Sasha Malamed

Ovarian innervation develops before initiation of folliculogenesis in the rat

SummarySympathetic neurotransmitters have been shown to be present in the ovary of the rat during early postnatal development and to affect steroidogenesis before the ovary becomes responsive to gonadotropins, and before the first primordial follicles are formed. This study was undertaken to determine if development of the ovarian innervation is an event that antedates the initiation of folliculogenesis in the rat, Rattus norvegicus. Serial sections of postnatal ovaries revealed a negligible frequency of follicles 24 h after birth (about 1 primordial follicle per ovary). Twelve hours later there were about 500 follicles per ovary, a number that more than doubled to about 1300 during the subsequent 12 h, indicating that an explosive period of follicular differentiation occurs between the end of postnatal days 1 and 2. Electron microscopy demonstrated that before birth the ovaries are already innervated by fibers containing clear and dense-core vesicles. Immunohistochemistry performed on either fetal (day 19) or newborn (less than 15h after birth) ovaries showed the presence of catecholaminergic nerves, identified by their content of immunoreactive tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. While some of these fibers innervate blood vessels, others are associated with primordial ovarian cells, thereby suggesting their participation in non-vascular functions. Since prefollicular ovaries are insensitive to gonadotropins, the results suggest that the developing ovary becomes subjected to direct neurogenic influences before it acquires responsiveness to gonadotropins.

Activation in vitro of glucose-6-phosphatase, inorganic pyrophosphate glucose phosphotransferase and related enzymes. Relationship to microsomal membrane structure

Abstract 1. 1. The elevation in enzymatic activity of certain microsomal enzymes caused in vitro by treatment with NH 4 OH or deoxycholate has been studied in relationship to the appearance of the membranes in corresponding electron micrographs. 2. 2. When freshly prepared liver microscomes were treated at 0° with NH 4 OH (about pH 9.8), activation of glucose-6-phosphatase (EC 3.1.3.9), inorganic pyrophosphatase, inorganic pyrophosphate-glucose phosphotransferase and inorganic pyrophosphate glycerol phosphotransferase was not instantaneous. 3. 3. Exposure to NH 4 OH for 15 to 30 min at 30° or contact for many hours at o° resulted in a maximum in vitro elevation of the enzyme activities that was approximately quantitatively equal to that observed instantaneously with such detergents as deoxycholate or Triton X-100. 4. 4. An electron microscopic study of NH 4 OH-activated microsomes at 0° showed that ribosomes were largely retained on the membranes when about 50% of maximum stimulation of enzymatic activities occurred. Longer exposure at 0° or brief warming at 30° with NH 4 OH, effecting full enzymatic activation, resulted in detachment of most of the ribosomes. The resulting membranes resembled those seen after exposure to inorganic pyrophosphate. 5. 5. The enzymatic activity of smooth microsomal membrane subfractions was capable of a percentage increase in vitro as great or greater than that of ribosome-rich rough membranes. 6. 6. Activation in vitro of these membrane enzymes is probably due to conformational changes in the membrane itself rather than to removal of ribosomes from their membrane attachment.

Ontogeny of pituitary growth hormone and growth hormone mRNA in the chicken.

Abstract The changes in pituitary growth hormone (GH) mRNA levels have been determined by Northern blot analysis and laser densitometry during embryonic development and posthatch growth of white Leghorn cockerels. Pituitary GH mRNA levels were observed to progressively increase between 18 days of embryonic development to a maximum at 4 weeks of age (posthatch). Subsequently, pituitary GH mRNA levels declined between 4 and 8 weeks of age, and between 12 weeks of age and adulthood. Pituitary GH contents showed increases during embryonic development and posthatch growth that paralleled the rise in GH mRNA. The decline in pituitary GH mRNA levels between 4 weeks of age and adulthood occurs when GH secretion has been observed previously to decline.

Growth hormone secretion from chicken adenohypophyseal cells in primary culture: Effects of human pancreatic growth hormone-releasing factor, thyrotropin-releasing hormone, and somatostatin on growth hormone release

A primary culture of chicken adenohypophyseal cells has been developed to study the regulation of growth hormone (GH) secretion. Following collagenase dispersion, cells were exposed for 2 hr to vehicle (control) or test agents. Human pancreatic (tumor) growth hormone-releasing factor (hpGRF) and rat hypothalamic growth hormone-releasing factor stimulated GH release to similar levels. GH release was increased by the presence of dibutyryl cyclic AMP. Thyrotropin-releasing hormone (TRH) alone did not influence GH release; however, TRH plus hpGRF together exerted a synergistic (greater than additive) effect, increasing GH release by 100 to 300% over the sum of the values for each secretagogue acting alone. These relationships between TRH and hpGRF were further examined in cultured cells exposed to secretagogues for two consecutive 2-hr incubations. TRH pretreatment enhanced subsequent hpGRF-stimulated GH release by about 80% over that obtained if no secretagogue was present during the first incubation. In other experiments, somatostatin (SRIF) alone did not alter GH secretion. However, SRIF reduced hpGRF-stimulated GH release to levels found in controls. Furthermore, GH release stimulated by the presence of both TRH and hpGRF was lowered to control values by SRIF. The results of these studies demonstrate that a primary culture of chicken adenohypophyseal cells is a useful model for the study of GH secretion. Indeed, these results suggest that TRH and hpGRF regulate GH secretion by mechanisms which are not identical.

Electron microscopic observations of terminals of functionally identified afferent fibers in cat spinal cord

Using the method of intra-axonal injection of horseradish peroxidase, functionally identified afferent fibers from three slowly adapting (Type I) receptors and one Pacinian corpuscle in the glabrous skin of the hind paw of the cat were stained. Electron microscopic observation of the terminals of these fibers revealed predominantly axodendritic asymmetric synapses containing round, clear vesicles. Multiple synapses on a single dendrite were observed, separated by as little as 900 mm from one another.

Steroid control of steroidogenesis in isolated adrenocortical cells: molecular and species specificity

The molecular and species specificity of glucocorticoid suppression of corticosteroidogenesis was investigated in isolated adrenocortical cells. Trypsin-isolated cells from male rat, domestic fowl and bovine adrenal glands were incubated with or without steroidogenic agents and with or without steroids. Glucocorticoids were measured by radioimmunoassay or fluorometric assay after 1-2 h incubation. Glucocorticoids suppressed ACTH-induced steroidogenesis of isolated rat cells with the following relative potencies: corticosterone greater than cortisol = cortisone greater than dexamethasone. The mineralocorticoid, aldosterone did not affect steroidogenesis. Suppression by glucocorticoids was acute (within 1-2 h), and varied directly with the glucocorticoid concentration. Testosterone also suppressed ACTH-induced steroidogenesis. Glucocorticoid-type steroids have equivalent suppressive potencies, thus suggesting that these steroids may induce suppression at least partly by a common mechanism. Although corticosterone caused the greatest suppression, testosterone was more potent. The steroid specificity of suppression of cyclic AMP (cAMP)-induced and ACTH-induced steroidogenesis were similar, suggesting that suppression is not solely the result of interference with ACTH receptor function or the induction of adenylate cyclase activity. Exogenous glucocorticoids also suppressed ACTH-induced steroidogenesis of cells isolated from domestic fowl and beef adrenal glands, thus suggesting that this observed suppression may be a general mechanism of adrenocortical cell autoregulation.

Glucocorticoid control of steroidogenesis in isolated rat adrenocortical cells

The role of end-product glucocorticoids in the regulation of corticosteroidogenesis in isolated adrenocortical cells was investigated. Trypsin-isolated cells from male rat adrenal glands were incubated with or without corticotropin (ACTH) and with or without corticosterone. Endogenous corticosterone production was determined by radioimmunoassay at the end of incubation. Cessation of ACTH-induced corticosterone production was apparent after 2-4 h of incubation. The suppression occurred later with lower cell concentrations. Corticosterone production was partially restored after washing the suppressed cells. Supernatant fluid from suppressed cell suspensions also suppressed steroidogenesis of a fresh population of cells. However, the suppressing property of the supernatant fluid was abolished after the removal of corticosterone by charcoal-dextran treatment, suggesting that corticosterone or other steroids caused the suppression. Exogenous corticosterone induced suppression over a wide range of ACTH concentrations, but did not change the half-maximal steroidogenic concentration of ACTH, indicating that the suppression does not change the sensitivity of the cells to ACTH. Suppression occurred within 30-60 min after corticosterone had been added to the incubation medium either at the start of incubation or while steroidogenesis was in progress. Suppression varied directly with the concentration of exogenous corticosterone. These data indicate that glucocorticoids can directly and acutely suppress corticosteroidogenesis and thus control adrenocortical function in concert with other regulators such as ACTH and Ca2+.

Polyhormonal regulation of avian and mammalian corticosteroidogenesis in vitro

1. The combined actions of ACTH, corticosterone and prolactin (PRL) in the acute regulation of corticosteroidogenesis were investigated using isolated adrenocortical cells from intact and hypophysectomized (hypox) rats (Rattus norvegicus) and from intact male domestic fowl (Gallus gallus domesticus). 2. Exogenous corticosterone suppressed to about 50% ACTH-induced corticosterone production of cells from either species. This suppression, in part, was due to corticosterone degradation. 3. oPRL, in the presence or absence of ACTH, raised corticosterone production of hypox rat cells, but not intact rat and domestic fowl cells. 4. In addition, oPRL counteracted the corticosterone-induced suppression of net ACTH-stimulated corticosterone production of hypox rat and intact domestic fowl cells, but not intact rat cells. 5. The potency of oPRL with domestic fowl cells was 4 times that with hypox rat cells. 6. Furthermore, in domestic fowl cells, the effect of oPRL was Ca2+-dependent.

Age-related changes of the somatotrophs of the domestic fowl Gallus gallus

SummaryThe ultrastructure of the somatotrophs of the caudal pituitary of the domestic fowl was studied quantitatively. Two age groups of male chickens were compared: 4–6 weeks and 24–30 weeks post-hatching. With age, somatotrophs decreased from about 40% to about 30% of the pituitary cell population. Their volume density decreased similarly. Mean volume of a somatotroph was the same in young and adult animals. Because the granule volume density of the somatotrophs was unchanged, but the somatotroph volume density of the gland declined, the granule volume density of the caudal pituitary gland dropped in parallel with that of the somatotrophs. Thus the volume of the gland comprised of somatotroph granules fell about 32%: from 6.57% to 4.45%. This lowered pool of stored hormone may be linked to the lowered circulating levels of growth hormone found in older animals by other investigators.The granule volume density of the somatotrophs was unchanged but the numerical density approximately doubled; thus the mean granule size decreased by 47% with age. The relationship of the size reduction of the granules to the lowered plasma growth hormone levels is not understood at present.

Isolated adrenocortical cells of the domestic fowl (Gallus domesticus): Steroidogenic and ultrastructural properties

Isolated adrenocortical cells from White Leghorn chickens (Gallus domesticus) were compared to those from rats (Rattus norvegicus). Cells were prepared from collagenase-dispersed adrenal glands of sexually mature male animals. Corticosterone was measured by radioimmunoassay after incubation for 2 h with steroidogenic agents. Of the four ACTH analogues used, three were 6-17 times more potent with rat cells than with fowl cells (potencies were indicated by half-maximal steroidogenic concentrations). However, 9-tryptophan (O-nitrophenylsulfenyl) ACTH was 8 times more potent with fowl cells than with rat cells, thus suggesting that ACTH receptor differences exist between the two cell types. In addition, cAMP analogues were 10 times more potent with rat cells than with fowl cells suggesting that fowl corticosteroidogenesis is less dependent on cAMP than is rat corticosteroidogenesis. At equal cell concentrations, rat cells secreted 20-40 times more corticosterone than did chicken cells when they were maximally stimulated. Although rat cells converted 8 times more pregnenolone to corticosterone than did fowl cells, the half-maximal steroidogenic concentration for pregnenolone-supported corticosterone synthesis was the same for both cell types (about 5 microM). This suggests that fowl cells have lower steroidogenic enzyme content rather than lower steroidogenic enzyme activity. An unusual feature seen in the isolated fowl adrenocortical cells was an abundance of intracellular filaments.