ABSTRACT

Untreated serum exhibited two forms of insulin-like growth factor-I (IGF-I)-binding protein complexes during gel chromatography: one of M _r 150 000 and the other of M _r 40 000–45 000. The majority of the immunoreactive IGF-I was associated with the M _r 150 000 complex. Following acid-ethanol extraction of serum, the binding activity at M _r 150 000 disappeared and a reduced binding activity appeared in the albumin size range. Acid incubation of serum was slightly less effective than acid-ethanol extraction in reducing the binding activity. Acid-ethanol-extracted or acid-incubated serum were parallel to IGF-I standard in the dose–response displacement of iodinated IGF-I. Gel filtration of serum with 1 mol acetic acid/l almost completely separated IGF-I and the binding proteins. Binding-protein fractions from gel filtration interfered with the immunoreactivity of IGF-I with its antibodies, causing a non-parallel displacement curve in the radioimmunoassay (RIA). Serum IGF-I could be isolated as a single peak by high performance C₁₈ reverse-phase liquid chromatography (HPLC). The concentrations of IGF-I measured in bovine sera by RIA were similar between acid gel filtration and HPLC; the concentrations by acid-ethanol extraction and acid incubation being about 30% smaller than those measured with former methods. The lower concentration of IGF-I measured in bovine serum with acid-ethanol extraction or acid incubation appears to be due to interference of IGF-binding proteins not removed by either treatment.

Journal of Endocrinology (1990) 127, 139–148

ABSTRACT

To gain insight into the involvement and interactions of the insulin-like growth factors (IGFs) and oestrogen in mammary growth and differentiation, the temporal expression of mammary mRNAs encoding components of the IGF system in pregnant and pseudopregnant pigs was examined. Pseudopregnant pigs received 5 mg oestradiol valerate or vehicle daily from day 45 after oestrus and underwent mammary biopsy on days 60, 90 or 112. In mammary tissue of pregnant pigs, steady-state levels of the mRNAs encoding IGF-I, IGF-II and type-I IGF receptor as well as the levels of the membrane-associated type-II IGF receptor were higher during the early phase of mammogenesis (≤day 45) than during the subsequent stages of mammary development. Mammary IGF-I, IGF-II and type-I receptor mRNAs were expressed at their lowest levels around day 90 of pregnancy (20–40% of those for day 30 of pregnancy) coincident with the onset of β-casein mRNA accumulation. Mammary IGF-binding protein-2 (IGFBP-2) mRNA levels increased twofold during the latter half of pregnancy, whereas the amount of IGFBP-3 mRNA declined after day 30 to undetectable levels by midpregnancy. Pseudopregnant pigs had reduced levels of these mRNAs (except for IGF-II) relative to their pregnant counterparts and this was associated with premature differentiation of mammary tissue as reflected by an earlier onset of β-casein mRNA accumulation in the former. The administration of oestradiol valerate decreased the levels of IGF-I and type-I IGF receptor mRNAs by day 60 of pseudopregnancy, but the reverse was evident by day 112. Oestradiol administration increased β-casein mRNA levels in pseudopregnant pigs, but had no effect on mammary IGFBP-2 and IGFBP-3 mRNA levels. Mammary IGF content was greater in late pregnancy (≥day 90) and pseudopregnancy than at early pregnancy. Serum IGF-I and IGF-II levels declined steadily during pregnancy and this was similar to, but not correlated with, mammary IGF mRNA levels, whereas in pseudopregnant pigs, serum IGF concentrations did not change temporally or in response to oestradiol. Serum IGFBP-2 levels were unaltered during pregnancy or pseudopregnancy, but serum IGFBP-3 levels declined after day 60 of pregnancy. In pseudopregnant pigs, serum IGFBP-3 levels did not change temporally, but declined after oestradiol treatment. Results indicate that mammary IGF-I and type-I IGF receptor systems are down-regulated during pregnancy-associated differentiation of this tissue and in response to oestrogen. Locally produced (autocrine and paracrine) IGFs are likely to mediate mammogenesis, whereas oestrogen stimulates mammary differentiation and lactogenesis in the pig. However, the high mammary IGF content and the reciprocal expression of mammary IGFBP-2 and IGFBP-3 mRNAs during late pregnancy suggests the involvement of IGFs in lactogenesis as well.

Journal of Endocrinology (1993) 137, 473–483

Sphingomyelin synthase 2 (SMS2) is an enzyme that catalyzes the conversion of phosphatidylcholine and ceramide to sphingomyelin and diacylglycerol, and it is crucial to cellular lipid metabolism. Using the technique of subtraction hybridization, we have isolated a full-length cDNA encoding SMS2 from rat testes, which shared 93 and 87% identity at the nucleotide level with SMS2 in mice and humans respectively. A specific polyclonal antibody was prepared against a 20 amino acid peptide of NH₂-FSWPLSWPPGCFKSSCKKYS-COOH near the C-terminus of SMS2. Studies by RT-PCR and immunoblotting have shown that the expression of SMS2 was limited to late round spermatids and elongating spermatids, but it was not detected in late elongate spermatids and Sertoli cells. Furthermore, SMS2 was shown to associate with the developing acrosome beginning in late round spermatid through elongating spermatids (but not late elongate spermatids) and the cell membrane in studies using fluorescent microscopy and immunohistochemistry. These data were further confirmed by studies using immunogold electron microscopy. The expression of SMS2 in the seminiferous epithelium is stage-specific with its highest expression detected in the acrosome region in late round spermatids from stages VIII–IX, and also in the acrosome in elongating spermatids with diminished intensity in stages X–V; however, it was not found in the acrosome in elongate spermatids in stages VI–VIII. Collectively, these results suggest that SMS2 may play a crucial role in the lipid metabolism in acrosome formation and the plasma membrane restructuring from late round spermatids to early elongating spermatids.

The short heterodimer partner (SHP) (NR0B2) is an orphan nuclear receptor whose function in pancreatic β-cells is unclear. Mitochondrial uncoupling protein (UCP2) in β-cells is upregulated in obesity-related diabetes, causing impaired glucose-stimulated insulin secretion (GSIS). We investigated whether SHP plays a role in UCP2-induced GSIS impairment. We overexpressed SHP in normal islet cells and in islet cells overexpressing UCP2 by an adenovirus-mediated infection technique. We found that SHP overexpression enhanced GSIS in normal islets, and restored GSIS in UCP2-overexpressing islets. SHP overexpression increased the glucose sensitivity of ATP-sensitive K⁺ (K_ATP) channels and enhanced theATP/ADP ratio. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, GW9662, did not block the SHP effect on GSIS. SHP overexpression also corrected the impaired sensitivity of UCP2-overexpressing β-cells to methylpyruvate, another energy fuel that bypasses glycolysis and directly enters the Krebs cycle. K_ATP channel inhibition mediated by dihydroxyacetone, which gives reducing equivalents directly to complex II of the electron transport system, was similar in Ad-Null-, Ad-UCP2- and Ad-UCP2+Ad-SHP-infected cells. The mitochondrial metabolic inhibitor sodium azide totally blocked the effect of SHP overexpression on GSIS. These results suggest that SHP positively regulates GSIS in β-cells and restores glucose sensitivity in UCP2-overexpressing β-cells by enhancing mitochondrial glucose metabolism, independent of PPARγ activation.

ABSTRACT

A continuous line of somatostatin-producing medullary thyroid carcinoma cells was established from a transplantable tumour in BALB/c mice. Virtually all of the somatostatin immunoreactivity co-chromatographed with somatostatin 14. The tumour cells replicated in spinner cultures with a doubling time of approximately 4 days, and the concentration of somatostatin released into the culture medium increased in proportion to the number of cells. Two-to threefold increases in amounts of stored and released somatostatin were observed after treatment of the cells with bromodeoxyuridine. This cell line might be valuable for studies of somatostatin regulation in normal and neoplastic C-cells, and for other studies of C-cell biology which require a mouse model.

J. Endocr. (1986) 110, 309–313

The timely restructuring of the blood–testis barrier (BTB) that facilitates the migration of preleptotene and leptotene spermatocytes from the basal to the adluminal compartment in the seminiferous epithelium of adult rat testes, which occurs at late stage VII through early stage VIII of the epithelial cycle, is a crucial cellular event of spermatogenesis. However, the regulation of BTB dynamics at the biochemical level remains elusive. In this study, tumor necrosis factor α (TNFα), a secretory product of Sertoli and germ cells in rat testes, was shown to affect junction dynamics in vivo. Following an acute administration of recombinant TNFα directly to adult rat testes in vivo at 0.5 and 2 μg/testis (with a body weight ~300 g), this treatment significantly and transiently disrupted the BTB. It also transiently inhibited the steady-state protein levels of occludin, zonula occludens-1, and N-cadherin, but not junction adhesion molecule-A, α-, and β-catenin in testes at the BTB site as illustrated by immunoblottings, immunohistochemistry, electron microscopy, and fluorescent microscopy. This transient disruption of the BTB integrity induced by TNFα treatment was further demonstrated by a functional test to assess the passage of a fluorescent dye (e.g. fluorescein-5-isothiocyanate) from the systemic circulation to the adluminal compartment. Additionally, both the phosphorylated-Ser/Thr protein kinase activated by MAP kinase kinase (p-p38) and phosphorylated-externally regulated kinase (p-ERK) mitogen -activated protein kinase-signaling pathways were transiently activated. Collectively, these data coupled with the recently published in vitro studies have illustrated that the BTB is likely utilizing a novel mechanism in which localized production of TNFα by Sertoli and germ cells into the microenvironment at the basal compartment facilitates the timely restructuring (‘opening’?) of the BTB during spermatogenesis to facilitate germ cell migration.

In adult rat testes, blood–testis barrier (BTB) restructuring facilitates the migration of preleptotene spermatocytes from the basal to the adluminal compartment that occurs at stage VIII of the epithelial cycle. Structural proteins at the BTB must utilize an efficient mechanism (e.g. endocytosis) to facilitate its transient ‘opening’. Dynamin II, a large GTPase known to be involved in endocytosis, was shown to be a product of Sertoli and germ cells in the testis. It was also localized to the BTB, as well as the apical ectoplasmic specialization (apical ES), during virtually all stages of the epithelial cycle. By co-immunoprecipitation, dynamin II was shown to associate with occludin, N-cadherin, zonula occludens-1 (ZO-1), β-catenin, junctional adhesion molecule-A, and p130Cas, but not nectin-3. An in vivo model in rats previously characterized for studying adherens junction (AJ) dynamics in the testes by adjudin (formerly called AF-2364, 1-(2,4-dichlorobenzyl)-1H-indazole-3-car-hohydrizide) treatment was used in our studies. At the time of germ cell loss from the seminiferous epithelium as a result of adjudin-induced AJ restructuring without disrupting the BTB integrity, a significant decline in the steady-state dynamin II protein level was detected. This change was associated with a concomitant increase in the levels of two protein complexes at the BTB, namely occludin/ZO-1 and N-cadherin/β-catenin. Interestingly, these changes were also accompanied by a significant increase in the structural interaction of dynamin II with β-catenin and ZO-1. β-Catenin and ZO-1 are adaptors that structurally link the cadherin- and occludin-based protein complexes together at the BTB in an ‘engaged’state to reinforce the barrier function in normal testes. However, β-catenin and ZO-1 were ‘disengaged’ from each other but bound to dynamin II during adjudin-induced AJ restructuring in the testis. The data reported herein suggest that dynamin II may assist the ‘disengagement’ of β-catenin from ZO-1 during BTB restructuring. Thus, this may permit the occludin/ZO-1 complexes to maintain the BTB integrity when the cadherin/catenin complexes are dissociated to facilitate germ cell movement.