Assessment of the mechanism by which prolactin stimulates progesterone production by early corpora lutea of pigs

Abstract

Previously, we reported that administration of prolactin (PRL) during the early luteal phase in sows increases plasma progesterone concentrations. In the current study, we searched for the mechanisms by which PRL exerts this luteotrophic effect. The objectives of the study were (1) to examine the effect of PRL and/or low-density lipoproteins (LDL) on progesterone production by porcine luteal cells derived from early corpora lutea, and (2) to assess the ability of PRL to activate phosphoinositide-specific phospholipase C (PI-PLC) and protein kinase C (PKC) in these luteal cells. Ovaries with early corpora lutea (day 1-2 of the oestrous cycle) were obtained from the slaughterhouse. Progesterone production by dispersed luteal cells was measured after treatment with PRL, phorbol 12-myristate 13-acetate or inhibitors of PKC in the presence or absence of LDL. LDL increased progesterone concentration in the incubation medium (304·5 vs 178·6 ng/ml in control, P<0·05). PRL augmented LDL-stimulated progesterone secretion by luteal cells (to 416 ng/ml, P<0·05), but PRL alone did not affect progesterone production (209·6 ng/ml, P>0·05). Staurosporine, a PKC inhibitor, inhibited progesterone secretion stimulated by the combined action of LDL and PRL; however, such inhibition was not demonstrated when cells were treated with the PKC inhibitor, H-7. PKC activation was assessed by measuring the specific association of [ 3 H]phorbol dibutyrate ( 3 H-PDBu) with luteal cells after treatment with PRL or ionomycin (a positive control). PRL and ionomycin increased 3 H-PDBu-specific binding in early luteal cells by 28 5·5% (within 5 min) and 70·2 19·3% (within 2 min) over control binding respectively (P<0·05). In addition, PRL did not augment the LDLstimulated progesterone production in PKC-deficient cells. In contrast with PKC, total inositol phosphate accumulation, as well as intracellular free calcium concentrations, were not affected by PRL in the current study. We conclude that PRL, in the presence of LDL, stimulates progesterone production by early corpora lutea in vitro.Moreover, PRL appears to activate PKC, but not PI-PLC, in these cells. Thus intracellular transduction of the PRL signal may involve activation of PKC that is not dependent on PI-PLC.

Effect of neonatal or adult heat acclimation on plasma fT3 level, testicular thyroid receptors expression in male rats and testicular steroidogenesis in vitro in response to triiodothyronine treatment

“…The assay sensitivity was 0.3 nmol/l and intra-assay variation coefficient was 5%. Concentrations of A4, T and E2 in media were measured by RIA (Ciereszko et al 1998, Szafranska et al 2002. The intra-and inter-assay coefficients of variance were as follows: for A4 -2% and 13.3%, respectively; for T -2.1% and 5.1%, respectively; and for E2 -1.4% and 10.6%, respectively.…”

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters steroid secretion but does not affect cell viability and the incidence of apoptosis in porcine luteinised granulosa cells

“…The medium concentration of steroid hormones was measured by a 3 H-radioimmunoassay (RIA). The validation of this assay for use in experiments with porcine ovarian cells has been reported previously (Ciereszko et al, 1998). Specificity of the anti-progesterone (SO/91/4) and anti-oestradiol antibodies (BS/88/733) is described elsewhere (Ciereszko et al, 2001;Szafrań-ska et al, 2002).…”

The effect of the estrous cycle on the expression of prepro-orexin gene and protein and the levels of orexin A and B in the porcine pituitary

“…Measurement of plasma progesterone level To confirm correctness of the evaluation of the estrous cycle phase, the level of progesterone (P 4 ) was determined according to the method described by Ciereszko et al (1998). Validity of the assay was confirmed by parallelism between the standard curves and a series of dilutions of the samples.…”