Tissue-specific effects of castration and ovariectomy on murine epidermal growth factor and its mRNA.

The polypeptide mitogen, epidermal growth factor (EGF), was originally isolated from mouse submaxillary gland (SMG). In mice, these glands are sexually dimorphic; the SMG of male animals typically contains up to 400 pmol EGF/mg protein whereas EGF concentrations in the SMG of female mice are only 5-20 pmol/mg protein. When mice were castrated at 8 weeks of age, EGF mRNA levels in the SMG fell rapidly to the low levels observed in control female animals. Although the concentration of EGF in the SMG, measured by radioimmunoassay, also fell to very low levels (13.8 +/- 0.7 (S.E.M.) pmol/mg protein) in the castrated mice, the rate of decrease was considerably slower than that of the mRNA. In contrast to the loss of EGF and EGF mRNA from mice following castration, ovariectomy led to a rise in EGF mRNA levels in SMG, with a maximal increase (approximately 100-fold) 4-6 weeks after ovariectomy. Concentrations of EGF in the SMG also rose markedly in the ovariectomized animals, from a control value of 5.4 +/- 0.8 to 34.7 +/- 7.9 pmol/mg protein at 6 weeks. In mice, the kidney displays the second highest level of EGF gene expression. However, in contrast to the effects on SMG, kidney EGF mRNA was not affected by either castration or ovariectomy. These results provide further evidence for the tissue-specific control of EGF gene expression in response to steroid hormones.

Characterization of the high-affinity receptors on Swiss 3T3 cells which mediate the binding, internalization and degradation of the mitogenic peptide bombesin.

Bombesin and bombesin-related peptides such as gastrin-releasing peptide (GRP) stimulate DNA synthesis and proliferation of Swiss 3T3 cells in culture. We have used 125I-labelled [Tyr4]bombesin and 125I-labelled GRP to characterize and identify the receptors for these peptides on Swiss 3T3 cells. The binding of 125I-[Tyr4]bombesin, which retained full biological activity, was maximal between 20 and 30 min incubation at 37 degrees C, after which continued incubation led to a decline in cell-associated radioactivity. This decline was markedly slowed by the presence of lysosomal enzyme inhibitors. Specificity of the binding site was indicated by the competitive inhibition of binding by bombesin-related peptides, but not by unrelated peptides and growth factors. Scatchard analysis of binding data indicated a single class of high-affinity receptors. The calculated value for the dissociation constant (Kd) was 2.1 nM and each cell possesses approx. 240,000 receptors. Because [Tyr4]bombesin has no free amino group, 125I-GRP was used in chemical cross-linking studies. When disuccinimidyl suberate was used to covalently couple 125I-GRP to the cells, two major radiolabelled complexes were detected with molecular masses of approx. 80,000-85,000 and 140,000. The binding of 125I-[Tyr4]bombesin to the cells was pH-dependent with maximal binding at pH 6.5-7.5 and effectively no specific binding at pH values below 4.5. At 37 degrees C, cell-associated 125I-[Tyr4]bombesin quickly became resistant to removal by acidic buffers, suggesting its rapid transfer to an intracellular compartment. However, pre-incubation with unlabelled [Tyr4]bombesin did not induce down-regulation of bombesin receptors as measured by the subsequent binding of 125I-[Tyr4]bombesin. In contrast with the Swiss 3T3 cells, specific binding of 125I-[Tyr4]bombesin was not detectable in two cell lines which are biologically unresponsive to bombesin-related peptides.

Is embryonic mortality increased in normal female rats mated to subfertile males?

Normal female rats were mated to control males or males which were subjected to unilateral testicular heating (43 degrees C for 30 min), irradiation (500 R), efferent duct ligation, arterial ligation or castration; in all males, the contralateral ductus deferens was ligated. All treatments caused reduced fertility and eventually infertility, as judged by the percentage of females becoming pregnant; the infertility was temporary after heating and irradiation. During the periods of reduced fertility, the numbers of fetuses per pregnant female and the fetus/corpus luteum ratios were reduced. In subsequent experiments, after heating of the testis, there was not only failure of fertilization despite the presence of normal numbers of spermatozoa in the uterus, but also an increased rate of embryonic degeneration in normal females. These results provide evidence that the male, while still fertile, can affect the fecundity of the female and the rate of embryo mortality.

Protein kinase C-mediated negative-feedback inhibition of unstimulated and bombesin-stimulated polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells.

Bombesin-related peptides stimulate a rapid increase in polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells. These peptides generate an increase in the efflux of 45Ca2+ from pre-labelled cells, a response consistent with an inositol trisphosphate-mediated mobilization of intracellular Ca2+. The bombesin-stimulated release of cellular 45Ca2+ is inhibited by tumour-promoting phorbol esters (e.g. 12-O-tetradecanoylphorbol 13-acetate, TPA). Although there are several possible sites of action at which this effect might occur, our results indicate that TPA induces an uncoupling of bombesin-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) without decreasing cellular binding of bombesin. In cultured cells, protein kinase C can be down-modulated by a prolonged incubation of the cells with phorbol esters. Such pretreatment greatly decreased the inhibitory effect of TPA on bombesin-stimulated PIP2 hydrolysis, suggesting that this action of the phorbol ester is mediated via protein kinase C. Since diacylglycerol is an endogenous activator of protein kinase C and a direct product of PIP2 hydrolysis, these results suggest that protein kinase C inhibition of polyphosphoinositide hydrolysis may function as a negative-feedback pathway. Cells in which protein kinase C has been down-modulated show elevated basal and bombesin-stimulated production of inositol phosphates, providing evidence that such a feedback loop limits polyphosphoinositide turnover in both unstimulated and mitogen-stimulated cells.

Transport of free and conjugated steroids from the boar testis in lymph, venous blood and rete testis fluid.

In 12 anaesthetized boars the concentrations of oestrone sulphate and dehydroepiandrosterone sulphate (DHAS) were 15- to 35-fold higher in lymph collected from a vessel in the spermatic cord than in testicular venous blood plasma from a vein in the spermatic cord. The concentrations of testosterone, total unconjugated oestrogens and dehydroepiandrosterone (DHA) were about twofold higher in lymph. The concentrations of all steroids studied were higher in testicular venous blood plasma than in arterial blood plasma (testosterone about sixfold; total unconjugated oestrogens about fourfold; oestrone sulphate about threefold; DHA and DHAS about twofold), but the concentrations of testosterone, total unconjugated oestrogens and oestrone sulphate in rete testis fluid were comparable to those in arterial blood plasma. Lymph flow from the pig testis was about 7% of plasma flow so that about 80% of the oestrone sulphate and DHAS produced by the testis leaves the organ in the lymph; the comparable values for testosterone, total unconjugated oestrogen and DHA were about 20%. In the 90-min period following an injection of human chorionic gonadotrophin there were substantial increases in the concentration of testosterone and smaller increases in the other steroids in arterial and spermatic venous blood plasma and in testicular lymph, but not in rete testis fluid; there were also small increases in lymph flow, but no change in blood flow.

The effects of long-term administration of either a crude inhibin preparation or an antiserum to FSH on serum hormone levels, testicular function and fertility of adult male rats.

Adult male rats were given either daily injections of ram rete testis fluid for periods of up to 70 days or injections of an antiserum against FSH every 3 days for 90 days. Compared with the control groups, the rats injected with ram rete testis fluid had lowered serum FSH levels, but only at treatment periods of 30 days and less. The levels of LH and testosterone in serum, testicular fluid secretion, sperm counts, testis weights and fertility were not affected by rete testis fluid treatment. The rats injected with anti-FSH serum exhibited an impairment of fertility which was never complete and evident only after 49 days of treatment. After 90 days of anti-FSH treatment, testis weight and free serum FSH were reduced, but sperm counts, testicular fluid secretion and serum levels of LH and testosterone were not affected.

Secretory activity of goat mammary glands during pregnancy and the onset of lactation.

1. The volume of the udder and the composition of the secretion have been followed in five goats through pregnancy to the onset of lactation. 2. During the middle of pregnancy udder volume was minimal and there was little or no fluid in the teats. 3. Two stages of commencing secretory activity (lactogenesis) were defined. In the first, starting up to ten weeks pre-partum, udder volume increased and the fluid in the teats changed from an extracellular-fluid-like to a milk-like composition and acquired a high concentration of immunoglobulins. Four goats accumulated several litres of a pre-colostral fluid with a high [lactose] 6-7 weeks pre-partum. 4. Comparison of the rate of increase in udder volume with previous data for the rate of increase in empty udder volume in pregnant goats showed that the rate of secretion, even in the last few days of pregnancy, was only a few per cent of the rate immediately after parturition. 5. In the last 2-3 days of pregnancy there was a three to elevenfold increase in [citrate] in the secretion; this heralded the onset of copious secretion at about the time of parturition. 6. The changes in mammary gland activities are discussed in relation to changes in plasma hormone concentrations during pregnancy.