In vivo versus in vitro oviductal glycoprotein (OGP) association with the zona pellucida (ZP) in the hamster and baboon.

The goal of this study was to determine if differences exist between in vivo vs. in vitro OGP association with the ZP and to quantitate those differences. Ovarian oocytes were harvested 12.5 or 27 hr post-hCG from hyperstimulated hamsters or baboons, respectively. Hamster and baboon ovarian oocytes were incubated in vitro in media +/- homologous OGP (100 or 200 microg/100 microl) or in some studies with 100 microl oviductal fluid for 3, 6, or 24 hr at 37 degrees C. Some of the baboon ovarian oocytes were transferred immediately after harvesting to the ampulla of both oviducts using a tom cat catheter and retrieved after a 3 hr in situ incubation. Hamster oviductal oocytes were collected 3, 6, and 24 hr following ovulation. After incubation or oocyte retrieval from the oviduct, cumulus cells were removed, oocytes were washed extensively and binding of OGP to the ZP was examined by immunofluorescence. Fluorescence intensity was quantified using densitometric scanning of photographic negatives with the background of each negative as an internal control. In all studies, OGP association with the ZP was significantly greater in vivo than in vitro (P < 0.05). In vitro OGP association with the ZP did not significantly increase with incubation time or OGP concentration; however, a small nonsignificant increase in OGP association with the ZP in the oviduct was detected over time. Differences did not appear to be due to depletion of OGP from the in vitro incubation media, since Western blot analysis of the media showed that OGP was still present. Although OGP concentration in vivo is unknown, Western blots showed similar intensity comparing 100 microg of OGP media and oviductal fluid. Immunolocalization of OGP using laser confocal microscopy showed regional differences in OGP binding. The outer half of the zona pellucida had significantly more OGP bound than the inner half on oviductal oocytes. No regional differences were detected for in vitro incubated oocytes. In conclusion, OGP association with the ZP is greater in vivo vs. in vitro, suggesting that one must be cautious in designing and evaluating in vitro studies of OGP function.

Enhancement of bovine oocyte fertilization in vitro with a bovine oviductal specific glycoprotein.

The purpose of this study was to determine the effect of a partially purified bovine oviductal glycoprotein (bOGP) on fertilization rates of bovine oocytes. The effect of albumin (control protein) or bOGP at 100 micrograms ml-1 during the 16-18 h fertilization period was evaluated in a standard IVF system using a sperm concentration between 0.5 and 0.125 x 10(6) spermatozoa ml-1. bOGP maintained a higher (P < 0.05) fertilization rate (62.0% versus 31.2%) at 0.125 x 10(6) spermatozoa ml-1 compared with the albumin control. The enhancement of fertilization by bOGP was blocked by the inclusion of a specific antibody to bOGP, whereas the antibody with albumin had no effect. A 2 h gamete preincubation step was subsequently included in the IVF procedure (0.125 x 10(6) spermatozoa ml-1) to determine whether the effect of bOGP was mediated through an interaction with the oocyte, the spermatozoon or both. When oocytes were preincubated with bOGP the fertilization rates were higher (P < 0.05) than with the albumin control (oocytes and spermatozoa exposed to albumin), whereas preincubation of spermatozoa with bOGP did not affect fertilization rates. There was no synergistic effect of preincubating oocytes and spermatozoa with bOGP. The increase in fertilization rate achieved by preincubating oocytes with bOGP was blocked with a specific antibody to bOGP. These results suggest that the increase in fertilization rates observed when bOGP is included during the 16-18 h fertilization period are primarily mediated through the interaction of bOGP with the oocyte since the same facilitatory effect was observed with a 2 h preincubation of oocytes before IVF. The ability to block these effects with a polyclonal antibody specifically generated against bOGP shows that this biological activity is due to bOGP. In summary, bOGP enhances fertilization in bovine oocytes whether it is included during preincubation or insemination and this appears to be due to a direct effect on the oocyte.

Regulation of the glycosylated beta-lactoglobulin homolog, glycodelin [placental protein 14:(PP14)] in the baboon (Papio anubis) uterus.

In vitro studies indicate that glycodelin (PP14) synthesis by the human endometrium increases dramatically at the time of implantation and early pregnancy. It has been postulated that this protein may have an immunosuppressive function. Due to the limitations associated with in vivo studies in the human, this study was undertaken to study the regulation of the baboon glycodelin homolog in vivo during the menstrual cycle and early pregnancy. In nonpregnant baboons, between days 10-12 postovulation (n = 3) the mid and apical regions of the glandular epithelium showed a distinct punctate staining pattern, which increased between days 12-18 of pregnancy (n = 3). Between days 25-60 of pregnancy, staining intensity in the glandular epithelium decreased. The decrease was more apparent at the implantation site compared with the nonimplantation site. The immunostaining correlated with the synthesis of radiolabeled baboon glycodelin in explant culture. Northern blot analysis demonstrated two messenger RNA (mRNA) transcripts [1.0 and 1.7 kilobases (kb)] in the baboon uterus compared with a single 1.0-kb transcript in the human, and mRNA expression was consistent with protein localization and synthesis. The protein and mRNA expression was consistently higher in the deeper glands of the functionalis and basalis during early pregnancy. Because the increased expression of glycodelin in the baboon endometrium coincided with peak levels of CG, a simulated pregnant baboon model was used to confirm hormonal regulation. Exogenous human CG (hCG) followed by estrogen and progesterone treatment in intact and ovariectomized baboons up-regulated glycodelin expression between days 18-25 postovulation (n = 10). By day 32 postovulation (n = 3), glycodelin synthesis decreased. Estrogen and progesterone treatment in the absence of exogenous hCG did not result in an increase of glycodelin synthesis. Analysis of uterine flushings from hCG-treated animals revealed that a minimum of 7 days of hCG treatment was required for glycodelin to be detectable in the uterine lumen. These studies indicate that a posttranslationally modified glycodelin homolog is synthesized by the baboon uterus during early pregnancy and appears to be regulated directly by CG. This pattern of synthesis is comparable with that observed with in vitro studies in the human. Because glycodelin expression is associated with CG secretion, we suggest that this protein may have a functional role during implantation in the primate. Thus, the baboon may serve as a nonhuman primate model to elucidate the function of this protein in vivo.

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. METHOD OF STUDY: Antibodies against a 17-amino-acid sequence of the OGP core protein (amino acids 52-68) and the denatured hamster OGP protein were generated, characterized, and tested in an in vitro sperm binding assay. RESULTS: Sperm binding was significantly decreased (P < 0.05) when oviductal oocytes were incubated for 2 hr with 4 or 8 mg/ml of immune IgG of both antibodies when compared with normal rabbit IgG. A fluorescence assay showed binding of both antibodies to the endogenous OGP associated with the ZP of ovulated hamster oocytes. CONCLUSIONS: These results suggest that OGP may be a potential immunocontraceptive target because both antibodies significantly decreased sperm binding to the ZP of oviductal oocytes. Immunocontraception may be accomplished by attempting to generate active immunity to a recombinant OGP, to the region selected in this study (amino acids 52-68) or to some other region of the core protein.

Immunologic and molecular characterization of an estrogen-dependent glycoprotein in the rhesus (Macaca mulatta) oviduct.

The objective of this study was to detect and characterize a secreted oviduct-specific glycoprotein (OGP) in the rhesus macaque (Macaca mulatta) and to compare the characteristics of this OGP to those previously characterized in baboons and women. Oviducts were obtained from untreated ovariectomized rhesus and from ovariectomized rhesus either treated with estradiol (E2) for 14 days or treated sequentially with E2 for 14 days and then with E2 plus progesterone (P4) for an additional 14 days. Segments of oviducts were either fixed for morphological analysis, cultured for OGP synthesis and release, or frozen for RNA analysis. The proteins present in the culture media were separated by one-dimensional SDS-PAGE, and OGP was detected on Western blots using polyclonal antibodies generated against the reduced form of baboon OGP or a 17-amino acid segment of the baboon core protein. Cross-reacting antigens were present in the 120-kDa region, identical to what was observed for baboon and human OGP. Indirect immunogold localization of OGP on thin sections demonstrated specific clustering of gold particles over the apical secretory granules of the secretory cells of the oviductal epithelium. A cDNA was generated using RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE), and sequenced. The total transcript was 2237 nucleotides in length plus a poly(A) tail. The largest open reading frame was 624 amino acids, which would produce a protein of 69.3 kDa. The nucleotide sequence was more than 95% identical to the nucleotide sequences of baboon and human OGP. Northern blots revealed a single message at 2.4 kilobases (kb) in oviduct samples obtained from E2-treated rhesus. This message was absent in oviducts obtained from untreated ovariectomized and from sequential E2 plus P4-treated rhesus macaques. In summary, the rhesus oviduct synthesizes and secretes an OGP in the presence of E2 that is immunologically and structurally similar to the baboon and human OGP. The presence of a highly homologous glycoprotein in several primates suggests a similar function for OGP in the reproductive process.

Immunocytochemical localization of transforming growth factor alpha, 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 alpha, epidermal growth factor and epidermal growth factor receptor in the endometrium of ovariectomized cats treated with oestradiol-17 beta 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 alpha 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.

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.

Characteristics of an oviductal glycoprotein and its potential role in fertility control.

At the time of ovulation the lining epithelium of the mammalian oviduct consists of columnar ciliated and secretory cells. These mature cells are dependent on ovarian steroids in carnivores. Oestradiol induces differentiation of these cells and maintains their mature functional state, and progesterone induces dedifferentiation. The secretory cells synthesize and secrete an oestrogen-dependent high molecular weight glycoprotein. The cDNAs encoding oviductal glycoproteins from several species have been sequenced and show high similarity. The human cDNA hybridized with a single message on northern blots of total oviduct RNA obtained from oestradiol-treated cats (about 2.3 kb) and dogs (about 2.1 kb). This glycoprotein is the major nonserum protein present in the oviductal lumen at the time of ovulation, fertilization and early embryonic development. The glycoproteins associate with the zona pellucida of oviductal eggs in all species studied to date. Recent studies suggest that the bovine glycoprotein facilitates sperm capacitation and significantly increases the ability of bovine spermatozoa to fertilize bovine oocytes in vitro, that the hamster glycoprotein increases the sperm penetration rate of the zona pellucida by three times and that the human glycoprotein increases sperm binding to the zona pellucida by three times. All of the evidence for a biological function for this glycoprotein is derived from studies performed in several different species at reproductive stages before fertilization. The biological actions of this glycoprotein suggest a potential role for the glycoprotein in fertility control. Specifically, purified or recombinant glycoprotein may improve success in IVF procedures by enhancing binding of spermatozoa to the zona pellucida and improving fertilization rates. The glycoprotein may also be a potential immunocontraceptive target since antibodies generated against the oviductal glycoprotein may prevent fertilization by preventing binding of spermatozoa to the zona pellucida.

The baboon oviduct: characteristics of an oestradiol-dependent oviduct-specific glycoprotein.

The baboon oviductal epithelium differentiates into a tall columnar epithelium consisting of ciliated and secretory cells during the follicular phase of the menstrual cycle in response to rising oestradiol levels. The apical tips of these secretory cells are filled with membrane-bound secretory granules. During the luteal phase when progesterone levels are elevated, the epithelium regresses and deciliation occurs. Analysis of secretory proteins obtained from explant culture media by SDS-PAGE followed by fluorography or Western blots has revealed that the baboon oviduct synthesizes and secretes a high molecular weight glycoprotein during the follicular phase of the cycle. Immunocytochemistry demonstrated that this oviductal glycoprotein is localized to the secretory granules of epithelial secretory cells, is oviduct specific, and that following secretion the oviductal glycoprotein binds to the zona pellucida and perivitelline space of ovulated oocytes and embryos within the oviduct. Similar proteins have been characterized in other mammalian species. cDNA data show that the complete coding sequence is 2228 bp for a protein of 623 amino acids. A Genbank search showed that baboon oviductal glycoprotein has high homology to other oviductal glycoprotein sequences at both the nucleotide and amino acid levels. Studies conducted to date probing the biological function of oviductal glycoprotein indicate that this protein plays a role in prefertilization reproductive events (sperm capacitation; sperm-zona binding; zona penetration). Additional experiments are needed to reveal a specific function and mechanism for this molecule.

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.

Regional distribution and hormonal control of estrogen-dependent oviduct-specific glycoprotein messenger ribonucleic acid in the baboon (Papio anubis).

Our objective in this study was to complete the sequence of the baboon oviductal glycoprotein, examine the hormonal regulation of the oviductal glycoprotein mRNA, and determine whether there was a regional variation within the oviduct in the level of oviductal glycoprotein mRNA expression. Finally, because of the structural similarity of the amino terminal end of the oviductal glycoprotein to chitinases, we sought to determine whether the oviductal glycoprotein functions as a glycosyl hydrolase. The total transcript length of the baboon oviductal glycoprotein was determined to be 2228 nucleotides in length plus a poly(A) tail. The largest open reading frame was 623 amino acids, which would produce a protein of 69.3 kDa. The first 420 amino acids were highly homologous to the amino acid sequence of other oviductal glycoproteins, but the remainder of the sequence differed considerably from that of all other species except the human. Although the N-terminal region exhibited sequence similarity to chitinases, the oviductal glycoprotein did not exhibit any activity towards typical chitinase substrates. The oviductal glycoprotein mRNA levels were elevated to approximately the same extent in the fimbria, ampulla, and isthmus of the oviduct after estradiol treatment and in the late follicular stage of the menstrual cycle. The oviductal glycoprotein mRNA levels were lower in the early follicular stage and early luteal stage and were not detectable in the late luteal stage or in progesterone-treated baboons. These results indicate that the oviductal glycoprotein mRNA is induced by estradiol and is present at the highest levels at the time of fertilization. Although there is structural homology with chitinases, no such glycosyl hydrolase activity could be detected. However, the common structure of the N-terminal region of the oviductal glycoproteins implies that it has the same, as yet unknown, function in all species.

Association of oviduct-specific glycoproteins with human and baboon (Papio anubis) ovarian oocytes and enhancement of human sperm binding to human hemizonae following in vitro incubation.

The objectives of this study were 1) to determine whether or not human and baboon oviduct-specific glycoproteins (human OGP, baboon OGP) would associate with ovarian oocytes during in vitro incubation in a manner similar to that detected in vivo for oviductal oocytes and 2) to determine whether the association of OGP with ovarian oocytes influenced sperm binding. In vitro association of OGP with ovarian oocytes was assessed by indirect immunofluorescence assay using a polyclonal antibody prepared against human or baboon OGP. Human and baboon ovarian oocytes incubated in culture media containing OGP showed association of OGP with the zona pellucida (ZP) as detected by bright fluorescence. A similar pattern of fluorescence was observed in baboon oviductal oocytes (positive control). No fluorescence of the ZP was detected from ovarian oocytes incubated with culture medium alone. The pattern of fluorescence for ovarian oocytes incubated with OGP and serum albumin, the major oviductal fluid protein, was similar to that for oocytes incubated with OGP alone. A modified hemizona assay was used to assess whether association of human OGP with human ovarian oocytes influenced sperm binding. The number of sperm bound to hemizonae in the presence of human OGP was significantly greater (p < 0.01) than the number bound to hemizonae in the control culture medium. Addition of antibodies specific for human OGP to the incubation medium 1 h prior to addition of gametes blocked the enhancement of sperm binding seen in the presence of human OGP alone. Finally, human hemizona assays conducted in the presence of baboon OGP resulted in a significant decrease (p < 0.05) in the number of sperm bound per zona compared with that in culture medium alone despite high homology between human and baboon OGP. These results 1) suggest that human OGP associates with ovulated oocytes in vivo; 2) support the hypothesis that association of OGP with the ZP may play a role in fertilization, possibly through enhancing the binding of sperm to the ZP within the oviduct; and 3) suggest that a homologous system (i.e., gametes and oviductal glycoprotein from the same species) is necessary for study of the function of oviductal glycoproteins.

Smooth muscle myosin II and alpha smooth muscle actin expression in the baboon (Papio anubis) uterus is associated with glandular secretory activity and stromal cell transformation.

The objective of this study was to investigate the localization and hormonal regulation of smooth muscle myosin II (SMM II) and alpha smooth muscle actin (alpha SMA) in the baboon uterus, since cytoskeletal proteins are involved in secretory function and morphological transformation. Uterine tissue was obtained from baboons 1) during the menstrual cycle, 2) following steroid treatment of ovariectomized baboons, 3) during pregnancy (Days 14-60 postovulation [PO]), and 4) during simulated pregnancy (Days 18-32 PO). Tissues were processed for immunocytochemical localization of SMM II or alpha SMA with specific polyclonal or monoclonal antibodies, respectively. SMM II stained all smooth muscle cells of blood vessels and myometrium regardless of treatment. Glandular epithelial staining was present only in endometrium obtained during the luteal phase or following estrogen and progesterone treatment. Staining intensity was greater in the basalis than in the functionalis. The number of glands staining positive for SMM II on Days 18-32 of pregnancy and simulated pregnancy was variable. Glandular stain was absent after Day 32 PO. These immunocytochemical data were confirmed by immunoblot analysis of glandular cytosolic extracts. Stromal staining for SMM II was present under the luminal epithelium during simulated pregnancy (Days 18-32), on Day 25 of steroid treatment in the simulated-pregnant controls, and in nonimplantation sites during pregnancy. In contrast, alpha SMA staining was low or absent in all uterine cell types in ovariectomized baboons. Under estrogen-dominated conditions (follicular phase and estrogen treatment), alpha SMA staining was present in smooth muscle cells, and this staining persisted throughout the remaining treatment periods. Glandular epithelial staining for alpha SMA was absent in all treatment groups. However, alpha SMA staining in stromal fibroblasts underneath the luminal epithelium was evident as early as Day 14 of pregnancy and Day 18 of simulated pregnancy. The number of stromal fibroblasts that stained positive increased in the surface region of the functionalis between Days 18 and 32 PO, and the staining extended throughout the upper functionalis region. There was a decrease in the number of positively stained stromal fibroblasts, particularly at the implantation site, between Days 32 and 40 of pregnancy. By Days 50-60 of pregnancy, this staining was almost absent. The induction of alpha SMA in stromal fibroblasts in the functionalis region in pregnant baboons was confirmed by immunoblot analysis of stromal cell cytosol extracts. We conclude that the progesterone-induced glandular expression of SMM II may be involved in uterine secretory function and that alpha SMA expression in stromal fibroblasts during pregnancy and after long-term steroid treatment is associated with the decidualization process.

A human oviduct-specific glycoprotein: synthesis, secretion, and localization during the menstrual cycle.

The major objective of this study was to examine the hormonal regulation of a human oviduct-specific glycoprotein (huOGP) throughout the menstrual cycle and in all regions of the human oviduct. Regulation of synthesis and secretion was examined at both the protein (Western immunoblots and immunocytochemistry) and mRNA (Northern and slot blots) levels and correlated with changes in the morphological features of the oviductal epithelial cells throughout the cycle. Immunoblot analysis of oviductal fluid and explant culture media from all regions of the oviduct demonstrated that huOGP is primarily found during the follicular stage of the cycle and is not present in serum, follicular fluid, or uterine endometrium. Moreover, two-dimensional (2-D) immunoblots showed that all major isoelectric variants of huOGP observed on 2-D fluorographs are immunologically related. Light microscopic immunocytochemistry localized huOGP to oviductal secretory cells in both ampulla and isthmic regions, with the most intense immunoperoxidase staining seen in midcycle samples. Using an indirect immunogold technique at the electron microscopic level, huOGP was specifically localized to secretory granules of the ampullary and isthmic nonciliated epithelial cells. The ultrastructural characteristics of these secretory cells during the mid to late follicular phase of the cycle suggested elevated protein synthetic activity. In addition, mRNA expression for huOGP was elevated in all regions of the oviduct in midcycle specimens. Collectively, these data indicate that huOGP is a major tissue-specific, stage-specific secretory product of the human oviduct during the periovulatory stage of the cycle and support the hypothesis that huOGP synthesis and secretion may be regulated by fluctuations in the levels of estrogen and progesterone.

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.

Secretory proteins and growth factors of the baboon (Papio anubis) uterus: potential roles in pregnancy.

The primate endometrium undergoes distinct morphological changes during the menstrual cycle. These alterations are regulated by the steroid hormones, estrogen and progesterone. Several lines of evidence suggest that some of these hormonally induced changes may be modulated by growth factors. Our studies have focused on characterizing the secretory activity of the uterine endometrium associated with these hormonally regulated morphological changes during the menstrual cycle and pregnancy in the baboon. Additionally, we have also attempted to study the regulation of specific growth factors and their receptors. In this review we present evidence to indicate that growth factor receptors for insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF), and secretory proteins, insulin-like growth factor binding protein-1 (IGFBP-1) and retinol binding protein (RBP), which are present in the glandular epithelium during the menstrual cycle, undergo cell-specific changes in gene expression at the implantation site during pregnancy. We postulate that these alterations in growth factor receptor and secretory protein expression are conceptus modulated and may play important regulatory roles during trophoblast invasion and decidualization.

Complementary deoxyribonucleic acid cloning and molecular characterization of an estrogen-dependent human oviductal glycoprotein.

A 120-kDa oviduct-specific glycoprotein is synthesized and secreted into the oviductal lumen during estrogen dominance in the human. The objective of this investigation was to clone, sequence, and characterize the cDNA to this core protein. Rapid amplification of cDNA ends was used to clone a contiguous 3' CDNA end and 5' cDNA end. The total length of the cDNA was determined to be 2.2 kb by sequence analysis and exhibited a 92% sequence identity with the comparable overlapping baboon cDNA (1.2 kb). A high degree of homology was found to the N-terminal sequence of hamster oviductin and the partial sequence of a homologous baboon and bovine oviduct glycoprotein. Northern blots revealed a single mRNA species of 2.4 kb. Using RNA from various tissues of an estrogen-treated baboon, we found that the mRNA for the oviductal glycoprotein was present only in the oviduct. Hybridization was detected to an mRNA of similar size from oviductal tissue of the baboon, hamster, and mouse and to an mRNA of slightly smaller size in the rabbit, cow, and cat but not to any mRNA species from rat oviductal RNA. Slot-blot analysis showed that the message was present in significantly greater (p < 0.05) concentrations in RNA from oviductal tissue from the late follicular stage than from the early follicular, early or late luteal, or postpartum stages. In conclusion, we have isolated the complete cDNA for a human oviductal glycoprotein. The presence of significantly greater amounts of the mRNA during the late follicular phase of the menstrual cycle is consistent with the proposed estrogen control. The mRNA for the oviductal glycoprotein is present only in the oviduct of an estrogen-treated baboon, and a cross-hybridizing RNA is found in oviductal RNA from various mammals.

Epidermal growth factor, transforming growth factor-alpha, and epidermal growth factor receptor localization in the baboon (Papio anubis) oviduct during steroid treatment and the menstrual cycle.

OBJECTIVES: Polypeptide growth factors may modulate the actions of estrogen (E2) and progesterone (P) in reproductive tissues in an autocrine/paracrine manner. The objective of this study was to determine whether the baboon oviduct contains epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and EGF receptor (EGF-R) and whether changes in their expression are correlated with various hormonal states. METHODS: Oviductal tissue was obtained from adult female baboons (Papio anubis) after oophorectomy and steroid treatment, and during the menstrual cycle. Ampullary regions were fixed in Bouin's fixative and embedded in paraffin for immunocytochemistry using rabbit polyclonal antibodies against EGF and EGF-R, and mouse monoclonal antibody against TGF alpha. RESULTS: Both EGF and EGF-R were present in all tissue compartments (most strongly in the epithelium, followed by smooth muscle and stroma) at all reproductive stages and showed similar staining patterns. However, the most intense immunoreactive product was found in the tissue obtained from the E2-treated and late follicular phase animals. At this time, intense staining was present in the apical regions of the mature ciliated cells, whereas the stain was dispersed uniformly over the cytoplasm of all other cell types. Immunoreactive TGF alpha was limited primarily to the nonciliated epithelial cells, and staining was most intense in the E2-treated and late follicular phase tissues. Transforming growth factor-alpha formed intense perinuclear deposits in the mature secretory cells, an area that corresponds to the Golgi region. No immunoreactive product was observed for any of these proteins when preimmune serum was substituted for the primary antibody or when the primary antibody was preabsorbed with antigen. CONCLUSION: In summary, EGF, TGF alpha, and EGF-R are present in the ampulla of the baboon oviduct. Moreover, the localization and intensity of immunoreactive product are dependent on cell type and hormonal state. These data are consistent with the concept that EGF, TGF alpha, and EGF-R may be regulated by E2 and P and thus may play a role in cell differentiation and function. In addition, the specific localization of TGF alpha suggests that this growth factor may be synthesized for release from the secretory cells and thus may also function as a modulator of gamete/embryo viability and development.

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-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. 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 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. RESULTS: 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. CONCLUSIONS: 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.