Harold G. Verhage

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.

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)

Progesterone Suppression of the Estradiol Receptor in the Reproductive Tract of Macaques, Cats, and Hamsters

Few biological phenomena are so well described and yet so poorly understood as the periodic morphological changes that occur in the mammalian reproductive tract during estrous or menstrual cycles.

Species-specific effect of oviductal glycoproteins on hamster sperm binding to hamster oocytes

The secretory cells of the oviductal epithelium secrete a high‐ molecular‐weight glycoprotein (OGP). OGPs from different mammalian species show similar immunological characteristics, their cDNAs show high homologies, and they associate with the zona pellucida of oviductal oocytes in vivo. The purpose of this study was to determine the effect of OGP obtained from different species on the binding of hamster sperm to hamster oocytes. Hamster oocytes were inseminated (30 min) in the presence or absence of homologous or heterologous OGPs, and sperm bound/oocyte were counted after removing loosely attached sperm. Ovarian oocytes had an average of 2.9 ± 0.6 sperm bound/oocyte, whereas oviductal oocytes had 36.3 ± 2.7. Hamster OGP (0.1 mg/ml) significantly increased sperm binding to ovarian oocytes twofold and had no effect on sperm bound/oviductal oocytes. Human OGP (0.5 mg/ml) significantly decreased sperm binding to ovarian oocytes (0.9 ± 0.3 sperm bound/oocyte). This effect was dose dependent for oviductal oocytes and could be blocked by preincubating human OGP with a specific antibody to human OGP. The presence of baboon and cow OGP during the insemination of hamster oviductal oocytes also resulted in a significant decrease in sperm bound/oocyte, whereas the addition of hamster OGP to hamster oviductal oocytes had no effect. These results show that homologous OGP enhances sperm binding to the ZP, whereas heterologous OGP inhibits that effect. Thus, our results suggest that OGP plays a role in the species‐specific characteristics of sperm/ZP interaction, and that one must use a homologous system (OGP and gametes from the same species) to study the biological effect of OGP. Mol Reprod Dev 46:201–207, 1997.

Cytosol and nuclear progesterone receptor in cat uterus and oviduct.

Abstract Binding of progesterone (P) to a specific cytosol receptor and the in vivo translocation of the cytosol receptor complex to the nucleus was studied in the cat uterus and oviduct. Cytosol and nuclear fractions were prepared from the reproductive tracts of ovariectomized animals treated for 14 days with estradiol (E 2 ) and from those which were subsequently infused with a P solution for 15 min. In vitro cytosol incubation with [ 3 H]-P included cortisol to eliminate binding to CBG. The specific binding of [ 3 H]-P reached equilibrium within 90 min at 0°C and was stable for at least 4 h in both tissues. Competitive binding studies revealed the following binding affinities for the cytosol receptor; R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) = P ⪢ corticosterone = E 2 = testosterone. Scatchard analysis of [ 3 H] P binding in cytosols from E 2 -treated animals yielded a K D of 3.2 × 10 −8 M in the uterus and 2.5 × 10 −8 M in the oviduct. The P binding protein had a sedimentation coefficient of 6.8 S. The content of nuclear receptor was assessed by an exchange assay using R5020. Equilibrium was reached within 4 h at 22°C and remained unchanged for another 2h. The relative binding affinities for the nuclear receptor were; R5020 > P > corticosterone > testosterone > E 2 . The K D was 8.2 × 10 −9 M in uterine nuclei and 6.0 × 10 −9 M in oviduct nuclei from E 2 + P treated animals. Following P-infusion the decrease in the quantity of specifically bound [ 3 H]-P in the cytosol, and the increase in the quantity of specifically bound [ 3 H]-R5020 in the nuclei, were significant in both tissues.

Gain of OGP, an Estrogen-Regulated Oviduct-Specific Glycoprotein, Is Associated with the Development of Endometrial Hyperplasia and Endometrial Cancer

Purpose: Lesions in the endometrium are difficult to differentially diagnose. The present study examined whether oviduct-specific glycoprotein is differentially expressed in normal, hyperplastic, and malignant endometrium. Experimental Design: The expression of oviduct-specific glycoprotein was characterized by immunohistochemical methods with whole sections of endometrium from 90 women. An endometrial cancer tissue microarray with 200 cases of endometrial cancer was also assessed for oviduct-specific glycoprotein, estrogen receptor, and PTEN expression. Results: In normal endometrium, there was focal oviduct-specific glycoprotein expression in the basalis layer, where the stem cells reside, in 10 of 15 cases. On average, atypical hyperplastic endometria stained more intensely than hyperplastic endometria (P = 0.017), whereas the percentage of positively stained cells was not significantly different. The mean staining indices (intensity × percentage of positive cells score) for hyperplasia and atypical hyperplastic were 4.7 and 5.5 and were significantly higher than staining indices seen in normal cycling endometria or well-differentiated endometrioid endometrial carcinomas (P < 0.0001 and P < 0.001, respectively). The endometrial cancer tissue microarray showed that of 139 endometrioid endometrial carcinomas, 11 cases were strongly oviduct-specific glycoprotein positive, whereas the other 128 cases were negative or weakly positive. Analysis of Kaplan-Meier curves with log-rank statistics showed a trend toward significance, with strong oviduct-specific glycoprotein staining serving as a predictor of good prognosis (P = 0.1). There was a significant positive correlation between oviduct-specific glycoprotein staining and loss of PTEN in the cases of endometrial cancer (P = 0.004). Conclusions: Oviduct-specific glycoprotein may be a useful diagnostic adjunct to more accurately classify premalignant and early malignant change in the endometrium, improving on the current irreproducible histologic classification system.

Immunocytochemical localization of transforming growth factor α, epidermal growth factor and epidermal growth factor receptor in the cat endometrium and placenta

This study was undertaken to determine the immunocytochemical localization of transforming growth factor α, epidermal growth factor and epidermal growth factor receptor in the endometrium of ovariectomized cats treated with oestradiol-17β and/or progesterone and in the endometrium and placenta of pregnant cats. Specific immunostaining was observed for all three antibodies. Moderate immunostaining for transforming growth factor α was observed in the epithelium of ovariectomized and oestrogen-treated cats. Dark epithelial staining was observed throughout pregnancy. The epithelial cells in progesterone-treated and peri-implantation animals contained dense deposits of reaction product, which were not reduced in intensity when immunoabsorbed antiserum was used. For epidermal growth factor, light--moderate epithelial staining was observed in ovariectomized and steroid-treated animals, and this increased in pregnant cats. Stromal staining for both the transforming and the epidermal growth factors was limited in steroid-treated animals and increased as pregnancy continued. Dark staining for epidermal growth factor receptor was observed in the epithelium and stroma in all the animals studied. The tips of surface epithelial convolutions in the non-implantation sites were always more darkly stained than in other regions of the surface epithelium. Staining in the placental trophoblast was limited to the syncytiotrophoblast for the two growth factors and the cytotrophoblast for the receptor during most of pregnancy and was absent late in pregnancy. The placental maternal giant cells contained specific immunoreactivity for all the immunogens from the middle of pregnancy to term. This study demonstrates that the two growth factors and the epidermal growth factor receptor are present in the endometrium and placenta of cats and suggests that these growth factors may play an autocrine/paracrine role during reproduction

Characterization of Antibodies Generated Against a Conserved Portion of Oviductal Glycoprotein (OGP) and Endogenous Hamster OGP and Their Ability to Decrease Sperm Binding to the Zona Pellucida In Vitro

PROBLEM: The effect of antibodies generated against hamster oviductal glycoprotein (OGP) on sperm binding to the zona pellucida (ZP) was evaluated.

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.

Steroid-Induced Proteins of the Primate Oviduct and Uterus: Potential Regulators of Reproductive Function

The biological requirements of the mammalian conceptus during the initial stages of development must be met by oviductal and uterine secretions since they constitute the primary environmental contact between the developing embryo and its mother prior to implantation. Both the mammalian oviduct and uterine endometrium are target tissues for the ovarian steroids and each responds by undergoing cyclic changes in morphology and secretory activity. These cyclic changes prepare the oviduct and uterus to receive and nourish the gametes during fertilization, early embryonic development and during the implantation process. Secretory products from these two tissue compartments are necessary to maintain gamete viability, however, their specific role in primate reproduction has yet to be determined. Studies to determine critical reproductive events during these very early stages of embryonic development and pregnancy obviously are very difficult, if not impossible, to perform in humans. Therefore, we have attempted to utilize the baboon (Papio anubis) as a non-human primate model to identify hormonally regulated secretory proteins of the oviduct and uterus and to determine similarities and dissimilarities in the secretory profile of these two tissue compartments in the baboon and human. This chapter summarizes our findings which suggests potential paracrine roles for specific, hormonally regulated proteins of the baboon and human oviduct and uterus.