Urszula Kosior-Korzecka
Hormonal changes and expression of leptin receptor mRNA in pituitary celi s of sheep with ACTH induced ovarian cysts 
The aim of the study was to estimate the contribution of leptin and leptin receptors to LH secretion disorders in the pituitary that lead to follicular cyst development. The experiments were conducted on 52 ewes which were divided into two groups that differed from each other in body mass and endogenous leptin level. Next, in the respective group the experimental and controi sheep were separated. The experimental sheep obtained ACTHl-24 (0.01 mg/kg b.w.) from the 11th to the 16th day of the oestrous cycle, whereas the control sheep – physiological salt solution. During the experiment, leptin, LH and ACTH concentrations were analysed in the plasma of the sheep. On the 10th day of the estrous cycle the activity of ovaries was estimated (ovarian cyst/corpus luteum). Then, the pituitaries were isolated from all ewes to prepare cell culture. The pituitary cells were incubated in vitro with leptin, ACTH and with factors regulating the synthesis and/or release of leptin (insulin, oestradiol, testosterone). During the culture the LH secretion and NO release were analysed. At the same time, mRNA from the pituitary cells was isolated to study the leptin receptor expression (OB-Ra and OB-Rb) on the level of transcription. After the detection of both isoforms using RT -PCR, the changes in the level of the short form mRNA (OB-Ra mRNA) was analysed by RPA and the changes in the level of the long form mRNA (OB-Rb) by Real-Time PCR. During the experiment 80% of the experimental sheep with a lower body mass (48.98 ±1.06 kg) and a lower leptin level (5.20 ±0,89 ng/m1) and 100% of the experimental sheep with a higher body mass (55.10 ±0.48 kg) and higher plasma leptin level (9.38 ±1.87 ng/ml) developed ovarian cysts. Ali observed cysts were classified as follicular. In the control sheep, instead, l to 3 corpora lutea were found on the ovaries. However, no cysts were observed in this group. The obtained results show that in sheep hyperleptinaemia predisposes to the development of ACTH-induced ovarian cysts. A very high positive correlation was found between the concentration of endogenous leptin during the period of ACTH administration and the frequency of ovarian cyst development In vitro, leptin in concentrations 10-8, 10-7 i 10-6 M/l stimulated GnRH-induced LH secretion and NO release, whereas in higher doses (l0-5 M/l) both the secretion of LH and NO from ovine pituitary cells decreased. However, the inhibition of NO synthesis by L-NAME eliminated the stimulatory effect of leptin on LH secretion. Leptin (10-10–10-5 M/l) caused a drop in mRNA for the both isoforms of the leptin receptor. After the incubation of cells with 10-5 M/l leptin, the quantity of mRNA for OB-Ra and OB-Rb was at the bottom limit of detection. The observed decrease in leptin receptors mRNA is the reason for the suppresion of LH secretion by a high level of leptin which in the physiological range of concentrations stimulates gonadotropin release. Similarly, ACTH (10-10–10-6 M/l) exerted an inhibitory effect on LH secretion and decreased the mRNA level, both for the short and long form of leptin receptor, simultaneously causing the loss of stimulatory action of leptin on LH. Almost a full positive correlation was found between the level of mRNA for the short form and LH secretion under the influence of ACTH (r = 0.99, after 6, 12 and 24 h of incubation with corticotropin). Simultaneously, a very high positive correlation was found between the level of mRNA for the long form and LH secretion under the same condition (r = 0.88, 0.85 and r = 0.84, respectively after 6, 12 and 24 h of incubation with ACTH). The mRNA level for OB-Rb decreased also under the influence of dexamethasone (10-10–10-7 M/l). 10-8–10-5 M/l insulin, instead, stimulated mRNA for the long form of leptin receptor expression. But insulin in concentration 10-10–10-9 M/l,similarly to oestradiol (10-10–10-6 M/l) and testosterone (10-10–10-6 M/l), had no effect on the expression level of OB-Rb. 
To sum up, the high leptin level increases the frequency of ACTH-induced follicular cysts development in sheep. Both leptin and ACTH in high concentrations lower LH secretion from ovine pituitary cells in vitro and cause a suppression of preovulatory LH surge in vivo. The drop in GnRH-induced LH secretion from pituitary cells under the influence of high concentrations of leptin and ACTH in vitro is at least partially caused by a decrease in mRNA expression for the short (OB-Ra) and long (OB-Rb) form of the leptin receptor. In the case of OB-Ra, it is related to an inability of leptin to stimulate NO synthesis, especially because NO is required for the manifestation of the stimulatory effect of leptin on LH secretion, which was demonstrated during the experiment, using the NOS inhibitor – L-NAME. 
Taking into account the obtained results, it is possible to claim that the accretion of the leptin level and a drop in the expression of the leptin receptor in pituitary cells initi­ate signalling changes that cause the inhibition of ovulation and afterwards follicular cyst development in sheep.