Prepubertal changes in the synthesis, storage and release of the somatostatin in the hypothalamus of female lambs: a morphofunctional study.

It is assumed that hypothalamic somatostatin plays a role in the preovulatory phase of the oestrous cycle in sheep. The aim of the study was to investigate the processes of synthesis, storage and release of somatostatin in hypothalamic neurons, in immature female lambs, in the period approaching to puberty. Experiments were carried out on 10 prepubertal (17 weeks old) and 10 peripubertal (32 weeks old) ovary-intact lambs. Morphofunctional changes in the somatostatin neurons were assayed with immunohistochemistry and hybridisation in situ. Computer image analysis was used to determine the density of both reactions and the percentage of the area exhibiting immunohistochemical staining. These parameters express the content of immunoreactive (ir) somatostatin and expression of mRNA for pre-pro-somatostatin (PPS). Two populations of ir somatostatin perikarya were localized in the hypothalamus: a very large number of perikarya in the periventricular (PEV) nucleus, and single cell bodies in the arcuate (ARC) nucleus. Only ir somatostatin fibres, but no perikarya were seen in the ventromedial (VM) nucleus and preoptic area. The analysis of mRNA PPS showed perikarya filled with silver grains localized in the PEV, ARC and VM. There were differences in the content of ir somatostatin and the intensity of the PPS mRNA signal between the two periods investigated. In the median eminence, the content of ir somatostatin in the terminals decreased in the peripubertal compared to the prepubertal group (P<0.001). In the PEV, the content of ir somatostatin in the perikarya and the expression of PPS mRNA decreased in the peripubertal compared to the prepubertal group (P<0.001). In the ARC, the content of ir somatostatin in the perikarya increased (P<0.001), but expression of PPS mRNA decreased (P<0.001) in the peripubertal compared to the prepubertal group. There were no differences in the expression of PPS mRNA in the VM. We concluded, that the different secretory activity of the two hypothalamic populations of somatostatin neurons can be related to their different physiological functions in the prepubertal period of female lambs.

Regulation by estradiol of hypothalamic somatostatin gene expression: possible involvement of somatostatin in the control of luteinizing hormone secretion in the ewe.

In the ewe, the mediobasal hypothalamus (MBH) is the primary central site for estradiol to generate the preovulatory GnRH/LH surges and sexual behavior. This area contains numerous neurons expressing the estradiol receptor alpha, distributed in the ventromedial nucleus (VMN) and the infundibular nucleus (IN). A large proportion of these neurons express somatostatin, making this neuropeptide a potential candidate for transmission of the estradiol signal to the GnRH neurons located in the preoptic area. We tested this hypothesis using ovariectomized ewes that had been subjected to an artificial estrous cycle. In the first experiment, 22 h after progesterone removal, ewes received estradiol (treated ewes) or empty implants (control ewes) for 4 h and then were killed. Using in situ hybridization, we showed that this short estradiol treatment increased the somatostatin mRNA amount by about 50% in the VMN and 42% in the IN. In the second experiment, preovulatory estradiol signal was replaced by somatostatin intracerebroventricular (ICV) administration. This treatment abolished LH pulsatility and dramatically decreased the mean basal level of LH secretion while it did not affect the mean plasma GH concentration. We demonstrated that an increase in somatostatin mRNA occurs at the time of the negative feedback effect of estradiol on LH secretion during the early stage of the GnRH surge induction. As ICV somatostatin administration inhibits the pulsatile LH secretion by acting on the central nervous system, we suggest that somatostatin synthesized in the MBH could be involved in the estradiol negative feedback before the onset of the preovulatory surge.

Early decrease of proopiomelanocortin but not neuropeptide Y mRNA expression in the mediobasal hypothalamus of the ewe, during the estradiol-induced preovulatory LH surge.

In sheep, the mediobasal hypothalamus (MBH) has been shown to be the primary central site of estradiol (E2) action that induces both the preovulatory surge and sexual behaviour. However, the nature of the neurotransmitters or neuromodulators synthesized in the MBH during E2 stimulation remains to be clearly defined. After the cloning of the ovine cDNA sequences and using in situ hybridization, hypothalamic proopiomelanocortin (POMC), and preproneuropeptide Y (preproNPY) mRNA expression was studied in ovariectomized ewes that received a sequential treatment of progesterone and E2. As we showed that an exposition to E2 only for 4h well in advance on the LH surge onset is sufficient to induce the preovulatory surge and estrous behaviour, mRNA expression was evaluated in ewes treated with 6x30-mm E2 implants (experimental group) or with empty implants (control group) and slaughtered 4h after the start of the E2 treatment. Our results demonstrate that this short E2 treatment significantly decreased both the mean number of silver grains per POMC-containing cell (35%) and the mean number of POMC-cells (38%) in the ovine infundibular nucleus, whereas the treatment had no effect on preproNPY mRNA expression. These observations suggest that a reduction of POMC gene transcription could participate to the early neural mechanism of E2 feedback.