Partial purification and characterization of proteins with growth promoting activities from ovine mammary gland secretions.
Developmental regulation of growth promoting activities in mammary secretions of pregnant Awassi ewes was defined, and growth factors contained in these secretions were partially purified and characterised. Mammary secretions from pregnant ewes enhanced fibroblast cell (AKR-2B) and mammary cell (CID-9 cell strain) proliferation to levels comparable to that induced by 10% Foetal calf serum. Major milk proteins in mammary secretions collected from pregnant ewes one month prior to lambing up to one week after lambing, were resolved by SDS-PAGE, while gelatinases were resolved by zymography. Gelatinase activity was noted prior to P134 and decreased thereafter to reach a minimum during lactation. This decrease was concomitant with the onset of casein production. It is during this critical developmental period that highest growth promoting activity in mammary secretions was detected. Secretions with highest growth promoting activity were fractionated by ion exchange and gel filtration chromatography. Two heat-resistant, trypsin/chymotrypsin sensitive, growth-promoting activities were characterised. The first, designated ovine mammary derived growth factor-1 (oMDGF-1), had around a 30 kDa molecular weight and eluted at 0.65 M NaCl gradient on cation ion exchange chromatography. The second, oMDGF-2, eluted under gel filtration conditions at a molecular weight of 50 kDa and 150 kDa. oMDGF-1 induced changes in Connexin 43, but not in beta-casein mRNA expression by CID-9 mammary cells. In conclusion, growth factor activities in ewe mammary secretions peak during gestation at a period that overlaps maximal gelatinase expression and precedes milk protein synthesis. The factors modulate mammary cell function and may play a role in mammary gland development.
Talhouk
R S
RS
Department of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut, Lebanon. rtalhouk@aub.edu.lb
Maa'ni
F A
FA
Kalaa'ni
N
N
Zoubian
G S
GS
Simaa'n
C J
CJ
Abi-Sai'd
M
M
Hamadeh
S
S
Barbour
E
E
El-Sabban
M E
ME
D000818:Animals; D015152:Blotting, Northern; D002364:Caseins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D002455:Cell Division; D002478:Cells, Cultured; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D018031:Connexin 43 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D018093:Gelatinases / Q000096:biosynthesis / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D006133:Growth Substances / Q000096:biosynthesis / Q000302:isolation & purification* / Q000502:physiology; D007774:Lactation; D008321:Mammary Glands, Animal / Q000254:growth & development / Q000378:metabolism*; D051379:Mice; D008894:Milk Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D011247:Pregnancy; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D012756:Sheep / Q000502:physiology*
D002364:Caseins; D018031:Connexin 43; D006133:Growth Substances; D008894:Milk Proteins; D012313:RNA; D018093:Gelatinases