Authors:Anna Ptak, Ewa L. Gregoraszczuk and J. Rząsa
This study was conducted to investigate the interactions between growth hormone (GH) and insulin-like growth factor-I (IGF-I) on progesterone (P4) secretion by porcine luteal cells cultured in vitro. Cells isolated from corpora lutea (CL) collected at three different periods of the luteal phase (CL1 - early luteal phase; CL2 - middle luteal phase and CL3 - late luteal phase) were incubated with different doses of GH (10, 100 or 200 ng/ml). After 48 h cultures were terminated and the media were frozen until further P4 concentration analysis. GH (100 ng/ml) increased P4 secretion by CL1 and CL2 and had no effect on CL3. In separate studies these cells were treated for 48 h with IGF-I alone or with GH combined with IGF-I. IGF-I alone increased basal P4 secretion only by cells collected from CL1 while concurrent treatment with GH had no effect on P4 secretion by any type of CL. To investigate the possible mechanism of GH and IGF-I mediated induction of P4 secretion, an inhibitory study was conducted. In this experiment, luteal cells collected from CL1 were cultured in the absence or presence of cycloheximide (an inhibitor of protein synthesis) or actinomycin D (an inhibitor of DNA transcription). Cycloheximide or actinomycin D completely blocked the stimulatory effect of both GH and IGF-I on P4 production but did not reduce basal progesterone secretion suggesting involvement of gene transcription and translation in the GH and IGF-I action on luteal cells. Additionally, the activity of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) under the influence of GH added alone or together with IGF was measured by the conversion of pregnenolone to progesterone. Stimulation of P4 secretion in P5-treated cells in GH-stimulated cultures was not observed, however, high stimulatory effect was noted in IGF-I treated cultures. In conclusion, the present studies indicate that there is direct and cycle stage dependent influence of GH and IGF-I on steroidogenesis in porcine luteal cells. It is suggested that both IGF and GH may exert some regulatory action during CL development in the pig.
Authors:Ewa L. Gregoraszczuk, Agnieszka Rak, Anna Wójtowicz, Anna Ptak, Tatiana Wojciechowicz and K. W. Nowak
Leptin (L) is recognised as an important regulator of puberty and a factor which controls reproduction. Whole pig ovarian follicles were incubated with different doses of leptin (2, 20 and 200 ng/ml) added alone or in combination with 100 ng/ml of GH or 50 ng/ml of IGF-I. The expression of the functional long form leptin receptor (Ob-Rb) mRNA was examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in follicular cells cultured with GH or IGF-I. Both GH and IGF-I increased leptin receptor expression in prepubertal pig ovaries. In separate experiments, the action of leptin on ovarian follicular steroidogenesis and cell apoptosis was examined. After 24 h of incubation with leptin alone or in combination with GH or IGF-I, oestradiol (E2) levels were determined in the culture medium while follicular tissue was used for the estimation of caspase-3 activity. Leptin increased E2 secretion and significantly diminished caspase-3 activity at all doses used. Both GH and IGF-I stimulated oestradiol secretion and decreased caspase-3 activity. No differences were demonstrable in oestradiol secretion and caspase-3 activity between cells treated with GH plus leptin and GH alone or cells treated with IGF-I plus leptin as compared to cultures treated with GH or IGF-I alone. However, GH diminished leptin-stimulated oestradiol secretion while IGF-I was without effect on it. Both GH and IGF-I reversed the anti-apoptotic action of leptin. In conclusion, we infer that (1) leptin directly affects ovarian function in prepubertal animals by its action on oestradiol secretion and cell apoptosis, (2) GH and IGF-I modulate the action of leptin, and (3) at least in part, the direct effect of GH/IGF-I on leptin production is due to an action on leptin receptor expression.