Insulin-like growth factors in sheep uterine fluids: concentrations and relationship to ovine trophoblast protein-1 production during early pregnancy

Uterine luminal fluids (ULFs) from Days 10, 12, 14, and 16 cyclic (C) and pregnant (Px) ewes were analyzed for presence of insulin-like growth factors (IGF-I, IGF-II) and other mitogenic factor(s). IGF content and non-IGF mitogenic activity were measured by IGF RIAs after removal of IGF binding proteins and by stimulatory effects on DNA synthesis of density arrested AKR-2B cells, respectively. ULF IGF-I content was not different between days, but differences in IGF-I between C and Px groups at Day 16 (C greater than Px) were found (p less than 0.05). ULF IGF-II content was not different between C and Px ewes; however, differences among days (p less than 0.01) were apparent. In both C and Px ewes, Day 14 ULF had highest IGF-II content (C: 4.60 +/- 0.98 ng/ml, Px: 5.39 +/- 1.38 ng/ml). In Day 12 and Day 14 (C and Px) ULF, IGF-II concentration was about 10-fold greater than that of IGF-I. AKR-2B mitogenic activity in ULF differed among days (p less than 0.01), but not between C and Px ewes. Highest activity was observed for Day 14 and Px ULF, whereas lowest activity was for Day 10 C and Day 16 Px ULF. Sephadex G-200 gel-filtration chromatography of ULF from Day 14 Px ewes demonstrated mitogenic activity in the column void volume fractions and in the 30-kDa size range of eluted proteins. Day 13 conceptuses were cultured in serum-free medium to define the effect of exogenous IGFs on ovine trophoblast protein-1 (oTP-1) secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Horse conceptuses secrete insulin-like growth factor-binding protein 3

Insulin-like growth factor-I (IGF-I) promotes early embryonic development in several species. In the rabbit, IGF-I binds to the embryonic coats from Day 3 of development onward by a 38-kDa protein that is probably insulin-like growth factor-binding protein 3 (IGFBP3). In the present study, ligand, Western, and Northern blot analyses were used to demonstrate the presence of IGF-I-binding activity, several immunoreactive IGFBP3 proteins, and IGFBP3 mRNA in horse conceptuses with particularly large amounts of immunoreactive IGFBP3 in the conceptus capsule. In addition, immunoprecipitation of radiolabeled proteins showed that cultured horse conceptuses secreted IGFBP3 into the culture medium. Endometrial samples from mares also contained IGFBP3 mRNA and protein; but there was no evidence of secretion of IGFBP3 into the uterine lumen by ligand blot analysis, and there was evidence of only very small amounts by Western blot analysis. These results indicate that the horse conceptus secretes significant quantities of IGFBP3 toward the conceptus capsule from as early as Day 10 after ovulation. Thus, most of the IGFBP3 contained within the capsule, which binds IGF-I to this special extracellular matrix of the preimplantation horse conceptus, is likely to be embryonic in origin. IGFBP3 in the horse conceptus capsule may enhance or modulate the action of IGFs on the developing conceptus.     

Effect of the association of IGF-I, IGF-II, bFGF, TGF-β1, GM-CSF, and LIF on the development of bovine embryos produced in vitro

This study examined the influence of the following growth factors and cytokines on early embryonic development: insulin-like growth factors I and II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-β), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-β1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-β1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these growth factors and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; P < 0.05) on Day 8 after in vitro fertilization and similar results to use of SOF + 10% fetal calf serum (38% and 16%, at the same stages, respectively). The averages of total cells, inner cell mass cells, and trophectoderm cells of exclusively in vitro Day-8 blastocysts for pooled GF-CYK treatments were higher than those for SOF and similar to those for fetal calf serum. The presence of these growth factors and cytokines in the embryo culture medium therefore has a combined stimulatory action on embryonic development; in particular through an increase in hatching rate and in the number of cells of both the inner cell mass and trophoblast. These results are the first to demonstrate that use of a combination of recombinant growth factors and cytokine, as IGF-I, IGF-II, bFGF, TGF-β1, LIF, and GM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the biosecurity of embryo transfer.     

Protein synthesis and degradation in non-cultured and in-vitro cultured rabbit blastocysts

In 'pulse-chase' experiments synthesis and half-lives of leucine-labelled proteins were determined in rabbit blastocysts. Embryos were either non-cultured controls or were cultured for 24 h or 48 h in Ham's F-10 medium supplemented with homologous serum or uterine flushings. In control blastocysts protein synthesis increased by a factor of 10 between Day 4 and Day 5. Half-lives of newly synthesized proteins were 32 h in Day-4 and 99 h in Day-5 control blastocysts. In-vitro culture of Day-4 blastocysts led to dramatically shortened half-lives, amounting to 6-10 h. Blastocysts developing in uterine flushing-supplemented media differed significantly from those cultured in serum-supplemented media. Protein synthesis was enhanced and protein degradation was normal for culture times up to 24 h. These results demonstrate (1) that half-lives of proteins in rabbit blastocysts increase with advancing embryonic age, and (2) that a characteristic feature of the altered metabolism of cultured blastocysts is a dramatically accelerated protein degradation, which (3) can be prevented for some time by supplementation of the culture medium with uterine secretions.     

Glucocorticoid influence on porcine granulosa cell IGF-I and steroid hormone production in vitro

The effect of cortisol on granulosa cell (GC) insulin-like growth factor I (IGF-I) synthesis, and IGF-mediated steroid production was examined at various stages of follicle maturation. Granulosa cells were recovered from gilts on Days 14, 18, and 20 of the estrous cycle, while luteinizing GC were recovered on Day 21, just prior to ovulation. The cells were cultured in serum-free medium with increasing concentrations of cortisol (0, 1, 10, and 100 microg/mL) for 5 d with or without IGF-I stimulation (10 ng/mL). During culture all cells were supplemented with FSH and androstenedione (A4). Cellular IGF-I, progesterone (P4) and estradiol-17beta (E2) production was determined by specific radioimmunoassays (RIA), and cell proliferation was assessed. Granulosa cell IGF-I and steroid hormone synthesis increased (P<0.05) with follicle maturation. Direct exposure to high cortisol concentrations, however, altered both IGF-I synthesis and action. Cortisol treatment lowered (P<0.05) IGF-I production by GC recovered on Days 18, 20, and 21. Furthermore, it reduced (P<0.05) IGF-stimulated P4 synthesis at all stages and decreased (P<0.05) IGF-stimulated E2 synthesis by cells recovered on Day 14. In contrast, cortisol enhanced (P<0.05) FSH-stimulated P4 production by GC collected on Days 14 and 18. The opposing effects on FSH and IGF-I action indicate that cortisol did not promote an overall suppressive effect on cell function, nor did it impair cell proliferation. Hence, these results demonstrate that elevated cortisol concentrations can disrupt both IGF-I synthesis and IGF-mediated actions by porcine GC under in vitro conditions, and that specific disruptions are dependent on the stage of follicle maturation.     

Electrophoretic characterization of the plasminogen activator produced by bovine blastocysts.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and zymography were used to determine the tissue source and to characterize the types of plasminogen activator (PA) produced by bovine blastocysts. Day 12-14 blastocysts were collected at slaughter from oestrus-synchronized, superovulated and artificially inseminated Holstein cows. In Expt 1, blastocysts were cultured for 24 h in Ham's F-12 in a humidified atmosphere of 5% CO2 in air at 37 degrees C. After culture, blastocysts and medium were recovered and stored separately at -20 degrees C. In Expt 2, embryonic discs were separated from trophoblast by microdissection. Intact blastocysts, embryonic discs and trophoblast were then cultured for 24 h and recovered as in Expt 1. In both experiments, embryonic tissues and media were electrophoresed with PA and molecular mass standards. Polyacrylamide gels were laid onto casein-agar gel plates (zymograms) and incubated at room temperature for 24-48 h. Caseinolytic zones in zymograms containing plasminogen were evidence of PA. In Expt 1, bovine blastocysts contained and secreted light and heavy forms of PA (47.0 +/- 1.0 and 86.1 +/- 0.7 kDa, respectively). In Expt 2, intact blastocysts and trophoblast produced both forms of PA (41.5 +/- 1.5 and 92.2 +/- 2.7 kDa) but PAs were not detected in embryonic discs. The results suggest that Day 12-14 bovine blastocysts produce urokinase-type PA (41.5-47.0 kDa) and a form of high molecular mass (86.1-92.2 kDa) that is either a novel tissue-type PA or a PA inhibitor which complexes with the lighter form.     

Insulin-like growth factor-I as a survival factor for the bovine preimplantation embryo exposed to heat shock

Insulin-like growth factor-I (IGF-I) is a survival factor for preimplantation mammalian embryos exposed to stress. One stress that compromises preimplantation embryonic development is elevated temperature (i.e., heat shock). Using bovine embryos produced in vitro as a model, it was hypothesized that IGF-I would protect preimplantation embryos by reducing the effects of heat shock on total cell number, the proportion of blastomeres that undergo apoptosis, and the percentage of embryos developing to the blastocyst stage. In experiment 1, embryos were cultured with or without IGF-I; on Day 5 after insemination, embryos >or=16 cells were cultured at 38.5 degrees C for 24 h or were subjected to 41 degrees C for 9 h followed by 38.5 degrees C for 15 h. Heat shock reduced the total cell number at 24 h after initiation of heat shock and increased the percentage of blastomeres that were apoptotic. Effects of heat shock were less for IGF-I-treated embryos. Experiment 2 was conducted similarly except that embryos were allowed to develop to Day 8 after insemination. The percentage reduction in blastocyst development for heat-shocked embryos compared with those maintained at 38.5 degrees C was less for embryos cultured with IGF-I than for control embryos. Heat shock reduced the total cell number in blastocysts and increased the percentage of blastomeres that were apoptotic, whereas IGF-I-treated embryos had increased total cell number and a reduced percentage of apoptosis. Taken together, these results demonstrate that IGF-I can serve as a survival factor for preimplantation bovine embryos exposed to heat shock by reducing the effects of heat shock on development and apoptosis.     

Development of bovine and porcine embryonic teratomas in athymic mice

Inner cell masses (ICM) and embryonic discs from bovine and porcine blastocysts of various ages were transplanted under the kidney capsule of athymic (nude) mice to evaluate growth of teratocarcinomas containing both differentiated tissues and undifferentiated stem cells. Inner cell masses were isolated immunosurgically from Day 8, Day 9 and Day 10 porcine blastocysts and from Day 8, Day 10 and Day 12 bovine blastocysts. Embryonic discs were mechanically dissected from Day 11 and Day 12 porcine embryos and from Day 14 bovine embryos. Day 6 egg cylinders were dissected from embryos and from hybrid embryos of a cross between BALB/C and an outbred strain of mouse. Two to four ICM, embryonic discs or egg cylinders were transplanted under the kidney capsule of each athymic host. After 8 weeks, graft hosts were killed and their tumors removed, fixed and prepared for histological and immunohistochemical examination. Embryonic teratomas developed at high frequency from murine egg cylinders and from Day 11 and Day 12 porcine and Day 14 bovine embryos. Tumors were observed only infrequently from younger bovine and porcine blastocysts. Murine embryonic tumors were composed of numerous differentiated cell types of ectodermal, mesodermal and endodermal origins, but representation of the three embryonic germ layers was somewhat more restricted in bovine and porcine embryonic tumors. No undifferentiated stem cells were detected in tumors of any of the three species. These results demonstrate that teratomas will develop from bovine and porcine embryos when grafted to an immunocompromised host, but the presence of undifferentiated teratocarcinoma stem cells from these species has yet to be achieved.     

A factor contained in plasma is required for IGF binding protein-1 to potentiate the effect of IGF-I on smooth muscle cell DNA synthesis.

One of the forms of the insulin-like growth factor (IGF) binding proteins present in human amniotic fluid has been shown to potentiate the growth-promoting effect of IGF-I markedly. This study was undertaken to determine the cellular and hormonal factors that modulate this potentiation and to determine whether this protein would potentiate the effects of other mitogens. Although the combination of the IGFBP-1 (20 ng/ml) and IGF-I (10 ng/ml) induced a five- to sixfold increase in DNA synthesis compared with IGF-I alone, this response required the simultaneous addition of IGF-I with 0.1% platelet-poor plasma (PPP). If PPP was omitted from the incubation medium, no increase above the effect that was obtained with IGF-I alone was noted. Substitution of cerebrospinal fluid (CSF) for PPP permitted a full mitogenic response, although substitution with amniotic fluid resulted in no enhancement. The factor contained in PPP was heat and acid stable. If the binding protein was co-incubated with fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), or epidermal growth factor (EGF), a slight inhibition of the cellular response to each of these factors was detected. Co-incubation of IGF-I with the IGF-binding protein plus these other peptide growth factors resulted in no further enhancement of DNA synthesis above the level observed with IGF-I and the binding protein alone. Likewise, addition of plasma proteins such as transferrin or albumin did not result in a further enhancement of the DNA synthesis response to IGF-I plus binding protein, and these proteins could not substitute for PPP or IGFBP-1. Transient exposure of the cultures (2 hr) to the binding protein plus IGF-I resulted in a submaximal DNA synthesis response, and the binding protein had to be present continuously to achieve a maximal effect. These studies indicate that a factor contained in plasma and CSF is required for a maximal cellular response to IGFBP-1 plus IGF-I, and this factor does not appear to be a well-defined mitogen.     

Insulin-like growth factor (IGF) family genes are aberrantly expressed in bovine conceptuses produced in vitro or by nuclear transfer

Embryos produced through somatic cell nuclear transfer (NT) or in vitro production (IVP) are often associated with increased abortion and abnormalities thought to arise from disruptions in normal gene expression. The insulin-like growth factor (IGF) family has a major influence on embryonic, fetal and placental development; differences in IGF expression in NT- and IVP-derived embryos may account for embryonic losses during placental attachment. In the present study, expression of IGF-I, IGF-II, IGF-I receptor (IGF-IR), and IGF-IIR mRNAs was quantitated in Day 7 and 25 bovine embryos produced in vivo, by NT, IVP, or parthenogenesis, to further understand divergent changes occurring during development. Expression of the IGF-I gene was not detected in Day 7 blastocysts for any treatment. However, there were no differences (P>0.10) among Day 7 treatments in the amounts of IGF-IR, IGF-II, and IGF-IIR mRNA. For Day 25 conceptuses, there was higher expression of IGF-I mRNA for NT and IVP embryonic tissues than for in vivo embryonic tissues (P<0.05). Furthermore, embryonic tissues from NT-derived embryos had higher expression of IGF-II mRNA than IVP embryonic tissues (P<0.05). Placental expression of IGF-IIR mRNA was greater for NT-derived than in vivo-derived embryos (P<0.05). There were no differences in IGF-IR mRNA across all treatments and tissues (P>0.10). In conclusion, these differences in growth factor gene expression during early placental attachment and rapid embryonic growth may directly or indirectly contribute to increased losses and abnormalities in IVP- and NT-derived embryos.     

Changes in water potential and its components during shoot formation in tobacco callus

Addition of plant growth regulators (5 nM NAA and 5μM BAP) to a defined basal medium stimulated adventitious bud formation of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) cotyledon explants in culture. Cytoplasmic soluble proteins synthesized during early stages of adventitious bud formation were analyzed by electrophoresis of ³H- and ¹⁴C-leucine labeled proteins on SDS polyacrylamide gels. Increased synthesis of low molecular weight proteins (16,000 to 20,000 daltons) was detected after 2 days in culture and reached a maximal level at day 4. When cotyledon explants cultured on bud medium for 2 days were transferred to callus medium (which suppressed adventitious bud formation), suppression of the synthesis of low molecular weight proteins was also observed, suggesting that these proteins may be associated with early stages of adventitious bud formation.     

Impact of peptide growth factors on the culture of small preantral follicles of domestic cats

Small preantral follicles (40 to 90 microm) of domestic cats were cultured in the presence or absence of epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (bFGF) for 5 d. The success of culture was estimated by in vitro incorporation of Brom-desoxyuridine (BrdU) into the oocytes and granulosa cells. Addition of EGF (4, 20, or 100 ng/ml) to the culture medium had no significant effect on the incidence of in vitro DNA synthesis. After supplementation with IGF-I and bFGF, BrdU-incorporation into the follicles and oocytes increased in correspondence to the concentration used, with 20 ng/ml IGF-I and 10 ng/ml bFGF giving the highest effect. In medium containing EGF, the IGF-I-induced increase in BrdU incorporation was suppressed, while the effect of bFGF was not decreased. Simultaneous addition of IGF-I and bFGF did not result in a further increase in DNA synthesis in the oocytes and granulosa cells. We conclude that bFGF mainly induces the proliferation of granulosa cells while IGF-I is involved in cellular activation of oocytes, which is modulated by EGF.     

Effects of growth hormone and insulin-like growth factor-I on development of in vitro derived bovine embryos

The objectives of this study were to determine whether the addition of growth hormone (GH) to maturation medium and GH or insulin-like growth factor-I (IGF-I) to culture medium affects development of cultured bovine embryos. We matured groups of 10 cumulus-oocyte complexes (COCs) in serum-free TCM-199 medium containing FSH and estradiol with or without 100 ng/ml GH. After fertilization, we transferred groups of 10 putative zygotes to 25 microl drops of a modified KSOM medium containing the following treatments: non-specific IgG (a control antibody, 10 microg/ml); GH (100 ng/ml) + IgG (10 microg/ml, GH/IgG); IGF-I (100 ng/ml) + IgG (10 microg/ml, IGF/IgG); antibody to IGF-I (10 microg/ml, anti-IGF); GH (100 ng/ml) + anti-IGF (10 microg/ml GH/anti-IGF); IGF-I (100 ng/ml) + anti-IGF (10 microg/ml, IGF/anti-IGF); no further additions (control). We repeated the experiment six times. Adding GH to the maturation medium increased cleavage rates at Day 3 compared to control (87.3 +/- 1.2% > 83.9 +/- 1.2%; P < 0.05) but had no effects on blastocyst development at Day 8. At Day 8, blastocyst development was greater (P < 0.01) for GH/IgG (24.8 +/- 2.5%) and IGF/IgG (33.7 +/- 2.5%) than for IgG (16.1 +/- 2.1%) and greater for IGF/IgG than for GH/IgG (P < 0.02). Blastocyst development at Day 8 did not differ between anti-IGF (20.4 +/- 1.8%) and GH/anti-IGF (24.1 +/- 1.9%) or IGF/anti-IGF (17.7 +/- 1.9%), but it was greater for GH/anti-IGF than for IGF/anti-IGF (P < 0.05). The Day 8 blastocysts of GH/IgG and IGF-I/IgG groups had a higher (P < 0.01) number of cells than the IgG group. The addition of anti-IGF-I eliminated the effects of IGF-I on cell number but did not alter GH effects. In conclusion, both GH and IGF-I stimulate embryonic development in cattle and GH effects may likely involve IGF-I-independent mechanisms.     

Stimulation of procollagenase synthesis in human rheumatoid synovial fibroblasts by mononuclear cell factor/interleukin 1

In order to define mechanisms regulating the synthesis of procollagenase in human rheumatoid synovial fibroblasts, the proteins synthesized by cultured cells were labeled with [35S]methionine. Labeled medium proteins were analyzed by SDS-PAGE directly and after immunocomplexing with a specific antibody to human fibroblast collagenase. Labeling of both the predominant form of the enzyme (Mr approximately 55 000) as well as a minor species (Mr approximately 61 000) was increased following incubation with the monokine, mononuclear cell factor/interleukin 1. The approximately 61 kDa form of the procollagenase appears to be a glycosylated form of the approximately 55 kDa precursor based on binding to Con A-Sepharose and decrease in the approximately 61 kDa form after culture in the presence of tunicamycin. Thus, mononuclear cell factor, homologous with interleukin 1, partially purified from monocyte conditioned medium increased incorporation of [35S]methionine into several medium proteins, including those complexed by the anticollagenase antibody. In the presence of mononuclear cell factor/interleukin 1, labeling of the procollagenase was increased 12-14-fold over control cultures incubated with medium alone. Therefore, one of the mechanisms involved in increase of collagenase activity in the medium of cultured synovial fibroblasts in the presence of mononuclear cell factor/interleukin 1 is a stimulation of enzyme protein synthesis.     

Stimulation of cartilage-matrix proteoglycan synthesis by morphologically transformed chondrocytes grown in the presence of fibroblast growth factor and transforming growth factor-beta

The effects of transforming growth factor-beta (TGF-beta) on the synthesis of cartilage-matrix proteoglycan by cultured rabbit chondrocytes were examined. Rabbit chondrocytes were seeded at low density and exposed to a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 0.5% fetal bovine serum, 1% bovine serum albumin, 50 micrograms/ml ascorbic acid, and 2 x 10(-7) M hydrocortisone (Medium A). Various combinations of TGF-beta, insulin-like growth factor-I (IGF-I), and fibroblast growth factor (FGF) were also added to Medium A, and the chondrocytes were grown to confluency. Chondrocytes grown with TGF-beta or FGF alone became flat or fibroblastic, those grown with FGF and TGF-beta became very elongated and formed distinct foci, and those grown with FGF and IGF-I showed the spherical configuration characteristic of overtly differentiated chondrocytes. Nevertheless, the incorporation of 3H with glucosamine into the large, chondroitin sulfate proteoglycan synthesized by cultures with FGF and TGF-beta was similar to that in cells grown with FGF and IGF-I and five times that in cells cultured with FGF alone. The increases in incorporation of 3H reflected real increases in proteoglycan synthesis, because chemical analyses showed an increase in the accumulation of macromolecules containing uronic acid in cultures with FGF and TGF-beta or with FGF and IGF-I. However, FGF in combination with either TGF-beta or IGF-I had little effect on the incorporation of 3H into small proteoglycans or hyaluronic acid. These results indicate that chondrocytes morphologically transformed with TGF-beta and FGF fully express the differentiated proteoglycan phenotype rather than the transformed glycosaminoglycan phenotype.     

Oocyte-secreted factor(s) determine functional differences between bovine mural granulosa cells and cumulus cells

Cumulus cells and mural granulosa cells (MGC) are phenotypically different and there is now evidence suggesting that the oocyte plays an active role in determining the fate of follicular somatic cells. This study investigates the role of oocyte-secreted factor(s) in the regulation of the growth and differentiation of cumulus and MGC. Bovine cumulus-oocyte complexes (COC) and MGC were cultured with various hormones for 18 h followed by a further 6-h pulse of [(3)H]thymidine as an indicator of follicular cell DNA synthesis. The COC incorporated 11 to 14 times more [(3)H]thymidine than MGC in either the absence or presence of 50 ng/ml insulin-like growth factor (IGF)-I. Purified porcine FSH (450 ng/ml) added together with IGF-I marginally increased (3)H incorporation in MGC relative to IGF-I alone but dramatically decreased incorporation in COC sixfold. Conversely, mean progesterone production in the presence of IGF-I + FSH was 13-fold higher from MGC than from COC, confirming a distinctive phenotype of cumulus cells. However, this phenotype was found to be dependent on the presence of the oocyte, as microsurgical removal of the oocyte (oocytectomy) resulted in an 11-fold decrease in [(3)H]thymidine incorporation in cumulus cells treated with IGF-I, elimination of the inhibitory effect of FSH on IGF-I-stimulated DNA synthesis, and led to a 2-fold increase in progesterone production in medium with IGF-I and FSH. All of these markers were completely restored to COC levels when oocytectomized complexes were cocultured with denuded oocytes (DO) at a concentration of 0.5 oocytes/microl, demonstrating that oocytes secrete a soluble factor(s) that promotes growth and attenuates cumulus cell progesterone secretion. In the presence of IGF-I, [(3)H]thymidine incorporation in MGC increased ninefold above control levels with the addition of DO. The addition of FSH to IGF-I-increased (3)H counts in MGC, however, led to a decrease in counts in MGC + DO as is also observed in COC. Furthermore, progesterone production was halved when DO were added to MGC cultures, most notably in the presence of IGF-I and/or FSH. These results provide further evidence that MGC and cumulus cells have distinctive phenotypes and that the oocyte is responsible for some of the characteristic features of cumulus cells. Bovine oocytes secrete a soluble factor(s) that simultaneously promotes growth and attenuates steroidogenesis in follicular somatic cells.     

Induction of the synthesis of the pregnancy-specific protein p70 in the endometrium by intramuscular injection of recombinant bovine interferon-alpha I1 in nonpregnant ewes.

We examined the effect of recombinant bovine interferon-alpha I1 (rboIFN-alpha I1) or recombinant bovine trophoblast protein-1 (rbTP-1) on protein synthesis by endometrial explants from Day-13 cyclic ewes and studied the ability of rboIFN-alpha I1 injected i.m. to influence subsequent protein secretion by endometrial tissue explants. In Expt 1, ewes were injected with either 2 mg rboIFN-alpha I1 or vehicle alone at 12 h intervals beginning on Day 11 of the oestrous cycle and ending on the morning of Day 13; 8 h after the last injection, ewes were hysterectomized and endometrial explant cultures were prepared. Explants were cultured for 24 h in leucine-deficient medium supplemented with 250 microCi L-[3H]leucine per culture. For Expt 2, additional explants were prepared from Expt 1 controls. Explants were cultured in the presence of 0, 20 or 200 ng/ml of either rboIFN-alpha I1 or rbTP-1 for 24 h in leucine-deficient medium supplemented with 250 microCi L-[3H]leucine per culture. Secreted proteins were analysed by two-dimensional electrophoresis and fluorography. There was a marked enhancement of a 70 kDa acidic protein, p70, in explants cultured in the presence of rboIFN-alpha I1 or rbTP-1. This polypeptide is a product of the gravid uterine horn from Day 14 to Day 20 of pregnancy and is a useful marker of the action of interferon-alpha (IFN-alpha) on endometrium. Enhanced production of p70 also occurred in ewes injected i.m. with rboIFN-alpha I1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-beta, insulin-like growth factor I, and fibroblast growth factor

Skeletal muscle satellite cells were cultured from mature rats and were treated in vitro with various combinations of transforming growth factor (TGF)-beta, fibroblast growth factor (FGF), and insulin-like growth factor I (IGF-I). In serum-free defined medium the following observations were made: TGF-beta depressed proliferation and inhibited differentiation; FGF stimulated proliferation and depressed differentiation; IGF-I stimulated proliferation to a small degree but demonstrated a more pronounced stimulation of differentiation. In evaluating combinations of these three factors, the differentiation inhibiting effect of TGF-beta could not be counteracted by any combination of IGF-I or FGF. The proliferation-depressing activity of TGF-beta, however, could not inhibit the mitogenic activity of FGF. Maximum stimulation of proliferation was observed in the presence of both FGF and IGF-I. The highest percentage fusion was also observed under these conditions, but differentiation with minimal proliferation resulted from treatment with IGF-I, alone. By altering the concentrations of TGF-beta, FGF, and IGF-I, satellite cells can be induced to proliferate, differentiate, or to remain quiescent.     

Effects of epidermal growth factor on early embryonic development after in vitro fertilization of oocytes collected from ewes treated with follicle stimulating hormone

Epidermal growth factor (EGF) has been shown to enhance the in vitro rate of blastocyst formation in several species. Follicular development was induced in ewes (n=15) by twice daily administration of FSH-P on Days 13 and 14 of the estrous cycle. Cumulus oocyte complexes (COCs) were collected from all visible follicles (n=25+/-2.4/ewe) on Day 15. COCs from each ewe were cultured separately for 24h in maturation medium (containing 10% serum, LH, FSH and estradiol) with (8.2+/-0.9 per ewe) or without (7.8+/-0.8 per ewe) EGF (10 ng/ml). Oocytes were then denuded by hyaluronidase treatment, and healthy oocytes were cultured in the presence of frozen-thawed semen in synthetic oviductal fluid (SOF) medium containing 2% sheep serum. After 18-20 h, zygotes were transferred to SOF medium without glucose and cultured for about 36 h until they reached the 4-8 cell stage. Embryos were transferred to SOF medium with glucose for further development. Medium was changed every other day until blastocyst formation on Day 8 of culture (Day 1=day of fertilization). The rate of embryonic development was evaluated throughout the culture period. After maturation, cumulus cells were more expanded in the presence than in the absence of EGF. The rates of fertilization (overall 75.7+/-3.9%) and morula formation (overall 40.6+/-7.1%) were similar (P>0.05) for COCs cultured with or without EGF. However, EGF increased (P<0.01) the number of blastocysts (1.4+/-0.1 versus 0.6+/-0.2 per ewe) and tended to increase (P<0.1) the rate of blastocyst formation (21.0+/-6.6% versus 13.4+/-4.3% per ewe). These data demonstrate that EGF increases blastocyst formation in FSH-treated ewes. Therefore, EGF is recommended as a supplement to maturation medium to enhance embryonic development in vitro in FSH-treated sheep.     

Insulin-like growth factor-I expression during early conceptus development in the pig

Porcine conceptuses (embryo and associated membranes) in utero undergo developmental morphological transformations coincident with structural and biochemical changes in the uterine endometrium during early gestation. To elucidate a possible role for insulin-like growth factor-I (IGF-I) in these events, porcine endometrial (Days 8, 10, 11, 12, 14, and 30) and conceptus (Days 12, 14, and 16) tissues were characterized for the presence of IGF-I peptide and mRNAs. The corresponding uterine luminal fluids (ULF) at these stages of pregnancy were also analyzed for immunoreactive IGF-I concentration. ULF IGF-I was lowest on Day 8, highest on Day 12, and declined by Day 14. In contrast, endometrial tissue IGF-I content remained constant during this period. Conceptus tissues contained less IGF-I than endometrial tissues; however, conceptus IGF-I values were maximum on Day 12 coincident with peak values for ULF IGF-I. Dot-blot hybridization analyses revealed temporal variation in steady-state levels of IGF-I mRNAs in endometrium. Highest levels of endometrial IGF-I mRNA were detected on Day 12 and were about 4-fold greater than on Day 30 of pregnancy. IGF-I mRNA expression in conceptus tissues on Days 12, 14, and 16 was the same and was significantly less than that in endometrium on Day 12. These results demonstrate the temporal variation of IGF-I mRNA abundance in uterine endometrium and of immunoreactive IGF-I in ULF and in conceptus tissues, with the developmental processes occurring in the conceptuses at early pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)