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Corticotropin-releasing hormone peptide and human first-trimester placental growth
Early Human Development 79 (2004) 77 – 80 www.elsevier.com/locate/earlhumdev
Short communication
Corticotropin-releasing hormone peptide and human first-trimester placental growth Mei Yee Choy*, Tse Ngong Leung, Tze K. Lau Department of Obstetrics and Gynaecology, The Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, SAR China Accepted 6 April 2004
Abstract The aim of this study was to determine whether corticotropin-releasing hormone (CRH) regulates human trophoblast cell growth. The results showed that exogenous CRH significantly stimulated human trophoblast proliferation in first-trimester primary cultures. In vivo, CRH was strongly immunolocalised to cytotrophoblastic cells in proliferative cell columns and in chorionic villi. We postulate that CRH may have an important role in early placental development and successful pregnancy. D 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: CRH; Human trophoblast cell growth; Chorionic villi
1. Introduction Corticotropin-releasing hormone (CRH), a 41-amino acid hypothalamic neuropeptide hormone is expressed in many peripheral sites including early gestation placenta in man [1,2]. Its biological role during early pregnancy is not fully understood. More recently, CRH has been found to promote human blastocyst implantation [3]. In other tissue systems, CRH is associated with the proliferative potential in certain neoplasms and possesses both mitogenic and proliferation-inhibiting properties in vitro. Early placental trophoblasts are also highly proliferative [4] and fetal well-being depends critically on the
* Corresponding author. Tel.: +852 26323099; fax: +852 26360008. E-mail address: [email protected] (M.Y. Choy). 0378-3782/$ - see front matter D 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2004.04.010
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M.Y. Choy et al. / Early Human Development 79 (2004) 77–80
regulated growth and activity of placental villous development [5]. Extravillous trophoblast proliferation and invasion of decidua is also crucial for successful pregnancy such that inadequate invasion resulting from defective trophoblast differentiation is associated with dysfunctional pregnancies [6]. The aims of this study were to localize CRH production in early placental tissue and to determine the effect of CRH on human first-trimester trophoblast proliferation in vitro.
2. Materials and methods 2.1. Immunolocalisation of CRH in first-trimester placenta Formalin-fixed paraffin-embedded first-trimester placentae (7 weeks) were immunostained for CRH using the indirect alkaline phosphatase method. Antigen was retrieved by microwave treatment, blocked with a non-serum protein block (Dako) and incubated with primary rabbit polyclonal CRH (1/50) [Phoenix Pharmaceuticals, CA, USA] for 2 h at RT. Non-immune rabbit IgG (Dako) was used as negative controls. Slides were incubated in biotinylated secondary goat anti-rabbit (1/300) for 30 min. Alkaline phosphatase-conjugated streptavidin (1/200) [Vector Laboratories, USA] was applied for 30 min and colour developed using a fast red substrate kit (Dako). Slides were mounted in glycerol. 2.2. Effect of CRH on human first-trimester trophoblast proliferation Ethical approval for the collection of human first-trimester placenta was obtained from the Ethical Committee of The Chinese University of Hong Kong. Patient consent was obtained prior to each collection. Placentae were collected from patients undergoing termination of normal pregnancies without medical complications. Trophoblast primary cultures were prepared according to the method of Choy and Manyonda [7]. A minimum of six experiments was analyzed. One placenta was used for each experiment. Day 2 cultures were treated with CRH (10 nM CRH) (Sigma) for 4 h. After incubation, slides were air-dried and immunohistochemically analysed for ki67 expression the indirect immunoperoxidase technique. Slides were fixed in a 1:1 mixture of cold acetone/methanol for 15 min at RT. Nonspecific endogenous peroxidase staining was blocked by treating cells with 1% H2O2 for 30 min before blocking with a protein block (Dako), for 10–15 min. Monoclonal anti-ki67 (1/50) applied for 1 h at RT, washed and secondary biotinylated rabbit anti-mouse (1/300) added for 30 min. After incubation in secondary antibody, streptavidin peroxidase (VectorLab) was added for 15 min and colour developed with a DAB (kit) for 5 min. Slides were dehydrated through alcohols and mounted in DPX. The proliferative index was determined by manual counting the number of ki67 positive cells in control (n=7) and CRH treated (n=6). A minimum of 500 cells per group was scored blind by one observer.
3. Results 3.1. Immunolocalisation of CRH to proliferating trophoblasts in vivo CRH was immunolocalized to cell clusters (arrows) in proliferating trophoblast cell columns (Fig. 1A,B) and in cytotrophoblastic cells of chorionic villi (Fig. 1C). Control rabbit IgG gave no positive staining (Fig. 1D).
M.Y. Choy et al. / Early Human Development 79 (2004) 77–80
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Fig. 1. CRH was immunolocalized (dark red staining, arrows) to clumps of cytotrophoblastic cells in cell columns [CC] (A, larger view B) and within the cytoplasm of villous cytotrophoblasts (C); control rabbit IgG (D). In trophoblast primary cultures, addition of CRH stimulated ki67 expression by 3.6 fold (E, F).
3.2. Effect of CRH on trophoblast proliferation in trophoblast primary cultures Addition of 20 nM CRH to trophoblast primary cultures increased ki67 expression by 3.6 folds (Fig. 1E,F). Percentage of ki67 positive cells for CRH treated (n=6) and control groups (n=7) was 50%F17 S.D. (Fig. 1E) and 14%F8 S.D. (Fig. 1F), respectively. Statistical analysis using Student’s t-test showed a significant difference of pb0.01.
4. Discussion The data in this study suggest that CRH is mitogenic for human first-trimester trophoblasts in vitro and is preferentially expressed by proliferative cytotrophoblasts in
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M.Y. Choy et al. / Early Human Development 79 (2004) 77–80
vivo. We postulate that in addition to its role in enhancing maternal tolerance during implantation, CRH also has an important part to play in early placental growth and development by stimulating trophoblastic cell proliferation.
References [1] Choy MY, Leung TN, Leung PS, Lau TK. First trimester human trophoblast production of placental corticotrophin-releasing hormone (CRH) is unresponsive to hypoxia in-vitro. Early Pregnancy, Biology and Medicine 2003 January;VI(1):235 – 47. [2] Frim DM, Emanuel RL, Robinson BG, Smas CM, Adler GK, Majzoub JA. Characterization and gestational regulation of corticotropin-releasing hormone messenger RNA in human placenta. J Clin Invest 1988; 82:287 – 92. [3] Makrigiannakis A, Zoumakis E, Kalantaridou S, Coutifaris C, Margioris AN, Coukos G, et al. Corticotropinreleasing hormone promotes blastocyst implantation and early maternal tolerance. Nat Immunol 2001; 2(11):1018 – 24. [4] Genbacev O. To proliferate or to divide—to be or not to be. Early Pregnancy 2001;5(1):63 – 4. [5] Kingdom J, Huppertz B, Seaward G, Kaufmann P. Development of the placental villous tree and its consequences for fetal growth. Eur J Obstet Gynecol Reprod Biol 2000;192(1):35 – 43. [6] Zhou Y, Damsky CH, Chiu K, Roberts JM, Fisher SJ. Preeclampsia is associated with abnormal expression of adhesion molecules by invasive cytotrophoblasts. J Clin Invest 1993;91(3):950 – 60. [7] Choy MY, Manyonda IT. The phagocytic activity of human first trimester extravillous trophoblast. Hum Reprod 1998;13(10):2941 – 9.
Short communication
Corticotropin-releasing hormone peptide and human first-trimester placental growth Mei Yee Choy*, Tse Ngong Leung, Tze K. Lau Department of Obstetrics and Gynaecology, The Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, SAR China Accepted 6 April 2004
Abstract The aim of this study was to determine whether corticotropin-releasing hormone (CRH) regulates human trophoblast cell growth. The results showed that exogenous CRH significantly stimulated human trophoblast proliferation in first-trimester primary cultures. In vivo, CRH was strongly immunolocalised to cytotrophoblastic cells in proliferative cell columns and in chorionic villi. We postulate that CRH may have an important role in early placental development and successful pregnancy. D 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: CRH; Human trophoblast cell growth; Chorionic villi
1. Introduction Corticotropin-releasing hormone (CRH), a 41-amino acid hypothalamic neuropeptide hormone is expressed in many peripheral sites including early gestation placenta in man [1,2]. Its biological role during early pregnancy is not fully understood. More recently, CRH has been found to promote human blastocyst implantation [3]. In other tissue systems, CRH is associated with the proliferative potential in certain neoplasms and possesses both mitogenic and proliferation-inhibiting properties in vitro. Early placental trophoblasts are also highly proliferative [4] and fetal well-being depends critically on the
* Corresponding author. Tel.: +852 26323099; fax: +852 26360008. E-mail address: [email protected] (M.Y. Choy). 0378-3782/$ - see front matter D 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2004.04.010
78
M.Y. Choy et al. / Early Human Development 79 (2004) 77–80
regulated growth and activity of placental villous development [5]. Extravillous trophoblast proliferation and invasion of decidua is also crucial for successful pregnancy such that inadequate invasion resulting from defective trophoblast differentiation is associated with dysfunctional pregnancies [6]. The aims of this study were to localize CRH production in early placental tissue and to determine the effect of CRH on human first-trimester trophoblast proliferation in vitro.
2. Materials and methods 2.1. Immunolocalisation of CRH in first-trimester placenta Formalin-fixed paraffin-embedded first-trimester placentae (7 weeks) were immunostained for CRH using the indirect alkaline phosphatase method. Antigen was retrieved by microwave treatment, blocked with a non-serum protein block (Dako) and incubated with primary rabbit polyclonal CRH (1/50) [Phoenix Pharmaceuticals, CA, USA] for 2 h at RT. Non-immune rabbit IgG (Dako) was used as negative controls. Slides were incubated in biotinylated secondary goat anti-rabbit (1/300) for 30 min. Alkaline phosphatase-conjugated streptavidin (1/200) [Vector Laboratories, USA] was applied for 30 min and colour developed using a fast red substrate kit (Dako). Slides were mounted in glycerol. 2.2. Effect of CRH on human first-trimester trophoblast proliferation Ethical approval for the collection of human first-trimester placenta was obtained from the Ethical Committee of The Chinese University of Hong Kong. Patient consent was obtained prior to each collection. Placentae were collected from patients undergoing termination of normal pregnancies without medical complications. Trophoblast primary cultures were prepared according to the method of Choy and Manyonda [7]. A minimum of six experiments was analyzed. One placenta was used for each experiment. Day 2 cultures were treated with CRH (10 nM CRH) (Sigma) for 4 h. After incubation, slides were air-dried and immunohistochemically analysed for ki67 expression the indirect immunoperoxidase technique. Slides were fixed in a 1:1 mixture of cold acetone/methanol for 15 min at RT. Nonspecific endogenous peroxidase staining was blocked by treating cells with 1% H2O2 for 30 min before blocking with a protein block (Dako), for 10–15 min. Monoclonal anti-ki67 (1/50) applied for 1 h at RT, washed and secondary biotinylated rabbit anti-mouse (1/300) added for 30 min. After incubation in secondary antibody, streptavidin peroxidase (VectorLab) was added for 15 min and colour developed with a DAB (kit) for 5 min. Slides were dehydrated through alcohols and mounted in DPX. The proliferative index was determined by manual counting the number of ki67 positive cells in control (n=7) and CRH treated (n=6). A minimum of 500 cells per group was scored blind by one observer.
3. Results 3.1. Immunolocalisation of CRH to proliferating trophoblasts in vivo CRH was immunolocalized to cell clusters (arrows) in proliferating trophoblast cell columns (Fig. 1A,B) and in cytotrophoblastic cells of chorionic villi (Fig. 1C). Control rabbit IgG gave no positive staining (Fig. 1D).
M.Y. Choy et al. / Early Human Development 79 (2004) 77–80
79
Fig. 1. CRH was immunolocalized (dark red staining, arrows) to clumps of cytotrophoblastic cells in cell columns [CC] (A, larger view B) and within the cytoplasm of villous cytotrophoblasts (C); control rabbit IgG (D). In trophoblast primary cultures, addition of CRH stimulated ki67 expression by 3.6 fold (E, F).
3.2. Effect of CRH on trophoblast proliferation in trophoblast primary cultures Addition of 20 nM CRH to trophoblast primary cultures increased ki67 expression by 3.6 folds (Fig. 1E,F). Percentage of ki67 positive cells for CRH treated (n=6) and control groups (n=7) was 50%F17 S.D. (Fig. 1E) and 14%F8 S.D. (Fig. 1F), respectively. Statistical analysis using Student’s t-test showed a significant difference of pb0.01.
4. Discussion The data in this study suggest that CRH is mitogenic for human first-trimester trophoblasts in vitro and is preferentially expressed by proliferative cytotrophoblasts in
80
M.Y. Choy et al. / Early Human Development 79 (2004) 77–80
vivo. We postulate that in addition to its role in enhancing maternal tolerance during implantation, CRH also has an important part to play in early placental growth and development by stimulating trophoblastic cell proliferation.
References [1] Choy MY, Leung TN, Leung PS, Lau TK. First trimester human trophoblast production of placental corticotrophin-releasing hormone (CRH) is unresponsive to hypoxia in-vitro. Early Pregnancy, Biology and Medicine 2003 January;VI(1):235 – 47. [2] Frim DM, Emanuel RL, Robinson BG, Smas CM, Adler GK, Majzoub JA. Characterization and gestational regulation of corticotropin-releasing hormone messenger RNA in human placenta. J Clin Invest 1988; 82:287 – 92. [3] Makrigiannakis A, Zoumakis E, Kalantaridou S, Coutifaris C, Margioris AN, Coukos G, et al. Corticotropinreleasing hormone promotes blastocyst implantation and early maternal tolerance. Nat Immunol 2001; 2(11):1018 – 24. [4] Genbacev O. To proliferate or to divide—to be or not to be. Early Pregnancy 2001;5(1):63 – 4. [5] Kingdom J, Huppertz B, Seaward G, Kaufmann P. Development of the placental villous tree and its consequences for fetal growth. Eur J Obstet Gynecol Reprod Biol 2000;192(1):35 – 43. [6] Zhou Y, Damsky CH, Chiu K, Roberts JM, Fisher SJ. Preeclampsia is associated with abnormal expression of adhesion molecules by invasive cytotrophoblasts. J Clin Invest 1993;91(3):950 – 60. [7] Choy MY, Manyonda IT. The phagocytic activity of human first trimester extravillous trophoblast. Hum Reprod 1998;13(10):2941 – 9.