The induction of baboon glycodelin expression by progesterone is not through Sp1.

Glycodelin is a major secretory product of the uterine glandular epithelial cells of the human and non-human primate during the late luteal phase of the menstrual cycle and early pregnancy. Since progesterone levels are elevated during these periods we sought to determine how progesterone modulates glycodelin gene expression. Co-transfection of various deletions of the baboon glycodelin promoter with the progesterone receptor (PR) into Ishikawa cells, a human endometrial cell line, revealed that full progesterone responsiveness is retained within the region -119/+48. In COS-1 cells, a kidney cell line, progesterone failed to elevate luciferase levels when various deletion constructs and the PR were co-transfected. Mutation of the Sp1 site in the -67/+48 region lowered basal expression but did not affect the ability of progesterone to increase expression of the luciferase reporter in Ishikawa cells. These findings suggest that Sp1 sites are not involved in the progesterone regulation of the baboon glycodelin gene. We propose that progesterone induces a factor that regulates glycodelin gene expression in the uterus since we failed to obtain a similar response in a non-uterine cell line.

Insulin-like growth factor binding protein-1 expression in baboon endometrial stromal cells: regulation by filamentous actin and requirement for de novo protein synthesis.

Stromal fibroblasts in the primate endometrium undergo dramatic morphological and biochemical changes in response to pregnancy. This transformation is characterized by the expression of insulin-like growth factor binding protein-1 (IGFBP-1). Stromal cells from the baboon endometrium of nonpregnant animals were cultured and subsequently treated with cytochalasin D to disrupt actin filaments. In response to cytochalasin D treatment, cells contracted and became rounded as early as 10 min after the initiation of treatment. When cytochalasin D was removed, cells reverted back to their original fibroblastic shape within 1 h. After cells were treated with cytochalasin D for 5 h, addition of (Bu)2cAMP and/or hormones (estradiol, medroxyprogesterone acetate, and relaxin) resulted in the expression of IGFBP-1 messenger RNA and protein within 24 h. Cells with an intact cytoskeleton did not express detectable levels of IGFBP-1 in response to hormones and/or (Bu)2cAMP. Furthermore, the addition of cycloheximide inhibited expression of IGFBP-1 in cytochalasin D-treated cells. Stromal cells were also isolated from early pregnant and simulated pregnant animals. Within 48 h, cells from both the pregnant and simulated pregnant animals produced IGFBP-1 in response to hormones and/or (Bu)2cAMP. In these studies, IGFBP-1 expression was also inhibited by cycloheximide. These studies suggest that induction of IGFBP-1 requires an intermediary protein and that alterations in the cytoskeleton may be involved.

Blastocyst invasion and the stromal response in primates.

One of the most remarkable processes associated with the establishment of pregnancy in the primate is the process of decidualization. This transformation of a stromal fibroblast to a fully differentiated decidual cell is required for implantation and embryo survival in early pregnancy. Although the morphological and biochemical characteristics of the primate decidual cell have been extensively studied, the precise cellular, biochemical and molecular signals required for this transformation have yet to be elucidated. During decidualization, stromal cells first proliferate and then differentiate. Based on our extensive in-vivo and ongoing in-vitro studies, we have suggested that the process of decidualization in the baboon can be divided into two distinct phases. The initial proliferative phase is characterized by the expression of the cytoskeletal protein alpha smooth muscle actin (alphaSMA) in the stromal fibroblasts and is independent of the presence of the conceptus. The second phase of differentiation is characterized by the expression of insulin-like growth factor binding protein-1 (IGFBP-1) and the down-regulation of alphaSMA in the decidualized stromal fibroblast. The expression of IGFBP-1 is dependent on the presence of the conceptus in vivo and is regulated by hormones and cAMP in vitro. We have postulated that, during the initial phase of stromal cell differentiation, alphaSMA expression is regulated by the interaction between stromal cell integrins with the secreted extracellular matrix proteins (ECM). In response to pregnancy a trophoblast 'factor', mediated by cAMP signal transduction, induces IGFBP-1 expression in decidualizing stromal fibroblasts. This induction of IGFBP-1 is associated with the disappearance of alphaSMA and de-novo protein synthesis. Our comparative studies suggest that the process of decidualization in the human and baboon involve similar mechanisms. However, the metabolic pathways required for decidualization in the two species appear to differ in their degree of sensitivity to external stimuli. This review focuses on the cellular events that may potentially regulate decidualization in the primate and its role in regulating trophoblast migration.

Implantation in the baboon: endometrial responses.

Blastocyst implantation in the baboon usually occurs between 8 and 10 days post ovulation. Changes that occur within this window of receptivity and immediately following implantation can be divided into three distinct phases. The first phase, regulated by estrogen and progesterone, is characterized primarily by changes in both the luminal and glandular epithelial cells in preparation for blastocyst apposition and attachment. The second phase is the further modulation of these steroid induced changes in both epithelial and stromal cells by embryonic signals. The final phase is associated with trophoblast invasion and the remodeling of the endometrial stromal compartment. During the initial phase, the actions of estrogen and progesterone are dependent on the presence of specific receptors. Estrogen up-regulates both its own receptor (ER) and the progesterone receptor (PR), while progesterone down-regulates this expression pattern. However, the pattern of progesterone-induced down-regulation of ER and PR is confined to the epithelial cells and demonstrates a gradient effect from the functionalis to the basalis. What is most intriguing is that the loss of epithelial PR is closely correlated with the establishment of uterine receptivity. Coincident with the changes in ER and PR expression, epithelial cells undergo alterations in their cytoskeletal architecture and secretory profile. These changes can be counteracted by PR antagonist treatment during the luteal phase. Although estrogen and progesterone play a critical role in establishing the initial phase of uterine receptivity, it is becoming increasingly evident that the embryo induces functional receptivity in ruminants and rodents. In our studies in the primate, we demonstrate that chorionic gonadotrophin when infused in a manner that mimics blastocyst transit, has physiological effects on the three major cell types in the uterine endometrium. The luminal epithelium undergoes endoreplication and distinct epithelial plaques are evident. The glandular epithelium responds by inducing transcriptional and post-translational modifications in the major secretory product, glycodelin. The stromal fibroblasts initiate their differentiation process into a decidual phenotype and are characterized by the expression of actin filaments. In phase three, blastocyst attachment to the surface epithelium and subsequent implantation is associated with local remodeling of the maternal stroma, smooth muscle, and endothelium of the blood vessels by the trophoblast. In addition, there is a gradual diminution of the epithelial plaques on the luminal surface although the glandular epithelium remains highly secretory. The most dramatic effect is on the stromal fibroblasts, which in response to embryonic stimuli, differentiate into decidual cells, the major cell type of the gestational endometrium. This differentiation is characterized by the expression of insulin-like growth factor binding protein-1 (IGFBP-1) in these cells. The cytokine IL-1 beta is one possible embryonic signal. COX-2 is the rate-limiting enzyme for prostaglandin biosynthesis and transcription of this enzyme in response to the embryonic stimulus (IL-1 beta) results in an increase in prostaglandin biosynthesis in stromal fibroblasts at the site of implantation. Prostaglandins and PGE2 in particular, binds to its specific receptor (EP2 or EP4) and activates adenyl cyclase. The resulting increase in intracellular levels of cAMP can now activate IGFBP-1 gene transcription at the site of implantation. In summary, our studies have demonstrated that chorionic gonadotrophin, when infused into non-pregnant baboons during the window of uterine receptivity can induce epithelial responses that are similar to those observed in a fertile cycle. Stromal differentiation is initiated; however, decidualization requires a signal from the conceptus.

Comparative studies on the in vitro decidualization process in the baboon (Papio anubis) and human.

The process of decidualization involves the morphological and functional transformation of stromal fibroblasts to decidual cells. The objective of this study was to define appropriate in vitro culture conditions required for decidualization of baboon stromal cells. Parallel studies were also done with human endometrial stromal cells for comparative analysis. Human stromal cells produced prolactin and insulin-like growth factor-binding protein (IGFBP)-1 in response to hormones (estradiol-17beta [36 nM], medroxyprogesterone acetate [1 microM], and relaxin [100 ng/ml]), and production was enhanced in the presence of 0.1 mM dibutyryl cAMP (dbcAMP). By contrast, baboon cells did not produce any detectable levels of prolactin, even in the presence of hormones and dbcAMP. IGFBP-1 expression in baboon stromal cells was detectable by Day 6 of hormone and dbcAMP treatment and increased exponentially thereafter. In both human and baboon stromal cells, alpha smooth muscle actin (alphaSMA) expression, an early marker for decidualization in the baboon in vivo, was induced spontaneously under normal culture conditions. Furthermore, a decrease in alphaSMA expression was observed in cells producing high levels of IGFBP-1. Human cells produced significant levels of IGFBP-1 (p < or = 0.01) in response to short-term dbcAMP treatment (48 h) after 2 and 12 days of hormone treatment. However, baboon stromal cells required 17 days of hormonal treatment before cells became responsive to short-term dbcAMP treatment (p < or = 0.01). Finally, human endometrial stromal cells expressed the protein kinase A regulatory subunits RIalpha, RIbeta, RIIalpha, and RIIbeta whereas baboon stromal cells expressed RIalpha, RIIalpha, and RIIbeta. No difference in the mRNA expression of these isoforms was observed in decidualized or nondecidualized cells of either human or baboon endometrium. Our observations indicate that baboon stromal cells can be induced to decidualize in vitro and that this requires dbcAMP in addition to hormones. This is the first report demonstrating in vitro decidualization in a nonhuman primate.

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.

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.

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.

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.

Detection of insulin-like growth factor binding protein-1 in cat implantation sites.

This study was undertaken to determine whether insulin-like growth factor binding protein-1 (IGFBP-1) was synthesized by the cat uterus and placenta during implantation and pregnancy. Endometrial and placental tissue explants from pregnant, pseudopregnant, and ovariectomized steroid-treated cats were cultured in the presence of 35S-methionine. Culture media proteins were separated by one-dimensional (1-D) and two-dimensional (2-D) SDS-PAGE, transferred to nitrocellulose, and immunostained using a rabbit polyclonal antibody against baboon IGFBP-1 and a murine monoclonal antibody to human IGFBP-1. The antibody cross-reacted with a protein with an M(r) = 30,000 and a pI = 5.1-5.4. Immunoreactive product was found in implantation site media from 16 days postcoitum (PC) through the end of pregnancy, and was confined to the superficial placental/junctional zone. Immunoreactivity was not detected in non-implantation site media until 7 wk PC and was never detected in serum or in media from liver, pseudopregnant endometrium, or endometrium from steroid-treated cats. Autoradiography and immunostaining of 2-D Western blots of culture media proteins demonstrated that implantation site and not non-implantation site tissue synthesized and released immunoreactive IGFBP-1 into the culture medium. 125Insulin-like growth factor-1 (IGF-1) specifically bound to this protein on 1-D Western ligand blots. Avidin-biotin immunocytochemistry utilizing the monoclonal antibody was used to localize IGFBP-1 in paraffin sections. Specific immunostaining was observed in the surface and glandular epithelium of the non-site endometrium throughout pregnancy, with stromal cell staining being detected later.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization and messenger ribonucleic acid levels of a progesterone-dependent endometrial secretory protein (cathepsin L) in the pregnant cat uterus.

The purpose of this study was to localize immunocytochemically a progesterone-dependent protein (PDP) and to determine PDP mRNA levels during the initial stage of the implantation period. Uterine tissue was collected from Day 0-18 postcoital animals. The tissue was processed for immunocytochemical localization of PDP, and the endometrial RNA was isolated and analyzed for PDP gene expression by slot-blot hybridization. PDP was detected immunocytochemically as early as Day 5 postcoitus in the epithelial cells of the deep uterine glands, and the intensity of immunostaining appeared to peak by Day 12 postcoitus. PDP was absent in the endometrium obtained from implantation sites after Day 16 postcoitus, but the synthesis of PDP was maintained in the endometrium obtained from nonimplantation sites. Immunogold electron microscopy demonstrated that PDP was present in electron-dense granules of the glandular epithelial cells. PDP mRNA was detectable in the endometrium at Day 5 postcoitus and peaked around Day 10 postcoitus. PDP mRNA was absent in the endometrium from implantation sites after Day 16 postcoitus, but was maintained in the endometrium from nonimplantation sites. In summary, the results of this study illustrate that PDP is synthesized within the epithelial cells of the deep uterine glands, packaged within membrane-bound secretory granules, and released into the uterine lumen. Also, the process of implantation alters the gene expression in a very localized way since PDP mRNA and PDP-positive granules were absent in the endometrial glands obtained from the implantation site within 1-2 days of the onset of implantation, whereas both PDP mRNA and PDP-positive granules were maintained in the endometrial glands from nonimplantation-site regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone-dependent cathepsin L proteolytic activity in cat uterine flushings.

Sequence analysis of a cDNA clone for the progesterone-dependent protein (PDP) of the cat uterus revealed that PDP may be cathepsin L. This study was undertaken to directly measure the cathepsin L activity in uterine flushings from pregnant and ovariectomized steroid-treated animals in order to confirm that PDP is cathepsin L. Optimum activity toward the substrate Z-Phe-Arg-NMec was observed at a pH of 5-6. Z-Phe-Phe-CHN2, a specific inhibitor of cathepsin L, significantly inhibited the proteolytic activity present in uterine flushings. Immunoabsorption of PDP from uterine flushings obtained from progesterone (P)-treated cats reduced cathepsin L proteolytic activity to levels observed in ovariectomized and estradiol (E2)-treated animals. In E2-primed and E2 + P-treated animals, proteolytic activity in uterine flushings was detectable after 7 days and peaked after 11-13 days of E2 + P treatment. This proteolytic activity was also dramatically increased before implantation (10-12 days after coitus) in pregnant cats. Thus, our data indicate that changes in cathepsin L activity in uterine flushings are correlated with changes in PDP, the uterine protein synthesized and released from the epithelial cells of the deep uterine glands. PDP, via its cathepsin L proteolytic activity, may play a role in the implantation process.

Cloning of a recombinant complementary DNA to a baboon (Papio anubis) estradiol-dependent oviduct-specific glycoprotein.

The estradiol-dominated baboon oviduct synthesizes and secretes a glycoprotein (mol wt, 120,000) that binds to oocyte zona pellucida in vivo. This glycoprotein separates into two major isoelectric variants (pl 8.0 and 4.5) on two-dimensional electrophoretic gels. This study was undertaken to further characterize the steroid-controlled gene expression of this glycoprotein. A recombinant cDNA library was prepared to poly(A)+ RNA isolated from oviducts obtained from estradiol-treated baboons. The library was screened with a polyclonal antibody prepared against the acidic component (pl 4.5) of the glycoprotein. A single positive plaque was purified. Digestion of the recombinant plasmid with EcoRI revealed fragments of 230 and 1000 basepairs. The antibody used to screen the library was epitope selected against the fusion protein produced by the purified recombinant phage. The epitope-selected antibody, when incubated with Western blots of oviductal explant culture medium obtained from estradiol-treated baboons, recognized both the acidic and the basic variants of the glycoprotein. Northern blot hybridization with a 32P-labeled insert indicated that a single message of 2.8 kilobases was present in oviducts obtained from estradiol-treated baboons; no hybridization was detectable to RNA from oviducts obtained from progesterone-treated baboons. A mRNA of comparable size was also found in human, hamster, rabbit, and mouse oviduct tissue. In vitro translation of oviductal poly(A)+ RNA from an estradiol-treated baboon followed by immunoprecipitation with the polyclonal antibody against the acidic component indicated that the protein component of the oviductal glycoprotein had a mol wt of 66,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and thyroid hormone regulation of albumin mRNA in Rana catesbeiana tadpole liver.

Thyroid hormones are responsible for the specific biochemical and structural changes that occur during amphibian metamorphosis. In this study we screened a series of cDNAs from a library constructed from T4-treated premetamorphic tadpole liver poly(A)+ RNA in order to identify a clone that could be used to study the influence of T3 on liver-specific gene expression during Rana catesbeiana metamorphosis. The cDNA from one clone exhibited a greater degree of hybridization to liver RNA from thyroid hormone-treated tadpoles than untreated tadpoles and no hybridization to RNA from tail fins of tadpoles of either group. On Northern blots, the mRNA to which the cDNA hybridized was 2.3 kilobases in size. The pattern of hybridization to genomic DNA digested by various restriction enzymes was consistent with the presence of a single gene. Using slot blot analysis we found that the mRNA levels first rose above basal levels only after 5 days of immersion of tadpoles in 12.5 micrograms/liter T3. The mRNA levels increased approximately 10-fold after 7 and 9 days of treatment. Frog livers had mRNA levels that were intermediate between those in untreated tadpoles and tadpoles immersed in T3 for 7 days. Sequence analysis revealed a significant degree of homology to serum albumin and alpha-fetoprotein. While it is known that serum albumin levels rise dramatically during metamorphosis in Rana species, presumably playing a critical role in maintaining water and electrolyte balance during the animals' terrestrial phase, the molecular basis of the induction has not been fully explained.

Molecular cloning and characterization of a progesterone-dependent cat endometrial secretory protein complementary deoxyribonucleic acid.

In the cat, a group of low molecular weight secretory proteins have previously been shown to appear in the endometrium after progesterone (P) administration to an estrogen (E2)-primed animal. Using a polyclonal antibody to these progesterone-dependent proteins (PDP) we have isolated a recombinant cDNA clone corresponding to the mRNA for PDP from a cDNA library prepared using poly(A+) RNA from the endometrium of P-treated E2-primed cats. Comparison of Western blots using the polyclonal antibody and epitope selected antibody demonstrated that the multiple molecular weight and isoelectric forms of the PDP are immunologically related and potentially products of the same gene. Northern analysis revealed that the mRNA for the PDP in the endometrium of P-treated E2-primed cats was 1.8 kilobases in length. Using slot blot analysis, we found that the PDP mRNA levels were low in the endometrium of ovariectomized animals and undetectable in E2-treated animals. With 1 day of P treatment the PDP mRNA levels were readily detectable and they peaked after 5 to 7 days of P treatment. No PDP mRNA was detectable in myometrium, oviduct, or ovary. Sequence analysis revealed that PDP had significant homology to human, rat, and mouse cathepsin L at the nucleotide (80%, 74%, and 73%, respectively) and amino acid (68%, 65%, and 63%, respectively) level. We suggest that PDP via its collagenolytic and elastolytic activities as a cathepsin L is responsible for preparing the endometrium for blastocyst implantation.

Immunological characterization and immunocytochemical localization of a progesterone-dependent cat endometrial secretory protein.

The major objective of this study was to make a polyclonal antibody to a previously described group of progesterone (P)-dependent low molecular weight secretory proteins of the cat uterus. Proteins present in uterine flushings obtained from a P-treated cat were partially purified using Sephadex G-75, separated on two-dimensional polyacrylamide gels, and transferred to nitrocellulose membranes. The region containing the polypeptides were cut out, solubilized in dimethyl sulfoxide, mixed with Freund's adjuvant, and injected at 2-wk intervals into a male rabbit. The antiserum used in this study was obtained 8 wk after the initial injection and crossreacted with antigens on Western blots of uterine flushings and uterine culture medium obtained from ovariectomized estradiol (E2)-primed cats treated with P, and from pregnant preimplantation animals. Polypeptides in three molecular weight regions crossreacted with the antisera (Mr approximately equal to 28,000, pI 5.5 6.0; Mr approximately equal to 36,000, pI 6.0 6.5; Mr approximately equal to 41,000, pI 5.5 6.0), and each region consisted of several isoelectric variants. The Mr approximately equal to 28,000 proteins were the dominant form observed in culture media, and the Mr approximately equal to 36,000 proteins were the major form present in uterine flushings. The antigens were not detected in uterine flushings or culture medium obtained from ovariectomized, E2-treated, and estrous animals. The antigens were also absent in serum and other reproductive and nonreproductive tract tissues. Immunocytochemical analysis demonstrated that antigen staining was limited to the epithelial cells of the deeper uterine glands of the P-dominated animal. Immunoperoxidase staining was diffuse throughout the cytoplasm of these epithelial cells. Thus, the epithelial cells of the uterine glands in the P-dominated cat synthesize and secrete a complex group of P-dependent, uterine-specific proteins that may have potential functional significance during early blastocyst development and implantation.

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.