Baboon Endometrium Research Articles

Symposium: reproduction in baboons. Secretory proteins of the baboon (Papio anubis) endometrium: regulation during the menstrual cycle and early pregnancy

The biological function of uterine endometrial secretory proteins in the primate remain to be elucidated. In general, during the luteal phase and under progesterone dominance, the glandular epithelial cells synthesize and secrete a number of proteins. Of these, placental protein 14 (PP14; now referred to as glycodelin) and insulin-like growth factor binding protein-1 (IGFBP-1) are the best characterized. Although induced by progesterone, their synthesis increases exponentially during pregnancy. In the baboon, glycodelin is immunolocalized to the mid functionalis and basal glands between days 10 and 12 post-ovulation. In response to either exogenous or blastocyst-secreted chorionic gonadotrophin, glandular synthesis increases markedly and remains elevated up to days 18-25 of pregnancy. The decrease in glycodelin in the endometrium is associated with glandular regression during the first third of pregnancy. In contrast, IGFBP-1 is only observed in the deep basal glands during the luteal phase. Following the establishment of pregnancy, IGFBP-1 synthesis switches from glandular to stromal and is correlated with the process of decidualization. IGFBP-1 synthesis continues to increase throughout gestation. We propose that glycodelin may have immunosuppressive properties and that IGFBP-1 may regulate trophoblast migration within the uterine endometrium.

The progressive rise in the expression of alpha crystallin B chain in human endometrium is initiated during the implantation window: modulation of gene expression by steroid hormones.

Human endometrium undergoes sequential changes during the menstrual cycle and becomes receptive to implantation during a defined period in the secretory phase. We attempted to identify the genes expressed during this period by representational difference analysis (RDA). When the cDNAs of a proliferative endometrium were used as the driver and the cDNAs of a post-ovulatory day 5 endometrium were used as the tester, a number of bands were identified by RDA. DNA of the cloned RDA products revealed that the majority of the clones contained a fragment of a cDNA identical to that of a crystallin B chain. Northern blot analysis showed that the expression of the alpha crystallin B chain mRNA was absent during the proliferative phase. The expression of the mRNA of alpha crystallin B chain first appeared in the secretory phase, progressively increased during this phase and peaked in the late secretory endometria. The pattern of expression of alpha crystallin B chain mRNA in the endometrium of mature cycling baboons (Papio anubis) was similar to that seen in human endometrium. As revealed by Western blot analysis, the expression of the alpha crystallin B chain protein in human endometrium followed a pattern of expression similar to its mRNA. At the cellular level, the immunoreactive protein first appeared in the surface epithelial cells of human endometrium within the implantation window without significant immunoreactivity in the underlying glandular cells. During the mid- and late secretory phases, the intensity of staining in the epithelial cells was enhanced and an intense immunoreactivity was developed in the glandular epithelium, alpha crystallin B chain was virtually an epithelial product and no immunoreactivity for this protein was detectable in the stromal cells, endothelial cells or lymphoid cells. The expression of alpha crystallin B chain could be regulated, by medroxy progesterone acetate as well as by oestrogen withdrawal, in human endometrial carcinoma cells (EnCa-101), transplanted to nude mice. Based on the data presented here, the known function of alpha crystallin B chain and its distinct pattern of expression in human endometrium, we suggest that this protein is an important factor within the molecular repertoire that makes endometrium receptive to implantation.

Distribution of integrins and the extracellular matrix proteins in the baboon endometrium during the menstrual cycle and early pregnancy.

Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in distribution in the endometrium during the menstrual cycle in women. Likewise the extracellular matrix (ECM) ligands for these receptors are likely to play a role in the establishment of a receptive endometrium. To develop primate models to study the role of these molecules in the cascade of molecular events leading to implantation, integrin expression and associated changes in ECM were investigated during the menstrual cycle and in early pregnancy in the baboon. Antibodies specific for the integrins (alpha(1-6) and alpha(v); beta1, beta3, and beta4) and ECM (laminin, collagen IV, fibronectin) were utilized. In addition, cytokeratin and alpha-smooth muscle actin were used as epithelial, stromal, and smooth muscle cell markers, respectively. Endometrium was obtained in duplicate or triplicate during the menstrual cycle and early pregnancy. Changes observed during the natural menstrual cycle were confirmed using ovariectomized, steroid-treated animals. Constitutively expressed integrins on the endometrial epithelium included the collagen/laminin receptors: alpha2, alpha3, alpha6, and beta4. The pattern of expression correlated well with the distribution of ECM in this tissue. Collagen IV was confined to the basement membrane of glandular epithelium and blood vessels. Laminin immunostaining was found in the basement membrane, mostly in the stroma of the basal region, in the glandular endometrium and vasculature. Fibronectin was present throughout the stroma but not in the basement membrane. The collagen receptor alpha1 beta1 and fibronectin receptor alpha4 beta1 appeared in the glandular epithelium in the luteal phase. As in the human, alpha1 and alpha4 disappeared from the glandular epithelium with the establishment of pregnancy. In contrast, the alpha4 beta3 vitronectin receptor appeared in the glandular epithelium only in pregnancy or following long-term steroid treatment with estrogen and progesterone but not during the time of uterine receptivity associated with the initial period of embryo attachment. Osteopontin, an ECM ligand for alpha(v) beta3, was coexpressed with this integrin in invading cytotrophoblasts, glandular epithelium, and decidualizing stromal cells. Decidualization in the baboon was associated with changes in integrin expression similar to those found in humans: there was an increase in alpha1, alpha3, alpha6, beta1, and alpha(v) beta3 in the decidualized stromal cells. Laminin and collagen IV expression also increased at the implantation site and throughout the endometrium. In contrast, fibronectin expression was most evident at the implantation site and corresponded to alpha5 expression on the invading cytotrophoblasts. In summary, marked similarities were found in the expression of ECM and the integrin receptors between the baboon and the human endometrium throughout the menstrual cycle and in pregnancy. Cycle-specific integrins, alpha1, and alpha4, were present on epithelial cells during the secretory phase. Delayed expression of alpha(v) beta3 in baboon endometrial glands correlated closely with the time of enhanced glandular secretory activity in this primate. The baboon appears to be an excellent model for the investigation of the role of integrins and ECM leading to successful implantation.

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.(35)S-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.

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.

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-alpha (TGF alpha), 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. 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 per group). The tissue was fixed in Bouin's solution and embedded in paraffin for immunocytochemistry using polyclonal antibodies against EGF and EGF-R and a monoclonal antibody to TGF alpha. Endometrial staining was located almost entirely in the glandular epithelium for TGF alpha 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 TGF alpha and EGF-R. A similar pattern was observed in the steroid-treated animals. In the endometrium from pregnant animals, EGF, TGF alpha, 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 TGF alpha 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 TGF alpha continued to be strong in the remaining epithelium through day 60. In placenta, EGF and EGF-R intensely stained the syncytiotrophoblast, but not the cytotrophoblast, whereas TGF alpha 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. This study demonstrates the presence of EGF, TGF alpha, and EGF-R in the endometrium during the cycle and early pregnancy. The detection of EGF, TGF alpha, and EGF-R in the stromal cells during pregnancy correlated with the onset of decidualization. We propose that EGF, TGF alpha, and EGF-R may play a role in glandular development during the cycle and in decidualization and implantation during early pregnancy.

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

The study was undertaken to characterize changes in uterine endometrial secretory activity and morphology in the baboon between Days 18 and 32 of pregnancy. Proteins synthesized by the implantation site endometrium (RI) and non-implantation site endometrium (RIII) and developing placenta were analyzed by two-dimensional PAGE and fluorography. The electrophoretic mobility and localization of chorionic gonadotropin (CG) in the baboon placenta, and insulin-like growth factor binding protein-1 (IGFBP-1) and retinol binding protein (RBP) in the baboon uterus were confirmed by Western blot analysis and immunocytochemistry. Placental protein synthesis was primarily characterized by the presence of radiolabeled CG at Days 18 and 25, which decreased by Day 32. Peak CG synthetic activity, immunolocalized to the syncytiotrophoblast, occurred during the formation of tertiary villi. Also associated with the development of mature villi was a corresponding increase in a basic protein (M(r) 23,000). The overall secretory pattern of the baboon endometrium was similar to that observed during the midluteal stage; however, the synthetic activity was enhanced during pregnancy. Radiolabeled and immunoreactive IGFBP-1 and RBP were detectable in the Day 32 functionalis and basalis, respectively, and their synthesis was higher in RI compared to RIII. The immunolocalization of IGFBP-1 in RI appeared to be directly related to the degree of decidualization. In addition, a novel group of low molecular-weight (M(r) 27,000-30,000) isoelectrovariant proteins was secreted predominantly by the basalis at Days 18 and 25 of pregnancy and disappeared by Day 32 in all but one animal. The synthesis of this group of proteins did not show regional specificity. These studies indicate that the baboon endometrium undergoes morphological and secretory changes during pregnancy that are associated with the development of a functional placenta and decidualized endometrium. Furthermore, these studies suggest that the conceptus/placenta may mediate specific changes in IGFBP-1 and RBP expression at the implantation site.

An Insulin-Like Growth Factor-Binding Protein in the Baboon (Papio anubis) Endometrium: Synthesis, Immunocytochemical Localization, and Hormonal Regulation*

The major secretory product of the baboon and human decidua during pregnancy is an insulin-like growth factor-binding protein (IGF-BP). This study was designed to determine the site and regulation of synthesis of this protein in the nonpregnant baboon based on our previous findings that this molecule is biochemically and immunologically similar in the two species during pregnancy. Endometrial tissue was obtained from cycling baboons and steroid-treated ovariectomized animals. Portions of the tissue were fixed for immunocytochemical analysis, cultured in the presence or absence of cyclohexamide and actinomycin-D, or snap-frozen in liquid nitrogen for RNA isolation. Immunostaining using monoclonal antibodies to IGF-BP indicated that the protein was localized predominantly in the epithelium of the deep glands and was most intense during the late luteal stage of the cycle. Immunoreactive product was also present in tissue from estrogen-primed progesterone-treated ovariectomized animals. However, the staining pattern was more variable and less intense than that in intact animals. Western blots of explant culture media showed the presence of an immunoreactive product only in those tissues that also demonstrated immunocytochemical localization. The absence of an immunoreactive band in medium obtained from tissue incubated in the presence of cyclohexamide suggested that this protein was synthesized de novo. The mRNA coding for IGF-BP appeared to be stable, as synthesis in explant cultures continued in the presence of actinomycin-D. The cDNA probe hybridized to a single message transcript of 1.65 kilobases. The presence of mRNA in tissues coding for this protein correlates with the immunochemical data relating to the site and hormonal regulation of its synthesis. The presence of this protein in the glandular epithelium of the baboon endometrium may have implications in the autocrine and/or paracrine regulation of trophoblast growth and penetration during implantation.

Decidua-associated antigens in the baboon.

Proteins associated with the uterine decidua (DE) may control critical events in pregnancy. This study was undertaken, therefore, to study DE-associated antigens in the baboon. Three antigens, designated 1, 2, and 3, were detected in the cytosols of all 17 specimens of the baboon DE by Ouchterlony’s immunodiffusion test (ID), using an antiserum against a glycoprotein fraction of baboon DE. Antigens 1 and 2 were also found in one sample of the cytosol of the term placenta and in cytosols of all 30 endometria from cycling baboons, but not in any of the 30 samples analyzed of sera from nonpregnant baboons. Only antigen 2 was detected in all 17 samples of sera from pregnant baboons. Antigen 3 was detected only in DE cytosols but not in any of the cytosols of 30 endometria from cycling animals, 30 sera from pregnant animals, or one sample of term placenta. In addition to the three antigens mentioned above, the baboon DE was found to contain a protein which was immunologically similar, but not identical, to the progestagen-dependent endometrial protein or PEP which we have previously identified in women. The PEP-like material was found in cytosols of 11 of the 17 specimens of DE and of one term placenta, and in one sample of amniotic fluid. However, it could not be detected in any of the cytosols of th 30 endometria from cycling baboons, 30 sera from nonpregnant or 17 sera from pregnant baboons. These observations strongly suggest that the development of baboon endometrium during pregnancy is associated with the increase in tissue concentration of at least two antigenic proteins, namely antigen 3 and a PEP-like component.

Nuclear receptor for oestrogen in the baboon endometrium: detection, characterization and variation in its concentration during the menstrual cycle.

The present paper describes studies conducted to detect and characterize the nuclear receptor for oestrogen in the baboon endometrium. Only 10% of the [3H]oestradiol nuclear receptor complexes were extracted with a 0.5 M-KCl solution. This solubilized receptor migrated as a 4.4S peak during 5-20% sucrose gradient centrifugation. The oestrogen receptor was not bound to oestrogen in the nuclei under normal physiological conditions. Using an unlabelled competitor addition technique with intact nuclei the variation in oestrogen-receptor concentration of baboon endometrium during the menstrual cycle was measured. This concentration increased slightly during the first week of the cycle, being maximal on day 7 before ovulation (2500 molecules/cell), then decreasing gradually, reaching the lowest level (300 molecules/cell) on day 5 after ovulation, where it remained until the end of the cycle.

The baboon as a primate model for the study of endometrium

A series of transcervical uterine biopsy specimens were obtained at various stages of the menstrual cycle from a colony of 11 normally cycling female baboons, Papio anubis and Papio cynocephalus. Morphologically, baboon endometrium appeared to be similar to human endometrium. Alkaline phosphatase activity was maximal throughout the preovulatory phase and during the late postovulatory period. During the preovulatory phase acid phosphatase was not demonstrable but increased after ovulation to reach maximal activity prior to menstruation. While differences exist between human and baboon endometria, the overall morphologic and histochemical changes are similar. In addition, the baboon's endometrium is readily accessible by transcervical uterine biopsy, thus making these animals valuable primate models for study of human reproductive problems.

Histochemical and ultrastructural effects of enovid E on the endometrium of the baboon.

Endometrium from a group of baboons treated with the oral contraceptive, Enovid E, was studied histologically, histochemically and ultrastructurally, and compared to endometrium from normally cycling animals. All endometria were obtained by transcervical uterine biopsy between 10 and 14 days of the treatment cycle or the normal menstrual cycle. Histologically, no discernible differences between the control and experimental endometria were apparent. While observable differences were not evident between the endometrial alkaline phosphatase and succinate dehydrogenase activities of control and experimental animals, there was an increased acid phosphatase activity in the Enovid E-treated baboon endometrium. Ultrastructurally, the glandular cells of treated animals appeared to be more physiologically advanced than did those from the control endometria. These advances were evident from the prominent Golgi complex, increased development of the endoplasmic reticulum and increases in the size, number and complexity of mitochondria. The functional correlates of these morphological and histochemical observations are discussed and compared to human endometrial studies.

Histology Of The Baboon Endometrium During The Menstrual Cycle And Pregnancy

The histologic characteristics of baboon endometrium during the menstrual cycle and pregnancy were studied and compared with those of human endometrium. Eight phases of endometrial change during the menstrual cycle are described. The samples were dated on the basis of sex skin changes. The basic histology of the baboon endometrium is similar to that of human endometrium, but some differences were observed. The growth of baboon endometrium is more sluggish, secretion is less intense, and many of the changes, especially in the stroma, are localized, less diffuse than they are in human endometrium. During pregnancy, decidual transformation in the baboon is much less intense than that in the human. No differences were observed between endometrium in association with preimplantation embryos and that from nonpregnant animals at comparable times following ovulation.

6A 1. Mechanism of action: Estrogens—I 184. Regulation of protein synthesis in baboon endometrium by endogenous estrogen and progestins

6A 1. Mechanism of action: Estrogens—I 184. Regulation of protein synthesis in baboon endometrium by endogenous estrogen and progestins

Effect of an intrauterine foreign body on [formula omitted] incorporation of acetate- 14C into endometrial macromolecules in baboons

The effect of an intrauterine foreign body (IUFB) on the incorporation of acetate-1- 14C into endometrial lipids, RNA and protein was studied in baboons. Thirty minutes after the intraluminal instillation of the radioprecursor, the control endometrium was found to contain about three times as much total radioactivity as the IUFB endometrium. However, the percentage of total tissue radioactivity which was incorporated into tissue macromolecules was 3–5 times greater in the IUFB than in the control endometrium. The tissue content of RNA and protein per unit tissue DNA was significantly lower and the specific activities of RNA and protein were significantly higher in the IUFB than in the control endometrium. These findings suggest that an IUFB may stimulate the turnover of macromolecules in baboon endometrium.