Interleukin-6 biosynthesis in human preovulatory follicles: some of its potential roles at ovulation.
In the present work we explored cellular sites of interleukin-6 (IL-6) biosynthesis in human follicular aspirates from patients undergoing in vitro fertilization therapy and the effects of this cytokine on oocyte fertilization and granulosa cell (GC) steroidogenesis. Biological IL-6 activity from 20-40 IU/mL was present in follicular fluids from 22 patients; it was also detected in 10 of 22 supernatants of cultured oocyte-cumulus complexes and in cumulus cell and GC cultures. Biological IL-6 activity in oocyte-cumulus complex cultures was not related to fertilization rates. Total ribonucleic acid was isolated from follicular aspirates and GC-enriched preparations. After reverse transcriptase and polymerase chain reaction cycles using oligonucleotide primers corresponding to known cDNA sequences for IL-6, a 126-basepair band characterized the amplification product of IL-6 transcripts on gel electrophoresis. To localize IL-6 messenger ribonucleic acid, in situ hybridization analysis was performed using a [35S]IL-6 riboprobe. The distribution of transcripts was more dense (15% vs. 3% stained cells) in GC-enriched preparations, which contained more than 95% GCs, than in original follicular preparations, which contained 20-40% viable GCs; it was not significantly modified by the presence of macrophage contaminants. The expression of IL-6 protein was assessed by positive immunohistological stainings. Biological IL-6 activity was higher, and in situ hybridization signals were more dense and more intense in 24-h GC cultures than in 72-h GC cultures, suggesting that IL-6 biosynthesis was transiently induced. Under experimental conditions of low IL-6 endogenous levels in cultures, adding recombinant human IL-6 from 10-200 IU/mL had no effect on progesterone production or aromatase activity in GC cultures free of macrophages, whereas in GC cultures including macrophage contaminants, stimulatory effects on basal and hCG-stimulated progesterone production and on basal and FSH-stimulated aromatase activities were observed. The present study provides strong support for the view that IL-6 is produced by GCs in the preovulatory follicle at the time of ovulation. In addition, we showed that IL-6 might be an intraovarian regulatory factor concerned with steroidogenesis.
Machelon
V
V
Institut National de la Santé et de la Recherche Médicale (INSERM), U-355, Clamart, France.
Emilie
D
D
Lefevre
A
A
Nome
F
F
Durand-Gasselin
I
I
Testart
J
J
D000328:Adult; D001141:Aromatase / Q000378:metabolism; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D005260:Female; D005307:Fertilization in Vitro; D015571:Follicular Fluid / Q000378:metabolism; D006107:Granulosa Cells / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D006080:Ovarian Follicle / Q000378:metabolism*; D010060:Ovulation / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011374:Progesterone / Q000096:biosynthesis; D012194:RNA-Directed DNA Polymerase; D011994:Recombinant Proteins / Q000494:pharmacology
D017931:DNA Primers; D015850:Interleukin-6; D011994:Recombinant Proteins; D011374:Progesterone; D001141:Aromatase; D012194:RNA-Directed DNA Polymerase