The aim of our in vitro experiments with isolated porcine ovarian follicles was to study the effects of gonadotropins, GH, IGF-I and oxytocin (OT) on release of ovarian steroid, OT, IGF-I, insulin-like growth factor-binding protein-3 (IGFBP-3), prostaglandin F (PGF), prostaglandin E (PGE) and cAMP. It was found that quarters of ovarian follicles cultured for 8 days produced significant amounts of progesterone, estradiol-17 beta, OT and IGFBP-3 with peaks of accumulation from the 3rd to the 8th day of culture. Addition of serum promoted progesterone, estradiol and OT release, whilst accumulation of IGFBP-3 was maintained to a greater extent in serum-free medium. GH (10 ng/ml or above) was able to inhibit androstenedione, OT, PGF and IGFBP-3, to stimulate IGF-I and cAMP, and to alter testosterone and PGE release by follicles cultured in serum-supplemented and/or serum-free medium. IGF-I (10 ng/ml or more) inhibited androstenedione and PGF secretion, stimulated testosterone, estradiol, OT and cAMP production, but did not influence progesterone, IGFBP-3 or PGE output in these conditions. OT (100 ng/ml) was able to inhibit androstenedione and to stimulate testosterone, IGF-I, PGF and PGE, but not estradiol or IGFBP-3 release. A stimulatory effect of LH on progesterone and OT and an inhibitory influence of LH on estradiol secretion in the serum-supplemented medium were observed. FSH in these conditions stimulated OT, but not progesterone or estradiol secretion. The use of this experimental model suggests the involvement of gonadotropins, OT, GH and IGF-I in the control of ovarian steroid and nonapeptide hormone, growth factor, growth factor-binding protein, prostaglandin and cyclic nucleotide production. The stimulatory effect of GH on IGF-I, and the stimulatory influence of IGF-I on OT, as well as coincidence of the majority of effects of IGF-I and OT, suggest the existence of a GH-IGF-I-OT axis. On the other hand, the different patterns of action of GH and IGF-I on OT, estrogen and IGFBP-3 suggest that part of the GH effect on ovarian cells is IGF-I independent.

The ability of GH to decrease fatness and insulin-regulated events such as lipogenic enzyme activities is well known in pigs. Nevertheless, the precise mechanism underlying these actions has not been elucidated yet. Expression of the transcription factor sterol regulatory element binding protein (SREBP)-1 has been reported as a key mediator of insulin action in rat hepatocytes and adipose cell lines. The present study aimed to determine whether the regulation of lipogenesis by GH and/or insulin in porcine adipocytes also involved SREBP-1. Isolated adipocytes, obtained from perirenal or s.c. adipose tissue samples of female pigs (51+/-0.4 kg; n=17), were cultured in serum-free medium in the absence or presence of these hormones for up to 4 days. Glucose incorporation and fatty acid synthase activity were increased by insulin in a dose-dependent manner in adipocytes of both sites. The increase was maximal at 1.7 and 17 nM in s.c. and perirenal adipocytes respectively, suggesting inter-depot differences in the regulation of lipogenesis by insulin. These insulin-stimulated events were decreased by GH (1 nM). No change in SREBP-1 mRNA levels was observed in response to GH and/or insulin. Taken together, these data indicate that the regulation of lipogenesis by insulin and GH appears to not involve changes in SREBP-1 mRNA levels in porcine adipocytes.

Abstract

We have examined the efficacy of various methods for reducing the interference of insulin-like growth factor-binding proteins (IGFBPs) with insulin-like growth factor-I (IGF-I) radioimmunoassays (RIAs) run on porcine sera. Acid–ethanol (AE) extraction, AE extraction followed by cryoprecipitation, glycyl–glycine (GG) extraction, GG extraction followed by Sephadex G-50 chromatography in 1 mol acetic acid/l (GG/G-50), and Sep-Pak chromatography were analysed. To provide a range of IGF-I and IGFBP levels, sera obtained from control, hypophysectomized, diabetic and somatotrophin-treated pigs were used. Recoveries of IGF-I added to sera prior to treatments other than Sep-Pak chromatography ranged from 85 to 105% and were not significantly different. In contrast, Sep-Pak chromatography gave extremely variable recoveries. ¹²⁵I-Labelled IGF-I ligand blotting showed that GG extraction followed by acid G-50 chromatography was by far the most effective method of removing or inactivating IGFBPs in porcine sera. Consequently, this procedure was used as a standard against which to compare other extraction procedures. GG extraction alone removed or inactivated low molecular weight binding proteins but appeared to have little effect on IGFBP-3. AE extraction reduced the level of IGFBP-3 but had little effect on lower molecular weight binding proteins. Even though none of the tested procedures completely removed or inactivated the binding proteins, all samples yielded IGF-I displacement curves that were parallel to that obtained for IGF-I standard. Despite yielding parallel displacement curves, sera extracted by various methods gave dramatically different apparent IGF-I levels when subjected to IGF-I RIA. IGF-I RIA of GG extracted sera yielded IGF-I values that were closest to those obtained for identical serum samples subjected to glycyl-glycine extraction followed by G-50 chromatography. For sera from control, hypophysectomized, diabetic and somatotrophin-treated pigs, the relationship of the IGF-I level in GG-extracted sera to that in GG-extracted, acid G-50 chromatographed (GG/G-50) sera was √GG=1·13√GG/G-50−0·23 (r ²=0·98). Consequently, GG extraction can be used to remove IGFBP interference with IGF-I RIAs of porcine sera from normal, hypophysectomized, diabetic and somatotrophin-treated animals.

Journal of Endocrinology (1994) 140, 229–237

ABSTRACT

The effect of transforming growth factor-β (TGF-β) on relaxin release by porcine large luteal cells (LLC) was examined by use of a reverse haemolytic plaque assay. In this assay, mixed luteal cells were co-cultured in monolayers with protein A-coupled sheep erythrocytes. In the presence of complement and porcine relaxin antiserum, a zone of haemolysis (a plaque) developed around relaxin-releasing LLCs. The rate of plaque development in time-course experiments and the average size of plaque areas were used to monitor the rate of relaxin release and cumulative amounts of hormone respectively. Monolayers were bathed in medium containing TGF-β alone, or in the co-presence of a stimulatory secretagogue (prostaglandin E₂; PGE₂).

Exposure of luteal cell-containing monolayers to TGF-β (1 ng/1–100 μg/l) elicited a dose-related inhibition in the rate of basal relaxin release. Minimal and maximal concentrations were approximately 10 ng/l and 10 μg/l respectively. Treatment with 1 μg TGF-β/l reduced the cumulative amount of relaxin released to 63 ± 6% of control values (mean ± s.d., P < 0·05, n = 6; averaged over the whole course of the experimental incubation). Exposure of monolayers treated with TGF-β to the relaxin-stimulatory secretagogue PGE₂ (0·1 μmol) resulted in a significant (P < 0·05) increase in the amount of relaxin released by TGF-β-suppressed LLCs, and restored rates of hormone release to control levels. This is evidence that TGF-β and PGE₂ interact antagonistically in the modulation of relaxin. The effect of TGF-β was strictly time-dependent. A period of at least 16 h of treatment with TGF-β was required to induce an inhibitory effect on relaxin.

These results implicate TGF-β as a novel inhibitor of relaxin release, and the presence of TGF-β in porcine luteal tissue suggests that such regulation may be achieved via paracrine or autocrine routes. It is possible that TGF-β interacts functionally during luteal life with other secretagogues to achieve integrated control of hormone release.

Journal of Endocrinology (1992) 135, 543–550

ABSTRACT

4-Oestren-3,17-dione (19-norandrostenedione) has been identified as a major steroid in porcine ovarian follicular fluid for the first time. This steroid was isolated by C₁₈ reverse-phase high performance liquid chromatography (HPLC) and its structure was established unambiguously by capillary gas chromatography–mass spectrometry. The mean concentration in pooled fluid obtained from prepubertal gilts determined by HPLC and capillary gas chromatography was 1·71 μmol/l. The level of this steroid in fluid obtained from large preovulatory follicles of sows exceeded 21·0 μmol/l on days 19 and 20 of the oestrous cycle. The high levels of this steroid within the follicle during the follicular phase might imply a possible biological role in follicular development and oocyte maturation.

J. Endocr. (1985) 107, 375–381

ABSTRACT

The incubation of female rat adenohypophysial cells in primary culture with porcine granulosa cell culture medium (GCM) led to the complete inhibition of responses of LH and FSH to LH releasing hormone (LHRH) as well as to the inhibition of spontaneous release of FSH. These effects of GCM suggest the specificity of the 'inhibin'-like activity of this material. Granulosa cell culture medium completely reversed the stimulatory effect of oestradiol-17β on the responses of LH and FSH to LHRH, as well as reversing the stimulatory effect of progesterone, oestradiol or a combination of both steroids on the spontaneous release of FSH, while not affecting the spontaneous release of LH. The antioestrogenic effects of progesterone observed on the response of LH to 0·3 nm-LHRH were amplified in the presence of GCM while the stimulatory effects of progesterone, oestradiol or both on the response of FSH to 0·3 nm-LHRH were completely reversed by the medium. Moreover, the presence of GCM led to an additive inhibitory effect with dihydrotestosterone on the response of LH to LHRH while it completely reversed the stimulatory effect of the androgen on spontaneous and LHRH-induced FSH release. The present data show that the presence of porcine granulosa cell 'inhibin' activity can exert marked interactions with sex steroids in the control of gonadotrophin secretion. This 'inhibin' activity reversed all the stimulatory effects and potentiated all the inhibitory effects of sex steroids on gonadotrophin secretion. Although the physiological role of 'inhibin' remains to be defined well, the importance of this activity is clearly demonstrated in anterior pituitary cells in culture.

J. Endocr. (1984) 100, 133–140

ABSTRACT

The regulation of thyroid follicular cell growth in vitro involves autocrine or paracrine actions of insulin-like growth factor-I (IGF-I), which are partially suppressed by transforming growth factor-β (TGF-β). Using subconfluent monolayers of porcine thyroid follicular cells, the aims of this study were to establish whether the actions of TGF-β involve changes in the synthesis of, or response to, IGF-I. We also investigated the extent to which inhibitory actions of iodide on IGF-I-dependent proliferation of thyroid follicular cells may be attributable to the production of TGF-β by follicular cells, as opposed to iodide-mediated autoregulation events.

Exposure of porcine thyroid follicular cells in subconfluent monolayer culture to TGF-β over a 7-day period reduced both IGF-I release and the incorporation of [met hyl-³H]thymidine into trichloroacetic acidprecipitable cellular material, while preincubation of cells with NaI (0 ·1 mmol/l) for 24 h prior to the addition of TSH reduced the stimulatory effect of the latter on IGF-I release over the following 7 days. Preincubation of cells with iodide also reduced basal (i.e. autonomous) [methyl-³H]thymidine incorporation. This effect was partially reversed when, following initial exposure to follicular cells, iodide-containing preincubation medium was immunoadsorbed with a neutralizing TGF-β antiserum, and subsequently readded to the cells. Furthermore, similar immunoadsorption of iodide-free preincubation medium resulted in an enhancement of the control level of [methyl-³H]thymidine incorporation when the treated medium was returned to the original cultures. The results of this study are consistent with the hypothesis that IGF-I and TGF-β are both produced by subconfluent thyroid follicular cells in vitro, and that the inhibitory action of TGF-β on follicular cell growth may involve a decrease in the thyroidal production of IGF-I. While the attenuating action of iodide on follicular cell proliferation may, in part, reflect an increased autocrine production of TGF-β, and a reduction by TGF-β of the growth response to IGF-I, these studies also provide evidence that the intrathyroidal actions of TGF-β include an attenuation of IGF-I biosynthesis.

Journal of Endocrinology (1991) 130, 3–9

ABSTRACT

An effort was made to identify the antigenic epitope(s) of porcine GH (pGH) capable of inducing antibodies which would enhance the growth-promoting activity of the hormone. Several peptide sequences of the pGH molecule were synthesized and the antibodies to these peptides were generated in rabbits. The majority of these antibodies were found to be immunoreactive with both intact pGH and their respective peptide antigens. The biological activity of the antibodies was tested in growth-retarded hypophysectomized rats. Daily injections with pGH alone significantly increased their growth rate and treatment with a combination of pGH and antibody specific to the peptide sequence 110–118 further heightened the somatogenic effect. Administration of this antibody alone did not enhance the growth rate of hypophysectomized rats, neither did antibodies raised to intact pGH or the other peptide sequences. The present findings suggest that antibody with a particular pGH epitope specificity is able to increase the somatogenic activity of pGH and that the peptide corresponding to pGH amino acids 110–118 may prove useful in the development of growth promoting vaccines.

Journal of Endocrinology (1990) 127, 481–485

ABSTRACT

Thyrotrophin (4-256 μU/ml) promoted an increase in the rate of release of radioiodine from the organic iodine pool of cultured porcine thyroid cells in follicular formations. This action of TSH was antagonized by low concentrations of epidermal growth factor (EGF; 0·1–5 nmol/l). The maximal effect of EGF was reached by 0·5 nmol/l. EGF (0·5–5 nmol/l) also inhibited the stimulatory effect of 8-chloro cyclic AMP (0·06–1·0 nmol/l) on radioiodine turnover. Exposure of thyroid cultures to media with a calcium concentration of 17·7 μmol/l (1% of normal) resulted in a very marked increase in the rate of release of radioiodine. The effect of TSH in low-calcium media was to inhibit the increased release of radioiodine, and EGF (0·5 nmol/l) antagonized this inhibitory effect of TSH. The calcium ionophore, A23187, stimulated radioiodine release in a dose-dependent fashion, and EGF (1·7 nmol/l) inhibited this response. Fluid transport in thyroid monolayers was stimulated by prostaglandin E₂ (PGE₂; 1 μmol/l). EGF (5 nmol/l) also stimulated fluid transport, but antagonized the effect of PGE₂ added subsequently. It was concluded that EGF exerted acute antagonistic effects on thyroid cell responses in vitro to cyclic AMP and agents promoting accumulation of cyclic AMP in time-frames too short for these inhibitory effects to be attributable to the dedifferentiative effect of the growth factor.

Journal of Endocrinology (1991) 128, 213–218

The present study was undertaken to determine the effect of GH administration on GH and IGF-I receptors in skeletal muscle compared with liver in growing pigs. Plasma IGF-I and GH-binding protein (GHBP) levels were also determined. Twelve Large White pigs (castrated males) were treated daily with 100 micrograms pituitary porcine GH (pGH) per kg body weight or vehicle for 41 days intramuscularly. Relative to controls, pGH administration increased plasma IGF-I concentrations by 3.3-fold. Administration of pGH had no effect on plasma GHBP levels. In liver, 125I-labelled bovine GH (bGH)-specific binding (P < 0.05) and GH receptor (GHR) mRNA levels (P < 0.05) were higher in pGH-treated than in control pigs. In longissimus dorsi (LD), 125I-labelled bGH specific binding did not differ significantly between the two groups while GHR mRNA levels (P < 0.05) were lower in pGH-treated than in control pigs. Administration of pGH had no effect on 125I-labelled bGH-specific binding and GHR mRNA levels in trapezius (TR). 125I-Labelled IGF-I-specific binding in liver was unaffected by pGH administration. Similarly, in liver, LD and TR, IGF-I receptor mRNA levels were not different between pGH-treated and control animals. It can be concluded that (1) GH binding and IGF-I receptor mRNA are not affected by GH in skeletal muscle, (2) GH influences GHR in a tissue-specific manner and (3) hepatic GHR and GHBP levels are not co-regulated.