Characterization of insulin-like growth factor binding proteins (IGFBPs) during gestation in mice: Effects of hypophysectomy and an IGFBP-specific serum protease activity

Paul J. Fielder, Gudmundor Thordarson, Frank Talamantes, Ronald (Ron) Rosenfeld

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

Characterization of insulin-like growth factor binding proteins (IGFBPs) and their pituitary regulation were investigated in intact and hypophysectomized (HX) pregnant and nonpregnant mice. Serum samples were obtained from pregnant mice killed on days 5, 8, 10, 12, 14, and 18 of gestation and fetal mice killed on day 18 of gestation. Some animals were HX or sham operated (SH) on days 9, 11, and 14 of gestation; HX and SH control animals were killed 3 days post surgery. Identification and relative quantities of IGFBPs were determined by Western ligand blotting (WLB) of whole serum, or immunoprecipitated and/or deglycosylated serum, using [125I] IGF-II as the ligand. Serum IGF-I and -II concentrations were determined in both HX and SH day 14 pregnant, nonpregnant, and day 18 fetal mouse sera. WLB analysis of nonpregnant mouse serum demonstrated IGFBPs with apparent Mr of approximately 45-40, 31-27, and 24 K. The 45-40 K IGFBPs appeared to be the mouse equivalent of IGFBP-3, and one of the 31-27 K IGFBPs appeared to be IGFBP-2. The other IGFBPs present are not yet fully identified. During pregnancy, the amount of IGFBP-3 present in serum (as measured by WLB) gradually decreased to 42%, 17%, and 10% of virgin levels on days 5, 8, and 10 of gestation. The amount of IGFBP-3 present in pregnant serum after day 10 of gestation was not measurable by scanning laser densitometry. Additionally, an IGFBP of approximately 27 K appeared during gestation; after treatment with Endoglycosidase F, this IGFBP decreased to 24 K. The major IGFBP in fetal serum appeared to be IGFBP-2, with lesser amounts of IGFBP-3 and the 27 and 24 K IGFBPs. Hypophysectomy significantly decreased IGFBP-3, IGF-I, and IGF-II in nonpregnant mouse serum, increased the amount of IGFBP2 found in pregnant mouse serum, and had no effect on IGF-I or -II concentration in day 14 pregnant mouse serum. Incubation of nonpregnant serum with late (day 17) pregnant serum for 5 h at 37 C, decreased the amount of IGFBP-3 (as measured by WLB) in the mixed sample to 93% of the amount present in the nonmixed sample. Iodinated recombinant IGFBP-3 ([125I]human (h)IGFBP-3) was incubated with different mouse serum samples, and proteolytic fragments were visualized after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Pregnant mouse serum (after day 10 of gestation) was able to proteolize [125I]hIGFBP-3 from 43 K to a fragment of approximately 25 K. However, nonpregnant serum and serum from early pregnancy (day 10 or before) lacked this ability. Hyphosectomy had no effect on the ability of nonpregnant or pregnant serum to proteolyze IGFBP-3. The protease activity found in pregnant mouse serum was inhibited by inclusion of protease inhibitors in the incubation mixtures. These data indicate that the major serum IGFBP decreases during gestation in mice and that this decrease is in part due to a serum protease activity that is specific for IGFBP-3.

Original languageEnglish (US)
Pages (from-to)2270-2280
Number of pages11
JournalEndocrinology
Volume127
Issue number5
StatePublished - Nov 1990
Externally publishedYes

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Insulin-Like Growth Factor Binding Proteins
Hypophysectomy
Peptide Hydrolases
Pregnancy
Insulin-Like Growth Factor Binding Protein 3
Serum
Insulin-Like Growth Factor II
Ligands
Western Blotting
Insulin-Like Growth Factor I
Insulin-Like Growth Factor Binding Protein 2
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Characterization of insulin-like growth factor binding proteins (IGFBPs) during gestation in mice : Effects of hypophysectomy and an IGFBP-specific serum protease activity. / Fielder, Paul J.; Thordarson, Gudmundor; Talamantes, Frank; Rosenfeld, Ronald (Ron).

In: Endocrinology, Vol. 127, No. 5, 11.1990, p. 2270-2280.

Research output: Contribution to journalArticle

Fielder, Paul J. ; Thordarson, Gudmundor ; Talamantes, Frank ; Rosenfeld, Ronald (Ron). / Characterization of insulin-like growth factor binding proteins (IGFBPs) during gestation in mice : Effects of hypophysectomy and an IGFBP-specific serum protease activity. In: Endocrinology. 1990 ; Vol. 127, No. 5. pp. 2270-2280.
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abstract = "Characterization of insulin-like growth factor binding proteins (IGFBPs) and their pituitary regulation were investigated in intact and hypophysectomized (HX) pregnant and nonpregnant mice. Serum samples were obtained from pregnant mice killed on days 5, 8, 10, 12, 14, and 18 of gestation and fetal mice killed on day 18 of gestation. Some animals were HX or sham operated (SH) on days 9, 11, and 14 of gestation; HX and SH control animals were killed 3 days post surgery. Identification and relative quantities of IGFBPs were determined by Western ligand blotting (WLB) of whole serum, or immunoprecipitated and/or deglycosylated serum, using [125I] IGF-II as the ligand. Serum IGF-I and -II concentrations were determined in both HX and SH day 14 pregnant, nonpregnant, and day 18 fetal mouse sera. WLB analysis of nonpregnant mouse serum demonstrated IGFBPs with apparent Mr of approximately 45-40, 31-27, and 24 K. The 45-40 K IGFBPs appeared to be the mouse equivalent of IGFBP-3, and one of the 31-27 K IGFBPs appeared to be IGFBP-2. The other IGFBPs present are not yet fully identified. During pregnancy, the amount of IGFBP-3 present in serum (as measured by WLB) gradually decreased to 42{\%}, 17{\%}, and 10{\%} of virgin levels on days 5, 8, and 10 of gestation. The amount of IGFBP-3 present in pregnant serum after day 10 of gestation was not measurable by scanning laser densitometry. Additionally, an IGFBP of approximately 27 K appeared during gestation; after treatment with Endoglycosidase F, this IGFBP decreased to 24 K. The major IGFBP in fetal serum appeared to be IGFBP-2, with lesser amounts of IGFBP-3 and the 27 and 24 K IGFBPs. Hypophysectomy significantly decreased IGFBP-3, IGF-I, and IGF-II in nonpregnant mouse serum, increased the amount of IGFBP2 found in pregnant mouse serum, and had no effect on IGF-I or -II concentration in day 14 pregnant mouse serum. Incubation of nonpregnant serum with late (day 17) pregnant serum for 5 h at 37 C, decreased the amount of IGFBP-3 (as measured by WLB) in the mixed sample to 93{\%} of the amount present in the nonmixed sample. Iodinated recombinant IGFBP-3 ([125I]human (h)IGFBP-3) was incubated with different mouse serum samples, and proteolytic fragments were visualized after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Pregnant mouse serum (after day 10 of gestation) was able to proteolize [125I]hIGFBP-3 from 43 K to a fragment of approximately 25 K. However, nonpregnant serum and serum from early pregnancy (day 10 or before) lacked this ability. Hyphosectomy had no effect on the ability of nonpregnant or pregnant serum to proteolyze IGFBP-3. The protease activity found in pregnant mouse serum was inhibited by inclusion of protease inhibitors in the incubation mixtures. These data indicate that the major serum IGFBP decreases during gestation in mice and that this decrease is in part due to a serum protease activity that is specific for IGFBP-3.",
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N2 - Characterization of insulin-like growth factor binding proteins (IGFBPs) and their pituitary regulation were investigated in intact and hypophysectomized (HX) pregnant and nonpregnant mice. Serum samples were obtained from pregnant mice killed on days 5, 8, 10, 12, 14, and 18 of gestation and fetal mice killed on day 18 of gestation. Some animals were HX or sham operated (SH) on days 9, 11, and 14 of gestation; HX and SH control animals were killed 3 days post surgery. Identification and relative quantities of IGFBPs were determined by Western ligand blotting (WLB) of whole serum, or immunoprecipitated and/or deglycosylated serum, using [125I] IGF-II as the ligand. Serum IGF-I and -II concentrations were determined in both HX and SH day 14 pregnant, nonpregnant, and day 18 fetal mouse sera. WLB analysis of nonpregnant mouse serum demonstrated IGFBPs with apparent Mr of approximately 45-40, 31-27, and 24 K. The 45-40 K IGFBPs appeared to be the mouse equivalent of IGFBP-3, and one of the 31-27 K IGFBPs appeared to be IGFBP-2. The other IGFBPs present are not yet fully identified. During pregnancy, the amount of IGFBP-3 present in serum (as measured by WLB) gradually decreased to 42%, 17%, and 10% of virgin levels on days 5, 8, and 10 of gestation. The amount of IGFBP-3 present in pregnant serum after day 10 of gestation was not measurable by scanning laser densitometry. Additionally, an IGFBP of approximately 27 K appeared during gestation; after treatment with Endoglycosidase F, this IGFBP decreased to 24 K. The major IGFBP in fetal serum appeared to be IGFBP-2, with lesser amounts of IGFBP-3 and the 27 and 24 K IGFBPs. Hypophysectomy significantly decreased IGFBP-3, IGF-I, and IGF-II in nonpregnant mouse serum, increased the amount of IGFBP2 found in pregnant mouse serum, and had no effect on IGF-I or -II concentration in day 14 pregnant mouse serum. Incubation of nonpregnant serum with late (day 17) pregnant serum for 5 h at 37 C, decreased the amount of IGFBP-3 (as measured by WLB) in the mixed sample to 93% of the amount present in the nonmixed sample. Iodinated recombinant IGFBP-3 ([125I]human (h)IGFBP-3) was incubated with different mouse serum samples, and proteolytic fragments were visualized after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Pregnant mouse serum (after day 10 of gestation) was able to proteolize [125I]hIGFBP-3 from 43 K to a fragment of approximately 25 K. However, nonpregnant serum and serum from early pregnancy (day 10 or before) lacked this ability. Hyphosectomy had no effect on the ability of nonpregnant or pregnant serum to proteolyze IGFBP-3. The protease activity found in pregnant mouse serum was inhibited by inclusion of protease inhibitors in the incubation mixtures. These data indicate that the major serum IGFBP decreases during gestation in mice and that this decrease is in part due to a serum protease activity that is specific for IGFBP-3.

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