This study on heifers (n = 27) compared the effects of a GnRH antagonist (Antarelix) and those of an agonist (Triptorelin) on gonadotropin release during the periovulatory period of the oestrous cycle. In three experiments (EXP I-III), an initial injection of GnRH analogs was given 48 h after a single PGF 2alpha pretreatment during the luteal phase, with a further five at 12 h intervals. A challenge by a GnRH agonist (Gonavet) was performed six hours after the last application of analogs. In EXP I (n = 9), heifers received six times 1.5 mg of Antarelix, 0.5 mg of Triptorelin, and mannitol (5%; control), respectively. In EXP II (n = 12), identical Antarelix and Triptorelin treatments were followed by a single injection of estradiol-17beta valerate (6 h after Gonavet). The dosage of Antarelix was increased to 5 mg for each injection in EXP III (n = 6). Measurement of LH in blood plasma frequently sampled was performed parallely by a competitive RIA method (EXP I + II) and by a sandwich-type electro-chemiluminescence-immunoassay (ECLIA) in EXP III. This non-isotopic technique was also used to additionally analyse FSH levels. Results of EXP I showed that the GnRH antagonist equally suppressed LH surges and ovulation. On the contrary, prior to suppression of LH levels due to down-regulation of pituitary GnRH receptors the agonist Triptorelin induced an initial increase in LH concentration which was followed by ovulation. In the control animals we observed endogenous LH surges as well as smaller elevations after the agonist (Gonavet) challenge. An increase was also observed in antagonist, but not in Triptorelin treated heifers. Pituitary GnRH receptors were not detectable in animals previously treated by the analogs, whereas concentrations between 2.2-21.0 fmol/mg protein were measured in controls. Results of EXP II confirmed the described effects of GnRH analogs. Additionally, it was shown that exogenous estradiol is able to release LH from the pituitary, although a previous treatment by a GnRH agonist had dropped the pulsatile gonadotropin secretion. Contrary to the LH pattern and despite elevated amounts of the antagonist, the mean concentration and pulse number of FSH were not influenced by the antagonist treatment (EXP III). These data confirmed that (a) the reversibly blocked pituitary function induced by a potent GnRH antagonist may be a useful tool to study gonadotropin-dependent final follicular growth as well as ovulation in cyclic heifers and (b) the novel non-isotopic ECLIA methods for the determination of FSH and LH provided practical alternatives to other immunoassay types.

In this study, the molecular masses and isoelectric characteristics of pituitary LH

In the following investigations, the LH secretion of cells from pituitaries in heifers on days 16-18 of their oestrous cycle (n = 14) was analysed. Cells were dissociated with trypsin and collagenase and maintained in a static culture system. For the estimation of LH release, the cells were incubated with various concentrations of mammalian GnRH (Lutrelef) for 6 h. To determine the action of Antarelix (GnRH antagonist), the cells were preincubated for 1 h with concentrations of 10 -5 or 10 -4 M Antarelix followed by 10 -6 M GnRH coincubation for a further 6 h. At the end of each incubation, the medium was collected for LH analysis. Parallel, intracellular LH was qualitatively detected by immunocytochemistry. Changes in the intensity of LH staining within the cells in dependence of different GnRH concentrations were not observed, but a significant increase LH secretion in pituitary cells was measured at 10 -6 M GnRH. Antarelix had no effect on basal LH secretion at concentrations of 10 -4 and 10 -5 M. After coincubation of pituitary cells with Antarelix and GnRH, Antarelix blocked the GnRHstimulated LH secretion with a maximal effect of 10 -4 M, but the staining of immunoreactive intracellular LH was detected at approximately the same level compared to the pituitary cells treated with exogenous GnRH alone. These data demonstrate that Antarelix is effective in influencing the GnRH-stimulated LH secretion of pituitary cells in vitro. After administration of Antarelix in vivo, the GnRH-stimulated LH secretion of cultured pituitary cells was not inhibited.

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