The role of QRFP43 in the secretory activity of the gonadotrophic axis in female sheep.

In mammals reproduction is regulated by many factors, among others by the peptides belonging to the RFamide peptide family. However, the knowledge concerning on the impact of recently identified member of this family (QRFP43) on the modulation of the gonadotrophic axis activity is still not fully understood and current research results are ambiguous. In the present study we tested the in vivo effect of QRFP43 on the secretory activity of the gonadotrophic axis at the hypothalamic-pituitary level in Polish Merino sheep. The animals (n = 48) were randomly divided into three experimental groups: controls receiving an icv infusion of Ringer-Locke solution, group receiving icv infusion of QRFP43 at 10 µg per day and 50 µg per day. All sheep received four 50 min icv infusions at 30 min intervals, on each of three consecutive days. Hypothalamic and pituitaries were collected and secured for further immunohistochemical and molecular biological analysis. In addition, during the experiment a blood samples have been collected for subsequent RIA determinations. QRFP43 was found to downregulate Kiss mRNA expression in the MBH and reduce the level of IR material in ME. This resulted in a reduction of GnRH IR material in the ME. QRFP43 increased plasma FSH levels while decreasing LH levels. Our findings indicate that QRFP43 inhibits the activity of the gonadotropic axis in the ovine at the level of the hypothalamus and may represent another neuromodulator of reproductive processes in animals.

Brain-derived neurotrophic factor (BDNF) affects the activity of the gonadotrophic axis in sheep.

This study aimed to examine the hypothesis that BDNF modulates the activity of the gonadotrophic axis in sheep. Central infusions of BDNF were administered to sexually mature Polish Merino sheep. The sheep were randomly divided into three groups: the control group received intracerebroventricular (ICV) infusions of the vehicle, the BDNF 10 group received ICV infusions of BDNF at 10 µg/480 µL/day, and the BDNF 60 group was infused with BDNF at 60 µg/480 µL/day. A series of four infusions on three consecutive days was performed. Blood samples were collected on days 0 and 3 of the infusions. Immediately after the experiment, all the sheep were slaughtered, and selected structures of the hypothalamus and pituitaries were collected for Real Time RT-qPCR analysis. The collected plasma samples, as well as parts of pituitaries were stored for radioimmunoassay analysis of LH and FSH. Central treatment with exogenous BDNF stimulated GnRH mRNA expression in the preoptic area, as well as GnRH-R mRNA in the pituitary. Furthermore, substantial changes in the KNDy mRNA expression in the mediobasal hypothalamus were observed after the ICV BDNF administration. Additionally, central BDNF infusion caused a decrease in LH concentration and a simultaneous increase in FSH concentration in peripheral blood. Neither pulse amplitude nor pulse frequency for any gonadotrophin was affected in both groups of sheep that received BDNF infusion. Our results revealed that exogenous BDNF affects GnRH and KNDy gene expression and changes in the LH and FSH pituitary cell secretory activities. These findings suggest that BDNF may participate in the mechanism modulating the activity of the gonadotrophic axis at the central level in female sheep.

Central obestatin administration affect the LH and FSH secretory activity in peripubertal sheep.

Obestatin - a 23 amino acid peptide is synthesized as another product of the ghrl gene and its synthesis occurs mainly in gastric mucosa cells. This hormone is involved in complex gut-brain neurohormonal networks, thereby can participates in the modulation of gonadotrophic axis activity. The aim of this study was to investigate the consequence of intracerebroventricular infusions of obestatin on LH and FSH pituitary cells secretory activity in peripubertal female sheep. Animals were randomly divided into two groups: the control group (n = 14) received intracerebroventricular infusions of Ringer-Lock solution (120 µL h-1), and the obestatin group (n = 14) was infused with obestatin (25 µg/120 µL h-1) diluted in Ringer-Lock solution. A series of four infusions was performed on three consecutive days. Blood samples were collected on day 0 and day 3. The sheep were slaughtered immediately after the end of the experiment. For molecular biological analysis, pituitaries from 7 sheep from each group (n = 7 + 7) were prepared and frozen in liquid nitrogen immediately after collection and then stored at -80 °C until Real Time RT-qPCR and RIA analyzes. For immunohistochemical analysis, pituitary tissues from the remaining animals (n = 7 + 7) was fixed in situ for further examination. Real-Time qPCR and immunohistochemistry analyses revealed substantial changes in the LH and FSH pituitary cells secretory activity in obestatin-infused sheep. Exogenous obestatin administration reduced LHß mRNA expression and increased the accumulation of immunoreactive LH in gonadotrophic cells of the adenohypophysis. These changes were accompanied by a decrease in the mean LH concentration in the peripheral blood resulting from the lower LH pulse amplitude. Moreover, an increase in both FSHß mRNA expression and FSH immunoreactivity and amount in pituitary cells were noted, while mean blood FSH concentration remained unchanged after obestatin treatment. The obtained results showed that exogenous obestatin affected LH secretory activity at the level of protein synthesis, accumulation and release as well as obestatin increase FSHß mRNA expression and accumulation of this hormone but at the same time have no effect on FSH release to blood. Thus, obestatin can participate in the neuroendocrine network, which modulates gonadotrophic axis activity in sheep.

Obestatin may affect the GnRH/KNDy gene network in sheep hypothalamus.

The effects of obestatin on gonadotrophic axis activity in ruminants have not yet been determined. The aim of this study was to investigate the effect of intracerebroventricular infusions of obestatin on the gonadotrophin-releasing hormone (GnRH) mRNA and protein expressions as well as on KNDy mRNA and kisspeptin (Kiss) peptide expressions in peripubertal female sheep. Animals were randomly divided into two groups: the control group received intracerebroventricular infusions of the vehicle, and the obestatin group was infused with obestatin (25 µg/120 µL h-1). The series of four 1-h infusions per day during three consecutive days were performed. After the end of the experiment parts of sheep brains were fixed in situ for immunohistochemical analysis, while the remaining brains were frozen for Real Time qPCR analysis. Substantial changes in the activity of the GnRH and KNDy gene network were observed in obestatin-infused sheep. In those animals an increase of GnRH mRNA expression in the preoptic area, a decrease of GnRH mRNA expression in the median eminence and an increase of GnRH immunoreactivity in the median eminence were found. Moreover, changes in the KNDy mRNA expression in mediobasal hypothalamus as well as decrease Kiss expression in arcuate nucleus and median eminence were observed. It was revealed that obestatin affects the GnRH and KNDy gene network as well as Kiss at the level of mRNA and protein expression. Thereby, it can be concluded that obestatin participates in the mechanism modulating gonadotrophic axis activity at the central level in peripubertal female sheep.

Obestatin stimulates the somatotrophic axis activity in sheep.

The effects of obestatin (an anorexigenic peripheral peptide) on somatotrophic axis activity in ruminants have not yet been determined. The aim of this study was to investigate the consequence of intracerebroventricular infusions of obestatin on the activity of the somatotrophic axis in peripubertal female sheep. Animals were randomly divided into two groups: control group received intracerebroventricular infusions of the vehicle, and the obestatin group was infused with obestatin (25 µg/120 µL h-1). The series of four hourly infusions on three consecutive days were performed. The blood samples were collected on day 0 and on day 3. Immediately after the end of experiment sheep were slaughtered. Parts of the brains were fixed in situ for further immunohistochemical analysis, while the remaining brains were frozen for Real Time RT-qPCR analysis. Substantial changes in the activity of the somatotrophic axis were observed in obestatin-infused sheep. In those animals obestatin evoked an increase in growth hormone-releasing hormone (GHRH) mRNA expression and a decrease in somatostatin mRNA expression in the anterior hypothalamic area. Moreover, a decrease in somatostatin immunoreactivity in the periventricular nucleus and an increase in somatostatin immunopositive fibers in the median eminence were noted. Changes in the GHRH and somatostatin activity are associated with an increase in growth hormone (GH) gene expression and in the amount of GH immunoreactive material stored in the somatotrophic pituitary cells. Consequently, an increase in GH concentration in the peripheral blood, due to an increase in the number of pulses was observed. It was revealed that obestatin affects the somatostatin/GHRH/GH system at the level of protein synthesis, accumulation and release. It is suggested that obestatin participates in the mechanism modulating somatotrophic axis activity at the central level by stimulating GH release through suppression of somatostatin output. Thereby, it can be concluded that obestatin may be involved in the modulation of growth processes in sheep.