Nerve growth factor-like immunoreactivities in rodent salivary glands and testis

Abstract

A series of polyclonal affinity-purified antibodies against mouse submandibular-gland nerve growth factor (NGF) are described. Using the submandibular gland of the male mouse and indirect immunofluorescence, the specificity and sensitivity of affinity-purified immunoglobulins and various other fractions from the immunized animals have been tested. It will be shown that affinity-purification schemes, including pre-purification of protein A-fractionated immunoglobulins to remove antibodies that bind to unrelated hydrophilic and hydrophobic proteins, significantly enhance the signal-to-noise ratio and specificity of the antibodies. The antibodies effectively detect NGF-like immunoreactivity in both fresh and fixed glandular tissue. Optimal fixation procedures are described. Fluorescence intensities are linearly correlated to log antibody concentration. By use of the best antibody fractions and optimal fixation protocols, the distribution of NGF-like immunoreactivity is described in eight different salivary glands (rat and mouse, male and female, submandibular and sublingual glands). In addition to the well-known large numbers of immunoreactive cells in the submandibular gland of the male mouse, immunoreactive cells were found in the sublingual gland of male mice and in the submandibular and sublingual glands of female mice. One antibody revealed a weak specific fluorescence also in the submandibular gland of the male mouse. In a survey of genital organs of male mice, one antibody revealed fluorescence in the germ cell line. We conclude that several polyclonal affinity-purified antibodies have been characterized that show a strong NGF-dependent binding to the secretory granules of tubular cells in the submandibular gland of male mice. These antibodies should make it possible to locate endogenous and perturbed NGF levels immunocytochemically, e.g., in the peripheral and central nervous system, where NGF concentrations may be several orders of magnitude lower than in the salivary glands.

Growth factors as autocrine and paracrine modulators of male gonadal functions

“…Surprisingly, the ß-NGF subunit or a closely related peptide has been shown to be present in spermatogenic cells and not detectable in the supporting somatic cells. In one case, ß-NGF was localized by using immunofluorescence microscopy, with an affinity-purified, polyclonal antibody, to the cytoplasm of mouse spermatogonia, primary spermatocytes and spermatids (Olson et al, 1987). In a more recent study, mouse spermatogonia were reported to be negative for ß-NGF (Ayer-LeLeviere et al, 1988).…”

Initiation of testicular tubulogenesis is controlled by neurotrophic tyrosine receptor kinases in a three-dimensional Sertoli cell aggregation assay

“…Recently, neurotrophic growths factors were reported to participate in the development of various non-neural organs, such as the dermatome, kidney, and ovary (Sainio et al 1994, Brill et al 1995, Dissen et al 1995. In the testis, Ngf mRNA was first detected in spermatocytes and early spermatids in the adult rodent testis where it acts as a survival factor for maturing spermatozoa (Olson et al 1987, Ayer-LeLievre et al 1988. At the onset of meiosis, DNA synthesis in stage VIII and IX preleptotene spermatocytes is thought to be under autocrine control through NGF (Parvinen et al 1992).…”

Role of thyroid hormone in the initiation of EGF (epidermal growth factor) expression in the sublingual gland of the postnatal mouse

“…Granular duct cells can be induced in the SLGs of adult female mice by the individual or combined actions of androgens, thyroid hormones, and/or adrenocortical hormones (Gresik and Barka 1983;. These granular duct cells in the SLG stain immunocytochemically positive for EGF (Gresik and Barka, 1983), NGF (Olson et al, 1987;Ayer-Le Lievre et al, 1989), renin (Bing et al, 1980;Gresik and Barka, 1983), and a true tissue kallikrein, mK1 (Kurabuchi et al, 2000), and NGF m-RNA has been detected in such cells by in situ hybridization (Ayer-Le Lievre et al, 1989;Humple et al, 1993). By the way, we have previously found by electron microscopy that many SD cells have minimal secretory granules, forming granular striated tubules (GSTs) in both sexes, and further suggested that the GCT-like granular cells hormonally developed from some of the GST cells in the SLG (Kurabuchi et al, 2000;.…”