Granulosa cells were prepared from small follicles (<3 mm) from the ovaries of 5-month-old gilts. They were cultured in plastic dishes coated with a synthetic adhesion peptide in a chemically defined medium supplemented with 2% serum substitute. After 3 days of culture, the cells reached confluence and expression of the LH receptor could be stimulated in a hormonally defined medium. LH receptor RNAs were estimated by autoradiography using Northern blots and dot blots of total cell RNA. LH receptor RNAs were hybridized with a homologous 32P-labelled random-primed DNA probe. The LH receptor was measured using 125I-labelled human chorionic gonadotrophin (hCG) as tracer.
Northern blots of LH receptor RNAs revealed a predominant signal of 4.4kb and two less-intense hybridization bands of 7.5 and 1.9kb. The 4.4kb band was used for quantification of LH receptor RNAs because it was the most intense and may be attributed to the full-length messenger RNA. In these conditions, after 72h stimulation, FSH (0.6 nm), insulin (5 μg/ml), oestradiol (30 nm) and deoxycorticosterone (0.3 nm) yielded high LH receptor RNA levels (eight times unstimulated cell level), while dibutyryl cyclic AMP (1 mm), cortisol (5.4 nm), thyroxine (100 nm) and epidermal growth factor (16pm) gave low LH receptor RNA levels (one to five times). However, the respective amounts of the receptor RNA did not give yield to the same proportion of LH receptor for every factor, indicating some post-transcriptional regulations.
The kinetic study of the production of the LH receptor obtained in a defined medium supplemented with FSH, oestradiol and insulin showed that the receptor appeared after 48 h of stimulation and reached a maximum of about 7000 receptors per cell at 72 h. The three hybridization bands on Northern blots evolved in parallel and appeared as early as 24 h. They were at maximal level from 24 to 48 h of stimulation. When the granulosa cells were pulse-treated for 2 h with cycloheximide (10 μg/ml), they exhibited a transient rise in LH receptor RNA content which was followed by a delayed receptor increase especially at 72 h of stimulation.
Taken together, these results indicate that the LH receptor in primary culture of granulosa cells seems to be regulated by different physiological factors both at the transcriptional and the translational levels.
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