James A. Dias

Inhibition of FSH action on granulosa cells by low molecular weight components of follicular fluid.

Immature female rats were injected with a single dose (10 IU) of pregnant mare serum gonadotropin to induce growth and maturation of ovarian follicles. Using such ovaries as a model, we tested the effects of low molecular weight subfractions of charcoal-absorbed bovine follicular fluid (FF-c) on (a) radioiodinated human FSH (125I-hFSH) binding to ovarian homogenates, (b) ovine FSH-stimulated adenylate cyclase activity in granulosa cell homogenates and (c) cAMP production by intact granulosa cells. The follicular fluid was fractionated by ultrafiltration through membranes of differing pore-sized into molecular weight components of 1000-5000 (passing Amicon H1P-5 hollow fibers but retained by Amicon UM-2 membrane) and 500-1000 (passing Amicon UM-2 membrane but retained by Amicon Um-05 membrane). These low molecular weight fractions inhibited 125I-hFSH binding to ovarian receptors, FSH-stimulated cAMP production by rat granulosa cells and FSH-stimulated, as well as fluoride-ion-stimulated adenylate cyclase activity in granulosa cell homogenates. Inhibition of FSH-stimulated adenylate cyclase activity by the FF subfractions was non-competitive as determined by double reciprocal plot analysis. Our results suggest that modulation of FSH effects on granulosa cells may be mediated by low molecular weight constituents of follicular fluid.

Evidence for the presence of follicle-stimulating hormone receptor antibody in human serum *

A male patient presented with polyostotic fibrous dysplasia, elevated serum gonadotropin levels, primary gonadal failure, and an immunoglobulin M (IgM) monoclonal gammopathy. When the patients serum was added to radioiodinated human follicle-stimulating hormone (125I-hFSH)-bovine testis membrane-receptor complex solubilized by detergent, followed by the addition of antihuman immunoglobulin G ((anti-hIgG), the preformed complex was precipitated. No such precipitation was seen when normal human or rabbit serum, serum from a patient with polyostotic fibrous dysplasia, or serum from other patients with polyclonal gammopathies were utilized. The patients serum did not precipitate free 125I-hFSH. Detergent-solubilized testis receptor not complexed to hFSH, as well as highly purified radioiodinated receptor were also precipitated when this serum was added followed by anti-hIgG. Our results indicate that serum from the patient contained antibodies to FSH testis receptor. It is possible that antibodies to gonadal receptors may exist in other patients, although it is not yet possible to assume a causal relationship between the presence of receptor antibodies and gonadal failure.

Effects of the polyamine spermine on binding of follicle-stimulating hormone to membrane-bound immature bovine testis receptors

The effect of the polyamine, spermine, on the interaction of human 125I-labeled FSH with membrane-bound receptors derived from bovine calf testes has been examined. Concentrations of spermine less than 0.01 M resulted in a slight but insignificant (P greater than 0.10) enhancement of FSH concentrations of 0.01 M and above caused a progressive reduction of FSH binding. Membrane receptors incubated in the presence of spermine at concentrations inhibitory to human 125I-FSH binding (0.01-0.04 M) resulted in an 8-50% decrease in the apparent FSH receptor concentration and a 10-65% decrease in the affinity constant as determined by computerized analysis of the isothermic ligand-binding data. Within the temperature range 4-20 degrees C, simultaneous addition of spermine (0.025 M) increased the reversibility of human 125I-FSH binding approx. 10% (P less than 0.005). Delayed addition of spermine (0.01-0.04 M) resulted in a dose-related dissociation of human 125I-FSH already bound to its receptor (P less than 0.05). However, preincubation of membrane receptors with spermine (0.002-0.04 M) at 4 degrees C or 34 degrees C followed by washing and addition of human 125I-FSH, resulted in an increase in hormone binding (P less than 0.05) over that of controls. If membrane receptor was incubated at 34 degrees C with spermine in the absence of radioligand, the usual loss of hormone binding was reduced (P less than 0.05), while membrane receptor incubated with spermine at 4 degrees C exhibited hormone binding greater (P less than 0.05) than that observed before treatment. Thus, the mechanism of inhibition of human 125I-FSH binding to membrane receptors appears to be correlated with an increase in reversibility of the membrane receptor-human 125I-FSH complex and is expressed as a decrease in the calculated receptor concentration and affinity constant of that interaction. Second, spermine appears to stabilize the membrane receptor in the absence of ligand, presumably through a membrane effect. These data suggest that spermine, and possibly other polyamines, which are endogenous to eukaryotic cells and undergo increases in concentration following stimulation by trophic hormone may play a role in the modulation of the ligand-membrane receptor interaction, in part, through direct effects on the membrane and/or the receptor.

High‐Affinity Binding of Fibronectin to Cultured Kupffer Cells

Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose‐dependent manner with the 125I‐fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I‐fibronectin to Kupffer cells. The phagocytosis of gelatinizedcoated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash‐removal of the fibronectin did not increase the uptake of gelatin‐coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I‐fibronectin as well as fibronectin‐coated sheep erythrocytes. The binding of 125I‐fibronectin‐gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high‐affinity (Kd = 7.46 × 10‐9 M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800–3,500 binding sites or putative “fibronectin receptors” per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin‐coated particles opsonized by fibronectin.

Hypothalamic-Adenohypophyseal Origin of Reproductive Failure in Mice following Chronic Infection with Toxoplasma gondii

Abstract Mice chronically infected with Toxoplasma gondii exhibited reproductive failure characterized by a constant diestrous vaginal cytology and ovarian and uterine atrophy. Chronically infected mice were treated with 20 ng of D-Leu6des-Gly-NH2-Pro-ethylamide (D-Leu6), a structural analog of luteinizing hormone-releasing hormone (LHRH), every 4 hr over a 12-hr period daily, for 3 days. Infected animals treated with D-Leu6 had greater pituitary weight (P < 0.01), ovarian weight (P < 0.01), and uterine weight (P < 0.025), than did infected control mice treated with saline. In addition, a change in vaginal cytology to estrus, metestrus, and proestrus of the D-Leu6-treated animals was observed, although a contiguity of normal estrous cycles and reproductive function was not determined. Comparable basal levels of serum luteinizing hormone (LH) were seen in infected mice and uninfected normal mice. However, the infected animals demonstrated a decreased pituitary responsiveness to D-Leu6 when monitored at 60 (P < 0.025) and 120 min (P < 0.010) following intraperitoneal administration of a bolus of 200 ng of the analog. Thus, the observed reproductive failure involves the readily releasable pool of pituitary LH, since basal LH is similar in both groups, and appears to be due to a dysfunction of the hypothalamic-adenohypophyseal axis.

Properties of Follicle Stimulating Hormone Binding Inhibitors Found in Physiological Fluids

Our interest in follicle stimulating hormone binding inhibitors (FSH-BI) stemmed from earlier studies on the presence of a low molecular weight testicular factor(s) in buffer or water extracts of mature rat testis which inhibited binding of 125I-FSH to gonadal receptors (Reichert and Abou-Issa, 1977). Shortly thereafter, we described the presence of similar factors in human serum (Reichert et al., 1979) and in bovine follicular fluid (Darga and Reichert, 1978). Most recently, low molecular weight components with FSH-BI activity have been detected in human seminal plasma (Dias et al., 1981). Our initial studies on these factors have been summarized in several reviews (Reichert, 1978; Reichert et al., 1981) and will not be discussed further at this time. Rather, we will present in this report a description of our latest efforts to characterize the FSH binding inhibitors found in human plasma and bovine follicular fluid.

Cellular Origin of Prolonged Induction of Ornithine Decarboxylase in the Rat Ovary

Abstract The temporal changes of ornithine decarboxylase (ODC) activity were investigated in the immature rat ovary following a single subcutaneous injection of pregnant mare serum gonadotropin (PMSG). A dose-response relationship was established. Maximal ODC activity was obtained at a dose of 25 IU of PMSG. This increase in ODC activity was accompanied by an increase of ovarian weight before reaching a maximum. A 250-fold increase of ODC activity was observed 1 day following a single dose of PMSG (50 IU). The enzyme specific activity only returned to the control level 4–5 days after hormone treatment. Immunoreactive ODC in immature, PMSG-primed rat ovaries and in heavily luteinized rat ovaries was localized utilizing the immunoperoxidase method and an antibody to ODC. Immunoreactive enzyme was confined to the cytoplasm of the granulosa cells but was not present in luteal cells. Thecal cells showed only weak immunostaining. This study provides clear evidence that the granulosa cell is the unique source of ODC activity in response to PMSG treatment. Furthermore, these data support the concept that polyamines play a role in granulosa cell proliferation and hence follicular development.

An enzyme immunoassay for human follicle-stimulating hormone

Abstract An enzyme immunoassay for human follicle-stimulating hormone (hFSH) has been developed utilizing highly purified hFSH coupled to glucose oxidase (EC 1.1.3.4) as the tracer, and antiserum to hFSH. Coupling of hormone and enzyme was done in the presence of glutaraldehyde and separation of bound hormone-enzyme complex from unbound was by double-antibody precipitation. Glucose oxidase activity was measured in buffer-reconstituted pellets derived from precipitation by second antibody. The hFSH-glucose oxidase conjugate reained immunologic activity after storage for 7 months at −20°C. Follicle-stimulating hormone activity, in a series of human pituitary fractions varying in potency from 125 to 3608 IU/mg as determined by a rat ovarian weight gain bioassay, gave similar potency estimates by enzyme immunoassay, with an index of discrimination of 1.10. The enzyme immunoassay proved useful for measurement of hormone mass in preparations of radiolabeled hFSH, allowing accurate calculation of specific radioactivity. The mean index of precision λ for a series of 20 enzyme immunoassay measurements was 0.079, with a detection limit of 4 mIU, about 1 ng of purified hFSH. The enzyme immunoassay gave results in good agreement with those from radioimmunoassay when applied to measurement of follicle-stimulating hormone levels in dialyzed human serum with an index of discrimination (enzyme immunoassay/radioimmunoassay) of 1.05. Recovery of varying amounts of hFSH added to a single serum pool was greater than 90%. Since the enzyme immunoassay does not require isotopes or specialized counting equipment, it may represent an attractive alternative to radioimmunoassay for laboratories with limited facilities.

BIOCHEMICAL PROPERTIES OF THE TESTICULAR FOLLITROPIN‐RECEPTOR SYSTEM*

The interaction of FSH with membrane receptors from rat and calf testis has been studied in some detail. The FSH receptor has been solubilized through use of the nonionic detergent Triton X-100 and highly purified by affinity chromatography on Affigel-10 coupled to ovine FSH. Hormone binding activity of the solubilized receptor has been preserved for extended periods through use of the structure-stabilizing agent glycerol. Other components of the FSH testes receptor system including the guanyl nucleotide binding protein and adenylate cyclase have been solubilized by nonionic detergents and also found to be stabilized by glycerol. FSH binding activity has been observed in testes cytosol and represents a putative class of receptors prepared from testes in the absence of detergent. The concentration of this buffer-soluble component decreased with age and increased concomitantly with loss of membrane receptors consequent to their down-regulation after administration of exogenous FSH. Phospholipids seem involved in the interaction of FSH with membrane-bound, detergent-solubilized, and buffer-soluble FSH binding activity. Phospholipids may maintain or stabilize a particular receptor conformation necessary for interaction with the hormone. A specific role for GTP seems indicated in regulation of FSH-stimulated adenylate cyclase activity in immature rat testis. Follitropin binding to testes receptor appears modulated by a variety of factors present in serum, testes extracts, follicular fluid, and seminal plasma, which are poorly understood at present. Inhibition of FSH binding by seminal plasma best-fit by a model proposing two hormone binding sites per receptor molecule, where binding to one site decreases the affinity of the other site for FSH. As a result of studies in this and other laboratories, the molecular endocrinology of FSH interaction with testis receptors is becoming increasingly understood.