Asgerally T. Fazleabas

A Baboon (Papio anubis) simulated-pregnant model: cell specific expression of insulin-like growth factor binding protein-1 (IGFBP-1), type I IGF receptor (IGF-1 R) and retinol binding protein (RBP) in the uterus

In order to test the hypothesis that the baboon conceptus/placenta regulates the synthesis of specific proteins in the endometrium, we developed a simulated-pregnant baboon model. Baboons (n=2–6/group) were treated with increasing amounts of human chorionic gonadotrophin (hCG) for 10 or 12 days beginning on day 6 or 7 PO. Uterine tissues were obtained at day 18 PO following 12 days of hCG treatment. Animals in the day 25 and 32 PO group were treated for 10 days with hCG. Following the hCG treatment, estradiol (E) and progesterone (P) implants were inserted subcutaneously. Control groups consisted of E and P treatment only (day 25 PO), or ovariectomy on day 6 or 7 PO followed by hCG plus E and P treatment (days 18 and 25 PO). Serum samples were obtained daily or once every 2 days and analysed for E and P by radioimmunoassay. hCG activity in serum was determined by a Leydig cell bioassay. Portions of the endometrial tissue were either subjected to organ explant culture, analysed by immunocytochemistry or extracted for RNA. Peripheral serum levels of hCG, E and P in the experimental groups fell within the 95% confidence interval limits of hormone concentrations achieved during pregnancy. The morphology of the endometrium in the hCG treated baboons and pregnant baboons was similar i.e., distended convoluted glands, many spiral artery beds, a loose vacuolized stroma, and increased collagen staining. However, in the absence of hCG (E+P treatment only) the glands tended to be straight rather than corkscrew-shaped, and decreased stromal vacuolization and collagen staining was evident.35S-methionine labeled proteins in explant culture conditioned media (TCM) were analysed by two-dimensional SDS-PAGE and fluorography. A comparable pattern of protein synthesis was apparent in all treatment groups except for a low molecular weight (27 000–30 000 daltons) group of polypetides which only was evident in TCM from the hCG treated baboons. A similar group of proteins are also secreted by the baboon endometrium during pregnancy. The immunocytochemical localization of estrogen (ER) and progesterone receptors (PR) was comparable to that observed in pregnant baboons. IGFBP-1 localization was confined to the glandular epithelium in the hCG treated groups (intact and ovariectomized) and was virtually undetectable in the E and P treated group. The intensity of IGFBP-1 staining was variable within each of the hCG treatment groups on days 18, 25 and 32 PO. This variability was also apparent by Western blot analysis, immunoassay of proteins in TCM and on Northern blots of total RNA from the same animals. In contrast, IGF-I R immunostaining was evident in both glandular and surface epithelium of all treatment groups. Expression of RBP was confined to the basal glands. The characteristic upregulation of RBP synthesis in the functionalis observed during early pregnancy was not apparent in any of the treatment groups. In summary, these studies indicate that exogenous hCG in conjunction with E and P, can induce the general morphological and biosynthetic changes the baboon endometrium undergoes during early pregnancy. In addition, this hormonal treatment is also capable of maintaining the epithelial expression of IGFBP-1, IGF-1 and RBP. However, other factors from the conceptus appear to be necessary to induce the cell specific changes in the expression of these three proteins that are observed during pregnancy.

A Baboon Model for Inducing Endometriosis

Endometriosis is a disease that is associated with severe pelvic pain and is a major cause of infertility in women. It is an enigmatic disease whose etiology and pathophysiology has been studied to a limited extent. The events associated with the establishment of the disease and mechanisms associated with infertility are difficult to assess in a systemic manner in women. In order to understand the early and progressive events associated with the establishment of the disease, we have developed a baboon model in which the disease can be induced. This induction manifests itself in a manner that recapitulates the spontaneous disease. The advantage of the induced model is that the progressive changes in both the ectopic and eutopic endometrium can be studied in a nonhuman primate model at specific times during the menstrual cycle and as the disease process continues.

Epidermal growth factor, transforming growth factor-alpha, and epidermal growth factor receptor localization in the baboon (Papio anubis) uterus during the menstrual cycle and early pregnancy.

OBJECTIVE: Marked alterations occur in the synthesis of endometrium-specific proteins during the first third of pregnancy in the baboon. Because epidermal growth factor (EGF) expression has been associated with proliferation in the human and mouse endometrium, we hypothesized that EGF, transforming growth factor-α (TGFα), and EGF receptor (EGF-R) expression in baboon endometrium may be modulated by the early invasive trophoblast and play a role in decidualization of the endometrial stroma. METHODS: Endometrial tissue was obtained from cycling baboons (n = 4-5 per time point), ovariectomized steroid-treated baboons (n = 4 per group), or from pregnant baboons on days 18-60 of pregnancy (n = 2-4 pergroup). The tissue was fixed in Bouins solution and embedded in paraffin for immunocytochemistry using polyclonal antibodies against EGF and EGF-R and a monoclonal antibody to TGFα. RESULTS: Endometrial staining was located almost entirely in the glandular epithelium for TGFα and EGF-R in the follicular phase animals, whereas EGF staining was strongest in the periglandular stroma. In the luteal phase, specific staining for EGF also was detected in the glands as well as the periglandular stroma. There appeared to be little difference in endometrial staining between the late follicular and mid-luteal phase for TGFα and EGF-R. A similar pattern was observed in the steroid-treated animals. In the endometrium from pregnant animals, EGF, TGFα, and EGF-R intensely stained the glandular epithelium on days 18, 25, and 32. Both EGF and EGF-R showed light stromal staining on days 18 and 25. Light stromal TGFα staining was present on day 25 and became moderately intense by day 32. By day 60, the most intense staining for EGF and EGF-R was stromal. Staining of TGFα continued to be strong in the remaining epithelium through day 60. In placenta, EGF and EGF-R intensely stained the syncytiotropho blast, but not the cytotrophoblast, whereas TGFα stained only the villous cytotrophoblast and intermediate cytotrophoblast within maternal blood vessels. There appeared to be no change in this staining pattern or intensity in the placenta throughout early pregnancy. CONCLUSIONS: This study demonstrates the presence of EGF, TGFα, and EGF-R in the endometrium during the cycle and early pregnancy. The detection of EGF, TGFα, and EGF-R in the stromal cells during pregnancy correlated with the onset of decidualization. We propose that EGF, TGFα, and EGF-R may play a role in glandular development during the cycle and in decidualization and implantation during early pregnancy. (J Soc Gynecol Invest 1994 ;1 :277-84)

Differential regulation of stromelysin-1 (matrix metalloproteinase-3) and matrilysin (matrix metalloproteinase-7) in baboon endometrium.

Objective: To investigate in vivo expression of matrix metalloproteinase enzymes (MMP), MMP-3 and MMP-7, by the baboon endometrium in relation to the process of tissue remodeling that accompanies mensttruation in the primate. Methods: Endometrial tissues and uterine fluids obtained from reproductively intact cycling baboons, cycling baboons treated with an anti-progestin, and ovariectomized steroid-treated baboons were evaluated for MMP mRNA expression and protein production by using multiple analytic techniques. Results: The pattern of MMP protein production matched the pattern of MMP mRNA expression. In intact cyclcing baboons, MMP mRNA expression and protein production were specific to cell type and stage of the menstrual cycle. Endometrial stroma expressed minimal amounts of MMP-3 during the proliferative and secretory stages, whereas endomterial epithelia expressed MMP-7 during the prolfierative stage. Expression of stromal MMP-3 and epithelial MMP-7 increased during early menses. Administration of anti-progestin beginning on the day of the LH surge increased MMP-7 but not MMP-3 expression at the mid secretory phase. Expression of MMP-3 and MMP-7 in the ovariectomized steroid-treated animals paralleled that of the intact cycling animals. Conclusions: These results highlight the similarity of human and baboon endometrial MMP-3 and MMP-7 production, providing further evidence for use of the baboon as a model for studying events associated with hormonally regulated chanes during the menstrual cycle. These results also demonstrate that ednometrial epithelial MMP-7 is suppressed by progresterone in the baboon endometrium, but endometrial stromal MMP-3 is regulated by a different mechanism.

Characterization of insulin-like growth factor binding proteins by two-dimensional polyacrylamide gel electrophoresis and ligand blot analysis

Insulin-like growth factor binding proteins (IGFBPs) in pregnant baboon serum and tissue culture media obtained following explant culture of uteri from pregnant baboons were characterized by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE) followed by Western ligand blot analysis using 125I-labeled IGF-I. IGFBP-1 (Mr 30,000; pI 4-4.2), IGFBP-2 (Mr 34,000, pI 5.7-6.2), IGFBP-3 (doublet Mr 42-48,000; pI 6.2-6.8), and IGFBP-4 (Mr 24,000; pI 5.7-6.0) were clearly separated from one another. The authenticity of IGFBP-1, -2, and -3 was verified by immunoprecipitation using polyclonal antibodies followed by ligand blotting. Specificity of 125I-labeled IGF-I binding to IGFBPs was also determined by competitive binding studies using unlabeled IGF-I and -II. This technique allows for the identification of IGFBPs in complex biological fluids on the basis of their characteristic Mr and pI with or without the availability of specific antibodies and can be done rapidly using the mini 2D SDS-PAGE systems.

Induction of insulin-like growth factor binding protein-1 expression in baboon endometrial stromal cells by cells of trophoblast origin

Objective: To determine the influence of the conceptus on the induction of decidualization in endometrial stromal cells from the baboon. Methods: For in vivo studies, implantation sites from day 22 of pregnancy in the baboon were analyzed for insulin-like growth factor-II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) mRNAs using in situ hybridization. For in vitro studies, Jeg-3 cells or primary cytotrophoblasts isolated from term placenta were cocultured with a monolayer of stromal cells from the baboon endometrium. Cytotrophoblasts were placed either directly on top of the stromal cell monolayer or in transwell cell culture inserts and cultured for 7 days. No exogenous hormones were added. Stromal cells were analyzed for IGFBP-1 mRNA and protein by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. Results: Examination of early implantation sites in the baboon revealed a high expression of IGF-II mRNA in the invading cytotrophoblasts. Conversely, the stromal cells of the endometrium directly adjacent to the cytotrophoblasts expressed IGFBP-1 mRNA. Endometrial stromal cells cocultured with Jeg-3 cells or primary cytotrophoblasts for 7 days expressed IGFBP-1 mRNA and protein. This expression occurred in both the direct coculture system and coculture using the cell culture inserts. Cytotrophoblasts also induced the expression of prolactin in stromal fibroblasts following coculture. Conclusions: The concepts is capable of inducing decidualization of endometrial stromal cells. This is shown in both the in vivo and in vitro systems.

A baboon model for simulating pregnancy.

Estrogen and progesterone secreted by the corpus luteum regulate the function of the uterine endometrium in preparation for pregnancy. Embryonic signals superimposed on this steroid hormone-primed uterus further modulate the uterine environment to make it conducive to pregnancy. Understanding the signaling mechanisms that initiate the embryonic-maternal dialog in humans is not feasible. In an effort to elucidate the role of chorionic gonadotropin as a mediator of endometrial function in addition to its luteotrophic role, we have developed a simulated pregnant model in the baboon. Infusion of chorionic gonadotropin in a manner that mimics blastocyst transit induces major changes in the morphology and secretory activity of the endometrium. This model provides a method by which the function of various embryonic factors on endometrial can be tested in an in vivo model.

Insulin-Like Growth Factor Binding Proteins: A Paradigm for Conceptus-Maternal Interactions in the Primate

Complex interactions between the developing blastocyst and the maternal endometrium occur during implantation. Enders and Schlafke (1) defined the morphological process of implantation into three distinct stages; 1) apposition of the trophoblast and uterine epithelium; 2) adhesion, which involves the formation of junctional complexes between the two cell types and 3) epithelial penetration, which include the spreading of trophoblasts within the endometrium, destruction of the uterine glands and interruption of the integrity of the maternal vascular system. The morphological changes that take place during the establishment of pregnancy have been described to a limited extent in the human in the classic studies of Hertig and Rock (2) and more extensively in the rhesus monkey (3,4). However, the absence of biochemical studies on the secretory changes that accompany the dynamic interactive processes between the blastocyst and maternal endometrium limits our understanding of the morphological events associated with implantation. In an effort to correlate morphological changes with specific secretory changes in the endometrium we have focused our efforts on characterizing the proteins synthesized by the baboon uterus during the menstrual cycle and early pregnancy.

Methods for evaluating histocompatibility antigen gene expression in the baboon.

A wide variety of techniques has been developed for qualitative and quantitative analysis of gene expression in human cells and tissues. Two commonly used methods are reverse-transcription (RT)-polymerase chain reaction (PCR) to analyze the transcribed messenger RNAs (mRNA) and immunohistochemistry to detect the translated proteins. These techniques can be modified and adapted for use in analyzing gene expression in animal models. In particular, as a result of the close phylogenetic relationship between humans and nonhuman primates, human reagents, especially antibodies, cross-react with nonhuman primate tissues. However, the results are not always satisfactory as some antibodies may cross-react with irrelevant antigens in these tissues. In this chapter, we describe the use of RT-PCR and immunohistochemical techniques to analyze expression of Paan-AG, a novel class lb major histocompatibility complex antigen in the olive baboon (Papio anubis) placenta. We used Paan-AG-specific primers to amplify Paan-AG transcripts from baboon placenta, and generated Paan-AG isoform-specific polyclonal antibodies for use in immunohistochemistry.

Implantation: Lessons from a Primate Model

Implantation is a complex spatio-temporal interaction between the genotypically different embryo and mother. This is a highly coordinated process that is activated when the trophoblast cells of the embryo establish contact with the maternal endometrium. The initiation of pregnancy requires a precisely timed synchrony between endometrial development and the implanting blastocyst. Under the influence of ovarian steroids, the uterine endometrium undergoes profound modifications in cellular differentiation. In primates, at the appropriate phase of the menstrual cycle, the uterus becomes “receptive” and enables the blastocyst to attach. This “receptive window” is initially dependent on estrogen and progesterone. However, further morphological and biochemical changes are induced within the uterus by signals from the developing embryo and following trophoblast invasion.