Identification and characterization of a rat decidual insulin-like growth factor-binding protein complementary DNA.

A partial cDNA which encodes the rat homolog of human placental protein-12, the low mol wt insulin-like growth factor-binding protein (IGFBP-1), has been isolated from a rat decidual cDNA library using low stringency hybridization with a human IGFBP-1 cDNA probe. The incomplete cDNA obtained from this library was used to screen a rat liver cDNA library from which a full-length cDNA was obtained. The predicted amino acid sequence of rat IGFBP-1 showed 66%, 29%, and 34% sequence identity with the human IGFBP-1, the human GH-dependent binding protein IGFBP-3, and rat IGFBP-2, the BP secreted by buffalo rat liver cells, respectively. The rat IGFBP-1 cDNA hybridized with a 1.6-kilobase transcript which was easily detected in uterine RNA from the pseudopregnant rat and RNA from the liver and kidney of adult rats. A low level of expression was apparent in the brain and the diestrous uterus. Of the tissues examined the order of abundance of IGFBP-1 mRNA was deciduoma tissue greater than or equal to liver much greater than kidney much greater than uterus greater than brain. The 1.6-kb mRNA was more abundant in RNA from neonatal rat liver than in that from maternal liver, but was not detected in total RNA (50 micrograms) from other neonatal rat tissues (kidney, lung, brain, and heart). Under stringent conditions, rat IGFBP-1 did not hybridize with RNA from the BRL-3A rat liver cell line. In food-deprived rats, hepatic IGFBP-1 mRNA was increased 10.0 +/- 2.2-fold compared to that in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

A serum-free medium for culturing lactogen dependent and autonomous NB2 node lymphoma cells.

A serum-free, hormone-free medium (SF2) was designed for the Nb2 rat lymphoma bioassay for lactogens as batches of horse serum (HS), which were commonly used, were found to be inconsistent in their suitability and to contain factors modulating the PRL-induced growth response of clone Nb2-11C. In a 3-day incubation with less than 500 pg/ml human GH (hGH), SF2 was better than the traditional medium in supporting Nb2-11C growth, although the comparative efficiency of SF2 decreased at higher hGH levels. Known growth factors (epidermal growth factor, fibroblast growth factor, platelet derived-growth factor, recombinant somatomedin-C, multiplication-stimulating activity) and insulin had no consistent effect on the cell growth in SF2 either in the presence or absence of hGH. Corticosterone (12.4-150 nM) was toxic to the Nb2-11C cells. SF2 could support the growth of Nb2-11C cells for at least 30 passages in the presence of 5 ng/ml hGH, and that of 2 spontaneously proliferating cell lines (Nb2-SP and Nb2-HSP) for the same length of time in the absence of lactogen. However, in all cases the growth rate in SF2 was lower than that seen in the presence of 10% HS. Long-term culture of Nb2-SP and Nb2-HSP cells in SF2 led to an increase of the growth rate with time. There was a change in the responsiveness of Nb2-SP cells to lactogens after long-term culture in SF2 which was only apparent in the presence of HS. After 10 passages in SF2, Nb2-11C cells showed no apparent changes in lactogen-induced growth response, cell phenotype, cell size, or binding capacity for [125I]hGH.

Molecular cloning and expression of rat placental lactogen-I complementary deoxyribonucleic acid.

A full-length cDNA clone for rat placental lactogen I (rPL-I) has been isolated from a phage lambda gt11 library containing cDNA synthesized from day 11 rat placental mRNA. By Northern blot analysis the rPL-I cDNA clone hybridizes to a 1.0-kilobase placental mRNA and appears as early as day 10 of gestation. Maximal expression of this mRNA was observed in day 11 and 12 placenta, and faint hybridization of the rPL-I cDNA was also detected in day 18 to term placenta. In contrast, the mouse clone hybridized to mRNA for mouse PL-I (mPL-I) only in day 10 mouse placenta (9). In vitro translation of rPL-I mRNA produced by transcription of the cDNA template yielded a 27-kDa polypeptide the size of the expected precursor protein which was immunoprecipitated by a monoclonal antibody to rPL-I. The rPL-I cDNA nucleotide sequence has been determined. The sequence is very similar to that for mPL-I and contains an open reading frame encoding a polypeptide of 230 amino acids compared to 224 for mPL-I. Comparison of the predicted primary translation product of rPL-I mRNA with that of mPL-I mRNA revealed that rPL-I shares 73% identity to mPL-I at the amino acid level. The predicted rPL-I protein shares 41% amino acid identity with rPL-II precursor, 24% with rat prolactin-like protein A, 26% with rat prolactin-like protein B, and 31% with rat PRL. In situ hybridization studies indicated that mRNA for rPL-I was present in a few rapidly dividing cells as early as day 8 of gestation, and by day 9 could be localized to giant cells which surround the conceptus.

Expression of rat prolactin-like protein B in deciduoma of pseudopregnant rat and in decidua during early pregnancy.

The rat PRL-like protein-B (rPLP-B) complementary DNA (cDNA), originally cloned from a late term placental library, hybridizes to transcripts in the deciduoma tissue artificially produced in pseudopregnant rats. The expression of rPLP-B in deciduoma is first observed 48 h (pseudopregnancy day 7) after the deciduogenic stimulus and increases to a maximum by 96-120 h (pseudopregnancy day 9-10). In situ hybridization studies show that rPLP-B hybridizes specifically to the antimesometrial cells of deciduoma tissue in day 9 pseudopregnant rats and to the homologous cells of the decidua that surround the embryo-trophoblastic structure at day 9 of pregnancy. A temporal study on the expression of rPLP-B during pregnancy shows that rPLP-B is concomitantly expressed by the decidual cells of maternal origin and the cytotrophoblast of fetal origin around day 13 of pregnancy.

Expression of insulin-like growth factor-I and insulin-like growth factor-binding protein-1 in the rat uterus during decidualization.

Decidualization of the uterus involves proliferation and differentiation of uterine cells. The effects of decidualization on uterine expression of insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) have been examined in the hypophysectomized-ovariectomized (hypox-ovx) rat and the pituitary-intact (ovx) rat. Decidualization was induced by uterine stimulation of animals treated with a combination of 17 beta-estradiol and progesterone. The patterns of change in uterine IGF-I mRNA and IGFBP-1 mRNA abundance were similar to hypox-ovx rats, hypox-ovx rats replaced with GH and T4, and ovx rats. The changes in IGF-I mRNA abundance were temporally related to 17 beta-estradiol injections. IGFBP-1 mRNA was undetectable early in the decidualization process and reached maximal levels on day 6. Mechanical separation of the deciduoma tissue from the underlying myometrium revealed that the deciduoma tissue was depleted in IGF-I mRNA, while the majority of the IGFBP-1 was located in the deciduoma tissue. The in situ hybridization technique was used to localize IGF-I and IGFBP-1 mRNA in the decidualized uterus. The majority of the IGF-I expression was localized to the outer stroma and smooth muscle cell layer, whereas IGFBP-1 mRNA was detected in uterine epithelial cells and stromal glands. These experiments demonstrated that uterine IGF-I and IGFBP-1 expression during the process of decidualization are pituitary independent. Furthermore, our observations support the hypothesis that the expression of IGFBP-1, a protein capable of inhibiting the mitogenic activity of IGF-I, in deciduoma tissue may inhibit paracrine IGF-1 actin and allow for the differentiation of stromal tissue.

Influence of growth factors on the plasminogen activator activity of avian granulosa cells from follicles at different maturational stages of preovulatory development.

Granulosa cells from the first (F1), third (F3) and fifth and sixth (F5-6) preovulatory follicles and the small yellow follicles (SYFs; diameter 6-8 mm) were cultured for 21 h in the absence and presence of murine and human epidermal growth factors, fibroblast growth factor, transforming growth factors alpha and beta-I (TGF alpha, TGF beta), platelet-derived growth factor and insulin-like growth factor-I at concentrations of 0.1-100 ng/ml. Plasminogen activator (PA) activities in the cell (PAc) and in the medium (PAm) were measured by fibrinolysis and fibrin overlay methods. Basal PAc and PAm activities were highest in cell cultures from the less mature follicles (F5-6 and SYF) and decreased as the follicles matured (F3 > F1). PAc activity was greater than PAm activity, irrespective of the stage of follicular development. All growth factors examined at the 100 ng/ml level were effective in increasing PAc and PAm activities in cultures of granulosa cells from F1 follicles. However, only TGF alpha was able to increase PA activities at lower concentrations. The stimulation of the PA activities of granulosa cells from F3 follicles was inconsistent. None of the growth factors significantly increased PA activities in granulosa cells from F5-6 follicles and SYFs, as determined by fibrinolysis. The major PAc and PAm species (characterized by fibrin overlay) had a molecular mass of about 35 kDa, which is characteristic of the urokinase type. Both assay methods detected a stimulatory effect of the growth factors on PA activities in the granulosa cells from F1 follicles. However, an increase in PA activities in cells from F3 and F5-6 follicles and SYFs was indicated only after fibrin overlay analysis. Tritiated thymidine was incorporated into the DNA of granulosa cells at all stages of follicular development and was enhanced by all growth factors, although TGF alpha and TGF beta were the most effective and had a ranked order of activity: F3, F5-6 > F1, SYF. The present findings show that, of the growth factors examined, TGF alpha may be an effective regulator of PA activity in avian granulosa cells during follicular development, in addition to its observed mitogenic action.

The physiological significance of androgen-induced ovulation in the hen.

The ovulation-inducing property of androgens in the laying hen was investigated. In a first experiment, four different androgens were injected subcutaneously into single-comb White Leghorn hens on the day of the last oviposition of a sequence. The hens were killed 10 h later and examined for the presence of an ovum in the oviduct. Testosterone induced ovulation in accordance to the dose injected (median effective dose, 966 +/- 193 microgram/hen) but the responses to 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol were not dose-related. The effect of 4-androstene-3, 17-dione was more like that of progesterone since it induced ovulation 2 h earlier than the three other androgens. The physiological significance of the ovulation response to an injection of testosterone was examined in more detail in experiment 2. Seven out of ten hens which were injected with 1 mg testosterone/kg body weight ovulated within 10 h after the injection. Blood samples were taken at hourly intervals and the concentrations of testosterone and progesterone were determined by radioimmunoassay. An injection of testosterone produced an increase in the concentration of testosterone in plasma which was considerably greater and occurred earlier than the preovulatory increase of testosterone in the control birds. The increase in the concentration of progesterone in the hens injected with testosterone was similar in magnitude but occurred earlier than the spontaneous preovulatory increase of progesterone in the control hens. The possible physiological role of testosterone in the ovulation cycle is discussed.

Stimulation of growth of Nb2 lymphoma cells by interleukin-2 in serum-free and serum-containing media.

The human recombinant alanine-125 analogue of interleukin-2 (IL-2) causes a dose-dependent mitogenic response in rat lymphoma Nb2-11C cloned cells when tested in serum-containing medium and serum-free medium. IL-2 and hGH elicit their growth stimulation through different receptors since IL-2 does not compete with 125I-hGH for binding to Nb2 cells and Met14hGH, an antagonist of hGH, inhibits the hGH-stimulated growth of Nb2 cells but not that caused by IL-2. The tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) potentiates the action of hGH on Nb2 cells grown in both serum-containing and in serum-free medium. TPA to a variable degree also potentiates IL-2-stimulated growth of Nb2 cells when cultured in medium containing serum but has no effect on cells grown in serum-free medium. In conclusion, IL-2 is a potent mitogen for Nb2-11C cells.

Biological determination of inhibin in rat ovarian-cell culture medium.

Rat ovarian-cell culture medium contains steroids and inhibin material that are able to influence basal and LHRH-stimulated secretion of FSH and LH from dispersed pituitary cells. Studies on procedures to remove steroids, such as ethyl acetate extraction and charcoal: dextran adsorption, showed that adsorption of the culture medium with 0.5 to 1.0% charcoal and 0.05 to 0.1% dextran, respectively, was the optimal method to eliminate most of the steroids in the medium without a detrimental effect on inhibin activity. The inclusion of serum or bovine albumin in the culture medium after the culture protected the inhibin from adsorption by the charcoal: dextran treatment. The inhibin present in the rat ovarian-cell culture medium treated with 1.0% charcoal: 0.1% dextran behaved like the inhibin reference standard (from rete testis fluid of the sheep: RTFS) in the basal secretion of FSH and the LHRH-stimulated secretion of FSH and LH in the in-vitro bioassay.

An in-vitro model for the study of inhibin production by rat ovarian cells.

A culture system for ovarian cells from immature rats injected with PMSG was developed to study the production of inhibin. The results showed that 2 X 10(6) cells per culture dish (60 X 15 mm) were necessary for sustained production of inhibin and that serum was an obligatory component in the medium. Serum from bovine fetuses was better than serum from newborn calves for inhibin production. The constituents responsible for the maintenance of inhibin production by fetal bovine serum could be adsorbed with 1% charcoal and 0.1% dextran.

[Simultaneous increase in the aromatization of testosterone into estrogens and secretion of inhibin by Sertoli cells in culture]. / Augmentation simultanée de l'aromatisation de la testostérone en oestrogènes et de la sécrétion d'inhibine par les cellules de Sertoli en culture.

17 beta oestradiol and inhibin production by Sertoli cells has been investigated in vitro. In basal conditions, 17 beta-oestradiol secretion is weak whereas inhibin production is undetectable on days 7 and 9 of culture. Addition of PMSG (FSH-like gonadotrophin) and testosterone to the culture medium induces a simultaneous increase in 17 beta-oestradiol and inhibin production. PMSG alone has no effect neither on inhibin nor on 17 beta-oestradiol secretion. Testosterone alone significantly increases 17 beta-oestradiol and inhibin production. Human chorionic gonadotrophin (LH-like gonadotrophin) does not modify inhibin secretion. Dihydrotestosterone stimulates inhibin production without affecting oestrogens secretion. Thus, stimulation of aromatization of testosterone into 17 beta-oestradiol is associated with an increase of inhibin production, but this effect seems to be due to a direct action of androgens.

Plasma concentrations of LH, progesterone, and corticosterone during ACTH- and corticosterone-induced ovulation in the hen (Gallus domesticus).

Corticosterone and ACTH were injected either 6 hr after ovulation of a mid-sequence follicle or 14 hr before the first ovulation of a sequence. Ovulation was not induced by injection of either hormone given 6 hr after ovulation but 12 of 15 hens injected with 1.5 mg of corticosterone and 6 of 13 hens injected with 10 IU of ACTH given 14 hr before the first ovulation of a sequence ovulated within 8 hr. Injection of either ACTH or corticosterone 6 hr after a mid-sequence ovulation was followed by a decline in the concentration of LH, whereas the concentration of progesterone remained stable. The concentration of both LH and progesterone was increased during 1-5 hr before an ovulation induced by an injection of either ACTH or corticosterone given 14 hr before the first ovulation of a sequence. The increase in the plasma concentration of corticosterone which was required to induced ovulation with either hormone was identical and not within the normal physiological range. It was concluded that the ovulation-inducing action of ACTH was mediated by its effect on corticosterone production and/or secretion by the adrenal gland, that a mature follicle capable of progesterone secretion must exist within the ovary before an injection of either ACTH or corticosterone can induce ovulation, and that the ovary is the most probable target tissue for corticosterone in the context of its ovulation-inducing action.

[Effects of inhibin on the in vitro synthesis of desoxyribonucleic acid in the rat testis]. / Effet de l'inhibine sur la synthèse de l'acide désoxyribonucléique du testicule du rat in vitro.

Three preparations of inhibin extracted from ram rete testis fluid (RTF) and from human seminal plasma (HSP) reduce tritiated thymidine incorporation into testicular desoxyribonucleic acids (DNA) in vitro. Effect of low molecular inhibin from RTF is dose-dependent. Castrated ram serum does not modify testicular DNA synthesis in vitro. Besides their suppressive action on follicle stimulating hormone (FSH) secretion in vivo and in vitro, these inhibin preparation display a direct inhibiting effect on testicular DNA synthesis and, thus, on mitotic activity. Identity between inhibin and testicular chalones are discussed.

Granulosa-thecal cell interactions in the regulation of plasminogen activator activity during ovarian follicular development in the hen.

In the present studies, hen granulosa and thecal cells from the first (F1) and fourth (F4) largest and developing large white follicles (LWF) were cultured alone or cocultured, and plasminogen activator (PA) activity was determined. The PA of the cells (PAc) and the medium (PAm) was measured through use of the chromogenic substrate Val-Leu-Lys-p-nitroanilide, and the total PA (PAt) was calculated. The PA activity of cultured granulosa cells from all stages of follicular development increased with time of culture. Granulosa cell PAc and PAm activity differed with follicular development: the LWF granulosa cells had the highest levels of activity, the F4 had intermediate levels, and the F1 cells had the lowest. Thecal cell PA activity increased during culture but was unaffected by the stage of follicular development. Cocultures of granulosa and thecal cells from F1 follicles exhibited PA activity 2- to 3-fold higher than the sum of the activities of granulosa and thecal cells cultured alone. The PA activity of granulosa cells from LWF was not affected by coculture with thecal cells. Conditioned medium from thecal cells (TCCM) of all stages of follicular development stimulated PAc activity of granulosa cells from F1 and F4 follicles. Conditioned medium from thecal cells of F4 and LWF caused small inhibitory effects on the PAc activity of granulosa cells from LWF. Zymographic analysis of the PA activity of F1 granulosa cell cultures indicates that the enzyme activity is associated with a molecular mass of about 33 kDa, which is consistent with that of urokinase type PA. Thecal cell PA activity was unaffected by granulosa cell-conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)

The phosphodiesterase inhibitors pentoxifylline and rolipram suppress macrophage activation and nitric oxide production in vitro and in vivo.

We studied the effects of the phosphodiesterase inhibitors pentoxifylline (PTX) and rolipram (ROL) on nitric oxide (NO) production by macrophages and correlated this with cellular cAMP levels. The RAW 264.7 cell line or mouse peritoneal macrophages were activated with lipopolysaccharide (LPS) and interferon gamma (IFN gamma), with or without ROL, PTX, cAMP analogues, or Forskolin. In vivo, peritoneal macrophages were stimulated with staphylococcal enterotoxin B with or without administration of ROL. Nitrite levels in culture and the total cellular cAMP levels were measured. ROL and PTX suppressed NO production of LPS/IFN gamma-stimulated macrophages. ROL (IC(50) = 68-74 microM) was about 40 times more potent than PTX (IC(50) = 2.4-2.9 mM). The suppression paralleled increased total cellular cAMP level (EC(50) = 68-72 microM) and was mimicked by other cAMP elevating agents. ROL and PTX suppressed inducible NO synthase at the mRNA level. The inhibition of NO production of macrophages by ROL or PTX could be beneficial in NO-mediated inflammatory and/or autoimmune disorders.

Up-regulation of urokinase plasminogen activator messenger ribonucleic acid and protein in hen granulosa cells by transforming growth factor alpha in vitro during follicular development.

The aim of the present study was to examine the regulatory role of transforming growth factor alpha (TGF alpha) on urokinase plasminogen activator (uPA) gene expression and protein levels in hen granulosa cells from different stages of ovarian follicular development in vitro. Granulosa cells from the first (F1), the second and third (F2-3), and the fourth, fifth, and sixth (F4-6) largest preovulatory follicles were cultured for 21 h in the absence and presence of TGF alpha (10 ng/ml). The uPA mRNA abundance and protein content were determined by Northern and Western blot analysis, respectively. Cell-associated and secreted PA activity was measured by a fibrinolysis assay and characterized by zymography. Hen granulosa cells produce a uPA with a molecular mass of about 35 kDa and a transcript size of approximately 2.5 kb. Basal uPA mRNA abundance, protein content, and activity were highest in granulosa cells from F4-6 follicles and decreased with follicular maturation. Granulosa cell uPA mRNA levels, protein content, and activity were increased in the presence of TGF alpha, reaching maximal levels in granulosa cells from less mature follicles, although the percentage of stimulation was higher in cells from late stages of follicular development. These findings clearly demonstrate specific expression of uPA in proliferatively active granulosa cells and responsiveness of uPA to TGF alpha at both transcriptional and translational levels. They support the concept that PA of the urokinase type plays an important role in extracellular matrix remodeling during TGF alpha-induced granulosa cell proliferation and ovarian follicular growth.