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Tanycytes in long-term ovariectomized ewes treated with estrogen exhibit ultrastructural features associated with increased cellular activity.

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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.
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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).
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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).
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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).
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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.
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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.
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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.
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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.
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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.
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Fig. 10. Irregular and rounded miniblebs are a frequen
feature in between microvilli on tanycytes from OVX E,I
ewes. TEM. X 21,800.
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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
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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-
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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.
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