Krzysztof Darlak

Effects of chronic food restriction and treatments with leptin or ghrelin on different reproductive parameters of male rats

The existence of a close relationship between energy status and reproductive function is well-documented, especially in females, but its underlying mechanisms remain to be fully unfolded. This study aimed to examine the effects of restriction of daily calorie intake, as well as chronic treatments with the metabolic hormones leptin and ghrelin, on the secretion of different reproductive hormones, namely pituitary gonadotropins and prolactin, as well as testosterone, in male rats. Restriction (50%) in daily food intake for 20 days significantly reduced body weight as well as plasma PRL and T levels, without affecting basal LH and FSH concentrations and testicular weight. Chronic administration of leptin to rats fed ad libitum increased plasma PRL levels and decreased circulating T, while it did not alter other hormonal parameters under analysis. In contrast, in rats subjected to 50% calorie restriction, leptin administration increased plasma T levels and reduced testis weight. Conversely, ghrelin failed to induce major hormonal changes but tended to increase testicular weight in fed animals, while repeated ghrelin injections in food-restricted males dramatically decreased plasma LH and T concentrations and reduced testis weight. In sum, we document herein the isolated and combined effects of metabolic stress (50% food restriction) and leptin or ghrelin treatments on several reproductive hormones in adult male rats. Overall, our results further stress the impact and complex way of action of different metabolic cues, such as energy status and key hormones, in reproductive function also in the male.

Role of ghrelin in regulating rabbit ovarian function and the response to LH and IGF-I.

The aim of these in vivo and in vitro studies was to examine the role of ghrelin in the control of plasma hormone concentrations, the proliferation, apoptosis and secretory activity of ovarian granulosa cells and the response of these cells to hormonal treatments. Female rabbits were injected with ghrelin (10 microg/animal/day for one week before ovulation induced by 25IU PMSG and 0.25IU LHRH). On the day of ovulation, blood samples were collected and analyzed for concentrations of progesterone (P(4)), testosterone (T), estradiol (E(2)), estrone-sulphate (ES), insulin-like growth factor I (IGF-I) and leptin (L) by RIA. Some control and ghrelin-treated animals were killed in the periovulatory period, their ovaries were weighed and granulosa cells were isolated and cultured for 2d. Cell proliferation (expression of PCNA) and apoptosis (expression of TdT) were evaluated by immunocytochemistry and TUNEL respectively. Secretion of P(4), T, E(2), IGF-I, and prostaglandin F (PGF) by granulosa cells cultured with and without LH or IGF-I (1, 10 or 100 ng/ml medium) was assessed by RIA. The remaining control and treated animals were kept until parturition, while the number, viability and body weight of pups were recorded. Ghrelin treatment increased rabbit plasma T and decreased ES concentrations but did not influence P(4), E(2), IGF-I or L. Granulosa cells from ghrelin-treated animals showed higher expression of PCNA and lower expression of TdT, than those from control animals. They also secreted less P(4), T, E(2), IGF-I and PGF than granulosa cells from untreated animals. Treatment of cultured granulosa cells with ghrelin (1, 10 or 100 ng/ml medium) either increased (at 1 ng/ml) or decreased (at 10 ng/ml) P(4) secretion, increased (at 100 ng/ml) or decreased (at 10 ng/ml) IGF-I secretion, decreased T (at 1 and 10 ng/ml) and OT (at 1 ng/ml) secretion, and increased (at 100 ng/ml) PGF secretion. LH treatment of cells from control animals stimulated P(4) (at 1 and 10 ng/ml), E(2), and IGF-I (both at 10 and 100 ng/ml), but not T secretion. IGF-I stimulated P(4) (all concentrations) and PGF (at 100 ng/ml) but suppressed T (all concentrations) and E(2) (at 1 and 10 ng/ml) secretion. Pre-treatment of animals with ghrelin stimulated, suppressed or even reversed subsequent LH and IGF-I effects on hormone secretion by cultured granulosa cells. Ghrelin injections did not affect ovarian weight or the number and body mass of pups born, although pup mortality was significantly lower in ghrelin-treated than in control mothers. These observations suggest that ghrelin is involved in the control of ovarian cell proliferation, apoptosis and secretion of hormones, as well as in the response of these cells to physiological stimulators such as LH and IGF-I.

Structure-activity studies of dermorphin. The role of side chains of amino acid residues on the biological activity of dermorphin

Seventeen analogues of dermorphin were synthesized and bio-assayed to determine the influence of side chains of the individual amino acid residues forming the sequence of dermorphin on the biological activity of this opioid peptide. Syntheses were carried out using solid-phase procedure, and the analogues obtained were purified by gel filtration on Sephadex G-10. Biological activities determined in guinea pig ileum (GPI) and mouse vas deferens (MVD) tests showed that the N-terminal tetrapeptide is responsible for the activity of dermorphin. Substitutions in the C-terminal fragment, particularly in position 5, for other amino acid residues results in substantial differentiation towards mu and delta receptors.

Dermorphin analogs: Resistance to in vitro enzymatic degradation is not always increased by additional D-amino acid substitutions

Dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and seven analogs were examined for their biostability towards rat brain homogenate; half-lives for the parent were 127 min and 48 min, respectively, with Gly4-Tyr5 cleavage confirmed by collection and identification of the N-terminal fragment. Surprisingly, several analogs with additional D-amino acid substitutions were cleaved more rapidly than the parent, suggesting the importance of remote secondary structural features for differential enzyme susceptibility.

Involvement of the transcription factor STAT1 in the regulation of porcine ovarian granulosa cell functions treated and not treated with ghrelin

The aim of our in vitro experiments was to study the role of the transcription factor STAT1 and the hormone ghrelin in controlling porcine ovarian function. The effects of treatment with ghrelin (0, 1, 10, 100 ng/ml), transfection-induced overexpression of transcription factor STAT1, and their combination on apoptosis (expression of apoptosis-related peptides caspase-3, BAX and anti-apoptotic peptide BCL2), proliferation (expression of proliferating cell nuclear antigene PCNA, proliferation-associated protein kinase MAPK/ERK1,2) and release of the hormones progesterone (P(4)), prostaglandin F (PGF) and oxytocin (OXT) in cultured porcine ovarian granulosa cells was evaluated using RIA, immunocytochemistry and SDS-PAGE-western immunoblotting. It was found that ghrelin, when given alone, increased the expression of proliferation-associated PCNA and MAPK/ERK1,2, decreased the accumulation of apoptosis-related substances caspase-3, BAX, BCL2, decreased P(4), and increased PGF and OXT release. Ghrelin tended to promote accumulation of STAT1 in both control and transfected cells, although in transfected cells ghrelin at 1 ng/ml decreased STAT1 accumulation. Transfection of porcine granulosa cells by a gene construct encoding STAT1 promoted the expression of STAT1 and apoptosis-related-BAX but the expression of BCL2 did not, and decreased the accumulation of proliferation-associated MAPK/ERK1,2 but not that of PCNA. It also promoted PGF and OXT but not P(4) release. Overexpression of STAT1 reversed the effect of ghrelin on STAT1, PCNA, PGF, OXT (from stimulatory to inhibitory), BCL2, P(4) (from inhibitory to stimulatory), prevented ghrelin effect on caspase-3 and BAX, but did not affect ghrelins effect on MAPK/ERK1,2 expression. These results suggest that ghrelin directly affects porcine ovarian cells function - stimulates proliferation, inhibits apoptosis and affects secretory activity. Furthermore, they demonstrated the involvement of the transcription factor STAT1 in controlling these functions, the promotion of some markers of apoptosis (BAX), inhibition of some markers of proliferation (MAPK/ERK1,2) and stimulation of PGF release. Finally, the obtained data failed to demonstrate that STAT1 is involved in mediating the action of ghrelin on ovarian cell functions.