A. W. Gustafson

The adrenal cortex during activity and hibernation in the male little brown bat, Myotis lucifugus lucifugus: Annual rhythm of plasma cortisol levels

Abstract Plasma cortisol concentrations were measured by radioimmunoassay in male little brown bats, Myotis lucifugus lucifugus (Chiroptera: Vespertilionidae), collected from a wild population throughout the entire annual cycle of activity (May–September) and hibernation (October–April). Two populations of corticosteroid levels were observed which corresponded to the physiological states of activity and hibernation, respectively: lower levels ( X = 0.46 ± 0.04 μ g/ml ) were measured during the months of activity whereas higher levels ( X = 0.81 ± 0.05 μ g/ml ) prevailed during the period of hibernation. The hibernating levels of cortical hormone for this bat are higher than those that have been measured in the plasma of other mammalian hibernators. These data suggest that the adrenal cortex in this species may be highly active during hibernation, a result that is at variance with the longstanding concept of “polyglandular involution” of the endocrine organs as a prerequisite for preparation and maintenance of hibernation in mammals. The findings also imply a seasonal “resetting” of feedback control in the brain-pituitary-adrenal axis with respect to ACTH in this species. Finally, it is suggested that elevated glucocorticoids would be necessary for the energetic requirements of hibernation and that periodic spontaneous arousals during hibernation may be important in permitting or accelerating hormonal action.

Luteinizing hormone-releasing hormone (LH-RH) cells and their projections in the forebrain of the batMyotis lucifugus lucifugus

Abstract Luteinizing hormone-releasing hormone (LH-RH) neurons and their projections were studied by immunocytochemistry in the brains of little brown bats (Myotis lucifugus lucifugus: Chiroptera: Vespertilionidae) as a first step in the study of relationships between these neurons and the seasonal reproductive events characteristic of this species. The majority of immunoreactive neurons in adult male, adult female, and fetal bats were ovoid bipolar cells with one thin and one thicker process, both of which gave rise to fine varicose fibers. LH-RH-immunoreactive perikarya were concentrated in the region of the arcuate nuclei in all bats examined. Perikarya were also consistently found dispersed in the mammillary region, anterior hypothalamus, preoptic areas, septum, diagonal band of Broca, and olfactory tracts; they were occasionally observed in the dorsal hypothalamus, organum vasculosum of the lamina terminalis (OVLT), habenula, amygdala, and cingulate gyrus. LH-RH-immunoreactive fibers projected heavily to the median eminence, infundibular stalk, and posterior pituitary. In extrahypothalamic areas, these fibers were especially abundant in the stria medullaris/habenula and stria terminalis/amygdala, but also contributed to the diagonal band of Broca and the olfactory tracts. Immunoreactive fibers that may be components of many different pathways clustered in the rostral septum and permeated the medial hypothalamus. LH-RH-containing fibers frequently entered the subfornical organ, but were observed less often in the OVLT and only occasionally in the pineal. The organization of the LH-RH system in the little brown bat resembles that of primates, but differs considerably from that in the rat. Anatomical characteristics of the LH-RH system in bats thus suggest that this animal may be a particularly suitable species for further study of neuroendocrine control of reproductive function as it may relate to primates, including humans.

Identification of a specific binding protein for sex steroids in the plasma of the male little brown bat, Myotis lucifugus lucifugus

A specific sex steroid-binding protein (SBP) was identified in the plasma of the male little brown bat, Myotis lucifugus lucifugus (Chiroptera: Vespertilionidae). Electrophoresis of bat plasma in 5% polyacrylamide gels yielded an active molecular species with a mobility distinctly different from albumin. This protein had high binding affinities for 5α-dihydrotestosterone (DHT), estradiol, and testosterone but low or negligible affinities for estrone, progesterone, androstenedione, or cortisol. The association constant for binding with DHT was calculated by Scatchard analysis to be 5.77 × 109 M−1. Using a DEAE-cellulose filter assay, the concentrations of SBP (mean ± SE) measured in males collected during the summer spermatogenic period were (i) 3.07 ± 0.43 × 10−7 M (early July), (ii) 1.64 ± 0.21 × 10−7 M (late July), and (iii) 2.07 ± 0.22 × 10−7 M (mid-August). The presence of SBP in the plasma of Myotis l. lucifugus, a species which has a wide seasonal variation in plasma testosterone levels with an extremely high summer peak, could provide valuable information on the physiological role of SBP. Previously, SBP has not been demonstrated in Chiroptera.

Evidence for diverse pathways of hypophysiotropic hormone transport in mammals

Comparative studies of mammalian hypothalamic-pituitary relationships have revealed striking variations in hypophysiotropic systems and in portal vascular architecture. Immunocytochemical studies indicate that mammalian GnRH, GHRH and somatostatin systems can project to all portions of the neurohypophysis (median eminence, infundibular stem and pituitary neural lobe). In rats, primary secretion sites are located within the median eminence and upper infundibular stem, whereas in bats, most projections extend into the lower infundibular stem and pituitary neural lobe. In ferrets and monkeys, sites of secretion appear to extend throughout the neurohypophysis, from median eminence to proximal neural lobe. In this review, these interspecific differences are examined in light of observed structural variations in portal vascular systems. Correlations suggest that hypophysiotropic hormones can be delivered to target cells in the pars distalis by diverse routes, with some species relying more heavily on long and others on short portal transport. These patterns may have important functional implications with respect to regulatory mechanisms operating within the hypothalamic-pituitary complex.

Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX.

SummaryCultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin.Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested.In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction or Heidenhain iron hematoxylin but without the latters’ length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.

Purification and characterization of the sex hormone-binding globulin in serum from Djungarian hamsters☆

A high-affinity sex hormone-binding globulin (SHBG) was purified from the serum of prepubertal Djungarian hamsters (Phodopus sungorus). A purification of more than 2000-fold with an overall yield of 23% was achieved without the use of androgen affinity chromatography. Two predominant variants (51 and 55 kDa) were resolved by denaturing polyacrylamide gel electrophoresis. Both variants participated in the binding of dihydrotestosterone (DHT) and had identical amino-terminal sequences. The sequences obtained for Djungarian hamster SHBG (dhSHBG) showed a high degree of identity with those of other mammals. The affinity of purified dhSHBG for DHT (2.5 x 10(9) M(-1) was similar to that measured in unfractionated serum. This protein was isolated as a dimer with a single calcium-dependent steroid-binding site and a major pI of 4.7. The described purification procedure yielded active dhSHBG from small volumes of prepubertal serum. These studies also provide the first direct structural evidence that a SHBG-like protein, not of testicular origin, is expressed by a rodent during prepubertal development.