Kathleen M. Donnelly

Modulation of the Action of Chorionic Gonadotropin in the Baboon (Papio anubis) Uterus by a Progesterone Receptor Antagonist (ZK 137.316)

Abstract Signals from the developing mammalian blastocyst rescue the corpus luteum (CL) and modulate the uterine environment in preparation for implantation and early pregnancy. Our previous studies demonstrated that both short- and long-term administration of chorionic gonadotropin (CG) markedly alters the morphology and the biochemical activity of the receptive endometrium. Because the effects of CG were superimposed on a progesterone-primed endometrium, this study was undertaken to determine if the inhibition of progesterone action by progesterone receptor antagonists (PRa) in intact and ovariectomized baboons would alter the action of CG on the endometrium at the time of uterine receptivity. In the short-term hCG-treated baboons, the PRa reduced the epithelial plaque reaction, completely inhibited α-smooth muscle actin (αSMA) expression in stromal fibroblasts, and induced the reappearance of the progesterone (PR) and estrogen (ERα) receptors in epithelial cells. However, this treatment protocol had no effect on the expression of glycodelin in the glandular epithelium. In contrast, glycodelin expression in addition to αSMA was suppressed in the ovariectomized animals. In the long-term hCG-treated baboons, the PRa had a similar effect on both αSMA, PR, and ER. In addition, this treatment also resulted in an inhibition of glycodelin expression in the glandular epithelium. These results indicate that blocking the action of progesterone on the endometrium even for a short period of time has a profound effect on the hCG-induced response in stromal fibroblasts. In contrast, for the diminution of glandular epithelial function in the presence of an ovary requires prolonged inhibition of progesterone action, suggesting a potential paracrine effect on the endometrium from the CL in response to hCG.

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.

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.

Interactions Between the Embryo and Uterine Endometrium During Implantation and Early Pregnancy in the Baboon (Papio anubis)

The establishment of pregnancy in all mammalian species requires a synchronous interaction between the implanting embryo and the maternal endometrium. The mammalian uterus is receptive to the implanting blastocyst for a specific period of time and this receptive window appears to be regulated primarily by ovarian steroids. Embryo implantation is the natural culmination of this period, and successful nidation requires the precise preparation of both the blastocyst and endometrium. A remarkable synchrony is achieved by continuous maternal/conceptus interaction even prior to trophoblast invasion. The internal lining of the uterus is a specialized interface where a complex combination of anatomic, biochemical, endocrinologic, and immunologic events occur to ensure successful embryonic development. It is apparent, therefore, that the biological requirements of the early mammalian conceptus must be met by uterine and oviductal secretions since they constitute the primary environmental contact between the developing embryo and its mother prior to implantation.

Embryo—Maternal Dialogue in the Baboon (Papio Anubis)

“Endometrial receptivity” has been defined as the period during the menstrual or estrous cycle that is most favorable for blastocyst implantation. However, in the absence of a blastocyst or a blastocyst “signal,” the true definition of a receptive uterus is difficult to ascertain. This is especially true for studies pertaining to the human endometrium. Towards this end, we have developed the baboon as a nonhuman primate model and attempted to identify changes in the receptive uterine endometrium both for the presence or absence of a conceptus. The window of receptivity in the baboon is between 8 and 11 days postovulation. For the purposes of our studies we have divided the receptive window into three phases. Phase I is characterized by the changes induced by estrogen and progesterone during the luteal phase of the normal menstrual cycle. Phase II is defined as modulation of the Phase I receptive endometrium by blastocyst signals. In the primate, chorionic gonadotrophin (CG) is the primary luteotrophic hormone and presumably the major embryonic “signal.” Phase III of the implantation window is induced by the blastocyst interacting with the receptive endometrium. This phase is characterized by rapid trophoblast migration and the onset of decidualization of stromal fibroblasts.