Anterior pituitary glands were collected from immature and mature (intact and castrated) male hamsters. The various species of FSH present within these glands were separated by Concanavalin A (Con A) chromatography and polyacrylamide gel isoelectric focusing (PAG-IEF) and measured by a specific FSH radioimmunoassay (RIA) as well as a radioreceptor assay (RRA). Two distinct forms of FSH (Con A unbound and bound) were separated by Con A chromatography and detected by both RIA and RRA. These two populations of FSH were present within anterior pituitary glands of all three animal models tested. Castration before collection of anterior pituitary glands reduced the ratio of Con A unbound: bound immunoreactive FSH. When measured by RRA this reduction was not observed. When homogenates of anterior pituitary glands obtained from mature animals were separated by PAG-IEF, six distinct species of FSH were observed by RIA with isoelectric points (pI) of 6·0, 5·7, 5·3, 5·0, 4·7 and 4·2–3·8. Homogenates of anterior pituitary glands obtained from immature male hamsters did not contain one of these species of FSH (pI value, 4·7). The relative contribution of some of the species of FSH to the total amount of detectable FSH differed depending upon the endocrine status of the animal. The species with pI value of 4·2–3·8 did not show any receptor-binding activity in any of the three models studied. The overall ratio of the activity of FSH measured by RRA compared with RIA was highest in anterior pituitary glands from intact mature and immature hamsters and lowest in anterior pituitary glands obtained from castrated animals. The RRA: RIA ratio for each species of FSH in all models tested declined as the isoelectric point of that species decreased. Thus, these results demonstrated the presence of multiple species of FSH within the anterior pituitary glands of immature and mature male hamsters. The relative proportions and receptor-binding activities of these species differed according to the isoelectric point and the pattern of hormone secretion at the time of collection of pituitary glands. Gonadal and other endocrine factors may influence not only the relative amount of each species of FSH but also the receptor-binding capacity of the FSH species synthesized by the anterior pituitary gland.

In the present study, we analyzed the role of Lys191 on function, structure, and dynamic behavior of the human GnRH receptor (hGnRHR) and the formation of the Cys14–Cys200 bridge, which is essential for receptor trafficking to the plasma membrane. Several mutants were studied; mutants lacked either the Cys14–Cys200 bridge, Lys191 or both. The markedly reduced expression and function of a Cys14Ser mutant lacking the 14–200 bridge, was nearly restored to wild-type/ΔLys191 levels upon deletion of Lys191. Lys191 removal resulted in changes in the dynamic behavior of the mutants as disclosed by molecular dynamics simulations: the distance between the sulfur- (or oxygen-) sulfur groups of Cys (or Ser)14 and Cys200 was shorter and more constant, and the conformation of the NH₂-terminus and the exoloop 2 exhibited fewer fluctuations than when Lys191 was present. These data provide novel information on the role of Lys191 in defining an optimal configuration for the hGnRHR intracellular trafficking and function.

GnRH agonists or antagonists are currently utilized as therapeutic agents in a number of diseases. A side-effect of prolonged treatment with GnRH analogues is hypoestrogenism. In this study, we tested the in vitro potency of different GnRH analogues originally found to be partial agonists (i.e. analogues with decreased efficacy for activating or stimulating their cognate receptor) as well as novel analogues, to identify compounds that might potentially be useful for partial blockade of gonadotrophin release. Cultured COS-7 cells transiently expressing the rat or human GnRH receptor (GnRHR) were exposed to increasing concentrations (10⁻⁸ to 10⁻⁵ M) of GnRH analogues (c(4–10)[Asp⁴,DNal⁶,Dpr¹⁰]-GnRH; c(4–10) [Dpr⁴,DNal⁶,Asp¹⁰]-GnRH; c(4–10)[Cys^4,10,DNal⁶]-GnRH; c[Eaca¹,DNal⁶]-GnRH; c[Gly¹,DNal⁶]-GnRH; c[βAla¹,DTrp⁶]-GnRH; c[Dava¹,DNal⁶]-GnRH; c[Gaba¹, DNal⁶]-GnRH), and the ability of these analogues to provoke or antagonize GnRH-stimulated inositol phosphate production was assessed.

With both human and rat GnRHRs, c[Eaca¹,DNal⁶]-GnRH, c[Gly¹,DNal⁶]-GnRH, c[βAla¹,DTrp⁶]-GnRH and c[Dava¹,DNal⁶]-GnRH exhibited partial agonist activity (35–87% of the maximal efficacy shown by 10⁻⁶ M GnRH), whereas c[Gaba¹,DNal⁶]-GnRH behaved as a partial agonist with the human GnRHR and as full agonist with the rat GnRHR. c(4–10)[Asp⁴, DNal⁶,Dpr¹⁰]-GnRH and c(4–10)[Dpr⁴,DNal⁶,Asp¹⁰]-GnRH exhibited full antagonist activity with both GnRHRs, and c(4–10) [Cys^4,10,DNal⁶]-GnRH was a weak, partial agonist with the human GnRHR and a full antagonist with the rat GnRHR. With the exception of c[Gaba¹,DNal⁶]-GnRH stimulation of the human GnRHR, and c[Dava¹,DNal⁶]-GnRH and c[Gaba¹, DNal⁶]-GnRH stimulation of the rat GnRHR, all partial agonists also exhibited antagonist activity in the presence of the exogenous full agonist.

The results demonstrate that structurally similar analogues display variable potencies and efficacies in vitro for a specific GnRHR as well as for the human versus the rat GnRHR. Their ultimate in vivo usefulness to treat clinical conditions in which complete suppression of gonadotroph activity is not required remains to be investigated.