Presence of stem cell factor in follicular fluid and its expression in the human ovary

Autor: Tomio Iwabe, Akiko Enatsu, Naoki Terakawa, Tasuku Harada, Masayuki Ito, Masahiro Tanikawa
Rok vydání: 2000
Předmět:
Zdroj: Fertility and Sterility. 73:1259-1260
ISSN: 0015-0282
DOI: 10.1016/s0015-0282(00)00528-8
Popis: Stem cell factor (SCF), a pleiotropic glycoprotein with a molecular weight of 25‐36 kd, is a ligand for the tyrosine kinase receptor encoded by the c-kit protooncogene. The SCF and c-kit are essential for germ cell development, melanogenesis, and hematopoiesis. Although several lines of evidence in experimental animals suggest that SCF and c-kit may also play essential roles in human ovaries, little information is available. We previously reported that c-kit receptor messenger RNA (mRNA) was expressed in human oocytes (1). In the present study, we examined the expressions of SCF, a ligand for c-kit, in human oocytes, granulosa, theca, and ovarian stromal cells. We also investigated the relationship between SCF and steroid hormone concentrations in follicular fluid. Fifty-five samples of follicular fluid were collected from nine women (mean age 6 SD, 37.0 6 5.1 years; range, 29 ‐ 43 years) who were undergoing oocyte pick up for IVF-ET. The patients undergoing IVF-ET were stimulated with a combination of a GnRH analog (Suprecur, Hoechst Marrion Roussel, Tokyo, Japan) and hMG; clinical pregnancy was achieved in two of the nine women. Human preovulatory granulosa cells were collected from each of the nine women who underwent IVF-ET. Ovarian tissue was obtained from three additional women with regular menstrual cycles who were undergoing benign gynecological surgery unrelated to an ovarian condition; theca internal layers were microdissected from the follicle wall. A patient who underwent IVF-ET for research purposes donated seven oocytes used in this study. All of the oocytes used in this study were metaphase II. The total RNA was extracted from the oocytes as well as from cultured granulosa, theca, and stromal cells by the guanidium thiocyanate method. Informed consent was obtained from all subjects. We performed a reverse transcription polymerase chain reaction (RT-PCR) study with a Gene Amp RNA PCR Core Kit (Perkin, Elmer, NJ). The specific primers for SCF were 59-CCAGAACCCAGGCTCTTTAC-3 9 (sense), and 59-AGGCTCCAAAAGCAAAGC-3 9 (antisense). The SCF primers were designed to span exon 6, producing a 316-base pair fragment for the secretable form of SCF and a 233-base pair fragment for the membrane-bound form that lacks exon 6. The specificity of the PCR product was confirmed by a Southern blot analysis using the biotinylated oligonucleotide internal probe. The concentration of soluble SCF in the follicular fluid was determined by means of ELISA kit (R & D Systems Inc., Minneapolis, MN). The concentrations of E2 and P in the follicular fluid were measured with the use of an enzyme immunosorbent assay kit (Johnson & Johnson, Clinical Diagnostics, Amersham, UK). The concentration of T was measured by use of a radioimmunoassay kit (Diagnostic Products Corporation, Los Angeles, CA).
Databáze: OpenAIRE
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