Expression of calbindin-d28k in human fallopian tubes

http://www.actaps.com.cn 567 Research Paper Expression of calbindin-d28k in human fallopian tubes XU Duo 1, ZHU Wei-Jie 1,*, WANG Zi-Neng 2 1 Center for Reproductive Immunology Research; 2 Medical College, Jinan University, Guangzhou 510632, China Abstract: The present study was aimed at investigating the expression of calbindin-d28k (CaBP-D28k) in human fallopian tube, which were collected from 33 childbearing age women undergoing abdominal hysterectomy with adnexectomy for benign disease in the pelvic cavity. These women had normal menstrual cycle and history of normal pregnancy. Isthmus, ampullary and umbrella segments of fallopian tubes were respectively collected. These specimens were divided into 6 groups based on their menstrual cycles: earlyproliferative stage (n=6), mid-proliferative stage (n=5), late-proliferative stage (n=5), early-secretory stage (n=7), mid-secretory stage (n=5) and late-secretory stage (n=5). The expressions of CaBP-D28k protein and mrna in fallopian tubes were determined by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) methods. Positive expressions of CaBP-D28k protein and mrna were observed in human fallopian tubes. There was no significant difference in the expression of CaBP-D28k protein among the isthmus, ampulla and umbrella segments in the same phase of menstrual cycle (P>0.05). However, in the menstrual cycle, the expression level of CaBP-D28k protein in the epithelium was the lowest during the early- and mid-proliferative stages and increased in both the late-proliferative and early-secretory stages (P<0.05), and then decreased in the mid- and late-secretory stages (P< 0.05). The expressed CaBP-D28k protein was disposed to gobbets or dispersed sheets in cytoplasm in the early- and mid- proliferative stages, and showed concentrated granules on the top of cells in the late-proliferative and early-, mid-secretory stages. Then in the latesecretory stage redistribution renewed as in the early- and mid-proliferative stages. The CaBP-D28k mrna obviously increased in the late-proliferative and early-secretory stages (P<0.05). These findings indicate that the expressions of CaBP-D28k protein and mrna exist in human fallopian tubes and exhibit a cyclic change. Key words: fallopian tube; calbindin-d28k; immunohistochemistry; reverse transcription polymerase chain reaction 钙结合蛋白 calbindin-d28k 在人输卵管组织的表达 许多 1, 朱伟杰 1,*, 王自能 2 暨南大学 1 生殖免疫研究中心 ; 2 医学院, 广州 510632 摘要 : 本研究旨在探讨育龄妇女输卵管中钙结合蛋白 calbindin-d28k (CaBP-D28k) 的表达和变化 33 例月经规律 有正常生育史的育龄妇女, 因子宫肌瘤 子宫腺肌症行子宫切除术 ( 一并切除输卵管 ), 输卵管取材均包括峡部 壶腹部和伞部, 按月经周期分为增生早期组 6 例, 增生中期组 5 例 增生晚期组 5 例, 分泌早期组 7 例, 分泌中期组 5 例和分泌晚期组 5 例 采用免疫组织化学法和逆转录聚合酶链反应法检测 CaBP-D28k 在人输卵管组织中的表达 结果显示, 人输卵管组织中存在 CaBP-D28k 蛋白及 mrna 表达 CaBP-D28k 蛋白表达于输卵管上皮细胞的胞浆中, 在月经周期同一时期, 输卵管峡部 壶腹部 伞部的表达无明显差别 (P>0.05) 在月经周期中,CaBP-D28k 蛋白表达在增生早 中期最低, 而在增生晚期和分泌早期明显增高 (P<0.05), 分泌中 晚期显著下降 (P<0.05) CaBP-D28k 蛋白阳性表达分布在月经周期中出现再分布, 在增生早 中期呈灶状或片状弥散分布在胞浆中, 增生晚期和分泌早 中期 CaBP-D28k 蛋白呈聚集的颗粒状集中在细胞顶部, 至分泌晚期则仍呈灶状或弥散状分布 ;CaBP-D28k mrna 表达也随月经周期发生变化, 在增生晚期和分泌早期明显增高 (P< 0.05) 结果表明, 人输卵管组织中存在 CaBP-D28k 蛋白及 mrna 表达, 且具有周期性变化 关键词 : 输卵管 ;calbindin-d28k; 免疫组织化学 ; 逆转录聚合酶链反应中图分类号 :R339.2 Received 2005-10-11 Accepted 2006-10-17 This work was supported by the Doctoral Research Grant of Jinan University. * Corresponding author. Tel: +86-20-85225718; E-mail: tzhuwj@jnu.edu.cn

568 Calbindin-D28k (CaBP-D28k) is a 28 kda vitamin D-dependent calcium-binding protein. CaBP-D28k contains EF hand motifs, which have high affinity for Ca 2+. CaBP-D28k is thought to buffer intracellular calcium levels and play several important physiological roles [1-4]. Previous investigations demonstrated that CaBP-D28k is involved in female reproduction. The expression of CaBP- D28k in human endometrium has been detected, suggesting that CaBP-D28k is potentially essential for the establishment of uterine receptivity and implantation in human [5]. The fallopian tube, an anatomical continuation of the uterus, has a close relation with many reproductive activities of the uterus. Although the relationship between CaBP-D28k and oviduct has been observed in some animals [6,7], the presence and localization of CaBP-D28k have not been studied in human fallopian tubes and the role of CaBP- D28k in human fallopian tubes is still unknown. In the present study, the expression and change of CaBP-D28k protein in human fallopian tubes were investigated by using immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) methods. 1 MATERIALS AND METHODS 1.1 Tissue preparation Human fallopian tubes and endometrial tissues were obtained from 33 childbearing age women (30~48 years old) undergoing abdominal hysterectomy with adnexectomy for the benign disease in the pelvic cavity. The study protocol was approved by the Institutional Review Board of Jinan University, and informed consent was obtained from all patients. These women had normal menstrual cycles (28~30 d) and history of normal pregnancy and birth. Their last menstruations could exactly be recalled and no sex hormones were used half a year before the operation. Only fallopian tubes showing normal histology were included in the study. 1.2 Sample preparation and grouping The sample of fallopian tubes was collected contained isthmus segment, ampulla segment and umbrella segment. Endometriums were obtained from the center of posterior wall close to the uterus bottom. Tissues (100~150 mg) selected for RT-PCR were snap frozen in 70 o C within 1 h after operation. Those used for immunohistochemistry and hematoxylin-eosin (HE) staining were fixed in 10% formalin for 24 h and embedded in paraffin, then 4-μmthick serial sections were made. According to the histological dating of the endometrium, the specimens of 33 fallopian tubes were divided into 6 groups: early-proliferative stage (n=6), mid-proliferative stage (n=5), late-proliferative stage (n=5), early-secretory stage (n=7), mid-secretory stage (n=5) and late-secretory stage (n=5). 1.3 Immunohistochemical analysis The kit of strept avidin-biotin complex (SABC) reagent (Boster Co., China) was adopted for immunohistochemical staining. The procedures of staining were as follows: the paraffin-embedded fallopian tube sections were routinely deparaffinized, hydrated by successive series of ethanol, rinsed in distilled water, and then incubated in 3% H 2 O 2 for 10 min at room temperature to quench endogenous peroxidases, repaired in 0.01 mol/l citrate-buffered saline (ph 6.0) with the microwave antigen for 10 min, incubated in normal rabbit serum for 15 min to suppress non-specific binding. With removal of the serum, sections were incubated with 1:100 dilution of CaBP-D28k mouse polyclonal antibody (Boster Co., China) at 4 ºC for 12 h, and subsequently incubated with the second antibody labeled by biotin at 37 ºC for 30 min. Then the samples were incubated with SABC at 37 ºC for 30 min, and visualized with diaminobenzidine. At last, sections were counterstained with hematoxylinm. Gradual dehydration with ethanol was followed by transparence by using dimethylbenzene and then the samples were sealed with a neutral gum. The negative control was performed with phosphate-buffered saline (PBS) instead of the first antibody. Meanwhile, the positive control was carried out in endometrium. 1.4 RT-PCR 1.4.1 Isolation of total RNA About 100 mg fresh-frozen human tubal tissues were rapidly triturated. Total RNA was extracted from the trituration using 1 ml TRIzol reagent (Invitrogen Co., USA). The quantity and quality of the isolated RNA was determined by OD 260 /OD 280 with DU 640 spectrophotometer (Beckman Co., USA). 1.4.2 cdna synthesis cdna was synthesized according to manufacturer s instructions of Reverse Transcription Kit (Ding Guo Co., China). Total RNA (1 μl) was reverse-transcribed in a 20 μl reaction mixture. 1.4.3 PCR amplification cdna was amplified with a PCR kit (Ding Guo Co., China) with following primers: CaBP-D28k (Biotechnology, CA, Shanghai): forward, 5 -TCCTGCTGCTCTTCCGATGCC- 3 ; reverse, 5 -ATGTATCCATTGCCGTCCT-3 (355 bp products). GAPDH (PCR kit, Ding Guo Co., China) primers were added to RT-PCR system as an inner control (579 bp products). Cycling parameters were listed as

XU Duo et al: Expression of Calbindin-D28k in Human Fallopian Tubes 569 follows: 2 min at 94 ºC pre-denaturation, 45 s at 94 ºC denaturation, 45 s at 56 ºC, 45 s at 72 ºC, 30 cycles, 5 min at 72 ºC final extension. 1.4.4 Electrophoresis The PCR reaction mixture (10 μl) and DNA marker (100 bp DNA Ladder, TaKaRa Co., Japan) were electrophoretically separated on the 1% agarose gel containing ethidium bromide, observed and photographed under ultraviolet light. The signal intensity was quantified by Fluorchem TM 8900 image analysis system. 1.5 Assessment of results and statistical analysis Cells stained weak-yellow and brown-yellow were CaBP- D28k-positive cells for immunohistochemical staining. The positive expression level of CaBP-D28k in human tubal epithelium was determined by Leica Q550 image analysis system and the positive unit (PU) was calculated. It was necessary to test each of the stained sections in 5 fields at random. Target amplification was quantified by product band at corresponding bp. Semiquantity of PCR product was determined by Fluorchem TM 8900 image analysis system. The ratio of integrated optical density of CaBP-D28k to GAPDH was used in assessment of relative contents of CaBP-D28k mrna. Data were expressed as mean±sd. SPSS 11.5 statistical software package was employed to process the data and the single-factor variance analysis was applied (bilateral α=0.05). 2 RESULTS 2.1 Expression of CaBP-D28k protein in human fallopian tubes The positive expression of CaBP-D28k protein was shown in all 33 tubal tissues. The positive cells were located in ciliated and nonciliated epithelial cells of tubal epithelium and the positive signals appeared in cytoplasm of these cells. In the same phase of menstrual cycle, there was no significant difference in the expression level of CaBP-D28k protein in the epithelium among isthmus, ampulla and umbrella segments (P>0.05) (Table 1). In the different phases of menstrual cycle, the expression level of CaBP-D28k protein in the epithelium was the lowest during the early-, mid-proliferative stages, and significantly increased in the late-proliferative and early-secretory stages (P<0.05). In the mid- and late-secretory stages, the expression level of CaBP-D28k decreased again (P<0.05) (Table 1, Fig.1). The distribution of CaBP-D28k protein in tubal epithelium was unsymmetrical in cytoplasm. During different phases of menstrual cycle, redistribution of the CaBP-D28k protein was observed. The expressed CaBP-D28k protein was disposed to gobbets or dispersed sheets in cytoplasm in the early- and mid-proliferative stages, and showed concentrated granules stained on the top of cells in the lateproliferative and early-, mid-secretory stages. Then the gobbets or dispersed sheets appeared again in cytoplasm in the late-secretory stage (Fig.2). Table 1. Expression of CaBP-D28k protein in human tubal epithelium throughout the menstrual cycle (PU) Group Isthmus Ampulla Umbrella Early-proliferative stage group (A) 4.12±1.06 * 4.05±1.26 * 4.18±1.20 * Mid-proliferative stage group (B) 4.85±1.78 * 4.72±1.81 * 4.92±2.04 * Late-proliferative stage group (C) 17.44±1.45 # 17.55±1.54 # 17.53±1.51 # Early-secretory stage group (D) 16.25±1.45 # 17.48±1.38 # 17.76±1.39 # Mid-secretory stage group (E) 9.89±2.03 + 9.98±2.25 + 9.94±2.24 + Late-secretory stage group (F) 8.71±2.18 + 8.60±2.20 + 8.57±1.97 + mean±sd. * P<0.05 vs C, D, E and F; # P<0.05 vs A, B, E and F; + P<0.05 vs A, B, C and D. Table 2. Expression of CaBP-D28k mrna in human tubal epithelium throughout the menstrual cycle (PU) Early-proliferative Mid-proliferative Late-proliferative Early-secretory Mid-secretory Late-secretory stage (A) stage (B) stage (C) stage (D) stage (E) stage (F) CaBP-D28k mrna 23.57±5.63 * 56.50±16.38 + 109.88±20.12 # 101.44±23.59 # 47.36±8.97 + 57.94±13.28 + mean±sd. * P<0.05 vs B, C, D, E and F; # P<0.05 vs A, B, E and F; + P<0.05 vs A, C and D.

570 Fig. 1. Expression of CaBP-D28k in human tubal epithelium. A: During the mid-proliferative stage. B: During the late-proliferative stage. C: During the early-secretory stage. D: During the mid-secretory stage. E: During the late-secretory stage. F: Negative control. Scale bar, 20 μm. Fig. 2. Location of CaBP-D28k in human tubal epithelium in the late-proliferative and mid-secretory stages. A: Positive expression of CaBP- D28k shows concentrated granules on the top of cells in the late-proliferative stage. B: Positive expression of CaBP-D28k shows gobbets or sheets dispersed in cytoplasm in the mid-secretory stage. Scale bar, 20 μm. Fig. 3. Expression of CaBP-D28k mrna: RT-PCR electrophoresis. Lane 1, marker; Lane 2, early-proliferative stage; Lane 3, mid-proliferative stage; Lane 4, late-proliferative stage; Lane 5, early-secretory stage; Lane 6, mid-secretory stage; Lane 7, late-secretory stage.

XU Duo et al: Expression of Calbindin-D28k in Human Fallopian Tubes 571 2.2 Expression of CaBP-D28k mrna in human fallopian tubes RT-PCR revealed that both characteristics of 355 bp and 579 bp bands were clearly detected in all 33 specimens (Fig.3), indicating that the CaBP-D28k mrna appeared in human fallopian tubes. The relative optical density of CaBP-D28k mrna revealed that throughout the menstrual cycle the expression level of CaBP-D28k mrna in the tubal tissues was the lowest during the early-proliferative stage and increased in the mid-proliferative and mid-, late-secretory stages (P< 0.05). It was the highest in the late-proliferative and earlysecretory stages (P<0.05)(Table 2). 3 DISCUSSION Recently, studies of CaBP-D28k in female reproduction have been reported [8,9]. The relation between CaBP-D28k and the physiological function of fallopian tube has also been observed. Initially, the expression of CaBP-D28k located in the distal isthmus was detected in the oviduct of the laying hen. There was a correlation between CaBP- D28k and eggshell buildup [6]. The expression of CaBP- D28k was found in mouse oviduct epithelium but not in rat oviduct. The expression of CaBP-D28k in mouse oviduct epithelium was not regulated by ovarian steroids [7]. However, up to now, studies on CaBP-D28k and the physiological function of fallopian tube are virtually lack. The expression of CaBP-D28k has not been studied in human fallopian tubes. Previous investigations demonstrated that CaBP-D28k had many functions, such as cell protection [10,11], activation of some enzymes and regulation of some genes [12-14]. The present study demonstrated that positive expressions of CaBP-D28k protein and mrna existed in human fallopian tubes. Positive expression of CaBP-D28k protein located in tubal ciliated and nonciliated epithelial cell cytoplasm. We presumed that CaBP-D28k could affect the function of these cells. CaBP-D28k possibly had cell protective effects on these cells and might potentially regulate ciliary beat of ciliated epithelial cells and secretions of nonciliated epithelial cells, which might affect the reproductive process. Moreover, in this study the change of CaBP-D28k expression level during the menstrual cycle was detected. The expression levels of CaBP-D28k protein and mrna were the lowest in the early-proliferative stage and the highest in the late-proliferative and early-secretory stages. These results suggest a potential regulation of CaBP-D28k expression by ovarian steroids. Especially the highest CaBP-D28k expression in the late-proliferative and early-secretory stages suggests that CaBP-D28k might relate to fertilization and early development of the embryo, because these are important stages for fertilization and early development of the embryo. A phenomenon that the distribution of CaBP-D28k protein in cytoplasm changed with the menstrual cycle was also found in this study. Previous investigations demonstrated that the change of protein distribution and the function of protein were correlated [15,16]. Fallopian tube is the important place for fertilization and early development of the embryo in the late-proliferative and early-, mid-secretory stages. Under such consideration, whether the redistribution of CaBP-D28k in the late-proliferative and early-, mid-secretory stages related to its physiological function needs further investigation. This study indicates that CaBP-D28k appears in human fallopian tubes. CaBP-D28k regulates the physiological function of the fallopian tubes by buffering intracellular calcium levels in tubal epithelium, and further affects some reproductive processes such as the maturation and transport of gametes and embryo, fertilization, and early development of the embryo. 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