The intrapituitary mechanisms underlying the inhibitory actions of hyperprolactinaemia on the reproductive axis remain unclear. Previous work on primary pituitary cultures revealed combined suppressive effects of prolactin (PRL) and dopamine on the gonadotrophin response to GnRH. However, whether these effects occur directly at the level of the gonadotroph and are accompanied by changes in gene expression is still unresolved. Here, aT 3 -1 and LbT2 cells were used to investigate the effects of PRL and dopamine on gonadotrophin synthesis and release in gonadotroph monocultures under basal and GnRH-stimulated conditions. PRL receptor and dopamine receptor mRNA expressions were first determined by RT-PCR in both cell lines. Then, PRL and the dopamine agonist bromocriptine (Br), alone or in combination, were shown to block the maximal a-subunit and LHb-subunit mRNA responses to a dose-range of GnRH. The LH secretory response was differentially affected by treatments. GnRH dose-dependently stimulated LH release, with a 4-5 fold increase at 10 K8 M GnRH. Unexpectedly, PRL or Br stimulated basal LH release, with PRL, but not Br, enhancing the LH secretory response to GnRH. This effect was, however, completely blocked by Br. These results reveal direct effects of PRL and dopamine at the level of the gonadotroph cell, and interactions between these two hormones in the regulation of gonadotrophin secretion. Moreover, uncoupling between LH synthesis and release in both the basal and the GnRH-stimulated responses to PRL and dopamine was clearly apparent.

Previous studies have provided evidence for a paracrine interaction between pituitary gonadotrophs and lactotrophs. Here, we show that GnRH is able to stimulate prolactin (PRL) release in ovine primary pituitary cultures. This effect was observed during the breeding season (BS), but not during the nonbreeding season (NBS), and was abolished by the application of bromocriptine, a specific dopamine agonist. Interestingly, GnRH gained the ability to stimulate PRL release in NBS cultures following treatment with bromocriptine. In contrast, thyrotropin-releasing hormone, a potent secretagogue of PRL, stimulated PRL release during both the BS and NBS and significantly enhanced the PRL response to GnRH during the BS. These results provide evidence for a photoperiodically modulated functional interaction between the GnRH/gonadotropic and prolactin axes in the pituitary gland of a short day breeder. Moreover, the stimulation of PRL release by GnRH was shown not to be mediated by the gonadotropins, since immunocytochemical, Western blotting, and PCR studies failed to detect pituitary LH or FSH receptor protein and mRNA expressions. Similarly, no gonadotropin receptor expression was observed in the pituitary gland of the horse, a long day breeder. In contrast, S100 protein, a marker of folliculostellate cells, which are known to participate in paracrine mechanisms within this tissue, was detected throughout the pituitaries of both these seasonal breeders. Therefore, an alternative gonadotroph secretory product, a direct effect of GnRH on the lactotroph, or another cell type, such as the folliculostellate cell, may be involved in the PRL response to GnRH in these species.

In a variety of species, the LH-secretory response to gonadotropin-releasing hormone (GnRH) is completely suppressed by the combined actions of prolactin (PRL) and dopamine (DA). In sheep, this effect is only observed under long days (nonbreeding season [NBS]). To investigate the level at which these mechanisms operate, we assessed the effects of PRL and bromocriptine (Br), a DA agonist, on the gonadotropin-secretory and mRNA responses to GnRH in pituitary cell cultures throughout the ovine annual reproductive cycle. As expected, the LH-secretory response to GnRH was only abolished during the NBS following combined PRL and Br application. Conversely, the LHB subunit response to GnRH was reduced during both the BS and NBS by the combined treatment and Br alone. Similar results were obtained in pars distalis-only cultures, indicating that the effects are pars tuberalis (PT)- independent. Further signaling studies revealed that PRL and Br alter the LH response to GnRH via convergence at the level of PLC and PKC. Results for FSH generally reflected those for LH, except during the BS where removal of the PT allowed PRL and Br to suppress the FSH-secretory response to GnRH. These data show that suppression of the LH-secretory response to GnRH by PRL and DA is accompanied by changes in mRNA synthesis, and that the photoperiodic modulation of this inhibition operates primarily at the level of LH release through alterations in PKC and PLC. Furthermore, the suppressive effects of PRL and DA on the secretion of FSH are photoperiodically regulated in a PT-dependent manner.