The in vitro synthesis of estrogen-dependent proteins by the baboon (Papio anubis) oviduct

This study was undertaken to determine the effect of estradiol and progesterone on the synthesis of oviduct-specific proteins and to determine if there were changes in the synthesis of these proteins in different regions of the oviduct. Ovariectomized females were either untreated, treated with estradiol for 7 or 14 days, or primed with estradiol for 14 days and then treated with estradiol plus progesterone or progesterone alone for either 7 or 14 days. Oviducts were incubated in the presence of labeled methionine or glucosamine for 24 h at 37 C, and the culture medium was then analyzed by one- and two-dimensional (2-D) polyacrylamide gel electrophoresis, followed by fluorography. A family of macromolecules [mol wt (Mr), 100,000-130,000] was only present in the animals treated with estradiol for 7 or 14 days. 2-D analysis demonstrated that two proteins, one basic and one acidic, were the major estradiol-responsive proteins in the 100,000-130,000 Mr region. In addition, an acidic protein in this region increased in intensity with estradiol treatment. All three proteins incorporated methionine and glucosamine. Since a steroid-responsive gradient is known to exist in the oviduct, oviducts were divided into fimbria, ampulla, and isthmic regions and cultured separately. 2-D analysis of the medium indicated that the ampulla synthesized both the acidic (Mr, 100,000-120,000) and basic (Mr, 120,000-130,000) proteins, whereas the acidic protein was dominant in the fimbria and the basic protein was dominant in the isthmus. Another acidic protein (Mr, 110,000) was only present in the fimbria and ampulla. This study clearly demonstrates that the estradiol-treated baboon oviduct synthesizes several proteins that were not detectable in the ovariectomized or progesterone-treated animal. Also, the different regions of the oviduct have different synthetic capabilities for these proteins. The electrophoretic characteristics of these proteins are similar to those we have observed at midcycle in the intact baboon and human oviduct.     

Localization of oxytocin and neurophysin in baboon (Papio anubis) corpus luteum by immunocytochemistry

Immunoreactive oxytocin is detectable in the corpora lutea of women and cynomolgus monkeys by radioimmunoassay. To localize the presence of oxytocin and neurophysin I in ovarian tissues of subhuman primates, three corpora lutea and ovarian stromal tissues and two Fallopian tubes obtained during the menstrual cycle of the baboon and decidua from two pregnant baboons were examined using highly specific antisera against either oxytocin or neurophysin I and peroxidase-antiperoxidase light microscopy immunohistochemistry. Oxytocin-like as well as neurophysin I-like immunoreactivities were found in some cells of all the corpora lutea only, but could not be demonstrated in ovarian stromal tissues, Fallopian tubes and decidua. Specificity of the immunocytochemical reaction was further confirmed by immunoabsorption of the antiserum with excess oxytocin or neurophysin, after which the immunoreactivities for both oxytocin and neurophysin in the luteal tissue were negative. Similar controls using normal rabbit serum gave no positive staining for either oxytocin or neurophysin. Counterstaining of the positive immunoreactivities for oxytocin and neurophysin I with Mayer's haematoxylin and eosin demonstrated clearly that the oxytocin and neurophysin I appeared as granular material mainly within the cytoplasm of the luteal cells. The localization of immunoreactive oxytocin and neurophysin I in the corpus luteum of the baboon demonstrates directly the presence of these two neurohypophysial peptides within primate luteal cells and suggests their local production.     

Heparin-binding EGF-like growth factor modulation by antiprogestin and CG in the baboon (Papio anubis)

The objectives of this study were to determine whether antiprogestin therapy or the infusion of human CG to mimic blastocyst transit in the baboon alters heparin-binding EGF-like growth factor expression during the window of implantation. During the menstrual cycle, heparin-binding EGF-like growth factor protein accumulation in the glandular epithelium was low in the proliferative phase and increased to maximal expression on d 5 and 10 postovulation. Stromal cells accumulated high levels of heparin-binding EGF-like growth factor in the proliferative phase, which decreased by d 5 postovulation. These transitional changes in both cell types were delayed when cycling baboons were treated with the antiprogestin ZK 137.316 during the luteal phase. The treatment with human CG had no effect on expression of heparin-binding EGF-like growth factor when compared with cycling baboons on d 10 postovulation and was comparable with that observed on d 18 and 22 of pregnancy. However, the superimposition of the antiprogestin with the human CG treatment also decreased expression in the epithelial cells. In summary, heparin-binding EGF-like growth factor accumulation in the epithelial glands is under the influence of progesterone and does not seem to be influenced by the paracrine secretion of trophoblast CG.     

Regulation of insulin-like growth factor-binding proteins in the baboon (Papio anubis) uterus during early pregnancy.

The baboon uterus begins to synthesize insulin-like growth factor-binding protein-1 (IGFBP-1) in the deep glands of the late secretory endometrium, and this protein then becomes the major secretory product of the term decidua. We hypothesized that the placenta and/or conceptus may regulate the synthesis and secretion of IGFBP-1 by decidualized stromal cells during pregnancy. To test this hypothesis, tissue was obtained from pregnant baboons on days 18, 25, and 32 postovulation. The uterus was separated into three regions: RI (directly below the implantation site), RII (adjacent to the implantation site), and RIII (opposite the implantation site). Portions of the tissue were fixed in Bouin's solution for immunocytochemistry, and the remainder was subdivided into functionalis, basalis, and myometrium and subjected to organ explant culture. The placenta was fixed or cultured separately. Ligand blot analysis of functionalis medium showed that the major IGFBP had a mol wt (Mr) of 29,000-31,000; however, a doublet of 37,000-43,000 Mr and a band at 24,000 Mr were also present. The functionalis from all regions expressed the majority of the IGFBPs, but basalis from RI tissue also secreted the same array of IGFBPs on days 25 and 32. Ligand blot analysis of placental medium proteins revealed a doublet at Mr 37,000-43,000 on days 25 and 32, but not on day 18. Immunoprecipitation followed by ligand blot analysis of medium proteins using polyclonal antibodies to IGFBP-1 and IGFBP-2 and -3 confirmed that IGFBP-1 and -2 were the predominant products of the endometrium and decidua, while IGFBP-3 was synthesized by the placenta. Immunocytochemistry with a monoclonal antibody to IGFBP-1 demonstrated intense glandular epithelial staining in all regions on days 18, 25, and 32. Stromal staining for IGFBP-1 was first evident on day 25 and was only present in stromal cells in intimate contact with the trophoblastic tissue. By day 32, IGFBP-1 expression was not limited to the endometrial-trophoblastic junction, but extended to the deeper stromal cells and included the perivascular regions. IGFBP-1 staining was most intense in RI, but stromal cells at the luminal surface and those surrounding the spiral arteries also showed some staining in RII and RIII on day 32. These studies suggest that the baboon placenta and/or conceptus regulate IGFBP expression in the uterine endometrium during the initial stages of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunological characterization and immunocytochemical localization of oviduct-specific glycoproteins in the baboon (Papio anubis)

Oviducts obtained from estradiol-treated ovariectomized baboons synthesize and release a family of high mol wt (100,000-130,000) glycoproteins during short term explant culture. The objective of this study was to make a polyclonal antibody to these glycoproteins and then use the antibody to determine the presence of the glycoproteins in oviduct flushings, tissue culture media, and tissues obtained from cycling and steroid-treated baboons. Oviduct culture medium proteins from estradiol-treated baboons were separated on one-dimensional polyacrylamide gels and transferred to nitrocellulose membranes. The region containing the glycoproteins was cut out, solubilized in dimethylsulfoxide, mixed with Freund's adjuvant, and injected at 2-week intervals into a male rabbit. The anti-serum used in this study was obtained 6 weeks after the initial injection and cross-reacted with antigens on Western blots of oviduct flushings and oviduct culture media obtained from follicular stage and estradiol-treated baboons. The antigens were absent in oviduct flushings obtained from luteal stage, ovariectomized and estradiol-primed baboons treated with estradiol and progesterone or progesterone alone. The antigens were not detected on Western blots of other reproductive and nonreproductive tract culture media or in serum obtained from follicular stage or estradiol-treated baboons. Immunoperoxidase staining was limited to discrete granules in the apical cytoplasm of secretory cells in oviducts obtained from follicular stage and estradiol-treated baboons. Thus, the secretory cells of the baboon oviduct synthesize and secrete a family of estradiol-dependent oviduct-specific glycoproteins that may have potential functional significance during fertilization and embryo development.     

Immunocytochemical localization of estrogen and progestin receptors in the baboon (Papio anubis) uterus during implantation and pregnancy.

Although estrogen and progesterone are essential for the establishment of pregnancy in primates, localization of their specific receptors in uterine cell types during pregnancy has not been investigated. Therefore, uteri were obtained from baboons during the menstrual cycle, after steroid treatment, or during early pregnancy on days 18, 25 and 32 postovulation. Uterine and placental tissues were also collected from baboons during late pregnancy. Tissues were processed for indirect immunocytochemical localization with specific monoclonal antibodies against estrogen receptor (ER; H222) and progestin receptor (PR; JZB39). Identification of specific cell types was confirmed by iron-hematoxylin/van Gieson and Gimori's stains. Specific staining for steroid receptors was detected only in the nucleus. In the absence of ovarian steroid hormones (ovariectomized baboons), ER were present in glandular epithelium, stroma, and myometrial smooth muscle cells (SMC). In contrast, PR were absent from all uterine cell types. In the estrogen-dominated (follicular and estradiol treatment) uterus, ER and PR were detected in the nuclei of glandular and surface epithelium, stroma, and myometrial SMC. Elevated progesterone levels (luteal or after progesterone treatment) resulted in a loss of nuclear ER in stroma and epithelium, except in the deep glandular epithelium in the basalis. PR was maintained in the stroma throughout the endometrium, but was detected only in the epithelium of the deep glands. The myometrial SMC contained both ER and PR. In early pregnancy, ER was absent from the glands and stroma as early as day 18 postovulation, but was present in the wall of spiral arteries, blood vessels, and myometrial SMC. On day 18 postovulation, staining for PR was absent from all glandular epithelium, but was maintained in the stroma surrounding the glands and spiral arteries, the wall of spiral arteries, blood vessels, and myometrial SMC. Stroma away from glandular epithelium contained few PR-positive cells. This staining pattern persisted throughout early pregnancy. No apparent differences in ER and PR localization were evident between the implantation and nonimplantation sites of the endometrium and myometrium. In late pregnancy, ER were only present in the SMC of the myometrium; however, PR were detected in stroma and myometrial SMC. The maternally derived decidua expressed PR, but not ER, in the majority of cells. In contrast, fetally derived tissues, placenta, and amnio-chorion, did not contain either ER or PR at any stage of pregnancy. Clearly, ER and PR persist in particular uterine cell compartments despite the continual high levels of progesterone in pregnancy and, thus, support a receptor-mediated mechanism for estrogen and progesterone regulation of implantation and pregnancy.     

Testicular steroidogenesis in the mature and immature baboon Papio anubis.

The following studies were undertaken to compare testicular steroidogenesis in the mature and immature baboon. Testicular fragments (50 mg) were incubated for 3 hr with [7-³H]pregnenolone, or with [7-³H]progesterone. The mature testis formed more testosterone (4.6%), androstenedione (1.6%), and progesterone (28.5%) from pregnenolone than did the immature testis (0.6, 0.5, and 26.1%). The immature testis formed more 17α-hydroxyprogesterone (34.7%) and 20α-dihydroprogesterone (23.2%) from pregnenolone than did the mature testis. Similar conversions were obtained in progesterone incubates. 5α-Androstanediol was identified only in mature incubates. These results suggest that the mature baboon testis has greater C₁₇-C₂₀ lyase, 17β-hydroxysteroid dehydrogenase, and 5α-reductase activities than the immature testis, while the immature testis has greater 20α-reductase activity.     

Zoonotic intestinal parasites in Papio anubis (baboon) and Cercopithecus aethiops (vervet) from four localities in Ethiopia

A total of 59 faecal samples from ranging Papio anubis (baboons) and another 41 from Cercopithecus aethiops (vervet) from the Rift Valley areas of Ethiopia were microscopically examined to determine the prevalence and species of major gastro-intestinal parasites of zoonotic importance. Faecal smears were prepared from fresh faecal samples, stained using modified Ziehl–Neelsen method and microscopically examined. About 3 gm of the dropping was also preserved separately in clean and properly labelled containers containing 10% formalin. The specimens were microscopically examined after formalin-ether concentration for ova, larvae, cysts and oocyst of intestinal parasites. The results of microscopic examination of faecal samples of baboons demonstrated the presence of Trichuris sp. (27.1%), Strongyloides sp. (37.3%), Trichostrongylus sp. (8.5%), Oesophagostomum sp. (10.2%), Schistosoma mansoni (20.3%), Entamoeba coli (83.1%), Entamoeba histolytica/dispar (16.9%), Blastocystis hominis (3.3%), Cyclospora sp. (13.3%) and Cryptosporidium sp. (11.9%). Likewise, the results of microscopic examination of faecal samples of vervets demonstrated the presence of Trichuris sp. (36.6%), Oesophagostomum sp. (4.9%), E. coli (61.0%), E. histolytica/dispar (24.4%), B. hominis (34.2%), Cyclospora sp. (22.0%) and Cryptosporidium sp. (29.3%). The presence of parasitic protozoa and helminths in baboons and vervets in the study areas is a high risk to human welfare because these non-human primates use the same water sources as humans and range freely in human habitats. An implication of such parasitic infection for the control programme is discussed.     

Evidence for the presence of the estrogen receptor in the ovary of the baboon (Papio anubis).

Although intraovarian estrogen has been firmly established as an important factor in the regulation of ovarian follicular development and function in the rat, an autocrine-paracrine role for estrogen in the primate ovary is not yet established. Immunocytochemical identification of an estrogen receptor in the monkey follicle was negative, but it was positive for the granulosa cells of antral follicles of the human ovary. In the present study baboon ovaries obtained during the follicular phase were examined for the presence of estrogen receptor by immunocytochemical analysis of frozen sections and Northern blot analysis of RNA extracts of the ovaries. Immunocytochemistry identified the estrogen receptor in the granulosa cells of healthy appearing and atretic or cystic-like antral follicles and in occasional, but rare, thecal cells. The ovaries contained a prominent mRNA species for the estrogen receptor, approximately 7 kilobases in size, which was present in relatively low abundance compared to that in the nonpregnant baboon uterus, but in a similar abundance to the estrogen receptor mRNA content of the pregnant endometrium. These studies are the first to report the presence of estrogen receptor mRNA in the ovary of a primate. These results in conjunction with the immunocytochemical studies firmly establish the presence of the estrogen receptor in the primate ovary and suggest an autocrine-paracrine role for intraovarian estrogen in primate ovarian physiology.     

Impact of estradiol on gamma-aminobutyric acid- and glutamate-mediated calcium responses of fetal baboon (Papio anubis) hippocampal and cortical neurons

High levels of maternal estrogens are likely to gain access to the fetal brain, yet little is known regarding the role of the steroid hormone 17beta-estradiol in neuronal differentiation and maturation of primate neurons. Previous research documented the presence of estrogen receptors during development in the hippocampus and cortex of the primate brain, but the functional significance of steroid exposure has not been widely investigated. Using both an in vitro preparation of primary hippocampal and frontal cortex neurons and Western blot analysis of fetal hippocampal and frontal cortex tissue, we documented the effects of in utero and acute in vitro exposure to 17beta-estradiol on the development of neuronal responsiveness to the amino acid transmitters gamma-aminobutyric acid (GABA) and glutamate in fetal baboon, Papio anubis, hippocampal, and cortical neurons. We found that in utero 17beta-estradiol exposure enhanced the excitatory action of the GABAergic system on immature cortical and hippocampal neurons, as manifest by increases in intracellular calcium after transient muscimol application and changes in the relevant ion cotransporters. Acute exposure to 17beta-estradiol in vitro had limited effect on GABAergic responses in cultured hippocampal and frontal cortex neurons. Moreover, there was limited effect of both prolonged in utero and acute estradiol on the response to glutamatergic system activation, consistent with previous findings in the rat. Along with documenting a prominent role for 17beta-estradiol in maturation of the GABAergic system, these findings increase our understanding of neuronal differentiation and maturation in the fetal primate brain.