corticotropin-releasing hormone peptide and human first-trimester placental growth
TRANSCRIPT
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
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ress: [email protected] (M.Y. Choy).
M.Y. Choy et al. / Early Human Development 79 (2004) 77–8078
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).
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).
M.Y. Choy et al. / Early Human Development 79 (2004) 77–80 79
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
M.Y. Choy et al. / Early Human Development 79 (2004) 77–8080
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
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