No abstract

The detection of noninfectious ovarian inflammation (oophoritis) and serum ovarian autoantibodies in a patient with premature ovarian failure is indicative of an autoimmune etiology. The mechanisms of autoimmune ovarian injury leading to loss of function are currently unknown. In this study we investigated the impact of oophoritis on ovarian function based on two murine autoimmune ovarian disease (AOD) models. AOD can be induced by thymectomy at Day 3 after birth (d3tx). D3tx mice develop ovarian inflammation and atrophy with loss of oocytes. In these mice, ovarian atrophy and not oophoritis correlated with abnormal estrous cyclicity. The second AOD model is induced by active immunization of adult mice with a murine ZP3 peptide (pZP3) in adjuvant. After active immunization, the zona pellucida antibody titer, not oophoritis, correlated with reduced fertility. To investigate the effect of oophoritis in the absence of antibody response or ovarian atrophy, pZP3-specific T cells were passively transferred into naive syngeneic mice. This recruited cytokine-producing cells into the ovaries so that elevated cytokine production and its effect on ovarian function could be examined. Recipients of pZP3-specific T cells developed severe granulomatous oophoritis, and the diseased ovaries had elevated ovarian mRNA levels of interferon-gamma, interleukin-1beta, and tumor necrosis factor alpha. Despite these changes, fertility rates and gonadotropin-induced follicular development remained essentially normal. Therefore, normal ovarian function is compatible with severe ovarian inflammation mediated by autoreactive T cells.

We have studied insulin-like growth factors (IGFs) and IGF-binding proteins released by human fibroblasts. Conditioned medium was obtained after incubation of 2 X 10(6) cells in 2 ml serum-free medium for 72 h. IGF binding protein was identified in aliquots of conditioned medium at 4 C for 16 h with [125]IGF II after charcoal separation. After gel filtration in neutral phosphate buffer through Sephadex G-150, the binding activity eluted with an apparent size greater than 100,000 daltons. After gel filtration through Bio-Rad P-100 in 1 M acetic acid, binding activity had a molecular size of about 50,000 daltons. When [125I]IGF-II bound to conditioned medium binding protein was cross-linked with disuccimidyl suberate and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, the complex had an estimated molecular size of 67,000 daltons. Competitive binding studies with labeled and unlabeled IGF-I and IGF-II showed that IGF-II was preferentially bound by fibroblast binding protein. The above findings are characteristic of serum binding protein but not shed IGF surface receptors. To eliminate possible interference from binding proteins in the IGF-I RIA and the IGF-II radioreceptor assay, conditioned medium was subjected to acid gel filtration, and the peptide fractions were pooled. We found that conditioned medium of seven fibroblast lines contained 0.20 +/- 0.06 ng/ml IGF-I. After the addition of 20 ng/ml human GH (hGH), the conditioned medium contained 0.48 +/- 0.09 ng/ml. These results are lower than those previously reported. One of the two lines of fibroblasts from patients apparently resistant to GH had a minimal increase in IGF-I in conditioned medium after hGH addition. We were able to detect IGF-II in fibroblast conditioned medium in concentrations of 4.4 to 21 ng/ml but there was no consistent response to GH either in the normal fibroblast lines or in fibroblasts obtained from children with short stature.

Levels of multiplication-stimulating activity (MSA) in fetal rat serum are high (2-4 micrograms/ml), suggesting that MSA may have a role in fetal growth. We now demonstrate that fibroblasts derived from rat embryos (REFs) have specific MSA receptors and respond to MSA with increased DNA synthesis. Two types of insulin-like growth factor (IGF) receptors were demonstrated by competitive binding of radioiodinated MSA, IGF-I, and IGF-II and by chemical cross-linking of [125I]iodo-MSA and [125I]iodo-IGF-I to REFs. One type of receptor (mol wt, 260,000 under reducing conditions) did not interact with insulin, and another type of receptor (mol wt, 130,000, under reducing conditions) was recognized by insulin. Scatchard analysis of [125I]iodo-MSA binding data was consistent with one class of noninteracting binding sites. A biological response of MSA, increased DNA synthesis, was demonstrated with autoradiography in REFs. During a 16-hr incubation, DNA synthesis was stimulated by normal rat serum, and platelet-poor plasma plus platelet-derived growth factor (PDGF), but not by serum from hypophysectomized (hypox) rats or hypophysectomized (hypox) platelet-poor plasma plus PDGF. However, when MSA was added to hypox serum or to hypox platelet-poor plasma plus PDGF, DNA synthesis was stimulated to the level achieved by normal rat serum. By contrast, during a longer cell multiplication experiment, REFs grew equally well in normal or hypox rat serum, raising the possibility that REFs may produce a MSA-like factor.