ABSTRACT

Anterior pituitary glands were removed from neonatally androgenized (100 μg testosterone propionate) female rats and normal controls at 5, 10, 18, 21, 30, 60 and 90 days of age, and the multiple forms of FSH present within them were separated by chromatofocusing (pH range 7·5–4·0). Additional pituitary glands from intact adult males (90 days old) were also studied for comparative purposes. All animal groups exhibited multiple forms of immunoactive FSH within a pH range of 7·5–4·0, as well as an additional FSH form obtained after the addition of 1·0 mol NaCl/l to the chromatofocusing column (salt peak). In animals 5–30 days old (controls and androgenized) the majority of FSH applied to the chromatofocusing columns was recovered within the salt peak (45-85% of total FSH immunoactivity recovered). However, as the animals aged, more FSH immunoactivity focused within less acidic regions (isoelectric point (pI) 5·9–5·0); pituitaries from animals 60 days old contained the greatest proportion of FSH focused within this pH range (controls, 39·2±0·6%; androgenized, 23·1 ±0·9% of total immunoactivity recovered; P < 0·03 vs animals 30 days old for both experimental groups). This shift towards less acidic FSH was attenuated in androgenized animals compared with the controls (P<0·01).

In control adult rats, the chromatofocusing distribution pattern of pituitary FSH varied according to the day of the oestrous cycle. Pituitary extracts from control rats decapitated during the morning of pro-oestrus, oestrus and day 1 of dioestrus exhibited the highest proportion of immunoactive FSH (23·2–28·8% of total) focused within a pH range of 5·9–5·0, whilst only 10·4–11·6% of FSH from androgenized rats and those on day 1 of dioestrus was recovered within this pH range (P<0·05). In control animals decapitated during the morning of prooestrus and oestrus, 10–26% of FSH focused within the most alkaline region (pI 7·5–6·0); the chromatofocusing pattern of pituitary FSH from the neonatally androgenized animals was characteristic, in that no more than one peak (1·5±0·5% of total) was detected in this alkaline region. In the adult male rats, the majority of pituitary FSH eluted from the chromatofocusing columns within a pH of 4·9–4·0 (52·4±1·2% of total FSH immunoactivity) and the salt peak (pH <4·0) (33·1 ±2·4 of total). All FSH isoforms obtained after chromatofocusing represented α and β dimers as disclosed by size exclusion chromatography.

The results strongly suggest that a cyclic or 'female' pattern of hypothalamic and gonadal secretion leads the anterior pituitary towards the production of less acidic FSH isoforms, whereas a tonic or 'androgenic' type of secretion, as that present in adult males and females with the androgen-induced anovulatory syndrome, leads more to the production of strongly acidic FSH isoforms. The finding of qualitative and quantitative differences among normally cycling and androgenized animals gives further support for the concept of the existence of a sexual dichotomy in terms of the type of FSH synthesized by the anterior pituitary gland.

Journal of Endocrinology (1990) 126, 323–332

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

Gestodene (17 alpha-ethynyl-13 beta-ethyl-17 beta-hydroxy-4, 15-gonadien-3-one) is the most potent synthetic progestin currently available and it is widely used as a fertility regulating agent in a number of contraceptive formulations because of its high effectiveness, safety and acceptability. The observation that contraceptive synthetic progestins exert hormone-like effects other than their progestational activities, prompted us to investigate whether gestodene (GSD) administration may induce oestrogenic effects, even though the GSD molecule does not interact with intracellular oestrogen receptors (ER). To assess whether GSD may exert oestrogenic effects through some of its neutral metabolites, a series of experimental studies were undertaken using GSD and three of its A-ring reduced metabolites. Receptor binding studies by displacement analysis confirmed that indeed GSD does not bind to the ER, whereas its 3 beta,5 alpha-tetrahydro reduced derivative (3 beta GSD) interacts with a relative high affinity with the ER. The 3 alpha,5 alpha GSD isomer (3 alpha GSD) also binds to the ER, though to a lesser extent. The ability of the A-ring reduced GSD derivatives to induce oestrogenic actions was evaluated by the use of two different molecular bioassays: (a) transactivation of a yeast system co-transfected with the human ER alpha (hER alpha) gene and oestrogen responsive elements fused to the beta-galactosidase reporter vector and (b) transactivation of the hER alpha-mediated transcription of the chloramphenicol acetyl transferase (CAT) reporter gene in a HeLa cells expression system. The oestrogenic potency of 3 beta GSD was also assessed by its capability to induce oestrogen-dependent progestin receptors (PR) in the anterior pituitary of castrated female rats. The results demonstrated that 3 beta GSD and 3 alpha GSD were able to activate, in a dose-dependent manner, the hER alpha-mediated transcription of both the beta-galactosidase and the CAT reporter genes in the yeast and HeLa cells expression systems respectively. In both assays the 3 beta derivative of GSD exhibited a significantly greater oestrogenic effect than its 3 alpha isomer, while unchanged GSD and 5 alpha GSD were completely ineffective. Neither 3 beta GSD nor 3 alpha GSD exhibited oestrogen synergistic actions. Interestingly, the pure steroidal anti-oestrogen ICI-182,780 diminished the transactivation induced by 3 beta GSD and 3 alpha GSD in the yeast expression system. Furthermore, administration of 3 beta GSD resulted in a significant increase of oestrogen-dependent PR in the anterior pituitaries of castrated rats in comparison with vehicle-treated animals. The characteristics of the 3 beta GSD-induced PR were identical to those induced by oestradio benzoate. The overall results demonstrate that 3 beta GSD and its 3 alpha isomeric alcohol specifically bind to the ER and possess a weak intrinsic oestrogenic activity, whereas unmodified GSD does not. The data contribute to a better understanding of the GSD mechanism of action and allow the hypothesis to be advanced that the slight oestrogenlike effects attributable to GSD are mediated by its non-phenolic, tetrahydro reduced metabolites.