Int J Clin Exp Pathol 2014;7(5):1967-1976 www.ijcep.com /ISSN:1936-2625/IJCEP0000127 Original Article Testosterone decreases the expression of endometrial pinopode and L-selectin ligand (MECA-79) in adult female rats during uterine receptivity period Helmy Mohd Mokhtar1, Nelli Giribabu1, Sekaran Muniandy2, Naguib Salleh1 1Department of Physiology, Faculty of Medicine, University of Malaya, 50603 Lembah Pantai, Kuala Lumpur, Malaysia; 2Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Lembah Pantai, Kuala Lumpur, Malaysia Received February 28, 2014; Accepted April 3, 2014; Epub April 15, 2014; Published May 1, 2014 Abstract: Pinopode, a progesterone-dependent endometrial projection which appears during uterine receptivity pe- riod, participates in blastocyst implantation. Blastocyst loosely attaches to pinopode via L-selectin ligand (MECA- 79). We hypothesized that pinopode and MECA-79 expressions were affected by testosterone. Therefore, the ef- fect of testosterone on pinopode and MECA-79 expressions during uterine receptivity period were investigated. Methods: Ovariectomized adult female rats received 8 days sex-steroid replacement intended to mimic hormonal changes in early pregnancy with day 6 to 8 represents uterine receptivity period. Testosterone (1 mg/kg/day) was injected together with flutamide or finasteride during the period of uterine receptivity. At the end of treatment, rats were sacrificed and uteri were removed. The existence of pinopodes in the endometrium was visualized by electron microscopy and uterine expression and distribution of MECA-79 protein were examined by Western blotting and im- munohistochemistry (IHC) respectively. Results: Abundant pinopodes and MECA-79 expressions were observed in rats received normal steroid replacement regime. Administration of testosterone during uterine receptivity period re- duced pinopodes and MECA-79 expressions, which were antagonized by flutamide and not finasteride.Conclusions : The decrease in uterine pinopodes and MECA-79 expressions during uterine receptivity period by testosterone may cause failure of blastocyst to implant in conditions associated with high level of this hormone. Keywords: Pinopode, testosterone, MECA-79, uterine receptivity Introduction In the uterus, pinopode displays several func- tions. In rodents, this projection is involved in Pinopodes are dome-like projections arising pinocytosis which assist the removal of fluid from the apical membrane of uterine luminal from the uterine lumen to initiate luminal clo- epithelia [1]. In rats, this projection appears at sure preceding blastocyst attachment [8]. day 4, peaking at day 5 and rapidly decline at Additionally, this bulb-like projection has been day 6 of early pregnancy, coincide with uterine reported to participate in embryo-endometrial receptivity or implantation window period [2]. interaction via L-selectin ligand [9] which con- Due to its existence within a limited time peri- tains a carbohydrate epitope, MECA-79 [10]. In od, pinopode has been used as a marker of humans, pinopodes were found to release uterine receptivity [3]. The appearance of secretory vesicles containing LIF into the uter- pinopodes coincides with the expression of ine lumen [11]. Pinopode expression is proges- other uterine receptivity markers such as leu- terone dependent [12]. A strong correlation kaemic inhibitory factor (LIF) [4], mucin, between pinopode with plasma P4 level and P4 L-selectin, integrin αvβ3 and heparin-binding receptor expression has been reported in the epidermal growth factor (HBEGF) [5, 6]. In uterus of humans [12], rats [1] and mice [13]. humans however, its role as uterine receptivity Estrogen however causes loss of pinopode marker is debatable [7]. expression [1]. Ovariectomized mice were found Testosterone, pinopode and MECA-79 in uterus to have high number of pinopodes, suggested water ad libitum. Ovariectomies were per- that the removal of E2 most probably cause the formed under isofluorane anesthesia at least increase in pinopode expression [14]. 14 days prior to initiating any hormone treat- ment. All procedures performed were approved In primates [15] and humans [16], the level of by the ethics committee with ethics number: testosterone increases at around the time of 2013-07-15/FIS/R/NS. All experiments were ovulation and in the late luteal phase of the carried out in accordance with international menstrual cycle. In mice, serum testosterone guideline outlined in the Guide for the Care and level was the highest before estrogen and Use of Laboratory Animals [24]. Steroids were luteinizing hormone (LH) peak which occur 24 dissolved in arachis oil and injected subcutane- hours before implantation [17]. Evidence that ously in a volume of 0.1 ml. To mimic the hor- testosterone is required for implantation was monal profile in early pregnancy, 17β-estradiol shown by Humprey [18] where the number of (E2) and progesterone (P4) were administered implantation sites were increased with an according to the established protocol by increase in plasma testosterone level. In Kennedy [25] however with slight modifica- female, testosterone is produced by the ovaries tions. In brief, the basic protocol involved injec- [19] and decidua and is required for decidual- tion of 1.0 µg/kg/day E for day 1 and day 2, ization [20]. In parallel to the increase in plas- 2 1.0 µg/kg/day E2 and 4 mg/kg/day P4 on day 3, ma testosterone level, high expression of uter- no treatment on the 4th and 5th day, 16 mg/kg/ ine androgen receptor was also reported in the day P4 and 0.5 µg/kg/day E2 on the next 3 days secretory phase of the cycle [21]. Although tes- (days 6-8). Description of the study design was tosterone is required for normal implantation shown in Figure 1. Vehicle treated animals [22], high level of this hormone could interfere (control) received 8 daily injections of arachis with the peri-implantation embryo and uterine oil. 1 mg/kg/day testosterone, regarded as developments resulting in early pregnancy loss supraphysiological dose in female [26] was [23]. The mechanism responsible for the administered for three (3) days from day 6-8 adverse testosterone effect on the uterus dur- which was considered as uterine receptivity ing implantation is however unknown. period with and without flutamide (5 mg/kg/ We hypothesize that testosterone could affect day) or finasteride (1 mg/kg/day) where both the pinopode and MECA-79 expressions in the inhibitors were administered 30 minutes prior uterus in early pregnancy which could interfere to the testosterone injection. with the blastocyst-endometrial interaction, Field emission scanning electron microscopy resulting in implantation failure. This study (FESEM) therefore investigated the effect of testoster- one on pinopode and MECA-79 expression dur- FESEM was performed to evaluate the pres- ing uterine receptivity period in rats which ence of pinopodes on the endometrial surface. could explain the adverse effect of testoster- For FESEM preparation, uteri were cut longitu- one on blastocyst implantation. dinally into 2 mm × 2 mm section and fixed at Materials and methods least 24 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C. The speci- Chemicals mens were air-dried and then placed on carbon planchets and examined with FEG Quanta 450 Testosterone propionate, 17β-estradiol and EDX-OXFORD, Field Emission Scanning Electron progesterone were purchased from Sigma- Microscope, Netherlands at 5 kV, under low Aldrich, St. Louis, USA. Other remaining chemi- vacuum and at working distances of 11.0 mm cals were analytical grades. and at magnifications of 3000 ×. Photographs were taken in slow scanning mode at 1280 × Animals and hormones treatment 1024 pixels. Adult female Wistar rats, purchased from Transmission electron microscopy (TEM) Animal House, University of Malaya, weighted 200-250 g were maintained under 12:12 light- Individual uteri were fixed in 2.5% glutaralde- ing conditions, with free access to food and hyde in 0.1 M phosphate buffer, pH 7.4, and 2 1968 Int J Clin Exp Pathol 2014;7(5):1967-1976 Testosterone, pinopode and MECA-79 in uterus Figure 1. Protocol of sex-steroid replacement regime. Schematic representation of time-course of steroid treatment protocol administered to ovariectomized rats to mimic the fluctuating steroid hormones level in early pregnancy. The animals were kept at 12 hours light and 12 hours dark schedule. D: day, T: testosterone. mm-cross sections of each uterine horn were Immunoperoxidase detection of MECA-79 cut and incubated overnight at 4°C in 2.5% glu- taraldehyde buffer. The buffer was removed, Uterine tissues were fixed overnight in 4% para- and the samples were rinsed three times, 15 formaldehyde (PFD) before processing and minutes each in 0.1 M phosphate buffer. dehydrated through increasing concentrations Samples were incubated in 1% osmium in 0.1 of ethanol, cleared in chloroform and were then M phosphate buffer, rinsed and dehydrated in a blocked in a paraffin wax. For IHC, tissues were series of ethanol (70%-100%) washes. Samples cut into 5 µm sections, deparaffinized in xylene, were incubated twice for 5 minutes in propyl- rehydrated in reducing concentration of etha- ene oxide and then transferred to a rotor for 1 h nol. Tri sodium citrate (pH 6.0) was used for at room temperature in a 1:1 mixture of propyl- antigen retrieval, while 1% H2O2 in phosphate ene oxide and epon [47% Embed 812, 31% buffered saline (PBS) was used to neutralize DDSA (dodenyl succinic anhydride), 19% NMA the endogenous peroxidase. Sections were (nadic methyl anhydride), 3% BDMA (benzyldi- then blocked in 5% BSA for the non-specific methylamine)], followed by an overnight incuba- binding, prior to incubation with rat monoclonal tion in 1:2 propylene oxide-epon, and finally primary antibody to MECA-79 (200 μg/0.1 ml, 100% epon for 2-3 hrs. Individual uterine sam- 1:500, Santa Cruz, SC 19602) at a dilution of ples were embedded in 100% epon in silicon 1:100 in 5% BSA at room temperature for an flat embedding molds, and capsules were hour.