Two-dimensional polyacrylamide gel electrophoresis of proteins synthesized and released by conceptuses and endometria from pony mares

Conceptuses were obtained from pony mares on each day of pregnancy between Days 12 and 28, and on Days 39, 45, 65 and 100. Endometrium was obtained from mares at Days 12, 14, 16, 18, 39, 45, 65 and 100 of pregnancy, and from non-pregnant mares during anoestrus, during transition into the breeding season, at oestrus, or during dioestrus. Tissues were incubated in vitro for 24 h with L-[3H]leucine. Proteins synthesized and released into the culture medium were analysed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and fluorography. Conceptuses obtained before Day 14 after ovulation released a characteristic pattern of labelled proteins. These included two groups of apparent isoelectric variants of relative molecular weights (Mr) 30,000-40,000 (pI values 4.5-5.5 and 6-7), one group of Mr approximately 22,000 (pI 6.5-7), and large protein(s) that did not enter the 10% polyacrylamide gel. After Day 14 the array of labelled proteins had changed and resembled that produced by isolated yolk sac at the later stages of pregnancy studied. Included amongst these were several acidic polypeptides with Mr 20,000 (pI 5-6). The endometrial samples released an array of non-dialysable polypeptides into the culture medium. Fluorograms could be assigned to one of three general groups, with endometrium from mares within each group producing similar patterns of labelled proteins. The first group consisted of anoestrous, transitional and ovariectomized mares, and mares at oestrus or Day 1 or Day 18 after ovulation. The second group was comprised of mares at Days 12-16 of dioestrus or Days 12-18 of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of polypeptides by the cervix of the baboon (Papio anubis)

Administration of oestradiol to ovariectomized baboons caused the epithelium of the cervix to differentiate into tall columnar cells that were ciliated or secretory. Administration of progesterone in the presence or absence of oestradiol altered the appearance of the lining epithelium, suggesting a decrease in secretory activity. Fluorographs of media from cultures of tissue from steroid-treated animals reflected changes in polypeptide biosynthesis which correlated with the morphological observations: 6 polypeptides (Mr 88,000-37,000; pI 5.5-6.0) were observed in all treatment groups and, except for relative changes in intensity, these polypeptides were electrophoretically similar to those synthesized by the endometrium. A new group of low molecular weight polypeptides (Mr 23,000-20,000, pI greater than 8.0-5.5) and a basic protein (Mr 160,000) were synthesized and released in the oestradiol-dominated animal. These polypeptides were distinct to the cervical mucosa since they were not observed in the endometrium or oviduct. Progesterone suppressed the synthesis of the low molecular weight acidic polypeptides (Mr 23,000-20,000; pI 6.1-5.5) but maintained the synthesis of the basic polypeptides (Mr 23,000-20,000; pI greater than 8). Treatment with progesterone +/- oestradiol did not appear to induce the synthesis of any new major polypeptides in the cervical epithelium. These results suggest that oestradiol induces the synthesis of a group of cervix-specific polypeptides and progesterone antagonizes the action of oestradiol in the baboon cervix.     

Immunological and molecular characterization of plasminogen activator inhibitors 1 and 2 in baboon (Papio anubis) placental tissues

Two major plasminogen activator inhibitors (PAI-1 and PAI-2) increase in the peripheral circulation during pregnancy in humans. PAI-1 is of vascular endothelial origin whereas PAI-2 is produced primarily by human placental tissues. This study was undertaken to determine a) if PAI-1 and PAI-2 are also present in the baboon and b) their association with pregnancy. Citrated plasma was obtained from pregnant baboons sequentially at 15 +/- 3-day intervals between Days 30 and 140 of pregnancy. PAI activity increased significantly (p less than 0.05) at Day 120 (15.3 IU/ml) and 140 (21.8 IU/ml) of gestation and returned to baseline (2.6 IU/ml) 48 h post cesarean section. Placental tissues obtained at cesarean section during the third trimester were either placed in explant culture, fixed for immunocytochemistry, or frozen for RNA extraction. Western blot analysis of tissue culture media (TCM) indicated that the polyclonal antibody to PAI-1 reacted with a major band (Mr 47 000) in TCM from placental tissues while the PAI-2 antibody reacted primarily with a doublet (Mr 67 000 and 69 000) in these same media. PAI-1 was immunocytochemically localized primarily in the chorioamniotic tissue (CAM-D) and PAI-2 was found predominantly in placental villi. Slot blot hybridization with cDNAs to PAI-1 and PAI-2 indicated that the mRNA for PAI-2 was found primarily in placental villi, whereas the mRNA for PAI-1 was present in all three tissue compartments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of a progesterone-induced plasmin/trypsin inhibitor from uterine secretions of pigs and its immunocytochemical localization in the pregnant uterus

The porcine uterus secretes a group of basic, low molecular weight protease inhibitors under the influence of progesterone, but not estrogen. One of these inhibitors (Mr approximately 14,500) which inhibits trypsin, plasmin, and chymotrypsin, but not other proteases tested, has been purified 10- to 15-fold from uterine secretions of pseudopregnant pigs using Sephadex G-100 chromatography, CM-cellulose ion exchange chromatography, and Sephadex G-50 or Bio-Gel P-10 chromatography. The inhibitor which is relatively heat- and pH-stable forms a 1:1 molar complex with trypsin which is not dissociated in sodium dodecyl sulfate except by boiling. Chymotrypsin appears to bind at the same site on the inhibitor as trypsin. The inhibitor is high in half-cysteine residues and basic amino acids, and appears not to be a glycoprotein. Antiserum has been raised against the purified inhibitor in rabbits and used to test its distribution in pigs using the immunoperoxidase-staining technique on tissue sections. The inhibitor is associated only with the glandular and surface epithelium of the uterus. Endometrial explants from pseudopregnant animals, cultured in presence of L-[3H]leucine, release the inhibitor in radioactive form indicating that it is a uterine product. The antiserum against the inhibitor cross-reacts with at least three other, basic, low molecular weights plasmin/trypsin inhibitors in porcine uterine secretions, suggesting that a family of isoinhibitors exists which may constitute up to 15% of the protein in porcine uterine secretions. The inhibitor(s) appears to coat and to be taken up by the trophoectoderm cells of the elongating blastocyst during pregnancy. It is suggested that the inhibitors may serve to protect the uterus from proteases released by the porcine trophoblast or to prevent degradation of essential macromolecules, such as uteroferrin, which have to be taken up by the conceptus.     

A baboon model for endometriosis: implications for fertility

Endometriosis is one of the most common causes of chronic pelvic pain and infertility in women in the reproductive age group. Although the existence of this disease has been known for over 100 years our current knowledge of its pathogenesis and the pathophysiology of its related infertility remains unclear. Several reasons contribute to our lack of knowledge, the most critical being the difficulty in carrying out objective long-term studies in women. Thus, we and others have developed a model of this disease in the non-human primate, the baboon (Papio anubis). Intraperitoneal inoculation of autologous menstrual endometrium results in the development of endometriotic lesions with gross morphological characteristics similar to those seen in the human. Multiple factors have been implicated in endometriosis-associated infertility. We have described aberrant levels of factors involved in multiple pathways important in the establishment of pregnancy, in the endometrium of baboons induced with endometriosis. Specifically, we have observed dysregulation of proteins involved in invasion, angiogenesis, methylation, cell growth, immunomodulation, and steroid hormone action. These data suggest that, in an induced model of endometriosis in the baboon, an increased angiogenic capacity, decreased apoptotic potential, progesterone resistance, estrogen hyper-responsiveness, and an inability to respond appropriately to embryonic signals contribute to the reduced fecundity associated with this disease.     

Notch1 mediates uterine stromal differentiation and is critical for complete decidualization in the mouse

Uterine receptivity implies a dialogue between the hormonally primed maternal endometrium and the free-floating blastocyst. Endometrial stromal cells proliferate, avert apoptosis, and undergo decidualization in preparation for implantation; however, the molecular mechanisms that underlie differentiation into the decidual phenotype remain largely undefined. The Notch family of transmembrane receptors transduce extracellular signals responsible for cell survival, cell-to-cell communication, and differentiation, all fundamental processes for decidualization and pregnancy. Using a murine artificial decidualization model, pharmacological inhibition of Notch signaling by γ-secretase inhibition resulted in a significantly decreased deciduoma. Furthermore, a progesterone receptor (PR)-Cre Notch1 bigenic (Notch1(d/d)) confirmed a Notch1-dependent hypomorphic decidual phenotype. Microarray and pathway analysis, following Notch1 ablation, demonstrated significantly altered signaling repertoire. Concomitantly, hierarchical clustering demonstrated Notch1-dependent differences in gene expression. Uteri deprived of Notch1 signaling demonstrated decreased cellular proliferation; namely, reduced proliferation-specific antigen, Ki67, altered p21, cdk6, and cyclinD activity and an increased apoptotic-profile, cleaved caspase-3, Bad, and attenuated Bcl2. The results demonstrate that the preimplantation uterus relies on Notch signaling to inhibit apoptosis of stromal fibroblasts and regulate cell cycle progression, which together promotes successful decidualization. In summary, Notch1 signaling modulates multiple signaling mechanisms crucial for decidualization and these studies provide additional perspectives to the coordination of multiple signaling modalities required during decidualization.     

Interaction of the conceptus and endometrium to establish pregnancy in mammals: role of interleukin 1β

Implantation and the establishment of pregnancy in mammals involves an intricate interplay of hormones, cytokines, growth factors, proteins, lipids, ions and the extracellular matrix between the uterine epithelium, stroma, immune cells and the conceptus trophectoderm. The divergent nature of implantation in the mouse, human and pig provides not only an interesting contrast in the establishment of pregnancy and early embryonic development but also intriguing similarities with regard to early endometrial-conceptus signaling. An interesting pro-inflammatory cytokine expressed in a number of mammalian species during the period of implantation is interleukin-1β (IL1B). The presence of IL1B might be involved with immunotolerance at the maternal-placental interface and has been proposed as one of the mediators in placental viviparity. The production of IL1B and other proinflammatory cytokines might play a role in establishing pregnancy through modulation of the nuclear factor kappa-B (NFKB) system in a number of species. A model for the regulation of cellular progesterone receptor expression and NFKB activation for endometrial receptivity and conceptus attachment is continuing to evolve and is discussed in the present review.