiti
Imo State Nigeria
bGeneral Hospital Mina
Niger State, Nigeria
cFederal Medical Center
Owerri, Imo State, Nigeria
dSpecialist Hospital Port Harcourt
Rivers State, Nigeria
eCollege of Pharmacy
Lyceum Northwestern University
Dagupan City, The Philippines
1. Tan XJ , Lang JH, Zheng WM, Leng JH, Zhu L. Ovarian steroid hormones differentially regulate thrombospondin-1 expression in cultured endometrial stromal cells: implications for endometriosis. Fertil Steril 2010; 93:328–31.
2. Berthois Y, Karzeneiienbogen JA, Karzeneiienbogen BS. Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proc Natl Acad Sci USA 1986; 83:2496-500.
3. Mirkin S, Archer DF. Effects of levonorgestrel, medroxyprogesterone acetate, norethindrone, progesterone, and 17 beta-estradiol on thrombospondin-1 mRNA in Ishikawa cells, Fertil Steril 2004; 82:220–2.
4. Sengupta K, Banerjee S, Saxena NK, Banerjee SK. Thombospondin-1 disrupts estrogen-induced endothelial cell proliferation and migration and its expression is suppressed by estradiol. Mol Cancer Res 2004; 2:150–8.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2010.03.038
The Authors Respond:
We thank Dr. Ajonuma and colleagues for their interest and comments regarding our recent report, “Ovarian steroid hormones differentially regulate thrombospondin-1 expression in cultured endometrial stromal cells: implication for endometriosis” (1). It is a good opportunity for us to clarify some points related to our study which were not expressed in the published correspondence.
As to the patients’ age range and endometriosis classification, we are regretful that we did not provide this information in the article. Since our study was based on cultured cells rather than patients, we did not recognize the importance of this information initially. In fact, the patients in our study were selected strictly, with the median age of 35 years old (range 25~44). The stage of endometriosis was determined according to the revised classification of The American Fertility Society (AFS) (2), with 5 patients of stage II, 6 of stage III, and 4 of stage IV. In order to harvest more stromal cells, all endometrial samples were collected in the proliferative phase of menstrual cycle.
We totally agree with Dr. Ajonuma about the possible effects of phenol red on the studies related to estrogenic activities. Indeed, the medium used in our study was phenol-free RPMI 1640 ( Invitrogen; Cat. No. 1835030), and we had stated this information in the legend of Figure 1 (…in serum free and hormone-depleted culture condition). However, due to some cursoriness, we did not emphasize it in the text.
With regard to the enquiry of time interval between stimulation and the peaks of TSP-1 messenger RNA and protein expressions in our study, we agree that an exogenous gene takes 6-8 hours to peak at mRNA level upon transfected into cells and its protein synthesis is usually detected at 24-48 hours. Endogenous gene and protein, however, can be much swifter in response to challenges via genomic or non-genomic pathway, depending on the nature of the stimuli and their receptors. Drug functions through nuclear-located receptor, like ovarian steroid hormones, generally stirs gene expression changes much faster than those functions through cell membrane-located receptor. In accordance with our results, Kanga et al (3) and Sengupta et al (4) also observed that TSP-1 mRNA and protein expression respond very sensitive to stimulations.
Anyway, our preliminary study mainly focused on observing effects of ovarian steroid hormones on the expression of thrombospondin-1 in endometrial stromal cells in vitro, and we were not able to explore the detailed mechanisms of these effects in the meantime. Further investigation to the mechanism is ongoing in our laboratory. In fact, when we were preparing the manuscript, we did notice the reports of Sengupta et al (4) and Mirkin et al (5), and we had included their findings in the discussion.
Xian-Jie Tan, M.D., Ph.D.
Jing-He Lang, M.D.
Department of Obstetrics and Gynecology
Peking Union Medical College Hospital,
Peking Union Medical College
Chinese Academy of Medical Sciences
Beijing, People’s Republic of China
1. Tan XJ, Lang JH, Zheng WM, Leng JH, Zhu L. Ovarian steroid hormones differentially regulate thrombospondin-1 expression in cultured endometrial stromal cells: implications for endometriosis. Fertil Steril 2010;93:328-31.
2. American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine Classification of Endometriosis 1996. Fertil Steril 1997;67:817–21.
3. Kanga JH, Kimb SA, Honga KJ. Induction of TSP1 gene expression by heat shock is mediated via an increase in mRNA stability. FEBS Letters 2006; 580: 510-6.
4. Sengupta K, Banerjee S, Saxena NK, Banerjee SK. Thombospondin-1 disrupts estrogen-induced endothelial cell proliferation and migration and its expression is suppressed by estradiol. Mol Cancer Res 2004;2:150-8.
5. Mirkin S, Archer DF. Effects of levonorgestrel, medroxyprogesterone acetate, norethindrone, progesterone, and 17beta-estradiol on thrombospondin-1 mRNA in Ishikawa cells. Fertil Steril 2004;82:220-2.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2010.03.037