OBJECTIVE: To characterize growth factor gene expression by passaged coculture cell lines demonstrated to enhance in vitro pre-embryo growth. DESIGN: Ribonucleic acids isolated from the isthmus, ampullary, and fimbriae portions of the human oviduct, and from buffalo rat liver cell monolayers were subjected to Northern analysis using probes for growth factors. SETTING: Academic tertiary care hospital. PATIENT(S): Two reproductive age women undergoing a hysterectomy and bilateral salpingectomy for benign gynecologic conditions consented to experimental use of their oviducts. INTERVENTION(S): Cell cultures were established from fresh human oviduct segments and commercially purchased buffalo rat liver cells. After two passages, total RNA was isolated from these confluent monolayers, fractionated on denaturing agarose gels, transferred to nylon membranes, and analyzed by Northern hybridization using complementary DNAs from epidermal growth factor (EGF), stem cell factor, also known as Kit-ligand, colony-stimulating factor-1 (CSF), leukemia inhibitory factor, and interleukin-6 (IL-6). Radioactively labeled probes were prepared by in vitro transcription or by 5' end labeling. After hybridization, blots were washed at increasing strigencies to remove nonspecifically bound radioactivity and subjected to autoradiography. RESULT(S): Human oviduct coculture cells express EGF (kit-ligand), CSF, leukemia inhibitory factor, and IL-6. Buffalo rat liver cells contain the messenger RNA transcripts for kit-ligand and CSF. CONCLUSION(S): Human oviduct and buffalo rat liver coculture cells express specific growth factors. These results support the theory that coculture systems may enhance pre-embryo growth via the production of embryotrophic factors. The identification of these ligands may provide the rationale for selecting specific growth factors for media supplementation as well as contribute to our understanding of the general mechanisms involved in regulating early embryonic growth and development.
OBJECTIVE: To characterize growth factor gene expression by passaged coculture cell lines demonstrated to enhance in vitro pre-embryo growth. DESIGN: Ribonucleic acids isolated from the isthmus, ampullary, and fimbriae portions of the human oviduct, and from buffalo rat liver cell monolayers were subjected to Northern analysis using probes for growth factors. SETTING: Academic tertiary care hospital. PATIENT(S): Two reproductive age women undergoing a hysterectomy and bilateral salpingectomy for benign gynecologic conditions consented to experimental use of their oviducts. INTERVENTION(S): Cell cultures were established from fresh human oviduct segments and commercially purchased buffalo rat liver cells. After two passages, total RNA was isolated from these confluent monolayers, fractionated on denaturing agarose gels, transferred to nylon membranes, and analyzed by Northern hybridization using complementary DNAs from epidermal growth factor (EGF), stem cell factor, also known as Kit-ligand, colony-stimulating factor-1 (CSF), leukemia inhibitory factor, and interleukin-6 (IL-6). Radioactively labeled probes were prepared by in vitro transcription or by 5' end labeling. After hybridization, blots were washed at increasing strigencies to remove nonspecifically bound radioactivity and subjected to autoradiography. RESULT(S): Human oviduct coculture cells express EGF (kit-ligand), CSF, leukemia inhibitory factor, and IL-6. Buffalo rat liver cells contain the messenger RNA transcripts for kit-ligand and CSF. CONCLUSION(S): Human oviduct and buffalo rat liver coculture cells express specific growth factors. These results support the theory that coculture systems may enhance pre-embryo growth via the production of embryotrophic factors. The identification of these ligands may provide the rationale for selecting specific growth factors for media supplementation as well as contribute to our understanding of the general mechanisms involved in regulating early embryonic growth and development.
Authors: L I Barmat; H C Liu; S D Spandorfer; A Kowalik; C Mele; K Xu; L Veeck; M Damario; Z Rosenwaks Journal: J Assist Reprod Genet Date: 1999-03 Impact factor: 3.412