Characterization of an Insulin-Like Growth Factor Binding Protein, Analogous to Human Pregnancy-Associated Secreted Endometrial α1-Globulin, in Decidua of the Baboon (Paplo Anubis) Placenta1

Abstract

The major secreted protein of the human decidua (pregnancy-associated endometrial alpha 1-globulin [alpha 1-PEG]), is an insulin-like growth factor-binding protein (IGF-BP) that is immunologically and biochemically similar to placental protein 12 (PP12) extracted from term human placenta. Since previous studies have demonstrated that the baboon and human endometrium synthesize and release a number of biochemically and immunologically related polypeptides in culture, this study was undertaken to further characterize a related IGF-BP in baboon placental tissues. Decidua, chorio-amniotic membranes with adhering decidua (CAM-D), and placental villi were obtained from pregnant baboons between Days 134 and 160 of gestation by Cesarean sections. Portions of tissue were either cultured in the presence of 35S-methionine, fixed for immunocytochemistry, or frozen in liquid nitrogen for cytosol extraction. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of tissue culture media (TCM) revealed that the major secretory product of the decidua and CAM-D was an acidic polypeptide (Mr 33,000). Western blot analysis and immunoprecipitation of TCM with murine monoclonal antibody (B2H10) against human alpha 1-PEG demonstrated that this molecule, secreted by the baboon decidua and CAM-D, but not the placental villi, was immunologically identical to the human IGF-BP. Immunocytochemical localization of IGF-BP was intense in the cytoplasm of stromal cells in decidua and CAM-D and absent in the placenta. Gel filtration of TCM and cytosol followed by screening of eluates for 125I-IGF-I binding resolved two peaks (Mr greater than 100,000 and 35,000) of specific IGF-BP in decidua and CAM-D. The 35,000 peak had 100-200 times the binding capacity of the Mr greater than 100,000 peak and a Kd of 1.14-1.83 nM. The eluates contained in the Mr 35,000 peak were also immunoreactive to alpha 1-PEG, as accessed by a polyclonal radioimmunoassay. Affinity cross-linking with 125I-IGF-I followed by sodium dodecyl sulfate-PAGE revealed an immunoreactive complex of Mr 36,000, confirming that the baboon protein represents a high affinity IGF-BP. These studies indicate that the hypertrophied stromal cells of the baboon decidua and CAM-D synthesize and release an IGF-BP as their major secretory product, analogous to the situation in humans. The results of this study suggest that this protein may play a role in the regulation of IGF action during pregnancy.

Secretory and Morphological Changes in the Baboon (Papio Anubis) Uterus and Placenta during Early Pregnancy1

“…2B and 3B). By Day 32 the overall secretory pattern was altered, and protein synthesis by both the functionalis and placenta was more similar to that observed in late pregnant tissues [17]. Figure 4 is a composite showing a representative fluorograph, a corresponding immunoblot, and the immunocytochemical localization of CG, IGFBP-1, and RBP in placental and endometrial tissues.…”

“…However, it is only when decidual synthesis of IGFBP-1 is induced at the implantation site [18] that it becomes a significant labeled product on fluorographs. As the stromal cells decidualize, IGFBP-1 synthesis increases, and this protein eventually becomes the major secretory product of the primate decidualized endometrium [17]. The synthesis and localization of RBP in the nonpregnant baboon endometrium is similar to that observed for IGFBP-1 [22].…”

“…However, it may be more than coincidental that the highest period of synthetic activity by both the endometrium and placenta occurs at the same time as the peak of measurable CG in the peripheral circulation [8] and also at the time when progesterone synthesis undergoes a luteal-placental shift in the baboon [23]. By Day 32, a mature placenta has been established in the baboon [9], and the protein secretory pattern of both the endometrium and placenta begins to resemble the pattern observed during the last third of pregnancy [17].…”

“…One possible explanation may be increased glycosylation of the CG molecule associated with the formation of the syncytium. The synthesis of the uncharacterized basic protein (Mr 23 000) is highest at Days 18 and 25, and this protein continues to be secreted in low amounts throughout pregnancy [17].…”

“…The Western blotting technique was carried out as described previously [16,17], and the immunoreactive product was visualized by use of the Bio-Rad Alkaline Phosphatase conjugated detection kit (CG and IGFBP-1) or ' 25 I-labeled protein-A (RBP).…”