B. Barenton

Photoperiodic control of growth hormone secretion and body weight in rams

This study was undertaken to assess the influence of photoperiod on growth hormone (GH) secretion in rams and its possible influence on body weight. Twenty young adult rams were divided into two groups. One was subjected to an annual (AR) and the other to a semestral (SR) light regime during the same 18-month period. In both groups, daylength (DL) varied gradually between 8 to 17 hr. Plasma prolactin (PRL) and GH profiles consisting of 6 hr samples were determined and animals were weighed throughout the course of the experiment. Maximal PRL secretion was observed with largest DL. In contrast, GH secretion increased during increasing DL but it began to decrease before maximal DL was reached in both light regimes. Mean GH secretion was maximal when the DL was about 11 hr in SR and between 8 to 12 hr in AR. Similarly, body weight increased when DL increased and plateaued during decreasing DL in both AR and SR animal groups. Significant (P less than 0.05) differences were observed throughout the course of the experiment according to the effects of decreasing or increasing DL in each group. Analysis of variance showed that the effect of DL on plasma PRL and GH levels and weight velocity (WV) was significant (P less than 0.05) in both light regimes. This suggests that in SR, plasma PRL and GH levels and WV vary according to a six month period.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cryptorchidism in the ram: changes in the concentrations of testosterone and estradiol and receptors for LH and FSH in the testis, and its histology

Cryptorchidism was induced in 5 pre-pubertal lambs and 7 adult rams, 5 months after surgery, testicular weight and membrane protein content were 4-fold lower than in the control. The total number of Leydig cells per testis was markedly decreased but their size was not changed. In contrast, the total number of Sertoli cells per testis was not affected but their nuclear size was smaller. Induced cryptorchidism had no effect on the length of seminiferous tubules; blood vessel volume was reduced; and the production of germ cells was completely disrupted. The number of LH receptors estimated per Leydig cell was not changed in pre-pubertal lambs but decreased 4-fold in adult rams. The number of FSH receptors calculated per Sertoli cell was reduced by 95% in both pre-pubertal and adult animals. No effect on the binding affinities of LH (Ka = 1 X 10(10) M-1) and FSH (Ka = 4.5 X 10(9) M-1) to their testicular receptors was observed. Although testicular concentrations of testosterone and estradiol-17 beta were increased, the total content of testosterone within the testis was increased only in pre-pubertal lambs. The estimated ratio of testosterone per Leydig cell was higher in cryptorchid animals than in controls, suggesting that, despite their reduction in number and the decrease of LH receptors, the Leydig cells of cryptorchid rams have an enhanced steroidogenic capacity. This study also confirms the important dysfunction of the Sertoli cells in cryptorchid rams.

Purification of chinook salmon (Oncorhynchus tshawytscha) GH for receptor study

A method for the purification of chinook Salmon (Oncorhynchus tshawytscha) GH, which retains its biological activity, is described. The biological activity was investigated with an established radioreceptor assay using liver membranes from pregnant rabbits and bovine GH as standard and labelled hormone. The enrichment of the preparation was checked with electrophoresis (SDS-PAGE). Extraction and further steps were carried out using low molarity alkaline buffer (pH 8–10, M = 100 mM). Three chromatography steps were performed (Concanavalin-A sepharose, Bio-gel P60, DEAE). Ion exchange chromatography was performed under isocratic conditions (using a 50 cm column). Two isoforms (sGH1 and sGH2) were isolated. The purification yield is 0.7% compared to lyophilized pituitaries. The molecule is homogeneous in SDS-PAGE. Contamination by prolactin, gonadotrophin and corticotrophin is negligible (< 0.5%). It could be demonstrated that the biological activity of the preparation is maintained since this preparation stimulates the growth of juvenile trout (Salmo gairdneri) and binds specifically (35%) to trout liver membranes.

Influence of photoperiod and protein diet on growth hormone secretion in rams.

Abstract Studies of sheep were undertaken to determine the effects of photoperiod and protein diet on growth hormone (GH) secretion. Rams were subjected to either a control (R1) or an inverted (R2) 6-month (semestral) light regime. In both light regimes day lengths varied gradually between 8 and 16 hr. Within each light regime group of animals, the rams received either a low (L) or a high (H) protein diet containing the same level of energy. Plasma GH profiles consisting of 13 hourly samples were determined at regular intervals corresponding to known day lengths. Analysis of variance indicated that (i) there was a significant effect of day length (P < 0.01) and protein diet (P < 0.05) on GH secretion, (ii) the two light regimes R1 and R2 were equivalent with respect to GH secretion, and (iii) there were no interactions among the three experimental factors. Although mean GH secretion was consistently higher in L groups than in H groups, there was a similar trend in all the animals of increasing GH secretion as day length was increased. GH secretion was maximum when the day length reached 13 hr 20 min in increasing photoperiods in L groups (15.6 ± 1.6 ng × h × ml-1) and 16 hr in H groups (13.0 ± 1.2 ng × h × ml-1). From these results we conclude that both an increasing day length and a deficiency in protein diet stimulate GH secretion in rams but the GH response to these two factors may involve different regulatory processes and may have different functions.

The Insulin-Like Growth Factor-II/Mannose-6-Phosphate Receptor: Structure, Function and Differential Expression

The insulin like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is a bifunc-tional binding protein that binds lysosomal enzymes bearing the M6P recognition marker and IGF-II at distinct binding sites (45, 52). In addition, transforming growth factor (TGF) beta precursor, thyroglobulin and proliferin, a protein which is expressed in rapidly proliferating cells are also recognized by this receptor (Table 1). In avian and amphibian cells the receptor lacks the binding site for IGF-II but serves as a binding protein for M6P bearing ligands (7,9,76). Almost all mammalian cells described until today express IGF-II/M6P receptors that bind both classes of ligands, namely M6P-containing glycoproteins and IGF-II (55–59).

Human IM-9 lymphoblasts as a model of the growth hormone-insulin-like growth factor axis: gene expression, and interactions of ligands with receptors and binding proteins

Human IM-9 lymphoblasts bind growth hormone (hGH) and insulin-like growth factors (IGFs). We have systematically examined the IM-9 cells as a valuable model of the interaction of hGH and the IGFs at the cellular level. Cells were cultured in medium with 10% serum and for a subset of experiments cultured in serum-free medium. Binding of [125I]hGH and [125I]IGF-I and -II to intact IM-9 cells was measured: unlabeled hGH inhibited binding of [125I]hGH (half max. 20 ng/ml). Binding of [125I]IGF-I was inhibited by IGF-I (half max. 7.5 ng/ml), IGF-II (half max. 60 ng/ml), and insulin and anti IGF-I receptor antibody (alpha IR3). [125I]IGF-II was inhibited by IGF-II (half max. 15 ng/ml), IGF-I (half max. 500 ng/ml), insulin (half max. 250 ng/ml) but not by alpha IR3. Crosslinking experiments with [125I]IGF-II and DSS as the crosslinking agent and analysis of radioligand-receptor complexes by SDS-PAGE under reducing conditions revealed that [125I]IGF-II bound to a 250 kDa and a 135 kDa receptor species. The latter possibly represents an insulin-type receptor whereas the 250 kDa species had the characteristics of the IGF-II/M6P receptor. When IM-9 cell conditioned medium was analyzed in ligand blotting experiments with either [125I]IGF-I or -II a 30 kDa IGFBP species was detected on the autoradiographs. Also, IGF-II immunoreactivity (approx. 1 ng/ml medium) was measured in the cell conditioned medium using an IGF-BP blocked RIA employing [125I]IGF-II. In a subset of experiments IM-9 cells were homogenized in 4 M guanidinium-thiocyanate and RNA extracted in 5.7 M CsCl. Denatured RNA was electrophoresed on 0.8% agarose gels and transferred to a nylon membrane, fixed and the blots hybridized with cDNA probes. Probes were labeled with [32P]dCTP using a random prime labeling procedure: a Pst I 700 bp fragment of the human IGF-I cDNA, a 554 bp Pst I-Sal I fragment of the IGF-II cDNA, a 614 bp Pst I fragment of the IGF-I receptor cDNA and a 663 bp Pst I fragment of the IGF-II/M6P receptor. Autoradiographs of Northern blots showed specific hybridization with the IGF-I probe at 3.7 kb and with the IGF-II probe at 5.3 kb. No signal was detected with the IGF-I receptor cDNA probe. Hybridization with the IGF-II/M6P receptor probe yielded a 9 kb RNA species.(ABSTRACT TRUNCATED AT 400 WORDS)

Rabbit slow and fast skeletal muscle-derived satellite myoblast phenotypes do not involve constitutive differences in the components of the insulin-like growth factor system.

The insulin‐like growth factor (IGF) system is actively involved in the control of proliferation and differentiation of several myogenic cell lines, and phenotypic differences between myoblasts are associated with modifications of the equilibrium of the components of the IGF system. To determine whether this observation is a physiologic feature that also concerns the phenotypes of ex vivo adult satellite myoblasts in primary cell culture, we investigated the IGF system in rabbit slow‐twitch muscle‐derived satellite myoblasts (SSM), which differ phenotypically from fast‐twitch muscle‐derived satellite myoblasts (FSM) by their proliferation and differentiation kinetics in vitro. The expression of IGF‐I and IGF‐II were similar in SSM and FSM as well as their concentrations measured in cell‐conditioned media. Ligand blotting of conditioned media samples indicated the presence of five IGF binding protein (IGFBP) species of Mr 37–40, 32, 30–31, 28, and 24 kDa. The 30–31 kDa doublet was visible in SSM‐conditioned medium only and associated with the presence of a 22‐kDa protein, which may represent a proteolytic fragment. In contrast, the 32‐kDa band was observed in FSM‐conditioned medium only. The other IGFBP moieties were present in both SSM‐ and FSM‐conditioned media. Cross‐linking experiments revealed the presence of the M6P/IGF‐II receptor on both SSM and FSM membranes. We also observed an IGF‐I receptor form bearing unusual high affinity for IGF‐II: the binding of [125I]IGF‐I on this receptor was preferentially displaced by IGF‐I but that of [125I]IGF‐II was mostly inhibited by IGF‐II, suggesting that the two tracers did not bind on the same epitopes. [125I]IGF‐II binding to this receptor was greater on SSM than on FSM membranes. Autophosphorylation of WGA‐purified receptors revealed an ∼400‐kDa band after SDS‐PAGE under nonreducing conditions, which corresponded to the α2β2 form of the IGF‐I receptor, and two β subunit moieties of Mr 101 and 105 kDa under reducing conditions in both SSM and FSM extracts. Phosphorylation of the 105‐kDa moiety was more intensively increased than that of the 101‐kDa protein after growth factor stimulation. Basal phosphorylation state of the two β subunits was similarly stimulated by IGF‐I and IGF‐II and less by insulin. Since both insulin and IGF‐I receptors were expressed in FSM and SSM, one of the two β subunits may actually correspond to that of the insulin receptor. We conclude that the IGF system is not considerably affected by the phenotypes of SSM and FSM. The differences observed, which mostly concern IGFBP species, more likely appear as regulatory adaptations than as phenotypic changes targeting the components of the IGF system.

Reevaluation of lipolytic activity of growth hormone in rabbit adipocytes.

The lipolytic activities of porcine pituitary fractions and purified growth hormone (GH) from human (h), porcine (p), ovine (o) and rabbit (Rb) origin as well as ovine placental lactogen (oPL), were compared to that of ACTH on rabbit adipocytes. All the GH preparations and oPL were equivalent in inhibiting the binding of labelled oGH to liver plasma membranes from pregnant rabbits. ACTH, and to a lesser extent porcine pituitary fractions and hGH, stimulated free fatty acid production by isolated adipocytes. The sensitivity of the adipocytes to these factors was increased when adenosine deaminase was added to the incubation medium. But, RbGH, pGH, oGH and oPL had no effect. We conclude that GH is not directly involved in the control of lipolysis in rabbit adipocytes and that the effect of hGH is rather due to a contamination of this preparation by other pituitary factors.

Effects of Temporary or Permanent Cryptorchidism on Leydig and Sertoli Cell Functions in the Lamb

Experimentally induced cryptorchidism results in a disruption of spermatogenesis although gonadotrophin levels increase. We wanted to know how the populations of Leydig and Sertoli cells evolve from a morphological point of view, whether the testis retains its sensitivity to gonadotrophins and androgens, whether the secretory activity of Leydig and Sertoli cells is modified by cryptorchidism, and whether the effects of cryptorchidism are reversible when testes are redescended into the scrotum.