It is now widely accepted that the mammary gland is under interconnected hormonal and local control. Growth factors are involved in the intercellular signalling of the gland. Our aim was the detection of transforming growth factors alpha (TGF-alpha) and beta 1 (TGF-beta 1) messenger RNA during mammogenesis, lactogenesis, galactopoiesis and involution in the bovine mammary gland (total n = 27). During these stages the RNA was assessed by means of ribonuclease protection assay and reverse transcription-polymerase chain reaction (RT-PCR). To study possible influences of oestrogen, progesterone and prolactin on growth factor expression, mammary RNA was obtained from heifers after induced mammogenesis and lactogenesis, with and without additional prolactin inhibition (total n = 20). Very low levels of TGF-alpha and TGF-beta 1 expression were detected during lactogenesis and galactopoiesis, increasing levels during mammogenesis of primigravid heifers, and highest levels during mammogenesis of virgin heifers and during involution. TGF-alpha expression after induced mammogenesis was greater than after induced lactogenesis or physiological mammogenesis during pregnancy. Furthermore, TGF-alpha mRNA contents increased after prolactin inhibition. TGF-beta 1 expression was almost equal after induced mammogenesis and lactogenesis, but greater than during the physiological mammogenesis and lactogenesis. In conclusion, it can be assumed that growth promoting TGF-alpha and growth inhibiting TGF-beta 1 are co-expressed in the bovine mammary gland. Higher mRNA contents of both factors during mammogenesis and involution may indicate autocrine or paracrine functions for these growth factors during proliferation and reorganisation of the mammary tissue.
A Plath, R Einspanier, F Peters, F Sinowatz and D Schams
J. W. SIEBERS, F. PETERS, MARIA TERESA ZENZES, J. SCHMIDTKE and W. ENGEL
Ovarian tissue of prenatal, newborn, and 5-day-old rats does not specifically bind 125Ilabelled HCG. Specific binding of HCG was first observed in ovaries of 10-day-old animals and binding increased with age. These results indicate that, contrary to rat testis, the HCG receptor in the rat ovary is not present during foetal and early postnatal development. Thus, the insensitivity of the ovary to endogenous and exogenous LH or HCG during this developmental period is due to the lack of specific receptors.
F. Bollengier, B. Velkeniers, E. Hooghe-Peters, A. Mahler and L. Vanhaelst
Prolactin and GH cells from rat pituitary glands were separated into three main fractions on discontinuous Percoll gradient layers. SDS-PAGE and subsequent immunoblotting of these fractions revealed that: (1) multiple rat prolactin (rPRL) molecular variants were present in total culture, Percoll layer 1 and 2; four variants were clear-cut: M r ∼23 000, M r doublet ∼25 000–26 000, M r ∼40 000 and M r ∼42 000; (2) cell cytosol from Percoll gradient layer 1 was particularly enriched in prolactin; (3) cells from gradient layer 1 secreted into the culture medium only prolactin in detectable amounts; (4) three distinct molecular forms of rat growth hormone (rGH) were recorded in layer 3: M r ∼36 000,24 000 and 20 000; the 20 000 variant was paramount; and (5) cells from layer 3 secreted both rPRL and rGH into the culture medium.
Reduction experiments showed that, on the one hand, 42 000 and 40 000 rPRL variants and, on the other hand, 36 000 rGH variants are disulphide-bridged dimers. An important finding was the presence of glycosylated rPRL and rGH: indeed Concanavalin A–Sepharose 4B affinity chromatography indicated that 26 000 rPRL and 24 000 rGH display a very strong affinity for lectin. Competitive inhibition tests showed that this affinity is specific and not due to hydrophobic binding. When rPRL was submitted to deglycosylation in conditions specific for O-linked glycoproteins, the 26 000 rPRL variant disappeared. The biological role of glycosylated rPRL is as yet unknown.
Journal of Endocrinology (1989) 120, 201–206
B M Lewis, I S Ismail, B Issa, J R Peters and M F Scanlon
We have studied the effects of glucose on the release of somatostatin (SS), TRH and GHRH from incubated hypothalami of normal and genetically diabetic, Goto-Kakizaki (GK) rats. The active isomer d-glucose caused a dose-related inhibition of SS, TRH and GHRH from normal rat hypothalami over a 20-min incubation period in vitro. In contrast, in GK rats the effects of glucose on TRH and SS were significantly reduced and the effects on GHRH were abolished. These data indicate that the sensitivity of SS-, TRH- and GHRH-producing hypothalamic neurones is reduced in diabetic rats. The effect is most pronounced for GHRH release as there was no change in the release of this peptide with increasing glucose concentrations. In conclusion, it appears that the diabetic state in GK rats causes differential desensitisation (GHRH>TRH and SS) of neuronal responses to subsequent changes in glucose concentrations in vitro. This may be due to alterations in the neurotransmitter control and/or a reduction in number, affinity or function of glucose transporters on these peptidergic neurones or other intermediary neuronal pathways.
Journal of Endocrinology (1996) 151, 13–17
C. A. Edwards, C. Dieguez, J. Ham, J. R. Peters and M. F. Scanlon
Recent data suggest that the response of GH to GH-releasing factor (GRF) is reduced following prior exposure to high concentrations of GRF. However, it is unknown whether this is due to alterations in GRF receptors, adenylate cyclase activity or the size of a GRF-releasable storage pool of GH. In order to clarify these questions we have compared the effects of pretreatment with GRF (10 nmol/l every 2 h for 12 h) with those of pretreatment with somatostatin (SRIF; 1 μmol/l), forskolin (10 μmol/l) and GRF plus SRIF (10 nmol/l and 1 μmol/l added together) on the subsequent responses of GH to GRF (1 pmol/l–10 nmol/l), cholera toxin (10 nmol/l), 3-isobutyl-1-methylxanthine (IBMX) (100 μmol/l) and forskolin (10 μmol/l). Experiments were performed on 4-day monolayer cultures of rat anterior pituitary cells. The cells were pretreated with test substances every 2 h for 12 h and incubated with GRF or forskolin for 3 h.
Per cent maximal (Bmax) GH responses to GRF (10 nmol/l) were reduced after pretreatment with both GRF (control, 173% of basal; GRF, 25% of basal; P < 0·001) and forskolin (98% of basal; P < 0·001), but were increased after pretreatment with SRIF (246% of basal; P < 0·02). However, GH responses after pretreatment with GRF plus SRIF were not significantly different from those of the control. The dose giving 50% maximum stimulation (ED50) of GRF action was unaltered by SRIF pretreatment (control 48·5 ± 9·2 (s.e.m.) pmol/l vs 140 ± 38 pmol/l; P > 0·05) or by forskolin pretreatment (140 ± 70 pmol/l; P > 0·05), but was increased by GRF pretreatment (900 ± 230 pmol/l; P < 0·05) and GRF plus SRIF pretreatment (1010 ± 730 pmol/l; P < 0·01). Growth hormone responses to forskolin (32% of basal after GRF pretreatment vs 121% of basal in the control saline-treated cells; P < 0·01), cholera toxin (40% of basal after GRF pretreatment vs 106% of basal in the control cells; P < 0·025) and IBMX (195% of basal in the GRF-pretreated cells vs 133% of basal in the control cells; P < 0·025) were also decreased after GRF pretreatment in comparison with controls. Responses of cAMP to 10 μmol forskolin/l were not different from controls after pretreatment with either GRF, SRIF or GRF plus SRIF. However, cAMP responses to 10 nmol GRF/l were reduced after pretreatment with GRF, SRIF, GRF plus SRIF or forskolin.
Reduction of Bmax GH responses to GRF, cholera toxin, IBMX and forskolin by GRF pretreatment, and the reversal of this action by combined SRIF plus GRF pretreatment, suggests that depletion of the GRF-sensitive releasable pool of GH may contribute to the desensitization phenomenon. In addition, however, the increase in ED50 of GRF following GRF or GRF plus SRIF pretreatment, as compared with control or SRIF pretreatment alone, together with decreased cAMP responses to GRF after GRF plus SRIF pretreatment, supports the view that uncoupling of the regulatory protein of adenylate cyclase, Ns, also plays a role in this phenomenon.
J. Endocr. (1988) 116, 185–190
E. MULDER, M. J. PETERS, W. M. O. VAN BEURDEN, M. GALDIERI, F. F. G. ROMMERTS, F. H. A. JANSZEN and H. J. VAN DER MOLEN
Department of Biochemistry (Division of Chemical Endocrinology), Medical Faculty, Erasmus University Rotterdam, Rotterdam, The Netherhnds
(Received 31 March 1976)
Studies on steroid hormone receptors in testicular tissue have previously revealed the presence of specific oestradiol receptors in interstitial tissue (Brinkmann, Mulder, Lamers-Stahlhofen, Mechielsen & van der Molen, 1972) and androgen receptors in tubular tissue (Hansson, McLean, Smith, Tindall, Weddington, Nayfeh, French & Ritzen, 1974; Mulder, Peters, van Beurden & van der Molen, 1974).
Recently methods for the preparation of isolated Leydig cells (Janszen, Cooke, van Driel & van der Molen, 1976) and Sertoli cells (Fritz, Rommerts, Louis & Dorrington, 1976) have become available. In the present study, binding of testosterone and oestradiol was investigated in such Leydig cell and Sertoli cell preparations.
Leydig cells were prepared, as described by Janszen et al. (1976), from adult rats 8 days after hypophysectomy. Sertoli cells were prepared, according to Fritz et al. (1976),