Effects of gonadotrophin-releasing hormone, recombinant human activin-A and sex steroid hormones upon the follicle-stimulating isohormones secreted by rat anterior pituitary cells in culture

Abstract

ABSTRACT FSH is produced and secreted from the anterior pituitary gland of rats in multiple molecular forms. At times of high gonadotrophin-releasing hormone (GnRH) and oestrogen output (e.g. the morning of the day of pro-oestrus) the pituitary increases the production of FSH isoforms with isoelectric point (pI) values >5·0, whilst sex steroid deprivation leads to the production of strongly acidic and less in-vitro biologically active FSH molecules. It is not known, however, whether sex steroids modulate the production of specific FSH isoforms by a direct action at the pituitary level or indirectly through altering the rate of synthesis and/or secretion of GnRH. In order to obtain some insight on this issue, we examined the charge heterogeneity of FSH secreted by cultured pituitary cells exposed to different FSH-releasing factors, oestradiol-17β and progesterone, alone or in different time-sequenced combinations. Anterior pituitary glands from 21-day-old female rats were enzymatically dispersed into a single cell suspension and cultured for 5 days. During days 1 to 3, cells were incubated in the absence of factors or steroid hormones; on days 3 to 4, cells were incubated in the absence (controls) or presence of either oestradiol17β (3·67 nmol/l) or oestradiol-17β plus progesterone (3·67 and 31·8 nmol/l respectively). Finally, during days 4 to 6, GnRH (10 nmol/l) or recombinant human activin-A (2 nmol/l) were added to half of all culture wells. Media from each cell group were concentrated and the several forms of secreted FSH were then separated by polyacrylamide gel isoelectric focusing (pH range 6·5–4·0) and quantitated. All media concentrates contained several forms of immunoactive secreted FSH focusing within a pH range of 6·44–4·23. A large amount (51–76%) of total FSH recovered focused within a pI range of 4·9–4·0 (area 3), whilst 20–43% and 4–8% of the total were identified within pi range of 5·9–5·0 (area 2) and 6·5–6·0 (area 1) respectively. Addition of GnRH to control or oestradiol-primed cells significantly increased the release of FSH isoforms recovered within area 2 compared with the remaining groups (per cent (±s.d.) FSH recovered within area 2 in groups treated with GnRH and those treated with oestradiol plus GnRH= 43·2±2·0 and 39·4±2·5 of total respectively; control groups and groups treated with oestradiol-17β, oestradiol-17β plus progesterone and activin-A = 32·1±1·2, 21·7±1·9, 19·7±5·0 and 21·5±4·0% of total respectively; P<0·05 compared with groups exposed to GnRH and oestradiol plus GnRH). The presence of progesterone in the culture media prevented this GnRH-mediated effect. Cells exposed to oestradiol-17β, oestradiol-17β plus progesterone and activin-A (with or without sex steroids) predominantly released FSH forms recovered within the most acidic area of the gel (area 3) (72·9±4·5, 76·6±8·6 and 70·9±5·9% of total respectively; P < 0·05 compared with GnRH-treated and oestradiol-17β plus GnRH-treated groups). There were no between-group differences in the amount of FSH recovered within area 1 (pI 5·6–6·0). FSH molecules that focused within area 2 exhibited a higher receptor-binding activity than those recovered from the most acidic region of the gel (radioreceptor assay/radioimmunoassay FSH activity ratio in area 2 = 2·56±0·29, area 3=0·83±0·03; P<0·01). We conclude that under in-vitro conditions GnRH selectively increases the release of less acidic FSH isoforms possessing an enhanced receptor-binding potency. It is suggested that oestradiol modulates the in-vivo production and secretion of specific FSH isoforms indirectly through temporal modifications in either the rate of synthesis and/or secretion of GnRH at the hypothalamic level or pituitary responsiveness to this releasing hormone. Journal of Endocrinology (1992) 134, 97–106