Abstract
OBJECTIVE:
High genistein doses have been reported to induce fluid accumulation in the uteri of ovariectomised rats, although the mechanism underlying this effect remains unknown. Because genistein binds to the oestrogen receptor and the cystic fibrosis transmembrane regulator mediates uterine fluid secretion, we hypothesised that this genistein effect involves both the oestrogen receptor and cystic fibrosis transmembrane regulator.
METHODS:
Ovariectomised adult female Sprague-Dawley rats were treated with 25, 50, or 100 mg/kg/day genistein for three consecutive days with and without the ER antagonist ICI 182780. One day after the final drug injection, the animals were humanely sacrificed, and the uteri were removed for histology and cystic fibrosis transmembrane regulator mRNA and protein expression analysis using real-time polymerase chain reaction and Western blotting, respectively. The cystic fibrosis transmembrane regulator protein distribution was analysed visually by immunohistochemistry.
RESULTS:
The histological analysis revealed an increase in the circumference of the uterine lumen with increasing doses of genistein, which was suggestive of fluid accumulation. Moreover, genistein stimulated a dose-dependent increase in the expression of cystic fibrosis transmembrane regulator protein and mRNA, and high-intensity cystic fibrosis transmembrane regulator immunostaining was observed at the apical membrane of the luminal epithelium following 50 and 100 mg/kg/day genistein treatment. The genistein-induced increase in uterine luminal circumference and cystic fibrosis transmembrane regulator expression was antagonised by treatment with ICI 182780.
CONCLUSION:
Genistein-induced luminal fluid accumulation in ovariectomised rats' uteri involves the oestrogen receptor and up-regulation of cystic fibrosis transmembrane regulator expression, and these findings reveal the mechanism underlying the effect of this compound on changes in fluid volume in the uterus after menopause.
Genistein; Cystic Fibrosis Transmembrane Regulator; Oestrogen Receptor
INTRODUCTION
Genistein, a phyto-oestrogen, is capable of binding to oestrogen receptor (ER)-α and
β, which are expressed in the uterus (11. Norrby M, Madej A, Ekstedt E, Holm L. Effects of genistein on oestrogen and
progesterone receptor, proliferative marker Ki-67 and carbonic anhydrase localisation in the uterus
and cervix of gilts after insemination. Anim Reprod Sci. 2013 Feb 6. pii:
S0378-4320(13)00034-1.). This compound
has also been reported to induce morphological changes (22. Diel P, Geis R-B, Caldarelli A, Schmidt S, Leschowsky UL, Voss A, et al. The
differential ability of the phytoestrogen genistein and of estradiol to induce uterine weight and
proliferation in the rat is associated with a substance specific modulation of uterine gene
expression. Molecular and Cellular Endocrinology. 2004;221(1-2):21-32,
http://dx.doi.org/10.1016/j.mce.2004.04.006.
http://dx.doi.org/10.1016/j.mce.2004.04....
),
luminal fluid secretion (33. Salleh N, Helmy MM, Fadila KN, Yeong SO. Isoflavone genistein induces fluid
secretion and morphological changes in the uteri of post-pubertal rats. Int J Med Sci.
2013;10(6):665-75.) and proliferation of the
endometrium, as evidenced by the increased expression of proliferative markers (33. Salleh N, Helmy MM, Fadila KN, Yeong SO. Isoflavone genistein induces fluid
secretion and morphological changes in the uteri of post-pubertal rats. Int J Med Sci.
2013;10(6):665-75.,44. Rimoldi GCJ, Seidlova-Wuttke D, Jarry H, Wuttke W. Effects of chronic genistein
treatment in mammary gland, uterus, and vagina. Environ Health Perspect. 2007;115(Suppl
1):62-8.) in the uteri of
ovariectomised adult rats. These genistein-mediated effects may have various implications on the
female reproductive system. For example, genistein-induced changes may help to restore some uterine
functions and reduce uterine atrophy after menopause (55. Santos E, Sampaio M, Cecon P, Jesus Simões M, Sartori M, Girão M.
Effects of soy isoflavones on the uterus and urethra of ovariectomized rats. Int Urogynecol J.
2010;21(1):111-6, http://dx.doi.org/10.1007/s00192-009-0995-6.
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). At
high doses, however, genistein may produce harmful effects because it can stimulate the development
of endometrial hyperplasia (33. Salleh N, Helmy MM, Fadila KN, Yeong SO. Isoflavone genistein induces fluid
secretion and morphological changes in the uteri of post-pubertal rats. Int J Med Sci.
2013;10(6):665-75.,66. Chandrareddy A, Muneyyirci-Delale O, McFarlane SI, Murad OM. Adverse effects of
phytoestrogens on reproductive health: A report of three cases. Complementary Therapies in Clinical
Practice. 2008;14(2):132-5, http://dx.doi.org/10.1016/j.ctcp.2008.01.002.
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). There is also evidence that genistein can affect female fertility by interfering with
the normal development of the reproductive system (77. Jefferson WN, Padilla-Banks E, Newbold RR. Disruption of the female reproductive
system by the phytoestrogen genistein. Reprod Toxicol. 2007;23:308-16,
http://dx.doi.org/10.1016/j.reprotox.2006.11.012.
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,88. Jefferson WN, Padilla-Banks E, Phelps JY, Cantor AM, Williams CJ. Neonatal
Phytoestrogen Exposure Alters Oviduct Mucosal Immune Response to Pregnancy and Affects
Preimplantation Embryo Development in the Mouse. Biol Reprod. 2012;87(1):10,1-10.) and the normal pattern of the reproductive cycle (77. Jefferson WN, Padilla-Banks E, Newbold RR. Disruption of the female reproductive
system by the phytoestrogen genistein. Reprod Toxicol. 2007;23:308-16,
http://dx.doi.org/10.1016/j.reprotox.2006.11.012.
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).
The reported effects of genistein on fluid secretion may affect the volume of fluid in the
uterus. Under conditions in which a low amount of fluid is present, such as in menopause (99. Inceboz U, Uyar Y, Baytur Y, Kandiloglu AR. Endometrial fluid in postmenopausal
women. Int J Gynaecol Obstet. 2009;107(2):154-5.), genistein may help to restore the uterine fluid volume.
Although changes in morphology and fluid secretion have been reported in rodent uteri, genistein has
been shown to have no significant effect on restoring normal endometrial morphology in humans (1010. D'Anna R, Cannata ML, Marini H, Atteritano M, Cancellieri F, Corrado F, et
al. Effects of the phytoestrogen genistein on hot flushes, endometrium, and vaginal epithelium in
postmenopausal women: a 2-year randomized, double-blind, placebo-controlled study. Menopause.
2009;16(2):301-6, http://dx.doi.org/10.1097/gme.0b013e318186d7e2.
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) and primates (1111. Wood CE, Appt SE, Clarkson TB, Franke AA, Lees CJ, Doerge DR, et al. Effects of
High-Dose Soy Isoflavones and Equol on Reproductive Tissues in Female Cynomolgus Monkeys. Biol
Reprod. 2006;75(3):477-86, http://dx.doi.org/10.1095/biolreprod.106.052142.
http://dx.doi.org/10.1095/biolreprod.106...
) after
menopause. The presence of fluid within the uterine cavity in the post-menopausal period is
typically associated with endometrial pathology, although increased fluid accumulation has also been
observed in healthy post-menopausal women (99. Inceboz U, Uyar Y, Baytur Y, Kandiloglu AR. Endometrial fluid in postmenopausal
women. Int J Gynaecol Obstet. 2009;107(2):154-5.). Therefore, the
function of fluid in the uterus post-menopause remains unknown. In females of reproductive age, a
normal amount of uterine fluid is important to sustain fertility (1212. Yang J, Ajonuma LC, Tsang LL, Lam SY, Rowlands DK, Ho LS, et al. Differential
expression and localization of CFTR and ENaC in mouse endometrium during pre-implantation. Cell Biol
Int. 2004;28(6):433-9, http://dx.doi.org/10.1016/j.cellbi.2004.03.011.
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), as any interference with the fluid volume can result in infertility (1313. Naftalin R, Thiagarajah, Pedley K, Pocock V, Milligan S. Progesterone
stimulation of fluid absorption by the rat uterine gland. Reproduction. 2002.
2002;123(5):633-8.). Before menopause, uterine fluid volume regulation is under the
control of sex steroids such as oestrogen (E2) and progesterone (P4) (1414. Salleh N, Baines DL, Naftalin RJ, Milligan SR. The Hormonal Control of Uterine
Luminal Fluid Secretion and Absorption. J Membr Biol. 2005;206(1):17-28,
http://dx.doi.org/10.1007/s00232-005-0770-7.
http://dx.doi.org/10.1007/s00232-005-077...
).
The cystic fibrosis transmembrane regulator (CFTR), a cAMP-dependent chloride channel, plays an
important role in mediating uterine fluid secretion (1515. Gholami K, Muniandy S, Salleh N. Progesterone downregulates oestrogen-induced
expression of CFTR and SLC26A6 proteins and mRNA in rats' uteri. J Biomed Biotechnol.
2012;2012:596084.,1616. Chan L, Tsang LL, Rowlands DK, Rochelle LG, Boucher RC, Liu CQ, et al.
Distribution and regulation of ENaC subunit and CFTR mRNA expression in murine female reproductive
tract. J Membr Biol. 2002;185(2):165-76,
http://dx.doi.org/10.1007/s00232-001-0117-y.
http://dx.doi.org/10.1007/s00232-001-011...
). CFTR mutations lead to cystic fibrosis, a disease associated
with defective Cl- and fluid secretion (1717. Marson FABC, Secolin R, Ribeiro AF, Ribeiro JD. Genetic interaction of GSH
metabolic pathway genes in cystic fibrosis. BMC Med Genet. 2013;10(14(1)):60,
http://dx.doi.org/10.1186/1471-2350-14-60.
http://dx.doi.org/10.1186/1471-2350-14-6...
). CFTR
has been reported to be expressed at the apical membrane of the endometrial epithelium (1414. Salleh N, Baines DL, Naftalin RJ, Milligan SR. The Hormonal Control of Uterine
Luminal Fluid Secretion and Absorption. J Membr Biol. 2005;206(1):17-28,
http://dx.doi.org/10.1007/s00232-005-0770-7.
http://dx.doi.org/10.1007/s00232-005-077...
), and its function is dependent on cyclic AMP (cAMP) (1818. Bargon J, Trapnell BC, Chu CS, Rosenthal ER, Yoshimura K, Guggino WB, et al.
Down-regulation of cystic fibrosis transmembrane conductance regulator gene expression by agents
that modulate intracellular divalent cations. Mol Cell Biol. 1992;12(4):1872-8.). In addition to secreting fluid and Cl-, CFTR has also been
reported to participate in cAMP-dependent HCO3- secretion (1919. Wang XF, Zhou CX, Shi QX, Yuan YY, Yu MK, Ajonuma LC, et al. Involvement of CFTR
in uterine bicarbonate secretion and the fertilizing capacity of sperm. Nat Cell Biol.
2003;5(10):902-6, http://dx.doi.org/10.1038/ncb1047.
http://dx.doi.org/10.1038/ncb1047...
). CFTR expression is under the influence of sex-steroids; this
protein is down-regulated by P4 and up-regulated by E2, during the proestrus
and estrus stages of the oestrous cycle in rats (1515. Gholami K, Muniandy S, Salleh N. Progesterone downregulates oestrogen-induced
expression of CFTR and SLC26A6 proteins and mRNA in rats' uteri. J Biomed Biotechnol.
2012;2012:596084.). The
effect of genistein on uterine CFTR expression is unknown, although this compound has been reported
to affect CFTR expression in the intestine (2020. Al-Nakkash L, Batia L, Bhakta M, Peterson A, Hale N, Skinner R, et al.
Stimulation of Murine Intestinal Secretion by Daily Genistein Injections: Gender-dependent
Differences. Cell Physiol Biochem. 2011;28(2):239-50,
http://dx.doi.org/10.1159/000331736.
http://dx.doi.org/10.1159/000331736...
,2121. Tuo B, Wen G, Seidler U. Differential activation of the HCO3- conductance
through the cystic fibrosis transmembrane conductance regulator anion channel by genistein and
forskolin in murine duodenum. Br J Pharmacol. 2009;158(5):1313-21.), kidney (2222. Schmidt A, Hughes LK, Cai Z, Mendes F, Li H, Sheppard DN, et al. Prolonged
treatment of cells with genistein modulates the expression and function of the cystic fibrosis
transmembrane conductance regulator. Br J Pharmacol. 2008;153(6):1311-23.), airways
(2323. Andersson C, Servetnyk Z, Roomans GM. Activation of CFTR by genistein in human
airway epithelial cell lines. Biochem Biophys Res Commun. 2003;308(3):518-22,
http://dx.doi.org/10.1016/S0006-291X(03)01436-0.
http://dx.doi.org/10.1016/S0006-291X(03)...
), and epididymis (2424. Leung GPH, Wong PYD. Activation of Cystic Fibrosis Transmembrane Conductance
Regulator in Rat Epididymal Epithelium by Genistein. Biol Reprod. 2000;62(1):143-9,
http://dx.doi.org/10.1095/biolreprod62.1.143.
http://dx.doi.org/10.1095/biolreprod62.1...
).
Genistein is widely consumed as a supplement to prevent or alleviate menopause-related symptoms
such as hot flashes, night sweats, and headaches (2525. Reed SD, Lampe JW, Qu C, Gundersen G, Fuller S, Copeland WK, et al.
Self-reported menopausal symptoms in a racially diverse population and soy food consumption.
Maturitas. 2013;75(2):152-8, http://dx.doi.org/10.1016/j.maturitas.2013.03.003.
http://dx.doi.org/10.1016/j.maturitas.20...
). In
addition, this compound has also been shown to assist in overcoming post-menopausal-related
complications such as osteoporosis (2626. Tsuang Y-H, Chen L-T, Chiang C-J, Wu L-C, Chiang Y-F, Chen P-Y, et al.
Isoflavones prevent bone loss following ovariectomy in young adult rats. J Orthop Surg Res.
2008;3(1):12, http://dx.doi.org/10.1186/1749-799X-3-12.
http://dx.doi.org/10.1186/1749-799X-3-12...
) and cardiovascular
diseases such as hypertension and ischaemic heart disease (2727. Squadrito F, Altavilla D, Squadrito G, Saitta A, Cucinotta D, Minutoli L, et al.
Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced
by ovariectomy in rats. Cardiovasc Res. 2000;45(2):454-62,
http://dx.doi.org/10.1016/S0008-6363(99)00359-4.
http://dx.doi.org/10.1016/S0008-6363(99)...
). Despite its wide use, the effect of genistein on the post-menopausal uterus remains
poorly characterised. However, limited data indicate that this compound can cause fluid accumulation
in the uteri of ovariectomised rats (33. Salleh N, Helmy MM, Fadila KN, Yeong SO. Isoflavone genistein induces fluid
secretion and morphological changes in the uteri of post-pubertal rats. Int J Med Sci.
2013;10(6):665-75.). Because the
underlying mechanism is unknown, the present study investigated the involvement of the ER and CFTR
in regulating this genistein-mediated effect. This study is of clinical significance because the
presence of fluid in the uterine cavity of post-menopausal women could be due to high genistein
consumption in addition to other factors known to cause fluid accumulation in the uterus during this
period.
METHODS
Animal preparation and hormone treatment
Three-month-old adult female Sprague-Dawley (SD) rats weighing approximately 225 g were housed in a clean and well-ventilated animal room with standardised housing conditions (lights on for 12 h from 06:00 h to 18:00 h; room temperature of 24°C; 5-6 animals per cage). Post-weaning, the animals were provided free access to a soy-free diet (Gold-Coin Pellets) and water free of dissolved endocrine-disrupting chemicals (EDCs). All procedures were approved by the Faculty of Medicine, Animal Care and Use Committee, University of Malaya, under ethics number FIS/01/12/2008 (NFK). Genistein (G-6055) was purchased from LC Laboratories (Woburn, MA, USA) at greater than 99% purity; this compound is a crystalline powder with a light yellowish colour.
Bilateral ovariectomy was performed under isoflurane anaesthesia at least ten days prior to drug
treatment to eliminate the effect of endogenous sex steroids (1414. Salleh N, Baines DL, Naftalin RJ, Milligan SR. The Hormonal Control of Uterine
Luminal Fluid Secretion and Absorption. J Membr Biol. 2005;206(1):17-28,
http://dx.doi.org/10.1007/s00232-005-0770-7.
http://dx.doi.org/10.1007/s00232-005-077...
). After surgery, the animals were given intramuscular injections of 0.1 ml Kombitrim
antibiotic to prevent post-surgical wound infection. The animals were divided into eight groups
(n = 6 per group). Group 1 was treated for three days with peanut oil (vehicle);
groups 2 to 7 received subcutaneous injections of genistein at doses of 25, 50, and 100 mg/kg/day
for three consecutive days with and without the ER antagonist ICI 182780 at 100 µg/kg/day; and
group 8 received ICI 182780 only at 100 µg/kg/day. The drugs were dissolved in peanut oil and
were then administered via subcutaneous injection behind the scruff of the neck.
Uterine morphological analyses
At the end of the 3-day period, the rats were sacrificed via anaesthetic overdose followed by cervical dislocation 24 h after the last drug treatment. The right uterine horns were immediately harvested and fixed in 4% paraformaldehyde (PFD) at 4°C for 4 to 5 h. A standardised region of the uterine horn for all animals (mid-portion) was processed using an automated tissue processing machine (Leica, Germany). Tissues were then embedded in paraffin wax using conventional methods, and sections with a thickness of 5 µm were prepared and mounted onto glass slides. The sections were then stained with haematoxylin and eosin (H&E) and visualised under a light microscope (Olympus, Japan). The circumference of the uterine lumen was measured using the NIS-Elements AR program. All images were captured using a Nikon Eclipse 80i camera that was attached to the light microscope.
Quantification of CFTR mRNA expression by real-time PCR
Immediately after the animals were sacrificed, the uteri were removed and placed into RNAlater
solution (Ambion, USA), which maintains RNA integrity prior to extraction. In this study, the
absolute quantification method, which correlates the PCR signal to a standard curve, was used to
determine the copy number of RNA (2828. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8,
http://dx.doi.org/10.1006/meth.2001.1262.
http://dx.doi.org/10.1006/meth.2001.1262...
). Absolute
quantification provides the exact copy number (2929. Watzinger F, Suda M, Preuner S, Baumgartinger R, Ebner K, Baskova L, et al.
Real-Time Quantitative PCR Assays for Detection and Monitoring of Pathogenic Human Viruses in
Immunosuppressed Pediatric Patients. J Clin Microbiol. 2004;42(11):5189-98,
http://dx.doi.org/10.1128/JCM.42.11.5189-5198.2004.
http://dx.doi.org/10.1128/JCM.42.11.5189...
) and can be
used to compare various treatment groups. In brief, the protocol for PCR included the following
steps. First, total RNA was freshly isolated from rat uteri using the RNeasy Plus Mini Kit (Qiagen,
USA). The total RNA concentration in the tissue samples was then determined using a Nanodrop
(Fisher-Scientific). Reverse transcription into cDNA was performed using a High-capacity RNA-to-cDNA
Kit (Applied Biosystems, USA). Taqman® real-time PCR was used to evaluate gene expression, with
B-actin and GAPDH serving as endogenous controls. The PCR program consisted of 2 min at 50°C
for UNG activity, 20 s at 95°C for activation of the ampliTaq gold DNA polymerase, 1 min of
denaturation at 95°C, 20 s for annealing, and extension at 60°C for 1 min. Denaturing and
annealing were performed for 40 cycles. All TaqMan assays were purchased from Applied Biosystem,
USA. Control reactions included amplifications performed on samples identically prepared with no
reverse transcriptase (-RT) or no added substrate (water control). The CFTR Primer was obtained from
Roche (USA) with id number Rn014559. Each reaction was performed in duplicate on RNA isolated from
three separate animals. The average crossover point (CT) was determined using Roche software and was
then converted into copy number per ng of total RNA by comparison to the standard curve. For
absolute quantification, standard cDNA (Roche, USA) was diluted to different concentrations, and a
standard curve was generated. Quantitative PCR was then performed as described above.
Quantification of CFTR protein expression by Western blotting
Snap-frozen uterine tissues were homogenised using a sonicator with PRO-PREP (Intron) extraction solution in the presence of protease inhibitors. Total cell protein was obtained by centrifugation at 13,000 g for 15 min at 4°C. After determination of the protein concentration, an equal amount of protein was loaded onto a 12% SDS-PAGE gel. The protein was then transferred onto a PVDF membrane and incubated in 5% BSA for 90 min. The blot was then exposed to the primary antibody at a 1:1,000 dilution. After incubation with a CFTR goat polyclonal antibody obtained from Santa Cruz, USA, the blot was incubated with HRP-conjugated secondary antibody and was finally visualised using Optic 4C (Bio RAD). B-actin (abCam, UK) was used as a loading control. Photos of the blots were captured, and the density of each band was determined using Image J software. The ratio of each band/β-actin was determined and was considered the expression level of each of the target proteins.
Localisation of CFTR protein distribution by immunohistochemistry (IHC)
Immunohistochemistry was performed to examine the CFTR protein distribution in the uterus. Uterine paraffin blocks from the various treatment groups were sectioned into 5 µm thick sections and mounted onto glass slides. The tissue sections were incubated in 10% H2O2 in methanol to quench the activity of endogenous peroxidase and were then incubated overnight with primary antibody (rat polyclonal IgG) against CFTR at a 1:100 dilution at 4°C in a humidified chamber. The tissues were then incubated with secondary antibody, i.e., biotinylated rabbit anti-rat IgG (Amersham, UK), at 1:500 for 1 h at room temperature followed by tertiary antibody, i.e., streptavidin-horseradish peroxidase (Amersham, UK), at 1:500 for 1 h at room temperature. The sites of antibody binding were visualised using DAB (diaminobenzidine HCl), which provided a dark brown stain. The sections were then counterstained with haematoxylin for nuclear staining.
Evaluation of immunostaining
The relative intensity of the immunoreactions at the luminal and glandular epithelium was
evaluated and graded blindly by three independent observers using a light microscope (Olympus,
Japan) at 40X and 100X magnifications. The staining intensity was estimated semi-quantitatively on a
scale of 0-3+ (+++) as follows: −, no detectable stain; −/+,
faint; +, moderate; ++, strong and +++, very intense staining, as
previously described (3030. Perrot-Applanat M, Deng M, Fernandez H, Lelaidier C, Meduri G, Bouchard P.
Immunohistochemical localization of estradiol and progesterone receptors in human uterus throughout
pregnancy: expression in endometrial blood vessels. J Clin Endocrinol Metab. 1994;78(1):216-24,
http://dx.doi.org/10.1210/jc.78.1.216.
http://dx.doi.org/10.1210/jc.78.1.216...
).
Statistical analyses
The results are expressed as the mean ± SEM, with n indicating the number of rats. Comparisons between groups were performed using one-way ANOVA. P-values <0.05 were considered statistically significant. Data assist v3 was used to analyse the real-time PCR (qPCR) results.
RESULTS
Effect of genistein treatment on uterine luminal circumference
Figure 1 shows images of uterine sections from ovariectomised rats pre-treated with genistein at various doses with and without the presence of the ER antagonist ICI 182780, whereas Table 1 presents the circumference of the uterine lumen from these treatment groups. Treatment with 25, 50, and 100 mg/kg/day genistein resulted in 1.19-, 3.94-, and 4.76-fold increases in uterine circumference, respectively, compared with the control. Administration of ICI 182780 significantly reduced the genistein-related increase in uterine luminal circumference. In particular, ICI 182780 administration resulted in 1.1-, 3.06-, and 2.17-fold decreases in uterine circumference in the groups receiving 25, 50, and 100 mg/kg/day genistein, respectively. ICI 182780 treatment alone did not cause any significant changes in uterine circumference compared with the control treatment.
Representative images of uterine sections from ovariectomised SD rats treated with (A) peanut oil (control), (B) 25G (25 mg/kg/day genistein), (C) 25G (25 mg/kg/day genistein) + ICI 182780, (D) 50G (50 mg/kg/day genistein), (E) 50G (50 mg/kg/day genistein + ICI 182780), (F) 100G (100 mg/kg/day genistein), (G) 100G (100 mg/kg/day genistein) + ICI 182780, or (H) ICI 182780 (100 μg/kg/day) only for 3 consecutive days. There was a clear increase in the luminal circumference following treatment with 50 and 100 mg/kg/day genistein, although these changes were antagonised by treatment with ICI 182780. Sections were stained with H&E, and the magnification is X4. L indicates the uterine lumen. (G-genistein), n = 6 per treatment group.
Measurement of the circumference of the uterine lumen in ovariectomised rats treated with peanut oil (control) and different doses of genistein (25, 50 and 100 mg/kg/day) with and without ICI 182780. The values represent the mean ± SEM, with n = 6 per treatment group. The circumference was increased with increasing doses of genistein. Administration of ICI 182780 reduced the uterine circumference in all genistein-treated groups.
CFTR mRNA expression analysis by real-time PCR (qPCR)
Based on the CT values obtained for each sample, CFTR mRNA expression was quantified using the
absolute quantification method. Absolute and relative quantifications have been shown to produce
comparable results (3131. Wong ML MJ. Real-time PCR for mRNA quantitation. Biotechniques.
2005;39(1):75-85, http://dx.doi.org/10.2144/05391RV01.
http://dx.doi.org/10.2144/05391RV01...
). Figure 2(a) shows the standard curve generated using standard cDNA. As shown in Figure 2(b), the absolute CFTR mRNA expression was determined
according to the standard curve from CT values obtained in each treatment group. CFTR
mRNA expression was markedly increased following 25, 50, and 100 mg/kg/day genistein treatment, and
these levels were 6.8-, 13.5-, and 26.75-fold greater, respectively, than the control.
Administration of ICI 182780 resulted in a significant inhibition of mRNA expression in all groups
receiving different doses of genistein.
Representative images of the uterine glandular and luminal epithelium under 20X magnification and the luminal epithelium under 100X magnification (in the upper right-hand corner) from ovariectomised SD rats treated with (A) control (vehicle-treated) (B) 25G (25 mg/kg/day genistein), (C) 25G (25 mg/kg/day genistein) + ICI 182780, (D) 50G (50 mg/kg/day), (E) 50G (50 mg/kg/day genistein) + ICI 182780, (F) 100G (100 mg/kg/day genistein), (G) 100G (100 mg/kg/day genistein) + ICI 182780, or (H) ICI 182780 (100 μg/kg/day) only for 3 consecutive days or (I) incubation with a non-immune peptide. There was a clear increase in the intensity of immunostaining in the group treated with 100 mg/kg/day genistein. L- uterine lumen, Gl- uterine gland, G-genistein. Sections were stained with H&E for nuclear staining; n = 6 per treatment group.
CFTR protein expression analysis by Western blotting
As shown in Figure 3, the expression of CFTR protein was increased with increasing doses of genistein. The ratio of CFTR/B-actin was 2.92- and 3.35-fold higher in the groups treated with 50 and 100 mg/kg/day genistein, respectively, compared with the group receiving 25 mg/kg/day genistein treatment. Meanwhile, ICI 182780 administration led to a significant decrease in genistein-induced CFTR protein expression in all treatment groups.
(a) Standard curve obtained from cDNA dilution. The y axis represents the absolute nuclear DNA copy number, and the x axis represents the CT value. The linear regression equation was given by y = 1E+06e-0.34x, R2 = 0.989. (b) CFTR mRNA expression evaluated by real-time PCR. The absolute copy number (per 1 ng mRNA used for real-time PCR) was calculated from the standard curve (a). The expression of CFTR mRNA was 26 times higher following treatment with 100 mg/kg/day genistein compared with the control. The absolute copy number obtained from each sample represents the mean of four assays with the standard deviation. C = control, ICI = ICI 182780, 25G = 25 mg/kg/day genistein, 25G (25 mg/kg/day genistein) + ICI 182780, 50G = 50 mg/kg/day genistein, 50G (50 mg/kg/day genistein) + ICI 182780, 100G = 100 mg/kg/day genistein, 100G (100 mg/kg/day genistein) + ICI 182780.
Analysis of CFTR protein distribution by IHC
Figure 4 shows images of the endometrium at 20× magnification and the luminal epithelium at 100× magnification, whereas Table 2 presents a semi-quantitative analysis of the staining intensity of CFTR in various treatment groups with and without ICI 182780. Our findings indicated that the intensity of immunostaining was highest following treatment with 50 and 100 mg/kg/day genistein, predominantly at the luminal and glandular epithelium, and these intensity values were decreased following ICI 182780 administration. CFTR immunostaining was highest at the apical membrane of the luminal epithelium following treatment with 50 and 100 mg/kg/day genistein. Mild to moderate staining could be observed following treatment with 25 mg/kg/day genistein, which was also reduced following ICI 182780 administration. Incubation with non-immune peptide resulted in no staining (negative control).
The level of CFTR protein expression in ovariectomised rats treated with different doses of genistein (25, 50, and 100 mg/kg/day) with and without ICI 182780. The expression of CFTR protein was increased in a dose-dependent manner with increasing doses of genistein, and this increase was significantly antagonised by ICI 182780. *p<0.05 compared with the respective treatment group without the presence of ICI 182780; n = 6 per group. The data are presented as the mean ± SEM. 25G = 25 mg/kg/day genistein, 25G (25 mg/kg/day genistein) + ICI 182780, 50G = 50 mg/kg/day genistein, 50G (50 mg/kg/day genistein) + ICI 182780, 100G = 100 mg/kg/day genistein, 100G (100 mg/kg/day genistein) + ICI 182780.
Semi-quantitative analysis of the intensity of CFTR immunostaining following different doses of genistein treatment (25, 50, and 100 mg/kg/day) with and without ICI 182780. The staining intensity was graded as follows: −, negative; −/+, faint; +, moderate; ++ strong and +++, very intense. The intensity was confirmed by 3 independent observers.
DISCUSSION
To the best of our knowledge, this study was the first to report the mechanism underlying
genistein-induced fluid accumulation in the uteri of ovariectomised rats. Our study confirmed the
previous observation that genistein at high doses (50 and 100 mg/kg/day) leads to a significant
increase in fluid volume in the uteri of ovariectomised rats (33. Salleh N, Helmy MM, Fadila KN, Yeong SO. Isoflavone genistein induces fluid
secretion and morphological changes in the uteri of post-pubertal rats. Int J Med Sci.
2013;10(6):665-75.). Although the significance of this effect is unknown, it could help to restore fluid in
the uterus after menopause. Because high-dose genistein has also been reported to cause an increase
in the thickness of the endometrium and myometrium in ovariectomised rats (33. Salleh N, Helmy MM, Fadila KN, Yeong SO. Isoflavone genistein induces fluid
secretion and morphological changes in the uteri of post-pubertal rats. Int J Med Sci.
2013;10(6):665-75.), this compound may therefore be used to block uterine atrophy resulting from a
lack of oestrogen. In other studies involving ovariectomised rats, high-dose genistein (100
mg/kg/day) was reported to increase bone mineral density (BMD) (3232. Gallo D, Zannoni GF, Apollonio P, Martinelli E, Ferlini C, Passetti G, et al.
Characterization of the pharmacologic profile of a standardized soy extract in the ovariectomized
rat model of menopause: effects on bone, uterus, and lipid profile. Menopause. 2005;12(5):589-600,
http://dx.doi.org/10.1097/01.GME.0000156348.61767.D5.
http://dx.doi.org/10.1097/01.GME.0000156...
) and partially regress atrophied mammary glands (3333. Gallo D, Zannoni GF, Martinelli E, Ferlini C, Fabrizi M, Riva A, et al.
Estradiol and phytoestrogens differently influence the rodent postmenopausal mammary gland.
Menopause. 2006;13(1):72-9, http://dx.doi.org/10.1097/01.gme.0000191208.05491.94.
http://dx.doi.org/10.1097/01.gme.0000191...
). In humans, consumption of a genistein-rich diet, including approximately 50 mg/day of
this compound, by women in the post-menopausal period has been reported to reduce the incidence of
vasomotor episodes such as hot flashes (3434. Jenks B, Iwashita S, Nakagawa Y, Ragland K, Lee J, Carson W, A pilot study on
the effects of S-equol compared to soy isoflavones on menopausal hot flash frequency. J Womens
Health (Larchmt). 2012;21(6): 674-82, http://dx.doi.org/10.1089/jwh.2011.3153.
http://dx.doi.org/10.1089/jwh.2011.3153...
). Genistein
supplementation has also been found to decrease the total cholesterol (3535. Yang T-S, Wang S-Y, Yang Y-C, Su C-H, Lee F-K, Chen S-C, et al. Effects of
standardized phytoestrogen on Taiwanese menopausal women. Taiwan J Obstet Gynecol.
2012;51(2):229-35.) and low density lipoprotein (LDL) (3636. Hwang J, Sevanian A, Hodis HN, Ursini F. Synergistic inhibition of LDL oxidation
by phytoestrogens and ascorbic acid. Free Radic Biol Med. 2000;29(1):79-89.37,
http://dx.doi.org/10.1016/S0891-5849(00)00322-1.
http://dx.doi.org/10.1016/S0891-5849(00)...
)
levels and to reduce the risk for breast cancer development (3737. Boucher B, Cotterchio M, Anderson LN, Kreiger N, Kirsh VA, Thompson LU. Use of
isoflavone supplements is associated with reduced postmenopausal breast cancer risk.
Int J Cancer. 2013;132(6):1439-50.) in women after menopause.
Our findings revealed that genistein-induced fluid accumulation in the uteri of ovariectomised
rats involves the ER, as evidenced from the antagonising effect of ICI 182780. The effect of
genistein on ER expression in the uterus has been previously reported. Administration of selective
soy extract (SSE) containing genistein at 50 and 100 mg/kg/day to ovariectomised rats was found to
enhance the expression of ER-β in the uterine stroma. This increase in ER-β expression
was shown to negatively modulate the expression of ER-α in the luminal epithelia (3838. Gallo D ZG, Fabrizi M, De Stefano I, Mantuano E, Scambia G. Comparative effects
of 17beta-estradiol and phytoestrogens in the regulation of endometrial functions in the rodent
uterus. J Endocrinol Invest. 2008;31(1):48-56.). Down-regulation of ER-α has also been reported in the
uteri of intact non-ovariectomised rats receiving chronic genistein treatment (3939. Zin S, Omar SZ, Khan NL, Musameh NI, Das S, Kassim NM. Effects of the
phytoestrogen genistein on the development of the reproductive system of Sprague Dawley rats.
Clinics. 2013;68(2):253-62, http://dx.doi.org/10.6061/clinics/2013(02)OA21.
http://dx.doi.org/10.6061/clinics/2013(0...
), suggesting that this effect may be due to the up-regulation of ER-β
expression by genistein. In view of these findings, we speculated that the effect of genistein as
observed in our study may be mediated via ER-β, although further studies are needed to confirm
this hypothesis.
In humans, the presence of fluid within the uterine cavity of post-menopausal women has been
associated with endometrial malignancy (4040. Zalel Y, Tepper R, Cohen I, Goldberger S, Beyth Y. Clinical significance of
endometrial fluid collections in asymptomatic postmenopausal women. J Ultrasound Med.
1996;15(7):513-5.) and cervical
stenosis (4141. Goldstein SR. The presence of endometrial fluid in asymptomatic postmenopausal
women is associated with clinically relevant cervical stenosis. J Ultrasound Med.
1997;16(3):208.). In most cases, however, there is no obvious
relationship between the neoplastic lesions and the amount of fluid within the uterus after
menopause (4242. Bedner R, Rzepka-Górska I. Diagnostic value of uterine cavity fluid
collection in the detection of pre-neoplastic lesions and endometrial carcinoma in the asymptomatic
post-menopausal women. Ginekol Pol. 1998;69(5):237-40.). In women who have no pathological lesions
either in the uterus or cervix, increased uterine fluid amount after menopause is almost always
associated with advancing age (4343. Gull B, Karlsson B, Wikland M, Milsom I, Granberg S. Factors influencing the
presence of uterine cavity fluid in a random sample of asymptomatic postmenopausal women. Acta
Obstet Gynecol Scand. 1998;77(7):751-7,
http://dx.doi.org/10.1080/j.1600-0412.1998.770710.x.
http://dx.doi.org/10.1080/j.1600-0412.19...
) and the co-existence of
medical conditions such as diabetes and hypertension (99. Inceboz U, Uyar Y, Baytur Y, Kandiloglu AR. Endometrial fluid in postmenopausal
women. Int J Gynaecol Obstet. 2009;107(2):154-5.). The
association between the use of hormone replacement therapy (HRT), i.e., oestrogen or selective ER
modulators (SERMs), and post-menopausal uterine fluid accumulation remains unknown. Because our
study found that genistein induced fluid accumulation in the uteri of ovariectomised rats, it is
possible that this compound may lead to increased fluid accumulation in the uterus during the
post-menopausal period. High-dose genistein intake should therefore be considered as one of the
causes for increased fluid levels in the uteri of post-menopausal women.
Our findings also revealed that CFTR is involved in mediating the genistein-induced increase in
the fluid amount in ovariectomised rats' uteri. In particular, we found that the expression of
CFTR mRNA and protein was markedly increased following treatment with 50 and 100 mg/kg/day
genistein. Up-regulation of CFTR mRNA and protein expression was dependent on the ER, suggesting
that this effect was mediated via a genomic pathway. Under the effect of genistein, CFTR was
expressed mainly at the apical membrane of the luminal epithelium, which suggests that it may
participate in fluid, Cl-, and HCO3- secretion into the uterine lumen. The importance of
CFTR in mediating uterine fluid secretion was demonstrated by Navis et al. (4444. Navis A, Marjoram L, Bagnat M. Cftr controls lumen expansion and function of
Kupffer's vesicle in zebrafish. Development. 2013;140(8):1703-12,
http://dx.doi.org/10.1242/dev.091819.
http://dx.doi.org/10.1242/dev.091819...
), in which deletion of the CFTR gene was found to inhibit luminal fluid
secretion. Our hypothesis that CFTR is involved in uterine fluid secretion is also supported by the
observation that a marked increase in uterine CFTR expression occurs in diseases related to
excessive fluid accumulation in the uterus, such as hydrosalpinx (4545. Yang JZ, Jiang X, Dong J, Guo J, Chen H, Tsang LL, et al. Abnormally enhanced
cystic fibrosis transmembrane conductance regulator-mediated apoptosis in endometrial cells
contributes to impaired embryo implantation in controlled ovarian hyperstimulation. Fertil Steril.
2011;95(6):2100-6.e2, http://dx.doi.org/10.1016/j.fertnstert.2011.02.036.
http://dx.doi.org/10.1016/j.fertnstert.2...
). Apart from the uterus, CFTR has been reported to mediate fluid secretion in the small
intestine (4646. Patanayindee J, Muanprasat C, Soodvilai S, Chatsudthipong V. Antidiarrheal
efficacy of a quinazolin CFTR inhibitor on human intestinal epithelial cell and in mouse model of
cholera. Indian J Pharmacol. 2012;44(5):619-23.), submucosal glands (4747. Lee RJ, Foskett JK. Why mouse airway submucosal gland serous cells do not
secrete fluid in response to camp stimulation. J Biol Chem. 2012;287(45):38316-26,
http://dx.doi.org/10.1074/jbc.M112.412817.
http://dx.doi.org/10.1074/jbc.M112.41281...
), and salivary glands (4848. Frizzell RA, Hanrahan JW. Physiology of epithelial chloride and fluid secretion.
Cold Spring Harb Perspect Med. 2012;2(6):a009563.). Therefore, it
is likely that the observed increase in fluid accumulation within the uterine lumen was due to
genistein-induced CFTR up-regulation. In addition to mediating fluid secretion, CFTR has also been
reported to be involved in Cl- and HCO3- secretion in the uterus
(4949. Chan HC, Shi QX, Zhou CX, Wang XF, Xu WM, Chen WY, et al. Critical role of CFTR
in uterine bicarbonate secretion and the fertilizing capacity of sperm. Mol Cell Endocrinol.
2006;250(1-2):106-13, http://dx.doi.org/10.1016/j.mce.2005.12.032.
http://dx.doi.org/10.1016/j.mce.2005.12....
), small intestine (2020. Al-Nakkash L, Batia L, Bhakta M, Peterson A, Hale N, Skinner R, et al.
Stimulation of Murine Intestinal Secretion by Daily Genistein Injections: Gender-dependent
Differences. Cell Physiol Biochem. 2011;28(2):239-50,
http://dx.doi.org/10.1159/000331736.
http://dx.doi.org/10.1159/000331736...
), and airways (5050. Derichs N, Mekus F, Bronsveld I, Bijman J, Veeze HJ, von der Hardt H, et al.
Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated residual chloride secretion does
not protect against early chronic Pseudomonas aeruginosa infection in F508del homozygous cystic
fibrosis patients. Pediatr Res. 2004;55(1):69-75,
http://dx.doi.org/10.1203/01.PDR.0000100758.66805.CE.
http://dx.doi.org/10.1203/01.PDR.0000100...
). As genistein has been reported
to induce CFTR-mediated Cl- secretion in the small intestine (2020. Al-Nakkash L, Batia L, Bhakta M, Peterson A, Hale N, Skinner R, et al.
Stimulation of Murine Intestinal Secretion by Daily Genistein Injections: Gender-dependent
Differences. Cell Physiol Biochem. 2011;28(2):239-50,
http://dx.doi.org/10.1159/000331736.
http://dx.doi.org/10.1159/000331736...
) and jejunum (5151. Baker MJ, Hamilton KL. Genistein stimulates electrogenic Cl− secretion in
mouse jejunum. Am J Physiol Cell Physiol. 2004;287(6):C1636-45,
http://dx.doi.org/10.1152/ajpcell.00236.2003.
http://dx.doi.org/10.1152/ajpcell.00236....
), it is likely that this
compound may also stimulate Cl- secretion in the uterus via CFTR.
This study investigated the acute effect of genistein on fluid accumulation and CFTR protein and
mRNA expression in the uterus of ovariectomised rats. Three days of genistein treatment was
sufficient to cause an increase in CFTR expression in the uterus. A similar duration of genistein
treatment in ovariectomised rats was also found to induce changes in the morphology of the uterus
and vagina (5252. Schmidt S, Degen G, Seibel J, Hertrampf T, Vollmer G, Diel P. Hormonal activity
of combinations of genistein, bisphenol A and 17β-estradiol in the female Wistar rat. Arch
Toxicol. 2006;80(12):839-45.) and affect the expression of several other
proteins, including proliferative cell nuclear antigen (PCNA) (22. Diel P, Geis R-B, Caldarelli A, Schmidt S, Leschowsky UL, Voss A, et al. The
differential ability of the phytoestrogen genistein and of estradiol to induce uterine weight and
proliferation in the rat is associated with a substance specific modulation of uterine gene
expression. Molecular and Cellular Endocrinology. 2004;221(1-2):21-32,
http://dx.doi.org/10.1016/j.mce.2004.04.006.
http://dx.doi.org/10.1016/j.mce.2004.04....
,33. Salleh N, Helmy MM, Fadila KN, Yeong SO. Isoflavone genistein induces fluid
secretion and morphological changes in the uteri of post-pubertal rats. Int J Med Sci.
2013;10(6):665-75.) and complement C3 (5353. Diel P, Smolnikar K, Schulz T, Laudenbach-Leschowski U, Michna H, Vollmer G.
Phytoestrogens and carcinogenesis—differential effects of genistein in experimental models of
normal and malignant rat endometrium. Human Reprod. 2001;16(5):997-1006,
http://dx.doi.org/10.1093/humrep/16.5.997.
http://dx.doi.org/10.1093/humrep/16.5.99...
) in the uterus and blood, respectively. In this study, the route of genistein
administration could represent the limiting factor for extrapolating these findings to humans. In
humans, this compound is usually consumed orally as a dietary supplement or as a component of the
diet. Moreover, the level of genistein in the blood is expected to be higher following subcutaneous
administration compared with oral administration, as orally administered genistein is metabolised by
the gut flora to 5-hydroxy-equol (5454. Matthies A, Loh G, Blaut M, Braune A. Daidzein and genistein are converted to
equol and 5-hydroxy-equol by human intestinal slackia isoflavoniconvertens in gnotobiotic rats.
J Nutr. 2012;142(1):40-6.). In addition, oral
genistein administration also undergoes metabolism in the liver (5555. Coldham N, Zhang AQ, Key P, Sauer MJ. Absolute bioavailability of
[14C] genistein in the rat; plasma pharmacokinetics of parent compound, genistein
glucuronide and total radioactivity. Eur J Drug Metab Pharmacokinet.
2002;27(4):249-58.). Thus, the lack of uterotopic responses observed in humans following oral genistein
consumption (1010. D'Anna R, Cannata ML, Marini H, Atteritano M, Cancellieri F, Corrado F, et
al. Effects of the phytoestrogen genistein on hot flushes, endometrium, and vaginal epithelium in
postmenopausal women: a 2-year randomized, double-blind, placebo-controlled study. Menopause.
2009;16(2):301-6, http://dx.doi.org/10.1097/gme.0b013e318186d7e2.
http://dx.doi.org/10.1097/gme.0b013e3181...
) is likely due to low plasma bioavailability
as the consequence of these first-pass effects.
In conclusion, genistein-induced fluid accumulation in the uteri of ovariectomised rats was shown to involve the ER and CFTR, and these findings reveal the underlying effect of this compound on the uterus after menopause. Although the significance of this effect is unknown, this compound may help to restore fluid within the uterine cavity and inhibit uterine atrophy. Finally, high-dose genistein consumption, rather than the presence of malignancy, should be considered a potential cause for fluid in the uteri of post-menopausal women.
This study was supported by the UMRG grant (RG404/12HTM) from the University of Malaya, Kuala Lumpur.
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No potential conflict of interest was reported.
Publication Dates
-
Publication in this collection
Feb 2014
History
-
Received
25 Apr 2013 -
Reviewed
6 June 2013 -
Accepted
18 July 2013