136 EFFECTS OF INSULIN-LIKE GROWTH FACTOR I (IGF-1) ON THE DEVELOPMENT AND APOPTOSIS OF PREIMPLANTATION BUFFALO (BUBALUS BUBALIS) EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 215
Author(s):  
F. Lu ◽  
T. Luo ◽  
H. Sun ◽  
N. Li ◽  
X. Liu ◽  
...  
Keyword(s):  
Growth Factor ◽  
Mrna Level ◽  
Bubalus Bubalis ◽  
Apoptotic Index ◽  
Mrna Expression Level ◽  
Blastocyst Development ◽  
Factor I ◽  
Igf I ◽  
Bax Gene

The aim of this study was to explore the effects of insulin-like growth factor-I (IGF-1) on the development and apoptosis of preimplantation buffalo (Bubalus bubalis) embryos derived from IVF or somatic cell nuclear transfer (SCNT) in order to improve the quality of in vitro embryo culture (IVC). Buffalo oocytes collected from ovaries at slaughter were cultured in the maturation medium (TCM-199 + 26.2 mmol L–1 NaHCO3 + 5 mmol L–1 HEPES + 5% FBS) for 22–24 h, and fertilized in vitro, or enucleated and reconstructed for SCNT. Embryos were then cultured in the culture medium (CM: TCM-199 + 3% FBS) supplemented with different concentrations of IGF-1. Blastocyst development was evaluated after 7 days of culture. A total of 1566 oocytes were used in this study. The experimental data were analyzed using two-way ANOVA, P < 0.05 was considered to be statistically significant. The results showed that the cleavage rates of IVF or SCNT embryos cultured with 0, 10, 50, or 100 ng mL–1 IGF-I, were not significantly different (P > 0.05). However, the blastocyst rate of IVF embryos cultured with 50 ng mL–1 IGF-1 was significantly increased compared to the 0 ng mL–1 group (35.1 v. 23.0%; P < 0.05), but not significantly different among the 0, 10, and 100 ng mL–1 groups (23.0 v. 28.2 and 26.5%; P > 0.05). In the same line, more SCNT embryos could develop to the blastocyst stage when cultured in the CM supplemented with 50 ng mL–1 IGF-I by comparison with the 0 ng mL–1 group (32.3 v. 20.2%; P < 0.05), but the blastocyst development decreased with 100 ng mL–1 (32.3 v. 21.4%; P < 0.05). Apoptosis and total cell number (TCN) of IVF/SCNT blastocysts were respectively detected by TUNEL or Hoechst 33342 staining. By comparison with the 0 ng mL–1 group, the TCN of IVF/SCNT blastocysts was significantly increased (IVF: 91.7 ± 6.9 v. 108.7 ± 3.9, SCNT: 76.3 ± 5.6 v. 92.8 ± 3.9; P < 0.05) and the apoptotic index was obviously decreased (IVF: 3.9 ± 0.7 v. 2.5 ± 0.7; 7.2 ± 0.5 v. 2.9 ± 0.5; P < 0.05) when the embryos were cultured in the CM with 50 ng mL–1 IGF-I. The result of RT-qPCR analysis showed that the mRNA expression level of the anti-apoptotic bcl-2 gene was distinctly enhanced, while the mRNA expression level of the pro-apoptotic bax gene was remarkably reduced in IVF/SCNT embryos cultured with 50 ng mL–1 IGF-I by comparison with the 0 ng mL–1 group (P < 0.05). These results demonstrated that supplementing CM with 50 ng mL–1 IGF-1 could improve the developmental competence of buffalo embryos, increase the TCN of blastocysts and decrease their apoptotic index, probably by down-regulating the mRNA level of pro-apoptotic bax gene and up-regulating the mRNA level of anti-apoptotic bcl-2 gene. This work was funded by the China High Technology Development Program (2011AA100607), China Natural Science Foundation (31072033), and Guangxi Science Foundation (2012GXNSFFA060004).

Longitudinal bone growth in vitro: effects of insulin-like growth factor I and growth hormone

1991 ◽  
Vol 124 (5) ◽  
pp. 602-607 ◽  
Author(s):  
Ben A. A. Scheven ◽  
Nicola J. Hamilton
Keyword(s):  
Growth Hormone ◽  
Growth Factor ◽  
Bone Growth ◽  
Long Bone ◽  
Long Bones ◽  
Insulin Like Growth Factor ◽  
Serum Free ◽  
Factor I ◽  
Igf I

Abstract. Longitudinal growth was studied using an in vitro model system of intact rat long bones. Metatarsal bones from 18- and 19-day-old rat fetuses, entirely (18 days) or mainly (19 days) composed of chondrocytes, showed a steady rate of growth and radiolabelled thymidine incorporation for at least 7 days in serum-free media. Addition of recombinant human insulin-like growth factor-I to the culture media resulted in a direct stimulation of the longitudinal growth. Recombinant human growth hormone was also able to stimulate bone growth, although this was generally accomplished after a time lag of more than 2 days. A monoclonal antibody to IGF-I abolished both the IGF-I and GH-stimulated growth. However, the antibody had no effect on the growth of the bone explants in control, serum-free medium. Unlike the fetal long bones, bones from 2-day-old neonatal rats were arrested in their growth after 1-2 days in vitro. The neonatal bones responded to IGF-I and GH in a similar fashion as the fetal bones. Thus in this study in vitro evidence of a direct effect of GH on long bone growth via stimulating local production of IGF by the growth plate chondrocytes is presented. Furthermore, endogenous growth factors, others than IGFs, appear to play a crucial role in the regulation of fetal long bone growth.

Effect of growth hormone and insulin-like growth factor-I on DNA synthesis and matrix production in rat epiphyseal chondrocytes in monolayer culture

1992 ◽  
Vol 133 (2) ◽  
pp. 291-NP ◽  
Author(s):  
C. Ohlsson ◽  
A. Nilsson ◽  
O. G. P. Isaksson ◽  
A. Lindahl
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Cell Density ◽  
Monolayer Culture ◽  
Culture Conditions ◽  
Factor I ◽  
Sulphate Uptake ◽  
Igf I ◽  
Matrix Production

ABSTRACT The influence of various culture conditions was studied on the effect of GH and insulin-like growth factor-I (IGF-I) on DNA and matrix synthesis in epiphyseal rat chondrocytes in monolayer culture. Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in 48-well culture plates and precultured for 10 days in Ham's F-12 medium supplemented with 1% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture, the medium was changed to Ham's F-12 medium supplemented with 1% serum from hypophysectomized rats, and the effect of GH and IGF-I on DNA synthesis ([3H]thymidine incorporation) and matrix production ([35S]sulphate uptake) was studied during an additional 96-h culture period. Isotopes were present during the last 24 h of culture. Both hGH and IGF-I stimulated DNA synthesis in a dose-dependent manner. A maximal effect of GH was seen at a concentration of 25 μg/l (60 ± 11% stimulation over control) and for IGF-I at 10 μg/l (162 ± 12%). The stimulatory effects of the same concentrations of human GH (hGH) and IGF-I on [35S]sulphate uptake were 135 ± 25 and 320 ± 42% respectively. In-vitro pulse labelling revealed that GH did not produce a response during the first 3 days of culture (after addition of GH) but was effective during days 4 and 5 of culture. In contrast, IGF-I was effective throughout the culture period. Pretreatment of cells with GH or IGF-I for 2·5 days showed that GH but not IGF-I produced a sustained effect on [3H]thymidine uptake. In order to study the influence of cell density on the effect of GH and IGF-I on DNA synthesis, the effect of added peptides was evaluated after different preculture periods (5–15 days). A maximal stimulatory effect of hGH was seen at a cell density of 150 000–300 000 cells/cm2. GH had no significant effect at a low (< 100 000 cells/cm2) or a high (>400 000 cells/cm2) cell density. The magnitude of the stimulatory effect of IGF-I was the same at densities between 10 000 and 250 000 cells/cm2, but was reduced at higher cell densities (over 250 000 cells/cm2). Chondrogenic properties of cells that had been cultured for 15 days were verified in vitro by positive alcian blue staining and identification of type II collagen, and in vivo by development of cartilage nodules in nude mice. The results from the present study clearly show that GH and IGF-I both stimulate DNA synthesis and matrix production in epiphyseal chondrocytes in monolayer culture. The results also demonstrate that expression of the effect of GH is highly dependent upon the culture conditions. Journal of Endocrinology (1992) 133, 291–300

Secretion of insulin-like growth factor-I (IGF-I) and IGF-binding proteins from bovine mammary tissue in vitro

1991 ◽  
Vol 128 (2) ◽  
pp. 219-228 ◽  
Author(s):  
P. G. Campbell ◽  
T. C. Skaar ◽  
J. R. Vega ◽  
C. R. Baumrucker
Keyword(s):  
Growth Factor ◽  
Binding Proteins ◽  
Conditioned Media ◽  
Mammary Tissue ◽  
Igf Binding Proteins ◽  
Factor I ◽  
Igf I ◽  
Igf Binding ◽  
Bovine Mammary Tissue

ABSTRACT In vitro, insulin-like growth factor-I (IGF-I) promotes both growth and development of bovine mammary tissue. In vivo, the effects of IGF-I may encompass endocrine, paracrine or autocrine mediation. We addressed the possibility of paracrine/autocrine effects of IGF-I in the mammary gland by examining the in-vitro secretion of IGF-I and IGF-binding proteins (IGFBPs) from bovine mammary tissue. Bovine mammary explants from pregnant non-lactating and lactating non-pregnant animals were found to synthesize and secrete IGF-I and IGFBPs. Mammary acini cultures, representative of mammary secretory epithelia, secreted both IGF-I and IGFBP, but synthesized only IGFBP. Concentrations of IGF-I in conditioned media from explants were 1·54 and 0·72 fmol/μg DNA for pregnant and lactating animals respectively. Concentrations of IGFBPs in conditioned media from explants were similar for both physiological states at 2529 pmol 125I-labelled IGF-I bound/pg DNA. Ligand/Western blotting procedures identified four IGFBPs of 29, 33, 37 and 44 kDa for acini cultures and five IGFBPs of 28, 31, 36, 44 and 46 kDa for explant cultures. Similar affinities for IGF-I and IGF-II were shown by IGFBP, using 125I-labelled recombinant human IGF-I as the competing ligand (median effective dose (ED50) of 0·085 pmol). When 125I-labelled bovine IGF-II was used as the ligand, only bovine IGF-II (ED50 of 0·25 pmol) inhibited binding. The addition of prolactin, insulin and cortisol, with or without GH, did not affect secretion of either IGF-I or IGFBP. This report describes the ability of normal mammary tissue to synthesize and secrete IGF-I and IGFBPs. Journal of Endocrinology (1991) 128, 219–228

Effects of insulin-like growth factor-I on aromatase cytochrome P450 activity and oestradiol biosynthesis in preimplantation porcine conceptuses in vitro

1991 ◽  
Vol 130 (2) ◽  
pp. 245-250 ◽  
Author(s):  
A. Hofig ◽  
F. A. Simmen ◽  
F. W. Bazer ◽  
R. C. M. Simmen
Keyword(s):  
Growth Factor ◽  
Aromatase Activity ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Cytochrome P450 Activity ◽  
Igf I ◽  
Growth Factor I ◽  
Oestradiol Production ◽  
P450 Activity

ABSTRACT The effects of insulin-like growth factor-I (IGF-I) on aromatase P450 activity and steroid production in preimplantation pig conceptuses were evaluated in vitro. Conceptuses recovered from gilts on days 10 and 12 of pregnancy were incubated for 6 h in modified Eagle's Minimum Essential Medium (MEM) plus IGF-I (0·1 μg/ml) or insulin (8·5 μg/ml), and conceptuses were monitored for their ability to convert [1,2-3H]β-testosterone into oestrogens. Aromatase activity of day-10 conceptuses was low and unaffected by IGF-I or insulin. In contrast, basal aromatase activity in day-12 conceptuses was about threefold higher and was further increased by IGF-I (P < 0·02), but was unaffected by insulin. To determine whether higher aromatase P450 activity was associated with increased oestradiol production, concentrations of oestradiol were determined by radioimmunoassay in culture medium of day-11 and -12 conceptuses, after incubation in MEM alone or in the presence of dehydroepiandrosterone (DHA, 1 μg/ml) with or without IGF-I (0·1 μg/ml) or insulin (0·1 or 8·5 μg/ml) for 24 h. Conceptuses in MEM plus DHA produced more oestradiol (P < 0·01) than those in MEM alone. Addition of IGF-I or insulin did not increase the effect of DHA. Basal oestradiol production was dependent on conceptus size; however, IGF-I or insulin did not affect basal or DHA-stimulated oestradiol production regardless of conceptus size. These findings demonstrate that IGF-I can modulate aromatase activity in vitro, without affecting overall de-novo steroidogenesis. Thus, the developmental increase in conceptus oestradiol production observed during early pregnancy in the pig may reflect synergistic interactions between IGF-I and other regulatory factors present within the conceptus and/or uterine environment. Journal of Endocrinology (1991) 130, 245–250

Insulin and growth hormone act synergistically to stimulate insulin-like growth factor-I production by cultured chicken hepatocytes

1991 ◽  
Vol 128 (3) ◽  
pp. 389-393 ◽  
Author(s):  
B. Houston ◽  
I. E. O'Neill
Keyword(s):  
Growth Factor ◽  
Insulin Like Growth Factor ◽  
Free Medium ◽  
In Vitro System ◽  
Factor I ◽  
Maximal Stimulation ◽  
Igf I ◽  
Growth Factor I ◽  
Stimulation Of

ABSTRACT Cultured chicken hepatocytes were used to investigate whether insulin and GH interact to regulate insulin-like growth factor-I (IGF-I) production in vitro. In the first set of experiments hepatocytes were preincubated for 6 h in hormone-free medium, and the effects of various combinations of insulin and GH on IGF-I production over the next 24 h were quantified by radioimmunoassay. Basal IGF-I production was 5·36 pg IGF-I/μg DNA and this was increased 1·31±0·13-fold (mean ± s.e.m.) by insulin, 1·90±0·24-fold by GH and 4·46±0·68-fold by a combination of insulin and GH. These results demonstrate that insulin and GH interact synergistically to stimulate IGF-I production in vitro. The synergism with GH occurred at physiological concentrations of insulin with half-maximal stimulation occurring at an insulin concentration of 6 ng/ml. In hepatocytes which had been exposed to insulin immediately before the start of the experiment, the presence of insulin was no longer required for maximal stimulation of IGF-I production by GH. This in-vitro system will facilitate the study of the molecular basis of the interaction between insulin and GH. Journal of Endocrinology (1991) 128, 389–393

A combination of insulin-like growth factor I (IGF-I) and FSH promotes proliferation of prepubertal bovine Sertoli cells isolated and cultured in vitro

2017 ◽  
Vol 29 (8) ◽  
pp. 1635 ◽  
Author(s):  
A. Dance ◽  
J. Kastelic ◽  
J. Thundathil
Keyword(s):  
Growth Factor ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Seminiferous Tubules ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Bull Calves ◽  
Igf I ◽  
Growth Factor I

Beef and dairy bull calves fed a low-nutrition diet during early life had decreased concentrations of circulating insulin-like growth factor I (IGF-I), delayed increases in testosterone, smaller testes and delayed puberty compared with those fed high-nutrition diets. Although IGF-1 has important roles in Sertoli cell function in rats and mice, this has not been well documented in bulls. The objectives of this study were to: (1) isolate Sertoli cells from bull calves at 8 weeks of age, (2) culture them in vitro and (3) determine the effects of IGF-I, FSH and a combination of both hormones on cell proliferation. For Sertoli cell isolation, minced testicular tissues were treated with collagenase followed by trypsin and hyaluronidase to digest seminiferous tubules and release Sertoli cells. In this study, Sertoli cells were successfully isolated from 8-week-old Holstein bull calves (n = 4) and these cells were cultured for up to 8 days. A combination of IGF-I and FSH increased proliferation (~18%) and therefore cell number (1.5-fold) of prepubertal bovine Sertoli cells in culture, providing clear evidence that IGF-I has a similar role in bovine Sertoli cells as reported in rodents.

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