The granin proteins secretogranin II (SgII) and chromogranin A (CgA) are commonly found associated with LH and/or FSH within specialised secretory granules in gonadotroph cells, and it is possible that they play an important role in the differential secretion of the gonadotrophins. In this study we have examined the regulation of the biosynthesis and secretion of SgII and CgA, in relation to LH secretion, in the LbetaT2 mouse pituitary gonadotroph cell line. Three experiments were carried out to investigate the effects of oestradiol (E2) and dexamethasone (Dex) in the presence and absence of GnRH (experiment 1), differing GnRH concentrations (experiment 2) and alterations in GnRH pulse frequency (experiment 3). In experiment 1, exposure to E2, Dex or E2+Dex, either with or without GnRH treatment, resulted in increased LH secretion. Steroids alone had no effect on LHbeta mRNA levels, but in the presence of GnRH LHbeta mRNA levels were increased in Dex- and E2+Dex-treated cells. GnRH receptor (GnRH-R) mRNA levels were up-regulated by Dex and E2+Dex, but were unaffected by GnRH. There were no steroid-induced changes in SgII or CgA mRNA, but increased levels of CgA mRNA were observed after GnRH treatment in cells cultured in the presence of Dex. In experiment 2, increasing concentrations of GnRH resulted in increases in LH secretion that were inversely dose-dependent. No changes in LHbeta, GnRH-R or SgII mRNA levels were observed, but there were dose-dependent increases in CgA mRNA levels. In experiment 3, GnRH was given as either 1 pulse/day or 4 pulses/day for 3 days. Both pulse regimes resulted in increased LH, SgII and CgA secretion compared with controls during the first 15 min pulse on day 3. Exposure to GnRH at 4 pulses/day increased LH and SgII secretion compared with controls during all 4 pulses, but secretion of both proteins was reduced during pulses 2-4 compared with pulse 1. CgA secretion also increased due to GnRH in pulse 1, but was decreased by GnRH treatment during pulse 2, and unchanged by GnRH during pulses 3 and 4. Total daily secretion of LH and SgII from cells given 1 pulse/day of GnRH increased compared with controls on all three treatment days, while total CgA secretion increased in response to GnRH on days 2 and 3 only. Intracellular levels of SgII, but not LH, decreased after GnRH treatment. In contrast, intracellular CgA was increased, but only after 4 pulses/day of GnRH. Levels of LHbeta, but not SgII, mRNA were increased by both pulse regimes, while CgA mRNA levels increased after 1 pulse/day of GnRH. These results indicate that there is a close correlation between the GnRH-stimulated release of LH and SgII from LbetaT2 cells, suggesting that SgII may have an influential role in the regulated secretion of LH, possibly by inducing LH aggregation to facilitate trafficking into secretory granules. CgA secretion does not appear to be closely associated with that of LH, but CgA expression does appear to be regulated by GnRH, which may indicate involvement in the control of LH secretion, possibly by influencing the proportion of LH in the different types of secretory granules.

SUMMARY

1. The defence mechanisms of the body are predominantly functions of the reticulo-endothelial system (RES). Stimulation of the RES leads to increased body defence indicated by increased phagocytic activity, raised serum γ-globulin and increased protection of experimental animals against virulent infections. The strongest RES stimulants appear to be oestrogens, natural and synthetic.

2. In the mouse, 0·002 mg. diethylstilboestrol daily is sufficient to produce significant prolongation of survival time against infection.

3. RES activity varies at different stages of the oestrous cycle and of pregnancy in both the rat and the mouse. The stages where the activity is greatest correspond with those in the human subject when the output of oestrogens is increased.

4. The results suggest that oestrogen is the natural stimulant of body defence in both the male and female, the latter having high oestrogen levels when protection against infection is normally most needed.

5. RES stimulation does not rise in step with oestrogenicity, and oestrogenic compounds have two separate biological activities—one which stimulates the RES to raise body defence, and one which acts on the reproductive organs; these two activities are apparently unrelated, although shared by the same molecule.

Chronic high caloric intake has contributed to the increased prevalence of pediatric obesity and related morbidities. Most overweight or obese children, however, do not present with frank metabolic disease but rather insulin resistance or subclinical precursors. The innate immune system plays a role in the pathophysiology of type 2 diabetes but how it contributes to early metabolic dysfunction in children on chronic high-fat diet (HFD) is unclear. We hypothesize that such inflammation is present in the pancreas of children and is associated with early insulin resistance. We used nonhuman primate (NHP) juveniles exposed to chronic HFD as a model of early pediatric metabolic disease to demonstrate increased pancreatic inflammatory markers before the onset of significant obesity or glucose dysregulation. Pancreata from 13-month-old Japanese macaques exposed to a HFD from in utero to necropsy were analyzed for expression of cytokines and islet-associated macrophages. Parameters from an intravenous glucose tolerance test were correlated with cytokine expression. Before significant glucose dysregulation, the HFD cohort had a twofold increase in interleukin 6 (IL6), associated with decreased first-phase insulin response and a sexually dimorphic (male) increase in IL1β correlating with increased fasting glucose levels. The number of islet-associated macrophages was also increased. Pancreata from juvenile NHP exposed to HFD have increased inflammatory markers and evidence of innate immune infiltration before the onset of significant obesity or glucose dysregulation. Given the parallel development of metabolic disease between humans and NHPs, these findings have strong relevance to the early metabolic disease driven by a chronic HFD in children.

We have shown previously that, in sheep primary pituitary cells, bone morphogenetic proteins (BMP)-4 inhibits FSHβ mRNA expression and FSH release. In contrast, in mouse LβT2 gonadotrophs, others have shown a stimulatory effect of BMPs on basal or activin-stimulated FSHβ promoter-driven transcription. As a species comparison with our previous results, we used LβT2 cells to investigate the effects of BMP-4 on gonadotrophin mRNA and secretion modulated by activin and GnRH. BMP-4 alone had no effect on FSH production, but enhanced the activin+GnRH-induced stimulation of FSHβ mRNA and FSH secretion, without any effect on follistatin mRNA. BMP-4 reduced LHβ mRNA up-regulation in response to GnRH (±activin) and decreased GnRH receptor expression, which would favour FSH, rather than LH, synthesis and secretion. In contrast to sheep pituitary gonadotrophs, which express only BMP receptor types IA (BMPRIA) and II (BMPRII), LβT2 cells also express BMPRIB. Smad1/5 phosphorylation induced by BMP-4, indicating activation of BMP signalling, was the same whether BMP-4 was used alone or combined with activin±GnRH. We hypothesized that activin and/or GnRH pathways may be modulated by BMP-4, but neither the activin-stimulated phosphorylation of Smad2/3 nor the GnRH-induced ERK1/2 or cAMP response element-binding phosphorylation were modified. However, the GnRH-induced activation of p38 MAPK was decreased by BMP-4. This was associated with increased FSHβ mRNA levels and FSH secretion, but decreased LHβ mRNA levels. These results confirm 1. BMPs as important modulators of activin and/or GnRH-stimulated gonadotrophin synthesis and release and 2. important species differences in these effects, which could relate to differences in BMP receptor expression in gonadotrophs.

Activins and inhibins, members of the transforming growth factor-beta family are able to stimulate and inhibit, respectively, FSH synthesis and release. Other members of this superfamily, the bone morphogenetic proteins (BMPs), may also affect FSH synthesis in the mouse. The aim of this work was to determine whether BMPs are expressed in the ovine pituitary and whether they play a role in the regulation of FSH release.

The mRNAs encoding BMP-2, BMP-4, BMP-7 and the oocyte-derived growth factor, growth differentiation factor (GDF)-9 were detected in the pituitaries of cyclic ewes by reverse-transcriptase PCR, as well as the mRNAs encoding the BMP type I receptors, BMPR-IA (activin-receptor-like kinase (ALK)-3) and BMPR-IB (ALK-6), and type II receptors (BMPR-II). Immunolabeling of pituitary sections revealed the presence of BMPR-IA (ALK-3) and BMPR-II in gonadotrope cells. To investigate the potential effects of BMPs on FSH secretion, ewe pituitary cell cultures were treated with BMP-4 (10⁻¹¹ M to 10⁻⁹ M) for 48 h. Interestingly, FSH release was decreased in a dose-dependent manner. At 10⁻⁹ M BMP-4 both FSH concentration and FSHβ mRNA expression were reduced by 40% of control values. In contrast, there was no inhibitory effect on either LH or LHβ mRNA expression. A similar result was found with BMP-6. BMP-4 triggered the phosphorylation of Smad1, suggesting that the effect of BMP-4 on FSH secretion is due to the activation of the BMPs signaling pathway. Furthermore, BMP-4 blocked the stimulatory effect of activin on both FSH release and FSHβ mRNA and amplified the suppression of FSH release and FSHβ mRNA levels induced by 17β-estradiol. These results indicate that a functional BMP system operates within the sheep pituitary, at least in vitro, to decrease FSH release and to modulate the effect of activin.