Tanycytes in long-term ovariectomized ewes treated with estrogen exhibit ultrastructural features associated with increased cellular activity.код для вставкиСкачать
\- THE ANATOMICAL RECORD 203579-187 (1982) Tanycytes in Long-term Ovariectomized Ewes Treated With Estrogen Exhibit UItrastructural Features Associated With Increased Cellular Activity PENELOPE W. COATES AND STEVEN L. DAVIS Department of Anatomy, Texas Tech University Health Sciences Center, Lubbock, Texas 79430 (I? WC.) and Department of Animal Science, University of Idaho, Moscow, Idaho 83843 (S.L.D.) ABSTRACT Ependymal tanycytes on the floor of the third ventricle of longterm ovariectomized (OVX) female sheep (ewes) and OVX ewes treated with 17p-estradiol (E&) were investigated with scanning and transmission electron microscopy (SEM and TEM) for evidence of differences in ultrastructure. Plasma concentrations of luteinizing hormone (LH)in the same ewes that were analyzed ultrastructurally were 45.1 t 28.7 nglml in the nontreated OVX group, and 2.2 t 1.3 nglml in the OVX E2Pgroup, confirming the widely reported negative feedback effect of estrogen on LH. With SEM, the most prominent difference between groups was that tanycytes on the floor of the third ventricle in OVX ewes had very few surface membrane modifications, particularly microvilli, and appeared relatively bare-surfaced compared to those from the estrogen-treated group. Tissues from estrogen-treated OVX ewes were, for the most part, covered with a fuzzy nap of delicate microvilli. Transmission electron microscopy confirmed and extended this impression. With TEM, tanycytes in E,P-treated ewes exhibited not only a more luxurious display of microvilli but also a much richer complement of welldeveloped organelles including rough endoplasmic reticulum, ribosomes, Golgi complexes,microtubules, and filaments than tanycytes in OVX alone ewes. Collectively, the fine structural data indicates a positive correlation between tanycytes displaying well-developed features associated with heightened cellular activity synthesis, secretion and/or absorption - and the presence of estrogen. + We have been interested in exploring the relationship between reproductive status and the ultrastructure of tanycytes in the third ventricle of sheep. Our previous investigations had shown that tanycytes in castrated rams (wethers)treated with testosterone propionate (TP)had a significantly different morphology compared to untreated wethers (Coates and Davis, 1979). Tanycytes in anestrous ewes were also different from tanycytes in anestrous ewes treated with estrogen and progesterone (Coates and Davis, 1977), and subtle changes were found comparing tanycytes from the different stages of the estrous cycle in ewes (Coates and Davis, 1980). In this paper, we report our findings on both scanning electron microscopy (SEM) and transmission electron microscopy (TEM)used to analyze fine structural changes in tanycytes of long-term ovariectomized (OVX) female sheep (ewes)treated with 17p-estradiol benzo- 0003-276X/82/2031-0179$03.00 0 1982 Alan R. Liss, Inc. ate (E$) compared with tanycytes from untreated OVX ewes, along with plasma concentrations of luteinizing hormone (LH) in the same animals. MATERIALS AND METHODS Eight mature ewes were ovariectomized (OVX)and then kept on a routine maintenance schedule for 8% months, at which time they were divided into two groups of four animals each. One group of ewes received daily injections of 10 pg estradiol benzoate (E,p) per kilogram body weight in a vegetable oil vehicle, over a 2-week period. The remaining OVX ewes served as the control group and received daily injections of vehicle alone for the same 2-week period. Received November 24, 1981: accepted December 30,1981. 180 F.W. COATES AND STEVEN L. DAVIS At the end of the 2-week treatment period and immediately prior to sacrifice, blood was drawn from each ewe for analysis of luteinizing hormone (LH)by a radioimmunoassay (RIA) method developed previously (Niswender et al., 1969). The animals were then sacrificed by intravenous injection of succinyl choline, followed by perfusion of the head with a combination of aldehydes in a phosphate buffer using a method previously developed for this purpose (Coates and Davis, 1977). For ultrastructural analysis by scanning electron microscopy (SEM) and transmission electron microscopy (TEM),the brain was removed from the cranial cavity and put into fresh cold fixative. The hypothalamic region was dissected out, and a sagittal section was made through the third ventricle. One side was selected for processing for SEM analysis of the ventral floor including the infundibular recess of all eight ewes. Areas from the ventral floormedian eminence of the other half were selected for TEM processing and analysis from four ewes (two randomly chosen OVX controls and two randomly chosen OVX E,P ewes). SEM and TEM preparative procedures followed protocols already established (Coates and Davis, 1979). + RESULTS RIA Plasma LH concentration in the OVX control group averaged 45.1 f 28.7 ngiml (range 129.0 to 0.35 ngiml). The level of LH in the OVX E,P ewes was 2.2 f 1.3 ngiml (range 6.0 to 0.68 ngiml). + SEM Surface features on the floor-median eminence of all specimens were analyzed with SEM. There is a wide expanse of predominantly nonciliated ventral floor easily distinguished from the heavy ciliated dorsolateral walls of the third ventricle. The sheep third ventricle typically presents an unusually large infundibular mouth which funnels down into the infundibular recess. For this study a zone which included the anterior and middle portions of the ventral floor encompassing the mouth and recess of the infundibulum was examined. Excluded for purposes of this report was the area of the third ventricular floor in and around the mammillary recess. SEM of OVX ewes The overall impression of the ventral floor in the group of OVX ewes was the relative absence of surface features, particularly microvilli, as compared with the E,@-treatedgroup. Although much of the floor surface was patchy with microvilli (Fig. l),many ependymal tanycyte cell surfaces were flat and relatively featureless (Fig. 2). Borders between such cells were hard to distinguish, although the presence of a single stubby cilium usually indicated the location of a cell (Fig. 2 ) . Where tanycyte surfaces did not display a scattering of microvilli, the surface was relatively smooth and bare (Fig. 2). Interspersed with these smooth, bare areas were regions where microvilli covered some adjacent ependymal surfaces as well, giving a checkerboard or patchy pattern (Fig. 1). SEM of OVX El@ewes The overall impression was of intense proliferation of microvilli: Surfaces of ependymal tanycytes on the ventral floor of the gonadal steroid-treated ewes for the most part covered with microvilli over broad expanses (Fig. 3). These microvilli were plentiful, long, and slender, thus giving a distinctly fuzzy appearance to the floor (Figs. 3,4).Occasional clusters of cilia were observed (Fig. 3), but single individual cilia and cell borders were all but obscured by the surrounding carpet of microvilli (Figs. 3,4).Large regions of bare-surfaced tanycytes were substantially lacking. However, it was only on cells where microvilli appeared sparser that miniblebs were easily detected (Fig. 5). TEM of O V X ewes Due to the requirements of thin sectioning, only the cell bodies and most proximal portions of tanycytes were considered for TEM analysis. Many ependymal tanycytes exhibited an extremely attenuated profile as they lined the floor and stretched out for considerable distances parallel to the lumen of the third ventricle. Tanycytes such as these invariably lacked microvilli in quantity at their luminal surfaces. Other tanycytes were not attenuated but nevertheless were not rich in surface membrane modifications, i.e., microvilli. Internally, tanycytes showed poorly developed rough endoplasmic reticulum. Other organelles generally were not remarkable, although present (Fig. 6). + + TEM of OVX E l ewes Tanycytes from this group routinely displayed a proliferation of microvilli and small apical blebs at their luminal surface (Figs. 7-10). The microvilli were characteristically long, slender, and irregular in disposition (Fig. 8).They often originated as a clump from irregular protruding sites at the tanycyte surface (Fig. 9). Irregular and round miniblebs were Fig. 1. Ependymal tanycytes on the floor of the third ventricle of an OVX ewe exhibit a characteristic patchy appearance over widespread areas. Individual cells whose surfaces are relatively sparse of microvilli and miniblebs alternate with nearby cells whose surfaces retain these membrane modifications. SEM. X 5,000. Fig. 2. Many tanycytes on the floor of the third ventricle in OVX ewes exhibit fewer surface membranemodifications resulting in a relatively flat and featureless surface such as seen here. There are only minimal numbers of microvilli and miniblebs, and an occasional short stubby cilium. The remainder of the tanycyte surface is smooth. SEM. X 5,6000. + Fig. 3. Tanycytes from this OVX E,O ewe display SUP faces covered with microvilli, resulting in a fuzzy appem. ance of the floor of the third ventricle. SEM. X 2.700. fig. 4. At higher SEM magnification the cell borders and a single cilium are all but obscured by the plentiful microvilli. SEM. X 5.100. Fig. 5. Miniblebs are easily detected on these presumptive tanycyte surfaces from the infundibuIar recess of an OVX E,P ewe due to the lack of overwhelming numbers of microvilli. SEM. floor of third ventricle. X 4,900. + Fig. 6. Tanycytes from the floor of the third ventricle in this OVX ewe possess few microvilli and miniblebs a t their luminal surfaces. Although internal cell organelles are evident, they are not as well developed as in tanycytes from OVX + E,i3 ewes. TEM. X 10.500. + Fig. 7. Typical of tanycytes in OVX E,@ewes is the abundant display of microvilli on their luminal surfaces. This is the TEM correlateof the SEM imageof the fuzzy nap covering the ventricular floor. TEM. X 10.500. + Fig. 8. Microvilli on tanycytes of OVX E,P ewes are plentiful,long, slender, and irregular in disposition.TEM. X 20,000. Fig. 9. Some microvilli originate in clusters from common sites which are irregular small protrusions from the tanycyte surface. OVX E,p ewes. TEM. X 20,000. + Fig. 10. Irregular and rounded miniblebs are a frequen feature in between microvilli on tanycytes from OVX E,I ewes. TEM. X 21,800. + TANYCYTES IN OVX EWES observed in between microvilli (Fig. 10). Organelles in these tanycytes were typically well-developed as exemplified by numerous profiles of rough endoplasmic reticulum which often were dilated and filled with a moderately electron-dense product (Fig. 11). Golgi complexes were often prominent and enlarged (Fig. 12). Regions of free ribosomes and rosette formations were frequently observed (Fig. 11). Microtubules and bundles of filaments were plentiful in the cell bodies of these tanycytes (Fig. 11). DISCUSSION The concentration of plasma LH was approximately 20-fold higher for the group of control OVX ewes compared to the OVX E,P group. These data confirm that the E,P treatment was sufficient to produce the widely documented negative feedback influence of estradiol on LH secretion (Scaramuzzi et al., 1971; Butler et al., 1972; Reeves et al., 1972; Davis and Borger, 1974). Overall characterization of the normal sheep third ventricle by SEM was first accomplished by Kozlowski et al. (1973). In the present study, the SEM and TEM ultrastructural data are highly reminiscent of the appearance of ependymal tanycytes in wethers and TPtreated wethers which we previously reported (Coates and Davis, 1979). In these earlier experiments we found that most tanycytes in TPtreated wethers possessed a surface nap of delicate yet long microvilli which gave a fuzzy appearance to wide aspects of the floor (mouth) of the infundibular recess region, much like that exhibited by tanycytes in the present OVX E,P ewes. Conversely, surface features of tanycytes in untreated wethers resembled tanycytes of the present long-term OVX ewes in that, although microvilli were never totally absent, their amount was qualitatively less and they were distributed differently than in the TP- or E,P-treated animals. There were also similarities in the internal organization and development of organelles as revealed by TEM. Both the OVX E,P ewes and TP-treated wethers displayed better development of organelles conventionally associated with active synthesis, secretion, andlor absorption, which included well-developed rough endoplasmic reticulum, plentiful free ribosomes and ribosomal rosettes, microtubules and filaments, and prominent Golgi complexes. None of these alone can be used as an index of greater cellular activity, nor did every single + + + 185 tanycyte from each animal in either group exhibit all ultrastructural characteristics for that group. Nevertheless, taken as a whole, these data support the interpretation that tanycytes in sheep deprived of their normal gonadal steroid hormone complement are sensitive to treatment with oxogenous gonodal steroids and display ultrastructural features which are associated with heightened cellular activity (suggestive of synthetic activity andlor absorptionlsecretion). There is at least some evidence to support the notion that tanycytes in several mammalian species may be sensitive to the presence or absence of estrogen. Some tanycytes in rhesus monkeys display an altered morphology in relation to the menstrual cycle and are strikingly altered after ovariectomy and after injection of estrogen following ovariectomy (Knowles and Anand Kumar, 1969). Similar observations were made following OVX and estrogen replacement therapy (Coates, 1974)in another species of monkey, the pig tail macaque. Several reports on tanycytes in rats present evidence that collectively suggests tanycyte morphology varies with the estrous cycle, after ovariectomy and after estrogen treatment following ovariectomy (Kobayashi and Matsui, 1969; Brawer et al., 1974; Mitchell et al., 1975). Normally cyclic ewes exhibit subtle alterations in tanycytes during the estrous cycle (Coates and Davis, 1980). A recent report on the effects of estrogen in seasonally anovulatory mares even suggests localized areas of deciliation associated with estrogen treatment (Melrose and Douglas, 1981). Yet it appears that tanycytes in the third ventricle are responsive ultrastructurally, not only to estrogen but also to a variety of stimuli including testosterone (Knowles and Anand Kumar, 1969; Coates and Davis, 1979).monosodium glutamate (del Cerro et al., 1978), transiently to epinephrine and dopamine (Schechter and Weiner, 1972; Paul1 et al., 1978) and LH-RH (Bruni et al., 1977),and even viral invasion (Bleier and Marsh, 1978). I t is even conceivable that LH itself could influence tanycytes directly. Luteinizing hormone has been detected in the cerebrospinal fluid (CSF)of nonendocrine patients, albeit at low levels (Jordan et al., 1976). Even more interesting in this context is the presence of LH in the CSF of large domestic animals (seasonally anovulatory mares) following injection of saline or gonadotropin releasing hormone (GnRH) with and without steroid pretreatment (Douglas et al., 1979). Fig. 11. The cytoplasm of a tanycyte cell body from an OVX E,p ewe displays a rich development of cell organ elles often associated with synthesis and secretion: Dilated rough endoplasmic reticulum whose cisterna are filled with a moderately electron dense product, free ribosomes and ribosomal rosettes, microtubules. filaments, and mitochon- + + Fig. 12. This tanycyte from an OVX E,@ewe exhibits a distinctly prominent double Golgi complex along with a centriole. TEM. X 22,500. TANYCYTES IN OVX EWES Thus it appears that ependymal tanycytes on the floor of the third ventricle constitute a very plastic tissue which exhibits distinct ultrastructural changes in relation to, but not limited to, gonadal steroids. Nevertheless, if estrogen is involved, what might be the possible relationship of estrogen (or gonadal steroids) to tanycytes? Localization of estrogen to neurons in specific brain areas is now well established (Stumpf, 1970; Pfaff and Keiner, 1973). Furthermore, it has been shown that testosterone is converted to estrogens in brain neurons possessing the appropriate enzymes for estrification (Naftolin e t al., 1972), and is thus believed to exert its effects. Yet to date, there has been no clear-cut localization of either estrogen or testosterone in or on tanycytes. This suggests that 1)their possible presence in tanycytes is so short it cannot be captured by present technology; 2) gonadal steroids are somehow changed within tanycytes and thus cannot be recognized by present technology; or 3) their effect is indirect, possibly via an influence on neurons in areas, such as the arcuate nucleus, which are known to be involved in reproductive events. These possible neuronal influences can be transmitted to tanycytes via synapticlike contacts previously described (Knigge and Scott, 1970). ACKNOWLEDGMENTS Grateful acknowledgment is given to Mr. Chuan Teh, Dr. Mary Doohan, Mr. Sam Prien, and Mrs. Jane Moore-Schwartz for their assistance during the course of the studies. This work was supported by HD 12833 from the NIH and a grant from the Institute for Biomedical Research, TTUSM. LITERATURE CITED Bleier, R., and R. Marsh (1978)Reaction of third ventricular ependyma and supraependymal cells to vesicular stomatit i s virus. SEMiI978iJI: 29-37. Brawer, J.R., and P.S. Lin, and C. Sonnenschein (1974)Morphological plasticity during the estrous cycle in the rat: A scanning electron microscopic study. Anat. Rec., 179: 481-490. Bruni, J.E., D.G. Montemurro, and R.E. 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