Biomedical subjects
K F Roby
Publications and source records attributed to K F Roby.
Recapitulation of the pathway for trophoblast giant cell differentiation in vitro: stage-specific expression of members of the prolactin gene family.
The trophoblast giant cell lineage is characterized by endoreduplication and expression of members of the PRL gene family. This report describes the functional consequences following in vitro manipulation of a rat trophoblast cell line, termed Rcho-1. Rcho-1 cells can be cultured under conditions that promote proliferation or differentiation. Proliferation is maintained by culturing the cells in the presence of fetal bovine serum under subconfluent conditions. Differentiation is induced by growing the cells to confluence and removing the mitogenic source. Differentiation is characterized by continued synthesis of DNA in the absence of proliferation (endoreduplication) and the sequential expression of members of the PRL gene family. Western and Northern blot analyses demonstrated that placental lactogen-I (PL-I) was first expressed, followed sequentially by PL-II, PRL-like protein-A, and PRL-like protein-C. The ontogeny of expression of members of the PRL gene family by the Rcho-1 cells recapitulated the pattern of in situ expression by trophoblast giant cells of the junctional zone of the chorioallantoic placenta. A notable difference between in vivo trophoblast giant cell differentiation and in vitro Rcho-1 cell differentiation is the termination of PL-I expression in normal trophoblast giant cells developing in vivo and the continued expression of PL-I in differentiated Rcho-1 cell cultures. The Rcho-1 cell line provides a unique in vitro model for investigating the initiation and maintenance of the trophoblast giant cell differentiation pathway.
Mouse endometrial tumor necrosis factor-alpha messenger ribonucleic acid and protein: localization and regulation by estradiol and progesterone.
Tumor necrosis factor-alpha (TNF) messenger RNA (mRNA) and protein have been identified in the uteri of cycling rats, mice, and women. In this study, a mouse model was used to investigate the cell-specific expression and regulation of the TNF gene in the endometrium. Uteri from cycling and ovariectomized hormone-reconstituted mice were tested by in situ and Northern blot hybridizations as well as by immunohistochemistry. Cyclic variations were observed in TNF mRNA. Although weak to undetectable during proestrus, estrus, and diestrus-I, TNF mRNA was present in both epithelial and stromal cells on diestrus-II. TNF protein was observed in the endometrium on each day of the estrous cycle, primarily in the epithelial cells. Stromal TNF immunoreactivity was observed only during diestrus-I. Seven days after ovariectomy, TNF mRNA and protein were undetectable in the endometrium. Specific message and protein were restored in both epithelial and stromal cells after administration of 17 beta-estradiol (E2), progesterone (P4), and E2 plus P4. E2 treatment resulted in a biphasic pattern of TNF mRNA expression. mRNA was present in epithelial cells 1 and 6 h posttreatment, was not detectable after 24 h, and then was present in both the epithelium and stroma after 72 h. In contrast, TNF mRNA was detectable at all time points after P4 administration. TNF mRNA was localized to both the epithelium and stroma until 72 h of P4 treatment, when stromal TNF mRNA was no longer detectable. TNF mRNA was also present at all time points examined after the combination E2 and P4 treatment. TNF mRNA was invariably localized to the epithelium; however, stromal mRNA fluctuated over the course of treatment. TNF mRNA was in the stroma 1 and 24 h post-E2 plus P4 treatment, but was not present at the 6 and 72 h points. In general, the pattern of TNF protein matched that of the TNF mRNA, with a few exceptions. Twenty-four hours after E2 treatment, TNF mRNA was not detectable; however, TNF protein was present in the epithelium. The opposite was observed 24 h after E2 plus P4 administration when the epithelium and stroma contained TNF mRNA; however, no protein was observed. In addition, with the exception of 72 h of E2 treatment, stromal TNF protein was not abundant, although TNF mRNA was often detected. In summary, the results of this study indicate that both epithelial and stromal cells contain TNF mRNA and protein and that E2 and P4 exert individual influences on the cell-specific expression of TNF transcripts and protein.
Expression of HLA class II-associated peptide transporter and proteasome genes in human placentas and trophoblast cell lines.
Expression of HLA class I antigens is closely controlled in the placental trophoblast cells, which interface directly with maternal cells during pregnancy. In this study, the possibility that peptide transporter (TAP-1, TAP-2) or proteasome (LMP7) genes might be involved in regulating antigen expression in these or other cells that comprise placentas was investigated. Analysis by Northern blot hybridization showed that transcripts from all three genes were present in samples of first trimester and term placental RNA. TAP-1 and TAP-2 messages were consistently more abundant in early than in late gestation placentas, whereas the reverse was observed for LMP7 mRNA. Futher experiments were done on two trophoblast cell lines. One line, Jar, is negative for HLA class I, and the second, JEG-3, expresses HLA-G as well as other HLA class I genes. Both Jar and JEG-3 cells contained TAP-1, TAP-2 and LMP7 mRNA. With the exception of LMP7 in JEG-3 cells, message from all three genes was increased by treating the trophoblast cells with interferon-gamma. While no evidence was collected to support the postulate that the HLA class I negative status of some trophoblast cell subpopulations could be related to absent or dysfunctional TAP-1, TAP-2 or LMP7 mRNA, the data are consistent with the postulate that placental cell expression of HLA class I antigens could be influenced by the availability of peptide transporters and proteasome components.
Decidual prolactin-related protein. Identification, molecular cloning, and characterization.
In this report, we describe the isolation, molecular cloning, and characterization of a new member of the prolactin (PRL)-growth hormone (GH) family expressed in rat decidual tissue. A 29-kDa protein was isolated from medium conditioned by decidual explants. The protein possessed an affinity for concanavalin A and cross-reactivity with antibodies to two rat placental proteins, PRL-like protein-B (PLP-B) and PLP-C and with antibodies to human PRL. NH2-terminal sequencing of the isolated decidual protein indicated that it shared significant sequence identity with the NH2 terminus of PLP-C. The decidual protein was termed decidual prolactin-related protein (dPRP). A PLP-C cDNA was used to identify dPRP cDNAs from a rat decidual cDNA library. Nucleotide sequence analyses of the dPRP cDNAs predicted a mature protein of 239 amino acids, including a 28-amino acid signal sequence. The predicted dPRP amino acid sequence contains two putative N-linked glycosylation sites and 6 cysteine residues. The 6 cysteines are located in positions homologous to the cysteines of PLP-C and PRL. Additional sequence similarities with members of the PRL-GH family are evident. The dPRP gene was localized to rat chromosome 17, which also carries other members of the PRL gene family. Northern blot analysis showed that the dPRP cDNA clone specifically hybridized to a 1.0-kilobase mRNA. The relationship of dPRP with other members of the PRL-GH family and its putative role(s) in the physiology of pregnancy are discussed.
Heterologous expression and characterization of prolactin-like protein-A. Identification of serum binding proteins.
In this report, we describe the heterologous expression of prolactin-like protein-A (PLP-A) in Chinese hamster ovary (CHO) cells, the characterization of recombinant PLP-A, and the identification of serum PLP-A-binding proteins. CHO cell and native placental PLP-A showed similar immunoreactive characteristics and electrophoretic mobilities. N-terminal sequencing verified the identity and purity of the recombinant PLP-A species and the site of cleavage of the signal peptide from the mature secreted PLP-A species. Recombinant PLP-A lacked activity in standardized prolactin and growth hormone in vitro bioassays. Antibodies generated to recombinant PLP-A facilitated the cellular localization of PLP-A and the identification of high molecular weight PLP-A complexes. Cross-linking analyses of radioiodinated PLP-A with serum harvested from late gestation rats indicated the presence of two major cross-linked complexes migrating under reducing conditions at 130 and 250 kDa and two minor cross-linked complexes migrating at 70 and 110 kDa. Binding of PLP-A to serum proteins was specific for PLP-A and not effectively competed by other members of the prolactin/growth hormone family. The PLP-A binding species were also found in serum from non-pregnant female and male rats.
Recombinant porcine prorelaxin produced in Chinese hamster ovary cells is biologically active.
Although prorelaxin has a similar structure as proinsulin, the posttranslational processing of prorelaxin seems to be quite different from that of proinsulin. There are no pairs of basic residues flanking the relaxin moiety in most prorelaxins studied so far. Instead, the prorelaxins of many species contains a tetrabasic sequence (Arg-Lys-Lys-Arg) between the connecting peptide and the A-chain. This is the recognition sequence of furin. In order to study this possible processing by furin, we express the recombinant porcine prorelaxin in Chinese hamster ovary cells. The expected 19 kDa recombinant porcine prorelaxin was found to be constitutively secreted into the medium at a level of approximately 250 ng/ml. No conversion of the 19 kDa prorelaxin into the 6 kDa relaxin was observed. Unlike most prohormones which are biologically inactive, the recombinant prorelaxin was found to be biologically active in an in vitro bioassay.
Molecular cloning and characterization of prolactin-like protein C complementary deoxyribonucleic acid.
In this report, we describe the isolation and characterization of a full length cDNA clone for rat prolactin-like protein C (PLP-C) and describe the expression of PLP-C mRNA in the developing rat placenta. Nucleotide sequence analysis of the PLP-C cDNA clone predicted a mature protein of 238 amino acids, including a 30-amino acid signal sequence. The predicted PLP-C amino acid sequence contains seven cysteine residues, three tryptophan residues, and two putative N-linked glycosylation sites. Six of the cysteine residues in PLP-C are located in positions homologous to the cysteines of pituitary prolactin (PRL). Additional sequence similarities with pituitary PRL and other members of the rat placental PRL family are evident. The PLP-C gene was localized to rat chromosome 17. Northern blot analysis showed that the PLP-C cDNA clone specifically hybridized to a 1.0-kilobase mRNA. PLP-C mRNA was first detectable between days 13 and 14 of gestation, peaked by day 18 of gestation, and remained elevated until term. In situ hybridization analysis indicated that PLP-C mRNA was specifically expressed by spongiotrophoblast cells and some trophoblast giant cells in the junctional zone region of rat chorioallantoic placenta.
Identification and characterization of a new member of the prolactin family, placental lactogen-I variant.
This report describes the identification and characterization of a new member of the placental prolactin (PRL) family, termed placental lactogen-I variant (PL-Iv). PL-Iv was isolated from medium conditioned by late gestation placental explants. Rat PL-Iv was found to be closely related to rat PL-I. Amino-terminal sequence analysis indicated that PL-Iv shared approximately 88% sequence identity with the amino terminus of PL-I. PL-Iv proteins cross-reacted with antiserum to recombinant mouse PL-I and PL-Iv mRNA hybridized with a PL-I cDNA. Multiple PL-I and PL-Iv species were present in placental cytosol. Despite the structural similarities between PL-I and PL-Iv, distinct differences were also evident. Antibodies generated to the amino-terminal 19 amino acids of PL-Iv specifically recognized PL-Iv, while failing to recognize PL-I. Secreted PL-Iv had an affinity for concanavalin A, whereas secreted PL-I lacked affinity for the lectin. PL-I was predominantly secreted as a 36-40-kDa species and PL-Iv was predominantly secreted as a 33-kDa species. Furthermore, PL-I and PL-Iv were synthesized at different times during gestation and by different cell types. PL-I was synthesized by trophoblast giant cells during the first half of gestation, while PL-Iv was predominantly synthesized by spongiotrophoblast cells during the later stages of gestation. PL-Iv was shown to stimulate the proliferation of rat Nb2 lymphoma cells, an in vitro measure of lactogenic activity. In summary, PL-Iv shares structural similarities with PL-I; however, it shows other structural differences in addition to unique cell- and temporal-specific patterns of expression in the rat chorioallantoic placenta.
Generation and characterization of antipeptide antibodies to rat cytochrome P-450 side-chain cleavage enzyme.
In this report, we describe the generation of immunologic probes to rat P-450scc. Two regions of the P-450scc amino acid sequence were identified (internal domain: amino acids 421-441; carboxy terminal domain: amino acids 509-526), chemically synthesized and used as immunogens in rabbits. Antibody production was monitored by enzyme-linked immunoassay (EIA) and Western blot analyses. Antisera were successfully generated to each of the P-450scc regions that recognized the entire 49 kDa rat P-450scc protein. Antiserum directed to the internal domain of P-450scc showed broad species crossreactivity, whereas antiserum directed to the carboxy terminal domain of P-450scc crossreacted with only rat and mouse. Both antisera were useful for Western blot and immunocytochemical analyses of rat P-450scc expression. In addition to recognizing the major 49 kDa P-450scc protein, each antiserum also recognized lower molecular weight species. Antiserum directed to the internal domain of P-450scc specifically recognized a 42 kDa species, whereas antiserum directed to the carboxy terminal domain specifically recognized an 8 kDa species. We hypothesize that the two lower molecular weight immunoreactive species are generated by proteolytic cleavage of rat P-450scc between the internal and carboxy terminal epitopes.
Pregnancy and the prolactin family of hormones: coordination of anterior pituitary, uterine, and placental expression.
Explore the source record for details and available documents.
Identification and immunochemical characterization of a major placental secretory protein related to the prolactin-growth hormone family, prolactin-like protein-C.
Trophoblast cells of the rat chorioallantoic placenta synthesize and secrete a number of proteins structurally related to pituitary PRL. During the purification of one member of the placental PRL family, PRL-like protein-A (PLP-A), we identified a major contaminating protein with a similar mol wt but possessing a more acidic pI (5.9-6.1) and different immunoreactivities. After isolation by two-dimensional gel electrophoresis, the more acidic contaminating protein was electroeluted, and its N-terminal amino acid sequence was determined by gas phase sequencing. The N-terminal sequence showed considerable homology with members of the PRL family, including characteristic positioning of cysteine residues at amino acids 4 and 11. The newly identified protein species have been termed PLP-C based on their structural similarity with pituitary PRL. The protein was further characterized by the generation of specific immunological probes. Antibodies were generated to electrophoretically purified protein and to a chemically synthesized peptide representing amino acids 11-32 of its N-terminal sequence. Each antiserum specifically recognized two major species migrating at approximately 25 and 29 kDa, respectively. The 29-kDa species specifically bound to Concanavalin-A, while the 25-kDa species failed to bind to the lectin. Furthermore, the 29-kDa species could be converted to the 25-kDa species by enzymatic deglycosylation. The antisera have also been used to examine the cell- and temporal-specific patterns of expression. The immunoreactive protein species (25 and 29 kDa) were localized primarily to spongiotrophoblast cells present in the junctional zone of the chorioallantoic placenta. Expression was initiated after midgestation and increased during the remaining part of gestation. In summary, PLP-C is a major secretory protein produced by spongiotrophoblast cells during the second half of gestation.
Effects of tumor necrosis factor-alpha in vitro on steroidogenesis of healthy and atretic follicles of the rat: theca as a target.
Tumor necrosis factor-alpha (TNF), a pleiotropic cytokine localized within the ovary, alters follicular steroidogenesis. Preovulatory follicles dissected from ovaries of normal cyclic adult rats on the morning of proestrus exhibit steroidogenic and histological signs of atresia after 24 h of culture under the conditions of 5% CO2 and air. Follicles cultured for 24 h in 5% CO2 and 95% O2 appeared histologically and steroidogenically healthy. Under both culture conditions, human recombinant TNF (5 ng/ml) significantly increased the production of pregnenolone, progesterone, 20 alpha-dihydroprogesterone, and 17 alpha-hydroxyprogesterone by the follicles. Follicles cultured in 5% CO2 and air exhibited no change in androstenedione or estradiol production compared to control follicles incubated without TNF. In contrast, follicles cultured in 5% CO2 and 95% O2 responded to TNF with increased androstenedione and estradiol production. Separation of the thecal and granulosa compartments indicated that the increased progestin production observed in the whole follicle in response to TNF originated from the theca. TNF significantly inhibited basal and FSH-stimulated progesterone production from the granulosa of preovulatory follicles. Exogenous substrate added to whole follicles cultured in the presence or absence of TNF indicated that TNF enhanced the conversion of 25-hydroxycholesterol to pregnenolone. These studies reveal that TNF enhanced steroidogenesis in both healthy and atretic follicles and that this action of TNF is on the theca, where TNF increases the conversion of cholesterol to pregnenolone. The data imply that TNF has differential effects on thecal and granulosa steroidogenesis.
Immunological evidence for a human ovarian tumor necrosis factor-alpha.
Tumor necrosis factor-alpha (TNF alpha) was localized to follicular and luteal compartments of the human ovary using a biotin strept-avidin immunocytochemical technique with polyclonal antibodies to human recombinant TNF alpha. Immunoreactive TNF alpha (I-TNF) was observed in granulosa cells of healthy antral and atretic follicles and appeared to be secreted by the granulosa cells, since it was present in the follicular fluid surrounding antral and degenerating granulosa. Preantral follicles did not exhibit I-TNF. I-TNF was in the cytoplasm of large granulosa-lutein cells and small paraluteal cells. Granulosa cells cultured on an Immobilon membrane exhibited I-TNF in the cytoplasm and I-TNF released onto the membrane, which appeared as a dark-red stain after immunolocalization. Granulosa cells cultured on plastic slides exhibited I-TNF throughout the cytoplasm. To determine if I-TNF was secreted by granulosa cells, granulosa cell-conditioned medium was examined for the presence of TNF by a double sandwich enzyme-linked immunosorbant assay using both monoclonal and polyclonal antibodies to human recombinant TNF alpha. Conditioned medium contained up to 800 pg TNF/500,000 cells after 24 h of culture. I-TNF was also found (100-170 pg/mL) in human follicular fluid. Immunoblot assay of I-TNF in cultured granulosa revealed approximately 240 pg TNF/250,000 cells. The results indicate that the human ovary contains I-TNF, and thus, a paracrine and/or autocrine role of TNF in human ovarian follicular function seems likely.
Partial purification and characterization of an endothelial cell growth regulator from the bovine ovary.
An inhibitor of endothelial cell thymidine incorporation in vitro was partly purified from cow ovaries using ammonium sulphate (AS) precipitation. Supernatant fluid from the 100,000 g pellet of freshly homogenized ovaries was subjected to stepwise AS precipitation. Precipitates were collected sequentially at 40%, 60%, 80% and 95% saturation, and then each was dissolved, dialysed (Mr 8000 cutoff) and examined in tissue culture for effects on cellular thymidine incorporation by cow pulmonary artery endothelial cells (CPAE) and mouse fibroblasts (L929 and 3T3). The 80% AS precipitate (ppt.) inhibited the in-vitro uptake of [3H]thymidine by CPAE and L929 cells, but not 3T3 cells. Heparin-Sepharose (HS) chromatography of the 80% AS ppt. revealed that the inhibitory activity on CPAE and L929 cells did not bind to HS; the inhibitory fraction was found in the HS column breakthrough (80% BT). The 80% BT fraction reduced CPAE[3H]thymidine uptake as determined by autoradiography and increased cellular uptake of trypan blue. Serial fractions from Sephacryl S-200 exclusion chromatography of the 80% BT contained CPAE inhibitory activity in the Mr range 30,000-50,000. The inhibitory activity on endothelial cells and L929 fibroblasts and the non-reduced molecular weight range of that fraction are similar to those of tumour necrosis factor alpha (TNF alpha). The results indicate that the cow ovary contains a fraction that inhibits endothelial cell growth in vitro and may have important roles in follicular atresia and luteal regression.
Tumor necrosis factor alpha alters follicular steroidogenesis in vitro.
Preovulatory follicles from cyclic proestrous rats were incubated in vitro in M199 for up to 24 hours with various doses of human tumor necrosis factor alpha (TNF). Stepwise increases in progesterone (P) production (20-80 ng/ml/2 follicle/24 hours) were observed with 30-300 pM TNF; 3000 pM TNF reduced (p less than 0.001) P production compared to 300 pM TNF but it was still higher (p less than 0.001) than controls. Follicular androstenedione production was inhibited by low doses of TNF (30-60 pM TNF) and stimulated by 3000 pM TNF. TNF did not alter follicular estradiol (E2) production. The time course studies using 300 pM TNF revealed that follicular P production did not increase significantly until 24 hours in culture unlike LH (160 pM) which increased P by 6 hours. Preincubation of 30 pM TNF with a monoclonal antibody to human recombinant TNF alpha prevented the increase in follicular P production observed at 24 hours in TNF-treated controls. These results indicate that in vitro TNF stimulates follicular P production but only after a lag period and thus provide a basis for future studies elucidating the role of TNF in follicular development.
Trophoblast cell differentiation and organization: role of fetal and ovarian signals.
The rat chorioallantoic placenta is organized into two distinct zones (junctional and labyrinth) and expresses six members of the placental prolactin (PRL) family: placental lactogen-I (PL-I), PL-I variant (PL-Iv), PL-II, PRL-like protein-A (PLP-A), PLP-B, and PLP-C. These placental hormones are expressed in distinct cell- and temporal-specific patterns and can be used to monitor the state of differentiation of rat trophoblast cells. This study was initiated to examine the role of the fetus and maternal ovaries in the regulation of trophoblast cell differentiation and organization. Expression of the placental PRL family was monitored by Northern and Western blotting and immunocytochemical analysis. The roles of the fetus and maternal ovaries were examined by chemically induced fetal death (DFX, induced by intraamniotic injection of digoxin) and surgical removal of the ovaries (OVX), respectively. The endocrine differentiation of the placenta was assessed on day 19 of gestation (sperm positive = day 0, parturition = day 21). Day 10 of gestation was the earliest day that DFX could be reliably performed. Day 10 is a time point during pregnancy preceding the onset of expression of all members of the placental PRL family except PL-I. DFX on day 10 of gestation did not affect the endocrine differentiation of the trophoblast cells but did alter the organization of the chorioallantoic placenta. PL-II, PL-Iv, and the PLPs were all expressed in their appropriate molecular forms and cell types in placentas developing in the absence of fetal influence. The maternal ovaries, in the absence of the fetus, had two distinct actions on the placenta: ovarian signals were essential during a period at midgestation for the maintenance of the placenta and exposure of the developing placenta to ovarian signals during the second half of gestation, in the absence of the fetus, arrested labyrinth zone development. The active ovarian signals were progesterone and estrogen. We conclude that trophoblast cell differentiation occurs independent of the fetus and maternal ovaries. However, signals from both the fetus and maternal ovaries are required for normal organization of the chorioallantoic placenta.
Impaired ovulation by 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) in immature rats treated with equine chorionic gonadotropin.
Several studies have established that 2,3,7,8 tetrachloro-p-dioxin (TCDD) blocks ovulation. The main purpose of this study was to determine if induced ovulation was delayed temporarily by TCDD. The ovulation model used was that of the gonadotropin-primed intact or hypophysectomized rat. Immature intact female Sprague-Dawley rats (IIR) were given 32 microg TCDD/kg by gavage on day 24 of age. The next day equine chorionic gonadotropin (eCG) (5 IU) was injected sc to stimulate follicular development. The number of ova in the oviducts, the ovulation rate, and steroid concentrations were determined at 72, 96, 120, and 144 h after eCG. Immature female Sprague-Dawley rats (IHR) were hypophysectomized on day 23 of age. On day 26, the IHR were given 20 microg TCDD/kg by gavage. The next day eCG (10 IU) was injected sc to stimulate follicle development and at 52 h after eCG, 10 IU human chorionic gonadotropin (hCG) was given to induce ovulation. The same parameters as in IIR were determined in IHR at 72, 96, and 120 h after eCG. TCDD decreased body and ovarian weight gains in both IIR and IHR. In IIR, TCDD delayed ovulation by 24 to 48 h reducing the number of ova shed as well as the number of animals ovulating at 72 and 96 h after eCG. In IHR, however, TCDD reduced only the number of ova shed but caused no delay in ovulation. The IIR treated with TCDD had low levels of progesterone (P4) at 72 and 96 h after eCG but high levels of estradiol (E2) at the same time points. This sustained high level of E2 production coincided with a transient decrease in serum concentrations of androstenedione (A4). The alteration of steroid hormones by TCDD was restored to normal by 48 h after ovulation in IIR. Serum P4 concentration was not altered by TCDD in IHR at 72 h after eCG but was decreased thereafter. The delay in ovulation induced by TCDD in IIR indicates the disruption of the hypothalamus-pituitary-ovary axis during proestrus. The decrease in number of ova shed in IHR induced by exogenous gonadotropins indicates an additional direct ovarian effect of TCDD in blocking ovulation.