br Material and methods br Results br Discussion

Material and methods
Results
Discussion The inhibitory action of a GABA agonist, muscimol, on the level of mRNA encoding GnRH in the POA-hypothalamus [25] of follicular-phase sheep has suggested that at the hypothalamic level GABA may exert a suppressive effect on subsequent steps of the GnRH biosynthetic pathway. Our current study provides direct support for this suggestion, when similar muscimol-related changes in the level of post-translational product of the GnRH gene were determined in sheep. Specifying, our results demonstrated for the first time that muscimol infused into the third cerebral ventricle of follicular-phase sheep decreased levels of GnRH in the POA, AH and VMH, and SME. Thus, the possibility that decapeptide level is exclusively coupled with cytoplasmic GnRH mRNA cannot be excluded, even if studies on mice and rats suggested that GnRH biosynthesis regulated via post-transcriptional mechanism(s) into the mature GnRH form is not strictly related with GnRH mRNA levels [45,46]. However, it should be noted that GnRH mRNA amount in various parts of the ARP 100 results not only from the transcriptional activity of GnRH gene, but also from the stability of GnRH transcript and its utilization in GnRH biosynthesis. Hence, in some circumstances transcription and translation may be far from having a linear and simple relationship [47]. Since GnRH mRNA was not detected in the SME of sheep in our previous study [25], it is certain that the post-translational product of GnRH gene in this structure results from the axonal transport of the hormone from other hypothalamic areas. It is now indisputable that the most important source of hormone conveyed to SME are GnRH neurons located in POA, i.e., the hypothalamic structure where most of GnRH perikarya are located [48] and most of GnRH transcript is synthesized [49,50]. Therefore, it could be suspected that the content of GnRH protein should be definitely higher in the POA in comparison to other hypothalamic structures. Surprisingly, such a dependency was not observed in the present study. One probable reason for these apparently conflicting results may be due to the method (relative measurement vs total protein concentration) that we used in the experiment. Furthermore, our studies were conducted on sheep in the late follicular phase of the oestrous cycle, just before the preovulatory GnRH/LH surge, and it is thus probable that in this period the differences in the GnRH content in particular hypothalamic areas of this breed of sheep could be less pronounced. This last opinion seems to be more justified, especially that the level of GnRH in the hypothalamus of follicular-phase sheep is significantly higher as compared to anestrous animals [51]. In the present study, the blockade of GABAAR induced by bicuculline increased the level of GnRH in the POA-hypothalamus. We previously reported the existence of a similar relationship between the administration of bicuculline and GnRH transcript level in the POA, AH and VMH of follicular-phase sheep [25]. Importantly, the decrease or increase in GnRH amount in the POA-hypothalamus of muscimol- and/or bicuculline-treated ewes, respectively, was associated with similar changes in LH secretion. Although we did not measure the level of GnRH in the hypophyseal portal blood, the diminished LH secretion following muscimol treatment indicates that GABA also suppresses the release of GnRH into the portal circulation. Therefore, it seems that GABA could reduce GnRH/LH release via the inhibition of GnRH biosynthesis. Thus, the possibility that GABA-mediated mechanisms controlling GnRH gene expression are similar to that governing decapeptide release cannot be excluded, even if such a point of view is in contrast with the earlier studies in sheep, suggesting that neural mechanism(s) controlling GnRH release is/are distinct from those regulating GnRH gene expression [52,53].